O OH N O O
- ; y - ypp --y y g p p , 16
N N O O P OH O
N O P OH
N O P OH
(SM-041; nonyl (2-(4-pentadecanoylpiperazin-1-yl)e Nthyl) hydr Ooge Pn OH p Ohosphate), N O y y y O P O
y y g OH , 17
OH
HO
O
N O OH and salts and isomers thereof. In an aspect, the disclosure provides a compound having the following structure: 18
N N O O O PH O
( - ; nony (-(-(unecan--y)pperazn--y)ety) yrogen pospate), and salts and isomers thereof. In an aspect, the disclosure provides a compound of Formula III: salt or isomer thereof, where Y O O P O O- R7II) or a
Y is selected R9 N x from t x xhe group consisting of x N R8R9 N x xR9 N x
, 9 x 9 x x9 x
x
9 8
8 9 8 19
R
7 and R
9 are either the same or different and are independently selected from the group consisting of C
2-C
22 alkyl, C
2-C
22 alkenyl, and C
2-C
22 alkynyl , each of which is optionally substituted, optionally R
7, R
9, or R
7 and R
9 are branched, optionally R
7, R
9, or R
7 and R
9 are an optionally substituted cycloalkyl or R
7 and R
9 may join to form an optionally substituted cycloalkyl; R
8 is selected from the group consisting of branched or unbranched C
1-C
7 alkyl, C
2-C
7 alkenyl, and C
2-C
7 alkynyl, each of which is optionally substituted, and x is absent, 1, 2 or 3. In embodiments, R
7 and R
9 are the same. In embodiments, R
7 or R
9 are independently selected from the group consisting of C
4-C
12 alkyl, C
4-C
12 alkenyl, and C
4-C
12 alkynyl, each of which is optionally substituted, optionally wherein R
7 and R
9 are independently selected from the group of C
4-C
12 alkyl, C
4-C
12 alkenyl, and C
4-C
12 alkynyl, each of which is optionally substituted. In embodiments, R
8 is 0, 1, 2, 3, 4, 5, or 6. In embodiments, R
7 or R
9 are independently selected from the group consisting of branched or unbranched C
4-C
12 alkyl, C
4-C
12 alkenyl, and C
4-C
12 alkynyl, each of which is optionally substituted, and R
8 is 0, 1, 2, 3, 4, 5, or 6, optionally wherein R
7 and R
9 are independently selected from the group consisting of branched or unbranched C
4-C
12 alkyl, C
4-C
12 alkenyl, and C
4-C
12 alkynyl, each of which is optionally substituted, and R
2 is 0, 1, 2, 3, 4, 5, or 6. In embodiments, R
8 is 2, 4, or 6. In embodiments, R
7 is selected from the group consisting of branched or unbranched C
6- C
9 alkyl, C
6-C
9 alkenyl, and C
6-C
9 alkynyl, each of which is optionally substituted, R
9 is selected from the group consisting of branched or unbranched C
6-C
9 alkyl, C
6-C
9 alkenyl, and C
6-C
9 alkynyl, each of which is optionally substituted, and R
8 is 2, 3, 4, 5, or 6. In embodiments, R
8 is 2, 4, or 6.
In embodiments, R
7 and R
9 are independently optionally substituted C
6-C
9 alkyl, and R
2 is 2, 3, 4, 5, or 6, optionally wherein R
8 is 2, 4, or 6. In embodiments, R
7 is C
9, R
9 is C
6-C
17 alkyl, and R
8 is absent, 1, or 2. In embodiments, R
7 and R
9 are independently an alkyl selected from the group consisting of heptane, octane, nonane, decane, undecane, and dodecane, each of which is optionally substituted. In embodiments, one or more of R
7 and R
9 are independently an alkenyl selected from the group consisting of hept-1-ene, hept-2-ene, hept-3-ene, oct-1-ene, oct-2-ene, oct-3-ene, oct-4-ene, non-1-ene, non-2-ene, non-3-ene, non-4-ene, non-5-ene, dec-1-ene, dec-2-ene, dec-3-ene, dec-4- ene, dec-5-ene, dec-6-ene, undec-1-ene, undec-2-ene, undec-3-ene, undec-4-ene, undec-5-ene, undec-6-ene, undec-7-ene, dodec-1-ene, dodec-2-ene, dodec-3-ene, dodec-4-ene, dodec-5-ene, dodec-6-ene, dodec-8-ene, and an alkenyl group comprising two or more double bonds, each of which is optionally substituted. In embodiments, one or more of R
7 and R
9 are independently an alkynyl selected from the group consisting of hept-1-yne, hept-2-yne, hept-3-yne, oct-1-yne, oct-2-yne, oct-3-yne, oct-4-yne, non-1-yne, non-2-yne, non-3-yne, non-4-yne, non-5-yne, dec-1-yne, dec-2-yne, dec-3-yne, dec-4- yne, dec-5-yne, dec-6-yne, undec-1-yne, undec-2-yne, undec-3-yne, undec-4-yne, undec-5-yne, undec-6-yne, undec-7-yne, dodec-1-yne, dodec-2-yne, dodec-3-yne, dodec-4-yne, dodec-5-yne, dodec-6-yne, dodec-8-yne, and an alkynyl group comprising two or more triple bonds, each of which is optionally substituted. In an aspect, the disclosure provides a pharmaceutical composition comprising a lipid of Formula I: R1N m n X A O O O PH O R2 (I)
or a salt or isomer thereof, where 21
A is a bond, C
1-C
22 alkyl, C
2-C
22 alkenyl, C
2-C
22 alkynyl, or C
3-C
8 cycloalkyl, each of which is optionally substituted, X is N or CH, R
1 is C
5-C
22 alkyl, C
5-C
22 alkenyl, C
5-C
22 alkynyl, C
3-C
22 cycloalkyl, or C(O)C
4-C
21 alkyl, each of which is optionally substituted; R
2 is C
2-C
22 alkyl, C
2-C
22 alkenyl, C
2-C
22 alkynyl, or C
3-C
22 cycloalkyl, each of which is optionally substituted; and each of m and n is independently 0, 1, 2, or 3. In an aspect, the disclosure provides a pharmaceutical composition comprising a lipid of a compound of Formula II: R R33 N R5 A1 R6 O OH O P O R4 ,
or a salt or isomer thereof, where A
1 is C
1-C
22 alkyl, C
2-C
22 alkenyl, or C
2-C
22 alkynyl, each of which includes at least one substitution; or C
3-C
8 cycloalkyl or heterocycloalkyl, each of which is optionally substituted; R
3 is C
7-C
22 alkyl, C
7-C
22 alkenyl, C
7-C
22 alkynyl, or C
4-C
22 cycloalkyl, each of which is optionally substituted; R
4 is C
2-C
16 alkyl, C
2-C
16 alkenyl, C
2-C
16 alkynyl, or C
3-C
22 cycloalkyl, each of which is optionally substituted; and each of R
5 and R
6 is independently a bond, C
1-C
7 alkyl, C
2-C
7 alkenyl, or C
2-C
7 alkynyl, each of which is optionally substituted.
In embodiments of the pharmaceutical composition including Formula I, or salt or isomer thereof, R
1 and R
2 are the same. In embodiments of the pharmaceutical composition including Formula I, or salt or isomer thereof, R
1 is selected from the group consisting of C
5-C
12 alkyl, C
5-C
12 alkenyl, and C
5-C
12 alkynyl, each of which is optionally substituted and R
2 is selected from the group consisting of C
4- C
12 alkyl, C
4-C
12 alkenyl, and C
4-C
12 alkynyl, each of which is optionally substituted. In embodiments of the pharmaceutical composition including Formula I, or salt or isomer thereof, m is 2 and A is selected from the group consisting of C
2-C
6 alkyl, C
2-C
6 alkenyl, and C
2- C
6 alkynyl, each of which includes at least one substitution, or A is C
3-C
5 cycloalkyl. In embodiments of the pharmaceutical composition including Formula I, or salt or isomer thereof, R
1 is selected from the group consisting of branched or unbranched C
5-C
9 alkyl, C
5-C
9 alkenyl, and C
5-C
9 alkynyl, each of which is optionally substituted, and R
2 is selected from the group consisting of branched or unbranched C
4-C
9 alkyl, C
4-C
9 alkenyl, and C
4-C
9 alkynyl, each of which is optionally substituted. In embodiments of the pharmaceutical composition including Formula I, or salt or isomer thereof, m is 2 and A is selected from the group consisting of C
2-C
6 alkyl, C
2-C
6 alkenyl, and C
2- C
6 alkynyl, each of which includes at least one substitution, or A is C
3-C
5 cycloalkyl. In embodiments of the pharmaceutical composition including Formula I, or salt or isomer thereof, R
1 is selected from the group consisting of branched or unbranched C
6-C
9 alkyl, C
6-C
9 alkenyl, and C
6-C
9 alkynyl, each of which is optionally substituted, and R
2 is selected from the group consisting of branched or unbranched C
6-C
9 alkyl, C
6-C
9 alkenyl, and C
6-C
9 alkynyl, each of which is optionally substituted. In embodiments of the pharmaceutical composition including Formula I, or salt or isomer thereof, m is 2 and A is selected from the group consisting of C
2-C
6 alkyl, C
2-C
6 alkenyl, and C
2- C
6 alkynyl, each of which includes at least one substitution, or A is C
3-C
5 cycloalkyl. In embodiments of the pharmaceutical composition including Formula I, or salt or isomer thereof, R
1 and R
2 are independently optionally substituted C
6-C
9 alkyl, m is 2, and A is selected 23
from the group consisting of C
2-C
6 alkyl, C
2-C
6 alkenyl, and C
2-C
6 alkynyl, each of which includes at least one substitution, or A is C
3-C
5 cycloalkyl. In embodiments of the pharmaceutical composition including Formula I, or salt or isomer thereof, R
1 is optionally substituted C
6-C
9 alkyl, R
2 is optionally substituted C
9 alkyl, m is 2, and A is selected from the group consisting of C
2-C
6 alkyl, C
2-C
6 alkenyl, and C
2-C
6 alkynyl, each of which is includes at least one substitution, or A is C
3-C
5 cycloalkyl. In embodiments of the pharmaceutical composition including Formula I, or salt or isomer thereof, R
1 and R
2 are independently an alkyl selected from the group consisting of heptane, octane, nonane, decane, undecane, and dodecane, each of which is optionally substituted. In embodiments of the pharmaceutical composition including Formula I, or salt or isomer thereof, R
1 and R
2 are independently an alkenyl selected from the group consisting of hept-1-ene, hept-2-ene, hept-3-ene, oct-1-ene, oct-2-ene, oct-3-ene, oct-4-ene, non-1-ene, non-2-ene, non-3- ene, non-4-ene, non-5-ene, dec-1-ene, dec-2-ene, dec-3-ene, dec-4-ene, dec-5-ene, dec-6-ene, undec-1-ene, undec-2-ene, undec-3-ene, undec-4-ene, undec-5-ene, undec-6-ene, undec-7-ene, dodec-1-ene, dodec-2-ene, dodec-3-ene, dodec-4-ene, dodec-5-ene, dodec-6-ene, dodec-8-ene, and an alkenyl group comprising two or more double bonds, each of which is optionally substituted. In embodiments of the pharmaceutical composition including Formula I, or salt or isomer thereof, one or more of R
1 and R
2 are independently an alkynyl selected from the group consisting of hept-1-yne, hept-2-yne, hept-3-yne, oct-1-yne, oct-2-yne, oct-3-yne, oct-4-yne, non-1-yne, non- 2-yne, non-3-yne, non-4-yne, non-5-yne, dec-1-yne, dec-2-yne, dec-3-yne, dec-4-yne, dec-5-yne, dec-6-yne, undec-1-yne, undec-2-yne, undec-3-yne, undec-4-yne, undec-5-yne, undec-6-yne, undec-7-yne, dodec-1-yne, dodec-2-yne, dodec-3-yne, dodec-4-yne, dodec-5-yne, dodec-6-yne, dodec-8-yne, and an alkynyl group comprising two or more triple bonds, each of which is optionally substituted. In embodiments of the pharmaceutical composition including Formula II, or salt or isomer thereof, R
3 and R
4 are the same, optionally wherein R
5 and R
6 are the same.
In embodiments of the pharmaceutical composition including Formula II, or salt or isomer thereof, R
3 is selected from the group consisting of C
7-C
12 alkyl, C
7-C
12 alkenyl, and C
7-C
12 alkynyl, each of which is optionally substituted, R
4 is selected from the group consisting of C
4-C
12 alkyl, C
4-C
12 alkenyl, and C
4-C
12 alkynyl, each of which is optionally substituted, wherein R
5 and R
6 are independently selected from the group consisting of a bond, C
2-C
6 alkyl, C
2-C
6 alkenyl, or C
2-C
6 alkynyl, each of which is optionally substituted, optionally wherein R
5 and R
6 are both methyl group, or either R
5 or R
6 is a methyl group and the other is a bond. In embodiments of the pharmaceutical composition including Formula II, or salt or isomer thereof, A
1 is selected from the group consisting of optionally branched C
2-C
6 alkyl, C
2-C
6 alkenyl, and C
2-C
6 alkynyl, each of which includes at least one substitution, or A
1 is C
3-C
5 cycloalkyl. In embodiments of the pharmaceutical composition including Formula II, or salt or isomer thereof, A
1 is selected from the group consisting of optionally branched C
3-C
4 alkyl, C
3-C
4 alkenyl, and C
3-C
4 alkynyl, each of which includes at least one substitution, or A
1 is C
3 cycloalkyl. In embodiments of the pharmaceutical composition including Formula II, or salt or isomer thereof, R
3 is selected from the group consisting of branched or unbranched C
7-C
9 alkyl, C
7-C
9 alkenyl, and C
7-C
9 alkynyl, each of which is optionally substituted and R
4 is selected from the group consisting of branched or unbranched C
4-C
9 alkyl, C
4-C
9 alkenyl, and C
4-C
9 alkynyl, each of which is optionally substituted. In embodiments of the pharmaceutical composition including Formula II, or salt or isomer thereof, A
1 is selected from the group consisting of optionally branched C
2-C
6 alkyl, C
2-C
6 alkenyl, and C
2-C
6 alkynyl, each of which includes at least one substitution, or A
1 is selected from the group consisting of optionally branched C
3-C
4 alkyl, C
3-C
4 alkenyl, and C
3-C
4 alkynyl, each of which includes at least one substitution. In embodiments of the pharmaceutical composition including Formula II, or salt or isomer thereof, R
3 is selected from the group consisting of branched or unbranched C
7-C
9 alkyl, C
7-C
9 alkenyl, and C
7-C
9 alkynyl, each of which is optionally substituted, and R
4 is selected from the group consisting of branched or unbranched C
6-C
9 alkyl, C
6-C
9 alkenyl, and C
6-C
9 alkynyl, each of which is optionally substituted.
In embodiments of the pharmaceutical composition including Formula II, or salt or isomer thereof, A
1 is selected from the group consisting of optionally branched C
2-C
6 alkyl, C
2-C
6 alkenyl, and C
2-C
6 alkynyl, each of which includes at least one substitution, or A
1 is selected from the group consisting of optionally branched C
3-C
4 alkyl, C
3-C
4 alkenyl, and C
3-C
4 alkynyl, each of which includes at least one substitution. In embodiments of the pharmaceutical composition including Formula II, or salt or isomer thereof, R
3 is optionally substituted C
7-C
9 alkyl and R
4 is optionally substituted C
6-C
9 alkyl, wherein A
1 is selected from the group consisting of optionally branched C
2-C
6 alkyl, C
2-C
6 alkenyl, and C
2-C
6 alkynyl, each of which includes at least one substitution, or A
1 is selected from the group consisting of optionally branched C
3-C
4 alkyl, C
3-C
4 alkenyl, and C
3-C
4 alkynyl, each of which includes at least one substitution. In embodiments of the pharmaceutical composition including Formula II, or salt or isomer thereof, R
3 and R
4 are independently an alkyl selected from the group consisting of heptane, octane, nonane, decane, undecane, and dodecane, each of which is optionally substituted. In embodiments of the pharmaceutical composition including Formula II, or salt or isomer thereof, one or more of R
3 and R
4 are independently an alkenyl selected from the group consisting of hept-1-ene, hept-2-ene, hept-3-ene, oct-1-ene, oct-2-ene, oct-3-ene, oct-4-ene, non-1-ene, non- 2-ene, non-3-ene, non-4-ene, non-5-ene, dec-1-ene, dec-2-ene, dec-3-ene, dec-4-ene, dec-5-ene, dec-6-ene, undec-1-ene, undec-2-ene, undec-3-ene, undec-4-ene, undec-5-ene, undec-6-ene, undec-7-ene, dodec-1-ene, dodec-2-ene, dodec-3-ene, dodec-4-ene, dodec-5-ene, dodec-6-ene, dodec-8-ene, and an alkenyl group comprising two or more double bonds, each of which is optionally substituted. In embodiments of the pharmaceutical composition including Formula II, or salt or isomer thereof, one or more of R
3 and R
4 are independently an alkynyl selected from the group consisting of hept-1-yne, hept-2-yne, hept-3-yne, oct-1-yne, oct-2-yne, oct-3-yne, oct-4-yne, non-1-yne, non- 2-yne, non-3-yne, non-4-yne, non-5-yne, dec-1-yne, dec-2-yne, dec-3-yne, dec-4-yne, dec-5-yne, dec-6-yne, undec-1-yne, undec-2-yne, undec-3-yne, undec-4-yne, undec-5-yne, undec-6-yne, undec-7-yne, dodec-1-yne, dodec-2-yne, dodec-3-yne, dodec-4-yne, dodec-5-yne, dodec-6-yne,
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) dodec-8-yne, and an alkynyl group comprising two or more triple bonds, each of which is optionally substituted. In embodiments of the pharmaceutical composition including Formula II, or salt or isomer thereof, A
1 is an optionally substituted cycloalkyl and R
5 and R
6 are independently selected from the group consisting of optionally branched C
2-C
6 alkyl, C
2-C
6 alkenyl, and C
2-C
6 alkynyl, each of which is optionally substituted, optionally R
5 is absent, optionally R
6 is absent. In embodiments of the pharmaceutical composition including Formula II, or salt or isomer thereof, A
1 is an optionally substituted cycloalkyl having three members and R
5 and R
6 are independently selected from the group consisting of optionally branched C
2-C
6 alkyl, C
2-C
6 alkenyl, and C
2-C
6 alkynyl, each of which is optionally substituted, optionally R
5 is absent, optionally R
6 is absent. In an aspect, the disclosure provides a lipid particle comprising any of the above compounds. In embodiments, the lipid particle, further includes a therapeutic agent. In embodiments, the therapeutic agent is a nucleic acid. In an aspect, the disclosure provides a pharmaceutical composition comprising any of the above lipid particles and a pharmaceutically acceptable excipient, carrier, or diluent. In an aspect, the disclosure provides a nucleic acid-lipid particle for delivering a nucleic acid cargo to a lung tissue of a subject, the nucleic acid-lipid particle comprising nonyl (2-(4- (undecan-6-yl)piperazin-1-yl)ethyl) hydrogen phosphate (SM-037) comprising 30-70 mol % or about 40-60 mol % or about 50 mol % of the total lipid present in the nucleic acid-lipid particle. In embodiments, the nucleic acid-lipid includes a conjugated lipid that inhibits aggregation of particles comprising from 0.01 to 2% of the total lipid present, optionally wherein the conjugated lipid comprises a polyethyleneglycol (PEG)-lipid conjugate, optionally wherein the PEG of the PEG-lipid conjugate has an average molecular weight of from 550 Daltons to 3000 Daltons, optionally wherein the PEG-lipid conjugate is a PEG2000-lipid conjugate, optionally wherein the PEG2000-lipid conjugate comprises one or more of 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2k) and 1,2-distearoyl-rac-glycero-3- 27
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) methoxypolyethylene glycol-2000 (DSG-PEG2k), optionally wherein the PEG2000-lipid conjugate is 1,2-Dimyristoyl-rac–glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k), optionally wherein the nucleic acid-lipid particle comprises a PEG-lipid conjugate at a concentration selected from the group consisting of about 0.5 mol % of the total lipid present in the nucleic acid-lipid particle, about 1.0 mol % of the total lipid present in the nucleic acid-lipid particle, and about 1.5 mol % of the total lipid present in the nucleic acid-lipid particle. In embodiments, the PEG-lipid conjugate is DMG-PEG2k comprising about 1.5 mol % of the total lipid present in the nucleic acid-lipid particle. In embodiments, the nucleic acid-lipid includes one or more non-cationic lipids comprising from 20 mol % to 80 mol % of the total lipid present in the lipid-nucleic acid particle, optionally wherein the one or more non-cationic lipids comprise cholesterol or a derivative thereof. In embodiments, the nucleic acid-lipid includes cholesterol or a derivative thereof at a concentration range selected from the group consisting of 35 mol % to 45 mol % of the total lipid present in the nucleic acid-lipid particle, 45 mol % to 55 mol % of the total lipid present in the nucleic acid-lipid particle, and 55 mol % to 65 mol % of the total lipid present in the nucleic acid- lipid particle, optionally wherein the cholesterol or a derivative thereof is about 50% of the total lipid present in the nucleic acid-lipid particle. In embodiments, the nucleic acid-lipid includes a cationic lipid selected from the group consisting of Dimethyldioctadecylammonium, Bromide Salt (DDAB), N-(4-carboxybenzyl)-N,N- dimethyl-2,3-bis(oleoyloxy) propan-1-aminium (DOBAQ), 1,2-dioleoyl-3-trimethylammonium- propane or 18:1 TAP, a di-chain or gemini, cationic lipid (DOTAP), 1,2-di-O-octadecenyl-3- trimethylammonium propane, chloride salt (DOTMA), ethyl phosphatidylcholine (EPC), and trimethyl sphingosine. In embodiments, the nucleic acid-lipid includes a cationic lipid that has the following structure: 28
Attorney Ref.: BN00004.0144 O OME-013WO S O S (PCT Application) Z N 3 NH 3 NH
, CNHl
+ C NlH
+ H2N NH2 h
+ Caln- cholesterol or a derivative thereof, optionally wherein the one or more non-cationic lipid other than cholesterol or a derivative thereof comprises from 5 mol % to 20 mol % of the total lipid present in the lipid-nucleic acid particle, optionally wherein the one or more non-cationic lipid other than cholesterol or a derivative thereof comprises about 10 mol % of the total lipid present in the nucleic acid-lipid particle. In embodiments, the nucleic acid-lipid includes one or more non-cationic lipid other than cholesterol or a derivative thereof comprises a non-cationic lipid selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and β- sitosterol, optionally wherein the one or more non-cationic lipid other than cholesterol or a derivative thereof is dioleoylphosphatidylcholine (DOPC). In embodiments, the nucleic acid-lipid includes an ionizable phospholipid selected from the group consisting of 29
Attorney Ref.: BN00004.0144 OME-013WO O (PCT Application) N N O O PH O ,
O
N N O P O OH
, 30
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) N N O P O OH
O O
O O
( - ; nony (-(-(unecan--y)pperazn--y)exy) yrogen pospae), 31
Attorney Ref.: BN00004.0144 O O OME-013WO (PCT Application) O P OH
O O
N N O P OH
(SM-063; 2-(4-(dihexylamino)piperidin-1-yl)ethyl nonyl hydrogen phosphate), 32
Attorney Ref.: BN00004.0144 O OME-013WO (PCT Application O OH ) N N P O
- ; oy - -u ecae--y -,- aepa--y e y y oge pospae, N N O OH
(SM-108; nonyl (3-(4-(undecan-6-yl)piperazin-1-yl)propyl) hydrogen phosphate), N N O P OH
(SM-116; nonyl (3-(4-(undecane-6-yl)-1,4-diazepan-1-yl)propyl) hydrogen phosphate), 33
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application N N OO P O ) H
O
( - ; ( )--(-( exyamno)pper n--y)e y non--en--y yrogen pospae), O P O N
(SM-119; 2-(4-((dihexylamino)methyl)piperidin-1-yl)ethyl nonyl hydrogen phosphate), 34
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) N N O O P O OH
- ; - y y - - y pp --y y y g p p , N N O P OH
(SM-122; 2-(4-(dipentylamino)piperidin-1-yl)ethyl nonyl hydrogen phosphate), N N O P O
(SM-123; nonyl (2-(4-(tridecan-7-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate), 35
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) N N O O P OH O
; y y pp y y y g p p , N N O P O
(SM-126; decyl (2-(4-(tridecan-7-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate), and salts and isomers thereof. In embodiments, the nucleic acid-lipid includes a an ionizable phospholipid selected from the group consistin Og of N N O O P O OH
y y y y y g , 36
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application)
O OH N O O
- ; y - ypp --y y g p p , 37
Attorney Ref.: BN00004.0144 OME-013WO O (PCT Application) N N O O PH O
N O P OH
N O P OH
(SM-041; nonyl (2-(4-pentadecanoylpiperazin-1-yl)e Nthyl) hydr Ooge Pn OH p Ohosphate), N O
y y y y O P O y g OH , 38
Attorney Ref.: BN00004.0144 OME-013WO N O P O (PCT Application) O OH
HO
O
N O OH and salts and isomers thereof. In embodiments, the nucleic acid cargo comprises a synthetic or naturally occurring RNA or DNA, or derivatives thereof, optionally wherein the nucleic acid cargo is a modified RNA, optionally wherein the modified RNA is selected from the group consisting of a modified mRNA, a modified antisense oligonucleotide and a modified siRNA, optionally wherein the modified mRNA encodes a nucleic acid modulating controller. In embodiments, the nucleic acid cargo comprises one or more modifications selected from the group consisting of 2′-O-methyl modified nucleotides, a nucleotide comprising a 5′- phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, a 2′-deoxy-2′- fluoro modified nucleotide, a 5′-methoxy-modified nucleotide (e.g., 5′-methoxyuridine), a 2′- 39
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non- natural base comprising nucleotide; internucleoside linkages or backbones including phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. In embodiments, the lung tissue is selected from the group consisting of epithelium, endothelium, interstitial connective tissue, blood vessel, hematopoietic tissue, lymphoid tissue, and pleura. In embodiments, the nucleic acid-lipid particle comprises SM-037 at about 30 mol % of the total lipid present in the nucleic acid-lipid particle, cholesterol at about 50 mol % of the total lipid present in the nucleic acid-lipid particle, SM-005 at about 50 mol % of the total lipid present in the nucleic acid-lipid particle, and DMG-PEG2k at about 1.5 mol % of the total lipid present in the nucleic acid-lipid particle. In embodiments, intravenous administration of the nucleic acid-lipid particle to the subject results in expression of the nucleic acid cargo in cells of the lung tissue of the subject at a level that is at least two-fold higher than expression of the nucleic acid cargo in cells of liver, heart, spleen, ovary, pancreas and kidney of the subject, optionally wherein expression of the nucleic acid cargo in cells of the lung tissue of the subject is at least three-fold higher, optionally at least four-fold higher, optionally at least five-fold higher, optionally at least six-fold higher, optionally at least seven-fold higher, optionally at least eight-fold higher, optionally at least nine-fold higher, optionally at least ten-fold higher, optionally at least eleven-fold higher, optionally at least twelve- fold higher, optionally at least thirteen-fold higher, optionally at least fourteen-fold higher, optionally at least fifteen-fold higher, optionally at least twenty-fold higher, than expression of the nucleic acid cargo in cells of liver, heart, spleen, ovary, pancreas and kidney of the subject. 40
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) In embodiments, intravenous administration of the nucleic acid-lipid particle or pharmaceutical composition to the subject results in localization of the nucleic acid-lipid particle to the lung tissue of the subject at an at least two-fold higher concentration than the concentration of the nucleic acid-lipid particle in one or more other tissues of the subject selected from the group consisting of heart, spleen, ovaries and pancreas, optionally wherein at least three-fold, optionally at least four-fold, optionally at least five-fold, optionally at least six-fold higher concentration of the nucleic acid-lipid particle is located in lung as compared to one or more other tissues of the subject selected from the group consisting of heart, spleen, ovaries and pancreas. In embodiments, the nucleic acid-lipid particle or pharmaceutical composition is administered to treat a lung disease or disorder, optionally wherein the disease or disorder is selected from the group consisting of lung cancer, pneumonia, pulmonary fibrosis, COPD, asthma, bronchiectasis, sarcoidosis, pulmonary hypertension, emphysema, alpha-1 antitrypsin deficiency, aspergillosis, bronchiolitis, bronchitis, pneumoconiosis, a coronavirus, Middle Eastern Respiratory Syndrome, Severe Acute Respiratory Syndrome, cystic fibrosis, Legionnaire’s disease, influenza, pertussis, pulmonary embolism and tuberculosis. In an aspect, the disclosure provides a compound of Formula IV: R R1100 N R5 A1 R6 O OH 1
1 22 2 22 2 22 O P O R11V)
or a salt or isomer thereof, wherein A is C -C alkyl, C -C alkenyl, or C -C alkynyl, each of which includes at least one substitution; or C
3-C
8 cycloalkyl or heterocylcloalkyl, each of which is optionally substituted; R
10 is C
5-C
22 alkyl, C
5-C
22 alkenyl, C
5-C
22 alkynyl, or C
4-C
22 cycloalkyl, each of which is optionally substituted; R
11 is C
5-C
16 alkyl, C
5-C
16 alkenyl, C
5-C
16 alkynyl, or C
3-C
22 cycloalkyl, each of which is optionally substituted; and 41
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) each of R
5 and R
6 is independently a bond, C
1-C
7 alkyl, C
2-C
7 alkenyl, or C
2-C
7 alkynyl, each of which is optionally substituted. In embodiments, R
10 and R
11 are the same, optionally wherein R
5 and R
6 are the same. In embodiments, R
10 is selected from the group consisting of C
5-C
6 alkyl, C
5-C
6 alkenyl, and C
5-C
6 alkynyl, each of which is optionally substituted and R
11 is selected from the group consisting of C
5-C
12 alkyl, C
5-C
12 alkenyl, and C
5-C
12 alkynyl, each of which is optionally substituted, wherein R
5 and R
6 are independently selected from the group consisting of a bond, C
2- C
6 alkyl, C
2-C
6 alkenyl, or C
2-C
6 alkynyl, each of which is optionally substituted, optionally wherein R
5 and R
6 are both methyl group, or either R
5 or R
6 is a methyl group and the other is a bond. In embodiments, R
10 is selected from the group consisting of branched or unbranched C
5- C
6 alkyl, C
5-C
6 alkenyl, and C
5-C
6 alkynyl, each of which is optionally substituted, and R
11 is selected from the group consisting of branched or unbranched C
5-C
9 alkyl, C
5-C
9 alkenyl, and C
5- C
9 alkynyl, each of which is optionally substituted. In an aspect, the disclosure provides a compound selected from: N N O O P OH O
(SM-064; nonyl (2-(4-(undecane-6-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate), 42
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application N N OO P O ) H
O
( - ; ( )--(-( exyamno)pper n--y)e y non--en--y yrogen pospae), O P O N
(SM-119; 2-(4-((dihexylamino)methyl)piperidin-1-yl)ethyl nonyl hydrogen phosphate), 43
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) N N O O P O OH
- ; - y y - - y pp --y y y g p p , N N O P OH
(SM-122; 2-(4-(dipentylamino)piperidin-1-yl)ethyl nonyl hydrogen phosphate), or N N O P OH O
(SM-124; decyl (2-(4-(dihexylamino)piperidin-1-yl)ethyl) hydrogen phosphate). Definitions Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard 44
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) deviations of the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Unless otherwise clear from context, all numerical values provided herein are modified by the term "about." As used herein, "fully closed RNA" and "circular RNA" refer to closed-loop oligoribonucleotide molecules in which the free 3' and 5' ends found in linear RNA forms are joined together to form a closed-loop that appears to render them stable and long-lasting. Without wishing to be bound by theory, this is believed to be due to the lack of free ends making such fully closed/circular RNAs resistant to exonuclease digestion. Certain such closed-loop RNAs have recently been designed to include translatable sequences (e.g., mRNAs) in a format commercially referred to as "Endless RNA™" or "eRNA" (refer, e.g., to U.S. Publication Nos.2022/0257794 and 2022/0143062, and to U.S. Patent No. 10,953,033). Fully closed or circular RNAs can therefore refer to a mRNA that is circular and reads through continuously. Without wishing to be bound by theory, circular RNA has been described as a versatile synthetic RNA platform that instructs cells to express a desired therapeutic protein and, because of its natural stability, the protein expression is persistent for long periods of time (in contrast with the transient existence of linear translatable RNA). In addition, because of its lack of immunogenicity, circular RNA has also been described to allow for repeat redosing; and because of its inherent stability, circular RNA has also been described to allow for multiple routes of administration, including intravenous dosing, subcutaneous dosing, topical dosing, intratracheal administration, etc. The term "lipid" refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) "simple lipids" which include fats and oils as well as waxes; (2) "compound lipids" which include phospholipids and glycolipids; (3) "derived lipids" such as steroids. 45
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) As used herein, the term "cationic lipid" refers to any lipid species that carries a net positive charge at a selected pH such as, for example, physiological pH. A cationic lipid may have a head group that is always positively charged in aqueous solution (an "obligate cationic lipid"). For example and without limitation, an obligate cationic lipid may have a quaternary amine as a head group. Alternatively, a cationic lipid may have a head group that accepts a proton in solution such that the lipid exists predominantly as a cation below its pKa and predominantly as a neutral moiety above its pKa, e.g., it may have a pH-titratable amino head group (e.g., for an "ionizable lipid," as defined infra). For example and without limitation, an ionizable lipid may have a primary, secondary, or tertiary amine as a head group, (e.g., an alkylamino or dialkylamino head group). In some embodiments, the ionizable lipids comprise: a protonatable tertiary amine (e.g., pH- titratable) head group; C
18 hydrocarbon chains e.g., alkyl, alkenyl, or alkynyl chains, wherein each hydrocarbon chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and ether, ester, or ketal linkages between the head group and hydrocarbon chains. Examples of obligate cationic lipids include, but are not limited to, Dimethyldioctadecylammonium, Bromide Salt (DDAB), N-(4-carboxybenzyl)-N,N-dimethyl- 2,3-bis(oleoyloxy) propan-1-aminium (DOBAQ), 1,2-dioleoyl-3-trimethylammonium-propane or 18:1 TAP, a di-chain or gemini, cationic lipid (DOTAP), 1,2-di-O-octadecenyl-3- trimethylammonium propane, chloride salt (DOTMA), ethyl phosphatidylcholine (EPC), and trimethyl sphingosine A range of forms of the obligate cationic lipid EPC, as well as related forms of obligate cationic phosphatidylcholines, are commercially available. Ethyl phosphatidylcholine, 18:1 EPC (Cl Salt), also known as 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt), has the following structure: .
18:0 EPC (Cl Salt), also known as 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (chloride salt), has the following structure: 46
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) .
16:0 EPC (Cl Salt), also known as 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (chloride salt), has the following structure: .
14:0 EPC (Cl Salt), also known as 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (chloride salt), has the following structure: .
12:0 EPC (Cl Salt), also known as 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (chloride salt), has the following structure:
. 14:1 EPC (Tf Salt), also known as 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (Tf salt), has the following structure: 47
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) .
16:0-18:1 EPC (Cl Salt), also known as 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (chloride salt), has the following structure: .
18:1 EPC (Cl Salt), also known as 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt), has the following structure: .
16:0-18:0 PC, also known as 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, has the following structure: . 1
6:0/16:1(9Z)-PC, also known as 1-(1-enyl-palmitoyl)-2-palmitoleoyl-sn-glycero-3- phosphocholine, has the following structure: 48
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) .
16:0-18:2 PC, also known as 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, has the following structure: .
18:0-18:1(9Z)-PC, also known as 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, has the following structure: .
18:0-18:2(9Z, 12Z)-PC, also known as 1-Octadecanyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3- phosphocholine, has the following structure: .
18:1-18:2(9Z, 12Z)-PC, also known as 1-(9Z,12Z-octadecadienoyl)-2-(9Z-octadecenoyl)-glycero- 3-phosphocholine, has the following structure: 49
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application)
. As used herein, the term "ionizable lipid" or "ionizable cationic lipid" refers to a lipid that becomes cationic (protonated) as the pH is lowered below the pKa of the ionizable group of the lipid but is progressively more neutral at higher pH values. When a component of a lipid-nucleic acid particle, at pH values below the pKa, the lipid is then able to associate with negatively charged polynucleic acids. Certain examples of such ionizable lipids include lipids and salts thereof having one, two, three, or more fatty acid or fatty hydrocarbon chains and a pH-titratable amino head group (e.g., an alkylamino or dialkylamino head group). Exemplary ionizable lipids include, without limitation, 1,2-Dioleoyl-3-dimethylammonium-propane (DODAP), 9-Heptadecanyl 8- {(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), disulfanediylbis(ethane-2,1-diyl)bis(piperidine-1,4-diyl)bis(ethane-2,1-diyl)bis(oxy)bis(2- oxoethane-2,1-diyl)bis(4,1-phenylene) dioleate (SS-OP), Dimethyl Sphingosine, 3-(N—(N′,N′- dimethylaminoethane)-carbamoyl)cholesterol (DC-Cholesterol), C12-200; N4-Cholesteryl- Spermine HCl Salt (GL67); N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); 1,2-distearyloxy- N,N-dimethyl-3-aminopropane (DSDMA); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA); 1,2-dilinolenyloxy- N,N-dimethyl-3-aminopropane (DLenDMA); 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA); 1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinK-DMA); 1,2- dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DlinKC2-DMA) (also known as Dlin-C2K- 50
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) DMA, XTC2, and C2K); 2,2-dilinoleyl-4-(3-dimethylaminopropyl)[1,3]-dioxolane (Dlin-K-C3- DMA); 2,2-dilinoleyl-4-(4-dimethylaminobutyl)[1,3]-dioxolane (Dlin-K-C4-DMA); 1,2- dilinolenyloxy-4-(2-dimethylaminoethyl)- [1,3]-dioxolane (γ-Dlen-C2K-DMA); 1,2-di-γ- linolenyloxy-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (γ-Dlen-C2K-DMA); dilinoleylmethyl- 3-dimethylaminopropionate (Dlin-M-C2-DMA) (also known as MC2); (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (Dlin-M-C3-DMA) (also known as MC3); 3-(dilinoleylmethoxy)-N,N-dimethylpropan-1-amine (Dlin-MP-DMA) (also known as 1-B11); 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)- octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA); (2R) 2-({8-[(3β)-cholest-5-en- 3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (R- Octyl-CLinDMA); (2S) 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)- octadeca-9,12-dien-1-yloxy]propan-1-amine (S-Octyl-CLinDMA); (2S)-1-{7-[(3β)-cholest-5-en- 3-yloxy]heptyloxy}-3-[(4Z)-dec-4-en-1-yloxy]-N, N -dimethylpropan-2-amine; (2R)-1-{4-[(3β)- cholest-5-en-3-yloxy]butoxy}-3-[(4Z)-dec-4-en-1-yloxy]-N,N-dimethylpropan-2-amine; 1-[(2R)- 1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-yl]guanidine; 1-[(2R)-1-{7- [(3β)-cholest-5-en-3-yloxy]heptyloxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1- yloxy]propan-2-amine; 1-[(2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine; (2S)-1-({6-[(3β))-cholest-5-en-3- yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9Z)-octadec-9-en-1-yloxy]propan-2-amine; (3β)-3-[6- {[(2S)-3-[(9Z)-octadec-9-en-1-yloxyl]-2-(pyrrolidin-1-yl)propyl]oxy}hexyl)oxy]cholest-5-ene; (2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-amine; (2R)-1-({8-[(3β)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-(pentyloxy)propan-2-amine; (2R)-1-({8-[(3β)- cholest-5-en-3-yloxy]octyl}oxy)-3-(heptyloxy)-N,N-dimethylpropan-2-amine; (2R)-1-({8-[(3β)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(2Z)-pent-2-en-1-yloxy]propan-2-amine; (2S)-1-butoxy-3-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethylpropan-2-amine; (2S- 1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-3-[2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9- hexadecafluorononyl)oxy]-N,N-dimethylpropan-2-amine; 2-amino-2-{[(9Z,12Z)-octadeca-9,12- dien-1-yloxy]methyl}propane-1,3-diol; 2-amino-3-({9-[(3β,8ξ,9ξ,14ξ,17ξ,20ξ)-cholest-5-en-3- yloxy]nonyl}oxy)-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol; 2-ammo-3- ({6-[(3β,8ξ,9ξ,14ξ,17ξ,20ξ)-cholest-5-en-3-yloxy]hexyl}oxy)-2-{[(9Z)-octadec-9-en-1- yloxy]methyl}propan-1-ol; (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine; (17Z,20Z)- 51
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) N,N-dimethylhexacosa-17,20-dien-9-amine; (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-8- amine; (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine; (12Z,15Z)-N,N-dimethylhenicosa- 12,15-dien-4-amine; (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine; (15Z,18Z)-N,N- dimethyltetracosa-15,18-dien-7-amine; (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine; (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine; (14Z,17Z)-N,N-dimethyltricosa-14,17- dien-4-amine; (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-9-amine; (18Z,21Z)-N,N- dimethylheptacosa-18,21-dien-8-amine; (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7-amine; (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine; (22Z,25Z)-N,N-dimethylhentriaconta- 22,25-dien-10-amine; (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine; (18Z)-N,N- dimethylheptacos-18-en-10-amine; (17Z)-N,N-dimethylhexacos-17-en-9-amine; (19Z,22Z)-N,N- dimethyloctacosa-19,22-dien-7-amine; N,N-dimethylheptacosan-10-amine; (20Z,23Z)-N-ethyl- N-methylnonacosa-20,23-dien-10-amine; 1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1- yl]pyrrolidine; (20Z)-N,N-dimethylheptacos-20-en-10-amine; (15Z)-N,N-dimethylheptacos-15- en-10-amine; (14Z)-N,N-dimethylnonacos-14-en-10-amine; (17Z)-N,N-dimethylnonacos-17-en- 10-amine; (24Z)-N,N-dimethyltritriacont-24-en-10-amine; (20Z)-N,N-dimethylnonacos-20-en- 10-amine; (22Z)-N,N-dimethylhentriacont-22-en-10-amine; (16Z)-N,N-dimethylpentacos-16-en- 8-amine; (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine; (13Z,16Z)-N,N- dimethyl-3-nonyldocosa-13,16-dien-1-amine; N,N-dimethyl-1-[(1S,2R)-2- octylcyclopropyl]heptadecan-8-amine; 1-[(1S,2R)-2-hexylcyclopropyl]-N,N- dimethylnonadecan-10-amine; N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10- amine; N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine; N,N-dimethyl-1- [(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine; N,N- dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine; N,N-dimethyl-1-[(1R,2S)-2- undecylcyclopropyl]tetradecan-5-amine; N,N-dimethyl-3-{7-[(1S,2R)-2- octylcyclopropyl]heptyl}dodecan-1-amine; 1-[(1R,2S)-2-heptylcyclopropyl]-N,N- dimethyloctadecan-9-amine; 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine; N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine; (11E,20Z,23Z)-N,N- dimethylnonacosa-11,20,23-trien-10-amine; 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]- dioxane (Dlin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (Dlin-K-MPZ), 2,2-dioleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DO-K-DMA), 2,2-distearoyl-4- dimethylaminomethyl-[1,3]-dioxolane (DS-K-DMA), 2,2-dilinoleyl-4-N-morpholino-[1,3]- 52
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) dioxolane (Dlin-K-MA), 2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane chloride (Dlin-K- TMA.Cl), 2,2-dilinoleyl-4,5-bis(dimethylaminomethyl)-[1,3]-dioxolane (Dlin-K2-DMA), 2,2- dilinoleyl-4-methylpiperzine-[1,3]-dioxolane (D-Lin-K—N-methylpiperzine), Dlen-C2K-DMA, γ-Dlen-C2K-DMA, Dpan-C2K-DMA, Dpan-C3K-DMA, Dlen-C2K-DMA, γ-Dlen-C2K-DMA, Dpan-C2K-DMA, TLinDMA, C2-TLinDMA, C3-TLinDMA, 1,2-di-γ-linolenyloxy-N,N- dimethylaminopropane (γ-DLenDMA), 1,2-dilinoleyloxy-(N,N-dimethyl)-butyl-4-amine (C2- DLinDMA), 1,2-dilinoleoyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDAP), CP-LenMC3, CP-γ-LenMC3, CP-MC3, Cp-Dlen-C2K-DMA, CP-γDLen-C2K-DMA, CP-C2K-DMA, CP- DODMA, CP-DPetroDMA, CP-DLinDMA, CP-DLenDMA, CP-γDLenDMA, 1,2- dioeylcarbamoyloxy-3-dimethylaminopropane (DO-C-DAP), 1,2-dimyristoleoyl-3- dimethylaminopropane (DMDAP), 1,2-dioleoyl-3-trimethylaminopropane chloride (DOTAP.Cl), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (Dlin-C-DAP), 1,2-dilinoleyoxy-3- (dimethylamino)acetoxypropane (Dlin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (Dlin- MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3- dimethylarninopropane (Dlin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (Dlin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (Dlin-TMA.Cl), 1,2- dilinoleoyl-3-trimethylaminopropane chloride salt (Dlin-TAP.Cl), 1,2-dilinoleyloxy-3-(N- methylpiperazino)propane (Dlin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DlinAP), 3- (N,N-dioleylamino)-1,2-propanedio (DOAP), 1,2-dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (Dlin-EG-DMA), 3-dimethylamino-2-(cholest-5-en-3-beta- oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5′-(cholest-5-en-3- beta-oxy)-3′-oxapentoxy)-3-dimethy-1-(cis,cis-9′,1-2′-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N′-dioleylcarbamyl-3- dimethylaminopropane (DOcarbDAP), and 1,2-N,N′-dilinoleylcarbamyl-3- dimethylaminopropane (DLincarbDAP); as well as pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing. As used herein, the term "non-cationic lipid" refers to any uncharged, anionic, or zwitterionic lipid. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, diacylglycerols, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, 53
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids. In some embodiments, the non-cationic lipid used in the instant disclosure is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Distearoyl-sn- glycero-3-phosphocholine (DSPC), and/or 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In embodiments, the non-cationic lipid is cholesterol (CHE) and/or β-sitosterol. In some embodiments, the non-cationic lipid present in the lipid particles comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof. In certain embodiments, a lipid composition of the disclosure can include lipids such as "neutral lipids," "helper lipids," and/or "stealth lipids." "Neutral lipids" suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids. In some embodiments, neutral lipids disclosed herein may include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols. Other examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), palmitoyloleoyl phosphatidylcholine (POPC), dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of dioleoyl phosphatidylethanolamine (DOPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC). Without wishing to be bound by theory, neutral lipids have been described to function to stabilize and improve processing of the LNPs. "Helper lipids" are lipids that enhance transfection (e.g., transfection of the nanoparticle including the biologically active agent). Without wishing to be bound by theory, the mechanism by which the helper lipid enhances transfection includes enhancing particle stability. In certain embodiments, the helper lipid enhances membrane fusogenicity. Helper lipids include the above- referenced "neutral lipids," including but not limited to include, but are not limited to, 54
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), palmitoyloleoyl phosphatidylcholine (POPC), dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof, as well as steroids, and sterols. Helper lipids suitable for use in the present disclosure include, but are not limited to, neutral lipids, cholesterol, and PEG-cholesterol. In one embodiment, the helper lipid may be cholesterol. In one embodiment, the helper lipid may be PEG- cholesterol. "Stealth lipids" are lipids that alter the length of time the nanoparticles can exist in vivo (e.g., in the blood). Without wishing to be bound by theory, stealth lipids may assist in the formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipids used herein may modulate pharmacokinetic properties of the LNP. Stealth lipids suitable for use in a lipid composition of the disclosure include, but are not limited to, stealth lipids having a hydrophilic head group linked to a lipid moiety. Stealth lipids suitable for use in a lipid composition of the present disclosure and information about the biochemistry of such lipids can be found in Romberg et al, Pharmaceutical Research, Vol.25, No.1, 2008, pg.55-71 and Hoekstra et al, Biochimica et Biophysica Acta 1660 (2004) 41-52. In one embodiment, the hydrophilic head group of stealth lipid comprises a polymer moiety selected from polymers based on PEG (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N- vinylpyrrolidone), polyaminoacids and poly N-(2- hydroxypropyl)methacrylamide. Stealth lipids may comprise a lipid moiety. In some embodiments, the lipid moiety of the stealth lipid may be derived from diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independently comprising from about C
4 to about C
40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester. The dialkylglycerol or dialkylglycamide group can further comprise one or more substituted alkyl groups. In some embodiments, stealth lipids may comprise α-Methoxy-ω-(3- oxopropoxy), polyoxyethylene (Methoxy PEG, Aldehyde), PEG2k-DMG, PEG2k-DSG, PEG2k- 55
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) DSPE, PEG2K-DOPE, PEG5k-DOPE, Methoxy PEG aldehyde 20k, PEG2K-Cholesterol, and the like. The term "lipid nanoparticle" or "LNP" as used herein refers to different types of compositions of nano-scale particles, wherein the particles comprising lipids function as carriers across cell membranes and biological barriers and deliver compounds to targeted cells and tissues of humans and other organisms. As used herein, "lipid nanoparticles" of the instant disclosure may further comprise additional lipids and other components. Other lipids may be included for a variety of purposes, such as to prevent lipid oxidation or to attach ligands onto the lipid nanoparticle surface. Any of a number of lipids may be present in lipid nanoparticles of the present disclosure, including amphipathic, neutral, cationic, and anionic lipids. Such lipids can be used alone or in combination, and can also include bilayer stabilizing components such as polyamide oligomers (see, e.g., U.S. Pat. No.6,320,017), peptides, proteins, detergents, lipid-derivatives, such as PEG coupled to phosphatidylethanolamine and PEG conjugated to ceramides (see, e.g., U.S. Pat. No. 5,885,613). As used herein, a "PEG" conjugated lipid that inhibits aggregation of particles refers to one or more of a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, and a mixture thereof. In one aspect, the PEG-lipid conjugate is one or more of a PEG- dialkyloxypropyl (DAA), a PEG-diacylglycerol (DAG), a PEG-phospholipid, a PEG-ceramide, and a mixture thereof. In one aspect, the PEG-DAG conjugate is one or more of a PEG- dilauroylglycerol (C
12), a PEG-dimyristoylglycerol (C
14), a PEG-dipalmitoylglycerol (C
16), and a PEG-distearoylglycerol (C
18). In one aspect, the PEG-DAA conjugate is one or more of a PEG- dilauryloxypropyl (C
12), a PEG-dimyristyloxypropyl (C
14), a PEG-dipalmityloxypropyl (C
16), and a PEG-di stearyloxypropyl (C
18). In some embodiments, PEG is 2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (PEG-DMG or PEG2k-DMG) and/or 1,2-distearoyl-rac- glycero-3-methoxypolyethylene glycol-2000 (PEG-DSG). The term "N/P ratio" as used herein refers to the (N)itrogen-to-(P)hosphate molar ratio between the cationic amino lipid and negatively charged phosphate groups of the nucleic acid. The "polydispersity index" or "PDI" as used herein is a measure of the heterogeneity of a sample based on size. Polydispersity can occur due to size distribution in a sample or agglomeration or aggregation of the sample during isolation or analysis. 56
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) The "zeta potential" or "surface charge" as used herein refers to the degree of electrostatic repulsion between adjacent, similarly charged particles in a dispersion. For molecules and particles that are small enough, a high zeta potential will confer stability, i.e., the solution or dispersion will resist aggregation. As used herein, the term nucleic acid "cargo" refers to the intended nucleic acid for delivery to the cell or tissue (in embodiments, a therapeutic nucleic acid for delivery to the cell or tissue). As used herein, the term "nucleic acid-lipid nanoparticle" refers to lipid nanoparticles as described above that associate with or encapsulate one or more nucleic acids to deliver one or more nucleic acid cargoes to a tissue. As used herein, "encapsulated" can refer to a nucleic acid-lipid nanoparticle formulation that provides a nucleic acid with full encapsulation, partial encapsulation, association by ionic or van der Waals forces, or all of the aforementioned. In one embodiment, the nucleic acid is fully encapsulated in the nucleic acid-lipid nanoparticle. As used herein, "nucleic acid" refers to a synthetic or naturally occurring RNA or DNA, or derivatives thereof. In one embodiment, a cargo and/or agent of the instant disclosure is a nucleic acid, such as a double-stranded RNA (dsRNA). In one embodiment, the nucleic acid or nucleic acid cargo is a single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrid. For example, a double-stranded DNA can be a structural gene, a gene including control and termination regions, or a self-replicating system such as a viral or plasmid DNA. A double- stranded RNA can be, e.g., a dsRNA or another RNA interference reagent. A single-stranded nucleic acid can be, e.g., an mRNA, an antisense oligonucleotide, ribozyme, a microRNA, or triplex-forming oligonucleotide. In certain embodiments, the nucleic acid or nucleic acid cargo may comprise a modified RNA, wherein the modified RNA is one or more of a modified mRNA, a modified antisense oligonucleotide and a modified siRNA. In some embodiments, a nucleic acid cargo of the instant disclosure includes or is a modified mRNA that encodes a nucleic acid modulating controller. As used herein, the term "modified nucleic acid" refers to any non-natural nucleic acid, including but not limited to those selected from the group comprising 2′-O-methyl modified nucleotides, a nucleotide comprising a 5′-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, a 2′-deoxy-2′-fluoro modified nucleotide, a 5′-methoxy-modified nucleotide (e.g., 5′-methoxyuridine), a 2′-deoxy-modified nucleotide, a locked nucleotide, an 57
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide; internucleoside linkages or backbones including phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. As used herein, the term "nucleic acid modulating controller" refers to a mRNA that encodes for protein controller components, though reference to “nucleic acid modulating controller” can also refer to the mRNA-expressed protein controller components themselves. In certain embodiments, the mRNA-encoded protein controller components include Zinc-Finger proteins (ZFPs) or other forms of DNA or RNA binding domains (DBDs or RBDs) that are associated with (and optionally tethered to) one or more epigenetic regulators or nucleases (the epigenetic regulators or nucleases are generally referred to as effectors, effector domains, or effector moieties). Without wishing to be bound by theory, an advantage of a nucleic acid modulating controller as described herein is that it provides durable gene programming only at the confluence of (1) where the nucleic acid modulating controller-encoding mRNA is expressed, (2) where nucleic acid binding of the ZFP or other nucleic acid binding domain occurs and (3) where the associated effector domain is able to exert activity (i.e. where the effector domain is capable of changing the epigenomic state (e.g., in the instance of an epigenomic controller)). As used herein, the term "effector moiety" or "effector domain" refers to a domain that is capable of altering the expression of a target gene when localized to an appropriate site in a cell, e.g., in the nucleus of a cell. In some embodiments, an effector moiety recruits components of the transcription machinery. In some embodiments, an effector moiety inhibits recruitment of components of transcription factors or expression repressing factors. In some embodiments, an effector moiety comprises an epigenetic modifying moiety (e.g., epigenetically modifies a target DNA sequence). Specific examples of effector moieties include, without limitation, effectors capable of binding Krueppel-associated box (KRAB) domains (KRAB is a domain of around 75 amino acids that is found in the N-terminal part of about one third of eukaryotic Krueppel-type 58
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) C2H2 zinc finger proteins (ZFPs)) and the engineered prokaryotic DNA methyltransferase MQ1, among others. As used herein, "epigenetic modifying moiety" refers to a domain that alters: i) the structure, e.g., two-dimensional structure, of chromatin; and/or ii) an epigenetic marker (e.g., one or more of DNA methylation, histone methylation, histone acetylation, histone sumoylation, histone phosphorylation, and RNA-associated silencing), when the epigenetic modifying moiety is appropriately localized to a nucleic acid (e.g., by a targeting moiety). In some embodiments, an epigenetic modifying moiety comprises an enzyme, or a functional fragment or variant thereof, that affects (e.g., increases or decreases the level of) one or more epigenetic markers. In some embodiments, an epigenetic modifying moiety comprises a DNA methyltransferase, a histone methyltransferase, CREB-binding protein (CBP), or a functional fragment of any thereof. As used herein, the term "expression control sequence" refers to a nucleic acid sequence that increases or decreases transcription of a gene and includes (but is not limited to) a promoter and an enhancer. An "enhancing sequence" refers to a subtype of expression control sequence and increases the likelihood of gene transcription. A "silencing or repressor sequence" refers to a subtype of expression control sequence and decreases the likelihood of gene transcription. As used herein, the term "expression repressor" refers to an agent or entity with one or more functionalities that decreases expression of a target gene in a cell and that specifically binds to a DNA sequence (e.g., a DNA sequence associated with a target gene or a transcription control element operably linked to a target gene). In certain embodiments, an expression repressor comprises at least one targeting moiety and optionally one effector moiety. As used herein, the term "targeting moiety" means an agent or entity that specifically targets, e.g., binds, a genomic sequence element (e.g., an expression control sequence or anchor sequence; promoter, enhancer or CTCF site). In some embodiments, the genomic sequence element is proximal to and/or operably linked to a target gene (e.g., MYC). As used herein, "localization" refers to the position of a lipid, peptide, or other component of a lipid particle of the instant disclosure, within an organism and/or tissue. In some embodiments, localization can be detectible in individual cells. In some embodiments a label can be used for detecting localization, e.g., a fluorescent label, optionally a fluorescently labeled lipid, optionally Cy7. In some embodiments, the label of the lipid nanoparticle may be a quantum dot, or the lipid detectible by stimulated Raman scattering. In other embodiments, the label is any fluorophore 59
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) known in the art, i.e. with excitation and emission in the ultraviolet, visible, or infrared spectra. In some embodiments the localization is detected or further corroborated by immunohistochemistry or immunofluorescence. As used herein, the term "activity" refers to any detectable effect that is mediated by a component or composition of the instant disclosure. In embodiments, “activity” as used herein, can refer to a measurable (whether directly or by proxy) effect, e.g., of a cargo of the instant lipid particles of the disclosure. Examples of activity include, without limitation, the intracellular expression and resulting effect(s) of a nucleic acid cargo (e.g., a mRNA, a CRISPR/Cas system, a RNAi agent, a nucleic acid modulating controller, etc.), which can optionally be measured at a cellular, tissue, organ and/or organismal level. As used herein, "multidosing" refers to two or more doses of a lipid nanoparticle formulation given as part of a therapeutic regimen to a subject. As used herein, the term "subject" includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In many embodiments, subjects are mammals, particularly primates, especially humans. In some embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. In some embodiments (e.g., particularly in research contexts) subject mammals will be, for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like. As used herein, "administration" to a subject may include parenteral administration, optionally for intravenous injection, inhalation, intravenous, intra-arterial, intratracheal, topical, or involve direct injection into a tissue. The term "treating" includes the administration of compositions to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease (e.g., cancer, including, e.g., tumor formation, growth and/or metastasis), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease. As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of a lipid particle, optionally a nucleic-acid lipid nanoparticle (NLNP) and a 60
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) pharmaceutically acceptable carrier. As used herein, "pharmacologically effective amount," "therapeutically effective amount” or simply “effective amount” refers to that amount of nucleic acid effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to induce at least a 25% reduction in that parameter. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. As used herein, "lung tissue" may refer to any cell within the organ of the lung including but not limited to the group comprising the epithelium, endothelium, interstitial connective tissue, blood vessel, hematopoietic tissue, lymphoid tissue, and pleura. In preferred embodiments, the nucleic acid-lipid nanoparticle targets lung tissue. In some other embodiments, the nucleic acid- lipid nanoparticle may target other cells or tissues including but not limited to brain, nerve, skin, eye, pharynx, larynx, heart, vascular, hematopoietic (e.g., white blood cell or red blood cell), breast, liver, pancreas, spleen, esophagus, gall bladder, stomach, intestine, colon, kidney, urinary bladder, ovary, uterus, cervix, prostate, muscle, bone, thyroid, parathyroid, adrenal, and pituitary cells or tissues. As used herein, "localization" refers to the position of a lipid, peptide, or other component of a lipid particle of the instant disclosure, within an organism and/or tissue. In some embodiments, localization can be detectible in individual cells. In some embodiments a label can be used for detecting localization, e.g., a fluorescent label, optionally a fluorescently labeled lipid, optionally Cy7. In some embodiments, the label of the lipid nanoparticle may be a quantum dot, or the lipid detectible by stimulated Raman scattering. In other embodiments, the label is any fluorophore known in the art, i.e. with excitation and emission in the ultraviolet, visible, or infrared spectra. In some embodiments the localization is detected or further corroborated by immunohistochemistry or immunofluorescence. As used herein, the term "activity" refers to any detectable effect that is mediated by a component or composition of the instant disclosure. In embodiments, "activity" as used herein, can refer to a measurable (whether directly or by proxy) effect, e.g., of a cargo of the instant lipid 61
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) particles of the disclosure. Examples of activity include, without limitation, the intracellular expression and resulting effect(s) of a nucleic acid cargo (e.g., a mRNA, a CRISPR/Cas system, a RNAi agent, a nucleic acid modulating controller, etc.), which can optionally be measured at a cellular, tissue, organ and/or organismal level. As used herein, the term "lung disease or disorder" may include, without limitation, a disease or disorder selected from the following: lung cancer, pneumonia, pulmonary fibrosis, COPD, asthma, bronchiectasis, sarcoidosis, pulmonary hypertension, emphysema, alpha-1 antitrypsin deficiency, aspergillosis, bronchiolitis, bronchitis, pneumoconiosis, Coronaviruses, Middle Eastern Respiratory Syndrome, Severe Acute Respiratory Syndrome, cystic fibrosis, Legionnaire's disease, influenza, pertussis, pulmonary embolism, and tuberculosis. As used herein, a "joint diseases or disorder," may include, without limitation, a disease or disorder selected from the following: rheumatoid arthritis, psoriatic arthritis, gout, tendinitis, bursitis, Carpal Tunnel Syndrome, and osteoarthritis. As used herein, an "inflammatory disease or disorder," may include, without limitation, a disease or disorder selected from the following: inflammatory bowel disease, peritonitis, osteomyelitis, cachexia, pancreatitis, trauma induced shock, bronchial asthma, allergic rhinitis, cystic fibrosis, acute bronchitis, acute intense bronchitis, osteoarthritis, rheumatoid arthritis, infectious arthritis, post-infectious arthritis, gonocoele arthritis, tuberculous arthritis, arthritis, osteoarthritis, gout, spondyloarthropathies, ankylosing spondylitis, arthritis associated with vasculitis syndrome, nodular polyarteritis nervosa, irritable vasculitis, rugenic granulomatosis, rheumatoid polyposis myalgia, arthritis cell arteritis, calcium polycystic arthropathy, caustic gout, non-arthritic rheumatism, bursitis, hay fever, suppurative inflammation (e.g., tennis elbow), neuropathic joint disease, hemarthrosic, Henoch-Schlein purpura, hypertrophic osteoarthritis, multisized hemorrhoids, scoliosis, hemochromatosis, hyperlipoproteinemia, hypogammaglobulinemia, COPD, acute respiratory distress syndrome, acute lung injury, broncho-pulmonary dysplasia and systemic lupus erythematosus (SLE). As used herein, an "epidermal disease or disorder," may include, without limitation, a disease or disorder selected from the following: psoriasis, atopic dermatitis, scleroderma, eczema, rosacea, seborrheic dermatitis, melanoma, solar keratosis, ichthyosis, Grover's disease, common warts, keratoacanthoma, and seborrhoeic keratosis. 62
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural. Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another aspect. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. It is also understood that throughout the application, data are provided in a number of different formats and that this data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, "nested sub-ranges" that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction. The transitional term "comprising," which is synonymous with "including," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in 63
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) the claim. The transitional phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the disclosure. As used herein, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group having from 1 to 22 carbon atoms ("C
1–C
22 alkyl"). In some embodiments, an alkyl group may have 2 to 22 carbon atoms ("C
2-C
22 alkyl"). In some embodiments, an alkyl group may have 3 to 22 carbon atoms ("C
3–C
22 alkyl") and/or 4 to 22 carbons ("C
4-C
22 alkyl") and/or 5 to 22 carbons ("C
5-C
22 alkyl") and/or 7 to 22 carbon atoms ("C
7–C
22 alkyl"). In some embodiments, an alkyl group may have 7 to 18 carbon atoms ("C
7–C
18 alkyl") and/or 7 to 12 carbon atoms ("C
7– C
12 alkyl"). In some embodiments, an alkyl group has 7 to 8 carbon atoms ("C
7–C
8 alkyl"). In some embodiments, an alkyl group has 7 to 9 carbon atoms ("C
7–C
9 alkyl"). In some embodiments, an alkyl group may have 7 to 10 carbon atoms ("C
7–C
10 alkyl"). In some embodiments, an alkyl group has 7 to 11 carbon atoms ("C
7–C
11 alkyl"). In some embodiments, an alkyl group may have 8 to 12 carbon atoms ("C
8–C
12 alkyl"). In some embodiments, an alkyl group has 9 to 12 carbon atoms ("C
9–C
12 alkyl"). In some embodiments, an alkyl group has 10 to 12 carbon atoms ("C
10– C
12 alkyl"). In some embodiments, an alkyl group has 11 to 12 carbon atoms ("C
11–C
12 alkyl"). Additional examples of alkyl groups include n-heptyl (C
7), n-octyl (C
8), n-nonyl (C
9), n-decyl (C
10), n-undecyl (C
11), n-dodecyl (C
12), and the like. An "alkyl" group as used herein may be unsubstituted or optionally substituted. Unless otherwise specified, each instance of an alkyl group is independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") with one or more substituents. Suitable substituent groups may include, but are not limited to, hydroxyl, nitro, amino (e.g., —NH
2 or dialkyl amino), imino, cyano, halo (e.g., F, Cl, Br, I, and the like), haloalkyl (e.g., —CCl
3, —CF
3, and the like), thio, sulfonyl, thioamido, amidino, imidino, oxo, oxamidino, methoxamidino, imidino, guanidino, sulfonamido, carboxyl, formyl, alkyl, alkoxy, alkoxy-alkyl, alkylcarbonyl, alkylcarbonyloxy (e.g., —OCOR), aminocarbonyl, arylcarbonyl, aralkylcarbonyl, carbonylamino, heteroarylcarbonyl, heteroaralkyl-carbonyl, alkylthio, aminoalkyl, cyanoalkyl, carbamoyl (e.g., —NHCOOR— or —OCONHR—), urea (e.g., —NHCONHR—), cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, (═O), thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, nitro, amino, heterocycle, —CN, and the like. An 64
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) "alkyl" as used herein may be combined with other groups, such as those provided above, to form a functionalized alkyl. An "alkyl" group, as defined above, may further comprise 1 or more (e.g., 1, 2, 3, 4, etc.) heteroatoms (e.g., a "heteroalkyl" such as, e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus, and the like) within the parent chain, wherein the one or more heteroatoms are inserted between adjacent carbon atoms within the parent carbon chain and/or one or more heteroatoms are inserted between a carbon atom and the parent molecule, i.e., between the point of attachment. In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 22 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
1-C
22 alkyl"). In some embodiments, a heteroalkyl group refers to a saturated group having from 3 to 22 carbon atoms and/or 7 to 22 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
3-C
22 alkyl" and/or "hetero C
7–C
22 alkyl"). In some embodiments, a heteroalkyl group may have 7 to 18 carbon atoms and/or 7 to 12 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
7-C
18 alkyl" and/or "hetero C
7–C
12 alkyl"). In some embodiments, a heteroalkyl group may have 7 to 8 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
7-C
8 alkyl"). In some embodiments, a heteroalkyl group may have 7 to 9 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
7-C
9 alkyl"). In some embodiments, a heteroalkyl group has 7 to 10 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
7-C
10 alkyl"). In some embodiments, a heteroalkyl group has 7 to 11 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
7-C
11 alkyl"). In some embodiments, a heteroalkyl group has 8 to 12 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
8-C
12 alkyl"). In some embodiments, a heteroalkyl group has 9 to 12 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
9-C
12 alkyl"). In some embodiments, a heteroalkyl group has 10 to 12 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
10-C
12 alkyl"). In some embodiments, a heteroalkyl group has 11 to 12 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
11-C
12 alkyl"). As used herein, the term "alkenyl" includes a chain of carbon atoms, which is optionally branched, having from 2 to 22 carbon atoms and including at least one double bond (e.g., 1, 2, 3, 4, etc. carbon-carbon double bonds) ("C
2–C
22 alkenyl"). In some embodiments, an alkenyl group may have 3 to 22 carbon atoms ("C
3–C
22 alkenyl") and/or 4 to 22 carbons ("C
4-C
22 alkenyl") and/or 5 to 22 carbons ("C
5-C
22 alkenyl") and/or 7 to 22 carbon atoms ("C
7–C
22 alkenyl”). In some embodiments, an alkenyl group may have 7 to 18 carbon atoms ("C
7–C
18 alkenyl") and/or 7 to 12 carbon atoms ("C
7–C
12 alkenyl"). In some embodiments, an alkenyl group has 7 to 8 carbon atoms 65
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) ("C
7–C
8 alkenyl"). In some embodiments, an alkenyl group has 7 to 9 carbon atoms ("C
7–C
9 alkenyl"). In some embodiments, an alkenyl group may have 7 to 10 carbon atoms ("C
7–C
10 alkenyl"). In some embodiments, an alkenyl group has 7 to 11 carbon atoms ("C
7–C
11 alkenyl"). In some embodiments, an alkenyl group may have 8 to 12 carbon atoms ("C
8–C
12 alkenyl"). In some embodiments, an alkenyl group has 9 to 12 carbon atoms ("C
9–C
12 alkenyl"). In some embodiments, an alkenyl group has 10 to 12 carbon atoms ("C
10–C
12 alkenyl"). In some embodiments, an alkenyl group has 11 to 12 carbon atoms ("C
11–C
12 alkenyl"). Additional examples of alkenyl groups include n-heptyl (C
7), n-octyl (C
8), n-nonyl (C
9), n-decyl (C
10), n- undecyl (C
11), n-dodecyl (C
12), and the like. The one or more carbon-carbon double bonds may be internal (e.g., 2-butenyl) or terminal (e.g., 1- butenyl). Examples of C
2-4 alkenyl groups include ethenyl (C
2), 1-propenyl (C
3), 2-propenyl (C
3), 1-butenyl (C
4), 2-butenyl (C
4), butadienyl (C
4), and the like. Examples of C
2-6 alkenyl groups include the aforementioned C
2-4 alkenyl groups as well as pentenyl (C
5), pentadienyl (C
5), hexenyl (C
6), and the like. Additional examples of alkenyl include heptenyl (C
7), octenyl (C
8), octatrienyl (C
8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkenyl") or substituted (a "substituted alkenyl") with one or more substituents e.g., from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. In certain embodiments, the alkenyl group is unsubstituted C
3-C
22 alkenyl. In certain embodiments, the alkenyl group is substituted C
3-C
22 alkenyl. Exemplary substituents are listed above with respect to "alkyl" and may be used here with respect to "alkenyl" as well. The term "heteroalkenyl," as used herein, refers to an alkenyl group, as defined above, which further comprises one or more (e.g., 1, 2, 3, 4, etc.) heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus, and the like), wherein the one or more heteroatoms is inserted between adjacent carbon atoms within the parent carbon chain and/or one or more heteroatoms are inserted between a carbon atom and the parent molecule, i.e., between the point of attachment. In some embodiments, a heteroalkenyl group refers to an unsaturated group having 2 to 22 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
2-C
22 alkenyl"). In some embodiments, a heteroalkenyl group refers to an unsaturated group having from 7 to 18 carbon atoms and/or 7 to 12 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
7-C
18 alkenyl" or "hetero C
7–C
12 alkenyl"). In some embodiments, a heteroalkenyl group may have 7 to 8 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
7-C
8 alkenyl"). In some embodiments, a heteroalkenyl group may 66
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) have 7 to 9 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
7-C
9 alkenyl"). In some embodiments, a heteroalkenyl group has 7 to 10 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
7-C
10 alkenyl"). In some embodiments, a heteroalkenyl group has 7 to 11 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
7-C
11 alkenyl"). In some embodiments, a heteroalkenyl group has 8 to 12 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
8-C
12 alkenyl"). In some embodiments, a heteroalkenyl group has 9 to 12 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
9-C
12 alkenyl"). In some embodiments, a heteroalkenyl group has 10 to 12 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
10-C
12 alkenyl"). In some embodiments, a heteroalkenyl group has 11 to 12 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
11-C
12 alkenyl"). Additional examples of alkenyl groups include n-heptyl (C
7), n-octyl (C
8), n-nonyl (C
9), n-decyl (C
10), n-undecyl (C
11), n-dodecyl (C
12), and the like. The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of alkenyl include heptenyl (C
7), octenyl (C
8), octatrienyl (C
8), and the like. As used herein, the term "alkynyl" includes a chain of carbon atoms, which is optionally branched, and contains from 2 to 22 carbon atoms ("C
2–C
22 alkynyl"), including at least one carbon-carbon triple bond (i.e., C ^C). In some embodiments, an alkynyl group may have 3 to 22 carbon atoms ("C
3–C
22 alkynyl") and/or 7 to 22 carbon atoms ("C
7–C
22 alkynyl"). In some embodiments, an alkynyl group may have 7 to 18 carbon atoms ("C
7–C
18 alkynyl") and/or 7 to 12 carbon atoms ("C
7–C
12 alkynyl"). In some embodiments, an alkynyl group has 7 to 8 carbon atoms ("C
7–C
8 alkynyl"). In some embodiments, an alkynyl group has 7 to 9 carbon atoms ("C
7–C
9 alkynyl"). In some embodiments, an alkynyl group may have 7 to 10 carbon atoms ("C
7–C
10 alkynyl"). In some embodiments, an alkynyl group has 7 to 11 carbon atoms ("C
7–C
11 alkynyl"). In some embodiments, an alkynyl group may have 8 to 12 carbon atoms ("C
8–C
12 alkynyl"). In some embodiments, an alkynyl group has 9 to 12 carbon atoms ("C
9–C
12 alkynyl"). In some embodiments, an alkynyl group has 10 to 12 carbon atoms ("C
10–C
12 alkynyl"). In some embodiments, an alkynyl group has 11 to 12 carbon atoms ("C
11–C
12 alkynyl"). Alkynyl may be unsubstituted or substituted as described above for "alkyl" or as described in the various embodiments provided herein. Illustrative alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl, and the like. The term "heteroalkynyl," as used herein, refers to an alkynyl group, as defined above, which further comprises one or more (e.g., 1, 2, 3, 4, etc.) heteroatoms (e.g., oxygen, sulfur, 67
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) nitrogen, boron, silicon, phosphorus, and the like), wherein the one or more heteroatoms are inserted between adjacent carbon atoms within the parent carbon chain and/or one or more heteroatoms are inserted between a carbon atom and the parent molecule, i.e., between the point of attachment. In some embodiments, a heteroalkynyl group refers to an unsaturated group having 2 to 22 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
2-C
22 alkynyl"). In some embodiments, a heteroalkynyl group refers to an unsaturated group having from 7 to 18 carbon atoms and/or 7 to 12 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
7-C
18 alkynyl" or "hetero C
7–C
12 alkynyl"). In some embodiments, a heteroalkynyl group may have 7 to 8 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
7-C
8 alkynyl"). In some embodiments, a heteroalkynyl group may have 7 to 9 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
7-C
9 alkynyl"). In some embodiments, a heteroalkynyl group has 7 to 10 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
7-C
10 alkynyl"). In some embodiments, a heteroalkynyl group has 7 to 11 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
7-C
11 alkynyl"). In some embodiments, a heteroalkynyl group has 8 to 12 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
8-C
12 alkynyl"). In some embodiments, a heteroalkynyl group has 9 to 12 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
9-C
12 alkynyl"). In some embodiments, a heteroalkynyl group has 10 to 12 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
10-C
12 alkynyl"). In some embodiments, a heteroalkynyl group has 11 to 12 carbon atoms and 1, 2, 3, 4, etc. heteroatoms ("heteroC
11-C
12 alkynyl"). As used herein, "carbocyclyl" or "carbocyclic" refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 8 ring carbon atoms ("C
3-C
8 carbocyclyl") and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms ("C
3-C
7 carbocyclyl"). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms ("C
3-C
6 carbocyclyl"). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms ("C
4-C
6 carbocyclyl"). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms ("C
5-C
6 carbocyclyl"). In some embodiments, a carbocyclyl group has 5 to 8 ring carbon atoms ("C
5-C
8 carbocyclyl"). Exemplary C
3-C
6 carbocyclyl groups include, without limitation, cyclopropyl (C
3), cyclopropenyl (C
3), cyclobutyl (C
4), cyclobutenyl (C
4), cyclopentyl (C
5), cyclopentenyl (C
5), cyclohexyl (C
6), cyclohexenyl (C
6), cyclohexadienyl (C
6), and the like. Exemplary C
3-C
8 carbocyclyl groups include, without limitation, the aforementioned C
3-C
6 carbocyclyl groups as well as cycloheptyl (C
7), cycloheptenyl (C
7), cycloheptadienyl (C
7), 68
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) cycloheptatrienyl (C
7), cyclooctyl (C
8), cyclooctenyl (C
8), bicyclo[2.2.1]heptanyl (C
7), bicyclo[2.2.2]octanyl (C
8), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system ("bicyclic carbocyclyl") or tricyclic system ("tricyclic carbocyclyl") and can be saturated or can contain one or more carbon-carbon double or triple bonds. "Carbocyclyl" also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted (an "unsubstituted carbocyclyl") or substituted (a "substituted carbocyclyl") with one or more substituents. In certain embodiments, the carbocyclyl group is an unsubstituted C
3-C
10 carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C
3-C
10 carbocyclyl. In some embodiments, "carbocyclyl" or "carbocyclic" is referred to as a "cycloalkyl," i.e., a monocyclic, saturated carbocyclyl group having from 3 to 8 ring carbon atoms ("C
3-C
8 cycloalkyl"). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms ("C
3-C
6, cycloalkyl"). In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms ("C
4-C
6 cycloalkyl"). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms ("C
5-C
6 cycloalkyl"). In some embodiments, a cycloalkyl group has 5 to 8 ring carbon atoms ("C
5-C
8 cycloalkyl"). Examples of C
5-C
6 cycloalkyl groups include cyclopentyl (C
5) and cyclohexyl (C
5). Examples of C
3-C
6 cycloalkyl groups include the aforementioned C
5-C
6 cycloalkyl groups as well as cyclopropyl (C
3) and cyclobutyl (C
4). Examples of C
3-C
8 cycloalkyl groups include the aforementioned C
3-C
6 cycloalkyl groups as well as cycloheptyl (C
7) and cyclooctyl (C
8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an "unsubstituted cycloalkyl") or substituted (a "substituted cycloalkyl") with one or more substituents. In certain embodiments, the cycloalkyl group is an unsubstituted C
3-8 cycloalkyl. In certain embodiments, the cycloalkyl group is a substituted C
3-C
8 cycloalkyl. The term "heterocycle" or "heterocyclyl" refers to a saturated or an unsaturated aromatic or non-aromatic group having from 1 to 8 annular carbon atoms and from 1 to 4 annular heteroatoms, such as nitrogen, oxygen, sulfur, boron, phosphorus, silicon, and the like, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally 69
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) quaternized. A heterocycle group may have a single ring or multiple condensed rings. A heterocycle comprising more than one ring may be fused, spiro or bridged, or any combination thereof. In fused ring systems, one or more of the fused rings can be aryl or heteroaryl. Examples of heterocycle groups include, but are not limited to, dihydropyranyl, thiazolinyl, thiazolidinyl, tetrahydrothiophenyl, 2,3-dihydrobenzo[b]thiophen-2-yl, 4-amino-2-oxopyrimidin-1(2H)-yl, benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, N-oxides thereof, and the like. A "heterocycle" as disclosed herein may be optionally substituted with one or more substituents, including e.g., but not limited to, hydroxyl, nitro, amino (e.g., —NH
2 or dialkyl amino), imino, cyano, halo (e.g., F, Cl, Br, I, and the like), haloalkyl (e.g., —CCl
3, —CF
3, and the like), thio, sulfonyl, thioamido, amidino, imidino, oxo, oxamidino, methoxamidino, imidino, guanidino, sulfonamido, carboxyl, formyl, alkyl, alkoxy, alkoxy-alkyl, alkylcarbonyl, alkylcarbonyloxy (e.g., —OCOR), aminocarbonyl, arylcarbonyl, aralkylcarbonyl, carbonylamino, heteroarylcarbonyl, heteroaralkyl-carbonyl, alkylthio, aminoalkyl, cyanoalkyl, carbamoyl (e.g., —NHCOOR— or —OCONHR—), urea (e.g., — NHCONHR—), cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, (═O), thiocarbonyl, O-carbamyl, N-carbamyl, O- thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, nitro, amino, heterocycle, —CN, and the like. For example and without limitation, additional optional substituents include fluorine, chlorine, bromine, and iodine atoms and CF
3, CN, OH, =O, SH, =S, 70
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) NH
2, =NH, N
3 and NO
2 groups. Optional substituents also include C
1-C
10 alkyl, C
1-C
10 heteroalkyl, C
2-C
10 alkenyl, C
2-C
10 heteroalkenyl, C
2-C
10 alkynyl, C
2-C
10 hetero alkynyl, and the like. Exemplary substituents are F, Cl, Br, OH, SH, =O, NH2, amino, C
1-4 alkyl (e.g., methyl, ethyl, t-butyl), C
1-C
4 heteroalkyl cyclopropyl, SF
5, NO, NO
2, Nme2, CONH
2, Ch
2Nme2, NHSO
2Me, C(CH
3)
2CN, COMe, Ome, Sme, COOMe, COOEt, CH
2COOH, OCH
2COOH, COOH, SOMe, SO
2Me, cyclopropyl, SO
2NH
2, SO
2NHMe, SO
2CH
2CH
2OH, NHCH
2CH
2OH, CH
2CH
2OCH
3, SF
5, So
2Nme2, NO, NO
2, OCF
3, SO
2CF
3, CN or CF
3. In heterocycle groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocycle group can either be monocyclic ("monocyclic heterocycle") or a fused, bridged or spiro ring system such as a bicyclic system ("bicyclic heterocycle"), and can be saturated or can be partially unsaturated. Heterocycle bicyclic ring systems can include one or more heteroatoms in one or both rings. "Heterocycle" also includes ring systems wherein the heterocycle ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently optionally substituted, i.e., unsubstituted (an "unsubstituted heterocyclyl") or substituted (a "substituted heterocycle") with one or more substituents. In certain embodiments, the heterocycle group is unsubstituted 3-8 membered heterocycle. In certain embodiments, the heterocycle group is substituted 3-8 membered heterocycle. In some embodiments, a heterocycle group is a 3-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("5-10 membered heterocycle"). In some embodiments, a heterocycle group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-8 membered heterocycle"). In some embodiments, a heterocycle group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-6 membered heterocycle"). In some embodiments, the 5-6 membered 71
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) heterocycle has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocycle has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocycle has one ring heteroatom selected from nitrogen, oxygen, and sulfur. As used herein, the expression "optionally substituted" means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Heteroatoms, such as nitrogen, may have substituents, such as any suitable substituent described herein which satisfies the valencies of the heteroatoms and results in the formation of a stable moiety. The embodiments set forth below and recited in the claims can be understood in view of the above definitions. Other features and advantages of the disclosure will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description, given by way of example, but not intended to limit the disclosure solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, in which: FIGS. 1A-1I show an exemplary formula and exemplary structures of heterocycle ring cationic lipids as disclosed herein. FIG.1A shows an exemplary Formula I, Formula II, Formula 72
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) III, and Formula IV for heterocycle ring cationic lipids of the instant disclosure. FIG.1B shows an exemplary structure of nonyl (2-(4-(undecan-6-yl)piperazin-1-yl)ethyl) hydrogen phosphate (SM-037). FIG. 1C shows an exemplary structure of 2-(4-hexylpiperazin-1-yl)ethyl nonyl hydrogen phosphate (SM-033). FIG.1D shows an exemplary structure of nonyl (2-(4-(tridecan- 7-yl)piperazin-1-yl)ethyl) hydrogen phosphate (SM-052). FIG.1E shows an exemplary structure of 2-(4-(heptadecan-9-yl)piperazin-1-yl)ethyl nonyl hydrogen phosphate (SM-053). FIG. 1F shows an exemplary structure of nonyl (4-(4-(undecan-6-yl)piperazin-1-yl)butyl) hydrogen phosphate (SM-061). FIG. 1G shows an exemplary structure of nonyl (6-(4-(undecan-6- yl)piperazin-1-yl)hexyl) hydrogen phosphate (SM-058). FIG. 1H shows an exemplary structure of nonyl (4-(4-(tridecan-7-yl)piperazin-1-yl)butyl) hydrogen phosphate (SM-057). FIG.1I shows an exemplary structure of nonyl (6-(4-(tridecan-7-yl)piperazin-1-yl)hexyl) hydrogen phosphate (SM-059). FIG. 1J shows an exemplary structure of 2-(4-(dihexylamino)piperidin-1-yl)ethyl nonyl hydrogen phosphate (SM-063). FIG. 1K shows an exemplary structure of nonyl (2-(4- (undecane-6-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate (SM-064). FIG. 1L shows an exemplary structure of nonyl (3-(4-(undecan-6-yl)piperazin-1-yl)propyl) hydrogen phosphate (SM-108). FIG.1M shows an exemplary structure of nonyl (3-(4-(undecane-6-yl)-1,4-diazepan- 1-yl)propyl) hydrogen phosphate (SM-116). FIG. 1N shows an exemplary structure of (Z)-2-(4- (dihexylamino)piperidin-1-yl)ethyl non-3-en-1-yl hydrogen phosphate (SM-118). FIG.1O shows an exemplary structure of 2-(4-((dihexylamino)methyl)piperidin-1-yl)ethyl nonyl hydrogen phosphate (SM-119). FIG. 1P shows 2-butyloctyl (2-(4-(dihexylamino)piperidin-1-yl)ethyl) hydrogen phosphate (SM-121). FIG. 1Q shows an exemplary structure of 2-(4- (dipentylamino)piperidin-1-yl)ethyl nonyl hydrogen phosphate (SM-122). FIG. 1R shows an exemplary structure of nonyl (2-(4-(tridecan-7-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate (SM-123). FIG. 1S shows an exemplary structure of decyl (2-(4-(dihexylamino)piperidin-1- yl)ethyl) hydrogen phosphate (SM-123). FIG. 1T shows an exemplary structure of decyl (2-(4- (tridecan-7-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate (SM-126). FIG. 1U shows an exemplary structure of 2-(4-hexanoylpiperazin-1-yl)ethyl nonyl hydrogen phosphate (SM-031). FIG. 1V shows an exemplary structure of 2-((dioctylamino)methyl)cyclopropyl)methyl nonyl hydrogen phosphate (SM-032). FIG. 1W shows an exemplary structure of 3-(dioctylamino)-2- methylpropyl nonyl hydrogen phosphate (SM-034). FIG. 1X shows an exemplary structure of nonyl (2-(4-tetradecanoylpiperazin-1-yl)ethyl) hydrogen phosphate (SM-035). FIG.1Y shows an 73
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) exemplary structure of nonyl (1-octylpiperidin-4-yl) hydrogen phosphate (SM-036). FIG. 1Z shows an exemplary structure of nonyl (2-(4-stearoylpiperazin-1-yl)ethyl) hydrogen phosphate (SM-038). FIG.1AA shows an exemplary structure of nonyl (2-(4-palmitoylpiperazin-1-yl)ethyl) hydrogen phosphate (SM-039). FIG. 1AB shows an exemplary structure of 2-(4- heptadecanoylpiperazin-1-yl)ethyl nonyl hydrogen phosphate (SM-040). FIG. 1AC shows an exemplary structure of nonyl (2-(4-pentadecanoylpiperazin-1-yl)ethyl) hydrogen phosphate (SM- 041). FIG.1AD shows an exemplary structure of 1-(2-heptylnonyl)piperidin-4-yl nonyl hydrogen phosphate (SM-042). FIG. 1AE shows an exemplary structure of 1-(2-heptylnonyl)azetidin-3-yl nonyl hydrogen phosphate (SM-044). FIG. 1AF shows and exemplary structure of 1-(2- heptylnonyl)azepan-4-yl nonyl hydrogen phosphate (SM-045). FIG. 1AG shows an exemplary structure of 1-(2-heptylnonyl)pyrrolidin-3-yl nonyl hydrogen phosphate (SM-047). FIGS. 2A-2C show that SM-037 lipid nanoparticles (LNPs) robustly localized to and expressed mRNA cargo in the lungs of treated mice, when LNPs formulated with reporter mRNA as cargo were administered intravenously. FIG. 2A shows that SM-037-LNPs including SM- 005/SM-037/CHOL/PEG-DMG at a mole ratio of 30/50/50/1.5 displayed concentrated luciferase activity in mouse lungs for three different test subjects as viewed in a dorsal image, with observed effects persisting for 24h. FIG.2B shows the data for the same three subjects displayed in FIG. 2A in a ventral image. Cy7 signal distribution indicates LNP biodistribution, while the luminescence signal indicates reporter mRNA cargo expression and activity. Notably, lung levels of cargo mRNA expression were particularly robust, even as SM-037-LNPs distributed well to a number of tissues. FIG. 2C shows quantification of the observed Cy7-DOPE lipid luminescent biodistribution signal in harvested mouse organs. FIG. 2D shows the luminescence signal from the expression of mFluc mRNA in the mice organs - specifically, liver, lungs, kidney, and spleen. Strong specificity of SM-037-LNPs delivery to lungs (protein expression preferentially in lung) was observed relative to other organs in the subject. FIG. 3 shows that iterative optimization of first generation (GEN-1), second generation (GEN-2), and third generation (GEN-3) formulations led to LNP8 formulations possessing excellent physical properties, including good frozen stability, consistent small size (top row of bar 74
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) graphs), high encapsulation efficiency (middle row of bar graphs), and low PDI (bottom row of bar graphs). FIGS. 4A-4C show in vivo, ex vivo, and tissue staining for Fluc reporter expression, respectively. FIG. 4A shows concentrated luciferase activity in mouse lungs for three different test subjects as viewed in a dorsal image (left panel) and ventral image (right panel), at 6h. FIG. 4B shows the observed luminescent biodistribution signal in harvested mouse organs. FIG. 4C shows that the systemic lung-targeting LNP formulations of the instant disclosure exhibited strong Fluc reporter expression in airway epithelial cells and in endothelial cells, where images were stained with CY3-luciferase (orange), DAPI (nuclear, blue), and FITC-epithelial cell stain (green). DETAILED DESCRIPTION The present disclosure is based, at least in part, upon the discovery of novel phospholipids containing a heterocycle ring having a tertiary amine or amide having advantageous properties when used in lipid particles for the in vivo delivery of a therapeutic agent(s). In particular, the techniques herein provide lipid-based nanoparticle compositions and formulations capable of specifically targeting a cargo moiety (e.g., a nucleic acid cargo) to the lung and lung tissues of a subject, without requiring a ligand-based targeting strategy. SM-037 is an ionizable phospholipid which can be included in lipid-based nanoparticle compositions described herein which, upon systemic or local administration, has been remarkably effective in shifting the tropism of vectors specifically to lungs without requiring a further active-targeting component in the LNPs. The instant disclosure indicates the surprising structural affinity SM-037 possesses for lung tissues, which can be exploited for effective delivery of nucleic acid cargoes, including, e.g., expression of therapeutic mRNAs, upon systemic administration (e.g., via intravenous (IV) injection). Immunohistochemistry (IHC) evaluation of lung tissues also demonstrated successful delivery and expression of cargo mRNA in endothelial cells, epithelial cells, fibroblasts and macrophages using the SM-037 LNPs disclosed herein. The instant disclosure, therefore, significantly provides nucleic acid-lipid particles that offer particular advantages for repeated systemic administration to lung tissues. Traditional LNPs are composed of four main components. An ionizable or cationic lipid for mRNA encapsulation, amphipathic helper phospholipids for increased efficacy, cholesterol for 75
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) structural stability and polyethylene glycol (PEG)-lipids for steric stability. This first generation of LNPs can be considered as "one ionizable lipid-only LNPs," or "single LNPs." Conventionally, effective intracellular delivery materials have relied on an optimal balance of ionizable amines to bind and release RNAs (pKa between 6.0 and 6.5) and nanoparticle-stabilizing hydrophobicity. Thus, there has been an exhaustive focus on developing ionizable lipids, which have been proven to be highly effective delivery platforms for liver and hepatocytes. However, changing the chemical structure of the ionizable/cationic lipid to achieve different pKa values and generating libraries, although validated, is a time consuming, investment heavy and labor-intensive exercise. The present disclosure provides novel phospholipids containing a heterocycle ring having a tertiary amine or amide having the surprising ability to preferentially localize to and deliver associated nucleic acid cargoes to the lung of a subject, with delivery occurring to various types of tissue within the lung of a subject. Without wishing to be bound by theory, certain novel phospholipids containing a heterocycle ring having a tertiary amine or amide (e.g., SM-037) disclosed herein appear to be able to shift the tropism of LNP vectors disclosed herein specifically to lungs without requiring a further active-targeting component in the LNPs of the instant disclosure. Demonstrated herein is also the surprising structural affinity of SM-037 for lung tissues in mediating effective delivery of nucleic acid cargoes, in particular, expression of various reporter mRNAs, upon systemic administration (IV). The novel phospholipids containing a heterocycle ring having a tertiary amine or amide disclosed herein have the general structure set forth in Formula I, Formula II, Formula III, and Formula IV below and include the (R) and/or (S) enantiomers thereof. In embodiments, the techniques herein provide improved lipid-based compositions for the delivery of therapeutic agents, in particular, nucleic acid therapeutic agents. As disclosed herein, these lipid-based compositions are effective in increasing the efficiency of cargo release from lipid-based composition such as LNPs. Furthermore, the present disclosure demonstrates that the activity of these improved lipid-based compositions is dependent on the presence of certain novel phospholipids containing a heterocycle ring having a tertiary amine or amide disclosed herein. It is contemplated within the scope of the disclosure that the lipid-based compositions including the novel phospholipids containing a heterocycle ring having a tertiary amine or amide disclosed herein may be used for a variety of purposes such as, for example, the delivery of encapsulated therapeutic agents to cells, in vitro and/or in vivo. In this regard, the present disclosure 76
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) provides methods of treating diseases or disorders in a subject in need thereof by contacting the subject with the lipid-based compositions disclosed herein when combined with the suitable therapeutic agent such as, for example, nucleic acids (e.g., siRNA, ASO, tRNA, miRNA, mRNA, DNA, and the like), proteins, peptides, and other macromolecules. Nucleic acid therapy has well-known, tremendous potential to treat diseases at the gene level. However, safe and effective delivery systems are essential for nucleic acid therapeutics. Non-specific delivery to organs and tissues often results in off-site effects and toxicity. Delivery of therapeutics to a specific organ of interest is a well-recognized need in the development of lipid- nanoparticles, as well as in drug development generally. The concept of only targeting the cause of a disease without harming other parts of the body was described by Ehrlich 120 years ago. However, extant methods do not provide defined or well-known methodologies for developing nanoparticles targeting specific tissues without introducing additional ligand-based targeting strategies. Organ-specific targeting of lipid nanoparticles based on the structural affinity of the lipid to the tissue, as now disclosed herein, therefore meets a well-established need in terms of reducing off-site effects and toxicity. Lungs are one of the key target organs for gene therapy. Specific delivery to the lungs by avoiding activity in the other organs is vital to treat respiratory system related diseases effectively. The instant disclosure demonstrates that incorporation of SM-037 shifts the tropism of vectors specifically to lungs without requiring an active-targeting component in the LNPs. In embodiments, the lipid-based compositions disclosed herein are particularly useful for the delivery of nucleic acid therapeutics (e.g., siRNA, ASO, tRNA, miRNA, mRNA, DNA, and the like). The lipid-based compositions disclosed herein may be used to modulate the expression of target genes and proteins both in vitro and in vivo by contacting tissues/cells with a lipid-based composition including a lipid as disclosed herein carrying a cargo such as a therapeutic nucleic acid (e.g., an siRNA) that may reduce expression of a desired target gene. The techniques herein provide novel phospholipids containing a heterocycle ring having a tertiary amine or amide that enable the formulation of pharmaceutical compositions for the in vitro or in vivo delivery of therapeutic agents such as, for example, nucleic acids (e.g., siRNA, ASO, tRNA, miRNA, mRNA, DNA, and the like), proteins, peptides, and other macromolecules. Exemplary embodiments of the novel phospholipids containing a heterocycle ring having a tertiary amine or amide of the present disclosure, as well as lipid-based compositions comprising 77
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) the same, as well as their synthesis and use to deliver therapeutic agents is described in further detail below. Lipids The present disclosure provides novel phospholipids containing a heterocycle ring having a tertiary amine or amide of the general structure of Formula I shown in FIG. 1A. The novel phospholipids containing a heterocycle ring having a tertiary amine or amide have design features including a heterocycle ring backbone comprising an ionizable tertiary amine (e.g., head group), an electron withdrawing phosphate group, and a linker, wherein the linker connects the tertiary amine to the phosphate group. Exemplary novel phospholipids containing a heterocycle ring having a tertiary amine or amide as disclosed herein is shown in FIG.1B to FIG.1AG. Certain aspects of the present disclosure provide novel phospholipids containing a heterocycle ring having a tertiary amine or amide that may be advantageously used in lipid-based compositions of the present disclosure for the in vivo delivery of therapeutic agents to tissues/cells. It is contemplated within the scope of the disclosure that the novel phospholipids containing a heterocycle ring having a tertiary amine or amide comprises a racemic mixture or a mixture of one or more diastereomers. In some embodiments, the cationic lipid is enriched in one enantiomer, such that the cationic lipid comprises at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% enantiomeric excess. In some embodiments, the cationic lipid is enriched in one diastereomer, such that the cationic lipid comprises at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% diastereomeric excess. In some embodiments, the cationic lipid is chirally pure (e.g., comprises a single optical isomer). In some embodiments, the cationic lipid is enriched in one optical isomer (e.g., an optically active isomer), such that the cationic lipid comprises at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% isomeric excess. The disclosure provides the synthesis of novel phospholipids containing a heterocycle ring having a tertiary amine or amide of Formula I as a racemic mixture or in optically pure form. As used herein, the term "salts" includes any anionic and cationic complex, such as the complex formed between a cationic lipid disclosed herein and one or more anions. Examples of anions include, but are not limited to, inorganic and organic anions such as, e.g., hydride, fluoride, chloride, bromide, iodide, oxalate (e.g., hemioxalate), phosphate, phosphonate, hydrogen phosphate, dihydrogen phosphate, oxide, carbonate, bicarbonate, nitrate, nitrite, nitride, bisulfite, 78
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) sulfide, sulfite, bisulfate, sulfate, thiosulfate, hydrogen sulfate, borate, formate, acetate, benzoate, citrate, tartrate, lactate, acrylate, polyacrylate, fumarate, maleate, itaconate, glycolate, gluconate, malate, mandelate, tiglate, ascorbate, salicylate, polymethacrylate, perchlorate, chlorate, chlorite, hypochlorite, bromate, hypobromite, iodate, an alkylsulfonate, an arylsulfonate, arsenate, arsenite, chromate, dichromate, cyanide, cyanate, thiocyanate, hydroxide, peroxide, permanganate, and mixtures thereof. In particular embodiments, the salts of the cationic lipids disclosed herein are crystalline salts. As used herein, the term "alkyl" includes a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n- hexyl, and the like, while saturated branched alkyls include, without limitation, isopropyl, sec- butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, while unsaturated cyclic alkyls include, without limitation, cyclopentenyl, cyclohexenyl, and the like. As used herein, the term "alkenyl" includes an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include, but are not limited to, ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2- methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like. Cyclic alkenyls are also contemplated for the lipids of the instant disclosure. As used herein, the term "alkynyl" includes any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include, without limitation, acetylenyl, propynyl, 1-butynyl, 2- butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like. As used herein, the term "acyl" includes any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. The following are non-limiting examples of acyl groups: —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl. As used herein, the term "heterocycle" includes a monocyclic (e.g., 5-, 6-, 7-membered, and the like), bicyclic (e.g., 7-, 8-, 9-, 10-membered, and the like), or heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur 79
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include, but are not limited to, heteroaryls as defined below, as well as morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like. As used herein, the terms "optionally substituted alkyl," "optionally substituted alkenyl," "optionally substituted alkynyl," "optionally substituted acyl," and "optionally substituted heterocycle" mean that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O), two hydrogen atoms are replaced. In this regard, substituents include, but are not limited to, oxo, halogen, heterocycle, —cN, —NRxRy, — NRxC(═O)ry, —NRxSO2Ry, —C(═O)Rx, —c(═o)ORx, —c(═o)NRxry, —SOnRx, and — SOnNRxRy, wherein n is 0, 1, or 2, Rx and Ry are the same or different and are independently hydrogen, alkyl, or heterocycle, and each of the alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy, —NRxC(═O)ry, —NRxSO2Ry, —c(═o)ORx, —c(═o)ORx, —c(═o)NRxRy, —C(O-R1)(O-R2), —SOnRx, and —SOnNRxRy. The term "optionally substituted," when used before a list of substituents, means that each of the substituents in the list may be optionally substituted as described herein. As used herein, the term "halogen" includes fluoro, chloro, bromo, and iodo. In embodiments, the present disclosure provides a lipid of Formula I having the following structure: R1N m n X A O O PH O R2 (I)
O 80
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) or a salt or isomer thereof, where A is a bond, C
1-C
22 alkyl, C
2-C
22 alkenyl, C
2-C
22 alkynyl, or C
3- C
8 cycloalkyl, each of which is optionally substituted, X is N or CH, R
1 is C
5-C
22 alkyl, C
5-C
22 alkenyl, C
5-C
22 alkynyl, C
3-C
22 cycloalkyl, or C(O)C
4-C
21 alkyl, each of which is optionally substituted; R
2 is C
2-C
22 alkyl, C
2-C
22 alkynyl, or C
3-C
22 cycloalkyl, each of which is optionally substituted; and each of m and n is independently 0, 1, 2, or 3; or a compound of Formula II: R alt or isomer t R33 N R5 A1 R6 O O PH O R4I or a s , O I)
A
1 is C
1-C
22 alkyl, C
2-C
22 alkenyl, or C
2-C
22 alkynyl, each of which includes at least one substitution, or C
3-C
8 cycloaklkyl or heterocycloalkyl, each of which is optionally substituted, R
3 is C
7-C
22 alkyl, C
7-C
22 alkenyl, C
7-C
22 alkynyl, or C
4-C
22 cycloalkyl, each of which is optionally substituted; R
4 is C
2-C
16 alkyl, C
2-C
16 alkenyl, C
2-C
16 alkynyl, or C
3-C
22 cycloalkyl, each of which is optionally substituted; and each of R
5 and R
6 is independently a bond, C
1-C
7 alkyl, C
2-C
7 alkenyl, or C
2-C
7 alkynyl, each of which is optionally substituted. In some embodiments, R
1 and R
2 are each independently C
7-C
8 alkyl, C
7-C
9 alkyl, C
7-C
10 alkyl, C
7-C
11 alkyl, C
7-C
12 alkyl, C
7-C
13 alkyl, C
7-C
14 alkyl, C
7-C
15 alkyl, C
7-C
16 alkyl, C
8-C
9 alkyl, C
8-C
10 alkyl, C
8-C
11 alkyl, C
9-C
10 alkyl, C
9-C
11 alkyl, C
7-C
8 alkenyl, C
7-C
9 alkenyl, C
7-C
10 alkenyl, C
7-C
11 alkenyl, C
7-C
12 alkenyl, C
7-C
13 alkenyl, C
7-C
14 alkenyl, C
7-C
15 alkenyl, C
7-C
16 alkenyl, C
8-C
9 alkenyl, C
8-C
10 alkenyl, C
8-C
11 alkenyl, C
9-C
10 alkenyl, C
9-C
11 alkenyl, C
2-C
3 alkynyl, C
2-C
4 alkynyl, C
7-C
8 alkynyl, C
7-C
9 alkynyl, C
7-C
10 alkynyl, C
7-C
11 alkynyl, C
7-C
12 alkynyl, C
7-C
13 alkynyl, C
7-C
14 alkynyl, C
7-C
15 alkynyl, C
7-C
16 alkynyl, C
8-C
9 alkynyl, C
8-C
10 alkynyl, C
8-C
11 alkynyl, C
9-C
10 alkynyl, and/or C
9-C
11 alkynyl. In some embodiments, R
1 and R
2 are the same. In some embodiments, R
1 and R
2 are both C
6-9 alkyl. In some embodiments , R
3 and R
4 are each independently C
7-C
8 alkyl, C
7-C
9 alkyl, C
7-C
10 alkyl, C
7-C
11 alkyl, C
7-C
12 alkyl, C
7-C
13 alkyl, C
7-C
14 alkyl, C
7-C
15 alkyl, C
7-C
16 alkyl, C
8-C
9 alkyl, C
8-C
10 alkyl, C
8-C
11 alkyl, C
9-C
10 alkyl, C
9-C
11 alkyl, C
7-C
8 alkenyl, C
7-C
9 alkenyl, C
7-C
10 81
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) alkenyl, C
7-C
11 alkenyl, C
7-C
12 alkenyl, C
7-C
13 alkenyl, C
7-C
14 alkenyl, C
7-C
15 alkenyl, C
7-C
16 alkenyl, C
8-C
9 alkenyl, C
8-C
10 alkenyl, C
8-C
11 alkenyl, C
9-C
10 alkenyl, C
9-C
11 alkenyl, C
2-C
3 alkynyl, C
2-C
4 alkynyl, C
7-C
8 alkynyl, C
7-C
9 alkynyl, C
7-C
10 alkynyl, C
7-C
11 alkynyl, C
7-C
12 alkynyl, C
7-C
13 alkynyl, C
7-C
14 alkynyl, C
7-C
15 alkynyl, C
7-C
16 alkynyl, C
8-C
9 alkynyl, C
8-C
10 alkynyl, C
8-C
11 alkynyl, C
9-C
10 alkynyl, and/or C
9-C
11 alkynyl. In some embodiments, R
1 and R
2 are the same. In some embodiments, R
3 and R
4 are both C
6-9 alkyl. In some embodiments, R
1, R
2, R
3, and/or R
4 include 1, 2, 3, 4, 5, 6, or more sites of unsaturation that correspond to, for example, cis double bonds, trans double bonds, or combinations thereof, and may be located at specific positions in one or both of the unsaturated R
1 and R
2 side-chains. For those unsaturated side-chains where a double bond is located between hydrogen atoms and alkyl or alkylene chains, the chemical notation "E" refers to the trans double bond configuration and the chemical notation "Z" refers to the cis double bond configuration. As non-limiting examples, one or both R
1 and R
2 are C
8 alkyl groups containing any combination of double bonds in the cis and/or trans configuration at one or more positions, and/or are of any structure shown in the below Examples. Similarly, as non-limiting examples, one or both R
1 and R
2 are C
12 alkyl groups containing any combination of double bonds which can be characterized by either the "E" chemical notation and/or the "Z" chemical notation at one or more positions in the side-chain. In some embodiments, the positions of saturation in R
1 and R
2 are the same. In some embodiments, R
1 and R
2 are independently an alkenyl selected from the group consisting of hept-1-ene, hept-2-ene, hept-3-ene, oct-1-ene, oct-2-ene, oct-3-ene, oct-4-ene, non- 1-ene, non-2-ene, non-3-ene, non-4-ene, non-5-ene, dec-1-ene, dec-2-ene, dec-3-ene, dec-4-ene, dec-5-ene, dec-6-ene, undec-1-ene, undec-2-ene, undec-3-ene, undec-4-ene, undec-5-ene, undec- 6-ene, undec-7-ene, dodec-1-ene, dodec-2-ene, dodec-3-ene, dodec-4-ene, dodec-5-ene, dodec-6- ene, and dodec-8-ene. In some embodiments, R
3 and R
4 are independently an alkenyl selected from the group consisting of hept-1-ene, hept-2-ene, hept-3-ene, oct-1-ene, oct-2-ene, oct-3-ene, oct-4-ene, non- 1-ene, non-2-ene, non-3-ene, non-4-ene, non-5-ene, dec-1-ene, dec-2-ene, dec-3-ene, dec-4-ene, dec-5-ene, dec-6-ene, undec-1-ene, undec-2-ene, undec-3-ene, undec-4-ene, undec-5-ene, undec- 6-ene, undec-7-ene, dodec-1-ene, dodec-2-ene, dodec-3-ene, dodec-4-ene, dodec-5-ene, dodec-6- ene, and dodec-8-ene. 82
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) In some embodiments, R
1 and R
2, are independently an alkynyl selected from the group consisting of hept-1-yne, hept-2-yne, hept-3-yne, oct-1-yne, oct-2-yne, oct-3-yne, oct-4-yne, non- 1-yne, non-2-yne, non-3-yne, non-4-yne, non-5-yne, dec-1-yne, dec-2-yne, dec-3-yne, dec-4-yne, dec-5-yne, dec-6-yne, undec-1-yne, undec-2-yne, undec-3-yne, undec-4-yne, undec-5-yne, undec- 6-yne, undec-7-yne, dodec-1-yne, dodec-2-yne, dodec-3-yne, dodec-4-yne, dodec-5-yne, dodec- 6-yne, and dodec-8-yne. In some embodiments, R
3 and R
4, are independently an alkynyl selected from the group consisting of hept-1-yne, hept-2-yne, hept-3-yne, oct-1-yne, oct-2-yne, oct-3-yne, oct-4-yne, non- 1-yne, non-2-yne, non-3-yne, non-4-yne, non-5-yne, dec-1-yne, dec-2-yne, dec-3-yne, dec-4-yne, dec-5-yne, dec-6-yne, undec-1-yne, undec-2-yne, undec-3-yne, undec-4-yne, undec-5-yne, undec- 6-yne, undec-7-yne, dodec-1-yne, dodec-2-yne, dodec-3-yne, dodec-4-yne, dodec-5-yne, dodec- 6-yne, and dodec-8-yne. In some embodiments, the linker connecting the phosphate group and the amine group may include 1, 2, 3, 4, 5, 6, or more sites of unsaturation that correspond to, for example, cis double bonds, trans double bonds, or combinations thereof, and/or one or more triple bonds and may be located at specific positions within the linker. In embodiments, the present disclosure provides a lipid of Formula III having the following structure: Y O O P O O- R7II)
or a salt or isomer thereof, wherein Y is selected from the group consisting of 83
Attorney Ref.: BN00004.0144 OME-013WO (PCT Applicat R N x ion) 9 x x N R8 R9 N x x R9 N x
, 9 x x9 x x x 9 8 9 8
7 x
9 8 p 9y g oup consisting of C
2-C
22 alkyl, C
2-C
22 alkenyl, and C
2-C
22 alkynyl , each of which is optionally substituted, optionally R
7, R
9, or R
7 and R
9 are branched, optionally R
7, R
9, or R
7 and R
9 are an optionally substituted cycloalkyl or R
7 and R
9 may join to form an optionally substituted cycloalkyl; R
8 is selected from the group consisting of branched or unbranched C
1-C
7 alkyl, C
2-C
7 alkenyl, and C
2-C
7 alkynyl, each of which is optionally substituted, and x is absent, 1, 2 or 3. In embodiments, R
7 and R
9 are the same. In embodiments, R
7 or R
9 are independently selected from the group consisting of C
4-C
12 alkyl, C
4-C
12 alkenyl, and C
4-C
12 alkynyl, each of which is optionally substituted, optionally wherein R
7 and R
9 are independently selected from the group of C
4-C
12 alkyl, C
4-C
12 alkenyl, and C
4-C
12 alkynyl, each of which is optionally substituted. In embodiments, R
8 is 0, 1, 2, 3, 4, 5, or 6. In embodiments, R
7 or R
9 are independently selected from the group consisting of branched or unbranched C
4-C
12 alkyl, C
4-C
12 alkenyl, and C
4-C
12 alkynyl, each of which is optionally substituted, and R
8 is 0, 1, 2, 3, 4, 5, or 6, optionally wherein R
7 and R
9 are independently selected from the group consisting of branched or unbranched C
4-C
12 alkyl, C
4-C
12 alkenyl, and C
4-C
12 alkynyl, each of which is optionally substituted, and R
8 is 0, 1, 2, 3, 4, 5, or 6, optionally, R
8 is 2, 4, or 6. In embodiments, R
7 is selected from the group consisting of branched or unbranched C
6- C
9 alkyl, C
6-C
9 alkenyl, and C
6-C
9 alkynyl, each of which is optionally substituted, R
9 is selected from the group consisting of branched or unbranched C
6-C
9 alkyl, C
6-C
9 alkenyl, and C
6-C
9 84
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) alkynyl, each of which is optionally substituted, and R
8 is 2, 3, 4, 5, or 6, optionally R
8 is 2, 4, or 6. In embodiments, R
7 and R
9 are independently optionally substituted C
6-C
9 alkyl, and R
8 is 2, 3, 4, 5, or 6, optionally wherein R
8 is 2, 4, or 6. In embodiments, R
7 is C
9, R
9 is C
6-C
17 alkyl, and R
8 is absent, 1, or 2. In embodiments, R
7 and R
9 are independently an alkyl selected from the group consisting of heptane, octane, nonane, decane, undecane, and dodecane, each of which is optionally substituted. In embodiments, one or more of R
7 and R
9 are independently an alkenyl selected from the group consisting of hept-1-ene, hept-2-ene, hept-3-ene, oct-1-ene, oct-2-ene, oct-3-ene, oct-4-ene, non-1-ene, non-2-ene, non-3-ene, non-4-ene, non-5-ene, dec-1-ene, dec-2-ene, dec-3-ene, dec-4- ene, dec-5-ene, dec-6-ene, undec-1-ene, undec-2-ene, undec-3-ene, undec-4-ene, undec-5-ene, undec-6-ene, undec-7-ene, dodec-1-ene, dodec-2-ene, dodec-3-ene, dodec-4-ene, dodec-5-ene, dodec-6-ene, dodec-8-ene, and an alkenyl group comprising two or more double bonds, each of which is optionally substituted. In embodiments, one or more of R
7 and R
9 are independently an alkynyl selected from the group consisting of hept-1-yne, hept-2-yne, hept-3-yne, oct-1-yne, oct-2-yne, oct-3-yne, oct-4-yne, non-1-yne, non-2-yne, non-3-yne, non-4-yne, non-5-yne, dec-1-yne, dec-2-yne, dec-3-yne, dec-4- yne, dec-5-yne, dec-6-yne, undec-1-yne, undec-2-yne, undec-3-yne, undec-4-yne, undec-5-yne, undec-6-yne, undec-7-yne, dodec-1-yne, dodec-2-yne, dodec-3-yne, dodec-4-yne, dodec-5-yne, dodec-6-yne, dodec-8-yne, and an alkynyl group comprising two or more triple bonds, each of which is optionally substituted. In embodiments, the present disclosure provides a lipid of Formula IV having the following structure: 85
Attorney Ref.: BN00004.0144 OME-013WO R (PCT Appli R100 N R5 A1 R6 O OH cation) 1 O P O R11V) or a salt or isomer
A
1 is C
1-C
22 alkyl, C
2-C
22 alkenyl, or C
2-C
22 alkynyl, each of which includes at least one substitution; or C
3-C
8 cycloalkyl or heterocylcloalkyl, each of which is optionally substituted; R
10 is C
5-C
22 alkyl, C
5-C
22 alkenyl, C
5-C
22 alkynyl, or C
4-C
22 cycloalkyl, each of which is optionally substituted; R
11 is C
5-C
16 alkyl, C
5-C
16 alkenyl, C
5-C
16 alkynyl, or C
3-C
22 cycloalkyl, each of which is optionally substituted; and each of R
5 and R
6 is independently a bond, C
1-C
7 alkyl, C
2-C
7 alkenyl, or C
2-C
7 alkynyl, each of which is optionally substituted. In embodiments, R
10 and R
11 are the same, optionally wherein R
5 and R
6 are the same. In embodiments, R
10 is selected from the group consisting of C
5-C
6 alkyl, C
5-C
6 alkenyl, and C
5-C
6 alkynyl, each of which is optionally substituted and R
11 is selected from the group consisting of C
5-C
12 alkyl, C
5-C
12 alkenyl, and C
5-C
12 alkynyl, each of which is optionally substituted, wherein R
5 and R
6 are independently selected from the group consisting of a bond, C
2- C
6 alkyl, C
2-C
6 alkenyl, or C
2-C
6 alkynyl, each of which is optionally substituted, optionally wherein R
5 and R
6 are both methyl group, or either R
5 or R
6 is a methyl group and the other is a bond. In embodiments, R
10 is selected from the group consisting of branched or unbranched C
5- C
6 alkyl, C
5-C
6 alkenyl, and C
5-C
6 alkynyl, each of which is optionally substituted, and R
11 is selected from the group consisting of branched or unbranched C
5-C
9 alkyl, C
5-C
9 alkenyl, and C
5- C
9 alkynyl, each of which is optionally substituted. 86
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) In embodiments, the disclosure provides a compound selected fro O OH m: N N O P O
- ; oy - -u ecae--y -,- aepa--y e y y oge pospae, N HO
O
(SM-118; (Z)-2-(4-(dihexylamino)piperidin-1-yl)ethyl non-3-en-1-yl hydrogen phosphate), 87
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) O HO P O O N (
SM-119; 2-(4-((dihexylamino)methyl)piperidin-1-yl)ethyl nonyl hydrogen phosphate), N N O P OH
(SM-121; 2-butyloctyl (2-(4-(dihexylamino)piperidin-1-yl)ethyl) hydrogen phosphate), N N O P OH
88
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) (SM-122; 2-(4-(dipentylamino)piperidin-1-yl)ethyl nonyl hydrogen phosphate), or N N O O P OH O
- ; ecy - - e y a o p pe - -y e y y oge p osp a e . The compounds described herein may be prepared by known organic synthesis techniques, including the methods described in the below Examples. Lipid-based Compositions The techniques herein provide lipid-based compositions (e.g., LNPs and the like) comprising one or more of the novel phospholipids containing a heterocycle ring having a tertiary amine or amide or salts thereof described herein. In some embodiments, the lipid-based compositions of the disclosure further comprise one or more non-cationic lipids. In some embodiments, the lipid-based compositions further comprise one or more conjugated lipids capable of reducing or inhibiting particle aggregation. In some embodiments, the lipid-based compositions further comprise one or more active agents or therapeutic agents such as, for example, nucleic acids (e.g., siRNA, ASO, tRNA, miRNA, mRNA, DNA, and the like), proteins, peptides, and other macromolecules. As disclosed herein, lipid-based compositions include, but are not limited to, lipid nanoparticles, lipid vesicles (e.g., liposomes), and the like. As used herein, a lipid vesicle may include a structure having lipid-containing membranes enclosing an aqueous interior. In some embodiments, lipid-based compositions comprising one or more of the novel phospholipids containing a heterocycle ring having a tertiary amine or amide described herein may be used to encapsulate therapeutic agents such as, for example, nucleic acids, within the lipid vesicles. In some embodiments, lipid vesicles comprising one or more of the novel phospholipids containing 89
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) a heterocycle ring having a tertiary amine or amide described herein may be complexed with nucleic acids. The lipid-based compositions of the disclosure typically comprise a therapeutic agent, an ionizable lipid, a non-cationic lipid, and a conjugated lipid (e.g., a polyethylene glycol (PEG)- lipid) that inhibits aggregation of particles. In some embodiments, the therapeutic agent is fully encapsulated within the lipid portion of the lipid-based compositions such that the therapeutic agent is resistant to enzymatic degradation, e.g., by a nuclease or protease. In some embodiments, the lipid-based compositions described herein are substantially non-toxic to mammals such as humans. It is contemplated within the scope of the disclosure that the lipid-based compositions described herein typically have a mean diameter of from about 30 nm to about 250 nm, from about 40 nm to about 200 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, or from about 70 to about 90 nm. in some embodiments, the lipid-based compositions disclosed herein have a lipid:therapeutic agent (e.g., lipid:nucleic acid) ratio (mass/mass ratio) of from about 1:1 to about 1000:1, from about 1:1 to about 500:1, from about 2:1 to about 250:1, from about 3:1 to about 200:1, from about 5:1 to about 150:1, from about 5:1 to about 100:1, from about 5:1 to about 50:1, from about 5:1 to about 25:1, from about 5:1 to about 20:1, from about 5:1 to about 10:1, or from about 6:1 to about 9:1. Alternatively, the lipid- based compositions disclosed herein have a lipid:therapeutic agent (e.g., lipid:nucleic acid) ratio (mole/mole ratio) of from about 1:1 to about 30:1, from about 2:1 to about 20:1, from about 2:1 to about 15:1, from about 3:1 to about 10:1, from about 4:1 to about 9:1, from about 5:1 to about 8:1, or from about 6:1 to about 8:1. In some embodiments, the lipid-based compositions of the disclosure are nucleic acid-lipid particles that include an interfering RNA (e.g., dsRNA such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, and/or miRNA), an ionizable lipid (e.g., one or more lipids of Formulas I-XIX or salts thereof as set forth herein), a non-cationic lipid (e.g., mixtures of one or more phospholipids and cholesterol), and a conjugated lipid that inhibits aggregation of the particles (e.g., one or more PEG-lipid conjugates). The nucleic acid-lipid particle may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unmodified and/or modified interfering RNA molecules (e.g., siRNA). Nucleic acid-lipid particles and their method of preparation are described in, e.g., U.S. Pat. Nos.5,753,613; 5,785,992; 5,705,385; 5,976,567; 5,981,501; 6,110,745; and 6,320,017; and PCT Publication No. 90
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) WO 96/40964, the disclosures of which are each herein incorporated by reference in their entirety for all purposes. In the nucleic acid-lipid particles disclosed herein, the nucleic acid may be fully encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation. In preferred embodiments, a nucleic acid-lipid particle comprising a nucleic acid such as an interfering RNA may be fully encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation. In some embodiments, the nucleic acid may be complexed with the lipid portion of the particle. It is contemplated within the scope of the disclosure that the lipid-based compositions disclosed herein are substantially non-toxic to mammals such as humans. As used herein, the term "fully encapsulated" indicates that the nucleic acid in the nucleic acid-lipid particle is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free DNA or RNA. In a fully encapsulated system, preferably less than about 25% of the nucleic acid in the particle is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than about 10%, and most preferably less than about 5% of the nucleic acid in the particle is degraded. In some embodiments, the present disclosure provides a nucleic acid-lipid particle composition comprising a plurality of nucleic acid-lipid particles. In some instances, the nucleic acid-lipid particle composition comprises nucleic acid that is fully encapsulated within the lipid portion of the particles, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or range therein) of the particles have the nucleic acid encapsulated therein. 91
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) The techniques herein provide that the proportions of the components within the lipid- based compositions may be varied and the delivery efficiency of a particular formulation can be measured using, e.g., an endosomal release parameter (ERP) assay. It is contemplated within the scope of the disclosure that the lipid-based compositions disclosed herein have increased delivery efficiency due to enhanced endosomal release caused, at least in part, by the novel phospholipids containing a heterocycle ring having a tertiary amine or amide disclosed herein. According to the techniques herein, any one or more of the novel phospholipids containing a heterocycle ring having a tertiary amine or amide of Formula I may be used in the lipid-based compositions disclosed herein, either alone or in combination with one or more other cationic lipid species or non-cationic lipid species. Other obligate cationic lipids or salts thereof and/or ionizable lipids or salts thereof may also be included in the lipid-based compositions of the present disclosure In some embodiments, the novel phospholipids containing a heterocycle ring having a tertiary amine or amide disclosed herein comprise from about 40 mol % to about 90 mol %, from about 40 mol % to about 85 mol %, from about 40 mol % to about 80 mol %, from about 40 mol % to about 75 mol %, from about 40 mol % to about 70 mol %, from about 40 mol % to about 65 mol %, from about 40 mol % to about 60 mol %, from about 40 mol % to about 55 mol %, from about 50 mol % to about 90 mol %, from about 50 mol % to about 85 mol %, from about 50 mol % to about 80 mol %, from about 50 mol % to about 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, from about 50 mol % to about 60 mol % of the total lipid present in the particle. In some embodiments, the novel phospholipids containing a heterocycle ring having a tertiary amine or amide disclosed herein comprise from about 50 mol % to about 58 mol %, from about 51 mol % to about 59 mol %, from about 51 mol % to about 58 mol %, from about 51 mol % to about 57 mol %, from about 52 mol % to about 58 mol %, from about 52 mol % to about 57 mol %, from about 52 mol % to about 56 mol %, or from about 53 mol % to about 55 mol % of the total lipid present in the particle. In some embodiments, the cationic lipid comprises about 50 mol %, 51 mol %, 52 mol %, 53 mol %, 54 mol %, 55 mol %, 56 mol %, 57 mol %, 58 mol %, 59 mol %, 60 mol %, 61 mol %, 62 mol %, 63 mol %, 64 mol %, or 65 mol % (or any fraction thereof or range therein) of the total lipid present in the particle. In some embodiments, the ionizable lipid 92
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) comprises at least about 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 mol % of the total lipid present in the particle. In some embodiments, the ionizable lipid disclosed herein comprises from about 2 mol % to about 60 mol %, from about 5 mol % to about 50 mol %, from about 10 mol % to about 50 mol %, from about 20 mol % to about 50 mol %, from about 20 mol % to about 40 mol %, from about 30 mol % to about 40 mol %, or about 40 mol % of the total lipid present in the particle. One of skill in the art will appreciate that the percentage of ionizable lipid present in the lipid-based compositions of the disclosure is a target amount, and that the actual amount of cationic lipid present in the formulation may vary, for example, by about ±5 mol %. The lipid-based compositions disclosed herein may also include a variety of non-cationic lipids including, but not limited to, phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl- phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl- phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl- phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl- phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C
10-C
24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Other examples of non-cationic lipids may include, but are not limited to, sterols such as cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether, cholesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5α- 93
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. In preferred embodiments, the cholesterol derivative is a polar analogue such as cholesteryl-(4′-hydroxy)-butyl ether. In some embodiments, the non-cationic lipid comprises from about 10 mol % to about 60 mol %, from about 20 mol % to about 55 mol %, from about 20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, from about 25 mol % to about 50 mol %, from about 25 mol % to about 45 mol %, from about 30 mol % to about 50 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 35 mol % to about 45 mol %, from about 37 mol % to about 42 mol %, or about 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %, 43 mol %, 44 mol %, or 45 mol % (or any fraction thereof or range therein) of the total lipid present in the particle. As discussed above with respect to cationic lipids, one of skill in the art will also appreciate that the percentage of non-cationic lipid present in the lipid particles of the disclosure is a target amount, and that the actual amount of non-cationic lipid present in the formulation may vary, for example, by ±5 mol %. Lipid nanoparticles of any size may be used according to the instant disclosure. In certain embodiments of the instant disclosure, lipid nanoparticles have a size ranging from about 0.02 microns to about 0.4 microns, between about 0.05 and about 0.2 microns, or between 0.07 and 0.12 microns in diameter. In some embodiments, the LNPs may also comprise other cationic lipids including but not limited to, those comprising a protonatable tertiary amine (e.g., pH-titratable) head group; C
18 alkyl chains, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and ether, ester, or ketal linkages between the head group and alkyl chains. Such cationic lipids include, but are not limited to, 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), N,N- dioleyl-N,N-dimethylammonium chloride ("DODAC"); 3 -(N-(N',N'-dimethylaminoethane)- carbamoyl)cholesterol ("DC-Chol"), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethyl ammonium bromide ("DMRIE"), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy- N,N-dimethyl-3-aminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA, 1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2- dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA) (also known as DLin- 94
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) C2K-DMA, XTC2, and C2K), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)[1,3]-dioxolane (DLin- K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)[1,3]-dioxolane (DLin-K-C4-DMA), 1,2- dilinolenyloxy-4-(2-dimethylaminoethyl)- [1,3]-dioxolane (γ-DLen-C2K-DMA), 1,2-di-γ- linolenyloxy-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (γ-DLen-C2K-DMA), dilinoleylmethyl- 3-dimethylaminopropionate (DLin-M-C2-DMA) (also known as MC2), (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-M-C3-DMA) (also known as MC3) and 3-(dilinoleylmethoxy)-N,N-dimethylpropan-1-amine (DLin-MP-DMA) (also known as 1-B11). In some embodiments, the particles of the instant disclosure may include neutral lipids, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols. In other embodiments, LNPs may include anionic lipids, including but not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids. In some aspects, the non-cationic lipid used in the instant disclosure is 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero- 3-phosphocholine (DOPC), and/or 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC). In some aspects, one or more non-cationic lipid of the instant particles is cholesterol (CHE), β-sitosterol, and/or derivatives thereof. Cationic lipids disclosed herein may include, but are not limited to, the following exemplary cationic lipids: 1,2-DiLinoleyloxy-N,N-dimethylaminopropane. ("DLinDMA"), 1,2- Dilinolenyloxy-N,N-dimethylaminopropane ("DLenDMA"), dioctadecyldimethylammonium ("DODMA"), Distearyldimethylammonium ("DSDMA"), N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTAP"); 3 -(N-(N',N'- dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol") and N-(1,2-dimyristyloxyprop-3-yl)- N,N-dimethyl-N-hydroxyethyl ammonium bromide ("DMRIE"). For example, cationic lipids that have a positive charge at below physiological pH include, but are not limited to: DODAP, 95
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) DODMA, DMDMA, and SM-005. In some cases, the cationic lipids comprise a protonatable tertiary amine head group, C
18 alkyl chains, ether linkages between the head group and alkyl chains, and 0 to 3 double bonds. Such lipids include, e.g., DSDMA, DLinDMA, DLenDMA, and DODMA. In an exemplary embodiment, such lipids may include SM-005, and salts and isomer thereof. The chemical structure of SM-005 is shown below: (SM-0
Z N OSNH3
+ NH O S NH3 NH According to the techniques herein, "helper lipids" may inclu Cdle SM-037, C Sl
+ M-038 H2 ,N SM- NH2
+ Cl- 042, SM-044, SM-045, and/or SM-047. The chemical structures of these molecules are shown below: O O P O
(SM-037; nonyl (2-(4-(undecan-6-yl)piperazin-1-yl)ethyl) hydrogen phosphate); 96
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) N N O O P OH O
N O O P O OH
- ; - -epy oy ppe --y oy y oge pospae; O OH
(SM-044; 1-(2-heptylnonyl)azetidin-3-yl nonyl hydrogen phosphate); N H OO P
O (SM-045; 1-(2-heptylnonyl)azepan-4-yl nonyl hydrogen phosphate); and 97
Attorney Ref.: BN00004.0144 OME-013WO (PCT Applicati N O on) O P O OH
embodiments that employ PEG-conjugated lipids, the PEG-conjugated lipid is one or more of a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, and a mixture thereof. In one aspect, the PEG-lipid conjugate is one or more of a PEG-dialkyloxypropyl (DAA), a PEG-diacylglycerol (DAG), a PEG-phospholipid, a PEG-ceramide, and a mixture thereof. In one aspect, the PEG-DAG conjugate is one or more of a PEG-dilauroylglycerol (C
12), a PEG- dimyristoylglycerol (C
14), a PEG-dipalmitoylglycerol (C
16), and a PEG-distearoylglycerol (C
18). In one aspect, the PEG-DAA conjugate is one or more of a PEG-dilauryloxypropyl (C
12), a PEG- dimyristyloxypropyl (C
14), a PEG-dipalmityloxypropyl (C
16), and a PEG-di stearyloxypropyl (C
18). In some embodiments, PEG is 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000 (PEG-DMG) and/or 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG- DSG). In some embodiments, amphipathic lipids are included in particles of the instant disclosure. Amphipathic lipids may refer to any suitable material, wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Such compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids. Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatdylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Other phosphorus-lacking compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and β-acyloxyacids, can also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols. Also suitable for inclusion in the lipid particles of the instant disclosure are programmable fusion lipid formulations. Such formulations have little tendency to fuse with cell membranes and deliver their cargo until a given signal event occurs. This allows the lipid formulation to distribute 98
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) more evenly after injection into an organism or disease site before it starts fusing with cells. The signal event can be, for example, a change in pH, temperature, ionic environment, or time. In the latter case, a fusion delaying or "cloaking" component, such as an ATTA-lipid conjugate or a PEG- lipid conjugate, can simply exchange out of the lipid nanoparticle membrane over time. By the time the formulation is suitably distributed in the body, it has lost sufficient cloaking agent so as to be fusogenic. With other signal events, it is desirable to choose a signal that is associated with the disease site or target cell, such as increased temperature at a site of inflammation. In certain embodiments, it can be desirable to target the lipid nanoparticles of this disclosure further, using targeting moieties that are specific to a cell type or tissue. Targeting of lipid nanoparticles using a variety of targeting moieties, such as ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and monoclonal antibodies, has been previously described (see, e.g., U.S. Pat. Nos.4,957,773 and 4,603,044). The targeting moieties can comprise the entire protein or fragments thereof. Targeting mechanisms generally require that the targeting agents be positioned on the surface of the lipid nanoparticle in such a manner that the target moiety is available for interaction with the target, for example, a cell surface receptor. A variety of different targeting agents and methods are known and available in the art, including those described, e.g., in Sapra, P. and Allen, T M, Prog. Lipid Res.42(5):439-62 (2003); and Abra, R M et al., J. Lipid nanoparticle Res.12:1-3, (2002). Standard methods for coupling target agents can be used. For example, phosphatidylethanolamine, which can be activated for attachment of target agents, or derivatized lipophilic compounds, such as lipid-derivatized bleomycin, can be used. Antibody-targeted lipid nanoparticles can be constructed using, for instance, lipid nanoparticles that incorporate protein A (see, Renneisen, et al., J. Bio. Chem., 265:16337-16342 (1990) and Leonetti, et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451 (1990). Other examples of antibody conjugation are disclosed in U.S. Pat. No.6,027,726, the teachings of which are incorporated herein by reference. Examples of targeting moieties can also include other proteins, specific to cellular components, including antigens associated with neoplasms or tumors. Proteins used as targeting moieties can be attached to the lipid nanoparticles via covalent bonds (see, Heath, Covalent Attachment of Proteins to Lipid nanoparticles, 149 Methods in Enzymology 111-119 (Academic Press, Inc.1987)). Other targeting methods include the biotin-avidin system. 99
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) A variety of methods for preparing lipid nanoparticles are known in the art, including e.g., those described in Szoka, et al., Ann. Rev. Biophys. Bioeng., 9:467 (1980); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,946,787; PCT Publication No. WO 91/17424; Deamer and Bangham, Biochim. Biophys. Acta, 443:629-634 (1976); Fraley, et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352 (1979); Hope, et al., Biochim. Biophys. Acta, 812:55-65 (1985); Mayer, et al., Biochim. Biophys. Acta, 858:161-168 (1986); Williams, et al., Proc. Natl. Acad. Sci., 85:242-246 (1988); Lipid nanoparticles, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1; Hope, et al., Chem. Phys. Lip., 40:89 (1986); and Lipid nanoparticles: A Practical Approach, Torchilin, V. P. et al., ed., Oxford University Press (2003), and references cited therein. Suitable methods include, but are not limited to, sonication, extrusion, high pressure/homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small lipid nanoparticle vesicles, and ether-infusion methods, all of which are well known in the art. In some embodiments the disclosure, SM-037-LNPs were prepared using a microfluidic mixing process or a T-junction mixing process involving two fluid streams, one of which contained an aqueous solution of nucleic acid entities and the other had the organic solution of lipid components and/or ic molecules. Lipid/ components were prepared by combining a lipid according to the formula of 20-30 mol% of cationic lipids (e.g., SM-005), 30 to 50 mol% of a phospholipid such as SM-037 described herein, 30 to 50 mol% of a structural lipid such as cholesterol (Chol or CHE), and 0.3 to 5 mol% of a PEG-lipid (e.g., PEG-DMG) at a combined concentrations of about 10 to 30 mM in ethanol. Lipid components are combined to yield desired molar ratios (see e.g., Table 1) and diluted with aqueous solution of the nucleic acids to a final lipid concentration of between 3 to 15 mM. Nanoparticle compositions including the nucleic acids and lipid components are prepared by combining the organic solution containing the lipid/ components with the aqueous solution of nucleic acids with a total lipid to nucleic acid w/w ratio between about 10:1 and about 100:1. The lipid solution is rapidly injected using a NanoAssemblr microfluidic based system at flow rates between about 8 and about 12 mL/min into the nucleic acid aqueous solution with an aqueous to organic volume ratio between about 1:1 and about 4:1. The mixture is then immediately diluted with nuclease free water at 1:1 volume ratio. The diluted mixture is then processed using a buffer exchange column or a tangential flow filtration (TFF) system to exchange the solution with the 100
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) final desired buffer, such as Tris-HCl or a Tris/Acetate buffer, at neutral pH between 7.0 and 7.5 containing up to 15% of sucrose. The solution is then subsequently concentrated using a TFF or a centrifugation column with a filter. The concentrated solution is then sterile filtered and diluted to a desired concentration between about 0.1 mg/mL and about 1.0 mg/mL nucleic acid prior to freezing for storage. Lipid particles prepared according to methods as disclosed herein and as known in the art can in certain embodiments be stored for substantial periods of time prior to drug loading and administration to a patient. For example, lipid nanoparticles can be dehydrated, stored, and subsequently rehydrated and loaded with one or more active agents, prior to administration. Lipid nanoparticles may also be dehydrated after being loaded with one or more active agents. Dehydration can be accomplished by a variety of methods available in the art, including the dehydration and lyophilization procedures described, e.g., in U.S. Pat. Nos.4,880,635, 5,578,320, 5,837,279, 5,922,350, 4,857,319, 5,376,380, 5,817,334, 6,355,267, and 6,475,517. In one embodiment, lipid nanoparticles are dehydrated using standard freeze-drying apparatus, i.e., they are dehydrated under low pressure conditions. Also, the lipid nanoparticles can be frozen, e.g., in liquid nitrogen, prior to dehydration. Sugars can be added to the LNP environment, e.g., to the buffer containing the lipid nanoparticles, prior to dehydration, thereby promoting the integrity of the lipid nanoparticle during dehydration. See, e.g., U.S. Pat. No.5,077,056 or 5,736,155. Lipid nanoparticles may be sterilized by conventional methods at any point during their preparation, including, e.g., after sizing or after generating a pH gradient. Cargo-Loaded Lipid Particle Compositions In various embodiments, lipid particles of the instant disclosure may be used for many different applications, including the delivery of an active agent to a cell, tissue, organ or subject. For example, lipid nanoparticles of the instant disclosure may be used to deliver a therapeutic agent systemically via the bloodstream or to deliver a cosmetic agent to the skin. Accordingly, lipid nanoparticles of the instant disclosure and one or more active agents as cargo(es) are included in the instant disclosure. 101
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Lipid Particle Cargoes The instant disclosure describes lipid nanoparticles (i.e., a lipid nanoparticle comprising DOTAP) in combination with an active agent as a cargo. Active agents, as used herein, include any molecule or compound capable of exerting a desired effect on a cell, tissue, organ, or subject. Such effects may be biological, physiological, or cosmetic, for example. Active agents may be any type of molecule or compound, including e.g., nucleic acids, such as single- or double-stranded polynucleotides, plasmids, antisense RNA, RNA interference agents, including, e.g., DNA-DNA hybrids, DNA-RNA hybrids, RNA-DNA hybrids, RNA-RNA hybrids, short interfering RNAs (siRNA), micro RNAs (mRNA) and short hairpin RNAs (shRNAs); peptides and polypeptides, including, e.g., antibodies, such as, e.g., polyclonal antibodies, monoclonal antibodies, antibody fragments; humanized antibodies, recombinant antibodies, recombinant human antibodies, and Primatized™ antibodies, cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell surface receptors and their ligands; hormones; and small molecules, including small organic molecules or compounds. Therapeutic Agents As disclosed herein, therapeutic agents may include any molecule or compound capable of exerting a desired effect on a cell, tissue, tumor, organ, or subject. Therapeutic agents may be any type of molecule or compound including, but not limited to, nucleic acids, peptides, polypeptides, small molecules, and mixtures thereof. In some embodiments, the therapeutic agent may be a salt or derivative thereof. Therapeutic agents may be therapeutically active themselves, or they may be prodrugs, which become active upon further modification/alteration. In some embodiments, the lipid-based compositions described herein may be associated with a nucleic acid such as, for example, an siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, miRNA, antisense oligonucleotides, ribozymes, and immunostimulatory oligonucleotides. Nucleic acids associated with or encapsulated by LNPs may contain modifications including but not limited to those selected from the following group: 2′-O-methyl modified nucleotides, a nucleotide comprising a 5′-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, a 2′-deoxy-2′-fluoro modified nucleotide, a 5′-methoxy-modified 102
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) nucleotide (e.g., 5′-methoxyuridine), a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide; internucleoside linkages or backbones including phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2.′ In certain embodiments, the active agent is a mRNA or a vector capable expressing a mRNA in a cell. In embodiments, the active agent is a CRISPR/Cas system. Optionally, a LNP of the instant disclosure can be formulated to include, e.g., both a guide strand (gRNA) and a Cas enzyme as cargoes, thereby providing a self-contained delivery vehicle capable of effecting and controlling CRISPR-mediated targeting of a gene in a target cell. In certain featured embodiments, the active agent is a nucleic acid modulating controller (e.g., a mRNA that encodes protein controller components, as described above). In some embodiments, the active agent is a therapeutic agent, or a salt or derivative thereof. Therapeutic agent derivatives may be therapeutically active themselves or they may be prodrugs, which become active upon further modification. Thus, in one embodiment, a therapeutic agent derivative retains some or all of the therapeutic activity as compared to the unmodified agent, while in another embodiment, a therapeutic agent derivative lacks therapeutic activity. In various embodiments, therapeutic agents include agents and drugs, such as anti- inflammatory compounds, narcotics, depressants, anti-depressants, stimulants, hallucinogens, analgesics, antibiotics, birth control medication, antipyretics, vasodilators, anti-angiogenics, cytovascular agents, signal transduction inhibitors, vasoconstrictors, hormones, and steroids. In certain embodiments, the active agent is an oncology drug, which may also be referred to as an anti-tumor drug, an anti-cancer drug, a tumor drug, an antineoplastic agent, or the like. Examples of oncology drugs that may be used according to the instant disclosure include, but are not limited to, adriamycin, alkeran, allopurinol, altretamine, amifostine, anastrozole, araC, arsenic 103
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) trioxide, azathioprine, bexarotene, biCNU, bleomycin, busulfan intravenous, busulfan oral, capecitabine (Xeloda), carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin, cladribine, cyclosporin A, cytarabine, cytosine arabinoside, daunorubicin, cytoxan, daunorubicin, dexamethasone, dexrazoxane, dodetaxel, doxorubicin, doxorubicin, DTIC, epirubicin, estramustine, etoposide phosphate, etoposide and VP-16, exemestane, FK506, fludarabine, fluorouracil, 5-FU, gemcitabine (Gemzar), gemtuzumab-ozogamicin, goserelin acetate, hydrea, hydroxyurea, idarubicin, ifosfamide, imatinib mesylate, interferon, irinotecan (Camptostar, CPT- 111), letrozole, leucovorin, leustatin, leuprolide, levamisole, litretinoin, megastrol, melphalan, L- PAM, mesna, methotrexate, methoxsalen, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel, pamidronate, Pegademase, pentostatin, porfimer sodium, prednisone, rituxan, streptozocin, STI-571, tamoxifen, taxotere, temozolamide, teniposide, VM-26, topotecan (Hycamtin), toremifene, tretinoin, ATRA, valrubicin, velban, vinblastine, vincristine, VP16, and vinorelbine. Other examples of oncology drugs that may be used according to the instant disclosure are ellipticin and ellipticin analogs or derivatives, epothilones, intracellular kinase inhibitors and camptothecins. While LNP compositions of the instant disclosure generally comprise a single active agent, in certain embodiments, they may comprise more than one active agent. In other embodiments of the instant disclosure, the lipid nanoparticles of the instant disclosure have a plasma circulation half-life of at least 0.5, 0.8, 1.2, 1.5, 2.0, 4.0, 6.0, 8.0, or 12 hours. In some embodiments, lipid nanoparticles have a plasma drug half-life of at least 0.5, 0.8, 1.2, 1.5, 2.0, 4.0, 6.0, 8.0, or 12 hours. Circulation and blood or plasma clearance half-lives may be determined as described, for example, in U.S. Patent Publication No.2004-0071768-A1. The techniques herein further comprise lipid particles and/or pharmaceutical compositions in which a therapeutic agent such as, for example, nucleic acids (e.g., siRNA, ASO, tRNA, miRNA, mRNA, DNA, and the like), proteins, peptides, and other macromolecules, is enclosed within the lipid portion of the particle or composition so that it is protected from degradation. Such lipid particles and/or pharmaceutical compositions may be formed by any method known in the art including, but not limited to, a continuous mixing method, a direct dilution process, and an in- line dilution process. In some embodiments, lipid particles and/or pharmaceutical compositions may include any of the novel phospholipids containing a heterocycle ring having a tertiary amine or amide disclosed 104
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) herein, or salts thereof, alone or in combination with other cationic lipids and/or non-cationic lipids. In other embodiments, the non-cationic lipids may be egg sphingomyelin (ESM), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2- oleoyl-phosphatidylcholine (POPC), dipalmitoyl-phosphatidylcholine (DPPC), monomethyl- phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, 14:0 PE (1,2-dimyristoyl- phosphatidylethanolamine (DMPE)), 16:0 PE (1,2-dipalmitoyl-phosphatidylethanolamine (DPPE)), 18:0 PE (1,2-distearoyl-phosphatidylethanolamine (DSPE)), 18:1 PE (1,2- dioleoylphosphatidylethanolamine (DOPE)), 18:1 trans PE (1,2-dielaidoyl- phosphatidylethanolamine (DEPE)), 18:0-18:1 PE (1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE)), 16:0-18:1 PE (1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)), polyethylene glycol-based polymers (e.g., PEG 2000, PEG 5000, PEG-modified diacylglycerols, or PEG- modified dialkyloxypropyls), cholesterol, derivatives thereof, or combinations thereof. The lipid particles and/or pharmaceutical compositions disclosed herein may be formed using techniques know in the art such as, for example, continuous mixing in which the process of continuously introducing lipid and buffer solutions into a mixing area causes a continuous dilution of the lipid solution with the buffer solution, which has the effect of producing a lipid vesicle almost immediately upon mixing. By mixing an aqueous solution comprising a therapeutic agent with an organic lipid solution, the organic lipid solution may undergo a continuous stepwise dilution in the presence of the buffer solution to produce a therapeutic agent-lipid particle. Such particles may have a size of from about 30 nm to about 250 nm, from about 40 nm to about 200 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 nm to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, or 250 nm, or any intermediate value or sub-range therein. Once formed, the particles do not aggregate. According to the techniques herein, the particles may be sized to achieve a uniform particle size. 105
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) It is also contemplated within the scope of the disclosure that such particles may be prepared by a direct dilution process (e.g., forming a lipid vesicle solution and directly introducing it into a container having a controlled amount of dilution buffer) such as is described in U.S. Patent Publication No. 20070042031, the disclosure of which is herein incorporated by reference in its entirety for all purposes. The particles formed using the direct dilution processes typically have a size of from about 30 nm to about 250 nm, from about 40 nm to about 200 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 nm to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, or 250 nm, or any intermediate value or sub-range therein. Once formed, the particles do not aggregate. According to the techniques herein, the particles may be sized to achieve a uniform particle size. In some embodiments, non-lipid polycations which are useful to effect the lipofection of cells may be added to the present compositions. Examples of suitable non-lipid polycations include, hexadimethrine bromide (sold under the brand name POLYBRENE®, from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts of hexadimethrine. Other suitable polycations include, for example, salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine, polyallylamine, and polyethyleneimine. Addition of these salts is preferably after the particles have been formed. Kits The instant disclosure also provides lipid nanoparticles and variations thereof in kit form. The kit may comprise a ready-made formulation or a formulation that requires mixing before administration. The kit will typically comprise a container that is compartmentalized for holding the various elements of the kit. The kit will contain the lipid nanoparticle compositions of the instant disclosure or the components thereof, in hydrated or dehydrated form, with instructions for their rehydration and administration. In particular embodiments, a kit comprises at least one compartment containing a lipid nanoparticle of the instant disclosure that is loaded with an active 106
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) agent. In another embodiment, a kit comprises at least two compartments, one containing a lipid nanoparticle of the instant disclosure and the other containing an active agent. Of course, it is understood that any of these kits may comprise additional compartments, e.g., a compartment comprising a buffer, such as those described in U.S. Patent Publication No. 2004-0228909-A1. Kits of the instant disclosure, which comprise lipid nanoparticles comprising ionizable lipids (e.g., SM-037), may also contain other features of the kits described in U.S. Patent Publication No.2004- 0228909 A1. Further the kit may contain drug-loaded lipid nanoparticles in one compartment and empty lipid nanoparticles in a second compartment. Alternatively, the kit may contain a lipid nanoparticle of the instant disclosure, an active agent to be loaded into the lipid nanoparticle of the instant disclosure in a second compartment, and an empty lipid nanoparticle in a third compartment. In a particular embodiment, a kit of the instant disclosure comprises a therapeutic compound encapsulated in a lipid nanoparticle comprising SM-037, where SM-037 constitutes at least 20%, at least 50%, or at least 70% (molar basis) of total phospholipids present in the lipid nanoparticle, as well as an empty lipid nanoparticle. In one embodiment, the lipid nanoparticle containing therapeutic compound and the empty lipid nanoparticle are present in different compartments of the kit. Methods of Treatment The LNP compositions of the instant disclosure may be used to treat any of a wide variety of diseases or disorders, including, but not limited to, inflammatory diseases, cardiovascular diseases, nervous system diseases, tumors, demyelinating diseases, digestive system diseases, endocrine system diseases, reproductive system diseases, hemic and lymphatic diseases, immunological diseases, mental disorders, musculoskeletal diseases, neurological diseases, neuromuscular diseases, metabolic diseases, sexually transmitted diseases, skin and connective tissue diseases, urological diseases, and infections. In certain embodiments, the LNP compositions can be employed to treat or prevent a lung disease or disorder, including but not limited to a disease or disorder selected from the following: lung cancer, pneumonia, pulmonary fibrosis, COPD, asthma, bronchiectasis, sarcoidosis, pulmonary hypertension, emphysema, alpha-1 antitrypsin deficiency, aspergillosis, bronchiolitis, bronchitis, pneumoconiosis, Coronaviruses, Middle Eastern Respiratory Syndrome, Severe Acute 107
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Respiratory Syndrome, cystic fibrosis, Legionnaire's disease, influenza, pertussis, pulmonary embolism, and tuberculosis. In other embodiments, the LNP compositions of the instant disclosure can be used to treat or prevent a joint disease or disorder, including but not limited to a disease or disorder selected from the following: rheumatoid arthritis, psoriatic arthritis, gout, tendinitis, bursitis, Carpal Tunnel Syndrome, and osteoarthritis. In other embodiments, the LNP compositions of the instant disclosure can be used to treat or prevent an inflammatory disease or disorder, including but not limited to a disease or disorder selected from the following: inflammatory bowel disease, peritonitis, osteomyelitis, cachexia, pancreatitis, trauma induced shock, bronchial asthma, allergic rhinitis, cystic fibrosis, acute bronchitis, acute intense bronchitis, osteoarthritis, rheumatoid arthritis, infectious arthritis, post- infectious arthritis, gonocoele arthritis, tuberculous arthritis, arthritis, osteoarthritis, gout, spondyloarthropathies, ankylosing spondylitis, arthritis associated with vasculitis syndrome, nodular polyarteritis nervosa, irritable vasculitis, rugenic granulomatosis, rheumatoid polyposis myalgia, arthritis cell arteritis, calcium polycystic arthropathy, caustic gout, non-arthritic rheumatism, bursitis, hay fever, suppurative inflammation (e.g., tennis elbow), neuropathic joint disease, hemarthrosic, Henoch-Schlein purpura, hypertrophic osteoarthritis, multisized hemorrhoids, scoliosis, hemochromatosis, hyperlipoproteinemia, hypogammaglobulinemia, COPD, acute respiratory distress syndrome, acute lung injury, broncho-pulmonary dysplasia and systemic lupus erythematosus (SLE). In other embodiments, the LNP compositions of the instant disclosure can be used to treat or prevent an epidermal disease or disorder, including but not limited to psoriasis, atopic dermatitis, scleroderma, eczema, rosacea, seborrheic dermatitis, melanoma, solar keratosis, ichthyosis, Grover's disease, common warts, keratoacanthoma, and seborrhoeic keratosis. In one embodiment, the LNP compositions of the instant disclosure can be used to treat or prevent a type of cancer. In particular, these methods can be applied to cancers of the blood and lymphatic systems, including lymphomas, leukemia, and myelomas. Examples of specific cancers that may be treated according to the instant disclosure include, but are not limited to, Hodgkin's and non-Hodgkin's Lymphoma (NHL), including any type of NHL as defined according to any of the various classification systems such as the Working formulation, the Rappaport classification and, preferably, the REAL classification. Such lymphomas include, but are not limited to, low- 108
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) grade, intermediate-grade, and high-grade lymphomas, as well as both B-cell and T-cell lymphomas. Included in these categories are the various types of small cell, large cell, cleaved cell, lymphocytic, follicular, diffuse, Burkitt's, Mantle cell, NK cell, CNS, AIDS-related, lymphoblastic, adult lymphoblastic, indolent, aggressive, transformed and other types of lymphomas. The methods of the instant disclosure can be used for adult or childhood forms of lymphoma, as well as lymphomas at any stage, e.g., stage I, II, III, or IV. The various types of lymphomas are well known to those of skill, and are described, e.g., by the American Cancer Society (see, e.g., www3.cancer.org). The compositions and methods described herein may also be applied to any form of leukemia, including adult and childhood forms of the disease. For example, any acute, chronic, myelogenous, and lymphocytic form of the disease can be treated using the methods of the instant disclosure. In preferred embodiments, the methods are used to treat Acute Lymphocytic Leukemia (ALL). More information about the various types of leukemia can be found, inter alia, from the Leukemia Society of America (see, e.g., (www)leukemia.org). Additional types of tumors can also be treated using the methods described herein, such as neuroblastomas, myelomas, prostate cancers, small cell lung cancer, colon cancer, ovarian cancer, non-small cell lung cancer, brain tumors, breast cancer, and others. The LNP compositions of the instant disclosure may be administered as first line treatments or as secondary treatments. In addition, they may be administered as a primary chemotherapeutic treatment or as adjuvant or neoadjuvant chemotherapy. For example, treatments of relapsed, indolent, transformed, and aggressive forms of non-Hodgkin's Lymphoma may be administered following at least one course of a primary anti- cancer treatment, such as chemotherapy and/or radiation therapy. Administration of LNP Compositions LNP compositions of the instant disclosure are administered in any of a number of ways, including parenteral, intravenous, systemic, local, oral, intratumoral, intramuscular, subcutaneous, intraperitoneal, inhalation, or any such method of delivery. In one embodiment, the compositions are administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In a specific embodiment, the LNP compositions are administered by intravenous infusion or intraperitoneally by a bolus injection. For example, in one embodiment, a patient is given an intravenous infusion of the lipid nanoparticle-encapsulated 109
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) active agent through a running intravenous line over, e.g., 5-10 minutes, 15-20 minutes, 30 minutes, 60 minutes, 90 minutes, or longer. In one embodiment, a 60-minute infusion is used. In other embodiments, an infusion ranging from 6-10 or 15-20 minutes is used. Such infusions can be given periodically, e.g., once every 1, 3, 5, 7, 10, 14, 21, or 28 days or longer, preferably once every 7-21 days, and preferably once every 7 or 14 days. LNP compositions of the instant disclosure may be formulated as pharmaceutical compositions suitable for delivery to a subject. The pharmaceutical compositions of the instant disclosure will often further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, dextrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, compositions of the instant disclosure may be formulated as a lyophilizate. The concentration of drug and lipid nanoparticles in the pharmaceutical formulations can vary widely, i.e., from less than about 0.05%, usually at or at least about 2-5% to as much as 10 to 30% by weight and will be selected depend upon the particular drug used, the disease state being treated and the judgment of the clinician taking. Further, the concentration of drug and lipid nanoparticles will also take into consideration the fluid volume administered, the osmolality of the administered solution, and the tolerability of the drug and lipid nanoparticles. In some instances, it may be preferable to use a lower drug or lipid nanoparticle concentration to reduce the incidence or severity of infusion-related side effects. Suitable formulations for use in the instant disclosure can be found, e.g., in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17
th Ed. (1985). Often, intravenous compositions will comprise a solution of the lipid nanoparticles suspended in an acceptable carrier, such as an aqueous carrier. Any of a variety of aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% dextrose, and the like, and may include glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. Often, normal buffered saline (135-150 mM NaCl) or 5% dextrose will be used. These compositions can be sterilized by conventional sterilization techniques, such as filtration. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and 110
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may also contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. Additionally, the composition may include lipid-protective agents, which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free-radical quenchers, such as α-tocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable. The amount of active agent administered per dose is selected to be above the minimal therapeutic dose but below a toxic dose. The choice of amount per dose will depend on a number of factors, such as the medical history of the patient, the use of other therapies, and the nature of the disease. In addition, the amount of active agent administered may be adjusted throughout treatment, depending on the patient's response to treatment and the presence or severity of any treatment-associated side effects. In certain embodiments, the dosage of LNP composition or the frequency of administration is approximately the same as the dosage and schedule of treatment with the corresponding free active agent. However, it is understood that the dosage may be higher or more frequently administered as compared to free drug treatment, particularly where the LNP composition exhibits reduced toxicity. It is also understood that the dosage may be lower or less frequently administered as compared to free drug treatment, particularly where the LNP composition exhibits increased efficacy as compared to the free drug. Exemplary dosages and treatment for a variety of chemotherapy compounds (free drug) are known and available to those skilled in the art and are described in, e.g., Physician's Cancer Chemotherapy Drug Manual, E. Chu and V. Devita (Jones and Bartlett, 2002). Patients typically will receive at least two courses of such treatment, and potentially more, depending on the response of the patient to the treatment. In single agent regimens, total courses of treatment are determined by the patient and physician based on observed responses and toxicity. Combination Therapies In certain embodiments, LNP compositions of the instant disclosure can be administered in combination with one or more additional compounds or therapies, such as surgery, radiation treatment, chemotherapy, or other active agents, including any of those described above. LNP 111
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) compositions may be administered in combination with a second active agent for a variety of reasons, including increased efficacy or to reduce undesirable side effects. The LNP composition may be administered prior to, subsequent to, or simultaneously with the additional treatment. Furthermore, where a LNP composition of the instant disclosure (which comprises a first active agent) is administered in combination with a second active agent, the second active agent may be administered as a free drug, as an independent LNP formulation, or as a component of the LNP composition comprising the first drug. In certain embodiments, multiple active agents are loaded into the same lipid nanoparticles. In other embodiments, lipid nanoparticles comprising an active agent are used in combination with one or more free drugs. In particular embodiments, LNP compositions comprising an active agent are formed individually and subsequently combined with other compounds for a single co-administration. Alternatively, certain therapies are administered sequentially in a predetermined order. Accordingly, LNP compositions of the instant disclosure may comprise one or more active agents. Other combination therapies known to those of skill in the art can be used in conjunction with the methods of the instant disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Reference will now be made in detail to exemplary embodiments of the disclosure. While the disclosure will be described in conjunction with the exemplary embodiments, it will be understood that it is not intended to limit the disclosure to those embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the disclosure as defined by the appended claims. Standard techniques well known in the art or the techniques specifically described below were utilized. 112
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) EXAMPLES Example 1: Sy HN N Boc TEA1 (b3 (01 e.2q) e Onthesis of SM-031 q D) CClM ON N Boc DCHMCl/ 2d5iox °Cane 3 h ON NH K 8 B0Ir ( °0C11 eq OO) P K O 6 O2HC (1O.03 ( e3q)0 eq) CPME ON N O OP O OH
ep 1: 0e- 20 °C- 2 hu y - e 2 3 2 h OMG Oa oy p pe a e- -ca o y a e: -
OT
MS
GM
T0
03
51
7NX1 ( eq Cl
HN N Boc T 0E 2A0 (3 °C0 e 2q h)) DCM N N Boc To a solution of tert-butyl piperazine-1-carboxylate (8.5 g, 45.64 mmol, 1.0 eq) in DCM (100 mL) was added TEA (13.85 g, 136.91 mmol, 19.06 mL, 3.0 eq). Then hexanoyl chloride (7.37 g, 54.76 mmol, 7.65 mL, 1.2 eq) was added slowly to the mixture at 0 °C. The mixture was stirred at 20 °C for 2 h under N
2 atmosphere. The reaction mixture was quenched by addition MeOH (10 mL) at 0°C, and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® Silica Flash Column, Eluent of 0~20% EtOAc/PE gradient @ 100 mL/min) to get compound tert-butyl 4- hexanoylpiperazine-1-carboxylate (10.5 g, 33.23 mmol, 72.8% yield, 90.0% purity) as a white solid. 1H NMR (400 MHz, DMSO-d
6) δ = 3.45 - 3.38 (m, 4H), 3.32 - 3.30 (m, 2H), 3.29 - 3.23 (m, 2H), 2.29 (t, J = 7.6 Hz, 2H), 1.54 - 1.45 (m, 2H), 1.41 (s, 9H), 1.33 - 1.22 (m, 4H), 0.87 (t, J = 6.8 Hz, 3H). 113
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Step 2:O 1-pNiper Naz Binoc-1-ylhexan-1-one: ( DCHCl/dioxane EC843 O3-7N) NH 2 M 25 °C 3 h
o a so uton o tert-buty - exanoy p pe 3raz ne-1-carboxylate (9.5 g, 33.40 mmol, 1.0 eq) in DCM (40 mL) was added HCl/dioxane (4 M, 40 mL, 4.8 eq). The mixture was stirred at 25 °C for 3 h. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was triturated with PE/EtOAc=3/1 (30 mL) at 20 °C for 0.5 h to get compound 1-piperazin- 1-ylhexan-1-one (7 g, 30.76 mmol, 92.1% yield, 97.0% purity, HCl) as a white solid. 1H NMR (400 MHz, CDCl
3) δ = 10.06 (s, 2H), 4.08 - 3.76 (m, 4H), 3.37 - 3.31 (m, 4H), 2.32 (t, J = 7.6 Hz, 2H), 1.69 - 1.56 (m, 2H), 1.41 - 1.26 (m, 4H), 0.91 (t, J = 6.8 Hz, 3H). Step 3: 2-(4-hexanoylpiperazin-1-yl)ethyl nonyl hydrogen phosphate: (EC8433-15) ON NH K 8 B0Ir ( °0C11 e2q OO) h P K O 6 OH 2C (1O035 (3 eq0) eq) CPME ON N
O OP O OH
To a solution of 1-piperazin-1-ylhexan-1-one (1.05 g, 4.76 mmol, 1.1 eq, HCl) and 2- bromoethyl nonyl hydrogen phosphate (1.5 g, 4.53 mmol, 1.0 eq) in CPME (15 mL) was added K
2CO
3 (1.88 g, 13.59 mmol, 3 eq) and KI (75.19 mg, 452.92 umol, 0.1 eq). The mixture was stirred at 80 °C for 12 h. The reaction mixture was filtered and washed with EtOAc (30 mL). The filtrate was collected and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0~10% MeOH/DCM gradient @ 60 mL/min) and prep-HPLC (column: Phenomenex luna C
18 150*25mm* 10um;mobile phase: [water(FA)-ACN]; mixture of water(FA) and ACN containing 114
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 35%-65% CAN, 8min) to get compound SM-031, also known as 2-(4-hexanoylpiperazin-1- yl)ethyl nonyl hydrogen phosphate (321.70 mg, 738.28 umol, 16.3% yield, 99.73% purity) as an off-white solid. LCMS: [M+H]
+: 435.5 1H NMR (400 MHz, CDCl
3) δ = 4.43 - 4.21 (m, 2H), 4.06 - 3.83 (m, 6H), 3.42 - 2.94 (m, 6H), 2.31 (t, J = 7.6 Hz, 2H), 1.68 - 1.58 (m, 4H), 1.37 - 1.24 (m, 16H), 0.99 - 0.81 (m, 6H). Example 2: Synth 1 O O DMt-BE 1bu Oes O 0 (1K9. Ois of SM-032 O50 ( P1 e ° OCq O5) e 2q4) h O
O O EtOH H220 Pd °C/C 16 h HO O CBr4 D (C1M.5 e 0q-) 25 PP °Ch34 (1 h.5 eq) Br O ( e)r () e H 2 (O 3 O 4 O ))) e)
3 ( ( e) e) ( e) r 3a (r e)e
115
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Step 1: Ethyl 2-(benzyloxymethyl)cyclopropanecarboxylate (2): (EC5059-232/23 O O tB 1 Obu OO (1 O5 e Oq O 6) P) O
O O
1o a so ut on DM oE e 0tK90y (1 °C5 e 2-qd4) het oxyp osp ory 2acetate (81.9 g, 365.41 mmol, 72.50 mL, 1.5 eq) in DME (600 mL) was added t-BuOK (41.0 g, 365.41 mmol, 1.5 eq) at 0 °C under N
2 (a large quantity of white precipitate was formed). After addition, the mixture was stirred at 20 °C for 1 h, and then 2-(benzyloxymethyl)oxirane (40 g, 243.60 mmol, 37.04 mL, 1.0 eq) in DME (50 mL) was added dropwise at 20 °C. The resulting mixture was stirred in 90 °C oil bath for 23 h. The reaction mixture was quenched by addition NH
4Cl (300 mL) at 0 °C, and then diluted with EtOAc (300 mL) and extracted with EtOAc (300 mL ^ 3 ). The combined organic layers were washed with brine (400 mL), dried over Na
2SO
4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (330 g SepaFlash® Silica Flash Column,PE: EtOAc: 0~10%) to give compound ethyl 2- (benzyloxymethyl)cyclopropanecarboxylate (30.1 g, 128.47 mmol, 52.8% yield) as yellow oil. 1H NMR (400 MHz, CDCl
3) δ = 7.40 - 7.28 (m, 5H), 4.53 (s, 2H), 4.13 (q, J = 7.2 Hz, 2H), 3.48 - 3.43 (m, 1H), 3.42 - 3.35 (m, 1H), 1.80 - 1.70 (m, 1H), 1.60 - 1.55 (m, 1H), 1.27 (t, J = 7.2 Hz, 3H), 1.24 - 1.18 (m, 1H), 0.89 - 0.84 (m, 1H). Step 2: ethyl 2-(hydroxymethyl)cyclopropanecarboxylate (3): (EC5059
O O To a 2 solu Otion of EtOH H 20d °C/C 16 h HO 3 O O -238/241) 2 P ethyl 2-(benzyloxymethyl)cyclopropanecarboxylate (30 g, 128.05 mmol, 1.0 eq) in EtOH (100 mL) were added Pd/C (3 g, 10% purity) and Pd(OH)
2 (3.3 g, 23.74 mmol) under Argon. The suspension was degassed under vacuum and purged with H
2 several times. The mixture was stirred under H
2 (45 psi) at 35 °C for 24 h. The reaction mixture was filtered and the 116
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) filter cake was washed with MeOH (100 mL ^ 3). The filtrate was concentrated under reduced pressure to give residue. The residue was purified by flash silica gel chromatography (220 g SepaFlash® Silica Flash Column, PE : EtOAc : 0~20%) to give compound ethyl 2- (hydroxymethyl)cyclopropanecarboxylate (18.1 g, 125.55 mmol, 97.8% yield) as yellow oil. S HtOep 3: ethy Ol 2-(bro CmBromethyl)cyclopropanecarboxylate (4): (EC5059-242/243) D4 (C1.5 eq), PPh3 (1.5 eq) Br O
T 3o O a solution of etMhy 0l- 22-5( °hCyd 4r hoxymethyl)cycl 4 4o Opropanecarboxylate (18 g, 124.85 mmol, 1.0 eq) in DCM (600 mL) were added CBr (62.11 g, 187.28 mmol, 1.5 eq) and PPh
3 (49.12 g, 187.28 mmol, 1.5 eq) in portions at 0 °C under N
2. After addition, the mixture was stirred at 25 °C for 4 h (a large quantity of white precipitate was formed). The reaction mixture was filtered and the filter cake was washed with EtOAc (100 mL ^ 3). The filtrate was concentrated under reduced pressure to give residue. The residue was purified by flash silica gel chromatography (330 g SepaFlash® Silica Flash Column, PE:EtOAc: 0~10%) to give compound ethyl 2- (bromomethyl)cyclopropanecarboxylate (20.5 g, 99.00 mmol, 79.5% yield) as yellow oil. 1H NMR (400 MHz, CDCl
3) δ = 4.19 - 4.10 (m, 2H), 3.41 - 3.28 (m, 2H), 1.98 - 1.84 (m, 1H), 1.67 - 1.63 (m, 1H), 1.43 - 1.37 (m, 1H), 1.30 - 1.25 (m, 3H), 0.98 - 0.94 (m, 1H). Step 4: [2-(bromomethy Ll)AcHy (c1l.1o epropyl]methanol (5): (EC5059-245/246)
Br O O THF 0 °C 2q h) Br OH To a sol4ution of ethyl 2-(bromomethyl)cyclopr 5opanecarboxylate (20.2 g, 97.55 mmol, 1.0 eq) in THF (400 mL) was added LAH (4.07 g, 107.31 mmol, 1.1 eq) in portions at 0 °C under N
2. After addition, the mixture was stirred at 0 °C for 2 h. The reaction mixture was quenched by slowly addition 10% HCl solution (200 mL) at 0 °C under N
2, and extracted with EtOAc (300 mL 117
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) ^ 3). The combined organic layers were washed with brine (300 mL), dried over Na
2SO
4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (120 g SepaFlash® Silica Flash Column,PE:EtOAc: 0~20%) to give compound [2-(bromomethyl)cyclopropyl]methanol (5.3 g, 32.12 mmol, 33.1% yield) as yellow oil. 1H NMR (400 MHz, CDCl
3) δ = 3.9 (t, J = 4.8 Hz, 1H), 3.49 - 3.37 (m, 2H), 3.29 - 3.24 (m, 1H), 1.26 - 1.21 (m, 1H), 1.18 - 1.11 (m, 1H), 0.78 - 0.74 (m, 1H), 0.70 - 0.63 (m, 1H). Step 5: [2-(bromomethyl)cyclopropyl]methyl nonyl hydrogen phosphate (7): (EC5059- 251/252) Br OH 1) 5 (12)06 T e HH ( 3qO 1F))01 P 00 eO-%q2C)5 Hl 63 ° TCE (1A 10 ( h3 eq0) e TqE)A TH (1F2 eq)
O OP O OH
TEA (3.68 g, 36.36 mmol, 5.1 mClL 4,01 °.C22 e hq) was slow Blry added to POCl
3 (4.65 g, 30.30 mmol, 2.82 mL, 1.0 eq) in dry THF (100 mL) at 0 °C under N
2. Then nonan-1-ol (4.37 g, 30.30 mmol, 1.0 eq) in THF (50 mL) was added drop wise over 1 h and the resulting mixture was warmed to 20 °C was stirred for 1 h. When all the alcohol had reacted (checked by TLC), the mixture was cooled to 0 °C and a second portion of TEA (9.20 g, 90.89 mmol, 12.65 mL, 3.0 eq) was added, followed by [2-(bromomethyl)cyclopropyl]methanol (5 g, 30.30 mmol, 4.69 mL, 1.0 eq) in THF (50 mL) was added dropwise. The reaction mixture was stirred at 20 °C for 14 h. decomposed with HCl 10% (100 mL) and heated at 40 °C for 2 h. THF was removed under vacuum and the aqueous residue was extracted with EtOAc (100 mL ^ 3). The combined organic layers were washed with brine (120 mL), dried over Na
2SO
4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (80 g SepaFlash® Silica Flash Column,PE : EtOAc: 0~20%) to give compound [2-(bromomethyl)cyclopropyl]methyl nonyl hydrogen phosphate (5.1 g, 13.74 mmol, 45.5% yield) as yellow oil. 118
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Step 6: [2-[(dioctylamino)methyl]cyclopropyl]methyl nonyl hydrogen phosphate (SM-032): (EC5059-257/258) Br OOP O OH 7 CHC 7l37a/i0 (P2 °rCO0H e 1q/6M) heCN NH N O O PO OH
A mixture of N-octyloctan-1-amine (2.60 g, 10.77 mmol, 2.0 eq), [2- (bromomethyl)cyclopropyl]methyl nonyl hydrogen phosphate (2 g, 5.39 mmol, 1.0 eq) in MeCN (1 mL), CHCl
3 (1 mL) and i-PrOH (1 mL) was degassed and purged with N
2 for 3 times, and then the mixture was stirred at 60 °C for 16 hr under N
2 atmosphere. The reaction mixture was directly concentrated under reduced pressure to give a residue. The residue was diluted with DCM (80 mL) and washed with HCl solution (10%, 20 mL ^ 2), dried over Na
2SO
4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (80 g SepaFlash® Silica Flash Column,(DCM : MeOH: 0~5%, 2% NH
3•H
2O in MeOH) and prep-HPLC (column: Phenomenex luna C18 150 * 25mm * 10um;mobile phase: [water(FA)-ACN]; mixture of water(FA) and ACN containing 80%-100% CAN, 8min) to give SM-032, also known as 2-((dioctylamino)methyl)cyclopropyl)methyl nonyl hydrogen phosphate (480 mg, 0.83 mmol, 15.8% yield, FA) as colorless oil. LCMS: [M+H]
+: 533.1 1H NMR (400 MHz, CDCl
3) δ = 13.58 (s, 1H), 4.35 - 4.27 (m, 1H), 3.96 - 3.83 (m, 2H), 3.30 - 3.19 (m, 3H), 3.13 - 3.03 (m, 1H), 2.95 - 2.84 (m, 1H), 2.82 - 2.71 (m, 1H), 2.17 (t, J = 12.0 Hz, 1H), 1.87 - 1.71 (m, 2H), 1.69 - 1.59 (m, 4H), 1.45 - 1.22 (m, 34H), 0.98 - 0.81 (m, 9H), 0.70 - 0.65 (m, 1H), 0.60 - 0.55 (m, 1H). 119
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) ExHaNm 1 Npl Beoc 3: Synthes N DaC HBEOHA1( 0Oca-2A ( (201c.)0 °1 e (1q.)5 Ois of SM-0 eq N33 N BocH 4C0l °/dCiox 1a2n he N NH ) (
3C eq 3) h) e) Br3 ( 5 OH e) ( 2 e) 3 23 ( e) N NH
) ( tep : tert-buty)e) ( e) ( e) (e) S - exyp r Operaz ne- -carboxy ate: ( C5000- 8/ 9) H oc NaBH(a (.3 eq) oc
1 To a solu DtC HiEOonA 0Oc o2A (25c f0 °) tCe e (1 rqt 3)5 - h eq) butyl piperazine- 21-carboxylate (10 g, 53.69 mmol, 1.0 eq) and hexanal (5.92 g, 59.06 mmol, 7.09 mL, 1.1 eq) in DCE (100 mL) was added HOAc (6.45 g, 107.38 mmol, 6.14 mL, 2 eq) at 0 °C for 1 hour, then naBh(OAc)
3 (17.07 g, 80.54 mmol, 1.5 eq) was added at 0 °C. The mixture was stirred at 25 °C for 3 h. The mixture pH was adjusted to 8 with 1M NaOH. The mixture was extracted with EtOAc (3 ^ 150 mL). The combined organic phase was washed with brine (100 mL), dried with anhydrous Na
2SO
4, filtered and concentrated in vacuum to give residue. The residue was purified by column chromatography (SiO
2, PE/EtOAc = 20/1 to 1/1) to give compound tert-butyl 4-hexylpiperazine-1-carboxylate (3.3 g, 11.46 mmol, 31.0% yield, 93.9% purity) as a colorless oil. 1H NMR (400 MHz, CDCl
3) δ = 3.51 - 3.44 (m, 4H), 2.52 - 2.46 (m, 4H), 2.42 - 2.40 (m, 2H), 1.52 - 1.49 (m, 2H), 1.46 - 1.38 (m, 9H), 1.32 - 1.25 (m, 6H), 0.90 - 0.83 (m, 3H). 120
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Step 2: 1-hexylpiperaNzine N: ( BEocC500H0-220) 4C0l °/dCioxane N NH To
a so ut on 2 12 h o tert- uty - exy pperaz ne- -car ox 3y ate (3.3 g, 12.20 mmol, 1.0 eq) in DCM (30 mL) was added HCl/dioxane (4 M, 30 mL, 9.8 eq). The mixture was stirred at 40 °C for 12 h. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was triturated with PE/EtOAc=3/1 at 25 °C to give compound 1-hexylpiperazine (1.9 g, 11.16 mmol, 91.4% yield) as a white solid. 1H NMR (400 MHz, CDCl
3) δ = 9.95 - 9.90 (m, 1H), 3.64 - 3.53 (m, 2H), 3.48 - 3.31 (m, 4H), 3.30 - 3.18 (m, 2H), 3.13 - 3.00 (m, 2H), 1.81 - 1.65 (m, 2H), 1.32 - 1.27 (m, 6H), 0.87 (t, J = 6.8 Hz, 3H). Step 3: 2-bromoethyl nonyl hy HO Bdrogen phosphate: (EC5000-215/223) 1) 4 (12)03 e ( 3q1)) T01 PHe0Oq%F)Cr H 0 Tl3CE2 ( 5 l1A040 ° ( OH 0C3 e0qC 1) e hq T 2)EhA TH (1F2 eq) Br
O OP O OH
TEA (16.84 g, 166.37 mmol, 23.16 mL, 1.2 eq) was slowly added to POCl
3 (21.26 g, 138.64 mmol, 12.88 mL, 1.0 eq) in dry THF (200 mL) at 0 °C under N
2. Then nonan-1-ol (20 g, 138.64 mmol, 1.0 eq) in THF (200 mL) was added drop wise over 1 h and the resulting mixture was warmed to 20 °C was stirred for 1 hour. When all the alcohol had reacted (checked by TLC), the mixture was cooled to 0 °C and a second portion of TEA (42.09 g, 415.93 mmol, 57.89 mL, 3.0 eq) was added, followed by 2-bromoethanol (17.33 g, 138.64 mmol, 9.84 mL, 1.0 eq) in THF (200 mL) was added dropwise. The reaction mixture was stirred at 20 °C for 14 h. Decomposed with HCl 10% (150 mL) and heated at 40 °C for 2 h. THF was removed under vacuum and the aqueous residue was extracted with DCM (300 mL ^ 3). The organic layer was dried over Na
2SO
4, 121
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) filtered, reduced under vacuum. The residue was purified by column chromatography (SiO
2, DCM/MeOH = 1/0 to 10/1) and purified by prep-HPLC (column: Phenomenex luna C18150 * 25mm * 10um;mobile phase: [water(FA)-ACN]; mixture of water(FA) and ACN containing 50%- 80% CAN, 8min) to give compound 2-bromoethyl nonyl hydrogen phosphate (3.3 g, 9.96 mmol, 82.5% yield) as a yellow oil. 1H NMR (400 MHz, CDCl
3) δ = 10.34 - 10.30 (m, 1H), 4.32 - 4.21 (m, 2H), 4.04 (q, J = 6.8 Hz, 2H), 3.54 (t, J = 6.4 Hz, 2H), 1.73 - 1.62 (m, 2H), 1.39 - 1.23 (m, 12H), 0.89 (t, J = 6.8 Hz, 3H). Step Br
O 4: 2-(4-hexylpiperazin-1-yl)ethyl nonyl hydrogen phosphate: (EC5000-231/237) OP O OH K2CO3 C3 (1 (300 e eqq)) N KI (0 NH1eq) N N
O OP O OH
PME 80 °C 12 h - y y y g p p g, . , . q - hexylpiperazine (1.25 g, 6.04 mmol, 1 eq, HCl) in Cyclopentyl anisole (5 mL) was added K
2CO
3 (2.50 g, 18.12 mmol, 3.0 eq) and KI (100.25 mg, 603.90 umol, 0.1 eq). The mixture was stirred at 80 °C for 12 h. The reaction mixture was directly concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18150*25mm* 10um;mobile phase: [water(FA)-ACN]; mixture of water(FA) and ACN containing 30%-60% CAN, 8min) and purified by column chromatography (SiO
2, DCM/MeOH = 1/0 to 10/1) to give compound SM-033, also known s 2-(4-hexylpiperazin-1-yl)ethyl nonyl hydrogen phosphate (1.15 g, 2.71 mmol, 76.1% yield, 99.27% purity) as a yellow oil. LCMS: [M+H]
+:421.6 1H NMR (400 MHz, CDCl
3) δ = 4.13 - 4.09 (m, 2H), 3.88 - 3.79 (m, 2H), 3.11 - 2.68 (m, 12H), 1.66 - 1.54 (m, 4H), 1.31 - 1.22 (m, 18H), 0.90 - 0.83 (m, 6H). 122
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Exa HmOple 4: Sy 4 OH D N PCnPBMhS,t (h1.0e eqsis of SM-034 1)
30 (1 °C.0, e 1q2)) h )) 1 ( (10 e e
B)
r B ) POCl3 ( (
Or 1
H OH 0 e e 22 )) TEA (12 e)r OP OH 4 (15 e) NH N O O PO OH 3 ( e)
( e) To a solution of 2-methylproprane-1,3-diol (15 g, 166.44 mmol, 14.85 mL, 1.0 eq) in DCM (300 mL) was added PPh
3 (43.66 g, 166.44 mmol, 1.0 eq) and NBS (29.62 g, 166.44 mmol, 1.0 eq) at 0 °C. The mixture was stirred at 0 °C for 12 h. The reaction mixture was filtered and concentrated in vacuum to give residue. The residue was purified by column chromatography (SiO
2, PE/EtOAc = 30/1 to 3/1) to give compound 3-bromo-2-methyl-propan-1-ol (15 g, 98.03 mmol, 58.9% yield) as a colorless oil. 1H NMR (400 MHz, CDCl
3) δ = 3.65 - 3.53 (m, 2H), 3.49 - 3.45 (m, 2H), 2.04 - 1.96 (m, 1H), 1.02 (d, J = 6.8 Hz, 3H). 123
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Step 2: (3-bromo-2-methyl-propyl) nonyl hydrogen phosphate: (EC5000- ) (10 eq B 235/242) 11)r POCl3 ( O1H0 e 2q) TE
O OH
HO 32)) 120 (%1 T0 HH eCFql) 04 T-02E5 °AC °C (3201 h e hq) THFA (12 eq) Br OP O TEA (11.78 g, 116.46 mmol, 16.21 mL, 1.2 eq) was slowly added to POCl
3 (14.88 g, 97.05 mmol, 9.02 mL, 1.0 eq) in dry THF (150 mL) at 0 °C under N
2. Then nonan-1-ol (14 g, 97.05 mmol, 1.0 eq) in THF (150 mL) was added drop wise over 1 h and the resulting mixture was warmed to 20 °C was stirred for 2 h. When all the alcohol had reacted (checked by TLC), the mixture was cooled to 0 °C and a second portion of TEA (29.46 g, 291.15 mmol, 40.52 mL, 3.0 eq) was added, followed by 3-bromo-2-methyl-propan-1-ol (14.85 g, 97.05 mmol, 1.0 eq) in THF (150 mL) was added dropwise. The reaction mixture was stirred at 20 °C for 14 h. Decomposed with HCl 10% (150 mL) and heated at 40 °C for 2 h. THF was removed under vacuum and the aqueous residue was extracted with DCM (300 mL ^ 3). The organic layer was dried over Na
2SO
4, filtered, reduced under vacuum. The residue was purified by column chromatography (SiO
2, DCM/MeOH = 1/0 to 5/1) to give compound (3-bromo-2-methyl-propyl) nonyl hydrogen phosphate (8.2 g, 22.83 mmol, 70.7% yield) as a yellow oil. 1H NMR (400 MHz, CDCl
3) δ = 4.15 - 3.81 (m, 4H), 3.49 - 3.41 (m, 2H), 2.19 - 2.13 (m, 1H), 1.64 - 1.58 (m, 2H), 1.39 - 1.22 (m, 12H), 1.07 - 0.99 (m, 3H), 0.85 (t, J = 6.8 Hz, 3H). 124
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Step 3: [3-(dioctylamino)-2-methy 4l (-1p5r eopyl] nonyl hydrogen phosphate: (E
OC P5 O0H00-255/265) Br
O OP O OH CPME 8q0) °C 7 N2H h N OO
o a so uton o (3-bromo- -met y -propy ) nony ydrogen p osp ate (3 g, 8.35 mmo, 1.0 eq) in Cyclopentyl anisole (10 mL) was added N-octyloctan-1-amine (3.02 g, 12.53 mmol, 1.5 eq).The mixture was stirred at 80 °C for 72 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with MeCN (50 mL) and washed with HCl solution (10%, 10 mL ), dried over Na
2SO
4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO
2, DCM/MeOH = 1/0 to 10/1) and by prep-HPLC (column: Phenomenex luna C18150 * 25mm * 10um;mobile phase: [water(FA)-ACN]; mixture of water(FA) and ACN containing 80%-100% CAN, 8min) to give compound SM-034, also known as [3-(dioctylamino)-2-methyl-propyl] nonyl hydrogen phosphate (290 mg, 557.93 umol, 22.3% yield) as a yellow oil. LCMS: [M+H]
+:520.9 1H NMR (400 MHz, CDCl
3) δ = 12.63 - 12.49 (m, 1H), 4.11 - 4.06 (m, 1H), 3.94 - 3.86 (m, 2H), 3.82 - 3.75 (m, 1H), 3.13 - 2.85 (m, 5H), 2.71 - 2.66 (m, 1H), 2.34 - 2.30 (m, 1H), 1.65 - 1.59 (m, 6H), 1.33 - 1.22 (m, 32H), 1.00 (d, J = 6.8 Hz, 3H), 0.89 - 0.83 (m, 9H). 125
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Example 5: Synthesis of SM-035 HN N Boc EDCI (1 DI51Pa eEq (A1). (0 H3 eO0qB e)t (10 e Oq) OH O N N Boc DC HMCl/ 2d5io °xCan 1e6 h O N NH r DCM 020 °q) B 1 2 OO3 P ( O O C 16 h H 2 3 ( e) e) ( e) Step 1:
tert-butyl 4-tetradecanoylpiperazine-1-carboxylate (2): (EC5059-263/264) HN N Boc
1 EDCI (1 A mixture of DC t DeMI5 rPa eq (1)0 H eOqB)t (10 e Oq) OH N N tE- 0Ab2u0 (3t °y0Cl e 1q p6)ip herazine-1-carboxylate (3 g, 216.11 mmol, 1.0 B eocq), tetradecanoic acid (3.68 g, 16.11 mmol, 1.0 eq), DIPEA (6.25 g, 48.32 mmol, 8.42 mL, 3.0 eq), EDCI (4.63 g, 24.16 mmol, 1.5 eq), HOBt (2.18 g, 16.11 mmol, 1.0 eq) in DCM (100 mL) was degassed and purged with N
2 for 3 times, and then the mixture was stirred at 20 °C for 16 h under N
2 atmosphere. The combined reaction mixture was quenched by addition H
2O (150 mL), and then diluted with EtOAc (120 mL) and extracted with EtOAc (130 mL ^ 3 ). The combined organic layers were washed with brine (150 mL), dried over Na
2SO
4, filtered and concentrated under reduced pressure to crude product. The residue was purified by flash silica gel chromatography (220 g SepaFlash® Silica Flash Column, PE : EtOAc: 0~20%) to give compound tert-butyl 4-tetradecanoylpiperazine- 1-carboxylate (10.8 g, 26.77 mmol, 82.9% yield, 98.3% purity) as a light yellow solid. 1H NMR (400 MHz, CDCl
3) δ = 3.60 - 3.55 (m, 2H), 3.48 - 3.34 (m, 6H), 2.31 (t, J = 7.6 Hz, 2H), 1.68 - 1.56 (m, 2H), 1.46 (s, 9H), 1.31 - 1.22 (m, 20H), 0.87 (t, J = 6.4 Hz, 3H). 126
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Step 2: 1-piperazin-1-ylte Otradecan-1-one NN DC HMC (l3 2/d): (EC5059-265) 5io °xCan 1e6 h O N
NH o a sout o 2n o tert- uty B-otectra ecanoy p peraz ne- -car oxy ate ( 3 g, . mmol, 1.0 eq) in DCM (50 mL) was added HCl/dioxane (4 M, 15 mL, 3.9 eq). The mixture was stirred at 25 °C for 16 h. The reaction mixture was directly concentrated under reduced pressure to give crude product. The crude product was triturated with PE (100 mL ^ 2) at 25
oC for 30 min to give compound 1-piperazin-1-yltetradecan-1-one (4.5 g, 13.52 mmol, 89.3% yield, HCl) as a white solid 1H NMR (400 MHz, CDCl
3) δ = 3.90 - 3.77 (m, 4H), 3.29 - 3.16 (m, 4H), 2.44 (t, J = 7.6 Hz, 2H), 1.68 - 1.55 (m, 2H), 1.38 - 1.27 (m, 20H), 0.90 (t, J = 6.8 Hz, 3H). Step 5: nonyl 2-(4-tetradecanoylpiperazin-1-yl)ethyl hydrogen phosphate (SM-035-): (EC5059-266/269) O N NH Br2 OO3 P O 6 OH (15 eq) O N NO P O
KC COP (20 eq) KI (01 eq) O OH A mix 3 ME 80 °C 48 h ture of 1-piperazin-1-yltetradecan-1-one (2 g, 6.01 mmol, 1.0 O eMqGT,0 H64Cl), 2-bromoethyl nonyl hydrogen phosphate (2.98 g, 9.01 mmol, 1.5 eq), K
2CO
3 (1.66 g, 12.01 mmol, 2.0 eq), KI (99.72 mg, 600.69 umol, 0.1 eq) in methoxycyclopentane (15 mL) was degassed and purged with N
2 for 3 times, and then the mixture was stirred at 80 °C for 48 h under N
2 atmosphere. The reaction mixture was filtered and the filter was concentrated under reduced pressure to give residue. The residue was purified by flash Diol chromatography (80 g SepaFlash® Diol Column, DCM : MeOH : 0~5%) to give SM-035, also known as nonyl (2-(4-tetradecanoylpiperazin-1-yl)ethyl) hydrogen phosphate (990 mg, 1.81 mmol, 30.9% yield, 99.99% purity) as light yellow solid 127
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) LCMS: [M+H]
+: 547.8 1H NMR (400 MHz, CDCl
3) δ = 4.35 - 4.18 (m, 2H), 4.00 - 3.78 (m, 6H), 3.20 - 2.98 (m, 6H), 2.30 (t, J = 7.6 Hz, 2H), 1.63 – 1.58 (m, 4H), 1.34 - 1.23 (m, 32H), 0.87 (t, J = 6.4 Hz, 6H). Example 6: Sy BocN 1 OH 2) 11)n ( 4t 1 (01h. e0e q e) 3qs ) T) Hi 1 TEO Ps 0HA%OF (C o 3 H 0lf 0C-2 (l e1 S 0q.40M-036 32 °)0C e T °qCH 1)F h 2 TE 0 hA-20 (1 °.2C e 1q2) h Boc N O OP O OH DC HMCl/ 2d0iox °Cane 1 h HN O OP O OH (3 e) O 3 4
a( Stepe 1:cc) ( e) t (er et-)butyl 4-[hydroxy(nonoxy)phosphoryl]oxypiperidine-1-carboxylate (3): (EC8433- 24/30/32) BocN OH 2) 11) ( 41 (01 e0 e) 3q) T) HO 1 TE P0HA%OF (C3 H 0l30C2 ( 2 l e1040 °)C e TqH 1)F h TE 0A20 (1 °2C e 1q2) h Boc N
O OP O OH
TEA (8.42 g, 83.19 mmol, 11.58 m0LC, 12. h2 eq) was slowly added to POCl
3 (10.63 g, 69.32 mmol, 6.44 mL, 1.0 eq) in dry THF (100 mL) at 0 °C under N
2. Then nonan-1-ol (10 g, 69.32 mmol, 1.0 eq) in THF (100 mL) was added dropwise over 1 h at 0 °C. The resulting mixture was warmed to 20 °C and stirred for 1 h. When all the alcohol had reacted (checked by TLC), the mixture was cooled to 0 °C and a second portion of TEA (21.04 g, 207.97 mmol, 28.95 mL, 3.0 eq) was added, followed by tert-butyl 4-hydroxypiperidine-1-carboxylate (13.95 g, 69.32 mmol, 128
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 1.0 eq) in THF (100 mL) was added dropwise. The reaction mixture was stirred at 20 °C for 12 h. Decomposed with HCl 10% (200 mL) and heated at 40 °C for 2 h. THF was removed under vacuum and the aqueous residue was extracted with DCM (200 mL × 3). The organic layer was dried over Na
2SO
4, filtered, reduced under vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 220 g SepaFlash® Silica Flash Column, Eluent of 0~8% MeOH/DCM gradient @ 100 mL/min) and prep-HPLC (column: Phenomenex luna C18 150 * 25mm * 10um;mobile phase: [water(FA)-ACN]; mixture of water(FA) and ACN containing 60%-90% CAN, 8min) to get compound tert-butyl 4-[hydroxy(nonoxy)phosphoryl]oxypiperidine-1- carboxylate (3.6 g, 8.57 mmol, 34.9% yield, 97.0% purity) as a yellow gum. 1H NMR (400 MHz, CDCl
3) δ = 4.57 - 4.42 (m, 1H), 4.02 (q, J = 6.8 Hz, 2H), 3.75 - 3.57 (m, 2H), 3.36 - 3.23 (m, 2H), 1.96 - 1.82 (m, 2H), 1.81 - 1.71 (m, 2H), 1.70 - 1.63 (m, 2H), 1.46 (s, 9H), 1.37 - 1.33 (m, 2H), 1.31 - 1.23 (m, 10H), 0.92 - 0.86 (m, 3H). Step 2: Non
Oyl O 4H-piperidyl hydrogen phosphate (4): (EC8433
O-37 O)H
Boc N OP O HCl/dioxane HN OP O To a sol 3 DCM 20 °C 1 h ution of tert-butyl 4-[hydroxy(nonoxy)phosphoryl]ox 4ypiperidine-1-carboxylate (3.6 g, 8.83 mmol, 1.0 eq) in DCM (10 mL) was added HCl/dioxane (4 M, 10 mL, 4.5 eq). The mixture was stirred at 20 °C for 1 h. The reaction mixture was concentrated under reduced pressure to compound nonyl 4-piperidyl hydrogen phosphate (3.04 g, crude, HCl) as an off-white solid. 1H NMR (400 MHz, CDCl
3) δ = 9.40 - 8.84 (m, 2H), 4.76 - 4.36 (m, 5H), 4.10 - 3.87 (m, 2H), 3.43 - 3.30 (m, 2H), 2.29 - 2.12 (m, 2H), 1.75 - 1.60 (m, 2H), 1.47 - 1.18 (m, 12H), 0.89 (t, J = 6.8 Hz, 3H). 129
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Step 3: Nonyl (1-octyl-4-piperidyl) hydrogen phosphate (SM-036): (EC843
O3-50) HN
O OP O OH NaB HH 5(O (1A.c2)3 eq (1)2 e Oq) N OP O OH on o nony -pp DeCrEd/MyeOOAHc yd 0 (2 r20 o0 eq)
o a so 4ut g °eCn 1 p2 hosp ate ( . g, 3.90 m OmMoGT,06.02 eq) n eO (5 mL) and DCE (12 mL) were added HOAc (468.89 mg, 7.81 mmol, 446.56 uL, 2.0 eq) and octanal (600.67 mg, 4.68 mmol, 731.63 uL, 1.2 eq) at 0 °C. The mixture was stirred at 25 °C for 1 h. Then naBh(OAc)
3 (992.94 mg, 4.68 mmol, 1.2 eq) was added to the mixture at 0 °C. The resulting mixture was stirred at 25 °C for 11 h. The reaction mixture was filtered and washed with EtOAc (100 mL). The filtrate was collected and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18150*25mm* 10um;mobile phase: [water(FA)-ACN]; mixture of water(FA) and ACN containing 45%-75% CAN, 8min) to get compound SM-036, also known as nonyl (1-octyl-4-piperidyl) hydrogen phosphate (912.84 mg, 2.13 mmol, 54.6% yield, 98.00% purity) as a yellow solid. LCMS: [M+H]
+: 420.6 1H NMR (400 MHz, CD
3OD) δ = 4.54 - 4.22 (m, 1H), 3.96 - 3.78 (m, 2H), 3.62 - 3.38 (m, 2H), 3.28 - 2.99 (m, 4H), 2.35 - 2.14 (m, 2H), 2.04 - 1.82 (m, 2H), 1.80 - 1.67 (m, 2H), 1.67 - 1.57 (m, 2H), 1.42 - 1.27 (m, 22H), 1.03 - 0.83 (m, 6H). Example 7: Synthesis of SM-037
HN 1N Boc TH NFaB HH H
2(O 1OOaAA (c 01c ()23530 (0 e1 °q eC O5)q e) 1q2) h 2NN Boc D HCCMl/d 2io0x °aCne 3 h N NH Br K
2C CO OOP
3 PM ( O36 OEH0 (18 e00q) e °Cq K)I 1 (201 h eq) N N O OP O OH 1330 OMGT060
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Step 1: tert-butyl 4-(1-pentylhexyl)piperazine-1-carboxylate (2): (EC8433-25/29/34) HN N Boc NaBH( 1OaA (153 eq O) N N Boc
2cc () (1 e5 e)) To a solution of tert-butyl piperazine-1-carboxylate (10 g, 53.69 mmol, 1.0 eq) in THF (100 mL) and H
2O (10 mL) were added undecan-6-one (13.71 g, 80.54 mmol, 1.5 eq), AcOH (9.67 g, 161.07 mmol, 9.21 mL, 3.0 eq) and NaBH
3CN (5.06 g, 80.54 mmol, 1.5 eq). The mixture was stirred at 60 °C for 12 h under N
2 atmosphere. The reaction mixture was basified by addition 2 N NaOH solution (80 mL) at 0 °C, and then diluted with water (20 mL) and extracted with EtOAc (100 mL × 3). The combined organic layers were washed with brine (300 mL), dried over Na
2SO
4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0~7% EA/PE gradient @ 100 mL/min) and prep-HPLC (column: Phenomenex luna C18150 * 25mm * 10um;mobile phase: [water(FA)-ACN]; mixture of water(FA) and ACN containing 32%-62% CAN, 8min) to get compound tert-butyl 4-(1-pentylhexyl)piperazine-1-carboxylate (3.6 g, 9.83 mmol, 33.5% yield, 93.0% purity) as a yellow gum. 1H NMR (400 MHz, CDCl
3) δ = 3.55 - 3.40 (m, 4H), 2.65 - 5.55 (t, J = 4.8 Hz, 4H), 2.55 - 2.46 (m, 1H), 1.57 - 1.48 (m, 2H), 1.46 (s, 9H), 1.37 - 1.33 (m, 2H), 1.32 - 1.22 (m, 12H), 0.94 - 0.83 (m, 6H). Step 2: 1-(1-pentylhexyl)piperazine (3): (EC8433-40) 131
Attorney Ref.: BN00004.0144 OME-013WO NN Boc (PCT App D HCCMl/d 2io0x °aCne lication) 3 h N NH
o a so ut on o tert- uty -( -penty exy )p peraz ne-1-carboxylate (3.6 g, 10.57 mmol, 1.0 eq) in DCM (15 mL) was added HCl/dioxane (4 M, 15.00 mL, 5.7 eq). The mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated under reduced pressure to get compound 1-(1-pentylhexyl)piperazine (2.93 g, 10.58 mmol, 100.0% yield, HCl) as a yellow solid. Step 3: Nonyl 2-[4-(1-pentylhexyl)piperazin-1-yl]ethyl hydrogen phosphate: (EC8433-43/55) N NH Br K O 2COO3 P ( O36 OH0 (1 e0q) eq) N N
O OP O OH
CPME 80 °C KI 1 (201 h eq) To a solution of 1-(1-pentylhexyl)piperazine (1.25 g, 4.53 mm-ol-, 1-.0- eq, HCl) and 2- bromoethyl nonyl hydrogen phosphate (1.5 g, 4.53 mmol, 1.0 eq) in methoxycyclopentane (15 mL) was added K
2CO
3 (1.88 g, 13.59 mmol, 3.0 eq) and KI (75.19 mg, 452.92 umol, 0.1 eq) .The mixture was stirred at 80 °C for 12 h. The reaction mixture was filtered and washed with EtOAc (50 mL). The filtrate was collected and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0~10% MeOH/DCM gradient @ 80 mL/min) and prep-HPLC (column: Phenomenex luna C18 150 * 25mm * 10um;mobile phase: [water(FA)-ACN]; mixture of water(FA) and ACN containing 55%-85% CAN, 8min) to get compound SM-037, also known as nonyl 2-[4-(1-pentylhexyl)piperazin-1-yl]ethyl hydrogen phosphate (535.35 mg, 1.07 mmol, 23.7% yield, 98.30% purity) as a brown gum. LCMS: [M+H]
+: 491.7 132
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 1H NMR (400 MHz, CDCl
3) δ = 4.22 (s, 2H), 3.89 (q, J = 6.8 Hz, 2H), 3.55 - 3.20 (m, 4H), 3.20 - 3.01 (m, 6H), 2.78 - 2.61 (M, 1H), 1.77 - 1.50 (m, 4H), 1.44 - 1.23 (m, 26H), 0.97 - 0.80 (m, 9H). Example 8: Synthesis of S HN N Boc HATU D (121 eaq ()1.1 DI ePqE)A (30 eq) OM-038 OH O N N Boc DC HMC 0l/d-2io5x °aCne 12 h O N NH Br 1 23 OO ( P O O CM 20 °C 12 h 2 3 (H e) e) ( e) oc (a e () e) ( e O OH
) Boc A mixture of tert-butyl piperazine-1-carboxylate (2.5 g, 13.42 mmol, 1.0 eq), stearic acid (4.20 g, 14.77 mmol, 4.97 mL, 1.1 eq), HATU (6.12 g, 16.11 mmol, 1.2 eq) and DIPEA (5.20 g, 40.27 mmol, 7.01 mL, 3.0 eq) in DCM (30 mL), and then the mixture was stirred at 20 °C for 12 h under N
2 atmosphere. The reaction mixture was diluted with water 100 mL and extracted with DCM (100 mL ^ 3). The combined organic layers were washed with water, dried over Na
2SO
4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO
2, PE/EtOAc = 30/1 to 1/1) to give compound tert-butyl 4- octadecanoylpiperazine-1-carboxylate (12 g, 26.51 mmol, 75.0% yield) as a white solid. 133
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 1H NMR (400 MHz, CDCl
3) δ = 3.63 - 3.53 (m, 2H), 3.46 - 3.35 (m, 6H), 2.37 - 2.28 (m, 2H), 1.71 - 1.59 (m, 2H), 1.49 - 1.46 (m, 9H), 1.33 - 1.22 (m, 28H), 0.88 (t, J = 6.8 Hz, 3H). Step 2: 1-piperazin-1-yloctade
Ocan-1-one: (EC5000-260) NN DC HMC 0l/d-2io5x °aCne 12 O N To a solution 2 of tert-butyl 4-oct Baodc h ecanoylpiperazine-1-carboxylate (6 g, 133.25 mmol, 1.0 NH eq) in DCM (60 mL) was added HCl/dioxane (4 M, 60 mL, 18.1 eq) at 0 °C. The mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated under reduced pressure to give crude product. The crude product was triturated with PE/EtOAc = 3/1 at 25
oC to give compound 1-piperazin-1-yloctadecan-1-one (4.6 g, 11.82 mmol, 89.21% yield, HCl) as a white solid. 1H NMR (400 MHz, CD
3OD) δ = 3.82 (t, J = 5.2 Hz, 4H), 3.29 - 3.18 (m, 4H), 2.46 - 2.42 (m, 2H), 1.65 - 1.54 (m, 2H), 1.36 - 1.25 (m, 28H), 0.92 - 0.89 (m, 3H). Step 3: nonyl 2-(4-octad Oecanoylpipera
N To a so 3lution of1-pip NeH Br razi K2 nC- CO1P3 OOM (-3 P 6E O y0 Ozin-1-yl)ethyl hydrogen phosphate: (EC5000-262/271) (H18l e0oq5) ° e cC Kq) tI 2a (04d h1e eqc) O N an-1-one (2.35 g, 6.04 m OMGm OTMoSGMlT,0031688N.0X N1 eq, OO H P O O CHl) and 2- bromoethyl nonyl hydrogen phosphate (3.00 g, 9.06 mmol, 1.5 eq) in CPME (15 mL) was added K
2CO
3 (2.50 g, 18.12 mmol, 3.0 eq) and KI (100.26 mg, 604.00 umol, 0.1 eq). The mixture was stirred at 80 °C for 24 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO
2, DCM/MeOH = 40/1 to 1/1) and purified by prep-HPLC (column: Welch Xtimate C1100 * 30mm * 5um;mobile phase: [water(FA)-MeOH]; mixture of water(FA) and ACN containing 80%-100% CAN, 8min) to give compound SM-038, also known as nonyl 2-(4-octadecanoylpiperazin-1-yl)ethyl hydrogen phosphate (860 mg, 1.40 mmol, 65.4% yield, 98.15% purity) as an off-white solid. 134
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) LCMS: [M+H]
+:604.0 1H NMR (400 MHz, CDCl
3) δ = 4.33 - 4.30 (m, 2H), 4.03 - 3.85 (m, 6H), 3.42 - 2.98 (m, 6H), 2.31 (t, J = 7.2 Hz, 2H), 1.67 - 1.55 (m, 4H), 1.34 - 1.23 (m, 40H), 0.88 (t, J = 6.4 Hz, 6H). Example 9: Synthesis of S HN N Boc HAT DUC (1M50 e-1q2.)0 T eEqA) (20 eq) OM-039 1a ( OH O N N Boc DC HMCl 2/d0io °xCan 1e2 h O N NH Br 1 0 °C 16 h 2 3 OO P O O (H
23 ( e) e) Step 1: tert-butyl 4-hexadecanoylpiperazine-1-carboxylate (2): (EC5059-268/270 HN N Boc EDCI (151a e (10 eq) O O ) OH N
1 To a solution DC D oMIfPE p 0qA) a20l ( H3m °O0CB it et i 1q ( c6)1 h0 eq) N acid (6.88 g, 26.85 mmol, 8.208 mL, 1.0 eq) i Bnoc DCM (150 mL) were added HATU (15.31 g, 40.27 mmol, 1.5 eq) and TEA (5.43 g, 53.69 mmol, 7.47 mL, 2.0 eq) at 20 °C under N
2. After addition, the mixture was stirred at this temperature for 1 h, and then tert- butyl piperazine-1-carboxylate (5 g, 26.85 mmol, 1.0 eq) was added. The resulting mixture was stirred at 20 °C for 15 h. The combined reaction mixture was quenched by addition H
2O (150 mL), and then diluted with EtOAc (150 mL) and extracted with EtOAc (150 mL ^ 3 ). The combined organic layers were washed with brine (150 mL), dried over Na
2SO
4, filtered and concentrated under reduced pressure to crude product. The residue was purified by flash silica gel chromatography (220 g SepaFlash® Silica Flash Column,PE : EtOAc: 0~20%) to give compound 135
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) tert-butyl 4-hexadecanoylpiperazine-1-carboxylate (10.5 g, 24.73 mmol, 92.1% yield) as a yellow solid. 1H NMR (400 MHz, CDCl
3) δ = 3.57 - 3.52 (m, 2H), 3.39 - 3.34 (m, 2H), 2.30 (t, J = 7.6 Hz, 2H), 2.20 - 2.12 (m, 4H), 1.62 - 1.56 (m, 2H), 1.44 (s, 9H), 1.27 - 1.21 (m, 24H), 0.85 (t, J = 7.2 Hz, 3H). Step 2: 1-piperazin-1-ylhexad Oecan-1-one (3): (EC5059-271 N DC HMCl 2/d0io °x ) Cane O N
To a solutio 2 N Boc 12 h 3 NH n of tert-butyl 4-hexadecanoylpiperazine-1-carboxylate (5 g, 11.77 mmol, 1.0 eq) in DCM (50 mL) was added HCl/dioxane (4 M, 10.0 mL, 3.4 eq). The mixture was stirred at 20 °C for 16 h. The reaction mixture was directly concentrated under reduced pressure to give crude product. The crude product was triturated with PE (100 mL ^ 2) at 25
oC for 30 min to give compound 1-piperazin-1-ylhexadecan-1-one (3.5 g, 9.70 mmol, 82.3% yield, HCl) as a white solid. Step 5: 2-(4-hexadecanoylpiperazin-1-yl)ethyl nonyl hydrogen phosphate (SM-039-NX-1): (EC5059-266/269/27 O6)
N NH Br A mix 3ture of 1-piperazi CnP K OOM
2 PC 6 -E OO O (H1 1
38-0 (52 y ° eC0q) e lh 7q e2) hxadecan-1-one (2.5 g, 6 OM.9G OT O N N 3MSGMT m00369m6NXo1l, OO P 1 O O .0H eq, HCl), 2- bromoethyl nonyl hydrogen phosphate (3.44 g, 10.39 mmol, 1.5 eq), K
2CO
3 (1.91 g, 13.85 mmol, 2 eq), KI (114.96 mg, 692.51 umol, 0.1 eq) in methoxycyclopentane (20 mL) was degassed and purged with N
2 for 3 times, and then the mixture was stirred at 80 °C for 72 h under N
2 atmosphere. The reaction mixture was filtered and the filter was concentrated under reduced pressure to give residue. The residue was purified by flash Diol chromatography (80 g SepaFlash®Diol Column, DCM : MeOH : 0~5%) and normal prep-HPLC (column: Welch Xtimate C1100 * 30mm * 5um; 136
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) mobile phase: [water(FA)-MeOH]; mixture of water(FA) and ACN containing 80%-100% CAN, 8min) to give SM-039, also known as nonyl (2-(4-palmitoylpiperazin-1-yl)ethyl) hydrogen phosphate (1.5 g, 2.61 mmol, 65.2% yield) as a white solid LCMS: [M+H]
+: 547.8 1H NMR (400 MHz, CDCl
3) δ = 4.32 - 4.28 (m, 2H), 4.01 - 3.85 (m, 6H), 3.30 - 3.05 (m, 6H), 2.30 (t, J = 7.6 Hz, 2H), 1.66 - 1.58 (m, 4H), 1.32 - 1.23 (m, 36H), 0.88 (t, J = 7.2 Hz, 6H). Example 10: Synthesis of HN N Boc HATU (121a eq ()1. D1I ePqE)A (3 O SM-040 OH O N DCM HC 0l/-d2i5ox °aCne 12 h O N Br 1 OO P O DCM 20 °C 12 h0 eq) 2 N Boc 3 NH 2
3 ( OH ( e) e) ( e) oc
( ea () e) ( e) O OH Boc A mixture of tert-butyl piperazine-1-carboxylate (5 g, 26.85 mmol, 1.0 eq), heptadecanoic acid (7.99 g, 29.53 mmol, 1.1 eq), HATU (12.25 g, 32.21 mmol, 1.2 eq) and DIPEA (10.41 g, 80.54 mmol, 14.03 mL, 3.0 eq) in DCM (100 mL), and then the mixture was stirred at 20 °C for 12 h under N
2 atmosphere. The reaction mixture was diluted with water 100 mL and extracted with DCM (150 mL ^ 3). The combined organic layers were washed with water, dried over Na
2SO
4, 137
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO
2, PE/EtOAc = 30/1 to 1/1) to give compound tert-butyl 4- heptadecanoylpiperazine-1-carboxylate (11 g, 25.07 mmol, 93.4% yield) as a white solid. 1H NMR (400 MHz, CDCl
3) δ = 3.68 - 3.38 (m, 8H), 2.37 - 2.28 (m, 2H), 1.66 - 1.61 (m, 2H), 1.49 - 1.40 (m, 9H), 1.33 - 1.22 (m, 26H), 0.88 (t, J = 6.8 Hz, 3H). Step 2: 1-piperazin-1-ylheptad Oecan-1-one: (E N HCCl5/d0i00-266) O
N DCM 0-2o5x °aCne 12 h N N To a 2 Boc 3 H solution of tert-butyl 4-heptadecanoylpiperazine-1-carboxylate (6 g, 13.68 mmol, 1.0 eq) in DCM (60 mL) was added HCl/dioxane (4 M, 60 mL, 17.6 eq) at 0 °C. The mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated under reduced pressure to give crude product. The crude product was triturated with PE/EtOAc = 3/1 at 25
oC to give compound 1-piperazin-1-ylheptadecan-1-one (3.6 g, 9.60 mmol, 70.2% yield, HCl) as a white solid. Step 3: 2-(4-heptadecanoylpiperazin-1-yl)ethyl nonyl hydrogen phosphate: (EC5000- 269/276) O
N NH Br K2C To a sol 3ution of 1-piperazin CO O -P3O 1M ( P36 -E O O0H (1 y 8 e l0q5) e Kq)I (01 eq) O N N h °eCp 72ta hdecan-1-one (2.26 g, 6.04 O mMG OmTMSGoMTl0,046017N.X01 eq OO P , H O OHCl) and 2- bromoethyl nonyl hydrogen phosphate (3 g, 9.06 mmol, 1.5 eq) in CPME (15 mL) was added K
2CO
3 (2.50 g, 18.12 mmol, 3.0 eq) and KI (100.25 mg, 603.90 umol, 0.1 eq). The mixture was stirred at 80 °C for 72 h. The reaction mixture was filtered and concentrated under reduced pressure 138
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) to give a residue. The residue was purified by column chromatography (SiO
2, DCM/MeOH = 30/1 to 1/1) and purified by prep-HPLC (column: Phenomenex luna C18150*25mm* 10um;mobile phase: [water(FA)-ACN]; mixture of water(FA) and ACN containing 80%-100% CAN, 8min) to give compound SM-040, also known as 2-(4-heptadecanoylpiperazin-1-yl)ethyl nonyl hydrogen phosphate (1.9 g, 3.23 mmol, 82.6% yield) as a white solid. LCMS: [M+H]
+:590.0 1H NMR (400 MHz, CDCl
3) δ = 4.33 - 4.29 (m, 2H), 4.03 - 3.85 (m, 6H), 3.31 - 3.01 (m, 6H), 2.31 (t, J = 7.6 Hz, 2H), 1.67 - 1.57 (m, 4H), 1.35 - 1.23 (m, 38H), 0.88 (t, J = 6.8 Hz, 6H). Example 11: Synthesis of SM-04 HN 1a O 1 OH O HC O Br 1 N Boc OO P HATU O OH D (C1M2 e 0q (1)2.01 D ° eIPCqE) 1A6 (3 h0 eq) 2 N N Boc DCMl 2/d0io °xCan 1e2 h 3 N NH
2 (
3 ( e) e) Step 1: tert-butyl 4-pentadecanoylpiperazine-1-carboxylate (2): (EC8433-49) HN N Boc HATU ( 1a (11 eq) O OH O N
12 eq) DIPEA (30 eq) N To a solution DC oMf p 0e2n0t °aCde 16ca hnoic acid (5.01 g, 20.67 mmol, 1.1 eq) in DCM B (o7c0 mL) were added HATU (8.57 g, 22.55 mmol, 1.2 eq) and DIPEA (7.29 g, 56.38 mmol, 9.82 mL, 3.0 eq). The mixture was stirred at 25 °C for 0.25 h. After that, tert-butyl piperazine-1-carboxylate (3.5 g, 18.79 mmol, 1.0 eq) was added to the mixture at 0 °C. The resulting mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with water (100 mL) and extracted with DCM (100 mL 139
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) × 3). The combined organic layers were washed with brine (100 mL), dried over Na
2SO
4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, PE/EtOAc = 0/1 to 5/1) to get compound tert-butyl 4- pentadecanoylpiperazine-1-carboxylate (8 g, 18.70 mmol, 99.5% yield, 96% purity) as a white solid. 1H NMR (400 MHz, CDCl
3) δ = 3.63 - 3.56 (m, 2H), 3.50 - 3.37 (m, 6H), 2.44 - 2.21 (m, 2H), 1.67 - 1.60 (m, 2H), 1.48 (s, 9H), 1.36 - 1.23 (m, 22H), 0.98 - 0.81 (m, 3H). Step 2: 1-piperazin-1-ylpentadecan-1-one (2): (EC8433-54
O NN DC HMCl 2/d0ioxane ) O N To a solu 2 Boc °C 12 h 3 NH tion of tert-butyl 4-pentadecanoylpiperazine-1-carboxylate (8 g, 19.48 mmol, 1.0 eq) in DCM (25 mL) was added HCl/dioxane (4 M, 10 mL). The mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated under reduced pressure to give a crude product. The crude product was triturated with PE/EtOAc=3:1 (30 mL) at 25 ºC for 15 min to get compound 1- piperazin-1-ylpentadecan-1-one (6.36 g, 16.50 mmol, 84.7% yield, 90% purity, HCl) as a white solid. Step 3: 2-(4-pentadecanoylpipe
O razin-1-yl)ethyl hydrogen phosphate: (EC8433 N NH Br -59/63/64) CP K OM2OC P 6EO O ( OH1380 (53 ° eC0q) e 1q6) h O N NO P O -piperazin-1-ylpentadecan-1-one (2 g, 5.76 O O OH To a 3 solution of 1 M mGTm06o5l, 1 eq, HCl) in methoxycyclopentane (25 mL) were added K
2CO
3 (2.39 g, 17.29 mmol, 3 eq), 2-bromoethyl nonyl hydrogen phosphate (2.86 g, 8.65 mmol, 1.5 eq) and KI (478.42 mg, 2.88 mmol, 0.5 eq). The 140
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) mixture was stirred at 80 °C for 12 h. The reaction mixture was filtered and washed with EtOAc (50 mL). The filtrate was collected and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0~10% MeOH/DCM gradient @ 60 mL/min) and prep-HPLC (column: Welch Xtimate C1100 * 30mm * 5um; mobile phase: [water(FA)-MeOH]; mixture of water(FA) and ACN containing 70%-100% CAN, 8 min) to get compound SM-041, also known as nonyl 2-(4- pentadecanoylpiperazin-1-yl)ethyl hydrogen phosphate (1.67 g, 2.98 mmol, 55.7% yield, 99.86% purity) as a yellow gum. LCMS: [M+H]
+: 561.9 1H NMR (400 MHz, CDCl
3) δ = 4.37 - 4.22 (m, 2H), 4.06 - 3.93 (m, 2H), 3.91 - 3.82 (m, 4H), 3.37 - 3.01 (m, 6H), 2.30 (t, J = 7.6 Hz, 2H), 1.65 - 1.56 (m, 4H), 1.34 - 1.22 (m, 34H), 0.87 (t, J = 6.8 Hz, 6H). Example 12: 1 O TH tF-B Ou -
C7O P S l8Kh-2 Py (511 Pnthesis of SM-042 Pb °6hhC (1 eq 16)2 e hq) 2 O TH HFCl 2/d5io °Cxan 1e2 h 3 O N HaNBH(OA Oc D O) PC
3 OE ( O1H520 eq °C) H 1O24A h (c1 (02 e0q e)q) OM NG OTMSGMT0O04 O62P9N
O OX
H1 Ou
C P
lh P ( P Phh ( e) e)
To a suspension of methoxymethyl(triphenyl)phosphonium;chloride (1.14 g, 3.31 mmol, 1.5 eq) in THF (2.5 mL) stirring at -78 °C was added t-BuOK (1 M, 3.31 mL, 1.5 eq) in portions. 141
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) The reaction mixture was allowed to warm to 0 °C over 0.33 h, then cooled to -78 °C, after which a solution of pentadecan-8-one (0.5 g, 2.21 mmol, 1 eq) in THF (2 mL) was added. The reaction mixture was allowed to warm to 25 °C and stirred for 12 h. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with water (100 mL) and extracted with EtOAc (100 mL × 2). The combined organic layers were washed with brine (100 mL × 2), dried over Na
2SO
4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, PE/EtOAc = 0/1 to 20/1) to get compound 8-(methoxymethylene)pentadecane (500 mg, 1.97 mmol, 89.0% yield) as a white oil. 1H NMR (400 MHz, CDCl
3) δ= 5.74 (s, 1H), 3.52 (s, 3H), 2.03 (t, J = 7.2 Hz, 2H), 1.85 (t, J = 7.2 Hz, 2H), 1.37 - 1.32 (m, 4H), 1.32 - 1.25 (m, 16H), 0.89 (t, J = 6.4 Hz, 6H). Step 2: 2-Heptylnonanal (3): (EC8433-68) O TH HFCl 2/d5ioxane O
°C 12 h To a solution of 8-(methoxymethylene)pentadecane (500 mg, 1.97 mmol, 1 eq) in THF (10 mL) was added HCl/dioxane (4 M, 985.00 uL, 2 eq). The mixture was stirred at 25 °C for 12 h under N
2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with water (100 mL) and extracted with EtOAc (100 mL × 2). The combined organic layers were washed with brine (100 mL × 2), dried over Na
2SO
4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, PE/EtOAc=100/1 to 50/1) to get compound 2-heptylnonanal (450 mg, 1.68 mmol, 85.5% yield, 90% purity) as a white solid. 1H NMR (400 MHz, CDCl
3) δ= 9.56 (d, J = 3.2 Hz, 1H), 2.33 - 2.14 (m, 1H), 1.67 - 1.60 (m, 2H), 1.47 - 1.41 (m, 2H), 1.33 - 1.25 (m, 20H), 0.89 (t, J = 6.4 Hz, 6H). 142
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Step 3: [1-(2-Heptylnonyl)-4-piperidyl] nonyl hydrogen phosphate (SM-042-NX-1): (EC8433-70/81) O N HaNBH(OA Oc O) P O 3 ( O1H5 eq) HO 4A (c1 (02 e0q e) NO O
DCE 20 °C 12 hq)
P O O H To a solution of nonyl 4-piperidyl hydrogen phosphate (200 mg, 650.69 umol, 1 eq) in DCE (2 mL) and MeOH (1 mL) were added HOAc (78.15 mg, 1.30 mmol, 74.43 uL, 2 eq), 2- heptylnonanal (203.37 mg, 845.90 umol, 1.3 eq) and naBh(OAc)
3 (179.28 mg, 845.90 umol, 1.3 eq). The mixture was stirred at 20 °C for 12 h. The reaction mixture was filtered and washed with EtOAc (20 mL). The filtrate was collected and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0~10% MeOH/DCM gradient @ 45 mL/min) and prep-HPLC (column: Welch Xtimate C1100 * 30mm * 5um;mobile phase: [water(FA)-MeOH]; mixture of water(FA) and ACN containing 70%-100% CAN, 8 min) to get compound SM-042, also known as [1-(2-heptylnonyl)-4-piperidyl] nonyl hydrogen phosphate (158.32 mg, 297.68 umol, 60.9% yield, 99.99% purity) as a white solid. LCMS: [M+H]
+: 532.7 1H NMR (400 MHz, CD
3OD-d
4) δ = 4.54 - 4.23 (m, 1H), 3.97 - 3.76 (m, 2H), 3.61 - 3.38 (m, 2H), 3.30 - 2.96 (m, 4H), 2.32 - 2.11 (m, 2H), 2.08 - 1.92 (m, 2H), 1.91 - 1.82 (m, 1H), 1.68 - 1.57 (m, 2H), 1.44 - 1.27 (m, 36H), 1.04 - 0.78 (m, 9H). 143
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Example 13: Synthesis Boc 1 N OH2) 11) (1 (10.0 eq e) H of SM-044 2 3qO )) T T 1E P0HA%OF (C3 H 0l3-C022 (l01 e.q 4 °0)0C e ° TqC 1H) hF 2 TE h 0A2 (01°.C2 e 1q2) h Boc N O OP O OH 3 DCM 2 T5FA °C 3 h HN O OP O OH 4 aac( ( ec))
3 ( O ( e e)e)c ( e) ) ( HO3
oc TEA (8).4 (2 g, e e 8)) 3).18 ( ( mm eo) e) ( e) l, 11.58 mL, 1.2 eq)o wcas slowly added to POCl
3 (10.63 g, 69.32 mmol, 6.44 mL, 1.0 eq) in dry THF (100 mL) at 0 °C under N
2. Then nonan-1-ol (10 g, 69.32 mmol, 1.0 eq) in THF (100 mL) was added drop wise over 1 h at 0 °C. The resulting mixture was warmed to 20 °C and stirred for 1 h. When all the alcohol had reacted (checked by TLC), the mixture was cooled to 0 °C and a second portion of TEA (21.04 g, 207.96 mmol, 28.95 mL, 3.0 eq) was added, followed by tert-butyl 3-hydroxyazetidine-1-carboxylate (12.01 g, 69.32 mmol, 1.0 eq) in THF (100 mL) was added dropwise. The reaction mixture was stirred at 20 °C for 12 h. Decomposed with HCl 10% (200 mL) and heated at 40 °C for 2 h. THF was removed under vacuum and the aqueous residue was extracted with DCM (200 mL × 3) . The organic layer was dried over Na
2SO
4, filtered, reduced under vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0~10% DCM/MeOH 144
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) gradient @ 100 mL/min) to get compound tert-butyl 3- [hydroxy(nonoxy)phosphoryl]oxyazetidine-1-carboxylate (14 g, 33.21 mmol, 47.9% yield, 90% purity) as a yellow oil. 1H NMR (400 MHz, CDCl
3) δ = 5.11 - 4.84 (m, 1H), 4.22 - 4.18 (m, 2H), 4.05 - 3.99 (m, 4H), 1.77 - 1.61 (m, 2H), 1.44 (s, 9H), 1.40 - 1.23 (m, 12H), 0.89 (t, J = 6.8 Hz, 3H). Step 2: 1-(1-pentylhexyl)piperazine (3): (EC8433-11 Boc N
O OP O OH TFA 6)
O P O OH
To a solu 3tion of tert-butyl 3-[ DhCyMdr 2o5x °Cy( 3no h HN noxy)phosph
Ooryl]ox 4yazetidine-1-carboxylate (5 g, 13.18 mmol, 1.0 eq) in DCM (20 mL) was added TFA (15.40 g, 135.06 mmol, 10 mL, 10.3 eq). The mixture was stirred at 25 °C for 3 h. The reaction mixture was concentrated under reduced pressure to get compound azetidin-3-yl nonyl hydrogen phosphate (5.18 g, 13.17 mmol, 99.9% yield, TFA) as a yellow oil. Step 3: Nonyl 2-[4-(1-pentylhexyl)piperazin-1-yl]ethyl hydrogen phosphate: (EC8433- 120/122) HN O OP O OH NaBH(OAc) O 3 ( 51 (21 e2 e)q) HOAc (20 N
O OP O OH
NaOAc (3 e) DCM/MeOH 20C e 12) h To a solution of azetidin-3-yl nonyl hydrogen phosphate (1.2 g, 3.05 mmol, 1.0 eq, TFA) in DCM (10 mL) and MeOH (5 mL) were added NaOAc (750.81 mg, 9.15 mmol, 3.0 eq), HOAc (366.41 mg, 6.10 mmol, 348.96 uL, 2.0 eq), 2-heptylnonanal (880.19 mg, 3.66 mmol, 1.2 eq) and naBh(OAc)
3 (775.91 mg, 3.66 mmol, 1.2 eq). The mixture was stirred at 20 °C for 12 h. The reaction mixture was filtered and washed with EtOAc (20 mL). The filtrate was collected and 145
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0~10% MeOH/DCM gradient @ 80 mL/min) and prep-HPLC (column: Welch Xtimate C1100 * 30mm * 5um;mobile phase: [water(FA)-MeOH]; mixture of water(FA) and ACN containing 70%-100% CAN, 8 min) to get compound SM-044, also known as [1-(2-heptylnonyl)azetidin-3-yl] nonyl hydrogen phosphate (1.01 g, 2.01 mmol, 63.2% yield) as a white solid. LCMS: [M+H]
+: 504.5 1H NMR (400 MHz, CDCl
3) δ = 5.52 - 4.92 (m, 1H), 4.79 - 4.44 (m, 2H), 4.01 - 3.70 (m, 4H), 2.98 - 2.84 (m, 2H), 1.67 - 1.52 (m, 3H), 1.38 - 1.22 (m, 36H), 0.88 (t, J = 6.8 Hz, 9H). Example 14: Synthesis of SM-045 Boc N 1 OH 1) 1( 21)01A eq 3 ())1 H 1 P0O0O% eCq) Hl3 TC TH (1lEF0A 400 e (-3q °2C)00 T ° e 2CEq h)A 11 T (A1 hH2F eq) Boc N O OP O OH 2 DC HMCl/ 2d0io °xCan 2e h HN O OP O OH 3 NaBH(OA DcC)3E (1052 e0q °)C H O 1O2A hc 3A (20 eq),
Step 1: tert -butyl 4-[hydroxy(nonoxy)phosphoryl]oxyazepane-1-carboxylate (2): (EC8433- 115/121) 1) 1(10 eq) H POOCl3 (10 eq) TEA 1 (A12 eq
O OH
Boc N 1 OH 2) 1A 3 ()1100% eq) H TC THlEFA 400 (32C00 ° e 2Cq h) 1 T hHF) Boc N OP O 146
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) TEA (6.73 g, 66.55 mmol, 9.26 mL, 1.2 eq) was slowly added to POCl
3 (8.50 g, 55.46 mmol, 5.15 mL, 1.0 eq) in dry THF (80 mL) at 0 °C under N
2. Then nonan-1-ol (8 g, 55.46 mmol, 1.0 eq) in THF (80 mL) was added drop wise over 1 h at 0 °C. The resulting mixture was warmed to 20 °C and stirred for 1 h. When all the alcohol had reacted (checked by TLC), the mixture was cooled to 0 °C and a second portion of TEA (16.84 g, 166.37 mmol, 23.16 mL, 3.0 eq) was added, followed by tert-butyl 4-hydroxyazepane-1-carboxylate (11.94 g, 55.46 mmol, 1 eq) in THF (80 mL) was added dropwise. The reaction mixture was stirred at 20 °C for 12 h. Decomposed with HCl 10% (200 mL) and heated at 40 °C for 2 h. THF was removed under vacuum and the aqueous residue was extracted with DCM (200 mL × 3) . The organic layer was dried over Na
2SO
4, filtered, reduced under vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0~10% MeOH/DCM gradient @ 100 mL/min) and purified by prep-HPLC (column: Phenomenex Luna C18 150 * 25mm * 10um;mobile phase: [water(FA)-ACN]; mixture of water(FA) and ACN containing 60%-90% CAN, 8min) to get compound tert-butyl 4-[hydroxy(nonoxy)phosphoryl]oxyazepane-1-carboxylate (1.9 g, 4.46 mmol, 8.0% yield, 98.9% purity) as a yellow oil. 1H NMR (400 MHz, CDCl
3) δ = 4.66 - 4.46 (m, 1H), 4.01 (q, J = 6.4 Hz, 2H), 3.60 - 3.48 (m, 1H), 3.46 - 3.40 (m, 1H), 3.38 - 3.28 (m, 2H), 2.06 - 1.89 (m, 4H), 1.87 - 1.78 (m, 1H), 1.71 - 1.63 (m, 3H O), 1.47 (s, 9H), 1.39 - 1.34 (m, 2H), 1.33 - 1.26 (m, 10H), 0.89 (t, J = 6.8 Hz, 3H). Step 2: azepan-4-yl nonyl hydrogen phosphate (3): (EC84
O33-129) N OP O OH DC HMCl/ 2d0io °xCane OP O OH
Boc 2 h HN To a solut 2ion of tert-butyl 4-[hydroxy(nonoxy)phosphoryl]ox 3yazepane-1-carboxylate (1.8 g, 4.27 mmol, 1.0 eq) in DCM (10 mL) was added HCl/dioxane (4 M, 5 mL, 4.7 eq). The mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated under reduced pressure to get compound azepan-4-yl nonyl hydrogen phosphate (1.53 g, crude, HCl) as a yellow oil. Step 3: Nonyl 2-[4-(1-pentylhexyl)piperazin-1-yl]ethyl hydrogen phosphate: (EC8433- 120/122) 147
Attorney Ref.: BN00004.0144 OME-013WO ( HN O OP O NaBH(OA DcC)3E (1052 e0q °)C H 1O2A 3 hcA (20 eq) OPCOT Application) OH O N HO P O
o a so u o o a epa - -y o y y oge p osp a e . g, . o, . eq, DCE (10 mL) and MeOH (5 mL) were added NaOAc (756.45 mg, 9.22 mmol, 3.0 eq), HOAc (369.17 mg, 6.15 mmol, 351.59 uL, 2.0 eq), 2-heptylnonanal (886.84 mg, 3.69 mmol, 1.2 eq) and naBh(OAc)
3 (781.78 mg, 3.69 mmol, 1.2 eq). The mixture was stirred at 20 °C for 4 h. The reaction mixture was filtered and washed with EtOAc (30 mL). The filtrate was collected and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0~10% MeOH/DCM gradient @ 45 mL/min) to get compound SM-045, also known as [1-(2-heptylnonyl)azepan-4-yl] nonyl hydrogen phosphate (972.79 mg, 1.75 mmol, 57.1% yield, 98.41% purity) as a yellow gum. LCMS: [M+H]
+: 546.4 1H NMR (400 MHz, CD
3OD-d
4) δ = 4.55 - 4.43 (m, 1H), 3.84 (q, J = 6.4 Hz, 2H), 3.72 - 3.36 (m, 2H), 3.31 - 3.07 (m, 2H), 3.04 (d, J = 6.4 Hz, 2H), 2.35 - 2.12 (m, 2H), 2.12 - 1.99 (m, 2H), 1.91 - 1.76 (m, 3H), 1.66 - 1.59 (m, 2H), 1.43 - 1.28 (m, 36H), 1.03 - 0.79 (m, 9H). 148
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Example 15: Synthesis of SM-047 Boc N 1 OH 1) 1( 21)01A eq 3 ())1 H 1 P0O0O% eCq) Hl T
3C TH (1lEFA0400 e (-3q °2C)00 T ° e 2CEq h)A 11 T (A1 hH2F eq) Boc N O OP O OH 2 H 2C5l/ °dCiox 1a hne HN O OP O OH 3 NaBH(OA DcC)3E (1052 e0q °)C H O 1O2A 3 hcA (20 eq),
(EC8433-124/126) OH 1) 1(10 eq) H POOCl3 TH (1F00 e-q2)0 T °CEA (12 eq) Boc
OP OH
Boc N 1 (7.582 g), 1A 73 ()1.18007% eq m H)C TmlEA 4o0 (,3 °C00 e 2q. h) 1 T hH mF N , . eq) was O O s ow y 2 2 added to OCl
3 (9.57 g, 62.39 mmol, 5.80 mL, 1.0 eq) in dry THF (100 mL) at 0 °C under N . Then nonan-1-ol (9 g, 62.39 mmol, 1.0 eq) in THF (100 mL) was added dropwise over 1 hour at 0 °C. The resulting mixture was warmed to 20 °C and stirred for 1 hour. When all the alcohol 1A had reacted (e.g., as checked by TLC), the mixture was cooled to 0 °C and a second portion of TEA (18.94 g, 187.17 mmol, 26.05 mL, 3.0 eq) was added, followed by tert-butyl 3-hydroxypyrrolidine-1-carboxylate (11.68 g, 62.39 mmol, 1.0 eq) in THF (100 mL) was added dropwise. The reaction mixture was stirred at 20 °C for 12 hours, and then decomposed with HCl 10% (200 mL) and heated at 40 °C for 2 hours. The THF was removed under vacuum and the aqueous residue was extracted with DCM (200 mL × 3). The organic layer was dried over Na
2SO
4, filtered, and reduced under vacuum. The residue was purified by prep-HPLC (column: Welch Xtimate C1 100 * 30mm * 5um; mobile phase: [water(FA)-ACN]; mixture of water(FA) and ACN containing 60%-90% CAN, 8 minutes) to get 149
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) compound tert-butyl 3-[hydroxy(nonoxy)phosphoryl]oxypyrrolidine-1-carboxylate (1.5 g, 3.81 mmol, 6.1% yield) as a yellow gum. 1H NMR (400 MHz, CDCl3) δ = 5.01 - 4.87 (m, 1H), 4.01 - 3.94 (m, 2H), 3.58 - 3.35 (m, 4H), 2.30 - 1.96 (m, 2H), 1.67 - 1.59 (m, 2H), 1.46 (s, 9H), 1.31 - 1.24 (m, 12H), 0.89 (t, J = 6.8 Hz, 3H). S Btoecp 2: nonyl pyrrolidin-3-yl hydrogen phosphate (3): (EC8433 N
O OP O OH H 2C5l/dioxane HN
O OP O OH-133)
2 , , °C.1 hq 3 of tert-butyl 3- [hydroxy(nonoxy)phosphoryl]oxypyrrolidine-1-carboxylate (928 mg, 2.36 mmol, 1.0 eq) in DCM (10 mL). The mixture was stirred at 25 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to compound nonyl pyrrolidin-3-yl hydrogen phosphate (777.86 mg, crude, HCl) as a yellow gum. Step 3: [1-(2-heptylnonyl)pyrrolidin-3-yl] nonyl hydrogen phosphate: (EC8433-134)
HN
O OP O OH NaBH(OAc)3 (15 e) H OOA 3cA (20 e) N O OP O OH NaOAc (580.42 mg, 7.08 mmol, 3.0 eq), HOAc (283.27 mg, 4.72 mmol, 269.78 uL, 2.0 eq), 2-heptylnonanal (680.47 mg, 2.83 mmol, 1.2 eq) and naBh(OAc)3 (599.85 mg, 2.83 mmol, 1.2 eq) were added to a solution of nonyl pyrrolidin-3-yl hydrogen phosphate (777.86 mg, 2.36 mmol, 1.0 eq, HCl) in DCM (6 mL) and MeOH (6 mL). The mixture was stirred at 25 °C for 12 hours. The reaction mixture was filtered and washed with EtOAc (20 mL). The filtrate was 150
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) collected and concentrated under reduced pressure to give a residue, which was then purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0~10% MeOH/DCM gradient @ 45 mL/min) and prep-HPLC (column: Welch Xtimate C1100 * 30 mm * 5 um; mobile phase: [water (FA)-MeOH]; mixture of water(FA) and ACN containing 70%-100% CAN, 8 min) to get compound SM-047, also known as [1-(2-heptylnonyl)pyrrolidin-3-yl] nonyl hydrogen phosphate (773.78 mg, 1.49 mmol, 63.32% yield, 99.94% purity) as a yellow gum. 1H NMR (400 MHz, CD3OD-d4) δ = 4.90 (s, 1H), 3.86 (q, J = 6.4 Hz, 2H), 3.83 - 3.67 (m, 2H), 3.45 - 3.32 (m, 1H), 3.27 - 3.16 (m, 2H), 3.14 - 3.08 (m, 1H), 2.49 - 2.15 (m, 2H), 1.89 - 1.75 (m, 1H), 1.63 (m, 2H), 1.43 - 1.27 (m, 36H), 1.04 - 0.80 (m, 9H). Example 16: Synthesis of SM-052 HN N Boc Na 1a (11 e Oq) N N Boc HCl/dioxane (50 eq) N NH Br KCO OO P ( O36 OH0 (1 e0q) eq K)I (01 eq) N N OOP OOH
Ste 1p 1: t DeC HB rEOHA( t 2Oc0A - ( °6c bC)0
31u e (12q)5 h eq) tyl 4-(trid 2 DCM 20 °C 16 h ecan-7-yl)piperazine 3-1-carbo
2 x CP
3 yMEla 80t °Ce 72 ( h2): (EC7126- O1M4G OTM2SGM)T005826NX1 HN N Boc NaBH(O 1Aac) (31 ( e1q5) O e) N N Boc
c ( e) To a solution of tert-butyl piperazine-1-carboxylate (42.3 g, 226.9 mmol, 3.0 eq), naBh(OAc)
3 (24.0 g, 113 mmol, 1.5 eq) and HOAc (27.3 g, 454 mmol, 26 mL, 6.0 eq) in DCE (200 mL) was added tridecan-7-one (15 g, 75.6 mmol, 1.0 eq). The mixture was stirred at 20 °C for 12 h under N
2. The reaction mixture was basified by addition 1 N NaOH solution (30 mL)to 151
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) pH=10 ~ 11 at 0 °C, then diluted with water (150 mL) and extracted with EtOAc (150 mL × 3). The combined organic layers were washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 220 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 2% EtOAc/PE gradient @100 mL/min) to give compound tert-butyl 4-(1- hexylheptyl)piperazine-1-carboxylate (9.22 g, 24.76 mmol, 32.7% yield, 99.0% purity) as a colorless oil. 1H NMR (400 MHz, CDCl
3) δ = 3.42 - 3.30 (m, 4H), 2.51 - 2.38 (m, 4H), 2.36 - 2.29 (m, 1H), 1.45 (s, 9H), 1.36 - 1.13 (m, 20H), 0.88 (t, J = 6.8 Hz, 6H). Step 2: 1-(tridecan-7-yl)piperazine (3): (EC7126-144)
N N Boc HC Dl/CdMioxa 2n0e °C (5106 e hq) N NH To a solution of tert-butyl 4-(1-hexylheptyl)piperazine-1-carboxylate (5 g, 13.6 mmol, 1.0 eq) in DCM (50 mL) was added HCl/dioxane (4 M, 17 mL, 5.0 eq). The mixture was stirred at 20 °C for 10 h. The reaction was concentrated to give compound 1-(1-hexylheptyl)piperazine (4 g, 12.99 mmol, 95.7% yield, 99.0% purity, HCl) as a yellow oil. 1H NMR (400 MHz, CDCl
3) δ = 4.16 - 4.00 (m, 2H), 3.52 - 3.50 (m, 2H), 3.15 - 3.05 (m, 1H), 2.01 - 1.90 (m, 2H), 1.71 - 1.60 (m, 2H), 1.54 - 1.11 (m, 20H), 0.90 - 0.85 (m, 6H). Step 3: 2-[4-(1-hexylheptyl)piperazin-1-yl]ethyl nonyl hydrogen phosphate (SM-052): (EC5059-403/EC7126-148) 152
Attorney Ref.: BN00004.0144 OME-013WO H B K O OH (PCT Application) N Nr2C COOP3 PM (36 OE0 (18 e00q) ° eCq K)I 7 (20 h1 eq) N N OO P O
OH
m xture o -bromoet y nony ydrogen p osp ate (3. 9 g, 0.5 mmo, . eq), -( - hexylheptyl)piperazine (3 g, 8.79 mmol, 1.0 eq, 2HCl), K
2CO
3 (3.64 g, 26.4 mmol, 3.0 eq), KI (146 mg, 879 umol, 0.1 eq) in methoxycyclopentane (15 mL) was degassed and purged with N
2 for 3 times, and then the mixture was stirred at 80 °C for 72 h under N
2. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 5% MeOH/DCM @ 50 mL/min) to give compound SM-052, also known as 2-[4-(1- hexylheptyl)piperazin-1-yl]ethyl nonyl hydrogen phosphate (952.44 mg, 1.83 mmol, 15.8% yield, 99.55% purity) as an orange solid. LCMS: [M+H]
+: 519.4 1H NMR (400 MHz, MeDH-d
4) δ = 4.16 - 4.07 (m, 2H), 3.89 (q, J = 6.8 Hz, 2H), 3.28 - 3.04 (m, 6H), 3.03 - 2.88 (m, 4H), 2.75 - 2.60 (m, 1H), 1.71 - 1.59 (m, 4H), 1.42 - 1.29 (m, 30H), 0.98 - 0.88 (m, 9H). Example 17: Synthesis of SM-053
HN N Boc Na 1a (11 eq) O N N Boc HC Dl/Cdioxane (50 eq) N NH Br KCO OO P ( O36 OH0 (1 e0q) eq K)I (01 eq) N N OOP OOH St 1ep 1: D tC HB eEOHA( r 0Oc2A t (20c) -0 °
3Cb e (1q 1)5 u2 e hq) tyl 4-(hepta 2 M 20 °C 3 h decan-9-yl)piperazine- 31-carbox
2 y ClP
3MaEt 8e0 °C ( 122 h): (EC7126-141) OMGT087 153
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) HN N Boc 1a (11 O 3 eq) N N Boc
a(c (c) e () e) To a solution of tert-butyl piperazine-1-carboxylate (32.9 g, 177 mmol, 3.0 eq), HOAc (21.2 g, 354 mmol, 20.2 mL, 6.0 eq) and naBh(OAc)
3 (18.74 g, 88.43 mmol, 1.5 eq) in DCE (200 mL) was stirred at 25 °C for 0.5 h, Then heptadecan-9-one (15 g, 58.95 mmol, 1.0 eq) was added and stirred at 25 °C for 12 h under N
2. The reaction mixture was basified by addition 2 N NaOH solution (30 mL) at 0 °C, and then diluted with water (300 mL) and extracted with EtOAc (300 mL × 3). The combined organic layers were washed with brine (300 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO
2, PE/EtOAc=1/0) to give compound tert-butyl 4- (1-octylnonyl)piperazine-1-carboxylate (11.32 g, 26.39 mmol, 44.8% yield, 99.0% purity) as a yellow oil. 1H NMR (400 MHz, CDCl
3) δ = 3.51 - 3.30 (m, 4H), 2.53 - 2.37 (m, 4H), 2.36 - 2.24 (m, 1H), 1.45 (s, 9H), 1.30 - 1.10 (m, 27H), 0.88 (t, J = 6.8 Hz, 6H). Step 2: 1-(heptadecan-9-yl)piperazine (3): (EC7126-143)
N N Boc HC Dl/CdMioxa 2n0e °C (5100 e hq) N NH To a solution of tert-butyl 4-(1-octylnonyl)piperazine-1-carboxylate (5 g, 11.8 mmol, 1.0 eq) in DCM (50 mL) was added HCl/dioxane (4 M, 14.7 mL, 5.0 eq). The mixture was stirred at 154
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 20 °C for 10 h. The reaction was concentrated to give compound 1-(heptadecan-9-yl)piperazine (4.5 g, 11.22 mmol, 95.3% yield, 90.0% purity, HCl) as a yellow oil. 1H NMR (400 MHz, CDCl
3) δ = 4.15 - 3.95 (m, 2H), 3.14 - 2.96 (m, 2H), 3.24 - 3.00 (m, 1H), 2.03 - 1.84 (m, 2H), 1.72 - 1.57 (m, 2H), 1.49 - 1.08 (m, 28H), 0.86 (t, J = 6.8 Hz, 6H). Step 3: 2-(4-(heptadecan-9-yl)piperazin-1-yl)ethyl nonyl hydrogen phosphate (SM-053-NX- 1): (EC7126-145/146/147) N NH Br K2C CO OOP3 PM ( O36 OH E0 (18 e0q) eq K)I (01 eq) N N OO P O OH
0 °C 12 h To a solution of 1-(1-octylnonyl)piperazine (3 g, 9.24 mmol, 1 eq) and 2-bromoethyl nonyl hydrogen phosphate (3.06 g, 9.24 mmol, 1 eq) in methoxycyclopentane (30 mL) was added K
2CO
3 (3.83 g, 27.7 mmol, 3 eq) and KI (153 mg, 924 umol, 0.1 eq). The mixture was stirred at 80 °C for 72 h. The reaction was filtered and the filtrate was concentrated. The residue was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 5% MeOH/DCM @ 50 mL/min) and further purified by prep-HPLC (FA condition; : Welch Xtimate C1 100 * 30 mm * 5 um; mobile phase: [water (FA) - MeOH]; mixture of water(FA) and ACN containing 70%-100% CAN, 8 min) to give SM-053, also known as 2-(4-(heptadecan-9- yl)piperazin-1-yl)ethyl nonyl hydrogen phosphate (600.5 mg, 1.03 mmol, 53.6% yield, 98.26% purity) as an orange solid. LCMS: [M+H]
+: 575.3 1H NMR (400 MHz, CDCl
3) δ = 4.15 - 4.05 (m, 2H), 3.87 (q, J = 6.8 Hz, 2H), 3.30 - 3.10 (m, 4H), 3.09 - 2.76 (m, 6H), 2.70 - 2.60 (m, 1H), 1.69 - 1.57 (m, 4H), 1.40 - 1.28 (m, 38H), 0.90 (t, J = 6.8 Hz, 9H). 155
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Example 18: Preparation of SM-037-LNP Formulations of Different Parameters SM-037-LNPs were formulated using a microfluidic mixer or a T-junction mixing of two fluid streams, one of which contained an aqueous solution of nucleic acid entities and the other had the organic solution of lipid components and/or IC molecules. Lipid/components were prepared by combining a lipid according to the formula of 20-30 mol% of cationic lipids (e.g., SM-005), 30 to 50 mol% of a phospholipid such as SM-037 described herein, 30 to 50 mol% of a structural lipid such as cholesterol (Chol), and 0.3 to 5 mol% of a PEG- lipid (e.g., PEG-DMG) at a combined concentrations of about 10 to 30 mM in ethanol. Lipid components are combined to yield desired molar ratios (see, e.g., Table 1) and diluted with aqueous solution of the nucleic acids to a final lipid concentration of between 3 to 15 mM. Nanoparticle compositions including the nucleic acids and lipid components are prepared by combining the organic solution containing the lipid/ components with the aqueous solution of nucleic acids with a total lipid to nucleic acid w/w ratio between about 10:1 and about 100:1. The lipid solution is rapidly injected using a NanoAssemblr microfluidic based system at flow rates between about 8 and about 12 mL/min into the nucleic acid aqueous solution with an aqueous to organic volume ratio between about 1:1 and about 4:1. The mixture is then immediately diluted with nuclease free water at 1:1 volume ratio. The diluted mixture is then processed using a buffer exchange column or a tangential flow filtration (TFF) system to exchange the solution with the final desired buffer, such as Tris-HCl or a Tris/Acetate buffer, at neutral pH between 7.0 and 7.5 containing up to 15% of sucrose. The solution is then subsequently concentrated using a TFF or a centrifugation column with a filter. The concentrated solution is then sterile filtered and diluted to a desired concentration between about 0.1 mg/mL and about 1.0 mg/mL nucleic acid prior to freezing for storage. Table 1. Formulation mixture examples F
ormulation Cationic lipid Phospholipid Structural Lipid PEG-lipid
156
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 8 23.0 38.3 38.3 0.4
rentially Target Lung Tissue SM-037-LNPs LNPs were prepared using a microfluidic mixing process. Briefly, lipid stocks of SM-005, SM-037, CHOL, and PEG-DMG were prepared in ethanol at 20 mg/ml concentration to a final mole ration of 30/50/50/1.5 for SM-005/SM-037/CHOL/PEG-DMG, respectively. In all initial formulations, the SM-037 mol percent was kept at 50% of total lipid in the particle. For the initial nucleic acid-particle formulations, lipids were mixed together for the given compositions in ethanol with a final lipid concentration of 5.8 mg/ml. Firefly luciferase mRNA (mFluc) was used as the mRNA in the aqueous phase at a concentration of 0.25 mg/ml. The mixing of two phases and LNP preparation was performed using a 2:1 aqueous to organic volume ratio, and at an 8 ml/min flow rate in a microfluidic chip with staggered herringbone structure. Resulting LNPs were subjected to purification and buffer exchange by tangential flow filtration (TFF) against PBS. Precise control of the characterization parameters enabled the preparation of SM-037-LNPs in the size range of 51-188 nm, with surface charges between 0-26 mV, and PDI values at or below 0.2. All formulations showed more than 98% of encapsulation efficiency (EE) calculated by Ribogreen assay using the manufacturer’s protocol. In vivo systemic, post-IV biodistribution of SM-037-LNPs as disclosed herein harboring mRNA cargoes was assessed in mice. SM-037-LNPs possessing varying surface charges (0-26 mV) and PEGylation values (0-1%) were specifically examined in intravenously LNP-injected C57BL/6 mice via both in vivo imaging (see, e.g., FIG.2A and FIG.2B) and ex vivo detection of delivery and cargo expression in harvested organs (see, e.g., FIG.2C). SM-037-LNP formulations possessing 50% (by mol) SM-037 were prepared as described above. LNPs were also fluorescently labeled with Cy7-DOPE in the formulation (0.5% mol). Briefly, mFluc mRNA-loaded SM-037- LNPs were administered to mice at 3 mg/kg dose intravenously. At 6 hours and at 24 hours post- administration, 150 mg/kg luciferin in PBS was injected intraperitoneally, and mice were anesthetized under isoflurane for live animal fluorescence and luminescence imaging. Cy7 signal distribution indicated LNP biodistribution, while the luminescence signal indicated reporter mRNA cargo activity. Notably, tested SM-037-LNPs demonstrated concentrated luciferase activity (and therefore both localization and expression) in mouse lungs (FIG.2A and FIG.2B). 157
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Major organs of treated mice were then examined ex vivo. As shown in FIG.2C, SM-037- LNP mRNA expression was highly specific to the lungs. Tested SM-037-LNP delivery and expression of associated mRNA cargoes was observed in the lungs at levels exceeding 90% of all luminescence signal detected. These data demonstrated that the lung selectivity observed herein for tested SM-037-LNP mRNA delivery and expression was not due to LNP surface charge alone, but also without wishing to be bound by theory, was likely caused by an apparent structural affinity between SM-037 and the lung epithelium. Body weight and liver function tests also indicated that the SM-037-LNPs of the instant disclosure were not toxic in vivo within 24 hours post-IV administration. Example 20: Exemplary LNP Compositions Several different LNP compositions were prepared and tested to determine their respective efficacies in delivering mRNA to targeted tissues and cells. As shown in Table 2, LNP compositions including SM-005, SM-008, SM-037, DOTAP, DDAB, Cholesterol, and PEG-DMG in various combinations were prepared. Table 2. Nanoparticle composition ID Lipid Components Lipid Composition Lipid/nucleic (m l%) id i ht r tio
Procedure for Preparing LNP Formulations Lipid nanoparticles disclosed herein may be formulated using a microfluidic mixer, a cross, or a T-junction by mixing two or three fluid streams containing nucleic acid cargo and lipid components, respectively. Lipid components were prepared by combining a lipid according to the following general formula: 20-30 mol% of cationic lipids (e.g., DOTAP, DDAB or SM-005), 30- 50 mol% of phospholipid (e.g., SM-037), 30-50 mol% of a structural lipid (such as, e.g., cholesterol), and 0.3-5 mol% of a PEG-lipid (e.g., PEG-DMG) at a combined concentrations at 158
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) about 10 to 30 mM in ethanol. The lipid mixture may then be diluted with ethanol and water to a final lipid concentration of between about 3 mM and about 75 mM. Lipid nanoparticles compositions including the nucleic acids and lipid components disclosed herein were prepared by rapidly mixing the organic solution containing the lipid components with the aqueous solution of nucleic acid cargo with a total lipid to nucleic acid w/w ratio between about 10:1 and about 100:1 by using either a NanoAssemblr microfluidic based system or an equivalent pump system at flow rates between about 8 mL/min and about 30 mL/min into the nucleic acid aqueous solution with an aqueous to organic volume ratio between about 1:1 and about 6:1. The resulting mixture was then immediately diluted with water to a final ethanol concentration between about 10% and about 20%. The diluted suspension was concentrated between about 2 and 10-fold before being buffer exchanged to a storage buffer containing between about 5-15% sugar (such as sucrose or trehalose), 10-100 mM of NaCl, 10-200 mM Tris-HCL, 10- 200 mM Tris-Base, and 10-200 mM sodium acetate between about a pH of 6.5-8.0 and having an osmolarity between about 200-400 mOsm/kg. The resulting mixture was then concentrated using a dead-end filtration on a spin column (MilliporeSigma, Amicon) and then sterile filtered using a 0.2 um sterile filter and diluted to a desired concentration between about 0.1 mg/mL and about 2.0 mg/mL nucleic acid prior to storing at temperature at – 80 °C, – 20 °C, or at 4 °C. The isolated LNPs were characterized to determine the encapsulation efficiency, average hydrodynamic size, and polydispersity index, as described below. Characterization of LNP Formulations A DynaPro® Plate Reader III (Wyatt Technology, Santa Barbara, CA, US) was used to determine the particle size and the polydispersity index (PDI). A Mobius™ (Wyatt Technology, Santa Barbara, CA, US) was used to determine the zeta potential of the LNP compositions. The nanoparticle formulations were diluted 50 to 100-fold in 1X buffer (Tris-HCl or Tris-Acetate buffer, 10-100 mM, pH 7.0 – 7.5) when determining particle size, PDI, and zeta potential. A QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, Calif.) was used to evaluate the encapsulation of mRNA by the nanoparticle composition. The samples 159
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) were diluted to a concentration of approximately 0.2 – 2 μg/ml in a TE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). Diluted samples were transferred to a polystyrene 96 well plate and equivalent volume of either TE buffer or 0.5 – 2% Triton X-100 solution was added to the wells. The RIBOGREEN® reagent was diluted 1:200 in TE buffer, and 2X volume of this solution was added to each well. The fluorescence intensity was measured using a fluorescence plate reader (Tecan Spark, Tecan Trading AG, Switzerland) at an excitation wavelength of about 485 nm and an emission wavelength of about 530 nm. The fluorescence values of the reagent blank were subtracted from that of each of the samples and the percentage of free mRNA was determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100). In vivo (Systemic Injection) Protocol – Bioluminescence To monitor how effectively various nanoparticle compositions deliver mRNA to targeted tissues and cells, different nanoparticle compositions including a particular mRNA (for example, a firefly luciferase mrNA (FLuc mRNA), TriLink BioTechnologies, San Diego, CA) were prepared and administered to rodent populations. Female BALB/c or C57BL/6 albino mice (~ 20 g) were administered intravenously through the tail veins using the nanoparticle compositions disclosed herein with a formulation such as those provided in Table 1 combined with FLuc mRNA. Dosages may range from 0.005 mg/kg to 5 mg/kg, where 5 mg/kg describes a dose including 5 mg of nucleic acid cargo in the nanoparticle composition for each 1 kg of body mass of the mouse. Bioluminescence was measured at 6 hours after the administration (100 µL of D- luciferin (30 mg/mL in PBS) was injected systemically 30 mins prior to imaging). Mice were then immediately sacrificed, and tissues (liver, lung, kidney, and spleen) were extracted for ex-vivo bioluminescence imaging as well as immunohistochemistry and/or immunofluorescence analysis for specific cell-type transfections. Immunohistochemistry/Immunofluorescence Protocol Lung tissues from in vivo bioluminescence study were harvested and fixed in 10% formalin solution before sending them out for immunohistochemistry and or immunofluorescence analysis. 160
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Tissues were fixed in 10% formalin solution and further dehydrated by using an alcohol solution (70 – 100%) and xylene solution for up to 24 hours and subsequently embedded in molten paraffin wax at 55 °C – 60 °C temperature. Thick tissue sections (e.g., about 5 to 15 µm) were then cut using a rotary microtome. The tissue slides were then incubated with appropriate antibody staining solutions and rinsed before imaging under a bright-field (immunohistochemistry) microscope or a fluorescent (immunofluorescence) microscope. Example 21: Iterative Optimization Led to a LNP Lead with Excellent Physical Properties For achieving lead LNPs for systemic delivery via intravenous injection, an iterative optimization process was followed to identify lead LNP formulations possessing excellent physical properties, including good frozen stability, consistent small size, high encapsulation efficiency, and low polydispersity index (PDI). Optimization of particle size consistency is shown in FIG.3, top series of bar graphs, which shows data for first, second, and third generation LNP8 formulations including SM-037, respectively. Briefly, a first generation LNP formulation showed 130 nM variability in particle size post-freeze/thaw (green bar; particle size up to 230 nM) versus pre-freeze/thaw (orange bar, 100 nM). A second generation LNP formulation showed 10 nM variability in particle size post- freeze/thaw (green bar; particle size up to 110 nM) versus pre-freeze/thaw (orange bar, 100 nM). However, a third generation LNP formulation showed no variability in particle size post- freeze/thaw (green bar; particle size up to 100 nM) versus pre-freeze/thaw (orange bar, 100 nM), indicating that a GEN-3 formulation produces particles of a very consistent small size. Optimization of encapsulation frequency is shown in FIG.3, middle series of bar graphs. A first generation LNP formulation showed ~70% nM encapsulation efficiency pre- and post- freeze/thaw (red and green bars; ~70% encapsulation). A second generation LNP formulation showed 90% nM encapsulation efficiency pre- and post-freeze/thaw (red and green bars; 90% encapsulation). However, a third generation LNP formulation showed >90% encapsulation efficiency pre- and post-freeze/thaw (red and green bars; >90% encapsulation), indicating that a GEN-3 formulation produces particles having very high encapsulation efficiency. 161
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) PDI was assessed for first, second, and third generation LNP formulations as shown in FIG.3, bottom series of bar graphs. First generation formulations showed variable PDI between 0.1 and 0.2 pre- and post-freeze/thaw, second generation formulations showed variable PDI less than 0.1 pre- and post-freeze/thaw, and third generation formulations showed consistent low PDI just under 0.1 pre- and post-freeze/thaw. Example 22: Lung-Targeting LNP8 Showed Strong FLuc Expression in Airway Epithelial Cells and Endothelial Cells In vivo systemic, post-IV biodistribution of LNPs as disclosed herein harboring mRNA cargoes was assessed in mice. Intravenously LNP-injected C57BL/6 mice were assessed via both in vivo imaging (see, e.g., FIG. 4A) and ex vivo detection of delivery and cargo expression in harvested organs (see, e.g., FIG.4B). Major organs of treated mice were then examined ex vivo. As shown in FIG.4B, systemic lung-targeting LNP formulations of the instant disclosure exhibited strong and preferential FLuc reporter expression in the lungs of treated subjects, as compared to corresponding liver, kidney and spleen organs of treated subjects. Immunohistochemistry analyses of lead LNP-treated lung sections revealed significant delivery of the FLuc nucleic acid payload to both airway epithelial cells and endothelial cells (FIG. 4C), where the luciferase reporter was targeted by a CY3-conjugated antibody, nuclei were stained with DAPI, and airway epithelial cells were imaged using a FITC-conjugated antibody. Example 23: Lipid Nanoparticle (LNP) formulation According to the techniques herein, nanoparticles were formulated using a microfluidic mixer, a cross, or a T-junction by mixing, for example, two or three fluid streams containing nucleic acid cargo and the lipid components respectively. Lipid components were prepared by combining a lipid according to the formula of 20-30 mol% of cationic lipids (e.g., DOTAP, DDAB, SM-005, and the like), 30 to 50 mol% of phospholipid (e.g., SM-037 and the like), 30 to 50 mol% of a structural lipid (e.g., cholesterol and the like), and 0.3 to 5 mol% of a PEG-lipid (e.g., PEG- DMG) at a combined concentration at about 10 to about 50 mM in ethanol. The lipid mixture was then diluted with ethanol and water to a final lipid concentration of between about 3 and about 75 mM. 162
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Nanoparticle compositions including the nucleic acids and lipid components were prepared by rapidly mixing the organic solution containing the lipid components with the aqueous solution of nucleic acid cargo with a total lipid to nucleic acid w/w ratio between about 10:1 and about 100:1 by using either a NanoAssemblr microfluidic based system or an equivalent pump system at flow rates between about 8 and about 30 mL/min into the nucleic acid aqueous solution with an aqueous to organic volume ratio between about 1:1 and about 6:1. The resulting mixture was then immediately diluted with water to a final ethanol concentration of between about 10% and about 20%. The diluted suspension was then buffer exchanged to a storage buffer containing between about 5-15% sugar (e.g., sucrose, trehalose and the like), about 10-100 mM of NaCl, about 10-200 mM Tris-HCL, about 10-200 mM Tris-Base, and about 10-200 mM sodium acetate between about a pH of 6.5-8.0 and having an osmolarity of between about 200-400 mOsm/kg. The resulting mixture was then concentrated using a dead-end filtration on a spin column (MilliporeSigma, Amicon) and then sterile filtered using a 0.2 um sterile filter and diluted to a desired concentration between about 0.1 mg/mL and about 2.0 mg/mL nucleic acid prior to storing at temperature at – 80 °C, – 20 °C, or at 4 °C. The isolated LNPs were characterized to determine the encapsulation efficiency, average hydrodynamic size, and polydispersity index, as described below. LNP cargos disclosed herein include, but are not limited to, mRNA cargo such as FLuc- mRNA (TriLink BioTechnologies). Cationic lipids disclosed herein include, but are not limited to, the following exemplary cationic lipids: 1,2-DiLinoleyloxy-N,N-dimethylaminopropane. ("DLinDMA"), 1,2- Dilinolenyloxy-N,N-dimethylaminopropane ("DLenDMA"), dioctadecyldimethylammonium ("DODMA"), Distearyldimethylammonium ("DSDMA"), N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTAP"); 3 -(N-(N',N'- dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol") and N-(1,2-dimyristyloxyprop-3-yl)- N,N-dimethyl-N-hydroxyethyl ammonium bromide ("DMRIE"). For example, cationic lipids that have a positive charge at below physiological pH include, but are not limited to: DODAP, 163
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) DODMA, DMDMA, and SM-005 (β-L-arginyl-2,3-diamino propionic acid-N-palmityl-N-oleyl- amide trihydrochloride). In some cases, the cationic lipids comprise a protonatable tertiary amine head group, C
18 alkyl chains, ether linkages between the head group and alkyl chains, and 0 to 3 double bonds. Such lipids include, e.g., DSDMA, DLinDMA, DLenDMA, and DODMA. In an exemplary embodiment, such lipids may include SM-005, and salts and isomer thereof. The chemical structure of SM-005 (β-L-arginyl-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide trihydrochloride) is shown below: (SM-0
Z N OS According to the techniques herein, "helper lipids" may include CNH ,l3
+ NH O but are not l C S N imlH3
+ ited to H2 ,N NH SM- NH2
+ Cl- 037, SM-038, SM-042, SM-044, SM-045, and/or SM-047. The chemical structures of these molecules are shown below: O O P O
(SM-037); 164
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) N N O O P OH O
(SM-045); and 165
Attorney Ref.: BN00004.0144 OME-013WO (PCT Appli N O P O cation) O OH
Exemplary sterols may include, but are not limited to, cholesterol. Exemplary PEG-lipids may include, but are not limited to, PEG-dilauroylglycerol, PEG- dimyristoylglycerol (PEG-DMG) (catalog # GM-020 from NOF, Tokyo, Japan), PEG- dipalmitoylglycerol, PEG-distearoylglycerol (PEG- DSPE) (catalog # DSPE-020CN, NOF, Tokyo, Japan), PEG- cholesterol (l-[8'-(Cholest-5-en-3[beta]-oxy)carboxamido-3',6'- dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), 1,2-dimyristoyl-sn- glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k- DMG) (cat. #880150P from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2- distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (cat. #880120C from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycerol, methoxypoly ethylene glycol (PEG2k-DSG; GS-020, NOF Tokyo, Japan), poly (ethylene glycol)-2000- dimethacrylate (PEG2k-DMA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000] (PEG2K, DOPE), 1,2-Dioleoyl-sn–glycero-3- phosphoethanolamine-polyethylene glycol methoxy (PEG, DOPE 2k, 5k, 20k). In some embodiments the PEG-lipid may include a stealth lipid such as, for example, α-Methoxy-ω-(3- oxopropoxy), polyoxyethylene (Methoxy PEG, Aldehyde), PEG2k-DMG, PEG2k-DSG, PEG2k- DSPE, PEG2K-DOPE, PEG5k-DOPE, Methoxy PEG aldehyde 20k, PEG2K-Cholesterol, and the like. Table 3. LNP compositions Lipid Components Mole % ratio
166
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) LNP5 SM-005/SM-037/Cholesterol/PEG-DMG 22.8/38/38/1.2 LNP6 SM-005/SM-038/Cholesterol/PEG-DMG 22.8/38/38/1.2 )
LNP characterization A DynaPro® Plate Reader III (Wyatt Technology, Santa Barbara, CA, US) was used to determine the particle size and the polydispersity index (PDI) of LNPs. A Mobius™ (Wyatt Technology, Santa Barbara, CA, US) was used to determine the zeta potential of the nanoparticle compositions. The nanoparticle formulations were diluted 50 to 100-fold in 1X buffer (Tris-HCl 167
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) or Tris-Acetate buffer, 10-100 mM, pH 7.0 – 7.5) in determining particle size, PDI, and zeta potential. A QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, Calif.) was used to evaluate the encapsulation of mRNA by the nanoparticle composition. The samples were diluted to a concentration of approximately 0.2 – 2 μg/ml in a TE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). Diluted samples were transferred to a polystyrene 96 well plate and equivalent volume of either TE buffer or 0.5 – 2% Triton X-100 solution was added to the wells. The RIBOGREEN® reagent was diluted 1:200 in TE buffer, and 2X volume of this solution was added to each well. The fluorescence intensity was measured using a fluorescence plate reader (Tecan Spark, Tecan Trading AG, Switzerland) at an excitation wavelength of about 485 nm and an emission wavelength of about 530 nm. The fluorescence values of the reagent blank were subtracted from that of each of the samples and the percentage of free mRNA was determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100). In vivo (systemic injection) protocol – bioluminescence To monitor how effectively various nanoparticle compositions were delivered mRNA to targeted tissues and cells, different nanoparticle compositions including a particular mRNA (for example, a firefly luciferase mrNA (FLuc mRNA), TriLink BioTechnologies, San Diego, CA, US) were prepared and administered to rodent populations. Female BALB/C or C57BL/6 albino mice (~ 20 g) were administered intravenously through the tail veins with the nanoparticle compositions disclosed herein having a formulation such as those provided in Table 3 combined with FLuc mRNA. Dose sizes may range from 0.005 mg/kg to 5 mg/kg, where 5 mg/kg describes a dose including 5 mg of nucleic acid cargo in the nanoparticle composition for each 1 kg of body mass of the mouse. IVIS Imaging: Bioluminescence was measured at 6 hours after the administration. All animals will be dosed with luciferin at 15 mg/mL via subcutaneous (SC) injection at 0.2 mL/animal. Whole body imaging was performed 5-15 minutes following D-Luciferin administration. 168
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Tissue Collection and Ex-vivo Imaging: Following perfusion, the lungs were collected and placed in a petri dish (typically one dish per group) and subjected to ex-vivo imaging sessions for bioluminescent signal. Tissue Fixation: Following ex vivo organ imaging, tissues will be placed in individual cassettes and fixed in 10% NBF for approximately 24 hours, then transferred into histology grade 70% EtOH until shipped ambient to histology facility at the completion of the study. Histology: All fixed tissues will be analyzed at the completion of the study. Ex vivo assays IHC protocol – FLuc HRP 1. Paraffin sections were deparaffinized and hydrated using the following steps at room temperature: • 15 min in xylene (repeat 2X) • 5 min in ethanol (repeat 3X at 100%, 100% and 75%, respectively) • 5 min in PBS (repeat 3X) 2. 250 mL of sodium citrate (H-3300) was placed into a slide holding container and filled with up to 12 slides. 3. Buffer solution was heated in pressure cooker (Bio SB) for approximately 15 min to 110°C. 4. The pressure was released manually (as needed). 5. The container was taken out and cooled down for 30 min. 6. The slides were washed for 5 min with running water in a retrieval container. 7. The slides were removed from the container and the tissue was circled using an oil pen. 169
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) For the following steps, use ImmPRESS HRP Horse Anti-Rabbit Kit (MP-7801) for anti- FLuc antibody. 8. The sections were incubated with BLOXALL for 10 minutes at room temperature to quench the endogenous peroxidase. 9. The sections were washed for 5 min (3X) with PBST washing buffer. 10. The sections were blocked with protein block solution (ab64226) for 30 minutes at RT. 11. The sections were washed for 5 min (1X) with PBST washing buffer. 12. The sections were incubated with primary antibody containing 3% protein blocking solution overnight at 4°C. 13. The sections were washed for 5 min (3X) with PBST washing buffer. 14. The sections were incubated with ImmPRESS Reagent for 30 min. 15. The sections were washed for 5 min (3X) with PBST washing buffer. 16. Equal volumes of ImmPACT DAB EqV Reagent 1 were combined with Reagent 2 and mixed well. 17. The sections were incubated in ImmPACT DAB EqV working solution until desired stain density developed for 2 min. 18. The sections were rinsed in tap water. 19. The sections were counterstained with hematoxylin, cleared and mounted. 170
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) TSA-DIF Protocol DAY 1 Paraffin sections were deparaffinized and hydrated using the following steps: 15 minutes in xylene twice; 5 minutes, 5 minutes, and 5 minutes in 100%, 100%, and 75% ethanol, respectively; and 5 minutes in PBS at room temperature repeated three times. 1. The slide container was filled with 250mL of Citrate Acid retrieval buffer (H-3300), the empty container will be filled with distilled water for the purpose of heating evenly. 2. The buffer solution was heated in a pressure cooker (Bio SB) for 15 minutes to a temperature of 110°C. 3. The container was taken out and cooled down for 30 minutes. 4. The slides were gently washed with running water in a retrieval container. 5. The slides were then removed from the container and the tissue was circled with a Hydrophobic Barrier PAP Pen (H-4000). 6. The sections were incubated in BLOXALL Blocking Solution for 10 minutes to quench the endogenous peroxidase. 7. The slides were then washed in the washing buffer for 5 minutes 3 times. 8. The sections were blocked with 2.5% Goat serum for 30 minutes. 9. Meanwhile, the first primary antibody was prepared according to Table 4. 10. Antibody was applied and then the slides were incubated at 4°C overnight. DAY 2 1. The slides were washed in washing buffer for 5 minutes 3 times. 2. The HRP goat anti rabbit secondary antibody was prepared with dilution 1:500 in TBS. 171
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 3. The HRP goat anti rabbit secondary antibody (Invitrogen 31460) was added and incubated for 1 hour at room temperature. 4. The slides were washed in the washing buffer for 5 minutes 3 times. 5. The TSA reagent dilution buffer was prepared. 6. The CY3-TSA reagent with 1:100 dilution was added to the buffer and incubated for 10 minutes. 7. The slides were washed in the washing buffer for 5 minutes 3 times. 8. The slides were then washed with pure water. 9. Antigen retrieval occurred via the same method described above for DAY 1. 10. The slides were washed in the washing buffer for 5 minutes 3 times. 11. 2.5% Goat Serum was added and then incubated for 30 minutes. 12. Meanwhile, the second primary antibody was prepared according to Table 3. 13. The antibody was then applied and incubated on the slides at 4°C overnight. Day 3 14. The slides were washed in the washing buffer for 5 minutes 3 times. 15. The HRP goat anti rabbit secondary antibody with dilution 1:500 was prepared in TBS. 16. The HRP goat anti rabbit secondary antibody (Invitrogen 31460) was added and incubated for 1 hour at room temperature 17. The slides were washed in the washing buffer for 5 minutes 3 times. 18. The TSA reagent dilution buffer were prepared. 19. The CY5-TSA reagent with 1:100 dilution with the buffer was added and incubated for 10 minutes. 172
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 20. The slides were washed in the washing buffer for 5 minutes 3 times. 21. The autofluorescence was quenched with 0.1% Sudan Black for 15 minutes. 22. The slides were washed with running water for 15 minutes. 23. The slides were stained with DAPI and a coverslip was added. Results LNP analytical characterization (size, size distribution and encapsulation) Table 4 presents the values for average particle size, polydispersity, and % EE for various LNP compositions. Size (nm) PDI %EE LNP1 92 0.214 98.4
173
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) LNP27 221.3 0.437 98.1 LNP28 121.9 0.184 98.7
Example 22: Synthesis of SM-061 O O P O OH
(SM-061; nonyl (4-(4-(undecan-6-yl)piperazin-1-yl)butyl) hydrogen phosphate) Synthesis of OMGT-S
HO 1) 2) 12 (1 (10 T0H eqF) 0 P-O25C °l
3C (12 O0HM-061-NX-1 Br 2 h eq) TEA (12 eq) O OP O OH KCO (30 eq 6) (N K1I0 (0 eq1 N) eHq) N N O OP O OH Step 11: 4-bromo 3 e b)q 1) u0 T%E t HA yC (3l0 l 40 eq n °C) T o 2H n hF 14 h yl hyd Brrogen ph 3osphate:
2 CP
3ME 80 °C 72 h SM061 174
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) TEA (12.63 g, 124.78 mmol, 17.37 mL, 1.2 eq) was slowly added to POCl
3 (15.94 g, 103.98 mmol, 9.66 mL, 1.0 eq) in dry THF (150 mL) at 0 °C under N
2. Then nonan-1-ol (15 g, 103.98 mmol, 1.0 eq) in THF (150 mL) was added dropwise over 1 h and the resulting mixture was warmed to 20 °C and stirred for 1 h. When all the alcohol 1 had reacted (monitored by TLC), the mixture was cooled to 0 °C and a second portion of TEA (31.57 g, 311.95 mmol, 43.42 mL, 3.0 eq) was added, followed by 4-bromobutan-1-ol (19.89 g, 103.98 mmol, 80% purity, 1.0 eq) in THF (150 mL). The reaction mixture was stirred at 20 °C for 14 h, then decomposed with HCl 10% (200 mL) and heated at 40 °C for 2 h. THF was removed under vacuum and the aqueous residue was extracted with EtOAc (300 mL ^ 3). The organic layer was dried over anh. Na
2SO
4, filtered, reduced under vacuum. The residue was purified by column chromatography (SiO
2, PE/EtOAc = 40/1 to 1/1 v/v) to give compound 4-bromobutyl nonyl hydrogen phosphate (15 g, 26.31 mmol, 26.3% yield, 63% purity) as a yellow oil.
1H NMR (400 MHz, CDCl
3) δ = 4.15 - 3.92 (m, 4H), 3.51 - 3.40 (m, 2H), 2.10 - 1.92 (m, 2H), 1.85 - 1.77 (m, 2H), 1.66 - 1.60 (m, 2H), 1.35 - 1.20 (m, 12H), 0.89 (t, J = 6.8 Hz, 3H). Step 2: SM061, (aka. nonyl 4-[4-(1-pentylhexyl)piperazin-1-yl]butyl hydro Ogen phosphate) O OP O OH K23 6 (N10 eq N)H N N OP O OH
r C COPM (3E08 e0)C KI 7 (20 h1 e) To a solution of 1-(1-pentylhexyl)piperazine (1.32 g, 4.78 mmol, 1.0 eq, HCl) and 4-bromobutyl nonyl hydrogen phosphate (3 g, 5.26 mmol, 63% purity, 1.1 eq) in CPME (15 mL) were added K
2CO
3 (1.98 g, 14.35 mmol, 3.0 eq) and KI (79.40 mg, 478.29 umol, 0.1 eq). The mixture was stirred at 80 °C for 72 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO
2, DCM/MeOH = 50/1 to 10/1 v/v) and then purified by prep-HPLC (column: Welch Xtimate C
1100 * 30 mm * 5 um; mobile phase: [water (FA)-MeOH]; B%: 70%-100%, 8 min) to give compound nonyl 4-[4-(1- 175
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) pentylhexyl)piperazin-1-yl]butyl hydrogen phosphate (1.2 g, 2.31 mmol, 60.0% yield, 99.99% purity) as a light yellow solid. LCMS: [M+H]
+:519.5
1H NMR (400 MHz, CDCl
3) δ = 3.97 - 3.80 (m, 4H), 3.46 - 3.44 (m, 2H), 2.96 - 2.74 (m, 8H), 2.44 -2.42 (m, 1H), 1.94 - 1.92 (m, 2H), 1.74 - 1.63 (m, 4H), 1.62 - 1.57 (m, 2H), 1.36 - 1.19 (m, 26H), 0.92 - 0.83 (m, 9H). Example 23: Synthesis of SM-058 O O P O OH
(SM-058; nonyl (6-(4-(undecan-6-yl)piperazin-1-yl)hexyl) hydrogen phosphate) Step 1: 6-bromohexyl nonyl hydrogen phosphate (2): TEA (8.42 g, 83.19 mmol, 11.58 mL, 1.2 eq) was slowly added to POCl
3 (10.63 g, 69.32 mmol, 6.44 mL, 1.0 eq) in dry THF (150 mL) at 0 °C under N
2. Then nonan-1-ol (10 g, 69.32 mmol, 1 eq) in THF (100 mL) was added drop wise over 1 h at 0 °C. The resulting mixture was warmed to 25 °C and stirred for 1 h. When all the alcohol had reacted (monitored by TLC), the mixture was cooled to 0 °C and a second portion of TEA (21.04 g, 207.97 mmol, 28.95 mL, 3.0 eq) was added, followed by 6-bromohexan-1-ol (12.55 g, 69.32 mmol, 9.10 mL, 1.0 eq) in THF (100 mL). The reaction mixture was stirred at 25 °C for 12 h, then decomposed with HCl 10% (150 mL) and heated at 40 °C for 2 h. THF was removed under vacuum and the aqueous residue was extracted 176
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) with DCM (150 mL × 3). The organic layer was dried over Na
2SO
4, filtered, reduced under vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 220 g SepaFlash® Silica Flash Column, Eluent of 0~5% MeOH/DCM gradient @ 100 mL/min) to get compound 6- bromohexyl nonyl hydrogen phosphate (8 g, 20.24 mmol, 14.8% yield, 98.0% purity) as a yellow gum.
1H NMR (400 MHz, CDCl
3) δ = 4.09 - 3.98 (m, 4H), 3.41 (t, J = 6.8 Hz, 2H), 1.92 - 1.83 (m, 2H), 1.75 - 1.64 (m, 4H), 1.53 - 1.40 (m, 4H), 1.40 - 1.21 (m, 12H), 0.96 - 0.83 (m, 3H). Step 2: SM058 (aka. Nonyl 2-[4-(1-pentylhexyl)piperazin-1-yl]ethyl hydrogen phosphate) To a solution of 1-(1-pentylhexyl)piperazine (1.0 g, 3.61 mmol, 1.0 eq, HCl) in methoxycyclopentane (20 mL) were added K
2CO
3 (1.50 g, 10.83 mmol, 3.0 eq), KI (299.63 mg, 1.81 mmol, 0.5 eq) and 6-bromohexyl nonyl hydrogen phosphate (1.40 g, 3.61 mmol, 1.0 eq). The mixture was stirred at 25 °C for 48 h. The reaction mixture was filtered and washed with EtOAc (50 mL). The filtrate was collected and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0~10% MeOH/DCM gradient @ 45 mL/min) and prep-HPLC (column: Welch Xtimate C
1100 * 30 mm * 5 um; mobile phase: [water (FA)-MeOH]; B%: 70%-100%, 8 min) to get compound nonyl 6-[4-(1-pentylhexyl)piperazin-1-yl]hexyl hydrogen phosphate (527.5 mg, 964.60 μmol, 22.9% yield, 99.99% purity) as a yellow oil. LCMS: [M+H]
+: 547.5
1H NMR (400 MHz, CDCl
3) δ = 3.98 - 3.91 (m, 2H), 3.91 - 3.85 (m, 2H), 3.62 - 3.40 (m, 2H), 3.15 - 3.0 (m, 2H), 2.98 - 2.88 (m, 2H), 2.85 - 2.58 (m, 4H), 2.47 - 2.36 (m, 1H), 1.86 - 1.75 (m, 2H), 1.72 - 1.62 (m, 4H), 1.60 - 1.45 (m, 6H), 1.40 - 1.22 (m, 26H), 0.98 - 0.82 (m, 9H). Example 24: Synthesis of SM-057 177
Attorney Ref.: BN00004.0144 O OH OME-013WO (PCT Application) O P O
( - ; nony ( -( -( r ecan- -y )p peraz n- -y ) u y ) y rogen p osp a e ) Step 1: tert-butyl 4-(1-hexylheptyl)piperazine-1-carboxylate (2) To a solution of tert-butyl piperazine-1-carboxylate (18.78 g, 100.83 mmol, 2.0 eq) and HOAc (9.08 g, 151.25 mmol, 8.65 mL, 3.0 eq) in DCE (150 mL) was added NaBH(OAc)
3 (21.37 g, 100.83 mmol, 2.0 eq). After addition, the mixture was stirred at 25 °C for 0.5 h, and then tridecan- 7-one (10 g, 50.42 mmol, 1.0 eq) was added. The resulting mixture was stirred at 25 °C for 15.5 h. The reaction mixture was basified by adding NaOH (60 mL, 1 N) till pH 10~11 at 0 °C, and then diluted with water (100 mL) and extracted with EtOAc (150 mL * 3). The combined organic layers were washed with brine (150 mL), dried over anh. Na
2SO
4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (220 g SepaFlash® Silica Flash Column, PE : EtOAc: 0~10%) to give compound tert-butyl 4-(1- hexylheptyl)piperazine-1-carboxylate (8.4 g, 22.79 mmol, 61.2% yield) as yellow oil.
1H NMR (400 MHz, CDCl
3) δ = 3.35 - 3.28 (m, 4H), 2.50 - 2.41 (m, 4H), 2.36 - 2.30 (m, 1H), 1.46 (s, 9H), 1.44 - 1.40 (m, 2H), 1.33 - 1.21 (m, 18H), 0.89 (t, J = 6.4 Hz, 6H). Step 2: 1-(1-hexylheptyl)piperazine (3): To a solution of tert-butyl 4-(1-hexylheptyl)piperazine-1-carboxylate (3 g, 8.14 mmol, 1.0 eq) in DCM (30 mL) was added HCl/dioxane (4 M, 9.00 mL, 4.4 eq). The mixture was stirred at 20 °C 178
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) for 3 h. The reaction mixture was directly concentrated under reduced pressure to give compound 1-(1-hexylheptyl)piperazine (2.4 g, crude, HCl) as a yellow solid. The crude product was used for next step without further purification. Step 3: SM-057 (aka.4-[4-(1-hexylheptyl)piperazin-1-yl]butyl nonyl hydrogen phosphate) A mixture of 1-(1-hexylheptyl)piperazine (2 g, 6.56 mmol, 1.0 eq, HCl), 4-bromobutyl nonyl hydrogen phosphate (2.83 g, 7.87 mmol, 1.2 eq), K
2CO
3 (2.72 g, 19.68 mmol, 3.0 eq), KI (108.87 mg, 655.87 umol, 0.1 eq) in methoxycyclopentane (15 mL) was degassed and purged with N
2 for 3 times, and then the mixture was stirred at 80 °C for 72 h under N
2 atmosphere. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give residue. The residue was purified by flash silica gel chromatography (40 g SepaFlash® Silica Flash Column, DCE : MeOH : 0~5%, twice) to give SM-057 (1.1 g, 2.00 mmol, 47.6% yield, 99.56% purity) as yellow oil. LCMS: [M+H]
+: 547.3
1H NMR (400 MHz, CDCl
3) δ = 3.94 - 3.89 (m, 2H), 3.83 - 3.81 (m, 2H), 3.67 - 3.34 (m, 2H), 3.05 - 2.60 (m, 8H), 2.45 - 2.30 (m, 1H), 1.98 - 1.83 (m, 2H), 1.73 - 1.65 (m, 2H), 1.62 -1.55 (m, 2H), 1.47 -1.39 (m, 2H), 1.32 - 1.22 (m, 30H), 0.95 - 0.80 (m, 9H). Example 25: Synthesis of SM-059 O O P O OH
179
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) (SM-059; nonyl (6-(4-(tridecan-7-yl)piperazin-1-yl)hexyl) hydrogen phosphate) Br H
OO P OO K2C COP3M (32E0 (18 eN00q) e °Cq K) NIH (01 eq) N N O OP O OH
To a solution of 1-(1-hexylheptyl)piperaz 4i8n he (1.0 g, 3.28 mmol, 1.0 SM0 e5q9, HCl) in methoxycyclopentane (15 mL) were added KI (272.19 mg, 1.64 mmol, 0.5 eq), K
2CO
3 (1.36 g, 9.84 mmol, 3.0 eq) and 6-bromohexyl nonyl hydrogen phosphate (1.27 g, 3.28 mmol, 1.0 eq.; see “Example 23 synthesis of SM-058” for preparation). The mixture was stirred at 25 °C for 24 h under N
2 atmosphere. The reaction mixture was filtered and washed with EtOAc (100 mL). The filtrate was collected and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0~10% MeOH/DCM gradient @ 45 mL/min) and by prep-HPLC (column: Welch Xtimate C1 100*30 mm*5 um; mobile phase: [water (FA)-MeOH]; B%: 70%-100%,8 min) to give SM059 (aka. 6-[4-(1-hexylheptyl)piperazin-1-yl]hexyl nonyl hydrogen phosphate) (536.56 mg, 933.38 μmol, 99.99% purity, 25.55% yield) as a yellow oil. LCMS: [M+H]
+: 575.4
1H NMR (400 MHz, CDCl
3) δ = 3.96 - 3.89 (m, 2H), 3.87 (q, J = 6.8 Hz, 2H), 3.62 - 3.37 (m, 2H), 3.18 - 3.02 (m, 2H), 2.97 - 2.88 (m, 2H), 2.87 - 2.65 (m, 4H), 2.52 - 2.42 (m, 1H), 1.82 - 1.73 (m, 2H), 1.69 - 1.60 (m, 4H), 1.56 - 1.44 (m, 6H), 1.42 - 1.15(m, 30H), 0.96 - 0.81 (m, 9H). Example 26: Common Synthesis Abbreviations and Intermediates As used herein, the following abbreviations may be used in the above- and below-described synthesis examples: anh.: anhydrous Bn: benzyl DCM: dichloromethane 180
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) DMAP: 4-dimethylaminopyridine EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EtOAc: ethyl acetate eq: equivalence FA: formic acid h: hour HATU: Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium HPLC: High-performance liquid chromatography LCMS: Liquid chromatography–mass spectrometry m: minute MeOH: methanol NMR: Nuclear magnetic resonance spectroscopy PE: petroleum ether Prep-HPLC: preparative HPLC TEA: triethylamine THF: tetrahydrofuran TLC: Thin layer chromatography TFA: trifluoroacetic acid. Common Intermediates Intermediate 1 is a common intermediate used in the above- and below-described synthesis examples. The synthesis of Intermediate 1 is shown below. Intermediate_1: 2-bromoethyl nonyl hydrogen phosphate: (EC5000-215/223) 181
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 1) 1 (10 eq) PO BCrl3 ( 210 OH eq) TEA
O OH
HO 2) 2 ( 31) T01eH0q%F) H 0 TC-E2lA04 ° (03C0C 1 e hq 2)h TH (1F2 eq) Br OP O Triethylamine (TEA; 16.84 g, 166.37 mmol, 23.16 mL, 1.2 eq) wasn selromwelya aed_ded to POCl
3 (21.26 g, 138.64 mmol, 12.88 mL, 1.0 eq) in dry THF (200 mL) at 0 °C under N2. Then nonan-1-ol (20 g, 138.64 mmol, 1.0 eq) in THF (200 mL) was added drop wise over 1 h and the resulting mixture was warmed to 20 °C and stirred for 1 hour. When all the alcohol had reacted (checked by TLC), the mixture was cooled to 0 °C and a second portion of TEA (42.09 g, 415.93 mmol, 57.89 mL, 3.0 eq) was added, followed by 2-bromoethanol (17.33 g, 138.64 mmol, 9.84 mL, 1.0 eq) in THF (200 mL) was added dropwise. The reaction mixture was stirred at 20 °C for 14 h; and decomposed with aq.10% HCl (150 mL) at 40 °C for 2 h. THF was removed under vacuum and the aqueous residue was extracted with DCM (300 mL, 3x). The organic layer was dried over anh. Na
2SO
4, filtered, reduced under vacuum. The residue was purified by column chromatography (SiO
2, DCM/MeOH = 1/0 to 10/1 v/v) and purified by prep-HPLC (column: Phenomenex luna C18150 * 25mm * 10um; mobile phase: [A/B water (with formic acid (FA))-ACN]; ACN_B%: from 50% to 80%, 8min) to give Intermediate 1 (aka 2-bromoethyl nonyl hydrogen phosphate) (3.3 g, 9.96 mmol, 82.5% yield) as a yellow oil.
1H NMR (400 MHz, CDCl
3) δ = 10.34 - 10.30 (m, 1H), 4.32 - 4.21 (m, 2H), 4.04 (q, J = 6.8 Hz, 2H), 3.54 (t, J = 6.4 Hz, 2H), 1.73 - 1.62 (m, 2H), 1.39 - 1.23 (m, 12H), 0.89 (t, J = 6.8 Hz, 3H). 182
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Example 27: Synthesis of SM-063 N N O O P OH O
1 NaBH(OAH D2cN)C
3E (3205 e N °qC) Bo H 1c (2OA hc e (q3)0 eq) 3 oc DCM 25o °xCan 1e2 h 4 Br K
2 OC O CO PP O 3 OMH (3E0 ( 8 e.0q) e °Cq K)I 7 (20 h1 eq)
Step 1: tert-butyl 4-(dihexylamino)piperidine-1-carboxylate: To a solution of tert-butyl 4-aminopiperidine-1-carboxylate (10 g, 49.93 mmol, 1.0 eq), NaBH(OAc)
3 (31.75 g, 149.79 mmol, 3.0 eq) and HOAc (9.00 g, 149.79 mmol, 8.58 mL, 3.0 eq) in DCE (150 mL) was added hexanal (12.50 g, 124.83 mmol, 14.99 mL, 2.5 eq). The mixture was stirred at 25 °C for 12 h. The pH was adjusted to 8 with NaOH. The mixture was extracted with EtOAc (3 × 150 mL). The combined organic phases were washed with water (100 mL), dried with anhydrous Na
2SO
4, filtered and concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO
2, DCM/MeOH = 30/1 to 10/1) to give compound tert-butyl 4- (dihexylamino)piperidine-1-carboxylate (20 g, 54.26 mmol, 66.7% yield) as a yellow oil. 1H NMR (400 MHz, CD
3OD-d
4) δ = 4.25 - 4.20 (m, 2H), 3.46 - 3.30 (m, 2H), 3.11 - 3.01 (m, 4H), 2.84 - 2.82 (m, 1H), 2.04 - 1.93 (m, 2H), 1.74 - 1.61 (m, 6H), 1.48 - 1.45 (m, 9H), 1.43 - 1.30 (m, 12H), 0.97 - 0.89 (m, 6H). 183
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Step 2: N,N-dihexylpiperidin-4-amine: To a solution of tert-butyl 4-(dihexylamino)piperidine-1-carboxylate (8 g, 21.70 mmol, 1.0 eq) in DCM (80 mL) was added HCl/dioxane (4 M, 80 mL, 14.7eq). The mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated under reduced pressure to give compound N,N- dihexylpiperidin-4-amine (6.6 g, 21.64 mmol, 99.7% yield, HCl) as a yellow oil, used without purification. Step 3: SM-063, aka.2-[4-(dihexylamino)-1-piperidyl]ethyl nonyl hydrogen phosphate: To a solution of N,N-dihexylpiperidin-4-amine (1.67 g, 5.49 mmol, 1.0 eq, HCl) and 2-bromoethyl nonyl hydrogen phosphate (2 g, 6.04 mmol, 1.1 eq) in CPME (20 mL) were added K
2CO
3 (2.28 g, 16.47 mmol, 3.0 eq) and KI (91.13 mg, 549.00 μmol, 0.1 eq). The mixture was stirred at 80 °C for 72 h. The reaction mixture was filtered and concentrated under reduced pressure to give residue. The residue was purified by column chromatography (SiO
2, DCM/MeOH = 40/1 to 10/1) and then purified by prep-HPLC (column: Welch Xtimate C1100 * 30 mm * 5 um; mobile phase: [A/B water (FA)-MeOH]; B%: 55%-85%, 2 min) to give compound SM-063, aka 2-[4-(dihexylamino)- 1-piperidyl]ethyl nonyl hydrogen phosphate (1.1 g, 2.12 mmol, 45.8% yield, 99.99% purity) as a light yellow gum. LCMS: [M+H]
+:519.4
1H NMR (400 MHz, CDCl
3) δ = 4.06 - 4.04 (m, 2H), 3.89 - 3.80 (m, 2H), 3.29 - 3.24 (m, 2H), 85 - 2.40 (m, 7H), 2.35 - 2.30 (m, 2H), 1.93 - 1.90 (m, 4H), 1.67 - 1.51 (m, 6H), 1.35 - 1.22 (m, 24H), 0.93 - 0.84 (m, 9H). 184
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Example 28: Synthesis of SM-064 N N O O P OH O
, HN 1oc 21 H)) cO 1oA (m1cp0 (d3 e01qa) eq ( N2)a5 DB eHCa Oq(E (O)A 240c5 e) ° °
3qCC) (304075 e5q h) h 2oc DCM 20o °xCane 16 h 3 Br K
2 OC O CO PP O 3 OMH (3E08 e0q) °C KI 7 (20 ( h11 e2q e)q)
Step 1: tert-butyl 4-(1-pentylhexyl)-1,4-diazepane-1-carboxylate To a solution of tert-butyl 1,4-diazepane-1-carboxylate (10 g, 49.93 mmol, 9.84 mL, 1.0 eq) and HOAc (6.00 g, 99.86 mmol, 5.72 mL, 2.0 eq) in DCE (150 mL) was added NaBH(OAc)
3 (15.87 g, 74.90 mmol, 1.5 eq). The mixture was stirred at 25 °C for 0.5 h, then undecan-6-one (10.20 g, 59.92 mmol, 1.2 eq) was added. The resulting mixture was stirred at 40 °C for 47.5 h. The reaction mixture was basified by the addition of NaOH (60 mL, 1 N) to pH=10~11 at 0 °C, diluted with water (100 mL) and extracted with EtOAc (150 mL, 3x). The combined organic layers were washed with brine (150 mL), dried over Na
2SO
4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (120 g SepaFlash® 185
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Silica Flash Column, PE : EtOAc: 0~10%) to give compound tert-butyl 4-(1- hexylheptyl)piperazine-1-carboxylate (8.4 g, 22.79 mmol, 61.2% yield) as yellow oil.
1H NMR (400 MHz, CDCl
3) δ = 3.63 - 3.56 (m, 1H), 3.48 - 3.32 (m, 4H), 2.69 - 2.55 (m, 4H), 1.76 - 1.64 (m, 2H), 1.46 (s, 9H), 1.33 - 1.28 (m, 12H), 0.92 - 0.88 (m, 10H). Step 2: 1-(1-pentylhexyl)-1,4-diazepane (3) To a solution of tert-butyl 4-(1-pentylhexyl)-1,4-diazepane-1-carboxylate (4 g, 11.28 mmol, 1.0 eq) in DCM (20 mL) was added HCl/dioxane (4 M, 20 mL, 7.1 eq). The mixture was stirred at 20 °C for 16 h. The reaction mixture was directly concentrated under reduced pressure to give compound 1-(1-pentylhexyl)-1,4-diazepane (3.1 g, crude, HCl) as a yellow oil. The crude product was used for next step without further purification. Step 3: SM-064, aka. nonyl 2-[4-(1-pentylhexyl)-1,4-diazepan-1-yl]ethyl hydrogen phosphate A mixture of 1-(1-pentylhexyl)-1,4-diazepane (2 g, 7.86 mmol, 1.0 eq), 2-bromoethyl nonyl hydrogen phosphate (3.12 g, 9.43 mmol, 1.2 eq), K
2CO
3 (3.26 g, 23.58 mmol, 3.0 eq), KI (130.48 mg, 0.79 mmol, 0.1 eq) in methoxycyclopentane (15 mL) was degassed and purged with N
2 for 3 times, and then the mixture was stirred at 80 °C for 72 h under N
2 atmosphere. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give residue. The residue was purified by flash silica gel chromatography two times (40 g SepaFlash® Silica Flash Column, DCE: MeOH: 0~10%) to give SM-064, aka nonyl 2-[4-(1-pentylhexyl)-1,4-diazepan-1-yl]ethyl hydrogen phosphate (1.4 g, 2.77 mmol, 58.3% yield, 99.86% purity) as yellow oil. LCMS: [M+H]
+: 505.3
1H NMR (400 MHz, CDCl
3) δ = 4.21 - 4.12 (m, 2H), 3.89 (q, J = 6.8 Hz, 2H), 3.29 - 3.24 (m, 2H), 3.20 - 3.16 (m, 2H), 3.02 - 2.95 (m, 2H), 2.91 - 2.83 (m, 2H), 2.62 - 2.55 (m, 1H), 2.18- 2.04 (m, 2H), 1.68 - 1.59 (m, 2H), 1.54 - 1.45 (m, 2H), 1.42 - 1.16 (m, 28H), 0.93 - 0.85 (m, 9H). 186
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Example 29: Synthesis of SM-108 O N N O P OH O
Ste 1po 1c: te) cro 2mtO)p c-ocbm (6pdu0a 1 eaqt) 6y(0 OC °lCc)
31a 40 (55 ( h. - e(q e 1)q) -pentyl 2hexyocoxane l)piper ° (. eq) Br O O P azine-1-c -carboxylate 3arboxyla
2t Ce
3 ( ( O OH. : 8 e e0qq)) °C (6 eq) To a solution of tert-butyl piperazine-1 (10 g, 53.69 mmol, 1.0 eq) in DCE SM (101800 mL) were added NaBH(OAc)
3 (17.07 g, 80.54 mmol, 1.5 eq) and HOAc (19.35 g, 322.15 mmol, 18.44 mL, 6.0 eq) at 20 °C. After addition, the mixture was stirred at this temperature for 0.5 h. Then undecan-6-one (10.06 g, 59.06 mmol, 1.1 eq) was added in the mixture. The mixture was stirred at 60 °C for 15 h. The pH was adjusted to 8 with 2 M NaOH. The mixture was extracted with DCM (2 * 100 mL). The combined organic phase was washed with water (100 mL), dried with anhydrous Na
2SO
4, filtered and concentrated in vacuum to give residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 10% EtOAc/PE @ 80 mL/min) to give compound tert-butyl 4-(1-pentylhexyl)piperazine-1-carboxylate (2.5 g, 5.14 mmol, 9.6% yield, 70.0% purity) as a colorless oil. 1H NMR (400 MHz, CDCl
3) δ = 3.49 - 3.25 (m, 4H), 2.44 - 2.39 (m, 4H), 2.32 (t, J = 6.4 Hz, 1H), 1.46 - 1.34 (m, 9H), 1.33 - 1.15 (m, 16H), 1.15 - 0.72 (m, 6H). 187
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Step 2: 1-(1-pentylhexyl)piperazine: To a solution of tert-butyl 4-(1-pentylhexyl)piperazine-1-carboxylate (5.5 g, 16.15 mmol, 1.0 eq) in DCM (30 mL) was added HCl/dioxane (4 M, 20.19 mL, 5.0 eq). The mixture was stirred at 20 °C for 3 h. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was triturated with PE at 20 °C for 1 h to give compound 1-(1- pentylhexyl)piperazine (2.2 g, 7.15 mmol, 44.3% yield, 90.0% purity, 2HCl) as yellow gum.
1H NMR (400 MHz, CDCl
3) δ = 11.30 (s, 1H), 10.16 (s, 1H), 9.53 (s, 1H), 3.95 - 3.85(m, 5H), 3.73 - 3.50 (m, 2H), 3.45 (s, 2H), 1.93 (s, 2H), 1.82 - 1.49 (m, 2H), 1.45 - 1.10 (m, 12H), 0.97 - 0.91 (m, 6H). Step 3: SM-108, aka. nonyl (3-(4-(undecan-6-yl)piperazin-1-yl)propyl) hydrogen phosphate: Preparation of 3-bromopropyl nonyl hydrogen phosphate was similar to the synthesis of Itermediate_1, however, 3-bromopropanol was used instead of 2-bromoethanol. To a solution of 1-(1-pentylhexyl)piperazine (700 mg, 2.53 mmol, 1.0 eq, 2HCl) in methoxycyclopentane (10 mL) were added 3-bromopropyl nonyl hydrogen phosphate (960.00 mg, 2.78 mmol, 1.1 eq) and K
2CO
3 (1.05 g, 7.58 mmol, 3.0 eq) and KI (41.97 mg, 252.81 μmol, 0.1 eq). The mixture was stirred at 80 °C for 16 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 10% MeOH/DCM @ 60 mL/min) and prep-HPLC (column: C1 250 * 80 mm, 10 μm; mobile phase: A/B [water(NH
4HCO
3) - ACN]; gradient: 52% - 82% B over 10 min) to give SM-108 (aka nonyl nonyl (3-(4-(undecan-6-yl)piperazin-1-yl)propyl) hydrogen phosphate) (252.83 mg, 500.88 μmol, 36.1% yield, 99.99% purity) as an off-white solid. LCMS: [M+H]
+:505.3
1H NMR (400 MHz, CDCl
3) δ = 4.01 - 3.98 (m, 2H), 3.90 - 3.82(m, 2H), 3.44 - 3.31 (m, 2H), 3.07 -3.01 (m, 2H), 2.99 - 2.93 (m, 2H), 2.90 - 2.40 (m, 4H), 2.39 - 2.24 (m, 1H), 2.15 - 2.04 (m, 2H), 1.71 - 1.63 (m, 2H), 1.58 - 1.47 (m, 2H), 1.45 - 1.09 (m, 26H), 1.15 - 0.62 (m, 9H). 188
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Example 30: Synthesis of SM-116 O N N O P OH O
, oc 1 NH 12) H) 1 cOo (A1mc0p (d3 eq01)a e Nq (1)aB2 DaHC e ((qOM)A 42 ec00)q3) ° ° (CC15610 e hq h) 2 N N Boc DCM 25ox °Cane 12 h 3
N NHr K
2C
CO
P 3 M (3
Ea0 (
8 e
0q)
° e
C Kq)I
1 (0
2 h1 eq)
Step 1: tert-butyl 4-(1-pentylhexyl)-1,4-diazepane-1-carboxylate: To a solution of tert-butyl 1,4-diazepane-1-carboxylate (20 g, 99.86 mmol, 19.69 mL, 1.0 eq) and HOAc (17.99 g, 299.58 mmol, 17.15 mL, 3.0 eq) in DCM (300 mL) was added NaBH(OAc)
3 (31.75 g, 149.79 mmol, 1.5 eq). After addition, the mixture was stirred at 20 °C for 1 h, and then undecan-6-one (20.41 g, 119.83 mmol, 1.2 eq) was added to the mixture. The mixture was stirred at 40 °C for 60 h. The reaction mixture was diluted with water (100 mL) and extracted with DCM (150mL, 2x). The combined organic layers were washed with water, dried over Na
2SO
4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO
2, PE/EtOAc = 40/1 to 10/1) to give compound tert-butyl 4-(1-pentylhexyl)- 1,4-diazepane-1-carboxylate (3.7 g, 10.40 mmol, 15.4% yield, 99.7% purity) as a yellow oil.
1H NMR (400 MHz, CDCl
3) δ = 3.47 - 3.28 (m, 4H), 2.70 - 2.52 (m, 4H), 2.40 (t, J = 6.4 Hz, 1H), 1.76 - 1.65 (m, 2H), 1.46 (s, 9H), 1.36 - 1.20 (m, 16H), 0.88 (t, J = 6.8 Hz, 6H). 189
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Step 2: 1-(1-pentylhexyl)-1,4-diazepane: To a solution of tert-butyl 4-(1-pentylhexyl)-1,4-diazepane-1-carboxylate (3.7 g, 10.44 mmol, 1.0 eq) in DCM (10 mL) was added HCl/dioxane (2 M, 26.09 mL, 5.0 eq). The mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated under reduced pressure to give compound 1-(1-pentylhexyl)-1,4-diazepane (3 g, 10.31 mmol, 98.8% yield, HCl) as yellow gum. Step 3: nonyl (3-(4-(undecan-6-yl)-1,4-diazepan-1-yl)propyl) hydrogen phosphate: To a solution of 1-(1-pentylhexyl)-1,4-diazepane (1.5 g, 5.16 mmol, 1.0 eq, HCl) and 3- bromopropyl nonyl hydrogen phosphate (2.14 g, 6.19 mmol, 1.2 eq) in methoxycyclopentane (20 mL) were added K
2CO
3 (2.14 g, 15.47 mmol, 3.0 eq) and KI (85.59 mg, 515.62 μmol, 0.1 eq). The mixture was stirred at 80 °C for 12 h. The reaction mixture was directly concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 11.1% MeOH/DCM gradient @ 80 mL/min) and purified by prep-HPLC (column: Welch Xtimate C1100 * 30mm * 5um; mobile phase: [A/B water(FA) - MeOH]; gradient: MeOH (B) from 55% to 85% over 8 min) to give SM-116, (aka nonyl (3-(4-(undecan-6-yl)-1,4-diazepan-1-yl)propyl) hydrogen phosphate) (748 mg, 1.44 mmol, 49.9% yield, 99.99% purity) as a yellow gum. LCMS: [M+H]
+:519.4
1H NMR (400 MHz, CDCl
3) δ = 4.06 - 3.94 (m, 2H), 3.92 - 3.83 (m, 2H), 3.13 (s, 2H), 3.05 - 3.00 (m, 4H), 2.85 (s, 2H), 2.73 (t, J = 6.4 Hz, 2H), 2.41 (s, 1H), 2.10 - 1.92 (m, 4H), 1.68 - 1.56 (m, 2H), 1.38 - 1.20 (m, 28H), 0.98 - 0.81 (m, 9H). 190
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Example 31: Synthesis of SM-118 O N N O HO P
O
SHOtep 11: 2-brom 2)o 2e ( 31)0t 1 e B h0q%r)y- T HEClA °l
3 O ( 4Hy y y y y g ) ( eq) ( [30(0 °CZ e eqq 2)) ) h T-H ( eq) nF 1o4 hn-3 B-reny HOl P] O 23 hy 3drogen phosp KCh COPaM (3Et0e 8 e0q:) °C KI 3 (06 h1 eq) N TEA (8.54 g, 84.37 mmol, 11.74 mL, 1.2 eq) was slowly added to POCl
3 (10.78 g, 7 SM011.830 mmol, 6.55 mL, 1.0 eq) in dry THF (100 mL) at 0 °C under N
2. Then (Z)-non-3-en-1-ol (10 g, 70.30 mmol, 1.0 eq) in THF (100 mL) was added dropwise over 1 h and the resulting mixture was warmed to 20 °C stirred for 1 h. After all the alcohol reacted (checked by TLC), the mixture was cooled to 0 °C and a second portion of TEA (21.34 g, 210.91 mmol, 29.36 mL, 3.0 eq) was added, followed by 2-bromoethanol (8.79 g, 70.30 mmol, 4.98 mL, 1.0 eq) in THF (100 mL) dropwise. The reaction mixture was stirred at 20 °C for 14 h; then decomposed with aq. HCl (10%,150 mL) and heated at 40 °C for 2 h. THF was removed under vacuum and the aqueous residue was extracted with DCM (200 mL, 3x). The organic layer was dried over Na
2SO
4, filtered, reduced under vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 30% MeOH/DCM @ 100 mL/min) and prep- HPLC (column: Welch Xtimate C1100 * 30 mm * 5 um; mobile phase: A/B [water (FA) - MeOH]; 191
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) gradient: MeOH from 60% - 90% over 12 min) to give 2-bromoethyl [(Z)-non-3-enyl] hydrogen phosphate (4.3 g, 12.41 mmol, 16.3% yield, 95.0% purity) as a yellow oil.
1H NMR (400 MHz, CDCl
3) δ = 8.12 (s, 1H), 5.75 - 5.40 (m, 1H), 5.35 - 5.24 (m, 1H), 4.59 - 4.18 (m, 2H), 4.09 - 3.95 (m, 2H), 3.53 - 3.46 (m, 2H), 2.46 - 2.19 (m, 2H), 2.04 - 1.92 (m, 2H), 1.55 - 1.06 (m, 6H), 0.89 (t, J = 6.8 Hz, 3H). Step 2: SM-118, aka. (Z)-2-(4-(dihexylamino)piperidin-1-yl)ethyl non-3-en-1-yl hydrogen phosphate: To a solution of N,N-dihexylpiperidin-4-amine (1 g, 3.28 mmol, 1.0 eq, 2HCl) in methoxycyclopentane (15 mL) were added 2-bromoethyl [(Z)-non-3-enyl] hydrogen phosphate (1.19 g, 3.61 mmol, 1.1 eq) and K
2CO
3 (1.36 g, 9.84 mmol, 3.0 eq) and KI (54.44 mg, 327.93 μmol, 0.1 eq). The mixture was stirred at 80 °C for 32 h. The reaction mixture was filtered and concentrated under reduced pressure to give residue. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 30% MeOH/DCM @ 80 mL/min) and prep-HPLC (column: Welch Xtimate C1100 * 30 mm * 5 um; mobile phase: A/B [water(FA) - MeOH]; gradient: MeOH from 50% - 80% B over 8 min) to give SM-118 (aka (Z)-2-(4-(dihexylamino)piperidin-1-yl)ethyl non-3-en-1-yl hydrogen phosphate) (153.73 mg, 297.47 μmol, 18.2% yield, 99.99% purity) as a colorless gum LCMS: [M+H]
+:517.3
1H NMR (400 MHz, CDCl
3) δ = 5.62 - 5.25 (m, 2H), 4.06 - 3.95 (m, 2H), 3.87 - 3.56(m, 2H), 3.25 -3.17 (m, 2H), 3.01 - 2.50 (m, 7H), 2.49 - 2.19 (m, 4H), 2.03 - 1.96 (m, 2H), 1.89 - 1.64 (m, 4H), 1.54 - 1.49 (m, 4H), 1.46 - 1.15 (m, 18H), 0.98 - 0.65 (m, 9H). 192
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Example 32: Synthesis of SM-1 O HO 19 P O O N (
- ; -( -(( exy am no)me y )p per n- -y )e y nony
Step 11: H tOeAcr (3t0- ebqa)u ( D.CtM
3 eyq 2)0l °C 415-5[ h(dihexyl 2amino)meotxahyl]piper 3 Br O idine-1-ca
2 O P O O r
3Hy rogen p osp a e) oca(c) ( eq) oc °ne b (ao ( e.qx) ° eqy) (la eqt)e: SM119 To a solution of N-hexylhexan-1-amine (10.03 g, 54.10 mmol, 12.61 mL, 1.0 eq) in DCM (100 mL) were added HOAc (9.75 g, 162.31 mmol, 9.29 mL, 3.0 eq) and tert-butyl 4-formylpiperidine- 1-carboxylate (15 g, 70.33 mmol, 1.3 eq) at 0 °C. After addition, the mixture was stirred at this temperature for 0.5 h, and then NaBH(OAc)
3 (17.20 g, 81.15 mmol, 1.5 eq) was added in the mixture. The mixture was stirred at 20 °C for 15 h. The pH was adjusted to 8 with 2 M NaOH. The mixture was extracted with DCM (100 mL, 2x). The combined organic phase was washed with water (100 mL), dried with anhydrous Na
2SO
4, filtered and concentrated in vacuum to give residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 50% EtOAc/PE @ 100 mL/min) to give compound tert-butyl 193
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 4-[(dihexylamino)methyl]piperidine-1-carboxylate (17 g, 42.21 mmol, 78.0% yield, 95.0% purity) as a colorless oil.
1H NMR (400 MHz, CDCl
3) δ = 4.25 - 3.91 (m, 2H), 2.68 (t, J = 12.0 Hz, 2H), 2.33 (t, J = 7.2 Hz, 4H), 2.17 (d, J = 6.8 Hz, 2H), 1.73 (d, J = 12.8 Hz, 2H), 1.56-1.50 (m, 1H), 1.48 (s, 9H), 1.37 (d, J = 6.0 Hz, 4H), 1.34 - 1.15 (m, 12H), 1.12 - 0.90 (m, 2H), 0.89 (t, J = 6.4 Hz, 6H). Step 2: N-hexyl-N-(4-piperidylmethyl)hexan-1-amine: To a solution of tert-butyl 4-[(dihexylamino)methyl]piperidine-1-carboxylate (3 g, 7.84 mmol, 1.0 eq) in DCM (10 mL) was added HCl/dioxane (2 M, 20 mL, 5.1 eq).The mixture was stirred at 20 °C for 16 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was without purification to give compound N-hexyl-N-(4-piperidylmethyl)hexan-1-amine (2.7 g, 6.84 mmol, 87.2% yield, 90.0% purity, as HCl salt) as a colorless gum.
1H NMR (400 MHz, CDCl
3) δ = 10.88 (s, 1H), 9.71 - 9.10 (m, 2H), 6.18 (s, 1H), 3.57 (d, J = 10.8 Hz, 2H), 3.42 - 2.68 (m, 8H), 2.51 - 2.08 (m, 3H), 2.02 - 1.56 (m, 6H), 1.32 (s, 12H), 0.88 (s, 6H). Step 3: 2-(4-((dihexylamino)methyl)piperidin-1-yl)ethyl nonyl hydrogen phosphate: To a solution of N-hexyl-N-(4-piperidylmethyl)hexan-1-amine (1.3 g, 3.66 mmol, 1.0 eq, 2HCl) in methoxycyclopentane (20 mL) were added 2-bromoethyl nonyl hydrogen phosphate (1.33 g, 4.02 mmol, 1.1 eq) and K
2CO
3 (1.52 g, 10.97 mmol, 3.0 eq) and KI (60.72 mg, 365.76 μmol, 0.1 eq). The mixture was stirred at 80 °C for 72 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 30% MeOH/DCM @ 80 mL/min) and purified by prep-HPLC (column: Welch Xltimate C4100 * 30 * 10 um; mobile phase: A/B [water (FA) - MeOH]; gradient: MeOH from 57% - 87% over 8 min) to give SM-119, aka. 2-(4-((dihexylamino)methyl)piperidin-1-yl)ethyl nonyl hydrogen phosphate (228.1 mg, 428.09 μmol, 9.1% yield, 99.99% purity) as a yellow gum. LCMS: [M+H]
+:533.5 194
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application)
1H NMR (400 MHz, CDCl
3) δ = 4.17 - 4.02 (m, 2H), 3.92 - 3.81 (m, 2H), 3.48 (d, J = 9.2 Hz, 2H), 3.05 (s, 2H), 2.57 (s, 2H), 2.38 - 2.30 (m, 4H), 2.29 (s, 2H), 1.95 (d, J = 9.8 Hz, 2H), 1.59 - 1.49 (m, 5H), 1.48 - 1.12 (m, 28H), 0.99 - 0.75 (m, 9H). Example 33: Synthesis of SM-121 N N O P OH
- ; - y y ) ( eq)- Br °3 ( OH- - y p p - -y y y g p p eq) ( eq) N ( N eH
Step 11: 2-brom 2)o 5 (e130t) eh 1q0)y% TEl HAC 2l (34-00b e °Cqu) 2 TtH hyFl 1o4 hr ctyl hyd Oro Oge 3n phospha K2 tCe CO:P3M (3E08 e0q) °C KI 1 (02 h1q e)q) N N SM12 O1P O TEA (6.52 g, 64.40 mmol, 8.96 mL, 1.2 eq) was slowly added to POCl
3 (8.23 g, 53.67 mmol, 5.00 mL, 1.0 eq) in dry THF (100 mL) at 0 °C under N
2. Then 2-butyloctan-1-ol (10 g, 53.67 mmol, 1.0 eq) in THF (100 mL) was added drop wise over 1 h and the resulting mixture was warmed to 20 °C was stirred for 1 h. When all the alcohol had reacted (checked by TLC), the mixture was cooled to 0 °C and a second portion of TEA (16.29 g, 161.00 mmol, 22.41 mL, 3.0 eq) was added, followed by 2-bromoethanol (6.71 g, 53.67 mmol, 3.80 mL, 1.0 eq) in THF (100 mL) was added dropwise. The reaction mixture was stirred at 20 °C for 14 h, decomposed with HCl 10% (150 mL) and heated to 40 °C for 2 h. THF was removed under vacuum and the aqueous residue was extracted with DCM (200 mL * 3). The organic layer was dried over Na
2SO
4, filtered, reduced under vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 220 g SepaFlash® Silica Flash Column, Eluent of 0~5.3% MeOH/DCM gradient @ 80 mL/min) to give 195
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) compound 2-bromoethyl 2-butyloctyl hydrogen phosphate (11.9 g, 31.34 mmol, 58.5% yield, 98.3% purity) as a yellow oil.
1H NMR (400 MHz, CDCl
3) δ = 7.05 - 6.91 (m, 1H), 4.38 - 4.16 (m, 2H), 4.04 - 3.84 (m, 2H), 3.66 - 3.43 (m, 2H), 1.64 (s, 1H), 1.28 (s, 16H), 0.98 - 0.82 (m, 6H). Step 2: 2-butyloctyl (2-(4-(dihexylamino)piperidin-1-yl)ethyl) hydrogen phosphate: To a solution of N,N-dihexylpiperidin-4-amine (1.4 g, 4.59 mmol, 1.0 eq, HCl) and 2-bromoethyl 2-butyloctyl hydrogen phosphate (2.06 g, 5.51 mmol, 1.2 eq) in methoxycyclopentane (20 mL) were added K
2CO
3 (1.90 g, 13.77 mmol, 3.0 eq) and KI (76.21 mg, 459.11 μmol, 0.1 eq). The mixture was stirred at 80 °C for 12 h. The reaction mixture was directly concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0~11.1% MeOH/DCM gradient @ 80 mL/min) and purified by prep-HPLC (column: Welch Xtimate C1100 * 30mm* 5um; mobile phase: [A/B water(FA) - MeOH]; gradient: 55% - 85% B over 8 min) to give SM-121 (aka 2-butyloctyl (2-(4- (dihexylamino)piperidin-1-yl)ethyl) hydrogen phosphate) (640 mg, 1.14 mmol, 35.6% yield, 99.98% purity) as a yellow gum. LCMS: [M+H]
+:561.5
1H NMR (400 MHz, CDCl
3) δ = 4.02 (s, 2H), 3.74 (t, J = 5.2 Hz, 2H), 3.23 (d, J = 3.2 Hz, 2H), 2.89 - 2.48 (m, 7H), 2.37 - 2.12 (m, 2H), 1.86 (s, 4H), 1.64 - 1.43 (m, 5H), 1.27 (d, J = 8.4 Hz, 28H), 0.89 (s, 12H). Example 34: Synthesis of SM-122 N N O P OH
196
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) (SM-122; 2-( 1 14).- 1 ((1.d0 eiq B)p,r POe 2Cnl OH (t1.y0 elq)a, TmEA (1i.2n eqo))piperid H2N N Boc Oin-1-yl)ethyl nonyl hydrogen phosphate) HO 2) 2 ( 31 T)0H 1F e0q%) 0-2 T H5ECA °l
3C ( 4320 h eq) THF 14 h Br HOO P O
O NaBH(OAc)
3 (25 e) HOA 5c (20 e0) °C 2 hN N Boc DC 3 HMCl/ 2d0ioxCan 1e2 hN NH Br K
2C OO O
3 P ( O3 O 3H0 (1 e1) e K)I (01 e) N N O OP O OH TEA (8.42 g, 83.19 mmol, 11.58 mL, 1.2 eq) was slowly added to POCl
3 (10.63 g, 69.32 mmol, 6.46 mL, 1.0 eq) in dry THF (100 mL) at 0 °C under N
2. Then nonan-1-ol (10 g, 69.32 mmol, 1.0 eq) in THF (100 mL) was added dropwise over 1 h and the resulting mixture was warmed to 20 °C was stirred for 1 h. When all the alcohol had reacted (checked by TLC), the mixture was cooled to 0 °C and a second portion of TEA (21.04 g, 207.97 mmol, 28.95 mL, 3.0 eq) was added, followed by 2-bromoethanol (8.66 g, 69.32 mmol, 4.91 mL, 1.0 eq) in THF (100 mL) was added dropwise. The reaction mixture was stirred at 20 °C for 14 h. Decomposed with HCl 10% (150 mL) and heated at 40 °C for 2 h. THF was removed under vacuum and the aqueous residue was extracted with DCM (200 mL * 3). The organic layer was dried over Na
2SO
4, filtered, reduced under vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 30% MeOH/DCM @ 100 mL/min) and prep- HPLC (column: YMC Triart C18 70 * 250 mm * 7 um; mobile phase: [water (FA) - ACN]; gradient: 35% - 65% B over 20 min) to give compound 2-bromoethyl nonyl hydrogen phosphate (4.1 g, 11.76 mmol, 27.8% yield, 95.0% purity) as a yellow gum.
1H NMR (400 MHz, CDCl
3) δ = 4.32 - 4.26 (m, 2H), 4.07-3.91 (m, 2H), 3.67 - 3.36 (m, 2H), 1.70 -1.64(m, 2H), 1.43 - 1.20 (m, 12H), 0.89 (t, J = 6.8 Hz, 3H). Step 2: tert-butyl 4-(dipentylamino)piperidine-1-carboxylate: To a solution of tert-butyl 4-aminopiperidine-1-carboxylate (20 g, 99.86 mmol, 1.0 eq) in DCM (300 mL) were added HOAc (11.99 g, 199.72 mmol, 11.43 mL, 2.0 eq) and NaBH(OAc)
3 (52.91 g, 249.65 mmol, 2.5 eq) at 20 °C. And then pentanal (18.92 g, 219.70 mmol, 23.36 mL, 2.2 eq) 197
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) was added at 0°C. The mixture was stirred at 20 °C for 16 h. The pH was adjusted to 8 with 2 M NaOH. The mixture was extracted with DCM (2 * 100 mL). The combined organic phase was washed with water (100 mL), dried with anhydrous Na
2SO
4, filtered and concentrated in vacuum to give residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 30% MeOH/DCM @ 100 mL/min) to give compound tert-butyl 4-(dipentylamino)piperidine-1-carboxylate (32 g, 92.09 mmol, 92.2% yield, 98.0% purity) as a yellow oil.
1H NMR (400 MHz, CDCl
3) δ = 4.14 - 4.02 (m, 2H), 2.92 - 2.47 (m, 3H), 2.46 - 2.21 (m, 4H), 1.68 -1.56 (m, 2H), 1.52 - 1.17 (m, 23H), 0.89 (t, J = 7.2 Hz, 6H). Step 3: N,N-dipentylpiperidin-4-amine: To a solution of tert-butyl 4-(dipentylamino)piperidine-1-carboxylate (5 g, 14.68 mmol, 1.0 eq) in DCM (20 mL) was added HCl/dioxane (2 M, 25 mL, 3.4 eq).The mixture was stirred at 20 °C for 16 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was without purification to give compound N,N-dipentylpiperidin-4-amine (3.5 g, 9.49 mmol, 64.7% yield, 85.0% purity, 2HCl) as a white gum.
1H NMR (400 MHz, CDCl
3) δ =10.78 (s, 1H), 9.79 - 9.24 (m, 2H), 3.98 - 3.91 (m, 1H), 3.86 - 3.54 (m, 3H), 3.53 - 3.29 (m, 5H), 3.28 - 3.13 (m, 2H), 3.11 - 2.82 (m, 2H), 2.14 - 1.64 (m, 4H), 1.37 (s, 8H), 0.92 (t, J = 6.4 Hz, 6H). Step 4: 2-(4-(dipentylamino)piperidin-1-yl)ethyl nonyl hydrogen phosphate: To a solution of N,N-dipentylpiperidin-4-amine (1.5 g, 4.79 mmol, 1.0 eq, 2HCl) in methoxycyclopentane (15 mL) were added 2-bromoethyl nonyl hydrogen phosphate (1.74 g, 5.27 mmol, 1.1 eq) and K
2CO
3 (1.98 g, 14.36 mmol, 3.0 eq) and KI (79.47 mg, 478.70 μmol, 0.1 eq) .The mixture was stirred at 80 °C for 16 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 30% EtOAc/PE @ 100 mL/min) and prep-HPLC (column: Welch Xltimate C4100 * 30 * 10 um; mobile phase: A/B [water (FA) - MeOH]; gradient: 55% - 85% of MeOH over 8 min) to give SM-122 (aka 2- 198
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) (4-(dipentylamino)piperidin-1-yl)ethyl nonyl hydrogen phosphate) (483.47 mg, 982.32 μmol, 20.5% yield, 99.70% purity) as a yellow gum. LCMS: [M+H]
+:491.2
1H NMR (400 MHz, CDCl
3) δ =4.04 - 3.91 (m, 2H), 3.85 - 3.61 (m, 2H), 3.25 - 3.14 (m, 2H), 2.99 - 2.42 (m, 7H), 2.38 - 2.21 (m, 2H), 1.91 - 1.84 (m, 4H), 1.77 - 1.42 (m, 6H), 1.41 - 1.11 (m, 20H), 1.09 - 0.64 (m, 9H). Example 35: Synthesis of SM-123 N N O O P OH O
(SM-123; nonyl (2-(4-(tridecan-7-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate) Boc N 1 NH 12 H))O 1 cAo (1cm (03d e0q 1) eaq N) (1a DB5CHa eM( (O)A 24c0 e0)
3q ° °C) (C105356 eq h h) 2
NBoc DCM 2d0o °xCan 1e2 h 3 N NHr K2C COP3M (3Ea0 ( 8 e0q) ° eC Kq)I 2 (405 h eq)
199
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Step 1: tert-butyl 4-(1-hexylheptyl)-1,4-diazepane-1-carboxylate: To a solution of tert-butyl 1,4-diazepane-1-carboxylate (7.0 g, 34.95 mmol, 6.89 mL, 1.0 eq) in DCM (70 mL) were added NaBH(OAc)
3 (11.11 g, 52.43 mmol, 1.5 eq) and HOAc (6.30 g, 104.85 mmol, 6.00 mL, 3.0 eq). The mixture was stirred at 25 °C for 0.5 h. Then tridecan-7-one (10.40 g, 52.43 mmol, 1.5 eq) was added to the reaction mixture at 25 °C. The resulting mixture was stirred at 40 °C for 35.5 h. The reaction mixture was basified by addition NaOH solution (2 N, 30 mL) at 0 °C, and then diluted with water (30 mL) and extracted with EtOAc (60 mL * 3). The combined organic layers were washed with brine (200 mL), dried over Na
2SO
4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 220 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 30% EtOAc/PE gradient @ 100 mL/min) to get compound tert-butyl 4-(1-hexylheptyl)-1,4-diazepane-1- carboxylate (3.2 g, 8.33 mmol, 19.7% yield, 99.6% purity) as a yellow oil.
1H NMR (400 MHz, CDCl
3) δ = 3.50 - 3.30 (m, 4H), 2.70 - 2.53 (m, 4H), 2.40 (t, J = 6.4 Hz, 1H), 1.76 - 1.65 (m, 2H), 1.46 (s, 9H), 1.44 - 1.42 (m, 2H), 1.30 - 1.26 (m, 18H), 0.88 - 0.86 (m, 6H). Step 2: benzyl 5-aminopentanoate: To a solution of tert-butyl 4-(1-hexylheptyl)-1,4-diazepane-1-carboxylate (3.2 g, 8.36 mmol, 1.0 eq) in DCM (10 mL) was added HCl/dioxane (2 M, 12.55 mL, 3.0 eq). The mixture was stirred at 25 °C for 12 h. The reaction mixture concentrated under reduced pressure to give compound 1-(1- hexylheptyl)-1,4-diazepane (1.0 g, 3.53 mmol, 42.2% yield, 99.6% purity) as a yellow oil. Step 3: nonyl (2-(4-(tridecan-7-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate To a solution of 1-(1-hexylheptyl)-1,4-diazepane (1 g, 3.14 mmol, 1.0 eq, HCl) and 2-bromoethyl nonyl hydrogen phosphate (1.25 g, 3.76 mmol, 1.2 eq) in methoxycyclopentane (12 mL) were added K
2CO
3 (1.30 g, 9.41 mmol, 3.0 eq) and KI (260.21 mg, 1.57 mmol, 0.5 eq). The mixture was stirred at 80 °C for 24 h. The reaction mixture was filtered and washed with EtOAc (50 mL). The filtrate was collected and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 20% MeOH/DCM gradient @ 80 mL/min) and prep-HPLC (column: Welch Xltimate C4100 * 30 * 10 um; mobile phase: A/B [water (FA) - MeOH]; gradient: 65% - 95% of MeOH 200
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) over 8 min) to get SM-123 (aka nonyl (2-(4-(tridecan-7-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate) (650 mg, 1.22 mmol, 38.9% yield, 99.99% purity) as a yellow gum. LCMS: [M+H]
+: 533.4
1H NMR (400 MHz, CDCl
3) δ = 4.14 - 4.05 (m, 2H), 3.82 (q, J = 6.4 Hz, 2H), 3.28 - 2.93 (m, 6H), 2.93 - 2.67 (m, 4H), 2.47 (s, 1H), 2.01 (s, 2H), 1.60 - 1.51 (m, 2H), 1.45 - 1.35 (m, 2H), 1.31 - 1.15 (m, 30H), 0.90 - 0.71 (m, 9H). Example 36: Synthesis of SM-124 N N O P OH O
(SM-124; decyl (2-(4-(dihexylamino)piperidin-1-yl 1a( DcH )ethyl) hydrogen phos )C
23NM ( 25 N Boc oc o O e °qC) 12 hc ( eq) 3 °xane 4 Brph 2 Oat O C P Oe P3 OH) M (E ( 8 e0q.) °C eq 1) (2 h eq)
H Step 1: tert-butyl 4-(dihexylamino)piperidine-1-carboxylate: To a solution of tert-butyl 4-aminopiperidine-1-carboxylate (10 g, 49.93 mmol, 1.0 eq) and HOAc (9.00 g, 149.79 mmol, 8.58 mL, 3.0 eq) in DCM (100 mL) was added NaBH(OAc)
3 (31.75 g, 149.79 mmol, 3.0 eq). After addition, the mixture was stirred at 25 °C for 1 h, and then hexanal (12.50 g, 124.83 mmol, 14.99 mL, 2.5 eq) was added in the mixture. The mixture was stirred at 25 201
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) °C for 11 h. The pH was adjusted to 8 with NaOH. The mixture was extracted with EtOAc (3 * 150 mL). The combined organic phase was washed with water (100 mL), dried with anhydrous Na
2SO
4, filtered and concentrated in vacuum to give residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 11.1% MeOH/DCM gradient @ 80 mL/min) to give compound tert-butyl 4-(dihexylamino)piperidine-1- carboxylate (7.3 g, 19.80 mmol, 39.7% yield) as a yellow oil.
1H NMR (400 MHz, CDCl
3) δ = 4.16 - 4.11 (m, 2H), 2.68 - 2.53 (m, 3H), 2.49 - 2.34 (m, 4H), 1.69 (d, J = 12.4 Hz, 2H), 1.46 (s, 9H), 1.43 - 1.34 (m, 6H), 1.33 - 1.22 (m, 12H), 0.89 (t, J = 6.8 Hz, 6H). Step 2: N,N-dihexylpiperidin-4-amine: To a solution of tert-butyl 4-(dihexylamino)piperidine-1-carboxylate (7.3 g, 19.80 mmol, 1.0 eq) in DCM (20 mL) was added HCl/dioxane (2 M, 29.71 mL, 3.0 eq). The mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated under reduced pressure to give compound N,N- dihexylpiperidin-4-amine (5 g, 16.40 mmol, 82.8% yield, HCl) as a yellow gum and used without purification. Step 3: decyl (2-(4-(dihexylamino)piperidin-1-yl)ethyl) hydrogen phosphate: The preparation of 2-bromoethyl decyl hydrogen phosphate was similar to Intermediate-1, decan- 1-ol was used instead of nonan-1-ol. To a solution of N,N-dihexylpiperidin-4-amine (1 g, 3.28 mmol, 1.0 eq, HCl) and 2-bromoethyl decyl hydrogen phosphate (1.25 g, 3.61 mmol, 1.1 eq) in methoxycyclopentane (20 mL) were added K
2CO
3 (1.36 g, 9.84 mmol, 3.0 eq) and KI (54.44 mg, 327.93 μmol, 0.1 eq). The mixture was stirred at 80 °C for 12 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 17.6% MeOH/DCM gradient @ 80 mL/min) and purified by prep-HPLC (column: Welch Xtimate C1100 * 30mm * 5um; mobile phase: A/B [water(FA) - MeOH]; gradient: 50% - 80% of MeOH over 8 min) to give SM-124 (aka decyl (2- (4-(dihexylamino)piperidin-1-yl)ethyl) hydrogen phosphate (925 mg, 1.74 mmol, 46.3% yield, 99.99% purity) as a white gum. 202
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) LCMS: [M+H]
+:533.5 1H NMR (400 MHz, CD
3OD-d
4) δ = 4.01 (d, J = 6.4 Hz, 2H), 3.85 (d, J = 6.4 Hz, 2H), 3.24 (d, J = 11.2 Hz, 2H), 2.96 (s, 1H), 2.89 - 2.68 (m, 6H), 2.40 (s, 2H), 1.92 (d, J = 11.6 Hz, 2H), 1.80 - 1.68 (m, 2H), 1.66 - 1.52 (m, 6H), 1.41 - 1.25 (m, 26H), 0.99 - 0.80 (m, 9H). Example 37: Synthesis of SM-126 N N O O P O OH
- ; ecy)- Br- OHeca - -y - , - a epa - -y e y y oge p osp a e 1.) (. ( e 3q) e) 1q0)-% H
3C (l (.40 e e °qCq)) 2 h (. eq)r 3
Sotcep 1: 2)) c-o (cmb ( er) eoa) (ama e( (o)ec e)
3 Br OO P O OH ( e) t) (h ey)l decyl hydroocgen poxahne 23 osphate: ( e) ( e) TEA (7.67 g, 75.81 mmol, 10.55 mL, 1.2 eq) was slowly added to POCl
3 (9.69 g, 63.18 mmol, 5.89 mL, 1.0 eq) in dry THF (80 mL) at 0 °C under N
2. Then decan-1-ol (10 g, 63.18 mmol, 12.06 mL, 1.0 eq) in THF (60 mL) was added drop wise over 1 h at 0 °C. The resulting mixture was warmed to 25 °C and stirred for 1 h. When all the alcohol had reacted (checked by TLC), the mixture was cooled to 0 °C and a second portion of TEA (19.18 g, 189.54 mmol, 26.38 mL, 3.0 eq) was added, followed by 2-bromoethanol (7.90 g, 63.18 mmol, 4.49 mL, 1.0 eq) in THF (60 203
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) mL) was added dropwise. The reaction mixture was stirred at 25 °C for 12 h. Decomposed with HCl 10% (100 mL) and heated at 40 °C for 2 h. THF was removed under vacuum and the aqueous residue was extracted with EtOAc (100 mL*3). The organic layer was dried over Na
2SO
4, filtered, reduced under vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 330 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 15% MeOH/DCM gradient @ 100 mL/min) and flash silica gel chromatography (ISCO®; 220 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 30% MeOH/DCM @ 100 mL/min) to give 2-bromoethyl decyl hydrogen phosphate (20 g, 78.21 mmol, 63.0% yield, 85.0% purity) as a yellow gum.
1H NMR (400 MHz, CDCl
3) δ = 4.28 - 4.24 (m, 2H), 4.19 - 3.94 (m, 2H), 3.54 (t, J = 6.4 Hz, 2H), 1.84 - 1.57 (m, 2H), 1.41 - 1.18 (m, 14H), 0.89 (t, J = 6.4 Hz, 3H). Step 2: tert-butyl 4-(1-hexylheptyl)-1,4-diazepane-1-carboxylate: To a solution of tert-butyl 1,4-diazepane-1-carboxylate (8.08 g, 40.33 mmol, 7.95 mL, 1.0 eq) in DCM (150 mL) were added HOAc (7.27 g, 121.00 mmol, 6.93 mL, 3.0 eq) and NaBH(OAc)
3 (12.82 g, 60.50 mmol, 1.5 eq) and tridecan-7-one (8 g, 40.33 mmol, 1.0 eq). The mixture was stirred at 20 °C for 24 h. The pH was adjusted to 8 with 2 M NaOH. The mixture was extracted with DCM (2 * 100 mL). The combined organic phase was washed with water (100 mL), dried with anhydrous Na
2SO
4, filtered and concentrated in vacuum to give residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 50% EtOAc/PE @ 100 mL/min) to give compound tert-butyl 4-(1-hexylheptyl)-1,4- diazepane-1-carboxylate (1.57 g, 3.90 mmol, 9.7% yield, 95.0% purity) as a colorless gum.
1H NMR (400 MHz, CDCl
3) δ = 3.62 - 3.19 (m, 4H), 2.84 - 2.48 (m, 4H), 2.45 - 2.40 (m, 1H), 1.70 -1.64 (m, 2H), 1.46 (s, 9H), 1.35 - 1.24 (m, 20H), 0.89 (t, J = 6.8 Hz, 6H). Step 3: 1-(1-hexylheptyl)-1,4-diazepane: To a solution of tert-butyl 4-(1-hexylheptyl)-1,4-diazepane-1-carboxylate (1.57 g, 4.10 mmol, 1.0 eq) in DCM (5 mL) was added HCl/dioxane (2 M, 10 mL, 4.9 eq). The mixture was stirred at 20 °C for 16 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was without purification to give compound 1-(1-hexylheptyl)-1,4-diazepane (1.46 g, 3.90 mmol, 95.1% yield, 95.0% purity, as HCl salt) as a colorless gum. 204
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application)
1H NMR (400 MHz, CDCl
3) δ = 11.13 (s, 1H), 10.30 (s, 1H), 9.78 (s, 1H), 4.45 - 3.85 (m, 3H), 3.84 - 3.27 (m, 5H), 3.19 - 3.04 (m, 1H), 2.98 - 2.42 (m, 2H), 2.38 - 2.21 (m, 1H), 2.11 - 2.01 (m, 2H), 1.69 - 1.59 (m, 2H), 1.54 - 1.15 (m, 16H), 0.89 (t, J = 6.4 Hz, 6H). Step 4: decyl (2-(4-(tridecan-7-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate: To a solution of 1-(1-hexylheptyl)-1,4-diazepane (1.46 g, 4.11 mmol, 1.0 eq, 2HCl) in methoxycyclopentane (10 mL) were added 2-bromoethyl decyl hydrogen phosphate (1.56 g, 4.52 mmol, 1.1 eq) and K
2CO
3 (1.70 g, 12.32 mmol, 3.0 eq) and KI (68.19 mg, 410.77 μmol, 0.1 eq). The mixture was stirred at 80 °C for 16 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0 ~ 30% EtOAC/PE @ 100 mL/min) and prep-HPLC (column: Welch Xtimate C1100 * 30 mm * 5 um; mobile phase: A/B [water (FA) - MeOH]; gradient: 60% - 90% B over 8 min) to give SM-126, aka decyl (2-(4- (tridecan-7-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate (565.21 mg, 1.03 mmol, 25.2% yield, 99.99% purity) as a yellow gum. LCMS: [M+H]
+:547.5
1H NMR (400 MHz, CD
3OD-d
4) δ = 4.15 - 3.94 (m, 2H), 3.89 - 3.74 (m, 2H), 3.31 - 2.90 (m, 10H), 2.84 - 2.69 (m, 1H), 2.18 - 1.82 (m, 2H), 1.71 - 1.54 (m, 4H), 1.49 - 1.15 (m, 32H), 1.09 - 0.70 (m, 9H). All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. One skilled in the art would readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the disclosure. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the disclosure, are defined by the scope of the claims. 205
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) In addition, where features or aspects of the disclosure are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group. The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," and "including," are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure. Embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the techniques herein. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure. Thus, it should be understood that although the present disclosure provides preferred embodiments, optional features, 206
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the description and the appended claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the techniques disclosed herein without departing from the scope and spirit of the disclosure. Thus, such additional embodiments are within the scope of the present disclosure and the following claims. The present disclosure teaches one skilled in the art to test various combinations and/or substitutions of chemical modifications described herein toward generating conjugates possessing improved contrast, diagnostic and/or imaging activity. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying conjugates possessing improved contrast, diagnostic and/or imaging activity. The disclosure contemplates that skilled artisans may employ such variations as appropriate, and the disclosure may be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims. 207
A is a bond, C -C alkyl, C -C alkenyl, C -C 2 alkynyl, or C3-C8 cycloalkyl, each of which is optionally substituted; X is N or CH; R1 is C5-C22 alkyl, C5-C22 alkenyl, C5-C22 alkynyl, C3-C22 cycloalkyl, or C3-C22 cycloalkyl, or C(O)C4-C21 alkyl, each of which is optionally substituted; R2 is C2-C22 alkyl, C2-C22 alkenyl, C2-C22 alkynyl, or C3-C22 cycloalkyl, each of which is optionally substituted; and each of m and n is independently 0, 1, 2, or 3. In an aspect, the disclosure provides a c R3 OoHmpound of Formula II: R3 N R5 A1 R6 O 1 1 22 2 22 O P O R4 ,
or a salt or isomer thereof, where A is a C -C alkyl, C -C alkenyl, or C2-C22 alkynyl, each of which includes at least one substitution; or C3-C8 cycloalkyl or heterocycloalkyl, each of which is optionally substituted; R3 is C7-C22 alkyl, C7-C22 alkenyl, C7-C22 alkynyl, or C4-C22 cycloalkyl, each of which is optionally substituted;
R4 is C2-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, or C3-C22 cycloalkyl, each of which is optionally substituted; and each of R5 and R6 is independently a bond, C1-C7 alkyl, C2-C7 alkenyl, or C2-C7 alkynyl, each of which is optionally substituted. In embodiments of Formula I, or salt or isomer thereof, R1 and R2 are the same. In embodiments of Formula I, or salt or isomer thereof, R1 is selected from the group consisting of C5-C12 alkyl, C5-C12 alkenyl, and C5-C12 alkynyl, each of which is optionally substituted and R2 is selected from the group consisting of C4-C12 alkyl, C4-C12 alkenyl, and C4- C12 alkynyl, each of which is optionally substituted. In embodiments of Formula I, or salt or isomer thereof, m is 2 and A is selected from the group consisting of C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A is C3-C5 cycloalkyl. In embodiments of Formula I, or salt or isomer thereof, R1 is selected from the group consisting of branched or unbranched C5-C9 alkyl, C5-C9 alkenyl, and C5-C9 alkynyl, each of which is optionally substituted, and R2 is selected from the group consisting of branched or unbranched C4-C9 alkyl, C4-C9 alkenyl, and C4-C9 alkynyl, each of which is optionally substituted. In embodiments of Formula I, or salt or isomer thereof, m is 2 and A is selected from the group consisting of C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A is C3-C5 cycloalkyl. In embodiments of Formula I, or salt or isomer thereof, R1 is selected from the group consisting of branched or unbranched C6-C9 alkyl, C6-C9 alkenyl, and C6-C9 alkynyl, each of which is optionally substituted, and R2 is selected from the group consisting of branched or unbranched C6-C9 alkyl, C6-C9 alkenyl, and C6-C9 alkynyl, each of which is optionally substituted. In embodiments of Formula I, or salt or isomer thereof, m is 2 and A is selected from the group consisting of C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A is C3-C5 cycloalkyl.
In embodiments of Formula I, or salt or isomer thereof, R1 and R2 are independently C6-C9 alkyl, which is optionally substituted, m is 2, and A is selected from the group consisting of C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A is C3-C5 cycloalkyl. In embodiments of Formula I, or salt or isomer thereof, R1 is optionally substituted C6-C9 alkyl, R2 is optionally substituted C9 alkyl, m is 2, and A is selected from the group consisting of C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A is C3-C5 cycloalkyl. In embodiments of Formula I, or salt or isomer thereof, R1 and R2 are independently an alkyl selected from the group consisting of heptane, octane, nonane, decane, undecane, and dodecane, each of which is optionally substituted. In embodiments of Formula I, or salt or isomer thereof, one or more of R1 and R2 are independently an alkenyl selected from the group consisting of hept-1-ene, hept-2-ene, hept-3-ene, oct-1-ene, oct-2-ene, oct-3-ene, oct-4-ene, non-1-ene, non-2-ene, non-3-ene, non-4-ene, non-5- ene, dec-1-ene, dec-2-ene, dec-3-ene, dec-4-ene, dec-5-ene, dec-6-ene, undec-1-ene, undec-2-ene, undec-3-ene, undec-4-ene, undec-5-ene, undec-6-ene, undec-7-ene, dodec-1-ene, dodec-2-ene, dodec-3-ene, dodec-4-ene, dodec-5-ene, dodec-6-ene, dodec-8-ene, and an alkenyl group comprising two or more double bonds, each of which is optionally substituted. In embodiments of Formula I, or salt or isomer thereof, one or more of R1 and R2 are independently an alkynyl selected from the group consisting of hept-1-yne, hept-2-yne, hept-3- yne, oct-1-yne, oct-2-yne, oct-3-yne, oct-4-yne, non-1-yne, non-2-yne, non-3-yne, non-4-yne, non- 5-yne, dec-1-yne, dec-2-yne, dec-3-yne, dec-4-yne, dec-5-yne, dec-6-yne, undec-1-yne, undec-2- yne, undec-3-yne, undec-4-yne, undec-5-yne, undec-6-yne, undec-7-yne, dodec-1-yne, dodec-2- yne, dodec-3-yne, dodec-4-yne, dodec-5-yne, dodec-6-yne, dodec-8-yne, and an alkynyl group comprising two or more triple bonds, each of which is optionally substituted. In embodiments of Formula II, or salt or isomer thereof, R3 and R4 are the same, optionally wherein R5 and R6 are the same.
In embodiments of Formula II, or salt or isomer thereof, R3 is selected from the group consisting of C7-C12 alkyl, C7-C12 alkenyl, and C7-C12 alkynyl, each of which is optionally substituted, R4 is selected from the group consisting of C4-C12 alkyl, C4-C12 alkenyl, and C4-C12 alkynyl, each of which is optionally substituted, wherein R5 and R6 are independently selected from the group consisting of a bond, C2-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, each of which is optionally substituted, optionally wherein R5 and R6 are both methyl group, or either R5 or R6 is a methyl group and the other is a bond. In embodiments of Formula II, or salt or isomer thereof, A1 is selected from the group consisting of optionally branched C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A1 is C3 cycloalkyl. In embodiments of Formula II, or salt or isomer thereof, A1 is selected from the group consisting of optionally branched C3-C4 alkyl, C3-C4 alkenyl, and C3-C4 alkynyl, each of which includes at least one substitution, or A1 is C3 cycloalkyl. In embodiments of Formula II, or salt or isomer thereof, R3 is selected from the group consisting of branched or unbranched C7-C9 alkyl, C7-C9 alkenyl, and C7-C9 alkynyl, each of which is optionally substituted, and R4 is selected from the group consisting of branched or unbranched C4-C9 alkyl, C4-C9 alkenyl, and C4-C9 alkynyl, each of which is optionally substituted. In embodiments of Formula II, or salt or isomer thereof, A1 is optionally branched C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl or A1 is optionally branched C3-C4 alkyl, C3-C4 alkenyl, and C3-C4 alkynyl. In embodiments of Formula II, or salt or isomer thereof, R3 is selected from the group consisting of branched or unbranched C7-C9 alkyl, C7-C9 alkenyl, and C7-C9 alkynyl, each of which is optionally substituted, and R4 is selected from the group consisting of branched or unbranched C6-C9 alkyl, C6-C9 alkenyl, and C6-C9 alkynyl, each of which is optionally substituted. In embodiments of Formula II, or salt or isomer thereof, A1 is selected from the group consisting of optionally branched C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which
includes at least one substitution, or A1 is selected from the group consisting of optionally branched C3-C4 alkyl, C3-C4 alkenyl, and C3-C4 alkynyl, each of which includes at least one substitution. In embodiments of Formula II, or salt or isomer thereof, R3 is optionally substituted C7-C9 alkyl and R4 is optionally substituted C6-C9 alkyl, wherein A1 is selected from the group consisting of optionally branched C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A1 is selected from the group consisting of optionally branched C3-C4 alkyl, C3-C4 alkenyl, and C3-C4 alkynyl, each of which includes at least one substitution. In embodiments of Formula II, or salt or isomer thereof, R3 and R4 are independently an alkyl selected from the group consisting of heptane, octane, nonane, decane, undecane, and dodecane, each of which is optionally substituted. In embodiments of Formula II, or salt or isomer thereof, one or more of R3 and R4 are independently an alkenyl selected from the group consisting of hept-1-ene, hept-2-ene, hept-3-ene, oct-1-ene, oct-2-ene, oct-3-ene, oct-4-ene, non-1-ene, non-2-ene, non-3-ene, non-4-ene, non-5- ene, dec-1-ene, dec-2-ene, dec-3-ene, dec-4-ene, dec-5-ene, dec-6-ene, undec-1-ene, undec-2-ene, undec-3-ene, undec-4-ene, undec-5-ene, undec-6-ene, undec-7-ene, dodec-1-ene, dodec-2-ene, dodec-3-ene, dodec-4-ene, dodec-5-ene, dodec-6-ene, dodec-8-ene, and an alkenyl group comprising two or more double bonds, each of which is optionally substituted. In embodiments of Formula II, or salt or isomer thereof, one or more of R3 and R4 are independently an alkynyl selected from the group consisting of hept-1-yne, hept-2-yne, hept-3- yne, oct-1-yne, oct-2-yne, oct-3-yne, oct-4-yne, non-1-yne, non-2-yne, non-3-yne, non-4-yne, non- 5-yne, dec-1-yne, dec-2-yne, dec-3-yne, dec-4-yne, dec-5-yne, dec-6-yne, undec-1-yne, undec-2- yne, undec-3-yne, undec-4-yne, undec-5-yne, undec-6-yne, undec-7-yne, dodec-1-yne, dodec-2- yne, dodec-3-yne, dodec-4-yne, dodec-5-yne, dodec-6-yne, dodec-8-yne, and an alkynyl group comprising two or more triple bonds, each of which is optionally substituted. In embodiments of Formula II, or salt or isomer thereof, A1 is an optionally substituted cycloalkyl and R5 and R6 are independently selected from the group consisting of optionally branched C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which is optionally substituted, optionally R5 is absent, optionally R6 is absent.
In embodiments of Formula II, or salt or isomer thereof, A1 is an optionally substituted cycloalkyl having three members and R5 and R6 are independently selected from the group consisting of optionally branched C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which is optionally substituted, optionally R5 is absent, optionally R6 is absent. In an aspect, the disclosure provides a compound selected from the group consisting of:
08),
drogen phosphate; SM-017),
hydrogen phosphate; SM-022),
- y y - - - y ydrogen phosphate; SM-023),
N O O N O PH O ,
O
N N O P O OH
,
N N O P O OH
O O
O O
( - ; nony (-(-(unecan--y)pperazn--y)exy) yrogen pospae),
O O
N N O P OH
(SM-063; 2-(4-(dihexylamino)piperidin-1-yl)ethyl nonyl hydrogen phosphate),
N N O O P OH O
- ; oy - -u ecae--y -,- aepa--y e y y oge pospae, N N O OH
(SM-108; nonyl (3-(4-(undecan-6-yl)piperazin-1-yl)propyl) hydrogen phosphate), N N O P OH
(SM-116; nonyl (3-(4-(undecane-6-yl)-1,4-diazepan-1-yl)propyl) hydrogen phosphate),
N O P O N HO O
( - ; ( )--(-( exyamno)pper n--y)e y non--en--y yrogen pospae), O P O N
(SM-119; 2-(4-((dihexylamino)methyl)piperidin-1-yl)ethyl nonyl hydrogen phosphate),
N N O O P O OH
- ; - y y - - y pp --y y y g p p , N N O P OH
(SM-122; 2-(4-(dipentylamino)piperidin-1-yl)ethyl nonyl hydrogen phosphate), N N O P O
(SM-123; nonyl (2-(4-(tridecan-7-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate), 14
N N O O P OH O
; y y pp y y y g p p , N N O P O
(SM-126; decyl (2-(4-(tridecan-7-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate), and salts and isomers thereof. In an aspec Ot, the disclosure provides a compound O selected from the group consisting of: N N O P O OH
, 15
A is a bond, C -C alkyl, C2-C22 alkenyl, C2-C22 alkynyl, or C3-C8 cycloalkyl, each of which is optionally substituted, X is N or CH, R1 is C5-C22 alkyl, C5-C22 alkenyl, C5-C22 alkynyl, C3-C22 cycloalkyl, or C(O)C4-C21 alkyl, each of which is optionally substituted; R2 is C2-C22 alkyl, C2-C22 alkenyl, C2-C22 alkynyl, or C3-C22 cycloalkyl, each of which is optionally substituted; and each of m and n is independently 0, 1, 2, or 3. 2. The compound of Formula I of claim 1, or salt or isomer thereof, wherein R1 and R2 are the same. 3. The compound of Formula I of claim 1, or salt or isomer thereof, wherein R1 is selected from the group consisting of C5-C12 alkyl, C5-C12 alkenyl, and C5-C12 alkynyl, each of which is optionally substituted, and R2 is selected from the group consisting of C4-C12 alkyl, C4-C12 alkenyl, and C4-C12 alkynyl, each of which is optionally substituted. 4. The compound of Formula I of claim 3, or salt or isomer thereof, wherein m is 2 and A is selected from the group consisting of C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A is C3-C5 cycloalkyl. 208
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 5. The compound of Formula I of claim 1, or salt or isomer thereof, wherein R1 is selected from the group consisting of branched or unbranched C5-C9 alkyl, C5-C9 alkenyl, and C5-C9 alkynyl, each of which is optionally substituted, and R2 is selected from the group consisting of branched or unbranched C4-C9 alkyl, C4-C9 alkenyl, and C4-C9 alkynyl, each of which is optionally substituted.. 6. The compound of Formula I of claim 5, or salt or isomer thereof, wherein m is 2 and A is selected from the group consisting of C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A is C3-C5 cycloalkyl. 7. The compound of Formula I of claim 1, or salt or isomer thereof, wherein R1 is selected from the group consisting of branched or unbranched C6-C9 alkyl, C6-C9 alkenyl, and C6-C9 alkynyl, each of which is optionally substituted, and R2 is selected from the group consisting of branched or unbranched C6-C9 alkyl, C6-C9 alkenyl, and C6-C9 alkynyl, each of which is optionally substituted. 8. The compound of Formula I of claim 7, or salt or isomer thereof, wherein m is 2 and A is selected from the group consisting of C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A is C3-C5 cycloalkyl. 9. The compound of Formula I of claim 1, or salt or isomer thereof, wherein R1 and R2 are independently C6-C9 alkyl, which is optionally substituted, m is 2, and A is selected from the group consisting of C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A is C3-C5 cycloalkyl. 10. The compound of Formula I of claim 1, or salt or isomer thereof, wherein R1 is optionally substituted C6-C9 alkyl, R2 is optionally substituted C9 alkyl, m is 2, and A is selected from the group consisting of C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A is C3-C5 cycloalkyl. 11. The compound of Formula I of claim 1, or salt or isomer thereof, wherein R1 and R2 are independently an alkyl selected from the group consisting of heptane, octane, nonane, decane, undecane, and dodecane, each of which is optionally substituted. 209
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 12. The compound of Formula I of claim 1, or salt or isomer thereof, wherein one or more of R1 and R2 are independently an alkenyl selected from the group consisting of hept-1-ene, hept-2- ene, hept-3-ene, oct-1-ene, oct-2-ene, oct-3-ene, oct-4-ene, non-1-ene, non-2-ene, non-3-ene, non- 4-ene, non-5-ene, dec-1-ene, dec-2-ene, dec-3-ene, dec-4-ene, dec-5-ene, dec-6-ene, undec-1-ene, undec-2-ene, undec-3-ene, undec-4-ene, undec-5-ene, undec-6-ene, undec-7-ene, dodec-1-ene, dodec-2-ene, dodec-3-ene, dodec-4-ene, dodec-5-ene, dodec-6-ene, dodec-8-ene, and an alkenyl group comprising two or more double bonds, each of which is optionally substituted. 13. The compound of Formula I of claim 1, or salt or isomer thereof, wherein one or more of R1 and R2 are independently an alkynyl selected from the group consisting of hept-1-yne, hept-2- yne, hept-3-yne, oct-1-yne, oct-2-yne, oct-3-yne, oct-4-yne, non-1-yne, non-2-yne, non-3-yne, non-4-yne, non-5-yne, dec-1-yne, dec-2-yne, dec-3-yne, dec-4-yne, dec-5-yne, dec-6-yne, undec- 1-yne, undec-2-yne, undec-3-yne, undec-4-yne, undec-5-yne, undec-6-yne, undec-7-yne, dodec- 1-yne, dodec-2-yne, dodec-3-yne, dodec-4-yne, dodec-5-yne, dodec-6-yne, dodec-8-yne, and an alkynyl group comprising two or more triple bonds, each of which is optionally substituted. 14. A compound selected from the group consisting of: O O P O
y y y y y g ), 210
Attorney Ref.: BN00004.0144 O OME-013WO (PCT Applicati O P OH on) N O
N N O O P O OH
O P OH
, 211
Attorney Ref.: BN00004.0144 O OME-013WO (PCT Application) O P O OH
O O
212
Attorney Ref.: BN00004.0144 O O OME-013WO (PCT Application) O P OH
O O
N N O P OH
(SM-063; 2-(4-(dihexylamino)piperidin-1-yl)ethyl nonyl hydrogen phosphate), 213
Attorney Ref.: BN00004.0144 OME-013WO (PCT Applicat O O ion) N N O PH O
- ; oy - -u ecae--y -,- aepa--y e y y oge pospae, N N O OH
(SM-108; nonyl (3-(4-(undecan-6-yl)piperazin-1-yl)propyl) hydrogen phosphate), N N O P OH
214
Attorney Ref.: BN00004.0144 OME-013WO (PCT N OH Application) N O O P O
( - ; nony (-(-(r ecan--y)-,- azepan--y)e y) yrogen pospae), N N O P O
(SM-126; decyl (2-(4-(tridecan-7-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate), and salts and isomers thereof. 15. A compoun Od selected from the group consisting N N O of: O P O OH
- ; - -eaoyppea --y e y oy y oge pospae, 215
O Attorney Ref.: BN00004.0144 OME-013WO N N O P O (PCT Application)
O OH
N N O P O
N O P OH O
y y y y Ny g O P OH O, 216
O Attorney Ref.: BN00004.0144 OME-013WO N (PCT Application)
N O O P OH O , N O O P O OH
O OH
N HO P
O , 217
Attorney Ref.: BN00004.0144 OME-013WO (PCT Applicatio N O P O n) O OH ers
e eo . 16. A compound having the following structure: N N O O P O
(SM-037), and salts and isomers thereof. 17. A compound of Formula II: R R33 N R5 A1 R6 O OH O P O R4II)
or a salt or isomer thereof, wherein A1 is C1-C22 alkyl, C2-C22 alkenyl, or C2-C22 alkynyl, each of which includes at least one substitution; or C3-C8 cycloalkyl or heterocylcloalkyl, each of which is optionally substituted; 218
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) R3 is C7-C22 alkyl, C7-C22 alkenyl, C7-C22 alkynyl, or C4-C22 cycloalkyl, each of which is optionally substituted; R4 is C2-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, or C3-C22 cycloalkyl, each of which is optionally substituted; and each of R5 and R6 is independently a bond, C1-C7 alkyl, C2-C7 alkenyl, or C2-C7 alkynyl, each of which is optionally substituted. 18. The compound of Formula II of claim 17, or salt or isomer thereof, wherein R3 and R4 are the same, optionally wherein R5 and R6 are the same. 19. The compound of Formula II of claim 17, or salt or isomer thereof, wherein R3 is selected from the group consisting of C7-C12 alkyl, C7-C12 alkenyl, and C7-C12 alkynyl, each of which is optionally substituted and R4 is selected from the group consisting of C4-C12 alkyl, C4-C12 alkenyl, and C4-C12 alkynyl, each of which is optionally substituted, wherein R5 and R6 are independently selected from the group consisting of a bond, C2-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, each of which is optionally substituted, optionally wherein R5 and R6 are both methyl group, or either R5 or R6 is a methyl group and the other is a bond. 20. The compound of Formula II of claim 19, or salt or isomer thereof, wherein A1 is selected from the group consisting of optionally branched C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A1 is C3-C5 cycloalkyl. 21. The compound of Formula II of claim 19, or salt or isomer thereof, wherein A1 is selected from the group consisting of optionally branched C3-C4 alkyl, C3-C4 alkenyl, and C3-C4 alkynyl, each of which includes at least one substitution, or A1 is C3 cycloalkyl. 22. The compound of Formula II of claim 17, or salt or isomer thereof, wherein R3 is selected from the group consisting of branched or unbranched C7-C9 alkyl, C7-C9 alkenyl, and C7-C9 alkynyl, each of which is optionally substituted, and R4 is selected from the group consisting of branched or unbranched C4-C9 alkyl, C4-C9 alkenyl, and C4-C9 alkynyl, each of which is optionally substituted. 219
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 23. The compound of Formula II of claim 22, or salt or isomer thereof, wherein A1 is optionally branched C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl or A1 is optionally branched C3-C4 alkyl, C3-C4 alkenyl, and C3-C4 alkynyl. 24. The compound of Formula II of claim 17, or salt or isomer thereof, wherein R3 is selected from the group consisting of branched or unbranched C7-C9 alkyl, C7-C9 alkenyl, and C7-C9 alkynyl, each of which is optionally substituted, and R4 is selected from the group consisting of branched or unbranched C6-C9 alkyl, C6-C9 alkenyl, and C6-C9 alkynyl, each of which is optionally substituted. 25. The compound of Formula II of claim 24, or salt or isomer thereof, wherein A1 is selected from the group consisting of optionally branched C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A1 is selected from the group consisting of optionally branched C3-C4 alkyl, C3-C4 alkenyl, and C3-C4 alkynyl, each of which includes at least one substitution. 26. The compound of Formula II of claim 17, or salt or isomer thereof, wherein R3 is optionally substituted C7-C9 alkyl, and R4 is optionally substituted C6-C9 alkyl, wherein A1 is selected from the group consisting of optionally branched C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A1 is selected from the group consisting of optionally branched C3-C4 alkyl, C3-C4 alkenyl, and C3-C4 alkynyl, each of which includes at least one substitution. 27. The compound of Formula II of claim 17, or salt or isomer thereof, wherein R3 and R4 are independently an alkyl selected from the group consisting of heptane, octane, nonane, decane, undecane, and dodecane, each of which is optionally substituted. 28. The compound of Formula II of claim 17, or salt or isomer thereof, wherein one or more of R3 and R4 are independently an alkenyl selected from the group consisting of hept-1-ene, hept- 2-ene, hept-3-ene, oct-1-ene, oct-2-ene, oct-3-ene, oct-4-ene, non-1-ene, non-2-ene, non-3-ene, non-4-ene, non-5-ene, dec-1-ene, dec-2-ene, dec-3-ene, dec-4-ene, dec-5-ene, dec-6-ene, undec- 1-ene, undec-2-ene, undec-3-ene, undec-4-ene, undec-5-ene, undec-6-ene, undec-7-ene, dodec-1- 220
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) ene, dodec-2-ene, dodec-3-ene, dodec-4-ene, dodec-5-ene, dodec-6-ene, dodec-8-ene, and an alkenyl group comprising two or more double bonds, each of which is optionally substituted. 29. The compound of Formula II of claim 17, or salt or isomer thereof, wherein one or more of R3 and R4 are independently an alkynyl selected from the group consisting of hept-1-yne, hept-2- yne, hept-3-yne, oct-1-yne, oct-2-yne, oct-3-yne, oct-4-yne, non-1-yne, non-2-yne, non-3-yne, non-4-yne, non-5-yne, dec-1-yne, dec-2-yne, dec-3-yne, dec-4-yne, dec-5-yne, dec-6-yne, undec- 1-yne, undec-2-yne, undec-3-yne, undec-4-yne, undec-5-yne, undec-6-yne, undec-7-yne, dodec- 1-yne, dodec-2-yne, dodec-3-yne, dodec-4-yne, dodec-5-yne, dodec-6-yne, dodec-8-yne, and an alkynyl group comprising two or more triple bonds, each of which is optionally substituted. 30. The compound of Formula II of claim 17, or salt or isomer thereof, wherein A1 is an optionally substituted cycloalkyl and R5 and R6 are independently selected from the group consisting of optionally branched C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which is optionally substituted, optionally R5 is absent, optionally R6 is absent. 31. The compound of Formula II of claim 22, or salt or isomer thereof, wherein A1 is an optionally substituted cycloalkyl having three members and R5 and R6 are independently selected from the group consisting of optionally branched C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which is optionally substituted, optionally R3 is absent, optionally R4 is absent. 32. A compound selected from the group consisting of:
; y y y p py y y y g p p , 221
O Attorney Ref.: BN00004.0144 OH OME-013WO (PCT Application) N O P O
N HO O
222
Attorney Ref.: BN00004.0144 HO OME-013WO (PCT Application) O P O O N (
SM-119; 2-(4-((dihexylamino)methyl)piperidin-1-yl)ethyl nonyl hydrogen phosphate), N N O P OH
N N O P OH
223
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) N N O O P OH O
; y y pp y y y g p p , and isomers thereof. 33. A compound of Formula III: or a salt or isomer thereof, whereinY O O P O O- R7II)
Y is selected fr R N xom t x xhe group consisting of 9 x N R8R9 N x xR9 N x , 9 x 9 x x9 x
R x 9 8 8 9 8 7 and R9 are either the same or different and are independently selected from the group consisting of C2-C22 alkyl, C2-C22 alkenyl, and C2-C22 alkynyl, each of which is optionally substituted, optionally R7, R9, or R7 and R9 are branched, optionally R7, R9, or R7 and R9 224
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) are an optionally substituted cycloalkyl or R7 and R9 may join to form an optionally substituted cycloalkyl; R8 is selected from the group consisting of branched or unbranched C1-C7 alkyl, C2-C7 alkenyl, and C2-C7 alkynyl, each of which is optionally substituted, and x is absent, 1, 2 or 3. 34. The compound of claim 33, or salt or isomer thereof, wherein R7 and R9 are the same. 35. The compound of claim 33, or salt or isomer thereof, wherein R7 or R9 are independently selected from the group consisting of C4-C12 alkyl, C4-C12 alkenyl, and C4-C12 alkynyl, each of which is optionally substituted, optionally wherein R7 and R9 are independently selected from the group of C4-C12 alkyl, C4-C12 alkenyl, and C4-C12 alkynyl, each of which is optionally substituted. 36. The compound of claim 35, or salt or isomer thereof, wherein R8 is 0, 1, 2, 3, 4, 5, or 6. 37. The compound of claim 33, or salt or isomer thereof, wherein R7 or R9 are independently selected from the group consisting of branched or unbranched C4-C12 alkyl, C4-C12 alkenyl, and C4-C12 alkynyl, each of which is optionally substituted, and R8 is 0, 1, 2, 3, 4, 5, or 6, optionally wherein R7 and R9 are independently selected from the group consisting of branched or unbranched C4-C12 alkyl, C4-C12 alkenyl, and C4-C12 alkynyl, each of which is optionally substituted, and R8 is 0, 1, 2, 3, 4, 5, or 6. 38. The compound of claim 37, or salt or isomer thereof, wherein R8 is 2, 4, or 6. 39. The compound of claim 33, or salt or isomer thereof, wherein R7 is selected from the group consisting of branched or unbranched C6-C9 alkyl, C6-C9 alkenyl, and C6-C9 alkynyl, each of which is optionally substituted, R9 is selected from the group consisting of branched or unbranched C6- C9 alkyl, C6-C9 alkenyl, and C6-C9 alkynyl, each of which is optionally substituted, and R8 is 2, 3, 4, 5, or 6. 40. The compound of claim 39, or salt or isomer thereof, wherein R8 is 2, 4, or 6. 225
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 41. The compound of claim 33, or salt or isomer thereof, wherein R7 and R9 are independently optionally substituted C6-C9 alkyl, and R8 is 2, 3, 4, 5, or 6, optionally wherein R8 is 2, 4, or 6. 42. The compound of claim 33, or salt or isomer thereof, wherein R7 is C9, R9 is C6-C17 alkyl, and R8 is absent, 1, or 2. 43. The compound of claim 33, or salt or isomer thereof, wherein R7 and R9 are independently an alkyl selected from the group consisting of heptane, octane, nonane, decane, undecane, and dodecane, each of which is optionally substituted. 44. The compound of claim 33, or salt or isomer thereof, wherein one or more of R7 and R9 are independently an alkenyl selected from the group consisting of hept-1-ene, hept-2-ene, hept- 3-ene, oct-1-ene, oct-2-ene, oct-3-ene, oct-4-ene, non-1-ene, non-2-ene, non-3-ene, non-4-ene, non-5-ene, dec-1-ene, dec-2-ene, dec-3-ene, dec-4-ene, dec-5-ene, dec-6-ene, undec-1-ene, undec- 2-ene, undec-3-ene, undec-4-ene, undec-5-ene, undec-6-ene, undec-7-ene, dodec-1-ene, dodec-2- ene, dodec-3-ene, dodec-4-ene, dodec-5-ene, dodec-6-ene, dodec-8-ene, and an alkenyl group comprising two or more double bonds, each of which is optionally substituted. 45. The compound of claim 33, or salt or isomer thereof, wherein one or more of R7 and R9 are independently an alkynyl selected from the group consisting of hept-1-yne, hept-2-yne, hept-3- yne, oct-1-yne, oct-2-yne, oct-3-yne, oct-4-yne, non-1-yne, non-2-yne, non-3-yne, non-4-yne, non- 5-yne, dec-1-yne, dec-2-yne, dec-3-yne, dec-4-yne, dec-5-yne, dec-6-yne, undec-1-yne, undec-2- yne, undec-3-yne, undec-4-yne, undec-5-yne, undec-6-yne, undec-7-yne, dodec-1-yne, dodec-2- yne, dodec-3-yne, dodec-4-yne, dodec-5-yne, dodec-6-yne, dodec-8-yne, and an alkynyl group comprising two or more triple bonds, each of which is optionally substituted. 46. A pharmaceutical composition comprising a lipid of Formul R1N lt or isomer thereof, wherein n X A O O PH a I: m O R2 (I)
or a sa O 226
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) A is a bond, C1-C22 alkyl, C2-C22 alkenyl, C2-C22 alkynyl, or C3-C8 cycloalkyl, each of which is optionally substituted, X is N or CH, R1 is C5-C22 alkyl, C5-C22 alkenyl, C5-C22 alkynyl, C3-C22 cycloalkyl, or C(O)C4-C21 alkyl, each of which is optionally substituted; R2 is C2-C22 alkyl, C2-C22 alkenyl, C2-C22 alkynyl, or C3-C22 cycloalkyl, each of which is optionally substituted; and each of m and n is independently 0, 1, 2, or 3. 47. The pharmaceutical composition of claim 46, including Formula I or salt or isomer thereof, wherein R1 and R2 are the same. 48. The pharmaceutical composition of claim 46, including Formula I or salt or isomer thereof, wherein R1 is selected from the group consisting of C5-C12 alkyl, C5-C12 alkenyl, and C5-C12 alkynyl, each of which is optionally substituted and R2 is selected from the group consisting of C4- C12 alkyl, C4-C12 alkenyl, and C4-C12 alkynyl, each of which is optionally substituted. 49. The pharmaceutical composition of claim 49, including Formula I or salt or isomer thereof, wherein m is 2 and A is selected from the group consisting of C2-C6 alkyl, C2-C6 alkenyl, and C2- C6 alkynyl, each of which includes at least one substitution, or A is C3-C5 cycloalkyl. 50. The pharmaceutical composition of claim 46, including Formula I or salt or isomer thereof, wherein R1 is selected from the group consisting of branched or unbranched C5-C9 alkyl, C5-C9 alkenyl, and C5-C9 alkynyl, each of which is optionally substituted, and R2 is selected from the group consisting of branched or unbranched C4-C9 alkyl, C4-C9 alkenyl, and C4-C9 alkynyl, each of which is optionally substituted. 51. The pharmaceutical composition of claim 50, including Formula I or salt or isomer thereof, wherein m is 2 and A is selected from the group consisting of C2-C6 alkyl, C2-C6 alkenyl, and C2- C6 alkynyl, each of which includes at least one substitution, or A is C3-C5 cycloalkyl. 227
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 52. The pharmaceutical composition of claim 46, including Formula I or salt or isomer thereof, wherein R1 is selected from the group consisting of branched or unbranched C6-C9 alkyl, C6-C9 alkenyl, and C6-C9 alkynyl, each of which is optionally substituted, and R2 is selected from the group consisting of branched or unbranched C6-C9 alkyl, C6-C9 alkenyl, and C6-C9 alkynyl, each of which is optionally substituted. 53. The pharmaceutical composition of claim 52, including Formula I or salt or isomer thereof, wherein m is 2 and A is selected from the group consisting of C2-C6 alkyl, C2-C6 alkenyl, and C2- C6 alkynyl, each of which includes at least one substitution, or A is C3-C5 cycloalkyl. 54. The pharmaceutical composition of claim 46, including Formula I or salt or isomer thereof, wherein R1 and R2 are independently optionally substituted C6-C9 alkyl, m is 2, and A is selected from the group consisting of C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A is C3-C5 cycloalkyl. 55. The pharmaceutical composition of claim 46, including Formula I or salt or isomer thereof, wherein R1 is optionally substituted C6-C9 alkyl, R2 is optionally substituted C9 alkyl, m is 2, and A is selected from the group consisting of C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A is C3-C5 cycloalkyl. 56. The pharmaceutical composition of claim 46, including Formula I or salt or isomer thereof, wherein R1 and R2 are independently an alkyl selected from the group consisting of heptane, octane, nonane, decane, undecane, and dodecane, each of which is optionally substituted. 57. The pharmaceutical composition of claim 46, including Formula I or salt or isomer thereof, wherein one or more of R1 and R2 are independently an alkenyl selected from the group consisting of hept-1-ene, hept-2-ene, hept-3-ene, oct-1-ene, oct-2-ene, oct-3-ene, oct-4-ene, non-1-ene, non- 2-ene, non-3-ene, non-4-ene, non-5-ene, dec-1-ene, dec-2-ene, dec-3-ene, dec-4-ene, dec-5-ene, dec-6-ene, undec-1-ene, undec-2-ene, undec-3-ene, undec-4-ene, undec-5-ene, undec-6-ene, undec-7-ene, dodec-1-ene, dodec-2-ene, dodec-3-ene, dodec-4-ene, dodec-5-ene, dodec-6-ene, dodec-8-ene, and an alkenyl group comprising two or more double bonds, each of which is optionally substituted. 228
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 58. The pharmaceutical composition of claim 46, including Formula I or salt or isomer thereof, wherein one or more of R1 and R2 are independently an alkynyl selected from the group consisting of hept-1-yne, hept-2-yne, hept-3-yne, oct-1-yne, oct-2-yne, oct-3-yne, oct-4-yne, non-1-yne, non- 2-yne, non-3-yne, non-4-yne, non-5-yne, dec-1-yne, dec-2-yne, dec-3-yne, dec-4-yne, dec-5-yne, dec-6-yne, undec-1-yne, undec-2-yne, undec-3-yne, undec-4-yne, undec-5-yne, undec-6-yne, undec-7-yne, dodec-1-yne, dodec-2-yne, dodec-3-yne, dodec-4-yne, dodec-5-yne, dodec-6-yne, dodec-8-yne, and an alkynyl group comprising two or more triple bonds, each of which is optionally substituted. 59. A pharmaceutical composition comprising a lipid o Of Formula II: R R33 N R5 A1 R6 OH 1 1 22 O P O R4II) or a salt or isomer th
ereof, wherein A is C -C alkyl, C2-C22 alkenyl, or C2-C22 alkynyl, each of which includes at least one substitution; or C3-C8 cycloalkyl or heterocycloalkyl, each of which is optionally substituted; R3 is C7-C22 alkyl, C7-C22 alkenyl, C7-C22 alkynyl, or C4-C22 cycloalkyl, each of which is optionally substituted; R4 is C2-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, or C3-C22 cycloalkyl, each of which is optionally substituted; and each of R5 and R6 is independently a bond, C1-C7 alkyl, C2-C7 alkenyl, or C2-C7 alkynyl, each of which is optionally substituted. 60. The pharmaceutical composition of claim 59, including Formula II or salt or isomer thereof, wherein R3 and R4 are the same, optionally wherein R5 and R6 are the same. 229
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 61. The pharmaceutical composition of claim 59, including Formula II or salt or isomer thereof, wherein R3 is selected from the group consisting of C7-C12 alkyl, C7-C12 alkenyl, and C7- C12 alkynyl, each of which is optionally substituted, R4 is selected from the group consisting of C4-C12 alkyl, C4-C12 alkenyl, and C4-C12 alkynyl, each of which is optionally substituted, wherein R5 and R6 are independently selected from the group consisting of a bond, C2-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, each of which is optionally substituted, optionally wherein R5 and R6 are both methyl group, or either R5 or R6 is a methyl group and the other is a bond. 62. The pharmaceutical composition of claim 61, including Formula II or salt or isomer thereof, wherein A1 is selected from the group consisting of optionally branched C2-C6 alkyl, C2- C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A1 is C3-C5 cycloalkyl. 63. The pharmaceutical composition of claim 62, including Formula II or salt or isomer thereof, wherein A1 is selected from the group consisting of optionally branched C3-C4 alkyl, C3- C4 alkenyl, and C3-C4 alkynyl, each of which includes at least one substitution, or A1 is C3 cycloalkyl. 64. The pharmaceutical composition of claim 59, including Formula II or salt or isomer thereof, wherein R3 is selected from the group consisting of branched or unbranched C7-C9 alkyl, C7-C9 alkenyl, and C7-C9 alkynyl, each of which is optionally substituted and R4 is selected from the group consisting of branched or unbranched C4-C9 alkyl, C4-C9 alkenyl, and C4-C9 alkynyl, each of which is optionally substituted. 65. The pharmaceutical composition of claim 64, including Formula II or salt or isomer thereof, wherein A1 is selected from the group consisting of optionally branched C2-C6 alkyl, C2- C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A1 is selected from the group consisting of optionally branched C3-C4 alkyl, C3-C4 alkenyl, and C3-C4 alkynyl, each of which includes at least one substitution. 66. The pharmaceutical composition of claim 59, including Formula II or salt or isomer thereof, wherein R3 is selected from the group consisting of branched or unbranched C7-C9 alkyl, C7-C9 alkenyl, and C7-C9 alkynyl, each of which is optionally substituted, and R4 is selected from 230
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) the group consisting of branched or unbranched C6-C9 alkyl, C6-C9 alkenyl, and C6-C9 alkynyl, each of which is optionally substituted. 67. The pharmaceutical composition of claim 66, including Formula II or salt or isomer thereof, wherein A1 is selected from the group consisting of optionally branched C2-C6 alkyl, C2- C6 alkenyl, and C2-C6 alkynyl, each of which is optionally substituted, or A1 is selected from the group consisting of optionally branched C3-C4 alkyl, C3-C4 alkenyl, and C3-C4 alkynyl, each of which includes at least one substitution. 68. The pharmaceutical composition of claim 59, including Formula II or salt or isomer thereof, wherein R3 is optionally substituted C7-C9 alkyl and R4 is optionally substituted C6-C9 alkyl, wherein A1 is selected from the group consisting of optionally branched C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which includes at least one substitution, or A1 is selected from the group consisting of optionally branched C3-C4 alkyl, C3-C4 alkenyl, and C3-C4 alkynyl, each of which includes at least one substitution. 69. The pharmaceutical composition of claim 59, including Formula II or salt or isomer thereof, wherein R3 and R4 are independently an alkyl selected from the group consisting of heptane, octane, nonane, decane, undecane, and dodecane, each of which is optionally substituted. 70. The pharmaceutical composition of claim 59, including Formula II or salt or isomer thereof, wherein one or more of R3 and R4 are independently an alkenyl selected from the group consisting of hept-1-ene, hept-2-ene, hept-3-ene, oct-1-ene, oct-2-ene, oct-3-ene, oct-4-ene, non- 1-ene, non-2-ene, non-3-ene, non-4-ene, non-5-ene, dec-1-ene, dec-2-ene, dec-3-ene, dec-4-ene, dec-5-ene, dec-6-ene, undec-1-ene, undec-2-ene, undec-3-ene, undec-4-ene, undec-5-ene, undec- 6-ene, undec-7-ene, dodec-1-ene, dodec-2-ene, dodec-3-ene, dodec-4-ene, dodec-5-ene, dodec-6- ene, dodec-8-ene, and an alkenyl group comprising two or more double bonds, each of which is optionally substituted. 71. The pharmaceutical composition of claim 59, including Formula II or salt or isomer thereof, wherein one or more of R3 and R4 are independently an alkynyl selected from the group consisting of hept-1-yne, hept-2-yne, hept-3-yne, oct-1-yne, oct-2-yne, oct-3-yne, oct-4-yne, non- 1-yne, non-2-yne, non-3-yne, non-4-yne, non-5-yne, dec-1-yne, dec-2-yne, dec-3-yne, dec-4-yne, 231
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) dec-5-yne, dec-6-yne, undec-1-yne, undec-2-yne, undec-3-yne, undec-4-yne, undec-5-yne, undec- 6-yne, undec-7-yne, dodec-1-yne, dodec-2-yne, dodec-3-yne, dodec-4-yne, dodec-5-yne, dodec- 6-yne, dodec-8-yne, and an alkynyl group comprising two or more triple bonds, each of which is optionally substituted. 72. The pharmaceutical composition of claim 59, including Formula II or salt or isomer thereof, wherein A1 is an optionally substituted cycloalkyl and R5 and R6 are independently selected from the group consisting of optionally branched C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which is optionally substituted, optionally R5 is absent, optionally R6 is absent. 73. The pharmaceutical composition of claim 72, including Formula II or salt or isomer thereof, wherein A1 is an optionally substituted cycloalkyl having three members and R5 and R6 are independently selected from the group consisting of optionally branched C2-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, each of which is optionally substituted, optionally R5 is absent, optionally R6 is absent. 74. A lipid particle comprising a compound of claims 1-45. 75. The lipid particle of claim 74, further comprising a therapeutic agent. 76. The lipid particle of claim 75, wherein the therapeutic agent is a nucleic acid. 77. A pharmaceutical composition comprising a lipid particle of claim 76 and a pharmaceutically acceptable excipient, carrier, or diluent. 78. A nucleic acid-lipid particle for delivering a nucleic acid cargo to a lung tissue of a subject, the nucleic acid-lipid particle comprising nonyl (2-(4-(undecan-6-yl)piperazin-1-yl)ethyl) hydrogen phosphate (SM-037) comprising about 30-70 mol % or about 40-60 mol % or about 50 mol % of the total lipid present in the nucleic acid-lipid particle. 79. The nucleic acid-lipid particle of claim 78, comprising a conjugated lipid that inhibits aggregation of particles comprising from 0.01 to 2% of the total lipid present, optionally wherein the conjugated lipid comprises a polyethyleneglycol (PEG)-lipid conjugate, optionally wherein the PEG of the PEG-lipid conjugate has an average molecular weight of from 550 Daltons to 3000 232
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) Daltons, optionally wherein the PEG-lipid conjugate is a PEG2000-lipid conjugate, optionally wherein the PEG2000-lipid conjugate comprises one or more of 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2k) and 1,2-distearoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DSG-PEG2k), optionally wherein the PEG2000-lipid conjugate is 1,2-Dimyristoyl-rac–glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k), optionally wherein the nucleic acid-lipid particle comprises a PEG-lipid conjugate at a concentration selected from the group consisting of about 0.5 mol % of the total lipid present in the nucleic acid-lipid particle, about 1.0 mol % of the total lipid present in the nucleic acid-lipid particle, and about 1.5 mol % of the total lipid present in the nucleic acid-lipid particle. 80. The nucleic acid-lipid particle of claim 79, wherein the PEG-lipid conjugate is DMG- PEG2k comprising about 1.5 mol % of the total lipid present in the nucleic acid-lipid particle. 81. The nucleic acid-lipid particle of claims 79-80, comprising one or more non-cationic lipids comprising from 20 mol % to 80 mol % of the total lipid present in the lipid-nucleic acid particle, optionally wherein the one or more non-cationic lipids comprise cholesterol or a derivative thereof. 82. The nucleic acid-lipid particle of claim 81, comprising cholesterol or a derivative thereof at a concentration range selected from the group consisting of 35 mol % to 45 mol % of the total lipid present in the nucleic acid-lipid particle, 45 mol % to 55 mol % of the total lipid present in the nucleic acid-lipid particle, and 55 mol % to 65 mol % of the total lipid present in the nucleic acid-lipid particle, optionally wherein the cholesterol or a derivative thereof is about 50% of the total lipid present in the nucleic acid-lipid particle. 83. The nucleic acid-lipid particle of claims 79-82, further comprising a cationic lipid selected from the group consisting of Dimethyldioctadecylammonium, Bromide Salt (DDAB), N-(4- carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy) propan-1-aminium (DOBAQ), 1,2-dioleoyl-3- trimethylammonium-propane or 18:1 TAP, a di-chain or gemini, cationic lipid (DOTAP), 1,2-di- O-octadecenyl-3-trimethylammonium propane, chloride salt (DOTMA), ethyl phosphatidylcholine (EPC), and trimethyl sphingosine. 84. The nucleic acid-lipid particle of claims 79-82, further comprising a cationic lipid that has the following structure: 233
Attorney Ref.: BN00004.0144 OME-013WO (PCT Applicatio Z O O n) NSNH3 NH S3 NH
, Cl+ C NlH+ H2N NH2 p+ Cild- other than cholesterol or a derivative thereof, optionally wherein the one or more non-cationic lipid other than cholesterol or a derivative thereof comprises from 5 mol % to 20 mol % of the total lipid present in the lipid-nucleic acid particle, optionally wherein the one or more non-cationic lipid other than cholesterol or a derivative thereof comprises about 10 mol % of the total lipid present in the nucleic acid-lipid particle. 86. The nucleic acid-lipid particle of claim 85, wherein the one or more non-cationic lipid other than cholesterol or a derivative thereof comprises a non-cationic lipid selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and β- sitosterol, optionally wherein the one or more non-cationic lipid other than cholesterol or a derivative thereof is dioleoylphosphatidylcholine (DOPC). 87. The nucleic acid-lipid particle of claim 79-86, comprising an ionizable phospholipid selected from the group consisting of 234
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) N O O N O PH O ,
O
N N O P O OH
, 235
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) N N O P O OH
O O
O O
( - ; nony (-(-(unecan--y)pperazn--y)exy) yrogen pospae), 236
Attorney Ref.: BN00004.0144 O OME-013WO (PCT Application) O P O OH
O O te),
(SM-063; 2-(4-(dihexylamino)piperidin-1-yl)ethyl nonyl hydrogen phosphate), 237
Attorney Ref.: BN00004.0144 OME-013WO (PCT Applicat O O ion) N N O PH O
- ; oy - -u ecae--y -,- aepa--y e y y oge pospae, N N O OH
(SM-108; nonyl (3-(4-(undecan-6-yl)piperazin-1-yl)propyl) hydrogen phosphate), N N O P OH
238
Attorney Ref.: BN00004.0144 O OME-013WO P OH (PCT Application) N N O O
( - ; nony (-(-(r ecan--y)-,- azepan--y)e y) yrogen pospae), N N O P O
(SM-126; decyl (2-(4-(tridecan-7-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate), and salts and isomers thereof. 88. The nucleic acid-lipid particle of claim 79-86, comprising an ionizable phospholipid selected from the g Oroup consisting of N N O O P O OH
( - ; -(-exanoypperazn--y)e y nony yrogen pospae), 239
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application)
O OH N O O
- ; y - ypp --y y g p p , 240
Attorney Ref.: BN00004.0144 OME-013WO OH (PCT Application) N N O O P O
N O P OH
N O P OH
(SM-041; nonyl (2-(4-pentadecanoylpiperazin-1-yl)e Nthyl) hydr Ooge Pn OH p Ohosphate), N O
y y y y O P O y g OH , 241
Attorney Ref.: BN00004.0144 OME-013WO N O P O (PCT Application) O OH
HO
O
N N HO O
242
Attorney Ref.: BN00004.0144 HO OME-013WO (PCT Application) O P O O N (
SM-119; 2-(4-((dihexylamino)methyl)piperidin-1-yl)ethyl nonyl hydrogen phosphate), N N O P OH
N N O P OH
243
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) N N O O P OH O
; y y p p y y y g p p , and salts and isomers thereof. 89. The nucleic acid-lipid particle of claims 79-88, wherein the nucleic acid cargo comprises a synthetic or naturally occurring RNA or DNA, or derivatives thereof, optionally wherein the nucleic acid cargo is a modified RNA, optionally wherein the modified RNA is selected from the group consisting of a modified mRNA, a modified antisense oligonucleotide and a modified siRNA, optionally wherein the modified mRNA encodes a nucleic acid modulating controller. 90. The nucleic acid-lipid particle of claims 79-89, wherein the nucleic acid cargo comprises one or more modifications selected from the group consisting of 2′-O-methyl modified nucleotides, a nucleotide comprising a 5′-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, a 2′-deoxy-2′-fluoro modified nucleotide, a 5′-methoxy-modified nucleotide (e.g., 5′- methoxyuridine), a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′- amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide; internucleoside linkages or backbones including phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. 244
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 91. The nucleic acid-lipid particle of claims 79-90, wherein the lung tissue is selected from the group consisting of epithelium, endothelium, interstitial connective tissue, blood vessel, hematopoietic tissue, lymphoid tissue, and pleura. 92. The nucleic acid-lipid particle of claims 79-91, wherein the nucleic acid-lipid particle comprises SM-037 at about 30 mol % of the total lipid present in the nucleic acid-lipid particle, cholesterol at about 50 mol % of the total lipid present in the nucleic acid-lipid particle, SM-005 at about 50 mol % of the total lipid present in the nucleic acid-lipid particle, and DMG-PEG2k at about 1.5 mol % of the total lipid present in the nucleic acid-lipid particle. 93. The nucleic acid-lipid particle of claims 79-92, wherein intravenous administration of the nucleic acid-lipid particle to the subject results in expression of the nucleic acid cargo in cells of the lung tissue of the subject at a level that is at least two-fold higher than expression of the nucleic acid cargo in cells of liver, heart, spleen, ovary, pancreas and kidney of the subject, optionally wherein expression of the nucleic acid cargo in cells of the lung tissue of the subject is at least three-fold higher, optionally at least four-fold higher, optionally at least five-fold higher, optionally at least six-fold higher, optionally at least seven-fold higher, optionally at least eight-fold higher, optionally at least nine-fold higher, optionally at least ten-fold higher, optionally at least eleven- fold higher, optionally at least twelve-fold higher, optionally at least thirteen-fold higher, optionally at least fourteen-fold higher, optionally at least fifteen-fold higher, optionally at least twenty-fold higher, than expression of the nucleic acid cargo in cells of liver, heart, spleen, ovary, pancreas and kidney of the subject. 94. The nucleic acid-lipid particle claims 79-93, wherein intravenous administration of the nucleic acid-lipid particle or pharmaceutical composition to the subject results in localization of the nucleic acid-lipid particle to the lung tissue of the subject at an at least two-fold higher concentration than the concentration of the nucleic acid-lipid particle in one or more other tissues of the subject selected from the group consisting of heart, spleen, ovaries and pancreas, optionally wherein at least three-fold, optionally at least four-fold, optionally at least five-fold, optionally at least six-fold higher concentration of the nucleic acid-lipid particle is located in lung as compared to one or more other tissues of the subject selected from the group consisting of heart, spleen, ovaries and pancreas. 245
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) 95. The nucleic acid-lipid particle claims 79-93, wherein the nucleic acid-lipid particle or pharmaceutical composition is administered to treat a lung disease or disorder, optionally wherein the disease or disorder is selected from the group consisting of lung cancer, pneumonia, pulmonary fibrosis, COPD, asthma, bronchiectasis, sarcoidosis, pulmonary hypertension, emphysema, alpha- 1 antitrypsin deficiency, aspergillosis, bronchiolitis, bronchitis, pneumoconiosis, a coronavirus, Middle Eastern Respiratory Syndrome, Severe Acute Respiratory Syndrome, cystic fibrosis, Legionnaire’s disease, influenza, pertussis, pulmonary embolism and tuberculosis. 96. A compound of Formula IV: R or a salt or isomer t R1100 N R5 A1 R6 O OH ereo , w ere n 1 1 22 2 22 O P O R11V)
A is C -C alkyl, C -C alkenyl, or C2-C22 alkynyl, each of which includes at least one substitution; or C3-C8 cycloalkyl or heterocylcloalkyl, each of which is optionally substituted; R10 is C5-C22 alkyl, C5-C22 alkenyl, C5-C22 alkynyl, or C4-C22 cycloalkyl, each of which is optionally substituted; R11 is C5-C16 alkyl, C5-C16 alkenyl, C5-C16 alkynyl, or C3-C22 cycloalkyl, each of which is optionally substituted; and each of R5 and R6 is independently a bond, C1-C7 alkyl, C2-C7 alkenyl, or C2-C7 alkynyl, each of which is optionally substituted. 97. The compound of Formula II of claim 96, or salt or isomer thereof, wherein R10 and R11 are the same, optionally wherein R5 and R6 are the same. 98. The compound of Formula II of claim 96, or salt or isomer thereof, wherein R10 is selected from the group consisting of C5-C6 alkyl, C5-C6 alkenyl, and C5-C6 alkynyl, each of which is 246
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) optionally substituted and R11 is selected from the group consisting of C5-C12 alkyl, C5-C12 alkenyl, and C5-C12 alkynyl, each of which is optionally substituted, wherein R5 and R6 are independently selected from the group consisting of a bond, C2-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, each of which is optionally substituted, optionally wherein R5 and R6 are both methyl group, or either R5 or R6 is a methyl group and the other is a bond. 99. The compound of Formula II of claim 96, or salt or isomer thereof, wherein R10 is selected from the group consisting of branched or unbranched C5-C6 alkyl, C5-C6 alkenyl, and C5-C6 alkynyl, each of which is optionally substituted, and R11 is selected from the group consisting of branched or unbranched C6-C10 alkyl, C6-C10 alkenyl, and C6-C10 alkynyl, each of which is optionally substituted. 100. A compound selected from the group consisting O O oHf: N N O P O
(SM-064; nonyl (2-(4-(undecane-6-yl)-1,4-diazepan-1-yl)ethyl) hydrogen phosphate), 247
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application N N OO P O ) H O
( - ; ( )--(-( exyamno)pper n--y)e y non--en--y yrogen pospae), O P O N
(SM-119; 2-(4-((dihexylamino)methyl)piperidin-1-yl)ethyl nonyl hydrogen phosphate), 248
Attorney Ref.: BN00004.0144 OME-013WO (PCT Application) N N O O P O OH
- ; - y y - - y pp --y y y g p p , N N O P OH
(SM-122; 2-(4-(dipentylamino)piperidin-1-yl)ethyl nonyl hydrogen phosphate), and N N O P OH O
(SM-124; decyl (2-(4-(dihexylamino)piperidin-1-yl)ethyl) hydrogen phosphate). 249