EA046244B1 - MODIFIED AMINE LIPIDS - Google Patents

Description

Cross references to related applications

This application claims benefit from U.S. Provisional Patent Application No. 62/775,783, filed December 5, 2018, the entire contents of which are incorporated herein by reference.

State of the art

Lipid nanoparticles containing ionizable amine-containing lipids can serve as vehicles for the delivery of biologically active agents, in particular polynucleotides such as RNA, mRNA and guide RNAs into cells. LNP compositions containing ionizable lipids can facilitate the delivery of oligonucleotide agents across cell membranes and can be used to introduce gene editing components and compositions into living cells. Biologically active agents that are particularly difficult to deliver into cells include proteins, nucleic acid drugs and derivatives thereof, especially drugs that include relatively large oligonucleotides such as mRNA. Of particular interest are compositions for delivering promising genome editing technologies into cells, for example, for delivering components of the CRISPR/Cas9 system (for example, mRNA encoding a nuclease and associated guide RNA (gRNA)).

Compositions are needed to deliver CRISPR/Cas protein and nucleic acid components into a cell, such as a patient cell. In particular, compositions for delivering mRNA encoding the protein component of CRISPR and for delivering CRISPR guide RNAs are of particular interest. Compositions with useful in vitro and in vivo delivery properties that can stabilize and deliver RNA components are also of particular interest.

Summary

Presented herein are amine-containing lipids useful for the preparation of lipid nanoparticle (LNP) compositions. Such LNP compositions may have properties useful for delivering nucleic acid cargo, such as CRISPR/Cas gene editing components, into cells.

In some embodiments, the present disclosure relates to a compound having the structure of formula (IA):

where independently for each case

X ¹ represents C _1-3 alkylene or '

X ² is selected from O, NH, NMe and a bond, with the proviso that when X ² is O, R ² together with a nitrogen atom and either R ¹ or a carbon atom X ³ forms a 4-membered, 5-membered or 6-membered -membered ring,

X ³ represents C _2-4 alkylene,

X ⁴ represents a C1 alkylene or bond,

X ⁵ represents a C1 alkylene or bond,

R ¹ represents C _1-3 alkyl, optionally substituted with a hydroxyl group,

R ² represents C _1-3 alkyl, optionally substituted with a hydroxyl group, or

R ² and R ¹ , or R ² and the carbon atom X ³ , together with the nitrogen atom to which they are attached, form a 4-membered, 5-membered or 6-membered ring,

Y ¹ is selected from the bond, -CH=CH-, -(C=O)O- and -O(C=O)-,

Y ² is selected from -CH2-CH=CH- and C3-C4 alkylene,

R ³ is selected from H, C _5-7 cycloalkyl, C ₈ -C ₁₀ alkenyl, and C _3-18 alkyl,

R ⁴ represents C _4-8 alkyl, or a pharmaceutically acceptable salt thereof.

In some embodiments, the present disclosure relates to a compound having the structure of formula (I):

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where independently for each case,

X ¹ represents C _1-3 alkylene,

X ² is selected from O, NH, NMe and a bond, with the proviso that when X ² is O, R ² together with a nitrogen atom and either R ¹ or a carbon atom X ³ forms a 4-membered, 5-membered or 6-membered -membered ring,

X ³

X ⁴

X ⁵

R ¹ _R 2

R ² is a C2-4 alkylene, is a C1 alkylene or bond, is a C1 alkylene or bond, is a C1-2 alkyl, optionally substituted with a hydroxyl group, is a C1-2 alkyl, optionally substituted with a hydroxyl group, or and R ¹ , or R ² and the carbon atom X ³ , together with the nitrogen atom to which they are attached, form

5-membered or 6-membered ring,

Y ¹ is selected from -CH=CH-, -(C=O)O- and -O(C=O)-,

R ³ is selected from H, C _5-7 cycloalkyl, and C _3-18 alkyl,

R ⁴ represents C4-7 alkyl, or a pharmaceutically acceptable salt thereof.

In certain embodiments, X ¹ is a linear C2 alkylene. In other embodiments, X ¹ is a C1 alkylene.

In some other embodiments, X ² is NH or O.

In certain embodiments, X ³ is C _2-3 alkylene.

In some other embodiments, R ² and carbon atom X ³ together with the nitrogen atom to which they are attached form a 4-6 membered ring.

In some other embodiments, R ² is C _1-2 alkyl.

In some other embodiments, R ¹ and R ² together with the nitrogen atom to which they are attached form a 4-6 membered ring.

In some other embodiments, R ¹ and R ² together with the nitrogen atom to which they are attached form a 5-membered ring.

IN

IN

IN

IN

IN

IN

IN

In some other embodiments, some other embodiments, some other embodiments, some other embodiments, some other embodiments, some other embodiments,

R ¹

R ³

R ³

R ³

R ³

R ⁴ in certain other embodiments, X in certain other embodiments, X ⁵ is C _1-2 alkyl.

is a linear C _4-16 alkyl.

is a branched C _6-10 alkyl.

represents C _5-7 cycloalkyl.

represents C _8-10 alkenyl.

is a linear C5-6 alkyl.

represents a connection.

is a C1 alkylene.

In certain embodiments, the compound is a compound of formula (IA); and Y2-R ⁴ is -CH ₂ -CH=CH-R ⁴ .

In some other embodiments, Y ¹ is selected from the bond, -CH=CH- and -O(C=O)-.

In certain embodiments, the compound is a compound of Formula II:

where the variables are defined as for formula (IA).

In certain embodiments, the compound is a compound of formula III;

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where the variables are defined as for formula (IA).

Typical compounds of formulas (IA) or (I) include:

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or a pharmaceutically acceptable salt thereof.

In some embodiments, the invention provides a lipid nanoparticle (LNP) composition containing any compound described herein in a lipid component.

In some embodiments, the invention relates to any LNP composition described herein, wherein the lipid component further includes an auxiliary lipid, a polyethylene glycol (PEG) lipid, and a neutral lipid.

In some embodiments, the invention relates to any LNP composition described herein, wherein the composition further comprises a nucleic acid.

In some embodiments, the invention relates to any LNP composition described herein, wherein the composition has a nitrogen/phosphorus (N/P) ratio of 3-10.

In some embodiments, the invention relates to any LNP composition described herein where the N/P ratio is 6±1.

In some embodiments, the invention provides any LNP composition described herein further comprising an RNA component, wherein the RNA component comprises mRNA.

In some embodiments, the invention relates to any LNP composition described herein, wherein the RNA component comprises a sequence encoding an RNA-guided DNA binding agent, such as Cas nuclease mRNA.

In some embodiments, the invention relates to any LNP composition described herein, wherein the RNA component includes gRNA.

In some embodiments, the invention relates to the use of an LNP composition to edit a gene in a subject.

In some embodiments, the invention relates to the use of an LNP composition to produce a gene editing drug in a subject.

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Brief description of the figures

In fig. 1A-1C show in vivo editing at the TTR locus and compounds tested in the study. In fig. Figure 1A shows the chemical structures of the tested compounds. Fig. 1B shows the percentage change in TTR in mouse liver cells after delivery. In fig. Figure 1C shows serum TTR levels (μg/mL) after delivery.

Fig. 2 is a graph showing the percentage change in TTR in mouse liver cells following delivery using LNPs containing a compound of formula (IA) or a control compound. Dose response data are also shown.

In fig. 3A-3B show results after delivery using LNPs containing a compound of formula (IA). Fig. 3A is a graph showing the percentage change in TTR in mouse liver cells after delivery. Fig. 3B is a graph showing serum TTR (μg/ml) after delivery.

In fig. 4A-4C show in vivo editing at the TTR locus and compounds tested in the study. Fig. 4A illustrates the chemical structures of the test compounds. Fig. 4B shows the percentage change in TTR in mouse liver cells after delivery. In fig. Figure 4C shows serum TTR levels (μg/mL) postpartum.

In fig. 5A-5C show compounds of formula (IA) and delivery results using these compounds. Fig. 5A illustrates the chemical structures of the test compounds. Fig. 5B shows the percentage change in TTR in mouse liver cells after delivery. In fig. Figure 5C shows serum TTR levels (µg/ml) after delivery.

In fig. 6A-6C show compounds of formula (IA) and delivery results using these compounds. Fig. 6A illustrates the chemical structures of the test compounds. Fig. 6B shows the percentage change in TTR in mouse liver cells after delivery. In fig. Figure 6C shows serum TTR levels (μg/mL) after delivery.

In fig. 7A-7C show compounds of formula (IA) and delivery results using these compounds. Fig. 7A illustrates the chemical structures of the test compounds. Fig. 7B shows the percentage change in TTR in mouse liver cells after delivery. In fig. Figure 7C shows serum TTR levels (μg/mL) after delivery.

In fig. 8A-8C show compounds of formula (IA) and delivery results using these compounds. Fig. 8A illustrates the chemical structures of test compounds where n=1, 2 or 3. FIG. 8B shows the percentage change in TTR in mouse liver cells after delivery. In fig. 8C shows serum TTR levels (μg/ml) after delivery.

In fig. 9A-9C show compounds of formula (IA) and delivery results using these compounds. Fig. 9A illustrates the chemical structures of the test compounds. Fig. 9B shows the percentage change in TTR in mouse liver cells after delivery. In fig. 9C shows serum TTR levels (μg/ml) after delivery.

In fig. 10A-10C show compounds of formula (IA) and delivery results using these compounds. Fig. 10A illustrates the chemical structures of the test compounds. Fig. 10B shows the percentage change in TTR in mouse liver cells after delivery. In fig. 10C shows serum TTR levels (μg/ml) after delivery.

In fig. 11A-11D show compounds of formula (IA) and delivery results using these compounds. Fig. 11A and FIG. 11B illustrates the chemical structures of the test compounds. Fig. 11C shows the percentage of TTR editing in mouse liver cells after delivery. In fig. 11D shows serum TTR levels (μg/ml) after delivery.

In fig. 12A-12B show compounds of formula (IA) and delivery results using these compounds. Fig. 12A shows the percentage change in TTR in rat liver cells after delivery. In fig. 12B shows serum TTR levels (μg/ml) after delivery.

In fig. 13A-13B show compounds of formula (IA) and delivery results using these compounds. Fig. 13A illustrates the chemical structures of the test compounds. Fig. 13B shows the percentage of TTR editing in HUH7 cells after delivery.

Detailed description

The present invention relates to lipids, particularly ionizable lipids, that can be used to deliver biologically active agents, including nucleic acids, such as CRISPR/Cas (cargo) RNA components, into a cell, as well as methods for making and using such compositions. Lipids and their pharmaceutically acceptable salts are provided, optionally in the form of compositions containing lipids, including LNP compositions. In some embodiments, the LNP composition may contain a biologically active agent, for example an RNA component and a lipid component, which includes a compound of formula (IA), (I), (II) or (III) as defined herein. In some embodiments, the RNA component includes a nucleic acid. In some embodiments, lipids are used to deliver a biologically active agent, for example a nucleic acid, such as mRNA, to a cell, such as a liver cell. In some embodiments, the RNA component includes gRNA and not

- 25 046244 necessarily mRNA encoding a class 2 Cas nuclease, and optionally gRNA or nucleic acids encoding gRNA. Gene editing techniques and methods for creating engineered cells using these compositions are also presented.

Compositions of lipid nanoparticles.

Disclosed herein are various LNP compositions for delivering biologically active agents such as nucleic acids, for example, mRNA and gRNA, including CRISPR/Cas cargo. Such LNP compositions include an ionizable amine lipid along with a neutral lipid, a PEG lipid, and an auxiliary lipid. A lipid nanoparticle or LNP refers, without limitation, to a particle that includes multiple (ie, more than one) LNP components physically linked to each other by intermolecular forces.

Lipids.

The invention provides lipids that can be used in LNP compositions.

In some embodiments, the present disclosure relates to a compound having the structure of formula (IA):

where independently for each case

X ¹ represents C _1-3 alkylene or

X ² is selected from O, NH, NMe and a bond, with the proviso that when X ² is O, R ² together with a nitrogen atom and either R ¹ or a carbon atom X ³ forms a 4-membered, 5-membered or 6-membered -membered ring,

X ³ represents C _2-4 alkylene,

X ⁴ represents a C1 alkylene or bond,

X ⁵ represents a C1 alkylene or bond,

R ¹ represents C _1-3 alkyl, optionally substituted with a hydroxyl group,

R ² represents C _1-3 alkyl, optionally substituted with a hydroxyl group, or

R ² and R ¹ , or R ² and the carbon atom X ³ , together with the nitrogen atom to which they are attached, form a 4-membered, 5-membered or 6-membered ring,

Y ¹ is selected from the bond, -CH=CH-, -(C=O)O- and -O(C=O)-,

Y ² is selected from -CH ₂ -CH=CH- and C3-C4 alkylene,

R ³ is selected from H, C _5-7 cycloalkyl, C ₈ -C ₁₀ alkenyl, and C ₃ - ₁₈ alkyl,

R ⁴ represents C _4-8 alkyl, or a pharmaceutically acceptable salt thereof.

In some embodiments, the present disclosure relates to a compound having the structure of formula (I):

where independently for each case,

X ¹ represents C _2-3 alkylene,

X ² is selected from O, NH, NMe and a bond, with the proviso that when X ² is O, R ² together with a nitrogen atom and either R ¹ or a carbon atom X ³ forms a 4-membered, 5-membered or 6-membered -membered ring,

X ³ represents C _2-4 alkylene,

X ⁴ represents a C1 alkylene or bond,

X ⁵ represents a C1 alkylene or bond,

R ¹ represents C _1-2 alkyl, optionally substituted with a hydroxyl group,

R ² is C _1-2 alkyl, optionally substituted with a hydroxyl group, or

R ² and R ¹ , or R ² and the carbon atom X ³ , together with the nitrogen atom to which they are attached, form a 5-membered or 6-membered ring,

Y ¹ is selected from -CH=CH-, -(C=O)O- and -O(C=O)-,

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R ³ is selected from H, C _5-7 cycloalkyl, and C _3-18 alkyl,

R ⁴ represents C _4-7 alkyl, or a pharmaceutically acceptable salt thereof.

In certain embodiments, X ¹ is a linear C ₂ alkylene. In other embodiments, X ¹ is a C1 alkylene.

In some other embodiments, X ² is NH or O.

In certain embodiments, X ³ is C _2-3 alkylene.

In some other embodiments, R ² and carbon atom X ³ together with the nitrogen atom to which they are attached form a 4-6 membered ring.

In some other embodiments, R ² is C _1-2 alkyl.

In some other embodiments, R ¹ and R ² together with the nitrogen atom to which they are attached form a 4-6 membered ring.

In some other embodiments, R ¹ and R ² together with the nitrogen atom to which they are attached form a 5-membered ring.

In some other embodiments, R ¹ is C _1-2 alkyl.

In some other embodiments, R ³ is linear C _4-16 alkyl.

In some other embodiments, R ³ is a branched C _6-10 alkyl.

In some other embodiments, R ³ is C _5-7 cycloalkyl.

In some other embodiments, R ³ is C _8-10 alkenyl.

In some other embodiments, R ⁴ is linear C _5-6 alkyl.

In some other embodiments, X ⁴ represents a link.

In some other embodiments, X ⁵ is a C1 alkylene.

In certain embodiments, the compound is a compound of formula (IA); and Y ² -R ⁴ is -CH ₂ -CH=CH-R ⁴ .

In some other embodiments, Y ¹ is selected from the bond, -CH=CH- and -O(C=O)-.

In certain embodiments, the compound is a compound of Formula II:

where the variables are defined as for formula (IA).

In certain embodiments, the compound is a compound of Formula III;

where the variables are defined as for formula (IA).

Typical compounds of formulas (IA) or (I) include:

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or a pharmaceutically acceptable salt thereof.

In some embodiments, at least 75% of the compound of formula (IA), (I), (II) or (III) lipid compositions formulated as described herein is cleared from the subject's plasma within 8, 10, 12. 24, or 48 hours, or 3, 4, 5, 6, 7 or 10 days after administration. In some embodiments, at least 50% of the lipid compositions containing a compound of formula (IA), (I), (II) or (III) as disclosed herein are cleared from the subject's plasma within 8, 10, 12. 24. or 48 hours, or 3, 4, 5, 6, 7 or 10 days after administration, which can be determined, for example, by measuring lipid (for example, compounds of formula (IA), (I), (II) or (III)), RNA (eg, mRNA) or other component in plasma. In some embodiments, the encapsulated lipid is measured versus the free lipid, RNA, or nucleic acid in the lipid composition.

Lipid clearance can be measured as described in the literature. See Maier, M.A., et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther. 2013, 21(8), 1570-78 (Maier). For example, at Maier, LNP-siRNA systems containing siRNAs targeting luciferases were administered to six- to eight-week-old male C57B1/6 mice at a dose of 0.3 mg/kg by intravenous bolus injection through the lateral tail vein. Blood, liver, and spleen samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, and 168 h postdose. Mice were perfused with saline before tissue collection, and blood samples were processed to obtain plasma. All samples were processed and analyzed by LC-MS. In addition, Maier describes a procedure for assessing toxicity following administration of LNP-siRNA compositions. For example, siRNA targeting luciferase was administered to male Sprague-Dawley rats at 0, 1, 3, 5, and 10 mg/kg (5 animals per group) via a single intravenous bolus injection of 5 ml/kg. After 24 hours, about 1 ml of blood was obtained from the jugular vein of conscious animals and the serum was isolated. 72 hours after dosing, all animals were sacrificed for necropsy. Clinical signs, body weight, serum chemistry, organ weights, and histopathology were assessed. Although Maier describes methods for evaluating siRNA-LNP compositions, these methods can be used to evaluate the clearance, pharmacokinetics and toxicity of administration of lipid compositions, such as the LNP compositions of the present invention.

In some embodiments, lipid compositions using compounds of formula (IA), (I), (II) or (III) disclosed herein exhibit an increased rate of clearance compared to alternative ionizable amine lipids. In some such embodiments, the clearance rate is the rate of lipid clearance, e.g., the rate at which a compound of formula (IA), (I), (II) or (III) is eliminated

- 49 046244 from blood, serum or plasma. In some embodiments, the clearance rate is the clearance rate of cargo (eg, a biologically active agent), eg, the rate at which a cargo component is removed from blood, serum, or plasma. In some embodiments, the clearance rate is the rate of RNA clearance, eg, the rate at which mRNA or gRNA is cleared from blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNP is cleared from blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNP is removed from tissue, such as liver tissue or spleen tissue. It is desirable that a high rate of elimination may result in a safety profile without significant side effects and/or reduced accumulation of LNP in the circulation and/or tissues.

The compounds of formula (IA), (I), (II) or (III) of the present invention can form salts depending on the pH of the environment in which they are found. For example, in a slightly acidic environment, compounds of formula (IA), (I), (II) or (III) can be protonated and thus carry a positive charge. Conversely, in a slightly alkaline environment, such as, for example, blood, where the pH is approximately 7.35, compounds of formula (IA), (I), (II) or (III) may not be protonated and thus have no charge. In some embodiments, the compounds of formula (IA), (I), (II), or (III) of the present invention may be advantageously protonated at a pH of at least about 9. In some embodiments, the compounds of formula (IA), (I) , (II) or (III) of the present invention may preferably be protonated at a pH of at least about 10.

The pH at which a compound of formula (IA), (I), (II) or (III) is preferentially protonated is related to its own pKa. In preferred embodiments, a salt of a compound of formula (IA), (I), (II) or (III) of the present invention has a pKa in the range of about 5.1 to about 8.0, more preferably from about 5.5 to about 7 .5, for example from about 6.1 to about 6.3. In other preferred embodiments, a salt of a compound of formula (IA), (I), (II) or (III) of the present invention has a pKa in the range of about 5.3 to about 8.0, for example, from about 5.6 to about 7.2, from about 5.7 to about 6.5. In other embodiments, a salt of a compound of formula (IA), (I), (II) or (III) of the present invention has a pKa in the range of about 5.7 to about 6.4, for example, from about 5.8 to about 6.2. In other preferred embodiments, a salt of a compound of formula (IA), (I), (II) or (III) of the present invention has a pKa in the range of about 5.7 to about 6.5, for example, from about 5.8 to about 6.4. Alternatively, a salt of a compound of formula (IA), (I), (II) or (III) of the present invention has a pKa in the range of about 5.8 to about 6.5. In some embodiments, the pKa of the protonated form of a compound of formula (IA), (I), (II) or (III) is from about 5.5 to about 6.0. A salt of a compound of formula (IA), (I), (II) or (III) of the present invention may have a pKa in the range of about 6.0 to about 8.0, preferably from about 6.0 to about 7.5. The pKa of a salt of a compound of formula (IA), (I), (II) or (III) may be an important factor in the formulation of LNPs, since LNPs formulated with certain lipids have been found to have a pKa range from about 5.5 to about 7 ,0 are effective for delivering cargo in vivo, for example to the liver. In addition, LNPs formulated with certain lipids having a pKa in the range of about 5.3 to about 6.4 have been found to be effective for delivery in vivo, such as to tumors. See for example WO 2014/136086.

In some embodiments, the invention provides an enantioenriched compound of formula (IA), (I), (II) or (III). The level of enantioenrichment of a compound can be expressed as enantiomeric excess (ee). Its compound can be measured by dividing the difference in the fractions of enantiomers by the sum of the fractions of enantiomers. For example, if a compound is found to contain 98% (B) enantiomer and 2% (R) enantiomer, then its compound is (98-2)/(98+2) or 96%. In some embodiments, a compound of formula (IA), (I), (II) or (III) has about 30% or more, 40% or more, 50% or more, 60% or more, 70% its or more, about 80% of it, about 85%, about 88% of it, about 90% of it, about 91%, about 92% of it, about 93%, about 94%, about 95%, about 96% of it, about 97% ee, about 98%, about 99% or higher, about 99% ee, even if that % ee is greater than the % ee of the starting material, for example 0% ee (racemic). In some embodiments, the compound of formula (IA), (I), (II) or (III) is enantioenriched. In some embodiments, the compound of formula (IA), (I), (II) or (III) is enantiopure. In embodiments of the invention where a compound has more than one stereocenter, that stereocenter may be enriched in a significant manner independent of the stereochemical arrangement/enrichment of any other stereocenters on the molecule. For example, a compound described herein may have 30% de or more, 40% de or more, 50% de or more, 60% de or more, 70% de or more, 80% de or more, 90% de or more, 95% or more, or even 98% or more at a particular stereocenter.

Additional lipids.

Neutral lipids suitable for use in the lipid composition of the present invention include, for example, a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use herein include

- 50 046244 including, but not limited to, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine ( PSPC), 1,2-diarachidoyl-c-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-diicosenoyl-c-glycero-3-phosphocholine (DEPC), palmitoyl oleoyl phosphatidylcholine ( POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoyl phosphatidylcholine distearoyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In some embodiments, the neutral phospholipid may be selected from distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidyl ethanolamine (DMPE), preferably distearoylphosphatidylcholine (DSPC).

Accessory lipids include steroids, sterols and alkyl resorcinols. Auxiliary lipids suitable for use herein include, but are not limited to, cholesterol, 5-heptadecylresorcinol and cholesterol hemisuccinate. In some embodiments, the auxiliary lipid may be cholesterol or a derivative thereof, such as cholesterol hemisuccinate.

PEG lipids can influence the lifespan of nanoparticles in vivo (for example, in the blood). PEG lipids can aid the preparation process by, for example, reducing particle aggregation and controlling particle size. The PEG lipids used here can modulate the pharmacokinetic properties of LNP. Typically, a PEG lipid includes a lipid moiety and a PEG-based polymer moiety (sometimes called poly(ethylene oxide)) (PEG moiety). PEG lipids suitable for use in a lipid composition with a compound of formula (IA), (I), (II) or (III) of the present invention, and information on the biochemistry of such lipids can be found in Romberg et al., Pharmaceutical Research 25 (1 ), 2008, pp. 55-71 and Hoekstra et al., Biochimica et Biophysica Acta 1660 (2004) 41-52. Additional suitable PEG lipids are described, for example, in WO 2015/095340 (page 31, line 14 - page 37, line 6), WO 2006/007712 and WO 2011/076807 (clandestine lipids).

In some embodiments, the lipid moiety may be derived from a diacylglycerol or diacylglycamide, including groups containing a dialkylglycerol or dialkylglycamide group having an alkyl chain length independently containing from about _C4 to about _C40 saturated or unsaturated carbon atoms, wherein the chain may contain one or more several functional groups, such as, for example, an amide or an ester. In some embodiments, the alkyl chain length is from about C ₁₀ to C ₂₀ . The dialkylglycerol or dialkylglycamide group may further contain one or more substituted alkyl groups. The lengths of the chains can be symmetrical or asymmetrical.

Unless otherwise specified, the term PEG as used herein means any polyethylene glycol or other polyalkylene ether polymer, such as an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some embodiments, the PEG moiety is not substituted. Alternatively, the PEG moiety may be substituted, for example, with one or more alkyl, alkoxy, acyl, hydroxy or aryl groups. For example, the PEG moiety may include a PEG copolymer such as PEG-polyurethane or PEG-polypropylene (see, for example, J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); alternatively, the PEG moiety may be a PEG homopolymer. In some embodiments, the PEG moiety has a molecular weight of from about 130 to about 50,000, such as from about 150 to about 30,000, or even from about 150 to about 20,000. Likewise, the PEG moiety may have a molecular weight of from about 150 to about 15,000 , from about 150 to about 10,000, from about 150 to about 6,000, or even from about 150 to about 5,000. In some preferred embodiments of the invention, the PEG moiety has a molecular weight of from about 150 to about 4,000, from about 150 to about 3,000, from about 300 to about 3000, from about 1000 to about 3000, or from about 1500 to about 2500.

In some preferred embodiments, the PEG moiety is PEG-2K, also called PEG 2000, which has an average molecular weight of about 2000 daltons. PEG-2K is represented here by the following formula (II), where n is 45, which means that

l. Lor ΐ^Ίη , . . . . ^<llL

However, other PEG embodiments known in the art may be used, including, for example, those in which the number-averaged degree of polymerization is about 23 subunits (n=23) and/or 68 subunits (n=68). In some embodiments, n may range from about 30 to about 60. In some embodiments, n may range from about 35 to about 55. In some embodiments, n may range from about 35 to about 55.

- 51 046244 n may be in the range of about 40 to about 50. In some embodiments, n may be in the range of about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl, such as methyl.

In any of the embodiments described herein, the PEG lipid may be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (catalog number GM020 from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-distearoylglycerol ( PEG-DSPE) (catalog number DSPE-020CN, NOF, Tokyo, Japan), PEG-dilauryl glycamide, PEG dimyristyl glycamide, PEG-dipalmitoyl glycamide and PEG distearoyl glycamide, PEG cholesterol (1[8'-(Cholest-5-en-3 [beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methylpoly(ethylene glycol), PEG-DMB (3,4-ditetradecoxybenzyl-[omega]-methyl-poly(ethylene glycol)ether), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-\-[methoxy(polyethylene glycol)-2000] (PEG2k-EMO) (Cat. No. 880150P from Avanti Polar Lipids, Alabaster, AL, USA), 1,2- distearoyl-sn-glycero-3phosphoethanolamine-M-[methoxy(polyethylene glycol)-2000] (TOr2k-DSPE) (cat. no. 880120C from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2kDSG; GS-020, NOF Tokyo, Japan), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA) and 1,2distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In some such embodiments, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In other embodiments, the PEG lipid may be PEG2k-DSPE. In some embodiments, the PEG lipid may be PEG2k-DMA. In yet other embodiments, the PEG lipid may be PEG2k-C.'-AML. In some embodiments, the PEG lipid may be compound S027 disclosed in WO2016/010840 (paragraphs [00240] to [00244]). In some embodiments, the PEG lipid may be PEG2k-DSΛ. In other embodiments, the PEG lipid may be PEG2k-C11. In some embodiments, the PEG lipid may be PEG2k-C14. In some embodiments, the PEG lipid may be PEG2xC16. In some embodiments, the PEG lipid may be PEG2k-C18.

Cationic lipids suitable for use in the lipid composition herein include, but are not limited to, H.'H-dioleyl-'H.'N-dimethylammonium chloride (DODAC), KH-distearyl-KH-dimethylammonium bromide (DDAB) , N(1-(2,3-dioleyloxy)propyl)-N,^Ntrimethylammonium chloride (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N-(1-(2,3dioleyloxy) propyl)-H,H,H-trimethylammonium chloride (DOTMA), 1,2-dioleoylcarbamyl-3dimethylammonium propane (DOCDAP), 1,2-dilineyl-3-dimethylammonium propane (DLINDAP), dilauryl(C12:0) trimethyl ammonium propane (DLTAP), Dioctadecylamidoglycyl spermine (DOGS), DC-Hoi, dioleyloxy-H-[2-(sperminecarboxamido)ethyl]-H,H-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3- dimethyl-hydroxyethyl ammonium bromide (DMRIE), 3-dimethylamino-2(Cholest-5-ene-3-beta-hydroxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), N,Ndimethyl-2,3-dioleyloxy)propylamine (DODMA), 2-[5'-(cholest-5-en-3[beta]-oxy)-3'-oxapentoxy)-3dimethyl-1-(cis,cis -9', 1 -2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4dioleyloxybenzylamine (DMOBA) and N-N^'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP). In one embodiment, the cationic lipid is DOTAP or DLTAP.

Anionic lipids suitable for use herein include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidyl ethanolamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine cholesterol hemisuccinate (CHEMS), and lysylphosphatidylglycerol.

Lipid compositions.

The present invention provides a lipid composition containing at least one compound of formula (IA), (I), (II) or (III) or a salt thereof (eg, a pharmaceutically acceptable salt thereof) and at least one other lipid component. Such compositions may also contain a biologically active agent, optionally in combination with one or more other lipid components. In some embodiments, the lipid compositions comprise a lipid component and an aqueous component containing a biologically active agent.

In one embodiment of the invention, the lipid composition contains a compound of formula (IA), (I), (II) or (III) or a pharmaceutically acceptable salt thereof and at least one other lipid component. In another embodiment of the invention, the lipid composition further comprises a biologically active agent, optionally in combination with one or more other lipid components. In another embodiment of the invention, the lipid composition is in the form of liposomes. In another embodiment of the invention, the lipid composition is in the form of lipid nanoparticles (LNPs). In another embodiment of the invention, the lipid composition is suitable for delivery to the liver.

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In one embodiment of the invention, the lipid composition contains a compound of formula (IA), (I), (II) or (III) or a pharmaceutically acceptable salt thereof and another lipid component. Such other lipid components include, but are not limited to, neutral lipids, auxiliary lipids, PEG lipids, cationic lipids and anionic lipids. In some embodiments, the lipid composition comprises a compound of formula (IA), (I), (II) or (III), or a pharmaceutically acceptable salt thereof, and a neutral lipid, for example DSPC, optionally with one or more additional lipid components. In another embodiment of the invention, the lipid composition contains a compound of formula (IA), (I), (II) or (III) or a pharmaceutically acceptable salt thereof and an auxiliary lipid, for example cholesterol, optionally with one or more additional lipid components. In a further embodiment of the invention, the lipid composition contains a compound of formula (IA), (I), (II) or (III) or a pharmaceutically acceptable salt thereof and a PEG lipid, optionally with one or more additional lipid components. In a further embodiment of the invention, the lipid composition contains a compound of formula (IA), (I), (II) or (III) or a pharmaceutically acceptable salt thereof and a cationic lipid, optionally with one or more additional lipid components. In a further embodiment of the invention, the lipid composition contains a compound of formula (IA), (I), (II) or (III) or a pharmaceutically acceptable salt thereof and an anionic lipid, optionally with one or more additional lipid components. In a sub-embodiment of the invention, the lipid composition contains a compound of formula (IA), (I), (II) or (III) or a pharmaceutically acceptable salt thereof, an auxiliary lipid and a PEG lipid, optionally with a neutral lipid. In a further sub-embodiment of the invention, the lipid composition contains a compound of formula (IA), (I), (II) or (III) or a pharmaceutically acceptable salt thereof, an auxiliary lipid, a PEG lipid and a neutral lipid.

Compositions containing lipids of Formula (IA), (I), (II) or (III), or pharmaceutically acceptable salts thereof, or lipid compositions thereof, may be in various forms, including, but not limited to, particulate delivery agents, including microparticles, nanoparticles and transfection agents that are used to deliver various molecules into cells. Particular compositions are effective for transfection or delivery of biologically active agents. Preferred biologically active agents are RNA and DNA. In further embodiments of the invention, the biologically active agent is selected from mRNA, gRNA and DNA. gRNA can be dgRNA or sRNA. In some embodiments, the cargo includes an mRNA encoding an RNA-guided DNA binding agent (eg, a Cas nuclease, a class 2 Cas nuclease, or Cas9), a gRNA, or a nucleic acid encoding a gRNA, or a combination of mRNA and gRNA.

Representative compounds of formula (IA), (I), (II) or (III) for use in the above lipid compositions are given in Examples 2-48, 50-97, 99-103 and 105-113.

In some embodiments, the compound of formula (IA) is compound 2. In some embodiments, the compound of formula (IA) is compound 3. In some embodiments, the compound of formula (IA) is compound 4. In some embodiments, the compound of formula (IA) is compound 5. In some embodiments, the compound of formula (IA) is compound 6. In some embodiments, the compound of formula (IA) is compound 7. In some embodiments, the compound of formula (IA) is compound 8. In some embodiments, the compound of formula (IA) is compound 9. In some embodiments, the compound of formula (IA) is compound 10.

In some embodiments, the compound of formula (IA) is compound 11. In some embodiments, the compound of formula (IA) is compound 12. In some embodiments, the compound of formula (IA) is compound 13. In some embodiments, the compound of formula (IA ) is compound 14. In some embodiments, the compound of formula (IA) is compound 15. In some embodiments, the compound of formula (IA) is compound 16. In some embodiments, the compound of formula (IA) is compound 17. In some embodiments, the compound of formula (IA) is compound 17. in some embodiments, the compound of formula (IA) is compound 18. In some embodiments, the compound of formula (IA) is compound 19. In some embodiments, the compound of formula (IA) is compound 20.

In some embodiments, the compound of formula (IA) is compound 21. In some embodiments, the compound of formula (IA) is compound 22. In some embodiments, the compound of formula (IA) is compound 23. In some embodiments, the compound of formula (IA ) is compound 24. In some embodiments, the compound of formula (IA) is compound 25. In some embodiments, the compound of formula (IA) is compound 26. In some embodiments, the compound of formula (IA) is compound 27. In some embodiments, the compound of formula (IA) is compound 27. implementation options

- 53 046244 the compound of formula (IA) is compound 28. In some embodiments, the compound of formula (IA) is compound 29. In some embodiments, the compound of formula (IA) is compound 30.

In some embodiments, the compound of formula (IA) is compound 31. In some embodiments, the compound of formula (IA) is compound 32. In some embodiments, the compound of formula (IA) is compound 33. In some embodiments, the compound of formula (IA ) is compound 34. In some embodiments, the compound of formula (IA) is compound 35. In some embodiments, the compound of formula (IA) is compound 36. In some embodiments, the compound of formula (IA) is compound 37. In some embodiments, the compound of formula (IA) is compound 37. in some embodiments, the compound of formula (IA) is compound 38. In some embodiments, the compound of formula (IA) is compound 39. In some embodiments, the compound of formula (IA) is compound 40.

In some embodiments, the compound of formula (IA) is compound 41. In some embodiments, the compound of formula (IA) is compound 42. In some embodiments, the compound of formula (IA) is compound 43. In some embodiments, the compound of formula (IA ) is compound 44. In some embodiments, the compound of formula (IA) is compound 45. In some embodiments, the compound of formula (IA) is compound 46. In some embodiments, the compound of formula (IA) is compound 47. In some embodiments, the compound of formula (IA) is compound 47. in some embodiments, the compound of formula (IA) is compound 48. In some embodiments, the compound of formula (IA) is compound 50.

In some embodiments, the compound of formula (IA) is compound 51. In some embodiments, the compound of formula (IA) is compound 52. In some embodiments, the compound of formula (IA) is compound 53. In some embodiments, the compound of formula (IA ) is compound 54. In some embodiments, the compound of formula (IA) is compound 55. In some embodiments, the compound of formula (IA) is compound 56. In some embodiments, the compound of formula (IA) is compound 57. In some embodiments, the compound of formula (IA) is compound 57. in some embodiments, the compound of formula (IA) is compound 58. In some embodiments, the compound of formula (IA) is compound 59. In some embodiments, the compound of formula (IA) is compound 60.

In some embodiments, the compound of formula (IA) is compound 61. In some embodiments, the compound of formula (IA) is compound 62. In some embodiments, the compound of formula (IA) is compound 63. In some embodiments, the compound of formula (IA ) is compound 64. In some embodiments, the compound of formula (IA) is compound 65. In some embodiments, the compound of formula (IA) is compound 66. In some embodiments, the compound of formula (IA) is compound 67. In some embodiments, the compound of formula (IA) is compound 67. in some embodiments, the compound of formula (IA) is compound 68. In some embodiments, the compound of formula (IA) is compound 69. In some embodiments, the compound of formula (IA) is compound 70.

In some embodiments, the compound of formula (IA) is compound 71. In some embodiments, the compound of formula (IA) is compound 72. In some embodiments, the compound of formula (IA) is compound 73. In some embodiments, the compound of formula (IA ) is compound 74. In some embodiments, the compound of formula (IA) is compound 75. In some embodiments, the compound of formula (IA) is compound 76. In some embodiments, the compound of formula (IA) is compound 77. In some embodiments, the compound of formula (IA) is compound 77. in some embodiments, the compound of formula (IA) is compound 78. In some embodiments, the compound of formula (IA) is compound 79. In some embodiments, the compound of formula (IA) is compound 80.

In some embodiments, the compound of formula (IA) is compound 81. In some embodiments, the compound of formula (IA) is compound 82. In some embodiments, the compound of formula (IA) is compound 83. In some embodiments, the compound of formula (IA ) is compound 84. In some embodiments, the compound of formula (IA) is compound 85. In some embodiments, the compound of formula (IA) is compound 86. In some embodiments, the compound of formula (IA) is compound 87. In some embodiments, the compound of formula (IA) is compound 87. in some embodiments, the compound of formula (IA) is compound 88. In some embodiments, the compound of formula (IA) is compound 89. In some embodiments, the compound of formula (IA) is compound 90.

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In some embodiments, the compound of formula (IA) is compound 91. In some embodiments, the compound of formula (IA) is compound 92. In some embodiments, the compound of formula (IA) is compound 93. In some embodiments, the compound of formula (IA ) is compound 94. In some embodiments, the compound of formula (IA) is compound 95. In some embodiments, the compound of formula (IA) is compound 96. In some embodiments, the compound of formula (IA) is compound 97. In some embodiments, the compound of formula (IA) is compound 97. in some embodiments, the compound of formula (IA) is compound 99. In some embodiments, the compound of formula (IA) is compound 100.

In some embodiments, the compound of formula (IA) is compound 101. In some embodiments, the compound of formula (IA) is compound 102. In some embodiments, the compound of formula (IA) is compound 103. In some embodiments, the compound of formula (IA ) is compound 105. In some embodiments, the compound of formula (IA) is compound 106. In some embodiments, the compound of formula (IA) is compound 107. In some embodiments, the compound of formula (IA) is compound 108. In some embodiments, the compound of formula (IA) is compound 108. in some embodiments, the compound of formula (IA) is compound 109. In some embodiments, the compound of formula (IA) is compound 110.

In some embodiments, the compound of formula (IA) is compound 111. In some embodiments, the compound of formula (IA) is compound 112. In some embodiments, the compound of formula (IA) is compound 113.

In some embodiments, the compound is not Compound 1. In some embodiments, the compound is not Compound 49. In some embodiments, the compound is not Compound 114.

LNP compositions.

Lipid compositions can be presented as LNP compositions. Lipid nanoparticles may be, for example, microspheres (including single-layer and multilayer vesicles, for example, liposomes - lipid bilayers of the lamellar phase, which in some embodiments of the invention are substantially spherical, and in more specific embodiments of the invention may include an aqueous core, to for example, containing part of RNA molecules), dispersed phase in an emulsion, micelles or internal phase in suspension.

LNPs have a size of from about 1 to about 1000 nm, from about 10 to about 500 nm, from about 20 to about 500 nm, in a further embodiment from about 50 to about 400 nm, in a further embodiment from about 50 to about 300 nm, in a further embodiment from about 50 to about 200 nm, and in a further embodiment from about 50 to about 150 nm, and in another further embodiment from about 60 to about 120 nm. Preferably, the LNPs have a size of from about 60 nm to about 100 nm. The average dimensions (diameters) of fully formed LNP can be measured by dynamic light scattering on a Malvern Zetasizer. The LNP sample is diluted in phosphate-buffered saline (PBS) so that the emission intensity is approximately 200-400 kHz/s. Data are presented as a weighted average of the intensity index.

Embodiments of the present invention relate to lipid compositions described in accordance with the respective molar ratios of the lipid components in the composition. All mole percentage numbers are given as fractions of the lipid component of the lipid composition or, more specifically, LNP compositions. In some embodiments of the invention, mol. The % of a compound of formula (IA), (I), (II) or (III) can be from about 30 mol.% to about 70 mol.%. In some embodiments, the mol % of a compound of formula (IA), (I), (II) or (III) may be at least 30 mol %, at least 40 mol %, at least 50 mol %. % or at least 60 mol.%.

In some embodiments, the mole % of the neutral lipid can be from about 0 mole % to about 30 mole %. In some embodiments, the neutral lipid mole% may be from about 0 mole% to about 20 mole%. In some embodiments, the mole% neutral lipid may be about 10 mole%. In some embodiments, the mole% neutral lipid may be about 9 mole%.

In some embodiments, the mole percentage of the auxiliary lipid can be from about 0 mol.% to about 80 mol.%. In some embodiments, the mol % of the auxiliary lipid may be from about 20 mol % to about 60 mol %. In some embodiments, the mole % of the auxiliary lipid may be from about 30 mol.% to about 50 mol.%. In some embodiments, the mol % of the auxiliary lipid may be from 30 mol % to about 40 mol %, or from about 35 mol % to about 45 mol %. In some embodiments, the mole % of the auxiliary lipid is adjusted depending on the concentration of the compound of formula (IA), (I), (II) or (III), neutral lipid and/or

- 55 046244 PEG lipid to provide the lipid component up to 100 mol.%.

In some embodiments, the mole % of the PEG lipid can be from about 1 mole % to about 10 mole %. In some embodiments, the mole % of the PEG lipid can be from about 1 mole % to about 4 mole %. In some embodiments, the mol % of the PEG lipid may be from about 1 mol % to about 2 mol %. In some embodiments, the mole % of the PEG lipid may be about 1.5 mole %.

In various embodiments of the invention, the LNP composition contains a compound of formula (IA), (I), (II) or (III) or a salt thereof (such as a pharmaceutically acceptable salt thereof (eg, as disclosed herein)), a neutral lipid ( eg DSPC), auxiliary lipid (eg cholesterol) and PEG lipid (eg PEG2k-DMG). In some embodiments, the LNP composition contains a compound of formula (IA), (I), (II) or (III) or a pharmaceutically acceptable salt thereof (eg, as described herein), DSPC, cholesterol and PEG lipid. In some such embodiments, the LNP composition contains a PEG lipid containing DMG, such as PEG2k-DMG. In some preferred embodiments of the invention, the LNP composition contains a compound of formula (IA), (I), (II) or (III) or a pharmaceutically acceptable salt thereof, cholesterol, DSPC and PEG2k-DMG.

In some embodiments, lipid compositions, such as LNP compositions, contain a lipid component and a nucleic acid component, for example an RNA component, and a molar ratio of a compound of formula (IA), (I), (II) or (III) to nucleic acid. Embodiments of the present invention also provide lipid compositions having a specific molar ratio between the positively charged amino groups of pharmaceutically acceptable salts of compounds of formula (IA), (I), (II) or (III) (N) and the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This can be represented mathematically by the N/P equation. In some embodiments, a lipid composition, such as an LNP composition, may contain a lipid component that includes a compound of formula (IA), (I), (II) or (III) or a pharmaceutically acceptable salt thereof; and a nucleic acid component, wherein the N/P ratio is from about 3 to 10. In some embodiments, the LNP composition may contain a lipid component that includes a compound of formula (IA), (I), (II) or (III) or thereof a pharmaceutically acceptable salt; and an RNA component in which the N/P ratio is from about 3 to 10. For example, the N/P ratio may be about 4-7. Alternatively, the N/P ratio may be about 6, for example 6±1 or 6±0.5.

In some embodiments of the invention, the aqueous component contains a biologically active agent. In some embodiments, the aqueous component includes a polypeptide, optionally in combination with a nucleic acid. In some embodiments, the aqueous component contains a nucleic acid, such as RNA. In some embodiments, the aqueous component is a nucleic acid component. In some embodiments, the nucleic acid component includes DNA and may be referred to as a DNA component. In some embodiments, the nucleic acid component includes RNA, and may be referred to as an RNA component. In some embodiments, the aqueous component, such as an RNA component, may contain mRNA, such as mRNA encoding an RNA-guided DNA binding agent.

In some embodiments, the RNA-guided DNA binding agent is a Cas nuclease. In some embodiments, the aqueous component may contain mRNA encoding Cas9. In some embodiments, the aqueous component may comprise gRNA. In some compositions containing mRNA encoding an RNA-guided DNA binding agent, the composition further comprises a gRNA nucleic acid, such as gRNA. In some embodiments, the aqueous component contains an RNA- and gRNA-driven DNA binding agent. In some embodiments, the aqueous component comprises Cas nuclease mRNA and gRNA. In some embodiments, the aqueous component comprises Cas class 2 nuclease mRNA and gRNA.

In some embodiments, a lipid composition, such as an LNP composition, may contain an mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, a compound of formula (IA), (I), (II) or (III), or a pharmaceutically acceptable salt thereof , an auxiliary lipid, optionally a neutral lipid and a PEG lipid. In some compositions containing mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, the accessory lipid is cholesterol. In other compositions containing mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, the neutral lipid is DSPC. In additional embodiments containing mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, such as Cas9, the PEG lipid is PEG2k-DMG. In specific compositions containing mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, and a compound of formula (IA), (I), (II) or (III) or a pharmaceutically acceptable salt thereof. In some compositions, the composition further comprises gRNA, such as dgRNA or sRNA.

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In some embodiments, the lipid composition, such as an LNP composition, may contain gRNA. In some embodiments, the composition may contain a compound of formula (IA), (I), (II) or (III) or a pharmaceutically acceptable salt thereof, a gRNA, an auxiliary lipid, an optional neutral lipid, and a PEG lipid. In some LNP compositions containing gRNA, the accessory lipid is cholesterol. In some compositions containing gRNA, the neutral lipid is DSPC. In additional embodiments containing gRNA, the PEG lipid is PEG2k-DMG. In some compositions, the gRNA is selected from dgRNA and sgRNA.

In some embodiments, the lipid composition, such as an LNP composition, contains mRNA encoding an RNA-guided DNA binding agent and a gRNA, which may be an sRNA, in an aqueous component and a compound of formula (IA), (I), (II) or (III) in the lipid component. For example, the LNP composition may contain a compound of formula (IA), (I), (II) or (III) or a pharmaceutically acceptable salt thereof, mRNA encoding Cas nuclease, mRNA, auxiliary lipid, neutral lipid and PEG lipid. In some compositions containing mRNA encoding Cas nuclease and gRNA, the accessory lipid is cholesterol. In some compositions containing mRNA encoding Cas nuclease and gRNA, the neutral lipid is DSPC. In additional embodiments containing mRNA encoding a Cas nuclease and gRNA, the PEG lipid is PEG2k-DMG.

In some embodiments, lipid compositions, such as LNP compositions, include an RNA-guided DNA binding agent, such as a Cas class 2 mRNA and at least one gRNA. In some embodiments, the LNP composition includes a ratio of gRNA to RNA-guided DNA binding agent mRNA, such as Cas class 2 nuclease mRNA, of about 1:1 or about 1:2. In some embodiments, the ratio is from about 25:1 to about 1:25, from about 10:1 to about 1:10, from about 8:1 to about 1:8, from about 4:1 to about 1:4 , or from about 2:1 to about 1:2.

Lipid compositions disclosed herein, such as LNP compositions, may include a template nucleic acid, such as a DNA template. The template nucleic acid may be delivered with or separately from lipid compositions containing a compound of formula (IA), (I), (II) or (III), or a pharmaceutically acceptable salt thereof, including as LNP compositions. In some embodiments, the template nucleic acid may be single-stranded or double-stranded, depending on the desired repair mechanism. The template may have regions of homology with the target DNA, for example in the target DNA sequence and/or in sequences adjacent to the target DNA.

In some embodiments, LNPs are formed by mixing an aqueous solution of RNA with an organic solvent-based lipid solution. Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, acetate buffer, ethanol, chloroform, diethyl ether, cyclohexane, tetrahydrofuran, methanol, isopropanol. For example, the organic solvent could be 100% ethanol. A pharmaceutically acceptable buffer can be used, for example, to administer LNP in vivo. In some embodiments, a buffer is used to maintain the pH of the LNP-containing composition at or above pH 6.5. In some embodiments, a buffer is used to maintain the pH of the LNP-containing composition at or above pH 7.0. In some embodiments, the composition has a pH in the range of about 7.2 to about 7.7. In additional embodiments, the composition has a pH in the range of about 7.3 to about 7.7, or in the range of about 7.4 to about 7.6. In additional embodiments, the composition has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of the composition can be measured using a pH microprobe. In some embodiments, a cryoprotectant is included in the composition. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO and ethylene glycol. Exemplary compositions may include up to 10% of a cryoprotectant, such as, for example, sucrose. In some embodiments, the composition may contain Tris-sucrose saline solution (TSS). In some embodiments, the LNP composition may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% cryoprotectant. In some embodiments, the LNP composition may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% sucrose. In some embodiments, the LNP composition may include a buffer. In some embodiments, the buffer may comprise phosphate buffer (PBS), Tris buffer, citrate buffer, and mixtures thereof. In some exemplary embodiments of the invention, the buffer contains NaCl. In some embodiments, the buffer does not contain NaCl. Exemplary amounts of NaCl may range from about 20 mM to about 45 mM. Exemplary amounts of NaCl may range from about 40 mM to about 50 mM. In some embodiments, the amount of NaCl is about 45 mM. In some embodiments, the buffer is a Tris buffer. Approximate amounts of Tris can range from about 20 mM to about 60 mM. Approximate amounts of Tris may range from about 40 mM to about 60 mM. IN

- 57 046244 In some embodiments, the amount of Tris is about 50 mM. In some embodiments, the buffer contains NaCl and Tris. Some exemplary embodiments of LNP compositions contain 5% sucrose and 45 mM NaCl in Tris buffer. In other illustrative embodiments, the compositions contain sucrose at about 5 w/v%, about 45 mM NaCl, and about 50 mM Tris at pH 7.5. The amount of salt, buffer and cryoprotectant can be varied so as to maintain the osmolality of the entire composition. For example, final osmolality can be maintained at less than 450 mOsm/L. In additional embodiments, the osmolality is between 350 and 250 mOsm/L. Some embodiments of the invention have a final osmolality of 300±20 mOsm/L or 310±40 mOsm/L.

Some embodiments of the invention use microfluidic mixing, mixing or cross-mixing of an aqueous solution of RNA and a lipid solution in an organic solvent. In some embodiments, the flow rate, connection size, connection geometry, connection shape, tubing diameter, solutions and/or concentrations of RNA and lipids may vary. LNP or LNP compositions can be concentrated or purified, for example, by dialysis, centrifugal filter, tangential flow filtration or chromatography. LNPs can be stored, for example, in the form of a suspension, emulsion or lyophilized powder. In some embodiments, the LNP composition is stored at 2-8°C, in some embodiments, the LNP composition is stored at room temperature. In additional embodiments of the invention, the LNP composition is stored frozen, for example, at -20°C or -80°C. In other embodiments, the LNP composition is stored at a temperature ranging from about 0°C to about -80°C. LNP compositions can be thawed before use, for example, on ice, at room temperature or at 25°C.

LNPs may be, for example, microspheres (including single-layer and multilayer vesicles, for example, liposomes - lipid bilayers of the lamellar phase, which in some embodiments of the invention are substantially spherical, and, in more specific embodiments, may contain an aqueous core, to for example, including a significant part of RNA molecules), dispersed phase in an emulsion, micelles or internal phase in suspension.

Preferred lipid compositions, such as LNP compositions, are biodegradable in that they do not accumulate to cytotoxic levels in vivo at a therapeutically effective dose. In some embodiments, the compositions do not induce an innate immune response that results in significant side effects at therapeutic dose levels. In some embodiments, the compositions provided herein do not cause toxicity at therapeutic dose levels.

In some embodiments, the LNPs disclosed herein have a polydispersity index (PDI) that can range from about 0.005 to about 0.75. In some embodiments, the LNPs have a PDI that can range from about 0.01 to about 0.5. In some embodiments, the LNP has a PDI that can range from about zero to about 0.4. In some embodiments, the LNP has a PDI that can range from about zero to about 0.35. In some embodiments, the LNP has a PDI that can range from about zero to about 0.35. In some embodiments, the LNP PDI may range from about zero to about 0.3. In some embodiments, the LNP has a PDI that can range from about zero to about 0.25. In some embodiments, the LNP PDI may range from about zero to about 0.2. In some embodiments, the LNP has a PDI that may be less than about 0.08, 0.1, 0.15, 0.2, or 0.4.

The LNPs disclosed herein have a size (eg, Z-average diameter) of about 1 to about 250 nm. In some embodiments, the LNPs range in size from about 10 nm to about 200 nm. In additional embodiments, the LNPs have a size of from about 20 nm to about 150 nm. In some embodiments, the LNPs range in size from about 50 nm to about 150 nm. In some embodiments, the LNPs have a size of from about 50 nm to about 100 nm. In some embodiments, the LNPs range in size from about 50 nm to about 120 nm. In some embodiments, the LNPs have a size of from about 60 nm to about 100 nm. In some embodiments, the LNPs range in size from about 75 nm to about 150 nm. In some embodiments, the LNPs range in size from about 75 nm to about 120 nm. In some embodiments, the LNPs have a size of from about 75 nm to about 100 nm. Unless otherwise stated, all sizes mentioned herein are the average sizes (diameters) of fully formed nanoparticles measured by dynamic light scattering on a Malvern Zetasizer. A sample of nanoparticles is diluted in phosphate-buffered saline (PBS) so that the emission intensity is approximately 200-400 kHz/s. Data are presented as a weighted average of the intensity index (Z-mean diameter).

In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 50% to about 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 50% to about 95%. IN

- 58 046244 Some implementations of LNP are formed with an average encapsulation efficiency ranging from about 70% to about 90%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 90% to about 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 75% to about 95%.

Cargo.

The cargo delivered by the LNP composition may be a biologically active agent. In some embodiments, the cargo is or includes one or more biologically active agents, such as mRNA, guide RNA, nucleic acid, expression vector, template nucleic acid, RNA-guided DNA binding agent, antibody (e.g., monoclonal, chimeric, humanized, nanobody and their fragments, etc.), cholesterol, hormone, peptide, protein, chemotherapeutic and other types of antitumor agents, small molecule drug, vitamin, cofactor, nucleoside, nucleotide, oligonucleotide, enzymatic nucleic acid, antisense nucleic acid, triplex-forming oligonucleotide, antisense DNA or RNA composition, chimeric DNA:RNA composition, allozyme, aptamer, ribozyme, baits and their analogues, plasmids and other types of vectors, as well as small nucleic acid molecule, PHKi agent, short interfering nucleic acid (siNA), short interfering RNA (miRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA) and self-replicating RNA (encoding replicase enzymatic activity and the ability to control its own replication or amplification in vivo) molecules, peptide nucleic acid (PNA ), locked nucleic acid ribonucleotide (LNA), morpholine nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), si-siRNA (small internally segmented interfering RNA) and iRNA (asymmetric interfering RNA). The above list of biologically active agents is exemplary only and is not intended to be limiting. Such compounds may be purified or partially purified, may be naturally occurring or synthetic, and may be chemically modified.

The cargo delivered by the LNP composition may be an RNA, such as an mRNA molecule encoding a protein of interest. For example, mRNA is included to express a protein such as green fluorescent protein (GFP), an RNA-guided DNA binding agent, or a Cas nuclease. LNP compositions are provided that include Cas nuclease mRNA, for example class 2 Cas nuclease mRNA, which enables expression in the cell of a class 2 Cas nuclease, such as the Cas9 or Cpf 1 protein. In addition, the cargo may contain one or more guide RNAs or nucleic acids , encoding guide RNAs. A template nucleic acid, for example, for repair or recombination, may also be included in the composition, or the template nucleic acid may be used in the methods described herein. In a sub-embodiment, the cargo comprises mRNA that optionally encodes Streptococcus pyogenes Cas9 and S. pyogenes gRNA. In a further sub-embodiment of the invention, the cargo includes mRNA that optionally encodes Neisseria meningitidis Cas9 and nme gRNA.

mRNA refers to a polynucleotide and includes an open reading frame that can be translated into a polypeptide (i.e., it can serve as a substrate for translation by the ribosome and aminoacylated tRNAs). The mRNA may include a phosphate-sugar backbone, including ribose residues or analogues thereof, for example, 2'-methoxy-ribose residues. In some embodiments, the sugars of the phosphate sugar backbone of the mRNA consist primarily of ribose residues, 2'-methoxyribose residues, or a combination thereof. Typically, mRNAs do not contain significant amounts of thymidine residues (e.g., 0 residues or less than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6% , 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2% or 0.1% thymidine). The mRNA may contain modified uridines at some or all of the uridine positions.

CRISPR/Cas cargo.

In some embodiments, the disclosed compositions contain mRNA encoding an RNA-guided DNA binding agent, such as a Cas nuclease. In specific embodiments, the disclosed compositions contain mRNA encoding a class 2 Cas nuclease, such as S. Pyogenes Cas9.

As used herein, the term RNA-driven DNA binding agent means a polypeptide or complex of polypeptides having RNA-DNA binding activity, or a DNA binding subunit of such a complex, wherein the DNA binding activity is sequence specific and dependent on the sequence of the RNA. Typical RNA-guided DNA binding agents include Cas cleavages/nickases and their inactivated forms (dCas DNA binding agents). As used herein, the term Cas nuclease covers Cas cleavages, Cas nickases, and dCas DNA binding agents. Cas-cleaving/nickase and dCas DNA-binding agents include the Csm or Cmr complex of the type III CRISPR system, its subunit Cas10, Csm1 or Cmr2, the Cascade complex of the type I CRISPR system, its subunit Cas3, and class 2 Cas nucleases. In this context, a class 2 Cas nuclease is a single-stranded polypeptide with an RNA-guided DNA-binding amino acid.

- 59 046244 activity. Class 2 Cas nucleases include class 2 Cas cleavage/nickases (e.g., H840A, D10A, or N863A variants), which additionally have RNA-guided DNA cleavage or nickase activity, and class 2 dCas DNA-binding agents, in which cleavage/nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (for example, variants N497A, R661A, Q695A, Q926A), HypaCas9 (for example, variants N692A, M694A, Q695A, H698A), eSPCas9(1.0) (for example, variants K81OA, K1OOZA, R1060A) and eSPCas9(1.1) (for example, variants K848A, K1OOZA, R1060A) and their modifications. The Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9 and contains a RuvC-like nuclease domain. Zetsche Cpf1 sequences are included by reference in their entirety. See, for example, Zetsche, Tables S1 and S3. See, for example, Makarova et al., Nat RevMicrobiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

As used herein, the term ribonucleoprotein (RNP) or RNP complex refers to a guide RNA together with an RNA-guided DNA-binding agent, such as a Cas nuclease, e.g., Cascleavage, Cas-nickase, or a dCas DNA-binding agent (eg, Cas9). In some embodiments, the guide RNA directs an RNA-guided DNA binding agent, such as Cas9, to a target sequence, and the guide RNA hybridizes to and binds to the target sequence; in cases where the agent is a cleavage or a nickase, the binding may be followed by a cleavage or a successful pairing in a cross.

In some embodiments of the present invention, a cargo for an LNP composition includes at least one guide RNA comprising guide sequences that direct an RNA-guided DNA binding agent, which may be a nuclease (e.g., a Cas nuclease such as Cas9), to a target DNA. The gRNA can direct a Cas nuclease or a class 2 Cas nuclease to a target sequence on a target nucleic acid molecule. In some embodiments, the gRNA binds to a class 2 Cas nuclease and provides cleavage specificity. In some embodiments, the gRNA and Cas nuclease may form a ribonucleoprotein (RNP), for example, a CRISPR/Cas complex, such as a CRISPR/Cas9 complex. In some embodiments, the CRISPR/Cas complex may be a type II CRISPR/Cas9 complex. In some embodiments, the CRISPR/Cas complex may be a type V CRISPR/Cas complex, such as a Cpf1/guide RNA complex. Cas nucleases and related gRNAs can be paired. The gRNA scaffold structures that bind to each class 2 Cas nuclease vary depending on the specific CRISPR/Cas system.

Guide RNA, gRNA, and simply guide are used interchangeably herein to refer to the related guide nucleic acid for an RNA-guided DNA-binding agent. gRNA can be either sgRNA (also known as CRISPR RNA) or a combination of sgRNA and trRNA (also known as tracrRNA). Guide RNAs may include modified RNAs, as described herein. sgRNA and trRNA can be linked as one RNA molecule (single guide RNA, sgRNA) or as two separate RNA molecules (double guide RNA, dgRNA). Guide RNA or gRNA is one of each type. The trRNA may be a natural sequence or a trRNA sequence with modifications or variations compared to naturally occurring sequences.

As used herein, a guide sequence refers to a sequence in a guide RNA that is complementary to a target sequence and functions to direct the guide RNA to the target sequence for binding or modification (eg, cleavage) by an RNA-guided DNA binding agent. A guide sequence may also be referred to as a targeting sequence or spacer sequence. The guide sequence may be 20 base pairs in length, for example in the case of Streptococcus pyogenes (ie Spy Cas9) and related Cas9 homologs/orthologues. Shorter or longer sequences can also be used as guides, for example 15, 16, 17, 18, 19, 21, 22, 23, 24 or 25 nucleotides in length. In some embodiments, the target sequence is located, for example, on a gene or chromosome and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between the guide sequence and its corresponding target sequence may be about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the guide sequence and the target region may be 100% complementary or identical over a region of at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides. In other embodiments of the invention, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3 or 4 mismatches, where the total length of the target sequence is at least 17, 18, 19, 20 or more base pairs. In some embodiments, the guide sequence and the target region may contain 1-4 mismatches, where the guide sequence contains at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and target

- 60 046244 region may contain 1, 2, 3 or 4 mismatches where the guide sequence contains 20 nucleotides.

Target sequences for RNA-guided DNA-binding proteins, such as Cas proteins, include both the positive and negative strands of genomic DNA (i.e., the target sequence and the reverse complement of the sequence), since the nucleic acid substrate for the Cas protein is a double-stranded nucleic acid acid. Accordingly, when a guide sequence is said to be complementary to a target sequence, it is understood that the guide sequence can direct the gRNA to bind to the reverse complement of the target sequence. Thus, in some embodiments of the invention, where the guide sequence binds the reverse complement of the target sequence, the guide sequence is identical to certain nucleotides of the target sequence (eg, a target sequence that does not include PAM), except for the replacement of U with T in the guide sequence.

The length of the target sequence may depend on the CRISPR/Cas system and the components used. For example, different class 2 Cas nucleases from different bacterial species have different optimal target sequence lengths. Accordingly, the targeting sequence may include 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, 35, 40, 45, 50 or more than 50 nucleotides in length. In some embodiments, the target sequence length is 0, 1, 2, 3, 4, or 5 nucleotides longer or shorter than the natural CRISPR/Cas system guide sequence. In some embodiments, the Cas nuclease and the gRNA scaffold will be derived from the same CRISPR/Cas system. In some embodiments, the targeting sequence may contain or consist of 18-24 nucleotides. In some embodiments, the targeting sequence may contain or consist of 19-21 nucleotides. In some embodiments, the targeting sequence may contain or consist of 20 nucleotides.

In some embodiments, the ssRNA is a Cas9 ssRNA capable of mediating RNA-controlled DNA cleavage by the Cas9 protein. In some embodiments, the ssRNA is a Cpf1 ssRNA capable of mediating RNA-controlled DNA cleavage by the Cpf1 protein. In some embodiments, the gRNA contains sgRNA and tracr-RNA sufficient to form an active complex with the Cas9 protein and RNA-mediated DNA cleavage. In some embodiments, the gRNA contains sgRNAs sufficient to form an active complex with the Cpf1 protein and RNA-mediated DNA cleavage. See Zetsche 2015.

Some embodiments of the invention also provide nucleic acids, for example, expression cassettes encoding the gRNA described herein. Guide RNA nucleic acid is used herein to refer to a guide RNA (eg, sgRNA or dgRNA) and a guide RNA expression cassette, which is a nucleic acid encoding one or more guide RNAs.

Modified RNAs.

In some embodiments, lipid compositions, such as LNP compositions, contain modified nucleic acids, including modified RNA.

Modified nucleosides or nucleotides may be present in RNA, for example gRNA or mRNA. gRNA or mRNA containing, for example, one or more modified nucleosides or nucleotides is called modified RNA to describe the presence of one or more non-natural and/or naturally occurring components or configurations that are used instead of or in addition to the canonical residues A, G, C and U. In some embodiments, the modified RNA is synthesized with a non-canonical nucleoside or nucleotide, referred to herein as modified.

Modified nucleosides and nucleotides may include one or more of: (i) changes, for example, replacement of one or both non-bonding phosphate oxygen atoms and/or one or more linking phosphate oxygen atoms in a phosphodiester backbone bond (exemplary backbone modification); (ii) changing, for example, replacing a moiety of the ribose sugar, for example the 2'hydroxyl in the ribose sugar (exemplary sugar modification); (iii) complete replacement of the phosphate moiety with dephospho linkers (a typical backbone modification); (iv) modifying or replacing a naturally occurring nitrogenous base, including a non-canonical nitrogenous base (exemplary base modification); (v) replacement or modification of the ribose phosphate backbone (exemplary backbone modification); (vi) modification of the 3'-end or 5'-end of the oligonucleotide, for example, removal, modification or replacement of a terminal phosphate group or conjugation of a fragment, cap or linker (such modifications of the 3' or 5' cap may include a sugar and/or modification of the main chain); and (vii) modifying or replacing sugar (exemplary sugar modification).

Some embodiments of the invention include modification of mRNA, gRNA or nucleic acid. Some embodiments of the invention comprise modification of the 5' end of the mRNA, gRNA or nucleic acid. Some embodiments of the invention contain

- 61 046244 modification of the 3'-end of mRNA, gRNA or nucleic acid. The modified RNA may contain modifications at the 5' end and 3' end. Modified RNA may contain one or more modified residues in non-terminal locations. In some embodiments, the gRNA includes at least one modified residue. In some embodiments, the mRNA includes at least one modified residue.

Unmodified nucleic acids may be subject to degradation, for example, by intracellular nucleases or nucleases found in serum. For example, nucleases can hydrolyze phosphodiester bonds of nucleic acids. Accordingly, in one aspect, the RNAs (eg, mRNA, gRNA) described herein may contain one or more modified nucleosides or nucleotides, for example, to impart stability to intracellular or serum-based nucleases. In some embodiments, the modified gRNA molecules described herein may exhibit a reduced innate immune response when introduced into a population of cells both in vivo and ex vivo. The term innate immune response includes the cellular response to exogenous nucleic acids, including single-stranded nucleic acids, which includes the induction of expression and release of cytokines, especially interferons, and cell death.

Accordingly, in some embodiments, the RNA or nucleic acid in the disclosed LNP compositions contains at least one modification that imparts increased or improved stability to the nucleic acid, including, for example, increased resistance to in vivo nuclease digestion. As used herein, the terms modification and modified, as such terms relate to the nucleic acids provided herein, include at least one change that preferably increases stability and makes the RNA or nucleic acid more stable (e.g., resistant to digestion by nucleases) than the wild-type or naturally occurring version of RNA or nucleic acid. As used herein, the terms stable and stable refer to the nucleic acids of the present invention and, in particular with respect to RNA, refer to increased or improved resistance to degradation, for example, by nucleases (i.e., endonucleases or exonucleases) that are typically capable of degrading such RNA. Increased stability may include, for example, less sensitivity to hydrolysis or other degradation by endogenous enzymes (eg, endonucleases or exonucleases) or conditions in the target cell or tissue, thereby increasing or enhancing the residence of such RNA or nucleic acid in the target cell, tissue, subject and/or cytoplasm. The stabilized RNA or nucleic acid molecules provided herein exhibit longer half-lives compared to their naturally occurring, unmodified counterparts (eg, the wild-type version of mRNA). Also by the terms modification and modified are meant the terms relating to the mRNA of the LNP compositions disclosed herein and changes that improve or enhance the translation of mRNA nucleic acids, including, for example, the inclusion of sequences that act in initiating protein translation (eg, Kozak consensus sequence). (Kozak, M., Nucleic Acids Res 15 (20):8125-48 (1987)).

In some embodiments, the RNA or nucleic acid of the LNP compositions described herein has been chemically or biologically modified to make it more stable. Examples of modifications to RNA, such as mRNA, include base depletion (eg, by deletion or substitution of one nucleotide for another) or base modification, eg, chemical modification of the base. The phrase chemical modifications as used herein includes modifications that introduce chemical compositions that differ from those observed in natural RNA or nucleic acids, for example, covalent modifications such as the introduction of modified nucleotides (for example, nucleotide analogues or the inclusion side groups that do not naturally occur in such RNA or nucleic acid molecules).

In some embodiments of backbone modification, the phosphate group of the modified residue may be modified by replacing one or more oxygen atoms with another substituent. In addition, a modified residue, for example, a modified residue present in a modified nucleic acid, may include complete replacement of an unmodified phosphate moiety with a modified phosphate group, as described herein. In some embodiments, backbone modification of the phosphate backbone may include changes that result in either an uncharged linker or a charged linker with an asymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrophosphonates, phosphoramidates, alkyl or arylphosphonates, and phosphotriesters. The phosphorus atom in the unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygen atoms with one of the above atoms or groups of atoms can make the phosphorus atom chiral. A stereogenic phosphorus atom can have an R configuration (here Rp) or an S configuration (here Sp). The frame can also be modified

- 62 046244 by replacing the bridging oxygen (that is, the oxygen that binds the phosphate to the nucleoside) with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene phosphonates). The replacement can occur at either the connecting oxygen or at both connecting oxygen atoms. The phosphate group can be replaced by non-phosphorus connectors in some backbone modifications. In some embodiments, the charged phosphate group may be replaced by a neutral moiety. Examples of moieties that may replace the phosphate group may include, but are not limited to, methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioester, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formoacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

mRNA.

In some embodiments, the composition or composition disclosed herein comprises an mRNA containing an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease or a class 2 Cas nuclease, as described herein. In some embodiments, an mRNA is provided, used, or administered comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease or a class 2 Cas nuclease. The mRNA may contain one or more of a 5' cap, a 5' untranslated region (UTR), a 3' UTR, and a polyadenine tail. The mRNA may contain a modified open reading frame, for example, to encode a nuclear localization sequence or to use alternative codons to encode a protein.

The mRNA in the disclosed LNP compositions may encode, for example, a secreted hormone, enzyme, receptor, polypeptide, peptide, or other protein of interest that is normally secreted. In one embodiment, the mRNA may optionally have chemical or biological modifications that, for example, improve the stability and/or half-life of such mRNA or that improve or otherwise facilitate protein production.

Additionally, suitable modifications include changes to one or more nucleotides of the codon such that the codon codes for the same amino acid but is more stable than the codon found in the wild-type version of the mRNA. For example, an inverse relationship between RNA stability and the abundance of cytidine (C) and/or uridine (U) residues has been demonstrated, and RNA lacking C and U residues has been found to be resistant to most RNases (Heidenreich, et al., J Biol Chem 269, 2131-8 (1994). In some embodiments, the number of C and/or U residues in the mRNA sequence is reduced. In another embodiment of the invention, the number of C and/or U residues is reduced by replacing one codon encoding a particular amino acid with another codon encoding the same or a related amino acid. Putative modifications of mRNA nucleic acids also include the incorporation of pseudouridines. Incorporation of pseudouridines into the nucleic structure of mRNA can increase stability and translational ability, as well as reduce immunogenicity in vivo. See, for example, Kariko, K., et al., Molecular Therapy 16 (11): 1833-1840 (2008). Substitutions and modifications to the mRNA can be made by methods well known to one skilled in the art.

The restrictions on reducing the number of C and U residues in a sequence are likely to be greater in the coding region of the mRNA compared to the untranslated region (i.e., it will likely be impossible to remove all C and U residues present in a message while still maintaining the ability of the message to encode the desired amino acid sequence). However, the degeneracy of the genetic code makes it possible to reduce the number of C and/or U residues present in a sequence while maintaining the same coding capacity (i.e., depending on which amino acid is encoded by a codon, several different possibilities for modifying RNA sequences are possible).

The term modification also includes, for example, the inclusion of non-nucleotide linkages or modified nucleotides in the mRNA sequence (eg, modification of one or both 3' and 5' ends of an mRNA molecule encoding a functionally secreted protein or enzyme). Such modifications include the addition of bases to the mRNA sequence (for example, the inclusion of a polyA tail or a longer polyA tail), changes to the 3'-UTR or 5'-UTR, complexation of the mRNA with an agent (for example, a protein or complementary nucleic acid molecule). acids), as well as the inclusion of elements that change the structure of the mRNA molecule (for example, forming secondary structures).

The poly-A tail is thought to stabilize natural messengers. Therefore, in one embodiment, a long poly-A tail can be added to the mRNA molecule, which makes the mRNA more stable. Poly-A tails can be added using a variety of techniques recognized in the art. For example, long poly-A tails can be added to synthetic or in vitro transcribed mRNA using poly-A polymerase (Yokoe, et al., Nature Biotechnology. 1996; 14: 1252-1256). The transcription vector can also encode long poly-tails. Additionally, poly-A tails can be added by transcription directly from pro

- 63 046244 PCR ducts. In one embodiment, the length of the poly A tail is at least about 90, 200, 300, 400, or at least 500 nucleotides. In one embodiment of the invention, the length of the poly-Tail is adjusted to control the stability of the modified mRNA molecule and thus protein transcription. For example, since the length of the poly-A tail can influence the half-life of the mRNA molecule, the length of the poly-A tail can be adjusted to change the level of nuclease resistance of the mRNA and thus control the time course of protein expression in the cell. In one embodiment of the invention, the stabilized mRNA molecules are sufficiently resistant to degradation in vivo (eg, by nucleases) such that they can be delivered to the target cell without a carrier.

In one embodiment, the mRNA may be modified to include 3' and/or 5' untranslated (UTR) sequences that are not naturally found in wild-type mRNA. In one embodiment, a 3' and/or 5' flanking sequence that naturally flanks the mRNA and encodes a second unrelated protein may be included in the nucleotide sequence of the mRNA molecule encoding the therapeutic or functional protein to modify it. For example, 3' or 5' sequences from mRNA molecules that are stable (eg, globin, actin, GAPDH, tubulin, histone or citric acid cycle enzymes) may be included in the 3' and/or 5' region of the sense molecule mRNA nucleic acid to increase the stability of the sense mRNA molecule. See, for example, US2003/0083272.

More detailed descriptions of mRNA modifications can be found in US2017/0210698A1, pages 57-68, the contents of which are incorporated herein.

Template nucleic acid.

The compositions and methods disclosed herein may include a template nucleic acid. The template can be used to modify or insert a nucleic acid sequence at or near a target site for an RNA-guided DNA-binding protein, such as a Cas nuclease, for example a class 2 Cas nuclease. In some embodiments of the invention, the methods include introducing a template into a cell. In some embodiments, a single template may be provided. In other embodiments, two or more templates may be provided so that editing can occur on two or more target sites. For example, different templates may be provided for editing one gene in a cell or two different genes in a cell.

In some embodiments, the template can be used in homologous recombination. In some embodiments, homologous recombination may result in integration of a template sequence or a portion of a template sequence into a target nucleic acid molecule. In other embodiments, the template can be used in homology-directed repair, which involves the invasion of a DNA strand into a cleavage site in a nucleic acid. In some embodiments, homology-directed repair may result in the inclusion of a template sequence in the edited target nucleic acid molecule. In yet other embodiments, the template can be used in non-homologous end joining-mediated gene editing. In some embodiments, the template sequence is not similar to a nucleic acid sequence adjacent to the cleavage site. In some embodiments of the invention, a template or part of a template sequence is included. In some embodiments, the template includes inverted terminal repeat (ITR) flanking sequences.

In some embodiments, the template sequence may correspond to, contain, or consist of an endogenous sequence of the target cell. It may also or alternatively correspond to, contain or consist of an exogenous sequence of the target cell. As used herein, the term endogenous sequence refers to a sequence that is native to a cell. The term exogenous sequence refers to a sequence that is not native to a cell, or a sequence whose natural location in the cell's genome is elsewhere. In some embodiments, the endogenous sequence may be the genomic sequence of the cell. In some embodiments, the endogenous sequence may be a chromosomal or extrachromosomal sequence. In some embodiments, the endogenous sequence may be a plasmid sequence of the cell.

In some embodiments, the template comprises ss,3HK or dS'^K containing inverse terminal repeat (ITR) flanking sequences. In some embodiments, the template is in the form of a vector, plasmid, minicircle, nanocircuit, or PCR product.

In some embodiments, the nucleic acid is purified. In some embodiments, the nucleic acid is purified using a precipitation method

- 64 046244 denius (for example, LiCl precipitation, alcohol precipitation or an equivalent method, for example, as described in this document). In some embodiments, the nucleic acid is purified using a chromatography-based method, such as an HPLC-based method or an equivalent method (eg, as described herein). In some embodiments, the nucleic acid is purified using both a precipitation method (eg, LiCl precipitation) and an HPLC-based method. In some embodiments, the nucleic acid is purified by tangential flow filtration (TFF).

The compounds or compositions usually, but not necessarily, include one or more pharmaceutically acceptable excipients. The term excipient includes any ingredient other than the compound(s) of the present invention, other lipid component(s) and biologically active agent. An excipient may provide functional (eg, drug release rate control) and/or non-functional (eg, processing aid or diluent) characteristics to the compositions. The choice of excipient will depend largely on factors such as the specific route of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

Formulations for parenteral administration are typically aqueous or oily solutions or suspensions. If the formulation is aqueous, excipients such as sugars (including but not limited to glucose, mannitol, sorbitol, etc.), salts, carbohydrates and buffering agents (preferably pH 3 to 9), but for some applications they may be more suitably formulated in a sterile non-aqueous solution or in a dried form for use in combination with a suitable vehicle such as sterile pyrogen-free water (WFI).

Although the inventions have been described in conjunction with the illustrated embodiments, it should be understood that they are not intended to limit the inventions to these embodiments. Rather, the inventions are intended to cover all alternatives, modifications and equivalents, including equivalents of specific features, that may be included in the invention as defined by the appended claims.

Both the foregoing general description and the detailed description and the following examples are illustrative and explanatory only and are not limiting of the ideas. The section headings used herein are for organizational purposes only and should in no way be construed as limiting the intended subject matter. In the event that any literature incorporated by reference conflicts with any term defined in this specification, that specification shall control. All ranges shown in the application cover endpoints unless otherwise noted.

Definitions.

It should be noted that when used in this application, the singular forms include references to the plural unless the context clearly dictates otherwise. Thus, for example, a composition reference includes a plurality of compositions, and a cell reference includes a plurality of cells, and the like. The use of or is inclusive and means and/or unless otherwise specified.

Unless specifically stated in the above description, embodiments in the description that repeat containing various components are also considered to consist of or consist substantially of the listed components; embodiments in the description that repeat being composed of various components are also considered to contain or consist substantially of the listed components; embodiments of the invention in the description that talk about various components are also considered as with the listed components; and embodiments in the specification that repeat consisting essentially of various components are also considered to be composed of or containing the listed components (this interchangeability does not apply to the use of these terms in the claims).

Numeric ranges include numbers that define a range. Measured and measured values are considered approximate, subject to significant figures and uncertainty associated with measurement. As used in this application, the terms approximately and approximately have their meanings as understood in the art; using one over the other does not necessarily imply a different scope of application. Unless otherwise indicated, the figures used in this application, with or without a modifying term such as about or approximate, should be understood to include normal variance and/or fluctuation as would be appreciated by one skilled in the art. In some embodiments, the term approximately or approximately refers to a range of values that are 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 (more or less) of the stated reference value, unless otherwise stated or otherwise not obvious from context (unless such number exceeds 100% of the possible value).

The term contact as used herein means the establishment of a physical connection.

- 65 046244 communication between two or more objects. For example, contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and the nanoparticle are designed to physically communicate together. Methods for contacting cells with external objects both in vivo and ex vivo are well known in the field of biology. For example, contacting the nanoparticle composition and a mammalian cell located within the mammal can be accomplished by various routes of administration (eg, intravenous, intramuscular, intradermal, and subcutaneous) and may involve varying amounts of the nanoparticle composition. Moreover, the nanoparticle composition can contact more than one cell of a mammal.

The term delivery as used here means the delivery of an object to the recipient. For example, delivery of a therapeutic and/or prophylactic agent to a subject may involve administering to the subject a nanoparticle composition, including the therapeutic and/or prophylactic agent (eg, by an intravenous, intramuscular, intradermal, or subcutaneous route). Introduction of the nanoparticle composition into a mammalian or mammalian cell may involve contacting one or more cells with the nanoparticle composition.

As used herein, the term encapsulation efficiency refers to the amount of therapeutic and/or prophylactic agent that becomes part of the nanoparticle composition, relative to the initial total amount of therapeutic and/or prophylactic agent used in preparing the nanoparticle composition. For example, if 97 mg of therapeutic and/or prophylactic agent is encapsulated in a nanoparticle composition from the total 100 mg of therapeutic and/or prophylactic agent originally provided in the composition, the encapsulation efficiency can be 97%. As used herein, the term encapsulation may refer to complete, substantial or partial enclosure, limitation, encirclement or enclosure.

In this context, the term biodegradable is used to refer to materials that, when introduced into cells, are broken down by cellular mechanisms (eg, enzymatic degradation) or hydrolysis into components that the cells can reuse or utilize without significant toxic effect(s) on the cells. In some embodiments, the components generated by the degradation of the biodegradable material do not cause inflammation and/or other adverse effects in vivo. In some embodiments of the invention, biodegradable materials are broken down enzymatically. Alternatively or additionally, in some embodiments, the biodegradable materials are destroyed by hydrolysis.

In this context, the N/P ratio is the molar ratio of ionizable (in the physiological pH range) nitrogen atoms in a lipid (eg, a compound of Formula I) to phosphate groups in RNA, eg, in a nanoparticle composition comprising a lipid component and RNA.

The compositions may also include salts of one or more compounds. The salts may be pharmaceutically acceptable salts. As used herein, the term pharmaceutically acceptable salts refers to derivatives of the described compounds in which the parent compound is modified by converting an existing acidic or basic moiety to its salt form (eg, by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, salts of mineral or organic acids of basic moieties such as amines; alkaline or organic salts of acid residues such as carboxylic acids; etc. Typical acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, etc. Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like, as well as non-toxic ammonium, quaternary ammonium and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and the like. Pharmaceutically acceptable salts of the present invention include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from a starting compound that contains a basic or acidic moiety by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of a suitable base or acid in water or an organic solvent, or a mixture thereof; Non-aqueous media are generally preferred, such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, ^17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.

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H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., JouPHKl of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

The term polydispersity index as used herein is a ratio that describes the uniformity of particle size distribution in a system. A small value, such as less than 0.3, indicates a narrow particle size distribution. In some embodiments, the polydispersity index may be less than 0.1.

As used herein, the term transfection refers to the introduction of a species (eg, RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo or in vivo.

As used herein, the term alkyl means a branched or straight-chain saturated hydrocarbon group containing from 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, c-butyl, m-butyl, n-pentyl, isopentyl, c-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. The alkyl group may be cyclic or acyclic. The alkyl group may be branched or straight-chain (ie, linear). The alkyl group may also be substituted or unsubstituted (preferably unsubstituted). For example, an alkyl group may be substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfoxo, sulfonate, carboxylate, or thiol, as described herein. A lower alkyl group is an alkyl group containing from one to six (eg, one to four) carbon atoms.

As used herein, the term alkenyl refers to an aliphatic group containing at least one carbon-carbon double bond, and is intended to include both unsubstituted alkenyls and substituted alkenyls, the latter of which refers to alkenyl moieties having hydrogen substituents on one or more atoms carbon of the alkenyl group. Such substituents may occur on one or more carbon atoms that are included or not included in one or more double bonds. Moreover, such substituents include all substituents provided for alkyl groups, as discussed below, except where stability is unacceptable. For example, it is contemplated that the alkenyl group may be substituted with one or more alkyl, carbocyclyl, aryl, heterocyclyl or heteroaryl groups. Examples of alkenyl groups include, but are not limited to, vinyl (-CH= _CH2 ) _, allyl ( _-CH2CH = _CH2 ) _{, cyclopentyl} ( _-C5H7 ) and 5-hexenyl ( _{-CH2CH2CH2CH2} CH= _CH2 ).

An alkylene group refers to a divalent alkyl radical, which may be branched or unbranched (ie, linear). Any of the above monovalent alkyl groups can be converted to alkylene by abstraction of the second hydrogen atom from the alkyl. Typical alkylenes include C _2-4 alkylene and C _2-3 alkylene. Typical alkylene groups include, but are not limited to -CH(CH3)-, -C(CH3)2-, -CH2CH2-, _-CH2CH (CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, - CH2CH2CH2CH2- and the like. The alkylene group may also be substituted or unsubstituted. For example, the alkylene group may be substituted with one or more groups, including, but not limited to, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfoxo, sulfonate, sulfonamide, urea, amide, carbamate, ester, carboxylate or thiol, as described herein.

The term alkenylene includes divalent, straight or branched, unsaturated acyclic hydrocarbyl groups having at least one carbon-carbon double bond and, in one embodiment, no carbon-carbon triple bonds. Any of the above monovalent alkenyl groups can be converted to alkenylene by abstraction of the second hydrogen atom from the alkenyl group. Typical alkenylenes include C _2-6 alkenylenes.

The term C _xy when used in combination with a chemical moiety such as alkyl or alkylene means to include groups that contain x to y carbon atoms in the chain. For example, the term Cx _-y alkyl refers to substituted or unsubstituted saturated hydrocarbon groups, including straight and branched alkyl and alkylene groups, which contain x to y carbon atoms in the chain.

Incorporation of information into the description of the invention by reference.

The contents of articles, patents and patent applications, and all other documents and electronically available information mentioned or cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication had been specifically and individually cited to include via link. Applicant reserves the right to physically include in this application any materials and information from any such articles, patents, patent applications or other physical and electronic documents.

- 67 046244

Examples

Basic information.

All reagents and solvents were purchased and used as received from commercial suppliers or synthesized according to the specified procedures. All intermediates and final compounds were purified using silica gel flash column chromatography. NMR spectra were recorded on a Bruker or Varian 400 MHz spectrometer, and NMR data were collected in CDCl3 at ambient temperature. Chemical shifts are given in parts per million (ppm) relative to CDCl3 (7.26). Data for 1H NMR are presented as follows: chemical shift, multiplicity (br=broad, 8=singlet, d=doublet, t=trinlet, dd=doublet doublet, dt=doublet triplet, q=quartet, m=multinlet, ddd=doublet doublets of doublets, td=triplet of doublets, tt triplet of triplets, tdd=triplet of doublets of doublets, dddd=doublet of doublets of doublets of doublets), coupling constant and integration. MS data were recorded on a Waters SQD2 mass spectrometer with an electrospray ionization (ESI) source. The purity of the final compounds was determined by UPLC-MS-ELS using a Waters Acquity H-Class liquid chromatography instrument equipped with an SQD2 mass spectrometer with photodiode array (PDA) and evaporative light scattering (ELS) detectors.

The pKa of each amine lipid was determined according to the method described by Jayaraman, et al. (Angewandte Chemie, 2012) with the following adaptations. pKa was determined for the unformulated amine lipid in ethanol. Stock lipid solutions (2.94 mM) were diluted with sodium phosphate buffers (0.1 M, Boston Bioproducts) of varying pH (pH range: 4.5–9.0), yielding a final lipid concentration of ca. 100 µM. TNS {6-(p-toluidino)-2naphthalene sulfonic acid sodium salt} was added to the test samples, incubated, and fluorescence intensity was measured using excitation and emission wavelengths of 321 nm and 448 nm, respectively. The recorded data were normalized and the corresponding pKa values were obtained from the sigmoidal fit.

Table 1 Connections

Compound (pKa) Structure 1 (6.35) ABOUT V° % 2 (5.28) ABOUT % 3 (5.89) O V ⁰ %

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8 (7.54) ( / \ / (1 > (I ) / ( \ ^c= x \ p 4 VW o=x '—X /— ⁷ ° == ° z — / 9 (6.32) i OD == y _o y V° X ° X / / ) \ \ i) / ^x / ) 10 (6.05) 0 /X)__________ V o % eleven (5.60) ( / \ / (! < \ ( x ^Q= ( X / ° / p4_ (W ^o= x ⁴ —x )— ⁷ ° )>

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- 71 046244

16 (5.72) ( / \ ) (1 /(( ( ^ο= λ ( / ^ο / ?л_ 0=4 ^o= \ '—\ /— ^z ° o Vo 17 (6.18) 0 ν θ % 18 (5.83) 0 ν ° % Ο'>/--,-^·---·^'------'''-..,-·'-' ⁼ ·''-..,-''· ⁼ '-'--..,--'''·----·'^'--. 19 (5.99) 0 %

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- 73 046244

- 74 046244

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32 (5.64) 0 'JJ'''-'Ό Ao % 33 (5.95) 0 ' Ν ' Ό A) % 34 (6.36) Ν o - ^ν /·ν ^Λ ο Η 1 V ο % ο''\- ^ζχ /'··- 35 (6.35) ( / / |ι (ι \ ) ( \ ^ο= \ ( ?Λ_ (jA == Ε°

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36 (6.13) 0 θγ-Q% 37 (5.36) y g o - 2 - g V Η,^λ >° 2° } l’ / )=° / ) \ \ 1 1) / 2) 38 (5.62) ( / / (1 II \ / ( \ ^o= \ \ °h ? °H o=<'^, g^ o g— l—o $ ° —Ζ \ 39 (6.32) \ ΛΆ ° / 0-2 3Ζ ° _GS G5 Ch?° \ ° \ / )=° / ) \ \ 1) / / )

- 77 046244

40 (6.34) = g O—? IZ У° } с° / / ) \ 1) / \ι| 41 (6.51) o G oA IZ )\ \II 1) / ) ) 42 (6.52) PU' )\ 1) / ) ) 43 (7.33) ί / / (| / / (1 o > o—. ί o A Q=( i )— ⁷ O ZX Vc $ “ r\

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44 (6.45) 1 n.. 0 ''Ν^Ό n X V ⁰ % 45 0 (5.79) G ^N V θ Ογ-Ο^ % o''- ^zx - ^z 46 (6.16) Ό. ABOUT / mA, Η 1 V θ % f47 (5.99) 'ν' O )—7 O

- 79 046244

- 80 046244

- 81 046244

57 (6.18) 0 O-( ZZ :% V’ 0 O 0 58 0 (6.32) •nA, η Λ 0 O O' cr 0 59 O (6.34) X A N O n 1 0 O O ^:? Sx_ 0 0 60 0 (6.22) , ^N. A h y 0 °Α ^ό 0 O 0

- 82 046244

61 (6.29) > ⁰ .- ^ν --'-ν ^Λ ο η JL jT ° ° Ο ^? Ό-.,— _O Ό 0 62 (6.32) w Ο IZ у \ > ^c о Vo s \ ° \ / )= ^o / ) < / ^=0 63 (6.32) A ° -—A Η 1 0 % 64 (6.30) ° o V ) ( ( / ^o= \ ( X ° / / ( Pa oV }— ^/ o °K ' ^— \ P ZI it > ° r\

- 83 046244

- 84 046244

- 85 046244

73 (6.41) ( / / |ι (i \ ) ( / ° / i o=\ '—\ )— ⁷ ° Z= Vo 4 ° \ 74 (6.44) ( / ) (I / 1 75 (6.17) 0 % 76 (6.74) ΑΊ ⁰ Η I V ο %

- 86 046244

77 (7.23) XZ m )\ j) / ) ) 78 (5.93) O OA xz \ ° \ / )=° / )\ j) / / ) 79 (4.96) ΟΟ’ζΧ Η 1 v° % 80 (6.41) \ Λ\ ° / =Z Vo-5 // )\ \I) j) / /

- 87 046244

- 88 046244

85 (7.02) ( ) ) (I (1 \ < ( J ° jj ==( A, V o ,= r ^; $ ° O 86 (6.23) ) - z O >40° / .0 / ) \ il I) / ) ) 87 (ND) 1° 0A 1 H l _ . , v ° 88 (ND) 1 ⁰ _r ^N —Ν ^Λ Ο ' - - °0 0 ^ϋ ·γ4Z=WX %

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89 (6.76) N I .... ~ ... V o % 90 (6.80) o $ ⁰ O-# iz 3,H · 3° Vo ) Wo V ) \ \ i) I) / \ ) 91 (6.48) 1 ⁰ v ° % ₀ Ww-www^=wa=%w^w% 92 (5.96) g 7 <? _x g-<^Л ^> ^o Vo Vo / o / / 1° / ) \ \ 1 I) / ) )

- 90 046244

- 91 046244

- 92 046244

102 (5.16) 0 θγ^ % 103 (6.09) w o $ O-^ IZ V >o 5—0 ( O ( / /—\ )\ i) / \ ) 105 (6.53) - ^Λ 'Ν'3 ⁿ Λ I ⁰ ' Ύ^ο % 106 (6.80) ί / ? ί II\ \ ( oLs4 ζτ λ-ο > ° σ

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107 % ⁰ (6.35) -- ^N N'^'O Η I V o % 108 (6.52) u= ? O-f IZ c _z --( \--4 Vo Vo ) Vo ( ° \ / v° / //( ) / \ ( I) J / / ) 109 /V ° (6.91) v ⁿ -^ _n A _o ^H V V o % 110 Λ^Ί o (7.01) o \ oD ΖΖ ⁷ : . ) \ ) )

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Example 1 - Comparative Compound 1.

Intermediate 1a: 4-oxohexadecanoic acid

A solution of succinic anhydride (3.0 g, 1.0 equiv.) in THF (0.1 M) was cooled to -78°C, followed by the addition of dodecylmagnesium bromide (1.0 M solution in hexane, 0.5 equiv.) dropwise. within 5 min. The reaction was maintained at -78°C for at least 18 hours, after which it was quenched by the addition of water and 1 M HCl. The reaction mixture was concentrated in vacuo and the resulting aqueous layer was extracted with DCM three times. The combined DCM layers were then extracted 2 times with 2 M NaOH, and the combined aqueous layers were acidified to pH 1 with concentrated HCl. The mixture was then extracted 3 times with DCM. The combined DCM layers were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo to give the crude material. The crude material was triturated with hexane three times and the resulting precipitate was filtered to give the product as a white solid (1.31 g, 33%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 2.72 (ddd, J=7.0, 5.8, 1.1 Hz, 2H), 2.67-2.60 (m, 2H), 2, 44 (t, J=7.5 Hz, 2H), 1.59 (q, J=7.4 Hz, 2H), 1.26 (d, J=2.7 Hz, 18H), 0.91- 0.85 (m, 3H).

Intermediate 1b: 2-oxopropane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12dienoate)

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To a solution of dihydroxyacetone (1.55 g, 1.0 eq.) in DCM (0.1 M) was added linoleic acid (2.05 eq.), DMAP (0.2 eq.), DIPEA (2.4 eq. ) and EDCI (2.4 eq.). The reaction mixture was stirred at room temperature for at least 18 hours. Once complete, the mixture was quenched by adding water, and the organic layer was washed 1 time with 1 M HCl and 1 time with 5% NaHCO3. The organic layer was then dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography (EtOAc/hexanes) to the product as a colorless oil (8.0 g, 75%).

1H NMR (400 MHz, CDCl ₃ ) δ 5.44-5.28 (m, 8H), 4.75 (s, 4H), 2.82-2.74 (m, 4H), 2.42 (t , J=7.5 Hz, 4H), 2.05 (q, J=6.7 Hz, 8H), 1.67 (q, J=7.4 Hz, 4H), 1.40-1.24 (m, 28H), 0.95-0.86 (m, 6H).

Intermediate 1c: 2-hydroxy-3-[(9Z,12Z)-octadeca-9,12-dienoyloxy]propyl (9Z,12Z)-octadeca-9,12-dienoate

A solution of intermediate 1b (8.1 g, 1.0 equiv.) in 4:2:1 THF/water/toluene (0.05-0.1 M) was cooled to 0-5°C followed by the addition of NaBH4 (1 .7 eq.). The reaction mixture was stirred for at least 1 hour at 0-25°C. Once complete, the mixture was quenched by adding water and EtOAc, and the mixture was extracted with 3x EtOAc. The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude material was purified by column chromatography (EtOAc/hexanes) to give the product as a colorless oil (5.9 g, 73%).

1H NMR (400 MHz, CDCl3) δ 5.46-5.26 (m, 8H), 4.23-4.10 (m, 4H), 2.81-2.73 (m, 4H), 2, 42 (d, J=4.8 Hz, 1H), 2.38-2.31 (m, 4H), 2.05 (q, J=6.7 Hz, 8H), 1.68-1.57 (m, 4H), 1.42-1.23 (m, 29H), 0.93-0.85 (m, 6H).

Intermediate 1d: 2-((4-oxohexadecanoyl)oxy)propane-1,3-diyl (9Z,9'Z,12Z,12'Z)bis(octadeca-9,12-dienoate)

To a solution of intermediate 1c (1.0 equiv) in DCM (0.2 M), intermediate 1a (1.0-1.2 equiv), Et3N or DIPEA (1.5-4.5 equiv) was added. , DMAP (0.1-0.2 equiv.) and EDCI (1-2 equiv.). The reaction mixture was stirred at room temperature for at least 12 hours and then quenched by adding water. The mixture was washed 1 time with 1 M HCl and 1 time with 5% NaHCO3. The organic layer was then dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude material was purified by column chromatography (EtOAc/hexanes) to give the product as a colorless oil (780 mg, 79%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.45-5.28 (m, 8H), 5.24 (tt, J=5.9, 4.3 Hz, 1H), 4.29 (dd, J=11.9, 4.3 Hz, 2H), 4.15 (dd, J=11.9, 5.8 Hz, 2H), 2.84-2.74 (m, 4H), 2.71 (t, J=6.8 Hz, 2H), 2.59 (t, J=6.3 Hz, 2H), 2.43 (t, J=7.5 Hz, 2H), 2.32 (t , J=7.6 Hz, 4H), 2.05 (q, J=6.8 Hz, 8H), 1.60 (dd, J=14.7, 7.3 Hz, 6H), 1.44 -1.20 (m, 49H), 0.88 (td, J=6.9, 4.3 Hz, 9H).

Intermediate 1e: 2-((4-oxohexadecanoyl)oxy)propane-1,3-diyl (9Z,9'Z,12Z,12'Z)bis(octadeca-9,12-dienoate)

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A solution of intermediate 1d (1.43 g, 1.0 equiv) in 4:2:1 THF/water/toluene (0.05-0.1 M) was cooled to 0-5°C followed by the addition of NaBH ₄ ( 5.0 eq.). The reaction was maintained at 0-25°C for at least 2 hours. The reaction mixture was then diluted with water and EtOAc, and the resulting two-phase mixture was extracted with 3x EtOAc. The combined organic layers were dried over Na ₂ SO4, filtered and concentrated in vacuo. The crude material was purified by column chromatography (EtOAc/hexanes) to give the alcohol intermediate as a colorless oil.

The resulting alcohol was immediately resolubilized in DCM (0.1 M) followed by the addition of pyridine (2-3 equiv.) and 4-nitrophenylchloroformate (1-1.5 equiv.). The reaction mixture was stirred for at least 1 hour at room temperature. The mixture was quenched by adding water and extracted three times with DCM. The combined DCM layers were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude material was purified by column chromatography (EtOAc/hexanes) to give the product as a colorless oil (910 mg, 54%).

^1H NMR (400 MHz, CDCl3) δ 8.37-8.25 (m, 2H), 7.48-7.40 (m, 2H), 5.49-5.25 (m, 9H), 4 ,96-4.84 (m, 1H), 4.34 (dd, J=12.0, 4.3 Hz, 2H), 4.18 (dd, J=11.9, 5.7 Hz, 2H ), 2.79 (t, J=6.4 Hz, 4H), 2.49 (dd, J=8.4, 6.7 Hz, 2H), 2.33 (tt, J=7.6, 4.2 Hz, 4H), 2.16-1.95 (m, 10H), 1.81-1.71 (m, 1H), 1.65 (ddq, J=15.2, 7.5, 4.8, 4.2 Hz, 6H), 1.50-1.13 (m, 50H), 0.90 (td, J=6.8, 3.7 Hz, 9H).

Compound 1: 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9 ,12-dienoate)

To a solution of intermediate 1e (250 mg, 1.0 equiv.) in MeCN (0.1 M) was added pyridine (3.0 equiv.), 3-(dimethylamino)propan-1-ol (1.5-3. 0 equivalent) and DMAP (1.0 equivalent). The reaction mixture was stirred for at least 2 hours at room temperature. Heptane (0.1 M) was then added to the reaction mixture, and the heptane layer was washed 3 times with MeCN. The combined MeCN layers were extracted three times with heptane, and the combined heptane layers were washed one final time with MeCN. The heptane was then removed in vacuo and the crude material was purified by column chromatography (MeOH/DCM) to give the product as a colorless oil (100 mg, 41%).

^1H NMR (400 MHz, CDCl3) δ 5.46-5.22 (m, 9H), 4.72 (tdd, J=7.5, 5.3, 4.0 Hz, 1H), 4.29 (dd, J=12.0, 4.4 Hz, 2H), 4.23-4.10 (m, 4H), 2.84-2.73 (m, 4H), 2.62-2.27 (m, 13H), 2.05 (q, J=6.9 Hz, 8H), 2.01-1.84 (m, 4H), 1.68-1.50 (m, 7H), 1, 43-1.21 (m, 50H), 0.88 (td, J=6.9, 4.1 Hz, 9H). MS: 1001.2 m/z [M+H].

Example 2 - compound 2.

Compound 2: 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9 ,12-dienoate)

Compound 2 was synthesized in 64% yield from intermediate 1e and 3-97 046244 (diethylamino)propan-1-ol using the method used for Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.43-5.20 (m, 9H), 4.72 (tdd, J=7.5, 5.3, 4.0 Hz, 1H), 4.29 (dd, J=11.9, 4.4 Hz, 2H), 4.23-4.10 (m, 4H), 2.81-2.73 (m, 4H), 2.57-2.36 (m, 8H), 2.31 (td, J=7.6, 1.3 Hz, 4H), 2.05 (q, J=6.8 Hz, 8H), 1.97-1.77 ( m, 4H), 1.61 (t, J=7.4 Hz, 6H), 1.40-1.21 (m, 49H), 1.01 (t, J=7.1 Hz, 6H), 0.88 (td, J=6.9, 4.1 Hz, 9H). MS: 1029.7 m/z [M+H].

Example 3 - compound 3.

Compound 3: 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane1,3-diyl (9Z,9'Z, 12Z, 12^)-bis (octadeca-9,12-dienoate)

Compound 3 was synthesized in 79% yield from intermediate 1e and 3((methyl)ethylamino)propan-1-ol using the method used for Example 1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.49-5.20 (m, 9H), 4.77-4.67 (m, 1H), 4.29 (dd, J=11.9, 4 .4 Hz, 2H), 4.16 (tdd, J=11.9, 6.2, 2.5 Hz, 4H), 2.77 (t, J=6.4 Hz, 4H), 2.70 -2.37 (m, 7H), 2.37-2.25 (m, 7H), 2.13-1.82 (m, 12H), 1.69-1.48 (m, 7H), 1 .42-1.20 (m, 46H), 1.14 (s, 2H), 0.88 (td, J=6.8, 3.9 Hz, 7H). MS: 1015.7 m/z [M+H].

Example 4 - compound 4.

Compound 4: 2-((4-(((2-(dimethylamino)ethoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9 ,12-dienoate)

Compound 4 was synthesized in 93% yield from intermediate 1e and 2 dimethylaminoethan-1-ol using the method used for Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.42-5.31 (m, 8H), 5.25 (tt, J=5.8, 4.3 Hz, 1H), 4.73 (tdd, J =7.6, 5.5, 4.0 Hz, 1H), 4.29 (dd, J=11.9, 4.4 Hz, 4H), 4.14 (ddd, J=11.9, 5 ,8, 3.4 Hz, 2H), 2.77 (td, J=6.0, 1.1 Hz, 4H), 2.70 (d, J=16.4 Hz, 1H), 2.49 -2.29 (m, 11H), 2.05 (q, J=6.8 Hz, 8H), 1.99-1.81 (m, 2H), 1.67-1.50 (m, 8H ), 1.40-1.21 (m, 48H), 0.88 (td, J=7.0, 4.2 Hz, 9H). MS: 987.6 m/z [M+H].

Example 5 - compound 5.

Compound 5: 2-((4-(((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane1,3-diyl (9Z,9'Z, 12Z, 12^)-bis (octadeca-9,12-dienoate)

ABOUT

Compound 5 was synthesized in 54% yield from intermediate 1e and 3 (pyrrolidin-1-yl)propan-1-ol using the method used for Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.45-5.21 (m, 9H), 4.72 (tdd, J=7.3, 5.5, 4.2 Hz, 1H), 4.29 (dd, J=11.9, 4.4 Hz, 2H), 4.17 (dddd, J=15.3, 9.1, 7.3, 3.7 Hz, 4H), 2.77 (td , J=6.0, 1.1 Hz, 4H), 2.57 (d, J=8.8 Hz, 6H), 2.40 (dt, J=8.7, 6.5 Hz, 2H) , 2.31 (td, J=7.6, 1.2 Hz, 4H), 2.05 (q, J=6.8 Hz, 8H), 1.98-1.83 (m, 5H), 1.80 (p, J=3.2 Hz, 4H), 1.68-1.50 (m, 7H), 1.41-1.18 (m, 48H), 0.96-0.81 ( m, 9H). MS: 1027.6 m/z [M+H].

Example 6 - compound 6.

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Example 6: 2-((4-((((1-ethylpiperidin-4-yl)oxy)carbonyl)oxy)hexadecanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12'Z )-6uc(oKTageka-9,12-dienoate)

Compound 6 was synthesized in 53% yield from intermediate 1e and 1ethylpiperidin-4-ol using the same method as Example 1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.42-5.21 (m, 9H), 4.72 (tdd, J=7.6, 5.3, 4.0 Hz, 1H), 4, 64 (d, J=5.1 Hz, 1H), 4.29 (dd, J=11.9, 4.4 Hz, 2H), 4.14 (ddd, J=11.9, 5.8, 4.1 Hz, 2H), 2.84-2.70 (m, 6H), 2.51-2.36 (m, 4H), 2.31 (td, J=7.6, 1.4 Hz , 4H), 2.24 (d, J=23.1 Hz, 2H), 2.11-1.93 (m, 11H), 1.92-1.73 (m, 4H), 1.70- 1.52 (m, 8H), 1.431.21 (m, 48H), 1.09 (t, J=7.2 Hz, 3H), 0.88 (td, J=7.0, 4.2 Hz , 9Н). MS: 1028.2 m/z [M+H].

Example 7 - compound 7.

Compound 7: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)hexadecanoyl)oxy)propan-1,3diyl (9Z,9'Z, 12Z, 12^)-bis (octadeca-9,12-dienoate)

Compound 7 was synthesized in 66% yield from intermediate 1e and Ν1-3τηλ-Ν1 methylethane-1,2-diamine using the method used for Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.47-5.08 (m, 10H), 4.76 (s, 1H), 4.28 (ddd, J=11.9, 4.4, 1, 4 Hz, 2H), 4.15 (ddd, J=11.9, 5.9, 3.3 Hz, 2H), 3.24 (q, J=6.0 Hz, 2H), 2.82- 2.74 (m, 4H), 2.53-2.26 (m, 10H), 2.21 (s, 3H), 2.05 (q, J=6.8 Hz, 8H), 1.98 -1.74 (m, 2H), 1.68-1.41 (m, 8h), 1.41-1.19 (m, 49H), 1.04 (t, J=7.2 Hz, 3H ), 0.95-0.82 (m, 9H). MS: 1001.1 m/z [M+H].

Example 8 - compound 8.

Compound 8: 2-((4-(((3-(dimethylamino)propyl)carbamoyl)oxy)hexadecanoyl)oxy)propane-1,3diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9 ,12-dienoate)

Compound 8 was synthesized in 55% yield from intermediate 1e and N1,N1 dimethylpropane-1,3-diamine using the method used for Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.46 (t, J=5.8 Hz, 1H), 5.43-5.28 (m, 8H), 5.24 (tt, J=5.8 , 4.4 Hz, 1H), 4.75 (s, 1H), 4.28 (ddd, J=11.9, 4.5, 2.2 Hz, 2H), 4.15 (ddd, J= 11.9, 5.8, 1.6 Hz, 2H), 3.23 (h, J=6.4 Hz, 2H), 2.822.73 (m, 4H), 2.48-2.20 (m , 14H), 2.05 (q, J=6.8 Hz, 8H), 1.97-1.42 (m, 12H), 1.42-1.20 (m, 48H), 0.88 ( td, J=6.9, 4.1 Hz, 9H). MS: 1000.6 m/z [M+H].

Example 9 - compound 9.

Compound 9: 2-((4-((((1-methylpyrrolidin-2-yl)methyl)carbamoyl)oxy)hexadecanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12^) -bis(octadeca-9,12-dienoate)

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Compound 9 was synthesized in 65% yield from intermediate 1e and (1methylpyrrolidin-2-yl)methanamine using the method used for Example 1.

^1H NMR (400 MHz, chloroform-d) δ 5.45-5.28 (m, 8H), 5.25 (ddd, J=10.2, 5.8, 4.4 Hz, 1H), 5 ,19-5.11 (m, 1H), 4.76 (s, 1H), 4.28 (dd, J=11.9, 4.4 Hz, 2H), 4.14 (ddd, J=11 ,9, 5.9, 3.0 Hz, 2H), 3.44-3.31 (m, 1H), 3.193.02 (m, 2H), 2.84-2.72 (m, 4H), 2.46-2.16 (m, 12H), 2.05 (q, J=6.9 Hz, 8H), 1.97-1.66 (m, 6H), 1.66-1.42 ( m, 8H), 1.42-1.20 (m, 49H), 0.88 (td, J=7.0, 4.2 Hz, 9H). MS: 1013.1 m/z [M+H].

Example 10 - compound 10.

Compound 10: 2-((4-((1,4-dimethylpiperidin-4-carbonyl)oxy)hexadecanoyl)oxy)propane-1,3diyl (9Z,9'Z, 12Z,12^)-bis(octadeca-9 ,12-dienoate)

A solution of intermediate 1d (360 mg, 1.0 equiv) in 4:2:1 THF/water/toluene (0.05 M) was cooled to 0°C followed by the addition of NaBH4 (5.0 equiv). The reaction was maintained at 0°C for 18 hours. The reaction mixture was then diluted with water and EtOAc, and the resulting biphasic mixture was extracted with 3x EtOAc. The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude material was purified by column chromatography (EtOAc/hexanes) to give the alcohol intermediate as a colorless oil.

The resulting crude alcohol was immediately resolubilized in DCM (0.1 M) followed by the addition of 1,4-dimethylpiperidine-4-carboxylic acid (1.0 eq), DMAP (0.1 eq), DIPEA (4.0 eq). ) and EDCI (1.5 eq.). The reaction mixture was stirred at room temperature. After 16 hours, additional 1,4-dimethylpiperidine-4-carboxylic acid (1.0 equiv), DMAP (0.05 equiv), DIPEA (2.0 equiv), and EDCI (1.5 equiv) were added to the reaction mixture. .) and the reaction mixture was heated to 40°C. After another two hours, the reaction was quenched by adding water and DCM, and the organic layer was washed 1 time with 1 M HCl, 1 time with 5% NaHCO ₃ and 1 time with brine. The resulting organic layer was dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude material was purified by column chromatography (MeOH/DCM) to give the product as a rose oil (94 mg, 22%).

1H NMR (400 MHz, CDCl ₃ ) δ 5.46-5.22 (m, 9H), 4.92 (ddd, J=11.6, 7.5, 4.7 Hz, 1H), 4.30 (dd, J=11.9, 4.4 Hz, 2H), 4.14 (ddd, J=12.0, 5.8, 1.9 Hz, 2H), 2.82-2.74 (m , 4H), 2.67 (s, 2H), 2.41-2.25 (m, 9H), 2.14 (d, J=14.2 Hz, 4H), 2.05 (q, J= 6.8 Hz, 8H), 1.98-1.78 (m, 3H), 1.66-1.49 (m, 9H), 1.43-1.18 (m, 50H), 0.88 (td, J=6.9, 4.3 Hz, 9H).

Example 11 - compound 11.

Compound 11: 2-((4-((4-(diethylamino)butanoyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9 ,12-dienoate)

Compound 11 was synthesized in 17% yield from intermediate 1d and 3-(diethylamino)butanoic acid using the same method as Example 10.

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1H NMR (400 MHz, CDC13) δ 5.46 (t, J=5.8 Hz, 1H), 5.43-5.28 (m, 8H), 5.24 (tt, J=5.8, 4.4 Hz, 1H), 4.75 (s, 1H), 4.28 (ddd, J=11.9, 4.5, 2.2 Hz, 2H), 4.15 (ddd, J=11 ,9, 5.8, 1.6 Hz, 2H), 3.23 (h, J=6.4 Hz, 2H), 2.822.73 (m, 4H), 2.48-2.20 (m, 14H), 2.05 (q, J=6.8 Hz, 8H), 1.97-1.42 (m, 12H), 1.42-1.20 (m, 48H), 0.88 (td , J=6.9, 4.1 Hz, 9H). MS: 1013.9 m/z [M+H].

Example 12 - compound 12.

Compound 12: 2-((4-(((2-(dimethylamino)ethyl)carbamoyl)oxy)hexadecanoyl)oxy)propane-1,3diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9 ,12-dienoate)

Compound 12 was synthesized in 67% yield from intermediate 1e and N1,N1dimethylethane-1,2-diamine using the method used for Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.44-5.29 (m, 8H), 5.29-5.18 (m, 2H), 4.75 (s, 1H), 4.34-4 ,24 (m, 2H), 4.15 (ddd, J=11.9, 5.8, 3.6 Hz, 2H), 3.25 (q, J=5.8 Hz, 2H), 2, 77 (td, J=6.2, 1.2 Hz, 4H), 2.47-2.35 (m, 4H), 2.35-2.28 (m, 4H), 2.24 (s, 6H), 2.05 (q, J=7.1 Hz, 8H), 1.98-1.73 (m, 4H), 1.65-1.42 (m, 7H), 1.40-1 .20 (m, 48H), 0.88 (td, J=7.0, 4.3 Hz, 9H). MS: 987.2 m/z [M+H].

Example 13 - compound 13.

Compound 13: 2-((4-(((3-(diethylamino)propyl)carbamoyl)oxy)hexadecanoyl)oxy)propane-1,3diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9 ,12-dienoate)

Compound 13 was synthesized in 62% yield from intermediate 1e and N1,N1 diethylpropane-1,3-diamine using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.93 (s, 1H), 5.44-5.28 (m, 8H), 5.24 (ddd, J=5.8, 4.5, 1, 3 Hz, 1H), 4.74 (s, 1H), 4.28 (ddd, J=11.9, 4.5, 2.7 Hz, 2H), 4.14 (ddd, J=11.9 , 5.8, 1.2 Hz, 2H), 3.24 (tt, J=13.2, 6.6 Hz, 2H), 2.812.71 (m, 4H), 2.57 (d, J= 7.7 Hz, 6H), 2.38 (ddd, J=8.9, 6.8, 3.2 Hz, 2H), 2.35-2.28 (m, 4H), 2.05 (q , J=6.6 Hz, 8H), 1.94-1.85 (m, 2H), 1.79 (dq, J=14.8, 8.1 Hz, 2H), 1.73-1, 56 (m, 7H), 1.56-1.43 (m, 3H), 1.40-1.20 (m, 47H), 1.06 (t, J=7.1 Hz, 6H), 0 .88 (td, J=7.0, 4.3 Hz, 9H). MS: 1029.2 m/z [M+H].

Example 14 - compound 14.

Compound 14: 2-((4-(((4-(diethylamino)butoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9 ,12-dienoate)

Compound 14 was synthesized in 60% yield from intermediate 1e and N1,N1dimethylethane-1,2-diamine using the method used for Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.45-5.30 (m, 9H), 5.28 (tt, J=5.8, 4.3 Hz, 1H), 4.78-4.69 (m, 1H), 4.31 (dd, J=11.9, 4.4 Hz, 2H), 4.22-4.09 (m, 4H), 2.79 (td, J=6.1 , 1.1 Hz, 4H), 2.63 (q, J=7.2 Hz, 4H), 2.55 (t, J=7.6 Hz,

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2H), 2.47-2.38 (m, 2H), 2.38-2.30 (m, 4H), 2.07 (q, J=7.1 Hz, 8H), 2.02-1 .85 (m, 2H), 1.76-1.51 (m, 11H), 1.451.21 (m, 49H), 1.08 (t, J=7.2 Hz, 6H), 0.95- 0.82 (m, 9H). MS: 1044.1 m/z [M+H].

Example 15 - compound 15.

Compound 15: 2-((4-(((2-(diethylamino)ethyl)carbamoyl)oxy)hexadecanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca -9,12-dienoate)

Compound 15 was synthesized in 75% yield from intermediate 1e and N1,N1diethylethane-1,2-diamine using the method used for Example 1.

^1H NMR (400 MHz, CDCls) δ 5.44-5.29 (m, 9H), 5.25 (ddd, J=5.8, 4.4, 1.4 Hz, 1H), 4.75 (s, 1H), 4.28 (ddd, J=11.9, 4.4, 1.2 Hz, 2H), 4.14 (ddd, J=11.9, 5.8, 3.0 Hz , 2H), 3.23 (q, J=5.7 Hz, 2H), 2.80-2.73 (m, 4H), 2.57 (q, J=7.0 Hz, 6H), 2 .38 (ddd, J=9.0, 6.6, 4.7 Hz, 2H), 2.34-2.28 (m, 4H), 2.05 (q, J=6.8 Hz, 8H ), 1.90 (h, J=9.0, 7.9 Hz, 1H), 1.80 (p, J=7.3, 6.5 Hz, 1H), 1.66-1.44 ( m, 7H), 1.40-1.20 (m, 53H), 1.03 (t, J=7.1 Hz, 6H), 0.88 (td, J=6.9, 4.3 Hz , 9H). MS: 1014.9 m/z [M+H].

Example 16 - compound 16.

Compound 16: 2-((4-(((2-(1-methylpyrrolidin-2-yl)ethoxy)carbonyl)oxy)hexadecanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12 ^)-bis(octadeca-9,12-dienoate)

Compound 16 was synthesized in 78% yield from intermediate 1e and 2-(1methylpyrrolidin-2-yl)ethan-1-ol using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.43-5.28 (m, 9H), 5.25 (tt, J=5.8, 4.3 Hz, 1H), 4.72 (qd, J =7.6, 4.6 Hz, 1H), 4.29 (dd, J=11.9, 4.4 Hz, 2H), 4.26-4.09 (m, 4H), 3.14 ( t, J=8.8 Hz, 1H), 2.82-2.74 (m, 4H), 2.45-2.28 (m, 9H), 2.22 (td, J=11.5, 10.9, 6.0 Hz, 2H), 2.15-1.91 (m, 12H), 1.69-1.48 (m, 9H), 1.42-1.20 (m, 50H) , 0.88 (td, J=6.9, 4.1 Hz, 9H). MS: 1027.9 m/z [M+H].

Example 17 - compound 17.

Intermediate 17a: Hexadecylmagnesium bromide

To a solution of Mg (15.92 g, 2 equiv.) in THF (300 ml) was added I ₂ (0.01 equiv.), then 1-bromohexadecane (1.0 equiv.) was added dropwise to the above at 80°C. in THF (100 ml). The mixture was stirred for 6 hours at 80°C. The product was obtained in quantitative yield as a brown liquid and was immediately used in the next step.

Intermediate 17b: 4-oxoicosanoic acid

A solution of succinic anhydride (54 g, 1.0 eq.) in THF (1.2 M) was cooled to -78° C. followed by the addition of intermediate 17a. The reaction mixture was kept at -78°C for 12 hours before pouring into sat. NH ₄ Cl and extracted three times with EtOAc. The combined organic phases were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated. Purification by column chromatography gave the product as a white solid (4 g, 2%).

1H NMR (400 MHz, CDCl ₃ ) δ 4.11 (t, J=6.7 Hz, 1H), 2.80-2.62 (m, 4H), 2.46 (t, J=7.5 Hz, 1H), 1.63 (dq, J=14.7, 7.1 Hz, 2H), 1.49-1.20 (m, 27H), 0.90 (t, J=6.7 Hz , 3H).

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Intermediate 17c: 2-((4-oxoicosanoyl)oxy)propane-1,3-diyl (9Z,9'Z,12Z,12'Z)bis(octadeca-9,12-dienoate)

Intermediate 17c was synthesized in 53% yield from intermediate 17b and intermediate 1c using the method used for intermediate 1d.

1H NMR (400 MHz, CDCl3) δ 5.43-5.21 (m, 9H), 4.29 (dt, J=11.9, 4.1 Hz, 2H), 4.14 (dd, J= 11.9, 5.9 Hz, 2H), 4.06 (t, J=6.8 Hz, 1H), 2.73 (dt, J=22.5, 6.5 Hz, 6H), 2, 67-2.54 (m, 4H), 2.42 (t, J=7.5 Hz, 2H), 2.31 (t, J=7.6 Hz, 4H), 2.04 (q, J =6.9 Hz, 9H), 1.61 (q, J=7.9, 7.3 Hz, 8H), 1.42-1.15 (m, 55H), 0.88 (td, J= 6.7, 4.0 Hz, 9H).

Intermediate 17d: 2-((4-(((4-nitrophenoxy)carbonyl)oxy)icosanoyl)oxy)propane1,3-diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9,12 -dienoate)

Intermediate 17d was synthesized in 29% yield from intermediate 17c using the same method as intermediate 1e.

1H NMR (400 MHz, CDCl3) δ 8.33-8.24 (m, 2H), 7.43-7.34 (m, 2H), 5.45-5.23 (m, 8H), 4, 92-4.82 (m, 1H), 4.31 (dd, J=12.0, 4.3 Hz, 2H), 4.20-4.09 (m, 2H), 2.76 (t, J=6.3 Hz, 4H), 2.47 (dd, J=8.3, 6.7 Hz, 2H), 2.30 (td, J=7.6, 4.2 Hz, 4H), 2.04 (q, J=7.2 Hz, 9H), 1.61 (ddt, J=11.5, 7.4, 3.7 Hz, 4H), 1.43-1.22 (m, 49H), 0.88 (td, J=6.9, 3.8 Hz, 9H).

Compound 17: 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)icosanoyl)oxy)propane-1,3diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9 ,12-dienoate)

Compound 17 was synthesized in 74% yield from intermediate 17d and 3-(dimethylamino)propan-1-ol using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.42-5.22 (m, 10H), 4.72 (tdd, J=7.5, 5.4, 4.0 Hz, 1H), 4.36 -4.25 (m, 2H), 4.23-4.08 (m, 4H), 2.82-2.71 (m, 4H), 2.50-2.28 (m, 9H), 2 .25 (s, 6H), 2.09-1.97 (m, 9H), 1.91-1.80 (m, 3H), 1.70-1.47 (m, 7H), 1.40 -1.20 (m, 57H), 0.96-0.82 (m, 9H). MS: 1058.1 m/z [M+H].

Example 18 - compound 18.

Compound 18: 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)icosanoyl)oxy)propan-1,3diyl (9Z,9'Z, 12Z, 12^)-bis (octadeca-9,12-dienoate)

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Compound 18 was synthesized in 77% yield from intermediate 17d and 3((methyl)ethylamino)propan-1-ol using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.45-5.22 (m, 10H), 4.72 (qd, J=7.7, 4.7 Hz, 1H), 4.29 (dd, J =12.0, 4.4 Hz, 2H), 4.20-4.10 (m, 4H), 2.83-2.73 (m, 4H), 2.51-2.36 (m, 6H ), 2.35-2.28 (m, 4H), 2.24 (s, 3H), 2.10-1.99 (m, 9H), 1.91-1.82 (m, 3H), 1.66-1.52 (m, 6H), 1.42-1.19 (m, 60H), 1.06 (t, J=7.1 Hz, 3H), 0.88 (td, J= 6.9, 4.1 Hz, 9H). MS: 1072.3 m/z [M+H].

Example 19 - compound 19.

Compound 19: 2-((4-(((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)icosanoyl)oxy)propan-1,3diyl (9Z,9'Z, 12Z, 12^)-bis (octadeca-9,12-dienoate)

Compound 19 was synthesized in 67% yield from intermediate 17d and 3(pyrrolidin-1-yl)propan-1-ol using the same method as Example 1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.44-5.22 (m, 9H), 4.72 (tt, J=7.5, 4.5 Hz, 1H), 4.29 (dd, J=11.9, 4.4 Hz, 2H), 4.24-4.10 (m, 4H), 2.83-2.72 (m, 4H), 2.57 (d, J=8, 3 Hz, 6H), 2.40 (dt, J=8.6, 6.7 Hz, 2H), 2.31 (td, J=7.6, 1.2 Hz, 4H), 2.04 ( dd, J=8.5, 5.3 Hz, 8H), 2.00-1.86 (m, 4H), 1.86-1.78 (m, 4H), 1.53 (ddt, J= 14.1, 8.3, 4.7 Hz, 7H), 1.44-1.20 (m, 56H), 0.88 (td, J=6.9, 4.2 Hz, 9H). MS: 1084.2 m/z [M+H].

Example 20 - compound 20.

Compound 20: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)icosanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12^) -bis(octadeca-9,12-dienoate)

Compound 20 was synthesized in 73% yield from intermediate 17d and 3(pyrrolidin-1-yl)propan-1-ol using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.44-5.17 (m, 10H), 4.75 (s, 1H), 4.28 (ddd, J=11.9, 4.4, 1, 3 Hz, 2H), 4.15 (ddd, J=11.9, 5.8, 3.4 Hz, 2H), 3.25 (d, J=6.2 Hz, 2H), 2.81- 2.73 (m, 4H), 2.50-2.36 (m, 6H), 2.36-2.28 (m, 4H), 2.22 (s, 3H), 2.05 (q, J=6.8 Hz, 8H), 1.94-1.71 (m, 4H), 1.66-1.20 (m, 63H), 1.05 (t, J=7.1 Hz, 3H ), 0.88 (td, J=6.9, 4.2 Hz, 9H). MS: 1056.6 m/z [M+H].

Example 21 - compound 21.

Compound 21: 2-((4-((((1-methylpyrrolidin-2-yl)methyl)carbamoyl)oxy)icosanoyl)oxy)propan1,3-diyl (9Z,9'Z, 12Z,12'Z)- bis(octadeca-9,12-dienoate)

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Compound 21 was synthesized in 85% yield from intermediate 17d (1methylpyrrolidin-2-yl)methanamine using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.43-5.30 (m, 8H), 5.29-5.18 (m, 2H), 4.76 (s, 1H), 4.28 (dd , J=11.9, 4.4 Hz, 2H), 4.21-4.07 (m, 2H), 3.46-3.30 (m, 1H), 3.11 (ddt, J=22 ,3, 8.9, 4.3 Hz, 2H), 2.80-2.72 (m, 4H), 2.45-2.15 (m, 12H), 2.04 (dd, J=8 ,5, 5.3 Hz, 9H), 1.96-1.77 (m, 4H), 1.71 (tq, J=8.4, 5.1, 4.0 Hz, 2H), 1, 65-1.45 (m, 8H), 1.391.20 (m, 60H), 0.88 (td, J=6.9, 4.4 Hz, 9H). MS: 1068.8 m/z [M+H].

Example 22 - compound 22.

Intermediate 22a: (2)-9-(non-2-en-1-yloxy)-9-oxononanoic acid

To a solution of nonanedioic acid (25 g, 1.3 eq.) in THF (100 ml) at 0° C., oxalyl chloride (1.0 eq.) was added dropwise over one minute. DMF (0.1 eq) was then added dropwise over one minute. The reaction mixture was stirred at 0°C for 10 min, then at room temperature for 10 min. The reaction mixture was then cooled to 0°C followed by the addition of cis-2nonen-1-ol (1.0 equiv.) in THF (100 ml). The reaction mixture was stirred at 0°C for 5 minutes, then stirred at room temperature for 16 hours. Upon completion, the reaction mixture was concentrated in vacuo, suspended with DCM and filtered. The filtrate was concentrated and purified by column chromatography (80 g, MeOH/DCM) to give the product as a pale yellow oil (15 g, 47%), which was used directly in subsequent steps.

Intermediate 22b: 9,9'-di((2)-non-2-en-1-yl)O'1,O1-(2-oxopropan-1,3-diyl)di(nonanedioate)

Intermediate 22b was synthesized in 73% yield by combining dihydroxyacetone and intermediate 22a using the method used for intermediate 1b.

Intermediate 22c: O'1,O1-(2-hydroxypropane-1,3-diyl)9,9'-di((2)-non-2-en-1yl)di(nonanedioate)

Intermediate 22c was synthesized in 71% yield from intermediate 22b using the same method as intermediate 1c, except the reaction was complete after 45 min.

1H NMR (400 MHz, CDCl ₃ ) δ 5.64 (dtt, J=11.0, 7.3, 1.3 Hz, 2H), 5.52 (dtt, J=10.9, 6.8, 1.5 Hz, 2H), 4.674.58 (m, 4H), 4.23-4.05 (m, 5H), 2.51 (d, J=4.6 Hz, 1H), 2.32 ( dt, J=18.1, 7.6 Hz, 8H), 2.15-2.00 (m, 4H), 1.61 (td, J=10.0, 2.7 Hz, 8H), 1 .42-1.20 (m, 28H), 0.93-0.83 (m, 6H).

Intermediate 22d: 9,9'-di((2)-non-2-en-1-yl)O'1,O1-(2-((4oxohexadecanoyl)oxy)propan-1,3-diyl)di( nonanedioate)

- 105 046244

Intermediate 22d was synthesized in 38% yield from intermediate 22c and intermediate 1a using the method used for intermediate 1d.

1H NMR (400 MHz, CDCl3) δ 5.66 (dtt, J=11.0, 7.3, 1.3 Hz, 2H), 5.54 (dtt, J=11.0, 6.9, 1 .5 Hz, 2H), 5.26 (tt, J=5.9, 4.3 Hz, 1H), 4.64 (dd, J=6.9, 1.3 Hz, 4H), 4.31 (dd, J=11.9, 4.3 Hz, 2H), 4.17 (dd, J=11.9, 5.9 Hz, 2H), 2.74 (t, J=6.7 Hz, 2H), 2.68-2.56 (m, 2H), 2.45 (t, J=7.5 Hz, 2H), 2.33 (td, J=7.6, 6.0 Hz, 8H ), 2.19-2.07 (m, 4H), 1.70-1.57 (m, 12H), 1.43-1.23 (m, 46H), 0.98-0.84 (m , 9H).

Intermediate 22e: O'1,O1-(2-((4-(((4-nitrophenoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl)9,9'-di((2) -non-2-en-1-yl)di(nonanedioate)

Intermediate 22e was synthesized in 51% yield from intermediate 22d using the same method as intermediate 1e.

^1H NMR (400 MHz, CDCl ₃ ) δ 8.38-8.31 (m, 1H), 8.31-8.24 (m, 2H), 7.54-7.47 (m, 1H), 7.44-7.36 (m, 2H), 5.64 (dtt, J=11.0, 7.3, 1.3 Hz, 2H), 5.51 (dtt, J=11.0, 6 ,8, 1.5 Hz, 2H), 5.26 (tt, J=5.8, 4.3 Hz, 1H), 4.86 (ddd, J=11.8, 7.5, 4.8 Hz, 1H), 4.65-4.57 (m, 4H), 4.36-4.25 (m, 2H), 4.21-4.10 (m, 2H), 2.50-2, 44 (m, 2H), 2.30 (td, J=7.5, 4.5 Hz, 8H), 2.13-2.05 (m, 4H), 1.62 (d, J=37, 2 Hz, 9H), 1.46-1.18 (m, 49H), 0.94-0.80 (m, 9H).

Compound 22: O'1,O1-(2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propan-1,3-diyl)9,9'-di(( 2)-non-2-en-1-yl)di(nonanedioate) o

O.,J y o o % o

Compound 22 was synthesized in 54% yield from intermediate 22e and 3(dimethylamino)propan-1-ol using the same method as Example 1.

1H NMR (400 MHz, CDCl3) δ 5.71-5.58 (m, 2H), 5.52 (dtt, J=10.9, 6.8, 1.5 Hz, 2H), 5.25 ( tt, J=5.7, 4.4 Hz, 1H), 4.72 (ddd, J=11.7, 7.4, 4.5 Hz, 1H), 4.62 (dd, J=6, 8, 1.3 Hz, 4H), 4.34-4.25 (m, 2H), 4.25-4.08 (m, 4H), 2.39 (ddd, J=12.9, 8, 4, 6.4 Hz, 4H), 2.34-2.21 (m, 13H), 2.09 (tdd, J=7.4, 5.7, 1.5 Hz, 4H), 2.02 -1.67 (m, 7H), 1.69-1.51 (m, 11H), 1.42-1.19 (m, 45H), 0.97-0.78 (m, 8H). MS: 1065.7 m/z [M+H].

Example 23 - compound 23.

Compound 23: O'1,O1 -(2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)hexadecanoyl)oxy)propan-1,3-diyl)9,9' -di((2)-non-2-en-1-yl)di(nonanedioate)

- 106 046244

Compound 23 was synthesized in 59% yield from intermediate 22e and NI-ethylN-methylethane-1,2-diamine using the method used in Example 1.

1H NMR (400 MHz, CDCl3) δ 5.72-5.59 (m, 2H), 5.52 (dtt, J=11.0, 6.8, 1.5 Hz, 2H), 5.36- 5.18 (m, 2H), 4.75 (s, 1H), 4.62 (dd, J=6.9, 1.2 Hz, 4H), 4.28 (dd, J=11.9, 4.4 Hz, 2H), 4.14 (ddd, J=11.9, 5.8, 3.5 Hz, 2H), 3.35-3.20 (m, 2H), 2.56-2 .43 (m, 4H), 2.38 (ddd, J=8.7, 6.6, 4.1 Hz, 2H), 2.34-2.19 (m, 11H), 2.10 (qd , J=7.4, 1.5 Hz, 4H), 1.96-1.72 (m, 3H), 1.63 (d, J=6.9 Hz, 11H), 1.41-1, 18 (m, 48H), 1.07 (t, J=7.1 Hz, 3H), 0.93-0.81 (m, 9H). MS: 1065.2 m/z [M+H].

Example 24 - compound 24.

Compound 24: O'1,O1-(2-((4-((((1-methylpyrrolidin-2-yl)methyl)carbamoyl)oxy)hexadecanoyl)oxy)propan-1,3-diyl)9,9' -di((2)-non-2-en-1-yl)di(nonanedioate)

Compound 24 was synthesized from intermediate 22e and (1-methylpyrrolidin-2yl)methanamine using the method used in Example 1.

1H NMR (400 MHz, CDCls) δ 5.71-5.59 (m, 2H), 5.52 (dtt, J=10.9, 6.8, 1.5 Hz, 2H), 5.24 ( ddd, J=8.8, 5.8, 4.4 Hz, 2H), 4.76 (s, 1H), 4.62 (dd, J=6.9, 1.3 Hz, 4H), 4 .28 (dd, J=11.9, 4.4 Hz, 2H), 4.14 (ddd, J=11.9, 5.8, 3.2 Hz, 2H), 3.39 (q, J =11.2, 10.1 Hz, 1H), 3.10 (t, J=14.0 Hz, 2H), 2.50-2.20 (m, 15H), 2.10 (qd, J= 7.4, 1.5 Hz, 4H), 1.97-1.67 (m, 6H), 1.61 (d, J=7.3 Hz, 12H), 1.41-1.18 (m , 47H), 0.97-0.79 (m, 9H). MS: 1076.7 m/z [M+H].

Example 25 - compound 25.

Intermediate 25a: 5-oxoheptadecanoic acid

Intermediate 25a was synthesized in 64% yield from a solution of glutaric anhydride and dodecylmagnesium bromide using the method used in Intermediate 1a.

1H NMR (400 MHz, CDCls) δ 2.75 (t, J=6.7 Hz, 1H), 2.49 (t, J=7.2 Hz, 2H), 2.44-2.35 (m , 4H), 1.90 (p, J=7.2 Hz, 2H), 1.56 (t, J=7.3 Hz, 2H), 1.26 (d, J=4.0 Hz, 19H ), 0.88 (t, J=6.7 Hz, 3H).

Intermediate 25b: 2-((5-oxoheptadecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)bis(octadeca-9,12-dienoate)

Intermediate 25b was synthesized in 55% yield from intermediate 25a and intermediate 1c using the method used for intermediate

- 107 046244 connections 1d.

1H NMR (400 MHz, CDCI3) δ 5.45-5.19 (m, 9H), 4.30 (dd, J=11.9, 4.3 Hz, 2H), 4.12 (dd, J= 11.9, 5.8 Hz, 2H), 2.76 (t, J=6.4 Hz, 4H), 2.46 (t, J=7.2 Hz, 2H), 2.42-2, 24 (m, 8H), 2.04 (q, J=6.8 Hz, 8H), 1.88 (p, J=7.2 Hz, 2H), 1.57 (dp, J=21.3 , 7.2 Hz, 6H), 1.43-1.18 (m, 41H), 0.87 (td, J=6.8, 4.1 Hz, 8H).

Intermediate 25c: 2-((5-(((4-nitrophenoxy)carbonyl)oxy)heptadecanoyl)oxy)propan-1,3-diyl (9Z,9'Z,12Z,12'Z)-bis(octadeca- 9,12-dienoate)

Intermediate 25c was synthesized in 23% yield from intermediate 25b using the method used for intermediate 1e.

^1H NMR (400 MHz, CDCl ₃ ) δ 8.31-8.24 (m, 2H), 7.42-7.35 (m, 2H), 5.44-5.19 (m, 9H), 4.83 (q, J=5.7 Hz, 1H), 4.31 (ddd, J=12.0, 4.4, 2.9 Hz, 2H), 4.14 (ddd, J=11, 9, 5.8, 2.1 Hz, 2H), 2.76 (t, J=6.3 Hz, 4H), 2.34 (dt,

J=30.7, 7.0 Hz, 6H), 2.04 (q, J=6.9 Hz, 8H), 1.80-1.52 (m, 14H), 1.29 (dd, J =19.3, 12.3 Hz, 44H), 0.88 (td, J=6.8, 3.7 Hz, 9H).

Compound 25: 2-((5-(((3-(dimethylamino)propoxy)carbonyl)oxy)heptadecanoyl)oxy)propane-1,3diyl (9Z,9'Z,12Z,12'Z)-bis(octadeca- 9,12-dienoate)

Compound 25 was synthesized in 94% yield from intermediate 25c and 3(dimethylamino)propan-1-ol using the same method as Example 1.

1H NMR (400 MHz, CDO3) δ 5.46-5.19 (m, 9H), 4.74-4.65 (m, 1H), 4.29 (ddd, J=11.9, 4.4 , 1.7 Hz, 2H), 4.25-4.07 (m, 4H), 2.82-2.72 (m, 4H), 2.39-2.28 (m, 8H), 2, 22 (s, 6H), 2.05 (q, J=6.8 Hz, 8H), 1.84 (dq, J=8.2, 6.7 Hz, 2H), 1.73-1.50 (m, 14H), 1.41-1.18 (m, 50H), 0.88 (td, J=7.0, 4.2 Hz, 9H). MS: 1016.11 m/z [M+H].

Example 26 - compound 26.

Compound 26: 2-((5-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)heptadecanoyl)oxy)propan1,3-diyl (9Z,9'Z,12Z,12'Z)- bis(octadeca-9,12-dienoate)

Compound 26 was synthesized in 84% yield from intermediate 25c and 3((methyl)ethylamino)propan-1-ol using the same method as Example 1.

1H NMR (400 MHz, CDO3) δ 5.46-5.20 (m, 10H), 4.69 (dq, J=7.5, 5.5 Hz, 1H), 4.29 (ddd, J= 11.9, 4.4, 1.6 Hz, 2H), 4.25-4.10 (m, 4H), 2.83-2.72 (m, 4H), 2.45-2.38 ( m, 4H), 2.37-2.28 (m, 6H), 2.21 (s, 3H), 2.05 (q, J=6.8 Hz, 8H), 1.88-1.79 (m, 2H), 1.74-1.52 (m, 13H), 1.44-1.22 (m, 51H), 1.04 (t, J=7.2 Hz, 3H), 0, 88 (td, J=6.9, 4.1 Hz, 9H). MS: 1030.1 m/z [M+H].

Example 27 - compound 27.

Compound 27: 2-((5-(((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)heptadecanoyl)oxy)propane- 108 046244

1,3-diyl (9Z,9'Z, 12Z, 12'Z)-6uc(oKTageka-9,12-dienoate)

Compound 27 was synthesized in 79% yield from intermediate 25c and 3(pyrrolidin-1-yl)propan-1-ol using the same method as Example 1.

^1H NMR (400 MHz, CDCl3) δ 5.44-5.21 (m, 10H), 4.69 (dt, J=7.1, 5.3 Hz, 1H), 4.29 (ddd, J =11.9, 4.3, 1.6 Hz, 2H), 4.25-4.10 (m, 4H), 2.81-2.73 (m, 4H), 2.52 (dd, J =14.8, 7.1 Hz, 6H), 2.33 (dt, J=13.4, 7.2 Hz, 6H), 2.05 (q, J=6.8 Hz, 8H), 1 .89 (dq, J=10.8, 6.8 Hz, 2H), 1.82-1.74 (m, J=3.8, 3.3 Hz, 4H), 1.71-1.53 (m, 13H), 1.40-1.21 (m, 49H), 0.88 (td, J=6.9, 4.2 Hz, 9H). MS: 1041.8 m/z [M+H].

Example 28 - compound 28.

Compound 28: 2-((5-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)heptadecanoyl)oxy)propan-1,3diyl (9Z,9'Z, 12Z,12^)-bis (octadeca-9,12-dienoate)

Compound 28 was synthesized in 87% yield from intermediate 25c and NI-ethyl1-methylethane-1,2-diamine using the same method as Example 1.

1H NMR (400 MHz, CDQ3) δ 5.43-5.21 (m, 10H), 5.12 (s, 1H), 4.80-4.65 (m, 1H), 4.29 (dt, J=11.9, 4.5 Hz, 2H), 4.19-4.09 (m, 2H), 3.24 (d, J=4.8 Hz, 2H), 2.83-2.72 (m, 4H), 2.44 (dt, J=14.6, 6.6 Hz, 4H), 2.38-2.27 (m, 6H), 2.20 (s, 3H), 2, 04 (dd, J=7.8, 5.7 Hz, 8H), 1.74-1.42 (m, 15H), 1.42-1.19 (m, 50H), 1.04 (t, J=7.1 Hz, 3H), 0.88 (td, J=7.0, 4.4 Hz, 9H). MS: 1014.1 m/z [M+H].

Example 29 - compound 29.

Compound 29: 2-((5-((((1-methylpyrrolidin-2-yl)methyl)carbamoyl)oxy)heptadecanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12^) -bis(octadeca-9,12-dienoate)

Compound 29 was synthesized in 93% yield from intermediate 25c and (1methylpyrrolidin-2-yl)methanamine using the same method as Example 1.

^1H NMR (400 MHz, CDCl3) δ 5.43-5.21 (m, 11H), 5.08 (d, J=8.8 Hz, 1H), 4.74 (s, 1H), 4, 29 (dt, J=11.9, 4.6 Hz, 2H), 4.13 (ddd, J=11.9, 5.8, 4.5 Hz, 2H), 3.39 (q, J= 11.3, 10.6 Hz, 1H), 3.08 (d, J=25.9 Hz, 2H), 2.822.73 (m, 4H), 2.39-2.18 (m, 12H), 2.05 (q, J=6.8 Hz, 9H), 1.91-1.82 (m, 1H), 1.62 (dddd, J=27.4, 20.2, 13.2, 6 .7 Hz, 18H), 1.42-1.20 (m, 52H), 0.88 (td, J=6.9, 4.4 Hz, 9H). MS: 1026.8 m/z [M+H].

Example 30 - compound 30.

Intermediate 30a: 3-hydroxypropane-1,2-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12dienoate)

- 109 046244

To a solution of glycerol (1.59 g, 1.0 eq) in DCM (0.1 M) was added linoleic acid (2.05 eq), DMAP (0.2 eq), DIPEA (2.4 eq). ), EDCI (2.4 eq.) and 1 ml DMF sequentially. The reaction mixture was stirred at room temperature for 65 hours. Once complete, the mixture was quenched by adding water and the organic layer was washed 1 time with 1 M HCl and 1 time with 5% NaHCO3. The organic layer was then dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude material was purified by column chromatography (EtOAc/hexanes) to the product as a colorless oil.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.43-5.28 (m, 8H), 5.13-5.04 (m, 1H), 4.32 (dd, J=11.9, 4 ,6 Hz, 1H), 4.23 (dd, J=11.9, 5.6 Hz, 1H), 3.73 (ddd, J=6.5, 5.0, 1.3 Hz, 2H) , 2.82-2.73 (m, 4H), 2.33 (dt, J=8.8, 7.5 Hz, 4H), 2.03 (dq, J=13.1, 6.6 Hz , 9H), 1.69-1.57 (m, 4H), 1.44-1.23 (m, 29H), 0.96-0.83 (m, 6H).

Intermediate 30b: 3-((4-oxohexadecanoyl)oxy)propane-1,2-diyl (9Z,9'Z,12Z,12'Z)bis(octadeca-9,12-dienoate)

Intermediate 30b was synthesized in 66% yield from intermediate 30a and intermediate 1a using the method used in intermediate 1d.

1H NMR (400 MHz, CDCl3) δ 5.43-5.26 (m, 9H), 4.29 (ddd, J=11.8, 4.3, 2.1 Hz, 2H), 4.15 ( ddd, J=11.9, 7.7, 6.0 Hz, 2H), 2.82-2.74 (m, 4H), 2.71 (td, J=6.3, 5.6, 2 ,4 Hz, 2H), 2.62-2.55 (m, 2H), 2.43 (t, J=7.5 Hz, 2H), 2.31 (td, J=7.6, 5, 4 Hz, 4H), 2.05 (q, J=6.9 Hz, 8H), 1.66-1.54 (m, 8H), 1.41-1.19 (m, 47H), 0, 88 (td, J=7.0, 4.4 Hz, 9H).

Intermediate 30c: 3-((4-(((4-nitrophenoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,2-diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9 ,12-dienoate)

Intermediate 30c was synthesized in 40% yield from intermediate 30b using the same method as intermediate 1e.

Compound 30: 9-dodecyl-2-methyl-7,12-dioxo-6,8,13-trioxa-2-azahexadecane-15,16-diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca -9,12-dienoate)

Compound 30 was synthesized in 84% yield from intermediate 30c and 3

- 110 046244 ((methyl)ethylamino)propan-1-ol using the method used in example 1.

¹ H NMR (500 MHz, CDCl ₃ ) δ 5.44-5.20 (m, 8H), 4.76-4.67 (m, 1H), 4.29 (ddd, J=11.9, 4 ,3, 1.7 Hz, 2H), 4.22-4.11 (m, 4H), 2.77 (t, J=6.7 Hz, 4H), 2.45-2.28 (m, 8H), 2.22 (s, 6H), 2.05 (q, J=6.9 Hz, 8H), 2.00-1.79 (m, 4H), 1.69-1.51 (m , 6H), 1.40-1.17 (m, 48H), 0.88 (td, J=6.9, 4.8 Hz, 8H). MS: 1002.2 m/z [M+H].

Example 31 - compound 31.

Intermediate 31a: 4-oxododecanoic acid

Intermediate 31a was synthesized in 9% yield from a solution of succinic anhydride and octylmagnesium bromide using the method used in intermediate 1a.

1H NMR (400 MHz, CDCl ₃ ) δ 2.71 (t, J=6.4 Hz, 2H), 2.62 (t, J=6.7 Hz, 2H), 2.43 (t, J= 7.5 Hz, 2H), 1.57 (p, J=7.1 Hz, 2H), 1.26 (d, J=3.6 Hz, 10H), 0.87 (t, J=6, 7 Hz, 3H).

Intermediate bis(octadeca-9,12-dienoate)

31b: 2-((4-oxododecanoyl)oxy)propane-1,3-diyl (9Z,9'Z,12Z, 12'Z)-

Intermediate 31b was synthesized in 38% yield from intermediate 31a and intermediate 1c using the method used in intermediate 1d.

1H NMR (400 MHz, CDCl ₃ ) δ 5.47-5.19 (m, 8H), 4.28 (dd, J=11.9, 4.3 Hz, 2H), 4.15 (dd, J =11.9, 5.9 Hz, 2H), 2.77 (t, J=6.4 Hz, 4H), 2.71 (t, J=6.5 Hz, 2H), 2.59 (t , J=6.5 Hz, 2H), 2.43 (t, J=7.5 Hz, 2H), 2.32 (t, J=7.6 Hz, 4H), 2.04 (q, J =6.9 Hz, 8H), 1.67-1.53 (m, 6H), 1.40-1.20 (m, 37H), 0.88 (td, J=6.8, 5.0 Hz, 9H).

Intermediate 31c: 2-((4-(((4-nitrophenoxy)carbonyl)oxy)dodecanoyl)oxy)propane1,3-diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9,12 -dienoate)

Intermediate 31c was synthesized in 68% yield using the same method as intermediate 1e.

1H NMR (400 MHz, CDCl ₃ ) δ 8.26-8.16 (m, 2H), 7.40-7.30 (m, 2H), 5.39-5.05 (m, 9H), 4 .80 (tdd, J=7.5, 5.4, 4.0 Hz, 1H), 4.49-4.04 (m, 4H), 2.77-2.63 (m, 4H), 2 .45-2.18 (m, 6H), 2.08-1.85 (m, 10H), 1.74-1.46 (m, 6H), 1.41-1.11 (m, 40H) , 0.81 (td, J=6.9, 2.4 Hz, 9H).

Compound 31: 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9 ,12-dienoate)

Compound 31 was synthesized in 71% yield from intermediate 31c and 3-111046244 (dimethylamino)propan-1-ol using the method used in Example 1.

^1H NMR (400 MHz, CDO3) δ 5.42-5.22 (m, 9H), 4.72 (tdd, J=7.4, 5.3, 4.0 Hz, 1H), 4.34 -4.26 (m, 2H), 4.23-4.10 (m, 4H), 2.82-2.72 (m, 4H), 2.45-2.36 (m, 4H), 2 ,31 (ddd, J=8.0, 7.1, 1.2 Hz, 4H), 2.25 (s, 6H), 2.05 (q, J=6.6 Hz, 8H), 1, 99-1.91 (m, 1H), 1.91-1.82 (m, 3H), 1.67-1.53 (m, 6H), 1.40-1.21 (m, 41H), 0.94-0.82 (m, 9H). MS: 945.6 m/z [M+H].

Example 32 - compound 32.

Compound 32: 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)dodecanoyl)oxy)propan-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis( octadeca-9,12-dienoate)

Compound 32 was synthesized in 71% yield from intermediate 31c and 3(diethylamino)propan-1-ol using the method used in Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.43-5.28 (m, 9H), 5.25 (ddd, J=5.8, 4.4, 1.5 Hz, 1H), 4.72 (tdd, J=7.6, 5.4, 4.0 Hz, 1H), 4.29 (dd, J=11.9, 4.4 Hz, 2H), 4.25-4.08 (m , 5H), 2.77 (td, J=6.1, 1.1 Hz, 4H), 2.64-2.53 (m, 6H), 2.40 (dt, J=8.7, 6 ,8 Hz, 2H), 2.31 (ddd, J=8.0, 7.2, 1.1 Hz, 4H), 2.10-2.01 (m, 10H), 1.95 (dddd, J=11.1, 9.2, 6.9, 3.5 Hz, 1H), 1.91-1.81 (m, 3H), 1.69-1.49 (m, 7H), 1, 39-1.20 (m, 42H), 1.05 (t, J=7.2 Hz, 6H), 0.88 (td, J=7.0, 4.9 Hz, 9H). MS: 974.0 m/z [M+H].

Example 33 - compound 33.

Compound 33: 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)dodecanoyl)oxy)propan1,3-diyl (9Z,9'Z,12Z,12'Z)- bis(octadeca-9,12-dienoate)

Compound 33 was synthesized in 79% yield from intermediate 31c and 3((methyl)ethylamino)propan-1-ol using the method used in Example 1.

1H NMR (400 MHz, CDO3) δ 5.45-5.21 (m, 10H), 4.72 (tdd, J=9.5, 6.7, 4.1 Hz, 1H), 4.38- 4.25 (m, 2H), 4.25-4.10 (m, 4H), 2.77 (td, J=6.2, 1.1 Hz, 4H), 2.51-2.38 ( m, 6H), 2.37-2.27 (m, 5H), 2.24 (d, J=2.4 Hz, 3H), 2.05 (q, J=7.1 Hz, 8H), 1.97-1.83 (m, 4H), 1.70-1.50 (m, 7H), 1.39-1.21 (m, 41H), 1.06 (t, J=7.2 Hz, 3H), 0.940.84 (m, 9H). 960.1 m/z [M+H].

Example 34 - compound 34.

Compound 34: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propan-1,3diyl (9Z,9'Z,12Z,12'Z)- bis(octadeca-9,12-dienoate)

Compound 34 was synthesized in 59% yield from intermediate 31c and NI-ethylM-methylethane-1,2-diamine using the method used in Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.44-5.13 (m, 10H), 4.75 (s, 1H), 4.28 (ddd, J=11.9, 4.4, 1, 4 Hz, 2H), 4.15 (ddd, J=11.9, 5.8, 3.3 Hz, 2H), 3.25 (q, J=5.7 Hz, 2H), 2.83- 2.73 (m, 4H), 2.53-2.35 (m, 6H), 2.35-2.27 (m, 4H), 2.21 (d, J=3.3 Hz, 3H) , 2.05 (q, J=6.8 Hz, 8H), 1.96-1.74 (m, 3H), 1.66-1.43 (m, 7H), 1.41-1.21 (m, 41H), 1.05

- 112 046244 (t, J=7.1 Hz, 3H), 0.88 (td, J=6.9, 5.6 Hz, 9H). 944.7 m/z [M+H].

Example 35 - compound 35.

Compound 35: 2-((4-((((1-methylpyrrolidin-2-yl)methyl)carbamoyl)oxy)dodecanoyl)oxy)propane1,3-diyl (9Z,9'Z, 12Z, 12^)-bis (octadeca-9,12-dienoate)

Compound 35 was synthesized in 71% yield from intermediate 31c and (1methylpyrrolidin-2-yl)methanamine using the method used in Example 1.

^1H NMR (400 MHz, CDC13) δ 5.44-5.21 (m, 10H), 4.86-4.66 (m, 1H), 4.28 (dd, J=11.9, 4, 4 Hz, 2H), 4.14 (ddd, J=11.9, 5.8, 3.1 Hz, 2H), 3.48-3.35 (m, 1H), 3.22-3.00 (m, 2H), 2.83-2.71 (m, 4H), 2.47-2.19 (m, 12H), 2.04 (q, J=6.9 Hz, 8H), 1, 96-1.67 (m, 6H), 1.67-1.42 (m, 8H), 1.41-1.19 (m, 41H), 0.95-0.84 (m, 9H). 956.5 m/z [M+H].

Example 36 - compound 36.

Compound 36: 2-((4-(((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3diyl (9Z,9'Z, 12Z, 12^)-bis (octadeca-9,12-dienoate)

Compound 36 was synthesized in 34% yield from intermediate 31c and 3(pyrrolidin-1-yl)propan-1-ol using the method used in Example 1.

1H NMR (400 MHz, CDCI3) δ 5.43-5.22 (m, 9H), 4.72 (tdd, J=7.4, 5.4, 4.0 Hz, 1H), 4.29 ( dd, J=11.9, 4.3 Hz, 2H), 4.25-4.08 (m, 4H), 2.82-2.72 (m, 4H), 2.67 (t, J= 7.6 Hz, 5H), 2.47-2.27 (m, 6H), 2.09-1.91 (m, 11H), 1.91-1.80 (m, 4H), 1.68 -1.47 (m, 7H), 1.47-1.19 (m, 42H), 0.88 (td, J=7.0, 4.8 Hz, 9H). 971.3 m/z [M+H].

Example 37 - compound 37.

Compound 37: 2-((4-(((2-(ethyl(methyl)amino)ethyl)(methyl)carbamoyl)oxy)dodecanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9,12-dienoate)

Compound 37 was synthesized in 67% yield from intermediate 31c and NI-ethyl\1,\2-dimethyl.thene111-1,2-diamine using the method used in Example 1.

1H NMR (400 MHz, CDC13) δ 5.44-5.18 (m, 8H), 4.78 (s, 1H), 4.28 (dt, J=11.9, 4.3 Hz, 2H) , 4.14 (dd, J=11.9, 5.8 Hz, 2H), 3.44-3.24 (m, 2H), 2.91 (d, J=8.1 Hz, 3H), 2.81-2.71 (m, 4H), 2.60-2.21 (m, 12H), 2.05 (q, J=6.9 Hz, 7H), 1.98-1.46 ( m, 14H), 1.43-1.19 (m, 37H), 1.14-1.00 (m, 3H), 0.88 (td, J=7.0, 5.3 Hz, 8H) . 958.6 m/z [M+H].

Example 38 - compound 38.

Compound 38: 2-((4-(((2-(dimethylamino)ethyl)(methyl)carbamoyl)oxy)dodecanoyl)oxy)propane1,3-diyl (9Z,9'Z, 12Z, 12^)-bis( octadeca-9,12-dienoate)

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Compound 38 was synthesized in 83% yield from intermediate 31c and \1,\1,\2-trimethylethane-1,2-diamine using the method used in Example 1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.43-5.19 (m, 8H), 4.78 (s, 1H), 4.28 (dt, J=11.9, 4.1 Hz, 2H), 4.14 (dd, J=11.9, 5.8 Hz, 2H), 3.37 (d, J=23.8 Hz, 2H), 2.91 (d, J=6.8 Hz, 3H), 2.82-2.72 (m, 4H), 2.54-2.21 (m, 13H), 2.04 (q, J=6.7 Hz, 7H), 1.95 -1.44 (m, 14H), 1.42-1.21 (m, 37H), 0.88 (td, J=7.0, 5.3 Hz, 8H). 944.5 m/z [M+H].

Example 39 - compound 39.

Intermediate 39a: Decylmagnesium bromide

To a solution of Mg (10.99 g, 2 equiv.) and I ₂ (0.0001 equiv.) in THF (250 ml) was added dropwise 1 bromodecane (1.0 equiv.) at 80°C for 2 hours. After addition, the mixture was stirred at this temperature for 2 hours. The crude product decyl magnesium bromide was obtained as a black-brown solution, which was immediately used in the next step without further purification.

Intermediate 39b: 4-oxotetradecanoic acid

A solution of succinic anhydride (45 g, 1.0 eq.) in THF (1.1 M) was cooled to -78°C, followed by the addition of intermediate 39a. The reaction mixture was kept at -78°C for 2 hours before stirring at 15°C for 16 hours. The reaction was then quenched by adding us. NH ₄ Cl, diluted with H ₂ O and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated. Purification by column chromatography gave the product as a white solid. (7.8 g, 7%).

^1H NMR (400 MHz, CDO3) δ 2.74 (dd, J=6.9, 5.2 Hz, 2H), 2.70-2.63 (m, 2H), 2.46 (t, J =7.5 Hz, 2H), 1.671.54 (m, 2H), 1.29 (d, J=5.2 Hz, 14H), 0.90 (t, J=6.8 Hz, 3H).

Intermediate 39c: 2-((4-oxotetradecanoyl)oxy)propane-1,3-diyl (9Z,9'Z,12Z,12'Z)bis(octadeca-9,12-dienoate)

Intermediate 39c was prepared from intermediates 39b and intermediate 1c using the same method as intermediate 1d.

^1H NMR (400 MHz, CDO3) δ 5.42-5.18 (m, 9H), 4.28 (dd, J=11.9, 4.3 Hz, 2H), 4.14 (dd, J =11.9, 5.9 Hz, 2H), 2.74 (dt, J=22.1, 6.8 Hz, 6H), 2.66-2.56 (m, 2H), 2.43 ( t, J=7.5 Hz, 2H), 2.31 (t, J=7.6 Hz, 4H), 2.04 (q, J=6.8 Hz, 8H), 1.59 (tq, J=14.2, 7.5 Hz, 7H), 1.41-1.18 (m, 44H), 0.88 (td, J=6.9, 4.6 Hz, 9H).

Intermediate 39d: 2-((4-(((4-nitrophenoxy)carbonyl)oxy)tetradecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca- 9,12-dienoate)

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NaBH ₄ (2.5 eq.) was slowly added to a solution of intermediate 39c in MeOH (0.13 M) at 5°C. The mixture was stirred at this temperature for 2 hours. The reaction mixture was quenched by adding H ₂ O and then extracted twice with EtOAc. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to leave a residue. The residue was purified by column chromatography to give a colorless oil, which was immediately reduced in DCM (1 M) followed by the addition of 4-nitrophenyl chloroformate (1.5 equiv.) and pyridine (2 equiv.) at 0°C. The mixture was stirred at 20°C for 12 hours, after which the reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give intermediate 39d in 31% yield.

1H NMR (400 MHz, CDCl3) δ 8.33-8.22 (m, 2H), 7.46-7.33 (m, 2H), 5.43-5.23 (m, 9H), 4, 31 (dd, J=12.0, 4.3 Hz, 2H), 4.15 (dd, J=12.0, 5.8 Hz, 2H), 2.76 (t, J=6.3 Hz , 4H), 2.47 (t, J=7.5 Hz, 2H), 2.30 (td, J=7.6, 4.2 Hz, 4H), 2.04 (q, J=7, 3 Hz, 10H), 1.67 (dt, J=47.7, 6.1 Hz, 7H), 1.30 (tq, J=14.3, 4.9, 3.0 Hz, 53H), 0.88 (td, J=6.8, 3.5 Hz, 10H).

Compound 39: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)tetradecanoyl)oxy)propan-1,3diyl (92.9'2,122.12'2)-bis( octadeca-9,12-dienoate)

Compound 39 was synthesized in 78% yield from intermediate 39d and NI-ethylN-methylethane-1,2-diamine using the same method as Example 1.

^1H NMR (400 MHz, CDQ3) δ 5.49-5.17 (m, 9H), 4.75 (s, 1H), 4.28 (dd, J=11.9, 4.4 Hz, 2H ), 4.15 (ddd, J=11.9, 5.9, 3.3 Hz, 2H), 3.26 (q, J=6.0 Hz, 2H), 2.77 (t, J= 6.4 Hz, 4H), 2.52-2.27 (m, 10H), 2.23 (s, 3H), 2.05 (q, J=6.8 Hz, 8H), 1.95- 1.42 (m, 14H), 1.42-1.18 (m, 40H), 1.05 (t, J=7.1 Hz, 3H), 0.88 (td, J=6.8, 4.5 Hz, 7H). 972.9 m/z [M+H].

Example 40 - compound 40.

Intermediate 40a: 4-hydroxydecanoic acid

NaOH (2.0 eq) was added to a solution of 5-hexyltetrahydrofuran-2-one (15 g, 1.0 eq.) in EtOH (240 mL) and H ₂ O (80 mL). The reaction mixture was stirred at 25°C for 12 hours. The mixture was concentrated to remove EtOH. Then the pH was adjusted to 5 aq. 1 n. HCl and extracted three times with EtOAc. The combined organic phase was washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc/hexanes) to give the product as a white solid (48%).

Intermediate 40b: 4-oxodecanoic acid

To a solution of NaH2PO4 (17 eq.) and Na ₂ HPO ₄ (2.4 eq.) in H2O (1000 ml) was added intermediate 40a (10 g, 1.0 eq.). NaClO (200 ml, 6-8% Cl) was then added to the solution. The reaction mixture was stirred at 25°C for 12 hours. Then H3PO4 (200 ml, 85%) and silica gel (300

- 115 046244

G). The mixture was dried under reduced pressure and washed 3 times with EtOAc. The organic layer was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography (EtoAc/hexanes) to give the product as a white solid (80%).

^1H NMR (400 MHz, CDCl ₃ ) δ 2.70 (dd, J=7.0, 5.3 Hz, 2H), 2.63-2.58 (m, 2H), 2.43 (t, J=7.5 Hz, 2H), 1.58 (q, J=7.4 Hz, 2H), 1.33-1.19 (m, 6H), 0.90-0.83 (m, 3H ).

Intermediate bis(octadeca-9,12-dienoate)

40c: 2-((4-oxodecanoyl)oxy)propane-1,3-diyl (9Z,9'Z,12Z,12'Z)-

Intermediate 40c was synthesized in 40% yield from intermediate 40b and intermediate 1c using the same method as Example 1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.42-5.28 (m, 8H), 5.24 (tt, J=5.9, 4.3 Hz, 1H), 4.28 (dd, J=11.9, 4.3 Hz, 2H), 4.14 (dd, J=11.9, 5.9 Hz, 2H), 2.73 (dt, J=22.4, 6.7 Hz , 7H), 2.58 (t, J=6.5 Hz, 2H), 2.43 (t, J=7.5 Hz, 2H), 2.31 (t, J=7.6 Hz, 4H ), 2.04 (q, J=6.9 Hz, 8H), 1.59 (q, J=6.7 Hz, 7H), 1.40-1.22 (m, 35H), 0.88 (td, J=6.8, 4.0 Hz, 9H).

Intermediate 40d: 2-((4-(((4-nitrophenoxy)carbonyl)oxy)decanoyl)oxy)propane1,3-diyl(9Z,9'Z,12Z,12^)-bis(octadeca-9,12 -dienoate)

Intermediate 40d was synthesized in 31% yield from intermediate 40c using the same method as intermediate 1e.

^1H NMR (400 MHz, CDCl3) δ 8.37-8.27 (m, 2H), 7.43 (dd, J=9.1, 5.9 Hz, 2H), 5.45-5.24 (m, 9H), 4.34 (dd, J=12.0, 4.3 Hz, 2H), 4.18 (dd, J=11.9, 6.0 Hz, 2H), 2.79 ( t, J=6.3 Hz, 4H), 2.49 (t, J=7.5 Hz, 2H), 2.41-2.30 (m, 4H), 2.07 (q, J=6 .9 Hz, 9H), 1.83-1.58 (m, 7H), 1.48-1.12 (m, 37H), 1.00-0.82 (m, 9H).

Compound 40: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)decanoyl)oxy)propan-1,3-diyl (9Z,9'Z,12Z,12'Z )-bis(octadeca-9,12-dienoate)

Compound 40 was synthesized in 53% yield from intermediate 40d and Nl-ethyl10-methylthetan-1,2-diamine using the same method as Example 1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.44-5.19 (m, 9H), 4.76 (s, 1H), 4.32-4.22 (m, 2H), 4.15 ( ddd, J=11.9, 5.9, 3.4 Hz, 2H), 3.26 (s, 2H), 2.77 (t, J=6.5 Hz, 4H), 2.57-2 .36 (m, 6H), 2.36-2.27 (m, 4H), 2.23 (s, 3H), 2.05 (q, J=6.6 Hz, 8H), 1.98- 1.42 (m, 15H), 1.41-1.19 (m, 35H), 1.06 (t, J=7.2 Hz, 3H), 0.88 (td, J=9.7, 8.2, 4.2 Hz, 9H). MS: 916.7 m/z [M+H].

Example 41 - compound 41.

Intermediate 41a: 4-cyclohexyl-4-oxobutanoic acid

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To a solution of succinic anhydride (30 g, 1.0 eq.) in Et ₂ O (300 ml) was added CuI (0.1 eq.). The mixture was cooled to -20°C. Cyclohexylmagnesium bromide (1.0 eq.) was then added to the mixture. The reaction mixture was heated to 25°C and stirred for 14 hours. The reaction was quenched by adding 1 M aq. HCl and stirred for 15 minutes. The reaction mixture was then filtered and the filtrate was extracted 3 times with DCM. The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated. The crude residue was purified by column chromatography (EtOAc/hexanes) to give the product as a pale pink solid (13%).

1H NMR (400 MHz, CDCl ₃ ) δ 10.09 (s, 1H), 2.66 (dt, J=55.4, 6.5 Hz, 4H), 2.35 (td, J=10.8 , 9.3, 5.2 Hz, 1H), 1.75 (ddd, J=43.2, 36.6, 11.9 Hz, 5H), 1.46-1.09 (m, 5H).

Intermediate 41b: 2-((4-cyclohexyl-4-oxobutanoyl)oxy)propane-1,3-diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9,12-dienoate)

Intermediate 41b was synthesized in 34% yield from intermediate 41a and intermediate 1c using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.42-5.28 (m, 8H), 5.24 (ddd, J=10.2, 5.6, 4.2 Hz, 1H), 4.28 (dt, J=11.9, 4.6 Hz, 2H), 4.14 (ddd, J=11.9, 5.8, 2.3 Hz, 2H), 2.81-2.70 (m , 6H), 2.64-2.54 (m, 3H), 2.38-2.27 (m, 5H), 2.04 (q, J=6.8 Hz, 8H), 1.89- 1.55 (m, 11H), 1.40-1.20 (m, 34H), 0.88 (t, J=6.8 Hz, 6H).

Intermediate 41c: 2-((4-cyclohexyl-4-(((4-nitrophenoxy)carbonyl)oxy)butanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12^)-bis (octadeca-9,12-dienoate)

Intermediate 41c was synthesized in 14% yield from intermediate 41b using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 8.33-8.21 (m, 2H), 7.45-7.34 (m, 2H), 5.44-5.13 (m, 9H), 4 .70 (ddd, J=9.3, 6.1, 3.4 Hz, 1H), 4.50 (dd, J=12.3, 3.9 Hz, 1H), 4.35-4.10 (m, 4H), 2.76 (t, J=6.4 Hz, 4H), 2.50-2.40 (m, 2H), 2.38-2.22 (m, 4H), 2, 04 (q, J=6.9 Hz, 10H), 1.85-1.52 (m, 10H), 1.49-1.00 (m, 31H), 0.88 (t, J=6, 8 Hz, 6H).

Compound 41: 2-((4-cyclohexyl-4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)butanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z , 12^)-bis(octadeca-9,12-dienoate)

Compound 41 was synthesized in 60% yield from intermediate 41d and M-ethyl-Mmethylethane-1,2-diamine using the method used for intermediate 1e.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.45-5.09 (m, 9H), 4.60 (s, 1H), 4.28 (ddd, J=11.9, 4.5, 1 ,6 Hz, 2H), 4.15 (ddd, J=11.9, 5.8, 2.9 Hz, 2H), 3.25 (s, 2H), 2.77 (t, J=6, 5 Hz, 4H), 2.54-2.15 (m, 12H), 2.05 (q, J=6.7 Hz, 8H), 1.99-0.95 (m, 50H), 0, 93-0.80 (m, 6H). MS: 914.5 m/z [M+H].

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Example 42 - compound 42.

Intermediate 42a: 4-hydroxyoctanoic acid

Intermediate 42a was synthesized in 53% yield from 5-butyltetrahydrofuran-2one using the same method as intermediate 39b.

Intermediate 42b: 4-oxooctanoic acid

Intermediate 42b was synthesized in 61% yield from intermediate 42a using the method used for intermediate 38b.

¹ H NMR (400 MHz, CDCl ₃ ) δ 2.65 (ddd, J=7.0, 5.8, 1.1 Hz, 2H), 2.56 (ddd, J=7.1, 6.0 , 1.2 Hz, 2H), 2.38 (t, J=7.5 Hz, 2H), 1.56-1.44 (m, 2H), 1.30-1.17 (m, 2H) , 0.84 (t, J=7.3 Hz, 3H).

Intermediate 42c: 2-((4-oxooctanoyl)oxy)propane-1,3-diyl (9Z,9'Z,12Z,12'Z)bis(octadeca-9,12-dienoate)

Intermediate 42c was synthesized in 29% yield from intermediate 42b and intermediate 1c using the same method as Example 1.

^1H NMR (400 MHz, CDCl ₃ ) δ 5.35 (tq, J=18.2, 9.4, 7.6 Hz, 8H), 5.24 (t, J=5.3 Hz, 1H) , 4.28 (dd, J=12.0, 3.9 Hz, 2H), 4.14 (dd, J=12.1, 5.8 Hz, 2H), 2.74 (dt, J=20 ,9, 6.7 Hz, 6H), 2.59 (d, J=6.6 Hz, 2H), 2.43 (t, J=7.6 Hz, 2H), 2.31 (t, J =7.7 Hz, 4H), 2.04 (q, J=7.0 Hz, 8H), 1.60 (t, J=7.5 Hz, 8H), 1.31 (d, J=14 ,1 Hz, 29H), 0.88 (t, J=6.9 Hz, 9H).

Intermediate 42d: 2-((4-(((4-nitrophenoxy)carbonyl)oxy)octanoyl)oxy)propane1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9, 12-dienoate)

Intermediate 42d was synthesized in 51% yield from intermediate 40c using the same method as intermediate 1e.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.28 (dd, J=7.8, 4.7 Hz, 2H), 7.47-7.36 (m, 2H), 5.32 (dp, J=19.8, 9.2 Hz, 8H), 5.17 (s, 1H), 4.87 (d, J=7.3 Hz, 1H), 4.49 (d, J=12.2 Hz, 1H), 4.36-4.09 (m, 4H), 2.75 (d, J=6.7 Hz, 4H), 2.49 (dt, J=23.8, 7.9 Hz , 2H), 2.42-2.14 (m, 5H), 2.03 (q, J=7.2 Hz, 10H), 1.73 (s, 1H), 1.61 (dd, J= 23.8, 11.9 Hz, 6H), 1.31 (d, J=14.4 Hz, 30H), 0.89 (tt, J=9.5, 7.0, 2.7 Hz, 9H ).

Compound 42: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)octanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12^) -bis(octadeca-9,12-dienoate)

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Compound 42 was synthesized in 93% yield from intermediate 42d and Nl-ethylN-methylethane-1,2-diamine using the same method as Example 1.

1H NMR (400 MHz, CDCl3) δ 5.43-5.10 (m, 11H), 4.76 (s, 1H), 4.28 (ddd, J=11.9, 4.5, 1.3 Hz, 2H), 4.15 (ddd, J=11.8, 5.8, 3.3 Hz, 2H), 3.25 (s, 2H), 2.77 (t, J=6.5 Hz , 4H), 2.39 (ddd, J=8.7, 6.6, 4.3 Hz, 6H), 2.35-2.28 (m, 4H), 2.22 (s, 3H), 2.05 (q, J=6.7 Hz, 8H), 1.96-1.74 (m, 3H), 1.61 (s, 11H), 1.42-1.22 (m, 34h) , 1.10-0.99 (m, 3H), 0.95-0.83 (m, 9H).

Example 43 - compound 43.

Intermediate 43a: Sodium 4-hydroxybutanoate

To a mixture of tetrahydrofuran-2-one (10 g, 1.0 eq.) in H ₂ O (100 ml) was added NaOH (4.0 eq.), the mixture was degassed and purged 3 times with N2. The mixture was stirred at 25°C for 24 hours under N2 atmosphere. The reaction mixture was then concentrated under reduced pressure to give the crude residue as a colorless oil, which was used in the next step without further purification (12 g, crude).

Intermediate 43b: Benzyl 4-hydroxybutanoate

To a mixture of intermediate 43a (12 g, 1.0 equiv) in acetone (100 ml), TBAB (0.05 equiv) and benzyl bromide (1.2 equiv) were added, the mixture was degassed and purged 3 times with N2. The mixture was stirred at 60°C for 12 hours under N2 atmosphere. The reaction mixture was then concentrated under reduced pressure and the resulting residue was diluted with EtOAc and washed l times with l N each time. NaH _s O ₄ , NaHCO ₃ and H2O. The organic layer was dried over Na2SO4, filtered and concentrated, and the crude residue was purified by column chromatography (EtOAc/hexanes) to give the product as a colorless oil (43%).

Intermediate 43 c: benzyl 4-(((4-nitrophenoxy)carbonyl)oxy)butanoate

To a mixture of intermediate 43b (5 g, 1.0 equiv) in DCM (150 ml) was added 4-nitrophenyl chloroformate (1.0 equiv), the mixture was degassed and purged 3 times with N2. The reaction mixture was cooled to 0°C followed by the addition of pyridine (2.0 eq.). The reaction mixture was stirred at 25°C for 5 hours under N2 atmosphere. The reaction mixture was then concentrated under reduced pressure, diluted with hexane and filtered. The filtrate was washed 10 times with H ₂ O, and the combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated. The crude material was purified by column chromatography to give the product as a yellow oil (32%).

Intermediate 43 d: benzyl 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)butanoate

To a mixture of intermediate 43c (3 g, 1.0 equiv.) in MeCN (50 ml) was added N1-3τβλ-N1methylethane-1,2-diamine (2.5 equiv.), pyridine (3.0 equiv.) and DMAP (1.0 equiv.). The mixture was degassed and purged 3 times with N2, and the mixture was stirred at 70°C for 12 hours under an N2 atmosphere. The reaction mixture was then concentrated under reduced pressure and the crude residue was purified by column chromatography to give the product as a yellow oil (56%).

^1H NMR (400 MHz, CDCl3) δ 7.33-7.23 (m, 4H), 5.24 (d, J=12.8 Hz, 1H), 5.05 (s, 2H), 4, 04 (dt, J=12.9, 6.7 Hz, 2H), 3.19 (q, J=5.8 Hz, 2H), 3.00 (s, 1H), 2.39 (dt, J =10.4, 6.6 Hz, 6H), 2.13 (s, 3H), 1.89 (p, J=6.9 Hz, 2H), 0.98 (t, J=7.1 Hz , 3H).

Intermediate 43e: 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)butanoic acid

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Pd/C (10 wt%, 0.05 equiv) was added to a mixture of Intermediate 43d (1.5 g, 1.0 equiv) in MeOH (30 mL), and the mixture was degassed and purged 3 times with H ₂ . The reaction mixture was stirred at 25°C for 1 hour under H ₂ atmosphere, after which the filtrate was concentrated to obtain a crude residue, which was used in the next step without further purification.

1H NMR (400 MHz, CD3OD) δ 4.11 (t, J=6.3 Hz, 2H), 3.49 (t, J=6.0 Hz, 2H), 3.29-3.21 (m , 5H), 2.89 (s, 3H), 2.38 (t, J=7.2 Hz, 2H), 1.92 (p, J=6.7 Hz, 2H), 1.34 (t , J=7.3 Hz, 3H).

Compound 43: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)butanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z,12^) -bis(octadeca-9,12-dienoate)

Compound 43 was synthesized in 45% yield from intermediate 43 d and intermediate 1c using the method used for intermediate 1d.

1H NMR (400 MHz, CDCl ₃ ) δ 5.43-5.22 (m, 9H), 4.29 (dd, J=11.9, 4.4 Hz, 2H), 4.18-4.06 (m, 4H), 3.25 (q, J=5.8 Hz, 2H), 2.76 (t, J=6.4 Hz, 4H), 2.43 (dt, J=10.5, 7.3 Hz, 6H), 2.31 (t, J=7.6 Hz, 4H), 2.20 (s, 3H), 2.04 (q, J=6.9 Hz, 8H), 1 .95 (q, J=6.9 Hz, 2H), 1.59 (d, J=7.1 Hz, 7H), 1.40-1.22 (m, 26H), 1.04 (t, J=7.1 Hz, 3H), 0.88 (t, J=6.8 Hz, 6H).

Example 44 - compound 44.

Compound 44: 2-((4-(((( 1 -methylpyrrolidin-2-yl)methyl)carbamoyl)oxy)decanoyl)oxy)propane1,3-diyl(9Z,9'Z, 12Z,12^)-bis (octadeca-9,12-dienoate)

Compound 44 was synthesized in 35% yield from intermediate 40d and (1methylpyrrolidin-2-yl)methanamine using the method used for intermediate 1e.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.44-5.20 (m, 9H), 5.16 (s, 1H), 4.76 (s, 1H), 4.28 (dd, J= 11.9, 4.4 Hz, 2H), 4.15 (ddd, J=11.9, 5.8, 3.0 Hz, 2H), 3.39 (s, 1H), 3.10 (d , J=27.2 Hz, 2H), 2.81-2.73 (m, 4H), 2.43-2.18 (m, 11H), 2.05 (q, J=6.7 Hz, 8Н), 1.96-1.42 (m, 17Н), 1.30 (dqt, J=12.8, 8.8, 5.4 Hz, 36Н), 0.92-0.83 (m, 9H). MS: 928.9 m/z [M+H].

Example 45 - compound 45.

Compound 45: 2-((4-(((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)decanoyl)oxy)propan-1,3diyl (9Z,9'Z, 12Z, 12^)-bis (octadeca-9,12-dienoate)

Compound 45 was synthesized in 52% yield from intermediate 40d and 3(pyrrolidin-1-yl)propan-1-ol using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.44-5.21 (m, 9H), 4.72 (ddd, J=11.3, 8.8, 5.7 Hz, 1H), 4.29 (dd, J=11.9, 4.3 Hz, 2H), 4.25-4.10 (m, 4H), 2.81-2.74 (m, 4H), 2.59 (s, 5H ), 2.40 (dt, J=8.6, 6.4 Hz, 2H), 2.36-2.27 (m, 4H),

- 120 046244

2.05 (q, J=6.7 Hz, 8H), 1.98-1.78 (m, 9H), 1.67-1.51 (m, 7H), 1.41-1.21 ( m, 36H), 0.88 (td, J=7.0, 5.0 Hz, 9H). MS: 943.8 m/z [M+H].

Example 46 - compound 46.

Compound 46: 2-((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)hexadecanoyl)oxy)propane-1,3diyl (9Z,9'Z,12Z,12'Z)- bis(octadeca-9,12-dienoate)

Compound 46 was synthesized in 53% yield from intermediate 1e and 2-(pyrrolidin-1-yl)ethan-1-amine using the same method as Example 1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.55 (s, 1H), 5.46-5.21 (m, 9H), 4.75 (s, 1H), 4.28 (dd, J= 11.9, 4.4 Hz, 2H), 4.15 (ddd, J=11.9, 5.8, 3.6 Hz, 2H), 3.35 (d, J=6.0 Hz, 2H ), 2.81-2.60 (m, 9Н), 2.39 (ddd, J=8.6, 6.7, 3.8 Hz, 2Н), 2.31 (t, J=7.6 Hz, 4H), 2.05 (q, J=6.8 Hz, 8H), 1.84 (s, 7H), 1.66-1.19 (m, 55H), 0.88 (td, J =7.0, 4.3 Hz, 9H). MS: 1012.5 m/z [M+H].

Example 47 - compound 47.

Compound 47: 2-((4-((((1-methylpiperidin-4-yl)oxy)carbonyl)oxy)hexadecanoyl)oxy)propane1,3-diyl (9Z,9'Z, 12Z, 12^)-bis (octadeca-9,12-dienoate)

To a solution of intermediate 1e (250 mg, 1.0 eq) in MeCN (0.1 M) was added pyridine (3.0 eq), 1-methylpiperidin-4-ol (1.5 eq) and DMAP ( 1.0 eq.). The reaction mixture was stirred for 18 hours at room temperature. An additional portion of 1-methylpiperidin-4-ol (3.0 eq.) was added and the mixture was heated to 50°C. After 2 hours, additional DMAP (1.0 eq.) was added and the reaction was maintained for another 2 hours. Upon completion, heptane (0.1 M) was added to the reaction mixture and the heptane layer was washed 3 times with MeCN. The combined MeCN layers were extracted three times with heptane, and the combined heptane layers were washed one final time with MeCN. The heptane was then removed in vacuo and the crude material was purified by column chromatography (MeOH/DCM) to give the product as a colorless oil (62 mg, 32%).

1H NMR (400 MHz, CDCl ₃ ) δ 5.44-5.20 (m, 9H), 4.77-4.60 (m, 2H), 4.29 (dd, J=11.9, 4, 4 Hz, 2H), 4.14 (ddd, J=11.9, 5.8, 3.6 Hz, 2H), 2.83-2.66 (m, 6H), 2.46-2.26 (m, 11H), 2.08-1.71 (m, 18H), 1.71-1.47 (m, 8H), 1.40-1.21 (m, 47H), 0.88 (td , J=7.0, 4.2 Hz, 9H). MS: 1013.7 m/z [M+H].

Example 48 - compound 48.

Compound 48: 2-((4-(((3-(bis(2-hydroxyethyl)amino)propyl)carbamoyl)oxy)hexadecanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12 ^)-bis(octadeca-9,12-dienoate)

To a solution of intermediate 1e (250 mg, 1.0 equiv.) in MeCN (0.1 M) was added pyridine (3.0 equiv.) and 2-[(3-aminopropyl)(2-hydroxyethyl)amino]ethane- 1-ol (1.5 equiv.). The reaction mixture was stirred for 2 hours at room temperature. Upon completion, heptane (0.1 M) was added to the reaction mixture and the heptane layer was washed 3 times with MeCN. The combined MeCN layers were extracted three times with heptane, and the combined heptane layers were washed one final time with MeCN. Then heptane was removed in vacuum

- 121046244 IU and the crude material was purified by column chromatography (MeOH/DCM) to give the product as a colorless oil (97 mg, 48%).

1H NMR (400 MHz, CDCI3) δ 5.43-5.20 (m, 9H), 4.76 (s, 1H), 4.29 (ddd, J=11.9, 4.6, 1.8 Hz, 2H), 4.16 (dd, J=11.9, 5.8 Hz, 2H), 3.72 (t, J=5.0 Hz, 4H), 3.35-3.22 (m , 2H), 2.98-2.46 (m, 14H), 2.41-2.28 (m, 6H), 2.091.86 (m, 9H), 1.80 (dd, J=14.2 , 7.2 Hz, 3H), 1.67-1.42 (m, 7H), 1.42-1.18 (m, 46H), 0.88 (td, J=7.0, 4.3 Hz, 9H). MS: 1060.6 m/z [M+H].

Example 49 - comparative compound 49.

Compound 49: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 127)-octadeca-9, 12-dienoate

ABOUT %

Compound 49 was synthesized according to the methods described in WO 2015/095340 A1 (example 13).

1H NMR (CDCl ₃ , 400 MHz) δ 5.35 (m, 4H), 4.48 (t, J=5.6 Hz, 1H), 4.17 (m, 8H), 3.56 (m, 2H), 3.40 (m, 2H), 2.77 (t, J=6.6 Hz, 2H), 2.55 (q, J=7.2 Hz, 6H), 2.40 (m, 3H), 2.30 (t, J=7.6 Hz, 2H), 2.05 (q, J=6.8 Hz, 4H), 1.92 (m, 2H), 1.84 (m, 2H), 1.57 (m, 6H), 1.30 (m, 34H), 1.03 (t, J=7.2 Hz, 6H), 0.88 (m, 9H) ppm; MS: 853 m/z [M+H].

Example 50 - compound 50.

Compound 50: 2-((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12^) -bis(octadeca-9,12-dienoate)

Compound 50 was synthesized in 36% yield from intermediate 40d and 2(pyrrolidin-1-yl)ethan-1-amine using the same method as Example 1.

1H NMR (400 MHz, CDC13) δ 5.49-5.29 (m, 8H), 5.27-5.20 (m, 1H), 4.76 (s, 1H), 4.28 (dd, J=11.9, 4.4 Hz, 2H), 4.15 (ddd, J=11.9, 5.8, 3.8 Hz, 2H), 3.35 (s, 2H), 2.83 -2.44 (m, 10H), 2.39 (ddd, J=8.4, 6.7, 3.3 Hz, 2H), 2.36-2.25 (m, 4H), 2.05 (q, J=6.8 Hz, 8H), 1.98-1.43 (m, 15H), 1.42-1.20 (m, 33H), 0.89 (td, J=6.1 , 5.4, 0.9 Hz, 9H). MS: 928.6 m/z [M+H].

Example 51 - compound 51.

Compound 51: 2-((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propane-1,3diyl (9Z,9'Z, 12Z, 12^)-bis (octadeca-9,12-dienoate)

Compound 51 was synthesized in 27% yield from intermediate 31c and 2(pyrrolidin-1-yl)ethan-1-amine using the same method as Example 1.

1H NMR (400 MHz, CDC13) δ 5.55 (s, 1H), 5.44-5.28 (m, 8H), 5.28-5.22 (m, 1H), 4.76 (s, 1H), 4.28 (dd, J=11.9, 4.4 Hz, 2H), 4.15 (ddd, J=11.9, 5.8, 3.9 Hz, 2H), 3.37 (s, 2H), 2.83-2.51 (m, 10H), 2.39 (ddd, J=8.4, 6.8, 3.1 Hz, 2H), 2.35-2.27 (m, 4H), 2.05 (q, J=6.7 Hz, 8H), 1.97-1.41 (m, 17H), 1.41-1.20 (m, 40H), 0, 94-0.84 (m, 9H). MS: 957.0 m/z [M+H].

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Example 52 - compound 52.

Intermediate 52a: Benzyl 3-hydroxyundecanoate

OH oZr™

To a solution of THF (150 ml) at 66°C under N ₂ atmosphere, Zn (22.99 g, 1.0 equiv.) and nonanal (25 g, 1.0 equiv.) were added dropwise. Benzyl 2-bromoacetate (50.00 g, 1.24 eq.) was then slowly and carefully added dropwise to the reaction mixture. After complete addition of benzyl 2-bromoacetate, the mixture was cooled and stirred at 20°C for 2 hours. The reaction mixture was then concentrated under reduced pressure to remove THF, and the resulting residue was diluted with water and extracted with 3x EtOAc. The combined organic layers were concentrated under reduced pressure and purified by column chromatography to give the product as a yellow oil (25 g, 49%).

^1H NMR (400 MHz, CDCls) δ 7.33-7.24 (m, 5H), 5.09 (s, 2H), 3.95 (tt, J=7.7, 3.8 Hz, 1H ), 2.75 (d, J=3.9 Hz, 1H), 2.56-2.35 (m, 2H), 1.54-1.11 (m, 18H), 0.81 (t, J=6.7 Hz, 3H).

Intermediate 52b: Benzyl 3-(((4-nitrophenoxy)carbonyl)oxy)undecanoate

L L l

To a solution of intermediate 52a (25 g, 1.0 equiv) in DCM (250 ml) was added (4nitrophenyl)carbonochloridate (17.23 g, 1.0 equiv) and pyridine (13.80 ml, 2.0 equiv). .) in an N2 atmosphere. The mixture was stirred at 20°C for 2 hours. Upon completion, the reaction mixture was concentrated under reduced pressure to remove DCM, and the resulting residue was diluted with water and extracted with 3x EtOAc. The combined organic layers were concentrated under reduced pressure and purified by column chromatography to give the product as a colorless oil (12 g, 31%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.31-8.01 (m, 4H), 7.28 (s, 5H), 5.22-5.14 (m, 2H), 5.13- 5.01 (m, 4H), 2.68 (dd, J=14.8, 6.4 Hz, 3H), 1.75-1.64 (m, 2H), 1.30-1.19 ( m, 18H), 0.80 (t, J=6.6 Hz, 3H).

Intermediate 52c: Benzyl 3-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)undecanoate

Pyridine ( 7.41 ml, 3.0 eq.) and DMAP (3.74 g, 1.0 eq.). The mixture was stirred at 20°C for 12 hours. Upon completion, the reaction mixture was concentrated under reduced pressure to remove MeCN, and the resulting residue was diluted with water and extracted with 3x EtOAc. The combined organic layers were concentrated under reduced pressure and purified by column chromatography to give the product as a colorless oil. (6 g, 47%).

1H NMR (400 MHz, CDCl3) δ 7.39-7.28 (m, 5H), 5.11 (s, 4H), 3.21 (h, J=6.8 Hz, 2H), 2.62 (td, J=14.4, 13.6, 6.4 Hz, 2H), 2.50-2.39 (m, 4H), 1.59 (h, J=8.2 Hz, 2H), 1.25 (d, J=16.1 Hz, 14H), 1.03 (t, J=7.2 Hz, 3H), 0.86 (t, J=6.7 Hz, 3H).

Intermediate 52d: 3-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)undecanoic acid

Pd/C (2 g, 10% w/w) was added to a solution of intermediate 52c (6 g, 1.0 equiv) in MeOH (60 mL). The mixture was stirred at 20°C for 2 hours under an H ₂ atmosphere. The reaction mixture was then filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a brown oil.

1H NMR (400 MHz, CDCl ₃ ) δ 12.76 (s, 1h), 6.28 (t, J=5.8 Hz, 1H), 5.01 (p, J=6.1, 5.6 Hz, 1H), 3.30 (qq, J=13.8, 6.0 Hz, 2H), 2.70 (hd, J=12.1, 11.0, 5.4 Hz, 3H), 2 .37 (d, J=14.1 Hz, 4H), 1.51 (ddq, J=21.6, 14.2, 6.7 Hz, 2H), 1.34-1.14 (m, 11H ), 1.10 (t, J=7.1 Hz, 3H), 0.80 (t, J=6.7 Hz, 3H).

Compound 52: 2-((3-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)undecanoyl)oxy)propan-1,3diyl (9Z,9'Z,12Z,12'Z)- bis(octadeca-9,12-dienoate)

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To a solution of intermediate 52d (1.0-1.1 eq) and intermediate 1c (1.0-1.1 eq) in DCM (0.1-0.25 eq) was added DMAP (0.25 eq.), DIPEA (3.5 eq.) and EDCI (1.5 eq.) sequentially. The reaction mixture was stirred at 23°C for at least 16 hours. Additional DMAP (0.25 equiv.) was added to the reaction mixture and the reaction mixture was stirred at 23°C for an additional 6 hours. The reaction was then quenched by adding water, washed with 1 times 1 M HCl and 1 time 5% NaHCO ₃ . The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. Purification by column chromatography gave the product as a colorless oil (28%).

^1H NMR (400 MHz, CDCl ₃ ) δ 5.53-5.28 (m, 8H), 5.24 (s, 1H), 5.08 (s, 1H), 4.28 (ddd, J= 15.9, 11.9, 4.5 Hz, 2H), 4.15 (dt, J=13.4, 6.8 Hz, 2H), 2.77 (t, J=6.5 Hz, 4H ), 2.59 (tt, J=15.9, 9.0 Hz, 6H), 2.37-2.14 (m, 6H), 2.05 (q, J=6.8 Hz, 7H) , 1.59 (d, J=14.2 Hz, 12H), 1.46-0.97 (m, 42H), 0.96-0.81 (m, 9H). MS: 930.8 m/z [M+H].

Example 53 - compound 53.

Intermediate 53a: 2-(hydroxymethyl)propane-1,3-diyl(92.9'2,122.12'2)-bis(octadeca9,12-dienoate)

EDCI (13, 67 g, 1.0 eq.), DMAP (1.39 g, 0.16 eq.) and DIPEA (37.27 ml, 3.0 eq.). The mixture was stirred at 20°C for 2 hours. Upon completion, the reaction mixture was concentrated under reduced pressure to remove DCM, and the resulting residue was diluted with water and extracted with 3x EtOAc. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude residue was purified by column chromatography (EtOAc/hexanes) to give the product as a colorless oil (6.3 g, 11%).

1H NMR (400 MHz, CD3OD) δ 5.44-5.30 (m, 8H), 4.17 (qd, J=11.1, 6.0 Hz, 4H), 3.63 (d, J= 6.0 Hz, 2H), 2.80 (t, J=6.1 Hz, 4H), 2.35 (t, J=7.4 Hz, 4H), 2.21 (h, J=6, 0 Hz, 1H), 2.14-2.03 (m, 8H), 1.64 (p, J=7.4 Hz, 4H), 1.46-1.28 (m, 27H), 0, 93 (t, J=6.7 Hz, 6H).

Compound 53: 2-(11-methyl-5-octyl-3,7-dioxo-2,6-dioxa-8,11-diazatridecyl)propan-1,3-diyl (92.9'2,122.12'2) -bis(octadeca-9,12-dienoate)

Compound 53 was synthesized in 45% yield from intermediate 52d and intermediate 53a using the same method as 52.

1H NMR (400 MHz, CDCl ₃ ) δ 5.48-5.24 (m, 8H), 5.07 (t, J=6.5 Hz, 1H), 4.20-4.07 (m, 6H ), 2.77 (t, J=6.5 Hz, 4H), 2.62-2.46 (m, 4H), 2.45-2.37 (m, 2H), 2.34-2, 15 (m, 6H), 2.05 (q, J=6.8 Hz, 8H), 1.43-0.97 (m, 43H), 0.95-0.83 (m, 9H). MS: 944.8 m/z [M+H].

Example 54 - compound 54.

Intermediate 54a: Benzyl 4-hydroxydodecanoate

NaOH (1.1 equiv) was added to a solution of 5-octyltetrahydrofuran-2-one (20 g, 1.0 eq.) in water (200-250 ml). The mixture was stirred at 20°C for 24 hours under N2 atmosphere. Then the reaction mixture is conc.

- 124 046244 tririated under reduced pressure to remove the solvent - water. The resulting crude material was reconstituted in acetone (200 ml) followed by the addition of TBAB (0.05 eq) and BnBr (1.2-1.5 eq). The mixture was heated to 60°C and stirred for 12 hours under N2 atmosphere. Upon completion, the mixture was concentrated under reduced pressure and the resulting residue was poured into us. NaHCO3 solution. The product was extracted 3 times with EtOAc and the combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude residue was purified by column chromatography (EtOAc/hexanes) to give the product as a colorless oil (20 g, 39%).

^1H NMR (400 MHz, CDCl3) δ 7.45-7.29 (m, 5H), 5.12 (s, 2H), 3.65-3.57 (m, 1H), 2.51 (td , J=7.2, 2.7 Hz, 2H), 1.86 (dtd, J=14.6, 7.4, 3.5 Hz, 1H), 1.71 (ddd, J=14.2 , 8.6, 7.1 Hz, 1H), 1.61 (d, J=5.0 Hz, 1H), 1.52-1.17 (m, 17H), 0.88 (t, J= 6.7 Hz, 3H).

Intermediate 54b: Benzyl 4-(((4-nitrophenoxy)carbonyl)oxy)dodecanoate

To a solution of intermediate 54a (10 g, 1.0 equiv.) in DCM (80-100 ml) was added (4nitrophenyl)carbonochloridate (1.5 equiv.). The reaction mixture was degassed and purged 3 times with N ₂ and cooled to 0°C before adding pyridine (2.0 eq.). The reaction mixture was stirred at 20°C for 5 hours under N ₂ atmosphere. Upon completion, the mixture was concentrated under reduced pressure and the resulting residue was reduced in hexane and filtered. The resulting filtrate was washed 3 times with water, and the organic layer was washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by column chromatography (EtOAc/hexanes) to give the product as a colorless oil (9 g, 29%).

1H NMR (400 MHz, CDQ3) δ 8.27 (dd, J=9.8, 7.8 Hz, 2H), 7.50-7.30 (m, 8H), 5.12 (d, J= 8.3 Hz, 2H), 4.87 (ddt, J=12.2, 7.3, 4.1 Hz, 1H), 2.50 (t, J=7.5 Hz, 2H), 2, 16-2.06 (m, 1H), 1.99 (dq, J=14.8, 7.5 Hz, 1H), 1.73 (ddd, J=14.0, 12.0, 7.0 Hz, 1H), 1.63 (tt, J=8.6, 4.5 Hz, 1H), 1.45-1.19 (m, 12H), 0.94-0.84 (m, 3H) .

Intermediate 54c: Benzyl 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecanoate

To a solution of intermediate 54b (9 g, 1.0 equiv.) in MeCN (45-90 ml) was added N'-ethylU-methylethane-1,2-diamine (1.5-2.5 equiv.), pyridine ( 3-4 eq.) and DMAP (2.33 g, 1.0 eq.), and the resulting mixture was degassed and purged 3 times with N2. The reaction mixture was heated to 70°C and stirred for 2 hours under N ₂ atmosphere. Upon completion, the mixture was concentrated to remove the solvent, and the resulting residue was diluted with EtOAc, washed 3 times with sat. NaHCO3 and 3 times with water. The organic layer was then dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude residue was purified by column chromatography (EtOAc/hexanes) to give the product as a pale yellow oil (5 g, 60%).

1H NMR (400 MHz, CDQ3) δ 7.38-7.22 (m, 5H), 5.04 (s, 2H), 4.69 (s, 1H), 3.16 (q, J=6, 0 Hz, 2H), 2.452.27 (m, 6H), 2.13 (s, 3H), 1.95-1.71 (m, 2H), 1.40 (d, J=7.3 Hz, 2H), 1.34-1.09 (m, 13H), 0.96 (t, J=7.1 Hz, 3H), 0.80 (t, J=6.7 Hz, 3H).

Intermediate 54d: 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecanoic acid

Pd/C (1.8 g, 10% w/w) was added to a solution of intermediate 54c (6 g, 1.0 equiv) in MeOH (60 mL). The mixture was stirred at 20°C for 5 hours under an H ₂ atmosphere. The reaction mixture was then filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a brown oil (3.2 g, 67%).

^1H NMR (400 MHz, CD3OD) δ 4.76 (dp, J=8.1, 4.1 Hz, 1H), 3.65 (dt, J=14.0, 7.0 Hz, 1H), 3.28-3.10 (m, 4H), 3.07 (ddd, J=11.3, 7.5, 2.1 Hz, 1H), 2.79 (s, 3H), 2.27 ( ddd, J=14.0, 8.2, 4.4 Hz, 1H), 2.20-1.99 (m, 2H),

- 125 046244

1.86-1.74 (m, 1H), 1.71-1.56 (m, 1H), 1.48 (dq, J=13.7, 7.6, 6.8 Hz, 1H), 1.31 (dd, J=14.1, 7.0 Hz, 14H), 0.940.83 (m, 3H).

Compound 54: 2-(12-methyl-6-octyl-3,8-dioxo-2,7-dioxa-9,12-diazatetradecyl)propane-1,3-diyl (9Z,9'Z, 12Z, 12U) -bis(octadeca-9,12-dienoate)

Compound 54 was synthesized in 47% yield from intermediate 54d and intermediate 53a using the method used for compound 52.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.44-5.26 (m, 8H), 4.75 (s, 1H), 4.12 (d, J=6.0 Hz, 6H), 2 .77 (t, J=6.5 Hz, 4H), 2.55-2.34 (m, 6H), 2.34-2.19 (m, 6H), 2.05 (q, J=6 ,8 Hz, 8H), 1.97-1.70 (m, 4H), 1.61 (t, J=7.2 Hz, 10H), 1.41-1.00 (m, 43H), 0 .94-0.82 (m, 9H). MS: 959.1 m/z [M+H].

Example 55 - compound 55.

Intermediate 55a: Benzyl 5-hydroxytridecanoate

Intermediate 55a was prepared in 25% yield from 6-octyltetrahydropyran-2-one using the same method as intermediate 54a.

^1H NMR (400 MHz, CDCl3) δ 7.34 (qt, J=9.5, 4.5 Hz, 5H), 5.11 (s, 2H), 3.57 (s, 1H), 2, 39 (t, J=7.3 Hz, 2H), 1.85-1.65 (m, 2H), 1.60-1.11 (m, 18H), 0.98-0.80 (m, 3H).

Intermediate 55b: Benzyl 5-(((4-nitrophenoxy)carbonyl)oxy)tridecanoate

Intermediate 55b was prepared in 37% yield from intermediate 55a using the same method as intermediate 54b.

^1H NMR (400 MHz, CDCl ₃ ) δ 8.32-8.24 (m, 3H), 7.39 (dddd, J=20.3, 9.9, 5.7, 1.9 Hz, 8H ), 5.12 (s, 2H), 4.87-4.75 (m, 1H), 2.41 (t, J=6.8 Hz, 2H), 1.69 (dddt, J=33, 5, 23.3, 14.2, 6.0 Hz, 6H), 1.51-1.01 (m, 13H), 0.87 (t, J=6.6 Hz, 3H).

Intermediate 55c: Benzyl 5-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)tridecanoate

Intermediate 55c was prepared in 53% yield from intermediate 55b using the same method as intermediate 54c.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.41-7.18 (m, 5H), 5.04 (s, 2H), 4.65 (p, J=6.5 Hz, 1H), 3 ,19 (q, J=6.4, 5.9 Hz, 2H), 2.49-2.23 (m, 6H), 2.16 (d, J=29.0 Hz, 3H), 1, 69-1.54 (m, J=6.7 Hz, 2H), 1.44 (tt, J=13.6, 7.1 Hz, 4H), 1.37-1.05 (m, 14H) , 0.98 (t, J=7.1 Hz, 3H), 0.80 (t, J=6.6 Hz, 3H).

Intermediate 55d: 5-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)tridecanoic acid

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Pd/C (2.5 g, 10% w/w) was added to a solution of intermediate 55c (2.2 g, 1.0 equiv) in THF (20 mL). The mixture was stirred at 20°C for 5 hours under an H ₂ atmosphere. The reaction mixture was then filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a brown oil (1 g, 58%).

1H NMR (400 MHz, CDCl3) δ 12.81 (s, 1H), 5.93-5.72 (m, 1H), 4.61 (q, J=7.1 Hz, 1H), 3.68 -3.47 (m, 1H), 3.29-3.04 (m, 1H), 2.76 (tt, J=17.2, 9.1 Hz, 3H), 2.40 (s, 3H ), 2.34-2.06 (m, 2H), 1.85-0.87 (m, 22H), 0.80 (t, J=6.4 Hz, 3H).

Compound 55: 2-(13-methyl-7-octyl-3,9-dioxo-2,8-dioxa-10,13-diazapentadecyl)propan-1,3-diyl (9Z,9'Z, 12Z, 12' I)-bis(octadeca-9,12-dienoate)

Compound 55 was prepared in 58% yield from intermediate 53a and intermediate 55d using the same method as 52.

1H NMR (400 MHz, CDCh) δ 5.44-5.27 (m, 8H), 4.73 (s, 1H), 4.12 (d, J=6.0 Hz, 6H), 3.32 (s, 2H), 2.812.73 (m, 4H), 2.49 (d, J=45.9 Hz, 4H), 2.43-2.22 (m, 10H), 2.05 (q, J=6.8 Hz, 8H), 1.75-1.42 (m, 13H), 1.421.21 (m, 40H), 1.11 (s, 3H), 0.95-0.82 (m , 9H). MS: 972.8 m/z [M+H].

Example 56 - compound 56.

Intermediate 56a: 9-(decyloxy)-9-oxononanoic acid

To a mixture of nonanedioic acid (90 g, 1.0 equiv.) in THF (300-900 ml) at 0°C, DMF (0.1-0.2 equiv.) and (COCl) ₂ (1.0 eq.). The mixture was degassed and purged 3 times with N2, and then the mixture was stirred at 20°C for 2 hours under an _N2 atmosphere. Decan-1-ol (1.0 equiv.) was added dropwise to the reaction mixture at 0°C and stirred at 20°C for 2 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove the solvent, the resulting solution was poured into water and extracted three times with EtOAc. The combined organic layers were washed with brine, then dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude residue was purified by column chromatography (EtOAc/hexanes) to give the product as a white solid (20 g, 6%).

1H NMR (400 MHz, CDCh) δ 4.05 (t, J=6.7 Hz, 2H), 2.34 (t, J=7.5 Hz, 2H), 2.29 (t, J=7 .5 Hz, 2H), 1.62 (q, J=7.3 Hz, 6H), 1.41-1.15 (m, 19H), 0.88 (t, J=6.7 Hz, 3H ).

Intermediate 56b: 9,9'-didecyl O'1,O1-(2-oxopropane-1,3-diyl)di(nonanedioate)

To a mixture of 1,3-dihydroxypropan-2-one (1.37 g, 0.5 eq), EDCI (1.0 eq) and DMAP (1.0 eq) in DCM (100-200 ml) intermediate 56a (1.0 eq.) in portions at 0°C, and the mixture was degassed and purged 3 times with N ₂ . The mixture was stirred at 20°C for 5 hours under N ₂ atmosphere. The reaction mixture was concentrated under reduced pressure to remove the solvent, the resulting solution was poured into water and extracted three times with EtOAc. The combined organic layers were washed with brine, then dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by column chromatography (EtOAc/hexanes) to give the product as a white solid (10 g, 46%).

1H NMR (400 MHz, CDCh) δ 4.74 (d, J=2.1 Hz, 4H), 4.05 (t, J=6.8 Hz, 4H), 2.41 (t, J=7 .5 Hz, 4H), 2.28 (t, J=7.5 Hz, 4H), 1.62 (d, J=8.8 Hz, 12H), 1.39-1.19 (m, 39H ), 0.87 (t, J=6.7 Hz, 6H).

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Intermediate 56c: 9,9'-didecyl O'1,O1-(2-hydroxypropane-1,3-diyl)di(nonanedioate)

NaBH ₄ ( _1.5 equiv.). The mixture was stirred at 15-20°C for 5 hours under N2 atmosphere. Upon completion, the reaction mixture was diluted with water and extracted three times with EtOAc. The combined organic layers were washed with brine, then dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude residue was purified by column chromatography (EtOAc/hexanes) to give the product as a white solid (5.1 g, 38%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.15-4.03 (m, 5H), 3.98 (t, J=6.8 Hz, 4H), 2.44 (d, J=4, 8 Hz, 1H), 2.25 (dt, J=24.2, 7.5 Hz, 8H), 1.55 (q, J=7.1 Hz, 12H), 1.22 (d, J= 22.7 Hz, 38H), 0.81 (t, J=6.7 Hz, 6H).

Compound 56: 9,9'-didecyl O'1,O1-(2-((3-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)undecanoyl)oxy)propane-1,3 - diyl)di(nonanedioate)

Compound 56 was prepared in 36% yield from intermediate 52d and intermediate 56c using the same method as 52.

^1H NMR (400 MHz, CDCl3) δ 5.24 (t, J=5.2 Hz, 1H), 5.13-5.04 (m, 1H), 4.28 (ddd, J=14.5 , 11.9, 4.5 Hz, 2H), 4.14 (ddd, J=11.9, 8.3, 5.8 Hz, 2H), 4.05 (t, J=6.8 Hz, 4H), 3.31 (s, 2H), 2.58 (qd, J=15.3, 6.4 Hz, 6H), 2.30 (dt, J=10.3, 7.6 Hz, 10H ), 1.61 (dd, J=10.1, 4.1 Hz, 16H), 1.29 (dt, J=17.6, 3.3 Hz, 54H), 0.88 (td, J= 6.9, 1.9 Hz, 9H). MS: 1027.2 m/z [M+H].

Example 57 - compound 57.

Compound 57: 9,9'-didecyl O'1,O1-(2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propane-1,3 - diyl)di(nonanedioate)

Compound 57 was prepared in 55% yield from intermediate 54d and intermediate 56c using the same method as 52.

1H NMR (400 MHz, CDCl ₃ ) δ 5.28-5.21 (m, 1H), 4.76 (s, 1H), 4.28 (ddd, J=11.8, 4.4, 1, 2 Hz, 2H), 4.15 (ddd, J=11.9, 5.9, 4.6 Hz, 2H), 4.05 (t, J=6.8 Hz, 4H), 3.29 ( s, 2H), 2.38 (ddd, J=8.6, 6.9, 2.7 Hz, 5H), 2.35-2.12 (m, 10H), 1.98-1.73 ( m, 3H), 1.68-1.37 (m, 17H), 1.29 (dt, J=19.5, 4.1 Hz, 54H), 0.88 (td, J=6.9, 1.9 Hz, 9H). MS: 1040.8 m/z [M+H].

Example 58 - compound 58.

Compound 58: 9,9'-didecyl O'1,O1-(2-((5-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)tridecanoyl)oxy)propane-1,3- diyl)di(nonanedioate))

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Compound 58 was prepared in 60% yield from intermediate 55d and intermediate 56c using the same method as 52.

1H NMR (400 MHz, CDCl ₃ ) δ 5.27-5.20 (m, 1H), 4.73 (s, 1H), 4.28 (ddd, J=11.9, 4.4, 2, 8 Hz, 2H), 4.13 (ddd, J=11.9, 5.9, 2.6 Hz, 2H), 4.05 (t, J=6.7 Hz, 4H), 3.31 ( s, 2H), 2.39-2.09 (m, 12H), 1.62 (dq, J=13.8, 7.2, 6.5 Hz, 22H), 1.41-0.99 ( m, 54H), 0.88 (td, J=6.9, 2.0 Hz, 9H). MS: 1055.0 m/z [M+H].

Example 59 - compound 59.

Intermediate 59a: 9-oxo-9-(undecyloxy)nonanoic acid

Intermediate 59a was prepared in 41% yield from nonanedioic acid and undecan-1-ol using the same method as intermediate 56a.

1H NMR (400 MHz, CDCl3) δ 4.05 (t, J=6.7 Hz, 2H), 2.31 (dt, J=22.6, 7.5 Hz, 4H), 1.62 (q , J=7.1 Hz, 6H), 1.40-1.18 (m, 23H), 0.87 (t, J=6.7 Hz, 3H).

Intermediate 59b: O'1,O1-(2-oxopropane-1,3-diyl) 9,9'-diundecyl di(nonanedioate)

Intermediate 59b was prepared in 23% yield from 1,3-dihydroxypropan-2-one and intermediate 59a using the same method as intermediate 56b.

1H NMR (400 MHz, CDCl ₃ ) δ 3.98 (t, J=6.7 Hz, 2H), 2.35 (t, J=7.5 Hz, 2H), 2.22 (t, J= 7.5 Hz, 2H), 1.57 (dt, J=18.6, 6.9 Hz, 6H), 1.36-1.12 (m, 23H), 0.89-0.76 (m , 3H).

Intermediate 59c: O'1,O1-(2-hydroxypropane-1,3-diyl) 9,9'-diundecyl di(nonanedioate)

Intermediate 59c was prepared in 47% yield from intermediate 59b using the same method as intermediate 56c.

1H NMR (400 MHz, CDCl3) δ 4.23-4.00 (m, 9H), 2.52 (d, J=4.7 Hz, 1H), 2.31 (dt, J=24.2, 7.5 Hz, 8H), 1.62 (q, J=7.0 Hz, 13H), 1.29 (d, J=23.9 Hz, 43H), 0.88 (t, J=6, 7 Hz, 6H).

Compound

O'1,O1 -(2-((3-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)undecanoyl)59:

hydroxy)propane-1,3-diyl) 9,9'-diundecyl di(nonanedioate)

Compound 59 was prepared in 35% yield from intermediate 52d and intermediate 59c using the same method as 52.

1H NMR (400 MHz, CDCl ₃ ) δ 5.23 (s, 1H), 5.08 (d, J=6.5 Hz, 1H), 4.28 (ddd, J=15.0, 11.9 , 4.5 Hz, 2H), 4.14 (dt, J=13.1, 6.7 Hz, 2H), 4.05 (t, J=6.8 Hz, 4H), 2.58 (qd , J=15.4, 6.6 Hz, 4H), 2.30 (dt, J=10.4, 7.5 Hz, 10H), 1.60 (q, J=7.7, 6.9 Hz, 20H), 1.43-0.97 (m, 58H), 0.88 (td, J=6.9, 1.9 Hz, 9H). MS: 1054.9 m/z [M+H].

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Example 60 - compound 60.

Compound 60: O'1,O1-(2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propan-1,3-diyl) 9,9' -diundecyl di(nonanedioate)

Compound 60 was prepared in 79% yield from intermediate 54d and intermediate 59c using the same method as 52.

1H NMR (400 MHz, CDCl3) δ 5.25 (p, J=5.1 Hz, 1H), 4.76 (s, 1H), 4.28 (ddd, J=11.9, 4.5, 2.7 Hz, 2H), 4.15 (dt, J=11.7, 5.6 Hz, 2H), 4.05 (t, J=6.8 Hz, 4H), 2.38 (t, J=7.5 Hz, 5H), 2.35-2.11 (m, 10H), 1.98-1.41 (m, 23H), 1.41-0.96 (m, 57H), 0 .88 (td, J=6.9, 1.8 Hz, 9H). MS: 1068.9 m/z [M+H].

Example 61 - compound 61.

Compound 61: O'1,O1-(2-((5-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)tridecanoyl)oxy)propan-1,3-diyl) 9,9' -diundecyl di(nonanedioate)

Compound 61 was prepared in 61% yield from intermediate 55d and intermediate 59c using the same method as 52.

^1H NMR (400 MHz, CDCl3) δ 5.29-5.20 (m, 1H), 4.72 (d, J=7.4 Hz, 1H), 4.28 (ddd, J=11.9 , 4.4, 2.3 Hz, 2H), 4.14 (ddd, J=11.9, 5.9, 2.0 Hz, 2H), 4.05 (t, J=6.8 Hz, 4H), 2.82-2.13 (m, 15H), 1.82-1.39 (m, 21H), 1.390.97 (m, 56H), 0.88 (td, J=6.9, 1.9 Hz, 9H). MS: 1083.0 m/z [M+H].

Example 62 - compound 62.

Intermediate 62a: 1-((tert-butyldimethylsilyl)oxy)-3-hydroxypropan-2-one

TBSC1 (83, 66 g, 1.0 eq.), and the mixture was stirred at 20°C for 2 hours under N2 atmosphere. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue, which was purified by column chromatography (EtOAc/hexanes) to give the product as a colorless oil (15 g, 13%).

Intermediate 62b: 1-(3-((tert-butyldimethylsilyl)oxy)-2-oxopropyl) 9-undecylnonanedioate

To a mixture of intermediate 62a (10 g, 1.0 equiv), EDCI (9.38 g, 1.0 equiv) and DMAP (5.98 g, 1.0 equiv) in DCM (300 ml) at At 0° C., intermediate 59a (16.76 g, 1.0 eq.) was added and the mixture was stirred at 20° C. for 12 hours under N ₂ atmosphere. Upon completion, the reaction mixture was concentrated under reduced pressure to remove DCM, and the resulting residue was diluted with water. The crude material was extracted three times with EtOAc and the combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude residue was purified by column chromatography (EtOAc/hexanes) to give the product as a colorless oil (11 g, 43%).

1H NMR (400 MHz, CDCl3) δ 4.84 (s, 2H), 4.16 (s, 2H), 3.95 (t, J=6.8 Hz, 2H), 2.32 (t, J =7.5 Hz, 2H), 2.18 (t, J=7.5 Hz, 2H), 1.61-1.48 (m, 6H), 1.30-1.12 (m, 22H) , 0.82 (s, 9H), 0.78 (t, J=6.8 Hz, 3H).

Intermediate 62c: 1-(3-hydroxy-2-oxopropyl) 9-undecylnonanedioate

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To a mixture of intermediate 62b (11 g, 1.0 equiv) in THF (50 ml) was added HF-pyridine (3.75 ml, 8.0 equiv). The mixture was stirred at 20°C for 5 hours under N ₂ atmosphere. Upon completion, the reaction mixture was diluted with water and extracted three times with EtOAc. The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude residue was purified by column chromatography (EtOAc/hexanes) to give the product as a white solid (6 g, 70%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.70 (s, 2H), 4.30 (s, 2H), 3.98 (t, J=6.8 Hz, 2H), 2.95 (s , 1H), 2.36 (t, J=7.5 Hz, 2H), 2.22 (t, J=7.6 Hz, 2H), 1.57 (dt, J=20.3, 7, 1 Hz, 6H), 1.37-1.10 (m, 22H), 0.81 (t, J=6.7 Hz, 3H).

Intermediate 62d: 1-(3-(((92,122)-octadeca-9,12-dienoyl)oxy)-2-oxopropyl) 9-undecyl nonanedioate

To a solution of intermediate 62c (6 g, 1.0 equiv.) in DCM (100 ml) at 0°C was added EDCI (1.0 equiv.), DMAP (1.0 equiv.). The solution was degassed and purged 3 times with N ₂ followed by the addition of linoleic acid (4 ml, 1.0 equiv.). The mixture was stirred at 20°C for 12 h in an N2 atmosphere. Upon completion, the reaction mixture was concentrated under reduced pressure to remove DCM, and the resulting residue was diluted with water. The crude material was extracted three times with EtOAc and the combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude residue was purified by column chromatography (EtOAc/hexanes) to give the product as a colorless oil (7.5 g, 55%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.70 (s, 2H), 4.30 (s, 2H), 3.98 (t, J=6.8 Hz, 2H), 2.95 (s , 1H), 2.36 (t, J=7.5 Hz, 2H), 2.22 (t, J=7.6 Hz, 2H), 1.57 (dt, J=20.3, 7, 1 Hz, 6H), 1.37-1.10 (m, 22H), 0.81 (t, J=6.7 Hz, 3H).

Intermediate 62e: 1-(2-hydroxy-3-(((92,122)-octadeca-9,12-dienoyl)oxy)propyl) 9-undecyl nonanedioate

To a solution of intermediate 62d (7 g, 1.0 equiv.) in THF/toluene/water (4:1:2, 0.04 M) at 0°C was added NaBH ₄ (5 equiv.) and the mixture was stirred at 20 °C for 5 hours in N2 atmosphere. Upon completion, the mixture was quenched with sat. NH ₄ Cl at 5°C and extracted three times with EtOAc. The combined organic layers were washed twice with brine, dried over Na ₂ SO ₄ , filtered and concentrated to leave a residue. The crude residue was purified by column chromatography (EtOAc/hexagon) to give the product as a colorless oil (6 g, 85%).

1H NMR (400 MHz, CDQ3) δ 5.42-5.25 (m, 4H), 4.25-3.90 (m, 8H), 2.76 (t, J=6.4 Hz, 2H) , 2.55 (s, 1H), 2.30 (dt, J=24.5, 7.6 Hz, 6H), 2.03 (q, J=6.8 Hz, 4H), 1.61 ( q, J=7.0 Hz, 8H), 1.48-1.06 (m, 35H), 0.87 (td, J=6.8, 3.8 Hz, 6H).

Compound 62: 1 -(3-methyl-13 -((((9Z, 12Z)-octadeca-9,12-dienoyl)oxy)methyl)-9-octyl-7,11-dioxo8,12-dioxa-3, 6-diazatetradecane-14-yl) 9-undecyl nonanedioate

Compound 62 was prepared in 36% yield from intermediate 52d and intermediate 62e using the same method as 52.

1H NMR (400 MHz, CDCl ₃ ) δ 5.47-5.28 (m, 4H), 5.23 (t, J=5.2 Hz, 1H), 5.14-5.04 (m, 1H ), 4.28 (ddd, J=15.3, 11.9, 4.6 Hz, 2H), 4.14 (ddd, J=11.8, 7.5, 5.8 Hz, 2H), 4.05 (t, J=6.8 Hz, 2H), 3.38 (s, 2H), 2.77 (t, J=6.5 Hz, 2H), 2.58 (qd, J=15 ,2, 6.3 Hz, 5H), 2.37-2.22 (m, 7H), 2.05 (q, J=6.7 Hz, 4H), 1.62 (d, J=6, 8 Hz, 14H), 1.48

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0.97 (m, 50H), 0.97-0.79 (m, 9H). MS: 992.3 m/z [M+H].

Example 63 - compound 63.

Compound 63: 1-(3-methyl-14-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)-9-octyl-7,12-dioxo8,13-dioxa-3, 6-diazapentadecan-15-yl) 9-undecyl nonanedioate

Compound 63 was prepared in 79% yield from intermediate 54d and intermediate 62e using the same method as 52.

^1H NMR (400 MHz, CDCl3) δ 5.44-5.28 (m, 4H), 5.25 (ddd, J=5.8, 4.5, 1.4 Hz, 1H), 4.76 (s, 1H), 4.28 (dd, J=11.9, 4.4 Hz, 2H), 4.15 (ddd, J=11.9, 5.8, 4.2 Hz, 2H), 4.05 (t, J=6.8 Hz, 2H), 3.31 (s, 2H), 2.77 (t, J=6.7 Hz, 2H), 2.70-2.12 (m , 15H), 2.05 (q, J=6.9 Hz, 4H), 1.97-1.72 (m, 3H), 1.61 (h, J=6.5 Hz, 12H), 1 .40-1.19 (m, 47H), 1.11 (s, 3H), 0.96-0.81 (m, 9H). MS: 1006.9 m/z [M+H].

Example 64 - compound 64.

Compound 64: 1-(3-methyl-15-((((9Z, 12Z)-octadeca-9,12-dienoyl)oxy)methyl)-9-octyl-7,13-dioxo8,14-dioxa-3, 6-diazahexadecan-16-yl) 9-undecyl nonanedioate

Compound 64 was prepared in 79% yield from intermediate 55d and intermediate 62e using the same method as 52.

1H NMR (400 MHz, CDCl3) δ 5.48-5.28 (m, 4H), 5.24 (h, J=4.9, 4.4 Hz, 1H), 4.73 (s, 1H) , 4.29 (ddd, J=11.9, 4.4, 3.3 Hz, 2H), 4.14 (ddd, J=11.9, 5.9, 3.1 Hz, 2H), 4 .05 (t, J=6.8 Hz, 2H), 3.31 (s, 2H), 2.77 (t, J=6.5 Hz, 2H), 2.67-2.11 (m, 14H), 2.05 (q, J=6.8 Hz, 4H), 1.73-1.41 (m, 17H), 1.41-0.95 (m, 50H), 0.94-0 .82 (m, 9H). MS: 1021.0 m/z [M+H].

Example 65 - compound 65.

Compound 65: 2-((4-((((1-methylpyrrolidin-2-yl)methyl)carbamoyl)oxy)tetradecanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12^) -bis(octadeca-9,12-dienoate)

Compound 65 was prepared in 75% yield from intermediate 39d and 1-(1methylpyrrolidin-2-yl)methanamine using the same method as Example 1.

1H NMR (400 MHz, CDCl3) δ 5.45-5.28 (m, 8H), 5.25 (ddd, J=10.2, 5.7, 4.4 Hz, 1H), 4.76 ( s, 1H), 4.28 (dd, J=11.9, 4.4 Hz, 2H), 4.20-4.11 (m, 2H), 3.45 (s, 1H), 3.21 (s, 2H), 2.82-2.74 (m, 4H), 2.46-2.24 (m, 9H), 2.05 (q, J=6.8 Hz, 8H), 2, 01-1.42 (m, 16H), 1.41-1.20 (m, 45H), 0.88 (td, J=7.0, 4.7 Hz, 9H). MS: 984.9 m/z [M+H].

Example 66 - compound 66.

Compound 66: 2-((4-(((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)tetradecanoyl)oxy)propane1,3-diyl (9Z,9'Z, 12Z, 12^)-bis (octadeca-9,12-dienoate)

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Compound 66 was prepared in 65% yield from intermediate 39d and 3(pyrrolidin-1-yl)propan-1-ol using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.57-5.21 (m, 9H), 4.72 (dt, J=12.2, 5.2 Hz, 1H), 4.29 (dd, J =11.9, 4.4 Hz, 2H), 4.24-4.07 (m, 4H), 2.82-2.74 (m, 4H), 2.60 (d, J=102.7 Hz, 5H), 2.45-2.38 (m, 2H), 2.38-2.26 (m, 4H), 2.13-1.73 (m, 16H), 1.73-1, 43 (m, 9H), 1.41-1.16 (m, 42H), 1.04-0.74 (m, 9H). MS: 999.4 m/z [M+H].

Example 67 - compound 67.

Compound 67: 2-((4-((((1-methylpyrrolidin-2-yl)methyl)carbamoyl)oxy)octanoyl)oxy)propan1,3-diyl (9Z,9'Z, 12Z, 12^)-bis (octadeca-9,12-dienoate)

Compound 67 was prepared in 50% yield from intermediate 42d and 1-(1methylpyrrolidin-2-yl)methanamine using the same method as Example 1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.42-5.08 (m, 9H), 4.77 (s, 1H), 4.28 (ddd, J=11.8, 4.5, 1 .8 Hz, 2H), 4.204.12 (m, 2H), 3.48 (s, 1H), 3.33 (s, 2H), 2.77 (td, J=6.1, 1.2 Hz , 4Н), 2.63-2.23 (m, 1Ш), 2.05 (q, J=6.8 Hz, 9Н), 1.98-1.42 (m, 14H), 1.42- 1.22 (m, 32H), 0.96-0.81 (m, 9H). MS: 900.0 m/z [M+H].

Example 68 - compound 68.

Compound 68: 2-((4-(((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)octanoyl)oxy)propan-1,3diyl (9Z,9'Z, 12Z, 12'7)- bis(octadeca-9,12-dienoate)

Compound 68 was prepared in 43% yield from intermediate 42d and 3(pyrrolidin-1-yl)propan-1-ol using the same method as Example 1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.43-5.04 (m, 9H), 4.72 (tt, J=7.4, 5.1 Hz, 1H), 4.40-4, 10 (m, 6H), 2.81-2.74 (m, 4H), 2.54 (d, J=58.1 Hz, 5H), 2.40 (ddd, J=8.4, 6, 8, 4.4 Hz, 2H), 2.36-2.27 (m, 4H), 2.11-1.73 (m, 16H), 1.73-1.43 (m, 8H), 1 .43-1.21 (m, 31H), 0.99-0.77 (m, 9H). MS: 915.7 m/z [M+H].

Example 69 - compound 69.

Intermediate 69a: 4-oxononanoic acid

To a mixture of 5-pentyltetrahydrofuran-2-one (50 g, 1.0 equiv.) in 3:1 EtOH/H ₂ O (500 ml) was added NaOH (2.0 equiv.) in one portion at 15 °C under N2 atmosphere . The mixture was stirred at 15°C for 12 hours. Upon completion, the reaction mixture was concentrated under reduced pressure, and the resulting residue was extracted twice with DCM. The combined organic layers were dried over Na ₂ SO ₄ and concentrated to give a crude residue.

- 133 046244

The crude residue was then reconstituted in DCM (1 L) followed by the addition of DMP (3 eq) in one portion at 15°C in N ₂ . The mixture was stirred at 15°C for 4 hours. Once complete, the reaction was concentrated under reduced pressure and the solution was adjusted to pH 3 with 1 M HCl. The suspension was filtered and washed with H ₂ O. The resulting filter cake was dissolved in EtOAc, dried over Na ₂ SO ₄ , filtered and concentrated to give the product as a white solid (22 g, 45%).

1H NMR (400 MHz, CDCl ₃ ) δ 2.72 (dd, J=7.0, 5.4 Hz, 2H), 2.63 (ddd, J=7.2, 5.9, 1.2 Hz , 2H), 2.44 (t, J=7.4 Hz, 2H), 1.64-1.54 (m, 2H), 1.36-1.20 (m, 4H), 0.89 ( t, J=6.9 Hz, 3H).

Intermediate 69b: 2-((4-oxononanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)bis(octadeca-9,12-dienoate)

Intermediate 69b was synthesized in 74% yield from intermediate 69a and intermediate 1c using the method used for intermediate 1d.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.46-5.16 (m, 9H), 4.29 (dd, J=11.9, 4.3 Hz, 2H), 4.14 (dt, J=11.8, 6.0 Hz, 2H), 2.74 (dt, J=21.5, 6.5 Hz, 6H), 2.59 (t, J=6.6 Hz, 2H), 2.43 (t, J=7.5 Hz, 2H), 2.32 (t, J=7.6 Hz, 4H), 2.04 (dd, J=8.6, 5.3 Hz, 8H ), 1.60 (ddd, J=14.2, 8.4, 4.8 Hz, 6H), 1.50-1.06 (m, 32H), 0.89 (t, J=6.8 Hz, 9H).

Intermediate 69c: 2-((4-(((4-nitrophenoxy)carbonyl)oxy)nonanoyl)oxy)propane1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9, 12-dienoate)

To a solution of intermediate 69b (10 g, 1.0 eq) in THF (300 mL), toluene (150 mL) and H2O (75 mL) was added NaBH4 (5.0 eq) at 5°C. The mixture was stirred at 15°C for 3 hours under N2 atmosphere. Upon completion, Na ₂ SO ₄ was added to the reaction mixture at 0°C, and the resulting mixture was filtered and concentrated under reduced pressure. The resulting residue was purified by column chromatography to give the alcohol intermediate as a colorless oil.

The intermediate was immediately reduced in DCM (80 ml) followed by the addition of pyridine (1.67 eq) and (4-nitrophenyl)carbonochloridate (2 eq) at 0°C. The mixture was stirred at 0°C for 5 hours under N ₂ atmosphere. Upon completion, the reaction mixture was concentrated under reduced pressure to obtain a residue, which was purified by column chromatography to give the product as a colorless oil (4.05 g, 33%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.26-8.19 (m, 2H), 7.40-7.28 (m, 2H), 5.43-5.11 (m, 9H), 4.80 (tt, J=7.5, 4.4 Hz, 1H), 4.25 (dd, J=11.9, 4.3 Hz, 2H), 4.09 (dd, J=11, 9, 5.8 Hz, 2H), 2.70 (t, J=6.4 Hz, 4H), 2.40 (t, J=7.4 Hz, 2H), 2.33-2.16 ( m, 4H), 1.97 (q, J=6.8 Hz, 10H), 1.67 (ddt, J=13.4, 9.9, 4.7 Hz, 1H), 1.55 (dtd , J=15.7, 8.4, 7.4, 4.8 Hz, 5H), 1.43-1.16 (m, 36H), 0.83 (q, J=6.8 Hz, 9H ).

Compound 69: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)nonanoyl)oxy)propan-1,3-diyl (9Z,9'Z,12Z,12'Z )-bis(octadeca-9,12-dienoate)

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Compound 69 was prepared in 77% yield from intermediate 69c and K1-ethyl-K1methylethane-1,2-diamine using the same method as Example 1.

1H NMR (500 MHz, CDCl ₃ ) δ 5.35 (pt, J=8.8, 4.4 Hz, 8H), 5.25 (p, J=5.1 Hz, 1H), 4.76 ( dt, J=12.3, 6.7 Hz, 1H), 4.28 (dd, J=11.9, 4.5 Hz, 2H), 4.15 (dh, J=11.4, 5, 9 Hz, 2H), 3.31 (s, 2H), 2.77 (t, J=6.8 Hz, 4H), 2.62-2.45 (m, 4H), 2.38 (dt, J=9.0, 6.2 Hz, 2H), 2.30 (q, J=13.0, 10.4 Hz, 8H), 2.05 (q, J=7.0 Hz, 9H), 1.91 (tt, J=9.5, 4.5 Hz, 1H), 1.81 (dt, J=14.7, 7.8 Hz, 1H), 1.65-1.43 (m, 7H), 1.40-1.17 (m, 36H), 1.09 (q, J=8.8, 8.2 Hz, 3H), 0.88 (q, J=6.5 Hz, 9H ). MS: 902.6 m/z [M+H].

Example 70 - compound 70.

Compound 70: 2-((4-((((1-methylpyrrolidin-2-yl)methyl)carbamoyl)oxy)nonanoyl)oxy)propane1,3-diyl (9Z,9'Z, 12Z, 12^)-bis (octadeca-9,12-dienoate)

Compound 70 was prepared in 62% yield from intermediate 69c and 1-(1methylpyrrolidin-2-yl)methanamine using the same method as Example 1.

¹ H NMR (500 MHz, CDCl ₃ ) δ 5.45-5.29 (m, 8H), 5.25 (p, J=5.2 Hz, 1H), 4.76 (s, 1H), 4 .28 (dd, J=11.9, 4.5 Hz, 2H), 4.15 (dq, J=11.7, 5.8 Hz, 2H), 3.46 (d, J=25.9 Hz, 1H), 3.19 (s, 2H), 2.77 (t, J=6.8 Hz, 4H), 2.34 (dt, J=31.1, 8.3 Hz, 12H), 2.05 (q, J=7.0 Hz, 8H), 1.99-1.42 (m, 16H), 1.42-1.19 (m, 35H), 0.88 (q, J= 6.8 Hz, 9H). MS: 914.4 m/z [M+H].

Example 71 - compound 71.

Compound 71: 2-((4-(((3 -(pyrrolidin-1-yl)propoxy)carbonyl)oxy)nonanoyl)oxy)propan-1,3diyl (9Z,9'Z, 12Z, 12D-bis(octadeca -9,12-dienoate)

Compound 71 was prepared in 78% yield from intermediate 69c and 3(pyrrolidin-1-yl)propan-1-ol using the same method as Example 1.

1H NMR (500 MHz, chloroform-d) δ 5.50-5.28 (m, 8H), 5.25 (p, J=5.1 Hz, 1H), 4.72 (tt, J=7, 8, 4.5 Hz, 1H), 4.36-4.26 (m, 2H), 4.23-4.11 (m, 4H), 2.77 (t, J=6.7 Hz, 4H ), 2.41 (dt, J=15.0, 9.0 Hz, 2H), 2.31 (t, J=7.5 Hz, 5H), 2.05 (q, J=6.9 Hz , 8H), 2.00-1.75 (m, 9H), 1.72-1.48 (m, 8H), 1.43-1.20 (m, 35H), 1.00-0.75 (m, 9H). MS: 929.4 m/z [M+H].

Example 72 - compound 72.

Compound 72: 2-((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoyl)oxy)propan-1,3-diyl (9Z,9'Z,12Z,12'Z )-bis(octadeca-9,12-dienoate)

Compound 72 was prepared in 72% yield from intermediate 69c and 2-(pyrrolidin-1-yl)ethan-1-amine using the same method as Example 1.

1H NMR (500 MHz, CDCl3) δ 5.49 (s, 1H), 5.34 (dtt, J=18.0, 6.9, 3.9 Hz, 8H), 5.25 (p, J= 5.1 Hz, 1H), 4.76 (s, 1H), 4.28 (dd, J=11.9, 4.4 Hz, 2H), 4.15 (dt, J=11.6, 5 ,4 Hz, 2H), 3.40-3.27 (m, 2H), 2.80-2.50 (m, 11H), 2.45-2.35 (m, 2H), 2.31 ( t, J=7.6 Hz, 5H), 2.05 (q, J=7.0 Hz, 9H), 1.97-1.67 (m, 8H), 1.60 (q, J=7 .3 Hz, 8H), 1.33 (d, J=26.5 Hz, 36H), 0.88 (q, J=6.5 Hz, 9H). MS: 914.7 m/z [M+H].

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Example 73 - compound 73.

Intermediate 73a: 4-oxundecanoic acid

To a solution of 5-heptyltetrahydrofuran-2-one (20 g, 1.0 eq.) in 2:1 EtOH/H ₂ O (400 ml) was added NaOH (1.1 eq.). The mixture was degassed and purged 3 times with N2, and then the mixture was stirred at 20°C for 12 hours under an N2 atmosphere. Upon completion, the reaction mixture was concentrated under reduced pressure to remove the solvent, and 1N was added to the resulting residue. HCl to pH 3. The resulting suspension was filtered, and the filter cake was concentrated under reduced pressure to obtain a residue.

The resulting residue was reconstituted in DCM (200 ml) followed by the addition of DMP (3 eq.). The mixture was stirred at 20°C for 5 hours. When complete, the mixture was concentrated under reduced pressure and the resulting residue was diluted with H ₂ O and extracted with 3x EtOAc. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated. The crude material was purified by column chromatography to give the product as a white solid (10 g, 46%).

1H NMR (400 MHz, CDCl3) δ 2.65 (dd, J=7.0, 5.3 Hz, 2H), 2.56 (t, J=6.8 Hz, 2H), 2.37 (t , J=7.5 Hz, 2H), 1.51 (q, J=7.2 Hz, 2H), 1.27-1.13 (m, 8H), 0.86-0.76 (m, 3H).

Intermediate 73b: 2-((4-oxoundecanoyl)oxy)propane-1,3-diyl (9Z,9'Z,12Z,12'Z)bis(octadeca-9,12-dienoate)

Intermediate 73b was synthesized in 42% yield from intermediate 73a and intermediate 1c using the method used for intermediate 1d.

1H NMR (400 MHz, CDCl ₃ ) δ 5.28 (dddd, J=12.3, 10.7, 8.7, 5.5 Hz, 8H), 5.21-5.15 (m, 1H) , 4.22 (dd, J=11.9, 4.3 Hz, 2H), 4.13-4.05 (m, 2H), 2.70 (t, J=6.4 Hz, 4H), 2.64 (t, J=6.5 Hz, 2H), 2.52 (t, J=6.5 Hz, 2H), 2.36 (t, J=7.5 Hz, 2H), 2, 25 (t, J=7.6 Hz, 4H), 2.02-1.95 (m, 8H), 1.53 (h, J=7.0 Hz, 6H), 1.33-1.16 (m, 37H), 0.81 (td, J=6.8,4.7 Hz, 9H).

Intermediate 73 c: 2-((4-(((4-nitrophenoxy)carbonyl)oxy)undecanoyl)oxy)propane1,3-diyl(92,9'2,122,12'2)-bis(octadeca-9,12 -dienoate)

NaBH4 (5.0 eq) was added to a solution of intermediate 73b (25 g, 1.0 eq) in THF (500 mL), toluene (125 mL) and H2O (250 mL) at 0°C under N2. The mixture was stirred at 5°C for 1 hour under N ₂ atmosphere. When complete, the residue was poured into saturated NH ₄ Cl solution and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography to give the alcohol intermediate as a colorless residue.

The intermediate was immediately reconstituted in DCM (100 ml) followed by the addition of pyridine (2 eq) and (4-nitrophenyl)carbonochloridate (2 eq) at 0°C. The mixture was stirred at 20°C under N2 for 5 hours, after which the reaction mixture was concentrated under reduced pressure. The resulting residue was diluted with hexane and filtered. The filtrate was washed 3 times with water and 1 time

- 136 046244 brine, then dried over Na ₂ SO ₄ , filtered and concentrated. Purification by column chromatography gave the product as a colorless oil (5.2 g, 40%).

1H NMR (400 MHz, chloroform-d) δ 8.28 (d, J=8.8 Hz, 2H), 7.41 (dd, J=8.6, 6.1 Hz, 2H), 5.33 (tdd, J=23.5, 11.3, 6.1 Hz, 9H), 4.87 (tt, J=7.7, 4.6 Hz, 1H), 4.32 (dd, J=12 ,0, 4.4 Hz, 2H), 4.16 (dd, J=12.0, 5.8 Hz, 2H), 2.77 (t, J=6.4 Hz, 4H), 2.47 (t, J=7.5 Hz, 2H), 2.39-2.25 (m, 4H), 2.04 (q, J=6.9 Hz, 10H), 1.79-1.55 ( m, 7H), 1.48-1.15 (m, 36H), 0.89 (t, J=6.7 Hz, 9H).

Compound 73: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)undecanoyl)oxy)propan-1,3diyl (9Z,9'Z, 12Z,12^)-bis (octadeca-9,12-dienoate)

Compound 73 was prepared in 53% yield from intermediate 73c and (2aminoethyl)(ethyl)methylamine using the same method as Example 1.

1H NMR (500 MHz, CDCl3) δ 5.47-5.27 (m, 8H), 5.24 (q, J=5.1 Hz, 1H), 4.76 (s, 1H), 4.28 (dd, J=11.9, 4.4 Hz, 2H), 4.15 (dt, J=11.6, 5.4 Hz, 2H), 3.31 (s, 2H), 2.77 ( t, J=6.7 Hz, 4H), 2.61-2.35 (m, 6H), 2.31 (t, J=7.6 Hz, 7H), 2.05 (q, J=7 ,0 Hz, 8H), 1.90 (q, J=11.5, 9.0 Hz, 1H), 1.81 (dt, J=14.5, 7.6 Hz, 1H), 1.65 -1.41 (m, 9H), 1.391.19 (m, 38H), 1.10 (s, 3H), 0.88 (q, J=7.0 Hz, 9H). MS: 930.3 m/z [M+H].

Example 74 - compound 74.

Compound 74: 2-((4-((((1-methylpyrrolidin-2-yl)methyl)carbamoyl)oxy)undecanoyl)oxy)propane1,3-diyl (9Z,9'Z, 12Z,12^)-bis (octadeca-9,12-dienoate)

Compound 74 was prepared in 55% yield from intermediate 73c and 1-(1methylpyrrolidin-2-yl)methanamine using the same method as Example 1.

1H NMR (500 MHz, CDCl ₃ ) δ 5.34 (dtt, J=18.0, 6.9, 3.8 Hz, 8H), 5.25 (p, J=5.2 Hz, 1H), 4.81-4.72 (m, 1H), 4.28 (dd, J=11.9, 4.4 Hz, 2H), 4.15 (dt, J=11.7, 5.2 Hz, 2H), 3.43 (s, 1H), 3.17 (s, 2H), 2.77 (t, J=6.8 Hz, 4H), 2.34 (dt, J=31.2, 8 .3 Hz, 11H), 2.05 (q, J=7.0 Hz, 8H), 1.99-1.43 (m, 16H), 1.43-1.13 (m, 39H), 0 .88 (q, J=7.7, 7.2 Hz, 9H). MS: 942.8 m/z [M+H].

Example 75 - compound 75.

Compound 7 5: 2-((4-(((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)undecanoyl)oxy)propan-1,3diyl (9Z,9'Z, 12Z,12^)- bis(octadeca-9,12-dienoate)

Compound 75 was prepared in 73% yield from intermediate 73c and 3(pyrrolidin-1-yl)propan-1-ol using the same method as Example 1.

¹ H NMR (500 MHz, CDCl ₃ ) δ 5.46-5.28 (m, 8H), 5.25 (p, J=5.1 Hz, 1H), 4.72 (tt, J=7, 8, 4.5 Hz, 1H), 4.29 (dd, J=11.9, 4.4 Hz, 2H), 4.22-4.11 (m, 4H), 2.77 (t, J =6.8 Hz, 4H), 2.45-2.37 (m, 2H), 2.31 (t, J=7.5 Hz, 4H), 2.05 (q, J=7.0 Hz , 8H), 2.01-1.91 (m, 3H), 1.91-1.78 (m, 5H), 1.67-1.50 (m, 9H), 1.39-1.22 (m, 39H), 0.88 (q, J=6.6 Hz, 9H). MS: 957.4 m/z [M+H].

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Example 76 - compound 76.

Compound 76: 2-((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)undecanoyl)oxy)propan-1,3diyl (9Z,9'Z,12Z,12'Z)- bis(octadeca-9,12-dienoate)

Compound 76 was prepared in 81% yield from intermediate 73c and 2(pyrrolidin-1-yl)ethan-1-amine using the same method as Example 1.

1H NMR (500 MHz, CDCl ₃ ) δ 5.56 (s, 1H), 5.46-5.28 (m, 8H), 5.25 (p, J=5.2 Hz, 1H), 4, 76 (s, 1H), 4.28 (dd, J=11.9, 4.4 Hz, 2H), 4.15 (dt, J=11.7, 5.5 Hz, 2H), 3.43 -3.31 (m, 2H), 2.77 (t, J=6.7 Hz, 11H), 2.45-2.35 (m, 2H), 2.31 (t, J=7.6 Hz, 4H), 2.05 (q, J=7.0 Hz, 8H), 1.97-1.74 (m, 7H), 1.69-1.43 (m, 8H), 1.43 -1.09 (m, 39H), 0.88 (q, J=7.0 Hz, 9H). MS: 943.2 m/z [M+H].

Example 77 - compound 77.

Compound 77: 2-((4-(((2-(azetidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)propan-1,3-diyl (9Z,9'Z,12Z,12'Z )-bis(octadeca-9,12-dienoate)

Compound 77 was prepared in 36% yield from intermediate 40d and 2-(azetidin-1yl)ethan-1-amine using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.45-5.29 (m, 8H), 5.25 (h, J=4.6, 4.1 Hz, 1H), 4.75 (s, 1H ), 4.28 (ddd, J=11.9, 4.5, 1.9 Hz, 2H), 4.15 (ddt, J=11.9, 5.8, 3.6 Hz, 2H), 3.74-3.54 (m, 3H), 3.48-3.38 (m, 1H), 3.31 (d, J=6.3 Hz, 1H), 2.86 (s, 1H) , 2.81-2.73 (m, 4H), 2.41-2.26 (m, 7H), 2.05 (q, J=6.8 Hz, 8H), 1.97-1.87 (m, 2H), 1.87-1.77 (m, 2H), 1.60 (d, J=7.2 Hz, 8H), 1.44-1.15 (m, 37H), 0. 96-0.84 (m, 9H). MS: 914.7 m/z [M+H].

Example 78 - compound 78.

Compound 78: 2-((4-(((2-(piperidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)propan-1,3-diyl (9Z,9'Z,12Z,12'Z )-bis(octadeca-9,12-dienoate)

Compound 78 was prepared in 60% yield from intermediate 40d and 2-(piperidin1-yl)ethan-1-amine using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.47-5.28 (m, 8H), 5.25 (h, J=4.8 Hz, 1H), 4.76 (s, 1H), 4, 28 (ddd, J=11.9, 4.4, 1.3 Hz, 2H), 4.15 (dt, J=11.8, 5.7 Hz, 2H), 3.42 (s, 2H) , 2.80-2.72 (m, 4H), 2.44 (d, J=6.7 Hz, 2H), 2.39 (ddd, J=8.6, 6.9, 2.5 Hz , 2H), 2.36-2.27 (m, 5H), 2.05 (q, J=6.9 Hz, 8H), 1.95-1.41 (m, 18H), 1.41- 1.20 (m, 37H), 0.96-0.81 (m, 9H). MS: 942.4 m/z [M+H].

Example 79 - compound 79.

Compound 79: 2-((4-(((2-methyl-1-(pyrrolidin-1-yl)propan-2-yl)carbamoyl)oxy)decanoyl)oxy)propan-1,3-diyl (9Z,9 'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate)

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Compound 79 was prepared in 48% yield from intermediate 40d and 2-methyl-1(pyrrolidin-1-yl)propan-2-amine using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.42-5.21 (m, 9H), 4.72 (s, 1H), 4.28 (ddt, J=11.9, 4.4, 2, 2 Hz, 2H), 4.15 (ddd, J=11.9, 5.8, 4.6 Hz, 2H), 2.86-2.49 (m, 8H), 2.39 (td, J =8.2, 7.4, 2.2 Hz, 1H), 2.31 (td, J=7.6, 1.8 Hz, 4H), 2.05 (q, J=6.8 Hz, 8H), 2.00-1.45 (m, 17H), 1.45-1.03 (m, 41H), 0.98-0.77 (m, 9H). MS: 956.5 m/z [M+H].

Example 80 - compound 80.

Intermediate 80a: (5-methylhexyl)magnesium bromide.

To a solution of Mg (1.63 g, 1.2 equiv.) and I ₂ (0.1 equiv.) in THF (100 ml) was added 1-bromo-5-methylhexane (1.0 equiv.) at 15°C. The mixture was stirred at 40°C for 2 hours under N2 atmosphere. No further research. The reaction mixture was used directly in the next step. Bromo(5methylhexyl)magnesium in THF was obtained as a gray solution.

Intermediate 80b: Ethyl 9-methyl-4-oxodecanoate o

To a solution of ethyl 4-chloro-4-oxobutanoate (16 g, 1.0 eq) in THF (150 ml) was added CuI (1.0 eq) and intermediate 80a (2.0 eq) at -78° WITH. The mixture was stirred at -78°C for 2 hours under N2 atmosphere. Upon completion, the reaction mixture was quenched with water and extracted three times with EtOAc. The combined organic layers were washed 2 times with brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to leave a residue. The crude residue was purified by column chromatography to give the product (10 g, 45% yield) as a colorless oil.

1H NMR (400 MHz, CDCl ₃ ) δ 4.06 (q, J=7.1 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.50 (t, J= 6.8 Hz, 2H), 2.38 (t, J=7.5 Hz, 2H), 1.55-1.45 (m, 3H), 1.28-1.15 (m, 6H), 1.15-1.06 (m, 2H), 0.79 (d, J=6.6 Hz, 6H).

Intermediate 80c: 9-methyl-4-oxodecanoic acid O

HE

To a solution of intermediate 80b (10 g, 1.0 equiv) in EtOH (100 ml) was added KOH (3.0 equiv) at 20°C. The mixture was stirred at 50°C for 12 hours under N2 atmosphere. The reaction mixture was then concentrated to remove EtOH and the pH was adjusted to 5.5 with HCl (1 M). The resulting mixture was diluted with water and extracted twice with EtOAc. The combined organic layers were washed twice with brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude material was purified by column chromatography to give the product (4.4 g, 50%) as a white solid.

1H NMR (400 MHz, CDCl ₃ ) δ 2.71 (t, J=6.8 Hz, 2H), 2.63 (ddd, J=7.0, 5.8, 1.1 Hz, 2H), 2.44 (t, J=7.5 Hz, 2H), 1.53 (dq, J=19.8, 7.0, 6.6 Hz, 3H), 1.31-1.24 (m, 2H), 1.16 (dt, J=8.6, 6.7 Hz, 2H), 0.85 (d, J=6.6 Hz, 6H).

Intermediate 80d: 2-((9-methyl-4-oxodecanoyl)oxy)propane-1,3-diyl (9Z,9'Z, 12Z,12^)-bis(octadeca-9,12-dienoate)

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Intermediate 80d was synthesized in 40% yield from intermediate 80c and intermediate 1c using the method used for intermediate 1d.

1H NMR (400 MHz, CDCl ₃ ) δ 5.45-5.28 (m, 8H), 5.24 (p, J=5.1 Hz, 1H), 4.28 (dd, J=11.9 , 4.3 Hz, 2H), 4.14 (dd, J=11.9, 5.9 Hz, 2H), 2.76 (t, J=6.4 Hz, 4H), 2.71 (t , J=6.5 Hz, 2H), 2.59 (t, J=6.5 Hz, 2H), 2.43 (t, J=7.5 Hz, 2H), 2.31 (t, J =7.6 Hz, 4H), 2.04 (q, J=6.8 Hz, 8H), 1.67-1.50 (m, 8H), 1.39-1.21 (m, 29H) , 1.22-1.12 (m, 3H), 0.93-0.82 (m, 12H).

Intermediate 80e: 2-((9-methyl-4-(((4-nitrophenoxy)carbonyl)oxy)decanoyl)oxy)propan-1,3-diyl (9Z,9'Z,12Z,12'Z)- bis(octadeca-9,12-dienoate)

To a solution of intermediate 80d (1.5 g, 1.0 eq.) in THF (30 mL), toluene (15 mL) and H ₂ O (7.5 mL) was added NaBH4 (5.0 eq.) at 0 °C. The mixture was stirred at 5°C for 5 hours. After complete reduction, the reaction mixture was partitioned between THF and H ₂ O, and the resulting organic phase was separated, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the alcohol intermediate as a yellow oil.

The resulting intermediate was immediately reconstituted in DCM (10 ml) followed by the addition of pyridine (2.0 equiv.) and (4-nitrophenyl)carbonochloridate (2.0 equiv.) at 0°C. The mixture was stirred at 0°C for 5 hours. The reaction mixture was then filtered and concentrated under reduced pressure to obtain a crude residue, which was purified by column chromatography to obtain the product as a colorless oil.

^1H NMR (400 MHz, CDCl3) δ 8.25-8.18 (m, 2H), 7.38-7.28 (m, 2H), 5.39-5.15 (m, 9H), 4 .80 (tt, J=7.1, 4.3 Hz, 1H), 4.25 (dd, J=12.0, 4.3 Hz, 2H), 4.13-4.04 (m, 2H ), 2.70 (t, J=6.4 Hz, 4H), 2.40 (dd, J=8.3, 6.6 Hz, 2H), 2.33-2.19 (m, 4H) , 2.05-1.85 (m, 10H), 1.67 (ddt, J=14.6, 9.4, 4.8 Hz, 1H), 1.52 (dddq, J=32.6, 26.2, 13.2, 6.6, 6.1 Hz, 8H), 1.37-1.17 (m, 30H), 1.11 (dt, J=9.0, 6.5 Hz, 2H), 0.94-0.66 (m, 12H).

Compound 80: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)-9-methyldecanoyl)oxy)propane1,3-diyl (9Z,9'Z,12Z, 12^ )-bis(octadeca-9,12-dienoate)

Compound 80 was prepared in 68% yield from intermediate 80e and (2aminoethyl)(ethyl)methylamine using the same method as Example 1.

1H NMR (400 MHz, CDCl3) δ 5.43-5.22 (m, 9H), 4.76 (s, 1H), 4.28 (ddd, J=12.0, 4.5, 1.5 Hz, 2H), 4.15 (ddd, J=11.9, 5.8, 4.7 Hz, 2H), 3.37 (s, 3H), 2.77 (td, J=6.0, 1.3 Hz, 4H), 2.39 (ddd, J=8.4, 6.9, 2.4 Hz, 2H), 2.352.28 (m, 5H), 2.05 (q, J=6 ,8 Hz, 8H), 1.86 (ddt, J=37.3, 14.4, 7.1 Hz, 3H), 1.77-1.38 (m, 12H), 1.39-1, 19 (m, 34H), 1.15 (d, J=6.7 Hz, 3H), 0.94-0.79 (m, 12H). MS: 929.9 m/z [M+H].

Example 81 - compound 81.

Intermediate 81a: 1-bromo-6-methylheptane

To a solution of PPh3 (66.97 g, 1.33 eq.) in DCM (300 ml) was added Br ₂ (1.33 eq.) at 0°C. 6-methylheptan-1-ol (1.0 equiv) was then added in one portion. The mixture was stirred at 0°C for 2 hours under N ₂ atmosphere. Upon completion, the reaction mixture was diluted with H ₂ and the organic phase was separated. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. Pentane was added and the precipitated triphenylphosphine oxide was filtered off. The organic phase was concentrated under reduced pressure to obtain a residue, which was purified by column

- 140 046244 chromatography, obtaining the product as a yellow oil (30 g, 81%).

1H NMR (400 MHz, CDCI3) δ 3.41 (td, J=6.9, 1.1 Hz, 2H), 1.91-1.79 (m, 2H), 1.59-1.49 ( m, 1H), 1.45-1.35 (m, 2H), 1.34-1.25 (m, 3H), 1.21-1.12 (m, 2H), 0.86 (dd, J=6.6, 1.1 Hz, 6H).

Intermediate 81b: (6-methylheptyl)magnesium bromide.

BrMg

To a solution of Mg (4.53 g, 1.2 eq.) and I ₂ (0.1 eq.) in THF (300 ml) was added intermediate 81a (1.0 eq.) at 15°C. The mixture was stirred at 40°C for 2 hours under N2 atmosphere. No further research. The reaction mixture was used directly in the next step. Bromine (6methylheptyl)magnesium in THF was obtained as a gray solution.

Intermediate 81c: Ethyl 10-methyl-4-oxundecanoate

_EtO2C

To a solution of ethyl 4-chloro-4-oxobutanoate (25 g, 1.0 equiv.) and CuI (1.0 equiv.) in THF (200 ml) was added intermediate 81b (1.0 equiv.) at -78° WITH. The mixture was stirred at -78°C for 5 hours under N2 atmosphere. The reaction mixture was then diluted with water and extracted with EtOAc three times. The combined organic layers were separated, washed 2 times with brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to leave a residue. The residue was purified by column chromatography to give the product as a yellow oil (10 g, 27%).

^1H NMR (400 MHz, CDCI3) δ 4.06 (q, J=7.1 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.50 (t, J= 6.7 Hz, 2H), 2.37 (t, J=7.5 Hz, 2H), 1.51 (q, J=7.5 Hz, 2H), 1.43 (dq, J=13, 2, 6.6 Hz, 1H), 1.26-1.15 (m, 7H), 1.14-1.04 (m, 2H), 0.79 (d, J=6.6 Hz, 6H ).

Intermediate 81d: 10-methyl-4-oxundecanoic acid but _2c

A mixture of Intermediate 81c (10 g, 1.0 equiv) and KOH (3.0 equiv) in EtOH (50 ml) was degassed and purged 3 times with N2, and then the mixture was stirred at 50°C for 12 h under atmosphere N2. The mixture was concentrated to remove EtOH, then adjusted to pH 5.5 with HCl (1 M). The reaction mixture was then diluted with water and extracted 3 times with EtOAc. The combined organic layers were separated, washed 2 times with brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to leave a residue. The residue was purified by column chromatography to give the product as a white solid (4 g, 45%).

1H NMR (400 MHz, CDCI3) δ 2.65 (t, J=6.1 Hz, 2H), 2.56 (ddd, J=7.1, 5.9, 1.2 Hz, 2H), 2 .37 (t, J=7.5 Hz, 2H), 1.51 (q, J=7.3 Hz, 2H), 1.42 (dt, J=13.2, 6.6 Hz, 1H) , 1.20 (tt, J=4.9, 2.5 Hz, 4H), 1.14-1.05 (m, 2H), 0.79 (d, J=6.6 Hz, 6H).

Intermediate 81e: 2-((10-methyl-4-oxoundecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,2'Z)-bis(octadeca-9,12-dienoate)

Intermediate 81e was synthesized in 40% yield from intermediate 81d and intermediate 1c using the method used for intermediate 1d.

1H NMR (400 MHz, CDCI3) δ 5.39-5.20 (m, 8H), 5.17 (ddd, J=10.1, 5.8, 4.3 Hz, 1H), 4.21 ( dd, J=11.9, 4.3 Hz, 2H), 4.07 (dd, J=11.9, 5.9 Hz, 2H), 2.69 (t, J=6.4 Hz, 4H ), 2.64 (t, J=6.5 Hz, 2H), 2.52 (t, J=6.5 Hz, 2H), 2.36 (t, J=7.5 Hz, 2H), 2.24 (t, J=7.5 Hz, 4H), 1.97 (q, J=6.8 Hz, 8H), 1.66-1.46 (m, 7H), 1.33-1 .13 (m, 35H), 1.08 (q, J=6.9 Hz, 2H), 0.84-0.77 (m, 12H).

Intermediate 81f: 2-((10-methyl-4-(((4-nitrophenoxy)carbonyl)oxy)undecanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12'Z) - bis(octadeca-9,12-dienoate)

- 141 046244

To a solution of intermediate 81e (6 g, 1.0 eq.) in THF (120 mL), toluene (30 mL) and H ₂ O (60 mL) was added NaBH ₄ (5.0 eq.) at 0°C. The mixture was stirred at 5°C for 5 hours. After complete reduction, the reaction mixture was partitioned between THF and H ₂ O, and the resulting organic phase was separated, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the alcohol intermediate as a yellow oil.

The resulting intermediate was immediately reconstituted in DCM (10 ml) followed by the addition of pyridine (2.0 equiv.) and (4-nitrophenyl)carbonochloridate (2.0 equiv.) at 0°C. The mixture was stirred at 0°C for 1 hour. The reaction mixture was then filtered and concentrated under reduced pressure to give a crude residue, which was purified by column chromatography to give the product as a yellow oil (4.3 g, 59%).

1H NMR (400 MHz, CDCl ₃ ) δ 8.27-8.16 (m, 2H), 7.37-7.29 (m, 2H), 5.43-5.12 (m, 9H), 4 .80 (s, 1H), 4.25 (dd, J=11.9, 4.4 Hz, 2H), 4.14-4.06 (m, 2H), 2.70 (t, J=6 ,4 Hz, 4H), 2.40 (t, J=7.5 Hz, 2H), 2.24 (td, J=7.5, 4.1 Hz, 4H), 2.05-1.88 (m, 9H), 1.71-1.62 (m, 1H), 1.62-1.47 (m, 6H), 1.45-0.97 (m, 35H), 0.89-0 .66 (m, 12H).

Compound 81: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)-10-methylundecanoyl)oxy)propan-1,3-diyl (9Z,9'Z,12Z, 12'Z)-bis(octadeca-9,12-dienoate)

Compound 81 was prepared in 85% yield from intermediate 81f and (2aminoethyl)(ethyl)methylamine using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.42-5.21 (m, 9H), 4.76 (s, 1H), 4.33-4.23 (m, 2H), 4.15 (dt , J=11.8, 5.7 Hz, 2H), 3.38 (d, J=40.4 Hz, 3H), 2.77 (t, J=6.5 Hz, 4H), 2.38 (t, J=7.7 Hz, 2H), 2.32 (td, J=7.6, 1.1 Hz, 5H), 2.05 (q, J=6.7 Hz, 8H), 1 .96-1.78 (m, 3H), 1.68-1.43 (m, 12H), 1.43-1.20 (m, 36H), 1.14 (d, J=6.6 Hz , 3H), 0.93-0.80 (m, 12H). MS: 944.5 m/z [M+H].

Example 82 - compound 82.

Compound 82: 2-((9-methyl-4-(((2-(pyrrolidin-1 -yl)ethyl)carbamoyl)oxy)decanoyl)oxy)propane1,3-diyl (9Z,9'Z, 12Z, 12 ^)-bis(octadeca-9,12-dienoate)

Compound 82 was prepared in 86% yield from intermediate 80e and 2-(pyrrolidin-1yl)ethan-1-amine using the same method as Example 1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.87 (s, 1H), 5.43-5.23 (m, 9H), 4.76 (s, 1H), 4.28 (ddd, J= 12.0, 4.5, 2.6 Hz, 2H), 4.15 (ddd, J=11.9, 5.8, 4.6 Hz, 2H), 3.52-3.42 (m, 2H), 2.94 (s, 5H), 2.82-2.75 (m, 4H), 2.39 (ddd, J=8.4, 6.9, 2.0 Hz, 2H), 2 ,36-2.29 (m, 4H), 2.05 (q, J=6.8 Hz, 8H), 2.01-1.76 (m, 7H), 1.67-1.45 (m , 8H), 1.40-1.21 (m, 33H), 1.15 (s, 2H), 0.93-0.81 (m, 12H). MS: 942.3 m/z [M+H].

Example 83 - compound 83.

- 142 046244

Compound 83: 2-(( 10-methyl-4-(((2-(pyrrolidin-1 -yl)ethyl)carbamoyl)oxy)undecanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z , 12^)-bis(octadeca-9,12-dienoate)

Compound 83 was prepared in 87% yield from intermediate 81f and 2-(pyrrolidin1-yl)ethan-1-amine using the same method as Example 1.

1H NMR (400 MHz, CDCh) δ 5.99 (s, 1H), 5.45-5.19 (m, 9H), 4.76 (s, 1H), 4.28 (ddd, J=11, 9, 4.5, 3.2 Hz, 2H), 4.15 (ddd, J=11.9, 5.8, 4.7 Hz, 2H), 3.50 (d, J=6.3 Hz , 2H), 2.99 (s, 5H), 2.81-2.73 (m, 4H), 2.39 (ddd, J=8.2, 6.9, 1.8 Hz, 2H), 2.35-2.28 (m, 4H), 2.05 (q, J=6.8 Hz, 8H), 2.02-1.77 (m, 7H), 1.66-1.43 ( m, 8H), 1.411.20 (m, 35H), 1.14 (d, J=6.7 Hz, 2H), 0.95-0.81 (m, 12H). MS: 956.5 m/z [M+H].

Example 84 - compound 84.

Compound 84: 2-((4-(((1-methylazetidin-3-yl)carbamoyl)oxy)decanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12'7)-bis (octadeca-9,12-dienoate)

Compound 84 was prepared in 87% yield from intermediate 40d and 1methylazetidin-3-amine using the same method as Example 1.

1H NMR (500 MHz, CDCl ₃ ) δ 5.44-5.28 (m, 8H), 5.23 (p, J=5.2 Hz, 1H), 4.77 (d, J=7.0 Hz, 1H), 4.46 (s, 1H), 4.28 (dt, J=10.9, 5.2 Hz, 2H), 4.16 (ddd, J=8.7, 5.6, 2.8 Hz, 2H), 3.99 (s, 2H), 3.81 (s, 2H), 2.77 (t, J=6.7 Hz, 4H), 2.65 (s, 2H) , 2.40-2.29 (m, 6H), 2.05 (q, J=7.0 Hz, 8H), 1.93 (dtd, J=15.1, 7.6, 3.8 Hz , 1H), 1.83 (q, J=7.4 Hz, 1H), 1.66-1.44 (m, 7H), 1.40-1.17 (m, 37H), 0.88 ( q, J=7.0 Hz, 9H). MS: 900.4 m/z [M+H].

Example 85 - compound 85

Intermediate 85a: Benzyl 4-hydroxyoctanoate

To a solution of 5-butyltetrahydrofuran-2-one (20 g, 1.0 eq.) in H ₂ O (50 ml) was added NaOH (5.63 g, 1.0 eq.). The mixture was stirred at 25°C for 12 hours. Upon completion, the reaction mixture was concentrated under reduced pressure to remove H ₂ O.

The resulting residue was reconstituted in DMF (100 mL) followed by the addition of BnBr (14.08 g, 1.0 eq.). The mixture was stirred at 25°C for 2 hours. The reaction mixture was then concentrated under reduced pressure to remove DMF. The residue was diluted with H ₂ O (150 ml) and extracted with 3x EtOAc, dried over Na ₂ SO ₄ , filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the product as a yellow oil (15 g, 43%).

Intermediate 85b: Benzyl 4-(((4-nitrophenoxy)carbonyl)oxy)octanoate

To a mixture of intermediate 85a (15 g, 1.0 equiv) in DCM (150 ml), (4nitrophenyl)carbonochloridate (32.21 g, 5.0 equiv) was added under N2, and then pyridine ( 5.16 ml, 2.0 equiv.). The reaction mixture was stirred at 20°C for 1 hour under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with hexane and filtered. The filtrate was washed 3 times with H ₂ O. The organic layer was washed 1 time with salt

- 143 046244 solution, dried over Na ₂ SO ₄ and filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a colorless oil (12 g, 48%).

1H NMR (400 MHz, CDCl ₃ ) δ 8.31-8.17 (m, 2H), 7.42-7.30 (m, 6H), 5.13 (s, 2H), 4.87 (qd , J=7.6, 4.8 Hz, 1H), 2.50 (t, J=7.4 Hz, 2H), 2.11 (dtd, J=15.3, 7.8, 4.0 Hz, 1H), 2.00 (dt, J=14.7, 7.6 Hz, 1H), 1.82-1.69 (m, 1H), 1.63 (ddt, J=14.3, 8.5, 5.0 Hz, 1H), 1.45-1.27 (m, 4H), 1.01-0.83 (m, 3H).

Intermediate 85c: Benzyl 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoate

To a solution of intermediate 85b (4 g, 2.0 equiv) and 2-pyrrolidin-1-ylethanamine (550 mg, 1.0 equiv) in MeCN (20 ml) was added DMAP (59 mg, 0.1 equiv). ), then pyridine (1.17 ml, 3.0 eq.) at 0°C. The mixture was stirred at 20°C for 12 hours under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove MeCN (20 ml). The residue was diluted with H ₂ O and extracted with 3x EtOAc. The combined organic layers were concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a colorless oil (1.8 g, 48%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.32-7.23 (m, 5H), 5.03 (d, J=10.2 Hz, 3H), 4.69 (s, 1H), 3 .23 (dq, J=18.7, 5.9 Hz, 3H), 2.70 (s, 2H), 2.60-2.40 (m, 7H), 2.34 (td, J=8 ,3, 7.6, 2.7 Hz, 2H), 1.87 (d, J=10.5 Hz, 1H), 1.70 (t, J=3.8 Hz, 6H), 1.44 (d, J=24.4 Hz, 2H), 1.22 (q, J=11.9 Hz, 4H), 0.80 (t, J=4.2 Hz, 3H).

Compound 85: 2-((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12^) -bis(octadeca-9,12-dienoate)

To a solution of intermediate 85c (1.8 g, 1.0 equiv) in THF (9 mL) was added Pd/C (0.5 g, 10% w/w) under Ar. The mixture was purged with 3x H ₂ and then the mixture was stirred at 20°C for 5 hours under an atmosphere of H ₂ (15 psi). The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to remove THF.

The resulting crude material was dissolved in DCM (10 ml) followed by the addition of DMAP (65 mg, 0.16 equiv.), DIPEA (1.74 ml, 3.0 equiv.) and EDCI (638 mg, 1.0 equiv.) . Intermediate 1c (2.05 g, 1.0 equiv) was then added at 0°C. The mixture was stirred at 20°C for 16 hours under N ₂ atmosphere. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was diluted with H ₂ O and extracted with 2x EtOAc. The combined organic layers were concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a colorless oil (1.1 g, 50%).

1H NMR (400 MHz, CDCl ₃ ) δ 5.39-5.21 (m, 7H), 5.18 (p, J=5.0 Hz, 1H), 5.09 (s, 1H), 4, 76-4.65 (m, 1H), 4.22 (dd, J=12.0, 4.4 Hz, 2H), 4.08 (ddd, J=11.9, 5.9, 3.4 Hz, 2H), 3.21 (q, J=5.9 Hz, 2H), 2.70 (t, J=6.4 Hz, 4H), 2.51 (t, J=6.1 Hz, 2H), 2.43 (d, J=5.8 Hz, 4H), 2.32 (ddd, J=8.9, 6.6, 3.9 Hz, 2H), 2.25 (t, J =7.6 Hz, 4H), 1.98 (q, J=6.9 Hz, 7H), 1.91-1.66 (m, 6H), 1.63-1.35 (m, 8H) , 1.25 (tq, J=7.2, 4.6, 3.6 Hz, 30H), 0.82 (t, J=6.7 Hz, 9H). MS: 900.4 m/z [M+H].

Example 86 - compound 86.

Intermediate 86a: Ethyl 1-nonanoylcyclopropane-1-carboxylate

To a solution of diethylcyclopropane-1,1-dicarboxylate (15 g, 1.0 eq.) in THF (200 mL), a solution of bromo(octyl)magnesium (2 M, 40.28 mL, 1.0 eq.) was added dropwise at -78°C under N2. The reaction mixture was stirred at -78°C for 5 hours under N2 atmosphere. The reaction mixture was poured into aq. _NH4Cl . The aqueous phase was extracted 3 times with ethyl acetate. The combined organic phase was washed with brine, dried with anhydrous Na ₂ SO ₄ , filtered and the filtrate was concentrated in vacuo. The residue was purified by another portion of column chromatography to give the product as a colorless oil (20 g, 48%).

- 144 046244

1H NMR (400 MHz, CDCl ₃ ) δ 4.13 (q, J=7.1 Hz, 2H), 2.75 (t, J=7.4 Hz, 2H), 1.57-1.49 ( m, 2H), 1.35 (s, 4H), 1.21 (dd, J=8.7, 5.5 Hz, 16H), 0.81 (t, J=6.9 Hz, 6H).

Intermediate 86b: 1-nonanoylcyclopropane-1-carboxylic acid

To a solution of intermediate 86a (10 g, 1.0 equiv.) in EtOH (60 ml) and H ₂ O (30 ml) was added NaOH (2.36 g, 1.5 equiv.) and LiOH (1.41 g , 1.5 equiv.) under N2. The mixture was then stirred at 20°C for 12 hours under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was brought to pH 3 by adding 2 N. HCl. The mixture was then stirred at 20°C for 1 hour under N2 atmosphere. The mixture was extracted 3 times with EtOAc. The combined organic phase was washed with brine, dried with anhydrous Na ₂ SO ₄ , filtered and the filtrate was concentrated in vacuo. The collected product is a colorless oil (5 g, 28%).

1H NMR (400 MHz, CDCl ₃ ) δ 2.25 (t, J=7.3 Hz, 2H), 1.96 (q, J=4.4 Hz, 2H), 1.71 (q, J= 4.4 Hz, 2H), 1.641.55 (m, 2H), 1.26 (d, J=3.7 Hz, 11H), 0.87 (t, J=6.8 Hz, 3H).

Intermediate 86c: 2-((1-nonanoylcyclopropane-1-carbonyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12^)-bis(octadeca-9,12-dienoate)

To a solution of intermediate 86b (6 g, 1.0 equiv), intermediate 1C (16.36 g, 1.0 equiv), EDCI (6.10 g, 1.5 equiv) and DMAP (324 mg , 0.1 equiv.) in DCM (60 ml) was added Et ₃ N (5.54 ml, 1.5 equiv.) at 0°C. The mixture was stirred at 20°C for 12 hours under N2 atmosphere. It was then poured into water and extracted with EtOAc three times. The combined organic phase was washed with brine, dried with anhydrous Na ₂ SO ₄ , filtered and the filtrate was concentrated in vacuo to leave a residue. The residue was purified by column chromatography to give the product as a colorless oil (8 g, 37%).

1H NMR (400 MHz, CDCl ₃ ) δ 5.28 (dtt, J=15.4, 10.8, 5.8 Hz, 8H), 4.26 (dd, J=12.1, 4.1 Hz , 2H), 4.08 (dd, J=12.0, 6.1 Hz, 2H), 2.76 (t, J=7.4 Hz, 2H), 2.70 (t, J=6, 4 Hz, 4H), 2.24 (t, J=7.6 Hz, 4H), 1.98 (q, J=6.9 Hz, 8H), 1.54 (p, J=7.0 Hz , 9H), 1.43-1.33 (m, 4H), 1.33-1.15 (m, 40H), 0.85-0.79 (m, 9H).

Intermediate 86d: 2-((1-(1-hydroxynonyl)cyclopropane-1-carbonyl)oxy)propane1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12- dienoate)

A solution of intermediate 86c (8 g, 1.0 eq.) in 4:2:1 THF/water/toluene (0.05-0.1 M) was cooled to 0-5°C followed by the addition of NaBH ₄ (5. 0 eq.). The reaction was maintained at 0-25°C for at least 2 hours. The reaction mixture was then diluted with water and EtOAc, and the resulting two-phase mixture was extracted with 3x EtOAc. The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude material was purified by column chromatography (EtOAc/hexanes) to give the product as a colorless oil (4 g, 50%).

1H NMR (400 MHz, CDCl ₃ ) δ 5.33 (tdq, J=20.3, 10.0, 5.6, 4.9 Hz, 9H), 4.28 (dt, J=12.0, 4.0 Hz, 2H), 4.15 (ddd, J=12.1, 8.0, 6.0 Hz, 2H), 3.18-3.12 (m, 1H), 2.77 (t , J=6.4 Hz, 4H), 2.68 (s, 1H), 2.31 (t, J=7.6 Hz, 4H), 2.05 (q, J=6.9 Hz, 8H ), 1.68-1.48 (m, 9H), 1.40-1.16 (m, 43H), 0.98 (ddd, J=10.7, 6.7, 4.1 Hz, 1H ), 0.93-0.84 (m, 9H), 0.81 (ddd, J=9.7, 6.8, 4.2 Hz, 1H).

Intermediate 86e: 2-((1-(1-(((4-nitrophenoxy)carbonyl)oxy)nonyl)cyclopropane-1carbonyl)oxy)propane-1,3-diyl (9Z,9'Z,12Z,12' Z)-bis(octadeca-9,12-dienoate)

- 145 046244

To a solution of intermediate 86d (4 g, 1.0 equiv) in DCM (40 ml) was added (4nitrophenyl)carbonochloridate (1.95 g, 2.0 equiv), pyridine (781 µl, 2.0 equiv) at 0°C. The reaction mixture was then stirred at 20°C for 5 hours under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was reduced with petroleum ether and filtered. The filtrate was washed with H2O. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to leave a residue. The residue was purified by column chromatography to give the product (2.8 g, 59%) as a colorless oil.

1H NMR (400 MHz, CDCl ₃ ) δ 8.25-8.16 (m, 2H), 7.36-7.26 (m, 2H), 5.27 (tdd, J=17.5, 8, 5, 4.0 Hz, 9H), 4.84 (dd, J=9.1, 4.4 Hz, 1H), 4.24 (dt, J=11.9, 3.8 Hz, 2H), 4.08 (dd, J=11.8, 5.8 Hz, 2H), 2.70 (t, J=6.3 Hz, 4H), 2.23 (t, J=7.6 Hz, 4H ), 2.02-1.93 (m, 8H), 1.82 (qd, J=9.4, 4.9 Hz, 1H), 1.69 (ddt, J=14.4, 10.0 , 5.1 Hz, 1H), 1.53 (t, J=7.2 Hz, 5H), 1.35-1.16 (m, 41H), 1.07-1.01 (m, 1H) , 0.95-0.89 (m, 1H), 0.81 (td, J=6.8, 3.8 Hz, 9H).

Compound 86: 2-((1-(1-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)nonyl)cyclopropane-1carbonyl)oxy)propan-1,3-diyl (9Z,9' Z, 12Z, 12^)-bis(octadeca-9,12-dienoate)

Compound 86 was synthesized in 90% yield from intermediate 86e and NI-ethyl-N-methylethane-1,2-diamine using the method used in Example 1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.53-5.22 (m, 9H), 4.75 (dd, J=8.7, 4.5 Hz, 1H), 4.29 (ddd, J=22.5, 11.8, 4.3 Hz, 2H), 4.14 (dd, J=11.9, 5.9 Hz, 2H), 3.30 (s, 2H), 2.77 (t, J=6.5 Hz, 4H), 2.54 (d, J=13.7 Hz, 4H), 2.38-2.21 (m, 7H), 2.05 (q, J= 6.7 Hz, 8H), 1.60 (t, J=7.4 Hz, 4H), 1.40-1.17 (m, 40H), 1.12-1.00 (m, 4H), 0.91-0.85 (m, 9H). MS: 956.1 m/z [M+H].

Example 87 - compound 87.

Intermediate 87a: 5-hydroxy-1-methyl-5-octylpyrrolidin-2-one

To a solution of 1-methylpyrrolidine-2,5-dione (20 g, 1.0 eq.) in THF (150 mL) was added bromo(octyl)magnesium (1 M, 265 mL, 1.5 eq.) dropwise at 15 °C under N2. The mixture was stirred at 15°C for 12 hours under N2 atmosphere. The reaction mixture was quenched by adding NH4Cl at 0°C. It was then diluted with H ₂ O and extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a white solid (20 g, 50%).

1H NMR (400 MHz, CDCl ₃ ) δ 3.75 (s, 1H), 2.68 (s, 3H), 2.43 (ddd, J=16.4, 10.0, 6.4 Hz, 1H ), 2.24 (ddd, J=16.7, 10.0, 4.5 Hz, 1H), 2.13 (ddd, J=13.6, 10.0, 6.4 Hz, 1H), 1.93 (ddd, J=14.0, 10.0, 4.7 Hz, 1H), 1.73 (td, J=13.4, 3.7 Hz, 1H), 1.65-1, 53 (m, 1H), 1.35-1.07 (m, 12H), 0.88-0.76 (m, 3H).

Intermediate 87b: 4-hydroxy-M-methyldodecanamide

To a solution of intermediate 87a (15 g, 1.0 eq) in MeOH (150 mL) was added portionwise NaBH4 (4.99 g, 2.0 eq). The mixture was stirred at 15°C for 1 hour. The reaction mixture was quenched by adding NH ₄ Cl at 0°C, then it was diluted with H2O and extracted with 3x EtOAc. The combined organic layers were concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a white solid (8 g, 53%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.93 (s, 1H), 3.60-3.50 (m, 1H), 3.11 (s, 1H), 2.73 (d, J= 4.8 Hz, 3H), 2.36

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2.20 (m, 2H), 1.78 (dddd, J=14.3, 7.7, 6.5, 3.1 Hz, 1H), 1.58 (ddt, J=13.8, 8 ,9, 6.7 Hz, 1H), 1.46-1.09 (m, 14H), 0.87-0.77 (m, 3H).

Intermediates 87c: ^)-4-hydroxy^-methyldodecanamide

To a solution of intermediate 87b (1.0 eq) in Et ₂ O (0.2-1 M) was added Novozym 435 (0.89 eq) and vinyl acetate (2.0 eq). The mixture was stirred at 40°C for 96 hours. The reaction mixture was concentrated under reduced pressure to obtain a residue, which was purified by column chromatography. The material collected from the column was subjected to the above reaction conditions three times to obtain (R)-88c as a yellow solid (24% yield).

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.57 (s, 1H), 3.54 (tt, J=7.7, 3.6 Hz, 1H), 2.74 (d, J=4, 8 Hz, 3H), 2.64 (d, J=4.6 Hz, 1H), 2.28 (td, J=6.9, 3.2 Hz, 2H), 1.79 (dtd, J= 14.3, 7.0, 3.1 Hz, 1H), 1.60 (ddd, J=14.3, 8.8, 6.8 Hz, 2H), 1.47-1.12 (m, 14H), 0.87-0.74 (m, 3H).

Intermediate 87d: ^)-5-octyldihydrofuran-2(3H)-one

NaOH (10 equiv) was added to a solution of intermediate 87d (1.0 eq) in MeOH (0.1-1.0 M). The mixture was stirred at 80°C for at least 2 hours, after which HCl (1-2 M, 10.0 equiv.) was added and the mixture was stirred at 25°C for 1 hour. The reaction mixture was then diluted with H ₂ O and extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a yellow oil.

1H NMR (400 MHz, CDCl ₃ ) δ 4.41 (p, J=6.9 Hz, 1H), 2.46 (dd, J=9.5, 6.8 Hz, 2H), 2.25 ( dq, J=13.4, 6.7 Hz, 1H), 1.78 (dq, J=12.7, 9.3 Hz, 1H), 1.72-1.62 (m, 1H), 1 .60-1.47 (m, 1H), 1.45-1.10 (m, 14H), 0.81 (t, J=6.6 Hz, 3H).

Intermediate 87e: ^)-4-hydroxydodecanoic acid

NaOH (1.0-1.5 eq) was added to a solution of intermediate 87d (1.0 eq) in H ₂ O (0.1-0.5 M). The mixture was stirred at 20°C for at least 12 hours. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was used directly in the next step without further purification.

Intermediate 88f: benzyl ^)-4-hydroxydodecanoate

BnBr (1.0-1.5 eq) and TBAB (0.05 eq) were added to a solution of intermediate 87e (1.0 eq) in acetone (0.3 M). The mixture was stirred at 60°C for at least 12 hours. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was either (1) purified by column chromatography or (2) diluted with sat. NaHCO ₃ , extracted three times with EtOAc, dried over Na ₂ SO ₄ , filtered and concentrated before purification by column chromatography. Both methods gave the product as a yellow oil (54% yield).

Intermediate 87g: benzyl^)-4-(((4-nitrophenoxy)carbonyl)oxy)dodecanoate

To a solution of intermediate 87f (1.0 equiv.) and (4-nitrophenyl)carbonochloridate (3.0 equiv.) in DCM (0.3-1.2 M) was added pyridine (3.0 equiv.) at 0- 20°C. The mixture was stirred at 20°C for at least 1 hour. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was either (1) purified by column chromatography or (2) used in the next step without additional

- 147 046244 thread cleaning. The product was collected as a pale yellow oil (36%).

1H NMR (400 MHz, CDCl ₃ ) δ 8.22-8.13 (m, 2H), 7.29 (d, J=2.0 Hz, 2H), 5.06 (s, 2H), 4, 80 (tt, J=7.6, 4.5 Hz, 1H), 2.43 (t, J=7.5 Hz, 2H), 2.04 (ddt, J=15.3, 7.8, 3.9 Hz, 1H), 1.92 (dq, J=14.8, 7.4 Hz, 1H), 1.71-1.61 (m, 1H), 1.55 (ddd, J=14 ,3, 9.1, 5.2 Hz, 1H), 1.36-1.13 (m, 11H), 0.81 (t, J=6.6 Hz, 3H).

Intermediate 87h: benzyl oxy)dodecanoate^)-4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)-

Pyridine (3 .0 equivalent) and DMAP (1.0 equivalent). The mixture was stirred at 20°C for 12 hours. The reaction mixture was then concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a yellow oil (88%).

1H NMR (400 MHz, CDCl ₃ ) δ 7.60-7.23 (m, 5H), 5.04 (s, 2H), 4.77-4.64 (m, 1H), 3.15 (t , J=6.0 Hz, 2H), 2.36 (ddt, J=12.3, 8.2, 4.3 Hz, 5H), 2.13 (s, 3H), 1.96-1, 70 (m, 3H), 1.55-1.33 (m, 2H), 1.29-1.10 (m, 13H), 0.96 (t, J=7.1 Hz, 3H), 0 .80 (t, J=6.7 Hz, 4H).

Compound 87: 2-((^)-4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propane1,3-diyl (9Z,9'Z, 12Z, 12^ )-bis(octadeca-9,12-dienoate)

Pd/C (10% w/w, 1.0 equiv) was added to a solution of intermediate 87h (1.0 equiv) in THF (0.1-0.3 M). The mixture was stirred at 15-20°C for 5 hours under an H ₂ atmosphere (15 psi). Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to obtain a residue.

To a solution of residue and intermediate 1c (1.0 equiv) in DCM (10 ml) was added EDCI (1.0 equiv), DMAP (0.1 equiv) and Et ₃ N (1.05 equiv). The mixture was stirred at 15-20°C for at least 12 hours. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was diluted with H2O and extracted with 3x EtOAc. The combined organic layers were dried over Na ₂ SO ₄ , filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the product as a pale yellow oil (61%).

^1H NMR (400 MHz, CDCl ₃ ) δ 5.40-5.14 (m, 8H), 5.08 (s, 1H), 4.69 (s, 1H), 4.21 (dd, J= 11.9, 4.4 Hz, 2H), 4.08 (ddd, J=12.0, 5.9, 3.3 Hz, 2H), 3.17 (q, J=6.0 Hz, 2H ), 2.70 (t, J=6.4 Hz, 4H), 2.43-2.28 (m, 5H), 2.24 (t, J=7.6 Hz, 4H), 2.14 (s, 3H), 1.98 (q, J=6.8 Hz, 7H), 1.79 (ddd, J=42.6, 15.1, 7.5 Hz, 3H), 1.53 ( q, J=7.6 Hz, 8H), 1.33-1.06 (m, 37H), 0.97 (t, J=7.1 Hz, 3H), 0.81 (q, J=6 ,3 Hz, 9H).

MS: 944.1 m/z [M+H].

Example 88 - compound 88.

Intermediate 88a: ^)-1-(methylamino)-1-oxododecane-4-ylacetate

Intermediate 88a was synthesized in 23% yield from intermediate 87b using the same method as intermediate 87c.

1H NMR (400 MHz, CDCl ₃ ) δ 5.81 (s, 1H), 4.79 (tq, J=8.5, 3.8 Hz, 1H), 2.73 (d, J=4.8 Hz, 3H), 2.17-2.03 (m, 2H), 1.98 (s, 3H), 1.95-1.63 (m, 3H), 1.56-1.36 (m, 2H), 1.19 (d, J=8.4 Hz, 11H), 0.81 (t, J=6.8 Hz, 3H).

Intermediate 88b: ^)-5-octyldihydrofuran-2(3H)-one

Intermediate 88b was prepared from intermediate 88a using the same method as intermediate 87d.

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Intermediate 88c: benzyl(S)-4-rugpoKCugogeKaHoaT

Intermediate 88c was prepared from intermediate 88b using the methods used for intermediates 87e and 87f.

Intermediate 88d: benzyl^)-4-(((4-nitrophenoxy)carbonyl)oxy)dodecanoate

Intermediate 88d was prepared from intermediate 88c using the same method as intermediate 88g.

Intermediate carbamoyl)oxy)dodecanoate

^)-4-(((2-(ethyl(methyl)amino)ethyl)88е:

benzyl

Intermediate 88e was synthesized in 47% yield from intermediate 88e using the method used for intermediate 87h.

Compound 88: 2-((^)-4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propane-1,3diyl (9Z,9'Z, 12Z, 12' 2)-bis(octadeca-9,12-dienoate)

Compound 88 was synthesized in 42% yield from intermediate 88d using the same method as 87.

Example 89 - compound 89.

Intermediate 89a: 4-hydroxy-M-methyldecanamide

To a mixture of methamine hydrochloride (19.83 g, 2.0 eq.) in DCM (250 mL) was added AlMe ₃ (2 M, 152.72 mL, 2.08 eq.) dropwise at 0° C. under N2 atmosphere. The mixture was stirred at 0°C for 2 hours under N2 atmosphere. This solution was then added dropwise to a solution of 5-hexyltetrahydrofuran-2-one (25 g, 1.0 equiv) in DCM (100 ml) under N2. The mixture was stirred at 20°C for 12 hours under N2 atmosphere. The reaction mixture is quenched by adding HCl (1 N) at 0°C. The organic layer was separated and the aqueous layer was extracted with DCM three times. The combined organic phase was washed 3 times with brine, dried with anhydrous Na ₂ SO ₄ , filtered and the filtrate was concentrated in vacuo. The residue was purified by column chromatography to give the product as a white solid (67%).

^1H NMR (400 MHz, CD3OD) δ 3.50 (td, J=7.7, 3.8 Hz, 1H), 2.70 (s, 3H), 2.29 (ddt, J=25.9 , 16.6, 6.8 Hz, 2H), 1.77 (dddd, J=13.3, 9.2, 6.8, 3.9 Hz, 1H), 1.66-1.55 (m , 1H), 1.42 (h, J=4.0 Hz, 3H), 1.39-1.25 (m, 8H), 0.95-0.87 (m, 3H).

Intermediate 89b: ^)-1-(methylamino)-1-oxodecan-4-yl acetate

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Intermediate 89b was synthesized in 23% yield using the same method as intermediate 87c.

1H NMR (400 MHz, CDCl3) δ 5.70 (s, 1H), 4.79 (tt, J=8.4, 4.4 Hz, 1H), 2.74 (d, J=4.8 Hz , 3H), 2.11 (dp, J=21.3, 7.7, 7.2 Hz, 2H), 1.98 (s, 3H), 1.86 (tdd, J=13.6, 6 ,7, 3.2 Hz, 1H), 1.81-1.70 (m, 1H), 1.45 (ddp, J=19.0, 13.6, 6.6, 6.1 Hz, 2H ), 1.27-1.12 (m, 8H), 0.80 (t, J=6.7 Hz, 3H).

Intermediate 89c: ^)-5-hexyldihydrofuran-2(3H)-one

Intermediate 89c was synthesized in 78% yield from intermediate 89b using the same method as intermediate 87d.

1H NMR (400 MHz, CDCl3) δ 4.48 (ddd, J=13.7, 7.5, 5.6 Hz, 1H), 2.52 (dd, J=9.5, 6.9 Hz, 2H), 2.31 (dq, J=13.4, 6.7 Hz, 1H), 1.91-1.80 (m, 1H), 1.80-1.68 (m, 1H), 1 .58 (dq, J=13.8, 5.2 Hz, 1H), 1.51-1.21 (m, 8H), 0.87 (t, J=6.6 Hz, 3H).

Intermediate 89d: benzyl ^)-4-hydroxydecanoate

To a solution of intermediate 89c (3 g, 1.0 equiv) in EtOH (25 ml) and H ₂ O (5 ml) was added NaOH (1.1 equiv) under N2. The mixture was then stirred at 20°C for 12 hours under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove the solvent.

The resulting residue was subjected to the same method as intermediate 87f to give the product as a colorless oil (3 g, 50%).

Intermediate 89e: benzyl ^)-4-(((4-nitrophenoxy)carbonyl)oxy)decanoate

To a solution of intermediate 89d (3 g, 1.0 equiv) in DCM (30 ml) was added (4nitrophenyl)carbonochloridate (3.26 g, 1.5 equiv) under N2. Pyridine (1.74 mL, 2.0 equiv.) was then added to the mixture at 0°C. The reaction mixture was stirred at 20°C for 1 hour under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove the solvent to obtain a residue. The residue was diluted with hexane and filtered. The filtrate was washed 3 times with H ₂ O. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and the filtrate was concentrated under reduced pressure to leave a residue. The residue was purified by column chromatography to give the product as a colorless oil (2 g, 42%).

1H NMR (400 MHz, CD3OD) δ 8.23-8.13 (m, 2H), 7.37-7.18 (m, 7H), 5.39 (s, 1H), 5.03 (s, 2H), 4.47 (s, 1H), 4.00 (q, J=7.1 Hz, 2H), 2.42 (t, J=7.1 Hz, 2H), 2.00 (ddt, J=15.0, 7.6, 3.8 Hz, 1H), 1.85 (dt, J=14.7, 7.3 Hz, 1H), 1.57 (ddt, J=21.1, 14.0, 6.8 Hz, 2H), 1.35-1.16 (m, 8H), 0.91-0.75 (m, 3H).

Intermediate 89f: benzyl ^)-4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoate

OB η

To a solution of intermediate 89e (1.5 g, 1.0 eq) in MeCN (15 ml) was added 2-pyrrolidin-1-ylethanamine (1.16 g, 3.0 eq), pyridine (819 µl, 3.0 eq.), DMAP (413 mg, 1.0 eq.) under N ₂ atmosphere. The mixture was stirred at 20°C for 5 hours under N ₂ atmosphere. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with EtOAc and washed 5 times with 1 N. NaHCO3 and 3 times H2O. The organic layer was dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatograph

- 150 046244 fiey to obtain the product in the form of a pale yellow oil (0.65 g, 41%).

1H NMR (400 MHz, CD3OD) δ 7.41-7.26 (m, 5H), 5.11 (d, J=1.1 Hz, 2H), 4.73 (dd, J=10.9, 5.7 Hz, 1H), 3.26 (dd, J=6.7, 1.7 Hz, 2H), 2.79-2.57 (m, 7H), 2.41 (t, J=7 .4 Hz, 2H), 1.99-1.74 (m, 7H), 1.53 (s, 3H), 1.361.24 (m, 9H), 0.92-0.86 (m, 3H) .

Compound 89: 2-((^)-4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)propane-1,3diyl (9Z,9 'Z, 12Z,12^ )-bis(octadeca-9,12-dienoate)

Intermediate 89f (1.0 eq) was added to a suspension of Pd/C (10% w/w, 1.0-3.0 eq) in THF (0.2-0.5 M). The mixture was stirred at 15-25°C for at least 5 hours in an H ₂ atmosphere. The reaction mixture was filtered and the filtrate was concentrated to give a residue. The residue was purified by column chromatography to give the product as a thick brown oil.

The brown oil was immediately reconstituted in DCM (0.2-0.5 M) followed by the addition of intermediate 1c (1.0 equiv), EDCI (1.2 equiv), DMAP (0.1-0.2 equiv). ) and Et ₃ N (1.5 equiv.) at 0°C under N2. The mixture was stirred at 20°C for at least 12 hours under N2 atmosphere. The mixture was diluted with H ₂ O and extracted with 3x EtOAc. The combined organic phase was washed with brine, dried with anhydrous Na ₂ SO ₄ , filtered and the filtrate was concentrated in vacuo. The residue was purified by column chromatography to give the product as a colorless oil (23%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.49-5.22 (m, 8H), 5.18 (t, J=5.0 Hz, 1H), 5.08 (s, 1H), 4 .69 (s, 1H), 4.22 (dd, J=11.9, 4.4 Hz, 2H), 4.12-4.04 (m, 2H), 3.28-3.15 (m , 2H), 2.70 (t, J=6.5 Hz, 4H), 2.50 (d, J=6.4 Hz, 2H), 2.44 (s, 3H), 2.32 (s , 2H), 2.25 (t, J=7.6 Hz, 4H), 1.98 (q, J=6.9 Hz, 8H), 1.83 (s, 1H), 1.70 (s , 5H), 1.38-0.94 (m, 37H), 0.81 (q, J=6.6 Hz, 9H). MS: 928.4 m/z [M+H].

Example 90 - compound 90.

Intermediate 90a: Benzyl ^)-4-hydroxydecanoate

Intermediate 90a was synthesized in 27% yield from (5R)-5hexyltetrahydrofuran-2-one using the same method as intermediate 89d.

Intermediate 90b: Benzyl ^)-4-(((4-nitrophenoxy)carbonyl)oxy)decanoate

Intermediate 90b was synthesized in 57% yield from intermediate 90a using the same method as intermediate 89e.

Intermediate 90c: Benzyl ^)-4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoate

Intermediate 90c was synthesized in 47% yield from intermediate 90b using the same method as intermediate 89f.

Compound 90: 2-((^)-4-(((2-(pyrrolidin-1 -yl)ethyl)carbamoyl)oxy)decanoyl)oxy)propane-1,3diyl (9Z,9'Z, 12Z,12^ )-bis(octadeca-9,12-dienoate)

- 151 046244

Compound 90 was synthesized in 27% yield from intermediate 90c using the same method as 89.

Example 91 - compound 91.

Intermediate 91a: 4-hydroxydodecanoic acid

To a solution of 5-octyltetrahydrofuran-2-one (20 g, 1.0 eq.) in H ₂ O (200 ml) was added NaOH (4.03 g, 1.0 eq.). The mixture was stirred at 20°C for 12 hours under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove H ₂ O. This gave the crude product as a white solid (14 g, 65%).

^1H NMR (400 MHz, D2O) δ 3.55 (ddd, J=9.5, 7.5, 4.6 Hz 1H), 2.19 (qdd, J=15.0, 9.3, 6 ,3 Hz, 2H), 1.69 (dddd, J=13.9, 9.4, 6.4, 4.4 Hz, 1H), 1.59 (dddd, J=14.0, 9.2 , 7.7, 6.4 Hz, 1H), 1.46-1.30 (m, 3H), 1.23 (d, J=6.6 Hz, 11H), 0.86-0.77 ( m, 3H).

Intermediate 91b: Benzyl 4-hydroxydodecanoate

OH

O^J

OBn

BnBr (7.91 g, 1.0 eq) was added to a solution of intermediate 91a (10 g, 1.0 eq) in DMF (100 mL). The mixture was stirred at 20°C for 2 hours under N2 atmosphere. The mixture was diluted with H ₂ O and extracted with 2x EtOAc. The combined organic layers were concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a colorless oil (5.7 g, 40%).

1H NMR (400 MHz, CDCI3) δ 7.46-7.28 (m, 5H), 5.12 (s, 2H), 3.61 (h, J=4.6 Hz, 1H), 2.51 (td, J=7.3, 2.9 Hz, 2H), 1.86 (dtd, J=14.6, 7.4, 3.5 Hz, 1H), 1.76-1.59 (m , 3H), 1.42 (q, J=12.1, 9.1 Hz, 4H), 1.28 (d, J=11.9 Hz, 12H), 0.88 (t, J=6, 8 Hz, 3H).

Intermediate 91c: Benzyl 4-(((4-nitrophenoxy)carbonyl)oxy)dodecanoate

L 1 n

OBn

To a solution of intermediate 91b (10 g, 1.0 equiv) and (4-nitrophenyl)carbonochloridate (9.87 g, 1.05 equiv) in DCM (100 ml) was added pyridine (3.95 ml, 1.0 equiv). 5 equiv.) at 0°C. The mixture was stirred at 20°C for 1 hour under N ₂ atmosphere. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was diluted with H2O and extracted with 3x EtOAc. The combined organic layers were concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a colorless oil (7.5 g, 49%).

1H NMR (400 MHz, CDCI3) δ 8.30-8.21 (m, 2H), 7.48-7.26 (m, 7H), 5.22 (d, J=69.6 Hz, 2H) , 4.87 (tt, J=7.5, 4.5 Hz, 1H), 2.59-2.42 (m, 2H), 1.80-1.57 (m, 2H), 1.52 -1.16 (m, 13H), 0.88 (t, J=6.6 Hz 3H).

Intermediate 91d: Benzyl 4-(((2-(dimethylamino)ethyl)carbamoyl)oxy)dodecanoate

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To a solution of intermediate 91c (1.0 eq.) in MeCN (0.1-0.5 M) was added a diamine or amino alcohol (N,N-dimethylethane-1,2-diamine (2.0-3.0 eq. )), pyridine (3.0 equiv.) and DMAP (0.1 equiv.) at 0°C. The mixture was stirred at 15-25°C for at least 2 hours. Upon completion, the reaction mixture was concentrated under reduced pressure, diluted with H ₂ O and extracted with 3x EtOAc. The combined organic layers were concentrated to give a residue, which was purified by column chromatography to give the product as a yellow oil (79%).

^1H NMR (400 MHz, CDCl3) δ 7.46-7.28 (m, 5H), 5.08 (s, 1H), 4.76 (s, 1H), 3.23 (q, J=5 .9 Hz, 2H), 2.40 (ddt, J=12.0, 8.9, 4.5 Hz, 4H), 2.21 (s, 7H), 1.94 (d, J=9, 9 Hz, 1H), 1.82 (p, J=7.6 Hz, 1H), 1.61-1.40 (m, 3H), 1.39-1.16 (m, 14H), 0, 87 (t, J=6.7 Hz, 3H).

Compound 91: 2-((4-(((2-(dimethylamino)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca -9,12-dienoate)

Pd/C (10% w/w) was added to a solution of intermediate 91d in THF (0.1-0.5 M). The mixture was stirred at 25°C for at least 5 hours under an H ₂ atmosphere at 15 psi. The resulting product was diluted in THF and filtered to remove solids. The filtrate was concentrated in vacuo. The residue was purified by column chromatography to give the carboxylic acid intermediate as a brown oil.

From the resulting oil, compound 91 was subsequently synthesized in 15% yield using the method used in intermediate 90f

1H NMR (400 MHz, CDCl ₃ ) δ 5.44-5.26 (m, 8H), 5.27-5.20 (m, 1H), 5.12 (s, 1H), 4.75 (s , 1H), 4.28 (ddd, J=11.9, 4.5, 1.3 Hz, 2H), 4.14 (ddd, J=11.9, 5.8, 3.4 Hz, 2H ), 3.24 (q, J=5.7 Hz, 2H), 2.77 (t, J=6.7 Hz, 4H), 2.442.35 (m, 4H), 2.31 (t, J =7.6 Hz, 4H), 2.22 (s, 6H), 2.04 (q, J=6.9 Hz, 8H), 1.97-1.84 (m, 2H), 1.80 (dt, J=14.9, 7.7 Hz, 1H), 1.67-1.42 (m, 8H), 1.42-1.14 (m, 39H), 0.88 (td, J =6.8, 5.2 Hz, 9H). MS: 930.4 m/z [M+H].

Example 92 - compound 92.

Intermediate 92a: Benzyl 4-((((1-methylpiperidin-4-yl)oxy)carbonyl)oxy)dodecanoate

Intermediate 92a was synthesized in 94% yield from intermediate 91c and 1-methylpiperidin-4-ol using the method used for intermediate 91d.

1H NMR (400 MHz, CDCl ₃ ) δ 7.39-7.28 (m, 5H), 5.11 (s, 2H), 4.73 (dtd, J=11.8, 5.4, 4, 6, 2.7 Hz, 1H), 4.62 (tt, J=8.6, 4.2 Hz, 1H), 2.68 (s, 2H), 2.48-2.37 (m, 2H ), 2.29 (s, 5H), 2.01-1.84 (m, 4H), 1.78 (dtt, J=12.5, 8.2, 3.5 Hz, 2H), 1, 67-1.58 (m, 1H), 1.58-1.46 (m, 1H), 1.27 (d, J=15.6 Hz, 13H), 0.87 (t, J=6, 8 Hz, 3H).

Compound 92: 2-((4-((((1-methylpiperidin-4-yl)oxy)carbonyl)oxy)dodecanoyl)oxy)propan-1,3diyl (9Z,9'Z, 12Z, 12^)-bis (octadeca-9,12-dienoate)

- 153 046244

Compound 92 was synthesized in 28% yield from intermediate 92a using the same method as 91.

1H NMR (400 MHz, CDCl ₃ ) δ 5.39-5.21 (m, 8H), 5.21-5.16 (m, 1H), 4.65 (d, J=6.9 Hz, 1H ), 4.57-4.52 (m, 1H), 4.22 (dd, J=11.9, 4.3 Hz, 2H), 4.08 (ddd, J=11.9, 5.8 , 4.1 Hz, 2H), 2.70 (t, J=6.5 Hz, 4H), 2.61 (s, 2H), 2.362.29 (m, 2H), 2.29-2.18 (m, 7H), 2.14 (s, 2H), 1.98 (q, J=6.8 Hz, 8H), 1.93-1.84 (m, 4H), 1.83-1, 77 (m, 1H), 1.77-1.65 (m, 3H), 1.54 (t, J=7.3 Hz, 13H), 1.33-1.12 (m, 41H), 0 .81 (td, J=6.9, 5.3 Hz, 9H). MS: 957.4 m/z [M+H].

Example 93 - compound 93.

Intermediate 93a: benzyl 4-(((2-(azetidin-1-yl)ethyl)carbamoyl)oxy)dodecanoate

Intermediate 93a was synthesized in 80% yield from intermediate 91c and 2-(azetidin-1-yl)ethanamine using the method used for intermediate 92d.

1H NMR (400 MHz, CDCl ₃ ) δ 7.49-7.26 (m, 5H), 5.11 (s, 2H), 5.04 (s, 1H), 4.74 (s, 1H), 3.20 (t, J=7.0 Hz, 4H), 3.11 (q, J=5.8 Hz, 2H), 2.50 (t, J=5.9 Hz, 2H), 2, 41 (ddd, J=8.6, 6.7, 3.2 Hz, 2H), 2.10-2.00 (m, 2H), 1.961.88 (m, 1H), 1.82 (d, J=15.4 Hz, 4H), 1.46 (d, J=6.9 Hz, 3H), 1.34-1.18 (m, 14H), 0.87 (t, J=6.8 Hz, 3H).

Compound 93: 2-((4-(((2-(azetidin-1 -yl)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z,12^) -bis(octadeca-9,12-dienoate)

Compound 93 was synthesized in 18% yield from intermediate 93a using the method used for compound 91.

1H NMR (400 MHz, CDCl ₃ ) δ 5.36-5.22 (m, 8H), 5.18 (p, J=5.0 Hz, 1H), 4.98 (t, J=5.6 Hz, 1H), 4.68 (s, 1H), 4.22 (ddd, J=11.9, 4.5, 1.6 Hz, 2H), 4.08 (ddq, J=11.7, 5.8, 2.8 Hz, 2H), 3.12 (t, J=7.0 Hz, 4H), 3.05 (q, J=5.9 Hz, 2H), 2.70 (t, J=6.4 Hz, 4H), 2.43 (t, J=5.9 Hz, 2H), 2.31 (ddd, J=8.8, 6.6, 4.2 Hz, 2H), 2.25 (t, J=7.6 Hz, 4H), 1.99 (dq, J=10.0, 6.9 Hz, 10H), 1.88-1.68 (m, 3H), 1 .59-1.36 (m, 7H), 1.34-1.11 (m, 42H), 0.86-0.77 (m, 9H). MS: 942.1 m/z [M+H].

Example 94 - compound 94.

Intermediate 94a: benzyl 4-(((2-(piperidin-1-yl)ethyl)carbamoyl)oxy)dodecanoate SLL n A o3

ORP

Intermediate 94a was synthesized in 68% yield from intermediate 91c and 2-(piperidin-1-yl)ethan-1-amine using the same method as intermediate 91d.

1H NMR (400 MHz, CDCl ₃ ) δ 7.38-7.28 (m, 5H), 5.10 (s, 3H), 4.75 (s, 1H), 3.22 (t, J=6 ,0 Hz, 2H), 2.64 (s, 1H), 2.45-2.29 (m, 8H), 1.99-1.88 (m, 1H), 1.82 (dq, J= 15.0, 8.0 Hz, 1H), 1.48 (dt, J=53.9, 5.8 Hz, 9H), 1.34- 154 046244

1.17 (m, 12H), 0.86 (t, J=6.7 Hz, 3H).

Compound 94: 2-((4-(((2-(piperidin-1-yl)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12^) -bis(octadeca-9,12-dienoate)

Compound 94 was synthesized in 22% yield from intermediate 94a using the same method as 91.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.29 (qq, J=11.7,6.7 Hz, 8H), 5.18 (p, J=5.1 Hz, 1H), 5.08 (s, 1H), 4.69 (s, 1H), 4.22 (ddd, J=11.9, 4.5, 2.2 Hz, 2H), 4.08 (ddd J=11.9, 5.9, 3.6 Hz, 2H), 3.18 (q, J=5.9 Hz, 2H), 2.70 (t, J=6.5 Hz, 4H), 2.39-2, 18 (m, 12H), 1.98 (q, J=6.9 Hz, 8H), 1.91-1.79 (m, 1H), 1.79-1.68 (m, 1H), 1 .61-1.33 (m, 15H), 1.33-1.10 (m, 42H), 0.81 (q, J=6.4 Hz, 9H). MS: 970.2 m/z [M+H].

Example 95 - compound 95.

Intermediate 95a: Benzyl 4-(((4-(diethylamino)butoxy)carbonyl)oxy)dodecanoate

Intermediate 95a was synthesized in 83% yield from intermediate 91c and 4-(diethylamino)butan-1-ol using the same method as intermediate 92d.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.44-7.28 (m, 5H), 5.11 (s, 2H), 4.72 (tdd, J=7.6, 5.4, 4 .0 Hz, 1H), 4.11 (td, J=6.7, 3.8 Hz, 2H), 2.49 (q, J=7.1 Hz, 4H), 2.42 (dtd, J =7.7, 5.1, 4.4, 2.5 Hz, 4H), 1.99 (dddd, J=14.4, 8.9, 7.0, 4.1 Hz, 1H), 1 .89 (dtd, J=14.7, 8.4, 6.5 Hz, 1H), 1.64 (ddt, J=17.4, 10.5, 6.2 Hz, 3H), 1.56 -1.46 (m, 3H), 1.341.21 (m, 13H), 1.00 (t, J=7.1 Hz, 6H), 0.87 (t, J=6.8 Hz, 3H) .

Compound 95: 2-((4-(((4-(diethylamino)butoxy)carbonyl)oxy)dodecanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca -9,12-dienoate)

Compound 95 was obtained in 27% yield from intermediate 95a using the same method as 91.

1H NMR (400 MHz, CDCl3) δ 5.35 (qdd, J=10.6, 4.4, 1.7 Hz, 8H), 5.27-5.22 (m, 1H), 4.71 ( tt, J=7.6, 2.7 Hz, 1H), 4.28 (dd, J=11.9, 4.3 Hz, 2H), 4.13 (dtt, J=10.4, 6, 6, 3.7 Hz, 4H), 2.76 (t, J=6.6 Hz, 4H), 2.50 (q, J=7.2 Hz, 4H), 2.41 (ddd, J= 15.2, 8.0, 6.4 Hz, 4H), 2.31 (t, J=7.6 Hz, 4H), 2.04 (q, J=6.8 Hz, 8H), 1, 98-1.82 (m, 3H), 1.72-1.47 (m, 12H), 1.39-1.21 (m, 42H), 1.00 (t, J=7.1 Hz, 6H), 0.88 (td, J=6.8, 4.8 Hz, 9H). MS: 987.5 m/z [M+H].

Example 96 - compound 96.

Intermediate 96a: Benzyl 4-(((2-(diethylamino)ethyl)carbamoyl)oxy)dodecanoate

- 155 046244

Intermediate 96a was synthesized in 58% yield from intermediate 91c and X',X'-diethylethane-1,2-diamine using the same method as intermediate 91d. lH NMR (400 MHz, CDCI3) δ 7.39-7.28 (m, 5H), 5.19 (s, 1H), 5.11 (s, 2H), 4.76 (s, 1H), 3 .51 (s, 2H), 3.27-3.15 (m, 2H), 2.54 (dd, J=8.7, 5.9 Hz, 6H), 2.41 (ddd, J=9 ,0, 6.9, 2.6 Hz, 2H), 1.94 (d, J=7.5 Hz, 1H), 1.83 (p, J=7.3 Hz, 1H), 1.63 -1.40 (m, 3H), 1.35-1.17 (m, 13H), 1.01 (t, J=7.1 Hz, 6H), 0.87 (t, J=6.8 Hz, 3H).

Compound 96: 2-((4-(((2-(diethylamino)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z,12^)-bis(octadeca -9,12-dienoate)

Compound 96 was obtained in 42% yield from intermediate 96a using the same method as 91.

1H NMR (400 MHz, CDCI3) δ 5.29 (qq, J=11.4, 6.5 Hz, 8H), 5.18 (p, J=5.2 Hz, 1H), 5.08 (s , 1H), 4.69 (s, 1H), 4.21 (ddd, J=11.8, 4.5, 2.0 Hz, 2H), 4.08 (ddd, J=12.0, 5 ,9, 2.7 Hz, 2H), 3.12 (dt, J=13.0, 6.3 Hz, 2H), 2.70 (t, J=6.5 Hz, 4H), 2.45 (q, J=7.3 Hz, 6H), 2.28 (dt, J=27.2, 8.0 Hz, 6H), 1.98 (q, J=6.9 Hz, 8H), 1 .83 (s, 1H), 1.78-1.68 (m, 1H), 1.54 (t, J=7.2 Hz, 9H), 1.35-1.11 (m, 42H), 0.81 (q, J=6.3 Hz, 9H). MS: 958.4 m/z [M+H].

Example 97 - compound 97.

Compound 97: 2-((5-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)tridecanoyl)oxy)propan-1,3diyl (9Z,9'Z, 12Z,12D-bis(octadeca -9,12-dienoate)

Compound 96 was prepared in 75% yield from intermediate 55d and intermediate 1c using the same method as 52.

1H NMR (400 MHz, CDCI3) δ 5.42-5.28 (m, 8H), 5.27-5.22 (m, 1H), 5.12 (d, J=5.7 Hz, 1H) , 4.76-4.67 (m, 1H), 4.28 (dt, J=11.9, 4.5 Hz, 2H), 4.13 (ddd, J=12.0, 5.9, 4.5 Hz, 2H), 3.23 (h, J=7.2, 6.2 Hz, 2H), 2.76 (t, J=6.6 Hz, 4H), 2.43 (dt, J=14.5, 6.6 Hz, 4H), 2.35-2.27 (m, 6H), 2.19 (s, 3H), 2.04 (q, J=6.8 Hz, 8H ), 1.71-1.46 (m, 13H), 1.40-1.19 (m, 42H), 1.03 (t, J=7.1 Hz, 3H), 0.92-0, 83 (m, 9H). MS: 958.5 m/z [M+H].

Example 98.

Intermediate 98a: Ethyl 2,2-dimethyl-3-oxundecanoate.

To a solution of X-isopropylpropan-3-amine (20.0 ml, 1.0 equiv.) in THF (1-3 M) was added n-BuLi (2.5 M, 56.60 ml, 1.0 equiv.) at -78°C in an N ₂ atmosphere. The mixture was stirred at -78°C for 20 minutes. Ethyl 2-methylpropanoate (18.96 mL, 1.0 equiv) was then added at -78°C. The mixture was stirred at -78°C for another 20 minutes. To the above solution, nonanoyl chloride (26.60 ml, 1.0 equiv.) in THF (200 ml) was added dropwise. The mixture was allowed to warm to 15°C and stirred at 15°C for 12 hours under N2 atmosphere. The reaction mixture was quenched by adding ice water, filtered and concentrated under reduced pressure to obtain a residue. The residue was diluted with H ₂ O and extracted with 3x EtOAc. The organic phase was separated, washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a yellow oil (15 g, 41%).

1H NMR (400 MHz, CDCI3) δ 4.11 (q, J=7.2 Hz, 2H), 2.36 (t, J=7.3 Hz, 2H), 1.56-1.46 (m , 2H), 1.28 (s, 6H), 1.25-1.13 (m, 13H), 0.80 (t, J=6.7 Hz, 3H).

Intermediate 98b: 2,2-dimethyl-3-oxundecanoic acid.

- 156 046244

A mixture of Intermediate 98a (15 g, 1.0 eq), LiOH (2.10 g, 1.5 eq), KOH (4.92 g, 1.5 eq) in EtOH (120 ml) and H2O (40 ml) was degassed and purged 3 times with N2, and then the mixture was stirred at 40°C for 3 hours under an N2 atmosphere. The mixture was concentrated to remove EtOH. The pH was then adjusted to 5-6 with HCl (1 M). The mixture was partitioned between H2O and EtOAc. The organic phase was separated, washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a yellow oil (5 g, 37%).

1H NMR (400 MHz, chloroform-d) δ 2.46 (t, J=7.3 Hz, 2H), 1.58-1.47 (m, 2H), 1.33 (s, 6H), 1 .20 (s, 10H), 0.81 (t, J=6.6 Hz, 3H).

Example 99 - compound 99.

Intermediate 99a: 2-(((2,2-dimethyl-3-oxundecanoyl)oxy)methyl)propan-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9, 12-dienoate)

To a solution of intermediate 53a (12.16 g, 1.1 equiv) in THF (20 mL) was added intermediate 98b (4 g, 1.0 equiv), DBAD (8.07 g, 2.0 equiv). ) and PPh ₃ (9.19 g, 2.0 equiv.) at 0°C. The mixture was stirred at 0°C for 15 minutes. The mixture was then stirred at 15°C for 16 hours under N2 atmosphere. The reaction mixture was diluted with H ₂ O and extracted three times with EtOAc. The organic phase was separated, washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a yellow oil (3 g, 20%).

¹ H NMR (400 MHz, CDCl3) δ 5.27 (dtt, J=10.8, 8.7, 6.1 Hz, 8H), 4.10 (d, J=6.0 Hz, 2H), 4.03 (d, J=6.0 Hz, 4H), 2.70 (t, J=6.4 Hz, 4H), 2.37 (t, J=7.3 Hz, 2H), 2, 23 (t, J=7.6 Hz, 4H), 1.98 (q, J=6.8 Hz, 8H), 1.52 (tt, J=14.2, 7.4 Hz, 8H), 1.31-1.17 (m, 46H), 0.85-0.77 (m, 12H).

Intermediate 99b: 2-(((3-hydroxy-2,2-dimethylundecanoyl)oxy)methyl)propane-1,3diyl (9Z,9'Z, 12Z,12^)-bis(octadeca-9,12-dienoate )

To a solution of intermediate 99a (1.0 eq.) in 4:2:1 THP:H ₂ O:toluene (0.025-0.1) NaBH4 (5.0 eq.) was added at 0°C. The mixture was stirred at 15°C C for 5 hours under N2. The reaction mixture was partitioned between THF and H2O and the organic phase was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the product as a colorless oil (33%).

1H NMR (400 MHz, CDQ3) δ 5.41-5.25 (m, 8H), 4.20-4.07 (m, 7H), 3.63 (dd, J=10.2, 6.7 Hz, 1H), 2.76 (t, J=6.4 Hz, 4H), 2.40 (t, J=6.0 Hz, 2H), 2.30 (t, J=7.6 Hz, 4H), 2.04 (q, J=6.8 Hz, 8H), 1.59 (q, J=7.3 Hz, 6H), 1.40-1.22 (m, 40H), 1, 16 (d, J=4.5 Hz, 6H), 0.88 (td, J=6.8, 4.0 Hz, 9H).

Intermediate 99c: 2-(((2,2-dimethyl-3-(((4-nitrophenoxy)carbonyl)oxy)undecanoyl)oxy)methyl)propan-1,3-diyl (9Z,9'Z,12Z, 12'Z)-bis(octadeca-9,12-dienoate)

- 157 046244

Pyridine (191 µL, 2.0 equiv) was added to a solution of intermediate 99b (1 g, 1.0 equiv) and (4-nitrophenyl)carbonochloridate (478 mg, 2.0 equiv) in DCM (10 ml). at O°C. under N2. The mixture was stirred at 15-25°C for 5 hours under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a colorless oil (0.8 g, 67%).

1H NMR (400 MHz, CDCi ₃ ) δ 8.20 (d, J=8.8 Hz, 2H), 7.30 (d, J=8.9 Hz, 2H), 5.29 (dq, J= 13.1, 7.1, 6.1 Hz, 8H), 5.03 (dd, J=10.4, 2.3 Hz, 1H), 4.08 (dd, J=6.0, 2, 5 Hz, 6H), 2.69 (t, J=6.4 Hz, 4H), 2.35 (p, J=6.0 Hz, 1H), 2.23 (q, J=8.4, 7.7 Hz, 4H), 1.97 (q, J=6.9 Hz, 8H), 1.53 (h, J=7.1, 5.9 Hz, 8H), 1.36-1, 12 (m, 56H), 0.85-0.77 (m, 12H).

Compound 99: 2-(4,4,11-trimethyl-5-octyl-3,7-dioxo-2,6-dioxa-8,11-diazatridecyl)propan-1,3diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9,12-dienoate)

To a solution of intermediate 99c (1.0 eq) in MeCN (0.1 M) was added diamine (N'-ethylN-methylethane-1,2-diamine (3.0 eq)), DMAP (1.0 eq.) and pyridine (3.0 eq.) at 0°C. The mixture was stirred at 15°C for at least 2 hours under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was diluted with H ₂ O and extracted with 3x EtOAc. The organic phase was separated, washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a yellow oil (66%).

1H NMR (400 MHz, CDCi ₃ ) δ 5.28 (pt, J=11.5, 5.8 Hz, 8H), 4.90 (t, J=6.4 Hz, 1H), 4.11- 4.02 (m, 5H), 3.17 (dt, J=13.4, 7.3 Hz, 2H), 2.70 (t, J=6.4 Hz, 4H), 2.37 (dq , J=12.3, 6.2 Hz, 5H), 2.23 (t, J=7.6 Hz, 4H), 2.14 (s, 3H), 1.98 (q, J=6, 8 Hz, 7H), 1.54 (p, J=6.8 Hz, 4H), 1.37-1.06 (m, 49H), 0.97 (t, J=7.1 Hz, 3H) , 0.81 (q, J=6.9 Hz, 9H). MS: 973.5 m/z [M+H].

Example 100 - connection 100.

Intermediate 100a: Ethyl 2,2-dimethyl-3-oxononanoate

Intermediate 100a was synthesized in 26% yield from N-isopropylpropan-2amine, ethyl 2-methylpropanoate and heptanoyl chloride using the same method as intermediate 99a.

1H NMR (400 MHz, CDCi ₃ ) δ 4.14 (q, J=7.1 Hz, 2H), 2.40 (t, J=7.3 Hz, 2H), 1.60-1.47 ( m, 2H), 1.36-1.14 (m, 13H), 0.84 (t, J=6.8 Hz, 3H).

Intermediate 100b: 2,2-dimethyl-3-oxononanoic acid

A mixture of Intermediate 100a (20 g, 1.0 eq), LiOH (3.15 g, 1.5 eq), KOH (7.37 g, 1.5 eq) in EtOH (6 mL) and H ₂ O (2 ml) was stirred at 40°C for 4 hours under N2 atmosphere. The mixture was concentrated to remove EtOH. The pH was then adjusted to 5-6 with HCl (1 M). The mixture was partitioned between H2O and EtOAc and extracted twice with EtOAc. The organic phase was separated, washed with brine,

- 158 046244 dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a colorless oil (6 g, 34%).

1H NMR (400 MHz, CDCl ₃ ) δ 2.53 (t, J=7.3 Hz, 2H), 1.58 (p, J=7.2 Hz, 2H), 1.40 (s, 6H) , 1.34-1.23 (m, 6H), 0.92-0.81 (m, 3H).

Intermediate 100c: 2-(((2,2-dimethyl-3-oxononanoyl)oxy)methyl)propan-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9, 12-dienoate)

To a solution of intermediate 1c (19.06 g, 1.1 equiv) in THF (50 ml) was added PPh ₃ (14.41 g, 2.0 equiv) and DBAD (12.65 g, 2.0 equiv) .) and intermediate 100b (5.5 g, 1.0 equiv.) at 0°C. The mixture was stirred at 0°C for 20 minutes, the mixture was allowed to warm to 15°C and stirred at 15°C for 16 hours under N2. The reaction mixture was diluted with H ₂ O and extracted three times with EtOAc. The organic phase was separated, washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a yellow oil (4 g, 18%).

1H NMR (400 MHz, CDC13) δ 5.28 (tt, J=11.1, 5.4 Hz, 8H), 4.10 (d, J=6.0 Hz, 2H), 4.05 (dd , J=10.4, 6.0 Hz, 4H), 2.70 (t, J=6.4 Hz, 4H), 2.34 (dt, J=17.5, 6.6 Hz, 3H) , 2.23 (t, J=7.6 Hz, 4H), 1.98 (q, J=6.8 Hz, 8H), 1.61-1.44 (m, 8H), 1.39 ( d, J=14.2 Hz, 3H), 1.35-1.14 (m, 41H), 0.85-0.76 (m, 9H).

Intermediate 100d: 2-(((3-hydroxy-2,2-dimethylnonanoyl)oxy)methyl)propane-1,3diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9,12-dienoate )

To a solution of intermediate 100c (1.0 eq.) in 4:2:1 TI IF:112('):to.luo.l (0.025-0.1) NaBH ₄ (5.0 eq.) was added at 0° C. The mixture was stirred at 5° C. for 5 hours under N2. The reaction mixture was partitioned between THF and H ₂ O, and the organic phase was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography , obtaining the product in the form of a colorless oil (20%).

Intermediate 100e: 2-(((2,2-dimethyl-3-(((4-nitrophenoxy)carbonyl)oxy)nonanoyl)oxy)methyl)propan-1,3-diyl (9Z,9'Z, 12Z, 12^)-bis(octadeca-9,12-dienoate)

To a solution of intermediate 100d (0.4 g, 1.0 equiv) and (4-nitrophenyl)carbonochloridate (890 mg, 9.0 equiv) in DCM (5 ml) was added pyridine (119 µl, 3.0 equiv). .) at 0°C. The mixture was stirred at 15°C for 3 hours under N2 atmosphere. The mixture was then stirred at 25°C for 2 hours under N ₂ atmosphere. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a colorless oil (0.2 g, 42%).

1H NMR (400 MHz, CDC13) δ 8.21 (dd, J=9.3, 2.5 Hz, 2H), 7.31 (dd, J=9.4, 2.8 Hz, 2H), 5 .43-5.16 (m, 8H), 5.03 (dd, J=10.3, 2.3 Hz, 1H), 4.12-3.99 (m, 6H), 2.70 (t , J=6.4 Hz, 4H), 2.35 (h, J=6.0 Hz, 1H), 2.22 (t,

- 159 046244

J=7.6 Hz, 4H), 1.97 (t, J=6.9 Hz, 7H), 1.63-1.47 (m, 6H), 1.40-1.12 (m, 41H ), 0.82 (t, J=6.7 Hz, 9H).

Compound 100: 2-(((2,2-dimethyl-3 -(((2-(pyrrolidin-1 -yl)ethyl)carbamoyl)oxy)nonanoyl)oxy)methyl)propan-1,3-diyl (9Z, 9'Z, 12Z, 12^)-bis(octadeca-9,12-dienoate)

Compound 100 was synthesized in 90% yield from intermediate 100e and 2-pyrrolidin-1-ylethanamine using the same method as 99.

1H NMR (400 MHz, CDCl ₃ ) δ 5.28 (tdq, J=17.2, 12.4, 5.8, 5.3 Hz, 8H), 4.90 (t, J=6.4 Hz , 1H), 4.13-4.00 (m, 6H), 3.22 (ddq, J=19.5, 12.8, 6.3, 5.8 Hz, 2H), 2.70 (t , J=6.4 Hz, 4H), 2.51 (t, J=6.3 Hz, 2H), 2.44 (d, J=6.0 Hz, 4H), 2.34 (p, J =6.0 Hz, 1H), 2.23 (t, J=7.6 Hz, 4H), 1.98 (q, J=6.8 Hz, 8H), 1.70 (h, J=7 .5 Hz, 4H), 1.54 (p, J=7.0 Hz, 4H), 1.41-1.06 (m, 44H), 0.81 (dt, J=9.6, 6, 7 Hz, 9H). MS: 956.4 m/z [M+H].

Example 101 - connection 101.

Compound 101: 2-((4-(((3-(azetidin-1-yl)propoxy)carbonyl)oxy)decanoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z,12^) -bis(octadeca-9,12-dienoate)

Compound 101 was synthesized in 65% yield from intermediate 40d and 3-(azetidin-1-yl)propan-1-ol using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.45-5.28 (m, 8H), 5.25 (tt, J=5.8, 4.3 Hz, 1H), 4.71 (ddd, J =11.5, 7.6, 4.7 Hz, 1H), 4.29 (dd, J=11.9, 4.3 Hz, 2H), 4.15 (dddd, J=13.3, 7 ,2, 5.5, 3.2 Hz, 4H), 3.18 (t, J=7.0 Hz, 4H), 2.822.72 (m, 4H), 2.48 (t, J=7, 3 Hz, 2H), 2.40 (dt, J=8.8, 6.8 Hz, 2H), 2.31 (td, J=7.6, 1.2 Hz, 4H), 2.10- 1.99 (m, 10H), 1.99-1.84 (m, 2H), 1.77-1.50 (m, 15H), 1.42-1.21 (m, 37H), 0. 88 (td, J=6.9, 5.4 Hz, 9H). MS: 929.3 m/z [M+H].

Example 102 - connection 102.

Compound 102: 2-((4-(((3-(piperidin-1-yl)propoxy)carbonyl)oxy)decanoyl)oxy)propane-1,3diyl (9Z,9'Z, 12Z, 12^)-bis (octadeca-9,12-dienoate)

Compound 102 was synthesized in 72% yield from intermediate 40d and 3(piperidin-1-yl)propan-1-ol using the same method as Example 1.

1H NMR (400 MHz, CDCl ₃ ) δ 5.44-5.28 (m, 8H), 5.25 (ddd, J=5.8, 4.3, 1.5 Hz, 1H), 4.76 -4.69 (m, 1H), 4.37-4.24 (m, 2H), 4.24-4.10 (m, 4H), 2.82-2.71 (m, 4H), 2 .48-2.26 (m, 12H), 2.05 (q, J=6.8 Hz, 8H), 2.01-1.81 (m, 4H), 1.71-1.49 (m , 17H), 1.43 (s, 2H), 1.40-1.24 (m, 37H), 0.88 (td, J=6.9, 5.0 Hz, 9H). MS: 957.1 m/z [M+H].

Example 103 - compound 103.

Intermediate 103a: (2-ethylbutyl)magnesium bromide.

To a solution of Mg (1.1 equiv.) and I ₂ (0.1 equiv.) in THF (0.4-0.5 M) 3

- 160 046244 (bromomethyl)pentane (1.0 equiv.). The mixture was stirred at 40°C for at least 1 hour. The reaction mixture was used directly in the next step without additional purification.

Intermediate 103b: Ethyl 6-ethyl-4-oxooctanoate.

To a solution of ethyl 4-chloro-4-oxobutanoate (1.0 eq) in THF (0.3 M) was added CuI (1-2 eq) and intermediate 103a (1-2 eq) at -78 °C drop by drop. The mixture was stirred at -78°C for at least 1.5 hours. Once complete, saturated NH ₄ Cl was added and the reaction mixture was extracted with 3x EtOAc. The organic layer was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a brown oil (82%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.05 (q, J=7.1 Hz, 2H), 2.65 (t, J=6.6 Hz, 2H), 2.49 (d, J =13.1 Hz, 2H), 2.30 (d, J=6.8 Hz, 2H), 1.82-1.71 (m, J=6.5 Hz, 1H), 1.33-1 .13 (m, 7H), 0.78 (t, J=7.4 Hz, 6H).

Intermediate 103c: 6-ethyl-4-oxooctanoic acid

NaOH (1.5 equiv.), LiOH (1.5 equiv.) were added to a solution of intermediate 103c (1.0 equiv.) in 3:1 EH/H ₂ O (0.2 M). The mixture was stirred at 40°C for at least 1 hour. Upon completion, the pH of the reaction was adjusted to 3 with 1 M HCl, the reaction mixture was diluted with H ₂ O and extracted with 3x EtOAc. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a yellow oil (46%).

1H NMR (400 MHz, CDCl ₃ ) δ 2.69-2.60 (m, 2H), 2.56 (t, J=6.6 Hz, 2H), 2.30 (d, J=6.7 Hz, 2H), 1.75 (hept, J=6.4 Hz, 1H), 1.22 (qp, J=13.8, 6.6 Hz, 4H), 0.78 (t, J=7 ,4 Hz, 6H).

Intermediate 103d: 2-((6-ethyl-4-oxooctanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate)

To a solution of intermediate 103c (1.0 equiv) in DCM (0.2-0.5 M) was added intermediate 1c (1.0 equiv), EDCI (1.2 equiv), DMAP (0.1 -0.2 equiv.) and Et ₃ N (1.5 equiv.) at 0°C in an N2 atmosphere. The mixture was stirred at 20-25°C for at least 12 hours under N2 atmosphere. The mixture was diluted with H ₂ O and extracted with 3x EtOAc. The combined organic phase was dried with anhydrous Na ₂ SO ₄ , filtered and the filtrate was concentrated in vacuo. The residue was purified by column chromatography to give the product as a yellow oil (40%).

1H NMR (400 MHz, CDCl ₃ ) δ 5.40-5.21 (m, 8H), 5.18 (p, J=5.0 Hz, 1H), 4.23 (dt, J=11.9 , 5.0 Hz, 2H), 4.08 (dd, J=11.9, 5.9 Hz, 2H), 2.67 (dt, J=20.4, 6.5 Hz, 6H), 2 .51 (td, J=6.5, 2.9 Hz, 2H), 2.32-2.22 (m, 5H), 1.98 (q, J=6.8 Hz, 8H), 1, 75 (p, J=6.4 Hz, 1H), 1.53 (q, J=7.2 Hz, 4H), 1.35-1.09 (m, 31H), 0.80 (dt, J =16.7, 6.9 Hz, 12H).

Intermediate 103e: 2-((6-ethyl-4-(((4-nitrophenoxy)carbonyl)oxy)octanoyl)oxy)propan-1,3-diyl (9Z,9'Z,12Z,12'Z)- bis(octadeca-9,12-dienoate)

Intermediate 103e was synthesized in 46% yield from intermediate

- 161 046244 nia 103 d using the method applied for intermediate 1e.

1H NMR (400 MHz, CDCl ₃ ) δ 8.21 (d, J=8.1 Hz, 2H), 7.32 (d, J=8.4 Hz, 2H), 5.27 (tt, J= 14.1, 7.8 Hz, 8H), 4.89 (p, J=7.4 Hz, 1H), 4.32-4.02 (m, 4H), 2.70 (t, J=6 .5 Hz, 4H), 2.41 (t, J=7.5 Hz, 2H), 2.32-2.19 (m, 4H), 1.98 (q, J=7.2 Hz, 10H ), 1.69-1.40 (m, 7H), 1.39-1.16 (m, 36H), 0.82 (t, J=6.9 Hz, 12H).

Compound 103: 2-((6-ethyl-4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)octanoyl)oxy)propane1,3-diyl (9Z,9'Z, 12Z, 12 '7)-bis(octadeca-9,12-dienoate)

Compound 103 was synthesized in 98% yield from intermediate 103e and N'ethyl-N-methylethane-1,2-diamine using the method used in intermediate 91d.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.42-5.21 (m, 8H), 5.18 (q, J=5.0 Hz, 1H), 5.07 (s, 1H), 4 .79 (s, 1H), 4.21 (ddd, J=11.8, 4.4, 1.8 Hz, 2H), 4.08 (ddd, J=11.9, 5.8, 3, 4 Hz, 2H), 3.17 (d, J=6.2 Hz, 2H), 2.70 (t, J=6.4 Hz, 4H), 2.44-2.27 (m, 6H) , 2.24 (t, J=7.6 Hz, 4H), 2.13 (s, 3H), 1.98 (q, J=6.8 Hz, 8H), 1.84 (dq, J= 8.4, 4.4 Hz, 1H), 1.71 (dd, J=14.8, 7.5 Hz, 1H), 1.58-1.43 (m, 8H), 1.29-1 .16 (m, 38H), 0.96 (d, J=7.2 Hz, 3H), 0.80 (ddd, J=13.4, 6.7, 3.7 Hz, 13H). MS: 916.3 m/z [M+H].

Example 104.

Intermediate 104a: (3,7-dimethyloctyl)magnesium bromide

Intermediate 104a was synthesized from 1-bromo-3,7-dimethyloctane using the same method as intermediate 104a.

Intermediate 104b: Ethyl 7,11-dimethyl-4-oxododecanoate

Intermediate 104b was synthesized in 66% yield from intermediate 104a using the same method as intermediate 103b.

1H NMR (400 MHz, CDCl ₃ ) δ 4.11 (qd, J=7.1, 2.5 Hz, 2H), 2.71 (t, J=6.6 Hz, 2H), 2.56 ( t, J=6.6 Hz, 2H), 2.49-2.35 (m, 2H), 1.68-1.54 (m, 1H), 1.49 (dq, J=13.2, 6.6 Hz, 1H), 1.43-1.31 (m, 2H), 1.24 (td, J=8.0, 7.1, 3.6 Hz, 6H), 1.10 (dddd , J=16.9, 9.9, 4.7, 2.0 Hz, 3H), 0.84 (d, J=6.5 Hz, 9H).

Intermediate 104c: 7,11-dimethyl-4-oxododecanoic acid

Intermediate 104c was synthesized in 51% yield from intermediate 104b using the same method as intermediate 103c.

1H NMR (400 MHz, CDCl ₃ ) δ 2.72 (t, J=6.1 Hz, 2H), 2.63 (t, J=6.5 Hz, 2H), 2.50-2.36 ( m, 2H), 1.61 (ddt, J=11.8, 9.0, 4.3 Hz, 1H), 1.51 (hept, J=6.6 Hz, 1H), 1.45-1 .34 (m, 2H), 1.24 (ddt, J=16.2, 13.5, 4.2 Hz, 4H), 1.11 (ddt, J=16.0, 9.6, 5, 9 Hz, 3H), 0.91-0.81 (m, 9H).

Intermediate 104d: 2-((7,11-dimethyl-4-oxododecanoyl)oxy)propan-1,3-diyl

Intermediate 104d was synthesized in 58% yield from intermediate 104c using the same method as intermediate 103d.

- 162 046244

1H NMR (400 MHz, CDC13) δ 5.41-5.28 (m, 8H), 5.24 (ddd, J=10.2, 5.8, 4.3 Hz, 1H), 4.28 ( dd, J=11.9, 4.4 Hz, 2H), 4.14 (dd, J=11.9, 5.8 Hz, 2H), 2.74 (dt, J=17.5, 6, 5 Hz, 6H), 2.58 (t, J=6.5 Hz, 2H), 2.48-2.37 (m, 2H), 2.31 (t, J=7.6 Hz, 4H) , 2.04 (q, J=6.9 Hz, 8H), 1.60 (ddd, J=10.0, 5.6, 2.6 Hz, 6H), 1.54-1.47 (m , 1H), 1.42-1.19 (m, 34H), 1.10 (ddt, J=19.6, 10.0, 6.9 Hz, 4H), 0.93-0.80 (m , 15H).

Intermediate 104e: 2-((7,11-dimethyl-4-(((4-nitrophenoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diyl (9Z,9'Z, 12Z,12^) -bis(octadeca-9,12-dienoate)

Intermediate 104e was synthesized in 58% yield from intermediate 104d using the same method as intermediate 1e.

1H NMR (400 MHz, CDCl ₃ ) δ 8.27-8.11 (m, 2H), 7.36-7.29 (m, 2H), 5.36-5.16 (m, 9H), 4 .78 (ddq, J=12.5, 7.0, 3.4 Hz, 1H), 4.25 (dd, J=12.0, 4.3 Hz, 2H), 4.12-4.05 (m, 2H), 2.70 (t, J=6.3 Hz, 4H), 2.40 (t, J=7.5 Hz, 2H), 2.24 (tt, J=7.7, 4.0 Hz, 4H), 2.07-1.88 (m, 10H), 1.72-1.48 (m, 7H), 1.44 (ddd, J=13.3, 6.6, 1.3 Hz, 1H), 1.391.14 (m, 34H), 1.06 (dtd, J=8.6, 6.7, 4.7 Hz, 3H), 0.86-0.76 (m , 15H).

Example 105 - compound 105.

Compound 105: 2-((7,11-dimethyl-4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propan-1,3-diyl (9Z,9'Z , 12Z, 12^)-bis(octadeca-9,12-dienoate)

Compound 105 was synthesized in 62% yield from intermediate 104e and 2-pyrrolidin-1-ylethanamine using the same method as for intermediate 91d.

1H NMR (400 MHz, CDCl ₃ ) δ 5.38-5.22 (m, 8H), 5.22-5.15 (m, 1H), 5.08 (s, 1H), 4.67 (s , 1H), 4.22 (dd, J=12.0, 4.4 Hz, 2H), 4.08 (ddd, J=11.9, 5.9, 3.5 Hz, 2H), 3, 20 (t, J=6.0 Hz, 2H), 2.70 (t, J=6.4 Hz, 4H), 2.51 (t, J=6.1 Hz, 2H), 2.43 ( d, J=5.9 Hz, 4H), 2.32 (ddd, J=9.2, 6.7, 3.7 Hz, 2H), 2.25 (t, J=7.6 Hz, 4H ), 1.98 (q, J=6.8 Hz, 8H), 1.84 (d, J=7.5 Hz, 1H), 1.77-1.66 (m, 5H), 1.54 (p, J=7.1 Hz, 7H), 1.44 (p, J=6.6 Hz, 3H), 1.35-1.10 (m, 34H), 1.10-0.96 ( m, 5H), 0.86-0.74 (m, 15H). MS: 984.2 m/z [M+H].

Example 106 - compound 106.

Compound 106: 2-((6-ethyl-4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoyl)oxy)propane1,3-diyl (9Z,9'Z, 12Z, 12 ^)-bis(octadeca-9,12-dienoate)

Compound 106 was prepared in 78% yield from intermediate 103e and 2-pyrrolidin-1-ylethanamine using the same method as intermediate 91d.

1H NMR (400 MHz, CDCl ₃ ) δ 5.28 (qt, J=11.5, 6.0 Hz, 8H), 5.19 (q, J=5.0 Hz, 1H), 4.79 ( s, 1H), 4.22 (dd, J=12.2, 4.5 Hz, 2H), 4.08 (ddd, J=11.8, 5.8, 3.5 Hz, 2H), 3 .22 (q, J=5.9 Hz, 2H), 2.70 (t, J=6.4 Hz, 4H), 2.53 (t, J=6.2 Hz, 2H), 2.46 (s, 3H), 2.33 (dd, J=9.6, 7.0 Hz, 2H), 2.25 (t, J=7.6 Hz, 4H), 1.98 (q, J= 6.8 Hz, 7H), 1.89

- 163 046244

1.80 (m, 2H), 1.76-1.61 (m, 7H), 1.60-1.45 (m, 6H), 1.34-1.16 (m, 33H), 0. 87-0.72 (m, 12H). MS: 928.4 m/z [M+H].

Example 107 - compound 107.

Intermediate 107a: ^)-1-bromonon-3-ene

Br._ - To a solution of PPh ₃ (36.79 g, 1.33 eq.) in DCM (1 M) was added Br ₂ (7.23 ml, 1.33 eq.) at 0°C. (Z)-hoh3-en-1-ol (15 g, 1.0 equiv.) was added to the reaction mixture and the mixture was stirred at 0°C for 2 hours. The reaction mixture was quenched by adding H ₂ O at 0°C and three times extracted with EtOAc. The combined organic layers were concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a colorless oil (15 g, 69%).

^1H NMR (400 MHz, CDQ3) δ 5.47-5.30 (m, 2H), 3.64 (t, J=6.7 Hz, 2H), 2.18-2.06 (m, 2H ), 2.02 (q, J=6.7 Hz, 2H), 1.77 (s, 1H), 1.62 (p, J=6.9 Hz, 2H), 1.38-1.20 (m, 6H), 0.87 (t, J=6.8 Hz, 3H).

Intermediate 107b: ^)-non-3-en-1-ylmagnesium bromide

To a suspension of Mg (2.13 g, 1.2 eq.) in THF (150 ml) was added I ₂ (1.86 g, 0.1 eq.). Intermediate 107a (15 g, 1.0 equiv) was then added to the mixture and the mixture was stirred at 45°C for 2 hours. The reaction mixture was used directly in the next step without further purification.

Intermediate 107c: ethyl ^)-4-oxotridec-7-enoate

ABOUT

EY ₂ S

Intermediate 107c was synthesized in 75% yield from intermediate 107b using the same method as intermediate 103b.

1H NMR (400 MHz, CDQ3) δ 5.43-5.23 (m, 2H), 4.10 (qd, J=7.1, 3.2 Hz, 3H), 2.69 (t, J= 6.6 Hz, 2H), 2.55 (t, J=6.6 Hz, 2H), 2.47 (t, J=7.4 Hz, 2H), 2.30 (q, J=7, 3 Hz, 2H), 2.01-1.95 (m, 2H), 1.34-1.18 (m, 11H), 0.86 (t, J=6.8 Hz, 3H).

Intermediate 107d: ^)-4-oxotridec-7-enoic acid

Intermediate 107d was synthesized in 80% yield from intermediate 107c using the same method as for intermediate 103c.

1H NMR (400 MHz, CDQ3) δ 5.23 (ddt, J=34.8, 17.4, 7.8 Hz, 2H), 4.27-3.96 (m, 2H), 2.58 ( t, J=6.1 Hz, 2H), 2.38 (t, J=7.4 Hz, 2H), 2.31-2.08 (m, 4H), 1.92 (q, J=6 ,9 Hz, 2H), 1.75 (s, 2H), 1.21 (dq, J=15.0, 9.2, 8.0 Hz, 6H), 0.80 (t, J=6, 7 Hz, 3H).

Intermediate 107e: 2-((^)-4-oxotridec-7-enoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12- dienoate)

Intermediate 107e was synthesized in 80% yield from intermediate 107d and intermediate 1c using the method used in intermediate 104c.

1H NMR (400 MHz, CDQ3) δ 5.35-5.22 (m, 9H), 5.21-5.15 (m, 1H), 4.23 (dt, J=11.9, 4.8 Hz, 2H), 4.124.01 (m, 4H), 2.76-2.67 (m, 4H), 2.65 (t, J=6.5 Hz, 2H), 2.55-2.49 (m, 2H), 2.42 (t, J=7.5 Hz, 2H), 2.25 (t, J=7.6 Hz, 6H), 1.98 (q, J=7.2 Hz , 13H), 1.54 (p, J=7.6 Hz, 4H), 1.32-1.18 (m, 37H), 0.82 (td, J=7.0, 1.7 Hz, 9H).

Intermediate 107f: 2-((^)-4-(((4-nitrophenoxy)carbonyl)oxy)tridec-7enoyl)oxy)propane-1,3-diyl (9Z,9'Z,12Z,12'Z) -bis(octadeca-9,12-dienoate)

- 164 046244

Intermediate 107f was synthesized in 21% yield from intermediate 107e using the same method as for intermediate 1e.

1H NMR (400 MHz, CDCl ₃ ) δ 8.48-8.20 (m, 2H), 7.56-7.32 (m, 2H), 5.45-5.20 (m, 10H), 4 .88 (tt, J=7.9, 4.6 Hz, 1H), 4.30 (ddd, J=12.1, 10.3, 4.5 Hz, 2H), 4.15 (ddd, J =11.9, 5.8, 2.1 Hz, 2H), 2.74 (dt, J=19.6, 6.5 Hz, 5H), 2.59 (t, J=6.5 Hz, 1H), 2.48 (q, J=7.1 Hz, 2H), 2.41-2.24 (m, 5H), 2.15 (q, J=7.7 Hz, 1H), 2, 09-1.96 (m, 10H), 1.61 (q, J=12.7, 9.4 Hz, 5H), 1.39-1.20 (m, 30H), 0.99-0, 69 (m, 9H).

Compound 107: 2-((^)-4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)tridec-7enoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z,12'Z)-bis(octadeca-9,12-dienoate)

Compound 107 was synthesized in 56% yield from intermediate 107f and N'-ethylN-methylethane-1,2-diamine using the same method as for intermediate 91d.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.41-5.29 (m, 9H), 5.24 (p, J=5.0 Hz, 1H), 5.16 (d, J=5, 3 Hz, 1H), 4.81-4.74 (m, 1H), 4.28 (dd, J=11.9, 4.4 Hz, 2H), 4.14 (ddd, J=11.9 , 5.8, 3.3 Hz, 2H), 3.23 (q, J=5.9 Hz, 2H), 2.76 (t, J=6.4 Hz, 4H), 2.48-2 .36 (m, 6H), 2.31 (t, J=7.6 Hz, 4H), 2.20 (s, 3H), 2.02 (dq, J=20.4, 7.0 Hz, 12H), 1.96-1.87 (m, 2H), 1.81 (dd, J=14.7, 7.3 Hz, 1H), 1.65-1.55 (m, 7H), 1 .39-1.22 (m, 35H), 1.03 (t, J=7.1 Hz, 3H), 0.88 (td, J=7.0, 2.3 Hz, 9H). MS: 955.3 m/z [M+H].

Example 108 - compound 108.

Compound 108: 2-((^)-4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)tridec-7enoyl)oxy)propan-1,3-diyl (9Z,9'Z, 12Z,12'Z)-bis(octadeca-9,12-dienoate)

Compound 108 was synthesized in 51% yield from intermediate 107f and 2-pyrrolidin-1-ylethanamine using the same method as for intermediate 91d.

1H NMR (400 MHz, CDCl ₃ ) δ 5.43-5.26 (m, 9H), 5.24 (q, J=5.1 Hz, 1H), 4.78 (tt, J=8.6 , 4.6 Hz, 1H), 4.28 (dd, J=11.9, 4.4 Hz, 2H), 4.14 (ddd, J=11.9, 5.8, 3.5 Hz, 2H), 3.29 (q, J=5.9 Hz, 2H), 2.76 (t, J=6.4 Hz, 4H), 2.66-2.50 (m, 5H), 2, 39 (ddd, J=9.6, 6.7, 3.6 Hz, 2H), 2.31 (t, J=7.6 Hz, 4H), 2.01 (dq, J=20.4, 7.1 Hz, 12H), 1.87-1.72 (m, 5H), 1.59 (q, J=7.3 Hz, 6H), 1.40-1.15 (m, 31H), 0.88 (td, J=6.9, 2.2 Hz, 9H). MS: 967.3 m/z [M+H].

Example 109 - compound 109.

Intermediate 109a: 3-((tert-butyldimethylsilyl)oxy)-2-oxopropyl (9Z,12Z)octadeca-9,12-dienoate

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Intermediate 109a was synthesized in 63% yield from intermediate 62a and linoleic acid using the method used for intermediate 62b.

1H NMR (400 MHz, CDCl ₃ ) δ 5.31-5.19 (m, 4H), 4.84 (s, 2H), 4.16 (s, 2H), 2.67 (t, J=6 .7 Hz, 2H), 2.32 (t, J=7.5 Hz, 2H), 1.94 (q, J=6.9 Hz, 4H), 1.57 (q, J=7.4 Hz, 2H), 1.31-1.13 (m, 15H), 0.80 (d, J=15.1 Hz, 12H).

Intermediate 109b: 3-hydroxy-2-oxopropyl (92,122)-octadeca-9,12-dienoate

HF-pyridine (5.0 eq) was added to a solution of intermediate 109a (1.0 eq) in THF (180 mL). The mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove THF. The residue was diluted with H ₂ O and extracted with 3x EtOAc. The combined organic layers were dried over Na ₂ SO ₄ , filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the product as a colorless oil (66%).

1H NMR (400 MHz, CDCl ₃ ) δ 5.33 (dtd, J=12.5, 10.3, 9.5, 5.6 Hz, 4H), 4.74 (s, 1H), 4.36 (s, 1H), 2.76 (t, J=6.4 Hz, 2H), 2.41 (t, J=7.5 Hz, 1H), 2.37-2.27 (m, 1H) , 2.03 (q, J=6.9 Hz, 4H), 1.65 (p, J=7.1 Hz, 2H), 1.411.24 (m, 14H), 0.87 (t, J= 6.7 Hz, 3H).

Intermediate 109c: 3-(oleoyloxy)-2-oxopropyl (92,122)-octadeca-9,12-dienoate

Intermediate 109c was synthesized in 50% yield from intermediate 109b and oleic acid using the same method as 52.

¹ H NMR (400 MHz, CDQ3) δ 5.37-5.22 (m, 6H), 4.68 (s, 4H), 2.70 (t, J=6.4 Hz, 2H), 2, 35 (t, J=7.5 Hz, 4H), 2.02-1.91 (m, 8H), 1.60 (q, J=7.3 Hz, 4H), 1.33-1.17 (m, 33H), 0.81 (dt, J=7.0, 3.3 Hz, 6H).

Intermediate 109d: 2-hydroxy-3-(oleoyloxy)propyl(92,122)-octadeca-9,12-dienoate

Intermediate 109d was synthesized in 50% yield from intermediate 109c using the same method as intermediate 1c.

1H NMR (400 MHz, CDQ3) δ 5.37-5.21 (m, 6H), 4.15-4.00 (m, 4H), 2.70 (t, J=6.3 Hz, 2H) , 2.28 (t, J=7.6 Hz, 4H), 2.04-1.89 (m, 8H), 1.56 (t, J=7.4 Hz, 4H), 1.23 ( dt, J=13.9, 7.0 Hz, 34H), 0.82 (td, J=6.8, 3.8 Hz, 6H).

Compound 109: 3-(oleoyloxy)-2-((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoyl)oxy)propyl(92,122)-octadeca-9,12-dienoate

Compound 109 was synthesized from intermediate 109d and intermediate 85c using the same method as 85.

1H NMR (400 MHz, CDCi ₃ ) δ 5.35 (tdd, J=8.8, 6.5, 3.6 Hz, 5H), 5.28-5.22 (m, 1H), 5.16 (s, 1H), 4.29 (dd, J=11.9, 4.4 Hz, 2H), 4.15 (ddd, J=11.9, 5.8, 3.3 Hz, 2H), 3.34-3.22 (m, 2H), 2.77 (t, J=6.5 Hz, 2H), 2.55 (d, J=30.6 Hz, 5H), 2.39 (ddd , J=8.8, 6.6, 3.8 Hz, 2H), 2.31 (t, J=7.6 Hz, 4H), 2.09-1.97 (m, 7H), 1, 91 (d, J=7.2 Hz, 1H), 1.78 (d, J=6.0 Hz, 4H), 1.61 (t, J=7.2 Hz, 6H), 1.30 ( td, J=10.5, 9.4, 4.7 Hz, 30H), 0.91-0.87 (m, 6H). MS:

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902.3 m/z [M+H].

Example 110 - compound 110.

Intermediate 110a: 2-oxopropane-1,3-diyl dioleate

DMAP (1.1 equiv) and EDCI (1.0 equiv) were added to a solution of 1,3-dihydroxypropan-2-one (5 g, 0.5 equiv) in DCM (300 ml). Oleic acid (1.0 equiv) was then added to the above mixture and stirred at 25°C for 12 hours. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was diluted with H ₂ O and extracted with 3x EtOAc. The combined organic layers were dried over Na ₂ SO ₄ , filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the product as a colorless oil (8 g, 12%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.34-5.21 (m, 4H), 4.68 (s, 4H), 2.35 (t, J=7.5 Hz, 4H), 1 .94 (q, J=6.4 Hz, 8H), 1.60 (q, J=7.4 Hz, 4H), 1.31-1.14 (m, 38H), 0.89-0, 75 (m, 6H).

Intermediate 110b: 2-hydroxypropane-1,3-diyl dioleate

Intermediate 110b was synthesized in 47% yield from intermediate 110a using a method similar to that used for intermediate 1c.

1H NMR (400 MHz, CDCl ₃ ) δ 5.41-5.26 (m, 4H), 4.22-4.06 (m, 4H), 2.34 (t, J=7.6 Hz, 4H ), 2.01 (q, J=6.4 Hz, 7H), 1.63 (p, J=7.2 Hz, 4H), 1.28 (d, J=14.2 Hz, 33H), 0.90-0.81 (m, 6H).

Compound 110: 2-((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoyl)oxy)propane-1,3-diyl dioleate

Compound 110 was synthesized from intermediate 110b and intermediate 85c using the same method as compound 85.

1H NMR (400 MHz, CDCl ₃ ) δ 5.33-5.22 (m, 4H), 5.22-5.15 (m, 1H), 5.08 (s, 1H), 4.69 (s , 1H), 4.28-4.17 (m, 2H), 4.08 (ddq, J=12.2, 6.4, 2.9 Hz, 2H), 3.21 (d, J=6 .8 Hz, 2H), 2.51 (t, J=6.2 Hz, 2H), 2.43 (d, J=6.0 Hz, 4H), 2.32 (ddd, J=8.6 , 6.6, 3.7 Hz, 2H), 2.24 (t, J=7.6 Hz, 4H), 1.94 (q, J=6.7 Hz, 8H), 1.87-1 .78 (m, 1H), 1.70 (p, J=3.1 Hz, 5H), 1.61-1.36 (m, 9H), 1.22 (d, J=13.2 Hz, 45H), 0.82 (td, J=6.9, 3.7 Hz, 9H). MS: 904.2 m/z [M+H].

Example 111 - connection 111.

Compound 111-3-((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoyl)oxy)propan-1,2-diyl (9Z,9'Z,12Z,12'Z )-bis(octadeca-9,12-dienoate)

Compound 111 was synthesized from intermediate 85c and intermediate 30a using the same method as 85.

1H NMR (400 MHz, CDCl ₃ ) δ 5.43-5.27 (m, 8H), 5.24 (t, J=5.2 Hz, 1H), 5.14 (s, 1H), 4, 74 (s, 1H), 4.27 (ddd, J=12.2, 8.2, 4.2 Hz, 2H), 4.18-4.09 (m, 2H), 3.27 (q, J=5.9 Hz, 2H), 2.75 (t, J=6.4 Hz, 4H), 2.57 (t, J=6.2 Hz, 2H), 2.50 (s, 4H) , 2.37 (td, J=8.9, 7.8, 3.5 Hz, 2H), 2.30 (td, J=7.5, 4.4 Hz, 4H), 2.03 (q , J=6.9 Hz, 8H), 1.981.86 (m, 2H), 1.76 (h, J=3.1 Hz, 5H), 1.59 (tt, J=9.8, 5, 1 Hz, 7H), 1.38-1.21 (m, 30H), 0.87 (t, J=6.7 Hz, 9H). MS: 900.1 m/z [M+H].

Example 112 - connection 112.

Compound 112: 2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoyl)oxy)methyl)propane1,3-diyl (9Z,9'Z, 12Z, 12^ )-bis(octadeca-9,12-dienoate)

Compound 112 was synthesized from intermediate 86c and intermediate 53a using the same method as 84.

1H NMR (400 MHz, CDCl ₃ ) δ 5.44-5.28 (m, 8H), 5.19 (s, 1H), 4.75 (s, 1H), 4.12 (d, J=5 .9 Hz, 6H), 3.29 (q, J=6.1 Hz, 2H), 2.77 (t, J=6.4 Hz, 4H), 2.64-2.49 (m, 6H ), 2.38 (ddd, J=10.1, 8.5, 4.8 Hz, 3H), 2.30 (t, J=7.6 Hz, 4H), 2.05 (q, J= 6.8 Hz, 8H), 1.90 (s, 2H), 1.86-1.72 (m, 6H), 1.61 (t, J=7.3 Hz, 7H), 1.32 ( tq, J=7.6, 4.9, 3.7 Hz, 33H), 0.89 (t, J=6.7 Hz, 9H). MS: 914.1 m/z [M+H].

Example 113 - compound 113.

Intermediate 113a: benzyl 3-hydroxyheptanoate

Zn was poured into HCl (1 M) and stirred for 30 min. The mixture was filtered and the precipitate was washed with EtOH. The solid was dried under reduced pressure to obtain freshly prepared Zn. To a suspension of Zn (15.18 g, 2.0 eq.) in THF (50 ml), pentanal (1.0 eq.) and benzyl 2-bromoacetate (1.5 eq.) were added dropwise at 66°C for 0 ,5 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was diluted with H ₂ O and extracted with 3x EtOAc. The combined organic layers were concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a colorless oil (10 g, 36%).

1H NMR (400 MHz, CDCl ₃ ) δ 7.34 (d, J=4.1 Hz, 5H), 5.14 (s, 2H), 4.01 (tt, J=8.4, 3.6 Hz, 1H), 2.87 (s, 1H), 2.59-2.41 (m, 2H), 1.57-1.47 (m, 1H), 1.43 (qd, J=9, 6, 8.9, 4.6 Hz, 2H), 1.37-1.24 (m, 4H), 0.93-0.87 (m, 3H).

Intermediate 113b: Benzyl 3-(((4-nitrophenoxy)carbonyl)oxy)heptanoate

To a solution of intermediate 113a (8 g, 1.0 equiv.) and (4-nitrophenyl)carbonochloridate (2.0 equiv.) in DCM (10 ml) was added pyridine (3.0 equiv.) at 0°C. The mixture was stirred at 20°C for 2 hours under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was diluted with H ₂ O and extracted with 3x EtOAc. The combined organic layers were concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a colorless oil (8 g, 59% yield).

1H NMR (400 MHz, CDCl3) δ 8.30-8.21 (m, 2H), 7.39-7.28 (m, 7H), 5.16 (d, J=3.3 Hz, 2H) , 2.82-2.69 (m, 2H), 1.81-1.68 (m, 2H), 1.43-1.30 (m, 5H), 0.94-0.89 (m, 3H).

Intermediate 113c: Benzyl 3-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)heptanoate

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DMAP (0.1 equiv) and pyridine ( 3.0 equiv.) at 0°C. The mixture was stirred at 20°C for 2 hours. The reaction mixture was concentrated under reduced pressure to remove MeCN. The residue was diluted with H ₂ O and extracted with 3x EtOAc. The combined organic layers were concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a colorless oil (0.4 g, 85%).

1H NMR (400 MHz, CDCl ₃ ) δ 7.36-7.20 (m, 5H), 5.05 (s, 2H), 3.17 (dt, J=13.3, 6.4 Hz, 2H ), 2.56 (td, J=13.2, 11.4, 6.3 Hz, 2H), 2.50-2.37 (m, 6H), 1.72-1.63 (m, J =4.0, 3.5 Hz, 4H), 1.49 (s, 2H), 1.28-1.20 (m, 4H), 0.81 (q, J=4.2, 3.2 Hz, 3I).

Intermediate 113d: 3-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)heptanoic acid

Pd/C (10% w/w) was added to a solution of intermediate 113c (3.5 g, 1.0 equiv) in THF (10 mL). The mixture was purged 3 times with H ₂ and then the mixture was stirred at 20°C for 12 hours under an H ₂ atmosphere. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give the product as a colorless oil (2 g, 75%).

1H NMR (400 MHz, CDCl3) δ 10.00 (s, 1H), 6.46 (t, J=6.0 Hz, 1H), 5.02 (dt, J=10.0, 5.2 Hz , 1H), 3.43-3.25 (m, 3H), 2.92 (dt, J=21.7, 6.5 Hz, 6H), 2.37 (qd, J=14.9, 6 ,4 Hz, 2H), 1.87 (q, J=4.4, 2.9 Hz, 4H), 1.64-1.45 (m, 2H), 1.24 (h, J=7, 7, 6.7 Hz, 4H), 0.88-0.75 (m, 3H).

Compound 113: 2-(((3-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)heptanoyl)oxy)methyl)propane1,3-diyl (9Z,9'Z, 12Z,12^ )-bis(octadeca-9,12-dienoate)

Compound 113 was synthesized from intermediate 113c and intermediate 53a using the method used for compound 52.

1H NMR (400 MHz, CDCl ₃ ) δ 5.37-5.18 (m, 8H), 5.00 (q, J=6.4 Hz, 1H), 4.06 (p, J=6.5 , 6.1 Hz, 6H), 3.21 (p, J=7.3, 6.8 Hz, 2H), 2.70 (t, J=6.5 Hz, 4H), 2.55-2 .39 (m, 7H), 2.32 (p, J=6.0 Hz, 1H), 2.23 (t, J=7.6 Hz, 4H), 1.98 (q, J=6, 8 Hz, 7H), 1.76-1.63 (m, 5H), 1.54 (t, J=7.3 Hz, 6H), 1.33-1.15 (m, 30H), 0, 82 (t, J=6.7 Hz, 9H). MS: 900.1 m/z [M+H].

Example 114 - Comparative Compound 114.

Compound 114: 2-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)bis(octadeca-9,12 -dienoate)

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To a solution of intermediate 1c (250 mg, 1.0 equiv) in DCM (2 ml) was added pyridine (3.0 equiv) and 4-nitrophenyl chloroformate (1.1 equiv). The reaction mixture was stirred at 25°C for 2 hours, after which the reaction was quenched by adding water and extracted 3 times with DCM. The organic layers were dried over Na ₂ SO ₄ , filtered and concentrated. The resulting crude residue was reconstituted in MeCN (2 ml) followed by the addition of pyridine (3.0 equiv), Na-ethyl-N1-methylethane1,2-diamine (1.5 equiv) and DMAP (1.0 equiv). The reaction mixture was stirred at 25°C for 16 hours. Heptane was then added to the reaction mixture and the heptane layer was washed 3 times with MeCN. The combined MeCN layers were extracted three times with heptane, and the combined heptane layers were washed one final time with MeCN. The heptane was then removed in vacuo and the crude material was purified by column chromatography (MeOH/DCM) to give the product as a colorless oil (138 mg, 46%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.49-5.27 (m, 8H), 5.14 (p, J=5.0 Hz, 1H), 4.33-4.12 (m, 4H), 3.29 (q, J=5.7 Hz, 2H), 2.83-2.71 (m, 4H), 2.60-2.38 (m, 5H), 2.38-2 .16 (m, 7H), 2.04 (q, J=6.9 Hz, 8H), 1.69-1.56 (m, 4H), 1.42-1.22 (m, 28H), 1.07 (t, J=7.2 Hz, 3H), 0.96-0.85 (m, 6H).

Example 115 - Materials and methods.

LNP compositions.

LNPs were prepared using various amine lipids in a 4-component lipid system consisting of an ionizable lipid (eg, amine lipid), DSPC, cholesterol, and PEG-2k-DMG. In studies of percent liver change in mice, Cas9 mRNA and a chemically modified ssRNA targeting the mouse sequence were formulated in LNP at either a 1:1 w/w or 1:2 w/w ratio.

LNP composition - cross flow.

Lipid components were dissolved in 100% ethanol with the molar ratios of lipid components described below. Chemically modified ssRNA and Cas9 mRNA were combined and dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in a total cargo RNA concentration of approximately 0.45 mg/mL. LNPs were formulated with an N/P ratio of approximately 6, with a chemically modified ssRNA:Cas9 mRNA ratio of either 1:1 or 1:2 w/w, as described below.

LNPs were formed by mixing lipid in ethanol with two volumes of RNA solutions and one volume of water. The lipid in ethanol is mixed through a mixing cross with two volumes of RNA solution. A fourth water stream is mixed with the cross outlet stream through an integral tee, (see, for example, WO2016010840, Fig. 2.) The LNP was kept for 1 hour at room temperature and then diluted with water (approximately 1:1 v/v) . Diluted LNPs were concentrated using tangential flow filtration on a flat cartridge (Sartorius, 100 kDa MWCO) and then buffer exchanged by diafiltration into 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7.5 ( TSS). Alternatively, final buffer exchange in the TSS was completed using PD-10 desalting columns (GE). If necessary, the compositions were concentrated by centrifugation on Amicon 100 kDa centrifugal filters (Millipore). The resulting mixture was then filtered using a sterile 0.2 μm filter. The final LNP was stored at 4°C or -80°C until further use.

LNP composition analytics.

Dynamic light scattering (DLS) is used to characterize the polydispersity index (pdi) and size of the LNPs of the present invention. DLS measures the scattering of light that occurs when a sample is exposed to a light source. The PDI, as determined from DLS measurements, is the particle size distribution (around the mean particle size) in a population with a perfectly homogeneous population having a PDI of zero.

Electropheretic light scattering is used to characterize the surface charge of LNPs at a given pH. Surface charge or zeta potential is a measure of the magnitude of electrostatic repulsion/attraction between particles in an LNP suspension.

Asymmetric Flow Field Flow Fractionation - Multi-Angle Light Scattering (AF4MALS) is used to separate particles in a formulation by hydrodynamic radius and then measure the molecular weights, hydrodynamic radii, and root mean square radii of the fractionated particles. This allows estimation of molecular weight and size distribution, as well as secondary characteristics such as the Burchard-Stockmeyer plot (the ratio of the root mean square (rms) radius to the hydrodynamic radius over time, assuming the internal core density of the particle) and the root mean square conformation plot (the logarithm of the root mean square radius over compared to the logarithm of molecular weight, where the slope of the resulting linear fit gives the degree of compactness versus elongation).

Nanoparticle tracking analysis (NTA, Malvern Nanosight) can be used to determine particle size distribution as well as particle concentration. LNP samples are appropriately diluted and injected onto a microscope slide. The camera records scattered light as particles slowly move through the field of view. Once a video segment is captured, Nanoparticle Trajectory Analysis processes the video segment by tracking pixels and calculating the diffusion coefficient. This diffusion coefficient can be converted into the hydrodynamic radius of the part

- 170 046244 tsy. The instrument also counts the number of individual particles counted in the assay to determine particle concentration.

Cryo-electron microscopy (cryo-EM) can be used to determine the particle size, morphology and structural characteristics of LNPs.

Lipid composition analysis of LNPs can be determined using liquid chromatography followed by charged aerosol detection (LC-CAD). This analysis can provide a comparison of actual lipid content with theoretical lipid content.

LNP formulations were analyzed for average particle size, polydispersity index (pdi), total RNA content, RNA encapsulation efficiency, and zeta potential. LNP compositions can be further characterized using lipid analysis, AF4-MALS, NTA and/or cryo-EM. Average particle size and polydispersity are measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument. LNP samples were diluted in PBS buffer before measurement by DLS. Z-average diameter, which is an intensity-based measurement of average particle size, was presented along with number-average diameter and pdi. The Malvern Zetasizer instrument is also used to measure the zeta potential of LNP. Before measurement, samples are diluted 1:17 (50 µl per 800 µl) in 0.1X PBS, pH 7.4.

A fluorescence-based assay (Ribogreen®, ThermoFisher Scientific) is used to determine total and free RNA concentrations. Encapsulation efficiency is calculated as (Total RNA - Free RNA)/Total RNA. LNP samples are appropriately diluted with 1x TE buffer containing 0.2% Triton-X 100 for total RNA determination or 1x TE buffer for free RNA determination. Standard curves are prepared using the RNA stock solution used to prepare the formulations and diluting it in 1x TE ±0.2% Triton-X 100 buffer. The diluted RiboGreen® dye (according to the manufacturer's instructions) is then added to each of the standards and samples and incubated for approximately 10 min at room temperature in the absence of light. A SpectraMax M5 microplate reader (Molecular Devices) is used to read samples with excitation, auto-cutoff, and emission wavelengths set to 488 nm, 515 nm, and 525 nm, respectively. Total RNA and free RNA were determined from the corresponding standard curves.

Encapsulation efficiency is calculated as (Total RNA - Free RNA)/Total RNA. The same procedure can be used to determine the encapsulation efficiency of a DNA-based cargo component. For single-stranded DNA, you can use Oligreen dye, and for double-stranded DNA, you can use Picogreen dye.

AF4-MALS is used to analyze molecular weight and size distributions and secondary statistics of these calculations. LNPs are diluted appropriately and injected into the AF4 separation channel using an HPLC autosampler, where they are focused and then eluted with an exponential cross-flow gradient through the channel. All fluid is driven by an HPLC pump and a Wyatt Eclipse instrument. Particles exiting channel AF4 pass through a UV detector, a multi-angle light scattering detector, a quasi-elastic light scattering detector, and a differential refractive index detector. The raw data is processed using the Debye model to determine the molecular mass and root mean square radius from the detector signals.

Lipid components in LNPs are quantitatively analyzed using HPLC coupled with a charged aerosol detector (CAD). Chromatographic separation of the 4 lipid components is achieved by reverse phase HPLC. CAD is a destructive mass detector that detects all non-volatile compounds and the signal remains the same regardless of the structure of the analyte.

Cargo mRNA Cas9 and gRNA.

Cargo Cas9 mRNA was produced by in vitro transcription. Capped and polyadenylated Cas9 mRNA containing 1x NLS (SEQ ID NO: 3) or the sequence from table. 24 of document PCT/US2019/053423 (which is incorporated herein by reference), was produced by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. For example, plasmid DNA containing the T7 promoter and a 100 nucleotide poly(A/T) region can be linearized by incubation at 37°C for 2 hours with XbaI under the following conditions: 200 ng/μl plasmid, 2 units/ µl XbaI. (NEB) and 1x reaction buffer. XbaI can be inactivated by heating the reaction mixture at 65°C for 20 minutes. The linearized plasmid can be purified from enzymes and buffer salts using a silica gel spin column (Epoch Life Sciences) and analyzed on an agarose gel to confirm linearization. The IVT reaction to generate modified Cas9 mRNA can be carried out by incubation at 37°C for 4 hours under the following conditions: 50 ng/μl linearized plasmid; 2 mM each of GTP, ATP, CTP, and Na-methyl pseudo-UTP (Trilink); 10 mM ARCA (Trilink); 5 U/μl T7 RNA polymerase (NEB); 1 U/μl murine RNase inhibitor (NEB); 0.004 U/μL inorganic E. coli pyrophosphatase (NEB);

- 171 046244 and 1x reaction buffer. After a 4-hour incubation, TURBO DNase (ThermoFisher) was added to a final concentration of 0.01 U/μl, and the reaction mixture was incubated for an additional 30 minutes to remove template DNA. Cas9 mRNA was purified by LiCl precipitation.

The sgRNAs in the following examples were chemically synthesized.

LNP In Vivo Delivery.

Female CD-1 mice aged 6–10 weeks were used in each study. Animals were weighed and grouped according to body weight to prepare dosage solutions based on group average weight. LNP was administered via the lateral tail vein in a volume of 0.2 ml per animal (approximately 10 ml per kilogram of body weight). Animals were monitored periodically for side effects for at least 24 hours after dosing.

For studies measuring in vivo editing in the liver, female CD-1 mice were dosed at 0.1 mg/kg body weight unless otherwise stated. Animals were sacrificed after 6 or 7 days by exsanguination via cardiac puncture under isoflorane anesthesia. Liver tissue was collected from each animal for DNA extraction and analysis. Blood was collected into serum separator tubes or into tubes containing buffered sodium citrate for plasma as described here. Cohorts of mice were measured for editing using next generation sequencing (NGS).

NGS sequencing.

Briefly, to quantify the efficiency of editing at a target region of the genome, genomic DNA was isolated and deep sequencing was used to detect the presence of insertions and deletions introduced by gene editing.

PCR primers were designed around the target site (eg, B2M), and the genomic region of interest was amplified. Additional PCR was performed according to the manufacturer's protocols (Illumina) to add the required chemistry for sequencing. Amplicons were sequenced on an Illumina MiSeq instrument. Reads were mapped to the corresponding reference genome (eg GRCm38) after excluding those with low quality scores. The resulting files containing reads were mapped to a reference genome (BAM files), where reads that overlapped the target region of interest were selected and the number of wild-type reads versus the number of reads containing an insertion, substitution, or deletion was calculated.

Editing percentage (eg, editing efficiency or editing percentage) is defined as the total number of sequence reads with insertions or deletions compared to the total number of sequence reads, including the wild type.

Transthyretin (TTR) ELISA assay.

Blood was collected and serum isolated as indicated. Total serum levels of mouse TTR were determined using a mouse prealbumin (transthyretin) ELISA kit (Aviva Systems Biology, Cat. OKIA00111). Briefly, sera were serially diluted with the kit's sample diluent, for example to a final dilution of 10,000-fold and/or 2500-fold. The diluted sample was then added to the ELISA plates and then assayed according to the instructions. Serum TTR data from treatment groups are expressed as a percentage of vehicle control levels.

Example 116 - Evaluation of Lipid Efficiency by In Vivo Editing.

The inventors evaluated the in vivo editing efficiency of materials supplied with formulations including various amino-lipid compounds. Editing was measured using either G282 (SEQ ID NO: 1), which targets the mouse TTR gene, or G650 (SEQ ID NO: 2), which targets the mouse B2M gene. The lipids described above were evaluated for effectiveness in in vivo editing experiments. LNPs were formulated at an sRNA to Cas9 mRNA ratio of 1:1 and at an N/P ratio of about 6. Molar lipid concentrations in the lipid component of LNPs are expressed as molar% amino lipid/DSPC/cholesterol/PEG-2k-DMG, e.g. 50/ 10/38.5/1.5. The final LNPs were characterized to determine encapsulation efficiency, polydispersity index, and average particle size according to the analytical methods outlined above. Analysis of average particle size, polydispersity (PDI), total RNA content and RNA encapsulation efficiency are shown in Table. 2. The pKa values of the following ionizable lipids were also measured and presented in table. 1.

In experiment 9, presented below, a series of compounds were tested that are based on compound 7 and that differ in the inclusion of either a C ₆ -cycloalkyl or a C _4-12 -alkyl at the R ³ position of formula (1A), as shown in FIG. 1A. As shown in table. 3 and in fig. 1B and 1C, all six compounds tested were effective for in vivo editing. Additional R ³ groups in the series of compounds based on compound 7 are also tested in Example 119 below.

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Table 2 Composition Analytics

Conn. No. Composition ratio (mol %) Encapsulation (%) Z-Ave Size(nm) PDI Num Ave Size(nm) Exp. 1 50/10/38.5/1.5 99 79.25 0.085 57.00 1 3 50/10/38.5/1.5 98 83.58 0.042 67.38 1 7 50/10/38.5/1.5 98 87.93 0.052 70.97 1 8 50/10/38.5/1.5 98 94.18 0.066 71.54 1 9 50/10/38.5/1.5 97 83.72 0.049 67.39 1 thirty 50/10/38.5/1.5 98 116.20 0.040 97.58 1 49 50/9/38/3 99 86.06 0.033 71.23 1 5 50/10/38.5/1.5 97 88.86 0.003 73.37 2 6 50/10/38.5/1.5 98 68.98 0.075 50.12 2 10 50/10/38.5/1.5 98 68.38 0.079 51.46 2 49 50/9/38/3 98 75.34 0.052 59.17 2 eleven 50/10/38.5/1.5 98 92.55 0.063 70.42 3 12 50/10/38.5/1.5 98 73.87 0.051 56.58 3

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49 50/9/38/3 97 78.85 0.036 64.05 3 14 50/10/38.5/1.5 96 95.77 0.023 77.66 4 15 50/10/38.5/1.5 97 90.90 0.055 71.35 4 16 50/10/38.5/1.5 98 84.13 0.039 66.00 4 49 50/9/38/3 98 82.71 0.056 64.97 4 1 50/9/39.5/1.5 96 89.37 0.041 70.27 5 17 50/10/38.5/1.5 98 91.32 0.037 72.26 5 18 50/10/38.5/1.5 99 77.64 0.057 58.93 5 19 50/10/38.5/1.5 99 82.31 0.011 66.83 5 20 50/10/38.5/1.5 98 88.10 0.056 70.77 5 21 50/10/38.5/1.5 99 80.80 0.041 65.00 5 31 50/10/38.5/1.5 98 93.72 0.009 76.38 5 32 50/10/38.5/1.5 98 78.88 0.054 62.21 5 33 50/10/38.5/1.5 98 87.33 0.036 70.78 5 34 50/10/38.5/1.5 96 90.69 0.044 73.81 5 35 50/10/38.5/1.5 97 85.62 0.063 68.07 5 36 50/10/38.5/1.5 97 76.38 0.058 60.24 5 49 50/9/38/3 98 82.71 0.056 64.97 5 7 50/10/38.5/1.5 98 95.20 0.020 76.61 6 9 50/10/38.5/1.5 98 85.62 0.048 68.63 6 22 50/10/38.5/1.5 98 98.32 0.026 79.28 6 23 50/10/38.5/1.5 97 94.38 0.048 75.29 6 24 50/10/38.5/1.5 98 109.50 0.133 72.78 6 49 50/9/38/3 99 75.92 0.040 60.22 6 1 50/9/39.5/1.5 97 90.24 0.002 74.63 7 25 50/10/38.5/1.5 99 81.90 0.029 64.59 7 26 50/10/38.5/1.5 99 79.29 0.047 62.17 7 27 50/10/38.5/1.5 99 85.38 0.040 65.81 7 28 50/10/38.5/1.5 98 90.56 0.029 73.19 7 29 50/10/38.5/1.5 83 90.05 0.002 74.44 7 49 50/9/38/3 98 80.07 0.019 64.16 7 34 50/10/38.5/1.5 98 92.98 0.014 75.69 8 38 50/10/38.5/1.5 98 87.14 0.045 68.11 8 49 50/9/38/3 98 86.84 0.020 71.49 8

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7 50/10/38.5/1.5 97 89.00 0.034 73.67 9 34 50/10/38.5/1.5 97 85.93 0.043 68.07 9 39 50/10/38.5/1.5 98 89.19 0.004 73.72 9 40 50/10/38.5/1.5 97 98.55 0.023 81.09 9 41 50/10/38.5/1.5 98 109.00 0.036 88.36 9 42 50/10/38.5/1.5 98 113.80 0.014 93.70 9 43 50/10/38.5/1.5 97 136.60 0.014 113.90 9 49 50/9/38/3 99 86.26 0.017 70.81 9 49 50/9/38/3 96 83.65 0.031 67.22 9 114 50/10/38.5/1.5 95 120.3 0.011 100.5 9 34 50/10/38.5/1.5 98 92.98 0.014 75.69 10 38 50/10/38.5/1.5 98 87.14 0.045 68.11 10 49 50/9/38/3 98 86.84 0.020 71.49 10 49 50/9/38/3 96 83.65 0.031 67.22 10

Table Figure 3 shows the percentage of editing in mouse liver measured by NGS.

Table 3

Editing efficiency in mouse liver resulting in % deletion formation for G282 and G650 experiments and serum TTR levels for G282 experiments

Connection No. Editing Serum TTR Index Exp. Average % deletion SD N Serum TTR (µg/ml) SD Sample number (n) Serum TTR (%TSS) TSS 0.1 0.1 4 884 FROM 5 100% G282 1 1 13.1 8.2 5 671 201 5 76% G282 1 3 23.7 8.5 5 691 182 5 78% G282 1 7 45.3 8.3 5 270 132 5 31% G282 1 8 0.1 0.1 5 745 97 5 84% G282 1 9 44.1 13.3 5 339 184 5 38% G282 1 thirty 10.4 4.8 5 841 89 5 95% G282 1 49 33.5 8.9 5 388 103 5 44% G282 1 TSS 0.1 0.1 5 G650 2 5 17.5 4.4 5 G650 2 6 0.4 0.1 5 G650 2

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10 5.3 1.3 5 G650 2 49 10.2 2.8 4 G650 2 TSS 0.1 0.1 5 G650 3 eleven 9.2 1.2 5 G650 3 12 5.5 2.4 5 G650 3 49 15.7 5.5 5 G650 3 TSS 0.1 o,o 5 G650 4 14 20.6 8.7 5 G650 4 15 18.0 2.4 5 G650 4 16 10.1 1.7 5 G650 4 49 15.4 3.5 5 G650 4 TSS 0.2 0.1 5 G650 5 1 18.6 3.9 5 G650 5 17 12.3 4.4 5 G650 5 18 4.5 1.4 5 G650 5 19 13.0 4.1 5 G650 5 20 15.4 5.1 5 G650 5 21 12.1 1.4 5 G650 5 31 15.6 1.8 5 G650 5 32 H.6 2.0 5 G650 5 33 20.0 5.0 5 G650 5 34 44.8 8.3 5 G650 5 35 35.5 6.6 5 G650 5 36 30.0 8.4 5 G650 5 49 22.3 4.6 5 G650 5 TSS 0.0 o,o 5 G650 6 7 22.6 5.6 5 G650 6 9 20.3 2.0 5 G650 6 22 6.7 2.4 5 G650 6 23 7.3 2.0 5 G650 6 24 6.8 0.9 5 G650 6 49 H,4 2.2 5 G650 6 TSS o,o 0.1 5 G650 7 1 3.2 0.8 5 G650 7

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25 10.2 5.5 5 G650 7 26 5.2 1.4 5 G650 7 27 13.0 2.8 5 G650 7 28 17.8 2.5 5 G650 7 29 3.6 0.7 5 G650 7 49 20.5 4.3 5 G650 7 TSS 0.1 0 5 777 89 5 100% G282 8 34 50.4 6.3 5 101 62 5 13% G282 8 38 26.7 6.1 5 301 51 5 39% G282 8 49 38.6 8.0 5 247 82 5 32% G282 8 TSS 0.1 0.1 5 812 33 5 100% G282 9 7 46.1 6.8 5 271 73 5 33% G282 9 34 32.4 5.4 5 367 97 5 45% G282 9 39 36.5 7.3 5 335 99 5 41% G282 9 40 44.7 8.1 5 251 61 5 31% G282 9 41 28.8 15.9 5 393 190 5 48% G282 9 42 21.6 7.3 5 473 122 5 58% G282 9 49 15.8 6.6 5 602 60 5 74% G282 9 49 30.7 6.7 5 527 145 5 65% G282 9 114 0.8 1.1 5 637 49 5 78% G282 9 TSS 0.1 0 5 777 89 5 100% G282 10 34 50.4 6.3 5 101 62 5 13% G282 10 38 26.7 6.1 5 301 51 5 39% G282 10 49 38.6 8.0 5 247 82 5 32% G282 10 49 30.4 5.1 5 311 76 5 38% G282 10

Example 117 - dose-sensitivity editing in the liver.

To evaluate whether editing was dose dependent, experiments were performed in vivo at different LNP dose levels. Cas9 mRNA Example 115 was prepared as an LNP with an sRNA targeting TTR (G00282; SEQ ID NO: 1). These LNPs were formulated at a 1:1 ratio of ssRNA to Cas9 mRNA. LNPs were formulated using a cross-flow procedure with lipid molar compositions as described in Table. 4, with an N/P ratio of 6.0.

LNP formulations were analyzed for average particle size, polydispersity (pdi), total RNA content, and RNA encapsulation efficiency as described in Example 115.

Analysis of average particle size, polydispersity (PDI), total RNA content and RNA encapsulation efficiency are shown in Table. 4.

Table 4 Composition Analytics

Connection number Composition ratio (mol.%) Encapsulation (%) Z-average size (nm) PDI Numerical average size (nm) 7 50/10/38.5/1.5 97 85.09 0.033 68.39 9 50/10/38.5/1.5 97 74.83 0.033 59.99 49 50/9/38/3 98 84.93 0.007 69.51

Female CD-1 mice were administered i.v. at 0.1 mg/kg body weight or 0.3 mg/kg body weight and is evaluated for editing in the liver. The results are shown in Fig. 2 and in table. 5.

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Table 5

Dose-dependent editing of liver TTR and serum TTR levels

Connection number Dose (mg/kg) Average % deletion SD N TSS p/a 0.4 0.1 5 7 0.1 49.1 8.9 5 7 o, s 69.4 0.8 5 9 0.1 50.6 6.5 5 9 o, s 64.5 3.8 5 49 0.1 26.8 5.7 5 49 o, s 60.6 5.9 5

Example 118.

To evaluate dosing scalability, dose-response experiments were performed in vivo. Example 115 Cas9 mRNA was prepared as an LNP with gRNA targeting TTR (G00282; SEQ ID NO: 1). These LNPs were formulated, prepared and analyzed as described in Example 115 using a 1:1 w/w ratio of ssRNA to Cas9 mRNA. The component composition of these LNPs is described in table. 6.

Table 6

Composition analytics

Connection number Composition ratio (mol.%) Encapsulation (%) Z-average size (nm) PDI Numerical average size (nm) Connection 5 50/10/38.5/1.5 99 76.02 0.009 62.64 Connection 34 50/10/38.5/1.5 98 86.45 0.015 71.97 Connection 49 50/9/38/3 99 83.63 0.011 69.75

Female CD-1 mice were administered i.v. at 0.1 mg/kg body weight or 0.3 mg/kg body weight and is evaluated for editing in the liver. The results are shown in Fig. 3A, fig. 3B and in table. 7.

Table 7 Editing and Serum TTR Levels

Compound Number Dose (mg/kg) Editing Serum TTR Average % deletion SD N Serum TTR (µg/ml) SD N %TSS TSS TSS 0.1 0.0 5 818 118 5 100% 5 0.1 26.1 2.2 5 549 33 5 67% 5 0.3 52.5 5.1 5 233 100 5 28% 34 0.1 54.9 4.6 4 170 55 4 21% 34 0.3 68.3 1.5 5 45 12 5 5% 49 0.1 50.0 I.5 5 254 107 4 31% 49 0.3 67.7 2.5 5 40 13 3 5%

Example 119 - Evaluation of the efficiency of lipid delivery by in vivo editing.

We assessed the in vivo editing efficiency of materials supplied with variable tail length formulations in in vivo editing experiments. LNPs were formulated as described above with G502 (SEQ ID NO: 4), which targets the mouse TTR gene, which was used to measure editing. The formulations contained a 1:2 ratio of ssRNA to Cas9 mRNA. The characteristics of the final LNPs are shown in Table. 8. The pKa values of the ionizable lipids were measured and shown in Table. 1.

In experiment 4, a series of compounds based on compound 7 were tested, which differed by the inclusion of a C _5-12 alkyl at the R ³ position of formula (1A), as shown in FIG. 4A. As shown in table. 9 and FIG. 4B and 4C, all five compounds tested were effective for in vivo editing.

In experiment 8, a series of compounds based on compound 9 were tested, which differed by the inclusion of a C _5-12 alkyl at the R ³ position of formula (1A), as shown in FIG. 5A. Experiment 2 tested compound 287 of this series, which includes a C ₄ alkyl at the R ³ position. As shown in table. 9 and FIG. 5B and 5C, all compounds in this series were effective for in vivo editing.

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In experiment 3, a series of compounds based on compound 46 were tested, which differed by the inclusion of a C _5-8 alkyl at the R ³ position of formula (1A), as shown in FIG. 6A. Compound 46 includes a C12 alkyl at the R ³ position, and compounds with different tail lengths were tested. As shown in table. 9 and FIG. 6B and 6C, compounds with R ³ groups C5 (compound 72), C6 (compound 50), C ₇ (compound 76) and C ₈ (compound 51) were effective for in vivo editing.

In experiment 7, a series of compounds based on compound 5 were tested, which differed by the inclusion of a ^-^-alkyl at the R ³ position of formula (1A), as shown in FIG. 7A. Experiment 2 tested compound 66 of this series, which includes a C4 alkyl at the R ³ position. Compound 5 contains a C12 alkyl at the R ³ position and was shown to be effective for in vivo editing in Examples 116 and 118. As shown in table. 9 and Fig. 7B and 7C, all additional compounds tested in this series were effective for in vivo editing.

In experiment 1, a series of compounds based on compound 34 were tested that vary at the X ¹ and Y ¹ positions of formula (1A), as shown in FIG. 8A. Compound 52 and compound 34 share dilinole groups at the Y ¹ position and vary by the inclusion of C0-1 alkylene groups at the X ¹ position. Compound 53, compound 54, and compound 55 share triol groups at the Y ¹ position and vary by the inclusion of C0-2 alkylene groups at the X ¹ position. Compound 56, compound 57, and compound 58 share groups of 20 atoms at the Y ¹ position and vary by the inclusion of C0-1 alkylene groups at the X ¹ position. Compound 59, compound 60 and compound 61 share a group of 21 atoms at the ^Y1 position and vary by the inclusion of C0-2 alkylene groups at the ^X1 position. Compound 62, compound 63 and compound 64 share heterogeneous linoleic and 21 atom groups at the Y ¹ position and vary by the inclusion of C _0-2 alkylene groups at the X ¹ position. As shown in table. 9 and FIG. 8B and 8C, all compounds tested were effective for in vivo editing.

In experiment 5, a series of compounds based on compound 50 were tested that vary at the X ² positions of formula (1A) as shown in FIG. 9A. As shown in table. 9 and FIG. 9B and 9C, all compounds tested were effective for in vivo editing at a dose of 0.1 mg/kg.

In experiment 6, a series of compounds based on compound 34 was tested, as shown in FIG. 10A. Compound 86 and compound 99 differ from compound 34 at the X ¹ position of formula (1A). Compound 103 differs from compound 34 at the R ³ position of formula (1A). Compound 87 and compound 88 are R- and S-ethantiomers at the carbon atom between the R ³ and X ¹ positions of racemic compound 34. The data are presented in table. 9 and Fig. 10B and 10C. All compounds in this series tested in mice were effective for in vivo editing.

In experiment 9, a series of compounds based on compound 50 was tested, as shown in FIG. 11A. Compound 90 and compound 89 are R- and S-enantiomers at the carbon atom between the R ³ and X ¹ positions of racemic compound 50. Compound 100 and compound 107 are different from compound 50 and the R ³ and X ¹ positions. Connection 105, connection 106 and connection 108 are different from connection 50 at position R ³ . The data is presented in table. 9 and FIG. 11B and 11C. All compounds in this series tested in mice were effective for in vivo editing.

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Table 8

Composition analytics

Connection No. Composition ratio (mol %) Encapsulation (%) Z-average size (nm) PDI Numerical average size (nm) Exp. 49 50/9/38/3 98% 87.92 0.05 69.84 1 1 50/10/38.5/1.5 99% 68.63 0.04 54.84 1 34 50/10/38.5/1.5 98% 93.66 0.02 76.32 1 52 50/10/38.5/1.5 98% 90.75 0.03 74.35 1 53 50/10/38.5/1.5 99% 92.99 0.05 75.76 1 54 50/10/38.5/1.5 99% 94.16 0.04 75.05 1 55 50/10/38.5/1.5 97% 117.30 0.02 99.29 1 56 50/10/38.5/1.5 96% 112.50 0.03 91.81 1 57 50/10/38.5/1.5 98% 100.40 0.02 83.73 1 59 50/10/38.5/1.5 97% 109.90 0.03 88.36 1 60 50/10/38.5/1.5 97% 103.40 0.07 79.57 1 61 50/10/38.5/1.5 97% 102.00 0.06 79.7 1 62 50/10/38.5/1.5 97% 106.40 0.04 87.26 1 63 50/10/38.5/1.5 98% 106.00 0.04 82.18 1 64 50/10/38.5/1.5 97% 106.40 0.02 86.19 1 49 50/9/39.5/3 98% 88.36 0.014 73.13 2 44 50/9/39.5/1.5 99% 97.65 0.034 78.34 2 45 50/9/39.5/1.5 99% 102.70 0.021 85.54 2 67 50/9/39.5/1.5 99% 102.20 0.043 82.14 2 68 50/9/39.5/1.5 99% 122.30 0.051 100.4 2 49 50/9/38/3 93 77.07 0.03 63.19 3 50 50/10/38.5/1.5 98 94.33 0.02 76.57 3 51 50/10/38.5/1.5 98 96.78 0.02 80.57 3 72 50/10/38.5/1.5 98 106.80 0.02 88.38 3 76 50/10/38.5/1.5 98 94.32 0.01 78.11 3 49 50/9/38/3 98 81.83 0.026 66.26 4 7 50/10/38.5/1.5 98 87.61 0.002 73.07 4 39 50/10/38.5/1.5 98 89.83 0.015 73.52 4 40 50/10/38.5/1.5 97 101.10 0.027 81.51 4 69 50/10/38.5/1.5 96 108.90 0.017 88.76 4 73 50/10/38.5/1.5 97 95.81 0.029 76.59 4 49 50/10/38.5/1.5 98 83.00 0.03 65 5 50 50/10/38.5/1.5 98 103.00 0.06 77 5 77 50/10/38.5/1.5 98 104.00 0.08 78 5

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78 50/10/38.5/1.5 97 91.00 0.13 59 5 79 50/10/38.5/1.5 98 75.00 0.09 55 5 84 50/10/38.5/1.5 99 91.00 0.03 75 5 49 50/9/38/3 99% 90.00 0.02 74 6 34 50/10/38.5/1.5 98% 89.00 0.03 73 6 86 50/10/38.5/1.5 98% 87.00 0.04 68 6 99 50/10/38.5/1.5 99% 79.00 0.03 64 6 103 50/10/38.5/1.5 91% 102.00 0.01 82 6 87 50/10/38.5/1.5 98% 83.00 0.06 63 6 88 50/10/38.5/1.5 98% 88.00 0.02 73 6 49 50/9/38/3 93 77.07 0.026 63.19 7 66 50/10/38.5/1.5 98 79.79 0.035 65.07 7 36 50/10/38.5/1.5 98 75.9 0.058 58.34 7 75 50/10/38.5/1.5 98 89.78 0.02 73.46 7 45 50/10/38.5/1.5 98 95.26 0.012 77.92 7 71 50/10/38.5/1.5 97 104.5 0.035 86.63 7 49 50/9/38/3 98% 84.47 0.026 69.01 8 9 50/10/38.5/1.5 98% 64.83 0.074 48.82 8 65 50/10/38.5/1.5 99% 69.67 0.056 52.04 8 35 50/10/38.5/1.5 99% 81.51 0.068 62.82 8 74 50/10/38.5/1.5 99% 92.14 0.024 74.58 8 44 50/10/38.5/1.5 99% 104.3 0.034 85.76 8 70 50/10/38.5/1.5 99% 124.2 0.018 102 8 49 50/9/38/3 98 87 0.04 70 9 50 50/10/38.5/1.5 97 98 0.04 78 9 90 50/10/38.5/1.5 98 99 0.02 81 9 89 50/10/38.5/1.5 98 88 0.01 73 9 100 50/10/38.5/1.5 99 102 0.05 83 9 105 50/10/38.5/1.5 99 80 0.02 64 9 106 50/10/38.5/1.5 99 91 0.01 74 9 107 50/10/38.5/1.5 98 89 0.03 72 9 108 50/10/38.5/1.5 99 89 0.03 73 9 49 50/9/38/3 0.98 90 0.04 75 10 34 50/10/38.5/1.5 0.98 95 0.03 77 10 91 50/10/38.5/1.5 0.98 97 0.01 82 10 92 50/10/38.5/1.5 0.98 67 0.06 51 10 93 50/10/38.5/1.5 0.98 103 0.01 86 10 94 50/10/38.5/1.5 0.98 70 0.06 53 10 95 50/10/38.5/1.5 0.98 105 0.01 88 10 51 50/10/38.5/1.5 0.98 98 0.02 82 10 96 50/10/38.5/1.5 0.98 89 0.03 73 10 97 50/10/38.5/1.5 0.98 92 0.03 75 10

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Table 9 shows the percentage of editing in mouse liver measured by NGS.

Table 9

Editing efficiency in mouse liver resulting in % deletion formation for experiments with G502 and serum TTR levels

Editing Serum TTR Conn. No. Dose (mg/kg) % Editing SD N Serum a TTR (µg/ml) SD N %TSS Exp. TSS p/a od 0.00 5 1076.77 368.43 5 100.00 1 49 OD 37.58 10.65 5 501.07 107.20 5 46.53 1 1 OD 28.82 9.24 5 522.07 212.60 5 48.49 1 34 od 64.95 4.74 5 101.04 45.14 5 9.38 1 52 od 60.14 8.13 5 186.42 92.64 5 17.31 1 53 od 61.50 4.89 5 150.43 53.66 5 13.97 1 54 od 57.22 5.81 5 197.28 90.55 5 18.32 1 55 od 15.86 7.12 5 783.74 94.62 5 72.79 1 56 od 16.94 2.41 5 750.53 94.88 5 69.70 1 57 od 50.76 11.63 5 264.64 112.65 5 24.58 1 59 od 10.28 6.63 5 868.93 145.85 5 80.70 1 60 od 23.88 12.59 5 651.52 199.20 5 60.51 1 61 od 9.40 1.17 5 877.31 93.63 5 81.48 1 62 od 23.74 9.69 5 623.37 138.65 5 57.89 1 63 od 6.53 2.41 5 902.96 96.13 5 83.86 1 64 od 5.84 1.04 5 919.42 90.82 5 85.39 1 TSS p/a 0.24 0.05 5 1232.25 259.96 5 100.00 2 49 0.03 6.5 0.39 5 1128.34 197.52 5 91.56 2 4 0.03 8.6 1.00 5 900.69 291.61 5 73.09 2

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45 0.03 5.16 1.38 5 965.93 156.29 5 78.39 2 67 0.03 5.94 1.63 5 806 219.39 5 65.41 2 68 0.03 6.48 0.05 5 845.04 259.96 5 68.58 2 TSS n/a 0.18 0.04 5 1161.07 311.28 5 100.00 3 49 0.03 5.02 2.30 5 960.376 129.68 5 82.71 3 49 0.1 30.66 10.27 5 544.37 183.72 5 46.89 3 50 0.03 37.54 24.51 5 528.43 165.52 5 45.51 3 50 0.1 65.56 3.59 5 160.42 44.32 5 13.82 3 51 0.03 38.8 5.98 5 447.172 44.70 5 38.51 3 51 0.1 65.62 4.90 5 108,258 49.36 5 9.32 3 72 0.03 27.72 10.69 5 777 191.77 5 66.92 3 72 0.1 53.06 8.34 5 306,856 39.10 5 26.43 3 76 0.03 27.32 14.64 5 939.50 171.63 5 69.72 3 76 0.1 63.08 6.07 5 148,554 67.25 5 12.79 3 TSS n/a 0.24 0.05 5 690.45 84.84 5 100% 4 49 0.03 28.78 9.67 5 464.17 74.74 5 67% 4 49 0.1 56.82 4.70 5 154.53 60.30 5 22% 4 7 0.03 38.23 13.85 4 397.92 52.45 4 58% 4 7 0.1 66.72 5.63 5 66.59 45.44 5 10% 4 69 0.03 36.68 6.47 5 370.88 69.44 5 54% 4 69 0.1 67.38 5.13 4 85.93 27.44 4 12% 4 40 0.03 34.53 5.48 4 529.70 128.79 4 77% 4 40 0.1 69.44 2.12 5 64.65 21.93 5 9% 4 73 0.03 57.32 7.29 5 173.78 81.14 5 25% 4 73 0.1 70.76 2.03 5 30.62 14.97 5 4% 4 39 0.03 44.74 14.44 5 286.24 100.55 5 41% 4 39 0.1 66.66 4.44 5 39.85 20.49 5 6% 4 TSS n/a 0.16 0.05 5 809.30 145.45 5 100 5 49 0.03 16.98 10.77 5 592.00 150.58 5 73.1 5 49 0.1 40.32 11.31 5 379.70 106.18 5 46.9 5 77 0.03 5.18 0.79 5 552.80 84.36 5 68.3 5 77 0.1 10.36 3.05 5 652.20 179.50 5 80.6 5 78 0.03 7 2.04 5 807.50 97.21 5 99.8 5 78 0.1 24.2 7.13 5 537.90 74.96 5 66.5 5

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79 0.03 0.58 0.19 5 703.50 195.81 5 86.9 5 79 OD 3.34 1.51 5 812.20 203.80 5 100.4 5 84 0.03 11.24 3.37 5 649.70 161.07 5 80.3 5 84 0.1 18.86 9.21 5 592.10 98.42 5 73.2 5 50 0.03 23.92 3.08 5 567.70 202.13 5 70.1 5 50 0.1 49.6 14.63 5 241.90 104.88 5 29.9 5 TSS n/a 0.1 0.07 5 964.34 179.38 5 100.00 6 49 0.03 3.28 1.05 5 969.91 131.91 5 100.57 6 49 0.1 26.88 6.84 5 609.23 222.39 5 63.17 6 34 0.03 19.66 3.87 5 820.73 196.33 5 85.10 6 34 0.1 50.82 2.43 5 306.74 84.07 5 31.80 6 86 0.03 9.62 5.42 5 897.846 166.96 5 93.10 6 86 0.1 50.7 6.45 5 277,696 38.44 5 28.80 6 99 0.03 10.2 4.30 5 891.268 228.52 5 92.42 6 99 0.1 37.12 6.54 5 413.382 113.60 5 42.87 6 103 0.03 20.16 7.23 5 696.58 103.62 5 72.23 6 103 0.1 51.98 7.53 5 280.156 106.56 5 29.05 6 87 0.03 16.08 4.23 5 773.57 157.56 5 80.21 6 87 0.1 51.78 6.51 5 321.386 85.50 5 33.33 6 88 0.03 20.66 9.33 5 723.87 105.48 5 75.06 6 88 0.1 57.32 7.41 5 322,566 132.08 5 33.45 6 TSS n/a 0.16 0.05 5 1648.15 261.09 5 100 7 49 0.03 4.08 1.33 5 1459.99 292.22 5 88,584 7 49 0.1 27.5 8.04 5 860.90 343.62 5 52,234 7 66 0.03 27.64 8.25 5 901.24 307.33 5 54,682 7 66 0.1 55.26 4.08 5 324.25 115.30 5 19,674 7 36 0.03 9.96 0.82 5 1249.04 115.28 5 75,785 7 36 0.1 35.92 2.78 5 722.28 85.81 5 43,824 7 75 0.03 29.6 6.95 5 743.29 58.71 5 45,098 7 75 0.1 57.22 7.58 5 197.72 99.45 5 11,996 7 45 0.03 31.1 4.96 5 715.87 97.41 5 43.435 7 45 0.1 60.3 6.80 5 166.30 55.94 5 10.09 7 71 0.03 20.94 7.55 5 781.63 115.04 5 47.425 7 71 0.1 55.78 2.10 5 248.13 55.94 5 15,055 7

- 184 046244

TSS p/a 0.1 0.00 5 748.33 130.95 5 100.00 8 49 0.03 7.62 6.50 5 735.41 83.84 5 98.27 8 49 0.1 37.46 4.17 5 369.43 45.54 5 49.37 8 9 0.03 0.84 0.46 5 853.25 107.87 5 114.02 8 9 0.1 3.46 1.09 5 772.22 172.17 5 103.19 8 65 0.03 1.32 0.31 5 896.77 143.42 5 119.84 8 65 0.1 10.12 9.68 5 742.52 151.70 5 99.22 8 35 0.03 4.46 0.70 5 780.47 74.29 5 104.30 8 35 0.1 17.94 4.99 5 741.82 79.87 5 99.13 8 74 0.03 4.36 1.25 5 752.38 120.29 5 100.54 8 74 0.1 11.12 1.63 5 823.58 126.35 5 110.06 8 44 0.03 4.76 1,Yu 5 897.76 51.74 5 119.97 8 44 0.1 17.97 7.30 5 589.60 52.34 5 78.79 8 70 0.03 4.84 1.77 5 945.08 81.30 5 126.29 8 70 0.1 8.04 1.17 5 654.70 98.42 5 87.49 8 TSS p/a 0.1 0.00 5 964.50 154.71 5 100 9 49 0.03 20.54 12.51 5 677.31 194.76 5 70.22 9 50 0.03 21.74 8.62 5 687.70 218.46 5 71.3 9 90 0.03 26.02 5.64 5 578.26 188.44 5 59.95 9 89 0.03 31.02 2.76 5 469.27 63.53 5 48.65 9 100 0.03 31.3 6.03 5 450.19 47.05 5 46.68 9 105 0.03 33.86 8.75 5 462.70 120.85 5 47.97 9 106 0.03 11.58 3.69 5 795.41 161.67 5 82.47 9 106 0.03 1.98 1.16 5 855.49 158.34 5 88.7 9 107 0.03 26.98 4.62 5 564.55 102.79 5 58.53 9 108 0.03 27 4.04 5 550.89 102.92 5 57.12 9 TSS p/a 0.14 0.05 5 767.76 98.15 5 100% 10 49 0.03 10.33 2.15 4 704.2 126.26 4 92% 10 34 0.03 37.44 10.7 5 419.07 133.1 5 55% 10 91 0.03 22.7 6.77 5 681.26 63.6 5 89% 10 92 0.03 2.38 0.79 5 923.39 226.76 5 120% 10 93 0.03 12.68 1.94 5 765.38 95.14 5 100% 10 94 0.03 1.82 1.29 5 838.22 110.3 5 109% 10 95 0.03 37.96 13.54 5 589.77 283.04 5 77% 10 51 0.03 30.86 4.36 5 619.84 114.91 5 81% 10 96 0.03 28.04 3.67 5 540.39 120 5 70% 10 97 0.03 28.26 3.88 5 603.03 177.26 5 79% 10

Example 120 Editing lipid potency and tolerance in vivo.

To evaluate lipid efficacy, dose-response experiments were performed in vivo. Cas9 mRNA Example 115 was prepared as an LNP with gRNA targeting TTR (G00534; SEQ ID NO: 5). These LNPs were formulated, prepared and analyzed as described in Example 115 using a 1:2 w/w ratio of ssRNA to Cas9 mRNA.

The component composition of these LNPs is described in table. 10.

- 185 046244

Table 10 Composition Analytics

Compound Number Composition Encapsulation (%) Z-Average Size(nm) PDI Numerical average size (nm) 49 50/9/38/3 97 87.84 0.028 69.85 34 50/10/38.5/1.5 93 96.43 0.052 76.08 45 50/10/38.5/1.5 95 101.00 0.052 83.48 50 50/10/38.5/1.5 87 98.55 0.050 79.18

For efficacy, female Sprague Dawley rats were administered IV at 0.1 mg/kg or 0.03 mg/kg. 6-7 days after dosing, rats were killed by CO2 asphyxiation. Blood was collected into tubes with a serum separator. The liver biopsy was processed for gDNA and assessed for editing using the NGS methods described in Example 115. The results are presented in Table 1. 11 and fig. 12A and 12B. To ensure tolerability, female Sprague Dawley rats were dosed at 6 mg/kg and killed by CO2 asphyxiation 24 hours after dosing. Blood was collected into serum separator tubes and sent to an external CRO for quantification of liver enzymes using a liver enzyme panel. The results are presented in table. 12.

Table 11

Editing efficiency in rats providing % deletion formation for experiments with _________________G534 and serum TTR levels__________________

Conn. No. Dose (mg/kg) Editing Serum TTR Guides (gRNAs) exp % Edit roving SD N Serum TTR (µg/ml) SD N % TSS TSS 0.1 0.00 5 845.3 70.69 5 100% G534 1 49 0.03 4.76 1.49 5 790.9 82.94 5 94% G534 1 49 0.1 25.62 14.1 8 5 669.5 181.1 3 5 79% G534 1 34 0.03 21.22 6.54 5 514.2 119.3 6 5 61% G534 1 34 0.1 53.9 7.17 5 188.9 112.6 7 5 22% G534 1 45 0.03 10.76 8.80 5 910.4 73.77 5 110% G534 1 45 0.1 48.16 2.90 5 270.7 109.7 1 5 32% G534 1 50 0.03 26.9 9.04 5 581.7 76.89 5 69% G534 1 50 0.1 62.6 1.04 5 116.6 24.78 5 14% G534 1

Table 12

Rat Liver Enzyme Panel Results

Connection No. Doses (mg/kg) ALT (U/L) AST (U/L) Creatine kinase (U/L) GGT (U/L) ALP (U/L) Bilirubi h Total (mg/dl) TSS 47 72 132.2 0 318.4 ο,ι 49 6 408.5 534.8 157.5 2 784.3 0.6 34 6 179.6 349.6 184.8 3.4 542 0.2 45 6 163.2 681 1103 2.4 439.8 o, s 50 6 250.8 820.8 272 eleven 508.6 0.4

Example 121 - in vitro editing using alkyl compounds R ³ .

A series of compounds based on compound 46, which differ in the inclusion of a C _4-12 alkyl at the R ³ position of formula (1A) (as shown in Fig. 13A), were tested for in vitro editing. Human hepatocellular carcinoma cell line HUH7 (Japan Research Bank Collection

- 186 046244 bioresource cells, catalog JCRB0403) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were seeded at a density of 20,000 cells/well in a 96-well plate 24 h before transfection (~80% confluence during transfection). LNPs were formulated at a 1:2 w/w ratio of ssRNA to Cas9 mRNA using a cross-flow procedure with lipid molar compositions as described in Table 1 . 13, with an N/P ratio of 6.0. LNP compositions were analyzed for average particle size, polydispersity (pdi), total RNA content, and RNA encapsulation efficiency as described in Example 115. Analysis of average particle size, polydispersity (PDI), total RNA content, and RNA encapsulation efficiency are shown in Table 1. 13. LNP was diluted in the medium, incubated for 5 min at 37°C and pipetted onto the cells at a dose of 2000 ng of RNA per well. Each condition was examined in biological duplicate. Cells were harvested after 6 h of transfection. Genomic DNA was extracted using 50 μl/well of BuccalAmp DNA extraction solution (Epicenter, Cat. QE09050) according to the manufacturer's protocol. All DNA samples were subjected to PCR and subsequent NGS analysis for editing at the target locus G000502 as described above. As shown in table. 14 and fig. 13B, all compounds tested were effective for in vitro editing.

Table 13

Composition analytics

Connection No. Composition ratio (mol.%) Z-Average Size [nm] PDI Average number [nm] %E 49 50/9/38/3 93.14 0.015 74.82 98 34 50/10/38.5/1.5 93.01 0.009 75.88 97 46 50/10/38.5/1.5 94.54 0.003 76.76 99 51 50/10/38.5/1.5 97.21 0.009 80 98 76 50/10/38.5/1.5 98.79 0.024 81.12 97 50 50/10/38.5/1.5 102.8 0.021 84.16 97 72 50/10/38.5/1.5 111.4 0.018 91.47 94 85 50/10/38.5/1.5 117.1 0.045 93.05 96

Table 14

In vitro editing

Connection No. Editing SD 49 22.6 0.8 34 49.0 2.5 46 63.7 6.4 51 69.0 9.7 76 61.2 9.9 50 64.3 5.6 72 65.8 7.3 85 66.4 0.4

- 187 046244

Sequence table

Description Subsequence SEQ ID No. G00282 sgRNA targeting mouse TTR mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmG mCmUmAmGmAm AmAmUmAmGmCAAGUUAAAAUAAG GCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmA mGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmG mCmU*mU*m U*mU eleven G00650 mG*mA*mC*AAGCACCAGAAAGACCAGUUUUAGAmG 22 ssRNA targeting B2M person mRNA encoding Cas9 mCmUmAmGmAm AmAmUmAmGmCAAGUUAAAAUAAG GCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmA mGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmG mCmU*mU*mU*mU GGGUCCCGCAGUCGGCGUCCAGCGGCUCUGCUUGUU CGUGUGUGUGUCGUUGCAGGCCUUAUUCGGAUCCGC CACCAUGGACAAGAAGUACAGCAUCGGACUGGACAU CGGAACAAACAGCGUCGGAUGGGCAGUCAUCACAGA CGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCU GGGAAACACAGACAGACACAGCAUCAAGAAGAACCU GAUCGGAGCACUGCUGU UCGACAGCGGAGAAACAGC AGAAGCAACAAGACUGAAGAGAACAGCAAGAAGAAG AUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCA GGAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGA CAGCUUCUUCCACAGACUGGAAGAAAGCUUCCUGGU CGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUU CGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAA GUACCCGACAAUCUACCACCUGAGAAAGAAGCUGGU CGACAGCACAGACAAGGCAGACCUGAGACUGAUCUA CCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACA CUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGAC AUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGC AAGCGGAGUCGACGCAAA CAAUCCUGAGCGCAAG ACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGC ACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGG AAACCUGAUCGCACUGAGCCUGGGACUGACACCGAA CUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAA GCUGCAGCUGAGCAAGGACACAUACGACGACGACCU GGACAACCUGCUGGCACAGAUCGGAGACCAGUACGC AGAC CUGUUCCUGGCAGCAAAGAACCUGAGCGACGC AAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGA AAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAA GAGAUACGACGAACACCACCAGGACCUGACACUGCU GAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUA 33

- 188 046244

CAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUA CGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGA AUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAG AGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAA CGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACU GCACGCAAUCCUGA GAAGACAGGAAGACUUCUACCC GUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAU CCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCU GGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAG AAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA AGAAGUCGUCGACAAGGGAGCAAGCGCACAGCUU CAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCC GA ACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUA CGAAUACUUCACAGUCUACAACGAACUGACAAAGGU CAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUU CCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCU GCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCA GCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUU CGACAGCGUCGAAAUCAGCG GAGUCGAAGACAGAUU CAACGCAAGCCUGGGAACAUACCACGACCUGCUGAA GAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGA AAACGAAGACAUCCUGGAAGACAUCGUCCUGACACU GACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAG ACUGAAGACAUACGCACACCUGUUCGACGACAAGGU CAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUG G GGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCU GAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCA GCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGA CAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAG CCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCC GGCAAUCAAGAAGGGAAUCCUGCA GACAGUCAAGGU CGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAA GCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAA

- 189 046244

CCAGACACACAGAAGGGACAGAAGAACAGCAGAGA AAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAAC UGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAA ACACACAGCUGCAGAACGAAAAGCUGUACCUGUACU ACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGG AACUGGACAUCAACAGACUGAGCGACUACGACGUCG ACCACAUCGUCCCGCAGAGCUUCCU GAAGGACGACA GCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGA ACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAG UCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGC UGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACA ACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAAC UGGACAAGGCAGGAUUCAUCAAGACAGCUGGUCG AAACAAGACAGAUC ACAAAGCACGUCGCACAGAUCC UGGACAGCAGAAUGAACACAAAGUACGACGAAAACG ACAAGCUGAUCAGAAGUCAAGGUCAUCACACUGA AGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCC AGUUCUACAAGGUCAGAGAAAUCAACAACUACCACC ACGCACACGACGCAUACCUGAACGCAGUCGUCGGAA CAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCG AAUUCGUCUACGGAGACUACAAGGUCUACGACGUCA GAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAA AGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCA UGAACUUCUUCAAGACAGAAAUCACACUGGCAAACG GAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACG GAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAG ACUUCGCAACAGUCAGAAAGGUC CUGAGCAUGCCGC AGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAG GAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAA ACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGG ACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAG UCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAA AGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAAC UGCUGG GAAUCACAAUCAUGGAAAGAAGCAGCUUCG AAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAU

- 190 046244

ACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGC CGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAA AGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGG GAAACGAACUGGCACUGCCGAGCAAGUACGUCAACU UCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGG GAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCG UCGAACAGCACAAGCACUACCU GGACGAAAUCAUCG AACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGG CAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACA ACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAG AAAACAUCAUCCACCUGUUCACACUGACAAACCUGG GAGCACCGGCAGCAUUCAAGUACUUCGACACAACAA UCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCC UGGACGCAA CACUGAUCCACCAGAGCAUCACAGGAC UGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAG GAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGG UCUAGCUAGCCAUCACAUUUAAAAGCAUCUCAGCCU ACCAUGAGAAUAAGAGAAAGAAAAUGAAGAUCAAU AGCUUAUUCAUCUCUUUUUCUUUUUCGUUGGUGUAA AGCCAACACCCUGUCUAAAAAACAU AAAUUUCUUUA AUCAUUUUGCCUCUUUUCUCUGUGCUUCAAUUAAUA AAAAAUGGAAAGAACCUCGAGAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA G000502 ssRNA targeting mouse TTR mA*mC*mA*CAAAUACCAGUCCAGCGGUUUUAGAmG mCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAG GCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmA mGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmG mCmU*mU* mU*mU 44 G000534 ssRNA targeting rat TTR mA*mC*mG*CAAAUAUCAGUCCAGCGGUUUUAGAmG mCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAG GCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmA mGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmG mCmU*mU* mU*mU 55

2'-O-methyl modifications and phosphorothioate bonds as follows (m=2'-OMe;

*=phosphorothioate).

Claims (33)

CLAIM 1. Compound of formula IA (IA) where independently for each case X ¹ represents C _1-3 alkylene or X ² is selected from O, NH, NMe and a bond, with the proviso that when X ² is O, R ² together with a nitrogen atom and either R ¹ or a carbon atom X ³ forms a 4-membered, 5-membered or 6-membered -membered ring, X ³ represents C _2-4 alkylene, X ⁴ represents a C1 alkylene or bond, X ⁵ represents a C1 alkylene or bond, R ¹ represents C _1-3 alkyl, optionally substituted with a hydroxyl group, R ² is C _1-3 alkyl, optionally substituted with a hydroxyl group, or R ² and R ¹ , or R ² and the carbon atom X ³ , together with the nitrogen atom to which they are attached, form a 4-membered, 5-membered or 6-membered ring, Y ¹ is selected from the bond, -CH=CH-, -(C=O)O- and -O(C=O)-, Y ² is selected from -CH ₂ -CH=CH- and C ₃ -C ₄ alkylene, R ³ is selected from H, C _5-7 cycloalkyl, C ₈ -C _1o alkenyl, and C _3-18 alkyl, R ⁴ represents C _4-8 alkyl, or a pharmaceutically acceptable salt thereof. 2. A compound according to claim 1, wherein the compound is a compound of formula I where independently for each case X ¹ represents C2-3 alkylene, X ² is selected from O, NH, NMe and a bond, with the proviso that when X ² is O, R ² together with a nitrogen atom and either R ¹ or a carbon atom X ³ forms a 4-membered, 5-membered or 6-membered -membered ring, X ³ represents C2-4 alkylene, X ⁴ represents a C1 alkylene or bond, X ⁵ represents a C1 alkylene or bond, R ¹ represents C _1-2 alkyl, optionally substituted with a hydroxyl group, R ² is C _1-2 alkyl, optionally substituted with a hydroxyl group, or R ² and R ¹ , or R ² and the carbon atom X ³ , together with the nitrogen atom to which they are attached, form a 5-membered or 6-membered ring, Y ¹ is selected from -CH=CH-, -(C=O)O- and -O(C=O)-, R ³ is selected from H, C _5-7 cycloalkyl, and C _3-18 alkyl, R ⁴ represents C4-7 alkyl, or a pharmaceutically acceptable salt thereof. 3. A compound according to claim 1 or 2, where X ¹ represents C ₂ alkylene. 4. A compound according to claim 1, where X ¹ represents C1 alkylene. 5. The compound according to any of the previous paragraphs, where X ² represents NH or O. 6. The compound according to any of the previous paragraphs, where X ³ represents C2-3 alkylene. 7. A compound according to any of the previous paragraphs, where R ² and the carbon atom X ³ together with the nitrogen atom to which they are attached form a 4-6 membered ring. 8. A compound according to any of the previous paragraphs, wherein R ² represents C _1-2 alkyl. - 192 046244 9. A compound according to any one of claims 1-6, where R ¹ and R ² together with the nitrogen atom to which they are attached form a 4-6-membered ring. 10. A compound according to any one of claims 1 to 6, wherein R ¹ and R ² together with the nitrogen atom to which they are attached form a 5-membered ring. 11. A compound according to any one of claims 1-8, where R ¹ represents C _1-2 alkyl. 12. The compound according to any of the previous paragraphs, where R ³ represents a linear C _4-16 al kyl. 13. A compound according to any one of claims 1-11, where R ³ represents a branched C _6-10 alkyl. 14. A compound according to any one of claims 1 to 11, where R ³ represents C _5-7 cycloalkyl. 15. A compound according to any one of claims 1-11, where R ³ represents C _8-10 alkenyl. 16. The compound according to any of the previous paragraphs, where R ⁴ represents a linear C _5-6 al kyl. 17. Connection according to any of the previous paragraphs, where X ⁴ represents a connection. 18. A compound according to any of the previous paragraphs, wherein X ⁵ is a C1 alkylene. 19. A compound according to any of the previous claims, wherein the compound is a compound of formula (IA); and Y2-R ⁴ is -CH ₂ -CH=CH-R ⁴ . 20. The connection according to any of the previous paragraphs, where Y ¹ is selected from the connection, -CH=CH- and -O(C=O)-. 21. Connection by claim 1, where the compound is a compound of formula II: 22. Connection by claim 1, wherein the compound is a compound of formula III 23. The connection according to claim 1, where the connection is selected from: - 193 046244 - 194 046244 - 195 046244 - 196 046244 - 197 046244 - 198 046244 - 199 046244 - 200 046244 - 201 046244 - 202 046244 - 203 046244 - 204 046244 - 205 046244 - 206 046244 - 207 046244 - 208 046244 - 209 046244 - 210 046244 - 211 046244 - 212 046244 - 213 046244 - 214 046244 or a pharmaceutically acceptable salt thereof. 24. A lipid nanoparticle (LNP) composition comprising a compound according to any of the preceding claims in a lipid component. 25. The LNP composition according to claim 24, wherein the lipid component further includes an auxiliary lipid, a polyethylene glycol (PEG) lipid and a neutral lipid. 26. The LNP composition according to claim 24 or 25, characterized in that the composition additionally contains a nucleic acid. 27. The LNP composition according to claim 26, characterized in that the composition has a ratio of nitrogen atoms/phosphorus groups (N/P) of 3-10. 28. LNP composition according to claim 27, characterized in that the N/P ratio is 6±1. 29. The LNP composition according to any one of claims 24-28, further comprising an RNA component, wherein the RNA component contains mRNA. 30. The LNP composition according to claim 29, wherein the RNA component contains a sequence encoding an RNA-directed DNA binding agent, such as Cas nuclease mRNA. 31. The LNP composition according to claim 29 or 30, characterized in that the RNA component includes gRNA. 32. Use of the LNP composition according to any one of claims 24-31 for gene editing in a subject. 33. Use of the LNP composition according to any one of claims 24 to 31 for the production of a drug for gene editing in a subject.