WO2009075778A2 - Nonpeptidic inhibitors of cruzain - Google Patents

Abstract

Cruzain is the major cysteine protease of T. cruzi, which is the causative agent of Chagas' disease and is a promising target for the development of new chemotherapy. With the goal of developing potent nonpeptidic inhibitors of cruzain, the Substrate Activity Screening (SAS) method was used to screen a library of protease substrates initially designed to target the homologous human protease cathepsin S. Structure-based design was next used to further improve substrate cleavage efficiency by introducing additional binding interactions in the S3 pocket of cruzain. The optimized substrates were then converted to inhibitors by the introduction of cysteine protease mechanism-based pharmacophores. Inhibitor (38) was determined to be reversible even though it incorporated the vinyl sulfone pharmacophore that is well documented to give irreversible cruzain inhibition for peptidic inhibitors. The previously unexplored β-chloro vinyl sulfone pharmacophore provided mechanistic insight that led to the development of potent irreversible acyl- and aryl- oxymethyl ketone cruzain inhibitors. For these inhibitors, potency did not solely depend on leaving group pTa, with 2,3,5,6-tetrafluorophenoxy methyl ketone (54) identified as one of the most potent inhibitors with a second order inactivation constant of 147,000 s-1M-1. This inhibitor completely eradicated the T. cruzi parasite from mammalian cell cultures and consequently has the potential to lead to new chemo therapeutics for Chagas' disease.

Description

NONPEPTIDIC INHIBITORS OF CRUZAIN

CROSS REFERENCES TO PREVIOUS APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application Serial No. 60/992,982 filed on December 6, 2007 (Attorney Docket No. 061818-02-5078 PR), which is herein incorporated by reference in its entirety for all purposes.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under GM54051 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Chagas' disease (American trypanosomiasis), caused by the protozoan Trypanosoma cruzi, is the leading cause of heart disease in Latin America. Today, at least 12 million people are infected with the protozoa, resulting in more than 50,000 deaths each year. www.who.int/tdr/diseases/chagas/direction.htm Chemotherapy for Chagas' disease is unsatisfactory with current drugs, nifurtimox and benznidazole, having significant toxic side effects, de Castro, S. L. Acta tropica 1993, 53, 83-98. Due to the toxicity of current chemotherapy and emerging drug resistance, there is an urgent need for developing an effective therapy against Chagas' disease.

[0004] Cruzain, a cysteine protease of the papain family, is the primary cysteine protease of T. cruzi. It is involved in intracellular replication and differentiation and is essential at all stages of the parasite's life cycle. Harth, G., et al., MoI. Biochem. Parasitol. 1993, 58, 17-24. Recently, it has been demonstrated that T. cruzi infection can be cured in cell, mouse, and dog models by treatment with irreversible inhibitors of cruzain. Engel, J. C, et al, Antimicrob. Agents Chemother. 2005, 49, 5160-5161. Protozoa vulnerability to cruzain inhibition results from the lack of redundancy of this enzyme. Moreover, protozoa localization provides a means for preferential inhibition of cruzain over the highly homologous human papain superfamily cysteine proteases cathepsins B, L, K, S, F and V because the protozoa resides in the host cell cytoplasm whereas the cathepsins are located in the less accessible lysosomes. McKerrow, J. H. et al., Biorg. Med. Chem. 1999, 7, 639-644. For these reasons, cruzain is a highly attractive therapeutic target for the treatment of Chagas' disease. McKerrow, J. H. et al, Parasitology Today 1995, 11, 279-282.

[0005] Dipeptidyl vinyl sulfone 1 is the most advanced inhibitor of cruzain and is currently in pre-clinical trials (Figure 1). Jacobsen, W. et al, Drug Metab. Dispos. 2000, 28, 1343- 1351. Although this peptidic inhibitor has shown good efficacy with minimal toxicity, improved inhibitors would benefit the art. The current invention addresses these and other needs.

BRIEF SUMMARY OF THE INVENTION

[0006] Recently, Substrate Activity Screening (SAS) was developed as a new method for the rapid identification of nonpeptidic enzyme inhibitors. Wood, W. J. L., et al., J. Am. Chem. Soc. 2005, 127, 15521-15527; Salisbury, C. M., et al, ChemBioChem 2006, 7, 1034-1037; Patterson, A. W., et al, J. Med. Chem. 2006, 49, 6298-6307; Inagaki, H., et al, J. Med. Chem. 2007, 50, 2693-2699; Soellner, M. B., et al, J. Am. Chem. Soc. 2007, 129, 9613-9615. The SAS method consists of the identification of nonpeptidic substrate fragments, substrate optimization, and then conversion of optimal substrates to inhibitors. Significantly, the SAS method has successfully been applied to the papain superfamily protease cathepsin S, Wood, W. J. L., et al., J. Am. Chem. Soc. 2005, 127, 15521-15527; Salisbury, C. M., et al, ChemBioChem 2006, 7, 1034-1037; Patterson, A. W., et al, J. Med. Chem. 2006, 49, 6298- 6307; Inagaki, H., et al, J. Med. Chem. 2007, 50, 2693-2699; which has high homology to cruzain. Bromme, D.; McGrath, M. E. Protein Science 1996, 5, 789-791. Using a focused substrate library developed for cathepsin S as a starting point, the development of a new class of nonpeptidic 2,3,5,6-tetrafluorophenoxymethyl ketone inhibitors that exhibit potent inhibitory activity against cruzain as well as complete eradication of T. cruzi parasites in cell culture is reported here. This class of compounds represents a promising and novel inhibitor class for the treatment of Chagas' disease.

[0007] In one aspect, the invention provides a compound of the invention. In an exemplary embodiment, the invention provides a compound having a structure according to a formula described herein. In an exemplary embodiment, the compound has a structure according to the following formula:

wherein R¹ is a member selected from H, OR*, NR*R**, SR*, -S(O)R*, -S(O)₂R*, -S(O)₂NR*R**, nitro, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0008] In another aspect, the invention provides a method of inhibiting cruzain, said method comprising: (i) contacting the cruzain with a compound of the invention, in an amount effective to inhibit the cruzain, thereby inhibiting the cruzain.

[0009] In another aspect, the invention provides a method of killing or inhibiting the growth of a protozoa, said method comprising: (i) contacting said protozoa with a compound of the invention, in an amount effective to kill or inhibit the growth of said protozoa, thereby killing or inhibiting the growth of the protozoa.

[0010] In another aspect, the invention provides a method of treating a disease, said method comprising administering a therapeutically effective amount of a compound of the invention, to an animal suffering from the disease, thereby treating the disease.

[0011] In another aspect, the invention is a pharmaceutical composition comprising: a) a compound of the invention; b) a pharmaceutically acceptable excipient.

[0012] Other objects and advantages of the invention will be apparent to those of skill in the art from the detailed description that follows.

BRIEF DESCRIPTION OF FIGURES [0013] Figure 1. Most advanced inhibitor of cruzain.

[0014] Figure 2. Alignment of cathepsin S (PDB ID: 2H7J) and cruzain (PDB ID: 1F2C) amino acid sequences with the catalytic triad depicted in bolded font. Identical residues are indicated with " * " and similar residues are indicated with " . ". The figure was produced using Swiss-Pdb Viewer (http://ca.expasy.org/spdbv/).

[0015] Figure 3. (a) Crystal structure of cathepsin S (PDB ID: 2H7J) and (b) molecular replacement model of cruzain (PDB ID: 1F2C) with chloromethyl ketone inhibitor 14. The atoms are shaded according to element: protein carbons are green, inhibitor carbons are grey, nitrogens are blue, and oxygens are red. The figure was produced using PyMOL (www.pymol.orgy

[0016] Figure 4. Time-dependence of (a) vinyl sulfone 38 and (b) β-chloro vinyl sulfone 43.

[0017] Figure 5. Mechanism of inhibition of cysteine proteases by (a) vinyl sulfones and (b) β-chloro vinyl sulfones.

[0018] Figure 6. Effect of inhibitors at 5-10 μM on survival of J744 macrophages infected with T. cruzi parasites. Survival time is defined as the time before the cell monolayer is destroyed by the infection. Engel, J. C, et al., J. Exp. Med. 1998, 188, 725-734. (a) Treatment was stopped on day 14 due to compound toxicity, (b) Treatment was stopped on day 27 to distinguish between trypanostatic and trypanocidal inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0019] "Compound of the invention," as used herein refers to a compound described herein, as well as salts (e.g. pharmaceutically acceptable salts), solvates and hydrates of the compound.

[0020] MIC, or minimum inhibitory concentration, is the point where the compound stops more than 50% of cell growth, preferably 60% of cell growth, preferably 70% of cell growth, preferably 80% of cell growth, preferably 90% of cell growth, preferably 95% of cell growth, preferably 98% of cell growth, preferably 98% of cell growth, preferably 100% of cell growth, relative to an untreated control.

[0021] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents, which would result from writing the structure from right to left, e.g., -CH₂O- is intended to also recite -OCH₂-.

[0022] The term "poly" as used herein means at least 2. For example, a polyvalent metal ion is a metal ion having a valency of at least 2.

[0023] "Moiety" refers to a radical of a molecule that is attached to the remainder of the molecule. [0024] The symbol */VV\Λ _; whether utilized as a bond or displayed perpendicular to a bond, indicates the point at which the displayed moiety is attached to the remainder of the molecule.

[0025] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. C₁-Ci_O means one to ten carbons). In some embodiments, the term "alkyl" means a straight or branched chain, or combinations thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4- pentadienyl, 3-(l ,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.

[0026] The term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified, but not limited, by -CH₂CH₂CH₂CH₂-, and further includes those groups described below as "heteroalkylene." Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.

[0027] The terms "alkoxy," "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.

[0028] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom. In some embodiments, the term "heteroalkyl," by itself or in combination with another term, means a stable straight or branched chain, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom. In an exemplary embodiment, the heteroatoms can be selected from the group consisting of O, N, S and Si, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH₂- CH₂-O-CH₃, -CH₂-CH₂-NH-CH₃, -CH₂-CH₂-N(CH₃)-CH₃, -CH₂-S-CH₂-CH₃, -CH₂-CH₂,- S(O)-CH₃, -CH₂-CH₂-S(O)₂-CH₃, -CH=CH-O-CH₃, -CH₂-CH=N-OCH₃, and -CH=CH- N(CH₃)-CH₃. Up to two heteroatoms may be consecutive, such as, for example, -CH₂-NH- OCH₃. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH₂-CH₂-S- CH₂-CH₂- and -CH₂-S-CH₂-CH₂-NH-CH₂-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkyl enedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)₂R'- represents both -C(O)₂R'- and -R⁵C(O)₂-.

[0029] The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl", respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1 -cyclohexenyl, 3- cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1 -(1,2,5,6-tetrahydropyridyl), 1 -piperidinyl, 2-piperidinyl, 3-piperidinyl, 4- morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, and the like.

[0030] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(Ci-C₄)alkyl" is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0031] The term "aryl" means, unless otherwise stated, a polyunsaturated, aromatic, substituent that can be a single ring or multiple rings (preferably from 1 to 3 rings), which are fused together or linked covalently. The term "heteroaryl" refers to aryl groups (or rings) that contain from one to four heteroatoms. In an exemplary embodiment, the heteroatom is selected from N, O, S, and Si, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2- phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4- thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2- pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, 6-quinolyl, dioxaborolane, dioxaborinane and dioxaborepane. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

[0032] For brevity, the term "aryl" when used in combination with other terms (e.g. , aryloxy, arylthioxy, arylalkyl) includes those radicals in which an aryl group is attached through the next moiety to the rest of the molecule. Thus, the term "arylalkyl" is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, l-(3- nitrophenyl)ethyl and the like). A substituent such as benzyl or l-(3-nitrophenyl)ethyl can also be represented by 'substituted alkyl' wherein the ethyl radical is substituted with a 3- nitrophenyl moiety. The term "aryloxy" is meant to include those radicals in which an aryl group is attached to an oxygen atom. The term "aryloxyalkyl" is meant to include those radicals in which an aryl group is attached to an oxygen atom which is then attached to an alkyl group (e.g., phenoxymethyl, 3-(l-naphthyloxy)propyl, and the like).

[0033] For brevity, the term "heteroaryl" when used in combination with other terms (e.g., heteroaryloxy, heteroaryl thioxy, heteroaryl alkyl) includes those radicals in which a heteroaryl group is attached through the next moiety to the rest of the molecule. Thus, the term "heteroarylalkyl" is meant to include those radicals in which a heteroaryl group is attached to an alkyl group (e.g., pyridylmethyl and the like). The term "heteroaryloxy" is meant to include those radicals in which a heteroaryl group is attached to an oxygen atom. The term "heteroaryloxyalkyl" is meant to include those radicals in which an aryl group is attached to an oxygen atom which is then attached to an alkyl group, (e.g., 2-pyridyloxymethyl and the like). [0034] Each of the above terms (e.g. , "alkyl," "heteroalkyl," "aryl" and "heteroaryl") are meant to include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0035] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are generically referred to as "alkyl group substituents," and they can be one or more of a variety of groups selected from, but not limited to: -R', -OR', =O, =NR\ =N-OR\ -NR'R", -SR', -halogen, -SiR'R"R"\ -OC(O)R', - C(O)R', -CO₂R', -CONR'R", -OC(O)NR⁵R", -NR"C(O)R', -NR'-C(O)NR"R'", - NR"C(0)₂R', -NR'""-C(NR'R"R'")=NR"", -NR""-C(NR'R")=NR'", -S(O)R', -S(O)₂R', - S(O)₂NR⁵R", -NR"SO₂R', -CN, -NO₂, -N₃, -CH(Ph)₂, fluoro(C_rC₄)alkoxy, and 1IuOrO(C₁- C₄)alkyl, in a number ranging from zero to (2m '+I), where m' is the total number of carbon atoms in such radical. R', R", R'", R"" and R'"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1 -3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'", R"" and R'"" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7- membered ring. For example, -NR'R" is meant to include, but not be limited to, 1- pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g. , -CF₃ and -CH₂CF₃) and acyl (e.g., -C(O)CH₃, -C(O)CF₃, -C(O)CH₂OCH₃, and the like).

[0036] Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are generically referred to as "aryl group substituents." The substituents are selected from, for example: -R', -OR', =O, =NR', =N-0R', -NR'R", -SR', - halogen, -SiR'R"R"\ -OC(O)R', -C(O)R', -CO₂R', -CONR'R", -OC(O)NR⁵R", - NR"C(0)R\ -NR'-C(O)NR"R"', -NR"C(0)₂R\ -NR'""-C(NR'R"R'")=NR"", -NR""-C(NR'R")=NR⁵", -S(O)R⁵, -S(O)₂R⁵, -S(O)₂NR⁵R", -NR⁵⁵SO₂R', -CN, -NO₂, -N₃, - CH(Ph)₂, fluoro(Ci-C₄)alkoxy, and fluoro(Ci-C₄)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R', R", R'", R⁵⁵⁵⁵ and R'"" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'", R"" and R'"" groups when more than one of these groups is present.

[0037] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(O)-(CRR')_q-U-, wherein T and U are independently -NR-, -O-, -CRR'- or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH₂)_r-B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)₂-, -S(O)₂NR'- or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula - (CRR')_s-X-(CR"R'")d-, where s and d are independently integers of from 0 to 3, and X is -O- , -NR'-, -S-, -S(O)-, -S(O)₂-, or -S(O)₂NR'-. The substituents R, R', R" and R'" are preferably independently selected from hydrogen or substituted or unsubstituted (Ci-C₆)alkyl.

[0038] As used herein, the term "acyl" describes a substituent containing a carbonyl residue, C(O)R. Exemplary species for R include H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl.

[0039] "Ring" as used herein, means a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. A ring includes fused ring moieties. The number of atoms in a ring is typically defined by the number of members in the ring. For example, a "5- to 7- membered ring" means there are 5 to 7 atoms in the encircling arrangement. Unless otherwise specified, the ring optionally includes a heteroatom. Thus, the term "5- to 7- membered ring" includes, for example phenyl, pyridinyl and piperidinyl. The term "5- to 7- membered heterocycloalkyl ring", on the other hand, would include pyridinyl and piperidinyl, but not phenyl. The term "ring" further includes a ring system comprising more than one "ring", wherein each "ring" is independently defined as above.

[0040] As used herein, the term "fused ring system" means at least two rings, wherein each ring has at least 2 atoms in common with another ring. "Fused ring systems may include aromatic as well as non aromatic rings. Examples of "fused ring systems" are naphthalenes, indoles, quinolines, chromenes and the like.

[0041] As used herein, the term "heteroatom" includes atoms other than carbon (C) and hydrogen (H). Examples include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

[0042] The term "leaving group" means a functional group or atom which can be displaced by another functional group or atom in a substitution reaction, such as a nucleophilic substitution reaction. By way of example, representative leaving groups include triflate, chloro, bromo and iodo groups; sulfonic ester groups, such as mesylate, tosylate, brosylate, nosylate and the like; and acyloxy groups, such as acetoxy, trifluoroacetoxy and the like.

[0043] The term "amino-protecting group" means a protecting group suitable for preventing undesired reactions at an amino nitrogen. Representative amino-protecting groups include, but are not limited to, formyl; acyl groups, for example alkanoyl groups, such as acetyl, trichloroacetyl or trifluoroacetyl; alkoxycarbonyl groups, such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups, such as benzyloxycarbonyl (Cbz) and 9- fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups, such as benzyl (Bn), trityl (Tr), and l,l-di-(4'-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl (TMS) and tert- butyldimethylsilyl (TBS); and the like.

[0044] The term "hydroxy-protecting group" means a protecting group suitable for preventing undesired reactions at a hydroxy group. Representative hydroxy-protecting groups include, but are not limited to, alkyl groups, such as methyl, ethyl, and tert-butyl; acyl groups, for example alkanoyl groups, such as acetyl; arylmethyl groups, such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl (benzhydryl, DPM); silyl groups, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS); and the like.

[0045] The symbol "R" is a general abbreviation that represents a substituent group that is selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl and substituted or unsubstituted heterocycloalkyl groups. [0046] The term "derived from" includes its plain language meaning and also refers to a molecule that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, or 60% homologous to a referenced molecule. The molecules referred to in this definition include chains of RNA or DNA, oligonucleotides, polypeptides, or proteins of any length and composition. [0047] "Peptide" refers to a polymer in which the monomers are "amino acids" and are joined together through amide bonds, alternatively referred to as a polypeptide. When the amino acids are α-amino acids, either the L-optical isomer or the D-optical isomer can be used. Additionally, non-standard amino acids, e.g., amino acids that are not gene-encoded are also of use in the compounds of the invention. All of the amino acids used in the present invention may be either the D - or L -isomer. The L -isomers are generally preferred. In addition, other peptidomimetics are also useful in the present invention. For a general review, see, Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983).

[0048] The standard amino acids of use in the present invention include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Aside from the twenty standard amino acids, there are a vast number of "nonstandard amino acids". Two of these can be encoded in the genetic code, but are rather rare in proteins. Selenocysteine is incorporated into some proteins and pyrrolysine is used by some methanogenic bacteria in enzymes that they use to produce methane. Further examples of nonstandard amino acids include lanthionine, 2-aminoisobutyric acid, dehydroalanine and the neurotransmitter gamma-aminobutyric acid. Nonstandard amino acids often occur as intermediates in the metabolic pathways for standard amino acids - for example ornithine and citrulline occur in the urea cycle, part of amino acid catabolism. Nonstandard amino acids are also formed through modifications to standard amino acids. For example, homocysteine is formed through the transsulfuration pathway or by the demethylation of methionine via the intermediate metabolite S-adenosyl methionine, while dopamine is synthesized from 1- DOPA, and hydroxyproline is made by a posttranslational modification of proline. Other non-standard amino acids of use in the compounds of the invention include the β-amino acids. Additional non-standard amino acids are β-alanine, phenylglycine and homoarginine

[0049] By "effective" amount of a drug, formulation, or permeant is meant a sufficient amount of a active agent to provide the desired local or systemic effect. A "pharmaceutically effective" or "therapeutically effective" amount refers to the amount of drug needed to effect the desired therapeutic result.

[0050] The term "pharmaceutically acceptable salts" is meant to include salts of the compounds of the invention which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0051] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compounds in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0052] In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs of the compounds or complexes described herein readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment.

[0053] Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain ^• compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0054] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are encompassed within the scope of the present invention. The graphic representations of racemic, ambiscalemic and scalemic or enantiomerically pure compounds used herein are taken from Maehr, J. Chem. Ed. 1985, 62: 114-120. Solid and broken wedges are used to denote the absolute configuration of a stereocenter unless otherwise noted. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are included.

[0055] Compounds of the invention can exist in particular geometric or stereoisomeric forms. The invention contemplates all such compounds, including cis- and trøns-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, as falling within the scope of the invention. Additional asymmetric carbon atoms can be present in a substiruent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

[0056] Optically active (R)- and (5)-isomers and d and / isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If, for instance, a particular enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis, or by derivatization with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as an amino group, or an acidic functional group, such as a carboxyl group, diastereomeric salts can be formed with an appropriate optically active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means known in the art, and subsequent recovery of the pure enantiomers. In addition, separation of enantiomers and diastereomers is frequently accomplished using chromatography employing chiral, stationary phases, optionally in combination with chemical derivatization (e.g., formation of carbamates from amines). [0057] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

[0058] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable vehicle" refers to any formulation or carrier medium that provides the appropriate delivery of an effective amount of an active agent as defined herein, does not interfere with the effectiveness of the biological activity of the active agent, and that is sufficiently non-toxic to the host or patient. Representative carriers include water, oils, both vegetable and mineral, cream bases, lotion bases, ointment bases and the like. These bases include suspending agents, thickeners, penetration enhancers, and the like. Their formulation is well known to those in the art of cosmetics and topical pharmaceuticals. Additional information concerning carriers can be found in Remington: The Science and Practice of Pharmacy, 21 st Ed., Lippincott, Williams & Wilkins (2005) which is incorporated herein by reference.

[0059] The term "excipients" is conventionally known to mean carriers, diluents and/or vehicles used in formulating drug compositions effective for the desired use.

[0060] "Biological medium," as used herein refers to both in vitro and in vivo biological milieus. Exemplary in vitro "biological media" include, but are not limited to, cell culture, tissue culture, homogenates, plasma and blood. In vivo applications are generally performed in mammals, preferably humans.

[0061] "Inhibiting" and "blocking," are used interchangeably herein to refer to the partial or full blockade of enzyme.

Compounds

[0062] In one aspect, the invention provides a compound of the invention. In an exemplary embodiment, the invention provides a compound having a structure according to a formula described herein. In another exemplary embodiment, the invention provides a compound described herein. In an exemplary embodiment, the compound has a structure according to the following formula:

wherein R¹ is a member selected from H, OR*, -C(O)R*, NR*R**, SR*, -S(O)R*, -S(O)₂R*, -S(O)₂NR*R**, nitro, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. Each R* and R** are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In an exemplary embodiment, R¹ is described herein. In an exemplary embodiment, R¹ is substituted or unsubstituted arylalkyl or substituted or unsubstituted heteroarylalkyl. In an exemplary embodiment, R¹ is a member selected from:

In another exemplary embodiment, R is a member selected from:

wherein L is a member selected from CH₂ and C(O). In an exemplary embodiment, R 1 is a member selected from:

In an exemplary embodiment, R¹ is a member selected from:

In an exemplary embodiment, R¹ is a member selected from:

wherein L is a member selected from CH₂ and C(O). R¹⁶ is a member selected from unsubstituted Ci-C₆ alkyl. In an exemplary embodiment, R¹⁶ is a member selected from methyl and ethyl. In an exemplary embodiment, R¹⁶ is a member selected from n-propyl and z^'-propyl. In an exemplary embodiment, R¹⁶ is a member selected from n-butyl, /-butyl and t- butyl.

In an exemplary embodiment, R¹ is a member selected from:

wherein L is a member selected from CH₂ and C(O). R¹⁷ is a member selected from unsubstituted Ci-C₆ alkyl. In an exemplary embodiment, R¹⁷ is a member selected from methyl and ethyl. In an exemplary embodiment, R¹⁷ is a member selected from n-propyl and /-propyl. In an exemplary embodiment, R¹⁷ is a member selected from n-butyl, /-butyl and t- butyl.

In an exemplary embodiment, R¹ is a member selected from:

wherein L is a member selected from CH₂ and C(O). X is a member selected from halogen. In an exemplary embodiment, X is a member selected from fluorine and chlorine. In an exemplary embodiment, X is a member selected from bromine and iodine. [0063] In an exemplary embodiment, the compound has a structure according to the following formula:

wherein R² is a member selected from substituted or unsubstituted Ci-C₆ alkyl, and substituted or unsubstituted C₃-C₆ cycloalkyl. R³ is a member selected from substituted or unsubstituted Ci-C₆ alkyl, and substituted or unsubstituted C₃-C₆ cycloalkyl. R⁴ is a member selected from H, OR*, -C(O)R*, NR*R**, SR*, -S(O)R*, -S(O)₂R*, -S(O)₂NR*R**, nitro, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. Each R* and R** are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In an exemplary embodiment, R² is /-propyl and R³ is n-butyl. In an exemplary embodiment, R⁴ is described herein. In an exemplary embodiment, R⁴ is:

O_{s /}O

X"^^S>h

In an exemplary embodiment, R⁴ is: o_wo

X Ph

In an exemplary embodiment, R⁴ is:

In an exemplary embodiment, R is:

In an exemplary embodiment, R⁴ is:

[0064] In an exemplary embodiment, the compound has a structure according to the following formula:

wherein R⁶ is a member selected from substituted or unsubstituted Ci-C₆ alkyl, and substituted or unsubstituted C₃-C₆ cycloalkyl. R⁷ is a member selected from substituted or unsubstituted Ci-C₆ alkyl, and substituted or unsubstituted C₃-C₆ cycloalkyl. R⁵ and R⁸ are members independently selected from H, OR*, -C(O)R*, NR*R**, SR*, -S(O)R*, -S(O)₂R*, -S(O)₂NR*R**, nitro, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. Each R* and R** are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In an exemplary embodiment, R⁵ and R⁸ are described herein. In an exemplary embodiment, R⁵ is substituted or unsubstituted arylalkyl or substituted or unsubstituted heteroarylalkyl. In an exemplary embodiment, wherein R⁵ is a member selected from:

In an exemplary embodiment, wherein R is a member selected from:

In an exemplary embodiment, wherein R⁸ is:

and R is a member selected from:

In an exemplary embodiment, wherein R⁸ is:

and R is a member selected from:

[0065] In an exemplary embodiment, the invention provides a compound having a structure according to a formula which is a member selected from:

wherein R⁹ is a member selected from H, OR*, -C(O)R*, NR*R**, SR*, -S(O)R*, -S(O)₂R*, -S(O)₂NR*R**, nitro, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. Each R* and R** are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

R¹⁰, R¹ ', R¹² and R¹³ are members independently selected from H, OR*, NR*R**, SR*, - S(O)R*, -S(O)₂R*, -S(O)₂NR*R**, nitro, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. Each R* and R** are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

In an exemplary embodiment, R is substituted or unsubstituted arylalkyl or substituted or unsubstituted heteroarylalkyl.

[0066] In an exemplary embodiment, the compound has a structure according to the following formula:

wherein R¹⁴ is a member selected from substituted or unsubstituted alkyl, and substituted or unsubstituted cycloalkyl. R¹⁵ is a member selected from substituted or unsubstituted alkyl, and substituted or unsubstituted cycloalkyl. In an exemplary embodiment, R¹⁴ and R¹⁵ are described herein. In an exemplary embodiment, R¹⁴ is a member selected from substituted or unsubstituted CpC₆ alkyl, and substituted or unsubstituted C₃-C₆ cycloalkyl. In an exemplary embodiment, R¹⁴ is a member selected from unsubstituted C₁-C₆ alkyl and unsubstituted C₃- C₆ cycloalkyl. In a specific exemplary embodiment, R¹⁴ is a member selected from z-propyl, /-butyl, cyc/o-propyl, cyc/o-butyl, cyc/o-pentyl, and cyclo-hexyl.

In an exemplary embodiment, R¹⁵ is a member selected from substituted or unsubstituted Ci- C₆ alkyl, and substituted or unsubstituted C₃-C₆ cycloalkyl. In an exemplary embodiment, R¹⁵ is a member selected from unsubstituted Ci-C₆ alkyl. In a specific exemplary embodiment, R¹⁵ is a member selected from methyl, ethyl, and H-butyl.

[0067] High correlation between substrate cleavage efficiency and inhibitory activity was observed in the previous development of cathepsin S inhibitors. Wood, W. J. L., et al. J. Am. Chem. Soc. 2005, 127, 15521-15527; Patterson, A. W., et. al., J. Med. Chem. 2006, 49, 6298- 6307. Substrate analogues were therefore first evaluated and optimized before conversion to inhibitors. A triazole-based substrate library consisting of more than 150 substrates was screened against cruzain. Substrate activity was measured by monitoring liberation of the 7- amino-4-methyl coumarin acetic acid (AMCA) fluorophore, which results from protease- catalyzed amide bond hydrolysis (Scheme 1).

Scheme 1. Fluorogenic substrate screening

Fluorogenic substrate library Fluorescent

[0068] Shown in Table 1 is the structure activity relationship (SAR) for a subset of substrates from the triazole library that exemplifies cruzain's substrate specificity requirements. The weakest substrate for which a signal could be detected was substrate 2 that incorporated a simple benzyl substituent on the triazole ring. A variety of more active hydroxyl substituted substrates were screened and the optimal aliphatic functionalities identified were the methyl and isopropyl substituents present in substrate 4. Replacement of the hydroxyl with a benzamide moiety in substrate 5 resulted in an increase in cleavage efficiency. The epimeric compounds 6 and 7 demonstrate that cruzain shows strong chiral recognition with epimer 7 being much more active. Substitutions on the benzamide moiety indicated that ortho substitutions are not well tolerated by cruzain (substrates 8 and 11). In contrast, meta and para substituents resulted in increases in substrate activity (substrates 9, 10 and 12, 13) with /?-methoxy substituted benzamide substrate 13 identified as the most efficient substrate from the screening of the initial triazole library.

Table 1. Representative Substrates from the Initial 1,2,3-Triazole Library

[0069] Structure-guided Substrate Optimization. Further optimization of substrate binding to cruzain was accomplished using structure-based design. Recently, a crystal structure was obtained of chloromethyl ketone inhibitor 14 bound to cathepsin S. Patterson, A. W., et al., J. Med. Chem. 2006, 49, 6298-6307 The amino acid sequences of cathepsin S and cruzain are 38% homologous and their active sites nearly identical (Figure 2). Bromme, D.; McGrath, M. E. Protein Science 1996, 5, 789-791. Taking into account the high homology between these two enzymes, molecular replacement was performed to model inhibitor binding to cruzain (Figure 3).

[0070] Due to the similarities in the active sites, the inhibitors are predicted to bind in a similar fashion with the n-butyl group in the Sl pocket, the methyl and isopropyl groups in the S2 pocket, and the benzamide moiety in the S3 pocket. The majority of prior inhibitor development for cruzain has focused on the Sl ', Sl, and S2 pockets. The S3 pocket of cruzain is largely unexplored with no previous reports of significant binding interactions in this pocket. Upon closer inspection of our molecular replacement model, key differences in the S3 pockets were noted. The S3 pocket of cathepsin S is small and well-defined whereas that of cruzain is large and open-ended. To take advantage of cruzain's larger S3 pocket, a focused library of substrate analogues incorporating planar heterocycles in place of the phenyl ring of the benzamide moiety was designed. Heterocycles were chosen with potential for hydrophobic interactions with the hydrophobic side of the pocket and with potential for hydrogen bonding interactions with the serine residue in the S3 pocket.

[0071] The synthesis of the 1 ,4-disubstituted-l,2,3-triazole substrates containing the AMCA fluorophore was accomplished on solid support (Scheme 2). All but one of the substrates of the focused library were more active than the unsubstituted benzamide substrate 7 (Table 2). The most potent substrates identified were quinoline 30 and benzothiazole 31 with 7-9 fold increases in cleavage efficiency. These substrates both contained a nitrogen atom para to the amide bond, which could be interacting with the serine residue in the S3 pocket (Figure 3b). When comparing naphthyl 20 and quinoline 30, a 4- fold increase in activity was observed presumably due to this polar interaction. Moreover, the 4-fold increase in cleavage efficiency of indole 29 relative to benzotriazole 21 demonstrated that hydrophobic interactions also contribute to binding. Substrate 18 incorporating a morpholine moiety was prepared because this substituent has led to potent vinyl sulfone inhibitors of cruzain. Palmer, J. T., et al., J. Med. Chem. 1995, 38, 3193-3196. However, this substituent resulted in a decrease in substrate cleavage efficiency.

Scheme 2. General Synthesis of Focused Library 1,2,3-Triazole Substrates"

a Reagents: (a) CuI, /-Pr₂EtN, THF, rt; (b) RCO₂H, triphosgene, THF, rt; (c) RCOH, NaBH(OAc)₃, AcOH, THF, rt; (d) CF₃CO₂H, CH₂Cl₂, rt. Table 2. Cleavage Efficiencies of 1,2,3 -Triazole Amide Substrates against Cruzain

[0072] A notable feature in the inhibitor binding model is the nonessential nature of the benzamide carbonyl (Figure 3b). Therefore, amine 33 corresponding to benzamide substrate 7 was prepared resulting in a 3-fold increase in cleavage efficiency (Table 3). To determine if amine substrate SAR correlated with the SAR trends observed for the corresponding amide substrates, additional amine analogues were synthesized and evaluated. High correlation was observed between the SAR for the amide and amine substrate series, resulting in the identification of quinoline amine substrate 36 and benzothiazole amine substrate 37 with 19- fold greater cleavage efficiency than unsubstituted benzamide 7.

Table 3. Comparison of 1,2,3 -Triazole Amide and Amine Substrate Activity against Cruzain

Cmpd ReI k_alIK_m Cmpd ReI W cat'K₁

[0073] Conversion of Substrates into Inhibitors. In an exemplary embodiment, the aminocoumarin group can be precisely oriented in the active site for amide bond hydrolysis to occur and can therefore be replaced with mechanism-based pharmacophores. The optimal quinoline amine substrate 36 was first converted to inhibitors to evaluate the effectiveness of different cysteine protease mechanism-based pharmacophores.

[0074] The vinyl sulfone pharmacophore was initially chosen because it has been incorporated in potent inhibitors of cruzain that have proven effective at eradicating Chagas' disease in both cell culture and animal models. Engel, J. C, et al., J. Exp. Med. 1998, 188, 125-114; Barr, S. C, et al., Antimicrob. Agents Chemother. 2005, 49, 5160-5161 ; Palmer, J. T., et al., J. Med. Chem. 1995, 38, 3193-3196; Roush, W. R., et al., J. Am. Chem. Soc. 1998, 120, 10994-10995. Vinyl sulfone inhibitor 38 was prepared via a Horner-Wadsworth- Emmons olefination (Scheme 3). Kinetic analysis of the vinyl sulfone inhibitor, surprisingly, indicated no time dependence and was consistent with competitive reversible inhibition (Figure 4a). Scheidt, K. A., et al., J. Biorg. Med. Chem. 1998, 6, 2477-2494. Scheme 3. Synthesis of Vinyl Sulfone Inhibitor 38^α

38 a Reagents: (a) CuI, /-Pr₂EtN, THF, rt; (b) diazomethane, THF, rt; (c) DIBAL-H, Et₂O, -78 ⁰C; (d) (OEt)₂P(O)CH₂SO₂Ph, LiBr, NEt₃, CH₃CN, -40 ⁰C.

[0075] To gain further insight, reversible nature of vinyl sulfone inhibitor 38 was investigated. Vinyl sulfones are thought to irreversibly alkylate cysteine proteases via a Michael addition followed by protonation of the α-carbon by the active site histidine to form a covalent thioether adduct (Figure 5a). Powers J. C, et al., Chem. Rev. 2002, 102, 4639- 4750. Two potential reasons for the reversibility of vinyl sulfone 38 are therefore either that the active site cysteine is not adding into the vinyl sulfone or that the active site histidine is not properly oriented for protonating the resulting anion. We postulated that a β-chloro vinyl sulfone could distinguish between these possibilities because cysteine protease inactivation could be accomplished via Michael addition followed by β-elimination of chloride ion thereby eliminating the need for anion protonation (Figure 5b). Although this particular pharmacophore has never before been investigated, it is analogous to previously characterized β-chloro α,β-unsaturated ester inhibitors. Govardhan, C. P.; Abeles, R. H. Arch. Biochem. Biophys. 1996, 330, 1 10-114.

[0076] β-Chloro vinyl sulfone inhibitor 43 was prepared according to the route depicted in Scheme 4 with the key step being conversion of ketosulfone 45 to vinyl chloride 46 via the vinyl triflate. Gratifyingly, time-dependence analysis and dilution experiments indicated that β-chloro vinyl sulfone inhibitor 43 was an irreversible inhibitor of cruzain (Figure 4b). This result suggests that the active site cysteine is adding into vinyl sulfone 38 and that the lack of a protonation event resulted in a reversible inhibitor. The β-chloro sulfone inhibitor 43 had a modest second order rate of inactivation constant of 805 s^" M^"1 (Table 4). Encouraged by this result, other pharmacophores that irreversibly inactivate cysteine proteases according to mechanisms that do not utilize a protonation step were explored. Scheme 4. Synthesis of β-Chloro Vinyl Sulfone Inhibitor 43^α

OH OMe

Bu Bu 40 44

a Reagents: (a) diazomethane, THF, rt; (b) methylphenylsulfone, H-BuLi, THF, 0 ⁰C to -78 ⁰C; (c) Tf₂O, /-Pr₂EtN, THF, -20 ⁰C to rt; (d) TBACl, THF, rt; (e) Na ascorbate, CuSO₄, 1 :1 H₂O:t-BuOH, 39, rt.

Table 4. Second-Order Inactivation Rates of Cruzain Inhibitors with Varying Pharmacophores

Cmpd R fc,_nact/K, (s-¹M-¹)^a pK_a"

o ^F3°γ^f]

50 157,000 ± 1,520 0.58"

O CF₃

a Tests were performed in quadruplicate (S.D. values included). b pK_a of the carboxylic acid or phenol leaving group. ⁰ No time- dependence observed. ^d Ref 19. ^e Ref 18.

[0077] Acyloxymethyl ketone inhibitors were designed by Krantz as more stable halomethyl ketone analogues. Smith, R. A., et al., J. Am. Chem. Soc. 1988, 110, 4429-4431; Krantz, A., et al., Biochemistry 1991, 30, 4678-4687. This pharmacophore has led to potent time-dependent inhibitors of cathepsins B, L, and S. Powers J. C, et al., Chem. Rev. 2002, 102, 4639-4750. Hence, the acyloxymethyl ketone pharmacophore were incorporated next. The synthesis of the acyloxymethyl ketone inhibitors required the preparation of azide intermediates 47a and 47b (Scheme 5). Beginning with L-norleucine azido acid 40, the common bromomethyl ketone precursor 48 was obtained in three steps. Displacement of the bromide followed by cyclization with propargyl amine 39 then afforded the acyloxymethyl ketone inhibitors 49 and 50 as mixtures of diastereomers.

Scheme 5. Synthesis of Aryl- and Acyl-oxymethyl Ketone Inhibitors"

α Reagents: (a) isobutyl chloroformate, N-methylmorpholine, THF, -40 ⁰C; (b) diazomethane, THF, 0 ⁰C; (c) HBr, THF, 0 ⁰C; (d) KF, DMF, 0 ⁰C; (e) Na ascorbate, CuSO₄, 1 :1 H₂O:f-BuOH, rt.

[0078] The 2,6-dimethyl acyloxymethyl ketone inhibitor 49 was initially investigated and observed to be an irreversible inhibitor of cruzain with a second-order rate constant of 2,690 S^-1M^"1 (Table 4). Acyloxymethyl ketone inhibitors of the cathepsins have shown a strong correlation between the leaving group pK_a and the rate of inactivation. Krantz, A. Methods Enzymol. 1994, 244, 656-671. Accordingly, the 2,6-bis-trifluoromethyl acyloxymethyl ketone inhibitor 50 was prepared and found to be 58-fold more potent than inhibitor 49 with a second-order rate constant of 157,000 8^"1M^"1 (Table 4). Inhibitors incorporating this pharmacophore were subsequently prepared corresponding to both the amides and amines of the benzothiazole and quinoline substrates (Scheme 5). There was good correlation between substrate activity and inhibitor potency with the amine inhibitors 50 and 51 being more potent than the amide inhibitors 52 and 53 (Table 5). Table 5. Second-Order Inactivation Rates of 1,2,3-Triazole Cruzain Inhibitors⁰

1 ,520" 54 147,000 ± 6,790⁶ 5,000^C 55 105,000 ± 2,910^c

a Tests were performed in quadruplicate (S D values included) ^bΛ,nact /K₁ (S-¹M^"1) ^C fc_ass (s-¹M-¹) ²⁴

[0079] The aryloxymethyl ketone pharmacophore has the same mechanism of inhibition as the acyloxymethyl ketone pharmacophore. It is more attractive, however, because it should be less prone to nucleophilic attack, cannot undergo hydrolysis, and has a lower molecular weight. This pharmacophore has proven to be particularly effective for caspase inhibition. Brady, K. D., et al., Biorg. Med. Chem. 1999, 7, 621-631; Brady, K. D. Biochemistry 1998, 37, 8508-8515.

[0080] In particular, Idun pharmaceuticals used 2,3,5,6-tetrafluorophenol as the leaving group in an aryloxymethyl ketone pan-caspase inhibitor that has progressed to Phase II clinical trials. Linton, S. D.; et al. J. Med. Chem. 2005, 48, 6779-6782. In contrast, there has only been one report of aryloxymethyl ketone inhibitors of a member of the papain superfamily, and only modest inhibition was observed. Smith, R. A. et al., J. Am. Chem. Soc. 1988, 110, 4429-4431. Nevertheless, we prepared 2,3,5,6-tetrafluorophenol aryloxymethyl ketone inhibitor 54 (Scheme 5). Unexpectedly, the aryloxymethyl ketone inhibitor 54 was equipotent to the acyloxymethyl ketone inhibitors with a second-order inactivation constant of 147,000 8^'1M^"1 despite the >10⁵ difference in the acidity of the corresponding leaving groups (pAT_a of 2,3,5,6-tetrafluorophenol = >5.5, pK_a of 2,6-bis-trifluoromethyl benzoic acid = 0.58) (Table 4). Inhibitor potency is clearly not solely dependent on the pK_a values of the leaving group with leaving group binding and/or orientation also playing a significant role. Brady, K. D., et al., Biorg. Med. Chem. 1999, 7, 621-631. [0081] The preparation of both the acyl- and aryl-oxymethyl ketone inhibitors resulted in epimerization alpha to the pharmacophore carbonyl. To investigate the configurational stability of the inhibitors in vivo, an alternative sequence for the preparation of diastereomerically pure aryloxymethyl ketone inhibitor 54 was developed (Scheme 6). Specifically, racemization through enolization was prevented by reducing the acyloxymethyl ketone 47c prior to the cycloaddition step. Alcohol 57 was then oxidized back to the acyloxymethyl ketone. The diastereomerically pure inhibitor 58 was then subjected to the assay buffer conditions at 37 ⁰C for several hours (Scheme 7). This resulted in complete racemization of the inhibitor, suggesting that in vivo the inhibitor will be able to funnel through the active diastereomer.

Scheme 6. Synthesis of Diastereomerically Pure Aryloxymethyl Ketone Inhibitor 58"

a Reagents: (a) NaBH₄, 95:5 THF:H₂O, 0 ⁰C to it; (b) Na ascorbate, CuSO₄, 1 :1 H₂O:t- BuOH, rt; (c) Dess-Martin periodonane, CH₂Cl₂, rt.

Scheme 7. Configurational Lability of Aryloxymethyl Ketone Inhibitor 54

37 °C

[0082] Additional analogs (73-81), similar to the Aryloxymethyl Ketone Inhibitor 54, were synthesized in an analogous way to that described above. Scheme 8. Synthesis of 2,3,5,6-tetrafluorophenoxymethyl ketone azides 47c and 61-62

Scheme 9. Synthesis of quinoline propargyl amines 39 and 68-72

16, R = /-Propyl 39, R = /-Propyl

63, R = cyc/o-Propyl 68, R = cyc/o-Propyl

64, R = /-Butyl 69, R = /-Butyl

65, R = cyc/o-Butyl 70, R = cyc/o-Butyl

66, R = cyc/o-Pentyl 71, R = cyc/o-Pentyl

67, R = cyc/o-Hexyl 72, R = cyc/o-Hexyl

Scheme 10. Synthesis of 1,2,3-triazole compounds 73-81

39, R₁ /-Propyl 47c, R = n-Bu

68, R₁ cyc/o-Propyl 61, R = Et 73, R₁ = /-Propyl, R₂ = Et

69, R₁ : /-Butyl 62, R = Me 74, R₁ = /-Propyl, R₂ = Me

70, R₁ ^■■ cyc/o-Butyl 75, R₁ = cyc/o-Propyl, R₂ = n-Bu 71, R₁ cyc/o-Pentyl 76, R₁ = /-Butyl, R₂ = n-Bu cyc/o-Hexyl 77, R₁ = /-Butyl, R₂ = Et

78, R₁ = cyc/o-Butyl, R₂ = n-Bu

79, R₁ = cyc/o-Butyl, R₂ = Et

80, R₁ = cyc/o-Pentyl, R₂ = n-Bu

81, R₁ = cyc/o-Hexyl, R₂ = n-Bu

[0083] To further improve the potency and/or pharmacokinetics of inhibitor 54, analogues were prepared varying the substituents that bind in the Sl and S2 pockets. The Sl pocket is solvent-exposed, which prompted exploring inhibitors with shorter alkyl chains. Truncating the n-Bu moiety to an ethyl, as in inhibitor 73, resulted in an equally potent inhibitor. However, further truncating the Sl -binding moeity to a methyl resulted in a significant decrease in inhibitory activity for inhibitor 74. The ring size of the substituent binding in the hydrophobic S2 pocket was explored with inhibitors 75 and 78-81. It was found that larger rings, such as the cyc/o-butyl and cyc/o-pentyl, as in inhibitors 78 and 80, respectively, resulted in ~4-fold increases in potency. Inhibitor 81, which incorporates a cyc/o-hexyl substituent, has reduced activity potentially reflecting unfavorable steric interactions with the S2 pocket. Inhibitor 75 containing the larger /-butyl substituent also increased the inhibitory activity by ~4-fold. These SAR studies resulted in the development of inhibitors 77 and 79 which are 4-fold more potent in addition to having a reduced molecular weight, log P, and number of rotatable bonds.

Table 6. Second-Order Inactivation Rates for Cruzain Inhibitors

second-order inactivation inhibitor Ri R₂ constant (8^"1M^"1)

54 /-propyl w-butyl 147,000 ± 7,000' ^b

73 /-propyl ethyl 122,000 ± 34,000 °

74 /-propyl methyl 9j400 ± 200⁶

75 cyc/o-propyl n-butyl 178,000 ± 23,000 °

76 /-butyl n-butyl 680,000 ± 160,000°

77 /-butyl ethyl 652,000 ± 13,000 *

78 ςyc/o-butyl n-butyl 547,000 ± 9,000 ^b

79 cyclo-bvXy\ ethyl 584,000 ± 162,000°

80 cyc/o-pentyl n-butyl 433,000 ± 6,000 ^b

81 cyclo-hexyl n-butyl 241,000 ± 67,000 °

^inact IK.\. /c_ass.

[0084] Cell Culture Assays. Acyloxymethyl ketone inhibitors 50-53 and aryloxymethyl ketone inhibitors 54-55 were tested for their effectiveness in eliminating T. cruzi infection in irradiated (9000 rad) J744 macrophages. Infected host cells died after 5 days without treatment (Figure 6). Notably, all of the inhibitors significantly delayed the parasites' intracellular replication at concentrations of 5-10 μM. Toxicity to the mammalian host cells was observed at 10 and 5 μM with the 2,6-bis-trifluoromethyl acyloxymethyl ketone inhibitors 50, 51, and 52. As a result, it was necessary to stop treatment by day 14 after which point the cells died. Acyloxymethyl ketone inhibitor 53 was quite effective at 10 μM in delaying T. cruzi replication, however, by day 23 the cell monolayer had been destroyed by the infection.

[0085] To distinguish between trypanostatic compounds that only delay parasite replication and trypanocidal compounds that effectively kill T. cruzi, treatment for the remaining aryloxymethyl ketone inhibitors was ended on day 27 and the cells were monitored for two more weeks. Most significantly, the quinoline aryloxymethyl ketone inhibitor 54 was trypanocidal at 10 μM and had completely eradicated the T. cruzi parasite with no parasites observed at day 40 post-infection. The performance of inhibitor 54 was comparable to vinyl sulfone 1, which is the most advanced inhibitor of cruzain to date.

[0086] A potent irreversible 2,3,5,6-tetrafluorophenoxymethyl ketone inhibitor 54 was developed that eradicates T. cruzi parasites in cell culture. A substrate library containing more than 150 triazole-based substrates developed for cathepsin S, was first evaluated against cruzain to define important structural features for efficient substrate cleavage. Subsequent optimization of the substrate scaffold in the S3 pocket was guided by structure-based design and led to nonpeptidic substrates with even greater cleavage efficiency.

[0087] Vinyl sulfone, β-chloro vinyl sulfone, acyl- and aryloxymethyl ketone pharmacophores were then explored in the conversion of the most efficient substrates to inhibitors. The β-chloro vinyl sulfone pharmacophore, which had not previously been reported, led to key mechanistic insight and ultimately resulted in the development of potent irreversible aryloxymethyl ketone inhibitors, a pharmacophore class that had previously been little explored against the papain superfamily.

[0088] Evaluation of 2,3,5,6-tetrafluorophenoxymethyl ketone inhibitor 54 in animal models of the disease is currently underway. The nonpeptidic nature of this potent class of inhibitors, coupled with their potent cell-based activity, makes these compounds very promising starting points for the development of chemotherapy for Chagas' disease.

Methods

[0089] In another aspect, the invention provides a method of inhibiting cruzain, said method comprising: (i) contacting the cruzain with a compound of the invention, in an amount effective to inhibit the cruzain, thereby inhibiting the cruzain.

[0090] In an exemplary embodiment, the invention is a method of killing or inhibiting the growth of a protozoa, said method comprising: (i) contacting said protozoa with a compound of the invention, in an amount effective to kill or inhibit the growth of said protozoa. In an exemplary embodiment, said protozoa belongs to the order of Trypanosomes. In an exemplary embodiment, said protozoa is a Trypanosoma. In an exemplary embodiment, the protozoa is Trypanosoma cruzi. In another exemplary embodiment, the protozoa is Trypanosoma brucei.

[0091] In an exemplary embodiment, the invention is a method of treating a disease described herein, said method comprising administering a therapeutically effective amount of a compound of the invention, to an animal suffering from the disease, thereby treating said disease. In an exemplary embodiment, the disease is associated with a protozoa. In an exemplary embodiment, the disease is associated with a Trypanosoma. In an exemplary embodiment, the disease is a trypanosomal disease, hi an exemplary embodiment, said trypanosomal disease is a trypanosomiasis (a disease in vertebrates caused by protozoa trypanosomes of the genus Trypanosoma). In an exemplary embodiment, the trypanosomiasis is a member selected from Chagas disease, sleeping sickness, Nagana, Surra, or Dourine (covering sickness). In an exemplary embodiment, the disease is associated with Trypanosoma cruzi. In an exemplary embodiment the disease is Chagas disease. In an exemplary embodiment, the disease is Human American trypanosomiasis. In another exemplary embodiment, the disease is associated with a Trypanosoma brucei. In an exemplary embodiment the disease is sleeping sickness. In an exemplary embodiment the disease is Human African trypanosomiasis. In an exemplary embodiment the disease is Nagana. In an exemplary embodiment, the animal is a mammal. In an exemplary embodiment, the animal is a cow. In an exemplary embodiment, the animal is a human.

Pharmaceutical Formulations

[0092] While compounds of the present invention can be administered as the raw chemical, it is preferable to present them as a pharmaceutical composition. Therefore, in another aspect, the invention is a pharmaceutical composition comprising: a) a compound of the invention; b) a pharmaceutically acceptable excipient. In an exemplary embodiment, the present invention provides a pharmaceutical composition comprising a compound described herein or a pharmaceutically acceptable salt, hydrate or solvate thereof, together with one or more pharmaceutical carrier and optionally one or more other therapeutic ingredients. The carrier(s) are "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The term "pharmaceutically acceptable carrier" includes vehicles, diluents, excipients and other elements appropriate for incorporation into a pharmaceutical formulation.

[0093] The formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal and topical (including dermal, buccal, sublingual and intraocular) administration, as well as those for administration by inhalation. The most suitable route may depend upon the condition and disorder of the recipient. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association a compound or a pharmaceutically acceptable salt or solvate thereof ("active ingredient") with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation. Oral formulations are well known to those skilled in the art, and general methods for preparing them are found in any standard pharmacy school textbook, for example, Remington: The Science and Practice of Pharmacy., A. R. Gennaro, ed. (1995), the entire disclosure of which is incorporated herein by reference.

[0094] Pharmaceutical compositions containing compounds of the invention may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. Preferred unit dosage formulations are those containing an effective dose, or an appropriate fraction thereof, of the active ingredient, or a pharmaceutically acceptable salt thereof. The magnitude of a prophylactic or therapeutic dose typically varies with the nature and severity of the condition to be treated and the route of administration. The dose, and perhaps the dose frequency, will also vary according to the age, body weight and response of the individual patient.

[0095] In general, the total daily dose ranges from about 0.1 mg per day to about 7000 mg per day, preferably about 1 mg per day to about 100 mg per day, and more preferably, about 25 mg per day to about 50 mg per day, in single or divided doses. In some embodiments, the total daily dose may range from about 50 mg to about 500 mg per day, and preferably, about 100 mg to about 500 mg per day.

[0096] Different therapeutically effective amounts may be applicable for different proliferative disorders, as will be readily known by those of ordinary skill in the art. Similarly, amounts sufficient to prevent, manage, treat or ameliorate such proliferative disorder, but insufficient to cause, or sufficient to reduce, adverse effects associated with the compounds of the invention are also encompassed by the above described dosage amounts and dose frequency schedules. Further, when a patient is administered multiple dosages of a compound of the invention, not all of the dosages need be the same. For example, the dosage administered to the patient may be increased to improve the prophylactic or therapeutic effect of the compound or it may be decreased to reduce one or more side effects that a particular patient is experiencing. [0097] In a specific embodiment, the dosage of the composition of the invention or a compound of the invention administered to prevent, treat, manage, or ameliorate a cell proliferative disorder or one or more symptoms thereof in a patient is 150 μg /kg, preferably 250 μg/kg, 500 μg /kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, or 200 mg/kg or more of a patient's body weight. In another embodiment, the dosage of the composition of the invention or a compound of the invention administered to prevent, treat, manage, or ameliorate a proliferative disorder or one or more symptoms thereof in a patient is a unit dose of 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7m g, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

[0098] It is further recommended that children, patients over 65 years old, and those with impaired renal or hepatic function, initially receive low doses and that the dosage is titrated based on individual responses and/or blood levels. It may be necessary to use dosages outside these ranges in some cases, as will be apparent to those in the art. Further, it is noted that the clinician or treating physician knows how and when to interrupt, adjust or terminate therapy in conjunction with individual patient's response.

[0099] It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

[0100] Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

[0101] A tablet may be made by compressing or molding a compound described herein optionally using one or more additional ingredient. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide sustained, delayed or controlled release of the active ingredient therein. Oral and parenteral sustained release drug delivery systems are well known to those skilled in the art, and general methods of achieving sustained release of orally or parenterally administered drugs are found, for example, in Remington: The Science and Practice of Pharmacy, pages 1660-1675 (1995).

[0102] Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Formulations for parenteral administration also include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit-dose of multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example saline, phosphate-buffered saline (PBS) or the like, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter or polyethylene glycol. Formulations for topical administration in the mouth, for example, buccally or sublingually, include lozenges comprising the active ingredient in a flavored basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerin or sucrose and acacia.

[0103] The pharmaceutically acceptable carrier may take a wide variety of forms, depending on the route desired for administration, for example, oral or parenteral (including intravenous). In preparing the composition for oral dosage form, any of the usual pharmaceutical media may be employed, such as, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents in the case of oral liquid preparation, including suspension, elixirs and solutions. Carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders and disintegrating agents may be used in the case of oral solid preparations such as powders, capsules and caplets, with the solid oral preparation being preferred over the liquid preparations. Preferred solid oral preparations are tablets or capsules, because of their ease of administration. If desired, tablets may be coated by standard aqueous or nonaqueous techniques. Oral and parenteral sustained release dosage forms may also be used.

[0104] Exemplary formulations, are well known to those skilled in the art, and general methods for preparing them are found in any standard pharmacy school textbook, for example, Remington, THE SCIENCE AND PRACTICE OF PHARMACY, 21 st Ed., Lippincott.

[0105] Since one aspect of the present invention contemplates the treatment of the disease/conditions with a combination of pharmaceutically active agents that may be administered separately, the invention further relates to combining separate pharmaceutical compositions in kit form. The kit comprises two separate pharmaceutical compositions: a compound of the present invention, and a second pharmaceutical compound. The kit comprises a container for containing the separate compositions such as a divided bottle or a divided foil packet. Additional examples of containers include syringes, boxes, bags, and the like. Typically, the kit comprises directions for the administration of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.

[0106] An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process recesses are formed in the plastic foil. The recesses have the size and shape of the tablets or capsules to be packed. Next, the tablets or capsules are placed in the recesses and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil which is opposite from the direction in which the recesses were formed. As a result, the tablets or capsules are sealed in the recesses between the plastic foil and the sheet. Preferably the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.

[0107] It may be desirable to provide a memory aid on the kit, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen which the tablets or capsules so specified should be ingested. Another example of such a memory aid is a calendar printed on the card, e.g., as follows "First Week, Monday, Tuesday, . . . etc . . . Second Week, Monday, Tuesday, . . . " etc. Other variations of memory aids will be readily apparent. A "daily dose" can be a single tablet or capsule or several pills or capsules to be taken on a given day. Also, a daily dose of a compound of the present invention can consist of one tablet or capsule, while a daily dose of the second compound can consist of several tablets or capsules and vice versa. The memory aid should reflect this and aid in correct administration of the active agents.

[0108] In another specific embodiment of the invention, a dispenser designed to dispense the daily doses one at a time in the order of their intended use is provided. Preferably, the dispenser is equipped with a memory-aid, so as to further facilitate compliance with the regimen. An example of such a memory-aid is a mechanical counter which indicates the number of daily doses that has been dispensed. Another example of such a memory-aid is a battery-powered micro-chip memory coupled with a liquid crystal readout, or audible reminder signal which, for example, reads out the date that the last daily dose has been taken and/or reminds one when the next dose is to be taken.

[0109] The materials, methods and devices of the present invention are further illustrated by the examples that follow. These examples are offered to illustrate, but not to limit the claimed invention.

EXAMPLES

General synthetic methods

[0110] Unless otherwise noted, all reagents were obtained from commercial suppliers and used without purification. Tetrahydrofuran (THF), diethyl ether, methylene chloride (CH₂Cl₂), and toluene were obtained from a Seca Solvent Systems by GlassContour (solvent dried over alumina under a N₂ atmosphere). Anhydrous DMF (water <50 ppm) was purchased from Acros. Diisopropylethylamine (/-Pr₂EtN) was distilled over CaH₂. Wang resin was purchased from Novabiochem (San Diego, CA), <9-(7-azabenzotriazol-l-yl)- N,JV,N',JV'-tetramethyluronium hexafluorophosphate (HATU) was purchased from PerSeptive Biosystems (Foster City, CA), p-toluenesulfonylmethylnitrosamide (Diazald) was purchased from Sigma- Aldrich. Fmoc-protected 7-amino-4-methyl coumarin acetic acid (Fmoc- AMCA) was synthesized according to a method analogous to the synthesis of 7-amino-4- carbamoylmethyl coumarin (AMC). www.who.int/tdr/diseases/chagas/direction.htm (S)-tert- Butanesulfinamide was provided by AllyChem Co. Ltd (Dalian, China). Propargyl amines 16 and 63-67 were synthesized as previously reported. Patterson, A. W.; Ellman, J. A. J. Org. Chem. 2006, 71, 7110-7112. Compounds 2-13, 15, 20, 33-34, 40, and 44 were synthesized as previously reported. Wood, W. J. L.; Patterson, A. W.; Tsuruoka, H.; Jain, R. K.; Ellman, J. A. J. Am. Chem. Soc. 2005, 727, 15521-15527. All solution-phase reactions were carried out in flame-dried glassware under an inert N₂ atmosphere. Solid-phase reactions were conducted in polypropylene cartridges equipped with 70 mm PE frits (Applied Separations, Allentown, PA) and Teflon stopcocks, and were rocked on an orbital shaker. Reverse-phase HPLC analysis and purification were conducted with an Agilent 1100 series instrument. ¹H, ¹³C, ¹⁹F NMR spectra were obtained on a Bruker AV-300, AVB-400, AVQ-400, or DRX-500 at room temperature. Chemical shifts are reported in ppm, and coupling constants are reported in Hz. ¹H resonances are referenced to CHCl₃ (7.26 ppm) or DMSO-d₆ (4.90 ppm), ¹³C resonances are referenced to CHCl₃ (77.23 ppm) or DMSOd₆ (39.50 ppm), and ¹⁹F resonances are referenced to CFCl₃ (0 ppm). IR spectra were recorded on a Nicolet MAGNA-IR 850 spectrometer. Melting points were determined on a Laboratory Devices Mel-Temp 3.0 and are reported uncorrected. Elemental analyses and high-resolution mass spectrometry analyses were performed by the University of California at Berkeley Microanalysis and Mass Spectrometry Facilities.

EXAMPLE 1 General synthetic methods for aminocoumarin substrates.

18 36 37

General synthesis of urea substrate 18 and amine substrates 36-37 (Procedure A) [0111] To resin 15 (0.35-0.65 mmol/g, 1 equiv), preswollen in THF, was added a 0.02M solution of a propargylamine intermediate (1-2.2 equiv) and /-Pr₂EtN (100 equiv) in THF. CuI (3 equiv) was then added and the mixture was shaken for 48 h. After removal of the solution, the resin was washed with three portions (20 mL) each of THF, CH₃OH, CH₃CN, THF, and CH₂Cl₂, and then the product was cleaved from support and purified following Procedure C (vide infra). . _u

General synthesis of amide substrates 19 and 22-31 (Procedure B)

[0112] To resin 15 (0.115 g, 0.0750 mmol), preswollen in THF, was added a 0.02 M solution of the HCl salt of propargylamine 16 (0.025 g, 0.17 mmol) and Z-Pr₂EtN (1.3 mL, 7.5 mmol) in THF (8.2 mL). CuI (0.043 g, 0.23 mmol) was then added and the mixture was shaken for 48 h. After removal of the solution, the resin was washed with three portions (20 mL) each of THF, CH₃OH, CH₃CN, and THF to afford support-bound amine intermediate 17. After washing derivatized resin 17, /-Pr₂EtN (8 equiv) was added. To a 0.1 M solution of carboxylic acid (3.3-3.5 equiv) in THF with triphosgene (1.1 equiv) was added 2,4,6- collidine (10 equiv). The resulting slurry was stirred for about 1 min and was then added to the cartridge containing resin 17. The resulting mixture was shaken for 4-12 h. After removal of the solution, the resin was washed with THF (20 mL) and the coupling was repeated two more times. After removal of the solution, the resin was washed with three portions (20 mL) each of THF, CH₃OH, THF, and CH₂Cl₂, and then the product was cleaved from support and purified following Procedure C (vide infra).

General procedure for support cleavage and purification of substrates (Procedure C) [0113] The resin was swollen in CH₂Cl₂. To the swollen resin was added a solution of 9:1 CH₂C1₂:(95% CF₃CO₂H, 2.5% H₂O, 2.5% triisopropylsilane), and the mixture was shaken 1 h. Upon removal of the solution, the resin was washed with one portion of the cleavage solution (5 mL) and three portions of CH₂Cl₂ (5 mL). The combined washes were concentrated under reduced pressure. The crude product mixture was purified by HPLC [preparatory reverse-phase Q₈ column (24.1 x 250 mm), CH₃CN/H₂O-0.1% CF₃CO₂H = 5:95 to 95:5 over 55 min; lOmL/min; 254 nm detection for 65 min] and lyophilized. The purity of each compound was confirmed by HPLC-MS analysis (Cl 8 column (2.1 x 150 mm); 0.4 mL/min; 254 nm detection in two solvent systems: CH₃CN/H₂O-0.1% CF₃CO₂H, 5:95 to 95:5 over 16 min, 95:5 for 2 min; CH₃OH/H₂O, 5:95 to 95:5 over 20 min, 95:5 for 10 min). Synthesis of aminocoumarin substrates

Urea substrate 18

[0114] Procedure A was followed using resin 15 (0.136 g, 0.0890 mmol), propargyl urea Sl (0.010 g, 0.044 mmol), /-Pr₂EtN (1.6 mL, 8.9 mmol), and CuI (0.050 g, 0.27 mmol) in THF (2.2 mL) to afford 8.4 mg (32%) of 18 as a white powder. ¹H NMR (400 MHz, DMSO- d₆): δ 0.63 (d, 3H, J= 6.8), 0.81-0.85 (m, 6H), 1.05-1.35 (m, 4H), 1.60 (s, 3H), 2.10-2.23 (m, 2H), 2.37 (s, 3H), 2.51 (overlap with solvent), 3.15-3.24 (m, 4H), 3.48-3.55 (M, 4H), 3.58 (s, 2H), 5.43 (dd, IH, J= 6.6, 8.6), 6.20 (s, IH), 7.49 (dd, IH, J= 1.6, 8.8), 7.73 (d, IH₅ J= 1.6), 7.81 (d, IH₃ J= 8.8), 8.03 (s, IH), 10.95 (s, IH), 12.40 (br s, IH). HRMS-FAB (m/z): [MNa]⁺ calcd for C₃₀H₄₀N₆O₇Na, 619.2856; found, 619.2855.

Amide substrate 19

[0115] Procedure B was followed using indole-6-carboxylic acid (0.042 g, 0.075 mmol), triphosgene (0.025 g, 0.080 mmol), 2,4,6-collidine (0.10 mL, 0.75 mmol), and /-Pr₂EtN (0.11 mL, 0.60 mmol) to afford 7.0 mg (15%) of 19 as a white powder. ¹H NMR (400 MHz, DMSO-d₆): δ 0.71 (d, 3H, J= 6.8), 0.83 (t, 3H, J= 7.4), 0.91 (d, 3H₅ J= 6.8), 1.05-1.37 (m, 4H), 1.78 (s, 3H), 2.13-2.24 (m, 2H), 2.36 (s, 3H), 2.72 (sept, IH₅ J= 6.8), 3.58 (s, 2H)₅ 5.46 (dd, IH, J= 7.2, 8.8), 6.47 (s, IH)₅ 7.42 (d, IH, J= 8.4), 7.47-7.52 (m, 2H), 7.56 (d, IH, J = 8.4), 7.72 (d, IH, J= 2.0), 7.80 (d, IH, J= 8.8), 7.86 (s, IH)₅ 7.94 (s, IH), 8.17 (s, IH), 10.96 (s, IH), 11.34 (s, IH). HRMS-FAB (m/z): [MNa]⁺ calcd for C₃₄H₃₈N₆O₆Na, 649.2751 ; found, 649.2744.

Amide substrate 21

[0116] To resin 15 (0.115 g, 0.0750 mmol), preswollen in THF, was added a 0.02 M solution of the HCl salt of propargylamine 16 (0.025 g, 0.17 mmol) and /-Pr₂EtN (1.3 mL, 7.5 mmol) in THF (8.2 mL). CuI (0.043 g, 0.23 mmol) was then added and the mixture was shaken for 48 h. After removal of the solution, the resin was washed with three portions (20 mL) each of THF, CH₃OH, CH₃CN, and THF to afford support-bound amine intermediate 17. Resin 17 was swollen with three portions (20 mL) of DMF. A 0.4 M solution of HATU (0.200, 0.530 mmol), 2,4,6-collidine (0.070 mL, 0.53 mmol), and benzotriazole-5-carboxylic acid (0.086 g, 0.53 mmol) in DMF (1.3 mL) was added to the resin, and the mixture was shaken for 48 h. After removal of the solution, the resin was washed with three portions (20 mL) each of DMF, THF, CH₃OH, THF, and CH₂Cl₂, and then the product was cleaved from support and purified following Procedure C to afford 7.9 mg (17%) of 21 as a white powder. ¹H NMR (500 MHz, DMSO-d₆): δ 0.74 (d, 3H, J = 6.5), 0.83 (t, 3H, J = 7.2), 0.93 (d, 3H, J = 6.5), 1.05-1.38 (m, 4H), 1.76 (s, 3H), 2.14-2.22 (m, 2H), 2.36 (s, 3H), 2.68 (sept, IH, J = 6.5), 3.58 (s, 2H), 5.45 (dd, IH, J= 7.5, 8.0), 7.49 (d, IH, J= 8.5), 7.72 (s, IH), 7.79-7.82 (m, 2H), 7.90 (br s, IH), 8.16 (s, IH), 8.25 (s, IH), 8.40 (br s, IH), 10.96 (s, IH), 12.38 (br s, IH). HRMS-FAB (m/z): [MNa]⁺ calcd for C₃₂H₃₆N₈O₆Na, 651.2656; found, 651.2660.

Amide substrate 22

[0117] Procedure B was followed using l-methyl-lH-indole-5-carboxylic acid (0.037 g, 0.060 mmol), triphosgene (0.020 g, 0.07 mmol), 2,4,6-collidine (0.080 mL, 0.60 mmol), and /-Pr₂EtN (0.09 mL, 0.48 mmol) to afford 2.3 mg (6%) of 22 as a white powder. ¹H NMR (400 MHz, DMSO-d₆): δ 0.71 (d, 3H, J= 6.8), 0.83 (t, 3H, J= 7.2), 0.91 (d, 3H, J= 6.8), 1.05-1.37 (m, 4H), 1.78 (s, 3H), 2.12-2.24 (m, 2H), 2.36 (s, 3H), 2.73 (sept, IH, J= 6.8), 3.58 (s, 2H), 3.80 (s, 3H), 5.43-5.51 (m, IH), 6.53 (d, IH, J= 3.2), 7.39 (d, IH, J= 2.8), 7.45 (d, IH, J= 8.8), 7.49 (d, IH, J= 8.8), 7.58 (d, IH, J= 8.8), 7.72 (d, IH, J= 2.0), 7.79 (d, IH, J = 8.8), 7.90 (s, IH), 8.05 (s, IH), 8.15 (s, IH), 10.96 (s, IH), 12.51 (br s, IH). HRMS-FAB (m/z): [MNa]⁺ calcd for C₃₅H₄₀N₆O₆Na, 663.2907; found, 663.2910.

Amide substrate 23

[0118] Procedure B was followed using 4-(lH-imidazol-l-yl)-benzoic acid (0.049 g, 0.26 mmol), triphosgene (0.025 g, 0.080 mmol), 2,4,6-collidine (0.10 mL, 0.75 mmol), and i- Pr₂EtN (0.11 mL, 0.60 mmol) to afford 18.4 mg (38%) of 23 as a white powder. ¹H NMR (400 MHz, DMSOd₆): δ 0.73 (d, 3H, J= 6.8), 0.83 (t, 3H, J= 7.2), 0.92 (d, 3H, J= 6.8), 1.10-1.38 (m, 4H), 1.75 (s, 3H), 2.14-2.23 (m, 2H), 2.36 (s, 3H), 2.67 (sept, IH₅ J = 6.8), 3.58 (s, 2H), 5.45 (dd, IH, J= 6.8, 8.4), 7.47 (dd, IH, J= 2.0, 8.8), 7.75 (d, IH, J= 2.0), 7.79-7.89 (m, 4H), 8.00 (d, 2H, J= 8.4), 8.16 (s, IH), 8.24 (s, IH), 8.32 (br s, IH), 9.62 (br s, IH), 11.00 (s, IH), 12.47 (br s, IH). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₅H₄₀N₇O₆, 654.3040; found, 654.3035.

Amide substrate 24

[0119] Procedure B was followed using 4-morpholinobenzoic acid (0.050 g, 0.24 mmol), triphosgene (0.025 g, 0.080 mmol), 2,4,6-collidine (0.10 mL, 0.75 mmol), and /-Pr₂EtN (0.11 mL, 0.60 mmol) to afford 31.4 mg (62%) of 24 as a white powder. ¹H NMR (400 MHz, DMSO-d₆): δ 0.68 (d, 3H, J= 6.8), 0.83 (t, 3H, J= 7.2), 0.88 (d, 3H, J= 6.8), 1.10-1.37 (m, 4H), 1.72 (s, 3H), 2.12-2.23 (m, 2H), 2.36 (s, 3H), 2.68 (sept, IH, J = 6.8), 3.15-3.21 (m, 4H), 3.57 (s, 2H), 3.69-3.75 (m, 4H), 5.44 (dd, IH, J= 6.8, 8.6), 6.94 (d, 2H, J= 8.8), 7.49 (dd, IH, J= 2.0, 8.8), 7.67 (d, 2H, J= 8.8), 7.72 (d, IH, J= 2.0), 7.75 (s, IH), 7.80 (d, IH, J = 8.8), 8.12 (s, IH), 10.95 (s, IH). HRMS-FAB (m/z): [MNa]⁺ calcd for C₃₆H₄₄N₆O₇Na, 688.2417; found, 688.2411.

Amide substrate 25

[0120] Procedure B was followed using piperonylic acid (0.043 g, 0.26 mmol), triphosgene

(0.025 g, 0.080 mmol), 2,4,6-collidine (0.10 mL, 0.75 mmol), and /-Pr₂EtN (0.1 1 mL, 0.60 mmol) to afford 24.2 mg (51%) of 25 as a white powder. ¹H NMR (400 MHz, DMSOd₆): δ 0.70 (d, 3H, J= 6.8), 0.83 (t, 3H, J= 7.2), 0.88 (d, 3H, J= 6.8), 1.10-1.36 (m, 4H), 1.71 (s, 3H), 2.13-2.22 (m, 2H), 2.36 (s, 3H), 2.65 (sept, IH, J = 6.8), 3.58 (s, 2H), 5.44 (dd, IH, J = 6.6, 8.8), 6.07 (s, 2H), 6.96 (d, IH, J = 8.0), 7.30 (d, IH, J = 1.2), 7.34-7.37 (m, IH), 7.49 (dd, IH, J= 2.0, 8.8), 7.72 (d, IH, J= 2.0), 7.80 (d, IH, J= 8.8), 7.86 (s, IH), 8.11 (s, IH), 10.95 (s, IH), 12.50 (br s, IH). HRMS-FAB (m/z): [MNa]⁺ calcd for C₃₃H₃₇N₅O₈Na, 654.2540; found, 654.2529.

Amide substrate 26

[0121] Procedure B was followed using 4-(2-methyl-4-thiazolyl)benzoic acid (0.046 g, 0.060 mmol), triphosgene (0.020 g, 0.070 mmol), 2,4,6-collidine (0.080 mL, 0.60 mmol), and i- Pr₂EtN (0.090 mL, 0.48 mmol) to afford 8.2 mg (20%) of 26 as a white powder. ¹H NMR (500 MHz, DMSOd₆): δ 0.72 (d, 3H, J= 6.8), 0.83 (t, 3H, J= 7.5), 0.91 (d, 3H, J= 6.5), 1.05-1.37 (m, 4H), 1.74 (s, 3H), 2.13-2.23 (m, 2H), 2.36 (s, 3H), 2.65-2.73 (m, 4H), 3.58 (s, 2H), 5.42-5.48 (m, IH), 7.50 (d, IH, J= 8.5), 7.73 (s, IH), 7.77-7.86 (m, 3H), 7.99 (d, 2H, J = 8.0), 8.06 (d, 2H, J= 7.0), 8.15 (s, IH), 10.97 (s, IH). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₆H₄IN₆O₆S, 685.2808; found, 685.2798.

Amide substrate 27

[0122] Procedure B was followed using l-benzofuran-5-carboxylic acid (0.041 g, 0.25 mmol), triphosgene (0.025 g, 0.080 mmol), 2,4,6-collidine (0.10 mL, 0.75 mmol), and i- Pr₂EtN (0.11 mL, 0.60 mmol) to afford 21.9 mg (47%) of 27 as a white powder. ¹H NMR (400 MHz, DMSO-d₆): δ 0.73 (d, 3H, J= 6.8), 0.83 (t, 3H, J = 12), 0.92 (d, 3H, J= 6.8), 1.06-1.38 (m, 4H), 1.75 (s, 3H), 2.13-2.22 (m, 2H), 2.36 (s, 3H), 2.69 (sept, IH₅ J = 6.8), 3.58 (s, 2H), 5.41-5.50 (m, IH), 7.06 (d, IH, J = 2.0), 7.50 (dd, IH, J= 2.0, 8.8), 7.63 (d, IH, J= 8.6), 7.70-7.75 (m, 2H), 7.80 (d, IH, J= 8.6), 8.04-8.08 (m, 2H), 8.09-8.12 (m, IH), 8.15 (s, IH), 10.95 (s, IH). HRMS-FAB (m/z): [MNa]⁺ calcd for C₃₄H₃₇N₅O₇Na 650.2591 ; found, 650.2587.

Amide substrate 28

[0123] Procedure B was followed using l-benzothiaphene-5-carboxylic acid (0.038 g, 0.060 mmol), triphosgene (0.020 g, 0.070 mmol), 2,4,6-collidine (0.080 niL, 0.60 mmol), and i- Pr₂EtN (0.09 mL, 0.48 mmol) to afford 10.9 mg (28%) of 28 as a white powder. ¹H NMR (500 MHz, DMSO-d₆): δ 0.74 (d, 3H, J= 6.5), 0.83 (t, 3H, J= 7.0), 0.93 (d, 3H, J= 6.5), 1.02-1.37 (m, 4H), 1.76 (s, 3H), 2.13-2.24 (m, 2H), 2.36 (s, 3H), 2.70 (sept, IH₅ J= 6.5), 3.58 (s, 2H), 5.41-5.50 (m, IH), 7.50 (d, IH, J= 8.5), 7.56 (d, IH, J= 5.5), 7.19-7.27 (m, 2H), 7.79 (d, IH, J= 9.0), 7.83 (d, IH, J= 5.5), 8.05 (d, IH, J= 8.5), 8.12 (s, IH), 8.16 (s, IH), 8.31 (s, IH), 10.97 (s, IH), 12.53 (br s, IH). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₄H₃₈N₅O₆S, 644.2543; found, 649.2553.

Amide substrate 29

[0124] Procedure B was followed using indole-5-carboxylic acid (0.041 g, 0.25 mmol), triphosgene (0.025 g, 0.080 mmol), 2,4,6-collidine (0.10 mL, 0.75 mmol), and /-Pr₂EtN (0.11 mL, 0.60 mmol) to afford 2.0 mg (4%) of 29 as a white powder. ¹H NMR (400 MHz, DMSOd₆): δ 0.71 (d, 3H, J= 6.8), 0.83 (t, 3H, J= 7.2), 0.91 (d, 3 H, J= 6.8), 1.08-1.37 (m, 4H), 1.76 (s, 3H), 2.13-2.25 (m, 2H), 2.36 (s, 3H), 2.72 (sept, IH, J = 6.8), 3.58 (s, 2H), 5.40- 5.49 (m, IH), 6.51-6.54 (m, IH), 7.38-7.43 (m, 2H), 7.47-7.56 (m, 2H), 7.72 (d, IH, J= 2.0), 7.79 (d, IH, J= 8.8), 7.88 (s, IH), 8.05 (s, IH), 8.16 (s, IH), 10.97 (s, IH), 11.30 (s, IH), 12.46 (br s, IH). HRMS-FAB (m/z): [MNa]⁺ calcd for C₃₄H₃₈N₆O₆Na, 649.2751; found, 649.2748.

Amide substrate 30

[0125] Procedure B was followed using 6-quinolinecarboxylic acid (0.045 g, 0.26 mmol), triphosgene (0.025 g, 0.080 mmol), 2,4,6-collidine (0.10 mL, 0.75 mmol), and /-Pr₂EtN (0.11 mL, 0.60 mmol) to afford 18.0 mg (38%) of 30 as a white powder. ¹H NMR (500 MHz, DMSO-d₆): δ 0.76 (d, 3H, J= 6.8), 0.83 (t, 3H, J= 7.2), 0.95 (d, 3H, J= 6.8), 1.05-1.37 (m, 4H), 1.78 (s, 3H)₅ 2.12-2.23 (m, 2H), 2.35 (s, 3H), 2.71 (sept, IH, J = 6.8), 3.57 (s, 2H), 5.41- 5.49 (m, IH), 7.48 (d, IH, J= 9.0), 7.68-7.76 (m, 2H), 7.78 (d, IH, J= 8.5), 8.07-8.16 (m, 2H), 8.18 (s, IH), 8.36 (s, IH), 8.51 (s, IH), 8.67 (d, IH₅ J= 8.5), 9.06 (m, IH)₅ 10.98 (s, IH). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₅H₃₉N₆O₆, 639.2931 ; found, 639.2918.

Amide substrate 31

[0126] Procedure B was followed using l^-benzothiazole-ό-carboxylic acid (0.044 g, 0.25 mmol), triphosgene (0.025 g, 0.080 mmol), 2,4,6-collidine (0.10 mL, 0.75 mmol), and i- Pr₂EtN (0.11 mL, 0.60 mmol) to afford 18.0 mg (37%) of 31 as a white powder. ¹H NMR (500 MHz₅ DMSO-d₆): δ 0.74 (d, 3H, J= 7.0), 0.82 (t, 3H, J= 7.2), 0.93 (d, 3H, J= 7.0), 1.05-1.37 (m, 4H), 1.76 (s, 3H), 2.12-2.24 (m, 2H), 2.35 (s, 3H), 2.67 (sept, IH₅ J = 7.0),

3.58 (s, 2H), 5.45 (dd, IH, J= 6.5, 8.5), 7.49 (dd, IH, J= 2.0, 9.0), 7.72 (d, IH₅ J= 2.0), 7.79 (d, IH, J= 9.0), 7.90 (dd, IH₅ J= 2.0, 8.5), 8.11 (d, IH, J= 8.5), 8.16 (s, IH), 8.20 (s, IH),

8.59 (d, IH, J= 1.0), 9.50 (s, IH), 10.97 (s, IH). HRMS-FAB (m/z): [MNa]⁺ calcd for C₃₃H₃₆N₆O₆SNa 667.2315; found, 667.2322.

Amine substrate 35

[0127] To resin 15 (0.115 g, 0.0750 mmol), preswollen in THF, was added a 0.02 M solution of the HCl salt of propargylamine 16 (0.025 g, 0.17 mmol) and /-Pr₂EtN (1.3 mL, 7.5 mmol) in THF (8.2 mL). CuI (0.043 g, 0.23 mmol) was then added and the mixture was shaken for 48 h. After removal of the solution, the resin was washed with three portions (20 mL) each of THF, CH₃OH, CH₃CN, and THF to afford support-bound amine intermediate 17. After washing derivatized resin 17, THF (2.0 mL, 0.8 M) was added. To this solution was added /?-anisaldehyde (0.102 g, 0.750 mmol) and acetic acid (0.040 mL, 0.75 mmol). After letting the mixture react for about 2 min, NaBH(OAc)₃ (0.159 g, 0.750 mmol) was added. The resulting mixture was shaken for 48 h. After removal of the solution, the resin was washed with three portions (20 mL) each of THF, CH₃OH, THF, and CH₂Cl₂. The product was cleaved from support and purified following Procedure C to afford 13.4 mg (30%) of the TFA salt 35 as a white powder. ¹H NMR (500 MHz, DMSOd₆): δ 0.71 (d, 3H, J= 7.0), 0.84 (t, 3H, J= 7.2), 1.03 (d, 3H, J= 7.0), 1.12-1.38 (m, 4H), 1.67 (s, 3H), 2.19-2.31 (m, 2H), 2.37 (s, 3H), 2.67 (sept, IH, J = 7.0), 3.58 (s, 2H), 3.74 (s, 3H), 4.01-4.08 (m, 2H), 5.60 (dd, IH, J= 6.0, 9.5), 6.93 (d, 2H, J= 8.5), 7.25 (d, 2H, J= 8.5), 7.49 (dd, IH, J= 2.0, 8.5), 7.78 (d, IH, J= 2.0), 7.82 (d, IH₅ J= 8.5), 8.59 (s, IH), 8.93-9.12 (m, 2H), 11.11 (s, IH), 12.51 (br s, IH). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₃H₄₂N₅O₆, 604.3135; found, 604.3124.

Amine substrate 36

[0128] Procedure A was followed using resin 15 (0.421 g, 0.150 mmol), propargyl amine 39 (0.057 g, 0.23 mmol), /-Pr₂EtN (2.6 mL, 15 mmol), and CuI (0.086 g, 0.45 mmol) in THF (8.2 mL) to afford 51.0 mg (54%) of the TFA salt 36 as a white powder. ¹H NMR (400 MHz, DMSOd₆): δ 0.75 (d, 3H, J= 6.6), 0.84 (t, 3H, J= 7.2), 1.07 (d, 3H, J= 6.6), 1.12-1.39 (m, 4H), 1.73 (s, 3H), 2.21-2.34 (m, 2H), 2.37 (s, 3H), 2.70 (sept, IH, J = 6.6), 3.59 (s, 2H), 3.98- 4.06 (m, IH), 4.31-4.39 (m, IH), 5.62 (dd, IH, J= 6.0, 9.4), 7.49 (dd, IH, J= 2.0, 8.8), 7.61 (dd, 1H, J= 4.4, 8.4), 7.75 (dd, 1H, J= 1.6, 8.8), 7.80-7.85 (m, 2H), 7.96 (d, 1H, J= 1.5), 8.06 (d, IH₅ J= 8.8), 8.40 (d, IH₅ J= 7.6), 8.65 (s, IH), 8.96 (dd, IH₅ J= 1.6, 4.0), 9.22-9.36 (m, 2H), 11.13 (s, IH), 12.65 (br s, IH). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₅H₄iN₆O₅, 625.3138; found, 625.3146.

Amine substrate 37

[0129] Procedure A was followed using resin 15 (0.421 g, 0.150 mmol), propargyl amine S6b (0.058 g, 0.23 mmol), /-Pr₂EtN (2.6 mL, 15 mmol), and CuI (0.086 g, 0.45 mmol) in THF (8.2 mL) to afford 34.0 mg (36%) of the TFA salt 37 as a white powder. ¹H NMR (500 MHz, DMSOd₆): δ 0.74 (d, 3H₅ J= 6.5), 0.84 (t, 3H, J= 7.0), 1.06 (d, 3H, J= 6.5), 1.12- 1.39 (m, 4H), 1.72 (s, 3H), 2.21-2.34 (m, 2H), 2.36 (s, 3H), 2.70 (sept, IH, J = 6.5), 3.58 (s, 2H), 3.94-4.02 (m, IH), 4.26-4.33 (m, IH), 5.64 (dd, IH₅ J= 6.0, 9.5), 7.53 (dd, 2H, J= 1.5, 8.5), 7.78 (d, IH, J= 1.5), 7.81 (d, IH, J= 8.5), 8.08-8.14 (m, 2H), 8.63 (s, IH), 9.31 (br s, 2H), 9.45 (s, IH), 11.21 (s, IH). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₃H₃₉N₆O₅S_, 631.2703; found, 631.2691.

Synthesis of propargyl intermediates.

S1

Propargyl urea Sl

[0130] To a 0.25 M solution of the HCl salt of propargyl amine 16 (0.074 g, 0.50 mmol) and triethylamine (0.21 mL, 1.5 mmol) in CHCl₃ (2 mL) was added 4-morpholinecarbonyl chloride (0.082 g, 0.55 mmol). After stirring the resulting solution for 1 h at room temperature, DMAP (0.003 g, 0.02 mmol) was added. The reaction mixture was then stirred at reflux for 2 h. The mixture was washed with saturated aqueous NH₄Cl (I x 10 mL) and saturated NaCl (1 x 10 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. Column chromatography (40-60% EtOAc/hexanes) afforded 10.0 mg (8%) of Sl as a clear oil. ¹H NMR (300 MHz, CDCl₃): δ 0.98 (d, 3H, J = 6.9), 1.02 (d, 3H, J= 6.9), 1.61 (s, 3H), 2.34 (s, IH), 2.44 (sept, IH, J= 6.9), 3.31 (t, 4H, J = 4.8), 3.68 (t, 4H, J= 5.0), 4.54 (br s, IH). MS (ESI): m/z 225 [MH]⁺.

NaBHa

MeOH, 0 °C

39, S6b

Methyl benzothiazole-6-carboxylate S2b

[0131] A 0.2 M stirring solution of benzothiazole-6-carboxylic acid in THF (17 mL) was cooled to 0 ⁰C. Excess diazomethane was introduced in situ from Diazald (2.51 g, 11.7 mmol), according to literature procedure. Lombardi, P. Chem. Ind. (London) 1990, 708. After addition of the diazomethane, the solution was stirred at 0 ⁰C for 30 min and then at room temperature for 30 min. The solvent was removed under reduced pressure to afford 0.621 g (96%) of S2b as a tan solid, mp 105-106 ⁰C. ¹H NMR (400 MHz, CDCl₃): δ 3.95 (s, 3H), 8.13-8.18 (m, 2H), 4), 7.93 (dd, IH, J= 0.8, 1.2), 9.14 (s, IH). ¹³C-NMR (100 MHz, CDCl₃): δ 52.6, 123.6, 124.4, 127.5, 127.6, 133.9, 156.2, 157.5, 166.7. HRMS-FAB (m/z): [MH]⁺ calcd for C₉H₈NO₂S, 194.0276; found, 194.0270.

6-Quinolinylmethanol S3a

[0132] A 0.2 M solution of methyl quinoline-6-carboxylate (0.300 g, 1.60 mmol) in THF (8 mL) and a 0.6 M solution of DIBAL (0.860 mL, 4.81 mmol) in THF (8 mL) were cooled in a -78 ⁰C acetone-dry ice bath. The DIBAL solution was cannula transferred to the methyl ester solution, and the resulting solution was stirred for 1 h at 0 ⁰C. The reaction was quenched at 0 ⁰C by adding methanol (8 mL) and then acetic acid (1.37 mL, 24.0 mmol). After the reaction mixture was stirred for 5 min, a saturated sodium tartrate solution (16 mL) was added. After stirring for 20 min, the reaction mixture was diluted with EtOAc (50 mL) and water (10 mL). The aqueous layer was extracted with EtOAc (3 x 50 mL). The organic washes were combined and extracted once with water (15 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. Column chromatography (50-90% EtOAc/hexanes) afforded 0.236 g (93%) of S3a as a faintly yellow oil. ¹H NMR (500 MHz, CDCl₃): δ 4.89 (s, 2H), 7.35 (dd, IH, J = 4.0, 8.0), 7.65 (dd, IH₅ J= 1.5, 9.0), 7.77 (s, IH), 8.01 (d, IH₅ J= 9.0), 8.08 (d, IH₅ J= 4.0), 8.80 (dd, IH, J= 1.5, 4.0). ¹³C-NMR (125 MHz, CDCl₃): δ 64.8, 121.5, 125.0, 128.3, 129.0, 129.4, 136.4, 139.9, 147.7, 150.2. HRMS-FAB (m/z): [MH]⁺ calcd for C₁₀H₁₀NO, 160.0762; found, 160.0766.

(Benzothiazol-ό-yl)-methanol S3b

[0133] The same procedure as for alcohol S3a (vide supra) was followed using methyl benzothiazole-6-carboxylate (0.300 g, 1.55 mmol) in THF (7.75 mL) and DIBAL (0.830 mL, 4.66 mmol) in THF (7.75 mL). The crude reaction mixture was purified by column chromatography (30-50% EtOAc/hexanes) to afford 0.212 g (83%) of S3b as a yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 4.75 (s, 2H), 7.37 (dd, IH, J= 1.2, 8.4), 7.85 (s, IH), 7.93 (d, IH, J= 8.4), 8.85 (s, IH). ¹³C-NMR (100 MHz, CDCl₃): δ 64.4, 1 19.9, 123.2, 125.5, 133.8, 139.3, 152.2, 154.4. HRMS-FAB (m/z): [MH]⁺ calcd for C₈H₇NOS, 165.0248; found, 165.0243.

Quinoline-6-carboxyaldehyde S4a

[0134] This procedure was adapted from Meyers. Myers, A. G.; Zhong, B.; Movassaghi, M.; Kung, D. W.; Lanman, B. A.; Kwon, S. Tetrahedron Lett. 2000, 41, 1359-1362. Dess- Martin periodinane (2.57 g, 6.05 mmol) was added to a 0.28 M solution of 6- quinolinylmethanol (0.459 g, 2.88 mmol) in water-saturated CH₂Cl₂ (10 mL). The reaction mixture was stirred for 10 min and then CH₂Cl₂ (3 x 1 mL) was added over 15 min. The reaction mixture was diluted with diethyl ether (10 mL), and a solution of sodium thiosulfate (7.87 g, 31.7 mmol) in 80% saturated aqueous NaHCO₃ (10 mL) was added. The mixture was stirred rapidly for 45 min. The layers were separated and the aqueous layer was extracted with ether (2 x 20 mL). The combined organic layers were washed sequentially with saturated aqueous NaHCO₃ (30 mL), water (2 x 30 mL), and saturated NaCl (2 x 30 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by column chromatography (30-60% EtOAc/hexanes) to afford 0.383 g (85%) of S4a as a white solid, mp 76.2-76.5 ⁰C. ¹H NMR (400 MHz, CDCl₃): δ 7.53 (dd, IH, J= 4.4, 8.4), 8.18-8.23 (m, 2H), 8.33 (dd, IH, J= 2.0, 8.4), 8.36 (s, IH), 9.05 (dd, IH, J= 2.0, 4.4), 10.20 (s, IH). ¹³C-NMR (100 MHz, CDCl₃): δ 122.4, 126.8, 127.8, 130.9, 133.8, 134.4, 137.6, 151.0, 153.3, 191.6. HRMS-FAB (m/z): [MH]⁺ calcd for Ci₀H₇NO, 157.0528; found, 157.0521.

Benzothiazole-6-carboxyaldehyde S4b

[0135] The same procedure as for aldehyde S4a (vide supra) was followed using S3b (0.175 g, 1.06 mmol), Dess-Martin periodinane (0.943 g, 2.22 mmol) in water-saturated CH₂Cl₂ (3.8 mL). The crude reaction mixture was purified by column chromatography (20- 50% EtOAc/hexanes) to afford 0.105 g (61%) of the desired aldehyde. ¹H NMR (400 MHz, CDCl₃): δ 8.02 (dd, IH, J= 1.6, 8.4), 8.24 (d, IH, J= 8.4), 8.50 (s, IH), 9.20 (s, IH), 10.13 (s, IH). ¹³C-NMR (I OO MHZ₅ CDCI₃): δ 124.5, 125.2, 127.0, 133.9, 134.6, 157.1, 158.5, 191.4. HRMS-FAB (m/z): [MH]⁺ calcd for C₈H₅NOS, 163.0092; found, 163.0085.

Propargyl imine S5a

[0136] The HCl salt of propargyl amine 16 (0.248 g, 1.50 mmol) was dissolved in water (1 mL) and basified to pH=l 1 with 1 M NaOH. The aqueous layer was extracted with toluene (3 x 1.5 mL). The organic layers were combined, dried over Na₂SO₄, and filtered. To the 0.25 M solution of propargyl amine 16 in toluene (4.5 mL) were added S4a (0.176 g, 1.12 mmol) and activated 4 A molecular sieves. The reaction mixture was stirred for 16 h and then filtered through a plug of celite. The celite was washed with CH₂Cl₂ (3 x 5 mL). The organic washes were concentrated to afford 0.237 g (85%) of S5a. ¹H NMR (400 MHz, CDCl₃): δ 0.91 (d, 3H, J= 6.8), 1.10 (d, 3H, J= 6.8), 1.53 (s, 3H), 2.05 (sept, IH, J= 6.8), 2.67 (s, IH), 7.43 (dd, IH, J= 4.4, 8.4), 8.11-8.13 (m, 2H), 8.21 (d, IH, J= 7.2), 8.28 (dd, IH, J= 2.0, 8.4), 8.83 (s, IH), 8.94 (dd, IH, J= 2.0, 4.4). ¹³C-NMR (100 MHz, CDCl₃): δ 18.0, 18.1, 28.3, 38.3, 66.3, 77.4, 84.1, 121.8, 128.26, 128.29, 129.5, 130.1, 134.9, 136.8, 149.7, 151.4, 157.4. HRMS-FAB (m/z): [MH]⁺ calcd for C_nH]₉N₂, 251.1548; found, 251.1543.

Propargyl imine S5b

[0137] The same procedure as for propargyl imine S5a (vide supra) was followed using the HCl salt of propargyl amine 16 (0.143 g, 0.970 mmol) and benzothiazole-6-carboxyaldehyde (0.105 g, 0.640 mmol) in toluene (2.6 mL) to afford 0.154 g (94%) of S5b. ¹H NMR (400 MHz, CDCl₃): δ 0.90 (d, 3H, J= 6.8), 1.10 (d, 3H, J= 6.8), 1.52 (s, 3H), 2.03 (sept, IH₅ J = 6.8), 2.68 (s, IH), 7.97 (dd, IH, J= 1.6, 8.4), 8.16 (d, IH₃ J= 8.4), 8.39 (d, IH₅ J= 1.6), 8.78 (s, IH), 9.03 (s, IH). ¹³C-NMR (100 MHz, CDCl₃): δ 17.96, 18.01, 28.3, 38.3, 66.2, 77.4, 84.0, 122.2, 123.7, 126.6, 134.2, 134.3, 154.8, 155.7, 157.3. HRMS-FAB (m/z): [MH]⁺ calcd for C₁₅H₁₇N₂OS, 257.1112; found, 257.1105.

Propargyl Amine 39

[0138] To a 0.2 M solution of the propargyl imine (0.162 g, 0.700 mmol) in methanol (3.5 mL) at 0 ⁰C was added sodium borohydride (0.053 g, 1.4 mmol). After stirring the reaction mixture at 0 ⁰C for 1 h, it was diluted with water (3 mL) and extracted with CH₂Cl₂ (3 x 10 mL). The organic layers were combined, dried over Na₂SO₄, filtered and concentrated. The crude reaction mixture was purified by column chromatography (30-40% EtOAc/hexanes) to afford 0.122 g (86%) of 39 as a white solid, mp 45.9-46.7 ⁰C. IR v_max (cm ¹): 3299, 2964, 2875, 2360, 2342, 2096. ¹H NMR (300 MHz, CDCl₃): δ 1.04 (d, 3H, J= 6.9), 1.07 (d, 3H, J = 6.9), 1.31 (s, 3H), 1.39 (br s, IH), 1.87 (sept, IH, J= 6.9), 2.39 (s, IH), 4.00 (d, IH₅ J = 12.6), 4.06 (d, IH, J= 12.6), 7.36 (dd, IH₅ J= 4.2, 8.1), 7.74 (dd, IH₅ J= 1.8, 8.7), 7.78 (s, IH), 8.05 (d, IH, J= 8.4), 8.11 (dd, IH, J= 1.2, 8.4), 8.86 (dd, IH, J= 1.5, 4.2). ¹³C-NMR (100 MHz, CDCl₃): δ 17.1, 18.1, 23.1, 36.5, 48.3, 57.4, 71.6, 88.0, 121.3, 126.4, 128.4, 129.6, 130.8, 136.0, 139.7, 147.9, 150.2. HRMS-FAB (m/z): [MH]⁺ calcd for C₁₇H₂₁N₂, 253.1704; found, 253.1710.

Propargyl amine S6b

[0139] The same procedure as for propargyl amine 39 (vide supra) was followed using S5b (0.154 g, 0.600 mmol) and sodium borohydride (0.045 g, 1.2 mmol) in methanol (3.0 mL). The crude reaction mixture was purified by column chromatography (40-50% EtOAc/hexanes) to afford 0.12O g (77%) of the desired propargyl amine as a pale yellow solid, mp 62.4-63.2 ⁰C. IR v_max (cm^"1): 3291, 3154, 2967, 2086. ¹H NMR (400 MHz, CDCl₃): δ 1.03 (d, 3H, J= 6.8), 1.06 (d, 3H, J= 6.8), 1.30 (s, 3H), 1.32 (br s, IH), 1.88 (sept, IH, J = 6.8), 2.38 (s, IH), 3.97 (d, IH, J= 12.4), 4.02 (d, IH, J= 12.4), 7.52 (dd, IH₅ J= 1.6, 8.4), 7.98 (d, IH, J= 0.8), 8.06 (d, IH, J= 8.4), 8.94 (s, IH). ¹³C-NMR (100 MHz, CDCl₃): δ 17.0, 18.1, 23.1, 36.5, 48.3, 57.4, 71.6, 87.9, 121.3, 123.5, 127.2, 134.0, 139.1, 152.5, 153.8. HRMS-FAB (m/z): [MH]⁺ calcd for Ci₅Hi₉N₂S, 259.1269; found, 259.1270.

16 S7a,b

Propargyl amide S7a

[0140] To a 0.17 M solution of the HCl salt of propargyl amine 16 (0.050 g, 0.34 mmol) in THF (2 rnL) was added /-Pr₂EtN (0.472 mL, 2.71 mmol), and to a 0.08 M solution of triphosgene (0.1 15 g, 0.390 mmol) was added 6-quinolinecarboxylic acid (0.205 g, 1.18 mmol) and 2,4,6-collidine (0.447 mL, 3.39 mmol) in THF (5 mL). The triphosgene mixture was then added to the propargyl amine mixture, and the resulting mixture stirred for 18 h. The reaction mixture was diluted with EtOAc (15 mL) and washed with water (1 x 5 mL), 10% citric acid (2 x 5 mL), and water (3 x 5 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. Column chromatography (30-50% EtOAc/hexanes) afforded 55.0 mg (61%) of S7a as an opaque oil. IR v_max (cm^"1): 3301, 3051, 2970, 2938, 2877, 2109, 1652. ¹H NMR (400 MHz, CDCl₃): δ 1.06 (d, 3H, J= 6.8), 1.09 (d, 3H, J= 6.8), 1.78 (s, 3H), 2.43 (s, IH), 2.68 (sept, IH, J= 6.8), 6.46 (s, IH), 7.43 (dd, IH, J= 4.4, 8.4), 7.98 (dd, IH, J= 2.0, 8.8), 8.09 (d, IH, J= 8.8), 8.18 (d, IH, J= 8.4), 8.22 (d, IH, J= 2.0), 8.94 (d, IH, J= 2.4). ¹³C-NMR (100 MHz, CDCl₃): δ 17.7, 18.2, 24.3, 34.8, 57.1, 72.0, 84.8, 122.1, 127.2, 127.6, 127.7, 130.1, 133.1, 137.2, 149.4, 152.1, 165.9. HRMS-FAB (m/z): [MH]⁺ calcd for C₁₇Hi₉N₂O, 267.1497; found, 267.1499.

Propargyl amide S7b

[0141] The same procedure as for propargyl amide S7a (vide supra) was followed using propargyl amine 16 (0.150 g, 1.01 mmol) and /-Pr₂EtN (1.41 mL, 8.13 mmol) in THF (6 mL) and triphosgene (0.347 g, 1.17 mmol), l ,3-benzothiazole-6-carboxylic acid (0.647 g, 3.61 mmol), and 2,4,6-collidine (1.34 mL, 10.2 mmol) in THF (15 mL). The reaction was stirred for 24 h. The crude reaction mixture was purified by column chromatography (30-50% EtOAc/hexanes) to afford 0.269 g (98%) of S7b as a pale yellow solid, mp 75-76 ⁰C. IR v_max (cm^"1): 3300, 3056, 2970, 2939, 2876, 2110, 1647. ¹H NMR (400 MHz, CDCl₃): δ 1.06 (d, 3H, J= 6.8), 1.09 (d, 3H, J= 6.8), 1.77 (s, 3H), 2.43 (s, IH), 2.67 (sept, IH₅ J= 6.8), 6.34 (s, IH), 7.83 (dd, IH, J = 1.6, 8.8), 8.13 (d, IH, J= 8.4), 8.42 (d, IH, J= 1.6), 9.10 (s, IH). ¹³C- NMR (IOO MHz, CDCl₃): δ 17.7, 18.2, 24.4, 34.8, 57.1, 72.0, 84.9, 121.7, 123.7, 124.7, 132.7, 134.3, 155.2, 156.8, 165.8. HRMS-FAB (m/z): [MH]⁺ calcd for Ci₅H_nN₂OS, 273.1062; found, 273.1057.

General synthetic methods for inhibitors.

General synthesis of 1,2,3-triazole compounds (Procedure Dl)

[0142] This procedure was adapted from Sharpless. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem. Int. Ed. 2002, 41, 2596-2599. To a 0.25 M suspension of alkyne (1 equiv) and azide (1 equiv) in a 1 :1 mixture of water and tert-butyl alcohol was added sodium ascorbate (1 equiv of a freshly prepared 1.0 M solution in water) followed by copper(II) sulfate pentahydrate (0.1 equiv of a freshly prepared 0.3 M solution in water). The heterogeneous mixture was stirred vigorously overnight. Water was added and extracted with EtOAc (3x). The organic layers were combined, washed with saturated NaCl (Ix), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by HPLC [preparatory reverse-phase Ci₈ column (24.1 x 250 mm), CH₃CN/H₂O-0.1% CF₃CO₂H = 5:95 to 95:5 over 55 min; 10 mL/min; 254 nm detection for 65 min] and lyophilized to afford the TFA salt of the product. The free amine of the product was obtained by dissolving the TFA salt of the product in saturated aqueous NaHCO₃ and extracting with CH₂Cl₂ (4x). The organic layers were combined, dried over Na₂SO₄, filtered, and concentrated under reduced pressure.

General synthesis of acyl- and aryl-oxymethyl ketone azides 47a-c (Procedure E) [0143] To a 0.2 M solution of benzoic acid or phenol (3.1 - 4.0 equiv) in DMF at 0 ⁰C was added potassium fluoride (3.0 - 4.0 equiv), and the reaction mixture was stirred for 10 min. Bromomethyl ketone 48 (1.0 equiv) was then added in a small amount of DMF. The reaction mixture was stirred at 0 ⁰C for 0.5-3 h. The reaction mixture was diluted with CH₂Cl₂ and washed with water (Ix), saturated NaHCO₃ (Ix), water (2x), and brine (Ix). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. Column chromatography afforded the pure product. Synthesis of inhibitors.

Carboxylic acid 41

[0144] Procedure Dl was followed using propargyl amine 39 (0.063 g, 0.25 mmol), azide 40 (0.040 g, 0.25 mmol), sodium ascorbate (0.25 mL, 0.25 mmol), copper(II) sulfate pentahydrate (0.084 mL, 0.025 mmol) in 1 :1 ΦuOH:H₂O (1.0 mL) to afford 0.151 g (89%) of the TFA salt 41 as a sticky yellow solid. ¹H NMR (300 MHz, DMSO-d₆): δ 0.74 (d, 3H, J = 6.9), 0.81 (t, 3H, J= 7.5), 0.97-1.39 (m, 4H), 1.06 (d, 3H₅ J = 6.9), 1.70 (s, 3H), 2.13-2.29 (m, 2H), 2.07 (sept, IH, J= 6.9), 3.92-4.07 (m, IH), 4.28-4.41 (m, IH), 5.49 (dd, IH, J= 6.3, 9.6), 7.64 (dd, IH, J= 4.1, 8.4), 7.74 (dd, IH, J= 1.8, 8.7), 7.97 (d, IH₅ J= 1.5), 8.07 (d, IH, J= 8.7), 8.46 (d, IH, J= 8.4), 8.54 (s, IH), 8.99 (d, IH, J= 4.1), 9.24-9.49 (br s, 2H). ¹³C- NMR (125 MHz, DMSO-d₆): δ 13.7, 15.7, 16.3, 17.8, 21.3, 27.5, 30.6, 33.6, 45.8, 62.5, 63.7, 122.1, 125.0, 127.4, 128.4, 130.3, 130.5, 131.8, 137.1, 145.4, 146.5, 150.9, 170.3. HRMS- FAB (m/z): [MH]⁺ calcd for C₂₃H₃₂N₅O₂, 410.2556; found, 410.2566.

Methyl ester 42

[0145] A 0.2 M stirred solution of carboxylic acid 41 (0.050 g, 0.095 mmol) in THF (0.5 mL) was cooled to 0 ⁰C. Excess diazomethane was introduced in situ from Diazald (0.164 g, 0.764 mmol), according to literature procedure. Lombardi, P. Chem. Ind. (London) 1990, 708. After addition of the diazomethane, the solution was stirred at 0 ⁰C for 15 min. The solvent was removed under reduced pressure. The crude reaction mixture was purified by column chromatography (1-5% MeOH/CH₂Cl₂) to afford 30.0 mg (75%) of 42 as a clear oil. ¹H NMR (500 MHz, CDCl₃): δ 0.81 (d, 3H, J = 7.0), 0.88 (t, 3H, J= 7.0), 1.02 (d, 3H, J = 7.0), 1.11-1.44 (m, 4H), 1.50 (s, 3H), 1.89 (br s, IH), 2.05-2.29 (m, 3H), 3.61 (d, IH₅ J = 12.5), 3.74-3.80 (m, 4H), 5.38 (dd, IH, J= 5.5, 10.0), 7.37 (dd, IH, J= 4.0, 8.5), 7.62 (s, IH), 7.68 (dd, IH₅ J = 1.5, 8.5), 7.74 (s, IH), 8.02 (d, IH₅ J= 8.5), 8.11 (d, IH, J= 8.5), 8.86 (d, IH, J= 4.0). ¹³C-NMR (125 MHz, CDCl₃): δ 14.0, 17.4, 18.1, 19.6, 22.1, 28.0, 32.7, 37.4, 47.5, 53.1, 58.6, 62.8, 121.0, 121.3, 126.2, 128.4, 129.5, 130.8, 136.0, 140.0, 147.8, 150.1, 153.5, 170.0. HRMS-FAB (m/z): [MH]⁺ calcd for C₂₄H₃₄N₅O₂, 424.2712; found, 424.2724.

Vinyl sulfone 38

[0146] A 0.2 M solution of methyl ester 42 (28 mg, 0.067 mmol) in diethyl ether (0.3 mL) and a 0.6 M solution of DIBAL (0.048 mL, 0.27 mmol) in diethyl ether (0.4 mL) were cooled in a -78 ⁰C acetone-dry ice bath. The DIBAL solution was cannula transferred to the methyl ester solution, and the resulting solution was stirred for 1 h at -78 ⁰C. The reaction was quenched at -78 ⁰C by adding methanol (0.4 mL) and then acetic acid (0.060 mL, 1.0 mmol). After the reaction mixture was stirred for 5 min, a saturated sodium tartrate solution (0.8 mL) was added. After stirring for 20 min, the reaction mixture was diluted with EtOAc (5 mL) and water (2 mL). The aqueous layer was extracted EtOAc (3 x 5 mL). The organic washes were combined and extracted once with water (5 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The aldehyde was taken on to the next step without purification.

[0147] To a round-bottom flask containing LiBr (4.8 mg, 0.055 mmol) and diethyl (phenylsulfonyl)methyl]phosphonate, Enders, D.; von Berg, S.; Jandeleit, B. Org. Synth. 2002, 78, 169-176. (13 mg, 0.046 mmol) was added acetonitrile (1.2 mL) and triethylamine (0.006 mL, 0.05 mmol). After cooling the reaction mixture to -40⁰C in an acetonitrile-dry ice bath, the aldehyde (0.018 g, 0.050 mmol) in acetonitrile (0.6 mL) was added dropwise. The reaction mixture was stirred for 36 h at -40 ⁰C, and then the reaction was quenched by adding IM HCl (0.4 mL) and water (0.8 mL) at 0 ⁰C. The aqueous layer was then basified to pH=10 with IM NaOH and extracted with EtOAc (5 x 2 mL). The organic layers were combined and washed with saturated NaCl (1 x 2 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by HPLC [preparatory reverse-phase C₁₈ column (24.1 x 250 mm), CH₃CN/H₂O- 0.1% CF₃CO₂H = 5:95 to 95:5 over 55 min; 10 mL/min; 254 nm detection for 65 min] and lyophilized to afford 4.6 mg (11 %; 2 steps) of a 1 : 1 mixture of diastereomers of the TFA salt 38 as a sticky clear solid. The inhibitor was > 99% pure as determined by HPLC-MS analysis (Cl 8 column (2.1 x 150 mm); 0.4 mL/min; 254 nm detection in two solvent systems: CH₃CN/H₂O-0.1% CF₃CO₂H, 5:95 to 95:5 over 16 min, 95:5 for 2 min; CH₃OH/H₂O, 5:95 to 95:5 over 20 min, 95:5 for 10 min). ¹H NMR (500 MHz, DMSO-d₆): δ 0.70 (d, 3H, J= 6.5), 0.76 (t, 1.5H₅ J= 7.2), 0.77 (t, 1.5H, J= 7.2), 0.88-1.31 (m, 4H), 1.04 (d, 3H, J= 6.5), 1.68 (s, 3H), 1.99-2.12 (m, 2H), 2.64-2.79 (m, IH), 4.01-4.09 (m, IH), 4.28-4.38 (m, IH), 5.51- 5.60 (m, IH), 6.94 (dd, 0.5H, J= 1.1, 15.1), 6.96 (dd, 0.5H, J= 1.1, 15.1), 7.17 (dd, 0.5H, J = 1.5, 15.1), 7.18 (dd, 0.5H, J= 1.5, 15.1), 7.58-7.66 (m, 3H), 7.70-7.75 (m, 2H), 7.83-7.87 (m, 2H), 7.96-7.99 (m, IH), 8.05 (d, 0.5H, J= 8.7), 8.06 (d, 0.5H, J= 8.7), 8.39-8.43 (m, IH), 8.55 (s, 0.5H), 8.57 (s, 0.5H), 8.96 (dd, IH, J= 2.0, 4.3), 9.23-9.40 (m, 2H). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₀H₃₈N₅O₂S, 532.2746; found, 532.2745.

THF

β-Keto sulfone 45

[0148] This procedure was adapted from Chun. Chun, J.; Li, G.; Byun, H. -S.; Bittman, R. J. Org. Chem. 2002, 67, 2600-2605. To a 0.6 M solution of methyl phenyl sulfone (1.30 g, 8.31 mmol) in THF (14 raL) at 0 ⁰C was added dropwise n-butyllithium (7.55 mL, 16.6 mmol). The reaction mixture was stirred at 0 ⁰C for 30 min and then cooled to -78 ⁰C. A 0.6 M solution of methyl ester 44 (0.711 g, 4.15 mmol) in THF (6.9 mL) was added dropwise. The reaction mixture was stirred at -78 ⁰C for 3 h. Saturated aqueous NH₄Cl (10 mL) was added and the product was extracted with EtOAc (3 x 20 mL). The organic layers were combined, washed with saturated NaCl, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by column chromatography (5- 25% EtOAc/hexanes) to afford 0.300 g (25%) of 45 as a pale yellow solid, mp 64-66 ⁰C. IR V_max (cm^"1): 2959, 2935, 2873, 2104, 1728, 1321, 1 155. ¹H NMR (300 MHz, CDCl₃): δ 0.91 (t, 3H, J= 7.2), 1.28-1.46 (m, 4H), 1.52-1.90 (m, 2H), 4.13 (dd, IH, J= 4.8, 8.4), 4.26 (d, IH, J= 13.8), 4.42 (d, IH, J= 13.8), 7.56-7.64 (m, 2H), 7.68-7.75 (m, IH), 7.88-7.93 (m, 2H). ¹³C-NMR (125 MHz, CDCl₃): δ 14.0, 22.4, 27.9, 29.9, 63.7, 68.6, 128.6, 129.6, 134.7, 138.7, 195.5. HRMS-FAB (m/z): [MLi]⁺ calcd for Ci₃H_nN₃O₃SLi, 302.1151; found, 302.1142. β-Chloro sulfone 46

[0149] The preparation of the vinyl triflate was adapted from Mastalerz. Mastalerz, H.; Vinet, V. Tetrahedron Lett. 1985, 26, 4315-4318. To a 0.1 M solution of β-keto sulfone 45 (0.137 g, 0.460 mmol) in CH₂Cl₂ (4.6 mL) at -20 ⁰C were added triflic anhydride (0.086 mL, 0.51 mmol) and /-Pr₂EtN (0.089 mL, 0.51 mmol). The reaction mixture was stirred for 1 h while warming to room temperature and then diluted with CH₂Cl₂ (10 mL). The organic layer was washed with water (1 x 5 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The J-Pr₂EtN triflate salt was triturated away with ether. The product was taken on to the next step without further purification.

[0150] To a 0.2 M solution of the vinyl triflate (0.098 g, 0.23 mmol) in THF (1.2 mL) at 0 ⁰C was added tetrabutyl ammonium chloride (0.192 g, 0.690 mmol). The reaction mixture was warmed to room temperature and stirred for 19 h. Saturated aqueous NH₄Cl (5 mL) was added and the aqueous layer was extracted with EtOAc (3 x 10 mL). The organic layers were combined, washed with saturated NaCl (10 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by column chromatography (5-15% EtOAc/hexanes) afforded 0.037 g (51%) of 46 as a pale yellow oil. IR v_max (cm ¹): 3041, 2958, 2863, 2100, 1604, 1324, 1149. ¹H NMR (400 MHz, CDCl₃): δ 0.86 (t, 3H, J = 6.8), 1.14-1.36 (m, 4H), 1.62-1.82 (m, 2H), 3.95-4.02 (m, IH), 6.90 (s, IH), 7.53-7.59 (m, 2H), 7.64-7.69 (m, IH), 7.96-8.01 (m, 2H). ¹³C-NMR (125 MHz, CDCl₃): δ 14.0, 22.2, 27.4, 31.9, 67.1, 128.2, 129.4, 130.0, 134.2, 140.5, 147.1. HRMS-FAB (m/z): [MLi]⁺ calcd for C₁₃H₁₆N₃O₂SClLi, 320.0812; found, 320.0815. β-Chloro vinyl sulfone 43

[0151] Procedure Dl was followed using propargyl amine 39 (0.025 g, 0.10 mmol), azide 46 (0.031 g, 0.10 mmol), sodium ascorbate (0.10 mL, 0.10 mmol), copper(II) sulfate pentahydrate (0.033 mL, 0.010 mmol) in 1 :1 tBuOH:H₂O (0.4 mL) to afford 26.6 mg (51%) of a 0.7:0.3 mixture of diastereomers of 43 as a clear oil. Olefin geometry was confirmed by NOE spectroscopy (vide infra). ¹H NMR (400 MHz, CDCl₃): δ 0.74 (d, 2.1H, J= 6.8), 0.75 (d, 0.9H, J= 6.8), 0.86 (t, 3H, J= 7.2), 0.98 (d, 3H, J= 6.8), 1.08-1.39 (m, 4H), 1.46 (s, 3H), 1.76 (br s, IH), 2.09-2.13 (m, 3H), 3.53 (d, IH, J= 12.8), 3.73 (d, IH₅ J= 12.8), 5.18-5.25 (m, IH), 6.80 (s, 0.3H), 6.81 (s, 0.7H), 7.38 (dd, IH, J= 4.4, 8.4), 7.42 (s, IH), 7.51 (t, 2H, J = 7.6), 7.58-7.68 (m, 2H), 7.71 (s, IH), 7.90-7.95 (m, 2H), 8.03 (d, IH, J= 8.4), 8.12 (d, IH, J= 8.0), 8.87 (dd, IH, J= 1.6, 4.4). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₀H₃₇N₅O₂SCl, 566.2357; found, 566.2357. Anal. Calcd for C₃₀H₃₆N₅O₂SCl: C, 63.64; H, 6.41 ; N, 12.37. Found: C, 63.30; H, 6.56; N, 11.99. 1) isobutylchloroformate tø-methylmorp holme HO-,

N₃ THF. -4Q °C N₃^^Br

2) CH₂N₂, THF, 0 °C KF, DMF, 0 ⁰C

B^"U 3) HBr, THF, 0 "C Bu Bu 40 48 47a-c

Bromomethyl ketone azide 48

[0152] This procedure was adapted from a prior publication. Chino, M.; Wakao, M.; Ellman, J. A. Tetrahedron 2002, 58, 6305-6310. Isobutylchloroformate (0.69O mL, 5.25 mmol) was added to a 0.1 M solution of azido acid 40 (0.750 g, 4.77 mmol) and N-methyl morpholine (0.580 mL, 5.25 mmol) in THF (48 mL) at -40 ⁰C. The reaction mixture was stirred for 20 min and then cannula filtered into a flask at 0 ⁰C to remove the white solid. Excess diazomethane, prepared from Diazald (3.17 g, 14.8 mmol), was introduced in situ, according to the literature procedure, Lombardi, P. Chem. Ind. (London) 1990, 708, while the flask was maintained at 0 ⁰C. After addition of the diazomethane, the reaction flask was stoppered and was maintained at 0 ⁰C in a refrigerator overnight. The reaction mixture was treated with 48% aqueous HBr (0.981 mL) and stirred for 15 min at 0 ⁰C. After addition of the HBr, N₂ gas evolution was observed. The reaction mixture was diluted with EtOAc (50 mL) and was then washed with 10 wt% citric acid (2 x 10 mL), saturated NaHCO₃ (2 x 20 mL), and saturated NaCl (1 x 10 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. Column chromatography (1-5% EtOAc/hexanes) afforded 0.908 g (81%) of 48 as a faintly yellow oil. The purified product was contaminated with 10% of the methyl ester as determined by ¹H NMR. The methyl ester, however, was unreactive under the subsequent reaction conditions and was therefore easily removed later in the synthetic sequence. Only the peaks for the desired product are reported in the NMR spectra. IR v_max (cπf¹): 2960, 2874, 2106, 1821, 1739. ¹H NMR (300 MHz, CDCl₃): δ 0.91 (t, 3H, /= 6.5), 1.30-1.51 (m, 4H), 1.68-1.92 (m, 2H), 3.99-4.18 (m, 3H). ¹³C-NMR (75 MHz, CDCl₃): δ 14.0, 22.4, 28.0, 30.9, 32.0, 66.2, 199.0. MS (ESI): m/z 205, 207 [(M- N₂)H]⁺.

Acyloxymethyl ketone azide 47a

[0153] Procedure E was followed using bromomethyl ketone 48 (0.10 g, 0.43 mmol), 2,6- dimethylbenzoic acid (0.257, 1.71 mmol), and potassium fluoride (0.0990 g, 1.71 mmol). The reaction mixture was stirred for 30 min. The crude reaction mixture was purified by column chromatography (1-5% EtOAc/hexanes) to afford 0.082 g (63%) of 47a as a clear oil. IR v_max (cm^"1): 2960, 2932, 2873, 2106, 1743, 1596. ¹H NMR (500 MHz, CDCl₃): δ 0.94 (t, 3H, J= 7.0), 1.32-1.52 (m, 4H), 1.79-1.97 (m, 2H), 2.41 (s, 6H), 4.02 (dd, IH, J= 5.0, 8.0), 5.04 (d, IH₅ J= 17.5), 5.11 (d, IH₅ J= 17.0), 7.05 (d, 2H, J= 7.5), 7.21 (t, IH, J= 7.5). ¹³C- NMR (125 MHz, CDCl₃): δ 14.0, 20.1, 22.4, 27.8, 30.9, 66.7, 66.8, 127.9, 130.0, 132.5, 135.9, 169.15, 200.4. HRMS-FAB (m/z): [MLi]⁺ calcd for C₆H_2IN₃O₃Li, 310.1743; found, 310.1749.

Acyloxymethyl ketone azide 47b

[0154] Procedure E was followed using bromomethyl ketone 48 (0.250 g, 1.07 mmol), 2,6- bis(trifluoromethyl)-benzoic acid (0.854, 3.31 mmol), and potassium fluoride (0.186 g, 3.20 mmol). The reaction mixture was stirred for 30 min. The crude reaction mixture was purified by column chromatography (1-10% EtOAc/hexanes) to afford 0.321 g (73%) of 47b as a clear oil. IR v_max (cm^'1): 2962, 2935, 2876, 2107, 1744, 1594. ¹H NMR (400 MHz, CDCl₃): δ 0.92 (t, 3H₃ J= 7.0), 1.29-1.51 (m, 4H), 1.75-1.96 (m, 2H), 4.04 (dd, IH, J= 4.8, 8.4), 5.06 (d, IH₅ J= 17.6), 5.13 (d, IH, J= 17.6), 7.76 (t, IH, J= 8.0), 7.96 (d, 2H, J= 8.0). ¹³C-NMR (100 MHz, CDCl₃): δ 14.0, 22.4, 27.8, 30.6, 66.4, 68.2, 122.9 (q, J= 273), 129.4, 129.7, 130.2 (q, J= 4.0), 131.0, 164.4, 199.5. ¹⁹F NMR (376 MHz, CDCl₃): δ -58.7 (s, 6F). HRMS-FAB (m/z): [MLi]⁺ calcd for C₁₆H₁₅N₃O₃F₆Li, 418.1178; found, 418.1178.

Aryloxymethyl ketone azide 47c

[0155] Procedure E was followed using bromomethyl ketone 48 (0.1O g, 0.43 mmol), 2,3,5,6-tetrafluorophenol (0.220, 1.32 mmol), and potassium fluoride (0.0740 g, 1.28 mmol). The reaction mixture was stirred for 3 h. The crude reaction mixture was purified by HPLC [preparatory reverse-phase Ci₈ column (24.1 x 250 mm), CH₃CN/H₂O-0.1% CF₃CO₂H = 5:95 to 95:5 over 55 min; 10 mL/min; 254 nm detection for 65 min] and lyophilized to afford 0.062 g (45%) of 47c as a clear oil. IR v_max (cm^'1): 2962, 2935, 2876, 2106, 1743, 1642, 1518. ¹H NMR (500 MHz, CDCl₃): δ 0.94 (t, 3H, J= 7.0), 1.32-1.51 (m, 4H), 1.74-1.83 (m, IH), 1.89-1.97 (m, IH), 4.14 (dd, IH, J= 4.5, 8.5), 5.00 (d, IH, J= 17.5), 5.05 (d, IH, J = 17.5), 6.78-6.85 (m, IH). ¹³C-NMR (125 MHz, CDCl₃): δ 13.9, 22.4, 28.0, 30.5, 65.8, 75.6 (t, J= 3.0), 100.2 (t, J= 18.0), 137.0-137.2 (m), 140.6 (dm, J= 247), 146.5 (dm, J= 247), 201.8. ¹⁹F NMR (376 MHz, CDCl₃): δ -156.3 - -156.2 (m, 2F), -138.9 - -138.4 (m, 2F). HRMS-FAB (m/z): [MLi]⁺ calcd for C₃H₁₃N₃O₂F₄Li, 326.1104; found, 326.1 106.

Acyloxymethyl ketone inhibitor 49

[0156] Procedure Dl was followed using propargyl amine 39 (0.025 g, 0.10 mmol), azide 47a (0.031 g, 0.10 mmol), sodium ascorbate (0.10 mL, 0.10 mmol), copper(II) sulfate pentahydrate (0.033 mL, 0.010 mmol) in 1:1 tBuOH:H₂O (0.4 mL) to afford 19.3 mg (35%) of a 1 :1 mixture of diastereomers of 49 as a clear oil. ¹H NMR (500 MHz, CDCl₃): δ 0.82 (d, 3H, J= 7.0), 0.86 (t, 1.5H, J= 7.0), 0.88 (t, 1.5H, J= 7.0), 1.02 (d, 1.5H, J= 6.5), 1.03 (d, 1.5H, J= 6.5), 1.11-1.43 (m, 4H), 1.50 (s, 1.5H), 1.52 (s, 1.5H), 1.85 (br s, IH), 2.08-2.24 (m, 2H), 2.27-2.36 (m, IH), 2.35 (s, 3H), 2.36 (s, 3H), 3.61 (d, 0.5H, J= 13.0), 3.62 (d, 0.5H, J= 13.0), 3.78 (d, IH, J= 13.0), 4.84 (d, 0.5H, J= 17.0), 4.85 (d, 0.5H, J = 17.0), 4.95 (d, IH, J= 17.0), 5.44 (dd, IH, J= 5.0, 10.0), 7.03 (d, 2H, J= 7.5), 7.21 (t, IH, J= 7.5), 7.349 (dd, 0.5H, J= 4.5, 8.5), 7.353 (dd, 0.5H, J= 4.5, 8.5), 7.60 (d, IH, J= 5.5), 7.676 (dd, 0.5H, J= 2.0, 9.0), 7.684 (dd, 0.5H, J= 2.0, 9.0), 7.73 (s, IH), 8.02 (dd, IH, J= 2.0, 9.0), 8.08 (d, IH, J= 8.0), 8.86 (d, IH, J= 4.5). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₃H₄₂N₅O₃, 556.3288; found, 556.3279. Anal. Calcd for C₃₃H₄₁N₅O₃: C, 71.32; H, 7.44; N, 12.60. Found: C, 70.91 ; H, 7.62; N, 12.56.

Acyloxymethyl ketone inhibitor 50

[0157] Procedure Dl was followed using propargyl amine 39 (0.019 g, 0.075 mmol), azide 47b (0.031 g, 0.075 mmol), sodium ascorbate (0.075 mL, 0.075 mmol), copper(II) sulfate pentahydrate (0.025 mL, 0.0075 mmol) in 1 :1 tBuOH:H₂O (0.3 mL) to afford 24.6 mg (49%) of a 1 :1 mixture of diastereomers of 50 as a white sticky solid. ¹H NMR (400 MHz, DMSO- d₆): δ 0.69-0.81 (m, 6H), 0.95 (d, 3H, J= 6.8), 1.11-1.33 (m, 4H), 1.39 (s, 3H), 2.05-2.34 (m, 4H), 3.51 (d, IH, J= 13.2), 3.68 (d, IH, J= 13.2), 5.13 (d, 0.5H, J= 17.2), 5.14 (d, 0.5H, J= 17.2), 5.27 (d, 0.5H, J= 17.2), 5.28 (d, 0.5H, J= 17.2), 5.71 (dd, IH, J= 4.4, 10.8), 7.46 (dd, 0.5H, J= 4.0, 8.4), 7.47 (dd, 0.5H, J= 4.0, 8.4), 7.67 (d, IH, J= 8.8), 7.80 (s, IH), 7.91 (d, IH, J= 8.8), 7.99 (t, IH, J= 8.0), 8.17 (s, IH), 8.21-8.30 (m, 3H), 8.80-8.85 (m, IH). ¹⁹F NMR (376 MHz, CDCl₃): δ -58.7 (s, 6F). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₃H₃₆N₅O₃F₆, 664.2722; found, 664.2710. Anal. Calcd for C₃₃H₃₅N₅O₃F₆: C, 59.72; H, 5.32; N, 10.55. Found: C, 59.58; H, 5.34; N, 10.51.

Acyloxy methyl ketone inhibitor 51

[0158] Procedure Dl was followed using propargyl amine S6b (0.021 g, 0.082 mmol), azide 47b (0.034 g, 0.082 mmol), sodium ascorbate (0.082 mL, 0.082 mmol), copper(II) sulfate pentahydrate (0.027 mL, 0.0082 mmol) in 1 :1 tBuOH:H₂O (0.33 mL) to afford 24.4 mg (44%) of a 1 : 1 mixture of diastereomers of 51 as a white sticky solid. The inhibitor was > 99% pure as determined by HPLC-MS analysis (Cl 8 column (2.1 x 150 mm); 0.4 mL/min; 254 nm detection in two solvent systems: CH₃CN/H₂O-0.1% CF₃CO₂H, 5:95 to 95:5 over 16 min, 95:5 for 2 min; CH₃OH/H₂O, 5:95 to 95:5 over 20 min, 95:5 for 10 min). ¹H NMR (500 MHz, CDCl₃): δ 0.79 (d, 3H, J= 7.0), 0.83 (t, 1.5H, J= 7.0), 0.85 (t, 1.5H, J= 7.0), 1.006 (d, 1.5H, J= 7.0), 1.010 (d, 1.5H, J= 7.0), 1.07-1.41 (m, 4H), 1.48 (s, 1.5H), 1.49 (s, 1.5H), 1.72 (br s, IH), 2.02-2.32 (m, 3H), 3.55 (d, 0.5H, J= 12.5), 3.56 (d, 0.5H, J= 12.5), 3.73 (d, IH, J = 12.5), 4.90-5.01 (m, 2H), 5.495 (dd, 0.5H, J= 5.0, 10.0), 5.500 (dd, 0.5H, J= 5.0, 10.0), 7.45 (dd, 0.5H, J= 1.5, 8.5), 7.46 (dd, 0.5H, J= 1.5, 8.5), 7.57 (s, 0.5H), 7.58 (s, 0.5H), 7.76 (t, IH, J= 8.5), 7.92-7.98 (m, 3H), 8.02 (d, 0.5H, J= 8.5), 8.03 (d, 0.5H, J= 8.5), 8.931 (s, 0.5H), 8.933 (s, 0.5H). ¹⁹F NMR (376 MHz, CDCl₃): δ -58.7 (s, 6F). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₁H₃₄N₅O₃F₆S, 670.2287; found, 670.2288.

Acyloxymethyl ketone inhibitor 52

[0159] Procedure Dl was followed using propargyl amide S7a (0.040 g, 0.15 mmol), azide 47b (0.062 g, 0.15 mmol), sodium ascorbate (0.15 mL, 0.15 mmol), copper(II) sulfate pentahydrate (0.050 mL, 0.015 mmol) in 1 :1 tBuOH:H₂O (0.6 mL) to afford 22.7 mg (22%) of a 0.6:0.4 mixture of diastereomers of 52 as a white sticky solid. ¹H NMR (400 MHz, CDCl₃): δ 0.80-0.89 (m, 6H), 0.95 (d, 1.8H, J= 6.8), 0.99 (d, 1.2H, J= 6.8), 1.10-1.43 (m, 4H), 1.97 (s, 1.2H), 1.98 (s, 1.8H), 2.06-2.16 (m, IH), 2.24-2.35 (m, IH), 2.96-3.08 (m, IH), 4.96 (s, 2H), 5.51 (dd, IH, J= 5.0, 10.0), 7.47 (dd, 1H, J= 4.0, 8.4), 7.63 (s, 0.6H), 7.70 (s, 0.4H), 7.71 (s, IH), 7.77 (t, IH₅ J= 8.0), 7.95 (d, 2H, J= 8.0), 8.09-8.13 (m, IH), 8.14 (s, 0.6H), 8.17 (s, 0.4H), 8.24-8.28 (m, IH), 8.31 (d, 0.6H, J= 2.0), 8.32 (d, 0.4H, J= 2.0), 8.98 (dd, IH, J= 1.6, 4.4) . ¹⁹F NMR (376 MHz, CDCl₃): δ -58.67 (s, 3.6F), -58.68 (s, 2.4F). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₃H₃₄N₅O₄F₆, 678.2515; found, 678.2521. Anal. Calcd for C₃₃H₃₃N₅O₄F₆: C, 58.49; H, 4.91; N, 10.33. Found: C, 58.39; H, 5.07; N, 10.24.

Acyloxymethyl ketone inhibitor 53

[0160] Procedure Dl was followed using propargyl amide S7b (0.041 g, 0.15 mmol), azide 47b (0.062 g, 0.15 mmol), sodium ascorbate (0.15 mL, 0.15 mmol), copper(II) sulfate pentahydrate (0.05O mL, 0.015 mmol) in 1 :1 tBuOH:H₂O (0.6 mL) to afford 22.2 mg (22%) of a 1 :1 mixture of diastereomers of 53 as a white sticky solid. ¹H NMR (400 MHz, CDCl₃): δ 0.79-0.88 (m, 6H), 0.94 (d, 1.5H, J= 6.8), 0.98 (d, 1.5H, J= 6.8), 1.10-1.47 (m, 4H), 1.96 (s, 3H), 2.05-2.33 (m, 2H), 2.93-3.05 (m, IH), 4.96 (m, 2H), 5.51 (dd, IH, J= 4.8, 10.0), 7.58 (s, 0.5H), 7.61 (s, 0.5H), 7.70 (s, IH), 7.77 (t, IH, J= 8.0), 7.92-7.99 (m, 3H), 8.16 (s, 0.5H), 8.18 (s, 0.5H), 8.47 (m, IH), 9.11 (s, IH). ¹⁹F NMR (376 MHz, CDCl₃): δ -58.68 (s, 3.6F), - 58.67 (s, 2.4F). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₁H₃₂N₅O₄SF₆, 684.2079; found, 684.2083. Anal. Calcd for C₃₁H₃₁N₅O₄SF₆: C, 54.46; H, 4.57; N, 10.24; S, 4.69. Found: C, 54.22; H, 4.43; N, 10.00; S, 4.80.

Aryloxymethyl ketone inhibitor 54

[0161] Procedure Dl was followed using propargyl amine 39 (0.020 g, 0.075 mmol), azide 47c (0.024 g, 0.075 mmol), sodium ascorbate (0.075 mL, 0.075 mmol), copper(II) sulfate pentahydrate (0.025 mL, 0.0075 mmol) in 1 :1 tBuOH:H₂O (0.3 mL) to afford 21.0 mg (49%) of a 1 :1 mixture of diastereomers of 54 as a pale yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 0.80 (d, 3H, J= 6.8), 0.85 (t, 1.5H, J= 7.0), 0.87 (t, 1.5H, J= 7.0), 1.02 (d, 3H, J= 6.8), 1.07-1.45 (m, 4H), 1.50 (s, 1.5H), 1.51 (s, 1.5H), 1.79 (br s, IH), 1.98-2.13 (m, IH), 2.19 (sept, IH, J= 6.8), 2.22-2.46 (m, IH), 3.59 (d, IH, J= 12.8), 3.77 (d, 0.5H, J= 12.8), 3.78 (d, 0.5H, J= 12.8), 4.93 (s, 2H), 5.66 (dd, 0.5H, J= 4.8, 10.4), 5.67 (dd, 0.5H, J= 4.8, 10.4), 6.76-6.84 (m, IH), 7.37 (dd, IH, J= 4.4, 8.4), 7.57 (s, 0.5H), 7.58 (s, 0.5H), 7.65- 7.70 (m, IH), 7.73 (s, IH), 8.01 (d, 0.5H, J= 8.4), 8.03 (d, 0.5H, J= 8.4), 8.09-8.13 (m, IH), 8.84- 8.88 (m, IH). ¹⁹F NMR (376 MHz, CDCl₃): δ -156.2 - -156.1 (m, 2F), -138.0 - -137.9 (m, 2F). HRMS-FAB (m/z): [MNa]⁺ calcd for C₃₀H₃₃N₅O₂F₄Na, 594.2468; found, 594.2453. Anal. Calcd for C₃₀H₃₃N₅O₂F₄: C, 63.04; H, 5.82; N, 12.25. Found: C, 62.74; H, 5.95; N, 11.92.

Aryloxymethyl ketone inhibitor 55.

[0162] Procedure Dl was followed using propargyl amine S6b (0.039 g, 0.15 mmol), azide 47c (0.048 g, 0.15 mmol), sodium ascorbate (0.15 mL, 0.15 mmol), copper(II) sulfate pentahydrate (0.050 mL, 0.015 mmol) in 1 :1 tBuOH:H₂O (0.6 mL) to afford 31.5 mg (36%) of a 0.6:0.4 mixture of diastereomers of 55 as a clear sticky oil. ¹H NMR (400 MHz, CDCl₃): δ 0.79 (d, 3H, J= 6.8), 0.87 (t, 3H, J= 7.2), 1.00 (d, 3H, J= 6.8), 1.07-1.45 (m, 4H), 1.480 (s, 1.8H), 1.485 (s, 1.2H), 1.90 (br s, IH), 1.98-2.35 (m, 3H), 3.55 (d, IH, J= 12.4), 3.73 (d, IH, J= 12.4), 4.92 (s, 2H), 5.66 (dd, IH, J = 4.8, 10.4), 6.76-6.84 (m, IH), 7.44 (dd, IH₅ J = 2.0, 8.4), 7.56 (s, 0.4H), 7.57 (s, 0.6H), 7.93 (s, IH), 8.02 (d, 0.4H, J= 8.4), 8.03 (d, 0.6H, J = 8.4), 8.93 (s, 0.4H), 8.94 (s, 0.6H). ¹⁹F NMR (376 MHz, CDCl₃): δ -156.2 - -156.1 (m, 2F), -138.0 - -137.9 (m, 2F). HRMS-FAB (m/z): [MH]⁺ calcd for C₂₈H₃₂N₅O₂F₄S, 578.2213; found, 578.2208. Anal. Calcd for C₂₈H_3IN₅O₂SF₄: C, 58.22; H, 5.41; N, 12.12. Found: C, 58.05; H, 5.63; N, 11.86.

Aryloxymethyl alcohol azide 56

[0163] This procedure was adapted from a prior publication. 10 To a 0.1 M solution of aryloxymethyl ketone azide 47c (0.051 g, 0.16 mmol) in 95:5 THF:H₂O (1.6 mL) at 0 ⁰C was added sodium borohydride (0.008 g, 0.21 mmol). The reaction mixture was warmed to room temperature and stirred for 45 min. It was then neutralized with aqueous IN HCl and extracted with EtOAc (3 x 5 mL). The organic extracts were washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The product was taken on to the next step without purification as a 0.6:0.4 mixture of diastereomers. ¹H NMR (500 MHz, CDCl₃): δ 0.91-0.97 (m, 3H), 1.33-1.63 (m, 4H), 1.69-1.78 (m, 2H), 2.36 (d, 0.6H, J= 4.5), 2.54 (d, 0.4H, J= 4.5), 3.45-3.50 (m, 0.6H), 3.52-3.58 (m, 0.4H), 3.91-4.02 (m, IH), 4.22- 4.31 (m, 1.6H), 4.34-4.39 (m, 0.4H), 6.78-6.86 (m, IH). HRMS-FAB (m/z): [MLi]⁺ calcd for Ci₃H₁₅N₃O₂F₄Li, 328.1254; found, 328.1260.

Aryloxymethyl alcohol 57

[0164] Procedure Dl was followed using propargyl amine 39 (0.020 g, 0.078 mmol), azide 56 (0.025 g, 0.078 mmol), sodium ascorbate (0.078 mL, 0.078 mmol), copper(II) sulfate pentahydrate (0.026 mL, 0.0078 mmol) in 1 :1 tBuOH:H₂O (0.31 mL). The crude reaction mixture was purified by column chromatography (50-80% EtOAc/hexanes) to afford 30.0 mg (67%) of a 0.6:0.4 mixture of diastereomers of 57 as a clear oil. ¹H NMR (400 MHz, CDCl₃): δ 0.82 (t, 3H, J = 7.2), 0.86-0.94 (m, 3H), 1.02-1.07 (m, 3H), 1.28-1.45 (m, 4H), 1.52 (s, 3H), 1.68 (br s, IH), 2.03-2.33 (m, 3H), 3.20-3.26 (m, 0.4H), 3.43-3.51 (m, 0.6H), 3.56-3.63 (m, IH), 3.74-3.85 (m, 1.6H), 3.97-4.05 (m, IH), 4.20-4.26 (m, 0.4H), 4.36-4.46 (m, IH), 4.65-4.72 (m, 0.4H), 4.78-4.85 (m, 0.6H), 6.76-6.85 (m, IH), 7.41 (dd, IH, J= 4.0, 8.0), 7.53 (s, 0.4H), 7.60 (s, 0.6H), 7.67-7.73 (m, IH), 7.76 (s, IH), 8.06 (d, IH, J= 8.4), 8.15 (d, IH, J= 8.0), 8.90 (dd, IH, J= 1.6, 4.0). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₀H₃₆N₅O₂F₄, 574.2795; found, 574.2805.

Aryloxymethyl ketone inhibitor 58

[0165] The same procedure as for aldehyde S4a was followed using aryloxymethyl alcohol 58 (0.016 g, 0.028 mmol), Dess-Martin periodinane (0.035 g, 0.084 mmol) in water-saturated CH₂Cl₂ (0.7 mL) to afford 12.0 mg (75%) of 58 as a single diastereomer as a clear oil. Chromatography resulted in racemization, thus the product was taken on to the racemization study without purification. ¹H NMR (500 MHz, CDCl₃): δ 0.80 (t, 3H, J= 7.0), 0.87 (t, 3H, J = 7.0), 1.01 (d, 3H, J= 7.0), 1.11-1.43 (m, 4H), 1.51 (s, 3H), 2.03-2.12 (m, IH), 2.19 (sept, IH, J= 7.0), 2.26-2.35 (m, IH), 3.58 (d, IH₅ J= 13.0), 3.77 (d, IH, J= 13.0), 4.92 (s, 2H), 5.66 (dd, IH, J= 5.0, 10.0), 7.17 (tt, IH, J= 7.0, 10.0), 7.37 (dd, IH, J= 4.0, 8.0), 7.58 (s, IH), 7.68 (dd, IH, J= 2.0, 8.5), 7.73 (s, IH), 8.03 (d, IH, J= 8.5), 8.12 (d, IH, J= 8.0), 8.86 (dd, IH, J= 1.5, 4.0).

Racemization study

[0166] Diastereomerically pure inhibitor 58 (0.012 mg, 0.021 mmol) was dissolved in DMSOd₆ (10.0 mL) and added to assay buffer (200 mL) consisting of a 100 mM solution of pH 6.3 sodium phosphate buffer with 400 mM of sodium chloride. The mixture was heated to 37 ⁰C and stirred for 3 hours. The aqueous layer was extracted with EtOAc (4 x 100 mL). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was then dissolved in CH₂Cl₂ and washed with water. The aqueous layer was backextracted with CH₂Cl₂ (2 x 5 mL). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to afford an oil. ¹H-NMR of the crude inhibitor indicated a 1 : 1 mixture of diastereomers.

*

General synthesis of bromomethyl ketone azides 48 and 59-60 (Procedure F).

[0167] This procedure was adapted from a prior publication. Chino, M.; Wakao, M.; Ellman, J. A. Tetrahedron 2002, 58, 6305-6310. Isobutylchloroformate (1.1 equiv) was added to a 0.1 M solution of the azido acid (1 equiv) and TV-methyl morpholine (1.1 equiv) in THF at -40 ⁰C. Pelletier, J. C; Lundquist, J. T. Org. Lett. 2001, 3, 781-783. The reaction mixture was stirred for 20 min and then cannula filtered into a flask at 0 ⁰C to remove the white solid. Excess diazomethane, prepared from Diazald (3.1 equiv), was introduced in situ, according to the literature procedure, while the flask was maintained at 0 ⁰C. Lombardi, P. Chem. Ind. (London) 1990, 708. After addition of the diazomethane, the reaction flask was stoppered and was maintained at 0 ⁰C in a refrigerator overnight. The reaction mixture was treated with 48% aqueous HBr (0.981 mL) and stirred for 15 min at 0 ⁰C. After addition of the HBr, N₂ gas evolution was observed. The reaction mixture was diluted with EtOAc (50 mL) and was then washed with 10 wt% citric acid (2 x 10 mL), saturated NaHCO₃ (2 x 20 mL), and saturated NaCl (1 x 10 mL). The organic layer was dried over Na₂Sθ₄, filtered, and concentrated under reduced pressure. The product was taken on to the next step without further purification.

Bromomethyl ketone azide 48.

[0168] Procedure F was followed using isobutylchloroformate (0.184 mL, 1.40 mmol), 2- azidohexanoic acid (0.200 g, 1.27 mmol), /V-methyl morpholine (0.154 mL, 1.40 mmol), Diazald (0.84 g, 3.94 mmol), and 48% aqueous HBr (0.262 mL) in THF (13 mL) to afford the crude bromomethyl ketone product as a pale yellow oil.

Bromomethyl ketone azide 59.

[0169] Procedure F was followed using isobutylchloroformate (0.663 mL, 5.11 mmol), 2- azidobutyric acid (0.600 g, 4.65 mmol), TV-methyl morpholine (0.562 mL, 5.11 mmol), Diazald (3.00 g, 13.95 mmol), and 48% aqueous HBr (1.05 mL) in THF (50 mL) to afford the crude product as a pale yellow oil.

O

N₃>^^Br

Bromomethyl ketone azide 60.

[0170] Procedure F was followed using isobutylchloroformate (0.297 mL, 2.29 mmol), 2- azidopropionic acid (0.400 g, 2.08 mmol), TV-methyl morpholine (0.251 mL, 2.29 mmol), Diazald (1.34 g, 6.24 mmol), and 48% aqueous HBr (0.47 mL) in THF (24 mL) to afford the crude product as a colorless oil.

General synthesis of 2,3,5,6-tetrafluorophenoxymethyl ketone azides 47c and 61-62 (Procedure G).

[0171] To a 0.6 M solution of 2,3,5,6-tetrafluorophenol (3.0-3.1 equiv) in DMF at 0 ⁰C was added potassium fluoride (3.0 equiv), and the reaction mixture was stirred for 10 min. The appropriate bromomethyl ketone 48, 59, or 60 (1.0 equiv) was then added in a small amount of DMF. The reaction mixture was stirred at 0 ⁰C for 3-7 h. The reaction mixture was diluted with CH₂Cl₂ and washed with water (Ix), saturated NaHCO₃ (Ix), water (2x), and brine (Ix). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. Column chromatography afforded the pure product.

Aryloxymethyl ketone azide 47c.

[0172] Procedure G was followed using bromomethyl ketone 48 (0.10 g, 0.43 mmol), 2,3,5,6-tetrafluorophenol (0.220 g, 1.32 mmol), and potassium fluoride (0.0740 g, 1.28 mmol) in DMF (2.2 mL). The reaction mixture was stirred for 3 h. The crude reaction mixture was purified by HPLC [preparatory reverse-phase Ci₈ column (24.1 x 250 mm), CH₃CN/H₂O-0.1% CF₃CO₂H = 5:95 to 95:5 over 55 min; 10 mL/min; 254 nm detection for 65 min] and lyophilized to afford 0.062 g (45%) of 47c as a clear oil. IR v_max (cm^"1): 2962, 2935, 2876, 2106, 1743, 1642, 1518. ¹H NMR (500 MHz, CDCl₃): δ 0.94 (t, 3H, J= 7.0), 1.32-1.51 (m, 4H), 1.74-1.83 (m, IH), 1.89-1.97 (m, IH), 4.14 (dd, IH, J= 4.5, 8.5), 5.00 (d, IH, J= 17.5), 5.05 (d, IH, J= 17.5), 6.78-6.85 (m, IH). ¹⁹F NMR (376 MHz, CDCl₃): δ - 156.3 - -156.2 (m, 2F), -138.9 - -138.4 (m, 2F). HRMS-FAB (m/z): [MLi]⁺ calcd for Ci₃Hi₃N₃O₂F₄Li, 326.1104; found, 326.1106.

Aryloxymethyl ketone azide 61.

[0173] Procedure G was followed using bromomethyl ketone 59 (0.48 g, 2.33 mmol), 2,3,5,6-tetrafluorophenol (1.16 g, 6.99 mmol), and potassium fluoride (0.406 g, 6.99 mmol) in DMF (7.0 mL). The reaction mixture was stirred for 3 h. The crude reaction mixture was purified by silica gel chromatography (Hexanes/Ethyl Acetate = 20/80) to afford 0.38O g (56%) of 61 as a clear oil.

Aryloxymethyl ketone azide 62.

[0174] Procedure G was followed using bromomethyl ketone 60 (0.17 g, 0.89 mmol), 2,3,5,6-tetrafluorophenol (0.442 g, 2.66 mmol), and potassium fluoride (0.155 g, 2.66 mmol) in DMF (2.2 mL). The reaction mixture was stirred for 3 h. The crude reaction mixture was purified by silica gel chromatography (Hexanes/Ethyl Acetate = 20/80) to afford 0.125 g (51%) of 62 as a clear oil.

16, R = /-Propyl 39, R = /-Propyl

63, R = cyc/o-Propyl 68, R = cyc/o-Propyl

64, R = /-Butyl 69, R = /-Butyl

65, R = cyc/o-Butyl 70, R = cyc/o-Butyl

66, R = cyc/o-Pentyl 71, R = cyc/o-Pentyl

67, R = cyc/o-Hexyl 72, R = cyc/o-Hexyl

General synthesis of quinoline propargyl amines 39 and 68-72 (Procedure H).

[0175] The HCl salt of propargyl amine 16 or 63-67 (1.0-1.4 equiv) was dissolved in water and basified to pH=l 1 with 1 M NaOH. The aqueous layer was then extracted with an appropriate volume of toluene to give a 0.25M solution of the free amine in toluene. The procedure is performed in this way because the free-based amine is volatile. The organic layer is dried over Na₂SO₄, and filtered. To the solution of propargyl amine 16 or 63-67 in toluene were added quinoline-6-carboxyaldehyde (1-1.1 equiv) and activated 4 A molecular sieves. The reaction mixture was stirred for 16 h and then filtered through a plug of celite. The celite was washed with CH₂Cl₂ (3x). The organic washes were concentrated to afford the crude imine.

[0176] To a 0.2 M solution of the propargyl imine (1 equiv) in methanol at 0 °C was added sodium borohydride (2 equiv). After stirring the reaction mixture at 0 ⁰C for 1 h, it was diluted with water and extracted with CH₂Cl₂ (3x). The organic layers were combined, dried over Na₂SO₄, filtered, and concentrated. The crude reaction mixture was purified by column chromatography to afford the pure product.

Quinoline propargyl amine 39.

[0177] Procedure H was followed using the HCl salt of propargyl amine 16 (0.280 g, 1.89 mmol) and quinoline-6-carboxyaldehyde (0.248 g, 1.58 mmol) in toluene (6.3 mL) followed by reduction with sodium borohydride (0.120 g, 3.2 mmol) in methanol (7.9 mL) to afford 0.262 g (66%) of 39 as a white solid, mp 45.9-46.7 ⁰C. IR v_max (cm^"1): 3299, 2964, 2875, 2360, 2342, 2096. ¹H NMR (300 MHz, CDCl₃): δ 1.04 (d, 3H, J= 6.9), 1.07 (d, 3H, J= 6.9), 1.31 (s, 3H), 1.39 (br s, IH), 1.87 (sept, IH, J= 6.9), 2.39 (s, IH), 4.00 (d, IH, J= 12.6), 4.06 (d, IH, J= 12.6), 7.36 (dd, IH, J= 4.2, 8.1), 7.74 (dd, IH, J= 1.8, 8.7), 7.78 (s, IH), 8.05 (d, IH, J= 8.4), 8.11 (dd, IH₅ J= 1.2, 8.4), 8.86 (dd, IH₅ J= 1.5, 4.2). ¹³C-NMR (IOO MHz, CDCl₃): δ 17.1, 18.1, 23.1, 36.5, 48.3, 57.4, 71.6, 88.0, 121.3, 126.4, 128.4, 129.6, 130.8, 136.0, 139.7, 147.9, 150.2. HRMS-FAB (m/z): [MH]⁺ calcd for C₁₇H₂iN₂, 253.1704; found, 253.1710.

Quinoline propargyl amine 68.

[0178] Procedure H was followed using the HCl salt of propargyl amine 63 (0.105 g, 0.720 mmol) and quinoline-6-carboxyaldehyde (0.113 g, 0.720 mmol) in toluene (3.0 mL) followed by reduction with sodium borohydride (0.054 g, 1.44 mmol) in methanol (4.0 mL) to afford 0.088 g (49%) of 68 as a white solid.

Quinoline propargyl amine 69.

[0179] Procedure H was followed using the HCl salt of propargyl amine 64 (0.066 g, 0.41 mmol) and quinoline-6-carboxyaldehyde (0.048 g, 0.30 mmol) in toluene (1.6 mL) followed by reduction with sodium borohydride (0.023 g, 0.60 mmol) in methanol (1.5 mL) to afford 0.053 g (66%) of 69.

Quinoline propargyl amine 70.

[0180] Procedure H was followed using the HCl salt of propargyl amine 65 (0.111 g, 0.700 mmol) and quinoline-6-carboxyaldehyde (0.110 g, 0.700 mmol) in toluene (3.0 mL) followed by reduction with sodium borohydride (0.053 g, 1.400 mmol) in methanol (4.0 mL) to afford 0.115 g (63%) of 70 as a white solid.

Quinoline propargyl amine 71.

[0181] Procedure H was followed using the HCl salt of propargyl amine 66 (0.050 g, 0.29 mmol) and quinoline-6-carboxyaldehyde (0.050 g, 0.32 mmol) in toluene (1.2 mL) followed by reduction with sodium borohydride (0.022 g, 0.58 mmol) in methanol (1.5 mL) to afford 0.041 g (51%) of 71 as a clear oil.

Quinoline propargyl amine 72.

[0182] Procedure H was followed using the HCl salt of propargyl amine 67 (0.145 g, 0.92 mmol) and quinoline-6-carboxyaldehyde (0.129 g, 0.69 mmol) in toluene (2.8 mL) followed by reduction with sodium borohydride (0.052 g, 1.38 mmol) in methanol (3.4 mL) to afford 0.080 g (40%) of 72.

39, R, /-Propyl 47c, R = n-Bu

68, R₁ cy c/o-Propyl 61, R = Et 73, R₁ = /-Propyl, R₂ = Et

69, R₁ /-Butyl 62, R = Me 74, R₁ = /-Propyl, R₂ = Me

70, R₁ cyc/o-Butyl 75, R₁ = cyc/o-Propyl, R₂ = n-Bu 71 , R₁ cyc/o-Pentyl 76, R₁ = /-Butyl, R₂ = n-Bu 72, R₁ cyc/o-Hexyl 77, R₁ = /-Butyl, R₂ = Et

78, R₁ = cyc/o-Butyl, R₂ = n-Bu

79, R₁ = cyc/o-Butyl, R₂ = Et

80, R₁ = cyc/o-Pentyl, R₂ = n-Bu

81, R₁ = cyc/o-Hexyl, R₂ = n-Bu

General synthesis of 1,2,3-triazole compounds (Procedure D2).

[0183] This procedure was adapted from Sharpless. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem. Int. Ed. 2002, 41, 2596-2599. To a 0.25 M suspension of alkyne (1.0-1.2 equiv) and azide (1 equiv) in a 1 :1 mixture of water and tert-butyl alcohol was added an aqueous solution of sodium ascorbate (1 equiv of a freshly prepared 1.0 M solution in water) followed by an aqueous solution of copper(II) sulfate (0.1 equiv of a freshly prepared 0.3 M solution in water prepared from copper(II) sulfate pentahydrate). The heterogeneous mixture was stirred vigorously overnight. Water was added and extracted with EtOAc (3x). The organic layers were combined, washed with saturated NaCl (Ix), dried over NaSO₄, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by HPLC [preparatory reverse-phase Ci₈ column (24.1 x 250 mm), CH₃CN/H₂O-0.1% CF₃CO₂H = 5:95 to 95:5 over 55 min; 10 mL/min; 254 nm detection for 65 min] and lyophilized to afford the TFA salt of the product. The free amine of the product was obtained by dissolving the TFA salt of the product in saturated aqueous NaHCO₃ and extracting with CH₂Cl₂ (4x). The organic layers were combined, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. OO F

Aryloxymethyl ketone inhibitor 73.

[0184] Procedure D2 was followed using propargyl amine 39 (0.030 g, 0.12 mmol), azide 61 (0.032 g, 0.11 mmol), 1 M aqueous sodium ascorbate (0.12 mL, 0.12 mmol), 0.3 M aqueous copper(II) sulfate (0.040 mL, 0.012 mmol) in 1 :1 tBuOH:H₂O (0.5 mL) to afford 33.6 mg (52%) of a 1 :1 mixture of diastereomers of 73 as a pale yellow oil. ¹H NMR (300 MHz, CDCl₃): δ 0.80 (d, 3H, J= 6.6), 0.93 (t, 1.5H, J = 7.5), 0.96 (t, 1.5H, J= 7.5), 1.02 (d, 3H, J= 6.6), 1.50 (s, 1.5H), 1.51 (s, 1.5H), 2.03-2.21 (m, 2H), 2.33-2.37 (m, IH), 3.59 (d, IH, J= 12.9), 3.77 (d, IH, J= 12.9), 4.92 (s, 2H), 5.57 (dd, IH, J = 5.1, 10.2), 6.73-6.85 (m, IH), 7.37 (dd, IH, J= 4.5, 8.4), 7.57 (s, 0.5H), 7.58 (s, 0.5H), 7.67- 7.69 (m, IH), 7.73 (s, IH), 8.02 (d, 0.5H, J= 8.7), 8.03 (d, 0.5H, J= 8.7), 8.11 (m, IH), 8.84-8.88 (m, IH). MS (ESI): m/z 544 [MH]⁺.

Aryloxymethyl ketone inhibitor 74. [0185] Procedure D2 was followed using propargyl amine 39 (0.030 g, 0.12 mmol), azide 62 (0.031 g, 0.1 1 mmol), 1 M aqueous sodium ascorbate (0.12 mL, 0.12 mmol), 0.3 M aqueous copper(II) sulfate (0.040 mL, 0.012 mmol) in 1 :1 tBuOH:H₂O (0.5 mL) to afford 27.1 mg (43%) of a 1 :1 mixture of diastereomers of 74 as a pale yellow oil. ¹H NMR (300 MHz, CDCl₃): δ 0.81 (d, 3H, J= 6.9), 1.02 (d, 3H, J= 6.9), 1.51 (s, 3H), 1.84 (d, 1.5H, J= 5.1), 1.86 (d, 1.5H, J= 5.1), 2.17-2.22 (m, IH), 3.61 (d, IH₅ J= 12.9), 3.78 (d, IH₅ J= 12.9), 4.87 (d, IH, J= 16.8), 4.95 (d, IH, J= 16.8), 5.74-5.80 (m, IH), 6.73-6.84 (m, IH), 7.37 (dd, IH, J= 4.5, 8.4), 7.54 (s, 0.5H), 7.55 (s, 0.5H), 7.66- 7.73 (m, 2H), 8.02 (d, 0.5H, J= 8.4), 8.04 (d, 0.5H, J= 8.4), 8.09-8.13 (m, IH), 8.85-8.88 (m, IH). MS (ESI): m/z 530 [MH]⁺.

Aryloxymethyl ketone inhibitor 75.

[0186] Procedure D2 was followed using propargyl amine 68 (0.030 g, 0.12 mmol), azide 47c (0.032 g, 0.10 mmol), 1 M aqueous sodium ascorbate (0.12 mL, 0.12 mmol), 0.3 M aqueous copper(II) sulfate (0.040 mL, 0.012 mmol) in 1 :1 tBuOH:H₂O (0.5 mL) to afford 33.4 mg (49%) of a 1 :1 mixture of diastereomers of 75 as a pale yellow oil. ¹H NMR (300 MHz, CDCl₃): δ 0.18-0.22 (m, IH), 0.41-0.50 (m, 3H), 0.85 (t, 1.5H, J= 7.0), 0.87 (t, 1.5H, J = 7.0),l .15-1.22 (m, IH), 1.25-1.40 (m, 4H), 1.54 (s, 3H), 2.02-2.08 (m, IH), 2.20-2.33 (m, IH), 3.74 (d, IH, J= 12.6), 3.87 (d, IH, J= 12.6), 4.93 (s, 2H), 5.63-5.68 (m, IH), 6.76-6.84 (m, IH), 7.37 (dd, IH, J= 4.5, 8.4), 7.61 (s, 0.5H), 7.62 (s, 0.5H), 7.65- 7.70 (m, IH), 7.73 (s, IH), 8.02 (d, 0.5H, J= 8.4), 8.05 (d, 0.5H, J= 8.4), 8.09-8.13 (m, IH), 8.84-8.88 (m, IH). MS (ESI): m/z 570 [MH]⁺.

Aryloxymethyl ketone inhibitor 76.

[0187] Procedure D2 was followed using propargyl amine 69 (0.017 g, 0.065 mmol), azide 47c (0.021 g, 0.065 mmol), 1 M aqueous sodium ascorbate (0.065 mL, 0.065 mmol), 0.3 M aqueous copper(II) sulfate (0.022 mL, 0.0065 mmol) in 1 :1 tBuOH:H₂O (0.26 mL) to afford 30.0 mg (79%) of a 1 :1 mixture of diastereomers of 76 as a pale yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 0.760 (d, 1.5H, J= 6.6), 0.762 (d, 1.5H, J= 6.6), 0.83-.92 (m, 6H), 1.09- 1.23 (m, IH), 1.23-1.45 (m, 3H), 1.61 (s, 1.5H), 1.62 (s, 1.5H), 1.68-1.80 (m, 2H), 1.82-1.88 (m, 2H), 1.98-2.12 (m, IH), 2.24-2.36 (m, IH), 3.61 (d, 0.5H, J= 12.6), 3.62 (d, 0.5H, J= 12.6), 3.76 (d, IH, J = 12.6), 4.92 (s, 2H), 5.668 (dd, 0.5H, J= 4.7, 10.4), 5.673 (dd, 0.5H, J = 4.7, 10.4), 6.75-6.85 (m, IH), 7.38 (dd, IH, J = 4.3, 8.2), 7.59 (s, 0.5H), 7.60 (s, 0.5H), 7.64-7.69 (m, IH), 7.72 (s, IH), 8.02 (d, 0.5H, J= 8.7), 8.03 (d, 0.5H, J= 8.7), 8.09-8.14 (m, IH), 8.85-8.89 (m, IH). ¹⁹F NMR (376 MHz, CDCl₃): δ -156.2 - -156.0 (m, 2F), -138.0 - -

137.8 (m, 2F). HRMS-FAB (m/z): [MH]⁺ calcd for C_3IH₃₆N₅O₂F₄, 586.2796; found, 586.2805. Anal. Calcd for C₃₁H₃₅N₅O₂F₄: C, 63.58; H, 6.02; N, 1 1.96. Found: C, 63.53; H, 6.18; N, 11.96.

Aryloxymethyl ketone inhibitor 77.

[0188] Procedure D2 was followed using propargyl amine 69 (0.018 g, 0.069 mmol), azide 61 (0.020 g, 0.069 mmol), 1 M aqueous sodium ascorbate (0.069 mL, 0.069 mmol), 0.3 M aqueous copper(II) sulfate (0.023 mL, 0.0069 mmol) in 1 :1 tBuOH:H₂O (0.28 mL) to afford 26.0 mg (68%) of a 1 :1 mixture of diastereomers of 77 as a pale yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 0.76 (d, 3H, J= 6.6), 0.88-0.99 (m, 6H), 1.60 (br s, IH), 1.61 (s, 1.5H), 1.62 (s, 1.5H), 1.70-1.80 (m, IH), 1.82-1.88 (m, 2H), 2.04-2.15 (m, IH), 2.30-2.42 (m, IH), 3.62 (d, IH, J= 12.7), 3.76 (d, IH, J= 12.7), 4.93 (s, 2H), 5.68 (dd, IH, J= 4.8, 10.3), 6.74-6.85 (m, IH), 7.37 (dd, IH, J= 4.1, 8.2), 7.59 (s, 0.5H), 7.60 (s, 0.5H), 7.64- 7.66 (m, 0.5H), 7.66- 7.69 (m, 0.5H), 7.72 (s, IH), 8.02 (d, 0.5H, J= 8.7), 8.03 (d, 0.5H, J= 8.7), 8.09-8.14 (m, IH), 8.84-8.90 (m, IH). ¹⁹F NMR (376 MHz, CDCl₃): δ -156.2 - -156.1 (m, 2F), -138.0 - -

137.9 (m, 2F). HRMS-FAB (m/z): [MH]⁺ calcd for C₂₉H₃₂N₅O₂F₄, 558.2485; found, 558.2487. Anal. Calcd for C₂₉H₃₁N₅O₂F₄: C, 62.47; H, 5.60; N, 12.56. Found: C, 62.37; H, 5.78; N, 12.35.

Aryloxymethyl ketone inhibitor 78.

[0189] Procedure D2 was followed using propargyl amine 70 (0.023 g, 0.086 mmol), azide 47c (0.027 g, 0.086 mmol), 1 M aqueous sodium ascorbate (0.086 mL, 0.086 mmol), 0.3 M aqueous copper(II) sulfate (0.026 mL, 0.0086 mmol) in 1 : 1 tBuOH:H₂O (0.34 mL) to afford 25.7 mg (51%) of a 1 : 1 mixture of diastereomers of 78 as a pale yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 0.84 (t, 1.5H, J= 7.2), 0.86 (t, 1.5H, J= 7.2), 1.09-1.23 (m, IH), 1.23-1.45 (m, 3H), 1.537 (s, 1.5H), 1.542 (s, 1.5H), 1.67-2.00 (m, 7H), 2.00-2.14 (m, IH), 2.24-2.37 (m, IH), 2.81 (quint, IH, J= 8.8), 3.63 (d, IH, J= 12.8), 3.788 (d, 0.5H, J= 12.8), 3.794 (d, 0.5H, J= 12.8), 4.92 (s, 2H), 5.661 (dd, 0.5H, J= 4.8, 10.4), 5.665 (dd, 0.5H, J= 4.8, 10.4), 6.75-6.81 (m, IH), 7.37 (dd, IH, J= 4.2, 8.4), 7.58 (s, 0.5H), 7.59 (s, 0.5H), 7.686 (dd, 0.5H, J= 4.4, 8.8), 7.691 (dd, 0.5H, J= 4.4, 8.8), 7.74 (s, IH), 8.02 (d, 0.5H, J= 8.8), 8.03 (d, 0.5H, J = 8.8), 8.09-8.14 (m, IH), 8.84-8.89 (m, IH). ¹⁹F NMR (376 MHz, CDCl₃): δ -156.2 - -156.1 (m, 2F), -138.0 - -137.9 (m, 2F). MS (ESI): m/z 584 [MH]⁺.

Aryloxymethyl ketone inhibitor 79.

[0190] Procedure D2 was followed using propargyl amine 70 (0.023 g, 0.086 mmol), azide 61 (0.025 g, 0.086 mmol), 1 M aqueous sodium ascorbate (0.086 mL, 0.086 mmol), 0.3 M aqueous copper(II) sulfate (0.026 mL, 0.0086 mmol) in 1 :1 tBuOH:H₂O (0.34 mL) to afford 32.3 mg (68%) of a 1 :1 mixture of diastereomers of 79 as a clear oil. ¹H NMR (400 MHz, CDCl₃): δ 0.93 (t, 1.5H, J= 7.2), 0.95 (t, 1.5H, J= 7.2), 1.53 (s, 1.5H), 1.54 (s, 1.5H), 1.64- 1.99 (m, 7H), 2.02-2.17 (m, IH), 2.30-2.43 (m, IH), 2.80 (quint, IH, J = 8.8), 3.627 (d, 0.5H, J= 12.8), 3.631 (d, 0.5H, J= 12.8), 3.785 (d, 0.5H, J= 12.8), 3.788 (d, 0.5H, J= 12.8), 4.93 (s, 2H), 5.57 (dd, 0.5H, J= 4.8, 9.6), 5.58 (dd, 0.5H, J= 4.8, 9.6), 6.75-6.85 (m, IH), 7.37 (dd, IH, J= 4.4, 8.4), 7.58 (s, 0.5H), 7.59 (s, 0.5H), 7.688 (dd, 0.5H, J= 3.6, 8.6), 7.693 (dd, 0.5H, J= 3.6, 8.6), 7.74 (s, IH), 8.02 (d, 0.5H, J= 8.6), 8.03 (d, 0.5H, J= 8.6), 8.09-8.14 (m, IH), 8.85-8.89 (m, IH). ¹⁹F NMR (376 MHz, CDCl₃): δ -156.3 - -156.1(m, 2F), -138.0 - - 137.8 (m, 2F). MS (ESI): m/z 556 [MH]⁺.

Aryloxymethyl ketone inhibitor 80.

[0191] Procedure D2 was followed using propargyl amine 71 (0.020 g, 0.072 mmol), azide 47c (0.025 g, 0.079 mmol), 1 M aqueous sodium ascorbate (0.072 mL, 0.072 mmol), 0.3 M aqueous copper(II) sulfate (0.022 mL, 0.0072 mmol) in 1 :1 ΦuOH:H₂O (0.29 mL) to afford 29.0 mg (67%) of a 1 :1 mixture of diastereomers of 80 as a pale yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 0.85 (t, 1.5H, J= 7.2), 0.87 (t, 1.5H, J= 7.2), 1.08-1.22 (m, IH), 1.22-1.53 (m, 9H), 1.57 (s, 1.5H), 1.58 (s, 1.5H), 1.64-1.81 (m, 3H), 1.99-2.12 (m, IH), 2.23-2.35 (m, IH), 2.42 (quint, IH, J= 8.8), 3.64 (d, IH, J= 12.8), 3.81 (d, IH, J= 12.8), 4.91 (s, 2H), 5.65 (dd, IH, J= 4.8, 10.4), 6.76-6.84 (m, IH), 7.38 (dd, IH, J= 4.0, 8.0), 7.58 (s, 0.5H), 7.59 (s, 0.5H), 7.69 (dd, 0.5H, J= 4.4, 8.8), 7.70 (dd, 0.5H, J= 4.4, 8.8), 7.74 (s, IH), 8.02 (d, 0.5H, J= 8.8), 8.03 (d, 0.5H, J= 8.8), 8.08-8.14 (m, IH), 8.83-8.89 (m, IH). ¹⁹F NMR (376 MHz, CDCl₃): δ -156.2 - -156.0 (m, 2F), -138.0 - -137.8 (m, 2F). MS (ESI): m/z 598 [MH]⁺.

Aryloxymethyl ketone inhibitor 81.

[0192] Procedure D2 was followed using propargyl amine 72 (0.024 g, 0.082 mmol), azide 47c (0.026 g, 0.082 mmol), 1 M aqueous sodium ascorbate (0.082 mL, 0.082 mmol), 0.3 M aqueous copper(II) sulfate (0.027 mL, 0.0082 mmol) in 1 :1 tBuOH:H₂O (0.33 mL) to afford 33.5 mg (67%) of a 1 :1 mixture of diastereomers of 81 as a pale yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 0.85 (t, 1.5H, J= 7.2), 0.87 (t, 1.5H, J= 7.2), 0.91-1.11 (m, 2H), 1.12-1.43 (m, 6H), 1.51 (s, 1.5H), 1.52 (s, 1.5H), 1.56-1.73 (m, 5H), 1.74-1.86 (m, 2H), 2.01-2.12 (m, 2H), 2.24-2.37 (m, IH), 3.58 (d, IH, J= 13.0), 3.77 (d, IH₅ J= 13.0), 4.92 (s, 2H), 5.62-5.69 (m, IH), 6.75-6.85 (m, IH), 7.37 (dd, IH, J= 4.2, 8.3), 7.55 (s, 0.5H), 7.56 (s, 0.5H), 7.65- 7.70 (m, IH), 7.72 (s, IH), 8.01 (d, 0.5H, J= 8.8), 8.02 (d, 0.5H, J= 8.8), 8.09-8.14 (m, IH), 8.85-8.89 (m, IH). ¹⁹F NMR (376 MHz, CDCl₃): δ -156.2 - -156.1 (m, 2F), -138.0 - -137.9 (m, 2F). HRMS-FAB (m/z): [MH]⁺ calcd for C₃₃H₃₈N₅O₂F₄, 612.2971; found, 612.2962. Anal. Calcd for C₃₃H₃₇N₅O₂F₄: C, 64.80; H, 6.10; N, 11.45. Found: C, 64.51; H, 6.46; N, 11.14.

Assay procedures

General assay procedure

[0193] Cbz-Phe-Arg-AMC was purchased from Bachem (Torrance, CA). The proteolytic cleavage of N-acyl aminocoumarins by cruzain was conducted in Dynatech Microfluor fluorescence 96-well microtiter plates, and readings were taken on a Molecular Devices

Spectra Max Gemini SX instrument. The excitation wavelength was 370 nm and the emission wavelength was 455 nm, with a cutoff of 435 nm for AMCA substrates; the excitation wavelength was 350 nm and the emission wavelength was 450 nm, with a cutoff of 435 nm for peptidyl-AMC substrates. The assay buffer consisted of a 100 mM solution of pH 6.3 sodium phosphate buffer with 400 mM of sodium chloride, 5 mM of DTT, 10 mM of EDTA, and 0.025% Triton-X 100.

Assay procedure for AMCA substrates

[0194] Assays were conducted at 37 °C in duplicate with and without the enzyme. In each well was placed 38 μL of enzyme solution and 2 μL of a DMSO substrate solution. Assays were performed at substrate concentrations that were at minimum 6-times less than the K_m for that substrate. Relative fluorescent units (RFU) were measured at regular intervals over a period of time (maximum 15 min). A plot of RFU versus time was made for each substrate with and without cruzain. The slope of the plotted line gave relative k_cg,_t/K_m of each substrate for cruzain.

Assay procedure for irreversible inhibitors

[0195] The f°^r inhibitors were determined under pseudo-first order conditions using the progress curve method. Bieth, J. G. Methods Enzymol. 1995, 248, 59-84. Assay wells contained a mixture of inhibitor and 0.5 μM Cbz-Phe-Arg-AMC (K_m = 1.1 μM) in buffer. Aliquots of cruzain were added to each well to initiate the assay. The final enzyme concentration was 0.1 nM. Hydrolysis of the AMC substrate was monitored fluorometrically for 45 min. To determine the inhibition parameters, time points for which the control ([I] = 0) was linear were used. For each inhibitor, a U₀^ was calculated for at least four different concentrations of inhibitors via a nonlinear regression of the data according to the equation P = (v,/&_obs)[l-exp(-&obst) (where product formation = P, initial rate = v,, time = t, and the first- order rate constant = £_obs)- If &obs varied linearly with [I], then the association constant &_ass was determined by linear regression analysis using k₀^ = (£_ass[I])/(l+[S]/AT_m) where [S] is the concentration of the substrate. If k_o\,_s varied hyperbolically with [I], then non-linear regression analysis was performed to determine A:_inact/AT, using k_o\,_% ⁼ ^inact[I]/([I]+^i*(l+[S]/AT_m)). Inhibition was measured in quadruplicate and the average of four assays is reported.

T. cruzi culture assay

[0196] Mammalian cells were cultured in RPMI-1640 medium supplemented with 5% heat-inactivated fetal calf serum (FCS) at 37 °C in 5% CO₂. The Y strain of T. cruzi was maintained by serial passage in bovine embryo skeletal muscle (BESM) cells. Infectious trypomastigotes were collected from culture supernatants. For inhibitor assays, J774 macrophages were irradiated (9000 rad) and plated onto twelve-well tissue culture plates 24 h prior to infection with about 10⁵ trypomastigotes/well. Parasites were removed 2 h postinfection, and the medium was supplemented with the appropriate cysteine protease inhibitor (10 μM) (n=3 per treatment). For inhibitors 50, 51, 52, and 55 the concentration was lowered to 5 μM on days 9, 7, 12, and 12, respectively. Treatment was ended on day 14 for inhibitors 50-52. Inhibitor stocks (20 mM) in DMSO were stored at 4 °C. Fresh RPMI medium with or without inhibitor was replaced every 48 h. Monolayers were treated for 27 days and maintained without inhibitor for up to 40 days. Cultures were monitored daily by contrast phase microscopy. Untreated J774 monolayers were used as a negative control. Monolayers treated the trypanocidal inhibitor, 10 μM TV-methyl piperazine-Phe-homoPhe- vinyl sulfone phenyl (JV-Pip-F-hF -VSPh), acted as a positive control. Engel, J. C; Doyle, P. S.; Hsieh, I.; McKerrow, J. H. J. Exp. Med. 1998, 188, 725-734. T. cruzi completed the intracellular cycle in 5 days in untreated controls but was unable to survive in macrophages treated with N-Pip-F-hF-VSPh (40 days). The comparative effectiveness of each inhibitor was estimated from plots of the duration of the intracellular cycle of T. cruzi (days) in treated vs untreated control wells.

[0197] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A compound having a structure according to the following formula:

wherein

R¹ is a member selected from H, OR*, NR*R**, SR*, -S(O)R*, -S(O)₂R*,

-S(O)₂NR*R**, nitro, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl wherein each R* and R** are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

2. The compound of claim 1, wherein R¹ is a member selected from:

3. A compound having a structure according to the following formula:

wherein R² is a member selected from substituted or unsubstituted Ci-C₆ alkyl, and substituted or unsubstituted C₃-C₆ cycloalkyl R³ is a member selected from substituted or unsubstituted Ci-C₆ alkyl, and substituted or unsubstituted C₃-C₆ cycloalkyl R⁴ is a member selected from H, OR*, NR*R**, SR*, -S(O)R*, -S(O)₂R*,

-S(O)₂NR*R**, nitro, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl wherein each R* and R** are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

4. The compound of claim 3, wherein R⁴ is a member selected from:

5. A compound having a structure according to the following formula:

wherein

R⁶ is a member selected from substituted or unsubstituted Ci-C₆ alkyl, and substituted or unsubstituted C₃-C₆ cycloalkyl

R⁷ is a member selected from substituted or unsubstituted Ci-C₆ alkyl, and substituted or unsubstituted C₃-C₆ cycloalkyl R⁵ and R⁸ are members independently selected from H, OR*, NR*R**, SR*,

-S(O)R*, -S(O)₂R*, -S(O)₂NR*R**, nitro, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl wherein each R* and R** are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

6. The compound of claim 5, wherein R⁵ is a member selected from:

7. The compound of claim 5, wherein R is a member selected from:

and

8. A compound having a structure according to a formula which is a member selected from:

and wherein

R⁹, R¹⁰, R¹¹, R¹² and R¹³ are members independently selected from H, OR*, NR*R**, SR*, -S(O)R*, -S(O)₂R*, -S(O)₂NR*R**, nitro, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl wherein each R* and R** are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

9. A pharmaceutical composition comprising: a) the compound of claim 1; b) a pharamaceutically acceptable excipient.

10. A method of treating a disease, said method comprising administering a therapeutically effective amount of the compound of claim 1, to an animal suffering from the disease, thereby treating said disease.

11. The method of claim 10, wherein said disease is Chagas disease.

12. A method of killing or inhibiting the growth of a protozoa, said method comprising:

(i) contacting said protozoa with the compound of claim 1, in an amount effective to kill or inhibit the growth of said protozoa.

13. The method of claim 12, wherein said protozoa is Trypanosoma cruzi.