WO2009075778A2 - Inhibiteurs non peptidiques de la cruazïne - Google Patents

Inhibiteurs non peptidiques de la cruazïne Download PDF

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Publication number
WO2009075778A2
WO2009075778A2 PCT/US2008/013400 US2008013400W WO2009075778A2 WO 2009075778 A2 WO2009075778 A2 WO 2009075778A2 US 2008013400 W US2008013400 W US 2008013400W WO 2009075778 A2 WO2009075778 A2 WO 2009075778A2
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substituted
unsubstituted
mmol
inhibitors
cruzain
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WO2009075778A3 (fr
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Jonathan A. Ellman
Katrien Brak
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • 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.
  • 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.
  • 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.
  • cruzain is a highly attractive therapeutic target for the treatment of Chagas' disease. McKerrow, J. H. et al, Parasitology Today 1995, 11, 279-282.
  • 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.
  • SAS Substrate Activity Screening
  • the SAS method consists of the identification of nonpeptidic substrate fragments, substrate optimization, and then conversion of optimal substrates to inhibitors.
  • 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.
  • the invention provides a compound of the invention.
  • the invention provides a compound having a structure according to a formula described herein.
  • the compound has a structure according to the following formula: wherein R 1 is a member selected from H, OR*, NR*R**, SR*, -S(O)R*, -S(O) 2 R*, -S(O) 2 NR*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.
  • 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.
  • 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.
  • 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.
  • the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising: a) a compound of the invention; b) a pharmaceutically acceptable excipient.
  • Figure 1 Most advanced inhibitor of cruzain.
  • FIG. 1 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/).
  • 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
  • Figure 4 Time-dependence of (a) vinyl sulfone 38 and (b) ⁇ -chloro vinyl sulfone 43.
  • Figure 5 Mechanism of inhibition of cysteine proteases by (a) vinyl sulfones and (b) ⁇ -chloro vinyl sulfones.
  • Figure 6 Effect of inhibitors at 5-10 ⁇ M 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.
  • Compound of the invention refers to a compound described herein, as well as salts (e.g. pharmaceutically acceptable salts), solvates and hydrates of the compound.
  • MIC or minimum inhibitory concentration
  • MIC 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.
  • 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., -CH 2 O- is intended to also recite -OCH 2 -.
  • poly as used herein means at least 2.
  • a polyvalent metal ion is a metal ion having a valency of at least 2.
  • Moiety refers to a radical of a molecule that is attached to the remainder of the molecule.
  • the symbol */VV ⁇ indicates the point at which the displayed moiety is attached to the remainder of the molecule.
  • 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. C 1 -Ci O means one to ten carbons).
  • 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.
  • 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.
  • 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.
  • alkylene by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified, but not limited, by -CH 2 CH 2 CH 2 CH 2 -, and further includes those groups described below as “heteroalkylene.”
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • heteroalkylene by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH 2 -CH 2 -S- CH 2 -CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 -.
  • heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkyl enedioxy, alkyleneamino, alkylenediamino, and the like).
  • 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.
  • heterocycloalkyl examples 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.
  • halo or halogen
  • haloalkyl by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl.
  • halo(Ci-C 4 )alkyl is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
  • 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.
  • heteroaryl refers to aryl groups (or rings) that contain from one to four heteroatoms.
  • 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
  • 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.
  • 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.
  • aryloxy is meant to include those radicals in which an aryl group is attached to an oxygen atom.
  • 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).
  • 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.
  • heteroarylalkyl is meant to include those radicals in which a heteroaryl group is attached to an alkyl group (e.g., pyridylmethyl and the like).
  • heteroaryloxy is meant to include those radicals in which a heteroaryl group is attached to an oxygen atom.
  • 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).
  • alkyl e.g., 2-pyridyloxymethyl and the like.
  • 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.
  • 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.
  • 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.
  • -NR'R is meant to include, but not be limited to, 1- pyrrolidinyl and 4-morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g. , -CF 3 and -CH 2 CF 3 ) and acyl (e.g., -C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH 3 , and the like).
  • substituents for the aryl and heteroaryl groups are generically referred to as "aryl group substituents.”
  • 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.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH 2 ) r -B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O) 2 -, -S(O) 2 NR'- 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.
  • 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) 2 NR'-.
  • the substituents R, R', R" and R'" are preferably independently selected from hydrogen or substituted or unsubstituted (Ci-C 6 )alkyl.
  • acyl describes a substituent containing a carbonyl residue, C(O)R.
  • 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.
  • Ring 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.
  • the term “5- to 7- membered ring” includes, for example phenyl, pyridinyl and piperidinyl.
  • the term “ring” further includes a ring system comprising more than one "ring”, wherein each "ring” is independently defined as above.
  • 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.
  • heteroatom includes atoms other than carbon (C) and hydrogen (H). Examples include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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-meth
  • 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.
  • 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.
  • 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.
  • amino acids are ⁇ -amino acids
  • either the L-optical isomer or the D-optical isomer can be used.
  • 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.
  • other peptidomimetics are also useful in the present invention.
  • 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.
  • alanine arginine
  • asparagine aspartic acid
  • cysteine glutamic acid
  • glutamine glycine
  • histidine isoleucine
  • leucine leucine
  • lysine methionine
  • phenylalanine proline
  • serine threonine
  • tryptophan tyrosine
  • valine valine.
  • 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
  • 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 ⁇ -amino acids. Additional non-standard amino acids are ⁇ -alanine, phenylglycine and homoarginine
  • an 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.
  • salts are 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.
  • 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.
  • pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
  • 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.
  • inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Compounds of the invention can exist in particular geometric or stereoisomeric forms.
  • the invention contemplates all such compounds, including cis- and tr ⁇ ns-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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C). 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.
  • 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.
  • excipients is conventionally known to mean carriers, diluents and/or vehicles used in formulating drug compositions effective for the desired use.
  • Bio medium 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.
  • the invention provides a compound of the invention.
  • the invention provides a compound having a structure according to a formula described herein.
  • the invention provides a compound described herein.
  • the compound has a structure according to the following formula: wherein R 1 is a member selected from H, OR*, -C(O)R*, NR*R**, SR*, -S(O)R*, -S(O) 2 R*, -S(O) 2 NR*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.
  • 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.
  • R 1 is described herein.
  • R 1 is substituted or unsubstituted arylalkyl or substituted or unsubstituted heteroarylalkyl.
  • R 1 is a member selected from:
  • R is a member selected from:
  • L is a member selected from CH 2 and C(O).
  • R 1 is a member selected from:
  • R 1 is a member selected from: In an exemplary embodiment, R 1 is a member selected from: wherein L is a member selected from CH 2 and C(O). R 16 is a member selected from unsubstituted Ci-C 6 alkyl. In an exemplary embodiment, R 16 is a member selected from methyl and ethyl. In an exemplary embodiment, R 16 is a member selected from n-propyl and z ' -propyl. In an exemplary embodiment, R 16 is a member selected from n-butyl, /-butyl and t- butyl.
  • R 1 is a member selected from: wherein L is a member selected from CH 2 and C(O).
  • R 17 is a member selected from unsubstituted Ci-C 6 alkyl. In an exemplary embodiment, R 17 is a member selected from methyl and ethyl. In an exemplary embodiment, R 17 is a member selected from n-propyl and /-propyl. In an exemplary embodiment, R 17 is a member selected from n-butyl, /-butyl and t- butyl.
  • R 1 is a member selected from: wherein L is a member selected from CH 2 and C(O).
  • X is a member selected from halogen.
  • X is a member selected from fluorine and chlorine.
  • X is a member selected from bromine and iodine.
  • the compound has a structure according to the following formula:
  • R 2 is a member selected from substituted or unsubstituted Ci-C 6 alkyl, and substituted or unsubstituted C 3 -C 6 cycloalkyl.
  • R 3 is a member selected from substituted or unsubstituted Ci-C 6 alkyl, and substituted or unsubstituted C 3 -C 6 cycloalkyl.
  • R 4 is a member selected from H, OR*, -C(O)R*, NR*R**, SR*, -S(O)R*, -S(O) 2 R*, -S(O) 2 NR*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.
  • 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.
  • R 2 is /-propyl and R 3 is n-butyl.
  • R 4 is described herein. In an exemplary embodiment, R 4 is:
  • R 4 is: o w o
  • R 4 is:
  • R is:
  • R 4 is:
  • the compound has a structure according to the following formula:
  • R 6 is a member selected from substituted or unsubstituted Ci-C 6 alkyl, and substituted or unsubstituted C 3 -C 6 cycloalkyl.
  • R 7 is a member selected from substituted or unsubstituted Ci-C 6 alkyl, and substituted or unsubstituted C 3 -C 6 cycloalkyl.
  • R 5 and R 8 are members independently selected from H, OR*, -C(O)R*, NR*R**, SR*, -S(O)R*, -S(O) 2 R*, -S(O) 2 NR*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.
  • 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.
  • R 5 and R 8 are described herein.
  • R 5 is substituted or unsubstituted arylalkyl or substituted or unsubstituted heteroarylalkyl.
  • R 5 is a member selected from:
  • R is a member selected from:
  • R 8 is: and R is a member selected from:
  • R 8 is:
  • R is a member selected from:
  • the invention provides a compound having a structure according to a formula which is a member selected from:
  • R 9 is a member selected from H, OR*, -C(O)R*, NR*R**, SR*, -S(O)R*, -S(O) 2 R*, -S(O) 2 NR*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.
  • R 10 , R 1 ', R 12 and R 13 are members independently selected from H, OR*, NR*R**, SR*, - S(O)R*, -S(O) 2 R*, -S(O) 2 NR*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.
  • R is substituted or unsubstituted arylalkyl or substituted or unsubstituted heteroarylalkyl.
  • the compound has a structure according to the following formula:
  • R 14 is a member selected from substituted or unsubstituted alkyl, and substituted or unsubstituted cycloalkyl.
  • R 15 is a member selected from substituted or unsubstituted alkyl, and substituted or unsubstituted cycloalkyl. In an exemplary embodiment, R 14 and R 15 are described herein. In an exemplary embodiment, R 14 is a member selected from substituted or unsubstituted CpC 6 alkyl, and substituted or unsubstituted C 3 -C 6 cycloalkyl.
  • R 14 is a member selected from unsubstituted C 1 -C 6 alkyl and unsubstituted C 3 - C 6 cycloalkyl.
  • R 14 is a member selected from z-propyl, /-butyl, cyc/o-propyl, cyc/o-butyl, cyc/o-pentyl, and cyclo-hexyl.
  • R 15 is a member selected from substituted or unsubstituted Ci- C 6 alkyl, and substituted or unsubstituted C 3 -C 6 cycloalkyl. In an exemplary embodiment, R 15 is a member selected from unsubstituted Ci-C 6 alkyl. In a specific exemplary embodiment, R 15 is a member selected from methyl, ethyl, and H-butyl.
  • Table 1 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇
  • ⁇ -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 ⁇ -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 ⁇ -chloro sulfone inhibitor 43 had a modest second order rate of inactivation constant of 805 s " M "1 (Table 4).
  • a Reagents (a) diazomethane, THF, rt; (b) methylphenylsulfone, H-BuLi, THF, 0 0 C to -78 0 C; (c) Tf 2 O, /-Pr 2 EtN, THF, -20 0 C to rt; (d) TBACl, THF, rt; (e) Na ascorbate, CuSO 4 , 1 :1 H 2 O:t-BuOH, 39, rt.
  • Reagents (a) isobutyl chloroformate, N-methylmorpholine, THF, -40 0 C; (b) diazomethane, THF, 0 0 C; (c) HBr, THF, 0 0 C; (d) KF, DMF, 0 0 C; (e) Na ascorbate, CuSO 4 , 1 :1 H 2 O:f-BuOH, rt.
  • 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 -1 M "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 "1 M "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 Inhibitors 0
  • 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.
  • 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).
  • 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.
  • a potent irreversible 2,3,5,6-tetrafluorophenoxymethyl ketone inhibitor 54 was developed that eradicates T. cruzi parasites in cell culture.
  • 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.
  • 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.
  • said protozoa belongs to the order of Trypanosomes.
  • said protozoa is a Trypanosoma.
  • the protozoa is Trypanosoma cruzi.
  • the protozoa is Trypanosoma brucei.
  • 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.
  • the disease is associated with a protozoa.
  • the disease is associated with a Trypanosoma.
  • 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).
  • the trypanosomiasis is a member selected from Chagas disease, sleeping sickness, Nagana, Surra, or Dourine (covering sickness).
  • the disease is associated with Trypanosoma cruzi.
  • the disease is Chagas disease.
  • the disease is Human American trypanosomiasis.
  • the disease is associated with a Trypanosoma brucei.
  • the disease is sleeping sickness.
  • the disease is Human African trypanosomiasis.
  • the disease is Nagana.
  • the animal is a mammal.
  • the animal is a cow.
  • the animal is a human.
  • the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising: a) a compound of the invention; b) a pharmaceutically acceptable excipient.
  • 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.
  • pharmaceutically acceptable carrier includes vehicles, diluents, excipients and other elements appropriate for incorporation into a pharmaceutical formulation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ g /kg, preferably 250 ⁇ g/kg, 500 ⁇ g /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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • kits 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.
  • 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.
  • 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.
  • 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.
  • a memory aid is a calendar printed on the card, e.g., as follows "First Week, Monday, Tuesday, . . . etc . . . Second Week, Monday, Tuesday, . . . " etc.
  • a “daily dose” can be a single tablet or capsule or several pills or capsules to be taken on a given day.
  • 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.
  • a dispenser designed to dispense the daily doses one at a time in the order of their intended use is provided.
  • the dispenser is equipped with a memory-aid, so as to further facilitate compliance with the regimen.
  • a memory-aid is a mechanical counter which indicates the number of daily doses that has been dispensed.
  • 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.
  • Tetrahydrofuran (THF), diethyl ether, methylene chloride (CH 2 Cl 2 ), and toluene were obtained from a Seca Solvent Systems by GlassContour (solvent dried over alumina under a N 2 atmosphere).
  • Anhydrous DMF (water ⁇ 50 ppm) was purchased from Acros.
  • Diisopropylethylamine (/-Pr 2 EtN) was distilled over CaH 2 .
  • Procedure A was followed using resin 15 (0.136 g, 0.0890 mmol), propargyl urea Sl (0.010 g, 0.044 mmol), /-Pr 2 EtN (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.
  • 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 /-Pr 2 EtN (0.11 mL, 0.60 mmol) to afford 7.0 mg (15%) of 19 as a white powder.
  • 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 /-Pr 2 EtN (0.09 mL, 0.48 mmol) to afford 2.3 mg (6%) of 22 as a white powder.
  • 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- Pr 2 EtN (0.11 mL, 0.60 mmol) to afford 18.4 mg (38%) of 23 as a white powder.
  • 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 /-Pr 2 EtN (0.11 mL, 0.60 mmol) to afford 31.4 mg (62%) of 24 as a white powder.
  • 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- Pr 2 EtN (0.090 mL, 0.48 mmol) to afford 8.2 mg (20%) of 26 as a white powder.
  • 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- Pr 2 EtN (0.11 mL, 0.60 mmol) to afford 21.9 mg (47%) of 27 as a white powder.
  • 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- Pr 2 EtN (0.09 mL, 0.48 mmol) to afford 10.9 mg (28%) of 28 as a white powder.
  • 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 /-Pr 2 EtN (0.11 mL, 0.60 mmol) to afford 2.0 mg (4%) of 29 as a white powder.
  • 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 /-Pr 2 EtN (0.11 mL, 0.60 mmol) to afford 18.0 mg (38%) of 30 as a white powder.
  • Procedure B was followed using l ⁇ -benzothiazole- ⁇ -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- Pr 2 EtN (0.11 mL, 0.60 mmol) to afford 18.0 mg (37%) of 31 as a white powder.
  • Procedure A was followed using resin 15 (0.421 g, 0.150 mmol), propargyl amine 39 (0.057 g, 0.23 mmol), /-Pr 2 EtN (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.
  • Procedure A was followed using resin 15 (0.421 g, 0.150 mmol), propargyl amine S6b (0.058 g, 0.23 mmol), /-Pr 2 EtN (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.
  • 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 NaHCO 3 (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 NaHCO 3 (30 mL), water (2 x 30 mL), and saturated NaCl (2 x 30 mL). The organic layer was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure.
  • 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 ⁇ uOH:H 2 O (1.0 mL) to afford 0.151 g (89%) of the TFA salt 41 as a sticky yellow solid.
  • 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 0 C 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 0 C.
  • the reaction was quenched at -78 0 C by adding methanol (0.4 mL) and then acetic acid (0.060 mL, 1.0 mmol).
  • 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: CH 3 CN/H 2 O-0.1% CF 3 CO 2 H, 5:95 to 95:5 over 16 min, 95:5 for 2 min; CH 3 OH/H 2 O, 5:95 to 95:5 over 20 min, 95:5 for 10 min).
  • 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:H 2 O (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).
  • the reaction mixture was diluted with EtOAc (50 mL) and was then washed with 10 wt% citric acid (2 x 10 mL), saturated NaHCO 3 (2 x 20 mL), and saturated NaCl (1 x 10 mL).
  • the organic layer was dried over Na 2 SO 4 , 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 1 H 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.
  • 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.
  • 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.
  • 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.
  • 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:H 2 O (0.4 mL) to afford 19.3 mg (35%) of a 1 :1 mixture of diastereomers of 49 as a clear oil.
  • 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:H 2 O (0.3 mL) to afford 24.6 mg (49%) of a 1 :1 mixture of diastereomers of 50 as a white sticky solid.
  • 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:H 2 O (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: CH 3 CN/H 2 O-0.1% CF 3 CO 2 H, 5:95 to 95:5 over 16 min, 95:5 for 2 min; CH 3 OH/H 2 O, 5:95 to 95:5 over 20 min, 95:5 for 10 min).
  • 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:H 2 O (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.
  • 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:H 2 O (0.6 mL) to afford 22.2 mg (22%) of a 1 :1 mixture of diastereomers of 53 as a white sticky solid.
  • 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:H 2 O (0.3 mL) to afford 21.0 mg (49%) of a 1 :1 mixture of diastereomers of 54 as a pale yellow oil.
  • Aryloxymethyl ketone inhibitor 55 is Aryloxymethyl ketone inhibitor 55.
  • 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:H 2 O (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.
  • 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:H 2 O (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.
  • Diastereomerically pure inhibitor 58 (0.012 mg, 0.021 mmol) was dissolved in DMSOd 6 (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 0 C 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 Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was then dissolved in CH 2 Cl 2 and washed with water. The aqueous layer was backextracted with CH 2 Cl 2 (2 x 5 mL). The combined organic layers were washed with brine, dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to afford an oil. 1 H-NMR of the crude inhibitor indicated a 1 : 1 mixture of diastereomers.
  • the reaction mixture was diluted with EtOAc (50 mL) and was then washed with 10 wt% citric acid (2 x 10 mL), saturated NaHCO 3 (2 x 20 mL), and saturated NaCl (1 x 10 mL).
  • the organic layer was dried over Na 2 S ⁇ 4 , filtered, and concentrated under reduced pressure. The product was taken on to the next step without further purification.
  • 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.
  • 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.
  • 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.
  • 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 0 C.
  • 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.
  • 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 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 was followed using the HC
  • 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.
  • 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.
  • 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.
  • the free amine of the product was obtained by dissolving the TFA salt of the product in saturated aqueous NaHCO 3 and extracting with CH 2 Cl 2 (4x). The organic layers were combined, dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. OO F
  • Aryloxymethyl ketone inhibitor 73 Aryloxymethyl ketone inhibitor 73.
  • 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:H 2 O (0.5 mL) to afford 33.6 mg (52%) of a 1 :1 mixture of diastereomers of 73 as a pale yellow oil.
  • 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:H 2 O (0.5 mL) to afford 27.1 mg (43%) of a 1 :1 mixture of diastereomers of 74 as a pale yellow oil.
  • Aryloxymethyl ketone inhibitor 75 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aryloxymethyl ketone inhibitor 75.
  • 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:H 2 O (0.5 mL) to afford 33.4 mg (49%) of a 1 :1 mixture of diastereomers of 75 as a pale yellow oil.
  • Aryloxymethyl ketone inhibitor 76 Aryloxymethyl ketone inhibitor 76.
  • 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:H 2 O (0.26 mL) to afford 30.0 mg (79%) of a 1 :1 mixture of diastereomers of 76 as a pale yellow oil.
  • Aryloxymethyl ketone inhibitor 77 Aryloxymethyl ketone inhibitor 77.
  • 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:H 2 O (0.28 mL) to afford 26.0 mg (68%) of a 1 :1 mixture of diastereomers of 77 as a pale yellow oil.
  • Aryloxymethyl ketone inhibitor 78 Aryloxymethyl ketone inhibitor 78.
  • 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:H 2 O (0.34 mL) to afford 25.7 mg (51%) of a 1 : 1 mixture of diastereomers of 78 as a pale yellow oil.
  • Aryloxymethyl ketone inhibitor 79 Aryloxymethyl ketone inhibitor 79.
  • 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:H 2 O (0.34 mL) to afford 32.3 mg (68%) of a 1 :1 mixture of diastereomers of 79 as a clear oil.
  • Aryloxymethyl ketone inhibitor 80 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aryloxymethyl ketone inhibitor 80.
  • 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 ⁇ uOH:H 2 O (0.29 mL) to afford 29.0 mg (67%) of a 1 :1 mixture of diastereomers of 80 as a pale yellow oil.
  • Aryloxymethyl ketone inhibitor 81 Aryloxymethyl ketone inhibitor 81.
  • 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:H 2 O (0.33 mL) to afford 33.5 mg (67%) of a 1 :1 mixture of diastereomers of 81 as a pale yellow oil.
  • 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
  • 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.
  • Assays were conducted at 37 °C in duplicate with and without the enzyme. In each well was placed 38 ⁇ L of enzyme solution and 2 ⁇ L 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 c g, t /K m of each substrate for cruzain.
  • RFU Relative fluorescent units
  • Mammalian cells were cultured in RPMI-1640 medium supplemented with 5% heat-inactivated fetal calf serum (FCS) at 37 °C in 5% CO 2 .
  • FCS heat-inactivated fetal calf serum
  • 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.
  • J774 macrophages were irradiated (9000 rad) and plated onto twelve-well tissue culture plates 24 h prior to infection with about 10 5 trypomastigotes/well.

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Abstract

La cruzaïne est la principale cystéine protéase de T. cruzi, laquelle est l'agent causal de la maladie de Chagas, et représente une cible prometteuse pour le développement d'une nouvelle chimiothérapie. Avec pour objectif de développer de puissants inhibiteurs non peptidiques de la cruzaïne, la méthode du criblage de l'activité du substrat a été utilisée pour cribler une bibliothèque de substrats de la protéase initialement conçus pour cibler l'homologue humain de la protéase cathepsine S. Un concept basé sur la structure a ensuite été utilisé pour améliorer davantage l'efficacité de clivage du substrat en introduisant des interactions de liaisons supplémentaires dans la poche S3 de la cruzaïne. Les substrats optimisés ont ensuite été convertis en inhibiteurs par l'introduction de pharmacophores basés sur le mécanisme de la cystéine protéase. Il a été établi que l'inhibiteur 38 est réversible même s'il comprend le pharmacophore de vinylsulfone qui est bien documenté pour produire une inhibition irréversible de la cruzaïne pour des inhibiteurs peptidiques. Le pharmacophore β-chlorovinylsulfone non étudié auparavant a offert une compréhension mécanistique qui a mené au développement de puissants inhibiteurs irréversibles de la cruzaïne à base d'acyl- et d'aryl-oxyméthyle de cétone. Pour ces inhibiteurs, l'efficacité ne dépend pas uniquement du groupe partant pΛTa, la 2,3,5,6-tétrafluorophénoxyméthylcétone 54 ayant été identifiée comme l'un des plus puissants inhibiteurs avec une constante d'inactivation de second ordre de 147 000 s-1M-1. Cet inhibiteur a complètement éradiqué le parasite T. cruzi des cultures de cellules de mammifère et, par conséquent, il possède le potentiel de mener à de nouvelles chimiothérapies pour la maladie de Chagas.
PCT/US2008/013400 2007-12-06 2008-12-05 Inhibiteurs non peptidiques de la cruazïne Ceased WO2009075778A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016172261A1 (fr) * 2015-04-20 2016-10-27 The Regents Of The University Of California Traitement de trypanosoma cruzi avec des inhibiteurs de la cathepsine s
CN113149970A (zh) * 2021-03-05 2021-07-23 朗捷睿(苏州)生物科技有限公司 一种8-(吡啶三氮唑)取代香豆素类化合物及其制备方法和应用

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BRPI0415010B8 (pt) * 2003-10-24 2021-05-25 Lilly Co Eli formas cristalinas iv e v de {2-[1-(3,5-bis-trifluorometilbenzil)-5-piridin-4-il-1h-[1,2,3]triazol-4-il]-piridin-3-il)-(2-clorofenil)-metanona, (2-clorofenil)-[2-(2-hidróxi-2-piridin-4-il-vinil)piridin-3-il]metanona, seus processos de preparação, e composição farmacêutica sólida

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016172261A1 (fr) * 2015-04-20 2016-10-27 The Regents Of The University Of California Traitement de trypanosoma cruzi avec des inhibiteurs de la cathepsine s
CN113149970A (zh) * 2021-03-05 2021-07-23 朗捷睿(苏州)生物科技有限公司 一种8-(吡啶三氮唑)取代香豆素类化合物及其制备方法和应用

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