AU2015203844A1 - Inhibitors of IAP - Google Patents

Abstract

Novel inhibitors of IAP that are useful as therapeutic agents for treating malignancies and have the general formula I: R2 0 R' R4 R5 0 N O Ph wherein R , R2, R , R , R and R6 are as described herein.

Description

INHIBITORS OF IAP [0000] The present application is a divisional application of Australian Application No. 2013202780, which is incorporated in its entirety herein by reference. [0001] The present invention relates to organic compounds useful for therapy and/or prophylaxis in a mammal, and in particular to inhibitors of IAP proteins useful for treating cancers. [0002] Apoptosis or programmed cell death is a genetically and biochemically regulated mechanism that plays an important role in development and homeostasis in invertebrates as well as vertebrates. Aberrancies in apoptosis that lead to premature cell death have been linked to a variety of developmental disorders. Deficiencies in apoptosis that result in the lack of cell death have been linked to cancer and chronic viral infections (Thompson et al., (1995) Science 267, 1456-1462). [0003] One of the key effector molecules in apoptosis are the caspases (csteine containing apartate specific proteases). Caspases are strong proteases, cleaving after aspartic acid residues and once activated, digest vital cell proteins from within the cell. Since caspases are such strong proteases, tight control of this family of proteins is necessary to prevent premature cell death. In general, caspases are synthesized as largely inactive zymogens that require proteolytic processing in order to be active. This proteolytic processing is only one of the ways in which caspases are regulated. The second mechanism is through a family of proteins that bind and inhibit caspases. [0004] A family of molecules that inhibit caspases are the Inhibitors of Apoptosis (IAP) (Deveraux et al., J Clin Immunol (1999), 19:388-398). IAPs were originally discovered in baculovirus by their functional ability to substitute for P35 protein, an anti-apoptotic gene (Crook et al. (1993) J Virology 67, 2168-2174). IAPs have been described in organisms ranging from Drosophila to human. Regardless of their origin, structurally, IAPs comprise one to three Baculovirus IAP repeat (BIR) domains, and most of them also possess a carboxyl-terminal RING finger motif. The BIR domain itself is a zinc binding domain of about 70 residues comprising 4 alpha-helices and 3 beta strands, with cysteine and histidine residues that coordinate the zinc ion (Hinds et al., (1999) Nat. Struct. Biol. 6, 648-651). It is the BIR domain that is believed to cause the anti-apoptotic effect by inhibiting the caspases and thus inhibiting apoptosis. As an example, human X-chromosome linked IAP (XIAP) inhibits caspase 3, caspase 7 and the Apaf-1-cytochrome C mediated activation of caspase 9 (Deveraux et al., (1998) EMBO J. 17, 2215 2223). Caspases 3 and 7 are inhibited by the BIR2 domain of XIAP, while the BIR3 domain of XIAP is responsible for the inhibition of caspase 9 activity. XIAP is expressed ubiquitously in most adult and fetal tissues (Liston et al, Nature, 1996, 379(6563):349), and is overexpressed in a number of tumor cell lines of the NCI 60 cell line panel (Fong et al, Genomics, - 1 - 200(A 70:113; Tamn et aL Clin, Cancer Res. 20(10. 6(5):1796). Overexpression of XIAP in tumor cells has been demonstrated to confer protection against a variety of pro apoptotic stimuli and promotes resistance to chemotherapy (La( asse et al, Oncogene, 1998, 17(25):3247), Consistent with this, a strong correlation between XIAP protein levels and survival has ben demonstrated for patients with 5 acute myelogenous leukemia (Tanmm et at supra). Down-regulation of XIAP expression by antisense oligonucleotides has been shown to sensitize tumor cells to death induced by a wide range of pro apopmotic agents, both in vitro and in rivo (Sasaki et aL Cancer Res, 2000, 60(20):5659: Lin et a, Biochem J, 2001, 353:299; Ha et at Clin. Cancer Res., 2003, 9(7):2826). Smac/DIABLO-derived peptides have also been demonstrated to sensitize a number of different turnr cell lines to apoptosis 10 induced by a variety of pro-apoptotic drugs (Arnt et aL J. BioL Chem., 2002, 277(46)44236; Fulda et at Nature Med., 2002, 8(8):808; Quo et al, Bxx.2002, 99(9):3419; Vucic et al J BioL (7hem,2002. 277(14):12275; Yang et at Cancer Res., 2003, 63(4):831). [00051 Melanoma TAP (ML-IAP) is an IAP not detectable in most normal adult tissues but is strongly upregulated in melanoma (Vucic et al_ (2f000) Current Bio 10: 1359-1366). Determination of protein 15 structure demonstrated significant homology of the MLAAP BIR and RING finger domains to corresponding domains present in human XIAP, (-MAP1 and C-IAP2. The BIR domain of M-IAP appears to have the most similarities to the BIR2 and BIR3 of XIAP, CIAPI and C-IAP2, and appears to he responsible for the inhibition of apoptosis, as determined by deletional analysis. Furthermore, Vucic et aL demonstrated that ML-IAP could inhibit chemotherapeutic agent induced apoptosis. 20 Agents such as adriamycin and 4-tertiary butylphenol (4-TB1) were tested in a cclI culture system of melanomas overexpivssing ML-AP anid the chemotherapeutic agents were significantly less effective in killing the cells when compared to a normal melanocyte control. The mechanism by which ML -IAP produces an anti-apoptotic activity is in part through inhihition of caspase 3 and 9. ML-IAP did not effectively inhibit caspases 1. 2, 6, or 8. 25 [0006] Since apoptosis is a striedy. controlled pathway with multiple interacting factors, the discovery that IAPs themselves are regulated was not unusual. In the fruit fly Drosophila, the Reaper (rpr), Head Involution Defective (hid) and GRIM proteins physically interact with and inhibit the anti-apoptotic activity of the Drosophila family of APs. In the mammal, the proteins SMAC/DIABLO act to block the IAPs and allow apoptosis to proeed. it was shown that during normal apoptosis, SMAC is 30 processed into an active form and is released from the mitochondria into the cytoplasm where it physically binds to IAPs and prevents the TAP from binding to a caspase. This inhibition of the IAP allows the caspase to remain active and thus proceed with apoptosis. Interestingly, sequence 2 homology between the JAP inhibitors shows that there is a four amino acid motif in the N-terminus of the processed, active proteins. This tetrapeptide appears to bind into a hydrophobic pocket in the BIR domain and disrupts the BIR domain binding to easpases ((hai et al., (2000) Nature 406:855-862, Liu et al, (2000) Nature 408:10)4-1008, Wu et al., (2000) Nature 408 1008-1012), 5 SUMMARY OF THE INVENTION [0007] In one aspect of the present invention there are provided novel inhibitors of lAP proteins having the general formula (I) R 0 R' eN AN LR

R

4 R5 0N 0 Ph ON NX 10 wherein [000$] R is (V cycloalkyl, [0009] Ph is phenyl, [0010]} t RIR Ri' R' and RWare each independently in each occurrence H or C< alkyl; or, [00111 a pharmaceutaly acceptable salt thereof. 15 [0012] Formula I includes all stereotsomers. [0013] In another aspect of the invention, there are provided compositions comprising compounds of formula I and a carrier, diluent or excipient. [0014] In another aspect of the invention, there is provided a method of inducing apoptosis in a cell comprising introducing into said cell a compound of formula L. 20 [0015} In another aspect of the invention, there is provided a method of sensitizing a cell to an apoptotic signal comprising introducing into said cell a compound of formula 1. 3 [0016] In another aspect of the invention, there is provided a method for inhibiting the binding of an 1AP protein to a caspase protein comprising contacting said IAP protein with a compound of formula . [00.17] In another aspect of the invention, there is provided a method for treating a disease or 5 condition associated with the overexpression of an TAP protein in a mamnnal, comprising administering to said mamnmal an effective amount of a compound of formula 1. [0018] In another aspect of the invention, there is provided a method for treating cancer, BRIEF DESCRIPTION OF THE DRAWINGS [0019] Figure 1 shows the efficacy of la alone, as well as in combination with Aponab, along with 10 efficacy data for prior art compound IHI alone and in combination with Apomab. in the Xenograft Model using Calu-6 lung adenocarcinoma cells. Ta was administered po. 1ll was administered i-v, The doses were selected to produce the maximum tolerated dose of the drug. [0020] Figure 2 shows the efficacy of la alone, as well as in combination with Apomab, along with efficacy data for prior art compound HI alone and in combination with Apornab, in the Xenograft 15 Model using Col25 colorectal adenocarcinoma cells, Ta was administered po. Il was administered i,v. The doses were selected to produce the maximum tolerated dose of the drug. [0021] The phrase "a" or "an" entity as used herein reters to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. As such, the terms "a" (or 20 "an"), "one or more", and "at least one" can be used interchangeably herein. [0022] As used in this specification, whether in a transitional phrase or in the body of the claim, the terms "comprise(s)" and "comprising" are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases "having at least" or "including at least". When used in the context of a process, the term "comprising" means that the process includes at least 25 the recited steps, but may include additional steps, When used in the context of a compound or composition, the term "compring" means that the compound or composition includes at least the recited features or components, but may also include additional features or components. 4 [00231 The term "independently" is used herein to indicate that a variable is applied in any one instance without regard to the presence or absence of a variable having that same or a different definition within. the same compound. Thus, in a compound in which R" appears twice and is defined as "independently carbon or nitrogen", both R"s can be carbon, both R"s can be nitrogen. or one R" 5 can be carbon and the other nitrogen. [0024] The term "optional" or "optionally" as used herein means that a subsequently described event or circumstance niay, but need not, occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, "optionally substituted" means that the optionally substituted moiety may incorporate a hydrogen or a substituent. 10 [0025] The term "about" is used herein to mean approximately, in the region of, roughly, or around, When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values sc forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%, [0026] As used herein, the recitation of a nunerical range for a variable is intended to convey that the 15 invention mnay be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value of the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variabl c an be equal to any real value of the numerical range, including the end-points of the range. As an example, a variable which is described as having values between 0 and 2, can be 0, 1 or 2 20 for variables which are inherently discrete, and can be 0-0, 0,1, 0.01, 0.001, or any other real value for variables which are inherently continuous. [0027] Compounds of formniula I exhibit tautonerisni. Tautonieric compounds can exist as two or more interconvertable species. Prototropic tautomers result from the migration of a covalently bonded hydrogen atom between two atons. Tautomers generally exist in equilibrium and attempts to isolate 25 an individual tautomers usually produce a mixture whose chemical and physical properties are consistent with a mixture of compounds. The position of the equilibrium is dependent on chemical features within the molecule, For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the keto fori predominates while; in phenols, the enol form predominates, Common prototropic tautoners include keto/enol (-C(=O)-CH - Z -C(-OH)=CI-), amide/imidic acid (-(=0) 30 NH- t -C(-OH )=N-) and amidine (-C(=NR)-NH- 1 -C(-NHR)=N-) tautomers. The latter two are 5 particularly common in heteroaryl and heterocyclic rings, The present invention encompasses all tautomeric forms of the compounds described herein. [0028] "Alkyl" mns a branched or unbranched saturated aliphatic hydrocarbon group having up to 6 carbon atoms unless otherwise specified, also when used as part of another tenn, foir example 5 "alkylamino." Examples of preferred alkyl groups include methyl, ethyl, n-propyl, isopropyl n-butyl, iso-butyI, sec-butyl, tert-butyl n -pentyl. 2-methylbuty 2;2limethylpropyL n-hexyl, 2-methylpentyl, 2.2-dimethylbuty and the like. The terms "lower alkyl" "CIC4 alkyl" and "alkyl of I to 4 carbon atoms" are synonymous and used interchangeably to muean methyl, cthyiL .1 -propyl isopropy Cyclopropyl I -butyl, se-buty or t-butyL 10 [0029] Cycloalkyl groups can be mono-. bi- or tricyclic aliphatic rings of 3 to 7 carbon atoms. Preferred groups iml ude cyclopropyl. cyclobutyl, cyclopenty and cyclohexyl groups and more preferred are cyclopropyl and cyclohexyl and most preferred is cyclohexyl. [0030] "Amino-protecting group" refers to a derivative of the groups commonly employed to block or protect an amino group while reactions are carried out on other functional groups on the compound, 15 Examples of such protecting groups include carbamates, ami~fides, alkyl and aryl groups, mines, as well as many N-heteroatom derivatives which can be removed to regenerate the desired amine group. Preferred amino protecting groups are Boc, Fmoc and Cbz, Further examples of these groups are found inT' WT Greene and P , A Wuts, "Protective Groups in Organic Synthesis". 2' ed, John Wiley & Sons, Inc. New York, NY, 1991, chapter 7; . Haslaim, "Protective Groups in Organic Chemistry", J. 20 G. W. McOmie, Ed Plenum Press. New York, NY, 1973, Chapter 5, and TW. Greene., "Protective Groups in Organic Synthesis", John Wiley and Sons, New York., NY, 1981 The term "protected amino" refers to an amino group substituted with one of the above amino-protecting groups, These groups can be used during synthesis. [0031] "Carboxy-protecting group" refers to one of the ester derivatives of the carboxylic acid group 25 commonly employed to block or protect the carboxylic acid group while reactions are carried out on other functional groups on the compound, Examples of such carboxylic acid protecting groups include 4-nitrobenzl 4-methoxybenzyl 3 4-dinethoxybenzy 2,4-imethoxybenzy, 2,4 6-timethoxybenzyl 2 4,6-trimethylbenzy pentamethylbenzyl 3,4-nthyenedioxybenzy benthydry 4,4'~ dimethoxybenzhydryl 2,2a44'-ttaehoxybenhdryl alkyl such as t-butyl or t-amyt trityl, 4 30 methoxytrityl 4,4 dimethoxytrityl 4,44"~ri-methoxytrityl 2-phenylprop-2-yl trimtylsilyL t 6 butyldimethylsilyL phenacyl, 2,2,2-trichloroethyl. beta-trimethylsily)ethy, beta- (di(n buryl)methylsiiy~ethyi, p-toluenesulfonylethyl.4-imtrobnzylsulftonylehyl, allyl, cinnamyl., 1 (rrimethyilhneihylprop- -en-3-yl, and like moieties. The species of carboxy-protecting group employed is not critical so long as the derivatized carboxylic acid is stable to the condition of 5 subsequent reaction(s) on other positions of the molecuLe and can be removed at the appropriate point without disrupting the reniinder of the molecule. In particular, it is important not to subject a carboxy protected molecule o strong nucleophilic bases, such as lithium hydroxidc or NaOI or reductive conditions employing highly activated metal hydrides such as LiAl-T. (Such harsh removal conditions are also to be avoided when removing anino-protecting groups and hydroxy-protecting groups. 10 Preferred carboxylic acid protecting groups are the alkyl (e,g. methyl, ethyl T-butyl), allyl, benzyl and p-nitrobenzyl groups. Similar carboxy-protecting groups used in the cephalos porin. penicillin and peptide arts can also be used to protect a carboxy group substituents. Further examples of these groups are found in T, W. Greene and P. G. M. Wuts, "Protective Groups in Organic Synthesis", 2" ed,, John Wiley & Sons, Inc., New York N.Y. 1991. chapter 5; E. Halam "Protetive Groups in Organic i5 Chemistry"., J. G. WMcOnmie, Ed, Plenum Press, New York, N Y, 1973, Chapter5, and 'TW, Greene, "Pro:tectivc Groups in Organic Synthesis". John Wiley and Sons New York, NY, 1981, Chapter 5. The term "protected carboxy" refers to a carboxy group substituted with one of the above carboxy protecting groups. These groups can be used during synthesis. [0032) "Hydroxy-protectng group refers to a derivative of the hydroxy group commonly employed 20 to block or protect the hydroxy group while reactions are carried out on other functional groups on the compound. Examples of such protecting groups include tetrahydropyranyloxy., benzoyl, acetoxy, carbamoyloxy, benzyL and silyethers (e. TBS, TBI)PS) groups. Further examples of these groups are found in T, W. Greene and P. G. M. Wuts, "Protective Groups in Organic Synthesis", 2 ed., John Viey & Sons. Inc., New York, NY, 1991, chapters 2-3; E Haslam, "Protective Groups in Organic 25 Chernistry", J. G. W,V McOmie, Ed- Plenum Press, New York, NY, 1973, Chapter 5, and T.W. Greene, "Protective Groups in Organic Synthesis", John Wiley and Sons, New York, NY, 198L The term. "protected hydroxy" refers to a hydroxy group substituted with one of the above hydroxy-protecting groups. These groups can be used during synthesis, [0033] "Inhibitor" means a compound which reduces or prevents the binding of IAP proteins to 30 caspase proteins or which reduces or prevents the inhibition of apoptosis by an IAP protein (e.g., c 7 IAPI, c-IAP2, X-IAI or ML-IAP). Alternatively. "inhibitor" means a compound which prevents the binding interaction of X~AAP with caspases or the binding interaction of M 1-AP with SMAC. "Pharmaccutically acceptable salts" include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and 5 properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid and the like, and organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, lieterocyclic, carboxylic, and sulfonic classes of organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyru vic acid, oxalic acid, 10 malic acid, maleic acid, ialoneic acid, succinic acid, funtaric acid, tartaric acid, citric acid. aspartic acid, ascorbic acid, glutamic acid, anthranific acid. benzoic acid, cinnamic acid. mandelic acid, enbonic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-itenesulfonic acid, salicyclic acid and the like. Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium lithium. amnimonium. calcium, magnesium, Iron, zinc, 15 copper, manganese, aluminum salts and the like. Particularly preferred are the aimonimn, potassium, sodium, calcium and magnesium salts. Salts derived from pharmaceutically acceptable organic nontoxic bases includes salts of primary, secondary, and tertiar amnes, substituted amines inchiding naturally occurring substituted anines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethyl.amine, tripropylamint, ethanolamine, 2 20 diethylaniinoethanol, trimeihamiine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrahamine, choline, betaine, ethylnediamine, glucosan.iine methylglucamine, theobromine, purines, piperiztne. pipernen N-ethylpiperidhne, polyamine resins and thc lk. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trinihamine, dicyclohexylamine, choline, and caffeine, Form tula i is also intended to encompass hydrates and solvates of the compounds, 25 [0034] The present invention provides novel compounds having the general formula I. 8 R2 0 R ' N NR

R

4

R

5 0 N h 0 Ph N CIO [0035] In a specific embodiment, R is cyclohexyl. In another specific embodiment, I is cyclopentyL In a particular embodimentit, R is oriented such that the amino acid, or amino acid analogue, which it comprises is in the I-configuration. 5 [0036] R and R 3 are independently 1- or C; alkyL In one embodiment R and RW are both I, In another embodiment Rc is methyl and R .is 1t [0037] R i H or C alkyL In a specific embodiment R4 is H or methyl i another embodiment R is methyL in another embodiment RC is oriented such that the amino acid, or aino acid analogue, which it comprises is in the L-tfguration. 10 [00381 R5 and RC are each independent H or C alkL)] In one embodiment, R5 and R4 are H or methyl In one embodiment, R" is H and RW is methyl In another embodiment, R is methyl and R> is 1, In another embodiment R and R' are both methyL in another embodinent, U' and R' are both IL. [0039] In another aspect of the present invention the compound according to formula I is (S)4--[(S)2 cy e 2 2 t m r n i c ] r n aacid (2-oxazo~2 15 yl4-phenythiazolk5-yl)-amide (Ia). 9 [0040] Compounds of the invention contain one or more asnimetric carbon atoms. Accordingly, the compounds may exist as steroisoners, including diastereomers, enantioniers or mixtures thereof, The syntheses of the compoundsi may employ racemates, diastereomers or enantiomers as starting materials or as intermediates. Diastere ieric compounds may be separated by chromatographic or 5 crystallization me thods Similarly, enantiomeric mixtures may be separated using the same techniques or others known in the art. Each of the asymmetric carbon atoms may be in the R or S configuration and both of these configurations are within the scope of the invention, Preferably, compounds of the invention have the following stereochemical configuration of formula lb wherein RW R R R R> and R' are as described herein. R2 0 R' eN A N N R R4 R5 N 0 Ph S N 10 [041] Compounds of formula II wherein A is an optionally substituted 5-me mber heterocycte comprising 1 to 4 heteroatois have been disclosed in US Publication No. 20060(0X1 470(0 In some compounds disclosed in this publication A is N-(4-phenylthiazol-5-yl.) R2 0 R1 R / N -) N-IrN NHMe N N R4 R 0 NR 6 Me A S N 15 [042] It has now been fund that compounds wherein A.is 24(.azol-2-yi)-4-phenylthiazol-5-yl per the invention afford an unexpected increase in potency and oral bioavailabilty. In addition, the compounds of the invention have generally lower side effects, including improved Jung toxicity, e.g. compared to compound I a compound disclosed in US Publication No. 20060014700. Figures 1 and 10 2 show a comparative activity in xenograft tumor models between iv administration of 1I1, compared to compound Ia of the present invention administered orally, SYNTHESIS [0043] Compounds of the invention are prepared using standard organic synthetic techniques from 5 commercially available starting materials and reagents. General techniques are disclosed in WO 98/46576 and U SP 7,244,851, which are incorporated by reference herein for the preparation methods disclosed therein. It will be appreciated that synthetic procedures employed in the preparation of compounds of the invention will depend on the particular substituents present in a compound and that various protection and deprotection may be required as is standard in organic synthesis. In a general 10 synthetic scheme compounds of the invention may be prepared using typical peptide chemistry techniques by coupling the amimo acid residue analogues with typical amide coupling procedures, In scheme 1, amine-protected amino acid residue analogues are coupled and deprotected sequentially to give the final compounds using peptide synthesis protocols. Scheme 1 R' HN Boo<S OH t Boc- No OPr 0 0 O OPr R NrU , 0 R 4 15

R

4 R2 0 R' 1) Pr removal

R

2 0 R' ,NN 00N L N R3 4 R RbHN R 4 5N R4 OP r R NRS [0044] It will be appreciated that the amino acid analogs may be coupled in any order and may be prepared using solid phase support which is routine in the art. 20 [0045] When compounds of the invention incorporate R or Ra substituents other than I-, they may also be prepared by substitution of a suitable acid intermediate which incorporates a leaving group i1 with a desired amine. For example Br-CH(R4))-OH is substituted with an amine RV-NW or R NH-R3 according to scheme 2, Scheme 2 O R2 O p2 O NH -BrN- , OR * R3 3Y OR, N R KOH RSR4 R4 R R 4 [00461 Alernatively, the substitution reaction introducing R 2 or R substituents may be performed as 5 a final step in the preparation of the compound as illustrated in scheme 3, Scheme 3 0 R, DMF 0 R 1 NH+Br N~y DMF ( yN Nr 0 0 NR- 04 N 5 A 0 [0047] In a particular embodiment, 2-bromopropionic acid is reacted with the appropriate amines dissolved in DMF and bubbled until substitution is complete to form N-substituted alanine residue. [0048] Amine substituted ring A compounds which serve as an intermediate for preparing compounds 10 of the invention am commercial available or else are prepared from commercially available reagents emloig standard organic chemistry techniques. 2-(OxazoL-2-y{l)-4-peytizl5aiecnb prepared by condensation of an saminophenylacetonitwile hydrochloride and oxazole-2-carbaldeyde in the presence u sulfur and 'TEA (Scheme 4). Scheme 4 NH 2

H

2 N CN S 8 4 ;o Ph N Nt N 15 UTILITY [0049] The compounds of the invention inhibit the binding of TAP proteins to caspases, in particular X-lAP binding interaction with caspases 3 and 7. The compounds also inhibit the binding of ME-lAP '12 to Siac protein. Accordingly, the compounds of the invention are useful for inducing apoptosis in cells or sensitizing cells to apoptotic signals, in particular cancer cells. Compounds of the invention are useful for inducing apoptosis in cells that overexpress IAP proteins (e.g, c-IAPI, c -IAP2, X-AP or MILAP) Alternatively, compounds of the invention are useful for inducing apoptosis in cells in 5 which the mitochondrial apoptotic pathway is disrupted such that release of Sniac from MIL-AP proteins is inhibited, for example by up regulation of Bcl-2 or down regulation of Bax/Bak. More broadly, the compounds can be used for the treatment of cancer. [0050] They are especially useful for the treatment of all cancer types which faid to undergo apoptosis. Examples of such cancer types include neuroblastoma, intestine carcinoma such as rectum 10 carcinoma, colon carcinoma, familiar adenonatous polyposis carcinoma and hereditary non polyposis colorectal cancer, esophageal carcinoma, labial carcinoma. jarynx carcinoma, hypopharynx carcinona, tong carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, medullary thyroid carcinoima, papillary thyroid carcinoma, renal carcinoma, kidney parenchym carcinoma, ovarian carcinoma, cervix carcinoma, uterine corpus carcinoma, endometium carcinoma, chorion I1S carcinonia, pancreatic carcinoma, prostate carcinoma, testis carcinona, breast carcinoma, urinary carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma meningioma, meduloblastonma and peripheral neuroectodermal tumors, Hodgkin lymphoma, non iodgkin lymphoma, Burkitt lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), adult T-cell leukemia lymphoma, 20 hepatocellular carcinoma, gall bladder carcinoma, bronchial carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, multiple myeloma. basalioma, teratonma, retinoblastoma, choroidea melanoma, sentinoma, rhabdomyo sarcon, craniopharyngeona, osteosarcoma, chondrosarcoma. myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma and plasmocytoma. Useful is treatment of solid tumors. Useful also is treatment of breast cancer, pancreatic adenocarcinoma or malignant 25 melanoma. [00511 Compounds of the invention are useful for sensitizing cells to apoptotic signals. Accordingly, the compounds may be administered prior to, concomitantly with, or following administration of radiation therapy or cytostatic or antineoplastic chemotherapy. Suitable cytostatic chemotherapy compounds include, but are not limited to (i) antimetabolites, such as cytarabine, 30 fludarabine, 5-fluo-o 2 deoxyuirinene, gemcitabine, hydroxyurea or methotrexate: (ii) DNA fragmenting agents, such as bleomycin. (iii) DNA-crosslinking agents, such as chlorambucil, cisplatin. cyclophosphamide or nitrogen mustard; (iv) intercalating agents such as adriamycin 13 doxorubicin) or mitoxantrone; (v) protein synthesis inhibitors, such as L-asparaginase, cycksheximide, puromycin or diphtheria toxin; (Vi) topoisomerase I poisons, such as camptothecin or topotecan; (vii) topoisomerase i poisons, such as etopoSdeC (VP'd) or ft niposide; (viii) microtubule-directed agents., such as colcemid, colchicine, paclitaxel, vinblastine or vincristine; (ix 5 kinase inhibitors such as flavopiridol, staurosporin, S115717 (Pt 57.14811) or UCN-01 (7 droxystauri (x) iiscellaneous investigational a tch as thioplatin, PS-341L phenyibuty rate, [-18- 0(11 or farnesyl transferase inhibitors (1>739749. L-744832); polyphenols such as quercetin, resveratrolk piceatannol, epigallocatechine gallate, theaflavins, flavanos, procyanidins, betulinic acid and derivatives thereof; (xi) hormones such as glucocorticoids or 10 fenretinide; (xii) hornsme antagonists., such as tanoxifm. finasteride or LHRH antagonists. in a preferred embodiment, compounds of the present invention are coadninistered with a cytostatic compound seleted from the group consisting of cisplatin, doxorubicin, pacltaxel, docetaxel and mitomycin C, Most preferred, the cytostatic compound is doxorubicin. Useful are combinations with 5-FU, gemcitabine. capecitabine, vinorelbine, bevacizumab, or taxanes. 15 [0052] Another class of active compounds which can be used in the present invention are those which are able to sensitize for or induce apoptosis by binding to death receptors ("death receptor agonists"). Such agonists of death receptors include death receptor ligands such as tumor necrosis factor a (TNF), tumor necrosis factor ti (TNF-8, lymphotoxin-a) , 1-t4 (lymphotoxin-), TRAIL (Apo2L, DR4 ligand ), CD95 (Fas. APO-1) ligand. TRAMP (DR3. Apo-3) ligand, DR6 ligand as well 20 as fragments and derivatives of any of said ligands. Preferably, the death receptor ligand is TNF-Q. More preferably the death receptor ligand is Apo2L/TRAIL. Furthermore, death receptors agonists comtprise agonistic antibodies to death receptors such as anti-CD95 antiboxty, anti-TRAILR.1 (DR4) antibody, anti -TRAIL-R2 (DR5) antibody, anti-TRAIL-R3 antibody, anti.-TRAIL-R4 antibody, anti DR6 antibody, anti-INF-R1 antibody and anti-TRAIP (DR3) antibody as well as fragments and 25 derivatives of any of said antibodies. [00531 For the purpose of sensitizing cells for apoptosis, the compounds of the present invention can be also used in combination with radiation therapy. The phrase "radiation therapy" refers to the use of electromagnetic or particulate radiation in the treatment of neoplasia, Radiation therapy is based on the principle that high-dose radiation delivered to a target area will result in the death of reproducing 30 cells in both tumor and normal tissues. The radiation dosage regimen is generally defined in terms of radiation absorbed dose (rad), time and fractionation, and must be carefully defined by t oncologit. The amount of radiation a patient receives will depend on various consideration but the two most ,14 important considerations are the location of the tumor in relation to other critical structures or organs of the body, and the extent to which thul mnor has spread. Examples of radiotherapeutic agents are provided in, but not limited to, radi"atIion therapy and is known in the art (Heliman, Principles f Radiation Therapy, Cancer, in Principles I and Practice of Oncology, 24875 (Devita et al 4th ed- vol 5 1, 1993). Recent advances in radiation therapy include three-dimensional conformal external beam radiation, intensity modulated radiation therapy (IMRT), stereotactic radiosurgery and brachytherapy (interstitial radiation therapy), the latter placing the source of radiation directly into the tumor as implanted "seeds". These newer treatment modalities deliver greater doses of radiation to the tumor, which accounts for their increased effectiveness when compared to standard external beam radiation 10 therapy, [00541 Ionizing radiation with beta-enitting radionuclides is considered the most useful for radiotherapeutic applications because of the moderate linear energy transfer (LTFI) of the ioniz ing particle (electron) and its intermediate range (typically several millimeters in tissue). Gamma rays deliver dosage at lower levels over much greater distances. Alpha particles represent the other 15 extreme, they deliver very high LET dosage, but have an extremely limited range and nsuxt, therefore, be in intimate contact with the cells of the tissue to be treated, In addition, alpha emitters are generally heavy metals, which limits the possible chemistry and presents undue hazards from leakage of radionuclide from the area to be treated. Depending on the tumor to be treated all kinds of emitters are conceivable within the scope of the present invention. 20 [00551 Furthermore, the present invention encompasses types of non-ionizing radiation like eg. ultraviolet (UV) radiation, high energy visible light. microwave radiation (hyperthermia therapy), infrared (IR) radiation and lasers, In a particular embldiment of the present invention UV radiation is applied. [0056] More generally, the compounds of the invention can be used in combination therapy, 25 "Combination therapy" includes the administration of the subject compounds in further combination with other biology call active ingredients (such as, but not limited to, a second and different antineoplastic agent) and non-drug therapies (such as, but not limited to, surgery or radiation treatment). For instance, the compounds of the invention can be used in combination with other pharmaceutically active compounds, preferably compounds that are able to enhance the effect of the 30 compounds of the invention, The coipounds of the invention can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the other drug therapy. In general, 15 a combination therapy envisions administration of two or more drugs during a single cycle or course of therapy. thus, in one aspect of the invention, the subject compounds may be administered in combination With one1 or more separate agents that mcdulate protein kinases involved in various 5 disease states or targets downstream) thereof. Examples of such kinases may include, but are not limited to: serine/treonine specific kinases, receptor tyrosine specific kinases and non-receptor tyrosine specific kinases. Serine/threonine kinases include mitogen activated protein kinases (MAPK), mneiosis specific kinase (MEK), RAF and aurora kinase. Examples of receptor kinase families include epidermal growth factor receptor (EGFR) (eg. HER2/neu, HER3, 111R4, ErbB, ErbB2, ErbB3, ErbB4 10 Xmrk, DER, Let23); fibroblast growth factor (Ff) receptor (e.g. FGF R1 .GFF-R2/BEK/CEK3. FGF R3/CEK2, FGF-R4/TKF, KGF-R); hepatocyte growth/scatter factor receptor (HGFR) (e.g. MET, RON, SEA, SEX); insulin receptor (eg. IGFO~R, P13K, AKTmTor); [ph (e.g. CEK5, CEK8, EBK, ECK, EEK, EH1K-1, EHK-2, ELK, EPHL. ERK, IIEK, MDK2, MDK5, SEK); Axi (e.g. Mer/Nyk Rse); RET; and platelet-derived growth factor receptor (PDOFR) (e.g. PDGFa-R, PDGt-R, CSFI-R/FMS, i5 SCF-R/C-Kf. VEGF-R/FLT, NEK/FLKL FLT [3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but are not iated to. BCR-A3L (e.g. p43A ARG); BTK (e.g. ITKIEMT, TEC); CSK, FAK. FPS, JAK, SRC, BMX, FER, CDK and SYK. In another aspect of the invention, the subject compounds may be administered in combination with one or more separate agents that modulate non-kinase biological targets or processes. Such 20 targets include histone deacetylases (HDAC), DNA methyltransferase (DNMT) heat shock proteins (e.g. TSI)b hedgehog inhibitors and proteosomes. In a preferred embodiment, subject compounds may be combined with antineoplastic agents (e.g. small nolecules, monoclonal antibodies, antisense RNA, and fusion proteins) that inhibit one or more biological targets such as Erivedge, Zolinza Tarceva. lressa, Tykerb, G(eevec, Sutent, Sprycel, 25 Nexavar, CNF2024, RG108, BMS387032, Affinitak Avastin, Herceptin, Erbitux, AG24322, PD325901, ZD6474, P1D184322, Obatodax, ABT737 and AEE788 Also included are monoccnal antibodies directed toward specific kinases and/or receptors, e g. those mentioned herein and others, Such combinations may enhance therapeutic efficacy over efficacy achieved by any of the agents alone and may prevent or delay the appearance of resistant mutational variants. 30 In certain preferred embodiments, the compounds of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents encompass a wide range of therapeutic treatments in the field of oncology. These agents are administered at various stages of the 16 disease for the purposes of shrinking tumors. destroying remaining cancer cells left over after surgery, inducing remission, maintain remission and/or alleviating symptoms relating to the cancer or its treatment. E Vxaiples of such agents (some of which ane also discussed above) include, but are not limited to, alkylating agents such as mustard gas derivative (Mechiorethamine. cylophosphamide, 5 chlorambucil, meilphalan, ifosfamide), ethylenimines (thiotepa, hexamethyinelanine), Alkylsulfonates (Busulfan), Hydrazines and Triazines (Attretamine, Procarbaine, Dacarbazine and Tmozokomide), Nitrosoureas (Carmustine, Iomuqmsie and Streptozocin), Ifosfamide and metal salts arboplatin, Cisplatin, and Oxaiplatin); plant alkaloidssuch asPodophyllotoxins (Etoposide and Teni.sopide), Taxanes (Paclitaxel and Docetaxet), Vinca alkaloids (Vinris tine, Vinblastine, V indesine and 10 Vinorelbine) and Canmptothecan analogs (Irinotecan and Topotecan); anti-tumor antibioticssuch asChromomyins (Dactinomycin and Plicanycin), Anthracyclines (Doxorubicin. Daunorubicin, Epinuicin, Mitoxantrone, Valrubicin and Idarubicin), and miscellaneous antibiotics such as Mitomycin, Actinomycin and Bleomycin anti-metabolitessuch asfolic acid antagonists (Methotrexate, Pemetrexed, Raltitrexed, Aminopterin), pyrimidine antagonists (5-luorouracil, Floxuridine, 15 Cytarahine, Capecitabine, and Gemcitabine), purine antagonists (6-Mercaptopurine and 6 Thioguanine) and adenosine deaminase inhibitors (Cladribin, H hidarabine, Mercaptopurine, Clofarabine, Thioguanine, Nelarabine and Pentostatin); topoisomerase inhibitors such as topoisomerase I inhibitors (honotecan, topotecan) and topoisomerase It inhibitors (Amrsacrine, etoposide, etoposide phosphate, teniposide); monoclonal antibodies (Alemtuzumab, 20 (emtumumabozogamicin, Rituximah Trastuzumab, britumornablioxetan, Cetuximab, Paniturumab, Tositumomah, Bevacizurnmab); and miscellaneous anti.-neoplastics such as rbonucleotidereductase inhibitors (Hydroxyurea); adrenocortical steroid inhibitor (Mitotane); enzymes (Asparaginase and Pegaspargase); ani-mitrotubule agents (Estramustine); and retinoids (Blexarotene, isotretinoin, Trednoin (ATRA), etc. 25 [00571 The invenuon also includes pharmaceutical compositions or medicaments containing the compounds of the invention and a therapeutically inert carrier, diluent or excipient, as well as methods of using the compounds of the invention to prepare such compositions and medicamaents. Typically, the compounds of formula I used in the methods of the invention are formulated by nixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically 30 acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenicat administration forml. The pit of the formulation depends mainly on the 17 particular use and the concentration of compound, but preferably ranges anywhere from about 3 to about 8, Formulation in an acetate buffer at pHT 5 is a suitable embodiment. [00581 The inhibitory compound for use herein is preferably sterile. The compound ordinarily will be stored as a solid comIposition, although lyophilized formulations or aqueous solutions are 5 acceptable. [00591 The composition of the invention will be formulated, dosed. and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the 10 scheduling of administration, and other factors known to medical practitioners. The "effective amount" of the compound to be administered will be governed by such considerations, and is the minimum amount necessary to treat the target diseases, e.g., to inhibit 1AP interaction with caspases. induce apoptosis or sensitize a malignant cell to an apoptotic signal. Such amount is preferably below the amount that is toxic to normal cells, or the marmniiml as a whole, 15 [00601 Generally, the compounds of this invention can be dosed once or multiple times daily. They can also be dosed on a continuous daily schedule without treatment breaks. They can also be thus dosed with treatment breaks. These options are also available when used with other agents or modaIuties. The initial pharmaceuticals effective amount of the compound of the invention administered pa enter ally per dose will be in the range of about 0.01-100 mg/kg, preferably about 0.1 20 to 20 m/kg of patient body weight per day, with the typical initial range of compound used being 03 to 15 i mg/kgday. Oral unit dosage forms, such as tablets and capsules, preferably contain from about 25 to about 1000 mg of the compound of the invention. Oral administration is preferred, On/Off dosi ng schedules can be used as are comnion for cancer trvatments, e,g, daily oral dosing for 1 2, 3, 4, etc., weeks, followed by a treatment break of 1, 2 etc,. weeks, followed by daily oral dosing for 1, 25 2, 3, 4, etc., weeks. etc. In a preferred option, the compounds of the invention are dosed daily in a range of about 25 to about 3000 mg per day, more preferably about 300 to about 1500 mg of compound per day. [00611 The compound of the invention may be administered by any suitable means, including oral, topical, transdermal, parenteral, subcutaneous, intraperitoneal, i ntrapulmonary, and intranasal, and, if 30 desired for local treatment, intralesional administration. Paremeral infusions include intramuscular, 18 intravenous, intraarterial, intraperitoneal, or subcutaneous administration. An example of a suitable oral dosage form is a tablet containing about 25mg, 50mg 100mg 250mg, or 500mg of the COmnII4OUnd of the invention compounded wiithabout 90-30 mg anhydroUs lactose, about 5~40 mg sodium croscarmellose, about 5-30mg polyvinylpyrrolidone (PVP) K3 0, and about 1-10 mg S magnesium stearate. The powdered ingredients are first mixed together and then mixed with a solution of the PVP. 'he resuluing Composition can he dried, granulated, mixed with the magnesium stearate and compressed to tablet form usikg conventional equipment, An aerosol formulation can be prepared by dissolving the compound, For example 5-400 mg, of the invention in a suitable buffer solution, e.g. a phosphate buffet adding a tonicifier, e.g. a salt such sodium chloride, if desired. The 10 solution is typically filtered, e.g, using a 0:2 micron filter, to remove impurities and contaminants. [0062] More generally, the compounds of this invention can be used in accordance with the disclosure given in WO 98/46576 and. SP 7,244,851, whose disclosures are incorporated by reference herein for their general guidance on how to use the compounds. EXAMPLES 15 [0063] The iv ention will be more fully understood by reference to the following examples. They should not, however, be construed as liniiting the scope of the invention. Abbreviations used herein are as follows: [0064] A N: acetonitrile; [00651 Chg: cyclohexylglycine; 20 [00661 DCM: dichloromethane [00671 DIPEA: diisopropylethylamine; [0068] DMAP: 4- dimethylaminopyridine; [0069] DME: 1,2-dinethoxyethane; [0070] DMF: dimethylformamide; 25 [00711 DMSO: dimethylsulfoxide [00721 EDC: 1-ethyl-3-(3-dimethylaminopropyljcarbodiimide; [0073] EEDQ; 2-ethoxy- I -ethoxycarbornyl-1;2-dihydroquinolcie 19 [0074] LCMS: liquid chromatography mass spectrometry; [00751 H ATU1 :0-(7-Azobenzotriazobl-viyl-1,3-t3etramethyluronium hexaflunorophosphate; [00761 HOt: Ndlydroxybenzotriazole [0077] HBTU: 2-(IR-Benzotriazol-1-yI)-1,3,3-Tetramethy uronium Hexafluorophosphate 5 [00781 tPLC: high performance liquid chromatography; [0079] NBS: N-bromosuccinamide; [0080] TASF: tris(dimethyiamino)sulfonium difluorotrimethylsiicate; [00811 TEA: triethylamine; [0082] TFA: trifl uoroacetate; i0 [0083} TF: tetrahydrofuran; [00841 etla~ape [0085] 1 [24Cyclohexyb2-(2-methylaminopropionylamino)-acetyl]pyrrolidine-2carboxylicacid (2 phenyi-2H1-pyrazol-3-yl)-amide Mie Met BOC NNQ&t oA N y M e O CO2H Pr ? NH NL NH1 N--Ph (10) (1 (12) 4 15 [0086] A solution of BocxL-M la (hg-Pro-OHt (47.0 mg, 0,107 mmol) and pyridine (26 Lt. 0.32 nmmol) in anhydrous dichloromethane (300pL ) was cooled to O'C and a solution of oxalyl chloride in dichloromethane: (54 pL, 2.0 M, 011 Immol) was added dropwise over 10 minutes. The mixture was stirred at OC for 15 minutes, then at ambient temperature for 45 minutes, and a solution of 5-amino- -1 phenylpyrazole (1.5.9 rug, 0,1(00 mmiol; TiCI America catalog # A01 74) and pyridine (15,5 p~L, 0,191 20 ninedl) in dichloromethane (0.5 ml) was added. The resulting mixture was stirred at ambient temperature for 16 hours, diluted with dichloromethane to 20 mL- and washed with 0.2 N aqueous sodium hydroxide (20 mL). The organic phase was dried (MgSO4) and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, 60% ethyl acetate in 20 hexanes, then 100% ethyl acetate) to yield a yellow oil: nm/z 581 (M+f*), The oil was treated with 5% trifluoroacetic acid in dichloromethane (2 mL), and after 18 hours the solvent was removed in vacuo, The resulting oil (29.3 rug, 57% yield over 2 steps) was further purified by reversed-phase lPLC to yield the product (TFA salt, 9-6 mg. 15% yield): m/ 481 (M+H*). 503 (M+Na*). 5 Refrential Exiamle 2 [0087] Acid fluoride coupling procedure Me ye N' O BoeN NJt NH N ~ BoeN N N. Me 0 CO 2 H AMe OI (13) (14) (15) [0088] A solution of kx'-MeAla-Chg-Pro-OH (23 mniol) and pyridine (6.9 uimo) in anhydrous DCM (23 ml) was cooled to 0"C and cyanuric fluoride (2 3 munol) added dropwise over 30 sec. The 10 mixture was stirred at 0 *C for 15 min. at RT for 5 h. and then quenched with water. The mixture was extracted three ftnes with DCM (total It0 ml), and the combined organic phases washed with brine and dried (NaSO4). Filtration and concentration in vacuo vielded the peptide acid fluoride as a clear, colorless\ oil used directly without further purification. [0089] A\ solution of the. crude acid fluoride (0.50 .mmol) and pyridine (IS mmoi) in DCM (2.5 ml) 15 was added to the solid amine (14, 0.50 mmol), and the resulting mixture stirred either at RI or at 50"C (sealed vessel), The mixture was poured into aqueous NaIC03 and then extracted three times with dichliororuethane (total 100 ml) The combined organic phases were washed with brine, dried (Na:SO4), filtered and concentrated in vacua. The crude peptide amide was used directly without further purification 20 [0090] Referential Exampie 3 [0091] 1-[2-Cyclohexy-2-methylainopropionlaino)acetyl]-pyrroiidine-2-carboxylic acid (4 phenyi-[1/2,3]thiadiazo[-5-y2)-amide 21 1) NBoc-(S)-Pro-OH 1) N-Boc-Chg-OH11 Ph Nil, HOBt,-EDCPCPh HO)t, E2DCLUCI, DIPEA DMF H2-DIPEA. DMF N 2) 4M HCI/dioxane N 2) 4M C /dioxane (16) MeCN (17) 1) BocN(%e)-Ala-&H Nit-CiePro-Nil Ph HOBIt, EDlOC. it N - - - DIPEA, DMF H(Me)N-A * N 2) 4M HC/dioxane S N (18) MeCN (19) [00921 step ':'o a solution jf Boc-ro-OH (2eg) HOut (1.eq) EDC-ICI (Lieg and DIPEA (5eq) in DMF (10- 15 ol) was added 4-phenyl- 1,234hiadiaiol-5-amine (16). The reaction, initially 5 mil(y exothernic, was heated to 75 "C and stirred overnight, cooled to RT and the DMF was partially removed in iacuo, The solution was diluted with 'tOAc (10-15 vol) followed by washing with 1M UCI (2x) NaHC1(O( (1 x) and brine (1x) (1: 1 aq/org). The organic layer was concentrated in vacuo and the resulting. solid was scurried in refluxing McCN (a minimum volume necessary for easy stirring) for 30 nfn and then cooled to RT. Sut.ion filtration gave Boc-protected conjugation product 10 as an off-white crystalline solid in ca, 77% yield. The Boc-proteced product was suspe ended in a solution of 4M HCI/dioxane (4-5eq acid) and MetCN (1 vol eq to the dioxane solution) and stirred at RT until LCMS indicated complete deprotection (ca. I h) The reaction mixture was concentrated in vacuo and the resulting solid was vigorously slurried in refluxing MeCN (a minimum volume necessary for easy stirring), cooled to RT, and the solid collected by suction filtration and washed with 15 cold MeCN until residual color was removed from the cake which afforded the HCI salt 3f (S)-N-(4 phenyI-1,2,3-thiadiazoi-5~yl)pyrrolidine~2-carboxamide (17) as an off-white solid in approximately quanthative yield. [0093] step 2: To a solution of 17 and DIPEA (5 eq) in DIMF (10-15 vol) was added the Boc-L-Chg (L5 eq), fOBt (L4 eq) and EDC-HCI (1.4 eq), The reaction was stirred for ca 2 h then diluted with 20 EtOAc (15 vol) and washed with IM IICI (2x), NaRCO. (lx), and brine (Ix) (1: 1 aq/org). The organic extract was dried (Na 2

SO

4 ), filtered and concentrated in vacuo. The resulting solid is scurried in EtOH/lexane (20:80) (a minimum volume necessary for easy stirring) and filtered to give Boe protected conjugate product as a fluffy white solid in ca, 80% yield, The Boc-protected was dissolved 2 2 in a solution of 4M ICI/dioxane (4-5 eq acid) and MeCN (0,25 volume eq to the dioxane solution) and stirred at RT for ca.1 hr. The reaction was concentrated to dryness with toluene (2x) (the same volume as the deprotection solution) to yield the 11Cl salt of (8)- 1-((S)-2-atmino-2-cyclohexylacetl)-N-(4 pheny 1 2,3~thiadiazoi-5-yl)pyrrolidine-2-carboxamide (18) as a white crystalline solid in 5 approximately quantitative yield. [0094) step 3: To a solution of 18 and DIPEA (5 eq.) in DMF (10-15 vol) was added the BoC-L-N methyl Ala (15 eq), HOt (1,4eg) and EDC~HCI (L4eg). The vacation was stirred for I h, diluted with EtOAc (15 vol) and washed with 1IM 11 (2x), NafHCO (lx), and brine (lx) (1:1 aq/org). The organic extract was dried (NaSO.4), filtered and concentrated in vacuo to give afford Boc-protected 10 conjugate product as a beige, foamy solid in ca. 85% yield. The Boc-protected product was dissolved in a solution of 4M ICI/dioxane (4-5 eq acid) and MeCN (0,25 volume eq to the dioxane solution) and stirred at RT for ca. I hr. The reaction was concentrated to dryness with toluene (2x) (sane volume as deprotection solution) and the resulting solid was scurried in a solution of MTBE/ItOAc (70:30) (minimal volune necessary for easy stirring), -iltered and collected to yield crude (S)-I~((S)-2 15 eyclohex 2-((S) 2-(methylamino)propanamidoacetyi)-N-(4-phenyl1,2,3thiadiazol-5-yl)pyrroiidine 2-carboxamide (d) as an off-white freeflowing solid. The crude I( salt was suspended in MeOH (4 vol minimum) and dissolved with stirring at 65C. Warm isopropyl acetate (6-8 vol) was added in two portions, keeping the temperature at ca. 60 'C then the solution was allowed to cool with stirring, Crystallization took place rapidly, the suspension was stirred at RT for several hours, then stirred at 20 04C for an hour before the solid was collected by suction filtration. The crude product was washed with MeOH/ilYOAc (1:4, 2 vol) and dried to 19 as a white/off-white crystalline solid in ea. 80% yield. [00951 Referential Example 4 [1096) 5-Amino-2-(oxazoi-2-yl)-4-phenylhi aiole [00971 A suspension of ataminophenylacetonitrie hydrochloride (1 52 g, 899 mmol), powdered 25 sulfur (289 rmg, 9.01, munol) and oxazole-2-carbaldehyde (873 mig, 8,99 mmol) in 1tOt (1 mL) was treated with TEA (1.88 ml-, 13,5 mniol), and the mixture stirred at 50 "C for 60 min. The cooled mixture was treated with aqueous hydroxylamine (1.00 ml, 50% wt, 15 nmnol) at RT overnight, filtered and concentrated in vacuo The residue was partitioned between Ei)Ac and aqueous NaTICO,. and the separated organic phase washed with brine, dried (Na.SO2) filtered and concentrated in vacuo 30 to a dark brown oil. The crude oil was preabsorbed onto SiO% and purifed by automated flash 23 chromatography eluting with a gradient of 5-70% ethyl acetate in hexanes to yield 5-amino-2-(oxazo 2-yl)-4-phenyithiazole (20, 159 mig, 7,3%). Example [009$] (S)-1-[(S)-2-Cyciohexy-2-((S)2.-methylamino-propionyl amino)-acetyi1 -pyrrolidine-2 5 carboxylic acid (2-oxazol-2-yl-4-phenyl-thiazo-5-y-amide (la) [0099] step I To a solution of oc-L-Proiine (4.5 0,02 mniol) and pvridine (8.45 mL- 0.104 nmoi) in DCM (20 ml) cooled in an ice bathwas added dropwise cyanuiic fluoride (5.35 ml, 0.0627 numl). After the addition the reaction became milky The solution wvas stirred at 0 "C for 10 min then warmed to RT and stirred for 4 h. The reaction was quenched with water, and thrice extracted with 10 ICM. The cornbined organic extracts were washed with brine, dried, and concentrated in vacuw to afford sert-butyl 2.(fluorocarbony~pyrn 1idine-I1-carboxylate. The crude acid fluoride was used in next coupling reaction inmediately. [001001 The freshly prepared acid fluoride (4,55 g, 0,02 mrmol) was dissolved in MeCN (20 mL), and treated with 20 (17 g, 0.007 rmol) and pyridine (2.82 niL, 0.035 mmofl) The reaction was heated to 15 50* overnight, The reaction mixture was quenched with sat. NaHTC0 3 and thrice extracted with EtOAc. The combined organic Layers were washed with brine, dried and concentrated, The crude product was purified by Sit) chromnatography eliting with an EtOAc/hexane gradient (50 to 80% EtOAc) to afford (S)-terb-butyl 2-(oxazoi2-y1)4-phenylthiazol-5-ylcarbamoyi)pyrrolidine-1 carboxylate (21). 20 [001011 Removal of the Boc protecting group and sequential coupling with ocUN-Chg-OH and H(Me)N-Ala-O1 were carried out in accord with procedures in steps 2 and 3 of referential example 3. Purification of the Chg coupling product also was accomplished by SiO 2 chromatography eluting with an EtOAc/hexane gradient (50 to 80% EtOAc). The crude Boc-protected tripeptide product was purified by ISCO (50~80% EtOAc/Iexane). After remove of the Boc group the final proluct was 25 purified by Prep HPLC to afford pure la: (M+H)*= ins 565.3 [001021 Other Compounds of this invention, e.g.. 24 [001031 1 ()~1(S12~cyclopopyl-2~((S-2-methyiamno-propionyiamino)-acety]-pyrrolidne-2~ carboxyii.c acid (2-oxazo1- 2 -yi-4-phnyl-thiazoi-5-yl)-amide; [001041 2. (S)-1-(S)-2-cyclopent-24(()2-methy mino-propioylamino)-ace tyl] -pyrrolidine-2 carboxylic acid (2-oxazol-2-yl-4-phenyl-thiazol-y-aide; [001051 3, (S)-1-[(S)-2-cycloheptyl-2-((-2-methylamino-propionylamino)-acetyl pyrrolidine-2 carboxylic acid (2 oxazol-2-yl-4-phenyl-thiaol5-y)-mide; [00106) 4. (S)-1-[S)-2cylohexyl-2-(S)-2Le1hylO-prOpionyiaminO)-acetylj-pyrOidine-2 carboxyic acid (-xazol 2-yl-4-phenyiadoly nade; [001071 5. (5)-1-{() 2 cyclohexyl-2-((S)2nmthylamino-propionylamino)-acetyll-pyrrolidine-2 10 carhoxylic acid (-oxavol-2-y'-4-phenyl-thiazol-5-yD)N-methyl-amide; and [001081 6. (S)~1-()-2~cyciohexyl-2-((S)-2~methylamino-propionylamino)-2-methyl-acetyl]~ pyrrolidine-2-carboxylic acid (2-oxazl-2-yl-4-phenyl-thiazol-5-yl-amide, [001091 can be prepared analogously to the compound of Example I using correspondingly analogous starting materials in the procdure of Example 1. 15 Example 3 [001101lAP inhibition assays [001111 In the following experiments was used a chimraeric BIR domain referred to as MLXB1R38G in which 11 of 110 residues correspond to those found in XIAP-B1R3, while the remainder correspond to .M-IAP-BIR. The chimeric protein MILXBIR3SG was shown to bind and inhibit caspase-9 20 significantly better than either of the native BIR domains, but hound Snac-based peptides and mature Smac with affinities similar to those of native ML-AP-BAIR. The improved caspase-9 inhibition of the chimeric BIR domain MLXBIR3SG has been correlated with increased inhibition of doxorubicin induced apoptosis when transfected into MC7 cells. [00112] MLXBIR3SG sequence: 25 [00113] MGSSHH11HHSSGLVPRGSHMLETEEIEEEGAGA TLSRGPAFPGMOSEELR LASPYD) WVPUTAEVPPE:LLA AAGiFFHTGHI~QDKV RCFFC1'YGGLQS WKRCODDPWI TElAK WFPGCQFLL RS KGQEYINN1LTISL (SEQ ID NO.: 1) [00114] TR-FR ET Peptide Binding Assay 5 [00115]Time-Resolved Fluorescence Resonance Energy 'Transfer competition experiments were performed on the Wallac Victor2 Multilabeled Counter Reader (Perkin Elmer Life and Analytical Sciences, Inc.) according to the procedures of Kolb et al (Journal of Bionolecular Screening, 1996., +(4):203), A reagent cocktail containing 300 nM his-tagged MI.XBIR3SG; 20 .nM biotinylated SMAC peptide (A VPI); 5 pg/niL anti-his allophycocyanin (XL665) (CISBio International); and 200 10 ngmL streptavidin-europium (Perkin Elmer) was prepared in reagent buffer (50 mM iTris [pH1 7.21 120 mM NaC, 0,1% bovine globulins, 5mM DTT and 0.05% octylgl ucoside). (Alternatively, this cocktail can be made using europium-labeled anti-Iis (Perkin Elmer) and streptavidin allophycocvanin (Perkin Elmer) at concentrations of 6.5 nM and 25nM, respectively). The reagent cocktail was incubated at room temperature for 30 minutes. After incubation, the cocktail was added 15 to 1:3 serial dilutions of an antagonist compound (starting concentration of 50 pM) in 384-well black FIA plates (Greiner Bio-One. Inc.). After a 90 minute incubation at room temperature, the fluorescence was read with filters for the excitation of europium (340 nm) and for the emission wavelengths of europium (61.5 nm) and a allophycocyanin (665 nm), Antagonist data were calculated as a ratio of the emission signal of allophycocyanin at 665 nm to that of the emission of europium at 20 615 nm (these ratios were multiplied by a factor of 10,000 for ease of data manipulation), The resulting values were plotted as a function of antagonist concentration and fit to a 4-parameter equation using Kaleidograph software (Synergy Software, Reading, PA). Indications of antagonist potency were determined from the 1Q50 values. Compounds of the invention where found to have lAP inhibitory activity which was demonstrated in this assay. 25 [00116]Fluorescence Polarization Peptide Binding Assay [001171Polarization experiments were performed on an Analyst f T1 96-384 (Molecular Devices Corp.) according to the procedure of Keating, S.M ., Marsters, J, Beresini, M, Ladner, C., Zioncheck K" Clark, K., Arellano, F- and Todary, .S.(2000) in Proceedings of SPETX : In Vitro Diagnostic Instrumentation (Cohn. G.E. Ed. ) pp 128-137 Bellinghaim, WA. Samples for fluorescence 30 polarization affinity measurements were prepared by addition of 1:2 serial dilutions starting at a final concentration of 5sM of MLXBIR3SG in polarization buffer (50 mM Ti.s [pH 7.21, 120 mM NaCi, 26 .1% bovine globulins 5mM DTT and 0.05% octylglucoside) to 5-carboxyflourescein-conjugated AVPdi-Phe-NI (AVP-diPhe-FAM) at 5 nM final concentration. OH 0 . HN j Me ,Me Ph Ph 0 o Ni NH NH NH e 0 H m 0 (CH NH 2 AVP-diPhe-FAM pmobe [00118]The reactions were read after an incubation time of 10 minutes at rom temperature with standard cut-off filters for the fluorescein fluorophoren (I = 485 nm; ^,= 530 rnm) in 96-well black I96 plate. (MoleculariDevices Corp,), Fluorescence values were plotted as a function of the protein concentration, and the 1C50s were obtained by fitting the data to a 4-parameter equation using 10 Kaleidograph software (Synergy software, Reading, PA). competitionn experiments were performed by addition of the MlLXBIR3SG at 30 nM to wells containing 5 nM of the AVP-diPhe-JAM probe as welt as 1:3 serial dilutions of antagonist compounds starting at a concentration of 300 pM in the polarization buffer, Samples were read after a 10-minute incubaton. Fluorescence polarization values were plotted as a function of the antagonist concentration, and the IC , values were obtained by fitting 15 the data to a 4-parameter equation using Kaleidograph software (Synergy software, Reading, PA). Inhibition constants (K) for the antagonists were deitermined from the WK5o values. Compounds of the invention where found to lave AP inhibitory activity which was demonstrated in this assay. For example, the value for K, for compound la is 0.014 (MLIAP-BIR), [001191 Exanple 4 20 [001201 Tumor Xenograft Studies (Figs. 1 and 2) [00121] All procedures involving animals wQe performed in accordance with the guidelines of the Genentech Institutional Animal Care and Use Committee. Cancer cells such as Human breast MDA NIB-231. colorectal, Colo205, or NS( CC Calu6 cancer cells were obtained from American Type Culture Collection (Manassas, VA), Cell were resuspended in 1113SS (Colo205) or the cell 25 suspension was mixed' 1:1 with Matrigel (BD Biosciences; MDA-MB-231, Calu6). The cells 27 (L5 x 10 fr MDA-MB-231; 5.0 x 10" for Colo205, Calu6) were then implanted subcutaneously into the right flank of female nude mice (Chades River Laboratories, Hollister, CA) aged 6-8 weeks. Tumor volumes wexr calculated using the mean diameter measured with vernier calipers using the formula $ = 0.5 x a x b", were a nd b are the largest and smallest perp ndicular tumor diameters, 5 respectively. TYen nice With the appropriate mean tumor volume were assigned randomly to each of six groups. The mean tumor volume + the standard error of the mean (SEM) for all s'ix groups was 168 t 3 mm, at the initiation of treatment (Day 0), The mice were observed on each day of the study, and tumor volutmes and body weights were measured twice each week. Percent tumor growth inhibition was calculated using the farnula %TG1 = 100 X (1 - tumor Volure&, 5 hnor Voumnes). 10 [001,221n this assay using human breast MDA-MB-23 1* X1 cells, Conund la of the invention has an MIED value of' 3.4 mg/kg (iv Qwk) This is five times less than the amount needed for prior art compound IIin the same assay under the same conditions (MED=1 8.6 mg/kg. The AUC for la is four times less than that for 11.1 for similar efficacy. [00123] The features disclosed in the foregoing description, or the following claims, expressed in their 15 specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be utilized for realizing the invention in diverse forms thereof. [001241The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity and understanding. It will he obvious to one of skill in the art that changes and 20 modifications may he practiced within the scope of the appended claims. Therefore. it is to be understood that the above description is intended to be illustrative and not restrictive. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the following appended Claims, along with the full scope of equivalents to which such claims are entitled, 25 [00125]The patents, published applications, and scientific literature referred to herein establish the knowledge of those skilled in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and. individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specifications shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or 28 phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter. 29

Claims (13)

1. A compound of formula I R2 0 R' N N R R 4 R 6 0 N 0 Ph S NN wherein Ph is phenyl; R' is Cn cycloalkyl; R2, Rit R- and Roare each independently in each occurrence H1 or C; aikyl; or a pharmaceutically acceptable salt thereof, Io 2. The compound of clain I which is (S)1~[(S)r2~cyclohexyl2~((S)2~methyamino propionylatino)-acetylj-pyrrolidine-2-carboxylic acid (2-oxazol-2-yi-4-phenyl-thiazol-5-yl) amide (La) or a pharmaceutically acceptable salt thereof.

3. A compound of claim I wherein R RW are each independently H or methyl.

4. A compound of claim 1 wherein R is cyclohexyl 15 5. A compound of claim 3 wherein R is cyclohexyl.

6. A compound of claim 3 wherein one of R2 and R is H and the other is methyl; or R 4 is methyl; or Rt and R' are each I.

7. A method for inhibiting the binding of an IAP protein to a caspase protein comprising contacting said LAP protein with a compound of claim 1. 20

8. A method for treating a disease or condition associated with the overexpression of an TAP in a manmal comprising administering to said mammal an effective amount of a compound of claimLi 30

9. A method of inducing apoptosis in a cell comprising introducing into said cell a compound of claim L1

10. A method of sensitizing a cell to an apoptotic signal comprising introducing into said cell a 5 compound of claim I 1L The method of claim 10, wherein said apoptotic signal is induced by contacting said cell with a compound selected from the group consisting of cytarabine, fludarabine, 5-fluoro-2' deoxyuiridine, gemeitabine, methotrexate, Ibieomycin, cispatin, cyclophosphamide, 10 adriamycin (doxorubicin), mitoxantrone, caniptothecin, topotecan, colcemid, colchicine, paclitaxel, vinblastine, vincristine, tamoxifen, finasteride, docetaxel and mitomycin C. 12, The method of claim 10, wherein said apoptotic signal is induced by contacting said cell with Apo2L/TRAIL, 15

13. A method for treating cancer. comprising administering to said mammal an effective amount of a compound of claim 1,

14. A method for inhibiting the binding of an TAP protein to a caspase protein comprising 20 contacting said LAP protein with a compound < claim 2.

15. A method for treating a disease or condition associated with the overexpression of an LAP in a mammal, comprising administering to said mammal an effective amount of a compound of claim. 25 16. A method of inducing apoptosis in a cell comprising introducing into said cell a compound of claim 2.

17. A method of sensitizing a cell to an apoptotic signal comprising introducing into said cell a compound of claim 2, 30 18, The method of claim 17, wherein said apoptotic signal is indticed by contacting said cell with a compound selected from the group consisting of cytarahine, fltdarabine, 5-fuoro-2' deoxyuiridine, gerncitahine, methotrexate, bleomnycin, cisplatin, cyclophosphamide, 31 adriamycin (doxorubicin), initoxantrone, camptothecin, top(tecan, colcenid, coichicine, paclitaxel, vinblastine, vincristine, tanoxifen, finasteride, docetaxel and intomycin C.

19. The method of claim 17, wherein said apoptotic signal is induced by contacting said cell with 5 Apo2:1/TR.All. 20, A method for treating cancer, comprising administering to said manmmal an effective amount of a compound of claim 2, t) 21. A phannaceutcal composition comprising the compound of claim 1 and at least one pharmaceutically acceptable carrier, diluent or excipient, 22, A pharmaceutcal composition compnsing the compound of claim 2 and at least one pharmaceutically acceptable carrier, diluent or excipient. 15 32