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WO2007101347A1 - Bir domain binding compounds - Google Patents

Bir domain binding compounds Download PDF

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Publication number
WO2007101347A1
WO2007101347A1 PCT/CA2007/000371 CA2007000371W WO2007101347A1 WO 2007101347 A1 WO2007101347 A1 WO 2007101347A1 CA 2007000371 W CA2007000371 W CA 2007000371W WO 2007101347 A1 WO2007101347 A1 WO 2007101347A1
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Prior art keywords
compound
aryl
alkyl
compounds
substituents
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PCT/CA2007/000371
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French (fr)
Inventor
James Jaquith
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Aegera Therapeutics Inc.
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Publication of WO2007101347A1 publication Critical patent/WO2007101347A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D223/00—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom
    • C07D223/02—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D223/06—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom not condensed with other rings with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D223/08—Oxygen atoms
    • C07D223/10—Oxygen atoms attached in position 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D223/00—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom
    • C07D223/14—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D223/16—Benzazepines; Hydrogenated benzazepines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D267/00—Heterocyclic compounds containing rings of more than six members having one nitrogen atom and one oxygen atom as the only ring hetero atoms
    • C07D267/02—Seven-membered rings
    • C07D267/08—Seven-membered rings having the hetero atoms in positions 1 and 4
    • C07D267/12—Seven-membered rings having the hetero atoms in positions 1 and 4 condensed with carbocyclic rings or ring systems
    • C07D267/14—Seven-membered rings having the hetero atoms in positions 1 and 4 condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D281/00—Heterocyclic compounds containing rings of more than six members having one nitrogen atom and one sulfur atom as the only ring hetero atoms
    • C07D281/02—Seven-membered rings
    • C07D281/04—Seven-membered rings having the hetero atoms in positions 1 and 4
    • C07D281/08—Seven-membered rings having the hetero atoms in positions 1 and 4 condensed with carbocyclic rings or ring systems
    • C07D281/10—Seven-membered rings having the hetero atoms in positions 1 and 4 condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • the present invention concerns constrained cyclic compounds, which are useful for treating proliferative disorders and disorders of dysregulated apoptosis, such as cancer.
  • Apoptosis typically occurs in the normal development and maintenance of healthy tissues in multicellular organisms. It is a complex process which results in the removal of damaged, diseased or developmentally redundant cells, in the absence of signs of inflammation or necrosis.
  • Intrinsic apoptotic pathways are known to be dysregulated, most particularly in cancer and lymphoproliferative syndromes, as well as autoimmune disorders such as multiple sclerosis, in neurodegenerative diseases and in inflammation. As well, alterations in a host apoptotic response have been described in the development or maintenance of viral and bacterial infections.
  • the caspases are a family of proteolytic enzymes from the class of cysteine proteases which are known to initiate and execute apoptosis. In normal cells, the caspases are present as inactive zymogens, which are catalytically activated following external signals, for example those resulting from ligand driven Death Receptor activation, such as cytokines or immunological agents, or by release of mitochondrial factors, such as cytochrome C following genotoxic, chemotoxic, or radiation-induced cellular injury.
  • the Inhibitors of Apoptosis Proteins (IAPs) constitute a family of proteins which are capable of binding to and inhibiting the caspases, thereby suppressing cellular apoptosis.
  • the IAPs are capable of inhibiting programmed cell death from a wide variety of triggers, which include loss of homeostatic, or endogenous cellular growth control mechanisms, as well as chemotherapeutic drugs and irradiation.
  • the IAPs contain one to three homologous structural domains known as baculovirus IAP repeat (BIR) domains. They may also contain a RING zinc finger domain at the C- Attorney Docket No. L80003375WO terminus, with a capability of inducing ubiquitinylation of IAP-binding molecules via its E3 ligase function.
  • BIR baculovirus IAP repeat
  • the human IAPs, XIAP, HIAP1 (also referred to as clAP2), and HIAP2 (clAP1 ) each have three BIR domains, and a carboxy terminal RING zinc finger.
  • IAP X chromosome-linked inhibitor of apoptosis
  • caspase-9 the initiator caspase
  • Caspase-3 and Caspase-7 the effector caspases
  • the Iinker-BIR2 domain of XIAP inhibits the activity of caspases-3 and -7.
  • the BIR domains have also been associated with the interactions of IAPs with tumor necrosis factor-receptor associated factor (TRAFs)-I and -2, and to TAB1 , as adaptor proteins effecting survival signaling through NFkB activation.
  • the IAPs thus function as a direct break on the apoptosis cascade, by preventing the action of, or inhibiting active caspases and by redirecting cellular signaling to a pro-survival mode.
  • neoplasia may be viewed as a failure of cancer cells to execute normal pathways of apoptosis.
  • Normal cells receive continuous feedback from their environment through various intracellular and extracellular factors, and "commit suicide" if removed from this context. This induction of apoptosis is achieved by activation of the caspase cascade.
  • Cancer cells gain the ability to overcome or bypass this apoptosis regulation and continue with inappropriate proliferation. The majority of treatments for cancer induce at least a partial apoptotic response in the cancer target cell, resulting in remission or initiation of tumor regression.
  • cancer cells may avoid apoptosis by the sustained over- expression of one or more members of the IAP family of proteins, as documented in many primary tumor biopsy samples, as well as most established cancer cell lines.
  • Epidemiological studies have demonstrated that over-expression of the various IAPs is associated with poor clinical prognosis and survival. For XIAP this is shown in cancers as diverse as leukemia and ovarian cancer.
  • Over expression of HIAP1 and HIAP2 resulting from the frequent chromosome amplification of the 11q21-q23 region, which encompasses both, has been observed in a variety of malignancies, including medulloblastomas, renal cell carcinomas, glioblastomas, and gastric carcinomas.
  • (X)IAP negative regulatory molecules such as XAF, appear to be tumor suppressors, which are very frequently lost in clinical cancers.
  • the IAPs may directly contribute to tumor progression and resistance to pharmaceutical intervention. Induction of apoptosis in cancer cells by the use of potent small molecules which bind to specific IAP domains is the subject of this invention.
  • the functions of the BIRs when ligand-bound, appear to direct the activation of the ubiquitin ligase RING function of the IAPs to a bound target, or the IAPs themselves, to cause proteosomal loss.
  • small molecule antagonists of this target should be excellent pro-apoptotic agents, with potential uses in cancer, various proliferative disorders and inflammation.
  • a mammalian mitochondrial protein namely Second Mitochondria-derived Activator of Caspases (SMAC) which antagonizes IAP function, binds mainly to the BIR 3 or 2 sites on respective IAPs via an AxPy amino-terminal tetrapeptide.
  • SMAC Second Mitochondria-derived Activator of Caspases
  • IAPs to inhibit caspases also bind the BIR domains of the analogous Drosophila IAPs via a short AxPy amino-terminal tetrapeptide, a sequence that fits into the BIR binding pocket and disrupts IAP-caspase interactions.
  • the overall topology of individual BIR domains is highly conserved between the human IAPs and between individual BIR domains of the human IAPs, each BIR being a zinc finger polypeptide domain, locked into a coordinated Zn atom by two cysteines and a histidine residue.
  • the X-ray crystallographic structures of XIAP BIR2 and BIR3 reveal a critical binding pocket for an AXPY motif on the surface of each BIR domain. There are alterations in the intervening amino acid sequences that form the binding pocket and groove in both BIR2 and BIR3.
  • the aforesaid compounds have been shown to target an isolated BIR3 domain of XIAP via displacement of a fluorescently-labeled probe and they appear to induce an apoptotic event in a select set of cancer cell lines with potency in the low micromolar-nanomolar range. These compounds displayed poor in-vivo activity, likely due to limited bioavailability and may therefore have limited therapeutic application.
  • IAP BIR domains represent an attractive target for the discovery and development of novel therapeutic agents, especially for the treatment of proliferative disorders such as cancer,
  • the compounds are less peptidic in character because the proline in the previously described compounds has been replaced with a constrained cyclic moiety and as such have pharmaceutically acceptable stability and bioavailability.
  • the compounds bind to XIAP BIR3 and therefore are useful for treating proliferative disorders.
  • Y is NH, O or S
  • R 1 , R 2 , R 3 and R 4 are independently selected from: 1) H, or
  • W is a 5, 6, or 7-membered cyclic amide ring, optionally including one or two O, S, or N heteroatoms, the cyclic amide ring being optionally fused with an aryl or a heteroaryl, wherein the aryl and the heteroaryl are optionally substituted with one or more R 11 substituents, and wherein the cyclic amide ring is optionally substituted with one or more R 7 substituents;
  • A is 1) -(C 1 -C 6 alkyl)-C(O)- or
  • G is a 5, 6 or 7 membered ring which optionally incorporates one or more heteroatoms selected from S, N or O, and which is optionally substituted with one or more R 13 substituents, and which is optionally fused with an aryl, the aryl being optionally substituted with one or more R 11 substituents; or
  • Q is an aryl or a heteroaryl, the aryl and the heteroaryl being optionally substituted with one or more R 11 substituents;
  • Attorney Docket No. L80003375WO R 5 and R 6 are each independently selected from
  • NC(Y)NR 9 R 10 wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl is optionally substituted with one or more R 11 substituents;
  • R 9 and R 10 are each independently 1) H, Attorney Docket No. L80003375WO
  • R 9 and R 10 together with the nitrogen atom to which they are bonded form a five, six or seven membered heterocyclic ring optionally substituted with one or more R 11 substituents;
  • R 12 is 1 ) haloalkyl
  • heterobicyclyl wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is optionally substituted with one or more R 7 substituents; and wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl is optionally substituted with one or more R 11 substituents;
  • R 13 and R 14 are each independently 1) H, or
  • R 13 and R 14 are combined to form a heterocyclic ring or a heterobicyclyl ring; or a prodrug, or a pharmaceutically acceptable salt, or labeled with a detectable label or an affinity tag, or a stereoisomer thereof.
  • Attorney Docket No. L80003375WO there is provided an intermediate compound represented by Formula l(ii):
  • PG 2 is a protecting group
  • R 1 , R 3 , R 7 , R 4 R 5 are as defined herein
  • Y' has the same meaning as Y
  • R a and R b may be combined to form an aromatic ring system wherein double bond may represent a single or double bond
  • R 7 , R 4 R 5 , Y', R a and R b are as defined herein.
  • a pharmaceutical composition comprising a compound, as described above, mixed with a pharmaceutically acceptable carrier, diluent or excipient.
  • a pharmaceutical composition adapted for administration as an agent for treating a proliferative disorder in a subject comprising a therapeutically effective amount of a compound, as described above.
  • a pharmaceutical composition comprising a compound of Formula I in combination with one or more death receptor agonists, for example, an agonist of TRAIL receptor.
  • a pharmaceutical composition comprising a compound of formula I in combination with any therapeutic agent that increases the response of one or more death receptor agonists, for example cytotoxic cytokines such as interferons.
  • cytotoxic cytokines such as interferons.
  • a method of preparing a pharmaceutical composition comprising: mixing a compound, as described above, with a pharmaceutically acceptable carrier, diluent or excipient.
  • a method of treating a disease state characterized by insufficient apoptosis comprising: administering to a subject in need thereof, a therapeutically effective amount of a pharmaceutical composition, as described above, so as to treat the disease state.
  • a method of modulating IAP function comprising: contacting a cell with a compound of the present invention so as to prevent binding of a BIR binding protein to an IAP BIR domain thereby modulating the IAP function.
  • a method of treating a proliferative disease comprising: administering to a subject in need thereof, a therapeutically effective amount of the pharmaceutical composition, as described above, so as to treat the proliferative disease.
  • a method of treating cancer comprising: administering to a subject in need thereof, a therapeutically Attorney Docket No. L80003375WO effective amount of the pharmaceutical composition, as described above, so as to treat the cancer.
  • a method of treating cancer comprising: administering to the subject in need thereof, a therapeutically effective amount of a pharmaceutical composition, as described above, in combination or sequentially with an agent selected from: a) an estrogen receptor modulator, b) an androgen receptor modulator, c) retinoid receptor modulator, d) a cytotoxic agent, e) an antiproliferative agent, f) a prenyl-protein transferase inhibitor, g) an HMG-CoA reductase inhibitor, h) an HIV protease inhibitor, i) a reverse transcriptase inhibitor, k) an angiogenesis inhibitor,
  • an agent selected from: a) an estrogen receptor modulator, b) an androgen receptor modulator, c) retinoid receptor modulator, d) a cytotoxic agent, e) an antiproliferative agent, f) a prenyl-protein transferase inhibitor, g) an HMG-CoA reductase inhibitor,
  • a PPAR-. ⁇ agonist a PPAR-. ⁇ agonist
  • m a PPAR-. ⁇ . agonist
  • n an inhibitor of inherent multidrug resistance
  • o an anti-emetic agent
  • p an agent useful in the treatment of anemia
  • q agents useful in the treatment of neutropenia
  • r an immunologic-enhancing drug.
  • a proteasome inhibitor t) an HDAC inhibitor;' u) an inhibitor of the chemotrypsin-like activity in the proteasome; or v) E3 ligase inhibitors; w) a modulator of the immune system such as, but not limited to, interferon-alpha, Bacillus Calmette-Guerin (BCG), and ionizing radition (UVB) that can induce the release of cytokines, such as the interleukins, TNF, or induce release of death receptor ligands such as TRAIL; x) a modulator of death receptors TRAIL and TRAIL agonists such as the humanized antibodies HGS-ETR1 and HGS-ETR2; or in combination or sequentially with radiation therapy, so as to treat the cancer.
  • BCG Bacillus Calmette-Guerin
  • UVB ionizing radition
  • TRAIL a modulator of death receptors TRAIL and TRAIL agonists
  • a method for the treatment or prevention of a proliferative disorder in a subject comprising: administering to the subject a therapeutically effective amount of the composition, described above.
  • the method further comprises administering to the subject a therapeutically effective amount of a chemotherapeutic agent prior to, simultaneously with or after administration of the composition.
  • the method further comprises administering to the subject a therapeutically effective amount of a death receptor agonist prior to, simultaneously with or after administration of the composition.
  • the death receptor agonist is TRAIL or the death receptor agonist is a TRAIL antibody.
  • the death receptor agonist is typically administered in an amount that produces a synergistic effect.
  • a probe in another aspect of the present invention, there is provided a probe, the probe being a compound of Formula I above, the compound being labeled with a detectable label or an affinity tag.
  • a method of identifying compounds that bind to an IAP BIR domain comprising: a) contacting an IAP BIR domain with a probe to form a probe:BIR domain complex, the probe being displaceable by a test compound; b) measuring a signal from the probe so as to establish a reference level; c) incubating the probe:BIR domain complex with the test compound; d) measuring the signal from the probe; e) comparing the signal from step d) with the reference level, a modulation of the signal being an indication that the test compound binds to the BIR domain, wherein the probe is a compound of Formula I labeled with a detectable label or an affinity label.
  • the compounds of the present invention are useful as BIR domain binding compounds in mammalian IAPs and are represented by Formula I.
  • the following are embodiments, Attorney Docket No. L80003375WO groups and substituents of the compounds according to Formula I, which are described hereinafter in detail.
  • n 0.
  • R 1 , R 2 , R 3 , R 4 , W and Q are as defined herein.
  • W is a 6 or 7-membered cyclic amide ring, optionally including one or two O, S, or N heteroatoms, the cyclic amide ring being optionally fused with an aryl or a heteroaryl, wherein the aryl and the heteroaryl are optionally substituted with one or more R 11 substituents, and wherein the cyclic amide ring is optionally substituted with one or more R 7 substituents.
  • W is a 7-membered cyclic amide ring, the cyclic amide ring being optionally fused with an aryl.
  • compounds of the present invention comprise compounds of Formula IB and IC: Attorney Docket No. L80003375WO
  • R 1 , R 2 , R 3 , R 4 , A and Q are as defined herein.
  • W is a 7-membered cyclic amide ring, including one O, S, or N heteroatoms, the cyclic amide ring being optionally fused with an aryl or a heteroaryl.
  • W is a 7-membered cyclic amide ring, including one O, S, or N heteroatoms, the cyclic amide ring being fused with an aryl.
  • compounds of the present invention comprise compounds of Formula ID and IE
  • R 1 , R 2 , R 3 , R 4 , A and Q are as defined herein.
  • R 1 is C 1 -C 6 alkyl. In one example, R 1 is CH 3 .
  • R 2 is H.
  • R 2 any and each individual definition of R 2 as set out herein may be combined with any and each individual definition of W 1 Core, R 1 , R 3 , R 4 , A and Q as set out herein.
  • R 3 is Ci-C 6 alkyl. In one example, R 3 is CH 3 .
  • R 3 any and each individual definition of R 3 as set out herein may be combined with any and each individual definition of W, Core, R 1 , R 2 , R 4 , A and Q as set out herein.
  • R 4 is H.
  • R 4 any and each individual definition of R 4 as set out herein may be combined with any and each individual definition of W, Core, R 1 , R 2 , R 3 , A and Q as set out herein.
  • A is -(C 1 -C 6 alkyl)-C(O)-.
  • A is -CH 2 C(O)-.
  • Q is NR 5 R 6 .
  • R 5 and R 6 In one subset of the aforesaid compounds, R 5 is H and R 6 is selected from: Attorney Docket No. L80003375WO
  • alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is optionally substituted with one or more R 7 substituents.
  • R 5 is H and R 6 is selected from: I ) ⁇ C 1 -C 6 alkyl, or
  • R 6 when R 5 is H is selected from the group consisting of:
  • Q is selected from the group consisting of:
  • Q may be substituted with a detectable label or an affinity tag.
  • the detectable label or the affinity tag may be located at any suitable position on the compound.
  • substituents are itself substituted with one or more substituents, it is to be understood that that the one or more substituents may be attached to the same carbon atom or different carbon atoms. Combinations of substituents and variables defined herein are allowed only if they produce chemically stable compounds.
  • substitution patterns and substituents on compounds of the present invention may be selected to provide compounds that are chemically stable and can be readily synthesized using the chemistry set forth in the examples and chemistry techniques well known in the art using readily available starting materials.
  • the term “comprising” is intended to mean that the list of elements following the word “comprising” are required or mandatory but that other elements are optional and may or may not be present .
  • alkyl is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, for example, C 1 -C 6 as in C 1 -C 6 - alkyl is defined as including groups having 1 , 2, 3, 4, 5 or 6 carbons in a linear or branched arrangement, and C 1 -C 4 as in C 1 -C 4 alkyl is defined as including groups having 1 , 2, 3, or 4 carbons in a linear or branched arrangement.
  • C 1 -C 6 as in C 1 -C 6 - alkyl is defined as including groups having 1 , 2, 3, 4, 5 or 6 carbons in a linear or branched arrangement
  • C 1 -C 4 as in C 1 -C 4 alkyl is defined as including groups having 1 , 2, 3, or 4 carbons in a linear or branched arrangement.
  • Examples of d-C ⁇ -alkyl and d-C 4 alkyl as defined above include, but are not limited to, methyl, ethyl, n-propyl, /-propyl, n-butyl, f-butyl, /-butyl, pentyl and hexyl.
  • alkenyl is intended to mean unsaturated straight or branched chain hydrocarbon groups having the specified number of carbon atoms therein, and in which at least two of the carbon atoms are bonded to each other by a double bond, and having either E or Z regeochemistry and combinations thereof.
  • C 2 -C 6 as in C 2 -C 6 alkenyl is defined as including groups having 2, 3, 4, 5, or 6 carbons in a linear or branched arrangement, at least two of the carbon atoms being bonded together by a double bond.
  • Examples of C 2 -C 6 alkenyl include ethenyl (vinyl), 1-propenyl, 2-propenyl, 1- butenyl and the like.
  • alkynyl is intended to mean unsaturated, straight chain hydrocarbon groups having the specified number of carbon atoms therein and in which at least two carbon atoms are bonded together by a triple bond.
  • C 2 -C 4 as in C 2 - C 4 alkynyl is defined as including groups having 2, 3, or 4 carbon atoms in a chain, at least two of the carbon atoms being bonded together by a triple bond.
  • Examples of such alynyls include ethynyl, 1-propynyl, 2-propynyl and the like.
  • cycloalkyl is intended to mean a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms therein, for example, C 3 - C 7 as in C 3 -C 7 cycloalkyl is defined as including groups having 3, 4, 5, 6, or 7 carbons in a monocyclic arrangement.
  • Examples of C 3 -C 7 cycloalkyl as defined above include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
  • cycloalkenyl is intended to mean a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms therein, for example, C 3 -C 7 as in C 3 -C 7 cycloalkenyl is defined as including groups having 3, 4, 5, 6, or 7 carbons in a monocyclic arrangement.
  • Examples of C 3 -C 7 cycloalkenyl as defined above include, but are not limited to, cyclopentenyl, and cyclohexenyl.
  • halo or halogen is intended to mean fluorine, chlorine, bromine and iodine. Attorney Docket No. L80003375WO
  • haloalkyl is intended to mean an alkyl as defined above, in which each hydrogen atom may be successively replaced by a halogen atom. Examples of haloalkyls include, but are not limited to, CH 2 F, CHF 2 and CF 3 .
  • aryl either alone or in combination with another radical, means a carbocyclic aromatic monocyclic group containing 6 carbon atoms which may be further fused to a second 5- or 6-membered carbocyclic group which may be aromatic, saturated or unsaturated.
  • Aryl includes, but is not limited to, phenyl, indanyl, 1-naphthyl, 2-naphthyl and tetrahydronaphthyl.
  • the fused aryls may be connected to another group either at a suitable position on the cycloalkyl ring or the aromatic ring. For example:
  • biphenyl is intended to mean two phenyl groups bonded together at any one of the available sites on the phenyl ring. For example:
  • heteroaryl is intended to mean a monocyclic or bicyclic ring system of up to ten atoms, wherein at least one ring is aromatic, and contains from 1 to 4 hetero atoms selected from the group consisting of O, N, and S.
  • the heteroaryl substituent may be attached either via a ring carbon atom or one of the heteroatoms.
  • heteroaryl groups include, but are not limited to thienyl, benzimidazolyl, benzo[b]thienyl, furyl, benzofuranyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, 2H- pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyi, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, napthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, Attorney Docket No.
  • L80003375WO isothiazolyl, isochromanyl, chromanyl, isoxazolyl, furazanyl, indolinyl, isoindolinyl, thiazolo[4,5-b]-pyridine, and
  • fluoroscein derivatives such as:
  • heterocycle As used herein, the term “heterocycle”, “heterocyclic” or “heterocyclyl” is intended to mean a 5, 6, or 7 membered non-aromatic ring system containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S.
  • heterocycles include, but are not limited to pyrrolidinyl, tetrahydrofuranyl, piperidyl, pyrrolinyl, piperazinyl,
  • imidazolidinyl morpholinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, and
  • heterocycle either alone or in combination with another radical, is intended to mean a heterocycle as defined above fused to another cycle, be it a heterocycle, an aryl or any other cycle defined herein.
  • heterobicycles include, but are not limited to, coumarin, benzo[d][1 ,3]dioxole, 2,3- dihydrobenzo[b][1 ,4]dioxine and 3,4-dihydro-2H-benzo[b][1 ,4]dioepine.
  • heteroaryl is intended to mean a monocyclic or bicyclic ring system of up to ten atoms, wherein at least one ring is aromatic, and contains from 1 to 4 hetero atoms selected from the group consisting of O, N, and S.
  • the heteroaryl substituent may be attached either via a ring carbon atom or one of the heteroatoms.
  • heteroaryl groups include, but are not limited to thienyl, benzimidazolyl, benzo[b]thienyl, furyl, benzofuranyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, 2H- pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, napthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, isothiazolyl, isochromanyl, chromanyl,
  • heterocycle is intended to mean a 5, 6, or 7 membered non-aromatic ring system containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S.
  • heterocycles include, but are not limited to pyrrolidinyl, tetrahydrofuranyl, piperidyl, pyrrolinyl, piperazinyl, imidazolidinyl, morpholinyl, imidazolinyl, pyrazolidinyl, and pyrazolinyl,
  • heteroatom is intended to mean O, S or N.
  • the term "detectable label” is intended to mean a group that may be linked to a compound of the present invention to produce a probe or to an IAP BIR domain, such that when the probe is associated with the BIR domain, the label allows either direct or indirect recognition of the probe so that it may be detected, measured and quantified.
  • the term "affinity tag” is intended to mean a ligand or group, which is linked to either a compound of the present invention or to an IAP BIR domain to allow another compound to be extracted from a solution to which the ligand or group is attached.
  • the term "probe” is intended to mean a compound of Formula I which is labeled with either a detectable label or an affinity tag, and which is capable of binding, either covalently or non-covalently, to an IAP BIR domain.
  • the probe When, for example, the probe is non-covalently bound, it may be displaced by a test compound.
  • the probe When, for example, the probe is bound covalently, it may be used to form cross-linked adducts, which may be quantified and inhibited by a test compound.
  • the term "optionally substituted with one or more substituents” or its equivalent term “optionally substituted with at least one substituent” is intended to mean that the subsequently described event of circumstances may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. The definition is intended to mean from zero to five substituents.
  • the substituent may be protected with a suitable protecting group (PG) that is stable to the reaction conditions used in these methods.
  • the protecting group may be removed at a suitable point in the reaction sequence of the method to provide a desired intermediate or target compound.
  • suitable protecting groups and the methods for Attorney Docket No. L80003375WO protecting and de-protecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which may be found in T. Greene and P. Wuts, Protecting Groups in Chemical Synthesis (3 rd ed.), John Wiley & Sons, NY (1999), which is incorporated herein by reference in its entirety.
  • a substituent may be specifically selected to be reactive under the reaction conditions used in the methods of this invention. Under these circumstances, the reaction conditions convert the selected substituent into another substituent that is either useful in an intermediate compound in the methods of this invention or is a desired substituent in a target compound.
  • the term "residue" when referring to ⁇ -amino acids is intended to mean a radical derived from the corresponding ⁇ -amino acid by eliminating the hydroxyl of the carboxy group and one hydrogen of the ⁇ -amino group.
  • GIn, Ala, GIy, He, Arg, Asp, Phe, Ser, Leu, Cys, Asn, and Tyr represent the residues of L-glutamine, L-alanine, glycine, L-isoleucine, L-arginine, L-aspartic acid, L-phenylalanine, L-serine, L- leucine, L-cysteine, L-asparagine, and L-tyrosine, respectively.
  • subject is intended to mean humans and non-human mammals such as primates, cats, dogs, swine, cattle, sheep, goats, horses, rabbits, rats, mice and the like.
  • prodrug is intended to mean a compound that may be converted under physiological conditions or by solvolysis to a biologically active compound of the present invention.
  • prodrug refers to a precursor of a compound of the invention that is pharmaceutically acceptable.
  • a prodrug may be inactive or display limited activity when administered to a subject in need thereof, but is converted in vivo to an active compound of the present invention.
  • prodrugs are transformed in vivo to yield the compound of the invention, for example, by hydrolysis in blood or other organs by enzymatic processing.
  • the prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in the subject (see, Bundgard, H., Design of Prodrugs (1985), pp.
  • prodrug includes any covalently bonded carriers which release the active compound of the invention in vivo when such prodrug is administered to a subject.
  • Prodrugs of a compound of the present invention may be prepared by modifying functional groups present in the compound of the invention in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to a parent compound of the invention.
  • the term "pharmaceutically acceptable carrier, diluent or excipient” is intended to mean, without limitation, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, emulsifier, or Attorney Docket No. L80003375WO encapsulating agent, such as a liposome, cyclodextrins, encapsulating polymeric delivery systems or polyethyleneglycol matrix, which is acceptable for use in the subject, preferably humans.
  • pharmaceutically acceptable salt is intended to mean both acid and base addition salts.
  • the term "pharmaceutically acceptable acid addition salt” is intended to mean those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as acetic acid,
  • salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2- diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.
  • basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine,
  • BIR domain binding is intended to mean the action of a compound of the present invention upon an IAP BIR domain, which blocks or diminishes the binding of IAPs to BIR binding proteins or is involved in displacing BIR binding proteins Attorney Docket No. L80003375WO from an IAP.
  • BIR binding proteins include, but are not limited to, caspases and mitochondrially derived BIR binding proteins such as Smac, 0mi/WTR2A and the like.
  • the term "insufficient apoptosis" is intended to mean a state wherein a disease is caused or continues because cells deleterious to the subject have not apoptosed. This includes, but is not limited to, cancer cells that survive in a subject without treatment, cancer cells that survive in a subject during or following anti-cancer treatment, or immune cells whose action is deleterious to the subject, and includes, neutrophils, monocytes and auto-reactive T-cells.
  • the term "therapeutically effective amount” is intended to mean an amount of a compound of Formula I which, when administered to a subject is sufficient to effect treatment for a disease-state associated with insufficient apoptosis.
  • the amount of the compound of Formula I will vary depending on the compound, the condition and its severity, and the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.
  • the term "treating" or "treatment” is intended to mean treatment of a disease-state associated with insufficient apoptosis, as disclosed herein, in a subject, and includes: (i) preventing a disease or condition associated with insufficient apoptosis from occurring in a subject, in particular, when such mammal is predisposed to the disease or condition but has not yet been diagnosed as having it; (ii) inhibiting a disease or condition associated with insufficient apoptosis, i.e., arresting its development; or (iii) relieving a disease or condition associated with insufficient apoptosis, i.e., causing regression of the condition.
  • treating cancer is intended to mean the administration of a pharmaceutical composition of the present invention to a subject, preferably a human, which is afflicted with cancer to cause an alleviation of the cancer by killing, inhibiting the growth, or inhibiting the metastasis of the cancer cells.
  • the term "preventing disease” is intended to mean, in the case of cancer, the post-surgical, post-chemotherapy or post-radiotherapy administration of a pharmaceutical composition of the present invention to a subject, preferably a human, which was afflicted with cancer to prevent the regrowth of the cancer by killing, inhibiting Attorney Docket No. L80003375WO the growth, or inhibiting the metastasis of any remaining cancer cells. Also included in this definition is the prevention of prosurvival conditions that lead to diseases such as asthma, MS and the like.
  • the term "synergistic effect" is intended to mean that the effect achieved with the combination of the compounds of the present invention and either the chemotherapeutic agents or death receptor agonists of the invention is greater than the effect which is obtained with only one of the compounds, agents or agonists, or advantageously the effect which is obtained with the combination of the above compounds, agents or agonists is greater than the addition of the effects obtained with each of the compounds, agents or agonists used separately. Such synergy enables smaller doses to be given.
  • apoptosis or "programmed cell death” is intended to mean the regulated process of cell death wherein a dying cell displays a set of well-characterized biochemical hallmarks that include cell membrane blebbing, cell soma shrinkage, chromatin condensation, and DNA laddering, as well as any caspase-mediated cell death.
  • BIR domain or “BIR” are used interchangeably throughout and are intended to mean a domain which is characterized by a number of invariant amino acid residue including conserved cysteines and one conserved hisitidine residue within the sequence Cys-(Xaa1) 2 Cys-(Xaa1)i 6 His-(Xaa1) 6-8 Cys.
  • amino acid sequence of the consensus sequence is: Xaa1-Xaa1-Xaa1-Arg-Leu-Xaa1-Thr-Phe-Xaa1-Xaa1-Trp - Pro-Xaa2-Xaa1-Xaa1-Xaa2-Xaa2-Xaa1-Xaa1-Xaa1-Leu-Ala-Xaa1-Ala-Gly-Phe-Tyr- Tyr-Xaa 1 -Gly-Xaa 1 -Xaa 1 -Asp-Xaa 1 -Val-Xaa 1 -Cys-Phe-Xaa 1 -Cys-Xaa 1 -Xaa 1 -Xaa 1 -Xaa 1 -Xaa1 -Xaa1 -Trp-Xaa1 -Xaa 1 -Xaa 1 -Asp-Xaa 1 -Xaa 1 -Xaa 1
  • ring zinc finger or "RZF” is intended to mean a domain having the amino acid sequence of the consensus sequence: Glu-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1- Xaa1- Xaa- 1 -Xaa2-Xaa 1 -Xaa 1 -Xaa 1 -Cys-Lys-Xaa3-Cys-Met-Xaa 1 -Xaa 1 -Xaa 1 -Xaa 1 -Xaa 1 -Xaa3-X- aa 1 -Phe-Xaa 1 -Pro-Cys-Gly-His-Xaa 1 -Xaa 1 -Xaa 1 -Cys-Xaa 1 -Xaa 1 -Cys-Ala- Xaa1-Xaa- 1-Xaai-Xaai-Xaai-Cys-Pro-Xaai-
  • IAP is intended to mean a polypeptide or protein, or fragment thereof, encoded by an IAP gene.
  • IAPs include, but are not limited to human or mouse NAIP (Birc 1), H1AP-1 (clAP2, Birc 3), HIAP-2 (clAP1 , Birc 2), XIAP (Birc 4), survivin (Birc 5), livin (ML-IAP, Birc 7), ILP-2 (Birc 8) and Apollon/BRUCE (Birc 6) (see for example US Patent Numbers 6,107,041 ; 6,133,437; 6,156,535; 6,541 ,457; 6,656,704;
  • IAP gene is intended to mean a gene encoding a polypeptide having at least one BIR domain and which is capable of modulating (inhibiting or enhancing) apoptosis in a cell or tissue.
  • the IAP gene is a gene having about 50% or greater nucleotide sequence identity to at least one of human or mouse NAIP (Birc 1), HIAP-1 (CIAP2, Birc 3), HIAP-2 (clAP1 , Birc 2), XIAP (Birc 4), survivin (Birc 5), livin (ML- IAP, Birc 7), ILP-2 (Birc 8) and Apollon/BRUCE (Birc 6).
  • the region of sequence over which identity is measured is a region encoding at least one BIR domain and a ring zinc finger domain.
  • Mammalian IAP genes include nucleotide sequences isolated from any mammalian source.
  • the term 11 IC 50 is intended to mean an amount, concentration or dosage of a particular compound of the present invention that achieves a 50% inhibition of a maximal response, such as displacement of maximal fluorescent probe binding in an assay that measures such response.
  • EC 50 is intended to mean an amount, concentration or dosage of a particular compound of the present invention that achieves a 50% inhibition of cell survival.
  • the term “modulate” or “modulating” is intended to mean the treatment, prevention, suppression, enhancement or induction of a function or condition using the compounds of the present invention.
  • the compounds of the present invention can modulate IAP function in a subject, thereby enhancing apoptosis by significantly reducing, or essentially eliminating the interaction of activated apoptotic proteins, such as caspase-3, 7 and 9, with the BIR domains of mammalian IAPs or by inducing the loss of XIAP protein in a cell.
  • enhancing apoptosis is intended to mean increasing the number of cells that apoptose in a given cell population either in vitro or in vivo.
  • cell populations include, but are not limited to, ovarian cancer cells, colon cancer cells, breast cancer cells, lung cancer cells, pancreatic cancer cells, or T cells and the like. It will be appreciated that the degree of apoptosis enhancement provided by an apoptosis- enhancing compound of the present invention in a given assay will vary, but that one skilled in the art can determine the statistically significant change in the level of apoptosis that identifies a compound that enhances apoptosis otherwise limited by an IAP.
  • enhancing apoptosis means that the increase in the number of cells undergoing apoptosis is at least 25%, more preferably the increase is 50%, and most preferably the increase is at least one-fold.
  • the sample monitored is a sample of cells that normally undergo insufficient apoptosis (i.e., cancer cells).
  • Methods for detecting the changes in the level of apoptosis are described in the Examples and include methods that quantitate the fragmentation of DNA, methods that quantitate the translocation phosphatoylserine from the cytoplasmic to the extracellular side of the membrane, determination of activation of the caspases and methods quantitate the release of cytochrome C and the apoptosis inhibitory factor into the cytoplasm by mitochondria.
  • proliferative disease or “proliferative disorder” is intended to mean a disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both.
  • cancers such as lymphoma, Attorney Docket No. L80003375WO leukemia, melanoma, ovarian cancer, breast cancer, pancreatic cancer, and lung cancer, and autoimmune disoders are all examples of proliferative diseases.
  • the term "death receptor agonist” is intended to mean an agent capable of stimulating by direct or indirect contact the pro apoptotic response mediated by the death- receptors.
  • an agonist TRAIL receptor Antibody would bind to TRAIL receptor (S) and trigger an apoptotic response.
  • other agent such as interferon-a could trigger the release of endogeneous TRAIL and/or up regulate the TRAIL receptors in such a way that the cell pro-apoptotic response is amplified.
  • the compounds of the present invention, or their pharmaceutically acceptable salts may contain one or more asymmetric centers, chiral axes and chiral planes and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms and may be defined in terms of absolute stereochemistry, such as (R)- or (S)- or, as (D)- or (L)- for amino acids.
  • the present invention is intended to include all such possible isomers, as well as, their racemic and optically pure forms.
  • Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC.
  • the racemic mixtures may be prepared and thereafter separated into individual optical isomers or these optical isomers may be prepared by chiral synthesis.
  • the enantiomers may be resolved by methods known to those skilled in the art, for example by formation of diastereoisomeric salts which may then be separated by crystallization, gas-liquid or liquid chromatography, selective reaction of one enantiomer with an enantiomer specific reagent. It will also be appreciated by those skilled in the art that where the desired enantiomer is converted into another chemical entity by a separation technique, an additional step is then required to form the desired enantiomeric form. Alternatively specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts, or solvents or by converting one enantiomer to another by asymmetric transformation.
  • Certain compounds of the present invention may exist in Zwitterionic form and the present invention includes Zwitterionic forms of these compounds and mixtures thereof.
  • the compounds of the present invention are useful as IAP BIR domain binding compounds and as such the compounds, compositions and method of the present invention include application to the cells or subjects afflicted with or having a predisposition towards developing a particular disease state, which is characterized by insufficient apoptosis.
  • the compounds, compositions and methods of the present invention are used to treat cellular proliferative diseases/disorders, which include, but are not limited to, i) cancer, ii) autoimmune disease, iii) inflammatory disorders, iv) proliferation induced post medical procedures, including, but not limited to, surgery, angioplasty, and the like.
  • the compounds of the present invention may also be useful in the treatment of diseases in which there is a defect in the programmed cell-death or the apoptotic machinery (TRAIL, FAS, apoptosome), such as multiple sclerosis, asthma, artherosclerosis, inflammation, autoimmunity and the like.
  • TRAIL programmed cell-death or the apoptotic machinery
  • FAS apoptosome
  • the treatment involves administration to a subject in need thereof a compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof.
  • the compounds, compositions and methods of the present invention are useful for the treatment of cancer including solid tumors such as skin, breast, brain, lung, testicular carcinomas, and the like. Cancers that may be treated by the compounds, compositions and methods of the invention include, but are not limited to the following:
  • the compounds of the present invention may be administered in pure form or in an appropriate pharmaceutical composition, and can be carried out via any of the accepted modes of Galenic pharmaceutical practice.
  • compositions of the present invention can be prepared by mixing a compound of the present invention with an appropriate pharmaceutically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral (subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), sublingual, ocular, rectal, vaginal, and intranasal.
  • compositions of the present invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a subject.
  • Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of the present invention in Attorney Docket No. L80003375WO aerosol form may hold a plurality of dosage units.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Company, Easton, Pa., 1990).
  • the composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease-state as described above.
  • a pharmaceutical composition of the present invention may be in the form of a solid or liquid.
  • the carriers are particulate, so that the compositions are, for example, in tablet or powder form.
  • the carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid or an aerosol, which is useful in, for example inhalatory administration.
  • the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
  • the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form.
  • a solid composition will typically contain one or more inert diluents or edible carriers.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
  • excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like
  • lubricants such as magnesium stearate or Sterotex
  • glidants such as colloidal silicon dioxide
  • sweetening agents such as sucrose or saccharin
  • a flavoring agent such as peppermint, methyl sal
  • the pharmaceutical composition when in the form of a capsule, e.g., a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil such as soybean or vegetable oil.
  • a liquid carrier such as polyethylene glycol or oil such as soybean or vegetable oil.
  • the pharmaceutical composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension.
  • the liquid may be for oral administration or for delivery by injection, as two examples.
  • preferred Attorney Docket No. L80003375WO composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • liquid pharmaceutical compositions of the present invention may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; encapsulating agents such as cyclodextrins, including, but not limited to, ⁇ , ⁇ , or ⁇ -hydroxypropylcyclodextins or Captisol; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine tetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or de
  • a liquid pharmaceutical composition of the present invention used for either parenteral or oral administration should contain an amount of a compound of the present invention such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of a compound of the present invention in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. For parenteral usage, compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of the compound of the present invention.
  • compositions may be further diluted at the time of administration; for example a parenteral formulation may be further diluted with a sterile, isotonic solution for injection such as 0.9 % saline, 5 wt % dextrose (D5W), Ringer ' s solution, or others.
  • a sterile, isotonic solution for injection such as 0.9 % saline, 5 wt % dextrose (D5W), Ringer ' s solution, or others.
  • the pharmaceutical composition of the present invention may be used for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and Attorney Docket No. L80003375WO alcohol, and emulsifiers and stabilizers.
  • Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.
  • Topical formulations may contain a concentration of the compound of the present invention from about 0.1 to about 10% w/v (weight per unit volume).
  • the pharmaceutical composition of the present invention may be used for rectal administration to treat for example, colon cancer, in the form, e.g., of a suppository, which will melt in the rectum and release the drug.
  • the composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient.
  • bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
  • the pharmaceutical composition of the present invention may include various materials, which modify the physical form of a solid or liquid dosage unit.
  • the composition may include materials that form a coating shell around the active ingredients.
  • the materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
  • the active ingredients may be encased in a gelatin capsule.
  • the pharmaceutical composition of the present invention in solid or liquid form may include an agent that binds to the compound of the present invention and thereby assists in the delivery of the compound.
  • Suitable agents that may act in this capacity include, but are not limited to, a monoclonal or polyclonal antibody, a protein or a liposome.
  • the pharmaceutical composition of the present invention may consist of dosage units that can be administered as an aerosol.
  • aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of compounds of the present invention may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One skilled in the art, without undue experimentation may determine preferred aerosols.
  • compositions of the present invention may be prepared by Attorney Docket No. L80003375WO methodology well known in the pharmaceutical art.
  • a pharmaceutical composition intended to be administered by injection can be prepared by admixing a compound of the present invention with sterile, distilled water so as to form a solution.
  • a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
  • Surfactants are compounds that non-covalently interact with the compound of the present invention so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.
  • the compounds of the present invention, or their pharmaceutically acceptable salts are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
  • a therapeutically effective daily dose may be from about 0.1 mg to about 40 mg/kg of body weight per day or twice per day of a compound of the present invention, or a pharmaceutically acceptable salt thereof.
  • Combination therapy may also be administered simultaneously with, prior to, or after administration of one or more of the therapeutic agents described below.
  • Such combination therapy may include administration of a single pharmaceutical dosage formulation which contains a compound of the present invention and one or more additional agents given below, as well as administration of the compound of the present invention and each of additional agent in its own separate pharmaceutical dosage formulation.
  • a compound of the present invention and a chemotherapeutic agent such as taxol (paclitaxel), taxotere, etoposide, cisplatin, vincristine, vinblastine, and the like
  • a chemotherapeutic agent such as taxol (paclitaxel), taxotere, etoposide, cisplatin, vincristine, vinblastine, and the like
  • a single oral dosage composition such as a tablet or capsule
  • each agent administered in separate oral dosage formulations or via intravenous injection can be administered to the patient either together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations or via intravenous injection.
  • the compounds of the present invention and one or more additional agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially; combination therapy is understood to include all these regimens.
  • these compounds may synergize with molecules that may stimulate the death receptor apoptotic pathway through a direct Attorney Docket No. L80003375WO or indirect manner, as for example, the compounds of the present invention may be used in combination with soluble TRAIL any agent or procedures that can cause an increase in circulating level of TRAIL, such as interferon-alpha or radiation.
  • the present invention also encompasses the use of the compounds of the present invention in combination with radiation therapy or one or more additional agents such as those described in WO 03/099211 (PCT/US03/15861), which is hereby incorporated by reference.
  • additional agents include, but are not limited to the following: a) an estrogen receptor modulator, b) an androgen receptor modulator, c) retinoid receptor modulator, d) a cytotoxic agent, e) an antiproliferative agent, f) a prenyl-protein transferase inhibitor, g) an HMG-CoA reductase inhibitor, h) an HIV protease inhibitor, i) a reverse transcriptase inhibitor, k) an angiogenesis inhibitor, I) a PPAR-. ⁇ agonist, m) a PPAR-. ⁇ .
  • a proteasome inhibitor such as Velcade and MG132 (7-Leu-Leu-aldehyde) (see He at al. in Oncogene (2004) 23, 2554-2558); t) an HDAC inhibitor, such as sodium butyrate, phenyl butyrate, hydroamic acids, cyclin tetrapeptide and the like (see Rosato et al,. Molecular Cancer Therapeutics 2003, 1273-
  • a modulator of the immune system such as interferon-alpha and ionizing radition (UVB) that can induce the release of cytokines, such as the interleukins, TNF, or induce release of Death receptor Ligands such as TRAIL; x) a modulator of death receptors TRAIL and TRAIL agonists such as the humanized antibodies HGS-ETR1 and HGS-ETR2; and or in combination or sequentially with radiation therapy, so as to treat the cancer.
  • UVB interferon-alpha and ionizing radition
  • Additional combinations may also include agents which reduce the toxicity of the aforesaid agents, such as hepatic toxicity, neuronal toxicity, nephprotoxicity and the like.
  • co-administration of one of the compounds of Formula I of the present invention with a death receptor agonist such as TRAIL, such as a small molecule or an antibody that mimics TRAIL may cause an advantageous synergistic effect.
  • TRAIL a death receptor agonist
  • the compounds of the present invention may be used in combination with any compounds that cause an increase in circulating levels of TRAIL.
  • Vinca alkaloids that can be used in combination with the nucleobase oligomers of the invention to treat cancer and other neoplasms include vincristine, vinblastine, vindesine, vinflunine, vinorelbine, and anhydrovinblastine.
  • Dolastatins are oligopeptides that primarily interfere with tubulin at the vinca alkaloid binding domain. These compounds can also be used in combination with the compounds of the invention to treat cancer and other neoplasms. Dolastatins include dolastatin-10 (NCS 376128), dolastatin-15, ILX651 , TZT-1027, symplostatin 1 , symplostatin 3, and LU 103793 (cemadotin).
  • Cryptophycins bind tubulin within the vinca alkaloid-binding domain and induce G2/M arrest and apoptosis. Any of these compounds can be used in combination with the compounds of the invention to treat cancer and other neoplasms.
  • microtubule disrupting compounds that can be used in conjunction with the compounds of the invention to treat cancer and other neoplasms are described in U.S. Pat. Nos. 6,458,765; 6,433,187; 6,323,315; 6,258,841 ; 6,143,721 ; 6,127,377; 6,103,698; Attorney Docket No. L80003375WO 6,023,626; 5,985,837; 5,965,537; 5,955,423; 5,952,298; 5,939,527; 5,886,025; 5,831 ,002;
  • Taxanes such as paclitaxel, doxetaxel, RPR 109881 A, SB-T-1213, SB-T-1250, SB-T-
  • Taxane analogs e.g., BMS-184476, BMS-188797
  • functionally related non-taxanes e.g., epothilones (e.g., epothilone A, epothilone B (EPO906), deoxyepothilone B, and epothilone B lactam (BMS-247550)
  • eleutherobin discodermolide, 2-epi-discodermolide, 2-des-methyldiscodermolide, 5-hydroxymethyldiscoder- molide, 19-des- aminocarbonyldiscodermolide, 9(13)-cyclodiscodermolide, and laulimalide
  • microtubule stabilizing compounds that can be used in combination with the compounds of the invention to treat cancer and other neoplasms are described in U.S. Pat. Nos. 6,624,317; 6,610,736; 6,605,599; 6,589,968; 6,583,290; 6,576,658; 6,515,017;
  • chemotherapeutic agents that may be administered with a compound of the present invention are listed in the following Table: Attorney Docket No. L80003375WO
  • SR-27897 (CCK A inhibitor, Sanofi- gemtuzumab (CD33 antibody, Wyeth Ayerst)
  • BCX-1777 PNP inhibitor, BioCryst
  • PG2 hematopoiesis enhancer, Pharmagenesis
  • tocladesine cyclic AMP agonist, Ribapharm
  • exisulind PDE V inhibitor, Cell Pathways
  • ranpirnase ribonuclease stimulant, Alfacell
  • ImmunolTM triclosan oral rinse, Endo
  • alvocidib CDK inhibitor, Aventis
  • CP-461 PDE V inhibitor, Cell Pathways
  • galarubicin RNA synthesis inhibitor, Dong- A) triacetyluridine (uridine prodrug, Wellstat)
  • CapCellTM CYP450 stimulant, Bavarian TransMID-107 .TM. (immunotoxin, KS
  • GCS-100 gal3 antagonist, GlycoGenesys LifeSciences
  • PCK-3145 apoptosis promotor, Procyon
  • G 17DT immunogen (gastrin inhibitor, Aphton) bortezomib (proteasome inhibitor, Millennium) seocalcitol (vitamin D receptor agonist, Leo) doranidazole (apoptosis promotor, PoIa) efaproxiral (oxygenator, Allos Therapeutics) SRL- 172 (T cell stimulant, SR Pharma) CHS-
  • TLK-286 glutthione S transferase inhibitor
  • PI-88 heparanase inhibitor, Progen
  • ODC inhibitor eflomithine
  • NIH eflomithine
  • NIH eflomithine
  • NIH eflomithine
  • NIH eflomithine
  • NIH eflomithine
  • NIH eflomithine
  • NIH eflomithine
  • NIH eflomithine
  • NIH tesmilifene
  • YM PT-100 growth factor agonist
  • Yamanouchi midostaurin (PKC inhibitor, Novartis) histamine (histamine H2 receptor agonist, apomine (apoptosis promotor, ILEX Oncology)
  • SR-31747 (IL-I antagonist, Sanofi-Synthelabo) ceflatonin (apoptosis promotor, ChemGenex)
  • Additional combinations may also include agents which reduce the toxicity of the aforesaid agents, such as hepatic toxicity, neuronal toxicity, nephprotoxicity and the like.
  • the compounds of the present invention may also be used in a method to screen for other compounds that bind to an IAP BIR domain.
  • the IAP is bound to a support, and a compound of the invention is added to the assay.
  • the compound of the invention may be bound to the support and the IAP is added.
  • the compound of the invention may be fluorescently or radioactively labeled and binding determined directly. For example, this may be done by attaching the IAP to a solid support, adding a detectably labeled compound of the invention, washing off excess reagent, and determining whether the amount of the detectable label is that present on the solid support. Numerous blocking and washing steps may be used, which are known to those skilled in the art.
  • the compounds of the invention may also be used as competitors to screen for additional drug candidates or test compounds.
  • drug candidate or “test compounds” are used interchangeably and describe any molecule, for example, protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, and the like, to be tested for bioactivity.
  • the compounds may be capable of directly or indirectly altering the IAP biological activity.
  • Drug candidates can include various chemical classes, although typically they are small organic molecules having a molecular weight of more than 100 and less than about 2,500 Daltons.
  • Candidate agents typically include functional groups necessary for structural interaction with proteins, for example, hydrogen bonding and lipophilic binding, and typically include at least an amine, carbonyl, hydroxyl, ether, or carboxyl group.
  • the drug candidates often include cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more functional groups.
  • Drug candidates can be obtained from any number of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or Attorney Docket No. L80003375WO readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means.
  • ком ⁇ онентs may be done by combining an IAP BIR domain and a probe to form a probe:BIR domain complex in a first sample followed by adding a test compound from a second sample. The binding of the test is determined, and a change or difference in binding between the two samples indicates the presence of a test compound capable of binding to the BIR domain and potentially modulating the lAP's activity.
  • the binding of the test compound is determined through the use of competitive binding assays.
  • the probe is labeled with a fluorescent label. Under certain circumstances, there may be competitive binding between the test compound and the probe. Test compounds which display the probe, resulting in a change in fluorescence as compared to control, are considered to bind to the BIR region.
  • test compound may be labeled. Either the test compound, or a compound of the present invention, or both, is added first to the IAP BIR domain for a time sufficient to allow binding to form a complex.
  • Formation of the probe:BIR domain complex typically require Incubations of between 4 0 C and 40 0 C for between 10 minutes to about 1 hour to allow for high-throughput screening. Any excess of reagents are generally removed or washed away. The test compound is then added, and the presence or absence of the labeled component is followed, to indicate binding to the BIR domain.
  • the probe is added first, followed by the test compound.
  • Displacement of the probe is an indication the test compound is binding to the BIR domain and thus is capable of binding to, and potentially modulating, the activity of IAP.
  • Either component can be labeled.
  • the presence of probe in the wash solution indicates displacement by the test compound.
  • the presence of the probe on the support indicates displacement.
  • the test compound may be added first, with incubation and washing, followed by the probe.
  • the absence of binding by the probe may indicate the test compound is bound to the BIR domain with a higher affinity.
  • the probe is detected on the Attorney Docket No. L80003375WO support, coupled with a lack of test compound binding, may indicate the test compound is capable of binding to the BIR domain.
  • Modulation is tested by screening for a test compound's ability to modulate the activity of IAP and includes combining a test compound with an IAP BIR domain, as described above, and determining an alteration in the biological activity of the IAP. Therefore in this case, the test compound should both bind to the BIR domain (although this may not be necessary), and alter its biological activity as defined herein.
  • Positive controls and negative controls may be used in the assays. All control and test samples are performed multiple times to obtain statistically significant results. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound probe determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.
  • the signals that are detected in the assay may include fluorescence, resonance energy transfer, time resolved fluorescence, radioactivity, fluorescence polarization, plasma resonance, or chemiluminescence and the like, depending on the nature of the label.
  • Detectable labels useful in performing screening assays in this invention include a fluorescent label such as Fluorescein, Oregon green, dansyl, rhodamine, tetramethyl rhodamine, texas red, Eu 3+ ; a chemiluminescent label such as luciferase; colorimetric labels; enzymatic markers; or radioisotopes such as tritium, I 125 and the like
  • Affinity tags which may be useful in performing the screening assays of the present invention include be biotin, polyhistidine and the like.
  • R 1 , R 3 , R 4 , R 5 , and R 7 are as defined herein, PG 1 and PG 2 are protecting groups, Y' is NH, O, S or CH 2 , and R a and R B may be combined to form an aromatic ring system wherein double bond may represent a single or double bond
  • Scheme 2 An example of these procedures for the synthesis of compound 1 is illustrated in Scheme 2.
  • Boc-(3S)-3-amino-1-carboxymethylcaprolactam, intermediate 1-1 was coupled to (R) -1 ,2,3,4-tetrahydronaphthyl-1 -amine using the amide coupling reagents HBTU and HOBt, to provide intermediate 1-2a.
  • Boc deprotection was carried out using 4N HCI in 1 ,4-dioxane to provide intermediate 1-2b as its HCI salt.
  • Intermediate 1-3 was prepared by the coupling of Boc-N-MeAla-OH to intermediate 1-2b ⁇ CI using the coupling agents HBTU and HOBt. Boc deprotection of intermediate 1-3 using 4N HCI in 1 ,4-dioxane provided compound 1 as its HCI salt.
  • Intermediate 3-3 was prepared by the coupling of Boc-MeAla-OH to intermediate 3-2b ⁇ CI using the coupling agents HBTU and HOBt. Boc deprotection of intermediate 3-3 using 4N HCI in 1 ,4-dioxane provided compound 3 as its HCI salt.
  • Boc f-butoxycarbonyl
  • DIPEA diisopropylethylamine
  • EDC 3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; Attorney Docket No. L80003375WC
  • EDTA ethylenediaminetetracetic acid
  • HBTU O-(benzotriazol-1-yl)-N, ⁇ /, ⁇ /', ⁇ /'-tetramethyluronium hexafluorophosphate
  • HCI hydrochloric acid
  • HOAc acetic acid
  • HOBt 1 -hydroxybenzotriazole
  • MgSO 4 magnesium sulfate
  • NaHCO 3 sodium hydrogen carbonate
  • Pd/C palladium on carbon
  • TEA triethylamine
  • Step i
  • Boc-(3S)-3-amino-1-carboxymethylcaprolactam (286 mg, 1.0 mmol) was dissolved in DMF (5 mL) and treated with HBTU (564 mg, 1.5 mmol), HOBt (202 mg, 1.5 mmol) and DIPEA (520 ⁇ L, 3.0 mmol). The reaction was stirred for 5 minutes before (R)-1 , 2,3,4- tetrahydronaphthyl-1 -amine (176 ⁇ L, 1.2 mmol) was added. The reaction was stirred for 16 hrs at room temperature before being partitioned between ethyl acetate (100 mL) and 10% citric acid (20 mL).
  • the organic phase was washed with 10 % citric acid (2 x 20 mL), saturated aqueous NaHCO 3 (3 x 20 mL), and brine (1 x 20 mL).
  • the organic fraction was dried over anhydrous MgSO 4 , filtered, and the volatiles removed under reduced pressure to provide 1-2a as a white solid, which was advanced to the next step without further purification.
  • Crude compound 1-2a was treated with 50 % TFA/CH 2 CI 2 for 3 hrs. Volatiles were removed under reduced pressure and the residue suspended in diethyl ether (5 mL). An oil was formed and so volatiles were removed and the residue treated with 4N HCI in 1 ,4- dioxane (3 mL) for 30 minutes. Volatiles were removed under reduced pressure to Attorney Docket No. L80003375WO provide a white solid which was triturated with diethyl ether (3 x 10 mL) to provide 1-
  • Step 3 Boc-Me-Ala-OH (253 mg, 1.25 mmol) was dissolved in DMF (5 mL) and treated with HBTU (564 mg, 1.5 mmol), HOBt (202 mg, 1.5 mmol) and DIPEA (700 ⁇ L, 4.0 mmol). The reaction was stirred for 5 minutes before crude 1-2b-HCI was added. The reaction was stirred for 16 hrs at room temperature before being partitioned between ethyl acetate (100 mL) and 10% citric acid (20 mL). The organic phase was washed with 10 % citric acid (2 x 20 mL), saturated aqueous NaHCO 3 (3 x 20 mL), and brine (1 x 20 mL).
  • Step i
  • Boc-Me-Ala-OH (63 mg, 0.313 mmol) was dissolved in DMF (5 mL) and treated with
  • HBTU 140 mg, 0.375 mmol
  • HOBt 50 mg, 0.375 mmol
  • DIPEA 96 ⁇ l_, 1.0 mmol
  • the reaction was stirred for 5 minutes before 3-2b HCI (104 mg, 0.25 mmol) was added.
  • the reaction was stirred for 16 hrs at room temperature before being partitioned between ethyl acetate (100 mL) and 10% citric acid (20 mL).
  • the organic phase was washed with 10 % citric acid (2 x 20 mL), saturated aqueous NaHCO 3 (3 x 20 mL), and brine (1 x 20 mL).
  • the organic fraction was dried over anhydrous MgSO 4 , filtered, and the volatiles removed under reduced pressure.
  • R 1 , R 2 and R 11 are define as herein;
  • R 7 H or C 1-6 alkyl optionally substituted with aryl or heteroaryl;
  • Q is NR 4 R 5 ;
  • R 4 is H ;
  • R 5 is chosen from:
  • aryl and heteroaryl moieties may be substituted by R 11 , and wherein R 11 and R 11 are independently defined as R 11 hereinabove, and wherein the alkyl may be further substituted by R 6 as defined hereinabove.
  • the compounds of the present invention reduce, or eliminate, the interaction of apoptotic proteins, such as caspase 3, caspase 7, and caspase 9, with the BIR domains of mammalian IAPs, which promote apoptosis of cancer cells as single agents or in combination with a chemotherapeutic agent. Also, the compounds may modulate IAP proteins in cells and induce the proteolytic processing of IAPs.
  • apoptotic proteins such as caspase 3, caspase 7, and caspase 9
  • the compounds may modulate IAP proteins in cells and induce the proteolytic processing of IAPs.
  • GST-XIAP BIR3RING XIAP coding sequence amino acids 246-497 cloned into PGEX2T1 via BamH1 and AVA I.
  • the plasmid was transformed into E. coli DH5 ⁇ for use in protein expression and purification.
  • BIR 3 HIAP2 coding sequence from amino acids 251-363 cloned into PGex4T3 via BamH1 and Xhol. The plasmid was transformed into E. coli DH5 ⁇ for use in protein expression and purification.
  • BIR 3 HIAP1 coding sequence from amino acids 236-349, cloned into PGex4T3 via BamH1 and Xhol. The plasmid was transformed into E. coli DH5 ⁇ for use in protein expression and purification.
  • GST- linker BIR 2 BIR3Ring XIAP coding sequence from amino acids 93-497 cloned into PGex4T1 via BamH1 and Xhol. Amino acids 93-497 were amplified from full length XIAP Attorney Docket No. L80003375WO in pGex4t3, using the primers: TTAATAGGATCCATCAACGGCTTTTATC and
  • GST-XIAP linker BIR 2 XIAP linker BIR 2 coding sequence from amino acids 93-497 cloned into pGex4T3 via BamHI and Xhol. The plasmid was transformed into E. coli DH5 ⁇ for use in protein expression and purification.
  • a fluorescent peptide probe Fmoc-Ala-Val-Pro-Phe-Tyr(t-Bu)-Leu-Pro-Gly(t-Bu)-Gly-OH was prepared using standard Fmoc chemistry on 2-chlorotrityl chloride resin (Int. J. Pept. Prot. Res. 38:555-561 , 1991). Cleavage from the resin was performed using 20% acetic acid in dichloromehane (DCM), which left the side chain still blocked.
  • DCM dichloromehane
  • the C-terminal protected carboxylic acid was coupled to 4'-(aminomethy)fluorescein (Molecular Probes, A-1351 ; Eugene, Oreg.) using excess diisopropylcarbodiimide (DIC) in dimethylformamide (DMF) at room temperature and was purified by silica gel chromatography (10% methanol in DCM).
  • the N-terminal Fmoc protecting group was removed using piperidine (20%) in DMF, and purified by silica gel chromatography (20% methanol in DCM, 0.5% HOAc).
  • the t-butyl side chain protective groups were removed using 95% trifluoroacetic acid containing 2.5% water and 2.5% triisopropyl silane to provide probe P1 (>95% pure).
  • Glutathione S-transferase (GST) tagged proteins were expressed in Escherichia coli strains DH5-alpha.
  • GST Glutathione S-transferase
  • LB Luria Broth
  • Each pellet obtained from a 1 liter culture received 10 mL of lysis buffer (50 mM Tris-HCI, 200 mM NaCI, 1 mM DTT, 1 mM PMSF, 2 mg/mL of lysosyme), was incubated at 4 0 C with Attorney Docket No. L80003375WO gentle shaking. After 20 minutes of incubation, the cell suspension was placed at -80 0 C overnight or until needed.
  • lysis buffer 50 mM Tris-HCI, 200 mM NaCI, 1 mM DTT, 1 mM PMSF, 2 mg/mL of lysosyme
  • His-tag proteins were expressed in the E. CoIi strain in E. coli AD494 cells using a pet28ACPP32 construct.
  • the soluble protein fraction was prepared as described above.
  • the supernatant was purified by affinity chromatography using chelating-Sepharose (Pharmacia) charged with NiSO 4 according to the manufacturer's instructions. Purity of the eluted protein was >90% pure as determined by SDS-PAGE. The protein concentration of purified proteins was determined from the Bradford assay.
  • Tecan Polarion instrument with the excitation filter set at 485 nm and the emission filter set at 535 nm.
  • concentration of the target protein was first established by titration of the selected protein in order to produce a linear dose-response signal when incubated alone in the presence of the fluorescent probe.
  • the compound's potency (IC 50 ) and selectivity was assessed in the presence of a fix defined- amount of target protein and fluorescent probe and a 10 point serial dilution of the selected compounds.
  • IC 50 the assays were run as followed: Attorney Docket No.
  • an in vitro assay is used where caspase-3 is inhibited by linker- BIR2 or linker BIR2-BIR3-RING of XIAP. Briefly, 1.5 uL of the enzyme, 0.25 uM-2 uM of GST-XIAP fusion protein (GST-linker BIR2, GST-linker BIR2BIR3RING) is co-incubated with serial dilutions of compound (8OuM-0.04uM). Caspase 3 activity is measured by overlaying 25 uL of a 0.4 nM DEVD- AMC solution. Final reaction volume is 100 uL.
  • caspase buffer 50 mM Hepes pH 7.4, 10OmM NaCI, 10% sucrose, 1mM EDTA, 10 mM DTT, 0.1% CHAPS (Stennicke, H.R., and Salvesen, G. S. (1997). Biochemical characteristics of caspase-3, -6, -7, and -8. J. Biol. Chem. 272, 25719-25723)).
  • the fluorescent AMC released from the caspase-3 hydrolysis of the substrate is measured in a TECAN spectrophotometer at 360 nm excitation and 444 nm emission, on a kinetic cycle of 30 minutes with readings taken every 2 minutes.
  • Caspase activity is calculated as V 0 of AMC fluorescence/sec. Caspase de-repression by our compounds are compared to caspase-3 alone and caspase 3 repressed by the presence of XIAP fusion protein.
  • Pull-down assay Attorney Docket No. L80003375WO Compounds are dissolved in DMSO at 20 mM and used as the stock solution. Prior to the pull-down assay the compound affinity-agarose beads are prepared as follows: 300 uL of avidine-agarose beads prepared as 50% slurry in buffer A are incubated with 4OuI compound 107 (2.5 mM final) for 6h at 4 0 C with shaking. Upon incubation the beads ware washed 3 times with buffer A.
  • cells such as MDA-MB-231 or 293A are collected and lyzed with a buffer A containing 20 mM TrisHCI, 150 mM NaCI, 10% Glycerol 1 % NP-40 and Protease inhibitor cocktail obtained from Sigma with a final protein concentration between 3 to 30 ug/uL.
  • the pull-down assay 60 uL of the compound-beads preparation are incubated with 300 ug cells lysate and 1OuL compound (0.36 mM final) or as control with 10 uL of buffer A to provide a final volume of 550 uL. The mixture was incubated at 4 0 C over night with shaking. The following day, the beads are washed three times with the buffer A and the final bead pellet is re-suspended in 50 uL of 2X Leamli buffer. The samples are then centrifuged and the supernatant is collected for western blot analysis directed against XIAP of the other lAP's.
  • a direct IAP binding molecule is used to identify IAP binding compounds by contacting the test compound with an IAP protein which is either fixed or non-fixed on a solid support.
  • the non-binding interacting molecules are washed away and the bound compounds are identified using analytical techniques that can either identify directly the compound such as mass spectrometry, surface plasma resonance or can a evaluate a change of conformation or structure in the target molecule induced by the test compound or using techniques that can monitor the molecular interaction between the test compound and the target molecule such as NMR and protein crystallography.
  • Cytochrome C (final concentrations), and incubating at 37 0 C for 25 minutes. All reactions and dilutions used S100 buffer (50 mM Pipes pH 7.0, 5OmM KCI, 0.5mM EGTA pH 8.0, 2 mM MgCI 2 supplemented with 1/1000 dilutions of 2 mg/mL Cytochalisin B, 2 mg/mL Chymostatin, Leupeptin, Pepstatin, Antipapin, 0.1 M PMSF, 1 M DTT). Final reaction volume is 3OuI. Caspase-3 activity is measured by overlaying 30 uL of a 0.4nM DEVD- AMC solution.
  • AMC cleavage is measured in a TECAN spectrophotometer at 360 nm excitation and 444 nm emission, on a kinetic cycle of 1 hour with readings taken every 5 minutes.
  • Caspase activity is calculated as V 0 of AMC fluorescence/sec. Caspase de- repression by compounds are compared to fully activated extract and activated extract repressed by the presence of XIAP fusion protein.
  • MDA-MB-231 and H460 cells are routinely done on MDA-MB-231 and H460 cells.
  • Cells are seeded in 96 well plates at a respective density of 5000 and 2000 cells per well and incubated at 37 0 C in presence of 5% CO 2 for 24 hours.
  • Selected compounds are diluted into the media at various concentration ranging from 0.01 uM up to 100 uM. Diluted compounds are added onto the MDA-MB-231 cells.
  • H460 cells the compounds are added either alone or in presence of 3 ng/ml of TRAIL. After 72 hours cellular viability is evaluated by MTS based assays.
  • a solution of [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] is added onto cells for a period of 1 to 4 hours. Upon incubation the amount of converted MTS is evaluated using a Tecan spectrophotometer set at 570 nm.
  • Detection of cell expressed XIAP and PARP are done by western blotting.
  • Cells are plated at 300,000 cells/well in a 60 mm wells (6 wells plate dish). The next day the cells are treated with selected compound at the indicated concentration. 24 hours later cells the trypsinized cells, pelleted by centrifugation at 1800 rpm at 4 0 C. The resulting pellet is Attorney Docket No. L80003375WO rinsed twice with cold TBS. The final washed pellet of cells is the lysed with 250 uL Lysis buffer (NP-40, glycerol, 1% of a protease inhibitor cocktail (Sigma)), placed at 4 0 C for 25 min with gentle shaking.
  • NP-40 uL Lysis buffer
  • glycerol glycerol
  • a protease inhibitor cocktail Sigma
  • the cells extract is centrifuged at 4 0 C for 10min at 10,000 rpm. Both the supernatant and the pellet are kept for western blotting analysis as described below. From the supernatant, the protein content is evaluated and about 50ug of protein is fractionated onto a 10% SDS-PAGE. Pellets are washed with the lysis buffer and re- suspend into 5OuI of Lamelli buffer 1X, boiled and fractionated on SDS-PAGE. Upon electrophoresis each gel is electro-transferred onto a nitrocellulose membrane at 0.6A for 2 hours. Membrane non-specific sites are blocked for 1 hours with 5% Skim milk in TBST (TBS containing 0.1% (v/v) Tween-20) at RT.
  • membranes are incubated overnight with primary antibodies raised against XIAP clone 48 obtained from Becton-Dickison) or PARP: obtained from Cell signal or caspase-3 or caspase-9 primary antibodies are incubated at 4 0 C with shaking at dilutions as follows:
  • the membranes Upon overnight incubation, the membranes receive three washes of 15 min in TBST then are incubated for 1 hour at room temperature in the presence of a secondary antibody coupled with HRP-enzyme (Chemicon) and diluted at 1/5 000. Upon incubation each membrane are washed three times with TBST and the immunoreactive bands are detected by addition of a luminescent substrate (ECL kit Amersham) and capture of signal on a X-RAY film for various time of exposure. Active compounds are shown to induce the cleavage of PARP and XIAP as well as to translocate XIAP into an insoluble compartment.
  • a hollow fiber in vivo model is used to demonstrate in vivo efficacy of selected compounds against selected cell lines as single agent therapy or in combination with selected cytotoxic agents.
  • selected cell lines are cultured and the fiber filled at a cell density of about 40,000 cells/fiber.
  • three fibers are implanted sub-cutaneous into 28-35 Nu/Nu CD-1 male mice.
  • mice start to receive daily injection via sub-cutaneous route of control vehicle or vehicle containing the selected compound at the appropriate concentration and/or injection of cytotoxic agent via Attorney Docket No. L80003375WO intra-peritoneal route.
  • Efficacy of the compound is defined as the difference between the vehicle-treated animal and the animal treated with the compound alone or the compound given in combination of the cytotoxic agent
  • mice Female CD-1 nude mice (approximately 20-25 g) are subcutaneously injected with 1x10 6 H460 cells in the right flank. Animals are balanced into groups based on tumor size and drug therapy begins when tumors are ⁇ 30-50 mm 3 . Animals that have no tumor or that are deemed outliers because of excessive tumor size at this time are removed from the study. The remaining animals receive Taxotere (or equivalent volume of vehicle) at 30 mg/kg, ip 2 times, one week apart. The compound is given two times per day (at 10 mg/kg, sc, approximately 6hrs apart), starting at the time of Taxotere, and continuing daily for the duration of the experiment. If dehydration occurred, animals receive sc fluids (0.5ml). Tumor size is measured three times per week. Health assessments are performed at the time of the compound's delivery.
  • Selected compounds are dissolved into normal saline (0.9% NaCI) at 10 mg/ml and injected at 40 mg/Kg under sub-cutaneous route of administration.
  • blood sample of three mice is taken and the plasma fraction is prepared and kept frozen until analysis by liquid chromatography/electrospray mass spectrometry (LC/MS).
  • LC/MS liquid chromatography/electrospray mass spectrometry
  • plasma samples are thawed and extracted by liquid-liquid extraction procedure using 75% acetonitrile-water solution. Each extracted sample is analyzed for the presence of the selected compound on an Agilent 1100 LC/MS equipped with a C18 reverse-phase column. Quantitation of the compound in the plasma is done relatively to a plasma standard curve using of the selected compound.
  • the calculated area under the curve (AUC) and the peak at maximum concentration (Cmax) were calculated using Kinetica Version 4.2 software (Innaphase).
  • the compounds exemplified in Table 1 were tested and found to have IC 5 os of ⁇ 50 ⁇ M using the fluorescence polarization assay. Each of the IC 50 S was calculated from Graph Pad.

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Abstract

Disclosed herein are compounds as resented by Formula (I): wherein R1,R2, R3, R4, n, W, A, and Q are as defined herein. The compounds of formula (I) are useful as antagonists of the IAPs via binding to BIR domain. The compounds are useful for application to the cells or subjects afflicted with or having a predisposition towards developing a particular disease state, which is characterized by insufficient apoptosis. Thus, the compounds and compositions thereof are used to treat cellular proliferative diseases/disorders, which include, cancer, autoimmune diseases, inflammatory disorders, proliferation induced post medical procedures, etc. The compounds may also be useful in the treatment of diseases in which there is a defect in the programmed cell-death or the apoptotic machinery, such as multiple sclerosis, artherosclerosis, inflammation, autoimmunity, etc.

Description

Attorney Docket No. L80003375WO
BIR DOMAIN BINDING COMPOUNDS
FIELD OF THE INVENTION The present invention concerns constrained cyclic compounds, which are useful for treating proliferative disorders and disorders of dysregulated apoptosis, such as cancer.
BACKGROUND OF THE INVENTION
Apoptosis, or programmed cell death, typically occurs in the normal development and maintenance of healthy tissues in multicellular organisms. It is a complex process which results in the removal of damaged, diseased or developmentally redundant cells, in the absence of signs of inflammation or necrosis.
Intrinsic apoptotic pathways are known to be dysregulated, most particularly in cancer and lymphoproliferative syndromes, as well as autoimmune disorders such as multiple sclerosis, in neurodegenerative diseases and in inflammation. As well, alterations in a host apoptotic response have been described in the development or maintenance of viral and bacterial infections.
The caspases are a family of proteolytic enzymes from the class of cysteine proteases which are known to initiate and execute apoptosis. In normal cells, the caspases are present as inactive zymogens, which are catalytically activated following external signals, for example those resulting from ligand driven Death Receptor activation, such as cytokines or immunological agents, or by release of mitochondrial factors, such as cytochrome C following genotoxic, chemotoxic, or radiation-induced cellular injury. The Inhibitors of Apoptosis Proteins (IAPs) constitute a family of proteins which are capable of binding to and inhibiting the caspases, thereby suppressing cellular apoptosis. Because of their central role in regulating Caspase activity, the IAPs are capable of inhibiting programmed cell death from a wide variety of triggers, which include loss of homeostatic, or endogenous cellular growth control mechanisms, as well as chemotherapeutic drugs and irradiation.
The IAPs contain one to three homologous structural domains known as baculovirus IAP repeat (BIR) domains. They may also contain a RING zinc finger domain at the C- Attorney Docket No. L80003375WO terminus, with a capability of inducing ubiquitinylation of IAP-binding molecules via its E3 ligase function. The human IAPs, XIAP, HIAP1 (also referred to as clAP2), and HIAP2 (clAP1 ) each have three BIR domains, and a carboxy terminal RING zinc finger. Another IAP, NAIP, has three BIR domains (BIR1 , BIR2 and BIR3), but no RING domain, whereas Livin, TsIAP and MLIAP have a single BIR domain and a RING domain. The X chromosome-linked inhibitor of apoptosis (XIAP) is an example of an IAP which can inhibit the initiator caspase, known as caspase-9, and the effector caspases, Caspase-3 and Caspase-7, by direct binding. It can also induce the removal of caspases through the ubiquitylation-mediated proteasome pathway via the E3 ligase activity of a RING zinc finger domain. It is via the BIR3 domain that XIAP binds to and inhibits caspase-9. The Iinker-BIR2 domain of XIAP inhibits the activity of caspases-3 and -7. The BIR domains have also been associated with the interactions of IAPs with tumor necrosis factor-receptor associated factor (TRAFs)-I and -2, and to TAB1 , as adaptor proteins effecting survival signaling through NFkB activation. The IAPs thus function as a direct break on the apoptosis cascade, by preventing the action of, or inhibiting active caspases and by redirecting cellular signaling to a pro-survival mode.
Progress in the cancer field has led to a new paradigm in cancer biology wherein neoplasia may be viewed as a failure of cancer cells to execute normal pathways of apoptosis. Normal cells receive continuous feedback from their environment through various intracellular and extracellular factors, and "commit suicide" if removed from this context. This induction of apoptosis is achieved by activation of the caspase cascade. Cancer cells, however, gain the ability to overcome or bypass this apoptosis regulation and continue with inappropriate proliferation. The majority of treatments for cancer induce at least a partial apoptotic response in the cancer target cell, resulting in remission or initiation of tumor regression. In many cases, however, residual cells which are apoptosis- resistant are capable of escaping therapy and continuing the process of oncogenic/genetic change, resulting in the emergence of highly drug-resistant, metastatic disease which overcomes our ability to effectively treat the disease. Furthermore, most cancer therapies, including radiation therapy and traditional chemotherapy do induce apoptosis in cancer cells, but cause additional cellular injury, due to their lack of specificity in inducing apoptosis solely in cancer cells. The need to improve the specificity/potency of pro- apoptosis agents used to treat cancer, and indeed other proliferative disorders, is important because of the benefits in decreasing the side effects associated with administration of these agents. Therefore, finding novel means of inducing apoptosis in Attorney Docket No. L80003375WO cancer cells is a highly desired medical need and its solution offers the possibility of entirely new treatments for cancer.
A growing body of data indicates that cancer cells may avoid apoptosis by the sustained over- expression of one or more members of the IAP family of proteins, as documented in many primary tumor biopsy samples, as well as most established cancer cell lines. Epidemiological studies have demonstrated that over-expression of the various IAPs is associated with poor clinical prognosis and survival. For XIAP this is shown in cancers as diverse as leukemia and ovarian cancer. Over expression of HIAP1 and HIAP2 resulting from the frequent chromosome amplification of the 11q21-q23 region, which encompasses both, has been observed in a variety of malignancies, including medulloblastomas, renal cell carcinomas, glioblastomas, and gastric carcinomas. (X)IAP negative regulatory molecules such as XAF, appear to be tumor suppressors, which are very frequently lost in clinical cancers. Thus, by their ability to suppress the activation and execution of the intrinsic mediators of apoptosis, the caspases, the IAPs may directly contribute to tumor progression and resistance to pharmaceutical intervention. Induction of apoptosis in cancer cells by the use of potent small molecules which bind to specific IAP domains is the subject of this invention.
We and others have demonstrated the critical importance of the individual BIR domains for affecting the antiapoptotic function of the IAPs. We have proposed that antagonists of the IAPs, which may bind to the individual BIR domains, would disrupt the antiapoptotic function of the IAPs. Indeed, individual BIRs serve as critical binding sites for the N- terminal Ser-Gly-Val-Asp, Ser-Gly-Pro-lle and Ala-Thre-Pro-lle residues of the Caspases 3, 7, and 9, respectively, and such binding is imperative for the Caspase-inhibitory function of the IAPs. The binding of N-terminal AxPy tetra-peptide residues to XIAP results in the release of the active caspases 3, 7 and 9. In the case of the other IAPs, the functions of the BIRs, when ligand-bound, appear to direct the activation of the ubiquitin ligase RING function of the IAPs to a bound target, or the IAPs themselves, to cause proteosomal loss. In either case, small molecule antagonists of this target should be excellent pro-apoptotic agents, with potential uses in cancer, various proliferative disorders and inflammation.
A mammalian mitochondrial protein, namely Second Mitochondria-derived Activator of Caspases (SMAC) which antagonizes IAP function, binds mainly to the BIR 3 or 2 sites on respective IAPs via an AxPy amino-terminal tetrapeptide. Four Drosophila death-inducing Attorney Docket No. L80003375WO proteins, Reaper, HID, Grim, and Sickle, which antagonize the ability of the Drosophila
IAPs to inhibit caspases, also bind the BIR domains of the analogous Drosophila IAPs via a short AxPy amino-terminal tetrapeptide, a sequence that fits into the BIR binding pocket and disrupts IAP-caspase interactions.
The overall topology of individual BIR domains is highly conserved between the human IAPs and between individual BIR domains of the human IAPs, each BIR being a zinc finger polypeptide domain, locked into a coordinated Zn atom by two cysteines and a histidine residue. The X-ray crystallographic structures of XIAP BIR2 and BIR3 reveal a critical binding pocket for an AXPY motif on the surface of each BIR domain. There are alterations in the intervening amino acid sequences that form the binding pocket and groove in both BIR2 and BIR3. Likewise, we have described homologous domains in the BIRs of other IAPs clAP1 and clAP2. This opens the possibility of obtaining various classes of natural and synthetic binding compounds which will have different specificity and binding affinities between each of the BIR domains for each of the IAPs. Discerning the way in which such compounds will affect the biological function of the IAPs in cancer cells vs normal cells is a major new challenge in the discovery of novel mechanism agents to treat cancer and other proliferative disorders where dysregulated IAP function is observed. It is our finding that certain classes of BIR binding compounds may bind to IAP BIRs, with unexpected selectivity and potency, resulting in distinct therapeutic advantages for certain structural classes.
A number of peptidic AxPy-like and heterocyclic modified AxPy peptidic compounds have been described which activate cellular Caspase 3 by reportedly binding to XIAP BIR3. For a recent reviews, see Elmore et al., Annual Reports in Medicinal Chemistry, 40 (2006) 245-262; Sun et al., Bioorg. Med. Chem. Let. 15 (2005) 793-797; Oost et al., J.Med.Chem., 2004, 47(18), 4417-4426; Park et al., Bioorg. Med. Chem. Lett. 15 (2005) 771-775; Franklin et al., Biochemistry, Vol. 42, No. 27, 2003, 8223-8231 ; Kip et al., Biochemistry 2002, 41, 7344-7349; Wu et al., Chemistry and Biology, Vol.10, 759-767 (2003); Glover et al., Analytical Biochemistry, 320 (2003) 157-169; United States published patent application number 20020177557; and United States published patent application number 20040180828; United States published patent application number US2006/0025347A1 ; United States published patent application number US2005/0197403A1 ; and United States published patent application number US2006/0194741 A1. Attorney Docket No. L80003375WO
The aforesaid compounds have been shown to target an isolated BIR3 domain of XIAP via displacement of a fluorescently-labeled probe and they appear to induce an apoptotic event in a select set of cancer cell lines with potency in the low micromolar-nanomolar range. These compounds displayed poor in-vivo activity, likely due to limited bioavailability and may therefore have limited therapeutic application.
Thus, IAP BIR domains represent an attractive target for the discovery and development of novel therapeutic agents, especially for the treatment of proliferative disorders such as cancer,
SUMMARY OF THE INVENTION
We have discovered a novel series of constrained cyclic compounds that enhance cellular apoptosis through IAP modulation. The compounds are less peptidic in character because the proline in the previously described compounds has been replaced with a constrained cyclic moiety and as such have pharmaceutically acceptable stability and bioavailability. The compounds bind to XIAP BIR3 and therefore are useful for treating proliferative disorders.
In one aspect of the present invention, there is provided a compound represented by Formula I:
Figure imgf000006_0001
wherein n is 0 or 1 ; m is 0, 1 or 2; Attorney Docket No. L80003375WO p is 1 or 2;
Y is NH, O or S;
R1, R2, R3 and R4 are independently selected from: 1) H, or
2) C1-C6 alkyl, optionally substituted with one or more R7 substituents;
W is a 5, 6, or 7-membered cyclic amide ring, optionally including one or two O, S, or N heteroatoms, the cyclic amide ring being optionally fused with an aryl or a heteroaryl, wherein the aryl and the heteroaryl are optionally substituted with one or more R11 substituents, and wherein the cyclic amide ring is optionally substituted with one or more R7 substituents;
A is 1) -(C1-C6 alkyl)-C(O)- or
2) -(C1-C6 alkyl)-; wherein the alkyl is optionally substituted with one or more R7 substituents;
Q is I ) NR5R6,
2) OR12,
3) S(O)mR12; or Q is
Figure imgf000007_0001
wherein G is a 5, 6 or 7 membered ring which optionally incorporates one or more heteroatoms selected from S, N or O, and which is optionally substituted with one or more R13 substituents, and which is optionally fused with an aryl, the aryl being optionally substituted with one or more R11 substituents; or
Q is an aryl or a heteroaryl, the aryl and the heteroaryl being optionally substituted with one or more R11 substituents; Attorney Docket No. L80003375WO R5 and R6 are each independently selected from
1 ) H,
2) haloalkyl,
3) ^C1-C6 alkyl, 4) <-C2-C6 alkenyl,
5) <-C2-C4 alkynyl,
6) <-C3-C7 cycloalkyl,
7) <-C3-C7 cycloalkenyl,
8) ^aryl, 9) <-heteroaryl,
10) *-heterocyclyl, 11) <— heterobicyclyl, 12) ^-C(O)-R12, 13) <-C(O)O-R12, 14) ^C(=Y)NR9R10, or
15) ^S(O)2-R12, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is optionally substituted with one or more R7 substituents; and wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl is optionally substituted with one or more R11 substituents;
R7 is
1 ) halogen,
2) NO2,
3) CN, 4) haloalkyl,
5) C1-C6 alkyl,
6) C2-C6 alkenyl,
7) C2-C4 alkynyl,
8) C3-C7 cycloalkyl, 9) C3-C7 cycloalkenyl,
10) aryl,
11 ) heteroaryl,
12) heterocyclyl,
13) heterobicyclyl, 14) OR8, Attorney Docket No. L80003375WO 15) S(O)mR8,
16) NR9R10 ,
17) NR8S(O)2R12,
18) COR8, 19) C(O)OR8,
2O) CONR9R10, 2I ) S(O)2NR9R10
22) OC(O)R8,
23) OC(O)Y-R12, 24) SC(O)R8, or
25) NC(Y)NR9R10, wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl is optionally substituted with one or more R11 substituents;
R8 is
1) H,
2) haloalkyl,
3) C1-C6 alkyl,
4) C2-C6 alkenyl, 5) C2-C4 alkynyl,
6) C3-C7 cycloalkyl,
7) C3-C7 cycloalkenyl,
8) aryl,
9) heteroaryl, 10) heterocyclyl,
11 ) heterobicyclyl, 12) R9R10NC(=Y), or
13) C1-C6 alkyl-C2-C4 alkenyl, or
14) C1-C6 alkyl-C2-C4 alkynyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is optionally substituted with one or more R7 substituents; and wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl is optionally substituted with one or more R11 substituents;
R9 and R10 are each independently 1) H, Attorney Docket No. L80003375WO
2) haloalkyl,
3) C1-C6 alkyl,
4) C2-C6 alkenyl,
5) C2-C4 alkynyl, 6) C3-C7 cycloalkyl,
7) C3-C7 cycloalkenyl,
8) aryl,
9) heteroaryl,
10) heterocyclyl, 11 ) heterobicyclyl,
12) C(O)R12,
13) C(O)Y-R12, or
14) S(O)2-R12, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is optionally substituted with one or more R7 substituents; and wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl is optionally substituted with one or more R11 substituents;
or R9 and R10 together with the nitrogen atom to which they are bonded form a five, six or seven membered heterocyclic ring optionally substituted with one or more R11 substituents;
11 is
1 ) halogen,
2) NO2,
3) CN,
4) B(OR13KOR14),
5) CrC6 alkyl,
6) C2-C6 alkenyl,
7) C2-C4 alkynyl,
8) C3-C7 cycloalkyl,
9) C3-C7 cycloalkenyl,
10) haloalkyl,
11 ) OR8,
12; ) NR9R10,
13) SR8, Attorney Docket No. L80003375WO
14) COR8,
15) C(O)O R8, 16)S(O)mR8, 17) CONR9R10, 18) S(O)2NR9R10,
19) aryl, 20) heteroaryl, 21 ) heterocyclyl, or 22) heterobicyclyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl is optionally substituted with one or more R7 substituents;
R12 is 1 ) haloalkyl,
2) C1-C6 alkyl,
3) C2-C6 alkenyl,
4) C2-C4 alkynyl,
5) C3-C7 cycloalkyl, 6) C3-C7 cycloalkenyl,
7) aryl,
8) heteroaryl,
9) heterocyclyl, or
10) heterobicyclyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is optionally substituted with one or more R7 substituents; and wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl is optionally substituted with one or more R11 substituents;
R13 and R14 are each independently 1) H, or
2) C1-C6 alkyl; or
R13 and R14 are combined to form a heterocyclic ring or a heterobicyclyl ring; or a prodrug, or a pharmaceutically acceptable salt, or labeled with a detectable label or an affinity tag, or a stereoisomer thereof. Attorney Docket No. L80003375WO In another aspect of the present invention, there is provided an intermediate compound represented by Formula l(ii):
Figure imgf000012_0001
1(B)
In another aspect of the present invention, there is provided an intermediate compound represented by Formula l(ii):
Figure imgf000012_0002
1(iii)
In another aspect of the present invention, there is provided an intermediate compound represented by Formula l(iv):
Figure imgf000012_0003
l(iv)
wherein PG2 is a protecting group, and R1, R3, R7, R4 R5, are as defined herein, and wherein Y' has the same meaning as Y, and wherein Ra and Rb may be combined to form an aromatic ring system wherein double bond may represent a single or double bond
In another aspect of the present invention, there is provided a process for producing compounds represented by Formula I, described hereinabove, the process comprising Attorney Docket No. L80003375WO treating the intermediate as represented by Formula l(iv) under conditions suitable to remove the PG2 so as to produce the compound of Formula I.
In another aspect of the present invention, there is provided an intermediate compound represented by Formula l(iii):
Figure imgf000013_0001
l(iii) wherein R7, R4 R5, Y', Ra and Rb are as defined herein.
In another aspect of the present invention, there is provided a process for producing compounds represented by Formula I, described hereinabove, the process comprising:
a) coupling an intermediate represented by Formula l(iii):
Figure imgf000013_0002
l(iii)
R1
, N ^ XO2H PG2 - and R3 , in a solvent; and b) removing the protecting group PG2 so as to form compounds of Formula 1 , wherein PG2 is a protecting group, and R1, R3, R7, R4 R5, Y', Ra and Rb are as defined herein.
In another aspect of the present invention, there is provided a pharmaceutical composition comprising a compound, as described above, mixed with a pharmaceutically acceptable carrier, diluent or excipient. Attorney Docket No. L80003375WO In another aspect of the present invention, there is provided a pharmaceutical composition adapted for administration as an agent for treating a proliferative disorder in a subject, comprising a therapeutically effective amount of a compound, as described above.
In another aspect of the present invention, there is provided a pharmaceutical composition comprising a compound of Formula I in combination with one or more death receptor agonists, for example, an agonist of TRAIL receptor.
In another aspect of the present invention, there is provided a pharmaceutical composition comprising a compound of formula I in combination with any therapeutic agent that increases the response of one or more death receptor agonists, for example cytotoxic cytokines such as interferons.
In another aspect of the present invention, there is provided a method of preparing a pharmaceutical composition, the method comprising: mixing a compound, as described above, with a pharmaceutically acceptable carrier, diluent or excipient.
In another aspect of the present invention, there is provided a method of treating a disease state characterized by insufficient apoptosis, the method comprising: administering to a subject in need thereof, a therapeutically effective amount of a pharmaceutical composition, as described above, so as to treat the disease state.
In another aspect of the present invention, there is provided a method of modulating IAP function, the method comprising: contacting a cell with a compound of the present invention so as to prevent binding of a BIR binding protein to an IAP BIR domain thereby modulating the IAP function.
In another aspect of the present invention, there is provided a method of treating a proliferative disease, the method comprising: administering to a subject in need thereof, a therapeutically effective amount of the pharmaceutical composition, as described above, so as to treat the proliferative disease.
In another aspect of the present invention, there is provided a method of treating cancer, the method comprising: administering to a subject in need thereof, a therapeutically Attorney Docket No. L80003375WO effective amount of the pharmaceutical composition, as described above, so as to treat the cancer.
In another aspect of the present invention, there is provided a method of treating cancer, the method comprising: administering to the subject in need thereof, a therapeutically effective amount of a pharmaceutical composition, as described above, in combination or sequentially with an agent selected from: a) an estrogen receptor modulator, b) an androgen receptor modulator, c) retinoid receptor modulator, d) a cytotoxic agent, e) an antiproliferative agent, f) a prenyl-protein transferase inhibitor, g) an HMG-CoA reductase inhibitor, h) an HIV protease inhibitor, i) a reverse transcriptase inhibitor, k) an angiogenesis inhibitor,
I) a PPAR-.γ agonist, m) a PPAR-.δ. agonist, n) an inhibitor of inherent multidrug resistance, o) an anti-emetic agent, p) an agent useful in the treatment of anemia, q) agents useful in the treatment of neutropenia, r) an immunologic-enhancing drug. s) a proteasome inhibitor; t) an HDAC inhibitor;' u) an inhibitor of the chemotrypsin-like activity in the proteasome; or v) E3 ligase inhibitors; w) a modulator of the immune system such as, but not limited to, interferon-alpha, Bacillus Calmette-Guerin (BCG), and ionizing radition (UVB) that can induce the release of cytokines, such as the interleukins, TNF, or induce release of death receptor ligands such as TRAIL; x) a modulator of death receptors TRAIL and TRAIL agonists such as the humanized antibodies HGS-ETR1 and HGS-ETR2; or in combination or sequentially with radiation therapy, so as to treat the cancer. Attorney Docket No. L80003375WO
In another aspect of the present invention, there is provided a method for the treatment or prevention of a proliferative disorder in a subject, the method comprising: administering to the subject a therapeutically effective amount of the composition, described above.
In another aspect of the present invention, the method further comprises administering to the subject a therapeutically effective amount of a chemotherapeutic agent prior to, simultaneously with or after administration of the composition.
In yet another aspect, the method further comprises administering to the subject a therapeutically effective amount of a death receptor agonist prior to, simultaneously with or after administration of the composition. The death receptor agonist is TRAIL or the death receptor agonist is a TRAIL antibody. The death receptor agonist is typically administered in an amount that produces a synergistic effect.
In another aspect of the present invention, there is provided a probe, the probe being a compound of Formula I above, the compound being labeled with a detectable label or an affinity tag.
In another aspect of the present invention, there is provided a method of identifying compounds that bind to an IAP BIR domain, the assay comprising: a) contacting an IAP BIR domain with a probe to form a probe:BIR domain complex, the probe being displaceable by a test compound; b) measuring a signal from the probe so as to establish a reference level; c) incubating the probe:BIR domain complex with the test compound; d) measuring the signal from the probe; e) comparing the signal from step d) with the reference level, a modulation of the signal being an indication that the test compound binds to the BIR domain, wherein the probe is a compound of Formula I labeled with a detectable label or an affinity label.
DETAILED DESCRIPTION OF THE INVENTION
The compounds of the present invention are useful as BIR domain binding compounds in mammalian IAPs and are represented by Formula I. The following are embodiments, Attorney Docket No. L80003375WO groups and substituents of the compounds according to Formula I, which are described hereinafter in detail.
n: In one subset of compounds of Formula I, n is 0.
Core:
Therefore for compounds of Formula I, the present invention comprises compounds of Formula IA:
Figure imgf000017_0001
wherein R1, R2, R3, R4, W and Q are as defined herein.
Any and each individual definition of Core as set out herein may be combined with any and each individual definition of R1, R2, R3, R4, W, A and Q as set out herein.
W:
In one subset of the aforesaid compounds, W is a 6 or 7-membered cyclic amide ring, optionally including one or two O, S, or N heteroatoms, the cyclic amide ring being optionally fused with an aryl or a heteroaryl, wherein the aryl and the heteroaryl are optionally substituted with one or more R11 substituents, and wherein the cyclic amide ring is optionally substituted with one or more R7 substituents.
In another subset, W is a 7-membered cyclic amide ring, the cyclic amide ring being optionally fused with an aryl.
Thus, compounds of the present invention comprise compounds of Formula IB and IC: Attorney Docket No. L80003375WO
Figure imgf000018_0001
IB IC
wherein R1, R2, R3, R4, A and Q are as defined herein.
In one alternative subset of the aforesaid compounds, W is a 7-membered cyclic amide ring, including one O, S, or N heteroatoms, the cyclic amide ring being optionally fused with an aryl or a heteroaryl.
In one alternative subset of the aforesaid compounds, W is a 7-membered cyclic amide ring, including one O, S, or N heteroatoms, the cyclic amide ring being fused with an aryl.
Thus, compounds of the present invention comprise compounds of Formula ID and IE
Figure imgf000018_0002
ID IE
wherein R1, R2, R3, R4, A and Q are as defined herein.
Any and each individual definition of W as set out herein may be combined with any and each individual definition of R1, R2, R3, R4, A and Q as set out herein.
R1: In one subset, R1 is C1-C6 alkyl. In one example, R1 is CH3.
Any and each individual definition of R1 as set out herein may be combined with any and each individual definition of W, Core, R2, R3, R4, A and Q as set out herein. Attorney Docket No. L80003375WO
R2:
In one example, R2 is H.
Any and each individual definition of R2 as set out herein may be combined with any and each individual definition of W1 Core, R1, R3, R4, A and Q as set out herein.
R3:
In one subset, R3 is Ci-C6 alkyl. In one example, R3 is CH3.
Any and each individual definition of R3 as set out herein may be combined with any and each individual definition of W, Core, R1, R2, R4, A and Q as set out herein.
R4: In one example, R4 is H.
Any and each individual definition of R4 as set out herein may be combined with any and each individual definition of W, Core, R1, R2, R3, A and Q as set out herein.
A:
In one subset of compounds of Formula I, A is -(C1-C6 alkyl)-C(O)-.
In one example, A is -CH2C(O)-.
Any and each individual definition of A as set out herein may be combined with any and each individual definition of W, Core, R1, R2, R3, R4, Q and A as set out herein.
Q:
In one subset of the aforesaid compounds, Q is NR5R6.
Any and each individual definition of Q as set out herein may be combined with any and each individual definition of W, Core, R1, R2, R3, R4 and A as set out herein.
R5 and R6: In one subset of the aforesaid compounds, R5 is H and R6 is selected from: Attorney Docket No. L80003375WO
1 ) haloalkyl,
2) +-C1-C6 alkyl,
3) -(-C2-C6 alkenyl,
4) <-C2-C4 alkynyl, 5) <-C3-C7 cycloalkyl,
6) +-C3-C7 cycloalkenyl,
7) ^aryl,
8) *-heteroaryl,
9) <-heterocyclyl, or 10) <-heterobicyclyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is optionally substituted with one or more R7 substituents.
In another subset of the aforesaid compounds, R5 is H and R6 is selected from: I ) ^C1-C6 alkyl, or
2) ^aryl, wherein the alkyl substituted with phenyl.
Examples of R6 when R5 is H is selected from the group consisting of:
Figure imgf000020_0001
Thus in one subset, Q is selected from the group consisting of:
Figure imgf000020_0002
In an alternative subset of the compounds described above, Q may be substituted with a detectable label or an affinity tag. The detectable label or the affinity tag, however, may be located at any suitable position on the compound. Attorney Docket No. L80003375WO
If a substituent is itself substituted with one or more substituents, it is to be understood that that the one or more substituents may be attached to the same carbon atom or different carbon atoms. Combinations of substituents and variables defined herein are allowed only if they produce chemically stable compounds.
One skilled in the art will understand that substitution patterns and substituents on compounds of the present invention may be selected to provide compounds that are chemically stable and can be readily synthesized using the chemistry set forth in the examples and chemistry techniques well known in the art using readily available starting materials.
It is to be understood that many substituents or groups described herein have functional group equivalents, which means that the group or substituent may be replaced by another group or substituent that has similar electronic, hybridization or bonding properties.
Definitions
Unless otherwise specified, the following definitions apply:
The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise.
As used herein, the term "comprising" is intended to mean that the list of elements following the word "comprising" are required or mandatory but that other elements are optional and may or may not be present .
As used herein, the term "consisting of is intended to mean including and limited to whatever follows the phrase "consisting of. Thus the phrase "consisting of indicates that the listed elements are required or mandatory and that no other elements may be present.
As used herein, the term "alkyl" is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, for example, C1-C6 as in C1-C6- alkyl is defined as including groups having 1 , 2, 3, 4, 5 or 6 carbons in a linear or branched arrangement, and C1-C4 as in C1-C4 alkyl is defined as including groups having 1 , 2, 3, or 4 carbons in a linear or branched arrangement. Attorney Docket No. L80003375WO Examples of d-Cβ-alkyl and d-C4 alkyl as defined above include, but are not limited to, methyl, ethyl, n-propyl, /-propyl, n-butyl, f-butyl, /-butyl, pentyl and hexyl.
As used herein, the term, "alkenyl" is intended to mean unsaturated straight or branched chain hydrocarbon groups having the specified number of carbon atoms therein, and in which at least two of the carbon atoms are bonded to each other by a double bond, and having either E or Z regeochemistry and combinations thereof. For example, C2-C6 as in C2-C6 alkenyl is defined as including groups having 2, 3, 4, 5, or 6 carbons in a linear or branched arrangement, at least two of the carbon atoms being bonded together by a double bond. Examples of C2-C6 alkenyl include ethenyl (vinyl), 1-propenyl, 2-propenyl, 1- butenyl and the like.
As used herein, the term "alkynyl" is intended to mean unsaturated, straight chain hydrocarbon groups having the specified number of carbon atoms therein and in which at least two carbon atoms are bonded together by a triple bond. For example C2-C4 as in C2- C4 alkynyl is defined as including groups having 2, 3, or 4 carbon atoms in a chain, at least two of the carbon atoms being bonded together by a triple bond. Examples of such alynyls include ethynyl, 1-propynyl, 2-propynyl and the like.
As used herein, the term "cycloalkyl" is intended to mean a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms therein, for example, C3- C7 as in C3-C7 cycloalkyl is defined as including groups having 3, 4, 5, 6, or 7 carbons in a monocyclic arrangement. Examples of C3-C7 cycloalkyl as defined above include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
As used herein, the term "cycloalkenyl" is intended to mean a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms therein, for example, C3-C7 as in C3-C7 cycloalkenyl is defined as including groups having 3, 4, 5, 6, or 7 carbons in a monocyclic arrangement. Examples of C3-C7 cycloalkenyl as defined above include, but are not limited to, cyclopentenyl, and cyclohexenyl.
As used herein, the term "halo" or "halogen" is intended to mean fluorine, chlorine, bromine and iodine. Attorney Docket No. L80003375WO As used herein, the term "haloalkyl" is intended to mean an alkyl as defined above, in which each hydrogen atom may be successively replaced by a halogen atom. Examples of haloalkyls include, but are not limited to, CH2F, CHF2 and CF3.
As used herein, the term "aryl", either alone or in combination with another radical, means a carbocyclic aromatic monocyclic group containing 6 carbon atoms which may be further fused to a second 5- or 6-membered carbocyclic group which may be aromatic, saturated or unsaturated. Aryl includes, but is not limited to, phenyl, indanyl, 1-naphthyl, 2-naphthyl and tetrahydronaphthyl. The fused aryls may be connected to another group either at a suitable position on the cycloalkyl ring or the aromatic ring. For example:
Figure imgf000023_0001
Arrowed lines drawn from the ring system indicate that the bond may be attached to any of the suitable ring atoms.
As used herein, the term "biphenyl" is intended to mean two phenyl groups bonded together at any one of the available sites on the phenyl ring. For example:
Figure imgf000023_0002
As used herein, the term "heteroaryl" is intended to mean a monocyclic or bicyclic ring system of up to ten atoms, wherein at least one ring is aromatic, and contains from 1 to 4 hetero atoms selected from the group consisting of O, N, and S. The heteroaryl substituent may be attached either via a ring carbon atom or one of the heteroatoms. Examples of heteroaryl groups include, but are not limited to thienyl, benzimidazolyl, benzo[b]thienyl, furyl, benzofuranyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, 2H- pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyi, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, napthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, Attorney Docket No. L80003375WO isothiazolyl, isochromanyl, chromanyl, isoxazolyl, furazanyl, indolinyl, isoindolinyl, thiazolo[4,5-b]-pyridine, and
fluoroscein derivatives such as:
Figure imgf000024_0001
As used herein, the term "heterocycle", "heterocyclic" or "heterocyclyl" is intended to mean a 5, 6, or 7 membered non-aromatic ring system containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Examples of heterocycles include, but are not limited to pyrrolidinyl, tetrahydrofuranyl, piperidyl, pyrrolinyl, piperazinyl,
imidazolidinyl, morpholinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, and
Figure imgf000024_0002
As used herein, the term "heterobicycle" either alone or in combination with another radical, is intended to mean a heterocycle as defined above fused to another cycle, be it a heterocycle, an aryl or any other cycle defined herein. Examples of such heterobicycles include, but are not limited to, coumarin, benzo[d][1 ,3]dioxole, 2,3- dihydrobenzo[b][1 ,4]dioxine and 3,4-dihydro-2H-benzo[b][1 ,4]dioepine.
As used herein, the term "heteroaryl" is intended to mean a monocyclic or bicyclic ring system of up to ten atoms, wherein at least one ring is aromatic, and contains from 1 to 4 hetero atoms selected from the group consisting of O, N, and S. The heteroaryl substituent may be attached either via a ring carbon atom or one of the heteroatoms. Examples of heteroaryl groups include, but are not limited to thienyl, benzimidazolyl, benzo[b]thienyl, furyl, benzofuranyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, 2H- pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, napthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, isothiazolyl, isochromanyl, chromanyl, isoxazolyl, furazanyl, indolinyl, and isoindolinyl, Attorney Docket No. L80003375WO As used herein, the term "heterocycle", "heterocyclic" or "heterocyclyl" is intended to mean a 5, 6, or 7 membered non-aromatic ring system containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Examples of heterocycles include, but are not limited to pyrrolidinyl, tetrahydrofuranyl, piperidyl, pyrrolinyl, piperazinyl, imidazolidinyl, morpholinyl, imidazolinyl, pyrazolidinyl, and pyrazolinyl,
As used herein, the term "heteroatom" is intended to mean O, S or N.
As used herein, the term "detectable label" is intended to mean a group that may be linked to a compound of the present invention to produce a probe or to an IAP BIR domain, such that when the probe is associated with the BIR domain, the label allows either direct or indirect recognition of the probe so that it may be detected, measured and quantified. As used herein, the term "affinity tag" is intended to mean a ligand or group, which is linked to either a compound of the present invention or to an IAP BIR domain to allow another compound to be extracted from a solution to which the ligand or group is attached.
As used herein, the term "probe" is intended to mean a compound of Formula I which is labeled with either a detectable label or an affinity tag, and which is capable of binding, either covalently or non-covalently, to an IAP BIR domain. When, for example, the probe is non-covalently bound, it may be displaced by a test compound. When, for example, the probe is bound covalently, it may be used to form cross-linked adducts, which may be quantified and inhibited by a test compound.
As used herein, the term "optionally substituted with one or more substituents" or its equivalent term "optionally substituted with at least one substituent" is intended to mean that the subsequently described event of circumstances may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. The definition is intended to mean from zero to five substituents.
If the substituents themselves are incompatible with the synthetic methods of the present invention, the substituent may be protected with a suitable protecting group (PG) that is stable to the reaction conditions used in these methods. The protecting group may be removed at a suitable point in the reaction sequence of the method to provide a desired intermediate or target compound. Suitable protecting groups and the methods for Attorney Docket No. L80003375WO protecting and de-protecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which may be found in T. Greene and P. Wuts, Protecting Groups in Chemical Synthesis (3rd ed.), John Wiley & Sons, NY (1999), which is incorporated herein by reference in its entirety. Examples of protecting groups used throughout include, but are not limited to Fmoc, Bn, Boc, CBz and COCF3. In some instances, a substituent may be specifically selected to be reactive under the reaction conditions used in the methods of this invention. Under these circumstances, the reaction conditions convert the selected substituent into another substituent that is either useful in an intermediate compound in the methods of this invention or is a desired substituent in a target compound.
Abbreviations for α-amino acids used throughout are as follows:
Figure imgf000026_0001
Attorne Docket No. L80003375WO
Figure imgf000027_0001
As used herein, the term "residue" when referring to α-amino acids is intended to mean a radical derived from the corresponding α-amino acid by eliminating the hydroxyl of the carboxy group and one hydrogen of the α-amino group. For example, the terms GIn, Ala, GIy, He, Arg, Asp, Phe, Ser, Leu, Cys, Asn, and Tyr represent the residues of L-glutamine, L-alanine, glycine, L-isoleucine, L-arginine, L-aspartic acid, L-phenylalanine, L-serine, L- leucine, L-cysteine, L-asparagine, and L-tyrosine, respectively.
As used herein, the term "subject" is intended to mean humans and non-human mammals such as primates, cats, dogs, swine, cattle, sheep, goats, horses, rabbits, rats, mice and the like.
As used herein, the term "prodrug" is intended to mean a compound that may be converted under physiological conditions or by solvolysis to a biologically active compound of the present invention. Thus, the term "prodrug" refers to a precursor of a compound of the invention that is pharmaceutically acceptable. A prodrug may be inactive or display limited activity when administered to a subject in need thereof, but is converted in vivo to an active compound of the present invention. Typically, prodrugs are transformed in vivo to yield the compound of the invention, for example, by hydrolysis in blood or other organs by enzymatic processing. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in the subject (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam). The definition of prodrug includes any covalently bonded carriers which release the active compound of the invention in vivo when such prodrug is administered to a subject. Prodrugs of a compound of the present invention may be prepared by modifying functional groups present in the compound of the invention in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to a parent compound of the invention.
As used herein, the term "pharmaceutically acceptable carrier, diluent or excipient" is intended to mean, without limitation, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, emulsifier, or Attorney Docket No. L80003375WO encapsulating agent, such as a liposome, cyclodextrins, encapsulating polymeric delivery systems or polyethyleneglycol matrix, which is acceptable for use in the subject, preferably humans.
As used herein, the term "pharmaceutically acceptable salt" is intended to mean both acid and base addition salts.
As used herein, the term "pharmaceutically acceptable acid addition salt" is intended to mean those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
As used herein, the term "pharmaceutically acceptable base addition salt" is intended to mean those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2- diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.
As used herein, the term "BIR domain binding" is intended to mean the action of a compound of the present invention upon an IAP BIR domain, which blocks or diminishes the binding of IAPs to BIR binding proteins or is involved in displacing BIR binding proteins Attorney Docket No. L80003375WO from an IAP. Examples of BIR binding proteins include, but are not limited to, caspases and mitochondrially derived BIR binding proteins such as Smac, 0mi/WTR2A and the like.
As used herein, the term "insufficient apoptosis" is intended to mean a state wherein a disease is caused or continues because cells deleterious to the subject have not apoptosed. This includes, but is not limited to, cancer cells that survive in a subject without treatment, cancer cells that survive in a subject during or following anti-cancer treatment, or immune cells whose action is deleterious to the subject, and includes, neutrophils, monocytes and auto-reactive T-cells.
As used herein, the term "therapeutically effective amount" is intended to mean an amount of a compound of Formula I which, when administered to a subject is sufficient to effect treatment for a disease-state associated with insufficient apoptosis. The amount of the compound of Formula I will vary depending on the compound, the condition and its severity, and the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.
As used herein, the term "treating" or "treatment" is intended to mean treatment of a disease-state associated with insufficient apoptosis, as disclosed herein, in a subject, and includes: (i) preventing a disease or condition associated with insufficient apoptosis from occurring in a subject, in particular, when such mammal is predisposed to the disease or condition but has not yet been diagnosed as having it; (ii) inhibiting a disease or condition associated with insufficient apoptosis, i.e., arresting its development; or (iii) relieving a disease or condition associated with insufficient apoptosis, i.e., causing regression of the condition.
As used herein, the term "treating cancer" is intended to mean the administration of a pharmaceutical composition of the present invention to a subject, preferably a human, which is afflicted with cancer to cause an alleviation of the cancer by killing, inhibiting the growth, or inhibiting the metastasis of the cancer cells.
As used herein, the term "preventing disease" is intended to mean, in the case of cancer, the post-surgical, post-chemotherapy or post-radiotherapy administration of a pharmaceutical composition of the present invention to a subject, preferably a human, which was afflicted with cancer to prevent the regrowth of the cancer by killing, inhibiting Attorney Docket No. L80003375WO the growth, or inhibiting the metastasis of any remaining cancer cells. Also included in this definition is the prevention of prosurvival conditions that lead to diseases such as asthma, MS and the like.
As used herein, the term "synergistic effect" is intended to mean that the effect achieved with the combination of the compounds of the present invention and either the chemotherapeutic agents or death receptor agonists of the invention is greater than the effect which is obtained with only one of the compounds, agents or agonists, or advantageously the effect which is obtained with the combination of the above compounds, agents or agonists is greater than the addition of the effects obtained with each of the compounds, agents or agonists used separately. Such synergy enables smaller doses to be given.
As used herein, the term "apoptosis" or "programmed cell death" is intended to mean the regulated process of cell death wherein a dying cell displays a set of well-characterized biochemical hallmarks that include cell membrane blebbing, cell soma shrinkage, chromatin condensation, and DNA laddering, as well as any caspase-mediated cell death.
As used herein, the term "BIR domain" or "BIR" are used interchangeably throughout and are intended to mean a domain which is characterized by a number of invariant amino acid residue including conserved cysteines and one conserved hisitidine residue within the sequence Cys-(Xaa1)2Cys-(Xaa1)i6His-(Xaa1)6-8Cys. Typically, the amino acid sequence of the consensus sequence is: Xaa1-Xaa1-Xaa1-Arg-Leu-Xaa1-Thr-Phe-Xaa1-Xaa1-Trp - Pro-Xaa2-Xaa1-Xaa1-Xaa2-Xaa2-Xaa1-Xaa1-Xaa1-Xaa1-Leu-Ala-Xaa1-Ala-Gly-Phe-Tyr- Tyr-Xaa 1 -Gly-Xaa 1 -Xaa 1 -Asp-Xaa 1 -Val-Xaa 1 -Cys-Phe-Xaa 1 -Cys-Xaa 1 -Xaa 1 -Xaa 1 - Xaa1 -Xaa1 -Xaa1 -Trp-Xaa1 -Xaa 1 -Xaa 1 -Asp-Xaa 1 -Xaa1 -Xaa1 -Xaa1 -Xaa1 -His-Xaa- 1 - Xaal-Xaal-Xaal-Pro-Xaal-Cys-Xaal-Phe-Val, wherein Xaa1 is any amino acid and Xaa2 is any amino acid or is absent. Preferably the sequence is substantially identical to one of the BIR domain sequences provided for XIAP, HIAP1, or HIAP2 herein. The BIR domain residues are listed below (see Genome Biology (2001) 1-10):
Figure imgf000030_0001
Attorney Docket No. L80003375WO
Figure imgf000031_0001
As used herein, the term "ring zinc finger" or "RZF" is intended to mean a domain having the amino acid sequence of the consensus sequence: Glu-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1- Xaa- 1 -Xaa2-Xaa 1 -Xaa 1 -Xaa 1 -Cys-Lys-Xaa3-Cys-Met-Xaa 1 -Xaa 1 -Xaa 1 -Xaa 1 -Xaa 1 - Xaa3-X- aa 1 -Phe-Xaa 1 -Pro-Cys-Gly-His-Xaa 1 -Xaa 1 -Xaa 1 -Cys-Xaa 1 -Xaa 1 -Cys-Ala- Xaa1-Xaa- 1-Xaai-Xaai-Xaai-Cys-Pro-Xaai-Cys, wherein Xaa1 is any amino acid, Xaa2 is GIu or Asp, and Xaa3 is VaI or lie.
As used herein, the term "IAP" is intended to mean a polypeptide or protein, or fragment thereof, encoded by an IAP gene. Examples of IAPs include, but are not limited to human or mouse NAIP (Birc 1), H1AP-1 (clAP2, Birc 3), HIAP-2 (clAP1 , Birc 2), XIAP (Birc 4), survivin (Birc 5), livin (ML-IAP, Birc 7), ILP-2 (Birc 8) and Apollon/BRUCE (Birc 6) (see for example US Patent Numbers 6,107,041 ; 6,133,437; 6,156,535; 6,541 ,457; 6,656,704;
6,689,562; Deveraux and Reed, Genes Dev. 13, 239-252, 1999; Kasof and Gomes, J. Biol. Chem., 276, 3238-3246, 2001 ; Vucic et al., Curr. Biol. 10, 1359-1366, 2000; Ashab et al. FEBS Lett., 495, 56-60, 2001 , the contents of which are hereby incorporated by reference).
As used herein, the term "IAP gene" is intended to mean a gene encoding a polypeptide having at least one BIR domain and which is capable of modulating (inhibiting or enhancing) apoptosis in a cell or tissue. The IAP gene is a gene having about 50% or greater nucleotide sequence identity to at least one of human or mouse NAIP (Birc 1), HIAP-1 (CIAP2, Birc 3), HIAP-2 (clAP1 , Birc 2), XIAP (Birc 4), survivin (Birc 5), livin (ML- IAP, Birc 7), ILP-2 (Birc 8) and Apollon/BRUCE (Birc 6). The region of sequence over which identity is measured is a region encoding at least one BIR domain and a ring zinc finger domain. Mammalian IAP genes include nucleotide sequences isolated from any mammalian source.
As used herein, the term 11IC50" is intended to mean an amount, concentration or dosage of a particular compound of the present invention that achieves a 50% inhibition of a maximal response, such as displacement of maximal fluorescent probe binding in an assay that measures such response. Attorney Docket No. L80003375WO
As used herein, the term "EC50" is intended to mean an amount, concentration or dosage of a particular compound of the present invention that achieves a 50% inhibition of cell survival.
As used herein, the term "modulate" or "modulating" is intended to mean the treatment, prevention, suppression, enhancement or induction of a function or condition using the compounds of the present invention. For example, the compounds of the present invention can modulate IAP function in a subject, thereby enhancing apoptosis by significantly reducing, or essentially eliminating the interaction of activated apoptotic proteins, such as caspase-3, 7 and 9, with the BIR domains of mammalian IAPs or by inducing the loss of XIAP protein in a cell.
As used herein, the term "enhancing apoptosis" is intended to mean increasing the number of cells that apoptose in a given cell population either in vitro or in vivo. Examples of cell populations include, but are not limited to, ovarian cancer cells, colon cancer cells, breast cancer cells, lung cancer cells, pancreatic cancer cells, or T cells and the like. It will be appreciated that the degree of apoptosis enhancement provided by an apoptosis- enhancing compound of the present invention in a given assay will vary, but that one skilled in the art can determine the statistically significant change in the level of apoptosis that identifies a compound that enhances apoptosis otherwise limited by an IAP. Preferably "enhancing apoptosis" means that the increase in the number of cells undergoing apoptosis is at least 25%, more preferably the increase is 50%, and most preferably the increase is at least one-fold. Preferably the sample monitored is a sample of cells that normally undergo insufficient apoptosis (i.e., cancer cells). Methods for detecting the changes in the level of apoptosis (i.e., enhancement or reduction) are described in the Examples and include methods that quantitate the fragmentation of DNA, methods that quantitate the translocation phosphatoylserine from the cytoplasmic to the extracellular side of the membrane, determination of activation of the caspases and methods quantitate the release of cytochrome C and the apoptosis inhibitory factor into the cytoplasm by mitochondria.
As used herein, the term "proliferative disease" or "proliferative disorder" is intended to mean a disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancers such as lymphoma, Attorney Docket No. L80003375WO leukemia, melanoma, ovarian cancer, breast cancer, pancreatic cancer, and lung cancer, and autoimmune disoders are all examples of proliferative diseases.
As used herein, the term "death receptor agonist" is intended to mean an agent capable of stimulating by direct or indirect contact the pro apoptotic response mediated by the death- receptors. For example, an agonist TRAIL receptor Antibody would bind to TRAIL receptor (S) and trigger an apoptotic response. On the other hand, other agent such as interferon-a could trigger the release of endogeneous TRAIL and/or up regulate the TRAIL receptors in such a way that the cell pro-apoptotic response is amplified.
The compounds of the present invention, or their pharmaceutically acceptable salts may contain one or more asymmetric centers, chiral axes and chiral planes and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms and may be defined in terms of absolute stereochemistry, such as (R)- or (S)- or, as (D)- or (L)- for amino acids. The present invention is intended to include all such possible isomers, as well as, their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC. The racemic mixtures may be prepared and thereafter separated into individual optical isomers or these optical isomers may be prepared by chiral synthesis. The enantiomers may be resolved by methods known to those skilled in the art, for example by formation of diastereoisomeric salts which may then be separated by crystallization, gas-liquid or liquid chromatography, selective reaction of one enantiomer with an enantiomer specific reagent. It will also be appreciated by those skilled in the art that where the desired enantiomer is converted into another chemical entity by a separation technique, an additional step is then required to form the desired enantiomeric form. Alternatively specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts, or solvents or by converting one enantiomer to another by asymmetric transformation.
Certain compounds of the present invention may exist in Zwitterionic form and the present invention includes Zwitterionic forms of these compounds and mixtures thereof.
Utilities Attorney Docket No. L80003375WO The compounds of the present invention are useful as IAP BIR domain binding compounds and as such the compounds, compositions and method of the present invention include application to the cells or subjects afflicted with or having a predisposition towards developing a particular disease state, which is characterized by insufficient apoptosis. Thus, the compounds, compositions and methods of the present invention are used to treat cellular proliferative diseases/disorders, which include, but are not limited to, i) cancer, ii) autoimmune disease, iii) inflammatory disorders, iv) proliferation induced post medical procedures, including, but not limited to, surgery, angioplasty, and the like.
The compounds of the present invention may also be useful in the treatment of diseases in which there is a defect in the programmed cell-death or the apoptotic machinery (TRAIL, FAS, apoptosome), such as multiple sclerosis, asthma, artherosclerosis, inflammation, autoimmunity and the like.
The treatment involves administration to a subject in need thereof a compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof. In particular, the compounds, compositions and methods of the present invention are useful for the treatment of cancer including solid tumors such as skin, breast, brain, lung, testicular carcinomas, and the like. Cancers that may be treated by the compounds, compositions and methods of the invention include, but are not limited to the following:
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
The compounds of the present invention, or their pharmaceutically acceptable salts or their prodrugs, may be administered in pure form or in an appropriate pharmaceutical composition, and can be carried out via any of the accepted modes of Galenic pharmaceutical practice.
The pharmaceutical compositions of the present invention can be prepared by mixing a compound of the present invention with an appropriate pharmaceutically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral (subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), sublingual, ocular, rectal, vaginal, and intranasal. Pharmaceutical compositions of the present invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a subject. Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of the present invention in Attorney Docket No. L80003375WO aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Company, Easton, Pa., 1990). The composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease-state as described above.
A pharmaceutical composition of the present invention may be in the form of a solid or liquid. In one aspect, the carriers) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid or an aerosol, which is useful in, for example inhalatory administration.
For oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
When the pharmaceutical composition is in the form of a capsule, e.g., a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil such as soybean or vegetable oil.
The pharmaceutical composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred Attorney Docket No. L80003375WO composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
The liquid pharmaceutical compositions of the present invention, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; encapsulating agents such as cyclodextrins, including, but not limited to, α, β, or δ-hydroxypropylcyclodextins or Captisol; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine tetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.
A liquid pharmaceutical composition of the present invention used for either parenteral or oral administration should contain an amount of a compound of the present invention such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of a compound of the present invention in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. For parenteral usage, compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of the compound of the present invention. Pharmaceutical compositions may be further diluted at the time of administration; for example a parenteral formulation may be further diluted with a sterile, isotonic solution for injection such as 0.9 % saline, 5 wt % dextrose (D5W), Ringer's solution, or others.
The pharmaceutical composition of the present invention may be used for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and Attorney Docket No. L80003375WO alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. Topical formulations may contain a concentration of the compound of the present invention from about 0.1 to about 10% w/v (weight per unit volume).
The pharmaceutical composition of the present invention may be used for rectal administration to treat for example, colon cancer, in the form, e.g., of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
The pharmaceutical composition of the present invention may include various materials, which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule.
The pharmaceutical composition of the present invention in solid or liquid form may include an agent that binds to the compound of the present invention and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include, but are not limited to, a monoclonal or polyclonal antibody, a protein or a liposome.
The pharmaceutical composition of the present invention may consist of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of compounds of the present invention may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One skilled in the art, without undue experimentation may determine preferred aerosols.
The pharmaceutical compositions of the present invention may be prepared by Attorney Docket No. L80003375WO methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by admixing a compound of the present invention with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the compound of the present invention so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.
The compounds of the present invention, or their pharmaceutically acceptable salts, are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. Generally, a therapeutically effective daily dose may be from about 0.1 mg to about 40 mg/kg of body weight per day or twice per day of a compound of the present invention, or a pharmaceutically acceptable salt thereof.
Combination therapy The compounds of the present invention, or pharmaceutically acceptable salts thereof, may also be administered simultaneously with, prior to, or after administration of one or more of the therapeutic agents described below. Such combination therapy may include administration of a single pharmaceutical dosage formulation which contains a compound of the present invention and one or more additional agents given below, as well as administration of the compound of the present invention and each of additional agent in its own separate pharmaceutical dosage formulation. For example, a compound of the present invention and a chemotherapeutic agent, such as taxol (paclitaxel), taxotere, etoposide, cisplatin, vincristine, vinblastine, and the like, can be administered to the patient either together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations or via intravenous injection. Where separate dosage formulations are used, the compounds of the present invention and one or more additional agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially; combination therapy is understood to include all these regimens. In addition, these compounds may synergize with molecules that may stimulate the death receptor apoptotic pathway through a direct Attorney Docket No. L80003375WO or indirect manner, as for example, the compounds of the present invention may be used in combination with soluble TRAIL any agent or procedures that can cause an increase in circulating level of TRAIL, such as interferon-alpha or radiation.
Thus, the present invention also encompasses the use of the compounds of the present invention in combination with radiation therapy or one or more additional agents such as those described in WO 03/099211 (PCT/US03/15861), which is hereby incorporated by reference.
Examples of such additional agents include, but are not limited to the following: a) an estrogen receptor modulator, b) an androgen receptor modulator, c) retinoid receptor modulator, d) a cytotoxic agent, e) an antiproliferative agent, f) a prenyl-protein transferase inhibitor, g) an HMG-CoA reductase inhibitor, h) an HIV protease inhibitor, i) a reverse transcriptase inhibitor, k) an angiogenesis inhibitor, I) a PPAR-.γ agonist, m) a PPAR-. δ. agonist, n) an inhibitor of inherent multidrug resistance, o) an anti-emetic agent, p) an agent useful in the treatment of anemia, q) agents useful in the treatment of neutropenia, r) an immunologic-enhancing drug. s) a proteasome inhibitor such as Velcade and MG132 (7-Leu-Leu-aldehyde) (see He at al. in Oncogene (2004) 23, 2554-2558); t) an HDAC inhibitor, such as sodium butyrate, phenyl butyrate, hydroamic acids, cyclin tetrapeptide and the like (see Rosato et al,. Molecular Cancer Therapeutics 2003, 1273-
1284);' u) an inhibitor of the chemotrypsin-like activity in the proteasome; v) E3 ligase inhibitors; Attorney Docket No. L80003375WO w) a modulator of the immune system such as interferon-alpha and ionizing radition (UVB) that can induce the release of cytokines, such as the interleukins, TNF, or induce release of Death receptor Ligands such as TRAIL; x) a modulator of death receptors TRAIL and TRAIL agonists such as the humanized antibodies HGS-ETR1 and HGS-ETR2; and or in combination or sequentially with radiation therapy, so as to treat the cancer.
Additional combinations may also include agents which reduce the toxicity of the aforesaid agents, such as hepatic toxicity, neuronal toxicity, nephprotoxicity and the like.
In one example, co-administration of one of the compounds of Formula I of the present invention with a death receptor agonist such as TRAIL, such as a small molecule or an antibody that mimics TRAIL may cause an advantageous synergistic effect. Moreover, the compounds of the present invention may be used in combination with any compounds that cause an increase in circulating levels of TRAIL.
Vinca Alkaloids and Related Compounds
Vinca alkaloids that can be used in combination with the nucleobase oligomers of the invention to treat cancer and other neoplasms include vincristine, vinblastine, vindesine, vinflunine, vinorelbine, and anhydrovinblastine.
Dolastatins are oligopeptides that primarily interfere with tubulin at the vinca alkaloid binding domain. These compounds can also be used in combination with the compounds of the invention to treat cancer and other neoplasms. Dolastatins include dolastatin-10 (NCS 376128), dolastatin-15, ILX651 , TZT-1027, symplostatin 1 , symplostatin 3, and LU 103793 (cemadotin).
Cryptophycins (e.g., cryptophycin 1 and cryptophycin 52 (LY355703)) bind tubulin within the vinca alkaloid-binding domain and induce G2/M arrest and apoptosis. Any of these compounds can be used in combination with the compounds of the invention to treat cancer and other neoplasms.
Other microtubule disrupting compounds that can be used in conjunction with the compounds of the invention to treat cancer and other neoplasms are described in U.S. Pat. Nos. 6,458,765; 6,433,187; 6,323,315; 6,258,841 ; 6,143,721 ; 6,127,377; 6,103,698; Attorney Docket No. L80003375WO 6,023,626; 5,985,837; 5,965,537; 5,955,423; 5,952,298; 5,939,527; 5,886,025; 5,831 ,002;
5,741 ,892; 5,665,860; 5,654,399; 5,635,483; 5,599,902; 5,530,097; 5,521,284; 5,504,191; 4,879,278; and 4,816,444, and U.S. patent application Publication Nos. 2003/0153505 A1 ; 2003/0083263 A1; and 2003/0055002 A1, each of which is hereby incorporated by reference.
Taxanes and Other Micortubule Stabilizing Compounds
Taxanes such as paclitaxel, doxetaxel, RPR 109881 A, SB-T-1213, SB-T-1250, SB-T-
101187, BMS-275183, BRT 216, DJ-927, MAC-321 , IDN5109, and IDN5390 can be used in combination with the compounds of the invention to treat cancer and other neoplasms. Taxane analogs (e.g., BMS-184476, BMS-188797) and functionally related non-taxanes (e.g., epothilones (e.g., epothilone A, epothilone B (EPO906), deoxyepothilone B, and epothilone B lactam (BMS-247550)), eleutherobin, discodermolide, 2-epi-discodermolide, 2-des-methyldiscodermolide, 5-hydroxymethyldiscoder- molide, 19-des- aminocarbonyldiscodermolide, 9(13)-cyclodiscodermolide, and laulimalide) can also be. used in the methods and compositions of the invention.
Other microtubule stabilizing compounds that can be used in combination with the compounds of the invention to treat cancer and other neoplasms are described in U.S. Pat. Nos. 6,624,317; 6,610,736; 6,605,599; 6,589,968; 6,583,290; 6,576,658; 6,515,017;
6,531 ,497; 6,500,858; 6,498,257; 6,495,594; 6,489,314; 6,458,976; 6,441 ,186; 6,441 ,025;
6,414,015; 6,387,927; 6,380,395; 6,380,394; 6,362,217; 6,359,140; 6,306,893; 6,302,838;
6,300,355; 6,291 ,690; 6,291 ,684; 6,268,381 ; 6,262,107; 6,262,094; 6,147,234; 6,136,808;
6,127,406; 6,100,411 ; 6,096,909; 6,025,385; 6,011 ,056; 5,965,718; 5,955,489; 5,919,815; 5,912,263; 5,840,750; 5,821 ,263; 5,767,297; 5,728,725; 5,721,268; 5,719,177; 5,714,513;
5,587,489; 5,473,057; 5,407,674; 5,250,722; 5,010,099; and 4,939,168; and U.S. patent application Publication Nos. 2003/0186965 A1; 2003/0176710 A1 ; 2003/0176473 A1 ;
2003/0144523 A1 ; 2003/0134883 A1 ; 2003/0087888 A1 ; 2003/0060623 A1 ;
2003/0045711 A1 ; 2003/0023082 A1; 2002/0198256 A1; 2002/0193361 A1 ; 2002/0188014 A1; 2002/0165257 A1 ; 2002/0156110 A1 ; 2002/0128471 A1 ;
2002/0045609 A1; 2002/0022651 A1 ; 2002/0016356 A1 ; 2002/0002292 A1 , each of which is hereby incorporated by reference.
Other chemotherapeutic agents that may be administered with a compound of the present invention are listed in the following Table: Attorney Docket No. L80003375WO
Figure imgf000044_0001
Figure imgf000045_0001
Attorne Docket No. L80003375WO
Figure imgf000046_0001
Attorne Docket No. L80003375WO
Figure imgf000047_0001
Attorney Docket No. L80003375WO
Miscellaneous agents
SR-27897 (CCK A inhibitor, Sanofi- gemtuzumab (CD33 antibody, Wyeth Ayerst)
Synthelabo) CCI-779 (mTOR kinase inhibitor, Wyeth)
BCX-1777 (PNP inhibitor, BioCryst) PG2 (hematopoiesis enhancer, Pharmagenesis) tocladesine (cyclic AMP agonist, Ribapharm) exisulind (PDE V inhibitor, Cell Pathways) ranpirnase (ribonuclease stimulant, Alfacell) Immunol™ (triclosan oral rinse, Endo) alvocidib (CDK inhibitor, Aventis) CP-461 (PDE V inhibitor, Cell Pathways) galarubicin (RNA synthesis inhibitor, Dong- A) triacetyluridine (uridine prodrug, Wellstat)
CV-247 (COX-2 inhibitor, Ivy Medical) AG-2037 (GART inhibitor, Pfizer) tirapazamine (reducing agent, SRI International) SN-4071 (sarcoma agent, Signature BioScience)
P54 (COX-2 inhibitor, Phytopharm) WX-UKl (plasminogen activator inhibitor,
N-acetylcysteine (reducing agent, Zambon) Wilex)
CapCell™ (CYP450 stimulant, Bavarian TransMID-107 .TM. (immunotoxin, KS
Nordic) Biomedix)
R-flurbiprofen (NF-kappaB inhibitor, Encore) PBI-1402 (PMN stimulant, ProMetic
GCS-100 (gal3 antagonist, GlycoGenesys) LifeSciences)
3CPA (NF-kappaB inhibitor, Active Biotech) PCK-3145 (apoptosis promotor, Procyon)
G 17DT immunogen (gastrin inhibitor, Aphton) bortezomib (proteasome inhibitor, Millennium) seocalcitol (vitamin D receptor agonist, Leo) doranidazole (apoptosis promotor, PoIa) efaproxiral (oxygenator, Allos Therapeutics) SRL- 172 (T cell stimulant, SR Pharma) CHS-
131-I-TM-601 (DNA antagonist, 828 (cytotoxic agent, Leo)
TransMolecular) TLK-286 (glutathione S transferase inhibitor,
PI-88 (heparanase inhibitor, Progen) Telik) eflomithine (ODC inhibitor, ILEX Oncology) trans-retinoic acid (differentiator, NIH) tesmilifene (histamine antagonist, YM PT-100 (growth factor agonist, Point
BioSciences) Therapeutics) minodronic acid (osteoclast inhibitor, MX6 (apoptosis promotor, MAXIA)
Yamanouchi) midostaurin (PKC inhibitor, Novartis) histamine (histamine H2 receptor agonist, apomine (apoptosis promotor, ILEX Oncology)
Maxim) bryostatin-1 (PKC stimulant, GPC Biotech) indisulam (p53 stimulant, Eisai) urocidin (apoptosis promotor, Bioniche) tiazofurin (IMPDH inhibitor, Ribapharm) CDA-II (apoptosis promotor, Everlife) aplidine (PPT inhibitor, PharmaMar) Ro-31-7453 (apoptosis promotor, La Roche) cilengitide (integrin antagonist, Merck KGaA) SDX-101 (apoptosis promotor, Salmedix) rituximab (CD20 antibody, Genentech) brostallicin (apoptosis promotor, Pharmacia)
SR-31747 (IL-I antagonist, Sanofi-Synthelabo) ceflatonin (apoptosis promotor, ChemGenex)
Additional combinations may also include agents which reduce the toxicity of the aforesaid agents, such as hepatic toxicity, neuronal toxicity, nephprotoxicity and the like.
Screening assays
The compounds of the present invention may also be used in a method to screen for other compounds that bind to an IAP BIR domain. Generally speaking, to use the compounds of the invention in a method of identifying compounds that bind to an IAP BIR domain, the IAP is bound to a support, and a compound of the invention is added to the assay. Alternatively, the compound of the invention may be bound to the support and the IAP is added. Attorney Docket No. L80003375WO
There are a number of ways in which to determine the binding of a compound of the present invention to the BIR domain. In one way, the compound of the invention, for example, may be fluorescently or radioactively labeled and binding determined directly. For example, this may be done by attaching the IAP to a solid support, adding a detectably labeled compound of the invention, washing off excess reagent, and determining whether the amount of the detectable label is that present on the solid support. Numerous blocking and washing steps may be used, which are known to those skilled in the art.
In some cases, only one of the components is labeled. For example, specific residues in the BIR domain may be labeled. Alternatively, more than one component may be labeled with different labels; for example, using I125 for the BIR domain, and a fluorescent label for the probe. The compounds of the invention may also be used as competitors to screen for additional drug candidates or test compounds. As used herein, the terms "drug candidate" or "test compounds" are used interchangeably and describe any molecule, for example, protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, and the like, to be tested for bioactivity. The compounds may be capable of directly or indirectly altering the IAP biological activity.
Drug candidates can include various chemical classes, although typically they are small organic molecules having a molecular weight of more than 100 and less than about 2,500 Daltons. Candidate agents typically include functional groups necessary for structural interaction with proteins, for example, hydrogen bonding and lipophilic binding, and typically include at least an amine, carbonyl, hydroxyl, ether, or carboxyl group. The drug candidates often include cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more functional groups.
Drug candidates can be obtained from any number of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or Attorney Docket No. L80003375WO readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means.
Competitive screening assays may be done by combining an IAP BIR domain and a probe to form a probe:BIR domain complex in a first sample followed by adding a test compound from a second sample. The binding of the test is determined, and a change or difference in binding between the two samples indicates the presence of a test compound capable of binding to the BIR domain and potentially modulating the lAP's activity.
In one case, the binding of the test compound is determined through the use of competitive binding assays. In this embodiment, the probe is labeled with a fluorescent label. Under certain circumstances, there may be competitive binding between the test compound and the probe. Test compounds which display the probe, resulting in a change in fluorescence as compared to control, are considered to bind to the BIR region.
In one case, the test compound may be labeled. Either the test compound, or a compound of the present invention, or both, is added first to the IAP BIR domain for a time sufficient to allow binding to form a complex.
Formation of the probe:BIR domain complex typically require Incubations of between 4 0C and 40 0C for between 10 minutes to about 1 hour to allow for high-throughput screening. Any excess of reagents are generally removed or washed away. The test compound is then added, and the presence or absence of the labeled component is followed, to indicate binding to the BIR domain.
In one case, the probe is added first, followed by the test compound. Displacement of the probe is an indication the test compound is binding to the BIR domain and thus is capable of binding to, and potentially modulating, the activity of IAP. Either component can be labeled. For example, the presence of probe in the wash solution indicates displacement by the test compound. Alternatively, if the test compound is labeled, the presence of the probe on the support indicates displacement.
In one case, the test compound may be added first, with incubation and washing, followed by the probe. The absence of binding by the probe may indicate the test compound is bound to the BIR domain with a higher affinity. Thus, if the probe is detected on the Attorney Docket No. L80003375WO support, coupled with a lack of test compound binding, may indicate the test compound is capable of binding to the BIR domain.
Modulation is tested by screening for a test compound's ability to modulate the activity of IAP and includes combining a test compound with an IAP BIR domain, as described above, and determining an alteration in the biological activity of the IAP. Therefore in this case, the test compound should both bind to the BIR domain (although this may not be necessary), and alter its biological activity as defined herein.
Positive controls and negative controls may be used in the assays. All control and test samples are performed multiple times to obtain statistically significant results. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound probe determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.
Typically, the signals that are detected in the assay may include fluorescence, resonance energy transfer, time resolved fluorescence, radioactivity, fluorescence polarization, plasma resonance, or chemiluminescence and the like, depending on the nature of the label. Detectable labels useful in performing screening assays in this invention include a fluorescent label such as Fluorescein, Oregon green, dansyl, rhodamine, tetramethyl rhodamine, texas red, Eu3+; a chemiluminescent label such as luciferase; colorimetric labels; enzymatic markers; or radioisotopes such as tritium, I125 and the like
Affinity tags, which may be useful in performing the screening assays of the present invention include be biotin, polyhistidine and the like.
SYNTHESIS AND METHODOLOGY
General methods for the synthesis of the compounds of the present invention are shown below and are disclosed merely for the purpose of illustration and are not meant to be interpreted as limiting the processes to make the compounds by any other methods.
Those skilled in the art will readily appreciate that a number of methods are available for the preparation of the compounds of the present invention.
As illustrated in Scheme 1 , acid 1(i) is treated with amide coupling agents, followed by HNR4R5 to provide amide 1(ii). Deprotection of PG1 provides amine 1(iii). Attorney Docket No. L80003375WO PG2(R1)NHR2CCO2H is treated with an amide coupling agents, followed by 1(iii) to provide amide 1(iv). Deprotection of PG2 provides compounds of formula I.
Figure imgf000052_0001
1(i)
1(ii) X1=PG1 -η de protect 1(iii); X1=H J PG1
Figure imgf000052_0002
1(Iv); X2=PG2 . de protect formula I; X2=H "*~ PG2
Scheme 1
In the above Scheme, R1, R3, R4, R5, and R7 are as defined herein, PG1 and PG2 are protecting groups, Y' is NH, O, S or CH2, and Ra and RB may be combined to form an aromatic ring system wherein double bond may represent a single or double bond An example of these procedures for the synthesis of compound 1 is illustrated in Scheme 2.
Boc-(3S)-3-amino-1-carboxymethylcaprolactam, intermediate 1-1 , was coupled to (R) -1 ,2,3,4-tetrahydronaphthyl-1 -amine using the amide coupling reagents HBTU and HOBt, to provide intermediate 1-2a. Boc deprotection was carried out using 4N HCI in 1 ,4-dioxane to provide intermediate 1-2b as its HCI salt. Intermediate 1-3 was prepared by the coupling of Boc-N-MeAla-OH to intermediate 1-2bΗCI using the coupling agents HBTU and HOBt. Boc deprotection of intermediate 1-3 using 4N HCI in 1 ,4-dioxane provided compound 1 as its HCI salt. O
Figure imgf000053_0001
1-3; X=Boc 4N HCI in 1, X=H-HCI 1 ,4-dioxane
Scheme 2
A similar procedure, wherein the amine used in the first coupling step was changed from (R)-1,2,3,4-tetrahydronaphthyl-1-amine to phenethylamine provides compound 2 as its HCI salt.
Figure imgf000053_0002
2-HCI
Compound 3 was prepared according to a similar manner described for compound 1 , as illustrated in Scheme 2. (R)-3-Boc-amino-5-(carboxymethyl)-2,3-dihydro-1 ,5- benzothiazepin-4(5H)-one, 3-1 , was coupled to (R)-1 ,2,3,4-tetrahydronaphthyl-1 -amine using the amide coupling reagents HBTU and HOBt, to provide intermediate 3-2a. Boc deprotection was carried out using 4N HCI in 1 ,4-dioxane to provide intermediate 3-2b as its HCI salt. Intermediate 3-3 was prepared by the coupling of Boc-MeAla-OH to intermediate 3-2bΗCI using the coupling agents HBTU and HOBt. Boc deprotection of intermediate 3-3 using 4N HCI in 1 ,4-dioxane provided compound 3 as its HCI salt.
Figure imgf000054_0001
3-3; X=Boc 4N HCI in compound 3; X=H HCI 1 ,4-dioxane
Scheme 3
A similar procedure, wherein the amine used in the first coupling step was changed from (R)-1 ,2,3,4-tetrahydronaphthyl-1 -amine to phenethylamine provides compound 4 as its HCI salt.
Figure imgf000054_0002
4-HCI
EXAMPLES
The following abbreviations are used throughout:
Boc: f-butoxycarbonyl;
CBz: benzyloxycarbonyl;
DCM: dichloromethane;
DIPEA: diisopropylethylamine;
DMAP: 4-(dimethylamino)pyridine;
DMF: N.N-dimethylforrnamide;
DTT: dithiothreitol;
EDC: 3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; Attorney Docket No. L80003375WC
EDTA: ethylenediaminetetracetic acid;
Fmoc: N-(9-fluorenylmethoxycarbonyl);
HBTU: O-(benzotriazol-1-yl)-N,Λ/,Λ/',Λ/'-tetramethyluronium hexafluorophosphate;
HCI: hydrochloric acid;
HOAc: acetic acid;
HOBt: 1 -hydroxybenzotriazole;
HPLC: high performance liquid chromatography;
LCMS: liquid chromatography-mass spectrometer;
MeOH: methanol;
MgSO4: magnesium sulfate;
MS: mass spectrum;
NaHCO3: sodium hydrogen carbonate;
Pd/C: palladium on carbon;
TEA: triethylamine; and
THF: tetrahydrofuran.
Compound 1
Step i :
Boc-(3S)-3-amino-1-carboxymethylcaprolactam (286 mg, 1.0 mmol) was dissolved in DMF (5 mL) and treated with HBTU (564 mg, 1.5 mmol), HOBt (202 mg, 1.5 mmol) and DIPEA (520 μL, 3.0 mmol). The reaction was stirred for 5 minutes before (R)-1 , 2,3,4- tetrahydronaphthyl-1 -amine (176 μL, 1.2 mmol) was added. The reaction was stirred for 16 hrs at room temperature before being partitioned between ethyl acetate (100 mL) and 10% citric acid (20 mL). The organic phase was washed with 10 % citric acid (2 x 20 mL), saturated aqueous NaHCO3 (3 x 20 mL), and brine (1 x 20 mL). The organic fraction was dried over anhydrous MgSO4, filtered, and the volatiles removed under reduced pressure to provide 1-2a as a white solid, which was advanced to the next step without further purification.
Step 2:
Crude compound 1-2a was treated with 50 % TFA/CH2CI2 for 3 hrs. Volatiles were removed under reduced pressure and the residue suspended in diethyl ether (5 mL). An oil was formed and so volatiles were removed and the residue treated with 4N HCI in 1 ,4- dioxane (3 mL) for 30 minutes. Volatiles were removed under reduced pressure to Attorney Docket No. L80003375WO provide a white solid which was triturated with diethyl ether (3 x 10 mL) to provide 1-
2bΗCI as a white solid.
Step 3: Boc-Me-Ala-OH (253 mg, 1.25 mmol) was dissolved in DMF (5 mL) and treated with HBTU (564 mg, 1.5 mmol), HOBt (202 mg, 1.5 mmol) and DIPEA (700 μL, 4.0 mmol). The reaction was stirred for 5 minutes before crude 1-2b-HCI was added. The reaction was stirred for 16 hrs at room temperature before being partitioned between ethyl acetate (100 mL) and 10% citric acid (20 mL). The organic phase was washed with 10 % citric acid (2 x 20 mL), saturated aqueous NaHCO3 (3 x 20 mL), and brine (1 x 20 mL). The organic fraction was dried over anhydrous MgSO4, filtered, and the volatiles removed under reduced pressure. The resulting residue was purified by silica gel chromatography, eluting with a 10-100 % THF/hexanes gradient to provide 1-3 as a semi-solid.
Step 4:
Intermediate 1-3 was stirred with 4N HCI in 1 ,4-dioxane (3 mL) for 3 hours. Volatiles were removed under reduced pressure to provide a white solid which was suspended in diethyl ether and filtered, washing with diethyl ether (3 x 2 mL), to provide compound 1 HCI as a white solid (250 mg, 57 % yield over 4 steps). MS (m/z) M+1=401.2.
Compound 2
Compound 2 was prepared in a similar manner described for compound 1 wherein (R)- 1 ,2,3,4-tetrahydronaphthyl-1-amino was changed to phenethylamine to provide compound 2 as its HCI salt. MS (m/z) M+1 =375.2.
Compound 3
Step i :
(R)-3-Boc-amino-5-(carboxymethyl)-2,3-dihydro-1 ,5-benzothiazepin-4(5H)-one, 3-1 , (352 mg, 1.0 mmol) was dissolved in CH2CI2 (5 mL) and treated with EDC (232 mg, 1.5 mmol), HOBt (202 mg, 1.5 mmol) and DIPEA (520 μL, 3.0 mmol). The reaction was stirred for 5 minutes before (R)-1 , 2,3,4-tetrahydronaphthalamine (176 μL, 1.2 mmol) was added. The reaction was stirred for 16 hrs at room temperature before being partitioned between ethyl acetate (100 mL) and 10% citric acid (20 mL). The organic phase was washed with 10 % citric acid (2 x 20 mL), saturated aqueous NaHCO3 (3 x 20 mL), and brine (1 x 20 mL). Attorney Docket No. L80003375WO The organic fraction was dried over anhydrous MgSO4, filtered, and the volatiles removed under reduced pressure. The resulting residue was purified by silica gel chromatography, eluting with a 10-100 % THF/hexanes gradient to provide 3-2a as a white solid (360 mg, 75 % yield).
Step 2:
Compound 3-2a (360 mg) was treated with 4N HCI in 1 ,4-dioxane (3 ml.) for 3 hours. Volatiles were removed under reduced pressure to provide a white solid which was triturated with diethyl ether (3 x 10 mL) to provide 3-2tvHCI as a white solid (230 mg, 88 % yield). MS (m/z) M+ 1=382.2.
Step 3:
Boc-Me-Ala-OH (63 mg, 0.313 mmol) was dissolved in DMF (5 mL) and treated with
HBTU (140 mg, 0.375 mmol), HOBt (50 mg, 0.375 mmol) and DIPEA (96 μl_, 1.0 mmol). The reaction was stirred for 5 minutes before 3-2b HCI (104 mg, 0.25 mmol) was added. The reaction was stirred for 16 hrs at room temperature before being partitioned between ethyl acetate (100 mL) and 10% citric acid (20 mL). The organic phase was washed with 10 % citric acid (2 x 20 mL), saturated aqueous NaHCO3 (3 x 20 mL), and brine (1 x 20 mL). The organic fraction was dried over anhydrous MgSO4, filtered, and the volatiles removed under reduced pressure. The resulting residue was purified by silica gel chromatography, eluting with a 10-100 % THF/hexanes gradient to provide 3-3 as a semisolid (115 mg, 83 % yield). MS (m/z) M+23=589.2.
Step 4: Intermediate 3-3 (115 mg) was stirred with 4N HCI in 1,4-dioxane (3 mL) for 3 hours. Volatiles were removed under reduced pressure to provide an oil. The residue was washed with diethyl ether (3 x 5 mL) and then dissolved in CH2CI2 (2 mL) and hexane (1 mL). Volatiles were removed to provide compound 3ΗCI as a white solid (70 mg, 72 % yield). MS (m/z) M+1 =467.2.
Compound 4
Compound 4 was prepared in a similar manner described for compound 3 wherein (R)- 1 ,2,3,4-tetrahydronaphthyl-1 -amine was changed to phenethylamine to provide compound 4 as its HCI salt. MS (m/z) M+1 =441.2. Attorney Docket No. L80003375WO
Representative compounds of the present invention were prepared by simple modification of the above procedures and are illustrated in Table 1 :
TABLE 1
Figure imgf000058_0001
Representative compounds of the present invention which can be prepared by simple modification of the above procedures are illustrated in Table 2:
Attorney Docket No. L80003375WO
TABLE 2
Figure imgf000059_0001
l-l l-m
R1, R2 and R11 are define as herein;
R7 = H or C1-6 alkyl optionally substituted with aryl or heteroaryl;
Q is NR4R5;
R4 is H ;
R5 is chosen from:
Figure imgf000059_0002
alkyl / alkyl
Figure imgf000059_0003
Figure imgf000059_0004
X=CH2, S, S(O)m or O n=0,1, or 2
Figure imgf000059_0005
Attorney Docket No. L80003375WO
Figure imgf000060_0001
wherein the aryl and heteroaryl moieties may be substituted by R11, and wherein R11 and R11 are independently defined as R11 hereinabove, and wherein the alkyl may be further substituted by R6 as defined hereinabove.
Assays
Without wishing to be bound by theory, it is believed that the compounds of the present invention, reduce, or eliminate, the interaction of apoptotic proteins, such as caspase 3, caspase 7, and caspase 9, with the BIR domains of mammalian IAPs, which promote apoptosis of cancer cells as single agents or in combination with a chemotherapeutic agent. Also, the compounds may modulate IAP proteins in cells and induce the proteolytic processing of IAPs.
Molecular constructs for expression
GST-XIAP BIR3RING: XIAP coding sequence amino acids 246-497 cloned into PGEX2T1 via BamH1 and AVA I. The plasmid was transformed into E. coli DH5α for use in protein expression and purification.
GST-HIAP2 (clAP-1) BIR 3: HIAP2 coding sequence from amino acids 251-363 cloned into PGex4T3 via BamH1 and Xhol. The plasmid was transformed into E. coli DH5α for use in protein expression and purification.
GST-HIAP1(clAP-2) BIR 3: HIAP1 coding sequence from amino acids 236-349, cloned into PGex4T3 via BamH1 and Xhol. The plasmid was transformed into E. coli DH5α for use in protein expression and purification.
GST- linker BIR 2 BIR3Ring: XIAP coding sequence from amino acids 93-497 cloned into PGex4T1 via BamH1 and Xhol. Amino acids 93-497 were amplified from full length XIAP Attorney Docket No. L80003375WO in pGex4t3, using the primers: TTAATAGGATCCATCAACGGCTTTTATC and
GCTGCATGTGTGTCAGAGG, using standard PCR conditions. The PCR fragment was TA cloned into pCR-2.1 (invitrogen). Linker BIR 2 BIR 3Ring was subcloned into pGex4T1 by BamHI/Xhol digestion. The plasmid was transformed into E. coli DH5α for use in protein expression and purification.
GST-XIAP linker BIR 2: XIAP linker BIR 2 coding sequence from amino acids 93-497 cloned into pGex4T3 via BamHI and Xhol. The plasmid was transformed into E. coli DH5α for use in protein expression and purification.
Synthesis of fluorescent probe for FP assay
A fluorescent peptide probe, Fmoc-Ala-Val-Pro-Phe-Tyr(t-Bu)-Leu-Pro-Gly(t-Bu)-Gly-OH was prepared using standard Fmoc chemistry on 2-chlorotrityl chloride resin (Int. J. Pept. Prot. Res. 38:555-561 , 1991). Cleavage from the resin was performed using 20% acetic acid in dichloromehane (DCM), which left the side chain still blocked. The C-terminal protected carboxylic acid was coupled to 4'-(aminomethy)fluorescein (Molecular Probes, A-1351 ; Eugene, Oreg.) using excess diisopropylcarbodiimide (DIC) in dimethylformamide (DMF) at room temperature and was purified by silica gel chromatography (10% methanol in DCM). The N-terminal Fmoc protecting group was removed using piperidine (20%) in DMF, and purified by silica gel chromatography (20% methanol in DCM, 0.5% HOAc). Finally, the t-butyl side chain protective groups were removed using 95% trifluoroacetic acid containing 2.5% water and 2.5% triisopropyl silane to provide probe P1 (>95% pure).
Expression and purification of recombinant proteins A. Expression of Recombinant Proteins
Glutathione S-transferase (GST) tagged proteins were expressed in Escherichia coli strains DH5-alpha. For expression of the XIAP-BIR's, clAP-1 , clAP-2 and Livin transformed bacteria were cultured overnight at 37 0C in Luria Broth (LB) medium supplemented with 50 ug/ml of ampicillin. The overnight culture was then diluted 25 fold into fresh LB ampicillin supplemented media and bacteria were grown up to A600 = 0.6 then induced with 1 mM isopropyl-D-1-thiogalactopyranoside for 3 hours. Upon induction, cells were centrifuged at 5000 RPM for 10 minutes and the media was removed. Each pellet obtained from a 1 liter culture received 10 mL of lysis buffer (50 mM Tris-HCI, 200 mM NaCI, 1 mM DTT, 1 mM PMSF, 2 mg/mL of lysosyme), was incubated at 4 0C with Attorney Docket No. L80003375WO gentle shaking. After 20 minutes of incubation, the cell suspension was placed at -80 0C overnight or until needed.
B. Purification of recombinant proteins For purification of recombinant proteins, the IPTG-induced cell lysate was thaw and 100 uL of DNAase solution (100 ug/ml_) was added to the cell lysate and incubated at 4 0C for 30 minutes with gentle rocking. Cell lysate was then passed four times through a Bio-Neb Cell disruptor device (Glas-col) set at 100 Psi with Nitrogen gas. The resulting cell extract was centrifuged at 4 0C at 15000 RPM in a SS-34 Beckman rotor for 30 minutes. The resulting supernatant from 500 mL cell culture was then mixed with 2 ml. of glutathione- Sepharose beads (Pharmacia) for 1 hour at 4 0C. Upon incubation, the beads were washed 3 times with 1X Tris-Buffered Saline (TBS). Elution of the retained proteins was done with 3 washes of 2 ml of 50 mM TRIS pH 8.0 containing 10 mM reduced glutathione. The eluted proteins were pooled and precipitated with 604g/liter of ammonium sulfate and the resulting pellet re-suspended into an appropriate buffer. As judged by SDS-PAGE the purified proteins were >90% pure. The protein concentration of purified proteins was determined from the Bradford method.
His-tag proteins were expressed in the E. CoIi strain in E. coli AD494 cells using a pet28ACPP32 construct. The soluble protein fraction was prepared as described above. For protein purification, the supernatant was purified by affinity chromatography using chelating-Sepharose (Pharmacia) charged with NiSO4 according to the manufacturer's instructions. Purity of the eluted protein was >90% pure as determined by SDS-PAGE. The protein concentration of purified proteins was determined from the Bradford assay.
Binding assay
Fluorescence polarization-based competition assay
For all assays, the fluorescence and fluorescence-polarization was evaluated using a
Tecan Polarion instrument with the excitation filter set at 485 nm and the emission filter set at 535 nm. For each assay, the concentration of the target protein was first established by titration of the selected protein in order to produce a linear dose-response signal when incubated alone in the presence of the fluorescent probe. Upon establishing these conditions, the compound's potency (IC50) and selectivity, was assessed in the presence of a fix defined- amount of target protein and fluorescent probe and a 10 point serial dilution of the selected compounds. For each IC50 curve, the assays were run as followed: Attorney Docket No. L80003375WO 25 uL/well of diluted compound in 50 mM MES buffer pH 6.5 were added into a black 96 well plate then 25 ul/well of bovine serum albumin (BSA) at 0.5 mg/ml in 50 mM MES pH 6.5. Auto-fluorescence for each compound was first assessed by performing a reading of the compound/BSA solution alone. Then 25 uL of probe P1 diluted into 50 mM MES containing 0.05 mg/ml BSA were added and a reading to detect quenching of fluorescein signal done. Finally 25 uL/well of the target or control protein (GST- BIRs) diluted at the appropriate concentration in 50 mM MES containing 0.05 mg/ml BSA were added and the fluorescence polarization evaluated.
Determination of IC50 and Inhibitory constants
For each assay the relative polarization-fluorescence units were plotted against the final concentrations of compound and the IC50 calculated using the Grad pad prism software and/or Cambridge soft. The kj value were derived from the calculated IC5O value as described above and according to the equation described in Nikolovska-Coleska, Z. (2004) Anal Biochem 332, 261-273.
Caspase-3 linker BIR2 or Linker- BIR2- BIR3-RING derepression assay
In order to determine the relative activity of a selected compound against BIR2, an in vitro assay is used where caspase-3 is inhibited by linker- BIR2 or linker BIR2-BIR3-RING of XIAP. Briefly, 1.5 uL of the enzyme, 0.25 uM-2 uM of GST-XIAP fusion protein (GST-linker BIR2, GST-linker BIR2BIR3RING) is co-incubated with serial dilutions of compound (8OuM-0.04uM). Caspase 3 activity is measured by overlaying 25 uL of a 0.4 nM DEVD- AMC solution. Final reaction volume is 100 uL. All dilutions are performed in caspase buffer (50 mM Hepes pH 7.4, 10OmM NaCI, 10% sucrose, 1mM EDTA, 10 mM DTT, 0.1% CHAPS (Stennicke, H.R., and Salvesen, G. S. (1997). Biochemical characteristics of caspase-3, -6, -7, and -8. J. Biol. Chem. 272, 25719-25723)).
The fluorescent AMC released from the caspase-3 hydrolysis of the substrate is measured in a TECAN spectrophotometer at 360 nm excitation and 444 nm emission, on a kinetic cycle of 30 minutes with readings taken every 2 minutes. Caspase activity is calculated as V0 of AMC fluorescence/sec. Caspase de-repression by our compounds are compared to caspase-3 alone and caspase 3 repressed by the presence of XIAP fusion protein.
Pull-down assay Attorney Docket No. L80003375WO Compounds are dissolved in DMSO at 20 mM and used as the stock solution. Prior to the pull-down assay the compound affinity-agarose beads are prepared as follows: 300 uL of avidine-agarose beads prepared as 50% slurry in buffer A are incubated with 4OuI compound 107 (2.5 mM final) for 6h at 4 0C with shaking. Upon incubation the beads ware washed 3 times with buffer A.
On the day of the pull-down assay, cells such as MDA-MB-231 or 293A are collected and lyzed with a buffer A containing 20 mM TrisHCI, 150 mM NaCI, 10% Glycerol 1 % NP-40 and Protease inhibitor cocktail obtained from Sigma with a final protein concentration between 3 to 30 ug/uL.
For the pull-down assay, 60 uL of the compound-beads preparation are incubated with 300 ug cells lysate and 1OuL compound (0.36 mM final) or as control with 10 uL of buffer A to provide a final volume of 550 uL. The mixture was incubated at 40C over night with shaking. The following day, the beads are washed three times with the buffer A and the final bead pellet is re-suspended in 50 uL of 2X Leamli buffer. The samples are then centrifuged and the supernatant is collected for western blot analysis directed against XIAP of the other lAP's.
Alternatively a similar protocol is performed with AVPI-Biotin and IPVA-Biotin probes.
Assay to evaluate direct IAP binding compounds
A direct IAP binding molecule is used to identify IAP binding compounds by contacting the test compound with an IAP protein which is either fixed or non-fixed on a solid support. The non-binding interacting molecules are washed away and the bound compounds are identified using analytical techniques that can either identify directly the compound such as mass spectrometry, surface plasma resonance or can a evaluate a change of conformation or structure in the target molecule induced by the test compound or using techniques that can monitor the molecular interaction between the test compound and the target molecule such as NMR and protein crystallography.
Cell-free assay
Caspase de-repression assay using cellular extracts (apoptosome)
100ug of 293 cell S100 extract and 0.25 uM-2 uM of GST-XIAP fusion protein (GST- BIR 3RING, GST- linker BIR2 BIR3RING) are co-incubated with serial dilutions of compound Attorney Docket No. L80003375WO (0.02 uM-40 uM). Extracts are activated by adding 1 mM dATP, 0.1 mM ALLN, 133 ug
Cytochrome C (final concentrations), and incubating at 37 0C for 25 minutes. All reactions and dilutions used S100 buffer (50 mM Pipes pH 7.0, 5OmM KCI, 0.5mM EGTA pH 8.0, 2 mM MgCI2 supplemented with 1/1000 dilutions of 2 mg/mL Cytochalisin B, 2 mg/mL Chymostatin, Leupeptin, Pepstatin, Antipapin, 0.1 M PMSF, 1 M DTT). Final reaction volume is 3OuI. Caspase-3 activity is measured by overlaying 30 uL of a 0.4nM DEVD- AMC solution. AMC cleavage is measured in a TECAN spectrophotometer at 360 nm excitation and 444 nm emission, on a kinetic cycle of 1 hour with readings taken every 5 minutes. Caspase activity is calculated as V0 of AMC fluorescence/sec. Caspase de- repression by compounds are compared to fully activated extract and activated extract repressed by the presence of XIAP fusion protein.
Cell Culture and Cell Death Assays
A. Cell culture MDA-MD-231 (breast) and H460 (lung) cancer cells are cultured in RPMH 640 media supplemented with 10% FBS and 100 units/mL of Penicillin and Steptomycin.
S. Assays
Survival assays are routinely done on MDA-MB-231 and H460 cells. Cells are seeded in 96 well plates at a respective density of 5000 and 2000 cells per well and incubated at 370C in presence of 5% CO2 for 24 hours. Selected compounds are diluted into the media at various concentration ranging from 0.01 uM up to 100 uM. Diluted compounds are added onto the MDA-MB-231 cells. For H460 cells, the compounds are added either alone or in presence of 3 ng/ml of TRAIL. After 72 hours cellular viability is evaluated by MTS based assays. A solution of [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] is added onto cells for a period of 1 to 4 hours. Upon incubation the amount of converted MTS is evaluated using a Tecan spectrophotometer set at 570 nm.
Cellular biochemistry:
A. Detection of XIAP and PARP/Caspase-S/Caspase-Θ
Detection of cell expressed XIAP and PARP are done by western blotting. Cells are plated at 300,000 cells/well in a 60 mm wells (6 wells plate dish). The next day the cells are treated with selected compound at the indicated concentration. 24 hours later cells the trypsinized cells, pelleted by centrifugation at 1800 rpm at 4 0C. The resulting pellet is Attorney Docket No. L80003375WO rinsed twice with cold TBS. The final washed pellet of cells is the lysed with 250 uL Lysis buffer (NP-40, glycerol, 1% of a protease inhibitor cocktail (Sigma)), placed at 4 0C for 25 min with gentle shaking. The cells extract is centrifuged at 4 0C for 10min at 10,000 rpm. Both the supernatant and the pellet are kept for western blotting analysis as described below. From the supernatant, the protein content is evaluated and about 50ug of protein is fractionated onto a 10% SDS-PAGE. Pellets are washed with the lysis buffer and re- suspend into 5OuI of Lamelli buffer 1X, boiled and fractionated on SDS-PAGE. Upon electrophoresis each gel is electro-transferred onto a nitrocellulose membrane at 0.6A for 2 hours. Membrane non-specific sites are blocked for 1 hours with 5% Skim milk in TBST (TBS containing 0.1% (v/v) Tween-20) at RT. For protein immuno-detection, membranes are incubated overnight with primary antibodies raised against XIAP clone 48 obtained from Becton-Dickison) or PARP: obtained from Cell signal or caspase-3 or caspase-9 primary antibodies are incubated at 40C with shaking at dilutions as follows:
XIAP clone 80 (Becton-Dickinson) 1/2500 PARP (Cell Signal) 1/2500
Caspase 3 (Sigma) 1/1500
Caspase 9 (Upstate) 1/1000
Upon overnight incubation, the membranes receive three washes of 15 min in TBST then are incubated for 1 hour at room temperature in the presence of a secondary antibody coupled with HRP-enzyme (Chemicon) and diluted at 1/5 000. Upon incubation each membrane are washed three times with TBST and the immunoreactive bands are detected by addition of a luminescent substrate (ECL kit Amersham) and capture of signal on a X-RAY film for various time of exposure. Active compounds are shown to induce the cleavage of PARP and XIAP as well as to translocate XIAP into an insoluble compartment.
Hollow fiber model
A hollow fiber in vivo model is used to demonstrate in vivo efficacy of selected compounds against selected cell lines as single agent therapy or in combination with selected cytotoxic agents. At day 1 , selected cell lines are cultured and the fiber filled at a cell density of about 40,000 cells/fiber. At the day of operation (day 4), three fibers are implanted sub-cutaneous into 28-35 Nu/Nu CD-1 male mice. On day 5, mice start to receive daily injection via sub-cutaneous route of control vehicle or vehicle containing the selected compound at the appropriate concentration and/or injection of cytotoxic agent via Attorney Docket No. L80003375WO intra-peritoneal route. Upon 7 days of non-consecutive treatments, the animals are sacrificed, each fiber is removed and the metabolic viability of the remaining cells determined by MTT assay. Efficacy of the compound is defined as the difference between the vehicle-treated animal and the animal treated with the compound alone or the compound given in combination of the cytotoxic agent
Combination anti-cancer therapy in vivo with taxotere and compound
Female CD-1 nude mice (approximately 20-25 g) are subcutaneously injected with 1x106 H460 cells in the right flank. Animals are balanced into groups based on tumor size and drug therapy begins when tumors are ~30-50 mm3. Animals that have no tumor or that are deemed outliers because of excessive tumor size at this time are removed from the study. The remaining animals receive Taxotere (or equivalent volume of vehicle) at 30 mg/kg, ip 2 times, one week apart. The compound is given two times per day (at 10 mg/kg, sc, approximately 6hrs apart), starting at the time of Taxotere, and continuing daily for the duration of the experiment. If dehydration occurred, animals receive sc fluids (0.5ml). Tumor size is measured three times per week. Health assessments are performed at the time of the compound's delivery.
SKOV-3 Human Ovarian Cancer Cell Line Xenograpt Study with Compound 3 Female CD-1 nude mice (approximately 20-25 g) were subcutaneously injected 5 x 106 SKOV-3 human ovarian tumor cells in 50% matrigel subcutaneously in the right flank. On day 55, when tumors were approximately 100 mm3, treatment was initiated with compound 3 on a 5 on/2 off treatment schedule for the duration of the experiment. Tumor size was measured with digital calipers and calculated as V= (a x b2)/2, wherein, a is the longest dimension and b is the width.
MDA-MB-231 Human Mammary Cancer Cell Line Xenograph Study with Compound 3
Female CD-1 nude mice (approximately 20-25g) were subcutaneously injected 1 x 106 MDA-MB-231 human mammary tumor cells in the right flank. On day 71 , when tumors were approximately 90mm3, treatment was initiated with compound 3 on a 5 on/2 off treatment schedule for the duration of the experiment. Tumor size was measured with digital calipers and calculated as V=(a x b2)/2, wherein, a is the longest dimension and b is the width. Attorney Docket No. L80003375WO Pharmacokinetic studies
Selected compounds are dissolved into normal saline (0.9% NaCI) at 10 mg/ml and injected at 40 mg/Kg under sub-cutaneous route of administration. At each selected time point, blood sample of three mice is taken and the plasma fraction is prepared and kept frozen until analysis by liquid chromatography/electrospray mass spectrometry (LC/MS). At the day of analysis, plasma samples are thawed and extracted by liquid-liquid extraction procedure using 75% acetonitrile-water solution. Each extracted sample is analyzed for the presence of the selected compound on an Agilent 1100 LC/MS equipped with a C18 reverse-phase column. Quantitation of the compound in the plasma is done relatively to a plasma standard curve using of the selected compound. Upon determining the plasma concentration for each time point, the calculated area under the curve (AUC) and the peak at maximum concentration (Cmax) were calculated using Kinetica Version 4.2 software (Innaphase).
The compounds exemplified in Table 1 were tested and found to have IC5os of <50 μM using the fluorescence polarization assay. Each of the IC50S was calculated from Graph Pad.
Other Embodiments From the foregoing description, it will be apparent to one of ordinary skill in the art that variations and modifications may be made to the invention described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the present invention.
All publications mentioned in this specification are hereby incorporated by reference.

Claims

Attorney Docket No. L80003375WOCLAIMS
We claim: 1. A compound as represented by Formula I:
Figure imgf000069_0001
wherein n is O or i ; m is 0, 1 or 2; p is 1 or 2; Y is NH, O or S;
R1, R2, R3 and R4 are independently selected from: 1 ) H, or 2) C1-C6 alkyl, optionally substituted with one or more R7 substituents;
W is a 5, 6, or 7-membered cyclic amide ring, optionally including one or two O, S, or N heteroatoms, the cyclic amide ring being optionally fused with an aryl or a heteroaryl, wherein the aryl and the heteroaryl are optionally substituted with one or more R11 substituents, and wherein the cyclic amide ring is optionally substituted with one or more R7 substituents;
A is
1 ) -(C1-C6 alkyl)-C(O)-, or 2) -(C1-C6 alkyl)-; wherein the alkyl is optionally substituted with one or more R7 substituents;
Q is
I ) NR5R6, 2) OR12, Attorney Docket No. L80003375WO 3) S(O)01R12; or
Q is
Figure imgf000070_0001
wherein G is a 5, 6 or 7 membered ring which optionally incorporates one or more heteroatoms selected from S, N or O, and which is optionally substituted with one or more R13 substituents, and which is optionally fused with an aryl, the aryl being optionally substituted with one or more R11 substituents; or
Q is an aryl or a heteroaryl, the aryl and the heteroaryl being optionally substituted with one or more R11 substituents;
R5 and R6 are each independently selected from 1 ) H,
2) haloalkyl, 3) ^C1-C6 alkyl,
4) <-C2-C6 alkenyl,
5) -(-C2-C4 alkynyl,
6) <-C3-C7 cycloalkyl,
7) <-C3-C7 cycloalkenyl, 8) <-aryl,
9) <— heteroaryl,
10) <-heterocyclyl,
11 ) t-heterobicyclyl,
12) ^C(O)-R12, 13) ^C(O)O-R12,
14) <-C(= Y)NR9R10, or
15) ÷-S(O)2-R12, wherein th alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is optionally substituted with one or more R7 substituents; and wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl is optionally substituted with one or more R11 substituents;
R7 is Attorney Docket No. L80003375WO
1) halogen,
2) NO2,
3) CN,
4) haloalkyl, 5) C1-C6 alkyl,
6) C2-C6 alkenyl,
7) C2-C4 alkynyl,
8) C3-C7 cycloalkyl,
9) C3-C7 cycloalkenyl, 10) aryl,
11) heteroaryl, 12) heterocyclyl, 13) heterobicyclyl, 14) OR8, 15) S(O)mR8,
16) NR9R10 ,
17) NR8S(O)2R12,
18) COR8,
19) C(O)OR8, 20) CONR9R10,
2I ) S(O)2NR9R10
22) OC(O)R8,
23) OC(O)Y-R12,
24) SC(O)R8, or 25) NC(Y)NR9R10, wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl is optionally substituted with one or more R11 substituents;
R8 is 1) 1-1,
2) haloalkyl,
3) C1-C6 alkyl,
4) C2-C6 alkenyl,
5) C2-C4 alkynyl, 6) C3-C7 cycloalkyl, Attorney Docket No. L80003375WO
7) C3-C7 cycloalkenyl,
8) aryl,
9) heteroaryl,
10) heterocyclyl, 11 ) heterobicyclyl,
12) R9R10NCC=Y), or
13) C1-C6 alkyl-C2-C4 alkenyl, or
14) C1-C6 alkyl-C2-C4 alkynyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is optionally substituted with one or more R7 substituents; and wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl is optionally substituted with one or more R11 substituents;
R9 and R10 are each independently
1) H, 2) haloalkyl,
3) C1-C6 alkyl,
4) C2-C6 alkenyl,
5) C2-C4 alkynyl,
6) C3-C7 cycloalkyl, 7) C3-C7 cycloalkenyl,
8) aryl,
9) heteroaryl,
10) heterocyclyl,
11) heterobicyclyl, 12) C(O)R12,
13) C(O)Y-R12, or
14) S(O)2-R12, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is optionally substituted with one or more R7 substituents; and wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl is optionally substituted with one or more R11 substituents;
or R9 and R10 together with the nitrogen atom to which they are bonded form a five, six or seven membered heterocyclic ring optionally substituted with one or more R11 substituents; Attorney Docket No. L80003375WO R11 Is
1 ) halogen,
2) NO2,
3) CN, 4) B(OR13XOR14),
5) C1-C6 alkyl,
6) C2-C6 alkenyl,
7) C2-C4 alkynyl,
8) C3-C7 cycloalkyl, 9) C3-C7 cycloalkenyl,
10) haloalkyl,
11) OR8,
12) NR9R10,
13) SR8, 14) COR8,
15) C(O)O R8, 16) S(O)mR8,
17) CONR9R10,
18) S(O)2NR9R10, 19) aryl,
20) heteroaryl,
21) heterocyclyl, or
22) heterobicyclyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl is optionally substituted with one or more R7 substituents;
12 is
1) haloalkyl,
2) Ci-C6 alkyl,
3) C2-C6 alkenyl,
4) C2-C4 alkynyl,
5) C3-C; f cycloalkyl,
6) C3-C7 cycloalkenyl,
7) aryl, Attorney Docket No. L80003375WO
8) heteroaryl,
9) heterocyclyl, or
10) heterobicyclyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is optionally substituted with one or more R7 substituents; and wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl is optionally substituted with one or more R11 substituents;
R13 and R14 are each independently
I) H, or 2) C1-C6 alkyl; or
R13 and R14 are combined to form a heterocyclic ring or a heterobicyclyl ring; or a prodrug, or a pharmaceutically acceptable salt, or labeled with a detectable label or an affinity tag, or a stereoisomer thereof.
2. The compound, according to claim 1 , in which n is 0.
3. The compound, according to claim 1, comprising compounds of Formula IA:
Figure imgf000074_0001
wherein R1, R2, R3, R4, W and Q are as defined in claim"! .
4. The compound, according to claim 1 , in which W is a 6 or 7-membered cyclic amide ring, optionally including one or two O, S, or N heteroatoms, the cyclic amide ring being optionally fused with an aryl or a heteroaryl, wherein the aryl and the heteroaryl are optionally substituted with one or more R11 substituents, and wherein the cyclic amide ring is optionally substituted with one or more R7 substituents.
5. The compound, according to claim 4, in which W is a 7-membered cyclic amide ring, the cyclic amide ring being optionally fused with an aryl. Attorney Docket No. L80003375WO
6. The compound, according to claim 1, comprising compounds of Formula IB:
Figure imgf000075_0001
IB
wherein R1, R2, R3, R4, A and Q are as defined in claim 1.
7. The compound, according to claim 1, comprising compounds of Formula IC:
Figure imgf000075_0002
IC wherein R1, R2, R3, R4, A and Q are as defined in claim 1.
8. The compound, according to claim 4, in which W is a 7-membered cyclic amide ring, including one O, S, or N heteroatoms, the cyclic amide ring being optionally fused with an aryl or a heteroaryl.
9. The compound, according to claim 8, in which W is a 7-membered cyclic amide ring, including one O, S, or N heteroatoms, the cyclic amide ring being fused with an aryl.
10. The compound, according to claim 1 , comprising compounds of Formula ID:
Figure imgf000075_0003
ID wherein R1, R2, R3, R4, A and Q are as defined in claim 1. Attorney Docket No. L80003375WO
11. The compound, according to claim 1 , comprising compounds of Formula IE:
Figure imgf000076_0001
IE wherein R1, R2, R3, R4, A and Q are as defined in claim 1.
12. The compound, according to claim 1 , in which R1 is CH3.
13. The compound, according to claim 1 , in which R2 is H.
14. The compound, according to claim 1 , in which R3 is CH3.
15. The compound, according to claim 1, in which R4 is H.
16. The compound, according to claim 1 , in which A is -(C1-C6 alkyl)-C(O) -.
17. The compound, according to claim 16, in which A is -CH2C(O)-.
18. The compound, according to claim 1, in which Q is NR5R6.
19. The compound, according to claim 1 , in which R5 is H and R6 is selected from: 1 ) haloalkyl,
2) ^-C1-C6 alkyl,
3) *-C2-C6 alkenyl, 4) ^C2-C4 alkynyl,
5) <-C3-C7 cycloalkyl,
6) <— C3-C7 cycloalkenyl,
7) ^aryl,
8) <-heteroaryl, 9) <— heterocyclyl, or
10) <— heterobicyclyl, Attorney Docket No. L80003375WO wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is optionally substituted with one or more R7 substituents.
20. The compound, according to claim 19, in which R5 is H and R6 is selected from:
1 ) +-Ci-C6 alkyl, or
2) -aryl, wherein the alkyl substituted with phenyl.
21. The compound, according to claim 20, in which R5 is H and R6 is selected from the group consisting of:
Figure imgf000077_0001
22. The compound, according to claim 1, in which Q is selected from the group consisting of:
Figure imgf000077_0002
23. A compound, according to claim 1 :
Figure imgf000077_0003
or a pharmaceutically acceptable salt thereof.
24. A compound, according to claim 1 :
Figure imgf000077_0004
or a pharmaceutically acceptable salt thereof.
Attorney Docket No. L80003375WO 25. A compound, according to claim 1 :
Figure imgf000078_0001
or a pharmaceutically acceptable salt thereof.
26. A compound, according to claim 1:
Figure imgf000078_0002
or a pharmaceutically acceptable salt thereof.
27. An intermediate compound represented by Formula l(iv):
Figure imgf000078_0003
l(iv)
wherein PG2 is a protecting group, and R1, R3, R7, R4 R5, and wherein Y' has the same meaning as Y, and wherein Ra and Rb may be combined to form an aromatic ring system wherein double bond may represent a single or double bond.
28. A process for producing compounds represented by Formula I, according to claim 1 , the process comprising treating the intermediate as represented by Formula l(iv) under conditions suitable to remove the PG2 so as to produce the compound of Formula I.
29. An intermediate compound represented by Formula Attorney Docket No. L80003375WO
Figure imgf000079_0001
l(iii) wherein R7, R4 R5, Y', Ra and Rb are as defined herein.
30. A process for producing compounds represented by Formula I, according to claim 1 , the process comprising:
a) coupling an intermediate represented by Formula l(iii):
Figure imgf000079_0002
l(iii)
R1 , N. ,CO2H and R3 , in a solvent; and b) removing the protecting group PG2 so as to form compounds of Formula 1 , wherein PG2 is a protecting group, and R1, R3, R7, R4 R5, Y', Ra and Rb are as defined herein.
31. A pharmaceutical composition comprising a compound, according to claim 1 , mixed with a pharmaceutically acceptable carrier, diluent or excipient.
32. A pharmaceutical composition adapted for administration as an agent for treating a proliferative disorder in a subject, comprising a therapeutically effective amount of a compound, according to claim 1.
33. A pharmaceutical composition comprising a compound of Formula I, according to claim 1 , in combination with one or more death receptor agonists.
Attorney Docket No. L80003375WO 34. The composition, according to claim 33, in which the death receptor agonist is an agonist of TRAIL receptor.
35. A pharmaceutical composition comprising a compound of Formula I, according to claim 1 , in combination with any therapeutic agent that increases the response of one or more death receptor agonists.
36. A method of preparing a pharmaceutical composition, the method comprising: mixing a compound, according to claim 1 , with a pharmaceutically acceptable carrier, diluent or excipient.
37. A method of treating a disease state characterized by insufficient apoptosis, the method comprising: administering to a subject in need thereof, a therapeutically effective amount of a pharmaceutical composition, according to claim 31 , so as to treat the disease state.
38. A method of modulating IAP function, the method comprising: contacting a cell with a compound, according to claim 1 , so as to prevent binding of a BIR binding protein to an IAP BIR domain thereby modulating the IAP function.
39. A method of treating a proliferative disease, the method comprising: administering to a subject in need thereof, a therapeutically effective amount of the pharmaceutical composition, according to claim 31 , so as to treat the proliferative disease.
40. A method of treating cancer, the method comprising: administering to a subject in need thereof, a therapeutically effective amount of the pharmaceutical composition, according to claim 31 , so as to treat the cancer.
41. A method of treating cancer, the method comprising: administering to the subject in need thereof, a therapeutically effective amount of a pharmaceutical composition, according to claim 31 , in combination or sequentially with an agent selected from: a) an estrogen receptor modulator, b) an androgen receptor modulator, c) retinoid receptor modulator, d) a cytotoxic agent, Attorney Docket No. L80003375WO e) an antiproliferative agent, f) a prenyl-protein transferase inhibitor, g) an HMG-CoA reductase inhibitor, h) an HIV protease inhibitor, i) a reverse transcriptase inhibitor, k) an angiogenesis inhibitor,
I) a PPAR-.γ agonist, m) a PPAR-.δ. agonist, n) an inhibitor of inherent multidrug resistance, o) an anti-emetic agent, p) an agent useful in the treatment of anemia, q) agents useful in the treatment of neutropenia, r) an immunologic-enhancing drug. s) a proteasome inhibitor; t) an HDAC inhibitor;' u) an inhibitor of the chemotrypsin-like activity in the proteasome; or v) E3 ligase inhibitors; w) a modulator of the immune system such as, but not limited to, interferon-alpha, Bacillus
Calmette-Guerin (BCG), and ionizing radition (UVB) that can induce the release of cytokines, such as the interleukins, TNF, or induce release of death receptor ligands such as TRAIL; x) a modulator of death receptors TRAIL and TRAIL agonists such as the humanized antibodies HGS-ETR1 and HGS-ETR2; or in combination or sequentially with radiation therapy, so as to treat the cancer.
42. A method for the treatment or prevention of a proliferative disorder in a subject, the method comprising: administering to the subject a therapeutically effective amount of the composition, according to claim 41.
43. The method, according to claim 42, further comprises administering to the subject a therapeutically effective amount of a chemotherapeutic agent prior to, simultaneously with or after administration of the composition.
Attorney Docket No. L80003375WO 44. The method, according to claim 42, further comprises administering to the subject a therapeutically effective amount of a death receptor agonist prior to, simultaneously with or after administration of the composition.
45. The method, according to claim 44, in which the death receptor agonist is TRAIL or the death receptor agonist is a TRAIL antibody.
46. The method, according to claim 45, in which the death receptor agonist is administered in an amount that produces a clinical response.
47. A probe, the probe being a compound of Formula I, according to claim 1, the compound being labeled with a detectable label or an affinity tag.
48. A method of identifying compounds that bind to an IAP BIR domain, the assay comprising: a) contacting an IAP BIR domain with a probe to form a probe:BIR domain complex, the probe being displaceable by a test compound; b) measuring a signal from the probe so as to establish a reference level; c) incubating the probe:BIR domain complex with the test compound; d) measuring the signal from the probe; e) comparing the signal from step d) with the reference level, a modulation of the signal being an indication that the test compound binds to the BIR domain, wherein the probe is a compound of Formula I, according to claim 1 , labeled with a detectable label or an affinity label.
49. Use of a therapeutically effective amount of a pharmaceutical composition, according to claim 31 , for the manufacture of a medicament for treating a disease state characterized by insufficient apoptosis.
50. Use of a therapeutically effective amount of the pharmaceutical composition, according to claim 31 , for the manufacture of a medicament for treating a proliferative disease.
51. Use of a therapeutically effective amount of the pharmaceutical composition according to claim 31 , for the manufacture of a medicament for treating cancer. Attorney Docket No. L80003375WO
52. Use of a therapeutically effective amount of a pharmaceutical composition, according to claim 31 , in combination or sequentially with an agent selected from: a) an estrogen receptor modulator, b) an androgen receptor modulator, c) retinoid receptor modulator, d) a cytotoxic agent, e) an antiproliferative agent, f) a prenyl-protein transferase inhibitor, g) an HMG-CoA reductase inhibitor, h) an HIV protease inhibitor, i) a reverse transcriptase inhibitor, k) an angiogenesis inhibitor,
I) a PPAR-.γ agonist, m) a PPAR-.δ. agonist, n) an inhibitor of inherent multidrug resistance, o) an anti-emetic agent, p) an agent useful in the treatment of anemia, q) agents useful in the treatment of neutropenia, r) an immunologic-enhancing drug. s) a proteasome inhibitor; t) an HDAC inhibitor;' u) an inhibitor of the chemotrypsin-like activity in the proteasome; or v) E3 ligase inhibitors; w) a modulator of the immune system such as, but not limited to, interferon-alpha, Bacillus
Calmette-Guerin (BCG), and ionizing radition (UVB) that can induce the release of cytokines, such as the interleukins, TNF, or induce release of death receptor ligands such as TRAIL; x) a modulator of death receptors TRAIL and TRAIL agonists such as the humanized antibodies HGS-ETR1 and HGS-ETR2; or in combination or sequentially with radiation therapy, for the manufacture of a medicament for treating cancer. Attorney Docket No. L80003375WO
53. Use of a therapeutically effective amount of the composition, according to claim
31 , for the manufacture of a medicament for the treatment or prevention of a proliferative disorder in a subject.
54. The use, according to claim 53, further comprises a therapeutically effective amount of a death receptor agonist prior to, simultaneously with or after administration of the composition.
55. The use, according to claim 54, in which the death receptor agonist is TRAIL or the death receptor agonist is a TRAIL antibody.
56. The use, according to claim 55, in which the death receptor agonist is administered in an amount that produces a clinical response.
57. An intermediate compound represented by Formula l(ii):
Figure imgf000084_0001
1(B) wherein PG1 is a protecting group, and R7, R4 R5, and wherein Y' has the same meaning as Y, and wherein Ra and Rb may be combined to form an aromatic ring system wherein double bond may represent a single or double bond.
58. An intermediate compound represented by Formula l(ii):
Figure imgf000084_0002
1(iii) Attorney Docket No. L80003375WO wherein R7, R4 R5, and wherein Y' has the same meaning as Y, and wherein Ra and Rb may be combined to form an aromatic ring system wherein double bond may represent a single or double bond.
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