WO2011041737A2 - Inhibiteurs de bcl-2 à gain de fonction - Google Patents

Inhibiteurs de bcl-2 à gain de fonction Download PDF

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WO2011041737A2
WO2011041737A2 PCT/US2010/051208 US2010051208W WO2011041737A2 WO 2011041737 A2 WO2011041737 A2 WO 2011041737A2 US 2010051208 W US2010051208 W US 2010051208W WO 2011041737 A2 WO2011041737 A2 WO 2011041737A2
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compound
bcl
cells
overexpressing
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WO2011041737A3 (fr
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David Hockenbery
Julian Simon
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Fred Hutchinson Cancer Research Center
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Priority to US13/437,795 priority Critical patent/US8865901B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom 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
    • C07D213/72—Nitrogen atoms
    • C07D213/76—Nitrogen atoms to which a second hetero atom is attached
    • C07D213/77—Hydrazine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C205/00—Compounds containing nitro groups bound to a carbon skeleton
    • C07C205/13—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by hydroxy groups
    • C07C205/20—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by hydroxy groups having nitro groups and hydroxy groups bound to carbon atoms of six-membered aromatic rings
    • C07C205/21—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by hydroxy groups having nitro groups and hydroxy groups bound to carbon atoms of six-membered aromatic rings having nitro groups and hydroxy groups bound to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C205/22—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by hydroxy groups having nitro groups and hydroxy groups bound to carbon atoms of six-membered aromatic rings having nitro groups and hydroxy groups bound to carbon atoms of the same non-condensed six-membered aromatic ring having one nitro groups bound to the ring
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    • C07C251/86—Hydrazones having doubly-bound carbon atoms of hydrazone groups bound to carbon atoms of six-membered aromatic rings
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/10—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C323/18—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton
    • C07C323/20—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton with singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
    • C07C39/12—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic with no unsaturation outside the aromatic rings
    • C07C39/15—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic with no unsaturation outside the aromatic rings with all hydroxy groups on non-condensed rings, e.g. phenylphenol
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
    • C07C43/23—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring containing hydroxy or O-metal groups
    • C—CHEMISTRY; METALLURGY
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/257—Ethers having an ether-oxygen atom bound to carbon atoms both belonging to six-membered aromatic rings
    • C07C43/295—Ethers having an ether-oxygen atom bound to carbon atoms both belonging to six-membered aromatic rings containing hydroxy or O-metal groups
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/02—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
    • C07C69/12—Acetic acid esters
    • C07C69/16—Acetic acid esters of dihydroxylic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/02—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
    • C07C69/12—Acetic acid esters
    • C07C69/18—Acetic acid esters of trihydroxylic compounds
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/02—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
    • C07C69/22—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety
    • C07C69/30—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety esterified with trihydroxylic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/44—Radicals substituted by doubly-bound oxygen, sulfur, or nitrogen atoms, or by two such atoms singly-bound to the same carbon atom
    • C07D213/53—Nitrogen atoms

Definitions

  • the present invention relates to compounds for modulating apoptosis in cells over expressing Bcl-2 Family member proteins.
  • the present invention also relates to pharmaceutical compositions containing these compounds and methods of using the compounds.
  • Bcl-2, BC1-XL, and potentially other family members confer marked resistance to the cytotoxic effects of available anticancer agents.
  • Bcl-2 survival proteins are overexpressed in comparison to normal tissue counterparts in a significant subset of common cancers.
  • Low-basal BC1-XL expression is a strong negative predictor of cell survival with diverse classes of chemotherapeutic agents in the sixty cell lines included in the National Cancer Institute (NCI) anticancer drug screen. For these reasons, small-molecule inhibitors of Bcl-2 and BC1-XL are being considered as a goal for molecularly targeted cancer therapy.
  • 2-methoxy antimycin A represents the first of a novel class of inhibitors that display gain-of- function cytotoxicity, defined as enhanced killing in a cell line overexpressing a Bcl-2 survival protein target compared with an isogenic control cell line.
  • BC1-XL expression shows a positive correlation with sensitivity to 2-MeAA when comparing five cancer cell lines with the highest Bcl-x L mRNA expression in the NCI anticancer drug screen to five cell lines with lowest BC1-X L expression, i.e., 2-MeAA is most cytotoxic against cells with the highest BC1-XL expression.
  • BC1-XL expression levels show a negative correlation with standard therapeutic agents, i.e., standard therapeutics are less cytotoxic against cells with high Bcl-x L expression levels. Schwartz 2007.
  • BC1-XL inhibitors Overexpression of Bcl-x L in multiple cancers correlates with resistance to chemotherapy and radiation therapy, and provides a rationale for development of small-molecule BC1-XL inhibitors. Based on knockout studies, non-neoplastic cells also require BC1-XL survival functions, particularly when challenged with cytotoxic agents.
  • One BC1-XL inhibitor, 2-MeAA was found to be cytotoxic in cells with excess exogenous BC1-XL but had less cytotoxicity in isogenic cell line pairs having basal levels of BC1-XL expression (Tzung et al. Nature New Biol 2001 ;3: 183-91 ; Hockenbery et al. U.S. Patent No. 7,241,804, issued July 10, 2007; and
  • Figure 1 shows EC50 values for FH279 in micromolar units as measured in the following cell lines: CFPAC-1, HPAF-II MIA PaCa, PaTu-1, PaTu-8988t, ASPC-1, FA-6, and PANC-1, as described in Example 4.
  • Figure 2 shows the cytotoxic effect of FH279 (in micromolar units) on TAMH cells overexpressing BC1-XL (Tabx2s, squares)) compared to TAMH cells transfected with a neo expression vector (Tamh.neo, circles), as described in Example 5.
  • Figure 3 shows the relative expression levels of Bcl-2 (Fig. 3 A), Bcl-x L (Fig. 3B), and MCL-1 (Fig. 3C) measured in five pancreatic cell lines, expressed as percentage of control, maximal values, as described in Example 5.
  • Figure 4 shows the average tumor growth in an animal model following treatment with FH611 versus a vehicle control, as described in Example 7.
  • the compounds, as described herein, may be substituted with any number of substituents or functional moieties.
  • substituted whether preceded by the term “optionally” or not, and substituents contained in formulas of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent.
  • substituents When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • the term "substituted" is contemplated to include all permissible substituents of organic compounds, h a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms.
  • Alkyl as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms. In some embodiments, the alkyl employed in the invention contains 1 to 6 carbon atoms.
  • alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2- dimethylpentyl, 2,3- dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.
  • Lower alkyl as used herein, is a subset of alkyl, in some embodiments preferred, and refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms.
  • Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n- propyl, iso-propyl, n-butyl, iso-butyl,- tert- butyl, and the like.
  • alkyl or “lower alkyl” is intended to include both substituted and unsubstituted alkyl or lower alkyl unless otherwise indicated and these groups may be substituted with groups selected from halo (e.g., haloalkyl), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy (thereby creating a polyalkoxy such as polyethylene glycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, mercapto, alkyl-S(0) m , haloalkyl- S(0) m , alkenyl-S(0) m , alken
  • cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano where m 0, 1, 2 or 3.
  • alkenyl refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms (or in lower alkenyl 1 to 4 carbon atoms) which include 1 to 4 double bonds in the normal chain.
  • the alkenyl employed in the invention contains 1 to 6 carbon atoms.
  • Representative examples of alkenyl include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene, and the like.
  • alkenyl or “lower alkenyl” is intended to include both substituted and unsubstituted alkenyl or lower alkenyl unless otherwise indicated and these groups may be substituted with groups as described in connection with alkyl and lower alkyl above.
  • Alkynyl refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms (or in lower alkynyl 1 to 4 carbon atoms) which include 1 to 4 triple bond in the normal chain.
  • the alkynyl employed in the invention contain 1 to 6 carbon atoms.
  • Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2- butynyl, 4-pentynyl, 3- pentynyl, and the like.
  • alkynyl or “lower alkynyl” is intended to include both substituted and unsubstituted alkynyl or lower alkynyl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and lower alkyl above.
  • Cycloalkyl refers to groups having 3 to 10 carbon atoms. In some embodiments, the cycloalkyl employed in the invention has 3 to 8 carbon atoms. Suitable cycloalkyls include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the case of other aliphatic, heteroaliphatic or hetercyclic moieties, may optionally be substituted with the same groups as set forth in connection with alkyl and lower alkyl above.
  • Heterocycloalkyl or “heterocycle”, as used herein alone or as part of another group, refers to a non-aromatic 3-, 4-, 5-, 6-, 7-, or 8- membered ring or a polycyclic group, including, but not limited to a bi- or tri-cyclic group comprising fused six-membered rings having between one and four heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) the nitrogen and sulfur heteroatoms may be optionally oxidized, (ii) the nitrogen heteroatom may optionally be quatemized, and (iii) may form a spiro ring or be fused with a cycloalkyl, aryl, heterocyclic ring, benzene or a
  • heterocycle employed in the invention have 3 to
  • heterocycles include, but are not limited to, l,4-dioxa-8- azaspiro[4,5]decane, morpholine, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazolone, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline,
  • Aryl as used herein alone or as part of another group, refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused ring system having one or more aromatic rings. In some embodiments, the aryl employed in the invention has 3 to 14 carbon atoms.
  • aryl include, azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like.
  • aryl is intended to include both substituted and unsubstituted aryl unless otherwise indicated and these groups may be optionally substituted with the same groups as set forth in connection with alkyl and lower alkyl above.
  • Aryl alkyl as used herein alone or as part of another groups refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
  • Representative examples of aryl alkyl include, but are not limited to, benzyl, 2- phenyl ethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, and the like.
  • Heteroaryl refers to a cyclic, aromatic hydrocarbon in which one or more carbon atoms have been replaced with heteroatoms such as O, N, and S. If the heteroaryl group contains more than one heteroatom, the heteroatoms may be the same or different. In some embodiments, the heteroaryl employed in the invention have 3 to 14 carbon atoms.
  • heteroaryl groups include pyridyl, pyrimidinyl, imidazolyl, thienyl, furyl, pyrazinyl, pyrrolyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, indolyl, isoindolyl, indolizinyl, triazolyl, pyridazinyl, indazolyl, purinyl, quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, isothiazolyl, and benzo[b]thienyl.
  • pyridyl pyrimidinyl, imidazolyl, thienyl, furyl, pyrazinyl, pyrrolyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, indolyl, isoin
  • heteroaryl groups are five and six membered rings and contain from one to three heteroatoms independently selected from O, N, and S.
  • the heteroaryl group, including each heteroatom can be unsubstituted or substituted with from 1 to 4 substituents, as chemically feasible.
  • Alkoxy refers to an alkyl or lower alkyl group appended to the parent molecular moiety through an oxygen or sulfur atom.
  • the alkoxy or thioalkyl group contains 1-10 carbon atoms.
  • the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 carbon atoms.
  • the alkyl group contains 1-6 carbon atoms.
  • the alkyl group contains 1-4 carbon atoms.
  • alkoxy include but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert- butoxy, neopentoxy and n-hexoxy and the like.
  • thioalkyl include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.
  • Halo as used herein alone or as part of another group, refers to any suitable halogen, including -F, -CI, -Br, and -I.
  • Amin or "amino group”, as used herein alone or as part of another group, refers to the radical -NH2.
  • An “optionally substituted” amine refers to -NH2 groups wherein none, one or two of the hydrogen(s) is replaced by a suitable substituent. Disubstituted amines may have substituents that are bridging, i.e., form a heterocyclic ring structure that includes the amine nitrogen.
  • Aminoalkyl group is intended to mean the radical -NHR3, where R3 is an alkyl group.
  • Haloalkyl refers to an alkyl group having one, two, or three halogen atoms attached thereto and is exemplified by such groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.
  • apoptosis refers to a regulated network of biochemical events which lead to a selective form of cell suicide, and is characterized by readily observable morphological and biochemical phenomena, such as the fragmentation of the deoxyribo-nucleic acid (DNA), condensation of the chromatin, which may or may not be associated with endonuclease activity, chromosome migration, margination in cell nuclei, the formation of apoptotic bodies,
  • DNA deoxyribo-nucleic acid
  • condensation of the chromatin which may or may not be associated with endonuclease activity
  • chromosome migration chromosome migration
  • margination in cell nuclei the formation of apoptotic bodies
  • mitochondrial swelling widening of the mitochondrial cristae, opening of the mitochondrial permeability transition pores and/or dissipation of the mitochondrial proton gradient and the like.
  • apoptosis refers to at least a 5-fold greater stimulation of apoptosis, at a given concentration of an agent, including a 2-methoxy antimycin derivative, in cells that over-express a Bcl-2 family member protein as compared with cells that do not over- express the Bcl-2 family member protein (e.g., a 5-fold lower LD 50 or IC50).
  • an agent including a 2-methoxy antimycin derivative
  • substantially non-toxic refers to an agent, including 2-MeAA, that induces apoptosis in at least about 50 percent of cells that over- express a Bcl-2 family member protein, but does not induce apoptosis in more than about 5%, more preferably less than 1%, of cells that do not over-express the Bcl-2 family member protein.
  • Bcl-2 family member protein(s) refers to an evolutionarily conserved family of proteins characterized by having one or more amino acid homology domains, BHl, BH2, BH3, and/or BH4.
  • the Bcl-2 family member proteins include Bcl-2, Bcl-x L , Bcl-w, Al, Mcl-l, Bax, Bak, Bad, Bcl-xs, Bid, and the like.
  • the "Bcl-2 family member proteins” further include those proteins, or their biologically active fragments, that have at least 70%, preferably at least 80%, and more preferably at least 90% amino acid sequence identity with a Bcl-2 family member protein.
  • anti-apoptotic Bcl-2 family member protein refers to Bcl-2, BC1-XL, BCI- W, Al, Mcl-l, and other proteins characterized by having one or more amino acid homology domains, BHl, BH2, BH3, and/or BH4, and that promote cell survival by attenuating or inhibiting apoptosis.
  • the "anti-apoptotic Bcl-2 family member proteins” further include those proteins, or their biologically active fragments, that have at least 70%, preferably at least 80%, and more preferably at least 90% amino acid sequence identity with an anti- apoptotic Bcl-2 family member protein.
  • identity refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using either a PILEUP or BLAST sequence comparison algorithm (see, e.g., J. MoT Evol. 35:351-360, 1987; Higgins and Sharp, CABIOS 5: 151-153, 1989; Altschul et al, J. Mol Biol 215:403-410, 1990; Zhang et al, Nucleic Acid Res. 26:3986-3990, 1998; Altschul et al, Nucleic Acid Res. 25:3389- 33402, 1997).
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see, generally, Ausubel et al, supra).
  • a mutant Bcl-2 family member amino acid sequence having one or more amino acid substitutions, additions, or deletions as compared to the wild-type protein may correspond to a second Bcl-2 family member amino acid sequence (e.g., the wild-type sequence or a functionally equivalent variant thereof) according to the convention for numbering the second Bcl-2 family member sequence, whereby the mutant sequence is aligned with the second Bcl-2 family member sequence such that at least 50%, typically at least 60%, more typically, at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% of the amino acids in a given sequence of at least 20 consecutive amino acids are identical. Because not all positions with a given "corresponding region" need be identical, non- matching positions within a corresponding region are herein regarded as "corresponding positions.”
  • a single amino acid substitution in one ("first") mutant Bcl-2 family member protein "corresponds" to a single amino acid substitution in a second mutant Bcl-2 family member protein (e.g., Bcl-x L ) where the corresponding substituted amino acid positions of the first and second mutant proteins are identical.
  • the phrase "no substantial effect on tertiary protein structure relative to the corresponding wild-type Bcl-2 family member protein” or “no substantial alteration of tertiary protein structure relative to the corresponding wild-type Bcl-2 family member protein” means that, when a C trace providing a position for each Co; carbon of the mutant protein is superimposed onto a Co; trace of the corresponding wild- type protein and an a carbon root mean square (RMS) difference root mean square deviation (RMSD) is calculated; i.e., the deviation of the mutant structure from that of the wild-type structure), the RMSD value is no more than about 1.0 A when calculated using the same structural modeling method, typically no more than about 0.75 A, even more typically no more than about 0.5 A, preferably no more than about 0.35 A, and even more preferably no more than about 0.25 A.
  • RMS carbon root mean square
  • biologically active refers to the ability of a molecule to modulate apoptosis, such as by binding to a Bcl-2 family member protein.
  • a biologically active molecule can modulate apoptosis by causing a change in the mitochondrial proton motive force gradient, by causing a change in mitochondrial swelling or the morphological
  • an effective amount or “effective” can designate an amount that maintains a desired physiological state, i.e., reduces or prevents significant decline and/or promotes improvement in the condition of interest.
  • an amount of an agent that effectively modulates the apoptotic state of an individual cell such that apoptosis is induced and/or the inappropriately regulated cell death cycle in the cell returns to a normal state.
  • the dosage will vary depending on the administration routes, symptoms and body weight of the patient but also depending upon the compound being administered.
  • terapéuticaally useful and “therapeutically effective” refer to an amount of an agent that effectively modulates the apoptotic state of an individual cell such that apoptosis is induced and/or the inappropriately regulated cell death cycle in the cell returns to a normal state.
  • diagnosis refers to an agent (e.g., an antimycin derivative) for detecting the induction or inhibition of apoptosis in a subject. These terms further include molecules useful for detecting diseases associated with apoptosis, or the susceptibility to such diseases, and for detecting over-expression or under- expression of a Bcl-2 family member protein.
  • an agent e.g., an antimycin derivative
  • over-expression and “under-expression” refer to an increase or decrease, respectively, in the levels of a Bcl-2 family member protein in a cell relative to the level of such a protein found in the same cell or a closely related non-malignant cell under normal
  • apoptosis-associated disease includes diseases, disorders, and conditions that are linked to an increased or decreased state of apoptosis in at least some of the cells of a subject.
  • diseases include neoplastic disease (e.g., cancer and other
  • proliferative diseases tumor formation, arthritis, inflammation, autoimmune disease, human immunodeficiency virus (HIV) immunodeficiency syndrome, neurodegenerative diseases, myelodysplastic syndromes (such as aplastic anemia), ischaemic syndromes (such as myocardial infarction), liver diseases which are induced by toxins (such as alcohol), alopecia, damage to the skin due to UV light, lichen planus, atrophy of the skin, cataract, and graft rejections and other premalignant and noneoplastic hyperproliferative disorders.
  • Apoptosis- associated diseases further include drug resistance associated with increased or decreased levels of an anti-apoptotic
  • Ri is hydroxyl, halide, or alkoxy
  • R 2 is H, N0 2 , halide, alkyl, or alkoxy
  • R 3 is H, or alkyl
  • R 4 is H, halide, or alkyl
  • R 5 is H, halide, or hydroxyl
  • A is CH or N
  • R 7 is H, N0 2 , or CF 3 ;
  • R 8 is H or halide
  • R 1 is hydroxyl, CI, methoxyl
  • R 2 is H, N0 2 , CI, Br, t-butyl, or methoxyl
  • R 3 is H, methyl, or t-butyl
  • R 4 is H, CI, Br, t-butyl, or methyl
  • R 5 is H, CI, or hydroxyl
  • R 7 is H, N0 2 , or CF 3;
  • R 8 is H, or CI. Most preferably when X is -NH-R 5 , R 5 is
  • A is N;
  • R 5 is
  • A is CH or N; or R 5 is
  • R 6 is H or CF 3; or R 5 is
  • R 6 is CF 3 .
  • R 6 is H.
  • Another embodiment of the invention is a compound consisting of the structure of Formulas II or III,
  • Ri, R 2 , and R 3 are each independently H, OH, alkoxy, or OAc; R , R 5 , and R 6 are each independently H, alkyl , or halide and A is CH 2 , O, S, SO, S0 2 , or none.
  • R 2 is H, OH, OMe, or OAc
  • R 4 is H, Me, t-butyl, or CI
  • R 5 is H, Me, or CI
  • R 6 is H
  • A is O or CH 2 .
  • Another aspect of the invention is a compound selected from the group consisting of:
  • Another aspect of the invention is a compound consisting of formula:
  • A is -S-CH2-; -SO-CH2-; or -S02-.
  • Another aspect of the invention is a compound consisting of formula
  • Another aspect of the invention is any one of the above compounds in which the active compound has an IC50 less than about 5 micromole/liter (5 ⁇ ) in cells overexpressing BC1-XL, and at least about 20-fold less cytotoxic in isogenic cells that are not overexpressing Bcl-x L .
  • the active compound has an IC50 less than about 5 micromole/liter (5 ⁇ ) in cells overexpressing BC1-XL, and at least about 20-fold less cytotoxic in isogenic cells that are not overexpressing Bcl-x L . Examples of such compound are:
  • a most preferred embodiment is an active compound that has an IC50 less than about 3 ⁇ in cells overexpressing Bcl-x L .
  • Examples of such compounds are:
  • Another aspect of the invention is a compound described above in which the active compound has an IC50 less than about 5 micromole/liter (5 ⁇ ) in cells overexpressing Bcl- and at least about 10-fold less cytotoxic in isogenic cells that are not overexpressing BC1-XL.
  • the active compound has an IC50 less than about 5 micromole/liter (5 ⁇ ) in cells overexpressing Bcl- and at least about 10-fold less cytotoxic in isogenic cells that are not overexpressing BC1-XL.
  • Examples of such compounds include:
  • the compound selected from this group has an IC50 less than about 3 ⁇ cells overexpressing Bcl-x L .
  • Examples of such compound include:
  • Another aspect of the invention is a compound described above in which the active compound has an IC50 less than about 5 micromole/liter (5 ⁇ ) in cells overexpressing Bcl- and at least about 5-fold less cytotoxic in isogenic cells that are not overexpressing Bcl-x L .
  • the active compound has an IC50 less than about 5 micromole/liter (5 ⁇ ) in cells overexpressing Bcl- and at least about 5-fold less cytotoxic in isogenic cells that are not overexpressing Bcl-x L .
  • Examples of such compounds include:
  • the compound of this group has an IC50 less than about 3 ⁇ in cells overexpressing BC1-XL.
  • Examples of such compounds include:
  • Another aspect of the invention is an active compound that has an IC50 less than about 3 micromole/liter (3 ⁇ ) in cells overexpressing BC1-XL, and at least about 3 -fold less cytotoxic in isogenic cells that are not overexpressing BC1-XL.
  • active compound that has an IC50 less than about 3 micromole/liter (3 ⁇ ) in cells overexpressing BC1-XL, and at least about 3 -fold less cytotoxic in isogenic cells that are not overexpressing BC1-XL. Examples of such compounds include:
  • the active compound has an IC50 less than about 3 ⁇ in cells overexpressing BC1-XL.
  • examples of such compounds include:
  • Another aspect of the invention is an active compound that has an IC50 less than about 5 micromole/liter (5 ⁇ ) in cells overexpressing BCI-XL, and at least about 2-fold less cytotoxic in isogenic cells that are not overexpressing BCI-XL-
  • this group of compounds include:
  • the active compound has an IC50 less than about 3 ⁇ in cells overexpressing such compound is:
  • Another aspect of the invention is a method for treating an apoptosis-associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an active compound selected from any group of compounds described above.
  • the compounds of the present invention are useful for treating cells in which the cell death signal is down-regulated and the affected cell has an inappropriately diminished propensity for cell death, which is referred to herein as being in a "decreased apoptotic state.”
  • the invention further provides methods for the administration to a subject, a therapeutically effective amount of an apoptosis-modulating compound of the invention to treat an apoptosis-associated disease in which it is desirable to induce apoptosis in certain types of cells, such as virus-infected or autoantibody-expressing cells.
  • a method of treating a cancer characterized by the over- expression of a Bcl-2 family member is provided.
  • the treatment of the cancer can include the treatment of solid tumors or the treatment of leukemias.
  • the cancer can be of the skin, breast, brain, cervix, testis, and the like. More particularly, cancers may include, but are not limited to, the following organs or systems: cardiac, lung, gastrointestinal, genitourinary tract, liver, bone, nervous system, gynecological, hematologic, skin, and adrenal glands.
  • the methods herein can be used for treating gliomas (Schwannoma, glioblastoma, astrocytoma), neuroblastoma, pheochromocytoma, paraganlioma, meningioma, adrenal cortical carcinoma, kidney cancer, vascular cancer of various types, osteoblastic osteocarcoma, prostate cancer, ovarian cancer, uterine leiomyomas, salivary gland cancer, choroid plexus carcinoma, mammary cancer, pancreatic cancer, colon cancer, B and T cell lymphomas, acute and chronic myeloid or lymphoid leukemias, and multiple myeloma.
  • gliomas Rosta, glioblastoma, astrocytoma
  • neuroblastoma pheochromocytoma
  • paraganlioma paraganlioma
  • meningioma adrenal cortical carcinoma
  • kidney cancer vascular cancer of various types
  • osteoblastic osteocarcoma prostate
  • treatment may include pre-malignant conditions associated with any of the above cancers (e.g., colon adenomas, myelodysplastic syndrome).
  • methods of treating a neurodegenerative disease characterized by the over-expression of a Bcl-2 family member are provided.
  • Neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis and other diseases linked to degeneration of the brain, such as Creutzfeldt- Jakob disease and expanded polyglutamine repeat diseases.
  • Expanded polyglutamine repeat diseases with which the present invention is concerned include, but are not limited to, Huntington's disease, dentatorubral pallidoluysian atrophy, spinobulbar muscular atrophy, and spinocerebellar ataxia types 1, 2, 3, 6 and 7. See, e.g., Burke et al. US Patent No. 6,632,616.
  • methods of treating arthritis, inflammation, autoimmune diseases, human immunodeficiency virus (HIV) immunodeficiency syndrome, myelodysplastic syndromes are provided.
  • the compounds used in embodiments of the invention will be substantially purified prior to administration.
  • the subject can be an animal, including, but not limited to, cows, pigs, horses, chickens, cats, dogs, and the like, and is typically a mammal, and in a particular embodiment human. In another specific embodiment, a non- human mammal is the subject.
  • apoptosis-modulating compounds are administered as therapeutic or pharmaceutical compositions by any suitable route known to the skilled artisan including, for example, intravenous, subcutaneous, intramuscular, intradermal, transdermal, intrathecal, intracerebral, intraperitoneal, intransal, epidural, and oral routes.
  • Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulations.
  • administration can be by injection or infusion into the cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • administration can be with one or more other components capable of promoting penetration of the derivative across the blood- brain barrier.
  • it can be desirable to introduce a compound into the target tissue by any suitable route, including intravenous and intrathecal injection.
  • Pulmonary administration can also be employed, such as, for example, by use of an inhaler or nebulizer, and formulation of the compound with an aerosolizing agent.
  • the compound is coadministered with an inhibitor of esterase activity to further stabilize the compound.
  • Pharmaceutical compositions can also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, caplets, lozenges, aqueous suspensions or solutions.
  • carriers that are commonly used include lactose and corn starch.
  • Lubricating aids such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried corn starch.
  • the agent can be combined with emulsifying and suspending aids. If desired, certain sweeteners, flavorants, or colorants can also be used.
  • the compounds of the present invention can be combined with any other tumor and/or cancer therapy.
  • the therapy can include, for example and not by way of limitation, surgery, radiation, and chemotherapy either individually or in any combination.
  • Chemotherapy can include any current known or yet to be discovered chemotherapeutic agent including but are not limited to Aceglatone; Aclarubicin; Altretamine; Aminoglutethimide; 5- Aminogleavulinic Acid; Amsacrine; Anastrozole; Ancitabine Hydrochloride; 17-1 A Antibody; Antilymphocyte Immunoglobulins; Antineoplaston AIO; Asparaginase; Pegaspargase; Azacitidine; Azathioprine; Batimastat; Benzoporphyrin Derivative; Bicalutamide; Bisantrene Hydrochloride; Bleomycin
  • Dichlorodiethylsulphide Didemnin B.; Docetaxel; Doxifiuridine; Doxorubicin Hychloride; Droloxifene; Echinomycin; Edatrexate; Elliptinium; Elmustine; Enloplatin; Enocitabine;
  • Fluorouracil Fluorouracil; Flutamide; Formestane; Fotemustine; Gallium Nitrate; Gencitabine; Gusperimus; Homoharringtonine; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine;
  • Improsulfan Tosylate Inolimomab; lnterleukin-2; Irinotecan; JM-216; Letrozole; Lithium Gamolenate; Lobaplatin; Lomustine; Lonidamine; Mafosfarnide; Meiphalan; Menogaril;
  • the compounds of the present invention can be administered locally to the area in need of treatment; this administration can be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application (e.g., in conjunction with a wound dressing after surgery), by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non- porous, or gelatinous material, including membranes such as silastic membranes, or fibers.
  • administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
  • the compounds of the invention can be delivered in a vesicle, in particular a liposome (see, e.g., Langer, Science 249:1527-33, 1990; Treat et al, In Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-65, 1989; Lopez-Berestein, supra, pp. 317-27).
  • a liposome see, e.g., Langer, Science 249:1527-33, 1990; Treat et al, In Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-65, 1989; Lopez-Berestein, supra, pp. 317-27).
  • the compounds of the invention can be delivered in a controlled release system.
  • a pump can be used (see, e.g., Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201, 1987; Buchwald et ah, Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med. 321 :574, 1989).
  • polymeric materials can be used (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida, 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York, 1984; Ranger and Peppas, J. Macro mol. Sci. Rev. Macromol. Chem. 23:61, 1983; see also Levy et al, Science 228: 190, 1985; During et al, Ann. Neurol. 25:351, 1989; Howard et al, J. Neurosurg. 71 :105, 1989).
  • a controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical
  • compositions comprise a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of the invention.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more typically in humans.
  • carrier refers to a diluent, adjuvant, excipient, stabilizer, vehicle, or any combination thereof, with which the agent is formulated for administration.
  • Pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • Water is a typical carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol
  • compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • composition includes an inhibiter of esterase activity as a stabilizing agent.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • suitable pharmaceutical carriers are described in, for example, Remington 's Pharmaceutical Sciences, by E.W. Martin.
  • Such compositions will contain a therapeutically effective amount of a compound of the invention, typically in purified form, together with a suitable amount of carrier so as to provide a formulation proper for administration to the subject.
  • the formulation should suit the mode of administration.
  • the compound of the present invention is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • pharmaceutical compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition can also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form. For example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
  • the compounds of the invention can be formulated as neutral or salt forms.
  • pharmaceutically acceptable salt refers to a salt form of a compound permitting its use or formulation as a pharmaceutical and which retains the biological effectiveness of the free acid and base of the specified compound and that is not biologically or otherwise undesirable. Examples of such salts are described in Handbook of Pharmaceutical Salts:
  • Examples of pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
  • salts also include sulfates, pyrosulfates, bisulfates, sulfites, bisulfltes, phosphates, monohydrogenphosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates,
  • pharmaceutically acceptable salt includes sodium, potassium, calcium, ammonium, trialkylarylammonium and tetraalkylammonium salts.
  • prodrugs of the compounds may be used in embodiments of the invention.
  • Pharmaceutically acceptable prodrugs as used herein refers to those prodrugs of the active compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable risk/benefit ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention.
  • prodrug refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V.
  • Examples include a prodrug that is metabolized in vivo by a subject to an active drug having an activity of active compounds as described herein, wherein the prodrug is an ester of an alcohol or carboxylic acid group, if such a group is present in the compound; an acetal or ketal of an alcohol group, if such a group is present in the compound; an N-Mannich base or an imine of an amine group, if such a group is present in the compound; or a Schiff base, oxime, acetal, enol ester, oxazolidine, or thiazolidine of a carbonyl group, if such a group is present in the compound, such as described in US Patent No. 6,680,324 and US Patent No. 6,680,322.
  • the amount of the compound of the invention that is combined with the carrier to produce a single dosage form will vary, depending upon the nature of that agent and the composition of the dosage form. It should be understood, however, that a specific dosage and treatment regime for any particular patient or disease state will depend upon a variety of factors, including the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the judgment of the treating physician, and the severity of the particular disease being treated. The amount of active agent will also depend upon the specific activity of the compound and whether that agent is co-administered with any other therapeutic or prophylactic ingredients.
  • Determination of therapeutically effective dosages is typically based on animal model studies and is guided by determining effective dosages and administration protocols that significantly reduce the occurrence or severity of the apoptosis- associated disease in model subjects (e.g., in the case of treatment of malignancies, a tumor xenograft model in mice can be used.
  • a non-limiting range for a therapeutically effective amount of the compounds is about 0.001 mg/kg and about 100 mg/kg body weight per day, and in more specific embodiments between about 0.001 mg/kg and about 50 mg/kg, between about 0.01 mg/kg and about 20 mg/kg. between about 0.1 and about 10 mg/kg, or between about 0.1 mg/kg and about 5 mg/kg body weight per day.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such containers can be a notice, in the form prescribed by a governmental agency, regulating the manufacture, use, or sale of pharmaceuticals or biological products. The government notice should reflect approval by the agency of manufacture, use, or sale for human administration.
  • cell lines over-expressing BC1-XL were prepared and tested according to Hockenberry et al. U.S. Patent No. 7,241,804, hereby incorporated by reference in its entirety.
  • Competitive binding of compounds with BH-3 peptide were also measured using BC1-XL as previously described. Id.
  • R] is H or alkyl
  • R 2 is H, halide, or alkoxy
  • R 3 is H, halide or alkyl
  • R 3 is H, alkyl or alkoxy
  • R 6 is H or CF 3 .
  • the structure of various substituents of the Formula II and III (below) are given in Table 2.
  • Example 2 The compounds described in Example 2 were tested for their ability to inhibit Bcl-2 gain- of-function in TAMH cells, TAMH cells overexpressing Gclx L (TAMH-Bcl-x L ), TAMH cells transfected with Neo vector (Neo), TAMH cells over expressing a variant of Bcl-x L (A142L) using the methods of Example 1. The results are shown in the following Tables.3-6.
  • the EC50 for the compound Compound 0216 was determined in a panel of pancreatic cell lines.
  • the panel included CFPAC-1, HPAF-II MIA PaCa, PaTu-1, PaTu-8988t, ASPC-1, FA-6, and PANC-1 cell lines.
  • Pancreatic cancer samples were obtained from patients, with appropriate patient consent and approval of the Fred Hutchinson Cancer Research Center Institutional Review Board. Primary cells were maintained in Iscove's medium supplemented with 10% bovine calf serum, 100 ng-ml "1 stem cell factor, and 50 ng-ml "1 interleukin-3.
  • Compound 216 was tested in TAMH cells overexpressing Bcl-xL compared to TAMH cells transfected with the neo expression vector as in Example 2.
  • the results ( Figure 2) show that compound cells overexpressing Bcl-xL are more sensitive to the apoptosis-inducing effects of compound 216. Without being bound to a mechanistic model for the mode of action of compound 216, these results suggest that heightened sensitivity to this compound is associated with levels of expression of the Bcl-xL gene.
  • Example 2 The ability of compounds from Example 2 to induce apoptosis against solid tumors in vitro was tested.

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Abstract

L'invention concerne des composés utiles dans le traitement d'une maladie associée à l'apoptose, lesquels s'avèrent particulièrement cytotoxiques contre les cellules tumorales qui surexpriment les Bcl-XL, et qui sont bien moins cytotoxiques envers les cellules isogéniques qui ne surexpriment pas les Bcl-XL. L'invention concerne également des procédés d'utilisation de ces composés pour traiter une maladie associée à l'apoptose.
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