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EP1963330A1 - Dimeres et produits d'addition de 6-[phenyl (substitue)] triazolopyrimidines utilisables en tant qu'agents anticancereux - Google Patents

Dimeres et produits d'addition de 6-[phenyl (substitue)] triazolopyrimidines utilisables en tant qu'agents anticancereux

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Publication number
EP1963330A1
EP1963330A1 EP06845599A EP06845599A EP1963330A1 EP 1963330 A1 EP1963330 A1 EP 1963330A1 EP 06845599 A EP06845599 A EP 06845599A EP 06845599 A EP06845599 A EP 06845599A EP 1963330 A1 EP1963330 A1 EP 1963330A1
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EP
European Patent Office
Prior art keywords
compound
mammal
formula
pharmaceutically acceptable
effective amount
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP06845599A
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German (de)
English (en)
Inventor
Yanzhong Wu
David M. Blum
Carl Beyer
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Wyeth LLC
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Wyeth LLC
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Publication of EP1963330A1 publication Critical patent/EP1963330A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to certain adducts and dimers of 6- [(substituted)phenylj-triazolopyrimidine compounds or pharmaceutically acceptable salts thereof, and compositions containing said compounds or pharmaceutically acceptable salts thereof, wherein said compounds are anti-cancer agents useful for the treatment of cancer in mammals, treatment or prevention of cancerous tumors that express multiple drug resistance (MDR) or are resistant because of MDR, a method of treating or inhibiting the growth of cancerous tumor cells and associated diseases in a mammal in need thereof by promotion of microtubule polymerization and a method of treating or inhibiting the growth of cancerous tumors in a mammal with inherent or acquired resistance to chemotherapeutic agents used in chemotherapy treatment and in particular antimitotic agents by administering an effective amount of a compound of the invention or pharmaceutically acceptable salts thereof.
  • MDR multiple drug resistance
  • Antimicrotubule drugs are a major category of anticancer agents (Rowinsky, E.K., and Tolcher, A.W. Antimicrotubule agents. In: V.T. Devita, Jr., S. Hellman, and S.A. Rosenberg (eds.), Cancer Principles and Practice, Ed. 6, pp. 431-452.
  • taxane-site agents that promote microtubule formation and stabilize microtubules
  • vinca/peptide-site agents which destabilize microtubules and often induce formation of abnormal polymers or aggregates at high concentrations
  • cholchicine-site agents that also destabilize microtubules and generally do not induce other polymers
  • Paclitaxel and its semisynthetic derivative docetaxel interfere with microtubule formation and stabilize microtubules.
  • Paclitaxel (Taxol®), is a diterpene isolated from the bark of the Western ( Pacific) yew, Taxus brevifolia and is representative of a new class of therapeutic agent having a taxane ring system. It was additionally found in other members of the Taxacae family including the yew of Canada (Taxus canadensis) found in Gaspesia, eastern Canada and Taxus baccata found in Europe whose needles contain paclitaxel and analogs and hence provide a renewable source of paclitaxel and derivatives.
  • Paclitaxel has been demonstrated to possess antineoplastic activity. More recently, it was shown that the antitumor activity of paclitaxel is due to a promotion of microtubule polymerization (Kumar, N., J. Biol. Chem. 256:10435-10441 (1981); Rowinsky, et al., J. Natl. Cancer Inst., 82:1247-1259 (1990); and Schiff, et al., Nature, 277:665-667 (1979)).
  • Paclitaxel has now demonstrated efficacy in several human tumors in clinical trials (McGuire, et al., Ann. Int. Med., 111:273-279 (1989); Holmes, et al., J. Natl. Cancer Inst., 83:1797-1805 (1991); Kohn et al., J. Natl. Cancer Inst., 86:18-24 (1994); and A. Bicker et al., Anti-Cancer Drugs, 4,141-148 (1993). [0006] Two taxane-site agents (paclitaxel and docetaxel) and three vinca/peptide- site agents (vinblastine, vincristine, and vinorelbine) are used clinically to treat various human cancers.
  • Taxanes have proven to be of greater utility against solid tumors (e.g., lung, breast, ovarian) than the vinca alkaloids, suggesting that agents that promote microtubule formation might be superior clinically to those that destabilize microtubules. Cholchicine-site agents are not used therapeutically. [0007] Despite the widespread clinical use of paclitaxel and docetaxel, these drugs have several limitations that create a need for improved agents. First, many tumors are inherently resistant (e.g., colon tumors) or become resistant after multiple cycles of treatment, at least in part due to the expression of drug transporters located in cancer cell membranes that pump the drugs out of cells and thereby decrease their efficacy (Gottesman, M. M. Mechanisms of cancer drug resistance. Annu.
  • paclitaxel and docetaxel have poor water solubility and paclitaxel must be formulated in Cremophor EL, a vehicle that induces serious hypersensitivity reactions (Li, C.L., Newman, R.A., and Wallace, S. Reformulating paclitaxel. Science & Medicine, Jan/Feb: 38-47, 1999).
  • paclitaxel is a natural product having a highly complex structure
  • docetaxel is a closely related semisynthetic derivative. Therefore, there is a need for compounds that are readily available through synthesis, are structurally different from taxanes and which have taxane-like effects on microtubule polymerization.
  • cytotoxic agents for use in cancer therapy. In particular, there is a need for cytotoxic agents that inhibit or treat the growth of tumors that have an effect similar to paclitaxel and interfere with the process of microtubule formation. Additionally, there is a need in the art for agents that accelerate tubulin polymerization and stabilize the assembled microtubules.
  • the compounds of the present invention are a new class of taxane-like agents that satisfy the hereinbefore-described needs, and that differ in significant ways from the previously known classes of antimicrotubule compounds.
  • the compounds of this invention bind at the vinca site of ⁇ -tubulin, yet they have many properties that are similar to taxanes and distinct from vinca-site agents.
  • the compounds of this invention enhance the polymerization of microtubule-associated protein (MAP)-rich tubulin in the presence of GTP at low compound:tubulin molar ratios, in a manner similar to paclitaxel and docetaxel.
  • MAP microtubule-associated protein
  • the compounds of this invention also induce polymerization of highly purified tubulin in the absence of GTP under suitable experimental conditions, an activity that is a hallmark of taxanes.
  • the compounds of the present invention are potently cytotoxic for many human cancer cell lines in culture, including lines that overexpress the membrane transporters MDR (P-glycoprotein), MRP, and MXR, thus making them active against cell lines that are resistant to paclitaxel and vincristine.
  • MDR membrane transporters
  • MRP membrane transporters
  • MXR membrane transporters
  • representative examples of this invention have high water solubility and can be formulated in saline.
  • Representative examples of this invention are active as anti-tumor agents in athymic mice bearing human tumor xenografts of lung and colon carcinoma, melanoma, and glioblastoma, when dosed either intravenously or orally.
  • the compounds of the present invention have been shown, for example, to have broad antitumor activity in in vivo xenograft models of human non-small cell lung cancer (NSCLC), colon cancer, breast cancer, melanoma and glioblastoma, including models which are resistant to taxanes or other microtubule-active compounds.
  • NSCLC non-small cell lung cancer
  • colon cancer colon cancer
  • breast cancer breast cancer
  • melanoma glioblastoma
  • these compounds are active when dosed on a once- weekly schedule by either IV or oral routes.
  • 6-[(substituted)phenyl]triazolopyrimidines are disclosed in U.S. Application No. 10/950,543 as filed on September 24, 2004, and published as U.S. Patent Publication No. US2005/0090508A1 on April 28, 2005 and as International Patent Publication No. WO2005/030775 on April 7, 2005. The description of these compounds and the methods of making and using same as set forth in the published application are hereby incorporated by reference in their entirety. [0020] For the present invention, it has been found that acid adducts and dimers of the 6-[(substituted)phenyl]triazolopyrimidines may be formed under specified conditions.
  • Such compounds will also be useful for enhancing the polymerization of microtubule-associated protein (MAP)-rich tubulin in the presence of GTP at low compound:tubulin molar ratios and for inducing polymerization of highly purified tubulin in the absence of GTP under suitable experimental conditions.
  • MAP microtubule-associated protein
  • These compounds are also potently cytotoxic for many human cancer cell lines in culture, including lines that overexpress the membrane transporters MDR (P-glycoprotein), MRP and MXR, and have broad antitumor activity in in vivo xenograft models of human non-small cell lung cancer (NSCLC) 1 colon cancer, breast cancer, melanoma and glioblastoma, including models which are resistant to taxanes or other microtubule-active compounds.
  • NSCLC non-small cell lung cancer
  • n is an integer of 2, 3, or 4;
  • X is -Cl, -F or -Br;
  • Y is -O-, -S-, -CH 2 - or -NR 4 -;
  • L 1 and L 2 are each independently -H 1 -F, -Cl, -Br, or -CF 3 ;
  • R 3 is -CF 3 or -C 2 F 5 ;
  • R 4 , R 5 r and R 6 are each independently -H or -(Ci-C 3 )-alky ⁇ ;
  • R 7 is -ZR 9 , wherein Z is -CO-, -NO-, -SO 2 -, Or-PO 2 H-; and R 9 is -H. -OH, -(Ci-C 5 ) -alkyl or -(C 2 -Cs) -alkenyl, said alkyl or alkenyl optionally substituted with one or more of -O-, halogen, -OH, -NH 2 , or a five- or six-membered saturated, partially saturated, or unsaturated cycloalkyl group in which one to three of the ring carbon atoms are optionally independently replaced with an N, O, or S atom and said cycloalkyl group optionally is substituted with a -CH 3 , -OH, or halogen; and
  • R 8 is (C 1 -C 3 ) -alkyl; or pharmaceutically acceptable salts or stereoisomers thereof.
  • the instant invention further provides compounds of Formula (II): NR 6 R 2
  • n is an integer of 2, 3, or 4;
  • X is -Cl, -F or -Br;
  • Y is -O-, -S-, -CH 2 - or -NR 4 -;
  • L 1 and L 2 are each independently -H, -F, -Cl, -Br, or -CF 3 ;
  • R 3 is -CF 3 or -C 2 F 5 ;
  • R 2 , R 4 , R 5 , R 6 and R 9 are each independently -H or -(CrCaJ-alkyl;
  • R 8 is -(Ci-C 3 )-alkyl; and pharmaceutically acceptable salts or stereoisomers thereof.
  • the present invention further provides a method of treating or inhibiting the growth of cancerous tumor cells and associated diseases in a mammal by administering an effective amount of the compounds of Formula (I) and pharmaceutically acceptable salts thereof in need thereof.
  • the present invention also provides a method of treating or inhibiting the growth of cancerous tumor cells and associated diseases in mammals in need thereof by interacting with tubulin and microtubules by promotion of microtubule polymerization which comprises administering to said mammal an effective amount of the compounds of Formula (I) or (H), or pharmaceutically acceptable salts thereof.
  • a method for the treatment or prevention of tumors that express multiple drug resistance (MDR) or are resistant because of MDR in a mammal in need thereof comprises administering to said mammal an effective amount of such compounds or pharmaceutically acceptable salts thereof.
  • This invention also provides a method of promoting tubulin polymerization in a tubulin containing system by contacting said tubulin containing system with an effective amount of a compound of Formula (I) or (II), or pharmaceutically acceptable salts thereof.
  • this invention provides a method of stabilizing microtubules in a tubulin containing system which comprises contacting said tubulin containing system with an effective amount of a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof.
  • a method of treating, inhibiting the growth of, or eradicating a tumor in a mammal in need thereof wherein said tumor is resistant to at least one chemotherapeutic agent comprises administering, to said mammal an effective amount of the compounds of Formula (I) or (II), or pharmaceutically acceptable salts thereof.
  • this invention provides a compound of Formula (I) in combination or association with a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition, which comprises an effective amount of a compound of Formula (I) and a pharmaceutically acceptable carrier.
  • alkyl means a straight or branched hydrocarbon chain moiety, preferably of 1 to 3 carbon atoms.
  • f-BOC as used herein means ferf-butoxy carbonyl.
  • aminoalkoxy means a moiety of the formula
  • aminoalkyl means a moiety of the formula
  • aminoalkylthio means a moiety of the formula
  • aminoalkylamino means a moiety of the formula
  • hydroxyalkoxy means a moiety of the formula
  • alkali metal hydroxide includes lithium, potassium or sodium hydroxide.
  • alkali metal carbonate includes lithium, potassium or sodium carbonate.
  • alkali metal hydride includes lithium, potassium or sodium hydride.
  • strong base means an alkali metal hydroxide, alkali metal carbonate and alkali metal hydride (e.g., sodium hydride).
  • (Ci-C 5 )-alkyr refers to a linear or branched, saturated hydrocarbon having from 1 to 5 carbon atoms, preferably 1 to 3 carbon atoms.
  • Representative (Ci-C 5 )-alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, and neopentyl.
  • the (C ⁇ CsJ-alkyl group is substituted with one or more of the following groups: halogen, -N 3 , -NO 2 , -CN, -OR', -SR', -SO 2 R', -SO 2 N(R') 2( -N(R 1 J 2 , -COR', -CO 2 R', -NR 1 CO 2 R 1 , -NR 1 COR', -NR'CONR', or -CON(R') 2r wherein each R' is independently hydrogen or unsubstituted (C ⁇ -C 5 )-a ⁇ ky ⁇ .
  • (C 2 -C 5 )-alkenyr refers to a linear or branched hydrocarbon having from 2 to 5 carbon atoms and having at least one carbon-carbon double bond. In one embodiment, the (C 2 -C 5 )-alkenyl has one or two double bonds.
  • the (C 2 -C 5 )-alkenyl moiety may exist in the E or Z conformation and the compounds of the present invention include both conformations.
  • Representative (C 2 -C 5 )-alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, sec- butenyl, tert-butenyl, pentenyl, isopentenyl, and neopentenyl.
  • the (C 2 -C 5 )-alkenyl group is substituted with one or more of the following groups: halogen, -N 3 , -NO 2 , -CN, -OR 1 , -SR', -SO 2 R', -SO 2 N(R 1 );,, -N(R') 2 , -COR', -CO 2 R', -NR 1 CO 2 R', -NR'COR', -NR'CONR', or -CON(R') 2 , wherein each R' is independently hydrogen or unsubstituted (Ci-Cs)-alkyl.
  • administer refers to either directly administering a compound or pharmaceutically acceptable salt of the compound or a composition to an animal, or administering a prodrug derivative or analog of the compound or pharmaceutically acceptable salt of the compound or composition to the animal, which can form an equivalent amount of active compound within the animal's body.
  • animal as used herein includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus. In one embodiment, the animal is a mammal. In another embodiment, the animal is a human.
  • aryl refers to an aromatic species containing 1 to 3 aromatic rings, either fused or linked.
  • the aryl group is substituted with one or more of the following groups: -V'-halogen, -V-N 3 , -V-NO 2 , -V-CN, -V-OR", -V-SR', -V-SO 2 R', -V-SO 2 N(R 1 J 2 , -V-N(R 1 J 2 , -V-COR', -V-CO 2 R 1 , -V-NR 1 CO 2 R", -V-NR'COR', -V-NR 1 CONR', or -V-CON(R')2.
  • each R' is independently hydrogen or unsubstituted (C 1 -Ce)-SlKyI; and wherein each V is independently a bond or (Ci-C 6 )-alkyl.
  • condition effective to refers to synthetic reaction conditions which will be apparent to those skilled in the art of synthetic organic chemistry.
  • cyclic group includes a cycloalkyl group and a heterocyclic group. Any suitable ring position of the cyclic group may be covalently linked to the defined chemical structure.
  • the cyclic group is substituted with one or more of the following groups: -V-hatogen, -V-N 3 , -V-NO 2 , -V-CN, -V'-OR', -V-SR', -V-SO 2 R', -V-SO 2 N(R 1 J 2 , -V-N(R') 2 , -V-COR', -V-CO 2 R', -V-NR 1 CO 2 R', -V-NR'COR', -V-NR'CONR 1 , or -V-CON(R') 2 , wherein each R' is independently hydrogen or unsubstituted (Ci-C 6 )-alkyl; and wherein each V is independently a
  • cycloalkyl group refers to a three- to seven- membered saturated or partially unsaturated carbon ring. Any suitable ring position of the cycloalkyl group may be covalently linked to the defined chemical structure.
  • exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
  • the cycloalkyl group is substituted with one or more of the following groups: -V-halogen, -V-N 3 , -V-NO 2 , -V-CN, -V-OR', -V-SR", -V-SO 2 R', -V-SO 2 N(R 1 J 2 , -V-N(R 1 J 2 , -V-COR', -V-CO 2 R', -V-NR 1 CO 2 R', -V-NR'COR', -V-NR'CONR', or -V-CON(R 1 J 2 , wherein each R' is independently hydrogen or unsubstituted (C,-C 6 )-alkyl; and wherein each V is independently a bond or (d-C 6 )-alkyl.
  • phenyl refers to a substituted or unsubstituted phenyl group.
  • the phenyl group is substituted with one or more of the following groups: -V'-halogen, -V-N 3 , -V-NO 2 , -V-CN, -V-OR 1 , -V-SR', -V-SO 2 R', -V-SO 2 N(R') 2 , -V-N(R') 2l -V-COR', -V-CO 2 R', -V-NR 1 CO 2 R', -V-NR 1 COR', -V-NR'CONR', or -V-CON(R') 2 , wherein each R' is independently hydrogen or unsubstituted (CrC ⁇ )-alkyl; and wherein each V is independently a bond or (CrCe ⁇ alkyl.
  • halogen refers to fluorine, chlorine, bromine, and iodine.
  • heterocyclic group refers to a three- to seven- membered saturated, partially saturated, or unsaturated cycloalkyl group in which one to four of the ring carbon atoms have been independently replaced with a N, O, or S atom. Any suitable ring position of the heterocyclic group may be covalently linked to the defined chemical structure.
  • heterocyclic groups include, but are not limited to, azepanyl, azetidinyl, aziridinyl, furanyl, furazanyl, homopiperazinyl, imidazolidinyl, Jmidazolinyl, isothiazolyl, isoxazolyl, morpholinyl, oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, piperazinyl, piperidinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazolyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyr
  • the heterocyclic group is substituted with one or more of the following groups: -V-halogen, -V-N 3 , -V-NO 2 , -V-CN, -V-OR', -V-SR', -V-SO 2 R', -V-SO 2 N(R') 2 , -V-N(R') 2l -V-COR', -V-CO 2 R', -V-NROO 2 R',
  • each R' is independently hydrogen or unsubstituted (d-C 6 )-alkyl; and wherein each V is independently a bond or (d-CfO-alkyl.
  • an effective amount refers to an amount of a compound or pharmaceutically acceptable salt of a compound that, when administered to an animal, is effective to prevent, to at least partially ameliorate, or to cure, a condition from which the animal suffers or is suspected to suffer.
  • carrier shall encompass carriers, excipients, and diluents.
  • prodrug means a compound which is convertible in vivo by metabolic means (e.g. by hydrolysis) to a compound of Formula (I) or
  • isolated and purified refers to a component separated from other components of a reaction mixture or a natural source.
  • the isolate contains at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% of the compound or pharmaceutically acceptable salt of the compound by weight of the isolate.
  • pharmaceutically acceptable salt refers to a salt of an acid and a basic nitrogen atom of a compound of the present invention.
  • Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, hydrochloride, bromide, hydrobromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, ⁇ apthalenesulfonate, propionate, succinate, fumarate, maleate, malonate, mandelate, malate, phthalate, and pa
  • substantially free of its corresponding opposite enantiomer means that the compound contains no more than about 10% by weight of its corresponding opposite enantiomer. In other embodiments, the compound that is substantially free of its corresponding opposite entantiomer contains no more than about 5%, no more than about 1%, no more than about 0.5%, or no more than about 0.1% by weight of its corresponding opposite enantiomer.
  • An enantiomer that is substantially free of its corresponding opposite enantiomer includes a compound that has been isolated and purified or has been prepared substantially free of its corresponding opposite enantiomer.
  • X is -Cl, -F or -Br
  • Y is -O-, -S-, -CH 2 - or -NR 4 -;
  • L 1 and L 2 are each independently -H, -F, -Cl, -Br, or -CF 3 ;
  • R 3 is -CF 3 or -C 2 F 5 ;
  • R 4 , R 5 , and R 6 are each independently -H or -(Ci-CsJ-alkyl;
  • R 7 is -ZR 9 , wherein 2 is -CO-, -NO-, -SO 2 -, Or-PO 2 H-; and R 9 is -H, -OH, -(C 1 -C 5 ) -alkyl or -(C 2 -C 5 )-alkenyl, said alkyl or alkenyl optionally substituted with one or more of -0-, halogen, -OH, -NH 2 , or a five- or six-membered saturated, partially saturated, or unsaturated cycloalkyl group in which one to three of the ring carbon atoms are optionally independently replaced with an
  • n is an integer of 2, 3, or 4;
  • X is -CI, -F or -Br
  • Y is -O-, -S-, -CH 2 - or -NR 4 -;
  • L 1 and L 2 are each independently -H, -F, -Cl, -Br, or -CF 3 ;
  • R 3 is -CF 3 or -C 2 F 5 ;
  • R 2 , R 4 , R 5 , R 6 , and R 9 are each independently -H or ⁇ GrC ⁇ -alkyl
  • R 8 is -(d-CsJ-alkyl; or pharmaceutically acceptable salts or stereoisomers thereof.
  • R 6 is preferably a -(Ci-C 3 )-alkyl, and even more preferably is — CH 3 .
  • R 7 is -ZR 9 , wherein Z is -CO- or -NO-; and R 9 is a -(Ci-CsJ-alkyl or -(C 2 -C 5 )-alkenyl, optionally substituted with one or more of O, halogen, -OH, -NH 2 , or a five- or six-membered saturated, partially saturated, or unsaturated cycloalkyl group in which one to three of the ring carbon atoms are optionally independently replaced with an N, O, or S atom and said cycloalkyl group optionally is substituted with a -CH 3 , -OH, or halogen. More preferably, Z is -CO- and R 9 is a -C 2 -C 4 alkyl or -(C 2 -C 4 )-alkenyl, having a CO 2 H group.
  • R 1 is R 3 R 5 CH-NH- (where R 3 and R 5 are as defined above) and R 7 is carboxyalkylcarbonyl containing a total of 3 to 6 carbon atoms or carboxyalkenylcarbonyl containing a total of 4 to 6 carbon atoms (preferably carboxyalkylcarbonyl containing a total of 3 to 6 carbon atoms, e.g. -CO-(CH 2 ) 2 -CO 2 H) and Y is -O-.
  • R 1 is R 3 R 5 CH-NH- (where R 3 and R 5 are as defined above), X is -Cl, L 1 and L 2 are -F, n is 3, R 2 is H or -(C 1 to C 3 ) alkyl, R 6 is -(C 1 to C 3 ) alkyl (preferably -CH 3 ), and Y is -O-.
  • these compounds will preferably have R 7 as carboxyalkylcarbonyl containing a total of 3 to 6 carbon atoms or carboxyalkenylcarbonyl containing a total of 4 to 6 carbon atoms (preferably carboxyalkylcarbonyl containing a total of 3 to 6 carbon atoms, "e.g. -CO-(CH 2 ) 2 - CO 2 H).
  • R 1 is
  • Preferred compounds of Formula (I) and Formula (II) will include R 3 as -CF 3 and/or R 5 as a -C 1 -C 3 alkyl.
  • Preferred substituents for L 1 and L 2 are each independently -F, -Cl, or -Br.
  • L 1 and L 2 are each -F.
  • Y is preferably -O-, n is preferably 3, and/or X is preferably -Cl.
  • a particularly preferred compound of Formula (I) is:
  • a further preferred compound of Formula (I) of the invention is:
  • a still further preferred compound of Formula (I) includes:
  • r is an integer from 0 to 5, inclusive.
  • Preferred compounds of Formula (II) include:
  • the compounds of this invention may contain an asymmetric carbon atom and some of the compounds of this invention may contain one or more asymmetric centers and may thus give rise to stereoisomers, such as enantiomers and diastereomers.
  • the stereoisomers of the instant invention are named according to the Cahn-lngold-Prelog System. While shown without respect to stereochemistry, the present invention includes all the individual possible stereoisomers; as well as the racemic mixtures and other mixtures of R and S stereoisomers (scalemic mixtures which are mixtures of unequal amounts of enantiomers) and pharmaceutically acceptable salts thereof.
  • the present invention encompasses all stereoisomers of the compounds whether free from other stereoisomers or admixed with other stereoisomers in any proportion and thus s includes, for instance, racemic mixture of enantiomers as well as the diastereomeric mixture of isomers.
  • the absolute configuration of any compound may be determined by conventional X-ray crystallography.
  • Optical isomers may be obtained in pure form by standard separation techniques or enantiomer specific synthesis. A compound of Formula (I) or Formula (II) that is substantially free of its corresponding opposite enantiomer may thus be obtained.
  • this invention encompasses all crystalline and hydrated forms of compounds of Formulas (I) and (II) and their pharmaceutically acceptable salts.
  • the pharmaceutically acceptable salts of the compounds of this invention are those derived from such organic and inorganic pharmaceutically acceptable salt forming acids as: lactic, citric, acetic, tartaric, fumaric, succinic, maleic, malonic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, benzenesulfonic, L-aspartic, R or S-mandelic, palmitic and similarly known acceptable acids.
  • a further salt is the trifluoroacetic acid salt (TFA).
  • the hydrochloride, fumarate and succinate salts are preferred.
  • the compounds of Formula (I) or (II) are reacted with a suitable pharmaceutically acceptable salt forming acid.
  • a suitable pharmaceutically acceptable salt forming acid As a representative example of pharmaceutically acceptable salt formation, the hydrochloride salt of 5-chloro-6- ⁇ 2,6-difluoro-4-[3-(methylamino)propoxy]phenyl ⁇ -N-[(1S)-2,2,2-trifluoro-1- methylethyl][1,2,4]triazolo[1,5-a3pyrimidin-7-amine, is neutralized with aqueous alkali metal hydroxide or aqueous alkali metal carbonate, and further reacted with a suitable pharmaceutically acceptable salt forming acid described hereinabove in a suitable solvent.
  • suitable solvents which may be used include: water, methanol, ethanol, isopropanol or combination thereof and the like. A preferred solvent is water.
  • pharmaceutically acceptable salts may form by heating compounds of Formula (I) or (II) in a suitable solvent, at about 30-100 0 C, preferably at about 65-75 0 C, until a clear solution forms. Upon cooling, the compound may be collected and dried.
  • Dihydrates may be formed by further contact with an atmosphere of water at about 80-100% relative humidity for about 24 hours at room temperature.
  • the compounds of this invention may be prepared from: (a) commercially available starting materials; (b) known starting materials which may be prepared as described in literature procedures or (c) new intermediates described .in the schemes and experimental procedures herein.
  • reactions are performed in a solvent appropriate to the reagents and materials employed and suitable for the transformation being effected. It is understood by those skilled in the art of organic synthesis that the various functionalities present on the molecule must be consistent with the chemical transformations proposed. This may necessitate judgment as to the order of synthetic steps. Appropriate consideration must be made as to the protection of reactive functional groups to prevent undesired side reactions. Substituents on the starting materials may be incompatible with some of the reaction conditions. Such restrictions to the substituents which are compatible with the reaction conditions will be apparent to one skilled in the art. Reactions are run under inert atmospheres where appropriate.
  • a starting material for preparing compounds of Formula (I) and (II) includes compounds of the Formula (III):
  • R 2 is preferably -H.
  • Such compounds are made as disclosed in U.S. Application No. 10/950,543 as filed on September 24, 2004 published as U.S. Patent Publication No. US2005/0090508A1 on April 28, 2005 and as International Patent Publication No. WO2005/030775 on April 7, 2005, the disclosure of which is incorporated by reference in its entirety for the compounds and methods of making same.
  • the second starting material is a compound of formula HOZR 9 , wherein Z is -CO-; -NO-, -SO 2 -, or -PO 2 H-; and R 9 is -H, -OH, -(d-C 5 )-alkyl or -(C 2 -C 5 )-alkenyl, said alkyl or alkenyl optionally substituted with one or more of O, halogen, -OH, -NH 2 , or a five- or six-membered saturated, partially saturated, or unsaturated cycloalkyl group in which one to three of the ring carbon atoms are optionally independently replaced with an N, O 1 or S atom and said cycloalkyl group optionally is substituted with a -CH 3 , -OH, or halogen.
  • r is an integer from 0 to 5.
  • the second starting material is a compound of formula:
  • a specific compound of Formula (I) can be prepared by the following reaction:
  • a compound of Formula (II) is prepared by a process comprising heating a mixture having a compound of formula (111):
  • a compound of Formula (II) may be produced as follows:
  • the present invention accordingly provides a pharmaceutical composition which comprises a compound of this invention in combination or association with a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition which comprises an effective amount of a compound of this invention and a pharmaceutically acceptable carrier.
  • the compounds of this invention are useful as agents for treating, inhibiting or controlling the growth of cancerous tumor cells and associated diseases in a mammal in need thereof.
  • the compounds of the invention are useful as agents for treating, inhibiting or controlling the growth of cancerous tumor cells and associated diseases in a mammal in need thereof by interacting with tubulin and microtubules and promoting microtubule polymerization.
  • the compounds of the invention are also useful for the treatment or prevention of cancerous tumors that express multiple drug resistance (MDR) or are resistant because of MDR.
  • MDR multiple drug resistance
  • tubulin containing system when contacting a tubulin containing system with an effective amount of a compound of Formula (I) or (II) results in the promotion of microtubule polymerization and further stabilizes microtubules and by promoting microtubule polymerization and stabilizing microtubules said compounds of Formula (I) and (II) are useful as agents for treating, inhibiting or controlling the growth of cancerous tumor cells and associated diseases.
  • the tubulin containing system may be in a tumor cell, thereby inhibiting neoplastic disease by administering an effective amount of a compound described in the present invention. Mammals may be treated and in particular, humans. Further, said tubulin containing system may be in a patient.
  • cancer refers to all types of cancers, or neoplasms or benign or malignant tumors.
  • Preferred cancers for treatment using methods provided herein include carcinoma, sarcoma, lymphoma, or leukemia.
  • carcinoma is meant a benign or malignant epithelial tumor and includes, but is not limited to, breast carcinoma, prostate carcinoma, non- small lung carcinoma, colon carcinoma, melanoma carcinoma, ovarian carcinoma, or renal carcinoma.
  • a preferred host is a human.
  • the effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration and severity of the condition being treated. However, in general satisfactory results are obtained when the compounds of the invention are administered in amounts ranging from about 0.10 to about 100 mg/kg of body weight per day. A preferred regimen for optimum results would be from about 1 mg to about 20 mg/kg of body weight per day and such dosage units are employed that a total of from about 70 mg to about 1400 mg of the active compound for a subject of about 70 kg of body weight are administered in a 24 hour period.
  • the dosage regimen for treating mammals may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A decidedly practical advantage is that these active compounds may be administered in any convenient manner such as by the oral, intravenous, intramuscular or subcutaneous routes.
  • the active compounds of the invention may preferably be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsules, or they may be compressed into tablets or they may be incorporated directly with the food of the diet.
  • these active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between 10 and 1000 mg of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, or saccharin may be added or a flavoring agent such " as peppermint, oil of wi ⁇ tergreen or cherry flavoring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose, or saccharin may be added or a flavoring agent
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts used.
  • these active compounds may be incorporated into sustained-release preparations and formulations.
  • active compounds may also be administered parenterally or intraperitoneally.
  • Solutions or suspensions of these active compounds as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth or microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be prepared against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid poly-ethylene glycol), suitable mixtures thereof, and vegetable oils.
  • Intravenous administration is a preferred manner of administration of compounds of the invention.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor® ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • polyol for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • the term providing an effective amount of a compound means either directly administering such compound, or administering a prodrug, derivative, or analog which will form an effective amount of the compound within the body.
  • Medium is RPMI-1640 with L-glutamine, supplemented with 10% heat- inactivated fetal calf serum, 100 units/ml penicillin, and 100 ⁇ g/ml streptomycin (Gibco, Grand Island, NY).
  • Microtubule-associated protein (MAP)-rich tubulin containing about 70% tubulin and 30% MAPs (#ML113), and highly purified tubulin (>99% pure, #TL238), both from bovine brain, are obtained from Cytoskeleton, Inc., Denver, CO.
  • PEM buffer 80 mM piperazine-N,N'-bis[2-ethanesulfonic acid], pH 6.9, 1 mM ethylene glycol-bis( ⁇ -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 mM magnesium chloride
  • GTP guanosine 5'-triphosphate
  • S1 parental line from a subclone of human colon carcinoma line LS174T
  • S1-M1 derived S1-M1-3.2
  • Dr. L. Greenberger, Wyeth Research Dr. Binran, S.K., He, H., Singh, M., Brown, E., Collins, K.I., Annable, T., and Greenberger, L.M.
  • KB-3-1 (herein called KB, cloned from a human epidermoid carcinoma) and the derived lines KB-8-5 and KB-V1, which express moderate and very high levels of the MDR1 (P-glycoprotein) drug transporter protein, respectively, are provided by Dr. M. Gottesman, National Cancer Institute (Shen, D.W., Cardarelli, C, Hwang, J., Cornwell, M., Richert, N., Ishii, S., Pastan, I., and Gottesman, M. M. Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to cholchicine, adriamycin, or vinblastine show changes in expression of specific proteins.
  • MDR1 P-glycoprotein
  • the assay which is sold in kit form by Promega (Madison, Wl; CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay), is based on the conversion by viable cells, but not by dead cells, of the tetrazolium salt, MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt), into a water-soluble colored formazan which is detected by spectrophotometry.
  • MTS 3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt
  • IC 50 values Compounds are tested at nine concentrations, in order to determine IC 50 values.
  • cells are harvested by trypsinization (or, in the case of non-adherent cells, by simple resuspension), washed, counted and distributed to wells of 96-well flat-bottom microtiter plates at 1000 cells per well in 200 ⁇ l_ of medium.
  • one row of wells on a separate plate received cells as above ("time 0" plate). All plates are incubated at 37°C in humidified 5% CO 2 in air for about 24 hr.
  • the MTS assay is run on the "time 0" plate. This produced the "time 0 MTS value" which is related to the number of viable cells per well at the time of drug addition.
  • the MTS assay is done on all wells of the experimental plates. The absorbance values of the quadruplicate sample wells are averaged and divided by the average of the "time 0" values. The average of control wells without drug, divided by the average "time 0" value, gives the maximal relative increase in MTS color yield due to cell growth during the final three days of culture.
  • MAP-rich tubulin is dissolved in ice-cold PEM buffer containing 1 mM GTP (GPEM buffer) at a concentration of 1.3 mg/mL
  • the solution is centrifuged at top speed in an Eppendorf model 5415C microcentrifuge (Brinkmann Instruments, Westbury, NY) for 10 min at 4°C immediately before use.
  • the tubulin solution is added to wells of a 1 /4-area 96-well plate (Costar No. 3696, Corning, Inc., Corning, NY) already containing the compounds of interest. Each compound is tested in duplicate at a final concentration of 0.3 ⁇ M in a volume of 110 ⁇ L per well. The final DMSO concentration in all wells is 0.3%.
  • Control reactions which received compound solvent only, are done in quadruplicate.
  • the plate is put in a SpectraMax Plus plate reader (Molecular Devices Corp. Sunnyvale, CA) thermostated at 24°C and the absorbance of each well at 340 nm, a measure of the appearance of turbidity due to tubulin polymer formation, is determined every minute for 60 minutes.
  • the absorbance at time 0 for each well is subtracted from each of the subsequent absorbance readings for that well, and then the duplicates are averaged.
  • the procedure with pure tubulin is similar except for the following changes. Pure tubulin is dissolved in cold PEM buffer containing 10% glycerol and no added GTP at a concentration of 1.5 to 1.8 mg/mL (15 to 18 ⁇ M).
  • the supernatant after centrifugation is dispensed to a 96-well plate already containing compounds. Each compound is tested in duplicate at six serial 3-fold dilutions starting at 24.3 ⁇ M. The plate reader is thermostated at 35°C.
  • the binding of examples of this invention to highly purified tubulin is studied by competitive inhibition methods.
  • the ⁇ -tubulin heterodimer contains binding sites for the three major classes of microtubule-active pharmacological agents: taxanes, vinca/peptide-site agents, and cholchicine-site agents.
  • taxanes a major class of microtubule-active pharmacological agents
  • vinca/peptide-site agents a major class of microtubule-active pharmacological agents
  • cholchicine-site agents To study possible competition at the vinca/peptide and cholchicine sites, incubations are done under conditions which do not favor polymerization because vinblastine and cholchicine bind preferentially to unpolymerized heterodimer.
  • polymerized tubulin microtubules
  • paclitaxel binds preferentially to microtubules.
  • each column effluent (containing tubulin and bound radioligand) is mixed with scintillation fluid and counted in a liquid scintillation spectrometer.
  • Controls included samples without competitor, and samples with unlabeled vincristine, cholchicine, or paclitaxel.
  • the ability of the competitor to inhibit the binding of the radioligand is expressed as a percentage of control binding in the absence of any competitor.
  • tubulin polymers formed with dideoxyguanosine nucleotides in the presence and absence of microtubule-associated proteins J. Biol. Chem., 259: 2501-2508, 1984. This solution is incubated for 30 min at 37°C to allow microtubules to form.
  • [ 3 H]paclitaxel final concentration of 2.1 ⁇ M, 1.2 Ci/mmol
  • competitor final concentration of 20 ⁇ M, except 5 ⁇ M for unlabeled paclitaxel
  • Controls included samples without competitor, and samples with unlabeled vincristine, cholchicine, or paclitaxel.
  • the reactions are then centrifuged at top speed in an Eppendorf 5415C microfuge for 20 min at room temperature in order to pellet the microtubule protein.
  • HeLa cells are harvested by trypsinization, washed, counted and distributed to wells of 12-well plates at 125,000 cells per well in 2 ml_ medium. Cells are cultured overnight. Compound dilutions are made in DMSO and 10 ⁇ L aliquots are added to each well to produce the desired final concentrations. Cells are continued in culture for 18 hr after compound addition, then cells in each well are harvested (taking care to recover both adherent and non-adherent cells) and processed using the CycleTEST PLUSTM kit (Becton Dickinson lmmunocytometry Systems, San Jose, CA). Flow cytometry is done with a FACSortTM instrument (Becton Dickinson).
  • mice of this invention are studied in the athymic mouse xenograft standard pharmacological test.
  • Female nu/nu mice in an outbred albino background are obtained from Charles River Laboratories (Wilmington, MA). Animals are injected subcutaneously on the flank with the desired tumor ceJI suspension. Several days later, mice with tumors of approximately 150 mm 3 are selected from those injected (staged) and randomly distributed into groups of 5-10. The day of staging is called day 0.
  • Tumor/Control is obtained by dividing the mean tumor volume of the treated group by the mean tumor volume of the control group on each measurement day.
  • a treatment dose is defined as active if it produced a statistically significant T/C of 0.50 or less.
  • a treatment dose is defined as toxic if more than 10% of the animals died from a compound-related toxicity.
  • COLO 205 is a human colon carcinoma cell line that is used for comparative testing of the examples of this invention and several reference compounds. This line is sensitive to paditaxel and vincristine.
  • the succinic acid salt of 5-Chloro-6- ⁇ 2,6-difluoro-4-[3- (methylamino)propoxy]phenyl ⁇ -N-[(1S)-2,2,2-trifluoro-1- methylethyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine was found to have an IC 50 of 17.5 nanomolar and the IC 50 of paditaxel was 3.3 + 1.0 nanomolar (mean + SD) in 20 independent assays, in good agreement with literature values.
  • KB, KB-8-5, and KB-V1 Cells express different amounts of the P-glycoprotein (MDR1) membrane pump which produces resistance to the action of many cytotoxic compounds, including paclitaxel and vincristine.
  • MDR1 P-glycoprotein
  • the parental KB line expresses no P-glycoprotein
  • KB-8-5 expresses moderate levels of the protein
  • KB-V1 expresses very high levels.
  • P-glycoprotein to recognize and export a potential cytotoxic agent can be inferred from the change in IC 50 values on these lines (Loganzo, F., Discafani, CM., Annable, T., Beyer, C 1 Musto, S., Hari, M., Tan, X., Hardy, C, Hernandez, R., Baxter, M., Singanallore, T., Khafizova, G., Poruchynsky, M.S., Fojo, T., Nieman, J.A., Ayral-Kaloustian, S., Zask, A., Andersen, R.J., and Greenberger, L.M.
  • HTI-286 a synthetic analogue of the tripeptide hemiasterlin, is a potent antimicrotubule agent that circumvents P-glycoprotein- mediated resistance in vitro and in vivo. Cancer Res., 63: 1838-1845, 2003). If a compound is recognized by P-glycoprotein, its IC 50 value will increase substantially (several hundred-fold) on going from KB to KB-8-5 to KB-V1 ; if a compound is not recognized, it will have similar IC 50 values (3-fold or less difference) on all three lines.
  • KB-8-5 cells are moderately resistant to paclitaxel (19-fold), vincristine (11 -fold), cholchicine (3.4-fold) and doxorubicin (3.0-fold).
  • Representative examples of this invention show less than a 2-fold change in IC 50 values.
  • KB-V1 cells are highly resistant to paclitaxel (>345-fold), vincristine (>156-fold), cholchicine (116-fold), mitoxantrone (77-fold), and doxorubicin (>130- fold).
  • Representative examples of this invention will show less than a 3-fold change in IC 50 compared to the parental KB line. This indicates that these compounds are not recognized by P-glycoprotein and, therefore, that these compounds completely overcome P-glycoprotein-mediated resistance to cell killing.
  • HL-60/ADR cells overexpress the multidrug resistance protein MRP1 which mediates resistance to some chemotherapeutics (Gottesman, M.M., Fojo, T., and Bates, S.E. Multidrug resistance in cancer: role of ATP-dependent transporters. Nature Rev. Cancer, 2: 48-58, 2002).
  • MRP1 multidrug resistance protein
  • the IC50 values of representative examples of this invention, as well as reference compounds, on HL-60/ADR are compared to values on the sensitive parental HL-60 line. The results will indicate that the compounds of this invention are not recognized by MRP1 and, therefore, overcome cellular resistance mediated by this transporter.
  • S1-M1 cells overexpress the MXR transporter which mediates resistance to some chemotherapeutics (Gottesman, M. M., Fojo, T., and Bates, S.E. Miltidrug resistance in cancer: role of ATP-dependent transporters. Nature Rev. Cancer, 2: 48-58, 2002).
  • the IC 50 values of representative compounds of this invention, as well as reference compounds, on S1-M1 are compared to values on the sensitive parental S1 line. If the cells show no resistance, this indicates that the compounds are not recognized by MXR and, therefore, overcome cellular resistance mediated by this transporter.
  • control reactions with MAP-rich tubulin show an S-shaped absorbance profile characterized by three phases: first, a lag phase during which no change in absorbance occurs; second, a polymerization phase in which absorbance increases; and third, a plateau phase in which absorbance has reached a maximum and little or no further change occurs.
  • Polymerization enhancers such as paclitaxel and docetaxel shorten or eliminate the lag phase, increase the rate of the polymerization phase, and often increase the height of the plateau.
  • Polymerization inhibitors such as vincristine and cholchicine reduce or prevent the absorbance increase.
  • the compounds of this invention have a taxane-like effect on the polymerization reaction.
  • the site on highly purified bovine brain tubulin to which compounds of this invention bind is determined by competitive inhibition studies with the radioactive ligands [ ⁇ vinblastine, [ 3 H]cholchicine, and [ 3 H]paclitaxel. If the tested compounds inhibit the binding of [ 3 H]vinblastine to tubulin heterodimer, but do not inhibit binding of ⁇ HJcholchicine to tubulin heterodimer or of [ 3 H]paclitaxel to microtubules, it is strong evidence that these compounds bind at the vinca/peptide site of tubulin and not at the cholchicine or taxa ⁇ e sites.
  • the tested compounds enhance the binding of [ 3 H]cholchicine above the control level, it suggests that the binding of these compounds to the vinca/peptide site may induce a conformational change in the protein molecule that results in enhanced cholchicine binding. This change appears not to be induced by vincristine itself. If the tested compounds do not reduce [ 3 H]paclitaxel binding to microtubules, it indicates that they neither compete with [ 3 H]paclitaxel for binding nor depolymerize the microtubules to which [ 3 H]paclitaxel binds.
  • This procedure measures the percentages of cells in a population that are in the G1 , S, and G2/M phases of the cell cycle. It utilizes staining of cell nuclei with propidium iodide and analysis by flow cytometry. The procedure also provides an estimate of apoptosis caused by drug treatment by measurement of the appearance of particles with sub-G1 amounts of DNA. At high concentrations (i.e., higher than about 5 X ICgo concentrations) microtubule-active compounds characteristically arrest cells in the G2/M phase of the cell cycle because of disruption of the microtubules that comprise the mitotic spindle.
  • Taxanes such as paclitaxel and docetaxel induce substantial apoptosis before a G2/M block is observed (Jordan, M.A., Wendell, K., Gardiner, S., Derry, W.B., Copp, H., and Wilson, L. Mitotic block induced in HeLa cells by low concentrations of paclitaxel (Taxol) results in abnormal mitotic exit and apoptotic cell death. Cancer Res., 56: 816-825, 1996); this is not the case with microtubule depolymerizers such as vincristine and cholchicine.
  • the compounds of the invention may be tested against LOX melanoma xenografts, DLD1 colon carcinoma, U-87 MG glioblastoma xenografts, A549 lung carcinoma, and LoVo human colon carcinoma xenografts.
  • Compounds of this invention show potent cytotoxic activity against multiple human cancer cell lines in culture, including lines that are resistant to paclitaxel and vincristine because of drug transporter overexpression.
  • the compounds of the invention enhance the initial rate of MAP-rich tubulin polymerization, in a manner reminiscent of taxanes and distinct from the inhibitory effects of depolymerizers such as vinca alkaloids and cholchicine.
  • Compounds of the invention also induce polymerization of pure tubulin in the absence of GTP.
  • Compounds of this invention further induce apoptosis in target cells at low concentrations (around cytotoxic IC 50 values) without cell cycle block, another property that is characteristic of taxanes but not vincas or cholchicine.
  • Representative compounds of the invention inhibit the growth of several human tumor xenografts in athymic mice, including tumors resistant to taxanes and vinca alkaloids.

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Abstract

La présente invention concerne certains produits d'addition et dimères de composés de 6-[phényl (substitué)] triazolopyrimidine ou leurs sels pharmaceutiquement acceptables, ainsi que des compositions contenant lesdits composés ou leurs sels pharmaceutiquement acceptables, lesdits composés étant des agents anticancéreux utilisables pour le traitement du cancer chez les mammifères. L'invention concerne également un procédé de traitement ou d'inhibition de la croissance de cellules tumorales cancéreuses et des maladies associées chez un mammifère ainsi qu'un procédé de traitement ou de prévention des tumeurs cancéreuses qui présentent une résistance à plusieurs médicaments (Multiple Drug Resistance; MDR) ou qui sont résistantes en raison d'une MDR chez un mammifère qui en a besoin, ledit procédé comprenant l'administration audit mammifère d'une quantité efficace desdits composés ou de leurs sels pharmaceutiquement acceptables. L'invention concerne également un procédé de traitement ou d'inhibition de la croissance de cellules tumorales cancéreuses et des maladies associées chez un mammifère qui en a besoin en favorisant la polymérisation des microtubules, ledit procédé comprenant l'administration audit mammifère d'une quantité efficace desdits composés ou de leurs sels pharmaceutiquement acceptables.
EP06845599A 2005-12-16 2006-12-15 Dimeres et produits d'addition de 6-[phenyl (substitue)] triazolopyrimidines utilisables en tant qu'agents anticancereux Withdrawn EP1963330A1 (fr)

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