CA2633962A1 - Dimers and adducts of 6-[(substituted) phenyl] triazolopyrimidines useful as anticancer agents - Google Patents

Abstract

Certain adducts and dimers of 6-[(substituted)phenyl]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, are disclosed. Also disclosed is a method of treating or inhibiting the growth of cancerous tumor cells and associated diseases in a mammal and a method for the treatment or prevention of cancerous tumors that express multiple drug resistance (MDR) or are resistant because of MDR, in a mammal in need thereof which method comprises administering to said mammal an effective amount of said compounds or pharmaceutically acceptable salts thereof. Also disclosed is 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 which comprises administering to said mammal an effective amount of said compounds and pharmaceutically acceptable salts thereof.

Description

DIMERS AND ADDUCTS OF
6-[(SUBSTITUTED)PHENYL]TRIAZOLOPYRlMIDI N ES
USEFUL AS ANTICANCER AGENTS
FIELD OF THE INVENTION

[0001] The present invention relates to certain adducts and dimers of 6-[(substituted)phenyl]-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.

BACKGROUND OF THE INVENTION

[0002] Most of the cytostatics in use today either inhibit the formation of essential .
precursors for biosynthesis of DNA or block DNA polymerases or interfere with the template function of DNA because DNA was the primary target for developing therapeutic drugs for chemotherapy. Unfortunately, inhibition of the formation of essential precursors for biosynthesis of DNA or blocking DNA polymerases or interference with the template function of DNA also affects normal tissues.

[00031 Antimicrotubule drugs are a major category of anticancer agents (Rowinsky, E.K., and Toicher, A.W. Antimicrotubule agents. In: V.T. Devita, Jr., S.
Heliman, and S.A. Rosenberg (eds.), Cancer Principles and Practice, Ed. 6, pp. 431-452.
Philadelphia: Lippincott Williams and Wilkins, 2001). They work by interfering with the function of cellular microtubules, particularly the mitotic spindle. The disruption of normal spindle function leads to apoptotic cell death.

[0004] Currently, there are three major classes of known antimicrotubule pharmacological agents. Each has a distinct binding region on j3-tubulin and distinct effects on microtubule function. These classes are: 1) taxane-site agents that promote microtubule formation and stabilize microtubules; 2) vinca/peptide-site agents, which destabilize microtubuies and often induce formation of abnormal polymers or aggregates at high concentrations; and 3) cholchicine-site agents that also destabilize microtubules and generally do not induce other polymers (Hamel, E.
Antimitotic natural products and their interactions with tubulin. Med. Res.
Rev., 16:
207-231, 1996). Most of the ligands for all three classes of sites are natural products or semi-synthetic derivatives of natural products.

[0005] Paclitaxel and its semisynthetic derivative docetaxel (Taxotere ) 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. The crude extract was tested for the first time during the 1960s and its active principle was isolated in 1971 and the chemical structure identified (M.C. Wani et al., J. Am. Chem. Soc., 93, 2325 (1971)).
Further, a wide range of activity over melanoma cells, leukemia, various carcinomas, sarcomas and non-Hodgkin lymphomas, as well as a number of solid tumors in animals, was shown through additional testing. Paclitaxel and its analogs have been produced by partial synthesis from 10-deacetylbaccatin III, a precursor obtained from yew needles and twigs, and by total synthesis (Holton, et al., J. Am. Chem.
Soc.
116:1597-1601 (1994) and Nicolaou, et al., Nature 367:630-634 (1994)).
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-(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 microtubuies. Choichicine-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. Rev.
Med., 53: 615-627, 2002). The best known of these transporters is P-glycoprotein.
Accordingly, there is a need for new agents with taxane-like effects on microtubule polymerization that are not substrates of P-glycoprotein or other such pumps and that therefore will overcome this cause of taxane resistance in patients.

[0008] Second, 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). Patients are typically premedicated with corticosteroids and antihistamines before administration of paclitaxel to minimize these toxicities. Accordingly, there is a need for new agents with taxane-like effects on microtubule polymerization that are highly water-soluble and can be administered in physiological saline or other suitable non-toxic vehicle.

[0009] Third, paclitaxel is a natural product having a highly complex structure, and 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.

[0010] Accordingly, there is still a need in the art for 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.

[00111 Accordingly, it would be advantageous to provide new compounds that provide a method of treating or inhibiting cell proliferation, neoplastic growth and malignant tumor growth in mammals by administering compounds that have paclitaxel like anticancer activity.

[0012] Additionally, it would be advantageous to provide new compounds that provide a method for treating or inhibiting growth of cancerous tumors that express multiple drug resistance (MDR) or are resistant because of MDR.

[0013] Further, it would be advantageous to provide new compounds that provide a method of treating or inhibiting the growth of cancerous tumors in a mammal with inherent or acquired resistance to chemotherapeutic agents and in particular antimitotic agents.

[0014] Described in the art is the preparation and use of substituted triazolopyrimidines in agriculture as fungicides in US Patent Nos.: 5,593,996;
5,756,509; 5,948,783; 5,981,534; 5,612,345; 5,994,360; 6,020,338; 5,985,883;
5,854,252; 5,808,066; 5,817,663; 5,955,252; 5,965,561; 5,986,135; 5,750,766;

6,117,865; 6,117,876; 6,124,301; 6,204,269; 6,255,309; 6,268,371; 6,277,856;
6,284,762; 6,297,251; 6,387,848; US Patent Application Publication Nos.
US2002/0045631A1; US2002/0061882A1; US20030055069A1 and International or European Publication Nos.: W098/46607; W098/46608; W099/48893;
W099/41255; W000/18227; WO01/35738A2; W002/46195A1; W002/067679A1;
W002/083676A1; EPO 834513A2; EPO 782997A2; EP0550113131; FR2784381A1;
EPO 989130A1; W098/41496; W094/20501; EPO 945453A1; EPO 562615A1; EPO
56261561; EP 550113A2; EP 943241131; EP 988790B1 and having the following general formula:

R4--<v N~

[0015] Disclosed in International Publication No. WO 02/02563 is the use of triazolopyrimidines as anticancer agents.

[0016] 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 (i-tubulin, yet they have many properties that are similar to taxanes and distinct from vinca-site agents. In particular, 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. 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.

[0017] 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. in particular, 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.

[0018] 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. For example, these compounds are active when dosed on a once-weekly schedule by either IV or oral routes.

[0019] 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. W02005/030775 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. 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), colon cancer, breast cancer, melanoma and glioblastoma, including models which are resistant to taxanes or other microtubule-active compounds.

SUMMARY OF THE INVENTION

[00211 In accordance with the present invention, there is provided compounds represented by Formula (I):

Ll Y (CHZ)nNR6R7 R' NN
N~ ~- ~ L2 <
N X
(I) wherein R' is "'_Mt ' or a-(Cs - C8)-cycloalkyl optionally substituted with Ra;
n is an integer of 2, 3, or 4;

X is -Cl, -F or -Br;

Y is -0-, -S-, -CH2- or -NR4-;

L' and L2 are each independently -H, -F, -CI, -Br, or -CF3;
R3 is -CF3 or -C2F5;

R4, R5, and R6 are each independently -H or -(C1-C3)-alkyl;

R' is -ZR9, wherein Z is -CO-, -NO-, -SO2-, or -PO2H-; and R9 is -H, -OH, -(C1-C5) -alkyl or -(CZ-C5) -alkenyl, said alkyl or alkenyl optionally substituted with one or more of -0-, halogen, -OH, -NH2, 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-CH3, -OH, or halogen; and R8 is (C1-C3) -alkyl; or pharmaceutically acceptable salts or stereoisomers thereof.

[0022] The instant invention further provides compounds of Formula (II):
(CH2)nNR6R2 Y

Rs LZ
R~ L~ Li N%
r N y\ N RI
N N_' ~ N
N

N~
(II) wherein R' is or a -(C6 - C8)-cycloalkyl optionally substituted with RB;
n is an integer of 2, 3, or 4;

X is -Cl, -F or -Br;

Y is -0-, -S-, -CH2- or -NR4-;

L' and L2 are each independently -H, -F, -Cl, -Br, or -CF3;

R3 is -CF3 or -C2F5;

R2, R4, R5, R6 and R9are each independently -H or -{Cj-C3)-alkyl;
R$ is --(Cj-C3)-alkyl; and pharmaceutically acceptable salts or stereoisomers thereof.

[0023] 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.

[0024] 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 (1) or (II), or pharmaceutically acceptable satts thereof.
[0025] Further provided is 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 which method comprises administering'to said mammal an effective amount of such compounds or pharmaceutically acceptable salts thereof.
[0026] 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 (1) or (11), or pharmaceutically acceptable salts thereof.

[0027] Additionally 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 (Il), or a pharmaceutically acceptable salt thereof.

[0028] Further provided is 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, which method comprises administering, to said mammal an effective amount of the compounds of Formula (I) or (II), or pharmaceutically acceptable salts thereof.

[0029] In yet a further aspect, this invention provides a compound of Formula (I) in combination or association with a pharmaceutically acceptable carrier. In particular, the present invention provides a pharmaceutical composition, which comprises an effective amount of a compound of Formula (I) and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF
ILLUSTRATIVE EMBODIMENTS OF THE INVENTION
(a) Definitions [0030] The term "alkyl" means a straight or branched hydrocarbon chain moiety, preferably of 1 to 3 carbon atoms.

[0031] The term "t-BOC" as used herein means tert-butoxy carbonyl.
[0032] The term "aminoalkoxy" means a moiety of the formula Rs 1-0-(CH2)rF-N\,,, [0033] The term "aminoalkyl" means a moiety of the formula ~-CH -(CH ~
2 2)ri %7 1~

[0034] The term "aminoalkylthio" means a moiety of the formula Rs [0035] The term "aminoalkylamino" means a moiety of the formula [0036] The term "hydroxyalkoxy" means a moiety of the formula 1_0-(CH2)6___0H
[0037] The term "alkali metal hydroxide" includes lithium, potassium or sodium hydroxide.

[0038] The term "alkali metal carbonate" includes lithium, potassium or sodium*
carbonate.

[0039] The term "alkali metal hydride" includes lithium, potassium or sodium hydride.
[0040] The term "strong base" means an alkali metal hydroxide, alkali metal carbonate and alkali metal hydride (e.g., sodium hydride).

[0041] The term "(Cj-C5)-alkyl" as used herein refers to a linear or branched, saturated hydrocarbon having from 1 to 5 carbon atoms, preferably I to 3 carbon atoms. Representative (C,-C5)-alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, and neopentyl.
In one embodiment, the (CI-C5)-alkyl group is substituted with one or more of the following groups: halogen, -N3, -NO2, -CN, -OR', -SR', -SO2R', -SO2N(R')2, -N(R')2, -COR', -CO2R', -NR'CO2R', -NR'COR', -NR'CONR', or -CON(R')2, wherein each R' is independently hydrogen or unsubstituted (Cti-CS)-alkyl.

[0042] The term "(C2-C5)-alkenyl" as used herein 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 (C2-C5)-alkenyl has one or two double bonds.
The (Cz-CS)-alkenyl moiety may exist in the E or Z conformation and the compounds of the present invention include both conformations. Representative (C2-Cs)-alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, sec-butenyl, tert-butenyl, pentenyl, isopentenyl, and neopentenyl. In one embodiment, the (C2-CO-alkenyl group is substituted with one or more of the following groups:
halogen, -N3, -NO2, -CN, -OR', -SR', -SO2R', -SO2N(R')2r -N(R')2, -COR', -CO2R', -NR'COzR', -NR'COR', -NR'CONR', or -CON(R')2, wherein each R' is independently hydrogen or unsubstituted (Cl-C5)-alkyl.

[0043] The term "administet", "administering", or "administration", as used herein 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.

[0044] The term "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.

[0045] The term "aryl" as used herein refers to an aromatic species containing 1 to 3 aromatic rings, either fused or linked. In one embodiment, the aryl group is substituted with one or more of the following groups: -V'-halogen, -V'-N3, -V'-N02, -V'-CN, -V'-OR', -V'-SR', -V'-SO2R', -V'-SOZN(R')Z, -V'-N(R')Z, -V'-COR', -V'-C02R', -V'-NR'CO2R', -V'-NR'COR', -V'-NR'CONR', or V'-CON(R')2, wherein each R' is independently hydrogen or unsubstituted (C,-C6)-alkyl; and wherein each V' is independently a bond or (CI-Cs)-alkyl.

[0046] The term "conditions effective to" as used herein refers to synthetic reaction conditions which will be apparent to those skilled in the art of synthetic organic chemistry.

[0047] The term "cyclic group" as used herein 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. In one embodiment, the cyclic group is substituted with one or more of the following groups: -V'-halogen, -V'-N3, -V'-N02, -V'-CN, -V'-OR', -V'-SR', -V'-SO2R', -V'-SO2N(R')2, -V'-N(R')2, -V'-COR', -V'-CO2R', -V'-NR'C02R', -V'-NR'COR', -V'-NR'CONR', or -V'-CON(R')2, wherein each R' is independently hydrogen or unsubstituted (C,-C6)-alkyl; and wherein each V' is independently a bond or (Cl-C6)-alkyl.

[0048] The term "cycloalkyl group" as used herein 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. In one embodiment, the cycloalkyl group is substituted with one or more of the following groups: -V'-halogen, -V'-N3, -V'-N02, -V'-CN, -V'-OR', -V'-SR', -V'-SO2R', -V'-SO2N(R')2, -V'-N(R')2, -V'-COR', -V'-C02R', -V'-NR'CO2R', -V'-NR'COR', -V'-NR'CONR', or -V'-CON(R')2: wherein each R' is independently hydrogen or unsubstituted (Cl-C6)-alkyl; and wherein each V' is independently a bond or (Cl-C6)-alkyl.

[0049] The term "phenyl" as used herein refers to a substituted or unsubstituted phenyl group. In one embodiment, the phenyl group is substituted with one or more of the following groups: -V'-halogen, -V'-N3, -V'-NO2, -V'-CN, -V'-OR', -V'-SR', V'-SO2R', -V'-SO2N(R')2r -V'-N(R')z, -V'-COR', -V'-CO2R', -V'-NR'CO2R', -V'-NR'COR', -V'-NR'CONR', or -V'-CON(R')2, wherein each R' is independently hydrogen or unsubstituted (CI-CB)-alkyl; and wherein each V' is independently a bond or (C,-Cs)-alkyl.

[0050] The term "halogen" as used herein refers to fluorine, chlorine, bromine, and iodine.

[0051] The term "heterocyclic group" as used herein 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, 0, or S atom. Any suitable ring position of the heterocyclic group may be covalently linked to the defined chemical structure. Exemplary heterocyclic groups include, but are not limited to, azepanyl, azetidinyl, aziridinyl, furanyl, furazanyl, homopiperazinyl, imidazolidinyl, imidazolinyl, isothiazolyi, isoxazolyl, morpholinyl, oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, piperazinyl, piperidinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazolyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl, tetrahydrofuranyl, thiadiazinyl, thiadiazolyl, thienyl, thienothiazoly{, thienooxazolyl, thienoimidazolyl, thiomorpholinyl, thiophenyl, triazinyl, and triazolyl. In one embodiment, the heterocyclic group is substituted with one or more of the following groups: -V'-halogen, -V'-N3, -V'-NOz, -V'-CN, -V'-OR', -V'-SR', -V'-SOZR', -V'-SO2N(R')2, -V'-N(R')2, -V'-COR', -V'-CO2R', -V'-NR'CO2R', -V'-NR'COR', -V'-NR'CONR', or -V'-CON(R')2, wherein each R' is independently hydrogen or unsubstituted (C,-C6)-alkyl; and wherein each V' is independently a bond or (CI-Cs)-alkyl.

[0052] The term "effective amount" as used herein 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_ [0053] The term "carrier", as used herein, shall encompass carriers, excipients, and diluents.

[0054] The term "prodrug", as used herein means a compound which is convertible in vivo by metabolic means (e.g. by hydrolysis) to a compound of Formula (I) or Formula (II).

[0055] The term "isolated and purified" as used herein refers to a component separated from other components of a reaction mixture or a natural source. In certain embodiments, 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.

[0056] The term "pharmaceutically acceptable salt" as used herein 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, napthalenesulfonate, propionate, succinate, fumarate, maleate, malonate, mandelate, malate, phthalate, and pamoate. The term "pharmaceutically acceptable salt" as used herein also refers to a salt of a compound of the present invention having an acidic functional group, such as a carboxylic acid functional group, and a base. Exemplary bases include, but are not limited to, hydroxide of alkali metals including sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, organic amines such as unsubstituted or hydroxyl-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine;
pyridine;
N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2- H-(Cj-Cr,)-alkylamine), such as N,N-dimethyl-N- (2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; morpholine; thiomorpholine;
piperidine; pyrrolidine; and amino acids such as arginine, lysine, and the like. The term "pharmaceutically acceptable salt" also includes a hydrate of a compound of the present invention.

[0057] The term "substantially free of its corresponding opposite enantiomer"
as used herein 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.

(b) Compounds and Pharmaceutically Acceptable Salts of Compounds of the Invention [0058] 'In accordance with the present invention, there is provided compounds represented by Formula (I):

Ll Y (CH2)nNRsR7 Rl p N-'N <

NN JC
(i) wherein R' is R$ N-H

or a -(C6 - C$)-cycloalkyl optionally substituted with R8;
X is -Cl, -F or -Br;

Y is -0-, -S-, -CH2- or -NR4-;

L' and L2 are each independently -H, -F, -CI, -Br, or -CF3;
R3 is -CF3 or -C2F5;

R4, R5, and R6 are each independently -H or -(C,-C3)-alkyl;

R' is -ZR9, wherein Z is -CO-, -NO-, -SO2-, or -PO2H-; and R9 is -H, -OH, -(C1-C5) -alkyl or -(C2-C5)-alkenyl, said alkyl or alkenyl optionally substituted with one or more of -0-, halogen, -OH, -NH2, 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, 0, or S atom and said cycloalkyl group optionally is substituted with a -CH3, -OH, or halogen; and Re is -(CI-C3)-alkyl; and pharmaceutically acceptable salts or stereoisomers thereof.
[0059] The instant invention further provides a compound of Formula (II):
(CH2)nNR6R2 Y
Rs R' Ll ' L2 L1 N~N
N
-~--~ ~ y\' 1 / N R1 N_ n N
N

N~
(II) wherein R' is or a - (Ce - C$) -cycloalkyl optionally substituted with R'3;

n is an integer of 2, 3, or 4;

X is -CI, -F or -Br;

Y is -0-, -S-, -CH2- or -NR4-;

L' and L2 are each independently -H, -F, -Cl, -Br, or -CF3;
R3 is -CF3 or -C2F5;

R2, R4, W. W. and R9are each independently -H or -(Cl-C3)-alkyl;
R$ is -(C,-C3)-alkyi; or pharmaceutically acceptable salts or stereoisomers thereof.

[0060] In the compounds of Formula (I) and (II), Rs is preferably a-(C,-C3)-alkyl, and even more preferably is -CH3.

[0061] In compounds of Formula (I), preferably R' is -ZR9, wherein Z is -CO-or -NO-; and R9 is a-(CI-C5)-alkyl or -(C2-C5)-alkenyl, optionally substituted with one or more of 0, halogen, -OH, -NH2, 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, 0, or S atom and said cycloalkyl group optionally is substituted with a-CH3i -OH, or halogen. More preferably, Z is -CO- and R9 is a -C2-C4 alkyl or -(C2-Ca)-alkenyl, having a group.

[0062] Particularly preferred are compounds having formula (I) in which R' is R3WCH-NH- (where R3 and R5 are as defined above) and R7 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-(CH2) 2-CO2H) and Y is -0-.

[0063] Further preferred are compounds having formula (i) or (II) in which R' is R3R5CH-NH- (where R3 and R5 are as defined above), X is -CI, L' and L2 are -F, n is 3, R2 is H or -(Cl to C3) alkyl, R6 is -(Cl to C3) alkyl (preferably -CH3), and Y is -0-.
For compounds of formula (f), these compounds will preferably have R' 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-(CH2) 2-CO2H).

[0064] In preferred compounds of Formula (I) and Formula (II), R' is J

.ruI
[0065] Preferred compounds of Formula (l) and Formula (ll) will include R3 as -and/or R5 as a-C,-C3 alkyl.

[0066] Preferred substituents for L' and L2 are each independently -F, -Cl, or -Br.
More preferably, L' and LZ are each -F.

[0067] In the compounds of Formula (I) and Formula (li), Y is preferably -0-, n is preferably 3, and/or X is preferably -Cl.

[0068] A particularly preferred compound of Formula (I) is:

OH
O
O

,-JI NHF
N-N
/
-- I
N N Cl F

(la) [0069] A further preferred compound of Formula (I) of the invention is:
OH

Nf. i I 0,~ N,, N,N

<NN CI F

(Ib) [0070] A still further preferred compound of Formula (I) includes:

CF3 ~
NH F O N y {CHph-H

F
N cl (I~) wherein r is an integer from 0 to 5, inclusive.

[0071] Preferred compounds of Formula (11) include:

F
I ~ HN

N.- \

~ F / I
N
N ~ \

~ F
N

(Ila) [0072] More particularly, a preferred compound of Forrnula (Il) is:

F HN/

N---.N
CF3 F I ~
\ ~N
N NH

N \
<

~
N ~
N CI

(Ilb) [0073] As evident from these compounds, 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-Ingold-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. Included in the scope of the present invention are (R) and (S) isomers of compounds of general Formula (I) and (II) having a chiral center and the racemates thereof. The present invention encompasses all stereoisomers of the compounds whether free from other stereolsomers or admixed with other stereoisomers in any proportion and thus 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.

[0074] Optical isomers may be obtained in pure form by standard separation techniques or enantiomer specific synthesis. A compound of Formula (I) or Formula (li) that is substantially free of its corresponding opposite enantiomer may thus be obtained.

[0075] Also, the polymorphs, hydrates and solvates of the compounds of the present invention are included within the scope of the invention.

[0076] It is understood that this invention encompasses all crystalline and hydrated forms of compounds of Formulas (1) and (t!) 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, pafmitic and similarly known acceptable acids. A further salt is the trifluoroacetic acid salt (TFA). In particular, the hydrochloride, fumarate and succinate salts are preferred.

[0077] To form the salt, the compounds of Formula (I) or (II) are reacted with 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-[(1 S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyrimidin-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.

[0078] Preferably, pharmaceutically acceptable salts may form by heating compounds of Formula (I) or (II) in a suitable solvent, at about 30-100 C, preferably at about 65-75 C, until a clear solution forms. Upon cooling, the compound may be collected and dried.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] A starting material for preparing compounds of Formula (I) and (II) includes compounds of the Formula (ill):

Ll Y (CH7)nNRsR2 Rl NN
N~- b2 <
N X

(111) The substituents are as prievously defined, and R2 is preferably -H.

[0083] 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 Apri128, 2005 and as international Patent Publication No.
W02005/030775 on April 7, 2005, the disclosure of which is incorporated by reference in its entirety for the compounds and methods of making same.

[0084] The second starting material is a compound of formula HOZR9, wherein Z
is -CO-; -NO-, -SO2-, or -P02H-; and R9 is -H, -OH, -(Cj-C5)-alkyl or -(C2-C5)-alkenyl, said alkyl or alkenyl optionally substituted with one or more of 0, halogen, -OH, -NH2, 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, 0, or S atom and said cycloalkyl group optionally is substituted with a -CH3, -OH, or halogen.

[0085] An example of the reaction to produce a specific compound of Formula (I) is as follows:

"K
NH N
N F + H-(CH2)rCO 0 N
N CI H-(CH2)r CO
~F3 I
Nl-F 0=,/\,N_r(CH2)r -H

<N..N 0 N N CI F

wherein r is an integer from 0 to 5.

[0086] Alternatively, the second starting material is a compound of formula:
ZI A

X(HqCHPC) Z2 wherein p is 1 or 2; q is 0 or 1; x is 1, 2 or 3; Z' and Z2 are each independently -CH2-, -CO-, -NO-, -SO2-, or -PO2H-; and A is an -0-, -S- or -N- atom.

For example, a specific compound of Formula (I) can be prepared by the following reaction:

oH
~ o 1-1 N~ ~( C~/\~N~ p ~N~ 0,./\,N~
N, ~ ~ i IV, .~ \

<N CI F + ~ p <NN CI F
O
[0087] To produce a compound of Formula (I), the two starting materials are mixed under appropriate conditions in the presence of an ether solvent, including but not limited to tetrahydrofuran (THF), diethyl ether, methyl t-butyl ether, and dioxane. The starting materials are mixed for a time sufficient to produce a compound of Formula (!). The mixture may be heated as necessary to facilitate the reaction.

[0088] A compound of Formula (II) is prepared by a process comprising heating a mixture having a compound of formula (!tl):

Ll Y (CH2)nNRsR2 R' NN
< N L2 N X

(Itl) in an appropriate solvent system to a temperature and for a time sufficient to produce a compound of Formula (II).

[0089] For example, a compound of Formula (II) may be produced as follows:

"I'NFf.
N_N H CF3 <1 F F ~
N N CI HN
+ N-N
CF:3 H CjF3 F NN
N~
O~.-/~N~ ~ ~= I O,/-,,N,., I -,P

N~N N~N (c) <NN CI F NN CI 29 Pharmaceutical Compositions [0090] The present invention accordingly provides a pharmaceutical composition which comprises a compound of this invention in combination or association with a pharmaceutically acceptable carrier. In particular, the present invention provides a pharmaceutical composition which comprises an effective amount of a compound of this invention and a pharmaceutically acceptable carrier.

[0091] Based on the results of standard pharmacological test procedures described herein, 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 muitiple drug resistance (MDR) or are resistant because of MDR.

[0092] In particular, 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. In the case of cancer treatment, it is believed that many neoplasias such as leukemia, lung cancer, colon cancer, thyroid cancer, ovarian cancer, renal cancer, prostate cancer and breast cancers may be treated by effectively administering effective amounts of the compounds of Formula (I) or (iI). Additionally, compounds of Formula (I) and (II) are useful for the treatment or prevention of cancerous tumors that express multiple drug resistance (MDR) or are resistant because of MDR. As used herein, 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. By 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.

[0093] 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.

[0094] 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.

[0095] 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.
For oral therapeutic administration, these active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, etixirs, 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.

[0096] 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 wintergreen or cherry flavoring.
When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose, as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, these active compounds may be incorporated into sustained-release preparations and formulations.

[0097] These active compounds may also be administered parenterally or intraperitonealiy. 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.

[0098] 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. In all cases, 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.

[0099] Intravenous administration is a preferred manner of administration of compounds of the invention. For intravenous administration, examples of non-limiting 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, propyiene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
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. In many cases, it will be preferable to include 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.

[00100] As used in accordance with this invention, 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.

[00101] In addition to the above utilities some of the compounds of this invention are useful for the preparation of other compounds of this invention.

(d) Standard Pharmacological Test Procedures [0100] Examples of this invention are evaluated in several standard pharmacological test procedures that showed that the compounds of this invention possess significant activity as promoters of microtubule polymerization and are antineoplastic agents.
Based on the activity in the standard pharmacological test procedures that follow, the compounds of this invention are, therefore, useful as anticancer agents.
Associated cancers are selected from the group consisting of breast, colon, lung, prostate, melanoma, epidermal, leukemia, kidney, bladder, mouth, larynx, esophagus, stomach, ovary, pancreas, liver, skin and brain. In particular, the compounds of this invention possess an effect similar to Paclitaxel.

Materials and Methods Cell Culture Media and Reagents [0101] Medium is RPMI-1640 with L-glutamine, supplemented with 10% heat-inactivated fetal calf serum, 100 units/mi penicillin, and 100 g/mi 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(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 mM
magnesium chloride) and guanosine 5'-triphosphate (GTP) are also obtained from Cytoskeleton. [3H)paclitaxel, specific activity 14.7 Ci/mmol, is purchased from Moravek Biochemicals (Brea, CA). [3H]vinblastine, specific activity 9.60 Ci/mmol and MicroSpin G-50 columns are obtained from Amersham Biosciences (Piscataway, NJ). [3H]cholchicine, specific activity 76.5 Ci/mmot, is obtained from New England Nuclear (Boston, MA). Other reagents are obtained from Sigma (St. Louis, MO).

1. Cell Lines [0102] Human cancer cell lines, unless othenrvise noted, are obtained from the American Type Culture Collection (Rockville, MD). The following drug-sensitive parental cell lines, and their derived drug-resistant counterparts, are obtained from the originators as listed: (a) S1 (parental line from a subclone of human colon carcinoma line LS174T) and derived S1-M1 -3.2 (herein called S1-M1) which expresses the MXR drug transporter protein, are provided by Dr. L.
Greenberger, Wyeth Research (Rabindran, S.K., He, H., Singh, M., Brown, E., Collins, K.I., Annable, T., and Greenberger, L.M. Reversal of a novel multidrug resistance mechanism in human colon carcinoma cells by fumitremorgin C. Cancer Res., 58:
5850-5858, 1998); (b) parental HL-60 human promyelocytic leukemia line and derived HL-60/ADR, which expresses the MRP1 drug transporter protein, are provided by Dr. M_ Center, University of Kansas (McGrath, T., and Center, M.S.
Adriamycin resistance in HL60 cells in the absence of detectable P-glycoprotein.
Biochem. Biophys. Res. Commun., 145: 1171-1176, 1987), via Dr. L. Greenberger, Wyeth Research; and (c) parental 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 choichicine, ad(amycin, or vinblastine show changes in expression of specific proteins. J. Biol. Chem., 261: 7762-7770, 1986) via Dr. L. Greenberger, Wyeth Research.

2. Cytotoxicity Standard Pharmacological Test Procedure [0103] The assay, which is sold in kit form by Promega (Madison, WI; CeIlTiter AQueous Non-Radioactive Cell Proliferation Assay), is based on the conversion by viable cells, but not by dead cells, of the tetrazollum salt, MTS (3-(4,5-di methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt), into a water-soluble colored formazan which is detected by spectrophotometry. Compounds are tested at nine concentrations, in order to determine IC50 values. For the test procedure, 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 pL of medium. In addition, 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% CO2 in air for about 24 hr.

[0104] On day 2, compounds for test are diluted and added to wells. Compounds are dissolved in DMSO at 10 mg/mL. For each compound, nine serial 2-fold dilutions are prepared in DMSO. Ten pL of each dilution in DMSO is transferred to 100 pL
of medium, mixed well, and then 5 pL of this dilution is transferred in quadruplicate to wells containing cells. The final high concentration of each compound is typically 5 pM. Plates are returned to the incubator for three days.

[0105] At the time of drug addition to the experimental plates, 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.

[0106] After three days of culture with test compounds (day 5 overall), the MTS
assay is done on atl 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. The average of control wells with high drug concentration, divided by the "time 0" value, gives the minimal relative color yield for cells that are completely killed. The nine values for each compound are plotted against concentration, and the concentration that produces a relative color yield half way between the maximum and minimum is taken as the IC50 value. The most potent compounds have the lowest 1C50 values.

3. Tubutin Polymerization Standard Pharmacological Test Procedure [0107] Two variations of this procedure are done, one using MAP-rich tubulin and one using pure tubulin.

10108] 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'0-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 pM in a volume of 110 pL 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.

[0109] 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 pM). 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 pM. The plate reader is thermostated at 35 C.

4. Competitive Binding Standard Pharmacological Test Procedure [0110] The binding of examples of this invention to highly purified tubulin is studied by competitive inhibition methods. The aJ3-tubulin heterodimer contains binding sites for the three major classes of microtubule-active pharmacological agents:
taxanes, vinca/peptide-site agents, and choichicine-site agents. To study possible competition at the vinca/peptide and choichicine sites, incubations are done under conditions which do not favor polymerization because vinblastine and choichicine bind preferentially to unpolymerized heterodimer. To study possible competition at the taxane site, on the other hand, polymerized tubulin (microtubuies) is used because paclitaxel binds preferentially to microtubules.

[0111] Highly purified tubulin is dissolved in PEM buffer without GTP and used at a final concentration of 1.0 to 1.3 mg/ml (10 to 13 pM). To the tubulin solution is added various concentrations of examples of this invention up to a highest concentration of 100 pM, and [3H]vinblastine or [3H]cholchicine at final concentrations of 100 nM or 50 nM, respectively. These solutions are incubated at 24 C for 1 hr and then applied to MicroSpin G-50 columns which are centrifuged for 2 min at 3000 rpm in an Eppendorf 5415C microfuge. An aliquot of 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.

[0112] For competition with [3H]paclitaxel, highly purified tubulin is dissolved in PEM
buffer containing 0.75 M glutamate and 25 pM dideoxy-GTP; final protein concentration is 0.25 to 0.35 mg/mL (2.5 to 3.5 pM). These conditions foster the rapid formation of short, stable microtubule polymers (Hamel, E., del Campo, A.A., and Lin, C.M. Stability of 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. Then [3H]paclitaxel (final concentration of 2.1 pM, 1.2 Ci/mmol) and competitor (final concentration of 20 pM, except 5 pM for unlabeled paclitaxel) are added to aliquots of the polymerized tubulin solution and incubation at 37 C is continued for another 30 min. 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. Triplicate aliquots of each supernatant are mixed with scintillation fluid and counted in a liquid scintillation spectrometer. From the amount of radioactivity in the supernatants and the measured total starting radioactivity, the amount of [3H]paclitaxel bound to pelleted microtubule protein is calculated. The ability of each competitor to inhibit radioligand binding to pelleted protein is expressed as a percent of controls without any competitor.

5. Cell Cycle Analysis Standard Pharmacological Test Procedure [0113] 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 NL 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 CycIeTEST PLUSTM kit (Becton Dickinson lmmunocytometry Systems, San Jose, CA). Flow cytometry is done with a FACSortTM instrument (Becton Dickinson).

6. Antitumor Activity in Athymic Mice Bearing Human Tumor Xenografts Standard Pharmacological Test Procedure [0114] The ability of compounds of this invention to inhibit tumor growth in animals is 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 cell suspension. Several days later, mice with tumors of approximately 150 mm3 are selected from those injected (staged) and randomly distributed into groups of 5-10. The day of staging is called day 0. Compounds of the invention, usually formulated in saline (exceptions are noted in tables), are administered to animals by intravenous injection or oral gavage on various schedules starting on day 0 or 1, as noted in the tables. The control group in each experiment is dosed with vehicle on the same schedule. Tumor size is measured every 3-7 days with calipers in two orthogonal dimensions, and tumor volume is calculated from the formula volume = [(length x width2)/2].

[0115) Tumor/Control (T/C) 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 p value s 0.05, determined by one-side Student's t-test, is required for statistical significance. A treatment dose is defined as toxic if more than 10% of the animals died from a compound-related toxicity.

Results 1. Cytotoxicity Standard Pharmacological Test Procedure 1.1. With COLO 205 Cells [0116] 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 paclitaxel and vincristine. The compound of Formula (Ia), for example, was found to have an IC50 value of 1.9 micromolar in each of two separate titrations.
In the same assay, the succinic acid salt of 5-Chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-[(1 S)-2,2,2-trifluoro-l-methylethyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine was found to have an IC50 of 17.5 nanomolar and the ICso of paclitaxel was 3.3 + 1.0 nanomolar (mean SD) in 20 independent assays, in good agreement with literature values.

1.2. With KB, KB-8-5, and KB-V1 Cells [0117] The KB lines express different amounts of the P-glycoprotein (MDR1) membrane pump which produces resistance to the action of many cytotoxic compounds, including paclitaxel and vincristine. The parental KB line expresses no P-glycoprotein, KB-8-5 expresses moderate levels of the protein, and KB-V1 expresses very high levels. The ability of P-glycoprotein to recognize and export a potential cytotoxic agent can be inferred from the change in ICSO values on these lines (Loganzo, F., Discafani, C.M., Annable, T., Beyer, C., 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 IC50 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 wiU have similar IC50 values (3-fold or less difference) on all three lines.
For example, as shown in Table 2, KB-8-5 cells are moderately resistant to paclitaxel (19-fold), vincristine (19-fold), choichicine (3.4-fold) and doxorubicin (3.0-fold).
Representative examples of this invention (Nos. 1, 2a, 4a, 20, 25, 30, 32) show less than a 2-fold change in IC50 values.

[0118] Even slight interactions of compounds with P-glycoprotein can be determined with the KB-V1 line, which expresses a level of this protein higher than is typically found in clinical samples from a variety of tumors (Goldstein, L.J., Galski, H., Fojo, T., Willingham, M., Lai, S.L., Gazdar, A., Pirker, R., Green, A., Crist, W., Brodeur, G.M., Lieber, M., Cossman, J., Gottesman, M.M., and Pastan, I. Expression of a multidrug resistance gene in human cells. J. Natl. Cancer Inst. (Bethesda), 81: 116-124, 1989). KB-V1 cells are highly resistant to paclitaxel (>345-fold), vincristine (>156-fold), choichicine (116-fold), mitoxantrone (77-fold), and doxorubicin (>130-fold). Representative examples of this invention will show less than a 3-fold change in IC50 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.

1.3. (With HL-60 and HL-60/ADR Cells) [0119] 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). The IC50 values of representative examples of this invention, as well as reference compounds, on HL-601ADR 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.

1.4. With S9 and S1-M1 Cells [0120] Sl -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 lCw 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.

2. Effects of Compounds on Polymerization of MAP-rich and Pure Tubulin in vitro [0121] In this assay, 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.

[0122] Pure tubulin without added GTP shows no polymerization in control reactions.
Docetaxel, and to a much lesser extent, paclitaxel, are able to induce polymerization of pure tubulin under these conditions. Several examples of this invention will also induce polymerization of pure tubulin without GTP in a manner similar to docetaxel.
3. Binding of Compounds to Tubulin _ [0123] 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 [3H]vinblastine, [3H]chofchicine, and [3H]paclitaxel. If the tested compounds inhibit the binding of [3H]vinblastine to tubulin heterodimer, but do not inhibit binding of CH]chofchicine to tubulin heterodimer or of [3H]paclitaxel to microtubules, it is strong evidence that these compounds bind at the vinca/peptide site of tubulin and not at the choichicine or taxane sites. If the tested compounds enhance the binding of [3H]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 [3H]paclitaxel binding to microtubules, it indicates that they neither compete with [3H]paclitaxel for binding nor depolymerize the microtubules to which [3H]paclitaxel binds.

4. Effect of Compounds on Cell Cycle Progression [0124] 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 IC50 concentrations) microtubule-active compounds characteristically arrest cells in the G2/M phase of the cell cycle because of disruption of the microtubuies that comprise the mitotic spindle. However, at lower concentrations (near ICSO
values) on some cell lines, e.g., HeLa, 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.
Representative examples of this invention are tested in this procedure after 18 hr of culture with cells at multiple concentrations to see if they followed the "stabilizer' (taxane) or "destabilizer" (vincristine, choichicine) pattern. Compounds within the scope of the invention will follow the "stabilizer" pattern.

5. In Vivo Anti-tumor Activity of Compounds [0125] A number of experiments with human tumor xenografts in athymic mice have been done to evaluate the ability of compounds of this invention to inhibit tumor growth in vivo.

[01126] 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.

[0127] 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 choichicine. 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 IC50 values) without cell cycle block, another property that is characteristic of taxanes but not vincas or chofchicine. Representative compounds of the invention inhibit the growth of several human tumor xenografts in athymic mice, including tumors resistant to taxanes and vinca alkaloids.

EXAMPLE

10128] The following example is useful for the preparation of a representative non-limiting example of a compound of this invention, which is useful as a promoter of microtubule polymerization and as an anticancer agent.

Example I

S-N-(3-{4-[5-Chloro-7-(2, 2,2-trifluoro-l-methyi-ethylamino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl]-3,5-difluoro-phenoxy}-propyl)-N-methyl-succinamic acid (Compound 1) [0129] A mixture 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 (10.0 g, 21.5 mmol) and succinic anhydride (2.58 g, 25.8 mmol) in THF (50 mL) is stirred for 1 h.
The solvent is removed by distillation to a residue and the residue is dissolved into NaOH (1 N, 100 mL). The solution is filtered through a pad of CELITEO 545. The filtrate is cooled to 10-15 C, and HCI (3N, -32 mL) is added dropwise until pH
reached 3-4. The mixture is stirred for 30 min at 0-5 C. The solid is filtered, washed with cold water (2x20 mL) and dried at by air flow to give a solid product of Compound 1 (11.1 g, 92 !0).

Claims (59)

1. A compound of Formula (I):

wherein R1 is or a -(C6 - C8) cycloalkyl optionally substituted with R8;
n is an integer of 2, 3, or 4;

X is -Cl, -F or -Br;

Y is -O-, -S-, -CH2- or -NR4-;

L1 and L2 are each independently -H, -F, -Cl, -Br, or -CF3;
R3 is -CF3 or -C2F5;

R4, R5, and R6 are each independently -H or -(C1-C3) -alkyl;

R7 is -ZR9, wherein Z is -CO-, -NO-, -SO2-, or -PO2H-; and R9 is -H, -OH, -(C1-C5) -alkyl or -(C2-C5)-alkenyl, said alkyl or alkenyl optionally substituted with one or more of -O-, halogen, -OH, -NH2, 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 -CH3, -OH, or halogen; and R8 is (C1-C3) -alkyl; or pharmaceutically acceptable salts or stereoisomers thereof.

2. The compound of claim 1, wherein R6 is -(C1-C3) alkyl.

3. The compound of claim 2, wherein R6 is -CH3.

4. The compound of claim 1, 2 or 3, wherein R7 is -ZR9, wherein Z is -CO- or -NO-;
and R9 is -C1-C5 alkyl, optionally substituted with one or more of O, halogen, -OH, -NH2, 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 -CH3, -OH, or halogen.

5. The compound of claim 4, wherein Z is -CO-.

6. The compound of claim 5, wherein R9 is - (CH2)2COOH or - (CH)2COOH.

7. The compound of any one of claims 1 to 6, wherein R1 is

8. The compound of claim 7, wherein R3 is -CF3 and R5 is -C1-C3 alkyl.

9. The compound of any one of claims 1 to 8, wherein L1 and L2 are each independently -F, -Cl, or -Br.

10. The compound of claim 9, wherein L1 and L2 are each -F.

11. The compound of any one of claims 1 to 10, wherein Y is O.

12. The compound ofany one of claims 1 to 11, wherein n is 3.

13. The compound of any one of claims 1 to 12, wherein X is -Cl.

14. The compound of claim 1, wherein the compound of Formula (I) is

15. The compound of claim 1, wherein the compound of Formula (I) is

16. The compound of claim 1, wherein the compound of Formula (I) is and r is an integer from 0 to 5, inclusive.

17. A compound of Formula (II):

wherein R1 is or a - (C6 - C8) -cycloalkyl optionally substituted with R8;
n is an integer of 2, 3, or 4;

X is -Cl, -F or -Br;

Y is -O-, -S-, -CH2- or -NR4-;

L1 and L2 are each independently -H, -F, -Cl, -Br, or -CF3;
R3 is -CF3 or -C2F5;

R2, R4, R5, R6 and R9are each independently -H or -(C1-C3) alkyl;
R8 is C1-C3 alkyl; or pharmaceutically acceptable salts or stereoisomers thereof.

18. The compound of claim 17, wherein R6 is -C1-C3 alkyl.

19. The compound of claim 18, wherein R6 is -CH3.

20. The compound of claim 17, 18 or 19, wherein R2 is -H.

21. The compound of any one of claims 17 to 20, wherein R2 is -(C1-C3) alkyl

22. The compound of any one of claims 17 to 21, wherein R1 is

23. The compound of claim 22, wherein R3 is -CF3 and R5 is -C1-C3 alkyl.

24. The compound ofany one of claims 17 to 23, wherein L1 and L2 are each independently -F, -Cl, or -Br.

25. The compound of claim 24, wherein L1 and L2 are -F.

26. The compound of any one of claims 17 to 25, wherein Y is -O-.

27. The compound of any one of claims 17 to 26, wherein n is 3.

28. The compound of claim 17 to 27, wherein X is -Cl.

29. The compound of claim 17, wherein the compound of Formula (II) is

30. The compound of claim 17, wherein the compound of Formula (II) is

31. A method of treating or inhibiting the growth of cancerous tumor cells and associated diseases in a mammal in need thereof comprising administering an effective amount of a compound of Formula (I) as defined in any one of claims to 16 or a pharmaceutically acceptable salt thereof to a mammal in need thereof.

32. A method of promoting tubulin polymerization in a tubulin containing system comprising contacting said tubulin containing system with an effective amount of a compound of Formula (I) as defined any one of claims 1 to 16 or a pharmaceutically acceptable salt thereof.

33. A method of stabilizing microtubules in a tubulin containing system comprising contacting said tubulin containing system with an effective amount of a compound of Formula (I) as defined in any one of claims 1 to 16 or a pharmaceutically acceptable salt thereof.

34. 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, which method comprises administering to said mammal an effective amount of a compound of Formula (I) as defined in any one of claims 1 to 16 or a pharmaceutically acceptable salt thereof.

35. 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, which method comprises providing to said mammal an effective amount of a compound of Formula (I) as defined in any one of claims 1 to 16 of a pharmaceutically acceptable salt thereof.

36. A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of Formula (I) as defined in any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier therefor.

37. A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of Formula (I) as defined in claim 14, 15 or 16 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier therefor.

38. A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of Formula (II) as defined in any one of claims 17 to 28 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier therefor.

39. A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of Formula (II) as defined in claim 29 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier therefor.

40. A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of Formula (II) as defined in claim 30 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier therefor.

41. A method of treating or inhibiting the growth of cancerous tumor cells and associated diseases in a mammal in need thereof comprising administering an effective amount of a compound of Formula (II) as defined in any one of claims 17 to 30 or a pharmaceutically acceptable salt thereof to a mammal in need thereof.

42. A method of promoting tubulin polymerization in a tubulin containing system comprising contacting said tubulin containing system with an effective amount of a compound of Formula (II) as defined in any one of claims 17 to 30 or a pharmaceutically acceptable salt thereof.

43. A method of stabilizing microtubules in a tubulin containing system comprising contacting said tubulin containing system with an effective amount of a compound of Formula (II) as defined in any one of claims 17 to 30 or a pharmaceutically acceptable salt thereof.

44. 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, which method comprises administering to said mammal an effective amount of a compound of Formula (II) as defined in any one of claims 17 to 30 or a pharmaceutically acceptable salt thereof.

45. 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, which method comprises providing to said mammal an effective amount of.

a compound of Formula (II) as defined in any one of claims 17 to 30 or a pharmaceutically acceptable salt thereof.

46. A method according to claim 31, wherein said mammal is a human.

47. A method according to claim 34, wherein said mammal is a human.

48. A method according to claim 35, wherein said mammal is a human.

49. A method according to claim 41, wherein said mammal is a human.

50. A method according to claim 44, wherein said mammal is a human.

51. A method according to claim 45, wherein said mammal is a human.

52. Use of a compound as claimed in any one of claims 1 to 16 or 17 to 30 for the manufacture of a medicament for of treating or inhibiting the growth of cancerous tumor cells and associated diseases in a mammal.

53. A pharmaceutical composition according to claim 37, further comprising a compound of Formula (II) as follows:

wherein R1 is or a -(C6 - C8) -cycloalkyl optionally substituted with R8;
n is an integer of 2, 3, or 4;

X is -Cl, -F or -Br;

Y is -O-, -S-, -CH2- or -NR4-;

L1 and L2are each independently -H, -F, -Cl, -Br, or -CF3;
R3 is -CF3 or -C2F5;

R2, R4, R5, R6, and R9are each independently -H or -(C1-C3) alkyl;
R8 is C1-C3 alkyl; or pharmaceutically acceptable salts or stereoisomers thereof.

54. A method of treating or inhibiting the growth of cancerous tumor cells and associated diseases in a mammal in need thereof comprising administering an effective amount of a composition as defined in claim 53 to a mammal in need thereof.

55. A method of promoting tubulin polymerization in a tubulin containing system comprising contacting said tubulin containing system with an effective amount of a compound of Formula (II) as defined in claim 53 to a mammal in need thereof.

56. A method of stabilizing microtubules in a tubulin containing system comprising contacting said tubulin containing system with an effective amount of a compound of Formula (II) as defined in claim 53 to a mammal in need thereof.

57. 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, which method comprises administering to said mammal an effective amount of a compound of Formula (11) as defined in claim 53 to a mammal in need thereof.

58. 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, which method comprises providing to said mammal an effective amount of a compound of Formula (II) as defined in claim 53 to a mammal in need thereof.

59. A method according to any one of claims 54, 57 and 58, wherein said mammal is a human.