Antitumor Compounds

Abstract

A compound of general formula I, where X has various meanings, for use in the treatment of cancer. JPEG2024539180000061.jpg39170

Description

[Technical field]

[0001] The present invention relates to novel microtubule inhibitors, pharmaceutical compositions containing them, processes for their preparation and their use as antitumor agents. [Background technology]

[0002] A novel class of polyketides was identified in the Madagascar sponge Lithoplocamia lithistoides and has the formula

[0003] [ka]

[0004] The compound PM060184 (PM184) is included.

[0005] WO2007 / 144423 discloses PM184 (compound 1) and other natural or synthetic polyketides. PM184 is a microtubule inhibitor that targets a protein called tubulin through a novel mechanism. PM184 interferes with cancer growth by preventing cell division of tumor cells through inhibition of mitosis. PM184 is currently in Phase II trials for hormone receptor positive HER2 negative locally advanced and / or metastatic breast cancer, and is also being tested in various solid tumors and other Phase II solid tumor trials.

[0006] PM184 inhibits microtubule shortening and growth to the same extent, interfering with microtubule mechanical instability and affecting cells in both interphase and mitosis. These effects are believed to result from a novel form of interaction with tubulin dimers, sharing a common tubulin binding site with rhizoxin and simultaneously preventing the binding of vinblastine. A unique binding mode of tubulin was preliminarily suggested in PM060184-resistant mutants of Aspergillus nidulans, involving an interaction with a novel locus in β-tubulin defined by the Asn100 position. This was later confirmed by X-ray crystallography, corroborating the discovery of the maytansine binding site as a novel pharmacophore. In addition to its effects on the microtubule network, the antitumor activity of PM060184 may also depend on related antiangiogenic properties, namely the inhibition of migration and invasion as described in human umbilical vein endothelial (HUVEC) cells. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2007 / 144423 [Non-patent literature]

[0008] [Non-Patent Document 1] Wuts, P.G.M. and Greene, T.W., Protecting Groups in Organic Synthesis, 4th ed., Wiley-Interscience [Non-Patent Document 2] Kocienski PJ, Protecting Groups, 3rd edition, Georg Thieme Verlag [Non-Patent Document 3] E. W. Martin, "Remington's Pharmaceutical Sciences" [Non-Patent Document 4] Skehan et al., J. Natl. Cancer Inst. 1990 [Non-Patent Document 5] Yamada, T et al. (1986) Establishment of a human pancreatic adenocarcinoma cell line (PSN-1) with amplifications of both c-myc and activated c-Ki-ras by a point mutation. Biochem. Biophys. Res. Commun. vol. 140, pp. 167-173 [Non-Patent Document 6] V. Vichai and K. Kirtikara (2006) Nature Protoc. 1, 1112-1116 [Non-Patent Document 7] Boyd MR and Paull KD. Drug Dev. Res. 1995, 34, 91-104. Summary of the Invention [Problem to be solved by the invention]

[0009] Despite the positive results obtained in clinical applications in chemotherapy, the oncology field continues to seek to identify new compounds with optimal activity, tumor selectivity profiles, reduced systemic toxicity, and / or improved pharmacokinetic properties. [Means for solving the problem]

[0010] In a first aspect of the present invention, a compound of formula I

[0011] [ka]

[0012] (Wherein, X=F or Me) The compound of formula (1) is provided.

[0013] In a further aspect of the invention, the compound of formula II

[0014] [ka]

[0015] The compound of formula (1) is provided.

[0016] In a further aspect of the invention, the compound of formula III

[0017] [ka]

[0018] The compound of formula (1) is provided.

[0019] In a further aspect of the invention there is provided a pharmaceutical composition comprising a compound according to the invention and a pharma- ceutically acceptable carrier.

[0020] In yet a further aspect of the invention there is provided a dosage form comprising a pharmaceutical composition according to the invention.

[0021] In a still further aspect of the invention there is provided a compound, pharmaceutical composition or dosage form according to the invention for use as a medicament.

[0022] In yet another aspect of the present invention, there is provided a compound, pharmaceutical composition or dosage form according to the present invention for use in treating cancer.The cancer may be a solid tumor.The cancer may be selected from lung cancer, including non-small cell lung cancer, colorectal cancer, breast cancer, pancreatic cancer and gastric cancer.

[0023] In yet another aspect of the present invention, there is provided a use of a compound, pharmaceutical composition or dosage form according to the present invention for the manufacture of a medicament for the treatment of cancer.The cancer may be a solid tumor.The cancer may be selected from lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, pancreatic cancer and gastric cancer.

[0024] In yet a further aspect of the present invention, there is provided a method for the prevention or treatment of cancer, comprising administering to a patient, particularly a human, in need thereof an effective amount of a compound according to the present invention, an effective amount of a pharmaceutical composition according to the present invention, or an effective amount of a dosage form according to the present invention. The cancer may be a solid tumor. The cancer may be selected from lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, pancreatic cancer, and gastric cancer.

[0025] In yet another aspect of the present invention, there is provided the use of a compound according to the present invention for the treatment of cancer, or preferably in the preparation of a medicament for the treatment of cancer.The cancer may be a solid tumor.The cancer may be selected from lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, pancreatic cancer and gastric cancer.

[0026] In yet a further aspect of the invention there is provided a method of inhibiting the proliferation of cancer cells comprising the step of contacting cancer cells with a compound according to the invention or a pharmaceutical composition according to the invention, or a dosage form according to the invention, optionally wherein the cancer cells are solid tumors, including a cancer selected from lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, pancreatic cancer and gastric cancer.

[0027] In yet another aspect of the present invention, a kit is provided comprising a therapeutically effective amount of a compound according to the present invention and a pharma- ceutically acceptable carrier, or a pharmaceutical composition according to the present invention, or a dosage form according to the present invention. The kit is for use in the treatment of cancer. The cancer may be a solid tumor. The cancer may be selected from lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, pancreatic cancer, and gastric cancer.

[0028] In yet a further aspect of the invention, there is provided a process for the preparation of a compound of formula I or a pharma- ceutically acceptable salt thereof, comprising reacting a compound of formula V with a suitable reagent for the synthesis of a carbamate to obtain a compound of formula I

[0029] [ka]

[0030] (Wherein, X=F or Me)

[0023] A method is provided comprising the step of obtaining a compound of formula (I).

[0031] In yet a further aspect of the invention there is provided intermediate compounds of formula V, VII, IX or XI which are useful in the preparation of compounds according to the invention. [Brief description of the drawings]

[0032] [Figure 1] FIG. 13 is a graph of median tumor growth curves for mice bearing H460 xenografts and treated with PM050489 (N=10). [Diagram 2] FIG. 1 is a graph of median tumor growth curves for mice (N=10) bearing H460 xenografts and treated with PM060184, Compound 2 (Comp. 2) and Compound 3 (Comp. 3). [Diagram 3] 1 is a graph of Kaplan-Meier survival curves for mice (N=10) bearing H460 xenografts and treated with PM060184, Compound 2 (Comp. 2) and Compound 3 (Comp. 3). [Figure 4] FIG. 1 is a graph of median tumor growth curves for mice (N=8) bearing HGC-27 xenografts and treated with PM060184, Compound 2 (Comp. 2) and Compound 3 (Comp. 3). [Diagram 5] 1 is a graph of Kaplan-Meier survival curves for mice (N=8) bearing HGC-27 xenografts and treated with PM060184, Compound 2 (Comp. 2) and Compound 3 (Comp. 3). [Figure 6] FIG. 1 is a graph of median tumor growth curves for mice (N=10) bearing HCT-116 xenografts and treated with PM060184, Compound 2 (Comp. 2) and Compound 3 (Comp. 3). [Figure 7]1 is a graph of Kaplan-Meier survival curves for mice (N=10) bearing HCT-116 xenografts and treated with PM060184, Compound 2 (Comp. 2) and Compound 3 (Comp. 3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The following applies to all aspects of the invention.

[0034] The compounds of the present invention may be in crystalline or amorphous form, either as free compounds or as solvates (e.g., hydrates), with all forms being intended to be within the scope of the present invention. Methods of solvation are generally known within the art.

[0035] In addition, the compounds presented herein may exist in isotopically labeled forms. All isotopically labeled forms of the compounds presented herein and mixtures thereof are considered to be within the scope of the present invention.

[0036] Protected forms of the compounds disclosed herein are considered to be within the scope of the present invention. Suitable protecting groups are well known to those skilled in the art. General reviews of protecting groups in organic chemistry are provided by Wuts, PGM and Greene TW, Protecting Groups in Organic Synthesis, 4th Edition, Wiley-Interscience, and by Kocienski PJ, Protecting Groups, 3rd Edition, Georg Thieme Verlag. These references provide sections on protecting groups for OH and amino groups. All of these references are incorporated by reference in their entirety.

[0037] Within the scope of the present invention, an OH-protecting group is defined as an O-linked moiety resulting from the protection of OH through the formation of a suitable protected OH group. Examples of such protected OH groups include ethers, silyl ethers, esters, sulfonates, sulfenates and sulfinates, carbonates, and carbamates. In the case of ethers, the protecting groups for OH are methyl, methoxymethyl, methylthiomethyl, (phenyldimethylsilyl)methoxymethyl, benzyloxymethyl, p-methoxybenzyloxymethyl, [(3,4-dimethoxybenzyl)oxy]methyl, p-nitrobenzyloxymethyl, o-nitrobenzyloxymethyl, [(R)-1-(2-nitrophenyl)ethoxy]methyl, (4-methoxyphenoxy)methyl, guaiacolmethyl, [(p-phenylphenyl)oxy]methyl, t-butoxymethyl, 4-pentenyloxymethyl, siloxymethyl, 2-methoxyethoxymethyl, 2-cyanoethoxymethyl, bis(2-chloroethoxy)methyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, menthoxy Methyl, O-bis(2-acetoxy-ethoxy)methyl, tetrahydropyranyl, fluorous tetrahydropyranyl, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl, 4-methoxy-tetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)-phenyl]-4-methoxypiperidin-4-yl, 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl, 1-(4-chlorophenyl)-4-methoxypiperidin-4-yl, 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-Methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-hydroxyethyl, 2-bromoethyl, 1-[2-(trimethylsilyl)ethoxy]ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 1-methyl-1-phenoxyethyl, 2,2,2-trichloroethyl, 1,1-dianisyl-2,2,2-trichloroethyl, 1,1,1,3,3,3-hexafluoro-2-phenylyl isopropyl, 1-(2-cyanoethoxy)ethyl, 2-trimethylsilylethyl, 2-(benzylthio)ethyl, 2-(phenylselenyl)ethyl, t-butyl, cyclohexyl, 1-methyl-1'-cyclopropylmethyl, allyl, prenyl, cinnamyl, 2-phenaryl, propargyl, p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, 2,4-dinitrophenyl, 2,3,5,6-tetrafluoro-4-(trifluoromethyl)phenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxy Benzyl, 2,6-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, pentadienylnitrobenzyl, pentadienylnitropiperonyl, halobenzyl, 2,6-dichlorobenzyl, 2,4-dichlorobenzyl, 2,6-difluorobenzyl, p-cyanobenzyl, fluorous benzyl, 4-fluorous alkoxybenzyl, trimethylsilylxylyl, p-phenylbenzyl, 2-phenyl-2-propyl, p-acylaminobenzyl, p-azidobenzyl, 4-azido-3-chlorobenzyl, 2-tolyl Trifluoromethylbenzyl, 4-trifluoromethylbenzyl, p-(methylsulfinyl)benzyl, p-silaneylbenzyl, 4-acetoxybenzyl, 4-(2-trimethylsilyl)ethoxymethoxybenzyl, 2-naphthylmethyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, 2-quinolinylmethyl, 6-methoxy-2-(4-methylphenyl)-4-quinolinemethyl, 1-pyrenylmethyl, diphenylmethyl, 4-methoxydiphenylmethyl, 4-phenyldiphenylmethyl, p,p'-Dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, tris(4-t-butylphenyl)methyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenyl-methyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxy)phenyldiphenylmethyl, 4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4''-tris(levulinoyloxyphenyl)methyl, 4,4',4''-tris(benzoyloxyphenyl)methyl, 4,4'-dimethoxy-3''-[N-(isopropyloxyphenyl)methyl] midazolylmethyl)]trityl, 4,4'-dimethoxy-3''-[N-(imidazolylethyl)carbamoyl]trityl, bis(4-methoxyphenyl)-1'-pyrenylmethyl, 4-(17-tetrabenzo[a,c,g,i]fluorenylmethyl)-4,4''-dimethoxytrityl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-phenylthioxanthyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, 4,5-bis(ethoxycarbonyl)-[1,3]-dioxolan-2-yl, benzisothiazolyl S,S-dioxide. In the case of silyl ethers, the protecting groups for OH are trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, diethylisopropylsilyl, dimethylhexylsilyl, 2-norbornyldimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl, di-t-butylmethylsilyl, bis(t-butyl)-1-pyrenylmethoxysilyl, tris(trimethylsilyl)silyl, (2-hydroxystyryl)dimethylsilyl, (2-hydroxystyryl)diisopropylsilyl, t-butylmethoxyphenylsilyl, t-butoxydiphenylsilyl, 1,1,3,In the case of esters, the protecting group for OH, together with the oxygen atom of the unprotected OH to which it is attached, can be selected from formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trichloroacetamidate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, phenylacetate, diphenylacetate, 3-phenylpropionate, bisfluorous chain type propanoyl, propanoyl), 4-pentenoate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, 5[3-bis(4-methoxyphenyl)hydroxymethylphenoxy]levulinate, pivaloate, 1-adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate, 4-bromobenzoate, 2,5-difluorobenzoate, p-nitrobenzoate, picolinate, nicotinate, 2-(azidomethyl)benzoate, 4-azido-butyrate, (2-azidomethyl)phenylacetate, 2 -{[(tritylthio)oxy]methyl}benzoate, 2-{[(4-methoxytritylthio)oxy]methyl}benzoate, 2-{[methyl(tritylthio)amino]methyl}benzoate, 2-{{[(4-methoxytrityl)thio]methylamino}methyl}benzoate, 2-(allyloxy)phenylacetate, 2-(prenyloxymethyl)benzoate, 6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoate, 6-(levulinyloxymethyl)-3-methoxy-4-nitrobenzoate, 4-benzyloxybutyrate, 4-trialkylsilyloxybutyrate, 4-acetoxy-2,2-dimethylbutyrate, 2,2-Dimethyl-4-pentenoate, 2-iodobenzoate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 4-(methylthio-methoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2-(chloroacetoxymethyl)benzoate, 2-[(2-chloroacetoxy)ethyl]benzoate, 2-[2-(benzyloxy)ethyl]benzoate, 2-[2-(4-methoxybenzyloxy)ethyl]benzoate, 2,6-dichloro-4-methylphenoate and forming an ester which may be selected from alkyl ether, alkyl diphenyl ether, alkyl ethersulfate ... In the case of sulfonates, sulfenates and sulfinates, the protecting group for OH together with the oxygen atom of the unprotected OH to which it is attached forms a sulfonate, sulfenate or sulfinate which may be selected from sulfate, allylsulfonate, methanesulfonate, benzylsulfonate, tosylate, 2-[(4-nitrophenyl)ethyl]sulfonate, 2-trifluoromethylbenzenesulfonate, 4-monomethoxytritylsulfenate, alkyl 2,4-dinitrophenylsulfenate, 2,2,5,5-tetramethylpyrrolidin-3-one-1-sulfinate and dimethylphosphinothioyl. In the case of carbonates, the protecting group for OH, together with the oxygen atom of the unprotected OH to which it is attached, is methyl carbonate, methoxymethyl carbonate, 9-fluorenylmethyl carbonate, ethyl carbonate, bromoethyl carbonate, 2-(methylthiomethoxy)ethyl carbonate, 2,2,2-trichloroethyl carbonate, 1,1-dimethyl-2,2,2-Trichloroethyl carbonate, 2-(trimethylsilyl)ethyl carbonate, 2-[dimethyl(2-naphthylmethyl)silyl]ethyl carbonate, 2-(phenylsulfonyl)ethyl carbonate, 2-(triphenylphosphonio)ethyl carbonate, cis-[4-[[(methoxytrityl)sulfenyl]oxy]tetrahydrofuran-3-yl]oxycarbonate, isobutyl carbonate, t-butyl carbonate, vinyl carbonate, allyl carbonate, cinnamyl carbonate, propargyl carbonate, p-chlorophenyl carbonate, p-nitrophenyl carbonate, 4-ethoxy-1-naphthyl carbonate, 6-bromo-7-hydroxycoumarin-4-ylmethyl carbonate, benzyl carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate In one embodiment, the carbonate may be selected from 2-(4-nitrophenyl)ethyl carbonate, 2-(2,4-dinitrophenyl)ethyl carbonate, 2-(2-nitrophenyl)propyl carbonate, 2-(3,4-methylenedioxy-6-nitrophenyl)propyl carbonate, 2-cyano-1-phenylethyl carbonate, 2-(2-pyridyl)amino-1-phenylethyl carbonate, 2-[N-methyl-N-(2-pyridyl)]amino-1-phenylethyl carbonate, phenacyl carbonate, 3′,5′-dimethoxybenzoin carbonate, methyldithiocarbonate and S-benzylthiocarbonate. In the case of carbamates, the protecting group for OH, together with the oxygen atom of the unprotected OH to which it is attached, forms a carbamate which may be selected from dimethylthiocarbamate, N-phenylcarbamate, and N-methyl-N-(o-nitrophenyl)carbamate.

[0038] Within the scope of the present invention, an amino-protecting group is defined as an N-linked moiety resulting from the protection of an amino group through the formation of a suitable protected amino group. Examples of protected amino groups include carbamates, ureas, amides, heterocyclic systems, N-alkylamines, N-alkenylamines, N-alkynylamines, N-arylamines, imines, enamines, N-metal derivatives, NN derivatives, NP derivatives, N-Si derivatives and NS derivatives. In the case of carbamates, the protecting group for the amino group, together with the amino group to which it is attached, is methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate, 2,6-di-t-butyl-9-fluorenylmethyl carbamate, 2,7-bis(trimethylsilyl)fluorenylmethyl carbamate, 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 17-tetrabenzo[a,c,g,i]fluorenylmethyl carbamate, 2-chloro-3-indenylmethyl carbamate, benz[f]inden-3-ylmethyl carbamate, 1,1-dioxobenzo[b]-thiophen-2-ylmethyl carbamate, 2-methylsulfonyl-3-phenyl-1-prop-2-enyl carbamate, 2, 7-Di-t-butyl-[9,(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate, 2,2,2-trichloroethyl carbamate, 2-trimethylsilylethyl carbamate, (2-phenyl-2-trimethylsilyl)ethyl carbamate, 2-phenylethyl carbamate, 2-chloroethyl carbamate, 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate, 1,1-dimethyl-2,2,2-trichloroethyl carbamate, 2-(2'-pyridyl)ethyl carbamate, 2-(4'-pyridyl)ethyl carbamate, 2,2-bis(4'-nitrophenyl)ethyl carbamate, 2-[(2-nitrophenyl)dithio]-1-phenylethyl carbamate, 2-(N,N-Dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate, fluorous BOC carbamate, 1-adamantyl carbamate, 2-adamantyl carbamate, 1-(1-adamantyl)-1-methylethyl carbamate, 1-methyl-1-(4-biphenylyl)ethyl carbamate, 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate, triisopropylsilyloxycarbamate, vinyl carbamate, allyl carbamate, prenyl carbamate, 1-isopropyl Allyl carbamate, cinnamyl carbamate, 4-nitrocinnamyl carbamate, 3-(3'-pyridyl)prop-2-enyl carbamate, hexadienyl carbamate, propargyl carbamate, 1,4-but-2-ynyl biscarbamate, 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyl dithiocarbamate, benzyl carbamate, 3,5-di-t-butylbenzyl carbamate, p-methoxybenzyl carbamate, p-nitrobenzyl carbamate, p-bromobenzyl carbamate, Dicarbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate, 4-trifluoromethylbenzyl carbamate, fluorous benzyl carbamate, 2-naphthylmethyl carbamate, 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 4-phenylacetoxybenzyl carbamate, 4-azidobenzyl carbamate, 4-azido-methoxybenzyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(Dihydroxyboryl)-benzyl carbamate, 5-benzoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, 2-(4-nitrophenylsulfonyl)ethyl carbamate, 2-(2,4-dinitrophenylsulfonyl)ethyl carbamate, 2-(4-trifluoromethylphenylsulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate, 2-phosphonioethyl carbamate, 2-[phenyl(methyl)sulfonio]ethyl carbamate, 1-methyl-1-(triphenylphosphonio)ethyl carbamate, 1,1-dimethyl-2-cyanoethyl carbamate, 2-dansylethyl carbamate, 2-(4-nitrophenyl)ethyl carbamate, 4-methylthiophenyl carbamate, 2,4-dimethylthiophenyl carbamate, m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate , 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, α-methylnitropiperonyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, 2-nitrophenylethyl carbamate, 6-nitroveratryl carbamate, 4-methoxyphenacyl carbamate, 3',5'-dimethoxybenzoin carbamate, 9-xanthenylmethyl carbamate, N-methyl-N-(o-nitrophenyl)ethyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropyl methyl carbamate, cyclobutyl carbamate, cyclopentyl carbamate, cyclohexyl carbamate, isobutyl carbamate, isobornyl carbamate, cyclopropyl methyl carbamate, p-decyloxybenzyl carbamate, diisopropyl methyl carbamate, 2,2-dimethoxy-carbonyl vinyl carbamate , o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethyl-carboxamido)propyl carbamate, butynyl carbamate, 1,1-dimethylpropynyl carbamate, 2-iodoethyl carbamate, 1-methyl-1-(4'-pyridyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,To form a carbamate, which may be selected from 6-trimethylbenzylcarbamate, isonicotinylcarbamate, 4-(trimethyl-ammonium)benzylcarbamate, p-cyanobenzylcarbamate, di(2-pyridyl)methylcarbamate, 2-furanylmethylcarbamate, phenylcarbamate, 2,4,6-tri-t-butylphenylcarbamate, 1-methyl-1-phenylethylcarbamate and S-benzylthiocarbamate. In the case of urea, the protecting group for the amino group may be selected from phenothiazinyl-(10)-carbonyl, N'-p-toluenesulfonylaminocarbonyl, N'-phenylaminothiocarbonyl, 4-hydroxyphenylaminocarbonyl, 3-hydroxytryptaminocarbonyl and N'-phenylaminothiocarbonyl. In the case of amides, the protecting group for the amino, together with the amino group to which it is attached, is formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, penta-4-enamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanylamide, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, 2,2-dimethyl-2-(o-nitrophenyl)acetamide, o-nitrophenoxyacetamide, 3-phenylpropan ... -(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 3-methyl-3-nitrobutanamide, o-nitrocinamide, o-nitrobenzamide, 3-(4-t-butyl-2,6-dinitrophenyl)-2,2-dimethylpropanamide, o-(benzoyloxime-tyl)benzamide, 2-(acetoxymethyl)benzamide, 2-[(t-butyldiphenylsiloxy)methyl]benzamide, 3-(3',6'-dioxo-2',4',5'-trimethylcyclohexa-1',4'-diene)-3,To form an amide, which may be selected from 3-dimethylpropionamide, o-hydroxy-trans-cinamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, acetoacetamide, 3-(p-hydroxyphenyl)propanamide, (N'-dithiobenzyloxycarbonylamino)acetamide and N-acetylmethionineamide. In the case of heterocyclic systems, the protecting group for the amino group, together with the amino group to which it is attached, may be selected from 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dichlorophthalimide, N-tetrachlorophthalimide, N-4-nitrophthalimide, N-thiodiglycoloyl, N-dithiasuccinimide, N-2,3-diphenylmaleimide, N-2,3-dimethylmaleimide, N-2,5-dimethylpyrrole, N-2,5-bis(triisopropylsiloxy)pyrrole, N-1,1, Forming a heterocyclic ring system which may be selected from 4,4-tetramethyldisilylazacyclopentane adduct, N-1,1,3,3-tetramethyl-1,3-disilaisoindoline, N-diphenylsilyldiethylene, N-5-substituted-1,3-dimethyl-1,3,5-triazacyclohexane-2-one, N-5-substituted-1,3-benzyl-1,3,5-triazacyclohexane-2-one, 1-substituted 3,5-dinitro-4-pyridone and 1,3,5-dioxazine. In the case of N-alkyl, N-alkenyl, N-alkynyl or N-arylamines, the protecting groups for the amino group are N-methyl, Nt-butyl, N-allyl, N-prenyl, N-cinnamyl, N-phenylallyl, N-propargyl, N-methoxymethyl, N-[2-(trimethylsilyl)ethoxy]methyl, N-3-acetoxypropyl, N-cyanomethyl, N-2-azanorbornene, N-benzyl, N-4-methoxybenzyl, N-2,4-dimethoxybenzyl, N-2-hydroxybenzyl, N-ferrocenylmethyl, N-2,4-dimethoxybenzyl, N-2-hydroxybenzyl, N-methyl-N-phenyl-, N-propargyl, N-methoxymethyl, N-[2-(trimethylsilyl)ethoxy]methyl, N-3-acetoxypropyl, N-cyanomethyl, N-2-azanorbornene, N-benzyl, N-4-methoxybenzyl, N-2,4-dimethoxybenzyl, N-2-hydroxybenzyl, N-methyl-N-phenyl- ...It may be selected from 4-dinitrophenyl, o-methoxyphenyl, p-methoxyphenyl, N-9-phenylfluorenyl, N-fluorenyl, N-2-picolylamine N'-oxide, N-7-methoxycoumar-4-ylmethyl, N-diphenylmethyl, N-bis(4-methoxyphenyl)methyl, N-5-dibenzosuberyl, N-triphenylmethyl, N-(4-methylphenyl)diphenylmethyl, and N-(4-methoxyphenyl)diphenylmethyl. In the case of imines, the protecting group for the amino group may be selected from N-1,1-dimethylthiomethylene, N-benzylidene, Np-methoxybenzylidene, N-diphenylmethylene, N-[2-pyridyl)mesityl]methylene, N-(N',N'-dimethylaminomethylene), N-(N',N'-dibenzylaminomethylene), N-(N'-t-butylaminomethylene), N,N'-isopropylidene, Np-nitrobenzylidene, N-salicylidene, N-5-chlorosalicylidene, N-(5-chloro-2-hydroxyphenyl)phenylmethylene, N-cyclohexylidene and Nt-butylidene. In the case of enamines, the protecting group for the amino group can be selected from N-(5,5-dimethyl-3-oxo-1-cyclohexenyl), N-2,7-dichloro-9-fluorenylmethylene, N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, N-(1,3-dimethyl-2,4,6-(1H,3H,5H)-trioxopyrimidin-5-ylidene)-methyl, N-4,4,4-trifluoro-3-oxo-1-butenyl and N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolidin-3-yl). In the case of N-metal derivatives, the protecting group for the amino group can be selected from N-borane, N-diphenylborinic ester, N-diethylborinic ester, N-9-borabicyclononane, N-difluoroborinic ester and 3,5-bis(trifluoromethyl)phenylboronic acid, including N-phenyl(pentacarbonylchromium)carbenyl, N-phenyl(pentacarbonyl-tungsten)carbenyl, N-methyl(pentacarbonylchromium)carbenyl, N-methyl(pentacarbonyltungsten)carbenyl, N-copper chelate, N-zinc chelate and 18-crown-6 derivatives. In the case of NN derivatives, the protecting group for the amino group, together with the amino group to which it is attached, forms a NN derivative, which may be selected from N-nitroamino, N-nitrosamino, amine N-oxide, azide, triazene derivatives and N-trimethylsilylmethyl-N-benzylhydrazine. In the case of NP derivatives, the protecting group for the amino group, together with the amino group to which it is attached, may be selected from diphenylphosphinamide, dimethylthiophosphinamide, diphenylthiophosphinamide, dialkyl phosphoramidate, , forming a NP derivative which can be selected from dibenzyl phosphoramidate, diphenyl phosphoramidate and iminotriphenylphosphorane. In the case of N-Si derivatives, NH 2The protecting group for is selected from t-butyldiphenylsilyl and triphenylsilyl. In the case of NS derivatives, the protected amino group can be selected from N-sulfenyl or N-sulfonyl derivatives. The N-sulfenyl derivative can be selected from benzenesulfenamide, 2-nitrobenzenesulfenamide, 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 1-(2,2,2-trifluoro-1,1-diphenyl)ethylsulfenamide, and N-3-nitro-2-pyridinesulfenamide. The N-sulfonyl derivatives are methanesulfonamide, trifluoromethanesulfonamide, t-butylsulfonamide, benzylsulfonamide, 2-(trimethylsilyl)ethanesulfonamide, p-toluenesulfonamide, benzenesulfonamide, o-anisylsulfonamide, 2-nitrobenzenesulfonamide, 4-nitrobenzenesulfonamide, 2,4-dinitrobenzenesulfonamide, 2-naphthalenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide, 2-(4-methylphenyl)-6-methoxy-4-methylsulfonamide, 9-anthracenesulfonamide, pyridine-2-sulfonamide, benzothiamide, The sulfonamide may be selected from azole-2-sulfonamide, phenacylsulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide, 2,4,6-trimethoxybenzenesulfonamide, 2,6-dimethyl-4-methoxy-benzenesulfonamide, pentamethylbenzenesulfonamide, 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide, 4-methoxybenzenesulfonamide, 2,4,6-trimethylbenzenesulfonamide, 2,6-dimethoxy-4-methylbenzenesulfonamide, 3-methoxy-4-t-butylbenzenesulfonamide, and 2,2,5,7,8-pentamethylchroman-6-sulfonamide.

[0039] Reference to these groups is mentioned merely as exemplary protecting groups for OH and amino groups, but should not be construed as limiting the scope of the invention, as further groups having said functionality may be known to those skilled in the art and these should be understood to be encompassed by the present invention.

[0040] Examples of suitable reagents for the synthesis of carbamates include, but are not limited to, trichloroacetyl isocyanate, trimethylsilyl isocyanate, and triphenylsilyl isocyanate.

[0041] In order to provide a more concise description, some of the quantitative expressions presented herein may not be modified with the term "about". Regardless of whether the term "about" is explicitly used, it is understood that all given amounts in this specification refer to actual given values, and also refer to the equivalent value of such given values ​​and the approximation of such given values ​​that can be reasonably estimated by those skilled in the art, including approximation based on experimental and / or measurement conditions.

[0042] The present invention relates to a compound of formula I

[0043] [ka]

[0044] (Wherein, X=F or Me) The present invention relates to a compound of the formula:

[0045] The present invention also relates to a compound of formula II

[0046] [ka]

[0047] The present invention relates to a compound of the formula:

[0048] The present invention also relates to a compound of formula III

[0049] [ka]

[0050] The present invention relates to a compound of the formula:

[0051] An important property of the above compounds is their biological activity, and in particular their cytotoxic activity. In this respect, it has been surprisingly found that the compounds of the present invention show enhanced antitumor activity over the prior art compounds PM060184 and PM050489. This is shown for PM060184 in an in vitro bioassay in Example 3, where it is seen that 2 and 3 both have surprisingly improved in vitro activity over the prior art compound PM060184. 2 is 3.8-8.2 times more active than PM060184, and 3 is 1.3-3.7 times more active than PM060184. This is seen across A549, HT29, MDA-MB-231 and PSN-1 cell lines. This is also shown in in vivo xenograft models in Examples 5-7, where 2 and 3 show a significant improvement in efficacy over PM060184 across a broad range of cancer models. In H460, HGC27 and HCT116 xenograft models (models of non-small cell lung, gastric and colorectal cancer), both 2 and 3 show statistically significant improvements in survival time as well as antitumor activity across all three cell lines.

[0052] This is also shown for PM050489 in an in vitro bioassay in Example 3, where it can be seen that both 2 and 3 have improved in vitro activity over the prior art compound PM050489. Compound 2 is 1.9-7.9 times more active than the reference compound PM050489. Compound 3 is 1.3-1.8 times more active than the reference compound PM050489. This is seen across A549, HT29, MDA-MB-231 and PSN-1 cell lines. This is also shown in in vivo xenograft models, with significantly improved efficacy over PM050489 in the H460 xenograft model. This is seen in Example 3 for in vitro data and in the Comparative Examples and Example 5 for in vivo data.

[0053] In a further embodiment of the present invention, a pharmaceutical composition is provided that comprises a compound according to the present invention and a pharma- ceutically acceptable carrier. Examples of administration forms include, but are not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, ocular and intranasal. Parenteral administration includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques. Preferably, the composition is administered parenterally. The pharmaceutical composition of the present invention can be formulated so that the compound according to the present invention is bioavailable upon administration of the composition to an animal, preferably a human. The composition can be in the form of one or more dosage units, for example, a tablet can be a single dosage unit, and a container of the compound according to the present invention can contain the compound in liquid or aerosol form and can hold single or multiple dosage units.

[0054] The pharma- ceutically acceptable carrier or vehicle can be particulate, so that the composition is, for example, in tablet or powder form. The carrier can be liquid, so that the composition can be, for example, an oral syrup or an injectable liquid. In addition, the carrier can be gaseous or liquid, to provide an aerosol composition, for example, useful in inhalation administration. Powders can also be used for inhalation dosage forms. The term "carrier" refers to a diluent, adjuvant, or excipient with which the compound according to the invention is administered. Such pharmaceutical carriers can be liquids, such as water or oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The carrier can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, disaccharides, cyclodextrin, cyclodextrin derivatives, and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one embodiment, when administered to animals, the compounds and compositions according to the present invention and pharma- ceutically acceptable carriers are sterilized.When the compounds according to the present invention are administered intravenously, water is a preferred carrier.Saline and aqueous dextrose and glycerol solutions can also be utilized as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, cyclodextrin, cyclodextrin derivatives, glycerol, propylene glycol, water, ethanol, etc.The compositions of the present invention can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.

[0055] When intended for oral administration, the compositions are preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included in the forms considered herein as either solid or liquid.

[0056] As a solid composition for oral administration, the composition can be formulated into the form of powder, granule, compressed tablet, pill, capsule, chewing gum, wafer, etc. Such solid composition typically contains one or more inert diluents. In addition, one or more of the following may be present: binder, such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose or gelatin; excipient, such as starch, cyclodextrin, cyclodextrin derivative, lactose or dextrin; disintegrant, such as alginic acid, sodium alginate, corn starch, etc.; lubricant, such as magnesium stearate; lubricant, such as colloidal silicon dioxide; sweetener, such as sucrose or saccharin; flavor, such as peppermint, methyl salicylate or orange flavor; and colorant.

[0057] When the composition is in the form of a capsule (eg, a gelatin capsule), it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or fatty oils.

[0058] The composition can be in the form of liquid, such as elixir, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or delivery by injection. When intended for oral administration, the composition can contain one or more of sweeteners, preservatives, dyes / colorants and flavor enhancers. In the composition for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers and isotonic agents can also be included.

[0059] Preferred routes of administration are parenteral, including but not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, intracerebral, intraventricular, intrathecal, intravaginal or transdermal. The preferred mode of administration is at the discretion of the practitioner and will depend in part on the site of the medical condition (e.g., site of cancer). In a more preferred embodiment, the compounds according to the invention are administered intravenously. Infusion times of up to 24 hours are preferably used, more preferably 1 to 12 hours, and most preferably 1 to 6 hours. Shorter infusion times are particularly desirable, as they allow the procedure to be performed without an overnight stay in hospital. However, infusions may be 12 to 24 hours or even longer if necessary. Infusions may be performed at appropriate intervals, for example, 1 to 4 weeks.

[0060] The liquid composition of the present invention, whether in solution, suspension or other similar form, can also contain one or more of the following: sterile diluent, such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic saline, fixed oil, such as synthetic mono- or diglyceride, polyethylene glycol, glycerin, or other solvent; antibacterial agent, such as benzyl alcohol or methyl paraben; and agent for adjusting tonicity, such as sodium chloride or dextrose. Parenteral composition can be enclosed in ampule, disposable syringe or multiple dose vial made of glass, plastic or other material. Physiological saline is a preferred adjuvant.

[0061] The amount of the compound according to the present invention that is effective in treating a particular disorder or condition depends on the nature of the disorder and can be determined by standard clinical techniques.In addition, in vitro or in vivo assays can be optionally employed to help identify optimal dosage ranges.The exact amount to be employed in the composition also depends on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and each patient's circumstances.

[0062] The composition contains an effective amount of the compound of the present invention so that a suitable dosage is obtained.The exact dosage of the compound varies according to the specific formulation, the mode of application, and the specific site, the host to be treated and the disease, such as cancer, and in that case, what type of tumor.Other factors such as age, body weight, sex, diet, administration time, excretion rate, host condition, drug combination, response sensitivity and disease severity should be taken into account.Administration can be carried out continuously or periodically within the maximum tolerated dose.

[0063] Typically, the amount is at least about 0.01% of the compound of the present invention, and may comprise at least 80% by weight of the composition. When intended for oral administration, this amount can vary from about 0.1% to about 80% by weight of the composition. A preferred oral composition may comprise about 4% to about 50% by weight of the compound of the present invention.

[0064] Preferred compositions of the invention are prepared so that a parenteral lyophilized dosage unit contains from about 0.005% to about 10% by weight of a compound of the invention.

[0065] For intravenous administration, an exemplary dose is about 10.5 mg / m per cycle. 2 ~about 84mg / m 2 Between (or about 10 mg / m 2 ~about 85mg / m 2 The total dose per cycle may be divided according to a dosing schedule. Exemplary doses include about 0.5 mg / m 2 / day ~ approx. 3mg / m 2 The daily dose range includes / day. Daily dose value can be integrated according to dosing schedule. To match a specific dosing schedule with exemplary dose, add the specific dose administered over the course of a cycle to calculate the total dose per cycle, and divide this total dose per cycle by the number of days in the cycle to obtain the daily dose.

[0066] The compounds of the invention may be administered by any convenient route, for example by infusion or bolus injection, via absorption through epithelial or mucocutaneous linings.

[0067] In certain embodiments, it may be desirable to administer one or more compounds or compositions of the invention locally to the area requiring treatment, hi one embodiment, administration may be by direct injection at the site (or former site) of the cancer, tumor, or neoplastic or pre-neoplastic tissue.

[0068] Pulmonary administration can also be employed, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant. In certain embodiments, the compounds of the invention can be formulated as a suppository, with traditional binders and carriers such as triglycerides.

[0069] The compositions can take the form of a solution, suspension, emulsion, tablet, pill, pellet, capsule, capsule containing liquid, powder, sustained release formulation, suppository, emulsion, aerosol, spray, suspension, or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in E. W. Martin, "Remington's Pharmaceutical Sciences."

[0070] Pharmaceutical compositions can be prepared using methodology well known in the pharmaceutical field.For example, the composition intended to be administered by injection can be prepared by combining the compound of the present invention with water or other physiologically suitable diluent, such as phosphate buffered saline, to form a solution.Surfactant can be added to facilitate the formation of a homogeneous solution or suspension.

[0071] Compositions comprising the compounds of the invention may be lyophilized. Compositions comprising the compounds of the invention are typically present in vials containing a particular amount of such compound.

[0072] The present inventors have found that the compounds of the invention and compositions of the invention are particularly effective in the treatment of cancer.

[0073] Thus, as explained above, the present invention provides a method of treating a patient in need thereof, particularly a human suffering from cancer, comprising administering to the affected individual a therapeutically effective amount of a compound or composition according to the present invention.The present invention provides a compound or composition for use as a medicament.The present invention provides a compound or composition for use in the treatment of cancer, and more preferably solid tumors, even more preferably selected from lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, pancreatic cancer and gastric cancer.

[0074] Thus, the compounds and compositions according to the invention are useful for inhibiting the proliferation or growth of tumor or cancer cells or for treating cancer in an animal.

[0075] The compounds and compositions according to the invention show excellent activity in the treatment of cancer, especially solid tumors such as lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, pancreatic cancer and gastric cancer.

[0076] In this application, "cancer" is meant to include tumors, neoplasms, and any other malignant disease that has malignant tissues or cells as its origin.

[0077] As used herein, the term "treat", unless otherwise indicated, means to reverse, attenuate, alleviate, or inhibit the progression of the disease or condition to which such term applies, or one or more symptoms of such disorder or condition. As used herein, the term "treatment", unless otherwise indicated, refers to the act of treating as "treat" is defined immediately above.

[0078] The compounds and compositions according to the invention can be administered to animals that have also undergone surgery as a treatment for cancer. In one embodiment of the invention, the additional method of treatment is radiation therapy.

[0079] In a particular embodiment of the present invention, the compound or composition according to the present invention is administered simultaneously with radiation therapy.In another particular embodiment, radiation therapy is administered before or after the administration of the compound or composition of the present invention, preferably at least 1 hour, 3 hours, 5 hours, 12 hours, 1 day, 1 week, 1 month, more preferably several months (e.g. up to 3 months) before or after the administration of the compound or composition of the present invention.

[0080] Any radiotherapy protocol can be used depending on the type of cancer to be treated. For example, but not limited to, X-ray radiation can be administered, especially high-energy megavoltage (radiation with energy greater than 1 MeV) can be used for deep tumors, and electron beam and orthovoltage x-ray radiation can be used for skin cancer. Gamma-ray emitting radioisotopes can also be administered, such as radioisotopes of radium, cobalt and other elements.

[0081] In a further embodiment of the invention, a kit is provided comprising a therapeutically effective amount of a compound according to the invention and a pharma- ceutically acceptable carrier.

[0082] In one embodiment, the kit according to this embodiment is for use in the treatment of cancer, more preferably a solid tumor, and more preferably a cancer selected from lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, pancreatic cancer and gastric cancer.

[0083] A further embodiment of the invention relates to a process for the preparation of a compound according to the invention, comprising reacting a compound of formula V with a suitable reagent for the synthesis of a carbamate to give a compound of formula I

[0084] [ka]

[0085] (Wherein, X is F or Me) A method is provided for obtaining a compound of formula:

[0086] In another embodiment, the method comprises reacting a compound of formula VI with a compound of formula VII, 3 is unsubstituted C 1 ~C 4 is an alkyl group, ProtOH is a protecting group for the hydroxy, and X is defined for formula I), followed by coupling with a compound of formula OH In one embodiment, the method for preparing a compound of formula I further comprises the step of: deprotecting a compound of formula VI with a compound of formula VII, wherein R 3 is unsubstituted C 1 ~C 4 is an alkyl group, ProtOH is a protecting group for the hydroxy, and X is as defined for formula I; OH A method is provided that includes deprotecting a group to obtain a compound of formula V, and converting the compound of formula V to a compound of formula I.

[0087] [ka]

[0088] In another embodiment, the method comprises reacting a compound of formula VIII, where R is as defined for formula VII, with a compound of formula IX, where Prot OH is a protecting group for hydroxy and X is as defined for formula I to obtain a compound of formula VII. In one embodiment, the method for preparing a compound of formula I further comprises coupling a compound of formula VIII, where R is as defined for formula VII, with a compound of formula IX, where Prot OHis a protecting group for hydroxy and X is as defined for formula I) to obtain a compound of formula VII, and converting the compound of formula VII to a compound of formula I.

[0089] [ka]

[0090] In another embodiment, the method comprises the step of: NH is a protecting group for amino) with a compound of formula XI, where ProtOH is a protecting group for hydroxy and X is as defined for formula I, followed by coupling NHProt NH In one embodiment, the method for preparing a compound of formula I further comprises the step of deprotecting a group to obtain a compound of formula IX. NH is a protecting group for amino) with a compound of formula XI, where ProtOH is a protecting group for hydroxy and X is as defined for formula I, followed by coupling NHProt NH A method is provided that includes deprotecting the group to obtain a compound of formula IX, and converting the compound of formula IX to a compound of formula I.

[0091] [ka]

[0092] In a further embodiment of the invention there is provided an intermediate of formula V, VII, IX or XI useful for the preparation of a compound of formula I. In a preferred embodiment there is provided an intermediate of formula V EXAMPLES

[0093] Compounds 5 and 17 can be prepared as described in Examples 6 and 5, respectively, of WO2007 / 144423.

[0094] Reference compounds PM050489 and PM060184 were prepared as described in WO2007 / 144423 (compounds 1 and 4, respectively).

[0095] Example 1 Synthesis of 2

[0096] [ka]

[0097] Ozone was then added to the CH 2 Cl 2 A solution of alkene 5 (compound 22b in WO2007144423) (113.5 g, 235 mmol) and anhydrous pyridine (28.5 mL, 352.6 mmol) in 1175 mL of ozone is bubbled through the solution until the solution turns blue-green. 2 Purge for 30 min at 400 C and then transfer the mixture to a Büchner funnel in CH 2 Cl 2 Filtration through a silica pad (1200 g) eluting with 50% water gives 6 as a colorless liquid (75.3 g, 68% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 9.67 (dd, J = 2.9, 2.1 Hz, 1H), 7.70-7.57 (m, 4H), 7.50-7.33 (m, 6H), 4.39 (dq, J = 6.8, 5.6 Hz, 1H), 3.63 (ddd, J = 10.5, 7.1, 5.6 Hz, 1H), 3.55 (dt, J = 10.4, 6.0 Hz, 1H), 2.58 (ddd, J = 15.9, 5.6, 2.1 Hz, 1H), 2.48 (ddd, J = 16.0, 5.7, 2.9 Hz, 1H), 1.84 (ddt, J = 13.8, 7.0, 5.7 Hz, 1H), 1.78-1.66 (m, 1H), 1.05 (s, 9H), 0.82 (s, 9H), 0.04 (d, J = 4.9 Hz, 6H). ESI-MS m / z: 493.3 [M+Na]+ .

[0098] [ka]

[0099] To a solution of ethyl 2-(diethoxyphosphoryl)-2-fluoroacetate (35.8 mL, 176 mmol) in THF (1280 mL) at −78° C., nBuLi (110 mL, 1.6 M in hexanes, 176 mmol) was added slowly. After stirring for 30 min, a solution of 6 (75.3 g, 160 mmol) in THF (320 mL) cooled to −78° C. was then added over 55 min. The reaction mixture was stirred at −78° C. for 2.5 h and NH 4 The mixture was quenched by the addition of an aqueous saturated solution of Cl (1.16 L). 2 The mixture was diluted with 200 mL of EtOAc (170 mL), EtOAc (440 mL) was added and the layers were separated. The aqueous layer was extracted with EtOAc (200 mL) and the combined organic layers were washed with anhydrous Na 2 SO 4 The crude was purified by flash chromatography (Hex:EtOAc 99:1) to give 7 as a colorless oil (70.5 g, 79% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 7.71-7.60 (m, 4H), 7.49-7.28 (m, 6H), 5.96 (ddd, J = 21.9, 8.6, 6.9 Hz, 1H), 4.23 (q, J = 7.1 Hz, 2H), 4.03 (q, J = 5.6 Hz, 1H), 3.65-3.45 (m, 2H), 2.83-2.45 (m, 2H), 1.86-1.70 (m, 1H), 1.67-1.58 (m, 1H), 1.30 (t, J = 7.2 Hz, 3H), 1.05 (s, 9H), 0.81 (s, 9H), 0.02 (s, 6H). 19 F-NMR (376 MHz, CDCl 3): δ -120.3 (dd, J = 21.8, 2.8 Hz). ESI-MS m / z: 581.2 [M+Na] + .

[0100] [ka]

[0101] To a solution of 7 (70.3 g, 126 mmol) in anhydrous THF (754 mL) was added DIBAL (277 mL, 1.0 M in toluene, 277 mmol) dropwise over 30 min at 0° C. The reaction mixture was stirred at 0° C. for 15 min and then at 23° C. for 3 h. The reaction mixture was cooled again to 0° C. and quenched with MeOH (73 mL). Aqueous citric acid 10% solution (960 mL) was added, the mixture was stirred for 10 min, and extracted with EtOAc (700 mL). The organic layer was washed with anhydrous Na 2 SO 4 The crude was purified by flash chromatography (Hex:EtOAc 93:7 to 80:20) to give 8 (64.68 g, 99% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 7.71-7.63 (m, 4H), 7.48-7.33 (m, 6H), 5.13 (ddd, J = 21.0, 8.8, 7.6 Hz, 1H), 3.97-3.85 (m, 3H), 3.77-3.53 (m, 2H), 2.23-2.02 (m, 2H), 1.77-1.59 (m, 2H), 1.05 (s, 9H), 0.84 (s, 9H), 0.01 (s, 6H). 19 F-NMR (376 MHz, CDCl 3 ): δ -111.2 (q, J = 21.0 Hz). ESI-MS m / z: 539.3 [M+Na] + .

[0102] [ka]

[0103] Anhydrous CH 2 Cl 2 To a solution of 8 (26.55 g, 51 mmol) in 100 mL of ethyl acetate was added DMAP (0.25 g, 2 mmol), Et 3 N (12.2 mL, 87 mmol) and finally p-toluenesulfonyl chloride (12.7 g, 67 mmol) were added successively at 0° C. The reaction mixture was stirred at 0° C. for 3 h and then cooled to 5° C. 2 The mixture was quenched with O (390 mL). The phases were separated and the aqueous layer was washed with CH 2 Cl 2 (150 mL). The combined organic layers were extracted with anhydrous Na 2 SO 4 The crude was purified by flash chromatography (Hex:EtOAc 99:1 to 90:10) to give pure 9 (26.7 g, 78% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 7.78-7.70 (m, 2H), 7.66-7.61 (m, 4H), 7.48-7.37 (m, 2H), 7.41-7.30 (m, 4H), 7.34-7.23 (m, 2H), 5.34-5.20 (m, 1H), 4.42 (s, 1H), 4.37 (s, 1H), 3.88 (m, 1H), 3.65-3.48 (m, 2H), 2.46-2.41 (s, 3H), 2.15-1.95 (m, 2H), 1.74-1.50 (m, 2H), 1.03 (s, 9H), 0.84 (s, 9H), 0.01(s, 6H). 19 F-NMR (376 MHz, CDCl 3 ): δ -109.6 (q, J = 20.3 Hz). ESI-MS m / z: 693.3 [M+Na] + .

[0104] [ka]

[0105] To a solution of 9 (26.65 g, 40 mmol) in anhydrous THF (260 mL) was added LiEt 3 BH (46.7 mL, 1.7 M in THF, 79 mmol) was added slowly over a period of 20 min. After stirring for an additional 2 h, the reaction was carefully quenched with a 10% aqueous citric acid solution (472 mL). The mixture was washed with EtOAc (300 mL) followed by H 2 The mixture was diluted with 200 mL of EtOAc and the layers were separated. The aqueous layer was extracted with EtOAc (100 mL) and the combined organic layers were washed with brine and anhydrous Na 2 SO 4 The crude was filtered through a silica pad (Hex:EtOAc 90:10) to remove traces of boron salts to give 10 (18.4 g, 93% yield), which was used in the next step without further purification. 1 H NMR (400 MHz, CDCl 3 ): δ 7.69-7.65 (m, 4H), 7.47-7.30 (m, 6H), 5.05-4.81 (m, 1H), 3.88 (p, J = 5.9 Hz, 1H), 3.70-3.55 (m, 2H), 2.14-1.89 (m, 2H), 1.76-1.56 (m, 5H), 1.05 (s, 9H), 0.84 (s, 9H), 0.01(s, 6H). 19 F-NMR (376 MHz, CDCl 3 ): δ -93.9 (dq, J = 21.8, 17.6 Hz). ESI-MS m / z: 523 [M+Na] + .

[0106] [ka]

[0107] CH 2 Cl 2:CH 3 To a solution of 10 (18.3 g, 37 mmol) in OH (85:15 400 mL) was added camphorsulfonic acid (3.4 g, 15 mmol) at 23 °C and the reaction mixture was stirred at 23 °C for 4 h. 3 (300 mL) of a saturated aqueous solution was added (pH = 9) and the layers were separated. The aqueous layer was diluted with CH 2 Cl 2 (100 mL), and the combined organic layer was extracted with anhydrous Na 2 SO 4 The mixture was dried at rt, filtered, concentrated and purified by flash chromatography (Hex:EtOAc 100:0 to 50:50) to give pure 11 (13.56 g, 96% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 7.78-7.63 (m, 4H), 7.50-7.32 (m, 6H), 4.91-4.73 (m, 1H), 3.96-3.89 (m, 1H), 3.81-3.62 (m, 2H), 2.22-1.94 (m, 2H), 1.89-1.76 (m, 1H), 1.69-1.56 (m, 4H), 1.06 (s, 9H). 19 F-NMR (376 MHz, CDCl 3 ): δ -93.2 (dq, J = 21.7, 17.7 Hz). ESI-MS m / z: 409.3 [M+Na] + .

[0108] [ka]

[0109] Anhydrous CH 2 Cl 2 To a solution of 11 (13.3 g, 34 mmol) in (210 mL) of NaHCO 3 (16.0 g, 191 mmol) and Dess-Martin periodinane (16.0 g, 38 mmol) were added successively at 23° C. The suspension was stirred at 23° C. for 1 h and cooled to 0° C.2 S 2 O 3 with an aqueous saturated solution of NaHCO 3 A mixture of aqueous saturated solution of H (50:50, 350 mL) was carefully added and stirred for an additional hour. 2 The mixture was diluted with 200 mL of HO and the layers were separated. The aqueous layer was diluted with CH 2 Cl 2 (100 mL), and the combined organic layer was extracted with anhydrous Na 2 SO 4 Drying at 40° C., filtration and concentration to dryness gave crude 12 (12.35 g, 93% yield) which was used in the next step without further purification. 1 H NMR (400 MHz, CDCl 3 ): δ 9.70 (dd, J = 2.7, 2.0 Hz, 1H), 7.72-7.62 (m, 4H), 7.49-7.34 (m, 6H), 4.89 (dtd, J = 21.4, 8.1, 1.0 Hz, 1H), 4.23-4.16 (m, 1H), 2.58-2.42 (m, 2H), 2.15-2.09 (m, 2H), 1.66 (dq, J = 17.6, 0.9 Hz, 3H), 1.05 (s, 9H). 19 F-NMR (376 MHz, CDCl 3 ): δ -91.9 (dq, J = 21.7, 17.3 Hz).

[0110] [ka]

[0111] To a yellow suspension of iodomethylenetriphenylphosphorane (1.63 g, 3.1 mmol) in anhydrous THF (21 mL) was added dropwise NaHMDS (3.1 mL, 1.0 M in THF, 3.1 mmol) at 23 °C. After stirring for an additional 15 min, the solution was cooled to -78 °C and 12 (0.79 g, 2.1 mmol) in anhydrous THF (4 mL) was added dropwise. The temperature was kept at -78 °C while the reaction mixture was stirred for 2 h. Hexane (45 mL) was added and the mixture was filtered through a silica pad eluting with Hex:EtOAc 75:25. Evaporation of the volatiles gave an oil which was purified by flash chromatography (Hex:EtOAc 100:0 to 90:10) to give 13 (0.67 g, 64% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 7.77-7.63 (m, 4H), 7.48-7.30 (m, 6H), 6.30-6.17 (m, 2H), 4.99-4.85 (m, 1H), 3.86 (p, J = 5.6 Hz, 1H), 2.35-2.24 (m, 2H), 2.10-1.94 (m, 2H), 1.66 (dq, J = 17.6, 0.8 Hz, 3H), 1.06 (s, 9H). 19 F-NMR (376 MHz, CDCl 3 ): δ -93.2 (dq, J = 21.8, 17.6 Hz). ESI-MS m / z: 531.2 [M+Na] + .

[0112] [ka]

[0113] Boc-tert-Leu-CONH 2 A flask containing (7.33 g, 31.9 mmol), copper(I) iodide (1.21 g, 6.4 mmol), and potassium carbonate (8.79 g, 63.7 mmol) was evacuated and filled with N 2(×3). N,N'-dimethylethylenediamine (1.35 mL, 12.7 mmol) and 13 (10.8 g, 21.2 mmol) in dry DMF (210 mL) were added at 23° C. The flask was sealed and heated at 90° C. for 17 h and cooled to 23° C. The reaction mixture was diluted with EtOAc (2 L) and H 2 The layers were separated and the organic layer was diluted with H2O (750 mL). 2 Wash with 2×750 mL of anhydrous NaCl. 2 SO 4 The mixture was dried at 40° C., filtered and concentrated in vacuo, and the residue was purified by silica gel flash chromatography (hexane:EtOAc 100:1 to 6:1) to give 14 as a colorless oil (7.56 g, 58% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 7.77-7.63 (m, 4H), 7.51-7.32 (m, 6H), 7.06 (d, J = 10.5 Hz, 1H), 6.71-6.62 (m, 1H), 5.23 (d, J = 8.3 Hz, 1H), 4.92 (dt, J = 21.7, 7.9 Hz, 1H), 4.79-4.67 (m, 1H), 3.88-3.69 (m, 2H), 2.13-1.93 (m, 4H), 1.67 (d, J = 17.5 Hz, 3H), 1.43 (s, 9H), 1.07 (s, 9H), 0.97 (s, 9H). ESI-MS m / z: 633.2 [M+Na] + .

[0114] [ka]

[0115] Trimethylsilyl trifluoromethanesulfonate (17.8 mL, 98.2 mmol) was dissolved in CH 2 Cl 2To a solution of 14 (7.5 g, 12.3 mmol) and 2,6-lutidine (14.3 mL, 123 mmol) in 1H-tetrahydrofuran (310 mL) was added dropwise at 0° C. The mixture was stirred at 0° C. for 5 min and at 23° C. for an additional 1 h. The reaction was monitored with NH 4 Quench with an aqueous saturated solution of Cl (100 mL) and CH 2 Cl 2 The layers were separated and the organic layer was washed with 1 M NaOH solution (100 mL), an aqueous saturated solution of NaCl (100 mL) and H 2 Wash with 2×50 mL of anhydrous NaSO. 4 The resulting oil was purified on silica gel (hexanes:EtOAc 10:1 to 2:1) to give 15 as a colorless oil contaminated with 2,6-lutidine (7.5 g, 120% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 7.82-7.55 (m, 4H), 7.49-7.30 (m, 6H), 7.14 (m, 1H), 6.66 (m, 1H), 4.93 (dt, J = 21.9, 7.9 Hz, 1H), 4.78 (m, 1H), 3.79 (m, 1H), 2.25-2.12 (m, 3H), 2.06-1.98 (m, 2H), 1.64 (d, J = 17.7 Hz, 3H), 1.08-0.93 (m, 18H). ESI-MS m / z: 511.3 [M+H] + .

[0116] [ka]

[0117] To a solution of 15 (6.3 g, 12.3 mmol) and (Z)-3-tributylstannylpropenoic acid (4.45 g, 12.3 mmol) in EtOAc (120 mL) was added DIPEA (5.37 mL, 30.8 mmol) and propylphosphonic anhydride (T3P®) (4.05 mL, 50% in EtOAc, 13.6 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 30 min, warmed to 23° C. and stirred for 3 h. The reaction mixture was stirred at H 2 The mixture was quenched with O (750 mL) and the layers were separated. The organic layer was washed with brine (750 mL) and anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated, and the residue was purified by flash chromatography (hexane:EtOAc 40:1 to 10:1) to give 16 as a colorless oil (6.08 g, 58% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 7.77-7.69 (m, 4H), 7.55-7.37 (m, 6H), 7.13-7.06 (m, 1H), 7.07 (d, J = 12.3 Hz, 1H), 6.83 (d, J = 12.3 Hz, 1H), 6.75-6.63 (m, 1H), 6.51 (d, J = 9.4 Hz, 1H), 5.00 (dt, J = 21.4, 7.9 Hz, 1H), 4.85-4.72 (m, 1H), 4.42 (d, J = 9.6 Hz, 1H), 4.17 (q, J = 7.2 Hz, 1H), 3.83 (dt, J = 10.3, 5.4 Hz, 1H), 2.27-1.98 (m, 3H), 1.78-1.68 (m, 3H), 1.60-1.43 (m, 6H), 1.38-1.24 (m, 6H), 1.10 (s, 9H), 1.01 (s, 9H), 0.96-0-87 (m, 15H). ESI-MS m / z: 877.3 [M+Na] + .

[0118] [ka]

[0119] CuTC (870 mg, 4.57 mmol) was added in one portion to a solution of 16 (2.60 g, 3.04 mmol) and 17 (compound 17a in WO2007144423) (1.06 g, 3.04 mmol) in N-methyl-2-pyrrolidone (30 mL) at 0° C. After stirring for 30 min at 0° C. and 2 h at 23° C., Al 2 O 3 The reaction mixture was filtered through a pad. The product was washed off using EtOAc (80 mL) and the solvent was evaporated. The residue was diluted with EtOAc (40 mL) and washed with 1.0 N HCl (3×20 mL). The combined organic layers were washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and evaporated in vacuum. The residue was purified by flash chromatography (Hexanes:EtOAc 2:1 to 1:1) to give 18 as a white foam (1.43 g, 58% yield). 1 H NMR (400 MHz, CDCl 3): δ 7.71-7.62 (m, 4H), 7.51-7.35 (m, 6H), 7.31-7.21 (m, 1H), 7.12 (d, J = 10.8 Hz, 1H), 6.90 (td, J = 11.5, 1.2 Hz, 1H), 6.65 (dd, J = 10.8, 9.1 Hz, 1H), 6.26-6.12 (m, 2H), 5.73-5.56 (m, 2H), 5.28 (d, J = 9.9 Hz, 1H), 4.94 (ddd, J = 21.5, 8.4, 7.2 Hz, 1H), 4.82-4.70 (m, 1H), 4.30 (d, J = 9.3 Hz, 1H), 4.21 (ddd, J = 10.5, 7.7, 5.1 Hz, 1H), 3.83-3.76 (m, 1H), 3.66 (s, 3H), 2.94-2.77 (m, 1H), 2.49-2.32 (m, 2H), 2.20-1.98 (m, 4H), 1.84 (s, 3H), 1.68 (d, J = 17.5 Hz, 3H), 1.16 (d, J = 6.7 Hz, 3H), 1.06 (s, 9H), 1.00 (s, 9H). ESI-MS m / z: 785.3 [M+H] + , 807.3 [M+Na] + .

[0120] [ka]

[0121] A solution of TBAF (3.57 mL, 1.0 M in THF, 3.57 mmol) was added dropwise to a solution of 18 (1.40 g, 1.78 mmol) in THF (27 mL) at 23 °C. After stirring for 2 h, the reaction was cooled to 0 °C with NaHCO 3 (20 mL). The aqueous layer was extracted with EtOAc (3×20 mL) and the combined organic layers were washed with anhydrous Na 2 SO 4The residue was dried at 40° C., filtered and evaporated. Flash chromatography of the residue (Hex:EtOAc 2:1 to 1:1) afforded 19 as a white foam (680 mg, 70% yield). 1 H NMR (400 MHz, CD 3 OD): δ 7.24 (td, J = 11.6, 1.2 Hz, 1H), 6.95 (td, J = 11.6, 1.2 Hz, 1H), 6.64 (d, J = 9.1 Hz, 1H), 6.29-6.14 (m, 1H), 5.92 (d, J = 11.4 Hz, 1H), 5.87 (t, J = 4.7 Hz, 1H), 5.34 (dt, J = 9.9, 1.5 Hz, 1H), 5.06 (dtd, J = 21.7, 8.0, 1.1 Hz, 1H), 4.92 (dt, J = 9.1, 7.5 Hz, 1H), 4.42 (s, 1H), 4.32 (dt, J = 8.1, 7.3 Hz, 1H), 3.64 (s, 3H), 3.66-3.59 (m, 1H), 2.95-2.86 (m, 1H), 2.55-2.43 (m, 2H), 2.41-2.02 (m, 4H), 1.93-1.78 (m, 6H), 1.15 (d, J = 6.6 Hz, 3H), 1.03 (s, 9H). ESI-MS m / z: 547.2 [M+H] + .

[0122] [ka]

[0123] CH 2 Cl 2 To a solution of 19 (670 mg, 1.23 mmol) in (70 mL) was added trichloroacetyl isocyanate (174 μL, 1.47 mmol) at 23 °C. The reaction was stirred at 23 °C for 30 min and then added Al 2 O 3 (9g, before H 2 O (activated with 15% w / w) was added with stirring for 30 min. Then additional alumina Al2 O 3 (4.5 g) was added and stirred for 45 min. The reaction mixture was filtered and 2 Cl 2 :CH 3 After rinsing using a mixture of OH 10:1 and evaporation of the filtrate under reduced pressure, the product was purified by column chromatography (hexane:EtOAc 1:1 to 1:2) to give pure 2 (470 mg, 65% yield). 1 H NMR (400 MHz, CD 3 OD): δ 7.24 (t, J = 11.6 Hz, 1H), 6.95 (td, J = 11.5, 1.1 Hz, 1H), 6.69 (d, J = 9.0 Hz, 1H), 6.19 (d, J = 11.6 Hz, 1H), 5.93 (d, J = 11.6 Hz, 1H), 5.87 (t, J = 4.7 Hz, 1H), 5.35 (d, J = 9.9 Hz, 1H), 5.02 (ddd, J = 21.2, 8.6, 7.5 Hz, 1H), 4.88-4.80 (m, 1H), 4.59 (m, 1H), 4.47 (s, 1H), 4.32 (m, 1H), 3.64 (s, 3H), 2.91 (dt, J = 9.8, 6.8 Hz, 1H), 2.58-2.41 (m, 3H), 2.42-2.13 (m, 3H), 1.91-1.86 (m, 6H), 1.15 (d, J = 6.7 Hz, 3H), 1.04 (s, 9H). ESI-MS m / z: 590.2 [M+H] + .

[0124] Example 2 Synthesis of 3

[0125] [ka]

[0126] To a suspension of isopropyltriphenylphosphonium iodide (60.6 g, 117 mmol) in THF (1 L) at -78°C was slowly added sodium bis(trimethylsilyl)amide (140 mL, 1.0 M in THF, 140 mmol) via an addition funnel over a period of 30 min at 0°C. After stirring for 10 min, the reaction mixture was cooled to -78°C, and then a solution of 6 (55 g, 117 mmol) in THF (500 mL) cooled to -78°C was added for 1 h. The reaction mixture was stirred at -78°C for 1 h, warmed to 0°C and stirred for 30 min. Hexane (500 mL) was added and the resulting slurry was filtered through silica and washed with additional hexane (1 L). The filtrate was evaporated under reduced pressure to give pure 20 (53.1 g, 83% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 7.81-7.60 (m, 4H), 7.46-7.26 (m, 6H), 5.10-4.87 (m, 1H), 3.86 (m, 1H), 3.71-3.56 (m, 2H), 2.10 (m, 2H), 1.69 (m, 2H), 1.60 (s, 3H), 1.38 (s, 3H), 1.04 (s, 9H), 0.84 (s, 9H), -0.02 (s, 6H).

[0127] [ka]

[0128] CH 2 Cl 2 :CH 3 To a solution of 20 (53.37 g, 107 mmol) in OH (85:15, 2300 mL) was added camphorsulfonic acid (7.48 g, 32 mmol) at 23° C. The reaction mixture was stirred for 3 h at 23° C. NaHCO 3 A saturated aqueous solution of 1,2-dichloromethane was added (pH 9) and the layers were separated. The combined organic layers were washed with anhydrous Na 2 SO 4It was dried at 40° C., filtered and the solvent was evaporated. The resulting oil was purified by column chromatography (Hex:EtOAc 100:1) to give pure 21 (31.88 g, 78% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 7.77-7.65 (m, 4H), 7.51-7.34 (m, 6H), 4.92-4.86 (m, 1H), 3.96-3.90 (m, 1H), 3.85-3.77 (m, 1H), 3.75-3.59 (m, 1H), 2.23-2.12 (m, 2H), 2.11-2.01 (m, 1H), 1.94-1.81 (m, 1H), 1.66-1.60 (m, 1H), 1.57 (s, 3H), 1.35 (s, 3H), 1.06 (s, 9H).

[0129] [ka]

[0130] Anhydrous CH 2 Cl 2 To a solution of 21 (25.3 g, 66.2 mmol) in (660 mL) of NaHCO 3 (16.7 g, 199 mmol) and Dess-Martin periodinane (42.11 g, 99 mmol) were added successively at 23° C. The suspension was stirred for 1 h and cooled to 0° C. 2 S 2 O 3 with an aqueous saturated solution of NaHCO 3 A mixture of aqueous saturated solution of H (50:50, 350 mL) was carefully added and stirred for an additional hour. 2 The mixture was diluted with 200 mL of HO and the layers were separated. The aqueous phase was diluted with CH 2 Cl 2 (100 mL), and the combined organic layer was extracted with anhydrous Na 2 SO 4 Drying at 40° C., filtration and concentration to dryness afforded 22 (24.6 g, 98%) which was used in the next step without further purification. 1H NMR (400 MHz, CDCl 3 ): δ 9.71 (d, J = 2.8 Hz, 1H), 7.77-7.57 (m, 4H), 7.54-7.31 (m, 6H), 5.05-4.90 (m, 1H), 4.21-4.19 (m, 1H), 2.49-2.43 (m, 2H), 2.25-2.15 (m, 2H), 1.61 (s, 3H), 1.40 (s, 3H), 1.05 (s, 9H).

[0131] [ka]

[0132] To a suspension of iodomethylenetriphenylphosphorane (24.6 g, 64.7 mmol) in anhydrous THF (670 mL) was added dropwise at 23° C. over a period of 10 min. NaHMDS (90.6 mL, 1.0 M in THF, 90.6 mmol) was then added dropwise at 23° C. to a pre-cooled solution of 22 (24.6 g, 64.7 mmol) in anhydrous THF (100 mL). The temperature was kept at −78° C. while the reaction mixture was stirred for 3 h. Hexane (1 L) was added and the mixture was filtered through Celite® eluting with hexane (1 L). Evaporation of the volatiles gave an oil which was purified by flash chromatography (Hex:EtOAc 50:1) to give 23 (21.71 g, 67% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 7.76-7.60 (m, 4H), 7.51-7.31 (m, 6H), 6.36-6.14 (m, 2H), 5.09-4.93 (m, 1H), 3.95-3.80 (m, 1H), 2.36-2.24 (m, 2H), 2.13 (m, 2H), 1.63 (s, 3H), 1.43 (s, 3H), 1.06 (s, 9H).

[0133] [ka]

[0134] Boc-tert-Leu-CONH 2 A flask containing (5.16 g, 22.4 mmol), copper(I) iodide (850 mg, 4.5 mmol), and potassium carbonate (6.20 g, 44.8 mmol) was evacuated and filled with N 2 (×3). N,N'-Dimethylethylenediamine (0.95 mL, 9.0 mmol) and 23 (7.54 g, 14.9 mmol) in dry DMF (150 mL) were added at 23° C. The flask was sealed and heated at 90° C. for 17 h and cooled to 23° C. The reaction mixture was diluted with EtOAc (2 L) and H 2 The layers were separated and the organic layer was diluted with H2O (500 mL). 2 Wash with 2×O (3×500 mL) and anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated in vacuo, and the residue was purified by flash chromatography on silica gel (hexane:EtOAc 100:1 to 6:1) to give 24 (4.56 g, 52% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 7.74-7.63 (m, 4H), 7.48-7.31 (m, 6H), 7.00 (d, J = 10.7 Hz, 1H), 6.66 (t, J = 9.9 Hz, 1H), 5.32-5.20 (m, 1H), 5.07-4.97 (m, 1H), 4.82 (m, 1H), 3.92-3.67 (m, 2H), 2.24-1.96 (m, 4H), 1.66 (s, 3H), 1.43 (s, 9H), 1.43 (s, 3H), 1.05 (s, 9H), 0.96 (s, 9H). 13 C NMR (75 MHz, CDCl 3): δ 168.6, 156.1, 136.1, 135.0, 134.4, 134.1, 129.9, 127.8, 121.7, 120.4, 109.0, 73.0, 58.6, 35.5, 32.9, 28.5, 27.2, 26.7, 26.0, 19.5, 18.6, 18.0.

[0135] [ka]

[0136] Trimethylsilyl trifluoromethanesulfonate (8.82 mL, 48.73 mmol) was dissolved in CH 2 Cl 2 To a solution of 24 (4.55 g, 7.50 mmol) and 2,6-lutidine (6.11 mL, 52.48 mmol) in 2,4-dichloromethane (75 mL) was added dropwise at 0° C. The mixture was stirred at 0° C. for 5 min and at 23° C. for an additional 1 h. The reaction was then cooled to 37° C. with NH 4 Quench with an aqueous saturated solution of Cl (100 mL) and CH 2 Cl 2 The layers were separated and the organic layer was washed with 1 M NaOH solution (100 mL), aqueous saturated solution of NaCl (100 mL) and H 2 Wash with 2×50 mL of anhydrous NaSO. 4 Drying at 40° C., filtration and concentration gave crude 25 as a colorless oil contaminated with 2,6-lutidine (4.2 g, 111% yield), which was used in the next without further purification. 1 H NMR (500 MHz, CDCl 3): δ 8.44 (d, J = 10.7 Hz, 1H), 7.70-7.62 (m, 4H), 7.44-7.31 (m, 6H), 6.65-6.57 (m, 1H), 5.00 (td, J = 6.6, 6.1, 1.2 Hz, 1H), 4.89 (m, 1H), 3.80-3.76 (m, 1H), 3.66-3.62 (m, 1H), 2.32-2.24 (m, 1H), 2.16-2.09 (m, 3H), 1.59 (s, 3H), 1.40 (s, 3H), 1.04 (s, 9H), 1.00 (s, 9H).

[0137] [ka]

[0138] To a solution of 25 (1.19 g, 2.35 mmol) and (Z)-3-tributylstannylpropenoic acid (850 mg, 2.35 mmol) in EtOAc (23 mL) was added DIPEA (1.02 mL, 5.87 mmol) and propylphosphonic anhydride (1.54 mL, 50% in EtOAc, 2.58 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 30 min, warmed to 23° C. and stirred for 1 h. The reaction mixture was stirred at H 2 The mixture was quenched with 200 mL of O (50 mL) and the layers were separated. The organic layer was washed with brine (50 mL) and anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated, and the residue was purified by flash chromatography (hexane:EtOAc 40:1 to 10:1) to give 26 as a colorless oil (1.15 g, 75% yield). 1 H NMR (400 MHz, CDCl 3): δ 7.74-7.65 (m, 4H), 7.55-7.31 (m, 6H), 7.02 (d, J = 12.2 Hz, 1H), 6.91 (d, J = 11.0 Hz, 1H), 6.75 (d, J = 12.3 Hz, 1H), 6.68-6.60 (m, 1H), 6.22 (d, J = 9.4 Hz, 1H), 5.03 (t, J = 7.2 Hz, 1H), 4.88-4.77 (m, 1H), 4.30 (d, J = 9.5 Hz, 1H), 3.85-3.75 (m, 1H), 2.19-1.95 (m, 4H), 1.67 (s, 3H), 1.62-1.35 (m, 6H), 1.46 (s, 3H), 1.38-1.18 (m, 6H), 1.05 (s, 9H), 0.95 (s, 9H), 0.94-0.70 (m, 15H). ESI-MS m / z: 873.3 [M+Na] + .

[0139] [ka]

[0140] CuTC (720 mg, 3.79 mmol) was added in one portion to a solution of 26 (2.15 g, 2.53 mmol) and 17 (compound 17a in WO2007144423) (880 mg, 2.53 mmol) in N-methyl-2-pyrrolidone (25 mL) at 0° C. After stirring for 30 min at 0° C. and 2 h at 23° C., Al 2 O 3 The reaction mixture was filtered through a pad. The product was washed off using EtOAc (65 mL) and the solvent was evaporated. The residue was diluted with EtOAc (75 mL) and washed with 1.0 N HCl (3×20 mL). The combined organic layers were washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and evaporated in vacuum. The residue was purified by flash chromatography (Hexanes:EtOAc 2:1 to 1:1) to give 27 as a white foam (1.59 g, 80% yield). 1H NMR (400 MHz, CDCl 3 ): δ 7.74-7.62 (m, 4H), 7.50-7.33 (m, 6H), 7.33-7.20 (m, 1H), 7.13 (d, J = 10.7 Hz, 1H), 6.88 (td, J = 11.5, 1.2 Hz, 1H), 6.63 (t, J = 9.8 Hz, 1H), 6.27 (d, J = 9.2 Hz, 1H), 6.16 (d, J = 11.8 Hz, 1H), 5.73-5.57 (m, 2H), 5.27 (d, J = 9.8 Hz, 1H), 5.01 (t, J = 7.1 Hz, 1H), 4.95-4.75 (m, 1H), 4.34-4.25 (m, 1H), 4.25-4.15 (m, 1H), 3.79 (t, J = 5.8 Hz, 1H), 3.65 (s, 3H), 2.91-2.76 (m, 1H), 2.48-2.34 (m, 2H), 2.25-2.08 (m, 4H), 1.83 (d, J = 1.4 Hz, 3H), 1.65 (s, 3H), 1.43 (s, 3H), 1.15 (d, J = 6.6 Hz, 3H), 1.04 (d, J = 1.4 Hz, 9H), 0.98 (s, 9H). ESI-MS m / z: 781.4 [M+H] + .

[0141] [ka]

[0142] A solution of TBAF (4.07 mL, 1.0 M in THF, 4.07 mmol) was added dropwise to a solution of 27 (1.59 g, 2.04 mmol) in THF (31 mL) at 23 °C. After stirring for 2 h, the reaction was quenched with NaHCO 3 (45 mL). The aqueous layer was extracted with EtOAc (3×20 mL) and the combined organic layers were washed with anhydrous Na 2 SO 4It was dried at 40° C., filtered and evaporated. Flash chromatography of the residue (Hex:EtOAc 2:1 to 1:1) afforded 28 as a white foam (806 mg, 73% yield). 1 H NMR (400 MHz, CD 3 OD): δ 7.24 (t, J = 11.5 Hz, 1H), 6.95 (t, J = 11.6 Hz, 1H), 6.62 (d, J = 9.0 Hz, 1H), 6.18 (d, J = 11.6 Hz, 1H), 5.92 (d, J = 11.6 Hz, 1H), 5.86 (t, J = 4.7 Hz, 1H), 5.34 (d, J = 9.0 Hz, 1H), 5.20 (d, J = 7.1 Hz, 1H), 4.99-4.89 (m, 1H), 4.41 (s, 1H), 4.37-4.23 (m, 1H), 3.63 (s, 3H), 3.65-3.59 (m, 1H), 2.94-2.86 (m, 1H), 2.46 (m, 2H), 2.39-2.26 (m, 2H), 2.26-2.12 (m, 2H), 1.86 (s, 3H), 1.70 (s, 3H), 1.61 (s, 3H), 1.14 (d, J = 6.6 Hz, 3H), 1.02 (s, 9H). ESI-MS m / z: 543.3 [M+H] + .

[0143] [ka]

[0144] CH 2 Cl 2 To a solution of 28 (600 mg, 1.11 mmol) in (66 mL) was added trichloroacetyl isocyanate (157 μL, 1.32 mmol) at 23 °C. The reaction was stirred at 23 °C for 30 min and then added Al 2 O 3 (9g, before H 2 O (activated with 15% w / w) was added with stirring for 30 min. Then additional alumina Al 2O 3 (4.5 g) was added and stirred for 45 min. The reaction mixture was filtered and 2 Cl 2 :CH 3 After rinsing using a mixture of OH 10:1 and evaporation of the filtrate under reduced pressure, the product was purified by column chromatography (Hexane:EtOAc 1:1 to 1:2) to give pure 3 (590 mg, 91% yield). 1 H NMR (400 MHz, CD 3 OD: δ 8.70 (d, J = 10.5 Hz, 1H), 7.33 (d, J = 11.6 Hz, 1H), 6.90 (t, J = 11.5 Hz, 1H), 6.81 (t, J = 9.7 Hz, 1H), 6.52 (d, J = 9.3 Hz, 1H), 6.16 (d, J = 11.5 Hz, 1H), 5.71 (d, J = 11.6 Hz, 1H), 5.63 (d, J = 6.6 Hz, 1H), 5.37-5.04 (m, 4H), 4.93-4.73 (m, 1H), 4.53-4.34 (m, 2H), 4.34-4.19 (m, 1H), 3.66 (s, 3H), 2.93-2.77 (m, 1H), 2.57-2.34 (m, 3H), 2.34-2.25 (m, 2H), 2.22-2.06 (m, 1H), 1.82 (s, 3H), 1.62 (s, 3H), 1.59 (s, 3H), 1.15 (d, J = 6.5 Hz, 3H), 1.04 (s, 9H). ESI-MS m / z: 586.4 [M+H] + .

[0145] If necessary, further guidance on the general synthesis of compounds 2 and 3 can be obtained from WO2007 / 144423.

[0146] Example 3 In vitro bioassays for detection of antitumor activity The purpose of this assay is to evaluate the in vitro cytostatic (ability to slow or stop the growth of tumor cells) or cytotoxic (ability to kill tumor cells) activity of the samples being tested.

[0147] [Table 1]

[0148] Assessment of cytotoxic activity using the SRB colorimetric assay A colorimetric assay using the sulforhodamine B (SRB) reaction was adapted to provide a quantitative measure of cell proliferation and viability (using the technique developed by Skehan et al., J. Natl. Cancer Inst. 1990 and following the protocol detailed in V. Vichai and K. Kirtikara (2006) Nature Protoc. 1, 1112-1116).

[0149] The assay format employs 96-well cell culture microplates. All cell lines used in this study were obtained from the American Type Culture Collection (ATCC) unless otherwise stated, and were derived from different types of human cancers.

[0150] Cells were cultured in 10-well plates supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U / mL penicillin, and 100 U / mL streptomycin at 37°C and 5% CO 2 The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (for A549, HT-29, and MDA-MB-231) or RPMI (for PSN-1) at 98% and humidity. For experiments, cells were harvested from subconfluent cultures using trypsinization and resuspended in fresh medium before counting and plating.

[0151] Cells were plated at 5 × 10 per well in 150 μL aliquots. 3Cells were seeded in 96-well microtiter plates and allowed to adhere to the plate surface for 18 hours (overnight) in drug free medium. One control (untreated) plate of each cell line was then fixed (as described below) and used for zero-hour baseline. Culture plates were then treated with test compounds (50 μL aliquots of 4× concentrated compound stock solutions made in complete culture medium) using 10-fold dilutions (concentrations spanning the range of 10-0.00262 μg / mL) and triplicate cultures (final concentration of DMSO is 1%). After 72 hours of treatment, antitumor efficacy was measured by using the SRB method. Briefly, cells were washed twice with PBS, fixed in 1% glutaraldehyde solution for 15 minutes at room temperature, rinsed twice in PBS, and stained in 0.4% SRB solution for 30 minutes at room temperature. Cells were then rinsed several times with 1% acetic acid solution and air-dried at room temperature. SRB was then extracted in 10 mM trizma base solution and absorbance was measured at 490 nm in an automated spectrophotometer plate reader. The effects on cell proliferation and survival were estimated by applying the NCI algorithm (Boyd MR and Paull KD. Drug Dev. Res. 1995, 34, 91-104).

[0152] Using the mean ± SD of the triplicates, dose-response curves were automatically generated into a four-parameter logistic curve using nonlinear regression analysis. Three reference parameters were calculated by automatic interpolation (NCI algorithm). GI 50 = concentration of compound resulting in 50% cell growth inhibition compared to control cultures.

[0153] Table 1 shows the biological activity (GI 50 ) data (GI 50 The following shows examples of

[0154] [Table 2]

[0155] It can be seen that the compounds of the present invention have surprisingly improved activity over the comparative compounds in all cell lines. The compounds of the present invention are shown to have high activity in vitro, particularly low nanomolar activity across a variety of different cell lines. This demonstrates that the compounds according to the present invention exhibit high cytotoxicity against cancer cells and are useful in the treatment of cancer. Importantly, the compounds of the present invention exhibit improved activity when compared to the reference compounds. In particular, compound 2 is 3.8-8.2 times more active than reference compound PM060184. In addition, compound 3 is 1.3-3.7 times more active than reference compound PM060184. In addition, compound 2 is 1.9-7.9 times more active than reference compound PM050489. In addition, compound 3 is 1.3-1.8 times more active than reference compound PM050489.

[0156] Example 4 Determination of MTD and MTMD Example 4.1 Determination of MTD and MTMD in female athymic nude Fox1 nu / nu mice Female athymic nude Fox1 nu / nu mice (Envigo) were used for all experiments. Animals (N=10 / cage) were housed in individually ventilated cages (Sealsafe Plus®, Techniplast SPA) with a 12-h light / dark cycle, 21-23°C, and 40-60% humidity. Mice were fed irradiated standard rodent chow (Tecklad 2914C) and sterile water ad libitum. Animals were acclimated for 5 days and then individually identified by tattoo. Animal protocols were reviewed and approved by the regional Institutional Animal Care and Use Committees.

[0157] Mice were randomly assigned to experimental groups and administered intravenously either once for MTD (Maximum Tolerated Dose) determination or once a week for three consecutive weeks for MTMD (Maximum Tolerated Multiple Dose) determination studies. Animals were administered compounds dissolved in white formulations or experimental formulations at different concentrations. Dose was always 10 mL / kg. Once administered, animals were monitored for clinical signs of systemic toxicity, weight changes and mortality up to 14 days after administration.

[0158] The MTD results are summarized in Table 2.

[0159] [Table 3]

[0160] The MTMD results are summarized in Table 3.

[0161] [Table 4]

[0162] Comparative Example In vivo xenografting

[0163] [Table 5]

[0164] H460 cells were maintained in vitro at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (Sigma-Aldrich, Co.). Cultured cells were passaged every 3-5 days upon reaching confluence. Each animal received 5 × 10 cells suspended in 0.05 mL of a solution consisting of 50% Matrigel (Corning Incorporated Life Sciences) and 50% medium without serum or antibiotics at 4-6 weeks of age. 6H460 cells were implanted subcutaneously (in the right flank using a 26G needle and 1 cc syringe).

[0165] The antitumor activity of PM050489 was evaluated in one human tumor model, NSCLC (H460) xenografted immunosuppressed mice. All treatments were administered intravenously at their MTD.

[0166] Tumor is approximately 150-200 mm 3 When the tumor-bearing animals (N=10 / group) were randomly assigned to the following treatment groups: PM050489(0.08mg / kg) placebo

[0167] Treatments were administered intravenously once a week (days 0 and 7) for two consecutive weeks.

[0168] Tumor dimensions and body weights were recorded three times a week starting on the first day of treatment (day 0). Treatments resulting in >20% mortality and / or 20% net body weight loss were considered toxic. Tumor volume was calculated using the formula (a b 2 ) / 2, where a and b are the longest and shortest diameters, respectively. Animals were cultured until their tumors reached approximately 2,000 mm 3 Mice were euthanized when the tumor volume reached 0.01 mg / kg and / or when severe necrosis was observed. The median, mean, and SD of tumor volume were calculated on each measurement day.

[0169] Antitumor efficacy was calculated using ΔT / ΔC (%) and tumor growth delay (TGD). ΔT / ΔC was defined as the percentage of change in tumor volume for each treatment (T) and placebo (C) group. ΔT / ΔC was calculated immediately before the second dose and at the fourth week of the study. TGD was expressed as the percentage of volume delay in the treatment group and was expressed as [(D t -D c ) / D c ] · 100 (in the formula, D t and D. c The treatment and control groups were 1,000 mm 3The time to achieve a specific volume fixed as t is calculated by applying the median time in days to achieve a specific volume fixed as t.

[0170] The tolerability of the treatment was assessed by monitoring body weight gain, clinical signs, and evidence of local damage at the injection site.

[0171] All placebo-treated animals died or were sacrificed for ethical reasons between days 0 and 10. Tumors in this group had a calculated doubling time of 2.6 days (95% CI, 2.2 to 3.2).

[0172] PM050489 treatment (0.08 mg / kg) was terminated early due to severe necrosis in the tumor mass and / or loss of body weight. Antitumor efficacy parameters are shown in Table 7.

[0173] [Table 6]

[0174] Tumor growth curves are shown in FIG.

[0175] (Examples 5 to 7) In vivo xenografting Female athymic nu / nu mice (Harlan Laboratories Models, SL, Barcelona, ​​Spain or Envigo, Spain) were used for all experiments. Animals were housed in individually ventilated cages (Sealsafe® Plus, Techniplast SPA) with a maximum of 10 animals per cage, with a 12-h light / dark cycle, 21-23°C, and 40-60% humidity. Mice were fed irradiated standard rodent chow (Tecklad 2914C) and sterile water ad libitum. Animals were acclimated for at least 5 days before tumor implantation with tumor cell suspension.

[0176] [Table 7]

[0177] H460 cells were cultured in vitro at 37°C and 5% CO 2 The cells were maintained in Dulbecco's modified Eagle's medium (Sigma-Aldrich, Co.) at 4-6 weeks of age. Cultured cells were passaged every 3-5 days upon reaching confluence. Each animal received 5 × 10 cells suspended in 0.05 mL of a solution consisting of 50% Matrigel® (Corning Incorporated Life Sciences) and 50% medium without serum or antibiotics. 6 H460 cells were implanted subcutaneously (in the right flank using a 26G needle and 1 cc syringe).

[0178] HGC27 cells were cultured in vitro at 37°C and 5% CO 2 The cells were maintained in Iscove's modified Dulbecco's medium (Sigma Aldrich, Co.) at 4-6 weeks of age. Cultured cells were passaged every 3-5 days once they reached confluence. Each animal received 5 × 10 cells suspended in 0.05 mL of a solution consisting of 50% Matrigel® (Corning Incorporated Life Sciences), 50% medium without serum or antibiotics. 6 HGC-27 cells were implanted subcutaneously (in the right flank using a 26G needle and 1 cc syringe).

[0179] HCT116 cells were cultured in vitro at 37°C and 5% CO 2 The cells were maintained in McCoy's 5A medium (Sigma Aldrich, Co.). Cultured cells were passaged every 3-5 days once they reached confluence. Each animal received 5 × 10 cells at 4-6 weeks of age suspended in 0.05 mL of a solution consisting of 50% Matrigel® (Corning Incorporated Life Sciences) and 50% medium without serum or antibiotics. 6 HCT116 cells were implanted subcutaneously (in the right flank using a 26G needle and 1 cc syringe).

[0180] The antitumor activity of compounds 2 and 3 was compared with PM060184 in immunosuppressed mice xenografted with three different human tumor models, NSCLC (H460), gastric (HGC-27) and colon (HCT-116), demonstrating that PM060184 was highly active. All treatments were administered intravenously weekly at their MTD, 1 / 4MTD and 1 / 16MTD (high, medium and low doses).

[0181] In short, - Tumor cells grown in culture were implanted subcutaneously into 4- to 6-week-old athymic nu / nu female mice. - When tumors reached a certain volume, tumor-bearing animals (N=8-10 / group) were randomly assigned to treatment groups. - Tumor dimensions and body weights were recorded 2-3 times per week starting on the first day of treatment (day 0). Treatments resulting in >20% mortality and / or 20% net body weight loss were considered toxic. Tumor volumes were calculated using the formula (a·b 2 ) / 2, where a and b are the longest and shortest diameters, respectively. Animals were cultured until their tumors reached approximately 2,000 mm 3 Mice were euthanized when they reached 100% and / or showed severe necrosis. - Median tumor volumes were calculated on each measurement day. - Antitumor efficacy was assessed using two-tailed Mann-Whitney U tests comparing tumor volume data from arms at week 1, 2, 3, or later, as appropriate, of the study. Data are presented as median and interquartile range (IQR). Complete tumor regression (CR) defined as a tumor volume <63 mm for 2 or more consecutive measurements 3 This is defined as the case where - The tolerability of the treatment was assessed by monitoring body weight gain, clinical signs of systemic toxicity, as well as evidence of local damage at the injection site.

[0182] PM060184 was provided in the form of freeze-dried vials of lyophilized product, each vial was reconstituted with water for infusion to a concentration of 2.5 mg / mL.

[0183] Compounds 2 and 3 were prepared in powder form and solubilized with hydroxy-propyl-β-cyclodextrin (HPβCD) to reach concentrations of 1 mg / ml or 0.2 mg / ml, respectively.

[0184] The placebo was provided in the form of a lyophilized cake containing 1.2 g of hydroxy-propyl-β-cyclodextrin (HPβCD), which was reconstituted with water for infusion.

[0185] In these experiments, compounds 2, 3 and PM060184, as well as placebo, were administered intravenously once weekly for three consecutive weeks, on days 0, 7 and 14 where possible.

[0186] Example 5 In vivo studies to determine the efficacy of compounds 2 and 3 in the NSCLC xenograft model, H460 Experiments were performed as previously described. Tumors were approximately 160-170 mm 3 When the tumor-bearing animals (N=10 / group) were randomly assigned to the following treatment groups:

[0187] [Table 8]

[0188] Treatment began on day 0.

[0189] Animals in the placebo group were randomly assigned to receive a 10-mg dose of 100 mg / kg of placebo for 12 days after tumor volume reduction (>2,000 mm 3 ) and / or were sacrificed due to tumor necrosis. In this experiment, H460 tumors had a doubling time of 2.7 days.

[0190] Treatment with compounds 2 and 3 caused severe but reversible mean weight loss in tumor-bearing animals. Compound 2 (2.0 mg / kg) treatment recorded mean weight loss (approximately -13.4%) on day 9, and compound 3 (1.0 mg / kg) recorded (approximately -18.5%) on day 9. When animals were treated with 16 mg / kg PM060184, moderate weight loss was recorded on day 12 (approximately -6.8%). All treated animals returned to normal when treatment was stopped. No other clinical signs of systemic toxicity were observed.

[0191] Tumor growth curves are shown in Figure 2. The administered treatments showed dose-dependent antitumor activity in animals bearing H460 tumor xenografts. Treatments administered at high or medium doses showed very strong or strong antitumor activity, respectively, while low doses showed limited or no antitumor activity in this model. 100% of animals treated with the highest dose of compound 2 (2.0 mg / kg) or compound 3 (1.0 mg / kg) had complete tumor remission during the experiment. The average time frame during which animals had tumor remission was 8.8 days for animals treated with compound 2 (2.0 mg / kg) and 7.8 days for animals treated with compound 3 (1.0 mg / kg). For the groups treated with 2.0 mg / kg and 0.5 mg / kg Compound 2, tumor regression lasted for more than 48 and 30.9 days, respectively, and for the group treated with 1.0 mg / kg Compound 3, tumor regression lasted for more than 45.1 days. Four of 10 animals treated with 16 mg / kg PM060184 experienced complete tumor regression lasting 2 days.

[0192] The placebo group had a mean mean score of 1241 (1025-1506) mm on the 7th day, respectively. 3 On days 7, 14, 21, 28, and 35, PM160184-treated animals had median (IQR) tumor volumes of 100.1 (80.6-119.0), 97.8 (74.5-149.3), 129.4 (83.5-172.3), 631.0 (454.0-873.9), and 1470 (1470-1666) mm, respectively. 3On the same day, compound 2-treated animals had median (IQR) tumor volumes of 114.7 (97.2-131.6), 80.9 (61.1-109.3), 53.5 (33.4-59.7), 95.0 (73.2-119.1), and 505.4 (428.9-583.7) mm, respectively. 3 On days 7, 14, 21, 28, and 35, compound 3-treated animals had median (IQR) tumor volumes of 123.9 (93.4-157.6), 80.3 (71.0-83.5), 59.3 (38.7-62.5), 77.6 (55.3-85.1), and 374.8 (257.4-438.2) mm, respectively. 3 The median tumor volume was 1.0 (IQR) of 1.0 mg / kg. Compared to placebo, PM060184, compound 2 and 3 treated animals administered at higher doses (16, 2 or 1 mg / kg, respectively) experienced highly potent and statistically significant tumor reductions on day 7, the last measurement day in the placebo-treated group, which was euthanized. Compared to PM060184 (16 mg / kg), compound 2 at 2.0 mg / kg produced more potent and statistically significant antitumor activity in this model, as did compound 3 at 1.0 mg / kg. When compounds 3 and 2 were compared after administration at the corresponding MTD (1.0 mg / kg or 2.0 mg / kg, respectively), the antitumor activity of compound 3 produced statistically significant greater antitumor activity than compound 2 in this model (Table 8).

[0193] [Table 9]

[0194] [Table 10]

[0195] Kaplan-Meier survival curves are shown in FIG.

[0196] Treatment with PM060184 at either dose statistically significantly (p<0.0001) increased survival time compared to the placebo-treated group, with median survival times of 37, 30, or 16 days, respectively, compared to 12 days for placebo treatment.

[0197] Compound 2 treatment at all doses assayed statistically significantly (p<0.0001) increased survival time compared to the placebo-treated group, with median survival times of 45.5, 40, or 14 days, respectively, compared to 12 days for placebo treatment. At the high and mid doses, Compound 2 treatment produced a statistically significant increase in survival time compared to the PM060184-treated group (p=0.0002 and P<0.0001).

[0198] Compound 3 treatment at 1.0 mg / kg (high dose), 0.25 mg / kg (medium dose) and 0.0625 mg / kg (low dose) statistically significantly increased survival time compared to the placebo treatment group, with median survival times of 45.5, 40 or 14 days compared to 12 days for placebo treatment (p<0.0001, p<0.0001 and p=0.0049, respectively). When comparing high, medium or low doses of compound 3 treatment with PM060184 treatment, compound 3 treatment groups produced statistically significant increases in survival time compared to the PM060184 treatment group at the high, medium and low doses, respectively (p<0.0001, p=0.0001 and p=0.0219).

[0199] Example 6 In vivo study to determine the effects of compounds 2 and 3 in the gastric xenograft model, HGC-27 Experiments were performed as previously described. Tumors were approximately 165-175 mm 3 When the tumor-bearing animals (N=8 / group) were randomly assigned to the following treatment groups:

[0200] [Table 11]

[0201] Treatment began on day 0.

[0202] Animals in the placebo group were randomly assigned to receive a 10-mg dose of 100 mg / kg of placebo for 14 days after tumor volume reduction (>2,000 mm 3 ) and / or were sacrificed due to tumor necrosis. In this experiment, HGC-27 tumors had a doubling time of 2.9 days.

[0203] Treatment with compounds 2 and 3 caused a severe but reversible mean weight loss in tumor-bearing animals. Compound 2 (2.0 mg / kg) treatment recorded a mean weight loss of approximately -13.1% on day 2, and compound 3 (1.0 mg / kg) recorded approximately -13.1% on day 2. When animals were treated with 16 mg / kg PM060184, a slight weight loss was recorded on day 2 (approximately -4.1%). All treated animals returned to normal when treatment was stopped. No other clinical signs of systemic toxicity were observed.

[0204] Tumor growth curves are shown in Figure 4. All treatments showed potent and dose-dependent antitumor activity in HGC-27 tumor xenografts. 100% of animals treated with the highest dose of compound 2 (2.0 mg / kg) or compound 3 (1.0 mg / kg) were in complete tumor remission during the experiment. The mean time frame during which animals treated with 2.0 mg / kg compound 2 remained in tumor remission was more than 29.1 days, and for animals treated with 1.0 mg / kg compound 3 it was more than 31.5 days, with the last observation day being day 63. In addition, 3 of 8 animals treated with 16 mg / kg (high dose) PM060184 achieved complete remission lasting an average of 18 days. The placebo-treated group had tumor remissions of 1139 (985.9-1364) and 2008 (2008-2008) mm on days 7 and 14, respectively. 3 On days 7, 14, 21, 28, and 35, animals treated with PM060184 at 16.0 mg / kg had median (IQR) tumor volumes of 250.7 (212.8-433.0), 124.8 (102.8-213.2), 88.5 (68.2-118.3), 114.4 (63.7-155.1), and 267.3 (100.4-414.0) mm, respectively. 3On the same day, animals treated with Compound 2 at 2.0 mg / kg had median (IQR) tumor volumes of 313.6 (237.3-482.4), 143.2 (96.1-223.6), 75.0 (51.6-75.0), 42.5 (37.0-63.9), and 32.9 (20.4-36.6) mm, respectively. 3 On days 7, 14, 21, 28, and 35, compound 3-treated animals at 1.0 mg / kg had median (IQR) tumor volumes of 366.0 (300.1-479.8), 177.1 (142.3-256.0), 75.0 (65.6-84.4), 66.7 (38.2-73.7), and 27.3 (21.4-41.4) mm, respectively. 3 The median (IQR) tumor volume was 1.01 mg / kg. Compared to placebo, all compound-treated animals at the highest dose experienced very strong and statistically significant tumor reductions on day 7, the last measurement day in the placebo-treated group, which was euthanized. No differences were observed between groups treated with PM060184 versus compounds 2 and 3 (high dose) during the treatment administration period, but once treatment was stopped, groups treated with 2 or 3 experienced large statistically significant differences in antitumor activity, p=0.0047 on day 28 and p=0.0070 on day 35 for the group treated with 2 mg / kg 2, and p=0.0379 on day 28 and p=0.0129 on day 35 for the group treated with 1 mg / kg 3, compared to the group treated with PM060184 at 16 mg / kg (Table 10).

[0205] [Table 12]

[0206] [Table 13]

[0207] Kaplan-Meier survival curves are shown in FIG.

[0208] PM060184 treatment at 16.0 mg / kg (p=0.0001), 4.0 mg / kg (p<0.0001), or 1.0 mg / kg (p=0.0103) statistically significantly increased survival time compared to the placebo treatment group.

[0209] Compound 2 treatment statistically significantly increased survival time at 2.0 mg / kg (p=0.0002), 0.5 mg / kg (p<0.0001) and 0.125 mg / kg (p=0.0281) compared to the placebo treatment group. When comparing high and medium doses of PM060184 treatment with compound 2 treatment, the compound 2 treatment group produced a statistically significant increase in survival time compared to the PM060184 treatment group (p=0.0007), and when comparing medium doses of compound 2 and 3 treatment, the compound 2 treatment group produced a statistically significant (p=0.0004) increase in survival time compared to the compound 3 treatment group. For clarity, the high dose lines for compound 2 and 3 are overlaid.

[0210] Compound 3 treatment statistically significantly increased survival time compared to the placebo treatment group at 1.0 mg / kg (p<0.0001) or 0.25 mg / kg (p<0.0001). Comparing compound 3 treatment with PM060184 treatment, both at high doses, compound 3 treatment groups produced a statistically significant (p=0.0007) increase in survival time compared to the PM060184 treatment group at the high, medium, and low doses.

[0211] Example 7 In vivo studies to determine the efficacy of compounds 2 and 3 in the colon xenograft model, HCT-116 Experiments were performed as previously described. Tumors were approximately 195-205 mm 3 When the tumor-bearing animals (N=10 / group) were randomly assigned to the following treatment groups:

[0212] [Table 14]

[0213] Treatment began on day 0.

[0214] Animals in the placebo group were randomly assigned to receive a 10-mg dose of 100 mg / kg of placebo for treatment with a tumor volume >2,000 mm between days 12 and 19. 3 ) and / or were sacrificed due to tumor necrosis. In this experiment, HCT-116 tumors had a doubling time of 3.9 days.

[0215] Treatment with compounds 2 and 3 caused a severe but reversible mean weight loss in tumor-bearing animals. Compound 2 (2.0 mg / kg) treatment recorded a mean weight loss on day 9 (approximately -13.5%) and compound 3 (1.0 mg / kg) recorded a mean weight loss on day 9 (approximately -16.2%). When animals were treated with 16 mg / kg PM060184, a slight weight loss was recorded on day 2 (approximately -3.2%). All treated animals returned to normal values ​​when treatment was terminated. No other clinical signs of systemic toxicity were observed.

[0216] Tumor growth curves are shown in Figure 6. All treatments showed potent and dose-dependent antitumor activity in HGC-27 tumor xenografts. During the experiment, 100% of animals treated with the highest dose of compound 2 (2.0 mg / kg) or compound 3 (1.0 mg / kg) were in complete tumor remission. The average time frame during which animals treated with 2.0 mg / kg compound 2 remained in tumor remission was 25.8 days, and 23.7 days for animals treated with 1.0 mg / kg compound 3. Moreover, 6 / 10 animals treated with 16 mg / kg (high dose), 4 / 10 animals treated with 0.5 mg / kg (medium dose) compound 2, and 1 / 10 animals treated with 0.25 mg / kg (medium dose) compound 3 reached complete remission lasting 5.8 days, 9 days, and 2 days, respectively. The placebo group had mean mean scores of 836.3 (737.2-911.9) and 1299 (1141-1420) mm on days 7 and 14, respectively. 3On days 7, 14, 21, 28, and 35, animals treated with PM060184 at 16.0 mg / kg had median (IQR) tumor volumes of 140.5 (123.0-157.3), 59.2 (50.7-84.2), 63.8 (43.6-80.0), 265.7 (195.2-284.3), and 650.2 (516.7-732.6) mm, respectively. 3 On the same day, animals treated with Compound 2 at 2.0 mg / kg had median (IQR) tumor volumes of 135.5 (114.1-154.5), 57.2 (47.1-62.9), 25.0 (13.5-34.0), 13.5 (13.5-32.0), and 13.5 (13.5-13.5) mm, respectively. 3 On days 7, 14, 21, 28, and 35, compound 3-treated animals at 1.0 mg / kg had median (IQR) tumor volumes of 121.6 (86.3-164.8), 55.6 (16.2-82.3), 32.0 (4.0-40.0), 13.5 (4.0-13.5), and 13.5 (13.5-13.5) mm, respectively. 3 The median (IQR) tumor volume was 1.11 mm. Compared to placebo, all compound-treated animals at the highest dose experienced very strong and statistically significant tumor reductions from day 7 through day 14, the last measurement day in the placebo-treated group, when the animals were euthanized. No differences were observed between groups treated with PM060184 versus compounds 2 and 3 (high dose) during the treatment administration period, but upon termination of treatment, groups treated with 2 or 3 experienced a statistically significant large difference in antitumor activity, p=0.0002 from days 21 to 35 for the group treated with 2 mg / kg compound 2, and p=0.0005 on day 21, p=0.0002 on day 28, and p<0.0001 on day 35 for the group treated with 1 mg / kg compound 3, compared to the 16 mg / kg PM060184-treated group (Table 12).

[0217] [Table 15]

[0218] [Table 16]

[0219] Kaplan-Meier survival curves are shown in FIG.

[0220] Treatment with PM060184 at 16.0 mg / kg (p<0.0001), 4.0 mg / kg (p<0.0001), or 1.0 mg / kg (p=0.0047) statistically significantly increased survival time compared to the placebo treatment group. The median survival time for placebo treatment was 17.5 days compared to 47, 43, or 19 days for the PM060184 treatment groups (high, medium, or low doses, respectively).

[0221] Compound 2 treatment statistically significantly increased survival time compared to the placebo treatment group at 2.0 mg / kg (p<0.0001), 0.5 mg / kg (p<0.0001), but not at 0.125 mg / kg. When comparing both high and medium doses of PM060184 treatment with compound 2 treatment, the compound 2 treatment group produced a statistically significant increase in survival time compared to the PM060184 treatment group (p=0.0012 or p<0.0001, respectively), and when comparing medium and low doses of compound 2 and 3 treatment, the compound 2 treatment group produced a statistically significant increase in survival time compared to the compound 3 treatment group, p=0.0033 and p=0.0070, respectively.

[0222] Compound 3 treatment statistically significantly increased survival time compared to the placebo-treated group at 1.0 mg / kg (p<0.0001), 0.25 mg / kg (p<0.0001), but not at 0.125 mg / kg. Comparing high and mid doses of Compound 3 treatment with PM060184 treatment, the Compound 3 treatment group produced a statistically significant increase in survival time compared to the PM060184 treatment group, p<0.0001 and p=0.0076, respectively.

[0223] In conclusion, it can be seen that both compounds 2 and 3 have surprisingly improved activity over the prior art compounds PM060184 and PM050489.

[0224] In vitro, compound 2 is 3.8-8.2 times more active than PM060184, and compound 3 is 1.3-3.7 times more active than PM060184 across A549, HT29, MDA-MB-231 and PSN-1 cell lines. This can be seen in Example 3.

[0225] In vivo, compounds 2 and 3 show significant efficacy improvement over PM060184 across a wide range of cancer xenograft models. In H460, HGC27 and HCT116 xenograft models (models of non-small cell lung cancer, gastric cancer and colorectal cancer), both compounds 2 and 3 show statistically significant improvement in antitumor activity as well as survival time across all three cell lines. See Examples 5-7.

[0226] This is also shown for PM050489 in an in vitro bioassay in Example 3, where it can be seen that both 2 and 3 have improved in vitro activity over the prior art compound PM050489. Compound 2 is 1.9-7.9 times more active than the reference compound PM050489. Compound 3 is 1.3-1.8 times more active than the reference compound PM050489. This is seen across A549, HT29, MDA-MB-231 and PSN-1 cell lines. This is also shown in in vivo xenograft models, with significantly improved efficacy over PM050489 in the H460 xenograft model. This is seen in Example 3 for in vitro data and in the Comparative Examples and Example 5 for in vivo data.

[0227] (References) TIFF2024539180000054.tif214157

Claims

1. Formula I 【Chemical 1】 (wherein X=F or Me) Compound.

2. Formula II 【Chemistry 2】 The compound of claim 1.

3. Formula III 【Chemistry 3】 The compound of claim 1.

4. 10. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier.

5. A dosage form comprising the pharmaceutical composition of claim 4.

6. A pharmaceutical composition for treating cancer, comprising a compound according to any one of claims 1 to 3.

7. The pharmaceutical composition according to claim 6, wherein the cancer is a solid tumor.

8. The pharmaceutical composition described in claim 7, wherein the cancer is selected from lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, pancreatic cancer and gastric cancer.

9. An in vitro method for inhibiting cancer cell proliferation, comprising contacting cancer cells with a compound according to any one of claims 1 to 3, or a pharmaceutical composition according to claim 4, or a dosage form according to claim 5.

10. The in vitro method of claim 9, wherein the cancer cells are solid tumor cells.

11. The in vitro method of claim 10, wherein the cancer cells are selected from lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, pancreatic cancer and gastric cancer.

12. 10. A kit comprising a therapeutically effective amount of a compound according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier, or a pharmaceutical composition according to claim 4, or a dosage form according to claim 5.

13. The kit of claim 12, further comprising instructions for using the compound in the treatment of cancer.

14. A process for the preparation of a compound of formula I, comprising reacting a compound of formula V with a suitable reagent for the synthesis of a carbamate to form a compound of formula I 【Chemistry 4】 (wherein X=F or Me) obtaining a compound of formula (I):

15. Compounds of formula V, VII, IX or XI useful for preparing compounds of formula I 【Chemistry 5】 (In the formula, R 3 is unsubstituted C 1 ~C 4 is an alkyl group, ProtOH is a protecting group for the hydroxy, and X is defined for formula I. An intermediate compound.

16. Formula V 【Chemistry 6】 (wherein X=F or Me) 15. The intermediate compound of claim 14,

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