Patent Application
20050004211
1. Field of the Invention
This invention relates to pharmaceutical compositions comprising aryl-substituted acyclic enediyne compounds, in particular 6-aryl-hexen-1,5-diynes and 1,6-diaryl-hexen-1,5-diynes, which are found to have inhibitory activities against topoisomerase I or act as a S phase or G2/M phase blocker.
2. Description of the Related Art
A series of alkaloids containing enediyne cores which were isolated from Streptomyces, have a manifold of biological activities (Walkers, S. ; Valentine, K. G. ; Kahne, D. J. Am. Chem. Soc. 1990, 112, 6428; Dark, L., Iwasawa, N., Danishefsky, S., Crother, D. M., Proc. Natl. Acad. Sci. USA. 1991, 88, 7464; Povirk, L. F. ; Goldberg, I. H.; Biochemistry. 1980, 19, 4773; and Kappen, L. S., Goldberg, I. H., Nucleic Acid. Res. 1978, 5, 2959) owing to the generation of radicals. Several biologically active synthetic enediynes are also observed in the formation of radicals. However, besides formation of biradical intermediates, little attentions has been paid to other feasible reaction modes by which enediynes could act and the relative biological activities that enediynes could exhibit, in spite of reports of novel biradical reactions that have begun to surface (Wendi, D. M. ; Kerwin, S. M. J Am. Chem. Soc. 1997, 119, 1464; Tarli, A., Wang, K. K., J. Org. Chem. 1997, 62, 8841; and Xu, S. L., Moore, H. W., J. Org. Chem. 1992, 57, 326).
Generation of Radicals from Enediynes
Topoisomerases are important enzymes highly associated with the separation of DNA strands in many cellular metabolic progresses by altering the topological state of duplex DNA. Topoisomerases can be classified into two types based on their mode of cleaving duplex DNA (J. C. Wang, Annu. Rev. Biochem. 1996, 65, 635): topo I makes a transient nick on a single-strand of DNA and does not require an energy cofactor (M. Gupta, A. Fujimori, Y Ponmmier, Biochem. Biophys. Acta. 1995, 1262, 1), whereas topo II acts by nicking both strand of the DNA and hydrolyzes ATP during its catalytic cycle (J. C. Wang, Annu. Rev. Biochem. 1985, 54, 665-697; and S. J. Froelich-Ammon, N. Osheroff, J. Biol. Chem. 1995, 270, 21429). Topoisomerase I can be isolated from some cellular organisms, including nucleus and mitochondria. Moreover, topo I is present throughout the cell, and its activity varies less than topo II during cell cycle (M. M. Heck, W. N. Hittelman, W C. Earnshaw, Proc. Natl. Acad. Sci. USA. 1988, 85, 1086), which makes topo I inhibitor an attractive target of anticancer, antibacterial, and antiviral drug development.
Although a series of synthetic acyclic enediynes, i.e. 2-(6-substituted-3-hexen-1,5-diynyl)benzonitriles, provided the cytotoxicities on KB and Hep2,2,15 and significant topo I inhibitory properties (C. F. Lin, P. C. Hsieh, W. D. Lu, H. F. Chiu, M. J. Wu, J. Bioorg. Med. Chem. 2001, 9, 1707) in low micro-molar concentration ranges (see the following Scheme 1), the precision relationships between the cytotoxicities and these unique structures were still under investigation. This prompted us to further investigate the structure-activity-relationships (SAR) of new enediyne compounds.
On the other hand, in our earlier research work, we reported that treatment of 2-(6-substituted-3-hexen-1,5-diynyl)benzonitriles with sodium methoxide in methanol at reflux gives phenanthridinones and substituted biphenyl derivatives (M.-J. Wu et al. (1999), Org. Lett., 1, 767). Although the chemical yields are low, this reaction constitutes a novel cycloaromatization of enediynes (for examples, for nonradical cycloaromatization of enediynes, reference may be made to P. Magnus et al. (1993), J. Am. Chem. Soc., 115, 12627 and H. Sugiyama et al. (1992), Tetrahedron Lett. 1992, 33, 515) and a new synthetic approach to phenanthridinones and biphenyls.
Since phenanthridinones (Lewis, J. R. (1997), Nat. Prod. Rep., 14, 303), biphenyls (Bringmann, G.; Water, R.; Weirich, R. Angew. Chem., Int. Ed. Engl. 1990, 29, 977; Acton, U.; Goltner, C.; Mullen, K. Chem. Ber. 1992, 125, 2325; Rosin, C.; Franzin, L.; Rafaeli, A.; Salvadori, P. Synthesis 1992, 6, 503; Tamao, K.; Yamamoto, H.; Matsumoto, H.; Miyake, N.; Hayashi, Y.; Kumada, M. Tetrahedron Lett. 1977, 47, 1389; and Miyashita, A.; Karino, H.; Shimamura, J. I.; Chiba, T.; Nagano, K.; Nohira, H.; Takaya, H. Chem. Lett. 1989, 1849), and structurally related compounds are of current interest in the pharmaceutical area and for the preparation of new materials, methods for the selective preparation of these compounds have considerable value.
A proposed mechanism for the formation of phenanthridinones is methoxide addition to the cyano group, followed by an anionic cascade cycloaromatization. In contrast, methoxide addition to C2 of the acetylenic moiety and the same type of cycloaromatization will give biphenyls (see the following Scheme 2).
We believe that the low yields in these reactions are due to poor regioselectivity in the nucleophilic addition of methoxide to the conjugated system. Thus, we anticipated that introducing a polar aprotic solvent into the reaction mixture would increase the nucleophilicity and the hardness of the nucleophile and therefore promote regioselectivity in the nucleophilic addition step to increase the chemical yield.
Accordingly, in the first aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I):
The compounds of formula (I) are found to have cytotoxicity against tumor/cancer cells, such as leukemia cancer cells, non-small-cell lung cancer cells, colon cancer cells, CNS cancer cells, melanoma cancer cells, ovarian cancer cells, renal cancer cells, prostate cancer cells and breast cancer cells, in particular human oral epidermoid carcinoma cell, human cevix epitheloid carcinoma cell, human colon adenocarcinoma cell, human lung large cell carcinoma cell, and human hepatoma cell. Therefore, in the second aspect, the present invention provides a method for inhibiting the growth of tumor/cancer cells in a subject, comprising administering to a subject in need of such treatment a therapeutically effective amount of the pharmaceutical composition as described above.
The compounds of formula (I) are found to have inhibitory activities against topoisomerase I or act as a S phase or G2/M phase blocker. Therefore, the pharmaceutical composition of this invention may be used to treat a subject afflicted with a tumor/cancer by inhibiting topoisomerase I activities or blocking the S phase or G2/M phase of the tumor/cancer cells, or in the formulation of an anticancer, antibacterial or antiviral drug.
The above and other features and advantages of the present invention will become apparent in the following detailed description of the preferred embodiments with reference to the accompanying drawing, of which:
In our earlier research work (C. F. Lin et al. (2001), J. Bioorg. Med. Chem., 9, 1707-1711), we screened a series of 2-(6-substituted-3(Z)-hexen-1,5-diynyl)benzonitriles which showed potent cytotoxicity with KB cell (see the following Table 1). The high cytotoxicity of these 2-(6-substituted-3(Z)-hexen-1,5-diynyl) benzonitriles promoted our further studies toward this unexpected biological behavior.
It is considered that these molecules comprise an enediyne structure, but unlike the accustomed mode of enediynes (Jones, R. R., Bergman, R. G., J. Am. Chem. Soc. 1972, 94, 660; Bergman, R. G., Acc. Chem. Res. 1973, 6, 25; Lockhart, T. P., Comita, P B., Bergman, R. G., J. Am. Chem. Soc. 1981, 103, 4082; Myers, A. G., Kuo, E. Y, Finny, N. S., J. Am. Chem. Soc. 1989, 111, 8057; and Myers, A. G., Tetrahedron Lett. 1987, 28, 4493) which generate biradicals and facilitate the decease of cells (scheme A), there is no initiating factor to promote the enediynes to form active biradicals via Myers cycloaromatization (path a). Similarly, according to Bergman's reports, the acyclic enediynes cannot cycloaromatize to produce biradical intermediates under 37° C. (path b).
From an overview of the possible mechanisms which anticancer drugs would proceed, the most probable pathway of 2-(6-substituted-3(Z)-hexen-1,5-diynyl)benzonitriles to induce the death of cancer cells is the physiological enzyme inhibitors, especially the topological enzymes, i.e. the topoisomerase, which are essential for DNA replication, transcription, repair, recombination, and chromosome segregation (Champoux, J. J. in DNA Topology and its Biological Effects, Wang, J. C. and Cozzarelli, N. R. Eds. (Cold Spring Harbor Labortory, Cold Spring Harbor, N. Y., 1990). pp217-242; and Wang, J. C., Annu. Rev. Biochem. 1996, 65, 635). Exploration of the requisite fundamental mainstay of these compounds and the precision relationships between the cytotoxicities and these unique structures are still under investigation.
To further investigate the biological activities of 2-(6-substituted-3(Z)-hexen-1,5-diynyl)benzonitriles, new aryl-substituted acyclic enediyne compounds, in particular 6-aryl-hexen-1,5-diynes and 1,6-diaryl-hexen-1,5-diynes, were designed and synthesized by modification of the enediyne core and the aryl group(s) bearing on it, and their cytotoxicities were evaluated in terms of cytotoxic activities against human solid tumor cells, the topoisomerase I inhibitory activities and the cell cycle analysis.
Therefore, this invention provides a pharmaceutical composition comprising a compound of formula (I)
In a preferred embodiment of this invention, the compound of formula (I) is one where both R1 and R2 are H.
In another preferred embodiment of this invention, the compound of formula (I) is one where R1 and R2 together form a moiety represented by the formula
Preferably, the compound of formula (I) is one where R3 represents a substituted or unsubstituted alkyl having 4-20 carbon atoms, and more preferably 4-10 carbon atoms, or an aryl group having 3-20 carbon atoms, and more preferably 3-10 carbon atoms.
In a preferred embodiment of this invention, R3 represents: butyl, t-butyl, pentyl, hexyl, heptyl or octyl, each of which is optionally substituted with one to three substituents selected from the group consisting of halo, cyano, amino, nitro, carbonyl, carboxyl, hydroxy, C1-C6 alkyl, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 alkanoyl, and C1-C6 alkanoyloxy; thienyl, pyridinyl, piperidyl, pyrazinyl, cyclohexenyl, triisopropylsilyl, pyrrolyl, pyrrolidinyl, pyrrolinyl, pyridazinyl, pyrimidinyl, furanyl, uracilyl or pyrazolyl, each of which is optionally substituted with one to three substituents selected from the group consisting of halo, cyano, amino, nitro, carbonyl, carboxyl, hydroxy, C1-C6 hydroxyalkyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 alkanoyl, C1-C6 alkanoyloxy and phenyl; (C5-C8 aryl)oxyalkyl, such as tetrahydropyranyloxyalkyl; or a phenyl group which is unsubstituted or substituted with one to three substituents selected from the group consisting of halo, cyano, amino, nitro, carbonyl, carboxyl, hydroxy, hydroxycarbonyl, C1-C6 alkyl, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 alkanoyl, C1-C6 alkanoyloxy and t-butyldimethylsilyloxy.
In a more preferred embodiment of this invention, R3 represents: butyl, t-butyl, pentyl, tetrahydropyranyloxyalkyl, 2-thienyl, 3-thienyl, 5-methylthienyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-pyrazinyl, pyrazinyl, pyrazolyl, 3-pyridazinyl, 2-furanyl, 3-furanyl, 2-uracilyl, 2,4-dimethylpyrimidinyl, 1-(4-phenyl)pyrrolyl, 1-(4-phenyl)pyrrolidinyl, o-cyanophenyl, m-cyanophenyl, p-cyanophenyl, o-cyanomethylphenyl, m-cyanomethylphenyl, p-cyanomethylphenyl, p-chlorophenyl, 2-acetylphenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-formylphenyl, 3-formylphenyl, 4-formylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-anilinyl, 4-anilinyl, 3-aminomethylphenyl, 2-chloromethylphenyl, 1,3,5-trichlorophenyl, 2-hydroxycarbonylphenyl, 3-hydroxycarbonylphenyl, 4-hydroxycarbonylphenyl, 2-methylhydroxylphenyl, 3-methylhydroxylphenyl, 4-methylhydroxylphenyl, p-trifluoromethylphenyl, m-trifluoromethylphenyl, o-trifluoromethylphenyl, 2,3-dimethylphenyl or 2-thioanisyl.
In a further preferred embodiment of this invention, R3 is an aryl group selected from o-cyanophenyl, m-cyanophenyl, p-cyanophenyl, p-trifluoromethylphenyl, m-trifluoromethylphenyl, o-trifluoromethylphenyl,
Preferably, the compound of formula (I) is one where R4 represents a substituted or unsubstituted aryl group having 3-20 carbon atoms, and more preferably 3-10 carbon atoms.
In a preferred embodiment of this invention, R4 represents: thienyl, pyridinyl, piperidyl, pyrazinyl, cyclohexenyl, triisopropylsilyl, pyrrolyl, pyrrolidinyl, pyrrolinyl, pyridazinyl, pyrimidinyl, furanyl, uracilyl or pyrazolyl, each of which is optionally substituted with one to three substituents selected from the group consisting of halo, cyano, amino, nitro, carbonyl, carboxyl, hydroxy, C1-C6 hydroxyalkyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 alkanoyl, C1-C6 alkanoyloxy and phenyl; (C5-C8 aryl)oxyalkyl, such as tetrahydropyranyloxyalkyl; or a phenyl group which is unsubstituted or substituted with one to three substituents selected from the group consisting of halo, cyano, amino, nitro, carbonyl, carboxyl, hydroxy, hydroxycarbonyl, Cl-C6 alkyl, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 alkanoyl, C1-C6 alkanoyloxy and t-butyldimethylsilyloxy; or a group represented by the formula
where n=1 or 2.
In a more preferred embodiment of this invention, R4 represents: tetrahydropyranyloxyalkyl, 2-thienyl, 3-thienyl, 5-methylthienyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-pyrazinyl, pyrazinyl, pyrazolyl, 3-pyridazinyl, 2-furanyl, 3-furanyl, 2-uracilyl, 2,4-dimethylpyrimidinyl, 1-(4-phenyl)pyrrolyl, 1-(4-phenyl)pyrrolidinyl, o-cyanophenyl, m-cyanophenyl, p-cyanophenyl, o-cyanomethylphenyl, m-cyanomethylphenyl, p-cyanomethylphenyl, p-chlorophenyl, 2-acetylphenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-formylphenyl, 3-formylphenyl, 4-formylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-anilinyl, 4-anilinyl, 3-aminomethylphenyl, 2-chloromethylphenyl, 1,3,5-trichlorophenyl, 2-hydroxycarbonylphenyl, 3-hydroxycarbonylphenyl, 4-hydroxycarbonylphenyl, 2-methylhydroxylphenyl, 3-methylhydroxylphenyl, 4-methylhydroxylphenyl, p-trifluoromethylphenyl, m-trifluoromethylphenyl, o-trifluoromethylphenyl, 2,3-dimethylphenyl, 2-thioanisyl, or a group represented by the formula
where n=1 or 2.
In a further preferred embodiment of this invention, R4 is an aryl group selected from: o-cyanophenyl, m-cyanophenyl, p-cyanophenyl, p-trifluoromethylphenyl, m-trifluoromethylphenyl, o-trifluoromethylphenyl,
represented by the formula
where n=1 or 2.
In a preferred embodiment of this invention, the compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof is one having the following formula (IA):
In a further preferred embodiment of this invention, the compound of formula (IA) is one where R3 is an aryl group selected from 2-thienyl, 3-thienyl, 5-methylthienyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-pyrazinyl, pyrazinyl, pyrazolyl, 3-pyridazinyl, 2-furanyl, 3-furanyl, 2-uracilyl, 2,4-dimethylpyrimidinyl, 1-(4-phenyl)pyrrolyl, 1-(4-phenyl)pyrrolidinyl, o-cyanophenyl, m-cyanophenyl, p-cyanophenyl, o-cyanomethylphenyl, m-cyanomethylphenyl, p-cyanomethylphenyl, p-chlorophenyl, 2-acetylphenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-formylphenyl, 3-formylphenyl, 4-formylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-anilinyl, 4-anilinyl, 3-aminomethylphenyl, 2-chloromethylphenyl, 1,3,5-trichlorophenyl, 2-hydroxycarbonylphenyl, 3-hydroxycarbonylphenyl, 4-hydroxycarbonylphenyl, 2-methylhydroxylphenyl, 3-methylhydroxylphenyl, 4-methylhydroxylphenyl, p-trifluoromethylphenyl, m-trifluoromethylphenyl, o-trifluoromethylphenyl, 2,3-dimethylphenyl, and 2-thioanisyl.
In a further more preferred embodiment of this invention, the compound of formula (IA) is one where R3 is an aryl group selected from o-cyanophenyl, m-cyanophenyl, p-cyanophenyl, p-trifluoromethylphenyl, m-trifluoromethylphenyl, o-trifluoromethylphenyl,
In a preferred embodiment of this invention, the compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof is one having the following formula (IB):
In a more preferred embodiment of this invention, the compound of formula (IB) is one where R3 represents: butyl, t-butyl, pentyl, tetrahydropyranyloxyalkyl, 2-thienyl, 3-thienyl, 5-methylthienyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-pyrazinyl, pyrazinyl, pyrazolyl, 3-pyridazinyl, 2-furanyl, 3-furanyl, 2-uracilyl, 2,4-dimethylpyrimidinyl, 1-(4-phenyl)pyrrolyl, 1-(4-phenyl)pyrrolidinyl, o-cyanophenyl, m-cyanophenyl, p-cyanophenyl, o-cyanomethylphenyl, m-cyanomethylphenyl, p-cyanomethylphenyl, p-chlorophenyl, 2-acetylphenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-fornylphenyl, 3-formylphenyl, 4-formylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-anilinyl, 4-anilinyl, 3-aminomethylphenyl, 2-chloromethylphenyl, 1,3,5-trichlorophenyl, 2-hydroxycarbonylphenyl, 3-hydroxycarbonylphenyl, 4-hydroxycarbonylphenyl, 2-methylhydroxylphenyl, 3-methylhydroxylphenyl, 4-methylhydroxylphenyl, p-trifluoromethylphenyl, m-trifluoromethylphenyl, o-trifluoromethylphenyl, 2,3-dimethylphenyl or 2-thioanisyl.
In a further more preferred embodiment of this invention, the compound of formula (IB) is one where R3 is butyl, pentyl, tetrahydropyranyloxymethyl or tetrahydropyranyloxypropyl, or an aryl group selected from o-cyanophenyl, m-cyanophenyl, p-cyanophenyl, p-trifluoromethylphenyl, m-trifluoromethylphenyl, o-trifluoromethylphenyl,
In a preferred embodiment of this invention, the compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof is one having the following formula (IC):
In a more preferred embodiment of this invention, the compound of formula (IC) is one where R4 represents: tetrahydropyranyloxyalkyl, 2-thienyl, 3-thienyl, 5-methylthienyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-pyrazinyl, pyrazinyl, pyrazolyl, 3-pyridazinyl, 2-furanyl, 3-furanyl, 2-uracilyl, 2,4-dimethylpyrimidinyl, 1-(4-phenyl)pyrrolyl, 1-(4-phenyl)pyrrolidinyl, o-cyanophenyl, m-cyanophenyl, p-cyanophenyl, o-cyanomethylphenyl, m-cyanomethylphenyl, p-cyanomethylphenyl, p-chlorophenyl, 2-acetylphenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-formylphenyl, 3-formylphenyl, 4-formylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-anilinyl, 4-anilinyl, 3-aminomethylphenyl, 2-chloromethylphenyl, 1,3,5-trichlorophenyl, 2-hydroxycarbonylphenyl, 3-hydroxycarbonylphenyl, 4-hydroxycarbonylphenyl, 2-methylhydroxylphenyl, 3-methylhydroxylphenyl, 4-methylhydroxylphenyl, p-trifluoromethylphenyl, m-trifluoromethylphenyl, o-trifluoromethylphenyl, 2,3-dimethylphenyl, 2-thioanisyl, or a group represented by the formula
where n=1 or 2.
In a further preferred embodiment of this invention, the compound of formula (IC) is one where R4 is an aryl group selected from: o-cyanophenyl, m-cyanophenyl, p-cyanophenyl, p-trifluoromethylphenyl, m-trifluoromethylphenyl, o-trifluoromethylphenyl,
or a group represented by the formula
where n=1 or 2.
In a further preferred embodiment of this invention, the compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof is one having the following formula (ID):
In a more preferred embodiment of this invention, the compound of formula (ID) is one where Ar1 and Ar2 independently represent: tetrahydropyranyloxyalkyl, 2-thienyl, 3-thienyl, 5-methylthienyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-pyrazinyl, pyrazinyl, pyrazolyl, 3-pyridazinyl, 2-furanyl, 3-furanyl, 2-uracilyl, 2,4-dimethylpyrimidinyl, 1-(4-phenyl)pyrrolyl, 1-(4-phenyl)pyrrolidinyl, o-cyanophenyl, m-cyanophenyl, p-cyanophenyl, o-cyanomethylphenyl, m-cyanomethylphenyl, p-cyanomethylphenyl, p-chlorophenyl, 2-acetylphenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-formylphenyl, 3-formylphenyl, 4-formylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-anilinyl, 4-anilinyl, 3-aminomethylphenyl, 2-chloromethylphenyl, 1,3,5-trichlorophenyl, 2-hydroxycarbonylphenyl, 3-hydroxycarbonylphenyl, 4-hydroxycarbonylphenyl, 2-methylhydroxylphenyl, 3-methylhydroxylphenyl, 4-methylhydroxylphenyl, p-trifluoromethylphenyl, m-trifluoromethylphenyl, o-trifluoromethylphenyl, 2,3-dimethylphenyl or 2-thioanisyl.
In a more preferred embodiment of this invention, the compound of formula (ID) is one where Ar1 and Ar2 independently represent an aryl group selected from o-cyanophenyl, m-cyanophenyl, p-cyanophenyl, p-trifluoromethylphenyl, m-trifluoromethylphenyl, o-trifluoromethylphenyl,
In a most preferred embodiment of this invention, the compound of formula (ID) is one where Ar1 and Ar2 are identical and represent an aryl group selected from o-cyanophenyl, m-cyanophenyl, p-cyanophenyl, p-trifluoromethylphenyl, m-trifluoromethylphenyl, o-trifluoromethylphenyl,
In a still further preferred embodiment of this invention, the compound of formula (I) is
where n is 1 or 2.
According to this invention, the compounds of formula (I) may be in their free form or in the form of a pharmaceutically acceptable salt or solvate thereof.
Illustrative pharmaceutically acceptable salts include metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, manganese salt, iron salt and aluminum salt; mineral acid addition salts such as hydrochloride, hydrobromide, hydroiodide, sulfate and phosphate; organic acid addition salts such as benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, oxalate, maleate, fumarate, tartrate and citrate; and those with amino acids, such as arginine, aspartic acid and glutamic acid.
In addition, the compound of formula (I) of the present invention may also exist as a stereoisomer or in the form of solvates represented by the hydrate. Therefore, it is contemplated that these stereoisomers and solvates fall within the technical concept of the present invention.
This invention also provides a convenient and efficient process for producing a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof:
According to this invention, acyclic enediyne compounds 1-43 of formula (I) having structures shown below can be produced from the process of this invention using a corresponding 1,2-disubstituted-dihaloethene compound A1 of formula (IIA) as a starting material based on the following synthesis scheme 3. Coupling reaction (K. Sonogashira et al. (1997), Chem. Comm., 291) of the compound A1 with 2-substitutedacetylene (R3=alkyl, aryl) using palladium as a catalyst in an ether solution at 25° C. for 4 hours gave compound A2. Likewise, compound A2 was coupled with trimethylsilylacetylene to give compound A3. Treatment of compound A3 with TBAF in THF solution produced the desilylated compound A4. Finally, acyclic enediyne compounds 1-43 are produced from palladium-catalyzed coupling reaction of corresponding compounds A4 with various aryl iodides.
Alternatively, compounds of formula (IB), such as compounds 24-33 listed above, may be produced according to the following synthesis scheme 4, in which the percent yield of each compound is parenthesized.
THP = 3,4,5,6-Tetrahydro-2H-pyran-2-yl
Alternatively, compounds of formula (ID) of this invention, such as compounds 34-43 listed above, may be produced from the coupling reaction of 1,2-disubstituted-dihaloethene with a selected 2-aryl-1-ethyne according to the following synthesis scheme 5:
The compounds of formula (I) according to this invention have been 10 demonstrated to exhibit inhibitory activities against the growth of several solid tumor cell lines, in particular human oral epidermoid carcinoma cell, human cervix epitheloid carcinoma cell, human colon adenocarcinoma cell, human lung large cell carcinoma cell, and human hepatoma cell. Further in vitro tests show that the compounds of formula (I) may inhibit the activity of topoisomerase I or block the S phase or G2/M phase of tumor cells' cell cycle. Therefore, the present invention envisions the application of the compounds of formula (I) in the manufacture of pharmaceutical compositions.
Optionally, the pharmaceutical composition according to this invention may additionally comprise a pharmaceutically acceptable carrier widely employed in the art for the manufacture of medicaments. For example, the pharmaceutically acceptable carrier can include one or more than one of the following reagents: solvents, disintegrating agents, binders, excipients, lubricants, absorption delaying agents and the like.
The pharmaceutical composition according to this invention may be administered parenterally or orally in a suitable pharmaceutical form. Suitable pharmaceutical forms include sterile aqueous solutions or dispersions, sterile powders, tablets, troches, pills, capsules, and the like.
In addition, the active compounds of the present invention may be incorporated into sustained-release preparations and formulations. Optionally, the pharmaceutical composition according to this invention may be administered alone or in conjunction with an additional anticancer agent, such as such as Mitomycin, Adriamycin, Actinomycin, cis-platin and the like.
In addition, the compounds of formula (I) may be used in the synthesis of phenanthridinones, benzo[c]phenanthridinones and biaryls, which in turn may be developed into useful pharmaceuticals, by anionic cycloaromatization of said compounds with sodium methoxide in refluxing methanol in the presence of a polar aprotic solvent, such as DMSO, HMPA, THF, or 18-crown-6, according to schemes described in M. J. Wu et al. (2002), J. Org. Chem., 67, 5907-5912, the whole disclosure of which is incorporated herein by reference.
The present invention will be described in more detail with reference to the following examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the present invention.
Method A:
To a degassed solution of (trimethylsilyl)acetylene (12 mmole) in Et2O (25 mL) containing CuI (3.2 mmole) and n-BuNH2 (34 mmole) in Et2O (25 mL) was added a degassed solution of cis-1,2-dichloroethylene or 1,2-diiodobenzene (12 mmole) containing Pd(PPh3)4 (0.8 mmole) in Et2O (25 mL). The resulting reaction mixture was stirred for 6 hrs, and quenched with saturated aqueous NH4Cl solution. The aqueous layer was extracted with EtOAc (50 mL), and the combined organic extracts were washed with saturated aqueous Na2CO3 solution (40 mL) and dried over anhydrous MgSO4. After filtration and removal of solvent in vacuo, the residue was purified by column chromatography on silica gel (hexanes as eluent) to give the desired products.
Method B:
To a stirred solution of 2-(2-trimethylsilyl-1-ethynyl)iodobenzene (5.6 mmol) in dry DMF (40 mL) was added Pd(PPh3)4 (0.22 mmol), followed by Zn(CN)2 (1.25 mmol). The resulting reaction mixture was degassed, heated to reflux and stirred for 5 hrs. After cooling to room temperature, the reaction mixture was quenched with 2 N aqueous NH4OH and extracted with EtOAc. The combined organic extracts were washed with brine and dried over anhydrous MgSO4.
Method C:
After filtration and removal of solvent, K2CO3 (5 eq) was then added to a stirred solution of 2-(2-trimethylsilyl-1-ethynyl)benzonitrile in dry MeOH (20 mL). The resulting reaction mixture was degassed and stirred for 2 hrs at room temperature. After filtration and removal of solvent, the reaction mixture was quenched with saturated aqueous NaHCO3 solution. The aqueous layer was extracted with EtOAc. The combined organic extracts were washed with brine, and dried over anhydrous MgSO4. After filtration and removal of solvent, the residue was purified by column chromatography on silica gel (eluent=hexane/EA, 15:1) to give the desired products.
After filtration and removal of solvent, then to a stirred solution of 2-(2-trimethylsilyl-1-ethynyl)benzonitrile in dry MeOH (20 mL) was added K2CO3 (5 eq). The resulting reaction mixture was degassed and stirred for 2 hrs at room temperature. Then filtration and removal of solvent before quenched then reaction mixture was quenched with saturated aqueous NaHCO3 solution. The aqueous layer was extracted with EtOAc. The combined organic extracts were washed with brine and dried over anhydrous MgSO4. After filtration and removal of solvent, the residue was purified by column chromatography on silica gel (eluent=hexane/EA, 15:1) to give the desired products.
A degassed solution of 1,2-disubstituted-dihaloethene (12 mmol) in dry ether (30 mL) containing Pd(PPh3)4 (0.8 mmol) and CuI (3.2 mmol) was added to a solution of 2-substituted-1-ethene (24 mmol) containing n-butylamine (34 mmol). The resulting solution was stirred at 25° C. for 6 hrs, quenched with saturated aqueous NH4Cl and Na2CO3 solutions, and extracted with EtOAc. The organic layer was separated and dried over MgSO4. After filtration, the solvent was evaporated in vacuo. The residue was purified by flash chromatography to give the products.
To a degassed solution of 1,3,4-trisubstituted-6-trimethylsilyl-3-hexen-1,5-diynes (1 mmol) in dry THF (15 mL), TBAF (1.2 mmol) was added to the solution and stirred at 25° C. for 6 hrs, quenched with saturated aqueous NaCl solution, and extracted with EtOAc. The organic layer was separated and dried over MgSO4. After filtration, the solvent was evaporated in vacuo. The residue was purified by flash chromatography to give the products.
A degassed solution of (Z)-3-hexen-1,5-diyne (2.3 mmol) in dry ether (5 mL) containing Pd(PPh3) (0.1 mmol) and CuI (0.6 mmol) was added to a solution of aryl iodide (4.5 mmol) containing n-butylamine (5 mmol). The resulting solution was stirred for at 25° C. 6 hrs, quenched with saturated aqueous NH4Cl and Na2CO3 solutions, and extracted with EtOAc. The organic layer was separated and dried over MgSO4. After filtration, the solvent was evaporated in vacuo. The residue was purified by flash chromatography to give the products.
4-trimethylsilyl-1-chorobuten-3-yne (a1) was synthesized using cis-1,2-dichloroethylene and (trimethylsilyl)acetylene as the starting materials according to Method A of the above-described General Synthesis Procedures I, and gives a brown oil in 40% yield.
The title compound was synthesized as a yellow oil in 54% yield using 1,2-diiodobenzene and (trimethylsilyl)acetylene as the starting materials according to Method A of the above-described General Synthesis Procedures I.
The title compound may be prepared from compound (a2) of Example 2 in two steps according to the above synthesis scheme. According to Method B of the above-described General Synthesis Procedures I, compound (a2) is converted to 2-(2-trimethylsilylethynyl)benzonitrile, which is then dissolved into dry methanol with K2CO3 to give a white solid in 73% yield (Method C of the above-described General Synthesis Procedures I).
Alternatively, 2-ethynylbenzonitrile (a3) may be prepared according to the procedures set forth in M. J. Wu et al. (1999), Organic Letters, 1 (5): 767-768, which is incorporated herein by reference in its entirety. Specifically, palladium-catalyzed coupling reaction of trimethylsilylacetylene with 1,2-diiodobenzene produced 2-(2-trimethylsilylethynyl)iodobenzene in 58% yield; 2-(2-trimethylsilylethynyl)iodobenzene was then coupled with Zn(CN)2 using palladium(0) as a catalyst to give 2-(2-trimethylsilylethynyl)benzonitrile in 45% yield; and finally, 2-(2-trimethylsilylethynyl)benzonitrile was treated with tetrabutylammonium fluoride to give 2-ethynylbenzonitrile (a3) in 97% yield.
The coupling reaction of cis-1,2-dichloroethylene and 1-hexyne according to Method A of the above-described General Synthesis Procedures I gave (Z)-1-chloro-1-octen-3-yne (a4) as an oil in 65% yield.
Detected properties of the title compound:
1H NMR (CDCl3, 400 MHz): δ 6.28 (dd, 1H, J=7.3, 0.4 Hz), 5.84 (dt, 1H, J=7.3 2.2 Hz), 2.38 (td, 2H, J=7.0, 2.2 Hz), 1.59-1.42 (m, 4H), 0.92 (t, 3H, J=7.3 Hz).
MS (EI): 142 (M+, 32), 86 (53), 49 (56), 35 (25).
HRMS (EI) calcd for C8H11Cl: 142.0548, found 142.0550.
According to Method A of the above-described General Synthesis Procedures I, the coupling reaction of compound (a4) from Example 4 with trimethylsilylacetylene using palladium as a catalyst gave the title compound (a5) as an oil in 53% yield.
Detected properties of the title compound:
1H NMR (CDCl3, 200 MHz): δ 8.81-8.71 (m, 2H), 2.41 (t, 2H, J=6.8 Hz), 1.56-1.44 (m, 4H), 0.92 (t, 3H, J=6.8 Hz), 0.19 (s, 9H).
13C NMR (CDCl3, 50 MHz): δ 121.5, 118.1, 102.2, 101.7, 99.5, 78.1, 30.7, 21.9, 19.4, 13.6, −0.10.
MS (EI): 204 (M+, 91), 189 (100), 145 (32), 131 (28).
HRMS (EI) calcd for C13H20Si: 204.1331, found 204.1335.
Treatment of compound (a5) from Example 5 with TBAF according Method C of the above-described General Synthesis Procedures I produced the title compound in 80% yield.
Compound (a5) (3.02 g, 14.80 mmol) was dissolved in dry methanol (10 ml), and the solution was stirred with K2CO3 (1.0 g) at room temp. for 1.5 hrs. After the evaporation of methanol in vacuo, the reaction was quenched with saturated aqueous NaHCO3 solution, and the resultant solution was extracted with EtOAc. The organic layer was separated and dried over MgSO4. After filtration, the solvent was evaporated in vacuo. The residue was purified by flash chromatography to give the title compound as an oil in 80% yield.
Detected properties of the title compound:
1H NMR (CDCl3, 200 MHz): δ 5.88-5.75 (m, 2H), 3.28 (s, 1H), 2.41 (t, 2H, J=7.0 Hz), 1.58-1.40 (m, 4H), 0.92 (t, 3H, J=7.0 Hz).
13C NMR (CDCl3, 50 MHz): δ 122.4, 116.9, 99.6, 83.6, 80.9, 77.8, 30.5, 21.8, 19.4, 13.5.
Compounds 1-4 listed above may be synthesized according to the following scheme, and the detected characteristics thereof are described below.
A. 2-(7-(Tetrahydropyranyloxy)-3(Z)-hepten-1,5-diynyl)-benzonitrile 1
Obtained as an oil.
1H NMR (CDCl3, 400 MHz): δ 7.59-7.66 (m, 2H), 7.54 (td, 1H, J=7.5, 1.3 Hz), 7.41 (td, 1H, J=7.5, 1.3 Hz), 6.10 (d, 1H, J=10.8 Hz), 6.02 (td, 1H, J=10.8, 2.0 Hz), 4.87 (t, 1H, J=3.5 Hz), 4.56 (dd, 1H, J ═6.3, 1.9 Hz), 4.49 (dd, 1H, J=6.3, 1.9 Hz), 3.82-3.88 (m, 1H), 3.50-3.55 (m, 1H), 1.50-1.83(m, 6H).
13C NMR (CDCl3, 100 MHz): δ 132.8, 132.7, 132.2, 128.6, 128.5, 121.7, 121.3, 118.6, 96.8, 94.8, 92.7, 92.5, 82.9, 61.9, 55.0, 51.0, 30.2, 25.3, 18.9.
MS (EI) [m/z (relativeintensity)]: 291 (M+, 1.8), 190 (100), 85 (45).
HRMS calcd for C19H17NO2: 291.1260, found 291.1255.
B. 2-(9-(Tetrahydropyranyloxy)-3(Z)-nonen-1,5-diynyl)-benzonitrile 2
Obtained as an oil.
1H NMR (CDCl3, 400 MHz): δ 7.67-7.63 (m, 2H), 7.58-7.50 (m, 2H), 7.43-7.35 (m, 2H), 6.03 (d, 1H, J ═10.9 Hz), 5.96 (dt, 1H, J ═10.9, 1.7 Hz), 4.58 (t, 1H, J=3.8 Hz), 3.90-3.76(m, 2H), 3.57-3.46 (m, 2H), 2.60 (td, 2H, J ═7.3, 1.7 Hz), 1.95-1.48 (m, 8H).
13C NMR (CDCl3, 100 MHz): δ 132.8, 132.7, 132.6, 132.4, 132.2, 128.4, 122.5, 117.1, 115.0, 100.4, 98.7, 93.2, 91.7, 78.3, 66.0, 62.1, 30.6, 28.7, 25.4, 19.5, 16.8;
MS (EI) [m/z(relative intensity)] 319 (M+, 11), 235 (68), 216 (100), 203 (38), 190 (71).
HRMS calcd for C21H21NO2: 319.1573, found 319.1576.
C. 2-(3(Z)-Decen-1,5-diynyl)benzonitrile 3
Obtained as an oil.
1H NMR (CDCl3, 400 MHz): δ 7.66 (dd, 1H, J=7.8, 1.0 Hz), 7.59-7.49 (m, 2H), 7.43-7.35 (m, 1H), 6.03-5.98 (m, 2H), 2.47 (td, 2H, J=6.8, 1.5 Hz), 1.61-1.39 (m, 4H), 0.89 (t, 3H, J=7.0 Hz).
13C NMR (CDCl3, 100 MHz): δ 132.7, 132.6, 132.2, 128.3, 127.1, 122.7, 117.3, 116.9, 115.0, 101.2, 93.3, 91.4, 78.1, 30.6, 21.9, 19.6, 13.6.
MS (EI) [m/z (relative intensity)]: 233 (M+, 22), 218 (55), 204 (100), 190 (73), 164 (36); HRMS calcd for C17H15N: 233.1205, found 233.1210.
D. 2-(3(Z)-Undecen-1,5-diynyl)benzonitrile 4
Obtained as an oil.
1H NMR (CDCl3, 400 MHz): δ 7.67 (dd, 1H, J=7.2, 1.1 Hz), 7.57-7.53 (m, 2H), 7.49-7.36 (m, 1H), 6.06-5.99 (m, 2H), 2.47 (td, 2H, J=6.9, 1.5 Hz), 1.64-1.25 (m, 6H), 0.85 (t, 3H, J) 6.8 Hz).
13CNMR(CDCl3, 100 MHz): δ 132.7, 132.6, 132.2, 128.3, 127.1, 122.7, 117.3, 116.9, 115.0, 101.3, 93.3, 91.4, 78.1, 31.0, 28.2, 22.2, 19.9, 13.9.
MS (EI) [m/z (relative intensity)]: 247 (M+, 14), 217 (49), 204 (100), 190 (68);
HRMS calcd for C18H17N: 247.1362, found 247.1357.
The Title compound may be produced from compound a3 of Example 3 and 4-chloro-1-phenyl-3-buten-1-yne according to the procedures for the production of compounds 1-4 as set forth in Example 7.
The Title compound may be produced by the coupling reaction of compound a6 from Example 6 and iodobenzene according to the above-described General Procedures IV Alternatively, the Title compound may be produced from compound a4 of Example 4 and phenylacetylene according to the procedures for the production of compounds 1-4 as set forth in Example 7.
Detected Properties of the Title Compound:
Obtained as an oil in 67% yield.
1HNMR(CDCl3, 200 MHz): δ 7.49-7.43 (m, 2H), 7.35-7.29 (m, 3H), 5.94 (d, 1H, J=10.8 Hz), 5.87 (d, 1H, J=10.8 Hz), 2.45 (t, 2H, J=6.8 Hz), 1.63-1.43 (m, 4H), 0.9 (t, 3H, J=7.0 Hz).
13CNMR (CDCl3, 50 MHz): δ 131.7 128.3, 128.2, 123.2, 120.3, 118.1, 99.4, 96.0, 87.2, 78.4, 30.7, 21.9, 19.5, 13.6.
MS (EI) [m/z (relative intensity)]: 208 (M+, 67), 179 (26), 178 (63), 165 (100), 163 (28), 152 (27), 139 (39), 115 (28).
HRMS calcd for C16H16, Mr=208.1255, found 208.1252.
2-(2-(2-Alkynylphenyl)ethynyl)benzonitriles 7-10 may be synthesized by the coupling reaction of 2-ethynylbenzonitrile (a3) and corresponding 2-(2-sustituted-1-ethynyl)iodobenzenes according to the aforesaid Scheme 4. The obtained yields varied from 40% to 98%.
Specifically, a degassed solution of a selected 2-alkynyliodobenzene (12 mmol) in dry ether (30 mL) containing Pd(PPh3)4 (0.8 mmol) and CuI (3.2 mmol) was added to a solution of 2-ethynylbenzonitrile (24 mmol) containing n-butylamine (34 mmol). The resulting solution was stirred at 25° C. for 6 hrs, quenched with saturated aqueous NH4Cl and Na2CO3 solutions, and extracted with EtOAc. The organic layer was separated and dried over MgSO4. After filtration, the solvent was evaporated in vacuo. The residue was purified by flash chromatography to give the product.
A. 2-(2-(3-Tetrahydro-pyranyl-5-oxy-1-propynylphenyl)ethynyl)benzonitrile 7
Obtained as an oil in 98% yield.
1H NMR (CDCl3, 200 MHz): δ 7.84-7.37 (m, 8H), 5.08-5.04 (m, 1H), 4.70 (d, 2H, J=2.2 Hz), 4.21-3.93 (m, 1H), 3.69-3.61 (m, 1H). 1.93-1.58 (m, 6H).
13C NMR (CDCl3, 50 MHz): δ 132.7, 132.6, 132.5, 132.3, 132.2, 128.9, 128.4, 128.2, 127.1, 125.6, 124.9, 117.4, 115.1, 96.8, 94.4, 89.1, 84.0, 61.9, 54.9, 30.3, 25.4, 19.0.
HRMS (EI) calcd for C23H19NO2: 341.1414, found 341.1452.
B. 2-(2-(3-Tetrahydro-pyranyl-5-oxy-1-pentynylphenyl)ethynyl)benzonitrile 8
Obtained as an oil in 89% yield.
1H NMR (CDCl3, 200 MHz): δ 7.70-7.51 (m, 4H), 7.47-7.34 (m, 2H), 7.31-7.28 (m, 2H), 4.59-4.56 (m, 1H), 3.95-3.81 (m, 1H), 3.62-3.47 (m, 1H), 2.64 (t, 2H, J=7.4 Hz), 1.97-1.46 (m, 10H).
13C NMR (CDCl3, 50 MHz): δ 132.7, 132.6, 132.5, 132.3, 132.1, 132.0, 128.9, 128.2, 127.4, 127.3, 126.4, 124.3, 117.5, 115.2, 98.8, 94.9, 88.6, 79.3, 66.1, 62.2, 30.7, 28.9, 25.5, 19.5, 16.6.
HRMS (EI) calcd for C25H23NO2: 369.1725, found 369.1729.
C. 2-(2-(2-Hexynylphenyl)ethynyl)benzonitrile 9
Obtained as an oil in 62% yield.
1H NMR (CDCl3, 200 MHz): δ 7.71-7.52 (m, 4H), 7.47-7.37 (m, 2H), 7.33-7.25 (m, 2H), 2.52 (t, 2H, J=7.6 Hz), 1.66-1.43 (m, 4H), 0.89 (t, 3H, J=7.6 Hz).
HRMS (EI) calcd for C21H17N: 283.1326, found 283.1326.
D. 2-(2-(2-Heptynylphenyl)ethynyl)benzonitrile 10
Obtained as an oil in 40% yield.
1H NMR (CDCl3, 200 MHz): δ 7.70-7.53 (m, 4H), 7.47-7.34 (m, 2H), 7.30-7.25 (m, 2H), 2.51 (t, 2H, J=7.6 Hz), 1.69-1.25 (m, 6H), 0.85 (t, 3H, J=7.6 Hz).
HRMS (EI) calcd for C21H17N: 297.1515, found 297.1518.
The title compounds 11 and 12 may be respectively produced by the coupling reaction of compound a6 from Example 6 and 1,4-diiodobenzene and 4,4′-diiodobiphenyl according to the above scheme.
A. 1,4-Bis-(3-(Z)-Dodecen-1,5-diynyl)benzene 11
Obtained as an oil in 78% yield.
1H NMR (CDCl3, 200 MHz): δ 7.41 (s, 2H), 6.02-5.58 (m, 4H), 2.45 (t, 2H, J=7.2 Hz), 1.70-1.47 (m, 8H), 0.90 (t, 6H, J=7.2 Hz).
13C NMR (CDCl3, 50 MHz): δ 131.5, 122.2, 120.8, 117.9, 99.9, 95.7, 89.2, 78.4, 30.7, 21.9, 19.6, 13.6.
HRMS (EI) calcd for C26H26: 338.2035, found 338.2017.
B. 4,4′-Bis-(3-(Z)-Dodecen-1,5-diynyl)biphenyl 12
Obtained as an oil in 51 % yield.
1H NMR (CDCl3, 200 MHz: δ 7.58-7.51 (m, 8H), 5.98-5.86 (m, 4H), 2.45 (td, 4H, J=7.2, 2.0 Hz), 1.65-1.48 (m, 8H), 0.91 (t, 6H, J=7.4 Hz).
13C NMR (CDCl3, 50 MHz): δ 140.1, 132.2, 126.85, 122.6, 120.4, 116.1, 99.6, 95.9, 88.3, 78.5, 31.5, 30.7, 21.9, 14.2.
HRMS (EI) calcd for C32H30 414.2349, found 414.2346.
A series of 1-aryl dec-3-ene-1,5-diynes 12-23 may be produced by the coupling reaction of compound a6 from Example 6 and aryl iodides according to the above-described General Procedures IV.
A. 4-(3-(Z)-Dodecen-1,5-diynyl)benzochloride 13
Obtained as an oil in 31 %.
1H NMR (CDCl3, 200 MHz): δ 7.36 (d, 2H, J=8.8 Hz), 7.31 (d, 2H, J=8.6 Hz), 5.97-5.84 (m, 2H), 2.45 (t, 2H, J=6.8 Hz), 1.61-1.45 (m, 4H), 0.9 (t, 3H, J=7.0 Hz).
13C NMR (CDCl3, 50 MHz): δ 134.4, 132.8, 128.6, 121.7, 120.7, 117.7, 99.7, 94.7, 88.1, 78.3, 30.7, 21.8, 19.5, 13.5.
MS (EI) 242 (M+, 100), 201 (16), 199 (40), 192 (78), 165 (63), 164 (45), 163 (52).
HRMS (EI) calcd for C16H15Cl: 242.0859, found 242.0863.
B. 4-(3-(Z)-Dodecen-1,5-diynyl)benzonitrile 14
Obtained as an oil in 46%.
1H NMR (CDCl3, 200 MHz): δ 7.61 (d, 2H, J=6.4 Hz), 7.53 (d, 2H, J=8.4 Hz), 5.95 (d, 2H, J=1.6 Hz), 2.44 (t, 2H, J=6.8 Hz), 1.59-1.44 (m, 4H), 0.9 (t, 3H, J=7.2 Hz).
13CNMR(CDCl3, 50 MHz): δ 132.0, 131.9, 128.1, 122.2, 118.4, 117.1, 111.5, 100.6, 93.9, 91.3, 78.2, 30.5, 21.8, 19.4, 13.5.
MS (EI) 233 (M+, 51), 203 (51), 190 (100), 177 (35), 164 (46), 140 (28).
HRMS (EI) calcd for C17H15N: 233.1206, found 233.1205.
C. 2-(3-(Z)-Dodecen-1,5-diynyl)pyridine 15
Obtained as an oil in 84% yield.
1H NMR (CDCl3, 200 MHz): δ 8.58 (dt, 1H, J=5.0, 1.0 Hz), 7.63 (td, 1H, J=7.6, 1.8 Hz), 7.42 (td, 1H, J=7.6, 1.0 Hz), 7.23-7.16 (m, 2H), 5.99-5.93 (m, 2H), 2.43 (td, 2H, J=7.0, 1.6 Hz), 1.92-1.43 (m, 4H), 0.87 (t, 3H, J=7.0 Hz).
13C NMR (CDCl3, 50 MHz): δ 150.5, 143.3, 135.9, 127.2, 122.7, 122.1, 117.3, 100.3, 94.7, 86.8, 78.2, 30.6, 21.8, 19.5, 13.5.
MS (EI): 209 (M+, 19), 180 (100), 78 (18), 51 (18).
HRMS (EI) calcd for C15H15N: 209.1209, found 209.1205.
D. 2-(3-(Z)-Dodecen-1,5-diynyl)thiophene 16
Obtained as an oil in 53% yield.
1H NMR (CDCl3, 200 MHz): δ 7.29 (d, 1H, J=5.2 Hz), 7.22 (d, 1H, J=2.6 Hz), 7.00 (t, 1H, J=5.2 Hz), 5.94 (d, 1H, J=10.4 Hz), 5.86 (d, 1H, J=10.8 Hz), 2.45 (d, 2H, J=6.8 Hz), 1.63-1.47 (m, 4H), 0.92 (t, 3H, J=6.8 Hz).
13C NMR(CDCl3, 50 MHz): δ 132.1, 127.7, 127.2, 123.3, 120.1, 117.7, 99.8, 91.2, 89.1, 78.4, 30.6, 21.9, 19.5, 13.5.
MS (EI): 214 (M+, 100), 184 (41), 171 (80), 165 (53).
HRMS (EI) calcd for C14H14S: 214.0811, found 214.0817.
E. 2-(3-(Z)-Dodecen-1,5-diynyl)pyrazine 17
Obtained as an oil in 99% yield.
1H NMR (CDCl3, 200 MHz): δ 8.66 (d, 1H, J=1.6 Hz), 8.55 (s, 1H), 8.45 (d, 1H, J=2.4 Hz), 5.98 (m, 2H), 2.44 (t, 2H, J=6.8 Hz), 1.58-1.43 (m, 4H), 0.88 (t, 3H, J=7.0 Hz).
13C NMR (CDCl3, 50 MHz): δ 147.8, 144.4, 142.6, 140.3, 123.5, 116.5, 101.3, 91.7, 90.8, 78.1, 30.5, 21.8, 19.5, 13.5.
MS (EI): 210 (M+, 39), 181 (100), 168 (14), 127 (17).
HRMS (EI) calcd for C14H14N2: 210.1154, found 210.1158.
F. 4-(3-(Z)-Dodecen-1,5-diynyl)trifluoromethylbenzene 18
Obtained as an oil in 46% yield.
1H NMR (CDCl3, 200 MHz): δ 7.57 (s, 4H), 5.95 (d, 2H, J=1.6 Hz), 2.46 (t, 2H, J=6.8 Hz), 1.58-1.49 (m, 4H), 0.90 (t, 3H, J=7.0 Hz).
13C NMR (CDCl3, 50 MHz): δ 146.9, 134.6, 131.8, 127.0, 125.3, 121.6, 117.4, 112.1, 100.2, 89.4, 78.3, 30.6, 21.9, 19.5, 13.5.
MS (EI): 276 (M+, 100), 233 (59), 207 (31), 192 (24), 165 (47), 49 (43).
HRMS (EI) calcd for C17H15F3: 276.1129, found 276.1174.
G. 3-(3-(Z)-Dodecen-1,5-diynyl)trifluoromethylbenzene 19
Obtained as an oil in 46% yield.
1H NMR (CDCl3, 200 MHz): δ 7.72 (s, 1H), 7.61 (d, 1H, J=7.6 Hz), 7.55 (d, 1H, J=8.0 Hz), 7.45 (t, 1H, J=7.6 Hz), 5.97 (d, 1H, J=10.8 Hz), 5.92 (d, 1H, J=10.8 Hz), 2.46 (t, 2H, J=6.8 Hz), 1.62-1.45 (m, 4H), 0.90 (t, 3H, J=7.2 Hz).
13C NMR (CDCl3, 50 MHz): δ 146.9, 134.6, 128.8, 128.5, 124.8, 124.8, 124.2, 121.5, 117.4, 100.1, 94.2, 88.6, 78.3, 30.6, 21.8, 19.5, 13.4.
MS (EI): 276 (M+, 100), 261 (30), 246 (34), 233 (84), 207 (47), 183 (62), 178 (37), 165 (68).
HRMS (EI) calcd for C17H15F3: 276.1125, found 276.1127.
H. 2-(3-(Z)-Dodecen-1,5-diynyl)trifluoromethylbenzene 20
Obtained as an oil in 33% yield.
1H NMR (CDCl3, 200 MHz) δ 7.64 (td, 2H, J=7.8, 1.8 Hz), 7.39-7.37 (m, 2H) 6.02-5.87 (m, 2H) 2.45 (t, 2H, J=7.0 Hz), 1.60-1.51 (m, 4H) 0.90 (t, 3H, J=7.0 Hz).; 13C NMR(CDCl3, 50 MHz) δ 146.3, 134.2, 131.2, 128.0, 125.8, 125.7, 121.6, 117.5, 112.1, 100.3, 92.5, 91.5, 78.0, 30.6, 21.9, 19.5, 13.5.
MS (EI) 276 (M+, 100), 233 (34), 232 (29), 221 (48), 214 (22), 183 (29), 165 (14).
HRMS (EI) calcd for C17H15F3 276.1174, found 276.1174.
I. 4-nitro-1-(3-(Z)-dodecen-1,5-diynyl)benzene 21
Obtained as an oil in 59% yield.
1H NMR (CDCl3, 200 MHz): δ 8.16 (d, 2H, J=9.0 Hz), 7.60 (d, 2H, J=9.0 Hz), 5.97 (s, 2H), 2.45 (t, 2H, J=6.4 Hz), 1.61-1.45 (m, 4H), 0.9 (t, 3H, J=7.0 Hz).
3CNMR(CDCl3, 50 MHz): δ 147.0, 132.1, 123.5, 122.6, 118.3, 117.0, 100.9, 93.6, 92.2, 78.2, 30.6, 21.8, 19.5, 13.5.
MS (EI): 253 (M+, 100), 238 (16), 210 (16), 192 (14), 165 (13), 163 (21).
HRMS (EI) calcd for C16H15NO2: 253.1105, found 253.1103.
J. 2-(3-(Z)-Dodecen-1,5-diynyl)benzoate 22
Obtained as an oil in 31% yield.
1H NMR (CDCl3, 200 MHz): δ 7.95 (d, 1H, J=7.8 Hz), 7.57 (d, 1H, J=7.4 Hz), 7.46-7.31 (m, 2H), 6.00 (d, 1H, J=10.8 Hz), 5.91 (d, 1H, J=10.8 Hz), 3.91 (s, 2H), 2.45 (t, 2H, J=6.8 Hz), 1.62-1.42 (m, 4H), 0.87 (t, 3H, J=7.0 Hz).
13CNMR (CDCl3, 50 MHz): δ 166.5, 134.3 131.6, 131.5, 130.3, 127.9, 123.7, 120.8, 118.1, 99.6, 94.6, 78.4, 52.0, 30.6, 21.9, 19.5, 13.5.
MS (EI): 266 (M+, 71), 237 (75), 224 (51), 223 (70), 209 (82), 191 (21), 181 (49), 176 (24).
HRMS (EI) calcd for C18H18O2: 266.1305, found 266.1307.
K. 4-(3-(Z)-Dodecen-1,5-diynyl)acetophenone 23
Obtained as an oil in 96% yield.
1H NMR (CDCl3, 200 MHz): δ 7.92 (d, 2H, J=8.4 Hz), 7.54 (d, 2H, J=8.4 Hz), 5.99-5.90 (m, 2H), 2.60 (s, 3H), 2.45 (t, 2H, J=7.2 Hz), 1.63-1.46 (m, 4H), 0.91 (t, 3H, J=7.2 Hz).
13C NMR (CDCl3, 50 MHz): δ 197.2, 136.3, 131.7, 128.2, 128.1, 121.6, 117.6, 100.3, 95.0, 90.4, 78.4, 30.7, 26.6, 21.9, 19.5, 13.6.
MS (EI): 250 (M+, 100), 235 (37), 165 (29).
HRMS (EI) calcd for C18H18O: 250.1372, found 250.1358.
The Title compound may be produced in two steps using compound a1 from Example 1 and compound a3 from Example 3 as starting materials. Firstly, compound a1 and compound a3 were reacted according to Method A set forth in the above-described General Synthesis Procedures I to form 2-(6-trimethylsilyl-3-hexen-1,5-diynyl)benzonitrile, which was subsequently dissolved into into dry methanol with K2CO3 to give a brown oil in 53% yield.
The above-indicated 2-(6-aryl-3-hexen-1,5-diynyl)benzonitriles 24-33 may be produced by the coupling reaction of 2-(3-hexen-1,5-diyne)benzonitrile from Example 13 with various aryl iodides.
The above-indicated 1,6-diaryl-3-hexen-1,5-diynes 34-43 may be produced by the coupling reaction of 1,2-disubstituted-dihaloethene with a selected 2-aryl1-ethyne. Specifically, a degassed solution of 1,2-disubstituted-dihaloethene (12 mmol in dry ether (30 mL) containing Pd(PPh3)4 (0.8 mmol) and CuI (3.2 mmol) was added to a solution of the selected 2-aryl-1-ethyne (24 mmol) containing n-butylamine (34 mmol). The resulting solution was stirred at 25° C. for 4 hrs, quenched with saturated aqueous NH4Cl and Na2CO3 solutions, and extracted with EtOAc. The organic layer was separated and dried over MgSO4. After filtration, the solvent was evaporated in vacuo. The residue was purified by flash chromatography to give the products.
In order to determine the biological activities of the aryl-substituted acyclic enediynes 1-43 of formula (I) according to the present invention, the following pharmaceutical activity assay was performed.
A. In Vitro Anticancer Assay (Cytotoxicity):
The cytotoxicities of Compounds were evaluated using five human solid tumor cells (KB, Hela, DLD, NCI, and Hepa) and/or the NCI's full panel of 60 human cancer cell lines derived from nine cancer cell types, including: leukemia (CCRF-CEM, HL-60 (TB), MOLT-4, K-562, RPMI-826, and SR); non-small cell lung cancer (A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H332M, NCI-H460, and NCI-H522); colon cancer (COLO 205, HCC-2998, HCT-116, HCT-15, HT29, KM12, and SW-620); CNS cancer (SF-268, SF-295, SF-539, SNB-19, SNB-75, and U251); melanoma (LOX IMVI, MALME-3M, M14, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-62, and UACC-257); ovarian cancer (IGROVI, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, and SK-OV-3); renal cancer (786-0, ACHN, CAKI-1, RXF 393, SN12C, TK-10, and UO-31); prostate cancer (PC-3 and DU-145); and breast cancer (MCF7, MCF7/ADR-RES, MDA-MB-231/ATCC, HS 578T, MDA-MB-435, MDA-N, and T-47D).
For each compound, dose-response curves for each tumor cell were measured with five different drug concentrations, and the concentrations causing 50% cell-growth inhibition (GI50) compared with the control were calculated (A. Monks, D. Scudiero, P. Skehaan, R. Shoemaker, K. Paull, D. Vistica, C. Hose, J. Langley, P. Cronise, A. Vaigro-Wolff, M. Gray-Goodrich, H. Campbell, J. Mayo, M. Boyd, J. Natl. Cancer Inst. 1991, 83, 757).
B. General Topoisomerase I Course Assay:
All samples were kept in 23 μl total volume; (a) negative control (DNA alone): contained 10× Topo I buffer (2 μl) and pGEM9zf(−) DNA (1 μg/μl), 0.1% BSA (2 μl) and d-H2O (18 μl); (b) positive control (DNA+Topo I): contained 10× Topo I buffer (2 μl) and pGEM9zf(−) DNA (1 μg/μl), 1 units/μl of topoisomerase I, 0.1% BSA (2 μl) and d-H2O (17 μl); (c) camptothecin control (DNA+Topo I+camptothecin): contained 10× Topo I buffer (2 μl) and pGEM9zf(−) DNA (1 μg/μl), 1 units/μl of topoisomerase I, 0.1% BSA (2 μl), d-H2(15 μl) and camptothecin (2 μl, dissolved in DMSO, final conc. of DMSO was 8.7% v/v); (d) experiments for compounds 7-10, 11-12 and 13-23 (DNA+Topo I+compounds): contained 10× Topo I buffer (2 μl)and pGEM9zf(−) DNA(1 μg/μl), 1 units/μl of topoisomerase I, 0.1% BSA (2 μl), d-H2O (15 μl) and compounds (2 μl, dissolved in DMSO, final conc. of DMSO was 8.7% v/v). (a)-(d) were well-mixed before incubation. The tubes were incubated in 37° C. water bath for 30 min.
C. Gel Electrophoresis:
The reaction mixtures were proceeded by using 2% agarose gel in standard TBE buffer (1×,0.06 M Tris, 0.06 M boric acid, 0.5 M EDTA), which had previously been added 2 μl of loading buffer containing 0.25% bromophenol blue, 0.25% xylene cyanol, 5% SDS, and 0.25% sucrose. The gels were run at 50 volts for 1.5 hr, and stained with ethidium bromide for 20 min. Then, after placing in a UV box, photographic images were made of the gels, using Polaroid 665 films.
D. Cell Cycle Analysis:
Samples of 1×106 Hep G2 cells were plated on a 6 cm tissue culture dish, and the cells were allowed to recover 24 hrs before any treatment. Thereafter, DMSO or a test compound (1 μM, 10 μM or 50 μM) was added to the cells for 1 hr in complete medium at 37° C., washed twice with PBS, and incubated in fresh media for the incubation time. Cell were harvested with trypsin and washed twice with PBS. Samples were fixed in 70% ethanol and stored at 4° C. for at least 24 hrs and then washed once with Mcllvaine's buffer (0.2M Na2HPO4, 0.1M citric acid, pH=7.5) and once again with PBS. Samples were stained with PI (propidium iodide) staining solution (PBS containing 100 μg/ml, RNase A and 10 μg/ml PI [Sigma]), processed on a Coulter Elite flow cytometry, and the data analyzed with the Mutiplus AV program.
A. Cytotoxicity:
Compounds of this invention were subjected to in vitro assay to determine whether or not they exhibit the activity of inhibiting the growth of any of five human solid tumor cells (KB, Hela, DLD, NCI, and Hepa) and/or the NCI's full panel of 60 human tumor cell lines derived from 9 cancer cell types.
The IC50 values of compounds 1-23 with five human solid tumor cells (KB, Hela, DLD, NCI, and Hepa) are summarized in Table 2. It can be seen from Table 2 that 2-(6-substituted-3-hexen-1,5-diynyl)benzonitriles 1-5 demonstrated to have a tendency of inhibiting the growth of Hepa cell. Among these 23 compounds, 2-(6-phenyl-3-hexen-1,5-diynyl)benzonitrile 5 exhibited the highest cytotoxic activity against Hepa cell at 1.09 μg/ml. 2-(2-(2-alkynylphenyl)-ethynyl)-benzonitriles 8-10 and 1-aryl-3-dodecen-1,5-diynes 13-22 also had the same tendency with DLD cell line, in which 4-nitro-1-(3-(Z)-dodecen-1,5-diynyl)-benzene 21 showed selective potency against DLD cell at the low IC50 value of 1.66 μg/ml. 2-(3-(Z)-Dodecen-1,5-diynyl)trifluoromethylbenzene 20 was more active than the other derivatives against Hela cell (IC50 value=2.15 μg/ml).
*“—” means IC50 >20 μg/ml; the standard of cytotoxicity test is doxorubicin whose ED50 ≈ 0.1 μg/ml;
“n.d.” means not detectable.
KB: human oral epidermoid carcinoma.
Hela: human cervix epitheloid carcinoma.
DLD (DLD-1): human colon adenocarcinoma.
NCI (NCI-H661): human lung large cell carcinoma.
Hepa: human hepatoma.
From the cytotoxic assay, enediynes 1-5 were shown to have a tendency of cytotoxic activity with Hepa cell, and the longer the alkyl chain, the higher was the revealed cytotoxic activity. When the substituent at 6-position is a phenyl group, it provides the strongest activity against Hepa cell line. In addition, enediynes 13-22 could induce the growth inhibition of DLD cells, and among these compounds, compound 21 showed highest biological activity. Comparing the results of derivatives 15 (Ar=pyridinyl) and 17 (Ar=pyrazinyl) shown in Table 2, this suggests that the increase in the number of nitrogen atoms in the aromatic gave lower IC50 value and wider spectrums of cytotoxicity. Compound 6 and it's dimer 12 have a similar structure and remain selective toxicity against KB cell.
The 1,6-diaryl-3-hexen-1,5-diynes 24-43 were subjected to a preliminary cytotoxicity test using three tumor cells (Breast cancer cell MCF-7, non-small cell lung cancer cell NCI-H460, and CNS cancer cell SF-286) and the results are shown in Table 3.
*Compounds which reduced the growth of any one of the tested tumor cells to approximately 32% or less were subjected to further evaluation using the NCI's full panel of 60 tumor cell lines.
As can be seen from Table 3, compounds 26, 27, 30, 32, 34, 37, 38, 40 and 41 passed the preliminary screening test and, therefore, they were further subjected to the NCI's full panel screen of 60 cancer cell lines. The detail data of the cytotoxic activities of compounds 26, 27, 30, 32, 34, 37, 38, 40 and 41 are outlined in Table 4, 5 and 6, respectively.
*The IC50 value was the average value of all the cancer cell lines involved in the various kinds of human cancer cells.
aThe concentration produced 50% reduction in cell growth.
bThe concentration produced 50% cells killed.
According to the cytotoxicities observed in the NCI screening data, the compounds of this invention displayed good activities in growth inhibition of ten usual human cancers, including leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, breast cancer and liver cancer. The IC50 values of several of the compounds were 10−7 M or 10−8 M, and the acute toxicities (LC50 values) of most active compounds were larger than 10−4 M (data collected by NCI).
B. Evalution of Inhibitory Concentration of Camptothecin for Gel Electrophoresis.
Camptothecin, which showed significant topo I inhibition, was widely used as the standard for the comparison of activity to the novel compounds. Regarding the modest concentration of camptothecin that would exhibit explicit suppression of topo I and used in the following experimental section as comparison, various concentrations were prepared, and the results were obtained by agarose gel electrophresis. It was suggested that camptothecin showed less topo I inhibitory activity when the concentration was 26.1 μM. On the other hand, 43.5 μM of camptothecin exhibited observable inhibition of topo I. Therefore, the 43.5 μM was used as the standard concentration of camptothecin, when the inhibition of topo I was proceeded (data not shown).
C. Inhibition Tests of the Compounds 7-23 for Topoisomerase I:
The cleavage of supercoiled DNA by topo I in the presence of 2-(2-(2-alkynylphenyl)ethynyl)benzonitriles 7-10, 1,4-bis(3-decen-1,5-diynyl) benzene 11, 4,4′-bis(3-decen-1,5-diynyl)biphenyl 12, and 1-aryl-3-decen-1,5-diynes 13-23 were evaluated by gel electrophoresis and the results are shown in
Supercoiled pGEM9zf(−) DNA was treated with 0.025 μg/μl topoisomerase I and compounds 7-10 and compounds 11-12 (the unshown results of compounds 7 and 8 display no Topo I inhibition at 8.7 and 87 μM), then analyzed on a 2% agarose gel. In panel (a), lane 1: DNA+topo I; lane 2: DNA only; lane 3: DNA+topo I+camptothecin (43.5 μM); lanes 4-6. DNA+topo I+compound 10; lanes 7-9: DNA+topo I+compound 9. Each group of three lanes contained 0.87, 8.7, 87 μM of the analogs, respectively. In panel (b), lane 1: DNA+topo I; lane 2: DNA only; lanes 3-4: DNA+topo I+compound 11; and lanes 5-6: DNA +topo I+compound 12. Each group of both lanes contained 8.7, 87 μM of the analogs, respectively.
In panel (a) of
In panel (a) of
Supercoiled pGEM9zf(−) DNA was treated with 0.025 μg/μl topoisomerase I and compounds 13-23, and then analyzed on a 2% agarose gel. In panel (a), lane 1: DNA only; lane 2: DNA+topo I; lanes 3-7: DNA+topo I+compounds 13-17 (87 μM). In panel (b), lanes 1-6: DNA+topo I+compounds 18-23 (87 μM).
In panel (a) of
D. Cell Cycle Assay:
To obtain more information about the effect(s) of the acyclic enediynes according to this invention upon the growth of cells, human hepatoma Hep G2 cells or K-562 cells were used, and the growth characteristic of said cells were measured subsequent to the treatment with a test compound.
As shown in
There are two essential factors that may facilitate the enediyne derivatives against topo I, i.e. the enediyne core and the cyano group substituted on the o-position of benzene. The enediynes 2-5 reveal inhibition of topo I (C. F. Lin, P. C. Hsieh, W. D. Lu, H. F. Chiu, M. J. Wu, J. Bioorg. Med. Chem. 2001, 9, 1707). Replacement of the enediyne core with 1,2-diethynyl benzene lowers the inhibition abilities of topo I, which may be due to the alteration of conformation of the original enediyne core, or because of the lowered ability of 1,2-diethynyl benzene to form a complex with topo I as compared with the original enediyne core.
All unwinding DNAs detected in panel (b) of
In view of the above, the acyclic enediynes according to this invention displayed selective inhibitory effects upon the growth of a variety of human tumor cells, and some of them revealed inhibitory effects with topoisomerase I, although there seems to be no significant relationship between the cytotoxicity and topo I.
On the other hand, it was considered that the intact cancer cells owned complete biological functions. Hence, it is possible that other compensatory pathways in the cancer cells would be active when topoisomerase I was inhibited during the periods of DNA replication. The data shown in
Although the picture concerning the cytotoxicities of the compounds according to this invention is still incomplete, we discovered that several new lead compounds of enediynes provided potent activities against a variety of human solid tumor cells and topo I, and the cell cycle test of compound 5 indicates that the enediyene structures block the replication of DNA.
Referring to FIGS. 4 and 5, when K-562 cells were treated with compounds 26, 27, 30, 32, 34, 37, 38, 40 and 41 for 72 hrs, an accumulation of G2/M stage cells in these samples is observed. In addition, as shown in FIGS. 6 and 7, the percentage of cells at the G2/M phase increased from 17% to 84% after treatment of K-562 cells with compounds 26, 27, 30, 32, 34, 37, 38, 40 and 41, respectively. Further, the nine compounds caused the formation of apoptotic K-562 cells at 50 μM, and the proportion of apoptotic cells varied from 21.3% to 40.0% (FIGS. 8 and 9).
In general, there are two types of cellular death: necrosis and apoptosis. Necrotic cells were killed by external or tumor necrotic factors (TNF), while apoptotic cells participated in their own destruction. The presentation of apoptotic effect excluded the TNF for providing cytotoxicities to cancer cells in the presence of compounds 24-43. Usually, the apoptotic process was very complex. An important part of this phenomenon could be mediated by deregulation in cell cycle progression governed by a family of cyclin-dependent kinases (CDKs), although much more experimental evidence is necessary to support this hypothesis. It is found in this invention that 3,4-disubstituted-1,6-diaryl-3-hexen-1,5-diynes 24-43 may act as G2/M phase blocker. Investigation of the actual mechanism of acyclic 1,6-diaryl-3-hexen-1,5-diynes 24-43 in inhibiting the growth of tumor/cancer cells is still ongoing.
In this invention, we provided new 6-aryl-3-hexen-1,5-diynes of formula (I), which are found for the first time to have inhibitory activities against a variety of human tumor cells at low concentration (the highest IC50 value is 0.1 μM or 1 μg/ml), and whose activities do not arise from the formation of biradical intermediates as usually known. The main drawback of the existing anticancer drugs commonly used in clinical therapies is the occurrence of serious side effects subsequent to the administration of the drugs, which may undesirably result in the death of normal cells. Most of the LC50 values of these compounds are higher than 100 μM, which means that these compounds display the growth inhibition of human tumor cell lines and cause almost no normal cell death (caused lower side-effect). It is an important advancement for cancer therapies.
On the other hand, rigmarole synthetic procedures are frequent problems for most antitumor drugs. However, the acyclic 6-aryl-3-hexen-1,5-diynes of formula (I) according to this invention are easy to prepare. Moreover, by modification of the active structures, active anticancer or antivirus compounds with less side effects could be conveniently developed in the future.
All literature references cited in the present specification are hereby incorporated by reference in their entirety. In case of conflict, the present description, including definitions, will prevail.
While the invention has been described with reference to the above specific embodiments, it is apparent that numerous modifications and variations can be made without departing from the scope and spirit of this invention. It is therefore intended that this invention be limited only as indicated by the appended claims.
This application claims the benefits of U. S. Provisional Patent Application Ser. No. 60/483, 887, entitled “1,3,4-Trisubstituted-6-aryl-hexen-1,5-diynes, Their Preparation Processes, And Pharmaceutical Compositions Comprising The Same” and filed on Jun. 30, 2003.
| Number | Date | Country | |
|---|---|---|---|
| 60483887 | Jun 2003 | US |
