Synthesis, cytotoxicity, and antitumor activity of lantadene-A congeners.

Five new derivatives of the pentacyclic triterpenoid lantadene A (= 22beta-angeloyloxy-3-oxoolean-12-en-28-oic acid; 1) from the leaves of Lantana camara L. were synthesized, characterized, and screened for their cytotoxicities against four human cancer cell lines. The three most-potent compounds, i.e., 1, 4, and 6, with IC50 values in the range of ca. 20-29 microM, were further studied for their in vivo tumor-inhibitory potential upon oral administration in two-stage squamous cell carcinogenesis, using female Swiss albino mice, papilloma being induced by 7,12-dimethylbenz[a]anthracene (DMBA), and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). The results are discussed in terms of structure-activity relationship.

Synthesis and structure determination of the adducts formed by electrochemical oxidation of 1,2,3,4-Tetrahydro-7,12-dimethylbenz[a]anthracene in the presence of deoxyribonucleosides or adenine.

Study of DNA adducts formed with aromatic hydrocarbons is part of the strategy to elucidate the mechanisms of tumor initiation by these compounds. 1,2,3,4-Tetrahydro-7,12-dimethylbenz[a]anthracene (THDMBA) is of special interest because it allows discrimination between the pathways of bioactivation by one-electron oxidation and monooxygenation. To study and identify adducts formed biologically, synthetic adducts are needed as reference standards. THDMBA was electrochemically oxidized in the presence of deoxyadenosine (dA), adenine (Ade), deoxyguanosine (dG), or deoxycytidine (dC). In the presence of dA, four adducts were isolated: 7-methyl-1,2,3,4-tetrahydrobenz[a]anthracene-12-CH2-N7Ade (7-MTHBA-12-CH2-N7Ade, 3.6%), 12-MTHBA-7-CH2-N7Ade (4.2%), 7-MTHBA-12-CH2-N6dA (5.8%), and 12-exo-methylene-7-MTHBA-7-N6dA (22.8%); a dehydrogenated product, 7,12-di-exo-methylene-THBA (44.2%), was also obtained. In the presence of Ade, nine adducts were synthesized: 7-MTHBA-12-CH2-N7Ade (1.1%), 12-MTHBA-7-CH2-N7Ade (2.4%), 7-MTHBA-12-CH2-N1Ade (10.2%), 12-MTHBA-7-CH2-N1Ade (13.2%), 7-MTHBA-12CH2-N3Ade (1.7%), 12-MTHBA-7-CH2-N3Ade (1.7%), 7-exo-methylene-12-MTHBA-12-N3Ade (11.2%), 12-exo-methylene-7-MTHBA-7-N3Ade (27.9%), and 12-exo-methylene-7-MTHBA-7-N6Ade (12.1%), as well as the dehydrogenated product 7,12-di-exo-methylene-THBA (16.7%). In the presence of dG, three adducts were produced: 7-MTHBA-12-CH2-N7Gua (24.2%), 12-MTHBA-7-CH2-N7Gua (12.2%), and 7-MTHBA-12-CH2-N2dG (3.7%), as well as the dehydrogenated product 7,12-di-exo-methylene-THBA (38.9%). Anodic oxidation in the presence of dC yielded a large amount of 7,12-di-exo-methylene-THBA (80.4%), but no adducts. The structure of the adducts was elucidated by using UV, NMR, and MS. The N-7 positions in dG, dA, and Ade, the 2-NH2 in dG, and the N-1 position in Ade form exclusively methyl-linked adducts. In contrast, the 6-NH2 group of dA and Ade and the N-3 of Ade prefer to attack the meso-anthracenic positions rather than the methyl groups. The order of reactivity of dG and dA in the formation of methyl-linked THDMBA adducts agrees well with that previously found for 7,12-dimethylbenz[a]anthracene [RamaKrishna et al. (1992) J. Am. Chem. Soc. 114, 1863-1874.

Biologically active dihydrodiol metabolites of polycyclic aromatic hydrocarbons structurally related to the potent carcinogenic hydrocarbon 7,12-dimethylbenz[a]anthracene.

Syntheses of the trans-dihydrodiol derivatives implicated as the proximate carcinogenic metabolites of the polycyclic hydrocarbons cholanthrene, 6-methylcholanthrene, benz[a]anthracene, and 7- and 12-methylbenz[a]anthracene are described. These compounds are useful models for research to determine the molecular basis of the strong enhancement of carcinogenicity consequent upon methyl substitution in nonbenzo bay molecular sites and meso regions of polycyclic hydrocarbons. Synthesis of the bay region anti-diol epoxide derivative of cholanthrene, its putative ultimate carcinogenic metabolite, is also described. Tumorigenicity assays indicate that the 9,10-dihydrodiol derivatives of cholanthrene and its 3- and 6-methyl derivatives are all potent tumor initiators on mouse skin. The most active member of the series is the dihydrodiol derivative of 6-methylcholanthrene, which contains a bay region methyl group. The ability of the dihydrodiols 3a-c and the trans-3,4-dihydrodiol of 7,12-dimethylbenz[a]anthracene (3d) to induce chromosomal aberrations in rat bone marrow cells was also examined. The observed order of activity was 3d greater than 3c greater than 3b greater than 3a. These findings are consistent with the hypothesis that the diol epoxide metabolites of these dihydrodiols are the active carcinogenic forms of the parent hydrocarbons.

Crystal structure of a carcinogen:nucleoside adduct.

The product of reaction between the carcinogen, 7-bromomethyl-12-methylbenz[a]anthracene, and 2'-deoxyadenosine, i.e., N6-(12-methylbenz[a]anthracenyl-7-methyl)deoxyadenosine, has been prepared and characterized, and its structure has been determined by X-ray crystallographic techniques. The major structural features are: (a) the adenine and polycyclic aromatic hydrocarbon residues lie nearly perpendicular to one another; (b) the conformation about the glycosidic bond is syn, rather than anti, and an internal hydrogen bond between the deoxyribose 5'-hydroxyl group and N(3) of the adenine residue is present; and (c) the more planar anthracene portion of the hydrocarbon is stacked between adenine residues of other molecules throughout the crystal.

Tumorigenicity of 7,12-dimethylbenz[a]anthracene, its hydroxymethylated derivatives and selected dihydrodiols in the newborn mouse.

The newborn mouse lung adenoma model has been shown to be a sensitive test for studying the tumorigenicity of bay region diol epoxides and their precursor dihydrodiols. When a total dose of 28 nmol of 7,12-dimethylbenz[a]anthracene (DMBA) or its derivatives was injected i.p. into the preweaning mice, it was found that the 3,4-dihydrodiols of both DMBA and 7-hydroxymethyl-12-methylbenz[a]anthracene caused 13.3 and 4.1 times more lung adenomas than DMBA, respectively. The mice treated with the 5,6- and 8,9-dihydrodiols of DMBA, 7-hydroxymethyl-12-methylbenz[a]anthracene and its 5,6- and 8,9- and 10,11-dihydrodiols, 7-methyl-12-hydroxymethylbenz[a]anthracene and 7,12-dihydroxymethylbenz[a]anthracene developed a level of lung adenomas/mouse less than 2-fold higher than that found in the DMSO-treated control group. Liver tumors also developed in some of the mice. The percentage of mice with liver tumors also indicated that the 3,4-dihydrodiols of both DMBA and 7-hydroxymethyl-12-methylbenz[a]anthracene were more tumorigenic than DMBA itself. These data indicate that the 3,4-dihydrodiols of both DMBA and its 7-hydroxymethyl derivative may be proximate carcinogenic metabolites of DMBA in the newborn mouse.

Synthesis and mutagenicity of A-ring reduced analogues of 7,12-dimethylbenz[a]anthracene.

The synthesis and mutagenicity of two derivatives of 7,12-dimethylbenz[a]anthracene (DMBA; 1), i.e., 1,2-H2DMBA (4) and 1,2,3,4-H4DMBA (5), are reported. These analogues (4 and 5) represent dihydro and tetrahydro A-ring reduced forms of DMBA, a region in the parent hydrocarbon (1) proposed to be involved in metabolism to the ultimate carcinogen. The synthesis for 4 without isolation of intermediates from the tosylhydrazone of 1,2,3,4-tetrahydrobenz[a]anthracene-4,7,12-trione (10) by successive reaction with 8 molar equiv of CH3Li, HI, and NaBH4 represents a novel approach to this hydrocarbon now available in sufficient quantity for biological studies. Interestingly, both of these reduced analogues 4 and 5 exhibited mutagenic activity in the Ames assay in the presence or absence of microsomal activation for strains TA98 and TA100. In these strains, DMBA was active only in the presence of S-9 fraction. In the plasmid-deficient strain TA1537, only tetrahydro analogue 5 exhibited mutagenic activity both in the absence and presence of S-9 fraction.

Synthesis and carcinogenic activity of 5-fluoro-7-(oxygenated methyl)-12-methylvenz[a]anthracenes.

Treatment of 7,12-benz[a]anthraquinone (2) with methylmagnesium iodide or methyllithium yields mixtures of cis- and trans-7,12-dihydro-7,12-dihydroxy-7,12-dimethylbenz[a]anthracenes (3a,b), in which the ratio of cis to trans lies in the 3--4:1 region. Each isomer afforded high yields of 7-chloromethyl-12-methylbenz[a]anthracene (5) on treatment with hydrogen chloride in ethyl acetate. Similarly, 5-fluoro-7,12-benz[a]anthraquinone (8) afforded a mixture of cis- and trans-5-fluoro-7,12-dihydro-7,12-dihydroxy-7,12-dimethylbenz[a]anthracenes (9) which yielded 7-chloromethyl-5-fluoro-12-methylbenz[a]anthracene (10) on treatment with HCl. The chloromethyl compounds, 5 and 10, yielded 7-acetoxymethyl-12-methylbenz[a]anthracene (6) and 7-acetoxymethyl-5-fluoro-12-methylbenz[a]anthracene (11) on treatment with acetate ion. Hydrolysis of 6 and 11 yielded 7-hydroxymethyl-12-methylbenz[a]anthracene (7) and 5-fluoro-7-hydroxymethyl-12-methylbenz[a]anthracene (12), respectively. Since neither 11 nor 12 is appreciably carcinogenic, the carcinogenic metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) probably does not involve attack at the 7-methyl group.