[CANCER RESEARCH 33, 832-837, April 1973] Correlation between Carcinogenicity and Chemical Structure in Cyclopenta[a]phenanthrenes M. M. Coombs. T. S. Bhatt, and C. J. Croft Chemistry Department, Imperial Cancer Research Fund, Lincoln 's Inn Fields, London, WC2A 3PX, England ¡M.M. C..T.S.B.], and the National Institute for Medical Research, Mill Hill, London, NW7IAA, England ¡C.J. C./ SUMMARY steroids, but they also retain oxygen functions at positions commonly oxygenated in this class of natural products. This The relationship between structure and carcinogenicity in fact has led to speculation that abnormal steroid metabolism fifteen new cyclopenta[a]phenanthrene derivatives, all closely by mammalian cells might produce endogenous carcinogens. related to the potent carcinogen [15,16-dihydro-l 1-methyl- Recently, Bischoff (2) reviewed work on carcinogenic hydro cyclopenta[a]phenanthren-17-one (compound VI)], was carbons in connection with the carcinogenic effects of tested by mouse skin painting. Each mouse received 30 /zg of steroids. the compound on the dorsal skin twice weekly for 1 year, and In a recent paper, Hill et al. (17) proposed a connection the experiment was concluded at the end of the 2nd year. between the higher incidence of carcinoma of the colon in Nine compounds were active carcinogens. The chrysene Western Europe and North America compared with that in analog (1,2,3,4-tetrahydro-l 1-methylchrysen-l-one) displayed East Africa, Asia, and South America, and the higher levels of the same high potency as did the ketone (VI), while the dietary steroids consumed in the former areas. Furthermore, parent, unsubstituted chrysene was inactive. The 11-methyl- they demonstrated a higher anaerobe-to-aerobe ratio in the 17-ol derived from compound VI and the 1l-methoxy-7- bacterial flora of feces from people living in these areas and methyl-17-ketone also possessed marked activity. The 7- concluded that these differences in intestinal flora were methyl-17-ketone was much less active than VI while the 2-, brought about by differences in diet. They suggested that the 3-, 4-, and 6-methyl isomers and the 11,12-dihydro derivative etiology of cancer of the colon could be understood if certain Were inactive. The 11-ethyl homolog was also much less active gut bacteria were able to generate carcinogens from steroid than VI and the 11-«-butyl homolog was almost without precursors. activity. The positional isomer 15,16-dihydro-l 1-methyl- There has been little work on the modification of steroids cyclopenta [a] phenanthren-15-one was only weakly active, but by anaerobic bacteria. However, Hill et al. recently showed moderate carcinogenicity was associated with the 6-methoxy that Clostridium spp. are able to effect nuclear dehydrogena- and 16-hydroxy dérivâtesofVI. tion and C-10 demethylation of androgens to yield 1,3,5(10)- The results are discussed in connection with the possible oestratrienes (15) and -tetrenes (private communication). mode of action of these compounds and in relation to Similar reactions are brought about by a number of other hypothetical routes of steroid degradation, especially by microorganisms not known to inhabit the gut, notably Bacillus anaerobic gut bacteria. cyclo-oxidans (19), Pseudomonas testosteronii (23), and Norcardia restrictus (22), while aromatization of ring B of 7-dehydrocholesterol was observed with the protozoon Acanthamoeba castallanii (18). A remarkably varied and INTRODUCTION increasingly large number of chemical transformation of steroids are known to be carried out by microorganisms (4, Strong carcinogenic activity has been observed in certain 24), including hydroxylation of the 18-methyl group by a derivatives of cyclopenta[a]phenanthrene' which bear oxygen Aspergillus niger strain (1). This is of importance, since it at C-ll and C-17 (8). These compounds not only have the could lead to loss of the angular methyl group under mild same carbon ring system as have the naturally occurring conditions, thus possibly allowing the aromatization of ring C with the formation of compounds of the cyclopenta[a]phen- 'The term cyclopenta[a]phenanthrene (RRI 4781) (21) is properly anthrene series (8). reserved for the fully unsaturated hydrocarbon of which XXI is the 15//-isomer; XX is therefore the 16,17-dihydro derivative. Except in These new developments indicate the need to delineate the formal chemical names, the term is used in this paper loosely to imply structural limits associated with carcinogenicity in compounds both types of structure. The ring positions are numbered according to which could arise as steroid transformation products. In this the steroid convention, as indicated. study, we investigated the connection between carcinogenicity n and chemical structure in a number of new cyclopenta[a] phenanthrene derivatives related to the potent carcinogen 15,16-dihydro-l l-methyl-cyclopenta[a] phenanthren - 17 - one (VI). In particular, the consequences of positional isomerism XXI of the methyl group and ketone function, of partial reduction Received November 9, 1972; accepted January 18, 1973. of the conjugated system, of the introduction of additional 832 CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1973 American Association for Cancer Research. Carcinogenic Cyclopenta[ a] phenanthrencs oxygen functions, and of varying the substituent at C-ll have received attention. The carcinogenicity of the related chry- senes, XV and XVI, has also been studied. The structures of these compounds are illustrated in Chart 1. MATERIALS AND METHODS Chemistry. With the exception of VII and VIII, the synthesis of Compounds I to XVI is described elsewhere (5-7, 9, 10). UV spectra (log e in parentheses) were recorded for ethanol solutions on a Perkin-Elmer Model 402 spectrophoto- meter and for infrared spectra as Nujol mulls on a Perkin-Elmer Model 257 spectrophotometer. TLC2 was carried out on glass plates (20 x 10 cm) coated with Merck Silica Gel G to a thickness of 0.25 mm, and these were dried for a minimum of 18 hr at room temperature before use. Preparation of 15,16-dihydro-l l-ethylcyclopenta[a jphenan- thren-17-one (VII). A solution of 17,17-ethylenedioxy-ll, 12,13,14,15,16-hexahydro-ll-oxocyclopenta[a]phenanthrene (4.25 g) (5) in dry benzene (30 ml) was added drop- wise to a solution of ethyl magnesium bromide [pre pared from magnesium turnings (0.72 g), ethyl bromide VII vni IX (3.27 g), and anhydrous ether (50 ml)], and the mixture was heated under reflux for 6 hr. The cooled reaction mixture was shaken with ice-cold M H2S04, the organic layer was washed with aqueous NaHCOs and with water, and the mixture was dried over anhydrous Na2 SCU. Evaporation gave an amber gum that possessed only weak infrared carbonyl absorption at 6 urn. This gum was dissolved in a mixture of nitrobenzene (15 ml) and glacial acetic acid (60 ml) and was heated under reflux for 45 min with concentrated HC1 (15 ml). After dilution with water, the nitrobenzene was removed in steam. Extraction of the tarry residue with chloroform gave a brown gum that was purified by chromatography on a column (40 x 2 cm in diameter) of WoëlmGrade III alumina, eluting with benzene. Fractions homogeneous by TLC (CH2C12) were combined to yield a pale fawn solid (2.9 g) which recrystallized from ethanol as large, pale brown platelets ( 1.7 g) of the 11-ethyl-17-ketone (VII), m.p., 129-130°. C19H160 Required: C 87.65, H 6.2 Found: C 87.4, H 5.95 \„ax, 264.5 (4.91), 289 (4.47), 302 (4.33), 360 (3.51), and 378 (3.54) nm; i^max, 5.88 (aryl ketone), 12.38, 13.36, 13.90, XV XVI and 14.54 /¿m. Chart 1. Compounds tested by skin-painting experiments. Preparation of 15,16-Dihydro-ll-n-butylcyclopenta[a]- phenanthren-17-one (Vili). A sample of the above oxoketal (1.48 g) was added to a 0.54 M solution (10.0 ml) of «-butyl already described. The dark brown gum obtained after removal lithium in ether, contained in a flask fitted with a water-cooled of the nitrobenzene in steam was purified by chromatography, as outlined above, to give fawn crystals [from ethanol (150 condenser under a dry nitrogen atmosphere. The solvent mg)] of the 11-H-buty 1-17-ketone (VIII), m.p., 113-114°. boiled and the solid dissolved, yielding a yellow solution. After 18 hr at ambient temperature, this solution was treated first C2 iHjoO with M H2SÛ4, then with a boiling mixture of nitrobenzene (6 ml), acetic acid (24 ml), and concentrated HC1 (6 ml), as Required: C 87.45, H 7.0 Found: C 87.35, H 6.6 1The abbreviation used is: TLC, thin-layer chromatogiaphy. Xinax, 264.5 (4.90), 289.5 (4.45), 302 (4.32), 360 (3.41), and APRIL 1973 833 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1973 American Association for Cancer Research. M. M. Coombs, T. S. Bhatt, and C. J. Croft 20 dehydrogenation of the 17-ol (XII) to the ketone (VI) was demonstrated as follows. Compound XII (50 jug), dissolved in CH pure dimethyl sulfoxide (20 jul), was added to a suspension of rat liver microsomes [prepared from 400 mg of fresh liver by -15 the method of Gelboin (14)] in phosphate-buffered saline (3.0 00 ml) that contained NADPH (20 mg). After incubation at 37° for 30 min with shaking in air, the mixture was extracted with ethyl acetate and the extract was examined by TLC in the -10 solvent systems, tolueneiethyl acetate:methanol (15:5:1, by volume) and benzene:methanol:glacial acetic acid (11:2:1, by volume).