Chem. Res. Toxicol. 1988,1, 349-355
349
Synthesis and Tumor- Initiating Activities of Dimethylchrysenest Shantu Amin, George Balanikas, Keith Huie, and Stephen S. Hecht* Division of Chemical Carcinogenesis, American Health Foundation, 1 Dana Road, Valhalla, New York 10595 Received June 6, 1988
Previous studies have shown that 5-methylchrysene (5-MeC) is more carcinogenic on mouse skin than the other methylchrysenes and that the structural requirements favoring tumorigenicity of methylated polynuclear aromatic hydrocarbons are the presence of a bay region methyl group and free peri position, both adjacent to an unsubstituted angular ring. The purpose of this study was t o extend these structure-activity relationships to dimethylchrysenes. The following dimethylchrysenes were synthesized: 1,Ei-dimethylchrysene (1,5-diMeC), 5,6-diMeC, 5,7-diMeC, 5,12-diMeC, 1,6-diMeC, 6,7-diMeC, and 6,12-diMeC. Bioassays of these compounds for tumor-initiating activity on mouse skin demonstrated that all were significantly less tumorigenic than 5-MeC; only 5,6-diMeC had significant tumorigenic activity. Since the relatively low activities of 5,7-diMeC and 5,6-diMeC were unexpected on the basis of the structural requirements (anti-5,7stated above, anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5,7-dimethylchrysene diMeC-l,2-diol-3,4-epoxide) was synthesized. Its mutagenicity in Salmonella typhimurium and reactivity with calf thymus DNA were compared to those of the major ultimate carcinogen of It was strongly mutagenic (2500 revertants/nmol), 5-MeC, anti-5-MeC-1,2-diol-3,4-epoxide. (7200 revertants/nmol). Its reactivity although less active than anti-5-MeC-l,2-diol-3,4-epoxide The results of with calf thymus DNA was similar to that of anti-5-MeC-1,2-diol-3,4-epoxide. this study demonstrate that the structural requirements which favor tumorigenicity of monomethylchrysenes are not sufficient for high tumorigenicity of dimethylchrysenes.
structural requirements were incomplete, since 5,6-diMeC Introduction and 5,7-diMeC were both significantly less tumorigenic Our previous studies of the monomethylchrysenes and than was 5-MeC. In order to better understand the low their fluorinated derivatives have indicated that the tumorigenicity of 5,7-diMeC,we also synthesized its susstructural requirements favoring tumorigenicity on mouse pect ultimate carcinogen, anti-5,7-diMeC-192-diol-3,4-epskin are the presence of a bay region methyl group and a oxide, and assessed its mutagenicity in Salmonella typhfree peri position both adjacent to an unsubstituted animurium TA 100 and binding to calf thymus DNA. gular ring (1,2). Among the monomethylchrysenes, only 5-MeC' fulfills these requirements, and it is the only Experimental Section strongly tumorigenic isomer (2,3). Among the fluorinated Apparatus. NMR spectra were determined in CDCISwith a derivatives of 5-MeC, high tumorigenicity is observed only Jeol Model FX9OQ spectrometer and a Bruker AM 360 specwhen fluorine is not substituted in the 1-4 ring or at the trometer. UV spectra were run on a Hewlett-Packard Model 12-position (see Figure 1)(4). These results are consistent with the identification of anti-5-MeC-l(R),2(S)-diol-3- 8452A diode array spectrophotometer. MS were determined with a Hewlett-Packard Model 5988A instrument. High-resolution (S),4(R)-epoxideas an ultimate carcinogen of 5-MeC and MS were determined on a VG 70-250 double-focusing magnetic with the uniquely tumorigenic properties of this diol epsector instrument, a t the Rockefeller University Mass Spectrooxide which has a methyl group and epoxide ring in the metric Biotechnology Resource. All starting materials were obsame bay region (5, 6). However, limited information is tained from Aldrich Chemical Co., Milwaukee, WI, unless stated available on the mouse skin tumorigenicity of dimethylotherwise. chrysenes. In previous comparative studies, we have obSynthesis. (A) 2-(4-Methyl-l-naphthyl)-l-phenyl-2propanol (7). To a stirred solution of 1.84 g (0.01 mol) of 4 in served that 5,ll-diMeC is highly tumorigenic whereas 50 mL of EtzO a t 0 "C under Nzwas added dropwise benzyl5,12-diMeC is inactive (1,7). These results are consistent magnesium chloride (13.0 mL, 1 M in THF, 0.013 mol). The with the structural requirements stated above. Mouse skin reaction mixture was stirred under N2 overnight while warming tumorigenicity assays of 5,6-diMeC and 5,7-diMeC indito room temperature, poured into dilute HCl, and extracted with cated that these compounds were weakly tumorigenic, but EGO. The combined EGO extracts were washed with H20, dried comparative studies with other methylchrysene or di(MgS04), and concentrated to afford 2.2 g of a light yellow oil. methylchrysene isomers were not performed (8, 9). In Chromatography on silica gel, with elution by CHzC12/hexane, order to obtain more information on the structural regave 7 as a thick oil (1.7g, 61.1%). NMR 6 1.6 (s, 3 H),2.1 (e, quirements favoring tumorigenicity of dimethylchrysenes, 1H, OH), 2.5 (9, 3 H),3.2 (d, 2 H), 6.9-7.6 (m, 10 H), 7.8-8.1 (m, 1 H);MS m / e (relative intensity) 185 (M', loo), 169 (36), 141 we have synthesized 1,5-diMeC, 1,6-diMeC, 5,6-diMeC, (30), 91 (35). 5,7-diMeC, 6,7-diMeC, and 6,12-diMeC and have compared their tumorigenic activities on mouse skin, as well as assessing their tumorigenic potency compared to that of Abbreviations: 5-MeC, 5-methylchrysene; anti-B-MeC-l(R),2(S)5-MeC. The results showed that the previously stated dioL3(S),4(R)-epoxide, anti-l(R),2(S)-dihydroxy-3(S),4(R)-epoxy'This is paper 118 in the series "A Study of Chemical Carcinogenesis". 0893-228~/88/2701-0349$01.50/0
1,2,3,4-tetrahydro-5methylchrysene; 5,11-diMeC, 5,ll-dimethylchrysene; 5,12-diMeC,5,12-dimethylchrysene;anti-5,7-diMeC-1,2-diol-3,4-epoxide,
anti-l,2-dihydroxy-3,4-epoxy-l,2,3,4-tetrahydro-5,7-dimethylchrysene; TPA, 12-0-tetradecanoylphorbol13-acetate.
0 1988 American Chemical Society
350 Chem. Res. Toxicol., Vol. I , No. 6, 1988 12
7
1
6
Figure 1. Chrysene ring system. By use of the above procedure, alcohols 5 (70% yield) and 6 (50% yield) were synthesized by reaction of the appropriately substituted Grignard reagent with either 3 or acetonaphthone, respectively. NMR of 5: 6 1.8 (s, 3 H), 2.1 (s, 1 H, OH), 2.25 (s, 3 H), 3.5 (d, 2 H), 6.9-7.9 (m, 11 H). NMR of 6: 6 1.75 (s,3 H), 2.1 (s, 1 H, OH), 2.3 (s, 3 H), 3.3 (d, 2 H), 6.9-8.1 (m, 11 H). (B) 2- (4-Methylnapht hy1)-1-phenylpropene ( 10). Alcohol 7 (1.3 g, 0.005 mol) was dissolved in benzene (150 mL), and 5 mg of p-toluenesulfonic acid was added. The mixture was heated under reflux for 2 h, using a Dean-Stark trap. The usual workup gave a mixture of 10 and its exo-methylene isomer (1.1g, 82%), which was used without further purification in the next step. NMR 6 2.8 (s, 3 H, aromatic CH3), 2.9 (s, 1.5 H, vinylic CH3 of lo), 4.0 (s,0.5 H, CH2 of exo isomer), 5.5 (d, 0.5 H, =CH, of exo isomer), 6.8-7.7 (m, 10.5 H, 10 H, aromatic + 0.5 H, olefinic), 8.15-8.2 (m, 1H); MS m / e (relative intensity) 258 (M+, 93.3), 243 (1001, 167 (100). By use of the above procedure, alkenes 8 (75% yield) and 9 (64% yield) were prepared from the corresponding alcohols. NMR of 8: 6 2.6 (e, 3 H, aromatic CH,), 2.7 (s, 1.2 H, vinylic CH, of 8), 3.7 (s, 0.7 H, CH2 of exo isomer), 5.15 (s, 0.7 H, =CH, of exo isomer), 6.9-8.15 (m, 11.4 H). NMR of 9: 6 2.7 (s, 3 H, aromatic CH3 + s, 1H, vinylic CH3 of 9), 3.75 (s,0.8 H, CH, of exo isomer), 5.25 (s, 0.8 H, =CH2 of exo isomer), 7.0-7.5 (m, 8.3 H, 8 H, aromatic + 0.3 H, olefinic), 7.6-8.1 (m, 3 H). (C) 5,12-DiMeC. Photolysis of 10 (0.25 g, 0.001 mol) for 6 h under conditions as described below for the preparation of 1,6diMeC gave 5,12-diMeC, which was purified by column chromatography on silica gel, mp 112-113 "C [lit. mp 113 "C ( l o ) ] (38%). NMR 6 2.8 (s, 3 H), 3.2 (s,3 H), 7.5-7.8 (m, 6 H), 8.1-8.25 (m, 1 H), 8.6 (s, 1 H, HIJ, 8.65-8.8 (m, 1 H), 8.9-9.1 (m, 1 H); MS m / e (relative intensity) 256 (M+, loo), 239 (45.3). Highresolution MS: calculated for M+, 256.125; found, 256.126. In a similar manner, 1,5- and 5,7-diMeC were prepared from the corresponding alkenes 8 and 9 in 30% and 34% yields, respectively. (D) 1,5-DiMeC. Mp 117-118 "C. NMR 6 2.65 (s, 3 H), 3.4 (s, 3 H), 7.25-7.5 (m, 6 H), 7.75 (s, 1 H, H6), 7.85-8.2 (m, 3 H, H,, Hlo, and Hll). High-resolution MS: calculated for M - H, 255.117; found, 255.122. (E)5,7-DiMeC. Mp 165-166 "C [lit.' mp 168.5-169 "C (S)]. NMR 6 2.7 (s, 3 H), 3.45 (s, 3 H), 7.15-7.6 (m, 6 H), 7.8 (s, 1 H, He), 7.83-8.1 (m, 3 H, H,,Hlo, and Hll). High-resolution MS: calculated for M+, 256.124; found, 256.123. (F) Methyl 2-P henyl-3-(4-met hyl- 1-naphthyl) propenoate (15). A solution of phenylacetic acid (11) (2.7 g, 0.02 mol), 4methyl-1-naphthaldehyde (13) (3.4 g, 0.02 mol), 5 mL of triethylamine, and 5 mL of acetic anhydride was heated with stirring at 140 OC for 8 h. After cooling, the dark solution was diluted with H20 (150 mL) and acidified with 50 mL of concentrated HC1. The resulting suspension was extracted with CH2C12(2 X 150 mL). The CH2C12layer was washed with HzO, dried (MgSO,), and concentrated to afford 1.8 g of crude 2-pheny1-3-(4-methyl-lnaphthy1)propionicacid, which was used in the next step without further purification. MS m / e (relative intensity) 288 (M', 19.9), 243 (44.3). A mixture of the above acid (1.8 g, 0.0062 mol), KzCO3 (2.5 g, 0.18 mol), and dimethyl sulfate (1.5 g, 0.012 mol) in 150 mL of acetone was heated under reflux for 2 h. The reaction mixture was then filtered, and the K2C03was washed several times with CH2C12.The organic layer was washed with HzO, dried (MgS04), and evaporated to give the crude ester. This was purified by chromatography on silica gel with elution by hexane/CH,C12 (80:20) to give 1.2 g (63%) of a pure yellow oil 15. NMR 6 2.3 (s, 3 H, CH3), 4.0 (s, 3 H, OCH,), 6.9-7.5 (m, 4 H), 7.55-7.7 (m, 4 H), 7.9-8.2 (m, 3 H), 8.5 (s, 1 H); MS m / e (relative intensity)
Amin et al. 302 (M', 57.2), 243 (loo), 228 (49.0). By use of this method, alkene 16 was prepared in 40% yield from o-methylphenylacetic acid (12) and 1-naphthaldehyde (la), mp 112-114 "C. NMR 6 2.3 (s, 3 H, CH,), 4.0 (s, 3 H, OCH,), 6.9-7.4 (m, 7 H), 7.5-8.0 (m, 4 H), 8.6 (s, 1 H); MS m / e (relative intensity) 302 (M', 70.6), 243 (100). In a similar manner, alkene 27 was prepared in 27% yield from 6-methoxy-1-naphthylaceticacid (26) and o-tolualdehyde (24). NMR 6 2.45 (s, 3 H, CH,), 3.71 (5, 3 H, COOCH,), 3.92 (s,3 H, OCH,), 7.0-7.4 (m, 8 H), 7.68 (dd, 2 H), 8.3 (s, 1 H); MS m / e (relative intensity) 332 (M', loo), 273 (68.1), 241 (22.5). ( G ) 12-Carbomethoxy-6-methylchrysene(17). Photolysis of 15 (0.9 g, 0.003 mol) for 6 h, under conditions as described for the preparation of 1,6-dimethylchrysene, gave 17, which was purified by crystallization from ethanol, 0.44 g (50%); mp 110-112 "C. NMR 6 2.9 (s, 3 H, CH,), 4.1 (s, 3 H, OCH,), 7.6-7.8 (m, 4 H), 8.1-8.2 (dd, 2 H), 8.45 (s, 1 H), 8.75-8.85 (m, 1 H), 9.0-9.1 (m, 1 H), 9.4 (s, 1 H); MS m / e (relative intensity) 300 (M', 90.5), 269 (50). Similarly,6-carbomethoxy-7-methylchrysene (18) was prepared from alkene 16 in 55% yield, mp 137-138 "C. NMR 6 2.75 (s, 3 H, CH,), 4.1 (s, 3 H, OCH,), 7.5-7.9 (m, 4 H), 7.95-8.2 (m, 2 H), 8.5-8.9 (m, 4 H); MS m / e (relative intensity) 300 (M', 76.8), 286 (52.0), 269 (100). Likewise, 8-methoxy-11-carbomethoxy-1-methylchrysene (28) was prepared from 27 in 50% yield, mp 162-163 "C. NMR 6 2.75 (s, 3 H, CH,), 4.0 (9, 6 H, OCH,), 7.25-7.35 (m, 2 H, H7 and Hg), 7.48 (d, 1 H, Hz, 52-3 = 6.9 Hz), 7.65 (dtd, 1 H, H3,52-3 = 53-4 = 6.9 Hz), 7.95 (d, 1 H, H,j, J w = 9 Hz), 8.1 (d, 1 H, Hg, 5m = 9 Hz), 8.45 (s, 1 H, H12), 8.6 (d, 1 H, H4, 53-4 = 7.0 Hz), 8.7 (d, 1 H, Hlo,Jg_lo = 9.1 Hz); MS m / e (relative intensity) 330 (M', 100), 299 (31.5). (H) 12-(Hydroxymethyl)-6-methylchrysene (19). A solution of 17 (0.5 g, 0.0016 mol) in 25 mL of dry THF was added dropwise to a suspension of L N H 4 (0.1 g, 0.0027 mol) in 50 mL of dry THF. The mixture was stirred for 2 h at room temperature, poured into H20,and extracted with EhO. The organic layer was washed with HzO and dried (MgSO,). Removal of the solvent afforded alcohol 19, which was used in the next step without further purification. NMR 6 2.95 (s,3 H), 3.7 (b S, 1 H, OH), 5.4 (s,2 H, CHJ, 7.4-7.7 (m, 4 H), 7.9-8.1 (m, 2 H), 8.5-8.8 (m + s, 4 H); MS m/e (relative intensity) 272 (60.3), 258 (20.5), 254 (100). High-resolution MS: calculated for M+, 272.119; found, 272.117. In a similar manner, ester 18 (0.6 g, 0.002 mol) was converted to 6-(hydroxymethyl)-7-methylchrysene(21), mp 159-160 "C. NMR 6 3.0 (9, 3 H), 3.8 (bs, 1 H, OH), 5.4 (s, 2 H, CHZ), 7.4-7.8 (m, 4 H), 7.9-8.1 (m, 2 H), 8.5-8.9 (m + s, 4 H); MS m / e (relative intensity) 272 (M+,88.5), 258 (37.3), 254 (100). High-resolution MS: calculated for M+, 272.119; found, 272.114. By the same method, 8-methoxy-ll-(hydroxymethyl)-lmethylchrysene (29) was prepared in 88% yield from the corresponding ester 28, mp 245-248 "C. NMR 6 2.78 (s, 3 H, CH,), 3.3 (b s, 1 H, OH), 4.0 (s, 3 H, OCH3),5.2 (s, 2 H, CHzOH), 7.3 (dd, 1 H, Hg, Jg-10 = 9.2 Hz, 57-9 = 2.9 Hz), 7.5 (d, 1 H, H,, 52-3 = 6.9 Hz), 7.58 (d, 1 H, H7,57-g = 2.9 Hz), 7.6 (dd, 1 H, H3,52-3 = 53-4 = 8.5 Hz), 8.0 (d, 1H, He, 5~ = 9.0 Hz), 8.35 (9, 1H, Hlz), 8.7 (d, 1 H, H4), 8.8 (d, 1 H, Hg, 55-6= 9.0 Hz), 8.98 (d, 1 H, Hlo); MS m / e (relative intensity) 302 (M', 100), 284 (12.8), 273 (24.5). (I) 6-Methylchrysene-l2-carboxaldehyde (20). A solution of 19 (0.27 g, 0.001 mol) in 20 mL of CHZClzwas added dropwise during 10 min to a stirred suspension of 0.43 g pyridinium chlorochromate in 50 mL of CH2ClZ. The mixture was stirred for 3 h at room temperature. The usual workup gave the crude aldehyde 20 (0.24 g, 89%) which was purified by elution of hexane/CHzC1, through silica gel. NMR 6 2.9 (s, 3 H), 7.4-7.8 (m, 4 H), 7.9-8.1 (m, 2 H), 8.4-8.8 (m, 3 H, H4, Hlo and Hll), 9.1 (s, 1 H, HJ, 10.8 (s, 1 H, CHO). MS m / e (relative intensity) 270 (M+, 20.5), 231 (58.7), 216 (100). By the same procedure, 6-(hydroxymethyl)-7-methylchrysene (21) (0.27 g, 0.001 mol) was converted to 7-methylchrysene-6carboxaldehyde (22),mp 154-156 "C. NMR 6 2.85 (s,3 H), 7.5-8.2 (m, 4 H), 7.9-8.2 (m, 2 H), 8.5-8.9 (m, 3 H, H4,Hlo, and Hll), 9.1 (s, 1 H, H5), 10.8 (s, 1 H, CHO); MS m / e (relative intensity) 270 (M', 28.5), 231 (59.5), 216 (100). Similarly,l-methyl-8-methoxyc~sene-1l-carboxaldehyde (30) was prepared in 95% yield from the corresponding alcohol 29,
Tumor-Initiating Activities of Dimethylchrysenes mp 164-165 "C. NMR 6 2.85 (s, 3 H, CH3), 4.0 (s, 3 H, OCH3), 7.35 (dd, 1 H, H9,57-9 = 2.3 Hz, 54-10 = 9.0 Hz), 7.4 (d, 1 H, H7, 57-9= 2.3 Hz), 7.5 (d, 1 H, H2,52-3 = 6.9 Hz), 7.7 (t, 1H, H3, 52-3 = 7.1 Hz), 7.95 (d, 1 H, He, 5S-B = 9 Hz), 8.0 (d, 1 H, H 5 , 5 = ~ 9.2 Hz), 8.58 (d, 1H, H4,53-4 = 8.5 Hz), 8.6 (s, 1H, Hlz), 8.65 (d, 1 H, Hlo, Jsl0= 9.1 Hz), 10.6 (s, 1 H, CHO); MS m/e (relative intensity) 300 (M', loo), 285 (17.6), 272 (8.8). (J) 6,12-DiMeC. A mixture containing aldehyde 20 (0.13 g, 0.5 mmol), hydrazine (1mL), and KOH (0.11 g, 2 mmol) in 25 mL of diethylene glycol was heated under reflux for 2 h. After cooling, 5 mL of concentrated HC1 was added, and the mixture was extracted with CH2C12(3 X 100 mL). The organic layer was washed with H 2 0 ,dried (MgS04),and concentrated to give 0.1 g of 6,12-diMeC which was recrystallized from ethanol; mp 236-237 "C [lit.mp 238 "C (IO)]. NMR 6 2.8 (s, 6 H, CH3),7.5-7.7 (m, 4 H), 8.0-8.2 (m, 2 H), 8.45 (s, 2 H, H5 and Hll), 8.7-8.9 (m, 2 H, H4 and Hlo); MS m/e (relative intensity) 256 (M', 100). High-resolution MS: calculated for M', 256.124; found, 256.122. In a similar manner, 6,7-diMeC was prepared from the corresponding aldehyde 22 in 50% yield, mp 194-196 "C. NMR 6 2.8 (s, 6 H, CH3), 7.5-8.2 (m, 6 H), 8.5-8.9 (m + s, 4 H, H4, H5, Hlo, and Hll); MS m/e (relative intensity) 256 (M+, loo), 239 (59.8). High-resolution MS: calculated for M', 256.124; found, 256.120. Likewise, 2-methoxy-5,7-dimethylchrysene (31) was prepared from the corresponding aldehyde 30 in 56% yield, mp 176-177 "C. NMR 6 2.8 (s, 3 H,CH3),3.2 (s, 3 H, CH3),4.0 (s, 3 H, OCH3), 7.2-7.3 (m, 2 H, H1 and H3), 7.4 (d, 1 H, HE,JS9= 6.9 Hz), 7.5 (dd, 1H, Hg, 58-9= 54-10 = 7.1 Hz), 7.89 (d, 1H, H12,Jii-i~ = 9.0 Hz), 7.98 (9, 1 H, He), 8.58 (d, 1 H, H11, 511-12 = 9.0 Hz), 8.7 (d, 1 H, Hlo), 8.8 (d, 1 H, H,); MS m/e (relative intensity) 286 (M', loo), 271 (11.5).
(K)l-(4-Methyl-l-naphthyl)-2-(2-methylphenyl)ethylene
(25). To a stirred solution of the salt 23 (1.0 g, 0.002 mol) and o-tolualdehyde (0.24 g, 0.002 mol) in 50 mL of methanol was added sodium methoxide (0.11 g, 0.002 mol). The mixture was stirred at room temperature for 1 h, diluted with H 2 0 ,dried (MgS04), and concentrated. Silica gel chromatography of the resulting oil with elution by hexane gave 25 (0.25 g, 48%). NMR 6 2.4 (s, 6 H, CH3), 6.9-7.4 (m, 8 H), 7.5-8.1 (m, 4 H); MS m/e (relative intensity) 258 (M+, loo), 243 (48.5), 167 (100). (L) 1,6-DiMeC. Dry air was bubbled through a solution of 25 (0.12 g, 0.46 mmol) and 5 mg of Iz in dry benzene. This was irradiated with a Havonia 450-W medium-pressure lamp, using a Pyrex filter. The reaction was followed by TLC; after 10 h, 60% of the alkene had cyclized. Removal of the solvent gave a liquid which was purified by chromatography on silica gel with elution by hexane to give 40 mg of 1,6-diMeC. It was recrystallized from MeOH to give pure 1,6-diMeC (30 mg, 25%), mp 144-145 "C. NMR 6 2.8 (s, 3 H, CH3), 2.9 (s, 3 H, CH3), 7.3-7.8 (m, 4 H, H2, H3, Hg, and H9),8.0-8.2 (m d, 2 H, H7 and H12),8.5-8.9 (m + s, 4 H, H,, H5, Hlo, and Hll); MS m/e (relative intensity) 256 (M', loo), 239 (30.5). High-resolution MS: calculated for M+, 256.125; found, 256.126. (M) 2-Hydroxy-5,7-dimethylchrysene(32). To a stirred solution of 31 (0.23 g, 0.8 mmol) in 100 mL of CH2Clzwas added dropwise, over a period of 5 min, a solution of boron tribromide (1.6 mL, 1 M in CH2C12)at 0 OC under N2. The mixture was stirred for 12 h at room temperature and poured into ice-cold H20. The organic layer was collected, washed with HzO, dried (MgS04), and recrystallized from CH2ClZ/hexane to give pure 2-hydroxy5,7-dimethylchrysene (32) which was used in the next step without further purification. MS m/e (relative intensity) 272 (M+, loo), 244 (72.4). (N) 5,7-Dimethylchrysene-1,2-dione(33). A solution of Fremy's salt (0.58 g, 0.0021 mol) in 35 mL of 0.17 M KH2P04was added to a stirred solution of 32 (0.21 g, 0.77 mmol) and Adogen 464 (3 drops) in 130 mL of CH2C12/benzene(16:84). The reaction mixture was stirred for 12 h at room temperature, poured into H20,and extracted with benzene (3 X 50 mL). The organic layer was washed with H20,dried (MgS04),and concentrated, affording crude 33 (0.11 g, 50%), which was recrystallized from CH2C12/ hexane, mp 197-199 OC. NMR 6 2.8 ( s , 3 H, CH3), 3.0 ( s , 3 H, CH,), 6.5 (d, 1 H, H,,J3+ = 10.8 Hz), 7.5-7.6 (m, 2 H, HEand HB),8.0 (9, 1 H, He), 8.3 (d, 1 H, H11,511-12 = 8.6 Hz), 8.48 (d, 1 H, Hi09 J+lo = 7.9 Hz), 8.55 (d, 1 H, H4, 53-4 = 10.8 Hz), 8.8 (d,
+
Chem. Res. Toxicol., Vol. 1, No. 6,1988 351 1H, Hlz, 511-12 = 8.7 Hz); MS m/e (relative intensity) 286 (M', 12.5), 258 (100). (0)1,2-Dihydro-l,2-dihydroxy-5,7-dimethylchrysene (34). NaBH4 (0.5 g) was added to a stirred suspension of quinone 33 (0.1 g, 0.34 mmol) in 100 mL of ethanol, and the reaction mixture was stirred at room temperature for 48 h. The resulting light yellow solution was poured into HzO and extracted with ethyl acetate. The organic phase was washed with H20,dried (MgS04), and evaporated to dryness. The crude diol was purified by chromatography on Florisil with elution by CH2Clz and CH2C12/ethylacetate (6535) to give the pure diol 34 (30 mg, 31%), mp 182-183 "C. NMR 6 2.7 (s, 3 H, CH,), 3.0 (s, 3 H, CHJ, 4.3 (b s, 1 H, OH), 4.5-4.56 (m, 2 H, H2 and OH), 4.6 (dd, 1 H, H1, 51-2= 11.7 Hz, J1aH = 5.2 Hz), 6.15 (d, 1 H, H3,53-4 = 10.4 Hz), 7.3-7.45 (m, 3 H, H4,HE,and H9), 7.75 (s, 1H, He), 7.95 (d, 1 H, H12, 511-12 = 8.5 Hz), 8.5 (d, 1 H, Hlo, J+lo = 8.7 Hz), 8.7 (d, 1 H, Hll, 511-12 = 8.4 Hz); MS m/e (relative intensity) 290 (M', 54.5), 272 (76.8), 244 (36.8). (P) anti-5,7-DiMeC-l,2-diol-3,4-epoxide (35). A mixture of 10 mg (0.34 mmol) of diol 34,100 mg of m-chloroperbenzoic acid, and 20 mL of dry THF was stirred under N2 for 5 h. The reaction mixture was diluted with 100 mL of EtzO, washed with 2% aqueous NaOH (3 x 20 mL) and HzO, and dried (K2CO3). Evaporation of the solvent gave white crystals of 35, which were recrystallized from Et20/CH2C12,yielding 8 mg, 96%. NMR 6 2.7 (s, 3 H, CH3), 2.95 (s, 3 H, CH3), 3.74 (dd, 1H, H3), 3.82 (m, 1 H, Hz), 4.61 (d, 1 H, Hi, 51-2 = 8.2 Hz), 4.9 (d, 1H, H4,53-4 = 3.5 Hz), 5.4 (dd, 2 H, OHl and OHz), 7.45-7.55 (m, 2 H, HEand Hg), 7.9 (s,1 H, He), 8.25 (d, 1 H, H12,511-12 = 8.8 Hz), 8.7 (d, 1 H, Hie, 54-10 = 7.4 Hz), 8.85 (d, 1 H, Hi,, 511-12 = 8.6 Hz). MS m/e (relative intensity) 288 (18), 273 (8), 139 (44), 44 (100). High-resolution MS: calculated for M+, 306.125; found, 306.125. ( Q ) 5,g-DiMeC. 5,g-DiMeC was prepared in 70% yield from chrysene-5,6-dione(36) and methylmagnesium iodide as described ( I I ) , mp 114 "C (lit. mp 114 "C). NMR 6 2.6 (s, 3 H), 2.9 ( 8 , 3 H), 7.3-7.6 (m, 4 H), 7.8-8.2 (m, 3 H), 8.4-8.8 (m, 3 H); MS m/e (relative intensity) 256 (M+, 100),239 (56.8). High-resolution M S calculated for M+, 256.124; found, 256.125. Other Materials. 5-MeC and anti-5-MeC-l,2-diol-3,4-epoxide were synthesized ( 3 , I 2 ) . TPA was obtained from Consolidated Midland Co., Brewster, NY. Calf thymus DNA and enzymes used for its hydrolysis were obtained from Sigma Chemical Co., St. Louis, MO. Poly(dG) was purchased from P-L Biochemicals, Milwaukee, WI. Bioassay for Tumor-Initiating Activity. Each group consisted of 20 female CD-1 mice obtained at the age of 28-35 days from Charles River Breeding Laboratories, Inc., Kingston, NY. The animals were housed under standard conditions as previously described ( 5 ) . At the age of 50-55 days, each mouse received a single initiating dose of 33 nmol of the appropriate compound in 0.1 mL of acetone. Ten days later promotion began by application of 2.5 wg of TPA in 0.1 mL of acetone, three times weekly for 20 weeks. Mice were shaved when necessary, and tumors were counted weekly. Mutagenicity Assays. S. typhimurium strain TA 100 was kindly provided by Dr. Bruce N. Ames, University of California, Berkeley. Racemic diol epoxides were dissolved in DMSO, and the assays were performed as described with preincubation (13, 14). Reported mutagenicity values are the means of triplicate assays. Background revertants (per plate) have not been subtracted. Reactions of Diol Epoxides with Calf Thymus DNA and Poly(dG). A solution of the appropriate diol epoxide (2 mg) in 1 mL of T H F was added to a solution of 10 mg of calf thymus DNA in 10 mL of 0.05 M Tris-HC1 buffer, pH 7.0. The solutions were incubated at 37 "C for 24 h and then extracted twice with 10 mL of EtOAc. The DNA was precipitated with 30 mL of cold ethanol, collected by centrifugation, washed with ethanol and acetone, and dried. For hydrolysis of DNA, a solution of 30 000 units of micrococcal endonuclease (EC 3.1.4.7) and 90 units of spleen phosphodiesterase I1 (EC 3.1.4.18) in 20 mL of 20 mM sodium succinate and 10 mM CaClZ,pH 6.0, buffer was prepared. The solution was added to DNA such that the final concentration was 2 mg of DNA/mL. It was incubated at 37 OC for 6 h. One milliliter was removed and mixed with 1 mL of 0.1 M Tris-HC1 buffer, pH 9.0, and 2 units of alkaline phosphatase (EC 3.1.3.1),
352 Chem. Res. Toxicol., Vol. 1, No. 6, 1988
Amin et al. Scheme I
A
- -
I
Ri
I
COCH3
C CH3
CHI
MeOH
D
27
24
34
35 C) hv, Iz, PhH; d) LAH, THF; e) PCC, CHzCI,; f) NHzNHz, KOH; g) BBr3, CHZC12; h) (KS03hN0, KH2P04, CH2C12, PhH, Adogen 464; i) NaBH4, EIOH; i)m-CIPBA, THF
a) AC20, EIJN; b) (CH30)2S02;
type 111-S from Escherichia coli. The mixture was incubated at 37 O C for 16 h and then analyzed by HPLC as described previously (15). Extents of reaction were calculated by adding the areas of all the adduct peaks eluted from HPLC. Reactions with poly(dG) were carried out by adding a solution of 1mg of diol epoxide in 0.5 mL of THF to a solution of 5 units of poly(dG) in 2 mL of 0.05 M Tris-HC1 buffer, pH 7.0. The mixtures were incubated at 37 "C overnight. Two milliliters of 0.01 M Tris-MgC12 buffer, pH 7.0, was then added, and the resulting mixtures were hydrolyzed enzymatically and analyzed by HPLC as described in reference 15.
for preparation of dimethylchrysenes (16). We applied this method for the preparation of 1,5-diMeC, 5,7-diMeC, and 5,12-diMeC. As illustrated in Scheme IA, the appropriately substituted 1-acetonaphthone was reacted with either benzylmagnesium chloride (1) or (0-methylbenzy1)magnesium chloride (2) to give the alcohols 5-7. These were dehydrated and cyclized photochemically. These syntheses of 5,7-diMeC and 5,12-diMeC were more convenient and proceeded in higher yield than those previously reported
Results The syntheses of the dimethylchrysenes are summarized in Scheme IA-C. As in our earlier studies on the syntheses of various substituted chrysenes, we have found that the photochemical approach pioneered by Mallory for phenanthrene syntheses is the most flexible and facile method
The starting materials for the preparation of 6,lkdiMeC and 6,7-diMeC were phenylacetic acid (11) and omethylphenylacetic acid (12), which were condensed with 1-naphthaldehydes 13 and 14, giving the alkenes 15 and 16 (Scheme IB). Esterification and photocyclization gave the esters 17 and 18. The photocyclization reaction generally proceeds in higher yield for carbomethoxy-substi-
(8, 10).
Chem. Res. Toxicol., Vol. 1, No.6,1988 353
Tumor-Initiating Activities of Dimethylchrysenes Table I. Tumor-Initiating Activity of Dimethylchrysenes on Mouse Skin" % of skin tumor skin tumors/ compd bearing mice mouse 5-MeC 854' 2.4 1,5-diMeC 5 0.5 5,7-diMeC 20 0.25 5,6-diMeC 5OC 0.85 1,6-diMeC 0 0 6,7-diMeC 5 0.05 6,la-diMeC 0 0 acetone 5 0.05
r
aGroups of 20 female CD-1 mice (age 50-55 days) were shaved and treated with a single dose of 33 nmol of each compound in 0.1 mL of acetone. Ten days later, each group was treated three times weekly with 2.5 p g of TPA in 0.1 mL of acetone, for 20 weeks. Significantly more active than all other compounds, P < 0.01; x 2 test. 'Significantly more active than control, P < 0.01; xz test.
tuted olefins such as 15 and 16 than for the corresponding methyl-substituted compounds (17). Compounds 17 and 18 were converted to 6,12-diMeC and 6,7-diMeC by a sequence consisting of reduction to the corresponding alcohols, oxidation to the aldehydes with pyridinium chlorochromate, and Wolff-Kishner reduction. We have found that this sequence avoids side reactions and proceeds in good yields (17). Reaction of the phosphonium salt 23 with o-tolualdehyde followed by photocyclization provided 1,6-diMeC (Scheme IC). The synthesis of anti-5,7-diMeC-l,2-diol-3,4-epoxide (35) is summarized in Scheme ID. The key intermediate was 2-hydroxy-5,7-dimethylchrysene (321,which was prepared by the same photochemical strategy illustrated in Scheme IB. Oxidation of 32 with Fremy's salt as reported for other polynuclear aromatic hydrocarbons (la),followed by reduction with NaBH,, gave the trans-1,2-dihydrodio134, which was converted to the anti-diol epoxide 35 by oxidation with m-chloroperbenzoic acid. The stereoselectivity of the latter reaction is well established for diequatorial dihydrodiols (18). 5,6-DiMeC was prepared by reaction of chrysene-5,6dione with methylmagnesium iodide followed by aromatization with P and HI, as previously reported (11). All compounds had UV, MS, and NMR spectral data in agreement with their structures, and all were greater than 99% pure according to HPLC analysis. The results of the bioassay for tumor-initiating activity on mouse skin are summarized in Table I. Only 5-MeC and 5,6-diMeC had significant activity compared to controls (P < 0.01). 5-MeC was significantly more tumorigenic than all other compounds (P < 0.01). AU skin tumors were papillomas. The mutagenic activities in S. typhimurium TA 100 of anti-5-MeC-1,2-diol-3,4-epoxide and anti-5,7-diMeC-1,2diol-3,4-epoxide were compared, as illustrated in Figure 2. Both compounds were highly mutagenic. On the basis of the linear portions of the dose-response curves, anti5-MeC-1,2-diol-3,4-epoxide (7200 revertants/nmol) was more mutagenic than anti-5,7-diMeC-1,2-diol-3,4-epoxide (2500 revertants/nmol). Anti-5-MeC-1,2-diol-3,4-epoxide also appeared to be more toxic than anti-5,7-diMeC-1,2diol-3,4-epoxide. HPLC analyses of enzymatic hydrolysates of calf thymus DNA that had been allowed to react for 24 h with either anti-5-MeC-1,2-diol-3,4-epoxide or anti-5,7-diMeC-1,2diol-3,4-epoxide gave chromatograms similar to that previously published for reaction of racemic anti-5-MeC1,2-diol-3,4-epoxidewith DNA (15). The two major peaks formed from racemic anti-5-MeC-l,2-diol-3,4-epoxide have been identified by NMR and MS as diastereomeric de-
0.5 nmollplate
1.o
Figure 2. Comparative mutagenic activities toward S. typhi(0) and murium TA 100 of anti-5,7-diMeC-1,2-diol-3,4-epoxide anti-5-MeC-1,2-diol-3,4-epoxide (0). Background revertants (15l/plate) have not been subtracted.
5-MeC
5.MeC.1 R,PRdIoI
5MeC-1 R,ZSdiol-l$4RepoxIde
Figure 3. Major pathway of metabolic activation of 5-MeC in mouse epidermis.
oxyguanosine adducts resulting from reaction of N2 of deoxyguanosine with C-4 of the epoxide ring (15,19,20). The two major adduct peaks formed upon reaction of anti-5,7-diMeC-1,2-diol-3,4-epoxide with DNA coeluted with those formed upon its reaction with poly(dG), indicating that they are also deoxyguanosineadducts. On the basis of the integrated peak areas of these adducts, the relative extents of reaction of anti-5-MeC-1,2-diol-3,4-epoxide and anti-5,7-diMeC-1,2-diol-3,4-epoxide with DNA were virtually identical.
Discusslon
-
-
The major pathway of metabolic activation of 5-MeC in mouse skin is 5-MeC 5-MeC-l(R),B(R)-diol 5MeC-l(R),2(S)-dio1-3(S),4(R)-epoxide (Figure 3) (6). Tumorigenicity, mutagenicity, and DNA binding studies have all shown that 5-MeC-l(R),2(S)-diol-3(S),4(R)-epoxide, which has a methyl group in the same bay region as the epoxide ring, is responsible for most of the biological activity of 5-MeC (6, 15). In contrast, 6-MeC-l(R),2(S)diol-3(S),4(R)-epoxide, which is formed stereoselectively in the metabolism of 6-MeC, is not appreciably tumorigenic or mutagenic and binds less extensively to DNA than These comdoes 5-MeC-l(R),2(S)-dio1-3(S),4(R)-epoxide. parative studies clearly showed that the key factor in the high tumorigenic activity of 5-MeC was its ability to form a bay region diol epoxide with the epoxide ring and the methyl group in the same bay region. These observations are consistent with the structural requirements favoring tumorigenicity of methylated and fluorinated chrysenes. Thus, in the present study, the relatively low tumorigenic activities of 1,5-diMeC, 1,6-diMeC,6,7-diMeC, and 6,12diMeC were not surprising. In 1,5-diMeC,the metabolic pathway leading to 5MeC-1(R),2(S)-diol-3(S),4(R)-epoxide would be blocked by the methyl group, as we have previously observed for l-fluoro-5-MeC ( 4 ) . In 1,6-diMeC, 6,7-diMeC, and 6,12-diMeC there is no methyl group in the bay region and the formation of an exceptionally tumorigenic diol epoxide is not possible. The low tumori-
354 Chem. Res. Toxicol., Vol. 1, No. 6, 1988
genicity of 6,12-diMeC contrasts to the exceptional activity of 5,11-diMeC, its symmetrical isomer with two bay region methyl groups (1). The low tumorigenicity of 5,12-diMeC has previously been explained by inhibition of 1,2-diol formation by the peri 12-methyl group (21). However, the relatively low tumorigenic activities of 5,6-diMeC and 5,7-diMeC were unexpected on the basis of these considerations. Both compounds can potentially form diol epoxides with the methyl group and epoxide ring in the same bay region, as in 5-MeC. Previous studies have shown that 6-fluoro-5-MeCand 7-fluoro-5-MeCare both strong tumorigens, with activity equal to or greater than that of 5-MeC ( 4 ) . These results suggest either that the methyl groups in the 6- or 7-positions of 5-MeC are affecting its metabolism in such a way as to decrease the extent of formation of the corresponding l(R),2(S)-diol3(S),4(R)-epoxidesor that the intrinsic activity of the diol epoxides, if formed, are less than that of 5-MeC-l(R),2(S)-diol-3(S),4(R)-epoxide. To investigate the intrinsic activity of anti-5,7-diMeC-1,2-diol-3,4-epoxide, we synthesized it and assayed its mutagenicity and binding to DNA. It was strongly mutagenic, although less active than anti-5-MeC-l,2-diol-3,4-epoxide. Its extent of reaction with DNA appears to be similar to that of anti-5-MeC-1,2diol-3,4-epoxide. These results do not allow us to predict its tumorigenicity, although in our previous studies the mutagenicity and tumorigenicity of methylchrysenediol epoxides have been correlated (6, 15). Molecular shape could affect biological properties of the dimethylchrysenes. Deformation from planarity, as observed in 5-MeC and 7,12-dimethylbenz[a]anthracene,is associated with high tumorigenic activity, and it has been suggested that this is a key factor in metabolic activation as well as in DNA interactions of the corresponding diol epoxides (22-25). Deformations from planarity are greater in 5,6-diMeC than in 5-MeC, according to their X-ray crystal structures (23). This does not correlate with their tumorigenic activities. In order to gain further insight into the mechanisms associated with methylchrysene tumorigenesis, it will be important to compare the relative tumorigenic properties of the 1,2-diol-3,4-epoxidesof 5-MeC, 5,6-diMeC, and 5,7-diMeC. These studies, as well as metabolism experiments with the parent hydrocarbons,are currently in progress.
Acknowledgment. This study was supported by National Cancer Institute Grant CA44377. Registry No. 1, 6921-34-2; 2, 552-45-4; 3, 67757-61-3; 4, 28418-86-2; 5, 117022-13-6; 6, 117022-14-7; 7, 117022-15-8; 8, 117022-16-9; 9, 117022-17-0; 10, 117022-18-1; 11, 103-82-2; 12, 644-36-0; 13, 33738-48-6; 14, 66-77-3; 15, 117022-19-2; 16, 117022-20-5; 17, 117022-21-6; 18, 117022-22-7; 19, 117022-23-8; 20,22228-78-0; 21,117022-24-9; 22, 117022-25-0; 23, 53156-51-7; 24,529-20-4; 25,117022-26-1; 26,87901-81-3; 27,117022-27-2; 28, 117022-28-3; 29, 117022-29-4; 30, 117022-30-7; 31, 117022-31-8; 32, 117022-32-9;33,117022-33-0; 34, 117022-34-1;35,117022-35-2; 5,12-diMeC, 14250-05-6; 1,5-diMeC, 117022-36-3; 5,7-diMeC, 52171-92-3; 6,12-diMeC, 14207-77-3; 6,7-diMeC, 117022-38-5; l,B-diMeC, 117022-39-6;5-MeC, 3697-24-3; 5,6-diMeC, 3697-27-6; p-TsOH, 104-15-4; acetonaphthone, 941-98-0; 2-phenyl-344methyl-1-naphthy1)propionic acid, 117022-37-4; anti-5-MeC-1,2diol-3,4-epoxide, 117066-42-9; benzyl chloride, 100-44-7.
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Amin et al. carcinogenic methylated polynuclear aromatic hydrocarbons”. Ace. Chem. Res. 19, 174-180. (3) Hecht, S. S., Bondinell, W. E., and Hoffmann, D. (1974) “Chrysene and methylchrysenes: presence in tobacco smoke and carcinogenicity”. J. Natl. Cancer Inst. (U.S.) 53, 1121-1133. (4) Hecht, S. S., LaVoie, E. J., Mazzarese, R., Hirota, N., Ohmori, T., and Hoffmann, D. (1979) “Comparative mutagenicity, tumor-initiating activity, carcinogenicity, and in vitro metabolism of fluorinated 5-methylchrysenes”. JNCI, J . Natl. Cancer Inst. 63,855-861. (5) Hecht, S. S., Radok, L., Amin, S., Huie, K., Melikian, A. A., Hoffmann, D., Pataki, J., and Harvey, R. G. (1985) ’Tumorigenicity of 5-methylchrysene dihydrodiols and dihydrodiol epoxides in newborn mice and on mouse skin”. Cancer Res. 45, 1449-1452. (6) Hecht, S. S., Amin, S., Huie, K., Melikian, A. A,, and Harvey, R. G. (1987) “Enhancing effect of a bay region methyl group on tumorigenicity in newborn mice and mouse skin of enantiomeric bay region diol epoxides formed stereoselectively from methylchrysenes in mouse epidermis”. Cancer Res. 47, 5310-5315. (7) Hecht, S. S., Hirota, N., Loy, M., and Hoffmann, D. (1978) “Tumor initiating activity of fluorinated 5-methylchrysenes”. Cancer Res. 38, 1694-1698. (8) Coombs, M. M., Bhatt, T. S., Hall, M., and Croft, C. J. (1974) “The relative carcinogenic activities of a series of 5-methylchrysene derivatives”. Cancer Res. 34, 1315-1318. (9) Badger, G . M., Cook, J. W., Hewett, C. L., Kennaway, E. L., Kennaway, N. M., Martin, R. H., and Robinson, A. M. (1940) “The production of cancer by pure hydrocarbons V”. R o c . R. Soc. London, B, 439-467. (10) Cagniant, D., and Delpine, M. M. (1963) “Synthsse de chrysgnes mono-et dimgthyles sur les regions K, et K,”. C. R. Hebd. Seances Acad. Sci. 256, 5590-5593. (11) Koneiczny, M., and Harvey, R. G. (1980) “Reductive methylation of polycyclic aromatic quinones”. J. Org. Chem. 45, 1308-1310. (12) Harvey, R. G., Pataki, J., and Lee, H. (1986) ’Synthesis of the dihydrodiol and diol epoxide metabolites of chrysene and 5methylchrysene”. J . Org. Chem. 51, 1407-1412. (13) Ames, B. N., McCann, J., and Yamasaki, E. (1975) “Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian microsome mutagenicity test”. Mutat. Res. 31, 347-364. (14) Yahagi, T., Nagao, M., Seino, Y., Matsushima, T., Sugimura, T., and Okada, M. (1977) “Mutagenicities of N-nitrosamines in Salmonella”. Mutat. Res. 48, 121-130. (15) Melikian, A. A., Amin, S., Huie, K., Hecht, S. S., and Harvey, R. G. (1988) “Reactivity with DNA bases and mutagenicity toward S. typhimurium of methylchrysene diol epoxide enantiomers”. Cancer Res. 48, 1781-1787. (16) Mallory, F. B., and Mallory, C. W. (1984) “Photocyclization of stilbenes and related molecules”. Org. React. (N.Y.) 30, 1-456. (17) Amin, S., Camanzo, J., and Hecht, S. S. (1982) “Identification of metabolites of 5,ll-dimethylchrysene and 5J2-dimethylchrysene and the influence of a peri-methyl group on their formation”. Carcinogenesis (London) 3, 1159-1163. (18) Harvey, R. G . (1985) “Synthesis of dihydrodiol and diol epoxide metabolites of carcinogenic polycyclic hydrocarbons”. In Polycyclic Hydrocarbons and Carcinogenesis (Harvey, R. G., Ed.) pp 35-62, American Chemical Society, Washington, DC. (19) Melikian, A. A., Amin, S., Hecht, S. S., Hoffmann, D., Pataki, J., and Harvey, R. G. (1984) “Identification of the major adducts formed by reaction of 5-methylchrysene anti-dihydrodiol epoxides with DNA in vitro”. Cancer Res. 44, 2524-2529. (20) Reardon, D. B., Hilton, B. D., Roman, J. M., Prakash, A., Lee, H., Harvey, R. G., and Dipple, A. (1987) “Characterization of deoxyadenosine and deoxyguanosine adducts formed in DNA by the racemic anti-1,2-dihydrodiol-3,4-epoxideof 5-methylchrysene”. Carcinogenesis (London) 8, 1317-1322. (21) Amin, S., Camanzo, J., and Hecht, S. S. (1982) “Identification of metabolites of 5,11-dimethylchrysene and 5,12-dimethylchrysene and the influence of a peri-methyl group on their formation”. Carcinogenesis (London) 3, 1159-1163. (22) Kashino, S., Zacharias, D. E., Prout, C. K., Carrell, H. L. Glusker, J. P., Hecht, S. S., and Harvey, R. G. (1984) “Structure of 5-methylchrysene, CI9H,,“. Acta Crystallogr., Sect. C Cryst. Struct. Commun. C40, 536-540. (23) Zacharias, D. E., Kashino, S., Glusker, J. P., Harvey, R. G., Amin, S., and Hecht, S. S. (1984) “The bay region geometry of some 5-methylchrysenes: steric effects in 5,6- and 5,12-di-
Tumor-Initiating Activities of Dimethylchrysenes methylchrysenes”. Carcinogenesis (London) 5, 1421-1430. (24) Kashino, S.,Zacharias, D. E., Peck, R. M., Glusker,J. p., Bhatt, T. s., and Coombs, M. M.(1986) “Bay region distortions in cyclopenta[a]phenanthrenes”. Cancer Res. 46, 1817-1829.
Chem. Res. Toxicol., Vol. 1, No. 6,1988 355 (25) DiGiovanni, J., Diamond, L., Harvey, R. G., and Slaga, T. J. (1983) ‘Enhancement of the skin tumor-initiatingactivity of polycyclic aromatic hydrocarbons by methyl substitution at nonbenzo bay region positions”. Carcinogenesis (London)4,403-407.
