Stereochemistry of the major rat liver microsomal metabolites of the

Mar 28, 1991 - Colin C. Duke,1, Trevor W. Hambley,* Gerald M. Holder,*^ Corina 0. Navascues,*. Sarah Roberts-Thomson,f and Yuerong Yef. Department of ...
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Chem. Res. Toxicol. 1991,4, 546-555

546

Stereochemistry of the Major Rat Liver Microsomal Metabolites of the Carcinogen 7-Methylbenz[ c ]acridine Colin C. Duke,? Trevor W. Hambley,i Gerald M. Holder,**?Corina 0. Navascues,? Sarah Roberts-Thomson,? and Yuerong Yet Department of Pharmacy and School of Chemistry, University of Sydney, NSW 2006, A ust ra1ia Received March 28, 1991 The major metabolites of the carcinogen 7-methylbenz[c]acridine (7MBAC), trans-5,g-di-

hydro-5,6-dihydroxy-7-methylbenz[c]acridine (7MBAC-5,6-DHD), and trans-8,9-dihydro-8,9dihydroxy-7-methylbenz[ clacridine (7MBAC-8,g-DHD) were characterized as their enantiomers after separation of their bis-(+)-(1R,2S,4S)-endo-1,4,5,6,7,7-hexachlorobicyclo[2.2.l]hept-5ene-2-carboxylic acid [ (+)-HCA] esters and hydrolysis. The synthetic precursor, trans-3,4dihydroxy-7-methyl-l,2,3,4-tetrahydrobenz[c]acridine (7MBAC-3,4-THD), was similarly separated into enantiomers, and the dihydrodiol trans-3(S),4(S)-dihydro-3,4-dihydroxy-7-methylbenz[c]acridine (7MBAC-3,4-DHD) was prepared from 7MBAC-3(S),4(S)-THD. Absolute configurations were assigned by the chiral exciton coupling of the bis-p(dimethy1amino)benzoate of 7MBAC-3(R),4(R)-THD7and by the semiempirical methods based on the biaryl chromophores of the enantiomers of 7MBAC-5,6-DHD and of the methanolysis products of the 5,6-oxide of 7MBAC which were resolved as their (+)-HCA esters. X-ray crystallography was used for 7MBAC-8@),9(S)-DHDbis-(+)-HCA ester, and assignments were correlated with chiral exciton coupling of the bis-C(dimethy1amino)cinnamatesof 7MBAC-5(R),G(R)-DHD and 7MBAC8(S),9(S)-DHD. The stereochemical compositions of four metabolites (three dihydrodiols and 7MBAC-5,6-oxide) formed in incubations with rat liver microsomes from control and induced liver were determined by normal-phase separations of bis-(+)-HCA esters, and by chiral stationary-phase separation of the 5,6-oxide methanolysis products. The 3(R),4(R)-enantiomer of 7MBAC-3,4-dihydrodiol predominated, 74-98% enantiomeric purity, and purity for the oxide varied from about 71 % 5(R),6(S)-oxide for control microsomes to about 28% 5(R),G(S)-oxide for liver microsomes obtained from 3-methylcholanthrene-pretreated rats.

I ntroductlon Following very extensive interest in the biological oxidation of polycyclic aromatic hydrocarbons which possess carcinogenic properties when examined in rodent bioassays (1,2),interest has moved to structurally analogous compounds which possess a heteroatom (3). Many such compounds are found as air pollutants in urban environments ( 4 ) , and they have also been characterized as components of tobacco smoke found in smoke condensate (5,6). Additionally, other large groups of toxic substances to which humans are exposed are the nitroaromatics which are found as air pollutants (7)and the food mutagens formed from proteins by pyrolysis during cooking processes (8). Among objectives of investigations are the recognition of proximate and ultimate carcinogens formed by oxidative metabolism of the parent hydrocarbon, tumorigenicity, and mutagenicity tests to establish the relative importance of the proximate and ultimate carcinogens, and the characterization of the stereochemistry of the metabolic events involved in the bioactivation. The stereochemistry has been shown to be important because the metabolic activation steps are highly sterically selective and because rodent liver microsomal activities favor the formation of the more tumorigenic products. Such metabolites are (-1 - 7( R ),8(R)-dihydro-7,8-dihydroxybenzo[a ]pyrene and (+)-7(R),8(S),9(S),lO(R)-tetrahydrobenzo[a]pyrene-7,gdiol 9,lO-epoxide formed selectively from benzo[a]pyrene (BP)' through the 7(R),8(S)-oxideand (-)-7(R),8(R)-dihydroxy-

* Address correspondence to this author. Department of Pharmacy. *School of Chemistry.

7,8-dihydro-BP, respectively (2, 9, 10). Much of that stereoselectivemetabolism of the polycyclic hydrocarbons has been summarized in recent reviews (2, 11). The metabolism of the carcinogen 7-methylbenz[c]acridine (1) (7MBAC) has been extensively described (12-15). The major metabolite formed in both liver microsomal preparations and in hepatocyte incubations is tra~-8,9-dihydro-8,9-dihydroxy-7-methylbenz[c]acridine (2) (7MBAC-8,9-DHD),2other dihydrodiols being the 5,6(3) and 3,4-isomers (4) (7MBAC-5,6-DHD and 7MBAC3,4-DHD, respectively). In microsomal incubations the 3,4-dihydrodiol is only a minor metabolic product, constituting about 3 4 % of the total metabolites formed under substrate-saturating conditions. It has been shown that the 3,4-dihydrodiol is a proximate carcinogen of 7MBAC (16) and that the most probable ultimate carcinogen is a uic-diol epoxide derived from it (15). The evidence for the latter is derived from mutagenicity studies with synthesized metabolites. Although the K-region dihydrodiol is not a major metabolite, the related oxide, 7MBAC-5,6-oxide, is formed and accumulates in substantial amounts because Abbreviations: BP, benzo[a]pyrene; 7MBAC-3,4-DHD, trans-3,4dihydro-3,4-dihydroxy-7-methylbenz[c]acridine; 7MBAC, 7-methylbenz[clacridine; 7MBAC-5,6-DHD, trans-5,6-dihydro-5,6-dihydroxy-7methylbenz[c]acridine; 7MBAC-8,9-DHD, trans-8,9-dihydro-8,9-dihydroxy-7-methylbenz[c]acridine; HCA, endo-1,4,5,6,7,7-hexachlorobicyclo[2.2.l]hept-5-ene-2-carboxylicacid; MC, 3-methylcholanthrene; PB, phenobarbital; CIMS, chemical ionization maas spectrometry;TCPO, 3,3,3-trichloropropene1,a-oxide;DMSO, dimethyl sulfoxide;CD, circular dichroic; CSP, chiral stationary phase. Dihydrodiol metabolites formed by mammals from aromatic hydrocarbons always possess the trans configuration; this reflects the catalytic competence of epoxide hydrolase. For the remainder of this paper all uic-dihydrodiols referred to have this stereochemistry.

1991 American Chemical Society

Stereochemistry of 7-Methylbenz[c]acridine

Metabolites

of its reduced susceptibility to hydration catalyzed by epoxide hydrase. The work reported in this paper relates to these four metabolites; they are resolved, the absolute configurations of their enantiomers are determined, and the stereoselectivity of their metabolic formation from 7MBAC in incubations with rat liver microsomes is determined. Among other polycyclic heteroaromatic polycyclic compounds, a similar study was recently reported for dibenz[aj]acridine (In,and such results have also been obtained for dibenz[c,h] acridine (3).

Experimental Sectlon Instrumentation. Proton NMR spectra were recorded on a JEOL FX9OQ or a Bruker WM-400 spectrometer. Chemical ionization mass spectra were obtained on a Finnigan TSQ-46 mass spectrometer with methane or ammonia as reagent gas. HPLC was performed with Beckman-Altex Model 421 gradient system with a Jasco UVIDEC-V detector a t 270 nm. For some experiments a Hewlett-Packard diode array 1040 spectrophotometric detector was employed. Circular dichroic (CD) spectra were measured with a Jasco J-5OOc spectropolarimeter using methanol as solvent unless otherwise stated. A P a c h d 1900 'I'riCarb liquid scintillation spectrometer was used for radiochemical determinations. High-resolution mass spectra were obtained on an AEI MS-9 instrument, and composition matches were acceptable within 3 parts per thousand (m/z) of the calculated values. Chemicals.

[SH]Methyl-7-methylbenz[c]acridine(18),

Chem. Res. Toxicol., Vol. 4, No. 5, 1991 547

trans -S(R),6(R )-Dihydrobis[[I-(dimethylamino)cinnamoyl]oxy]-7-methylbenz[c]acridine. trans-5(R),G(R)Dihydroxy-5,6-dihydro-7-methylbenz[c]acridine (1.5 mg) and 1- [4-(dimethylamino)cinnamoyl]imidazole (5 mg) were dissolved in freshly distilled dried T H F (2 mL). NaH (50% in oil, 8 mg) was then added to the solution under N2 The mixture was stirred under N2 gas a t room temperature for 2 h. The reaction was completed by this time as shown by TLC. T H F was removed under reduced pressure, and the product was partitioned between EtOAc (20 mL) and 20% aqueous NaCl(20 mL). The EtOAc extract was dried (Na8OJ and concentrated, and then the residue was purified by chromatography on silica gel (3 cm X 0.3 cm i.d.) (gradient, EtOAc/petroleum ether, bp 70-75 "C, 1:19, to EtOAc) to yield a yellow solid (1.5 mg): 'H NMR (1.5 mg/0.2 mL, CDC1,) 6 2.82 (8, 3 H), 2.96 ( ~ , H), 6 2.98 (5, 6 H), 6.02 (d, 1Hp), 5.96 (d, 1 Hp), 6.32 (d, 1 H, H5), 6.56 (d, 2 H), 6.58 (d, 2 H), 6.80 (d, 1 H, He), 7.20-8.25 (m, aromatic protons), 8.78 (bd, 1 H, Hl); J5,6= 3.3 Hz, JaS = 15.8 Hz, J2,,3,(of cinnamate) = 9 Hz; CIMS, m/e (relative intensity) 624 (M + 1,66) 433 (loo), 244 (go), 192 (75), 174 (95); CD spectrum in CHC13, Atw = -19.9, AtM7 = +5.0 (M-' cm-'); in THF, Atm = -11.1, AtM = +3.5, Ata7 = -40.6 (M-l cm-'1. trans -3,4-Dihydroxy-1,2,3,4-tetrahydro-7-methylbenz[clacridine. This has not been previously characterized and was prepared as follows. Hydrolysis of trans-3,4-diacetoxy-1,2,3,4tetrahydro-7-methylbenz[c]acridine (19) (25.8 mg) as described previously gave trans-3,4-dihydroxy-1,2,3,4-tetrahydro-7methylbenz[c]acridine (17.6 mg, 89%) as pale yellow crystals: mp

215-218 "C dec; 'H NMR (3 mg/0.2 mL, tetrahydrofuran-d8/D20, 91)6 1.83-2.42 (m, 2 H), 3.17-3.96 (m, 3 H), 4.56 (d, Hl), 7.39-7.83 = 7 Hz; UV spectrum in MeOH (m, 3 H), 8.08-8.36 (m, 3 H); J3,4 [&, nm (e-)], 360 (8600), 258 (144OOO);high-resolution electron impact MS, m / z 279.126 (C18H17N02requires 279.126).

7MBAC, 7MBAC-3,4-DHD,7MBAC-5,6-DHD,7MBAC-8,9-DHD, and 7-methylbenz[c]acridine 5,6-oxide (7MBAC-5,6-oxide) (19, 20) were available from previous studies. The [,H]7MBAC was Enantiomers of trans -3,4-Dihydroxy1,2,3,4-tetrahydrogreater than 96% radiochemically pure as determined by HPLC 7-methylbenz[c]acridine. The silver salt of (+)-HCA (504 mg) on a 10-pm Hibar RP-8 reverse-phase column (250 X 4.0 mm i.d., was reacted with 1,2-dihydro-7-methylbenz[c]acridine (127 mg, E. Merck, Darmstadt, Germany) eluted with 20-35% methanol 85% purity) according to the procedure described for 1,2-diin water over 15 min, then 35-70% over 70 min, and finally hydrodibenz[aj]acridine (17). A mixture of the two diastereo70-100% over 5 min with a flow rate of 1.0 mL/min. Melting isomeric bis-(+)-HCA esters (136 mg, 33%) was isolated from the points (mp) are uncorrected. In chemical ionization maim spectra reaction by chromatography (gradient, hexane/EtOAc, 4 9 1 to methane was used as reagent gas unless otherwise stated. Pu423). The bis-HCA esters were separated by preparative HPLC rifications were effected by using short column vacuum chromatography on silica gel (21). (+)-(lR,2S,4S)-end0-1,4,5,6,7,7- (Lichrosorb, HPLC Technology, Macclesfield, U.K., 5-pm silica, 22.5 mm i.d. X 250 mm, 7.5 mL/min hexane/EtOAc, 91). The Hexachlorobicyclo[2.2.1]hept-5-ene-2-carboxylic acid (5)(HCA) less polar diastereoisomer was identified as traw3(S),4(S)-biswas prepared as described (22). Other chemicals were of the [ [ [ (1R,2S,4S)-endo-1,4,5,6,7,7-hexachlorobicyclo[2.2.l]hept-5highest grade available. clacridine: Resolution of (*)-trans-5,6-Dihydro-5,6-dihydroxy-7- en-2-yl]carbonyl]oxy]-1,2,3,4-tetrahydro-7-methylbenz[ 'H NMR (1.9 mg/0.2 mL, CDCla 6 2.3-2.9 (m, 6 H), 3.13 (s,7-Me), methylbenz[clacridine. (+)-HCA (514 mg) was converted to 3.5-3.9 (m, 4 H), 5.38 (m, H3), 6.17 (d, H4), 7.25-8.35 (m, 6 H); its acid chloride by refluxing with thionyl chloride for 2 h. The J34= 4.4 Hz; CIMS (reagent gas NH,), m/e (relative intensity) residue after evaporation of the excess reagent was dissolved in 35C1/37C1isotope cluster; MH+, 941 (3) 36c15/37c17,939 (10) dry T H F (2.0 mL) and added to a solution of (*)-7MBAC-5,635C4/37C43,937 (26) 35C1d37C15,935 (49) s5C18/37C14,933 (100) DHD (82 mg) and 4-(dimethylamino)pyridine (228 mg) in dry 35c$/37c13, 931 (91) 35C110/ Cl2, 929 (50) 36C111/aC11, 927 (13) W112, T H F (20 mL). After stirring for 0.5 h, the crude products were 246 (44). Hydrolysis of the less polar diastereoisomer (49.7 mg), isolated by partitioning of the reaction mixture between EtOAc as described previously gave trans-3(S),4(S)-dihydroxy-1,2,3,4and 20% aqueous NaCl and separated into two fractions by tetrahydro-7-methylbenz[c]acridine(14.8 mg, 99% 1. chromatography (gradient, hexane to hexane/EtOAc, 91). The The more polar diastereoisomer (53.3 mg) was identified as less polar diastereoisomer (37 mg) was identified as transtrans-3(R),4(R)-dihydroxy-1,2,3,4-tetrahydro-7-methylbenz[c]5(R),6(R)-dihydro-5,6-bis[[[ (1R,2S,4S)-endo-1,4,5,6,7,7acridine bis-(+)-HCA ester: 'H NMR (3.6 mg/0.2 mL, CDC1,) hexachlorobicyclo[2.2.l]hept-5-en-2-yl]carbonyl]oxy]-76 2.3-2.8 (m, 6 H), 3.12 (s, 7-Me), 3.5-3.9 (m, 4 H), 5.42 (m, H3), methylbenz[c]acridine: lH NMR (37 mg/0.3 mL, CDCl,) 6 6.20 (d, H4), 7.25-8.35 (m, 6 H); J3,4 = 3.7 Hz. CIMS (reagent 2.52 (m, 4 H), 2.81 ( 8 , 7-Me), 3.20-3.50 (m, 2 H), 6.16 (d, H5), 6.74 gas NH3) was the same as for the less polar diastereoisomer. (d, He), 7.4-7.8 (m, 5 H), 8.0-8.2 (m, 2 H), 8.73 (m, Hi); J5,e = Hydrolysis of the more polar diastereoisomer (53.2 mg), as de3.5 Hz. Hydrolysis occurred when the bis-(+)-HCA ester of scribed previously, gave trans-3(R),4(R)-dihydroxy-1,2,3,45(R),6(R)-7MBAC-5,6-DHD(30 mg) dissolved in THF (5 mL) was tetrahydro-7-methylbenz[c]acridine(14.9 mg, 94%): circular treated with 10% aqueous NaOH (0.125 mL) and MeOH (0.625 dichroic spectrum, AcB5 +1.0, At257 -29.5 (M-' cm-'). mL). The product, ~~U~-~(R),~(R)-~MBAC-~,~-DHD, was isotrans-3(R),4(R)-Dihydroxy-1,2,3,4-tetrahydro-7-methylbenzlated with EtOAc and purified by short column vacuum chro[clacridine (3.4 mg) was treated with 4-(dimethy1amino)benzoyl matography: circular dichroic spectrum, At242 -41.6, At214 + 26.2 chloride (11.2 mg) by the method described previously to give (0.1, MeOH) -211O. The more polar diaster(M-' cm-'); [alZsD after chromatography (gradient, hexane/CH2C12,1:1,to CH2C12, eoisomer (98 mg) was identified as trans-5(S),6(S)-dihydro5,6-dihydro-5,6-bis[ [[ (lR,25,4S)-endo-1,4,5,6,7,7-hexa- then CH2C12to CH2C12/EtOAc,19:l) a yellow gum (6.3 mg): 'H NMR (6.3 mg/0.25 mL of CDC13) 6 2.20-2.65 (m, 2 H), 2.97 (s, chlorobicyclo[2.1.1]hept-5-en-2-yl]carbonyl]oxy]-7-methylbenz[c]acridine: 'H NMR (98 mg/0.3 mL, CDCl,) 6 2.53 (m, 6 H), 3.01 (s, 6 H), 3.08 (s, 7-methyl), 3.79 (m, 2 H), 5.66 (m, H3), 6.55 (d, 2 H3, or 2 H3"), 6.60 (d, 2 H, or 2 H3,), 6.66 (d, H4), 7.50 4 H), 2.85 (8, 7-Me), 3.33-3.52 (m, 2 H), 6.10 (d, H5), 6.56 (d, He), 7.43-7.87 (m, 5 H), 8.03-8.25 (m, 2 H), 8.71 (m, Hl); J5,$ = 3.5 (d, H5), 7.55-7.80 (m, Hlo and HIJ, 7.85 (d, Hz, or H2"), 7.97 (d, H , or H r ) , 8.09 (d, He), 8.25 (m, Ha and HI,,); J3,4 = 5.5 Hz, J5,6 Hz. Hydrolysis as above afforded trans-5(S),G(S)-7MBAC-5,6DHD: CD spectrum, At242 +38.7, At214 -28.5 (M-' cm-'); [ a I z 5 ~ = 9.3 Hz, Jr,3,= J2,,,3,,= 9.1 Hz; 'H NMR (saturated solution in (0.1, MeOH) +215O. CD,OD) J3A= 6.1 Hz; CIMS m/z (relative intensity) 574 (MH',

548 Chem. Res. Toxicol., Vol. 4, No. 5, 1991

Duke et al.

EtOAc) to yield a pale yellow solid (1.5 mg): 'H NMR (400 MHz, 25), 409 (loo), 274 ( l l ) , 246 (57), 245 (21), 244 (131, 166 (50),148 1.5 mg/0.2 mL, CDClJ 6 2.79 (e, 3 H, 7-methyl), 2.98 (e, 6 H), (nm), (41,360 (10600), 315 (19); UV spectrum in MeOH [A, 2.99 ( ~ , H), 6 5.64 (dd, Hg), 6.15 (d, HJ, 6.11 (d, Hd), 6.62 (d, 2 (60300), 257 (155800); CD spectrum in MeOH, Ac323 -40.2, Atzss Ha), 6.60 (d, 2 Ha,), 6.67 (ddd, Hie), 6.72 (dd, H8), 7.26 (d, Hi,), +42.5 (M-l cm-'). 7.33 (d, 2 Hb), 7.36 (d, Hy), 7.58 (d, HB), 7.63 (d, Hp), 7.73-7.69 trans -3(5),4(S)-Dihydro-3,4-dihydroxy-7-methylbenz[clacridine. trans-3(S),4(S)-Dihydroxy-7-methyl-1,2,3,4-tetra- (m, H2 and H3), 7.84 (d, H5), 7.90 (dd, H4),7.96 (d, Hs), 9.39 (bd, Hi); J1,2 = 7.7 Hz, J3,4 = 7.1 Hz, J5,6 = 9.1 HZ, J8,e = 2.0 Hz, J10,ll hydro-7-methylbenz[c]acridine(5 mg)was acetylated, brominated = 9.7 Hz, J9,lo = 5.6 Hz, J8,10 = 1.1Hz, Jus = Jds = 15.5 Hz, J b b a t the 1-position, dehydrobrominated, and then hydrolyzed to give trans-3(S),4(S)-dihydro-3,4-dihydroxy-7-methylbenz[c]- = J.',bt = 8.8 Hz; CIMS, m/e (relative intensity) 624 (M + 1, 18), 433 (23), 244 (loo),192 (37) 174 (50); CD spectrum (CHC13),Atm acridine (1mg) as previously described (19). A sample (- mi= +5.5, AcW = -6.0 (M-' cm-') (see Figure 6). crogram range) was converted to its (-)-HCA diester (see derivMethanolysis of 7MBAC-5,6-oxide. Racemic 7MBAC-5,6atization below),and it cochromatographed on TLC with the more oxide (38 mg) was allowed to stand overnight at room temperature polar component of the (-)-HCA esters of racemic trans7MBAC-3,4-DHD. in anhydrous MeOH (60 mL) in which sodium (0.6 g) had been E n a n t i o m e r s of trans-8,9-Dihydro-8,9-dihydroxy-7- dissolved. After removal of most of the solvent under reduced pressure, the products were partitioned between EtOAc (25 mL) methylbenz[c]acridine. (*)-trans-7MBAC-8,9-DHD (14.3 mg) and 20% aqueous NaCl(20 mL). The combined organic phases and 4-(dimethy1amino)pyridine(46.8 mg) dissolved in dry T H F obtained after reextraction of the aqueous layer with EtOAc (20 (3 mL) were treated with the acid chloride prepared from (+)mL) were dried, the solvent was evaporated, and the residue was HCA, dissolved in dry T H F (2 mL) for 30 min. The crude separated by chromatography (gradient, CH2C12/EtOAc,91 to products isolated by extraction with EtOAc were dissolved in 2:l). The first fraction was trans-5,6-dihydro-6-hydroxy-5CH2C12 (-3 mL), and chromatography (gradient, hexane to methoxy-7MBAC (19 mg): CIMS (methane), m/z (relative hexane/EtOAc, 9:l) allowed the bis esters to be separated from abundance) 292 (MH', loo), 274 (59), 260 (30); UV spectrum in 4-(dimethylamino)pyridine before preparative HPLC on a 5-rm MeOH [A- (nm), (01,269 (29 100 M-' cm-'). The second fraction silica column (25 X 2.25 cm i.d.) with 6.6% EtOAc in petroleum from the column was trans-5,6-dihydro-5-hydroxy-6-methoxyfraction, bp 70-75 OC. Two products were readily separated from 7MBAC (21 mg): 'H NMR (in MeOH-d4) 6 2.88 (s, 7-methyl), each other. The first (16.7 mg) was trans-8(R),9(R)-dihydro8,9-bis[[ [ (1R,2S,4S)-endo-1,4,5,6,7,7-hexachlorobicyclo[2.2.1]-3.42 (s, 0-methyl), 5.07 and 5.11 (d, H5 and H6), 7.47-7.86 (m, 5 H), 8.07-8.24 (m, 2 H), 8.56 (m, Hl); J5,8 = 3.4 Hz; CIMS hept-5-en-2-yl]carbonyl]oxy]-7-methylbenz[c]acridine:mp (methane), m/z (relative intensity) 292 (MH', loo), 274 (45), 260 208-210 OC; 'H NMR (16.7 mg/0.3 mL) 6 2.57 (2 d, 4 H, H3,and (4O);Wspectrum in MeOH [A- (nm), (41, 269 (26000 M-lcm-l). H3"), 2.78 (s, 7-methyl), 3.47 (t, H20, 3.54 (t, H20, 5.51 (dd, Hg), The 'H NMR spectra of these isomers in CDC13 were identical 6.53 (m, Ha, Hlo), 7.29 (d, Hll), 7.67-8.02 (m, 5 H), 9.35 (m, HJ; with those reported (23). Each fraction was converted to its J 8 , g = 2.4 Hz, J8,10 = 1.3 Hz, J910 = 5.4 Hz, Jlo,ll = 9.7 Hz, JT,3, (+)-HCA esters as described above, and the diastereomers were and = 5.4 and 5.8 Hz,CIMS (reagent gas NH,),m/e (relative separated by chromatography (gradient, hexane/EtOAc, 9:l to intensity) 3sCl/37Clisotope cluster; MH+, 940 (2) 35c15/37c17, 938 6:l). (10)35C&/37C&, 936 (25) w17/37c15, 934 (48),3SC18/nC~,932 (loo), The diastereoisomers from trans-5,6-dihydro-6-hydroxy-5,3C 1'#w l 930 (89) 36cl10/37C12, 928 (48) 3sC111/37C1,926 (12) 36cl12, methoxy-7MBAC (10.5 mg) afforded two products after chro592 (5), 590 (18), 588 (40), 586 (59), 584 (271,244 (68). The second (16.8 mg) was trans-8(S),9(S)-dihydro-8,9-bis[[[(lR,2S,4S)- matography. The less polar product (6.6 mg) was identified as [ [ [ (lR,BS,4S)-endo-1,4,5,6,7,7-hexaendo-1,4,5,6,7,7-hexachlorobicyclo[2.2.1]hept-5-en-2-y1]- trans-5(R),6(R)-dihydro-6chlorobicyclo[ 2.2.11hept-5-en-2-ylcarbonylIoxy]-5-methoxy-7carbonyl]oxy]-7-methylbenz[c]acridine:mp 213-214 "C; 'H NMR methylbenz[c]acridine: 'H NMR (6 mg/0.3 mL) 6 2.48 (m, H3, (16.8 mg/0.3 mL, CDC1,) 2.22-2.70 (m, 4 H, Hv and H3"), 2.78 and H3,,),2.77 (s, 7-methyl), 3.27 (s, 5-OMe), 3.40 (dd, H2,),4.48 (s, 7-methyl), 3.52 and 3.62 (2 dd, 2 H2,),5.48 (dd, Hg), 6.48 (m, H8 and Hlo),7.29 (d, H,,), 7.68-8.03 (m, 5 H), 9.32 (m, HI); J8,9 (d, H5), 6.72 (d, H6), 7.38-7.82 (m, 5 H), 7.96-8.24 (m, 2 H), 8.70 (m, Hi); J 5 , 6 = 3.5 Hz,J2,,3,= 7.7 Hz,J2,,3,,= 5.3 Hz; CIMS, m/z = 2.3 Hz, J g , l o = 5.4 Hz, Jlo,ll = 9.8 Hz, J2,,3, = 4.8 Hz, J2,,3tt = (relative intensity) 35C1/37C1isotope cluster, MH+, 622 (0.6) 3.0 Hz; CIMS (reagent gas NH3), m/e (relative intensity) 36clf7Cl 35c13/37c13,620 (1.3) 36c1,/37c12, 618 (2.2) 36c15/37C1,616 (l.l),w&, 938 (3) 35Cl#7C&, 936 isotope cluster; MH', 940 (1)35c15/37C17, 547 (1.2), 545 (0.9), 302 (12), 274 (loo), 272 (18), 244 (72). Hy(8) 35C17/37C15,934 (14) 35C18/37C14,932 (29) 35c1g/37c13, 930 (25) drolysis, as described previously, and purification by chroma35c11~/37c12, 928 (13) 35c111/37c1, 926 (3) 35C112,592 (7), 590 (27), tography (solvent gradient, CH2C12/EtOAc, 9:l to 4:l) gave 588 (64), 586 (loo), 584 (51), 244 (32). Crystallization from petrans-5(R),6(R)-dihydro-6-hydroxy-5-methoxy-7-methylbenz[c]troleum spirit afforded needles which were examined by X-ray acridine (3.1mg): CD spectrum, Acvw -44.6, At211 +43.7 (M-l cm-'). diffraction. On the analytical Spheri-10 silica HPLC system the The more polar product (9.5 mg) was identified as trans8(R),9(R)-and 8(S),S(S)-isomers eluted at 11.4 and 17.4 min, 5(S),6(S)-dihydro-6-[[ [ (1R,2S,4S)-endo-1,4,5,6,7,7-hexachlororespectively. Hydrolysis was effected a t room temperature by using aqueous NaOH in THF/MeOH. bicyclo[2.2.11 hept-5-en-2-yl]carbonyl]oxy]5-methoxy-7-methylbenz[c]acridine: 'H NMR (9 mg/0.3 mL) 6 2.42 (m, H3" and Hy), Crystal data: a = 13.766(4)A, b = 14.144(5)A, c = 19.505(5) 2.81 (s, 7-methyl), 3.27 (s, 5-OMe), 3.43 (dd, H2.), 4.46 (d, Hs), A, V = 1864 A3, CMH19Cl12N04, orthorhombic space group P212121, 6.61 (d, Hs), 7.26-7.84 (m, 5 H), 7.97-8.24 (m, 2 H), 8.71 (m, HI); MoKa radiation (A = 0.71069 A), 2822 reflections collected, 1378 J5,6 = 3.3 Hz, J2,,3,= 8.4 Hz, J2t,3t1 = 4.7 Hz. CIMS was virtually with I > 2.5a(O used in the refinement. Data were collected on identical with that of the less polar diastereoisomeric product. an Enraf Nonius CAD4 diffractometer; the structure was solved Hydrolysis and chromatography, as described previously, gave by direct methods using SHELXS-86 and refined by blocked matrix trans-5(S),6(S)-dihydro-6-hydroxy-5-methoxy-7-methylbenz[c]least squares using SHELX-76 to a final R of 0.058, R, 0.062. The acridine (4.5 mg): CD spectrum, Aevw +43.7, AeZu -45.0 (M-l cm-'). largest peak in a final difference map was 0.4 e A-3 in height (see Treatment of racemic trans-5,6-dihydro-5-hydroxy-6-methalso supplementary material). oxy-7MBAC (9.6 mg) with the acid chloride of (+)-HCA, as detrans -8( S ),9( S )-Dihydro-d,g-bis[[4-(dimethylamino)scribed previously, afforded two products after chromatography. trans -cinnamoyl]oxy]-7-methylbenz[ c ]acridine. transThe less polar product (7.4 mg) was identified as trans-5(R),G(R)8(S),S(S)-Dihydro-8,9-dihydroxy-7-methylbenz[c]acridine (1.0 mg) and 4-(dimethylamino)-transs-cinnamic acid imidazole ester (4 mg) dihydro-5- [ [ [ (1R,2S,4S)-endo-1,4,5,6,7,7-hexachlorobicyclo[2.2.l]hept-5-en-2-yl]carbonyl]oxy]-6-methoxy-7-methylbenz[c]were dissolved in freshly dried T H F (2 mL). NaH (50% in oil, acridine: 'H NMR (7 mg/0.3 mL) 6 2.47 (d, 2 H30, 2.85 (s, 8 mg) was then added to the solution under N,. The mixture was 7-methyl), 3.32 (t,H2,),3.43 (s, 6-OMe), 5.06 (d, He), 6.28 (d, H5), stirred under N2 gas a t room temperature for 2 h. The reaction 7.44-7.85 (m, 5 H), 8.00-8.24 (m, 2 H), 8.70 (m, Hl); J5,s = 3.2 was completed by this time as shown by TLC (EtOAc/petroleum Hz,Jr,3,= 6.5 Hz; CIMS, m/z (relative intensity) 35Cl/rrC1isotope ether, bp 70-75 "C, 1:2). The reaction mixture was partitioned cluster, MH+, 622 (2.1) 35C13/37C13, 620 (5.0) 35c14/37c12,618 (7.5) between 20% aqueous NaCl and EtOAc. The EtOAc extract was 35C15/37C1, 616 (3.9) %C&,588 (Ll),586 (2.0), 584 (1.4), 549 (1.2), dried (Na2S04)and evaporated to dryness. The residue was 547 (2.3), 545 (2.1), 302 (ll),284 (5), 274 (100), 272 (24), 244 (91). purified by vacuum chromatography on silica gel (3 cm X 3 cm Hydrolysis and chromatography, as described previously, gave i.d.; gradient, EtOAc/petroleum ether, bp 70-75 "C, L19, to

Stereochemistry of 7-Methylbenz[clacridine Metabolites

trans-5(R),6(R)-dihydro-5-hydroxy-6-methoxy-7-methylbenz [c]acridine (3.5 mg): CD spectrum,Aew -44.0, A$,, +41.6 (M-l an-*). The more polar product (8.9 mg) was identified as trans-5(S) ,6(S)-dihydro-5-[ [ [ (1R,2S,4S)-endo-1,4,5,6,7,7-hexachlorobicyclo[2.2.11 hept-5-en-2-yl]carbonyl] oxyl-6-methoxy-7-methyL

Chem. Res. Toxicol., Vol. 4,No.5, 1991 549 Table I. Separation of (+)-HCA Esters of 7MBAC Metabolites or Metabolite Derivatives on a Spberi-10 Normal-Phase Column retention time. min compound R,R-isomer S,S-isomer 7MBAC-3,4-DHD" 16.4 14.2 7MBAC-5,6-DHDb 14.6 20.5 7MBAC-8,9-DHDb 11.4 17.4

benz[c]acridine: 'H NMR (8 mg/0.3 mL) 6 2.36 (m, HBtand Hr), 2.82 (s, 7-methyl), 3.36 (m, HzJ, 3.44 (s, 6-OMe), 5.02 (d, H&, 6.24 (d, H6), 7.44-7.85 (m, 5 H), 8.00-8.24 (m, 2 H), 8.70 (m, HI); J5,e = 3.2 Hz. CIMS was virtually identical with that of the less polar product. Hydrolysis and chromatography, as described previously, gave trans-5(S),6(S)-dihydro-5-hydroxy-6-methoxy-7-methyl- "6% EtOAc in hexane fraction at 0.8 mL/min was used as eluant. b5% EtOAc in hexane fraction at 1.0 mL/min was used. benz[c]acridine (4.2 mg): CD spectrum, AcZu +44.7, Ac213-43.3 (M-l cm-'). and where the minor metabolite was present in sufficient amount Methylation of 5(R),G(R)-trans-7MBAC-5,6-DHD. Enfor reliable area measurement. The retention times and chroantiomerically pure 5(R),6(R)-7MBAC-5,6-DHD(6.3 mg) dwolved matographic conditions are shown in Table I. 7MBAC-5,6-oxide in dry T H F (18 mL) was stirred at room temperature with NaH metabolite fractions were treated with 0.4 M sodium methoxide (35.4 mg, 50% in oil) and methyl iodide (0.18 mL) for 3 h. The in MeOH (50 pL) a t 60 "C for 2 h. The reaction was diluted with reaction was terminated when monomethylation products were CH2Cl2/EtOAc (2:l) and worked up as described below under maximal as shown by TLC. Partitioning of the products between derivatization. EtOAc and 20% aqueous NaCl followed by drying of the organic Derivatization. A portion of the dihydrodiol fraction isolated phase and then solvent evaporation gave the products transfrom the liver microsomal incubations (equivalent to about 5 pg) 5(R),6(R)-dihydro-5hydroxy-6-methoxy-7-methyl~nz[c]acridine, trans-5(R),6(R)-dihydro-6-hydroxy-5-methoxy-7-methylbenz[c]- in anhydrous tetrahydrofuran (THF, 20 pL) was treated with 20 pL of reagent containing p(dimethy1amino)pyridine (25.0 mg) acridine, 5-hydroxy-7-methylbenz[c]acridine(20), and the 5and (+)-HCA chloride (50.0mg) dissolved in anhydrous THF (1.0 (R),G(R)-dimethoxy derivative. These products were separated mL). After 5 min the reaction mixture was diluted with by CSP HPLC and identified by cochromatography with products CH2CI2/EtOAc(91 v/v, 1 mL) and vacuum filtered through TLC obtained by methanolysis of 7MBAC-5,6-oxide. grade silica gel (5 X 30 mm bed), and the silica gel was washed Animals. Male Wistar rats (150-200 g) were either untreated with fresh solvent (2 X 1 mL). The residue obtained after or pretreated by intraperitoneal injection of 3-methylcholanthrene evaporation of the combined eluants was chromatographed on (MC) (20 mg/kg) in corn oil daily for 2 days, or phenobarbital a 10-pm Spheri-10 column (25 X 0.46 cm i.d., Brownlee) using (PB), (60,80, and 100 mg/kg, respectively) in isotonic saline daily 6% v/v EtOAc in hexane fraction (1.0 mL/min) as the mobile for 3 consecutive days. Animals were fasted for 24 h after the phase. Measurement of peak heights and radioactivity allowed last inducer dose and killed, and liver microsomes (24) were the relative quantitation of both enantiomers. Retention times prepared as described. Microsomal suspensions were stored a t are shown in Table I. -80 "C until required and were used within 6 months of prepaChiral Stationary-Phase Chromatography. The four ration. Determinations of microsomal protein were by the method isomeric methanolysis products of racemic 7MBAC-5,6-oxidewere of Lowry e t al. (25), using bovine serum albumin as standard. separated on a Pirkle ionically bonded (R)-N-(3,5-dinitroMicrosomal Metabolism. [3H]7MBACwas incubated with benzoy1)phenylglycinecolumn (RegisChemical Co., Morton Grove, liver microsomal protein a t 37 "C in potassium phosphate buffer IL) using a 3.3% EtOH and 1.7% acetonitrile in hexane as the (0.1 M, pH 7.4) in the presence of magnesium chloride (4.5 mM), mobile phase (1.0 mL/min). The enantiomers of 5,6-dihydro-6glucose 6-phosphate (1.5 mM), glucose-6-phosphatedehydrogenase hydroxy-5-methoxy-7MBAC emerged at 35.2 min [5(R),6(R)]and (1 IU/mL), and NADP (0.5 mM). The substrate [40 pM, with 38.4 min [5(S),6(S)]with a resolution value of 2.1, while those (2-3) X los dpm per incubation] was added in acetone (50 pL), of 5,6-dihydro&hydroxy-6-methoxy-7MBACemerged at 41.4 min and the total volume of the incubation mixture was 1.0 mL. For [5(R),6(R)J and 43.4 min [5(S),6(S)J with a resolution value of (TCPO) one experiment, 1.9 mM 3,3,3-trichloropropene-1,2-oxide 1.3 and were not completely separated. was added to the incubation mixture. For preparations with control microsomes, and microsomes prepared from PB- and MC-pretreated animals, protein concentrations were 0.5,0.2, and Results 0.2 mg/mL and incubation times were 15, 10, and 10 min, reEnantiomers of 7MBAC-5,6-DHD. Racemic spectively. Metabolites were extracted with EtOAc (3 x 2.0 mL), 7MBAC-5,6-DHD (20)was converted to its (+)-HCA esthe combined organic extracts were dried and evaporated to ters, and the latter were separated chromatographically dryness, and the residue was dissolved in DMSO (20 pL) before by vacuum chromatography before hydrolysis back t o the analysis by separation of metabolites by methanol/water gradient p u r e enantiomers. The purity of the separated bis-(+)reverse-phase HPLC using the system described above. Metabolites from up to 6 incubations were pooled, and those fractions HCA esters was checked by HPLC on silica, and each gave that possessed the retention times and UV spectra of 7MBACa single peak. T h e value of J5,6of the dihydrodiol, 3.1 Hz, 5,6-DHD, 7MBAC-8,9-DHD, 7MBAC-5,6-oxide, and 7MBACand those of J5,6 in each of the diastereoisomeric bis3,4-DHD were collected for stereochemical analysis. The 3,4(+)-HCA esters, 3.5 Hz, indicated that a similar confordihydrodiol peak overlapped with that of 7-(hydroxymethy1)mation was present around t h e 5,6 carbon-carbon bond. benz[c]acridine 5,6-oxide (1.9, and this fraction was rechromaB y use of Karplus' relationships (26) the dihedral angle tographed on a 5-pm Spheri-5 silica column (250 X 4.6 mm i.d., between H5and H6 is calculated t o be about 70'. Brownlee Laboratories) using 2% EtOH in cyclohexane as eluant Of the two possible conformations of t h e 5,6-dihydrodiol (1.0 mL/min) to separate these. Retention times were 33.0 and with either a P or a M skew sense [Figure 1,shown for t h e 15.6 min, respectively. The HPLC eluant was monitored a t 270 nm, and a Hewlett5(R),6(R)-isomer] in which the H5 t o H6dihedral angle is Packard high-speed spectrophotometric detedor, Model HP1040A close t o 180' with quasi-diequatorial substituents, or 60' ( h e a r diode array), was used to obtain the metabolite UV spectra with quasi-diaxial groups, respectively, t h e latter is known as they were eluted from the reverse-phase HPLC. Their identities to be preferred from t h e coupling constant between H, and were thus confirmed. He.A small dihedral angle with t h e 5(S),G(S)-enantiomer The pooled dihydrodiol metabolite fractions were evaporated is only possible with P helicity or skew sense (Figure IC). to dryness under vacuum and dried overnight over phosphorus The CD spectra of t h e enantiomers (Figure 2) c a n be repentoxide, and the metabolites were converted to their bis esters garded as those of biaryls (20-22), being in this case subwith (+)-HCA chloride. The pairs of diastereoisomers were then s t i t u t e d 2-phenylquinolines. T h e CD spectrum of the separated by normal-phase HPLC to allow quantitation by radihydrodiol from the less polar diastereoisomer displayed diochemical determinations (liquid scintillation counting), and by peak area ratios when no synthetic carrier metabolite was used a strong negative Cotton effect at 242 n m a n d a weaker

Duke et al.

550 Chem. Res. Toxicol., Vol. 4, No. 5, 1991 a

OH

OH

k c

OH

I 320

I

I

1

1

1

LO 0 WAVELENGTH ( n m )

L50

Figure 3. CD spectrum in CHC&of 5(R),G(R)-dihydro-5,6-bis[ [4- (dimethy1amino)cinnamoylloxy]-7-methylbenz[c]acridine. OH

Figure 1. Conformations of the 5(R),G(R)-enantiomer of tram-5,6-dihydro-5,6-dihydroxy-7-methylbenz[c]acridine showing M helicity (a) and P helicity (b) (left-handedand right-handed skew sense, respectively),and that of the B(S),G(S)-isomerwith P helicity (c). Carbon 6 is closer to the viewer in these perspectives.

Figure 2. CD spectra of the enantiomers of trans-5,6-dihydro5,6-dihydroxy-7MBAC. The structure of the B(R),G(R)-enantiomer is shown. positive Cotton effect at 214 nm. This, taken with the 'H NMR evidence for the smaller dihedral angle, indicates conclusively that the biaryl conformation possessed the A4 skew sense and that this compound, and its precursor diester, had the 5(R),6(R)configuration. The enantiomeric (5S,6S)-7MBAC-5,6-DHD afforded a strong positive Cotton effect at 242 nm and a weaker negative one at 214 nm (Figure 2). Although the dihedral angle between the hydroxyl groups was large and would be expected to be so for its diesters, the 5(R),G(R)-enantiomer was converted to its bi~-4-(dimethylamino)cinnamate ester. Signals due to the

H5 and H6atoms in the 'H NMR were seen at 6.32 and 6.80 ppm, respectively, with a coupling constant of 3.3 Hz. While this indicated a rather large dihedral angle between the ester functions (an angle of near 60° between the hydrogen atoms), it did not prevent exciton coupling between these dipoles. The weak negative and positive Cotton effects displayed respectively at 385 and 347 nm in the CD spectrum measured in chloroform (Figure 3) indicated the configuration from a priori considerations to be 5(R),6(R) (30),the same as that assigned by the semiempirical method. The CD spectrum was also measured in THF (not shown). In addition to the Cotton effects seen at long wavelength, a strong extreme expected for the biaryl chromophore was present as expected -39.4 M-' cm-') and was comparable to that of the 5(R),6(R)-dihydrodiol (A6242 -41.6 M-' cm-'). Absolute Configuration of the Enantiomers of 7MBAC-5,6-oxide, Racemic 7MBAC-5,6-oxideafforded, on reaction with sodium methoxide, two products in approximately equal amounts. The less polar was identified as trans-5,6-dihydro-6-hydroxy-5-methoxy-7-methylbenz[clacridine (6) from the methine signals in the 'H NMR spectrum at 4.49 and 5.40 ppm (doublets, J = 3.3 Hz, the latter being broader) due to the methines attached to methoxy and hydroxy, respectively. An NOE effect on the 5.40 ppm signal was observed upon irradiation of the 7methyl signal at 2.81 ppm, indicating that the hydrogen giving rise to it was at position 6 in close proximity to the 7-methyl group. By similar arguments the more polar compound was trans-5,6-dihydro-5-hydroxy-6-methoxy7MBAC (7). These compounds have previously been characterized as primary photooxidation products of 7MBAC formed in methanol by pathways that did not involve the intermediacy of the 5,6-oxide (23). Both mono ethers 6 and 7 were converted to their (+)-HCA esters which were readily separated by vacuum chromatography and then hydrolyzed to the enantiomers of 6 and 7. The enantiomers of both 6 and 7 were also separable from one another and the isomeric monomethyl esters (7 and 6) by chiral stationary-phase chromatography; both ionically bonded and covalently bonded (R)-N-(3,5-dinitrobenzoy1)phenylglycine [ (R)-DNBPG-I and ( R ) DNBPG-C, respectively] afforded superior separations to those obtained with an (S)-DNBLeu-I CSP column, and of the two (R)-DNBPG columns the ionically bonded was preferred. For trans-5,6-dihydro-6-hydroxy-5-methoxydibenz[aj]acridine, analogous to 6, separation of the en-

Chem. Res. Toxicol., Vol. 4, No. 5, 1991 551

Stereochemistry of 7-Methylbenz[c]acridine Metabolites solvent MeOH

50 LO

30 20

3 -10 -20

-30 -LO

-50

Figure 5. X-ray crystallographic structure of trans-8(S),S(S)dihydro-8,9-bis[[ [ (+)-(1R,2S,4S)-1,4,5,6,7,7-hexachlorobicyclo[2.2.l]hept-5-en-2-yl]carbonyl]oxy]-7-methylbenz[c]acridine

1

(GSH2).

coupling occurred between the two 4-(dimethylamino)benzoate groups. The chiral exciton coupling seen at 299 and 323 nm as positive and negative Cotton effects, reWAVELENGTH l n m ) spectively, and the relatively large J3,.,of 6.1 Hz in the 'H Figure 4. CD spectrum of trans-3(R),4(R)-bis[[p-(dimethylamino)benzoyl]oxy]-7-methyl-l,2,3,4-tetrahydrobenz[c]acridine. NMR spectrum allowing a dihedral angle of about 110' to be assigned between the ester functions made the assignment of a 3(R),4(R) configuration to this isomer antiomers by CSP chromatography was not achieved, mandatory (30).The chromophore in the diester possesses however (31). a left-handed skew sense. The enantiomeric parent comThe couplings between H6 and HE in the 'H NMR pound, trans-7MBAC-3(S),4(S)-THD, was converted to spectra of about 3.2 Hz in the four bis esters show that the dihydrodiol(19) and its bis-(-)-HCA ester chromatosimilar quasi-diaxial conformations exist for the 5- and graphed with the second emerging peak at 25.6 min when 6-substituent groups as those determined for the parent racemic 7MBAC-3,4-DHD was similarly derivatized and compounds of -3.3 Hz (23).The CD spectra of the enchromatographed. antiomers of 6 and 7 (see supplementary material, Figures Resolution and Absolute Configuration of the En8 and 9) allowed the absolute configurations to be deterantiomers of trans-8,9-Dihydro-8,9-dihydroxy-7mined by the semiempirical method as used with other methylbenzr c ]acridine. Racemic trans-7MBAC-8,9biaryls and described above for the 7MBAC-5,6-DHD DHD was converted to its bis-(+)-HCA derivatives which enantiomers (Figure 1). The CD spectra were very similar were separated by preparative HPLC. Their JE,svalues to those of the 5,6-dihydrodiol enantiomers but with a (3.2 Hz) in their 'H NMR spectra indicated that the hyCotton effect at lower wavelength (-215 nm) of approxdrogens at the 8- and 9-positions were quasi-diequatorial, imately the same absolute magnitude as that seen at -242 the large substituent groups adopting the quasi-diaxial nm. The isomer of 6 obtained from the more rapidly conformation. After crystallization, the second fraction eluting (+)-HCA ester shows a negative first Cotton effect from preparative HPLC was subjected to X-ray crystalat 242.5 nm (AE -44 M-' cm-l) and an equally strong lographic examination to obtain the atomic coordinates positive effect at about 215 nm. This M helicity when and exact molecular structure (Figure 5). The absolute taken together with the quasi-diaxial conformation of the configuration of the (+)-HCA (5) was known to be that substituent groups indicates that the configuration is 5shown in Chart I, that is, having the l(R),2(S),4(S) con(R),6(R). Its enantiomer displays the expected mirror figuration? and this allowed the structural interpretation image CD spectrum. Similarly, the enantiomer of 7 which of the more polar bis-(+)-HCA ester, which was therefore was derived from the more rapidly eluting (+)-HCA ester assigned the 8(S),9(S) stereochemistry. possessed the same M helicity and thus the 5(R),6(R) The structure consists of neutral molecules packed with configuration (Figure la). These assignments were conno unusual close contacts. The molecule consists of an firmed by partial methylation of 5(R),6(R)-7MBAC-5,6approximately planar group with HCA groups disposed on DHD to afford a mixture of two monomethylated comeither side of this plane. The three-ring conjugated aropounds (single enantiomers of 6 and 7). Chromatography matic system is planar to within 0.06 A, and directly atof this on (R)-DNBPG-I gave peaks chromatographing at tached atoms are 0.12 A out of this plane. The ester group 35.2 and 41.4 min, indicating consistency between condefined by O(l), 0(2), C(15), and C(16) is planar to within clusions drawn for the 5,bdihydrodiols and their mono0.022 A, but the other is significantly distorted from plamethyl ethers. This agreement is to be expected since the narity (up to 0.075 A). O(4) of this group is disordered over absolute configurations were confirmed by fundamentally two sites (6040), and it may be that the apparent nonthe same method. planarity may not be real but may rather be a consequence trans -3,4-Dihydroxy-7-met hyl- 1,2,3,4-tetrahydroof the movement of other atoms associated with this disbenz[c ]acridine. Racemic trans-7MBAC-3,4-THD was order. separated into its enantiomers via the bis-(+)-HCA esters. The tetrahydrodiol obtained from the more polar (+)-HCA diester was converted to its bis-p-(dimethylamin0)benzoate C. C. Duke, D. Cutler, S. Carter, and G. M. Holder, unpublished ester, and its CD spectrum (Figure 4) showed that exciton observations (manuscript in preparation). 300

Loo

552 Chem. Res. Toxicol., Vol. 4,No.5, 1991

Duke et al.

Chart I

@:

I

I

I

I

I

'

@ 00

'"00, 9

& 3

I

6-

OH

OH

HO OH

2

2-

qI&T 4

-

-6 I

320

I

1

LOO

I

L50

Stereochemistry of 7-Methylbenz[c]acridine Metabolites

Chem. Res. Toxicol., Vol. 4, No. 5, 1991 553

Table 111. Enantiomeric Composition of [SH17MBAC-5,6-oxideFormed in Rat Liver Microsomal Incubationsa liver microsomal DreDaration control PB induced MC induced metabolite expt no. R,Sb enant purity % metabd R,S enant purity % metab R,S enant purity % metab 7MBAC-5,6-oxide 1' 78.41 56.8 1.0 (24.4) 38.9 22.2* 2.1 (5.6) 2 86.2f 72.4 17.4 (43.1) 73.6' 47.2 5.7 (57.4) 37.0' 26.0* 3.6 (58.0) 85.38 70.6 72.58 45.0 35.58 29.0* The metabolite was reacted with sodium methoxide, and the products were then separated by CSP chromatography on an ionically bonded (R)-N-(3,5-nitrobenzoyl)phenylglycine column. The predominant enantiomer has the 5(S),6(R)configuration determined from the relative amounts of the 5(R),6(R)-. and 5(S),G(S)-enantiomers of trans-5,6-dihydro-6-hydroxy-5-methoxy-7-methylbenz[c]acridine. CEnantiomeric purity. dMetabolism is shown as the percent of total metabolites present as the metabolite and the percent of substrate metabolized (in parentheses) as determined by the fraction of radioactivity emerging from the reverse-phase HPLC column before the unchanged substrate. 'This experiment was conducted in the presence of 1.9 mM TCPO to inhibit expoxide hydrase. 'Determination by radioactivity measurements. 8Determined by measurement of peak area ratios. No synthetic carrier added. *The 5(S)$(R)-isomer was predominant.

The enantiomeric selectivity of formation of the 5,6oxide was determined by sodium methoxide treatment of the metabolite followed by CSP-HPLC as described above. The ratios (shown in Table 111) of the 5(R),6(S)- to 5(S),6(R)-enantiomers were determined from the first two peaks to emerge from the CSP column, these being the 5(R),6(R)- and 5(S),G(S)-isomersof trans-5,6-dihydro-6hydroxy-5-methoxy-7MAC. The enantiomers are formed through bimolecular nucleophilic substitution by methoxide ion at C5 on 7MBAC-5(S),G(R)-oxideand 7MBAC5(R),G(S)-oxide, respectively. The base-line separation achieved for these compounds allowed accurate determination of the enantiomeric ratio of the oxide produced by both peak area and radioactivity determination (Table 111). The third and fourth peaks to emerge from the CSP column correspond to ethers formed by methoxide attack at C6 and allowed the quantitation to be verified by peak area measurements. Peak 3 was the 5(R)-hydroxy-G(R)methoxy derivative from the 5(R),G(S)-oxidewhile peak 4 was its enantiomer. The proportion of the 5(R),6(S)oxide determined from peaks 3 and 4 was, for experiment 2,83.2, 73.6, and 34.470, respectively, for liver microsomes from control, PB-pretreated, and MC-pretreated animals. This excellent agreement was not found in radioactivity determinations which were about 10% less (76.8,64.2, and 29.296, respectively). The resolution value for the separation of the 5-hydroxy-6-methoxy enantiomers was 1.3 and perhaps accounts for this discrepancy if the isotopically labeled ethers possess a fractionally greater capacity factor. Because the l0,ll-dihydrodiol is a minor metabolite formed in very small proportions, and 1,Zdihydrodiol has not been detected (12, 32), information on these compounds was not sought. However, 7MBAC-10,ll-DHD (+)-HCA derivatives were readily separable on the silica column.

Discussion The enantiomers of the dihydrodiols which are the major metabolic products of 7MBAC have now been prepared and their absolute configurations assigned, either by X-ray diffraction or by the semiempirical method involving the biaryl chromophores produced by reduction at the 5,6positions. By a similar method, the enantiomeric K-region oxides have also been characterized through their methanolysis products. The assignments of the stereochemistry of the 8,9-dihydrodioland 5,6-dihydrodiolwere supported by the nonempirical approach using chiral exciton coupling of bis-C(dimethylamino)cinnamates, although in these examinations, the conformations were not optimal for strong coupling because the steric interactions between the 7-methyl group and the peri-6- or 8-substituted [4-(dimethylamino)cinnamoyl]oxy group forced the ester groups to lie in quasi-diaxial positions in each compound. The

electric dipole-dipole interaction in the 5,6-dihydrodiolwas stronger, with an amplitude of about 25 (AAc), affording increased confidence to the configurational assignment when compared with that of the 8,g-isomer. Nevertheless, that the result obtained from the nonempirical chiral exciton coupling for the latter was in agreement with the X-ray crystallographic structure lends confidence to such conclusions even when they are based on weak dipolar interactions. Such a result would not have been obtainable with the p(dimethy1amino)benzoate esters or the pphenylbenzyl ethers (33)for which the UV maxima are at lower wavelengths because of interference by other chromophores in the compounds. The stereochemical results obtained for 7MBAC-5,6oxide for all liver microsomes examined are broadly in agreement with those obtained previously with dibenz[ajlacridine ( 17). The 5(R),G(S)-enantiomerpredominated for the latter with both control and PB-induced preparations, and there was a reversal to the predominance of the 5(S),G(R)-isomer in experiments with liver microsomes from MC-pretreated animals. However, for the dibenzacridine the enantiomeric purity was high at about 90% for MC-induced rat preparations in contrast to about 25% for 7MBAC. The latter result was independent of the presence of the epoxide hydrase inhibitor, TCPO, which substantially inhibited metabolism but had no impact upon the ratio of the two enantiomers, or on the proportion of oxide formed in relatively long incubations of up to 30 min. This indicates that epoxide hydrase catalyzed opening of the two enantiomers is not selectively inhibited by TCPO. The predominant metabolically produced K-region oxides of benzo[a]pyrene [4(S),5(R)-oxide] (34) and benz[alanthracene [5(S),G(R)-oxide](341, formed by MC-induced microsomes, and of 7,12-dimethylbenz[a]anthracene [5(S),G(R)-oxide](35),benzo[a]pyrene (36),and benz[a]anthracene (37),formed by a reconstituted cytochrome P-45Oc system, possessed the same absolute configuration as the predominant enantiomer from the nitrogenous heterocyclic systems, 7MBAC and dibenz[aj]acridine formed by MC-induced preparations. The heterocyclic system, dibenz[a,h]acridine,is extensively converted to one of its K-region oxides, but the absolute configuration and the stereoselectivity of this has not been elucidated (38). For the formation of benzo[c]phenanthrene 5(S),G(R)-oxide both microsomal and reconstituted cytochrome P-45Oc data indicated a lower enantiomeric excess (7,11,39).In all cases the (S,R)-oxide predominated. Microsomes from uninduced and PB-induced animals generally have not shown the reversal of the stereoselective preference compared with those from MC-pretreated animals. In this work the predominant K-region oxide formed reversed to the (S,R)-enantiomer when the MCinduced preparation was used. This was not seen with

554 Chem. Res. Toxicol., Vol. 4, No. 5, 1991

benz[a]anthracene (34, 37), benzo[a]pyrene (34), 12methylbenz[a]anthracene (401, or other compounds apart from chrysene (11,41) and dibenz[aj]acridine (17),which behave similarly to 7MBAC. For these other compounds (11) the K-region oxide metabolite has a higher enantiomeric purity when formed from MC-induced preparations. For naphthalene (42), dibenz[a,h]anthracene (43), triphenylene (44), benz[a]anthracene (34,37,45), benzo[clphenanthrene (46), and chrysene (41) the stereochemistry of the metabolic epoxidation has been elucidated at non-K-regions. The dihydrodiols of 7-methylbenz[c]acridine formed metabolically are produced with a stereoselectivity consistent with that observed with other polycyclic hydrocarbons non-K-region dihydrodiols. Benzo[a]pyrene-7(R),8(R)-dihydrodiol (2, 11, 47), benz[a]anthracene-8(R),S(R)-dihydrodiol and its 3(R),4(R)- and 10,ll-dihydrodiol derivatives (45, 481, phenanthrene-l(R),2(R)dihydrodiol and -3(R),4(R)-dihydrodiol (49, 50), chrysene-l(R),2(R)-dihydrodiol and -3(R),4(R)-dihydrodiol (4+51), benzo [cl phenanthrene-3(R),4(R)-dihydrodiol(52, 53), dibenz [a,h]anthracene-3(R),4(R)-dihydrodiol (54), 6-fluorobenzo[c]phenanthrene-3(R),4(R)-dihydrodiol(55), and 6-fluorobenzo[a]pyrene-7(R),8(R)-dihydrodiol(56)are the predominant M-region dihydrodiols formed by MCinduced or PCB-induced rat liver microsomes. If, as has been demonstrated for other PAH (2, 11), there is predominant attack of water catalyzed by epoxide hydrase on the allylic rather than benzylic carbon, then the observations of the predominant R,R configuration of 7-methylbenz[c]acridine-8,9-dihydrodioland -3,4-dihydrodioldemand that the 7(R),8(S)-oxideand 3(S),4(R)-oxideare the major oxidation product formed in a selective fashion at each site. The stereoselectivity of these reactions for 7methylbenz[c]acridine was not substantially affected by the pretreatment used in the present study while with chrysene (51) and benzo[c]phenanthrene (53), products formed by control and PB-induced preparations were either optically inactive or showed a small excess of the S,S-enantiomers. For the K-region dihydrodiol of 7 methylbenz[c]acridinethe pattern followed that found in benz[a]anthracene (11,34,48),benzo[a]pyrene (11,57), chrysene (51),and dibenz[a,h]anthracene (58) in which the R,R-enantiomers were predominant regardless of the cytochrome P-450~present (constitutive, PB induced, or MC induced). With several compounds a lack of planarity causes, to varying extents, the preferred production of the K-region S,S-enantiomer (11). Thus with benzo[c]phenanthrene (39), 12-methylbenz[a]anthracene(40), and 7,12-dimethylbenz[a]anthracene(59) all preparations afforded a major amount of the 5(S),G(S)-dihydrodiol.

Duke et al. and details of least-squares plane calculations (Tables Sl-S7) (14 pages); a table of observed and calculated structure factors for GSH2 (9 pages). Ordering information is given on any current masthead page.

References (1) Yang, S. K., and Silverman, B. D., Eds. (1988) Polycyclic Aro-

matic Hydrocarbon Carcinogenesis: Structure-Activity Relationships, Vols. I and 11, CRC Press, Boca Raton (2) Thakker, D. H., Yagi, H., Levin, W., Wood, A. W., Conney, A. H., and Jerina, D. M. (1985) Polycyclic aromatic hydrocarbon: metabolic activation to ultimate carcinogens. In Bioactioation of Foreign Compounds (Anders, M. W., Ed.) pp 177-242, Academic Press, New York. (3) Lehr, R. E., Wood, A. W., Levin, W., Conney, A. H., and Jerina, D. M. (1988) Benzacridines and dibenzacridines: metabolism, mutagenicity, and carcinogenicity. In Polycyclic Aromatic Hydrocarbon Carcinogenesis: Structure-Actiuity Relationships (Yang, S. K., and Silverman, B. D., Eds.) Vol. I, pp 31-58, CRC Press, Boca Raton. (4) Epstein, S. S. (1967) Carcinogenicity of organic extracts of atmospheric pollutants. J. Air Pollut. Control Assoc. 17, 728-729. (5) van Duuren, B. L., Bilbao, J. A., and Joseph, C. A. (1960) The carcinogenic nitrogen heterocycles found in cigarette smoke condensate. J. Natl. Cancer Inst. 25,5341. (6) Schmeltz, I., and Hoffmann, D. (1977) Nitrogen containing compounds in tobacco and tobacco smoke. Chem. Reo. 77, 295-31 1. (7) Fu, P. P., Chou, M. W., and Beland, F. A. (1988) Effect of nitro substitution on the in vitro metabolic activation of polycyclic aromatic hydrocarbons. In Polycyclic Aromatic Hydrocarbon Carcinogenesis: Structure-Actioity Relationships Vol. 11, (Yang, S. K., and Silverman, B. D., Eds.) pp 37-65, CRC Press, Boca Raton. (8) Sugimura, T. (1988) New environmental carcinogens in daily life. Trends Pharmacol. Sci. 9, 205-209. (9) Yang, S. K., McCourt, D. W., Roller, P., and Gelboin, H. V. (1976) Enzymatic conversion of benzo[a]pyrene leading predominantly to the diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10tetrahydrobenzo[a]pyrene through a single enantiomer of r-7,t-8dihydroxy-7,8-dihydrobenzo[a]pyrene.Proc. Natl. Acad. Sci. U.S.A. 73, 2594-2598. (10) Thakker, D. R., Yagi, H., Akagi, H., Koreeda, M., Lu, A. H. Y., Levin, W., Wood, A. W., Conney, A. H., and Jerina, D. M. (1977) Metabolism of benzo[a]pyrene. VI. Stereoselective metabolism of benzo[alpyrene and benzo[a]pyrene 7,&dihydrodiol to diol epoxides. Chem.-Biol. Interact. 16, 281-300. (11) Yang, S. K. (1988) Stereoselectivity of cytochrome P-450 isozymes and epoxide hydrolase in the metabolism of polycyclic hydrocarbons. Biochem. Pharmacol. 37,61-70. (12) Boux, L. J., Duke, C. C., Holder, G. M., Ireland, C. M., and Ryan, A. J. (1983) Metabolism of 7-methylbenz[c]acridine:comparison of rat liver and lung microsomal preparations and identification of some minor metabolites. Carcinogenesis 4, 1429-1435. (13) Boux, L. J., and Holder, G. M. (1985) The metabolism of the carcinogen 7-methylbenz[c]acridine by hepatocytes isolated from untreated and induced rats. Xenobiotica 15, 11-20. (14) Wright, D. J., Robinson, H. K., Holder, G . M., and Ryan, A. J. (1985) Metabolism of the carcinogen 7-methylbenz[c]acridine in Acknowledgment. Assistance with NMR by Fu the rat. Xenobiotica 15, 825-834. Shanlin, Ken Jones, and Dr. Jaques Nemorin and with (15) Gill, J. H., Bonin, A. M., Podobna, E., Baker, R. S. U., Duke, mass spectral work by Michael Smythe and Bruce Tattam C. C., Rosario, C. A., Ryan, A. J., and Holder, G. M. (1986) 7and financial support from the National Health and Methylbenz[c]acridine: mutagenicity of some of its metabolites Medical Research Council are gratefully acknowledged. and derivatives and the identification of trans-7-methylbenz[c]acridine-3,4-dihydrodiolas a microsomal metabolite. CarcinoRegistry No. 1, 3340-94-1; (*)-2, 135271-29-3; (R,R)-2, genesis 7, 23-31. 117019-97-3; (S,S)-2,117019-98-4; (*)-3, 135271-30-6; (R,R)-3, (16) Chang, R. L., Levin, W., Wood, A. W., Shirai, N., Duke, C. C., 117019-93-9; (S,S)-3, 117019-94-0; (*)-4, 135271-31-7; (R,R)-4, Jerina, D. M., Holder, G. M., and Conney, A. H. (1986) High 117019-90-6; (S,S)-4, 117019-91-7; (5R96S)-6, 135271-32-8; tumorigenicity of the 3,4-dihydrodiol of 7-methylbenz[c]acridine (5S,6R)-6, 135271-33-9. on mouse skin and in the newborn mouse. Cancer Res. 46, 4552-4555. Supplementary Material Available: X-ray data for the (17) Duke, C. C., Holder, G. M., Rosario, C. A., and Ryan, A. J. bis-(+)-HCA ester of B(S),S(S)-dihydro-8,9-dihydroxy-7(1989) Stereochemistry of the major rodent liver microsomal memethylbenz[c]acridine (Figure 7), CD spectra of the enantiomers tabolites of the carcinogen dibenz[aj]acridine. Chem. Res. Toxof trans-5,6-dihydro-6-hydroxy-5-methoxy-7-methylbenz[c]- icol. 1, 294-303. acridine (Figure 8) and trans-5,6-dihydro-5-hydroxy-6-meth- (18) Boux, L. J., and Holder, G. M. (1985) The activation and DNA oxy-7-methylbenz[c]acridine(Figure 9), and positional parameters, binding of 7MBAC catalyzed by mouse liver microsomes. Cancer bond lengths, bond angles, thermal parameters, torsion angles, Lett. 25, 333-342.

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