Chem. Res. Toxicol. 1995, 8, 203-208
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The Stereochemistry of the Major Rat Hepatic Microsomal Metabolites of 7,9=Dimethylbenz[c]acridine and 7,1O=Dimethylbenz[c]acridine Yuerong Ye, Colin C. Duke, and Gerald M. Holder" Department of Pharmacy, University of Sydney, NSW 2006, Australia Received August 9, 1994@ The monofunctionalized dihydrodiol metabolites of 7,9-dimethylbenz[c]acridineand 7,lOdimethylbenz[c]acridine formed in incubations with rat liver microsomes from untreated and phenobarbital and 3-methylcholanthrene-pretreated rats were isolated by reversed-phase high performance liquid chromatography. The relative amounts of each enantiomer were determined by HPLC of diastereoisomeric esters with (+)-(lR,2$,4$)-endo-1,4,5,6,7,7hexachlorobicyclo[2.2.llhept-5ene-2-carboxylic acid (HCA). For the K-region dihydrodiols, absolute configurations were determined from their circular dichroic spectra using the empirical method. The absolute configuration of 3,4-dihydrodiol of 7,9-dimethylbenz[clacridinewas determined by the exciton chirality method from the CD spectrum of its bis4-(dimethylamino)benzoate ester. For the 8,9-dihydrodiol of 7,10-dimethylbenz[clacridinethe absolute configurations were tentatively assigned by normal-phase HPLC comparison of the (+)-HCA esters with literature data. I n every case the R,R-configuration predominated with optical purities >86% for non-Kvegion dihydrodiols and 56-68% for the K-region dihydrodiols.
Introduction The polycyclic aromatic hydrocarbons (PAH)l and their nitrogen containing analogues have been extensively studied as environmental contaminants associated with the particulate fraction in air (1-5). They are produced by combustion of organic fossil fuels and during smoking of tobacco (6). Concern with the group follows the existence of so many potent animal carcinogens among them, and the extensive work and observations that have led to the postulation of the bay-region theory of chemical carcinogenesis for these materials (7-9). Metabolic activation occurs to afford a dihydrodiol, which possesses a double bond adjacent to the bay region of the molecule, and oxidation of this alkene affords species which are highly electrophilic and can alkylate macromolecules. For the activation steps with PAH (7-9) and polycyclic azaaromatic compounds such as 7-methylbenz[clacridine (7MBAC)(10)and dibenz[ajlacridine (DBAJAC)(111,the activation passes through a dihydrodiol (proximate carcinogen) which is formed in rodent liver preparations in a highly stereoselective fashion with the R,R absolute configuration predominating. Epoxidation of the bayregion alkene double bond in these dihydrodiols from PAH also occurs in a stereoselective way with predominant oxygen attack occurring from one face of the dihydrodiol to afford the anti-diol epoxide (or diol epoxide 2) as the major product from the R,R-enantiomers (8). The mirror image (S,S)-dihydrodiol is not formed metabolically in significant amounts, and when it is introduced as a substrate, it is attacked from the same face of the molecule as its enantiomer t o afford the syn-diol epoxide
(or diol epoxide 1). Usually, only one of the 4 diastereoisomeric diol epoxides is highly tumorigenic (8). Among the benz[c]acridines, methylated compounds are more carcinogenic than the parent compound and the dimethylated and trimethylated compounds among the most potent. The metabolites of the highly carcinogenic dimethylated benzacridines, 7,9-dimethylbenz[clacridine (7,g-DMBAC) and 7,10-dimethylbenz[clacridine (7,lODMBAC) (12)(Figure l),formed by methylcholanthrene (3MC)-pretreated rats have been characterized,2 and the metabolite profiles have been described for both control and induced rat liver microsome^.^ In the present paper, the stereochemistry of the isolated metabolites is investigated.
Experimental Procedures Caution. All compounds in this study are potential carcinogenic agents and should be handled in an appropriate manner. Chemicals. [9-meth~l-~H]7,9-DMBAC and [10-methyL3H]7,10-DMBAC (13) with specific radioactivities of 1180 and 202 mCi/mmol, respectively, were '98% radiochemically pure by HPLC. They were diluted with unlabeled compounds before use. (+)-(1R,2S,4S)-endo-1,4,5,6,7,7-Hexachlorobicyclo[2.2.11hept-5-ene-2-carboxylicacid ((+)-HCA) was converted to its acyl chloride by heating under reflux with thionyl chloride (14). Material used was greater than 99% optically pure as determined by its reaction with D-ribonolactone acetonide (14).3MC and sodium phenobarbitone (PB) were from Sigma Chemical Co. (St. Louis, MO., USA) and glucose 6-phosphate, glucose-6phosphate dehydrogenase, and NADP were from Boehringer Mannheim (Sydney, Australia). 4-(Dimethylamino)benzoicacid and 4-(dimethylamino)pyridinewere obtained from Aldrich Chemical Co. (Milwaukee, WI). Silica gel was 60 H grade for TLC from E. Merck (Darmstadt, Germany). Instrumentation. Proton NMR spectra were recorded on a Bruker W.M. (400 MHz) spectrometer in methanol& Chemical ionization mass spectra were obtained on a Finnigan TSQ46 mass spectrometer with methane as reagent gas. Circular
* To whom correspondence should be addressed. Phone: 61-2-6922363; Fax: 61-2-552-3760;E-mail:
[email protected]. Abstract published in Advance ACS Abstracts, January 15, 1995. Abbreviations: PAH, polycyclic aromatic hydrocarbons; 3MC, 3-methylcholanthrene; PB, phenobarbital sodium; DHD, dihydrodiol; 7MBAC, 7-methylbenz[clacridine;7,9-DMBAC, 7,9-dimethylbenz[clacridine; 7,10-DMBAC,7,10-dimethylbenz[clacridine;DBAJAC, diben~[ajlacridine; HCA, ~+~-~1R,2S,4S~-endo-1,4,5,6,7,7-hexachlorobicyclo-Y. Ye, C. C. Duke, and G. M. Holder, unpublished results. [2.2.llhept-5-ene-2-carboxylic acid. Y. Ye, C. E. Scharping, and G. M. Holder, unpublished results. 0893-228x/95/2708-0203~09.00/0 0 1995 American Chemical Society
204 Chem. Res. Toxicol., Vol. 8, No. 2, 1995
Ye et al.
A
OH
I
"q3fy4 R2
YR
4 7MBAC:
R1 = H; R2 = H; R3 = CH3
OH
OH
B
I
7,g-DMBAC: R 1 = H; R2 = CH3; R3 = CH3 7,lO-DMBAC: R1 = CH3; R2 = H; R3 = CH3
Figure 1. Structures of 7-methylbenz[clacridine(7MBAC), 7,9dimethylbenz[c]acridine (7,9-DMBAC),and 7,lO-dimethylbenz[clacridine (7,lO-DMBAC). dichroic spectra of optically active compounds were measured on a JASCO J-500C spectropolarimeter with a DP-BOON data system (Japan Spectroscopic Co. Ltd., Tokyo, Japan). The spectra were recorded over 200-400 nm and the cell path length was 1 mm. A Beckman System Gold HPLC included a 168 linear array spectrophotometric detector which allowed W spectra to be recorded during elution. A wavelength of 270 nm was chosen to monitor the eluent. Animals. Male Sprague-Dawley rats (150-200 g) were either untreated or pretreated by intraperitoneal injection with 3MC (20 mgkg as a 10 mg/mL solution in corn oil) daily for 2 days, or with PB (60, 80, and 100 mgkg in water) for 3 days, respectively. Animals were fasted for 24 h after last inducer dose and then sacrificed. Liver microsomes were prepared (15) and stored at -80 "C until required. Microsomal Metabolism of 7,9-DMBAC and 7,lO-DMBAC. Incubations (25 mL) contained r3H17,9-DMBACor r3H17,10-DMBAC (40 pM,1 x lo6 dpm/mL added in acetone (
