CHIRALITY 2:141-149 (1990)
Enantioselective Aliphatic Hydroxylations of Racemic l-Hydroxy-3-methylcholanthreneby Rat Liver Microsomes MAGANG SHOU AND SHEN K. YANG Department of Pharmacology, F. Edward Hkbert School of Medicine, Uniformed Services University of the Health Sciences, Bethesdu, Maryland 208144799
ABSTRACT
Enantiomeric pairs of l-hydroxy-3-hydroxymethylcholanthrene (1-OH-3-OHMC), 3-methylcholanthrene (3MC) trans- and cis-l,2-diols, and 1hydroxy-3-methylcholanthrene(1-OH-3MC) were resolved by HPLC using a covalently bonded (R)-N-(3,5-dinitrobenzoyl)phenylglycine chiral stationary phase (Pirkle type 1A) column. The absolute configuration of an enantiomeric 3MC trans-l,2-diol was established by the exciton chirality CD method following conversion to a bis-p-Nfl-dimethylaminobenzoate. Incubation of an enantiomeric 1OH-3MC with rat liver microsomes resulted in the formation of enantiomeric 3MC trans- and cis-1,2-diols;the absolute configurations of the enantiomeric 1-OH-3MC and 3MC cis-l,2-diol were established on the basis of the absolute configuration of an enantiomeric 3MC trans-l,2-diol. Absolute configurations of enantiomeric 1OH-3-OHMCwere determined by comparing their CD spectra with those of enantiomeric 1-OH-3MC. The relative amount of three aliphatic hydroxylation products formed by rat liver microsomal metabolism of racemic 1-OH-3MC was 1OH-3-OHMC > 3MC cis-l,2-diol > 3MC trans-1,2-diol. Enzymatic hydroxylation at Cz of racemic 1-OH-3MC was enantioselective toward the 1S-enantiomer over the 1R-enantiomer (-3/1); hydroxylation at the C3-methyl group was enantioselective toward the 1R-enantiomer over the 1S-enantiomer (-58/42). Rat liver microsomal C,-hydroxylation of racemic 1-OH-3MCresulted in a 3MC trans-l,2-diol with a (lS,2S)/(lR,2R)ratio of 63/37 and a 3MC cis-l,2-diol with a (lS,2R)/(lR,2S) ratio of 12/88, respectively.
KEY WORDS: 3-methylcholanthrene, l-hydroxy-3-methylcholanthrene,l-hydroxy-3-hydroxymethylcholanthrene,3-methylcholanthrene trans-1,2-diol, 3-methylcholanthrene cis-l,2-diol, highperformance liquid chromatography, chiral stationary phase, resolution of enantiomers, absolute configuration, circular dichroism spectra, enantioselective hydroxylation, rat liver microsomes INTRODUCTION
3-Methylcholanthrene (3MC, Fig. 11, a potent mutagen and carcinogen, is metabolized to a complex mixture of nontoxic and mutagenic/tumorigenic products by mammalian drug-metabolizing enzyme systems (ref. 1and references therein). 3MC is also widely used as a cytochrome P-450 isozyme inducer in experimental animals.' C1 and C, positions of 3MC are major sites of oxidative metabolism, resulting in the initial formation of 1-OH-3MCand 2-OH-3MC.3-6 1-OH-3MC and 2-OH-3MC are further metabolized to mutagenid tumorigenic and detoxification product^.'*'^^ In the metabolism of 3MC by rat liver microsomes, both 3MC trans- and cis-l,2-diols are detected as metabolite^.^-^ In rat liver microsomal metabolism of either 1OH-3MC or 2-OH-3MC, 3MC tmns-1,2-diol is detected, but very little 3MC cis-1,2-diol is 0
1990 Wiley-Liss,Inc.
In the metabolism of 3MC by rat liver microsomes, 1-OH-3MC is enriched in t h e (1s)-enantiomer (53-73%), whereas 2-OH-3MC is enriched in the 2Senantiomer (86-98%); the exact enantiomeric compositions are dependent on the extent of their further metabolism and whether the rats were pretreated with an enzyme inducer." In this report, we describe results on (1) the CSP HPLC resolution of enantiomeric 1OH-3MC, l-OH-3-OHMC, and 3MC trans- and cisReceived for publication February 16,1990;accepted March 9,1990. Address reprint requests to S.K. Yang at the address given above. Abbreviations: 3MC, 3-methylcholanthrene;l-OH-BMC, l-hydroxy3-methylcholanthrene;2-OH-3MC,2-hydroxy-3-methylcholanthrene; 3 - O H M C ,3-hydroxymethylcholanthrene; l-OH-3-OHMC,1hydroxy-3-hydroxymethylcholanthrene; 3 M C trans-l,2-diol,trans1,2-dihydroxy-3-methylcholanthrene;3 M C cis-l,2-diol,cis1,2-dihydroxy-3-methylcholanthrene;3MC-l-one,3-methylcholanthrene-l-one;PB, phenobarbital;CSP, chiral stationary phase; (R)-DNBPG,(R)-N-(3,5dinitrobenzoyl)phenylglycine;G6-P, glucose 6-phosphate;solvent A, ethanoUacetonitrile ( 2 4v/v).
142
SHOU A N D YANG
11
12
8
7
'f 6
benz[a]anthracene
X 3-methylcholanthrene (X = H)
Fig. 1. Structure and numbering system of benz[alanthracene,3-methylcholanthrene(3MC,X and l-hydroxy-3-methylcholanthrene(l-OH-BMC,X = OH).
=
H),
1,2-diols, (2) the elucidation of the absolute configura- of acetone and 200 ml of ethyl acetate. The organic tion of the resolved enantiomers, and (3)the combined solvent extract was dried with anhydrous MgSO,, filuse of reversed-phase, normal-phase, and CSP HPLC tered, and evaporated to dryness under reduced presmethods in the study of enantioselective hydroxyla- sure. The residue was redissolved in THF/methanol (l! tions at the C2-carbon and the C3-methyl group of ra- 1, v/v) for reversed-phase HPLC separation of metabocemic 1-OH-3MC. lites. MATERIALS AND METHODS High-PerformanceLiquid Chromatography Materials HPLC was performed using a Waters Associates 3MC, NADP+, glucose 6-phosphate (G-6-P), and G- (Milford, MA) liquid chromatograph consisting of a 6-P dehydrogenase were purchased from Sigma Chem- Model 6000A solvent delivery system, a Model M45 ical Co. (St. Louis, MO). 1-OH-3MC and 3MC-l-one solvent delivery system, a Model 660 solvent programwere synthesized according to procedures described by mer, and a Model 440 absorbance (254 or 280 nm) deSims" and Cavalieri et a1.12 3MC cis-1,2-diol was pre- tector. Samples were injected via a Valco Model N60 pared by oxidation of 3-methylcholanthrylene with loop injector (Valco Instruments, Houston, TX). RetenOsO, in pyridine. 3MC trans-1,2-diol was prepared by tion times and area under chromatographic peaks were refluxing a benzene solution of 3MC cis-1,2-diol con- determined with a Hewlett-Packard Model 3390A intaining a molar excess of 2,3-dichloro-5,6-dicy- tegrator. ano-1,4-benzoquinone(Aldrich Chemical Co., Milwaukee, WI); the resulting product (3MC 1,2-quinone)was Reversed-phase HPLC Products formed in the incubation of racemic or (1R)reduced to 3MC trans-1,2-diol with NaBH,. Enantiomers of 1-OH-3MC were obtained by CSP HPLC res- OH-3MC by rat liver microsomes were separated using olution of racemic 1-OH-3MC on a covalently bonded a Waters Associates RCM-100 Radial Compression Module fitted with a Nova-Pak C18 4 pm cartridge (8 (R)-DNBPG column." mm i.d. x 10 cm). The column was eluted with a 50 min Rat Liver Microsomes linear gradient of methanoVwater (213, v/v) to methaMale Sprague- Dawley rats (Charles River Breeding nol a t a flow rate of 2 ml/min. Bis-p-N,N-dimethylLaboratories, Wilmington, MA) weighing 100- 120 g aminobenzoate (retention time 34.8 rnin), a product were treated intraperitoneally with PB (75 mg/kg body formed by reaction of an enantiomeric 3MC trunsweight, injected in 0.5 ml of water) once daily on each 1,2-diol with p-N,N-dimethylaminobenzoylchloride, of three consecutive days. The rats were sacrificed the was isolated on a DuPont Zorbax ODS column (6.2 mm day following the last injection of the drug. Liver mi- i.d. x 25 cm) by elution with a 30 rnin linear gradient crosomes were prepared13 and microsomal protein was of methanovwater (l/l,v/v) to methanol at a flow rate determined', with bovine serum albumin as protein of 2 ml/min. Under these chromatographic conditions, 3MC trans-1,2-diol and two monoesters 11- and 2standard. (p-N,N-dimethylaminobenzoate)of 3MC truns-1,2Incubation of I-OH-3MC With Rat Liver Microsomes diol] had retention times of 18.6, 29.9, and 32.3 A 100 ml reaction mixture contained 100 mg protein min, respectively. equivalent of rat liver microsomes, 5 mmol Tris-HC1 (pH 7.5), 0.3 mmol of MgC12, 10 units of G-6-P dehy- Normal-phase HPLC Metabolite peaks separated by reversed-phase HPLC drogenase (type XII, Sigma Chemical Co.), 10 mg of NADP+ ,and 48 mg of G-6-P. The reaction mixture was as described above were further purified by normalpre-incubated at 37°C for 5 min in a water shaker bath. phase HPLC using a Zorbax silica column (6.2 mm i.d. 1-OH-3MC [or (lR)-OH-3MC, 8 pmol in 4 ml of x 25 cm), which was packed by Phenomenex (Ranch acetone1 was then added and the mixture was incu- Palos Verdes, CA) using porous silica microparticles (7 bated for 30 min. Residual 1-OH-3MC and its metabo- pm). The column was eluted with various percentages lites were extracted by sequential additions of 100 ml of solvent A (ethanol/acetonitrile, 211, v/v>in hexane at
ENANTIOSELECTIVE METABOLISM OF 1-HYDROXY-3-METHYLCHOLANTHRENE
143
data system by electron impact with a solid probe a t 70 eV and 250°C ionizer temperature. Ultraviolet-visible absorption spectra of samples were determined using a 1cm path length quartz cuvette on a DW2000 U V M S scanning spectrophotometer (slit 2 nm and scan rate 2 Chiral stationary phase HPLC ndsec; SLM Instruments, Inc., Urbana, IL). CD specEnantiomeric pairs of 3MC trans- and cis-l,2-diols tra of samples in a quartz cell of 1 cm path length a t and 1-OH-3-OHMC were resolved on a CSP column room temperature (-23°C) were measured using a (4.6 mm i.d. x 250 mm Rexchrom reversible Pirkle Jasco Model 500A spectropolarimeter equipped with a covalent phenylglycine column; Regis Chemical Co., Model DP500 data processor. The concentration of the Morton Grove, IL) packed with spherical particles of 5 sample is indicated by AAz/ml(absorbance units a t bm diameter of y-aminopropylsilanized silica to which wavelength A2 per ml of solvent). CD spectra are ex(R)-N-(3,5-dinitrobenzoyl)phenylglycine[(RbDNBPG] pressed by ellipticity (@Ai/AAZ,in millidegrees) for sowas covalently bonded. The mobile phase (7 or 10% of lutions that have an absorbance of 1.0 A,, unit per ml solvent A in hexane) was eluted a t a flow rate of 2 of solvent a t wavelength A2 (usually the wavelength of ml/min. maximal absorption)." Absolute Configuration of Enantiomeric 3MC trans-lf-Diols RESULTS AND DISCUSSION CSP HPLC Separation and CD Spectra of Enantiomeric The enantiomer of 3MC trans-l,2-diol (-35 Azss 3MC l%-Diols units, enantiomeric excess 96%) more strongly retained on the covalently bonded (R)-DNBPG column Enantiomeric pairs of synthetic 3MC trans- and ciswas dissolved in tetrahydrofuran and reacted with pNJV-dimethylaminobenoyl chloride in the presence of 1,2-diols were separated by HPLC on a covalently a catalyst (-2mg) p-NJV-dimethy1amin0pyridine.l~bonded (R)-DNBPG column (Fig. 2). The mobile The resulting dibenzoate was isolated by reversed- phases, a t a flow rate of 2 ml/min, were 10%and 7% of solvent A in hexane, respectively. Resolved enantiophase HPLC as described above. mers are indicated in Figure 2 as tl and t2 for the Spectral Analysis trans isomers and c l and c2 for the cis isomers, respecMass spectral analysis was performed on a Finnigan tively. UV-VIS absorption and CD spectra of resolved Model 4000 gas chromatograph-mass spectrometer enantiomers are shown in Figure 3. Absolute configu2 ml/min. These chromatographic conditions allowed the removal of impurities and/or other metabolites closely eluted in the individual fractions collected in the reversed-phase mode.
A
B
tl
cl
h
E
c2
C
0
00
c!
I
1s,2s
1 R.2R
W
0
z a
m a 0 cn m
a
-
7% Solvent A in hexane
1
20
1
1
24
1
1
28
1
I
32
I
20
24
28
32
RETENTION TIME (min) Fig. 2. CSP HPLC separation of enantiomeric 3MC tmns-l,2-diols (A) and 3MC cis-1,2-diols (B). A covalently bonded (R)-DNBF'Gcolumn was used and the mobile phase was 10%and 7% (v/v) of solvent A in hexane, respectively,at a flowrate of 2 mumin. See text on the assignment of absolute configuration of resolved enantiomers.
144
SHOU AND YANG
A ..'i.
.., . . .... .,, . .::: .... .. .: :.. ..
uv
H,C
1 R,2R
(12)
.
1 s,2s (11) I
l
l
I
250
I
I
I
1
1
1
I
1
I
I
I
350
WAVELENGTH (nm) Fig. 3. UV-VISabsorption and CD spectra of enantiomeric 3MC trans-l,2-diols (A) and 3MC cisl,2-diols (B). Enantiomers were resolved as described in Figure 2. See text on the assignment of absolute configurations.
rations of resolved enantiomers are indicated in Figure 3 and have been established in this study (see below).
OH-3MC were separated by reversed-phase HPLC (Fig. 5). 3MC cis-l,2-diol and 1-OH-3-OHMC were among the most abundant metabolites formed. The Absolute Configuration of an Enantiomeric AUC of 3MC trans-l,2-diol was about one-third of that 3MC trans-lf-Diol of 3MC cis-1,2-diol (Fig. 5). The metabolically formed The W-VIS absorption and CD spectra of the bis- 3MC tram- and cis-1,2-diols were identical to the corp-Nfl-dimethylaminobenzoatederived by reaction of responding synthetic compounds with respect to UVthe more strongly retained enantiomer of 3MC trans- VIS absorption (Fig. 3) and mass spectra [M+ at mlz 1,2-diol on the covalently bonded (R)-DNBPG column 300 and a characteristic fragment ion at mlz 282 (loss (peak t2, Fig. 2A) are shown in Figure 4. The UV-VIS of HzO)l, and retention times on reversed-phase HPLC absorption spectrum indicated characteristic absorp- (Fig. 5). The identification of 1-OH-3-OHMCwas based tion bands due to both benz[alanthracene (Ama 294 on its UV-VIS absorption spectrum (Fig. 61, which innm) and two p-N,N-dimethylaminobenzoate(Arna 319 dicated an intact benz[alanthracene nucleus, and by nm) chromophores. A strongly negative CD band at mass spectral analysis [M+ at mlz 300 and character321 nm in the CD chirality spectrum of the bis- istic fragment ions at mlz 282 (loss of H,O), 265 (loss of p-Nfl-dimethylaminobenzoatewas due to electronic HzO and OH), and 253 (loss of HzO and CHO)]. The transition dipole-dipole interactions between the two l-OH-3-OHMC, one of the major metabolites of 1benzoate groups. This characteristic negative CD band OH-3MC identified in this study, was not found to be a at 321 nm indicated that the enantiomeric 3MC trans- metabolite of either 1-OH-3MC or 3MC in earlier 1,2-diol enantiomer under study has a 1R,2R absolute studies. 13-6,9-12,16 Peaks a and b in Figure 5, with an AUC ratio of ~tereochemistry.'~ -63137, were diastereomeric trans-9,lO-dihydrodiols Reversed-Phase HPLC Separation of Metabolites derived from racemic 1-OH-3MC.These two diastereoof 1-0H-3MC meric 9,lO-dihydrodiols were identical to 1-OH-3MC Metabolites formed in the incubation of racemic 1- 9,lO-dihydrodiol-a and 1-OH-3MC 9,lO-dihydrodiol-b
ENANTIOSELECTIVE METABOLISM OF 1-HYDROXY-3-METHYLCHOLANTHRENE
145
meric ratio [(peak cl):(peak c2)I of 12/88. In principle, 1-OH-3-OHMC may be further hydroxylated at C2 to form both trans- and cis-1,2-diols. These metabolites, if formed, should be minor chromatographic peaks in Figure 5 and are yet to be found. EnantioselectiveHydrarylation at C,-Methgl Group of Racemic I-0H-3MC The metabolically formed l-OH-3-OHMC,isolated as described in Figure 5, was further purified by normalphase HPLC using 15% of solvent A in hexane as the eluent. Enantiomers of 1-OH-3-OHMC were separated by CSP HPLC on a covalently bonded (R)-DNBPG column (Fig. 7). The enantiomeric ratio was found to be 58/42 in favor of the less strongly retained enantiomer (Fig. 7). An optically pure and less strongly retained enantiomer was obtained by repetitive chromatography and it had a CD spectrum (Fig. 6) with Cotton effects closely similar to those of an optically pure (1R)OH-3MC. The absolute stereochemistry of enantiomeric 1-OH-3MChas been established by two different 321 methods; one method is described in this study (see below) and the other method is described in an earlier report." The results indicated that hydroxylation a t WAVELENGTH (nm) the C,-methyl group of racemic l-OH-3MC, catalyzed Fig. 4. UV absorption (- - -) and CD (-, 1.0AzS4/ml,methanol) by cytochromes P-450 in liver microsomes from PBspectra of a bis- p-NJV-dimethylaminobenzoatederivedfrom an enan- treated rats, was enantioselective toward the 1Rtiomeric 3MC truns-l,2-diol (enantiomeric excess 96%).W-VIS ab- enantiomer. The l-OH-3-OHMC,formed during the rat sorption spectra of two monoesters of 3MC tmns-l,2diol had a lower liver microsomal metabolism of optically pure (1R)value of absorbance at 315 nm relative to that at 294 nm. OH-3MC, had a CD spectrum similar to (Fig. 6)and a retention time identical to (Fig. 7) the less strongly previously described by Thakker et al.5.16Peak c was retained enantiomer of 1-OH-3-OHMC on CSP HPLC. identified as 3MC-l-one, which was identical to the Absolute Configuration of Enantiomeric 1-OH-3MC and authentic compound with respect to UV-VIS absorp3MC cis-If-Diol tion and mass spectra (Mf at mlz 282) as well as retention time on reversed-phase HPLC. In an earlier 3MC trans- and cis-1,2-diols were two of the major report," 3MC-1-one was found by thin-layer chroma- rat liver microsomal metabolites of 1-OH-3MC (Fig. 5). tography to be a metabolite of 1-OH-3MC.In two other We took advantage of these r a t liver microsome~ t u d i e s ,however, ~,~ 3MC-1-one was not recognized as a catalyzed hydroxylation reactions to elucidate the abproduct formed in rat liver microsomal metabolism of solute configurations of enantiomeric 1-OH-3MC and 1-OH-3MC.Because of the large number (>14) of other 3MC cis-1,2-diol. A 1-OH-3MC enantiomer, which metabolites formed, the stereochemistry of peaks a and was less strongly retained on the covalently bonded b as well as the identities of unmarked chromato- (R)-DNBPGcolumn, was used as the substrate in the in graphic peaks in Figure 5 will be fully described in a vitro incubation. The 3MC trans and cis-l,2-diols formed in the metabolism of the 1-OH-3MC enantiolater report. The relative amount of three aliphatic hydroxylation mer were isolated as described in Figure 5. CSP HPLC products was 1-OH-3-OHMC > 3MC cis-l,2-diol > analysis indicated that they coeluted with peaks tl and 3MC trans-1,2-diol with an AUC ratio of 70/48/15. c l of Figure 2, respectively. Since peak t2 in Figure 2A These results are in contrast to earlier report^^,^ indi- has been established to be the lR,2R-enantiomer, the cating that very little of the 3MC cis-1,2-diol was 1-OH-3MC from which the lS,aS-diol was derived was formed in the metabolism of 1-OH-3MC by rat liver easily established to be the 1R-enantiomer (see strucmicrosomes. 3MC trans-l,a-diol and cis-1,2-diol were tures in Fig. 8). Consequently the cis-1,2-diol, which formed with an AUC ratio of 24/76 in the metabolism of was derived from the (lR)-OH-SMC,was deduced to be racemic 1-OH-3MC (Fig. 5). Upon further purification the lS,2R-enantiomer (see structures in Fig. 8). It by normal-phase HPLC by using 15%of solvent A in should be pointed out that, due to the addition of a hexane as the eluent, the metabolically formed 3MC chiral center at C2, the hydroxyl group with 1R desigtrans-1,2-diol was subsequently analyzed by CSP nation in 1-OH-3MC is changed to 1s (and vice versa) HPLC according to the conditions described in Figure 2 in the enantiomeric 3MC trans- and cis-1,2-diols (Fig. and was found to have an enantiomeric ratio [(peak 8). These results were consistent with the earlier findtl):(peak t2)l of 63/37. Similarly, the metabolically ing that the more strongly retained enantiomer of 1formed 3MC cis-l,2-diol was found to have an enantio- OH-3MC on the covalently bonded (R)-DNBPGcolumn
c
146
SHOU AND YANG
UOH,C
L 'I
n
E c U v)
cu
a
W
0
no-
\
U
z a
rn a: 0 v) rn
C
a
I
I
10
I
I
I
ih,
I
I
20 30 RETENTION TIME (min)
I
I
40
Fig. 5. Reversed-phaseHPLC separation of products formed in the metabolism of racemic 1-OH-3MC by liver microsomes from PB-treated rats. Peaks a and b correspond to the diastereomeric 1-OH-3MC 9,lO-dihydrodiol-a and 1-OH- 3MC 9,lO-dihydrodiol-b,respectively, reported by Thakker et aL5316Peak c contains the 3MC-1-one.
was the 1s enantiomer." In the earlier study," the absolute configuration of 1s-enantiomer was determined by analysis of the CD spectrum of its p-nitrobenzoate derivative (the exciton chirality CD method). As can be seen in Figure 5, the AUC ratio of (trans1,2-diol)/(cis-1,2-diol)formed in the metabolism of racemic 1-OH-3MC was -1/3. However, when (1R)OH-3MC was used as the substrate in the rat liver microsomal incubation, the AUC ratio of (trans1,2-diol)/(cis-1,2-diol) was 71/29 (chromatogram not shown). This result suggested that, in the metabolism of racemic l-OH-3MC, the trans-1,2-diol was mainly derived from the (lR)-OH-SMC, whereas the cis1,2-diol was mainly derived from the (W-OH-3MC. Further analysis of the data obtained by using racemic 1-OH-3MC as the substrate in the in vitro incubation with rat liver microsomes (see below) confirmed the enantioselective/stereoselective hydroxylation reactions.
analysis (Fig. 2), respectively, and the results are summarized in Figure 9. The percentage of 1s-enantioselective C2-hydroxylation of racemic 1OH-3MC can be calculated by (the percentage of lR,2R-diol in the metabolically formed trans-l,2-diol) x (the percentage of trans-l,2-diol in the sum of transand cis-l,2-diols) + (the percentage of lR,aS-diol in the metabolically formed cis-l,2-diol) x (the percentage of cis-l,2-diol in the sum of trans- and cis-l,2-diols). The percentage of (lRI-OH-3MC enantioselective Czhydroxylation can be similarly calculated. The results of these calculations are summarized in Figure 9, which indicated that the C,-hydroxylation of racemic 1-OH-3MC was enantioselective toward t h e 1Senantiomer over the 1R-enantiomer in a ratio of -311. It can be seen in Figure 9 that, in the C,-hydroxylation of (lR)-OH-3MC, the trans-hydroxylation (i.e., the formation of trans-l,2-diol) was more prevalent than the cis-hydroxylation (i.e., the formation of cis1,2-diol) by a ratio of -15/9. In contrast, in the C2Enantioselective C,-Hydmylution of hydroxylation of (lS)-OH-SMC the cis-hydroxylation Racemic 1-0HJMC was more prevalent than the trans-hydroxylation by a The relative amounts and enantiomeric ratios of ratio of -6719. Thus the metabolism of racemic 1trans-l,2-diol and cis-l,2-diol, formed in the metabo- OH-3MC was not only substrate enantioselective, but lism of racemic 1-OH-3MC by liver microsomes from also stereoselective in the C,-hydroxylation of an enanPB-treated rats, were determined by AUC in the re- tiomeric I-OH-3MC. The (lR)-OH-SMC underwent versed-phase HPLC analysis (Fig. 5) and CSP HPLC (trans)-C,-hydroxylation more favorably than the (cis)-
ENANTIOSELECTIVE METABOLISM OF 1-HYDROXY-3-METHYLCHOLANTHFLEm
147
the remaining 1-OH-3MC (recovered by reversedphase HPLC) following incubation of racemic 1:: uv OH-3MC with rat liver microsomes was also found to be enriched in the 1R-enantiomer. Thus the overall metabolism of racemic 1-OH-3MC was enantioselective toward the IS-enantiomer. The same W e n antioselective metabolism was found regardless of . . 1R . . ... whether liver microsomes from untreated, PB-treated, .. .. ... . or 3MC-treated rats were used." Pretreatment of rats . .. with PB and 3MC induces different forms of cytochrome P-450.2 In this study, two aliphatic hydrox_... : ylation reactions by rat liver microsomes had been an. . .. .... alyzed. Hydroxylation a t the C3-methyl group of racemic 1-OH-3MC was found to be enantioselective toward the lR-enantiomer. In contrast, hydroxylation a t C2-carbon of racemic 1-OH-3MC was enantioselective toward the 1S-enantiomer. Preliminary results indicated that peak a in Figure 5 was derived predominantly from the 1S-enantiomer, whereas peak b was derived mainly from the 1R-enantiomer. Since peaks a and b in Figure 5 as well as l-OH-3-OHMC,3MC trans1,2-diol,and 3MC cis-1,2-diol accounted for the majority of the metabolites formed, the results of this study I I I I I I I I I I I , I I I I , I are consistent with the earlier findings" on the sub250 300 350 strate enantioselectivity in rat liver microsomal meWAVELENGTH (nrn) Fig. 6. UV-VIS absorption and CD spectra of (lR)-OH-3-OHMC, tabolism of racemic 1-OH-3MC. which was less strongly retained on the covalently bonded (R)CONCLUSION DNBFG column (see Fig. 7), and the CD spectrum of (lR)-OH-&MC :_
whose absolute configuration has been established by two independent methods in this and an earlier study."
10% Solvent A
30 40 RETENTION TIME (min) Fig. 7. CSP HPLC separation of enantiomeric 1-OH-3-OHMC. A covalently bonded (R)-DNBPG column was used and the mobile phase was 10%(v/v) of solvent A in hexane at a flow rate of 2 mumin. See text on the assignment of absolute configuration of resolved enantiomers.
C,-hydroxylation, whereas the (lSI-OH-3MC underwent (cis)-C,-hydroxylation more favorably than the (trans)-C,-hydroxylation. In a recent report," the enantiomeric composition of
Enantiomeric pairs of l-OH-SMC, 1-OH-3-OHMC, 3MC truns-1,2-diol, and 3MC cis-l,2-diol were resolved by CSP HPLC on a covalently bonded (R)-DNBPG (Pirkle type 1A) column and their absolute configurations were established. Hydroxylation at the C3-methyl group of racemic 1-OH-3MC was enantioselective toward the 1R-enantiomer over the 1S-enantiomer in a ratio of -58142. Hydroxylation a t C2-carbonof racemic 1-OH-3MCresulted in the formation of 3MC truns- and cis-l,2-diols in a ratio of -113. The C,-hydroxylation was enantioselective toward the 1S-enantiomer over the 1R-enantiomer in a ratio of -311. In the C2hydroxylation of (lS)-OH-SMC, cis-l,2-diol and truns1,2-diol were formed in a ratio of -6719. In comparison, cis-1,2-diol and truns-1,2-diol were formed with a ratio of -9115 in the C2-hydroxylation of (lR)-OH-SMC. 3MC truns-l,2-diol formed in the metabolism of racemic 1-OH-3MC by liver microsomes from phenobarbital-treated rats was found to have a (lS,2S)I(lR,2R) enantiomer ratio of -63137 whereas the 3MC cis1,2-diol formed had a (lS,2R)I(lR,2S) enantiomer ratio of -121aa. ACKNOWLEDGMENTS
We thank Mr. Henry Weems for mass spectral analysis. This work was supported by U S . Public Health Service Grant CA29133. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences. The experiments reported herein were conducted according to the principles set forth in the "Guide for the Care and Use
148
SHOU AND YANG
cis-l,2-diol (76%)
trans-l,2-diol (24%)
+
H3C
HO\\Z
1 S,2S (63%) (tl)
@
H3C
P-450
+ H3C
OH
1R,2R (37%) (12)
‘‘.0 H
@
H3C
HO
1s
-
1S,2R (12%) (cl)
OH
1R,2S (88%) (c2)
Fig. 8. Absolute configurations of trans- and cis-1,2-diols derived from C,-hydroxylation of (1R)OH-3MC and (lS)-OH-SMC.Note the change of stereochemical designation (1s+ 1R and 1s + lR, respectively) of the hydroxyl group at C, upon C,-hydroxylation. The corresponding chromatographic peak in Figure 2 and CD spectrum (Fig. 3) are indicated under each structure. The enantiomeric compositions (determined as described in Figure 2) of tmns-1,2-diol (24% of all the l,2diols formed) and cis-1,2-diol (76% of all the 1,Zdiols formed), formed in rat liver microsomal metabolism of racemic 1-OH-3MCand isolated as described in Figure 5, are indicated in the parentheses.
1R-selective (24.2%)
1S-selective (75.8%)
H3C
P-450
1 R (50%)
1S,2S (15.1%)
1R,2S (66.9%)
1 S,2R (9.1%)
1R,2R (8.9%)
____)
H3C
1S (50%) Fig. 9. Summary of the analysis in the degree of substrate enantioselectivity and product stereoselectivity in rat liver microsomal metabolism of racemic 1-OH-3MC.
of Laboratory Animals,” Institute of Animal Resources, National Research Council, DHEW Publication No. (NIH) 78-23. LITERATURE CITED 1. Sims, P., Grover, P.L. Involvement of dihydrodiols and diol-
epoxides in the metabolic activation of polycyclic hydrocarbons other than benzo[alpyrene. In: Polycyclic Hydrocarbons and Cancer, Vol. 3. Gelboin, H.V., Ts’o, P.O.P., eds. New York: Academic Press, 1981: 117-181.
2. Lu, A.Y.H., West, S.B. Multiplicity of mammalian microsomal cytochromesP-450. Pharmacol. Rev. 31:277-295,1980. 3. Stoming, T.A., Bornstein, W., Bresnick, E. The metabolism of 3-methylcholanthrene by rat liver microsomes- A reinvestigation. Biochem. Biophys. Res. Commun. 79:461-469, 1977. 4. Tierney, B., Bresnick, E., Sims, P., Grover, P.L. Microsomal and nuclear metabolism of 3-methylcholanthrene. Biochem. Pharmacol. 28:2607-2610, 1979. 5. Thakker, D.R., Levin, W., Stoming, T.A., Conney, A.H., Jerina, D.M. Metabolism of 3-methylcholanthrene by rat liver mi-
ENANTIOSELECW E METABOLISM OF 1-HYDROXY-3-METHYLCHOLANTHRENE
6. 7.
8.
9. 10.
149
crosomes and a highly purified monooxygenase system with and 11. Sims, P. The metabolism of 3-methylcholanthrene and some rewithout epoxide hydrase. In: Carcinogenesis, Vol. 3, Polynuclear lated compounds by rat-liver homogenates. Biochem. J. 98215Aromatic Hydrocarbons. Jones, P.W., Freudenthal, R.I. eds. New 228,1966. York Raven, 1978: 253-264. 12. Cavalieri, E., Roth, R., Althoff, J., Grandjean, C., Patil, K., Eastman, A,, Bresnick, E. Metabolism and DNA binding of 3Marsh, S., McLaughlin, D. Carcinogenicity and metabolic profiles methylcholanthrene. Cancer Res. 394316-4321, 1979. of 3-methylcholanthrene oxygenated derivative at the 1 and 2 Wood, A.W., Chang, R.L., Levin, W., Thomas, P.E., Ryan, D., positions. Chem.-Biol. Interact. 22:69-81, 1978. Stoming, T.A., Thakker, D.R., Jerina, D.M., Conney, A.H. Meta- 13. Alvares, A.P., Schilling, G. Garbut, A., Kuntzman, R. Studies on bolic activation of 3-methylcholantrene and its metabolites to the hydroxylation of 3.4-benzpyrene by hepatic microsomes. Efproducts mutagenic to bacterial and mammalian cells. Cancer fect of albumin on the rate of hydroxylation of 3,4-benzpyrene. Res. 383398-3404,1978. Biochem. Pharmacol. 19:1449-1455, 1977. 14. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. Protein Levin, W., Buening, M.K., Wood, A.W., Chang, R.L., Thakker, D.R., Jerina, D.M., Conney, A.H. Tumorigenic activity of 3measurement with the Folin phenol reagent. J. Biol. Chem. methylcholanthrene metabolites on mouse skin and in newborn 193~265-275,1951. mice. Cancer Res. 39:3549-3553, 1979. 15. Harada, N., Nakanishi, K. Circular Dichroic Spectroscopy. ExciGardiner, E.M., Stoming, T.A. The metabolism of l-hydroxy- and ton Coupling in Organic Stereochemistry. Mill Valley, C A Uni2-hydroxy-3-methylcholanthrene by liver microsomes: Effect of versity Science Books, 1983. enzyme inducing agents. Cancer. Lett. 24:103-110, 1984. 16. Thakker, D.R., Levin, W., Wood,A.W., Conney, A.H., Stoming, Shou, M., Yang, S.K.1- and 2-Hydroxy-3-methylcholanthrene: T.A., J e r i n a , D.M. Metabolic formation of l,g,lO-trihyRegioselective and stereoselective formations in the metabolism droxy-9,10-dihydro-3-methylcholanthrene: A potential proximate of 3-methylcholanthrene and enantioselective disposition in rat carcinogen from 3-methylcholanthrene. J. Am. Chem. SOC. liver microsomes. Carcinogenesis, in press. 100645-647, 1978.
Enantioselective Aliphatic Hydroxylations of Racemic l-Hydroxy-3-methylcholanthreneby Rat Liver Microsomes MAGANG SHOU AND SHEN K. YANG Department of Pharmacology, F. Edward Hkbert School of Medicine, Uniformed Services University of the Health Sciences, Bethesdu, Maryland 208144799
ABSTRACT
Enantiomeric pairs of l-hydroxy-3-hydroxymethylcholanthrene (1-OH-3-OHMC), 3-methylcholanthrene (3MC) trans- and cis-l,2-diols, and 1hydroxy-3-methylcholanthrene(1-OH-3MC) were resolved by HPLC using a covalently bonded (R)-N-(3,5-dinitrobenzoyl)phenylglycine chiral stationary phase (Pirkle type 1A) column. The absolute configuration of an enantiomeric 3MC trans-l,2-diol was established by the exciton chirality CD method following conversion to a bis-p-Nfl-dimethylaminobenzoate. Incubation of an enantiomeric 1OH-3MC with rat liver microsomes resulted in the formation of enantiomeric 3MC trans- and cis-1,2-diols;the absolute configurations of the enantiomeric 1-OH-3MC and 3MC cis-l,2-diol were established on the basis of the absolute configuration of an enantiomeric 3MC trans-l,2-diol. Absolute configurations of enantiomeric 1OH-3-OHMCwere determined by comparing their CD spectra with those of enantiomeric 1-OH-3MC. The relative amount of three aliphatic hydroxylation products formed by rat liver microsomal metabolism of racemic 1-OH-3MC was 1OH-3-OHMC > 3MC cis-l,2-diol > 3MC trans-1,2-diol. Enzymatic hydroxylation at Cz of racemic 1-OH-3MC was enantioselective toward the 1S-enantiomer over the 1R-enantiomer (-3/1); hydroxylation at the C3-methyl group was enantioselective toward the 1R-enantiomer over the 1S-enantiomer (-58/42). Rat liver microsomal C,-hydroxylation of racemic 1-OH-3MCresulted in a 3MC trans-l,2-diol with a (lS,2S)/(lR,2R)ratio of 63/37 and a 3MC cis-l,2-diol with a (lS,2R)/(lR,2S) ratio of 12/88, respectively.
KEY WORDS: 3-methylcholanthrene, l-hydroxy-3-methylcholanthrene,l-hydroxy-3-hydroxymethylcholanthrene,3-methylcholanthrene trans-1,2-diol, 3-methylcholanthrene cis-l,2-diol, highperformance liquid chromatography, chiral stationary phase, resolution of enantiomers, absolute configuration, circular dichroism spectra, enantioselective hydroxylation, rat liver microsomes INTRODUCTION
3-Methylcholanthrene (3MC, Fig. 11, a potent mutagen and carcinogen, is metabolized to a complex mixture of nontoxic and mutagenic/tumorigenic products by mammalian drug-metabolizing enzyme systems (ref. 1and references therein). 3MC is also widely used as a cytochrome P-450 isozyme inducer in experimental animals.' C1 and C, positions of 3MC are major sites of oxidative metabolism, resulting in the initial formation of 1-OH-3MCand 2-OH-3MC.3-6 1-OH-3MC and 2-OH-3MC are further metabolized to mutagenid tumorigenic and detoxification product^.'*'^^ In the metabolism of 3MC by rat liver microsomes, both 3MC trans- and cis-l,2-diols are detected as metabolite^.^-^ In rat liver microsomal metabolism of either 1OH-3MC or 2-OH-3MC, 3MC tmns-1,2-diol is detected, but very little 3MC cis-1,2-diol is 0
1990 Wiley-Liss,Inc.
In the metabolism of 3MC by rat liver microsomes, 1-OH-3MC is enriched in t h e (1s)-enantiomer (53-73%), whereas 2-OH-3MC is enriched in the 2Senantiomer (86-98%); the exact enantiomeric compositions are dependent on the extent of their further metabolism and whether the rats were pretreated with an enzyme inducer." In this report, we describe results on (1) the CSP HPLC resolution of enantiomeric 1OH-3MC, l-OH-3-OHMC, and 3MC trans- and cisReceived for publication February 16,1990;accepted March 9,1990. Address reprint requests to S.K. Yang at the address given above. Abbreviations: 3MC, 3-methylcholanthrene;l-OH-BMC, l-hydroxy3-methylcholanthrene;2-OH-3MC,2-hydroxy-3-methylcholanthrene; 3 - O H M C ,3-hydroxymethylcholanthrene; l-OH-3-OHMC,1hydroxy-3-hydroxymethylcholanthrene; 3 M C trans-l,2-diol,trans1,2-dihydroxy-3-methylcholanthrene;3 M C cis-l,2-diol,cis1,2-dihydroxy-3-methylcholanthrene;3MC-l-one,3-methylcholanthrene-l-one;PB, phenobarbital;CSP, chiral stationary phase; (R)-DNBPG,(R)-N-(3,5dinitrobenzoyl)phenylglycine;G6-P, glucose 6-phosphate;solvent A, ethanoUacetonitrile ( 2 4v/v).
142
SHOU A N D YANG
11
12
8
7
'f 6
benz[a]anthracene
X 3-methylcholanthrene (X = H)
Fig. 1. Structure and numbering system of benz[alanthracene,3-methylcholanthrene(3MC,X and l-hydroxy-3-methylcholanthrene(l-OH-BMC,X = OH).
=
H),
1,2-diols, (2) the elucidation of the absolute configura- of acetone and 200 ml of ethyl acetate. The organic tion of the resolved enantiomers, and (3)the combined solvent extract was dried with anhydrous MgSO,, filuse of reversed-phase, normal-phase, and CSP HPLC tered, and evaporated to dryness under reduced presmethods in the study of enantioselective hydroxyla- sure. The residue was redissolved in THF/methanol (l! tions at the C2-carbon and the C3-methyl group of ra- 1, v/v) for reversed-phase HPLC separation of metabocemic 1-OH-3MC. lites. MATERIALS AND METHODS High-PerformanceLiquid Chromatography Materials HPLC was performed using a Waters Associates 3MC, NADP+, glucose 6-phosphate (G-6-P), and G- (Milford, MA) liquid chromatograph consisting of a 6-P dehydrogenase were purchased from Sigma Chem- Model 6000A solvent delivery system, a Model M45 ical Co. (St. Louis, MO). 1-OH-3MC and 3MC-l-one solvent delivery system, a Model 660 solvent programwere synthesized according to procedures described by mer, and a Model 440 absorbance (254 or 280 nm) deSims" and Cavalieri et a1.12 3MC cis-1,2-diol was pre- tector. Samples were injected via a Valco Model N60 pared by oxidation of 3-methylcholanthrylene with loop injector (Valco Instruments, Houston, TX). RetenOsO, in pyridine. 3MC trans-1,2-diol was prepared by tion times and area under chromatographic peaks were refluxing a benzene solution of 3MC cis-1,2-diol con- determined with a Hewlett-Packard Model 3390A intaining a molar excess of 2,3-dichloro-5,6-dicy- tegrator. ano-1,4-benzoquinone(Aldrich Chemical Co., Milwaukee, WI); the resulting product (3MC 1,2-quinone)was Reversed-phase HPLC Products formed in the incubation of racemic or (1R)reduced to 3MC trans-1,2-diol with NaBH,. Enantiomers of 1-OH-3MC were obtained by CSP HPLC res- OH-3MC by rat liver microsomes were separated using olution of racemic 1-OH-3MC on a covalently bonded a Waters Associates RCM-100 Radial Compression Module fitted with a Nova-Pak C18 4 pm cartridge (8 (R)-DNBPG column." mm i.d. x 10 cm). The column was eluted with a 50 min Rat Liver Microsomes linear gradient of methanoVwater (213, v/v) to methaMale Sprague- Dawley rats (Charles River Breeding nol a t a flow rate of 2 ml/min. Bis-p-N,N-dimethylLaboratories, Wilmington, MA) weighing 100- 120 g aminobenzoate (retention time 34.8 rnin), a product were treated intraperitoneally with PB (75 mg/kg body formed by reaction of an enantiomeric 3MC trunsweight, injected in 0.5 ml of water) once daily on each 1,2-diol with p-N,N-dimethylaminobenzoylchloride, of three consecutive days. The rats were sacrificed the was isolated on a DuPont Zorbax ODS column (6.2 mm day following the last injection of the drug. Liver mi- i.d. x 25 cm) by elution with a 30 rnin linear gradient crosomes were prepared13 and microsomal protein was of methanovwater (l/l,v/v) to methanol at a flow rate determined', with bovine serum albumin as protein of 2 ml/min. Under these chromatographic conditions, 3MC trans-1,2-diol and two monoesters 11- and 2standard. (p-N,N-dimethylaminobenzoate)of 3MC truns-1,2Incubation of I-OH-3MC With Rat Liver Microsomes diol] had retention times of 18.6, 29.9, and 32.3 A 100 ml reaction mixture contained 100 mg protein min, respectively. equivalent of rat liver microsomes, 5 mmol Tris-HC1 (pH 7.5), 0.3 mmol of MgC12, 10 units of G-6-P dehy- Normal-phase HPLC Metabolite peaks separated by reversed-phase HPLC drogenase (type XII, Sigma Chemical Co.), 10 mg of NADP+ ,and 48 mg of G-6-P. The reaction mixture was as described above were further purified by normalpre-incubated at 37°C for 5 min in a water shaker bath. phase HPLC using a Zorbax silica column (6.2 mm i.d. 1-OH-3MC [or (lR)-OH-3MC, 8 pmol in 4 ml of x 25 cm), which was packed by Phenomenex (Ranch acetone1 was then added and the mixture was incu- Palos Verdes, CA) using porous silica microparticles (7 bated for 30 min. Residual 1-OH-3MC and its metabo- pm). The column was eluted with various percentages lites were extracted by sequential additions of 100 ml of solvent A (ethanol/acetonitrile, 211, v/v>in hexane at
ENANTIOSELECTIVE METABOLISM OF 1-HYDROXY-3-METHYLCHOLANTHRENE
143
data system by electron impact with a solid probe a t 70 eV and 250°C ionizer temperature. Ultraviolet-visible absorption spectra of samples were determined using a 1cm path length quartz cuvette on a DW2000 U V M S scanning spectrophotometer (slit 2 nm and scan rate 2 Chiral stationary phase HPLC ndsec; SLM Instruments, Inc., Urbana, IL). CD specEnantiomeric pairs of 3MC trans- and cis-l,2-diols tra of samples in a quartz cell of 1 cm path length a t and 1-OH-3-OHMC were resolved on a CSP column room temperature (-23°C) were measured using a (4.6 mm i.d. x 250 mm Rexchrom reversible Pirkle Jasco Model 500A spectropolarimeter equipped with a covalent phenylglycine column; Regis Chemical Co., Model DP500 data processor. The concentration of the Morton Grove, IL) packed with spherical particles of 5 sample is indicated by AAz/ml(absorbance units a t bm diameter of y-aminopropylsilanized silica to which wavelength A2 per ml of solvent). CD spectra are ex(R)-N-(3,5-dinitrobenzoyl)phenylglycine[(RbDNBPG] pressed by ellipticity (@Ai/AAZ,in millidegrees) for sowas covalently bonded. The mobile phase (7 or 10% of lutions that have an absorbance of 1.0 A,, unit per ml solvent A in hexane) was eluted a t a flow rate of 2 of solvent a t wavelength A2 (usually the wavelength of ml/min. maximal absorption)." Absolute Configuration of Enantiomeric 3MC trans-lf-Diols RESULTS AND DISCUSSION CSP HPLC Separation and CD Spectra of Enantiomeric The enantiomer of 3MC trans-l,2-diol (-35 Azss 3MC l%-Diols units, enantiomeric excess 96%) more strongly retained on the covalently bonded (R)-DNBPG column Enantiomeric pairs of synthetic 3MC trans- and ciswas dissolved in tetrahydrofuran and reacted with pNJV-dimethylaminobenoyl chloride in the presence of 1,2-diols were separated by HPLC on a covalently a catalyst (-2mg) p-NJV-dimethy1amin0pyridine.l~bonded (R)-DNBPG column (Fig. 2). The mobile The resulting dibenzoate was isolated by reversed- phases, a t a flow rate of 2 ml/min, were 10%and 7% of solvent A in hexane, respectively. Resolved enantiophase HPLC as described above. mers are indicated in Figure 2 as tl and t2 for the Spectral Analysis trans isomers and c l and c2 for the cis isomers, respecMass spectral analysis was performed on a Finnigan tively. UV-VIS absorption and CD spectra of resolved Model 4000 gas chromatograph-mass spectrometer enantiomers are shown in Figure 3. Absolute configu2 ml/min. These chromatographic conditions allowed the removal of impurities and/or other metabolites closely eluted in the individual fractions collected in the reversed-phase mode.
A
B
tl
cl
h
E
c2
C
0
00
c!
I
1s,2s
1 R.2R
W
0
z a
m a 0 cn m
a
-
7% Solvent A in hexane
1
20
1
1
24
1
1
28
1
I
32
I
20
24
28
32
RETENTION TIME (min) Fig. 2. CSP HPLC separation of enantiomeric 3MC tmns-l,2-diols (A) and 3MC cis-1,2-diols (B). A covalently bonded (R)-DNBF'Gcolumn was used and the mobile phase was 10%and 7% (v/v) of solvent A in hexane, respectively,at a flowrate of 2 mumin. See text on the assignment of absolute configuration of resolved enantiomers.
144
SHOU AND YANG
A ..'i.
.., . . .... .,, . .::: .... .. .: :.. ..
uv
H,C
1 R,2R
(12)
.
1 s,2s (11) I
l
l
I
250
I
I
I
1
1
1
I
1
I
I
I
350
WAVELENGTH (nm) Fig. 3. UV-VISabsorption and CD spectra of enantiomeric 3MC trans-l,2-diols (A) and 3MC cisl,2-diols (B). Enantiomers were resolved as described in Figure 2. See text on the assignment of absolute configurations.
rations of resolved enantiomers are indicated in Figure 3 and have been established in this study (see below).
OH-3MC were separated by reversed-phase HPLC (Fig. 5). 3MC cis-l,2-diol and 1-OH-3-OHMC were among the most abundant metabolites formed. The Absolute Configuration of an Enantiomeric AUC of 3MC trans-l,2-diol was about one-third of that 3MC trans-lf-Diol of 3MC cis-1,2-diol (Fig. 5). The metabolically formed The W-VIS absorption and CD spectra of the bis- 3MC tram- and cis-1,2-diols were identical to the corp-Nfl-dimethylaminobenzoatederived by reaction of responding synthetic compounds with respect to UVthe more strongly retained enantiomer of 3MC trans- VIS absorption (Fig. 3) and mass spectra [M+ at mlz 1,2-diol on the covalently bonded (R)-DNBPG column 300 and a characteristic fragment ion at mlz 282 (loss (peak t2, Fig. 2A) are shown in Figure 4. The UV-VIS of HzO)l, and retention times on reversed-phase HPLC absorption spectrum indicated characteristic absorp- (Fig. 5). The identification of 1-OH-3-OHMCwas based tion bands due to both benz[alanthracene (Ama 294 on its UV-VIS absorption spectrum (Fig. 61, which innm) and two p-N,N-dimethylaminobenzoate(Arna 319 dicated an intact benz[alanthracene nucleus, and by nm) chromophores. A strongly negative CD band at mass spectral analysis [M+ at mlz 300 and character321 nm in the CD chirality spectrum of the bis- istic fragment ions at mlz 282 (loss of H,O), 265 (loss of p-Nfl-dimethylaminobenzoatewas due to electronic HzO and OH), and 253 (loss of HzO and CHO)]. The transition dipole-dipole interactions between the two l-OH-3-OHMC, one of the major metabolites of 1benzoate groups. This characteristic negative CD band OH-3MC identified in this study, was not found to be a at 321 nm indicated that the enantiomeric 3MC trans- metabolite of either 1-OH-3MC or 3MC in earlier 1,2-diol enantiomer under study has a 1R,2R absolute studies. 13-6,9-12,16 Peaks a and b in Figure 5, with an AUC ratio of ~tereochemistry.'~ -63137, were diastereomeric trans-9,lO-dihydrodiols Reversed-Phase HPLC Separation of Metabolites derived from racemic 1-OH-3MC.These two diastereoof 1-0H-3MC meric 9,lO-dihydrodiols were identical to 1-OH-3MC Metabolites formed in the incubation of racemic 1- 9,lO-dihydrodiol-a and 1-OH-3MC 9,lO-dihydrodiol-b
ENANTIOSELECTIVE METABOLISM OF 1-HYDROXY-3-METHYLCHOLANTHRENE
145
meric ratio [(peak cl):(peak c2)I of 12/88. In principle, 1-OH-3-OHMC may be further hydroxylated at C2 to form both trans- and cis-1,2-diols. These metabolites, if formed, should be minor chromatographic peaks in Figure 5 and are yet to be found. EnantioselectiveHydrarylation at C,-Methgl Group of Racemic I-0H-3MC The metabolically formed l-OH-3-OHMC,isolated as described in Figure 5, was further purified by normalphase HPLC using 15% of solvent A in hexane as the eluent. Enantiomers of 1-OH-3-OHMC were separated by CSP HPLC on a covalently bonded (R)-DNBPG column (Fig. 7). The enantiomeric ratio was found to be 58/42 in favor of the less strongly retained enantiomer (Fig. 7). An optically pure and less strongly retained enantiomer was obtained by repetitive chromatography and it had a CD spectrum (Fig. 6) with Cotton effects closely similar to those of an optically pure (1R)OH-3MC. The absolute stereochemistry of enantiomeric 1-OH-3MChas been established by two different 321 methods; one method is described in this study (see below) and the other method is described in an earlier report." The results indicated that hydroxylation a t WAVELENGTH (nm) the C,-methyl group of racemic l-OH-3MC, catalyzed Fig. 4. UV absorption (- - -) and CD (-, 1.0AzS4/ml,methanol) by cytochromes P-450 in liver microsomes from PBspectra of a bis- p-NJV-dimethylaminobenzoatederivedfrom an enan- treated rats, was enantioselective toward the 1Rtiomeric 3MC truns-l,2-diol (enantiomeric excess 96%).W-VIS ab- enantiomer. The l-OH-3-OHMC,formed during the rat sorption spectra of two monoesters of 3MC tmns-l,2diol had a lower liver microsomal metabolism of optically pure (1R)value of absorbance at 315 nm relative to that at 294 nm. OH-3MC, had a CD spectrum similar to (Fig. 6)and a retention time identical to (Fig. 7) the less strongly previously described by Thakker et al.5.16Peak c was retained enantiomer of 1-OH-3-OHMC on CSP HPLC. identified as 3MC-l-one, which was identical to the Absolute Configuration of Enantiomeric 1-OH-3MC and authentic compound with respect to UV-VIS absorp3MC cis-If-Diol tion and mass spectra (Mf at mlz 282) as well as retention time on reversed-phase HPLC. In an earlier 3MC trans- and cis-1,2-diols were two of the major report," 3MC-1-one was found by thin-layer chroma- rat liver microsomal metabolites of 1-OH-3MC (Fig. 5). tography to be a metabolite of 1-OH-3MC.In two other We took advantage of these r a t liver microsome~ t u d i e s ,however, ~,~ 3MC-1-one was not recognized as a catalyzed hydroxylation reactions to elucidate the abproduct formed in rat liver microsomal metabolism of solute configurations of enantiomeric 1-OH-3MC and 1-OH-3MC.Because of the large number (>14) of other 3MC cis-1,2-diol. A 1-OH-3MC enantiomer, which metabolites formed, the stereochemistry of peaks a and was less strongly retained on the covalently bonded b as well as the identities of unmarked chromato- (R)-DNBPGcolumn, was used as the substrate in the in graphic peaks in Figure 5 will be fully described in a vitro incubation. The 3MC trans and cis-l,2-diols formed in the metabolism of the 1-OH-3MC enantiolater report. The relative amount of three aliphatic hydroxylation mer were isolated as described in Figure 5. CSP HPLC products was 1-OH-3-OHMC > 3MC cis-l,2-diol > analysis indicated that they coeluted with peaks tl and 3MC trans-1,2-diol with an AUC ratio of 70/48/15. c l of Figure 2, respectively. Since peak t2 in Figure 2A These results are in contrast to earlier report^^,^ indi- has been established to be the lR,2R-enantiomer, the cating that very little of the 3MC cis-1,2-diol was 1-OH-3MC from which the lS,aS-diol was derived was formed in the metabolism of 1-OH-3MC by rat liver easily established to be the 1R-enantiomer (see strucmicrosomes. 3MC trans-l,a-diol and cis-1,2-diol were tures in Fig. 8). Consequently the cis-1,2-diol, which formed with an AUC ratio of 24/76 in the metabolism of was derived from the (lR)-OH-SMC,was deduced to be racemic 1-OH-3MC (Fig. 5). Upon further purification the lS,2R-enantiomer (see structures in Fig. 8). It by normal-phase HPLC by using 15%of solvent A in should be pointed out that, due to the addition of a hexane as the eluent, the metabolically formed 3MC chiral center at C2, the hydroxyl group with 1R desigtrans-1,2-diol was subsequently analyzed by CSP nation in 1-OH-3MC is changed to 1s (and vice versa) HPLC according to the conditions described in Figure 2 in the enantiomeric 3MC trans- and cis-1,2-diols (Fig. and was found to have an enantiomeric ratio [(peak 8). These results were consistent with the earlier findtl):(peak t2)l of 63/37. Similarly, the metabolically ing that the more strongly retained enantiomer of 1formed 3MC cis-l,2-diol was found to have an enantio- OH-3MC on the covalently bonded (R)-DNBPGcolumn
c
146
SHOU AND YANG
UOH,C
L 'I
n
E c U v)
cu
a
W
0
no-
\
U
z a
rn a: 0 v) rn
C
a
I
I
10
I
I
I
ih,
I
I
20 30 RETENTION TIME (min)
I
I
40
Fig. 5. Reversed-phaseHPLC separation of products formed in the metabolism of racemic 1-OH-3MC by liver microsomes from PB-treated rats. Peaks a and b correspond to the diastereomeric 1-OH-3MC 9,lO-dihydrodiol-a and 1-OH- 3MC 9,lO-dihydrodiol-b,respectively, reported by Thakker et aL5316Peak c contains the 3MC-1-one.
was the 1s enantiomer." In the earlier study," the absolute configuration of 1s-enantiomer was determined by analysis of the CD spectrum of its p-nitrobenzoate derivative (the exciton chirality CD method). As can be seen in Figure 5, the AUC ratio of (trans1,2-diol)/(cis-1,2-diol)formed in the metabolism of racemic 1-OH-3MC was -1/3. However, when (1R)OH-3MC was used as the substrate in the rat liver microsomal incubation, the AUC ratio of (trans1,2-diol)/(cis-1,2-diol) was 71/29 (chromatogram not shown). This result suggested that, in the metabolism of racemic l-OH-3MC, the trans-1,2-diol was mainly derived from the (lR)-OH-SMC, whereas the cis1,2-diol was mainly derived from the (W-OH-3MC. Further analysis of the data obtained by using racemic 1-OH-3MC as the substrate in the in vitro incubation with rat liver microsomes (see below) confirmed the enantioselective/stereoselective hydroxylation reactions.
analysis (Fig. 2), respectively, and the results are summarized in Figure 9. The percentage of 1s-enantioselective C2-hydroxylation of racemic 1OH-3MC can be calculated by (the percentage of lR,2R-diol in the metabolically formed trans-l,2-diol) x (the percentage of trans-l,2-diol in the sum of transand cis-l,2-diols) + (the percentage of lR,aS-diol in the metabolically formed cis-l,2-diol) x (the percentage of cis-l,2-diol in the sum of trans- and cis-l,2-diols). The percentage of (lRI-OH-3MC enantioselective Czhydroxylation can be similarly calculated. The results of these calculations are summarized in Figure 9, which indicated that the C,-hydroxylation of racemic 1-OH-3MC was enantioselective toward t h e 1Senantiomer over the 1R-enantiomer in a ratio of -311. It can be seen in Figure 9 that, in the C,-hydroxylation of (lR)-OH-3MC, the trans-hydroxylation (i.e., the formation of trans-l,2-diol) was more prevalent than the cis-hydroxylation (i.e., the formation of cis1,2-diol) by a ratio of -15/9. In contrast, in the C2Enantioselective C,-Hydmylution of hydroxylation of (lS)-OH-SMC the cis-hydroxylation Racemic 1-0HJMC was more prevalent than the trans-hydroxylation by a The relative amounts and enantiomeric ratios of ratio of -6719. Thus the metabolism of racemic 1trans-l,2-diol and cis-l,2-diol, formed in the metabo- OH-3MC was not only substrate enantioselective, but lism of racemic 1-OH-3MC by liver microsomes from also stereoselective in the C,-hydroxylation of an enanPB-treated rats, were determined by AUC in the re- tiomeric I-OH-3MC. The (lR)-OH-SMC underwent versed-phase HPLC analysis (Fig. 5) and CSP HPLC (trans)-C,-hydroxylation more favorably than the (cis)-
ENANTIOSELECTIVE METABOLISM OF 1-HYDROXY-3-METHYLCHOLANTHFLEm
147
the remaining 1-OH-3MC (recovered by reversedphase HPLC) following incubation of racemic 1:: uv OH-3MC with rat liver microsomes was also found to be enriched in the 1R-enantiomer. Thus the overall metabolism of racemic 1-OH-3MC was enantioselective toward the IS-enantiomer. The same W e n antioselective metabolism was found regardless of . . 1R . . ... whether liver microsomes from untreated, PB-treated, .. .. ... . or 3MC-treated rats were used." Pretreatment of rats . .. with PB and 3MC induces different forms of cytochrome P-450.2 In this study, two aliphatic hydrox_... : ylation reactions by rat liver microsomes had been an. . .. .... alyzed. Hydroxylation a t the C3-methyl group of racemic 1-OH-3MC was found to be enantioselective toward the lR-enantiomer. In contrast, hydroxylation a t C2-carbon of racemic 1-OH-3MC was enantioselective toward the 1S-enantiomer. Preliminary results indicated that peak a in Figure 5 was derived predominantly from the 1S-enantiomer, whereas peak b was derived mainly from the 1R-enantiomer. Since peaks a and b in Figure 5 as well as l-OH-3-OHMC,3MC trans1,2-diol,and 3MC cis-1,2-diol accounted for the majority of the metabolites formed, the results of this study I I I I I I I I I I I , I I I I , I are consistent with the earlier findings" on the sub250 300 350 strate enantioselectivity in rat liver microsomal meWAVELENGTH (nrn) Fig. 6. UV-VIS absorption and CD spectra of (lR)-OH-3-OHMC, tabolism of racemic 1-OH-3MC. which was less strongly retained on the covalently bonded (R)CONCLUSION DNBFG column (see Fig. 7), and the CD spectrum of (lR)-OH-&MC :_
whose absolute configuration has been established by two independent methods in this and an earlier study."
10% Solvent A
30 40 RETENTION TIME (min) Fig. 7. CSP HPLC separation of enantiomeric 1-OH-3-OHMC. A covalently bonded (R)-DNBPG column was used and the mobile phase was 10%(v/v) of solvent A in hexane at a flow rate of 2 mumin. See text on the assignment of absolute configuration of resolved enantiomers.
C,-hydroxylation, whereas the (lSI-OH-3MC underwent (cis)-C,-hydroxylation more favorably than the (trans)-C,-hydroxylation. In a recent report," the enantiomeric composition of
Enantiomeric pairs of l-OH-SMC, 1-OH-3-OHMC, 3MC truns-1,2-diol, and 3MC cis-l,2-diol were resolved by CSP HPLC on a covalently bonded (R)-DNBPG (Pirkle type 1A) column and their absolute configurations were established. Hydroxylation at the C3-methyl group of racemic 1-OH-3MC was enantioselective toward the 1R-enantiomer over the 1S-enantiomer in a ratio of -58142. Hydroxylation a t C2-carbonof racemic 1-OH-3MCresulted in the formation of 3MC truns- and cis-l,2-diols in a ratio of -113. The C,-hydroxylation was enantioselective toward the 1S-enantiomer over the 1R-enantiomer in a ratio of -311. In the C2hydroxylation of (lS)-OH-SMC, cis-l,2-diol and truns1,2-diol were formed in a ratio of -6719. In comparison, cis-1,2-diol and truns-1,2-diol were formed with a ratio of -9115 in the C2-hydroxylation of (lR)-OH-SMC. 3MC truns-l,2-diol formed in the metabolism of racemic 1-OH-3MC by liver microsomes from phenobarbital-treated rats was found to have a (lS,2S)I(lR,2R) enantiomer ratio of -63137 whereas the 3MC cis1,2-diol formed had a (lS,2R)I(lR,2S) enantiomer ratio of -121aa. ACKNOWLEDGMENTS
We thank Mr. Henry Weems for mass spectral analysis. This work was supported by U S . Public Health Service Grant CA29133. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences. The experiments reported herein were conducted according to the principles set forth in the "Guide for the Care and Use
148
SHOU AND YANG
cis-l,2-diol (76%)
trans-l,2-diol (24%)
+
H3C
HO\\Z
1 S,2S (63%) (tl)
@
H3C
P-450
+ H3C
OH
1R,2R (37%) (12)
‘‘.0 H
@
H3C
HO
1s
-
1S,2R (12%) (cl)
OH
1R,2S (88%) (c2)
Fig. 8. Absolute configurations of trans- and cis-1,2-diols derived from C,-hydroxylation of (1R)OH-3MC and (lS)-OH-SMC.Note the change of stereochemical designation (1s+ 1R and 1s + lR, respectively) of the hydroxyl group at C, upon C,-hydroxylation. The corresponding chromatographic peak in Figure 2 and CD spectrum (Fig. 3) are indicated under each structure. The enantiomeric compositions (determined as described in Figure 2) of tmns-1,2-diol (24% of all the l,2diols formed) and cis-1,2-diol (76% of all the 1,Zdiols formed), formed in rat liver microsomal metabolism of racemic 1-OH-3MCand isolated as described in Figure 5, are indicated in the parentheses.
1R-selective (24.2%)
1S-selective (75.8%)
H3C
P-450
1 R (50%)
1S,2S (15.1%)
1R,2S (66.9%)
1 S,2R (9.1%)
1R,2R (8.9%)
____)
H3C
1S (50%) Fig. 9. Summary of the analysis in the degree of substrate enantioselectivity and product stereoselectivity in rat liver microsomal metabolism of racemic 1-OH-3MC.
of Laboratory Animals,” Institute of Animal Resources, National Research Council, DHEW Publication No. (NIH) 78-23. LITERATURE CITED 1. Sims, P., Grover, P.L. Involvement of dihydrodiols and diol-
epoxides in the metabolic activation of polycyclic hydrocarbons other than benzo[alpyrene. In: Polycyclic Hydrocarbons and Cancer, Vol. 3. Gelboin, H.V., Ts’o, P.O.P., eds. New York: Academic Press, 1981: 117-181.
2. Lu, A.Y.H., West, S.B. Multiplicity of mammalian microsomal cytochromesP-450. Pharmacol. Rev. 31:277-295,1980. 3. Stoming, T.A., Bornstein, W., Bresnick, E. The metabolism of 3-methylcholanthrene by rat liver microsomes- A reinvestigation. Biochem. Biophys. Res. Commun. 79:461-469, 1977. 4. Tierney, B., Bresnick, E., Sims, P., Grover, P.L. Microsomal and nuclear metabolism of 3-methylcholanthrene. Biochem. Pharmacol. 28:2607-2610, 1979. 5. Thakker, D.R., Levin, W., Stoming, T.A., Conney, A.H., Jerina, D.M. Metabolism of 3-methylcholanthrene by rat liver mi-
ENANTIOSELECW E METABOLISM OF 1-HYDROXY-3-METHYLCHOLANTHRENE
6. 7.
8.
9. 10.
149
crosomes and a highly purified monooxygenase system with and 11. Sims, P. The metabolism of 3-methylcholanthrene and some rewithout epoxide hydrase. In: Carcinogenesis, Vol. 3, Polynuclear lated compounds by rat-liver homogenates. Biochem. J. 98215Aromatic Hydrocarbons. Jones, P.W., Freudenthal, R.I. eds. New 228,1966. York Raven, 1978: 253-264. 12. Cavalieri, E., Roth, R., Althoff, J., Grandjean, C., Patil, K., Eastman, A,, Bresnick, E. Metabolism and DNA binding of 3Marsh, S., McLaughlin, D. Carcinogenicity and metabolic profiles methylcholanthrene. Cancer Res. 394316-4321, 1979. of 3-methylcholanthrene oxygenated derivative at the 1 and 2 Wood, A.W., Chang, R.L., Levin, W., Thomas, P.E., Ryan, D., positions. Chem.-Biol. Interact. 22:69-81, 1978. Stoming, T.A., Thakker, D.R., Jerina, D.M., Conney, A.H. Meta- 13. Alvares, A.P., Schilling, G. Garbut, A., Kuntzman, R. Studies on bolic activation of 3-methylcholantrene and its metabolites to the hydroxylation of 3.4-benzpyrene by hepatic microsomes. Efproducts mutagenic to bacterial and mammalian cells. Cancer fect of albumin on the rate of hydroxylation of 3,4-benzpyrene. Res. 383398-3404,1978. Biochem. Pharmacol. 19:1449-1455, 1977. 14. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. Protein Levin, W., Buening, M.K., Wood, A.W., Chang, R.L., Thakker, D.R., Jerina, D.M., Conney, A.H. Tumorigenic activity of 3measurement with the Folin phenol reagent. J. Biol. Chem. methylcholanthrene metabolites on mouse skin and in newborn 193~265-275,1951. mice. Cancer Res. 39:3549-3553, 1979. 15. Harada, N., Nakanishi, K. Circular Dichroic Spectroscopy. ExciGardiner, E.M., Stoming, T.A. The metabolism of l-hydroxy- and ton Coupling in Organic Stereochemistry. Mill Valley, C A Uni2-hydroxy-3-methylcholanthrene by liver microsomes: Effect of versity Science Books, 1983. enzyme inducing agents. Cancer. Lett. 24:103-110, 1984. 16. Thakker, D.R., Levin, W., Wood,A.W., Conney, A.H., Stoming, Shou, M., Yang, S.K.1- and 2-Hydroxy-3-methylcholanthrene: T.A., J e r i n a , D.M. Metabolic formation of l,g,lO-trihyRegioselective and stereoselective formations in the metabolism droxy-9,10-dihydro-3-methylcholanthrene: A potential proximate of 3-methylcholanthrene and enantioselective disposition in rat carcinogen from 3-methylcholanthrene. J. Am. Chem. SOC. liver microsomes. Carcinogenesis, in press. 100645-647, 1978.
