Chem. Res. Toxicol. 1992,5, 157-162
157
Articles Microsomal Metabolism of Cyclopenta[ cdlpyrene. Characterization of New Metabolites and Their Mechanism of Formation Yousif Sahali, Hoonjeong Kwon, Paul L. Skipper, and Steven R. Tannenbaum* Department of Chemistry and Division of Toxicology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received May 13, 1991 Oxidation of cyclopenta[cd]pyrene (CPP) by mouse and human liver microsomes was used to produce several previously undescribed metabolites, which were separated and isolated by reversed-phase HPLC. Three of these, 3,4-dihydroCPP-c-3,4-diol, 4-hydroxy-3,4-dihydroCPP, and 4-oxo-3,4-dihydroCPP, were fully characterized by GC-MS and UV spectroscopic analysis as well as by total synthesis. Two additional pairs of metabolites were identified as isomeric tetrahydrotetrols and dihydrotriols by GC-MS analysis of their trimethylsilyl derivatives. Their UV spectra were recorded and found to agree with the structure assignments. The tetrahydrotetrols were further characterized by the fact that either 3,4- or 9,lO-trans-dihydrodiol could serve as their precursor, indicating that they are the two diastomeric 3,4,9,10-tetrahydroCPP-t-3,4-t-9,1O-tetrols.The dihydrotriols were shown to possess t-3,4-dihydrodiol functionality. As found previously using rat liver microsomes, the most abundant metabolite was 3,4-dihydroCPP-t-3,4-diol. It was produced with one enantiomer in severalfold excess over the other, and the major enantiomer was shown to have 3R,4R c o n f i i a t i o n by exciton chirality circular dichroism. Microsomal oxidation of [4-2H]CPP,which was synthesized for this study, was used to determine the mechanisms of formation of 4-OXO- and 4-hydroxy-3,4-dihydroCPP. The ketone was produced without detectable retention of deuterium label, eliminating the NIH shift as a possible mechanism. The alcohol was shown to arise by NADPH-dependent reduction of both the ketone and another intermediate presumed to be the 3,4-epoxide. 4-Hydroxy-3,4dihydroCPP is the first known example of a monohydroxy dihydro metabolite formed from a fully unsaturated cyclopenta fused polynuclear aromatic hydrocarbon.
Introduction Cyclopenta[cd]pyrene (CPP)' is a common environmental pollutant which is produced during combustion of coal, gasoline, and diesel fuel. It is both a mutagen (1) and a carcinogen (2). The ultimate carcinogenic form of CPP is not known since no DNA adduct structures have yet been determined. Epoxidation of the 3,4 double bond which forms the cyclopenta ring appears to be the major microsomal metabolic pathway, as evidenced by the preponderance of 3,4-dihydrodiol among the products of microsomal metabolism ( 3 , 4 ) ,and it has been suggested that the 3,4-epoxide may be the ultimate carcinogen derived from CPP. The mutagenicity of several oxygenated derivatives of CPP (5), though, casts some doubt on the role of CPP 3,4-oxide as a DNA-binding metabolite. For example, cis-3,4-dihydroxy-3,4-dihydroCPP is more mutagenic than the parent hydrocarbon, as is 3-hydroxy-3,4-dihydroCPP. The isomeric trans-3,4-diol and the related Chydroxy and 3- or 4-keto derivatives are also strong mutagens in the Salmonella typhimurium strain TM677 forward mutation assay. Incubation of CPP with rat liver microsomes has been reported to yield two products, the trans-3,4-dihydrodiol *Author to whom correspondence should be addressed.
and the trans-9,lO-dihydrodiol (4). The formation of 3,4-dihydrodiol suggests that the 3,4-oxide is transiently formed, and the formation of a 9,lO-dihydrodiol indicates that oxidation is not limited to the 3,4 double bond in CPP. Since CPP is a mouse carcinogen but ita carcinogenicity for the rat is unknown, we chose to determine the metabolism of CPP by mouse liver microsomes in further detail with the goal of identifying additional metabolites from which the identities of genotoxic alkylating species might be inferred. We &o had access to a limited amount of human liver microsomes and investigated their metabolism of CPP as well. A qualitative comparison of mouse and human microsomal metabolism is reported herein, and a more thorough and quantitative comparison will be reported elsewhere. During the course of this investigation of the metabolism of CPP which used mouse and human hepatic microsomal enzymes instead of rat microsomes we identified two monooxygenated metabolites, 4-hydroxy-3,4-dihydroCPP (1) and 4-oxo-3,4-dihydroCPP (2). The formation of 2 under nonphysiological conditions by rearrangement of CPPE has previously been reported (3,s). The mechanism ~
~
~
~~~~~
Abbreviations: CD, circular dichroism;CPP, cyclopenta[cd]pyrene; CPPE, cyclopenta[cd]pyrene3,4-epoxide;EI, electron ionization;GCMS, gas chromatography-mass spectrometry;PAH, polynuclear aromatic hydrocarbon; PMS, postmitochondrialsupernatant.
oa93-22axp2/ 2705-0i57$03.00/0 0 1992 American Chemical Society
158 Chem. Res. Toxicol., Vol. 5, No.2, 1992
was not determined but it was suggested that 2 was formed via an NIH shift. Formation of I, though, was clearly the result of a novel metabolic path since no other monohydroxydihydro derivative of the five-membered ring of a fully unsaturated CyclopenWAH has been reported. We were thus interested in the mechanism of formation of this structure. Metabolism of specifically deuterated CPP was chosen as a means to determine the mechanisms. Since the most electrophilic center of CPP 3,4-epoxideis C(3) ( I ) , it was necessary to introduce deuterium at C(4). None of the published syntheses of CPP are helpful in this regard (ref 7, and references therein), so we developed an alternate synthesis which is reported in this paper. We also report metabolic studies using the specifically deuterated CPP which reveal the mechanisms of formation of 1 and 2.
Experimental Sectlon Chemicals. [G-,H]CPP was obtained from the NCI chemical repository maintained by Chemsyn Science Laboratories (Lenexa,
KS) and purified by HPLC prior to use. Glucose 6-phosphate, &NADP+,and glucose-&phosphatedehydrogenasewere obtained from Sigma Chemical Co. (St. Louis, MO). trans-3,4-Dihydroxy-3,4-dihydroCPP was prepared by NaBH4reduction of 3,4-dioxo-3,4-dihydroCPP as described previously (7).The cisdihydrodiol was also isolated from this preparation by HPLC. This heretofore undescribed compound was characterized by 'H-NMR [(CD3COCD3)8.35-8.00 (m,8 H), 5.71 (s, 1 H), 5.66 (s, 1 H), 4.75 (s, 2 H); cf. trans-3,4-dihydroxy-3,4-dihydroCPP: 8.35-8.05 (m, 8 H), 5.68 (8, 1H), 5.59 (s, 1 H), 5.19 (8,2 H)] and by mass spectroscopy of the TMS derivative (M+ 404). 3-oxowere prepared 3,4-dihydroCPP and 3-hydroxy-3,4-dihydroCPP as described previously (7). (A) 4 4 Bromoacetyl)-l,2,3,6,7,8-hexahydropyrene. A1Br3 (5.2 g, 19.2 mmol) was added over 30 min to a solution of 1,2,3,6,7,8-hexahydropyrene(2 g, 9.6 mmol) and bromoacetyl bromide (0.84 mL, 9.6 mmol) in methylene chloride (100 mL) at 0 "C. The cooling bath was removed 15 min after completion of the AlBr, addition, and the reaction mixture was stirred for an additional 30 min at room temperature. The reaction mixture was quenched with ice water (100 mL), and the organic layer was separated, washed with 10% NaHC0, and water, dried over MgS04, and concentrated under reduced pressure to give 2.85 g (87% yield) crude product which was used directly for the next step. The melting point of a sample purified by TLC (silica) was 109-111 "C. 'H-NMR (CDCl,) 7.45-7.28 (m, 3 H), 4.54 (s,2 H), 3.3 (t, 2 H), 3.15 (m, 6 H), 2.12 (m, 4 H); MS, m / z 330 (7.7, 81Br - M+), 328 (8.2, 79Br- M+), 249 (loo), 235 (8). (B) 4-(Bromoacety1)pyrene. A suspension of 3 (2.5 g, 7.6 mmol) in a solution of DDQ (5.18 g, 22.8 mmol) in benzene was stirred for 3 h at 80 "C. The reaction mixture was then cooled to room temperature and filtered. The solid was washed with benzene, and the combined filtrate and washings were concentrated under reduced pressure. The crude product was passed over a basic alumina column which was eluted with benzene. This partially purified material was rechromatographed on silica gel with benzene to give 1.8 g of 4 (73%): mp 123-124 "C; 'H-NMR (CDC13) 8.32-7.95 (m, 9 H), 4.77 (s,2 H); MS, m/z 324 (10, slBr - M+), 322 (81Br- M'), 229 (loo), 201 (53). (C) 4-0xo-3,4-dihydrocyclopenta[cd]pyrene($a). A solution of 4 (250 mg, 0.77 mmol) in C C 4 (250 mL) was irradiated with 350-nm light in the presence of lithium carbonate for 5 h at room temperature in a Southern New England Ultraviolet Co. reactor. The reaction mixture was washed with water (2 X 150 mL), dried over MgSO,, filtered, and concentrated to give 170 mg of crude product. The crude product was purified by preparative TLC (silica) using benzene as eluent to give 80 mg of 2 (43% yield): mp 218-220 "C; 'H-NMR (CDC13)8.35-7.95 (m, 8 H), 5.48 (8, 2 H); MS, m/z 242 (100, M+), 214 (79), 213 (65). (D) 4-Hydroxy-3,4-dihydrocyclopenta[ cdlpyrene (IC). A solution of 2 (70 mg, 0.29 "01) in anhydrous ether (0.5 mL) was added slowly to a suspension of LiAlH, (8 mg, 0.17 mmol) in anhydrous ether (1mL) cooled to 0 "C. The cooling bath was removed and the reaction was stirred for 3 h at room temperature.
Sahali et al. The reaction mixture was quenched in the usual manner, THF (2 mL) was added, and the mixture was stirred for 30 min and filtered. The ether-THF solution was dried over MgSO,, filtered, and concentrated. The resulting product was purified by preparative TLC (silica) to give 62 mg of product (87% yield): 'HNMR (CDC1,) 8.25-7.85 (m, 8 H), 5.97 (t, J = 7 Hz, 1 H), 4.11 (dd, J = 18, J = 7 Hz, 1H), 3.55 (dd, J = 18, J = 7 Hz, 1H); MS, m / z 244 (100, Mi), 226 (501, 215 (38).
(E) [4-2H]-4-Hydroxy-3,4-dihydrocyclopenta[cd]pyrene (la). LiAlD, was substituted for LiAlH, in the above synthesis; otherwise all conditions were the same. 'H-NMR (CDC13) 8.25-7.85 (m, 8 H), 4.11 (d, J = 18 Hz, 1H), 3.56 (d, J = 18 Hz, 1 H); MS, m / z 245 (100, M+), 227 (51), 216 (36). (F)[4-2H]Cyclopenta[cd]pyrene. A solution of la (60 mg, 0.24 mmol) and TsOH (50 pg) in dry benzene (3 mL) was heated for 45 min at reflux. The reaction was cooled to room temperature, washed with 5% NaOH and water, dried over MgSO,, and concentrated to give 52 mg of crude product which was purified by column chromatography (silica gel) with hexane as eluent to give 44 mg of product (80% yield): mp 174-176 "C; 'H-NMR (CDC13) 8.41-8.10 (m, 8 H), 7.42 (s, 1H); MS, m/z 227 (100, M+), 113 (19). Oxidation of Alcohol 1. The alcohol isolated by reversedphase HPLC (ca. 0.6 nmol based on absorbance) was dissolved in CH2C12(0.1 mL) and added to a reaction vial containing CH,Cl, (0.1 mL) and a few grains of PCC. The mixture was stirred for 3 h at room temperature. Ether (0.2 mL) was added and the mixture was passed over a small column of Fluormil. The solvent was evaporated and the residue was subjected to GC-MS analysis. The peak corresponding to the ketone produced spectra identical with those described earlier for 2a. Instrumentation and Chromatography. 'H-NMR spectra were obtained with a Varian XL-300 instrument. GC-MS and other low-resolution MS analyses were performed using a Hewlett-Packard 5987A mass spectrometer equipped with a standard E1 source and 15-m X 0 . 2 5 " DB-5 fused silica capillary column (J & W, Folsom, CA). Samples were introduced either by direct insertion probe or by GC. Trimethylsilylderivatives were prepared by treatment with 5 pL of Trisyl-Z (Pierce, Rockford, IL) for 3-5 min at room temperature. A Jasco-500 spectropolarimeter was used for CD measurements. Chromatography of metabolites was performed with a Hewlett Packard H P 1090 equipped with a diode array detector. For preparative work a pBondapak C18(10- X 300-mm) column from Waters (Milford, MA) and the following gradient at the flow rate of 3 mL/min were used an isocratic phase of 10% MeOH in H20 for 3 min, and then a linear gradient to 37% MeOH at 5 min, to 55% MeOH at 10 min, to 68.5% MeOH at 25 min, and then to 100% MeOH at 40 min. Radioactive peaks were collected and rechromatographed on an analytical pBondapak C18column (3.9 x 300 mm) using a flow rate of 1.5 mL/min and the same gradient as above for all but one of the components. To resolve the two earliest eluting peaks, the solvent program was a 3-min isocratic phase of 10% MeOH in H20 followed by a linear gradient to 37% MeOH at 15 min. Enantiomeric resolution was performed using a Pirkle Ionic 1-A 5-pm spherical silica HPLC column (Regis Chemical Co., Morton Grove, IL) with (R)-(-)-N-(3,Bdinitrobenzoyl)-a-phenylglycinestationary phase. The elution solvent was 12.5% (v/v) of ethanol/acetonitrile (2:l v/v) in hexane, and the flow rate was 1.8 mL/min. Microsomal Incubations. Uninduced male CD-1 mice were used to produce mouse liver microsomes. Human liver microsomes from 3 donors (HL105, HL110, and HL116) were provided by Dr. F. P. Guengerich of Vanderbilt University. The substrate specificity of two of these (HL105 and HL110) has previously been described (8). Incubations were carried out at 37 "C for 3 h in phosphate buffer, pH 7.4, containing 10 mM MgC12, 0.7 mM @-NADP+,5 mM glucose 6-phosphate, 0.5 unit/mL glucose-6phosphate dehydrogenase, 0.5 mg/mL microsomal protein, and 12 pM CPP or 5 fiM dihydrodiol or 4-oxo-3,4-dihydroCPP. Reactions were terminated by either adding ethyl acetate, separating the organic layer, and further extraction with ethyl acetate or loading onto a C18 BondElut (Analytchem International, Harbor City, CA) disposablecolumn. After washing out unbound material with 3 mL of H20, bound material was sequentially eluted with 3 mL of 50/50 MeOH/H20, 3 mL of 90/10 MeOH/H20, and 1 mL of MeOH. All the eluates or organic extract were combined,
Chem. Res. Toxicol., Vol. 5, No. 2, 1992 159
Microsomal Metabolism of Cyclopenta[cdlpyrene
1 a
4
N
2
m
'OH
OH
0.
;0 . C
m
n
0.
n a
0.
L 0 H
0
6 c 0.
--
Time
28 (min.)
40
30
Figure 1. Reversed-phaseHPLC profile of the organic solvent ext,ractableproducts formed from incubation of CPP with mouse liver microsomes. Incubation of CPP with human microsomes produced a qualitatively similar pattern. The numbered peaks were the only regions of the chromatogram in which radioactivity eluted when [G-3H]CPPwas used. Peak 7 is unmetabolized CPP. dried with MgS04, concentrated under reduced pressure, and analyzed by HPLC.
Results Identification of Metabolites. The reversed-phase HPLC profles of metabolites from both mouse and human microsomal activation were qualitatively similar. A representative chromatogram is illustrated in Figure 1. Several new metabolites were isolated and characterized in addition to the known t-3,4-dihydrodiol,which was the major metabolite from enzymatic activation of CPP with both human and mouse microsomes. The t-9,lO-dihydrodiol, which has been isolated from rat liver microsomal activation, was not observed in our study. Several oxidized CPP derivatives, not including the dihydrotriols or tetrahydrotetrols, were synthesized and used as standards in characterizing the metabolites. 4Oxo-3,CdihydroCPP, 4-hydroxy-3,4-dihydroCPP, and the cis and trans isomers of 3,4-dihydroxy-3,4-dihydroCPP were prepared as described in the Experimental Section. 3-0xo-3,4-dihydroCPP and 3-hydroxy-3,I-dihydroCPP were prepared as described (7). Peak 6 was identified as 4-oxo-3,4-dihydroCPP. The HPLC retention time of this metabolite was the same as that of authentic material and distinguished it from the 3-OXO isomer. The UV-visible absorption of this compound was quite characteristic and clearly different from that of the $OXO isomer. Capillary GC also readily distinguished the metabolite from 3-oxo-3,4-dihydroCPP on the basis of retention time. Ita ELMS characteristics (M+ at m/z 242, fragment ions at m/z 214, 213) were in complete agreement with those of the synthetic standard. The metabolite represented by peak 5 had a UV-visible absorption spectrum characteristic of the pyrene chromophore, indicating that the ethylenic bridge in CPP had become saturated. In addition, the HPLC elution order between CPP and the 3,4-dihydrodiols suggested a nonphenolic monohydroxylated derivative, either 3- or 4hydroxy-3,4-dihydroCPP. These two compounds were separable with the system used, and the HPLC retention time of peak 5 agreed closely with that of 4-hydroxy-3,4dihydroCPP. The UV absorbance maximum at 344 nm also distinguished the metabolite from the 3-hydroxy isomer, which has a maximum absorbance at 342 nm. GC-ELMS was also useful to distinguish the two isomers. Although the two have nearly identical retention times,
OH "OH
. 300
.
.
Wavelength
.
,
4 00 (nm)
.
.
.
. * , 500
Figure 2. UV-vis spectrum of the metabolite isolated aa peak 1. Peak 2 produced essentially the identical spectrum. they could, in fact, be resolved. Their spectra also displayed different fragmentation patterns, and the spectrum of the methyl derivative of the metabolite clearly resembled that of the 4-isomer better than that of the 3-isomer. The UV-visible absorption spectra of the metabolites contained in peaks 3 and 4 also showed saturation of the ethylenic bridge of CPP and preservation of the pyrene nucleus. The GC-ELMS analysis of the trimethylsilyl derivatives of the metabolites were indicative of a diol structure (M+ 404) in each case. Thus, each metabolite was compared to authentic 3,4-dihydrodiols. The cis stereochemistry was assigned to peak 4 and the trans stereochemistry to peak 3 on the basis of HPLC retention time, UV-visible spectra, and EI-MS spectra. The two more polar metabolites contained in peaks 1 and 2 were initially characterized as tetrahydrotetrols of CPP by the ELMS of their trimethylsilyl derivatives: m/z 582 (100, M+), 567 (27, M+ - CHS), 493 (59, M+ TMSOH). The UV-visible absorption spectrum (Figure 2) of these metabolites was similar to the UV spectrum of the polyaromatic hydrocarbon phenanthrene, an indication that metabolism occurred at C(3), C(4), C(9), and C(l0). Since there were sufficient amounts of neither the tetrol metabolites nor synthetic standards for NMFt analysis, the following experiments were performed in order to fully determine the position as well as the relative stereochemistry of the vicinal hydroxyl groups: 1. Pure 3,4-dihydroCPP-t-3,4-diol was incubated with mouse liver microsomes to give tetrols with known trans stereochemistry
160 Chem. Res. Toxicol., Vol. 5, No. 2, 1992
Sahali et al. Scheme I
4
3
2a
w
w
la
5
at the C(3)-C(4) position. 2. Pure 9,lO-dihydroCPP-t9,lO-diol (isolated from incubation of CPP with rat liver 0.071 microsomes) was incubated with mouse liver microsomes to give tetrahydrotetrols with known trans stereochemistry at the C(9)-C(lO)positions. Each of the two incubations a 0.05 yielded the same two products, which were identical by HPLC and UV-visible spectroscopy with the two tetrahydrotetrols produced by microsomal metabolism of CPP. 0.03 was incubated with 3. Pure 3,4-dihydroCPP-c-3,4-diol 0 = 0.02 mouse liver microsomes to give metabolites which showed different HPLC retention times and slight shift in UV 0.01 spectrum. 9,10-DihydroCPP-9,10-diol was not available in the cis configuration,so we were unable to rule out that 0 . 0 0 1 1 3 00 4 00 5 00 the tetrahydrotetrols produced by microsomal oxidation Wavelength ( n m ) of CPP had 9,lO-cis stereochemistry. This possibility is Figure 3. Typical UV-vis of metabolites X1 and X2. considered very unlikely, though, because in all cases where both cis- and trans-dihydrodiols were available, we have been able to separate them chromatographically. nation of absolute configuration by the exciton chirality circular dichroism method. Taken together, these results indicate that peaks 1and 2 were isomers of 3,4,9,10-tetrahydroCPP-3,4,9,10-tetrol Subjecting synthetic racemic t-3,4-dihydrodiols to with 3,4-trans and 9,lO-trans stereochemistry. It was not chromatography on a column of (R)-(-)-N-(3,5-dinitropossible to relate the stereochemistry of the 3 and 4 posbenzoyl)-a-phenylglycineionically bonded to an aminoitions with that of the 9 and 10 positions. propylated silica support resulted in very good resolution Two additional metabolites (peaks X1 and X2) were of the two enantiomers. Applying the same chiral resoobserved which eluted, by reversed-phase HPLC, between lution conditions, metabolically formed t-3,4-dihydrodiols dihydrodiols and tetrahydrotetrols. Their UV-visible from both human and mouse microsomes were analyzed. spectra were indicative of a pyrene phenol structure Both human and mouse P450s were enantioselective, (Figure 3). Both metabolites were also formed when producing enantiomeric excesses of 80% and 85%, re3,4-dihydroCPP-t-3,4-diol was used as substrate in mispectively. The major enantiomer was more retained by crosomal incubations and are therefore presumed to have the column. The two enantiomers were purified, and the this functionality. EI-MS analysis of the trimethylsilylated absolute configuration of each was assigned by applying derivatives indicated that the metabolites each had three the exciton chirality CD method (9,lO). hydroxy groups: m / 492 ~ (loo%,M+), 477 (9, M+ - CHJ, It was not possible to assign the absolute configurations 403 (39, M+ - TMSOH). Thus, the structures are t-3,4of the t-3,4-dihydrodiols as such because their CD curves dihydroCPP-3,4,x-triol, where x indicates an unknown were very weak. The bis[p-(N&-dimethylamino)benzoyl] position of hydroxylation to a phenol. Insufficientmaterial derivatives were thus synthesized to increase the intensity for 'H-NMR precluded positional assignment of the pheof the CD curves. The dibenzoates were prepared by renolic hydroxyl. acting each resolved enantiomer with p(dimethy1Absolute Configuration of 3,4-DihydroCPP- t -3,4amino) benzoyl chloride in the presence of silver cyanide diols. The cytochromes P450, like the vast majority of in benzene (11). The CD curves of the derivatives are other enzymes, usually metabolize prochiral molecules shown in Figure 4. The strong intensities and locations enantioaelectively. CPP is a prochiral molecule which upon of the extrema (at 296 and 325 nm) of the CD curves epoxidation could form two enantiomeric CPPEs. The indicate very strongly that they arise from coupling beinstability of CPPE under the conditions of the metabotween the two p(dimethy1amino)benzoate groups. The lism experiments, even in the presence of epoxide hydroCD spectrum of the major enantiomer produced from both lase inhibitor, as well as in protic solvents makes it difficult human and mice microsomes exhibited a positive Cotton to determine directly the enantioselectivity of CPPE effect, from which it may be concluded that the configuformation. On the other hand, 3,4-dihydroCPP-t-3,4-diols ration is 3R,4R. formed from hydrolysis of CPPE are very stable and are Synthesis of Specifically Deuterated CPP. [4-2good candidates for resolution of enantiomers by Pirkle HICPP was prepared as outlined in Scheme I. The key type HPLC chiral column chromatography and determiintermediate in this procedure is the 4-ketone 2, which has
Chem. Res. Toxicol., Vol. 5, No. 2, 1992 161
Microsomal Metabolism of Cyclopenta[cdlpyrene
220
400
Wavelength (nm) Figure 4. CD cwes of the bis[p-(NJV-dimethy1amino)benzoyll derivatives of the major (R$) and minor (S,S) enantiomers of 3,4-dihydroCPP-t-3,4-diol.
previously only been prepared by rearrangement of CPPE. The obvious alternative approach to 2, cyclization of 1pyrenylacetic acid, has not been accomplished despite previous efforts (12, 13). Acylation of 1,2,3,6,7,8-hexahydropyrenewith bromoacetyl bromide proceeded readily in the presence of ABr3 to give the bromomethyl ketone in high yield. This was, without purification, treated with DDQ in benzene to produce the fully aromatic bromomethyl ketone, which was isolated in good yield (73%) after chromatography on silica gel. Ring closure to the pentacyclic structure was accomplished by irradiation in CC14 with 350-nm light. This procedure was prompted by the successful photochemical cyclization of 1-(bromoacety1)pyrene to 3-oxo-3,4-dihydroCPP (14) and the failure of several attempts in the present instance by the use of AlBr3. Thermal cyclization failed when the reaction was conducted in CH2C12,CH3NO2, CS2, or CH2C1CH2C1. In benzene some 4-oxo-3,4dihydroCPP was produced, but the major product resulted from reaction with benzene. The photochemical procedure gave a satisfactory yield (43%), and the major impurities were the reduction product 4-acetylpyrene and starting material. Deuterium was introduced by reduction of 2 with LAID4 to yield the alcohol la. Treatment of la with toluenesulfonic acid in dry benzene produced the desired [4-2HICPP. Both reactions proceeded in high yield.
Dlscusslon Mouse and human liver microsomes activated CPP in a qualitatively similar pattern, which was slightly different from that produced by rat liver microsomes. One product, 9,10-dihydroCPP-t-9,10-diol, which was observed in the metabolite mixture isolated from incubation of CPP with rat liver microsomes, was not found with mouse and human microsomes. The absence of the 9,lO-dihydrodiol should not necessarily be taken as evidence that it is not formed. Microsomal incubations were run for 3 h to maximize the formation of terminal pioducts; previous studies with rat microsomes used shorter incubation times. The major metabolite from all three liver microsomes of rat, mouse , mouse, and human is 3,4-dihydroCPP-t-3,4diol, and the configuration of the major metabolite enantiomer is 3R,4R, as described earlier. A variety of synthetic oxygenated CPP derivatives, including some of the compounds that are reported herein as metabolites, have been tested for mutagenic activity (5), although not all of them were known as metabolites at that time. All tested oxygenated compounds were mutagenic in the presence of rat PMS, and none other than CPPE were mutagenic in the absence of PMS. The mutagenic
activities of the t-9,lO-dihydrodiol and the 9,lO-epoxide were not reported. The two tetrahydrotetrols that were characterized are diastereoisomers of 3,4,9,1O-tetrahydroCPP- t-3,4-t-9,10tetrol as deduced from the experiments described in the Results section. The presence of tetrahydrotetrols suggests that a diol epoxide species is formed. The formation of the tetrols may provide a basis for explaining the considerable mutagenic activity of the oxygenated CPP derivatives tested in the presence of rat PMS such as c- and t-3,4-dihydrodiols,3- and 4-hydroxy-3,4-dihydroCPP, and others. Thus, each of these may be a good substrate for epoxidation at the 9,lO position, and the epoxide may then be the ultimate mutagenic form. The 4-hydroxy metabolite in which the ethylenic bridge forming the cyclopenta ring is saturated is the product of an unusual metabolic path. To the best of our knowledge it is the first metabolite of this type formed from fully unsaturated cyclopenta fused PAH (15-19). To elucidate the mechanism of its formation, mouse liver microsomes were used to produce metabolites of [4-2H]CPPand the fate of the isotopic label was determined. Both 1 and 2 were isolated from the incubations by HPLC. The amounts of each of these metabolites isolated were insufficient for direct 'H-NMR positional analysis of the deuterium label. Nevertheless, it was possible by a combination of mass spectral analysis and chemical conversion of 1 to 2 to determine the fate of the deuterium label. It should be noted first that the following conclusions are only valid if 3,CdihydroCPP is not a metabolite of CPP because the same results could be observed if 1 and 2 were successitre products of oxidation of 3,4-dihydroCPP. 3Methylcholanthrene, a polycyclic aromatic hydrocarbon with a saturated cyclopenta ring, is, in fact, metabolized in just this way (15). However, neither we nor others have any evidence for the formation of 3P-dihydroCPP from CPP. No dehterium was detectable in 2 by MS, indicating that most of the 4-oxo-3,4-dihydroCPP was formed through a since this mechanism other than the NIH shift (20,21), mechanism requires the 1,2 migration of deuteride from C(4) to C(3). The possibility that deuterium could be lost after formation of that from [3-2H]-4-oxo-3,4-dihydroCPP ketone by an NIH shift mechanism was ruled out by mwuring the rate of exchange of deuterium into 2a under the conditions of incubation with microsomes. The alternative mechanism, illustrated in Scheme 11,involves the loss of a deuteron from C(4) to produce an enolic double bond, followed by tautomerism to the ketone. Approximately 50% of the deuterium was lost from 1. The remaining deuterium was shown to still be at C(4) by pyridinium chlorochromate (PCC) oxidation of 1 to 2, which resulted in the loss of all the label. Thus, metabolically formed 4-hydroxy-3,4-dihydroCPP was composed of a nearly equal mixture of la and IC and none of lb. The deuterium-labeledalcohol la could not have arisen through a ketone intermediate. Thus, it may be inferred that la is formed by reduction of CPP 3,4-epoxide or the carbocation thereof. There is precedent for such a reduction. It has been reported, for example, that benzo[a]pyrene diol epoxide can be reduced nonenzymatically by NADPH to 7,8,9trihydroxy-7,8,9,1O-tetrahydrobenzo [a ]pyrene (22). The nonlabeled alcohol IC almost certainly arises by reduction of the ketone 2a. When synthetic 4-oxo-3,4-dihydroCPP was incubated with microsomes under the conditions used for metabolism of CPP, it was reduced to the alcohol in high yield.
Sahali et al.
162 Chem. Res. Toricol., Vol. 5, No. 2, 1992 Scheme I1
@< ti
H
/
I
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BD I O H
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In summary, it appears that the metabolite 4-hydroxy3,4-dihydroCPP is produced by two reductive pathways, one operating on the epoxide CPPE and the other operating on the epoxide rearrangement product 4-oxo-3,4dihydroCPP. The presence of 4hydroxy- as well as 4-oxo-3,4-dihydroCPP in microsomal incubations of CPP is also interesting in that it provides the first experimental support from a metabolic system for the proposal by Gold et al. (1) that C(3) in CPPE is the reactive center. It thus seems likely that adducts of CPPE to DNA and proteins would be formed through nucleophilic attack at C(3) if CPPE reaches those target sites before undergoing further transformation. Our finding of tetrahydrotetrol metabolites, in conjunction with earlier mutagenicity studies, also suggests the possibility that one or another diol epoxide of CPP formed by oxidation at the 3,4,9 and 10 positions may play an important role in effecting the genotoxicity of CPP. Acknowledgment. This work was supported by grants from the National Institutes of Health (ES01640 and ES02109).
References (1) Eisenstadt, E., Gold, A. (1978) Cyclopenta[cd]pyrene: A highly
mutagenic Dolvcvclic aromatic hvdrocarbon. Proc. Natl. Acad. Sci. ij.S.A.-75; i667-1669. (2) Cavalieri. E.. Roean. E., Toth. B., Munhall, A. (1981) Carcinogenicity ofthe' en&onmental pollutants cyclopenteno[cd]pyrene and cyclopentano[cd]pyrene in mouse skin. Carcinogenesis 2, 277-281. (3) Gold, A., Eisenstadt, E. (1980) Metabolic activation of cyclopenta[cd]pyrene to 3,4-epoxycyclopenta[cd]pyrene by rat liver microsomes. Cancer Res. 40, 3940-3944.
