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Analysis of the epoxide of 7,12-dimethylbenz(a)anthracene and its hydrolysis products by gas chromatography
ANALYTICAL
BIOCHEMISTRY
71, 125-132 (1976)
Analysis of the Epoxide of 7,12-Dimethylbenz(A)Anthracene and Its Hydrolysis Products by Gas Chromatography C. E. MORREAL, Department
of Breast
T. L. DAO, AND A. J. SPIES
Surgery. Roswell Buffalo, New York
Park Memorial 14203
Institute,
Received August 8, 1975; accepted October 17, 1975 Column chromatography and electron-capture gas chromatography have been applied to the separation and quantitative analysis of the K-region epoxide of 7,12-dimethylbenz(a)anthracene, S-hydroxy-7,12-dimethylbenz(a)anthracene, and trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz(a)anthracene. Deactivation of chromatographic alumina with water permitted quantitative elution of the three compounds. In a solution of acetone and water, the epoxide was hydrolyzed to the 5,6-diol, but none of the 5-hydroxy derivative was formed.
Epoxides have been implicated as possible metabolites responsible for the carcinogenic properties of certain polycyclic hydrocarbons (1). In studies concerning the metabolism of polycyclic hydrocarbons, the presence of phenols and dihydrodiols in metabolic pools has generally been accepted as evidence of the intermediate formation of epoxides, the hydrolysis of which have been shown to occur both in the presence of and in the absence of hepatic enzymes (2-6). The quantitative disappearance of precursor epoxide in aqueous media has been reported for the oxides of phenanthrene by high pressure liquid chromatography (20). Also, hydrolysis products have been determined by fluorescence and by thin-layer chromatography (5-9). In aqueous media, benzene oxide, phenanthrene-9,10-oxide (pepoxide), and dibenz(a,h)anthracene-5,6-oxide (DBA-epoxide) have been reported to spontaneously rearrange to phenols (9,lO). The nonenzymatic conversion of benzene oxide to phenol is catalyzed by protein (11). Goh and Harvey reported that the decomposition of the epoxide of 7,12dimethylbenz(a)anthracene (DMBA-epoxide) in aqueous acetone was “virtually complete in 72 hr at ambient temperature,” but data to support this claim that the epoxide can remain intact for this period of time in water are not revealed (12). Sims (9) investigated the conversion of DMBA-epoxide into DMBA-501 and trans-DMBA-diol after 24 hr reflux in aqueous acetone. Knowledge of the extent to which water alone converts reactive intermediates such as epoxides into polar hydrolysis products is an aid in the understanding of the reactions of these compounds in physiological media. 125 Copyright 0 1976 by Academic Press. Inc. All rights of reproductmn I” any form reserved.
126
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This paper reports the development of a sensitive method which quantitatively detects the presence of the epoxide of the powerful mammary carcinogen DMBA and also provides a means of separating this possible metabolite from its potential products of hydrolysis, 7,12-dimethylhydroxybenz(a)anthracene @MBA-5-01) and trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz(a)anthracene @runs-DMBA-diol). MATERIALS
AND METHODS
Aluminum oxide chromatographic powder was obtained from the J. T. Baker Co. and was deactivated by the addition of 0.5 ml water to 9.5 g of the powder and shaking for 5 min to obtain “5% deactivated alumina.” Cis-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz(a)anthracene was prepared by the method of Cook and Schoental (13) and converted into the ~runs isomer by oxidation to the quinone (14) and reduction with LiAlH, (12). 1,4-Dimethyl-2-phenylnaphthalene-3,2’-dicarboxaldehyde was prepared according to the methods of Hadler and Kryger (15) and converted into the epoxide of DMBA by the method of Newman (16) as modified by Sims (9). DMBA-5-01 was prepared by the reaction of 100 mg cis5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz(a)anthracene and 500 mg pyridine hydrochloride in 5 ml acetic acid at 80°C for 45 min. The reaction mixture was diluted with 20 ml HZ0 and extracted with 2 x 10 ml ether. The ether was washed with 3 x 5 ml HzO, dried with Na,SO,, and evaporated. The residue was applied to a 1 x 10 cm column of 10% water-deactivated alumina and eluted with 3% acetone in hexane. Fractions of 10 ml were collected, the DMBA-5-01 appearing in fractions 4-7. Evaporation of the solvent gave 76 mg (76%) DMBA-5-01 which was free of any contamination of unreacted DMBA-5,6-dihydrodiol as evidenced by thin-layer chromatography. The compound was identical in all respects to a sample of the material prepared by sublimation of the cis-DMBAdiol from acidic alumina according to the method of Newman and Olson (19). Hydrolysis of DMBA-epoxide. A solution of 10 pg of the epoxide in 0.2 ml acetone was diluted with 0.8 ml HzO, mixed thoroughly, and allowed to stand at room temperature (22°C) for 1, 5, 15, 30, 60, and 120 min. The solutions were extracted with 2 x 2 ml CHZCIP, and the combined organic layers were decanted from a small amount of water which was extracted with 1 ml CH&l,. The organic layer was dried with Na,SO, from which it was decanted. The Na,S04 was washed with 1 ml CHQ,, and the combined CH,Cl, was evaporated to dryness. The residue was dissolved in 50 ~1 benzene. Separation of DMBA derivatives. The solution of the epoxide and its hydrolysis products was applied to a 5 cm by 5 mm column of alumina which had been 5% deactivated with water. Elution was carried out in 2 min under slight pressure with 8 ml of 3% dioxane in hexane. Then elu-
DMBA
HYDROLYSlS
127
tion was continued without pressure with 8 ml of 5% acetone in hexane which was also 1% in acetic acid and 8 ml of water-saturated ether. These solvent systems elute DMBA-epoxide, DMBA-5-01 and truns-DMBAdial, respectively. Gas chromatography. The derivatives were converted into the heptafluorobutyrate esters by addition of 0.1 ml of 50% heptafluorobutyric anhydride in acetone. The solutions were kept at room temperature (22°C) for 30 min, evaporated under Nnrogen, and diluted with heptane. Gas-liquid chromatography was carried out with an 8 ft x 2 mm silanized glass column packed with 3% OV17 on 100-200 mesh Gas Chrom Q (Applied Science Laboratories) with 35 cm3/min N, on a Packard 7400 Series Gas Chromatograph with a Nickel-63 electron capture detector and a Model 878 electrometer set at 4 x lo-lo A. Inlet and detector temperatures were 26O”C, and column temperature was 255°C. Sensitivity, precision, and accuracy. Blank runs showed no peaks when derivatized samples were subjected to gas chromatography. Quadruplicate analysis of samples representing 2 ng DMBA-epoxide were analyzed by gas chromatography. When quantitated by measurement of the product of peak height and width at half height, the following unit areas were obtained: 5.12, 4.75, 5.25, and 5.38 cm2. Similar analysis of quadruplicate samples of 2.5 ng of trans-DMBA-diol gave area values of 3.65, 3.31, 3.53, and 3.60 cm2. The linearity of the method was demonstrated by analysis of varying amounts of DMBA epoxide and trans-DMBA-diol. These results, shown in Figs. 1 and 2, illustrate excellent correlation between analyzed sample and detector response in the 1- 10 ng range studied. RESULTS
The investigation of active and partially deactivated alumina led to the selection of 5% water-deactivated alumina as the best adsorbent for the separation of DMBA-epoxide, DMBA-5-01, and trans-DMBA-diol. When alumina which was not deactivated with water was used, large losses in recovery of the compounds were experienced, regardless of the polarity of the solvent used. With 5% deactivated adsorbent, no difficulty was experienced in the quantitative recovery of the DMBA-epoxide or the DMBA-5-01, but only partial elution was achieved with the transDMBA-diol, even when elution was conducted with polar solvents such as ethyl acetate or ethanol-benzene. This behavior was probably due to a tendency of polar solvents to revitalize the alumina by removal of the water (17) causing retention of part of the material on the column. This problem was overcome by replacing eluted water by utilization of ether saturated with water. When the alumina was deactivated further (8%, lo%, 15%), DMBA-5-01 and trans-DMBA-diol were easily eluted, but increasing conversion of the epoxide to the trans-DMBA-diol occurred
128
MORREAL,
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4
5
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7
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FIG. 1. Linearity of the gas chromatographic analysis of varying amounts of DMBAepoxide. Area represents product of peak height and width at half height.
directly on the column. A slight amount of hydrolysis (about 1%) occurs on the 5% deactivated column when elution is carried out by gravity, but this problem was overcome by fast elution under slight pressure. Thus, when 10 pg DMBA-epoxide was applied to a 5% deactivated column and eluted in 2 min followed by gravity elution of the solvent systems which elute the DMBA-5-01 and the trans-DMB’A-diol, no peaks were present in the diol fraction, even when 1000th of the sample was injected into the gas chromatograph. Less than 0.3 ng of the heptafluorobutyrate derivative can be measured accurately by these methods; therefore, if there is any conversion to the trans-DMBA-diol on the 5% deactivated column from which the epoxide is eluted under pressure, it would have to be less than 0.3%. Following the selection of 5% deactivated alumina as the best adsorbent for the separation of DMBA-epoxide followed by DMBA-5-01 and thentruns-DMBA-diol, 10,wgsamplesof each compound were applied 26 24 22 20. I6 0 2 Q
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I6 14. 12. IO a642I
2
3
4
5
6
7
8
9
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FIG. 2. Linearity of the gas chromatographic analysis of varying amounts of rrans-DMBAdial. Area represents product of peak height and width at half height.
DMBA
129
HYDROLYSIS
to separate columns and eluted with 3% dioxane in hexane, 5% acetone in hexane which was also 1% in acetic acid, and ether saturated with water. No overlap was observed, and quantitative recovery in the appropriate fraction was experienced for each of the three compounds. When the acetic acid was omitted from the second eluting system, quantitative recovery of DMBA-5-01 was not possible. The stability of DMBA-epoxide in water was studied by dissolving samples of the epoxide in aqueous acetone followed by extraction, column chromatography and gas chromatography. The results of these hydrolysis experiments are shown in Fig. 3 and demonstrate that the half-life of the epoxide in water is 7 min. There are low but measurable levels of epoxide still present in the aqueous medium after 2 hr. No material was detected in any of the middle fractions, indicating an absence of any conversion either by hydrolysis or rearrangement to DMBA-5-01. DISCUSSION Truns-DMBA-diol reacts with heptafluorobutyric anhydride in acetone to give an ester which gives a peak with a retention time of 10.2 min by gas chromatographic analysis. It was assumed that this derivative was the diheptafluorobutyrate of the diol. This interpretation was given further credence when the same derivative was formed from DMBA-5,6-oxide. Again, the epoxide forms only a single derivative, and this compound has a retention time of 10.2 min under the same conditions under which the Trans-DMBA-dial was treated. The structure of this compound was also presumed to be the diheptafluorobutyrate ester of the trans-DMBA-diol
MINUTES
FIG. (X -
3. Hydrolysis of DMBA-epoxide x), tram-DMBA-dial.
in aqueous
acetone.
(0 -
0).
DMBA-epoxide;
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AND
SPlESS
because of (a) the identical retention times of the derivatives from the two compounds and (b) the known formation of diesters from epoxides which have been treated with anhydrides (18). The identical retention time of the product of reaction of the isomeric cis-5,6-diol of DMBA was accepted as coincidence, even though cis and tram isomers and their derivatives usually have distinctly different retention times on gas chromatographic analysis. It was obvious that these interpretations of the data were faulty when the DMBA-5-01 became available and was also found to be converted by heptafluorobutyric anhydride into a derivative which had a retention time identical to that of the other three compounds. Thus, all of the components form the mono-heptafluorobutyrate of DMBA-5-01. The cis and truns DMBA dihydro dials share one property in common: Both can revert to totally aromatic structures by the simple removal of the elements of water. Thus, reaction with the reagent heptafluorobutyric anhydride, of which heptafluorobutyric acid is a product, can cause acid-catalyzed dehydration to DMBA-5-01 which then forms a mono-heptafluorobutyric acid ester. Also, mono esterification can be followed by acid catalyzed dehydration to the same DMBA-5-01 derivative.
Epoxides usually give trans-diesters when treated with anhydrides, but in the present case, a compound with full conjugated aromatic character can be formed by mono esterification. In the case of the epoxide, derivative formation to the heptafluorobutyrate is probably a concerted reaction.
The confusion over the reactions of these compounds was further compounded by the impossibility of obtaining DMBA-5-01 completely free of the cis diol of DMBA from which it is formed. The method of Newman and Olson by which this 5-01 is formed by sublimation from acidic alumina gave, in our hands, an equal mixture of both compounds because cisDMBA-diol also sublimes easily. Consistent formation of the DMBA-5-01
DMBA
HYDROLYSIS
131
was observed by reaction of the cis-diol with pyridine hydrochloride in acetic acid. A small amount of unreacted cis-diol is totally separated from the DMBA-5-01 by column chromatography to give chromatography pure (by tic) DMBA-5-01. The absence of any DMBA-S-01 as a hydrolysis or rearrangement product of the epoxide was an unusual development, since previous reports of hydration of the epoxides of phenanthrene and dibenz(a,h)anthracene showed exclusive phenol formation (3-5%) over a 24-hr period rather than conversion to dihydrodiols (10). The remainder of the material was presumed to be unreacted epoxide due to lack of fluorescence in the medium until reaction with acid was initiated. It was thus claimed that more than 90% of the epoxide remained intact in aqueous media over this period of time. The possibility of the prior conversion to a nonfluorescing dihydrodiol which would dehydrate in acid to the phenol was not investigated, Studies have shown that the nature of products formed in the solvolysis of phenanthrene oxides is pH dependent, the higher pH media favoring diol formation (20). It is difficult to reconcile the results of the present investigation with that reported previously by Sims (9) who found both DMBA-5-01 and Iruns-DMBA-diol following 24-hr reflux of DMBA-epoxide in aqueous acetone. It is possible that long exposure to heat causes dehydration of the diol to the phenol. Ambient temperature hydrolysis of DMBA-epoxide is, by the results of the present report, much faster (2 hr) than that reported earlier (72 hr) by Goh and Harvey (12). The techniques described in this report provide a means of separating and quantitatively detecting three derivatives of the powerful mammary carcinogen DMBA. Application of these methods to in vitro studies of DMBA metabolism are currently under way. ACKNOWLEDGMENT The authors are grateful for the support of the National Institute of Health (Grant No. 015162) for the financial assistance which made this work possible.
REFERENCES 1. Boyland. E. (1950) Symp. Biochem. SOC. 5,40. 2. Sims, P. (1970) Biochem. Pharmacol. 19, 795. 3. Keysell, G. R., Booth, J., Sims, P., Grover, P. L.. and Hewer. A. (1972) Biochem. J. 129, 41P. 4. Boyland, E., and Sims, P. (1967) Biochem. J. 104, 394. 5. Swaisland. A. J., Grover. P. L., and Sims, P. (1973) Biochem. Pharmacol. 22, 1547. 6. Jerina, D. M., Daly, J. W., Witkop. B., Saltzman-Nirenberg, P.. and Udenfriend, S. (1968) J. Amer. Chem. Sot. 90, 6525. 7. Boyland, E., and Sims, P. (1965) Biochem. J. 95, 788. 8. Boyland. E., and Sims. P. (1965) Biochem. J. 97, 7.
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9. Sims, P. (1973) B&hem. J. 131,405. 10. Grover, P. L., and Sims, P. (1970) Biochem. Pharmacol. 19, 225 1. 11. Jerina, D., Daly, J., Witkop, B., Saltzman-Nirenberg, P., and Udenfriend, S. (1968) Archs. Biochem. Biophys. 128, 176. 12. Goh, S. H., and Harvey, R. G. (1973)J. Amer. Chem. Sot. 95, 242. 13. Cook, J. W., and Shoental, R. (1948)J. Chem. Sot., 170. 14. Newman, M. S., and Davis, C. C. (1967)J. Org. Chem. 32, 66. 15. Hadler, H. I., and Kryger, A. C. (1960) J. Org. Chem. 25, 1896. 16. Newman, M. S., and Blum, S. (1964) J. Amer. Chem. Sot. 86, 5598. 17. Hesse, G., and Roscher, G. (1964) Z. Anal. Chem. 200, 3. 18. Rosowsky, A. (1964) in Heterocyclic Compounds with Three and Four-Membered Rings (Weissberger, A., ed.) Part 1, p. 437, Interscience Publishers, New York. 19. Newman, M. S., and Olson, D. R. (1974) J. Amer. Chem. Sot. 96,6207. 20. Bruice, P. Y., Bruice, T. C., Selander, H. G., Yagi, H., and Jerina, D. M. (1974) J. Amer. Chem. Sot. 96, 6814.
BIOCHEMISTRY
71, 125-132 (1976)
Analysis of the Epoxide of 7,12-Dimethylbenz(A)Anthracene and Its Hydrolysis Products by Gas Chromatography C. E. MORREAL, Department
of Breast
T. L. DAO, AND A. J. SPIES
Surgery. Roswell Buffalo, New York
Park Memorial 14203
Institute,
Received August 8, 1975; accepted October 17, 1975 Column chromatography and electron-capture gas chromatography have been applied to the separation and quantitative analysis of the K-region epoxide of 7,12-dimethylbenz(a)anthracene, S-hydroxy-7,12-dimethylbenz(a)anthracene, and trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz(a)anthracene. Deactivation of chromatographic alumina with water permitted quantitative elution of the three compounds. In a solution of acetone and water, the epoxide was hydrolyzed to the 5,6-diol, but none of the 5-hydroxy derivative was formed.
Epoxides have been implicated as possible metabolites responsible for the carcinogenic properties of certain polycyclic hydrocarbons (1). In studies concerning the metabolism of polycyclic hydrocarbons, the presence of phenols and dihydrodiols in metabolic pools has generally been accepted as evidence of the intermediate formation of epoxides, the hydrolysis of which have been shown to occur both in the presence of and in the absence of hepatic enzymes (2-6). The quantitative disappearance of precursor epoxide in aqueous media has been reported for the oxides of phenanthrene by high pressure liquid chromatography (20). Also, hydrolysis products have been determined by fluorescence and by thin-layer chromatography (5-9). In aqueous media, benzene oxide, phenanthrene-9,10-oxide (pepoxide), and dibenz(a,h)anthracene-5,6-oxide (DBA-epoxide) have been reported to spontaneously rearrange to phenols (9,lO). The nonenzymatic conversion of benzene oxide to phenol is catalyzed by protein (11). Goh and Harvey reported that the decomposition of the epoxide of 7,12dimethylbenz(a)anthracene (DMBA-epoxide) in aqueous acetone was “virtually complete in 72 hr at ambient temperature,” but data to support this claim that the epoxide can remain intact for this period of time in water are not revealed (12). Sims (9) investigated the conversion of DMBA-epoxide into DMBA-501 and trans-DMBA-diol after 24 hr reflux in aqueous acetone. Knowledge of the extent to which water alone converts reactive intermediates such as epoxides into polar hydrolysis products is an aid in the understanding of the reactions of these compounds in physiological media. 125 Copyright 0 1976 by Academic Press. Inc. All rights of reproductmn I” any form reserved.
126
MORREAL,
DA0
AND
SPIESS
This paper reports the development of a sensitive method which quantitatively detects the presence of the epoxide of the powerful mammary carcinogen DMBA and also provides a means of separating this possible metabolite from its potential products of hydrolysis, 7,12-dimethylhydroxybenz(a)anthracene @MBA-5-01) and trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz(a)anthracene @runs-DMBA-diol). MATERIALS
AND METHODS
Aluminum oxide chromatographic powder was obtained from the J. T. Baker Co. and was deactivated by the addition of 0.5 ml water to 9.5 g of the powder and shaking for 5 min to obtain “5% deactivated alumina.” Cis-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz(a)anthracene was prepared by the method of Cook and Schoental (13) and converted into the ~runs isomer by oxidation to the quinone (14) and reduction with LiAlH, (12). 1,4-Dimethyl-2-phenylnaphthalene-3,2’-dicarboxaldehyde was prepared according to the methods of Hadler and Kryger (15) and converted into the epoxide of DMBA by the method of Newman (16) as modified by Sims (9). DMBA-5-01 was prepared by the reaction of 100 mg cis5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz(a)anthracene and 500 mg pyridine hydrochloride in 5 ml acetic acid at 80°C for 45 min. The reaction mixture was diluted with 20 ml HZ0 and extracted with 2 x 10 ml ether. The ether was washed with 3 x 5 ml HzO, dried with Na,SO,, and evaporated. The residue was applied to a 1 x 10 cm column of 10% water-deactivated alumina and eluted with 3% acetone in hexane. Fractions of 10 ml were collected, the DMBA-5-01 appearing in fractions 4-7. Evaporation of the solvent gave 76 mg (76%) DMBA-5-01 which was free of any contamination of unreacted DMBA-5,6-dihydrodiol as evidenced by thin-layer chromatography. The compound was identical in all respects to a sample of the material prepared by sublimation of the cis-DMBAdiol from acidic alumina according to the method of Newman and Olson (19). Hydrolysis of DMBA-epoxide. A solution of 10 pg of the epoxide in 0.2 ml acetone was diluted with 0.8 ml HzO, mixed thoroughly, and allowed to stand at room temperature (22°C) for 1, 5, 15, 30, 60, and 120 min. The solutions were extracted with 2 x 2 ml CHZCIP, and the combined organic layers were decanted from a small amount of water which was extracted with 1 ml CH&l,. The organic layer was dried with Na,SO, from which it was decanted. The Na,S04 was washed with 1 ml CHQ,, and the combined CH,Cl, was evaporated to dryness. The residue was dissolved in 50 ~1 benzene. Separation of DMBA derivatives. The solution of the epoxide and its hydrolysis products was applied to a 5 cm by 5 mm column of alumina which had been 5% deactivated with water. Elution was carried out in 2 min under slight pressure with 8 ml of 3% dioxane in hexane. Then elu-
DMBA
HYDROLYSlS
127
tion was continued without pressure with 8 ml of 5% acetone in hexane which was also 1% in acetic acid and 8 ml of water-saturated ether. These solvent systems elute DMBA-epoxide, DMBA-5-01 and truns-DMBAdial, respectively. Gas chromatography. The derivatives were converted into the heptafluorobutyrate esters by addition of 0.1 ml of 50% heptafluorobutyric anhydride in acetone. The solutions were kept at room temperature (22°C) for 30 min, evaporated under Nnrogen, and diluted with heptane. Gas-liquid chromatography was carried out with an 8 ft x 2 mm silanized glass column packed with 3% OV17 on 100-200 mesh Gas Chrom Q (Applied Science Laboratories) with 35 cm3/min N, on a Packard 7400 Series Gas Chromatograph with a Nickel-63 electron capture detector and a Model 878 electrometer set at 4 x lo-lo A. Inlet and detector temperatures were 26O”C, and column temperature was 255°C. Sensitivity, precision, and accuracy. Blank runs showed no peaks when derivatized samples were subjected to gas chromatography. Quadruplicate analysis of samples representing 2 ng DMBA-epoxide were analyzed by gas chromatography. When quantitated by measurement of the product of peak height and width at half height, the following unit areas were obtained: 5.12, 4.75, 5.25, and 5.38 cm2. Similar analysis of quadruplicate samples of 2.5 ng of trans-DMBA-diol gave area values of 3.65, 3.31, 3.53, and 3.60 cm2. The linearity of the method was demonstrated by analysis of varying amounts of DMBA epoxide and trans-DMBA-diol. These results, shown in Figs. 1 and 2, illustrate excellent correlation between analyzed sample and detector response in the 1- 10 ng range studied. RESULTS
The investigation of active and partially deactivated alumina led to the selection of 5% water-deactivated alumina as the best adsorbent for the separation of DMBA-epoxide, DMBA-5-01, and trans-DMBA-diol. When alumina which was not deactivated with water was used, large losses in recovery of the compounds were experienced, regardless of the polarity of the solvent used. With 5% deactivated adsorbent, no difficulty was experienced in the quantitative recovery of the DMBA-epoxide or the DMBA-5-01, but only partial elution was achieved with the transDMBA-diol, even when elution was conducted with polar solvents such as ethyl acetate or ethanol-benzene. This behavior was probably due to a tendency of polar solvents to revitalize the alumina by removal of the water (17) causing retention of part of the material on the column. This problem was overcome by replacing eluted water by utilization of ether saturated with water. When the alumina was deactivated further (8%, lo%, 15%), DMBA-5-01 and trans-DMBA-diol were easily eluted, but increasing conversion of the epoxide to the trans-DMBA-diol occurred
128
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4
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7
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FIG. 1. Linearity of the gas chromatographic analysis of varying amounts of DMBAepoxide. Area represents product of peak height and width at half height.
directly on the column. A slight amount of hydrolysis (about 1%) occurs on the 5% deactivated column when elution is carried out by gravity, but this problem was overcome by fast elution under slight pressure. Thus, when 10 pg DMBA-epoxide was applied to a 5% deactivated column and eluted in 2 min followed by gravity elution of the solvent systems which elute the DMBA-5-01 and the trans-DMB’A-diol, no peaks were present in the diol fraction, even when 1000th of the sample was injected into the gas chromatograph. Less than 0.3 ng of the heptafluorobutyrate derivative can be measured accurately by these methods; therefore, if there is any conversion to the trans-DMBA-diol on the 5% deactivated column from which the epoxide is eluted under pressure, it would have to be less than 0.3%. Following the selection of 5% deactivated alumina as the best adsorbent for the separation of DMBA-epoxide followed by DMBA-5-01 and thentruns-DMBA-diol, 10,wgsamplesof each compound were applied 26 24 22 20. I6 0 2 Q
-
I6 14. 12. IO a642I
2
3
4
5
6
7
8
9
IO
v
FIG. 2. Linearity of the gas chromatographic analysis of varying amounts of rrans-DMBAdial. Area represents product of peak height and width at half height.
DMBA
129
HYDROLYSIS
to separate columns and eluted with 3% dioxane in hexane, 5% acetone in hexane which was also 1% in acetic acid, and ether saturated with water. No overlap was observed, and quantitative recovery in the appropriate fraction was experienced for each of the three compounds. When the acetic acid was omitted from the second eluting system, quantitative recovery of DMBA-5-01 was not possible. The stability of DMBA-epoxide in water was studied by dissolving samples of the epoxide in aqueous acetone followed by extraction, column chromatography and gas chromatography. The results of these hydrolysis experiments are shown in Fig. 3 and demonstrate that the half-life of the epoxide in water is 7 min. There are low but measurable levels of epoxide still present in the aqueous medium after 2 hr. No material was detected in any of the middle fractions, indicating an absence of any conversion either by hydrolysis or rearrangement to DMBA-5-01. DISCUSSION Truns-DMBA-diol reacts with heptafluorobutyric anhydride in acetone to give an ester which gives a peak with a retention time of 10.2 min by gas chromatographic analysis. It was assumed that this derivative was the diheptafluorobutyrate of the diol. This interpretation was given further credence when the same derivative was formed from DMBA-5,6-oxide. Again, the epoxide forms only a single derivative, and this compound has a retention time of 10.2 min under the same conditions under which the Trans-DMBA-dial was treated. The structure of this compound was also presumed to be the diheptafluorobutyrate ester of the trans-DMBA-diol
MINUTES
FIG. (X -
3. Hydrolysis of DMBA-epoxide x), tram-DMBA-dial.
in aqueous
acetone.
(0 -
0).
DMBA-epoxide;
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SPlESS
because of (a) the identical retention times of the derivatives from the two compounds and (b) the known formation of diesters from epoxides which have been treated with anhydrides (18). The identical retention time of the product of reaction of the isomeric cis-5,6-diol of DMBA was accepted as coincidence, even though cis and tram isomers and their derivatives usually have distinctly different retention times on gas chromatographic analysis. It was obvious that these interpretations of the data were faulty when the DMBA-5-01 became available and was also found to be converted by heptafluorobutyric anhydride into a derivative which had a retention time identical to that of the other three compounds. Thus, all of the components form the mono-heptafluorobutyrate of DMBA-5-01. The cis and truns DMBA dihydro dials share one property in common: Both can revert to totally aromatic structures by the simple removal of the elements of water. Thus, reaction with the reagent heptafluorobutyric anhydride, of which heptafluorobutyric acid is a product, can cause acid-catalyzed dehydration to DMBA-5-01 which then forms a mono-heptafluorobutyric acid ester. Also, mono esterification can be followed by acid catalyzed dehydration to the same DMBA-5-01 derivative.
Epoxides usually give trans-diesters when treated with anhydrides, but in the present case, a compound with full conjugated aromatic character can be formed by mono esterification. In the case of the epoxide, derivative formation to the heptafluorobutyrate is probably a concerted reaction.
The confusion over the reactions of these compounds was further compounded by the impossibility of obtaining DMBA-5-01 completely free of the cis diol of DMBA from which it is formed. The method of Newman and Olson by which this 5-01 is formed by sublimation from acidic alumina gave, in our hands, an equal mixture of both compounds because cisDMBA-diol also sublimes easily. Consistent formation of the DMBA-5-01
DMBA
HYDROLYSIS
131
was observed by reaction of the cis-diol with pyridine hydrochloride in acetic acid. A small amount of unreacted cis-diol is totally separated from the DMBA-5-01 by column chromatography to give chromatography pure (by tic) DMBA-5-01. The absence of any DMBA-S-01 as a hydrolysis or rearrangement product of the epoxide was an unusual development, since previous reports of hydration of the epoxides of phenanthrene and dibenz(a,h)anthracene showed exclusive phenol formation (3-5%) over a 24-hr period rather than conversion to dihydrodiols (10). The remainder of the material was presumed to be unreacted epoxide due to lack of fluorescence in the medium until reaction with acid was initiated. It was thus claimed that more than 90% of the epoxide remained intact in aqueous media over this period of time. The possibility of the prior conversion to a nonfluorescing dihydrodiol which would dehydrate in acid to the phenol was not investigated, Studies have shown that the nature of products formed in the solvolysis of phenanthrene oxides is pH dependent, the higher pH media favoring diol formation (20). It is difficult to reconcile the results of the present investigation with that reported previously by Sims (9) who found both DMBA-5-01 and Iruns-DMBA-diol following 24-hr reflux of DMBA-epoxide in aqueous acetone. It is possible that long exposure to heat causes dehydration of the diol to the phenol. Ambient temperature hydrolysis of DMBA-epoxide is, by the results of the present report, much faster (2 hr) than that reported earlier (72 hr) by Goh and Harvey (12). The techniques described in this report provide a means of separating and quantitatively detecting three derivatives of the powerful mammary carcinogen DMBA. Application of these methods to in vitro studies of DMBA metabolism are currently under way. ACKNOWLEDGMENT The authors are grateful for the support of the National Institute of Health (Grant No. 015162) for the financial assistance which made this work possible.
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