Convenient syntheses of dibenzo[c,p]chrysene and its possible proximate and ultimate carcinogens: In vitro metabolism and DNA adduction studies

Article abstract of DOI:10.1021/jo040291k

Dibenzo[c,p]chrysene (DB[c,p]C) is the only hexacyclic polycyclic aromatic hydrocarbon having two Cord regions, both in different chemical environments. Its environmental presence and relative tumorigenic potency are not known due to the lack of synthetic

Full text of DOI:10.1021/jo040291k

Convenient Syntheses of Dibenzo[c,p]chrysene and Its Possible
Proximate and Ultimate Carcinogens: In Vitro Metabolism and
DNA Adduction Studies
Arun K. Sharma,* Jyh-Ming Lin, Dhimant Desai, and Shantu Amin
Penn State Milton S. Hershey Medical Center, Penn State College of Medicine, Department of
Pharmacology, H078, 500 University Drive, Hershey, Pennsylvania 17033
aks14@psu.edu
Received December 20, 2004
Dibenzo[c,p]chrysene (DB[c,p]C) is the only hexacyclic polycyclic aromatic hydrocarbon having two
fjord regions, both in different chemical environments. Its environmental presence and relative
tumorigenic potency are not known due to the lack of synthetic standards. We report here the
synthesis of dibenzo[c,p]chrysene (1), its proximate carcinogens, i.e., trans-1,2-dihydroxy-1,2-dihydro-
DB[c,p]C (2) and trans-11,12-dihydroxy-11,12-dihydro-DB[c,p]C (3), and possible ultimate carcino-
gens, i.e., anti-trans-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-DB[c,p]C (4) and anti-trans-11,12-
dihydroxy-13,14-epoxy-11,12,13,14-tetrahydro-DB[c,p]C (5). The syntheses of 1 and the appropriately
methoxy-substituted DB[c,p]C (12 and 27), key intermediates for the synthesis of its proximate
and ultimate metabolites, were tried first using a Suzuki cross-coupling reaction. However, the
cyclization of olefins (10 and 11) gave poor yields of the desired products. An alternate method was
thus developed employing a photochemical approach. The in vitro metabolism of DB[c,p]C was
established with the S9 fraction of liver homogenate from phenobarbital/â-naphthoflavone-induced
Sprague-Dawley rats. The major dihydrodiol formed was identified as the fjord region 11,12-
dihydroxy-11,12-dihydro-DB[c,p]C, while the major and minor phenols were identified as 11-hydroxy-
DB[c,p]C and 12-hydroxy-DB[c,p]C, respectively. Further, the DNA adduction studies with the calf
thymus DNA led to a mixture of dA and dG adducts for both fjord region diol epoxides (4 and 5).
Interestingly, the dA to dG ratio for 1,2-dihydroxy-3,4-epoxide was much higher (3.2) compared to
that of 11,12-dihydroxy-13,14-epoxide (0.5).
Introduction
counterparts, having the same number of rings. For
example, 7,12-dimethylbenz[a]anthracene, 5-methylchry-
sene (5-MeC), and 11-methylbenzo[a]pyrene, having a
methyl group in the bay region, are more carcinogenic
than their respective parent PAHs.3 Further, the fjord
region diol epoxides (DEs) derived from benzo[c]phenan-
Polycyclic aromatic hydrocarbons (PAHs), the ubiqui-
tous environmental pollutants,1 require metabolic activa-
tion to electrophilic reactive metabolites in order to exert
their mutagenic and tumorigenic activity.2 Structural
features have been reported to be the key factor in
determining the potency of a PAH as a carcinogen. It has
been observed that substituted PAHs with greater steric
hindrance are more tumorigenic than their less hindered
(2) (a) Gelboin, H. V. Physiol. Rev. 1980, 34, 1107. (b) Conney, A.
H. Cancer Res. 1982, 42, 4875. (c) Cooper, C. S.; Grover, P. L.; Sims,
P. Prog. Drug. Metab. 1983, 7, 295. (d) Guengerich, F. P. Carcinogenesis
2000, 21, 345-351. (e) Cavalieri, E. L.; Rogan, E. G. One-electron
oxidation in aromatic hydrocarbon carcinogenesis. In Polycyclic Hy-
drocarbons and Carcinogenesis; Harvey, R. G., Ed.; American Chemical
Society: Washington, D.C., 1985; pp 289-305. (f) Penning, T. M.;
Burczynski, M. E.; Hung, C.-F.; McCoull, K. D.; Palackal, N. T.;
Tsuruda, L. S. Chem. Res. Toxicol. 1999, 12, 1.
* To whom correspondence should be addressed. Tel: 717-531-1450.
Fax: 717-531-5013.
(1) Harvey, R. G. Polycyclic Aromatic Hydrocarbons: Chemistry and
Carcinogenicity; Cambridge University Press: Cambridge, England,
1991.
10.1021/jo040291k CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/25/2005
4962
J. Org. Chem. 2005, 70, 4962-4970

Synthesis, Metabolism, and DNA Adduction of Dibenzo[c,p]chrysene
threne (B[c]P), benzo[g]chrysene (B[g]C), and dibenzo-
[a,l]pyrene (DB[a,l]P) are more tumorigenic in rodents
than the bay region 7,8-dihydroxy-9,10-epoxy-7,8,9,10-
tetrahydrobenzo[a]pyrene (B[a]P-DE).4 The steric hin-
drance due to the fjord region causes a deviation from
planarity of a molecule, and this stereochemical effect
has been associated with carcinogenicity. Dipple et al.
have proposed that this effect may be due to an increased
number of binding sites of active diol epoxides (DEs) to
dexoyadenosine (dA) sites in DNA.5 For example, the bay
region (+)- and (-)-anti-B[a]P-DE bind predominantly
to deoxyguanosine (dG),6 while the sterically hindered
fjord region DEs of B[c]P,7,8 B[g]C,8,9 and DB[a,l]P10 bind
more extensively to dA than to the dG residues in DNA
to form stable adducts. The fjord PAH diol epoxide-N6-
dA adducts have been reported to be much more resistant
to repair by nucleotide excision repair enzymes in human
cell extracts than bay region B[a]P-DE N6-dA adducts.11
This was also supported by greater thermal stability of
duplexes with fjord PAH-N6-dA lesions relative to those
with bay region B[a]P-DE N6-dA adducts.12 Theoretical
calculations have attributed the increased activity of
sterically hindered PAHs with fjord region to the greater
instability of their epoxide ring upon protonation.13
The molecules studied so far contained only one fjord
region, and little is known about the biological activity
of PAHs containing two active sites, e.g., a fjord and a
FIGURE 1.
bay region14 or two fjord regions15 in the same molecule.
Our recent studies on benzo[c]chrysene (B[c]C), having
a bay and fjord region, have shown that the fjord region
diol epoxide, i.e., B[c]C-9,10-diol-11,12-epoxide, is a much
more potent rat mammary gland carcinogen than the bay
region B[c]C-1,2-diol-3,4-epoxide.14 In continuation of our
pursuits toward the understanding of the structure-
carcinogenicity relationship of sterically hindered PAHs,
we have chosen to study dibenzo[c,p]chrysene (DB[c,p]C).
It belongs to a class of cata-condensed16 hexacyclic PAHs
and attracted our attention because of its unique struc-
ture; it contains two fjord regions in the molecule causing
it to deviate from planarity which has been shown by
theoretical calculations.17 It incorporates the structures
of three potent tumorigenic PAHs, i.e., B[c]P, B[c]C, and
B[g]C, in its structure as shown by structures 1a, 1b,
and 1c, respectively (Figure 1). In view of all this, we
hypothesized that DB[c,p]C may be a potent carcinogen.
However, its environmental presence, metabolism pat-
tern, and carcinogenicty are not known due to the lack
of synthetic standards of DB[c,p]C and its potential active
metabolites. Thus, as a beginning in this direction, we
report here the syntheses of DB[c,p]C (1); the possible
proximate carcinogens trans-1,2-dihydroxy-1,2-dihydro-
DB[c,p]C (2) and trans-11,12-dihydroxy-11,12-dihydro-
DB[c,p]C (3), required in connection with the metabo-
lism studies; and possible ultimate carcinogens, i.e.,
anti-trans-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-
DB[c,p]C (4) and anti-trans-11,12-dihydroxy-13,14-epoxy-
11,12,13,14-tetrahydro-DB[c,p]C (5) (Figure 2) to study
the DNA adduct formation pattern.
(3) (a) Huggins, C. B.; Pataki, J.; Harvey, R. G. Proc. Natl. Acad.
Sci. U.S.A. 1967, 58, 2253. (b) DiGiovanni, J.; Diamond, L.; Harvey,
R. G.; Slaga, T. J. Carcinogenesis 1983, 4, 403. (c) Hecht, S. S.; Amin,
S.; Huie, K.; Melikan, A. A.; Harvey, R. G. Cancer Res. 1987, 47, 5310.
(d) Iyer, R. P.; Lyga, J. W.; Secrist, J. A., III; Daub, G. H.; Slaga, T. J.
Cancer Res. 1980, 40, 1073.
(4) (a) Hecht, S. S.; El-Bayoumy, K.; Rivenson, A.; Amin, S. Cancer
Res. 1994, 54, 21. (b) Levin, W.; Wood, A. W.; Chang, R. L.; Ittah, Y.;
Croisy-Delcey, M.; Yagi, H.; Jerina, D. M.; Conney, A. H. Cancer Res.
1980, 40, 3910-3914. (c) Amin, S.; Desai, D.; Dai, W.; Harvey, R. G.;
Hecht, S. S. Carcinogenesis 1995, 16, 2813. (d) Amin, S.; Krzeminski,
J.; Rivenson, A.; Kurtzke, C.; Hecht, S. S.; El-Bayoumy, K. Carcino-
genesis 1995, 16, 1971.
(5) (a) Dipple, A. In DNA Adducts: Identification and Biological
Significance; Hemminki, K., Dipple, A., Segerback, D., Kadulbar, F.,
Shuker, D., Bartsch, H., Eds.; IARC: Lyon, France, 1994; pp 107-
129. (b) Szeliga, J.; Lee, H.; Harvey, R. G.; Page, J. E.; Ross, H. L.;
Routledge, M. N.; Hilton B. D.; Dipple, A. Chem. Res. Toxicol. 1994, 7,
420.
(6) (a) Conney, A. H. Cancer Res. 1982, 42, 4875. (b) Geacintov, N.
E.; Cosman, M.; Hingerty, B. E.; Amin, S.; Broyde, S.; Patel, D. J.
Chem. Res. Toxicol. 1997, 10, 112. (c) Dipple, A.; Moschel, R. C.; Bigger,
C. A. H. In Chemical Carcinogens, 2nd ed.; Searle, C. E., Ed.; ACS
Monograph No. 182; American Chemical Society: Washington, DC,
1984; pp 63-84. (d) Harvey, R. G.; Geacintov, N. E. Acc. Chem. Res.
1988, 21, 66.
(7) Dipple A.; Pigott, M. A.; Agarwal, S. K.; Yagi, H.; Sayer, J. M.;
Jerina, D. M. Nature 1987, 327, 535.
(8) Giles, A. S.; Seidel, A.; Phillips, D. H. Chem. Res. Toxicol. 1995,
8, 591.
(9) (a) Szeliga, J.; Page, J. E.; Hilton, B. D.; Kiselyov, A. S.; Harvey,
R. G.; Dunayevskiy, Y. M.; Vouros, P.; Dipple, A. Chem. Res. Toxicol.
1995, 8, 1014. (b) Herndon, W. C.; Nowal, P. C.; Connor, D. A.; Lin, P.
J. Am. Chem. Soc. 1992, 114, 41.
Results and Discussion
(10) (a) Li, K. M.; Todorovic, R.; Rogan, E. G.; Cavalieri, E. L.; Ariese,
F.; Suh, M.; Jankowlak, R.; Small, G. J. Biochemistry 1995, 34, 8043.
(b) Jankowiak, R.; Ariese, F.; Hewer, A.; Luch, A.; Zamzow, D.; Hughes,
N. C.; Phillips, D.; Seidel, A.; Platt, K. L.; Oesch, F.; Small, G. J. Chem.
Res. Toxicol. 1998, 11, 674. (c) Ralston, S. L.; Coffing, S. L.; Seidel, A.;
Luch, A.; Platt, K. L.; Baird, W. M. Chem. Res. Toxicol. 1997, 10, 687.
(d) Baird, W. M.; Ralston, S.; Luch, A.; Platt, K. L.; Seidel, A. Polycyclic
Aromat. Compd. 1996, 10, 235. (e) Ralston, S. L.; Seidel, A.; Luch, A.;
Platt, K. L.; Baird, W. M. Carcinogenesis 1995, 16, 2899.
(11) Buterin, T.; Hess, M. T.; Luneva, N.; Geacintov, N. E.; Amin,
S.; Kroth, H.; Seidel, A.; Naegeli, H. Cancer Res. 2000, 60, 1849.
(12) Ruan, Q.; Kolbanovskiy, A.; Zhuang, P.; Chen, J.; Krzeminski,
J.; Amin, S.; Geacintov, N. E. Chem. Res. Toxicol. 2002, 15, 249.
(13) Borosky, G. L. J. Org. Chem. 1999, 64, 7738.
A synthesis of DB[c,p]C has been reported in the
literature starting from self-condensation of tetralone via
(14) Amin, S.; Lin, J.-M.; Krzeminski, J.; Boyiri, T.; Desai, D.; El-
Bayoumy, K. Chem. Res. Toxicol. 2003, 16, 227.
(15) Harvey, R. G.; Zhang, J.-T.; Pataki, J. J. Org. Chem. 1998, 63,
6405.
(16) Harvey, R. G. Polycyclic Aromatic Hydrocarbons; Wiley-VCH:
New York, 1997; p 189.
(17) (a) Herdon, W. C.; Nowak, P. C.; Connor, D. A.; Lin, P. J. Am.
Chem. Soc. 1992, 114, 41. (b) Herdon, W. C.; Connor, D. A.; Lin, P.
Pure Appl. Chem. 1990, 62, 435.
J. Org. Chem, Vol. 70, No. 13, 2005 4963

Sharma et al.
(87%) yield of the adduct 9 which on Wittig condensation
resulted in high yield of a olefins 11 as a mixture of cis
and trans isomers. However, the cyclization using any
of the above methods gave not more than (10-17%) yields
of the cyclized product 12.
We have recently reported the synthesis of various
PAHs, e.g., N[1,2-a]P,22 N[1,2-e]P,23 B[c]C derivatives,24
and DMBA derivatives,24 through a similar approach,
and the cyclization with methanesulfonic acid worked
very well to give high yields of the cyclized product. The
only difference is that in all of the earlier cases, the
cyclization occurred to form the K-region of the PAH
whereas in the present case we were trying to cyclize the
olefins 10 and 11 to form the fjord region. The reaction
was extremely quick, perhaps because the K-region (5-
position) in compounds 10 and 11 involved in the cy-
clization is electron rich. However, due to steric reasons
the cyclization is not favored to form the desired six-
membered ring.
FIGURE 2.
a spiral intermediate.18 However, this method cannot be
applied to the syntheses of fjord region dihydrodiol
metabolites of DB[c,p]C. We intended to exploit the
Suzuki cross-coupling19 approach for their syntheses.
Suzuki coupling has recently been introduced to the PAH
synthesis,20-25 and we also have successfully exploited
this approach for the synthesis of N[1,2-a]P,22 N[1,2-e]P,23
B[c]C,24 DMBA,24 DB[a,l]P,25 and their metabolites. The
synthetic sequence of DB[c,p]C is depicted in Scheme 1.
Chrysene-6-boronic acid (7) was obtained by treatment
of 6-bromochrysene (6) with n-BuLi and triisopropyl
borate employing a literature method.21 Suzuki cross-
coupling reaction of boronic acid 7 with 2-bromobenzal-
dehyde in the presence of CsF and a catalytic amount of
Pd(PPh3)4 resulted in the formation of aldehyde 8 in 82%
yield. Treatment of 8 with (methoxymethyl)triphenylphos-
phonium chloride using PhLi as a base afforded the olefin
10, which cyclized readily on treatment with concentrated
sulfuric acid in methylene chloride (at 0 °C) to give DB-
[c,p]C (1), but in a very low (∼19%) yield. Various
attempts to improve the yield failed. The reactions in the
presence of methanesulfonic acid or BF3-etherate gave
even lower yields of the cyclized product. An attempt to
cyclize olefin 10 in the presence of PTSA resulted in the
formation of aldehyde 13 in quantitative yield. The
intermediate for the synthesis of 1,2-dihydroxy-1,2-
dihydro-DB[c,p]C, i.e., the 2-methoxy-DB[c,p]C, was also
obtained in a very low yield. Coupling of boronic acid 7
with 2-bromo-5-methoxybenzaldehyde gave a very good
DB[c,p]C and 2-methoxy-DB[c,p]C thus obtained were
characterized on the basis of NMR and high-resolution
MS data. Their 1H NMR spectra showed, apart from
other protons, the six downfield protons (H4, H5, H8, H9,
H14, and H15) characteristic of the bay and fjord regions.
Interestingly, the melting point of DB[c,p]C (152-153 °C)
differed substantially from that reported in the literature
(200-202 °C).18 An alternate synthetic approach was
thus necessary to unequivocally confirm the structure as
well as to improve the overall yield. For this purpose,
we were attracted to the photochemical approach since
it has been shown to work better in the case of such
sterically hindered molecules.26 The synthetic approach
is shown in Scheme 2. Condensation of 2-naphthaldehyde
(14) with ethylphenyl acetate in the presence of lithium
diisopropylamide (LDA) gave the olefins 16 as a mixture
of cis and trans isomers.27 Photocyclodehydrogenation of
the olefins 16 took place smoothly in the presence of
catalytic amount of I2 to yield 5-ethoxycarbonylbenzo[c]-
phenantherene (18). Reduction of 18 by LiAlH4 yielded
the alcohol 20, which was oxidized to the aldehyde
derivative 22 by treatment with pyridinium chlorochro-
mate (PCC). The Wittig condensation of 22 with benzyl-
triphenylphosphonium chloride resulted in the formation
of the olefins 24 as a mixture of cis and trans isomers in
an ∼1:1 ratio, which was easily photocyclized to furnish
1
the desired DB[c,p]C in very good (86%) yield. The H
NMR, MS, and the melting point of DB[c,p]C were
identical to the those obtained by the Suzuki cross-
coupling reaction. This suggested that the compound
reported previously in the literature18 was either not DB-
[c,p]C but some other rearranged product or the reported
melting point was incorrect.
The key intermediates for the synthesis of fjord region
dihydrodiols 2 and 3, i.e., the appropriately substituted
methoxy derivatives of DB[c,p]C, were also obtained
using photochemical methods (Scheme 2). 2-Methoxy-DB-
[c,p]C was obtained from 6-methoxy-2-naphthaldehyde
15 using a similar reaction sequence. Treatment of 15
with ethylphenyl acetate gave the olefin 17, which on
(18) Burnham, J. W.; Melton, R. G.; Eisenbraun, E. J. J. Org. Chem.
1973, 38, 2783.
(19) For recent reviews, see: (a) Hassan, J.; Sevignon, M.; Gozzi,
C.; Schulz, E.; Lamaire, M. Chem. Rev. 2002, 102, 1359. (b) Kotha, S.;
Lahiri, K.; Kashinath, D. Tetrahedron 2002, 58, 9633. (c) Suzuki, A.
J. Organomet. Chem. 1999, 576, 147. (d) Miyaura, N.; Suzuki, A. Chem.
Rev. 1995, 95, 2457. (e) Suzuki, A. Pure Appl. Chem. 1994, 66, 213.
(20) (a) Kumar, S. J. Chem. Soc., Perkin Trans. 1 1998, 3157. (b)
Zhang, F. J.; Cortez, C.; Harvey, R. G. J. Org. Chem. 2000, 65, 3952.
(c) Kumar, S. Synthesis 2001, 841. (d) Kumar, S. J. Org. Chem. 1997,
62, 8535.
(21) Rice, J. E.; Cai, Z.-W. J. Org. Chem. 1993, 58, 1415.
(22) Desai, D.; Sharma, A. K.; Lin, J.-M.; Krzeminski, J.; Pimental,
M.; El-Bayoumy, K.; Nesnow, S.; Amin, S. Chem. Res. Toxicol. 2002,
15, 964.
(23) Desai, D.; Sharma, A. K.; Lin, J.-M.; El-Bayoumy, K.; Amin,
S.; Pimentel, M.; Nesnow, S. Polycyclic Aromat. Compd. 2002, 22, 267.
(24) Sharma, A. K.; Kumar, S.; Amin, S. Polycyclic Aromat. Compd.
2002, 22, 277.
(25) Sharma, A. K.; Kumar, S.; Amin, S. J. Org. Chem. 2004, 69,
3979.
(26) Krzeminski, J.; Lin, J.-M.; Amin, S.; Hecht, S. S. Chem. Res.
Toxicol. 1994, 7, 125.
(27) The reaction did not go to completion. However, the olefin
mixture was separated easily from the unreacted starting materials
by silica gel column chromatography.
4964 J. Org. Chem., Vol. 70, No. 13, 2005

Synthesis, Metabolism, and DNA Adduction of Dibenzo[c,p]chrysene
SCHEME 1
SCHEME 2
photocyclization afforded 5-ethoxycarbonyl-10-methoxy-
benzo[c]phenanthrene (19). LiAlH4 reduction of 19 gave
alcohol 21, which was then oxidized to aldehyde 23 using
PCC. The Wittig reaction of 23 with benzyltriphenylphos-
phonium chloride resulted in the formation of the olefins
25 as a mixture of cis and trans isomers in an ∼1:1 ratio,
which was photocyclized to furnish the desired 2-meth-
oxy-DB[c,p]C (12) in very good (87%) yield. The product
was characterized on the basis of its 1H NMR and HRMS.
Demethylation of 12 with BBr3 in CH2Cl2 led to the
formation of 2-hydroxy-DB[c,p]C (28), which was oxidized
to DB[c,p]C-1,2-dione (29) by treatment with Fremy’s salt
[(SO3K)2NO] (Scheme 3). Reduction of quinone 29 with
NaBH4 furnished the trans-1,2-dihydroxy-1,2-dihydro-
DB[c,p]C (2) in excellent yield. The product was charac-
1
terized on the basis of its H NMR and MS. The trans
stereochemistry of the dihydrodiol 2 was established on
the basis of the large coupling constant of 10.5 Hz
J. Org. Chem, Vol. 70, No. 13, 2005 4965

Sharma et al.
SCHEME 3
SCHEME 4
FIGURE 3. HPLC profile of in vitro metabolites of DB[c,p]C:
peak 1, DB[c,p]C-11,12-dihydrodiol; peak 3, 12-hydroxy-DB-
[c,p]C; peak 4, 11-hydroxy-DB[c,p]C; peak 5, DB[c,p]C.
BBr3 (Scheme 4). As expected, 11-hydroxy-DB[c,p]C on
reaction with Fremy’s salt resulted exclusively in the
formation of DB[c,p]C-11,12-dione 31. NaBH4 reduction
of the dione 31 gave the trans-11,12-dihydroxy-11,12-
1
dihydro-DB[c,p]C. The structure was confirmed by H
NMR as well as by MS, and the trans stereochemistry of
the dihydrodiol was confirmed based on the coupling
constant (10.5 Hz) between H-11 and H-12, which was
found by resolving the multiplet signal (δ 4.50-4.60) for
H11 and H12 through decoupling experiments.
1
between H1 and H2 in its H NMR spectrum seen by
resolving the multiplet signal (δ 4.52-4.60) of H1 and
H2 through decoupling experiments.
11-Methoxy-DB[c,p]C or 12-methoxy-DB[c,p]C would
be a potential intermediate required for the synthesis of
11,12-dihydroxy-11,12-dihydro-DB[c,p]C. However, our
synthetic strategy suited the synthesis of 11-methoxy
derivative 27 (Scheme 2). The synthesis of 12-methoxy-
DB[c,p]C by this method would require the reaction of
aldehyde 22 with 3-methoxybenzyltriphenylphosphonium
chloride, which according to our experience with chrysene
derivatives28 was expected to result in a mixture of 12-
and 14-methoxy-DB[c,p]C. On the other hand, in the case
of the 11-methoxy derivative, there is a probability of
formation of a mixture of 11,12-dione and 11,14-dione on
the oxidation of 11-hydroxy-DB[c,p]C (Scheme 4). How-
ever, the chances for such a mixture were minimum in
this particular case due to the steric factors.26
Thus, the Wittig reaction between aldehyde 22 and
2-methoxybenzyltriphenylphosphonium chloride gave the
olefin mixture 26, which on photocyclization resulted in
the formation of 11-methoxy-DB[c,p]C (27) (Scheme 2).
We did not observe the formation of DB[c,p]C by elimina-
tion of methoxy group as observed by Melory et al. in
the photocyclization of stillbenes.29 Compound 27 was
then converted to the 11-hydroxy-DB[c,p]C (30) using
After preparation of the synthetic standards, e.g., DB-
[c,p]C, its hydroxy derivatives 28 and 30, and two critical
fjord region dihydrodiols 2 and 3, we carried out the
metabolism of DB[c,p]C with phenobarbital/â-naphthofla-
vone-induced male Sprague-Dawley rat S9 liver homo-
genate. The profile of the metabolites by reversed-phase
HPLC is shown in Figure 3. Peak 1 was identified as DB-
[c,p]C-11,12-dihydrodiol (3) on the basis of the identical
retention time (tR 28.8 min) and identical UV as the
synthetic standard. Similarly, peak 4 was assigned as
11-hydroxy-DB[c,p]C (30) on the basis of the identical
retention time (tR 38.1 min) and identical UV as the
synthetic standard. None of the peaks in the metabolism
trace identified with the synthetic standards of DB[c,p]C-
1,2-dihydrodiol (2) or 2-hydroxy-DB[c,p]C (28). We then
carried out the dehydration of 1,2-dihydrodiol by warm-
ing in benzene in the presence of p-toluenesulfonic acid,
which led to the formation of a mixture of 1-hydroxy-
DB[c,p]C and 2-hydroxy-DB[c,p]C. Using this mixture,
we did not detect 1-hydroxy-DB[c,p]C as a metabolite.
Dehydration of DB[c,p]C-11,12-dihydrodiol (3), under
similar conditions, led to a mixture of 11-hydroxy-DB-
[c,p]C and 12-hydroxy-DB[c,p]C. 11-Hydroxy-DB[c,p]C (tR
38.1) in this mixture coeluted with the synthetic standard
and peak 4 while the 12-hydroxy-DB[c,p]C (tR 36.8)
coeluted with peak 3 in the metabolism trace. Peak 5
coeluted with DB[c,p]C, and peak 2 did not match with
(28) El-Bayoumy, K.; Desai, D.; Boyiri, T.; Rosa, J.; Krzeminski, J.;
Sharma, A. K.; Pittman, B.; Amin, S. Chem. Res. Toxicol. 2002, 15,
972.
(29) Mellory, F. B.; Rudolph, M. J.; Oh, S. M. J. Org. Chem. 1989,
54, 4619.
4966 J. Org. Chem., Vol. 70, No. 13, 2005

Synthesis, Metabolism, and DNA Adduction of Dibenzo[c,p]chrysene
FIGURE 4. DNA adduct pattern of DB[c,p]C-1,2-diol-3,4-
epoxide.
FIGURE 5. DNA adduct pattern of DB[c,p]C-11,12-diol-13,-
14-epoxide.
any of the synthetic standards and thus was not assigned.
The metabolites obtained were thus identified as 11-
hydroxy-DB[c,p]C, 12-hydroxy-DB[c,p]C, and DB[c,p]C-
11,12-dihydrodiol (Figure 3). This has been an interesting
observation because only one terminal ring (ring B,
Figure 1) is involved in in vitro metabolism, although
both active rings A and B apparently have similar
reactive sites to offer.
It is known that diol epoxides derived from more
distorted fjord regions give a higher dA/dG adduct ratio
on treatment with calf thymus DNA compared to bay
region diol epoxides. This has also been true for B[c]C,
which possesses both a bay and a fjord region in the same
molecule; the dA/dG adduct ratio for the fjord region DE
was ∼0.4 compared to ∼0.1 for the bay region.14 DB[c,p]C
having two fjord regions in different chemical environ-
ment, as also indicated by its interesting metabolism
pattern, was thus thought to be an interesting candidate
to study the DNA adduction pattern with diol epoxides
4 and 5 derived from rings A and B of DB[c,p]C,
respectively. In view of this we converted dihydrodiols 2
and 3 to the corresponding 1,2-diol-3,4-epoxide 4 and 11,-
12-diol-13,14-epoxide 5, respectively, using m-CPBA
(Schemes 3 and 4). The reactions were performed under
N2 atmosphere, and the two epoxides were obtained in
good yields. These were characterized on the basis of their
1H NMR and MS.
The formation pattern of in vitro adducts with calf-
thymus DNA and epoxides 4 and 5 was then examined.
First, the nucleoside markers were prepared from ep-
oxides 4 and 5 with 2′-dG-5′-monophosphate and 2′-dA-
5′-monophosphate. The modified deoxyribonucleotides
were enzymatically hydrolyzed to deoxyribonucleosides,
which were analyzed by HPLC. Then, the epoxides 4 and
5 were incubated with calf-thymus DNA and the modified
DNA was isolated and digested enzymatically to give
nucleoside adducts. The HPLC analysis for 1,2-diol-3,4-
epoxide 4 is shown in Figure 4. A pair of dG adduct and
a pair of dA adduct peaks were identified by comparison
of their retention times to those of the standard markers.
The dA/dG ratio calculated from the peak areas was
found to be 3.2, which is comparable to that of DB[a,l]P
(dA/dG 3-4), the most potent PAH known. The HPLC
analysis for 11,12-diol-13,14-epoxide 5 (Figure 5) also
showed a pair of dG adduct and a pair of dA adduct peaks
which were identified by comparison of their retention
times to those of the standard markers. The dA/dG ratio
in this case was found to be 0.5 which is similar to the
dA/dG ratio for fjord region 3,4-dihydroxy-1,2-epoxy-
1,2,3,4-tetrahydrobenzo[c]phenanthrene (0.5-0.7); yet it
is higher7 than the dA/dG ratio of 0.05 for the bay region
7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]py-
rene.30 These observations are consistent with the trend
that fjord region diol epoxides give enhanced levels of dA
adducts.
Conclusion
It is observed that the synthesis of DB[c,p]C and its
metabolites by Suzuki coupling sequence is indeed pos-
sible but the overall yield is low. The photochemical
cyclization approach has proved to be a better method
in the present case where steric hindrance restricts
cyclization by other methods. The method is convenient
and gave high overall yields of DB[c,p]C and its possible
proximate and ultimate carcinogenic metabolites. DB-
[c,p]C would be a good standard for its identification in
the environment and will be useful for the metabolism
studies in vivo. The fjord region dihydrodiols 2 and 3 and
the hydroxy compounds 28 and 30, served to identify the
metabolites in vitro, and will be useful as standards to
identify the products of the metabolism of DB[c,p]C in
vivo, and for the comparison of their toxicity and tum-
origenicity with other known carcinogens. The dA/dG
adduct ratio (0.5) for diol epoxide 5 was similar to B[c]C
fjord region diol epoxide (0.5-0.7) which is expected from
their similar structural features. However, the dA/dG
adduct ratio (3.2) for diol epoxide 4 makes it second only
(30) Cheng, S. C.; Hilton, B. D.; Roman, J. M.; Dipple, A. Chem.
Res. Toxicol. 1989, 2, 334.
J. Org. Chem, Vol. 70, No. 13, 2005 4967

Sharma et al.
(m, 2H), 8.30 (d, 0.59H, J ) 7.9 Hz), 8.61 (s, 0.41H, H5, cis),
8.62 (s, 0.59H, H5, trans), 8.74 (d, 1H, J ) 7.6 Hz), 8.78 (d,
1H, J ) 9.2 Hz), 8.86 (d, 1H, J ) 8.5 Hz). HRMS: m/z calcd
for C27H20O 360.1509, found 360.1514.
to DB[a,l]P-11,12-diol-13,14-epoxide to have more than
75% dA adduct. On the basis of these observations, DB-
[c,p]C may be considered as a carcinogenic PAH.
6-[2-(â-Methoxyethenyl)-4-methoxyphenyl]chrysene
(11). Yield: 75%. 1H NMR: δ 3.19 (s, 1.5H), 3.72 (s, 1.5H), 3.95
(s, 1.5H), 3.96 (s, 1.5H), 4.84 (d, 0.5H, J ) 7.5 Hz, cis), 5.44 (d,
0.5H, J ) 13.1 Hz, trans), 5.86 (d, 0.5H, J ) 7.5 Hz, cis), 6.90-
6.93 (m, 1H), 6.99 (d, 0.5H, J ) 13.1 Hz, trans), 7.12 (d, 0.5H,
J ) 2.6 Hz), 7.31 (d, 0.5H, J ) 8.5 Hz), 7.33 (d, 0.5H, J ) 8.2
Hz), 7.51-7.56 (m, 1H), 7.61-7.73 (m, 4H), 7.93 (d, 0.5H, J )
2.6 Hz), 7.99-8.05 (m, 2H), 8.61 (s, 0.5H), 8.62 (s, 0.5H), 8.73-
8.79 (m, 2H), 8.85 (d, 1H, J ) 8.2 Hz). MS: m/z 390 (M+).
Experimental Section
Caution! DB[c,p]C and its derivatives described in this
paper may be carcinogenic. Therefore, appropriate safety
procedures should be followed when working with these
compounds.
Chrysene-6-boronic Acid (7). To a solution of 6-bromo-
chrysene (6) (6.14 g, 20 mmol) at -78 °C under N2 was added
n-BuLi (2.5 M in hexanes, 17.6 mL, 44 mmol) dropwise, and
the reaction mixture was stirred at the same temperature for
1 h. Triisopropylborate (7.52 g, 9.23 mL, 40 mmol) was then
added in one portion, and the reaction was stirred at -78 °C
for 30 min. It was then warmed to room temperature and
stirred for another 1 h. The reaction mixture was then diluted
with ether (400 mL) and acidified with 10% HCl (200 mL).
The ether extract was washed with water and dried over
MgSO4. Removal of solvent gave a white solid which was
filtered and washed with hexanes/ether (9/1) to give nearly
pure 7 (4.2 g, 77%) which was used as such for further
reactions. 1H NMR: δ 7.65-7.79 (m, 4H), 8.09 (d, 1H, J ) 7.9
Hz), 8.12 (d, 1H, J ) 9.2 Hz), 8.59 (d with fine splitting,
1H, J ) 7.6 Hz), 8.62 (br s, 2H), 8.88 (d, 1H, J ) 9.2 Hz),
8.94 (d, 1H, J ) 7.9 Hz), 8.99 (d, 1H, J ) 8.2 Hz), 9.09 (s, 1H,
H5).
Dibenzo[c,p]chrysene (1). To a solution of olefin 10 (0.20
g, 0.56 mmol) in anhydrous CH2Cl2 (10 mL) was added concd
H2SO4 (0.05 mL in 0.5 mL of CH2Cl2) dropwise under N2, and
the mixture was stirred at room temperature for 10 min. It
was then poured into ice-cold H2O, and the organic layer was
separated and washed successively with 10% NaHCO3 solution
and with water. The organic layer was dried (MgSO4) and
concentrated, and the crude residue was purified via silica gel
column chromatography (EtOAc/hexanes 1:99) to give 1 (35
mg, 19%) as a pale yellow crystalline solid. Mp: 152-153 °C
1
(lit.18 mp 200-202 °C). H NMR: δ 7.59-7.77 (m, 6H), 7.95
(d, 1H, J ) 8.9 Hz), 8.03-8.07 (m, 3H), 8.69 (d, 1H, J ) 8.9
Hz), 8.79 (dd, 1H, J ) 7.9 and 1.3 Hz), 8.83 (d, 1H, J ) 8.9
Hz), 8.91 (dd, 1H, J ) 8.9 and 1.6 Hz), 9.04-9.07 (m, 2H).
HRMS: calcd for C26H16 328.1247, found 328.1245.
2-Methoxy-DB[c,p]C (12). Yield: 17%. Mp: 116-117 °C.
1H NMR: δ 4.03 (s, 3H), 7.32 (dd, 1H, J ) 9.2 and J ) 2.6
Hz), 7.38 (d, 1H, J ) 2.6 Hz), 7.58-7.76 (m, 4H), 7.86 (d, 1H,
J ) 8.9 Hz), 8.00-8.04 (m, 2H), 8.65 (d, 1H, J ) 8.9 Hz), 8.76
(d, 1H, J ) 8.2 Hz), 8.81 (d, 1H, J ) 8.9 Hz), 8.89 (dd, 1H, J
) 9.2 and 1.6 Hz), 8.95 (d, 1H, J ) 9.2 Hz), 8.97 (dd, 1H, J )
7.9 and 1.6 Hz). High-resolution MS: m/z calcd for C27H18O
358.1352, found 358.1363.
6-(2-Formylphenyl)chrysene (8). A mixture of chrysene-
6-boronic acid (7) (1.2 g, 4.4 mmol), 2-bromobenzaldehyde (0.74
g, 0.47 mL, 4.0 mmol), cesium fluoride (1.21 g, 8.0 mmol), and
Pd(PPh3)4 (0.18 g, 0.16 mmol) in anhydrous DME (50 mL) was
refluxed for 14 h. The mixture was brought to room temper-
ature, and the reaction was quenched with ice-cold H2O. The
aqueous layer was extracted with CH2Cl2 (3 × 40 mL), and
the combined organic layers were washed with H2O and dried
over anhydrous MgSO4. Concentration in vacuo provided a
residue which was purified by silica gel column chromatogra-
phy (EtOAc/hexanes 1:99) to give 8 (1.2 g, 82%) as a white
crystalline solid. Mp: 184-185 °C. 1H NMR: δ 7.54-7.80 (m,
8H), 8.03 (dd, 1H, J ) 7.6 and 2.0 Hz), 8.08 (d, 1H, J ) 9.2
Hz), 8.19 (dd, 1H, J ) 7.9 and 1.0 Hz), 8.66 (s, 1H), 8.71 (d,
1H, J ) 8.9 Hz), 8.78 (d, 1H, J ) 9.2 Hz), 8.89 (d, 1H, J ) 8.5
Hz), 9.74 (s, 1H). HRMS: m/z calcd for C25H16O 332.1196,
found 332.1191.
5-Ethoxycarbonylbenzo[c]phenanthrene (18). To a so-
lution of ethyl phenylacetate (4.1 g, 25 mmol) in THF (50 mL)
at 0 °C was added LDA (2 M solution in THF, heptane,
ethylbenzene, 18.75 mL, 37.5 mmol) dropwise, and the mixture
was stirred at 0 °C for 2 h. A solution of 2-naphthaldehyde
(14) (3.9 g, 25 mmol) in THF (50 mL) was then added, and
the reaction mixture was refluxed for 2 h. It was then cooled
to rt, and ice-cold water was added, acidified with dilute HCl,
and extracted with EtOAc. The organic layer was washed with
water and dried over anhydrous MgSO4. Concentration in
vacuo gave a residue that was purified on a silica gel column
chromatography (EtOAc/hexanes 1:99) to yield a mixture of
cis and trans isomers of olefin 16 (4.25 g, 56%). A stirred
solution of 16 (1.51 g, 5.0 mmol) and I2 (cat.) in benzene was
irradiated with a Hanovia 450 W medium-pressure lamp, with
a Pyrex filter, for 8 h, while the dry air was bubbled through
the solution. Removal of solvent gave a residue which was
purified by silica gel column chromatography (EtOAc/hexanes
2/98) to give 18 (1.35 g, 89%) as a white crystalline solid. Mp:
96-97 °C. 1H NMR: δ 1.53 (t, 3H, J ) 7.2 Hz), 4.56 (q, 2H, J
) 7.2 Hz), 7.62-7.74 (m, 4H), 7.86 (d, 1H, J ) 8.5 Hz), 7.93
(d, 1H, J ) 8.5 Hz), 8.03 (dd, 1H, J ) 7.2 and J ) 2.0 Hz),
8.53 (s, 1H), 9.01-9.10 (m, 3H). HRMS: m/z calcd for C21H16O2
300.1145, found 300.1146.
6-(2-Formyl-4-methoxyphenyl)chrysene (9). Yield: 87%.
1
Mp: 165-166 °C. H NMR: δ 3.99 (s, 3H), 7.33 (dd, 1H, J )
8.5 and 2.9 Hz), 7.50 (d, 1H, J ) 8.5 Hz), 7.55-7.59 (m, 1H),
7.64-7.76 (m, 5H), 8.02 (dd, 1H, J ) 6.9 and 1.9 Hz), 8.07 (d,
1H, J ) 9.2 Hz), 8.64 (s, 1H), 8.70 (d, 1H, J ) 7.5 Hz), 8.77 (d,
1H, J ) 9.2 Hz), 8.88 (d, 1H, J ) 8.5 Hz), 9.70 (s, 1H). HRMS:
m/z calcd for C26H18O2 362.1301, found 362.1304.
6-[2-(â-Methoxyethenyl)phenyl]chrysene (10). A sus-
pension of anhydrous (methoxymethyl)triphenylphosphonium
chloride (4.8 g, 14.0 mmol) in freshly distilled Et2O (110 mL)
was cooled to -78 °C under N2. To this mixture was added
PhLi (1.8 M in cyclohexane/ether/70:30, 5.8 mL, 10.5 mmol),
dropwise, and the mixture was stirred for 30 min. The reaction
mixture was then warmed to -30 °C, stirred for another 30
min, and again cooled to -78 °C. A solution of 8 (0.93 g, 2.8
mmol) in THF (90 mL) was then added dropwise/ and the
mixture was left overnight at room temperature. The reaction
was quenched with 1 N HCl; the mixture was washed several
times with water and extracted with ethyl acetate (3 × 25 mL).
The combined organic layers were dried (MgSO4), filtered, and
concentrated. The resulting residue was purified on a silica
gel column chromatography (EtOAc/hexanes 1:99) to yield a
mixture of cis and trans isomers of olefin 10 (0.79 g, 78%) as
5-Ethoxycarbonyl-10-methoxybenzo[c]phenan-
1
threne (19). Yield: 83%. Mp: 123-124 °C. H NMR: δ 1.51
(t, 3H, J ) 7.2 Hz), 4.01 (s, 3H), 4.54 (q, 2H, J ) 7.2 Hz), 7.33
(dd, 1H, J ) 9.2 and J ) 2.6 Hz), 7.36 (d, 1H, J ) 2.6 Hz),
7.65-7.72 (m, 2H), 7.83 (s, 2H), 8.51 (s, 1H), 8.95 (d, 1H, J )
9.2 Hz), 8.98-9.03 (m, 2H). HRMS: m/z calcd for C22H18O3
330.1250, found 330.1254.
5-Hydroxymethylbenzo[c]phenanthrene (20). To a well-
stirred suspension of LiAlH4 (0.76 g, 20 mmol) in anhydrous
ether (60 mL) at 0 °C was added a solution of 18 (1.2 g, 4.0
mmol) in ether (50 mL) dropwise. After the addition was
complete, the reaction mixture was warmed to rt and stirred
1
a clear viscous oil. H NMR: δ 3.18 (s, 1.77H, trans), 3.71 (s,
1.23H, cis), 4.85 (d, 0.41H, J ) 7.2 Hz, cis), 5.45 (d, 0.59H, J
) 12.8 Hz, trans), 5.85 (d, 0.41H, J ) 7.2 Hz, cis), 6.97 (d,
0.59H, J ) 12.8 Hz, trans), 7.30-7.72 (m, 8.41H), 7.99-8.06
4968 J. Org. Chem., Vol. 70, No. 13, 2005

Synthesis, Metabolism, and DNA Adduction of Dibenzo[c,p]chrysene
for another 1 h. It was then diluted with ether, poured into
Dibenzo[c,p]chrysene (1). A stirred solution of 24 (0.15
ice-cold water, and acidified with 10% HCl. The organic layer
was separated, and the aqueous layer was extracted again with
ether. The combined ether extracts were washed with water
and dried over anhydrous MgSO4. Removal of solvent afforded
a crude residue which was purified on a silica gel column
chromatography (EtOAc/hexanes 30:70) to give (0.96 g, 93%)
of 18 as a white crystalline solid. Mp: 156-157 °C. 1H NMR:
δ 5.28 (s, 2H), 7.61-7.71 (m, 4H), 7.81 (d, 1H, J ) 8.5 Hz),
7.88 (s, 1H), 7.91 (d, 1H, J ) 8.5 Hz), 8.02 (dd, 1H, J ) 7.6
and J ) 1.3 Hz), 8.27 (dd, 1H, J ) 8.2 and J ) 2.6 Hz), 9.07
(d, 1H, J ) 8.5 Hz), 9.14 (dd, 1H, J ) 9.2 and 2.3 Hz). HRMS:
m/z calcd for C19H14O 258.1039, found 258.1047.
g, 0.46 mmol) and I2 (cat.) in dry benzene was irradiated with
a Hanovia 450W medium-pressure lamp, with a Pyrex filter,
for 3 h, while the dry air was bubbled through the solution.
Removal of solvent gave a residue that was purified by
chromatography on silica gel (EtOAc/hexanes 1:99) to give pure
1 (0.13 g, 86%) as a pale yellow crystalline solid. Mp: 152-
153 °C.
11-Methoxy-DB[c,p]C (27). Yield: 95%. Mp: 125-127 °C.
1H NMR: δ 4.10 (s, 3H), 7.00 (d, 1H, J ) 7.8 Hz), 7.54 (dd,
1H, J ) 8.5 and 7.8 Hz), 7.61-7.76 (m, 4H), 7.92 (d, 1H, J )
8.9 Hz), 8.05 (dd, 1H, J ) 7.2 and 2.3 Hz), 8.48 (d, 1H, J ) 8.5
Hz), 8.54 (d, 1H, J ) 9.2 Hz), 8.68 (d, 1H, J ) 9.2 Hz), 8.80
(dd, 1H, J ) 7.6 and 1.6 Hz), 8.83 (d, 1H, J ) 8.9 Hz), 9.05 (d,
2H, J ) 7.9 Hz). HRMS: m/z calcd for C27H18O 358.1352, found
358.1361.
2-Hydroxy-DB[c,p]C (28). To a stirred solution of 2-meth-
oxy-DB[c,p]C (12) (0.63 g, 1.75 mmol) in CH2Cl2 (35 mL) at
room temperature was added dropwise a solution of BBr3 (1
M solution in CH2Cl2, 3.5 mL, 3.5 mmol) under N2 over 10
min. After continuous stirring for 10 h at room temperature,
the reaction was quenched with ice-cold H2O. The organic layer
was washed several times with water and dried over MgSO4.
Removal of the solvent gave the crude solid that was purified
by silica gel column chromatography (EtOAc/hexanes 8:92) to
yield 28 (0.45 g, 75%) as a off-white solid. Mp: 215-216 °C.
1H NMR: δ 7.25 (dd, 1H, J ) 9.2 and 2.9 Hz), 7.38 (d, 1H, J
) 2.9 Hz), 7.58-7.76 (m, 4H), 7.81 (d, 1H, J ) 9.2 Hz), 8.01-
8.05 (m, 2H), 8.67 (d, 1H, J ) 9.2 Hz), 8.77 (dd, 1H, J ) 8.2
and 1.3 Hz), 8.80 (d, 1H, J ) 8.9 Hz), 8.89 (dd, 1H, J ) 9.2
and 1.9 Hz), 8.95 (d, 1H, H4, J ) 9.2 Hz), 8.96 (dd, 1H, J )
8.2 and 1.3 Hz). HRMS: m/z calcd for C26H16O 344.1196, found
344.1198.
5-Hydroxymethyl-10-methoxybenzo[c]phenanthrene
1
(21). Yield: 95%. Mp: 138-139 °C. H NMR: δ 4.00 (s, 3H),
5.25 (s, 2H), 7.31 (dd, 1H, J ) 9.2 and J ) 2.6 Hz), 7.35 (d,
1H, J ) 2.6 Hz), 7.64-7.71 (m, 2H), 7.77 (d, 1H, J ) 8.5 Hz),
7.81 (d, 1H, J ) 8.5 Hz), 7.83 (s, 1H), 8.24-8.27 (m, 1H), 8.96
(d, 1H, J ) 9.2 Hz), 9.04-9.07 (m, 1H). HRMS: m/z calcd for
C20H16O2 288.1145, found 288.1147.
5-Formylbenzo[c]phenanthrene (22). To a stirred sus-
pension of PCC (0.97 g, 4.5 mmol) in CH2Cl2 (50 mL) at rt
was added a solution of alcohol 20 (0.77 g, 3.0 mmol) in CH2-
Cl2 (80 mL) dropwise. The resulting mixture was stirred for 4
h, diluted with CH2Cl2, washed with 10% HCl and water, and
dried (MgSO4). Concentration in vacuo provided a residue that
was purified on a silica gel column chromatography (EtOAc/
hexanes 2:98) to yield 0.70 g (91%) of 22 as a pale yellow
crystalline solid. Mp: 133-134 °C. 1H NMR: δ 7.70-7.78 (m,
4H), 7.90 (d, 1H, J ) 8.5 Hz), 7.94 (d, 1H, J ) 8.5 Hz), 8.03-
8.06 (m, 1H), 8.32 (s, 1H), 9.05-9.08 (m, 2H), 9.44 (dd, 1H, J
) 8.2 and J ) 2.0 Hz), 10.47 (s, 1H). HRMS: m/z calcd for
C19H12O 256.0883, found 256.0886.
5-Formyl-10-methoxybenzo[c]phenanthrene (23). Yield:
89%. Mp: 109-110 °C. 1H NMR: δ 4.02 (s, 3H), 7.34 (dd, 1H,
J ) 9.2 and J ) 2.6 Hz), 7.37 (d, 1H, J ) 2.6 Hz), 7.70-7.78
(m, 2H), 7.87 (s, 2H), 8.28 (s, 1H), 8.93-8.99 (m, 2H), 9.43 (dd,
1H, J ) 7.9 and J ) 2.0 Hz), 10.43 (s, 1H). HRMS: m/z calcd
for C20H14O2 286.0988, found 286.0992.
11-Hydroxy-DB[c,p]C (30). Yield: 80%. Mp: 166-167 °C.
1H NMR: δ 6.97 (d, 1H, J ) 7.6 Hz), 7.46 (dd, 1H, J ) 8.5 and
7.9 Hz), 7.62-7.76 (m, 4H), 7.92 (d, 1H, J ) 8.9 Hz), 8.05 (dd,
1H, J ) 7.2 and 2.0 Hz), 8.46 (d, 1H, J ) 9.2 Hz), 8.49 (d, 1H,
J ) 8.5 Hz), 8.69 (d, 1H, J ) 9.2 Hz), 8.79 (d, 1H, J ) 7.6),
8.82 (d, 1H, J ) 8.9 Hz), 9.04 (d, 2H, J ) 7.9 Hz). HRMS: m/z
calcd for C26H16O 344.1196, found 344.1199.
DB[c,p]C-1,2-dione (29). To a stirred suspension of the
hydroxy derivative 28 (0.34 g, 1.0 mmol) in a mixture of CH2-
Cl2/C6H6/THF (25:75:3) (103 mL) and Adogen 464 (3 drops)
were added an aqueous solution of KH2PO4 (0.17 M, 75 mL)
and Fremy’s salt (0.80 g, 3.0 mmol). The reaction mixture was
stirred at room temperature for 1.5 h and then diluted with
CH2Cl2. The organic layer was separated, washed with water,
dried (MgSO4), and filtered. The crude residue obtained after
removal of the solvent was washed several times with a
mixture of acetone/hexane (1:9) to give 29 (0.30 g, 84%) as a
dark red solid. Mp: 222-224 °C. 1H NMR: δ 6.50 (d, 1H, J )
10.5 Hz), 7.61-7.69 (m, 3H), 7.79 (dd, 1H, J ) 8.2 and 7.2
Hz), 8.02 (dd, 1H, J ) 7.2 and 1.6 Hz), 8.07 (d, 1H, J ) 8.9
Hz), 8.12 (d, 1H, J ) 7.8 Hz), 8.28 (d, 1H, J ) 8.5 Hz), 8.34 (d,
1H, J ) 10.5 Hz), 8.49 (d, 1H, J ) 8.9 Hz), 8.57 (d, 1H, J )
8.2 Hz), 8.68 (d, 1H, J ) 7.9 Hz), 8.85 (d, 1H, J ) 8.5 Hz).
HRMS: m/z calcd for C26H14O2 358.0988, found 358.1003.
5-(â-Phenylethenyl)benzo[c]phenanthrene (24). To a
mixture of 22 (0.154 g, 0.6 mmol) and (phenylmethyl)triph-
enylphosphomium chloride (0.47 g, 1.2 mmol) was added a
solution of NaOH (0.06 g, 1.44 mmol) in water (0.5 mL). The
resulting mixture was stirred for 15 min, diluted with CH2-
Cl2, washed with 10% HCl and water, and dried (MgSO4).
Concentration in vacuo provided a residue that was purified
on a silica gel column chromatography (EtOAc/hexanes 1:99)
to yield 0.179 g (90%) of 24 as a viscous mass which solidifies
as white solid containing a mixture (1:1) of cis and trans
1
isomers. H NMR: δ 6.94 (d, 0.5 H, J ) 12.1 Hz), 7.50-7.36
(m, 4H), 7.42-7.47 (m, 1H), 7.61-7.75 (m, 6H), 7.83-8.04 (m,
3H), 8.07 (s, 0.5H), 8.31 (dd, 0.5H, J ) 8.2 and 1.3 Hz), 8.42
(dd, 0.5H, J ) 7.2 and 2.3 Hz), 9.09 (d, 0.5H, J ) 8.5 Hz),
9.13-9.19 (m, 1.5H). HRMS: m/z calcd for C26H18 330.1408,
found 330.1416.
5-(â-Phenylethenyl)-10-methoxybenzo[c]phenan-
1
threne (25). Yield: 95%. H NMR: δ 4.01 (s, 1.5H, OCH3),
4.02 (s, 1.5 H), 6.92 (d, 0.5 H, J ) 12.1 Hz), 7.06-7.19 (m,
3H), 7.25-7.37 (m, 3.5H), 7.42-7.46 (m, 1H), 7.60-7.73 (m,
4H), 7.77 (d, 0.5H, J ) 8.5 Hz), 7.83 (s, 1H), 7.94 (d, 0.5H, J
) 16.1 Hz), 8.28 (dd, 0.5H, J ) 8.2 and 1.3 Hz), 8.37-8.40 (m,
0.5H), 8.99 (d, 0.5H, J ) 9.2 Hz), 9.02-9.11 (m, 1.5H).
HRMS: m/z calcd for C27H20O 360.1509, found 360.1514.
5-[â-(2-Methoxyphenyl)ethenyl]benzo[c]phenan-
threne (26). Yield: 87%. 1H NMR δ 3.83 (s, 1.5H, OCH3), 3.95
(s, 1.5 H, OCH3), 6.46 (ddd, 0.5H, J ) 7.6, 7.6 and 1.0 Hz),
6.85 (d, 0.5H, J ) 8.2 Hz), 6.93 (dd, 0.5H, J ) 7.5 and 1.6 Hz),
6.98 (d, 0.5H, J ) 8.2 Hz), 7.04-7.11 (m, 1H), 7.17-7.18 (m,
1H), 7.30-7.35 (m, 0.5H), 7.59-8.03 (m, 9H), 8.09 (s, 0.5H),
8.32 (dd, 0.5H, J ) 8.2 and 1.6 Hz), 8.43 (dd, 0.5H, J ) 7.5
and 2.3 Hz), 9.09 (d, 0.5H, J ) 8.5 Hz), 9.12-9.16 (m, 1.5H).
HRMS: m/z calcd for C27H20O 360.1509, found 360.1513.
DB[c,p]C-11,12-dione (31). Yield: 82%. Mp: 234-236 °C.
1H NMR: δ 6.48 (d, 1H, J )10.5 Hz), 7.66-7.75 (m, 4H), 7.93
(d, 1H, J ) 8.9 Hz), 7.98 (d, 1H, J ) 8.9 Hz), 8.05 (d, 1H, J )
9.5 Hz), 8.23 (d, 1H, J ) 10.5 Hz), 8.33 (d, 1H, J ) 8.5 Hz),
8.63 (d, 1H, J ) 9.5 Hz), 8.67 (d, 1H, J ) 8.5 Hz), 8.88
(unresolved d, 1H, J ) 9.5 Hz), 8.95 (unresolved d, 1H, J )
9.2 Hz). HRMS: m/z calcd for C26H14O2 358.0988, found
358.1006.
trans-1,2-Dihydroxy-1,2-dihydro-DB[c,p]C (2). To a sus-
pension of dione 29 (0.11 g, 0.3 mmol) in ethanol (400 mL)
was added NaBH4 (0.34 g, 9.0 mmol) in several portions over
10 min. The mixture was stirred for 24 h at room temperature
while a stream of oxygen was bubbled through the solution.
The solution was then concentrated under reduced pressure
to one-fourth of its original volume. This concentrate was
J. Org. Chem, Vol. 70, No. 13, 2005 4969

Sharma et al.
diluted with ice-cold water and extracted with EtOAc (3 × 40
mL). The organic layer was dried (MgSO4), filtered, and
concentrated, and the resulting residue was washed with a
mixture of ether/hexanes (1:9) to give dihydrodiol 2 (0.081 g,
74%). Silica gel column chromatography (EtOAc/CH2Cl2/Et3N
30:70:0.5) gave pure dihydrodiol 2. Mp: 197-199 °C. 1H NMR
(DMSO-d6): δ 4.52-4.60 (m, 2H, H1, H2), 5.40 (d, 1H, OH,
JOH,2 ) 4.2 Hz), 5.75 (d, 1H, OH, JOH,1 ) 4.6 Hz), 6.23 (d, 1H,
H3, J3,4 ) 10.2 Hz), 7.11 (d, 1H, H4, J4,3 )10.2 Hz), 7.65-7.77
(m, 4H), 7.93 (d, 1H, J ) 8.2 Hz), 8.01-8.14 (m, 2H), 8.38 (d,
1H, J ) 8.2 Hz), 8.71-8.77 (m, 3H), 8.82 (d, 1H, J ) 8.2 Hz).
HRMS: m/z calcd for C26H18O2 362.1301, found 362.1293.
trans-11,12-Dihydroxy-11,12-dihydro-DB[c,p]C (3). Yield:
90%. Mp: 200-202 °C. 1H NMR (DMSO-d6): δ 4.50-4.60 (m,
2H), 5.36 (d, 1H, J ) 4.6 Hz), 5.70 (d, 1H, J ) 5.3 Hz), 6.20
(dd, 1H, J ) 10.2 and 1.6 Hz), 7.00 (d, 1H, J )10.2 Hz), 7.64-
7.73 (m, 4H), 7.94 (d, 1H, J ) 8.2 Hz), 7.99 (d, 1H, J ) 8.9
Hz), 8.10 (d, 1H, J ) 7.9 Hz), 8.30 (d, 1H, J ) 8.9 Hz), 8.75 (d,
1H, J ) 8.5 Hz), 8.81 (br d, 2H, J ) 7.9 Hz), 8.89 (d, 1H, J )
7.9 Hz). HRMS: m/z calcd for C26H18O2, 362.1301, found
362.1291.
and 8 mL of the 9000 g supernatant from livers of phenobar-
bital/â-naphthoflavone-induced male Sprague-Dawley rats.31
The mixture was extracted with EtOAc (3 × 30 mL), dried
(MgSO4), filtered, and concentrated at reduced pressure to give
a residue that was dissolved in methanol. The metabolites of
DB[c,p]C were analyzed by HPLC on a 4.6 × 250 mm (5 µm)
Vydac C18 reversed-phase column (Separation Group, Hes-
peria, CA) with solvent A, H2O, or solvent B, methanol, using
a gradient program from A/B (50:50) to A/B (0:100) over 40
min.
Preparation of DNA Adducts. anti-DB[c,p]CDE (4/5) (1
mg) in 1.0 mL of THF was added to a solution of calf thymus
DNA (10 mg) in 10 mL of 10 mM Tris-HCl buffer, pH 7. This
mixture was incubated at 37 °C. The DNA was isolated and
enzymatically hydrolyzed to deoxyribonucleosides as described
in the literature.30 Modified deoxyribonucleosides were ana-
lyzed by HPLC.
Preparation of the Standard 2′-dGuo or 2′-dAdo Ad-
ducts from Diol Epoxide. Approximately 100 mg each of
2′-dGuo-5′-monophosphate or 2′-dAdo-5′-monophosphate were
dissolved in 10 mL of 10 mM Tris-HCl buffer (pH 7.0). Then
anti-DB[c,p]CDE (4/5) (1 mg) in 1.0 mL of acetone/THF (50/
50) was added, and the solution was incubated overnight at
37 °C. The modified deoxyribonucleotides were separated from
unmodified deoxyribonucleotides on Sep-Pak C18 cartridges.
They were then enzymatically hydrolyzed to the corresponding
deoxyribonucleosides,32 and analyzed by HPLC with a Beck-
man Ultrasphere ODS 5-µm column (4.6 × 250 mm; reversed-
phase) using the following solvent system: 46% MeOH in H2O
for 10 min, followed by a linear gradient from 46 to 60% MeOH
in H2O over 20 min, then 90% MeOH in H2O for 30 min at a
flow rate of 1 mL/min.
anti-trans-1,2-Dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-
DB[c,p]C (4). A solution of dihydrodiol 2 (54 mg, 0.15 mmol)
and m-CPBA (0.26 g, 1.5 mmol) in freshly distilled THF (30
mL) was stirred at room temperature under N2 and monitored
by normal-phase HPLC using analytical Licrosorb Si60 column
(5 µm) (E. Merck, Darmstadt, Germany) with hexane/THF (75:
25) in isocratic program at a flow rate of 1.0 mL/min. After 2
h of stirring, the mixture was diluted with 150 mL of ether,
washed with cold 2% NaOH (2 × 100 mL) and water (3 × 150
mL), and then dried (K2CO3), filtered, and concentrated at
room temperature. The concentrate was washed with a
mixture of hexane/ether (3:1, 50 mL) and dried in vacuo to
1
yield diol epoxide 4 (0.041 g, 72%) as a pale yellow solid. H
Acknowledgment. This study was supported by
NCI Contract No. NO2-CB-37025-48 and Cancer Center
Support Grant No. CA-17613. Further support has been
provided by the Penn State Cancer Institute of the Penn
State College of Medicine.
NMR (DMSO-d6): δ 3.77 (dd, 1H, H3, J ) 4.3 and 1.5 Hz),
3.95 (dd, 1H, H2, J ) 8.5 and 1.5 Hz), 4.68 (d, 1H, H4, J ) 4.3
Hz), 4.95 (d, 1H, H1, J ) 8.5 Hz), 7.62-7.69 (m, 4H), 7.75-
7.79 (m, 1H), 8.06-8.09 (m, 2H), 8.61 (d, 1H, J ) 7.9 Hz), 8.66
(d, 1H, J ) 8.9 Hz), 8.71 (d, 1H, J ) 7.9 Hz), 8.85 (d, 1H, J )
8.9 Hz), 8.87 (d, 1H, J ) 9.8 Hz). HRMS: m/z calcd for
C26H18O3 378.1250, found 378.1232.
Supporting Information Available: Experimental pro-
cedures for compounds 3, 5, 9, 11, 12, 19, 21, 23, 25-27, 30,
and 31 and copies of the 1H NMR spectra for compounds 1-3,
5, 8-12, and 18-31. Also includes the HPLC traces for
incubation of diol epoxides 4 and 5 with dA, dG, and DNA.
This material is available free of charge via the Internet at
http://pubs.acs.org.
anti-trans-11,12-Dihydroxy-13,14-epoxy-11,12,13,14-
1
tetrahydro-DB[c,p]C (5). Yield: 77%. H NMR (DMSO-d6):
δ 3.79 (dd, 1H, H13, J ) 4.3 and 1.3 Hz), 3.93 (dd, 1H, J ) 8.9
and 1.3 Hz), 4.69 (d, 1H, J ) 4.3 Hz), 4.94 (d, 1H, J ) 8.9 Hz),
7.65-7.78 (m, 4H), 8.07 (d, 1H, J ) 8.9 Hz), 8.11 (d, 1H, J )
8.5 Hz), 8.15 (d, 1H, J ) 7.9 Hz), 8.58 (d, 1H, J ) 8.9 Hz),
8.81-8.87 (m, 2H), 8.93 (d with fine splitting, 1H, J ) 7.9 Hz),
9.01 (d, 1H, J ) 8.2 Hz). HRMS: m/z calcd for C26H18O3
378.1250, found 378.1239.
JO040291K
In Vitro Metabolism of DB[c,p]C with Phenobarbital/
â-Naphthoflavone-Induced Male Sprague-Dawley Rat
S9 Liver Homogenate. DB[c,p]C (2 mg in 200 µL DMSO)
was incubated at 37 °C for 20 min, in the presence of cofactors
(31) Desai, D.; Krzeminski, J.; Lin, J.-M.; Chaddha, A.; Miyata, N.;
Yagi, H.; Jerina, D. M.; Amin, S. Polycyclic Aromat. Compod. 1999,
16, 255.
(32) Baird, W. M.; Brookes, P. Cancer Res. 1973, 33, 2378.
4970 J. Org. Chem., Vol. 70, No. 13, 2005