Synthesis of dibenzo[def,p]chrysene, its active metabolites, and their 13C-labeled analogues

Article abstract of DOI:10.1021/ol7029323

Dibenzo[def,p]chrysene (DBC) Is a highly carcinogenic polycyclic aromatic hydrocarbon suspected to be Involved in initiation of lung cancer in smokers. Efficient new syntheses of DBC, Its active metabolites [DBC diol (1), DBC dione (2), DBC diol epoxlde (3)], and their previously unknown ¹³C ₂-labeled analogues are reported. The ¹³C ₂-labeled analogues are required as standards for sensitive methods of analysis of their DNA adducts in human cells using stable isotope dilution liquid chromatography/tandem mass spectrometry.

Full text of DOI:10.1021/ol7029323

ORGANIC
LETTERS
2008
Vol. 10, No. 6
1059-1062
Synthesis of Dibenzo[def,p]chrysene, Its
Active Metabolites, and Their
13C-Labeled Analogues
Daiwang Xu,† Yazhen Duan,† Ian A. Blair,‡ Trevor M. Penning,§ and
Ronald G. Harvey*,†
Ben May Department for Cancer Research, UniVersity of Chicago,
Chicago, Illinois 60637, and Centers for Cancer Pharmacology and Excellence in
EnVironmental Toxicology, UniVersity of PennsylVania School of Medicine,
Philadelphia, PennsylVania 19104
rharVey@huggins.bsd.uchicago.edu
Received December 19, 2007
ABSTRACT
Dibenzo[def,p]chrysene (DBC) is a highly carcinogenic polycyclic aromatic hydrocarbon suspected to be involved in initiation of lung cancer
in smokers. Efficient new syntheses of DBC, its active metabolites [DBC diol (1), DBC dione (2), DBC diol epoxide (3)], and their previously
unknown 13C2-labeled analogues are reported. The 13C2-labeled analogues are required as standards for sensitive methods of analysis of their
DNA adducts in human cells using stable isotope dilution liquid chromatography/tandem mass spectrometry.
Polycyclic aromatic hydrocarbons (PAHs) have recently been
designated as human carcinogens by the WHO.1 They are
the most potent class of carcinogens commonly present in
urban environments.2-4 PAHs are formed in combustion of
organic matter,2-4 and significant levels are present in
tobacco smoke,5-7 auto and diesel engine emissions,4 and
charbroiled, smoked, and fried foods.4 Current evidence
indicates that PAHs are activated metabolically to reactive
forms that attack DNA leading to mutations and cancer.
Three activation pathways have been proposed: the diol
epoxide path (mediated by cytochrome P-450 enzymes),8 the
radical-cation path (mediated by peroxidase),9 and the
quinone path (mediated by aldo-keto reductase).10
Dibenzo[def,p]chrysene (DBC)11 (Figure 1) is the most
potent PAH carcinogen currently known.12 It is present in
cigarette smoke condensate13 and vehicle exhaust conden-
sate.13 In connection with studies aimed at elucidation of
† University of Chicago.
‡ Center for Cancer Pharmacology.
§ Center for Excellence in Environmental Toxicology.
(1) Straif, K.; Boan, R.; Grosse, Y.; Secretan, B.; Ghissassi, F. E.;
Cogliano, V. Nat. Oncol. 2005, 6, 931-932.
(2) Harvey, R. G. Polycyclic Aromatic Hydrocarbons: Chemistry and
Carcinogenicity; Cambridge University Press: Cambridge, U.K., 1991.
(3) Harvey, R. G. Polycyclic Aromatic Hydrocarbons; Wiley-Inter-
science: New York, 1997; Chapter 1.
(7) World Health Organization. Tobacco or Health: A Global Status
Report; WHO: Geneva, 1997; pp 10-48.
(8) Harvey, R. G. Acc. Chem. Res. 1981, 14, 218-226. Conney, A. H.
Cancer Res. 1082, 42, 4875-4910.
(4) International Agency for Research on Cancer. Polynuclear Aromatic
Compounds, Part 1, Chemical, EnVironmental and Experimental Data;
Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to
Humans, Vol. 32; IARC: Lyon, France, 1983.
(5) International Agency for Research on Cancer. Tobacco Smoke and
InVoluntary Smoking; Monographs on the Evaluation of the Carcinogenic
Risk of Chemicals to Humans, Vol. 83; IARC: Lyon, France, 2004.
(6) Pfiefer, G. P.; Denissenko, M. F.; Olivier, M.; Tretyakova, N.; Hecht,
S.; Hainaut, P. Oncogene 2002, 21, 7435-7451.
(9) Cavalieri, E. L.; Rogan, R. E. Xenobiotica 1995, 25, 677-688.
(10) Palackal, N. T.; Burczynski, M. E.; Harvey, R. G.; Penning, T. M.
Biochemistry 2001, 40, 10901-10910. Palackal, N. T.; Lee, S. H.; Harvey,
R. G.; Blair, I. A.; Penning, T. M. J. Biol. Chem. 2002, 277, 24799-24808.
Penning, T. M.; Ohnishi, S. T.; Ohnishi, T.; Harvey, R. G. Chem. Res.
Toxicol. 1996, 9, 84-92.
(11) The name dibenzo[def,p]chrysene accords with IUPAC nomenclature
rules and Chemical Abstracts, but the older name dibenzo[a,l]pyrene is still
in use. See ref 3 for a condensed version of the rules of PAH nomenclature.
10.1021/ol7029323 CCC: $40.75
© 2008 American Chemical Society
Published on Web 02/20/2008

Scheme 1. Synthesis of 9- and 10-Methylbenzo[g]chrysene
Figure 1. Structures of DBC and its active metabolites.
the role of DBC in human cancer, we required 13C-labeled
analogues of DBC and its active metabolites. These com-
pounds were needed as standards for sensitive methods of
stable isotope dilution liquid chromatography/tandem mass
spectrometric analysis of the metabolites and DNA adducts
of DBC.14,15
Although several syntheses of DBC16 and its active
metabolites17 have been described, they involve multistep
procedures that are not adaptable to synthesis of the13C-
labeled analogues. We now report convenient new syntheses
of DBC and its active carcinogenic metabolites [DBC trans-
11,12-dihydrodiol (1), DBC 11,12-dione (2), and the DBC
anti- and syn-11,12-diol-13,14-epoxides (3)]. Also reported
is synthesis of 13C-labeled DBC (13C-DBC) and the 13C-
labeled analogues of 1-3 (13C-1, 13C-2, and 13C-3) by
modification of this synthetic approach.
furnish a product whose 1H NMR spectrum was more
consistent with the structure of the 10-methyl isomer (7b)
rather than 7a. Particularly revealing was the appearance of
the methyl proton signal at δ 2.82, not shifted to higher field
as expected for a methyl group in a crowded bay region.
1
Also, the H NMR spectrum of 7b matched closely that
reported for this compound.19 Evidently, migration of the
methyl group occurred during or subsequent to cyclodehy-
dration. Methyl migration in acid-catalyzed reactions of
PAHs is well known.20
A brief study of this reaction was undertaken to optimize
conditions for synthesis of 7a. Reaction of 6 with 20% MsOH
at 90 °C for 3 days (Table 1) furnished equal amounts of 7a
Synthesis of DBC. Pd-catalyzed Suzuki coupling of
2-bromophenylacetone (4) with 9-phenanthrylboronic acid
(5) took place smoothly in the presence of Na2CO3 in DME
to provide 2-(9-phenanthryl)phenylacetone (6) (82%) (Scheme
1). Attempted cyclodehydration of 6 to 9-methyl-benzo[g]-
chrysene (7a) failed to take place under the usual conditions
for this type of reaction (20% MSA in CH2Cl2 at room
temperature for 2 days).18 However, reaction took place under
more vigorous conditions (50% MSA at 85-90 °C) to
Table 1. Cyclodehydration of 6
time (h)
temp (°C)
catalysta
7a (%)
7b (%)
48
72
2
48
20
24
30
40
66
rt
20% MSA
20%MSA
50% MSA
Hf(OTf)4
TiCl4
TiCl4
TiCl4
TiCl4
TiCl4
0
20
0
0
20
70
10
0
0
0
76
80
90
90
90
rt
rt
rt
0
45
60
72
6
(12) (a) Higginbotham, S.; RamaKrishna, N. S. V.; Johansson, S. I.;
Rogan, E. G.; Cavalieri, E. L. Carcinogenesis 1993, 14, 875-878. (b)
Cavalieri, E. L.; Higginbotham, S.; RamaKrishna, N. S. V.; Devanesan, P.
D.; Todorovic, R.; Rogan, E. G.; Salmasi, S. Carcinogenesis 1991, 12,
1939-1944. (c) Masuda, Y.; Kagawa, R. Chem. Pharm. Bull. 1972, 20,
2736-2737. (d) Lacassagne, A.; Buu-Hoi, F.; Zajdela, F. A. Naturwissen-
schaften 1968, 55, 43.
rt
rt
0
a MSA ) methanesulfonic acid
(13) Seidel, A.; Frank, H.; Behnke, A.; Schneider, D.; Jacob, J. Polycyclic
Aromat. Compd. 2004, 24, 759-771.
and 7b (40%). Similar reaction of 6 with Hf(OTf)4 as catalyst
gave 7b (10%) as the sole product. Reaction of 6 in the
presence of TiCl4 at room temp for times up to 30 h afforded
mixtures of 7a and unreacted 6 with no detectable 7b. At
(14) Ruan, Q.; Gelhaus, S.; Penning, T. M.; Harvey, R. G.; Blair, I. A.
Chem. Res. Toxicol. 2007, 20, 424-431.
(15) Ruan, Q.; Kim, H.; Jiang, H.; Penning, T. M.; Harvey, R. G.; Blair,
I. A. Rapid Commun. Mass Spectrom. 2006, 20, 1369-1380.
(16) (a) Gill, H. S.; Kole, P. I.; Wiley, J. C.; Li, K. M.; Higginbotham,
S.; Rogan, E. G.; Cavelieri, E. L. Carcinogenesis 1994, 15, 2455-2460.
(b) Luch, A.; Glatt, H.; Platt, K. L.; Oesch, F.; Seidel, A. Carcinogenesis
1994, 15, 2507-2516. (c) Sharma, A. K.; Kumar, S.; Amin, S. J. Org.
Chem. 2004, 69, 3979-3982.
(17) (a) Zhang, J.-T.; Harvey, R. G. Tetrahedron 1999, 55, 625-636.
(b) Krzeminski, J.; Lin, J.-M.; Amin, S.; Hecht, S. S. Chem. Res. Toxicol.
1994, 7, 125-129.
(18) Harvey, R. G.; Pataki, J.; Cortez, C.; DiRaddo, P.; Yang, C. J. Org.
Chem. 1991, 56, 1210-1217.
(19) Tinnemans, A. H. A.; Laarhoven, W. H. J. Am. Chem. Soc. 1974,
96, 4617-4622.
(20) Lindow, D. F.; Harvey, R. G. J. Am. Chem. Soc. 1971, 93, 3786-
3787. Harvey, R. G.; Halonen, M. Can. J Chem. 1967, 45, 2630-2632.
1060
Org. Lett., Vol. 10, No. 6, 2008

30 h, the yield of 7a was 72%, and 10% of 6 was recovered.
With increasing times, the ratio of 6 continued to decrease,
but the ratio of 7a also began to decrease. At the same time,
the ratio of 7b increased, and by 66 h, it was the sole product.
The most practical strategy for synthesis of 7a was to quench
reaction while a small amount of 6 remained and 7b was
not yet detectable (∼30 h), taking advantage of the fact that
separation of 6 from 7a is easy, while separation of 7a from
7b is difficult.
Scheme 3. Syntheses of 13C-DBCa
Transformation of 7a to DBC was carried out via the
sequence in Scheme 2. Bromination of 7a with NBS gave
Scheme 2. Synthesis of Dibenzo[def,p]chrysene
9-bromomethylbenzo[g]chrysene (8a), which was converted
to 9-formylbenzo[g]chrysene (8b) by treatment with DMSO
and NaHCO3.21 Compound 8a was somewhat unstable, but
satisfactory yields of 8b were obtainable by use of 8a directly
without purification. Wittig reaction of 8b with methoxy-
methylenetriphenylphosphine afforded 9-(2-methoxy-vinyl)-
benzo[g]chrysene (9) as a mixture of the E- and Z-isomers
(1:1 by NMR) (90%). Acid-catalyzed cyclization of 922
furnished DBC (75%).
a a: R ) H; b: R ) OMe.
Wittig reaction of 13C2-8b with 13C-labeled MeOCHdPPh3
followed by acid-catalyzed cyclization to yield 13C3-DBC.
A similar procedure was employed for introduction of 13C
in synthesis of 13C2-benzo[a]pyrene.25
Synthesis of Active Metabolites of DBC (1-3) and
their 13C2-Labeled Analogues. The active metabolites of
DBC were synthesized by modification of the method
employed for synthesis of 13C2-DBC (Scheme 4). The initial
synthetic target was 10-hydroxy-DBC (19b), previously
shown to be a synthetic precursor of all three metabolites
(1-3).16,17
Cross-coupling of 2-bromo-5-methoxybenzaldehyde with
phenanthrene-9-boronic acid (5) provided 2-(9-phenanthryl)-
5-methoxybenzaldehyde (10b) (62%). Wittig reaction of 10b
with ethylenetriphenylphosphine (generated from reaction of
EtPPh3Br with t-BuOK) provided 13 (89%) as a mixture of
E- and Z- isomers. Epoxidation of 13 with Oxone/acetone24
furnished 14 (89%), which was converted to 2-(9-phenan-
thryl)-5-methoxyphenylacetone (15) (70%) on stirring with
Hf(OTf)4 for 15 min at ambient temp.
TiCl4-catalyzed cyclodehydration of 15 was monitored by
TLC to determine the optimum time for minimization of
rearrangement of 9-methyl-12-methoxybenzo[g]chrysene (16)
to the 10-methyl isomer. The optimum time was only ∼3 h
(compared to ∼30 h for analogous reaction of 6). The
increased rate of cyclization is likely a consequence of
stabiization of the reaction intermediate by the electron-
donating methoxy group. Bromination of 16 with NBS
Synthesis of 13C2-DBC. Because13C-labeled 2-bromo-
phenylacetone (4) was unavailable, this synthesis was carried
out by a modified procedure (Scheme 3). The starting
compound, 2-(9-phenanthryl)benzaldehyde (10a), was pre-
pared by Pd-catalyzed coupling of 2-formylphenyl-boronic
acid with 9-bromophenanthrene. Wittig reaction of 10a with
13C2-EtPPh3Br23 and t-BuOK gave 11 as a mixture of E-
and Z-isomers (8:1 by NMR analysis). Epoxidation of 11
with Oxone/acetone24 gave 12 (87%), and it underwent
rearrangement in the presence of Hf(OTf)4 to furnish13C2-
2-(9-phenanthryl)phenylacetone (13C2-6) (74%). Cyclization
of 13C2-6 with TiCl4 by the procedure used for synthesis of
7a gave13C2-7a, and this was converted to13C2-DBC by the
sequence for DBC.
Should a higher level of 13C-labeling be desired, an
additional 13C-atom may be incorporated into 13C-DBC via
(21) Chen, H.; Luzy, J.-P.; Gresh, N.; Garbay, C. Eur. J. Chem. 2006,
2329-2335.
(22) Harvey, R. G.; Lim, K.; Dai, Q. J. Org. Chem. 2004, 69, 1372-
1373. Harvey, R. G.; Dai, Q.; Ran, C.; Penning, T. M. J. Org. Chem. 2004,
69, 2024-2032.
(23) 13C2-EtPPh3Br was prepared by addition of a solution of PPh3 (4.77
g, 18.2 mmol) in toluene (8 mL) to 13C2-bromoethane (1 g, 9.1 mmol).
The mixture was stirred at 120 °C for 2 days and then cooled to room
temperature. The precipitate was filtered, washed with cold benzene (2 ×
10 mL), and dried under vacuum to afford 13C2-EtPPh3Br.
(24) Hashimoto, N.; Kanda, A. Org. Process Res. DeV. 2002, 6, 405.
(25) Harvey, R. G.; Dai, Q.; Ran, C.; Lim, K.; Blair, I.; Penning, T.
Polycyclic Aromat. Compd. 2005, 25, 371-391.
Org. Lett., Vol. 10, No. 6, 2008
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by reaction with DMSO and NaHCO3. Wittig reaction of
17 with methoxymethylenetriphenylphosphine provided 18
as a mixture of E- and Z-isomers that were used directly in
the next step. Cyclization of 18 took place at 0 °C in the
presence of MSA to furnish 12-methoxy-DBC (19a). Dem-
ethylation of 19a by treatment with BBr3 provided 12-
hydroxy-DBC (19b) in good overall yield.
Scheme 4. Synthesis of 12-Hydroxy-DBC (19b)
Syntheses of the active carcinogenic metabolites of DBC
(1-3) from 19b were carried out by the procedures described
previously.16,17 Thus, oxidation of 19b with Fremy’s reagent
provided DBC 11,12-dione (2), and reduction of 2 with
NaBH4 afforded DBC trans-11,12-dihydrodiol (1). The latter
was, in turn, converted to the corresponding anti- and syn-
11,12-diol-13,14-epoxide isomers (3) by the established
procedures.17
The 13C2-labeled analogues of the active carcinogenic
metabolites of DBC (13C-1,13C-2, and13C-3) were synthesized
from13C2-19b by procedures analogous to those employed
for synthesis of the corresponding unlabeled compounds.13C2-
19b was prepared from 10b via Wittig reaction with13C2-
EtPPh3Br and subsequent steps analogous to those described
for synthesis of unlabeled 19b from 10b (Scheme 4).
Acknowledgment. This investigation was supported by
NIH Grants (P01 CA 92537, R01 CA 039504, R01 ES
015857, and P30 ES 013508).
Supporting Information Available: Experimental details
and spectroscopic data. This material is available free of
charge via the Internet at http://pubs.acs.org.
afforded 9-bromomethyl-12-methoxybenzo[g]chrysene which
was converted to 9-formyl-12-methoxybenzo[g]chrysene (17)
OL7029323
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Org. Lett., Vol. 10, No. 6, 2008