Synthesis of 13C2-benzo[a]pyrene and its 7,8-dihydrodiol and 7,8-dione implicated as carcinogenic metabolites

Article abstract of DOI:10.1016/j.tetlet.2008.05.033

Synthesis of the ¹³C₂-labelled analogues of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene and its active metabolites is described. The method entails Pd-catalyzed Suzuki-Miyaura coupling of a naphthalene boronic aci

Full text of DOI:10.1016/j.tetlet.2008.05.033

Tetrahedron Letters 49 (2008) 4531–4533
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of 13C2-benzo[a]pyrene and its 7,8-dihydrodiol and 7,8-dione
implicated as carcinogenic metabolites
Chongzhao Ran a, Daiwang Xu a, Qing Dai a, Trevor M. Penning b,c, Ian A. Blair b,c, Ronald G. Harvey a,
*
a Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637, United States
b Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, United States
c Center for Excellence in Environmental Toxicology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, United States
a r t i c l e i n f o
a b s t r a c t
Synthesis of the 13C2-labelled analogues of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]-
pyrene and its active metabolites is described. The method entails Pd-catalyzed Suzuki–Miyaura coupling
of a naphthalene boronic acid with 2-bromobenzene-1,3-dialdehyde followed by Wittig reaction of the
product with 13CH2@PPh3.
Article history:
Received 24 April 2008
Revised 6 May 2008
Accepted 7 May 2008
Available online 11 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Several advances have been made recently in understanding
PAH activation in human cells susceptible to tumourigenesis. An
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous envi-
ronmental pollutants that are produced in combustion of fossil
fuels and other organic matter.1–3 They occur commonly in auto
and diesel engine exhaust,4 tobacco smoke5–7 and smoked and
charbroiled meats.1–3 PAHs have been designated as human carcin-
ogens by the WHO,2 and they are implicated in the causation of hu-
man lung cancer.5–10
Benzo[a]pyrene (BP) is the prototype PAH carcinogen. It is enzy-
matically activated to metabolites that react with DNA leading to
mutations. The most studied activation path involves cytochrome
P-450 [CYP] mediated formation of a dihydrodiol (BP-7,8-diol) fol-
lowed by its oxidation to a diol epoxide (BPDE).3,11 A second path-
way entails aldo-keto reductase-mediated oxidation of BP-7,8-diol
to a catechol that enters into a redox cycle with O2 to form a qui-
none (BP 7,8-dione) and reactive oxygen species (ROS) that attack
DNA.12,13 A third pathway has also been proposed that entails per-
oxidase-mediated oxidation of BP to a radical-cation that reacts
with DNA to form depurinated adducts.14,15 However, the relative
importance of these pathways for human cancer is not certain.
investigation of metabolic activation of BP in human lung A549
cells has provided evidence that the AKR-mediated pathway to
generate the BP quinone and ROS is operative in these cells.16
And human bronchoalveolar H358 cells were recently introduced
as a model for study of PAH metabolism in normal human lung
cells.17,18 In this connection, development of a stable isotope dilu-
tion liquid chromatography tandem mass spectrometric method
for analysis of the metabolites of BP and its nucleoside adducts
was also described.19 In order to improve the scope and sensitivity
of this methodology, 13C-labelled analogues of BP and its active
metabolites with at least two 13C-atoms in the aromatic ring sys-
tem are needed as authentic standards.
2. Results and discussion
We now report synthesis of 13C2-benzo[a]pyrene (13C2-BP),
13C2-BP-7,8-diol, and 13C2-BP-7,8-dione required as standards for
LC–MS/MS analysis of the patterns of BP metabolism and DNA ad-
duct formation in human cells. Syntheses of all twelve of the
mono-13C-labelled isomers of BP with a 13C-atom at each of the
peripheral carbon atoms of the BP ring system were previously re-
ported.20 However, the classical synthetic methods employed were
not adaptable to preparation of the 13C2-labelled BP analogues.
Synthesis of 13C2-BP was carried out by a modified version of
the new synthetic approach to BP recently reported.21b This meth-
od involves Pd-catalyzed Suzuki–Miyaura coupling of naphthalene
2-boronic acid (1a) with 2-bromobenzene-1,3-dialdehyde (2) to
provide a dialdehyde product (3a) (Scheme 1). In the original ap-
proach the next step entailed double Wittig reaction of 3a with
(methoxymethylene)triphenylphosphine (CH3OCH@PPh3)22 to
provide the unlabelled di(methoxyvinyl compound (4b). However,
the 13C-labelled methoxymethyl halide (CH3O13CH2X) needed as
O
HO
O
HO
OH
OH
O
BPDE
BP-7,8-diol
BP-7,8-dione
* Corresponding author. Tel.: +1 773 702 6998; fax: +1 773 702 3184.
E-mail address: rharvey@huggins.bsd.uchicago.edu (R. G. Harvey).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.05.033

4532
C. Ran et al. / Tetrahedron Letters 49 (2008) 4531–4533
t-BuOK
13CH2=PPh3
13CH3PPh3
13CH3X + PPh3
X
OHC
OHC
Br
Pd(PPh3)4
Na2CO3
13CH2=PPh3
B(OH)2
+
CHO
R
R
CHO
1a: R = H
b: R = OMe
3a: R = H
b: R = MeO
2
O
R'
[O]
acid
catalyst
R
R
R
O
R'
6a: R = H (13C2-BP)
b: R = MeO
c: R = HO
5a: R = H
b: R = MeO
4a: R = R' = H
b: R = H; R' = MeO
c: R = MeO; R = H
Scheme 1.
the starting compound for preparation of CH3O13CH@PPh3 was
unavailable. Fortunately, 13CH3I (99 atom%) was readily obtainable,
and it was used for preparation of 13C-methylene)triphenylphos-
phine (13CH2@PPh3) by reaction with PPh3 followed by treatment
of the resulting phosphonium salt with n-BuLi.23 Double Wittig
reaction of 13CH2@PPh3 with 3a afforded a diolefin (4a) with 13C-
atoms in both vinyl groups.24 Although the di(methoxyvinyl) ana-
logue (4b) was found previously to undergo cyclization smoothly
in the presence of an acid catalyst,21a analogous reaction of 4a gave
principally polymeric products. This difficulty was avoided by con-
version of 4a to the bis-epoxide (5a) by treatment with Oxone/ace-
tone followed by acid-catalyzed cyclization and dehydration to
provide 13C2-BP (6a).25 The 1H NMR spectrum of 6a was in good
agreement with that of unlabelled BP. Its 13C NMR spectrum exhib-
ited characteristic peaks at d 122.1 and 128.1, confirming the pres-
ence of 13C atoms at the C-5 and C-11-positions of the BP ring
system.
Because the yield of 6a was found to be dependent upon reac-
tion conditions, a brief study of the influence of conditions on yield
was undertaken using unlabelled 5a. The findings are summarized
in Table 1. Significantly higher yields were obtained from reactions
carried out at 70 °C than from those conducted at ambient temper-
ature. The optimum yield of BP (94%) was obtained from reaction
of 5a at 70 °C with InCl3 (5%) as the catalyst. Good yields of BP were
also obtained from reactions carried out with Hf(OTf)4 and
methanesulfonic acid catalysts (84% and 80%, respectively).
13C2-8-Methoxy-BP (6b), needed as the starting compound for
synthesis of the oxidized metabolites of BP, was prepared by an
analogous sequence (Scheme 1). The dialdehyde 3b needed for this
purpose was prepared by Pd-catalyzed Suzuki coupling of 6-
methoxynaphthalene-2-boronic acid (1b) with 2, as previously de-
scribed.21a Reaction of 3b with 13CH2@PPh3 furnished 13C2-2-meth-
oxy-6-(2,6-divinylphenyl)naphthalene (4b). The 1H NMR spectrum
of 4b was in good agreement with that of its unlabelled analogue
4a,21a and its 13C NMR spectrum exhibited a peak at d 114.6, con-
firming the presence of 13C-atoms in the vinyl groups. Compound
4b was converted to the bis-epoxide (5b) by treatment with Ox-
one/acetone.25 Cyclization of 5b took place smoothly in the pres-
ence InCl3 at 70 °C to provide 13C2-8-MeO-BP (6b). The 1H NMR
spectrum of 6b was in good agreement with this structural assign-
ment.21a Its 13C NMR spectrum contained peaks at d 122.0 and
128.1, closely similar to the signals found for 13C-BP with 13C in
the C-5 and C-11-positions. Demethylation of 6b with HI/HOAc
took place smoothly to furnish 13C2-8-HO-BP (6c) (97%).26
Table 1
Effects of reaction variables on cyclization of 5a to 6a
Catalysta
25 °C
70 °C
Time (h)
Yield (%)
Time (h)
Yield (%)
Hf(OTf)4
Cu(OTf)2
In(OTf)3
Ce(OTf)4
InCl3
BiOClO4
ZnCl4
MSA
12
12
12
12
12
12
12
12
52
1.5
3.0
3.0
1.5
3.0
6.0
6.0
3.0
84
31
46
51
<10
<10
13
<10
<10
<10
<10
The 13C2-labelled oxidized metabolites of 13C2-BP were synthe-
sized from 6c via procedures analogous to those employed for syn-
thesis of the unlabelled analogues (Scheme 2). Thus, oxidation of
6c with o-iodoxybenzoic acid (IBX) in DMF21a took place smoothly
to furnish the o-quinone, 13C2-BP-7,8-dione (80%).27 Reduction of
94
<10
<10
80
a
The molar ratio of the catalyst used was 5% of 5a. MSA = methanesulfonic acid.
NaBH4
IBX
O2
HO
O
HO
O
HO
6c
13C2-BP-7,8-dione
13C2-BP-7,8-diol
Scheme 2.

C. Ran et al. / Tetrahedron Letters 49 (2008) 4531–4533
4533
13C2-BP-7,8-dione with NaBH4/O2
dihydro-7,8-dihydroxybenzo[a]pyrene
furnished 13C2-trans-7,8-
13C2-BP-7,8-diol).29 This
21a,28
16. Park, J.; Mangal, D.; Tacka, K. A.; Quinn, A. M.; Harvey, R. G.; Blair, I. A.; Penning,
T. M. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 6846–6851.
17. Jiang, H.; Gelhaus, S. L.; Mangal, D.; Harvey, R. G.; Blair, I. A.; Penning, T. M.
Chem. Res. Toxicol. 2007, 20, 1331–1341.
18. Ruan, Q.; Gelhaus, S.; Penning, T. M.; Harvey, R. G.; Blair, I. A. Chem. Res. Toxicol.
2007, 2, 424–431.
19. Ruan, Q.; Kim, H. Y. H.; Jiang, H.; Penning, T. M.; Harvey, R. G.; Blair, I. A. Rapid
Commun. Mass Spectrom. 2006, 20, 1369–1380.
20. Bodine, R. S.; Hylarides, M.; Daub, G. H.; VanderJagt, D. L. J. Org. Chem. 1978, 43,
4025–4028; Simpson, J. E.; Daub, G. H.; VanderJagt, D. L. J. Labelled Compd.
Radiopharm. 1980, 17, 895; Klassen, S. E.; Daub, G. H.; VanderJagt, D. L. J. Org.
Chem. 1983, 48, 4361–4366; Bodine, R. S.; Daub, G. H.; VanderJagt, D. L. J.
Labelled Compd. Radiopharm. 1984, 22, 475–485.
21. (a) Harvey, R. G.; Dai, Q.; Ran, C.; Penning, T. M. J. Org. Chem. 2004, 69, 2024–
2032; (b) Harvey, R. G.; Lim, K.; Dai, Q. J. Org. Chem. 2004, 69, 1372–1373.
22. CH3OCH@PPh3 was prepared by reaction of CH3OCH2Br with PPh3 and
treatment of the resulting phosphonium salt with t-BuOK (Ref. 21a).
23. 13CH2@PPh3 was prepared via reaction of 13CH3I (99 atom%) with PPh3 and
treatment of the phosphonium salt with n-BuLi. Thus, to a suspension of
13CH3PPh3I (2.0 g, 4.9 mmol) in anhydrous THF (20 mL) at À78 °C under argon
was added a solution of n-BuLi (2.0 mL of a 2.5 M solution in THF, 5.0 mmol).
The solution was stirred at À78 °C for 20 min, then the cooling bath was
removed, and stirring was continued for an additional 30 min.
(
dihydrodiol may be readily converted to the 13C2-anti- and syn-diol
epoxide isomers by the procedures previously described for syn-
thesis of the corresponding unlabelled compounds.30
The syntheses described in preceding paragraphs provide con-
venient access to the 13C2-labelled analogues of BP and its pre-
sumed carcinogenic metabolites. Synthesis of the 13C2-labelled
analogues of the potent carcinogenic PAH dibenzo[def,p]chrysene
and its corresponding active metabolites by a different synthetic
approach was recently reported by us.31 In principle, these meth-
ods are potentially applicable to synthesis of 13C2-labelled ana-
logues of a wide range of other PAH carcinogens and their
oxidized metabolites.
Acknowledgement
This investigation was supported by NIH Grants (P01 CA 92537,
R01 CA 039504, R01 ES 015857 and P30 ES 013508).
24. 13C2-2-(2,6-divinylphenyl)naphthalene (4a) was synthesized by reaction of 3a
with 13CH2@PPh3 prepared as described above. To the solution of 13CH2@PPh3
was added a solution of 3a (0.70 g, 2.45 mmol) in THF (10 mL), and the solution
was stirred for 30 min when TLC indicated reaction to be complete.
Conventional work-up and flash chromatography on silica gel eluted with
hexane/EtOAc (15:1) gave 4a (0.68 g, 98%) as a colourless oil; the 1H NMR
spectrum was in agreement with that for unlabelled 4a; 13C NMR (CDCl3) d
114.7. This compound was unstable and deteriorated on standing.
25. 13C2-Benzo[a]pyrene (6a) was synthesized from 4a via conversion to the bis-
References and notes
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epoxide (5a) and acid-catalyzed cyclization. To
a solution of 4a (0.29 g,
1.0 mmol) in EtOAc/acetone/H2O (20:10:10) was added NaHCO3 (0.63 g,
7.5 mmol), and to this solution was added dropwise a solution of Oxone
(5.4 g, 9.0 mmol) in (30 mL) over a 4 h period. Stirring was continued for an
additional 3 h, then the solvent was evaporated. The product was dissolved in
EtOAc and purified by flash chromatography to provide 5a (290 mg). This was
dissolved in CHCl3 (7.2 mL), InCl3 (11 mg) was added, and the mixture was
heated at reflux for 12 h. Flash chromatography of the product provided 6a
(70%); the 1H NMR spectrum was in generally good agreement with that of
unlabelled BP; 13C NMR (CDCl3) d 122.1 and 128.1.
26. A suspension of 6b (40 mg, 0.14 mmol) in 57% HI (5 mL) and HOAc (5 mL) was
stirred at 140 °C until TLC showed reaction to be complete (1.5 h). Then the
solution was cooled to room temperature, and poured into ice water (50 mL) to
afford 6c (37 mg, 97%) which was used directly in the next step.
27. The 13C NMR spectrum of 13C2-BP-7,8-dione exhibited characteristic signals at
d 122.9 and 128.8, corresponding to the 13C-atoms in the C-5 and C-11-
positions.
28. Sukumaran, K. B.; Harvey, R. G. J. Org. Chem. 1980, 45, 4407–4413.
29. The 13C NMR spectrum of 13C2-BP-7,8-diol showed signals at d 122.5 and 127.6
corresponding to the 13C-atoms in the C-5 and C-11-positions.
30. Harvey, R. G. In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R. G., Ed.;
ACS Symp. Series, No. 283; Amer. Chem. Soc.: Washington, DC, 1985; pp 35–62.
31. Xu, D.; Duan, Y.; Blair, I. A.; Penning, T. M.; Harvey, R. G. Org. Lett. 2008, 10,
1058–1062.