Synthesis of ¹³C₂-Benzo[a]pyrene and its 7,8-Dihydrodiol and 7,8-Dione Implicated as Carcinogenic Metabolites

Abstract

Synthesis of the ¹³C₂-labelled analogues of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene and its active metabolites are 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.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental 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 smoke,^6–8 and smoked and charbroiled meats.^1–3 PAHs have been designated as human carcinogens by the WHO,² and they are implicated in the causation of human lung cancer.^6,9,10

Benzo[a]pyrene (BP) is the prototype PAH carcinogen. It is enzymatically 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) followed by its oxidation to a diol epoxide (BPDE).^3,11 A second pathway 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 quinone (BP 7,8-dione) and reactive oxygen species (ROS) that attack DNA.^12,13 A third pathway has also been proposed that entails peroxidase-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.

Several advances have been made recently in understanding PAH activation in human cells susceptible to tumorigenesis. An 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 dilution 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 system are needed as authentic standards.

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 adduct 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 reported.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 method 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 approach 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 methoxymethy halide (CH₃O¹³CH₂X) needed as 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)triphenylphosphine (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) analogue (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 conversion of 4a to the bis-epoxide (5a) by treatment with Oxone/acetone 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 exhibited characteristic peaks at δ 122.1 and 128.1, confirming the presence of ¹³C atoms at the C-5 and C-11-positions of the BP ring system.

Scheme 1.

Because the yield of 6a was found to be dependent upon reaction 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 temperature. 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).

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	12	52	1.5	84
Cu(OTf)2	12	<10	3.0	31
In(OTf)3	12	<10	3.0	46
Ce(OTf)4	12	13	1.5	51
InCl3	12	<10	3.0	94
BiOClO4	12	<10	6.0	<10
ZnCl4	12	<10	6.0	<10
MSA	12	<10	3.0	80

^aThe molar ratio of the catalyst used was 5% of 5a. MSA = methanesulfonic acid.

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 described.^21a Reaction of 3b with ¹³CH₂=PPh₃ furnished ¹³C₂-2-methoxy-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 δ 114.6, confirming the presence of ¹³C-atoms in the vinyl groups. Compound 4b was converted to the bis-epoxide (5b) by treatment with Oxone/acetone.²⁵ Cyclization of 5b took place smoothly in the presence InCl₃ at 70 °C to provide ¹³C₂-8-MeO-BP (6b). The ¹H NMR spectrum of 6b was in good agreement with this structural assignment.^21a Its ¹³C NMR spectrum contained peaks at δ 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

The ¹³C₂-labelled oxidized metabolites of ¹³C₂-BP were synthesized from 6c via procedures analogous to those employed for synthesis of the unlabelled analogues (Scheme 2). Thus, oxidation of 6c with o-iodoxybenzoic acid (IBX) in DMF^21a took place smoothly to furnish the o-quinone, ¹³C₂-BP-7,8-dione (80 %).²⁷ Reduction of ¹³C₂-BP-7,8-dione with NaBH₄/O₂^{21a, 28} furnished ¹³C₂-trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (¹³C₂-BP- 7,8-diol).²⁹ This dihydrodiol may be readily converted to the ¹³C₂-anti- and syn-diol epoxide isomers by the procedures previously described for synthesis of the corresponding unlabelled compounds.³⁰

Scheme 2.

The syntheses described in preceding paragraphs provide convenient access to the ¹³C₂-labelled analogues of BP and its presumed 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 methods are potentially applicable to synthesis of ¹³C₂-labelled analogues of a wide range of other PAH carcinogens and their oxidized metabolites.