A New Strategy for the Synthesis of Polychlorinated Biphenyl

neoplastic nodules, and hepatocellular carcinoma) (1,2). Chlorobiphenyls are potential ... Abbreviations: PCB, polychlorinated biphenyl; BSTFA, bis(tr...
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Chem. Res. Toxicol. 1995, 8, 92-95

92

A New Strategy for the Synthesis of Polychlorinated Biphenyl Metabolites Udo Bauer, Anthony R. Amaro, and Larry W. Robertson* Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40506-0054 Received June 28, 1994@

A general and high-yielding synthetic route is presented by which a variety of chlorinated dihydroxybiphenyl metabolites may be synthesized. These synthetic standards will permit the investigation of novel pathways of polychlorinated biphenyl activation. The biphenyldiols were synthesized via palladium-catalyzed cross-coupling of dimethoxyphenylboronic acids with aryl bromides in the presence of a base. The formed dimethoxybiphenyls were demethylated using boron tribromide to yield the chlorinated dihydroxybiphenyls. These compounds were characterized by IR, NMR, and GC/MS.

Introduction Polychlorinated biphenyls (PCBs)l are abundant and persistent pollutants in the global ecosystem. Various toxic effects have been attributed to individual chlorobiphenyls, commercial PCB mixtures, and environmental residues. These toxic effects include immune suppression, endocrine disruption, nervous system effects, and especially hepatic effects (enzyme iqduction,hypertrophy, neoplastic nodules, and hepatocellular carcinoma) (1,2). Chlorobiphenyls are potential substrates for cytochrome P450 (3-15). Chlorinated biphenyls with fewer chlorine atoms are generally more rapidly metabolized (3,8,11,12), but tetra-, penta-, and hexachlorobiphenyls also undergo significant metabolism (7,8,11-15).Arene oxides have been implicated as reactive metabolic intermediates which can react with cellular macromolecules, resulting in toxicity, but other mechanisms of activation such as oxygen addition and rearrangement may also be operative (6-8, 1I). While monohydroxylated chlorobiphenyls are generally the predominant metabolites, catechols have also been identified as microsomal oxidation products (3,8-11).Hepatic microsomes very rapidly yielded at least 9 polar metabolites from 2,2',5-trichlorobiphenyl, while fewer metabolites of 2,2',5,5'-tetrachlorobiphenyl and 2,2',4,5,5'-pentachlorobiphenylwere more slowly formed (12). However, in extended incubations, the monohydroxylated metabolites of the trichlorobiphenyl were greatly reduced, presumably having been further metabolized (13). In vivo studies of tri-,penta-, and hexachlorobiphenyls in rats provided further evidence that PCBs are metabolized to catechols (14,15). Chlorinated monohydroxybiphenyls are relatively potent xenoestrogens (16),but the effects of chlorinated dihydroxybiphenyls, which may also be generated in extrahepatic tissues from primary hepatic products, are unknown. The carcinogenicity of catecholestrogens generated in extrahepatic tissues appears to be related to redox cycling and oxidative stress (17). The catechols of chlorobiphenyls could similarly cycle through quinones * Address correspondence to this author at the Graduate Center for Toxicology, 206 Funkhouser Building, University of Kentucky, Lexington, KY 40506-0054. Telephone: (606) 257-3952; Fax: (606) 3231059; E-Mail [email protected]. @Abstractpublished in Advance ACS Abstracts, November 15,1994. Abbreviations: PCB, polychlorinated biphenyl; BSTFA, bis(trimethylsilv1)trifluoroacetamide:TEA, triethylamine: DMF, dimethylformimide.

Scheme 1

1

2

3

to produce oxidative stress and/or bind to cellular macromolecules (18,19).Preliminary data from our laboratory show that mono- and dichlorobiphenyls may be metabolically activated to intermediates which form several adducts with 2'-deoxyguanine 3'-monophosphate (20). In order to further study the metabolic formation and toxic properties of chlorinated dihydroxybiphenyls, we have developed a method to synthesize various monoand dihydroxybiphenyls. The synthesis of biaryls is a problem familiar to many organic chemists and in general of primary interest due to the fact that the biaryl axis is the central building block in a very large number of natural products (21). Synthetic approaches to unsymmetrical biaryls are based on classical Pschorr, Ullmann, and Gomberg-Bachmann methodologies (22);however, these classical procedures are accompanied by disadvantages such as low regioselectivity, very high reaction temperatures, or modest yields. In recent years novel and useful procedures for the construction of unsymmetrical biaryls have appeared. Among these, the cross-coupling tactic, which employs ArMgX, ArZnX, and ArB(0H)z intermediates, has become an important synthetic methodology (23-25). We wish to report here a new and general synthetic route leading to a variety of mono- and dichlorinated dihydroxybiphenyls. The reaction employs palladium-catalyzed crosscoupling of boronic acids with aryl bromides and subsequent demethylation. The synthetic approach is outlined in Scheme 1.

Ewerimental Section All experiments were carried out under a nitrogen atmosphere. "he IR spectra were obtained using a Perkin-Elmer 1600 FT-IR spectrophotometer. "he lH NMR spectra were recorded on a Varian VXR-400s spectrometer by using CDzClz and acetone-& (Aldrich Chemical Co., Milwaukee, WI) as solvents and internal standards. GC/MS spectra were recorded

0893-228d95/2708-0092$09.00/00 1995 American Chemical Society

Synthesis of Dihydroxylated PCB Metabolites on a Finnigan INCOS 50 and Kratos Concept 1H by using a fused silica capillary column (DB-5MS 15 m x 0.25 mm and OV-1, 25 m, J&W Scientific, Folsom, CA). For GCMS all dihydroxybiphenyls were silylated with BSTFNpyridine (1:l). The melting points were determined on a MEL-TEMP apparatus and are uncorrected. Preparative thin-layer chromatography was carried out on Whatman silica gel glass-backed plates of 1-mm thickness (Fisher Scientific,Pittsburgh, PA). All anhydrous solvents were obtained from Aldrich Chemical Co. (Milwaukee, WI) and were used without further purification. The boronic acids (1)were prepared by the published procedures (26). Caution: Synthetic PCBs and metabolites should be consideredpotentially toxic and hazardous and therefore should be handled in an appropriate manner. GeneralProcedure for the Palladium-CatalyzedCoupling of Arylboronic Acids with Bromobenzenes. To a stirred solution of bromobenzene (2)(10 mmol) and Pd(PPhd4 in 20 mL of toluene under nitrogen atmosphere were (0.3 "01) added 10 mL of a 2 M aqueous solution of NazC03 and 11mmol of arylboronic acid (1) in 5 mL of ethanol. The mixture was stirred vigorously and warmed to 80 "C for 6 h, cooled, and then treated with 30% H202 (1mL) for 2 h. This mixture was diluted with 20 mL of diethyl ether and was extracted with 2 N NaOH (10 mL) and water (20 mL x 2); the organic phase was dried over MgSO4 and concentrated under reduced pressure. 3-Chloro-3',4'-dimethoxybiphenyl(3a).2.0 g (79%yield), colorless oil; TLC: Rf=0.5 (diethyl etherlpetroleum ether, 1:3); IR (neat): 3062,2999,2955,2834,1596,1562,1518,1473,1251, 1218,1172,1144,1027 cm-l; lH NMR (400 MHz, CDzCb): 6 = 3.87, 3.90 (29, 6H, OC&), 6.95 (d, J = 8.2 Hz, lH, 5'-H), 7.09 (d, J = 2.2 Hz, lH, 2'-H), 7.14 (dd, J = 8.2, 2.2 Hz, lH, 6'-H), 7.29 (ddd, J = 8.0, 2.0, 1.2 Hz, lH, 4-H), 7.36 (dt, J = 8.0, 0.4 Hz, lH, 5-H), 7.47 (ddd, J = 7.8, 1.8, 1.2 Hz, lH, 6-H), 7.57 (mc, J 2.0, 0.5 Hz, lH, 2-H); MS (70 eV): m l z (%) = 248 (100) [M+l, 233 (42) [M+ - CH31,205 (34) [M+ - CzH30). 3,4-Dichloro-3,4'-dimethoxybiphenyl (3b). 2.3 g (82% yield), white solid from diethyl etherlpetroleum ether, 1:5; mp 93-94 "C; IR (KBr): 3056,2989,2933,2833,1608,1586,1517, 1467,1248,1135,1025cm-l; lH NMR (400 MHz, CDzClz): 6 = 3.87, 3.90 (29, 6H, OCHs), 6.94 (d, J = 8.4 Hz, lH, 5'-H), 7.05 (d, J = 2.0 Hz, lH, 2'-H), 7.12 (dd, J = 8.4, 2.0 Hz, lH, 6'-H), 7.42 (dd, J = 8.4, 2.4 Hz, lH, 6-H), 7.49 (d, J = 8.0 Hz, lH, 5-H), 7.67 (d, J = 2.4 Hz, lH, 2-H); MS (70 eV): m l z (%) = 282 (100) [M+l, 267 (45) [M+ - CH31,239 (27) [M+ - CzH301, 204 (47) [M+ - CzH3C101. 3,5-Dichloro-3',4'-dimethoxybiphenyl(34. 2.2 g (78% yield), white solid from diethyl ether; mp 106-107 "C; IR (KBr): 3061, 2939, 2839, 1592, 1558, 1514, 1253, 1025 cm-'; 'H NMR (400 MHz, CDZClz): 6 = 3.87, 3.90 ( 2 ~6H, , OCH3), 6.94 (d, J = 8.4 Hz, lH, 5'-H), 7.05 (d, J = 2.0 Hz, lH, 2'-H), 7.12 (dd, J = 8.4, 2.0 Hz, lH, 6'-H), 7.30 (t, J = 2.0 Hz, lH, 4-H), 7.46 (d, J = 2.0 Hz, 2H, 2,6-H); MS (70 eV): mlz (%) = 282 (100) [M+], 267 (42) [M' - CH31, 239 (22) [M+ - CzH301, 204 (30) [M+ - CzH3C101. 3,6-Dichloro-2,6'-dimethoxybiphenyl (3d). 2.2 g (79% yield), white solid from diethyl etheripetroleum ether, 1:5; mp 102-103 "C; IR (KBr): 3056, 3011, 2956, 2922, 2833, 1583, 1558,1503,1218,1040cm-l; lH NMR (400 MHz, CDZC12): 6 = 3.76, 3.78 (28, 6H, OCH3), 6.85 (d, J = 3.0 Hz, lH, 6'-H), 6.89 (dd, J = 8.9, 3.0 Hz, lH, 4'-H), 6.94 (d, J = 8.9 Hz, lH, 3'-H), 7.33 (t, J = 1.9 Hz, lH, 4-H), 7.44 (d, J = 1.9 Hz, 2H, 2,6-H); MS (70 ev): mlz (%) = 282 (58) [M+l, 267 (23) [M+ - CH31, 232 (100) [M+ - CH3C1), 217 (28) [M+ - CzHsCl]. 3-Chloro-2,3'-dimethoxybiphenyl(3e).1.5 g (62%yield), colorless oil; TLC: Rf= 0.9 (diethyl etherlpetroleum ether, 1:2); IR (neat): 3065,2997,2934,2834,1588,1564,1467,1262,1229, 1122, 1034, 1006 cm-'; 'H NMR (400 MHz, CDzC12): 6 = 3.60, 3.90 ( 2 ~6H, , OCH3), 6.91 (dd, J = 7.9, 1.5 Hz, lH, 4'-H), 6.97 (dd, J = 7.9, 1.5 Hz, lH, W-H), 7.12 (t, 7.9 Hz, lH, 5'-H), 7.35 (m,2H, 4,5-H), 7.43 (dt, J = 7.2, 1.7 Hz, lH, 6-H), 7.53 (a, J 1.8,0.5 Hz, lH, 2-H); MS (70 eV): mlz (%) = 248 (95) [M+l, 233 (37) [M+ - CH31, 198 (100) [M+ - CH3ClI.

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Chem. Res. Toxicol., Vol. 8, NO.1, 1995 93

3,5-Dichloro-2',3-dmethoxybiphenyl (30. 2.3 g (82% yield), white solid from diethyl etherlpetroleum ether, 1:3; mp 116-118 "C; IR (KBr): 3089, 3006, 2962, 2933, 2836, 1585, 1556, 1479, 1457, 1402, 1273, 1131, 1001 cm-l; lH NMR (400 MHz, CD2Clz): 6 = 3.63,3.89 ( 2 ~6H, , OCH3), 6.89 (dd, J = 8.1, 1.3 Hz, lH, 4'-H), 6.98 (dd, J = 8.1, 1.3 Hz, lH, 6'-H), 7.12 (t, J = 8.1 Hz, lH, 5'-H), 7.35 (t, J = 1.9 Hz, lH, 4-H), 7.45 (d, J = 1.9 Hz, 2H, 2,6-H); MS (70 eV): m l z (%) = 282 (50) [M+],267 (22) [M+ - CH3],232 (100) [M+ - CH3Cl1,217 (25) [M+ - C2H6Cll. General Procedurefor the Demethylation Reaction. To a stirred solution of boron tribromide 0.7 g (2.8 mmol) in CH2Cl2 (20 mL) was added dropwise a solution of compound (3)(1.4 mmol) in CHzCl2 (5 mL) for 1 h at -10 "C. The mixture was allowed to warm up to room temperature slowly over 12 h and was hydrolyzed with ice-cold water (20 mL). The CHzClz layer was separated, and the aqueous layer was backwashed with diethyl ether (20 mL x 2). The combined organic fractions were dried over MgS04, filtered, and concentrated under reduced pressure. 3-Chloro-3,4-dihydroxybiphenyl(4a). 0.26 g (83%yield), white solid from CHCldpetroleum ether, 1:5; mp 83-84 "C; IR (KBr): 3419,3233, 1595, 1568, 1530, 1470, 1395, 1274, 1172, 1117 cm-l; lH NMR (400 MHz, acetone-de): 6 = 6.91 (d, J = 8.2 Hz, lH, 5'-H), 7.01 (dd, J = 8.2, 2.2 Hz, lH, 6'-H), 7.14 (d, J = 2.4 Hz, lH, 2'-H), 7.28 (ddd, J = 7.9, 2.0, 1.0 Hz, lH, 4-H), 7.39 (td, J = 7.8, 0.5 Hz, lH, 5-H), 7.49 (ddd, J = 7.8, 1.8, 1.0 Hz, lH, 6-H), 7.55 (m,J 2.0, 0.5 Hz, lH, 2-H), 8.09 ( 8 , 2H, OH); 13C NMR (100 MHz, acetone-ds): 6 = 114.75, 116.60, 119.44, 125.63, 126.98, 127.07, 131.13, 132.29, 134.97, 144.08, 146.35; MS (70 ev): m l z (%) = 364 (38) [M+l, 276 (11)[M+ C4H12Si1, 73 (100). 3,4-Dichloro-3,4-dhydroxybiphenyl (4b). 0.29 g (81% yield), white solid from CHCldpetroleum ether, 1:4; mp 128129 "C; IR (KBr): 3456, 3320, 1609, 1556, 1524, 1463, 1378, 1311, 1213, 1117 cm-l; lH NMR (400 MHz, acetone-de): 6 = 6.91 (d, J = 8.2 Hz, lH, 5'-H), 7.03 (dd, J = 8.2, 2.4 Hz, lH, 6'-H), 7.14 (d, J = 2.2 Hz, lH, 2'-H), 7.51 (dd, J = 8.0, 2.0 Hz, lH, 6-H), 7.56 (dd, J = 8.5, 0.3 Hz, lH, 5-H), 7.71 (dd, J = 2.2, 0.3 Hz, lH, 2-H), 8.15 (s,2H, OH); 13CNMR (100 MHz, acetonede): 6 = 114.68, 116.65, 119.44, 127.04, 128.86, 130.48, 131.12, 131.54, 132.90, 142.52, 146.41, 146.56;MS (70 eV): m l z (%) = 398 (25) [M+l, 73 (100). 3,5-Dichloro-3,4-dihydroxybiphenyl (4c). 0.26 g (74% yield), white solid from CHCldpetroleum ether, 1:3; mp 105106 "C; IR (KBr): 3555, 3364, 3061, 1588, 1554, 1515, 1372, 1305, 1206, 1111 cm-l; lH NMR (400 MHz, acetone-de): 6 = 6.92 (d, J = 8.2 Hz, lH, 5'-H), 7.05 (dd, J = 8.2, 2.2 Hz, lH, 6'-H), 7.15 (d, J = 2.0 Hz, lH, 2'-H), 7.34 (t, J = 1.9 Hz, lH, 4-H), 7.51 (d, J = 1.9 Hz, 2H, 2,6-H), 8.18 (8,2H, OH); 13CNMR (100 MHz, acetone-de): 6 = 114.80, 116.66, 119.64, 125.67, 126.64,130.77,135.76,145.44,146.45,146.91;MS (70 ev): mlz (%) = 398 (23) [M+l, 73 (100). 3,5-Dichloro-2,5'dihydroxybiphenyl (4d). 0.30 g (83% yield), white solid from CHCldpetroleum ether, 1:3; mp 123124 "C; IR (KBr): 3319,1590,1554,1512,1296,1198 cm-l; lH NMR (400 MHz, acetone-de): 6 = 6.74 (dd, J = 8.6, 3.0 Hz, lH, 4'-H), 6.82 (d, J = 3.0 Hz, lH, 6'-H), 6.85 (d, J = 8.7 Hz, lH, 3'-H), 7.38 (t, J = 1.9 Hz, lH, 4-H), 7.57 (d, J = 1.9 Hz, 2H, 2,6-H), 7.99 (s, 2H, OH); 13CNMR (100 MHz, acetone-de): 6 = 117.13, 117.22, 117.96, 126.56, 126.96, 128.57, 134.93, 143.21, 147.80, 151.57; MS (70 eV): m l z (%) = 398 (38) [M+l,383 (12) [M+ - CH31,348 (18)[M+ - CH3Cll,275 (16) [M+ - C4H12SiC11, 73 (100). 3-Chloro-2,3-dihydroxybiphenyl(4e). 0.15 g (50%yield), white solid from CHCldpetroleum ether, 1:4; mp 132-134 "C; IR (KBr): 3496,3400,3056,1628,1589,1470,1321,1211,1070 cm-1; lH NMR (400 MHz, acetone&): 6 = 6.77 (t, J = 7.8 Hz, lH, 5'- H), 6.84 (dd, J = 7.8, 1.8 Hz, lH, 4'-H), 6.88 (dd, J = 7.8, 1.8Hz, lH, 6'-H), 7.32 (ddd, J = 8.0, 2.2, 1.2 Hz, lH, 4-H), 7.41 (td, J = 8.0, 0.4 Hz, lH, 5-H), 7.54 (ddd, J = 7.7, 1.6, 1.2 Hz,lH, 6-H), 7.65 (mc,J 2.0, 0.4 Hz, lH, 2-H), 8.20 (s, 2H, OH); 13C NMR (100 MHz, acetone-de): 6 = 115.45, 120.57, 122.03, 127.37, 127.72, 128.44, 129.90, 130.45, 134.10, 141.64,

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Bauer et al.

94 Chem. Res. Toxicol., Vol. 8, No. 1, 1995

Table 1. Synthesis of Dihydroxybiphenyls via Palladium-Catalyzed Cross-Couplingand Demethylation Reactions enny

boronicaad

aryl bromide

cross-coupling product

yield (%)

demethylation product

yield (%)

Me0 79

H

O

W

'

83

4.

3. HO

Me0 02 3b

HO-&)-Cl

81

CI

4b

'CI HO

CI 74

78

WC1

HO

Me0 4

79

QB(0Hh

OH 4d

CI

OH

CI

83

l b OMe

-0

OMe

HO

C1

50

62 4.

82

31

143.43,145.83;M S (70em: m l z (%) = 364 (16)[M+l, 349 (4)

CI

products 3. Although the coupling products were obtained when aqueous Na2C03 was used as a base, the S,S-Dichloro-2,3'-dhydroxybiphenyl (40. 0.21 g (60% reaction can also be carried out successfully in the yield),white solid from CHCldpetroleum ether, 1:3;mp 142presence of 3 M aqueous NaOH or 1M aqueous TlzC03. 143 "C;IR (KBr): 3509, 3349, 3063, 1629, 1589,1560,1476, Under these conditions the yields could not be improved, 1326,1299,1186,1098 cm-I; 'H N M R (400MHz, acetone-d& but 6=6.78(t,J=7.8H~,lH,5'-H),6.87(dd,J=7.8,1.7H~,lH, TlzC03caused a significant shortening of the reaction time (2 h). On the other hand, TEA in DMF was not 4'-H), 6.92(dd, J = 7.8,1.7Hz,lH,6-H),7.40 (t, J = 1.9Hz, effective in attaining good yields of the cross-coupling lH,4-H),7.61(d, J = 1.9 Hz,2H,2,6-H)8.29( 8 , 2H,OH);I3C products 3, and the reaction did not proceed at a NMR (100M H z , acetone&): 6 = 116.10,120.71,121.85,126.35, 127.03,128.54,134.99,143.05,143.75,146.06; MS (70ev): mlz detectable rate in the absence of a base. These results (%) = 398 (21)[M+],383 (8)[M+- CH31,73 (100). indicate the important role of the base in the crosscoupling by acceleration of the transmetalation step, as Results and Discussion shown in a number of recently reported palladiumcatalyzed couplings (25,27). In addition, a highly selecThe cross-coupling reactions between the arylboronic tive coupling of the boron reagent through the carbonacids 1 and bromides 2 were conducted in the presence bromine bond was accomplished in all reactions, which of 3 mol % of tetrakis(triphenylphosphine)palladium(O) is in agreement with the order of reactivity I 1 Br >> C1 and 2 M aqueous NaZC03 and were generally complete commonly observed in the palladium-catalyzed crosswithin 6 h in toluene a t reflux, affording the dimethoxycoupling reactions of boron compounds with organic biphenyls 3. Subsequent demethylation of 3 was perhalides (25,28,29). The structures of products 3 and 4, formed with boron tribromide in CHzClz, which after which according to our knowledge have not been reported acidic workup afforded the chlorinated dihydroxybiphein the literature, were determined by a combination of nyls 4 in good yields. IR, NMR, and GC/MS. The dihydroxybiphenyls 4 can Selected results, summarized in Table 1,indicate that be kept in organic solvents, e.g., DMSO, for several all cross-coupling reactions proceeded smoothly without months at -10 "C under a nitrogen atmosphere without any electronic or steric effects of the substituents in the decomposition. boronic acids or bromobenzenes to give the desired

[M+- CH31,73 (100).

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Synthesis of Dihydroxylated PCB Metabolites

Since, the boronic acids were conventionally prepared by the reaction of aryl-Grignard or lithium reagents with trimethylborate followed by hydrolysis and acidification (26),it can be concluded that the present procedure is a convenient and straightforward method for the construction of various chlorinated dihydroxybiphenyls. In addition, the synthetic approach is also applicable for the generation of chlorinated monohydroxybiphenyls (results not shown) and offers therefore a powerful and general strategy for the preparation of PCB metabolites.

Acknowledgment. This research was supported by National Cancer Institute Grant CA57423. A.R.A. was supported by a training grant from the National Institute of Environmental Health Sciences (ES07266). The authors sincerely thank Dr. Larry Hansen for his valuable suggestions and improvements in the manuscript.

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