Synthesis, absolute configuration, and bacterial mutagenicity of the 8 stereoisomeric vicinal diol epoxides at the terminal benzo ring of carcinogenic dibenz[ a,h ]anthracene

Article abstract of DOI:10.1021/tx200398b

The synthesis of the 8 possible stereoisomeric diol epoxides (DEs) at the terminal benzo ring of carcinogenic dibenz[a,h]anthracene (DBA) is reported. trans-3,4-Dihydroxy-3,4-dihydro-DBA (1) afforded the 4 bay region DEs: the enantiomeric pairs of the ant

Full text of DOI:10.1021/tx200398b

ARTICLE
pubs.acs.org/crt
Synthesis, Absolute Configuration, and Bacterial Mutagenicity of the
8 Stereoisomeric Vicinal Diol Epoxides at the Terminal Benzo Ring of
Carcinogenic Dibenz[a,h]anthracene
Heinz Frank,†,‡,|| Mario Funk,†,§ Franz Oesch,† and Karl L. Platt*,†
†Institute of Toxicology, University Medical Center, Johannes Gutenberg-University Mainz, Obere Zahlbacher Strasse 67,
55131 Mainz, Germany
S Supporting Information
b
ABSTRACT: The synthesis of the 8 possible stereoisomeric diol epoxides (DEs) at the
terminal benzo ring of carcinogenic dibenz[a,h]anthracene (DBA) is reported. trans-3,4-
Dihydroxy-3,4-dihydro-DBA (1) afforded the 4 bay region DEs: the enantiomeric pairs of
the anti diastereomers (+)-3/(ꢀ)-3 and of the syn diastereomers (ꢀ)-4/(+)-4, respec-
tively. trans-1,2-Dihydroxy-1,2-dihydro-DBA (2) served as precursor of the 4 reverse DEs:
the enantiomeric pairs of the anti diastereomers (+)-5/(ꢀ)-5 and of the syn diastereomers
(ꢀ)-6/(+)-6, respectively. The transformation of the olefinic double bond in the
enantiomeric trans-dihydrodiols to epoxides was achieved by either (i) oxidation with
m-chloroperoxybenzoic acid or (ii) formation of a bromohydrin with N-bromoacetamide/
H2O followed by dehydrobromination with an anion exchange resin. Because of the
pseudodiequatorial conformation of the hydroxyl groups in 1, both reactions proceeded
highly stereoselectively, while the stereoselectivity was impaired by the pseudodiaxial
conformation of the hydroxyl groups in 2. Diastereomers and racemic compounds were
efficiently separated without derivatization by HPLC on achiral or chiral stationary phases, respectively. The absolute configura-
tions of the DEs were deduced from the absolute configuration of 1 and 2 considering the regio- and stereoselectivity of the
subsequent reactions and resulted in (+)-(1R,2S,3S,4R)-3/(ꢀ)-(1S,2R,3R,4S)-3, (ꢀ)-(1S,2R,3S,4R)-4/(+)-(1R,2S,3R,4S)-4,
(+)-(1R,2S,3S,4R)-5/(ꢀ)-(1S,2R,3R,4S)-5, and (ꢀ)-(1R,2S,3R,4S)-6/(+)-(1S,2R,3S,4R)-6. The bacterial mutagenicity of the 8
stereoisomeric DEs was determined in histidine-dependent strains TA98 and TA100 of Salmonella typhimurium in the absence of a
metabolizing system. In general, the bay region DEs of DBA were stronger mutagens than the reverse DEs. In strain TA98, the syn
diastereomers of bay region DEs were stronger mutagens than their anti isomers, while in the case of reverse DEs the anti
diastereomers were more potent than their syn isomers. In strain TA100, all syn diastereomers surpassed the bacterial mutagenicity
of their anti isomers. Concerning the bay region DEs of DBA, this corresponds to the situation described for benzo[a]pyrene: of the
4 enantiomeric bay region DEs of DBA and benzo[a]pyrene, the syn diastereomer with [(R,S)-diol (R,S)-epoxide] absolute
configuration is the most potent mutagen in both bacterial strains, while the anti isomer with [(S,R)-diol (R,S)-epoxide]
configuration is the weakest mutagen.
’ INTRODUCTION
the genotoxicity of the parent hydrocarbon because of its
substantial metabolic formation6,8,9 and its strong mutagenic11,12
and carcinogenic activity.13,14 The occurrence of several 1,2,3,4-
tetrahydroxy derivatives of DBA during the biotransformation of
3,4-dihydrodiol 18,15,16 as well as of trans-1,2-dihydroxy-1,2-
dihydrodibenz[a,h]anthracene (1,2-dihydrodiol, 2)9,17 indicates
the formation of vicinal diol epoxides as metabolic precursors.
Chromatographic evidence exists for the formation of anti- and
syn-diol epoxides (3 and 4, Scheme 1) from 3,4-dihydrodiol 1.16
The biotransformation of 1,2-dihydrodiol 2 to its anti- and syn-
diol epoxides (5 and 6, Scheme 1) can only be established
indirectly from the occurrence of r-1,t-2,t-3,c-4- and r-1,t-2,c-3,
t-4-tetrahydroxy-1,2,3,4-tetrahydro-DBA as microsomal metabo-
lites of 2.9 In order to account for the genotoxic properties of
Polycyclic aromatic hydrocarbons (PAHs) play an important role
as environmental contaminants due to their chemical stability and
because many of them exhibit strong genotoxicity.1 Like most
chemical carcinogens, PAHs have to be transformed enzymatically
to chemically reactive metabolites in order to exert their mutagenic
and carcinogenic potential.2 Vicinal diol epoxides with the oxirane ring
occupying a sterically hindered bay region have been considered as
important genotoxic metabolites of PAHs since the mid-1970s.3,4
These electrophilic metabolites are prone to nucleophilic attack by
water and consequently hydrolyze in the predominantly aqueous
environment of biological systems to tetrahydroxy derivatives,5 which
therefore indicate the metabolic formation of vicinal diol epoxides.
The carcinogenic PAH dibenz[a,h]anthracene (DBA) is en-
zymatically converted at its terminal benzo ring to trans-
dihydrodiols.6ꢀ10 The trans-3,4-dihydroxy-3,4-dihydrodibenz-
[a,h]anthracene (3,4-dihydrodiol, 1) plays an important role in
Received: September 12, 2011
Published: November 16, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/tx200398b Chem. Res. Toxicol. 2011, 24, 2258–2268
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Chemical Research in Toxicology
ARTICLE
Scheme 1. Chemical Structures of the 8 Stereoisomers of
Vicinal Diol Epoxides at the 1,2,3,4-Positions of Dibenz[a,h]-
anthracene
4(3H)-one19 by the method described by McCaustland and Engel20 for
the synthesis of the corresponding derivative of benzo[a]pyrene (BaP).
The 1,2-dihydrodiol (()-2 was obtained as reported elsewhere.21 The
chiral stationary phases based on silica-bound (ꢀ)-2-(2,4,5,7-tetranitro-
fluoren-9-ylideneaminooxy)propionic acid ((ꢀ)-TAPA) or (ꢀ)- and
(+)-2-(2,5,9,11-tetranitro-7H-benzo[c]fluoren-7-ylideneaminooxy)pro-
pionic acid ((ꢀ)-/(+)-TBFPA) were prepared as previously described.22,23
Pure m-chloroperoxybenzoic acid (MCPBA) used for the epoxidations
was obtained by extensive washing of commercial MCPBA (77%; Fluka,
Neu-Ulm, Germany) with phosphate buffer (1 M, pH 7.5)24 and drying.
N-Bromoacetamide (NBA), N-bromosuccinimide (NBS), the anion
exchange resin Amberlite IRA-400(Cl), and deuterated solvents for
NMR spectroscopy were provided by Aldrich, Steinheim, Germany.
LiCl and Li2CO3 were delivered by Fluka, Neu-Ulm, Germany. All other
chemicals and the achiral stationary phases for HPLC were supplied by
Merck, Darmstadt, Germany. All chemicals used were of analytical or
HPLC grade.
Analytical Methods. Preparative HPLC was conducted with
system 830 (Du Pont, Bad Homburg, Germany), analytical HPLC with
model 3500 B (Spectra Physics, Darmstadt, Germany), monitoring the
UV absorbance at 254 nm. Melting point determinations were carried
1
out in unsealed capillary tubes and are uncorrected. H NMR spectra
were recorded on system AM 400 (Bruker, Karlsruhe, Germany) at 400
MHz or with a Bruker WH 90 at 90 MHz. 1H chemical shifts (ppm) are
reported relative to tetramethylsilane. In the case of racemic compounds
and their two enantiomers, a 1H NMR spectrum is displayed just once
since the spectra of the three compounds are identical. UV/vis absor-
bance spectra were measured in ethanol with a MPS-2000 spectro-
photometer (Shimadzu, D€usseldorf, Germany). Specific rotations were
determined in THF at 589 nm (sodium D-line) and 20 °C with a 241 LC
polarimeter (Perkin-Elmer, Frankfurt, Germany). Mass spectra were run
on a MAT CH 711 (Varian, Darmstadt, Germany) using the electron
impact ionization method (EI).
(()-trans-3,4-Bis(benzoyloxy)-3,4-dihydrodibenz[a,h]anthracene
[(()-7]. To a solution of (()-trans-3,4-bis(benzoyloxy)-1,2,3,4-
tetrahydrodibenz[a,h]anthracene (1.2 g, 2.3 mmol) in CCl4 at
60 °C, NBS (0.6 g, 3.0 mmol) and benzoyl peroxide (5 mg) were
added. The mixture was irradiated with a UV lamp (300 W; Ultra
Vitalux, Osram, M€unchen, Germany) for 20 min and heated at reflux
for 40 min. After evaporation, the residue was dissolved in hexa-
methylphosphoric triamide (HMPT; 150 mL), LiCl (5.0 g, 118 mmol),
and Li2CO3 (7.5 g, 102 mmol) were added, and the suspension was
heated to 100 °C under an argon atmosphere for 2.5 h. The mixture
was cooled to room temperature, poured into 500 mL ice water, and
extracted with ethyl acetate (2 ꢁ 250 mL). The organic layer was
washed successively with water, 5% aqueous acetic acid, water, 10%
aqueous NaHCO3 solution and water, dried with MgSO4, and
concentrated under reduced pressure. Purification by chromatogra-
phy on silica (chloroform/n-hexane 8:2, v/v) and final trituration
with boiling methanol yielded (()-7 (0.88 g, 74%) as a pale yellow
solid: mp 233 °C (239ꢀ240 °C).24 1H NMR (400 MHz; Me2SO-d6/
D2O) δ 9.49 (s, 1H, H7), 9.06 (s, 1H, H14), 8.99 (d, 1H, H8, J8,9 = 7.9
Hz), 8.27ꢀ7.47 (m, 17H, H1, Haromat.), 6.78 (d, 1H, H4, J3,4 = 7.2
Hz), 6.52 (dd, 1H, H2, J1,2 = 9.1 Hz, J2,3 = 3.3 Hz), 6.14 (dd, 1H, H3,
J3,4 = 7.2 Hz, J2,3 = 3.3 Hz); EI-MS m/z (rel intensity, %) 520 (8)
[M]+, 398 (43) [MH ꢀ C6H5CO ꢀ H2O]+, 105 (100) [C6H5CO]+.
(()-trans-3,4-Dihydroxy-3,4-dihydrodibenz[a,h]anthracene [(()-1].
The methanolysis of (()-7 (0.6 g, 1.15 mmol) was carried out according
to ref 24 and afforded (()-1 (0.34 g, 94%) as a pale yellow solid: mp
265 °C (278ꢀ280 °C).24 1H NMR (400 MHz; acetone-d6/Me2SO-d6
[3:2, v/v]/D2O) δ 9.33 (s, 1H, H7), 8.92 (d, 1H, H8, J8,9 = 7.9 Hz), 8.79
(s, 1H, H14), 8.14 (d, 1H, H12, J12,13 = 8.6 Hz), 7.92ꢀ7.59 (m, 6H,
these reactive metabolites of DBA, their synthetic prepara-
tion was required. Since not only the formation of the trans-
dihydrodiols of DBA8ꢀ10,12 but also the mutagenicity of the
enantiomers12 is highly stereoselective, differences in the geno-
toxicity of their metabolites, the enantiomeric diol epoxides,
could be expected. Therefore, the 8 possible stereoisomers of
the vicinal diol epoxides at the terminal benzo ring of DBA
(Scheme 1) were synthesized. The 3,4-dihydrodiol 1 afforded the
4 bay region diol epoxides: the enantiomeric pair of the anti
diastereomers (+)-3/(ꢀ)-3 and the enantiomeric pair of the syn
diastereomers (ꢀ)-4/(+)-4 (Scheme 1). The 1,2-dihydrodiol 2
served as precursor of the 4 reverse diol epoxides: the enantio-
meric pair of the anti diastereomers (+)-5/(ꢀ)-5 and the enan-
tiomeric pair of the syn diastereomers (ꢀ)-6/(+)-6 (Scheme 1).
The absolute configuration of the 8 stereoisomeric diol
epoxides was deduced from the absolute configuration of the
starting trans-dihydrodiols 1 and 218 considering the regio- and
stereoselectivity of the subsequent reactions. Finally, as a stan-
dardized indicator of genotoxic activity the bacterial mutagenicity
in Salmonella typhimurium of the 8 stereoisomeric diol epoxides
was determined.
’ EXPERIMENTAL PROCEDURES
Caution: PAHs and their derivatives are potentially hazardous and
should be handled in accordance with NIH Guidelines for the Laboratory
Use of Chemical Carcinogens.
Chemicals. (()-trans-3,4-Bis(benzoyloxy)-1,2,3,4-tetrahydrodibenz-
[a,h]anthracene was prepared from 1,2-dihydrodibenz[a,h]anthracene-
H
5,6,9ꢀ11,13), 7.42 (dd, 1H, H1, J1,2 = 10.1 Hz, J1,3 = 2.4 Hz), 6.22 (dd,
1H, H2, J1,2 = 10.1 Hz, J2,3 = 2.4 Hz), 4.84 (d, 1H, H4, J3,4 = 11.5 Hz),
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dx.doi.org/10.1021/tx200398b |Chem. Res. Toxicol. 2011, 24, 2258–2268

Chemical Research in Toxicology
ARTICLE
4.47ꢀ4.43 (dt, 1H, H3, J3,4 = 11.5 Hz, J2,3 = J1,3 = 2.4 Hz). UV/vis:
λmax (nm) (ε [Mꢀ1 cmꢀ1]): 225 (24 400), 233 (22 800), 247 (16
900), 277 (46 300), 286 (63 500), 295 (55 400), 337 (1 400), 348
(3 100), 369 (4 200), 387 (2 000); EI-MS m/z (rel intensity, %) 312
(100) [M]+, 294 (91) [M ꢀ H2O]+, 266 (87) [M ꢀ H2O ꢀ CO]+, 265
(78) [M ꢀ H2O ꢀ CHO]+.
mg, 73%), [α]D20 = ꢀ17° (c = 0.18 mg/mL), the second fractions (+)-8
(35.2 mg, 90%), [α]D20 = +24° (c = 0.49 mg/mL), as white solids.
(()-t-3,c-4-Dihydroxy-r-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,h]-
anthracene [(()-4]. Dehydrobromination of (()-8 (92 mg, 0.22
mmol) with the anion exchange resin Amberlite IRA-400 according to
the method of Yagi et al.25 yielded (()-4 (37.0 mg, 50%) as a white
solid: mp 214ꢀ216 °C (dec). 1H NMR (400 MHz; acetone-d6/
Me2SO-d6 [3:2, v/v]/D2O) δ 9.39 (s, 1H, H7), 8.95 (s, 1H, H14),
8.93 (d, 1H, H8, J8,9 = 7.7 Hz), 8.24 (d, 1H, H13, J12,13 = 8.7 Hz),
7.96ꢀ7.89 (m, 2H, H11,12), 7.73 (d, 1H, H5, J5,6 = 9.3 Hz), 7.70ꢀ7.61
(+)-(3S,4S)-3,4-Dihydroxy-3,4-dihydrodibenz[a,h]anthracene [(+)-1]
and (ꢀ)-(3R,4R)-3,4-Dihydroxy-3,4-dihydrodibenz[a,h]anthracene
[(ꢀ)-1]. The separation of (()-1 (238 mg, 0.73 mmol) into the
enantiomers was performed by preparative HPLC (dichloromethane/
methanol 4:1, v/v; 21 mL/min) in20 mg portions using a chiral stationary
phase based on silica-bound (ꢀ)-TBFPA (5 μm; 16 ꢁ 250 mm) as
previously described.23 The first eluted fraction contained (ꢀ)-1 as the
major product and the second fraction (+)-1. The latter was purified by
3-fold rechromatography under the conditions described above and final
chromatography on Florisil (50 g; chloroform/diethyl ether 9:1, v/v) to
yield a pale yellow solid: (+)-1 (42.7 mg, 38%), [α]D20 = +202° (c =
0.61 mg/mL). Because of the tailing of (ꢀ)-1 during chromatographic
separation, the first fraction had to be enantiomerically purified by
application of a chiral stationary phase based on silica-bound (+)-TBFPA
(5 μm; 16 ꢁ 250 mm; dichloromethane/methanol 4:1, v/v; 18 mL/min),
which improved the separation by inversion of the elution order of the
enantiomers. 2-Fold rechromatography and final purification with Florisil
as described above afforded a pale yellow solid: (ꢀ)-1 (49.7 mg, 44%),
[α]D20 = ꢀ193° (c = 0.56 mg/mL).
(m, 3H, H6,9,10), 4.84 (d, 1H, H1, J1,2 = 4.2 Hz), 4.73 (d, 1H, H4, J3,4
=
5.9 Hz), 4.00 (d, 1H, H3, J3,4 = 5.9 Hz), 3.84ꢀ3.83 (m, 1H, H2). UV/
vis: λmax (nm) (ε [Mꢀ1 cmꢀ1]): 223 (41 900), 235 (33 100), 262 (47
600), 270 (60 500), 280 (103 600), 291 (126 000), 336 (7 600), 351 (8
000), 363 (5 700), 389 (1 500); EI-MS m/z (rel intensity, %) 328 (10)
[M]+, 310 (70) [M ꢀ H2O]+, 282 (20) [M ꢀ H2O ꢀ CO]+, 281 (45)
[M ꢀ H2O ꢀ CHO]+, 254 (22) [M ꢀ H2O ꢀ 2CO]+, 253 (41) [M ꢀ
H2O ꢀ CO ꢀ CHO]+, 252 (52) [M ꢀ H2O ꢀ 2CHO]+.
(+)-(1R,2S,3R,4S)-3,4-Dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrodibenz
[a,h]anthracene [(+)-4] and (ꢀ)-(1S,2R,3S,4R)-3,4-Dihydroxy-1,2-
epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene [(ꢀ)-4]. Dehydrobro-
mination of (ꢀ)-8 (25 mg, 0.061 mmol) as described above yielded (+)-
4 (15.7 mg, 78%) as a white solid: [α]D20 = +103° (c = 0.48 mg/mL).
Similarly, the reaction of (+)-8 (32.4 mg, 0.079 mmol) afforded (ꢀ)-4
(21.7 mg, 84%): [α]D20 = ꢀ77° (c = 0.64 mg/mL).
(ꢀ)-(1R,2R)-1,2-Dihydroxy-1,2-dihydrodibenz[a,h]anthracene [(ꢀ)-2]
and (+)-(1S,2S)-1,2-Dihydroxy-1,2-dihydrodibenz[a,h]anthracene [(+)-2].
Compound (()-2 (107 mg, 0.34 mmol) was separated in 21 mg
portions by preparative HPLC (dichloromethane/methanol 7:3, v/v;
33 mL/min) using a chiral stationary phase based on silica-bound (ꢀ)-
TAPA (5 μm; 16 ꢁ 250 mm) as previously described.23 The first eluted
fraction afforded (ꢀ)-2 (52.6 mg), the second fraction (+)-2 (50.6 mg),
as crude products. Final purification of the first fraction was carried out
(()-c-3,t-4-Dihydroxy-r-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,h]-
anthracene [(()-3]. Epoxidation of (()-1 (114 mg, 0.36 mmol) with
MCPBA carried out as described24 yielded (()-3 (67.1 mg, 56%) as a
white solid: mp 264 °C (dec) (196ꢀ197 °C).24 1H NMR (400 MHz;
acetone-d6/Me2SO-d6 [3:2, v/v]/D2O) δ 9.37 (s, 1H, H7), 9.04 (s,
1H, H14), 8.92 (d, 1H, H8, J8,9 = 8.1 Hz), 8.22 (d, 1H, H13, J12,13 = 8.8
Hz), 7.94ꢀ7.88 (m, 3H, H5,11,12), 7.70 (d, 1H, H6, J5,6 = 9.1 Hz),
7.68ꢀ7.60 (m, 2H, H9,10), 5.08 (d, 1H, H1, J1,2 = 4.5 Hz), 4.59 (d, 1H,
by chromatography on Florisil (50 g; chloroform/diethyl ether 9:1, v/v)
H4, J3,4 = 8.6 Hz), 3.92 (d, 1H, H3, J3,4 = 8.6 Hz), 3.83 (d, 1H, H2, J1,2
=
20
4.5 Hz). UV/vis: λmax (nm) (ε [Mꢀ1 cmꢀ1]): 203 (21 100), 222 (32
000), 236 (24 000), 262 (31 500), 271 (38 700), 281 (69 700), 292 (87
000), 337 (5 800), 353 (6 300), 369 (4 200), 389 (1 200); EI-MS m/z
(rel intensity, %) 310 (100) [M ꢀ H2O]+, 282 (8) [M ꢀ H2O ꢀ CO]+,
281 (16) [M ꢀ H2O ꢀ CHO]+, 254 (6) [M ꢀ H2O ꢀ 2CO]+, 252
(16) [M ꢀ H2O ꢀ 2CHO]+.
to yield pure (ꢀ)-2 (39.7 mg, 74%) as a pale yellow solid: [α]D
=
ꢀ468° (c = 2.33 mg/mL), mp 241 °C (257ꢀ259 °C).24 1H NMR (400
MHz; acetone-d6/D2O) δ 9.31 (s, 1H, H7), 8.92 (d, 1H, H8, J8,9 = 8.0
Hz), 8.87 (s, 1H, H14), 8.15 (d, 1H, H12, J12,13 = 8.5 Hz), 7.94ꢀ7.60 (m,
5H, H5,6,9ꢀ11), 7.44 (d, 1H, H13, J12,13 = 8.5 Hz), 6.75 (d, 1H, H4, J3,4
=
9.5 Hz), 6.30ꢀ6.26 (m, 1H, H3, J2,3 = 5.3 Hz, J1,3 = 0.8 Hz, J3,4 = 9.5 Hz),
5.56 (s, 1H, H1, J1,2 = 0 Hz), 4.52ꢀ4.43 (dd, 1H, H2, J2,3 = 5.3 Hz, J2,4
=
(+)-(1R,2S,3S,4R)-3,4-Dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrodibenz-
[a,h]anthracene [(+)-3] and (ꢀ)-(1S,2R,3R,4S)-3,4-Dihydroxy-1,2-
epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene [(ꢀ)-3]. Epoxidation
of (ꢀ)-1 (20 mg, 0.064 mmol) as described above afforded (+)-3 (17.2
mg, 82%): [α]D20 = +43° (c = 0.58 mg/mL). By an analogous reaction of
(+)-1 (19.2 mg, 0.061 mmol), (ꢀ)-3 (18.2 mg, 90%) was obtained:
[α]D20 = ꢀ37° (c = 0.48 mg/mL).
1.7 Hz). UV/vis: λmax (nm) (ε [Mꢀ1 cmꢀ1]): 258 (36 700), 267 (32
000), 280 (46 200), 293 (83 700), 307 (110 400), 329 (6 300), 345 (7
000), 363 (6 000), 383 (2 500); EI-MS m/z (rel intensity, %) 312 (2.2)
[M]+, 294 (100) [M ꢀ H2O]+, 265 (39) [M ꢀ CHO]+.
Pure (+)-2 (41.4 mg, 77%) [α]D20 = +406° (c = 2.17 mg/mL) was
obtained by similar treatment of the second fraction.
(()-t-2-Bromo-r-1,t-3,c-4-trihydroxy-1,2,3,4-tetrahydrodibenz[a,h]-
anthracene [(()-8]. According to the procedure described by Yagi
et al.,25 (()-1 (100 mg, 0.32 mmol) was transformed with NBA (50 mg,
0.36 mmol) to the bromotriol (()-8 (105 mg, 80%), which was obtained
as a white solid: mp 163 °C (dec). 1H NMR (90 MHz; acetone-d6/D2O)
δ 9.34 (s, 1H, H7), 8.99ꢀ8.91 (m, 1H, H8), 8.82 (s, 1H, H14), 8.23 (d,
1H, H13, J12,13 = 8.8 Hz), 8.00ꢀ7.65 (m, 6H, H5,6,9ꢀ12), 5.92 (d, 1H, H1,
J1,2 = 2.6 Hz), 4.96ꢀ4.79 (m, 2H, H3,4), 4.42ꢀ4.30 (m, 1H, H2); EI-MS
m/z (rel intensity, %) 374 (92) [M (81Br) ꢀ 2 ꢁ H2O]+, 372 (100) [M
(79Br) ꢀ 2 ꢁ H2O]+, 263 (51) [M ꢀ 2H2O ꢀ HBr ꢀ CHO]+.
(+)-(1S,2R,3S,4R)-2-Bromo-1,3,4-trihydroxy-1,2,3,4-tetrahydrodibenz
[a,h]anthracene [(+)-8] and (ꢀ)-(1R,2S,3R,4S)-2-Bromo-1,3,4-trihy-
droxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene [(ꢀ)-8]. Separation of
the racemic bromotriol (()-8 (78 mg, 0.19 mmol) was performed by
preparative HPLC (dichloromethane/methanol 3:2, v/v; 24 mL/min) in
26 mg portions using a chiral stationary phase based on silica-bound (ꢀ)-
TAPA (5 μm; 16 ꢁ 250 mm). The first eluted fractions gave (ꢀ)-8 (28.3
(ꢀ)-(1R,2S,3R,4S)-1,2-Dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrodibenz
[a,h]anthracene [(ꢀ)-6] and (+)-(1R,2S,3S,4R)-1,2-Dihydroxy-3,4-
epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene [(+)-5]. Epoxida-
tion of (ꢀ)-2 (100 mg, 0.32 mmol) with MCPBA by the procedure
employed for the preparation of (()-3 gave a mixture (73 mg) of syn
and anti diastereomers.24 Separation was accomplished in 10 mg
portions of the crude product by HPLC (LiChrosorb Si 60, 5 μm,
16 ꢁ 250 mm; THF/diethyl ether/n-heptane 3:3:2, v/v/v; 4.8 mL/min).
The first eluted fraction afforded the syn diastereomer (ꢀ)-6 (4.81 mg,
yielded the anti diastereomer (+)-5 (10.2 mg, 9.7%), [α]D20 = +200° (c =
0.20 mg/mL), as white powders. (+)-5: 1H NMR (400 MHz; acetone-
d6/Me2SO-d6 [3:2, v/v]/D2O) δ 9.52 (s, 1H, H7), 9.33 (s, 1H, H14),
8.91 (d, 1H, H8, J8,9 = 8.2 Hz), 8.16 (d, 1H, H13, J12,13 = 8.6 Hz),
7.93ꢀ7.87 (dd, 1H, H11, J10,11 = 7.2 Hz, J9,11 = 1.2 Hz), 7.82 (d, 1H, H6,
J5,6 = 9.0 Hz), 7.70ꢀ7.59 (m, 4H, H5,9,10,12), 5.07 (d, 1H, H1, J1,2 = 7.1
Hz), 4.16 (d, 1H, H4, J3,4 = 4.4 Hz), 4.15ꢀ4.13 (dd, 1H, H2, J1,2 = 7.1 Hz,
20
4.6%), [α]D = ꢀ171° (c = 0.14 mg/mL), and the second fraction
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J2,3= 1.8 Hz), 3.84ꢀ3.82 (dd, 1H, H3, J3,4 = 4.4 Hz, J2,3= 1.8 Hz). UV/vis:
λmax (nm) (ε [Mꢀ1 cmꢀ1]) 222 (70 400), 239 (62 200), 262 (86 000),
272 (107 900), 282 (192 400), 293 (245 000), 337 (13 300), 354 (12
900), 391 (2 700). EI-MS m/z (rel intensity, %) 310 (100) [M ꢀ H2O]+,
281 (7) [M ꢀ H2O ꢀ CHO]+, 252 (10) [M ꢀ H2O ꢀ 2 ꢁ CHO]+.
(+)-(1S,2R,3S,4R)-1,2-Dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrodibenz
[a,h]anthracene [(+)-6] and (ꢀ)-(1S,2R,3R,4S)-1,2-Dihydroxy-3,4-
epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene [(ꢀ)-5]. Epoxidation
of (+)-2 (100 mg, 0.32 mmol) as described above yielded a mixture
(69 mg) of syn and anti diastereomers. HPLC separation on LiChrosorb
Si60 as outlined above afforded as the first eluted fraction the syn
(11400),390(2500);EI-MSm/z(relintensity,%) 310(100) [Mꢀ H2O]+,
281 (7) [M ꢀ H2O ꢀ CHO]+, 252 (10) [M ꢀ H2O ꢀ 2 ꢁ CHO]+.
(+)-(1S,2R,3S,4R)-1,2-Dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrodibenz
[a,h]anthracene [(+)-6]. Dehydrobromination of (+)-9 (29.9 mg, 0.073
mmol) and purification as described above afforded (+)-6 (9.36 mg,
39%) as a white solid: [α]D20 = +167° (c = 0.21 mg/mL).
(+)-(1R,2S,3S,4R)-3-Bromo-1,2,4-trihydroxy-1,2,3,4-tetrahydrodibenz
[a,h]anthracene [(+)-10] and (ꢀ)-(1S,2R,3R,4S)-3-Bromo-1,2,4-trihy-
droxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene [(ꢀ)-10]. Bromotriol
(()-10 (80 mg, 0.20 mmol) was separated by preparative HPLC under
the conditions described for (()-9. The first eluted fraction afforded
20
20
diastereomer (+)-6 (5.44 mg, 5.2%), [α]D = +142° (c = 0.15 mg/
(+)-10 (24.7 mg, 62%), [α]D = +119° (c = 0.97 mg/mL), and
mL), and as the second fraction the anti diastereomer (ꢀ)-5 (8.00 mg,
the second fraction afforded (ꢀ)-10 (31.4 mg, 79%), [α]D20 = ꢀ119°
20
7.6%), [α]D = ꢀ102° (c = 0.23 mg/mL). Both diastereomers were
(c = 0.56 mg/mL).
obtained as white powders.
(+)-(1R,2S,3R,4R)-1,4-Dihydroxy-2,3-epoxy-1,2,3,4-tetrahydrodibenz
[a,h]anthracene [(+)-11] and (+)-(1R,2S,3S,4R)-1,2-Dihydroxy-3,4-
epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene [(+)-5]. Dehydrobro-
mination of (+)-10 (22.8 mg, 0.056 mmol) with the anion exchange
resin Amberlite IRA-400 according to Yagi et al.25 yielded a mixture of the
regioisomeric epoxides (+)-11 and (+)-5. HPLC separation (LiChrosorb
Si 60, 5 μm, 16 ꢁ 250 mm; THF/diethyl ether/n-heptane 3:3:2, v/v/v;
5.0 mL/min) afforded as the first eluted fraction (+)-11 (3.66 mg, 20%):
(()-t-3-Bromo-r-1,t-2,c-4-trihydroxy-1,2,3,4-tetrahydrodibenz[a,h]
anthracene [(()-9)] and (()-c-3-Bromo-r-1,t-2,t-4-trihydroxy-1,2,3,4-
tetrahydrodibenz[a,h]anthracene [(()-10]. According to the proce-
dure described by Yagi et al.,25 (()-2 (250 mg, 0.80 mmol) was
transformed with NBA (125 mg, 0.91 mmol) to a mixture (297 mg) of
the diastereomeric bromotriols (()-9 and (()-10. Separation was
performed by reversed-phase HPLC (LiChrosorb RP-18, 10 μm, 16 ꢁ
250 mm; methanol/water 3:2, v/v; 17 mL/min). The first eluted fraction
contained (()-9 (81.2 mg, 25%) and the second fraction (()-10 (83.5
mg, 26%). Both diastereomers were obtained as white solids:
[α]D20 = +120° (c = 0.10 mg/mL). H NMR (400 MHz; acetone-d6/
1
Me2SO-d6 [3:2, v/v]/D2O) δ 9.39 (s, 1H, H7), 9.04 (s, 1H, H14), 8.93 (d,
1H, H8, J8,9 = 8.0 Hz), 8.23 (d, 1H, H13, J12,13 = 8.7 Hz), 7.95 (d, 1H, H6,
1
(()-9: mp 223 °C (dec). H NMR (90 MHz; Me2SO-d6/D2O) δ
J5,6 = 9.0 Hz), 7.91ꢀ7.88 (m, 2H, H5,11), 7.72ꢀ7.60 (m, 3H, H9,10,12),
9.33 (s, 1H, H7), 9.00ꢀ8.89 (m, 1H, H8), 8.73 (s, 1H, H14), 8.20 (d, 1H,
H13, J12,13 = 8.8 Hz), 7.98ꢀ7.67 (m, 6H, H5,6,9ꢀ12), 5.40 (d, 1H, H1,
J1,2 = 3.6 Hz), 4.98 (d, 1H, H4, J3,4 = 8.6 Hz), 4.82ꢀ4.69 (dd, 1H, H3,
J3,4 = 8.6 Hz, J2,3 = 2.0 Hz), 4.40ꢀ4.33 (m, 1H, H2); EI-MS m/z (rel
intensity, %) 374 (72) [M (81Br) ꢀ 2 ꢁ H2O]+, 372 (71) [M (79Br) ꢀ
2 ꢁ H2O]+, 265 (30) [M ꢀ 2 ꢁ H2O ꢀ Br ꢀ CO]+, 263 (34) [M ꢀ 2 ꢁ
H2O ꢀ HBr ꢀ CHO]+.
5.02 (d, 1H, H2, J2,3 = 4.0 Hz), 4.65 (d, 1H, H1, J1,2 = 4.2 Hz), 4.49 (t, 1H,
H4, J3,4 = 3.4 Hz), 3.81 (t, 1H, H3, J3,4 = 3.4 Hz). As the second fraction,
(+)-5 (1.18 mg, 6.5%), [α]D20 = +189° (c = 0.10 mg/mL), was obtained.
(ꢀ)-(1S,2R,3S,4S)-1,4-Dihydroxy-2,3-epoxy-1,2,3,4-tetrahydrodibenz
[a,h]anthracene [(ꢀ)-11] and (ꢀ)-(1S,2R,3R,4S)-1,2-Dihydroxy-3,4-
epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene [(ꢀ)-5]. Dehydrobro-
mination of (ꢀ)-10 (28.7 mg, 0.070 mmol) affording a mixture of the
regioisomeric epoxides (ꢀ)-11 and (ꢀ)-5 and their separation were
performed as describedabove. The first fraction yielded (ꢀ)-11 (6.40 mg,
28%), [α]D20 = ꢀ43° (c = 0.31 mg/mL), and the second fraction yielded
(ꢀ)-5 (2.82 mg, 12%), [α]D20 = ꢀ114° (c = 0.14 mg/mL).
(()-10: mp 134 °C (dec). 1H NMR (90 MHz; Me2SO-d6/D2O) δ
9.35 (s, 1H, H7), 8.93 (s, 1H, H14), 9.00ꢀ8.87 (m, 1H, H8), 8.22 (d, 1H,
H13, J12,13 = 9.1 Hz), 8.00ꢀ7.67 (m, 6H, H5,6,9ꢀ12), 5.35 (d, 1H, H1,
J1,2 = 3.8 Hz), 4.96 (d, 1H, H4, J3,4 = 5.0 Hz), 4.11ꢀ4.02 (m, 2H, H2,3);
EI-MS m/z (rel intensity, %) 374 (19) [M (81Br) ꢀ 2 ꢁ H2O]+, 372
(19) [M (79Br) ꢀ 2 ꢁ H2O]+, 265 (11) [M ꢀ 2 ꢁ H2O ꢀ Br ꢀ CO]+,
263 (13) [M ꢀ 2 ꢁ H2O ꢀ HBr ꢀ CHO]+.
Mutagenicity Studies. The bacterial mutagenicity was deter-
mined by employing histidine-dependent strains TA98 and TA100 of
Salmonella typhimurium (generously provided by Dr. B. N. Ames,
University of California, Berkeley, CA) as described by Maron and
Ames26 with only minor modifications16 omitting an exogenous meta-
bolizing system. The mutagenicity test was performed according to the
plate incorporation protocol.26 The test compounds were dissolved in
30 μL/plate N,N-dimethylformamide immediately before use. The
specific mutagenicity was calculated from the slope of the initial linear
part of the doseꢀresponse curve.
(+)-(1S,2R,3S,4R)-3-Bromo-1,2,4-trihydroxy-1,2,3,4-tetrahydrodibenz
[a,h]anthracene [(+)-9] and (ꢀ)-(1R,2S,3R,4S)-3-Bromo-1,2,4-trihy-
droxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene [(ꢀ)-9]. Bromo-
triol (()-9 (80 mg, 0.20 mmol) was separated into the enantiomers in
10 mg portions by preparative HPLC (dichloromethane/methanol 3:2, v/v;
16 mL/min) using a chiral stationary phase based on silica-bound (ꢀ)-
TAPA (5 μm; 16 ꢁ 250 mm). In the first fraction, (ꢀ)-9 (29.2 mg, 73%),
[α]D20 =ꢀ16° (c= 0.48 mg/mL), was collected, and in the second fraction,
(+)-9 (35.7 mg, 89%), [α]D20 = +17° (c = 0.76 mg/mL), was collected.
(ꢀ)-(1R,2S,3R,4S)-1,2-Dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrodibenz
[a,h]anthracene [(ꢀ)-6]. Dehydrobromination of the enantiomeric
bromotriol (ꢀ)-9 (24.8 mg, 0.061 mmol) with the anion exchange
resin Amberlite IRA-400 performed according to Yagi et al.,25 and
final purification by HPLC (LiChrosorb Si 60, 5 μm, 16 ꢁ 250 mm;
THF/diethyl ether/n-heptane 3:3:2, v/v/v; 5.3 mL/min) afforded (ꢀ)-6
(5.37 mg, 27%) as a white solid: [α]D20 = ꢀ187° (c = 0.23 mg/mL). 1H
NMR (400 MHz; acetone-d6/Me2SO-d6 [3:2, v/v]/D2O) δ 9.44 (s, 1H,
H7), 8.97 (d, 1H, H8, J8,9 = 8.0 Hz), 8.81 (s, 1H, H14), 8.26 (d, 1H, H13,
’ RESULTS AND DISCUSSION
Synthesis. The diol epoxides 3, 4, 5, and 6 (Scheme 1) were
prepared from the 3,4-dihydrodiol 1 or the 1,2-dihydrodiol 2 by
employing two reactions: either epoxidation with MCPBA
or dehydrobromination of suitable bromotriols as shown in
Schemes 2 and 3.
The 3,4-dihydrodiol (()-1 was obtained by the classical
stepwise construction of the DBA nucleus19,27 via trans-3,4-
bis(benzoyloxy)-1,2,3,4-tetrahydrodibenz[a,h]anthracene,24 its
allylic bromination, and dehydrobromination to the dihydrodi-
benzoate (()-7 followed by methanolysis. The overall yield
(70%) for these three steps, a considerable improvement over the
published yield (36%),24 was achieved by using LiCl/Li2CO3 in
HMPT28 as previously reported for dehydrobromination of
J12,13 = 8.5 Hz), 7.97 (d, 1H, H6, J5,6 = 9.1 Hz), 7.93 (d, 1H, H11, J10,11
=
7.7 Hz), 7.79 (d, 1H, H5, J5,6 = 9.1 Hz), 7.75 (d, 1H, H12, J12,13 = 8.5 Hz),
7.72ꢀ7.63 (m, 2H, H9,10), 5.47 (t, 1H, H1), 4.61 (t, 1H, H2, J1,2 = 2.3 Hz,
J2,3 = 1.2Hz), 4.25 (d, 1H, H4, J3,4 =3.9Hz), 3.96ꢀ3.94 (m, 1H, H3). UV/
vis: λmax (nm) (ε [Mꢀ1 cmꢀ1]): 222 (56 000), 240 (50 700), 262
(74 700), 271 (94 600), 282 (170 000), 293 (221 000), 336 (11 100), 352
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Scheme 2. Synthesis of the Stereoisomers of trans-3,4-Dihy-
droxy-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene
(Bay Region Diol Epoxides)a
Scheme 3. Synthesis of the Stereoisomers of trans-1,2-Dihy-
droxy-3,4-epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene
(Reverse Diol Epoxidesa
a Chemical structures show only the terminal benzo ring of dibenz-
[a,h]anthracene. Reagents and/or chromatographic methods are indicated
in italics: MCPBA, m-chloroperoxybenzoic acid; NBA, N-bromoaceta-
mide; (ꢀ)-TAPA, silica-bound (ꢀ)-2-(2,4,5,7-tetranitrofluoren-9-ylide-
neaminooxy)propionic acid; (ꢀ)-TBFPA, silica-bound (ꢀ)-2-(2,5,9,
11-tetranitro-7H-benzo[c]fluoren-7-ylideneaminooxy)propionic acid.
1,2-diacetoxy-4,5-dibromocyclohexane in the synthesis of ben-
zene dihydrodiol.29 The racemic 3,4-dihydrodiol (()-1 was
separated into its enantiomers by chiral HPLC using silica-bound
(ꢀ)-TBFPA as stationary phase.23 Because of massive tailing, the
first eluting (ꢀ)-1 was contaminated with the later eluting
enantiomer (+)-1. Therefore, (ꢀ)-1 was purified using silica-
bound (+)-TBFPA, i.e., the chiral stationary phase of opposite
absolute configuration.
Epoxidation of the 1,2-double bond in the enantiomerically
pure 3,4-dihydrodiols (ꢀ)-1 and (+)-1 with MCPBA20,24,30
afforded highly stereoselective anti-diol epoxides (+)-3 and
(ꢀ)-3, respectively (Scheme 2) as a result of the hydrogen
bonding in the transition state between the allylic hydroxyl group
and the peroxy acid.31 Since the hydroxyl groups in 1 exist
predominantly in the pseudodiequatorial conformation,24 the
more distant benzylic hydroxyl group exerts no cis-directing
effect on the attack of the peroxy acid.
For the synthesis of the syn-diol epoxides (+)-4 and (ꢀ)-4, the
3,4-dihydrodiol (()-1 was reacted with NBA in water leading to
bromotriol (()-8 followed by dehydrobromination with the
anion exchange resin Amberlite IRA-400 according to the
method of Yagi et al.25,30 (Scheme 2). The diastereoselective
formation of the syn-diol epoxide from the trans-dihydrodiol is
the result of several regio- and stereoselective reactions: First, the
electrophilic Br+ formed from protonated NBA adds stereose-
lectively to the 1,2-double bond in a fashion similar to that of the
peroxy acid by the cis-directing effect of the allylic hydroxyl
group;32 the subsequent attack of water is regiospecific with
respect to the benzylic position of the hydroxyl group and
stereospecific with respect to the trans-opening of the cyclic
bromonium ion; finally, the intramolecular nucleophilic substitu-
tion of bromine can only take place by the adjacent alkoxide in
trans-position, i.e., by the benzylic hydroxyl group. Thus, the
bromohydrin is converted into the epoxide with retention of
configuration at the hydroxyl-bearing carbon and inversion at the
bromine-carrying one.32 The dehydrobromination leading to
a Chemical structures show only the terminal benzo ring of dibenz[a,h]-
anthracene. Reagents and/or chromatographic methods are indicated in
italics: MCPBA, m-chloroperoxybenzoic acid; NBA, N-bromoacetamide;
(ꢀ)-TAPA, silica-bound (ꢀ)-2-(2,4,5,7-tetranitrofluoren-9-ylideneami-
nooxy)propionic acid.
enantiomerically pure syn-diol epoxides (+)-4 and (ꢀ)-4 re-
quired the bromotriols (ꢀ)-8 and (+)-8, which were obtained in
high yields by preparative HPLC of (()-8 on a chiral stationary
phase based on silica-bound (ꢀ)-TAPA.22,23
For the synthesis of enantiomerically pure anti- and syn-diol
epoxides of 1,2-dihydrodiol, (()-2 was very efficiently separated
by preparative HPLC on a chiral stationary phase based on silica-
bound (ꢀ)-TAPA into (ꢀ)-2 and (+)-2.23 Epoxidation of the
3,4-double bond in 1,2-dihydrodiol 2 with MCPBA24 afforded a
mixture of the diastereomeric anti- and syn-diol epoxides 5 and 6
(Scheme 3). This is the result of the pseudodiaxial conformation
of the hydroxyl groups in bay region trans-dihydrodiols33 such as
the 1,2-dihydrodiol 2.24 Here, not only the cis-directing effect of
the allylic but also that of the benzylic hydroxyl group leads to the
entry of the oxiranyl oxygen in the anti- and syn-position with
respect to the benzylic hydroxyl group. The mixture of anti- and
syn-diol epoxides could be separated into the diastereomers by
preparative HPLC on silica gel as described,24 however, only when a
modified mobile phase was employed. The anti-andsyn-diol epoxides
5 and 6 were obtained in a ratio of about 2:1 (3:1).24
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Table 1. Vicinal Coupling Constants J of the Protons at the Hydroxyl Group-Bearing Carbon Atoms in trans-3,4- and -1,2-
Dihydrodiols of Dibenz[a,h]anthracene and in Their anti- and syn-Diol Epoxides
vicinal coupling constants J of the protons at the hydroxyl group-bearing carbon atoms
position of the
hydroxyl groups
trans-dihydrodiols
anti-diol epoxides
syn-diol epoxides
3,4
1
3
4
J3,4 = 11.5 Hz (11 Hz)24
J3,4 = 8.6 Hz (9 Hz)24
J3,4 = 5.9 Hz
6
1,2
2
5
J1,2 = 0 Hz (2 Hz)24
J1,2 = 7.1 Hz (0 Hz)24
J1,2 = 2.3 Hz
Since the syn-diol epoxide 6 was obtained in a rather low yield
by peroxy acid epoxidation, its synthesis from 1,2-dihydrodiol via
the bromotriol was attempted (Scheme 3). As in the case of
epoxidation of the 1,2-dihydrodiol with peroxy acid, the cis-
directing effect of the allylic and of the benzylic hydroxyl group
on the entry of the bromine atom was observed leading to a 1:1
mixture of bromotriols 9 and 10 after reaction of 2 with NBA in
the presence of water25,30 (Scheme 3). This mixture was
separated by preparative reversed-phase HPLC into the diastero-
meric racemic bromotriols (()-9 and (()-10. Very efficient
separation of the racemates into the enantiomers was then
achieved by preparative HPLC on a chiral stationary phase based
on silica-bound (ꢀ)-TAPA (Scheme 3).
considerable alteration of the coupling constant by this change in
solvent composition37 seems to be unlikely. For J1,2 in 5, the
value of 7.1 Hz is more plausible since J1,2 of the reverse anti-diol
epoxide of benz[a]anthracene with a terminal benzo ring struc-
turally identical to that of 5, determined in acetone-d6, was
reported as 6.8 Hz.5 In syn-diol epoxides, the benzylic hydroxyl
group and the oxirane oxygen are on the same side of the ring and
can thus undergo intramolecular hydrogen bonding,35 resulting
in a preference for the conformation in which the hydroxyl
groups are pseudodiaxial.5,36 This is illustrated by the reverse syn-
diol epoxide 6 with J1,2 = 2.3 Hz (Table 1; Supporting Informa-
tion). However, when the oxirane is part of a highly hindered bay
or fjord region the expected preferred pseudodiaxial conforma-
tion of the hydroxyl groups in syn-diol epoxides is shifted to
the pseudodiequatorial conformation. Reported examples are the
syn-diol epoxides of BaP (J7,8 = 6.0 Hz),38 benz[a]anthracene
(J3,4 = 7.0 Hz),39 benzo[c]phenanthrene (J3,4 = 9.0 Hz),36 and
now syn-diol epoxide 4 with a coupling constant J3,4 = 5.9 Hz
(Table 1; Supporting Information).
Absolute Configuration. The absolute configuration of the 8
enantiomeric and hence optically active diol epoxides (Scheme 1)
was deduced from the absolute configuration of their synthetic
precursors, the 3,4- and 1,2-dihydrodiols 1 and 2, considering the
regio- and stereoselectivity of the subsequent reactions discussed
above. The (R,R) configuration has been established for the en-
antiomers of both trans-dihydrodiols with negative optical rotation,
i.e., (ꢀ)-1 and (ꢀ)-2, by two spectroscopic methods;18 conse-
quently, (+)-1 and (+)-2 are assigned the (S,S) configuration.
Therefore, the bay region anti-diol epoxide (+)-3 prepared
from (ꢀ)-1 has the configuration (1R,2S,3S,4R) and its enan-
tiomer (ꢀ)-3 (1S,2R,3R,4S) (Scheme 2). The use of racemic
bromotriol (()-8 for the preparation of the enantiomeric syn-
diol epoxides of 1 (Scheme 2) required an additional experiment:
(ꢀ)-(3R,4R)-1 was stereospecifically transformed with NBA in
aqueous THF as described above, yielding bromotriol 8 with
positive optical rotation and hence (1S,2R,3S,4R) configuration
(Scheme 2); the same absolute configuration has the syn-diol
epoxide (ꢀ)-4, while its enantiomer (+)-4 is assigned the
(1R,2S,3R,4S) configuration.
Dehydrobromination of bromotriols (ꢀ)-9 and (+)-9 af-
forded stereospecifically the syn diastereomers of the 3,4-epox-
ides (ꢀ)-6 and (+)-6 (Scheme 3). Since in bromotriol 10 the
hydroxyl groups in 2- and 4-position are trans to the bromine
atom, nucleophilic displacement of bromine in 3-position by
both adjacent alkoxides resulted in a mixture of the 2,3-epoxide
11 and the anti diastereomers of the 3,4-epoxide 5 (Scheme 3) in
a ratio of about 5:2. The predominant formation of the 2,3-
epoxide is in agreement with an observation in the case of BaP
where the 8,9-epoxide was the main product of dehydrobromi-
nation of an analogous bromotriol.34
Both synthetic pathways to the anti and syn diastereomers of
the diol epoxides of the 1,2-dihydrodiol (Scheme 3) complement
each other. Starting with racemic 1,2-dihydrodiol 2, peroxy acid
epoxidation yielded the anti diastereomer 5 with an overall yield
of 13% compared to 7% of the syn diastereomer 6, whereas with
the bromotriol method, the syn diastereomer 6 was obtained with
14% overall yield in contrast to 3% of the anti diastereomer 5.
Conformation. The preferred conformation of the diol ep-
oxides can be deduced from the magnitude of the vicinal coupling
constants (J) for the carbinol hydrogens obtained by 1H NMR.35
As in the case of trans-dihydrodiols of a series of PAHs,33 large
values of J, i.e., 8ꢀ9 Hz, indicate a pseudodiequatorial conforma-
tion and low values, i.e., 0ꢀ3 Hz, a pseudodiaxial conformation of
the vicinal hydroxyl groups. anti-Diol epoxides have a relatively
strong preference for the conformation in which the hydroxyl
groups adopt pseudodiequatorial positions.5,36 This is demonstrated
by the bay region anti-diol epoxide 3 with J3,4 = 8.6 Hz (Table 1;
Supporting Information). For the reverse anti-diol epoxide 5, the
diagnostic coupling constant J1,2 wasreportedas0Hz,24 characteristic
for a pseudodiaxial conformation of the vicinal hydroxyl groups.
However, on reinvestigation we found J1,2 = 7.1 Hz for
5 (Table 1; Supporting Information), which indicates a prefer-
ence for the pseudodiequatorial conformation. Although in both
1H NMR experiments different solvents were used, acetone-d6 in
ref 24 and acetone-d6/Me2SO-d6 [3:2, v/v]/D2O in this study, a
Analogous considerations apply to the absolute configuration
of the reverse diol epoxides from 1,2-dihydrodiol 2. Thus, the
anti-diol epoxide with positive optical rotation, (+)-5, obtained
by peroxy acid epoxidation of (ꢀ)-(1R,2R)-2, is associated with
(1R,2S,3S,4R) absolute configuration, while the accompanying
syn-diol epoxide with negative optical rotation, (ꢀ)-6, is assigned
the (1R,2S,3R,4S) configuration (Scheme 3). Finally, the en-
antiomers of these diol epoxides, (ꢀ)-5 and (+)-6 possess
(1S,2R,3R,4S) and (1S,2R,3S,4R) absolute configurations,
respectively.
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Figure 1. Bacterial mutagenicity in strain TA98 of Salmonella typhimurium of the 8 stereoisomers of vicinal diol epoxides at the 1,2,3,4-positions of
dibenz[a,h]anthracene. Data are presented as the mean values ( SD from three independent experiments, each conducted in triplicate.
A perfect correspondence exists between the absolute config-
uration and the signs of optical rotation of the enantiomeric bay
region diol epoxides of DBA (this study) and those of BaP,40
benz[a]anthracene,41 and chrysene.41
upon heterolytic cleavage of the benzylic CꢀO bond of the
oxirane ring. It could indeed be verified that a quantitative
relationship exists between mutagenic activity and the calculated
value of ΔEdeloc/β: when this quantum chemical parameter was
plotted against the logarithm of relative bacterial mutagenicity of
tetrahydroepoxides49 or diol epoxides50 of various PAHs, a
satisfying linear correlation was obtained. As anticipated, the
ΔEdeloc/β value of 0.7384 calculated for the bay region diol
epoxides of DBA is high in contrast to the lower value of 0.59351
for the reverse diol epoxides.
Mutagenicity. The genotoxicity of the 8 stereoisomeric diol
epoxides was determined as bacterial mutagenicity by the rever-
sion of histidine-dependent strains TA98 and TA100 of Salmo-
nella typhimurium to histidine prototrophy42 in the absence of a
metabolizing system employing the plate incorporation protocol.26
Strain TA98 is reverted by frameshift mutagens, while TA100 detects
mutagens causing base-pair substitutions.26 As reported for
BaP,34,43,44 benz[a]anthracene,45 chrysene,46ꢀ48 and phenanthrene,46
the bacterial mutagenicity of bay region diol epoxides of DBA was
in all cases stronger in strain TA100 than in TA98 (Figure 1ꢀ4).
Remarkably, reverse diol epoxides of BaP34,44 and chrysene48 are
stronger mutagens in strain TA98 than in TA100. In the case of
DBA, this difference was only observed for the reverse anti-diol
epoxide (ꢀ)-5 (Figure 1ꢀ4). The decline in the number of his+-
revertant colonies at higher concentrations of (ꢀ)-4 (cf. Figures 1
and 2) is a result of the toxicity of the syn-diol epoxide as indicated
by a thinning of the hisꢀ background lawn.26
The purity of the stereoisomeric diol epoxides prepared in this
study requires some comments. The 1H NMR spectra indicate a
chemical purity of >95%, though the optical purity, especially of
(ꢀ)-4 and (ꢀ)-5, seems to be lower as demonstrated by the
values of optical rotation. Obviously, diol epoxides (ꢀ)-4 and
(ꢀ)-5 are somewhat contaminated with (+)-4 and (+)-5,
respectively. Since (ꢀ)-4 and (ꢀ)-5 are much stronger muta-
gens than (+)-4 and (+)-5 (cf. Figure 3), a higher purity of (ꢀ)-4
and (ꢀ)-5 had not changed this difference but only enhanced its
extent.
The 8 stereoisomeric diol epoxides of DBA constitute 4
diastereomeric pairs of anti and syn isomers (Scheme 1).
In strain TA98, the syn diastereomers of bay region diol epoxides
are stronger mutagens than their anti isomers: (ꢀ)-4(R,S,R,S) >(+)-
3 (R,S,S,R), and (+)-4 (S,R,S,R) > (ꢀ)-3 (S,R,R,S) (Figure 3), while
in the case of reverse diol epoxides, the anti diastereomers are
more potent than their syn isomers, (+)-5 (R,S,S,R) > (ꢀ)-6 (R,
S,R,S), and (ꢀ)-5 (S,R,R,S) > (+)-6 (S,R,S,R) (Figure 3). When
the absolute configurations of diol epoxides of different poly-
cyclic aromatic hydrocarbons are compared, the configuration is
given beginning with the absolute configuration of the benzylic
carbon bearing a hydroxyl group while omitting the numbering
of the carbon atoms, e.g., (R,S,S,R) stands for [(R,S)-diol (S,R)-
epoxide]. In strain TA100, all syn diastereomers surpass the
bacterial mutagenicity of their anti isomers (Figure 4). Concern-
ing the bay region diol epoxides, this corresponds to the situation
described for BaP.52 Of the 4 enantiomeric bay region diol
epoxides of DBA and BaP, the syn diastereomer with (R,S,R,S)
absolute configuration is the most potent mutagen in both strains
of Salmonella typhimurium, while the anti isomer with (S,R,R,S)
configuration is the weakest mutagen. In contrast to DBA and
BaP, the anti diastereomers of the bay region diol epoxides of
chrysene47 and benz[a]anthracene45 with (R,S,S,R) absolute con-
figuration were by far the strongest mutagens for strain TA100.
Finally, among the 4 stereoisomers of the structurally closely
related fjord region diol epoxide of benzo[c]phenanthrene, again
the anti diastereomer, in this case with (S,R,R,S) absolute config-
uration, was the strongest bacterial mutagen in strains TA98 and
TA100.53 A more pronounced correlation between the absolute
With the exception of (ꢀ)-5, the bay region diol epoxides of
DBA turned out to be stronger mutagens than the reverse diol
epoxides (Figures 3 and 4). This was also observed in the case of
the diol epoxides of BaP34,44 and chrysene48 and is obviously a
consequence of the stronger electrophilic reactivity of the oxirane
moiety in bay region vs reverse diol epoxides. As proposed by
Jerina et al.,4 the quantum chemical parameter ΔEdeloc/β repre-
sents the ease of formation of a resonance stabilized carbocation
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Figure 2. Bacterial mutagenicity in strain TA100 of Salmonella typhimurium of the 8 stereoisomers of vicinal diol epoxides at the 1,2,3,4-positions of
dibenz[a,h]anthracene. Data are presented as the mean values ( SD from three independent experiments, each conducted in triplicate.
Figure 3. Specific mutagenicity in strain TA98 of Salmonella typhimur-
ium of the 8 stereoisomers of vicinal diol epoxides at the 1,2,3,4-positions
of dibenz[a,h]anthracene.
Figure 4. Specific mutagenicity in strain TA100 of Salmonella typhi-
murium of the 8 stereoisomers of vicinal diol epoxides at the 1,2,3,4-
positions of dibenz[a,h]anthracene.
configuration of bay (and fjord) region diol epoxides and geno-
toxicity was observed for their carcinogenic activity: of the 16
configurational isomers studied from BaP,54,55 chrysene,56 benz-
[a]anthracene,57 and benzo[c]phenanthrene,58 the one with the
highest activity in two murine tumor models was the anti diaster-
eomer with (R,S,S,R) absolute configuration.
same order: (ꢀ)-(3R,4R)-1 > (+)-(3S,4S)-1 > (+)-(1S,2S)-2 >
(ꢀ)-(1R,2R)-2.12 Assuming that diol epoxides are responsible
for the genotoxicity of the trans-dihydrodiols of DBA, the high
bacterial mutagenicity of the bay region diol epoxides and the
low mutagenicity of the reverse diol epoxides of DBA determined
in this study could have been predicted from the results of previ-
ous work.12
Both DBA and BaP belong to the carcinogenic PAHs, and
DBA like BaP60 is metabolized predominantly to the most
genotoxic enantiomer of the 8 possible stereoisomeric diol
epoxides. However, in contrast to BaP, DBA was demonstrated
to be transformed additionally to bisdiol epoxides16 that ob-
viously play a more important role than simple diol epoxides in
the covalent binding of this PAH to DNA in vivo.61
As in the case of other PAHs,59 the microsomal biotransfor-
mation at the terminal benzo ring of DBA to trans-dihydrodiols,
the precursors of vicinal diol epoxides, proceeds highly regio- and
stereoselectively:8ꢀ10,12 (ꢀ)-(3R,4R)-1 was obtained with 11%
of total metabolic conversion succeeded by its enantiomer
(+)-(3S,4S)-1 with 5%, (ꢀ)-(1R,2R)-2 with 4% and its enantio-
mer (+)-(1S,2S)-2 with 1%.12 The bacterial mutagenicity of the
enantiomeric 1,2- and 3,4-dihydrodiols of DBA follows nearly the
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’ ASSOCIATED CONTENT
Metabolic Activation in Mutagenesis Testing (de Serres, F. J., Fouts, J. R.,
Bend, J. R., and Philpot, R. M., Eds.) pp 159ꢀ177, Elsevier/North-
Holland Biomedical Press, Amsterdam.
(5) Sayer, J. M., Whalen, D. L., Friedman, S. L., Paik, A., Yagi, H.,
Vyas, K. P., and Jerina, D. M. (1984) Conformational effects in the
hydrolyses of benzo-ring diol epoxides that have bay-region diol groups.
J. Am. Chem. Soc. 106, 226–233.
(6) Sims, P. (1970) Qualitative and quantitative studies on the
metabolism of a series of aromatic hydrocarbons by rat-liver prep-
arations. Biochem. Pharmacol. 19, 795–818.
(7) MacNicoll, A. D., Burden, P. M., Rattle, H., Grover, P. L., and
Sims, P. (1979) The formation of dihydrodiols in the chemical or
enzymic oxidation of dibenz[a,c]anthracene, dibenz[a,h]anthracene and
chrysene. Chem.-Biol. Interact. 27, 365–379.
(8) Nordqvist, M., Thakker, D. R., Levin, W., Yagi, H., Ryan, D. E.,
Thomas, P. E., Conney, A. H., and Jerina, D. M. (1979) The highly
tumorigenic 3,4-dihydrodiol is a principal metabolite formed from
dibenzo[a,h]anthracene by liver enzymes. Mol. Pharmacol. 16, 643–655.
(9) Platt, K. L., and Reischmann, I. (1987) Regio- and stereoselective
metabolism of dibenz[a,h]anthracene: Identification of 12 new micro-
somal metabolites. Mol. Pharmacol. 32, 710–722.
(10) Mushtaq, M., Weems, H. B., and Yang, S. K. (1989) Stereo-
selective formations of enantiomeric K-region epoxide and trans-dihy-
drodiols in dibenz[a,h]anthracene metabolism. Chem. Res. Toxicol. 2,
84–93.
Supporting Information. 1H NMR spectra of (()-3,
S
b
(()-4, (+)-5, and (ꢀ)-6. This material is available free of charge
via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 0049-6131-17-9260. Fax: 0049-6131-230506. E-mail:
platt@uni-mainz.de.
Present Addresses
‡Biochemical Institute for Environmental Carcinogens, Prof. Dr.
Gernot Grimmer-Foundation, Lurup 4, D-22927 Grosshansdorf,
Germany
§Evonik Industries AG, Kirschenallee, D-64293 Darmstadt,
Germany
Funding Sources
We are grateful for financial support by the Deutsche
Forschungsgemeinschaft (SFB 302).
Notes
Deceased.
(11) Wood, A. W., Levin, W., Thomas, P. E., Ryan, D., Karle, J. M.,
Yagi, H., Jerina, D. M., and Conney, A. H. (1978) Metabolic activation of
dibenzo(a,h)anthracene and its dihydrodiols to bacterial mutagens.
Cancer Res. 38, 1967–1973.
’ ACKNOWLEDGMENT
We express our appreciation to A. Stieglitz for valuable
technical assistance. Thanks are also due to C. B€ohres and
A. Vierengel for recording of the mass and NMR spectra,
respectively.
(12) Platt, K. L., Schollmeier, M., Frank, H., and Oesch, F. (1990)
Stereoselective metabolism of dibenz(a,h)anthracene to trans-dihydro-
diols and their activation to bacterial mutagens. Environ. Health Perspect.
88, 37–41.
(13) Buening, M. K., Levin, W., Wood, A. W., Chang, R. L., Yagi, H.,
Karle, J. M., Jerina, D. M., and Conney, A. H. (1979) Tumorigenicity of
the dihydrodiols of dibenzo(a,h)anthracene on mouse skin and in
newborn mice. Cancer Res. 39, 1310–1314.
(14) Slaga, T. J., Gleason, G. L., Mills, G., Ewald, L., Fu, P. P., Lee,
H. M., and Harvey, R. G. (1980) Comparison of the skin tumor-
initiating activities of dihydrodiols and diol-epoxides of various poly-
cyclic aromatic hydrocarbons. Cancer Res. 40, 1981–1984.
(15) Lecoq, S., Chalvet, O., Strapelias, H., Grover, P. L., Phillips, D. H.,
and Duquesne, M. (1991) Microsomal metabolism of dibenz[a,
c]anthracene, dibenz[a,h]anthracene and dibenz[a,j]anthracene to bis-dihy-
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than dihydrodiol oxides, are the principal microsomal metabolites of
’ DEDICATION
This work is dedicated to the memory of Donald M. Jerina
(1940ꢀ2011), the founder of the bay region theory of polycyclic
aromatic hydrocarbon carcinogenesis.
’ ABBREVIATIONS
BaP, benzo[a]pyrene; DBA, dibenz[a,h]anthracene; 1,2-dihydro-
diol, trans-1,2-dihydroxy-1,2-dihydrodibenz[a,h]anthracene; 3,4-
dihydrodiol, trans-3,4-dihydroxy-3,4-dihydrodibenz[a,h]anthracene;
HMPT, hexamethylphosphoric triamide; MCPBA, m-chloroperoxy-
benzoic acid; NBA, N-bromoacetamide; NBS, N-bromosuccinimide;
PAH, polycyclic aromatic hydrocarbon; (ꢀ)-TAPA, (ꢀ)-2-(2,4,5,
7-tetranitrofluoren-9-ylideneaminooxy)propionic acid; (ꢀ)-TBFPA,
(ꢀ)-2-(2,5,9,11-tetranitro-7H-benzo[c]fluoren-7-ylideneaminooxy)-
propionic acid
tumorigenic
trans-3,4-dihydroxy-3,4-dihydrodibenz[a,h]anthracene.
Chem. Res. Toxicol. 7, 89–97.
(17) Chou, M. W., Fu, P. P., and Yang, S. K. (1981) Metabolic
conversion of dibenz[a,h]anthracene (()trans-1,2-dihydrodiol and
chrysene (()trans-3,4-dihydrodiol to vicinal dihydrodiol epoxides. Proc.
Natl. Acad. Sci. U.S.A. 78, 4270–4273.
(18) Schollmeier, M., Frank, H., Oesch, F., and Platt, K. L. (1986)
Assignment of absolute configuration to metabolically formed trans-
dihydrodiols of dibenz[a,h]anthracene by two distinct spectroscopic
method. J. Org. Chem. 51, 5368–5372.
(19) Karle, J. M., Mah, H. D., Jerina, D. M., and Yagi, H. (1977)
Synthesis of dihydrodiols from chrysene and dibenzo[a,h]anthracene.
Tetrahedron Lett. 4021–4024.
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