Bioactivation of the carcinogen 11-methoxy-16,17-dihydro-15H-cyclopenta[a]phenanthrene

Article abstract of DOI:10.1016/S1383-5718(99)00215-6

The title compound is a more potent carcinogen than would be anticipated from its simple phenanthrene structure lacking further D-ring conjugation. In vitro it undergoes microsomal metabolism to yield as major metabolites its 15- and 17-alcohols and its 16,17-diol; other minor metabolites are also derived from attack at the 5-membered ring, but no evidence of aromatic oxidation is apparent. The title compound is a weak mutagen in the Ames' test with Salmonella typhimurium TA100, but only with microsomal bio-activation. The 17-ol and 16,17-diol are inactive, with or without biological activation. By contrast the 15-alcohol, a rather reactive compound, is a strong mutagen both in the presence and absence of the bio-activation system. This, therefore, may be the proximate carcinogen, and its structural analogy to the naturally occurring hepato-carcinogen safrole is noted. Copyright (C) 2000 Elsevier Science B.V.

Full text of DOI:10.1016/S1383-5718(99)00215-6

Mutation Research 465 Ž2000. 85–90
www.elsevier.comrlocatergentox
Community address: www.elsevier.comrlocatermutres
Bioactivation of the carcinogen
w x
1
1-methoxy-16,17-dihydro-15H-cyclopenta a phenanthrene
a
b,)
a
a
Fenton S. Catterall , Maurice M. Coombs , Costas Ioannides , Kim Walton
a
Molecular Toxicology Group, School of Biological Sciences, UniÕersity of Surrey, Guildford, Surrey GU2 5XH, UK
Department of Chemistry, School of Physical Sciences, UniÕersity of Surrey, Guildford, Surrey GU2 5XH, UK
b
Received 24 August 1999; received in revised form 27 October 1999; accepted 28 October 1999
Abstract
The title compound is a more potent carcinogen than would be anticipated from its simple phenanthrene structure lacking
further D-ring conjugation. In vitro it undergoes microsomal metabolism to yield as major metabolites its 15- and
1
7-alcohols and its 16,17-diol; other minor metabolites are also derived from attack at the 5-membered ring, but no evidence
of aromatic oxidation is apparent. The title compound is a weak mutagen in the Ames’ test with Salmonella typhimurium
TA100, but only with microsomal bio-activation. The 17-ol and 16,17-diol are inactive, with or without biological
activation. By contrast the 15-alcohol, a rather reactive compound, is a strong mutagen both in the presence and absence of
the bio-activation system. This, therefore, may be the proximate carcinogen, and its structural analogy to the naturally
occurring hepato-carcinogen safrole is noted. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Bioactivation; Carcinogen; 11-Methoxy-16,17-dihydro-15H-cyclopentawaxphenanthrene
1
. Introduction
Ž2b. is a strong carcinogen, similar in potency to the
classical polycyclic hydrocarbon benzowaxpyrene w4x.
Carcinogenicity in the cyclopentawaxphenanthrene
Moreover, like the latter it is bioactivated through
metabolism to its trans-3,4-dihydro-3,4-dihydroxy-
anti-1,2-epoxide which is the proximate carcinogen
w5x.
Not unexpectedly the 11-methoxy-17-ketone Ž2c.
is also a moderately strong carcinogen w6x, but here
there is a difference because the 11-methoxy hydro-
carbon Ž1c. is almost equally active w7x. This is all
the more surprising because the 17-methyl derivative
Ž3. of the latter did not give rise to tumours w8x. It
series is induced by correct Želectron releasing. bay-
region substitution w1x and is enhanced by a conju-
gated endocyclic or exocyclic double bond at C-17
w2x. Thus, whereas neither the unsubstituted
hydrocarbon 16,17-dihydro-15H-cyclopentaw ax-
phenanthrene Ž1a. nor its 17-ketone derivative
Ž15,16-dihydrocyclopentawaxphenanthren-17-one, 2a.
is carcinogenic, the 11-methyl hydrocarbon Ž1b. is
weakly active w3x whilst the 11-methyl-17-ketone
therefore seemed interesting and important to exam-
ine this matter further, and the in vitro microsomal
)
Ž .
metabolism of the 11-methoxy hydrocarbon 1c has
Corresponding author. Tel.: q44-1483-87-6827; fax: q44-
1
1
483-87-6851
now been studied with this in view.
383-5718r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S1383-5718Ž99.00215-6

8
6
F.S. Catterall et al.rMutation Research 465 (2000) 85–90
2
2
2
. Materials and methods
0.36 mCirmg. identical in all respects with the
non-radioactive compound.
.1. Chemistry
2
.1.3. 16,17-Dihydro-15-hydroxy-11-methoxy-15H-
.1.1. ImproÕed preparation of 16,17-dihydro-11-
cyclopenta[a]phenanthrene (4)
methoxy-15H-cyclopenta[a]phenanthrene (1c)
This compound was prepared by sodium tetrahy-
droborate reduction of the corresponding 15-ketone
ŽScheme 1. w9x. HPLC revealed that it exists as two
epimers ŽRRT 0.75 and 0.83, RRTsrelative reten-
tion time, relative to that of Ž1c.s1.00. the ratio of
which seemed to depend largely upon the tempera-
ture at which it had been previously kept. It formed
white crystals which on being stored in a tightly
stoppered glass tube at room temperature for some
months became brown, and TLC disclosed the for-
mation of a substantial amount of less polar impuri-
ties. Previously w9x, difficulty had been experienced
1
6,17-Dihydro-17-hydroxy-11-methoxy-15H-
cyclopentawaxphenanthrene Ž1.32 g, 5 mmole. was
stirred with dry tetrahydrofuran Ž20 ml. protected
from moisture in an ice bath. Pyridine-sulphur triox-
ide complex Ž1.2 g, 7.5 mmole. was added giving a
cream suspension which thickened on being stirred
in ice for 1.5 h, then at room temperature for an
additional 1 h. The flask was again cooled in ice
during the dropwise addition of a solution of lithium
aluminium hydride Ž1.15 g, 3 mmole. in dry tetrahy-
drofuran, and stirring was continued at room temper-
ature overnight giving a greyish-yellow suspension.
Water Ž1.1 ml. was added cautiously followed by
in obtaining correct combustion figures after purifi-
cation by crystallisation; it was surprising to discover
that sublimation at 1008Cr0.1 Torr, giving a pure
white crystalline solid, was a better method: found
C, 81.6; H, 5.95. C H O requires C, 81.8; H,
1
5% aqueous sodium hydroxide Ž1.1 ml. and more
water Ž3.3 ml., and finally ether Ž100 ml. was added
to the yellow suspension. The off-white precipitate
was filtered off, washed with more ether, and the
combined ether solutions were washed with water,
dried over magnesium sulphate, and evaporated to
give an orange gum Ž1.21 g.. This was extracted
several times with boiling hexane Ž50 ml., decanting
from the brown residue; evaporation of the hexane
left a white crystalline mass Ž0.75 g. of the 11-
methoxy hydrocarbon Ž1c. with UV and NMR spec-
tra as already described w9x.
1
8
16
2
6.1%; lmax Žlog10 ´. 215 Ž4.12., 228 Ž4.44., 253
Ž4.78., 273 Ž4.50., 294 Ž4.13., 305 Ž4.19., 325 Ž3.31.,
341 Ž3.51., 357 Ž3.63. nm.
2.1.4. 16,17-Dihydro-11,15-dimethoxy-15H-cyclo-
penta[a]phenantrene (7)
16,17-Dihydro-11,15-dimethoxy-15H-cyclopenta-
waxphenantrene Ž7, Scheme 2. metabolite RRT 0.90,
C H O : mass spectrum wmassrcharge Ž% relative
1
9
18
2
abundance.x 278 Ž17, molecular ion Mq., 248 Ž52,
1
4
2
.1.2. Preparation of the [methyl- C] labeled 11-
methoxy hydrocarbon (1c)
Mq –OCH ., 247 Ž100, Mq –OCH ., 246 Ž27,
2
3
Mq –OCH –H., 233 Ž12, Mq –OCH –CH ., 215
3
2
3
1
4
Using a gas line w Cxmethyl iodide Ž1 mCi. was
Ž21, M q –OCH –H–OCH ., 205 Ž10, M q
3
3
condensed into a flask containing 15,16-dihydro-11-
hydroxycyclopentawaxphenanthren-17-one Ž248 mg.,
anhydrous potassium carbonate Ž207 mg., and dry
acetonitrile Ž20 ml., and the mixture was stirred at
–OCH –CH –CO.; nmr ŽCDCl . d 9.64 Ž1H, d,
2 3 3
H-1., 7.96 Ž1H, d J6,7 9 Hz, H-7., 7.89 Ž1H, d J3,4
9
Hz, H-4., 7.80 Ž1H, d J6,7 9 Hz, H-6., 7.64–7.44
ambient temperature with exclusion of moisture for 3
days. The solid was removed by filtration, washed
with dichloromethane, and the combined solutions
1
4
were evaporated to leave the wmethyl- Cx11-
methoxy-17-ketone Ž2c. as a pale yellow solid Ž152
mg, 0.39 mCirmg.. This was converted into the
1
4
wmethyl- Cx-11-methoxy hydrocarbon Ž1c. as de-
scribed above. The final purification was by semi-
preparative HPLC; it formed white crystals Ž77 mg,
Scheme 1. Compounds mentioned in the text.

F.S. Catterall et al.rMutation Research 465 (2000) 85–90
87
parative column with a linear gradient of 30% aque-
ous methanol changing to 100% methanol over 60
min at a flow rate of 2 mlrmin. Aliquots of the
separate fractions were assayed for radioactivity by
scintillation counting and their UV spectra were
recorded. Like peaks from several runs were pooled,
evaporated to dryness at reduced pressure below
Scheme 2. Major metabolites of the carcinogenic 11-methoxy
hydrocarbon Ž1c. separated by HPLC. The RRT values are their
retention times relative to that of the unchanged hydrocarbon.
4
08C, and the residue was dissolved in CDCl3 for
Ž2H, m, H-2,3., 7.34 Ž1H, s, H-12., 5.47 Ž1H, dd
NMR analysis.
J15,16 2.5, 5.5, H-15., 4.13 Ž3H, s, 11-OCH ., 3.41
3
Ž3H, s, 15-OCH ., 3.32 Ž1H, m, H-16., 3.05 Ž1H, m,
2.3. Mutagenicity tests
3
H-17., 2.41 Ž1H, m, H-16., 2.36 Ž1H, m, H-17. ppm
from tetramethylsilane.
The mutagenicity of the 11-methoxy hydrocarbon
Ž1c. and its metabolites Ž4.–Ž6. was determined us-
2
.2. Incubation and isolation of metabolites
ing the Ames test in the presence and absence of a
microsomal activation system using Salmonella ty-
phimurium TA100 w11x. This strain was used because
Male adult Wistar albino rats Ž180–200 g. were
purchased from B & K Universal, Hull, West York-
shire, UK. Induction of the CYP 1 family was
achieved by administration of a single interperitoneal
dose of Aroclor 1254 Ž500 mgrkg., dissolved in
corn oil Ž200 mgrml.; the animals were sacrificed
it was found to be more easily reverted by the title
hydrocarbon than TA 98. The activation system con-
tained 10% vrv of the S9 Ž25% wrv. liver prepara-
tion. All tests were repeated to ensure reproducibil-
ity.
on the 5th day following administration. Livers were
immediately excised, and the postmitochondrial ŽS9.
fraction Žfor use as the activation system in the
mutagenicity assays. and the microsomal fraction
Žfor use in the metabolism studies. were prepared as
previously described w10x.
3. Results
The preparation of 11-methoxy-16,17-dihydro-
15H-cyclopentawaxphenanthrene Ž1c. was improved
The incubation mixture contained phosphate
buffer Ž0.1 M, pH 7.4, 6 ml., NADP Ž4 mM, 1 ml.,
glucose-6-phosphate Ž5 mM, 1 ml., glucose-6-phos-
phate dehydrogenase Ž20 units., and microsomes
Ž25% wrv, 2 ml., all in a 250-ml conical flask open
by avoidance of strong acid which causes some
elimination and dimension during catalytic hy-
drogenolysis of the 17-ketone w9x. Use was made of
Cory and Achiwa’s method w12x, conversion of the
17-alcohol to its sulphate ester with the sulphur
trioxide-pyridine complex followed by in situ reduc-
tion of the ester with lithium aluminium hydride, to
to the atmosphere and shaken in a water bath at
3
78C. The reaction was started by addition of the
1
4
substrate Ž1c. Ž1 mg in 100 ml of dimethylsulph-
oxide., and terminated at 30 min by cooling the flask
in ice and adding ice-cold ethyl acetate Ž10 ml..
Repeated extractions with six to eight lots of ethyl
acetate were carried out without delay, the extracts
were pooled, dried over magnesium sulphate, and
evaporated to dryness below 408C under reduced
pressure. The residue was dissolved in ethanol Ž1–2
ml. and stored at room temperature to await HPLC
give the pure hydrocarbon in a 60% yield. C-label-
ing was required to allow quantitation of the hydro-
carbon metabolites; this was readily achieved by
methylation of the 17-keto-phenol corresponding to
1
4
Ž2c. with C-methyl iodide followed by deoxygena-
1
4
tion of the wmethyl Cx-11-methoxy-17-ketone as
described.
The most notable feature in the spectrum of
metabolites from the 11-methoxy hydrocarbon Ž1c.
disclosed by HPLC ŽFig. 1. was the lack of polar
separation.
This was carried out with a Waters 660 solvent
programmer, 6000 A pumps, and UV detector set at
compounds. In the case of the corresponding 17-ke-
tones Ž2a. and Ž2b., the in vitro metabolism of which
has been studied in detail Ž2,5., although the 15-ols
2
54 nm, on a Whatman Partisil 10 ODS-1 semi-pre-

8
8
F.S. Catterall et al.rMutation Research 465 (2000) 85–90
A involvement was also seen. It is thought that the
very limited ring A oxidation may be the main
reason for the low biological activity of these hydro-
carbons as compared with their 17-keto derivatives.
A study w14x of the mutagenicity of the chemically
synthesised trans-3,4-dihydrodiols and trans-3,4-di-
hydro-3,4-dihydroxy-syn and anti-1,2-epoxides of
the hydrocarbons Ž1a. and Ž1b. showed that muta-
genicity increased markedly in the order: hydrocar-
bon -3,4-diol<3,4-diol-1,2-epoxide, with the lat-
ter powerfully active in the absence of biological
activation. Thus, it seems probable that these hydro-
carbons are activated in the same way as the corre-
sponding 17-ketones.
In the present study, having all the ring D ketones
and secondary alcohols of Ž1c. available and fully
characterised from a recent chemical synthesis w9x
greatly assisted identification of the major metabo-
lites of this 11-methoxy hydrocarbon. A comparison
of the relative retention times and UV spectra of
these peaks ŽFig. 1A. with those of the synthetic
alcohols indicated that these were the 15-ol Ž4., the
1
7-ol Ž5. and the 16,17-diol Ž6..
By repeating this incubation several times and
pooling the major peaks of like RRT enough of each
was obtained to compare their NMR spectra with
those of the synthetic alcohols, and in this way the
identities of the 15-ol, 17-ol and trans-16,17-diol
were positively confirmed. UV spectra of RRT 0.55
and the other less abundant peaks showed that they
were all derived from ring D ŽRRT 0.33 was an
artefact since it was not radioactive.. When the
Fig. 1. Metabolites of the 11-methoxy hydrocarbon Ž1c. separated
by HPLC: ŽA. within 2 h of extraction from the incubation
mixture; ŽB. 26 h after 2 h of extraction from the incubation
mixture Žfor details see the text.. The numbers are the retention
times relative to that of Ž1c.s1.00. At 2 and 26 h the radioactiv-
ity associated with each fraction Žas a percentage of the total
radioactivity recovered. was, respectively, as follows: RRT 1.00
Žunmetabolised substrate 1c. 28.8, 29.1; RRT 0.90 Ž15-methoxy 7.
ethanolic solution of metabolites was left for some
1
.5, 17.4; RRT 0.83 and 0.75 Ž15-ol epimers 4. 21.7, 10.2; RRT
0
.78 Ž17-ol 5. 7.4, 7.3; RRT 0.66 Ž16,17-diol 6. 9.0, 9.2; RRT
0
.55 ŽD-ring diol?. 5.9, 5.6.
are major metabolites there is also substantial oxida-
tion at ring A giving 1,2- and 3,4-dihydrodiols with
further conversion into 3,4-dihydroxy-1,2-epoxides
and tetrols. A preliminary study w13x of the microso-
mal metabolism of the unsubstituted hydrocarbon
Ž1a. showed that bio-oxidation of this compound
Fig. 2. Radioactivity in peaks at RRT 0.90, 0.83 and 0.75 Žsee Fig.
1
. as a function of time of storage of the ethanolic metabolite
was largely confined to ring D, although some ring
mixture before injection on to the HPLC column.

F.S. Catterall et al.rMutation Research 465 (2000) 85–90
89
time before injection into the HPLC column it was
noticed that the amount of the 15-ol epimers dimin-
ished while the small peak at RRT 0.90 ŽFig. 1A.
increased. This was studied systematically over 26 h
ŽFig. 2., at the end of which RRT 0.90 was the most
abundant metabolite ŽFig. 1B..
The new metabolite RRT 0.90 had a UV spectrum
almost identical with that of the 15-ol from which it
formed; its structure was readily established from its
NMR and mass spectra as 16,17-dihydro-11,15-di-
methoxy-15H-cyclopentawaxphenanthrene Ž7.. It is
not obvious how methylation of the 15-ol can take
place in the ethanolic solution of the metabolite
mixture, but the fact that it does occur emphasises
the reactivity of this alcohol.
cinogenicity, but no mutagenic response was evident
in the absence of the activating system. The 17-ol
was inactive with or without the microsomal system
as was the 16,17-diol, whereas the 15-ol Ž4. was a
strong mutagen both in the absence or presence of
the activation system. The fact that of all the major
metabolites the 15-ol is a potent direct acting muta-
gen makes it a likely candidate as the proximate
carcinogen.
4. Discussion
Several chemical differences were noted between
the 11-methoxy and 11-methyl hydrocarbons and
ascribed to enhanced electron release from the
methoxy group in the former w9x. C-15 is attached
para to the methoxy group in the aromatic ring and
hence acquires electron density from the latter by
mesomeric electron shift, so that C-15 is an espe-
cially electron rich centre. This no doubt accounts
for the lability of the CŽ15.–O bond; C-17 is at-
The importance of this 15-ol became more clear
when these compounds were tested in the Ames test
with Salmonella typhimurium TA100 ŽFig. 3.. With
a microsomal activation system from Aroclor 1254-
induced rats the 11-methoxy hydrocarbon Ž1c. was
weakly mutagenic, in keeping with its known car-
tached meta to the methoxy group and therefore
cannot acquire electron density by this mechanism.
Biologically the result is that the 17-ol Ž5. is not
mutagenic, whereas the isomeric 15-ol Ž4. is a strong
direct acting mutagen. Our attention was first drawn
to this compound Ž4. when we found that it exists as
a pair of epimers, readily interconverting under the
influence of factors such as temperature, presumably
by carbon–oxygen bond cleavage and reformation.
This compound also seemed to be unusually reactive,
decomposing slowly on being kept under ambient
conditions with the formation of less polar materials,
which however do not include the 15-methyl ether
Ž7.. The presence of the 15-ol as a major metabolite
was expected because biological 15-hydroxylation is
Fig. 3. Mutagenic potential of 11-methoxy-16,17-dihydro-15H-
cyclopentawaxphenanthrene Ž1c. and its 15-hydroxy Ž4., 17-hy-
droxy Ž5. and 16,17-dihydroxy Ž6. metabolites. Mutagenicity was
evaluated using the Ames test using S. typhimurium TA100, in
the presence or absence of an activating system derived from the
liver of Aroclor 1254-treated rats. Results are presented as mean
commonly found with other cyclopentawaxphenanth-
renes 2 . The observation that 4 was converted
spontaneously at an appreciable rate into this methyl
ether Ž7. when the metabolite mixture was allowed
w x
Ž .
to stand at room temperature before chromatographic
"
S.D. of triplicates; the spontaneous reversion rate of 99"6 has
separation was unexpected; it suggests that a reactive
already been subtracted. The methoxy hydrocarbon Ž1c., the 17-ol
Ž5. and the 16,17-diol Ž6. were all inactive in the absence of
microsomal activation Ždata not shown.. The steeper decline in the
number of revertants with increasing dose for the 15-ol with
activation indicates that there is a deactivation system also present
in the microsomal preparation.
methylating agent methyl folate? is co-extracted
from the microsomal system by the ethyl acetate.
However, irrespective of the mechanism, the fact
that it occurs points again to the reactivity of the
15-ol. The role of this alcohol became apparent
Ž
.

9
0
F.S. Catterall et al.rMutation Research 465 (2000) 85–90
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w3x A. Butenandt, H. Dannenberg, Untersuchungen u¨ ber die
krebserzengende Wirksamkeit der Methylhomologen des
1
,2-Cyclopentaphenanthrenes, Arch. Geschwulstforsch.
6
Scheme 3. Bio-activation of safrole Ž8.. The ultimate carcinogen
Ž10. is formed by loss of OH-from Ž9. leaving, on the benzylic
carbon atom, a positive charge which is partially stabilised by the
adjacent conjugated system.
Ž1953. 1–7.
4
w x
M.M. Coombs, T.S. Bhatt, S. Young, The carcinogenicity of
15,16-dihydro-11-methylcyclopentawaxphenanthren-17-one,
Br. J. Cancer 40 Ž1979. 914–921.
w5x M.M. Coombs, T.S. Bhatt, A.-M. Kissonerghis, J.A. Allen,
when, of the major metabolites Ž4.–Ž6., it alone was
found to be a strong direct acting mutagen, whereas
the parent 11-methoxy hydrocarbon Ž1c. was less
active and then only after bio-activation.
C.W. Vose, Identification of the proximate and ultimate
forms of the carcinogen 15,16-dihydro-11-methylcyclo-
pentawaxphenanthren-17-one, Cancer Res. 39 Ž1979. 4160–
4
165.
6 T.S. Bhatt, S.T. Hadfield, M.M. Coombs, Carcinogenicity
w x
This situation bears a strong structural analogy to
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ylenedioxybenzene, 8. which is known to be meta-
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X
bolised to its 1-hydroxy derivative Ž9. w15x ŽScheme
cyclopentawaxphenanthrenes, Carcinogenesis 9 Ž1988. 1669–
3
.. It is thought that bio-activation of the latter Ž9.
1
672.
occurs through conversion to its sulphate ester with
cleavage of the C–O bond to give the ultimate
carcinogen Ž10. w16x. In the present case it is reason-
able to conclude that the 15-ol Ž4. itself is the active
compound. This unexpected mode of activation of a
polycyclic aromatic compound is thus the direct
result of strong electron release from the para 11-
methoxy group, already implicated in certain unusual
chemical reactions of Ž1c. w9x. It may serve to ex-
plain the higher than expected carcinogenicity of this
compound although of course this explanation needs
confirmation by an independent in vivo study of the
comparative carcinogenic potentials of the 11-
methoxy hydrocarbon and its 15-ol.
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w11x D.M. Maron, B.N. Ames, Revised methods for the Salmonella
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3
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spectrum of Ž7. and Mr. J. Bloxsidge for the NMR
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