Novel spiro-quinone formation from 3′-hydroxydiethylstilbestrol after oxidation with silver oxide

Article abstract of DOI:10.1016/j.tetlet.2005.04.131

The human carcinogen diethylstilbestrol (DES) is metabolized into 3′-hydroxydiethylstilbestrol (3′-OH-DES) (1). Chemical oxidation of the catechol metabolites with silver oxide in CH₂Cl₂ affords a novel spiro-quinone (3) in quantitative yield. Protection of the phenolic OH group followed by oxidation gives 4″-OCH₃-DES- 3′,4′-Q (5) in excellent yield.

Full text of DOI:10.1016/j.tetlet.2005.04.131

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 4449–4451
Novel spiro-quinone formation from
30-hydroxydiethylstilbestrol after oxidation with silver oxide
Muhammad Saeed, Eleanor Rogan and Ercole Cavalieri*
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA
Received 22 March 2005; revised 28 April 2005; accepted 28 April 2005
Abstract—The human carcinogen diethylstilbestrol (DES) is metabolized into 30-hydroxydiethylstilbestrol (30-OH-DES) (1).
Chemical oxidation of the catechol metabolites with silver oxide in CH2Cl2 affords a novel spiro-quinone (3) in quantitative yield.
Protection of the phenolic OH group followed by oxidation gives 400-OCH3-DES-30,40-Q (5) in excellent yield.
Ó 2005 Elsevier Ltd. All rights reserved.
Diethylstilbestrol (DES) is a synthetic estrogen, which
was used from 1940 to 1971 as a treatment to prevent
spontaneous abortions in women. It is known to be a
human carcinogen based on sufficient evidence of car-
cinogenicity in both animals1 and humans.2 Despite
intensive investigation, the mechanistic details of carci-
nogenesis by this synthetic estrogen still remain obscure.
In addition to being a potent estrogen, DES is exten-
sively biotransformed in numerous mammalian species
to oxidative metabolites, some of which are capable of
damaging cellular macromolecules.3 Both hormonal
effects and metabolic activation are thought to act
in concert in the process of DES-induced carcinogene-
sis.4,5 DES, analogous to natural estrogens, is mainly
metabolized to the corresponding catechol, 30-OH-
DES,6,7 which can be further oxidized in situ to a reac-
tive ortho-quinone, DES-30,40-Q. The electrophilic
quinone can react with DNA to form depurinating
adducts8–10 that may lead to cancer initiating muta-
tions.11,12
Analysis of depurinating adducts in vivo and in vitro
requires the availability of synthesized depurinating
adducts. In the past, we have synthesized standard
depurinating adducts of natural13,14 and synthetic estro-
gens9,15 by oxidation of the catechol estrogens to the
corresponding ortho-quinones, followed by their reac-
tion with 20-deoxyguanosine (dG) or adenine (Ade) via
a 1,4-Michael addition. To synthesize the standard dep-
urinating adducts of 30-OH-DES (1), we initially fol-
lowed the same strategy of oxidizing the catechol 1
with Ag2O to the corresponding quinone, DES-30,40-Q
(2) and subsequent reaction of the latter with dG or
Ade. Quite surprisingly, we could not detect any adduct
after the reaction mixture was analyzed at different time
1
points by mass spectrometry (MS). The H NMR anal-
ysis of the quinone solution formed by reacting the
catechol 1 with Ag2O in CDCl3 ruled out the formation
of DES-30,40-Q (2) and instead we observed the forma-
tion of a sterically crowded unreactive spiro-quinone
(3) in almost quantitative yield (Scheme 1).16 In this
1
3''
6''
OH
OH
4''
1
2
3
2''
1''
2
2'
5'
2'
3
6
O
O
Ag2O (6 eq)
CH2Cl2, rt
HO
HO
O
O
3'
1'
6'
5''
3'
1'
6'
4
5
4
5
1''
4'
2''
3''
4'
6
5'
6''
4''
5''
1
2
3
O
Scheme 1. Synthesis of spiro-quinone (3) from 30-OH-DES (1).
Keywords: Diethylstilbestrol; Catechol estrogen; Oxidation; Depurinating adducts; spiro-Quinone.
*
Corresponding author. Tel.: +1 402 559 7237; fax: +1 402 559 8068; e-mail: ecavalie@unmc.edu
0040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.04.131

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M. Saeed et al. / Tetrahedron Letters 46 (2005) 4449–4451
communication, we report the formation of a novel
spiro-quinone (3) after oxidation of 1. This finding is
potentially relevant in understanding the mechanism of
tumor initiation by DES.
experiment, indicated the attachment of C-100 with C-60
(Fig. 1). The remaining 13C and 1H signals were also as-
signed and confirmed with the help of APT, HSQC, and
HMBC experiments (Fig. 1).
The structure of 3 was elucidated by detailed analyses.17
The HR-FAB gave a molecular ion peak at m/z
280.31742, corresponding to C18H16O3 (calculated mass
280.31784). Disappearance of characteristic d, d, and dd
The rationale for the production of spiro-quinone (3)
entails the initially formed DES-30,40-Q, which in the
tautomeric form 30-OH-DES-40,400-Q, affords a series
of rearrangements to form, initially, a spiro-catechol
(4) as shown in Scheme 2. Further oxidation of this com-
pound leads to the spiro-quinone (3). The reaction con-
ditions were modified to try to stop the reaction at DES-
30,40-Q, but, we were not able to block the formation of
the spiro-quinone (3). Moreover, the complete forma-
tion of 3 required excess of the oxidant Ag2O
(ꢀ6 equiv). Use of 2 equiv of Ag2O produced only a
mixture of products in which spiro-catechol (4) was a
major product (Table 1, Scheme 2).
1
signals in H NMR and appearance of two singlets at d
6.26 and 6.12 ppm indicated a substitution at C-60 took
place. On the other hand, disappearance of one aro-
matic signal in 13C NMR with concomitant appearance
of an unusual high-field signal at d 58.8 ppm indicated
that one of the aromatic ring sp2 carbons had been con-
verted to an sp3 carbon. Detailed 1- and 2-D NMR spec-
troscopy, including APT, HSQC, and HMBC
experiments, was performed to assign the chemical shifts
1
in H and 13C spectra. The signal at d 58.8 ppm, in the
APT spectrum of 3, was found to be due to a quaternary
carbon and was further confirmed by the absence of
direct 1H/13C correlation in the HSQC experiment.
Alternatively, the conversion of the quinone of DES to
the spiro-compound can be blocked by using a catechol
in which the phenolic ring is protected. Hence, oxidation
of the methoxylated catechol 5 with Ag2O yielded the yel-
lowish green quinone, 400-OCH3-DES-30,40-Q (6) (Scheme
3). Similarly, oxidation of 30-OH-hexestrol(HES) (7), in
which the central double bond C3–C4 is saturated, with
Ag2O formed the corresponding ortho-quinone, HES-
30,40-Q (8) (Scheme 3). These results suggest that the cen-
tral double bond renders the ortho-quinone of DES, DES-
30,40-Q, unstable. This finding is very important with
respect to elucidation of the mechanism of tumor initia-
tion by the synthetic estrogen DES. In fact, formation
of the depurinating adducts at the N-3 position of adenine
and N-7 position of guanine occurs only after treatment
of the catechol, 30-OH-DES, with lactoperoxidase in the
presence of DNA.18 This result suggests that the
1
Furthermore, the long range H/13C correlations of the
carbon at d 58.8 ppm with the protons at d 6.12 (H-
50), 6.65 (H-200), and 6.48 ppm (H-300), in the HMBC
O
58.8
6.65
H
O
6.12
H
H 6.48
O
3
Figure 1. Important long range 1H/13C correlations in spiro-quinone
(3).
OH
HO
OH
O
O
Ag2O (6 eq)
CH2Cl2, rt
HO
HO
O
O
2
1
O
O
HO
O
O
O
H
O
O
HO
HO
Ascorbic
acid
HO
H
O
Ag2O (6 eq)
O
O
O
3
4
Scheme 2. Sequential conversion of DES-30,40-Q (2) to spiro-compounds (3 and 4).

M. Saeed et al. / Tetrahedron Letters 46 (2005) 4449–4451
Table 1. Oxidation of catechols with Ag2Oa
4451
Catechol
Ag2O (equiv)
Solvent
Product
Time (min)
Yield (%)
30-OH-DES (1)
6
2
6
6
6
6
CH2Cl2
CH2Cl2
spiro-Quinone (3)
spiro-Catechol (4)
10
25
30
30
20
30
99
30
70
40
85
99
30-OH-DES (1)
30-OH-DES (1)
CH3COCH3
CH2Cl2
CH3CN
spiro-Quinone (3)
30-OH-400-OCH3-DES (5)
30-OH-400-OCH3-DES (5)
30-OH-HES (7)
400-OCH3-DES-30,40-Q (6)
400-OCH3-DES-30,40-Q (6)
HES-30,40-Q (8)
CH3COCH3
a The reactions were run at room temperature with the exception of 30-OH-400-OCH3-DES (5), which was at 0 °C.
OCH3
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O
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HO
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Ag2O (6 eq)
CH3CN, 0 o
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6
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OH
OH
O
HO
HO
Ag2O (6 eq)
O
CH3COCH3, rt
7
8
Scheme 3. Synthesis of ortho-quinones 6 and 8 from catechols 5 and 7,
respectively.
precursor 30-OH-DES intercalates into DNA to form a
physical complex. Subsequently, lactoperoxidase cata-
lyzes the oxidation to DES-30,40-Q in situ, enabling
reaction with DNA. In conclusion, the instability of the
DES-30,40-Q indirectly proves the importance of the
preliminary physical complex of the 30-OH-DES in the
formation of DNA adducts.
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16. 30-OH-DES (1) was efficiently oxidized to spiro-quinone
(3) under mild oxidation conditions. Typically, a solution
of 30-OH-DES (1) (1 mmol) in CH2Cl2 (10 mL) was stirred
at room temperature and Ag2O (6 mmol, Aldrich Chem-
ical Co.) was added. After 10 min, TLC indicated
complete consumption of 1. The dark orange colored
solution was filtered and the solvent was evaporated under
a stream of nitrogen to afford almost pure 3.
17. Spectroscopic data of 3: 1H NMR (CDCl3): d 6.65 (d,
J = 10.2 Hz, 2H, H-200,600), 6.48 (d, J = 10.2 Hz, 2H,
H-300,500), 6.26 (s, 1H, H-20), 6.12 (s, 1H, H-50), 2.47 (q,
J = 7.81 Hz, 2H, CH2), 2.17 (q, J = 7.81 Hz, 2H, CH2),
1.19 (t, J = 7.81 Hz, 3H, CH3), 1.08 (t, J = 7.81 Hz, 3H,
Acknowledgements
We thank Dr. P. Keifer, Eppley Institute, for helping to
obtain 2-D NMR data. This research was supported by
US Health Service Grant PO1 CA49210 from the Na-
tional Cancer Institute. Core support in the Eppley
Institute is provided by grant P30 CA36727 from the
National Cancer Institute.
References and notes
13
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CH3); C NMR (CDCl3): d 184.9 (C-400), 179.3 (C-40),
179.2 (C-30), 159.7 (C-4), 156.6 (C-10), 155.8 (C-60), 146.5
(CH-200,600), 144.1 (C-3), 130.9 (CH-300,500), 124.7 (CH-50),
115.7 (CH-20), 58.8 (C-100), 20.9 (CH2), 18.8 (CH2), 14.9
(CH3), 13.3 (CH3).
18. Saeed, M.; Rogan, E.; Cavalieri, E. Proc. Am. Assoc.
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