Synthesis and aromatase inhibitory activity of novel pyridine-containing isoflavones

Article abstract of DOI:10.1021/jm0306024

Aromatase, a cytochrome P450 hemoprotein that is responsible for estrogen biosynthesis by conversion of androgens into estrogens, has been an attractive target in the treatment of hormone-dependent breast cancer. As a result, a number of synthetic steroidal or nonsteroidal aromatase inhibitors have been successfully developed. In addition, there are several classes of natural products that exert potent activities in aromatase inhibition, with the flavonoids being most prominent. Previous studies have exploited flavone and flavanone scaffolds for the development of new aromatase inhibitors. In this paper, we describe the design, synthesis, and biological evaluation of a novel series of 2-(4′-pyridylmethyl)thioisoflavones as the first example of synthetic isoflavone-based aromatase inhibitors.

Full text of DOI:10.1021/jm0306024

4032
J . Med. Chem. 2004, 47, 4032-4040
Syn th esis a n d Ar om a ta se In h ibitor y Activity of Novel P yr id in e-Con ta in in g
Isofla von es
Young-Woo Kim, J ohn C Hackett, and Robert W. Brueggemeier*
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University,
500 West 12th Avenue, Columbus, Ohio 43210
Received December 2, 2003
Aromatase, a cytochrome P450 hemoprotein that is responsible for estrogen biosynthesis by
conversion of androgens into estrogens, has been an attractive target in the treatment of
hormone-dependent breast cancer. As a result, a number of synthetic steroidal or nonsteroidal
aromatase inhibitors have been successfully developed. In addition, there are several classes
of natural products that exert potent activities in aromatase inhibition, with the flavonoids
being most prominent. Previous studies have exploited flavone and flavanone scaffolds for the
development of new aromatase inhibitors. In this paper, we describe the design, synthesis,
and biological evaluation of a novel series of 2-(4′-pyridylmethyl)thioisoflavones as the first
example of synthetic isoflavone-based aromatase inhibitors.
In tr od u ction
Flavonoids are a diverse group of plant-derived
chemicals that are produced by various higher plants,1
which can therefore be found in numerous food sources
such as fruits, vegetables, legumes, and whole grains.2
Compounds in this class have shown a wide variety of
biological activities such as antiviral, antiinflammatory,
antibacterial, antifungal, and anticancer activities.3 In
particular, due to their structural and functional simi-
larities to endogenous estrogens, flavonoids have at-
tracted considerable interest as alternative estrogens,
termed phytoestrogens, and extensively studied for their
potential role in many estrogen-dependent diseases
including breast cancer. In fact, numerous flavonoids
have shown interesting pharmacological activities in
breast cancer biology, including binding affinities for
estrogen receptors,4 antiproliferative activities,5 and
inhibitory activities against aromatase enzyme.6
F igu r e 1. Chemical structures and aromatase inhibitory
activities of two isoflavones (genistein, biochanin A) and one
flavone (chrysin).6c
kinases, DNA topoisomerases, and protein kinase C
(PKC). It inhibits angiogenesis and induces apoptosis
and cell cycle arrest in the G2-phase.12
Genistein (GEN, Figure 1) is the most abundant
isoflavone in soybeans. Some researchers suggest that
GEN may be responsible for the relatively low incidence
of hormone-dependent cancers in certain regions with
high consumption of soy foods.7 Indeed, GEN has been
one of the most widely studied natural products and has
shown a number of important biological activities in
breast cancer biology. GEN displays moderate binding
affinities to estrogen receptors.4c GEN exhibits potent
antiproliferative activity in many breast cancer cell
lines, and these effects are mediated by several mech-
anisms of action.8 Furthermore, it is known that GEN
exerts many beneficial effects of endogenous estrogens
on several tissues such as bone9 and cardiovascular
system.10 In addition to estrogenic activities, GEN has
demonstrated a variety of other interesting biological
activities. It exerts antioxidant activity and is a potent
scavenger of hydrogen peroxide.11 GEN has also shown
inhibitory abilities against various enzymes involved in
tumor development and growth such as protein tyrosine
Inspired by the versatility of genistein, our research
group has been investigating exploitation of the isofla-
vone nucleus as a potential privileged structure13 for
the development of new therapeutic agents for hormone-
dependent breast cancer.14 As part of this effort, we
became interested in development of new isoflavone
derivatives targeting aromatase (CYP19), a cytochrome
P450 hemoprotein that is responsible for estrogen
biosynthesis by conversion of androgens into estro-
gens.15 Aromatase has been a particularly attractive
target for inhibition in the treatment of hormone-
dependent breast cancer since the aromatization is the
last step in steroid biosynthesis and the rate-limiting
step for estrogen synthesis.16 In general, isoflavones are
considered less effective than flavones or flavanones, of
which chrysin is a representative (Figure 1), in terms
of aromatase inhibition.6c,17 For this reason, to the best
of our knowledge, there has been no medicinal chemistry
effort to develop isoflavone-based aromatase inhibitors.
Nonetheless, we envisioned that the desired degree of
* To whom correspondence should be addressed: Phone: 614-292-
5231, Fax: 614-292-2435, E-mail: Brueggemeier.1@osu.edu.
10.1021/jm0306024 CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/02/2004

Novel Isoflavones as Aromatase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 4033
inhibitory activity against aromatase could be achieved
by chemical modification of functional groups around
the isoflavone nucleus. In the present paper, we wish
to report the first examples of synthetic isoflavones as
potent aromatase inhibitors.
In h ibitor Design
Most nonsteroidal aromatase inhibitors developed to
date are reversible inhibitors that compete with the
natural substrates, androstenedione and testosterone.
Strong evidence for the binding of flavones to the active
site of aromatase was obtained by difference spectral
absorption studies by Kellis and Vickery.18 They re-
ported R-naphthoflavone could displace androstenedione
from the aromatase active site and induce a spectrum
consistent with the low-spin state of iron. Ibrahim and
Abul-Hajj later reported reduction of the flavone 4-keto
group was detrimental to aromatase inhibition by these
compounds.19 Based on data obtained from site directed
mutagenesis studies and ligand docking into a homology
model of the aromatase protein, a binding orientation
was predicted in which the A and C rings of the flavone
(Figure 1) mimic the C and D rings of the steroid
substrate, respectively. Therefore, the 2-phenyl sub-
stituent is oriented in a region similar to that occupied
by the A ring of the steroid. This analysis places the
flavone 4-keto functionality in the same position as the
steroid 19-angular methyl group with respect to the
heme iron.6c Deductions based on the modeling results
obtained by other research groups, assuming the isofla-
vones bind to the aromatase active site in an orientation
reminiscent of the flavones, we hypothesize the transla-
tion of the phenyl group from the 2 to 3 position of the
flavone core may diminish the ability of the 4-keto
functionality to adequately coordinate to the heme iron
and introduce unfavorable steric interactions within the
enzyme active site.
Competitive nonsteroidal aromatase inhibitors usu-
ally possess a heteroatom that interferes with hydroxyl-
ation of steroids by coordinating with the heme iron of
aromatase. Although several heteroatoms, such as
sulfur, oxygen, and nitrogen, are known to show abilities
to bind to heme iron, the majority of compounds in this
class possess a nitrogen-containing heterocyclic moiety
such as imidazole, triazole, pyrimidine, or pyridine.19
We have envisioned that introduction of the appropriate
heme-coordinating functional group to the 2-position of
the isoflavone could result in a ligand whose binding
mode is now biased to mimic the predicted binding mode
of the flavones. Such an orientation would place the
3-phenyl functionality in the region occupied by the
steroid A-ring, with the 4-keto functionality now point-
ing away from the heme.
F igu r e 2. Chemical structures of the target 2,4′,7-trisubsti-
tuted isoflavones.
unfavorable for aromatase inhibitory activity. For ex-
ample, GEN is 10-fold less potent than its 4′-methoxy
analogue, biochanin A (BCA, Figure 1).6c Therefore, we
were interested in nonpolar alternatives to the 4′-OH
such as methoxy, methyl, and hydrogen. In fact, it was
proposed that isoflavones without the 4′-hydroxyl group
might more efficiently inhibit aromatase.4c With regard
to the 7-position, a 7-hydroxyl group appears to be the
most promising, as its presence is almost ubiquitous
among biologically active natural isoflavones. However,
7-methoxy analogues might also be interesting to in-
vestigate the consequences of masking the hydroxyl
group at the position.
Ch em istr y
The synthesis of the target compounds was carried
out as outlined in Scheme 1 and Scheme 2. The starting
deoxybenzoins 1a -c are commercially available or can
be easily prepared by published procedure.21 The 4-hy-
droxyl groups of compounds 1 were selectively protected
with a methyl or benzyl group under Mitsunobu reaction
conditions to give monoalkyl ethers 2a -e in excellent
yields (Scheme 1). The treatment of each deoxybenzoin
2 was treated with carbon disulfide and an appropriate
alkyl halide in a THF-aqueous NaOH solution in the
presence of 10 mol % of tetrabutylammonium hydro-
gensulfate (n-Bu4N‚HSO4) gave the corresponding
2-(alkylthio)isoflavone 3 in good to excellent yields.
Dealkylation reactions of selected compounds 3 were
performed with boron tribromide in dichloromethane
yielding hydroxy compounds 4a -f (Scheme 2). All
attempts to prepare the 7-hydroxy-4′-methoxy analogue
4g from 3k using typical debenzylation procedures (i.e.
catalytic hydrogenation reactions with various hydrogen
sources in the presence of palladium on carbon) failed
presumably due to catalyst poisoning of the sulfide
group of 3k . The use of 1 equiv of boron tribromide at a
low temperature also proved to be inefficient, only
providing a mixture of 3k , 4f, and 4g. However, we
found that BF3‚OEt2-Me2S reagent is mild enough to
achieve the selective removal of benzyl group, leaving
the 4′-methoxy group intact to give 4g in a good yield.
This reaction condition was originally reported as a mild
alternative debenzylation method in order to avoid
undesirable 1,4-conjugate addition of ethanethiol to
substrates containing a Michael acceptor.22
On the basis of this rationale, we designed a library
of 2,4′,7-trisubstituted isoflavones as shown in Figure
2. In this study, we have focused on the synthesis of
isoflavones containing a (pyridylmethyl)thio moiety at
the 2-position, which would be easily prepared via the
phase transfer catalysis procedure developed in our
laboratory.14a We were also interested in the preparation
of isoflavones containing a non-nitrogenated moiety at
the same position such as benzylthio or allylthio group
for comparison. Previous studies have indicated that the
presence of hydroxyl group at 4′-position seems to be
Biologica l Eva lu a tion
The aromatase assay was performed according to the
modified method of the procedure previously reported
by our laboratory, in which human placental mi-
crosomes were used as the aromatase source.23 IC50
values of the compounds were determined from dose-
response curves (Figure 3 for compounds 3j, 4f, and 4g)

4034 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Sch em e 1. Synthesis of Isoflavones 3a -k
Kim et al.
Sch em e 2. Synthesis of Isoflavones 4a -g
Ta ble 1. Aromatase Inhibitory Activities of Isoflavones 3a -k
and 4a -g
log IC50
compd R1
R2
R3
IC50 (µM) (nM) ((SE)a
3a
3b
3c
3d
3e
3f
3g
3h
3i
OMe OBn allyl
OMe OBn benzyl
>100
>100
>100
>100
-
-
-
-
H
OMe allyl
H
OMe benzyl
H
H
H
Me
OMe (4′-pyridyl)methyl 1.6
OMe (3′-pyridyl)methyl 9.2
OMe (2′-pyridyl)methyl >100
OMe (4′-pyridyl)methyl 3.0
3.21 ( 0.11
3.96 ( 0.16
-
3.48 ( 0.05
3.49 ( 0.02
2.33 ( 0.03
2.72 ( 0.11
-
OMe OMe (4′-pyridyl)methyl 3.1
H OBn (4′-pyridyl)methyl 0.21
OMe OBn (4′-pyridyl)methyl 0.53
F igu r e 3. Aromatase inhibitory activities of compounds 3j
(1), 4f (9), and 4g (2). Aminoglutethimide (0) and biochanin
A (O) were used as references. Error bars represent standard
error (n ) 3), and the data were statistically analyzed by a
nonlinear regression analysis method.
3j
3k
4a
4b
4c
4d
4e
4f
4g
AG
BCA
OH
OH
H
H
H
OH
OH
OH
OH
OH
OH
allyl
benzyl
benzyl
(4′-pyridyl)methyl 0.61
(3′-pyridyl)methyl 3.6
(4′-pyridyl)methyl 0.28
(4′-pyridyl)methyl 0.22
2.8
NDb
NDb
NDb
-
-
2.79 ( 0.11
3.56 ( 0.06
2.44 ( 0.07
2.34 ( 0.04
3.45 ( 0.05
4.53 ( 0.06
and are listed in Table 1. The IC50 values of (()-
aminoglutethimide (AG) and BCA were also determined
in our assay system for comparison. To examine the
mode of aromatase inhibition and more accurately
distinguish relative potencies, kinetic studies for se-
lected compounds (3e, 3h -j, 4f, and 4g) were also
performed, and their apparent Ki values are listed in
Table 2 with apparent Km values and Ki/Km ratios.
Lineweaver-Burk plot of compound 3j, the most potent
analogue in this series, is shown in Figure 4.
OH
OMe OH
34
a
IC50 values were calculated by a nonlinear regression analysis
(GraphPad Prism). Each dose-response curve contained 10 con-
centrations, each in triplicate. Not determined.
b
might play a role in inhibiting aromatase. It is obvious
that the position of the nitrogen atom in the pyridyl
moiety is important for inhibitory activity. 4′-Pyridyl
analogue 3e is ∼6-fold more potent that the 3′-pyridyl
analogue 3f, whereas the 2′-pyridyl analogue 3g has no
observable inhibitory activity in our assay system. This
trend in activity is echoed in the 7-hydroxy analogues
of this series; the 4′-pyridyl analogue 4d is also ∼6-fold
more potent than 3′-pyridyl analogue 4e. These results
suggest that the position of the pyridyl nitrogen influ-
ences inhibitory activity. One possible explanation is
that the pyridyl nitrogen may coordinate the active site
Resu lts a n d Discu ssion
In screening assay studies, analogues lacking a py-
ridyl group (3a -d and 4a -c) showed no significant
inhibition in enzyme assays containing up to 100 µM of
the compound. Therefore, dose-response studies of
these compounds were not pursued. On the other hand,
most of the pyridine-containing analogues showed in-
hibitory activities, suggesting that the functionality

Novel Isoflavones as Aromatase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 4035
Ta ble 2. Enzyme Kinetic Parameters for Selected Isoflavones
and Reference Compounds
4′-methoxy analogue 4g, but ∼2-fold greater than that
of the 7-hydroxy-4′-hydrogen analogue 4d . This result
suggests the presence of a proton acceptor in the 4′
position in the hydroxy analogues may be beneficial for
aromatase activity.
apparent Ki
(µM) ((SE)a
apparent Km
(µM) ((SE)a
compd
Ki/Km
3e
3h
3i
3j
4f
4g
AG
BCA
0.90 ( 0.04
1.16 ( 0.07
1.69 ( 0.18
0.22 ( 0.02
0.31 ( 0.02
0.26 ( 0.02
1.41 ( 0.10
12.0 ( 5b
0.11 ( 0.06
0.12 ( 0.01
0.11 ( 0.01
0.13 ( 0.01
0.11 ( 0.07
0.10 ( 0.02
0.09 ( 0.01
-
8.18
9.67
15.4
1.69
2.82
2.60
15.7
-
To our surprise, 7-benzyloxy analogue 3k , originally
prepared as a precursor of 4g, displayed promising
inhibitory activity. This observation was unexpected
because the compound was assumed to be too bulky to
fit within the enzyme active site. This led us to
synthesize the 7-benzyloxy-4′-hydrogen analogue 3j with
the hypothesis that 7-benzyloxy analogues would dis-
play similar structure-activity relationships at the 4′
position as observed in the 7-methoxy series. Indeed,
the elimination of the 4′-methoxy resulting in 3j proved
effective in producing a compound with enhanced aro-
matase inhibitory activity. In addition, 3j exhibits ∼8-
fold improvement compared to the corresponding 7-meth-
oxy analogue 3e. Finally, we obtained IC50 values in our
assay system for the isoflavone lead compound, BCA,
and the extensively studied aromatase inhibitor AG.
The most potent compound in this series in terms of
data obtained from dose-response studies, 3j, demon-
strated 162- and 13-fold improvements in aromatase
inhibition over these compounds, respectively.
a
Apparent Km, apparent Ki, and SE values were calculated by
weighted regression analysis.24 See ref 6c.
b
F igu r e 4. Lineweaver-Burk plot of aromatase inhibition by
compound 3j. Various concentrations of androstenedione (10-
300 nM) were incubated with microsomal enzyme preparations
at inhibitor concentrations of 0 nM (9), 100 nM (2), 500 nM
(1), or 2000 nM ([). Each point represents the average of three
determinations with less than 10% variation from the mean.
Enzyme kinetic studies were also conducted with the
most potent compounds of this series identified in the
dose-response studies. Compounds 3j, 4f, and 4g
demonstrated competititive-type inhibition with the
androstenedione substrate (Figure 3, plots for 4f and
4g not shown). The computed relative inhibitory potency
ratios reflected the same trend (3j > 4g > 4f) deter-
mined in the dose-response studies. As reflected by the
relative potency, 3j is the most potent compound within
this series and is more potent than AG. This result is
in agreement within the estimated experimental un-
certainty of the result obtained from the dose-response
studies. Compound 3j also demonstrates 50-fold en-
hancement in potency compared to the natural product
lead, BCA. The present enzyme kinetic results empha-
size the importance of these structural modifications for
optimization of aromatase inhibition by isoflavones.
heme iron. A method to probe this potential interaction
is to obtain difference spectra with the active compounds
and the enzyme preparation. Interestingly, we are
unable to obtain spectra supportive of Type II binding
with human placental microsomes or with CYP19 bacu-
lovirus-transfected insect cell microsomes (B-D Bio-
sciences, Woburn, MA).
The 7-hydroxy analogues 4d and 4e exhibit greater
inhibitory activity than the corresponding 7-methoxy
analogues 3e and 3f, respectively. This result suggests
the presence of a hydrogen bond donor at the C-7
position may elicit a favorable interaction with the
enzyme. With regard to the 4′ substituent, the presence
of a hydrogen atom, as in 3e, at this position is ∼2-fold
more favorable in achieving aromatase enzyme activity
compared to the 4′-methyl- and 4′-methoxy-substituted
analogues 3h and 3i. The IC50 values of 3h and 3i are
quite similar, and it is difficult to cast judgment about
the superiority of one compound over another based on
dose-response data alone. Toward this goal, we con-
ducted enzyme kinetic studies (Table 2) of compounds
3e, 3h , and 3i to verify the IC50 results and to more
accurately discern the relative activities of these ana-
logues. These compounds demonstrated typical competi-
tive-type inhibition with androstenedione in Lineweaver-
Burk plots (data not shown). As computed from the
relative potencies (Ki/ Km), 4′-methyl analogue 3h is 37%
more active than the 4′methoxy analogue, 3i. Interest-
ingly, this structure-activity relationship does not
translate to the 7-hydroxy analogues. In this case, the
4′-methoxy analogue is more potent than the 4′-
hydrogen analogue 4d . In addition, the potency of 4′,7-
dihydroxy analogue 4f was comparable to the 7-hydroxy-
Con clu sion s
To date, the isoflavone ring system has been consid-
ered an inappropriate scaffold for development of aro-
matase inhibitors. This study has shown that aromatase
inhibitory activity can be achieved in the isoflavone
nucleus by introducing functional groups with the
potential to coordinate the heme iron. The structure-
activity relationships indicate that the binding modes
of 7-protected analogues 3e-k might be different from
those of 7-hydroxy analogues 4d -g. Hydrogen-bonding
potential appears to be important in the 7-hydroxy
analogues 4d -g for their aromatase inhibitory activity,
whereas hydrophobic interactions appear to play a role
in 7-protected analogues 3e-k . Among the tested isofla-
vones, compounds 3j, 4f, and 4g show more potent
aromatase inhibitory activities than the others. Based
on the kinetic studies, it is clear that these compounds
compete at the active site of aromatase with the natural
substrate, androstenedione. While investigations are
currently underway to evaluate the exact nature of the
enzyme-ligand interactions, these compounds could be

4036 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Kim et al.
g, 31.0 mmol) and methanol (1.32 mL, 32.55 mmol), 6.85 g
(81%) of the title compound was obtained as a white solid: mp
101-102 °C (lit. 104 °C);24b IR (KBr) 1637, 1611, 1513, 1459,
new leads for the development of more potent inhibitors
in this series. Especially of great interest as a new lead
is compound 3j, containing a benzyl group that seems
to be a key feature for the inhibitory activity. Therefore,
one can envision that introduction of a proper function-
ality on the phenyl ring of the 7-benzyloxy group of 3j
may confer more potent aromatase inhibitory activity.
Based on this hypothesis, synthetic efforts for structural
optimization of 3j are also currently underway.
1346, 1301, 1237, 1174, 1148, 1026, 797, 787, 626, 522 cm-1
;
1H NMR (400 MHz, CDCl3) δ 12.72 (br s, 1H), 7.73 (d, J ) 8.8
Hz, 1H), 7.17 (d, J ) 8.6 Hz, 2H), 6.86 (d, J ) 8.6 Hz, 2H),
6.40-6.44 (m, 2H), 4.13 (s, 2H), 3.81 (s, 3H), 3.77 (s, 3H); 13
C
NMR (100 MHz, CDCl3) δ202.73, 166.55, 166.27, 159.07,
132.42, 130.78, 126.73, 114.61, 113.53, 108.21, 101.42, 55.99,
55.67, 44.37.
1-[2-Hyd r oxy-4-(p h en ylm eth oxy)p h en yl]-2-p h en yleth -
a n on e (2d ). Using the previous procedure and starting from
1-(2,4-dihydroxyphenyl)-2-phenylethanone (7.08 g, 31.0 mmol)
and benzyl alcohol (3.37 mL, 32.55 mmol), 8.62 g (87%) of the
title compound was obtained as a white solid: mp 106-108
°C (lit. 104-105 °C);24c IR (KBr) 1620, 1572, 1500, 1389, 1352,
1270, 1230, 1192, 1134, 1000, 974, 830, 760, 729, 697, 628,
561 cm-1; 1H NMR (400 MHz, CDCl3) δ 12.69 (br s, 1H), 7.48
(d, J ) 8.7 Hz, 1H), 7.25-7.39 (m, 10H), 6.49-6.51(m, 2H),
5.07 (s, 2H), 4.20 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 202.41,
166.20, 165.68, 136.22, 134.79, 132.53, 129.76, 129.17, 129.14,
128.76, 127.95, 127.51, 113.77, 108.75, 102.47, 70.66, 45.28;
HRMS calculated for C21H18NaO3 (M + Na)+ 341.1154, found
341.1136.
Exp er im en ta l Section
Unless otherwise noted, chemicals were commercially avail-
able and used as received without further purification. Mois-
ture sensitive reactions were carried out under a dry argon
atmosphere in flame-dried glassware. Solvents were distilled
before use under argon. Tetrahydrofuran was distilled from
sodium metal in the presence of benzophenone; dichlo-
romethane was distilled from calcium hydride. Thin-layer
chromatography was performed on precoated silica gel F254
plates (Whatman). Silica gel column chromatography was
performed using silica gel 60A (Merck, 230-400 Mesh).
Melting points were determined in open glass capillaries using
a Thomas-Hoover apparatus and are uncorrected. Infrared
spectra were recorded on a Nicolet Prote´ge´ 460 spectrometer
using KBr pellets. High-resolution electrospray ionization
mass spectra were obtained on the Micromass QTOF Electro-
spray mass spectrometer at The Ohio State Chemical Instru-
mentation Center. All the NMR spectra were recorded on a
Bruker DPX 250, or Bruker DRX 400 model spectrometer in
either DMSO-d6 or CDCl3. Chemical shifts (δ) for 1H NMR
spectra are reported in parts per million to residual solvent
protons. Chemical shifts (δ) for 13C NMR spectra are reported
in parts per million relative to residual solvent carbons.
Gen er a l P r oced u r e for th e P r ep a r a tion of 4-Alk oxy-
d eoxyben zoin s (2a -e). To a solution of 2-aryl-1-(2,4-dihy-
droxyphenyl)ethanone (31.0 mmol) and alcohol (32.55 mmol)
in THF (150 mL) was added triphenylphosphine (8.538 g, 32.55
mmol), followed by diisopropyl azodicarboxylate (6.41 mL,
32.55 mmol) at 0 °C, and the resulting yellow solution was
stirred at 0 °C for 10 min. The solvent was removed under
reduced pressure, and the oily residue was directly purified
by silica gel column chromatography (eluting with EtOAc:
hexane, 1:4) and recrystallization (EtOAc and hexane) to yield
desired product.
1-[2-Hydr oxy-4-(ph en ylm eth oxy)ph en yl]-2-(4-m eth oxy-
p h en yl)eth a n on e (2e). Using the previous procedure and
starting from 1-(2,4-dihydroxyphenyl)-2-(4-methoxyphenyl)-
ethanone (8.0 g, 31.0 mmol) and benzyl alcohol (3.37 mL, 32.55
mmol), 10.23 g (95%) of the title compound was obtained as a
white solid: mp 97-98 °C (lit. 93-95 °C);24d IR (KBr) 1635,
1611, 1512, 1496, 1387, 1351, 1289, 1227, 1173, 1131, 1029,
994, 948, 842, 830, 791, 745, 725, 695, 535 cm-1; 1H NMR (250
MHz, CDCl3) δ 12.73 (s, 1H), 7.75 (d, J ) 9.6 Hz, 1H), 7.33-
7.40 (m, 5H), 7.18 (d, J ) 8.5 Hz, 2H), 6.87 (d, J ) 8.5 Hz,
2H), 6.53-6.49 (m, 2H), 5.07 (s, 2H), 4.14 (s, 2H), 3.78 (s, 3H);
13C NMR (69.3 MHz, CDCl3) δ 202.77, 166.19, 165.63, 159.10,
136.26, 132.51, 130.82, 129.15, 128.77, 127.97, 126.71, 114.63,
113.73, 108.71, 102.48, 70.65, 55.69, 44.38; HRMS calculated
for C22H20NaO4 (M + Na)+ 371.1259, found 371.1265.
Gen er a l p r oced u r e for t h e p r ep a r a t ion of 2-a lk yl-
th ioisofla von es (3a -k ) fr om 2′-h yd r oxyd eoxyben zoin s.
To a stirred mixture of a deoxybenzoin (1 mmol), carbon
disulfide (0.6 mL, 10 mmol), alkyl halide (2.2 mmol), and
n-Bu4N‚HSO4 (34 mg, 0.1 mmol) in THF (3 mL) and water (1
mL) was slowly added 10 M solution of NaOH in water (1.2
mL, 12 mmol) at room temperature. A slight exothermic
reaction and a color change of the mixture were observed. The
resulting mixture was vigorously stirred at room temperature
for several hours, and the product was extracted with EtOAc
(2 × 10 mL). The combined organic layer was washed with
water (10 mL) and then with brine (10 mL), dried over MgSO4,
and filtered. The filtrate was concentrated under reduced
pressure, and the residue was purified by silica gel column
chromatography (eluting with MeOH/CHCl3 or EtOAc/hexane)
and recrystallization (EtOAc/hexane) to yield desired product.
1-(2-Hydr oxy-4-m eth oxyph en yl)-2-ph en yleth an on e (2a).
Using the previous procedure and starting from 1-(2,4-dihy-
droxyphenyl)-2-phenylethanone (7.08 g, 31.0 mmol) and metha-
nol (1.32 mL, 32.55 mmol), 6.60 g (88%) of the title compound
was obtained as a white solid: mp 87-88 °C (lit. 92 °C);24a IR
(KBr) 1635, 1620, 1589, 1437, 1350, 1291, 1230, 1206, 1127,
1021, 958, 802, 739, 729, 551 cm-1; 1H NMR (400 MHz, CDCl3)
δ 12.71 (br s, 1H), 7.74 (d, J ) 8.7 Hz, 1H), 7.31-7.34 (m, 2H),
7.23-7.26 (m, 3H), 6.40-6.44 (m, 2H), 4.20 (s, 2H), 3.81 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 202.38, 166.61, 166.30,
134.82, 132.47, 129.76, 129.16, 127.50, 113.58, 108.27, 101.44,
56.00, 45.27.
3-(4-Meth oxyp h en yl)-7-(p h en ylm eth oxy)-2-[(p r op en -2-
yl)th io]-4H-1-ben zop yr a n -4-on e (3a ). Using 1-[2-hydroxy-
4-(phenylmethoxy)phenyl]-2-(4-methoxyphenyl)ethanone (0.348
g, 1.0 mmol) as a starting deoxybenzoin and allyl bromide
(0.208 mL, 2.4 mmol) as an alkyl halide, 0.396 g (92%) of the
title compound was obtained as a white solid: mp 101-102
°C; IR (KBr) 1618, 1509, 1438, 1363, 1342, 1291, 1247, 1176,
1152, 1101, 1030, 944, 821, 740, 696 cm-1; 1H NMR (250 MHz,
CDCl3) δ 8.13 (d, J ) 8.8 Hz, 1H), 7.33-7.46 (m, 5H), 7.25 (d,
J ) 8.7 Hz, 2H), 7.02 (dd, J ) 8.9, 2.3 Hz, 1H), 6.95 (d, J )
8.7 Hz, 2H), 6.89 (d, J ) 2.3 Hz, 1H), 5.87 (ddt, J ) 16.9, 10.0,
6.9 Hz, 1H), 5.25 (dd, J ) 16.9, 1.2 Hz, 1H), 5.11-5.06 (m,
3H), 3.82 (s, 3H), 3.69 (d, J ) 6.9 Hz, 2H); 13C NMR (62.9 MHz,
CDCl3) δ 174.34, 163.40, 163.10, 159.93, 158.38, 136.17, 133.30,
132.30, 129.20, 128.84, 128.48, 127.95, 124.64, 122.88, 119.22,
117.86, 114.96, 114.36, 101.29, 71.01, 55.65, 34.59; HRMS
1-(2-H yd r oxy-4-m et h oxyp h en yl)-2-(4-m et h ylp h en yl)-
eth a n on e (2b). Using the previous procedure and starting
from 1-(2,4-dihydroxyphenyl)-2-(4-methylphenyl)ethanone (7.51
g, 31.0 mmol) and methanol (1.32 mL, 32.55 mmol), 7.38 g
(93%) of the title compound was obtained as a white solid: mp
(EtOAc/hexane) 71-72 °C; IR (KBr) 1639, 1623, 1590, 1516,
1508, 1439, 1388, 1355, 1268, 1231, 1205, 1131, 1033, 1010,
957, 800, 780, 571, 504, 491 cm-1; 1H NMR (400 MHz, CDCl3)
δ 12.72 (br s, 1H), 7.73 (d, J ) 8.6 Hz, 1H), 7.24-7.14 (m, 4H),
6.43-6.40 (m, 2H), 4.15 (s, 2H), 3.81 (s, 3H), 2.31 (s, 3H); 13
C
NMR (100 MHz, CDCl3) δ 202.63, 166.56, 166.28, 137.14,
132.47, 131.71, 129.87, 129.61, 113.60, 108.20, 101.43, 55.98,
44.89, 21.48; HRMS calculated for C16H16NaO3 (M + Na)+
279.0997, found 279.0989.
1-(2-Hydr oxy-4-m eth oxyph en yl)-2-(4-m eth oxylph en yl)-
eth a n on e (2c). Using the previous procedure and starting
from 1-(2,4-dihydroxyphenyl)-2-(4-methoxyphenyl)ethanone (8.0
calculated for
453.1123. Anal. (C26H22O4S‚0.3H2O) C, H.
C
26H22NaO4S (M + Na)+ 453.1137, found

Novel Isoflavones as Aromatase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 4037
3-(4-Met h oxyp h en yl)-7-(p h en ylm et h oxy)-2-[(p h en yl-
m eth yl)th io]-4H-1-ben zop yr a n -4-on e (3b). Using 1-[2-hy-
droxy-4-(phenylmethoxy)phenyl]-2-(4-methoxyphenyl)ethan-
one (0.348 g, 1.0 mmol) as a starting deoxybenzoin and benzyl
bromide (0.274 mL, 2.3 mmol) as an alkyl halide, 0.427 g (89%)
of the title compound was obtained as a white solid: mp 131-
132 °C; IR (KBr) 1618, 1508, 1438, 1364, 1247, 1176, 1029,
6.79 (d, J ) 2.1 Hz, 1H), 4.27 (s, 2H), 3.91 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 174.11, 164.24, 162.30, 158.42, 150.24,
149.35, 136.81, 132.93, 132.29, 130.96, 128.86, 128.80, 128.41,
124.11, 123.46, 117.60, 114.66, 100.10, 56.34, 33.21; HRMS
calculated for C22H17NNaO3S (M + Na)+ 398.0827, found
398.0840. Anal. (C22H17NO3S) C, H, N.
7-Met h oxy-3-p h en yl-2-[(2-p yr id ylm et h yl)t h io]-4H -1-
ben zop yr a n -4-on e (3g). Using 1-(2-hydroxy-4-methoxyphen-
yl)-2-phenylethanone (0.242 g, 1.0 mmol) as a starting deoxy-
benzoin and 2-(bromomethyl)pyridine hydrobromide (0.557 g,
2.2 mmol) as an alkyl halide, 0.345 g (92%) of the title
compound was obtained as a white solid: mp 168.5-169 °C;
IR (KBr) 1634, 1617, 1586, 1546, 1502, 1431, 1373, 1344, 1252,
1202, 1153, 1106, 1016, 943, 831, 782, 752, 698, 661 cm-1; 1H
NMR (400 MHz, DMSO-d6) δ 8.47 (ddd, J ) 4.9, 1.7, 0.9 Hz,
1H), 7.88 (d, J ) 8.8 Hz, 1H), 7.74 (dt, J ) 7.7, 1.8 Hz, 1H),
7.49 (d, J ) 7.8 Hz, 1H), 7.30-7.39 (m, 3H), 7.34 (ddd, J )
7.6, 4.9, 0.9 Hz, 1H), 7.17-7.19 (m, 3H), 7.03 (dd, J ) 8.8, 2.4
Hz, 1H), 4.54 (s, 2H), 3.88 (s, 3H); 13C NMR (100 MHz, DMSO-
d6) δ 173.33, 164.34, 163.86, 158.54, 157.30, 150.20, 137.88,
133.14, 131.46, 129.03, 128.86, 127.75, 124.04, 123.43, 122.40,
117.23, 115.46, 101.23, 57.02, 37.39; HRMS calculated for
1
822, 697 cm-1; H NMR (250 MHz, CDCl3) δ 8.14 (d, J ) 8.9
Hz, 1H), 7.22-7.48 (m, 10H), 7.21 (d, J ) 8.7 Hz, 2H), 7.03
(dd, J ) 8.9, 2.3 Hz, 1H), 6.93 (d, J ) 8.7 Hz, 2H), 6.89 (d, J
) 2.3 Hz, 1H), 5.17 (s, 2H), 4.28 (s, 2H), 3.81 (s, 3H); 13C NMR
(62.9 MHz, CDCl3) δ 174.33, 163.61, 163.09, 159.91, 158.38,
136.61, 136.17, 132.25, 129.33, 129.25, 129.18, 128.88, 128.49,
128.15, 127.96, 124.52, 122.42, 117.82, 115.03, 114.36, 101.25,
71.01, 55.65, 36.17; HRMS calculated for C30H24NaO4S (M +
Na)+ 503.1293, found 503.1258. Anal. (C30H24O4S) C, H.
7-Meth oxy-3-ph en yl-2-[(pr open -2-yl)th io]-4H-1-ben zopy-
r a n -4-on e (3c). Using 1-(2-hydroxy-4-methoxyphenyl)-2-phen-
ylethanone (0.242 g, 1.0 mmol) as a starting deoxybenzoin and
allyl bromide (0.190 mL, 2.2 mmol) as an alkyl halide, 0.314
g (96%) of the title compound was obtained as a white solid
(Method B): mp 117-118 °C; IR (KBr) 1635, 1615, 1585, 1546,
1503, 1435, 1373, 1345, 1252, 1197, 1108, 1017, 942, 922, 831,
753, 701, 662 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.13 (d, J )
8.9 Hz, 1H), 7.30-7.44 (m, 5H), 6.96 (dd, J ) 8.9, 2.4 Hz, 1H),
6.81 (d, J ) 2.3 Hz, 1H), 5.83-5.94 (m, 1H), 5.27 (dd, J ) 16.9,
1.2 Hz, 1H), 5.14 (dd, J ) 10.1, 0.8 Hz, 1H), 3.91 (s, 3H), 3.70
(d, J ) 6.9 Hz, 2H); 13C NMR (62.9 MHz, CDCl3) δ 174.21,
164.12, 163.40, 158.50, 133.20, 132.61, 131.08, 128.82, 128.68,
128.39, 123.35, 119.27, 117.69, 114.51, 100.13, 56.31, 34.54;
HRMS calculated for C19H16NaO3S (M + Na)+ 347.0718, found
347.0705. Anal. (C19H16O3S‚0.2H2O) C, H.
C
22H17NNaO3S (M + Na)+ 398.0827, found 398.0819. Anal.
(C22H17NO3S) C, H, N.
7-Met h oxy-3-(4-m et h ylp h en yl)-2-[(4′-p yr id ylm et h yl)-
t h io]-4H-1-b en zop yr a n -4-on e (3h ). Using 1-(2-hydroxy-4-
methoxyphenyl)-2-(4-methylphenyl)ethanone (0.256 g, 1.0 mmol)
as a starting deoxybenzoin and 4-(bromomethyl)pyridine hy-
drobromide (0.557 g, 2.2 mmol) as an alkyl halide, 0.335 g
(86%) of the title compound was obtained as a white solid: mp
157-160 °C; IR (KBr) 1628, 1598, 1585, 1543, 1497, 1434,
1373, 1343, 1254, 1198, 1182, 1099, 1016, 936, 837, 814 cm-1
;
7-Meth oxy-3-p h en yl-2-[(p h en ylm eth yl)th io]-4H-1-ben -
zop yr a n -4-on e (3d ). Using 1-(2-hydroxy-4-methoxyphenyl)-
2-phenylethanone (0.242 g, 1.0 mmol) as a starting deoxyben-
zoin and benzyl bromide (0.262 mL, 2.2 mmol) as an alkyl
halide, 0.365 g (97%) of the title compound was obtained as a
white solid: mp 153-154 °C; IR (KBr) 1636, 1617, 1586, 1546,
1502, 1438, 1373, 1341, 1252, 1205, 1106, 1016, 942, 831, 699,
1H NMR (400 MHz, CDCl3) δ 8.52 (d, J ) 5.6 Hz, 2H), 8.08 (d,
J ) 8.9 Hz, 1H), 7.26 (d, J ) 5.5 Hz, 2H), 7.21 (d, J ) 7.8 Hz,
2H), 7.14 (d, J ) 7.9 Hz, 2H), 6.92 (dd, J ) 8.9, 2.1 Hz, 1H),
6.69 (d, J ) 2.0 Hz, 1H), 4.20 (s, 2H), 3.87 (s, 3H), 2.35 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 174.20, 164.15, 161.79,
158.32, 150.46, 146.37, 138.72, 130.75, 129.67, 129.17, 128.46,
124.05, 123.46, 117.58, 114.44, 100.14, 56.29, 34.79, 21.85;
HRMS calculated for C23H19NNaO3S (M + Na)+ 412.0983,
found 398.1004. Anal. (C23H19NO3S) C, H, N.
1
661 cm-1; H NMR (400 MHz, CDCl3) δ 8.12 (d, J ) 8.9 Hz,
1H), 7.22-7.41 (m, 10H), 6.95 (dd, J ) 8.9, 2.4 Hz, 1H), 6.81
(d, J ) 2.3 Hz, 1H), 4.30 (s, 2H), 3.91 (s, 3H); 13C NMR (62.9
MHz, CDCl3) δ 174.22, 164.10, 163.54, 158.48, 136.51, 132.52,
131.04, 129.32, 129.17, 128.81, 128.66, 128.39, 128.17, 122.97,
117.70, 114.49, 100.19, 56.31, 36.17; HRMS calculated for
7-Meth oxy-3-(4-m eth oxyp h en yl)-2-[(4′-p yr id ylm eth yl)-
th io]-4H-1-ben zop yr a n -4-on e (3i). Using 1-(2-hydroxy-4-
methoxyphenyl)-2-(4-methoxyphenyl)ethanone (0.272 g, 1.0
mmol) as a starting deoxybenzoin and 4-(bromomethyl)-
pyridine hydrobromide (0.557 g, 2.2 mmol) as an alkyl halide,
0.332 g (82%) of the title compound was obtained as a white
solid: mp 140-141 °C; IR (KBr) 1622, 1609, 1549, 1510, 1434,
1369, 1343, 1288, 1250, 1199, 1180, 1099, 1024, 961, 945, 835,
C
23H18NaO3S (M + Na)+ 397.0874, found 397.0856. Anal.
(C23H18O3S) C, H.
7-Met h oxy-3-p h en yl-2-[(4′-p yr id ylm et h yl)t h io]-4H -1-
ben zop yr a n -4-on e (3e). Using 1-(2-hydroxy-4-methoxyphen-
yl)-2-phenylethanone (0.242 g, 1.0 mmol) as a starting deoxy-
benzoin and 4-(bromomethyl)pyridine hydrobromide (0.557 g,
2.2 mmol) as an alkyl halide, 0.305 g (81%) of the title
compound was obtained as a white solid: mp 136-137 °C; IR
(KBr) 1634, 1622, 1600, 1549, 1497, 1433, 1369, 1257, 1200,
1
821, 778 cm-1; H NMR (400 MHz, CDCl3) δ 8.53 (d, J ) 5.8
Hz, 2H), 8.08 (d, J ) 8.9 Hz, 1H), 7.27 (d, J ) 5.7 Hz, 2H),
7.18 (d, J ) 8.6 Hz, 2H), 6.92-6.94 (m, 3H), 6.69 (d, J ) 2.1
Hz, 1H), 4.21 (s, 2H), 3.88 (s, 3H), 3.80 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 174.29, 164.14, 161.82, 160.05, 158.32, 150.42,
146.46, 132.16, 128.48, 124.24, 124.06, 123.11, 117.57, 114.42,
114.40, 100.13, 56.29, 55.65, 34.82; HRMS calculated for
1
1098, 1067, 1013, 943, 831, 775, 756, 703, 658, 570 cm-1; H
NMR (400 MHz, CDCl3) δ 8.53 (dd, J ) 4.5, 1.5 Hz, 2H), 8.09
(d, J ) 8.9 Hz, 1H), 7.33-7.42 (m, 3H), 7.24-7.27 (m, 4H),
6.93 (dd, J ) 8.9, 2.4 Hz, 1H), 6.70 (d, J ) 2.3 Hz, 1H), 4.21
(s, 2H), 3.87 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 174.09,
164.21, 161.95, 158.33, 150.45, 146.38, 132.22, 130.95, 128.89,
128.86, 128.46, 124.04, 123.58, 117.59, 114.50, 100.16, 56.31,
34.79; HRMS calculated for C22H17NNaO3S (M + Na)+ 398.0827,
found 398.0818. Anal. (C22H17NO3S‚0.2H2O) C, H, N.
7-Met h oxy-3-p h en yl-2-[(3-p yr id ylm et h yl)t h io]-4H -1-
ben zop yr a n -4-on e (3f). Using 1-(2-hydroxy-4-methoxyphen-
yl)-2-phenylethanone (0.242 g, 1.0 mmol) as a starting deoxy-
benzoin and 3-(bromomethyl)pyridine hydrobromide (0.557 g,
2.2 mmol) as an alkyl halide, 0.334 g (89%) of the title
compound was obtained as a white solid: mp 151.5-152 °C;
IR (KBr) 1635, 1617, 1585, 1547, 1503, 1438, 1427, 1373, 1344,
1253, 1203, 1108, 1017, 943, 831, 754, 701, 662 cm-1; 1H NMR
(400 MHz, CDCl3) δ 8.62 (br s, 1H), 8.49 (d, J ) 4.3 Hz, 1H),
8.10 (d, J ) 8.9 Hz, 1H), 7.69 (d, J ) 7.9 Hz, 1H), 7.35-7.41
(m, 3H), 7.23-7.28 (m, 3H), 6.95 (dd, J ) 8.9, 2.1 Hz, 1H),
C
23H19NNaO4S (M + Na)+ 428.0932, found 428.0949. Anal.
(C23H19NO4S) C, H, N.
3-P h en yl-7-(ph en ylm eth oxy)-2-[(4′-pyr idylm eth yl)th io]-
4H-1-ben zop yr a n -4-on e (3j). Using 1-[2-hydroxy-4-(phenyl-
methoxy)phenyl]-2-phenylethanone (0.318 g, 1.0 mmol) as a
starting deoxybenzoin and 4-(bromomethyl)pyridine hydro-
bromide (0.557 g, 2.2 mmol) as an alkyl halide, 0.386 g (86%)
of the title compound was obtained as a pale yellow solid: mp
169-170 °C; IR (KBr) 1619, 1599, 1584, 1540, 1491, 1440,
1372, 1344, 1259, 1196, 1157, 1099, 991, 943, 836, 819, 781,
747, 695 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.51 (dd, J ) 4.5,
1.6 Hz, 2H), 8.11 (d, J ) 8.9 Hz, 1H), 7.34-7.45 (m, 8H), 7.22-
7.26 (m, 4H), 7.03 (dd, J ) 8.9, 2.3 Hz, 1H), 6.78 (d, J ) 2.3
Hz, 1H), 5.15 (s, 2H), 4.19 (s, 2H); 13C NMR (100 MHz, CDCl3)
δ 174.03, 163.22, 161.99, 158.23, 150.61, 146.23, 136.06,
132.19, 130.94, 129.25, 128.90, 128.88, 128.57, 127.88, 123.94,
123.59, 117.80, 115.11, 101.25, 71.03, 34.78; HRMS calculated

4038 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Kim et al.
for C28H21NNaO3S (M + Na)+ 474.1140, found 474.1136. Anal.
(C28H21NO3S‚0.1H2O) C, H, N.
title compound was obtained as a white solid: mp 215-217
°C; IR (KBr) 3413, 1610, 1559, 1486, 1453, 1375, 1269, 1250,
1219, 1194, 1105, 970, 947, 848, 699 cm-1; 1H NMR (400 MHz,
DMSO-d6) δ 10.80 (br s, 1H), 7.81 (d, J ) 8.7 Hz, 1H), 7.20-
7.37 (m, 8H), 7.13-7.19 (m, 2H), 6.94 (d, J ) 2.2 Hz, 1H), 6.87
(dd, J ) 8.7, 2.2 Hz, 1H), 4.38 (s, 2H); 13C NMR (100 MHz,
DMSO-d6) δ 173.31, 163.47, 163.20, 158.54, 138.11, 133.26,
131.46, 129.76, 129.41, 128.99, 128.77, 128.24, 128.08, 122.21,
3-(4-Meth oxyph en yl)-7-(p h en ylm eth oxy)-2-[(4′-p yr id yl-
m eth yl)th io]-4H-1-ben zop yr a n -4-on e (3k ). Using 1-[2-hy-
droxy-4-(phenylmethoxy)phenyl]-2-(4-methoxyphenyl)eth-
anone (0.348 g, 1.0 mmol) as a starting deoxybenzoin and
4-(bromomethyl)pyridine hydrobromide (0.557 g, 2.2 mmol) as
an alkyl halide, 0.402 g (84%) of the title compound was
obtained as a white solid: mp 169.5-170.5 °C; IR (KBr) 1616,
1539, 1509, 1440, 1414, 1371, 1342, 1294, 1251, 1197, 1173,
1156, 1100, 1029, 991, 943, 824, 735, 697, 665 cm-1; 1H NMR
(400 MHz, DMSO-d6) δ 8.44 (d, J ) 5.8 Hz, 2H), 7.87 (d, J )
8.8 Hz, 1H), 7.35-7.49 (m, 7H), 7.30 (d, J ) 2.3 Hz, 1H), 7.08-
7.12 (m, 3H), 6.93 (d, J ) 8.7 Hz, 2H), 5.26 (s, 2H), 4.39 (s,
2H), 3.75 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 173.39,
163.30, 162.91, 159.90, 158.31, 150.68, 147.69, 136.95, 132.64,
129.46, 129.08, 128.82, 127.86, 124.79, 124.70, 122.37, 117.32,
115.95, 114.55, 102.25, 70.95, 55.96, 34.03; HRMS calculated
for C29H23NNaO4S (M + Na)+ 504.1245, found 504.1238. Anal.
(C29H23NO4S‚0.2H2O) C, H, N.
Gen er a l P r oced u r e for Dea lk yla tion Usin g Bor on
Tr ibr om id e (4a -g). To a stirred solution of 2-substituted
7-alkoxy-3-aryl-4H-1-benzopyran-4-one (0.5 mmol) in CH2Cl2
(10 mL) was slowly added a 1.0 M solution of BBr3 (2.0 mL, 2
mmol) at 0 °C, and the resulting suspension was allowed to
warm to room temperature and stirred overnight. After being
cooled to 0 °C, the reaction mixture was quenched with water
and concentrated under reduced pressure. The residue was
suspended in a mixture of water and EtOAc, and the insoluble
product was collected by filtration. The filtrate was extracted
with EtOAc twice (2 × 20 mL), and the combined organic layer
was washed with brine, dried over MgSO4, filtered, and
concentrated under reduced pressure to give additional prod-
uct. The combined solid was purified by silica gel column
chromatography (eluting with MeOH/CHCl3) and/or directly
applied to recrystallization.
116.20, 115.97, 102.79, 35.39; HRMS calculated for C22H16
-
NaO3S (M + Na)+ 383.0718, found 383.0710. Anal. (C22H16O3S‚
0.4H2O) C, H.
7-H yd r oxy-3-p h en yl-2-[(4′-p yr id ylm et h yl)t h io]-4H -1-
ben zop yr a n -4-on e (4d ). Using the previous procedure and
starting from 7-methoxy-3-phenyl-2-[(4′-pyridylmethyl)thio]-
4H-1-benzopyran-4-one (0.165 g, 0.439 mmol), 0.098 g (62%)
of the title compound was obtained as a pale yellow solid: mp
229-230 °C (decomposed); IR (KBr) 3427, 1617, 1584, 1544,
1504, 1417, 1366, 1260, 1191, 1102, 1015, 969, 945, 843, 701
1
cm-1; H NMR (400 MHz, DMSO-d6) δ 10.80 (br s, 1H), 8.48
(d, J ) 4.0 Hz, 2H), 7.80 (d, J ) 8.4 Hz, 1H), 7.33-7.40 (m,
5H), 7.16 (d, J ) 7.0 Hz, 2H), 6.86-6.88 (m, 2H), 4.38 (s, 2H);
13C NMR (100 MHz, DMSO-d6) δ 173.28, 163.19, 162.72,
158.48, 150.65, 147.82, 133.12, 131.45, 129.06, 128.87, 128.09,
124.61, 122.50, 116.14, 116.01, 102.71, 34.05; HRMS calculated
for C21H15NNaO3S (M + Na)+ 384.0670, found 384.0667. Anal.
(C21H15NO3S‚0.2H2O) C, H, N.
7-H yd r oxy-3-p h en yl-2-[(3-p yr id ylm et h yl)t h io]-4H -1-
ben zop yr a n -4-on e (4e). Using the previous procedure and
starting from 7-methoxy-3-phenyl-2-[(3-pyridylmethyl)thio]-
4H-1-benzopyran-4-one (0.181 g, 0.482 mmol), 0.082 g (47%)
of the title compound was obtained as a yellow solid: mp 194-
197 °C (decomposed); IR (KBr) 3053, 1618, 1584, 1542, 1502,
1462, 1366, 1268, 1219, 1187, 1102, 970, 946, 843, 782, 754,
1
701 cm-1; H NMR (400 MHz, DMSO-d6) δ 10.82 (br s, 1H),
8.73 (br s, 1H), 8.53 (d, J ) 4.4 Hz, 1H), 8.03 (d, J ) 7.8 Hz,
1H), 7.81 (d, J ) 8.7 Hz, 1H), 7.53 (dd, J ) 7.8, 5.1 Hz, 1H),
7.30-7.39 (m, 3H), 7.13-7.15 (m, 2H), 6.95 (d, J ) 2.1 Hz,
1H), 6.89 (dd, J ) 8.7, 2.1 Hz, 1H), 4.46 (s, 2H); 13C NMR (100
MHz, DMSO-d6) δ 173.51, 163.12, 162.83, 158.55, 148.07,
147.00, 140.30, 136.09, 132.92, 131.36, 129.15, 128.99, 128.13,
125.64, 122.61, 116.10, 116.04, 102.79, 32.23; HRMS calculated
for C21H15NNaO3S (M + Na)+ 384.0670, found 384.0674. Anal.
(C21H15NO3S‚0.3H2O) C, H, N.
7-Hyd r oxy-3-(4-h yd r oxyp h en yl)-2-[(p r op en -2-yl)th io]-
4H-1-ben zop yr a n -4-on e (4a ). Using the previous procedure
and starting from 3-(4-methoxyphenyl)-7-(phenylmethoxy)-2-
[(propen-2-yl)thio]-4H-1-benzopyran-4-one (0.179 g, 0.416 mmol),
0.123 g (91%) of the title compound was obtained as a pale
yellow solid: mp 230-232 °C (decomposed); IR (KBr) 3231,
1610, 1561, 1539, 1512, 1497, 1439, 1376, 1245, 1220, 1194,
1
1174, 1103, 972, 949, 928, 846, 827, 809 cm-1; H NMR (400
7-Hyd r oxy-3-(4-h yd r oxyp h en yl)-2-[(4′-p yr id ylm eth yl)-
th io]-4H-1-ben zop yr a n -4-on e (4f). Using the previous pro-
cedure and starting from 3-(4-methoxyphenyl)-7-(phenyl-
methoxy)-2-[(4′-pyridylmethyl)thio]-4H-1-benzopyran-4-one
(0.173 g, 0.36 mmol), 0.117 g (86%) of the title compound was
obtained as a pale yellow solid: mp > 240 °C (decomposed);
IR (KBr) 3430, 1621, 1607, 1560, 1513, 1499, 1369, 1263, 1234,
1194, 1171, 1107, 950, 807 cm-1; 1H NMR (400 MHz, DMSO-
d6) δ 10.74 (br s, 1H), 9.52 (br s, 1H), 8.76 (d, J ) 6.4 Hz, 2H),
7.94 (d, J ) 6.4 Hz, 2H), 7.78 (d, J ) 9.0 Hz, 1H), 6.97 (d, J )
8.5 Hz, 2H), 6.86-6.88 (m, 2H), 6.76 (d, J ) 8.5 Hz, 2H), 4.58
(s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 173.49, 163.15,
161.71, 158.42, 158.17, 157.23, 144.66, 132.61, 128.07, 126.94,
123.07, 122.47, 116.14, 115.91, 115.89, 102.83, 34.15; HRMS
calculated for C21H15NNaO4S (M + Na)+ 400.0619, found
400.0627. Anal. (C21H15NO4S) C, H, N.
7-Hyd r oxy-3-(4-m eth oxyp h en yl)-2-[(4′-p yr id ylm eth yl)-
th io]-4H-1-ben zop yr a n -4-on e (4g). To a stirred suspension
of 3-(4-methoxyphenyl)-7-(phenylmethoxy)-2-[(4′-pyridylmeth-
yl)thio]-4H-1-benzopyran-4-one (0.200 g, 0.415 mmol) in Me2S
(3.0 mL) and CH2Cl2 (3.0 mL) was slowly added BF3‚OEt2 (1.52
mL, 12 mmol) at room temperature. The resulting yellow
solution was vigorously stirred at room-temperature overnight.
After cooling to 0 °C, the reaction mixture was quenched with
water and concentrated under reduced pressure. The residue
was suspended in a mixture of water and EtOAc, and the
insoluble product was collected by filtration. The filtrate was
extracted with EtOAc twice (2 × 25 mL), and the combined
organic layer was washed with brine, dried over MgSO4,
filtered, and concentrated under reduced pressure to give
additional product. The combined solid was purified by silica
MHz, DMSO-d6) δ 10.72 (br s, 1H), 9.50 (br s, 1H), 7.81 (d, J
) 9.3 Hz, 1H), 6.99 (d, J ) 8.6 Hz, 2H), 6.85-6.88 (m, 2H),
6.74 (d, J ) 8.6 Hz, 2H), 5.84 (ddt, J ) 16.9, 9.9, 6.9 Hz, 1H),
5.27 (dd, J ) 16.9, 1.4 Hz, 1H), 5.09 (dd, J ) 9.9, 1.4 Hz, 1H),
3.75 (d, J ) 6.9 Hz, 2H),; 13C NMR (100 MHz, DMSO-d6) δ
173.61, 163.10, 163.07, 158.51, 158.01, 134.75, 132.67, 128.07,
123.49, 122.50, 119.27, 116.24, 115.83, 115.80, 102.70, 34.24;
HRMS calculated for C18H14NaO4S (M + Na)+ 349.0511, found
349.0529. Anal. (C18H14O4S‚0.1H2O) C, H.
7-Hydr oxy-3-(4-h ydr oxyph en yl)-2-[(ph en ylm eth yl)th io]-
4H-1-ben zop yr a n -4-on e (4b). Using the previous procedure
and starting from 3-(4-methoxyphenyl)-7-(phenylmethoxy)-2-
[(phenylmethyl)thio]-4H-1-benzopyran-4-one (0.147 g, 0.305
mmol), 0.106 g (92%) of the title compound was obtained as a
white solid: mp 261-264 °C (decomposed); IR (KBr) 3280,
1625, 1605, 1592, 1508, 1459, 1375, 1246, 1228, 1189, 1174,
1111, 966, 948, 843, 821, 694 cm-1; 1H NMR (400 MHz, DMSO-
d6) δ 10.75 (br s, 1H), 9.49 (br s, 1H), 7.79 (d, J ) 8.7 Hz, 1H),
7.35-7.37 (m, 2H), 7.18-7.29 (m, 3H), 6.94 (d, J ) 8.5 Hz,
2H), 6.91 (d, J ) 2.2 Hz, 1H), 6.86 (dd, J ) 8.7, 2.2 Hz, 1H),
6.72 (d, J ) 8.5 Hz, 2H), 4.36 (s, 2H); 13C NMR (100 MHz,
DMSO-d6) δ 173.51, 163.25, 163.06, 158.48, 158.01, 138.18,
132.61, 129.75, 129.40, 128.20, 128.06, 123.38, 121.96, 116.23,
115.86, 115.81, 102.73, 35.38; HRMS calculated for C22H16
-
NaO4S (M + Na)+ 399.0667, found 399.0656. Anal. (C22H16O4S‚
0.5H2O) C, H.
7-Hyd r oxy-3-p h en yl-2-[(p h en ylm eth yl)th io]-4H-1-ben -
zop yr a n -4-on e (4c). Using the previous procedure and start-
ing from 7-methoxy-3-phenyl-2-[(phenylmethyl)thio]-4H-1-
benzopyran-4-one (0.223 g, 0.596 mmol), 0.189 g (88%) of the

Novel Isoflavones as Aromatase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 4039
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gel column chromatography (eluting with MeOH/CHCl3) and
then recrystallized from ethanol to give a white solid (0.136
g, 84%): mp 197-200 °C; IR (KBr) 3400, 1623, 1606, 1561,
1512, 1455, 1363, 1293, 1247, 1219, 1180, 1106, 1076, 1023,
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loss caused by estrogen deficiency. Endocrinology 1999, 140,
1893-1900. (b) Yamaguchi, M.; Sugimoto, E. Stimulatory effect
of genistein and daidzein on protein synthesis in osteoblastic
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plasma lipids in normocholesterolemic, premenopausal women.
Am. J . Clin. Nutr. 2000, 71, 1462-1469.
1003, 968, 856, 822 cm-1 1H NMR (400 MHz, DMSO-d6) δ
;
10.76 (br s, 1H), 8.79 (d, J ) 6.4 Hz, 2H), 8.01 (d, J ) 6.5 Hz,
2H), 7.80 (d, J ) 8.5 Hz, 1H), 7.11 (d, J ) 8.7 Hz, 2H), 6.94 (d,
J ) 8.7 Hz, 2H), 6.86-6.89 (m, 2H), 4.56 (s, 2H), 3.73 (s, 3H);
13C NMR (100 MHz, DMSO-d6) δ 173.45, 163.22, 161.74,
159.96, 158.48, 158.09, 144.07, 132.68, 128.10, 127.18, 124.80,
122.28, 116.13, 115.98, 114.57, 102.87, 56.00, 34.12; HRMS
calculated for C22H18NO4S (M + H)+ 392.0956, found 392.0962.
Anal. (C22H17NO4S) C, H, N.
P r ep a r a tion of Hu m a n P la cen ta l Micr osom es. Human
term placentas were processed immediately after delivery from
The Ohio State University Hospitals at 4 °C. The placenta was
washed with normal saline, and connective and vascular tissue
was removed. Microsomes were prepared from the remaining
tissue using the method described by Kellis and Vickery.18
Microsomal suspensions were stored at -80 °C until required.
In h ibition Stu d y. Inhibition of human placental aro-
matase was determined by monitoring the amount of 3H2O
released as the enzyme converts [1â-3H]androst-4-ene-3,17-
dione to estrone. Ten inhibitor concentrations ranging from
100 nM to 50 µM were evaluated. Aromatase activity assays
were carried in 0.1 M potassium phosphate buffer (pH 7.0)
with 5% propylene glycol. All samples contained a NADPH
regenerating system consisting of 2.85 mM glucose-6-phos-
phate, 1.8 mM NADP+ and 1.5 units of glucose-6-phosphate
dehydrogenase (Sigma, St. Louis, MO). Samples contained 100
nM androst-4-ene-3,17-dione (400 000-450 000 dpm). Reac-
tions were initiated with the addition of 50 µg of microsomal
protein. The total incubation volume was 2.0 mL. Incubations
were allowed to proceed for 15 min in a shaking water bath
at 37 °C. Reactions were quenched by the addition of 2.0 mL
of chloroform. Samples were then vortexed and centrifuged
for 5 min and the aqueous layer was removed. The aqueous
layer was subsequently extracted twice in the same manner
with 2.0 mL chloroform. A 0.5 mL aliquot of the final aqueous
layer was combined with 5 mL 3a70B scintillation cocktail
(Research Products International Corp., Mt. Prospect, IL) and
the amount of radioactivity determined. Each sample was run
in triplicate, and background values were determined with
microsomal protein inactivated by boiling. Samples containing
50 µM (() aminoglutethimide (Sigma, St. Louis, MO) were
used a positive control. IC50 sigmoidal dose-response data
were analyzed with the Graphpad Prism (Version 3.0) pro-
gram.
Kin etic Stu d y. Enzyme kinetic studies of compounds 3e,
3h -j, 4f, and 4g were conducted to investigate the nature of
aromatase inhibition. Michaelis-Menten enzyme kinetic pa-
rameters were determined by varying the concentration of
androst-4-ene-3,17-dione from 10 to 300 nM in the prescence
of a fixed concentration of 0, 100, 500, and 2000 nM inhibitor.
Assay conditions were the same as those described in the IC50
studies except reactions were initiated by the addition of 15
µg microsomal protein. Analysis of the enzyme kinetic data
was performed with the weighted linear regression analysis
previously described by Cleland.25
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tyrosine kinase inhibitor genistein on mammalian DNA topo-
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breast carcinoma cell lines characterized by P21WAF1/CIP1
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69, 44-54.
Ack n ow led gm en t. This research was supported by
a USAMRMC Breast Cancer Program Predoctoral Fel-
lowship grant DMAD17-99-1-9342 (Y.W.K.), Predoctoral
Fellowship grant DMAD17-02-1-0529 (J .C.H.), and Idea
Grant DMAD17-00-1-0388 (R.W.B.).
Su p p or tin g In for m a tion Ava ila ble: Elemental analyses
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
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