Synthesis and characterization of azole isoflavone inhibitors of aromatase

Article abstract of DOI:10.1016/j.bmc.2005.03.050

The synthesis and biological evaluation of a series of 2-azole and 2-thioazole isoflavones as potential aromatase inhibitors are described. Differences in inhibitory activity of triazole and imidazole inhibitors are rationalized with density functional th

Full text of DOI:10.1016/j.bmc.2005.03.050

Bioorganic & Medicinal Chemistry 13 (2005) 4063–4070
Synthesis and characterization of azole isoflavone
inhibitors of aromatase
John C Hackett, Young-Woo Kim, Bin Su and Robert W. Brueggemeier*
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University,
500 W. 12th Avenue, Columbus, OH 43210, USA
Received 29 January 2005; revised 26 March 2005; accepted 28 March 2005
Available online 25 April 2005
Abstract—The synthesis and biological evaluation of a series of 2-azole and 2-thioazole isoflavones as potential aromatase inhibitors
are described. Differences in inhibitory activity of triazole and imidazole inhibitors are rationalized with density functional theory to
expose a key difference in the electronic structure of these molecules. In addition, difference binding spectra of inhibitors to immu-
noaffinity-purified aromatase produces classical Type II spectra consistent with coordination of the nitrogen lone pair electrons to
the aromatase P450 heme.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
breast cancer.3 Tamoxifen increases long-term survival,
reduces recurrences, and has few side effects. In addition
to tamoxifenÕs antagonistic effects in breast tissue, it also
behaves as a weak or partial agonist in other tissues.
While exhibiting antagonistic activity in the breast, the
partial agonist effects in other parts of the body have
lead to the formation of secondary tumors of the liver
and uterus.4 A second pharmacological approach is to
block estradiol synthesis catalyzed by the cytochrome
P450 enzyme aromatase (CYP19). Aromatase has been
a particularly attractive target for inhibition in the treat-
ment of hormone-dependent breast cancer since the aro-
matization of androgen substrates is the terminal and
rate-limiting step in estrogen biosynthesis.5–8
Estrogens are important in the growth of breast cancers
in both pre- and post-menopausal women and, estrogen
sensitivity in breast cancer increases with patient age.
Two-thirds of breast cancer tumors in post-menopausal
women are positive for estrogen and/or progesterone
receptors, compared to pre-menopausal women where
the presence of these receptors appears in less than half
of tumors. The endogenous ligands for these receptors,
estrogens and progestins, stimulate cell proliferation di-
rectly by increasing the rate of early response genes and
indirectly through the stimulation of growth factors.1
Such dependence on sex steroids for tumor growth
makes hormonal therapy an inviting strategy for
treatment.2
In addition to the large number of aromatase inhibitors
that have arisen from medicinal chemistry efforts in
pharmaceutical and academic laboratories,5–9 a signifi-
cant number of natural product aromatase inhibitors
have been identified.10–12 The flavonoids are one of the
largest classes of naturally occurring aromatase inhibi-
tors.10 Generally, flavones and flavanones have higher
aromatase inhibitory activity than isoflavones. Chrysin
and Biochanin A (BCA) are two of the most potent
examples of flavone and isoflavone identified to date,
respectively (Fig. 1). The flavone, chrysin, is 20-fold
more potent than BCA in terms of IC50 values.11,12 This
large difference in potency is the likely reason why there
has been little effort to develop aromatase inhibitors on
an isoflavone scaffold.
Two general strategies have developed for the treatment
of hormone-dependent breast cancer. The first strategy
is to block estrogen receptor action by disrupting its
interaction with and activation by estradiol. Antiestro-
gens, such as tamoxifen, bind to the estrogen receptor
and interfere with the transcription of estrogen-induced
genes. The efficacy of tamoxifen has been established in
the treatment of post-menopausal, hormone-responsive
Keywords: Aromatase; Inhibitors; Isoflavones; Density functional
theory; Difference spectra.
*
Corresponding author. Tel.: +1 614 292 5231; fax: +1 614 292
3113; e-mail: brueggemeier.1@osu.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.03.050

4064
J. C Hackett et al. / Bioorg. Med. Chem. 13 (2005) 4063–4070
2-position of the isoflavone nucleus is beneficial for aro-
OMe
OH
O
O
matase inhibition. In addition, a hydrophobic function-
ality in the 7-position of the isoflavone and the reduced
necessity of the 40-substituent were important in achiev-
ing desirable affinity for the enzyme.
HO
Biochanin A
(apparent Ki = 12 5 µM)
We have now focused on the synthesis of azole isoflav-
ones, which were easily prepared by the phase transfer
catalysis developed in our laboratory.15,16 In this series,
we are interested in exploring the potential contribution
of the imidazole, triazole, thioimidazole, and thiotriaz-
ole toward aromatase inhibition. Herein, we describe
diversification of the 2-position of isoflavones and exam-
ine their contributions to the structure–activity relation-
ships of azole isoflavone aromatase inhibitors.
OH
O
HO
O
Chrysin
(apparent Ki = 2.6 0.1 µM)
O
2. Results and discussion
BnO
O
S
N
The 2-(methylsulfonyl)isoflavones 1a–d were prepared
from the corresponding 2-(methylthio)isoflavones
with mCPBA using the method previously described
by our laboratory.15,16 Azole isoflavones 2a–d were pre-
pared from 2-(methylsulfonyl)isoflavones 1a–d and thio-
azole isoflavones 3a–d were prepared from 1a in a single
step in moderate to good yields by displacing the 2-
methylsulfonyl group with various nucleophiles (Scheme
1).
7-Benzyloxy-2-(4-pyridylmethylthio)isoflavone
(apparent Ki = 0.22 0.02 µM)
Figure 1. Chemical structures and aromatase inhibitory activities of
biochanin A (isoflavone),14 chrysin (flavone),12 and the 7-benzyloxy-2-
(4-pyridylmethylthio)isoflavone reported by our laboratory.14
Isoflavones have diverse pharmacological activities,13
and we envisioned introduction of the proper functional
groups on the isoflavone core could result in the desired
aromatase activity. Non-steroidal aromatase inhibitors
developed to date are competitive inhibitors possessing
a heteroatom that interferes with steroid hydroxylation
by coordinating to the heme iron at the P450 active site.
Although several heteroatoms, such as sulfur, oxygen,
and nitrogen are capable of coordinating with the iron
atom, the majority of these compounds possess a nitro-
gen-containing heterocycle such as imidazole, triazole,
pyrimidine, or pyridine.9 In our previous work, we dem-
onstrated that the introduction of a 2-(4-pyridylmeth-
yl)thio functionality onto the isoflavone nucleus (Fig.
1) afforded a 160-fold enhancement in potency com-
pared to the natural product lead, BCA.14 This study
demonstrated proof of the principle that introduction
of appropriate nitrogen-containing heterocycle at the
Evaluation of the compounds for aromatase inhibitory
activity was performed using the tritiated water release
aromatase assay with human placental microsomes as
the aromatase source, as previously reported by our lab-
oratory.14 IC50 values of the compounds were deter-
mined in dose–response studies (Table 1). The IC50
values for ( )-aminoglutethimide (AG) and BCA were
also determined for comparison. Kinetic studies were
undertaken on promising compounds to examine their
mode of aromatase inhibition. The apparent Ki values
for each compound, assay-specific apparent Km values,
and Ki/Km ratios are listed in Table 2. The Linewe-
aver–Burk plot of compound 2c is shown in Figure 2.
In dose–response studies (Table 1), 2-thioimidazole
(3b,c) and 2-thiotriazole (3d) isoflavone analogs
Scheme 1. Synthesis of azole isoflavones 2a–d and 3a–d.

J. C Hackett et al. / Bioorg. Med. Chem. 13 (2005) 4063–4070
4065
Table 1. IC50 values for aromatase inhibition by azole isoflavones and
reference compounds
compared to triazole analogs was also observed in other
non-steroidal aromatase inhibitors.9 Recanatini and co-
workers previously postulated the coordinating ability
of the nitrogen heterocycle is related to the distribution
of the highest-occupied molecular orbital (HOMO) and
the resulting electron density over the potentially iron-
coordinating heterocycle. This postulate was used to
partially rationalize the observed differences in potency
between imidazomethyl-and triazomethyl-xanthones.17
To test this hypothesis, we fully optimized the geome-
tries of 2a and 3a with the B3LYP18 hybrid density func-
tional and 6-31G(d) basis set in the Gaussian 9819 suite
of programs. The optimized geometries of these mole-
cules share similar conformational preferences and
may bind similarly to the aromatase active site (Fig.
3). As expected, the HOMO of the imidazole analog
2a is more densely localized on the nitrogen heterocycle
than in 3a. Difference spectra of this pair of compounds
were obtained with immunoaffinity-purified human pla-
cental aromatase (Hauptman–Woodward Medical Re-
search Institute, Buffalo, NY). Both compounds
produce classical Type II spectra consistent with an in-
crease in concentration of low-spin iron as a result of
azole ligation. Compound 2a and 3a have absorption
minima at 394 and 391 nm, respectively, while both
spectra have maxima at 430 nm (Fig. 4). The smaller
change in absorbance induced by compound 3a is
consistent with triazole being a weaker ligand for the
IC50 (lM)
Log IC50 (nM) ( S.E.)a
2a
2b
0.77
2.0
2.89 0.04
3.29 0.04
2.71 0.03
3.67 0.09
4.26 0.09
2c
2d
0.52
4.7
3a
3b
18
>50
>50
>50
2.8
3c
3d
AG
BCA
3.45 0.05
4.53 0.06
34
a IC50 values were calculated by nonlinear regression analysis in
GraphPad Prism. Data for each dose-response curve were obtained
by evaluating aromatase inhibition at ten concentrations of the
compound, each in triplicate.
Table 2. Enzyme kinetic parameters for imidazole isoflavones and
reference compounds
a
Apparent Kia
Apparent Km
Ki/Km
2a
2b
0.68 0.04
1.82 0.15
0.25 0.02
4.40 0.34
1.41 0.10
12 5b
0.10 0.00
0.10 0.01
0.07 0.00
0.12 0.01
0.09 0.01
6.8
18
2c
2d
3.6
37
AG
BCA
16
a Values were calculated by weighted regression analysis13 and
expressed in lM standard error.
b Ref. 11.
10000
7500
5000
2500
0
0.000
0.005
0.010
0.015
0.020
[Androstenedione] (nM-1)
Figure 2. Lineweaver–Burk plot of aromatase inhibition by compound
2c. Androstenedione at concentrations of 50–500 nM was incubated
with microsomal enzyme preparations with inhibitor concentrations of
0 nM (j), 100 nM (m), 500 nM (s), and 2000 nM (h). Each point
represents the mean of three determinations standard error.
displayed poor or no inhibitory activity, with IC50 val-
ues greater than 50 lM. In contrast to the thioazole ana-
logs, compounds with the heterocycle attached directly
to the isoflavone scaffold showed promising inhibitory
activity. The consequence of the nature of the azole het-
erocycle is exemplified by comparing the IC50 values of
7-methoxy analogs 2a and 3a. The imidazole analog
2a is 23-fold more potent than the corresponding tria-
zole. The increased potency of imidazole derivatives
Figure 3. B3LYP/6-31G(d) optimized geometries of compound 2a
(top) and compound 3a (bottom) displaying the surfaces of the
highest-occupied molecular orbital (HOMO). A contour value of
0.05 e/B3 was used.

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J. C Hackett et al. / Bioorg. Med. Chem. 13 (2005) 4063–4070
3. Conclusion
0.06
0.04
0.02
0.00
-0.02
2a
3a
Our recent work on 2-(4-pyridylmethylthio)isoflavones
demonstrated for the first time that effective isoflav-
one-based aromatase inhibitors can be constructed by
the introduction of appropriate nitrogen-containing het-
erocycle. This study further extends this concept that
aromatase inhibitory activity can be achieved with the
isoflavone nucleus by introduction of an appropriate
heme-coordinating nitrogen heterocycle at the 2-posi-
tion of the isoflavone. Comparable potency to the 2-
(4-pyridylmethylthio)isoflavones can be obtained by
introducing an imidazole moiety at the 2-position. En-
zyme kinetic analyses of imidazole isoflavones reveal
these compounds interact competitively with the aroma-
tase active site. As revealed by DFT calculations and the
difference spectra of ligand binding, the superiority of
this heterocycle to achieve aromatase inhibition may
lie in the electronic structure of the nitrogen heterocycle
itself. While investigations are currently underway to re-
solve further aspects of the enzyme–ligand interactions,
these compounds are additional leads in our repertoire
of potent aromatase inhibitors.
360 380 400 420 440 460 480 500
(nm)
λ
Figure 4. Type II difference spectra of immunoaffinity-purified human
placental aromatase induced by 50 lM compound 2a (blue) and
compound 3a (red).
heme iron. Taken together with the density functional
theory (DFT) calculations, these observations explain
the superior potency of the imidazole analog relies on
the availability of the nitrogen lone pair electrons to
coordinate with the aromatase P450 heme.
In general, imidazole isoflavone inhibitors presented in
this work share similar structure–activity relationships
to those reported for 2-(4-pyridylmethyl)thioisoflav-
ones.14 Regardless of the 7-substituent, a decrease in po-
tency was observed when a methoxy group was
introduced at the 40-position. Additionally, the relative
decrease in potency is dependent on the nature of the
7-substituent. 40,7-Dimethoxy analog, 2b, is 2.6-fold less
potent than the 7-methoxy analog 2a. The loss of
potency is more pronounced when considering the 7-
benzyloxy analogs. The presence of the 40-methoxy in
2d results in a nine-fold loss of activity when compared
to 2c. 40,7-Dimethoxy-(4-pyridylmethylthio)isoflavone
and 7-benzyloxy-40-methoxy-2-(4-pyridyl-methylthio)iso-
flavone are 1.2- and 2.5-times less active than their 40-H
analogs. This observation may indicate the nature of the
40-substituent may be a more important factor in the
imidazole isoflavones in terms of aromatase inhibition.
4. Experimental section
4.1. Chemistry
Chemicals were commercially available and used as re-
ceived without further purification, unless otherwise
noted. Moisture sensitive reactions were carried out un-
der a dry argon atmosphere in flame-dried glassware.
Solvents were distilled before use under argon. Tetrahy-
drofuran was distilled from sodium metal in the pres-
ence of benzophenone; dichloromethane was distilled
from calcium hydride. Thin layer chromatography was
performed on pre-coated silica gel F254 plates (What-
man). 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. Infra-
´ ´
red spectra were recorded on a Nicolet Protege 460
spectrometer using KBr pellets. High-resolution electro-
spray ionization mass spectra were obtained on the
Micromass QTOF Electrospray mass spectrometer at
The Ohio State Chemical Instrumentation Center. All
the NMR spectra were recorded on a Bruker DPX
250, or Bruker DRX 400 model spectrometer in either
In enzyme kinetic studies, imidazole isoflavones 2a–d
demonstrated typical competitive-type inhibition in the
Lineweaver–Burk plots (Fig. 2 for 2c; plots for 2a, b, d
not shown) supporting their inhibition of aromatase
by competing with the natural substrate for the active
site. Ki/Km ratios of imidazole isoflavones were calcu-
lated as relative inhibitory potency and the same activity
trend is observed as in the dose–response studies. Com-
pound 2c demonstrates 48-fold enhancement in potency
compared to the natural product lead, BCA. As reflected
by its relative potency, imidazole analog 2c is 4.4-fold
more potent than the widely characterized inhibitor
AG, which is in agreement within the expected experi-
mental uncertainty of the result determined in the
dose–response studies (5.4-fold enhancement in potency
compared to AG). The results of the present enzyme ki-
netic studies emphasize the importance of these struc-
tural modifications for optimization of aromatase
inhibition by isoflavones.
1
DMSO-d6 or CDCl3. Chemical shifts (d) for H NMR
spectra are reported in parts per million to residual sol-
vent protons. Chemical shifts (d) for 13C NMR spectra
are reported in parts per million relative to residual sol-
vent carbons.
4.2. General method A for the preparation of 2-(alkyl-
sulfonyl)isoflavones (1a–d)
A mixture of 7-alkoxy-3-aryl-2-(methylthio)-4H-1-benzo-
pyran-4-one (8.0 mmol) and 3-chloroperoxybenzoic acid
(mCPBA) (4.14 g, 24 mmol) in CH2Cl2 (80 mL) were
stirred under reflux for 2 h. After the solvent was re-
moved under reduced pressure, the residue was

J. C Hackett et al. / Bioorg. Med. Chem. 13 (2005) 4063–4070
4067
suspended in diethyl ether and hexane (1:1, 100 mL),
sonicated, and placed in a refrigerator overnight. The
insoluble solid was collected by filtration, washed with
diethyl ether and hexane (1:1) several times, and recrys-
tallized from ethyl acetate to give desired product.
was obtained as a white solid: mp 226–227 ꢁC; HRMS
calculated for C24H20NaO6S (M+Na)+ 459.0878, found
459.0868. IR (KBr) 1648, 1624, 1609, 1578, 1569, 1510,
1439, 1332, 1292, 1248, 1162, 1138, 1101, 1030, 972, 840,
1
827, 774, 698, 613, 536, 525 cmꢀ1; H NMR (250 MHz,
DMSO-d6) d 7.95 (d, J = 8.9 Hz, 1H), 7.33–7.49 (m,
6H), 7.17–7.21 (m, 3H), 6.94 (d, J = 8.7 Hz, 2H), 5.29
(s, 2H), 3.77 (s, 3H), 3.27 (s, 3H); 13C NMR
4.2.1. 7-Methoxy-2-(methylsulfonyl)-3-phenyl-4H-1-benzo-
pyran-4-one (1a). Compound 1a was synthesized from 7-
methoxy-2-(methylthio)-3-phenyl-4H-1-benzopyran-4-
one (2.39 g, 8.0 mmol) using General Method A, and
2.59 g (98%) of the title compound was obtained as a
pale yellow solid: mp 208–210 ꢁC; HRMS calculated
for C17H14NaO5S (M+Na)+ 353.0460, found 353.0459.
IR (KBr) 1648, 1622, 1604, 1569, 1436, 1326, 1256,
1203, 1155, 1134, 1100, 1052, 1029, 962, 825, 772, 755,
(62.9 MHz, DMSO-d6)
d 176.57, 164.60, 160.32,
157.75, 157.32, 136.79, 132.83, 129.46, 129.10, 128.77,
127.95, 124.45, 121.67, 117.41, 117.38, 113.97, 101.67,
71.14, 55.96, 42.51.
4.3. General Method B for nucleophilic substitution
reactions of 2-(alkylsulfonyl)isoflavones (2a–d and 3a–d)
1
583 cmꢀ1; H NMR (400 MHz, DMSO-d6) d 7.94 (d,
J = 8.9 Hz, 1H), 7.36–7.38 (m, 3H), 7.34 (d,
J = 2.3 Hz, 1H), 7.25–7.27 (m, 2H), 7.12 (dd, J = 8.9,
2.3 Hz, 1H), 3.91 (s, 3H), 3.30 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) d 176.39, 165.70, 157.72, 157.47,
131.39, 129.98, 129.26, 128.41, 127.86, 124.68, 117.28,
117.03, 101.73, 57.30, 42.46.
A solution of 7-alkoxy-3-aryl-2-(methylsulfonyl)-4H-1-
benzopyran-4-one (1.0 mmol) in DMF (1 mL) was
added to a stirred solution of a nucleophile (as a sodium
salt purchased or generated in situ by the treatment of
sodium hydride) in DMF (3 mL) at 0 ꢁC. After stirring
at 0 ꢁC for 0.5 h, most reactions were completed accord-
ing to TLC. The reaction mixture was allowed to warm
to room temperature over 1 h, then cooled to 0 ꢁC, and
quenched with saturated aqueous NH4Cl solution. After
the volatile solvents were removed in vacuo, the residual
solid was suspended in a mixture of water and EtOAc,
ultrasonicated for 5 min, and cooled to 0 ꢁC. Insoluble
solid was collected by filtration and the filter cake was
washed with EtOAc/hexane mixture to give desire prod-
uct. The filtrate was extracted with EtOAc twice
(2 · 10 mL), and the combined organic layer was
washed with brine, dried over MgSO4, filtered, and con-
centrated under reduced pressure. The remnant was
purified by silica gel column chromatography (eluting
with MeOH/CHCl3) to collect additional product. The
combined solid was further purified by recrystallization.
4.2.2. 7-Methoxy-3-(4-methoxyphenyl)-2-(methylsulfon-
yl)-4H-1-benzopyran-4-one (1b). Compound 1b was syn-
thesized from 7-methoxy-3-(4-methoxyphenyl)-2-(meth-
ylthio)-4H-1-benzopyran-4-one
(2.63 g,
8.0 mmol)
using General Method A, and 2.68 g (93%) of the title
compound was obtained as a pale yellow solid: mp
227–228.5 ꢁC; HRMS calculated for C18H16NaO6S
(M+Na)+ 383.0565, found 383.0569. IR (KBr) 1646,
1618, 1610, 1570, 1509, 1438, 1337, 1311, 1261, 1246,
1203, 1176, 1141, 1100, 1024, 973, 955, 849, 773, 588,
1
539, 520, 482 cmꢀ1; H NMR (400 MHz, DMSO-d6) d
7.94 (d, J = 8.9 Hz, 1H), 7.33 (d, J = 2.3 Hz, 1H), 7.19
(d, J = 8.6 Hz, 2H), 7.11 (dd, J = 8.9, 2.3 Hz, 1H), 6.94
(d, J = 8.7 Hz, 2H), 3.91 (s, 3H), 3.77 (s, 3H), 3.28 (s,
3H); 13C NMR (100 MHz, DMSO-d6) d 176.59, 165.65,
160.31, 157.75, 157.43, 132.81, 127.88, 124.37, 121.66,
117.24, 116.96, 113.96, 101.69, 57.27, 55.96, 42.44.
4.3.1. 2-(1H-Imidazol-1-yl)-7-methoxy-3-phenyl-4H-1-benzo-
pyran-4-one (2a). Compound 2a was synthesized from 7-
methoxy-2-(methylsulfonyl)-3-phenyl-4H-1-benzopyran-
4-one (0.330 g, 1.0 mmol) and sodium salt of imidazole
(0.180 g, 2.0 mmol, purchased) using General Method
B, and 0.231 g (73%) of the title compound was obtained
as a white solid (recrystallized from EtOAc): mp 217–
219 ꢁC; HRMS calculated for C19H15N2O3 (M+H)+
319.1082, found 319.1060. IR (KBr) 1640, 1619, 1575,
1492, 1441, 1404, 1342, 1266, 1255, 1201, 1104, 1053,
4.2.3. 2-(Methylsulfonyl)-3-phenyl-7-(benzyloxy)-4H-1-
benzopyran-4-one (1c). Compound 1c was synthesized
from 2-(methylthio)-3-phenyl-7-(benzyloxy)-4H-1-benzo-
pyran-4-one (3.0 g, 8.0 mmol) using General Method A,
and 3.12 g (96%) of the title compound was obtained as
a white solid: mp 198–199 ꢁC; HRMS calculated for
C23H18NaO5S (M+Na)+ 429.0773, found 429.0761. IR
(KBr) 1651, 1620, 1568, 1499, 1440, 1332, 1256, 1161,
1140, 1113, 1027, 1005, 975, 832, 775, 744, 700,
1
1016, 953, 908, 834, 784, 754, 738, 703, 648 cmꢀ1; H
NMR (400 MHz, DMSO-d6) d 7.98 (d, J = 8.9 Hz,
1H), 7.71 (s, 1H), 7.31–7.33 (m, 3H), 7.28 (d,
J = 2.4 Hz, 1H), 7.17–7.20 (m, 3H), 7.12 (dd, J = 8.9,
2.4 Hz, 1H), 6.93 (s, 1H), 3.89 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) d 176.84, 165.15, 156.61, 151.04,
138.23, 131.39, 131.04, 129.92, 129.21, 129.00, 127.81,
120.31, 116.99, 166.20, 116.18, 101.65, 57.14. Elemental
analysis: Calcd for C19H14N2O3 (%): C, 71.69; H, 4.43;
N, 8.80. Found: C, 71.56; H, 4.49; N, 8.78.
1
616 cmꢀ1; H NMR (400 MHz, DMSO-d6) d 7.95 (d,
J = 8.9 Hz, 1H), 7.26–7.48 (m, 11H), 7.19 (dd, J = 8.9,
2.0 Hz, 1H), 5.28 (s, 2H), 3.29 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) d 176.39, 164.66, 157.72, 157.37,
136.77, 131.94, 131.40, 129.99, 129.46, 129.27, 129.10,
128.77, 128.42, 127.94, 124.77, 117.45, 102.71, 71.16,
42.53.
4.2.4. 3-(4-Methoxyphenyl)-2-(methylsulfonyl)-7-(benzyl-
oxy)-4H-1-benzopyran-4-one (1d). Compound 1d was
synthesized from 2-(methylthio)-3-phenyl-7-(benzyloxy)-
4H-1-benzopyran-4-one (3.24 g, 8.0 mmol) using Gen-
eral Method A, and 3.39 g (97%) of the title compound
4.3.2. 2-(1H-Imidazol-1-yl)-7-methoxy-3-(4-methoxyphen-
yl)-4H-1-benzopyran-4-one (2b). Compound 2b was syn-
thesized from 7-methoxy-3-(4-methoxyphenyl)-2-(meth-
ylsulfonyl)-4H-1-benzopyran-4-one (0.36 g, 1.0 mmol)

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J. C Hackett et al. / Bioorg. Med. Chem. 13 (2005) 4063–4070
and sodium salt of imidazole (0.180 g, 2.0 mmol, pur-
chased) using General Method B, and 0.324 g (93%) of
the title compound was obtained as a white solid (recrys-
tallized from EtOH): mp 232–233 ꢁC; HRMS calculated
for C20H16N2NaO4 (M+Na)+ 371.1008, found 371.1015.
IR (KBr) 1639, 1611, 1578, 1515, 1441, 1405, 1342,
1291, 1253, 1201, 1174, 1054, 1024, 831, 816,
4.3.5. 7-Methoxy-3-phenyl-2-(1H-1,2,4-triazol-1-yl)-4H-
1-benzopyran-4-one (3a). Compound 3a was synthesized
from 7-methoxy-2-(methylsulfonyl)-3-phenyl-4H-1-benzo-
pyran-4-one (0.165 g, 0.5 mmol) and sodium salt of
1,2,4-triazole (0.091 g, 1.0 mmol, purchased) using Gen-
eral Method B, and 0.144 g (90%) of the title compound
was obtained as a white solid (recrystallized from
EtOAc): mp 188.5–189.5 ꢁC; HRMS calculated for
C18H13N3NaO3 (M+Na)+ 342.0855, found 342.0843.
IR (KBr) 1643, 1619, 1575, 1502, 1429, 1337, 1262,
1217, 1203, 1124, 1104, 1060, 993, 920, 837, 831, 785,
747 cmꢀ1 1H NMR (400 MHz, CDCl3) d 8.15 (d,
;
J = 8.9 Hz, 1H), 7.62 (s, 1H), 7.12 (d, J = 8.7 Hz,
2H), 7.00–7.03 (m, 2H), 6.96 (s, 1H), 6.89–6.91 (m,
3H), 3.92 (s, 3H), 3.80 (s, 3H); 13C NMR (100 MHz,
CDCl3) d 177.20, 164.98, 160.18, 156.18, 150.21,
137.22, 131.71, 130.38, 128.41, 122.28, 118.77, 117.12,
115.46, 115.01, 114.43, 100.46, 56.41, 55.67. Elemental
analysis: Calcd for C20H16N2O4 (%): C, 68.96; H, 4.63;
N, 8.04. Found: C, 68.73; H, 4.72; N, 7.94.
1
755, 705, 662 cmꢀ1; H NMR (400 MHz, DMSO-d6) d
8.63 (s, 1H), 8.21 (s, 1H), 8.01 (d, J = 8.9 Hz, 1H),
7.28–7.29 (m, 4H), 7.11–7.16 (m, 3H), 3.89 (s, 3H); 13C
NMR (100 MHz, DMSO-d6) d 176.56, 165.48, 156.70,
153.62, 150.08, 147.29, 130.79, 130.67, 129.01, 128.92,
127.98, 119.39, 117.14, 116.57, 101.69, 57.25. Elemental
analysis: Calcd for C18H13N3O3 (%): C, 67.71; H, 4.10;
N, 13.16. Found: C, 67.60; H, 4.14; N, 13.06.
4.3.3. 2-(1H-Imidazol-1-yl)-3-phenyl-7-(benzyloxy)-4H-
1-benzopyran-4-one (2c). Compound 2c was synthesized
from 2-(methylsulfonyl)-3-phenyl-7-(benzyloxy)-4H-1-
benzopyran-4-one (0.41 g, 1.0 mmol) and sodium salt
of imidazole (0.180 g, 2.0 mmol, purchased) using Gen-
eral Method B, and 0.323 g (82%) of the title compound
was obtained as a white solid (recrystallized from
EtOAc): mp 152–153 ꢁC; HRMS calculated for
C25H19N2O3 (M+H)+ 395.1395, found 395.1396. IR
(KBr) 1633, 1610, 1572, 1494, 1441, 1402, 1389, 1336,
1251, 1193, 1150, 1103, 1094, 1051, 1017, 991, 908,
4.3.6. 2-(1H-Imidazolyl-2-thio)-7-methoxy-3-phenyl-4H-
1-benzopyran-4-one (3b). Compound 3b was synthesized
from 7-methoxy-2-(methylsulfonyl)-3-phenyl-4H-1-benzo-
pyran-4-one (0.165 g, 0.5 mmol), 2-mercaptoimidazole
(0.153 g, 1.5 mmol), and sodium hydride (0.036 g,
1.5 mmol) using General Method B, and 0.156 g (89%)
of the title compound was obtained as a white solid
(recrystallized from EtOH): mp 248–250 ꢁC (decom-
posed); HRMS calculated for C19H14N2NaO3S
(M+Na)+ 373.0623, found 373.0640. IR (KBr) 3434,
1622, 1589, 1537, 1437, 1376, 1349, 1331, 1260, 1205,
1
839, 790, 740, 699 cmꢀ1; H NMR (400 MHz, CDCl3)
d 8.17 (d, J = 8.9 Hz, 1H), 7.58 (s, 1H), 7.35–7.45 (m,
8H), 7.19–7.21 (m, 2H), 7.10 (dd, J = 8.9, 2.3 Hz, 1H),
6.99 (s, 1H), 6.97 (d, J = 2.3 Hz, 1H), 6.92 (s, 1H),
5.19 (s, 2H); 13C NMR (100 MHz, CDCl3) d 176.90,
164.06, 156.12, 150.35, 137.19, 135.85, 130.53, 130.41,
129.47, 129.26, 129.13, 128.96, 128.49, 127.91, 118.77,
117.32, 116.08, 114.82, 101.58, 71.14. Elemental analy-
sis: Calcd for C25H18N2O3 (%): C, 76.13; H, 4.60; N,
7.10. Found: C, 76.24; H, 4.71; N, 7.10.
1104, 1028, 939, 831, 784, 760, 711 cmꢀ1 1H NMR
;
(400 MHz, DMSO-d6) d 12.95 (br s, 1H), 7.91 (d,
J = 8.9 Hz, 1H), 7.38–7.47 (m, 4H), 7.33–7.35 (m, 2H),
7.11 (s, 1H), 7.04 (dd, J = 8.9, 2.3 Hz, 1H), 6.55 (d,
J = 2.3 Hz, 1H), 3.81 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) d 173.85, 164.59, 160.91, 158.42, 132.57,
131.69, 131.48, 130.14, 129.27, 129.19, 127.97, 123.41,
122.38, 117.12, 115.75, 100.79, 56.99. Elemental analy-
sis: Calcd for C19H14N2O3S (%): C, 71.69; H, 4.43; N,
8.80. Found: C, 71.56; H, 4.49; N, 8.78.
4.3.4. 2-(1H-Imidazol-1-yl)-3-(4-methoxyphenyl)-7-(benz-
yloxy)-4H-1-benzopyran-4-one (2d). Compound 2d was
synthesized from 3-(4-methoxyphenyl)-2-(methylsulfo-
nyl)-7-(benzyloxy)-4H-1-benzopyran-4-one (0.71 g,
1.63 mmol) and sodium salt of imidazole (0.30 g,
3.25 mmol, purchased) using General Method B, and
0.604 g (87%) of the title compound was obtained as a
white solid (recrystallized from EtOAc): mp 160–
161 ꢁC; HRMS calculated for C26H21N2O4 (M+H)+
425.1501, found 425.1511. IR (KBr) 1637, 1613, 1577,
1512, 1439, 1403, 1339, 1293, 1245, 1194, 1175, 1098,
4.3.7. 7-Methoxy-2-(1-methyl-1H-imidazolyl-2-thio)-3-phen-
yl-4H-1-benzopyran-4-one (3c). Compound 3c was syn-
thesized from 7-methoxy-2-(methylsulfonyl)-3-phenyl-
4H-1-benzopyran-4-one (0.165 g, 0.5 mmol), 2-mer-
capto-1-methylimidazole (0.171 g, 1.5 mmol), and so-
dium hydride (0.036 g, 1.5 mmol) using General
Method B, and 0.175 g (96%) of the title compound
was obtained as a white solid (recrystallized from
1055, 1018, 908, 823, 748, 700, 654 cmꢀ1 1H NMR
;
EtOAc):
172–173 ꢁC;
HRMS
calculated
for
C20H17N2O3S (M+H)+ 365.0960, found 365.0955. IR
(KBr) 1640, 1618, 1590, 1499, 1437, 1368, 1344, 1253,
(400 MHz, CDCl3) d 8.16 (d, J = 8.9 Hz, 1H), 7.61 (s,
1H), 7.36–7.45 (m, 5H), 7.12 (d, J = 8.7 Hz, 2H), 7.09
(dd, J = 8.9, 2.3 Hz, 1H), 7.00 (s, 1H), 6.96 (d,
J = 2.3 Hz, 1H), 6.95 (s, 1H), 6.90 (d, J = 8.7 Hz, 2H),
5.18 (s, 2H), 3.80 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 177.17, 163.99, 160.19, 156.09, 150.23, 137.22, 135.87,
131.70, 130.38, 129.25, 128.94, 128.49, 127.90, 122.25,
118.77, 117.31, 116.01, 115.02, 114.47, 101.54, 71.12,
55.67. Elemental analysis: Calcd for C26H20N2O4 (%):
C, 73.57; H, 4.75; N, 6.60. Found: C, 73.54; H, 4.82;
N, 6.55.
1198, 1099, 1016, 939, 783, 756, 698 cmꢀ1; H NMR
1
(400 MHz, DMSO-d6) d 7.90 (d, J = 9.0 Hz, 1H), 7.51
(d, J = 1.0 Hz, 1H), 7.40–7.48 (m, 3H), 7.34–7.36 (m,
2H), 7.11 (d, J = 1.2 Hz, 1H), 7.03 (dd, J = 9.0, 2.3 Hz,
1H), 6.57 (d, J = 2.3 Hz, 1H), 3.82 (s, 3H), 3.68 (s,
3H); 13C NMR (100 MHz, DMSO-d6) d 173.88,
164.60, 160.85, 158.42, 132.59, 131.89, 131.46, 131.06,
129.30, 129.22, 127.98, 126.94, 123.53, 117.15, 115.70,
100.96, 57.02, 34.68. Elemental analysis: Calcd for

J. C Hackett et al. / Bioorg. Med. Chem. 13 (2005) 4063–4070
4069
C20H16N2O3S (%): C, 65.92; H, 4.43; N, 7.69. Found: C,
65.68; H, 4.46; N, 7.62.
Louis, MO) were used a positive control. IC50 dose–re-
sponse data were analyzed with the Graphpad Prism
(Version 3.0) program.
4.3.8. 7-Methoxy-3-phenyl-2-(1H-1,2,4-triazolyl-3-thio)-
4H-1-benzopyran-4-one (3d). Compound 3d was synthe-
sized from 7-methoxy-2-(methylsulfonyl)-3-phenyl-4H-
1-benzopyran-4-one (0.165 g, 0.5 mmol), 1H-1,2,4-tri-
azole-3-thiol (0.152 g, 1.5 mmol), and sodium hydride
(0.036 g, 1.5 mmol) using General Method B, and
0.075 g (43%) of the title compound was obtained as a
pale yellow solid (recrystallized from EtOH): mp 246–
247 ꢁC; HRMS calculated for C18H13N3NaO3S
(M+Na)+ 374.0575, found 374.0586. IR (KBr) 1612,
1582, 1502, 1440, 1379, 1349, 1262, 1201, 1185, 1104,
4.4.3. Kinetic study. Enzyme kinetic studies of com-
pounds 2a–d were conducted to investigate the nature
of aromatase inhibition. Michaelis-Menten enzyme
kinetic parameters were determined by varying the con-
centration of androst-4-ene-3,17-dione from 50 to
500 nM in the presence of a fixed concentrations of 0,
100, 500, 2000 nM (2a,c) and 0, 2000, 5000, 10,000 nM
(2b,d). Assay conditions were the same as those de-
scribed in the IC50 studies except reactions were initiated
by the addition of 15 lg microsomal protein. Analysis of
the enzyme kinetic data was performed with the
weighted linear regression analysis previously described
by Cleland.21
1014, 969, 939, 830, 783, 758, 700 cmꢀ1
;
1H NMR
(400 MHz, DMSO-d6) 8.69 (s, 1H), 7.91 (d,
d
J = 8.9 Hz, 1H), 7.36–7.45 (m, 3H), 7.32–7.34 (m, 2H),
7.04 (dd, J = 8.9, 2.3 Hz, 1H), 6.73 (d, J = 2.3 Hz, 1H),
3.81 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 174.51,
164.83, 159.47, 158.67, 146.80, 132.74, 131.27, 129.33,
129.17, 127.96, 126.01, 117.02, 116.01, 100.81, 56.99. Ele-
mental analysis: Calcd for C18H13N3O3S (%): C, 61.53;
H, 3.73; N, 11.96. Found: C, 61.27; H, 3.76; N, 11.86.
4.4.4. Determination of P450 difference spectra. Immuno-
affinity-purified human placental cytochrome P450 aro-
matase (Hauptman-Woodward Medical Research
Institute, Buffalo, NY) was diluted to 7.0 lg/mL with
0.1 M Tris Buffer (pH 7.4) in a 50 lL quartz UV cell,
and the absorbance was scanned from 350 to 500 nm.
0.1 M Tris Buffer (pH 7.4) was used as a reference. Com-
pounds 2a and 3a were introduced into the sample cell in
1 lL of ethanol for a final concentration of 50 lM, and
the spectrum was rescanned at the aforementioned
wavelength range. Multiple scans were taken to ensure
spectrum stability for aromatase and aromatase/inhibi-
tor mixtures. Absorbance values of aromatase were sub-
tracted from the aromatase/inhibitor values to generate
the difference spectrum.
4.4. Enzymology
4.4.1. Preparation of human placental microsomes. Hu-
man 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. Micro-
somes were prepared from the remaining tissue using
the method described by Kellis and Vickery.20 Micro-
somal suspensions were stored at ꢀ80 ꢁC until required.
4.4.2. Inhibition study. Inhibition of human placental
aromatase was determined by monitoring the amount
Acknowledgements
3
of H2O released as the enzyme converts [1b-3H]and-
This research was supported by a USAMRMC Breast
Cancer Program grants Pre-doctoral Fellowship
DMAD17-02-1-0529 (J.C.H.), DMAD17-99-1-9342
(Y.W.K.), and Idea Grant DMAD-17-00-1-0388
(R.W.B.). The authors are indebted to the Ohio Super-
computer Center for computational resources.
rost-4-ene-3,17-dione to estrone. Ten inhibitor concen-
trations ranging from 100 nM to 50 lM 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-
phosphate, 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). Reactions were initiated with
the addition of 50 lg microsomal protein. The total
incubation volume was 2.0 mL. Incubations were al-
lowed 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 re-
moved. 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 Prod-
ucts 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 lM ( ) aminoglutethimide (Sigma, St.
References and notes
1. Dickson, R. B.; Lippman, M. E. Endocrine Rev. 1995, 16,
559.
2. McGuire, W. L. In Hormones and Cancer; Iacobelli, S.,
King, R. J. B., Lidner, H. R., Lippman, M. E., Eds.;
Raven: New York, 1980; Vol. 15, pp 337–344.
3. Early Breast Cancer TrialistÕs Group Lancet 1992, 339, 1.
4. Jordan, V. C. Ann. Rev. Pharmacol. Toxicol. 1995, 35,
195.
5. Brodie, A.; Lu, Q.; Long, B. J. Steroid. Biochem. Mol.
Biol. 1999, 69, 205.
6. Santen, R. J.; Harvey, H. A. Endocr. Relat. Cancer 1999,
6, 75.
7. Brueggemeier, R. W. Am. J. Ther. 2001, 8, 333.
8. Brueggemeier, R. W. Expert Rev. Anticancer Ther. 2002, 2,
181.
9. Recanatini, M.; Cavalli, A.; Valenti, P. Med. Res. Rev.
2002, 22, 282.

4070
J. C Hackett et al. / Bioorg. Med. Chem. 13 (2005) 4063–4070
´
10. Hodek, P.; Trefil, P.; Stiborova, M. Chem. Biol. Interact.
2002, 139, 1.
16. Kim, Y.-W.; Mobley, J. A.; Brueggemeier, R. W. Bioorg.
Med. Chem. Lett. 2003, 13, 1475.
11. Kao, Y.-C.; Zhou, C.; Sherman, M.; Laughton, C. A.;
Chen, S. Environ. Health Perspect. 1998, 106, 85.
12. Kellis, J. T., Jr.; Vickery, L. E. Science 1984, 225,
1032.
13. Ren, W.; Qiao, Z.; Wang, H.; Zhu, L.; Zhang, L. Med.
Res. Rev. 2003, 23, 519.
14. Kim, Y.-W.; Hackett, J. C; Brueggemeier, R. W. J. Med.
Chem. 2004, 47, 4032.
15. Kim, Y.-W.; Brueggemeier, R. W. Tetrahedron Lett. 2002,
43, 6113.
17. Recanatini, M.; Bisi, A.; Cavalli, A.; Belluti, F.; Gobbi, S.;
Rampa, A.; Valenti, P.; Palzer, M.; Palusczak, A.;
Hartmann, R. W. J. Med. Chem. 2001, 44, 672.
18. Stevens, P. J.; Devlin, F. J.; Chablowski, C. F.; Frisch, M.
J. J. Phys. Chem. 1994, 98, 11623.
19. Gaussian 98, Revision A.11.3. Frisch, M. J. et al. Gauss-
ian, Inc. Pittsburgh, PA, 2002.
20. Kellis, J. T., Jr.; Vickery, L. E. J. Biol. Chem. 1987, 262,
4413.
21. Cleland, W. W. Method. Enzymol. 1979, 63, 103.