Synthesis of some novel androstanes as potential aromatase inhibitors

Article abstract of DOI:10.1016/j.steroids.2010.12.013

Novel pyrazole and isoxazole derivatives (6-9) were synthesized as a aromatase inhibitors. Pyrazole was synthesized from hydrazine hydrate and isoxazoles from hydroxylamine hydrochloride under different conditions. Molecular docking studies were carried out for the synthesized compounds. The best score was obtained for the compound (9) followed by compound (6) while compound (8) afforded poorest of the score. Aromatase inhibitory activity for compound (6) having pyrazole ring at 2,3 position showed highest activity followed by nitrile derivative (9). Isomeric forms of isoxazole (7 and 8) showed very poor activity compared to fadrozole and aminoglutethimide. Preliminary kinetic studies have shown that both of the active compounds (6 and 9) are reversible inhibitors of the enzyme.

Full text of DOI:10.1016/j.steroids.2010.12.013

Steroids 76 (2011) 464–470
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of some novel androstanes as potential aromatase inhibitors
Mange Ram Yadava,∗, Prafulla M. Sabalea, Rajani Giridhara, Christina Zimmerb, Jorg Haupenthalb,
Rolf W. Hartmannb
a Pharmacy Department, Faculty of Technology and Engineering, Kalabhavan, The M.S. University of Baroda, Vadodara 390 001, Gujarat, India
b Pharmaceutical and Medicinal Chemistry, Saarland University & Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C 2 3, D-66123 Saarbrücken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 May 2010
Received in revised form 4 November 2010
Accepted 31 December 2010
Available online 6 January 2011
Novel pyrazole and isoxazole derivatives (6–9) were synthesized as a aromatase inhibitors. Pyrazole was
synthesized from hydrazine hydrate and isoxazoles from hydroxylamine hydrochloride under different
conditions. Molecular docking studies were carried out for the synthesized compounds. The best score
was obtained for the compound (9) followed by compound (6) while compound (8) afforded poorest of
the score. Aromatase inhibitory activity for compound (6) having pyrazole ring at 2,3 position showed
highest activity followed by nitrile derivative (9). Isomeric forms of isoxazole (7 and 8) showed very poor
activity compared to fadrozole and aminoglutethimide. Preliminary kinetic studies have shown that both
of the active compounds (6 and 9) are reversible inhibitors of the enzyme.
Keywords:
Androstane
Aromatase inhibitors
Breast cancer
© 2011 Elsevier Inc. All rights reserved.
Molecular modeling
1. Introduction
postmenopausal women the main source is adipose tissue and
muscle [5]. Aromatase (CYP19) is the cytochrome P450 enzyme
Estrogens are essential regulators of many physiological pro-
cesses including maintenance of the female sexual organs, the
reproductive cycle and numerous neuroendocrine functions. Along
with these normal physiological functions, these hormones also
play crucial roles in some disease states, particularly in breast
cancer, where through binding to their target receptor, they pro-
mote proliferation of breast cancer cells [1]. Breast cancer is the
most common cancer diagnosed in women, and despite advances
in treatment, remains the second most common cause of death
in women in the Western world. Generally, it is thought of as a
disease of older women; however 22% of the cases occur in those
below the age of 50 [2]. Worldwide, more than one million women
develop breast cancer each year with nearly half of these diagnoses
occurring in the Unites States and Europe. Moreover, nearly 40% of
these women die of their diseases [3]. Approximately two-thirds of
postmenopausal breast cancer patients have estrogen-dependent
breast cancer, which contains estrogen receptors (ERs) and requires
estrogen for tumor growth [4].
responsible for the conversion of androgens including androstene-
dione and testosterone, to the estrogen products, estrone and
estradiol [6]. Expression of aromatase is highest in or near the breast
tumor cells [7,8]. Endocrine-deprivation therapy for breast cancer
is based on the knowledge that certain tumors require estrogen
for their continued growth [9]. Antagonizing estrogen action is a
popular treatment strategy because ER overexpression is observed
in about 70% breast cancers [10–12]. Nonsteroidal antiestrogens
like tamoxifen are the most commonly used drugs in this category.
Apart from its side effects, the use of tamoxifen beyond five years is
not indicated leaving a large population of breast cancer survivors
at risk for relapse [13]. An alternative strategy of hormone depri-
vation is to block specifically estrogen biosynthesis, irrespective of
site of production [14]. Aromatase has been a particularly attrac-
tive target for inhibition in the treatment of hormone-dependant
breast cancer since the aromatization of androgen substrates is the
terminal and rate limiting step in estrogen biosynthesis [15].
Over the past two decades substantial efforts have been made
towards developing potent inhibitors of aromatase [16]. Aromatase
inhibitors have been subdivided into two main groups accord-
ing to their mechanism of action and structure. Type-I inhibitors
are steroidal in nature. These steroidal inhibitors associate to the
substrate-binding site of the enzyme and might inactivate it irre-
versibly. Such mechanism-based inactivators of aromatase are very
specific and show prolonged effects as neosynthesis of the enzyme
is necessary for estrogen formation. The type-II inhibitors are azoles
chemically. They are competitive reversible inhibitors and may lack
Production of estrogens takes place in many tissues throughout
the body including the ovaries, adipose tissue, muscle, liver, breast
tissue and malignant breast tumors. In premenopausal women the
ovaries are the main source of circulating estrogens while in the
∗
Corresponding author.
E-mail addresses: mryadav11@yahoo.co.in, mryadav-phar@msu.ac.in
(M.R. Yadav).
0039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.12.013

M.R. Yadav et al. / Steroids 76 (2011) 464–470
465
specificity [14]. However, until today several classes of potent and
2. Experimental
selective type-II inhibitors have been described [16–20]. Among
those, three third generation aromatase inhibitors, anastrozole (1)
[21], letrozole (2) [22] and the steroidal compound exemestane
(3) [23] have been approved by US-FDA for use in breast cancer,
with various other clinical applications being currently tested after
these products had became commercially available [24–26]. Aro-
matase inhibitors can be utilized in several adjuvant settings [27].
Sufficient data are available to recommend the use of aromatase
inhibitors as better alternatives to tamoxifen for adjuvant ther-
apy [28]. Furthermore, these inhibitors have also been shown to
be clinically effective in the management of other estrogen depen-
dant pathological processes such as endometriosis [29], prostate
hyperplasia and prostate cancer [30]. In addition, unlike tamox-
ifen that has mixed estrogen agonist and antagonist properties,
the aromatase inhibitors have no estrogenic agonist activity [31].
2.1. General
Melting points were determined using a VEEGO make micropro-
cessor based melting point apparatus having silicone oil bath and
are uncorrected. IR spectra (wave numbers in cm−1) were recorded
on a BRUKER ALPHA T FT-IR spectrophotometer using KBr discs.
NMR spectra were recorded on BRUKER AVANCE II 400 MHz instru-
ment in CDCl3 with TMS as internal standard for 1H NMR. Chemical
shift values are mentioned in ı, ppm. Chromatographic separations
were performed on silica gel columns. The progress of all reactions
was monitored by TLC on 2 cm × 5 cm pre-coated silica gel 60 F254
plates of thickness of 0.25 mm (Merck). The chromatograms were
visualized under UV (254 nm) and iodine vapours. The term “dried”
O
Me
N
N
N
N
N
Me
N
CN
O
NC
Me
CH2
Me
Me
CN
NC
Me
(2)
(1)
(3)
Treatment with aromatase inhibitors is generally well tolerated
with low incidence of serious side effects. However short-term
events like hot flushes, vaginal dryness, musculoskeletal pain and
headache have been observed [2,32]. Accordingly there is need for
new, potent, more selective and less toxic CYP19 inhibitors [33].
Eventually new aromatase inhibitors could also be superior to the
current compounds regarding the acquirement of resistance [1].
Mechanism-based inhibitors have distinct advantages in drug
design strategy because these inhibitors are highly enzyme specific,
produce prolonged inhibition and often exhibit minimum toxicitiy
[4]. Exemestane (3), a third generation steroidal mechanism-based
aromatase inhibitor has not been found to affect cortisol or aldos-
terone secretion at baseline or in response to adrenocorticotropic
hormone at any dose [34]. The apparent lack of cross-resistance
between exemestane (3) and other aromatase inhibitors is a major
benefit of the drug [35]. Of the three agents, exemestane (3) is
the most recently FDA approved drug. It may differ from the oth-
ers as the steroidal structure potentially protects bone and lipid
metabolism from estrogen ablation [11].
In the steroidal moieties, effective aromatase enzyme inhibition
is observed with an androstane skeleton possessing an A/B trans
ring junction, a ketone functionality at the C-3 position, unsat-
uration in the steroid nucleus (4-ene, 4,6-diene or 1,4,6-triene
functions) and either a 17-ketone or 17␤-hydroxyl group [4]. In
light of the above discussion it was planned to synthesize some
novel steroidal derivatives with the given structure (A) wherein the
2,3 position of the ring-A is fused to five-membered heterocyclic
rings like isoxazole (X = O/N and Y = N/O) and pyrazole (X = Y = N).
refers to the use of anhydrous sodium sulfate. All reagents used
were of analytical reagent grade.
2.2. Chemical
2.2.1. 2-Formyl 4,17ˇ-dihydroxy-4-androsten-3-one (5)
4-Hydroxytestosterone (4) [36] (0.5 g, 0.016 mol) and sodium
methoxide (1.0 g) in dry pyridine (15 ml) were stirred under nitro-
gen at −5 ◦C for 20 min. Freshly distilled ethyl formate (1.0 ml) was
added to the reaction mixture maintaining the temperature at 0 ◦C.
The reaction mixture was left in refrigerator for 72 h with occasional
shaking. The reaction mixture was acidified with conc. HCl, diluted
with cold water and extracted with solvent ether. The solvent ether
extract was washed with water and extracted with 10% NaOH solu-
tion. The alkaline solution was washed with solvent ether, acidified
with conc. HCl and extracted with solvent ether. The ether layer so
obtained was dried and evaporated slowly, to afford compound (5)
(0.2 g, 40%, mp 208–212 ◦C) after crystallization.
UV (MeOH): 374 nm (log Є4.22), UV (Alk. MeOH): 379 nm
(log Є4.38). IR (KBr): 2942, 2844, 1695, 1640, 1417, 1173, 1070,
939. 1H NMR: ı 10.29 (s, 1H); 3.64–3.68 (t, 1H); 2.77–2.81 (d, 1H);
2.29–2.33 (dd, 1H), 2.12–2.16 (d, 1H); 1.21–1.35 (m, 4H); 0.89–1.11
(m, 6H); 0.86 (s, 3H); 0.75 (s, 3H).
2.2.2. 17ˇ-Hydroxy-4-oxo-5˛-androstano[2,3-d]pyrazole (6)
Compound (5) (0.5 g, 0.0015 mol) and hydrazine hydrate
(0.09 ml, 0.0018 mol) were refluxed in aldehyde-free ethanol
(25 ml) for 2 h. Excess ethanol was removed and the reaction
mixture was poured into cold water, filtered and dried. The
dried residue afforded compound (6) after recrystallization from
methanol (0.2 g, 50%, mp 245–249 ◦C).
OH
Me
Me
UV (MeOH): 270 nm (log Є4.31). IR (KBr): 3388, 3258, 3319,
2944, 2844, 1634, 1545, 1447, 1259, 1074, 959. 1H NMR: ı 12.5
(s, 1H); 7.24 (s, 1H); 3.51–3.55 (t, 1H); 2.70–2.74 (d, 1H); 2.51–2.53
(dd, 1H); 2.20–2.24 (d, 1H); 1.19–1.38 (m, 6H); 0.92–1.18 (m, 4H);
0.68 (s, 3H); 0.64 (s, 3H).
X
Y
OH
(A)

466
M.R. Yadav et al. / Steroids 76 (2011) 464–470
2.2.3. 17ˇ-Hydroxy-4-oxo-5˛-androstano[3,2-c]isoxazole (7)
Compound (5) (0.5 g, 0.0015 mol) and hydroxylamine
hydrochloride (0.25 g, 0.036 mol) were stirred in aldehyde-
free ethanol (25 ml) containing a few drops of pyridine for 2 h
at room temperature and left overnight. Excess of ethanol was
recovered under reduced pressure and the reaction mixture was
poured into water, filtered and dried. The dried residue afforded
compound (7) after recrystallization from ether (0.3 g, 60%, mp
210–212 ◦C).
UV (MeOH): 310 nm (log Є3.80), UV (Alk. MeOH): 362 nm
(log Є3.89). IR (KBr): 3300, 2941, 2844, 1676, 1451, 1397, 1250,
1001, 944. 1H NMR: ı 8.29 (s, 1H); 3.55–3.58 (t, 1H); 2.82–2.86 (d,
1H); 2.27 (s, 3H); 2.20–2.25 (dd, 1H); 1.83–1.87 (d, 1H); 1.06–1.23
(m, 6H); 0.86–1.03 (m, 4H); 0.85 (s, 3H); 0.71 (s, 3H).
assigned by the Gasteiger–Huckel method with a distance depen-
dent dielectric function until a root mean square (rms) deviation of
0.001 kcal/mol A was achieved. The lowest energy conformer thus
˚
obtained was subsequently used for docking studies.
2.3.2. Docking studies
The crystal structure of human placental aromatase (pdb code:
3EQM) [39] obtained from the Protein Data Bank (USA) was refined
to remove water molecules. The bond orders and formal charges
were adjusted prior to docking. Docking was performed using
GLIDE software according to the previously reported protocol
except for scaling of van der Waals radii which was modified (scal-
ing factor 0.60).
2.4. Biological
2.2.4. 17ˇ-Acetoxy-4-oxo-5˛-androstano[2,3-d]isoxazole (8)
Compound (5) (0.5 g, 0.0015 mol) was heated with hydrox-
ylamine hydrochloride (0.25 g, 0.004 mol) in glacial acetic acid
(10 ml) for 8 h at 80 ◦C and left overnight. Glacial acetic acid was
removed under reduced pressure and the reaction mixture was
poured into water, filtered and dried. The dried residue afforded
compound (8) after recrystallization from methanol (0.25 g, 60%,
mp 221–225 ◦C).
UV (MeOH): 256 nm (log Є4.29). UV (Alk. MeOH): 336 nm
(log Є4.11). IR (KBr): 2947, 2842, 1732, 1694, 1452, 1364, 1240,
1171, 1042, 937, 906. (1H NMR): ı 8.26 (s, 1H); 4.61–4.65 (t, 1H);
2.81–2.85 (d, 1H); 2.56–2.60 (d, 1H); 2.45–2.49 (dd, 1H); 2.05 (s,
3H); 0.89 (s, 3H); 0.81 (s, 3H).
2.4.1. Aromatase inhibiting activity
2.4.1.1. Enzyme preparation. The enzyme was obtained from the
microsomal fraction of freshly delivered human term placental tis-
sue as per the procedure described by Thompson and Siiteri [40].
The isolated microsomes were suspended in minimum volume of
phosphate buffer (0.05 M, pH 7.4, 20% glycerol). Additionally DTT
(dithiothreitol, 10 mM) and EDTA (1 mM) were added to protect the
enzyme from degradation. The enzyme preparation was stored at
−70 ◦C.
2.4.1.2. Assay.
2.4.1.2.1. Normal test procedure. The assay was performed
according to our procedure [41]. Each incubation tube contained
[1␤-3H] androstenedione (0.08 ␮Ci, 15 nM), unlabeled androstene-
dione, (485 nM) NADP, (2 mM), glucose-6-phosphate, (20 mM)
glucose-6-phosphate-dehydrogenase (0.4 units) and inhibitor in
phosphate buffer (0.05 m, pH 7.4). The test compounds were
dissolved in DMSO and diluted with buffer. The final DMSO con-
centration in the control and inhibitor incubation was 2%. Each
tube was preincubated for 5 min at 30 ◦C in water bath. Micro-
somal protein was added to start the reaction (0.1 mg). The total
volume of each incubation was 0.2 ml. The reaction was termi-
nated by the addition of cold solution of mercuric chloride (1 mM,
200 ␮l). After addition of Norit A (Serva, Heidelberg, Germany)
(2%, 200 ␮l), the vials were shaken for 20 min and centrifuged at
1500 × g for 5 min to separate the charcoal-absorbed steroids. The
supernatant was assayed for 3H2O by counting in a scintillation
mixture using PerkinElmer-Wallac ␤-Counter. The calculation of
the IC50 value was performed by plotting the percent inhibition
vs. the concentration of inhibitor on a semi-log plot. From this the
molar concentration causing 50% inhibition was calculated.
2.4.1.2.2. Inhibition of aromatase by irreversibly binding com-
pounds. The assay was performed similar to that of the normal
test procedure. A preincubation of the aromatase containing
microsomes was performed along with a regenerating system
(2 mM NADP, 20 mM glucose-6-phosphate, 0.4 units of glucose-
6-phosphate-dehydrogenase) and inhibitor in phosphate buffer
(0.05 M, pH 7.4) for 30 min at 30 ◦C. The test compounds were
dissolved in DMSO and diluted with buffer. The final DMSO con-
centration in the control and inhibitor incubation was 2%. After
preincubation an aqueous dextran coated charcoal (DCC) suspen-
sion (2%) (Sigma, St. Louis, MO) was added followed by a shaking
step for 20 min at 4 ◦C. After full-speed centrifugation 200 ␮l of the
supernatant were supplemented with 50 ␮l of regenerating system
and 50 ␮l substrate (15 nM [1␤-3H]androstenedione (0.08 ␮Ci),
485 nM unlabeled androstenedione) to start the enzymatic reaction
at 30 ◦C. After several time points (8, 16, and 24 min) 50 ␮l of the
sample were stopped by the addition of 100 ␮l of a cold 1 mM HgCl2
solution. After addition of 100 ␮l of Norit A (2%) (Serva, Heidel-
berg, Germany), the vials were shaken for 20 min and centrifuged
2.2.5. 2-Cyano-3,17ˇ-dihydroxy-5˛-androst-2-en-4-one (9)
Compound (8) (0.2 g, 0.0006 mol) was stirred with sodium
methoxide (0.5 g) in dry THF (10 ml) under nitrogen atmosphere
at room temperature for 30 min. The reaction mixture was
poured into water, acidified with conc. HCl and extracted with
dichloromethane (3× 25 ml). The combined organic extracts were
washed, dried and the solvent evaporated to yield the crude prod-
uct. Purification by column chromatography (5 g silica gel, ethyl
acetate–n-hexane, 8:2) afforded compound (9) 0.12 g, 55%; mp
235–237 ◦C after recrystallization from acetone-n-hexane.
UV (MeOH): 286 nm (log Є4.29). UV (Alk. MeOH): 336 nm
(log Є4.12). IR (KBr): 3127, 2966, 2847, 2210, 1691, 1450, 1380,
1172, 1067, 947. 1H NMR: ı 3.58–3.62 (t, 1H); 2.85 (t, 1H); 2.46–2.50
(d, 1H); 2.39–2.44(d, 1H); 2.29–2.33 (dd, 1H); 0.90 (s, 3H); 0.73 (s,
3H).
2.3. Molecular modeling studies
Molecular modeling studies were performed on a Silicon Graph-
ics Fuel Workstation running on the IRIX 6.5 operating system using
SYBYL 7.0 molecular modeling software from Tripos, Inc. [37] and
GLIDE from Schrodinger Inc., USA [38] installed on Microsoft Win-
dows XP Professonal (version 2002) based Intel core2 Duo 2.53 GHz
PC (with 3.0 GB memory).
2.3.1. Molecular structure generation
All compounds were built from the fragments in the SYBYL
database. A set of low energy conformations for each molecule
under study were generated by dynamics using simulated anneal-
ing technique with Tripos force fields in SYBYL. The molecules
were heated to 700 K followed by cooling to 300 K. Time spent for
annealing was 1000 fs. Time increament for dynamics computa-
tions was 0.5 fs and coupling time for temperature regulation was
2.0 fs. Ten consecutive cycles were calculated. Repeating the cycle
many times and collecting the low energy structure results in a
set of low energy conformations. The lowest energy conformers
thus, obtained were further minimized using the conjugate gradi-
ent method in SYBYL 7.0 using Tripos force field, atomic charges

M.R. Yadav et al. / Steroids 76 (2011) 464–470
467
OH
Me
OH
Me
OH
Me
O
Me
Me
Me
a
b
H
N
N
H
O
O
H
O
OH
(5)
OH
(6)
(4)
c
d
OH
Me
OH
OAc
Me
Me
Me
e
Me
H
Me
NC
HO
O
N
N
O
H
O
H
O
O
(7)
(8)
(9)
a: Ethyl formate, sodium methoxide, pyridine
b: Hydrazine hydrate, ethanol
c: Hydroxylamine hydrochloride, ethanol, pyridine d: Hydroxylamine hydrochloride gl. acetic acid
e: Sodium methoxide, THF
Scheme 1.
at 1500 × g for 5 min to separate the charcoal-absorbed steroids.
The supernatant was assayed for 3H2O by counting in a scintillation
mixture using a LKB-Wallac ␤-counter. Exemestane was used as a
positive control that irreversibly binds to aromatase, as a negative
control (not binding irreversibly) aminoglutethimide was used. The
inhibition values after the three different incubation times were
related to the DMSO control, averaged and compared with the inhi-
bition values after performance of the normal test procedure using
the same inhibitor concentration.
been depicted as structure (5). Compound (5) can exist in various
tautomeric forms (like ‘B’ and ‘C’). Form ‘B’ seems to be the domi-
nant one as inferred from the spectral data [UV (MeOH): 374 nm
(log Є4.22). UV (Alk. MeOH): 379 nm (log Є4.38). IR: 1695 cm−1
(4-C O) and 1640 cm−1 (2-CHO). PMR: ı 10.29 (s, 1H 2-CHO);
2.77–2.81(d, 1H, 1␣/␤-H); 2.29–2.33 (dd, 1H, 5␣-H); 2.12–2.16 (d,
1H, 1␣/␤-H)]. Molecular modeling studies also indicated higher sta-
bility of form (B) over form (C). Both of the structures (5B and 5C)
were minimized as per the protocol mentioned in Section 2.
The energy calculated for structures (B) (23.771 kcal/mol) and
(C) (25.771 kcal/mol) clearly favored the dominance of form (B)
over form (C).
OH
OH
Me
Me
H
H
O
H
H
Me
Me
O
O
H
H
O
H
O
H
O
(B)
(5)
(C)
3. Results and discussion
According to the objective of this work, the 2-formyl derivative
(5) was reacted with hydrazine hydrate in ethanol under refluxing
conditions to obtain the desired pyrazole (6). Structure of com-
pound (6) is in conformity with the spectral data obtained for the
compound.
3.1. Chemical
4,17␤-Dihydroxy-4-androsten-3-one [36] (4) was used as the
starting material for the synthesis of the desired products as
per Scheme 1. Formylation of compound (4) with ethyl formate
under basic conditions offered the desired product which has
OH
Me
OH
Me
OH
Me
Me
Me
Me
Alkaline
O
N
O
N
N
Neutral/Acidic
N
H
O
H
HO
O
O
(7)
(D)
(6)

468
M.R. Yadav et al. / Steroids 76 (2011) 464–470
Table 1
G-Score and enzyme inhibition data of the compounds.
Compd.
G-score
% Inhibitiona of aromatase in normal
test procedure at
IC50 (nM)
% Inhibitiona of aromatase after
irreversible binding at
0.5 ␮M conc.
2.0 ␮M conc.
2.0 ␮M Conc.
6
7
8
9
−7.77
46.1 11.8
1.3 2.2
0.0 0.0
78.7 2.6
–
–
53.6 5.0
88.9 0.8
512.0 93.3
–
–
1019.8 157.5
153.9 14.7
12.3 2.1
–
–
2.0 3.5
66.3 0.6
−6.90
−4.44
−7.90
−6.99
37.0 10.3
3
Exemestane
Aminogluthemide
Androstenedione
Formestane
Control
–
–
–
–
–
3.6 1.7
30 ␮M
2.0 2.6
−7.13
−6.96
–
–
–
–
–
–
–
–
–
–
a
The given values are mean values of at least three experiments.
[UV (MeOH): 270 nm (log Є4.31). IR: 1634 cm−1 (4-C O). PMR;
2.70–2.74 (d, 1H, 1␣/␤-H); 2.51–2.53 (dd, 1H, 5␣-H); 2.20–2.24
(d, 1H, 1␣/␤-H)]. It is worth noting that the UVmax of compound
(6) does not show a bathochromic shift in alkaline medium and
the vibrational band of 4-keto moved towards lower frequency
(1634 cm−1). Both of these effects could be explained by the
thermodynamic stabilization of the system due to intramolecular
hydrogen bonding. Treatment of compound (5) with hydroxy-
lamine hydrochloride in presence of few drops of pyridine in
ethanol afforded the isoxazole (7). [UV (MeOH): 310 nm (log Є3.80).
UV (Alk. MeOH): 362 nm (log Є3.89). IR: 1676 cm−1 (4-C O). PMR:
ı; 2.82–2.86 (d, 1H, 1␣/␤-H); 2.20–2.25 (dd, 1H, 5␣-H); 1.83–1.87
(d, 1H, 1␣/␤-H)]. A big bathochromic shift in its UV spectrum in
alkaline medium is anticipated due to its conversion into the anion
(D).
Treatment of compound (5) with hydroxylamine hydrochlo-
ride under acidic conditions offered the isomeric isoxazole (8). [UV
(MeOH): 256 nm (log Є4.29). UV (Alk. MeOH): 336 nm (log Є4.11).
IR: 1694 cm−1 (4-C O). PMR: ı 2.81–2.85(d, 1H, 1␣/␤-H); 2.56–2.60
(d, 1H, 1␣/␤-H); 2.45–2.49 (dd, 1H, 5␣-H)]. Alkaline hydrolysis of
isoxazole (8) yielded the desired nitrile derivative (9). [UV (MeOH):
286 nm (log Є4.29), UV (Alk. MeOH): 336 nm (log Є4.12); IR: 2210
(CN stretching), 1691 cm−1 (4-Oxo); PMR: ı 2.46–2.50 (d, 1H; 1␣/␤-
H), 2.39–2.44(d, 1H; 1␣/␤-H), 2.29–2.33 (dd, 1H; 5␣-H)]. Both of
the compounds (8 and 9) convert to the same chrom-ophoric sys-
tem (as shown in structure E) in alkaline medium. Intramolecular
hydrogen bonding explains a slight shift in vibrational group fre-
quency of nitrile in compound (9).
that the best score was obtained for compound (9) followed by
compound (6) while compound (8) afforded poorest of the score
(−4.44).
The crystal structure of human placental aromatase [40] shows
that the bound ligand- androgen makes a hydrogen bond with
the backbone amide of Met 374. Similar hydrogen bonding was
observed with oxygen of 17-hydroxyl group for compounds (6) and
(9) (Fig. 1), which indicates that both the compounds bind with the
active site in a similar way as the natural substrate (3). Moreover,
compounds (6) and (9) showed additional hydrogen bonding of C-4
carbonyl group with amino acid residue Thr 310 (Fig. 1). Compound
(8) did not show the hydrogen bonding with Met 374 or Thr 310.
This could be one of the probable reasons for poor docking score for
compound (8). Compound (7) showed hydrogen bonding with Met
374 with oxygen of C-4 hydroxyl group (instead of C-17) which is
contrary to the hydrogen bonding of the natural substrate, flipping
the conformer in opposite direction. Thus, poor binding score of
the compound (7) could be because of its unfavorable positioning
in contrast to the natural substrate in the active site. Compound (6)
and (9) showed good contacts with Ala 306, The 310, Trp 224, IIe
133, Phe 134, Val 370, Leu 372, Val 373, Met 374 and Ser 478, active
site residues of aromatase.
Takahashi et al. have reported that hydrophobic amino acids
such as Ile 132, Ile 133, Ile 305, Phe 148, Met 303, and Ala 306 may
play a critical role in the binding to the active site [42]. Similar type
of observation was made with the compound (6) and compound
(9) as shown in Fig. 2a and b, respectively. Both of the compounds
are having good contacts with ␤-carbon of Ala 306 through the
OH
Me
OH
Me
OAc
Me
Me
Me
H
O
Me
H
+
N
N
H
OH
N
H
O
O
OH
H
H
O
O
O
(E)
(9)
(8)
hydrogen attached to position-3 of the A-ring while, this feature is
missing in compounds (7) and (8) (Fig. 2c). This could be one of the
reasons for the good binding score of both the compounds (6 and
9).
3.2. Molecular modeling
It was envisaged to perform molecular modeling studies of
the synthesized compounds (6–9). Structures of these compounds
were minimized as per the protocol given in Section 2. The energy
minimized structures were docked independently into the active
site of the aromatase enzyme. Similarly, the two known inhibitors
exemestane (3) and formestane and the substrate androstenedione
were also docked. Glide scores for the enzyme–drug complexes for
all of them were calculated (Table 1). It is evident from the data
3.3. Biological
All of the four synthesized compounds (6–9) were eval-
uated of their aromatase inhibiting activity. The assay was
performed by monitoring the enzyme activity by measur-

M.R. Yadav et al. / Steroids 76 (2011) 464–470
469
Fig. 1. Docked conformations of (A) compound 6 and (B) compound 9 along with the important amino acid residues of human placental aromatase. Both the compounds are
forming two hydrogen bonds.
Fig. 2. Interaction of (a) compound (6) and (b) compound (9) with ␤-carbon of Ala 306. NH of compound (6) and OH of compound (9) orients to the hydrophilic pocket where
as (c) compound (8) dislocates from the hydrophilic pocket.
ing the 3H2O formed from [1␤-3H] androstenedione dur-
ing aromatization. The activity profile of the compounds
is indicated in Table 1. IC50 values of aminoglutethimide and fadro-
zole were determined to be 30 ␮M and 30 nM in the test system
utilized for the evaluation of the test compounds. It is ample clear
from Table 1 that the pyrazole derivative (6) showed the highest
activity followed by nitrile derivative (9). Both of the isomeric isox-
azoles were found to be inactive. It is worth noting that both of the
active compounds (6 and 9) have hydrogen bond donating groups
attached to position 3 of the A-ring while, this feature is missing
in the two inactive derivatives (7 and 8). There is a high proba-
bility that these compounds are binding to a site in the aromatase
enzyme which acts as a hydrogen bond acceptor thereby increasing
the stability of the enzyme–inhibitor complex. Exemestane (3) does
not have hydrogen bond donor group in ring A or B. As discussed
earlier, exemestane (3) is a mechanism-based inhibitor as it has
electrophilic sites in ring A and B which can lead to covalent bond
formation between the enzyme and the drug. Compounds (6 and
9) do not have such active sites indicating that these compounds
should be acting as reversible competitive inhibitors.
coated charcoal (DCC) to remove unbound test compounds. The
enzyme preparation so obtained was incubated with the substrate
[1␤-3H]androstenedione and the enzyme activity evaluated by
measuring the concentration of 3H2O using a scintillation counter.
The experiment clearly indicated (Table 1) that both the com-
pounds (6 and 9) bind to the enzyme in a reversible manner. This
is contrary to the binding of exemestane (3) with the enzyme.
4. Conclusion
The novel pyrazole (6), isoxazole (7 and 8) and nitrile (9) deriva-
tives were synthesized. Molecular docking studies were carried
out for the synthesized compounds showed good docking score
for compound (9) followed by compound (6) while compound (8)
afforded poorest of the score. Aromatase inhibitory activity for
compound (6) having pyrazole ring at 2,3 position showed the high-
est activity followed by nitrile derivative (9). Isomeric forms (7 and
8) of isoxazole showed very poor activity compared to fadrozole
and aminoglutethimide.
It is worth noting that both of these compounds (6 and 9) bear
a hydrogen bond donating group at position 3 of the A-ring. This
feature is lacking in compounds (7 and 8) which show poor binding
affinity for the enzyme. Preliminary enzyme binding studies indi-
cate reversible binding of test compounds (6 and 9) to the enzyme
contrary to exemestane (3) which is a known irreversible inhibitor
of aromatase enzyme.
The normal test procedure used for the determination of IC50
values would assess both, reversible as well as irreversible bind-
ing of the test compounds to the aromatase enzyme. Hence, it was
decided to perform a separate test which would assess whether the
test compounds were binding to the enzyme irreversibly. The test
compounds were allowed to interact with aromatase enzyme in
a physiological environment followed by treatment with dextran-

470
M.R. Yadav et al. / Steroids 76 (2011) 464–470
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