Steroidal carbonitriles as potential aromatase inhibitors

Article abstract of DOI:10.1016/j.steroids.2012.04.010

Estrogens, responsible for the growth of hormone-dependant breast cancer are biosynthesized from androgens involving aromatase enzyme in the last rate limiting step. Inhibition of aromatase is an efficient approach for the prevention and treatment of brea

Full text of DOI:10.1016/j.steroids.2012.04.010

Steroids 77 (2012) 850–857
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Steroidal carbonitriles as potential aromatase inhibitors
a
a
b
b
Mange Ram Yadav a, , Prafulla M. Sabale , Rajani Giridhar , Christina Zimmer , Rolf W. Hartmann
⇑
a Pharmacy Department, Faculty of Technology and Engineering, Kalabhavan, The M.S. University of Baroda, Vadodara 390 001, India
b Pharmaceutical and Medicinal Chemistry, Saarland University & Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C 2 3, D-66123 Saarbrucken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Estrogens, responsible for the growth of hormone-dependant breast cancer are biosynthesized from
androgens involving aromatase enzyme in the last rate limiting step. Inhibition of aromatase is an efficient
approach for the prevention and treatment of breast cancer. Novel 4-phenylthia derivatives (2, 3 and 7)
have been synthesized as aromatase inhibitors. The synthesized compounds (2, 3 and 7) exhibited notice-
able enzyme inhibiting activity. Kinetics study of these compounds (2, 3, and 7) showed negligible inhibi-
tion of the enzyme under conditions conducive for irreversible inhibition of the enzyme. Introduction of
unsaturation at C-4, C-1 & 4 or C-4 & 6 (compounds 5, 9 and 11) was observed to not be an effective strat-
egy for entrancing aromatase inhibiting activity in 17-oxo-16b-carbonitrile derivatives. The D-seco deriv-
atives (13–15 and 17) having unsaturation at C-4, C-1 & 4 or C-4 & 6 along with carbonitrile function in
ring-D showed complete loss of aromatase inhibiting activity.
Received 29 November 2011
Received in revised form 1 March 2012
Accepted 10 April 2012
Available online 21 April 2012
Keywords:
Aromatase inhibitors
Breast cancer
4-Phenylthia derivatives
Carbonitrile
Ó 2012 Elsevier Inc. All rights reserved.
D
-Seco derivatives
Azasteroids
1. Introduction
Over the past few decades, considerable attempts have been
made toward developing potent inhibitors of aromatase [12,13].
Cancer is the second most important disease leading to death in
both developing and developed countries according to WHO [1]. In
females, breast cancer is the most frequently diagnosed and lead-
ing cause of cancer deaths worldwide, accounting for 23% of the
total new cancer cases and 14% of the total cancer deaths. About
half of the breast cancer cases and 60% of the deaths are estimated
to occur in economically developing countries [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 this
disease [3]. Approximately two-thirds of postmenopausal breast
cancer patients have estrogen-dependent breast cancer, which
contains estrogen receptors (ERs) and requires estrogens for its
growth [4,5]. Production of estrogens takes place in many tissues
of the body, including the ovaries, adipose tissue, muscle, liver,
breast tissue and malignant breast tumors [5]. In pre-menopausal
women the ovaries are the main source of circulating estrogens but
in postmenopausal women the main source is adipose tissue and
muscle [6]. Aromatase is a cytochrome P450 dependent enzyme
that catalyzes the aromatization of androgens to estrogens by
three sequential oxidation steps, each one requiring one mol each
of oxygen and NADPH [7,8]. The third step oxidatively cleaves the
C10–C19 bond, resulting in aromatization of the steroid A-ring and
release of formic acid [9–11].
Aromatase inhibitors that have been used clinically can be catego-
rized either by generations or by their mechanism of action. They
may be described as first-generation [aminoglutethimide (A) and
testolactone (B)], second-generation [formestane (C) and fadrozole
(D)] and third-generation [anastrozole (E), letrozole (F) and exe-
mestane (G)] [14–19] inhibitors according to the order of their clin-
ical development (Fig. 1). They can also be classified as type I or
type II according to their mechanism of action. Type I and type II
inhibitors are also known as steroidal and nonsteroidal inhibitors
respectively [20,21].
Clinical studies initially confirmed that administration of the first
generation inhibitor aminoglutethimide, caused regression of hor-
mone-dependent breast cancer in women [22]. This lead served as
the thrust to develop second and third generation inhibitors result-
ing in the development of compounds that are 1000–10,000-fold
more effective than aminoglutethimide [13]. Further, the third gen-
eration agents are more specific for the aromatase enzyme, associ-
ated with less side effects and are adequately long acting to be
administered on a once a day basis. The third generation aromatase
inhibitors can be divided into two classes: the competitive inhibi-
tors that bind reversibly to the active site of the enzyme and the
inactivators that destroy the enzyme by binding covalently to it
[13]. Most type I steroidal inhibitors are competitive inhibitors that
have structures similar to androgens. Certain steroidal agents inac-
tivate the enzyme irreversibly by blocking the substrate-binding
site, and are therefore known as aromatase inactivators. Irreversible
steroidal inhibitors such as exemestane form permanent bond with
⇑
Corresponding author.
E-mail address: mryadav11@yahoo.co.in (M.R. Yadav).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.04.010

M.R. Yadav et al. / Steroids 77 (2012) 850–857
851
O
Me
Me
Me
O
O
O
N
Me
NH2
Me
N
O
N
H
O
O
OH
(A)
(C)
(B)
(D)
N
N
N
O
Me
N
N
N
Me
Me
NC
Me
CN
CN
O
NC
CH2
(G)
Me
Me
(E)
(F)
Fig. 1. Steroidal and nonsteroidal aromatase inhibitors.
the aromatase enzyme. Type II inhibitors are usually nonsteroidal
and their action is reversible. Two of the most commonly used third
generation aromatase inhibitors anastrozole (E) and letrozole (F) are
nonsteroidal competitive inhibitors that inhibit the enzyme by
reversible binding to it [23].
Inhibition of aromatase enzyme is an efficient approach for the
prevention and treatment of breast cancer [24]. Besides attempts
to develop novel nonsteroidal compounds, there is a focus on the
development of steroidal compounds as potential aromatase inhib-
itors also [25–27]. Potent steroidal aromatase inhibitors are mainly
the C-1, C-2, C-4, C-6, C-16 and C-19 substituted or C-2 and C-19
bridged steroidal compounds, i.e. the A-, B- or D-ring substituted/
modified steroids. New analogs of formestane have been synthe-
sized and their biological activity investigated in an attempt to find
new aromatase inhibitors and to gain insight into their structure–
activity relationships [28].
tion at C-1, C-4, and C-6 positions in some of the targeted com-
pounds with the hope that introduction of unsaturation at these
positions could increase binding affinity of the derivatives to the
enzymes [29,30] as unsaturation could change the geometry of
the molecules for better binding to the active site in the enzyme.
It was envisaged to evaluate the synthesized compounds for their
aromatase inhibiting activity and determine their kinetics.
2. Experimental
2.1. General
Melting points were determined using a VEEGO make micro-
processor-based melting point apparatus having silicone oil bath
and are uncorrected. IR spectra (wave numbers in cmÀ1) were
recorded on a BRUKER ALPHA T FT-IR spectrophotometer using
KBr discs. 1H NMR spectra were recorded on BRUKER AVANCE II
400 MHz instrument in CDCl3 with TMS as internal standard.
Chemical shift values are mentioned in d, ppm. Chromatographic
separations were performed on silica gel columns. The microanal-
yses for C, H and N were performed on Thermo Scientific FLASH
2000 organic elemental analyzer. Progress of all the reactions
was monitored by TLC on 2 cm  5 cm pre-coated silica gel 60
F254 plates (Merck) of 0.25 mm thickness. The chromatograms
were visualized under UV (254 nm) and iodine vapors. The term
‘‘dried’’ refers to the use of anhydrous sodium sulfate. All reagents
used were of analytical reagent grade.
Extra units of unsaturation in ring A and/or B of several steroi-
dal 4-oxoandrostenes and
3-oxo-1,4-dienes, 3-oxo-4,6-dienes and 3-oxo-1,4,6-trienes showed
relatively high anti-aromatase activity [29,30]. In -seco deriva-
tives the 17-keto group showed higher anti-aromatase activity in
comparison to the 17-hydroxy function. The -secomethyl deriva-
D-homo-lactones like 3-oxo-4-enes,
D
D
tive of 4-androstendione showed the highest activity and compet-
itive type of inhibition of the enzyme [31,32].
In our previous publications [33,34] we have described the syn-
thesis of some novel A-ring fused heterocyclic systems and A- and
D-ring substituted/modified androstanes, and their evaluation for
aromatase inhibiting activity. It was observed that compound with
pyrazole ring fused to 2,3-positions of ring-A offered the highest
activity followed by the 2-carbonitrile substituted derivative. Both
of these compounds exhibited competitive inhibition in kinetics
studies, contrary to our expectations. Another very important out-
come of the study was that introduction of basic or neutral nitro-
gen in ring-A or ring-D of the androstane skeleton was highly
deleterious for the molecules for aromatase inhibiting activity [34].
Some nonsteroidal aromatase inhibitors like anastrozole (E),
letrozole (F) contain carbonitrile functionality. 2-Carbonitrile
2.2. Chemical
2.2.1. 17b-Hydroxy-4-phenylthia-4-androsten-3-one (2)
17b-Hydroxy-4n,5-oxido-5n-androstan-3-one (1) was prepared
by the reported method [36]. A solution of 17b-hydroxy-4n,5-oxi-
do-5n-androstan-3-one (1) (0.5 g, 0.0016 mol) in dioxane (10 ml)
was stirred with thiophenol (0.26 g, 0.0024 mol) and anhydrous
potassium carbonate (0.32 g, 0.0234 mol) for 3 h at room tempera-
ture under nitrogen atmosphere. The reaction mixture was poured
into water (100 ml) and extracted with dichloromethane (4 Â
25 ml). The combined organic extract was washed with water, dried
and solvent removed to afford the crude product. The crude product
so obtained was further purified by passing it through a column of
silica gel. The column was first run with hexane followed by hex-
ane–ethyl acetate (9:1). The solid was crystallized from methanol
to afford 17b-hydroxy-4-phenylthia-4-androsten-3-one (2) (0.32
derivative, 2-cyano-3,17b-dihydroxy-5a-androstan-2-en-4-one re-
ported earlier from this laboratory [33] showed significant aroma-
tase inhibiting activity. 4-Thioalkyl/aryl derivatives have also been
reported [35] to possess good aromatase inhibiting activity. Keep-
ing these observations in mind it was planned to introduce simul-
taneously cyano group in ring-D and 4-phenylthia function in
ring-A. It was of interest to see the impact of both of these func-
tionalities on aromatase inhibiting activity, when combined in a
single molecular entity. It was also planned to introduce unsatura-
g, 55%), m.p. 159–61 °C. UV (MeOH): 250 nm (logꢀ 3.67), IR (KBr):

852
M.R. Yadav et al. / Steroids 77 (2012) 850–857
3499, 1689, 1609, 1532 and 744. 1H NMR: 7.11–7.15 (m, 2H), 7.00–
7.04 (m, 3H), 3.55–3.60 (t, 1H), 1.22 (s, 3H) and 0.74 (s, 3H). Calcu-
lated for C25H32O2S: C 75.71, H 8.13. Found: C 76.02, H 8.45. MS: m/
z 397.9 (M+).
(5.0 ml) was added slowly to the above solution with stirring.
The reaction mixture was stirred for 2 h at 10 °C, poured into water
(100 ml) and extracted with dichloromethane (4 Â 25 ml). The
combined extract was washed with water, dried and the solvent
removed to afford the crude product. The crude product so
obtained was purified by passing through a column of silica gel
eluting with hexane first to remove excess thiophenol and then
with hexane–ethyl acetate (9:1). The solid so obtained was crystal-
lized from methanol to afford crystals of 16b-cyano-17b-hydroxy-
4-phenylthia-4-androsten-3-one (7) (0.28 g, 61%), m.p 145–47 °C.
2.2.2. 4-(4-Aminophenyl)thia-17b-hydroxy-4-androsten-3-one (3)
A solution of 17b-hydroxy-4n,5-oxido-5n-androstan-3-one [36]
(1) (0.5 g, 0.0016 mol) in dry tetrahydrofuran (10 ml) was stirred
with p-aminothiophenol (0.29 g, 0.0021 mol) and anhydrous
potassium carbonate (0.32 g, 0.0023 mol) for 3 h at room temper-
ature under nitrogen atmosphere. The reaction mixture was
poured into water (100 ml), acidified slightly with hydrochloric
acid and extracted with dichloromethane (4 Â 25 ml). The com-
bined extract was washed with water, dried and solvent removed
to afford a crude product. The crude product so obtained was puri-
fied by passing through a column of silica gel using first chloroform
and then chloroform–methanol (9:1) as eluents. The solid so
obtained was crystallized from methanol to afford 4-(4-aminophe-
nyl)thia-17b-hydroxy-4-androsten-3-one (3) (0.33 g, 51%), m.p.
UV (MeOH): 250 nm (log
ꢀ 3.74). IR (KBr): 3336, 2236, 1672,
1563, 1475, 1289, 1108, 1022 and 992. 1H NMR: 7.01–7.44 (m,
5H), 3.56–3.61 (m, 2H), 1.25 (s, 3H) and 0.73 (s, 3H). Calculated
for C26H31NO2S: C 74.07, H 7.41, N 3.32. Found: C 74.26, H 7.22,
N 3.18.
2.2.6. 16b-Cyano-1,4,6-androstatriene-3,17-dione (8)
16b-Cyano-3b-hydroxy-5-androsten-17-one (4) (0.5 g, 0.0016
mol) in dry dioxane (8 ml) was heated with 2,3-dichloro-5,6-dic-
yanobenzoquinone (DDQ) (1.5 g, 0.0066 mol) for 20 h at 100 °C
on an oil bath. The reaction mixture was cooled and diluted with
ethyl acetate (25 ml) and poured into saturated solution of sodium
bicarbonate (400 ml). The aqueous layer was extracted with ethyl
acetate (3 Â 25 ml). The combined organic layer was washed with
saturated solution of sodium bicarbonate, dried and the solvent re-
moved under vacuum. The crude residue so obtained was passed
through activated neutral alumina, using hexane–ethyl acetate
(9:1) as an eluent to afford crystals of 16b-cyano-1,4,6-androstatri-
ene-3,17-dione (8) (0.10 g, 20%), m.p. 185–86 °C. UV (MeOH): 296
223–25 °C. UV (MeOH): 260 nm (logꢀ 3.18), IR (KBr): 3493, 3360,
1677, 1634, 1492, 1294 and 817. 1H NMR: 6.97–7.01 (m, 2H),
6.48–6.51 (m, 3H), 3.55–3.59 (t, 1H), 1.21 (s, 3H) and 0.72 (s,
3H). Calculated for C25H33NO2S: C 72.95, H 8.08, N 3.40. Found: C
72.76, H 8.25, N 3.24. MS: m/z 412.1 (M+)
2.2.3. 16b -Cyano-4-androstene-3,17-dione (5)
16b-Cyano-3b-hydroxy-5-androsten-17-one (4) was prepared
by the reported method [37] using androstenolone as the starting
material. A solution of aluminium i.propoxide (2.0 g, 0.0097 mol)
in dry toluene (20 ml) was added drop by drop during azeotropic
distillation to a solution of 16b-cyano-3b-hydroxy-5-androsten-
17-one [37] (4) (2.0 g, 0.0064 mol) in dry toluene (125 ml) and
cyclohexanone (15 ml). Distillate (15–20 ml) was collected further
after complete addition of the aluminium i.propoxide solution. The
reaction mixture was refluxed for 2 h and allowed to stand over-
night. Water (1.0 ml) was added to precipitate out aluminium
hydroxide. The organic layer was filtered and the precipitate was
washed with toluene. The combined organic layer was subjected
to steam-distillation until complete removal of organic solvents
was effected. The aqueous layer was extracted with chloroform
(3 Â 50 ml) and the combined chloroform layer was washed with
water, dried and the solvent recovered. The residue so obtained
was crystallized from hexane–ethyl acetate to afford crystals
of 16b-cyano-4-androstene-3,17-dione (5) (1.0 g, 50%), m.p.
(logꢀ 4.28), 254 (shoulder peak) (logꢀ 2.11) and 221 (logꢀ 3.58). IR
(KBr): 2243, 1754, 1660, 1602, 1290 and 892.
2.2.7. 16b-Cyano-1,4-androstadiene-3,17-dione (9)
A mixture of 16b-cyano-3b-hydroxy-5-androsten-17-one (4)
(0.5 g, 0.0016 mol) in dry dioxane (8 ml) was refluxed with DDQ
(1.1 g, 0.0048 mol) for 8 h on an oil bath. The reaction mixture
was cooled and diluted with ethyl acetate (30 ml), mixed well
and poured into a saturated solution of sodium bicarbonate
(500 ml). The aqueous layer was extracted with ethyl acetate
(3 Â 20 ml). The combined organic layer was washed with satu-
rated solution of sodium bicarbonate twice, dried and the solvent
removed under vacuum. The crude product so obtained was passed
through activated neutral alumina, with ethyl acetate as an eluent.
The product so obtained was further purified by passing through
neutral alumina using hexane–ethyl acetate (9:1) as the eluent.
The white solid so obtained was crystallized with hexane–ethyl
acetate to get a mixture of 16b-cyano-1,4-androstadiene-3,17-
dione (8) and compound (9) (0.10 g, 20%), m.p. 170–72 °C. UV
181–83 °C. UV (MeOH): 240 nm (log
ꢀ 4.07). (Alk.MeOH): 244
(logꢀ 4.1). IR (KBr): 2237, 1753, 1661, 1615, 1455, 1010 and 734.
1H NMR: 5.76 (s, 1H), 3.06–3.11 (t, 1H), 1.23 (s, 3H) and 1.08 (s,
3H). MS: m/z 312 (M+).
(MeOH): 246 (log
ꢀ 3.98) and 296 (shoulder peak) (logꢀ 3.75),
2.2.4. 16b-Cyano-4n,5-oxido-5n-androstane-3,17-dione (6)
Cold solutions of sodium hydroxide (20%, 2.0 ml) and hydrogen
peroxide (30%, 3.0 ml) were added simultaneously drop by drop to
a stirred solution of 16b-cyano-4-androstene-3,17-dione (5) (1.0 g,
0.00305 mol) in methanol (25 ml) at À5 °C. The reaction mixture
was allowed to stand for 1 h at À5 °C, diluted with water
(200 ml) and extracted with dichloromethane (4 Â 25 ml). The
combined extract was washed with water, dried and the solvent
recovered to afford 16b-cyano-4n,5-oxido-5n-androstane-3,17-
dione (6) (0.5 g, 57%) which was used as such for the next step
without further purification.
(Alk. MeOH): 260 (log
ꢀ 4.17) and 296 nm (shoulder peak) (logꢀ
3.79). IR (KBr): 2235, 1751, 1647, 1294 and 888. 1H NMR: 7.01–
7.06 (m), 6.24–6.38 (m), 6.00–6.11 (m), 3.06–3.15 (m), 1.28 (s),
1.26 (s), 1.16 (s) and 1.11 (s). MS: m/z 307.9 (M+) and 309.9 (M+).
2.2.8. 16b-Cyano-3-ethoxy-3,5-androstadien-17-one (10)
A solution of 16b-cyano-4-androstene-3,17-dione (5) (0.5 g,
0.0016 mol) in dry dioxane (8.0 ml) was stirred with p-toluenesul-
fonic acid (50 mg) and triethyl orthoformate (1.5 ml, 0.009 mol) for
2 h at room temperature whereby the reaction mixture turned
greenish. Freshly prepared mixture of water (10 ml) and pyridine
(1.0 ml) was added to the reaction mixture and it was cooled to
0 °C. The yellow sticky solid so formed was extracted with dichlo-
romethane (3 Â 20 ml), washed with water containing pyridine
(0.5 ml), dried and the solvent removed under vacuum to yield a
yellow sticky solid (0.45 g, 83%) which was used as such for the
next step without further purification.
2.2.5. 16b-Cyano-17b-hydroxy-4-phenylthia-4-androsten-3-one (7)
Thiophenol (0.5 g, 0.00454 mol) was stirred with sodium hy-
dride (0.1 g, 0.00434 mol) in dry dioxane (5.0 ml) under nitrogen
atmosphere at 10 °C.
A solution of 16b-cyano-4n,5-oxido-5n-
androstane-3,17-dione (6) (0.5 g, 0.00152 mol) in dry dioxane

M.R. Yadav et al. / Steroids 77 (2012) 850–857
853
2.2.9. 16b-Cyano-4,6-androstadiene-3,17-dione (11)
solution of 16b-cyano-3-ethoxy-3,5-androstadien-17-one
255 (log
ꢀ
3.97) and 295 nm (log
ꢀ
4.09). IR (KBr): 2235, 1680,
A
1604, 1229, 893 and 734. 1H NMR: 7.01–7.03 (d, 1H), 6.46–6.47
(dd, 1H), 6.29–6.33 (dd, 1H), 6.07–6.10 (m, 2H), 2.79–3.00 (m,
2H), 1.57(s, 3H) and 1.21(s, 3H). Calculated for C19H20N2O: C
78.05, H 6.88, N 9.58. Found: C 77.94, H 6.43, N 9.39. MS: m/z
292.5 (M+).
(10) (0.5 g, 0.015 mol) in aqueous acetone (95%, 5.0 ml) was stirred
with a solution of DDQ (0.42 g, 0.0018 mol) in aqueous acetone
(95%, 2.0 ml) for 5–6 min at room temperature. The reaction mix-
ture was diluted with aqueous acetone (95%, 20 ml), poured onto
a column of neutral alumina and eluted with acetone until the yel-
low band moved to the base of the column. The solvent was
removed and the crude product so obtained was further purified
by passing through activated neutral alumina using hexane–ethyl
acetate (9:1) as the eluent. The solid so obtained was crystallized
with hexane–ethyl acetate to get crystals of 16b-cyano-4,6-andro-
stadiene-3,17-dione (11) (0.130 g, 29%), m.p. 192–94 °C. UV
2.2.13. 3-Oxo-16,17-seco-1,4-androstadiene-16,17-dinitrile (15)
A solution of 3b-hydroxy-16,17-seco-5-androstene-16,17-dinit-
rile (12) (0.5 g, 0.0017 mol) in dry dioxane (10 ml) and DDQ (1.0 g,
0.0045 mol) was refluxed for 8 h on oil bath and the reaction mix-
ture was diluted with ethyl acetate (80 ml). The organic layer was
washed with saturated solution of sodium carbonate, dried and the
solvent removed. The crude residue so obtained was passed
through neutral alumina using hexane–ethyl acetate (9:1) as the
eluent. The white solid so obtained was crystallized from
hexane–ethyl acetate to afford crystals which were characterized
to be a mixture of 3-oxo-16,17-seco-1,4-androstadiene-16,17-
dinitrile (15) and compound (14) (0.16 g, 32%), m.p. 174–76 °C.
(MeOH): 281 (log
ꢀ 4.38), (Alk. MeOH): 278 nm (logꢀ 4.47). IR
(KBr): 2243, 1751, 1652, 1417, 1206, 1150, 876 and 733. 1H
NMR: 6.21–6.24 (d, 1H), 6.08–6.10 (dd, 1H), 5.73 (s, 1H), 3.10–3.15
(m, 1H), 1.15 (s, 3H) and 1.14 (s, 3H). Calculated for C20H23NO2: C
77.64, H 7.48, N 4.50. Found: C 77.31, H 7.53, N 4.59. MS: m/z
309.9 (M+).
UV (MeOH): 243 (log
ꢀ 4.15) and 295 nm (shoulder peak) (logꢀ
3.77). IR (KBr): 2232, 1654, 1624, 1604, 890, 734 and 701. 1H
NMR: 7.03 (s), 7.00 (s), 6.98 (s), 6.43–6.46 (dd), 6.26–6.33 (m),
6.07–6.12 (m), 1.57 (s), 1.53 (s), 1.27 (s) and 1.22 (s). MS: m/z
292.9 (M+) and 294.9 (M+).
2.2.10. 3b-Hydroxy-16,17-seco-5-androstene-16,17-dinitrile (12)
A solution of 3b-acetoxy-16,17-seco-5-androstene-16,17-dinit-
rile [38] (2.0 g, 0.0059 mol) and potassium hydroxide (0.4 g,
0.007 mol) in methanol (50 ml) was stirred at room temperature
for 30 min. Two-third of the solvent was removed under reduced
pressure, the reaction mixture was poured into water (300 ml)
and acidified with hydrochloric acid (5%). White precipitate so ob-
tained was filtered off, washed with water, dried and crystallized
from methanol to afford crystals of 3b-hydroxy-16,17-seco-5-
androstene-16,17-dinitrile (12) (1.41 g, 80%), m.p. 168–69 °C. IR
(KBr): 3455, 2235, 1053 and 734. 1H NMR: 5.35–5.37 (m, 1H),
3.50–3.60 (m, 1H), 2.66–2.73 (m, 2H), 1.43 (s, 3H) and 1.02 (s,
3H). MS: m/z 316 (M + 18).
2.2.14. 3-Ethoxy-16,17-seco-3,5-androstadiene-16,17-dinitrile (16)
A
solution of 3-oxo-16,17-seco-4-androstene-16,17-dinitrile
(13) (0.5 g, 0.0017 mol), triethyl orthoformate (1.5 ml, 0.009 mol)
and p-toluenesulfonic acid (0.05 g) in dry dioxane (8 ml) was stir-
red for 2 h at room temperature. The greenish reaction mixture
was treated with a solution of water (10 ml) and pyridine (1 ml)
and the resulting yellowish sticky solid was extracted with dichlo-
romethane (3 Â 25 ml). The combined organic layer was washed
with water containing pyridine (0.2 ml), dried and the solvent
removed to get yellowish sticky solid of 3-ethoxy-16,17-seco-3,5-
androstadiene-16,17-dinitrile (0.54 g, 93%) (16) which was used
as such without purification for next step.
2.2.11. 3-Oxo-16,17-seco-4-androstene-16,17-dinitrile (13)
Aluminium i.propoxide (2.0 g, 0.0097 mol) in dry toluene
(20 ml) was added drop by drop during azeotropic distillation to
a solution of 3b-hydroxy-16,17-seco-5-androstene-16,17-dinitrile
(12) (2 g, 0.0067 mol) in dry toluene (125 ml) and cyclohexanone
(15 ml). The reaction mixture was further refluxed for 3 h and
allowed to stand overnight. Water (1 ml) was added to precipitate
excess of aluminium i.propoxide and the organic layer was filtered
and the slurry washed with toluene. The combined filtrate was
subjected to steam-distillation until complete removal of organic
solvents was effected. Aqueous layer was extracted with chloro-
form (3 Â 50 ml) and the combined chloroform layer was washed
with water, dried and recovered. The residue so obtained was crys-
tallized from hexane–ethyl acetate to afford crystals of 3-oxo-
16,17-seco-4-androstene-16,17-dinitrile (13) (1.0 g. 50%), m.p.
2.2.15. 3-Oxo-16,17-seco-4,6-androstadiene-16,17-dinitrile (17)
A
solution of 3-ethoxy-16,17-seco-3,5-androstadiene-16,17-
dinitrile (16) (0.5 g, 0.0016 mol) in aqueous acetone (95%, 5.0 ml)
was stirred with a solution of DDQ (0.45 g, 0.002 mol) in aqueous
acetone (95%, 2.0 ml) for 6–8 min at room temperature. The reac-
tion mixture was diluted with aqueous acetone (95%, 20 ml),
poured onto a column of neutral alumina and eluted with acetone
until the yellow band moved to the base of the column. The organic
solvent was removed from the eluent to get a crude product which
was further purified by passing through a column of neutral alu-
mina using hexane–ethyl acetate (9:1) as an eluent. The solid so
obtained was crystallized from hexane–ethyl acetate to obtain
3-oxo-16,17-seco-4,6-androstadiene-16,17-dinitrile (17) (0.21 g,
140–42 °C. UV (MeOH): 238 nm (logꢀ 4.32). IR (KBr): 2235, 1670,
and 1432. 1H NMR: 5.77 (s, 1H), 2.68–2.83 (m, 1H), 1.50 (m, 3H)
and 1.16 (s, 3H). Calculated for C19H24N2O: C 76.99, H 8.15, N
9.45. Found: C 77.12, H 8.22, N 9.31. MS: m/z 269.9 (M+).
47%), m.p. 185–86 °C. UV (MeOH): 279 nm (log
ꢀ 4.43). IR (KBr):
2244, 2231, 1666, 1621, 1273, 870, 755 and 735. 1HNMR: 6.28–
6.31 (d, 1H), 6.13–6.16 (dd, 1H), 5.76 (s, 1H), 2.79–3.01 (m, 2H),
1.54 (s, 3H) and 1.15 (s, 3H). Calculated for C19H22N2O: C 77.52,
H 7.53, N 9.52. Found: C 77.24, H 7.68, N 9.26. MS: m/z 294.9 (M+).
2.2.12. 3-Oxo-16,17-seco-1,4,6-androstatriene-16,17-dinitrile (14)
A solution of 3b-hydroxy-16,17-seco-5-androstene-16,17-dinit-
rile (12) (0.5 g, 0.0017 mol) in dry dioxane (10 ml) and DDQ (1.5 g,
0.0067 mol) was refluxed for 20 h on oil bath. The reaction mixture
was diluted with ethyl acetate (80 ml). The organic layer was
washed several times with saturated solution of sodium carbonate.
The organic extract was dried, solvent recovered and the crude res-
idue so obtained was passed through a column of neutral alumina
with hexane–ethyl acetate (9:1) as the eluent. The white solid so
obtained was crystallized from hexane–ethyl acetate to afford
crystals of 3-oxo-16,17-seco-1,4,6-androstatriene-16,17-dinitrile
2.3. Biological
2.3.1. Human placental microsomal aromatase assay
The synthesized compounds were screened for aromatase
inhibiting activity in human placental microsomal assay. As human
term placenta is a rich source of aromatase enzyme, the assay is a
measure of test compounds to bind aromatase enzyme in presence
of natural substrates testosterone and androstenedione.
(14) (0.18 g, 35%), m.p. 214–16 °C. UV (MeOH): 221 (log
ꢀ 4.01),

854
M.R. Yadav et al. / Steroids 77 (2012) 850–857
2.3.1.1. 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 [39].
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 À70 °C.
enhancement in aromatase inhibiting activity. So, we thought of
introducing carbonitrile group in ring-D and to observe its impact
on the aromatase inhibiting activity of the molecule. To achieve the
synthesis of target compound (7), preliminary setting of the reac-
tion conditions was done using testosterone as the starting mate-
rial. Testosterone was converted into oxirane derivative (1) using
the reported procedure [36]. The oxirane epimers (1) were treated
with thiophenol in presence of anhydrous potassium carbonate to
obtain 17b-hydroxy-4-phenylthia-4-androsten-3-one (2) as de-
picted in Scheme 1. The 4-phenylthia derivative showed sharp
peak at 250 nm in methanol in its UV spectrum and characteristic
IR bands appeared at 3499 (ÀOH) and 1689 cmÀ1 (–C@O). It of-
fered characteristic NMR signals at 7.11–7.15 (m, 2H; Ar-H),
2.3.1.2. Aromatase inhibition assay. The assay was performed by
measuring the 3H2O formed from [1b-3H]androstenedione during
aromatization [40]. Each incubation tube contained [1b-3H]andro-
stenedione (0.08 lCi, 15 nM), unlabeled androstenedione (485
7.00–7.04 (m, 3H; Ar-H) and 3.55–3.60 (t, 1H; 17a-H). The mass
nM), NADP (2 mM), glucose-6-phosphate (20 mM), glucose-6-
phosphate dehydrogenase (0.4 units) and the test compounds (in
three different concentrations for determining the IC50 value with-
in the linear range of the log-dose response curve, i.e. 20–80% inhi-
bition) in phosphate buffer (0.05 M, pH 7.4). The test compounds
were dissolved in DMSO and diluted with buffer. The final DMSO
concentration in the control and inhibitor incubation was 2%.
Microsomal protein (0.1 mg) was added to start the reaction. Each
tube was incubated for 5 min at 30 °C in water bath. The total vol-
ume for each incubation was 0.2 ml. The reaction was terminated
by the addition of cold solution of mercuric chloride (1 mM,
spectrum showed a peak at m/z 397.9 (M+) confirming the com-
pound (2). The oxirane epimers (1) when treated with p-amino-
thiophenol in basic medium in tetrahydrofuran under inert
atmosphere afforded 4-(4-aminophenylthia)-17b-hydroxy-4-andr-
osten-3-one (3). The thioether (3) showed UVmax at 260 nm in
methanol and prominent peaks appeared at 3493 (ÀNH2) and
1677 cmÀ1 (–C@O) in its IR spectrum. Characteristic NMR signals
appeared at 6.97–7.01 (d, 2H; Ar-H), 6.48–6.51 (d, 2H; Ar-H) and
3.55–3.59 (t, 1H; 17
a-H). Its mass spectrum showed peak at m/z
412.1 (M+).
For the preparation of the target compound (7), synthesis was
planned as given in Scheme 2. Androstenolone was converted into
16b-cyano-3b-hydroxy-5-androsten-17-one (4) by the reported
procedure [37]. Compound (4) showed UVmax at 266 nm in meth-
anol and characteristic bands appeared at 3490 (3-OH), 1754
(17-C@O) and 2250 cmÀ1 (-CN) in its IR spectrum. In its NMR spec-
trum signals appeared at d 5.36–5.38 (m, 1H; 6-CH), 3.50–3,62 (m,
200 ll). After addition of 200 ll Norit A (2%), the vials were shaken
for 20 min and centrifuged at 1500g for 5 min to separate the char-
coal-adsorbed steroids. Aliquots of the supernatant were assayed
for 3H2O by counting in a scintillation mixture using a b-Counter.
The calculation of the IC50 values was performed by plotting the
percent inhibition vs. the concentration of inhibitor on a semi-log
plot.
1H; 3a-CH) and 3.07–3.09 (dd, 1H; 16a-CH). Oppenauer oxidation
of the 3b-hydroxy-16b-nitrile (4) was carried out using aluminium
i.propoxide in cyclohexanone-toluene system to afford 16b-cyano-
4-androstene-3,17-dione (5). The compound (5) showed UVmax at
2.3.2. Test for irreversible inhibition of aromatase
The assay was performed similar to that of the normal test pro-
cedure. 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 the 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 concentration
in the control and inhibitor incubation was 2%. After preincubation
an aqueous dextran-coated charcoal (DCC) suspension (2%) (Sigma,
St. Louis, MO) was added followed by a shaking step for 20 min at
4 °C to adsorb unbound inhibitor. After full-speed centrifugation,
240 nm in methanol confirming the formation of
a,b-unsaturated
ketone. The compound (5) showed characteristics IR bands at
1661 (3-C@O), 1753 (17-C = O) and 2237 cmÀ1 (16-CN). It gave
NMR signals at d 5.76 (s, 1H; 4-CH) and 3.06–3.11 (t, 1H; 16-CH),
and its mass spectrum showed peak at m/z 312 (M+) confirming
its structure (5).
Treatment of 16b-cyano-3,17-dione (5) with alkaline hydrogen
peroxide gave the oxirane (6), which was found to be a mixture of
a,b-epimers. The mixture showed no absorption in its UV spectrum
at 240 nm. The oxirane mixture (6) on treatment with thiophenol
and anhydrous potassium carbonate under various temperature
conditions did not yield the desired compound. But, when the oxi-
rane (6) was stirred with thiophenol and sodium hydride in dry
dioxane under nitrogen atmosphere at À10 °C it gave 16b-cyano-
17b-hydroxy-4-phenylthia-4-androsten-3-one (7). We expected
the 17-oxo function to be retained but a reduced product (7) was
obtained. This could be because of the presence of slight excess
of sodium hydride present in the reaction mixture.
200
erating system and 50
(0.08 Ci) and 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 samples were stopped by the addition of
100 l of a cold 1 mM HgCl2 solution. After addition of 100 l of
l
l of the supernatant was supplemented with 50
ll of regen-
l
l substrate (15nM [1b-3H]androstenedione
l
l
l
l
Norit A (2%) (Serva, Heidelberg, Germany), the vials were shaken
for 20 min and centrifuged at 1500g for 5 min to separate the char-
coal-adsorbed steroids. The supernatant was assayed for 3H2O by
counting in a scintillation mixture using a b-counter. Exemestane
was used as a positive control that irreversibly binds to aromatase.
Aminoglutethimide was used as a negative control (not binding
irreversibly). The inhibition values after the three different incuba-
tion times were related to the DMSO control.
The compound (7) showed UVmax at 250 nm in methanol and
characteristic IR peaks appeared at 3336 (–OH), 2236 (ÀCN) and
OH
OH
Me
Me
a/b
Me
O
Me
K2CO3
3. Results and discussion
O
O
(2) X= H
(3) X = NH2
(1)
S
3.1. Chemical
X
a = C6H5SH; b = C6H4SH(p-NH2)
In our earlier studies [33] we have introduced carbonitrile
group in ring-A. The carbonitrile containing compound showed
Scheme 1.

M.R. Yadav et al. / Steroids 77 (2012) 850–857
855
O
O
O
Me
Me
Me
Me
Me
CN
Me
Me
CN
Me
CN
d
e
d
(4)
(9)
(8)
O
O
HO
a
O
O
O
Me
Me
Me
Me
CN
CN
CN
CN
Me
CN
b
(10)
(6)
(5)
C2H5O
O
O
O
O
c
d
OH
O
Me
Me
a =Al isopropoxide/Cyclohexanone
Me
b= H2O2/NaOH; c = C6H5SH, NaH
(7)
e = pTSA/triethyl orthoformate
d = DDQ;
(11)
O
S
Scheme 2.
1672 cmÀ1 (3-C@O). It offered characteristic NMR signals at d 7.01–
7.44 (m, 5H) for aromatic protons and 3.61–3.56 (m. 2H) for pro-
tons at C16 and C17 positions. Since p-aminothiaphenyl derivative
(3) did not offer better enzyme inhibiting activity over the thiaphe-
nyl derivative (2), the idea of synthesizing p-aminothiaphenyl
derivative with carbonitrile function in ring-D was dropped.
3b-Hydroxy-16b-nitrile (4) was dehydrogenated by refluxing it
with 3.3 equivalents of DDQ in anhydrous dioxane for 18 to 20 h to
afford 16b-cyano-1,4,6-androstatriene-3,17-dione (8) in very poor
yield. The compound showed UVmax at 221, 254 (shoulder peak)
and 296 nm and characteristic IR bands appeared at 2243 (-CN),
1754 (17-C@O) and 1660 cmÀ1 (3-C@O). 3b-Hydroxy-16b-nitrile
(4) when dehydrogenated by refluxing it with 1.3 equivalents of
DDQ in anhydrous dioxane for 8 to 10 h showed a single spot in
TLC. The product showed UVmax at 246 and 296 nm (shoulder
peak). IR peaks appeared at 1647 (3-C@O), 1751 (17-C@O) and
2235 cmÀ1 (16-CN). But, PMR spectrum of this product indicated
it to be a 50: 50 mixture of compounds (8 and 9).
DDQ in aqueous acetone to afford 16b-cyano-4,6-androstadiene-
3,17-dione (11). The dienone (11) showed UVmax at 281 nm in
methanol and characteristic IR bands appeared at 1652 (3-C@O),
1751 (17-C@O) and 2243 cmÀ1 (16-CN). In its NMR spectrum sig-
nals appeared at d 6.21–6.24 (dd, 1H; 7-CH), 6.08–6.10 (d; 1H;
6-CH), 5.73 (s, 1H; 4-CH) and 3.10–3.15 (m, 1H; 16-CH). Its mass
spectrum showed peak at m/z 309.9 (M+), confirming the
compound.
In order to introduce some degree of flexibility to the nitrile
group it was planned to break open the ring-D and introduce
unsaturation in ring-A and/or ring-B as illustrated in Scheme 3.
3b-Acetoxy-16,17-seco-5-androstene-16,17-dinitrile [38] became
handy for this purpose. Alkaline hydrolysis of 3b-acetoxy-16,17-
seco-5-androstene-16,17-dinitrile under mild reaction conditions
offered 3b-hydroxy-16,17-seco-5-androstene-16,17-dinitrile (12).
Compound (12) showed IR peaks at 3455 (–OH) and, 2235 cmÀ1
(–CN). Characteristic NMR signals appeared at d 5.35–5.37 (m,
1H; 6-CH), 3.50–3.60 (m, 1H; 3a-CH) and 2.66–2.73 (m, 2H; 15-
For the preparation of 4,6-diene derivative (11) the 16b-cyano-
3,17-dione (5) was treated with triethyl orthoformate in presence
of p-toluenesulfonic acid in dry dioxane for 1 h to afford 16b-cya-
no-3-ethoxy-3,5-androstadien-17-one (10) as an intermediate
which showed UVmax (MeOH) at 262 nm. The intermediate
3-enolether (10) was dehydrogenated with 1.2 equivalents of
CH2). Its mass spectrum showed peak at m/z 316 (M+18). Oppe-
nauer oxidation of the 3b-hydroxy-16,17-dinitrile (12) was carried
out using aluminium i.propoxide in cyclohexanone-toluene system
to afford 3-oxo-16,17-seco-4-androstene-16,17-dinitrile (13). The
compound showed UVmax at 238 nm for the
a,b-unsaturated
ketone which got confirmed by the presence of IR peak at
Me
Me
CN
Me
CN
CN
CN
CN
Me
c
Me
Me
a
CN
O
HO
EtO
(16)
b
(13)
(12)
b
b
Me
Me
Me
CN
CN
CN
CN
CN
CN
Me
Me
Me
O
O
O
(17)
(15)
(14)
a =Al isopropoxide/Cyclohexanone'
c = pTSA/ Triethyl orthoformate
b = DDQ;
Scheme 3.

856
M.R. Yadav et al. / Steroids 77 (2012) 850–857
Table 1
Biological activity (% inhibition of aromatase enzyme) of compounds at 5
lM concentration.
Compd. No
2
3
5
7
9
11
13
14
15
17
Exe
% Inhibition S.D.
89.5 7.2
81.3 10.4
0.7 1.2
96.0 3.7
0.0 0.0
0.0 0.0
0.9 1.5
0.0 0.0
1.0 1.3
1.5 2.7
96.1 2.2
The given values are mean values of at least three experiments.
1670 cmÀ1 (C@O). It gave characteristic signals at d 5.77 (s, 1H; 4-
CH) and 2.68–2.83 (m, 2H; 15-CH2) in its PMR spectrum. Its mass
spectrum showed peak at m/z 269.9 (M+), confirming the com-
pound (13).
(13–15 and 17) having a fractured androstane skeleton with bro-
ken D-ring were totally devoid of aromatase inhibiting activity.
The compounds (2, 3, and 7) bearing phenylthia group at C-4 posi-
tion exhibited significant enzyme inhibiting activity. Compound
(7) having phenylthia at C-4 position and nitrile group at 16-posi-
tion was found to be the most potent compound in the single dose
assay. It looks that phenylthia group at C-4 position and suitably
placed nitrile group [33] are favorable pharmacophores for aroma-
tase inhibiting activity. But, carbonitrile group all alone could not
endow aromatase inhibiting activity. Even introduction of unsatu-
ration at C-4, C-1 & 4 or C-4 & 6 along with carbonitrile function at
C-16 failed to incorporate aromatase inhibiting activity as ob-
served for compounds (5, 9 and 11).
The three active compounds (2, 3, and 7) were further evaluated
at three concentrations in order to determine their IC50 values.
Exemestane was used as standard drug for comparison. As is evi-
dent from Table 2, the aimed 16b-carbonitrile derivative (7) was
found to be equipotent to exemestane in inhibiting the enzyme.
The three potent compounds (2, 3, and 7), have been further
investigated for their ability for irreversible inhibition at 2.0 and
For the preparation of the 1,4,6-triene derivative (14) the 3b-hy-
droxy derivative (12) was dehydrogenated by refluxing it with 3.3
equivalents of DDQ in anhydrous dioxane for 18 to 20 h. The com-
pound showed UVmax at 221, 255 and 295 nm in methanol and
gave characteristic IR bands at 1680 (3-C@O), and 2235 cmÀ1
(–CN). It gave NMR signals at d 7.01–7.03 (d, 1H; 1-CH), 6.46–
6.47 (dd, 1H; 7-CH), 6.29–6.33 (d, 1H; 2-CH), 6.07–6.10 (m, 2H;
4-CH and 6-CH), and 2.79–3.00 (m, 2H; 15-CH2). Its mass spectrum
showed peak at m/z 292.5 (M+). Further, it was tried to obtain 1,4
diene derivative (15) by dehydrogenating 3b-hydroxy derivative
(12) with DDQ. The 3b-hydroxy derivative (12) was dehydrogenat-
ed by refluxing it with 1.3 equivalents of DDQ in anhydrous diox-
ane for 8 to 10 h to offer a product which showed single spot in
TLC. Its UV spectrum showed a peak at 243 nm with a shoulder
peak at 295 nm. But, the product was found to be a mixture of
compounds (14 and 15) as its PMR gave signals for vinylic protons
at C-1, 2, 4, 6 and 7 positions.
20 lM concentration using exemestane as reference compound.
For the preparation of 3-oxo-4,6-diene derivative (17), the en-
one (13) was treated with triethyl orthoformate in presence of p-
toluenesulfonic acid in dry dioxane for 1 h to yield (16) as an inter-
mediate which showed UVmax at 262 nm in methanol. The 3-enole-
ther intermediate (16) was dehydrogenated using 1.2 equivalents
of DDQ in aqueous acetone to afford the desired 3-oxo-16,17-
seco-4,6-androstadiene-16,17-dinitrile (17). The compound showed
UVmax at 279 nm in methanol. Characteristic NMR signals appeared
at d 6.28–6.31 (d, 1H; 6-CH), 6.13–6.16 (dd, 1H; 7-CH), 5.76 (s, 1H;
4-CH) and 2.79–3.01 (m, 2H; 15-CH2) and its mass spectrum
showed peak at m/z 294.9 (M+).
The results are given in Table 2. Unlike exemestane the test com-
pounds (2, 3, and 7) have shown negligible inhibition of the
enzyme under conditions which measures the capacity of a com-
pound for irreversible inhibition of the enzyme. Exemestane, an
irreversible inhibitor of the enzyme, has shown appreciable activity
under these conditions.
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