Synthesis and preliminary screening of novel a- and D-ring modified steroids as aromatase inhibitors

Article abstract of DOI:10.2174/157018011797655322

Estrogens are responsible for the growth of hormone-dependant breast cancer. Regulation of estrogen biosynthesis is considered as a potential therapeutic strategy for the control of breast cancer. The enzyme aromatase catalyzes conversion of androgens int

Full text of DOI:10.2174/157018011797655322

Letters in Drug Design & Discovery, 2011, 8, 943-950
943
Synthesis and Preliminary Screening of Novel A- and D-Ring Modified
Steroids as Aromatase Inhibitors
Mange Ram Yadav*,a, Prafulla M. Sabalea, Rajani Giridhara, Dharmendra Bariaa, Christina Zimmerb
and Rolf W. Hartmannb
aPharmacy Department, Faculty of Technology and Engineering, Kalabhavan, The M.S. University of Baroda,
Vadodara-390 001, India
bPharmaceutical and Medicinal Chemistry, Saarland University & Helmholtz Institute for Pharmaceutical Research
Saarland (HIPS), Campus C 2 3, D-66123 Saarbrucken, Germany
Received May 5, 2011: Revised August 8, 2011: Accepted August 16, 2011
Abstract: Estrogens are responsible for the growth of hormone-dependant breast cancer. Regulation of estrogen
biosynthesis is considered as a potential therapeutic strategy for the control of breast cancer. The enzyme aromatase
catalyzes conversion of androgens into estrogens in the last step of estrogen biosynthesis. Inhibition of aromatase is
adopted as an efficient approach for the prevention and treatment of breast cancer. Many steroidal and nonsteroidal
aromatase inhibitors have been developed and are used clinically for breast cancer therapy. In this report, it has been tried
to incorporate some structural features (six membered lactam ring) of nonsteroidal aromatase inhibitors like
aminoglutethimide and rogletimide into the A-/D-ring of the steroid nucleus with certain additional features responsible
for binding to the enzyme aromatase. Some ring-A [17ꢀ-hydroxy-4-(4-substitutedphenyl)-4-aza-5-androsten-3-one] and
ring-D modified steroidal compounds [4-substituted-17a-aza-D-homo-4-androstene-3,17-dione, 4-substituted-17-aza-D-
homo-4-androstene-3,16,17a-trione, 4-substituted-17a-methyl-17a-aza-D-homo-4-androsten-3-one] were synthesized and
screened for binding to the aromatase enzyme. None of the synthesized compounds showed promising aromatase
inhibiting activity leading to the conclusion that structurally the original androstane ring skeleton should not be tampered
with, for obtaining good aromatase inhibiting activity.
Keywords: A-/D-Heterosteroids, Androstanes, Aromatase, Aromatase Inhibitors, Azasteroids, Breast cancer.
1. INTRODUCTION
complex responsible for the conversion of androgens to
estrogens. Aromatase catalyzes conversion of androgens into
estrogens in the last step of estrogen biosynthesis [5].
Aromatase inhibitor testolactone [6] (1), is a leading
irreversible aromatase inhibitor of very low potency.
Aminoglutethimide (4) and 4-hydroxyandrostenedione (2)
have demonstrated therapeutic effectiveness in the treatment
of hormone-dependent breast tumors in both animals [7,8]
and humans [9-13]. Compounds that inhibit the enzyme
aromatase, such as exemestane [14] (3), rogletimide [15] (5),
anastrozole [16] (6), letrozole [17] (7), vorozole [18] (8) and
many other steroidal and nonsteroidal compounds have
applications in the treatment of advanced estrogen-dependent
breast cancer [18]. Inhibition of aromatase is an efficient
approach for the prevention and treatment of breast cancer
[19].
Breast cancer, the most common form of cancer
diagnosed in women, remains the second most common
cause of death in women in the Western world despite
advances made in its treatment. Generally considered to be
the disease of older women, 22 % of the breast cancer cases
occur in females below the age of 50 [1]. More than one
million women develop breast cancer each year worldwide
with nearly half of these diagnoses occurring in the Unites
States and Europe. Still worse is the fact that nearly 40 % of
these women die of their disease [2]. Approximately two-
thirds of postmenopausal breast cancer patients have
estrogen-dependent breast cancer, which contains estrogen
receptors (ERs) and requires estrogens for tumor growth [3].
The estrogen synthase (aromatase) enzyme system is
responsible for the biosynthesis of estrogens in humans.
Though estrogens are vital for normal growth and
development, but they also promote the growth of certain
breast cancers [4]. Approximately 30-50 % of breast cancers
are considered to be hormone-dependent. Consequently,
regulation of estrogen biosynthesis has advanced as a
potential therapeutic strategy, resulting into development of
active-site inhibitors having potential for the control of
breast cancer. Aromatase is a cytochrome P-450 enzyme
Potent steroidal aromatase inhibitors are mainly the C-1,
C-2, C-4, C-6, C-19 substituted and C-2 and C-19 bridged
steroidal compounds. New analogs of formestane have been
synthesized and their biological activity investigated in an
attempt to find new aromatase inhibitors and to gain insight
into their structure-activity relationships (SAR) [20, 21].
In the steroids, effective aromatase enzyme inhibition is
observed in compounds having androstane skeleton posses-
sing an A/B trans ring junction and a ketone functionality at
the C-3 position [3], while nonsteroidal inhibitors have a
suitably positioned hetero atom to interact strongly with
heme iron. With the exception of testolactone (1) D-
modified steroids have hardly received any research attention
*Address correspondence to this author at the Pharmacy Department,
Faculty of Technology and Engineering, Kalabhavan,The M.S. University
of Baroda, Vadodara-390 001, India; Tel: +912652434187;
Fax: +912552418927; E-mail: mryadav11@yahoo.co.in
1570-1808/11 $58.00+.00
© 2011 Bentham Science Publishers

944 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 10
Yadav et al.
O
O
Me
Me
Me
O
O
Me
Me
Me
O
O
O
CH2
OH
(1)
(2)
(3)
Me
O
Me
O
NH2
N
O
O
N
N
H
H
(5)
(4)
N
N
N
N
N
N
N
N
N
Me
N
N
Me
NC
Me
CN
N
CN
NC
Cl
Me
Me
(8)
(7)
(6)
as aromatase inhibitors. Aiming to inhibit aromatase activity,
we planned to synthesize some A- and D-ring modified
steroidal compounds and to perform preliminary screening
for their aromatase inhibiting activity.
2.1. Chemical
2.2.1. 17ꢀ-Hydroxy-4-aza-5-androsten-3-one [22] (10a)
17ꢂ-Hydroxy-4-nor-5-oxo-3,5-seco-3-androstanoic acid
[23] (9) (0.5 g, 1.62 mM) was heated with urea (0.19 g, 3.24
mM) in ethylene glycol at 150 °C under nitrogen atmosphere
for 2 hrs with constant stirring. The reaction mixture was
poured into water (50 ml), acidified with conc. hydrochloric
acid (5 ml) and stirred for 15 min. The crude product was
filtered out and dried. The solid so obtained was crystallized
from methanol to afford compound (10a) [22] (0.31 g, 65
%), m.p. 285-88 °C (Lit; [22] 289-91 °C). UV (MeOH): 231
(log ꢃ 3.33). IR (KBr): 3197, 1666, 1623, 1551, 1386, 1199
and 1056. NMR: 8.38 (s, 1H), 4.87-4.89 (m, 1H), 3.58-3.62
(t, 1H), 1.09 (s, 3H) and 0.76 (s, 3H).
Aminoglutethimide (4) and rogletimide (5) have amide
nitrogen in the six membered ring. To strike a structural
resemblance between these derivatives (4, 5) and the
steroidal skeleton, it was thought to incorporate nitrogens in
both A- and D-rings independently in the steroidal skeleton
as amide/imide functions. Such a structural change in the
steroidal system could cause higher binding of the modified
steroidal derivatives to the aromatase enzyme with retention
of selectivity of steroids for the enzyme.
2. EXPERIMENTAL
2.1. General
General Procedure for 17ꢀ-hydroxy-4-(4-substituted-
phenyl)-4-aza-5-androsten-3-ones
Melting points were determined using a VEEGO make
microprocessor-based melting point apparatus having
silicone oil bath and are uncorrected. IR spectra (wave
numbers in cmꢀ1) were recorded on a BRUKER ALPHA T
FT-IR spectrophotometer using KBr discs. NMR spectra
were recorded on BRUKER AVANCE II 400 MHz
Compound (9) (1.62 mM) was heated with different
aromatic amines (2.4 mM) in presence of DMAP (25 mg) as
catalyst at 160 °C under nitrogen atmosphere for 2 hrs. The
reaction mixture was poured into water (50 ml) acidified
with conc. hydrochloric acid (5 ml) and stirred for 15 min.
The crude product was filtered out, dried and purified by
passing through a silica gel column using hexane-ethyl
acetate, (9:1) as an eluent. The solid so obtained was
crystallized from methanol to afford the desired compound.
1
instrument in CDCl3 with TMS as internal standard for H
NMR. Chemical shift values are mentioned in ꢁ, ppm.
Chromatographic separations were performed on silica gel
columns. The microanalyses for C, H and N were performed
on Thermo Scientific FLASH 2000 organic elemental
analyzer. The progress of all reactions was monitored by
TLC on 2 cm x 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” refers to the use of anhydrous sodium sulfate. All
reagents used were of analytical reagent grade.
2.2.2. 17ꢀ-Hydroxy-4-phenyl-4-aza-5-androsten-3-one (10b)
Yield (0.19 g, 32 %), m.p. 263-65 °C. UV (MeOH): 229
(log ꢃ 3.23). IR (KBr): 3457, 1641, 1387 and 1235. NMR:
7.33-7.37 (m, 2H), 7.23-7.27 (m, 1H), 7.00-7.03 (m, 2H),
4.30-4.32 (m, 1H), 3.57-3.61 (t, 1H), 2.65-2.9 (m, 2H), 1.21
(s, 3H) and 0.72 (s, 3H). Calculated for C24H31NO2: C 78.87,

Synthesis and Antimicrobial Evaluation of Isonicotinic Acid Hydrazides
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 10 945
H 8.55, N 3.83. Found: C 78.02, H 8.71, N 3.54. MS: m/z
367.1 (M+).
3H) and 1.21 (s, 3H). Calculated for C25H31NO2S: C 73.31,
H 7.63, N 3.42. Found: C 73.54, H 7.91, N 3.29. MS: m/z
410.1 (M+).
2.2.3. 17ꢀ-Hydroxy-4-(4-fluorophenyl)-4-aza-5-androsten-
3-one (10c)
2.2.7.
stene-3,17-dione (15)
4-(4-Aminophenylthia)-17a-aza-D-homo-4-andro-
Yield (0.23 g, 38 %), m.p. 266-68 °C. UV (MeOH): 228
(log ꢀ 3.37).IR (KBr): 3479, 1642, 1504, 1398 and 1209.
NMR: 6.96-7.06 (m, 4H), 4.35-4.42 (m, 1H), 3.62-3.71 (t,
1H), 2.57-2.62 (m, 2H), 1.19 (s, 3H) and 0.72 (s, 3H).
Calculated for C24H30FNO2: C 75.17, H 7.88, N 3.65. Found:
C 74.89, H 7.50, N 3.85. MS: m/z 384.1 (M+).
p-Aminothiophenol (0.3 g, 2.4 mM) was stirred with
sodium hydride (0.056 g, 2.4 mM) in dry dioxane (5.0 ml)
under nitrogen atmosphere at 20°C. A solution of the
epoxide [24] (12) (0.5 g, 1.6 mM) in dry dioxane (3.0 ml)
was added slowly to the above solution with stirring. The
reaction mixture was stirred for 8 hrs at room temperature.
The reaction mixture was poured into water (100 ml),
acidified with hydrochloric acid and extracted with
dichloromethane (4 x 25 ml). The combined extract was
washed with water, dried and solvent recovered to afford the
crude product. The crude product so obtained was purified
by passing through a column of silica gel eluting first with
chloroform and then with chloroform-methanol (9.5: 0.5).
The solid so obtained was crystallized from methanol to
afford yellowish crystals of compound (15) (0.2 g, 30 %),
m.p. 210-13 °C. UV (MeOH): 260 (log ꢀ 4.3). IR (KBr):
3444, 1653, 1494, 1395, 1159, 820, 750 and 519. NMR:
7.06-7.09 (d, 2H), 6.54-6.55 (d, 2H), 6.29 (b, 1H), 1.25 (s,
3H) and 1.24 (s, 3H). Calculated for C25H32N2O2S: C 70.72,
H 7.60, N 6.60. Found: C 70.35, H 7.69, N 6.93. MS : m/z
425.1 (M+).
2.2.4. 17ꢀ-Hydroxy-4-(4-tolyl)-4-aza-5-androsten-3-one (10d)
Yield (0.2 g, 35 %), m.p. 214 -17 °C. UV (MeOH): 229
(log ꢀ 3.35). IR (KBr): 3199, 1675, 1651, 1510, 1390, 1217
and 804. NMR: 7.21-7.23 (d, 2H), 6.94-6.97 (d, 2H), 4.40-
4.42 (m, 1H), 3.63-3.68 (t, 1H), 2.36 (s, 3H), 1.24 (s, 3H)
and 0.79 (s, 3H). Calculated for C25H33NO2: C 79.11, H 8.76,
N 3.69. Found: C 79.43, H 8.39, N 3.96. MS: m/z 380.1
(M+).
2.2.5. 4-Hydroxy-17a-aza-D-homo-4-androstene-3,17-dione
[24] (13)
The epoxide (12) was prepared as per the reported
method [24,25]. To a chilled solution of epoxide (12) (0.6 g,
1.9 mM) in glacial acetic acid (6 ml) a chilled mixture of
conc. sulphuric acid (0.8 ml, 11.4 mM) in glacial acetic acid
(2.0 ml) was added with occasional shaking and the reaction
mixture was allowed to stand for 18 hrs at 0-5 °C. The
reaction mixture was poured into water (100 ml) and basified
with potassium hydroxide solution (5 %). The resulting
alkaline mixture was acidified with glacial acetic acid
followed by neutralization with excess of sodium
bicarbonate solution (5 %) and extracted with chloroform (3
x 25 ml). the combined chloroform extract was washed with
sodium bicarbonate solution (5 %), dried and the solvent
recovered to afford crude product which was crystallized
from methanol to obtain crystals of compound [24] (13) (0.3
g, 50 %), m.p. 262-65°C (Lit; [24] 264-66 °C). UV (MeOH):
276 (log ꢀ 3.19). (Alk. MeOH): 315 (log ꢀ 4.1).IR (KBr):
3458, 3183, 1681, 1391, 1238, 1163 and 964. NMR: 6.05 (s,
1H), 1.13 (s, 3H) and 1.10 (s, 3H). MS: m/z 318.0 (M+).
2.2.8. 4ꢁ,5-Oxido-17-aza-D-homo-5ꢁ-androstane-3,16,17a-
trione (20)
Cold solutions of sodium hydroxide (20 %, 1.5 ml, 7.6
mM) and hydrogen peroxide (30 %. 2 ml, 1.74 mM) were
added simultaneously drop by drop during stirring to a
chilled
solution
of
16,17a-dioxo-17-aza-D-homo-4-
androsten-3-one [26] (19) (1.0 g, 3.17 mM) in methanol (25
ml). The reaction mixture was allowed to stand for 3 hrs at 0
°C, diluted with water (200 ml) and extracted with
dichloromethane (4 x 25 ml). The combined extract was
washed with water, dried and the solvent recovered to afford
the crude product 4ꢁ,5-oxido-17-aza-D-homo-5ꢁ-androstane-
3,16,17a-trione (20) (0.6 g, 60 %), which was used as such
for the next step without purification. UV (MeOH):
Transparent at 239. IR (KBr): 3384, 1689, 1444, 1373, 1275
and 1054.
2.2.6.
4-Phenylthia-17a-aza-D-homo-4-androstene-3,17-
dione (14)
2.2.9. 4-Hydroxy-17-aza-D-homo-4-androstene-3,16,17a-
trione (21)
Thiophenol (0.250 g, 4.4 mM) was stirred with sodium
hydride (0.105 g, 4.4 mM) in dry dioxane (5.0 ml) under
nitrogen atmosphere at 20 °C. A solution of the epoxide [24]
(12) (0.7 g, 2.21 mM) in dry dioxane (5.0 ml) was added
slowly to the above solution with stirring. The reaction
mixture was stirred for 6 hrs at room temperature, poured
into water (100 ml) and extracted with dichloromethane (4 x
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 crystallized from methanol to
afford yellowish crystals of the desired compound (14) (0.6
g, 68 %), m.p. 222-24 °C. UV (MeOH): 249 (log ꢀ 3.91). IR
(KBr): 3195, 1678, 1654, 1396, 1232, 1157, 1068, 743, 465
and 427. NMR: 7.08- 7.22 (m, 5H), 6.05 (b, 1H), 1.30 (s,
The reaction was performed as described under
compound (13) using 4ꢁ,5-oxido-17-aza-D-homo-5ꢁ-
androstane-3,16,17a-trione (20) (1.0 g, 3.0 mM). The
reaction mixture was poured into water (100 ml) and
extracted with dichloromethane (4 x 25 ml). The combined
extract was washed with sodium bicarbonate solution (5 %),
dried and solvent recovered to afford a crude product which
was crystallized from methanol to afford 4-hydroxy-17-aza-
D-homo-4-androstene-3,16,17a-trione (21) (0.5 g, 50 %),
m.p 240-45 °C. UV (MeOH): 276 (log ꢀ 3.58), (Alk.
MeOH): 315 (log ꢀ 3.53). IR (KBr): 3390, 3298, 1688, 1658,
1433, 1384, 1280 and 1031. NMR: 7.62 (b, 1H; exchanged
with D2O), 6.03 (s, 1H; exchanged with D2O), 1.16 (s, 3H)
and 1.12 (s, 3H). Calculated for C19H25NO4: C 68.86, H 7.60,

946 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 10
Yadav et al.
N 4.23. Found: C 68.43, H 7.46, N 4.61. MS: m/z 332.1
(M+).
androsten-3-one [24] (21) (0.31 g, 52 %), m.p. 227-30 °C
(Lit; [24] 227-29 °C). UV (MeOH): 274 (log ꢀ 4.18), (Alk.
MeOH): 334 (log ꢀ 4.46). IR (KBr): 3431, 1672, 1620, 1369,
1163, 1089 and 987. NMR: 6.05 (s, 1H), 1.14 (s, 3H) and
0.88 (s, 3H). MS: m/z 318.1 (M+).
2.2.10. 16,17a-Dioxo-4-phenylthia-17-aza-D-homo-4-and-
rosten-3-one (22)
The reaction was performed as described under
compound (14) using epoxide (20) (0.5 g, 1.5 mM). The
reaction mixture was stirred for 3 hrs at room temperature,
poured into water (100 ml) and extracted with
dichloromethane (4 x 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
crystallized from methanol to afford yellow crystals of
16,17a-dioxo-4-phenylthia-17-aza-D-homo-4-androsten-3-
one (22) (0.3 g, 68 %), m.p 125-27 °C. UV (MeOH): 250
(log ꢀ 3.77). IR (KBr): 3229, 1695, 1686, 1580, 1437, 1276,
1113 and 1024. NMR: 7.63 (b, 1H; exchanged with D2O),
7.12-7.19 (d, 2H), 7.01-7.05 (m, 3H), 1.17 (s, 3H) and 0.86
(s, 3H). Calculated for C25H29NO3S: C 70.80, H 6.90, N
3.31. Found: C 70.42, H 7.11, N 3.63. MS: m/z 424.1 (M+).
2.2.13. 17a-Methyl-4-phenylthia-17a-aza-D-homo-4-andro-
sten-3-one (27)
The reaction was performed as described under
compound (14) using 17a-methyl-4ꢁ,5-oxido-17a-aza-D-
homo-5ꢁ-androstan-3-one [24] (25) (0.4g, 1.3 mM). The
reaction mixture was stirred for 6 hrs at room temperature,
poured into water (100 ml) and extracted with
dichloromethane (4 x 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
crystallized from methanol to afford yellowish crystals of
desired compound (27) (0.2 g, 52 %), m.p. 173-75 °C. UV
(MeOH): 251 (log ꢀ 4.03). IR (KBr): 1689, 1562, 1474,
1378, 1282 1106, 746, 808 and 693. NMR: 7.00-7.19 (m,
5H), 1.19 (s, 3H) and 1.00 (s, 3H). Calculated for
C26H35NOS: C 76.24, H 8.61, N 3.42. Found: C 76.38, H
8.55, N 3.64. MS: m/z 410.1 (M+).
2.2.11.
4-(4-Aminophenylthia)-17-aza-D-homo-4-andro-
stene-3,16,17a-trione (23)
The reaction was performed as described under
compound (15) using epoxide (20) (0.5 g, 1.58 mM).The
reaction mixture was poured into water (100 ml), acidified
with hydrochloric acid (5 ml) and extracted with
dichloromethane (4 x 25 ml). The combined extract was
washed with water, dried and the solvent recovered to afford
the crude product. The crude product so obtained was
purified by passing through a column of silica gel eluting
first with chloroform and then with chloroform-methanol
(9:1). The solid so obtained was crystallized from methanol
to afford yellowish crystals of 4-(4-aminophenylthia)-17-
aza-D-homo-4-androstene-3,16,17a-trione (23) (0.28 g,
59.66 %), m.p 120-25 °C. UV (MeOH): 258 (log ꢀ 3.07). IR
(KBr): 3531, 3243, 1694, 1373, 1267, 1064 and 829. NMR:
7.21-7.23 (d, 2H), 6.95-6.97 (d, 2H), 1.25 (s, 3H) and 0.82
(s, 3H). Calculated for C25H30N2O3S: C 68.46, H 6.89, N
6.39. Found: C 68.15, H 6.71, N 6.23. MS: m/z 456.6 (M+
18).
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.
2.3.1.2. Enzyme Preparation
The enzyme was obtained from the microsomal fraction
of freshly delivered human term placental tissue as per the
procedure described by Thompson and Siiteri [29]. The
isolated microsomes were suspended in minimum volume of
phosphate buffer (0.05 M, pH 7.4, 20 %). 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.
2.2.12. 4-Hydroxy-17a-methyl-17a-aza-D-homo-4-andro-
sten-3-one [24] (26)
2.3.1.2. Aromatase Inhibition Assay
To a chilled solution of 17a-methyl-4ꢁ,5-oxido-17a-aza-
D-homo-5ꢁ-androstan-3-one [24,25] (25) (0.7 g, 2.0 mM) in
glacial acetic acid (6 ml) a chilled mixture of conc. sulphuric
acid (1.4 ml, 14.0 mM) in glacial acetic acid (2.0 ml) was
added with occasional shaking and the reaction mixture was
allowed to stand for 18 hrs at 0-5 °C. The reaction mixture
was poured into water (100 ml) and basified with potassium
hydroxide solution (5 %). The resulting alkaline mixture was
acidified with glacial acetic acid followed by neutralization
with excess of sodium bicarbonate solution (5 %) and
extracted with chloroform (3 x 25 ml). The combined
chloroform extract was washed with sodium bicarbonate
solution (5 %), dried and the solvent recovered to afford
crude product which was crystallized from methanol to
obtain crystals of 4-hydroxy-17a-methyl-17a-aza-D-homo-4-
The assay was performed by measuring the 3H2O formed
from [1ꢂ-3H] androstenedione during aromatization [30].
Each incubation tube contained [1ꢂ-3H] androstenedione
(0.08 ꢃCi), unlabeled androstenedione (485 nM), NADP (2
mM), glucose-6-phosphate (20 mM), glucose-6-phosphate
dehydrogenase (0.4 units), and the inhibitor (0.05 nM) in
phosphate buffer (pH 7.4). The test compounds had been
dissolved in DMSO and diluted with buffer. The final
DMSO concentration in the control and inhibitor incubation
was 2%. Each tube was preincubated for 5 min at 30 °C in a
shaking water bath. Microsomal protein (0.1 mg) was added
to start the reaction. The total volume for each incubation
was 0.2 ml. The reaction was terminated by withdrawing 100
μl aliquots at 0, 7, 14, and 21 min and pipetting them into

Synthesis and Antimicrobial Evaluation of Isonicotinic Acid Hydrazides
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 10 947
OH
Me
OH
Me
a
Me
Me
(10a) R= H,
(10c) R= p-Ph-F, (10d) R= p-Ph-Me
(10b) R= Ph,
HOOC
O
O
N
R
(9)
(10)
a =Urea/Aromatic Amine
Scheme 1.
200 μl of a cold mercuric chloride solution (1 mM). After
addition of an aqueous dextran-coated charcoal (DCC)
suspension (200 μl, 2%), the vials were shaken for 20 min
and centrifuged at 1500 x g for 5 min to separate the
charcoal-adsorbed steroids. Aliquots of the supernatant were
assayed for 3H2O by counting in a scintillation mixture using
Perkin Elmer-Wallac ꢀ-Counter.
was treated with p-fluoroaniline in presence of DMAP, it
yielded 17ꢀ-hydroxy-4-(4-fluorophenyl)-4-aza-5-androsten-
3-one (10c). [IR: 3479 (17-OH) and 1642 cm-1 (4-C=O
lactam). It offered characteristic NMR signals at ꢁ 6.96-7.06
(m, 4H) for aromatic protons]. When compound (9) was
cyclized with p-toluidine it offered 17ꢀ-hydroxy-4-(4-tolyl)-
4-aza-5-androsten-3-one (10d). [IR: 3476 (17- OH ) and
1642 cm-1 (3-C=O). PMR: ꢁ 7.21-7.23 (d, 2H; Ar-H), 6.94-
6.97 (d, 2H; Ar-H), 4.40-4.42 (m, 1H; 6-CH), 3.63-3.68 (t,
1H; 17-H) and 2.36 (s, 3H; Ar-CH3)].
3. RESULTS AND DISCUSSION
3.1. Chemical
For the synthesis of D-ring modified steroids two
different schemes (Schemes 2 & 3) were followed. For
Scheme 2 17a-aza-D-homo-4-androstene-3,17-dione [24]
(11) was used as a starting material. Various D-ring
modified 4-androstene steroids were synthesized as depicted
in Scheme 2. Treatment of the enone (11) with alkaline
hydrogen peroxide afforded the epoxide, 4ꢃ,5-oxido-17a-
aza-D-homo-5ꢃ-androstane-3,17-dione (12), which upon
acid treatment offered the 4-hydroxy-17a-aza-D-homo-4-
androsten-3,17-dione [24] (13). The epoxide (12), when
stirred with thiophenol in dry dioxane with sodium hydride
afforded the 4-phenylthia-17a-aza-D-homo-4-androstene-
3,17-dione (14). [IR: 3195 (secondary-N-H), 1678 (-C=O)
and 1654 (17-C=O) cm-1. PMR: ꢁ 7.08-7.22 (m, 5H; Ar-H)
and 6.05 (b, 1H; NH)]. The oxirane (12) when stirred with p-
aminothiophenol in dry dioxane with sodium hydride
For the synthesis of A-ring modified steroidal
compounds
17ꢀ-hydroxy-5-oxo-A-nor-3,5-seco-3-andro-
stanoic acid [23] (9) was used as the starting material for the
synthesis of 4-aza-5-ene derivatives of androstane as shown
in Scheme 1. Compound (9) was condensed with urea in
ethylene glycol at 150 °C to afford 17ꢀ-hydroxy-4-aza-5-
androsten-3-one [22] (10a). [IR: 3197 (N-H str) and 1666
cm-1 (4-C=O lactam). PMR ꢁ: 8.38 (s, 1H; 4-N-H), 4.87-4.89
(m, 1H; 6-CH) and 3.58-3.62 (t, 1H; 17ꢂ-CH)]. Fusion of
compound (9) with aniline in presence of dimethylamino-
pyridine (DMAP) as catalyst at 160 °C under nitrogen
atmosphere, afforded 17ꢀ-hydroxy-4-phenyl-4-aza-5-andro-
sten-3-one (10b). [IR: 3457 (17- OH ) and 1641 cm-1 (3-
C=O). PMR: ꢁ 7.33-7.37 (m, 2H; Ar-CH), 7.23-7.27 (m, 1H;
Ar-CH), 7.00-7.03 (m, 2H; Ar-CH), 4.30-4.32 (m, 1H; 6-
CH) and 3.57-3.61 (t, 1H; 17ꢂ-CH)]. When compound (9)
H
H
N
H
N
Me
Me
Me
N
O
O
O
Me
Me
O
a
b
Me
O
O
O
(11)
(13)
(12)
d
OH
c
H
H
Me
Me
N
O
N
O
Me
Me
O
O
(15)
(14)
S
S
a = H2O2, NaOH b = H2SO4
c = C6H5SH d = C6H4SH(p-NH2)
NH2
Scheme 2.

948 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 10
Yadav et al.
O
O
O
Me
Me
Me
NH
NH
Me
NOH
Me
Me
O
O
HO
RO
O
(16)
R= Ac
R= H
(17)
(18)
(19)
a
O
O
O
Me
Me
Me
NH
NH
NH
Me
O
Me
Me
b
c
O
O
O
O
O
O
OH
S
(22)
(21)
(20)
d
O
Me
NH
Me
O
O
S
(23)
NH2
a = H2O2, NaOH b = H2SO4
c = C6H5SH d = C6H4SH(p-NH2)
Scheme 3.
afforded the targeted 4-(4-aminophenylthia)-17a-aza-D-
homo-4-androstene-3,17-dione (15). [IR: 3444 (-N-H) and
1653 cm-1 (-C=O). PMR: ꢀ 7.06-7.09 (d, 2H; Ar-H), 6.54-
6.55 (d, 2H; Ar-H), 6.29 (b, 1H; NH), 1.25 (s, 3H) and 1.24
(s, 3H)].
at room temperature it gave 4-(4-aminophenylthia)-17-aza-
D-homo-4-androstene-3,16,17a-trione (23). [ IR: 1694 cm-1
(3-C=O). PMR: ꢀ 7.21-7.23 (m, 2H; Ar-2H), 6.95-6.97 (m,
2H; Ar-2H)].
It was also planned to have basic nitrogen in the D-ring
It was planned to synthesize 17-aza derivatives with
substitution at position 4. Beckmann’s rearrangement of 3ꢁ-
hydroxy-16-oximino-5-androsten-17-one [27] (16) was
effected with acetic anhydride and acetic acid to afford 3ꢁ-
in place of the acidic amide. Scheme 4 was followed for this
purpose.
17a-Methyl-17a-aza-D-homo-4-androsten-3-one
[24] (24) on treatment with alkaline hydrogen peroxide
afforded the epoxide (25). Treatment of the oxirane (25) with
conc. sulphuric acid in glacial acetic acid gave 4-hydroxy-
17a-methyl-17a-aza-D-homo-4-androsten-3-one (26). IR:
3431 (-OH) and 1672 (3-C=O) cm-1, PMR: ꢀ 6.05 (s, 1H;
OH)]. The epoxide (25) was stirred with thiophenol in dry
dioxane and sodium hydride under inert environment at
room temperature to afford 17a-methyl-4-phenylthia-17a-
aza-D-homo-4-androsten-3-one (27). [IR: 1689 (3-C=O)
cm-1. PMR: ꢀ 7.00-7.19 (m, 5H; Ar-H)].
acetoxy-17-aza-D-homo-5-androstene-16,17a-dione
[28]
(17). Alkaline hydrolysis and Oppenauer oxidation of
compound (17) gave 16,17a-dioxo-17-aza-D-homo-4-
androsten-3-one [26] (19). The enone (19) on treatment with
alkaline hydrogen peroxide afforded the epoxide (20) which
shows no absorption at 240 nm in its UV spectrum due to the
absence of ꢂ,ꢁ-unsaturated system. Epoxide (20) upon acidic
treatment afforded 4-hydroxy-17-aza-D-homo-4-androstene-
3,16,17a-trione (21). [UV (MeOH): 276 (log ꢃ 3.58), (Alk.
MeOH): 315 (log ꢃ 3.53). IR: 3390 (-OH) and 1688 cm-1 (3-
C=O). PMR: ꢀ 7.62 (b, 1H; -NH), 6.03 (s, 1H; OH)]. When
the epoxide (20) was stirred with thiophenol in basic
medium, it afforded 16,17a-dioxo-4-phenylthia-17-aza-D-
homo-4-androsten-3-one (22). [IR: 1695 (3-C=O) and 1686
cm-1 (16, 17-C=O). PMR: ꢀ 7.63 (b, 1H; -NH), 7.12-7.19 (d,
2H; -Ar-H), 7.01-7.05 (m, 3H; -Ar-H)]. When the epoxide
(20) was treated with p-aminothiophenol and sodium hydride
3.2 Biological
All of the synthesized compounds (10a-10d, 13-15, 20-
23, 26 and 27) were evaluated for their aromatase inhibiting
activity. The assay was performed by monitoring the enzyme
activity by measuring the 3H2O formed from [1ꢁ-3H]
androstenedione during aromatization. The activity profile of
the compounds is indicated in Table 1. Rings A and D in

Synthesis and Antimicrobial Evaluation of Isonicotinic Acid Hydrazides
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 10 949
Me
Me
N
Me
Me
N
Me
a
Me
O
O
O
(24)
b
(25)
Me
Me
c
Me
N
Me
N
Me
Me
O
O
S
OH
(26)
(27)
a = H2O2/NaOH b = H2SO4
c = C6H5SH
Scheme 4.
compounds (10, 13-15, 20-23) were modified to make them
look structurally similar to nonsteroidal compounds (4, 5).
This change was effected with a view so that such structural
modification might cause a change in binding mode affinity
of these compounds to aromatase enzyme. As evident from
the results given in Table 1, these compounds lost their
binding affinity almost completely, to the enzyme.
Restoration of basicity to the nitrogen atom in compounds
(26 and 27) did not cause any impact as far as binding
affinity to the enzyme was concerned.
In our earlier report [21] we have reported some ring-A
substituted compounds having the original androstane ring
skeleton intact. Some of these compounds showed good
aromatase inhibiting properties. In the present study, it has
been observed that whenever a nitrogen atom (with or
without basicity) has been introduced into the androstane
ring skeleton by replacement of carbon at C-4, C-16 and C-
17 positions, total loss of binding affinity to the enzyme
resulted. This clearly shows that in the newly synthesized
compounds the original androstane skeleton must be
maintained for binding to the aromatase enzyme. Any future
designing process for the development of potential
aromatase inhibitors must take into consideration the fact
that any tampering of the original androstane ring skeleton
would most likely result in drastic reduction/abolition of
aromatase inhibiting activity.
Table 1. Biological Activity (% Inhibition of Aromatase
Enzyme) of Compounds
Compd.
Conc. μM
% Inhibition
10a
5.0
5 .0
5.0
5 .0
0.5
0.5
0.5
0.5
0.5
5.0
5 .0
5.0
5 .0
5.0
0.9±1.6
0.6±1.0
4.6±4.4
2.3±2.3
0.0±0.0
0.3±0.5
0.7±1.2
4.9±3.3
2.0±2.8
11.0±1.5
2.3±1.8
0.0±0.1
0.0±0.0
96.1±2.2
10b
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