Steroidal pyrazolines and pyrazoles as potential 5α-reductase inhibitors: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.steroids.2014.09.004

Taking pregnenolone as the starting material, two series of pyrazolinyl and pyrazolyl pregnenolones were synthesized through different routes. The synthesis of the analogs of both series is multistep and proceeds in good overall yields. While the key step in the synthesis of pyrazolinyl pregnenolones is the heterocyclization of benzylidine derivatives (3) in presence of hydrazine hydrate, it is the condensation of 3β-hydroxy-21-hydroxymethylidenepregn-5-en-3β-ol-20-one (5) with phenylhydrazine in the synthesis of pyrazolyl derivatives. Compounds of both the series were tested for their 5α-reductase inhibitory activities. Amongst all the compounds screened for their 5α-reductase inhibitory activities, compound 4b, 4c and 6b were found to be the most active.

Full text of DOI:10.1016/j.steroids.2014.09.004

Steroids 92 (2014) 13–19
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Steroidal pyrazolines and pyrazoles as potential 5
inhibitors: Synthesis and biological evaluation
a-reductase
a
c
Abid H. Banday a,b, , Shameem A. Shameem , Salika Jeelani
⇑
a Department of Chemistry, Islamia College of Science and Commerce, Srinagar 190009, India
b Department of Chemistry and Biochemistry, University of Arizona, Tucson 85721, USA
c Department of Chemistry, University of Kashmir, Hazratbal, Srinagar 190002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 May 2014
Received in revised form 23 July 2014
Accepted 15 September 2014
Available online 30 September 2014
Taking pregnenolone as the starting material, two series of pyrazolinyl and pyrazolyl pregnenolones were
synthesized through different routes. The synthesis of the analogs of both series is multistep and pro-
ceeds in good overall yields. While the key step in the synthesis of pyrazolinyl pregnenolones is the het-
erocyclization of benzylidine derivatives (3) in presence of hydrazine hydrate, it is the condensation of
3b-hydroxy-21-hydroxymethylidenepregn-5-en-3b-ol-20-one (5) with phenylhydrazine in the synthesis
of pyrazolyl derivatives. Compounds of both the series were tested for their 5a-reductase inhibitory
Keywords:
activities. Amongst all the compounds screened for their 5
a-reductase inhibitory activities, compound
Pregnenolone
Pyrazoline
Pyrazoles
4b, 4c and 6b were found to be the most active.
Ó 2014 Published by Elsevier Inc.
Benzylidines
5a-reductase inhibition
1. Introduction
defined targets, such as 5
explored for treatment of cancer.
The enzyme steroid 5 -reductase (5AR) catalyses the NADPH-
a-reductase (5AR), are being actively
Steroids represent a pharmacologically active class of molecules
associated with variety of physiological functions. Steroidal deriv-
atives in which ring D is modified with heterocyclic rings have
been of great pharmaceutical interest [1]. Steroids as well as their
derivatives have the potential to be developed as drugs for the
treatment of a large number of diseases including cardiovascular
[2], autoimmune diseases [3], brain tumors, breast cancer, prostate
cancer, osteoarthritis, etc. [4]. The promise of using steroids for
development of lead molecules lies in the regulation of a variety
of biological processes by these molecules and being a fundamen-
tal class of signaling molecules [5]. Though steroids and steroid
based molecules have been used as active pharmaceutical agents
against various diseases, there has recently been a surge in the
exploitation of these molecules against cancer. Unfortunately
despite the recent advances in the early diagnosis, prevention
and therapy, cancer still remains a challenge as it affects millions
of people world over and is one of the leading causes of death
[6,7]. It thus necessitates the development of new drugs against
this dreadful disease which remains the primary focus of various
research groups throughout the world. Emerging new molecularly
a
dependant reductive conversion of testosterone to dehydrotestos-
terone. The higher 5AR activity leads to increased levels of
dihydrotestosterone in the peripheral tissues, which is implicated
in the pathogenesis of prostate cancer, acne and male pattern bald-
ness [8]. However the deficiency of 5AR in males results in an
incomplete differentiation of external genitalia at birth [9]. It has
been established that there are two genes encoding two distinct
isozymes of 5AR that are differentially expressed in human tissues
and are referred to as type I 5AR (5AR1) and type II 5AR (5AR2)
[10]. While the former is expressed predominantly in the skin
and liver, the later is expressed mainly in prostate, seminal vesi-
cles, liver and epididymis [11]. A number of steroidal [12] and
non-steroidal [13] inhibitors have been tested against 5AR. The
two most important steroid based 5AR inhibitors are Finasteride
(PROSCAR, Merck) and dutasteride (Avodart, GlaxoSmithKline).
Finasteride, a type II-selective inhibitor, was the first 5AR inhibitor
approved in the United States for the treatment of prostate cancer
and benign prostatic hyperplasia (BPH). Dutasteride has no selec-
tivity and acts as an inhibitor against type I and type II 5AR.
However both the approved drugs suffer from serious side effects
such as erectile dysfunction, abnormal ejaculation, impotence,
abnormal sexual function, decreased sexual desire, gynecomastia
etc. [14]. Recent literature precedents indicate various steroidal
⇑
Corresponding author at: Department of Chemistry, Islamia College of Science
and Commerce, Srinagar 190009, India. Fax: +91 194 2429014.
E-mail address: abidrrl@gmail.com (A.H. Banday).
http://dx.doi.org/10.1016/j.steroids.2014.09.004
0039-128X/Ó 2014 Published by Elsevier Inc.

14
A.H. Banday et al. / Steroids 92 (2014) 13–19
D-ring heterocyclic derivatives as inhibitors of 5AR and 17
a
-
distinguish the product and could easily be differentiated are
reported).
hydroxylase/C17-20-lyase of which 17-imidazolyl, pyrazolyl, pyraz-
olinyl, isoxazolyl, oxazolyl and thiazolyl derivatives are very potent
[24]. Taking inputs from these literature precedents to obtain the
skeleton structure required for 5AR inhibitory activity, we, in con-
tinuation of our research program directed towards the develop-
ment of steroid based lead molecules [15], designed synthesis of
two series of novel pyrazolinyl and pyrazolyl analogs from preg-
nenolone. Further, as it is well established that 17b-heterocycles
are more active towards the 5AR inhibition [24], we also designed
the heterocycles on the same lines with b-orientation at the 17-
position. All the synthesized pregnenolone derivatives were evalu-
ated for their 5AR inhibitory activity. It was observed that com-
pounds 4b, 4c and 6b exhibit excellent activities and are the
most potent of all the screened compounds.
2.2.1.1. 1-(4,5-Dihydro-3-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,
17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phe-
nanthren-17-yl)-5-phenylpyrazol-1-yl) ethanone (4a). Colourless
25
solid powder. Yield 76%. M.p: 194–197 °C. [
a]
ꢁ85.9 (c 1 in
D
CHCl3). IR (KBr, cmꢁ1): 3384, 2926, 1717, 1646, 1404, 1042, 699.
1H NMR (CDCl3, 400 MHz): d 0.67 (s, 3H), 1.06 (s, 3H), 1.82–1.90
(m, 6H), 2.17 (s, 3H), 2.65 (t, 1H, J = 8.8), 2.79 (m, 2H), 3.26 (m,
1H), 3.49 (m, 1H), 5.33 (s, 1H), 5.44 (m, 1H), 7.15 (d, 2H, J = 6.5),
7.22–7.32 (m, 3H). 13C NMR (CDCl3, 400 MHz): d 14.85, 20.85,
22.40, 23.32, 25.83, 31.13, 33.04, 33.48, 37.99, 38.72, 39.94,
43.68, 45.30, 47.69, 51.53, 53.18, 57.94, 60.56, 73.11, 122.80,
126.78, 128.84, 130.28, 142.29, 160.63, 164.62. ESI-MS: 483
(M++Na). Anal. Calcd. for C30H40N2O2: C, 78.22; H, 8.75; N, 6.08;
found C, 78.47; H, 8.83; N, 6.21.
2. Experimental
2.2.1.2. 1-(5-(3-Fluorophenyl)-4,5-dihydro-3-((10R,13S)2,3,4,7,8,9,10,
11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-
2.1. General methods
cyclopenta[a]phenanthren-17-yl) pyrazol-1-yl)ethanone (4b). Col-
25
ourless solid. Yield 79%. M.p: 168–171 °C. [
a]
ꢁ40.2 (c 1 in
Melting points were recorded on Buchi melting point apparatus
D-545; IR spectra (KBr discs) were recorded on Bruker Vector 22
instrument. NMR spectra were recorded on Bruker DPX200 instru-
ment in CDCl3 with TMS as internal standard for protons and sol-
vent signals as internal standard for carbon spectra. Chemical
shift values are mentioned in d (ppm) and coupling constants are
given in Hz. Mass spectra were recorded on EIMS (Shimadzu)
and ESI-esquire 3000 Bruker Daltonics instrument. The progress
of all reactions was monitored by TLC on 2 ꢀ 5 cm pre-coated silica
gel 60 F254 plates of thickness of 0.25 mm (Merck). The chromato-
grams were visualized under UV 254–366 nm and iodine.
D
CHCl3). IR (KBr, cmꢁ1): 3408, 2936, 1718, 1448, 1021, 756. 1H
NMR (CDCl3, 400 MHz): d 0.67 (s, 3 H), 1.06 (s, 3H), 1.82–1.90
(m, 6H), 2.15 (s, 3H), 2.67 (t, 1H, J = 8.8), 2.77 (m, 2H), 3.26 (m,
1H), 3.35 (m, 1H), 5.30 (s, 1H), 5.39 (m, 1H), 6.80–6.91 (m, 2H),
7.30–7.34 (m, 2H). 13C NMR (CDCl3, 400 MHz): d 12.24, 18.40,
19.95, 20.82, 23.58, 28.60, 30.59, 31.03, 35.54, 36.27, 37.38,
41.23, 45.07, 50.69, 55.38, 57.66, 70.64, 111.11, 113.16, 120.30,
129.37,139.86, 143.79, 159.69, 164.58, 167.71. ESI-MS: 479
(M++H). Anal. Calcd. for C30H39FN2O2: C, 75.28; H, 8.21; N, 5.85;
found C, 75.43; H, 8.03; N, 6.04.
2.2.1.3. 1-(5-(4-Fluorophenyl)-4,5-dihydro-3-(10R,13S)2,3,4,7,8,9,10,
11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-
2.2. Chemical synthesis
cyclopenta[a]phenanthren-17-yl) pyrazol-1-yl)ethanone (4c). Col-
25
2.2.1. General procedure for the synthesis of pyrazoline derivatives
(4a–4j)
ourless solid powder. Yield 82%. M.p: 222–225 °C. [
a
]
D
ꢁ58.0 (c
1 in CHCl3). IR (KBr, cmꢁ1): 3375, 3166, 2928, 1721, 1404, 1042,
756. 1H NMR (CDCl3, 400 MHz): d 0.65 (s, 3 H), 1.05 (s, 3H),
1.82–1.90 (m, 6H), 2.17 (s, 3H), 2.76 (m, 2H), 3.26 (m,1H), 3.35
(m, 1H), 5.35 (s, 1H), 5.37 (m, 1H), 7.04 (d, 2H, J = 8.6), 7.14 (d,
2H, J = 8.4). 13C NMR (CDCl3, 400 MHz): d 12.34, 19.40, 19.65,
20.82, 23.58, 28.60, 30.59, 31.03, 35.54, 36.27, 38.38, 41.23,
42.91, 45.07, 51.69, 56.38, 57.66, 70.64, 111.11, 113.16, 120.30,
129.37,139.86, 143.79, 159.15, 162.71, 164.56, 167.71. ESI-MS:
479 (M++H). Anal. Calcd. for C30H39FN2O2: C, 75.28; H, 8.21; N,
5.85; found C, 75.51; H, 8.05; N, 6.09.
To a solution of pregnenolone 1 (0.316 g, 1 mmol, 1 eq.) in eth-
anol (10 ml) was added a conc. aq. solution of KOH (2 eq.). Then
aldehyde 2 (1.2 eq.) was charged into the reaction mixture to get
the corresponding benzylidine derivative 3. After completion, the
reaction mixture was precipitated with water. The precipitate
was filtered, dried and recrystallized from EtOAc:Hexane to give
product as solid white powder. It is to be mentioned that when
non-aromatic aldehydes were used, the product was formed in a
very minor quantity and that too not stable enough at ambient
conditions. Thus the study was restricted to the use of aromatic
aldehydes only. The condensation product 3 (1.0 g, 2.4 mmol)
was refluxed in ethanol in the presence of hydrazine hydrate
(0.24 g, 4.8 mmol) so as to yield the desired pyrazolines. However
the products thus obtained were very unstable and they decom-
posed even at ambient temperature conditions probably because
of the inherent instability associated with pyrazolines. The solvent
thus used was replaced by acetic acid so as to ensure the formation
of N-acetyl pyrazoline 4 (0.99 g, 2.2 mmol, 90%) which was highly
stable. The product was precipitated by charging the reaction mass
into excessive amounts of ice-cold water. After filtration under
suction, the product was obtained in high yields as colorless pow-
der which was later dried in vaccuo. However we observed a dia-
stereomeric mixture of pyrazolines which was separated through
the method discussed in the experimental section to yield the
desired isomer. The same procedure was followed for the synthesis
of all other analogs. Spectral data of various compounds is given as
under (Most of the peaks due to steroidal skelton were merged and
could not be differentiated. Thus d values of only those peaks that
2.2.1.4. 1-(4,5-Dihydro-3-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,
17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phe-
nanthren-17-yl)-5-p-tolylpyrazol-1-yl)ethanone (4d). Greyish pow-
25
der. Yield 79%. M.p: 230–233 °C. [
a]
ꢁ33.3 (c 1 in CHCl3). IR
D
(KBr, cmꢁ1): 3416, 2936, 1719, 1642, 1455, 1087, 756. 1H NMR
(CDCl3, 400 MHz): d 0.61 (s, 3 H), 1.08 (s, 3H), 1.82–1.92 (m, 6H),
2.05 (s, 3H), 2.22 (s, 3H), 2.67 (t, 1H, J = 8.8), 2.77 (m, 2H), 3.40
(m, 1H), 3.48 (m, 1H), 5.36 (m, 2H), 7.80 (d, 4H, J = 6.3). 13C NMR
(CDCl3, 400 MHz): d 12.87, 20.54, 22.46, 23.38, 25.83, 31.13,
33.04, 33.19, 33.48, 37.99, 38.72, 39.94, 43.68, 45.30, 47.38,
47.69, 51.53, 53.18, 57.94, 60.56, 73.11, 122.80, 126.78, 128.84,
130.28, 142.29, 161.65, 162.53. ESI-MS: 497 (M++Na). Anal. Calcd.
for C31H42N2O2: C, 78.44; H, 8.92; N, 5.90; found C, 78.67; H,
8.73; N, 6.13.
2.2.1.5. 1-(4,5-Dihydro-3-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,
17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phe-
nanthren-17-yl)-5-o-tolylpyrazol-1-yl) ethanone (4e). Colourless

A.H. Banday et al. / Steroids 92 (2014) 13–19
15
25
solid. Yield 83%. M.p: 224–226 °C. [
a]
ꢁ63.4 (c 1 in CHCl3). IR
23.32, 25.83, 31.13, 31.04, 33.19, 33.48, 37.99, 38.72, 39.94,
43.68, 45.30, 47.38, 43.69, 51.53, 53.18, 57.94, 60.56, 73.11,
122.80, 126.78, 128.84, 130.28, 142.29, 160.73, 164.62. ESI-MS:
491 (M++H). Anal. Calcd. for C31H42N2O3: C, 75.88; H, 8.63; N,
5.71; found C, 75.99; H, 8.41; N, 5.65.
D
(KBr, cmꢁ1): 3406, 2941, 1729, 1642, 1455, 1077, 753. 1H NMR
(CDCl3, 400 MHz): d 0.61 (s, 3 H), 1.09 (s, 3H), 1.84–1.93 (m, 6H),
2.10 (s, 3H), 2.66 (t, 1H, J = 8.8), 2.74 (m, 2H), 3.40 (m, 1H), 3.44
(m, 1H), 5.22 (s, 1H), 5.29 (m, 1H), 6.84 (m, 1H), 7.06 (m, 3H).
13C NMR (CDCl3, 400 MHz): d 11.76, 14.85, 20.85, 22.40, 23.32,
25.83, 31.13, 33.04, 33.19, 33.48, 37.99, 36.72, 39.84, 43.68,
45.80, 47.35, 47.69, 51.53, 53.18, 57.94, 60.74, 73.11, 123.80,
126.78, 127.84, 130.28, 145.29, 161.63, 166.62, ESI-MS: 497
(M++Na). Anal. Calcd. for C31H42N2O2: C, 78.44; H, 8.92; N, 5.90;
found C, 78.27; H, 8.85; N, 6.09.
2.2.1.10. 1-(5-(2-Chlorophenyl)-4,5-dihydro-3-((10R,13S)-2,3,4,7,8,9,
10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-
1H-cyclopenta[a]phenanthren-17-yl)
(4j). Colourless white powder. Yield 79%. M.p: 228–230 °C. [
pyrazol-1-yl)ethanone
25
a
]
D
ꢁ45.6 (c 1 in CHCl3). IR (CHCl3, cmꢁ1): 3386, 2944, 1720, 1640,
1492, 1407, 1090, 962, 756. 1H NMR (CDCl3, 400 MHz): d 0.67 (s,
3 H), 1.08 (s, 3H), 1.81–1.88 (m, 6H), 2.04 (s, 3H), 2.57–2.65 (m,
2H), 3.20 (m, 1H), 3.50 (m, 1H), 5.34–5.42 (m, 2H), 7.09 (d, 2H,
J = 8.4), 7.37 (d, 2H, J = 8.4). 13C NMR (CDCl3, 400 MHz): d 12.27,
12.46, 18.40, 19.61, 20.80, 23.36, 23.55, 30.53, 30.72, 31.01,
35.53, 36.25, 37.51, 41.17, 45.11, 49.23, 50.68, 55.46, 57.52,
70.66, 120.33, 125.82, 127.98, 132.14, 139.80, 158.60, 166.63,
167.71, 173.88. ESI-MS: 517.4 (M++Na). Anal. Calcd. for C30H39ClN2-
O2: C, 72.78; H, 7.94, Cl, 7.16; N, 5.66; found C,72.96; H, 8.21; Cl,
7.01; N, 5.79.
2.2.1.6. 1-(4,5-Dihydro-3-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,
17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phe-
nanthren-17-yl)-5-m-tolylpyrazol-1-yl) ethanone (4f). Colourless
25
solid powder. Yield 74%. M.p: 220–222 °C. [
a]
ꢁ25.5 (c 1 in
D
CHCl3). IR (KBr, cmꢁ1): 3374, 2939, 1719, 1638, 1404, 1041, 756,
702. 1H NMR (CDCl3, 400 MHz): d 0.67 (s, 3 H), 1.03 (s, 3H),
1.81–1.92 (m, 6H), 2.19 (s, 3H), 2.20–2.23 (m, 3H), 3.40 (m, 1H),
3.47 (m, 1H), 5.35 (m, 2H), 7.03 (d, 1H, J = 7.19), 7.17 (d, 1H,
J = 7.19). 13C NMR (CDCl3, 400 MHz): d 12.35, 20.85, 22.40, 23.32,
25.83, 31.13, 33.04, 33.19, 33.48, 37.99, 39.72, 39.94, 43.68,
45.30, 46.38, 47.69, 51.53, 53.18, 57.94, 60.56, 73.11, 122.80,
126.78, 128.84, 131.35, 143.23, 161.63, 167.62. ESI-MS: 475
(M++H). Anal. Calcd. for C31H42N2O2: C, 78.44; H, 8.92; N, 5.90;
found C, 78.24; H, 8.84; N, 6.11.
2.2.2. General procedure for the synthesis of pyrazolyl pregnenolones
All the pyrazolyl pregnenolone derivatives were prepared as per
known literature precedents [16]. The method described by
Schneider et al. [16] involves the cyclization reaction of 3b-
hydroxy-21-hydroxymethylidenepregn-5-en-20-one 5 [17] with
phenylhydrazine or its p-substituted derivatives. Compound 5
(2.07 g, 6 mmol) was suspended in CH2Cl2 (50 ml) and phenylhydr-
azine hydrochloride or one of its p-substituted derivatives (1.1
equivalent) was added to the homogenous mixture. This was fol-
lowed by the dropwise addition of BF3ꢂOEt2 (50%) (2 mmol,
0.25 ml). The reaction mixture was stirred for 5 h. After the disap-
pearance of the starting material as monitored by TLC, saturated
NaHCO3 solution (100 ml) was added and the mixture was stirred
until bubbling ceased. The organic layer was washed with water,
dried over anhydrous Na2SO4 and concentrated in vacuo. The res-
idue was chromatographed on silica gel starting with CH2Cl2/hex-
ane (1:1, v/v) as eluant, followed by CH2Cl2/hexane (2:1, v/v) and
CH2Cl2 to afford the desired pyrazolyl pregnenolone derivatives
6a–d. The original work by Schneider et al. [16] may be consulted
for further details and the complete spectral data.
2.2.1.7. 1-(5-(furan-2-yl)-4,5-dihydro-3-((10R,13S)-2,3,4,7,8,9,10,11,
12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-
cyclopenta[a]phenanthren-17-yl)pyrazol-1-yl)ethanone
(4g). Col-
ꢁ55.8 (c 1 in
25
ourless Powder. Yield 82%. M.p: 227–229 °C. [
a]
D
CHCl3). IR (KBr, cmꢁ1): 3386, 2936, 1727, 1647, 1451, 1043, 754.
1H NMR (CDCl3, 400 MHz): d 0.67 (s, 3H), 1.02 (s, 3H), 1.81–1.90
(m, 6H), 2.17 (s, 3H), 3.10–3.15 (m, 3H), 3.52 (m, 1H), 5.30
(s,1H), 5.37 (s, 1H), 5.51(m,1H), 6.28 (m, 2H), 7.30 (s,1H). 13C
NMR (CDCl3, 400 MHz): d 13.85, 20.85, 22.90, 22.32, 25.84, 31.13,
33.04, 33.19, 33.48, 37.99, 38.82, 39.94, 44.68, 45.30, 47.38,
48.69, 51.78, 53.18, 57.94, 60.56, 73.11, 122.80, 126.78, 128.84,
130.28, 142.29, 160.61, 164.62. ESI-MS: 473 (M++Na). Anal. Calcd.
for C28H38N2O3: C, 74.63; H, 8.50; N, 6.22; found C, 74.47; H,
8.71; N, 6.47.
2.2.1.8. 1-(4,5-Dihydro-3-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,
17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phe-
nanthren-17-yl)-5-(4-methoxyphenyl)
(4h). Colourless solid. Yield 84%. M.p: 211–215 °C. [
pyrazol-1-yl)ethanone
3. Determination of 5
a-reductase inhibitory activity
25
a]
ꢁ48.4 (c
D
1 in CHCl3). IR (KBr, cmꢁ1): 3406, 2930, 2871, 1719, 1642, 1419,
1041, 757. 1H NMR (CDCl3, 400 MHz): d 0.67 (s, 3H), 1.01 (s, 3H),
1.82–1.88 (m, 6H), 2.03 (s, 3H), 2.53–2.73 (m, 2H), 3.20 (m, 1H),
3.29 (m, 1H), 3.76 (s, 3H), 5.34 (m, 2H), 6.82 (d, 2H, J = 8.1), 7.05
(d, 2H, J = 8.1). 13C NMR (CDCl3, 400 MHz): d 13.38, 20.85, 22.44,
23.32, 25.83, 31.13, 33.04, 33.19, 34.48, 37.99, 38.72, 39.94,
43.68, 45.30, 47.38, 47.69, 51.55, 53.18, 57.94, 60.56, 73.11,
122.80, 126.78, 127.84, 130.28, 142.29, 160.63, 165.62. ESI-MS:
491 (M++H). Anal. Calcd. for C31H42N2O3: C, 75.88; H, 8.63; N,
5.71; found C, 75.63; H, 8.81; N, 5.87.
3.1. Type I 5 -reductase inhibitory activity
a
Human prostate was homogenized in 2 volumes of medium A
(20 mM sodium phosphate, pH 6.5 containing 0.32 M sucrose,
0.1 mM dithiothreitol Sigma–Aldrich, Inc.) with a tissue homoge-
nizer. Homogenates were centrifuged at 1500 g for 20 min
[18,19] in a SW 60 Ti rotor (Beckman Instruments, Palo Alto, CA).
The pellets were separated, suspended in medium A and kept at
70 °C. The suspension, 5 mg of protein/mL for human prostates,
determined by the Bradford’s Method [20] was used as source of
5a-reductase.
2.2.1.9. 1-(4,5-Dihydro-3-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,
17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phe-
The enzyme 5a-reductase was assayed as previously described
[18,19]. The reaction mixture for human prostate contained: 1 mM
dithiothreitol, sodium phosphate buffer 40 mM, at pH 6.5, 2 mM,
NADPH, 2 nM [1,2,6,7-3H] T [20] in a final volume of 1 mL. The
reaction in duplicate was started when it was added to the
enzymatic fraction (500 lg protein in a volume of 80 lL) incubated
at 37 °C for 60 min [19] and stopped by mixing with 1 mL of
dichloromethane; this was considered as the end point. Incubation
without tissue was used as a control. The mixture (incubation
nanthren-17-yl)-5-(2-methoxyphenyl)
(4i). Colourless solid. Yield 80%. M.p: 211–112 °C. [
pyrazol-1-yl)ethanone
25
a
]
D
ꢁ45.1 (c
1 in CHCl3). IR (KBr, cmꢁ1): 3399, 2936, 2871, 1719, 1642, 1419,
1039, 757.1H NMR (CDCl3, 400 MHz): d 0.66 (s, 3H), 1.01 (s, 3H),
1.81–1.88 (m, 6H), 2.04 (s, 3H), 2.54–2.73 (m, 2H), 3.20 (m, 1H),
3.29 (m, 1H), 3.83 (s, 3H), 5.35 (m, 2H), 6.85–6.93 (m, 2H), 7.24–
7.33 (m, 2H). 13C NMR (CDCl3, 400 MHz): d 13.67, 20.85, 22.40,

16
A.H. Banday et al. / Steroids 92 (2014) 13–19
medium/ dichloromethane) was agitated on a vortex for 1 min and
the dichloromethane phase was separated and placed in another
tube. This procedure was repeated 4 more times. The dichloro-
methane extract was evaporated to dryness under a nitrogen
stream and suspended in 50 lL of methanol that was spotted on
HPTLC Keiselgel 60 F254 plates. T and DHT were used as carriers
and were applied in different lanes on both lateral sides of the
plates (T, T + DHT and DHT). The plates were developed in chloro-
form–acetone 9:1 and were air-dried; the chromatography was
repeated 2 more times. The DHT steroid carriers were detected
using phosphomolibdic acid reagent and T with an UV lamp
(254 nm). After the plates were segmented in areas of 1 cm each,
they were cut off and the strips soaked in 5 mL of Ultima Gold
(Packard). The radioactivity was determined in a scintillation
counter (Packard tri-carb 2100 TR). The radioactivity content in
the segment corresponding to T and DHT carriers was identified.
The radioactivity that has identical chromatographic behavior as
the DHT standard was considered as the DHT transformation. Con-
trol incubations, chromatography separations and identifications,
were carried out in the same manner as described above except
that the tubes did not contain tissue. The DHT transformation
yields were calculated from the strips, taking into account the
entire radioactivity in the plate.
behavior as the DHT standard was considered as the DHT transfor-
mation in the presence of the tested compounds. Control incuba-
tions, chromatography separations and identifications, were
carried out in the same manner as described above except that
these tubes did not contain tissue. The DHT transformation yields
were calculated from the strips, taking into account the entire
radioactivity in the plate.
4. Results and discussion
The reductive conversion of testosterone (T) to dihydrotestos-
terone (DHT) by 5a-reductase catalysis is proposed to involve the
formation of a binary complex between the enzyme and NADPH,
followed by the formation of a ternary complex with the substrate
T. This leads to the formation of a delocalized carbocation due to
the activation of the enone system by a strong interaction with
an electrophilic residue (E+) present in the active site.
Direct hydride transfer from NADPH to the
a-face of the delo-
calized carbocation generates enolate of DHT which leads to a
selective reduction at C-5. Protonation on the b-face at C-4 of the
enolate, which is coordinated with NADP+ on the
a-face, generates
the ternary E-NADP+-DHT complex. However after the departure of
DHT, a binary complex of NADP+-enzyme is formed which finally
releases NADP+ to leave the enzyme free for further catalytic cycles
(Fig. 2). Three different types of inhibitors could be conceived
according to the kinetic mechanism of testosterone reduction as
shown in Fig. 1: type A inhibitors which are competitive with the
cofactor (NADPH) and the substrate (T) and interact with the free
enzyme; type B inhibitors which are competitive with the sub-
strate and fit the enzyme-NADPH complex, and type C inhibitors
which fits the enzyme-NADP+ complex exhibiting uncompetitive
mechanism versus the substrate [22,23]. Recently several steroid
based D-ring heterocyclic analogs have been evaluated as efficient
3.2. Type II 5a-reductase inhibitory activity
According to a convenient method already reported in the liter-
ature [21], human embryonic kidney cells (HEK293) over-express-
ing type II 5a-reductase were grown in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% FBS. Stable HEK293 cells
(1 ꢀ 106) were planted in 24-well cell plate and incubated in 5%
CO2 incubator for 24 h at 37 °C. Stable cells were suspended in
50 mM sodium phosphate buffer (pH 5.5) and lysed by sonication
for 1 min. The reaction mixture for type II 5
10 L (10 g total protein) of cell extract, 65
[60 mM sodium phosphate, pH 5.5, 50 mM KCl, 1 mM NADPH,
and 1 Ci [1,2,6,7-3H]testosterone (65.0 Ci/mmol, Amersham)],
and 1 M and 10 M of synthesized compounds. The reaction mix-
tures were incubated at 37 °C for 1 h, followed by steroids extrac-
tion with 250 L of stop solution (70% cyclohexane, 30% ethyl
acetate, 40 g/mL T and 40 g/mL DHT). Solvent was dried and ste-
roids were dissolved with 20 L of chloroform, spotted onto thin
a-reductase contained
5a-reductase inhibitors acting through one of the mechanisms as
l
l
lL of reaction buffer
discussed above. This is particularly true of nitrogenous heterocy-
clic analogs at the D-ring of steroids including the very important
class of pregnanes. Pyrazolyl and pyrazolinyl analogs are of special
significance as these analogs have been found to show interesting
biological potential probably because of their better interaction
with the target receptors. Taking inspiration from the number of
reported biological activities associated with structurally related
analogs, we devised a new synthetic design for the pyrazoline
derivatives and used an already reported method [16] for the syn-
thesis of the pyazolyl derivatives. Thus, we, in continuation of our
efforts towards the synthesis of novel D-ring heterocycles, herein
report efficient and simple synthesis of D-ring pyrazolinyl and pyr-
azolyl derivatives of 20-keto pregnenanes and their evaluation as
potential anticancer agents. The preparation of the latter involves
the following synthetic approach.
l
l
l
l
l
l
l
layer chromatography (TLC) plate (Merck, Darmastadt, Germany)
and developed in 80% toluene, 20% acetone. TLC plate was then
exposed to Hyperfilm 3H (Amersham, RPN 535B) for three days.
3.3. Determination of IC50 values of pregnenolone derivatives 4a–j and
6a–d in human prostatic 5a-reductase
In order to calculate the IC50 values (the concentration of preg-
nenolone derivatives 4a–j and 6a–d or Finasteride required to inhi-
bit 5a
-reductase activity (type I and type II) by 50%), six series of
4.1. Synthesis of the pyrazolyl derivatives 4(a–j)
tubes containing increasing concentrations of these steroids
(10ꢁ10–10ꢁ4 M) were incubated in triplicate, in the presence of
1 mM of dithiothreitol, 40 mM sodium phosphate buffer pH of
The stereochemistry of the benzylidine product 3 at the double
bond was proved to be trans by the coupling constant value of
16 Hz of the vicinal protons. Compound 3 (1.0 g, 2.4 mmol) was
refluxed in ethanol in the presence of hydrazine hydrate (0.24 g,
4.8 mmol) so as to yield the desired pyrazolines. However the
products thus obtained were very unstable and they decomposed
even at ambient temperature conditions probably because of the
inherent instability associated with pyrazolines. The solvent thus
used was replaced by acetic acid so as to ensure the formation of
N-acetyl pyrazoline 4 (0.99 g, 2.2 mmol, 90%) (Scheme 1) which
was highly stable. The product was precipitated by charging the
reaction mass into excessive amounts of ice-cold water. After fil-
tration under suction, the product was obtained in high yields as
6.5; 2 mM NADPH, 2 nM [1,2,6,7-3H]T and 500
lg of protein from
enzymatic fraction in a final volume of 1 mL. The reaction was car-
ried out in triplicate at 37 °C for 60 min; 1 mL of dichloromethane
was added to stop the reaction. The extraction and the chromato-
graphic procedures were carried out as described above.
The plates were segmented in areas of one cm each, cut off and
the strips were soaked in 10 mL of Ultima Gold (Packard) upon
completion of chromatography. The radioactivity was determined
in a scintillation counter (Packard tri-carb 2100 TR). The radioac-
tivity content in the segments corresponding to T and DHT carriers
was identified. The fraction that has identical chromatographic

A.H. Banday et al. / Steroids 92 (2014) 13–19
17
OH
OH
5α-Reductase
NADP+
NADPH
O
O
H
5α−Reductase Inhibitors
Testosterone (T)
Fig. 1. Site of action of 5
Dihydrotestosterone (DHT)
a
-Reductase inhibitors.
OH
OH
OH
δ+
δ-
-
Enz+
Enz+
O
O
O
H
H
Fig. 2. 5
a
-reduction of 3-keto-D4 steroids.
O
Ar
1'
H
5'
N
2'
O
H
H
Ar
H3C
N
O
4'
3'
H
H
H
Ar-CHO
2
NH2NH2.H2O
AcOH/ Reflux
EtOH/ KOH
HO
HO
HO
3
1
4 (5'SIsomer)
Scheme 1. Synthesis of D-ring substituted pyrazolinyl pregnenolones.
colorless powder which was later dried in vaccuo. In principle, the
ring closure would result in two diastereomers at the 50 position of
the pyrazoline ring. However the TLC (Hexane:EtOAc, 7:3) did not
show two different spots. The two spots resolved only when the
TLC was run in methyl tert-butyl ether as eluant. The two diaste-
reomers were isolated in their acetyl forms and the major product
was the 50S isomer. All the pyrazolinyl derivatives were deacety-
lated to give the required products 4a–j and the same procedure
was followed for the synthesis of all analogs.
Na2SO4 and concentrated in vacuo. The residue was chromato-
graphed on silica gel starting with CH2Cl2/hexane (1:1, v/v) as elu-
ent, followed by CH2Cl2/hexane (2:1, v/v) and CH2Cl2 to afford
pyrazolyl pregnenolone derivatives 6a–d (Scheme 2).
4.3. In vitro biological results
The in vitro biological activity of pregnenolone derivatives 4a–j
and 6a–d was determined for human 5a-reductase activity. The
radioactive zone that had identical chromatographic behavior as
the standard T (Rf value of 0.56) corresponds to 70% of the
accounted radioactivity in the plate. The radioactivity contained
in the zone corresponding to DHT standard (Rf value of 0.67) of
the experimental chromatogram was identified as the transformed
DHT and corresponds to 27% of the total radioactivity accounted in
the plate. This result was considered to be 100% of the activity of
4.2. Synthesis of the pyrazolyl derivatives 6(a–d)
Compound 5 [17] (2.07 g, 6 mmol) was suspended in CH2Cl2
(50 ml) and phenylhydrazine hydrochloride or one of its p-substi-
tuted derivatives (1.1 equivalent) was added, followed by the drop
wise addition of BF3ꢂOEt2 (50%) (2 mmol, 0.25 ml). The reaction
mixture was stirred for 6 h. After the disappearance of the starting
material (TLC monitoring), saturated NaHCO3 solution (100 ml)
was added and the mixture was stirred until bubbling ceased.
The organic layer was washed with water, dried over anhydrous
5a-reductase for the development of inhibition plots. Unmodified
[3H]T was identified (Rf value of 0.56) from control incubations
which did not contain tissue and had identical chromatographic
behavior as the non labeled standard. The radioactivity contained
OH
HC
Ar
H3C
O
N
N
H
HC
O
H
H
Ar-NHNH .HCl
2
BF3.Et2O
1
5
6
HO
HO
HO
Scheme 2. Synthesis of D-ring substitued pyrazolyl pregnenolones.

18
A.H. Banday et al. / Steroids 92 (2014) 13–19
Table 1
5. Conclusion
Nature of aryl group (Ar-) in pregnenolone derivatives 4a–j and 6a–d.
Entry
Nature of Ar
Entry
Nature of Ar
A series of D-ring substituted pyrazolinyl pregnenolone (4a–j)
and pyrazolyl pregnenolone (6a–d) derivatives were synthesized
4a
4h
OMe
and screened for their 5
data it was found that all the compounds exhibit promising inhib-
itory activity especially against type I human 5 -reductase. How-
a-reductase inhibitory activity. From the
4b
4c
4i
4j
F
a
ever the compounds 4b, 4c and 6b were found to be the most
active in this study.
MeO
Cl
F
Acknowledgement
4d
4e
6a
6b
AHB thanks UGC, India for the award of Raman postdoctoral fel-
lowship under Singh-Obama 21st century knowledge initiative.
Cl
4f
6c
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