Heterocyclizations of pregnenolone: Novel synthesis of thiosemicarbazone, thiophene, thiazole, thieno[2,3-b]pyridine derivatives and their cytotoxicity evaluations

Article abstract of DOI:10.1016/j.steroids.2012.09.004

Pregnenolone (1) was used as a template to develop new anticancer compounds. Ring D modification of 1 through its reaction with 4-phenyl-3-thiosemicarbazide gave the thiosemicarbazone derivative 3. The latter compound underwent heterocyclization reactions

Full text of DOI:10.1016/j.steroids.2012.09.004

Steroids 77 (2012) 1560–1569
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Heterocyclizations of pregnenolone: Novel synthesis of thiosemicarbazone,
thiophene, thiazole, thieno[2,3-b]pyridine derivatives and their cytotoxicity
evaluations
b
c
d
Rafat M. Mohareb a, , Wagnat W. Wardakhan , Gamal A. Elmegeed , Rehab M.S. Ashour
⇑
a Chemistry Department, Faculty of Science, Cairo University, Cairo, Egypt
b National Organization of Drug Control & Research (NODCAR), P.O. 29, Dokki, Giza, Egypt
c Hormones Department, National Research Centre, Dokki, Cairo, Egypt
d Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 June 2012
Received in revised form 4 September 2012
Accepted 8 September 2012
Available online 12 October 2012
Pregnenolone (1) was used as a template to develop new anticancer compounds. Ring D modification of 1
through its reaction with 4-phenyl-3-thiosemicarbazide gave the thiosemicarbazone derivative 3. The
latter compound underwent heterocyclization reactions to give the thiazolyl hydrazonoandrostane and
pyrazolyl semicarbazidoandrostane derivatives 5a–d, and 9a–d, respectively. On the other hand com-
pound 1 reacted with either malononitrile or ethyl cyanoacetate to give the Knoevenagel condensated
products 11a and 11b, respectively. Compounds 11a,b afforded the thiophenyl pregnane derivatives
12a and 12b, respectively, their reactivity toward some chemical reagents was studied. The cytotoxicity
of the newly synthesized heterocyclic steroids against three human tumor cell lines namely breast ade-
nocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and CNS cancer (SF-268) were studied.
Some of tested compounds were found to exhibit much higher inhibitory effects toward the three tumor
cell lines than the reference drug, doxorubicin.
Keywords:
Cytotoxicity
Pregnenolone
Thiosemicarbazone
Thiazole
Thiophene
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
which exhibit diverse biological activities. Up to now, several ste-
roidal derivatives have been investigated as new curative agents
Steroids have been the prime focus of research throughout the
scientific history [1–4]. But the recent past has seen an exhaustive
focus of research being diverted toward these biologically impor-
tant molecules [5–10]. This is pertinently true of the rational semi
synthetic modifications of steroidal molecules. Probably, it is
because of the various advantages associated with steroid based
chemotherapeutics [11–13]. These compounds turn out to be
non-toxic, less vulnerable to multi-drug resistance (MDR) and
highly bioavailable because of being capable of penetrating the cell
wall. Although various modifications of steroids including derivati-
zation, cyclization, heterocyclization, etc. have been tried [8,14–17]
but as far the literature precedents are concerned, little efforts
have been made toward the efficient synthesis and simultaneous
biological analysis of steroid based heterocyclic derivatives. Ste-
roids form an important class of biologically active compounds
for cancers, and other diseases [18–20]. Several modified steroids,
bearing heterocyclic systems as a part of rings-A and -D, have
exhibited diverse biological activities, such as anti-microbial,
anti-inflammatory, hypotensive, hypocholesterolemic, and diuretic
activities. Among these derivatives, 16,17-condensed steroidal pyr-
azolines have immense pharmacological significance [21–24].
Pregnenolone (1), a naturally occurring steroid, is known as a pre-
cursor to other hormones, including cortisone, estrogen, testoster-
one, and progesterone [25,26]. Previously different pregnenolone
derivatives have been synthesized, and evaluated for various
biological activities. The pregnenolone analogs, hydroxylated at
C-20, are known to effect calcium-dependent processes, and also
affect the degree of depolarization of smooth muscles [27]. The
hemisuccinate of pregnenolone-derivative significantly increases
the perfusion pressure, and vascular resistance in isolated rat heart
[28]. The nitrochlorambucil ester of pregnenolone exhibited a sig-
nificant cytotoxic activity toward brain posterior fossa, medulla-
blastoma (Daoy), and lung large cell carcinoma (H460) cell lines
[29]. In the present work we investigated some heterocyclization
reactions of pregnenolone together with anti-tumor evaluation of
the newly synthesized products toward cancer cell lines.
⇑
Corresponding author. Tel.: +20 1223406896; fax: +20 2 357 28843.
E-mail address: raafat_mohareb@yahoo.com (R.M. Mohareb).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.09.004

R.M. Mohareb et al. / Steroids 77 (2012) 1560–1569
1561
2.1.3. 1-(3b-Hydroxy-pregn-5-ene-20E-ylidene)-2-(3,4-
diphenylthiazol-2(3H)-ylidene)azine (5a)
2. Experimental
HPLC purity = 97.1% (C-18 NovaPak column; MeOH:H2O/85:15),
tr = 15 min; yellow crystals from ethanol (68%), m.p. 177–180 °C;
IR (KBr) cmꢁ1: 3465–4332, 3040, 2936, 1650, 1633, 1405, 1047;
1H NMR (DMSO): d 0.61 (s, 3H), 1.03 (s, 3H), 1.62–1.92 (m, 6H),
2.24–2.38 (m, 3H), 2.83 (t, J = 8.05 Hz, 1H); 3.50 (m, 1H); 6.78 (s,
J = 16.23 Hz, 1H), 5.38 (s, 1H), 6.89 (s, 1H), 7.27–7.38 (m, 10H);
13C NMR (500 MHz, CDCl3): d 13.5, 19.5, 21.8, 22.9, 24.6, 31.1,
31.7, 31.8, 37.3, 43.8, 45.1, 48.6, 48.8, 48.9, 49.3, 49.3, 50.1, 57.0,
71.6, 119.0, 119.3, 120.6, 121.3, 121.7, 126.8, 127.4, 128.3, 128.4,
130.1, 134.8, 140.6, 141.7, 168.6, 172.4; MS: m/e = 565 (M+); Anal.
Calcd. for C36H43N3OS: C, 76.42; H, 7.66; N, 7.43; S, 5.67. Found C,
76.28; H, 7.92; N, 7.51; S, 5.65.
2.1. Synthetic methods, analytical and spectral data
The starting steroid, pregnenolone, was purchased from Sigma
Company, USA. All solvents were dried by distillation prior to
using. Melting points were recorded on Buchi melting point appa-
ratus D-545; IR spectra (KBr disks) were recorded on Bruker Vector
22 instrument. NMR spectra were recorded on Bruker DPX200
instrument in CDCl3 and DMSO-d6 with TMS as internal standard
for protons and solvent signals as internal standard for carbon
spectra. Chemical shift values are mentioned in d (ppm). Mass
spectra were recorded on EIMS (Shimadzu) and ESI-esquire 3000
Bruker Daltonics instrument. Elemental analyses were carried out
by the Microanalytical Data Unit at the National Research Center,
Giza, Egypt and the Microanalytical Data Unit at Cairo University,
Giza, Egypt. 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 purity of tested compounds was determined
by HPLC (Waters Associates, Milford, MA, USA) by using an ultravi-
olet detector (215 nm) where all compounds showed more than
90% purity. All described compounds showed the characteristic
spectral data of cyclopentanoperhydrophenanthrene nuclei of
pregnene and androstane series were similar to those reported in
the literature [30,31]. For the nomenclature of steroid derivatives,
we used the definitive rules for the nomenclature of steroids pub-
lished by the Joint Commission on the Biochemical Nomenclature
(JCBN) of IUPAC [32,33].
2.1.4. 1-(3b-Hydroxy-pregn-5-ene-20E-ylidene)-2-(3-phenyl-4-(4-
chloro-pheny)lthiazol-2(3H)-ylidene)azine (5b)
HPLC purity = 98.2% (C-18 NovaPak column; MeOH:H2O/90:10),
tr = 24 min; pale yellow crystals from 1,4-dioxan (68%), m.p. 188–
191 °C; IR (KBr) cmꢁ1: 3466–4321, 3053, 2932, 1653, 1631, 1408,
1037; 1H NMR (DMSO): d 0.62 (s, 3H), 1.04 (s, 3H), 1.64–1.90 (m,
6H), 2.23–2.38 (m, 3H), 2.83 (t, J = 7.99 Hz, 1H); 3.52 (m, 1H);
6.74 (d, J = 16.03 Hz, 1H), 5.34 (s, 1H), 6.87 (s, 1H), 7.26–7.39 (m,
9H); 13C NMR (500 MHz, DMSO): d 13.5, 19.5, 21.8, 22.9, 24.6,
31.1, 31.7, 31.8, 37.3, 43.8, 45.1, 48.6, 48.8, 48.9, 49.3, 49.3, 50.1,
57.0, 71.6, 106.3, 119.3, 120.6, 121.3, 121.7, 126.8, 127.4, 128.3,
128.4, 130.1, 134.8, 140.7, 141.8, 143.2, 168.7, 173.0; MS:
m/e = 599 (M+); Anal. Calcd. for C36H42ClN3OS: C, 72.03; H, 7.05;
Cl, 5.91. N, 7.00; S, 5.34. Found C, 72.26; H, 7.12; Cl, 6.22; N,
7.22; S, 5.57.
2.1.1. 1-(3b-Hydroxy-pregn-5-ene-20E-ylidene)-4-
2.1.5. 1-(3b-Hydroxy-pregn-5-ene-20E-ylidene)-2-(3-phenyl-4-(4-
bromopheny)-lthiazol-2(3H)-ylidene)azine (5c)
phenylthiosemicarbazone (3)
To a solution of pregnenolone (1) (0.316 g, 1 mmol) in ethanol
(30 mL) 4-phenyl-3-thiosemicarbazide (0.167 g, 1 mmol) was
added. The reaction mixture was heated at 60 °C for 30 min then
left to cool. The solid product formed upon keeping the reaction
mixture overnight was collected by filtration. The precipitate was
filtered, dried and monitored through TLC for the purity. Thin
layer chromatography revealed just a single spot which proved
the presence of a single product. EtOAc:Hexane to give product
as solid white powder. HPLC purity = 98% (C-18 NovaPak column;
MeCN:MeOH:H2O/45:20:35), tr = 19 min; white crystals from
EtOAc:hexane (88%), m.p. 120–123 °C; IR (KBr) cmꢁ1: 3425–
4322, 3010, 2938, 1804, 1637, 1403, 1041; 1H NMR (CDCl3): d
0.63 (s, 3H), 1.00 (s, 3H), 1.61–1.90 (m, 6H), 2.20–2.38 (m, 3H),
2.82 (t, J = 8.6, 1H), 3.51 (m, 1H); 5.33 (s, 1H), 6.78 (d, J = 14.7,
1H), 7.29–7.37 (m, 5H), 8.22, 8.30 (2s, 2H); 13C NMR (500 MHz,
CDCl3): d 13.4, 19.5, 21.9, 22.9, 24.6, 31.1, 31.7, 31.9, 37.2, 43.8,
45.1, 48.6, 48.8, 48.9, 49.3, 49.3, 50.1, 57.2, 71.5, 119.0, 120.6,
121.3, 121.7, 126.8, 128.3, 128.4, 130.1, 134.8, 140.6, 141.7,
168.3, 180.3; MS: m/e = 465 (M+); Anal. Calcd. for C28H39N3OS:
C, 72.21; H, 8.44; N, 9.02; S, 6.89. Found C, 72.09; H, 8.73; N,
9.31; S, 7.05.
HPLC purity = 96.8% (C-18 NovaPak column; MeOH:H2O/85:15),
tr = 18 min; pale yellow crystals from 1,4-dioxan (79%), m.p. 203–
206 °C; IR (KBr) cmꢁ1: 3471–4320, 3060, 2935, 1650, 1638, 1406,
1039; 1H NMR (DMSO): d 0.62 (s, 3H), 1.06 (s, 3H), 1.64–1.92 (m,
6H), 2.22–2.39 (m, 3H), 2.83 (t, J = 8.6, 1H), 3.52 (m, 1H); 5.36 (s,
1H), 6.74 (d, J = 14.7, 1H), 6.86 (s, 1H), 7.26–7.39 (m, 9H); 13C
NMR (500 MHz, DMSO): d 13.4, 19.6, 21.8, 22.9, 24.6, 31.1, 31.7,
31.8, 37.3, 43.9, 45.1, 48.6, 48.8, 48.9, 49.3, 49.6, 50.1, 57.1, 71.6,
106.8, 119.3, 120.8, 121.4, 121.9, 126.9, 127.4, 128.6, 128.6,
130.4, 134.8, 140.7, 142.0, 143.2, 168.8, 172.1; MS: m/e = 643
(M+); Anal. Calcd. for C36H42BrN3OS: C, 67.07; H, 6.57; N, 6.52; S,
4.97. Found C, 66.94; H, 6.42; N, 6.72; S, 4.87.
2.1.6. 1-(3b-Hydroxy-pregn-5-ene-20E-ylidene)-2-(3-phenyl-4-(4-
methoxypheny)lthiazol-2(3H)-ylidene)azine (5d)
HPLC purity = 98.2% (C-18 NovaPak column; MeOH:H2O/90:10),
tr = 18 min; pale yellow crystals from 1,4-dioxan (88%), m.p. 166–
168 °C; IR (KBr) cmꢁ1: 3468–4322, 3048, 2938, 1652, 1632, 1410,
1044; 1H NMR (DMSO): d 0.63 (s, 3H), 1.06 (s, 3H), 1.64–1.92 (m,
6H), 2.22–2.38 (m, 3H), 2.83 (t, J = 7.95 Hz, 1H), 3.02 (s, 2H), 3.52
(m, 1H), 5.33 (s, 1H), 6.76 (d, J = 16.08 Hz, 1H), 7.28–7.35 (m,
9H); 13C NMR (500 MHz, DMSO): d 13.5, 18.6, 19.8, 21.6, 22.8,
24.6, 31.1, 31.7, 31.8, 37.3, 43.8, 45.1, 48.5, 48.7, 49.0, 49.3, 49.6,
50.2, 55.08, 57.1, 71.6, 119.0, 119.5, 120.8, 121.6, 121.9, 126.9,
127.4, 128.6, 128.6, 130.4, 134.8, 139.9, 142.3, 143.2, 168.5,
172.2; MS: m/e = 595 (M+); Anal. Calcd. for C37H45N3O2S: C,
74.58; H, 7.61; N, 7.05; S, 5.38. Found C, 74.74; H, 7.09; N, 7.04;
S, 5.67.
2.1.2. General procedure for the synthesis of the thiazole derivatives
5a–d
To a solution of compound 3 (0.465 g, 1 mmol) in ethanol (30 mL)
either phanacylbromide (0.20 g, 1 mmol), 4-chlorophenacylbromide
(0.23 g, 1 mmol), 4-bromophenacylbromide (0.280 g, 0.01 mol)
or 4-methoxyphenacylbromide (0.23 g, 0.01 mol) were added. The
reaction mixture was heated under reflux for 2 h then was left to
cool. The formed solid product, in each case, upon pouring
onto ice/water followed by the addition of few drops of sodium
carbonate solution, was collected by filtration and crystallized from
the appropriate solvent.
2.1.7. General procedure for the synthesis the pentan-3-ylidene
derivatives 7a,b
To a solution of compound 3 (0.465 g, 1 mmol) in 1,4-dioxan
(20 mL) either acetylacetone (0.01 g, 1 mmol) or ethyl acetoacetate

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R.M. Mohareb et al. / Steroids 77 (2012) 1560–1569
(0.13 g, 0.01 mol) was added. The whole reaction mixture was
heated in a boiling water bath for around 12 h till evolution of
H2S was seized. The solid formed upon pouring onto ice/water
was collected by filtration and crystallized from the appropriate
solvent.
2.1.12. 1-(3b-Hydroxy-pregn-5-ene-20E-ylidene)-4-phenyl-3-(3-
methyl-5-hydroxypyrazolo)-semicarbazide (9b)
HPLC purity = 96.8% (C-18 NovaPak column; MeCN:MeOH:H2O/
70:20:10), tr = 25 min; Colorless crystals from ethanol (84%), m.p.
110–112 °C; IR (KBr) cmꢁ1: 3466–4342, 3055, 2924, 1652, 1636,
1403, 1046; 1H NMR (DMSO): d 0.62 (s, 3H), 1.04 (s, 3H), 1.62–
1.93 (m, 6H), 2.21–2.38 (m, 3H), 2.67 (s, 3H), 2.86 (t, J = 8.21 Hz,
1H), 3.50 (m, 1H), 5.32 (s, 1H), 6.74 (d, J = 16.04 Hz, 1H), 7.26–
7.39 (m, 5H), 8.12, 8.34 (2s, 2H), 10.27 (s, 1H); 13C NMR
(500 MHz, DMSO): d 13.6, 19.5, 21.8, 22.0, 22.8, 22.9, 24.6, 31.1,
31.7, 31.9, 37.3, 43.8, 44.0, 45.6, 48.6, 48.8, 48.9, 49.3, 49.3, 50.1,
57.0, 71.6, 119.8, 120.5, 121.6, 121.7, 126.4, 127.8, 128.0, 128.8,
128.4, 130.4, 134.6, 140.9, 141.6, 154.3, 164.4, 168.2; MS:
m/e = 529 (M+); Anal. Calcd. for C32H43N5O2: C, 72.56; H, 8.18; N,
13.22. Found C, 72.60; H, 8.22; N, 13.48.
2.1.8. 1-(3b-Hydroxy-pregn-5-ene-20E-ylidene)-4-phenyl-3-(2,4-
dioxopentan-3-ylidene)semi-hydrazide (7a)
HPLC purity = 99.0% (C-18 NovaPak column; MeCN:MeOH:H2O/
55:20:25), tr = 23 min; colorless crystals from ethanol (80%), m.p.
127–129 °C; IR (KBr) cmꢁ1: 3480–4336, 3066, 2938, 1688, 1684,
1652, 1633, 1405, 1048; 1H NMR (DMSO): d 0.61 (s, 3H), 1.03 (s,
3H), 1.62–1.92 (m, 6H), 2.24–2.38 (m, 3H), 2.63, 2.69 (2s, 6H),
2.85 (t, J = 8.34 Hz, 1H), 3.50 (m, 1H), 5.34 (s, 1H), 6.74 (d,
J = 15.99 Hz, 1H), 7.26–7.39 (m, 5H), 8.26, 8.32 (2s, 2H); 13C NMR
(500 MHz, DMSO): d 13.5, 19.5, 21.8, 22.0, 22.3, 22.9, 24.6, 26.0,
26.3, 31.1, 31.7, 31.8, 37.3, 43.8, 45.1, 48.6, 48.8, 48.9, 49.3, 49.3,
50.1, 57.0, 71.6, 119.3, 120.6, 121.3, 121.7, 126.8, 127.4, 128.6,
128.4, 130.1, 132.1, 134.5, 140.9, 141.5, 162.3, 164.6, 168.8,
172.6; MS: m/e = 531 (M+); Anal. Calcd. for C33H45N3O3: C, 74.54;
H, 8.53; N, 7.90. Found C, 74.28; H, 8.35; N, 7.83.
2.1.13. 1-(3b-Hydroxy-pregn-5-ene-20E-ylidene)-4-phenyl-3-(3,5-
dimethyl-1-phenyl-pyrazolo)semicarbazide (9c)
HPLC purity = 97.5% (C-18 NovaPak column; MeCN:MeOH:H2O/
60:20:20), tr = 22 min; Colorless crystals from ethanol (55%), m.p.
193–196 °C; IR (KBr) cmꢁ1: 3448–4348, 3048, 2931, 1660, 1656,
1632, 1411, 1046; 1H NMR (DMSO): d 0.62 (s, 3H), 1.04 (s, 3H),
1.63–1.90 (m, 6H), 2.25–2.38 (m, 3H), 2.68, 2.73 (2s, 6H), 2.87 (t,
8.33 Hz, 1H), 3.50 (m, 1H), 5.38 (s, 1H), 6.72 (d, 15.78 Hz, 1H),
7.26–7.34 (m, 5H), 8.11 (s, 1H); 13C NMR (500 MHz, DMSO): d
13.6, 19.8, 21.4, 22.6, 22.3, 23.2, 24.6, 31.1, 31.4, 31.9, 37.3, 43.9,
45.3, 48.6, 48.8, 48.9, 49.8, 49.3, 50.2, 57.0, 71.3, 119.6, 120.8,
121.0, 121.4, 124.2, 125.4, 127.4, 128.6, 128.4, 130.1, 134.5,
135.0, 140.9, 141.3, 148.6, 164.2, 168.4; MS: m/e = 603 (M+); Anal.
Calcd. for C39H49N5O: C, 77.57; H, 8.18; N, 11.60 Found C, 77.49; H,
8.24; N, 11.80.
2.1.9. 1-(3b-Hydroxy-pregn-5-ene-20E-ylidene)-4-phenyl-3-(ethyl 3-
oxo-butanoato-2-ylidene)semihydrazide (7b)
HPLC purity = 87.1.0% (C-18 NovaPak column; MeCN:MeOH:H2-
O/45:30:25), tr = 20 min; Colorless crystals from ethanol (69%),
m.p. 158–160 °C; IR (KBr) cmꢁ1: 3483–4338, 3050, 2929, 1692,
1686, 1650, 1634, 1402, 1044; 1H NMR (DMSO): d 0.62 (s, 3H),
1.03 (s, 3H), 1.13 (t, 3H, 7.11 Hz, CH3), 1.62–1.93 (m, 6H), 2.22–
2.37 (m, 3H), 2.64 (s, 3H), 2.86 (t, J = 8.23 Hz, 1H), 3.50 (m, 1H),
4.22 (q, 7.11 Hz, 2H), 5.38 (s, 1H), 6.74 (d, J = 15.98 Hz, 1H), 7.26–
7.39 (m, 5H), 8.26, 8.31 (2s, 2H); 13C NMR (500 MHz, DMSO): d
13.5, 16.5, 19.3, 19.5, 21.8, 22.0, 22.3, 22.9, 24.6, 31.1, 31.7, 31.8,
37.3, 43.8, 44.0, 45.1, 48.6, 48.8, 48.9, 49.3, 49.3, 50.1, 57.0, 71.6,
119.3, 120.8, 121.4, 121.7, 126.9, 127.6, 128.8, 128.4, 130.4,
132.6, 134.6, 140.9, 141.6, 167.8, 168.4. MS: m/e = 561 (M+); Anal.
Calcd. for C34H47N3O4: C, 72.69; H, 8.43; N, 7.48. Found C, 72.73; H,
8.39; N, 7.57.
2.1.14. 1-(3b-Hydroxy-pregn-5-ene-20E-ylidene)-4-phenyl-3-(3-
methyl-5-hydroxy-1-phenyl-pyrazolo)semicarbazide (9d)
HPLC purity = 98.1% (C-18 NovaPak column; MeCN:MeOH:H2O/
55:35:10), tr = 26 min; yellow crystals from ethanol (89%), m.p.
150–153 °C; IR (KBr) cmꢁ1: 3489–4312, 3050, 2928, 1659, 1633,
1413, 1044; 1H NMR (DMSO): d 0.62 (s, 3H), 1.05 (s, 3H), 1.61–
1.92 (m, 6H), 2.20–2.39 (m, 3H), 2.68 (s, 3H), 2.87 (t, J = 7.91 Hz,
1H), 3.50 (m, 1H), 5.32 (s, 1H), 6.78 (d, J = 16.09 Hz, 1H), 7.28–
7.41 (m, 10H), 8.05 (s, 1H), 10.27 (s, 1H); 13C NMR (500 MHz,
DMSO): d 13.6, 19.5, 21.8, 22.0, 22.8, 22.9, 24.6, 31.1, 31.7, 31.9,
37.3, 43.8, 44.0, 45.6, 48.6, 48.8, 48.9, 49.3, 49.3, 50.1, 57.0, 71.6,
119.8, 120.5, 121.6, 121.7, 123.4, 123.9, 126.4, 127.8, 128.0,
128.4, 128.8, 128.4, 130.4, 134.6, 140.9, 141.7, 154.6, 164.6,
168.0; MS: m/e = 605 (M+); Anal. Calcd. for C38H47N5O2: C, 75.34;
H, 7.82; N, 11.56. Found C, 75.77; H, 8.02; N, 11.68.
2.1.10. General procedure for the synthesis the pyrazole derivatives
9a–d
To a solution of either 7a (0.531 g, 1 mmol) or 7b (0.527 g,
0.01 mol) in ethanol (40 mL) either hydrazine hydrate (0.05 g,
1 mmol) or phenylhydrazine (0.108 g, 1 mmol) was heated under
reflux for 1 h then poured onto ice/water containing one drop of
hydrochloric acid. The solid product formed, in each case was col-
lected by filtration and crystallized from the appropriate solvent.
2.1.15. General procedure for the synthesis of the ylidene derivatives
11a,b
To a dry solid of pregnenolone 1 (0.316 g, 1 mmol), either mal-
ononitrile (0.066 g, 1 mmol) or ethyl cyanoacetate (0.113 g,
1 mmol) was added followed by ammonium acetate (0.250 g).
The whole reaction mixture was heated in a boiling water bath
for 8 h then left to cool. The formed solid product was triturated
with diethyl ether and the formed solid product, in each case,
was collected by filtration.
2.1.11. 1-(3b-Hydroxy-pregn-5-ene-20E-ylidene)-4-phenyl-3-(3,5-
dimethylpyrazolo-)semicarbazide (9a)
HPLC purity = 97.6% (C-18 NovaPak column; MeCN:MeOH:H2O/
45:30:25), tr = 21 min; Colorless crystals from ethanol (71%), m.p.
133–135 °C; IR (KBr) cmꢁ1: 3488–4316, 3050, 2935, 1650, 1630,
1411, 1046; 1H NMR (DMSO): d 0.62 (s, 3H), 1.03 (s, 3H), 1.63–
1.90 (m, 6H), 2.25–2.38 (m, 3H), 2.65, 2.72 (2s, 6H), 2.86 (t,
J = 8.25 Hz, 1H), 3.50 (m, 1H), 5.33 (s, 1H), 6.74 (d, J = 16.01 Hz,
1H), 7.24–7.36 (m, 5H), 8.05, 8.31 (2s, 2H); 13C NMR (500 MHz,
DMSO): d 13.5, 19.6, 21.9, 22.0, 22.3, 22.9, 24.6, 31.1, 31.4, 31.9,
37.3, 43.8, 45.3, 48.6, 48.8, 48.9, 49.8, 49.3, 50.1, 57.0, 71.6,
119.3, 120.6, 121.3, 124.2, 125.4, 127.4, 128.6, 128.4, 130.1,
134.5, 140.9, 141.5, 144.2, 148.0, 164.6, 172.6; MS: m/e = 527
(M+); Anal. Calcd. for C33H45N5O: C, 75.10; H, 8.59; N, 13.27. Found
C, 75.29; H, 8.59; N, 13.42.
2.1.16. 2-(3b-Hydroxy-pregn-5-ene-20-ylidene)malononitrile (11a)
HPLC purity = 98.0% (C-18 NovaPak column; MeOH:H2O/80:20),
tr = 16 min; pale yellow crystals from ethanol (80%), m.p. 160–
163 °C; IR (KBr) cmꢁ1: 3494–4326, 2923, 2223, 2220, 1645, 1630,
1414, 1048; 1H NMR (DMSO): d 0.61 (s, 3H), 1.02 (s, 3H), 1.63–
1.88 (m, 6H), 2.22–2.37 (m, 3H), 2.85 (t, J = 8.09 Hz, 1H), 3.52 (m,
1H), 5.36 (s, 1H), 6.74 (d, J = 15.69 Hz, 1H); 13C NMR (500 MHz,
DMSO): d 13.5, 19.6, 22.0, 22.3, 22.9, 24.4, 31.1, 31.4, 31.9, 37.3,

R.M. Mohareb et al. / Steroids 77 (2012) 1560–1569
1563
43.8, 45.3, 48.6, 48.8, 48.9, 49.8, 49.3, 50.1, 57.0, 71.6, 89.6, 116.8,
117.3, 119.3, 120.6, 122.8, 129.5, 168.2; MS: m/e = 364 (M+); Anal.
Calcd. for C24H32N2O: C, 79.08; H, 8.85; N, 7.68. Found C, 79.19; H,
8.69; N, 7.88.
aniline or its derivative (1 mmol) in concentrated hydrochloric acid
(3 mL, 18 N) with continuous stirring] was added with stirring. The
reaction mixture was kept at room temperature for 1 h and the
formed solid product, in each case, was collected by filtration.
2.1.17. 2-Cyano-3-(3b-hydroxy-pregn-5-ene-20E-yl)but-2-enoate
(11b)
2.1.22. 2-Amino-4-(3b-hydroxy-androst-5-ene-17-yl)-5-
(phenylazo)thiophene-3-carbonitrile (14a)
HPLC purity = 96.4.0% (C-18 NovaPak column; MeOH:H2O/
85:15), tr = 24 min; pale yellow crystals from ethanol (65%), m.p.
85–87 °C; IR (KBr) cmꢁ1: 3485–4322, 2923, 2221, 1680, 1652,
1630, 1414, 1043; 1H NMR (DMSO): d 0.63 (s, 3H), 1.02 (s, 3H),
1.12 (t, J = 6.72 Hz, 3H), 1.61–1.82 (m, 6H), 2.21–2.37 (m, 3H),
2.88 (t, J = 8.09 Hz, 1H); 3.53 (m, 1H), 4.22 (q, J = 6.72 Hz, 2H),
5.36 (s, 1H), 6.72 (s, J = 16.21 Hz, 1H), 13C NMR (500 MHz, DMSO):
d 13.5, 16.3, 19.6, 22.0, 22.3, 22.9, 24.4, 31.1, 31.4, 31.9, 37.3, 42.1,
43.8, 45.3, 48.6, 48.8, 48.9, 49.8, 49.2, 50.1, 57.1, 71.3, 88.3, 116.2,
117.3, 118.8, 119.7, 129.6, 168.2, 172.0; MS: m/e = 411 (M+); Anal.
Calcd. for C26H37NO3: C, 75.87; H, 9.06; N, 3.40. Found C, 79.09; H,
8.69; N, 3.65.
HPLC purity = 98.8% (C-18 NovaPak column; MeOH:H2O/80:20),
tr = 19 min; orange crystals from ethanol (86%), m.p. 188–191 °C;
IR (KBr) cmꢁ1: 3474–4326, 3062, 2920, 2220, 1653, 1633, 1414,
1046; 1H NMR (DMSO): d 0.64 (s, 3H), 1.04 (s, 3H), 1.63–1.86 (m,
6H), 2.21–2.38 (m, 3H), 2.86 (t, J = 7.94 Hz, 1H), 3.54 (m, 1H),
4.20 (s, 2H), 4.77 (s, 2H), 5.33 (s, 1H), 6.79 (s, J = 15.93 Hz, 1H),
7.28–7.39 (m, 5H); 13C NMR (500 MHz, DMSO): d 13.4, 19.2, 22.5,
22.3, 22.6, 24.8, 31.1, 31.2, 31.9, 37.3, 43.9, 45.4, 48.6, 49.8, 49.3,
50.1, 57.0, 71.6, 116.5, 119.3, 120.6, 121.0, 122.8, 129.5, 133.5,
144.6; MS: m/e = 500 (M+); Anal. Calcd. for C30H36N4OS: C, 71.96;
H, 7.25; N, 11.19; S, 6.40. Found C, 71.87; H, 7.04; N, 10.98; S, 6.54.
2.1.23. 2-Amino-4-(3b-hydroxy-androst-5-ene-17-yl)-5-(4-
methylphenylazo)thiophene-3-carbonitrile (14b)
2.1.18. General procedure for the synthesis of the thiophene
derivatives 12a,b
HPLC purity = 97.4% (C-18 NovaPak column; MeOH:H2O/85:15),
tr = 17 min; Orange crystals from ethanol (86%), m.p. 188–191 °C;
IR (KBr) cmꢁ1: 3491–4346, 3055, 2926, 2225, 1652, 1630, 1414,
1048; 1H NMR (DMSO): d 0.62 (s, 3H), 1.06 (s, 3H), 1.60–1.87 (m,
6H), 2.23–2.37 (m, 3H), 2.88 (t, J = 8.47 Hz, 1H), 2.92 (s, 3H), 3.56
(m, 1H); 4.21 (s, 2H), 4.78 (s, 2H), 5.39 (s, 1H), 6.75 (d,
J = 16.18 Hz, 1H), 7.03–7.36 (m, 4H); 13C NMR (500 MHz, DMSO):
d 13.1, 18.3, 19.3, 22.7, 22.2, 22.8, 24.6, 31.2, 31.2, 31.9, 37.6,
43.9, 45.7, 48.2, 48.4, 48.6, 49.8, 49.9, 50.2, 57.0, 61.5, 71.3,
116.7, 119.3, 119.2, 120.2, 120.5, 121.4, 122.2, 125.3, 129.1,
133.8, 144.4; MS: m/e = 514 (M+); Anal. Calcd. for C31H38N4OS: C,
72.34; H, 7.44; N, 10.88; S, 6.23. Found C, 72.48; H, 7.34; N,
10.73; S, 6.44.
To a solution of either 11a (0.364 g, 1 mmol) or 11b (0.431 g,
1 mmol) in 1,4-dioxan (30 mL) containing catalytic amount of tri-
ethylamine, sulfur (0.032 g, 1 mmol) was added. The reaction mix-
ture was heated under reflux for 30 min then left at room
temperature for one night then poured onto ice/water and the
formed solid product was collected by filtration.
2.1.19. 2-Amino-4-(3b-hydroxy-androst-5-ene-17-yl)thiophene-3-
carbonitrile (12a)
HPLC purity = 97.2.0% (C-18 NovaPak column; MeOH:H2O/
80:20), tr = 18 min; yellow crystals from AcOH (69%), m.p. 197–
200 °C; IR (KBr) cmꢁ1: 3444–4316, 3030, 2920, 2222, 1656, 1630,
1414, 1046; 1H NMR (DMSO): d 0.61 (s, 3H), 1.02 (s, 3H), 1.63–
1.88 (m, 6H), 2.22–2.37 (m, 3H), 2.85 (t, J = 8.11 Hz, 1H), 3.52 (m,
1H), 4.77 (s, 2H), 5.35 (s, 1H), 6.78 (d, J = 15.66 Hz, 1H), 6.99 (s,
1H); 13C NMR (500 MHz, DMSO): d 13.3, 19.2, 22.0, 22.3, 22.9,
24.4, 31.1, 31.4, 31.9, 37.3, 43.8, 45.3, 48.6, 48.8, 48.9, 49.8, 49.3,
50.1, 57.0, 71.6, 116.5, 119.3, 122.8, 129.5, 133.5, 138.9, 140.3,
144.8; MS: m/e = 396 (M+); Anal. Calcd. for C24H32N2OS: C, 72.68;
H, 8.13; N, 7.06; S, 8.09. Found C, 72.83; H, 6.89; N, 7.18; S, 7.84.
2.1.24. 2-Amino-4-(3b-hydroxy-androst-5-ene-17-yl)-5-(4-
chlorophenylazo)thiophene-3-carbonitrile (14c)
HPLC purity = 98.8% (C-18 NovaPak column; MeOH:H2O/80:20),
tr = 23 min; Orange crystals from ethanol (69%), m.p. 222–225 °C;
IR (KBr) cmꢁ1: 3496–4324, 3052, 2924, 2220, 1650, 1631, 1416,
1049; 1H NMR (DMSO): d 0.60 (s, 3H), 1.03 (s, 3H), 1.62–1.86 (m,
6H), 2.24–2.38 (m, 3H), 2.86 (t, J = 7.93 Hz, 1H); 3.54 (m, 1H);
4.24 (s, 2H), 4.75 (s, 2H), 5.33 (s, 1H), 6.72 (d, J = 16.07 Hz, 1H),
7.28–7.38 (m, 4H); 13C NMR (500 MHz, DMSO): d 13.0, 18.6, 18.9,
19.4, 22.3, 22.5, 22.8, 24.6, 31.1, 31.2, 31.9, 37.6, 43.9, 45.4, 48.2,
48.4, 48.2, 49.5, 49.9, 50.2, 56.8, 61.6, 71.4, 116.8, 119.3, 119.6,
120.4, 120.6, 121.8, 122.3, 129.5, 133.6, 144.9; MS: m/e = 534
(M+); Anal. Calcd. for C30H35ClN4OS: C, 67.33; H, 6.59; N, 10.47;
S, 5.99. Found C, 67.08; H, 6.84; N, 10.67; S, 6.04.
2.1.20. Ethyl 2-Amino-4-(3b-hydroxy-androst-5-ene-17-
yl)thiophene-3-carboxylate (12b)
HPLC purity = 97.8% (C-18 NovaPak column; MeOH:H2O/85:15),
tr = 21 min; pale yellow crystals from AcOH (73%), m.p. 105–
107 °C; IR (KBr) cmꢁ1: 3439–4312, 3040, 2922, 1687, 1650, 1632,
1416, 1048; 1H NMR (DMSO): d 0.63 (s, 3H), 1.02 (s, 3H), 1.11 (t,
3H), 1.14 (t, J = 7.41 Hz, 3H), 1.61–1.82 (m, 6H), 2.21–2.37 (m,
3H), 2.88 (t, 1H), 3.53 (m, 1H); 4.22 (q, J = 7.41 Hz, 2H), 4.72 (s,
2H), 5.35 (s, 1H), 6.71 (s, J = 15.78 Hz, 1H), 7.07 (s, 1H); 13C NMR
(500 MHz, DMSO): d 13.5, 16.3, 19.6, 22.5, 22.3, 22.9, 24.4, 31.1,
31.4, 31.9, 37.3, 42.4, 43.6, 45.2, 48.6, 48.8, 48.9, 49.8, 49.2, 50.1,
57.1, 71.3, 119.4, 122.6, 129.2, 138.4, 140.8, 147.3, 162.8; MS:
m/e = 443 (M+); Anal. Calcd. for C26H37NO3S: C, 70.39; H, 8.41; N,
3.16; S, 7.23. Found C, 70.41; H, 8.52; N, 3.26; S, 7.44.
2.1.25. Ethyl 2-amino-4-(3b-hydroxy-androst-5-ene-17-yl)-5-
(phenylazo)thiophene-3-carboxylate (14d)
HPLC purity = 98.4% (C-18 NovaPak column; MeOH:H2O/
90::10), tr = 22 min; Orange crystals from ethanol (87%), m.p.
160–163 °C; IR (KBr) cmꢁ1: 3481–4320, 3054, 2922, 1653, 1635,
1412, 1050; 1H NMR (DMSO): d 0.62 (s, 3H), 1.02 (s, 3H), 1.14 (t,
J = 6.94 Hz, 3H), 1.60–1.85 (m, 6H), 2.24–2.39 (m, 3H), 2.82 (t,
1H); 3.53 (m, 1H); 4.21 (s, 2H), 4.24 (q, J = 6.94 Hz, 2H), 4.75 (s,
2H), 5.36 (s, 1H), 6.74 (d, J = 16.42 Hz, 1H), 7.26–7.39 (m, 5H);
13C NMR (500 MHz, DMSO): d 13.1, 16.2, 18.5, 18.9, 19.4, 22.2,
22.5, 22.6, 24.6, 31.0, 31.2, 32.3, 37.5, 43.6, 45.4, 48.2, 48.4, 48.2,
49.3, 49.8, 50.3, 56.6, 71.6, 119.4, 119.6, 120.6, 120.9, 121.8,
122.6, 129.5, 134.5, 143.2, 162.4; MS: m/e = 547 (M+); Anal. Calcd.
for C32H41N3O3S: C, 70.17; H, 7.54; N, 7.67; S, 5.85. Found C, 70.33;
H, 7.64; N, 7.71; S, 6.13.
2.1.21. General procedure for the synthesis of the arylazothiophene
derivatives 14a–f
To a solution of either compound 12a (0.396, 1 mmol) or 12b
(0.443 g, 1 mmol) in ethanol (30 mL) containing sodium
acetate (2.5 g), either benzenediazonium chloride (0.01 mol), 4-
methylbenzenediazonium chloride (1 mmol) or 4-chlorobenze-
nediazonium chloride (1 mmol) [prepared by adding sodium nitrite
solution (0.007 g, 1 mmol) to a cold solution of the appropriate

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R.M. Mohareb et al. / Steroids 77 (2012) 1560–1569
2.1.26. Ethyl 2-amino-4-(3b-hydroxy-androst-5-ene-17-yl)-5-(4-
methylphenylazo)thiophene-3-carboxylate (14e)
6.90 (s, 1H), 10.33 (s, 1H); 13C NMR (500 MHz, DMSO): d 13.0,
19.4, 22.5, 22.4, 22.6, 24.4, 30.8, 31.2, 31.4, 37.3, 43.7, 45.4, 48.4,
48.8, 49.6, 50.3, 57.2, 61.7, 71.8, 116.8, 119.3, 120.6, 122.2, 129.3,
130.3, 133.6, 134.8, 142.3, 144.6, 161.5; MS: m/e = 463 (M+); Anal.
Calcd. for C27H33N3O2S: C, 69.94; H, 7.17; N, 9.06; S, 6.92.
HPLC purity = 98.8% (C-18 NovaPak column; MeOH:H2O/85:15),
tr = 23 min; orange crystals from ethanol (69%), m.p. 144–146 °C;
IR (KBr) cmꢁ1: 3467–4322, 3053, 2920, 1655, 1635, 1415, 1052;
1H NMR (DMSO): d 0.63 (s, 3H), 1.01 (s, 3H), 1.15 (t, J = 5.94 Hz,
3H), 1.62–1.88 (m, 6H), 2.25–2.38 (m, 3H), 2.80 (t, J = 7.83 Hz,
1H), 2.90 (s, 3H), 3.53 (m, 1H), 4.22 (s, 2H), 4.23 (q, J = 5.94 Hz,
2H), 4.73 (s, 2H), 5.36 (s, 1H), 6.72 (d, J = 15,72 Hz, 1H), 7.28–
7.38 (m, 4H); 13C NMR (500 MHz, DMSO): d 13.0, 16.6, 18.5, 18.9,
19.4, 22.2, 22.8, 22.9, 24.2, 31.4, 31.8, 32.4, 37.5, 43.8, 45.4, 48.1,
48.8, 49.3, 49.8, 50.3, 56.6, 61.6, 71.6, 119.4, 119.9, 120.6, 120.9,
122.0, 122.6, 129.5, 134.8, 143.8, 154.8, 164.2; MS: m/e = 561
(M+); Anal. Calcd. for C33H43N3O3S: C, 70.55; H, 7.72; N, 7.48; S,
5.71. Found C, 70.82; H, 7.59; N, 7.33; S, 5.90.
2.1.31. Ethyl 4,6-diamino-3-(3b-hydroxy-androst-5-ene-17-
yl)thieno[2,3-b]pyridine-5-carboxylate (15c)
HPLC purity = 98.0% (C-18 NovaPak column; MeOH:H2O/80:20),
tr = 18 min; pale yellow crystals from ethanol (71%), m.p. 164–
166 °C; IR (KBr) cmꢁ1: 3492–4316, 3021, 2928, 1655, 1636, 1417,
1040; 1H NMR (DMSO): d 0.60 (s, 3H), 1.02 (s, 3H), 1.12 (t,
J = 6.63 Hz, 3H), 1.63–1.89 (m, 6H), 2.22–2.37 (m, 3H), 2.86 (t,
J = 7.83 Hz, 1H); 3.53 (m, 1H); 4.21 (s, 2H), 4.25 (q, J = 6.63 Hz,
2H), 4.49, 4.69 (2s, 4H), 5.38 (s, 1H), 6.80 (s, J = 16.06 Hz, 1H),
6.92 (s, 1H), 13C NMR (500 MHz, DMSO): d 13.1, 16.6, 19.3, 22.7,
22.4, 22.6, 24.4, 30.8, 31.2, 31.9, 37.3, 42.8, 43.9, 45.4, 48.6, 48.9,
49.6, 50.2, 57.2, 61.7, 71.8, 119.0, 116.8, 119.3, 120.6, 122.8,
129.5, 130.3, 133.8, 136.4, 138.8, 140.3, 147.3, 164.2; MS:
m/e = 509 (M+); Anal. Calcd. for C29H39N3O3S: C, 68.34; H, 7.71;
N, 8.24; S, 6.29. Found C, 68.41; H, 7.65; N, 8.38; S, 6.44.
2.1.27. Ethyl 2-amino-4-(3b-hydroxy-androst-5-ene-17-yl)-5-(4-
chlorophenylazo)thiophene-3-carboxylate (14f)
HPLC purity = 98.8% (C-18 NovaPak column; MeOH:H2O/80:20),
tr = 25 min; orange crystals from ethanol (84%), m.p. 203–206 °C;
IR (KBr) cmꢁ1: 3477–4352, 3056, 2922, 1655, 1635, 1417, 1052;
1H NMR (DMSO): d 0.64 (s, 3H), 1.02 (s, 3H), 1.14 (t, J = 7.41 Hz,
3H), 1.64–1.86 (m, 6H), 2.26–2.39 (m, 3H), 2.83 (t, J = 8.05 Hz,
1H), 2.92 (s, 3H), 3.56 (m, 1H), 4.20 (s, 2H), 4.24 (q, J = 7.41 Hz,
2H), 4.77 (s, 2H), 5.34 (s, 1H), 6.74 (d, J = 15.89 Hz, 1H), 7.28–
7.39 (m, 4H); 13C NMR (500 MHz, DMSO): d 13.1, 16.8, 18.5, 18.9,
19.4, 22.5, 22.8, 22.9, 24.2, 31.6, 31.7, 32.6, 37.3, 43.6, 45.6, 48.2,
48.6, 49.0, 49.4, 50.5, 56.6, 61.8, 71.6, 119.6, 119.9, 120.4, 120.9,
122.1, 122.6, 129.6, 133.6, 144.0; MS: m/e = 581 (M+); Anal. Calcd.
for C32H40ClN3O3S: C, 66.02; H, 6.93; N, 7.22; S, 5.51. Found C,
65.89; H, 6.79; N, 7.60; S, 5.82.
2.1.32. Ethyl 6-amino-3-(3b-hydroxy-androst-5-ene-17-yl)-4-
hydroxythieno[2,3-b]pyridine-5-carboxylate (15d)
HPLC purity = 97.0% (C-18 NovaPak column; MeOH:H2O/90:10),
tr = 24 min; pale yellow crystals from ethanol (66%), m.p. 188–
191 °C; IR (KBr) cmꢁ1: 3496–4310, 3010, 2925, 1650, 1636, 1418,
1040; 1H NMR (DMSO): d 0.61 (s, 3H), 1.02 (s, 3H), 1.12 (t,
8.52 Hz, 3H), 1.60–1.86 (m, 6H), 2.22–2.36 (m, 3H), 2.86 (t, 1H);
3.56 (m, 1H); 4.21 (s, 2H), 4.23 (q, 2H), 4.69 (s, 2H), 5.35 (s, 1H),
6.81 (d, J = 16.07 Hz, 1H), 6.93 (s, 1H), 10.2 (s, 1H); 13C NMR
(500 MHz, DMSO): d 13.1, 16.6, 19.3, 22.7, 22.4, 22.6, 24.4, 30.8,
31.2, 31.9, 37.3, 42.8, 43.9, 45.6, 48.6, 48.9, 49.6, 50.1, 57.2, 61.7,
71.6, 119.3, 120.6, 122.8, 130.1, 133.8, 139.5, 143.7, 148.8, 164.3;
MS: m/e = 510 (M+); Anal. Calcd. for C29H38N2O4S: C, 68.20; H,
7.50; N, 5.49; S, 6.28. Found C, 68.46; H, 7.81; N, 5.79; S, 6.51.
2.1.28. General procedure for the synthesis of thethieno[2,3-b]pyridine
derivatives 15a–d
To a solution of either compound 12a (0.396, 1 mmol) or 12b
(0.443 g, 1 mmol) in 1,4-dioxan (30 mL) containing triethylamine
(0.25 mL) either malononitrile (0.066 g, 0.01 mol) or ethyl cya-
noacetate (0.11 g, 1 mmol) was added. The reaction mixture was
heated under reflux for 2 h then left to cool and evaporated under
vacuum. The remaining product was triturated with diethyl ether
and the formed solid product in each case, was collected by
filtration.
2.1.33. General procedure for synthesis of the 3-
oxobutanamido)thiophene derivatives 16a,b
To a dry solid of either compound 12a (0.396, 1 mmol) or 12b
(0.443 g, 1 mmol) ethyl acetoacetate (0.130 g, 1 mmol) was added.
The reaction mixture was heated in an oil bath at 120 °C for 8 h
then was left to cool and the solidified product was triturated with
diethyl ether and the formed solid product, in each case, was col-
lected by filtration.
2.1.29. 4,6-Diamino-3-(3b-hydroxy-androst-5-ene-17-yl)thieno[2,3-
b]pyridine-5-carbonitrile (15a)
HPLC purity = 97.2% (C-18 NovaPak column; MeOH:H2O/80:20),
tr = 19 min; pale yellow crystals from ethanol (90%), m.p. >300 °C;
IR (KBr) cmꢁ1: 3488–4336, 2936, 2227, 1655, 1636, 1412, 1046; 1H
NMR (DMSO): d 0.61 (s, 3H), 1.02 (s, 3H), 1.63–1.89 (m, 6H), 2.23–
2.39 (m, 3H), 2.84 (t, J = 8.04 Hz, 1H); 3.53 (m, 1H); 4.21 (s, 2H),
4.68, 4.80 (2s, 4H), 5.34 (s, 1H), 6.79 (d, J = 15.84 Hz, 1H), 6.93 (s,
1H), 13C NMR (500 MHz, DMSO): d 13.1, 19.2, 22.5, 22.4, 22.6,
24.4, 30.8, 31.2, 31.9, 37.3, 43.9, 45.4, 48.6, 48.9, 49.8, 50.1, 57.2,
61.7, 71.8, 116.8, 119.3, 120.6, 122.8, 129.5, 130.3, 134.6, 142.1,
144.2, 161.8; MS: m/e = 462 (M+); Anal. Calcd. for C27H34N4OS: C,
70.09; H, 7.41; N, 12.11; S, 6.93. Found C, 70.15; H, 7.25; N,
12.32; S, 6.84.
2.1.34. 4-(3b-Hydroxy-androst-5-ene-17-yl)-2-(3-
oxobutanamido)thiophene-3-carbonitrile (16a)
HPLC purity = 98.4% (C-18 NovaPak column; MeCN:MeOH:H2O/
65:25:10), tr = 18 min; pale yellow crystals from ethanol (55%),
m.p. >300 °C; IR (KBr) cmꢁ1: 3459–4312, 3039, 2924, 2222, 1688,
1683, 1658, 1633, 1418, 1040; 1H NMR (DMSO): d 0.60 (s, 3H),
1.01 (s, 3H), 1.62–1.84 (m, 6H), 2.02 (s. 2H), 2.23–2.36 (m, 3H),
2.88 (t, J = 7.83 Hz, 1H), 3.54 (m, 1H), 3.88 (s, 2H), 4.24 (s, 2H),
4.68 (s, 2H), 5.38 (s, 1H), 6.66 (d, J = 15.78 Hz, 1H), 6.88 (s, 1H),
8.30 (s, 1H); 13C NMR (500 MHz, DMSO): d 13.0, 19.7, 22.5, 22.4,
22.6, 24.4, 29.6, 30.8, 31.0, 31.4, 37.3, 43.7, 45.4, 48.4, 48.8, 49.6,
50.3, 50.9, 57.2, 61.7, 71.3, 116.6, 119.3, 121.3, 133.6, 136.2,
142.3, 149.2, 164.7, 169.1; MS: m/e = 480 (M+); Anal. Calcd. for C28-
H36N2O3S: C, 69.97; H, 7.55; N, 5.83; S, 6.67. Found C, 70.11; H,
7.42; N, 5.63; S, 6.82.
2.1.30. 6-Amino-3-(3b-hydroxy-androst-5-ene-17-yl)-4-
hydroxythieno[2,3-b]pyridine-5-carbonitrile (15b)
HPLC purity = 96.8% (C-18 NovaPak column; MeOH:H2O/85:15),
tr = 22 min; pale yellow crystals from ethanol (73%), m.p. 273–
276 °C; IR (KBr) cmꢁ1: 3441–4316, 2922, 2224, 1652, 1638, 1418,
1042; 1H NMR (DMSO): d 0.60 (s, 3H), 1.01 (s, 3H), 1.62–1.86 (m,
6H), 2.24–2.36 (m, 3H), 2.85 (t, J = 8.41 Hz, 1H), 3.53 (m, 1H),
4.24 (s, 2H), 4.66 (s, 2H), 5.36 (s, 1H), 6.66 (d, J = 15.85 Hz, 1H),
2.1.35. Ethyl 4-(3b-hydroxy-androst-5-ene-17-yl)-2-(3-
oxobutanamido)thiophene-3-carboxylate (16b)
HPLC purity = 99.1% (C-18 NovaPak column; MeCN:MeOH:H2O/
70:20:10), tr = 21 min; pale yellow crystals from ethanol (55%),

R.M. Mohareb et al. / Steroids 77 (2012) 1560–1569
1565
O
H
S
C
H
H
NHNH2
+
Ph NH
HO
2
1
a
S
C
H
N
N
NH Ph
H
H
H
HO
Ph
N
3
Ar
N
S
N
b
H
ArCOCH2Br
+
3
H
H
4a, Ar =C6H5
b, Ar = 4-Cl-C6H4
c, Ar = 4-Br-C6H5
d. Ar = 4-CH3O-C6H4
HO
5a, Ar =C6H5
b, Ar = 4-Cl-C6H4
c, Ar = 4-Br-C6H5
d. Ar = 4-CH3O-C6H4
Scheme 1. Synthesis of pregnenolone derivatives. Reagents and conditions: (a) ethanol, 60 °C, (b) ethanol, reflux 2 h.
m.p. 231–233 °C; IR (KBr) cmꢁ1: 3473–4352, 3030, 2921, 1688,
1683–1680, 1658, 1633, 1418, 1042; 1H NMR (DMSO): d 0.64 (s,
3H), 1.01 (s, 3H), 1.12 (t, J = 6.88 Hz, 3H), 1.59–1.86 (m, 6H), 2.01
(s. 2H), 2.20–2.38 (m, 3H), 2.92 (t, J = 8.04 Hz, 1H), 3.56 (m, 1H),
3.88 (s, 2H), 4.21 (q, J = 6.88 Hz, 2H), 4.24 (s, 2H), 4.68 (s, 2H),
5.33 (s, 1H), 6.63 (d, J = 16.11 Hz, 1H), 6.86 (s, 1H), 8.33 (s, 1H);
13C NMR (500 MHz, DMSO): d 13.0, 16.3, 19.7, 22.7, 22.4, 22.8,
24.4, 29.8, 30.6, 31.2, 31.6, 37.3, 42.0, 43.7, 45.4, 48.4, 48.8, 49.6,
50.3, 50.9, 57.2, 61.7, 71.3, 119.3, 121.8, 133.6, 136.2, 143.2,
149.8, 163.8, 164.5, 189.4; MS: m/e = 527 (M+); Anal. Calcd. for
purchased from Cambrex (New Jersey, USA). Dimethyl sulfoxide
(DMSO), doxorubicin, penicillin, streptomycin and sulforhodamine
B (SRB) were purchased from Sigma Chemical Co. (Saint Louis,
USA).
2.2.2. Cell cultures
Three human tumor cell lines, MCF-7 (breast adenocarcinoma),
NCI-H460 (non-small cell lung cancer), and SF-268 (CNS cancer)
were used. MCF-7 was obtained from the European Collection of
Cell Cultures (ECACC, Salisbury, UK) and NCI-H460 and SF-268
were kindly provided by the National Cancer Institute (NCI, Cairo,
Egypt). They grow as monolayer and routinely maintained in
RPMI-1640 medium supplemented with 5% heat inactivated FBS,
2 mM glutamine and antibiotics (penicillin 100 U/mL, streptomy-
C30H41NO5S: C, 68.28; H, 7.83; N, 2.65; S, 6.08. Found C, 68.50; H,
7.55; N, 2.91; S, 5.83.
2.2. In vitro cytotoxic assay
cin 100 lg/mL), at 37 °C in a humidified atmosphere containing
2.2.1. Chemicals
5% CO2. Exponentially growing cells were obtained by plating
1.5 ꢀ 105 cells/mL for MCF-7 and SF-268 and 0.75 ꢀ 104 cells/mL
for NCI-H460, followed by 24 h of incubation. The effect of the
Fetal bovine serum (FBS) and L-glutamine, were purchased from
Gibco Invitrogen Co. (Scotland, UK). RPMI-1640 medium was

1566
R.M. Mohareb et al. / Steroids 77 (2012) 1560–1569
O
C
O
C
X
H3C
N
C
C
NH
NHPh
O
O
a
H
+
3
R
H
H
HO
6a,X = CH3
b, X = OC2H5
7a, X = CH3
b, X = OC2H5
RNHNH2
8a, R = H
b, R = Ph
b
N
N
NR
H3C
Y
C
NHPh
N
H
H
H
HO
9a, R = H, Y = CH3
b, R = H, Y = OH
c, R = Ph, Y = CH3
d, R = Ph, Y = OH
Scheme 2. (a) 1,4-Dioxan, water bath, 12 h; (b) ethanol, reflux 1 h.
vehicle solvent (DMSO) on the growth of these cell lines was eval-
uated in all the experiments by exposing untreated control cells to
the maximum concentration (0.5%) of DMSO used in each assay.
The 1H NMR and 13C NMR spectra of 3 were the basis of its struc-
ture elucidation. The 1H NMR spectrum showed beside the
expected signals for pregnenolone moiety a multiplet at d 7.29–
7.37 indicating the phenyl protons and two singlets (D2O
exchangeable) at d 8.22, 8.30 ppm indicating the NH groups. More-
over, the 13C NMR spectrum showed beside the characteristic sig-
nal for the C6H5 group, two signals at d 168.3 and 180.3 indicating
the C@N and C@S groups, respectively. Compound 3 reacted with
2.2.3. Tumor cell growth assay
The effects of compounds 3–16a–b on the in vitro growth of hu-
man tumor cell lines were evaluated according to the procedure
adopted by the National Cancer Institute (NCI, USA) in the ‘In vitro
Anticancer Drug Discovery Screen’ that uses the protein-binding
dye sulforhodamine B to assess cell growth [34]. Briefly, exponen-
tially, cells growing in 96-well plates were then exposed for 48 h to
five serial concentrations of each compound, starting from a max-
the
x-bromoacetophenone derivatives to give the 20-thiazolyl
hydrazonopregnenolone derivatives 5a–d (Scheme 1), their struc-
tures were based on analytical and spectral data (see Section 2).
On the other hand, the reaction of compound 3 with either acetyl-
acetone (6a) or ethylacetoacetate (6b) in ethanol solution under
reflux gave the condensated products 7a and 7b, respectively.
The latter products were good candidates for pyrazolyl steroids
formation through their respective reactions with hydrazines.
Thus, the reaction of either 7a or 7b with either hydrazine hydrate
(8a) or phenylhydrazine (8b) gave the pyrazolyl semicarbazido-
pregnenolone derivatives 9a–d, respectively (Scheme 2). The ana-
lytical and spectral data of such compounds are in analogy with
their respective structures. Thus the 1H NMR spectrum of 9a
showed two singlets at d 2.65, 2.72 indicating the two CH3 groups,
a multiplet at d 7.24–7.36 corresponding to the phenyl protons and
two singlets (D2O exchangeable) at d 8.05, 8.31 ppm indicating the
two NH groups. Moreover, the 13C NMR spectrum showed d at 22.9,
24.6 indicating the two CH3 groups and two signals at d 164.6 and
172.6 indicating the two C@N groups.
imum concentration of 150 lM. Following this exposure period
adherent cells were fixed, washed, and stained. The bound stain
was solubilized and the absorbance was measured at 492 nm in
a plate reader (Bio-Tek Instruments Inc., Powerwave XS, Wincoski,
USA). For each test compound and cell line, a dose–response curve
was obtained and the growth inhibition of 50% (GI50), correspond-
ing to the concentration of the compounds that inhibited 50% of
the net cell growth, was calculated as described elsewhere [35].
Doxorubicin was used as a positive control and tested in the same
manner.
3. Results and discussion
3.1. Chemistry
Next we studied the reaction of the acetyl group present in preg-
nenolone (1) toward active methylene reagents in the aim of forma-
tion of condensated products followed by their heterocyclization
The reaction of pregnenolone (1) with 1-phenyl-3-thiosemicar-
bazide gave the 20-thiosemicarbazonopregnenolone derivative 3.

R.M. Mohareb et al. / Steroids 77 (2012) 1560–1569
1567
Scheme 3. (a) NH4Ac, water bath, 8 h; (b) 1,4-dioxan, Et3N, S8, reflux 30 min; (c) ethanol, NaOAc, 0–5 °C.
into thiophene derivatives in a similar way to the reported Gewald’s
thiophene synthesis [36–39]. Thus, the reaction of 1 with either mal-
ononitrile (10a) or ethyl cyanoacetate (10b) in dry ammonium ace-
tate at 120 °C gave the Knoevenagel condensated products 11a and
11b, respectively [40,41]. The reaction of either 11a or 11b with
sulfur in 1,4-dioxan solution containing a catalytic amount of
triethylamine gave the 17-thiophenyl androstene derivatives 12a
and 12b, respectively. The 1H NMR and 13C NMR spectra were the
basis of their structures elucidation. Thus, the 1H NMR spectrum of
12a showed the presence of a singlet at d 4.77 ppm indicating the
presence of NH2 group and a singlet at d 6.99 ppm corresponding
to the thiophene H-5. Moreover, the 13C NMR spectrum showed
the presence of a CN group at d 116.5 and the characteristic
signals of the thiophene C at d 138.9, 140.3, and 144.8.
Encouraged by the excellent yields for 12a and 12b a series of
novel arylhydrazono derivatives were obtained with the aim of
improving the inhibitory effect against the tested cancer cell lines.
Moreover, in order to establish a relationship between the struc-
ture and activity for this type of compounds we have studied the
affects of substitution on the aryl azo compounds as well as the
effect of CN group in case of 12a and the ester group in case of
12b. Thus, the reaction of either 12a or 12b with either of the aryl-
diazonium salts namely the benzenediazonium chloride 13a, the 4-
methylphenyldiazonium salt 13b or the 4-chlorophenyl diazonium
salts 13c in 0–5 °C gave the aryl hydrazono derivatives 14a-f,
respectively (Scheme 3). On the other hand, the reaction of either
12a or 12b with either malononitrile (10a) or ethyl cyanoacetate
(10b) gave the 17-thieno[2,3-b]pyridinyl androstene derivatives
15a–d, respectively. Finally, the reaction of either 12a or 12b with
ethyl acetoacetate (6b) gave the 2-oxobutanamidothiophenyl
androstene derivatives 16a and 16b, respectively (Scheme 4).
Most of compounds synthesized in this work could exist in two
stereoisomeric forms E or Z. Trials to obtain pure isomers were car-
ried out by flash chromatography of crude compounds (0.5 mmol)
in hexane/acetone mixture (3:1). Thin layer chromatography
examination of the reaction mixture revealed minor products
probably due to the presence of other isomers. In all experiments
our compounds were recovered from the low mobility phases
and thus considered to be in the (E)-isomeric forms [42]. The
extracts of the low chromatographic mobility for compounds 3,
5a–d, 7a,b, 9a–d and 11b which are the (E) isomers showed high
purity (higher than 95%) toward HPLC by using an ultraviolet
detector (215 nm). It is important to note that the presence of
the diene moiety in the azole derivatives 5a–d and 9a–d, orient
such compounds toward the more stable transforms.
3.2. In vitro evaluation of cytotoxic activity
The effect of compounds 3–16a,b was evaluated on the in vitro
growth of three human tumor cell lines representing different tu-
mor types, namely, breast adenocarcinoma (MCF-7), non-small cell
lung cancer (NCI-H460) and CNS cancer (SF-268) after a continu-
ous exposure for 48 h. The results are summarized in Table 1. All
of the tested compounds were able to inhibit the growth of the
tested human tumor cell lines in a dose-dependent manner. The
results indicated through Table 1 revealed that compounds 5b,
5c, 9b, 9d, 11a, 12a, 14c, 14f, 15b, 15d, 16a and 16b showed the
highest inhibitory effect against all the three tumor cell lines.

1568
R.M. Mohareb et al. / Steroids 77 (2012) 1560–1569
NH2
X
N
S
Y
a
H2C
X
CN
12a,b
+
H
H
H
10a, X = CN
b, X = COOC2H5
HO
15a, X = CN, Y = NH2
b, X = CN, Y = OH
c, X = COOEt, Y = NH2
d, X = COOEt, Y = OH
HN COCH2COCH3
X
S
O
O
b
H
12a,b
+
OEt
H
6b
H
HO
16a, X = CN
b, X = COOC2H5
Scheme 4. (a) 1,4-Dioxan, Et3N, reflux 2 h; (b) oil bath, 120 °C, 8 h.
Compounds 9b, 9d, 11a, 12a, 14c, 14f and 16a showed high inhib-
Table 1
itory effects against non-small cell lung cancer (NCI-H460) and
breast adenocarcinoma (MCF-7), which are much higher than the
reference doxorubicin. Compounds 9a, 9c, 15a and 15c showed
the lowest inhibitory effect. The rest of the compounds showed a
moderate growth inhibitory effect.
Effect of compounds 3–16a,b on the growth of three human tumor cell lines.
Compound
GI50
MCF-7
(l )
mol Lꢁ1
NCI-H460
SF-268
3
5a
4.1 0.6
6.6 0.4
8.3 1.4
8.3 0.8
6.3 1.5
4.1 0.8
5b
5c
5d
0.6 0.4
0.2 0.2
4.6 2.9
0.2 0.08
0.6 0.02
6.9 1.8
0.8 0 .4
0.4 0.08
2.8 1.6
3.3. Structure–activity relationship
Comparing the cytotoxicity of 20-thiazolyl hydrazonopregneno-
lone derivatives 5a, 5b, 5c and 5d, it is obvious that the cytotoxicity
of compounds 5b and 5c are much higher than those of compounds
5a and 5b as the p-chloro group in 5b and the p-bromo group in 5c
are responsible for the increasing cytotoxicity toward the three cell
lines. On the other hand the cytotoxicity of compound 7b is higher
than that of 7a the reason is due to the presence of COOEt group in
7b. Comparing the cytotoxicity of the pyrazolyl semicarbazido-
pregnenolone derivatives 9a, 9b, 9c and 9d, it is obvious that com-
pounds 9b and 9d showed high inhibitory effects through the three
cancer cell lines which are higher than those of compounds 9a and
9c. The presence of the electron withdrawing OH group in 9b and
9d is responsible for such reactivity. Similarly, the presence of the
CN group in compounds 11a, 12a and 16a is responsible for the
high cytotoxicity activity toward the three cancer cell lines while
compounds 11b, 12b and 16b with the COOEt group showed lower
cytotoxicity.
7a
7b
9a
9b
9c
9d
22.7 17.5
8.1 0.6
12.2 4.8
4.5 0.8
16.0 4.3
6.7 1.6
50.1 0.7
0.01 0.002
38.0 1.8
0.02 0.006
0.01 0.006
11.9 0.9
0.02 0.007
4.1 0.7
23.2 4.8
0.06 0.004
44.0 0.8
0.01 0.004
0.01 0.006
12.6 1.8
0.03 4.8
2.2 0.8
18.4 1.8
0.04 0.006
20.5 1.1
0.03 0.001
0.03 0.005
10.8 0.8
0.04 0.008
2.4 0.6
11a
11b
12a
12b
14a
14b
14c
14d
14e
14f
15a
15b
15c
15d
16a
16b
Doxorubicin
8.1 0.7
6.2 2.8
4.4 2.8
10.1 0.7
0.01 0.007
14.1 0.7
22.1 2.3
0.03 0.002
30.1 2.6
2.1 0.4
50.1 12.4
2.1 0.3
0.01 0.003
2.1 0.4
18.2 4.8
0.02 0.008
12.2 4.8
20.2 4.8
0.02 0.004
33.2 4.6
2.2 0.9
23.2 4.8
1.2 0.2
0.01 0.002
4.2 4.8
12.4 1.8
0.04 0.08
10.4 1.8
22.4 2.8
0.02 0.001
22.4 4.4
1.4 0.6
18.4 1.6
2.4 0.4
0.04 0.005
2.4 0.6
Comparing the arylazo derivatives 14a–f it is clear that the
p-chloro group present in compounds 14c and 14f is responsible
for their high cytotoxicity among the six compounds. Considering
the cytotoxicity of the thieno[2,3-b]pyridinyl androstene deriva-
tives 15a–d, it is clear that the presence of the OH group in
compounds 15b and 15d is responsible for such high activity.
0.04 0.008
0.09 0.008
0.09 0.007
Results are given in concentrations that were able to cause 50% of cell growth
inhibition (GI50) after a continuous exposure of 48 h and show means SEM of
three-independent experiments performed in duplicate.

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1569
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R.M. Mohareb would like to express his deepest thanks to the
Alexander von Humboldt Foundation in Bonn for affording his a
fellowship in Germany, München during summer, 2012 for doing
research and completing this work.
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