Heterocyclic ring extension of androstenedione: Synthesis and cytotoxicity of fused pyran, pyrimidine and thiazole derivatives

Article abstract of DOI:10.1016/j.steroids.2014.04.011

The reaction of androstenedione with either malononitrile or ethyl cyanoacetate and aromatic aldehydes 2a-c gave the pyran derivatives 4a-f, respectively. On the other hand, the reaction of androstenedione with thiourea and the aromatic aldehydes 2a-c gave the pyrimidine derivatives 6a-c, respectively. Compound 6b reacted with 2-bromo-1-arylethanone derivatives 7a-d to give the indeno[2,1-e]thiazole derivatives 8a-d. Some of the produced compounds were used for further heterocyclization reactions. The cytotoxicity of the newly obtained products was evaluated against some cancer cell lines and a normal cell line.

Full text of DOI:10.1016/j.steroids.2014.04.011

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Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
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Heterocyclic ring extension of androstenedione: Synthesis and
cytotoxicity of fused pyran, pyrimidine and thiazole derivatives
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c,d
Rafat M. Mohareb a, , Nermeen S. Abbas , Mahmoud A. Abdelaziz
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a Department of Chemistry, Faculty of Science, Cairo University, Giza, Egypt
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b Department of Chemistry, Faculty of Science, Helwan University, Cairo, Egypt
c Preparatory Year Department, AL-Ghad International Colleges for Health Sciences, Tabuk Male, Saudi Arabia
d Basic Science Department, Modern Academy For Engineering and Technology in Maadi, Egypt
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a r t i c l e i n f o
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The reaction of androstenedione with either malononitrile or ethyl cyanoacetate and aromatic aldehydes
2a-c gave the pyran derivatives 4a-f, respectively. On the other hand, the reaction of androstenedione
with thiourea and the aromatic aldehydes 2a-c gave the pyrimidine derivatives 6a-c, respectively.
Compound 6b reacted with 2-bromo-1-arylethanone derivatives 7a-d to give the indeno[2,1-e]thiazole
derivatives 8a-d. Some of the produced compounds were used for further heterocyclization reactions.
The cytotoxicity of the newly obtained products was evaluated against some cancer cell lines and a
normal cell line.
Received 21 January 2014
Received in revised form 5 April 2014
Accepted 14 April 2014
Available online xxxx
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Keywords:
Androstenedione
Pyran
Pyrimidine
Thieno[2,3-b]pyridine
Cytotoxicity
Ó 2014 Elsevier Inc. All rights reserved.
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1. Introduction
supplements are used for their alleged ability to enhance athletic
performance by virtue of their metabolic conversion to testoster-
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Steroidal compounds are widely existent in natural world and
display a variety of biological activities [1–5]. Some steroidal
compounds have been used as traditional medicines, such as, anti-
bacterium and hormone kind medication. Besides the naturally
occurring substances, the majority of steroidal drugs are semi-syn-
thetic compounds [6–9]. The introduction of heteroatom, heterocy-
cle or replacement of one or more atoms in the structure of the
maternal steroids often results in alterations of its biological prop-
erties, for example, enhancing the cytotoxicity against some
tumour cell lines [10–15]. Increasing the selectivity and minimiz-
ing the side effects are still the priority of the medicinal chemists.
Due to their diverse biological properties and wide applications,
heterocyclic compounds have gained plenty of attention. These
moieties exist not only in naturally occurring compounds, like
alkaloids, vitamins, hormones and antibiotics, but also in pharma-
ceutical synthetic herbicides and dyes [16]. Nitrogen containing
heterocyclic systems have prevailed for decades for their various
applications [17].
one. In vivo, androstenedione is the immediate biosynthetic pre-
cursor of both testosterone and estrone supra-physiological
levels of androgenic steroids are known to have adverse health
consequences in humans including: endocrine disruption
(e.g., masculinization in females), hepatotoxicity (peliosis hepati-
tis, cholestatic jaundice, hepatocellular adenomas), and cardiovas-
cular toxicity (e.g., decreased HDL). Because of these potential
health risks, the USFood and Drug Administration mandated that
products containing androstenedione could no longer be sold and
distributed as dietary supplements (FDA, 2004). Although frank
hepatotoxicity in humans is associated with both the therapeutic
and illicit use of 17-a-alkylated anabolic–androgenic steroids
[18], no clear association with hepatotoxicity has been established
for non-alkylated steroids such as androstenedione. To our knowl-
edge there is reported works concerning the ring D extension of
androstenedione. Thus, our main aim in this work is to study the
heterocyclization of androstenedione together with studying the
cytotoxicity of the newly synthesized products against cancer
and normal cell lines. Chemical modification of the steroid D-ring
provides a way to alter the functional groups, sizes and stereo-
chemistry of the D-ring, and numerous structure–activity relation-
ships have been established by such synthetic alterations [19–21].
Steroids bearing heterocycles fused to the D-ring of the steroid
nucleus have been of pharmaceutical interest [22–24]. This study
Androstenedione is one of many naturally-occurring steroidal
compounds in common use as dietary supplements. These
⇑
Corresponding author.
E-mail address: raafat_mohareb@yahoo.com (R.M. Mohareb).
http://dx.doi.org/10.1016/j.steroids.2014.04.011
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: Mohareb RM et al. Heterocyclic ring extension of androstenedione: Synthesis and cytotoxicity of fused pyran, pyrimidine
and thiazole derivatives. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.04.011

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was focused on the efficient synthesis of androstenedione possess-
ing pyran, pyrimidine and thiazole ring systems.
hexane (86%), m.p. 180–182 °C; IR (KBr) cmꢁ1: 3485, 3430, 3054,
2936, 2220, 1683, 1636, 1567; 1H-NMR (CDCl3): d 0.86, 1.04 (2s,
6H), 1.33–2.84 (m, 10H), 2.90–2.93 (m, 4H), 4.22 (s, 2H, NH2),
5.11, 5.21 (2s, 2H), 5.38 (s, 1H), 6.11 (s, 1H), 6.41 (d, 1H,
J = 2.20 Hz), 6.85–7.38 (m, 4H); 13C-NMR (CDCl3): d 17.2,17.6,
19.9, 23.1, 25.3, 29.2, 30.4, 33.9, 34.4, 34.8, 41.9, 43.6, 44.1, 53.0,
117.8, 124.5, 134.6, 145.8, 147.3, 150.2, 190.3. MS: m/e = 441
(M+, 48%); Analysis Calcd for C28H31N3O2: C, 76.16; H, 7.08; N,
9.52%. Found: C, 76.08; H, 6.88; N, 9.36%.
Compound 4c: HPLC purity = 89% (C-18 NovaPak column;
MeOH:H2O/82:18), tr = 20 min; pale yellow crystals from EtOAc:
hexane (84%), m.p. 169–171 °C; IR (KBr) cmꢁ1: 3530, 3443, 3054,
2936, 2222, 1636, 1560; 1H-NMR (CDCl3): d 0.84, 1.03 (2s, 6H),
1.35–2.87 (m, 10H), 2.89–2.92 (m, 4H), 4.48 (s, 2H, D2O exchange-
able), 5.13, 5.20 (2s, 2H), 5.68 (s, 1H), 6.10 (s, 1H), 6.45 (d, 1H,
J = 3.03 Hz), 7.32–7.44 (m, 3H); 13C-NMR (CDCl3): d 17.2, 17.9
19.5, 22.9, 23.3, 26.9, 27.4, 32.4, 38.8, 40.5, 45.3, 110.8,
128.9,134.2, 136.2, 148.1, 190.4; MS: m/e = 446 (M+, 28%); Analysis
Calcd for C27H30N2O2S: C, 72.61; H, 6.77; N, 7.27; S, 7.18%. Found: C,
72.82; H, 6.59; N, 6.48; S, 7.49%.
Compound 4d: HPLC purity = 88% (C-18 NovaPak column;
MeOH:H2O/70:30), tr = 21 min; pale yellow crystals from EtOAc:
hexane (88%), m.p. 244–247 °C; IR (KBr) cmꢁ1: 3595–3420, 3054,
2936, 2223, 1636, 1562; 1H-NMR (CDCl3): d 0.83, 1.60 (2s, 6H),
1.30–2.88 (m, 10H), 2.72–2.92 (m, 4H), 5.11, 5.20 (2s, 2H), 5.68
(s, 1H), 6.10 (s, 1H), 6.40 (d, 1H, J = 3.32 Hz), 7.29–7.41 (m, 5H),
8.22 (s, 1H, D2O exchangeable); 13C-NMR (CDCl3): d 17.4, 17.8,
19.5, 22.8, 23.2, 26.6, 27.8, 31.6, 38.8, 40.4, 45.4, 112.9, 117.4,
120.7, 123.5, 128.4, 129.3, 137.2, 143.0, 148.3, 190.4. MS:
m/e = 441 (M+, 32%); Analysis Calcd for C29H31NO3: C, 78.88; H,
7.08; N, 3.17%. Found: C, 79.03; H, 6.89; N, 3.28%.
Compound 4e: HPLC purity = 80% (C-18 NovaPak column;
MeOH:H2O/90:10), tr = 18 min; yellow crystals from EtOAc: hex-
ane (86%), m.p. 102–104 °C; IR (KBr) cmꢁ1: 3587–3432, 3056,
2938, 2225, 1638, 1562; 1H-NMR (CDCl3): d 0.86, 1.7 (2s, 6H),
1.33–2.89 (m, 10H), 2.91–2.89 (m, 4H), 5.09, 5.18 (2s, 2H), 5.65
(s, 1H), 6.12 (s, 1H), 6.48 (d, 1H, J = 2.77 Hz), 7.29–7.41 (m, 4H),
8.21 (s, 1H, D2O exchangeable); 13C-NMR (CDCl3): d 17.3, 17.5,
19.5, 23.0, 23.3, 26.6, 27.6, 32.2, 38.4, 40.7, 45.8, 110.7,
128.6,135.7, 138.0, 150.3, 190.6; MS: m/e = 442 (M+, 33%); Analysis
Calcd for C28H30N2O3: C, 75.99; H, 6.83; N, 6.33%. Found: C, 76.03;
H, 6.77; N, 6.52%.
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2. Experimental
2.1. Synthetic methods, analytical and spectral data
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The starting steroid, androstenedione (1), was purchased from
Sigma Company, USA. All solvents were dried by distillation prior
to using. Melting points were recorded on Buchi melting point
apparatus D-545; 13C NMR and 1H NMR spectra were recorded
on Bruker DPX200 instrument in CDCl3 and DMSO with TMS as
internal standard for protons and solvent signals as internal stan-
dard 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 Ludwig-Maxim-
ilians-Universitaet-Muenchen, Germany. The progress of all reac-
tions was monitored by TLC on 2 ꢀ 5 cm pre-coated silica gel 60
F254 plates of thickness of 0.25 mm (Merck). The nomenclature
of the newly synthesized compounds were according to the Chem-
BioDraw Ultra12.
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2.2. Chemical syntheses
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2.2.1. (6aR,6bS,8aS,13aS,13bR)-10-amino-6a,8a-dimethyl-4-oxo-12-
phenyl-1,2,4,5,6,6a,6b-,7,8,8a,12,13,13a,13b-tetradecahydronaphtho-
[20,10:4,5]indeno[1,2-b]pyran-11-carbonitrile (4a), (6aR,6bS,8aS,13aS,
13bR)-10-amino-6a,8a-dimethyl-4-oxo-12-pyridyl-1,2,4,5,6,6a,6b,-
7,8,8a,12,13,13a,13b-tetradecahydronaphtho[20,10:4,5]indeno[1,2-
b]pyran-11-carbonitrile (4b), (6aR,6bS,8aS,13aS,13bR)-10amino-
6a,8a-dimethyl-4-oxo-12-thienyl-1,2,4,5,6,-6a,6b,7,8,8a,12,13,13a,
13b-tetradecahydronaphtho[20,10:4,5]indeno[1,2-b]pyran-11-
carbonitrile (4c), (6aR,6bS,8aS,13aS,13bR)-10-hydroxy-6a,8a-
dimethyl-4-oxo-12-phenyl-1,2,4,5,6,6a,6b,7,8,8a,12,13,13a,13b-
tetradecahydronaphtho[20,10:4,5]indeno[1,2-b]pyran-11-carbonitrile
(4d), (6aR,6bS,8aS,13aS,13bR)-10-hydroxy-6a,8a-dimethyl-4-oxo-12-
pyridyl-1,2,4,5,6,6a,6b,7,8,8a,12,13,13a,13b-tetradecahydron-
aphtho[20,10:4,5]indeno[1,2-b]pyran-11-carbonitrile (4e) and
(6aR,6bS,8aS,13aS,13bR)-10-hydroxy-6a,8a-dimethyl-4-oxo-12-
thienyl-1,2,4,5,6,6a,6b,7,8,8a,12,13,13a,13b-tetradecahydrona-
phtho[20,10:4,5]indeno[1,2-b]pyran-11-carbonitrile (4f)
General procedure: To a solution of androstenedione (0.286 g,
1 mmol) in absolute ethanol (40 mL) containing triethylamine
(0.025 mL), either of malononitrile (0.066 g, 1 mmol) or ethyl cya-
noacetate (0.113 g, 1 mmol) and either of the aromatic aldehydes
namely benzaldehyde (106 g, 1 mmol), pyridine-3-aldehyde
(0.107 g, 1 mmol) or thiophene-2-aldehyde (0.112 g, 1 mmol) were
added. The reaction mixture was heated under reflux for 1 h and
the formed solid product produced from the hot solution was col-
lected by filtration. Thin layer chromatography revealed just a sin-
gle spot which proved the presence of a single product.
Compound 4a: HPLC purity = 90% (C-18 NovaPak column;
MeOH:H2O/70:30), tr = 20 min; pale yellow crystals from EtOAc:
hexane (89%), m.p. 220–224 °C; IR (KBr) cmꢁ1: 3540, 3423, 3057,
2932, 2222, 1667, 1563; 1H-NMR (CDCl3): d 0.84, 1.01 (2s, 6H),
1.33–2.86 (m, 10H), 2.89–2.95 (m, 4H), 4.23 (s, 2H, NH2), 5.01,
5.20 (2s, 2H), 5.37 (s, 1H), 6.11 (s, 1H), 6.44 (d, 1H, J = 2.3 Hz),
6.85–7.38 (m, 5H); 13C-NMR (CDCl3): d 17.0,17.9, 19.8, 23.1, 25.3,
29.2, 30.2, 33.7, 34.4, 34.8, 41.8, 43.8, 44.0, 53.2, 117.8, 127.8,
124.2, 125.9, 128.6, 129.3, 144.3, 146.2, 148.9, 190.8. MS: m/
e = 440 (M+, 28%); Analysis Calcd for C29H32N2O2: C, 79.06; H,
7.32; N, 6.36%. Found: C, 79.22; H, 7.41; N, 6.55%.
Compound 4f: HPLC purity = 84% (C-18 NovaPak column;
MeOH:H2O/88:12), tr = 23 min; yellow crystals from EtOAc: hex-
ane (77%), m.p. 220–224 °C; IR (KBr) cmꢁ1: 3554–3428, 3053,
2936, 2223, 1635, 1560; 1H-NMR (CDCl3): d 0.83, 1.03 (2s, 6H),
1.30–2.89 (m, 10H), 2.87–2.91 (m, 4H), 5.11, 5.20 (2s, 2H), 5.64
(s, 1H), 6.11 (s, 1H), 6.49 (d, 1H, J = 3.63 Hz), 7.26–7.43 (m, 3H),
8.23 (s, 1H, D2O exchangeable); 13C-NMR (CDCl3): d 17.0, 17.9,
19.8, 23.0, 23.0, 26.6, 27.6, 32.3, 38.6, 40.2, 45.9, 110.8, 117.4,
123.8, 128.5, 129.3, 137.6, 143.0, 148.6, 190.2; MS: m/e = 447
(M+, 24%); Analysis Calcd for C27H29NO3S: C, 72.45; H, 6.53; N,
3.13; S, 7.16%. Found: C, 72.30; H, 6.68; N, 3.44; S, 6.09%.
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2.2.2. (6bS,aS,13aS,13bR)-10-mercapto-8a-methyl-12-phenyl-
5,6,6a,6b,7,8,8a,9,12,13,-13a,13b-dodecahydro-1H-naphtho-
[20,10:4,5]indeno[1,2-d]pyrimidin-4(2H)-one (6a),
(6bS,aS,13aS,13bR)-10-mercapto-8a-methyl-12-(pyridine-3-yl)-
5,6,6a,6b,7,8,8a,9,12,-13,13a,13b-dodecahydro-1H-naphtho-
[20,10:4,5]indeno[1,2-d]pyrimidin-4(2H)-one (6b), and (6bS,aS,
13aS,13bR)-10-mercapto-8a-methyl-12-(thienyl-2-yl)-5,6,6a,6b,7,
8,8a,9,12,13,13a,-13b-dodecahydro-1H-naphtho[20,10:4,5]indeno[1,2-
d]pyrimidin-4(2H)-one (6c)
General procedure: To a solution of androstenedione (0.286 g,
1 mmol) in absolute ethanol (40 mL) containing triethylamine
(0.025 mL) and thiourea (0.76 g, 0.01 mol) either of benzaldehyde
Compound 4b: HPLC purity = 92% (C-18 NovaPak column;
MeOH:H2O/80:20), tr = 22 min; pale yellow crystals from EtOAc:
Please cite this article in press as: Mohareb RM et al. Heterocyclic ring extension of androstenedione: Synthesis and cytotoxicity of fused pyran, pyrimidine
and thiazole derivatives. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.04.011

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(106 g, 1 mmol), pyridine-3-aldehyde (0.107 g, 1 mmol) or thio-
phene-2-aldehyde (0.112 g, 1 mmol) were added. The reaction
mixture was heated under reflux for 1 h and the formed solid prod-
uct produced from the hot solution was collected by filtration. Thin
layer chromatography revealed just a single spot which proved the
presence of a single product.
6.28 (s, 1H), 6.43 (d, 1H, J = 3.03 Hz), 7.28–7.41 (m, 9H); 13C-NMR
(CDCl3): d 17.3,17.8, 19.9, 23.4, 25.6, 29.4, 30.1, 33.7, 34.6, 34.8,
41.8, 43.9, 44.2, 53.0, 120.3, 122.8, 126.5, 127.1, 128. 8, 133.4,
144.8, 146.2, 149.2, 172.3, 176.2, 190.1. MS: m/e = 533 (M+, 44%);
Analysis Calcd for C34H35N3OS: C, 76.51; H, 6.61; N, 7.87; S, 6.01%.
Found: C, 76.38; H, 6.51; N, 6.39; S, 6.08%.
Compound 8b: HPLC purity = 81% (C-18 NovaPak column;
MeOH:H2O/80:20), tr = 18 min; yellow crystals from EtOAc: hex-
ane (80%), m.p. 266–268 °C; IR (KBr) cmꢁ1: 3053, 2931, 1635,
1567; 1H-NMR (CDCl3): d 0.89, 1.04 (2s, 6H), 1.32–2.88 (m, 10H),
2.85–2.96 (m, 4H), 5.12, 5.22 (2s, 2H), 5.35 (s, 1H), 6.10 (s, 1H),
6.25 (s,1H), 6.42 (d, 1H, J = 3.03 Hz), 7.28–7.41 (m, 8H); 13C-NMR
(CDCl3): d 17.1,17. 6, 19.9, 23.6, 25.6, 29.4, 30.1, 33.8, 34.6, 34.8,
41.5, 43.9, 44.0, 53.2, 120.4, 122.6, 126.3, 127.2, 128. 8, 133.4,
144.9, 146.3, 149.6, 172.1, 176.3, 190.4. MS: m/e = 568 (M+, 56%);
Analysis Calcd for C34H34ClN3OS: C, 71.87; H, 6.03; N, 7.40; S,
5.64%. Found: C, 71.66; H, 5.93; N, 7.44; S, 5.82%.
Compound 8c: HPLC purity = 86% (C-18 NovaPak column;
MeOH:H2O/90:10), tr = 20 min; Orange from EtOAc: hexane
(83%), m.p. 210–212 °C; IR (KBr) cmꢁ1: 3057, 2930, 1637, 1569;
1H-NMR (CDCl3): d 0.87, 1.04 (2s, 6H), 1.30–2.88 (m, 10H), 2.87–
2.98 (m, 4H), 3.18 (s, 3H), 5.10, 5.22 (2s, 2H), 5.35 (s, 1H), 6.10 (s,
1H), 6.28 (s,1H), 6.40 (d, 1H, J = 3.81 Hz), 7.28–7.40 (m, 8H); 13C-
NMR (CDCl3): d 17.0,17.8, 19.6, 23.6, 25.6, 26.8, 29.7, 30.0, 33.8,
34.6, 34.8, 41.6, 43.9, 44.1, 53.0, 120.6, 122.7, 126.2, 127.0, 128.
6, 135.2, 144.9, 148.2, 149.9, 172.4, 176.3, 190.1. MS: m/e = 547
(M+, 20%); Analysis Calcd for C35H37N3OS: C, 76.75; H, 6.81; N,
7.67; S, 5.85%. Found: C, 76.80; H, 6.73; N, 8.03; S, 5.88%.
Compound 8d: HPLC purity = 87% (C-18 NovaPak column;
MeOH:H2O/80:20), tr = 22 min; Orange from EtOAc: hexane
(80%), m.p. 190–193 °C; IR (KBr) cmꢁ1: 3054, 2932, 1637, 1569;
1H-NMR (CDCl3): d 0.87, 1.03 (2s, 6H), 1.30–2.88 (m, 10H), 2.87–
2.98 (m, 4H), 3.11 (s, 3H), 5.11, 5.22 (2s, 2H), 5.36 (s, 1H), 6.11
(s, 1H), 6.41 (d, 1H, J = 3.44 Hz), 6.29 (s,1H), 7.25–7.43 (m, 8H);
13C-NMR (CDCl3): d 17.0,17.8, 19.6, 23.6, 25.6, 29.7, 30.0, 33.8,
34.6, 34.8, 41.6, 43.9, 44.1, 53.0, 120.6, 122.9, 125.1, 127.0, 128.
6, 135.2, 146.2, 148.2, 149.9, 172.4, 176.3, 190.12. MS: m/e = 563
(M+, 28%); Analysis Calcd for C35H37N3O2S: C, 74.57; H, 6.62; N,
7.45; S, 5.69%. Found: C, 72.41; H, 6.83; N, 7.44; S, 5.64%.
Compound 6a: HPLC purity = 88% (C-18 NovaPak column;
MeOH:H2O/75:25), tr = 23 min; yellow crystals from EtOAc: hex-
ane (86%), m.p. 190–192 °C; IR (KBr) cmꢁ1: 3455–3225, 3053,
2930, 1635, 1562; 1H-NMR (CDCl3): d 0.86, 1.03 (2s, 6H), 1.31–
2.88 (m, 10H), 2.90–2.97 (m, 4H), 5.09, 5.22 (2s, 2H), 5.38 (s, 1H),
6.10 (s, 1H), 6.18 (s,1H), 6.47 (d, 1H, J = 2.3 Hz), 7.03–7.39 (m,
5H), 8.30 (s, 1H, D2O exchangeable); 13C-NMR (CDCl3):
d
17.0,17.9, 19.8, 23.1, 25.3, 29.2, 30.2, 33.7, 34.4, 34.8, 41.8, 43.8,
44.0, 53.2, 126.8, 127.0, 128.6, 129.3, 144.3, 146.2, 148.9, 172.3,
190.3. MS: m/e = 432 (M+, 39%); Analysis Calcd for C27H32N2OS: C,
74.96; H, 7.46; N, 6.48; S, 7.41%. Found: C, 74.88; H, 7.38; N,
6.72; S, 7.63%.
Compound 6b: HPLC purity = 85% (C-18 NovaPak column;
MeOH:H2O/78:22), tr = 21 min; yellow crystals from EtOAc: hex-
ane (86%), m.p. 144–146 °C; IR (KBr) cmꢁ1: 3455–3232, 3057,
2932, 1675, 1520; 1H-NMR (CDCl3): d 0.88, 1.04 (2s, 6H), 1.30–
2.84 (m, 10H), 2.90–2.95 (m, 4H), 5.12, 5.20 (2s, 2H), 5.34 (s, 1H),
6.11 (s, 1H), 6.18 (s, 1H), 6.40 (d, 1H, J = 3.55 Hz), 7.03–7.39 (m,
4H), 8.30 (s, 1H, D2O exchangeable); 13C-NMR (CDCl3):
d
17.0,17.9, 19.8, 23.1, 25.3, 29.2, 30.2, 33.7, 34.4, 34.8, 41.8, 43.8,
44.0, 53.2, 126.8, 127.4, 128.7, 129.3, 144.3, 145.2, 147.9, 172.3,
190.1. MS: m/e = 433 (M+, 24%); Analysis Calcd for C26H31N3OS: C,
71.02; H, 7.21; N, 9.69; S, 7.39%. Found: C, 71.17; H, 7.29; N,
9.88; S, 7.44%.
Compound 6c: HPLC purity = 84% (C-18 NovaPak column;
MeOH:H2O/80:20), tr = 18 min; yellow crystals from EtOAc: hex-
ane (87%), m.p. 133–135 °C; IR (KBr) cmꢁ1: 3544–3426, 3050,
2962, 1680, 1562; 1H-NMR (CDCl3): d 0.86, 1.03 (2s, 6H), 1.32–
2.89 (m, 10H), 2.83–2.902 (m, 4H), 5.19, 5.22 (2s, 2H), 5.64 (s,
1H), 6.11 (s, 1H), 6.18 (s, 1H), 6.47 (d, 1H, J = 3.22 Hz), 7.31–7.40
(m, 3H), 8.22 (s, 1H, D2O exchangeable); 13C-NMR (CDCl3): d
14.6, 19.5, 22.4, 23.5, 26.5, 27.4, 32.4, 38.8, 40.5, 45.0, 110.3,
128.4,135.4, 138.5, 149.6, 172.0, 190.2; MS: m/e = 438 (M+, 33%);
Analysis Calcd for C25H30N2OS2: C, 68.45; H, 6.89; N, 6.39; S,
14.62%. Found: C, 68.79; H, 6.83; N, 6.52; S, 14.87%.
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2.2.4. (6aR,6bS,8aS,12aS,12bR)-10-amino-6a,8a-dimethyl-4-oxo-
2,4,5,6,6a,6b,7,-8,8a,12,12a,12b-dodecahydro-1H-naph-
tho[20,10:4,5]indeno[2,1-b]thiophene-9-carbonitrile (10a) and ethyl
(6aR,6bS,-8aS,12aS,12bR)-10-amino-6a,8a-dimethyl-4-oxo-
2,4,5,6,6a,-6b,7,8-,8a,12,12a,12b-dodecahydro-1H-naphtho-
[20,10:4,5]indeno[2,1-b]thiophene-9-carbonitrile (10b)
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2.2.3. (6bS,8aS,15aS,15bR)-8a-methyl-10-phenyl-14-(pyridine-3-
yl)dodecahydronaphtho-[20,10:4,5]indeno[2,1-e]thiazolo[3,2-a]-
pyrimidin-4(2H)one (8a), (6bS,8aS,15aS,15bR)-8a-methyl-10-phenyl-
14-(4-chlorophenyl)dodecahydronaphtho-[20,10:4,5]indeno[2,1-e]-
thiazolo[3,2-a]pyrimidin-4(2H)one (8b), (6bS,8aS,15aS,15bR)-8a-
methyl-10-phenyl-14-(4-methylphenyl)dodecahydronaphtho-
[20,10:4,5]indeno[2,1-e]thiazolo[3,2-a]pyrimidin-4(2H)one (8c) and
(6bS,8aS,15aS,15bR)-8a-methyl-10-phenyl-14-(4-methoxyphenyl)-
dodecahydronaphtho[20,10:4,5]indeno[2,1-e]thiazolo[3,2-a]pyrimidin-
4(2H)one (8d)
General procedure: To a solution of compound 6b (0.419 g,
1 mmol) in ethanol (40 mL) either 2-bromo-1-phenylethanone
(0.20 g,1 mmol), 2-bromo-1-(4-chlorophenyl)ethanone (0.23 g,
1 mmol), 2-bromo-1-(4-methylphenyl)ethanone (0.21 g, 1 mmol)
or 2-bromo-1-(4-methoxyphenyl)ethanone (0.23 g, 1 mmol) was
added. The reaction mixture, in each case, was heated under reflux
for 1 h then poured onto ice/water and the formed solid product
was collected by filtration. Thin layer chromatography revealed
just a single spot which proved the presence of a single product.
Compound 8a: HPLC purity = 85% (C-18 NovaPak column;
MeOH:H2O/80:20), tr = 16 min; yellow crystals from EtOAc: hex-
ane (88%), m.p. 230–233 °C; IR (KBr) cmꢁ1: 3057, 2932, 1638,
1566; 1H-NMR (CDCl3): d 0.88, 1.04 (2s, 6H), 1.30–2.85 (m, 10H),
2.88–2.95 (m, 4H), 5.13, 5.20 (2s, 2H), 5.39 (s, 1H), 6.09 (s, 1H),
General procedure: To a solution of androstenedione (0.286 g,
1 mmol) in 1,4-dioxane (20 mL) containing triethylamine
(0.50 mL) each of elemental sulphur (0.032 g, 1 mmol) and either
malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (0.113 g,
0.01 mol) were added. The reaction mixture, in each case, was
heated under reflux for 1 h then poured onto ice/water containing
few drops of hydrochloric acid and the formed solid product was
collected by filtration. Thin layer chromatography revealed just a
single spot which proved the presence of a single product.
Compound 10a: HPLC purity = 82% (C-18 NovaPak column;
MeOH:H2O/75:25), tr = 22 min; yellow crystals from EtOAc: hex-
ane (88%), m.p. 170–173 °C; IR (KBr) cmꢁ1: 3477, 3326 (NH2),
3054, 2930, 2220, 1677, 1638, 1566; 1H-NMR (CDCl3): d 0.86,
1.06 (2s, 6H), 1.34–2.85 (m, 10H), 2.88–2.95 (m, 4H), 4.83 (s, 2H),
5.12, 5.20 (2s, 2H), 5.39 (s, 1H), 6.11 (s, 1H), 6.28 (s, 1H), 6.43 (d,
1H, J = 3.03 Hz); 13C-NMR (CDCl3): d 17.2,17.6, 19.8, 23.6, 25.2,
29.4, 30.0, 33.7, 34.6, 34.9, 41.8, 43.9, 44.4, 53.0, 118.3, 128.0,
133.4, 144.8, 146.2, 190.2. MS: m/e = 366 (M+, 30%); Analysis Calcd
for C22H26N2OS: C, 72.09; H, 7.15; N, 7.64; S, 8.75%. Found: C,
72.22; H, 6.87; N, 7.49; S, 8.59%.
Please cite this article in press as: Mohareb RM et al. Heterocyclic ring extension of androstenedione: Synthesis and cytotoxicity of fused pyran, pyrimidine
and thiazole derivatives. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.04.011

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Compound 10b: HPLC purity = 86% (C-18 NovaPak column;
17.6, 18.3, 19.9, 20.3, 23.1, 25. 9, 26.8, 29.4, 30.3, 33.9, 34.6, 34.6,
41.8, 43.9, 44.2, 53.2, 56.3, 61.2, 127.4, 129. 0, 130.8, 149.3,
149.8, 172.6, 174.6, 188.1, 190.0. MS: m/e = 499 (M+, 53%); Analysis
Calcd for C28H35NO5S: C, 67.58; H, 7.09; N, 2.81; S, 6.44%. Found: C,
68.37; H, 7.44; N, 2.94; S, 6.38%.
MeOH:H2O/82:18), tr = 20 min; yellow crystals from EtOAc: hex-
ane (89%), m.p. 230–233 °C; IR (KBr) cmꢁ1: 3480, 3334 (NH2),
3051, 29330, 1679, 1638, 1560; 1H-NMR (CDCl3): d 0.88, 1.04 (2s,
6H), 1.16 (t, 3H, J = 7.20 Hz), 1.32–2.87 (m, 10H), 2.86–2.95 (m,
2H), 4.80 (s, 2H), 4.25 (q, 2H, J = 7.20 Hz), 5.11, 5.21 (2s, 2H), 5.39
(s, 1H), 6.11 (s, 1H), 6.28 (s,1H), 6.40 (d, 1H, J = 3.63 Hz); 13C-
NMR (CDCl3): d 17.0, 17.6, 19.8, 20.3, 23.6, 25.2, 29.4, 30.0, 33.9,
34.68, 34.9, 41.8, 43.9, 44.4, 53.0, 56.2, 128.0, 133.4, 144.8, 146.2,
190.2. MS: m/e = 413 (M+, 20%); Analysis Calcd for C24H31NO3S: C,
69.70; H, 7.56; N, 3.39; S, 7.75%. Found: C, 69.62; H, 7.41; N,
3.60; S, 7.80%.
Compound 12d: HPLC purity = 83% (C-18 NovaPak column;
MeOH:H2O/78:22), tr = 20 min; yellow crystals from EtOAc: hex-
ane (83%), m.p. 267–269 °C; IR (KBr) cmꢁ1: 3477–3320 (NH),
3053, 2932, 1725, 1686, 1668, 1638, 1563; 1H-NMR (CDCl3): d
0.84, 1.04 (2s, 6H), 1.14, 1.17 (2t, 6H, J = 7.23, 6.53 Hz), 1.32–2.89
(m, 10H), 2.80–2.95 (m, 2H), 4.22,4.26 (2q, 4H, J = 7.23, 6.53 Hz),
4.82 (s, 2H), 5.06, 5.23 (2s, 2H), 5.42 (s, 1H), 6.10 (s, 1H), 6.20
(s,1H), 6.45 (d, 1H, J = 2.69 Hz), 8.28 (s, 1H); 13C-NMR (CDCl3): d
17.0, 17.8, 18.3, 19.6, 19.9, 20.3, 23.1, 25. 9, 26.8, 29.4, 30.3, 33.9,
34.6, 34.6, 41.8, 43.9, 44.2, 53.2, 56.3, 57.2, 127.4, 129.0, 130.4,
149.3, 150.0, 172.6, 174.1, 188.4, 190.2. MS: m/e = 527 (M+, 28%);
Analysis Calcd for C29H37NO6S: C, 66.01; H, 7.07; N, 2.65; S, 6.08%.
Found: C, 66.22; H, 7.09; N, 2.64; S, 6.46%.
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2.2.5. N-((6aR,6bS,8aS,12aS,12bR)-9-cyano-6a,8a,12b-trimethyl-4-
oxo-2,4,5,6,6a,7,8,8a,-12,12a,12b-dodecahydro-1H-naphtho-
[20,10:4,5]indeno[2,1-b]thiophen-10-yl)-3-oxobutanamide (12a), ethyl
3-(((6aR,6bS,8aS,12aS,12bR)-9-cyano-6a,8a,12b-trimethyl-4-oxo-
2,4,5,6,6a,7,8,8a,-12,12a,12b-dodecahydro-1H-naphtho-
[20,10:4,5]indeno[2,1-b]thiophen-10-yl)amino)-3-oxopropanoate
(12b), (6aR,6bS,8aS,12aS,12bR)-9-cyano-6a,8a,12b-trimethyl-4-oxo-
10-(3-oxobutanamido)-2,4,5,6,6a,7,8,8a,-12,12a,12b-dodecahydro-
1H-naphtho[20,10:4,5]indeno[2,1-b]thiophene-9-carboxylate (12c)
and (6aR,6bS,8aS,12aS,-12bR)-ethyl 10-(3-ethoxy-3-oxopro-
panamido)-6a,8a,12b-trimethyl-4-oxo-2,4,5,6,6a,7,8,8a,-12,12a,12b-
dodecahydro-1H-naphtho[20,10:4,5]indeno[2,1-b]thiophene-9-
carboxylate (12d)
General procedure: Equimolecular ratio of either compound 10a
(3.66 g, 0.01 mol) or 10b (0.413 g, 1 mmol) and either ethyl aceto-
acetate (0.130 g, 1 mmol) or diethylmalonate (0.160 g, 1 mmol) in
1,4-dioxane (20 mL) was heated under reflux for 2 h. The solid
product, formed in each case, upon pouring onto ice/water was col-
lected by filtration. Thin layer chromatography revealed just a sin-
gle spot which proved the presence of a single product.
Compound 12a: HPLC purity = 89% (C-18 NovaPak column;
MeOH:H2O/80:20), tr = 21 min; yellow crystals from EtOAc: hex-
ane (80%), m.p. 180–183 °C; IR (KBr) cmꢁ1: 3477–3338, 3057,
2930, 2222, 1720, 1689, 1670, 1639, 1563; 1H-NMR (CDCl3): d
0.85, 1.03 (2s, 6H), 1.32–2.89 (m, 10H), 2.84–2.96 (m, 2H), 3.11
(s, 3H), 4.86 (s, 2H), 5.10, 5.19 (2s, 2H), 5.39 (s, 1H), 6.11 (s, 1H),
6.46 (d, 1H, J = 2.72 Hz), 6.25 (s,1H), 8.30 (s, 1H); 13C-NMR (CDCl3):
d 17.2,17.8, 19.5, 20.3, 23.8, 25.6, 29.4, 30.3, 33.7, 34.6, 34.8, 41.7,
43.9, 44.2, 53.2, 61.8, 127.8, 128.5, 130.6, 149.2, 149.2, 172.3,
188.4, 190.1. MS: m/e = 450 (M+, 30%); Analysis Calcd for C26H30N2-
O3S: C, 69.30; H, 6.71; N, 6.22; S, 7.12%. Found: C, 70.19; H, 6.53; N,
6.43; S, 7.13%.
Compound 12b: HPLC purity = 83% (C-18 NovaPak column;
MeOH:H2O/88:12), tr = 23 min; yellow crystals from EtOAc: hex-
ane (84%), m.p. 155–157 °C; IR (KBr) cmꢁ1: 3483–3330, 3054,
2931, 2220, 1722, 1686, 1671, 1636, 1562; 1H-NMR (CDCl3): d
0.86, 1.03 (2s, 6H), 1.13 (t, 3H, J = 6.94 Hz), 1.30–2.89 (m, 10H),
2.82–2.95 (m, 2H), 4.25 (q, 2H, J = 6.94 Hz), 4.86 (s, 2H), 5.11,
5.19 (2s, 2H), 5.39 (s, 1H), 6.10 (s, 1H), 6.22 (s,1H), 6.48 (d, 1H,
J = 3.49 Hz), 8.28 (s, 1H); 13C-NMR (CDCl3): d 17.1, 17.6, 18.3,
19.9, 20.3, 23.1, 25. 9, 29.4, 30.3, 33.7, 34.6, 34.8, 41.8, 43.9, 44.2,
53.2, 56.8, 61.8, 127.4, 128. 8, 130.2, 149.0, 149. 8, 172.3, 188.6,
190.3. MS: m/e = 480 (M+, 26%); Analysis Calcd for C27H32N2O4S:
C, 67.47; H, 6.71; N, 5.83; S, 6.67%. Found: C, 68.04; H, 6.70; N,
5.83; S, 6.69%.
Compound 12c: HPLC purity = 86% (C-18 NovaPak column;
MeOH:H2O/90:10), tr = 22 min; yellow crystals from EtOAc: hex-
ane (80%), m.p. 180–183 °C; IR (KBr) cmꢁ1: 3456–3333 (NH),
3056, 2930, 1720, 1688, 1670, 1638, 1560; 1H-NMR (CDCl3): d
0.83, 1.05 (2s, 6H), 1.16 (t, 3H, J = 7.83 Hz), 1.32–2.89 (m, 10H),
2.62 (s, 3H), 2.80–2.95 (m, 2H), 4.22 (q, 2H, J = 7.83 Hz), 4.86 (s,
2H), 5.09, 5.19 (2s, 2H), 5.39 (s, 1H), 6.11 (s, 1H), 6.22 (s, 1H),
6.48 (d, 1H, J = 3.49 Hz), 8.28 (s, 1H); 13C-NMR (CDCl3): d 17.1,
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2.2.6. N-((6aR,6bS,aS,12aS,12bR)-9-cyano-6a,8a,12b-trimethyl-4-
oxo-2,4,5,6,6a,6b,-7,8,8a,12,12a,12b-dodecahydro-1H-naphtho-
[20,10:4,5]indeno[2,1-b]thiophen-10-yl)-3oxo-2-(2-
phenylhydrazono)butanamide (14a), N-((6aR,6bS,aS,12aS,12bR)-9-
cyano-6a,8a,12b-trimethyl-4-oxo-2,4,5,6,6a,6b,7,8,8a,12,-12a,12b-
dodecahydro-1H-naphtho[20,10:4,5]-indeno-[2,1-b]thiophen-10-yl)-
3oxo-2-(2-p-tolylhydrazono)butanamide (14b), N-
((6aR,6bS,aS,12aS,12bR)-9-cyano-6a,8a,12b-trimethyl-4-oxo-2,4,5,
6,6a,6b,7,8,8a,12,-12a,12b-dodecahydro-1H-naphtho-
[20,10:4,5]indeno[2,1-b]thiophen-10-yl)-3oxo-2-(2-(4- chlorophenyl)
hydrazono)butmide (14c), ethyl 3-(((6aR,6bS,8aS,12aS,12bR)-9-
cyano-6a,8a,12b-trimethyl-4-oxo-2,4,5,6,6a,6b,7,8,8a,12,-12a,12b-
dodecahydro-1H-naphtho-[20,10:4,5]indeno[2,1-b]thiophen-10-
yl)amino-3-oxo-2-(2-phenylhydrazono)propanoate (14d), ethyl 3-
(((6aR,6bS,8aS,12aS,12bR)-9-cyano-6a,8a,12b-trimethyl-4-oxo-
2,4,5,6,6a,6b,-7,8,8a,12,-12a,12b-dodecahydro-1H-naphtho-
[20,10:4,5]indeno[2,1-b]thiophen-10-yl)amino-3-oxo-2-(2-p-
tolylhydrazono)propanoate (14e) and ethyl 3-(((6aR,6bS,
8aS,12aS,12bR)-9-cyano-6a,8a,12b-trimethyl-4-oxo-
2,4,5,6,6a,6b,7,8,8a,12,-12a,12b-dodecahydro-1H-naphtho-
[20,10:4,5]indeno[2,1-b]thiophen-10-yl)amino-3-oxo-2-(2-(4-chloro-
phenyl)-hydrazono)propanoate (14f)
General procedure: To a solution of either compound 12a
(0464 g, 0.01 mol) or 12b (0.492 g, 1 mmol) in ethanol (30 mL)
containing sodium acetate (2.5 g), either benzenediazonium chlo-
ride (0.01 mol), 4-methylbenzenediazonium chloride (1 mmol) or
4-chlorobenzenediazonium chloride (1 mmol) [prepared by adding
sodium nitrite solution (0.007 g, 1 mmol) to a cold solution of the
appropriate aniline or its derivative (1 mmol) in concentrated
hydrochloric acid (3 mL, 18 M) with continuous stirring] was
added with stirring. The reaction mixture was kept at room tem-
perature for 1 h and the formed solid product, in each case, was
collected by filtration. Thin layer chromatography revealed just a
single spot which proved the presence of a single product.
Compound 14a: HPLC purity = 84% (C-18 NovaPak column;
MeOH:H2O/77:23), tr = 24 min; yellow crystals from EtOAc: hex-
ane (82%), m.p. 130–132 °C; IR (KBr) cmꢁ1: 3465–3332, 3054,
2932, 2227, 1718, 1686, 1670, 1639, 1567; 1H-NMR (CDCl3): d
0.86, 1.04 (2s, 6H), 1.30–2.87 (m, 10H), 2.86–2.96 (m, 2H), 3.06
(s, 3H), 5.08, 5.17 (2s, 2H), 5.34 (s, 1H), 6.11 (s, 1H), 6.23 (s,1H),
6.48 (d, 1H, J = 3.82 Hz), 7.28–7.38 (m, 5H), 8.27, 8.32 (2s, 2H);
13C-NMR (CDCl3): d 17.20,17.9, 19.5, 20.3, 23.6, 25.8, 29.4, 30.0,
33.9, 34.4, 34.8, 41.7, 43.9, 44.2, 117.9, 128.0, 128. 3, 130.8,
149.0, 149.6, 172.0, 188.5, 190.2. MS: m/e = 554 (M+, 21%); Analysis
Calcd for C32H34N4O3S: C, 69.29; H, 6.18; N, 10.10; S, 5.78%. Found:
C, 69.48; H, 6.39; N, 10.22; S, 5.83%.
Please cite this article in press as: Mohareb RM et al. Heterocyclic ring extension of androstenedione: Synthesis and cytotoxicity of fused pyran, pyrimidine
and thiazole derivatives. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.04.011

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Compound 14b: HPLC purity = 84% (C-18 NovaPak column;
MeOH:H2O/80:20), tr = 22 min; yellow crystals from EtOAc: hex-
ane (82%), m.p. 188–190 °C; IR (KBr) cmꢁ1: 3443–3330, 3056,
2930, 2223, 1716, 1687, 1671, 1639, 1563; 1H-NMR (CDCl3): d
0.85, 1.05 (2s, 6H), 1.32–2.87 (m, 10H), 2.83–2.99 (m, 2H), 3.08,
3.12 (2s, 6H), 5.18 (s, 2H), 5.14, 5.35 (2s, 2H), 6.20 (s,1H), 6.49 (d,
1H, J = 3.42 Hz), 7.26–7.39 (m, 4H), 8.23, 8.34 (2s, 2H); 13C-NMR
(CDCl3): d 17.22,17.9, 19.5, 20.3, 23.8, 25.8, 29.4,,31.8, 30.0, 33.9,
34.4, 34.8, 41.7, 43.9, 44.0, 117.6, 128.4, 128.8, 130.8, 149.0,
149.8, 172.0, 188.6, 190.3. MS: m/e = 568 (M+, 40%); Analysis Calcd
for C33H36N4O3S: C, 69.69; H, 6.38; N, 9.85; S, 5.50%. Found: C,
70.17; H, 6.70; N, 9.93; S, 5.77%.
2.2.7. (6aR,6bS,8aS,14aS,14bR)-10-Acetyl-9-amino-6a,8a-dimethyl-
6,6a,6b,7,8,8a,-12,14,14a,14b-decahydro-1H-
naphtho[200,100:40,50]indeno[10,20:4,5]thieno[2,3-b]pyridine-4,11-
(2H,5H)-dione (15a), (6aR,6bS,8aS,14aS,14bR)-ethyl 9-amino-6a,8a-
dimethyl-6,6a,6b,7,8,8a,12,14,-14a,14b-decahydro-1H-
naphtho[200,100:40,50]indeno-[10,20:4,5]-thieno[2,3-b]pyridine-10-
carboxylate (15b), (6aR,6bS,8aS,14aS,14bR)-10-Acetyl-9-hydroxy-
6a,8a-dimethyl-6,6a,6b,7,8,8a,12,14,14a,14b-decahydro-1H-naphtho-
[200,100:40,50]-indeno[10,20:4,5]-thieno[2,3-b]pyridine-4,11-(2H,5H)-
dione (15c), (6aR,6bS,8aS,14aS,14bR)-ethyl 9-hydroxy-6a,8a-
dimethyl-6,6a,6b,7,8,8a,12,14,14a,14b-decahydro-1H-naphtho-
[200,100:40,50]indeno-[10,20:4,5]thieno[2,3-b]pyridine-10-carboxylate
(15d)
Compound 14c: HPLC purity = 80% (C-18 NovaPak column;
MeOH:H2O/80:20), tr = 18 min; yellow crystals from EtOAc: hex-
ane (78%), m.p. 256–259 °C; IR (KBr) cmꢁ1: 3450–3326, 3054,
2931, 2220, 1718, 1684, 1670, 1633, 1563; 1H-NMR (CDCl3): d
0.85,1.05 (2s, 6H), 1.30–2.87 (m, 10H), 2.84–2.97 (m, 2H), 3.07
(s, 3H), 5.07, 5.15 (2s, 2H), 5.37 (s, 1H), 6.10 (s, 1H), 6.21
(s,1H), 6.46 (d, 1H, J = 3.09 Hz), 7.28–7.37 (m, 4H), 8.27, 8.36
General procedure: A solution of either 12a (0464 g, 0.01 mol),
12b (0.492 g, 1 mmol) in ethanol (30 mL), 12c (0.511 g, 1 mmol)
or 12d (0.541 g, 1 mmol) in absolute ethanol (40 mL) containing
triethylamine (0.50 g) was heated under reflux for 6 h. The mixture
was evaporated under vacuum, the remaining product was tritu-
rated with diethyl ether and the formed solid product was col-
lected by filtration. Thin layer chromatography revealed just a
single spot which proved the presence of a single product.
Compound 15a: HPLC purity = 86% (C-18 NovaPak column;
MeOH:H2O/90:10), tr = 19 min; yellow crystals from EtOAc: hex-
ane (83%), m.p. 233–235 °C; IR (KBr) cmꢁ1: 3488–3328, 3053,
2931, 1720, 1689, 1672, 1634, 1561; 1H-NMR (CDCl3): d 0.87,
1.05 (2s, 6H), 1.31–2.86 (m, 10H), 2.82–2.93 (m, 2H), 3.09 (s, 3H),
4.82 (s, 2H), 5.11, 5.20 (2s, 2H), 5.36 (s, 1H), 6.10 (s, 1H), 6.26
(s,1H), 6.41 (d, 1H, J = 4.07 Hz), 8.31 (s, 1H); 13C-NMR (CDCl3): d
17.0,17.68, 19.5, 20.3, 23.8, 25.6, 29.6, 30.3, 33.7, 34.6, 34.8, 41.7,
43.9, 44.2, 53.2, 123.6, 127.8, 128.7, 130.9, 133.4, 136.0, 149.6,
172.83, 188.3, 190.4. MS: m/e = 450 (M+, 26%); Analysis Calcd for
C26H30N2O3S: C, 69.30; H, 6.71; N, 6.22; S, 7.12%. Found: C,
69.25; H, 6.69; N, 6.04; S, 7.39%.
Compound 15b: HPLC purity = 80% (C-18 NovaPak column;
MeOH:H2O/86:14), tr = 21 min; yellow crystals from EtOAc: hex-
ane (83%), m.p. 196–198 °C; IR (KBr) cmꢁ1: 348o-3312, 3052,
2933, 1720, 1684, 1671, 1636, 1560; 1H-NMR (CDCl3): d 0.85,
1.02 (2s, 6H), 1.12 (t, 3H, J = 7.09 Hz), 1.30–2.90 (m, 10H), 2.80–
2.95 (m, 2H), 4.21 (q, 2H, J = 7.09 Hz), 4.63 (s, 2H), 5.19, 5.24 (2s,
2H), 5.35 (s, 1H), 6.10 (s, 1H), 6.24 (s,1H), 6.45 (d, 1H,
J = 3.11 Hz), 8.25 (s, 1H); 13C-NMR (CDCl3): d 17.4, 17.6, 18.3,
19.9, 20.6, 23.1, 25. 9, 29.4, 30.0, 33.7, 34.6, 34.8, 41.8, 43.9, 44.2,
53.1, 56.5, 127.4, 128. 8, 130.2, 130.9, 133.3, 136.5, 149.3, 149. 8,
172.6, 188.2, 190.2. MS: m/e = 480 (M+, 36%); Analysis Calcd for C27-
H32N2O4S: C, 67.47; H, 6.71; N, 5.83; S, 6.67%. Found: C, 67.24; H,
6.66; N, 5.53; S, 6.49%.
Compound 15c: HPLC purity = 83% (C-18 NovaPak column;
MeOH:H2O/90:10), tr = 19 min; yellow crystals from EtOAc: hex-
ane (82%), m.p. 166–168 °C; IR (KBr) cmꢁ1: 3558–3323, 3056,
2930, 1722, 1688, 1671, 1638, 1558; 1H-NMR (CDCl3): d 0.84,
1.04 (2s, 6H), 1.30–2.89 (m, 10H), 2.82–2.96 (m, 2H), 3.11 (s, 3H),
5.19, 5.26 (2s, 2H), 5.36 (s, 1H), 6.11 (s, 1H), 6.20 (s,1H), 6.43 (d,
1H, J = 3.49 Hz), 8.23 (s, 1H), 10.88 (s, 1H); 13C-NMR (CDCl3): d
17.3, 17.8, 18.4, 19.9, 20.1, 23.1, 25. 9, 26.8, 29.4, 30.2, 33.9, 34.6,
34.6, 41.8, 43.9, 44.2, 53.2, 56.3, 127.4, 129. 0, 130.8, 130.9,
134.2, 136.7, 149.6, 149. 8, 172.86, 188.10, 190.3. MS: m/e = 451
(M+, 23%); Analysis Calcd for C26H29NO4S: C, 69.15; H, 6.47; N,
3.10; S, 7.10%. Found: C, 68.95; H, 6.69; N, 2.88; S, 7.36%.
Compound 15d: HPLC purity = 86% (C-18 NovaPak column;
MeOH:H2O/80:20), tr = 22 min; yellow crystals from EtOAc: hex-
ane (86%), m.p. 173–176 °C; IR (KBr) cmꢁ1: 3540–3315, 3055,
2932, 1720, 1687, 1668, 1639, 1562; 1H-NMR (CDCl3): d 0.85,
1.02 (2s, 6H), 1.13 (t, 3H, J = 7.02 Hz), 1.34–2.89 (m, 10H), 2.80–
2.97 (m, 4H), 4.23 (q, 2H, J = 7.02 Hz), 5.07, 5.26 (2s, 1H), 5.40 (s,
1H), 6.43 (d, 1H, J = 3.73 Hz), 6.22 (s,1H), 8.26 (s, 1H),10.29 (s,
(2s, 2H); 13C-NMR (CDCl3):
d 17.20, 17.9, 19.5, 20.3, 23.8,
25.8, 29.4, 31.8, 30.3, 33.8, 34.4, 34.8, 41.7, 43.9, 44.2, 117.8,
128.6, 128.4, 130.8, 149.2, 149.8, 172.1, 188.7, 190.0. MS: m/
e = 5 (M+, 31%); Analysis Calcd for C32H33ClN4O3S: C, 65.24; H,
5.65; N, 9.51; S, 5.44%. Found: C, 65.53; H, 5.85; N, 9.37; S,
5.48%.
Compound 14d: HPLC purity = 83% (C-18 NovaPak column;
MeOH:H2O/85:15), tr = 17 min; yellow crystals from EtOAc: hex-
ane (77%), m.p. 230–233 °C; IR (KBr) cmꢁ1: 3442–3326, 3053,
2930, 1715, 1683, 1668, 1638, 1562; 1H-NMR (CDCl3): d 0.80,
1.04 (2s, 6H), 1.18 (t, 3H, J = 6.88 Hz), 1.31–2.90 (m, 10H), 2.81–
2.98 (m, 4H), 4.24 (q, 2H, J = 6.88 Hz), 5.15, 5.26 (2s, 2H), 5.36 (s,
1H), 6.10 (s, 1H), 6.20 (s,1H), 6.43 (d, 1H, J = 2.89 Hz), 7.30–7.39
(m, 5H), 8.24, 8.30 (2s, 2H); 13C-NMR (CDCl3): d 17.2, 17.6, 18.3,
19.8, 20.6, 23.1, 25. 9, 26.8, 29.4, 30.3, 33.9, 34.6, 34.6, 41.8, 43.9,
44.2, 53.4, 56.6, 127.4, 129.3, 132.6, 149.2, 149.6, 172.8, 188.3,
190.1. MS: m/e = 584 (M+, 39%); Analysis Calcd for C34H38N4O4S:
C, 67.78; H, 6.21; N, 9.58; S, 5.48%. Found: C, 68.03; H, 6.29; N,
9.53; S, 5.48%.
Compound 14e: HPLC purity = 86% (C-18 NovaPak column;
MeOH:H2O/85:15), tr = 22 min; yellow crystals from EtOAc: hex-
ane (80%), m.p. 177–179 °C; IR (KBr) cmꢁ1: 3461–3318, 3056,
2930, 1722, 1684, 1668, 1635, 1560; 1H-NMR (CDCl3): d 0.82,
1.05 (2s, 6H), 1.16 (t, 3H, J = 5.93 Hz), 1.33–2.90 (m, 10H), 2.81–
2.96 (m, 2H), 3.11 (s, 3H), 4.23 (q, 2H, J = 5.93 Hz), 5.17, 5.24
(2s, 2H), 5.38 (s, 1H), 6.11 (s, 1H), 6.20 (s, 1H), 6.22 (s,1H), 6.44
(d, 1H, J = 3.47 Hz), 7.27–7.40 (m, 4H), 8.21, 8.30 (2s, 2H); 13C-
NMR (CDCl3): d 17.1, 17.6, 18.3, 19.8, 20.3, 20.6, 23.1, 26.2,
26.6, 29.4, 30.4, 33.9, 34.6, 34.6, 41.8, 43.9, 44.6, 53.4,
56.8, 117.9, 127.6, 129.5, 133.8, 149.2, 149.6, 172.8, 188.6,
190.3. MS: m/e = 611 (M+, 40%); Analysis Calcd for C35H39N4O4S:
C, 68.71; H, 6.43; N, 9.16; S, 5.24%. Found: C, 68.52; H, 6.42; N,
9.36; S, 5.39%.
Compound 14f: HPLC purity = 83% (C-18 NovaPak column;
MeOH:H2O/85:15), tr = 20 min; yellow crystals from EtOAc: hex-
ane (83%), m.p. 103–105 °C; IR (KBr) cmꢁ1: 3453–3312, 3050,
2930, 1705, 1683, 1664, 1638, 1560; 1H-NMR (CDCl3): d 0.82,
1.02 (2s, 6H), 1.22 (t, 3H, J = 7.30 Hz), 1.33–2.90 (m, 10H), 2.81–
2.99 (m, 2H), 4.20 (q, 2H, J = 7.30 Hz), 5.16, 5.26 (2s, 2H), 5.36 (s,
1H), 6.22 (s,1H), 6.41 (d, 1H, J = 3.05 Hz), 7.28–7.39 (m, 4H), 8.25,
8.31 (2s, 2H); 13C-NMR (CDCl3): d 17.0, 17.6, 18.3, 19.8, 20.6,
23.1, 25. 9, 26.8, 29.4, 30.3, 33.9, 34.6, 34.6, 41.4, 43.9, 44.2, 53.4,
56.8, 117.1, 127.6, 129.7, 132.6, 149.0, 149.6, 172.3, 188.3, 190.2.
MS: m/e = 633 (M+, 22%); Analysis Calcd for C33H35ClN4O4S: C,
64.01; H, 5.70; N, 9.05; S, 5.18%. Found: C, 64.32; H, 5.55; N,
9.03; S, 5.22%.
Please cite this article in press as: Mohareb RM et al. Heterocyclic ring extension of androstenedione: Synthesis and cytotoxicity of fused pyran, pyrimidine
and thiazole derivatives. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.04.011

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1H); 13C-NMR (CDCl3): d 17.0, 17.8, 18.3, 19.6, 19.9, 20.3, 23.1, 25.
9, 26.8, 29.4, 30.3, 33.9, 34.6, 34.6, 41.8, 43.9, 44.2, 53.2, 56.3, 57.2,
127.4, 129.0, 130.4, 133.2, 134.8, 136.9, 149.2, 150.0, 172.4, 188.2,
190.0. MS: m/e = 481 (M+, 33%); Analysis Calcd for C27H31NO5S: C,
67.34; H, 6.49; N, 2.91; S, 6.66%. Found: C, 67.58; H, 6.69; N,
3.16; S, 6.82%.
Table 1
602
603
604
605
606
607
Cytotoxicity of the novel androstenedione derivatives 4e, 4f, 6b, 6c, 8b, 8d, 10a, 10b,
12d, 14d, 15b, 15c and 16 with significant activities against a variety of cancer cell
linesa [IC50 (nM)].
b
Compd no.
Cytotoxocity (IC50 in nM)
NUGC
DLDI
HA22T
HEPG2
HONE1
MCF
WI38
4e
4f
6b
6c
8b
33
69
180
40
60
181
35
1220
3505
154
87
876
1294
38
48
190
740
64
220
1464
60
240
488
760
66
2180
1549
36
120
69
234
82
33
2155
61
820
1260
275
238
1663
2739
96
366
446
837
328
227
4235
2345
630
1680
208
485
277
644
260
63
128
436
269
173
68
333
1189
254
1920
2270
2270
2411
2460
128
na
407
na
na
na
na
na
na
na
na
na
na
na
na
na
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
2.2.8. (6aR,6bs,8aS,12bR)-6a,8a-dimethyl-9-phenyl-10-thioxo-5,6,
6a,6b,7,8,8a,9,10,-12,12a,12b-dodecahydro-1H-napntho-
[20,10:4,5]indeno[1,2-d]thiazol-4(2H)-one (16)
8c
8d
2663
82
To a solution of androstenedione (0.286 g, 1 mmol) in 1,4-diox-
ane (20 mL) containing triethylamine (0.50 mL) each of elemental
sulphur (0.032 g, 1 mmol) and phenylisothio-cyanate (0.130 g,
1 mmol) was added. The whole reaction mixture was heated under
reflux for 3 h then left to cool overnight. The formed solid product
was collected by filtration. Thin layer chromatography revealed
just a single spot which proved the presence of a single product.
Compound 16: HPLC purity = 81% (C-18 NovaPak column;
MeOH:H2O/85:15), tr = 20 min; yellow crystals from EtOAc: hex-
ane (83%), m.p. 123–125 °C; IR (KBr) cmꢁ1: 3055, 2930, 1718,
1684, 1671, 1632, 1560, 1200–1195; 1H-NMR (CDCl3): d 0.86,
1.05 (2s, 6H), 1.30–2.88 (m, 10H), 2.82–2.95 (m, 2H), 5.11, 5.22
(2s, 2H), 5.38 (s, 1H), 6.10 (s, 1H), 6.28 (s,1H), 6.38 (d, 1H,
10a
10b
12d
14d
15b
15c
16
408
2073
436
122
2879
220
64
462
660
895
126
CHS 828
25
2315
2067
1245
15
18
a
NUGC, gastric cancer, DLDI, colon cancer, HA22T, liver cancer, HEPG2, liver
cancer; HONEI, nasopharyngeal carcinoma; HR, gastric cancer; MCF, breast cancer;
WI38, normal fibroblast cells.
b
The sample concentration produces a 50% reduction in cell growth.
O
H
H2C
X
CN
R
CHO
+
+
H
H
2a
, R = C6H5
b, R = 3-pyridyl
c, R= 2-thienyl
3a, X = CN
b, X = COOC2H5
O
1
EtOH/Et3N
X
O
CN
H
R
H
H
O
4a, X = NH2, R = C6H5
b
, X = NH2, R = 3-pyridyl
c
, X = NH2, R = 2-thienyl
d, X = OH, R = C6H5
e, X = OH, R = 3-pyridyl
f
, X = OH, R = 2-thienyl
Scheme 1. Synthesis of compounds 4a–f.
Please cite this article in press as: Mohareb RM et al. Heterocyclic ring extension of androstenedione: Synthesis and cytotoxicity of fused pyran, pyrimidine
and thiazole derivatives. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.04.011

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7
J = 4.22 Hz), 7.28–7.38 (m, 5H, C6H5); 13C-NMR (CDCl3): d 17.0,
17.6, 19.8, 20.4, 23.8, 25.6, 29.6, 30.3, 33.7, 34.6, 34.8, 41.5, 43.9,
44.6, 53.6, 122.3, 123.6, 127.8, 129.6, 130.9, 133.4, 149.8, 188.4,
190.2. MS: m/e = 435 (M+, 38%); Analysis Calcd for C26H29NOS2: C,
71.68; H, 6.71; N, 3.22; S, 14.72%. Found: C, 71.91; H, 6.55; N,
3.08; S, 14.64%.
and sulforhodamine B (SRB) were purchased from Sigma Chemical
Co. (Saint Louis, USA).
624
625
626
627
628
629
636
637
638
639
640
641
642
643
644
645
646
647
648
2.3.1.2. Cell cultures. Was obtained from the European Collection of
cell Cultures (ECACC, Salisbury, UK) and human gastric cancer
(NUGC and HR), human colon cancer (DLD1), human liver cancer
(HA22T and HEPG2), human breast cancer (MCF), nasopharyngeal
carcinoma (HONE1) and normal fibroblast cells (WI38) were kindly
provided by the National Cancer Institute (NCI, Cairo, Egypt). They
grow as monolayer and routinely maintained in RPMI-1640 med-
ium supplemented with 5% heat inactivated FBS, 2 mM glutamine
and antibiotics (penicillin 100 U/mL, streptomycin 100 lg/mL), at
37 °C in a humidified atmosphere containing 5% CO2. Exponentially
growing cells were obtained by plating 1.5 ꢀ 105 cells/mL for the
630
2.3. Biological evaluation
631
2.3.1. In vitro cytotoxic assay
2.3.1.1. Chemicals. Fetal bovine serum (FBS) and L-glutamine, were
purchased from Gibco Invitrogen Co. (Scotland, UK). RPMI-1640
medium was purchased from Cambrex (New Jersey, USA).
Dimethyl sulfoxide (DMSO), doxorubicin, penicillin, streptomycin
632 Q2
633
634
635
1
R-CHO
+
H2NCSNH2
+
2a, R = C6H5
5
b, R = 3-pyridyl
c
, R= 2-thienyl
EtOH/Et3N
SH
HN
N
H
Ar
H
H
O
6a, Ar = C6H5
b
, Ar = 3-pyridyl
c, Ar = 2-thienyl
R
S
N
N
H
EtOH
R-COCH2Br
6b
+
H
H
N
7a
, R = C6H5
O
b, R = 4-Cl-C6H4
c, R = 4-CH3-C6H4
d, R = -OCH3-C6H4
8a, R = C6H5
b, R = 4-Cl-C6H4
c, R = 4-CH3-C6H4
d
, R = 4-OCH3-C6H4
Scheme 2. Synthesis of compounds 6a–c and 8a–d.
Please cite this article in press as: Mohareb RM et al. Heterocyclic ring extension of androstenedione: Synthesis and cytotoxicity of fused pyran, pyrimidine
and thiazole derivatives. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.04.011

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No. of Pages 11, Model 5G
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649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
seven human cancer cell lines including cells derived from
0.75 ꢀ 104 cells/mL followed by 24 h of incubation. The effect of
the vehicle solvent (DMSO) on the growth of these cell lines was
evaluated in all the experiments by exposing untreated control
cells to the maximum concentration (0.5%) of DMSO used in each
assay.
in vitro cytotoxicity against six human cancer cell lines including
cells derived from human gastric cancer (NUGC), human colon can-
cer (DLD1), human liver cancer (HA22T and HEPG2), human breast
cancer (MCF), nasopharyngeal carcinoma (HONE1) and a normal
fibroblast cells (WI38). All of IC50 values were listed in Table 1.
Some heterocyclic compounds was observed with significant cyto-
toxicity against most of the cancer cell lines tested (IC50 = 10–
1000 nM). Normal fibroblasts cells (WI38) were affected to a
The heterocyclic androstenedione derivatives, prepared in this
study, were evaluated according to standard protocols for their
X
NH2
S
H
1
+
3a,b
S8
+
H
H
O
10a
b, X = COOEt
, X = CN
X
NHCOCH2Y
S
H
1,4-dioxane
H2C
Y
COOC2H5
10a,b
+
H
H
O
11a, Y = COCH3
b, Y = COOC2H5
12a, X = CN, Y = COCH3
b, X = CN, Y = COOC2H5
c, X = COOC2H5, Y = COCH3
d
, X = COOC2H5, Y = COOC2H5
X
NHCOC
Y
NNH
S
H
-
+
+
R
N
NCl
12a,b
H
H
R
13a, R = H
O
b, R = 4-CH3
c, R = 4-Cl
Y
X
CN
R
H
14
a
COCH3
COCH3
b
c
CN
4-CH3
4-Cl
H
COCH3
CN
CN
CN
CN
COOC2H5
d
e
COOC2H5
4-CH3
4-Cl
COOC2H5
f
Scheme 3. Synthesis of compounds 10a,b; 12a–d and 14a–f.
Please cite this article in press as: Mohareb RM et al. Heterocyclic ring extension of androstenedione: Synthesis and cytotoxicity of fused pyran, pyrimidine
and thiazole derivatives. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.04.011

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Y
O
R
NH
S
H
EtOH
Et3N
12a-d
H
H
O
15a, Y = COCH3, R = NH2
b, Y = COOC2H5, R = NH2
c, Y = COCH3, R = OH
d
, Y = COOC2H5, R = OH
Ph
N
S
S
H
1,4-dioxane
Et3N
1
+
S8
+
PhNCS
H
H
O
16
Scheme 4. Synthesis of compounds 15a–d and 16.
665
666
667
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693
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697
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700
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703
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707
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much lesser extent (IC50 > 10,000 nM). The reference compound
used is the CHS-828 which is a pyridyl cyanoguanidine anti-tumor
agent.
It is obvious that the high oxygen content of 12d is responsible
for its high cytotoxicity against the five cell lines NUGC, DLDI,
HA22T, HEPG2 and HONE1. The arylhydrazonothiophene deriva-
tive 12f with the combination of CN, COOEt and the4-chlorophenyl
moieties showed high potency towards the six cancer cell lines
among the group of derivatives 14a–f. The thieno[2,3-b]pyridine
derivatives 15a–d showed very low cytotoxicity although com-
pound 15b showed relatively high cytotoxicity against NUGC and
HEPG2 with IC50’s 876 and 660 nM, respectively. Finally the thia-
zole derivative 16 showed high cytotoxicity towards the six cancer
cell lines which is attributed to the high sulphur content of the
molecule. It is of great value to notice that the optimal cytotoxicity
against the six cancer cell lines were obtained through compounds
4e, 4f, 6b, 6c, 8b and 16.
It is very clear from our present finding that the newly synthe-
sized products with halogen substituted pattern OCH3, Cl or COOEt
show greater cytotoxic property. In every case it was observed that
molecules with electronegative substitutions as compounds 4e, 4f,
6b, 6c, 8b, 8d, 10a, 10b, 12d, 14d, 15b, 15c and 16 showed higher
cytotoxicity because them were either oxygen or chlorine substi-
tuted as well as comprised with similar structural features.
668
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671
672
673
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681
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685
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687
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691
2.3.1.3. Structure activity relationship. From Table 1 it is clear that
the cyclohexene moiety was found to be crucial for the cytotoxic
effect of cyclic compounds 4a–d to 16. Compounds 4e, 4f, 6b, 6c,
8b, 8d, 10a, 10b, 12d, 14d, 15b, 15c and 16 exhibited cytotoxic
effects against cancer cell lines, with IC50’s in the nM range which
were indicated through Table 1, the cytotoxicity of the rest of the
synthesized compounds were presented through the Supplemen-
tary Section. Comparing the cytotoxicity of the pyran derivatives
4a–f, it is obvious that the cytotoxicity of 4e the highest cytotoxic-
ity among the six compounds. The presence of the OH and the thi-
ophene groups are responsible for its high potency. Considering
the 4,5,6,7-tetrahydrobenzo[b]thiophene derivatives 5a–d, it is
clear that the cytotoxicity of 5c is higher than that of 5a, 5b and
5d. The high cytotoxicity of 5c is attributed to the presence of
the 4-OCH3 aryl moiety together with the 3-cyano group of the thi-
ophene moiety. On the other hand, considering the pyrimidine
derivatives 6a–c where compounds 6b and 6c showed high cyto-
toxicity which is attributed to the presence of the pyridyl and thio-
phenyl groups, respectively. Evaluation of the thiazolo[3,2-
a]pyrimidine derivatives 8a–d showed that the presence of the
4-chlorophenyl group in 8b and the 4-methoxyphenyl group 8d
are responsible for the high potency of these two compounds.
However, the 4-chlorophenyl derivative 4b is more potent that
8d towards HEPG2 and MCF cell lines.
713
714
3. Results and discussion
3.1. Chemistry
715
716
The present investigation emphasised mainly on two important
things, of these one is to the synthesis of heterocyclic compounds
Please cite this article in press as: Mohareb RM et al. Heterocyclic ring extension of androstenedione: Synthesis and cytotoxicity of fused pyran, pyrimidine
and thiazole derivatives. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.04.011

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bearing the androstenedione moiety and the other is to determine
their cytotoxicity against cancer and normal cell lines. Throughout
our program some of our synthesized products were good candi-
dates as target anticancer agents. The synthetic strategies adopted
for the synthesis of the intermediates and target compounds are
depicted in Schemes 1–4. Although much attention has been direc-
ted to study the biological importance of androstenedione [25–28],
no investigations have appeared in the literature to describe its
uses in one-pot investigation to form pyran and pyrimidine ring
systems. Therefore, the need to create novel androstenedione
derivatives for emerging drug targets is an active area of medicinal
chemistry. Recently, our research group was involved through a
series of heterocyclization of pregnenolone to form thiophene, pyr-
azole and pyridine derivatives as antitumor agents [29,30]. In the
present work, we studied the uses of androstenedione in heterocy-
clic chemistry through one pot reaction to form novel E-heterocy-
clic rings of androstenedione as potentially anticancer agents. The
obtained products were important in a different strategy, being
used to obtain new heterocyclic derivatives of androstenedione
together with the comparison of their cytotoxic activities towards
human cancer normal cell lines. Thus, the one pot reaction of
androstenedione (1) with either benzaldehyde (2a), pyridine-2-
aldehyde (2b) and thiophene-2-aldehyde (2c) and either malono-
nitrile (3a) or ethyl cyanoacetate (3b) gave the pyran derivatives
4a–f, respectively (Scheme 1). On the other hand, the reaction of
1 with either benzaldehyde (2a), pyridine-2-aldehyde (2b) and thi-
ophene-2-aldehyde (2c) and thiourea (5) gave the pyrimidine
derivatives 6a–c, respectively [31,32]. The analytical and spectral
data of compounds 6a–c were in agreement with their structures.
Compound 6b with its high yield encouraged us to study its reac-
tivity towards some chemical reagents. Thus, it reacted with either
2-bromo-1-phenylethanone (7a) 2-bromo-1-(4-chlorophenyl)eth-
anone (7b), 2-bromo-1-p-tolylethanone (7c) or 2-bromo-1-(4-
methoxyphenyl)ethanone (7d) to give the thiazolo[3,2-b]pyrimi-
dine derivatives 8a–d, respectively (Scheme 2). The structures of
the latter products were based on analytical and spectral data.
On the other hand, the reaction of androstenedione (1) with either
malononitrile (3a) or ethyl cyanoacetate (3b) and elemental sul-
phur gave the thiophene derivatives 10a and 10b, respectively.
The 2-amino group present in compounds 10a and 10b was capa-
ble for amide formation. Thus, the reaction of compounds 10a and
10b with either ethyl acetoacetate or diethyl malonate gave the
amide derivatives 12a–d, respectively. The elemental analyses
and spectral data were the basis of their structural elucidations.
Additionally, we focused on the azo coupling reactions of the 2-
amido derivatives 12a and 12b. Thus, the coupling reaction of
12a,b with either benzenediazonium chloride (13a), 4-meth-
ylbenzenediazonium chloride (13b) or 4-chlorobenzenediazonium
chloride (13c) gave the arylhydrazo derivatives 14a–f, respectively
(Scheme 3).
fused derivatives. The cytotoxicity of the newly synthesized prod-
ucts were evaluated against human gastric cancer (NUGC and HR),
human colon cancer (DLD1), human liver cancer (HA22T and
HEPG2), human breast cancer (MCF), nasopharyngeal carcinoma
(HONE1) and normal fibroblast cells (WI38). The results showed
that compounds 4e, 4f, 6b, 6c, 8b and 16 exhibited the optimal
cytotoxic effect against the six cancer cell lines, with IC50’s in the
nM range.
788
Appendix A. Supplementary data
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Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.steroids.
2014.04.011.
792
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Please cite this article in press as: Mohareb RM et al. Heterocyclic ring extension of androstenedione: Synthesis and cytotoxicity of fused pyran, pyrimidine
and thiazole derivatives. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.04.011