Facile synthesis and in vitro cytotoxic evaluation of novel thiadiazole, pyrazole, and dithiole-androstane derivatives

Article abstract of DOI:10.1080/10426500903008914

In the aim of identifying new steroidal cytotoxic agents with potential antiproliferative activity against hepatoma cell lines (Hep-G2), we synthesized modified steroids containing the thiadiazole, pyrazole, or dithiole moiety. Epiandrosterone 1 reacted with carbon disulfide and sodium hydride to furnish -oxoketene dithio-disodium salt 2. Treatment of 2 with the hydrazonoyl halides 5a-d produced the thiadiazole anellated androstanone 7a-d, respectively. The reaction of 1 with hydrazine hydrate produced the hydrazide adduct 8, which cyclized upon reflux in acetic acid to form the condensed pyrazoloandrostanone derivative 9. Interaction of 8 with carbon disulfide and sodium hydride formed the disodium salt 10, which reacted with ethylchloroacetate to furnish the final adduct, dithioloandrostane derivative, 13. Compounds 7a, 7d, 9, and 13 were examined for their cytotoxicity against a panel of hepatoma cell lines (Hep-G2) using MTT assay. The results provide that, at incubation time 72 h, in DMSO, compound 7d (50 mol/mL) showed the most significant cytotoxic effect at P 0.05. The higher dose (100 mol/mL) of compound 7d, at 48 h incubation, reversed the effect causing resistance and the growth rate return to the control level. Supplemental materials are available for this article. Go to the publisher's online edition of Phosphorus, Sulfur, and Silicon and the Related Elements to view the free supplemental file.

Full text of DOI:10.1080/10426500903008914

This article was downloaded by: [Florida State University]
On: 27 December 2014, At: 01:53
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Phosphorus, Sulfur, and Silicon and the
Related Elements
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/gpss20
Facile Synthesis and in Vitro Cytotoxic
Evaluation of Novel Thiadiazole,
Pyrazole, and Dithiole-Androstane
Derivatives
Nadia R. Mohamed a , Gamal A. Elmegeed b , Mervat M. Abdelhalim b
& Hanaa M. Rady c
a Photochemistry Department , National Research Center , Dokki,
Giza, Egypt
b Hormones Department , National Research Center , Dokki, Giza,
Egypt
c Chemistry of Natural Compounds Department , National Research
Center , Dokki, Giza, Egypt
Published online: 23 Mar 2010.
To cite this article: Nadia R. Mohamed , Gamal A. Elmegeed , Mervat M. Abdelhalim & Hanaa M. Rady
(2010) Facile Synthesis and in Vitro Cytotoxic Evaluation of Novel Thiadiazole, Pyrazole, and Dithiole-
Androstane Derivatives, Phosphorus, Sulfur, and Silicon and the Related Elements, 185:4, 848-856,
DOI: 10.1080/10426500903008914
To link to this article: http://dx.doi.org/10.1080/10426500903008914
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Phosphorus, Sulfur, and Silicon, 185:848–856, 2010
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500903008914
FACILE SYNTHESIS AND IN VITRO CYTOTOXIC
EVALUATION OF NOVEL THIADIAZOLE, PYRAZOLE,
AND DITHIOLE-ANDROSTANE DERIVATIVES
Nadia R. Mohamed,1 Gamal A. Elmegeed,2
Mervat M. Abdelhalim,2 and Hanaa M. Rady3
1Photochemistry Department, National Research Center, Dokki, Giza, Egypt
2Hormones Department, National Research Center, Dokki, Giza, Egypt
3Chemistry of Natural Compounds Department, National Research Center, Dokki,
Giza, Egypt
In the aim of identifying new steroidal cytotoxic agents with potential antiproliferative ac-
tivity against hepatoma cell lines (Hep-G2), we synthesized modified steroids containing the
thiadiazole, pyrazole, or dithiole moiety. Epiandrosterone 1 reacted with carbon disulfide
and sodium hydride to furnish α-oxoketene dithio-disodium salt 2. Treatment of 2 with the
hydrazonoyl halides 5a–d produced the thiadiazole anellated androstanone 7a–d, respec-
tively. The reaction of 1 with hydrazine hydrate produced the hydrazide adduct 8, which
cyclized upon reflux in acetic acid to form the condensed pyrazoloandrostanone derivative 9.
Interaction of 8 with carbon disulfide and sodium hydride formed the disodium salt 10, which
reacted with ethylchloroacetate to furnish the final adduct, dithioloandrostane derivative,
13. Compounds 7a, 7d, 9, and 13 were examined for their cytotoxicity against a panel of
hepatoma cell lines (Hep-G2) using MTT assay. The results provide that, at incubation time
72 h, in DMSO, compound 7d (50 µ mol/mL) showed the most significant cytotoxic effect at
P < 0.05. The higher dose (100 µ mol/mL) of compound 7d, at 48 h incubation, reversed the
effect causing resistance and the growth rate return to the control level.
Supplemental materials are available for this article. Go to the publisher’s online edition of
Phosphorus, Sulfur, and Silicon and the Related Elements to view the free supplemental
file.
Keywords Cytotoxicity; hepatoma; hydrazonoyl halides; pyrazole; steroids; thiadiazole
INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the most common malignancies world-
wide. It accounts for about 6% of all human cancers annually.1 Because of the multifocal
nature of liver carcinoma, most cancer patients are considered non-resectable. In these
patients, chemotherapy is the only choice of treatment. Unfortunately, the development of
drug resistance in the tumor after treatment is always a major obstacle to the successful
Received 24 February 2009; accepted 30 April 2009.
Address correspondence to Nadia R. Mohamed, Photochemistry Department, National Research Center
12622, Dokki, Giza, Egypt. E-mail: nadia ragab m@hotmail.com
848

THIADIAZOLE, PYRAZOLE, DITHIOLE-ANDROSTANE DERIVATIVES
849
management of liver cancer.2 Thus, developing new therapeutic agents that can overcome
drug resistance has become an urgent need for cancer patients.
Hybrid anticancer agents, which combine two active compounds in one, such as
steroidal alkylators, contain the steroidal moiety as biological vectors for cytotoxic agents
in order to diminish toxicity and to enhance specificity.3 These agents attain a duplicate
effect on cancer cells. A variety of steroids with unusual and interesting structures has been
synthesized and evaluated for their antitumor activity.4–6 In addition, the antitumor activ-
ities of many compounds containing a heterocyclic ring have been reviewed.7,8 Pyrazole,
thiadiazole, and dithiole rings represent molecular frameworks that serve as a platform for
developing pharmaceutical agents for various applications. Many derivatives of pyrazole,
thiadiazole, and dithiole have been proven as antitumor agents.9–11
In view of the above facts, we set a goal to prepare some new steroid derivatives by
combining them with a heterocyclic moiety of potent cytotoxic activity. In continuation of
our investigation of new aza and thia steroids and their condensed heterocyclic derivatives as
biologically active molecules,12–14 we report in this article the facile synthesis of some new
thiol, thiadiazole, pyrazole, and dithiole steroid derivatives. Furthermore, some compounds
were examined for their cytotoxicity against a panel of hepatoma cell lines (Hep-G2) using
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay.
RESULTS AND DISCUSSION
Chemistry
In a recent communication from this laboratory, we have reported the synthesis
of the non-isolable α-oxoketen dithio-disodium salt 2 via the reaction of 3β-hydroxy-
5α-androstan-3-one (epiandrosterone) 1 with carbon disulfide and 2 equivalent moles of
sodium hydride in dimethyl sulfoxide (DMSO) at 0◦C.13 The addition of the hydrazonoyl
halide, c-benzoyl-N-(4-chlorophenyl)hydrazonoyl chloride (3a) to the non-isolable salt 2
produced the corresponding androstane thiol deravative 4a (Scheme 1). The structure of 4a
was supported with the spectroscopic and microanalytical data. The 1H NMR spectrum of
structure 4a revealed beside the characteristic signals of androstane moiety, a singlet at δ =
1.23 ppm for the (SH), two multiplets at 7.20–7.60 and 7.67–7.90 ppm for the aromatic
protons, and two singlets at 11.30 and 11.80 for the (OH) and (NH) groups, respectively.
To generalize such a methodology, the previous reaction was carried out by the utility of
the hydrazonoyl halides 3b–d to form the corresponding androstan-ethanhydrazonothioate
derivatives 4b–d, respectively. All the spectroscopic and microanalytical data of compounds
4b–d were in accordance with the suggested structures (see the Materials and Methods
section). Many trials to cyclize compounds 4a–d under different conditions failed.
Carrying out the previous reaction with other types of hydrazonoyl halides including
electron-rich alkyl groups or a thiophene ring led to the formation of the corresponding
thiadiazole steroidal derivatives 7a–d, respectively (Scheme 2). The 1H NMR of the sug-
gested structures 7a–d reflect the disappearance of the characteristic signals of the (SH)
1
and (NH) groups. For example, the H NMR spectrum of 7a showed the appearance of
singlet at δ = 2.63 ppm for (CH3) group, multiplet at δ = 7.10–7.78 ppm for the aromatic
protons and a singlet at 11.58 for the (OH) group (see the Materials and Methods section).
With the aim to obtain the pyrazolo anellated androstane derivative, the 17-
hydrazonoandrostane derivative 8 was synthesized via the reaction of epiandrosterone 1

850
N. R. MOHAMED ET AL.
O
O
SNa
SNa
CS2
2NaH/ DMSO
HO
HO
2
1
COR
O
SC=N-NHAr
Cl
SH
RCO-C=N-NHAr
3a-d
HO
4a-d
3, 4
a
b
R
Ph
Ph
Ar
C6H4-Cl (P)
Ph
c
d
naphthyl
PhNH
Ph
C6H4-CH3 (P)
Scheme 1 Synthesis of compounds 4a–d.
with hydrazine hydrate under reflux.15 The pyrazoloandrostane derivative 9 was obtained
through the cyclization of compound 8 by heating in acetic acid under reflux (Scheme 3).
Interaction of hydrazonoandrostane derivative 8 with carbon disulfide and 2 equiva-
lent moles of sodium hydride in DMSO at 0◦C furnished the non-isolable dithio-disodium
O
COR
Cl
S
C
R-COC=N-NHAr
N
SH
2
NH Ar
5a-d
HO
6
COR
O
S
N
-H2S
N
Ar
HO
7a-d
5,7
a
R
CH3
Ar
Ph
b
CH3
C6H4-CH3 (P)
c
Ph
S
S
d
C6H4-Cl (P)
Scheme 2 Synthesis of compounds 7a–d.

THIADIAZOLE, PYRAZOLE, DITHIOLE-ANDROSTANE DERIVATIVES
851
O
N
NH2
NH2NH2
N
+
HO
HO
8
1
N
CH3
CH3COOH
HO
9
Scheme 3 Synthesis of compound 9.
salt 10. Addition of ethylchloroacetate to the non-isolable salt 10 yielded the corresponding
intermediate 11, which cyclized via the elimination of ethanol to give the dithioloandrostane
derivative 12 (Scheme 4). The structure of compound 12 was proved to isomerize to the
final form 13, since, in addition to the absence of CO group stretching in the IR spectrum,
the 1H NMR spectrum of the product revealed two singlets at 10.63 and 10.87 ppm for the
two OH groups.
SNa
N
NH2
N
N
SNa
2NaH
CS2
+
DMSO
HO
HO
8
10
ClCH2CO2Et
SCH2COOEt
SH
N
N
HO
11
-EtOH
S
S
S
N
N
N N
S
O
OH
HO
HO
12
13
Scheme 4 Synthesis of compound 13.

852
N. R. MOHAMED ET AL.
In Vitro Cytotoxic Activity
The cytotoxic effect of the tested compounds 7a, 7d, 9, and 13, against hepatoma
cell line (HepG2), was performed using the MTT assay, which is widely used as a screen-
ing method to measure cell viability and proliferation.16 The results are discussed in the
Supplemental Materials (available online, Figures S1 and S2)
There have been reported data that steroids at low concentrations exhibited increased
absorbance values in the MTT test over the control that could indicate a stimulating effect
of these compounds on cell proliferation and mitochondrial hydrogenase activity.17
CONCLUSION
In this study, we have described a straightforward and efficient synthesis of novel
steroid derivatives containing thiadiazole, pyrazole, or dithiole nucleus. We investigated
also the importance of incorporating heterocyclic moiety to the steroid nucleus to form
new effective anticancer hybrid molecules. The androstan-16-[(thiophene-2ꢁꢁ-carbonyl)-
3H-[1,3,4]thiadiazol-2ꢁ-ylidene] derivative 7d was the more potent cytotoxic agent against
hepatoma cell line (HepG2).
EXPERIMENTAL
The starting steroid (epiandrosterone) was purchased from Sigma Company, USA. All
anhydrous reactions were carried out under argon atmosphere, and solvents were dried by
distillation prior to use. All melting points were measured using an electrothermal apparatus
and are uncorrected. Hydrazonoyl halides were recrystallized and dried before use. The
IR spectra were recorded in (KBr discs) on a Shimadzu FT-IR 8201 PC spectrometer and
expressed in cm−1. The 1H NMR and 13C NMR spectra were recorded with a Jeol instrument
(Japan), at 270 and 125 MHz respectively, in DMSO-d6 or CDCl3 as solvent, and chemical
shifts were recorded in ppm relative to TMS. Mass spectra were recorded on a GCMS-QP
1000 Ex spectra mass spectrometer operating at 70 eV. Elemental analyses were carried out
by the Microanalytical Data Unit at National Research Center, Giza, Egypt. Reactions were
monitored on Merck aluminum thin layer chromatography (TLC) plates and visualized by
UV light (254 nm). For the nomenclature of steroid derivatives, we used the definitive rules
for the nomenclature of steroids published by the Joint Commission on the Biochemical
Nomenclature (JCBN) of IUPAC.18,19 The 1H NMR spectra of all compounds showed the
published characteristic data of the androstane series.20,21 The starting compound 8 was
prepared according to the published procedure.15
Synthesis of Compounds 4a–d and 7a–d
To a stirred solution of epiandrosetrone 1 (1.45 g, 5 mmol) in anhydrous DMSO
(30 mL), sodium hydride (60% suspension in mineral oil, 0.24 g, 10 mmol) and carbon
disulfide (0.38 g, 5 mmol) were added. The reaction mixture was stirred for 3 h at 0◦C, and
then the appropriate hydrazonoyl halide 3a–d (5 mmol) was added dropwise with stirring.
After completion of the addition, the mixture was stirred for further 3 h, left overnight at
0◦C, poured into an ice-water mixture, and finally the resulting solid product was collected
by filtration and crystallized from the appropriate solvent.

THIADIAZOLE, PYRAZOLE, DITHIOLE-ANDROSTANE DERIVATIVES
853
3β-Hydroxy-5α-androstan-16-ylidene-[(sulfanyl)methyl-(2ꢀ-oxo-2ꢀ-phenyl-
N-(p-chlorophenyl)ethanehydrazonothioate)]-17-one (4a). Orange crystals from
ethanol, yield 68% (2.12 g), mp 234–235◦C. IR (KBr, cm−1): ν = 3469–3340 (OH, NH),
2929, 2828 (CH3, CH2), 2590 (SH), 1729, 1685 (2C O), 1642 (C C); 1H NMR (DMSO,
ppm): δ = 0.81 (s, 3H, CH3–19), 1.08 (s, 3H, CH3–18), 1.23 (s, 1H, SH), 7.22–7.60 (m, 5H,
aromatic protons), 7.67–7.90 (m, 4H, aromatic protons), 11.30 (s, 1H, OH), 11.80 (s, 1H,
NH);. MS (EI): m/z (%): 622 (M+., 30), 105 (C6H5CO, 100); Calc for C34H38O3N2S2Cl
(622.27): C, 65.62; H, 6.15; N, 4.50; S, 10.30; Found: C, 65.43; H, 6.08; N, 4.63; S,
10.16%.
3β-Hydroxy-5α-androstan-16-ylidene[(sulfanyl)methyl-(2ꢀ-oxo-N,2ꢀ-diph
enyl-ethanhydrazonothioate)]-17-one (4b). Yellow crystals from ethanol, yield
78% (2.28 g), mp 196–197◦C; IR (KBr, cm−1): ν = 3480–3350 (OH, NH), 2997, 2950
(CH3, CH2), 2587 (SH), 1719, 1676 (2C O), 1649 (C C); 1H NMR (CDCl3, ppm): δ =
0.78 (s, 3H, CH3–19), 1.05 (s, 3H, CH3–18), 1.22 (s, 1H, SH), 7.24–7.30 (m, 5H, aromatic
protons), 7.45–7.95 (m, 5H, aromatic protons), 11.40 (s, 1H, OH), 12.14 (s, 1H, NH); MS
(EI): m/z (%): 586 (M+.−1, 37.2), 105 (C6H5CO, 100); Calc for C34H39O3N2S2 (587.83):
C, 69.74; H, 6.63; N, 4.76; S, 10.91; Found: C, 69.43; H, 6.48; N, 4.93; S, 11.12%.
3β-Hydroxy-5α-androstan-16-ylidene[(sulfanyl)methyl-(2ꢀ-naphthyl-2ꢀ-
oxo-N-phenylethanhydrazonothioate)]-17-one (4c). Red crystals from methanol,
yield 68% (2.2 g), mp 68–70◦C; IR (KBr, cm−1): ν = 4780–3420 (OH, NH), 2954, 2890
(CH3, CH2), 2592 (SH), 1720, 1678 (2C O), 1646 (C C); 1H NMR (DMSO, ppm): δ =
0.81 (s, 3H, CH3–19), 1.19 (s, 3H, CH3–18), 1.23 (s, 1H, SH), 7.02–7.32 (m, 5H, aromatic
protons), 7.59–8.07 (m, 6H, naphthyl protons), 8.50 (s, 1H, naphthyl proton), 11.49 (s, 1H,
OH), 12.20 (s, 1H, NH). MS (EI): m/z (%): 638 (M+., 23), 154 [(naphthyl-CO)-1, 100],
77 (C6H5, 37.5); Calc for C38H42O3N2S2 (638.90): C, 71.43; H, 6.62; N, 4.38; S, 10.03;
Found: C, 71.65; H, 6.43; N, 4.62; S, 10.21%.
3β-Hydroxy-5α-androstan-16-ylidene[(sulfanyl)methyl-(2ꢀ-oxo-2ꢀ-phenyl
imino-N-(p-methylphenyl)ethanhydrazonothioate)]-17-one (4d). Yellow crys-
tals from ethanol, yield 84% (2.52 g), mp 75–77◦C; IR (KBr, cm−1): ν = 3468–3360 (OH,
1
NH), 2928, 2866 (CH3, CH2), 2590 (SH), 1731, 1677 (2C O), 1642 (C C); H NMR
(CDCl3, ppm): δ = 0.90 (s, 3H, CH3–19),1.17 (s, 3H, CH3–18), 1.24 (s, 1H, SH), 2.61
(s, 3H, CH3), 7.18–7.37 (m, 5H, aromatic protons), 7.59–7.63 (m, 4H, aromatic protons),
11.73 (s, 1H, OH); MS (EI): m/z (%): 602 (M+., 28), 120 (C6H5NHCO, 100); Calc for
C34H40O3N3S2 (602.84): C, 67.79; H, 6.68; N, 6.47; S, 10.63; Found: C, 65.43; H, 6.08;
N, 4.63; S, 10.16%.
3β-Hydroxy-5α-androstan-16-(5ꢀ-acetyl-3ꢀ-phenyl-3ꢀH-[1ꢀ,3ꢀ,4ꢀ]thiadiazol-
2ꢀ-ylidene)-17-one (7a). Pale brown crystals from 1,4-dioxan, yield 73% (1.79 g), mp
135–137◦C; IR (KBr, cm−1): ν = 3468–3248 (OH, NH), 2931, 2860 (CH3, CH2), 1728,
1
1684 (2C O), 1648 (C C); H NMR (CDCl3, ppm): δ = 0.79 (s, 3H, CH3–19),1.10
(s, 3H, CH3–18), 2.63 (s, 3H, CH3), 7.10–7.78 (m, 5H, aromatic protons), 11.58 (s, 1H,
OH); 13C NMR (DMSO-d6, ppm). δ = 27.8 (C-1), 30.7 (C-2), 67.9 (C-3), 33.7 (C-4),
36.1 (C-5), 25.4 (C-6), 25.9 (C-7), 29.6 (C-8), 43.4 (C-9), 37.6 (C-10), 21.1 (C-11), 27.5
(C-12), 54.3 (C-13),39.3 (C-14), 11.3 (C-15), 126.9 (C-16), 202.4 (C-17), 17.2 (C-18),
21.2 (C-19), 136.2 (C-2ꢁ), 152.8 (C-5ꢁ), 200.3 (C O), 18.0 (CH3-acetyl), 146.0, 116.5,
129.1, 117.8 (C-phenyl). MS (EI): m/z (%): 491 (M+.−1, 32.7), 288 (C19H28O2, 100);
Calc for C29H36O3N2S (492.68): C, 70.69; H, 7.36; N, 5.68; S, 6.50; Found: C, 65.43; H,
6.08; N, 4.63; S, 10.16%.

854
N. R. MOHAMED ET AL.
3β-Hydroxy-5α-androstan-16-(5ꢀ-acetyl-3ꢀ-(p-chlorophenyl)-3ꢀH-[1ꢀ,3ꢀ,4ꢀ]
thiadiazol-2ꢀ-ylidene)-17-one (7b). Yellow crystals from ethanol, yield 67% (1.69 g),
mp 198–200◦C; IR (KBr, cm−1): ν = 3405 (OH), 3023, 2920 (CH3, CH2), 1704, 1678
1
(2C O), 1640 (C C); H NMR (CDCl3, ppm): δ = 0.82 (s, 3H, CH3–19), 1.08 (s, 3H,
CH3–18), 2.20 (s, 3H, CH3), 2.52 (s, 3H, CH3), 7.17 (d, 2H, aromatic protons), 7.34 (d, 2H,
aromatic protons), 11.45 (s, 1H, OH), MS (EI): m/z (%): 506 (M+., 47), 255 (C19H27,100);
Calc for C30H38O3N2S (506.71): C, 71.42; H, 7.55; N, 5.52; S, 6.32; Found: C, 65.43; H,
6.08; N, 4.63; S, 10.16%.
3β-Hydroxy-5α-androstan-16-(3ꢀ-phenyl-5ꢀ-(thiophene-2ꢀꢀ-yl-methanone)-
3ꢀH-[1ꢀ,3ꢀ,4ꢀ]thiadiazol-2ꢀ-ylidene)-17-one (7c). Pale yellow crystals from methanol,
yield 67% (1.87 g), mp 180–181◦C; IR (KBr, cm−1): ν = 3400 (OH), 2985, 2957 (CH3,
1
CH2), 1716,1678 (2C O), 1649 (C C); H NMR (CDCl3, ppm): δ = 0.76 (s, 3H,
CH3–19), 1.07 (s, 3H, CH3–18), 7.05–7.25 (m, 2H, aromatic protons), 7.40–7.60 (m, 3H,
thiophene protons), 7.80–7.95 (m, 3H, aromatic protons), 11.45 (s, 1H, OH);); 13C NMR
(DMSO-d6, ppm). δ = 27.2 (C-1), 31.2 (C-2), 67.5 (C-3), 34.7 (C-4), 37.0 (C-5), 25.5
(C-6), 26.3 (C-7), 29.0 (C-8), 42.8 (C-9), 37.4 (C-10), 20.4 (C-11), 28.0 (C-12), 55.6
(C-13), 39.8 (C-14), 12.6 (C-15), 125.0 (C-16), 203.6 (C-17), 16.5 (C-18), 20.5 (C-19),
136.0 (C-2ꢁ), 154.4 (C-5ꢁ), 184.3 (C O), 145.7, 137.6, 129.5, 135.9 (C-thiophene), 146.2,
115.3, 129.4, 118.3 (C-phenyl); MS (EI): m/z (%): 560 (M+., 48.3), 111 [thiophen-CO
(C5H3SO) 100]; Calc for C32H36O3N2S2 (560.78): C, 68.53; H, 6.47; N, 4.99; S, 11.43;
Found: C, 68.74; H, 6.25; N, 4.73; S, 11.19%.
3β-Hydroxy-5α-androstan-16-{3ꢀ-(p-chlorophenyl)-5ꢀ-(thiophene-2ꢀꢀ-yl-
methanone)-3ꢀH-[1ꢀ,3ꢀ,4ꢀ]thiadiazol-2ꢀ-ylidene}-17-one (7d). Orange crystals
from ethanol, yield 70% (2.28 g), mp 248–250◦C; IR (KBr, cm−1): ν = 3405 (OH), 2985,
2978 (CH3, CH2), 1716,1678 (2C O); 1645 (C C); 1H NMR (DMSO): δ = 0.78 (s, 3H,
CH3–19),1.03 (s, 3H, CH3–18), 7.14–7.27 (m, 2H, aromatic protons), 7.42–7.56 (m, 3H,
thiophene protons), 7.95–8.04 (m, 2H, aromatic protons), 11.50 (s, 1H, OH); 13C NMR
(DMSO-d6, ppm). δ = 26.2 (C-1), 31.5 (C-2), 66.9 (C-3), 33.7 (C-4), 37.4 (C-5), 25.9
(C-6), 26.3 (C-7), 29.5 (C-8), 42.6 (C-9), 37.4 (C-10), 20.0 (C-11), 28.6 (C-12), 56.4
(C-13), 39.3 (C-14), 12.8 (C-15), 124.6 (C-16), 201.6 (C-17), 17.5 (C-18), 21.5 (C-19),
135.3 (C-2ꢁ), 154.6 (C-5ꢁ), 185.2 (C O), 145.6, 137.9, 128.5, 136.4 (C-thiophene), 144.2,
116.3, 129.6, 123.0 (C-phenyl); MS (EI): m/z (%): 595 (M+., 58), 111 [thiophen-CO
(C5H3SO) 100]; Calc for C32H35O3N2S2Cl (595.23): C, 64.57; H, 5.92; N, 4.70; S, 10.77;
Found: C, 65.43; H, 6.08; N, 4.63; S, 10.16%.
Synthesis of 3β-Hydroxy-5ꢀ-methyl-5α-androstan[16,17:3ꢀ,4 ꢀ]pyrazole
(9)
To 17-hydrazonoandrostane derivative 8 (1.52 g, 5 mmol), 20 mL of glacial acetic
acid was added dropwise with stirring. The reaction was heated under reflux for 3 h until
all the starting materials had disappeared as indicated by TLC, and then poured into an
ice/water mixture (60 mL). The solid product that formed was collected by filtration, dried,
and crystallized from ethanol to give compound 9, pale brown crystals, yield 73% (1.19 g),
mp 60–62◦C; IR (KBr, cm−1): ν = 3450 (OH), 2942, 2851 (CH3, CH2); 1H NMR (DMSO):
δ = 0.78 (s, 3H, CH3–19),1.03 (s, 3H, CH3–18), 2.80 (s, 3H, CH3); 13C NMR (DMSO-d6,
ppm). δ = 27.4 (C-1), 30.2 (C-2), 67.0 (C-3), 34.9 (C-4), 36.8 (C-5), 25.3 (C-6), 25.9 (C-7),
29.4 (C-8), 42.5 (C-9), 39.0 (C-10), 22.4 (C-11), 27.6 (C-12), 45.6 (C-13), 43.8 (C-14),

THIADIAZOLE, PYRAZOLE, DITHIOLE-ANDROSTANE DERIVATIVES
855
25.6 (C-15), 34.0 (C-16), 167.2 (C-17), 16.3 (C-18), 21.2 (C-19), 164.0 (C-5ꢁ); MS (EI):
m/z (%): 328 (M+., 45.6), 55 (C2H3N2, 100); Calc for C21H32ON2 (328.50): C, 76.78; H,
9.81; N, 8.52; Found: C, 77.01; H, 9.67; N, 8.73%.
Synthesis of 3β-Hydroxy-5α-androstan-17-hydrazono-N-[(1ꢀ,3ꢀ)dithiol-
2ꢀ-ylidene]-4ꢀ-ol (13)
Sodium hydride(60% suspension inmineral oil, 0.24g, 10mmol) and carbondisulfide
(0.38 g, 5 mmol) were added to a stirred solution of 17-hydrazonoandrostane derivative 8
(1.52 g, 5 mmol) in anhydrous DMSO (40 mL). The reaction mixture was stirred for 4 h at
0◦C, and then ethyl chloroacetate (0.62 g, 5 mmol) was added dropwise with stirring. After
completion of the addition, the reaction mixture was stirred for further 2 h, left overnight
at 0◦C, poured into an ice/water mixture, and the resulting solid product was collected
by filtration and crystallized from ethanol to yield 66% (1.38 g) of dithioloandrostane
derivative 13, yellow crystals, mp 195–197◦C; IR (KBr, cm−1): ν = 3393 (OH), 2927,
2856 (CH3, CH2), 1649 (C C); 1H NMR (DMSO): δ = 0.78 (s, 3H, CH3–19), 1.12 (s, 3H,
CH3–18), 10.63, 10.87 (2s, 2H, 2OH); 13C NMR (DMSO-d6, ppm). δ = 28.0 (C-1), 30.2
(C-2), 68.0 (C-3), 34.5 (C-4), 36.7 (C-5), 25.4 (C-6), 26.7 (C-7), 29.4 (C-8), 43.1 (C-9),
38.6 (C-10), 20.8 (C-11), 27.5 (C-12), 42.5 (C-13), 46.8 (C-14), 26.6 (C-15), 23.8 (C-16),
165.6 (C-17), 16.8 (C-18), 21.0 (C-19), 163.4 (C-2ꢁ), 151.7 (C-4ꢁ), 87.3 (C-5ꢁ); MS (EI):
m/z (%): 420 (M+., 38.7), 255 (C19H27,100); Calc for C22H32O2N2S2 (420.64): C, 62.81;
H, 7.66; N, 6.65; S, 15.24; Found: C, 63.07; H, 7.85; N, 6.83; S, 15.47%.
In Vitro Cytotoxic Activity
The Supplemental Materials (available online) detail the materials and methods used
for the cytotoxic activity testing.22
Statistics
Statistical evaluation of the results was done with the analysis of variance (ANOVA)
using the MSTATC program.23 When significant differences (P < 0.05) were detected, the
least significant difference (LSD) test was used to separate the main values.24
REFERENCES
1. A. K. Nowak, P. K. Chow, and M. Findlay, Eur. J. Cancer, 40(10), 1474 (2004).
2. T.-H. Wu, R.-L. Yang, L.-P. Xie, H.-Z. Wang, L. Chen, S. Zhang, Y. Zhao, and R.-Q. Zhang,
Cancer Lett., 232, 199 (2006).
3. D. T. Trafalis, G. D. Geromichalos, C. Koukoulitsa, A. Papageorgiou, P. Karamanakos, and C.
Camoutsis, Breast Cancer Res. Treat., 97, 17 (2006).
4. D. T. Trafalis, G. D. Geromichalos, N. Bountouroglou, D. Koumbi, M. Kontos, D. Sougias, P.
Dalezis, P. Karamanakos, A. Papageorgiou, C. Camoutsis, and A. E. Athanassiou, J. Buon, 10(2),
227 (2005).
5. D. Thibeault, J. Roy, P. De Roy, and D. Poirier, Bioorg. Med. Chem., 16, 5062 (2008).
ˇ
6. E. A. Djurendic´, M. N. Sakacˇ, M. P. Zavisˇ, A. R. Gakovic´, J. J. Canadi, S. A. Andric´, O. R.
Klisuric´, V. V. Kojic´, G. M. Bogdanovic´, and K. M. Penov Gasˇi, Steroids, 73, 681 (2008).
7. M. N. Preobrazhenskaya, Chem. Heterocycl. Comp., 21, 13 (1985).

856
N. R. MOHAMED ET AL.
8. M. Kidwai, R. Venkataramanan, R. Mohan, and P. Sapra, Curr. Med. Chem., 9(12), 1209 (2002).
9. S. A. F. Rostom, M. A. Shalaby, and M. A. El-Demellawy, Eur. J. Med. Chem., 38, 959 (2003).
10. J. Matysiaka and A. Opolski, Bioorg. Med. Chem., 14, 4483 (2006).
11. M. K. Paul, R. Kumar, and A. K. Mukhopadhyay, Toxicol. Appl. Pharmacol., 226, 140 (2008).
12. N. R. Mohamed, G. A. Elmegeed, H. A. Abd-ElMalek, and M. Younis, Steroids, 70, 131 (2005).
13. M. M. Abdelhalim, M. M. T. El-Saidi, S. T. Rabee, and G. A. Elmegeed, Steroids, 72, 459 (2007).
14. H. Y. Hana, W. K. B. Khalil, A. I. Elmakawy, and G. A. Elmegeed, J. Steroid Biochem. Molecul.
Biol., 110, 248 (2008).
15. M. M. Abdelhalim, Bull. Fac. Pharm. Cario Univ., 45, 27 (2007).
16. T. Mosmann, J. Immunol. Meth., 65, 55 (1983).
17. A. Y. Misharin, A. R. Mehtiev, G. E. Morozevich, Y. V. Tkachev, and V. P. Timofeev, Bioorg.
Med. Chem., 16, 1460 (2008).
18. IUPAC, Joint Commission on Biochemical Nomenclature (JCBN), Pure Appl. Chem., 61, 1783
(1989).
19. IUPAC, Joint Commission on Biochemical Nomenclature (JCBN), Eur. J. Biochem., 186, 429
(1989).
20. E. Gacs-Baitz, L. Minuti, and A. Taticchi, J. Chem. Res., Synop., 7, 324 (1996).
21. N. S. Bhacca and D. H. William, Application of NMR Spectroscopy in Organic Chemistry (Holden
Day, San Fransisco, CA, 1964).
22. T. L. Jia, H. Z. Wang, L. P. Xie, X. Y. Wang, and R. Q. Zhang, Biochem. Pharmacol., 65, 709
(2003).
23. G. W. Snedecor and W. G. Cochran, Statistical methods, 6th ed. (Iowa State Univ. Press, Iowa,
USA, 1980), p. 593.
24. R. G. D. Steel and J. H. Torrie, Principles and Procedures of Statistics: A Biometrical Approach,
2nd ed. (McGraw-Hill, New York, 1981).