Synthesis and biological evaluation of some nitrogen containing steroidal heterocycles

Article abstract of DOI:10.1016/j.steroids.2010.09.001

epi-Androsterone 1 was converted into its hydrazone derivative through the reaction with hydrazine hydrate 80%. Hydrazonoandrostane derivative 2b reacted with hydrazonoyl halides in the presence of K₂CO₃ forming the corresponding hyd

Full text of DOI:10.1016/j.steroids.2010.09.001

Steroids 76 (2011) 78–84
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and biological evaluation of some nitrogen containing steroidal
heterocycles
Mervat M. Abdelhalima, Eman M. Kamelb, Samira T. Rabieb, Nadia R. Mohamedc,∗
a Hormones Department, National Research Centre, Dokki, Giza, Egypt
b Photochemistry Department, National Research Centre, Dokki, Giza, Egypt
c Chemistry Department, El-Aflaj Girls College, El-Kharj University Kingdom of Saudi Arabia, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
epi-Androsterone 1 was converted into its hydrazone derivative through the reaction with hydrazine
hydrate 80%. Hydrazonoandrostane derivative 2b reacted with hydrazonoyl halides in the presence of
K2CO3 forming the corresponding hydrazopyridazinoandrostane derivatives 6a–d. The 3␤-acetyl-17-
hydrazonoandrostane derivative 2b reacted with a halogen reagent, benzoyl chloride, to form the non-
cyclic 16-benzoylated hydrazone 9.
Received 15 December 2009
Received in revised form 4 September 2010
Accepted 7 September 2010
Available online 16 September 2010
On the other hand, compound 2b produced the corresponding pyridazinoandrostane derivatives 11
and 12 via its reaction with phenacyl bromide and chloroacetone respectively. Reaction of the hydrazono
derivative 2b with benzaldehyde in the presence of acetic acid drops led to the formation of the benzyli-
denehydrazonoandrostane derivative 13. The product 14, phosphinom-ethylenehydrazonoandrostane
was obtained by the reaction of the derivative 13 with trisdimethylaminophosphine in the presence of
dry benzene. The reaction of compound 2b with phenyl isothiocyanate followed by boiling in chloroacetic
acid or thioglycolic acid produced the pyrazoloandrostane derivatives 17 and 18 respectively. The bio-
logical activity of compounds 6a, 6d, 11, 12, and 15 was evaluated as inhibitor of growth in a human liver
carcinoma cell line and doxorubicine was used for comparison. Compounds 15 and 12 showed a higher
potency than the other tested compounds.
Keywords:
epi-Androsterone
Hydrazonoandrostane
Diazaandrostane
Pyridazinoandrostane
Hepatocelluar carcinoma (HCC)
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
decreased toxicity by increasing specificity [8]. A variety of steroids
with unusual and interesting structures have been synthesized and
Nitrogen containing steroid derivatives are common clinical
drugs in cancer chemotherapy [1,2]. Like almost all anticancer
drugs, their clinical use has been limited by the toxicity to nor-
mal tissues [3]. So, it is an important goal of cancer chemotherapy
to amplify the selective inhibition of tumor cells while decreas-
ing toxicity to normal tissues [4]. One approach for improving
the selectivity is fusing different nitrogen containing heterocycles
attached to the steroidal core. The formed cytotoxic adduct can tar-
get specific tumors. Many previous studies deal with the interaction
between steroids and their receptors in controlling the growth of
cancer cells [5]. Steroids can be used in steroid hormone depen-
dent tumors as carriers for cytotoxic war-heads [6]. On the other
hand, hepatocelluar carcinoma (HCC) is one of the most common
and dangerous types of cancer. It is the third leading cause of cancer
death [7]. Similarto otherhybrid anticanceragents, the heterocyclic
steroids presented include two principles in one molecule. Other
heterocycles have been investigated as biological vectors aiming at
evaluated for their antitumor activity [9,10]. Also, the antitumor
activity of many nitrogen containing steroid derivatives has been
reviewed [11]. Based on published results and in continuation of
our own work on nitrogen containing heterocyclic steroids [12,13],
we present here the synthesis of respective novel compounds and
their evaluation in a human liver carcinoma cell line.
2. Experimental
epi-Androsterone was purchased from Sigma Company, USA.
The appropriate precautions in handling moisture sensitive com-
pounds were undertaken. All melting points were measured using
an electrothermal apparatus in an open capillary tube and were cor-
rected. IR spectra expressed in (cm−1) were recorded in KBr pellets
on a Shimadzu FT-IR 8201 PC. Spectrophotometer. 1H NMR and 13
C
NMR spectra were recorded with JEOL 270 MHz (Japan) in DMSO-d6
as a solvent and the chemical shifts were recorded in ppm relative
to (TMS) as an internal standard. Mass spectra were recorded on
GCMS-QP 1000 ex spectra mass spectrometer operating at 70 eV.
Elemental analyses were carried out by the microanalytical unit at
the National Research Centre, Giza, Egypt.
∗
Corresponding author.
E-mail address: nadia ragab m@yahoo.com (N.R. Mohamed).
0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.09.001

M.M. Abdelhalim et al. / Steroids 76 (2011) 78–84
79
All described compounds showed the characteristic spectral
data of the androstane series skeleton, as shown in the previous
report [14]. 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 [15]. 3␤-Acetoxy-17-hydrazono-5␣-androstane derivative
2b was synthesized according to the previously published proce-
dure [16].
m/z: 522 (M+). Calc for C30H39N4O2Cl (522.5): C, 68.89%; H, 7.46%;
N, 10.71%; Cl, 6.79%. Found: C, 68.70%; H, 7.32%; N, 10.52%; Cl, 6.56%.
2.3. Reaction of hydrazono derivative 6a–d with halogenated
reagents
2.3.1. General procedure
An appropriate halogen reagent (0.005 ml) was added to the
ethanolic solution of compound 2b (1.73 g, 0.005 mol) in the pres-
ence of 3 ml piperidine. The reaction mixture was heated under
reflux about 3–5 h. The reaction mixture was triturated with cold
water. The resulting solid product was collected by filtration and
crystallized using ethanol.
2.1. 3ˇ-Acetoxy-17-hydrazono-5˛-androstane 2b
White crystals. Yield (1.12 g, 64%); mp 279280 ◦C. IR (KBr, cm−1
)
3480 (NH2); 2885 (C–H, aliphatic); 1720 (C O); 1670 (C N). 1H
NMR (DMSO): ı = 0.81 (s, 3H, Me-19); 1.90 (s, 3H, Me-18); 2.15
(s, 3H, COCH3); 5.91 (br, 2H, NH2). Ms m/z: 346 (M+). Calc for
2.3.2. 3ˇ-Acetoxy-16-benzoyl-17–benzoylhydrazono-5˛-
androstane (9)
C21H34N2O2 (346.26): C, 72.79%; H, 9.89%; N, 8.08%. Found: C,
Pale yellow powder. Yield (1.20 g, 64%), mp 230–231 ◦C. IR (KBr,
cm−1); 2930 (CH3); 2860 (CH2); 1720, 1690 (2C O); 1650 (C C).
1H NMR (DMSO): ı = 0.79 (s, 3H, Me-19); 1.20 (s, 3H, Me-18); 2.36
(s, 3H, COCH3) 7.12–7.81 (m, 11H, aromatic protons and NH). 13C
NMR (DMSO) ı: 19.6 (Me-18); 20.7 (Me-19); 21.0 (acetoxy); 22.4
(C-6); 27.0 (C-6); 29.1 (C-2); 34.0 (C-12); 35.4 (C-8); 39.7 (C-13);
40.9 (C-17); 60.6 (C-14); 128.0 (C C); 163.0 (C O); 170.0 (acetoxy);
195.3 (C O); Ms m/z: 554 (M+). Calc for C35H42N2O4 (554.72): C,
75.78%; H, 7.63%; N, 5.05%. Found: C, 75.72%; H, 7.59%; N, 5.10%.
72.79%; H, 9.73%; N, 8.10%.
2.2. General procedure for synthesis of 6a–d
A mixture of hydrazone derivative 2a (1.52 g, 0.005 mol) and
hydrazonoyl halide (0.005 mol) in absolute ethanol (30 ml) con-
taining K2CO3 (0.69 g, 0.005 mol) was stirred for 24 h, at room
temperature. The reaction mixture was poured onto ice/water and
the resulting solid was collected by filtration and crystallized using
ethanol.
2.3.3. 3ˇ-Acetoxy-6ꢀ-phenyl-1ꢀ,ˇꢀꢀ-dihydropyridazino
[3ꢀ,4ꢀ:16,17]-5˛-androstane (11)
2.2.1. 3ˇ-Hydroxy-3ꢀ-methyl-4ꢀ-(p-tolylhydrazono)-4ꢀ,
Pale yellow crystals. Yield (1.75 g, 78%), mp 155–157 ◦C. IR (KBr,
cm−1); 2927 (CH3); 2855 (CH2); 1653 (C C). 1H NMR (DMSO):
ı = 0.83 (s, 3H, Me-19); 1.21 (s, 3H, Me-18); 2.32 (s, 3H, COCH3);
4.46 (s, 1H, CH); 7.12–7.59 (m, 6 H, aromatic protons and NH). Ms,
m/z: 446 (M+). Calc for C29H38N2O2 (446.29): C, 77.99. %; H, 8.58%;
N, 6.27%. Found: C, 78.12%; H, 8.32%; N, 6.10%.
5ꢀ-dihydropyridazino-[5ꢀ,6ꢀ:16,17]-5˛-androstane (6a)
Pale yellow crystals. Yield (1.60 g, 69.5%) mp 180–181 ◦C. IR (KBr,
cm−1); 3353 (OH); 2926 (CH3); 2854 (CH2); 1673 (C C). 1H NMR
(DMSO): ı = 0.77 (s, 3H, Me-18); 1.27 (s, 3H, Me-19); 2.36 (s, 3H,
CH3); 2.42 (s, 3H, CH3); 7.09–7.50 (m, 4H, aromatic protons); 10.06
(s, 1H, OH); 9.7 (s, 1H, NH, D2O-exchangeable). 13C NMR (DMSO)
ı: 14.2 (C-3), 18.2 (C-18), 19.0 (C-19), 22.1 (C-21), 22.4 (C-11), 26.8
(C-16), 27.1 (C-6), 27.6 (C-7), 32.5 (C-1), 34.0 (C-12), 35.4 (C-8), 38.2
(C-4), 40.5 (C-15), 52.2 (C-9), 59.9 (C-14), 70.1 (C-3), 116.2, 129.3
(tolyl-C), 145 (C-5), 155, 164, 164.6 (pyridazine-C). Ms, m/z: 460
(M+) Calc for C29H40N4O (460.32): C, 75.61%; H, 8.75%; N, 12.18%.
Found: C, 75.23%; H, 8.59%; N, 12.11%.
2.3.4. 3ˇ-Acetoxy-6ꢀ-methyl-1ꢀ,3ꢀ-dihydropyridazino
[3ꢀ,4ꢀ:16,17]5˛-androstane (12)
Pale yellow crystals. Yield (1.40 g, 72%), mp 155–156 ◦C. IR (KBr,
cm−1); 2927 (CH3); 2855 (CH2); 1744 (C O);1653 (C N). 1H NMR
(DMSO): ı = 0.79 (s, 3H, Me-19); 1.21 (s, 3H, Me-18); 2.16 (s, 3H,
COCH3); 4. 31 (s, H, CH3); 4.21 (s, 1H, CH); 7.5 (s, 1H, NH). Ms, m/z:
384 (M+). Calc for C24H36N2O2 (384.55): C, 74.96%; H, 9.44%; N, 7.28
Found: C, 74.89; H, 9.31; N, 7.18.
2.2.2. 3ˇ-Hydroxy-3ꢀ-phenylamino-4ꢀ-(p-tolylhydrazono)-4ꢀ,
5ꢀ-dihydropyrida-zino[5ꢀ,6ꢀ:16,17]-5˛-androstane (6b)
Yellow crystals. Yield (1.92 g, 65%) mp 125–126 ◦C. IR (KBr,
cm−1); 2922 (CH3); 2865 (CH2); 1654 (C N). 1H NMR (DMSO):
ı = 0.78 (s, 3H, Me-19); 1.07 (s, 3H, Me-18); 2.30 (s, 3H, CH3);
7.10–7.80 (m, 9H, aromatic protons); 10.11 (s, 1H, NH, D2O-
exchangeable). Ms (E2), m/z: 537 (M+). Calc for C34H43N5O
(537.345): C, 75.94%; H, 8.06%; N, 13.02%. Found: C, 75.79%; H,
8.73%; N, 12.92%.
2.3.5. Synthesis of
3ˇ-acetoxy-17-benzylidenehydrazono-5˛-androstane (13)
To a solution of compound 2b (1.73 g, 0.005 mol) in 50 ml
ethanol containing few drops of acetic acid, benzaldehyde
(0.005 mol) was added. The reaction mixture was heated under
reflux for 3 hrs, then was poured onto ice water. The solid product
formed was collected and recrystallized using ethanol.
Yield (1.4 g, 66%) mp 95–97 ◦C, IR (KBr, cm−1), 2928 (CH3), 2875
(CH2), 1595 (C C). 1H NMR (DMSO): ı = 0.87 (s, 3H, Me-19); 1.07 (s,
3H, Me-18); 2.05 (s, 3H, COCH3); 7.61–7.78 (m, 5H, aromatic pro-
tons). Ms m/z: 434 (M+). Calc for C28H38N2O2 (434.61): C, 77.38%,
H, 8.81%; N, 6.45%, Found: C, 77.26%; H, 8.79%; N, 6.33%.
2.2.3. 3ˇ-Hydroxy-3ꢀ-phenyl-4ꢀ-(p-chlorophenylhydrazono)-4ꢀ,
5ꢀ-dihydropyrida-zino[5ꢀ,6ꢀ:16,17]-5˛-androstane (6c)
Yellow crystals. Yield (1.75 g, 64%) mp 109–110 ◦C. IR (KBr,
cm−1); 2927 (CH3); 2855 (CH2); 1654 (C C). 1H NMR (DMSO):
ı = 0.78 (s, 3H, Me-19); 1.07 (s, 3H, Me-18); 7.10–7.80 (m, 9H, aro-
matic protons,); 10.13 (s, 1H, NH, D2O-exchangeable). Ms m/z: 541
(M+). Calc for C33H39N4OCl (542.14): C, 72.97%; H, 7.24%; N, 10.32%;
Cl, 6.53%. Found: C, 72.59%; H, 7.29%; N, 10.35%; Cl, 6.24%.
2.3.6. Synthesis of 3ˇ-acetoxy-17-
({[bis(dimethylamino)phosphino]phenylmethylene}
hydrazono)-5˛-androstane (14)
2.2.4. 3ˇ-Hydroxy-3ꢀ-methyl-4ꢀ-(p-chlorophenylhydrazono)-
4ꢀ,5ꢀ-dihydropyrida-zino[5ꢀ,6ꢀ:16,17]-5˛-androstane (6d)
To a solution of compound 13 (2.17 g, 0.005 mol) in dry Ben-
zene 30 ml, Tris-dimethylamino-phosphine (0.82 g, 0.005 mol) was
added. The reaction mixture was stirred at room temperature. For
72 h, the solvent was evaporated under vacuum and the remain-
ing residue was triturated with benzene. The solid product was
collected by filtration.
Yellow crystals. Yield (1.9 g, 73%) mp 203–205 ◦C. IR (KBr, cm−1);
2927 (CH3); 2854 (CH2); 1596 (C N). 1H NMR (DMSO): ı = 0.91 (s,
3H, Me-19); 1.27 (s, 3H, Me-18); 2.50 (s, 3H, CH3); 7.10–7.40 (m,
4H, aromatic protons); 10.12 (s, 1H, NH, D2O-exchangeable) Ms

80
M.M. Abdelhalim et al. / Steroids 76 (2011) 78–84
NNH2
O
/3 hr
NH2NH2.H2O (99%)
RO
RO
2a,b
2a, R = H
1a,b
2b,R = OCOCH3
Scheme 1.
Yellow crystal yield (1.3 g, 50%) mp 96–98. IR (KBr, cm−1), 2980
COCH3); 3.10 (s, 2H, CH2); 5.11 (s, 1H, CH); 7.1–7.5 (m, 5H, aromatic
proton). Ms m/z: 508 (M+, 38%). Calc for C30H40N2O3S (508.72): C,
70.83%; H, 7.93%; N, 5.51%; S, 6.30%. Found: C, 70.65%; H, 7.78%; N,
5.46%; S, 6.23%.
(CH3), 2850 (CH2), 1720 (C O); 1640 (C C). 1H NMR (DMSO):
ı = 0.82 (s, 3H, Me-19); 1.22 (s, 3H, Me-18); 2.01 (s, 3H, COCH3);
2.41 (m, 12H, 4 CH3); 7.3–7.6 (m, 5H, aromatic protons). Ms, m/z:
552 (M+). Calc for C32H49N4O2P (552.73): C, 69.54%; H, 8.94%; N,
10.14%; P, 5.60%. Found: C, 69.32%; H, 8.69%; N, 10.12%; P, 5.56%.
2.3.8. Synthesis of
2.3.7. Synthesis of 3ˇ-acetoxy-17-thiazolidin-4ꢀ-oxo-
3ˇ-acetoxy-17-phenylthiosemicarbazide-5˛-androstane (16)
A mixture of compound 2b (1.73 g, 0.005 mol) and phenylisoth-
iocyanate (0.67 g, 0.005 mol) in dioxane (30 ml) was heated under
reflux for 5 h. The solvent was evaporated under vacuum. The
collected solid was crystallized using ethanol to give pale yellow
powder of mp 123–125 ◦C, IR (KBr, cm−1), 2987 (CH3), 2870 (CH2),
1720 (C O). 1H NMR (DMSO): ı = 0.84 (s, 3H, Me-19); 1.12 (s, 3H,
Me-18); 3.52 (s, 3H, COCH3); 7.28–7.51 (m, 6H, aromatic proton
e and NH). Ms m/z: 481 (M+). Calc for C28H39N3O2S (481.69): C,
69.82%; H, 8.16%; N, 8.72%; S, 6.66%. Found: C, 69.74%; H, 7.98%; N,
8.54%; S, 6.54%.
2ꢀ-phenyl-3ꢀ-ylimino-5˛-androstane (15)
To a solution of compound 13 (2.17 g, 0.005 mol) in dry ben-
zene (10 ml) thioglycolic acid (0.46 g, 0.005 ml) in 5 ml, benzene
was added. The reaction mixture was stirred at room temperature
for about 1 h, and then refluxed for 10 h. The solvent was evapo-
rated under vacuum and the remaining residue was triturated with
benzene/pet-ether (40–60). The solid formed was collected by fil-
tration. Yellow crystal yield (1.80 g, 70%) mp 105–109 ◦C, IR (KBr,
cm−1), 2980 (CH3), 2870 (CH2), 1700 (C O); 1640 (C C). 1H NMR
(DMSO): ı = 0.78 (s, 3H, Me-19); 1.21 (s, 3H, Me-18); 2.13 (s, 3H,
NNH2
O
NNH2
C
R
X
O
C
K2CO3
R
C
N
NHAr
+
EtOH/stirring
X= Br(Cl)
N
NHAr
HO
3a-d
4
2a
R
Ar
3a, CH3
b, PhNH
c, Ph
C6H4-CH3-p
C6H4-CH3-p
C6H4-Cl-p
C6H4-Cl-p
-H2O
d, CH3
HN
N
C
N
C
N
R
R
N
NHAr
N
NHAr
5
6
R
Ar
6a, CH3
C6H4-CH3-p
b, PhNH
c, Ph
d, CH3
C6H4-CH3-p
C6H4-Cl-p
C6H4-Cl-p
Scheme 2.

M.M. Abdelhalim et al. / Steroids 76 (2011) 78–84
81
N
N
H
NNH2
N
N
Ph
pip
EtOH
(or)
Ph
7
8
PhCOCl
+
AcO
H
N
N
COPh
2b
COPh
AcO
9
NH
NH
N
N
H
Ph
or
Ph
H
PhCOCH2Br
AcO
AcO
10
11
2b
NH
N
CH3
ClCH2COCH3
H
AcO
12
Scheme 3.
2.4. Synthesis of pyrazoloandrostane derivatives
2.4.1. General procedure
Compound 16 (2.40 g, 0.005 mol) in 10 ml benzene was added
to chloroacetic acid or thioglycolic acid in 10 ml dry benzene. The
reaction mixture was heated under reflux for 10 h. The solvent
was evaporated under vacuum; the solid formed was collected and
crystallized using ethanol.
2.4.3. 3ˇ-Acetoxy-3ꢀ-mercaptomethyl-N-phenyl-pyrazolo
[4ꢀ,5ꢀ:16,17]-5˛-androstane-2ꢀ-(4H)-carbothioamide (18)
Brown powder. Yield (1.8 g, 69%) mp 100–103 ◦C, IR (KBr, cm−1),
2989 (CH3), 2827 (CH2), 1700 (C O), 1635 (C C), 1290 (C S). 1H
NMR (DMSO): ı = 0.89 (s, 3H, Me-19); 1.12 (s, 3H, Me-18); 1.50 (s,
1H, SH); 2.01 (s, 3H, COCH3); 3.82 (s, 2H, CH2-S); 6.40 -7.00 (m, 6H,
aromatic proton and NH). Ms m/z: 537 (M+). Calc for C30H39N3O2S2
(537.78): C, 67.00%; H, 7.31%; N, 7.81%; S, 11.92%. Found: C, 66.98%;
H, 7.23%; N, 7.82%; S, 11.82%.
2.4.2. 3ˇ-Acetoxy-3ꢀ-chloromethl-
N-phenyl-pyrazolo[4ꢀ,5ꢀ:16,17]-5˛-andros-tane-2ꢀ-
(4H)-carbothioamide (17)
2.5. Bioassay
Brown crystals. Yield (1.90 g, 70%) mp 75–77 ◦C, IR (KBr, cm−1),
2980 (CH3), 2820 (CH2), 1720 (C O), 1630 (C C). 1H NMR (DMSO):
ı = 0.96 (s, 3H, Me-19); 1.08 (s, 3H, Me-18); 2.11 (s, 3H, COCH3); 4.52
(s, 1H, NH); 4.61 (s, 2H, CH2); 6.50–7.51 (m, 5H, aromatic proton).
Ms m/z: 540 (M+). Calc for C30H38ClN3O2S (540.16): C, 66.71%; H,
7.09%; N, 7.78%; Cl, 6.56%; S, 5.94%. Found: C, 66.54%; H, 6.99%; N,
7.65%; Cl, 6.30%; S, 5.64%.
2.5.1. Antitumor activity
Potential cytotoxicity of the compounds was tested using the
method of Skehan et al. [17]. Cells were plated in 96-multiwell
plates (104 cells/well) for 24 h before treatment with the com-
pounds allowing the attachment of cell to the wall of the plate.
Different concentrations of the test compound (0.0, 5.0, 12, 25.0
and 50.0 ␮g/ml) were added to the cell monolayer. Triplicate wells

82
M.M. Abdelhalim et al. / Steroids 76 (2011) 78–84
O
N
N
S
Ph
15
NMe
NMe
N
N
CHPh
NMe
P
HSCH2COOH
N
N
CPh
P
NMe
PhCHO
-H2O
NMe
-Me2NH
AcO
13
14
2b
S
C
H
N
H
N
N
Ph
PhNCS
dioxane
AcO
16
ClCH2COOH
H
O
S
N
CO
2
H
C
S
C
NHPh
H
S
N
C
NHPh
N
N
CH2Cl
CH2SH
AcO
17
AcO
18
Scheme 4.
were prepared for each individual dose. Monolayer cells were incu-
bated with the compounds for 48 h at 37 ◦C and in an atmosphere
containing 5% CO2. After 48 h, cells were fixed, washed and stained
with sulforhodamine B stain. Excess stain was washed with acetic
acid and attached stain was recovered with Tris EDTA buffer. Color
intensity was measured in an ELISA reader. The relation between
surviving fraction and the drug concentrations was plotted to get
the survival curve of each tumor cell line of the specified compound.
The cytotoxic potency is expressed as LC50. The reference doxoru-
bicin was used in the same concentrations as the test compound.
tion of hydrazonoyl halides to C16 of epi-androstane proceeded at
first via the release of HX to form the intermediate 4. The latter was
cyclized by the loss of water and isomerized to form the final iso-
lated product 6 (Scheme 2). The 1H NMR spectrum of 6a taken as
example revealed four singlets at ı = 0.77, 1.27, 2.36 and 2.42 ppm,
for the methyl group triplet at ı = 1.35 ppm for the C-15 proton
which was split by the C-16 proton, multiplet at ı = 7.09–7.50 ppm
for the aromatic protons, two singlets at ı = 9.7 and at ı10.06 ppm
for the NH and OH groups respectively. Also, the UV absorption
spectrum of compound 6a revealed its (ꢀmax) at 350 nm. This is
beside the characteristic signals of the hormone moiety. The IR, 13
C
NMR, mass spectra and the microanalytical data were in agreement
with the suggested structures 6a–d.
3. Results and discussion
The acetylated hydrazonoandrostane derivative 2b was allowed
to react with different aromatic halide reagents like benzoyl
chloride or phenacyl bromide for the synthesis of new types of
steroids attached to nitrogen heterocycles. The interaction with
benzoylchloride did not form the expected pyrazoloandrostane
derivative 7 or 8 but produced the unexpected additional adduct 9.
The structure of such product was elucidated on the basis of spec-
troscopic and microanalytical data. The 1H NMR spectrum revealed
the presence of the aromatic protons of the two phenyl groups at
ı = 7.12–7.81 ppm. Synthesis of dihydropyridazino steroids via the
17-Hydrazonoandrostane 2a, b (produced previously via the
reaction of epi-androsterone 1 with hydrazine hydrate 80% under
reflux [16]) was utilized for the synthesis of a variety of nitrogen
containing steroidal heterocycles [18,19]. All the spectroscopic and
microanalytical data were in accordance with the suggested struc-
tures 2a and 2b (cf. Section 2) (Scheme 1).
The behavior of the hydrazonoandrostane derivative 2a towards
hydrazonoyl halides was studied. The interaction of an equimolar
amount of 2a with different types of hydrazonoyl halides 3a–d was
carried out at room temperature in the presence of K2CO3. Addi-

M.M. Abdelhalim et al. / Steroids 76 (2011) 78–84
83
Table 1
Drug cytotoxicity.
Concentration (␮g/ml)
HEPG2-DOX
0.00
5.00
12.50
25.00
50.00
1.00
0.58
0.43
0.36
0.31
reaction of hydrazono steroid with ␣-halogenated compounds was
studied. The reaction of hydrazonoandrostane with phenacyl bro-
mide should produce either the cyclic pyridazino derivative 10 or
11. The 1H NMR spectrum showed the presence of the CH proton
at ı = 4.46 ppm, which means that its position is close to the phenyl
Curve 1. Cytotoxic activity of the standard, doxorubicine.
group and far from the NH group. Also the addition of D2O to the 1
H
NMR vial of this product resulted in the disappearance of the NH
signal and the CH proton did not sharpen up which confirmed the
distance between NH and OH protons. This is in accordance with
structure 11 (Scheme 3).
The use of chloroacetone as an alkyl halide reagent in the pre-
vious reaction yielded the corresponding pyridazinoandrostane
derivative 12 (Scheme 3).
The chemical activity of compound 2b was also examined with
other different organic reagents. It reacted with benzaldehyde to
yield the Schiff base derivative 13 that reacted with trisamiophos-
phine reagent to form the final adduct 14.
Curve 2. Cytotoxic activity of compounds 12 and 15.
Compound 13 also reacted with thioglycolic acid to cyclise and
form the corresponding thiazoloylandrostane derivative 15. The
structure of such adducts conformed to the spectroscopic data.
1H NMR spectra showed a singlet signal at ı = 3.1 ppm for the CH2
and other singlet at ı = 5.11 ppm for the CH group. Compound 2b
reacted with phenylisothiocyanate to produce the additional prod-
uct 16. The latter was cyclized on boiling with chloroacetic acid
and thioglycolic acid to form the corresponding products 17 and
18, respectively (Scheme 4). The structure of the newly synthe-
sized compounds was elucidated on the basis of the spectroscopic
and microanalytical data.
Table 2
Curve 3. Cytotoxic activity of compounds 6a, 6d and 11.
The cytotoxic activity of the investigated compounds.
Compounds
Concentration (␮g/ml)
HEPG2-R33
LC50
3.1. Bioactivity
3.1.1. Antitumor activity
0.00
5.00
12.50
25.00
50.00
0.00
1.00
0.80
0.45
0.27
0.31
1.00
0.87
0.44
0.29
0.36
1.00
0.60
0.38
0.20
0.24
1.00
0.55
0.24
0.26
0.26
1.00
0.45
0.34
0.23
0.25
6a
11.35
Evaluation of the anticancer activity of compounds (6a, 6d, 11,
12, and 15) was performed at the National Cancer Institute (NCI),
Cairo-Egypt. The tested compounds were evaluated for cytotoxic-
ity in the liver carcinoma cell line HEPG2. Different concentrations
of the investigated compounds were added to the cell monolayer
of tumor. A 48 h continuous drug exposure is used to estimate
growth [20]. Tables 1 and 2 indicate the cytotoxic activities of the
investigated compounds. It was found that compounds 12 and 15
are of the most active cytotoxic agents when compared with the
standard Doxorubicine. All investigated compounds exhibited the
highest anticancer activity at the concentration of 25,000 ␮g/ml.
These results are represented in Curves 1–3.
5.00
12.50
25.00
50.00
0.00
11.15
8.55
6.10
4.40
6d
11
12
15
5.00
12.50
25.00
50.00
0.00
5.00
12.50
25.00
50.00
0.00
4. Conclusion
5.00
12.50
25.00
50.00
The investigated compounds 12 and 15 exert the highest
antiproliferative activity in the human liver carcinoma cell line
HEPG2. The results indicated that these compounds revealed LC50

84
M.M. Abdelhalim et al. / Steroids 76 (2011) 78–84
values 6.10 and 4.40 ␮g/ml, respectively. The other tested com-
pounds 6a, 6d, and 11 showed LC50 values between 8.55 and
11.35 ␮g/ml which means that they have a moderate activity as
cytotoxic agents against HEPG2 cells.
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