Cytotoxicity and gene expression profiles of novel synthesized steroid derivatives as chemotherapeutic anti-breast cancer agents

Article abstract of DOI:10.1016/j.bmc.2011.09.033

Anti-cancer agents which combine two biologically active compounds in one such as steroidal heterocyclic derivatives attain both hormone and cytotoxic effects on cancer cells. The aim of the present study is to synthesize and evaluate new potential chemotherapeutic anti-breast cancer agents. Several pyridazino-, pyrimido-, quinazolo-, oxirano- and thiazolo-steroid derivatives were synthesized. The structure of the novel steroid derivatives was confirmed using the analytical and spectral data. The most structurally promising of the novel synthesized steroid derivatives, compounds 8, 12, 17, 20, 22c, 24c, 30a and 30b, were investigated individually as anti-breast cancer agents against human breast cancer cells (MCF-7) using sulforhodamine B (SRB) assay. The tested compounds 17, 20, 22c and 8 showed potent broad spectrum cytotoxic activity in vitro after 48 h incubation. Compound 17 (IC₅₀ = 2.5 μM) exhibited more inhibitory influence on MCF-7 growth than the reference drug doxorubicin (Dox) (IC₅₀ = 4.5 μM) after 48 h incubation. Also, the present study showed that all the tested steroid derivatives exhibited significant depletion with various intensities in gene expression of breast cancer related genes (VEGF, CYP19 and hAP-2γ). Noteworthy, compounds 17, 20 and 22c showed the most pronounced effect in this respect.

Full text of DOI:10.1016/j.bmc.2011.09.033

Bioorganic & Medicinal Chemistry 19 (2011) 6860–6872
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Cytotoxicity and gene expression profiles of novel synthesized steroid
derivatives as chemotherapeutic anti-breast cancer agents
Gamal A. Elmegeed a, , Wagdy K.B. Khalil b, Rafat M. Mohareb c,d, Hanaa H. Ahmed a,
⇑
Mervat M. Abd-Elhalim a, Ghada H. Elsayed a
a Hormones Department, National Research Centre, 12622 Dokki, Giza, Egypt
b Cell Biology Department, National Research Centre, Dokki, Giza, Egypt
c Organic Chemistry Department, Faculty of Pharmacy, October University of Modern Sciences and Arts (MSA), October City, Egypt
d Chemistry Department, Faculty of Science, Cairo University, Cario, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 June 2011
Revised 11 September 2011
Accepted 19 September 2011
Available online 22 September 2011
Anti-cancer agents which combine two biologically active compounds in one such as steroidal heterocy-
clic derivatives attain both hormone and cytotoxic effects on cancer cells. The aim of the present study is
to synthesize and evaluate new potential chemotherapeutic anti-breast cancer agents. Several pyridazi-
no-, pyrimido-, quinazolo-, oxirano- and thiazolo-steroid derivatives were synthesized. The structure of
the novel steroid derivatives was confirmed using the analytical and spectral data. The most structurally
promising of the novel synthesized steroid derivatives, compounds 8, 12, 17, 20, 22c, 24c, 30a and 30b,
were investigated individually as anti-breast cancer agents against human breast cancer cells (MCF-7)
using sulforhodamine B (SRB) assay. The tested compounds 17, 20, 22c and 8 showed potent broad spec-
Keywords:
Breast cancer
Cytotoxicity
Gene expression
Heterocycles
Steroids
trum cytotoxic activity in vitro after 48 h incubation. Compound 17 (IC50 = 2.5
itory influence on MCF-7 growth than the reference drug doxorubicin (Dox) (IC50 = 4.5
l
M) exhibited more inhib-
M) after 48 h
l
incubation. Also, the present study showed that all the tested steroid derivatives exhibited significant
depletion with various intensities in gene expression of breast cancer related genes (VEGF, CYP19 and
hAP-2
c
). Noteworthy, compounds 17, 20 and 22c showed the most pronounced effect in this respect.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
represent important experimental models for investigational
hormone-based therapies.
Breast cancer is reported to be one of the commonest cancers,
accounting for almost 20% of all malignancies world-wide, and
over half a million women develop breast cancer every year. Breast
cancer is the most common cause of cancer death among women
worldwide, and especially young women in the developing world.
If current rates of increase remain constant, a woman born today
has a 1 in 10 chance of developing breast cancer.1,2 It is well known
that breast tumors are steroid hormone dependent neoplasms and
Cancer is a complex disease and a single mono-functional drug
will not be effective for treating this most advanced disease. Com-
bined drugs that impact multiple targets simultaneously are better
at controllingcomplex disease systems, less prone to drug resistance
and are the standard of care in cancer treatment. In order to improve
the efficiency of using a two-drug cocktail, one approach involves
the use of the so-called hybrid drugs, which comprises the incorpo-
ration of two drugs in a single molecule with the intention of exert-
ing dual drug action.3 Heterocyclic rings represent molecular
framework that serve as a platform for developing pharmaceutical
agents for various applications. The antitumor activity of many com-
pounds containing heterocyclic ring have been reviewed.4,5 The
investigation of modified steroid derivatives condensed with vari-
ous heterocyclic rings has drawn great attention.6,7 Steroid hetero-
cycles anticancer agents aspire to attain both hormone and
cytotoxic effects on cancer cells. A variety of synthetic steroid deriv-
atives have been contrived. Most these derivatives can interact with
the steroid receptors because of a similarity of shape.8,9
Abbreviations: aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast
growth factor; CBMN, cytokinesis-block micronucleus; DMF, dimethylformamide;
DMSO, dimethylsulfoxide; Dox, doxorubicin; E2, estrogen; ER, estrogen receptor;
ER
a, estrogen receptor-alpha; ERb, estrogen receptor-beta; CYP19, human aroma-
tase; FBS, fetal bovine serum; hAP-2
c
, human activation protein-2 gamma; HER-2/
neu, human epidermal growth factor receptor 2; JCBN, joint commission on the
biochemical nomenclature; RT–PCR, reverse transcription–polymerase chain reac-
tion; RT, reverse transcription; SRB, sulforhodamine B; TLC, thin layer chromatog-
raphy; TCA, trichloroacetic acid; TGF-a, tumor growth factor-
a; TGF-b, tumor
growth factor-b; TNF-
a
, tumor necrosis factor- ; VEGF, vascular endothelial growth
a
Analysis of gene expression is increasingly important tool in
many research fields including early detection of breast cancer. Re-
verse transcription-polymerase chain reaction (RT–PCR) is a highly
factor.
⇑
Corresponding author. Tel.: +20 2 35682070; fax: +20 2 33370931.
E-mail address: gamalae@hotmail.com (G.A. Elmegeed).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.033

G. A. Elmegeed et al. / Bioorg. Med. Chem. 19 (2011) 6860–6872
6861
sensitive and specific method useful for the detection of rare tran-
scripts of limited amount of tumor cells.10
and pyridazine rings represent molecular frameworks that serve
as a platform for developing pharmaceutical agents for various
There is several genes play an important role in breast cancer
development. Vascular endothelial growth factor (VEGF) is a key
molecule in both tumor angiogenesis and the survival of tumor
endothelial cells.11 VEGF gene is expressed in the majority of tu-
mor types, including breast cancer.11 Aromatase activity in tumor
or surrounding tissue plays a significant role in promoting this type
of tumor growth due to the local production of estrogen, and thus
aromatase is an important target in the treatment of estrogen
receptor (ER) positive breast cancer.12 Human aromatase gene
(CYP19) expression in breast cancer cells is known to play an
important role in growing cancer cells.13 Breast cancer cell lines
that have abundant human epidermal growth factor receptor
(2HER-2/neu) protein concurrently over-express the human acti-
applications.17,18 Simple acetylation of 3b-hydroxy-5
17-one (1) (epi-androsterone) afforded 3b-acetoxy-5
a
a
-androstan-
-androstan-
17-one (3).19 Compound (3) fused with equimolar amount of
sulfadiazine 4 in the presence of glacial acetic acid to give the pyri-
midinyl benzene sulfonamide derivative 5 in 94% yield (Scheme 1).
Treatment of compound 5 with aqueous sodium hydroxide affor-
ded the corresponding isolable sodium salt 6. The reaction of
compound 6 with 2-bromoethanol (7) in dimethylformamide
(DMF) afforded the pyrimidinyl benzene sulfonamide ethanol
derivative 8 in 57% yield (Scheme 1).
The reaction of compound (3) with equimolar amount of
L-methionine (9) in ethanolic triethylamine solution afforded the
corresponding 2-androstanylideneamino-4-methylthiobutanoic
vation protein-2 gamma (hAP-2
c
) gene discreetly suggesting that
acid 10 in 71% yield (Scheme 2). Compound 10 reacted with hydra-
zine hydrate (11) in boiling ethanol solution to afford the corre-
sponding pyridazinyl androstane derivative 12 in 83% yield
(Scheme 2).
The azole moiety often shows some special biological activity
when it is introduced to some biologically active compounds.20,21
The basicity and hydrophilicity of an azole in theory might alter
the biological function of a steroid.22 Especially, 1,3-thiazoles are
an important class of antitumor agents.23,24 Bromination of com-
pound (3) using cupric bromide (13) in dry methanol afforded
deregulation of these proteins is the preceding event in the cascade
that culminate in mammary carcinoma.14
In view of the above facts, we set as a goal to prepare some new
potential chemotherapeutic anti-breast cancer agents through the
synthesis of new steroidal heterocyclic derivatives. The more
structurally promising agents were examined for their cytotoxicity
in vitro against breast cancer cell line (MCF-7) using SRB [sulforho-
damine B] assay. Moreover, the expression of VEGF, CYP19 and
hAP-2c genes in the breast cancer cells was evaluated after
treatment with the new agents.
the corresponding 3b-acetoxy-16-bromo-5a-androstan-17-one
(14) in 73% yield. Compound (14) reacted with equimolar amount
of cyanothioacetamide (15) in refluxing ethanol containing a cata-
lytic amount of piperidine to give the thiazoloandrostane deriva-
tive 17 in 60% yield. Also, compound (14) reacted with equimolar
amount of thiourea (16) in THF containing a catalytic amount of
piperidine to give the aminothiazoloandrostane derivative 18 in
50% yield (Scheme 3).
Transition metal complexes containing heterocyclic ligand are
commonly used in biological media as anticancer and play impor-
tant roles in processes such as catalysis of drug interaction with
2. Results and discussion
2.1. Chemistry
Sulfonamides are an important group of drugs. They exhibit a
broad spectrum of antibacterial activities. Recently, some
sulfonamide derivatives have been reported to have potential anti-
tumor effects.15,16 However, there are few literature references
available for steroid sulfonamide derivatives. Also, pyrimidine
O
O
H
H
CH3COCl
H
H
H
H
2
HO
AcO
H
H
3
1
O
O
N
N
N
O
S
NH
O
N
N
H
AcOH
3
+
H2N
S
N
H
5
4
H
NaOH
O
O
O
S
O
N
N
N
N
N
S
N
N
N
BrCH2CH2OH
7
H
Na
H
DMF
6
OH
8
H
H
Scheme 1.

6862
G. A. Elmegeed et al. / Bioorg. Med. Chem. 19 (2011) 6860–6872
in the IR spectrum of compound 17 shifted to lower frequency at
= 1630 cmꢀ1 upon coordination with copper. Also, the IR spec-
COOH
m
N
trum showed the characteristic broad band at m = 1379 for the nitro
H2N
COOH
complexes.28 Moreover, the 1H NMR spectrum showed the pres-
ence of a singlet at d = 2.0 (4H, D2O-exchangeable) for the two
NH2 groups and showed also D2O-exchangeable singlet (1H) at
8.20 for the NH group.
H
SCH3
EtOH/Et3N
+
H
3
H
AcO
N
H3CS
H
10
Introduction of the reactive oxirane ring in steroids results in dra-
matic changes in their biological activity.29 Thus, it was of significant
interest to trace the influence of replacement the ketone group by
spirooxirane moiety. We have attempted a straightforward synthe-
sis of spirooxiranoandrostane derivatives. epi-Androsterone (1) re-
acted with equimolar amount of either ethyl chloroacetate (21a),
chloroacetone (21b) or phenacylbromide (21c) in potassium tert-
butoxide to afford the corresponding spirooxiran-2,17b-androstane
derivatives 22a, 22b and 22c respectively (Scheme 4). Compounds
9
HO
N
NH
EtOH
H
NH2NH2.H2O
H
11
12
22a–c cyclized at the less hindered C-17a
face.30 Thin layer chroma-
Scheme 2.
tography examination of the reaction mixture revealed minor prod-
ucts probably due to the presence of other isomers. Isolation and
identification of these minor products is beyond the scope of this
study. To generalize such a methodology, the previous reactions
biomolecular activity.25 Many metallodrugs are pro-drugs and they
undergo ligand substitution and/or redox reactions before they
reach the target site.26 The pharmacological activity of
Cu(II)-thiazole complexes was found to be much higher than that
of the parent heterocycles.27 The reaction of compound 17 with
copper diethylenetriaminedinitrite [Cu(dien)(NO3)2] 19 in equimo-
lar ratio (1:1) afforded copper complex of 3b-acetoxy thiazol-
o[16,17:4,5]androstane derivative 20 in 73% yield (Scheme 3).
The resulted dark green compound is air stable crystalline powder
and was characterized by elemental analysis (cf. materials and
methods). The mass spectrum of compound 20 showed a molecu-
lar ion peak at m/z = 703 (3%). The IR spectrum showed absorption
with the a-haloketones 21a–c were carried out by utility of testos-
terone (23) under the same experimental conditions, to afford the
corresponding spirooxiran-2,3b-androstane derivatives 24a–c,
respectively.
The reaction of testosterone (23) with equimolar amount of
thionyl chloride (25) in anhydrous diethylether gave the corre-
sponding mixture of 17a- and 17b-chloroandrost-4-en-3-one 26.
Isolation and identification of this mixture is beyond the scope of
this study. Compound 26 reacted with either anthranilic acid
(27a) or 5-fluoroanthranilic acid (27b) in ethanol to give the
corresponding androstenylaminobenzoic acid derivatives 28a and
28b, respectively (Scheme 5). Compounds 28a,b reacted with equi-
molar amount of phenylisothiocyanate (29) in refluxing ethanol
bands at
m
= 3385–3320 cmꢀ1 for the NH2 and NH groups, and
showed also absorption band at
m
= 2220 cmꢀ1 for the cyano group.
The absorption band at 1642 cmꢀ1 which is attributed to the (C@N)
O
H
Br
dry methanol
CuBr2
+
3
H
13
H
AcO
H
14
S
S
NC
H2N
NH2
NH2
15
16
THF/ piperidine
EtOH/
piperidine
N
NH2
N
CN
S
S
H
H
H
NO3
H
18
17
NH
NH2
NO3
Cu
NH2
MeOH
N
CN
Cu(dien)(NO3)2
S
H
19
H
Scheme 3.
20

G. A. Elmegeed et al. / Bioorg. Med. Chem. 19 (2011) 6860–6872
6863
O
120
100
80
60
40
20
0
O
A
DOX
8
12
17
20
H
24 h
R
K, t-butanol
H
H
H
21a, ClCH2COOEt
b, ClCH2COCH3
c, PhCOCH2Br
H
H
HO
H
HO
22a, R=COOEt
b, R=COCH3
c, R=COPh
H
1
OH
OH
H
H
K, t-butanol
H
H
H
H
21a, ClCH2COOEt
b, ClCH2COCH3
c, PhCOCH2Br
O
O
24a, R=COOEt
b, R=COCH3
c, R=COPh
23
R
5
12.5
25
50
Conc. μg/mL
Scheme 4.
100
80
60
40
20
0
B
DOX
22c
24c
30a
30b
24 h
containing a catalytic amount of triethylamine to afford the corre-
sponding 17b-thioxoquinazolinandrost-4-en-3-one derivatives
30a,b, respectively (Scheme 5).
2.2. Biological assays
2.2.1. In vitro evaluation of cytotoxic activity
In this study, the most structurally promising compounds of the
novel synthesized steroid derivatives 8, 12, 17, 20, 22c, 24c, 30a
and 30b were investigated individually as anti-breast cancer
agents against the human breast cancer cells (MCF-7). The inhibi-
tion of proliferation of MCF-7 cells was determined using SRB assay
in comparison to the chemotherapeutic anti-breast cancer drug,
doxorubicin (Dox). The usage of DMSO as a solvent at a volume
5
12.5
25
50
of 100
l
L (the maximum volume used to dissolve the tested com-
Conc. μg/mL
pounds) had insignificant effect on the viability of MCF-7 cells
when treated for 24, 48 and 72 h.7
Figure 1. (A, B) Effects of tested compounds on MCF-7 cells at 24 h incubation time.
Dox significantly inhibited the growth of MCF-7 cells at all incu-
bation times as compared to control values (untreated MCF-7 cells).
After 24 h incubation, at 5, 12.5, 25 and 50
lg/mL concentrations
MCF-7 cells. Also, at concentrations 25 and 50 lg/mL, there was sig-
(Fig. 1A and B), all tested compounds showed significant inhibition
of MCF-7 cells growth as compared to the control values. As com-
pared to Dox values, at concentration 12.5 lg/mL, compounds 8,
nificant cell growth inhibition of MCF-7 cells by all tested com-
pounds when compared with Dox values. Compound 17 is the
most effective one in this respect with inhibition 91% at concentra-
17, 22c, 24c and 30a significantly inhibited the proliferation of
tion 12.5
l
g/mL and 85% at concentration 25 lg/mL.
Cl
OH
H2N
H
H
HOOC
R
SOCl2
H
H
H
H
25
27a, R=H
b, R=F
EtOH
O
O
23
26
O
HOOC
N
R
R
S
N
HN
EtOH/ Et3N
H
H
PhNCS
29
H
H
28a, R=H
b, R=F
30a, R=H
b, R=F
Scheme 5.

6864
G. A. Elmegeed et al. / Bioorg. Med. Chem. 19 (2011) 6860–6872
At 48 h incubation, (Fig. 2A and B) there was significant cell
growth inhibition of breast cancer cells by all tested compounds
120
100
80
60
40
20
0
A
DOX
8
72 h
at 5, 12.5, 25 and 50
trol values. As compared to Dox values, at concentration 5
compound 17 showed significant inhibition of proliferation of
MCF-7 cells. At concentration 12.5 g/mL, compounds 8, 12, 17,
20 and 22c significantly inhibited the growth of MCF-7 cells. Also,
at 25 and 50 g/mL concentrations, all tested compounds, except
compound 24c at concentration 25 g/mL and compound 8 at con-
centration 50 g/mL, showed significant inhibition of MCF-7 cells
lg/mL concentrations as compared to the con-
12
17
20
lg/mL,
l
l
l
l
growth. The incubation time 48 h is the best incubation time for
the cytotoxic effect of the tested compounds. At 72 h incubation,
at 5, 12.5, 25 and 50
lg/mL concentrations (Fig. 3A and B), all tested
compounds, except compound 20 at concentration of 5
l
g/mL
showed significant inhibition of MCF-7 cells growth as compared
to the control values. As compared to Dox values, compound 17 sig-
nificantly inhibited the cell proliferation of MCF-7 cells at concen-
5
12.5
25
50
Conc. μg/mL
tration of 5 lg/mL (93%) and at concentration of 12.5 lg/mL (91%).
120
100
80
60
40
20
0
B
Table 1 revealed the IC50 of the tested compounds. After 24 h
incubation time, Dox inhibition values were less than 50% with
the increase of its concentrations. After 48 h incubation time all
tested compounds showed high cytotoxic activity against breast
DOX
22c
24c
30a
30b
72 h
cancer cells (MCF-7). Compound 17 (IC50 = 2.5
effective as it exhibited more inhibitory effect than Dox
(IC50 = 4.5 M) after 48 h incubation time and showed similar effect
as Dox after 72 h incubation time (IC50 = 2.9 M). Also, after incuba-
tion time 48 h, the effect of compound 20 (IC50 = 4.7 M) was
approximately similar to Dox. Compounds 8 and 22c have nearly
lM) is the more
l
l
l
similar effect (IC50 = 7.5
lM and 7.3
l
M respectively) but they
100
A
DOX
8
12
17
20
5
12.5
25
50
48 h
Conc. μg/mL
80
60
40
20
0
Figure 3. (A, B) Effects of tested compounds on MCF-7 cells at 72 h incubation time.
Table 1
The in vitro cytotoxic activity of compounds on MCF-7 cells (IC50 in lM)
Compounds
24 h
48 h
72 h
Dox
8
12
—
4.5
7.5
9.9
2.9
10
11.5
2.9
14.7
18.5
2.9
17
2.5
20
14
4.7
7.3
16
12.5
11.5
17.3
17.3
12
17.5
16
22c
24c
30a
30b
17.1
15.1
13.1
17.7
5
12.5
25
50
Conc. μg/mL
100
B
DOX
22c
24c
30a
30b
48 h
80
60
40
20
exhibited less inhibition effect when compared to Dox. The results
revealed that the tested compounds 17 and 20 showed the higher
cytotoxic activity against breast cancer cells (MCF-7) and com-
pound 17 is better than Dox after 24 and 48 h incubation times.
2.2.1.1.Structure–activity relationship. The analysis of the struc-
ture–activity relationships indicates firstly that the cytotoxicity of
the tested compounds seems to be linked to the presence of het-
erocyclic moiety fused to the steroid moiety. This is identified
clearly in comparison with the published cytotoxic activity of some
steroids.31 At 48 h incubation time, the acetonitrilothiazolyl andro-
stane derivative 17 is the most cytotoxic compound followed by
the copper complex of thiazolyl androstane derivative 20, spiroox-
iranoandrostane derivative 22c and pyrimidinyl androstane deriv-
ative 8. The addition of copper complex to the acetonitrilothiazolyl
0
5
12.5
25
50
Conc. μg/mL
Figure 2. (A, B) Effects of tested compounds on MCF-7 cells at 48 h incubation time.

G. A. Elmegeed et al. / Bioorg. Med. Chem. 19 (2011) 6860–6872
6865
A
1
2
3
4
5
6
7
8
9
10
11
12
500 bp
VEGF
ß-actin
400 bp
200 bp
150 bp
1.6
1.4
1.2
1
B
a
a
ab
ab
ab
b
b
b
b
c
0.8
0.6
0.4
0.2
0
c
Control
DMSO
Doxo
8
12
17
20
22c
24c
30a
30b
Treatment
Figure 4. (A) RT–PCR confirmation of VEGF and b-actin genes expressed in breast cancer cell line (MCF-7). Lane 1 represents DNA marker, lane 2 represents control, lane 3
represents DMSO, lane 4 represents Dox, lane 5 represents compound 8, lane 6 represents compound 12, lane 7 represents compound 17, lane 8 represents compound 20,
lane 9 represents compound 22c, lane 10 represents compound 24c, lane 11 represents compound 30a, and lane 12 represents compound 30b. (B) Expression of VEGF gene
expressed in breast cancer cell line (MCF-7). The RNA recovery rate was estimated as the ratio between the intensity of VEGF gene and the b-actin gene. a,b,cMean values
within columns with unlike superscript letters were significantly different (P <0.05). a,b,ab,cMean values within columns with similar superscript letters were not significantly
different (P >0.05).
androstane moiety to form compound 20 decreased the cytotoxic
activity of the acetonitrilothiazolyl androstane moiety of com-
pound 17. The acetonitrilothiazole moiety of compound 17 is more
effective than the aminothiazole moiety of compound 18. The pres-
ence of spirooxirane ring attached to C-17 of steroid moiety (com-
pound 22c) is more effective than its presence at C-3 (compound
24c). The presence of fluorine atom attached to the quinazolinyl
androstene derivative (compound 30b) made no effective change
in the cytotoxicity of compound 30a. The addition of pyrimidine
ring to the steroid moiety (compound 8) is more effective than
the addition of pyridazine ring (compound 12). The results verified
the importance of the presence of thiazole, pyrimidine and oxirane
moieties as pharmacophores for the anti-cancer activity.
were down-regulated in breast cancer cell lines (MCF-7), treated
with Dox compared with control values (Figs. 4–6). It is known that
Dox in addition to its cytostatic effect has important adverse ef-
fects such as genotoxicity.32 Duffaud et al.33 reported that Dox
was a genotoxic drug and gave positive results with the cytokine-
sis-block micronucleus (CBMN) test and chromosome aberrations
test.
On the other hand, the results of the present study showed that
all the tested synthetic steroid derivatives showed significant de-
crease in gene expression of VEGF, CYP19 and hAP-2c genes com-
pared to control values. Compounds 17, 20 and 22c were more
effective as anticancer agents than the other steroid derivatives.
These compounds were significantly (P <0.001) decreased the level
expression of all selected genes compared with control values, the
level of expression was relatively similar to that of Dox. Moreover,
the other steroid derivatives 8, 12, 24c, 30a and 30b were also able
to decrease the expression level of all selected genes in comparison
to control values. However, the ability of these steroid derivatives
in decreasing the level of expression of tested genes was less than
that of compounds 17, 20 and 22c as well as Dox (Figs. 4–6). The
process of tumors growth is affected by several growth factors such
as aFGF (acidic fibroblast growth factor), bFGF (basic fibroblast
2.2.2. Molecular biology assay
2.2.2.1. Gene expression patterns. The cell line samples of 48 h
incubation time, the more effective incubation time in our in vitro
study, were selected for the gene expression analysis. The effect of
steroids derivatives 8, 12, 17, 20, 22c, 24c, 30a and 30b on VEGF,
CYP19 and hAP-2c genes were evaluated by calculation of the ratio
of its expression level to that of b-actin by semiquantitive analysis
and in comparison to Dox. The usage of DMSO as a solvent at a vol-
ume of 100
lL had insignificant effect on the expression levels of the
growth factor), TGF-
a
(tumor growth factor-a), TGF-b (tumor
selected genes compared with control values (Figs. 4–6).
growth factor-b), TNF-
a
(tumor necrosis factor-a), VEGF (vascular
The present results revealed that Dox decreased significantly
the expression levels of the selected genes which related to breast
endothelial growth factor), and their receptors act as stimulators
for various steps involved in this complex process. Among the var-
ious factors mentioned above, VEGF is considered to be the prime
cancer. The expression levels of VEGF, CYP19 and hAP-2
c genes

6866
G. A. Elmegeed et al. / Bioorg. Med. Chem. 19 (2011) 6860–6872
1
2
3
4
5
6
7
8
9
10
11
12
A
B
350 bp
300 bp
hAP-2- γ
200 bp
150 bp
ß-actin
1.8
1.6
1.4
1.2
1
a
a
b
b
b
b
b
c
c
c
c
0.8
0.6
0.4
0.2
0
Control
DMSO
Doxo
8
12
17
20
22c
24c
30a
30b
Treatment
Figure 5. (A) RT–PCR confirmation of hAP-2-
represents DMSO, lane 4 represents Dox, lane 5 represents compound 8, lane 6 represents compound 12, lane 7 represents compound 17, lane 8 represents compound 20,
lane 9 represents compound 22c, lane 10 represents compound 24c, lane 11 represents compound 30a, and lane 12 represents compound 30b. (B) Expression of hAP-2- gene
gene and the b-actin gene. a,b,cMean values
c and b-actin genes expressed in breast cancer cell line (MCF-7). Lane 1 represents DNA marker, lane 2 represents control, lane 3
c
expressed in breast cancer cell line (MCF-7). The RNA recovery rate was estimated as the ratio between the intensity of hAP-2-
c
within columns with unlike superscript letters were significantly different (P <0.05). a,b,cMean values within columns with similar superscript letters were not significantly
different (P >0.05).
regulator of angiogenesis, vasculogenesis and vascular permeabil-
ity in vivo.34
mitogen (MAP kinase), which induces angiogenesis and vascular
permeability in vivo. Therefore, VEGF could play a role with the
estrogen to enhance the tumor progression. Consequently, inhibi-
tion of the VEGF gene due to synthetic steroid derivatives treat-
ment may suppress the CYP19 gene activity.
VEGF is an endothelial cell-specific mitogen (MAP kinase),
which induces angiogenesis and vascular permeability in vivo.
Angiogenesis is a complex, multi-step process that results in the
formation of new blood vessels from preexisting vasculature.34
During tumor angiogenesis endothelial cells degrade the basement
membrane at the postcapillary venule by releasing enzymes, mi-
grate through the membrane to form a sprout, and finally prolifer-
ate to extend the blood vessel into the tumor. Therefore, we expect
that, the role of the current synthetic steroid derivatives on pre-
vention of tumor progression as a result of down regulation of
VEGF gene may be attributed to inhibition of the releasing en-
zymes which are essential to extend the blood vessel into the tu-
mor. In the present study the expression of CYP19 gene was
significantly decreased due to treatment with synthetic steroid
derivatives 17, 20 and 22c, and Dox compared to control. Kirma
et al.35 found that the over expression of CYP19 gene in the
mammary gland is promoted with secretion of estrogens (E2). E2
up-regulates aromatase expression by a nongenomic action of
Regarding hAP-2c gene, the present results found that expres-
sion of this gene was significantly decreased when treated with
the synthetic steroid derivatives especially 17, 20 and 22c, and
Dox compared to control. Breast cancer cell line (MCF-7) has abun-
dant HER-2/neu protein concurrently over-expressed hAP-2
c
gene.37 The regulation of hAP-2
c
expression was linked to HER-
2/neu over-expression-mediated mammary carcinoma cell line
(MCF-7).14 Over-expression of estrogen receptor (ERb) gene leads
to the elevation of HER2 expression in MCF-7 breast cancer cells.
Based on this information, VEGF gene may also interact with
CYP19 and hAP-2c genes under modulation of estrogen and/or
estrogen receptors resulting in regulation of tumor cells.
2.3. Conclusion
estrogen receptors (ER
a
) via cross-talk with MAP kinase growth
The present study described a facile synthesis of novel promis-
ing anticancer steroid derivatives and investigated also the impor-
tance of incorporating heterocyclic moiety to the steroid nucleus to
form new effective anticancer agents. The tested compounds
namely 17, 20, 22c and 8 dissolved in DMSO showed promising
cytotoxic activity in vitro after 48 h incubation. Compound 17
factor-mediated pathways. Where, secretion of estrogen causes in-
crease in aromatase expression through the phosphorylation of
MAP protein.36 This would suggest that a complex mechanism is
involved in regulating aromatase expression in breast cancer
tissue. As mentioned above, VEGF is an endothelial cell-specific

G. A. Elmegeed et al. / Bioorg. Med. Chem. 19 (2011) 6860–6872
6867
1
2
3
4
5
6
7
8
9
10
11
12
A
300 bp
250 bp
CYP19
ß-actin
200 bp
150 bp
1.8
1.6
1.4
1.2
1
B
a
a
b
b
b
b
bc
bc
c
c
c
0.8
0.6
0.4
0.2
0
Control DMSO
Doxo
8
12
17
20
22c
24c
30a
30b
Treatment
Figure 6. (A) RT–PCR confirmation of CYP19 and b-actin genes expressed in breast cancer cell line (MCF-7). Lane 1 represents DNA marker, lane 2 represents control, lane 3
represents DMSO, lane 4 represents Dox, lane 5 represents compound 8, lane 6 represents compound 12, lane 7 represents compound 17, lane 8 represents compound 20,
lane 9 represents compound 22c, lane 10 represents compound 24c, lane 11 represents compound 30a, and lane 12 represents compound 30b. (B) Expression of CYP19 gene
expressed in breast cancer cell line (MCF-7). The RNA recovery rate was estimated as the ratio between the intensity of CYP19 gene and the b-actin gene. a,b,cMean values
within columns with unlike superscript letters were significantly different (P <0.05). a,b,bc,cMean values within columns with similar superscript letters were not significantly
different (P >0.05).
(IC50 = 2.5
than Dox (IC50 = 4.5
l
M) exhibited more inhibition effect of MCF-7 growth
M) after 48 h incubation time. Also, our re-
were abbreviated by the letters: s-singlet, d-doublet, t-triplet, q-
quartet and m (multiplet, more than quartet). Mass spectra were
recorded on a GCMS-QP 1000 ex spectra mass spectrometer oper-
ating at 70 ev. Elemental analyses were carried by the Microanalyt-
ical Data Unit at the National Research Centre, Giza, Egypt and the
Microanalytical Data Unit at Cairo University, Giza, Egypt. The
reactions were monitored by thin layer chromatography (TLC)
which was carried out using Merck 60 F254 aluminum sheets
and visualized by UV light (254 nm). The mixtures were separated
by preparative TLC and gravity chromatography. All described
compounds showed the characteristic spectral data of cyclope-
ntanoperhydrophenanthrene nuclei of androstane series were sim-
ilar to those reported in literature.38,39 For the nomenclature of
steroid derivatives, we used the definitive rules for the nomencla-
ture of steroids published by the Joint Commission on the
Biochemical Nomenclature (JCBN) of IUPAC.40,41 Compound 3,
l
sults confirmed that, 48 h incubation time is the best incubation
time for the cytotoxic effect of the tested compounds. The present
study showed that all the tested synthetic steroid derivatives
showed significant decrease with various intensities in gene
expression of breast cancer related genes (VEGF, CYP19 and hAP-
2c). Compounds 17, 20 and 22c were more effective in this respect.
Finally, the antitumor activity displayed by these compounds may
be of interest for further studies of the mechanism of action and
toxicity profile of the promising tested compounds before applica-
tion in phase 1 of clinical study in the hope of finding more active
and selective anticancer agents.
3. Materials and methods
3.1. Chemistry
3b-acetoxy-5a-androstan-17-one was prepared by simple acetyla-
tion of epi-androsterone 1.19
Starting steroids, epi-androsterone and testosterone, were pur-
chased from Sigma Company, USA. All solvents were anhydrated
by distillation prior to using. All melting points were measured
using an Electrothermal apparatus and are uncorrected. The IR
spectra were recorded in (KBr discs) on a shimadzu FT-IR 8201
PC spectrometer and expressed in cmꢀ1. The 1H NMR and 13C
NMR spectra were recorded with Jeol instrument (Japan), at 270
and 125 MHz respectively, in DMSO-d6 as solvent and chemical
shifts were recorded in ppm relative to TMS. The spin multiplicities
3.1.1. Synthesis of 3b-acetoxy-17-ylideneamino(N-20-pyrimidy
lbenzenesulfonamide)-5
a-androstane (5)
To a suspension of 3b-acetoxy-5a-androstan-17-one (3) (0.33 g,
1 mmol) in glacial acetic acid (1.5 mL), equimolar amount of sulfa-
diazine 4 (0.25 g, 1 mmol) was added. The reaction mixture was
heated in an oil bath at 120 °C for 10 min and then left to cool at
room temperature. The fused result triturated with absolute ethanol
(5 mL) and the reaction mixture was heated again under reflux for

6868
G. A. Elmegeed et al. / Bioorg. Med. Chem. 19 (2011) 6860–6872
20 min. The solid product obtained upon cooling was collected by
filtration and crystallized from absolute ethanol to afford dark yel-
low crystals of compound 5, yield 0.53 g (94%), mp 220–222 °C, IR
triethylamine (2 mL) were heated under reflux for 10 h until all
the starting materials had disappeared as indicated by TLC. The
reaction mixture was allowed to stand overnight at room temper-
ature. The solid product that formed was collected by filtration
and crystallized from absolute ethanol to afford yellow crystals
of compound 10, yield 0.20 g (71%), mp 105–107 °C, IR (KBr,
(KBr, cmꢀ1):
m = 3354 (NH), 3037 (CH-aromatic), 2930, 2859 (CH-
aliphatic), 1734 (C@O, acetate), 1651 (C@N), 1583 (C@C), 1241
(SO2N). 1H NMR (DMSO-d6, ppm): d = 0.71 (s, 3H, CH3-19), 1.12 (s,
3H, CH3-18), 1.53 (m, 1H, C5-
aH), 2.06 (s, 3H, COCH3), 3.95 (m, 1H,
cmꢀ1):
m = 3424 (br, OH, carboxylate), 2965, 2830 (CH-aliphatic),
C3- H), 6.50 (t, 1H, J = 4.8 Hz, pyrimidine), 6.91 (d, 2H, J = 7.2 Hz,
a
1738, 1728 (2C@O), 1640 (C@N). 1H NMR (DMSO-d6, ppm):
d = 0.76 (s, 3H, CH3-19), 1.18 (s, 3H, CH3-18), 1.58 (m, 1H, C5-
aromatic-H), 7.53 (d, 2H, J = 7.2 Hz, aromatic-H), 8.40 (d, 2H,
J = 4.8 Hz, pyrimidine), 11.22 (s, 1H, SO2NH). 13C NMR (DMSO-d6,
ppm): d = 31.29 (C-1), 30.32, (C-2), 72.85 (C-3), 34.43 (C-4), 38.50
(C-5), 27.11 (C-6), 27.86 (C-7), 35.31(C-8), 50.56 (C-9), 44.00 (C-
10), 21.34 (C-11), 34.3 (C-12), 38.80 (C-13), 63.61 (C-14), 35.18 (C-
15), 33.64 (C-16), 184.3 (C-17), 19.3 (C-18), 20.02 (C-19), 170.37
(C@O), 21.10 (CH3-acetate), 112.67, 157.81, 158.74, 169.31 (C-
pyrimidine), 125.19, 129.76, 130.31, 153.51 (C-phenyl). M.S (EI):
m/z (%): 564 (M+Å, 33), 546 (C31H38N4O3S, 41), 504 (M+–CH3COOH,
40), 470 (C27H36NO4S, 35), 234 (C10H8N3O2S, 30), 109 (C8H3, 50),
67 (C3H3N2, 75), 55 (C2H3N2, 100). Calcd for C31H40N4O4S
(564.739): C, 65.90; H, 7.14; N, 9.92; S, 5.68. Found: C, 66.10; H,
7.34; N, 10.11; S, 5.93.
a
H), 1.90 (q, 2H, J = 4.3 Hz, CH2CH2S), 2.01 (s, 3H, COCH3), 2.07
(s, 3H, SCH3), 2.37 (t, 1H, J = 4.9 Hz, CHCOOH), 2.49 (t, 2H,
J = 4.8 Hz, CH2CH2S), 3.93 (m, 1H, C3- H), 8.21 (s, 1H, COOH).
a
13C NMR (DMSO-d6, ppm): d = 34.52 (C-1), 30.99, (C-2), 72.85
(C-3), 35.21 (C-4), 38.57 (C-5), 27.12 (C-6), 28.00 (C-7), 38.57
(C-8), 53.44 (C-9), 44.42 (C-10), 22.99 (C-11), 33.70 (C-12),
39.80 (C-13), 69.28 (C-14), 35.28 (C-15), 34.50 (C-16), 184.80
(C-17), 17.23 (C-18), 20.43 (C-19), 177.40 (C@O), 21.10 (CH3),
35.52 (C-10), 32.99, (C-20), 28.85 (C-30), 17.14 (SCH3). M.S (EI):
m/z (%): 463 (M+Å, 28), 418 (M+Å–COOH, 50), 416 (M+Å-SMe, 40),
301 (C20H29O2, 25), 61 (C2H5S, 100). Calcd for C26H41NO4S
(463.673): C, 67.35; H, 8.91; N, 3.02; S, 6.92. Found: C, 67.55;
H, 9.15; N, 3.25; S, 7.11.
3.1.2. Synthesis of 3b-acetoxy-17-ylideneamino(N-20-pyrimidyl
benzenesulfonamidesodium salt)-5
toxy-17-ylideneamino[20-(N-200-pyrimidylbenzenesulfonamide)
ethanol]-5 -androstane (8)
3.1.2.1. General procedure. To an aqueous solution of sodium
hydroxide (0.00354 g in 3.54 mL H2O), compound (0.5 g,
a-androstane (6) and 3b-ace
3.1.4. Synthesis of 3b-acetoxy-17-ylideneamino(10,40,50,60-
tetrahydro-30-hydroxypyridazin-40-yl)-5
a-androstane (12)
a
To a solution of compound 10 (0.2 g, 0.4 mmol) in absolute
ethanol (35 mL), hydrazine hydrate (11) (0.1 g, 2.1 mmol) was
added. The reaction mixture was heated under reflux for 15 h un-
til all the reactants had disappeared as indicated by TLC. The reac-
tion mixture poured with stirring over crushed ice. The solid
product that formed was collected by filtration and crystallized
from absolute ethanol to afford compound 12 as pale yellow crys-
5
0.88 mmol) was added, the solution was stirred and gently warmed
until all of compound 5 was dissolved and the pH of solution
dropped to about 8. The resulted solid product was collected by fil-
tration, dried and crystallized from ethanol (95%) to afford com-
pound 6 as yellow crystals, yield 0.45 g (86%). To a solution of
compound 6 (0.4 g, 0.6 mmol) in DMF (20 mL), 2-bromo ethanol 7
(0.085 g, 0.6 mmol) was added. The reaction mixture was heated
to 80 °C with stirring and maintained at this temperature for 8 h
with continued stirring. The reaction was monitored by TLC, con-
centrated under vacuum and poured over 50 mL H2O. The formed
solid product was filtered off, dried and crystallized from absolute
ethanol. Compound 8: Yellow crystals, yield 0.23 g (57%), mp
tals, yield 0.15 g (83%), mp 266–267 °C, IR (KBr, cmꢀ1):
m = 3390
(OH), 3365 (NH), 2937, 2856 (CH-aliphatic), 1732 (C@O, acetate),
1648 (C@N). 1H NMR (DMSO-d6, ppm): d = 0.75 (s, 3H, CH3-19),
1.07 (s, 3H, CH3-18), 1.42 (m, 1H, C5-aH), 1.54 (t, 1H, J = 6.4 Hz,
C-40 pyridazine), 2.03 (s, 3H, COCH3), 2.16 (t, 2H, J = 6.4 Hz, C-60
pyridazine), 2.20 (q, 2H, J = 6.4 Hz, C-50 pyridazine), 3.96 (m, 1H,
C3-aH), 4.55 (s, 1H, OH, D2O-exchangeable), 8.0 (s, 1H, 1NH,
D2O-exchangeable). 13C NMR (DMSO-d6, ppm): d = 34.52 (C-1),
30.99, (C-2), 72.85 (C-3), 35.21 (C-4), 38.57 (C-5), 27.12 (C-6),
28.00 (C-7), 38.57 (C-8), 53.44 (C-9), 44.42 (C-10), 22.99 (C-11),
33.70 (C-12), 39.80 (C-13), 69.28 (C-14), 35.28 (C-15), 34.50 (C-
16), 184.80 (C-17), 17.23 (C-18), 20.43 (C-19), 161.40 (C@O),
21.10 (CH3), 174.0, 60.30, 24.23, 48.50 (C-pyridazine). M.S (EI):
m/z (%): 429 (M+Å, 12), 369 (M+Å–CH3COOH, 14), 234 (C13H20N3O,
25), 111 (C4H5N3O, 12), 99 (C4H7N2O, 20), 55 (C3H3O, 100). Calcd
for C25H39N3O3 (429.595): C, 69.90; H, 9.15; N, 9.78. Found: C,
70.14; H, 9.42; N, 9.98.
205–206 °C, IR (KBr, cmꢀ1):
m = 3425 (OH), 3033 (CH-aromatic),
2936, 2861 (CH-aliphatic), 1733 (C@O, acetate), 1651 (C@N), 1585
(C@C), 1244 (SO2N). 1H NMR (DMSO-d6, ppm): d = 0.76 (s, 3H,
CH3-19), 1.17 (s, 3H, CH3-18), 1.47 (m, 1H, C5-aH), 2.01 (s, 3H,
COCH3), 3.86 (m, 1H, C3- H), 3.35, 3.98 (2t, 4H, J = 4.5 Hz, 2CH2-eth-
a
anol), 4.55 (t, 1H, J = 4.5 Hz, OH, D2O-exchangeable), 6.56 (dd, 1H,
J = 2.4 Hz, J = 4.2 Hz, pyrimidine), 7.58 (d, 2H, J = 9.0 Hz, aromatic-
H), 7.97 (d, 2H, J = 9 Hz, aromatic-H), 8.15 (dd, 1H, J = 2.4 Hz,
J = 4.2 Hz, pyrimidine), 8.45 (q, 1H, J = 2.4 Hz, pyrimidine). 13C
NMR (DMSO-d6, ppm): d = 31.83 (C-1), 29.01, (C-2), 73.43 (C-3),
34.43 (C-4), 38.57 (C-5), 27.11 (C-6), 27.86 (C-7), 35.83 (C-8), 51.1
(C-9), 44.53 (C-10), 21.87 (C-11), 34.3 (C-12), 38.8 (C-13), 63.61
(C-14), 35.18 (C-15), 33.64 (C-16), 184.3 (C-17), 19.3 (C-18), 20.02
(C-19), 170.37 (C@O), 21.60 (CH3), 51.12, 57.62 (CH2CH2OH),
112.67, 157.81, 158.74, 169.31 (C-pyrimidine), 125.19, 129.76,
130.31, 153.51 (C-phenyl). M.S (EI): m/z (%): 608 (M+Å, 35), 563
(M+–CH2CH2OH, 16), 548 (M+Å–CH3COOH, 14), 109 (C8H3, 30), 55
(C2H3N2, 100). Calcd for C33H44N4O5S (608.791): C, 65.10; H, 7.28;
N, 9.20; S, 5.27. Found: C, 64.87; H, 7.12; N, 9.01; S, 5.03.
3.1.5. Synthesis of 3b-acetoxy-16-bromo-5a-androstan-17-one
(14)
A mixture of compound (3) (0.33 g, 1 mmol) and cupric bromide
(13) (0.71 g, 3 mmol) in dry methanol (40 mL) was refluxed for
24 h. The formed light blue solution was left to cool at room tem-
perature and then poured over ice/water mixture. The formed solid
product was filtered off, dried and crystallized from methanol to
form compound 14 as pale green crystals. Yield 0.30 g (73%), mp
110–112 °C, IR (KBr, cmꢀ1):
1734,1728 (2C@O). 1H NMR (DMSO-d6, ppm): d = 0.77 (s, 3H,
CH3-19), 1.10 (s, 3H, CH3-18), 1.46 (m, 1H, C5- H), 2.45 (s, 3H,
H), 4.46 (t, 1H, J = 7.2 Hz, CH-16). M.S
m = 2936, 2860 (CH-aliphatic),
3.1.3. Synthesis of 2-(3b-acetoxy-5
a
-androstan-17-ylidenea
a
mino)-4-(methylthio)butanoic acid (10)
COCH3), 3.95 (m, 1H, C3-a
Equimolar amounts of 3b-acetoxy-5a-androstan-17-one (3)
(EI): m/z (%): 331 (M+Å-HBr, 30), 290 (C19H30O2, 26), 59 (C2H3O2,
100). Calcd for C21H31BrO3 (411.373): C, 61.31; H, 7.60. Found: C,
61.52; H, 7.78.
(0.20 g, 0.6 mmol) and
L-methionine (9) (0.09 g, 0.6 mmol) in
absolute ethanol (50 mL) containing
a
catalytic amount of

G. A. Elmegeed et al. / Bioorg. Med. Chem. 19 (2011) 6860–6872
6869
3.1.6. Synthesis of 3b-acetoxy-10,30-thiazolo[40,50:17,16]-
acetate), 1630 (C@N), 1590 (C@C), 1379 (NO3). 1H NMR (DMSO-
d6, ppm): d = 0.76 (s, 3H, CH3-19), 1.09 (s, 3H, CH3-18), 1.49 (m,
5a
-androstan-20-acetonitrile (17)
To a solution of compound 14 (0.15 g, 0.3 mmol), in absolute
1H, C5-
COCH3), 2.77 (m, 4H, 2CH2), 2.80 (m, 4H, 2CH2), 3.49 (s, 2H,
CH2CN), 3.90 (m, 1H, C3- H), 8.20 (s, 1H, NH, D2O-exchangeable).
aH), 2.0 (s, 4H, 2NH2, D2O-exchangeable), 2.43 (s, 3H,
ethanol (20 mL) containing
a catalytic amount of piperidine
(0.5 mL), cyanothioacetamide (15) (0.036 g, 0.3 mmol) was added.
The reaction mixture was heated under reflux for 5 h until all the
reactants had disappeared as indicated by TLC. The reaction mix-
ture was cooled, poured over ice/ water mixture, neutralized with
dilute hydrochloric acid and then left in a refrigerator at 4 °C for
24 h. The solid product that formed, was filtered off, dried and
crystallized of absolute ethanol to afford brown crystals of com-
pound 17, yield 0.09 g (60%), mp 170–171 °C, IR (KBr, cmꢀ1):
a
13C NMR (DMSO-d6, ppm): d = 32.70 (C-1), 29.01, (C-2), 73.60 (C-
3), 34.60 (C-4), 37.70 (C-5), 27.00 (C-6), 27.60 (C-7), 38.57 (C-8),
55.0 (C-9), 44.42 (C-10), 22.99 (C-11), 33.70 (C-12), 39.80 (C-13),
69.28 (C-14), 35.28 (C-15), 130.60 (C-16), 150.50 (C-17), 17.23
(C-18), 20.43 (C-19), 170.37 (C@O), 21.60 (CH3), 165.20 (C-20),
20.70 (CH2CN), 117.80 (CN), 41.70 (C-10), 51.01, (C-20). M.S (EI):
m/z (%): 703 (M+Å, 3), 507 (5), 332 (30), 273 (C19H29O, 100), 231
(C13H15N2S, 27). Calcd for C28H45CuN7O8S (703.317): C, 47.82; H,
6.45; N, 13.94; S, 4.56. Found: C, 47.55; H, 6.20; N, 13.70; S, 4.36.
m
= 2926, 2854 (CH-aliphatic), 2211 (CN), 1733 (C@O, acetate),
1642 (C@N), 1593 (C@C). 1H NMR (DMSO-d6, ppm): d = 0.87 (s,
3H, CH3-19), 1.16 (s, 3H, CH3-18), 1.57 (m, 1H, C5- H), 2.43 (s,
3H, COCH3), 3.49 (s, 2H, CH2CN), 3.87 (m, 1H, C3-
H). 13C NMR
a
a
3.1.9. Synthesis of spiro[oxiran-20,17b-5
a-androstane]
(DMSO-d6, ppm): d = 32.70 (C-1), 29.01, (C-2), 73.60 (C-3), 34.60
(C-4), 37.70 (C-5), 27.00 (C-6), 27.60 (C-7), 38.57 (C-8), 55.0 (C-
9), 44.42 (C-10), 22.99 (C-11), 33.70 (C-12), 39.80 (C-13), 69.28
(C-14), 35.28 (C-15), 130.60 (C-16), 150.50 (C-17), 17.23 (C-18),
20.43 (C-19), 170.37 (C@O), 21.60 (CH3), 165.20 (C-20), 20.70
(CH2CN), 117.80 (CN). M.S (EI): m/z (%): 411 (M+ꢁ-1, 8), 382 (M+ꢁ-
2CH3, 3), 347 (C21H30O2S, 5), 273 (C18H25O2, 85), 272 (C19H28O,
100), 55 (C3H3O, 68). Calcd for C24H32N2O2S (412.588): C, 69.87;
H, 7.82; N, 6.79; S, 7.77. Found: C, 69.60; H, 7.61; N, 6.59; S, 7.58.
derivatives (22a–c) and spiro[oxiran-20,3b-5
a-androstene]
derivatives (24a–c)
3.1.9.1. General procedure. To a solution of either epi-androster-
one (1) (0.29 g, 1 mmol) or testosterone (23) (0.28 g, 1 mmol) in
potassium tert-butoxide [prepeared by the reaction of dry tert-
butanol (30 mL) with potassium metal (0.5 g)],42 equimolar
amount of either ethyl chloroacetate (21a) (0.12 g, 1 mmol), chlo-
roacetone (21b) (0.09 g, 1 mmol), or phenacylbromide (21c)
(0.19 g, 1 mmol) was added. The reaction mixture, in each case,
was stirred at room temperature for 24 h and then heated for 6 h
in water bath at 70 °C. After cooling at room temperature, the reac-
tion mixture poured over ice/water mixture and the resulted semi-
solid was subjected to extraction with chloroform (2 ꢂ 30 mL). The
organic layer was dried over anhydrous magnesium sulfate and
then filtered. The oil product that formed in each case on removal
of the solvent in vacuum, was solidified by boiling in petroleum
ether (60–80 °C), collected by filtration and crystallized from the
appropriate solvent.
3.1.7. Synthesis of 3b-acetoxy-20-amino-10,30-thiazolo
[40,50:17,16]-5
a-androstane (18)
To a solution of compound 14 (0.15 g, 0.3 mmol) in anhydrous
tetrahydrofuran (20 mL) containing a catalytic amount of piperi-
dine (1.5 mL), equimolar amount of thiourea (16) (0.027 g,
0.3 mmol) was added with stirring. After complete addition, the
reaction mixture was stirred for 72 h at room temperature and
monitored by TLC. The reaction mixture poured over crushed ice
and then left in a refrigerator at 4 °C overnight. The solid product
was collected by filtration and crystallized from absolute ethanol.
Compound 18: Pale yellow crystals, yield 0.07 g (50%), mp 120–
3.1.9.2. Ethyl 3b-hydroxy-spiro[oxiran-20,17b-5
carboxylate (22a). Yellow crystals from absolute ethanol, yield
0.25 g (67%), mp 135–136 °C, IR (KBr, cmꢀ1):
= 3469 (OH), 2931,
2861 (CH-aliphatic), 1730 (C@O). 1H NMR (DMSO-d6, ppm):
d = 0.81 (s, 3H, CH3-19), 1.08 (s, 3H, CH3-18), 1.55 (m, 1H, C5- H),
1.27 (t, 3H, J = 7.4 Hz, ester-CH3), 2.02 (s, 1H, OH, D2O-exchange-
able), 3.47 (s, 1H, oxirane-CH), 3.85 (m, 1H, C3- H), 4.45 (q, 2H,
a
-androstan]-30-
122 °C, IR (KBr, cmꢀ1):
m
= 3420 (NH2), 2925, 2853 (CH-aliphatic),
m
1735 (C@O, acetate), 1643 (C@N), 1590 (C@C). 1H NMR (DMSO-
d6, ppm): d = 0.70 (s, 3H, CH3-19), 1.16 (s, 3H, CH3-18), 1.52 (m,
a
1H, C5-aH), 2.43 (s, 3H, COCH3), 3.93 (m, 1H, C3-aH), 4.38 (s, 2H,
NH2, D2O-exchangeable). 13C NMR (DMSO-d6, ppm): d = 32.70 (C-
1), 29.01, (C-2), 73.60 (C-3), 34.60 (C-4), 37.70 (C-5), 27.00 (C-6),
27.60 (C-7), 38.57 (C-8), 55.0 (C-9), 44.42 (C-10), 22.99 (C-11),
33.70 (C-12), 39.80 (C-13), 69.28 (C-14), 35.28 (C-15), 130.60 (C-
16), 150.50 (C-17), 17.23 (C-18), 20.43 (C-19), 170.37 (C@O),
21.60 (CH3), 165.20 (C-20), 20.70 (CH2CN), 117.80 (CN). M.S (EI):
m/z (%): 388 (M+Å, 23), 234 (C13H18N2S, 22), 178 (C9H11N2S, 2), 55
(C3H3O, 100). Calcd for C22H32N2O2S (388.567): C, 68.00; H, 8.30;
N, 7.21; S, 8.25. Found: C, 67.77; H, 8.08; N, 7.02; S, 8.04.
a
J = 7.4 Hz, ester-CH2). 13C NMR (DMSO-d6, ppm): d = 31.32 (C-1),
30.9 (C-2), 69.23 (C-3), 38.99 (C-4), 39.99 (C-5), 29.0 (C-6), 32.1
(C-7), 35.27 (C-8), 50.63 (C-9), 36.8 (C-10), 20.51 (C-11), 35.6 (C-
12), 44.36 (C-13), 53.86 (C-14), 27.1 (C-15), 22.5 (C-16), 71.4 (C-
17), 12.05 (C-18), 16.89 (C-19), 31.25 (C-10), 8.27 (C-20), 205.96
(C@O), 67.0 (C-oxirane). M.S (EI): m/z (%): 376 (M+Å, 24), 303
(M+Å-CO2Et, 26), 290 (C19H30O2, 100), 275 (C17H23O3, 14), 246
(C16H22O2, 34), 107 (56). Calcd for C23H36O4 (376.530): C, 73.37;
H, 9.64. Found: C, 73.15; H, 9.39.
3.1.8. Synthesis of 3b-acetoxy-N-(diethylenetriamine)copper(G)
dinitrite-10,30-thiazolo[40,50: 17,16]-5
a-androstane (20)
3.1.9.3. 30-Acetyl-spiro[oxiran-20,17b-5
Pale yellow crystals from dioxane, yield 0.25 g (73%), mp 125–126 °C,
IR (KBr, cmꢀ1):
= 3458 (OH), 2930, 2859 (CH-aliphatic), 1723
(C@O). 1H NMR (DMSO-d6, ppm): d = 0.71 (s, 3H, CH3-19), 1.06 (s,
3H, CH3-18), 1.46 (m, 1H, C5- H), 1.99 (s, 1H, OH, D2O-exchangeable),
2.14 (s, 3H, CH3), 3.44 (s, 1H, oxirane-CH), 3.90 (m, 1H, C3-
H). 13C
NMR (DMSO-d6, ppm): d = 32.22 (C-1), 30.5 (C-2), 70.03 (C-3),
38.45 (C-4), 39.29 (C-5), 29.2 (C-6), 32.46 (C-7), 35.20 (C-8), 51.33
(C-9), 36.8 (C-10), 20.53 (C-11), 35.6 (C-12), 44.33 (C-13), 53.86
(C-14), 27.1 (C-15), 22.5 (C-16), 72.7 (C-17), 12.05 (C-18), 17.59
(C-19), 218.23 (C@O), 34.26 (COCH3), 67.34 (C-oxirane), M.S (EI):
m/z (%): 346 (M+Å, 23), 327 (M+Å-COCH3, 16), 166 (C10H14O2, 2), 57
(C3H5O, 100). Calcd for C22H34O3 (346.504): C, 76.26; H, 9.89. Found:
C, 76, 03; 10.17.
a-androstan]-3b-ol (22b).
To a suspension of Cu(dien)(NO3)2 19 (0.14 g, 0.48 mmol) in
methanol (5 mL), a solution of compound 17 (0.2 g, 0.48 mmol)
in methanol (3 mL) was added dropwise with stirring. The reaction
mixture was stirred for 12 h at room temperature, until both of the
starting materials dissolved. The resulted blue solution was filtered
to remove any insoluble material. Anhydrous diethyl ether was
added to the filtrate dropwise with stirring until the clear solution
became cloudy. The reaction mixture was left for 24 h in a refriger-
ator at 4 °C, the solid product that formed, was isolated by filtra-
tion, washed with anhydrous diethyl ether several times, dried in
air and crystallized from methanol to afford compound 20 as dark
m
a
a
blue crystals yield, 0.25 g (73%). IR (KBr, cmꢀ1):
(NH2, NH), 2928, 2857 (CH-aliphatic), 2220 (CN), 1735 (C@O,
m = 3385–3320

6870
G. A. Elmegeed et al. / Bioorg. Med. Chem. 19 (2011) 6860–6872
3.1.9.4. 30-Benzoyl-spiro[oxiran-20,17b-5
a
-androstan]-3b-ol (22c).
3.1.10. Synthesis of 17-chloroandrost-4-en-3-one (26), 20-(3-
Orange crystals from dioxane, yield 0.30 g (75%), mp 155–156 °C, IR
(KBr, cmꢀ1):
= 3465 (OH), 3058 (CH-aromatic), 2945, 2861 (CH-ali-
phatic), 1729 (C@O), 1595 (C@C). 1H NMR (DMSO-d6, ppm): d = 0.70
(s, 3H, CH3-19), 1.06 (s, 3H, CH3-18), 1.47 (m, 1H, C5- H), 1.98 (s, 1H,
OH, D2O-exchangeable), 3.86 (m, 1H, C3- H), 4.38 (s, 1H, oxirane-
oxoandrost-4-en-17-yl-amino)benzoic acid (28a) and 20-(3-
oxoandrost-4-en-17yl-amino)-50-fluorobenzoic acid (28b)
To a solution of testosterone (23) (0.28 g, 1 mmol) in anhydrous
diethyl ether (20 mL) in ice bath, equimolar amount of thionyl
chloride (25) (0.12 g, 1 mmol) was added dropwise with stirring.
After complete addition, the reaction mixture was stirred in ice
bath for 5 h until all starting materials had disappeared as indi-
cated by TLC. Then, the reaction mixture was left at room temper-
ature over night. The solid product that formed was filtered off,
dried and crystallized from absolute ethanol to afford compound
26 as yellow crystals, yield 0.26 g (86%). Equimolar amounts of
compound 26 (0.31 g, 1 mmol) and anthranilic acid (27a)
(0.137 g, 1 mmol) or 5-fluoroanthranilic acid (27b) (0.15 g,
1 mmol) in absolute ethanol (50 mL) were refluxed for 6-8 h until
all the starting materials had disappeared as indicated by TLC. The
reaction mixture, then left to cool at room temperature, poured
over crushed ice and left in a refrigerator at 4 °C overnight. The
formed solid product, in each case, was collected by filtration
and crystallized from the appropriate solvent.
m
a
a
CH), 7.49-7.92 (m, 5H, aromatic-H). 13C NMR (DMSO-d6, ppm):
d = 31.32 (C-1), 30.9 (C-2), 69.23 (C-3), 38.99 (C-4), 39.99 (C-5), 29.0
(C-6), 32.1 (C-7), 35.27 (C-8), 50.63 (C-9), 36.8 (C-10), 20.51 (C-11),
35.6 (C-12), 44.36 (C-13), 53.86 (C-14), 27.1 (C-15), 22.5 (C-16), 71.4
(C-17), 12.05 (C-18), 16.89 (C-19), 197.05 (C = O), 67.0 (C-oxirane),
136.21, 128.7, 127.50, 133.2 (C-phenyl). M.S (EI): m/z (%): 408 (M+Å,
29), 303 (M+Å-COPh, 4), 247 (C17H27O, 4), 160 (C10H8O2, 5), 105 (PhCO,
67), 55 (C3H3O, 100). Calcd for C27H36O3 (408.573): C, 79.37; H, 8.88.
Found: C, 79.59; H, 9.17.
3.1.9.5. Ethyl 17b-hydroxy-spiro[oxiran-20,3b-androst-4-en]-30-
carboxylate (24a). Dark yellow crystals from absolute ethanol,
yield 0.23 g (62%), mp 135–137 °C, IR (KBr, cmꢀ1):
m = 3528 (OH),
3024 (CH-aromatic), 2939, 2874 (CH-aliphatic), 1736 (C@O),
1612 (C@C). 1H NMR (DMSO-d6, ppm): d = 0.72 (s, 3H, CH3-19),
1.18 (s, 3H, CH3-18), 1.36 (t, 3H, J = 7.5, ester-CH3), 1.97 (s, 1H,
Compound 28a: Dark yellow crystals from absolute ethanol, yield
0.35 g (87%), mp 110–112 °C, IR (KBr, cmꢀ1):
m = 3453 (NH), 3359
OH, D2O-exchangeable), 3.34 (t, 1H, C17
-
a
H, J = 8.8 Hz), 4.20 (s,
(OH), 3048 (CH-aromatic), 2938, 2865 (CH-aliphatic), 1686, 1675
(2C@O), 1613 (C@C). 1H NMR (DMSO-d6, ppm): d = 0.68 (s, 3H,
CH3-19), 1.15 (s, 3H, CH3-18), 2.52 (t, 1H, C17-H, J = 8.5 Hz), 4.51
(s, 1H, NH, D2O-exchangeable), 5.63 (s, 1H, C4-H), 6.53-7.40 (m,
4H, aromatic-H), 10.45 (s, 1H, OH, D2O-exchangeable). 13C NMR
(DMSO-d6, ppm): d = 35.12 (C-1), 34.20 (C-2), 198.22 (C-3), 123.0
(C-4), 171.06 (C-5), 32.60 (C-6), 31.90 (C-7), 36.20 (C-8), 49.9 (C-9),
34.9 (C-10), 23.01 (C-11), 35.40 (C-12), 40.92 (C-13), 52.90 (C-14),
24.70 (C-15), 28.40 (C-16), 65.10 (C-17), 16.20 (C-18), 22.50
(C-19), 170.50 (C@O), 113.0, 116.10, 131.10, 134.82, 151.20,
169.40 (C-phenyl). M.S (EI): m/z (%): 407 (M+Å, 28), 136 (C7H6NO2,
20), 91 (C6H5N, 25), 57 (C3H5O, 100). Calcd for C26H33NO3
(407.545): C, 76.62; H, 8.16; N, 3.44. Found: C, 76.82; H, 8.40; N, 3.65.
Compound 28b: Dark yellow crystals from absolute ethanol,
1H, oxirane-CH), 4.48 (q, 2H, J = 7.5, ester-CH2), 5.56 (s, 1H, C4-
H). 13C NMR (DMSO-d6, ppm): d = 31.95 (C-1), 31.27 (C-2), 57.70
(C-3), 123.90 (C-4), 140.03 (C-5), 33.0 (C-6), 32.1 (C-7), 35.12 (C-
8), 49.98 (C-9), 36.80 (C-10), 22.51 (C-11), 35.05 (C-12), 44.36 (C-
13), 53.42 (C-14), 27.1 (C-15), 22.50 (C-16), 79.80 (C-17), 11.18
(C-18), 16.89 (C-19), 31.25 (C-10), 8.27 (C-20), 205.96 (C@O),
63.40 (C-oxirane). M.S (EI): m/z (%): 374 (M+Å, 24), 356 (M+Å-H2O,
5), 301 (M+Å-CO2Et, 7), 289 (C19H29O2, 100), 271 (C19H27O, 2), 177
(C13H21, 0.6). Calcd for C23H34O4 (374.514): C, 73.76; H, 9.15.
Found: C, 73.56; H, 8.89.
3.1.9.6. 30-Acetyl-spiro[oxiran-20,3b-androst-4-en]-17b-ol (24b).
Pale yellow crystals from dioxane, yield 0.25 g (73%), mp 80–81 °C,
IR (KBr, cmꢀ1):
m
= 3422 (OH), 3024 (CH-aromatic), 2944, 2870
yield 0.4 g (90%), mp 100–101 °C, IR (KBr, cmꢀ1):
m = 3453 (NH),
(CH-aliphatic), 1720 (C@O), 1602 (C@C). 1H NMR (DMSO-d6, ppm):
d = 0.73 (s, 3H, CH3-19), 1.13 (s, 3H, CH3-18), 1.97 (s, 1H, OH, D2O-
3360 (OH), 3020 (CH-aromatic), 2924, 2844 (CH-aliphatic), 1690,
1672 (2C@O), 1595 (C@C). 1H NMR (DMSO-d6, ppm): d = 0.68 (s,
3H, CH3-19), 1.09 (s, 3H, CH3-18), 2.51 (t, 1H, C17-H, J = 8.8 Hz),
4.51 (s, 1H, NH, D2O-exchangeable), 5.63 (s, 1H, C4-H), 6.53-7.06
(m, 3H, aromatic-H), 10.45 (s, 1H, OH, D2O-exchangeable). 13C
NMR (DMSO-d6, ppm): d = 35.12 (C-1), 34.20 (C-2), 198.01 (C-3),
124.0 (C-4), 171.06 (C-5), 32.60 (C-6), 31.90 (C-7), 36.20 (C-8),
49.9 (C-9), 34.9 (C-10), 23.01 (C-11), 35.40 (C-12), 40.50 (C-13),
53.60 (C-14), 24.70 (C-15), 28.40 (C-16), 65.10 (C-17), 16.20 (C-
18), 22.50 (C-19), 172.20 (C@O), 115.0, 116.30, 121.10, 145.0,
151.30, 169.20 (C-phenyl). M.S (EI): m/z (%): 425 (M+Å, 36), 406
(M+Å-F, 25), 370 (C23H29FNO2), 139 (C7H4FO), 91 (C6H5N, 25), 55
(C3H3O, 100). Calcd for C26H33FNO3 (425.536): C, 73.38; H, 7.58;
N, 3.29. Found: C, 73.80; H, 7.75; N, 3.54.
exchangeable), 2.08 (s, 3H, CH3), 3.39 (t, 1H, C17-aH, J = 8.8 Hz),
4.32 (s, 1H, oxirane-CH), 5.57 (s, 1H, C4-H). 13C NMR (DMSO-d6,
ppm): d = 31.95 (C-1), 31.27 (C-2), 57.70 (C-3), 123.90 (C-4), 140.03
(C-5), 33.0 (C-6), 32.1 (C-7), 35.12 (C-8), 49.98 (C-9), 36.80 (C-10),
22.51 (C-11), 35.05 (C-12), 44.36 (C-13), 53.42 (C-14), 27.1 (C-15),
22.50 (C-16), 79.80 (C-17), 11.18 (C-18), 16.89 (C-19), 197.96
(C = O), 34.26 (COCH3), 63.40 (C-oxirane). M.S (EI): m/z (%): 344
(M+Å, 23), 301 (M+Å-COCH3, 55), 178 (C11H14O2, 25), 79 (C6H7, 100).
Calcd for C22H32O3 (344.488): C, 76.70; H, 9.36. Found: C, 76, 93; 9.61.
3.1.9.7. 30-Benzoyl-spiro[oxiran-20,3b-androst-4-en]-17b-ol (24c).
Orange crystals from dioxane, yield 0.32 g (80%), mp 120–122 °C, IR
(KBr, cmꢀ1):
m = 3526 (OH), 3058 (CH-aromatic), 2947, 2873 (CH-ali-
phatic), 1690 (C@O), 1613 (C@C). 1H NMR (DMSO-d6, ppm): d = 0.60
3.1.11. Synthesis of 17-[40(1H)-oxo-30-phenyl-20-thioxoquinaz
olin-1-yl] androst-4-en-3-one (30a), 17-[6-fluoro-40(1H)-oxo-30-
phenyl-20-thioxoquinazolin-1-yl]androst-4-en-3-one (30b)
3.1.11.1. General procedure. To a solution of either compound 28a
(0.21 g, 0.51 mmol) or compound 28b (0.22 g, 0.51 mmol), in abso-
lute ethanol (30 mL) containing a catalytic amount of triethylamine
(1 mL), phenyl isothiocyanate (29) (0.068 g, 0.51 mmol) was added.
The reaction mixture, in each case, was heated under reflux for 8–
10 h until all the reactants had disappeared as indicated by TLC.
The reaction mixture poured over crushed ice and then kept in a
refrigerator at 4 °C overnight. The solid product that formed in each
case, was filtered off, dried and crystallized from the proper solvent.
(s, 3H, CH3-19), 1.07 (s, 3H, CH3-18), 2.04 (s, 1H, OH, D2O-exchange-
able), 3.45 (t, 1H, C17-aH, J = 8.6 Hz), 4.41 (s, 1H, oxirane-CH), 5.56
(s, 1H, C4-H), 7.49–7.92 (m, 5H, aromatic-H). 13C NMR (DMSO-d6,
ppm): d = 31.95 (C-1), 31.27 (C-2), 57.70 (C-3), 123.90 (C-4), 140.03
(C-5), 33.0 (C-6), 32.1 (C-7), 35.12 (C-8), 49.98 (C-9), 36.80 (C-10),
22.51 (C-11), 35.05 (C-12), 44.36 (C-13), 53.42 (C-14), 27.1 (C-15),
22.50 (C-16), 79.80 (C-17), 11.18 (C-18), 16.89 (C-19), 197.96 (C@O),
63.40 (C-oxirane), 136.21, 127.70, 128.80, 133.20 (C-phenyl). M.S
(EI): m/z (%): 406 (M+Å, 29), 301 (M+Å-COPh, 5), 246 (C17H27O, 12), 124
(C8H12O, 41), 105 (PhCO, 23), 57 (C3H5O, 100). Calcd for C27H34O3
(406.557): C, 79.76; H, 8.43. Found: C, 79.99; H, 8.72.

G. A. Elmegeed et al. / Bioorg. Med. Chem. 19 (2011) 6860–6872
6871
Compound 30a: Pale yellow crystals from absolute ethanol, yield
0.20 g (74%), mp 308–310 °C, IR (KBr, cmꢀ1):
= 3048 (CH-aro-
cell pellet was suspended and seeded in 5 mL supplemented med-
ium in T25 Nunclon sterile tissue culture flasks. The cell suspen-
sion was incubated and followed up daily with replacing the
supplemented medium every 2–3 days. Incubation was continued
until a confluent growth was achieved and the cells were freshly
sub-cultured before each experiment.
The medium was discarded and the cell monolayer was washed
twice with 5 mL phosphate buffer saline. All the adherent cells
were dispersed from their monolayer by the addition of 1 mL tryp-
sin solution (0.025% trypsin w/v) for 2 min. The flask was left in the
incubator till complete detachment of all the cells and checked
with the inverted microscope (Olympus 1x70, Tokto, Japan). Tryp-
sin was inactivated by the addition of 5 mL of the supplemented
medium. The trypsin content was discarded by centrifugation at
1200 rpm for 10 min. The supernatant was discarded and the cells
were separated into single cell suspension by gentle dispersion
several times, then suspended and seeded in 5 mL supplemented
medium in T25 Nunclon sterile tissue culture flasks.
m
matic), 2976, 2846 (CH-aliphatic), 1683 (C@O), 1652 (C@O, amide),
1598 (C@C), 1199 (C@S). 1H NMR (DMSO-d6, ppm): d = 0.68 (s, 3H,
CH3-19), 1.15 (s, 3H, CH3-18), 2.55 (t, 1H, C17-H, J = 8.2 Hz), 5.63 (s,
1H, C4-H), 6.95–7.84 (m, 9H, aromatic-H). 13C NMR (DMSO-d6,
ppm): d = 35.12 (C-1), 33.60 (C-2), 198.01 (C-3), 123.23 (C-4),
171.06 (C-5), 32.60 (C-6), 31.90 (C-7), 36.20 (C-8), 49.90 (C-9),
34.90 (C-10), 23.01 (C-11), 35.05 (C-12), 41.40 (C-13), 53.40 (C-
14), 25.90 (C-15), 22.50 (C-16), 79.80 (C-17), 16.20 (C-18), 22.50
(C-19), 160.90 (C@O), 177.0 (C@S), 121.75, 123.94, 124.05,
124.79, 126.67, 128.97, 129.04, 132.17, 137.60, 159.0 (C-phenyl).
M.S (EI): m/z (%): 524 (M+Å, 35), 270 (C19H26O2, 24), 92 (C6H6N,
100), 77 (C6H5, 14). Calcd for C33H36N2O2S (524.716): C, 75.54; H,
6.92; N, 5.34; S, 6.11. Found: C, 75.32; H, 6.75; N, 5.62; S, 6.37.
Compound 30b: Pale yellow crystals from absolute ethanol,
yield 0.23 g (85%), mp 85–86 °C, IR (KBr, cmꢀ1):
m = 3029 (CH-aro-
matic), 2963, 2856 (CH-aliphatic), 1687 (C@O), 1661 (C@O, amide),
1529 (C@C), 1195 (C@S). 1H NMR (DMSO-d6, ppm): d = 0.68 (s, 3H,
CH3-19), 1.08 (s, 3H, CH3-18), 2.51 (t, 1H, C17-H, J = 8.7 Hz), 5.48 (s,
1H, C4-H), 6.79-7.64 (m, 8H, aromatic-H). 13C NMR (DMSO-d6,
ppm): d = 35.12 (C-1), 33.60 (C-2), 198.01 (C-3), 123.23 (C-4),
171.06 (C-5), 32.60 (C-6), 31.90 (C-7), 36.20 (C-8), 49.90 (C-9),
34.90 (C-10), 23.01 (C-11), 35.05 (C-12), 41.40 (C-13), 53.40 (C-
14), 25.90 (C-15), 22.50 (C-16), 79.80 (C-17), 16.20 (C-18), 22.50
(C-19), 160.90 (C@O), 177.0 (C=S), 121.75, 121.94, 123.05, 124.79,
128.67, 128.97, 129.04, 132.17, 137.60, 159.0 (C-phenyl). M.S
(EI): m/z (%): 542 (M+Å, 22), 523 (M+Å-F, 25), 312 (C17H3FN2OS,
26), 270 (C19H26O2, 24), 92 (C6H6N, 100), 77 (C6H5, 14). 55
(C3H3O, 100). Calcd for C33H35FN2O2S (542.707): C, 73.03; H,
6.50; N, 5.16; S, 5.91. Found: C, 73.29; H, 6.75; N, 5.43; S, 6.17.
3.2.3. Determination and counting of viable cells
Trypan blue solution (50 lL of 0.05%) was added to 50 lL of the
single cell suspension. The cells were examined under the inverted
microscope using the haemocytometer. Non stained (viable) cells
were counted using the following equation:
Viable cells=mL ¼ number of cells in 4 quarters
ꢂ 2 ðdilution factorÞ ꢂ 104=4
The cells were then diluted to give the concentration of single
cell suspension required for each experiment.
3.2.4. Growth inhibition assay
The cytotoxic effect of Dox and the tested compounds was
investigated using SRB assay. The human breast cancer (MCF-7)
cells were used when 90% confluence was reached in T25 flasks.
The cells were seeded in 96-well microplates, after the cell concen-
3.2. In vitro cytotoxic assay
3.2.1. Chemicals
Dimethylsulfoxide (DMSO), Sodium bicarbonate, Fetal Bovine
Serum (FBS), Penicillin/Streptomycin, Trypan blue, Trypsin, Sul-
phorhodamine-B (SRB), Trichloroacetic acid (TCA), Acetic acid, iso-
propanol, and Ethanol (70%) were purchased from Sigma Chemical
Co. (St. Louis, MO, USA). A stock solution of TCA 50% was prepared
trations were adjusted to (5 ꢂ 104–105 cells/well) in 100
lL RPMI-
1640 culture medium and warmed at 37 °C and supplemented
with penicillin/streptomycin and FBS. The cells were treated with
Dox or either one of the tested compounds which were dissolved
individually in DMSO in four concentrations (5, 12.5, 25, 50 lg/
mL) and re-incubated for 24, 48 and 72 h. Control cells were trea-
and stored 50 lL of the stock was added to 200 lL RPMI-1640
medium/well to yield a final concentration of 10% used for protein
ted with vehicle alone and for each drug concentration, 6 wells
precipitation.
were used. Then the cells were fixed with 50
acetic acid for 1 h at 4 °C. Wells were washed 5 times with distilled
water and stained for 30 min at room temperature with 50 L 0.4%
SRB dissolved in 1% acetic acid. The wells were then washed 4
times with 1% acetic acid. The plates were air-dried and the dye
lL cold 50% trichloro-
RPMI-1640 medium (Sigma Chemical Co., St. Louis, Mo, and
USA): The medium was used for culturing and maintenance of
the human tumor cell lines. It was prepared as follows: 10.4 g med-
ium was weighed, mixed with 2 g sodium bicarbonate, completed
to 1 L with distilled water and shaked carefully till complete disso-
lution. The medium was then sterilized by filtration in a Millipore
l
was solubilized with 100 lL/well of 10 mM tris base (pH 10.5)
for 5 min on a shaker (Orbital shaker OS 20, Boeco, Germany) at
1600 rpm. The optical density (O.D.) of each well was measured
spectrophotometrically at 564 nm with an ELIZA microplate reader
bacterial filter (0.22 lM). The prepared medium was kept in a
refrigerator (4 °C) and checked at regular intervals for contamina-
tion. Buffers: Tris base 10 mM (pH 10.5): It was used for SRB dye
solubilization. 121.1 g of tris base was dissolved in 1000 mL of dis-
tilled water and pH was adjusted by HCl acid (2 M).
(Meter tech.
R 960, USA). The results were expressed as percent of
cell growth inhibition compared with the control and calculated
with the following equation:43
ꢀ
ꢁ
3.2.2. Cell propagation and maintenance
Absorbance of treated
Absorbance of control
1 ꢀ
ꢂ 100
Breast cancer cell lines (MCF-7) were obtained in liquid nitro-
gen (ꢀ180 °C) from the American Type Culture Collection. The tu-
mor cell lines were maintained in the National Cancer Institute,
Cairo, Egypt. A cryotube containing frozen cells was taken out of
the liquid nitrogen container and then thawed in a water bath at
37 °C. The cryotube was opened under strict aseptic conditions
and its contents were supplied by 5 mL supplemented medium.
The tube was incubated for 2 h then its contents were centrifuged
at 1200 rpm for 10 min. The supernatant was discarded and the
3.3. Gene expression analysis
According to Brun et al.44 and Kronmiller et al.45 serial analysis
of gene expression via semi-quantitative reverse transcription
polymerase chain reaction (RT–PCR) were used for mRNA analysis
to determine the expression level of the selected genes.

6872
G. A. Elmegeed et al. / Bioorg. Med. Chem. 19 (2011) 6860–6872
3.3.1. First-strand cDNA synthesize using extracted RNA
References and notes
After cytotoxicity study, 48 h incubation time cell samples which
stored at ꢀ80 °C prior to extraction were used to extract total RNA.
The total RNA was isolated using the BioFlux RNA Extraction Kit
(China). Total RNA pellets were dissolved in DNase, RNase-free
water and quantitated using a spectrophotometer. The quality of
RNA samples was determined by electrophoresis through agarose
gels and staining with ethidium bromide; the 18S and 28S rRNA
bands were visualized under ultraviolet light. To synthesize the
1. Anderson, B. O.; Yip, C. H.; Smith, R. A.; Shyyan, R.; Sener, S. F.; Eniu, A.; Carlson,
R. W.; Azavedo, E.; Harford, J. Cancer 2008, 113, 2221.
2. Porter, P. N. Eng. J. Med. 2008, 358, 213.
3. Gediya, K. L.; Njar, C. V. Expert Opin. Drug Disc. 2009, 4, 1099.
4. Preobrazhenskaya, M. N. Chem. Heterocycl. Compd. 1985, 21, 13.
5. Kidwai, M.; Venkataramanan, R.; Mohan, R.; Sapra, P. Curr. Med. Chem. 2002, 9,
1209.
6. Mohamed, N. R.; Elmegeed, G. A.; Abdelhalim, M. M.; Rady, H. M. J. Phosphorus,
Sulfur Silicon 2010, 185, 848.
7. El-Far, M.; Elmegeed, G. A.; Eskander, E. F.; Rady, H. M.; Tantawy, M. A. Eur. J.
Med. Chem. 2009, 44, 3936.
first-strand cDNA, 5
samples was reverse transcribed into cDNA in a total volume of
20 L using 1
L oligo (poly(deoxythymidine)) primer.44 The com-
l
g of the complete Poly(A)+ RNA isolated from
8. Thibeault, D.; Roy, J.; De Roy, P.; Poirier, D. Bioorg. Med. Chem. 2008, 16, 5062.
ꢁ
´
´
´
9. Evgenija, A.; Djurendi, C.; Marija, N.; Sakaꢀc, M.; Zavis, M. P.; Gakovic, A. R.; Cꢀ
l
l
anadia, J. J.; Andric´, S. A.; Klisuric´, O. R.; Kojic´, V. V.; Bogdanovic, G. M.;
position of the reaction mixture consisted of 50 mM MgCl2, 10ꢂ re-
PenovGaꢀsi, K. M. Steroids 2008, 73, 681.
10. Marone, M.; Mozzetti, S.; De Ritis, D.; Pierelli, L.; Scambia, G. Biol. Proceed.
Online 2001, 3, 19.
verse transcription (RT) buffer (50 mM KCl; 10 mM Tris–HCl; pH
8.3), 200 U/
dNTP, and 50
l
L reverse transcriptase (RNase H free), 10 mM of each
M of oligo (dT) primer. The RT reaction was carried
11. Ferrara, N. Kidney Int. 1999, 56, 794.
l
12. Bruggemeier, R. W.; Hackett, J. C.; Diaz-Cruz, E. S. Endocr. Rev. 2005, 26, 331.
13. Su, B.; Mershon, S. M.; Stonerock, L. A.; Curley, R. W., Jr.; Bruggemeier, R. W. J.
Steroid Biochem. Mol. Biol. 2008, 109, 40.
14. Bosher, J. M.; Totty, N. F.; Hsuan, J. J.; Williams, T.; Hurst, H. C. Oncogene 1996,
13, 1701.
out at 25 °C for 10 min, followed by 1 h at 42 °C, and finished with
denaturation step at 99 °C for 5 min. Afterwards the reaction tubes
containing RT preparations were flash-cooled in an ice chamber un-
til being used for DNA amplification through RT-PCR.44,45
15. Ghorab, M. M.; Ragab, F. A.; Heiba, H. I.; Arafa, R. K.; El-Hossary, E. M. Eur. J.
Med. Chem. 2010, 45, 3677.
16. Alqasoumi, S. I.; Al-Taweel, A. M.; Alafeefy, A. M.; Noaman, E.; Ghorab, M. M.
Eur. J. Med. Chem. 2010, 45, 738.
17. Lattmann, E.; Ayuko, W. O.; Kinchinaton, D.; Langley, C. A.; Singh, H.; Karimi, L.;
Tisdale, M. J. J. Pharm. Pharmacol. 2003, 55, 1259.
3.3.2. RT–PCR assay
The first strand cDNA from different cell line samples was used
as templates for the semi-quantitative RT–PCR with a pair of spe-
18. Amr, A. E.; Mohamed, A. M.; Mohamed, S. F.; Abdel-Hafiz, N. A.; Hammam, A. G.
Bioorg. Med. Chem. 2006, 14, 5481.
19. Furniss, B. S.; Hannaford, A. J.; Rogers, V.; Smith, P. W. G.; Tatchell A. R. Vogel’s
Text Book Of Practical Organic Chemistry Including Qualitative Organic Analysis
(4th ed.), Longman Group Ltd, Longman House, Burnt Mill, Harlow, Essex,
CM20 2JE, England, 1978, p. 1076.
20. Tanitame, A.; Oyamada, Y.; Ofuji, K.; Fujimoto, M.; Suzuki, K.; Ueda, T., et al
Bioorg. Med. Chem. 2004, 12, 5515.
21. Hackett, J. C.; Kim, Y. W.; Su, B.; Brueggemeier, R. W. Bioorg. Med. Chem. 2005,
13, 4063.
cific primers in a 25 lL reaction volume. The quantitative values
of RT–PCR of VEGF (VEGF-F: 50-TCG GGC CTC CGA AAC CAT GA-30,
VEGF-R: 50-CCT GGT GAG AGA TCT GGT TC-30), Cyp19 (Cyp19-F:
50-TGT CTC TTT GTT CTT CAT GCT ATT TCT C-30, Cyp19-R: 50-TCA
CCA ATA ACA GTC TGG ATT TCC-30) and hAP-2
c
(hAP-2
c
-F: 50-
CCA GAC CTC ATC TTG GAG GAC-30, hAP-2
c
-R: 50-CAG CTG GAC
TCT GGT CTC CAG-30) genes were normalized on the bases of b-actin
(b-actin-F: 50-TTG CCG ACA GGA TGC AGA A-30, b-actin-R: 50-GCC
GAT CCA CAC GGA GTA CT-30) expression. The reaction mixture
for RT–PCR was consisted of 10 mM dNTP0s, 50 mM MgCl2, 10ꢂ
22. Wong, F.; Chen, C.; Chen, T.; Huang, J.; Fangd, H.; Yehb, M. Steroids 2006, 71, 77.
23. Raper, E. S. Coord. Chem. Rev. 1994, 129, 91.
24. Metzger, J. V.; Katritzky, A. B.; Rees, C. W.; Potts, K. T. Comprehensive
Heterocyclic Chemistry; Pergamon Press: Oxford, 1984. p. 235, Part 4B.
25. Desoize, B. Crit. Rev. Oncol. Hematol. 2002, 42, 213.
26. Ronconi, L.; Sadler, P. J. Coord. Chem. Rev. 2007, 251, 1633.
27. Bolos, C. A.; Papazisis, K. T.; Kortsaris, A. H.; Voyatzi, S.; Zambouli, D.;
Kyriakidis, D. A. J. Inorg. Biochem. 2002, 88, 25.
28. Chaviara, A. T.; Christidis, P. C.; Papageorgiou, A.; Chrysogelou, E.; Hadjipavlou-
Litina, D. J.; Bolos, C. A. J. Inorg. Biochem. 2005, 99, 2102.
29. Gella, I. M.; Gladkova, A. I.; Zolotukhina, V. A.; Karpenko, N. A. Pharm. Chem. J.
1995, 29, 753.
30. Cook, C. E.; Corley, R. C.; Wall, M. E. J. Org. Chem. 1968, 33, 2789.
31. Yoshida, S.; Honda, A.; Matsuzaki, Y.; Fukushima, S.; Tanaka, N.; Takagiwa, A.;
Fujimoto, Y.; Miyazaki, H.; Salen, G. Steroids 2003, 68, 73.
32. Dhawan, A.; Kayani, M. A.; Parry, J. M.; Parry, E.; Anderson, D. Mutagenesis
2003, 18, 487.
PCR buffer (50 mM KCl; 20 mM Tris–HCl; pH 8.3), 1 U/lL taq poly-
merase, and autoclaved water. The PCR cycling parameters of the
studied genes were performed as follows: VEGF: 40 cycles: 95 °C,
10 min; 94 °C, 30 s; 59 °C, 1 min; 68 °C, 2 min; 68 °C, 7 min;
CYP19: 50 cycles: 95 °C, 10 min; 95 °C, 15 s; 60 °C, 1 min; 72 °C,
1 min; 72 °C, 7 min, hAP-2-c: 20 cycles: 94 °C, 5 min; 94 °C,
1 min; 65 °C, 2 min; 72 °C, 30 s; 72 °C, 7 min, and b-actin: 40 cycles:
95 °C, 15 min; 94 °C, 15 s; 60 °C, 30 s; 72 °C, 1 min; 72 °C, 7 min.
The PCR products (VEGF: 400 bp; CYP19: 278 bp; hAP-2-c: 306 bp
and b-actin; 165 bp) were then loaded onto 2.0% agarose gel, with
PCR products derived from b-actin of the different samples.
33. Duffaud, F.; Orsière, T.; Baciuchka-Palmaro, M.; Digue, L.; Favre, R.; Botta, A.
Ann. Biol. Clin. (Paris) 1998, 56, 183.
3.4. Statistical analysis
34. Ramakrishnan, R.; Zell, J. A.; Malave, A.; Rathinavelu, A. Biochem. Biophys. Res.
Commun. 2000, 270, 709.
35. Kirma, N.; Gill, K.; Mandava, U.; Tekmal, R. R. Cancer Res. 2001, 61, 1910.
36. Yang, C.; Yu, B.; Zhou, D.; Chen, S. Oncogene 2002, 21, 2854.
37. Li, M.; Wang, Y.; Yu, Y.; Nishizawa, M.; Nakajima, T.; Ito, S.; Kannan, P. Gene
2002, 301, 43.
38. Gacs-Baitz, E.; Minuti, L.; Taticchi, A. Synop. 1996, 27, 324.
39. Frenkel, M.; Marsh, K.N. TRC Data Series: Spectral Data for Steroids, College
Station, Thermodynamics Research Center, Texas, TX, 1994, p 386.
40. IUPAC, Joint Commission on Biochemical Nomenclature (JCBN), Pure Appl.
Chem. 1989, 61, 1783.
The data were analyzed using version 11.0 of SPSS, 2001. All the
data are expressed as mean standard deviation. Analysis of the
data was done using Anova One Way to detect the significant dif-
ference among the studied compounds. A level of P >0.05 was de-
fined as statistically significant.
Acknowledgments
41. IUPAC, Joint Commission on Biochemical Nomenclature (JCBN), Eur. J. Biochem.
1989, 186, 429.
42. Johnson, W. S.; Schneider, W. P. Org. Synth. 1963, 4, 132.
The authors express sincere appreciation to Professor Dr. Selim
F. Estefan, Hormones Dept., National Research Centre, Egypt, for
critically reviewing the manuscript. The authors express their
thanks to National Cancer Institute, Cairo, Egypt for conducting
cytotoxic investigations in this study. The authors acknowledge
the financial support of the National Research Centre, Egypt (grant
no.: E-8040505-2008/2010).
´
´
43. Sakac, Mꢀ.; Gakovic, A.; Stojanovic, S.; Djurendic, E.; Kojic, V.; Bogdanovic, G.;
´
´
´
Penov Gasꢀi, K. Bioorg. Chem. 2008, 36, 128.
44. Brun, M. E.; Gasca, S.; Girard, C.; Bouton, K.; De Massy, B.; De Sario, A. Gene
2006, 371, 25.
45. Kronmiller, J. E.; Nguyen, T.; Berndt, W.; Wickson, A. Arch. Oral. Biol. 1995, 40,
831.