Development of new hetero-steroid hybrids with antiproliferative activity against MCF-7 breast cancer cells

Article abstract of DOI:10.1007/s00706-020-02716-0

Abstract: In continuation of our efforts to develop new antiproliferative agents that could be effective and selective in treatment of cancer, we designed and synthesized new hybrid structures containing an arylsulfonamide scaffold linked to a steroidal skeleton through a 1,2,3-triazole ring. Both in vitro cytotoxicity on breast MCF-7 cancer cells and human placental aromatase enzyme (pAROM) inhibition assays were performed on new hybrids. All new hybrids showed marked cytotoxic activity against breast MCF-7 cancer cells (IC₅₀ = 3.56–43.76?μM) in comparison to staurosporine (IC₅₀ = 4.06?μM). Tumor selectivity index was higher for some of the new hybrids on normal fibroblast (F-180) cells (TS = 1.5–25) in comparison to staurosporine (TS = 2.5). The p-nitro derivative exhibited the best inhibitory activity on the pAROM with an IC₅₀ of 64.6?ng/cm³, compared to hybrids unsubstituted derivative, p-bromo derivative, and letrozole (IC₅₀ = 375.14, 269.86, and 132.86?ng/cm³, respectively). Furthermore, the p-nitro hybrid arrested the cell cycle selectively at the G2/M phase, in addition to inducing both early and late apoptotic processes of breast MCF-7 cancer cells. Molecular docking studies were performed within pAROM to explore the binding modes of the new hybrids. Collectively, the antiproliferative profile of new hybrids indicates how good they are as promising leads for developing tumor-specific cytotoxins, and deserve further studies to optimize their structure and in vivo activity. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-020-02716-0

Monatshefte für Chemie - Chemical Monthly (2021) 152:137–149
https://doi.org/10.1007/s00706-020-02716-0
ORIGINAL PAPER
Development of new hetero‑steroid hybrids with antiproliferative
activity against MCF‑7 breast cancer cells
Muhamad Mustafa1 · Ahmed El‑Kardocy2 · Yaser A Mostafa3
Received: 9 July 2020 / Accepted: 19 November 2020 / Published online: 8 January 2021
© Springer-Verlag GmbH Austria, part of Springer Nature 2021
Abstract
In continuation of our eforts to develop new antiproliferative agents that could be efective and selective in treatment of
cancer, we designed and synthesized new hybrid structures containing an arylsulfonamide scafold linked to a steroidal
skeleton through a 1,2,3-triazole ring. Both in vitro cytotoxicity on breast MCF-7 cancer cells and human placental aro-
matase enzyme (pAROM) inhibition assays were performed on new hybrids. All new hybrids showed marked cytotoxic
activity against breast MCF-7 cancer cells (IC50 =3.56–43.76 μM) in comparison to staurosporine (IC50 =4.06 μM). Tumor
selectivity index was higher for some of the new hybrids on normal fbroblast (F-180) cells (TS=1.5–25) in comparison to
staurosporine (TS=2.5). The p-nitro derivative exhibited the best inhibitory activity on the pAROM with an IC50 of 64.6 ng/
cm3, compared to hybrids unsubstituted derivative, p-bromo derivative, and letrozole (IC50 =375.14, 269.86, and 132.86 ng/
cm3, respectively). Furthermore, the p-nitro hybrid arrested the cell cycle selectively at the G2/M phase, in addition to induc-
ing both early and late apoptotic processes of breast MCF-7 cancer cells. Molecular docking studies were performed within
pAROM to explore the binding modes of the new hybrids. Collectively, the antiproliferative profle of new hybrids indicates
how good they are as promising leads for developing tumor-specifc cytotoxins, and deserve further studies to optimize their
structure and in vivo activity.
Graphic abstract
Keywords Aromatase inhibitors · Steroidal hybrids · Anti-tumor agents · Flow cytometry · Molecular docking
Electronic supplementary material The online version of this
Introduction
article (https://doi.org/10.1007/s00706-020-02716-0) contains
supplementary material, which is available to authorized users.
Cancer is still globally a major health problem that afected a
* Yaser A Mostafa
large number of people around the world [1–3]. Its high mor-
bidity and mortality rates come next after cardiac disease
[4]. The pivotal role of the aromatase enzyme in the aroma-
tization of androgens to estrogens, and the correlation of the
high level of estrogens with stimulation of the progression
and metastasis of hormone-dependent breast cancer (HDBC)
in both pre- and post-menopausal women, targeted the use
of the apoptogenic activity of the aromatase inhibitors (AIs)
y3abdelh@uwaterloo.ca
1
Medicinal Chemistry Department, Faculty of Pharmacy,
Deraya University, Minia 61111, Egypt
2
Faculty of Pharmacy, Student Research Center, Assiut
University, Assiut 71526, Egypt
3
Pharmaceutical Organic Chemistry Department, Faculty
of Pharmacy, Assiut University, Assiut 71526, Egypt
Vol.:(0123456789)
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M. Mustafa et al.
Fig. 1 Chemical structures of tamoxifen and 4-adione
Fig. 3 Triazole AIs 7, designed by Touaibia group
Fig. 4 Pingaew indole AIs 8, bearing sulfonamide scafold
to drug therapy, also to decrease to large extent the risk
of drug-drug interactions and improve patient compliance
by reduction of medications’ number [13, 14]. Conjugates
as a class of DMLs are designed to have their molecular
scafolds of each biological target separated by a distinct
linker group [15, 16]. Dual aromatase-sulfatase inhibitors
(DASIs) are a widespread class of conjugates which tar-
geting estrogen receptors (ERs) [17]. Anastrazole (5) and
letrozole (6) are DASIs that clinically used as drugs for
the treatment of HDBC [17]. Triazoles are well-known
pharmacophores in a diverse range of biologically-active
molecules because of their potential biomolecular char-
acteristics such as the capability of H-bond formation
and metabolic stability [18, 19]. Touaibia group reported
an equipotent set of 1,4-disubstituted-1,2,3-triazoles 7,
designed-based on letrozole and anastrazole antiprolifera-
tive profle [20] (Fig. 3).
Fig. 2 Chemical structures of steroidal and non-steroidal AIs
a useful strategy in the treatment of HDBC [5–7]. Inter-
estingly, the selective inhibition of the aromatase enzyme
does not interfere with the other steroids biosynthesis and
generally well-tolerated with minimal side efects on uterus
[8, 9]. 4-Hydroxyandrostenedione (4-adione, 1) was found
to be one of the most potent AIs with higher efcacy and
fewer side efects than other remedies previously used for
the treatment of HDBC such as tamoxifen (2) [10] (Fig. 1).
Steroidal (type I) AIs such as fulvestrant (3), exemes-
tane (4); and non-steroidal (type II) AIs like anastrazole (5)
and letrozole (6); that clinically used and approved by the
US Food and Drug Administration (FDA) for treatment of
HDBC in post-menopausal women [11] (Fig. 2).
Discovery of novel cytotoxic agents using naturally bio-
active compounds as a building block is one of the attrac-
tive avenue in drug development these days. Moreover,
designed multiple ligands (DML) have proved their efec-
tiveness as an attractive alternative for either combination
therapies or single target ligands [12]. DMLs widely used
now to eliminate resistance developed by cancerous tissue
Pingaew et al. reported 34 new indoles 8, bearing sul-
fonamide moiety to explore the crucial structural features
that should be fulflled in building AIs (such as triazole
N-atom, sulfonamide moiety, –NO2 group, hydrophobicity,
H-bonding, etc.) and required for interaction with various
amino acid residues within aromatase enzyme active site
[21–23] (Fig. 4).
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Development of new hetero-steroid hybrids with antiproliferative activity against MCF-7…
139
Fig. 5 Ghorab non-steroidal
sulfonamide AIs
between the sulfonyl group oxygen and aromatic skeleton
with Met 374, Cys 437, Ala 306, and Ala 307 and their
correlation with inhibition of aromatase catalytic activity
[25].
Moreover, Ghosh et al. succeed to co-crystalize the
human placental aromatase enzyme (CYP19) with a highly
potent inhibitor, compound 11 (HDDG046), after using
the step-by-step chemical modifcation of both 4-adione
and exemestane skeletons as the main building block for
building such potent inhibitor [26] (Fig. 6).
From all these interesting fndings, we think likely to
design new conjugate in which the non-steroidal part of
recently reported pAROM inhibitors by Ghorab, Touaibia,
and Ghosh replaced by the steroidal ring of 17β-estradiol
(E2), while preserving both the sulfonamide and 1,2,3-tri-
azole moieties, could be a promising avenue to develop
potent human pAROM inhibitors which capable of ftting
better within pAROM active site and accommodate its
large hydrophobic pocket, as shown in Fig. 7.
Fig. 6 Steroidal inhibitor of pAROM (HDDG046, 11) built by Ghosh
On the same avenue and based on Pingaew fndings,
Ghorab et al. developed potent and selective aromatase
inhibitors, compounds 9 and 10, with apoptosis induction
capability [24] (Fig. 5). In addition, Ghorab performed
molecular docking studies within aromatase active site
to explore the H-bonding and hydrophobic interactions
Fig. 7 Design of the target hybrids 15a–15f from literature cytotoxic scafolds
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140
M. Mustafa et al.
In vitro cytotoxic activity
Results and discussion
Hybrids 15a–15f were tested for their cytotoxicity on
breast cancer MCF-7 cells. Being a matter of concentration
dependence, the percentages of cell viability declined signif-
icantly upon increasing the concentration of the new hybrids
15a–15f as shown in Table 1 (as shown in dose–response
curves in supporting information).
The intermediates and target hybrids were prepared
according to the sequence of reactions shown in Scheme 1.
Intermediate 13, the azidobenzenesulfonyl chloride, was
prepared from sulfanilic acid (12) according to reported
methods used for the preparation of various azides and
sulfonyl chlorides of diferent sulfonic acids [27, 28]. On
the other hand, one of the intermediates (intermediate
14a) was reported in 2011 by Mirian et al. [29], while
the other fve intermediates 14b–14f were reported by
our group [30]. The target 1,2,3-triazole sulfonamides
15a–15f were prepared in high yields via Cu(I)-catalyzed
azide-alkyne cycloaddition (CuAAC) reaction using the
catalytic CuSO4/Na ascorbate mixture in THF/H2O mix-
ture. Spectral characterization of hybrids 15a–15f was
All the new hybrids 15a–15f suppressed MCF-7 cells
viability by a percentage exceeds 50% at 100 μM concen-
tration. The MTT assay results demonstrated that hybrid 15d
which carries a nitro group at para position possessed the
best inhibitory activity on MCF-7 cells with an IC50 value of
3.57 μM compared to the reference drug staurosporine [stau-
rosporine (STS) was used as a clinically broad-spectrum
cytotoxic drug that has shown efectiveness in in vitro mod-
els of various tumors such as colon, leukemia, and breast
cancer; IC50 =4.06 μM], as shown in Table 1.
1
done using both H and 13C NMR, while their purity
was assessed via C,H,N elemental analyses and results
1
Hybrids 15a and 15b showed equipotency to each other
(IC50 values of 11.02 and 11.20 μM, respectively) which
is weaker then hybrid 15d and reference drug bust still in
the low micromolar range. Moreover, hybrids 15c, 15e, and
15f showed weaker inhibitory activity on MCF-7 than all
other hybrids and reference drug (IC50 = 37.9, 22.4, and
43.7 μM, respectively). The obtained results revealed that
the cytotoxic activity was relatively decreased with p-Br
and p-OCH3 groups (hybrids 15c and 15e, respectively).
Interestingly, incorporation of a NO2 group as with hybrid
15d boosted the cytotoxic activity signifcantly, although,
were within an acceptable range of 0.4. The H NMR
spectra of the prepared cycloadducts 15a–15f showed
the characteristic broad singlet signal of the sulfonamide
group above δ = 10.00 ppm (except with nitro derivatives
15d and 15f it appeared above 11.00 ppm), also, all the
new hybrids 15a–15f showed a common singlet signal at
8.61–8.66 ppm corresponding to the –CH of the newly
formed 1,2,3-triazole ring. Moreover, 13C NMR spectra
of all the new hybrids were fundamentally distinguishable
according to various anilines used.
Scheme 1
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Development of new hetero-steroid hybrids with antiproliferative activity against MCF-7…
141
Table 1 Dose-dependent
Compound
% Viabilitya
100 μM
inhibition of breast MCF-7
cancer cells via hybrids 15a–15f
25 μM
6.25 μM
1.56 μM
0.39 μM
Ref.b
15a
15b
15c
15d
15e
15f
28.48 0.007
34.22 0.003
33.24 0.011
41.21 0.008
28.63 0.012
39.19 0.007
45.01 0.005
35.92 0.005
39.82 0.005
44.87 0.004
54.79 0.005
36.27 0.004
50.73 0.004
54.27 0.004
47.51 0.006
54.30 0.008
53.12 0.008
62.63 0.008
45.45 0.005
58.23 0.002
61.09 0.005
56.21 0.008
68.23 0.005
65.11 0.008
76.77 0.006
53.65 0.004
67.27 0.005
71.50 0.004
66.85 0.004
76.63 0.005
75.88 0.005
82.15 0.007
66.99 0.011
78.04 0.009
81.62 0.004
aData were reported on MCF-7 cells as mean SD (n=3)
bStaurosporine was used as a positive control in the cytotoxicity screening
Table 2 IC50 of new hybrids 15a–15f on MCF-7 and F-180 cells
Table 3 IC50 data of hybrids 15a–15f using CYP19 screening assay
Compound
IC50/μMa
SI
Compound
% of inhibitiona
IC50/ng/cm3a
MCF-7b (column A)
F-180c (column B)
10b
1
0.1
0.01
Ref.d
15a
15b
15c
15d
15e
15f
4.1 0.13
11.0 0.36
11.2 0.36
37.9 1.24
3.6 0.11
22.4 0.73
43.8 1.43
10.2 0.41
34.1 0.08
66.2 0.18
80.6 0.54
89.3 0.33
100.8 0.14
63.1 0.26
2.5
3
6
Ref.c
15a
15b
15c
15d
15e
15f
90.73
81.08
82.78
82.09
69.79
63.75
–
77.90
–
41.51
31.21
36.99
–
58.92
–
22.57
10.50
22.22
–
28.84
–
0.13
0.37
0.27
–
0.06
–
d
2
–
25
4.5
1.5
91.14
–
–
–
–
–
–
aConcentration of test compounds in micromolar which inhibited 50%
of cells growth
aErrors are within 2%
bLog concentration used
bMCF-7 breast cancer cells
cLetrozole was used as a positive control in the human placental aro-
matase (CYP19) screening assay
cNormal fbroblast cells
dStaurosporine was used as a positive control in IC50 determination
dNot tested
changing the position of such NO2 group to the meta-posi-
tion decreased the inhibitory activity signifcantly as seen
with hybrid 15f.
than STS. On the other hand, hybrids 15c and 15f were
found to be slightly less selective cytotoxic agents than
staurosporine against breast MCF-7 cancer cells.
In vitro cytotoxic activity towards normal fbroblast
(F‑180) cells
Inhibition of aromatase enzyme
In an attempt to investigate the selectivity of the target
compounds on cancer cells, an in vitro cytotoxic assay
was performed on the noncancerous normal fbroblast cell
lines (F-180). The obtained IC50s showed that all the syn-
thesized hybrids 15a–15f were substantially tumor-specifc
cytotoxins than the reference drug, as shown in Table 2.
Tumor selectivity (TS) was calculated by dividing the
mean IC50 values against normal fbroblast cells (F-180)
by the mean IC50 against breast MCF-7 cancer cells
(TS = column B/column A), Table 2. Upon comparing the
calculated TS values, compound 15d showed the highest
selectivity towards MCF-7 cells among the test hybrids
even more the reference drug, staurosporine. Moreover,
hybrids 15a, 15b, and 15e showed better tumor selectivity
Based on various literature studies, inhibition of human
placental aromatase enzyme (CYP19) is one of the funda-
mental approaches in treating HDBCs [31, 32]. Hence, the
most potent hybrids 15a, 15b, and 15d were evaluated for
their ability to inhibit aromatase enzyme against the well-
known aromatase inhibitor letrozole (Table 3). All the test
hybrids showed a remarkable ability to inhibit the aro-
matase enzyme (a dose-dependent inhibition). Hybrid 15d
has the highest ability to inhibit CYP19 enzyme embed-
ded in MCF-7 cells with an IC50 of 64.6 ng/cm3, which is
almost 1.5 times the potency of letrozole (IC50 = 132.8 ng/
cm3). Hybrids 15a and 15b showed comparable inhibition
potency about half that of letrozole.
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M. Mustafa et al.
Table 4 Cell cycle distribution induced by hybrid 15d
value. The obtained data (as shown in Table 4) indicated that
hybrid 15d substantially arrested the G2/M phase compared
to the negative control (35.64% and 10.68%, respectively)
(Fig. 8).
Compound
Cell cycle phases
G0-G1
S
G2/M
Pre-G1 (sub-G1)
15d
Control
53.26
32.94
36.06
31.42
10.68
35.64
1.76
Induction of cellular apoptosis
18.43
Hybrid 15d showed a remarkable ability to induce apopto-
sis in MCF-7 cell lines, after staining with annexin V-FITC
and propidium iodide. At its IC50 concentration, hybrid 15d
initiated cellular apoptosis and dispersed cellular integrity,
as shown in Fig. 9. The percentage of early apoptotic cells
(the lower right quadrant) increased to declare 4.42% while
the negative control was 0.88%. Regarding cellular integrity
dispersion, 15d elevated the late apoptotic cells (the upper
right quadrant) up to 11.99% compared to only 0.51% for
Cell cycle analysis by fow cytometry
Regularly, antitumor agents abort the proliferation and the
growth of cancerous cells by arresting cell division at dif-
ferent checkpoints [33]. Treating cancer cells with potent
antitumor agents disengages the distinguish cells in diferent
phases of the cell cycle. In the current research, MCF-7 cells
were treated with the most potent sulfonamide 15d at its IC50
Fig. 8 Cell cycle analysis of MCF-7 cells treated with PI at test IC50s; a untreated (control) cells, b cells treated with hybrid 15d
Fig. 9 Contour diagram of Annexin V/PI fow cytometry: a untreated cells, b cells treated with hybrid 15d (lower left: live cells; lower right:
early apoptotic cells; upper right: late apoptotic cells; upper left: necrotic cells)
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Development of new hetero-steroid hybrids with antiproliferative activity against MCF-7…
143
the untreated cells. Necrosis was boosted to a lesser extent
(2.02%) compared to negative control = 0.37% on MCF-7
cells. Subsequently, hybrid 15d performed most of its activ-
ity through induction of apoptosis.
previously co-crystallized ligands such as androstenedione
(AE), exemestane, and HDDG046 as reference compounds.
All the intermolecular interaction energies (S in kJ/mol)
and amino acid residues with various functional groups of
new hybrids were summarized in Table 5. From the docking
data, all the docked hybrids 15a–15f successfully occupied
the active site with good energy scores ranging from−17.7
to−28.8 kJ/mol compared to−24.3,−23.5, and−31 kJ/mol
observed for AE, exemestane, and HDDG046, respectively.
The binding interactions of new hybrids 15a–15f with
amino acid residues were found to be either through hydro-
gen bonding or π–H interactions. It noteworthy that most
of the hydrogen bonding occurred through S=O functional
Molecular modelling studies
To study the possible binding modes of these new sul-
fonamides within the human placental aromatase (CYP19;
pAROM), molecular modelling studies were performed
using X-ray crystal structure data for aromatase obtained
from the protein data bank (PDB: ID 5JL9) [34] using
Table 5 Molecular modeling
data for the target hybrids
15a–15f, AE, exemestane, and
HDDG046 in pAROM (PDB:
ID 5JL9)
Compound
Si
H-bonding
(A)a
Other interactions
(B)b
(C)c
(D)d
AE
−24.3
−23.5
−31
Ala 306e
Arg 115e
Met 374e
C=O (3.31)
C=O (3.37)
C=O (2.82)
C=O (3.13)
C=O (3.32)
C=O (2.71)
C16 (3.29)
C=O (3.09)
C=O (3.39)
C=O (2.91)
S=O (3.85)
S=O (4.04)
C17–OH (3.20)
–
–
Exemestane
HDDG046
Ala 306e
Arg 115e
Met 374e
–
–
Leu 372f
Ala 306e
Arg 115e
Met 374e
Phe 221g
C24 (3.93)
15a
−28.2
Met 303f
Ala 306e
Cys 437f
Thr 310h
Triazole ring (3.89)
15b
15c
−26.6
−17.7
Met 374f
Ala 307e
Ph–OH (3.26)
Thr 310h
–
Triazole ring (3.99)
–
S=O (3.16)
Met 303f
Ser 199e
Ala 307e
NH (3.62)
S=O (3.09)
S=O (3.35)
15d
−28.8
Met 311f
Cys 437f
Met 303f
Arg 115e
Thr 310e
N+=O (3.50)
Cys 437h
Triazole ring (3.56)
N+–O− (3.53)
S=O (2.78/3.38)
Triazole-N-(2.98)
N+–O− (3.09)
15e
15f
−22
Met 303f
Ala 307e
Ser 199e
NH (3.52)
S=O (3.34)
S=O (3.15)
–
–
−21.8
Cys 437e
Ala 307e
Ala 438e
C17–OH (3.22)
Val 370h
Phenolic ring (3.79)
S=O (3.36)
C17–OH (3.19)
aH-bond forming residues and types
bFunction group forming H-bond and bond length (Å)
cOther interactions residues and types
dFunction group of interaction and bond length (Å)
eHydrogen acceptor
fHydrogen donor
gH–π
hπ–H
iEnergy score (δ kJ/mol)
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144
M. Mustafa et al.
group compared to its bioisostere (C=O) in the other
reported co-crystallized ligands (AE, exemestane, and
HDDG046), indicating the success of this bioisosteric
replacement in the design of these new hybrids. Hybrid
15d showed additional H-bond interactions with Met 311,
Cys 437, and Thr 310 residues through its p-NO2 group,
which helped in anchoring the whole molecule efciently
within the pAROM active site (Fig. 10), explaining its bit-
ter interaction energy compared to both AE and exemes-
tane reference compounds. Moreover, all the three reference
compounds shared a common H-bond interaction with Arg
115, which was found also with the most potent hybrid 15d
confrming its superior potency over its congers as an inhibi-
tor of the pAROM. Interestingly, changing the position of
the nitro group from para-position (as with hybrid 15d) to
the meta-position (as with hybrid 15f) leads to a remark-
able decrease in the binding energy score of hybrid 15f with
pAROM. Additionally, the π–H interaction with the active
site amino acid residues was found mainly through the tria-
zole ring N-atom of compounds 15a, 15b, and 15d, and to
lesser extent with a phenolic ring as with compound 15f,
while compounds 15c and 15e failed to show such interac-
tion, as shown in Table 5.
chemical company. Purifcation of various intermediates and
fnal hybrids was performed by fash chromatography using
Silicycle® silica gel and a mixture of EtOAc/hexane as an elu-
ent. NMR spectra were recorded on a JEOL ECA-500 II Spec-
trometer using DMSO-d6 (Faculty of Science, Almansura Uni-
versity, Almansura, Egypt), as a solvent, and chemical shifts
(δ) for 1H NMR spectra were recorded relative to TMS (Me4Si;
δ=0.00 ppm) as an internal standard, while 13C NMR spec-
tra were recorded relative to DMSO-d6 (δ=39.5 ppm, central
peak) residual peak and coupling constants (J) in Hertz (Hz).
High-resolution mass spectra (HRMS, electrospray ionization
ESI) were obtained on JEOL HX110 double-focusing mass
spectrometer, and elemental analyses (C, H, N) were measured
on Vario El-Elementar CHN Elemental Analyzer (Al Azahr
University, New City, and Cairo, Egypt) for all fnal hybrids
and were found within 0.4 of their calculated values. Finally,
melting points were determined on electrothermal melting
point apparatus (SMP1, UK).
Synthesis of 4‑azidobenzenesulfonyl chloride (13)
To 25 g sulfanilic acid (12, 1.44 mol) in 20 cm3 H2O, 0.58 g
sodium carbonate (5.47×10–3 mol) was added portion-wise
with continuous and vigorous stirring. After efervescence
subside, 0.84 g sodium nitrite (12.17×10–3 mol) was added
and stirring was continued till complete dissolution, followed
by a diazotization step using 1.4 cm3 conc. HCl diluted
with 3 cm3 H2O at 0–5 °C. A white precipitate was formed,
which was fltered under vacuum, and fnally washed with
8 cm3 cold water to aford the diazotized sulfanilic acid. To
a suspension of the former white precipitate (was used in
the next step without further purifcation) in 10 cm3 H2O,
was added as an aqueous saturated solution of sodium azide
(8 g in 20 cm3 H2O) portion-wise with vigorous stirring till
nitrogen gas evolution ceased. Sodium chloride was added
to salt-out the formed azido derivative of sulfanilic acid as a
white precipitate, which was fltered under vacuum, washed
with cold water, and fnally dried under vacuum for the next
step afording 20 g (70.7%). M.p.:>200 °C [28]. The dried
azido-derivative of sulfanilic acid was dissolved with stirring
in a mixture of 3 cm3 thionyl chloride/1 drop of DMF, and
heated for half an hour till boiling, after that, the remaining
thionyl chloride was removed under vacuum and the remain-
ing residue was extracted with dry ether (2× 5 cm3). The
etherial extract was concentrated under vacuum to aford
compound 13 as yellowish-white solid 15.9 g (73%). M.p.:
59–61 °C [27].
Conclusion
To summarize, a series of novel human placental aromatase
inhibitors containing a hybrid of the steroidal skeleton,
1,2,3-triazole ring, and a benzenesulfonamide scafold were
synthesized. The new hybrids showed a remarkable selective
inhibition for MCF-7 breast cancer cells compared to non-
cancerous fbroblast F-180 cells. Hybrid 15d was profoundly
tumor-specifc cytotoxin compared to staurosporine and
letrozole on MCF-7 cells and aromatase enzyme, respec-
tively. Additionally, its ability to abort the proliferation and
the growth of MCF-7 breast cancer cells at the G2/M phase
of the cell cycle with apoptosis induction mechanism. The
new hybrids 15a–15f complete occupied the pAROM active
site during molecular docking studies. Obviously, the ste-
roidal skeleton gave the extra ftting of the target molecules
within aromatase active site causing such strong inhibition
profle. Within the new hybrids, hybrids 15a, 15b, and 15d
could be considered as promising anti-proliferative aro-
matase inhibitors for the treatment of HDBC and deserves
further in vivo studies.
Experimental
General procedure for preparation of compounds
14a–14f
All solvents and reagents were of pure grades from Fluka®,
Sigma-Aldrich®, or Alfa-Aesar® and used without further puri-
fcation; ethinyl estradiol (EE) was obtained from Carbosynth®
In 1 cm3 dry pyridine, equimolar amounts of 13 and sub-
stituted aniline (1 × 10–5 mol) was mixed with stirring.
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Development of new hetero-steroid hybrids with antiproliferative activity against MCF-7…
145
Fig. 10 Binding of hybrids 15a–15f, AE, exemestane, and HDDG046 into the active site of pAROM (5JL9) as assessed by MOE software
1 3

146
M. Mustafa et al.
Fig. 10 (continued)
Stirring was continued at room temperature for 16 h, then
pyridine was removed azeotropically with toluene under
vacuum, followed by dissolving the residue in EtOAc
(2 × 5 cm3), collected extracts were dried with Na2SO4,
and finally concentrated under vacuum to afford cor-
responding sulfonamide derivatives 14a–14f. Physical
properties agree with those already published [30].
General procedure for preparation of hybrids
15a–15f
To a solution of ethinyl estradiol (EE, 0.67 mmol) in a
mixture of THF/H2O (12 cm3; 2:1), 14a–14f (0.67 mmol),
CuSO4 (0.06 mmol), and sodium ascorbate (0.12 mmol)
were added. The resulting mixture was stirred at room
1 3

Development of new hetero-steroid hybrids with antiproliferative activity against MCF-7…
147
temperature for 20–24 h, and the residue was extracted with
CH2Cl2 (3×10 cm3). The organic layer was washed twice
with water, brine, and dried over anhydrous Na2SO4.
7.42 (app. d, J=9 Hz, 2H, ArH), 7.06 (app. d, J=6.5 Hz,
2H, ArH), 6.91 (d, J=8.5 Hz, 1H, H-1), 6.44 (dd, J=3 Hz,
8.5 Hz, 1H, H-2), 6.39 (d, J=3 Hz, 1H, H-4), 5.34 (s, 1H),
2.67–2.63 (m, 2H, H-6), 2.38 (m, 1H), 2.10–1.98 (m, 2H),
1.84–1.78 (m, 3H), 1.62–1.20 (m, 6H), 1.05–1.03 (m,
1H), 0.94 (s, 3H, CH3, H-18) ppm; 13C NMR (DMSO-d6,
125 MHz): δ = 155.9 (C-3), 154.9, 139.7, 138.6, 137.2,
136.8, 132.5 (2 CHAr), 130.5, 128.7 (2 CHAr), 126.5, 122.3
(2 CHAr), 121.0, 120.2 (2 CHAr), 116.7, 114.9, 112.7, 81.1
(C-17), 47.8, 46.9, 43.1, 37.3, 32.8, 29.3, 27.2, 26.1, 23.6,
18.6, 14.4 (CH3, C-18) ppm; HRMS (ESI+): m/z calcd. for
C32H33BrN4O4S ([M+H]+) 648.1406, found 648.1413.
4‑[4‑(17α‑1,3,5(10)‑Estratrien‑3,17β‑diol)‑1H‑1,2,3‑triazol‑1
‑yl]‑N‑phenylbenzenesulfonamide (15a, C32H34N4O4S) Puri-
fcation was achieved using fash chromatography (ethyl
acetate/hexane, 1:3) which provided white solid (88%)
1
15a. M.p.: 212–214 °C; H NMR (DMSO-d6, 500 MHz):
δ=10.40 (brs, 1H, –NHSO2–), 8.96 (brs, 1H, ArOH), 8.65
(s, 1H, triazole = CH), 8.11 (d, J = 9 Hz, 2H, ArH), 7.91
(d, J = 9 Hz, 2H, ArH), 7.21 (t, J = 8 Hz, 2H, ArH), 7.07
(d, J=7.5 Hz, 2H, ArH), 7.01 (t, J=7 Hz, 1H, ArH), 6.91
(d, J=8.5 Hz, 1H, H-1), 6.44 (dd, J=2.5 Hz, 8.5 Hz, 1H,
H-2), 6.39 (d, J=2.5 Hz, 1H, H-4), 5.31 (s, 1H), 2.67 (m,
2H, H-6), 2.38 (m, 1H), 2.10–1.98 (m, 2H), 1.84–1.78 (m,
3H), 1.63–1.61 (m, 1H), 1.54–1.45 (m, 2H), 1.35–1.1.20 (m,
4‑[4‑(17α‑1,3,5(10)‑Estratrien‑3,17β‑diol)‑1H‑1,2,3‑
triazol‑1‑yl]‑N‑(4‑nitrophenyl)benzenesulfonamide (15d,
C32H33N5O6S) Purifcation was achieved using fash chro-
matography (ethyl acetate/hexane, 1:3) which provided
3H), 1.06–1.01 (m, 1H), 0.94 (s, 3H, CH3, H-18) ppm; 13
C
white solid (73%) 15d. M.p.: 223–224 °C; H NMR
1
NMR (DMSO-d6, 125 MHz): δ=155.8 (C-3), 154.8, 139.3,
139, 137.1, 130.4, 129.2 (2 CHAr), 128.6 (2 CHAr), 125.9,
120.9, 120.5 (2 CHAr), 120 (2 CHAr), 116.7, 114.9, 112.7,
81 (C-17), 47.7, 46.8, 43.1, 37.3, 32.7, 29.2, 27.1, 26.0, 21.8,
18.3, 14.3 (CH3, C-18) ppm; HRMS (ESI+): m/z calcd. for
C32H34N4O4S ([M+H]+) 570.2301, found 570.2309.
(DMSO-d6, 500 MHz): δ = 11.4 (s, 1H, –NHSO2–), 8.98
(brs, 1H, ArOH), 8.65 (s, 1H, triazole = CH), 8.14 (dd,
J = 9 Hz, 9 Hz, 4H, ArH), 8.05 (d, J = 8.5 Hz, 2H, ArH),
7.31 (d, J=9.5 Hz, 2H, ArH), 6.90 (d, J=8.5 Hz, 1H, H-1),
6.44–6.38 (m, 2H, H-2, H-4), 5.34 (s, 1H), 2.67 (m, 2H,
H-6), 2.40–2.25 (m, 2H), 2.10–1.78 (m, 4H), 1.63–1.12
(m, 7H), 1.08–0.93 (m, 4H) ppm; 13C NMR (DMSO-d6,
125 MHz): δ=167.1 (C–NO2), 155.9 (C-3), 139.9, 137.2,
132.0, 130.4, 128.8 (2 CHAr), 126.0, 125.5 (2 CHAr), 121.1,
120.4 (2 CHAr), 118.3 (2 CHAr), 116.4, 114.9, 112.7, 81.1
(C-17), 47.8, 46.9, 43.1, 37.3, 32.2, 29.3, 27.4, 23.3, 22.4,
18.6, 14.4 (CH3, C-18) ppm; HRMS (ESI+): m/z calcd. for
C32H33N5O6S ([M+H]+) 615.2152, found 615.2155.
4‑[4‑(17α‑1,3,5(10)‑Estratrien‑3,17β‑diol)‑1H‑1,2,3‑
triazol‑1‑yl]‑N‑(4‑chlorophenyl)benzenesulfonamide (15b,
C32H33ClN4O4S) Purifcation was achieved using fash chro-
matography (ethyl acetate/hexane, 1:3) which provided white
solid (77%) 15b. M.p.: 203–205 °C; 1H NMR (DMSO-d6,
500 MHz): δ = 10.55 (brs, 1H, –NHSO2–), 9.00 (brs, 1H,
ArOH), 8.61 (s, 1H, triazole =CH), 8.07 (d, J=9 Hz, 2H,
ArH), 7.88 (d, J = 9 Hz, 2H, ArH), 7.24 (t, J = 5 Hz, 2H,
ArH), 7.04 (t, J=8.5 Hz, 2H, ArH), 6.91 (d, J=8 Hz, 1H,
H-1), 6.44–6.39 (m, 2H, H-2, H-4), 5.34 (s, 1H), 2.66 (m,
2H, H-6), 2.38 (m, 1H), 2.15–1.77 (m, 5H), 1.62–1.20 (m,
4‑[4‑(17α‑1,3,5(10)‑Estratrien‑3,17β‑diol)‑1H‑1,2,3‑
triazol‑1‑yl]‑N‑(4‑methoxyphenyl)benzenesulfonamide
(15e, C33H36N4O5S) Purifcation was achieved using fash
chromatography (ethyl acetate/hexane, 1:3) which provided
white solid (68%) 15e. M.p.: 178–180 °C; 1H NMR (DMSO-
d6, 500 MHz): δ=10.01 (s, 1H, –NHSO2–), 8.97 (brs, 1H,
ArOH), 8.66 (s, 1H, triazole = CH), 8.11 (t, J = 7 Hz, 2H,
ArH), 7.82 (d, J = 9 Hz, 2H, ArH), 7.72 (dd, J = 9 Hz,
1.5 Hz, 2H, ArH), 6.90 (dd, J=2.5 Hz, 7.0 Hz, 2H, ArH),
6.80 (dd, J = 1.5 Hz, 9 Hz, 1H, H-1), 6.45–6.39 (m, 2H,
H-2, H-4), 5.32 (s, 1H), 3.66 (s, 3H, OCH3), 2.67 (m, 2H,
H-6), 2.40–2.25 (m, 2H), 2.10–1.75 (m, 5H), 1.62–1.15
(m, 7H), 0.94 (s, 3H, CH3, H-18) ppm; 13C NMR (DMSO-
d6, 125 MHz): δ=156.8 (C-3), 154.9, 144.6, 139.3, 138.6,
137.1, 136.1, 128.9, 128.6, 126.5, 124.0 (4 CHAr), 116.8,
114.9, 114.3 (4 CHAr), 112.7, 81.1 (C-17), 56.1 (OCH3),
47.8, 46.9, 43.1, 37.5, 32.4, 29.1, 27.3, 26.1, 22.4, 18.1,
13.9 (CH3, C-18) ppm; HRMS (ESI+): m/z calcd. for
C33H36N4O5S ([M+H]+) 600.2406, found 600.2415.
7H), 1.05–1.03 (m, 1H), 0.93 (s, 3H, CH3, H-18) ppm; 13
C
NMR (DMSO-d6, 125 MHz): δ=155.8 (C-3), 154.9, 139.7,
137.2, 131.6, 130.5, 129.0 (2 CHAr), 128.6 (2 CHAr), 126.1,
122.1 (2 CHAr), 121.0, 120.1 (2 CHAr), 116.7, 114.9, 112.7,
81.1 (C-17), 47.8, 46.9, 43.1, 37.8, 32.8, 29.3, 27.2, 26.1,
23.6, 20.3, 17.8, 14.4 (CH3, C-18) ppm; HRMS (ESI+):
m/z calcd. for C32H33ClN4O4S ([M+H]+) 604.1911, found
604.2001.
4‑[4‑(17α‑1,3,5(10)‑Estratrien‑3,17β‑diol)‑1H‑1,2,3‑
triazol‑1‑yl]‑N‑(4‑bromophenyl)benzenesulfonamide (15c,
C32H33BrN4O4S) Purification was achieved using flash
chromatography (ethyl acetate/hexane, 1:3) which pro-
1
vided white solid (91%) 15c. M.p.: 225–226 °C; H NMR
(DMSO-d6, 500 MHz): δ=10.56 (br s, 1H, –NHSO2–), 8.98
(br s, 1H, ArOH), 8.64 (s, 1H, triazole = CH), 8.13 (app.
d, J = 9 Hz, 2H, ArH), 7.92 (app. d, J = 7 Hz, 2H, ArH),
1 3

148
M. Mustafa et al.
4‑[4‑(17α‑1,3,5(10)‑Estratrien‑3,17β‑diol)‑1H‑1,2,3‑
triazol‑1‑yl]‑N‑(3‑nitrophenyl)benzenesulfonamide (15f,
C32H33N5O6S) Purifcation was achieved using fash chroma-
tography (ethyl acetate/hexane, 1:3) which provided white
solid (64%) 15f. M.p.: 231–232 °C; 1H NMR (DMSO-d6,
500 MHz): δ = 11.20 (brs, 1H,–NHSO–), 8.96 (brs, 1H,
ArOH), 8.65 (s, 1H, triazole =CH), 8.15 (d, J=9 Hz, 2H,
ArH), 7.95 (dd, J=8.5 Hz, 8.5 Hz, 3H, ArH), 7.88 (m, 1H,
ArH), 7.68 (m, 1H, ArH), 7.53 (m, 1H, ArH), 6.91 (m, 1H,
H-1), 6.45–6.39 (m, 2H, H-2, H-4), 5.33 (s, 1H), 2.67 (m,
2H, H-6), 2.38 (m, 1H), 2.10–1.78 (m, 5H), 1.63–1.40 (m,
3H), 1.36–1.15 (m, 5H), 1.02 (m, 1H), 0.93 (s, 3H, CH3,
H-18) ppm; 13C NMR (DMSO-d6, 125 MHz): δ = 165.9
(C–NO2), 154.9 (C-3), 139.8, 137.2, 130.9, 130.4, 128.7 (2
CHAr), 125.7, 125.5, 121.1, 120.4 (2 CHAr), 116.8, 114.9,
113.7, 112.7, 81.1 (C-17), 47.8, 46.9, 43.1, 37.8, 32.3,
29.3, 27.2, 25.5, 22.8, 18.6, 14.4 (CH3, C-18) ppm; HRMS
(ESI+): m/z calcd. for C32H33N5O6S ([M+H]+) 615.2152,
found 615.2158.
bufer, pH 7.4; 0.02 cm3 of 20 × NADPH-generating sys-
tem, 26 mM NADP+, 66 mM glucose-6-phosphate, and
66 mM MgCl2); and 0.001 cm3 of 100 μ/cm3 glucose-
6-phosphate dehydrogenase, were added and incubated
at 37 °C for 10 min. The reaction was initiated by adding
0.1 cm3 of enzyme/substrate mixture (0.8 cm3 of 50 mM
phosphate bufer, pH 7.4; 0.12 cm3 of 16 pmol/cm3 CYP19;
2×10–3 cm3 of 0.2 mM DBF) and 0.01 cm3 of each concen-
tration (10,000, 1000, 100, 10, and 0 ng/cm3) of test hybrids
15a–15f, negative control (10% DMSO), or letrozole (K983-
100-3) as positive control. Fluorescence was recorded at
λmax = 488/527 nm with cut-of 515 nm. Percentage (%)
inhibition was calculated using the following equation: %
inhibition=100−[(sample−blank)/(DMSO−blank)×100].
Molecular modelling studies
Molecular modeling and visualization processes were per-
formed within the aromatase active site using Molecular
Operating Environment (MOE: 2014.09) software (Chemi-
cal Computing Group, Montreal, QC, Canada). The co-crys-
tal structure was retrieved from the RCSB Protein Data Bank
(PDB code 3EQM). First, the compounds (AE, exemestane,
HDDG046, and hybrids 15a–15f) were prepared with the
standard protocol designated in the MOE program. How-
ever, the energy of the docked structures was minimized
using MMF94FX forcefeld with gradient RMS of 0.01 kcal/
mol, then the protein structure was prepared using the MOE
LigX protocol. To validate the docking study at the aro-
matase active site, the native ligand androstenedione was re-
docked into the binding site using the same set of parameters
as described above. The ligands were then docked into the
binding site using the triangle matcher placement method.
Refnement was carried out using Forcefeld and scored
using the Afnity δG scoring system. The resulting dock-
ing poses were visually inspected, and the pose of the lowest
binding free energy value was considered.
Cytotoxic studies
Cell cultures and MTT cytotoxic assays
The pure cultures breast cancer cell line (MCF-7; ATCC®
HTB-22) was obtained from American Type Culture Collec-
tion (ATCC®). Normal fbroblast cells (F180) were obtained
from VACSERA-Egypt. Cell viability was assessed by MTT
assay as described elsewhere [18].
Cell cycle analysis and assessment of apoptosis
The apoptotic efect of synthesized hybrids 15a–15f was
analyzed by propidium iodide (PI) fow cytometry kit for
cell cycle analysis (Annexin V Apoptosis Detection Kit®)
according to the manufacturer’s instructions. Data analysis
and nuclear morphology of apoptotic cells were examined
using BD FACS Calibur. Propidium iodide histograms of
normal and treated cells with the cell count on the y-axis
and the propidium iodide fuorescence intensity on the x-axis
were recorded. In addition, the cell cycle arrest caused by
hybrids 15a–15f in a concentration-dependent manner in
MCF-7 cells was examined.
Acknowledgements Special thanks to Prof. Gamal Abu-Rahmah,
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Minia University for his advises through the research work, also to Dr.
Abu-Baker Mostafa, Department of Pharmaceutical Organic Chemis-
try, Faculty of Pharmacy, Assiut University for his guidance through
chemistry part, and fnally, for Dr. A. Y. Desoky, Department of Chem-
istry, Faculty of Science, the University of Waterloo for running mass
spectral analysis for our group.
Aromatase enzyme inhibition assay
The assay was carried out using the Gentest kit (BioVision®,
catalog # K893-100) using the CYP19A (human microso-
mal and S9 fractions; EC 1.14.14.14; K983-100-1) enzyme
and O-benzylfuorescein benzyl ester (DBF; K983-100-
2) as a fuorimetric substrate according to manufacturer’s
instructions. Briefy, in a 96-well black plate, a mixture
of 0.1 cm3 the cofactor (0.08 cm3 of 50 mM phosphate
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