Aromatase inhibitors and apoptotic inducers: Design, synthesis, anticancer activity and molecular modeling studies of novel phenothiazine derivatives carrying sulfonamide moiety as hybrid molecules

Article abstract of DOI:10.1016/j.ejmech.2017.04.028

Hybrid molecules are used as anticancer agents to improve effectiveness and diminish drug resistance. So, the current study aimed to introduce twenty novel phenothiazine sulfonamide hybrids 5–22, 24 and 25 of promising anticancer activity. Compounds 11 an

Full text of DOI:10.1016/j.ejmech.2017.04.028

Accepted Manuscript
Aromatase inhibitors and apoptotic inducers: Design, synthesis, anticancer activity
and molecular modeling studies of novel phenothiazine derivatives carrying
sulfonamide moiety as hybrid molecules
Mostafa M. Ghorab, Mansour S. Alsaid, Nermin Samir, Ghada A. Abdel-Latif, Aiten M.
Soliman, Fatma A. Ragab, Dalal A. Abou El Ella
PII:
S0223-5234(17)30285-4
10.1016/j.ejmech.2017.04.028
EJMECH 9375
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 February 2017
Revised Date: 10 April 2017
Accepted Date: 12 April 2017
Please cite this article as: M.M. Ghorab, M.S. Alsaid, N. Samir, G.A. Abdel-Latif, A.M. Soliman,
F.A. Ragab, D.A. Abou El Ella, Aromatase inhibitors and apoptotic inducers: Design, synthesis,
anticancer activity and molecular modeling studies of novel phenothiazine derivatives carrying
sulfonamide moiety as hybrid molecules, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.04.028.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Aromatase inhibitors and apoptotic inducers: design, synthesis,
anticancer activity and molecular modeling studies of novel
phenothiazine derivatives carrying sulfonamide moiety as hybrid
molecules.
a,b*
a
c
d
Mostafa M. Ghorab , Mansour S. Alsaid , Nermin Samir , Ghada A. Abdel-Latif , Aiten M.
b
e
c,f
Soliman , Fatma A. Ragab , Dalal A. Abou El Ella
a
Department of Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi
Arabia.
b
Department of Drug Radiation Research, National Center for Radiation Research and Technology, Egyptian
Atomic Energy Authority, Cairo 113701, Egypt.
c
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Cairo 11566, Egypt.
d
Department of Pharmacology, Faculty of pharmacy, Misr International University, Cairo, Egypt.
e
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt.
f
Faculty of pharmacy, Nahda University, 62511 new Beni Suef (NUB), Egypt.
*
+
Correspondence author; e-mail: mmsghorab@yahoo.com, mghorab@ksu.edu.sa; Tel.: +966-53-4292-860; Fax:
966-01-4670-560.
Abstract
Hybrid molecules are used as anticancer agents to improve effectiveness and
diminish drug resistance. So, the current study aimed to introduce twenty novel phenothiazine
sulfonamide hybrids 5-22, 24 and 25 of promising anticancer activity. Compounds 11 and 13 revealed
more potent anticancer properties (IC 8.1 and 8.8 µM) than that of the reference drug (doxorubicin, IC
50
50
9.8 µM) against human breast cancer cell line (T47D). To determine the mechanism of their anticancer
=
activity, compounds 5, 6, 7, 11, 13, 14, 16, 17, 19 and 22 that showed promising activity on T47D, were
evaluated for their aromatase inhibitory effect. The study results disclose that the most potent aromatase
inhibitors 11 and 13 showed the lowest IC (5.67 µM and 6.7 µM), respectively on the target enzyme.
50
Accordingly, the apoptotic effect of the most potent compound 11 was extensively investigated and
-4
showed a marked increase in Bax level up to 55000 folds, and down-regulation in Bcl2 to 5.24*10 folds,
in comparison to the control. Furthermore, the effect of compound 11 on caspases 3, 8 and 9 was
evaluated and was found to increase their levels by 20, 34, and 8.9 folds, respectively, which indicates the
activation of both intrinsic and extrinsic pathways. Also, the effect of compound 11 on the cell cycle and
its cytotoxic effect were examined. Moreover, a molecular docking and computer aided ADMET studies
were adopted to confirm their mechanism of action.
Keywords: Phenothiazine; Benzenesulfonamide; Anticancer; Aromatase inhibitors; Apoptosis.
1

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1
. Introduction
Apoptosis is a programmed “self-automated” cell death with caspase-3 as a key player in
apoptotic process execution [1]. It is critical for the homeostasis that conserves cellular integrity.
Impairment of apoptosis signaling pathways may lead to several diseases including cancer [2]
.
Depending on the apoptogenic mechanisms of aromatase inhibitors [3, 4], the inhibition of
aromatase enzyme plays an important strategy in the treatment of cancer. Aromatase is
responsible for a key step in the biosynthesis of estrogens from androgens by aromatization[5].
High levels of estrogens stimulate the hormone-dependent breast cancer progression and
metastasis in both pre- and postmenopausal women [6]. Therefore, targeting critical apoptosis
regulators with a goal of apoptosis induction in cancer cells and suppression of estrogen
biosynthesis by aromatase inhibition is an attractive approach for the treatment of hormone-
sensitive breast cancer [4, 7, 8]. Aromatase can be competitively inhibited by various classes of
steroidal and non-steroidal compounds [9]. Novel potent, more selective, and less toxic
aromatase inhibitors (AIs) are necessary due to the side effects evolved from the prolonged use
of them [10].
In our previous work [11], we synthesized a novel series of chromene sulfonamide
hybrids based on structural diversification of a lead compound (A) (Fig.1) revealed by Pingaew
et al in 2015 that proved to have potent aromatase inhibitory activity (IC50= 0.2 µM) [12].
Where compound (B) methoxypyrimidinyl derivative of chromene benzenesulfonamide (Fig.1)
showed significant anticancer activity (IC50= 8.8 µM) higher than that of doxorubicin (IC50= 9.8
µM) against human breast cancer cell line (T47D). Also, it demonstrated the inhibitory effect on
the aromatase enzyme with IC = 4.6 µM and induced the levels of active caspase 3, caspase 8
5
0
and caspase 9. Moreover, it surprisingly boosted the Bax/Bcl2 ratio 5936 & 33000 folds,
respectively compared to the control. In addition, the molecular docking study of compound (B)
revealed that it formed a significant H-bond between its sulfonyl oxygen and Met 374 the
essential amino acid for the inhibitory activity and interacts with a large hydrophobic pocket
(Cys 437, Ala 306, and Ala 307) through its coumarinyl moiety [11]. As a continuation of our
previous work[13, 14], we attempted to produce another novel AIs using both compounds (A)
and (B) as leads. So, new analogs designed preserving the sulfonamide moiety 5-22 or sulfonyl
2
4 and 25 (blue dotted quadrant) and replacing coumarin ring with larger phenothiazine one (red
2

ACCEPTED MANUSCRIPT
dotted circle) to accommodate the large hydrophobic pocket present in the enzyme (Fig.1). To
prove our concept, we compared the binding mode of lead compound (B) with that of our newly
designed series and the results revealed that both have the same binding mode with the
aromatase enzyme as shown in Fig.2.
3

ACCEPTED MANUSCRIPT
O
O
O
N
N
N
O
O
S
N
O
O
Lead compound (A)
H
O
N
S
N
O
N
H
N
O
O
O
O
Lead compound (B)
O
NHR
S
H
O
H
O
N
N
R= H, acyclic, heterocyclic
S
Target compounds 5-22
NHR'
O
S
O
H
O
H
N
N
S
R'= H, 1-(10H-phenothiazin-2-yl)prop-2-en-1-one
Target compounds 24 and 25
Fig. 1. Structure based design of phenothiazine derivatives 5-22, 24 and 25 as AIs.
4

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Fig. 2. Binding mode of both lead compound (B) and one of the newly designed series, compound 18.
2
. Results and Discussion
2
.1. Chemistry
In this work, a novel series of phenothiazine derivatives carrying benzenesulfonamide
was synthesized. Thus, interaction of 1-(10H-phenothiazin-2-yl) ethanone 1 with
dimethylformamide-dimethyl acetal (DMF-DMA) 2 under reflux in dry xylene gave the strategic
starting material (E)-3-(dimethylamino)-1-(10H-phenothiazin-2-yl)-prop-en-1-one
3.
1
Enaminone 3 was assigned an E-configuration based on its H-NMR as the coupling constant of
the doublet signals for olefinic protons is 12.5 Hz correlated to the E-isomers[15]. Enaminone 3
was reacted with sulfa drugs 4 and/ or dapson 23 in absolute ethanol containing glacial acetic
acid (2:1)[16], the corresponding phenothiazine-sulfonamide derivatives 5-22, 24 and 25
(Schemes 2 and 3) were obtained. IR of 5-22 indicated the presence of characteristic bands for
1
NH, CO and SO groups. H-NMR of 5-22, 24 and 25 supported that these structures in (Z) form,
2
as the coupling constant of the doublet signal for olefinic protons equal to 10-10.5 Hz. Z form is
1
stabilized by intramolecular hydrogen bonding (Scheme 1). H-NMR of 5-22 indicated the
presence of two doublets at 5.7-6.4 ppm and 7.3-7.9 ppm assigned to CH=CH and CH-NH,
respectively, singlet at 8.1-8.9 ppm attributed to SO NH, singlet at 10.3-10.4 ppm due to NH
2
1
3
phenothiazine and singlet at 10.5-11.9 ppm for NH group. C-NMR of 5-22 exhibited signals at
4.7-95.8 ppm for CH=CH, 142.5-146.7 ppm for CH-NH and 185.1-189.5 ppm for CO group. In
9
addition, the interaction of compound 3 with dapson 23 in a molar ratio (1:1 mol) gave the
corresponding mono-substituted compound 24, while the bis-compound 25 was obtained in the
5

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same condition but in a molar ratio (2:1 mol) (Scheme 3). The IR of 24 revealed the presence of
-
1
-1
-1
bands at 3356, 3213 cm for (NH, NH ), 1635 cm for CO and 1363, 1197 cm for SO . while,
2
2
-1
-1
IR of 25 showed bands at 3410, 3354 cm for 4NH, 1635 cm for 2CO groups and 1390, 1197
-1
1
cm for SO
2
. H-NMR of 24 revealed two doublets at 6.1 and 7.5 ppm for CH=CH protons,
singlet at 6.2 ppm assigned to NH , singlet at 10.4 ppm for NH phenothiazine and singlet at 11.9
2
1
3
ppm attributed to NH group. C-NMR of 24 exhibited signals at 95.3, 144.8 ppm due to CH=CH
1
and 187.0 ppm for CO group. H-NMR of 25 revealed two doublets at 5.7 and 7.4 ppm due to
CH=CH, singlet at 10.4 ppm for 2NH phenothiazine, singlet at 11.9 ppm attributed to 2NH
1
3
groups. C-NMR of 25 exhibited signals at 95.3, 143.3 ppm for 2CH=CH and 185.1 for 2CO
groups.
Scheme 1: Synthetic pathway for compound 3.
6

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O
H
N
O
N
H N
2
S
NHR
O
S
EtOH:AcOH
:1
3
4
2
O
NHR
O
S
S
H
O
O
N
NHR
O
H
N
H
N
O
N
H
S
S
E-form
7: R =
Z-form
-22
5
NH
6
: R= COCH3
5
: R = H,
NH2
1
0: R =
8: R =
9: R =
N
N
N
N
O
O
N
N
S
N
11: R =
12: R =
13: R =
N
S
N
N
,
1
4: R =
15: R =
N
16: R =
N
N
N
O
N
N
N
N
19: R =
N
1
7: R =
N
18: R =
O
N
O
N
20: R =
N
O
2
1: R =
22: R =
N
N
N
H
O
Scheme 2: Synthetic pathway for compounds 5-22.
7

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Scheme 3: Synthetic pathways for compounds 24 and 25.
2
2
.2. Biological activity
.2.1. Anticancer activity against T47D human breast cancer cell line
The newly synthesized phenothiazine derivatives 3, 5-22, 24 and 25 were preliminarily
screened for their in vitro cytotoxic activity against T47D human breast cancer cell line utilizing
the Sulfo-Rhodamine-B (SRB) assay [17]. From the results obtained (Table 1), it is apparent that
all the tested phenothiazines exerted anticancer activity with IC values ranging from 8.1 µM -
5
0
1
35.8 µM. Sulfonamide group plays a major role in the anticancer activity as apparent from
comparing the IC50 value of compound 5 bearing unsubstituted sulfonamide and compound 3
bearing a terminal tertiary amine (IC = 67.6 µM, 135.8 µM), respectively.
5
0
Results showed that compounds 7, 11, 13 and 22 have significant anticancer activity (Table1)
with IC less than 25 µM. Among them compounds 11 with unsubstituted thiazolyl ring and 13
5
0
with unsubstituted pyridine exerted the highest activity having IC values of 8.1 and 8.8 µM,
5
0
respectively which are more potent than that of the reference drug used, Doxorubicin (IC50 9.8
µM).
8

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2
.2.2. Aromatase inhibitory activity
The aromatase inhibitory activity of the most potent compounds 5, 6, 7, 11, 13, 14, 16, 17, 19,
2 and 24 was determined. Results showed that most of the tested compounds have moderate to
2
high inhibitory activity ranging from 28.90 % to 72.22 %. Four compounds 7, 11, 13 and 22
showed percentage inhibition above 60% and their IC values were also recorded as 9.67, 5.67,
5
0
6
.70 and 8.90 µM, respectively (Table 1). Compound 11 bearing unsubstituted thiazole ring
showed the lowest IC50 value (5.67 µM), and 68.32% aromatase inhibition. Letrozole was used
as a reference drug with (29.5 µM), and 58% aromatase inhibition.
Table 1
Anticancer and aromatase inhibition activity of the newly synthesized compounds 3, 5–22, 24 and 25.
Compound
IC50
% inhibition of
IC50 of
No.
3
against T47D (µM)
aromatase enzyme
ND
aromatase enzyme (µM)
135.8
67.6
71.8
23.7
78.4
79.5
68.1
8.1
ND
ND
ND
9.67
ND
ND
ND
5.67
ND
6.7
5
28.9
6
40.52
72.22
ND
7
8
9
ND
1
1
1
1
1
1
1
1
1
1
2
2
0
1
2
3
4
5
6
7
8
9
0
1
ND
68.32
ND
71.4
8.8
68.86
30.82
ND
61.3
73.6
50.7
56.5
83.2
58.1
82.2
91.1
ND
ND
ND
ND
ND
ND
ND
ND
43.62
35.88
ND
44.94
ND
ND
9

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2
2
2
2
4
5
21.4
54.9
46.4
9.8
-
65.10
31.92
ND
-
8.9
ND
ND
-
Doxorubicin
Letrozole
58
29.5
*ND: Not Done
2
.2.3. Apoptosis studies
From the previous results, most of the synthesized phenothiazine-sulfonamide derivatives
proved to have aromatase inhibitory activity. As mentioned before, the inhibition of aromatase
enzyme leads consequently to the induction of apoptosis [3, 4], Therefore, to reveal the
proapoptotic potential of our target compounds, compound 11 with the lowest IC50 (5.67µM) on
the aromatase enzyme was chosen to explore its ability to induce the apoptosis cascade.
2.2.3.1. Activation of proteolytic caspases cascade
Activation of caspases plays a key role in the initiation and execution of the apoptotic process
18]. Among the caspases, caspase-3, a vital player that cleaves multiple proteins in the cells,
[
leading to apoptotic cell death [1]. The effect of compound 9 on caspase 3 was evaluated and
compared to vandetanib as a reference drug. It showed an increase in the level of active caspase
3
by 7 folds, compared to the control cells, and induced caspase 3 approximately twice that of
letrozole (Fig. 3). To trace compound 11 apoptotic mechanism whether it is through the
induction of the intrinsic or the extrinsic pathway or both, its effect on caspases 8 and 9 was also
evaluated. It has been found that compound 11 increased the levels of caspases 8 and 9 by 34,
and 8.9 folds, respectively compared to the control cells, which indicates activation of both
intrinsic and extrinsic pathways. (Table 2, Fig. 3).
Table 2
Effect of compound 11 on the active caspases 3, 8 and 9 in T47D cells.
Sample code
Caspase 3 ng/ml Caspase 8 ng/ml
0.418042 0.995
Caspase 9 ng/ml
10.5136
1
1
1
0

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Control
0.0208912
0.029
1.18753
Fig. 3. The effect of compound 11 on the active caspases 3 versus letrozole as a reference compound, and
on caspases 8 and 9 in T47D cells.
2.2.3.2. Effects on Bcl-2 family proteins
The B-cell lymphoma protein 2 (Bcl-2) family plays a key role in tumor progression or
inhibition of intrinsic apoptotic pathway triggered by mitochondrial dysfunction, with Bcl-2
itself and Bcl-X (also known as Bcl2l1) as anti-apoptotic and Bcl-2-associated X protein (BAX)
L
as pro-apoptotic proteins [19]. The effect of compound 11 on the expression levels of Bcl2, Bcl-
L
X and BAX was determined after treatment of T47D cells with the IC50 of compound 11, and
the results were illustrated in Table 3.
Compound 11 caused upregulation in the level of the proapoptotic protein; BAX by
approximately 55000 folds while it markedly downregulated the levels of the antiapoptotic
-4
proteins Bcl2 to 5.24*10 and Bcl-X to an undetectable level, compared to the control. From
L
literature survey, there is strong evidence that the ratio of pro-apoptotic BAX versus anti-
apoptotic Bcl-2 proteins indicates the susceptibility of cancer cells to undergo apoptosis more
accurately [20]. According to our results, compound 11 increased the BAX/Bcl2 ratio by
8
approximately 10 folds as compared to the control. These findings proved the proapoptotic
effect of compound 11.
Table 3
Effect of compound 11 on the expression of the gene of some apoptosis key markers.
1
1

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Sample code
BAX IU/ml
137,308
24.6
BCL2 IU/ml
298
BCL-X IU/ml
L
1
1
Below the detectable level
754,565
Control
568,019
2.2.3.3. Cell cycle analysis[21]
To gain further insight into the mode of action of compound 11, the most potent one, we
examined its effect at IC50 (8.1 µM) on the cell cycle by flow cytometry in T47D cells. The
results obtained following cell cycle analysis of both untreated control cells and cells treated with
compound 11 (Fig.4) interestingly showed a concentration-dependent change in the cell cycle
pattern. These results clearly demonstrate that a significant increase in the percentage of cell
population at the G2/M phase (56.3 %) was produced by treatment of the cells with compound
1
1 (Fig. 4A) when compared to untreated control (15.81%) (Fig.4 B). This implies that the cell
cycle of the treated cells was arrested at G2/M phase. Also, the cell population in G1 and S
phases were markedly decreased after treatment (7.4 % and 13.1 % versus 64.92% and 17.14 %,
respectively). From these results, it was disclosed that, cell cycle arrest in T47D cells in the
G2/M phase contributes to compound 11 cytotoxicity.
(
A)
(B)
Fig. 4. (A) Effect of compound 11 on the phases of the cell cycle in T47D cells. (B)
1
2

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Cell cycle phases in untreated cells.
2
.2.3.4. Cell viability test
The cytotoxic effect of compound 11, being the most potent synthesized compound, was tested
on the normal breast cells 184A1[22] using SRB assay. Compound 11 showed mild cytotoxic
effect with an IC of 391.3 µM. This result indicates the selectivity of compound 11 towards
5
0
breast cancer cells and its relative safety for normal breast cells (Fig. 5).
Fig.5. The effect of compound 11 on the viability percentage of 184A1 normal breast cells.
2
2
.3. Molecular Modeling studies
.3.1. Molecular docking
In general, hydrophobicity and H-bonding interactions are the important features for fitting
in the aromatase active site. The 3D ligand-protein interaction of the co-crystallized natural
substrate androstenedione (ASD) (Fig. 6) displayed hydrogen bonding of the CO group at
-1
position 17 with the amino acid Met 374, with an interaction energy= -44.54 Kcal mol [23].
1
3

ACCEPTED MANUSCRIPT
A
B
Fig. 6. (A) 3D ligand-protein interaction of the co-crystallized ASD with aromatase enzyme
o
(
PDB:3EQM). (B) Redocking of the co-crystallized ASD yielded RMSD of 0.448 A .
In order to investigate the binding mode of phenothiazines 5-22, 24 and 25 to the aromatase
enzyme, redocking of the co-crystallized ligand was performed in order to validate the docking
procedure. The redocked ligand was found to completely superimpose on the co-crystallized one
(
Fig. 6B). The target compounds were fit into the aromatase active site with low C-DOCKER
interaction energy. Compound 11 forms hydrogen bonds with Met 374 and Leu 372 using the
-1
sulfonamide oxygen and nitrogen, respectively with an interaction energy = -49.18 Kcal mol
Fig. 7). This further gives insight to the significant role of sulfonamide group to the anticancer
(
activity as early observed when comparing compound 3 (IC50=135.8 µM) which is devoid of a
sulfonamide group.
1
4

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Fig. 7. 3D ligand-protein interaction of compound 11 with aromatase enzyme (PDB:3EQM).
2
.3.2. ADMET studies
Computer aided ADMET study was done by using the software Accord for Excel (Accelrys
Discovery studio 2.5 software). These studies are solely based on the chemical structure of the
molecule. All the parameters calculated are tabulated (Table 4). The ADMET-Plot is a 2D plot
using calculated PSA_2D and A logp98 properties. Blood Brain Barrier (BBB) and Human
Intestinal Absorption (HIA) plots (Fig. 7) were drawn using all the compounds. In BBB plot all
the 20 compounds fall outside the 99% ellipse (undefined). Hence, these compounds may not be
able to penetrate the blood brain barrier, the chances of CNS side effects are low or absent. In
HIA plot 11 compounds fall outside the 99% confidence ellipse (low absorption), whereas the
remaining compounds fall inside the 99% ellipse. Hence compounds 5 and 6 have unsubstituted
sulfonamide and N-sulfonyl acetamide are expected to possess good human intestinal absorption.
Compounds 8, 11, 12, 13, 14, 15 and 17 showed moderate absorption as they have high
molecular weights. ADME Aqueous solubility logarithmic level of most of the compounds was
found to be 2 or 1, which indicates very little aqueous solubility. The hepatotoxicity score
predicts the hepatotoxic nature of the chemical compounds. The ADME.HEPATOTOX.PROB
values of most of the compounds lie in the range of 0.715-0.966. Hence the compounds are
likely to posses’ hepatotoxicity, further studies are necessary to determine the hepatotoxic dose
levels. The CYP2D6 score predicts the inhibitory and non-inhibitory character of the given query
chemical structure on Cytochrome P450 2D6 enzyme. All the compounds are predicted as non-
inhibitors of CYP2D6, so the side effects (i.e. liver dysfunction) are not expected upon
administration of these compounds. The plasma protein-binding model predicts whether a
compound is likely to be highly bound to carrier proteins in the blood. There is a high probability
that these compounds can reach the desired targets as all of the compounds showed more than
9
5% plasma protein binding. PSA is a key property that has been linked to drug bioavailability.
Thus, passively absorbed molecules with PSA > 140 are thought to have low bioavailability. All
the synthesized compounds have PSA ranging from 104.062-139.96 they theoretically should
present good passive oral absorption.
1
5

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Fig. 8. Human intestinal absorption (HIA) and Blood Brain Barrier plot for the newly synthesized compounds.
Table 4
Computer aided ADMET screening results of the prepared compounds.
AQ
BBB_
CYP
PPB_
A log
P98
Unk_Alog
P98
CPD
ID
Absorp_
Hepatox
Prob
d
g
SOl
Hepatox
ADEM_PSA_2D
b
a
Lev
e
f
Lev
c
2D6
Lev
LEV
5
6
7
8
9
4
4
4
4
4
4
4
4
4
4
4
4
0
0
2
1
2
2
1
1
1
1
1
2
2
2
2
2
1
1
1
1
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
0.953
0.887
0.947
0.92
0
0
0
0
0
0
0
0
0
0
0
0
2
2
2
2
2
2
2
2
2
2
2
2
3.586
3.576
3.421
4.23
0
0
0
0
0
0
0
0
0
0
0
0
104.062
107.633
139.96
114.148
114.148
106.942
101.594
112.855
101.594
112.855
112.855
112.855
0.907
0.947
0.94
4.716
5.72
1
1
1
1
1
1
1
0
1
2
3
4
5
6
4.559
4.57
0.92
0.953
0.894
0.913
0.715
4.757
4.111
4.394
4.676
1
6

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1
1
1
2
2
2
2
2
7
8
9
0
1
2
4
5
4
4
4
4
4
4
4
4
1
2
2
2
2
2
2
3
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0.92
0.887
0.887
0.821
0.953
0.94
0
0
0
0
0
0
0
0
2
2
2
2
2
2
2
2
4.368
4.095
5.157
4.636
5.013
5.372
5.237
9.036
0
0
0
0
0
0
0
0
112.855
121.785
130.715
130.715
116.649
112.855
104.062
120.444
0.96
0.966
a
BBB_Level; 4= undefined, 2= medium penetration, 1= high penetration
b
c
Absop_Level; 3= very low absorption, 2= low absorption, 1= moderate, 0= good absorption.
AQ SOl _LEV; 2= low solubility, 1= very low but soluble, 0= extremely low
d
Hepatotox_Level; 1= toxic, 0= non toxic
e
f
Cyp 2D6; 1= likely to inhibit, 0= non inhibitor
PPB (Plasma protein binding); 2= more than 95%, 1= more than 90%, 0= less than 90%
g
PSA (polar surface area); cpds must have log p value not greater than 5.0 to attain a reasonable probability of being
well absorbed.
2
.4. Structure Activity Relationship (SAR)
Replacement of the coumarin ring in lead compounds (A) and (B) with phenothiazine ring was
found to greatly enhance the cytotoxic activity [13]. The thiazolyl derivative 11 and the pyridinyl
derivative 13 revealed the most potent cytotoxic activity against human breast cancer cell line
(T47D) (IC50 8.1 and 8.8 µM), this gives an indication that the introduction of a substituted
sulfonamide group is more favorable than the unsubstituted and could enhance the activity. The
most potent cytotoxic compounds were screened for their aromatase inhibitory profile,
compounds 11 and 13 showed the highest percentage inhibition and the lowest IC towards the
5
0
1
7

ACCEPTED MANUSCRIPT
aromatase enzyme (IC50 5.67 and 6.70 µM). Followed by the quinoxalinyl derivative 22, that
showed moderate cytotoxicity (IC50 21.4 µM), but high percentage inhibition and low IC50
towards the aromatase enzyme (IC 8.90 µM). The mono and disubstituted derivatives 24 and
5
0
2
5 were found to possess moderate to poor cytotoxic activity and low percentage inhibition
towards the enzyme tested. Accordingly, the thiazolyl derivative 11 was investigated for its
apoptotic effect and showed a marked increase in Bax level, and down-regulation in Bcl2.
Furthermore, the effect on caspases 3, 8 and 9 was evaluated and was found to increase their
levels by 20, 34, and 8.9 folds, respectively, which indicates the activation of both intrinsic and
extrinsic pathways. Cell cycle analysis indicated that compound 11 arrested the cell cycle at
G2/M phase. Finally, according to computer aided ADMET studies, compounds 11 and 13 was
found to show moderate absorption in human intestine.
3
. Conclusion
Twenty novel phenothiazine derivatives bearing sulfonamide moiety were designed and
synthesized as aromatase inhibitors. All the newly prepared compounds were evaluated for their
in vitro anticancer activity against T47D cell line, where they showed anticancer activities with
IC values ranging from 8.1- 91.1 µM. In order to investigate their aromatase inhibitory effect,
5
0
eleven compounds were tested against the target enzyme, where compound 7 produced the
greatest inhibitory effect (72.2%) with IC = 9.67 µM, and compound 11 bearing unsubstituted
5
0
thiazole ring produced a good inhibitory effect (68.32 %) and the lowest IC value (5.67 µM).
50
Further investigations were performed to prove the proapoptotic effect of our target compounds.
Compound 11 showed a marked increase in BAX level and downregulation in Bcl2 and Bcl-XL
to undetectable levels, in comparison to the control. To figure out the apoptotic mechanism of
compound 11, its effect on caspases 3, 8 and 9 was evaluated and found to increase their levels
by activation of both intrinsic and extrinsic pathways of apoptosis. Finally, molecular modeling
studies were carried out. First, molecular docking which showed that phenothiazine series binds
to the target enzyme in the same pattern as androstenedione “the natural ligand” and that the
phenothiazine ring fits into the large hydrophobic pocket in the binding site. Second, computer
aided ADMET study was performed, compounds 5 and 6 having unsubstituted sulfonamide and
N-sulfonyl acetamide, respectively were expected to possess good human intestinal absorption.
1
8

ACCEPTED MANUSCRIPT
4
. Experimental
4
.1. Chemistry
Melting points (uncorrected) were determined in an open capillary on a Gallen Kamp
melting point apparatus (Sanyo Gallen Kamp, UK). Precoated silica gel plates (Kieselgel 0.25
mm, 60 F254, Merck, Germany) were used for thin layer chromatography. A developing solvent
system of chloroform/methanol (8:2) was used and the spots were detected by ultraviolet light.
IR spectra (KBr discs) were recorded using an FT-IR spectrophotometer (Perkin Elmer, USA).
1
H-NMR spectra were scanned on an NMR spectrophotometer (Bruker AXS Inc., Switzerland),
1
13
operating at 500 MHz for H and 125.76 MHz for C. Chemical shifts are expressed in (ppm) -
values relative to TMS as an internal standard, using DMSO-d as a solvent. Elemental analyses
6
were done on a model 2400 CHNSO analyzer (Perkin Elmer, USA). All the values were within ±
0
.4 % of the theoretical values. All reagents used were of AR grade. The starting material 1-
10H-phenothiazin-2-yl) ethanone 1 was purchased from sigma (USA) and was directly used for
the preparation of the target compounds.
(
4
.1.1. (Z)-3-(dimethylamino)-1-(10H-phenothiazin-2-yl) prop-2-en-1-one (3).
To a solution of 1 (2.41 g, 0.01 mol) in dry xylene (20 mL) dimethylformamide-
dimethylacetal (DMF-DMA) 2 (1.91g, 0.015 mol) was added and refluxed for 24 h. The obtained
solid was collected by filtration and crystallized from ethanol to give compound 3.
1
Yield, 81%; m.p. 247.0 ºC. IR: 3257 (NH), 3051 (arom.), 2970, 2846 (aliph.), 1637 (CO). H-
NMR: 3.1 (s, 6H, N(CH ) ), 5.7, 7.7 (2d, 2H, CH=CH, J= 12.5 Hz), 6.9-7.3 (m, 7H, Ar-H), 8.6
3
2
1
3
(
s, 1H, NH). C-NMR: 44.9 (2), 91.0, 113.1, 114.9 (2), 116.1, 120.4, 122.3, 126.1, 126.7, 128.2,
+
1
40.0, 142.1, 142.4, 154.6, 185.1. MS m/z (%): 296 (M ) (10.37), 227 (100). Anal. Calcd. for
C H N OS (296.39): C, 68.89; H, 5.44; N, 9.45. Found: C, 68.56; H, 5.11; N, 9.13.
17
16
2
4
.1.2. General procedure for the synthesis of benzenesulfonamide derivatives (5-22).
A mixture of 3 (2.96 g, 0.01mol) and sulfa drugs 4 (0.012 mol) in absolute ethanol (10 mL)
and glacial acetic acid (3mL) was refluxed for 18h. The solid product formed was collected by
filtration and crystallized from ethanol to give 5-22.
4
.1.2.1. (Z)-4-(3-Oxo-3-(10H-phenothiazin-2-yl) prop-1-enylamino)benzenesulfonamide (5)
1
9

ACCEPTED MANUSCRIPT
Yield, 61%; m.p. 299.4 ºC. IR: 3344, 3248 (NH
2
, NH), 3085 (arom.), 2943, 2863 (aliph.),
1
1
660 (2CO), 1334, 1186 (SO ). H-NMR: 6.0, 7.9 (2d, 2H, CH=CH, J= 10 Hz), 6.6-7.8 (m, 11H,
2
1
3
Ar-H), 8.7 (s, 1H, SO NH ), 10.3 (s, 1H, NH, phenothiazine), 11.9 (s, 1H, NH). C-NMR: 94.9,
2
2
1
1
12.7, 113.0 (2), 115.0, 115.5, 115.9, 116.4, 121.4, 122.5, 126.5, 127.9, 128.1, 128.3 (2), 128.4,
+
37.5, 141.8 (2), 143.3, 189.4. MS m/z (%): 423 (M ) (8.28), 198 (100). Anal. Calcd. for
21 17 3 3 2
C H N O S (423.51): C, 59.56; H, 4.05; N, 9.92. Found: C, 59.84; H, 4.33; N, 10.06.
4
.1.2.2. (Z)-N-4-(3-Oxo-3-(10H-phenothiazin-2-yl) prop-1-enylamino) phenylsulfonyl)acetamide
(6)
Yield, 84%; m.p. 278.8 ºC. IR: 3305, 3248 (NH), 3055 (arom.), 2931, 2836 (aliph.), 1737,
1
1
1
1
1
635 (2CO), 1363, 1190 (SO
2 3
). H-NMR: 1.9 (s, 3H, COCH ), 6.1, 7.5 (2d, 2H, CH=CH, J=
0.5 Hz), 6.7-7.4 (m, 11H, Ar-H), 8.7 (s, 2H, SO NH), 10.4 (s, 1H, NH phenothiazine), 10.5 (s,
2
1
3
H, NH). C-NMR: 23.7, 95.6, 112.7, 113.0 (2), 115.0, 115.3, 115.9, 116.3, 121.5, 122.1, 126.5,
+
26.6, 126.7, 128.3 (2), 128.4, 138.1, 141.7 (2), 142.5, 169.2, 189.5. MS m/z (%): 465 (M )
(
43.14), 267 (100). Anal. Calcd. for C H N O S (465.54): C, 59.34; H, 4.11; N, 9.03. Found:
23 19 3 4 2
C, 59.03; H, 4.00; N, 8.95.
4
.1.2.3.
(Z)-N-carbamimidoyl-4-(3-oxo-3-(10H-phenothiazine-2yl)
prop-1-enylamino)
benzenesulfonamide (7)
Yield, 77%; m.p. 285.4 ºC. IR: 3429, 3354, 3196 (NH
2
, NH), 3088 (arom.), 2954, 2854
1
(
aliph.), 1635 (CO), 1589 (CN), 1319, 1132 (SO ). H-NMR: 6.1, 7.9 (2d, 2H, CH=CH, J= 10.5
2
Hz), 6.9-7.8 (m, 12H, Ar-H), 8.1 (s, 1H, SO NH), 8.7 (s, 2H, NH ), 10.3 (s, 2H, NH,
2
2
1
3
phenothiazine + NH imino), 11.9 (s, 1H, NH). C-NMR: 94.7, 112.7 (2), 113.0, 115.0 (2),
1
1
15.9, 116.3, 121.4, 122.2, 126.5, 127.7, 127.9 (2), 128.3, 138.2, 141.8, 142.5, 143.8, 145.3,
+
58.5, 189.3. MS m/z (%): 465 (M ) (0.98), 388 (100). Anal. Calcd. for C22
H
19
N
5
O
3
S
2
(465.55):
C, 56.76; H, 4.11; N, 15.04. Found: C, 56.45; H, 4.00; N, 14.89.
.1.2.4. (Z)-N-(3-methylisoxazol-5-yl)-4-(3-oxo-3-(10H-phenothiazine-2yl) prop-1-enylamino)
4
benzenesulfonamide (8)
Yield, 85%; m.p. 270.5 ºC. IR: 3388, 3298, 3161 (NH), 3045 (arom.), 2912, 2871 (aliph.),
1
1
635 (CO), 1591 (CN), 1381, 1161 (SO ). H-NMR: 2.3 (s, 3H, CH ), 6.1, 7.4 (2d, 2H, CH=CH,
2
3
2
0

ACCEPTED MANUSCRIPT
2
J= 10.1 Hz), 6.2 (s, 1H, CH isoxazole), 6.7-7.3 (m, 11H, Ar-H), 8.7 (s, 1H, SO NH), 10.4 (s,
1
3
1
H, NH phenothiazine), 11.9 (s, 1H, NH). C-NMR: 12.5, 95.5, 100.4, 112.7, 115.0 (2), 115.7
(2), 115.9, 116.7, 121.5, 126.5, 126.7, 128.3, 129.1, 129.3, 132.1 (2), 138.8, 141.4 (2), 144.7,
+
1
58.0, 158.1, 189.5. MS m/z (%): 504 (M ) (18.53), 333 (100). Anal. Calcd. for C25
H
20
N
4
O
4
S
2
(504.58): C, 59.51; H, 4.00; N, 11.10. Found: C, 59.22; H, 3.87; N, 10.84.
4
.1.2.5.
(Z)-N-(3,4-Dimethylisoxazol-5-yl)-4-(3-oxo-3-(10H-phenothiazine-2-yl)
prop-1-
enylamino) benzenesulfonamide (9)
Yield, 69%; m.p. 277.5 ºC. IR: 3309, 3272 (NH), 3016 (arom.), 2823, 2765 (aliph.), 1634
1
(
CO), 1583 (CN), 1375, 1186 (SO ). H-NMR: 2.1, 2.2 (2s, 6H, 2CH ), 6.1, 7.5 (2d, 2H,
2
3
CH=CH, J= 10 Hz), 6.7-7.4 (m, 11H, Ar-H), 8.7 (s, 1H, SO
2
NH), 10.4 (s, 1H, NH,
1
3
phenothiazine), 11.9 (s, 1H, NH). C-NMR: 6.3, 10.7, 95.6, 100.5, 112.7, 113.0 (2), 115.0,
1
1
15.7, 115.9, 116.7, 121.5, 122.1, 126.5, 126.7, 128.3, 129.0, 129.2 (2), 138.8, 141.7 (2), 143.2,
+
56.1, 161.8, 187.1. MS m/z (%): 518 (M ) (9.43), 266 (100). Anal. Calcd. for C H N O S
2
6
22
4
4
2
(518.61): C, 60.21; H, 4.28; N, 10.80. Found: C, 62.54; H, 4.58; N, 11.15.
4
.1.2.6. (Z)-4-(3-Oxo-3-(10H-phenothiazine-2-yl) prop-1-enylamino)-N-(1-phenyl-1H-pyrazol-5-
yl)-benzenesulfonamide (10)
Yield, 88%; m.p. 258.9 ºC. IR: 3409, 3374 (NH), 3062 (arom.), 2816, 2742 (aliph.), 1635
1
(CO), 1589 (CN), 1390, 1157 (SO
2
). H-NMR: 5.9 (d, 2H, 2CH pyrazole, J= 10 Hz), 6.1, 7.5
(
2d, 2H, CH=CH, J= 10.5 Hz), 6.8-7.4 (m, 18H, Ar-H), 8.9 (s, 1H, SO NH), 10.4 (s, 1H, NH,
2
1
3
phenothiazine), 11.9 (s, 1H, NH). C-NMR: 95.5, 104.3, 112.8, 113.0 (2), 115.0, 115.6 (2),
1
1
16.6, 121.5, 122.1 (2), 124.7, 126.5, 126.6, 126.7, 127.9, 128.3 (2), 129.1, 132.2 (2), 135.0,
+
38.1, 140.0, 142.5 (2), 143.3, 145.5, 187.1. MS m/z (%): 565 (M ) (0.76), 488 (100). Anal.
23 5 3 2
Calcd. for C30H N O S (565.67): C, 63.70; H, 4.10; N, 12.38. Found: C, 63.43; H, 3.79; N,
1
2.02.
4
.1.2.7.
(Z)-4-(3-Oxo-3-(10H-phenothiazine-2-yl)
prop-1-enylamino)-N-(thiazol-2-yl)
benzenesulfonamide (11)
Yield, 90%; m.p. 282.4 ºC. IR: 3392, 3187 (NH), 3066 (arom.), 2946, 2852 (aliph.), 1637
1
(
CO), 1587 (CN), 1361, 1139 (SO ). H-NMR: 6.0, 7.3 (2d, 2H, CH=CH, J= 10.1 Hz), 6.7-7.2
2
2
1

ACCEPTED MANUSCRIPT
1
3
(
2
m, 13H, Ar-H), 8.7 (s, 1H, SO NH), 10.3 (s, 1H, NH phenothiazine), 11.9 (s, 1H, NH). C-
NMR: 95.0, 108.6, 112.7, 112.9 (2), 113.0, 115.5 (2), 116.4, 121.4, 122.3, 126.5, 126.7, 128.1,
+
1
28.2, 128.3 (2), 135.6, 141.8, 142.5 (2), 143.6, 169.9, 187.0. MS m/z (%): 506 (M ) (2.54), 351
18 4 3 3
(100). Anal. Calcd. for C24H N O S (506.62): C, 56.90; H, 3.58; N, 11.06. Found: C, 56.61; H,
3
.26; N, 10.78.
4
.1.2.8. (Z)-N-(5-Methyl-1,3,4-thiadiazol-2-yl)-4-(3-oxo-3-(10H-phenothiazine-2yl) prop-1-
enylamino) benzenesulfonamide (12).
Yield, 77%; m.p. 274.0 ºC. IR: 3304, 3198 (NH), 3034 (arom.), 2883, 2814, (aliph.), 1633
1
(
CO), 1589 (CN), 1363, 1149 (SO ). H-NMR: 2.4 (s, 3H, CH ), 6.1, 7.4 (2d, 2H, CH=CH, J=
2
3
1
1
1
0.5 Hz), 6.6-7.3 (m, 11H, Ar-H), 8.7 (s, 1H, SO
2
NH), 10.3 (s, 1H, NH phenothiazine), 11.9 (s,
1
3
H, NH). C-NMR: 16.5, 95.2, 112.7, 113.0 (2), 115.0, 115.6 (2), 116.6, 121.4, 122.0, 126.5,
26.7, 128.0, 128.2, 128.3 (2), 141.8, 142.5 (2), 143.4, 144.9, 168.2, 187.0. MS m/z (%): 521
+
(
M ) (33.62), 323 (100). Anal. Calcd. for C H N O S (521.63): C, 55.26; H, 3.67; N, 13.43.
24
19
5
3 3
Found: C, 54.91; H, 3.32; N, 13.11.
4
.1.2.9.
(Z)-4-(3-Oxo-3-(10H-phenothiazine-2yl)
prop-1-enylamino)-N-(pyridin-2-yl)
benzenesulfonamide (13)
Yield, 88%; m.p. 263.4 ºC. IR: 3450, 3358 (NH), 3100 (arom.), 2945, 2872 (aliph.), 1633
1
(CO), 1591 (CN), 1386, 1134 (SO
2
). H-NMR: 6.1, 7.4 (2d, 2H, CH=CH, J= 10 Hz), 6.6-7.3 (m,
1
3
1
9
1
5H, Ar-H), 8.5 (s, 1H, SO NH), 10.3 (s, 1H, NH phenothiazine), 11.9 (s, 1H, NH). C-NMR:
2
5.1, 112.7, 112.9, 113.0 (2), 114.0, 115.4, 115.7, 115.9, 116.4, 121.4, 122.3, 126.5, 126.7,
28.3, 128.9, 129.2 (2), 138.1, 141.7, 143.5 (2), 144.8, 144.9, 153.2, 187.0. MS m/z (%): 500
+
(
M ) (0.99), 267 (100). Anal. Calcd. for C H N O S (500.59): C, 62.38; H, 4.03; N, 11.19.
2
6
20
4
3 2
Found: C, 62.08; H, 3.83; N, 11.01.
4
.1.2.10.
(Z)-4-(3-Oxo-3-(10H-phenothiazine-2yl)
prop-1-enylamino)-N-(pyrimidin-2-yl)
benzenesulfonamide (14)
Yield, 69%; m.p. 309.9 ºC. IR: 3358, 3110 (NH), 3037 (arom.), 2941, 2870 (aliph.), 1633
1
(CO), 1585 (CN), 1373, 1195 (SO
2
). H-NMR: 6.0, 7.6 (2d, 2H, CH=CH, J= 10 Hz), 6.7-7.5 (m,
13
1
4H, Ar-H), 8.4 (s, 1H, SO NH), 10.4 (s, 1H, NH phenothiazine), 11.6 (s, 1H, NH). C-NMR:
2
2
2

ACCEPTED MANUSCRIPT
9
1
5.4, 112.6, 112.7 (2), 113.0, 115.0, 115.9 (2), 116.2, 121.5, 122.0, 125.4, 126.5, 126.7, 128.3,
+
30.0 (2), 141.7, 142.5 (2), 144.3, 157.4 (2), 158.8, 187.1. MS m/z (%): 501 (M ) (7.46), 342
(
100). Anal. Calcd. for C H N O S (501.58): C, 59.86; H, 3.82; N, 13.96. Found: C, 59.57; H,
25 19 5 3 2
3
.65; N, 13.60.
.1.2.11. (Z)-N-(4-Methylpyrimidin-2-yl)-4-(3-oxo-3-(10H-phenothiazine-2yl) prop-1-enylamino)
4
benzeneesulfonamide (15)
Yield, 90%; m.p. 302.0 ºC. IR: 3371, 3162 (NH), 3032 (arom.), 2960, 2870 (aliph.), 1635
1
(
CO), 1589 (CN), 1373, 1157 (SO ). H-NMR: 2.3 (s, 3H, CH ), 6.0, 7.5 (2d, 2H, CH=CH, J=
2
3
1
1
1
0.1 Hz), 6.6-7.4 (m, 13H, Ar-H), 8.3 (s, 1H, SO NH), 10.4 (s, 1H, NH phenothiazine), 11.9 (s,
2
1
3
H, NH). C-NMR: 23.7, 95.3, 112.4, 112.7, 113.0 (2), 115.0, 115.2, 115.8, 115.9, 121.5, 122.0,
25.4, 126.5, 126.7, 128.3, 130.2 (2), 141.8, 142.5 (2), 143.4, 153.4, 168.4, 168.6, 187.0. MS
+
m/z (%): 515 (M ) (6.93), 198 (100). Anal. Calcd. for C26
H
21
N
5
O
3
S
2
(515.61): C, 60.57; H,
4
.11; N, 13.58. Found: C, 60.24; H, 4.06; N, 13.27.
4
.1.2.12.
(Z)-N-(4,6-Dimethylpyrimidin-2-yl)-4-(3-oxo-3-(10H-phenothiazine-2yl)
prop-1-
enylam-ino) benzenesulfonamide (16)
Yield, 91%; m.p. 271.8 ºC. IR: 3360, 3116 (NH), 3100 (arom.), 2961, 2836 (aliph.), 1633
1
(
CO), 1587 (CN), 1371, 1192 (SO ). H-NMR: 2.2 (s, 6H, 2CH ), 6.0 (s, 1H, CH pyrimidine),
2
3
6
2
.4, 7.5 (2d, 2H, CH=CH, J= 10 Hz), 6.6-7.4 (m, 12H, Ar-H), 8.5 (s, 1H, SO NH), 10.3 (s, 1H,
1
3
NH phenothiazine), 11.9 (s, 1H, NH). C-NMR: 23.5 (2), 95.2, 112.7, 113.0, 113.9 (2), 114.9,
1
1
15.0, 115.8, 115.9, 121.5, 126.5, 126.7, 128.3, 130.5, 130.7, 133.5 (2), 141.7, 143.4 (2), 145.0,
+
67.8 (2), 172.5, 187.0. MS m/z (%): 529 (M ) (17.62), 262 (100). Anal. Calcd. for
C H N O S (529.63): C, 61.23; H, 4.38; N, 13.22. Found: C, 61.02; H, 4.10; N, 13.16.
27
23
5
3 2
4
.1.2.13. (Z)-N-(2, 6-Dimethylpyrimidin-2-yl)-4-(3-oxo-3-(10H-phenothiazine-2yl) prop-1-enyla-
mino) benzenesulfonamide (17)
Yield, 86%; m.p. 261.3 ºC. IR: 3372, 3210 (NH), 3100 (arom.), 2971, 2836 (aliph.), 1637
1
(
CO), 1589 (CN), 1373, 1199 (SO ). H-NMR: 2.2, 2.3 (2s, 6H, 2CH ), 6.0, 7.5 (2d, 2H,
2
3
CH=CH, J= 10 Hz), 6.6-7.4 (m, 12H, Ar-H), 8.7 (s, 1H, SO
2
NH), 10.3 (s, 1H, NH
1
3
phenothiazine), 11.9 (s, 1H, NH). C-NMR: 23.2, 23.9, 95.8, 105.0, 112.7, 113.0 (2), 115.0,
2
3

ACCEPTED MANUSCRIPT
1
1
15.3, 115.9, 116.2, 121.9, 126.5, 126.7, 128.3, 128.8, 128.9, 129.1 (2), 141.8, 142.5 (2), 143.5,
+
54.2, 163.8, 164.6, 187.0. MS m/z (%): 529 (M ) (34.36), 122(100). Anal. Calcd. for
C H N O S (529.63): C, 61.23; H, 4.38; N, 13.22. Found: C, 61.52; H, 4.76; N, 13.41.
27
23
5
3 2
4
.1.2.14. (Z)-N-(5-Methoxypyrimidin-2-yl)-4-(3-oxo-3-(10H-phenothiazine-2yl) prop-1-enyl-
amino) benzenesulfonamide (18)
Yield, 84%; m.p. 281.5 ºC. IR: 3363, 3294 (NH), 3100 (arom.), 2929, 2839 (aliph.), 1635
1
(CO), 1591 (CN), 1338, 1153 (SO
2
). H-NMR: 3.7 (s, 3H, OCH
3
), 6.0, 7.5 (2d, 2H, CH=CH, J=
1
1
1
0.5 Hz), 6.5-7.4 (m, 13H, Ar-H), 8.7 (s, 1H, SO NH), 10.4 (s, 1H, NH phenothiazine), 11.9 (s,
2
1
3
H, NH). C-NMR: 56.7, 95.3, 112.6, 112.7 (2), 113.0, 115.0, 115.8, 116.2, 121.4, 125.7, 126.5,
28.3, 129.8, 130.1, 133.3 (2), 141.7 (2), 142.5, 143.4 (2), 144.2, 144.9, 162.0, 187.0. MS m/z
+
(
%): 531(M ) (22.21), 203 (100). Anal. Calcd. for C H N O S (531.61): C, 57.74; H, 3.98; N,
26
21
5
4 2
1
3.17. Found: C, 57.46; H, 3.77; N, 12.84.
4
.1.2.15. (Z)-N-(2,6-Dimethoxypyrimidin-4-yl)-4- (3-oxo-3-(10H-phenothiazine-2yl) prop-1-
enyl-amino) benzenesulfonamide (19)
Yield, 80%; m.p. 199.4 ºC. IR: 3311, 3166 (NH), 3072 (arom.), 2949, 2899 (aliph.), 1637
1
(CO), 1589 (CN), 1363, 1155 (SO
2
). H-NMR: 3.6, 3.7 (2s, 6H, 2OCH
3
), 5.9 (s, 1H, CH
pyrimidine), 6.1, 7.5 (2d, 2H, CH=CH, J= 10 Hz), 6.7-7.4 (m, 11H, Ar-H), 8.5 (s, 1H, SO NH),
2
13
1
0.4 (s, 1H, NH phenothiazine), 11.9 (s, 1H, NH). C-NMR: 54.2, 55.0, 84.9, 95.5, 112.7, 112.9
(2), 113.0, 115.0, 115.8, 116.5, 121.5, 126.3, 126.5, 128.7, 129.6, 129.7, 129.8 (2), 141.7, 142.5
+
(
2), 143.3, 160.3, 164.7, 172.5, 187.2. MS m/z (%): 561(M ) (15.31), 77 (100). Anal. Calcd. for
C
27
H
23
N
5
O
5
S
2
(561.63): C, 57.74; H, 4.13; N, 12.47. Found: C, 57.96; H, 4.39; N, 12.81.
4
.1.2.16. (Z)-N-(5,6-Dimethoxypyrimidin-4-yl)-4- (3-oxo-3-(10H-phenothiazine-2yl) prop-1-
enyl-amino) benzenesulfonamide (20)
Yield, 78%; m.p. 141.2 ºC. IR: 3377, 3300 (NH), 3062 (arom.), 2945, 2861 (aliph.), 1635
1
(
CO), 1583 (CN), 1373, 1159 (SO ). H-NMR: 3.7, 3.8 (2s, 6H, 2OCH ), 6.0, 7.5 (2d, 2H,
2
3
CH=CH, J= 10 Hz), 6.6-7.4 (m, 11H, Ar-H), 8.1 (s, 1H, CH pyrimidine), 8.8 (s, 1H, SO NH),
2
1
3
1
1
0.4 (s, 1H, NH phenothiazine), 11.9 (s, 1H, NH). C-NMR: 54.5, 60.6, 95.4, 112.6, 112.7 (2),
13.0, 115.2, 115.9, 116.2, 122.0, 126.5, 126.7, 127.7, 128.3, 129.9, 130.1 (2), 133.5, 141.7,
2
4

ACCEPTED MANUSCRIPT
+
1
42.5 (2), 143.4, 150.9, 162.0, 172.2, 187.1. MS m/z (%): 561 (M ) (10.26), 93 (100). Anal.
Calcd. for C H N O S (561.63): C, 57.74; H, 4.13; N, 12.47. Found: C, 58.11; H, 4.38; N,
2
7
23
5
5 2
1
2.85.
4
.1.2.17.
(Z)-N-(1H-Indazol-6-yl)-4-(3-oxo-3-(10H-phenothiazine-2yl)
prop-1-enylamino)
benzenesulfonamide (21)
Yield, 85%; m.p. 259.3 ºC. IR: 3385, 4342 (NH), 3066 (arom.), 2966, 2841 (aliph.), 1635
1
(CO), 1591 (CN), 1348, 1186 (SO
2
). H-NMR: 6.0, 7.4 (2d, 2H, CH=CH, J= 10.5 Hz), 6.5-7.3
(
m, 15H, Ar-H + CH indazole), 8.7 (s, 1H, SO NH), 10.3 (s, 1H, NH phenothiazine), 11.9 (s, 1H,
2
1
3
NH), 12.8 (s, 1H, NH indazole). C-NMR: 91.0, 95.4, 112.7 (2), 113.1 (2), 113.2, 115.0, 115.3,
1
1
15.9, 116.5, 120.2, 121.5, 122.5, 126.5, 128.2, 129.0, 129.2, 133.4 (2), 136.6, 140.7, 142.1 (2),
+
43.3, 144.3, 145.3, 187.1. MS m/z (%): 539 (M ) (6.81), 181 (100). Anal. Calcd. for
28 21 5 3 2
C H N O S (539.63): C, 62.32; H, 3.92; N, 12.98. Found: C, 62.53; H, 4.28; N, 13.33.
4
.1.2.18.
(Z)-4-(3-Oxo-3-(10H-phenothiazine-2yl)
prop-1-enylamino)-N-(quinoxalin-2-yl)
benzenesulfonamide (22)
Yield, 78%; m.p. 231.5 ºC. IR: 3439, 3360 (NH), 3072 (arom.), 2979, 2841 (aliph.), 1635
1
(CO), 1591 (CN), 1346, 1190 (SO
2
). H-NMR: 5.9, 7.5 (2d, 2H, CH=CH, J= 10 Hz), 6.7-7.3 (m,
1
3
1
9
1
6H, Ar-H), 8.7 (s, 1H, SO NH), 10.4 (s, 1H, NH phenothiazine), 11.9 (s, 1H, NH). C-NMR:
2
5.4, 112.7, 113.0 (2), 114.9, 115.0, 115.8, 116.3, 120.4, 121.4, 122.4, 126.1, 126.5, 127.5,
28.2, 128.3, 129.1, 130.2, 130.5 (2), 131.1, 131.3, 138.8, 141.8, 142.1 (2), 146.7, 160.0, 185.1.
+
MS m/z (%): 551(M ) (16.70), 193 (100). Anal. Calcd. for C H N O S (551.64): C, 63.14; H,
2
9
21
5
3 2
3
.84; N, 12.70. Found: C, 63.50; H, 3.93; N, 12.98.
4
.1.3. (Z)-3-(4-Aminophenylsulfonyl) phenylamino)-1-(10H-phenothiazin-2-yl)prop-2-en-1-one
(24)
A mixture of 3 (2.96 g, 0.01 mol) and dapson 23 (2.48g, 0.01 mol) in absolute ethanol (10 mL)
and glacial acetic acid (5 mL) was refluxed for 13h. The obtained solid was crystallized from
dioxane to give 24.
Yield, 80%; m.p. 317.1 ºC. IR: 3356, 3213, 3153 (NH , NH), 3057 (arom.), 2941, 2861
2
1
(
aliph.), 1635 (CO), 1363, 1197 (SO ). HNMR: 6.1, 7.5 (2d, 2H, CH=CH, J= 10.5 Hz), 6.2 (s,
2
2
5

ACCEPTED MANUSCRIPT
1
3
2
9
2
H, NH ), 6.5-7.4 (m, 15H, Ar-H), 10.4 (s, 1H, NH phenothiazine), 11.9 (s, 1H, NH). CNMR:
5.3, 112.7, 113.0 (2), 113.4 (2), 115.0, 115.8, 115.9, 116.8, 121.4, 126.5, 126.7, 128.1, 129.5
+
(
4), 129.6, 129.7 (2), 141.7, 141.8 (2), 144.8, 153.8, 187.0. MS m/z (%): 499 (M ) (1.83), 483
21 3 3 2
(100). Anal . Calcd. for C27H N O S (499.60): C, 64.91; H, 4.24; N, 8.41. Found: C, 64.59; H,
4
.09; N, 8.14.
4
.1.4. (2Z,2'Z)-3, 3'-(4,4'-sulfonylbis(4,1-phenylene)bis(azanediyl))bis(1-(10H-phenothiazin-2-yl)
prop-2-en-1-one (25)
A mixture of 3 (5.92 g, 0.02 mol) and dapson 23 (2.48g, 0.01 mol) in absolute ethanol (20 mL)
and glacial acetic acid (5 mL) was refluxed for 18h. The obtained solid was crystallized from
acetic acid to give 25.
Yield, 79%; m.p. 233.3 ºC. IR: 3410, 3354 (NH), 3055 (arom.), 2917, 2880 (aliph.), 1635
1
(
2CO), 1390, 1197 (SO ). HNMR: 5.7, 7.4 (2d, 4H, 2CH=CH, J=12.5 Hz), 6.6-7.3 (m, 22H, Ar-
2
1
3
H), 10.4 (s, 2H, 2NH phenothiazine), 11.9 (s, 2H, 2NH). CNMR: 95.3 (2), 112.7 (2), 113.0 (4),
1
1
13.4 (2), 114.9 (2), 115.0 (2), 116.8 (2), 121.5 (2), 126.5 (2), 126.7 (2), 128.2 (2), 128.8 (6),
+
29.7 (2), 141.8 (2), 143.3 (4), 145.1 (2), 185.1 (2). MS m/z (%): 750 (M ) (49.8), 374 (100).
Anal. Calcd. for C H N O S (750.91): C, 67.18; H, 4.03; N, 7.46. Found: C, 67.48; H, 4.32;
4
2
30
4
4 3
N, 7.77.
4
4
.2. Biological evaluation
.2.1. SRB cytotoxicity assay
T47D breast cancer cells (obtained from National Cancer Institute, Cairo, Egypt) and 184A1
normal breast cells (obtained from VACSERA, Cairo, Egypt) were grown in RPMI-1640
-1
medium supplemented with 5% heat-inactivated fetal calf serum (FCS), 1 mmol L L-glutamine,
-1
and 50 µg mL gentamicin and maintained at 37 °C in a humidified atmosphere containing 5%
CO . The cells were maintained as “monolayer culture” by serial subculturing. Exponentially
2
growing cells were collected using 0.25% Trypsin-EDTA and seeded in 96-well plates at 1000-
2
000 cells/well in RPMI-1640 supplemented medium. After 24 h., cells were incubated for 48 h.
-1
with various concentrations (5, 12, 25 and 50 µmol L ) of the tested compounds. Following 48
h. treatment, the cells will be fixed with 10% trichloroacetic acid for 1 h. at 4 ºC. Wells were
2
6

ACCEPTED MANUSCRIPT
stained for 30 min at room temperature with 0.4% SRB dissolved in 1% acetic acid. The plates
were air dried for 24 h. and the dye was solubilized with Tris-HCl for 5 minutes on a shaker at
1
600 rpm. The optical density (OD) of each well was measured spectrophotometrically at 564
nm with an ELISA microplate reader (ChroMate-4300, FL, USA). The IC50 values were
calculated according to the equation for Boltzmann sigmoidal concentration–response curve
using the nonlinear regression fitting models (Graph Pad, Prism Version 5) [17].
4
.2.2. Aromatase assay
Aromatase activity and IC50 of the selected compounds were determined in T47D cells which
were obtained from American Type Culture Collection, T47D cells were cultured using DMEM
(
Invitrogen/Life Technologies) supplemented with 10% FBS (Hyclone), 10 ug/ml of insulin
Sigma), and 1% penicillin-streptomycin. All the other chemicals and reagents were from Sigma,
(
or Invitrogen. Plate cells (cells density 1.2 – 1.8 × 10,000 cells/well) in a volume of 100 µl
complete growth medium + 100 ul of the tested compound per well in a 96-well plate for 18–24
hours before the enzyme assay for aromatase. T47D cells were treated with serial dilutions of the
selected potent compounds for 24 hours and then the aromatase activity was determined as
previously mentioned.
4
.2.3. Effect on active caspase-3
The active caspase-3 level was measured by using Quantikine -Human active Caspase-3
Immunoassay (R&D Systems, Inc. Minneapolis, USA) according to the manufacturer protocol
by a method developed in our lab.
4
.2.4. Effect on caspase-8 & caspase-9
Human caspase-8 ELISA kit (EIA-4863) and human caspase-9 ELISA kit DRG
®
Caspase-9 (EIA-4860) (DRG International Inc., USA were used, and the procedure adopted by
our lab.
4
.2.5. PCR analysis and quantification of gene expression of BAX, Bcl-2, Bcl-X
L
RNeasy Mini Kit® (Qiagen Inc. Valencia, CA, USA) was used for the total RNA
extraction from cells. Reverse transcription was undertaken to construct cDNA library from
different treatments using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems,
2
7

ACCEPTED MANUSCRIPT
Foster City, CA). The archived cDNA libraries were then subjected to quantitative real-time
PCR reactions using cyber green fluorophore (Fermentas Inc., Glen Burnie, MD, USA). Primer
sequences were as follows: Bcl-2 forward primer GGG-TAC-GAT-AAC-CGG-GAG-AT and
reverse primer CTG-AAG-AGC-TCC-TCC-ACC-AC; BAX forward primer TCT-GAC-GGC-
AAC-TTC-AAC-TG and reverse primer TGG-GTG-TCC-CAA-AGT-AGG-AG; Bcl-XL
forward primer GGC GGA TTT GAA TCT CTT TCT C and reverse primer TTA TAA TAG
GGA TGG GCT CAA CC; reference housekeeping gene used was GAPDH with forwarding
primer TGC-ACC-ACC-AAC-TGC-TTA-G and reverse primer GAT-GCA-GGG-ATG-ATG-
TTC.
4
.3. Molecular docking
The crystal structure of the aromatase enzyme co-crystallized with the androstenedione
ASD) substrate was obtained from the Protein Data Bank (PDB ID: 3EQM). Docking study was
(
performed using Accelrys software (Discovery Studio 2.5) in the computer drug design lab in the
pharmaceutical chemistry department, Faculty of pharmacy, Ain Shams University. The protein
was prepared by prepare protein protocol. Our ligands were drawn as a database and prepared by
prepared ligand protocol during which it was refined using CHARMM force field with full
potential. CDOCKER protocol was then run, and different binding poses were ranked according
to the calculated interaction binding energies and the binding mode was analyzed for each
compound. Also, ADMET study was performed in the same lab using Accelrys software
(Discovery Studio 2.5). Some of the parameters that are calculated using Accord for Excel
includes Atom based Log P98 (A LogP 98), ADMET 2D polar surface area (ADMET 2D PSA),
Aqueous solubility (AQ SOl), Aqueous solubility level (AQ SOl LEV), Blood Brain Barrier
value (BBB), Blood Brain Barrier Level ( BBB LEV), Cytochrome P450 2D6 (CYP2D6),
Cytochrome P450 2D6 Probability (CYP PROB), Hepatotoxicity Level (HEPATOX LEV),
Hepatotoxicity Probability (HEPATOX PROB), Plasma protein binding logarithmic value (PPB
LOG) and Plasma protein binding logarithmic level (PPB LEV). The compounds structures were
drawn using DS Viewer ProSuite software and appended into Accord for Excel software then the
parameters were calculated.
Acknowledgment
2
8