Enhancement of the tail hydrophobic interactions within the carbonic anhydrase IX active site via structural extension: Design and synthesis of novel N-substituted isatins-SLC-0111 hybrids as carbonic anhydrase inhibitors and antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2018.10.068

Herein we report the design and synthesis of novel N-substituted isatins-SLC-0111 hybrids (6a-f and 9a-l). A structural extension approach was adopted via N-alkylation and N-benzylation of isatin moiety to enhance the tail hydrophobic interactions within the carbonic anhydrase (CA) IX active site. Thereafter, a hybrid pharmacophore approach was utilized via merging the pharmacophoric elements of isatin and SLC-0111 in a single chemical framework. As planned, a substantial improvement of inhibitory profile of the target hybrids (K_Is: 4.7–86.1 nM) towards hCA IX in comparison to N-unsubstituted leads IVa-c (K_Is: 192–239 nM), was achieved. Molecular docking of the designed hybrids in CA IX active site unveiled, as planned, the ability of N-alkylated and N-benzylated isatin moieties to accommodate in a wide hydrophobic pocket formed by T73, P75, P76, L91, L123 and A128, establishing strong van der Waals interactions. Hybrid 6c displayed good anti-proliferative activity under hypoxic conditions towards breast cancer MDA-MB-231 and MCF-7 cell lines (IC₅₀ = 7.43 ± 0.28 and 12.90 ± 0.34 μM, respectively). Also, 6c disrupted the MDA-MB-231 cell cycle via alteration of the Sub-G₁ phase and arrest of G₂-M stage. Additionally, 6c displayed significant increase in the percent of annexinV-FITC positive apoptotic cells from 1.03 to 18.54%. Furthermore, 6c displayed potent VEGFR-2 inhibitory activity (IC₅₀ = 260.64 nM). Collectively, these data suggest 6c as a promising lead molecule for the development of effective anticancer agents.

Full text of DOI:10.1016/j.ejmech.2018.10.068

Accepted Manuscript
Enhancement of the tail hydrophobic interactions within the carbonic anhydrase IX
active site via structural extension: Synthesis of novel N-substituted isatins-SLC-0111
hybrids as carbonic anhydrase inhibitors and antitumor agents
Wagdy M. Eldehna, Mahmoud F. Abo-Ashour, Alessio Nocentini, Radwan S. El-
Haggar, Silvia Bua, Alessandro Bonardi, Sara T. Al-Rashood, Ghada S. Hassan,
Paola Gratteri, Hatem A. Abdel-Aziz, Claudiu T. Supuran
PII:
S0223-5234(18)30946-2
DOI:
https://doi.org/10.1016/j.ejmech.2018.10.068
EJMECH 10854
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 September 2018
Revised Date: 11 October 2018
Accepted Date: 31 October 2018
Please cite this article as: W.M. Eldehna, M.F. Abo-Ashour, A. Nocentini, R.S. El-Haggar, S. Bua, A.
Bonardi, S.T. Al-Rashood, G.S. Hassan, P. Gratteri, H.A. Abdel-Aziz, C.T. Supuran, Enhancement of
the tail hydrophobic interactions within the carbonic anhydrase IX active site via structural extension:
Synthesis of novel N-substituted isatins-SLC-0111 hybrids as carbonic anhydrase inhibitors and
antitumor agents, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.10.068.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Enhancement of the tail hydrophobic interactions within the carbonic anhydrase
IX active site via structural extension: Synthesis of novel N-substituted isatins-
SLC-0111 hybrids as carbonic anhydrase inhibitors and antitumor agents
Wagdy M. Eldehna*, Mahmoud F. Abo-Ashour, Alessio Nocentini, Radwan S. El-Haggar,
Silvia Bua, Alessandro Bonardi, Sara T. Al-Rashood, Ghada S. Hassan, Paola Gratteri, Hatem
A. Abdel-Aziz, Claudiu T. Supuran*
A substantial improvement of inhibitory profile of the target hybrids (KIs: 4.7–86.1 nM) towards
hCA IX in comparison to N-unsubstituted leads IVa-c (KIs: 192–239 nM), was achieved.

ACCEPTED MANUSCRIPT
Enhancement of the tail hydrophobic interactions within the carbonic anhydrase
IX active site via structural extension: synthesis of novel N-substituted isatins-
SLC-0111 hybrids as carbonic anhydrase inhibitors and antitumor agents
Wagdy M. Eldehnaa,*, Mahmoud F. Abo-Ashourb, Alessio Nocentinic,d, Radwan S. El-Haggare,
Silvia Buac, Alessandro Bonardic,d, Sara T. Al-Rashoodf, Ghada S. Hassang, Paola Gratterid,
Hatem A. Abdel-Azizh, Claudiu T. Supuranc,**
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University,
Kafrelsheikh, Egypt
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr
City, Cairo 11829, Egypt
cDepartment of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of
Florence, Polo Scientifico, Via U. Schiff 6, 50019, Sesto Fiorentino, Firenze, Italy
dDepartment of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, Laboratory of
Molecular Modeling Cheminformatics & QSAR, University of Florence, Polo Scientifico, Via U. Schiff
ePharmaceutical Chemistry Department, Faculty of Pharmacy, Helwan University, Cairo, Egypt
6, 50019, Sesto Fiorentino, Firenze, Italy
fDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457,
Riyadh 11451, Saudi Arabia.
gDepartment of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516,
Egypt
hDepartment of Applied Organic Chemistry, National Research Center, Dokki, Giza, P.O. Box 12622,
Egypt
ABSTRACT
Herein we report the design and synthesis of novel N-substituted isatins-SLC-0111 hybrids
(6a-f and 9a-l). A structural extension approach was adopted via N-alkylation and N-
benzylation of isatin moiety to enhance the tail hydrophobic interactions within the carbonic
anhydrase (CA) IX active site. Thereafter, a hybrid pharmacophore approach was utilized via
merging the pharmacophoric elements of isatin and SLC-0111 in a single chemical framework.
As planned, a substantial improvement of inhibitory profile of the target hybrids (KIs: 4.7–86.1
nM) towards hCA IX in comparison to N-unsubstituted leads IVa-c (KIs: 192–239 nM), was
achieved. Molecular docking of the designed hybrids in CA IX active site unveiled, as planned,
the ability of N-alkylated and N-benzylated isatin moieties to accommodate in a wide
hydrophobic pocket formed by T73, P75, P76, L91, L123 and A128, establishing strong van
1

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der Waals interactions. Hybrid 6c displayed good anti-proliferative activity under hypoxic
conditions towards breast cancer MDA-MB-231 and MCF-7 cell lines (IC50 = 7.43±0.28 and
12.90±0.34 µM, respectively). Also, 6c disrupted the MDA-MB-231 cell cycle via alteration of
the Sub-G1 phase and arrest of G2-M stage. Additionally, 6c displayed significant increase in
the percent of annexinV-FITC positive apoptotic cells from 1.03 to 18.54%. Furthermore, 6c
displayed potent VEGFR-2 inhibitory activity (IC50 = 260.64 nM). Collectively, these data
suggest 6c as a promising lead molecule for the development of effective anticancer agents.
__________
Keywords: Anticancer Activity; Selective hCA IX and XII inhibitors; SLC-0111 hybrids; Tail
approach; VEGFR-2 inhibitors.
* Corresponding authors. E-mail addresses: wagdy2000@gmail.com (W.M. Eldehna),
claudiu.supuran@unifi.it (C.T. Supuran).
2

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1. Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread metalloenzymes in all organisms
which are necessary for CO2 hydration to bicarbonate and protons [1-3]. To date 16 diverse
isoforms have been identified, which have different tissue distribution and patterns of location,
thus cytosolic (I, II, III, VII, and XIII), membrane-bound (IV, IX, XII, and XIV), secreted (VI)
and mitochondrial (VA and VB) forms have been described in mammals [1-3]. CAs are
involved in many important physiological functions as respiration, pH regulation, ion transport,
bone resorption and secretion of gastric fluid juices. So, their inhibitors became an important
class of therapeutics during last decades. Some of novel applications of CAIs emerged, are
topically acting antiglaucoma, anticonvulsants and antitumor agents [4-7].
On account of its limited presence in normal tissues and high overexpression in a large
number of solid tumors, where it significantly contributes to survival and metastatic spread of
tumor cells, hCA IX has been known as a tumor-associated protein since its discovery in the
early 1990s. Thus, therapeutic strategies based on selective inhibition of CA IX have stood out
as an attractive tactic for discovering therapies for several human malignancies in the current
medical era [8-12]. The most exploited structural element enhances potency and selectivity of
sulfonamide-like CAIs is the “tail” that the zinc-binding inhibitors incorporate [13, 14].
Several tails with a diverse chemical nature has been employed to design selective CA IX and
hCA XII inhibitors [15-20].
Isatin, an endogenous compound in both human and other mammalian tissues and fluids,
[21, 22], is a privileged scaffold that is endowed with diverse assortment of biological actions,
to name just a few, anticancer [23-30] and CA inhibitory activities [31-38]. Interestingly, isatin
scaffold has been utilized as an effective tail for the design and development of promising
CAIs [31-38]. Exploring the structure activity relationships (SAR) for some reported isatin-
based CAIs, (compounds I-IV, Fig.1) highlighted that N-alkylation or N-benzylation of isatin
moiety could be an advantageous approach for the inhibitory activity towards hCA IX [31-34].
Incorporation of urea functionality as a linker between the zinc anchoring moiety and the
tail of the inhibitor emerged as an important trend in the design of selective sulfonamide CAIs
[39-45]. The most important synthesized ureido benzenesulfonamide is the clinical candidate
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SLC-0111 VI, (Fig. 1), which is currently in Phase I/II clinical trials for the treatment of
advanced, metastatic solid tumors [12, 45, 46]
Fig. 1. Structures and KI values against tumor-associated hCA IX isoform for some reported isatin-based
(I-V) carbonic anhydrase inhibitors and SLC-0111.
Recently, we described a series of ureidosubstituted benzenesulfonamides incorporating
isatin moieties (compounds IVa-c, Fig. 1) as CAIs. Although they displayed excellent
inhibition of hCA XII in the sub- to low-nanomolar range (KIs: 0.67 – 2.32 nM), they weakly
inhibited hCA IX (KIs: 192 – 239 nM) [37].
In light of the above findings and as a continuation of our research program to develop
effective isatin-based antitumor candidates targeting the tumor-associated isoforms hCA IX
and XII, it was thought worthwhile to extend our investigations around our study to improve
the potency of CAIs (IVa-c) towards the tumor-associated isoform hCA IX and also eliminate
or lower the inhibition of off-target hCA I which has a wide tissue distribution and, preferably,
should not be affected in the course of anticancer drugs development.
4

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Firstly, structural extension approach was adopted via N-alkylation and N-benzylation of isatin
moiety to enhance the tail hydrophobic interactions within the CA IX active site. Thereafter, a
hybrid pharmacophore approach was utilized via merging the pharmacophoric elements of isatin
and SLC-0111 in a single chemical framework to synthesize two series of novel N-substituted
isatins-SLC-0111 hybrids 6a-f and 9a-l (Fig. 2). Grafting halogen substituents (Cl and Br) at 5-
position of the isatin tail is to ensure different lipophilic environments which could be suitable
for the hydrophobic nature of the CA IX active site, and to carry out further elaboration of the
target scaffold to explore a valuable SAR.
Fig. 2. Design of the target N-substituted isatins-SLC-0111 hybrids 6a-f and 9a-l.
All the synthesized hybrids 6a-f and 9a-l were evaluated for their CA inhibitory effects
towards the physiologically relevant hCA isoforms, hCA I, II (cytosolic) as well as hCA IX
and XII (transmembrane, tumor-associated isoforms) using stopped-flow CO2 hydrase assay.
Furthermore, the most potent and selective hCA IX and XII inhibitors were examined for their
anti-proliferative activity towards breast cancer MDA-MB-231 and MCF-7 cell lines under
hypoxic conditions. Furthermore, hybrid 6c was examined for cell cycle disturbance and
apoptosis induction in MDA-MB-231 cells. Moreover, inhibitory activity of compounds 6c,
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6d, 9c and 9d against VEGFR-2 were evaluated. To point out the binding modes and
rationalize the trend of the inhibition profiles, molecular docking of the designed hybrids in CA
II and IX active sites was carried out (PDB; 5LJT and 5FL4, respectively).
2. Results and discussion
Preparation of the new N-substituted isatins-SLC-0111 hybrids 6a-f and 9a-l in this study is
illustrated in Schemes 1 and 2. The synthesis was initiated by N-alkylation of isatins 1a-c that
carried out via reaction with methyl iodide 2 or propyl iodide 3 in acetonitrile in the presence
of potassium carbonate to furnish N-alkylated isatin derivatives 4a-f. The first series of the
target hybrids was obtained by condensing 4-(3-(4-aminophenyl)ureido)benzenesulfonamide 5
with the appropriate N-substituted isatin derivative 4a-f in glacial acetic acid in 53-78% yield
(Scheme 1).
Scheme 1. Synthesis of target hybrids 6a-f; Reagents and conditions: (i) CH3CN, K2CO3, reflux 3 h, (ii)
Glacial acetic acid / reflux 3-4 h.
In Scheme 2, isatins 1a-c were benzylated with different benzyl bromides 7a-d in
refluxing dry acetonitrile in the presence of potassium carbonate as a base to afford N-
benzylated isatin derivatives 8a-l, which subsequently were condensed with the key
intermediate 5 in refluxing glacial acetic acid to produce the second group of the target hybrids
9a-l in good yields (70-83%) (Scheme 2).
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Scheme 2. Synthesis of target hybrids 9a-l; Reagents and conditions: (i) CH3CN, K2CO3, reflux 3 h, (ii)
Glacial acetic acid / reflux 4-7 h.
Postulated structure of the newly synthesized hybrids 6a-f and 9a-l were in full agreement
with their spectral and elemental analyses data.
1
It is worth highlighting that H NMR spectra of some compounds of both series (6 and 9)
unveiled their presence as E/Z geometrical isomers due to the exocyclic C=N double bond.
Their 1H NMR spectra revealed two sets of signals each belonging to the Z and E isomers. The
ratio between signals suggests that the Z-isomer is much more abundant than the E one (more
than 70% for most compounds); however this E/Z ratio is NMR solvent-dependent and stable
1
over time [37, 47]. For example, the HNMR spectrum of 9a showed two singlet signals
attributable to the protons of benzylic methylene function at δ 4.86 ppm (for E isomer) and δ
4.97 ppm (for Z isomer). The integration of the latter two signals showed that the E /Z ratio for
9a is 2.2:7.8.
2.2. Biological Evaluation
2.2.1. Carbonic anhydrase inhibition
The CA inhibitory effects of all the newly synthesized hybrids 6a-f and 9a-l were evaluated
towards the physiologically relevant hCA isoforms, hCA I, II (cytosolic) as well as hCA IX
and XII (transmembrane, tumor-associated isoforms) using an applied photophysics stopped-
flow instrument for assaying the CA-catalyzed CO2 hydration activity [48]. The inhibitory
activities were compared to acetazolamide (AAZ), a clinically used standard CA inhibitor, and
SLC-0111. The following SAR is evident from the data of Table 1:
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Table 1. Inhibition data of human CA isoforms hCA I, II, IX and XII with SLC-0111 and
hybrids 6a-f and 9a-l, determined by stopped-flow CO2 hydrase assay, using acetazolamide
(AAZ) as a standard drug.
KI (nM)*
Comp.
X
R
hCA I
898
hCA II
21.7
hCA IX
25.1
19.6
5.2
hCA XII
38.9
52.2
6.3
6a
H
H
6b
H
CH2CH3
H
939.4
406.9
607.2
1955.2
2956.1
641.3
2071.3
6567.1
2292
9
6c
Cl
Cl
Br
Br
H
823.8
262.3
57.9
6d
CH2CH3
H
8.2
7.8
6e
9.2
16.3
9.6
6f
CH2CH3
H
75
26.7
73.2
54.5
4.7
9a
131.4
114.9
81.4
80.9
3.6
9b
H
3-F
9c
H
4-CH3
4-CN
H
9.5
9d
H
77.3
9.7
8.5
9e
Cl
Cl
Cl
Cl
Br
Br
Br
Br
-
2496.4
5095.8
6147.9
8292.5
5890.7
4697
107.5
66.6
86.1
58.8
43
57.4
7.7
9f
3-F
9g
4-CH3
4-CN
H
221.5
460.4
246.4
336.1
440.1
611.5
960.0
12. 0
50.1
31.8
26
9h
34.7
41.2
61.2
26.4
36.9
45.0
25.2
9i
9j
3-F
1.3
9k
4-CH3
4-CN
-
4638.8
5842.3
5080
58.9
16.5
4.5
9l
SLC-0111
AAZ
-
-
250
5.6
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5-
10 % of the reported values).
8

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(i) The ubiquitous cytosolic isoform hCA I was inhibited by the prepared hybrids 6a-f and
9a-l with KI values ranging from 406.9 to 8292.5 nM (Table 1). Noteworthy, the N-alkylated
derivatives 6a-f showed a generally improved inhibitory profile towards hCA I in comparison
to their N-benzylated counterparts 9a-l. In particular 5-chloroisatin derivatives 6c and 6d
displayed the best hCA I inhibitory activity with KI values of 406.9 and 607.2 nM, respectively.
(ii) The in vitro kinetic data listed in Table 1 revealed that the physiologically dominant
isoform hCA II was inhibited by the prepared hybrids 6a-f and 9a-l with inhibition constants
ranging in the low-high nanomolar range, in detail, between 9 and 823.8 nM. Compound 6b
stood out as a single-digit nanomolar hCA II inhibitor that showed better activity (KI = 9 nM)
than the standard drug AAZ (KI = 12 nM against hCA II). Moreover, hybrids 6a, 6e, 6f, 9c, 9d
and 9f displayed good inhibitory activity against hCA II (KIs = 21.7, 57.9, 75, 81.4, 77.3 and
66.6 nM, respectively) whereas the remaining hybrids were weak potency inhibitors (KIs in the
range of 107.5-823.8 nM). It is noteworthy that N-alkylated hybrids which incorporate C5-
unsustituted isatin moiety (compounds 6a and 6b; KIs = 21.7 and 9 nM, respectively) or 5-Br
isatin moiety (compounds 6e and 6f; KIs = 57.9 and 75 nM, respectively) displayed enhanced
inhibitory activity than their N-benzylated analogues; compounds 9a-d (KIs: 77.3 – 131.4 nM)
and compounds 9i-l (KIs: 246.4 – 611.5 nM).
(iii) The tumor-associated isoform hCA IX was efficiently inhibited by all the prepared
hybrids 6a-f and 9a-l with KIs spanning in the nanomolar range: 4.7 – 86.1 nM. Superiorly,
hybrids 6c-e, 9c and 9d emerged as single-digit nanomolar hCA IX inhibitors with KIs = 5.2,
8.2, 9.2, 4.7 and 9.7 nM, respectively, which are more potent than the standard drug AAZ (KI =
25.2 nM) and SLC-0111 (KI = 45 nM). Also, hybrids 6a, 6b, 6f, 9g-i, 9k and 9l displayed
better inhibitory activity (KIs: 19.6 – 43 nM) than SLC-0111 towards hCA IX. It is noteworthy
that C-5 substitution of isatin moiety elicited an impact on the inhibitory activity within the
first series of derivatives (6a-f). Incorporation of 5-Cl isatin moiety (compounds 6c and 6d; KIs
= 5.2 and 8.2 nM, respectively) furnished the better inhibitory profile towards hCA IX in
comparison to unsubstituted isatin-based derivatives (compounds 6a and 6b; KIs = 25.1 and
19.6 nM, respectively) and 5-Br isatin-based derivatives (compounds 6e and 6f; KIs = 9.2 and
26.7 nM, respectively). Investigating the impact of substitution on the benzyl group within the
second series of derivatives (9a-l), suggested that such substitution is indispensable for hCA IX
9

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inhibitory activities. The order of activities of the substituted benzyl members in the second
series was decreased in the order of p-CH3 > p-CN > m-F, for unsubstituted (9b-d) and 5-Br
(9j-k) isatin-based derivatives. Whereas, the order of activities was decreased in the order of p-
CN > p-CH3 > m-F for 5-Cl (9f-h) isatin-based derivatives.
Interestingly, the adopted structural extension approach, via N-alkylation and N-
benzylation of isatin moiety, succeed to enhance the inhibitory profile of the target hybrids
(KIs: 4.7 – 86.1 nM) towards hCA IX in comparison to N-unsubstituted leads IVa-c (KIs: 192 –
239 nM) [37], as planned (Fig. 3).
300
250
200
150
100
50
0
Va
6a
6b
9a
9b
9c
9d
Vb
6c
6d
9e
9f
9g
9h
Vc
6e
6f
9i
9j
9k
9l
N-Alkylated
Hybrids
N-Benzylated Hybrids
N-Alkylated
Hybrids
N-Benzylated Hybrids
N-Alkylated N-Benzylated Hybrids
Hybrids
The tested Hybrid
Fig. 3. KI values for the tested hybrids (6a-f and 9a-l) and the leads IVa-c against tumor-
associated hCA IX.
(iv) The second tumor-associated isoform tested here was hCA XII. It was obvious from
the displayed results, in Table 1, that the newly synthesized hybrids 6a-f and 9a-l possessed
excellent to moderate inhibitory activity against hCA XII (KIs in the range of 1.3 – 80.9 nM).
In particular, hybrids 9b and 9j were found to be more potent hCA XII inhibitors (KIs = 3.6
and 1.3 nM, respectively) than AAZ (KI = 5.6 nM) and SLC-0111 (KI = 4.5 nM). Besides,
compounds 6c, 6d, 6f, 9c, 9d and 9f displayed single-digit nanomolar activity (KIs = 6.3, 7.8,
9.6, 9.5, 8.5 and 7.7 nM, respectively). As for hCA IX; substitution of the benzyl group is
advantageous for hCA XII inhibitory activity, except compound 9k. The order of hCA XII
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inhibitory activities of the substituted benzyl members in the second series was decreased in
the order of m-F > p-CN > p-CH3. It is worth stressing that grafting m-F substituent on the
benzyl group (9b, 9f and 9j) resulted in a considerable improvement of the activity with 7-fold
to 22-fold efficacy enhancement likened to the unsubstituted benzyl-based compound (9a, 9e
and 9i).
(v) As hCA I and hCAII isozymes are physiologically relevant, the selectivity is crucial
element for developing new hCA IX inhibitors. As a result of the inhibitory trends extrapolated
from the data in Table 1, interesting selectivity index (SI) arose for most hybrids herein
reported (Table 2). Regarding selectivity towards hCA IX over hCA II, the examined hybrids
possessed good selectivity indexes spanning in the range 5.2 – 158.4, apart from non-selective
hybrids 6a and 6b (SI = 0.9 and 0.5, respectively) and hybrids 6f, 9a, 9b, 9e and 9f which
displayed modest selectivity (SI: 1.1 – 2.8). Interestingly, all the tested hybrids 6a-f and 9a-l
displayed interesting selectivity towards hCA IX over hCA I (SI: 8.8 – 1397.3).
Table 2. Selectivity ratios for the inhibition of hCA IX and XII over hCA I, and II for hybrids
6a-f and 9a-l reported in the paper, and acetazolamide.
Selectivity ratio
Cpd.
I/IX
35.8
II/IX
I/XII
II/XII
0.9
23.1
0.6
6a
6b
6c
6d
6e
6f
47.9
78.3
0.5
158.4
32
18.0
64.6
0.2
130.8
33.6
3.6
74.1
77.9
212.5
110.7
8.8
6.3
120.0
307.9
7.9
2.8
7.8
1.8
1.6
9a
9b
9c
9d
9e
9f
38.0
2.1
575.4
691.3
269.7
43.5
31.9
8.6
1397.3
236.3
29.0
17.3
8.0
9.1
1.3
1.87
8.7
86.7
1.1
661.8
122.7
143.0
5.2
4.4
9g
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239.0
143.0
76.8
13.3
6.0
260.8
226.6
3613.1
78.8
14.5
9.5
9h
9i
5.5
258.5
7.5
9j
175.7
158.3
9.9
16.7
16.6
0.5
9k
354.08
44.6
37.1
2.2
9l
AAZ
2.2.2. In vitro anti-proliferative activity towards breast cancer
Breast cancer is the most frequently diagnosed malignancy among women, and the second
leading cause of cancer-related deaths in women. It is considered to be a diverse group of
diseases with different intrinsic tumor subtypes that have several treatment modalities and
long-term survival probabilities. It is well established that hCA IX is highly expressed in breast
malignancies, where numerous studies correlated the high hCA IX levels with prognosis and
therapy outcomes for breast cancer patients [6, 49].
Hybrids 6c, 6d, 9c and 9d displayed single-digit nanomolar inhibitory activities towards
hCA IX (KIs: of 4.7 – 9.7 nM) and hCA XII (KIs: of 6.3 – 9.5 nM), also they possessed good
selectivity towards hCA IX over hCA I (SI: 74.1 – 1397.3) and hCA II (SI: 8 – 158.4).
Accordingly, the four hybrids were selected to be examined for their anti-proliferative activity
towards two breast cancer MDA-MB-231 and MCF-7 cell lines, under hypoxix conditions
utilizing the MTT colorimetric assay as described by T. Mosmann [50]. A chemically-
induced hypoxia was established by the use of cobalt (II) chloride hexahydrate (CoCl2.6H2O)
as a chemical inducer of HIF-1α. The results were expressed as median growth inhibitory
concentration (IC50) values that represent the compound concentration required to produce a
50% inhibition of cell growth after 48 h of incubation, compared to the untreated controls
(Table 3).
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Table 3. In vitro anti-proliferative activity of hybrids 6c, 6d, 9c and 9d against breast MDA-
MB-231 and MCF-7 cancer cell lines.
IC50 (µM)a
Comp.
MDA-MB-231
7.43 ± 0.28
MCF-7
12.90 ± 0.34
7.36 ± 0.19
11.79 ± 0.82
5.97 ± 0.16
6.32 ± 0.21
6c
25.59 ± 1.17
48.93 ± 1.66
23.16 ± 1.02
8.50 ± 0.24
6d
9c
9d
Dox.
a IC50 values are the mean ± S.D. of three separate experiments.
The results of the MTT assay displayed in Table 3 revealed that the examined compounds
generally exhibited better anti-proliferative activity towards MCF-7 cell line (IC50 range: 5.97
± 0.16 – 12.90 ± 0.34
higher potency towards MDA-MB-231 cells (IC50 = 7.43 ± 0.28
12.90 ± 0.34 M). Concerning activity against MDA-MB-231 cells, compound 6c emerged as
µ
M) than MDA-MB-231 cells, except compound 6c which displayed
µ
M) than MCF-7 cells (IC50 =
µ
the most potent counterpart, whereas, the remaining derivatives 6d, 9c and 9d exhibited
moderate anti-proliferative activity with IC50 values equal 25.59 ± 1.17, 48.93 ± 1.66 and 23.16
± 1.02 µM, respectively.
Interestingly, hybrid 6c displayed dual anti-proliferative activity towards both MDA-MB-
231 and MCF-7 cancer cell lines, in addition, it exhibited interesting selectivity towards hCA
IX over hCA I (SI = 78.3), hCA IX over hCA II (SI = 158.4), hCA XII over hCA I (SI = 64.6)
and XII over hCA II (SI = 130.8). Thence, hybrid 6c was selected for further biological
evaluations to acquire mechanistic insights into the anti-proliferative activity of the target
hybrids.
2.2.3. Cell Cycle Analysis
In the current study, hybrid 6c was assessed for its impact on the cell cycle distribution in
MDA-MB-231 cells (Fig. 4). The results of the DNA flow cytometric analsis highlighted that
treatment of MDA-MB-231 cells with hybrid 6c at its IC50 concentration (7.43 ± 0.28) resulted
in a significant 8.4-fold increased percentage of MDA-MB-231 cells at Sub-G1, with
concurrent significant reduction in the G2-M phase by approximately 4.9-fold.
13

ACCEPTED MANUSCRIPT
6c
Control
Fig. 4. Effect of hybrid 6c on the phases of cell cycle of MDA-MB-231 cells. * Significantly
different from control at p < 0.05. (Two-way ANOVA test).
2.2.4. AnnexinV-FITC/Propidium Iodide Analysis of Apoptosis.
AnnexinV-FITC/PI dual staining assay [51] was carried out to evaluate the effect of hybrid
6c on both early and late apoptosis percentages in MDA-MB-231 cells (Fig. 5, Table 4). As
presented in Fig. 5, the assay outcomes indicates that treatment of MDA-MB-231 cells with
hybrid 6c led to a significant increase in the percentage of annexinV-FITC-positive apoptotic
cells, including both the early and late apoptotic phases (LR; from 0.77% to 7.28%, and UR;
from 0.26% to 11.26%), that represents about eightfold total increase in comparison with
untreated control (Table 4).
14

ACCEPTED MANUSCRIPT
6c
Control
Fig. 5. Effect of hybrid 6c on the percentage of annexin V-FITC-positive staining in MDA-MB-231
cells. The experiments were done in triplicates. The four quadrants identified as: LL, viable; LR, early
apoptotic; UR, late apoptotic; UL, necrotic.
Table 4. Distribution of apoptotic cells in the AnnexinV-FITC/PI dual staining assay in MDA-
MB-231 cells after treatment with hybrid 6c.
Early Apoptosis
(Lower Right %)
7.28
Late Apoptosis
(Upper Right %)
11.26
Total
(L.R % + U.R %)
18.54
Comp.
6c
Control
0.77
0.26
1.03
2.2.5. In vitro VEGFR-2 kinase inhibitory activity
On account of its significant contribution to the heterogeneity of the tumor
microenvironment, hypoxia is a considerable environmental stressor that orchestrates several
biological adaptations critical for cancer progression, including altered metabolism, pH
regulation and angiogenesis [9].
Angiogenesis, a process of sprouting of new capillary blood vessels from the quiescent
preexisting vasculature, is an important mediator of tumor progression [52]. Transcriptional
activation of hypoxia inducible factor-1α (HIF-1α) by hypoxia leads to increased expression of
several angiogenic factors that enable tumors make the angiogenic switch [9]. Members of the
vascular endothelial growth factor (VEGF) family have a prevalent role among the various
players involved in the angiogenesis process [53]. Accordingly, targeting tumor-associated
proteins involved in pH regulation (such as CA IX and hCA XII), synchronous with targeting
15

ACCEPTED MANUSCRIPT
proteins involved in hypoxia-induced angiogenesis (such as VEGFR-2) may furnish more
effective therapeutic approach for curbing different human malignancies. Noteworthy, such
dual targeting was encouraged by the previously reported good VEGFR-2 inhibitory activity of
the investigated indoline urea scaffold (IVb; IC50 = 310 ± 40 nM, Table 5) [54].
In this study, the most potent and selective hCA IX and XII inhibitors 6c, 6d, 9c and 9d
were selected to evaluate their inhibitory activity against VEGFR-2 by use of a colorimetric
Enzyme-Linked Immunosorbent Assay (ELISA). Sorafenib, a clinically approved VEGFR-2
inhibitor, was used as a reference drug. The results were reported as a 50% inhibition
concentration values (IC50) which determined as triplicate determinations from the standard
curve and displayed in Table 5
.
Table 5. IC50 values for the inhibitory activity of 6c, 6d, 9c and 9d against VEGFR-2.
Compound
IC50 (nM)a
VEGFR-2
260.64 ± 14.82
383.61 ± 21.34
462.38 ± 19.20
207.04 ± 13.52
310.00 ± 40.00
104.01 ± 8.06
6c
6d
9c
9d
IVbb
Sorafenib
a IC50 values are the mean ± SD of three separate experiments.
b Reference [54].
Results revealed that the examined hybrids possessed VEGFR-2 inhibitory activity with
IC50 values ranging from 207.04 to 462.38 nM. Compounds 6c and 9d emerged as the most
potent VEGFR-2 inhibitors in this study that displayed about 2-fold decreased potency (IC50 =
207.04 and 260.64 nM) to the reference drug Sorafenib (IC50 = 104.01 nM).
Of particular interest, this study is the first to our knowledge that developed novel
sulfonamides with potent and selective inhibitory activity against tumor-associated isozymes
hCA IX and XII, accompanied with promising VEGFR-2 inhibitory activity.
16

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2.3. Molecular modeling studies
To point out the binding modes and rationalize the trend of the inhibition profiles, hybrids
in Table 1 were submitted to docking within CA isozymes II and IX (PDB; 5LJT [55] and
5FL4 [56], respectively). As expected, docking solutions place the benzensulfonamide moiety
of all derivatives deeply into the active site region of both the isozymes, establishing two
hydrogen bonds with T199/T200 (hCA II/hCA IX). The sulfonamide negatively charged
nitrogen atom directly coordinates the zinc ion and the phenyl ring is involved in several
interactions with the hydrophobic region lined by V121, V143 and L198 (V121, V142, L199 in
hCA IX).
In hCA II compounds bearing aliphatic portions (methyl or propyl) on the isatin nitrogen,
orient differently than those substituted with benzyl groups (Fig. 6A). Docking poses of the
first series of
N-alkylated derivatives 6a-f show the N-alkyl moiety oriented toward a little
hydrophobic region defined by G132, Q136, P202, and L204. The middle phenyl ring makes
hydrophobic interaction with V131, V135, L198 and P202 (Fig. 6A). The isatine ring is
variably oriented depending on the substituent at position 5, namely a hydrogen (6a
, b),
chlorine (6c ) or bromine atom (6e ). The unsubstituted and chlorine bearing-hybrids orient
,
d
, f
roughly similar, but the chlorine atom of 6c establishes unfavorable contacts with Q136 side
chain that could account for the drop of its hCA II inhibition (Table 1). The increased
hindrance of the bromine atom does not allow 6e and 6f to occupy the same target portion. The
isatin, and in particular the middle phenyl portion of the hybrids are oriented towards P202,
where the hydrophobic contacts increase significantly thus restoring the inhibition in the
nanomolar range.
The size of the area described above does not allow the bulky N-benzylisatin tail of hybrids
9a-l to fit inside the pocket, resulting in the budging of the entire tail toward the hydrophilic
half of the cavity (Fig. 6B). The ureidic carbonyl group engages an H-bond with Q92 side
chain and the benzyl pendants lie within the cleft formed by L57, R58, E69, D71, D72 and I91.
The mainly hydrophilic nature of the pocket is not well suited for the hydrophobic
characteristics of the benzyl moiety located therein and a more or less marked drop of efficacy
against hCA II of these
N-alkylated derivatives occurs (depending on the substituents on the
benzyl and isatin aromatic rings, Table 1).
17

ACCEPTED MANUSCRIPT
Conversely, superimposable orientations are found for all studied derivatives in hCA IX. In
fact, all
N-alkylated and N-benzylated isatin moieties accommodate in a wide hydrophobic
pocket (devoid of hydrophilic residues instead present in the correspondent area of hCA II)
formed by T73, P75, P76, L91, L123 and A128 (Fig. 7). As a result, the alkylation and
bezylation of the isatin NH favors the binding of these type of derivatives to hCA IX, and
makes them more active towards this isoenzyme than other
N-unsubstituted derivatives
previously reported [37]. Moreover, the binding mode of halo-compounds 6c-f and 9e-l is
strengthened by a non-conventional hydrogen bond involving R129.
Fig. 6. Docking of A) 6c (red), 6e (violet) and 9j (green), B) 9g (green) and 9l (blue) in hCA II.
Fig. 7. Docking of A) 6d (light blue), 6e (violet) and 9j (green), B) 9g (green) and 9l (blue) in hCA IX.
18

ACCEPTED MANUSCRIPT
3. Conclusion
In summary, we reported the design and synthesis of two novel
0111 hybrids series 6a-f and 9a-l. A structural extension approach was adopted via N
alkylation and -benzylation of isatin moiety to enhance the tail hydrophobic interactions
N-substituted isatins-SLC-
-
N
within the carbonic anhydrases (CAs) IX active site. Thereafter, a hybrid pharmacophore
approach was utilized via merging the pharmacophoric elements of isatin and SLC-0111 in a
single chemical framework. The inhibition profile of the newly synthesized hybrids was
determined against the physiologically relevant hCA isoforms, hCA I, II (cytosolic) as well as
hCA IX and XII (transmembrane, tumor-associated isoforms) using stopped-flow CO2 hydrase
assay. Both tumor-associated isoforms hCA IX and XII were efficiently inhibited by all the
prepared hybrids with KIs spanning in the nanomolar ranges: 4.7 – 86.1 nM and 1.3 – 80.9 nM,
respectively. A significant enhancement of inhibitory activity of the target hybrids 6a-f and 9a-
l (KIs: 4.7–86.1 nM) towards hCA IX in comparison to N-unsubstituted leads IVa-c (KIs: 192–
239 nM) was achieved. Noteworthy, thirteen hybrids displayed better inhibitory activity (KIs:
4.7 – 43 nM) than SLC-0111(KIs = 45 nM) towards hCA IX. Molecular docking of the
designed hybrids in CA IX active site unveiled, as planned, the ability of
benzylated isatin moieties to accommodate in a wide hydrophobic pocket formed by T73, P75,
P76, L91, L123 and A128, establishing strong van der Waals interactions. Hybrids 6c 6d 9c
N-alkylated and N-
,
,
and 9d, with potent and selective IX/II inhibitory trend, were examined for their anti-
proliferative activity towards breast cancer MDA-MB-231 and MCF-7 cell lines under hypoxic
conditions. Compound 6c displayed good anti-proliferative activity towards both MDA-MB-
231 and MCF-7 cell lines (IC50 = 7.43±0.28 and 12.90±0.34 µM, respectively). Furthermore,
6c was examined for cell cycle disturbance and apoptosis induction in MDA-MB-231 cells.
The results outcomes revealed the ability of 6c to disrupt the MDA-MB-231 cell cycle via
alteration of the Sub-G1 phase and arrest of G2-M stage, and its ability to increase the percent
of annexinV-FITC positive apoptotic cells from 1.03 to 18.54%. Finally, hybrids 6c, 6d, 9c and
9d displayed good inhibitory activity against VEGFR-2 (IC50: 207 – 462 nM). This study is the
first to our knowledge that developed novel sulfonamides targeting tumor-associated proteins
involved in pH regulation (such as CA IX and hCA XII), synchronous with targeting proteins
involved in hypoxia-induced angiogenesis (such as VEGFR-2).
19

ACCEPTED MANUSCRIPT
4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were measured with a Stuart melting point apparatus and were uncorrected.
Infrared (IR) Spectra were recorded as KBr disks using Schimadzu FT-IR 8400S
spectrophotometer. NMR Spectra were recorded on a Varian Mercury and Bruker NMR
1
spectrometers. H spectrum was run at 400 MHz and 13C spectrum was run at 100 MHz in
deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts are expressed in values ppm using
the solvent peak as internal standard. All coupling constant (J) values are given in hertz. The
abbreviations used are as follows: s, singlet; d, doublet; m, multiplet. Elemental analyses were
carried out at the Regional Center for Microbiology and Biotechnology, Al-Azhar University,
Cairo, Egypt. Analytical thin layer chromatography (TLC) on silica gel plates containing UV
indicator was employed routinely to follow the course of reactions and to check the purity of
products.
4.1.2. Synthesis of compound 5
Compound
5 was prepared according to the literature procedures [57].
4.1.3. Synthesis of N-substituted isatin derivatives 4a-f and 8a-l.
Isatin derivatives 1a-c (0.005 mol) were stirred in acetonitrile (20 mL) with (0.007 mol) of
the appropriate methyl iodide 2, propyl iodide 3 or benzyl bromide 7a-d in the presence of
catalytic amount of potassium iodide with (0.010 mol) of dry potassium carbonate at reflux
temperature. The reaction was monitored with TLC. After complete of reaction, the mixture
was poured into ice-water, the formed solid was collected, washed with water and
recrystallized from ethanol-water to furnish the final compounds 4a-f and 8a-l. [58-61]
4.1.3.1. 5-Bromo-1-(3-fluorobenzyl)indoline-2,3-dione 8j
Red crystals (yield 75%); m.p. 171-173 °C; 1H NMR (DMSO-d6, 400 MHz)
benzylic protons), 6.90-7.74 (m, 7H, Ar-H); 13C NMR (DMSO-d6, 100 MHz)
δ
ppm: 4.94 (s, 2H,
ppm: 42.89
δ
20

ACCEPTED MANUSCRIPT
(benzylic carbon), 113.47, 114.49, 114.71, 114.92, 115.57, 120.21, 123.81, 127.12, 131.09,
138.70, 139.92, 149.38, 158.56, 161.62, 164.05, 182.11; Anal. Calcd. for C15H9BrFNO2
(334.14): C, 53.92; H, 2.71; N, 4.19; found C, 54.07; H, 2.69; N, 4.24.
4.1.3.2. 4-((5-Bromo-2,3-dioxoindolin-1-yl)methyl)benzonitrile 8l
Red crystals (yield 81%); m.p. 201-203 °C; 1H NMR (DMSO-d6, 400 MHz)
benzylic protons), 6.89 (s, 1H, Ar-H), 7.57-7.98 (m, 6H, Ar-H); 13C NMR (DMSO-d6, 100
MHz) ppm: 42.64 (benzylic carbon), 112.03, 113.36, 115.59, 119.17, 120.36, 127.10, 130.21,
δ ppm: 4.98 (s, 2H,
δ
131.10, 131.80, 132.79, 137.51, 139.85, 149.18, 158.70 (N-CO-CO-), 182.01 (N-CO-CO-);
Anal. Calcd. for C16H9BrN2O2 (341.16): C, 56.33; H, 2.66; N, 8.21; found C, 56.21; H, 2.68;
N, 8.17.
4.1.4. Synthesis of target compounds 6a-f and 9a-l.
The key intermediate
2 was refluxed with equivalent amount of different isatin derivatives 4a-f
or 8a-l in glacial acetic acid. The reaction was monitored with TLC. After complete of the
reaction, the formed solid was collected and washed with water and recrystallized from
DMF/ethanol to get the target compounds 6a-f and 9a-l respectively.
4.1.4.1. 4-{3-[4-((1-Methyl-2-oxoindolin-3-ylidene)amino)phenyl]ureido}benzenesulfonamide
6a
.
Orange powder (yield 73%); m.p. over 280 °C; IR (KBr,
ⱱ
cm-1): 3123-3400 broad band (NH,
ppm: 3.10,
=8 Hz), 6.96 (d, 2H, Ar-
=7.6 Hz), 7.19 (s, 2H, NH2, D2O exchangeable), 7.41 (t, 1H,
=7.6 Hz), 7.54-7.62 (m, 4H, Ar-H), 7.71 (d, 2H, Ar-H, =8.4 Hz), 8.88, 8.92 (s, 1H,
1
NH2), 1724, 1612 (2C=O), 1307, 1150 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
3.19 (s, 3H,
H, =8.4 Hz), 7.09 (d, 1H, Ar-H,
Ar-H,
N-CH3), 6.68 (d, 1H, Ar-H, J=8 Hz), 6.81 (t, 1H, Ar-H, J
J
J
J
J
NH, D2O exchangeable), 9.08, 9.10 (s, 1H, NH, D2O exchangeable); Anal. Calcd. for
C22H19N5O4S (449.49): C, 58.79; H, 4.26; N, 15.58; found C, 58.65; H, 4.22; N, 15.67.
4.1.4.2. 4-{3-[4-((2-Oxo-1-propylindolin-3-ylidene)amino)phenyl]ureido}benzenesulfonamide
6b.
Yellow powder (yield 70%); m.p. over 280 °C; IR (KBr,
ⱱ
cm-1): 3122-3395 broad band (NH,
1
NH2), 1724, 1610 (2C=O), 1300, 1150 (SO2); H NMR (DMSO-d6, 400 MHz)
δ ppm: 0.92 (t,
21

ACCEPTED MANUSCRIPT
3H, CH3-CH2,
Ar-H, =7.2 Hz), 6.81 (t, 1H, Ar-H,
3H, 1H Ar-H and 2H of NH2, D2O exchangeable), 7.41-7.52 (m, 1H, Ar-H), 7.59-7.66 (m, 4H,
Ar-H), 7.76 (d, 2H, Ar-H, =8.4 Hz), 8.92 (s, 1H, NH, D2O exchangeable), 9.12 (s, 1H, NH,
D2O exchangeable); 13C NMR (DMSO-d6, 100 MHz)
ppm: 11.68 (2CH3), 19.03, 20.5 (CH2),
41.29, 41.50 ( -CH2), 109.98, 110.71, 114.61, 115.74, 117.58, 117.98 (2C), 118.56, 119.20,
J=8.0 Hz), 1.59 (m, 2H, CH3-CH2), 3.70 (t, 2H,
N
-CH2,
J=8.0 Hz), 6.68 (d, 1H,
J
J=7.6 Hz), 7.01 (d, 2H, Ar-H,
J=8.4 Hz), 7.16-7.23 (m.
J
δ
N
119.85, 121.82, 122.44, 122.59, 123.12, 125.34, 127.32 (2C), 134.07, 134.67, 137.37, 137.49,
137.65, 137.87, 142.98, 143.31, 144.93, 146.07, 147.68, 151.34, 152.69 (aromatic carbon),
152.79, 154.22 (C=O of urea), 157.51, 162.82 (C=O of isatin); Anal. Calcd. for C24H23N5O4S
(477.54): C, 60.36; H, 4.85; N, 14.67; found C, 60.51; H, 4.89; N, 14.75.
4.1.4.3.
4-{3-[4-((5-Chloro-1-methyl-2-oxoindolin-3-
ylidene)amino)phenyl]ureido}benzenesulfonamide 6c
.
°
Orange red powder (yield 53%); m.p. over 280 C; IR (KBr,
ⱱ
cm-1): 3123-3350 broad band
ppm
=7.2
Hz), 7.23 (s, 2H, NH2, D2O exchangeable), 7.47-7.56 (m, 2H, Ar-H), 7.61-7.66 (m, 3H, Ar-H),
7.75 (d, 2H, Ar-H, =8.8 Hz), 8.95 (s, 1H, NH, D2O exchangeable), 9.12 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6, 100 MHz)
ppm: 19.03, 26.73 ( -CH3), 111.39, 112.15,
1
(NH, NH2), 1714, 1612 (2C=O), 1300, 1150 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
:
3.51 (s, 3H, N-CH3), 6.66 (s, 1H, Ar-H), 7.02 (d, 2H, Ar-H, J=8.4), 7.14 (d, 1H, Ar-H, J
J
δ
N
112.68, 116.84, 118.00 (2C), 118.43, 119.41, 119.75, 121.91, 123.15, 123.48, 124.20, 124.46,
126.34, 127.31, 127.43 (2C), 133.11, 133.85, 137.39, 137.93, 138.51, 142.34, 143.26, 144.30,
145.19, 147.00, 150.06, 152.64 (aromatic carbon) 152.74, 153.14 (C=O of urea), 157.43,
162.55 (C=O of isatin); Anal. Calcd. for C22H18ClN5O4S (483.93): C, 54.60; H, 3.75; N, 14.47;
found C, 54.83; H, 3.72; N, 14.41.
4.1.4.4.
4-{3-[4-((5-Chloro-2-oxo-1-propylindolin-3-
ylidene)amino)phenyl]ureido}benzenesulfonamide 6d
.
Orange powder (yield 63%); m.p. over 280 °C; IR (KBr,
ⱱ
cm-1): 3113-3378 broad band (NH,
ppm: 0.88 (t,
=7.2 Hz), 6.64 (s, 1H,
=8.8 Hz), 7.14-7.25 (m, 4H, 2H Ar-H and 2H of NH2, D2O
1
NH2), 1724, 1612 (2C=O), 1317, 1155 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
3H, CH3-CH2,
J=7.6 Hz), 1.55 (m, 2H, CH3-CH2), 3.67 (t, 2H, N-CH2, J
Ar-H), 7.01 (d, 1H, Ar-H,
J
22

ACCEPTED MANUSCRIPT
exchangeable), 7.43-7.54 (m, 2H, Ar-H), 7.57-7.69 (m, 3H, Ar-H), 7.71 (d, 2H, Ar-H, J=8.8
Hz), 8.95 (s, 1H, NH, D2O exchangeable), 9.10 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO-d6, 100 MHz) ppm: 11.61 (CH3), 20.66 (CH2), 41.62 ( -CH2), 111.66, 112.40,
δ
N
116.94, 117.78, 117.94, 117.98, 118.39 (2C), 119.35 (2C), 119.73 (2C), 122.07, 123.17,
123.57, 124.62, 126.21, 127.28 (2C), 133.86, 137.36, 137.89, 138.52, 142.30, 143.24, 144.34,
144.65, 146.45, 150.08, 152.62 (aromatic carbon), 152.74, 153.09 (C=O of urea), 157.30,
162.49 (C=O of isatin); Anal. Calcd. for C24H22ClN5O4S (511.98): C, 56.30; H, 4.33; N, 13.68;
found C, 56.47; H, 4.29; N, 13.77.
4.1.4.5.
4-{3-[4-((5-Bromo-1-methyl-2-oxoindolin-3-
ylidene)amino)phenyl]ureido}benzenesulfonamide 6e
.
°
Orange red powder (yield 78%); m.p. over 280 C; IR (KBr,
ⱱ
cm-1): 3105-3340 broad band
ppm
=8.4 Hz), 7.08 (d, 1H, Ar-H,
=8.0 Hz), 7.19 (s, 2H, NH2, D2O exchangeable), 7.21 (d, 1H, Ar-H, =8.8 Hz), 7.43 (d, 1H,
=8.8 Hz), 8.91, 8.96 (s, 1H,
1
(NH, NH2), 1714, 1610 (2C=O), 1300, 1150 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
:
3.10, 3.18 (s, 3H, N-CH3), 6.76 (s, 1H, Ar-H), 6.99 (d, 2H, Ar-H, J
J
J
Ar-H, J=8.8 Hz), 7.57-7.62 (m, 3H, Ar-H), 7.71 (d, 2H, Ar-H, J
NH, D2O exchangeable) 9.08, 9.10 (s, 1H, NH, D2O exchangeable); Anal. Calcd. for
C22H18BrN5O4S (528.38): C, 50.01; H, 3.43; N, 13.25; found C, 49.89; H, 3.45; N, 13.20.
4.1.4.6.
4-{3-[4-((5-Bromo-2-oxo-1-propylindolin-3-
ylidene)amino)phenyl]ureido}benzenesulfonamide 6f
.
°
Red powder (yield 77%); m.p. over 280 C; IR (KBr,
ⱱ
cm-1): 3123-3320 broad band (NH,
ppm: 0.88 (t,
=8.0 Hz), 6.48 (d, 1H,
=8.4 Hz), 6.79 (s, 1H, Ar-H), 7.01-7.10 (m, 2H, Ar-H), 7.12-7.23 (m, 3H, 1H Ar-H and
1
NH2), 1727, 1619 (2C=O), 1307, 1160 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
3H, CH3-CH2,
Ar-H,
J=8.0 Hz), 1.64 (m, 2H, CH3-CH2), 3.64 (t, 2H, N-CH2 J
J
2H of NH2, D2O exchangeable), 7.54-7.62 (m, 4H, Ar-H), 7.66-7.73 (m, 2H, Ar-H), 8.95 (s,
1H, NH, D2O exchangeable), 9.10 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6, 100
MHz)
δ ppm: 11.64 (CH3), 19.03, 20.68 (CH2), 41.46, 41.65 (N-CH2), 111.52, 112.08, 112.81,
113.93, 114.91, 117.40, 118.02 (2C), 118.41, 119.20, 119.43, 119.71, 123.31, 123.96, 124.22,
127.32 (2C), 127.45, 135.93, 136.65, 137.40, 137.95, 138.61, 142.28, 143.27, 144.34, 144.99,
146.80, 149.85, 152.64 (aromatic carbon), 152.75, 152.97 (C=O of urea), 157.19, 162.39 (C=O
23

ACCEPTED MANUSCRIPT
of isatin); Anal. Calcd. for C24H22BrN5O4S (556.44): C, 51.81; H, 3.99; N, 12.59; found C,
52.03; H, 3.95; N, 12.55.
4.1.4.7. 4-{3-[4-((1-Benzyl-2-oxoindolin-3-ylidene)amino)phenyl]ureido}benzenesulfonamide
9a
.
°
Red powder (yield 78%); m.p. over 280 C; IR (KBr,
ⱱ
cm-1): 3127-3395 broad band (NH,
ppm: 4.86,
=8.0 Hz),
7.00-7.04 (m, 3H, Ar-H), 7.18 (s, 2H, NH2, D2O exchangeable), 7.27-7.43 (m, 6H, Ar-H),
7.55-7.62 (m, 4H, Ar-H), 7.71 (d, 2H, Ar-H, =8.4 Hz), 8.94, 8.98 (2s, 1H, NH, D2O
1
NH2), 1715, 1612 (2C=O), 1317, 1150 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
4.97 (2s, 2H, benzylic proton), 6.73 (d, 1H, Ar-H, J=8.0 Hz), 6.80 (t, 1H, Ar-H, J
J
exchangeable), 9.12, 9.16 (2s, 1H, NH, D2O exchangeable); Anal. Calcd. for C28H23N5O4S
(525.58): C, 63.99; H, 4.41; N, 13.33; found C, 64.17; H, 4.38; N, 13.32.
4.1.4.8.
4-{3-[4-((1-(3-Fluorobenzyl)-2-oxoindolin-3-
ylidene)amino)phenyl]ureido}benzenesulfonamide 9b
.
°
Red powder (yield 70%); m.p. over 280 C; IR (KBr,
ⱱ
cm-1): 3123-3400 broad band (NH,
ppm: 5.08 (s,
=8 Hz), 6.89 (d, 1H,
=8.4 Hz), 7.18 (s, 2H, NH2,
=7.6 Hz), 7.38 (d, 1H, Ar-H, =8.8 Hz), 7.52-7.62 (m,
1
NH2), 1724, 1610 (2C=O), 1300, 1160 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
2H, benzylic proton), 6.73 (d, 1H, Ar-H,
Ar-H, =8 Hz), 6.99 (d, 2H, Ar-H, =8.8 Hz), 7.14 (d, 1H, Ar-H,
D2O exchangeable), 7.30 (t, 1H, Ar-H,
J=7.2 Hz), 6.78 (d, 1H, Ar-H, J
J
J
J
J
J
4H, Ar-H), 7.67-7.73 (m, 4H, Ar-H), 8.97 (s, 1H, NH, D2O exchangeable), 9.15 (s, 1H, NH,
D2O exchangeable); Anal. Calcd. for C28H22FN5O4S (543.57): C, 61.87; H, 4.08; N, 12.88;
found C, 61.75; H, 4.06; N, 12.83.
4.1.4.9.
4-{3-[4-((1-(4-Methylbenzyl)-2-oxoindolin-3-
ylidene)amino)phenyl]ureido}benzenesulfonamide 9c
.
Orange powder (yield 78%); m.p. over 280 °C; IR (KBr,
ⱱ
cm-1): 3120-3365 broad band (NH,
ppm: 3.27 (s,
=7.2 Hz), 6.80 (t, 1H, Ar-H,
=7.6 Hz), 6.98-7.10 (m, 3H, Ar-H), 7.17 (s, 2H, NH2, D2O exchangeable), 7.25-7.46 (m, 5H,
1
NH2), 1724, 1610 (2C=O), 1307, 1160 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
3H, -CH3), 4.98 (s, 2H, benzylic proton), 6.71 (d, 1H, Ar-H,
J
J
Ar-H), 7.54-7.67 (m, 4H, Ar-H), 7.71 (d, 2H, Ar-H, J=8.4 Hz), 8.97 (s, 1H, NH, D2O
13
exchangeable), 9.15 (s, 1H, NH, D2O exchangeable); C NMR (DMSO-d6, 100 MHz)
δ ppm:
24

ACCEPTED MANUSCRIPT
21.15, 21.75 (CH3), 42.62, 42.78 (benzylic carbon), 110.18, 110.95, 112.12, 112.15, 116.28,
117.52, 117.99 (2C), 119.30, 119.73, 121.52, 122.30, 122.76, 122.98, 123.47, 125.30, 125.34,
127.21 (2C), 130.36, 130.45, 131.32, 131.90, 132.72, 133.90, 134.52, 137.38, 137.60, 137.85,
138.01, 138.17, 142.85, 143.32, 144.83, 145.41, 146.94, 150.99, 152.69 (aromatic carbon),
152.75, 153.90 (C=O of urea), 157.75, 163.15 (C=O of isatin); Anal. Calcd. for C29H25N5O4S
(539.61): C, 64.55; H, 4.67; N, 12.98; found C, 64.72; H, 4.65; N, 13.04.
4.1.4.10.
4-{3-[4-((1-(4-Cyanobenzyl)-2-oxoindolin-3-
ylidene)amino)phenyl]ureido}benzenesulfonamide 9d
.
Orange powder (yield 75%); m.p. over 280 °C; IR (KBr,
ⱱ
cm-1): 3123-3387 broad band (NH,
ppm: 5.08 (s,
=7.2 Hz), 7.01-7.08
=7.2 Hz), 7.47 (d,
=8.4 Hz), 7.95 (s, 1H, Ar-
H), 8.95 (s, 1H, NH, D2O exchangeable), 9.13 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO-d6, 100 MHz) ppm: 42.52, 42.70 (benzylic carbon), 110.18, 110.90, 112.09, 112.14,
1
NH2), 1720, 1612 (2C=O), 1300, 1150 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
2H, benzylic proton), 6.78 (d, 1H, Ar-H,
(m, 3H, Ar-H), 7.23 (s, 2H, NH2, D2O exchangeable), 7.36 (t, 1H, Ar-H,
1H, Ar-H, =8.8 Hz), 7.58-7.66 (m, 5H, Ar-H), 7.75 (d, 3H, Ar-H,
J=7.2 Hz), 6.84 (t, 1H, Ar-H, J
J
J
J
δ
116.22, 117.59, 117.99 (2C), 118.52, 119.12, 119.34, 119.83, 121.42, 122.25, 122.76, 122.98,
123.47, 125.36, 125.36, 127.32 (2C), 130.36, 130.42, 131.38, 131.90, 132.72, 133.90, 134.50,
137.37, 137.58, 137.80, 138.00, 138.19, 142.91, 143.30, 144.88, 145.42, 146.94, 150.99,
152.69 (aromatic carbon), 152.80, 153.99 (C=O of urea), 157.78, 163.20 (C=O of isatin); Anal.
Calcd. for C29H22N6O4S (550.59): C, 63.26; H, 4.03; N, 15.26; found C, 63.37; H, 4.00; N,
15.31.
4.1.4.11.
4-{3-[4-((1-Benzyl-5-chloro-2-oxoindolin-3-
ylidene)amino)phenyl]ureido}benzenesulfonamide 9e
.
Dark red powder (yield 70%); m.p. over 280 °C; IR (KBr,
ⱱ
cm-1): 3123-3357 broad band (NH,
ppm: 4.98 (s,
1
NH2), 1724, 1619 (2C=O), 1304, 1150 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
2H, benzylic proton), 6.69 (s, 1H, Ar-H), 7.04-7.07 (m, 2H, Ar-H), 7.14 (s, 2H, Ar-H), 7.18 (s,
2H, NH2, D2O exchangeable), 7.31-7.39 (m, 3H, Ar-H), 7.54 (d, 2H, Ar-H, =8.8 Hz), 7.59-
7.62 (m, 2H, Ar-H), 7.66 (d, 2H, Ar-H, =8.8 Hz), 7.71 (d, 2H, Ar-H, =8.8 Hz), 9.03 (s, 1H,
J
J
J
25

ACCEPTED MANUSCRIPT
NH, D2O exchangeable), 9.17 (s, 1H, NH, D2O exchangeable); Anal. Calcd. for
C28H22ClN5O4S (560.03): C, 60.05; H, 3.96; N, 12.51; found C, 60.13; H, 3.95; N, 12.47.
4.1.4.12.
4-{3-[4-((5-Chloro-1-(3-fluorobenzyl)-2-oxoindolin-3-
ylidene)amino)phenyl]ureido}benzenesulfonamide 9f
.
Red powder (yield 75%); m.p. over 280 °C; IR (KBr,
ⱱ
cm-1): 3120-3340 broad band (NH, NH2),
ppm: : 4.96, 5.07 (s,
=8.0 Hz), 7.02-7.09 (m,
=8.0 Hz), 7.48 (d, 1H, Ar-
1
1724, 1601 (2C=O), 1307, 1170 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
2H, benzylic proton), 6.78 (d, 1H, Ar-H,
3H, Ar-H),7.24 (s, 2H, NH2, D2O exchangeable), 7.36 (t, 1H, Ar-H,
H, =8.0 Hz), 7.60-7.67 (m, 4H, Ar-H,), 7.76 (d, 3H, Ar-H, =8.0 Hz),7.93 (d, 1H, Ar-H,
J=8.0 Hz), 6.85 (t, 1H, Ar-H, J
J
J
J
J=8.0
13
Hz), 8.92, 8.95 (s, 1H, NH, D2O exchangeable), 9.12, 9.13 (s, 1H, NH, D2O exchangeable); C
NMR (DMSO-d6, 100 MHz)
δ ppm: 42.83, 42.98 (benzylic carbon), 111.85, 112.58, 114.57,
114.62, 114.79, 114.81, 114.97, 115.02, 117.36, 117.99 (2C), 118.03, 118.42, 119.53 (2C),
119.76 (2C), 122.17, 123.47, 123.76, 123.79, 123.82, 123.92, 124.69, 126.64, 127.33, 127.73
(2C), 131.13, 131.18, 131.21, 131.26, 133.04, 133.74, 137.42, 138.03, 138.71, 138.98,
139.05,139.07, 139.15, 142.29, 143.27, 144.07, 144.32, 145.82, 149.72, 152.65, 152.77, 152.88
(aromatic carbon),157.50, 161.61 (C=O of urea), 162.81, 164.03(C=O of isatin); Anal. Calcd. for
C28H21ClFN5O4S (578.02): C, 58.18; H, 3.66; N, 12.12; found C, 58.05; H, 3.67; N, 12.09.
4.1.4.13.
4-{3-[4-((5-Chloro-1-(4-methylbenzyl)-2-oxoindolin-3-
ylidene)amino)phenyl]ureido}benzenesulfonamide 9g
.
Orange powder (yield 83%); m.p. over 280 °C; IR (KBr,
ⱱ
cm-1): 3123-3388 broad band (NH,
ppm: 3.19 (s,
=8.0 Hz), 6.77 (s, 0.5H, Ar-H),
1
NH2), 1714, 1602 (2C=O), 1300, 1150 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
3H, -CH3), 4.78 (s, 2H, benzylic proton), 6.49 (d, 2H, Ar-H,
J
7.00-7.08 (m, 3.5H, Ar-H), 7.16 (s, 2H, NH2, D2O exchangeable), 7.54-7.63 (m, 5H, Ar-H),
7.67-7.73 (m, 4H, Ar-H), 8.24 (s, 1H, NH, D2O exchangeable), 8.86 (s, 1H, NH, D2O
exchangeable); Anal. Calcd. for C29H24ClN5O4S (574.05): C, 60.68; H, 4.21; N, 12.20; found
C, 60.42; H, 4.18; N, 12.26.
4.1.4.14.
4-{3-[4-((5-Chloro-1-(4-cyanobenzyl)-2-oxoindolin-3-
ylidene)amino)phenyl]ureido}benzenesulfonamide 9h
.
26

ACCEPTED MANUSCRIPT
Yellow powder (yield 73%); m.p. over 280 °C; IR (KBr,
ⱱ
cm-1): 3120-3320 broad band (NH,
NH2), 1724, 1617 (2C=O), 1307, 1150 (SO2); H NMR (DMSO-d6, 400 MHz) ppm: 4.96,
5.07 (2s, 2H, benzylic proton), 6.74 (s, 1H, Ar-H), 6.95 (d, 1H, Ar-H, =8 Hz), 7.03 (d, 1H,
Ar-H, =8 Hz), 7.24 (s, 2H, NH2, D2O exchangeable), 7.35 (d, 1H, Ar-H, =8), 7.45 (t, 1H, Ar-
H, =8.4 Hz), 7.56-7.67 (m, 5H, Ar-H), 7.76 (d, 4H, Ar-H, =7.6 Hz), 7.94 (s, 1H, Ar-H), 8.97,
9.01 (s, 1H, NH, D2O exchangeable), 9.12, 9.15 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO-d6, 100 MHz) ppm: 42.64, 42.80 (benzylic carbon), 111.77, 112.10, 112.15, 112.49,
1
δ
J
J
J
J
J
δ
117.52, 117.99, 118.03, 118.40, 119.12, 119.21, 119.54, 119.77, 122.16, 123.51, 124.08,
124.69, 126.65, 127.32, 127.73, 130.34, 130.39, 131.27, 131.31, 131.87, 131.93, 132.72,
133.00, 133.70, 137.41, 137.86, 137.94, 138.01, 138.67, 142.28, 143.26, 143.98, 144.32,
145.71, 149.78, 152.65 (aromatic carbon), 152.77, 152.93 (C=O of urea), 157.62, 162.93 (C=O
of isatin); Anal. Calcd. for C29H21ClN6O4S (585.04): C, 59.54; H, 3.62; N, 14.37; found C,
59.42; H, 3.58; N, 14.35.
4.1.4.15.
4-{3-[4-((1-Benzyl-5-bromo-2-oxoindolin-3-
.
ylidene)amino)phenyl]ureido}benzenesulfonamide 9i
Dark red powder (yield 71%); m.p. over 280 °C; IR (KBr,
ⱱ
cm-1): 3123-3350 broad band (NH,
ppm: 4.99 (s,
1
NH2), 1724, 1603 (2C=O), 1307, 1145 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
2H, benzylic proton), 6.71 (s, 1H, Ar-H), 7.06-7.12 (m, 2H, Ar-H), 7.18 (s, 2H, Ar-H), 7.19 (s,
2H, NH2, D2O exchangeable), 7.33-7.38 (m, 3H, Ar-H), 7.56 (d, 2H, Ar-H, =8.8 Hz), 7.61-
7.64 (m, 2H, Ar-H), 7.68 (d, 2H, Ar-H, =8.8 Hz), 7.74 (d, 2H, Ar-H, =8.8 Hz), 9.05 (s, 1H,
NH, D2O exchangeable), 9.18 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6, 100
MHz) ppm: 42.68, 42.82 (benzylic carbon), 111.79, 112.18, 112.21, 112.51, 117.52, 117.99,
J
J
J
δ
119.18, 119.25, 119.57, 119.80, 122.19, 123.55, 124.17, 124.75, 126.65, 127.35, 127.75,
130.34, 130.45, 131.29, 131.35, 131.89, 131.92, 132.72, 133.15, 133.74, 137.43, 137.89,
137.94, 138.01, 138.69, 142.32, 143.28, 143.98, 144.37, 145.75, 149.78, 152.67 (aromatic
carbon), 152.79, 152.96 (C=O of urea), 157.65, 162.97 (C=O of isatin); Anal. Calcd. for
C28H22BrN5O4S (604.48): C, 55.64; H, 3.67; N, 11.59; found C, 55.73; H, 3.65; N, 11.67.
4.1.4.16.
4-{3-[4-((5-Bromo-1-(3-fluorobenzyl)-2-oxoindolin-3-
ylidene)amino)phenyl]ureido}benzenesulfonamide 9j
.
27

ACCEPTED MANUSCRIPT
°
Red powder (yield 80%); m.p. over 280 C; IR (KBr,
ⱱ
cm-1): 3123-3400 broad band (NH,
ppm: 4.93,
1
NH2), 1704, 1610 (2C=O), 1300, 1142 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
5.04 (s, 2H, benzylic proton), 6.55-7.24 (m, 8H, 2H of NH2, D2O exchangeable and 6H, Ar-H),
7.40-7.78 (m, 9H, Ar-H), 9.03 (s, 1H, NH, D2O exchangeable), 9.17 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6, 100 MHz)
δ ppm: 42.95 (benzylic carbon), 112.33,
113.04, 114.32, 114.55, 114.77, 115.02, 115.28, 117.57, 117.83 (2C), 118.03, 118.41, 119.54,
119.71, 121.46, 123.49, 123.77, 124.28, 124.91, 127.32 (2C), 127.48, 131.18, 131.26, 135.86,
136.53, 137.41, 138.03, 138.71, 138.96, 139.03, 139.13, 142.27, 143.27, 144.32, 144.46,
146.18, 149.56, 152.65, 152.76, 152.79, 157.37 (aromatic carbon), 158.03, 161.60 (C=O of
urea), 162.68, 164.03 (C=O of isatin); Anal. Calcd. for C28H21BrFN5O4S (622.47): C, 54.03; H,
3.40; N, 11.25; found C, 53.87; H, 3.38; N, 11.24.
4.1.4.17.
4-{3-[4-((5-Bromo-1-(4-methylbenzyl)-2-oxoindolin-3-
ylidene)amino)phenyl]ureido}benzenesulfonamide 9k
.
Orange powder (yield 75%); m.p. over 280 °C; IR (KBr,
ⱱ
cm-1): 3129-3340 broad band (NH,
ppm: : 3.10,
=8.0 Hz), 6.63
(br s, 0.5H, Ar-H), 7.00-7.11 (m, 3.5H, Ar-H), 7.14-7.16 (m, 3H, 1H, Ar-H, 2H, NH2, D2O
exchangeable), 7.50-7.63 (m, 5H, Ar-H), 7.67 (d, 2H, Ar-H, =8.0 Hz), 7.71 (d, 1H, Ar-H,
=8.0 Hz), 8.24, 8.36 (2s, 1H, NH, D2O exchangeable), 8.86 ,8.99 (2s, 1H, NH, D2O
1
NH2), 1719, 1603 (2C=O), 1300, 1150 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
3.19 (2s, 3H, -CH3), 4.68, 4.78 (2s, 2H, benzylic proton), 6.49 (d, 2H, Ar-H,
J
J
J
exchangeable); Anal. Calcd. for C29H24BrN5O4S (618.51): C, 56.32; H, 3.91; N, 11.32; found
C, 56.27; H, 3.90; N, 11.28.
4.1.4.18.
4-{3-[4-((5-Bromo-1-(4-cyanobenzyl)-2-oxoindolin-3-
ylidene)amino)phenyl]ureido}benzenesulfonamide 9l
.
°
Yellow orange powder (yield 79%); m.p. over 280 C; IR (KBr,
ⱱ
cm-1): 3123-3400 broad
1
band (NH, NH2), 1714, 1622 (2C=O), 1317, 1159 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
ppm: 4.75, 4.79 (2s, 2H, benzylic proton), 6.49 (d, 2H, Ar-H,
6.99 (d, 2H, Ar-H, =8.0 Hz), 7.06 (d, 2H, Ar-H, =8.4 Hz), 7.17 (s, 2H, NH2, D2O
exchangeable), 7.54 (m, 4H, Ar-H), 7.66 (d, 2H, Ar-H, =8.8 Hz), 7.72 (d, 1H, Ar-H, =8.8
Hz), 8.25 (s, 1H, Ar-H), 9.07 (s, 1H, NH, D2O exchangeable), 9.15 (s, 1H, NH, D2O
J=8.4 Hz), 6.78 (s, 1H, Ar-H),
J
J
J
J
28