Amido/ureidosubstituted benzenesulfonamides-isatin conjugates as low nanomolar/subnanomolar inhibitors of the tumor-associated carbonic anhydrase isoform XII

Article abstract of DOI:10.1016/j.ejmech.2016.01.030

By using a molecular hybridization approach, two series of amido/ureidosubstituted benzenesulfonamides incorporating substituted-isatin moieties were synthesized. The prepared derivatives were in vitro evaluated for their inhibitory activity against human

Full text of DOI:10.1016/j.ejmech.2016.01.030

Accepted Manuscript
Amido/ureidosubstituted benzenesulfonamides-isatin conjugates as low nanomolar/
subnanomolar inhibitors of the tumor-associated carbonic anhydrase isoform XII
Wagdy M. Eldehna, Mohamed Fares, Mariangela Ceruso, Hazem A. Ghabbour,
Sahar M. Abou-Seri, Hatem A. Abdel-Aziz, Dalal A. Abou El Ella, Claudiu T. Supuran
PII:
S0223-5234(16)30031-9
10.1016/j.ejmech.2016.01.030
EJMECH 8320
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 August 2015
Revised Date: 7 December 2015
Accepted Date: 17 January 2016
Please cite this article as: W.M. Eldehna, M. Fares, M. Ceruso, H.A. Ghabbour, S.M. Abou-Seri, H.A.
Abdel-Aziz, D.A. Abou El Ella, C.T. Supuran, Amido/ureidosubstituted benzenesulfonamides-isatin
conjugates as low nanomolar/subnanomolar inhibitors of the tumor-associated carbonic anhydrase
isoform XII, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.01.030.
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ACCEPTED MANUSCRIPT
Graphical abstract
Amido/ureidosubstituted benzenesulfonamides-isatin conjugates as low
nanomolar/subnanomolar inhibitors of the tumor-associated carbonic
anhydrase isoform XII
Wagdy M. Eldehna,* Mohamed Fares, Mariangela Ceruso, Hazem A. Ghabbour, Sahar M.
Abou-Seri, Hatem A. Abdel-Aziz, Dalal A. Abou El Ella, Claudiu T. Supuran*
Two series of amido and ureido-sulfonamides conjugated with isatins were synthesized and evaluated for
their inhibitory activity against a panel of hCAs; I, II (cytosolic) and IX, XII (transmembrane)
H
F
Cl
Br
NO2
O O
S
O
NH2
O O
S
O
N
N
NH2
R
H
N
H N
2
O
H
Me
MeO
N
4
6a-g
H
K hCA XII = 2.7 nM
0.47 nM < K hCA XII < 1.76 nM
I
I

ACCEPTED MANUSCRIPT
Amido/ureidosubstituted benzenesulfonamides-isatin conjugates as low
nanomolar/subnanomolar inhibitors of the tumor-associated carbonic anhydrase
isoform XII
a
a
b
c
Wagdy M. Eldehna ,* Mohamed Fares , Mariangela Ceruso , Hazem A. Ghabbour , Sahar M.
d
c,e
f
b,g
Abou-Seri , Hatem A. Abdel-Aziz , Dalal A. Abou El Ella , Claudiu T. Supuran *
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr
City, Cairo, P.O. Box 11829, Egypt
b
Department of Chemistry, Laboratory of Bioinorganic Chemistry, University of Florence, Polo
Scientifico, Via della Lastruccia 3, 50019 Sesto Fiorentino (Firenze), Italy
c
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box
2
457, Riyadh 11451, Saudi Arabia
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini
d
Street, Cairo, P.O. Box, 11562, Egypt
e
Department of Applied Organic Chemistry, National Research Center, Dokki, Giza, P.O. Box 12622,
Egypt.
f
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Cairo,
Abbassia, P.O. Box 11566, Egypt
g
Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of
Florence, Polo Scientifico, Via U. Schiff 6, 50019 Sesto Fiorentino (Firenze), Italy
Abstract. By using a molecular hybridization approach, two series of amido/ureidosubstituted
benzenesulfonamides incorporating substituted-isatin moieties were synthesized. The prepared
derivatives were in vitro evaluated for their inhibitory activity against human carbonic anhydrase
(
hCA, EC 4.2.1.1) isoforms I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) isoforms.
All these isoforms were inhibited in variable degrees by the sulfonamides reported here. hCA I was
inhibited with K s in the range of 7.9–894 nM, hCA II in the range of 7.5–1645 nM (with one
I
compound having a K > 10 µM); hCA IX in the range of 5.0–240 nM, whereas hCA XII in the range
I
of 0.47–2.83 nM. As all these isoforms are involved in various pathologies, in which their inhibition
can be exploited therapeutically, the derivatives reported here may represent interesting extensions to
the field of CA inhibitors of the sulfonamide type.
_
___________
Keywords: Carbonic anhydrase; tumor-associated isoforms; amido/ureido sulfonamides; isatin.
Corresponding authors. Tel.: +2-02-2864-3342; fax: +2-02-2864-3332; e-mail:
wagdy2000@gmail.com (WME); Tel./fax:+39-055-4573729; e-mail: claudiu.supuran@unifi.it (CTS)
∗
1

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1-Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread metalloenzymes in all life kingdoms [1,2].
They catalyze the efficient interconversion between CO and bicarbonate, being involved in crucial
2
physiologic processes. Most mammals, including humans, possess two blood isoforms denominated
with a total concentration of these proteins as high as 0.2 mM [1]. The catalytic activity of the
human (h) isoform hCA I (slow blood enzyme) is much lower compared to that of hCA II and in
addition hCA I is inhibited by the chloride and bicarbonate present in the plasma, leaving a lot of
questions regarding the physiologic function of this isoform [1,2]. The high activity isoform hCA II
(rapid blood enzyme) which is one of the best catalysts known in nature, is involved in the secretion
of electrolytes in a multitude of tissues, such as the bicarbonate rich aqueous humor in the anterior
chamber of the eyes, the cerebrospinal fluid and in pH and CO homeostasis all over the body [1,2].
2
Dysregulation of the activity of these two isoforms in one or more tissues has important pathologic
consequences, such as glaucoma and oedema [1,2]. There are several types of cancer, in which CA II
was observed to be overexpressed - alone or together with other isoforms such as CA IX and XII
[1,2]. However most solid tumors overexpress two transmembrane isoforms, hCA IX and XII, which
are involved in tumor progression and metastases formation [1,2]. Thus, many of the hCA isoforms
are validated drug targets for various applications, such as diuretics, anticonvulsants, antiglaucoma
agents, and antitumor/antimetastatic drugs [1,2].
Benzenesulfonamides incorporating an amido or ureido moiety represent interesting classes of
CA inhibitors (CAIs) [1-5]. Akdemir et al. [3] developed a new class of substituted-phenylacetamido
benzenesulfonamides as potent inhibitors of the four physiologically relevant hCA isoforms, (hCA I,
I
II, IX and XII). Interestingly, the K values were in the nanomolar range for the tumor-associated
hCA IX/XII, in particular, compound I has excellent inhibitory action against hCA XI (0.32 nM) and
hCA XII (0.64 nM), Figure 1.
Figure 1
Moreover, incorporation of urea functionality in the aromatic/heterocyclic sulfonamides
represents the most recent and promising trend in the design of CAIs with selectivity profile for
inhibiting the transmembrane, tumor associated isoforms (CAs IX and XII) over the off-target
cytosolic CAs I and II [6-15]. SLC-0111 II, Figure 1, an ureido benzenesulfonamide derivative
discovered by one of our groups, is currently in clinical evaluations. SLC-0111 II is characterized by
2

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its selectivity towards inhibition of the transmembrane isoforms hCA IX/XII (over the cytosolic
isoforms hCA I/II), which attributable to the presence of the ureido functionality as linker between
the benzene sulfonamide fragment (which coordinates in deprotonated form at the sulfamoyl group
to the zinc ion from the CA active site) and the tail of the inhibitor. This ureido linker allows a great
flexibility to the tail of the molecule which imparts the possibility for the inhibitor to adopt a variety
of orientations when bound within the enzyme active site. These orientations allow the specific
interactions between the inhibitor tail and amino acid residues at the entrance of the active site
cavity, which is the most variable region in the various a-CA isoforms [16-22]. SLC-0111 II was
also able to block human breast cancer invasion, delay tumor growth and diminish the cancer stem
cell population in vivo. Furthermore, combination of SLC-0111 II with paclitaxel significantly
inhibits tumor growth and attenuates spontaneous lung metastasis in this aggressive orthotopic breast
cancer model [23]. Besides, recent study [24] has reported 2,4-dichloro-5-(3-(4-sulfamoylphenyl)
ureido)benzenesulfonamide (III) as a potent CA IX and XII inhibitor with K values of 15.8 and 3.6
I
nM, respectively, Figure 1.
Isatin is a privileged heterocyclic scaffold, as its derivatives possesses interesting biological
activity profiles and are well-tolerated in humans [25]. In 2015, a series of 2/3/4-[(2-oxo-1,2-
dihydro-3H-indol-3-ylidene)amino]benzenesulfonamides was synthesized, showing low nanomolar
inhibitory activity against the tumor associated CA isoforms IX and XII. Compound IV was one of
the potent analogs within this series [26]. In addition, the isatin-based 1,3,4-thiadiazole-2-thione
derivative V was found to inhibit the tumor-associated hCA IX with K value of 1.25 ꢀM [27],
I
Figure 1.
VEGFR inhibitors restrain tumor growth by suppressing tumor angiogenesis and blocking the
developing vasculature of tumors. Regrettably, resistance is common and individual responses are
variable; some patients exhibiting resistance from the outset (intrinsic resistance) and others going
on to develop resistance following a short time of disease control (acquired resistance) [28-31].
Recent studies have highlighted the possibility that anti-angiogenic therapy induces an increasingly
hypoxic tumor microenvironment, thence, increased expression of hypoxia-regulated genes and
proteins, including CA IX and XII, in response to treatment was observed. For example, CA-IX
expression correlates with poor prognosis in most tumor types and with worse outcome in
bevacizumab-treated metastatic colorectal cancer patients, malignant astrocytoma and recurrent
malignant glioma [32-34]. Continuing our interest in developing compounds which inhibit
enzymes/proteins overexpressed in hypoxic tumors, here we report two series of
benzenesulfonamides-isatin conjugates 6a-g and 10a-g incorporating amido or ureido linkers, which
3

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have been assayed for the inhibition of cytosolic and transmembrane CA isoforms involved in
various pathologies.
2
-Results and Discussion
2
.1. Chemistry
The synthetic pathways employed to prepare the targeted sulfonamides are depicted in schemes 1
and 2. Firstly, the reaction of 4-nitrobenzoyl chloride (1) with 4-aminobenzenesulfonamide (2) in
THF at ambient temperature in the presence of TEA afforded the corresponding 4-nitro-N-(4-
sulfamoylphenyl)benzamide (3) (Scheme 1). Reduction of the 4-nitrobenzamide derivative 3 using
Pd/C resulted in the formation of 4-amino-N-(4-sulfamoylphenyl)benzamide (4) in 85% yield. 4-((2-
Oxoindolin-3-ylidene)amino)-N-(4-sulfamoylphenyl)benzamides 6a-g were prepared via refluxing
4
-amino-N-(4-sulfamoylphenyl)benzamide 4 with isatins 5a-g, respectively, in glacial acetic acid
(Scheme 1).
Scheme 1
Next, 1-isocyanato-4-nitrobenzene (7) was treated with 4-aminobenzenesulfonamide (2) in
acetonitrile under reflux to give 4-(3-(4-nitrophenyl)ureido)benzenesulfonamide (8). Reduction of
the latter over Pd/C produced 4-(3-(4-aminophenyl)ureido)benzenesulfonamide (9) in 81% yield.
Finally, the reaction of compound 9 with isatins 5a-g afforded 4-(3-(4-((2-oxoindolin-3-
ylidene)amino)phenyl)ureido)benzenesulfonamides 10a-g, respectively (Scheme 2).
Scheme 2
The structures of amides 6a-g and ureas 10a-g were confirmed under the basis of their spectral
1
data and elemental analyses. It is worthy to mention that H NMR spectra of these compounds revealed
their presence as cis/trans conformers and E/Z geometrical isomers due to the amide group and
exocyclic -C=N- double bond, respectively (Figure 2) [35-38]. Moreover, it was reported that the
compounds containing imine bond are present in higher percentage as E isomer in dimethyl-d sulfoxide
6
solution. According to the literature, the ratio of the isomers is solvent-dependent [37, 38]. However, the
spectral data of 6a-g and 10a-g, in our hands, is similar to that reported for isatin imine E/Z isomers [26]
and isatin amide cis/trans conformers [39]. The latter facts noticed for some compounds of amides 6a-g
4

ACCEPTED MANUSCRIPT
and urea derivatives 10a-g through the appearance of additional NMR signals due to their isomerization.
1
We could identify some proton signals of isomers especially for NHs in H NMR spectra whereas the
1
3
overlapping of symmetrical carbon signals of isomers appeared in several cases in CNMR spectra. For
1
13
example, H and C NMR spectra of amide 6d revealed the signals of protons and carbons of its
1
possible isomers whereas H NMR spectra of amides 6f and 6g appeared their isomerization through the
1
signals of their 2NH protons. H NMR spectra of urea derivatives 10d and 10g showed their
isomerization through the integration and position of their three NHs (isatin NH and urea 2NHs),
1
respectively. For example, H NMR spectrum of 10d showed two sets of signals for its three D O
2
exchangeable NHs with the same ratio of isomerization at 8.89 and 8.95 (s, 1H, NH urea), 9.08 (s, 1H,
NH urea), 10.96 and 11.71 (s, 1H, NH isatin), respectively.
Figure 2
2
.2. Carbonic anhydrase inhibition
Sulfonamides 4, 9, 6a-g and 10a-g were screened for their ability to inhibit four physiologically
relevant hCA isoforms, hCA I, II (cytosolic) as well as hCA IX and XII (transmembrane, tumor-
associated isoforms). Table 1 shows inhibition data of the prepared conjugates and the sulfonamide
acetazolamide AAZ (as a standard inhibitor) against the four isoforms [42]. The following structure-
activity relationship (SAR) should be noted regarding the inhibition data of Table 1:
(
i) Isoform hCA I was inhibited by the prepared conjugates reported here with K s ranging between
I
7
.9 and 894 nM. Concerning activity of the amido analogs 4 and 6a-g, all of them showed better
activity (K s: 7.9–95.6 nM) than that of the standard drug AAZ (K of 250 nM against this isoform).
I
I
Among them, compounds 6b-6f emerged as efficient hCA I inhibitors with K s values of 9, 9.2, 7.9,
I
1
0.2 and 8.5 nM, respectively, with more than 25-fold increased activity than AAZ. As the precise
physiologic function of hCA I is largely unknown, these inhibitors may be valuable tools for
investigating this enzyme in more details. The effective inhibitors discovered here incorporated the
amide linker and the following substituents at the isatin ring: 5-halogens (F, Cl, Br), 5-nitro and 5-
methyl. All these derivatives were around one order of magnitude better hCA I inhibitors compared
to 6a which has hydrogen at the isatin ring, proving that substitution of isatin at 5-position led to
dramatic differences of biological activity. The compounds incorporating the ureido linker 9 and
5

ACCEPTED MANUSCRIPT
1
0a-g were on the other hand less effective as hCA I inhibitors, as reported in the original series of
derivatives to which SLC-0111 belongs [17, 18]. In fact these derivatives showed inhibition
constants in the range of 45.2–894 nM (Table 1).
Table 1
(ii) Isoform hCA II was effectively inhibited by most sulfonamides reported here, except 10d (K
I
>
1
0 µM) and 10b, 10c, 10e and 10g (K s in the range of 585–1645 nM) (Table 1). It may be thus
I
observed that among the ureido derivatives reported here only 9 and 10f were potent hCA II
inhibitors (K of 15.7 and 33 nM, respectively) whereas all other derivatives were weak or
I
ineffective inhibitors of this isoform, similar to the uerido-benzenesyulfonamides described earlier
by one of our groups [17,18]. This behavior has also been explained from the structural viewpoint,
since in the X-ray crystal structures of five such inhibitors bound to hCA II, a very high flexibility of
the arylureido tail has been evidenced, which led to very diverse orientations of the inhibitor within
the active site. As a consequence of this flexibility, the inhibitors belonging to this chemotype may
participate in a multitude of favorable and unfavorable interactions with amino acid residues from
the active site, leading thus to a versatile inhibitory behavior [17,18]. The compounds incorporating
the amide linker, 4 and 6a-6g showed a better hCA II inhibitory action. Incorporation of
I
unsubstituted isatin moiety led to compound 6a with moderate activity against hCA II (K of 116
nM), but its congeners incorporating halogens, nitro, methyl and methoxy substituents at the 5-
position of isatin ring were much more effective (K s in the range of 7.5–14.5 nM, Table 1).
I
(iii) The transmembrane isoform hCA IX was inhibited by all sulfonamides investigated here, with
K
I
s in the range of 5.0–240 nM. The best inhibitory activity against this isoform was observed for
counterparts 6b, 10e, 10f and 10g (K s = 5.0, 14.8, 16.7 and 8.5 nM, respectively) whereas the
I
remaining compounds were medium potency inhibitors (K s in the range of 159–240 nM). Notably,
I
grafting of nitro, methyl or methoxy groups at the 5-position of isatin moiety in the ureido series
1
0a-g, greatly enhanced the activity against hCA IX. Thence, the order of activities of the isatin-
substituted members in this series, was decreased in the order of OMe > NO > Me >>> halogens.
2
Differently, the SAR for the amido series 6a-g is difficult to interpret as compounds in this series
6

ACCEPTED MANUSCRIPT
showed effective activity, although rather similar derivatives (e.g., 6b and 6c) had a rather different
activity profile.
(
iv) The second transmembrane isoform investigated here, hCA XII, was highly inhibited by the
sulfonamides reported here, many of which were subnanomolar inhibitors. Superiorly, sulfonamides
, 6b-6g, 10c and 10e showed K s in the subnanomolar range of 0.47–0.71, nM whereas the
9
I
I
remaining derivatives were also highly effective single-digit nanomolar hCA XII inhibitors (K s in
the range of 1.17–2.83 nM). Thus, all compounds investigated here act as highly potent hCA XII
inhibitors, indiscriminately of the linker or substitution pattern at the isatin ring.
Finally, we can deduce that grafting of different substituents (as halogens, nitro, methyl and
methoxy) at the 5-position of isatin moiety is advantageous for the activity of the amido series 6a-g
against hCA I, II and XII, regardless of the electronic nature of substituent. Also, substitution of
isatin moiety in the ureido series 10a-g with an electron withdrawing group as 5-nitro or electron
donating group like 5-methyl or 5-methoxy is indispensable for the activity against hCA IX.
3
. Conclusion
In conclusion, we report here the synthesis of two series of amido/ureido-substituted
benzenesulfonamides incorporating substituted-isatin moieties, utilizing a molecular hybridization
approach. The prepared derivatives were in vitro evaluated for their inhibitory activity against a panel of
hCA I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) isoforms. All the tested isoforms
were inhibited in variable degrees by the prepared sulfonamides reported here. Best activity was
observed against the tumor-associated isoform hCA XII. Sulfonamides 9, 6b-6g, 10c and 10e displayed
I
outstanding potency with K s in the subnanomolar range of 0.47–0.71, nM. Moreover, the remaining
derivatives emerged as single-digit nanomolar hCA XII inhibitors (K s in the range of 1.17–2.83 nM).
I
The preliminary SAR study revealed the importance of various substituents at the 5-position of isatin for
activity of the amido series 6a-g against hCA I, II and XII, besides, the significance of grafting 5-nitro,
5
-methyl and 5-methoxy groups to the isatin ring for activity of the ureido series 10a-g against hCA IX.
Meanwhile, the electronic nature of such substituents has no effect on the enzyme inhibitory activity of
the compounds.
7

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4
4
4
. Experimental
.1. Chemistry
.1.1. General
Melting points were determined with a Stuart apparatus and are uncorrected. IR spectra were
-1
recorded on Schimadzu FT-IR 8400S spectrophotometer and expressed in wave number (cm ). The
NMR spectra were recorded on Varian Gemini-300BB at 300 MHz (Varian Inc., Palo Alto, CA) or
H
Bruker spectrophotometer at 400 MHz. Chemical shifts (δ ) are reported relative to TMS as internal
standard. All coupling constant (J) values are given in hertz. The abbreviations used are as follows:
s, singlet; d, doublet; m, multiplet. Microanalyses were carried out using Perkin Elmer PE 2400
CHN Elemental Analyzer. Reaction courses and product mixtures were routinely monitored by thin
layer chromatography (TLC) on silica gel precoated F254 Merck plates. All the starting materials and
reagents used were purchased from Sigma-Aldrich. Unless otherwise noted, all solvents and reagents
were used without further purification.
Compounds 3 [40], 6 [41], 10a-c [41] are previously reported.
4
.1.2. 4-Amino-N-(4-sulfamoylphenyl)benzamide (4)
The nitro derivative 3 (1.61 g, 5 mmol) was suspended in MeOH (25 mL) and 10% palladium on
carbon (0.5 g) was added. The reaction mixture was stirred at room temperature under a hydrogen
atmosphere for 3 h. Then, DMF (10 mL) was added to dissolve the product, the catalyst was filtered
off, and the filtrate was concentrated in vacuum. The residual solid was collected and washed with
-1
AcOEt–hexane to give 4 (1.23 g, 85%) as crystals: m.p. > 280 °C; IR (KBr, ν cm ): 3344, 3302,
1
3
238 (NH, NH ), 1648 (C=O) 1314, 1171, (SO ); H NMR (DMSO-d )
δ
ppm: 5.85 (s, 2H, D O
2
2
6
2
exchangable, NH ), 6.61 (d, 2H, J= 8.7 Hz, Ar-H), 7.23 (s, 2H, D O exchangable, SO NH ), 7.73
2
2
2
2
(d, 2H, J= 6.8 Hz, Ar-H), 7.75 (d, 2H, J= 6.8 Hz, Ar-H), 7.91 (d, 2H, J= 8.7 Hz, Ar-H), 10.06 (s,
1
3
1
1
1
H, D
2 6
O exchangable, NH); C NMR (DMSO-d , 75 MHz) δ: 113.07, 119.86, 126.87, 128.00,
30.05, 138.32, 143.31, 152.89, 166.0; Anal. calcd. For C H N O S (291.07): C, 53.60; H, 4.50; N,
1
3
13
3
3
4.42. Found C, 53.72; H, 4.54; N, 14.45.
4
.1.3. General procedure for preparation of target 4-((2-oxoindolin-3-ylidene)amino)-N-(4-
sulfamoylphenyl)benzamides 6a-g.
8

ACCEPTED MANUSCRIPT
A mixture of equimolar quantities of isatins 5a-g (1 mmol) and 4-Amino-N-(4-
sulfamoylphenyl)benzamide 4 (1 mmol) was refluxed in glacial acetic acid (10 mL) for 5 h. The
precipitate formed was collected by filtration while hot, washed with hot ethanol, dried and
crystallized from ethanol/DMF to afford compounds 6a-g with 41-65% yield.
4
.1.3.1. 4-(2-Oxoindolin-3-ylideneamino)-N-(4-sulfamoylphenyl)benzamide (6a): Yellow powder
-1
(
2
yield 59%); m.p. > 280 °C; IR (KBr, ν cm ): 3340, 3289, 3238 (NH, NH ), 1718, 1655 (2C=O),
1
1
342, 1156 (SO ); H NMR (DMSO-d )
δ
ppm: 6.39-7.20 (m, 4H, Ar-H), 7.26 (s, 2H, D O
2
6
2
exchangable, SO NH ), 7.35-7.97 (m, 6H, Ar-H), 8.10 (d, J= 7.8 Hz, 2H, Ar-H), 10.04, 10.45 and
2
2
1
0.56 (s, 1H, D
2 2
O exchangable, NH), 10.95, 11.01 and 11.13 (s, 1H, D O exchangable, NH); Anal.
calcd. For C H N O S (420.44): C, 59.99; H, 3.84; N, 13.33; Found C, 60.05; H, 3.81; N, 13.39.
2
1
16
4
4
4
.1.3.2. 4-(5-Fluoro-2-oxoindolin-3-ylideneamino)-N-(4-sulfamoylphenyl)benzamide (6b): Orange
-1
powder (yield 61%); m.p. > 280 °C; IR (KBr, ν cm ): 3350, 3304, 3247 (NH, NH ), 1733, 1653
2
1
(
2C=O), 1310, 1153 (SO ); H NMR (DMSO-d )
δ
ppm: 6.07 (d, J= 7.8 Hz, 1H, Ar-H), 6.60-7.25
NH ), 7.71-7.80 (m, 6H, Ar-H), 8.11 (d, J= 8.4
2
6
(
m, 2H, Ar-H), 7.27 (s, 2H, D
2
O exchangable, SO
2
2
Hz, 2H, Ar-H), 10.21, 10.38 and 10.59 (s, 1H, D O exchangable, NH), 10.48, 11.03 and 11.15 (s,
2
1
3
1
1
1
3
H, D O exchangable, NH); C NMR (DMSO-d , 75 MHz) δ: 117.08, 118.13, 119.79, 126.51,
2 6
28.43, 129.66, 130.09, 130.98, 138.09, 138.65, 142.15, 142.54, 143.61, 153.03, 163.23, 165.17,
68.77; Anal. calcd. For C H FN O S (438.43): C, 57.53; H, 3.45; N, 12.78; Found C, 57.71; H,
2
1
15
4
4
.49; N, 12.63.
4
.1.3.3. 4-(5-Chloro-2-oxoindolin-3-ylideneamino)-N-(4-sulfamoylphenyl)benzamide (6c): Orange
-1
powder (yield 60%); m.p. > 280 °C; IR (KBr, ν cm ): 3359, 3307, 3246 (NH, NH ), 1733, 1654
2
1
(2C=O), 1306, 1155 (SO
2
); H NMR (DMSO-d
6
)
δ
ppm: 6.32 (s, 1H, Ar-H), 6.60-7.20 (m, 2H, Ar-
H), 7.27 (s, 2H, D O exchangable, SO NH ), 7.43-8.00 (m, 6H, Ar-H), 8.12 (d, J= 8.4 Hz, 2H, Ar-
2
2
2
H), 10.23, 10.49 and 10.60 (s, 1H, D O exchangable, NH), 10.40, 11.04 and 11.17 (s, 1H, D O
2
2
1
3
exchangable, NH); C NMR (DMSO-d
6
, 75 MHz)
δ
: 116.77, 117.06, 119.80, 122.56, 124.73,
1
25.33, 126.52, 129.64, 130.90, 134.21, 138.51, 142.13, 145.08, 153.09, 154.08, 162.92, 165.10;
Anal. calcd. For C H ClN O S (454.88): C, 55.45; H, 3.32; N, 12.32; Found C, 55.33; H, 3.34; N,
2
1
15
4
4
1
2.38.
4
.1.3.4. 4-(5-Bromo-2-oxoindolin-3-ylideneamino)-N-(4-sulfamoylphenyl)benzamide (6d): Orange
-1
powder (yield 65%); m.p. > 280 °C; IR (KBr, ν cm ): 3351, 3298, 3249 (NH, NH ), 1731, 1678 (2C=O)
2
9

ACCEPTED MANUSCRIPT
1
1
303, 1154 (SO ); H NMR (DMSO-d ) δ ppm: 6.45-7.28 (m, 3H, Ar-H), 7.29 (s, 2H, D O exchangable,
2 6 2
SO NH ), 7.55-8.15 (m, 8H, Ar-H), 10.24, 10.51 and 10.62 (s, 1H, D O exchangable, NH), 10.41, 11.05
2
2
2
1
3
and 11.18 (s, 1H, D
2
O exchangable, NH); C NMR (DMSO-d
6
, 100 MHz) δ: 113.46, 114.24, 114.53,
1
17.67, 117.78, 118.60, 118.99, 120.21, 120.25, 120.33, 123.45, 125.90, 127.01, 128.07, 128.89,
28.94, 129.26, 130.14, 130.63, 131.37, 137.22, 137.41, 138.97, 139.01, 139.16, 142.65, 142.76,
42.79, 143.05, 145.60, 146.82, 152.90, 153.03, 153.63, 154.50, 158.58, 163.29, 165.60, 165.75,
65.94, 169.31; Anal. calcd. For C H BrN O S (499.34): C, 50.51; H, 3.03; N, 11.22; Found C, 50.26;
1
1
1
2
1
15
4
4
H, 3.09; N, 11.31.
4
.1.3.5. 4-(5-Nitro-2-oxoindolin-3-ylideneamino)-N-(4-sulfamoylphenyl)benzamide (6e): Yellow
-1
2
powder (yield 55%); m.p. > 280 °C; IR (KBr, ν cm ): 3318, 3176 (NH, NH ), 1747, 1659 (2C=O)
1
1
325, 1150 (SO ); H NMR (DMSO-d )
δ
ppm: 7.10 (d, 1H, J= 8.4 Hz, Ar-H); 7.22 (d, 2H, J= 8.1
2
6
Hz, Ar-H); 7.30 (s, 2H, D O exchangable, SO NH ), 7.81 (d, 2H, J= 7.8 Hz, Ar-H), 7.96 (d, 2H, J=
2
2
2
8
1
.1 Hz, Ar-H), 8.01 (s, 1H, Ar-H), 8.15 (d, 2H, J= 7.8 Hz, Ar-H), 8.28 (d, 1H, J= 8.4 Hz, Ar-H),
1
3
0.54 and 10.64 (s, 1H, D O exchangable, NH), 11.62 and 11.75 (s, 1H, D O exchangable, NH); C
2
2
NMR (DMSO-d , 75 MHz)
δ
: 115.55, 117.22, 119.86, 126.23, 127.60, 128.43, 129.68, 130.49,
6
1
31.10, 138.70, 141.56, 142.09, 152.51, 153.49, 158.68, 163.51, 164.96; Anal. calcd. For
C H N O S (465.44): C, 54.19; H, 3.25; N, 15.05; Found C, 54.31; H, 3.31; N, 14.92.
21
15
5
6
4
.1.3.6. 4-(5-Methyl-2-oxoindolin-3-ylideneamino)-N-(4-sulfamoylphenyl)benzamide (6f): Beige
-1
powder (yield 41%); m.p. > 280 °C; IR (KBr, ν cm ): 3315, 3247, 3213 (NH, NH ), 1742, 1655
2
1
(
2C=O) 1315, 1153 (SO ); H NMR (DMSO-d )
δ
ppm: 1.98 and 2.09 (s, 3H, CH ), 6.24 (s, 1H, Ar-
2
6
3
H), 6.77-7.26 (m, 2H, Ar-H), 7.26 (s, 2H, D
2 2 2
O exchangable, SO NH ), 7.71-8.01 (m, 6H, Ar-H),
8
.11 (d, J= 8.1 Hz, 2H, Ar-H), 10.05, 10.23 and 10.40 (s, 1H, D O exchangable, NH), 10.49, 10.57
2
1
3
and 10.91 (s, 1H, D O exchangable, NH); C NMR (DMSO-d , 75 MHz) δ: 20.4, 24.09, 115.59,
2
6
1
1
17.11, 117.29, 118.44, 125.76, 126.46, 128.43, 128.72, 129.24, 129.35, 129.58, 130.51, 130.59,
35.33, 142.18, 142.54, 145.01, 153.61, 155.03, 163.27, 165.22, 168.78; Anal. calcd. For
C H N O S (434.47): C, 60.82; H, 4.18; N, 12.90; Found C, 60.98; H, 4.12; N, 13.01.
22
18
4
4
4
.1.3.7. 4-(5-Methoxy-2-oxoindolin-3-ylideneamino)-N-(4-sulfamoylphenyl)benzamide (6g): Red
-1
powder (yield 49%); m.p. > 280 °C; IR (KBr, ν cm ): 3313, 3237 (NH, NH ), 1748, 1662 (2C=O)
2
1
1
6
310, 1150 (SO
2
); H NMR (DMSO-d
6
)
δ
ppm: 3.46 and 3.78 (s, 3H, OCH
3
), 5.90 (s, 1H, Ar-H),
.81-7.21 (m, 2H, Ar-H), 7.26 (s, 2H, D O exchangable, SO NH ), 7.71-8.00 (m, 6H, Ar-H), 8.10 (d,
2
2
2
1
0

ACCEPTED MANUSCRIPT
J= 8.1 Hz, 2H, Ar-H), 10.22, 10.57 and 10.71 (s, 1H, D O exchangable, NH), 10.40, 10.49 and 10.83
2
1
3
(
s, 1H, D
2
O exchangable, NH); C NMR (DMSO-d
17.26, 119.77, 126.50, 128.43, 128.75, 129.58, 129.81, 130.69, 138.47, 138.64, 140.60, 142.16,
53.56, 153.99, 155.22, 155.45, 163.27, 165.17; Anal. calcd. For C H N O S (450.47): C, 58.66;
6
, 75 MHz) δ: 55.00, 112.19, 116.04, 117.08,
1
1
2
2
18
4
5
H, 4.03; N, 12.44; Found C, 58.86; H, 4.06; N, 12.37.
4
.1.4. 4-(3-(4-Aminophenyl)ureido)benzenesulfonamide (9)
The compound 9 was prepared from 8 in a manner similar to that described for 4 to yield a white
-1
solid (81%): m.p. > 280 °C; IR (KBr, ν cm ): 3411, 3384, 3293 (NH, NH ), 1630 (C=O) 1310, 1146
2
1
(
SO ); H NMR (DMSO-d )
δ
ppm: 4.80 (s, 2H, D O exchangable, NH ), 6.51 (d, 2H, J= 8.7 Hz,
2
6
2
2
Ar-H); 7.07 (d, 2H, J= 8.7 Hz, Ar-H); 7.13 (s, 2H, D
2
2 2
O exchangable, SO NH ),7.55 (d, 2H, J= 8.7
Hz, Ar-H); 7.68 (d, 2H, J= 8.7 Hz, Ar-H); 8.23 (s, 1H, D O exchangable, NH), 8.84 (s, 1H, D O
2
2
14 4 3
exchangable, NH); Anal. calcd. For C13H N O S (306.08): C, 50.97; H, 4.61; N, 18.29. Found C,
5
1.08; H, 4.74; N, 18.45.
4
.1.5. General procedure
for
preparation
of
target
4-(3-(4-((2-oxoindolin-3-
ylidene)amino)phenyl)ureido)benzenesulfonamides 10a-g.
A mixture of 4-(3-(4-aminophenyl)ureido)benzenesulfonamide 9 (1 mmol) and the appropriate
isatin 5a-g (1 mmol) in glacial acetic acid (10 mL) was heated under reflux for 2 h, filtered while hot
and the precipitate was washed with ethanol. The solid product was collected and crystallized from
an ethanol/DMF to furnish compounds 10a-g with 57-73% yield.
4
.1.5.1. 4-(3-(4-(5-Bromo-2-oxoindolin-3-ylideneamino)phenyl)ureido)benzenesulfonamide (10d):
-1
2
Orange powder (yield 73%); m.p. > 280 °C; IR (KBr, ν cm ): 3123-3400 broad band (NH, NH ),
1
1
724, 1612 (2C=O),1307, 1150 (SO ); H NMR (DMSO-d )
δ
(ppm): 6.56-7.24 (m, 6H, 4H Ar-H
2
6
and 2H SO NH ), 7.44-7.76 (m, 7H, Ar-H), 8.91 and 8.98 (s, 1H, D O exchangeable, NH urea),
2
2
2
9
.10, 9.12 (s, 1H, D
2
2
O exchangeable, NH urea), 10.98 and 11.10 (s, 1H, D O exchangeable, NH
1
3
isatin); C NMR (DMSO-d , 75 MHz)
δ
: 112.84, 113.41, 117.41, 118.76, 119.12, 124.13, 126.77,
36.20, 136.86, 137.30, 137.77, 142.75, 143.95, 145.90, 152.14, 153.34, 163.15; Anal. calcd. For
S (514.35): C, 49.04; H, 3.14; N, 13.62; Found C, 48.98; H, 3.11; N, 13.69.
6
1
21 5 4
C H16BrN O
4
.1.5.2. 4-(3-(4-(5-Nitro-2-oxoindolin-3-ylideneamino)phenyl)ureido)benzenesulfonamide (10e):
-1
2
Yellow powder (yield 69%); m.p. > 280 °C; IR (KBr, ν cm ): 3200-3404 broad band (NH, NH ),
1
1
728, 1615 (2C=O), 1304, 1147 (SO ); H NMR (DMSO-d )
δ
(ppm): 7.05-7.12 (m, 2H, Ar-H),
2
6
1
1

ACCEPTED MANUSCRIPT
7
.20 (s, 2H, D O exchangeable, SO NH ), 7.34 (d, J =8.1 Hz, 1H, Ar-H), 7.48 (d, J =8.4 Hz, 1H, Ar-
2
2
2
H), 7.62-7.65 (m, 4H, Ar-H), 7.73 (d, J = 8.7 Hz, 2H, Ar-H), 8.27 (d, J = 8.7 Hz, 1H, Ar-H), 8.96
and 9.03 (s, 1H, D O exchangeable, NH urea), 9.12 (s, 1H, D O exchangeable, NH urea), 11.53 and
2
2
13
1
1.65 (s, 1H, D O exchangeable, NH isatin); C NMR (DMSO-d , 75 MHz) δ: 115.65, 117.12,
2 6
1
19.06, 120.10, 126.77, 128.98, 129.94, 136.90, 137.82, 138.48, 141.50, 142.71, 143.33, 150.09,
52.13, 163.91, 189.22; Anal. calcd. For C H N O S (480.45): C, 52.50; H, 3.36; N, 17.49; Found
1
2
1
16
6
6
C, 52.76; H, 3.41; N, 17.41.
4
.1.5.3. 4-(3-(4-(5-Methyl-2-oxoindolin-3-ylideneamino)phenyl)ureido)benzenesulfonamide (10f):
-
1
Biege powder (yield 57%); m.p. > 280 °C; IR (KBr, ν cm ): 3192-3415 broad band (NH, NH
2
),
),
1
1
685, 1614 (2C=O), 1310, 1146 (SO
2
); H NMR (DMSO-d
6
)
δ
(ppm): 2.01 and 2.29 (s, 3H, CH
3
6
.54-7.14 (m, 5H, Ar-H), 7.24 (s, 2H, D O exchangeable, SO NH ), 7.58-7.75 (m, 6H, Ar-H), 8.87
2 2 2
and 8.93 (s, 1H, D
2
O exchangeable, NH urea), 9.08 and 9.12 (s, 1H, D
2
O exchangeable, NH urea),
13
1
0.74 and 10.85 (s, 1H, D
2
O exchangeable, NH isatin); C NMR (DMSO-d , 75 MHz) : 21.03,
6
δ
1
11.75, 115.28, 116.29, 117.60, 119.34, 121.32, 122.10, 125.83, 127.25, 131.72, 135.14, 137.28,
43.19, 144.92, 152.57, 154.99, 164.20 ;Anal. calcd. For C H N O S (449.48): C, 58.79; H, 4.26;
1
2
2
19
5
4
N, 15.58; Found C, 58.71; H, 4.30; N, 15.66.
4
.1.5.4.
4-(3-(4-(5-Methoxy-2-oxoindolin-3-ylideneamino)phenyl)ureido)benzenesulfonamide
-1
(
10g): Red powder (yield 65%); m.p. > 280 °C; IR (KBr, ν cm ): 3270-3489 broad band (NH, NH ),
2
1
1
6
8
1
1
1
1
721, 1688 (2C=O), 1302, 1149 (SO ); H NMR (DMSO-d )
δ
(ppm): 3.49 and 3.77 (s, 3H, OCH3),
2
6
.18-7.17 (m, 5H, Ar-H), 7.21 (s, 2H, D O exchangeable, SO NH ), 7.42-7.76 (m, 6H, Ar-H), 8.86 and
2
2
2
.92 (s, 1H, D O exchangeable, NH urea), 9.09, 9.10 (s, 1H, D O exchangeable, NH urea), 10.65 and
2
2
1
3
0.77 (s, 1H, D O exchangeable, NH isatin); C NMR (DMSO-d , 75 MHz) δ: 55.74, 56.11, 107.63,
2
6
11.97, 112.10, 112.41, 114.65, 116.69, 117.57, 117.92, 117.97, 118.63, 119.15, 119.42, 119.93,
20.49, 121.43, 122.15, 123.14, 127.25, 127.30, 136.77, 137.33, 137.40, 137.64, 139.34, 140.78,
43.05, 143.30, 143.77, 144.98, 152.70, 152.80, 153.01, 154.42, 155.53, 155.62, 159.34, 164.20; Anal.
19 5 5
calcd. For C22H N O S (465.48): C, 56.77; H, 4.11; N, 15.05; Found C, 56.86; H, 4.09; N, 14.93.
4
.2 CA inhibitory assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed
CO hydration activity [42]. The enzymes are recombinant proteins prepared in our lab. Phenol red
2
1
2

ACCEPTED MANUSCRIPT
(at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of
5
2 4
57 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na SO (for maintaining constant the
ionic strength), following the initial rates of the CA-catalyzed CO hydration reaction for a period of
2
1
2
0-100 s. The CO concentrations ranged from 1.7 to 17 mM for the determination of the kinetic
parameters and inhibition constants. For each inhibitor at least six traces of the initial 5-10% of the
reaction have been used for determining the initial velocity. The uncatalyzed rates were determined
in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1
mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done thereafter
with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room
temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition
constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff
equation, as reported earlier [43-36], and represent the mean from at least three different
determinations. All CA isofoms were recombinant ones obtained in-house as reported earlier [43-
4
6].
Acknowledgements
The authors would like to extend their sincere appreciation to the Deanship of Scientific
Research at King Saud University for its funding of this research through the Research Group
Project no. RGP-VPP-321. Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Egyptian Russian University, Cairo, Egypt, is highly appreciated for supporting this research.
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Captions page
Figure 1. Structures of the reported carbonic anhydrase inhibitors I-V and the target conjugates 6a-g
and 10a-g.
Figure 2. Cis/Trans conformers around amide function and E/Z geometrical isomers around
-C=N- of compounds 6a-g.
2
Scheme 1. Reagents and conditions: (i) THF / TEA / r.t. 5h; (ii) H / Pd/C / MeOH / r.t. 3h;
(iii) Glacial acetic acid / reflux 5h.
3 2
Scheme 2. Reagents and conditions: (i) CH CN, reflux 2h; (ii) H / Pd/C / MeOH / r.t. 3h;
(iii) Glacial acetic acid / reflux 2h.
Table 1. Inhibition data of human CA isoforms hCA I, II, IX and XII with sulfonamides 4, 9, 6a-g
and 10a-g reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a
stopped flow CO hydrase assay.
2
1
9

ACCEPTED MANUSCRIPT
Table 1: Inhibition data of human CA isoforms hCA I, II, IX and XII with sulfonamides 4, 9, 6a-
g and 10a-g reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a
stopped flow CO hydrase assay.
2
K (nM)
I
Compound R
hCAI
78.4
94.9
95.6
9.0
hCA II
28.1
15.7
116
hCA IX
159
hCA XII
2.7
4
9
6
6
6
6
6
6
6
1
1
1
1
1
1
1
-
-
171
0.71
a
H
240
1.76
b
F
7.7
5.0
0.69
c
Cl
Br
NO
Me
9.2
8.4
194
208
237
201
175
217
235
239
192
14.8
16.7
8.5
0.54
0.58
0.69
0.63
0.47
2.32
2.83
0.67
2.31
0.64
1.17
1.62
5.7
d
7.9
7.5
e
2
10.2
8.5
9.9
f
9.1
g
MeO
H
29.3
679
894
85.9
462
62.8
45.2
387
250
14.5
157
585
1645
>10000
877
33.0
690
12
0a
0b
0c
0d
0e
0f
0g
F
Cl
Br
NO
Me
2
MeO
-
AAZ
25
*
%
Mean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5-10
of the reported values).

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Figure 1. Structures of the reported carbonic anhydrase inhibitors I-V and the target conjugates
6
a-g and 10a-g.

ACCEPTED MANUSCRIPT
R
O O
O
S
NH2
O O
S
O
N
N
Z
HN
R
H
NH2
N
E N
O
O
H
N
H
Trans, Z
Trans, E
R
O
O
H
HN
N
H
N
Z
N
E N
R
O
O
N
H
S O
O
O
S
H N
2
H N
2
O
Cis, Z
Cis, E
Figure 2. Cis/Trans conformers around amide function and E/Z geometrical isomers around
C=N- of compounds 6a-g.
-

ACCEPTED MANUSCRIPT
O
O
S
NH2
O
O
O
O
O
O
i
H N
2
S
N
+
N
H
NH2
Cl
O
2
N
O
3
1
ii
O
O O
S
R
O
O
O
O
S
NH2
NH2
O
N
N
N
H
H
R
5
a-g
N
H
O
iii
4
N
6a-g
H2N
H
5
, 6; a = H, b = F, c = Cl, d = Br, e = NO , f = CH , g = OCH
2 3
3
Scheme 1. Reagents and conditions: (i) THF / TEA / r.t. 5h; (ii) H / Pd/C / MeOH / r.t. 3h;
2
(iii) Glacial acetic acid / reflux 5h.

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Scheme 2. Reagents and conditions: (i) CH CN, reflux 2h; (ii) H / Pd/C / MeOH / r.t. 3h;
3
2
(iii) Glacial acetic acid / reflux 2h.

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Highlights
-
-
-
-
Different sulfonamides incorporating amido or ureido linkers were synthesized.
Inhibitory activity of these sulfonamides was evaluated toward hCA I, II, IX and XII.
9, 6b-g, 10c and 10e displayed outstanding potency against CA XII (K : 0.47–0.71 nM).
I
Other derivatives emerged as single-digit nanomolar CA XII inhibitors (1.17–2.83 nM).