Novel sulfonamides bearing pyrrole and pyrrolopyrimidine moieties as carbonic anhydrase inhibitors: Synthesis, cytotoxic activity and molecular modeling

Article abstract of DOI:10.1016/j.ejmech.2014.09.059

Novel pyrrole and pyrrolopyrimidine scaffold-based sulfonamides were designed and synthesized. The carbonic anhydrase (CA) inhibition ability of all derivatives was assessed against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumo

Full text of DOI:10.1016/j.ejmech.2014.09.059

European Journal of Medicinal Chemistry 87 (2014) 186e196
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel sulfonamides bearing pyrrole and pyrrolopyrimidine moieties
as carbonic anhydrase inhibitors: Synthesis, cytotoxic activity and
molecular modeling
, b,
Mostafa M. Ghorab a *, Mariangela Ceruso c, Mansour S. Alsaid a, Yassin M. Nissan d,
, e, **
Reem K. Arafa d, Claudiu T. Supuran c
a Pharmacognosy Department, College of Pharmacy, King Saud University, P.O.Box 2457, Riyadh 11451, Saudi Arabia
b Drug Radiation Research Department, National Center for Radiation Research & Technology, Atomic Energy Authority, Cairo, Egypt
c
ꢀ
Universita degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Firenze, Italy
d Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr Al-Aini st., P.O.Box 11562, Cairo, Egypt
e
ꢀ
Universita degli Studi di Firenze, Polo Scientifico, Dipartimento NEUROFABA, Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019
Sesto Fiorentino, Firenze, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel pyrrole and pyrrolopyrimidine scaffold-based sulfonamides were designed and synthesized. The
carbonic anhydrase (CA) inhibition ability of all derivatives was assessed against the human (h) cytosolic
isoforms hCA I and II and the transmembrane, tumor-associated isoforms hCA IX and XII. Some of these
sulfonamides were 6e8 fold more potent than the reference drug acetazolamide (AZA, Ki ¼ 5.7 nM))
against hCA XII showing subnanomolar activity. The in vitro cytotoxicity of these derivatives was eval-
Received 10 March 2014
Received in revised form
15 September 2014
Accepted 17 September 2014
Available online 19 September 2014
uated against MCF-7, where some derivatives were more cytotoxic than doxorubicin (IC50 ¼ 8.02
displaying IC50 values between 6.46 and 7.56 M. Docking of these sulfonamides with CA XII was per-
formed and their binding modes were comparable with that of AZA.
mM)
m
Keywords:
Pyrroles
Pyrrolopyrimidines
Sulfonamides
Carbonic anhydrase
Cytotoxic activity
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
selectivity of conventional chemotherapeutic agents for cancer
tissues, causing unwanted side effects. The second is the acquisition
of multiple-drug resistance by cancer cells, rendering them unre-
sponsive to conventional chemotherapeutic agents. Unwanted side
effects of antitumor drugs could be overcome with agents capable
of discriminating tumor cells from normal proliferative cells and
the resistance is minimized using combined modality approach
with different complementary mechanisms of action [4]. Pyr-
rolo(2,3-d)pyrimidines are purine analogs that display remarkable
biological activities, such as ant-inflammatory [5], anticancer
[6e10], antimicrobials [11,12], and antiviral [13e15]. Also, natural
products and synthetic drugs displaying a wide range of biological
activities were found to contain a pyrrole moiety as their key
skeleton [16,17], subsequently substituted pyrroles have become
one of the major targets in synthetic chemistry. On the other hand,
sulfonamides and their different derivatives are extensively used in
medicine due to their bioactivity as antibacterial agents and di-
uretics [11,12,18]. In addition, sulfonamides have been reported for
potent antitumor activity in vitro and in vivo against numerous
types of cancers [19,20]. A number of mechanisms of action were
Cancer is a disease of striking impact on the people's health in
the world today. It is the second leading cause of death in the world
after cardiovascular diseases and it is projected to be the primary
cause of death within the coming years [1,2]. The discovery of novel
small molecules with potential usefulness as potent, selective and
less toxic anticancer agents is still a major challenge to medicinal
chemistry researchers [3]. Despite the important advances ach-
ieved over recent decades in the research and development of
various cancerostatic drugs, current antitumor chemotherapy still
suffers from two major limitations, the first being the lack of
* Corresponding author. Department of Pharmacognosy, College of Pharmacy,
King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia.
** Corresponding author. Universita degli Studi di Firenze, Laboratorio di Chimica
Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Firenze,
ꢀ
Italy.
E-mail addresses: mmsghorab@yahoo.com (M.M. Ghorab), claudiu.supuran@
unifi.it (C.T. Supuran).
http://dx.doi.org/10.1016/j.ejmech.2014.09.059
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

M.M. Ghorab et al. / European Journal of Medicinal Chemistry 87 (2014) 186e196
187
Scheme 1. Synthesis of sulfonamide derivatives 5e13.
reported to be associated with the anticancer ability of sulfon-
2. Results and discussion
amides, the most prominent being their power to act as carbonic
anhydrase inhibitors (CAIs) [21]. Carbonic anhydrases (CA, EC
4.2.1.1) are a group of metalloenzymes that catalyze the hydration
of carbon dioxide (cellular waste product) to bicarbonate and a
proton [22e27]. This family of enzymes comprises five main clas-
2.1. Chemistry
The synthesis of the sulfonamide derivatives in this study is
depicted in Schemes 1e3. The synthesis of derivatives 5e13 is
displayed in Scheme 1. Reaction of sulfanilamide 1 with 2-bromo-
1-(4-bromophenyl)ethanone 2 yielded the corresponding 4-(2-(4-
bromophenyl)-2-oxoethylamino)benzenesulfonamide 3, which
upon reaction with malononitrile in absolute ethanol in presence of
sodium ethoxide afforded the pyrrole derivative 5 via the formation
of intermediate 4, followed by intramolecular cyclization. The
structure of compound 3 was verified by elemental analysis and
spectral data. The IR spectrum of compound 3 showed the presence
of the characteristic bands for the NH, NH2, C]O and SO2 groups.
Also, the 1H NMR spectrum indicated the presence of a singlet at
4.7 ppm, which could be assigned to the CH2 group. The IR spec-
trum of compound 5 exhibited bands for the NH2, C^N and SO2
functions. In addition, the 1H NMR spectrum of compound 5
revealed two D2O exchangeable signals, one at 6.1 ppm assigned to
the NH2 group and another at 7.9 ppm for the SO2NH2 group.
ses, viz.
a,
b
,
g,
d
and
z. Among the 16 known CA isoforms belonging
to class
a, the human cytosolic isoforms hCA I and hCA II are
ubiquitous in the body and are targets for anticonvulsant, diuretic
and anti-glucoma drugs. In addition, the transmembrane isoforms
hCA IX and hCA XII, possessing an extracellular active site, were
found to be associated with some types of cancers being induced by
tumor hypoxia. This being the case, these two isoforms justifiably
represent good candidates for antitumor drug design [23,28,29]. In
lights of the previous facts and based on our previous work in the
field of design and synthesis of sulfonamide based molecules as
cytotoxic agents [30e33], we herein report the results achieved
from the design and synthesis of novel pyrrole and pyrrolo(2,3-d)
pyrimidine derivatives carrying biologically active sulfonamide
moiety. All the newly synthesized compounds were subjected to
cytotoxic screening against the breast cancer cell line MCF-7.
Assessment of the in vitro enzyme-inhibitory capacity of these
novel sulfonamides against hCA I, II, IX and XII was performed. The
observed activity of these derivatives was compared with the well-
known CAI acetazolamide (AZA). Finally, molecular docking of all
compounds in the active site of CA XII was carried out in an attempt
to speculate the possible binding mode of these molecules in the
active site of the target enzyme.
The reaction of compound
5 with isothiocyanates under
different conditions was studied. Thus, reaction of compound 5
with various aryl isothiocyanates in refluxing absolute ethanol gave
the corresponding thiourea derivatives 6e12. On the other hand,
treatment of 5 with 1-isothiocyanato-4-methylbenzene in pyridine
furnished the corresponding pyrrolopyrimidine derivative 13. The
structures of compounds 6e13 were established through the IR
spectral data. While IR spectra of sulfonamides 6e13 showed the
presence of
a characteristic C]S band in the range of
1230e1271 cmꢀ1, only compounds 6e12 showed the presence of a

188
M.M. Ghorab et al. / European Journal of Medicinal Chemistry 87 (2014) 186e196
Scheme 2. Synthesis of sulfonamide derivatives 14, 15, 17, 18 and 21.
band corresponding to C^N which was absent in the case of de-
rivative 13.
disulfide in pyridine for 24 h (Scheme 2). The reaction proceeds via
the formation of intermediates 19 and 20 (Dimroth rearrangement)
[34]. On the other hand, Scheme 3 outlines the synthesis strategy
adopted for preparation of sulfonamides 22, 24e26, 30 and 31.
Compounds 22 and 24 were obtained via reaction of compound 5
with thioacetamide in trifluoroacetic acid. Compound 24 was
separated from the reaction by filtration while hot, and compound
22 was obtained from the filtrate after cooling of the mother liquor.
It is believed that a fraction of 22 underwent intramolecular
cyclization forming the intermediate 23 and elimination of one
molecule of ammonia that was followed by Dimroth rearrangement
[34] to furnish compound 24. The IR spectrum of 24 exhibited the
Scheme 2 outlines the synthesis protocol followed to obtain
sulfonamides 14, 15, 17, 18 and 21. First, 2-Arylsulfonylamino pyr-
role derivatives 14 and 15 were obtained by refluxing compound 5
with benzene sulfonyl chloride or toluene sulfonyl chloride in dry
benzene containing a few drops of pyridine. The structures of
compounds 14 and 15 were confirmed by the IR, 1H NMR and 13C
NMR spectra. The IR spectra revealed the presence of characteristic
bands for the C^N group at 2218 and 2210 cmꢀ1 for derivatives 14
and 15, respectively. The 1H NMR spectra of 14, 15 showed a singlet
at 12.0, 11.9 ppm assigned to the SO2NH proton of both compounds,
respectively.
Furthermore, fusion of compound 5 with urea or thiourea fur-
nished the condensed pyrrolopyrimidines 17 and 18, respectively.
Products 17 and 18 were formed via the formation of intermediate
16 through the loss of an ammonia molecule, followed by an
intramolecular addition to the cyano function to give the final
isolated products 17 and 18. The IR spectra of 17 and 18 exhibited
the disappearance of a band characteristic of the carbonitrile
functional group and the presence of bands in the range of
3354e3396 cmꢀ1 for the NH and NH2 groups. The target ring sys-
tem 21 was synthesized by reaction of compound 5 with carbon
absence of C^N band and presence of the C]S group at 1219 cmꢀ1
.
The 1H NMR spectrum of 24 revealed a singlet at 2.4 ppm due to
methyl group. In addition, the pyrrole derivative 25 was obtained
by the reaction of compound 5 with ethyl acetoacetate under
conditions of fusion. The IR spectrum of compound 25 revealed the
presence of bands characteristic for the C^N at 2200 cmꢀ1 and two
C]O functions at 1730 and 1690 cmꢀ1 1H NMR spectrum of 25
.
exhibited a singlet peak at 4.1 ppm corresponding to the CH2CO
group. Finally, the reaction of compound 5 with 4-bromobenzoyl
chloride in pyridine afforded the benzoyl derivative 26, while
fusion of compound 5 with benzoyl chloride or 4-chlorobenzoyl

M.M. Ghorab et al. / European Journal of Medicinal Chemistry 87 (2014) 186e196
189
Scheme 3. Synthesis of sulfonamide derivatives 22, 24e26, 30 and 31.
chloride only gave the cyclic compounds pyrrolopyrimidines 30
and 31, respectively. The mechanistic pathway to obtain 30 and 31
is suggested to proceed initially through the formation of the in-
termediate 27 followed by its intramolecular cyclization to furnish
the oxazine intermediate 28. Spontaneous Dimroth rearrangement
of 28 would give the intermediate pyrrolopyrimidine 29. Further,
reaction of 29 with a second molecule of benzoyl chloride would
eventually yield compounds 30 and 31 (Scheme 3). The structure of
the sulfonamide 26 was proved on the basis of its IR spectrum
which showed the presence of a C^N band at 2187 cmꢀ1 and a C]
O band at 1676 cmꢀ1. The structures of compounds 30 and 31 were
supported on the basis of their elemental analyses, IR, 1H NMR and
13C NMR spectra. The IR spectra of sulfonamides 30 and 31 revealed
the absence of C^N bands and presence of two C]O bands for each
compound in the range 1764 of 1691 cmꢀ1
.
2.2. Carbonic anhydrase inhibition
The CA inhibitory ability of the synthesized sulfonamides was
assessed against the cytosolic isoforms hCA I and II as well as the
membrane associated isoforms hCA IX and XII employing stopped
flow assay method and results are displayed in Table 1. The well-
known carbonic anhydrase inhibitor acetazolamide (AZA) was
used as the reference drug for this assay. Data obtained from the CA
inhibition assay led to the following inferences.

190
M.M. Ghorab et al. / European Journal of Medicinal Chemistry 87 (2014) 186e196
Table 1
calculated to measure their differential inhibitory activity towards
hCA I, II, IX and XII inhibition results.
isoforms hCA I/XII and hCA II/XII (Table 1). All the sulfonamides
derivatives displaying subnanomolar Ki values in this study showed
excellent selectivity profiles with SI values in the range of
10.95e2750 for the isoforms hCA I/XII and 9.89 to 866.18 for iso-
forms hCA II/XII. On the other hand, all other compounds that were
half as potent as or equipotent with AZA didn't display good
selectivity profile for the two isoforms hCA II and hCA XII as re-
flected in their SI values. However, the later derivatives exhibited
high hCA I/XII SI values of above 17, and compound 12 showing
exceptional high selectivity of >1204.81.
Compound Kia (nM)
SIb
hCA I
hCA II
hCA IX hCA XII hCA I/XII
hCA II/XII
5
6
7
8
84.7
198
377
479
414
483
887
283
812
1870
704
60.7
235
151
40.7
214
147
218
4.6
8.3
8.9
41.8
17.2
9.4
150
8.5
6.3
62.4
141
0.74
19.3
5.6
114.46
10.26
67.32
88.70
431.25
508.42
1095.06
422.39
1176.81
2750.00
8.88
1.25
0.68
2.50
1.42
23.52
17.09
22.95
>1204.81 1.12
>666.66
6.22
0.43
1.59
7.74
17.92
9.89
185.19
12.69
12.75
866.18
2.42
4.23
0.26
0.71
0.20
90.3
86.6
91.1
460
51.2
162
849
171
24.4
42.9
7.1
6.0
5.4
5.5
5.3
6.5
5.4
9
0.96
0.95
0.81
0.67
0.69
0.68
79.3
48.5
348
60.4
28.6
9.1
8.6
9.5
10
11
12
13
14
15
17
18
21
22
24
25
26
30
31
AZA
8.8
589
192
205
89.3
43.0
5.8
4.5
5.2
4.5
2.3. In vitro anticancer activity
The in vitro anticancer activity of the newly synthesized com-
pounds was evaluated against the breast cancer cell line MCF-7
employing Doxorubicin as the positive standard drug (results are
listed in Table 2). Most of the synthesized compounds showed
better or comparable activity to that of doxorubicin as a reference
drug. The o-aminocarbonitrile pyrrole derivative 5 showed IC50 of
0.49
0.60
0.47
>10,000 9.3
>10,000 >10,000 488
250 12.1 25.0
8.3
15.0
5.7
>666.66
2.12
43.86
7.56
of 8.02
thiocyanates to obtain the aryl thiourea derivatives 6e12, the ac-
tivity was enhanced in case of the phenyl derivative 6 (IC50 7.29 M)
and the p-methoxy derivative 7 (IC50 7.01 M). However, the ac-
m
M, which was better than that of Doxorubicin with IC50 value
m
M. Upon treatment of compound 5 with several aryl iso-
AZA (Acetazolamide), a well-known CAI, was used as a standard for comparison.
a
Ki presented is the mean from 3 different assays; errors are in the range of
5e10% of the reported values (data not shown).
m
b
SI (Selectivity index) is a ratio between the Ki values observed for two hCA
m
isoforms; low value index is indicative of weak selectivity.
tivity slightly decreased with the p-chloro (8), p-nitro (9), p-bromo
(10), p-iodo (11) and p-sulfamoyl (12) derivatives (IC50 values be-
tween 8.11 and 8.15 mM). This indicated that para-substitution with
(i) The cytosolic isoform hCA I was moderately to poorly
inhibited by the sulfonamide derivatives with Ki values in the
range of 40.7e1870 nM with the exception of derivatives 30
and 31 which were totally inactive displaying Ki values
>10,000 nM. The most active compounds against hCA I were
22, 17, 5, 25, 21, 6, 24, 26 and 18 arranged in descending order
of potency. It is noteworthy that all these derivatives were
more active than AZA.
(ii) With regards to the profiling assay against hCA II, eleven
compounds were found to be more potent inhibitors than
AZA (5e7, 10, 12, 13, 22, 24e26 and 30), eight compounds
were less active than AZA exhibiting moderate hCA II
inhibitory activity (8, 9, 11, 14, 15, 17, 18 and 21) and only 31
was totally inactive (Ki 10,000 nM).
electron withdrawing groups was not in favor of the cytotoxic ac-
tivity. On the other hand, the iminopyrrolopyrimidine derivative 13
displayed significantly improved cytotoxic activity being the most
active compound in this series with IC50 of 6.46
the formation of sulfonamidocarbonitrile pyrrole derivatives 14
and 15 (IC50 values of 7.01 and 7.56 M, respectively) enhanced the
mM. Furthermore,
m
cytotoxic activity giving more potent compounds than the refer-
ence drug doxorubicin. It is noteworthy that preparation of the
aminopyrrolopyrimidine derivatives 17 and 18 has markedly
Table 2
In vitro anticancer screening of the synthesized compounds against human breast
cancer cell line (MCF7).
(iii) The tumor associated target isoform hCA IX was highly
inhibited by sulfonamides 26, 24, 25, 22, 5, 30 and 17 (ar-
ranged in a descending manner with respect to inhibition
efficiency) showing Ki values lower than that of AZA. These
derivatives were 5 fold more potent to equipotent when
compared to AZA. On the other hand, the other derivatives
were moderate to very weak hCA III inhibitors.
(iv) Finally, excellent results were displayed by seven of the novel
sulfonamides (5, 9e14) in the profiling assay performed
against the transmembrane, tumor associated isozyme hCA
XII. These derivatives displayed potency at the subnanomolar
level being 6e8.5 fold more active than AZA against hCA XII.
On the other hand, while 7 and 8 were almost equipotent
with AZA, the counterparts 24e26 and 30 were almost half
as active as AZA. The remaining analogs were moderate to
very weak hCA XII inhibitors.
Compound Compound concentration (
10 25
Surviving fraction (mean S.E.M.)a
m
M)
IC50
M)
(
m
m
M
m
M
50
mM
100 mM
5
6
7
8
0.327 0.121 0.273 0.043 0.233 0.011 0.255 0.020 7.56
0.308 0.001 0.235 0.011 0.227 0.114 0.217 0.112 7.29
0.262 0.011 0.263 0.008 0.249 0.003 0.217 0.012 7.01
0.288 0.100 0.257 0.002 0.260 0.021 0.290 0.031 8.12
0.280 0.007 0.276 0.044 0.255 0.022 0.260 0.021 8.11
0.298 0.100 0.280 0.181 0.277 0.024 0.290 0.051 8.15
0.312 0.041 0.290 0.162 0.281 0.011 0.288 0.042 8.15
0.311 0.001 0.291 0.033 0.255 0.051 0.276 0.064 8.13
0.250 0.077 0.242 0.021 0.229 0.111 0.245 0.133 6.46
0.272 0.041 0.290 0.022 0.202 0.001 0.232 0.035 7.01
0.324 0.011 0.271 0.052 0.222 0.011 0.214 0.044 7.56
0.270 0.002 0.276 0.004 0.203 0.015 0.238 0.072 6.74
0.231 0.055 0.255 0.014 0.199 0.020 0.250 0.030 6.46
0.337 0.021 0.294 0.049 0.219 0.031 0.225 0.011 7.56
0.330 0.052 0.312 0.014 0.300 0.022 0.299 0.019 8.11
0.236 0.052 0.218 0.031 0.198 0.068 0.231 0.002 6.74
0.290 0.044 0.282 0.001 0.280 0.069 0.291 0.022 8.11
0.358 0.061 0.327 0.032 0.249 0.045 0.254 0.004 7.56
0.366 0.022 0.318 0.017 0.284 0.052 0.268 0.034 8.11
0.549 0.011 0.293 0.083 0.201 0.039 0.295 0.001 13.10
9
10
11
12
13
14
15
17
18
21
22
24
25
26
30
31
It is noteworthy that the most active compounds against the
tumor associated hCA XII isoform (5, 9e14) were less active against
isoforms hCA I and hCA II, an attribute that is desirable for cytotoxic
agents to guard against the occurrence of untoward side effects. For
that, the selectivity index (SI) of the tested compounds was
Doxorubicin 0.314 0.032 0.309 0.016 0.251 0.023 0.266 0.032 8.02
a
Each value is the mean of three values standard error.

M.M. Ghorab et al. / European Journal of Medicinal Chemistry 87 (2014) 186e196
191
improved the activity of these sulfonamides (IC50
6.46 M, respectively). On the other hand, the dithioxo derivative
21 displayed similar potency (IC50 ¼ 7.56 M) compared with the
starting material 5. With regards to the pyrrole derivatives 22, 25
and 26, the activity ranged between 7.56 and 8.11 M. However,
cyclization of 22 to the pyrrolopyrimidine derivative 24 had again
increased the activity to 6.74 M. Finally, regarding the 2-phenyl-3-
s
6.74 and
hCA XII inhibitors in this study 5 and 9e14 are listed in Table 3. The
rest of results can be found in the Supplementary material.
The docking scores for the newly synthesized compounds fall in
the range of (ꢀ11.44 to ꢀ36.52 kcal/mol). The best docking score
was assigned to compound 12 (ꢀ36.52 kcal/mol). On the other
hand, all of the newly synthesized compounds were capable of
interaction with the Zn atom, while coordinating with it by means
of the nitrogen atom of the NH2 group, in a similar manner to the
co-crystallized ligand (AZA). It was observed from the amino acid
interactions of the newly synthesized compound that interactions
with Thr 199 and Thr 200 were achieved only by compounds 5 and
9e14 and in a way comparable to that of AZA while, the rest of the
compounds, which displayed low activity in the hCA XII inhibition
assay failed to show interactions with these two amino acids in the
docking study.
¼
m
m
m
m
benzoylpyrrolopyrimidine derivatives, while the derivative
without substitution on both phenyl rings 30 showed nearly
equipotent activity with doxorubicin (IC50 ¼ 8.11
phenyl counterpart 31 exhibited much reduced activity
(IC50 ¼ 13.10 M).
mM) the p-chloro
a
m
2.4. Molecular modeling and docking results
In a further look to docking results, we can observe that the
most active compounds (5, 9e14) have shown excellent docking
scores (ꢀ35.27, e34.20, e31.06, e31.77, e36.52, e34.64
and ꢀ35.99, respectively), in addition to their interactions with the
Thr 199 and Thr 200 by at least one hydrogen bond, which can give
us a simulating illustration of their interactions with the active site
of the hCA XII. The amino acid interactions of the most active
compounds (12, 13 and 14) are illustrated in Figs. 2e4, respectively.
All the synthesized compounds reported here revealed prom-
ising CA inhibitory activity especially against hCA XII. Thus, it was
interesting to perform molecular docking within the active site of
this isozyme to study the differences in docking patterns and amino
acid interactions for the newly synthesized compounds. The pro-
tein data bank file (PDB ID: 1JD0) was selected for this purpose. The
file contains hCA XII co-crystallized with acetazolamide. All dock-
ing procedures were achieved by MOE (Molecular Operating
Environment) software 10.2008 provided by chemical computing
group, Canada. Docking on the active site of hCA XII was performed
for all synthesized sulfonamide derivatives 5e15, 17, 18, 21, 22,
24e26, 30 and 31. The docking protocol was verified by re-docking
of the co-crystallized ligand (acetazolamide) in the vicinity of the
active site of the enzyme with energy score (S) ¼ ꢀ28.90 kcal/mol
and root mean standard deviation (RMSD) ¼ 1.47. Acetazolamide
interacted with the active site of hCA XII by: two hydrogen bonds
between Thr 199 and the SO2 group and NH2; one hydrogen bond
between Thr 200 and N3 of the thidiazole ring and an interaction
between Zn and nitrogen atom of NH2 (coordination) (Fig. 1). All
synthesized compounds were docked with proper fitting to the
active site of hCA XII with good energy scores (S), supporting the
observed activity of these sulfonamides as hCA XII inhibitors. The
energy scores (S) and amino acid interactions of the most potent
3. Conclusion
A series of pyrrole and pyrrolo(2,3-d)pyrimidine derivatives
bearing a benzenesulfonamide motif were designed and synthe-
sized as potential carbonic anhydrase inhibitors (CAIs). The cyto-
toxic efficacy of these compounds was assessed against the human
breast cancer cell line MCF-7 and all sulfonamides were either more
potent than or equipotent to the positive control doxorubicin,
except compound 31. Also, the CA inhibitory activity of these
compounds was assessed against the human cytosolic isoforms
hCA I and hCA II and the transmembrane isoforms hCA IX and hCA
XII, which are over expressed in various types of tumors. The tested
sulfonamides showed variable degrees of activities against all four
evaluated hCA isoforms. Interestingly, sulfonamides 5 and 9e14
were 6e8 fold more potent than the well-known CAI acetazol-
amide (AZA) against hCA XII exhibiting subnanomolar Ki values.
These new sulfonamides also displayed high selectivity towards the
tumor associated isoform hCA XII as displayed by their hCA I/XII
and hCA II/XII SIs. The docking of all compounds was performed
with the hCA XII. The enzyme-ligand binding interactions of the
docked sulfonamides with the active site amino acids of hCA XII
were similar to those displayed by AZA, especially for compounds 5,
9e14. These results merit further future investigation of these new
sulfonamides as potential antitumor agents.
4. Experimental
4.1. Chemistry
Melting points (ꢁC, uncorrected) were determined in open
capillaries on a Gallenkamp melting point apparatus (Sanyo Gal-
lenkemp, Southborough, UK) and were uncorrected. Precoated TLC
plates, silica gel G F254 were used for thin layer chromatography
(Merck, Germany). Dichloromethane/methanol (9.5:0.5) mixture
was used as a developing solvent system for TLC and the spots were
visualized by ultraviolet light and/or iodine. The infra-red spectra
were recorded in KBr discs using an IR-470 Shimadzu spectrometer
(Shimadzu, Tokyo, Japan). The NMR spectra (in DMSO-d6) were
recorded on a Bruker AC-500 Ultra Shield NMR spectrometer
(Bruker, Flawil, Switzerland) at 500 MHz for the 1H NMR and
125 MHz for the 13C NMR using TMS as an internal Standard and
Fig. 1. 2D interaction of AZA inside the active site of hCA XII: (S) ¼ ꢀ28.89 kcal/mol
(RMSD) ¼ 1.47.

192
M.M. Ghorab et al. / European Journal of Medicinal Chemistry 87 (2014) 186e196
Table 3
4.1.1. 4-(2-(4-Bromophenyl)-2-oxoethylamino)benzenesulfonamide
3
Docking results of the synthesized sulfonamide derivatives with CA XII using MOE
software version 10.2008.
A mixture of sulfanilamide 1 (1.72 g, 0.01 mol) and 2-bromo-1-
(4-bromophenyl)ethanone 2 (2.77 g, 0.01 mol) was refluxed in
N,N0-dimethylformamide (20 mL) in the presence of catalytic
amount of triethylamine for 6 h. The solid obtained was filtered off
and recrystallized from ethanol to give 3. Yield: 89%; M.p.: 232.6 ꢁC;
IR (KBr, cmꢀ1): 3358, 3255 (NH, NH2), 3100 (CH arom.), 2970, 2863
(CH aliph.), 1685 (C]O), 1381, 1157 (SO2); 1H NMR (DMSO-d6, D2O,
Compound S (kcal/
mol)
Amino
acids
Interacting
groups
Type of
interaction
Length
(Å)
5
ꢀ35.27
Thr 199
Thr 199
NH2
SO2
H-bond (donor) 2.18
H-bond
(acceptor)
H-bond (donor) 2.88
2.74
His 94
His 94
NH2
SO2
H-bond
3.02
d): 4.7 (s, 2H, CH2), 6.6 (s, 1H, NH, D2O exchangeable), 6.7, 7.5 (2d,
(acceptor)
His 96
Lys 67
NH2
NH2
H-bond (donor) 3.35
Coordination
(Zn)
4H, AreH, AB system, J ¼ 7.1 Hz), 7.8, 8.0 (2d, 4H, AreH, AB system,
J ¼ 6.9 Hz); 13C NMR (DMSO-d6,
d): 49.4, 111.4 [2], 127.1, 127.7, 129.9
[2], 130.7 [2], 131.8 [2], 133.9, 150.8, 195.3; Anal. Calcd. for
9
ꢀ34.20
CN
H-bond
2.84
C14H13BrN2O3S (369.23): C, 45.54; H, 3.55; N, 7.59. Found: C, 45.54;
(acceptor)
Thr 199
Thr 199
NH2
SO2
H-bond (donor) 2.22
H, 3.31; N, 7.24.
H-bond
2.77
(acceptor)
His 94
NH2
NH2
H-bond (donor) 2.86
Coordination
(Zn)
10
11
ꢀ31.06
ꢀ31.77
Thr 199
Thr 199
NH2
SO2
H-bond (donor) 2.17
H-bond
2.86
(acceptor)
H-bond (donor) 2.80
Coordination
4.1.2. 4-(2-Amino-4-(4-bromophenyl)-3-cyano-1H-pyrrol-1-yl)
benzenesulfonamide 5
His 94
NH2
NH2
A mixture of compound 3 (3.69 g, 0.01 mol) and malononitrile
(0.66 g, 0.01 mol) in absolute ethanol (20 mL) containing sodium
ethoxide (0.5 g) was refluxed for 8 h. The reaction mixture was
cooled and acidified with dil. HCl. The solid obtained was filtered off
and recrystallized from dioxane to give 5. Yield: 78%; M.p.: 221.2 ꢁC;
IR (KBr, cmꢀ1): 3419, 3344, 3238 (NH2), 3095 (CH arom.), 2187
(Zn)
Thr 199
Thr 199
NH2
SO2
H-bond (donor) 2.17
H-bond
2.78
(acceptor)
H-bond (donor) 2.88
His 94
His 94
NH2
SO2
H-bond
3.00
(acceptor)
(C^N), 1342, 1176 (SO2). 1H NMR (DMSO-d6, D2O,
d): 6.1 (s, 2H, NH2,
His 96
Tyr 20
NH2
NH2
H-bond (donor) 3.37
Coordination
(Zn)
D2O exchangeable), 7.0 (s, 1H, CH pyrrole), 7.5e7.9 (m, 10H,
AreH þ SO2NH2); 13C NMR (DMSO-d6,
d): 70.5, 113.5, 117.5, 119.5,
12
13
ꢀ36.52
ꢀ34.64
SO2
H-bond
2.49
121.3 [2], 125.2, 127.3, 131.2 [2], 131.6 [2], 132.3 [2], 139.5, 142.9,
148.5; Anal. Calcd. for C17H13BrN4O2S (417.28): C, 48.93; H, 3.14; N,
13.43. Found: C, 48.71; H, 3.50; N, 13.16.
(acceptor)
H-bond (donor) 2.48
Thr 199
Thr 200
NH2
SO2
H-bond
2.52
(acceptor)
His 94
Lys 67
NH2
NH2
H-bond (donor) 3.68
Coordination
(Zn)
NH
H-bond
2.81
(acceptor)
Thr 199
Thr 199
NH2
SO2
H-bond (donor) 2.12
H-bond
2.79
(acceptor)
H-bond
(acceptor)
H-bond (donor) 3.83
Coordination
(Zn)
Thr 200
His 94
SO2
2.77
NH2
NH2
14
ꢀ35.99
Lys 67
Gln 92
SO2
SO2
H-bond
(acceptor)
H-bond
3.13
2.57
(acceptor)
Thr 199
Thr 200
NH2
SO2
H-bond (donor) 2.49
H-bond
2.58
(acceptor)
H-bond
His 94
SO2
3.68
(acceptor)
Coordination
(Zn)
NH2
peak multiplicities were designed as follows: s, singlet; d, doublet;
t, triplet; m, multiplet. Elemental analyses were performed on a
Carlo Erba 1108 Elemental Analyzer (Heraeus, Hanau, Germany).
Fig. 2. 2D enzyme-ligand interaction of compound 12 inside the active site of hCA XII:
energy score (S) ¼ ꢀ36.52 kcal/mol.

M.M. Ghorab et al. / European Journal of Medicinal Chemistry 87 (2014) 186e196
193
(C^N), 1330, 1161 (SO2), 1261 (C]S); 1H NMR (DMSO-d6,
d): 6.3 (s,
1H, CH pyrrole), 6.9e8.1 (m, 15H, AreH þ SO2NH2), 9.5, 10.4 (2s, 2H,
2NH, D2O-exchangeable); 13C NMR (DMSO-d6,
d): 113.5, 117.6, 119.5,
122.3, 123.5 [2], 124.2, 125.2, 126.7, 127.1 [2], 128.3 [2], 130.5 [2],
131.5 [2], 132.3 [2], 136.9, 138.8, 139.4, 148.5, 174.2; Anal. Calcd. for
C24H18BrN5O2S2 (552.46): C, 52.18; H, 3.28; N, 12.68. Found: C,
52.52; H, 3.01; N, 12.39.
4.1.3.2. 4-(4-(4-Bromophenyl)-3-cyano-2-(3-(4-methoxyphenyl)thi-
oureido)-1H-pyrrol-1-yl)benzenesulfonamide (7). Yield: 91%; M.p.:
170.7 ꢁC; IR (KBr, cmꢀ1): 3404, 3369, 3256 (NH, NH2), 3100 (CH
arom.), 2933, 2836 (CH aliph.), 2203 (C^N), 1330, 1161 (SO2), 1246
(C]S); 1H NMR (DMSO-d6,
d
): 3.7 (s, 3H, OCH3), 6.2 (s, 1H, CH
pyrrole), 6.6e8.0 (m, 14H, AreH þ SO2NH2), 9.8, 10.6 (2s, 2H, 2NH,
D2O-exchangeable); 13C NMR (DMSO-d6,
): 55.1, 113.3, 116.8 [2],
d
117.6, 119.5, 121.3, 122.3 [2], 123.9, 125.2, 126.7 [2], 127.1 [2], 130.4,
131.7 [2], 132.7 [2], 136.2, 139.5, 148.5, 155.1, 176.2; Anal. Calcd. for
C25H20BrN5O3S2 (582.49): C, 51.55; H, 3.46; N, 12.02. Found: C,
51.30; H, 3.10; N, 12.35.
4.1.3.3. 4-(4-(4-Bromophenyl)-2-(3-(4-chlorophenyl)thioureido)-3-
cyano-1H-pyrrol-1-yl)benzenesulfonamide (8). Yield: 81%; M.p.:
297.5 ꢁC; IR (KBr, cmꢀ1): 3404, 3387, 3276 (NH, NH2), 3056 (CH
arom.), 2200 (C^N), 1396, 1159 (SO2), 1255 (C]S), 736 (CeCl); 1H
Fig. 3. 2D enzyme-ligand interaction of compound 13 inside the active site of hCA XII:
energy score (S) ¼ ꢀ34.64 kcal/mol.
NMR (DMSO-d6,
d): 6.4 (s, 1H, CH pyrrole), 7.0e8.1 (m, 14H,
AreH þ SO2NH2), 9.7, 10.8 (2s, 2H, 2NH, D2O-exchangeable); 13C
NMR (DMSO-d6, d): 113.6, 117.6, 119.5, 121.3, 123.4 [2], 125.2, 126.7,
127.1 [2], 127.3 [2], 127.9 [2], 130.4 [2], 131.6, 132.3 [2], 132.6, 137.7,
139.5, 148.5, 179.8; Anal. Calcd. for C24H17BrClN5O2S2 (586.91): C,
49.11; H, 2.92; N, 11.93. Found: C, 49.42; H, 2.68; N, 11.58.
4.1.3.4. 4-(4-(4-Bromophenyl)-3-cyano-2-(3-(4-nitrophenyl)thiour-
eido)-1H-pyrrol-1-yl)benzenesulfonamide (9). Yield: 79%; M.p.:
177.5 ꢁC; IR (KBr, cmꢀ1): 3371, 3292, 3206 (NH, NH2), 3079 (CH
arom.), 2206 (C^N), 1378, 1161 (SO2), 1264 (C]S). 1H NMR (DMSO-
d6,
d
): 6.3 (s, 1H, CH pyrrole), 6.6e8.1 (m, 14H, AreH þ SO2NH2), 9.5,
10.6 (2s, 2H, 2NH, D2O-exchangeable); 13C NMR (DMSO-d6,
d):
112.3, 116.6, 119.3, 121.3, 123.6 [2], 124.1, 125.0 [2], 126.7, 127.3 [2],
130.6 [2], 131.4 [2], 132.4 [2], 135.5, 139.5, 142.8, 145.5, 148.5, 178.1;
Anal. Calcd. for C24H17BrN6O4S2 (597.46): C, 48.25; H, 2.87; N, 14.07.
Found: C, 48.56; H, 2.60; N, 14.28.
4.1.3.5. 4-(4-(4-Bromophenyl)-2-(3-(4-bromophenyl)thioureido)-3-
cyano-1H-pyrrol-1-yl)benzenesulfonamide (10). Yield: 66%; M.p.:
170.4 ꢁC; IR (KBr, cmꢀ1): 3402, 3370, 3180 (NH, NH2), 3080 (CH
arom.), 2203 (C^N),1332,1163 (SO2), 1253 (C]S); 1H NMR (DMSO-
d6,
d
): 6.4 (s, 1H, CH pyrrole), 7.1e8.1 (m, 14H, AreH þ SO2NH2), 9.1,
9.9 (2s, 2H, 2NH, D2O-exchangeable); 13C NMR (DMSO-d6,
d): 113.5,
116.8, 117.6, 119.5, 120.5, 123.4 [2], 124.0, 125.2, 126.8 [2], 127.3 [2],
130.4 [2], 131.6 [2], 132.3 [2], 132.6, 138.2, 139.5, 147.5, 175.2; Anal.
Calcd. for C24H17Br2N5O2S2 (631.36): C, 45.66; H, 2.71; N, 11.09.
Found: C, 45.37; H, 2.42; N, 11.26.
Fig. 4. 2D enzyme-ligand interaction of compound 14 inside the active site of hCA XII:
energy score (S) ¼ ꢀ35.99 kcal/mol.
4.1.3. General procedure for the synthesis of 4-(4-(4-bromophenyl)-
3-cyano-2-(3-substituted thioureido)-1H-pyrrol-1-yl)
benzenesulfonamides (6e12)
A mixture of compound 5 (4.17 g, 0.01 mol) and aryl iso-
thiocyanates (0.01 mol) in absolute ethanol (30 mL) was refluxed
for 6 h. The reaction mixture was cooled and the obtained solid was
recrystallized from dioxane to give compounds 6e12.
4.1.3.6. 4-(4-(4-Bromophenyl)-3-cyano-2-(3-(4-iodophenyl)thiour-
eido)-1H-pyrrol-1-yl)benzenesulfonamide (11). Yield: 61%; M.p.:
280.9 ꢁC; IR (KBr, cmꢀ1): 3386, 3320, 3236 (NH, NH2), 3066 (CH
arom.), 2202 (C^N),1386,1163 (SO2), 1257 (C]S); 1H NMR (DMSO-
d6,
d
): 6.5 (s, 1H, CH pyrrole), 7.3e8.2 (m, 14H, AreH þ SO2NH2), 9.9,
10.8 (2s, 2H, 2NH, D2O-exchangeable); 13C NMR (DMSO-d6,
d): 91.4,
113.6, 117.3, 119.6, 120.0, 120.6 [2], 121.6, 123.2, 126.1 [2], 126.8 [2],
130.6 [2], 131.7 [2], 132.6, 137.2, 137.3, 142.8 [2], 144.5, 178.3; Anal.
Calcd. for C24H17BrIN5O2S2 (678.36): C, 42.49; H, 2.53; N, 10.32.
Found: C, 42.13; H, 2.38; N, 10.66.
4.1.3.1. 4-(4-(4-Bromophenyl)-3-cyano-2-(3-phenylthioureido)-1H-
pyrrol-1-yl)benzene-sulfonamide (6). Yield: 89%; M.p.: 187.9 ꢁC; IR
(KBr, cmꢀ1): 3410, 3390, 3210 (NH, NH2), 3055 (CH arom.), 2200

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M.M. Ghorab et al. / European Journal of Medicinal Chemistry 87 (2014) 186e196
4.1.3.7. 4-(3-(4-(4-Bromophenyl)-3-cyano-1-(4-sulfamoylphenyl)-
1H-pyrrol-2-yl)thioureido)benzenesulfonamide (12). Yield: 72%;
M.p.: 183.0 ꢁC; IR (KBr, cmꢀ1): 3348, 3310, 3266 (NH, NH2), 2202
4 .1. 6 .1. 4 - ( 4 - A m i n o - 5 - ( 4 - b r o m o p h e n y l ) - 2 - o x o - 1, 2 -
dihydropyrrolo(2,3-d)pyrimidin-7-yl)benzenesulfonamide
(17).
Yield: 59%; M.p.: 204.7 ꢁC; IR (KBr, cmꢀ1): 3396, 3356, 3266 (NH,
(C^N), 1330, 1161 (SO2), 1272 (C]S); 1H NMR (DMSO-d6,
d
): 6.3 (s,
1H, CH pyrrole), 7.2e8.0 (m, 18H, AreH þ SO2NH2), 10.6, 10.9 (2s,
2H, 2NH, D2O-exchangeable); 13C NMR (DMSO-d6,
): 113.5, 117.6,
NH2), 1695 (C]O), 1595 (C]N), 1398, 1159 (SO2); 1H NMR (DMSO-
d6,
6.9e8.0 (m, 10H, AreH þ SO2NH2), 10.1 (s, 1H, NH, D2O-exhange-
able); 13C NMR (DMSO-d6,
): 112.4, 117.9, 120.3, 123.0 [2], 125.2,
d): 5.5 (s, 2H, NH2, D2O-exchangeable), 6.6 (s, 1H, CH pyrrole),
d
119.5, 121.3, 124.3 [2], 125.2, 126.5, 127.3 [2], 128.9 [2], 129.6 [2],
130.9 [2], 131.6 [2], 131.9, 132.3, 139.5, 142.8, 148.5, 179.6; Anal.
Calcd. for C24H19BrN6O4S3 (631.54): C, 45.64; H, 3.03; N, 13.31.
Found: C, 45.41; H, 3.29; N, 13.56.
d
126.5, 127.3 [2], 129.8 [2], 132.6 [2], 140.1, 140.9, 150.2, 155.5, 159.8;
Anal. Calcd. for C18H14BrN5O3S (460.30): C, 46.97; H, 3.07; N, 15.21.
Found: C, 46.60; H, 3.35; N, 15.46.
4.1.6.2. 4-(4-Amino-5-(4-bromophenyl)-2-thioxo-1,2-
4.1.4. 4-(5-(4-Bromophenyl)-4-imino-2-thioxo-3-p-tolyl-1,2,3,4-
tetrahydro-pyrrolo(2,3-d)pyrimidin-7-yl)benzenesulfonamide (13)
A mixture of 5 (4.17 g, 0.01 mol) and 1-isothiocyanato-4-
methoxybenzene (1.49 g, 0.01 mol) in pyridine (20 mL) was
refluxed for 16 h. The reaction mixture was poured onto ice water
and the obtained solid was recrystallized from acetic acid to give 13.
Yield: 69%; M.p.: 161.1 ꢁC; IR (KBr, cmꢀ1): 3390, 3336, 3276 (NH,
NH2), 3100 (CH arom.), 2960, 2910 (CH aliph.), 1595 (C]N), 1339,
dihydropyrrolo(2,3-d)pyrimidin-7-yl)benzenesulfonamide
(18).
Yield: 55%; M.p.: 241.1 ꢁC; IR (KBr, cmꢀ1): 3376, 3354, 3210 (NH,
NH2), 1624 (C]N), 1338, 1161 (SO2), 1281 (C]S); 1H NMR (DMSO-
d6,
7.0e7.9 (m, 10H, AreH þ SO2NH2), 11.9 (s, 1H, NH, D2O-exhange-
able); 13C NMR (DMSO-d6,
): 112.6, 119.6, 121.3, 124.0 [2], 126.6,
d): 6.1 (s, 2H, NH2, D2O-exchangeable), 6.7 (s, 1H, CH pyrrole),
d
129.5, 130.4 [2], 131.8 [2], 132.9 [2], 137.2, 139.8, 146.4, 157.8, 185.4;
Anal. Calcd. for C18H14BrN5O2S2 (476.37): C, 45.38; H, 2.96; N, 14.70.
Found: C, 45.71; H, 2.60; N, 14.43.
1161 (SO2), 1230 (C]S); 1H NMR (DMSO-d6,
d): 2.2 (s, 3H, CH3), 6.8
(s, 1H, CH pyrrole), 7.2 (s, 1H, NH, D2O-exchangeable), 7.3e7.9 (m,
14H, AreH þ SO2NH2), 9.8 (s,1H, NH imino, D2O-exchangeable); 13
C
4.1.7. 4-(5-(4-Bromophenyl)-2,4-dithioxo-1,2,3,4-
NMR (DMSO-d6, d): 20.4, 100.2, 116.8, 120.6, 123.2 [2], 123.7, 126.7,
tetrahydropyrrolo(2,3-d)pyrimidin-7-yl)benzenesulfonamide (21)
A mixture of 5 (4.17 g, 0.01 mol) and carbon disulfide (1.52 g,
0.02 mol) in dry pyridine (15 mL) was refluxed for 24 h. The reac-
tion mixture was poured onto ice water and the obtained solid was
recrystallized from ethanol to give 21. Yield: 59%; M.p.: 213.9 ꢁC; IR
(KBr, cmꢀ1): 3385, 3366, 3210 (NH, NH2), 3095 (CH arom.), 1329,
128.7 [2], 129.1 [2], 130.4 [2], 131.7 [2], 132.7, 133.4 [2], 136.1, 136.8,
139.5, 141.7, 161.5, 179.5; Anal. Calcd. for C25H20BrN5O2S2 (566.49):
C, 53.00; H, 3.56; N, 12.36. Found: C, 53.32; H, 3.21; N, 12.09.
4.1.5. General procedure for synthesis of N-(4-(4-Bromophenyl)-3-
cyano-1-(4-sulfamoyl-phenyl)-1H-pyrrol-2-yl)substituted
sulfonamides (14, 15)
A mixture of 5 (4.17 g, 0.01 mol) and sulfonyl chloride de-
rivatives (0.01 mol) in dry benzene containing 3 drops of pyridine
was refluxed for 7 h. The obtained solid was recrystallized from
dioxane to give 14 and 15, respectively.
1159 (SO2), 1234 (C]S); 1H NMR (DMSO-d6,
role), 7.1e8.2 (m, 10H, AreH þ SO2NH2), 8.6 (s, 2H, 2NH, D2O-
exchangeable); 13C NMR (DMSO-d6,
): 107.2, 119.9, 121.4, 124.6 [2],
d): 7.0 (s, 1H, CH pyr-
d
126.4, 126.6, 129.7 [2], 131.6 [2], 132.3 [2], 138.5, 142.1, 147.7, 178.0,
178.6; Anal. Calcd. for C18H13BrN4O2S3 (493.42): C, 43.82; H, 2.66;
N, 11.35. Found: C, 43.60; H, 2.38; N, 11.56.
4.1.8. N-(4-(4-bromophenyl)-3-cyano-1-(4-sulfamoyl)-1H-pyrrol-
2-yl)-ethanimidothioic acid (22) and 4-(5-(4-bromophenyl)-2-
methyl-4-thioxo-3,4-dihydropyrrolo(2,3-d) pyrimidin-7-yl)
benzenesulfonamide (24)
4.1.5.1. N-(4-(4-bromophenyl)-3-cyano-1-(4-sulfamoylphenyl)-1H-
pyrrol-2-yl)benzene-sulfonamide (14). Yield: 65%; M.p.: 349.8 ꢁC; IR
(KBr, cmꢀ1): 3406, 3380, 3266 (NH, NH2), 3066 (CH arom.), 2218
(C^N), 1334, 1161 (SO2); 1H NMR (DMSO-d6,
d
): 6.8 (s, 1H, CH
pyrrole), 7.0e8.6 (m, 15H, AreH þ SO2NH2), 12.0 (s, 1H, SO2NH,
D2O-exchangeable); 13C NMR (DMSO-d6,
): 107.2, 117.3, 119.0,
A mixture of 5 (4.17 g, 0.01 mol) and thioacetamide (0.75 g,
0.01 mol) in trifluoroacetic acid (10 mL) was refluxed for 14 h. The
reaction mixture was filtered while hot to give compound 24, while
compound 22 was obtained from the mother liquor after cooling.
22: Yield: 48%; M.p.: 142.5 ꢁC; IR (KBr, cmꢀ1): 3348, 3283 (NH2),
3100 (CH arom.), 2970, 2920 (CH aliph.), 2201 (C^N), 1595 (C]N),
d
121.8, 125.3 [2], 126.0, 126.7, 127.6 [2], 128.4 [2], 129.2 [2], 131.1 [2],
133.1, 134.6 [2], 136.7, 139.6, 143.1, 144.9; Anal. Calcd. for
C
23H17BrN4O4S2 (557.44): C, 49.56; H, 3.07; N, 10.05. Found: C,
49.36; H, 3.28; N, 10.31.
1330, 1161 (SO2); 1H NMR (DMSO-d6,
d): 0.9 (s, 3H, CH3), 1.3 (s, 1H,
SH, D2O-exchangeable), 6.4 (s, 1H, CH pyrrole), 7.0e8.0 (m, 10H,
4.1.5.2. N-(4-(4-bromophenyl)-3-cyano-1-(4-sulfamoylphenyl)-1H-
pyrrol-2-yl)-4-methyl-benzenesulfonamide (15). Yield: 61%; M.p.:
300.5 ꢁC; IR (KBr, cmꢀ1): 3385, 3270 (NH, NH2), 3066 (CH arom.),
2940, 2860 (CH aliph.), 2210 (C^N), 1338, 1161 (SO2); 1H NMR
AreH þ SO2NH2); 13C NMR (DMSO-d6,
d): 29.8, 113.6, 117.0, 117.6,
119.4, 121.3 [2], 125.2, 127.1, 127.3 [2], 131.6 [2], 132.3 [2], 139.5,
142.8, 148.5, 167.2; Anal. Calcd. for C19H15BrN4O2S2 (473.98): C,
48.00; H, 3.18; N, 11.79. Found: C, 48.31; H, 3.41; N, 11.46.
(DMSO-d6, d): 2.3 (s, 3H, CH3), 6.6 (s, 1H, CH pyrrole), 7.0e8.5 (m,
24: Yield: 45%; M.p.: 282.7 ꢁC; IR (KBr, cmꢀ1): 3375, 3263 (NH2),
3100 (CH arom.), 2960, 2847 (CH aliph.), 1593 (C]N), 1344, 1149
14H, AreH þ SO2NH2), 11.9 (s, 1H, SO2NH, D2O-exchangeable); 13C
NMR (DMSO-d6, d): 22.6, 113.0, 115.6, 119.0, 121.6, 122.8 [2], 124.6,
(SO2), 1291 (C]S); 1H NMR (DMSO-d6,
d
): 2.4 (s, 3H, CH3), 6.5 (s, 1H,
CH pyrrole), 6.9e8.1 (m, 10H, AreH þ SO2NH2), 13.4 (s, 1H, SH, D2O-
exchangeable); 13C NMR (DMSO-d6,
): 26.0, 107.9, 116.4, 121.8 [2],
125.4, 127.0 [2], 128.0 [2], 130.4 [2], 131.1 [2], 133.6 [2], 135.1, 135.7,
139.4, 142.6, 143.2; Anal. Calcd. for C24H19BrN4O4S2 (571.47): C,
50.44; H, 3.35; N, 9.80. Found: C, 50.19; H, 3.11; N, 9.48.
d
124.5, 129.8 [2], 130.8, 131.3 [2], 132.6 [2], 138.3, 143.0, 148.2, 158.6,
168.3, 182.6; Anal. Calcd. for C19H15BrN4O2S2 (475.38): C, 48.00; H,
3.18; N, 11.79. Found: C, 47.77; H, 3.50; N, 11.50.
4.1.6. General procedure for synthesis of 4-(4-amino-5-(4-
bromophenyl)-2-substituted-1,2-dihydropyrrolo(2,3-d)pyrimidin-
7-yl)benzenesulfonamides (17, 18)
A mixture of 5 (4.17 g, 0.01 mol) and urea or thiourea (0.01 mol)
was fused in an oil bath at 220 ꢁC for 1 h. The obtained solid was
recrystallized from ethanol to give 17 and 18, respectively.
4.1.9. N-(4-(4-bromophenyl)-3-cyano-1-(4-sulfamoylphenyl)-1H-
pyrrol-2-yl)-3-oxobutanamide (25)
A solution of 5 (4.17 g, 0.01 mol) in ethylacetoacetate (10 mL)
was refluxed for 1 h. The reaction mixture was cooled and

M.M. Ghorab et al. / European Journal of Medicinal Chemistry 87 (2014) 186e196
195
recrystallized from ethanol to give 25. Yield: 65%; M.p.: 105.7 ꢁC; IR
(KBr, cmꢀ1): 3344, 3230, 3170 (NH, NH2), 3086 (CH arom.), 2940,
2846 (CH aliph.), 2200 (C^N),1730, 1690 (2C]O), 1340, 1163 (SO2);
manner and subtracted from the total observed rates. Stock solu-
tions 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 or 4 ꢁC prior to assay, in
order to allow for the formation of the EeI complex. Data from
Table 1 were obtained after 15 min incubation of enzyme and in-
hibitor [35]. The inhibition constants were obtained by non-linear
least squares methods using PRISM 3, as reported earlier [36],
and represent the mean from three different determinations. All CA
isofoms were recombinant ones obtained in-house as reported
earlier [35,37e42].
1H NMR (DMSO-d6,
d
): 2.2 (s, 3H, CH3), 4.1 (s, 2H, CH2), 6.3 (s,1H, CH
pyrrole), 7.0e8.0 (m, 10H, AreH þ SO2NH2), 10.3 (s, 1H, NH, D2O-
exchangeable); 13C NMR (DMSO-d6,
): 30.0, 51.2, 113.5, 117.6, 119.5,
d
120.6, 121.4 [2], 125.5, 127.8, 129.1 [2], 131.9 [2], 132.3 [2], 139.5,
140.7,148.5,168.6, 201.6; Anal. Calcd. for C21H17BrN4O4S (501.35): C,
50.31; H, 3.42; N, 11.18. Found: C, 50.66; H, 3.10; N, 11.41.
4.1.10. 4-(Bromo-N-(4-(4-bromophenyl)-3-cyano-1-(4-
sulfamoylphenyl)-1H-pyrrol-2-yl)-benzamide (26)
A mixture of 5 (4.17 g, 0.01 mol) and 4-bromobenzoyl chloride
(2.21 g, 0.01 mol) in pyridine (10 mL) was refluxed for 4 h. The
reaction mixture was poured onto ice water and the obtained solid
was recrystallized from ethanol to give 26. Yield: 71%; M.p.:
205.4 ꢁC; IR (KBr, cmꢀ1): 3419, 3329, 3236 (NH, NH2), 3080 (CH
arom.), 2187 (C^N),1676 (C]O),1389,1163 (SO2); 1H NMR (DMSO-
4.3. In vitro anticancer activity
Human breast cancer cell line (MCF-7) was used in this study.
The cytotoxic activity was measured in vitro for the newly syn-
thesized compounds using the Sulforhodamine-B stain (SRB) assay
using the method of Skehan et al. [43]. The in vitro anticancer
screening was done by the pharmacology unit at the National
Cancer Institute, Cairo University, Egypt. Cells were plated in 96-
multiwell plate (104 cells/well) for 24 h before treatment with the
compound(s) to allow attachment of cells to the wall of the plate.
Test compounds were dissolved in dimethyl sulfoxide. Different
d6,
NH, D2O-exchangeable), 13.2 (s, 2H, SO2NH2, D2O-exchangeable);
13C NMR (DMSO-d6,
): 114.0, 118.0, 120.0, 121.9, 125.7 [2], 127.3 [2],
d): 6.2 (s, 1H, CH pyrrole), 7.0e8.0 (m, 12H, AreH), 10.9 (s, 1H,
d
127.6, 127.8 [2], 130.4 [2], 131.7 [2], 132.0 [2], 132.1 [2], 140.0 [2],
143.4, 149.0, 167.1; Anal. Calcd. for C24H16Br2N4O3S (600.28): C,
48.02; H, 2.69; N, 9.33. Found: C, 48.29; H, 2.41; N, 9.68.
concentrations of the compound under test (10, 25, 50, and 100 mM)
4.1.11. 4-(3-Benzoyl)-5-(4-bromophenyl)-4-oxo-2-phenyl-3,4-
dihydropyrrolo(2,3-d)pyrimidin-7-yl)benzenesulfonamide [30] and
4-(5-(4-bromophenyl)-3-(4-chlorobenzoyl)-2-(4-chloro-phenyl)-4-
oxo-3,4-dihydropyrrolo(2,3-d)pyrimidin-7-yl)benzenesulfonamide
[31]
were added to the cell's monolayer. Triplicate wells were prepared
for each individual concentration. Monolayer cells were incubated
with the compound(s) for 48 h at 37 ꢁC and in atmosphere of 5%
CO2. After 48 h, cells were fixed, washed and stained for 30 min
with 0.4% (wt/vol) SRB dissolved in 1% acetic acid. Excess unbound
dye was removed by four washes with 1% acetic acid and attached
stain was recovered with TriseEDTA buffer. Color intensity was
measured in an ELISA reader. The relation between surviving
fraction and drug concentration was plotted to get the survival
curve for breast tumor cell line after the specified time. The molar
concentration required to inhibit 50% of cell viability (IC50) was
calculated and compared with the reference drug doxorubicin (CAS,
25316-40-9). The surviving fractions were expressed as
means S.E.M.
A solution of 5 (4.17 g, 0.01 mol) in benzoyl chloride or p-
chlorobenzoyl chloride (10 mL) was refluxed for 10 h. The obtained
solid was recrystallized from dioxane to give 30 and 31,
respectively.
30: Yield: 61%; M.p.: 146.5 ꢁC; IR (KBr, cmꢀ1): 3310, 3290 (NH2),
3082 (CH arom.), 1764, 1710 (2C]O), 1593 (C]N), 1375, 1170 (SO2);
1H NMR (DMSO-d6,
d): 7.1 (s, 1H, CH pyrrole), 7.3e8.0 (m, 20H,
AreH þ SO2NH2); 13C NMR (DMSO-d6,
d): 113.6, 119.0, 121.7, 122.6
[2], 124.8, 127.0, 127.7 [2], 128.5 [2], 128.6 [2], 129.0, 129.2 [2], 129.8
[2], 130.2 [2], 130.9, 131.2 [2], 132.8, 133.2, 136.7, 138.6, 141.4, 159.2,
166.8, 167.2; Anal. Calcd. for C31H21BrN4O4S (625.49): C, 59.53; H,
3.38; N, 8.96. Found: C, 59.19; H, 3.71; N, 8.16.
4.4. Molecular modeling and docking
31: Yield: 65%; M.p.: 188.4 ꢁC; IR (KBr, cmꢀ1): 3381, 3296 (NH2),
3075 (CH arom.), 1712, 1691 (2C]O), 1620 (C]N), 1378, 1161 (SO2),
All the molecular modeling studies were carried out on an Intel
Pentium 1.6 GHz processor, 512 MB memory with Windows XP
operating system using Molecular Operating Environment (MOE,
10.2008) software. All the minimizations were performed with
MOE until an RMSD gradient of 0.05 kcal/molÅ with MMFF94X
force field and the partial charges were automatically calculated.
The X-ray crystallographic structure of CA isozyme XII complexed
with acetazolamide (PDB ID: 1JD0) was obtained from the protein
data bank. The enzymes were prepared for docking studies; where,
(i) The co-crystallized ligand molecule (AZA) was removed from the
enzyme active site. (ii) Hydrogen atoms were added to the structure
with their standard geometry. (iii) MOE Alpha Site Finder was used
for the active sites search in the enzyme structure and dummy
atoms were created from the obtained alpha spheres. (iv) The ob-
tained model was then used in predicting the ligand enzymes in-
teractions at the active site.
751 (CeCl); 1H NMR (DMSO-d6)
d: 7.4 (s, 1H, CH pyrrole), 7.5e7.9
(m, 18H, AreH þ SO2NH2); 13C NMR (DMSO-d6,
d): 113.7, 119.6,
121.7, 122.8 [2], 123.6, 124.0, 128.1, 128.6 [2], 128.9 [2], 129.0 [2],
129.5 [2], 129.6 [2], 131.0 [2], 131.8 [2], 133.2, 134.0 [2], 137.7, 138.5,
146.7, 157.2, 164.5, 166.3; Anal. Calcd. for
C31H19BrCl2N4O4S
(694.38): C, 53.62; H, 2.76; N, 8.07. Found: C, 53.36; H, 2.96; N, 8.37.
4.2. Carbonic anhydrase inhibition assay
An Applied Photophysics stopped flow instrument has been
used for assaying the CA catalyzed CO2 hydration activity. The
initial rates of the CA-catalyzed CO2 hydration reaction were
determined for a period of 10e100 s through measuring the
absorbance (lmax; 557 nm) of the color intensity obtained from a
solution containing 0.2 mM phenol red as an indicator, 20 mM
Hepes (pH 7.5) as a buffer, and 20 mM Na2SO4 (for maintaining
constant ionic strength). The CO2 concentrations ranged from 1.7 to
17 mM for the determination of the kinetic parameters and inhi-
bition constants. For each inhibitor at least six traces of the initial
5e10% of the reaction have been used for determining the initial
velocity. The uncatalyzed rates were determined in the same
Acknowledgments
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.

196
M.M. Ghorab et al. / European Journal of Medicinal Chemistry 87 (2014) 186e196
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