Pyrrolo and pyrrolopyrimidine sulfonamides act as cytotoxic agents in hypoxia via inhibition of transmembrane carbonic anhydrases

Article abstract of DOI:10.1016/j.ejmech.2019.112021

A series of novel sulfonamide derivatives bearing pyrrole and pyrrolopyrimidine scaffolds were synthesized and screened as carbonic anhydrase inhibitors. The inhibition activity of the synthesized compounds was evaluated against the cytosolic human carbon

Full text of DOI:10.1016/j.ejmech.2019.112021

Journal Pre-proof
Pyrrolo and pyrrolopyrimidine sulfonamides act as cytotoxic agents in hypoxia via
inhibition of transmembrane carbonic anhydrases
Omneya M. Khalil, Aliaa M. Kamal, Silvia Bua, Heba El Sayed Teba, Yassin M.
Nissan, Claudiu T. Supuran
PII:
S0223-5234(19)31179-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.112021
EJMECH 112021
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 December 2019
Revised Date: 25 December 2019
Accepted Date: 27 December 2019
Please cite this article as: O.M. Khalil, A.M. Kamal, S. Bua, H. El Sayed Teba, Y.M. Nissan, C.T.
Supuran, Pyrrolo and pyrrolopyrimidine sulfonamides act as cytotoxic agents in hypoxia via inhibition
of transmembrane carbonic anhydrases, European Journal of Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.ejmech.2019.112021.
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Ki hCA II = 3.8 nM
Ki hCA IX = 19.6 nM
IC50 (Normoxia) =
2.71 µM
IC50 (Hypoxia) =
8.80 µM
hCA II
hCA II
hCA IX
Hypoxia
Normoxia
HeLa cells

Pyrrolo and Pyrrolopyrimidine Sulfonamides act as Cytotoxic Agents in Hypoxia
via Inhibition of Transmembrane Carbonic Anhydrases
Omneya M. Khalil a, Aliaa M. Kamal a,b,*, Silvia Bua c, Heba El Sayed Teba b,*, Yassin M. Nissan d,e
, Claudiu T. Supuran c,*
a
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, 11562,
Cairo, Egypt.
b Organic Chemistry Department, Faculty of Pharmacy, October University for Modern Sciences and
Arts (MSA), 11787, Giza, Egypt.
c Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University
of Florence, Polo Scientifico, Via U. Schiff 6, 50019, Sesto Fiorentino (Firenze), Italy.
d
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, 11562, Cairo,
Egypt.
e
Pharmaceutical Chemistry Department, Faculty of Pharmacy, October University for Modern
Sciences and Arts (MSA), 11787, Giza, Egypt.
Keywords: cytotoxic; carbonic anhydrase inhibitor; pyrrolopyrimidine; sulfonamide.
*Corresponding author: Aliaa M. Kamal, Organic Chemistry Department, Faculty of Pharmacy,
Cairo University, 11562, Cairo, Egypt.
*Corresponding author: Heba El Sayed Teba, Organic Chemistry Department, Faculty of Pharmacy,
October University for Modern Sciences and Arts (MSA), 11787, Giza, Egypt.
Email: hteba@msa.eun.eg
*Corresponding author: Claudiu T. Supuran, Department of NEUROFARBA, Section of
Pharmaceutical and Nutraceutical Sciences, University of Florence, Polo Scientifico, Via U. Schiff 6,
50019, Sesto Fiorentino (Firenze), Italy.
Abstract
A series of novel sulfonamide derivatives bearing pyrrole and pyrrolopyrimidine scaffolds were
synthesized and screened as carbonic anhydrase inhibitors. The inhibition activity of the synthesized
compounds was evaluated against the cytosolic human carbonic anhydrase isoforms I and II and the
transmembranal isoforms IX and XII. Several candidates showed potent inhibitory activity against IX
and XII isoforms. Furthermore, ex vivo screening of cytotoxic selectivity and activity of the most
potent derivatives were carried out against normal cells (WI38) and cervical cancer cell line (HeLa)
under normal and hypoxic conditions using acetazolamide as reference drug. Compound 11b potency
was nearly three folds higher in hypoxic than normoxic condition whereas that of compound 11f was
nearly four folds higher in hypoxic vs. normoxic HeLa cells. All the screened derivatives exhibited
1

less potency on normal cells (WI38). Molecular docking was carried out to discover the possible
binding mode of compounds within the active site of isoform CA IX.
1. Introduction
According to the American cancer society, cancer was estimated to cause 1700 deaths daily [1].
The rapid increase in cancer cases and social burdens represent real crisis for public health and health
systems worldwide [2]. Using conventional non selective chemotherapeutic agents affects both cancer
and normal rapidly dividing cells causing many problems such as severe side effects, high patient
risks, repeated treatment and increased possibility of cancer cells to acquire multidrug resistance [3].
Sulfonamides represent an essential class of drugs having various biological activities such as
antibacterial [4], antiviral [5, 6], anti-inflammatory [7, 8], diuretic [9, 10], hypoglycemic [11],
antithyroid, protease inhibitors [8, 12], antioxidant, antiacetylcholinesterase [13], lactoperoxidase
inhibitors [14], butyrylcholinesterase inhibitors [15], and anti-cancer agents [16-19]. The mechanism
of action of sulfonamides as anti-cancer agents is by acting as carbonic anhydrase inhibitors (CAIs)
which is the same mechanism of action of acetazolamide (AZA), methazolamide and topiramate (Fig.
1) [20]. Sulfamates, sulfamides (sulfonamides isosteres) and sulfonamides, represent the main classes
of CAIs [21]. They all coordinate the zinc ion of CA enzyme with their terminal deprotonated
nitrogen atom [22].
Fig. 1: Sulfonamides with potent CA inhibition activity
Pyrrole and fused pyrrole compounds such as pyrrolopyrimidine derivatives possess a wide
spectrum of pharmacological activities mainly acting as anticancer agents [23, 24]. In previous work,
a combination of sulfonamide with pyrrole and pyrrolopyrimidine derivative exhibited promising
anticancer agents [25-29].
2

The metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the reversible hydration of CO2
to bicarbonate and protons in all living organisms [8, 30-38]. They are involved in various
physiological functions such as intra and extra cellular pH regulation, respiration, inorganic carbon
transport and bio mineralization [39-42]. Up till now, 15 isoforms of CAs were discovered in
humans, and they differ in tissue distribution, subcellular localization and catalytic activity. Five CAs
are cytosolic (CA I–III, VII and XIII), two are mitochondrial (CA VA and VB), one is secreted (CA
VI) and the others are membrane-bound (CA IV, IX, XII, and XIV) [22]. CA enzymes active site
contains a Zn+2 ion which is coordinated by three imidazole groups of histidines and one hydroxide
ion in a slightly distorted tetrahedral geometry [8]. Carbonic anhydrase IX (CA IX) is a cell surface
protein that is overexpressed in cancer cells, being involved in solid tumor acidification but it is not
expressed in most normal cells [22, 43, 44]. CA IX was first identified on the surface of the human
cervical carcinoma cell line (HeLa) [45]. Expression of CA IX is found in cancers of lung [46], colon
[47], breast [48], brain [49] and cervix [50] among others. Expression of CA IX is induced by
hypoxia, the enzyme functioning as a pro-survival factor which protects cancer cells against acidosis
and hypoxia by its ability to maintain an intracellular pH (pHi) favorable for survival and growth of
tumor cells [51]. Recently, the synthesis of new CA IX-selective inhibitors is receiving a considerable
attention to furnish a novel targeted strategy in cancer therapy [51-53].
Structure activity relationship of sulfonamides showed that the sulfamoyl group is essential for CA
inhibitory activity. Amino group of SO2NH2 must remain unsubstituted to retain both in vitro and in
vivo activities. Sulfamoyl group must be attached to aromatic ring and introduction of any additional
substituents to the ring decreases the activity [54].
In this study, we synthesized novel sulfonamide derivatives incorporating pyrrole and
pyrrolopyrimidine scaffolds. All the synthesized compounds were evaluated for their carbonic
anhydrase inhibition activity against four CA isoforms, the cytosolic, widespread hCA I and II, and
the transmembranal, tumor-associated CA IX and XII using the well-known CAI acetazolamide
(AZA). In vitro anticancer screening of the most potent derivatives was carried out against cervical
cell line (HeLa) under normal and hypoxic conditions. Docking study was carried out within the
active site of hCA IX isoenzyme. Synthesis of the target sulfonamides was represented in Schemes1
and 2.
2. Results and discussion
3

2.1. Chemistry
The synthesis of sulfonamide derivatives 4-7a-g is displayed in scheme 1. Reaction of
sulfanilamide 1 with 4-chlorophenacyl bromide 2 yielded the corresponding 4-(2-(4-chlorophenyl)-2-
oxoethylamino)benzenesulfonamide 3 which upon reaction with malononitrile in presence of a base
1
gave the pyrrole derivative 4. The H NMR spectrum revealed the presence of a singlet signal at δ
4.77 ppm due to CH2 group and a singlet D2O exchangeable signal at δ 6.60 corresponding to NH
group. The IR spectrum of compound 4 exhibited a characteristic absorption band of CN group at
1
2187 cm-1. In addition, the H NMR spectrum exhibited the appearance of a singlet D2O
exchangeable signal at δ 6.14 ppm assigned to NH2 group and a singlet signal at δ 7.03 ppm due to
CH of the pyrrole ring. Acid hydrolysis of compound 4 using 60% H2SO4 yielded the acid derivative
5 while condensation of compound 4 with different aromatic aldehydes yielded the corresponding
schiff’s bases 7a-g. IR spectrum of compound 5 showed the disappearance of CN group and
appearance of a broad absorption band at 3550-2720 cm-1 assigned to OH of the carboxylic group and
1
absorption band at 1683 cm-1 corresponding to C=O group. H NMR spectrum revealed the
appearance of a singlet D2O exchangeable signal at δ 9.85 ppm assigned to OH group of the
1
carboxylic group. The H NMR spectra of compounds 7a-g revealed the presence of singlet signals
for (N=CH) in the range of δ 8.88-9.41 ppm. Esterification of compound 5 was carried out using
absolute ethanol to yield the ester derivative 6. IR spectrum of 6 indicated the disappearance of the
broad absorption band of OH group and the shifting of the C=O band to 1723 cm-1. Moreover, the
1
presence of CH3 and CH2 groups was proved by H NMR spectrum by the characteristic triplet and
quartet signals at δ 1.11 and 3.77 ppm respectively with the same J values and by 13C NMR spectrum
at 14.57 and 61.51 ppm.
In scheme 2, reaction of amino cyanopyrrole derivative 4 with formic acid afforded the
pyrrolopyrimidinone 8 which underwent chlorination with phosphorous oxychloride followed by
reaction with different amines to afford compounds 9, 10 and 11a-g respectively. Cyclization and
formation of 8 was confirmed by IR spectrum which showed the appearance of C=O absorption band
1
at 1657 cm-1 in addition to the disappearance of CN band of its precursor. H NMR spectrum
exhibited the appearance of a singlet signal at δ 8.00 ppm corresponding to CH of pyrimidine and a
13
singlet D2O exchangeable signal at δ 12.31 ppm due to NH/OH proton. C NMR spectrum showed
the appearance of a signal at 158.93 ppm corresponding to C=O group. Structure of compound 9 was
4

1
proved via H NMR spectrum which showed the disappearance of the singlet D2O exchangeable
1
signal assigned to NH proton at δ 12.31 ppm. The H NMR spectrum of compound 10 revealed the
1
presence of a singlet D2O exchangeable signal due to NH proton at δ 12.30 ppm. The H NMR
spectra of 11a-g showed the characteristic signal of NH in the range of δ 12.31-12.33 ppm. For
compounds 11a-c, the appearance of signals at the range of δ 2.12-2.31 ppm confirmed the presence
of CH3 group. Compound 11d showed signal at δ 3.75 ppm corresponding to OCH3 group.
Appearance of signal at δ 4.72 ppm in compound 11g is due to CH2 group. 13C NMR spectra of
compound 11f showed the presence of signal at 158.92 ppm assigned to C=O. Compounds 12a-g
1
were synthesized via reacting the hydrazino derivative 10 with different aromatic aldehydes. The H
NMR spectra of 12a-g revealed the presence of singlet signals in the range of δ 8.33-9.01 ppm for
(N=CH) protons in addition to singlet D2O exchangeable signals in the range of δ 11.16-12.31 ppm
assigned to NH protons. Reacting compound 4 with triethylorthoformate gave the compound 13
1
which upon hydrazinolysis yielded the aminoimino pyrrolopyrimidine 14. H NMR spectrum of
compound 13 showed the appearance of triplet and quartet signals at δ 1.04 and 3.45 ppm
respectively with the same J values corresponding to CH3 and CH2 groups in addition to two singlet
signals at δ 8.54 and 8.71 ppm with integration of half proton each (N=CH proton) due to the
presence of two geometrical isomers in nearly equal ratios and that was confirmed with TLC.
1
Structure of compound 14 was proved on the basis of its H NMR spectrum which indicated the
appearance of a singlet D2O exchangeable signal at δ 6.14 ppm assigned to NH2 group and a singlet
signal at δ 7.96 ppm corresponding to CH of pyrimidine. To obtain the 2-methylpyrrolopyrimidinone,
compound 4 was refluxed with acetic anhydride. After short time, a new spot on TLC appeared and
after working up the reaction mixture, it was found that the newly formed compound was the
acetylated aminocyano derivative 15. Increasing reaction time up to 24 hours gave the
pyrrolopyrimidinone 16. IR spectrum of 15 showed the appearance of absorption band at 1730 cm-1
corresponding to C=O group while the 1H NMR spectrum indicated the presence of singlet signal at δ
2.51 corresponding to CH3 group in addition to a singlet D2O exchangeable signal at δ 10.24 ppm
assigned to NH group. Cyclization and formation of the pyrrolopyrimidinone 16 was confirmed by IR
via disappearance of the absorption band due to CN group at 2226 cm-1. 1H NMR spectrum revealed
the appearance of a singlet signal at δ 2.45 ppm corresponding to CH3 group in addition to a singlet
D2O exchangeable signal at δ 12.27 ppm assigned to NH group.
5

6

7

2.2.Biological evaluation
2.2.1. Carbonic anhydrase inhibition
Screening of CA inhibition activity of the synthesized sulfonamide derivatives was carried out by
a stopped flow assay method [55-62] and was used to assess the inhibitory activity against four
isoforms of the enzyme, hCA I and II (cytosolic isoforms) and hCA IX and XII (membrane
associated isoforms). The reference drug used for this assay was acetazolamide (AZA), the well-
known CA inhibitor. Results of the assay are displayed in Table 1.
8

Table 1: The inhibition activity of compounds 4-16 against hCA I, II, IX, and XII using AZA as
reference drug.
Compound
Kia (nM)
SIb
hCA I
7550.6
128.4
8339
2564.4
3851.5
1846
2035.8
217.3
160.8
5481.1
504.6
372.8
477.8
309.2
318.5
361.3
86.2
406.2
261.4
195.7
146.6
323.9
47.2
hCA II
9243.4
56.8
hCA IX
2874.1
75.2
hCA XII hCA II / IX hCA II / XII
88.5
54.1
151.9
247.9
207.8
40.1
71.8
73.5
68.8
239.6
71.6
66.5
78.9
28.7
36
3.22
0.76
2.81
0.44
0.84
0.87
1.86
0.10
0.08
2.38
2.26
0.62
1.77
0.18
0.83
1.23
0.35
0.49
0.19
0.68
0.35
0.80
0.05
0.31
0.54
0.48
0.83
1.42
0.95
6.22
1.86
0.5
104.45
1.05
34.53
5.45
10.98
21.05
20.36
1.76
1.30
26.95
1.80
0.71
1.92
0.07
0.78
0.76
0.50
0.59
0.08
1.06
0.44
0.96
0.06
2.98
4.41
4.20
8.28
7.87
4.42
39.83
1.43
2.19
4
5
6
7a
7b
7c
7d
7e
7f
7g
8
9
10
11a
11b
11c
11d
11e
11f
11g
12a
12b
12c
12d
12e
12f
12g
13
5245.3
1350.2
2281.8
844.3
1461.9
129.7
89.5
6456.5
129.1
47.1
151.8
1.9
28
34.3
23
34.4
3.8
88
55.5
127.5
9.8
907.4
1440.2
1213.1
2508.3
1444.2
142.2
5197.4
134.6
12.5
1868.9
3048.9
2707.7
975.4
785.1
1315.9
1136.4
2717.4
57.2
75.6
85.6
10.6
33.7
27.8
66.1
70.4
19.6
44.9
46
57.9
45.2
83.1
125.7
133
129
157.4
159.6
206.3
2966.6
2687.4
2553
3031.1
1016
149.9
835.6
72.3
165.4
304.3
326.7
288.9
303.1
183.6
32.2
130.5
93.8
5.7
2035.2
3380.2
2827.5
4744.7
2362.5
405.6
7812.7
518.5
250
14
15
16
AZA
25
a Ki is the mean from 3 different assays. (errors are in the range of ± 5-10% of the reported values)
b SI (selectivity index) is a ratio between the Ki values observed for two hCA isoforms (low value
index is indicative of weak selectivity).
Sulfonamides 5, 7e, 7f, 11d, 11g, 12a and 12c showed good activity against the cytosolic
isoform hCA I, superior to that of AZA ranging from 47.2 to 217.3 nM. Moreover, compounds 11a,
9

11f and 12c exhibited potent inhibitory effect towards the cytosolic isoform hCA II compared to AZA
ranging from 1.9 to 9.8 nM.
hCA IX was highly inhibited by compounds 11a and 11f with Ki values of 10.6 and 19.6 nM,
respectively compared to 25 nM for AZA. Compounds 5, 8, 9, 10, 11b, 11c, 11d, 11e and 16 showed
moderate activity in the range of 27.8-85.6 nM while other compounds showed poor activity towards
hCA IX. With regards to the tumor associated target isoform hCA XII, all compounds were less
potent than AZA. Sulfonamides 5, 7c, 11a, 11b, 11c, 11d, 11f, 11e and 14 exhibited moderate hCA
XII inhibitory activity. The remaining derivatives showed poor activity.
To measure the differential inhibitory activity of the tested candidates towards isoforms hCA
II/IX and hCA II/XII, the selectivity index (SI) was calculated (Table 1). The higher the inhibitory
activity against the tumor associated hCA IX and hCA XII isoforms over hCA II isoform, the more
effective and safe a drug would be. Compounds 4, 5, 6, 7b, 7c, 7d, 7g, 8, 9, 10, 11b, 11c, 11g, 12g,
13, 14, 15 and 16 exhibited high hCA II/IX SI values higher than that of AZA.
2.2.2. In vitro anticancer activity
It is worth to be mentioned that, HeLa, cervical cancer cells showed increased CA IX expression
upon hypoxic conditions. To study the efficacy of the synthesized compounds as anticancer agents
via carbonic anhydrase inhibition, the in vitro anticancer activity of the most potent hCA IX
inhibitors was screened against the cervical cancer cells (HeLa) under normal and hypoxic conditions
using AZA as reference drug applying MTT method [63-66]. Moreover, the toxicity of the
synthesized compounds was evaluated by measuring their IC50 against normal cells WI-38. Results
are listed in Table 2 and Figure 2.
Table 2: In vitro anticancer screening against cervical cancer cell line (HeLa) under normal and
hypoxic conditions & normal cell line (WI-38)
IC50 (µM)*
Compound
HeLa
WI-38
Normoxia
Hypoxia
8
40.22±1.66
8.86±0.35
32.65±1.61
6.65±0.25
69.57±4.27
26.56±1.51
11a
10

11b
11c
9.05±0.42
12.35±0.88
8.80±0.41
7.82±0.22
3.17±0.11
13.33±0.81
2.71±0.06
2.83±0.04
24.71±0.93
16.17±0.75
22.02±1.36
14.45±0.77
11f
AZA
*Each value is the mean of three values ± standard error
Fig. 2: Graph showing IC50 (µM) of the tested compounds against HeLa
Anticancer activity of the candidates was very promising as all the tested candidates showed
potency under hypoxic conditions greater than normoxic conditions. Compounds 11f and 11b were
nearly triple the potency when subjected to hypoxic conditions. The aryl amino pyrrolopyrimidine
derivative 11f was the most potent with IC50 2.71 µM under hypoxic conditions. It was obvious that
the presence of carboxylic group in o-position in 11f increased the activity. Replacement of the
carboxylic group with methyl group in different positions in compounds 11a-c decreased the activity
where the m-methyl derivative 11b is nearly with the same activity as AZA while the p-methyl
derivative 11a and the o-methyl derivative 11c showed lower activity with IC50 3.17, 6.65 and 13.33
µM, respectively. The pyrrolopyrimidinone 8 was the least active as anticancer agent among the
tested compounds.
11

The cytotoxic concentration that kills 50% of normal cells WI-38 for the test compounds was
determined by MTT cytotoxic assay. All the test compounds exhibited selectivity towards cancer
cells than normal cells.
2.3.Molecular modeling and docking results
Most of the synthesized compounds showed promising activity against hCA I, II, IX and XII
isoforms. The transmembranal, tumor associated isoenzyme hCA IX is our target enzyme as the
candidates showed excellent inhibition activity against this isoform. The molecular docking of the
newly synthesized compounds within the active site of hCA IX isoenzyme was performed and the
amino acids interactions and docking patterns were investigated using the protein data bank file (PDB
ID: AI3I). The file contains hCA IX enzyme co-crystallized with AZA. The docking procedures were
performed by Molecular Operating Environment (MOE, 2015.10) software. Docking setup was first
validated by self-docking of the co-crystallized ligand (acetazolamide) in the vicinity of the binding
site of the enzyme, with energy score (S) = -9.96 kcal/mol and root mean standard deviation (RMSD)
= 0.59. AZA interacts with the active site of hCA IX by one hydrogen bond between Thr 199 and
SO2 group, one hydrogen bond between Thr 200 and N3 of thiadiazole ring, one hydrogen bond
between Leu 198 and thiadiazole ring and two interactions between Zn and nitrogen atom of NH2
group and oxygen atom of SO2 group (coordination) (Fig. 3, 4).
12

Leu
198
Thr
200
4.34
444...33344
.21
4.3
44...222111
4.21
Thr
199
H
2.84
222...888444
2.84
Val
131
2.85
222...888555
2.85
N
N
Val
143
O
HN
S
S
-NH
O
O
Glu
106
2.05
222...000555
2.05
2.88
222...888888
2.88
ZN
262
His
119
Gln
92
Val
121
Pro
201
His
94
His
96
Trp
209
Fig. 3: 2D interaction diagram showing AZA docking pose interactions with CAIX binding site.
(a)
(b)
Fig. 4: a) 2D representation, b) 3D representation of the superimposition of the co-crystallized (red)
and the docking pose (green) of AZA in CA IX binding site with RMSD of 0.59 A°.
13

Docking of all the newly synthesized compounds was performed and showed proper fitting in the
active site of hCA IX with good energy scores (S), which supports the observed activity of these
sulfonamide derivatives as hCA IX inhibitors. The energy score (S) and amino acid interactions of
the most potent hCA IX inhibitors are listed in Table 3.
Table 3: Docking results of the synthesized sulfonamide derivatives with hCA IX isoform
S
Amino
acids
Interacting
groups
O (S)
NH (S)
Pyrrole
O (S)
Length
(A°)
2.29
2.63
3.37
2.81
3.02
1.74
3.22
2.63
1.77
2.11
3.24
4.52
2.44
3.04
2.80
2.15
2.77
3.13
3.41
2.84
2.20
4.40
3.56
2.80
2.88
2.05
4.34
2.85
2.84
Compound
Type of interaction
(kcal/mol)
-11.8161
Metal complex (Zn)
Metal complex (Zn)
H-bond (donor)
H-bond (acceptor)
Metal complex (Zn)
Metal complex (Zn)
H-bond (donor)
H-bond (acceptor)
Metal complex (Zn)
Metal complex (Zn)
H-bond (donor)
8
Gln92
Thr199
-12.0662
-12.4743
O (S)
11a
11b
NH (S)
Pyrrole
O (S)
Gln92
Thr199
O (S)
NH (S)
Pyrrole
Arene-H
O (S)
NH (S)
O (S)
NH (S)
Pyrrole
O (S)
O (S)
O (S)
NH (S)
Phenyl
O (S)
Gln92
Leu135
Thr199
Trp209
Pyrimidine
H-bond (acceptor)
H-bond (acceptor)
Metal complex (Zn)
Metal complex (Zn)
H-bond (donor)
H-bond (acceptor)
H-bond (acceptor)
Metal complex (Zn)
Metal complex (Zn)
Arene-H
H-bond (acceptor)
H-bond (acceptor)
Metal complex (Zn)
Metal complex (Zn)
Arene-H
-12.4018
-10.3689
-7.3279
11c
11f
Gln92
Thr199
Thr199
Gln92
Thr199
Thr200
O (S)
O (S)
NH (S)
Thiadiazole
O (S)
AZA
Leu198
Thr199
Thr200
H-bond (acceptor)
N (Thiadiazole) H-bond (acceptor)
All compounds showed interaction with the Zn ion through sulfonamide group in coordinate
bonds by the deprotonated sulfonamide moiety. Moreover, interactions with Thr 199 through
14

hydrogen bond were observed. Compound 11c formed two hydrogen bonds with Thr 199. Compound
11f formed a hydrogen bond with Thr 200 which mimics the pattern of interaction of AZA with the
enzyme. Moreover, all the docked compounds showed the same orientation within the active site of
CA IX suggesting the binding pattern of these derivatives within CA IX isoform (Fig.
5).ppppppppppppppppppppppppppppp
15

(a)
(b)
(c)
(d)
(e)
Fig. 5: 2D enzyme-ligand interaction of compounds (a) 8, (b) 11a, (c) 11b, (d) 11c, (e) 11f inside
the active site of hCA IX
16

3. Conclusion
In this work, a group of pyrrole and pyrrolopyrimidine sulfonamide derivatives were designed
and synthesized as cytotoxic agents. The new compounds were characterized by spectral procedures,
and microanalytical analyses. All synthesized compounds were tested as CAIs against hCA I and
hCA II (cytosolic isoforms) and hCA IX and hCA XII (transmembrane associated isoforms) using
AZA as the reference drug. All compounds showed activity against hCA IX isoform while showed
less activity against hCA XII than AZA. Sulfonamide 11a was 2 and half fold more potent than AZA
towards hCA IX with Ki value 10.6 nM. The most active CAIs were assessed against HeLa cell line
under normal and hypoxic conditions. The potency of cytotoxicity for compounds had elevated for all
tested derivatives except for 11b. The effect of tested compounds against WI-38 normal cells
revealed much higher IC50 values than HeLa cells. The docking of the synthesized compounds was
carried out and the docked derivatives showed enzyme-ligand binding interactions with the amino
acids within the active site of hCA IX isoform similar to AZA binding interactions. These results
support further studies about these new derivatives.
4. Experimental
4.1. Chemistry
Melting points (oC, uncorrected) were determined on Stuart apparatus and the given values
were uncorrected. Progress of the reaction was monitored by thin layer chromatography (TLC) using
aluminum sheets precoated with UV fluorescent silica gel (MERCK 60 F 254) and spots were
visualized by UV lamp. The solvent system used was dichloromethane: methanol (in different ratios).
The IR spectra (cm-1) were determined using KBr discs on a Shimadzu IR 8400s Spectrophotometer,
1
13
Microanalytical Unit, Faculty of Pharmacy, Cairo University, Cairo, Egypt. H-NMR and C-NMR
spectra were performed on Bruker 400-BB 400 MHz Spectrophotometer, microanalytical unit,
Faculty of Pharamacy, Cairo University, Cairo, Egypt, using tetramethylsilane (TMS) as internal
standard and chemical shift values were recorded in ppm on
δ scales. Peak multiplicities were
designed as follows: s, singlet; d, doublet; t, triplet; m, multiplet. Elemental analyses were performed
at the Regional Center for Mycology and Biotechnology, Faculty of Pharmacy, Al-Azhar University,
Cairo, Egypt.
4.1.1. 4-(2-(4-Chlorophenyl)-2-oxoethylamino)benzenesulfonamide (3)
17

A mixture of sulfanilamide 1 (1.72 g, 0.01 mol) and 4-chloro phenacyl bromide 2 (2.33 g, 0.01
mol) was refluxed in absolute ethanol (20 mL) in presence of catalytic amount of triethylamine for 6
h. The solid obtained was filtered and crystallized from dioxane to give compound 3.
Yield: 85%; M.p.: 205-207 °C; IR (KBr, cm-1): 3475, 3317, 3244 (NH, NH2), 3070 (CH arom.), 2931
1
(CH aliph.), 1685 (C=O), 1311, 1149 (SO2) cm-1. H NMR (DMSO-d6, D2O,
δ
): 4.77 (s, 2H, CH2),
6.60 (s, 1H, NH, D2O exchangeable), 6.93 (s, 2H, NH2, D2O exchangeable), 6.74- 7.53 (2d, 4H, Ar-
H, =8.80 Hz, =8.80 Hz), 7.64-8.10 (2d, 4H, Ar-H, =8.60 Hz, =8.60 Hz) ppm. Anal. Calcd. for
C14H13ClN2O3S (324.78): C, 51.77; H, 4.03; N, 8.63. Found: C, 51.95; H, 4.19; N, 8.90.
J
J
J
J
4.1.2. 4-(2-Amino-4-(4-chlorophenyl)-3-cyano-1H-pyrrol-1-yl)benzenesulfonamide (4)
Sodium ethoxide (sodium metal 0.5g in absolute ethanol 10 mL) was added to a mixture of
compound 3 (3.24 g, 0.01 mol) and malononitrile (0.66 g, 0.01 mol) in dioxane (20 mL) then was
refluxed for 8 h. The reaction mixture was cooled and acidified with dil. HCl till litmus paper is
acidic. The obtained solid was filtered and crystallized from dioxane to give compound 4
Yield: 72%; M.p.:192-194 °C; IR (KBr, cm-1): 3344, 3240 (NH2), 3093 (CH arom.), 2187 (C
1319, 1165 (SO2) cm-1. 1H NMR (DMSO-d6, D2O,
): 6.14 (s, 2H, NH2, D2O exchangeable), 7.03 (s,
1H, pyrrole), 7.34-7.97 (m, 10H, Ar-H + SO2NH2) ppm. C NMR (DMSO-d6,
≡N),
δ
13
δ
): 114.09, 118.10,
121.82, 125.72, 127.51, 127.64, 129.20, 130.34, 131.55, 132.51, 140.05, 143.4, 149.01 ppm. Anal.
Calcd. for C17H13ClN4O2S (372.83): C, 54.77; H, 3.51; N, 15.03. Found: C, 55.01; H, 3.70; N, 15.21.
4.1.3. 2-Amino-4-(4-chlorophenyl)-1-(4-sulfamoylphenyl)-1H-pyrrole-3-carboxylic acid (5)
Compound 4 (3.72 g, 0.01 mol) was heated with aqueous sulfuric acid (60 mL, 60% H2SO4) at
o
100 C for 24 h. The reaction mixture was poured into ice /water and the formed solid was
crystallized from ethanol to give compound 5.
Yield: 65%; M.p.: 242-244 °C; IR (KBr, cm-1): 3550-2720 (OH, NH2), 3130 (CH arom.), 1683
1
(C=O), 1328, 1171 (SO2) cm-1. H NMR (DMSO-d6, D2O,
δ
): 5.56 (s, 2H, NH2, D2O exchangeable),
7.58 (s, 1H, pyrrole), 7.04-8.04 (m, 10H, Ar-H + SO2NH2), 9.85 (s, 1H, OH, D2O exchangeable)
ppm. 13C NMR (DMSO-d6,
δ
): 113.10, 115.99, 125.12, 126.09, 127.89, 129.66, 130.09, 131.61,
18

138.53, 144.41, 159.25, 164.81, 166.95 ppm. Anal. Calcd. for C17H14ClN3O4S (391.83): C, 52.11; H,
3.60; N, 10.72. Found C, 52.38; H, 3.76; N, 10.95.
4.1.4. Ethyl-2-amino-4-(4-chlorophenyl)-1-(4-sulfamoylphenyl)-1H-pyrrole-3-carboxylate (6)
A mixture of compound 5 (3.91 g, 0.01 mol) and absolute ethanol (20 mL) was refluxed in the
presence of catalytic amount of conc. H2SO4 for 8 h. The reaction mixture was poured onto sodium
carbonate solution and the formed solid was crystallized from ethanol to give compound 6
Yield: 60%; M.p.: 269-271 °C; IR (KBr, cm-1): 3392, 3300 (NH2), 3096 (CH arom.), 2982 (CH
aliph.), 1723 (C=O), 1336, 1163 (SO2) cm-1. 1H NMR (DMSO-d6, D2O,
Hz, CH3), 3.77 (q, 2H,
= 7.04, 7.08 Hz, CH2), 7.34-7.98 (m, 13H, Ar-H + NH2 + SO2NH2) ppm. 13C
NMR (DMSO-d6, ): 14.57, 61.51, 118.00, 121.60, 127.90, 129.07, 129.40, 130.76, 131.43, 134.30,
δ): 1.11 (t, 3H, J = 7.04, 7.08
J
δ
136.30, 138.65, 140.50, 145.10, 165.90 ppm. Anal. Calcd. for C19H18ClN3O4S (419.88): C, 54.35; H,
4.32; N, 10.01. Found C, 54.62; H, 4.59; N, 10.27.
4.1.5. General procedures for synthesis of compounds (7a-g)
A mixture of compound 4 (3.72 g, 0.01 mol) and the appropriate aromatic aldehyde (0.01 mol) in
absolute ethanol (20 mL) and glacial acetic acid (1 mL) was refluxed for 2-6 h. The obtained solid
was filtered and crystallized from ethanol to give compounds 7a-g respectively.
4.1.5.1. 4-(2-(Benzylideneamino)-4-(4-chlorophenyl)-3-cyano-1H-pyrrol-1-yl) benzenesulfonamide
(7a)
Yield: 70% (2 h); M.p.: 257-259 °C; IR (KBr, cm-1): 3387, 3271 (NH2), 3101 (CH arom.), 2206
1
(C
≡
N), 1697 (C=N), 1338, 1157 (SO2) cm-1. H NMR (DMSO-d6, D2O,
δ): 7.51 (s, 1H, pyrrole),
13
7.54-7.99 (m, 15 H, Ar-H + SO2NH2), 9.14 (s, 1H, N=CH) ppm. C NMR (DMSO-d6,
δ
): 79.48,
117.27, 120.22, 124.66, 126.20, 127.09, 128.23, 129.50, 129.64, 129.66, 131.44, 132.65, 133.40,
135.44, 139.90, 143.61, 147.24, 164.04 ppm. Anal. Calcd. for C24H17ClN4O2S (460.94): C, 62.54; H,
3.72; N, 12.16. Found: C, 62.31; H, 3.94; N, 12.39.
4.1.5.2. 4-[4-(4-Chlorophenyl)-3-cyano-2-(4-methoxybenzylideneamino)-1H-pyrrol-1-yl]
benzenesulfonamide (7b)
19

Yield: 80% (2 h); M.p.: 248-250 °C; IR (KBr, cm-1): 3344, 3259 (NH2), 3101 (CH arom.), 2935 (CH
aliph.), 2206 (C
≡
N), 1593 (C=N), 1311, 1153 (SO2) cm-1. 1H NMR (DMSO-d6, D2O,
δ
): 3.86 (s, 3H,
13
OCH3), 7.50 (s, 1H, pyrrole), 7.09-7.98 (m, 14H, Ar-H + SO2NH2), 9.04 (s, 1H, N=CH) ppm. C
NMR (DMSO-d6, δ): 56.09, 78.94, 114.40, 115.20, 117.46, 119.69, 124.39, 126.12, 127.05, 128.18,
129.48, 131.75, 132.53, 137.10, 140.01, 143.48, 148.02, 163.43, 163.68 ppm. Anal. Calcd. for:
C25H19ClN4O3S (490.96): C, 61.16; H, 3.90; N, 11.41. Found: C, 60.89; H, 4.13; N, 11.57.
4.1.5.3. 4-[4-(4-Chlorophenyl)-3-cyano-2-(2-hydroxybenzylideneamino)-1H-pyrrol-1-
yl]benzenesulfonamide (7c)
Yield: 75% (2 h); M.p.: 287-289 °C; IR (KBr, cm-1): 3410, 3255, 3147 (OH, NH2), 3074 (CH arom.),
1
2206 (C
≡
N), 1600 (C=N), 1327, 1165 (SO2) cm-1. H NMR (DMSO-d6, D2O,
δ
): 7.53 (s, 1H,
pyrrole), 6.93-7.99 (m, 14H, Ar-H + SO2NH2), 9.41 (s, 1H, N=CH), 10.91 (s, 1H, OH, D2O
exchangeable) ppm. 13C NMR (DMSO-d6,
): 79.44, 117.24, 120.23, 120.90, 124.76, 126.47, 127.15,
δ
128.29, 129.51, 130.00, 131.46, 132.64, 134.00, 135.17, 139.82, 141.00, 143.87, 146.77, 160.06,
161.98 ppm. Anal. Calcd. for: C24H17ClN4O3S (476.93): C, 60.44; H, 3.59; N, 11.75. Found C, 60.67;
H, 3.80; N, 11.92.
4.1.5.4. 4-[4-(4-Chlorophenyl)-3-cyano-2-(4-hydroxy-3-methoxybenzylideneamino)-1H-pyrrol-1-yl]
benzenesulfonamide (7d)
Yield: 70% (6 h); M.p.: 232-234 °C; IR (KBr, cm-1): 3387 (OH), 3267, 3113 (NH2), 3074 (CH
1
arom.), 2974 (CH aliph.), 2210 (C
D2O,
1H, N=CH), 10.12 (s, 1H, OH, D2O exchangeable) ppm. C NMR (DMSO-d6,
≡
N), 1585 (C=N), 1338, 1161 (SO2) cm-1. H NMR (DMSO-d6,
δ
): 3.80 (s, 3H, OCH3), 7.50 (s, 1H, pyrrole), 6.92 – 7.98 (m, 13H, Ar-H + SO2NH2), 8.96 (s,
13
δ
): 56.08, 78.69,
111.62, 116.21, 117.51, 119.44, 124.35, 125.44, 126.14, 126.97, 128.17, 129.48, 131.61, 132.51,
140.04, 143.42, 148.07, 148.64, 152.36, 163.35 ppm. Anal. Calcd. for: C25H19 ClN4O4S (506.96): C,
59.23; H, 3.78; N, 11.05. Found C, 59.50; H, 3.91; N, 11.23.
4.1.5.5. 4-[4-(4-Chlorophenyl)-3-cyano-2-(3-nitrobenzylideneamino)-1H-pyrrol-1-yl]
benzenesulfonamide (7e)
Yield: 60% (6 h); M.p.: 238-240 °C; IR (KBr, cm-1): 3383, 3263 (NH2), 3089 (CH arom.),
2214(C
≡
N), 1593 (C=N), 1319, 1157 (SO2) cm-1. 1H NMR (DMSO-d6, D2O,
δ): 7.53 (s, 1H, pyrrole),
20

7.58 -8.68 (m, 14H, Ar-H + SO2NH2), 9.31 (s, 1H, N=CH) ppm. 13C NMR (DMSO-d6,
δ): 80.19,
100.08, 116.97, 121.00, 123.89, 125.00, 126.29, 127.13, 128.29, 129.54, 131.25, 131.32, 132.79,
135.05, 137.02, 139.75, 143.80, 146.03, 148.80, 161.60 ppm. Anal. Calcd. for C24H16ClN5O4S
(505.93): C, 56.98; H, 3.19; N, 13.84. Found C, 57.14; H, 3.46; N, 14.07.
4.1.5.6. 4-[4-(4-Chlorophenyl)-3-cyano-2-(4-nitrobenzylideneamino)-1H-pyrrol-1-yl]
benzenesulfonamide (7f)
Yield: 60% (6 h); M.p.: 294-296 °C; IR (KBr, cm-1): 3375, 3267 (NH2), 3074 (CH arom.), 2214
(C
≡
N), 1593 (C=N), 1327, 1165 (SO2) cm-1. 1H NMR (DMSO-d6, D2O,
δ): 7.53 (s, 1H, pyrrole), 7.58
13
-8.37 (m, 14H, Ar-H + SO2NH2), 9.29 (s, 1H, N=CH) ppm. C NMR (DMSO-d6,
δ
): 80.30, 116.99,
121.37, 124.74, 125.22, 126.37, 127.14, 128.31, 129.55, 130.50, 131.19, 132.84, 139.71, 140.91,
143.82, 145.83, 149.90, 161.10 ppm. Anal. Calcd. for: C24H16ClN5O4S (505.93): C, 56.98; H, 3.19;
N, 13.84. Found C, 57.11; H, 3.42; N, 13.75.
4.1.5.7. 4-[4-(4-Chlorophenyl)-3-cyano-2-(4-(dimethylamino)benzylideneamino)-1H-pyrrol-1-
yl]benzenesulfonamide (7g)
Yield: 65% (6 h); M.p.: 238-240 °C; IR (KBr, cm-1): 3348, 3251 (NH2), 3082 (CH arom.), 2904 (CH
aliph.), 2206 (C
≡
N), 1585 (C=N), 1311, 1153 (SO2) cm-1. 1H NMR (DMSO-d6, D2O,
δ
): 3.04 (s, 6H,
13
2CH3), 7.50 (s, 1H, pyrrole), 6.78-7.97 (m, 14H, Ar-H + SO2NH2), 8.88 (s, 1H, N=CH) ppm. C
NMR (DMSO-d6, δ): 40.60, 78.08, 111.52, 112.07, 117.87, 118.85, 122.73, 124.12, 126.00, 126.97,
128.10, 129.42, 131.66, 131.77, 132.36, 140.22, 143.22, 149.19, 153.87, 163.16 ppm. Anal. Calcd.
for C26H22ClN5O2S (504.00): C, 61.96; H, 4.40; N, 13.90. Found C, 61.82; H, 4.56; N, 14.13.
4.1.6. 4-(5-(4-Chlorophenyl)-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-7(4H)-yl) benzenesulfonamide (8)
A solution of compound 4 (3.72 g, 0.01 mol) in formic acid (20 mL) was refluxed for 6 h. The
reaction mixture was poured onto ice/cold water and the obtained solid was crystallized from ethanol
to give compound 8
Yield: 80%; M.p.: 264-266 °C; IR (KBr, cm-1): 3348, 3300, 3250 (NH, NH2), 3050 (CH arom.), 1657
1
(C=O), 1328, 1163 (SO2) cm-1. H NMR (DMSO-d6, D2O,
δ): 7.41-8.08 (m, 10H, Ar-H + SO2NH2),
13
7.45 (s, 1H, pyrrole), 8.00 (s, 1H, pyrimidine), 12.31 (s, 1H, NH/OH, D2O exchangeable) ppm. C
21

NMR (DMSO-d6, δ):106.90, 120.54, 122.12, 124.99, 127.20, 128.46, 130.47, 131.68, 132.44, 139.99,
142.80, 145.43, 148.73, 158.93 ppm. Anal. Calcd. for C18H13ClN4O3S (400.84): C, 53.94; H, 3.27; N,
13.98. Found: C, 54.08; H, 3.54; N, 14.19.
4.1.7. 4-(4-Chloro-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl) benzenesulfonamide (9)
A mixture of compound 8 (4.00 g, 0.01 mol) and phosphorous oxychloride (60 mL) was refluxed
for 8 h. The reaction mixture was cooled then poured onto ice/water. The formed solid was filtered
and crystallized from ethanol to give compound 9.
Yield: 85%; M.p.: 239-241 °C; IR (KBr, cm-1): 3352, 3232 (NH2), 3078 (CH arom.), 1330, 1161
(SO2) cm-1. 1H NMR (DMSO-d6, D2O,
8.05 (s, 1H, pyrimidine) ppm. 13C NMR (DMSO-d6,
δ
): 7.56 (s, 1H, pyrrole), 7.41-8.80 (m, 10H, Ar-H + SO2NH2),
): 116.07, 123.9, 124.97, 126.85, 127.06, 127.4,
δ
128.43, 129.36, 130.22, 132.51, 132.80, 133.15, 143.09, 151.45 ppm. Anal. Calcd. for
C18H12Cl2N4O2S (419.28): C, 51.56; H, 2.88; N, 13.36. Found: C, 51.82; H, 3.12; N, 13.59.
4.1.8. 4-(5-(4-Chlorophenyl)-4-hydrazinyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl) benzenesulfonamide
(10)
A mixture of compound 9 (4.19 g, 0.01 mol) and hydrazine hydrate (99%, 3mL) in absolute
ethanol (20 mL) was heated under reflux for 8 h. The reaction mixture was cooled, filtered and the
precipitate was dried and crystallized from dimetylformamide to give compound 10.
Yield: 75%; M.p.: 226-228 °C; IR (KBr, cm-1): 3350, 3217, 3190 (NH, NH2), 3078 (CH arom.),
1327, 1161 (SO2) cm-1. 1H NMR (DMSO-d6, D2O,
δ
): 7.58 (s, 1H, pyrrole), 8.00 (s, 1H, pyrimidine),
13
7.45-8.41 (m, 12H, Ar-H + NH2 + SO2NH2), 12.30 (s, 1H, NH, D2O exchangeable) ppm. C NMR
(DMSO-d6, δ):106.88, 120.55, 122.09, 125.01, 127.22, 128.45, 129.39, 131.70, 132.40, 139.98,
142.77, 145.41, 148.7, 158.93 ppm. Anal. Calcd. for C18H15ClN6O2S (414.87): C, 52.11; H, 3.64; N,
20.26. Found C, 52.38; H, 3.51; N, 20.17.
4.1.9. General procedures for synthesis of compounds (11a-e)
A mixture of compound 9 (4.19 g, 0.01 mol) and the appropriate amine (0.01 mol) in dry pyridine
(20 mL) was heated under reflux for 12 h and poured onto ice/cold water then acidified with HCl.
22

The obtained solid was filtered and crystallized from the appropriate solvent to give compounds 11a-
e respectively.
4.1.9.1. 4-(5-(4-Chlorophenyl)-4-(p-tolylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)
benzenesulfonamide (11a)
Yield: 80%; M.p.: 207-209 °C; IR (KBr, cm-1): 3348, 3217, 3194 (NH, NH2), 3078 (CH arom.), 2924
1
(CH aliph.), 1330, 1161 (SO2) cm-1. H NMR (DMSO-d6, D2O,
δ
): 2.12 (s, 3H, CH3), 7.48 (s, 1H,
pyrrole), 8.05 (s, 1H, pyrimidine), 6.46-8.58 (m, 14H, Ar-H + SO2NH2), 12.31 (s, 1H, NH, D2O
exchangeable) ppm. 13C NMR (DMSO-d6,
): 20.57, 106.89, 114.55, 120.55, 122.55, 124.50, 127.22,
δ
128.47, 129.42, 130.76, 131.70, 132.43, 136.66, 139.99, 141.89, 142.79, 145.43, 148.73, 158.93 ppm.
Anal. Calcd. for C25H20ClN5O2S (489.98): C, 61.28; H, 4.11; N, 14.29. Found C, 61.04; H, 4.32; N,
14.52.
4.1.9.2. 4-(5-(4-Chlorophenyl)-4-(m-tolylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)
benzenesulfonamide (11b)
Yield: 85%; M.p.: 201-203 °C; IR (KBr, cm-1): 3350, 3230, 3190 (NH, NH2), 3090 (CH arom.), 2950
1
(CH aliph.), 1330, 1160 (SO2) cm-1. H NMR (DMSO-d6, D2O,
δ
): 2.21 (s, 3H, CH3), 7.58 (s, 1H,
pyrrole), 8.13 (s, 1H, pyrimidine), 7.44-8.94 (m, 14H, Ar-H + SO2NH2), 12.33 (s, 1H, NH, D2O
exchangeable) ppm. 13C NMR (DMSO-d6,
): 21.24, 100.71, 119.04, 124.26, 125.01, 127.68, 128.44,
δ
129.36, 129.73, 130.47, 131.07, 133.25, 138.92, 142.42, 143.51, 144.50, 145.37, 146.64, 148.16,
152.20, 158.92 ppm. Anal. Calcd. for C25H20ClN5O2S (489.98): C, 61.28; H, 4.11; N, 14.29. Found
C, 60.99; H, 4.28; N, 14.50.
4.1.9.3. 4-(5-(4-Chlorophenyl)-4-(o-tolylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)
benzenesulfonamide (11c)
Yield: 80%; M.p.: 262-264°C; IR (KBr, cm-1): 3357, 3210, 3183 (NH, NH2), 3050 (CH arom.),
1
2910(CH aliph.), 1330, 1165 (SO2) cm-1. H NMR (DMSO-d6, D2O,
δ
): 2.31 (s, 3H, CH3), 7.60 (s,
1H, pyrrole), 8.04 (s, 1H, pyrimidine), 7.44-8.48 (m, 14H, Ar-H + SO2NH2), 12.32 (s, 1H, NH, D2O
exchangeable) ppm. 13C NMR (DMSO-d6,
): 21.00, 100.92, 106.85, 118.62, 120.55, 122.09, 124.23,
δ
124.99, 125.78, 126.88, 127.23, 128.46, 129.66, 130.82, 131.72, 133.07, 139.97, 142.75, 145.40,
23

148.71, 158.00 ppm. Anal. Calcd. for C25H20ClN5O2S (489.98): C, 61.28; H, 4.11; N, 14.29. Found
C, 61.13; H, 4.34; N, 14.43.
4.1.9.4. 4-(5-(4-Chlorophenyl)-4-((4-methoxyphenyl)amino)-7H-pyrrolo[2,3-d] pyrimidin-7-
yl)benzenesulfonamide (11d)
Yield: 75%; M.p.: 216-218 °C; IR (KBr, cm-1): 3475, 3344, 3224 (NH, NH2), 3074 (CH arom.), 2931
(CH aliph.), 1330, 1161 (SO2) cm-1. 1H NMR (DMSO-d6, D2O,
): 3.75 (s, 3H, OCH3), 7.58 (s, 1H,
pyrrole), 7.41-8.08 (m, 14H, Ar-H + SO2NH2), 8.59 (s, 1H, pyrimidine), 12.31 (s, 1H, NH, D2O
exchangeable) ppm. 13C NMR (DMSO-d6,
): 55.68, 106.88, 115.16, 120.59, 122.01, 123.83, 124.98,
δ
δ
127.24, 129.41, 130.46, 131.72, 132.34, 136.98, 139.99, 142.71, 145.34, 148.70, 149.74, 158.93 ppm.
Anal. Calcd. for C25H20ClN5O3S (505.98): C, 59.34; H, 3.98; N, 13.84. Found C, 59.60; H, 3.79; N,
14.05.
4.1.9.5. 4-(5-(4-Chlorophenyl)-4-((4-chlorophenyl)amino)-7H-pyrrolo[2,3-d] pyrimidin-7-
yl)benzenesulfonamide (11e)
Yield: 75%; M.p.: 234-236 °C; IR (KBr, cm-1): 3474, 3300, 3232 (NH, NH2), 3072 (CH arom.),
1328, 1161 (SO2) cm-1. 1H NMR (DMSO-d6, D2O,
): 7.58 (s, 1H, pyrrole), 7.44-8.80 (m, 14H, Ar-H
+ SO2NH2), 8.38 (s, 1H, pyrimidine), 12.31 (s, 1H, NH, D2O exchangeable) ppm. 13C NMR (DMSO-
d6, ): 101.40, 116.03, 117.55, 123.99, 125.00, 127.30, 128.99, 129.45, 130.46, 130.75, 132.59,
δ
δ
138.17, 140.25, 142.06, 148.88, 150.66, 152.14, 157.39 ppm. Anal. Calcd. for C24H17Cl2N5O2S
(510.40): C, 56.48; H, 3.36; N, 13.72. Found C, 56.73; H, 3.48; N, 13.98.
4.1.9.6. 2-((5-(4-Chlorophenyl)-7-(4-sulfamoylphenyl)-7H-pyrrolo[2,3-d] pyrimidin-4-
yl)amino)benzoic acid (11f)
Yield: 70%; M.p.: 229-231 °C; IR (KBr, cm-1): 3474, 3300, 3232 (OH, NH, NH2), 3074 (CH arom.),
1
1662 (C=O), 1327, 1161 (SO2) cm-1. H NMR (DMSO-d6, D2O,
δ
): 7.59 (s, 1H, pyrrole), 7.96-8.15
(m, 14H, Ar-H + SO2NH2), 8.34 (s, 1H, pyrimidine), 9.20 (s, 1H, OH, D2O exchangeable), 12.31 (s,
1H, NH, D2O exchangeable) ppm. 13C NMR (DMSO-d6,
): 101.25, 106.88, 117.88, 120.56, 122.07,
δ
124.99, 127.32, 128.44, 129.50, 130.46, 131.70, 132.38, 132.41, 132.73, 139.98, 142.42, 142.76,
145.39, 148.71, 156.21, 158.92 ppm. Anal. Calcd. for C25H18ClN5O4S (519.96): C, 57.75; H, 3.49; N,
13.47. Found C, 58.01; H, 3.71; N, 13.68.
24

4.1.9.7. 4-(4-(Benzylamino)-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)
benzenesulfonamide (11g)
Yield: 70%; M.p.: 140-142 °C; IR (KBr, cm-1): 3400, 3379, 3300 (NH, NH2), 3062 (CH arom.), 2970
1
(CH aliph.), 1327, 1161 (SO2) cm-1. H NMR (DMSO-d6, D2O,
δ
): 4.72 (s, 2H, CH2), 7.60 (s, 1H,
pyrrole), 7.24-8.34 (m, 15H, Ar-H + SO2NH2), 8.48 (s, 1H, pyrimidine), 12.31 (s, 1H, NH, D2O
exchangeable) ppm. 13C NMR (DMSO-d6,
): 44.47, 100.69, 101.72, 117.29, 118.58, 124.91, 127.28,
δ
127.51, 128.76, 130.84, 131.68, 132.88, 133.02, 135.05, 139.33, 140.29, 142.13, 143.20, 158.92 ppm.
Anal. Calcd. for C25H20ClN5O2S (489.98): C, 61.28; H, 4.11; N, 14.29. Found C, 61.45; H, 4.40; N,
14.50
4.1.10. General procedures for synthesis of compounds (12a-g)
A mixture of compound 10 (4.14 g, 0.01 mol) and the appropriate aromatic aldehyde (0.01 mol) in
absolute ethanol (20 mL) and small amount of glacial acetic acid (1 mL) was refluxed for 6 h. the
obtained solid was filtered off and recrystallized from the appropriate solvent to give compounds
12a-g respectively.
4.1.10.1. (E/Z) 4-(4-(2-Benzylidenehydrazinyl)-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d] pyrimidin-7-
yl)benzenesulfonamide (12a)
Yield: 65%; M.p.: 276-278 °C; IR (KBr, cm-1): 3350, 3317, 3300 (NH, NH2), 3070 (CH arom.),
1
1334, 1161 (SO2) cm-1. H NMR (DMSO-d6, D2O,
δ
): 6.85-8.20 (m, 15H, Ar-H + SO2NH2), 7.48 (s,
1H, pyrrole), 8.18 (s, 1H, pyrimidine), 8.33 (s, ½ H, ½ N=CH), 8.43 (s, ½ H, ½ N=CH), 11.16 (s, ½
13
H, ½ NH, D2O exchangeable), 11.97(s, ½ H, ½ NH, D2O exchangeable) ppm. C NMR (DMSO-d6,
δ
): 101.68, 103.22, 118.67, 120.47, 122.29, 124.27, 125.32, 126.69, 128.21, 130.82, 131.62, 133.03,
134.56, 136.06, 142.21, 143.48, 145.29, 149.83, 151.86, 152.73, 156.67 ppm. Anal. Calcd. for
C25H19ClN6O2S (502.98): C, 59.70; H, 3.81; N, 16.71. Found C, 59.87; H, 4.03; N, 16.89.
4.1.10.2. (E/Z) 4-(5-(4-Chlorophenyl)-4-(2-(4-methoxybenzylidene)hydrazinyl)-7H-pyrrolo[2,3-
d
]pyrimidin-7-yl)benzenesulfonamide (12b)
Yield: 75%; M.p.: 152-154 °C; IR (KBr, cm-1): 3479, 3350, 3309 (NH, NH2), 3074 (CH arom.), 2966
(CH aliph.), 1323, 1165 (SO2) cm-1. 1H NMR (DMSO-d6, D2O,
δ): 3.83 (s, 3H, OCH3), 7.05-8.08 (m,
25

14H, Ar-H + SO2NH2), 7.45 (s, 1H, pyrrole), 8.00 (s, 1H, pyrimidine), 8.63 (s, ¾ H, ¾ N=CH), 9.87
13
(s, ¼ H, ¼ N=CH), 12.31 (s, 1H, NH, D2O exchangeable) ppm. C NMR (DMSO-d6,
δ): 55.81,
106.83, 114.84, 114.95, 120.58, 122.03, 125.03, 126.88, 127.24, 128.45, 130.01, 130.48, 131.74,
132.33, 139.96, 142.69, 145.34, 148.69, 158.94, 161.01, 162.14, 164.70 ppm. Anal. Calcd. for
C26H21ClN6O3S (533.00): C, 58.59; H, 3.97; N, 15.77. Found C, 58.38; H, 4.11; N, 15.89.
4.1.10.3. 4-(5-(4-Chlorophenyl)-4-(2-(2-hydroxybenzylidene)hydrazinyl)-7
]pyrimidin-7-yl)benzenesulfonamide (12c)
H-pyrrolo[2,3-
d
Yield: 75%; M.p.: 213-215 °C; IR (KBr, cm-1): 3583, 3332, 3305 (OH, NH, NH2), 3008 (CH arom.),
1315, 1195 (SO2) cm-1. 1H NMR (DMSO-d6, D2O,
): 7.42 (s, 1H, pyrrole), 7.94 (s, 1H, pyrimidine),
6.80-7.78 (m, 14H, Ar-H + SO2NH2), 9.01 (s, 1H, N=CH), 11.13 (s, 1H, NH, D2O exchangeable),
11.40 (s, 1H, OH, D2O exchangeable) ppm. 13C NMR (DMSO-d6,
): 116.15, 116.98, 118.52, 119.51,
δ
δ
120.04, 120.16, 126.95, 128.69, 129.24, 131.43, 133.79, 142.89, 156.91, 159.03, 163.37 ppm. Anal.
Calcd. for C25H19ClN6O3S (518.97): C, 57.86; H, 3.69; N, 16.19. Found C, 58.09; H, 3.84; N, 16.45.
4.1.10.4. 4-(5-(4-Chlorophenyl)-4-(2-(4-hydroxy-3-methoxybenzylidene) hydrazinyl)-7
H-
pyrrolo[2,3- ]pyrimidin-7-yl)benzenesulfonamide (12d)
d
Yield: 70%; M.p.: 278-280 °C; IR (KBr, cm-1): 3475, 3332, 3236 (OH, NH, NH2), 3078 (CH arom.),
2939 (CH aliph.), 1334, 1161 (SO2) cm-1. 1H NMR (DMSO-d6, D2O,
): 3.80 (s, 3H, OCH3), 7.50 (s,
δ
1H, pyrrole), 6.87 – 8.12 (m, 13H, Ar-H + SO2NH2), 8.10 (s, 1H, pyrimidine), 8.58 (s, 1H, N=CH),
9.77 (s, 1H, OH, D2O exchangeable), 12.30 (s, 1H, NH, D2O exchangeable) ppm. 13C NMR (DMSO-
d6, δ): 55.98, 110.50, 116.15, 116.98, 118.52, 119.51, 120.04, 120.16, 123.96, 125.96, 126.95, 128.69,
129.24, 131.43, 133.79, 142.89, 148.44, 150.33, 156.91, 159.03, 161.10, 163.37 ppm. Anal. Calcd.
for C26H21ClN6O4S (549.00): C, 56.88; H, 3.86; N, 15.31. Found C, 57.04; H, 4.09; N, 15.48.
4.1.10.5. 4-(5-(4-Chlorophenyl)-4-(2-(3-nitrobenzylidene)hydrazinyl)-7H-pyrrolo [2,3-d]pyrimidin-7-
yl)benzenesulfonamide (12e)
Yield: 80%; M.p.: 170-172 °C; IR (KBr, cm-1): 3321, 3250, 3182 (NH, NH2), 3086 (CH arom.),
1
1346, 1161 (SO2) cm-1. H NMR (DMSO-d6, D2O,
δ
): 7.45-8.38 (m, 14H, Ar-H + SO2NH2), 7.78 (s,
1H, pyrrole), 8.71 (s, 1H, pyrimidine), 8.91 (s, 1H, N=CH), 12.30 (s, 1H, NH, D2O exchangeable)
ppm. 13C NMR (DMSO-d6,
δ
): 119.23, 121.79, 123.05, 126.22, 127.24, 127.93, 130.44, 131.06,
26

131.51, 134.90, 135.29, 135.67, 139.00, 148.61, 148.64, 160.90, 172.47. Anal. Calcd. for
C25H18ClN7O4S (547.97): C, 54.80; H, 3.31; N, 17.89. Found C, 54.96; H, 3.57; N, 17.69.
4.1.10.6. 4-(5-(4-Chlorophenyl)-4-(2-(4-nitrobenzylidene)hydrazinyl)-7H-pyrrolo [2,3-d]pyrimidin-7-
yl)benzenesulfonamide (12f)
Yield: 65%; M.p.: 285-287 °C; IR (KBr, cm-1): 3350, 3332, 3294 (NH, NH2), 3086 (CH arom.),
1
1346, 1161 (SO2) cm-1. H NMR (DMSO-d6, D2O,
δ
): 7.09-8.88 (m, 14H, Ar-H + SO2NH2), 7.57 (s,
1H, pyrrole), 8.18 (s, 1H, pyrimidine), 8.88 (s, 1H, N=CH), 12.27 (s, 1H, NH, D2O exchangeable)
ppm. 13C NMR (DMSO-d6,
): 101.60, 103.45, 118.87, 122.90, 124.56, 125.30, 128.98, 130.54,
δ
131.65, 133.80, 135.76, 136.06, 141.21, 143.32, 145.76, 149.83, 150.86, 152.73, 154.67 ppm. Anal.
Calcd. for C25H18ClN7O4S (547.97): C, 54.80; H, 3.31; N, 17.89. Found C, 55.03; H; 3.60; N, 17.81.
4.1.10.7. 4-(5-(4-Chlorophenyl)-4-(2-(4-(dimethylamino)benzylidene)hydrazinyl)-7
]pyrimidin-7-yl)benzenesulfonamide (12g)
H-pyrrolo[2,3-
d
Yield: 70%; M.p.: 230-232 °C; IR (KBr, cm-1): 3400, 3355, 3200 (NH, NH2), 3084 (CH arom.), 2911
1
(CH aliph.), 1301, 1160 (SO2) cm-1. H NMR (DMSO-d6, D2O,
δ
): 3.00 (s, 6H, 2CH3), 7.50 (s, 1H,
pyrrole), 7.30-8.55 (m, 14H, Ar-H + SO2NH2), 8.25 (s, 1H, pyrimidine), 8.88 (s, 1H, N=CH), 12.30
(s, 1H, NH, D2O exchangeable) ppm. 13C NMR (DMSO-d6,
): 40.81, 105.83, 113.80, 114.95,
δ
120.30, 121.03, 124.60, 125.58, 127.14, 128.45, 131.48, 132.53, 137.90, 141.92, 144.37, 147.69,
157.45, 161.13, 162.14, 163.70 ppm. Anal. Calcd. for C27H24ClN7O2S (546.04): C, 59.39; H, 4.43; N,
17.96. Found C, 59.51; H, 4.57; N, 18.12.
4.1.11. (E/Z)-Ethyl N-(4-(4-chlorophenyl)-3-cyano-1-(4-sulfamoylphenyl)-1H-pyrrol-2-
yl)formimidate (13)
A mixture of compound 4 (3.72 g, 0.01 mol) and triethylorthoformate (20 mL) was refluxed for 6
h. the reaction mixture was cooled and then poured onto ice /water. The formed solid was filtered and
crystallized from ethanol to give compound 13
Yield: 80%; M.p.: 181-183 °C; IR (KBr, cm-1): 3340, 3232 (NH2), 3075 (CH arom.), 2950 (CH
aliph.), 2201 (C
≡
N), 1330, 1160 (SO2) cm-1. 1H NMR (DMSO-d6, D2O,
δ): 1.04 (t, 3H, J = 7.00, 7.04
Hz, CH3), 3.45 (q, 2H,
J
= 7.00, 7.04 Hz, CH2), 7.68 (s, 1H, pyrrole), 7.47-8.04 (m, 10H, Ar-H +
27

13
SO2NH2), 8.54 (s, ½ H, N=CH), 8.71 (s, ½ H, N=CH) ppm. C NMR (DMSO-d6, δ): 14.54, 56.50,
113.99, 121.81, 125.72, 125.80, 126.27, 127.63, 128.07, 128.65, 129.54, 131.63, 132.41, 144.27,
149.10, 162.56, 162.98 ppm. Anal. Calcd. For C20H17ClN4O3S (428.89): C, 56.01; H, 4.00; N, 13.06.
Found C, 56.23; H, 4.19; N, 13.28.
4.1.12. 4-(3-Amino-5-(4-chlorophenyl)-4-imino-3H-pyrrolo[2,3-d]pyrimidin-7(4H)-
yl)benzenesulfonamide (14) T1
A mixture of compound 13 (4.28 g, 0.01 mol) and hydrazine hydrate (99%, 3mL) in absolute
ethanol (20 mL) was heated under reflux for 8 h. the reaction mixture was cooled and solid formed
was filtered and crystallized from the appropriate solvent to give compound 14
Yield: 75%; M.p.: 173-175 °C; IR (KBr, cm-1): 3344, 3249, 3215 (NH, NH2), 3093 (CH arom.),
1319, 1176 (SO2) cm-1. 1H NMR (DMSO-d6, D2O,
1H, pyrrole), 7.96 (s, 1H, pyrimidine), 7.42-8.50 (m, 11H, Ar-H + NH + SO2NH2). 13C NMR
(DMSO-d6, ): 114.84, 118.09, 121.82, 126.68, 127.97, 129.37, 131.74, 132.51, 133.07, 136.85,
δ): 6.14 (s, 2H, NH2, D2O exchangeable), 7.03 (s,
δ
140.05, 143.39, 149.01, 160.15. Anal. Calcd. For C18H15ClN6O2S (414.87): C, 52.11; H, 3.64; N,
20.26. Found C, 52.39; H, 3.81; N, 20.12.
N-(4-(4-Chlorophenyl)-3-cyano-1-(4-sulfamoylphenyl)-1H-pyrrol-2-yl) acetamide (15)
A solution of compound 4 (3.72 g, 0.01 mol) in acetic anhydride (20 mL) was refuxed for 5 min.
the reaction mixture was concentrated and the solid separated was crystallized from ethanol to give
compound (15)
Yield: 80%; M.p.: 170-172 °C; IR (KBr, cm-1): 3447, 3350, 3260 (NH, NH2), 3101 (CH arom.), 2970
(CH aliph.), 2226 (CN), 1730 (C=O), 1331, 1163 (SO2) cm-1. 1H NMR (DMSO-d6, D2O,
3H, CH3), 7.57 (s, 1H, pyrrole), 7.53-8.02 (m, 10H, Ar-H + SO2NH2), 10.24 (s, 1H, NH, D2O
exchangeable) ppm. 13C NMR (DMSO-d6,
): 22.84, 114.52, 115.63, 121.75, 123.50, 125.81, 127.56,
δ): 2.51 (s,
δ
129.62, 130.82, 131.44, 134.80, 138.61, 139.74, 145.02, 172.37 ppm. Anal. Calcd. For.
C19H15ClN4O3S (414.87): C, 55.01; H, 3.64; N, 13.50. Found C, 55.30; H, 3.76; N, 13.72.
4.1.13. 4-(5-(4-Chlorophenyl)-2-methyl-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-7(4H)-
yl)benzenesulfonamide (16)
28