Pyrazolylbenzo[d]imidazoles as new potent and selective inhibitors of carbonic anhydrase isoforms hCA IX and XII

Article abstract of DOI:10.1016/j.bmc.2016.04.061

Novel pyrazolylbenzo[d]imidazole derivatives (2a-2f) were designed, synthesized and evaluated against four human carbonic anhydrase isoforms belonging to α family comprising of two cytosolic isoforms hCA I and II as well as two transmembrane tumor associated isoforms hCA IX and XII. Starting from these derivatives that showed high potency but low selectivity in favor of tumor associated isoforms hCA IX and XII, we investigated the impact of removing the sulfonamide group. Thus, analogs 3a-3f without sulfonamide moiety were synthesized and biological assay revealed a good activity as well as an excellent selectivity as inhibitors for tumor associated hCA IX and hCA XII and the same was analyzed by molecular docking studies.

Full text of DOI:10.1016/j.bmc.2016.04.061

Accepted Manuscript
Pyrazolylbenzo[d]imidazoles as new potent and selective inhibitors of carbonic
anhydrase isoforms hCA IX and XII
Satish Kumar, Mariangela Ceruso, Tiziano Tuccinardi, Claudiu T. Supuran,
Pawan K. Sharma
PII:
S0968-0896(16)30315-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.04.061
BMC 12976
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 March 2016
27 April 2016
28 April 2016
Please cite this article as: Kumar, S., Ceruso, M., Tuccinardi, T., Supuran, C.T., Sharma, P.K.,
Pyrazolylbenzo[d]imidazoles as new potent and selective inhibitors of carbonic anhydrase isoforms hCA IX and
XII, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.04.061
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Pyrazolylbenzo[d]imidazoles as new potent and selective inhibitors of carbonic
anhydrase isoforms hCA IX and XII
Satish Kumara, Mariangela Cerusob, Tiziano Tuccinardic, Claudiu T. Supuranb*, Pawan K.
Sharmaa*
aDepartment of Chemistry, Kurukshetra University, Kurukshetra-136119, India.
bUniversità degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm 188, and Neurofarba
Department, Sezione di Scienze Farmaceutiche, Via U. Schiff 6, I-50019 Sesto Fiorentino
(Firenze), Italy.
cDepartment of Pharmacy, University of Pisa, Via Bonanno 6, 56126, Pisa, Italy
*Corresponding authors: Tel.: +91 9416457355; Fax: +91 1744 238277; e-mail:
pksharma@kuk.ac.in (PKS); Tel/Fax: +39-055-4573005, e-mail: claudiu.supuran@unifi.it (CTS)
Abstract
Novel pyrazolylbenzo[d]imidazole derivatives (2a-2f) were designed, synthesized and
evaluated against four human carbonic anhydrase isoforms belonging to α family comprising of two
cytosolic isoforms hCA I and II as well as two transmembrane tumor associated isoforms hCA IX
and XII. Starting from these derivatives that showed high potency but low selectivity in favor of
tumor associated isoforms hCA IX and XII, we investigated the impact of removing the
sulfonamide group. Thus, analogs 3a-3f without sulfonamide moiety were synthesized and
biological assay revealed a good activity as well as an excellent selectivity as inhibitors for tumor
associated hCA IX and hCA XII and the same was analyzed by molecular docking studies.
Key words: Carbonic anhydrase isoforms, pyrazolylbenzo[d]imidazoles, acetazolamide, inhibition
constant (Ki)
Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) constitute ubiquitous family of metalloenzymes
containing Zn2+ at their active sites and are widely distributed in living organisms. CAs are
particularly responsible for catalyzing the interconversion of carbon dioxide and water to produce
bicarbonate and protons playing a crucial role in various physiological processes such as respiration
and transport of CO2/bicarbonate between metabolizing tissues and the lungs, pH and CO2
homeostasis, electrolyte secretion, biosynthetic reactions such as gluconeogenesis, lipogenesis and
ureagenesis, bone resorption, calcification, tumorigenicity and many other physiologic or
pathologic processes.1-9
Out of 15 different isoforms of α-class of human associated CAs, hCA I and II are the
cytosolic forms distributed in eye, kidney, central nervous system and inner ear and have been

targeted for clinically used diuretics, antiglaucoma and anticonvulsant drugs.10- 12 hCA IX is a
multidomain protein existing in dimeric form being stabilized by a disulfide bond and is induced by
hypoxic environment in tumor cells being mediated by the hypoxia- inducible transcription factor-1
(HIF-1). hCA XII is also induced by hypoxia but its induction by HIF-1 is not yet well
established.Since hCA IX and XII have been established to contribute to pH regulation of tumor
cells, cell proliferation, cell adhesion and malignant cell invasion, they have been considered as
valuable markers for cancer and are being targeted for designing anticancer drugs.13-16Furthermore,
specifically targeting hCA IX and XII over the physiologically relevant isoforms hCA I and II can
be a promising strategy in developing anticancer drugs with minimum side effects.
Primary sulfonamides containing aromatic/heterocyclic compounds are the most widely
investigated category of carbonic anhydrase inhibitors (CAIs) that led to the development of new
classes of pharmaceutical agents including clinically used CAIs like acetazolamide(AZA),
methazolamide(MZA), ethoxzolamide(EZA)etc (Figure 1).Over the years, azoles also constitute
immensely important members of the biologically active aromatic heterocycles due to their
presence in various CA inhibitors.17 Recently, benzimidazole pharmacophore has been extensively
studied as an important scaffold for the development of novel anticancer agents.18,19 Omeprazole,
pantoprazole, lansoprazole, rabeprazole drugs bearing benzimidazole nucleus in their structure have
been reported to exhibit excellent anticancer activities besides having antiulcer properties (Figure
1).
Figure 1.Some clinically used CA drugs and potential lead compounds

In addition to benzimidazoles, pyrazoles are also known to be more potent and selective for
tumor targeting CA isoforms. Recently, our research group has reported the 4-functionalized
pyrazoles 1 which were found to be selective CA inhibitors against tumor isoforms hCA IX and
XII.20-23Motivated by the these findings coupled with our ongoing interest in heterocyclic scaffolds
of medicinal interest,24-30 we envisioned to synthesize pyrazolylbenzo[d]imidazoles 2a-2f bearing a
benzenesulfonamide moiety as novel scaffold for CA inhibition profile.Excellent inhibition but poor
selectivity profile of 2a-2f prompted us to study the effect of removing the sulfonamide group (3a-
3f) on CA inhibition potency as well as selectivity which was further analyzed by docking studies.
N
N
N
N
H
H
N
N
SO2NH2
H
N
N
R1
R1
3
2
Figure 2.Pyrazolylbenzo[d]imidazoles (2a-2f and 3a-3f) as CA inhibitors
The molecular docking of compound 2a having free SO2NH2 moiety in the active site of hCA XII
was performed in order to find out the possible binding interactions with target enzymes. The
molecular docking of compound 3a(without sulfonamide moiety) against hCA I, II, IX and XII was
also performed in order to find out the possible binding interactions of the selected compound in the
target enzymes which throw some light into the high selectivity of compounds 3 for tumor
associated isozymes hCA IX and XII when compared to compounds 2.
2. Results and discussion
2.1. Chemistry
Synthesis of desired pyrazolylbenzo[d]imidazoles (2)has been accomplished by the
condensation of appropriate 4-formylpyrazoles 10 with ortho-phenylenediamine (12) in refluxing
DMF/H2O using oxone as an oxidant (Scheme 1).Formylpyrazoles 10 in turn were prepared starting
from appropriate acetophenones and phenylhydrazines following our earlier reported procedure.28
In order to study the effect of free sulfonamide moiety on activity and selectivity pattern, analogs 3
(without SO2NH2 moiety) were also designed and synthesized following a similar protocol as
depicted in Scheme 1. It is worthwhile to mention here that synthesis and characterization of all

these pyrazolylbenzo[d]imidazoles (2 and 3) completed in our labs in 2013. While CA inhibition
studies and molecular docking studies of these compounds were underway,Reddy et al31 reported
the synthesis of a few pyrazolylbenzo[d]imidazoles (3a, 3c, 3d, 3e) although via a different route
using sodium metabisulphite. 4-Aminosulfonyl group present at para position of aromatic ring
attached to N-1 of pyrazole ring in case of hydrazones 7 also got protected under Vilsmeier Haack
conditions to afford intermediate 9 which was deprotected under basic conditions in THF-MeOH to
yield 4-formylpyrazoles 10 having free 4-aminosulfonyl group.28 The spectral data of target
compounds 2 and 3 were in full agreement with their proposed structures. IR spectra of these
compounds exhibits characteristic absorption band at ~ 3148 cm-1, 1597 cm-1 and ~ 1512 cm-
1corresponding to N-H stretch, C=N stretch and N-H bending respectively.
R
CH3
HN N C
R1
NHNH2
COCH3
N
N
(i)
(ii)
R1
+
CHO
R = SO2N=CHN(CH3)2
10 R = SO2NH2
11 R = H
R
R1
R
9
(iii)
7 R = SO2NH2
8 R = H
4 R = SO2NH2
5 R = H
6
R1
R
N
N
NH2
R
N
H
(iv)
N
R1
N
+
NH2
N
CHO
12
10 R = SO2NH2
11 R = H
2 R = SO2NH2
3 R = H
Substitution pattern of pyrazolylbenzo[d]imidazoles (2 and 3) and intermediates
R1
Compound
H
CH3
OCH3
F
Cl
Br
4 or 2,7,10 (R = SO2NH2), 3,8,11(R = H)
9 (R = SO2N=CHN(CH3)2)
a
b
c
d
e
f
Scheme 1. Synthesis of pyrazolylbenzo[d]imidazoles (2 and 3): (i) Ethanol-water, CH3COOH (catalytic), reflux, 30 min;
(ii) POCl3/DMF, 50-60 °C, 5-6h; (iii) NaOH/MeOH/THF overnight; (iv) oxone/DMF/H2O, reflux 2-3h.
Further, in case of compounds 2 a set of characteristic SO2 stretching bands appeared at ~
1335 cm-1 and 1157 cm-1. C5-H proton of the pyrazole ring resonates in the range δ 9.05-9.30 as a
singlet while a set of doublet of doublets corresponding to benzimidazole ring protons appeared at ~
1
δ 7.60 and ~ δ 7.25 in H NMR spectra of these compounds.13C NMR and mass spectral data
further supported and confirmed the structures of the target compounds 2 and 3.

2.2. Biological evaluation
2.2.1. In vitro carbonic anhydrase inhibition assay
All the newly synthesized compounds 2a-2f and 3a-3f were screened for their CA activity
by stopped-flow, CO2 hydrase assay against hCA I, II, IX and XII isoforms. CA inhibitory profile of
all newly synthesized compounds, including 2a-2f possessing sulfonamide group and, 3a-3f without
sulfonamide moiety against four human carbonic anhydrase isoforms hCA I, II, IX and XII was
assayed by using stopped flow assay method32and results obtained regarding CA inhibition profile
were presented in Table 1. Acetazolamide (AZA) was chosen as reference for the CA assay.
Biological assay data of the newly synthesized compounds 2a-2f and 3a-3f provides interesting
results.
The pyrazolylbenzo[d]imidazole molecules possessing the sulfonamide zinc binding group (2a-2f)
showed a nanomolar activity against all the tested CAs. All the compounds showed a very similar
activity against hCA I (Ki values between 32.4 and 87.6 nM) and hCA II (Ki values between 1.5 and
4.2 nM). For hCA IX compounds 2c and 2f showed a marked decrease of activity (Ki values of
554.0 and 641.5 nM, respectively) whereas the other four compounds showed Ki values from 24.9
to 74.7. Finally, with regards to hCA XII, compounds 2a and 2e showed a high potency, about ten-
fold higher than that of compound 2b. In terms of selectivity these compounds did not possess a
good profile, as shown in Table 1, the most promising compounds seemed to be 2a and 2e, that
showed only a good level of hCA XII selectivity against hCA I.
The removal of the sulfonamide zinc binding group determined an unexpected change of the
biological results. Very interestingly, all the compounds showed a general inactivity against hCA I
and hCA II, whereas they were able to maintain inhibition activity in the nanomolar range against
both hCA IX and hCA XII. Independently on the R1 substituent all the compounds were highly
selective and as shown in Table 1 all six compounds were active hCA IX and hCA XII inhibitors
with a selectivity ratio greater than 400 fold with respect to the cytosolic isoform hCA I and hCA II.
Overall, the analysis of the biological activity data (with respect to potency and selectivity) allowed
us to highlight that the removal of the sulfonamide zinc binding group for these
pyrazolylbenzo[d]imidazole molecules produced astonishing results in terms of selectivity for the
tumor associated transmembrane isoforms hCA IX and hCA XII. A possible explanation of this
behavior was investigated via in silico docking experiments using currently available crystal
structures of several CA isoforms.

Table 1.Carbonic anhydrase activity of pyrazolylbenzo[d]imidazoles 2 and 3 using stopped-flow, CO2
hydrase assay.
Compounds
Ki (nM)*
hCA II hCA IX hCA XII
Selectivity ratio
I/XII II/IX
0.10
hCA I
33.9
37.6
32.4
35.0
87.6
33.4
I/IX
1.36
II/XII
3.0
2.7
2.3
4.2
1.5
2.5
1.9
24.9
49.3
554.0
74.7
70.2
641.5
55.9
23.3
48.7
65.0
7.5
0.9
9.1
5.5
3.7
1.0
7.6
7.3
7.2
9.4
7.8
17.7
72.9
5.7
37.66
4.13
2a
2b
2c
0.76
0.058
0.46
0.046
0.0075
0.20
0.25
5.89
0.76
9.45
0.40
2d
2e
1.24
87.6
0.035
0.0029
>716
>1717
>821
>5128
>5333
>726
0.5
2.5
0.052
>716
>1717
>821
>615
>5333
608.8
10.0
4.39
0.25
2f
>40000 >40000
>40000 >40000
>40000 >40000
>40000 >40000
>40000 >40000
>5479
>5556
>4255
>5128
>2260
460.2
43.9
>5479
>5556
>4255
>5128
>2260
>549
2.2
3a
3b
3c
3d
3e
33550
250
>40000
12.1
55.1
25
3f
AZA
AZA = acetazolamide, reference compound, a standard sulfonamide CAI, is also provided for comparison.
* Mean from 3 different assays, errors were in the range of ± 5-10 % of the reported value.
2.3. Docking studies
The presence of the sulfonamide group in position 4 of the phenyl ring determines a
nanomolar inhibitory activity for all the four carbonic anhydrase subtypes. As shown in Figure 3,
the binding disposition of 2a into hCA XII is driven by the sulfonamide portion of the molecule that
acted as a zinc binding group, also showing two additional H-bond interactions with Thr227. The
phenylpyrazole scaffold of the compound showed lipophilic interactions with V147 and L225, the
benzimidazole system showed a H-bond interaction with Q117 whereas the phenyl ring was
directed towards the solvent exposed region of the enzyme without highlighting important
interactions.

Figure 3. Docking analysis of compound 2a into hCA XII
Compound 3a, which correspond to one of the most promising ligands reported in this
series, was docked using AUTODOCK 4.0 into a minimized structure of hCA I, II,IX and XII.
Figure 4 shows the docking of the compound into the four enzymes. In the hCA XII binding site,
the 3-phenyl ring of 3a showed a π-π stacking interaction with H119, the 1-phenyl ring was inserted
into a lipophilic cleft mainly delimited by T116, V147, A157, S161, L167 and L225, whereas the
benzimidazole system was directed towards the solvent and showed an H-bond with K97 (Figure
4A). As shown in Figure 4B, the compound showed into the hCA IX a binding disposition very
similar to that observed for hCA XII, with the π-π stacking interaction between H226 and the 3-
phenyl ring, the lipophilic interactions of the 1-phenyl ring into the lipophilic cleft mainly delimited
by L223,V253, V262, L266, L272 and L331 and the H-bond between the benzimidazole system of
3a and Q203 that corresponded to the K97 in hCA XII. In the hCA II binding site the lipophilic
cavity in which the 1-phenyl ring interacted in hCA IX and hCA XII was partially occluded by the
non-conserved F130 and for this reason the compound showed a completely different binding mode
with the lack of the above described interactions and both the 3-phenyl and benzimidazole rings that
were located near the solvent exposed entrance of the binding site. A very similar analysis could
also be carried out for the interaction of 3a into hCA I; the lipophilic cavity in which the 1-phenyl
ring interacted in hCA IX and hCA XII was partially occluded by the non-conserved F92
determining the impossibility for the compound to interact in this region. Furthermore, the presence
of the non conserved H201 that replaced a threonine residue (T227 in hCA XII)prevented the π-π
stacking interaction between H95 of the 3-phenyl ring. As a result, the compound was located near
the entrance of the binding site with the two phenyl rings that were exposed to the solvent showing
only lipophilic interactions with W6 and P203.

Figure 4.Docking analysis of compound 3a into hCA XII (A), hCA IX (B), hCA II (C) and hCA I
(D).
3. Conclusions
In the present study we have designed and synthesized two series of compounds bearing
benzimidazole as well as pyrazole ring in the same molecular frame and in vitro evaluated them
against four human carbonic anhydrase isoforms representing two cytosolic isoforms (hCA I and II)
and two transmembrane tumor associated (hCA IX and XII) isoforms. In general, compounds 2a-2f
bearing a sulfonamide group exhibited excellent to good CA inhibition against all the four isoforms
used in the present investigation. Furthermore, removing the sulfonamide group from these
compounds generated analogs 3a-3f that were found to be ineffective against cytosolic isoforms
hCA I and II as indicated from their high inhibition constant values (Ki ≥ 33550)and still active
against hCA IX and XII, thus rendering these compounds highly selective inhibitors of hCA IX and
XII over hCA I and II. The molecular docking study of compound 3a into the four CA isoforms
suggested that the removal of the sulfonamide zinc binding group allowed a high level of freedom
in terms of ligand disposition inside the binding site. In the hCA IX and hCA XII binding sites the
interaction of this compound seemed to be guided by π-π stacking and lipophilic interactions inside
binding site clefts. Due to the presence of non-conserved residues, the same binding disposition
could not be reproduced in the hCA I and II, thus suggesting a possible explanation of the
selectivity showed by this class of compounds. Finally, the results of this study could also suggest
that the removal of the sulfonamide zinc binding group could also be useful for other classes of
compounds.

4. Experimental protocols
Melting points were determined in open glass capillaries in an electrical melting point
apparatus and are uncorrected. The infrared (IR) spectra were recorded on ABB MB 3000 DTGS IR
1
13
instrument using the KBr pellet technique. H NMR and C NMR spectra were recorded either in
pure DMSO-d6 or in CDCl3 on Bruker NMR spectrometers at 300 MHz and 75.5 MHz respectively
1
using tetramethylsilane (TMS) as internal standard. Chemical shifts are expressed in δ, ppm. H
NMR data are reported in order: multiplicity (br, broad; s, singlet; d, doublet; t, triplet; m, multiplet;
ex, exchangeable by D2O), approximate coupling constant in Hertz,number of protons and type of
1
protons.The purity of the compounds was checked by H NMR and thin layer chromatography
(TLC) on silica gel plates using a mixture of petroleum ether and ethyl acetate as eluent. Iodine or
UV lamp was used as a visualizing agent. Mass spectra were recorded on JEOL-AccuTOF JMS-
T100LC Mass spectrometer having DART (Direct Analysis in Real Time) source in ES+ mode.
4.1 General procedure for synthesis of 2-(1,3-diaryl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazoles (2 and 3)
To a solution of 4-formylpyrazole 1028,1133 34 (1 mmol) and ortho-phenylenediamine 12(1.2
ʹ
mmol) in 10 ml of dimethylformamide was added oxone (1 mmol) and 1 mL of water. The reaction
mixture was refluxed for 1-2 h when colour of reaction changed from greenish to reddish brown.
The course of reaction was monitored by thin layer chromatography. Reaction mixture was poured
in water whereupon a brown solid precipitated out. The solid so precipitated out was filtered,
washed with hot water and dried to afford the target compound in good yield.
2-{1-(4-aminosulfonylphenyl)-3-phenyl-1H-pyrazol-4-yl}-1H-benzo[d]imidazole (2a)
m.p. 130-132 °C, yield 86%; IR (cm-1): 1597 (s, C=N stretch), 1504 (N-H bend), 1335 & 1157 (s,
SO2 stretch); 1H NMR (300 MHz, DMSO-d6): δ 9.21 (s, 1H, C5-H pyrazol), 8.19 (d, J = 8.4 Hz, 2H,
Ar-H) , 8.04 (d, J = 8.7 Hz, 2H, Ar-H), 7.90-7.88 (m, 2H, Ar-H), 7.60 (dd, J = 6.0, 3.3 Hz, 2H,
Benzim-H), 7.49 (s, ex, 2H, SO2NH2), 7.45-7.43 (m, 3H, Ar-H), 7.24 (dd, J = 6.0, 3.3 Hz, 2H,
13
Benzim-H); C NMR (75.5 MHz, DMSO-d6): δ 151.7, 145.6, 142.5, 141.5, 138.8, 132.2, 131.4,
129.2, 128.8, 128.6, 128.0, 122.7, 119.1, 115.3, 113.5; DART MS: m/z 416 ([M+H]+),
C22H17N5O2SH+Calcd. 416.
2-{1-(4-aminosulfonylphenyl)-3-(4-methylphenyl)-1H-pyrazol-4-yl}-1H-
benzo[d]imidazole (2b)
m.p. 192-194 °C, yield 87%; IR (cm-1): 1597 (s, C=N stretch), 1512 (N-H bend), 1335 & 1157 (s,
SO2 stretch); 1H NMR (300 MHz, DMSO-d6): δ 9.20 (s, 1H, C5-H pyrazol), 8.17 (d, J = 8.7 Hz, 2H,
Ar-H), 8.02 (d, J = 8.7 Hz, 2H, Ar-H), 7.76 (d, J = 8.1 Hz, 2H, Ar-H), 7.60 (dd, J = 6.0, 3.3 Hz, 2H,

Benzim-H), 7.49 (s, ex, 2H, SO2NH2), 7.27-7.23 (m, 4H, Ar-H, Benzim-H), 2.35 (s, 3H, CH3); 13C
NMR (75.5 MHz, DMSO-d6): δ 151.7, 145.6, 142.5, 141.5, 138.6, 131.4, 129.4, 128.5, 127.9,
127.2, 122.7, 119.0, 115.2, 113.3, 21.3 (CH3); DART MS: m/z 430 ([M+H]+), C23H19N5O2SH+
Calcd.430.
2-{1-(4-aminosulfonylphenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl}-1H-
benzo[d]imidazole (2c)
m.p. 172-174 °C, yield 83%; IR (cm-1): 1597 (s, C=N stretch), 1512 (s, N-H bend), 1366 & 1157 (s,
SO2 stretch); 1H NMR (300 MHz, DMSO-d6): δ 9.18 (s, 1H, C5-H pyrazol), 8.16 (d, J = 8.7 Hz, 2H,
Ar-H), 8.02 (d, J = 8.7 Hz, 2H, Ar-H), 7.85 (d, J = 8.4 Hz, 2H, Ar-H), 7.60 (dd, J = 6.0, 3.3 Hz, 2H,
Benzim-H), 7.48 (s, ex, 2H, SO2NH2), 7.24 (dd, J = 6.0, 3.0 Hz, 2H, Benzim-H), 7.01 (d, J = 8.7
Hz, 2H, Ar-H), 3.79 (s, 3H, OCH3); 13C NMR (75.5 MHz, DMSO-d6): δ 160.2, 151.4, 145.7, 142.4,
141.5, 131.4, 130.0, 127.9, 124.6, 122.7, 118.9, 115.2, 114.2, 113.1, 55.6 (OCH3); DART MS: m/z
446 ([M+H]+), C23H19N5O3SH+Calcd. 446.
2-{1-(4-aminosulfonylphenyl)-3-(4-flourophenyl)-1H-pyrazol-4-yl}-1H-
benzo[d]imidazole (2d)
m.p. 202-204 °C, yield 90%; IR (cm-1): 1597 (s, C=N stretch), 1512 (N-H bend), 1336 & 1157 (s,
SO2 stretch); 1H NMR (300 MHz, DMSO-d6): δ 9.22 (s, 1H, C5-H pyrazol), 8.17 (d, J = 8.1 Hz, 2H,
Ar-H), 8.04 (m, 4H, Ar-H), 7.59 (m 2H, Ar-H), 7.49 (s, ex, 2H, SO2NH2), 7.32-7.24 (m, 4H, Ar-H);
1
13C NMR (75.5 MHz, DMSO-d6): δ 162.9 (d, JCF = 246.9 Hz), 150.7, 145.5, 142.6, 141.4, 131.4,
3
2
130.9 (d, JCF = 8.3 Hz), 128.8, 128.0, 122.6, 119.0, 115.6 (d, JCF = 21.9 Hz), 113.6; DART MS:
m/z 434 ([M+H]+), C22H16N5O2SFH+Calcd. 434.
2-{1-(4-aminosulfonylphenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl}-1H-
benzo[d]imidazole(2e)
m.p. 188-190 °C, yield 84%; IR (cm-1): 1597 (C=N stretch), 1512 (N-H bend), 1335 & 1157 (s, SO2
stretch); 1H NMR (300 MHz, DMSO-d6): δ 9.22 (s, 1H, C5-H pyrazol), 8.17 (d, J = 9.0 Hz, 2H, Ar-
H), 8.03 (d, J = 8.7 Hz, 2H, Ar-H), 7.95 (d, J = 8.4 Hz, 2H, Ar-H), 7.60 (dd, J = 6.0, 3.3 Hz, 2H,
Ar-H), 7.53 (d, J = 8.4 Hz, 2H, Ar-H), 7.50 (s, ex, 2H, SO2NH2), 7.24 (dd, J = 6.0, 3.0 Hz, 2H, Ar-
H); 13C NMR (75.5 MHz, DMSO-d6): δ 150.4, 145.4, 142.7, 141.4, 133.9, 131.5, 131.2, 130.5,
128.8, 128.0, 122.7, 119.1, 115.2, 113.7; DART MS: m/z 450/452 ([M+H/M+2+H]+),
C22H16N5O2SClH+Calcd. 450/452.
2-{1-(4-aminosulfonylphenyl)-3-(4-bromophenyl)-1H-pyrazol-4-yl}-1H-
benzo[d]imidazole(2f)

m.p. 178-180 °C, yield 79%; IR (cm-1): 1597 (s, C=N stretch), 1504 (N-H bend), 1335 & 1157 (s,
SO2 stretch); 1H NMR (300 MHz, DMSO-d6): δ 9.22 (s, 1H, C5-H pyrazol), 8.17 (d, J = 8.4 Hz, 2H,
Ar-H), 8.03 (d, J = 8.7 Hz, 2H, Ar-H), 7.89 (d, J = 8.4 Hz, 2H, Ar-H), 7.66 (d, J = 8.4 Hz, 2H, Ar-
H), 7.59 (dd, J = 5.7, 3.0 Hz, 2H, Ar-H), 7.50 (s, ex, 2H, SO2NH2), 7.23 (dd, J = 5.7, 3.0 Hz, 2H,
13
Ar-H); C NMR (75.5 MHz, DMSO-d6): δ 150.5, 145.4, 142.7, 141.4, 131.7, 131.4, 130.8, 128.1,
128.0, 122.7, 119.1, 115.4, 113.2; DART MS: m/z 494/496 ([M+H/M+2+H]+),
C22H16N5O2SBrH+Calcd. 494/496.
2-(1,3-phenyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazole (3a)
m.p. 188-190 °C, yield 84%; IR (cm-1): 1597 (s, C=N stretch), 1504 (m, N-H bend); 1H NMR (300
MHz, DMSO-d6): δ 9.11 (s, 1H, C5-H pyrazol), 7.98 (d, J = 7.8 Hz, 2H, Ar-H), 7.89 (d, J = 6.9 Hz,
2H, Ar-H), 7.61-7.59 (m, 4H, Ar-H, Benzim-H), 7.44-7.42 (m, 4H, Ar-H), 7.24 (dd, J = 5.7, 3.3 Hz,
2H, Benzim-H); 13C NMR (75.5 MHz, DMSO-d6): δ 150.7, 145.6, 142.4, 141.2, 138.5, 132.6,
131.1, 129.7, 128.7, 128.6, 128.1, 122.7, 119.1, 115.3, 113.5; DART MS: m/z 337 ([M+H]+),
C22H16N4H+Calcd. 337.
2-(1-phenyl-3-(4-methylphenyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazole (3b)
m.p. 136-138 °C, yield 88%; IR (cm-1): 3148 (m, N-H stretch), 2978 (m, C-H stretch) 1597 (s, C=N
1
stretch), 1504 (m, N-H bend); H NMR (300 MHz, DMSO-d6): δ 9.19 (s, 1H, C5-H pyrazol), 7.96
(d, J = 7.8 Hz, 2H, Ar-H), 7.71 (d, J = 8.1 Hz, 2H, Ar-H), 7.66 (dd, J = 6.1, 3.3 Hz, 2H, Benzim-H),
7.58 (d, J = 7.5 Hz, 2H, Ar-H), 7.44 (d, J = 7 2 Hz, 2H, Ar-H), 7.22 (dd, J = 6.0, 3.3 Hz, 2H,
13
Benzim-H), 7.25 (d, J = 7.8 Hz, 2H, Ar-H), 2.34 (s, 3H, Ar-CH3): C NMR (75.5 MHz, DMSO-
d6): δ 151.2, 145.3, 139.3, 138.6, 131.8, 130.2, 129.5, 129.3, 128.4, 127.7, 123.8, 119.2, 115.0, 21.3
(CH3); DART MS: m/z 351 ([M+H]+), C23H18N4H+Calcd. 351.
2-(1-phenyl-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazole (3c)
1
m.p. 142-144 °C, yield 85%; IR (cm-1): 1597 (s, C=N stretch), 1512 (N-H bend); H NMR (300
MHz, DMSO-d6): δ 9.05 (s, 1H, C5-H pyrazol), 7.96 (d, J = 8.1 Hz, 2H, Ar-H), 7.86 (d, J = 8.7 Hz,
2H, Ar-H), 7.61-7.57 (m, 4H, Ar-H, Benzim-H), 7.44-7.33 (m, 2H, Ar-H), 7.22 (dd, J = 6.0, 3.3 Hz,
2H, Benzim-H), 7.00 (d, J = 8.7 Hz, 2H, Ar-H), 3.80 (s, 3H, OCH3): 13C NMR (75.5 MHz, DMSO-
d6): δ 159.9, 150.8, 149.3, 146.1, 139.3, 130.2, 129.8, 129.3, 127.5, 122.6, 119.2, 114.2, 112.5,
111.9, 55.5 (OCH3); DART MS: m/z 367 ([M+H]+), C23H18N4OH+Calcd. 367.
2-(1-phenyl-3-(4-fluorophenyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazole (3d)
m.p. 260-262 °C, yield 82%; IR (cm-1): 3148 (m, N-H stretch), 1597 (s, C=N stretch), 1512 (N-H
1
bend); H NMR (300 MHz, DMSO-d6): δ 9.30 (s, 1H, C5-H pyrazol), 7.96 (d, J = 7.5 Hz, 2H, Ar-
H), 7.91-7.86 (m, 2H, Ar-H), 7.70 (dd, 6.0, 3.0 Hz, 2H, Benzim-H), 7.61 (t, J = 7.5 Hz, 2H, Ar-H),

7.47-7.44 (m, 1H, Ar-H), 7.22 (dd, J = 6.0, 3.0 Hz, 2H, Benzim-H), 7.30 (t, J = 8.7 Hz, 2H, Ar-H);
1
13C NMR (75.5 MHz, DMSO-d6): δ 162.9 (d, JCF = 246.9 Hz), 150.4, 146.1, 141.9, 139.1, 135.3,
2
132.2, 130.8 (d, 3JCF = 8.3 Hz), 130.3, 128.0, 124.6, 119.4, 116.1 (d, JCF = 21.1 Hz), 114.8, 112.2;
DART MS: m/z 355 ([M+H]+), C22H15N4FH+Calcd. 355.
2-(1-phenyl-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazole (3e)
m.p. 168-170 °C, yield 86%; IR (cm-1): 3101 (m, N-H stretch), 1597 (C=N stretch), 1504 (N-H
bend); 1H NMR (300 MHz, DMSO-d6): δ 9.12 (s, 1H, C5-H pyrazole), 7.97 (d, J = 8.1 Hz, 4H, Ar-
H), 7.61-7.57 (m, 4H, Ar-H, SO2NH2), 7.51 (d, J = 8.4 Hz, 2H, Ar-H), 7.44-7.33 (m, 2H, Ar-H),
7.22 (dd, J = 6.0, 3.3 Hz, 2H, Benzim-H); 13C NMR (75.5 MHz, DMSO-d6): δ 150.5, 145.3, 142.6,
141.3, 133.9, 131.5, 131.1, 130.5, 128.8, 128.0, 122.8, 119.1, 115.4, 113.6; DART MS: m/z 371/373
([M+H/M+2+H]+), C22H15N4ClH+Calcd. 371/373.
2-(1-phenyl-3-(4-bromophenyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazole (3f)
m.p. 184-188 °C, yield 80%; IR (cm-1): 3140 (N-H, stretch), 1597 (C=N stretch), 1512 (N-H bend);
1H NMR (300 MHz, DMSO-d6): δ 9.13 (s, 1H, C5-H pyrazol), 7.98 (m, 4H, Ar-H), 7.61-7.57 (m,
4H, Ar-H), 7.51 (d, J = 8.4 Hz, 2H, Ar-H), 7.46-7.39 (m, 1H, Ar-H), 7.25 (dd, J = 6.0, 3.3 Hz, 2H,
13
Benzim-H); C NMR (75.5 MHz, DMSO-d6): δ 149.8, 145.6, 139.3, 133.7, 131.4, 131.0, 130.4,
130.2, 128.7, 127.7, 122.8, 119.2, 118.9, 115.2, 112.5; DART MS: m/z 415/417 ([M+H/M+2+H]+),
C22H15N4BrH+Calcd. 415/417
4.2 CA inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed
CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM
Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed
CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM
for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least
six traces of the initial 5–10% of the reaction have been used for determining the initial velocity.
The unanalyzed rates were determined in the same manner and subtracted from the total observed
rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions
up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were
preincubated together for 15 min–72 h at room temperature (15 min) or 4 °C (all other incubation
times) prior to assay, in order to allow for the formation of the E–I complex. Data from Table 1
were obtained after 15 min incubation as there was no difference of inhibitory power when the
enzyme and inhibitors were kept for longer periods in incubation.35The inhibition constants were
obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as

reported earlier,36 and represent the mean from at least three different determinations. All CA
isofoms were recombinant ones obtained in-house as reported earlier.37-40
4.3 Docking methodologies.
The crystal structures of hCA I (pdb code 1AZM41), hCA II (pdb code 3HSA42), hCA IX
(pdb code 3IAI43), and hCA XII (pdb code 1JD044) were taken from the Protein Data Bank.45After
adding hydrogen atoms and removing complex ligands, the four proteins were minimized using
Amber 1146 software and parm03 force field at 300 K. The four proteins were placed in a
rectangular parallelepiped water box, an explicit solvent model for water, TIP3P, was used and the
complexes were solvated with a 10 Å water cap. Sodium ions were added as counter ions to
neutralize the system. Two steps of minimization were then carried out; in the first stage, we kept
the protein fixed with a position restraint of 500 kcal/mol•Å2 and we solely minimized the positions
of the water molecules. In the second stage, we minimized the entire system through 5000 steps of
steepest descent followed by conjugate gradient (CG) until a convergence of 0.05 kcal/Å•mol.
Automated docking was carried out by means of the AUTODOCK 4.0 program;47Autodock Tools48
was used in order to identify the torsion angles in the ligand, add the solvent model and assign the
Kollman atomic charges to the protein. The ligand charge was calculated using the Gasteiger
method.A grid spacing of 0.375 Å and a distance-dependent function of the dielectric constant were
used for the energetic map calculations. Using the Lamarckian Genetic Algorithm, the docked
compound was subjected to 100 runs of the Autodock search, using 500000 steps of energy
evaluation and the default values of the other parameters. Cluster analysis was performed on the
results using an RMS tolerance of 2.0 Å and the best docked conformation was taken into account.
Acknowledgements
One of the authors (Satish Kumar) is grateful to the University Grants Commission, New
Delhi, India for the award of Senior Research Fellowships. The authors are thankful to
Sophisticated Analytical Instrument Facility, Central Drug Research Institute, Lucknow for
providing Mass spectra. Work from CTS lab was financed by an EU project of the FP7 programme
(Metoxia).
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Graphical Abstract
N
N
N
N
H
H
N
N
SO2NH2
H
N
N
R1
R1
Excellent inhibition against all
isoforms hCA I, II, IX and XII
poor selectivity
Good inhibition against
isoforms hCA IX and XII
high selectivity
3
2

Research Highlights
 12 novel pyrazolylbenzo[d]imidazoles were designed and synthesized.
 Their inhibition potential was evaluated against hCA I, II, IX and XII.
 Compounds having sulfonamide moiety were better inhibitors with poor selectivity.
 Selectivity against tumor isoforms hCA IX abd XII enhanced on removing sulfonamide.
 Inhibition results were promising as compared to antitumor drug acetazolamide.