Synthesis of benzamide derivatives with thiourea-substituted benzenesulfonamides as carbonic anhydrase inhibitors

Article abstract of DOI:10.1002/ardp.202000230

The novel compounds with the chemical structure of N-({4-[N′-(substituted)sulfamoyl]phenyl}carbamothioyl)benzamide (1a–g) and 4-fluoro-N-({4-[N′-(substituted)sulfamoyl]phenyl}carbamothioyl)benzamide (2a–g) were synthesized as potent and selective human carbonic anhydrase (hCA) I and hCA II candidate inhibitors. The aryl part was changed to sulfacetamide, sulfaguanidine, sulfanilamide, sulfathiazole, sulfadiazine, sulfamerazine, and sulfametazine. The K_i values of compounds 1a–g were in the range of 20.73 ± 4.32 to 59.55 ± 13.07 nM (hCA I) and 5.69 ± 0.43 to 44.81 ± 1.08 nM (hCA II), whereas the K_i values of compounds 2a–g were in the range of 13.98 ± 2.57 to 75.74 ± 13.51 nM (hCA I) and 8.15 ± 1.5 to 49.86 ± 6.18 nM (hCA II). Comparing the K_i values of the final compounds and acetazolamide, compound 1c with the sulfanilamide moiety (K_i = 5.69 ± 0.43 nM, 8.8 times) and 2f with the sulfamerazine moiety (K_i = 8.15 ± 1.5 nM, 6.2 times) demonstrated promising and selective inhibitory effects against the hCA II isoenzyme, the main target protein in glaucoma. Furthermore, compounds 1d (K_i = 20.73 ± 4.32, 4 times) and 2d (K_i = 13.98 ± 2.57, 5.9 times), which have the sulfathiazole moiety, were found as potent hCA I inhibitors. Compounds 1c and 2f can be considered as the lead compounds determined in the present study, which can be investigated further to alleviate glaucoma symptoms.

Full text of DOI:10.1002/ardp.202000230

Received: 6 July 2020
Revised: 10 September 2020
Accepted: 16 September 2020
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DOI: 10.1002/ardp.202000230
F U L L P A P E R
Synthesisofbenzamidederivativeswiththiourea‐substituted
benzenesulfonamides as carbonic anhydrase inhibitors
Mehtap Tugrak1
| Halise Inci Gul1
| Yeliz Demir2
| Ilhami Gulcin3
1Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Ataturk University,
Erzurum, Turkey
Abstract
The novel compounds with the chemical structure of N‐({4‐[N′‐(substituted)
sulfamoyl]phenyl}carbamothioyl)benzamide (1a–g) and 4‐fluoro‐N‐({4‐[N′‐
2Department of Pharmacy Services, Nihat
̈
Delibalta Gole Vocational High School,
Ardahan University, Ardahan, Turkey
(substituted)sulfamoyl]phenyl}carbamothioyl)benzamide (2a–g) were synthesized
as potent and selective human carbonic anhydrase (hCA) I and hCA II candidate
inhibitors. The aryl part was changed to sulfacetamide, sulfaguanidine, sulfanilamide,
sulfathiazole, sulfadiazine, sulfamerazine, and sulfametazine. The Ki values of com-
pounds 1a–g were in the range of 20.73 4.32 to 59.55 13.07 nM (hCA I) and
5.69 0.43 to 44.81 1.08 nM (hCA II), whereas the Ki values of compounds 2a–g
were in the range of 13.98 2.57 to 75.74 13.51 nM (hCA I) and 8.15 1.5 to
49.86 6.18 nM (hCA II). Comparing the Ki values of the final compounds and
acetazolamide, compound 1c with the sulfanilamide moiety (Ki = 5.69 0.43 nM,
8.8 times) and 2f with the sulfamerazine moiety (Ki = 8.15 1.5 nM, 6.2 times) de-
monstrated promising and selective inhibitory effects against the hCA II isoenzyme,
the main target protein in glaucoma. Furthermore, compounds 1d (Ki = 20.73 4.32,
4 times) and 2d (Ki = 13.98 2.57, 5.9 times), which have the sulfathiazole moiety,
were found as potent hCA I inhibitors. Compounds 1c and 2f can be considered as
the lead compounds determined in the present study, which can be investigated
further to alleviate glaucoma symptoms.
3Department of Chemistry, Faculty of Science,
Ataturk University, Erzurum, Turkey
Correspondence
Mehtap Tugrak and Halise I. Gul, Department
of Pharmaceutical Chemistry, Faculty of
Pharmacy, Ataturk University, Erzurum 25040,
Turkey.
Email: mehtaptugrak@hotmail.com (M. T.) and
incigul1967@yahoo.com (H. I. G.)
K E Y W O R D S
benzamide, carbonic anhydrase, enzyme inhibitors, sulfonamides, thiourea
applications.[9,10] Well‐known sulfonamide‐based CA inhibitors in-
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1
INTRODUCTION
clude acetazolamide, ethoxzolamide, and methazolamide. Benzene-
Carbonic anhydrases (CAs; E.C. 4.2.1.1) are metalloenzymes with
important roles in various diseases. They are considered as target
proteins in diuretic, epilepsy, glaucoma, cancer, and obesity. The ir-
regular expression of CA isoenzymes leads to pathological problems.
Among human CAs (hCA), common hCA I and hCA II isoenzymes in
the human body are well‐known and most‐studied targets. hCA I
plays role in edema, whereas hCA II is the main target protein in
glaucoma.[1–6]
sulfonamides and sulfonamides exhibit inhibition effects through
interaction with Zn2+ ion at the active site of the enzyme and amino
acid residues are the proposed effect mechanism.[7–9,11,12] Although
primary sulfonamides are known as the most valuable pharmaco-
phoric group for CA inhibitors, secondary sulfonamides were also
reported with their selective and effective CA inhibitory activity
against several CAs isoenzymes with a similar effect mechanism as
primary sulfonamides.[13–15] Several CA inhibitors with primary or
secondary benzenesulfonamide are presented in Figure 1. From this
perspective, we designed novel compounds that incorporated both
primary and secondary sulfonamide pharmacophores as novel CA I
Several primary sulfonamides and their derivatives are known as
an attractive and versatile compound class of CA inhibitors.[7,8] They
are mainly employed as antiglaucoma and diuretics agents in clinic
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Arch Pharm. 2020;e2000230.
wileyonlinelibrary.com/journal/ardp © 2020 Deutsche Pharmazeutische Gesellschaft
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FIGURE 1 Structures of some carbonic anhydrase inhibitors[16,17]
and CA II inhibitors in the present study to observe their effects on
bioactivity.
isoenzymes.[25–29] Despite the significant SLC‐0111 behavior as se-
lective CA IX inhibitor, many compounds were reported to exhibit
unselective inhibition toward CA isoenzymes. This unfavorable case
leads to several side effects. Thus, a new drug design strategy, that is,
the tail approach, was preferred in the design of novel CAs inhibitors
in the present study.
Furthermore, compounds derived from urea and its sulfur ana-
logue thiourea have continuously been used in medicinal chemistry
to design new bioactive compounds due to their promising physico-
chemical and pharmacological features. Also, these compounds have
many biological activities including anticancer, antiviral, and anti-
microbial activities, and they also acted as 15‐lipoxygenase inhibitors,
polo‐like kinase 1 (Plk1), polo‐box domain (PBD) inhibitors, anti‐
Alzheimer's disease agents, CA inhibitors, and so forth.[18–24]
Primary sulfonamide‐based urea derivative SLC‐0111, designed
by the tail‐approach method, exhibited selective inhibition toward
CA IX and CA XII isoenzymes associated with cancer against targets
CA I and CA II. SLC‐0111 (Figure 1) was referred to in clinical trials
related with hypoxic tumors.[25–27] It was suggested that the ureido
moiety of SLC‐0111 may lead to an increase in flexibility of the tail of
the compound, which may cause various several conformational
changes at the active site of the enzyme. These flexible conforma-
tions allow several favorable interactions between inhibitor and CAs
Several drug molecules such as encorafenib, dacomitinib, and
lorlatinib that include a fluorine atom have been known with their
significant clinical pharmacological uses.[30] It was considered that
the substitution of fluorine would lead to more potent compounds
with increased resistance against drug metabolism, as it regulates the
reactivity and stability of the compounds.[30] Furthermore, fluorine
atom modulates pKa, lipophilicity, and hydrophobic interactions that
affect the physicochemical properties, which play a role in the
pharmacokinetic process of the compound. Due to the significant
molecular properties of fluorine, it has been considered in novel drug
design strategies.[10,31]
In the present study, we combined primary or secondary benze-
nesulfonamide and thioureido pharmacophores in a molecule with the

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Inhibitory potency of the compounds
toward CAs
tail approach drug design to investigate promising enzyme inhibitors.
N‐({4‐[N′‐(Substituted)sulfamoyl]phenyl}carbamothioyl)benzamides (1a–g)
and 4‐fluoro‐N‐({4‐[N′‐(substituted)sulfamoyl]phenyl}carbamothioyl)
benzamides (2a–g) were designed and synthesized, and their enzyme
inhibitory activities against hCA I and hCA II were investigated to
discover possible promising drug candidate/s in the current study.
2.2
Compounds 1a–g and 2a–g were tested against slow cytosolic hCA I
and the fast cytosolic hCA II isoenzymes, as the main CA inhibitor
classes had a sulfonamide group. The findings concerning the com-
pounds are presented in Table 1.
The CA isoenzymes were effectively inhibited by compounds
1a–g. IC50 values of the compounds were 13.07–31.5 nM against
both isoenzymes. IC50 values were calculated in the range of
17.77–31.50 nM (hCA I) and 13.07–24.75 nM (hCA II), whereas IC50
values of the reference drug AZA were 48.78 nM (hCA I) and 43.76
(hCA II). Compounds 1a–g were 1.5–2.7 times and 2.1–3.3 times
more potent when compared with reference drug AZA against hCA I
and hCA II, respectively, based on the IC50 values. The compound
N‐({4‐[N′‐(thiazol‐2‐yl)sulfamoyl]phenyl}carbamothioyl)benzamide (1d)
was the best inhibitor against hCA I and hCA II isoenzymes, with IC50
values of 17.77 and 13.07 nM, respectively.
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2
RESULTS AND DISCUSSION
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2.1
Chemistry
In the present study, we synthesized two series of benzamide deri-
vatives with thiourea‐substituted benzenesulfonamides in two steps
(Scheme 1). First, aroyl isothiocyanate derivatives were synthesized
by the reaction of aroyl chlorides (benzoyl chloride for 1a–g and 4‐
fluorobenzoyl chloride for 2a–g series) and potassium thiocyanate in
acetone. The intermediates were not isolated and used without any
further purification for the next step. Then, aroyl isothiocyanates
were reacted with substituted sulfonamides (sulfacetamide [1a, 2a],
sulfaguanidine [1b, 2b], sulfanilamide [1c, 2c], sulfathiazole [1d, 2d],
sulfadiazine [1e, 2e], sulfamerazine [1f, 2f], and sulfamethazine [1g,
2g]) to obtain final compounds 1a–g and 2a–g (Scheme 1). All final
compounds 1a–g and 2a–g were characterized by spectral methods.
Compounds 1a, 1b, 2a, 2b, and 2f have been reported for the first
time in the present study. Compounds 1c, 2c, 1d, 2d, 1e, 2e, 1f, 1g,
Ki values of compounds 1a–g were also in the range of 20.73 4.32
to 59.55 13.07 nM (hCA I) and 5.69 0.43 to 44.81 1.08 nM (hCA II),
whereas AZA's Ki values were 82.13 4.56 nM (hCA I) and
50.27 3.75 nM (hCA II). Compound 1d, N‐({4‐[N′‐(thiazol‐2‐yl)
sulfamoyl]phenyl}carbamothioyl)benzamide, was the best inhibitor
against hCA I, with Ki = 20.73 4.32 nM, whereas compound 1c, N‐({4‐
sulfamoylphenyl}carbamothioyl)benzamide, was found as the best CA
inhibitor in the series against hCA II, with a Ki value of 5.69 0.43 nM.
IC50 values of the compound 2a–g were in the range of
23.90–33.0 nM (hCA I) and 21.66–32.48 nM (hCA II), whereas for the
reference drug AZA, IC50 values were 48.78 nM (hCA I) and 43.76
nM (hCA II). Compounds 2a–g with 4‐fluorophenyl moiety also ex-
hibited significant CA inhibition potential. CA inhibitory effects of the
compounds 2a–g were higher when compared with AZA. The IC50
values were 1.5–2.0 times and 1.3–2.0 times more potent against
hCA I and hCA II, respectively. Compound 2d, 4‐fluoro‐N‐({4‐[N′‐
(thiazol‐2‐yl)sulfamoyl]phenyl}carbamothioyl)benzamide, can be
and 2g with bioactivities have been reported only in
studies.[32–35]
a few
1H NMR (nuclear magnetic resonance) spectral data were
presented for compound 1b as follows: Signals of protons attached
with thiourea moiety were at δ 12.78 and 12.14 ppm as a singlet,
whereas the signal of the proton on the sulfamoyl group was ob-
served at 11.73 as a singlet. Methyl protons on the acetyl group
were observed at δ 1.96 ppm as a singlet. Besides, 13C NMR
spectra revealed that aryl and aliphatic carbon peaks of the
compounds were observed in the expected areas. High‐resolution
mass spectrometry (HRMS) data also confirmed the molecular
weight of the compound 1b (calculated [M+Na]+: 400.0396 and
found [M+Na]+: 400.0405).
considered as the potent compound, with IC50 = 23.9 and IC50
21.66 nM against hCA I and hCA II, respectively.
=
On the basis of the Ki values, compounds 2a–g strongly inhibited
hCA I isoenzyme, with Ki values of 13.98 2.57 to 75.74 13.51 nM,
SCHEME 1 The synthesis of compounds
1a–g and 2a–g. Reaction conditions: (i) KSCN,
acetone, reflux, 1.5 hr; (ii) acetone,
reflux, 4–8 hr

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TABLE 1 Carbonic anhydrase (CA)
enzyme inhibition results of compounds
IC50
K
i
2
2
Compounds
hCA I (nM)
31.50
22.35
20.38
17.77
23.10
24.75
26.65
28.88
30.88
24.75
23.90
30.13
33.00
29.87
48.78
r
hCA II (nM)
19.25
16.90
13.59
13.07
18.24
21.00
24.75
23.90
32.48
25.67
21.66
31.50
30.13
26.65
43.76
r
hCA I (nM)
hCA II (nM)
44.81 1.08
24.95 5.90
5.69 0.43
1a–g and 2a–g
1a
.9779
.9919
.9745
.9796
.9798
.9832
.9975
.9840
.9655
.9718
.9727
.9804
.9834
.9952
.9878
.9918
.9746
.9927
.9727
.9730
.9768
.9878
.9761
.9738
.9869
.9813
.9729
.9741
.9801
.9813
38.51 7.83
30.91 6.62
40.38 8.15
20.73 4.32
46.55 4.56
54.26 3.89
59.55 13.07
64.31 13.16
75.74 13.51
19.88 1.04
13.98 2.57
45.20 1.24
30.17 2.35
55.95 10.72
82.13 4.56
1b
1c
1d
17.93 1.02
38.77 6.77
16.61 0.87
12.19 2.24
25.83 1.99
42.99 9.57
49.86 6.18
12.49 1.04
18.92 1.63
8.15 1.50
1e
1 f
1 g
2a
2b
2c
2d
2e
2f
2g
47.96 7.91
50.27 3.75
Acetazolamide
whereas AZA's Ki value was 82.13 4.56 nM against hCA I iso-
enzyme. Thus, hCA I isoenzyme inhibition potency of the compounds
was 5.9–1.1 times more than AZA. Compound 2d can be considered
as a promising inhibitor against hCA I isoenzyme in the series, with
Ki = 13.98 2.57 nM.
sulfonamide. However, compounds sulfadiazine (1e), sulfamerazine
(1f), and sulfamethazine (1g) which have six‐membered pyrimidine
ring were less effective when compared with 1c. Among pyrimidine
derivatives, it could be suggested that substitution of the additional
methyl groups on the ring led to unfavorable results. Thus, methyl
groups caused steric hindrance in the compounds, which affected the
interaction at the active side of the enzyme. However, the acetyl and
guanidine substitution in compounds 1a and 1b led to an increased
activity when compared with 1c. The SAR data indicated that a five‐
membered ring and small groups led to positive effects on hCA I
inhibitory potency of the benzamide derivatives with thiourea‐
substituted benzenesulfonamides. Also, in series 1a–g against hCA II,
the most potent compound was the sulfanilamide derivative 1c in
contrast to the hCA I results. Surprisingly, in this series against hCA
II, sulfamerazine‐bearing 1f and sulfamethazine‐bearing 1g deriva-
tives, except sulfadiazine‐bearing 1e, on six‐membered pyrimidine
significantly inhibited the hCA II, whereas they were not considerably
effective against hCA I. Thus, methyl groups on pyrimidine enhanced
the inhibitory activity against hCA II as compared with their effects
on hCA I. Enzyme selectivity of the drugs or drug candidates is one of
the crucial properties of enzyme‐targeted diseases. For instance, the
compound that targets glaucoma should selectively inhibit the hCA II
enzyme. Among 1a–g series, compound 1c with primary sulfonamide
was approximately eightfold more selective toward hCA II, which the
main target of glaucoma. This suggested that 1c could be considered
as a potential candidate compound in this series.
Furthermore, for compounds 2a–g, Ki values were 8.15 1.5 to
49.86 6.18 nM against cytosolic hCA II isoenzyme, whereas AZA's
Ki value was 50.27 3.75 nM against hCA II isoenzyme. 4‐Fluoro‐N‐
({4‐[N′‐(4‐methylpyrimidin‐2‐yl)sulfamoyl]phenyl}carbamothioyl)
benzamide (2f) was the strongest inhibitor in the series, with a
Ki value of 8.15 1.5 nM against hCA II isoenzyme.
Compounds 1c with phenyl moiety and 2c with 4‐fluorophenyl
moiety have been reported for hCA II inhibitory effects in the
literature.[33] In previous study findings, compounds 1c and 2c have
been found to exhibit inhibitory effects against hCA II, with IC50
values of 110 nM and 580 nM, respectively, whereas in the present
study, these values are 13.59 nM (1c) and 25.67 nM (2c). However,
the differences could be due to the employed techniques. Un-
substituted compound 1c was more effective when compared with its
fluorinated analog 2c, similar to our findings, against hCA II.
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2.3
Structure–activity relationships (SAR)
The following SAR results could be determined on the basis of
compounds' Ki values against hCA I and hCA II. Initially, in series
1a–g against hCA I, the most potent compound was the thiazole‐
bearing sulfathiazole derivative 1d. Compounds 1a, 1b, and 1d
bearing secondary sulfonamides were found more effective inhibitors
than sulfanilamide moiety‐bearing compound 1c, which has a primary
The SAR results of compounds 2a–g bearing fluorophenyl could
be summarized as follows. Compound 2d with sulfathiazole core was
the strongest inhibitor in the series. This situation was similar to hCA
I findings. The substitution of the fluorine atom in compound 2d

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increased the activity by 1.5 times when compared with its corre-
sponding phenyl analog 1d. When primary sulfonamide derivative 1c
converted to its fluorinated analog 2c, the activity was also increased
two times. When pyrimidine ring‐bearing compounds 2e–g were
considered, fluorination of the compounds slightly increased the ac-
tivity as compared with their non‐fluorinated analogs 1e–g. However,
sulfacetamide (2a) and sulfaguanidine (2b) derivatives exhibited a
lower activity against hCA I when compared with their corresponding
analogs 1a and 1b. The SAR data revealed that fluorination of the
compounds generally increased the enzyme inhibitory activity
against hCA I.
TABLE 2 Druglikeness properties of compounds 1a–g and 2a–g,
based on Lipinski's RO5
Compounds Formula
MW (g/mol) nHBA nHBD LogP
1a
1b
1c
1d
1e
1f
C16H15N3O4S2
377.44
377.44
335.40
418.51
413.47
427.50
441.53
4
4
4
4
5
5
5
5
5
5
5
6
6
6
3
4
3
3
3
3
3
3
4
3
3
3
3
3
1.85
1.18
1.76
2.33
1.87
2.22
2.49
2.00
1.52
1.91
2.75
2.31
2.58
2.78
C
C
15H15N5O3S2
14H13N3O3S2
C17H14N4O3S3
C
C
18H15N5O3S2
19H17N5O3S2
1g
2a
2b
2c
2d
2e
2f
C20H19N5O3S2
The phenyl analog of compound 1c with sulfanilamide moiety
was the most effective inhibitor against hCA II. However, it is in-
teresting to note that the fluorinated derivative 2c significantly de-
creased the enzyme activity against hCA II by about 10‐fold when
compared with 1c. The most powerful inhibitor against hCA II, 2f,
with sulfamerazine moiety, increased the activity via fluorination
when compared with phenyl analog 1f. Four fluorinated compounds
(2a, 2d, 2e, and 2f) exhibited an increased activity against hCA II.
Briefly, fluorination of the phenyl ring led to either an increase or
decrease in the inhibition activity against both isoenzymes. Thus, the
impact of fluorine was variable. The overall analysis of the results
demonstrated that compounds 1c with sulfanilamide and 2f with
sulfamerazine could be considered as the most promising and se-
lective hCA II inhibitors, which could be further analyzed in future
research.
C
C
C
C
C
16H14FN3O4S2 395.43
15H14FN5O3S2 395.43
14H12FN3O3S2 353.39
17H13FN4O3S3 436.50
18H14FN5O3S2 431.46
C19H16FN5O3S2 445.49
C20H18FN5O3S2 459.52
2g
Note: Lipinski filter: MW ≤ 500, MlogP ≤ 4.15, N or O ≤ 10, NH or
OH ≤ 5.[36]
Abbreviations: MW, molecular weight; nHBA, number of H‐bond acceptors;
nHBD, number of H‐bond donors; RO5, rule of five.
Druglikeness properties of the oral drugs are identified by
Lipinski's rule of five (RO5).[36] Thus, we calculated suitable para-
meters of the compounds to determine whether they were compa-
tible with the RO5. On the basis of the analysis (Table 2), the
following values were determined: logP (lipophilicity, 1.18–2.78),
nHBD (number of H‐bond donors, 3–4), nHBA (number of H‐bond
acceptors, 4–6), and MW (molecular weight, 335.4–459.52). All
synthesized compounds were found compatible with Lipinski's RO5.
inhibitors. However, sulfathiazole derivatives 1d and 2d were more
effective inhibitors when compared with 1e and 2e with sulfamer-
azine moiety. It appears that the sulfathiazole compounds (1d and
2d), with a five‐membered ring, have a significant inhibitory effect on
both isoenzymes in terms of Ki values. The SAR data also indicated
that fluorination of the phenyl ring led to variable effects against
both isoenzymes. All synthesized compounds were also found com-
patible with Lipinski's RO5.
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EXPERIMENTAL
3
CONCLUSION
4
|
Chemistry
In the present study, two series of benzamide derivatives with
thiourea‐substituted benzenesulfonamides (1a–g and 2a–g) were
designed and synthesized as potent and selective CA inhibitors.
Spectral techniques confirmed the proposed chemical structure of
the compounds. Ki values of compounds 1a–g were in the range of
20.73 4.32 to 59.55 13.07 nM (hCA I) and 5.69 0.43 to
44.81 1.08 nM (hCA II), whereas Ki values of compounds 2a–g with
4‐fluorobenzoyl chloride were in the range of 13.98 2.57 to
75.74 13.51 nM (hCA I) and 8.15 1.5 to 49.86 6.18 nM (hCA II).
Compounds 1c (Ki = 5.69 0.43 nM, 8.8 times) with sulfanilamide
moiety and 2f (Ki = 8.15 1.5 nM, 6.2 times) with sulfamerazine
moiety exhibited promising and selective hCA II isoenzyme inhibition
potential, which is the main target protein in glaucoma. Furthermore,
compounds 1d (Ki = 20.73 4.32, 4 times) and 2d (Ki = 13.98 2.57,
5.9 times) with sulfathiazole moiety were found as potent hCA I
4.1
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4.1.1
General
The chemical structures of the final compounds were confirmed by
the NMR spectra: 1H NMR (400 MHz), 13C NMR (100 MHz; Varian
Mercury Plus spectrometer; Varian Inc., Palo Alto, CA), and HRMS
(Shimadzu, Kyoto, Japan). The original spectra are provided as Sup-
porting Information. Chemical shifts (δ) are reported in ppm and
coupling constants (J) are expressed in hertz (Hz). Mass spectra
(HRMS) for the compounds were taken using a liquid chromato-
graphy ion trap time‐of‐flight tandem mass spectrometer (Shimadzu)
equipped with an electrospray ionization (ESI) source, operating in
both positive and negative ionization modes. Shimadzu's LCMS So-
lution software was used for data analysis. Melting points were

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TUGRAK ET AL.
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determined using an Electrothermal 9100/IA9100 instrument (Bibby
Scientific Limited, Staffordshire, UK) and were uncorrected. Reac-
tions were monitored by thin‐layer chromatography (TLC) using silica
gel 60 HF254 (Merck KGaA). Chloroform/methanol (4.8:0.2) solvent
mixture was used as the TLC solvent system. Dimethyl sulfoxide
(DMSO)‐d6 (Merck) was used as an NMR solvent.
C
14H13N3O3S2; calculated [M–H]−: 334.0326; found [M–H]–:
334.0330.
N‐({4‐[N′‐(Thiazol‐2‐yl)sulfamoyl]phenyl}carbamothioyl)‐
benzamide (1d)
Cream color solid, mp: 245–246°C, yield 84%. 1H NMR (DMSO‐d6,
ppm, 400 MHz), δ 12.79 (s, 1H, NH), 12.71 (s, 1H, NH), 11.69 (s, 1H,
NH), 7.99–7.97 (m, 2H, Ar‐H), 7.91–7.82 (m, 4H, Ar‐H), 7.69–7.65
(m, 1H, Ar‐H), 7.56–7.52 (m, 2H, Ar‐H), 7.29–7.27 (m, 1H, Ar‐H), and
6.86–6.85 (m, 1H, Ar‐H); 13C NMR (DMSO‐d6, ppm, 100 MHz) δ
179.7 (C═S), 169.4 (C═O), 168.6, 141.6, 139.9, 133.7, 132.5, 129.2,
128.9, 126.9, 124.9, 124.7, and 108.8; HRMS (ESI‐MS)
C17H14N4O3S3; calculated [M+Na]+: 441.0120; found [M+Na]+:
441.0129.
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
|
4.1.2
General procedure for the synthesis of
compounds 1a–g (Scheme 1)
A solution of benzoyl chloride (1 mmol) in acetone (20 ml) was added
dropwise to a suspension of potassium thiocyanate (1 mmol) in
acetone (30 ml), and the reaction mixture was refluxed for 1.5 hr to
afford isothiocyanates. The reaction mixture was checked with TLC
and benzoyl isothiocyanate derivative was formed in a reaction
medium. After completion of the reaction, the substituted sulfona-
mide derivative (1 mmol) was added and the mixture was stirred
under reflux for 4–8 hr. Upon completion of the reaction (checked
with TLC), the resulting precipitate was collected by filtration and
recrystallized from dimethylformamide/ethanol/H2O (4.2:0.6:0.2) to
obtain the pure products 1a–g.[23]
N‐({4‐[N′‐(Pyrimidin‐2‐yl)sulfamoyl]phenyl}carbamothioyl)‐
benzamide (1e)
Light cream color solid, mp: 250–252°C, yield 88%. 1H NMR (DMSO‐
d6, ppm, 400 MHz), δ 12.75 (s, 1H, NH), 11.70 (s, 1H, NH), 8.54–8.52
(m, 2H, Ar‐H), 8.03–7.97 (m, 7H, Ar‐H, NH), 7.69–7.65 (m, 1H, Ar‐H),
7.56–7.52 (m, 2H, Ar‐H), and 7.08–7.05 (m, 1H, Ar‐H); 13C NMR
(DMSO‐d6, ppm, 100 MHz) δ 179.7 (C═S), 168.6 (C═O), 158.9, 157.3,
142.3, 137.8, 133.7, 132.5, 129.2, 128.9, 128.7, 124.3, and 116.3;
HRMS (ESI‐MS) C18H15N5O3S2; calculated [M+Na]+: 436.0509;
found [M+Na]+: 436.0502.
N‐({4‐[N′‐(Acetyl)sulfamoyl]phenyl}carbamothioyl)benzamide (1a)
White color solid, mp: 235–237°C, yield 80%. 1H NMR (DMSO‐d6,
ppm, 400 MHz), δ 12.78 (s, 1H, NH), 12.14 (s, 1H, NH), 11.73 (s, 1H,
NH), 8.03–7.94 (m, 6H, Ar‐H), 7.68–7.66 (m, 1H, Ar‐H), 7.57–7.54
(m, 2H, Ar‐H), and 1.96 (s, 3H, –CH3); 13C NMR (DMSO‐d6, ppm,
100 MHz) δ 179.8 (C═S), 169.3 (C═O), 168.6, 143.0, 136.5, 133.7,
132.5, 129.2, 128.9, 128.8, 124.5, and 23.7; HRMS (ESI‐MS)
C16H15N3O4S2, Calculated [M+Na]+: 400.0396; Found [M+Na]+:
400.0405.
N‐({4‐[N′‐(4‐Methylpyrimidin‐2‐yl)sulfamoyl]phenyl}carbamothioyl)‐
benzamide (1f)
Light white color solid, mp: 231–232°C, yield 80%. 1H NMR (DMSO‐
d6, ppm, 400 MHz), δ 12.75 (s, 1H, NH), 11.69 (s, 1H, NH), 8.35–8.33
(m, 1H, Ar‐H), 8.03–7.94 (m, 7H, Ar‐H, NH), 7.68–7.65 (m, 1H, Ar‐H),
7.56–7.53 (m, 2H, Ar‐H), 6.92–6.91 (m, 1H, Ar‐H), and 2.33 (s, 3H,
CH3); 13C NMR (DMSO‐d6, ppm, 100 MHz) δ 179.7 (C═S), 168.6
(C═O), 158.7, 157.2, 156.9, 142.1, 138.0, 133.7, 132.5, 129.2, 128.9,
124.1, and 23.7; HRMS (ESI‐MS) C19H17N5O3S2; calculated [M+H]+:
428.0846; found [M+H]+: 428.0834.
N‐({4‐[N′‐(Diaminomethylene)sulfamoyl]phenyl}carbamothioyl)‐
benzamide (1b)
White color solid, mp: 242–243°C, yield 85%. 1H NMR (DMSO‐d6,
ppm, 400 MHz), δ 12.70 (s, 1H, NH), 11.68 (s, 1H, NH), 8.00–7.95
(m, 2H, Ar‐H), 7.86–7.84 (m, 2H, Ar‐H), 7.80–7.78 (m, 2H, Ar‐H),
7.69–7.65 (m, 1H, Ar‐H), 7.57–7.53 (m, 2H, Ar‐H), and 6.75 (bs, 4H,
NH2); 13C NMR (DMSO‐d6, ppm, 100 MHz) δ 179.8 (C═S), 162.8
(C═O), 158.6, 142.3, 140.8, 133.7, 132.6, 129.2, 128.9, 126.6, and
124.6; HRMS (ESI‐MS) C15H15N5O3S2; calculated [M+H]+: 378.0689;
found [M+H]+: 378.0693.
N‐({4‐[N′‐(4,6‐Dimethylpyrimidin‐2‐yl)sulfamoyl]phenyl}‐
carbamothioyl)benzamide (1g)
White color solid, mp: 216–218°C, yield 84%. 1H NMR (DMSO‐d6, ppm,
400 MHz), δ 12.74 (s, 1H, NH), 11.69 (s, 1H, NH), 8.03–7.92 (m, 7H,
Ar‐H), 7.68–7.64 (m, 1H, Ar‐H), 7.56–7.52 (m, 2H, Ar‐H), 6.76 (s, 1H, NH),
and 2.26 (s, 6H, CH3); 13C NMR (DMSO‐d6, ppm, 100 MHz) δ 179.2
(C═S), 168.1 (C═O), 158.9, 157.2, 156.0, 142.3, 141.4, 133.2, 132.0,
128.69, 128.66, 128.4, 123.4, and 22.7; HRMS (ESI‐MS) C20H19N5O3S2;
calculated [M+H]+: 442.1002; found [M+H]+: 442.1006.
N‐({4‐Sulfamoylphenyl}carbamothioyl)benzamide (1c)
White color solid, mp: 234–236°C, yield 86%. 1H NMR (DMSO‐d6,
ppm, 400 MHz), δ 12.72 (s, 1H, NH), 11.70 (s, 1H, NH), 8.00–7.98
(m, 2H, Ar‐H), 7.93–7.85 (m, 4H, Ar‐H), 7.70–7.66 (m, 1H, Ar‐H),
7.57–7.53 (m, 2H, Ar‐H), and 7.41 (s, 2H, NH2); 13C NMR (DMSO‐d6,
ppm, 100 MHz) δ 179.9 (C═S), 168.7 (C═O), 141.8, 141.4, 133.7,
132.5, 129.2, 128.9, 126.7, and 124.8; HRMS (ESI‐MS)
|
4.1.3
General procedure for the synthesis of
compounds 2a–g (Scheme 1)
A solution of fluorobenzoyl chloride (1 mmol) in acetone (20 ml)
was added dropwise to a suspension of potassium thiocyanate

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TUGRAK ET AL.
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(1 mmol) in acetone (30 ml), and the reaction mixture was re-
fluxed for 1.5 hr to afford isothiocyanates. The reaction mixture
was checked with TLC and benzoyl isothiocyanate derivative was
formed in a reaction medium. After completion of the reaction,
the substituted sulfonamide derivative (1 mmol) was added and
the mixture was stirred under reflux for 3–6 hr. Upon completion
of the reaction (checked with TLC), the resulting precipitate was
collected by filtration and recrystallized from dimethylforma-
mide/ethanol/H2O (4.2:0.6:0.2) to obtain the pure products
2a–g.[23]
4‐Fluoro‐N‐({4‐[N′‐(pyrimidin‐2‐yl)sulfamoyl]phenyl}carbamothioyl)‐
benzamide (2e)
White color solid, mp: 240–241°C, yield 86%. 1H NMR (DMSO‐d6, ppm,
400 MHz), δ 12.69 (s, 1H, NH), 11.75 (s, 1H, NH), 8.54–8.48 (m, 2H,
Ar‐H), 8.08–7.95 (m, 7H, Ar‐H, NH), 7.40–7.36 (m, 2H, Ar‐H), and
7.08–7.06 (m, 1H, Ar‐H); 13C NMR (DMSO‐d6, ppm, 100 MHz) δ 179.2
(C═S), 166.9 (C═O), 166.2, 163.7, 158.3, 156.8, 141.8, 137.3, 131.8,
128.2, 123.8, 115.6, and 115.4; HRMS (ESI‐MS) C18H14N5O3FS2; cal-
culated [M+H]+: 432.0595; found [M+H]+: 432.0603.
4‐Fluoro‐N‐({4‐[N′‐(4‐methylpyrimidin‐2‐yl)sulfamoyl]phenyl}‐
carbamothioyl)benzamide (2f)
4‐Fluoro‐N‐({4‐[N′‐(acetyl)sulfamoyl]phenyl}carbamothioyl)‐
benzamide (2a)
Light cream color solid, mp: 235–236°C, yield 86%. 1H NMR (DMSO‐
d6, ppm, 400 MHz), δ 12.69 (s, 1H, NH), 11.75 (s, 1H, NH), 8.32
(dd, 1H, Ar‐H, J = 9.8, 4.5 Hz), 8.08–7.93 (m, 7H, Ar‐H, NH), 7.40–7.36
(m, 2H, Ar‐H), 6.92–6.91 (m, 1H, Ar‐H), and 2.33 (s, 3H, CH3); 13C
NMR (DMSO‐d6, ppm, 100 MHz) δ 179.2 (Carddp═S), 166.9 (C═O),
166.2, 163.7, 156.4, 141.6, 137.5, 131.8, 131.7, 128.4, 123.6, 115.6,
115.4, and 23.2; HRMS (ESI‐MS) C19H16N5O3FS2; calculated [M+H]+:
446.0751; found [M+H]+: 446.0747.
Light white color solid, mp: 247–248°C, yield 90%. 1H NMR (DMSO‐
d6, ppm, 400 MHz), δ 12.72 (s, 1H, NH), 12.14 (s, 1H, NH), 11.78
(s, 1H, NH), 8.09–8.06 (m, 2H, Ar‐H), 8.02–7.94 (m, 4H, Ar‐H),
7.41–7.36 (m, 2H, Ar‐H), and 1.95 (s, 3H, –CH3); 13C NMR (DMSO‐d6,
ppm, 100 MHz) δ 179.3 (C═S), 168.8 (C═O), 166.9, 142.5, 136.0,
131.8, 131.7, 128.3, 123.9, 115.6, 115.4, and 23.2; HRMS (ESI‐MS)
C16H14N3O4FS2; calculated [M+Na]+: 418.0302; found [M+Na]+:
418.0284.
4‐Fluoro‐N‐({4‐[N′‐(4,6‐dimethylpyrimidin‐2‐yl)sulfamoyl]phenyl}‐
carbamothioyl)benzamide (2g)
4‐Fluoro‐N‐({4‐[N′‐(diaminomethylene)sulfamoyl]phenyl}‐
carbamothioyl)benzamide (2b)
White color solid, mp: 210–211°C, yield 84%. 1H NMR (DMSO‐d6, ppm,
400 MHz), δ 12.69 (s, 1H, NH), 11.74 (s, 1H, NH), 8.08–8.01 (m, 5H,
Ar‐H), 7.92 (d, 2H, Ar‐H, J = 7.2 Hz), 7.40–7.35 (m, 2H, Ar‐H), 6.76 (s, 1H,
NH), and 2.26 (s, 6H, CH3); 13C NMR (DMSO‐d6, ppm, 100 MHz) δ 179.1
(C═S), 167.0 (C═O), 166.2, 163.7, 155.9, 141.4, 137.9, 131.8, 131.7,
128.7, 123.4, 115.6, 115.4, and 22.7; HRMS (ESI‐MS) C20H18N5O3FS2;
calculated [M+H]+: 460.0908; found [M+H]+: 460.0915.
Light cream color solid, mp: 240–241°C, yield 82%. 1H NMR
(DMSO‐d6, ppm, 400 MHz), δ 12.64 (s, 1H, NH), 11.73 (s, 1H, NH),
8.09–8.06 (m, 2H, Ar‐H), 7.85–7.78 (m, 4H, Ar‐H), 7.41–7.37
(m, 2H, Ar‐H), and 6.75 (bs, 4H, NH2); 13C NMR (DMSO‐d6, ppm,
100 MHz) δ 179.2 (C═S), 167.1 (C═O), 163.7, 158.1, 141.8, 140.3,
131.8, 128.6, 126.1, 124.1, and 115.6; HRMS (ESI‐MS)
C15H14N5O3FS2; calculated [M+H]+: 396.0595; found [M+H]+:
396.0569.
|
4.2
Pharmacological/biological assays
4‐Fluoro‐N‐({4‐sulfamoylphenyl}carbamothioyl)benzamide (2c)
White color solid, mp: 215–216°C, yield 80%. 1H NMR (DMSO‐d6,
ppm, 400 MHz), δ 12.67 (s, 1H, NH), 11.76 (s, 1H, NH), 8.09–8.06
(m, 2H, Ar‐H), 7.92–7.85 (m, 3H, Ar‐H), and 7.42–7.37 (m, 5H, Ar‐H, NH);
13C NMR (DMSO‐d6, ppm, 100 MHz) δ 179.9 (C═S), 167.6 (C═O),
166.7, 164.2, 141.8, 132.3, 129.1, 126.8, 124.8, and 116.1; HRMS
(ESI‐MS) C14H12N3O3FS2; calculated [M–H]–: 352.0231; found
[M–H]–: 352.0228.
|
4.2.1
Carbonic anhydrase enzyme assay
The purification of cytosolic CA isoenzymes (CA I and CA II) was
previously described with a simple one‐step method by Sepharose‐
4BL‐tyrosine‐sulfanilamide affinity chromatography.[37] The protein
quantity in the column effluents was determined spectro-
photometrically at 280 nm. Sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS‐PAGE) was applied with a BioRad Mini Gel
system, Mini‐PROTEAN system, Bio‐Rad Laboratories, Inc., after
purification of both CA isoenzymes. Briefly, it was performed in ac-
rylamide for the running (10%) and the stacking gel (3%) contained
SDS (0.1%), respectively. The increase in absorbance of the reaction
medium was spectrophotometrically recorded at 348 nm. Activities
of CA isoenzymes were determined according to a method by Ver-
poorte et al.[38] Also, the quantity of protein was determined at
595 nm according to the Bradford method.[39] Bovine serum albumin
was used as standard protein. The experimental procedure was based
on the procedures reported in the literature.[4–6,10,40,41]
4‐Fluoro‐N‐({4‐[N′‐(thiazol‐2‐yl)sulfamoyl]phenyl}carbamothioyl)‐
benzamide (2d)
Light cream color solid, mp: 239–240°C, yield 88%. 1H NMR (DMSO‐
d6, ppm, 400 MHz), δ 12.80 (s, 1H, NH), 12.66 (s, 1H, NH), 11.74
(s, 1H, NH), 8.07–8.05 (m, 2H, Ar‐H), 7.90–7.82 (m, 4H, Ar‐H), 7.38
(t, 2H, Ar‐H, J = 7.4 Hz), 7.28 (s, 1H, Ar‐H), and 6.86 (s, 1H, Ar‐H); 13C
NMR (DMSO‐d6, ppm, 100 MHz) δ 179.7 (C═S), 169.4 (C═O), 167.5,
164.2, 166.8, 141.6, 139.9, 132.3, 129.1, 126.9, 124.7, 116.1, and
108.8; HRMS (ESI‐MS) C17H13N4O3F S3; calculated [M+H]+:
437.0207; found [M+H]+: 437.0187.

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|
4.2.2
Calculation of IC50 and Ki values
[19] J. Moeker, K. Teruya, S. Rossit, B. L. Wilkinson, M. Lopez, L. F. Bor-
naghi, A. Innocenti, C. T. Supuran, S. A. Poulsen, Bioorg. Med. Chem.
2012, 20, 2392.
An activity (%)–compound graph was drawn to calculate the CA in-
hibition potential of the compounds. The IC50 values were obtained
from activity (%) versus compound plots. Three different con-
centrations were used to calculate Ki values. The Lineweaver–Burk
plots[42] were drawn and calculations were performed as described in
detail before.[4–6,10,40,41]
[20] E. Domínguez‐Álvarez, D. Łażewska, Z. Szabó, S. Hagenow, D. Reiner,
M. Gajdács, G. Spengler, H. Stark, J. Handzlik, K. Kieć‐Kononowicz,
ChemistrySelect 2019, 4, 10943.
[21] V. Ravichandran, S. Shalini, K. S. Kumar, H. Rajak, R. K. Agrawal, Lett.
Drug Des. Discov. 2019, 16, 618.
[22] A. Saeed, S. Zaib, A. Pervez, A. Mumtaz, M. Shahid, J. Iqbal, Med.
Chem. Res. 2013, 22, 3653.
[23] M. Mahdavi, M. S. Shirazi, R. Taherkhani, M. Saeedi, E. Alipour, F. H.
Moghadam, A. Moradi, H. Nadri, S. Emami, L. Firoozpour, A. Shafiee,
A. Foroumadi, Eur. J. Med. Chem. 2014, 82, 308.
ACKNOWLEDGMENTS
The authors would like to thank Dr. C. Kazaz and Dr. B. Anil (Ataturk
University) for NMRs.
[24] T. X. Yun, T. Qin, Y. Liu, L. H. Lai, Eur. J. Med. Chem. 2016, 124, 229.
[25] Y. Lou, P. C. McDonald, A. Oloumi, S. Chia, C. Ostlund, A. Ahmadi,
Cancer Res. 2011, 71, 4733.
[26] F. Pacchiano, M. Aggarwal, B. S. Avvaru, A. H. Robbins, A. Scozzafava,
R. McKenna, C. T. Supuran, Chem. Commun. 2010, 46, 8371.
[27] P. C. McDonald, S. Chia, P. L. Bedard, Q. Chu, M. Lyle, L. Tang,
M. Singh, Z. Zhang, C. T. Supuran, D. J. Renouf, S. Dedhar, Am. J. Clin.
Oncol. 2020, 43, 484.
CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.
ORCID
[28] S. M. Monti, C. T. Supuran, G. de Simone, Expert Opin. Ther. Pat. 2013,
23, 737.
Mehtap Tugrak
Halise Inci Gul
Yeliz Demir
http://orcid.org/0000-0002-6535-6580
https://orcid.org/0000-0001-6164-9602
https://orcid.org/0000-0003-2013-7162
https://orcid.org/0000-0001-5993-1668
[29] P. C. McDonald, J. Y. Winum, C. T. Supuran, S. Dedhar, Oncotarget
2012, 3, 84.
Ilhami Gulcin
[30] H. B. Mei, J. L. Han, S. Fustero, M. Medio‐Simon, D. M. Sedgwick,
C. Santi, R. Ruzziconi, V. A. Soloshonok, Chem. Eur. J. 2019, 25, 11797.
[31] C. Yamali, D. O. Ozgun, H. I. Gul, H. Sakagami, C. Kazaz, N. Okudaira,
Med. Chem. Res. 2017, 26, 2015.
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section.
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How to cite this article: Tugrak M, Gul HI, Demir Y, Gulcin I.
Synthesis of benzamide derivatives with thiourea‐substituted
benzenesulfonamides as carbonic anhydrase inhibitors. Arch
Pharm. 2020;e2000230.
[17] F. M. Awadallah, S. Bua, W. R. Mahmoud, H. H. Nada, A. Nocentini,
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https://doi.org/10.1002/ardp.202000230