Novel synthesized SLC-0111 thiazole and thiadiazole analogues: Determination of their carbonic anhydrase inhibitory activity and molecular modeling studies

Article abstract of DOI:10.1016/j.bioorg.2019.04.002

In the presented work, we report the design and synthesis of novel SLC-0111 thiazole and thiadiazole analogues (11a–d, 12a–d, 16a–c and 17a–d). A bioisosteric replacement approach was adopted to replace the 4-fluorophenyl tail of SLC-0111 with thiazole and thiadiazole ones, which were thereafter extended with lipophilic un/substituted phenyl moieties. All the newly synthesized SLC-0111 analogues were evaluated in vitro for their inhibitory activity towards a panel of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) isoforms (hCA I, II, IX and XII), using a stopped-flow CO₂ hydrase assay. All the examined isoforms were inhibited by the primary sulfonamide derivatives (11a–d and 12a–d) in variable degrees with the following K_I ranges: 162.6–7136 nM for hCA I, 9.0–833.6 nM for hCA II, 7.9–153.0 nM for hCA IX, and 9.4–94.0 nM for hCA XII. In particular, compounds 12b and 12d displayed 5.5-fold more potent inhibitory activity (K_Is = 8.3 and 7.9 nM, respectively) than SLC-0111 (K_I = 45 nM) towards hCA IX. Molecular docking study was carried out for 12d within the hCA IX (PDB 3IAI) active site, to justify its inhibitory activity.

Full text of DOI:10.1016/j.bioorg.2019.04.002

Accepted Manuscript
Novel synthesized SLC-0111 thiazole and thiadiazole analogues: Determination
of their carbonic anhydrase inhibitory activity and molecular modeling studies
Mahmoud F. Abo-Ashour, Wagdy M. Eldehna, Alessio Nocentini, Hany S.
Ibrahim, Silvia Bua, Hatem A. Abdel-Aziz, Sahar M. Abou-Seri, Claudiu T.
Supuran
PII:
S0045-2068(19)30356-6
DOI:
https://doi.org/10.1016/j.bioorg.2019.04.002
YBIOO 2908
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 March 2019
31 March 2019
2 April 2019
Please cite this article as: M.F. Abo-Ashour, W.M. Eldehna, A. Nocentini, H.S. Ibrahim, S. Bua, H.A. Abdel-Aziz,
S.M. Abou-Seri, C.T. Supuran, Novel synthesized SLC-0111 thiazole and thiadiazole analogues: Determination of
their carbonic anhydrase inhibitory activity and molecular modeling studies, Bioorganic Chemistry (2019), doi:
https://doi.org/10.1016/j.bioorg.2019.04.002
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Novel synthesized SLC-0111 thiazole and thiadiazole analogues:
Determination of their carbonic anhydrase inhibitory activity and
molecular modeling studies
Mahmoud F. Abo-Ashoura, Wagdy M. Eldehnab,*, Alessio Nocentinic, Hany S. Ibrahima,
Silvia Bua c, Hatem A. Abdel-Azizd, Sahar M. Abou-Serie,*, Claudiu T. Supuranc,**
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University,
Badr City, Cairo 11829, Egypt
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University,
Kafrelsheikh, P.O. Box 33516, Egypt
cDepartment of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences,
University of Florence, Polo Scientifico, Via U. Schiff 6, 50019, Sesto Fiorentino, Firenze, Italy
dDepartment of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622,
Egypt
eDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini
Street, Cairo, P.O. Box 11562, Egypt
ABSTRACT
In the presented work, we report the design and synthesis of novel SLC-0111 thiazole
and thiadiazole analogues (11a-d, 12a-d, 16a-c and 17a-d). A bioisosteric replacement
approach was adopted to replace the 4-fluorophenyl tail of SLC-0111 with thiazole and
thiadiazole ones, which were thereafter extended with lipophilic un/substituted phenyl
moieties. All the newly synthesized SLC-0111 analogues were evaluated in vitro for their
inhibitory activity towards a panel of the metalloenzyme carbonic anhydrase (CA, EC
4.2.1.1) isoforms (hCA I, II, IX and XII), using a stopped-flow CO2 hydrase assay. All
the examined isoforms were inhibited by the primary sulfonamide derivatives (11a-d and
12a-d) in variable degrees with the following KIs ranges: 162.6–7136 nM for hCA I, 9.0–
833.6 nM for hCA II, 7.9–153.0 nM for hCA XI, and 9.4–94.0 nM for hCA XII. In
particular, compounds 12b and 12d displayed 5.5-fold more potent inhibitory activity
(KIs = 8.3 and 7.9 nM, respectively) than SLC-0111 (KI = 45 nM) towards hCA IX.
1

Molecular docking study was carried out for 12d within the hCA IX (PDB 3IAI) active
site, to justify its inhibitory activity.
__________
Keywords: Carbonic anhydrase inhibitors; Molecular docking; SLC-0111 analogues;
Thiazoles and thiadiazoles; Ureido-benzenesulfonamide.
* Corresponding authors. E-mail addresses: wagdy2000@gmail.com (W.M. Eldehna),
saharshaarawy_69@yahoo.com (S.M. Abou-Seri), claudiu.supuran@unifi.it (C.T.
Supuran).
2

1. Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes acting as efficient
catalysts for the reversible hydration of carbon dioxide to bicarbonate [1, 2]. 16 different
α-CA isoforms have been isolated in mammals, which differ in their subcellular
localization and tissue distribution, and play crucial physiological roles in various
processes such as lipogenesis, gluconeogenesis, ureagenesis and tumorigenicity [3-6].
Recently, different carbonic anhydrases become validated drug targets for various
applications, such as CA VII and XIV for antiepileptic drugs, CA II, IV and XII for
antiglaucoma drugs, CA II, IV, XII and XIV for diuretics and CA IX and XII isoforms
for cancer therapies, especially in hypoxic tumors [3, 4].
SLC-0111 (I, Fig. 1) is an ureido substituted benzenesulfonamide derivative that
possesses selective inhibitory action towards CA isoforms IX and XII over the off-target
ubiquitous hCA I and hCA II. SLC-0111 displayed significant anticancer/antimetastatic
action in animal models with good pharmacokinetic profile. SLC-0111 is currently in
Phase I/II clinical trials for the treatment of metastatic solid hypoxic tumors [7-9]. The
ureido linker in SLC-0111 has an important role because it donates a great flexibility to
the tail of the molecule which imparts the possibility for the inhibitor to adopt a diversity
of orientations when bound within the enzyme active site. These orientations permit the
specific interactions between the inhibitor tail and amino acid residues at the entrance of
the active site cavity that represents the most variable region in several α-CA isoforms
[10-15].
Recently, SLC-0111 has been adopted as a lead compound for designing other
molecules with potential selective and potent CA IX inhibitory activity. Two studies have
reported the design and synthesis of novel series of SLC-0111 hybrids via merging SLC-
0111 and the pharmacophoric elements of isatin in a single chemical framework
(compound II, Fig. 1). Also, the bioisosteric replacement strategy was adopted through
substitution of the ureido spacer in SLC-0111 with different linkers [16-20] such as
enaminone, thioureido, selenoureido, cyanoguanidine or 1,3-triazene linkers (compounds
III-VII, respectively, Fig. 1). The inhibition profiles of these analogues displayed loss of
selectivity against the tumor-associated hCA IX over the cytosolic hCA II isoform
3

through significant improvement of the inhibitory activity against hCA II and/or with
worsening of effectiveness towards hCA IX.
Furthermore, several SLC-0111 analogues were developed either via grafting
different substituents, rather than p-fluoro, within the phenyl tail (as compound VIII, Fig.
1), or via replacement of 4-fluorophenyl tail with different polycyclic tails (as compound
IX, Fig. 1) [21].
Taking the above into account, the presented work reports the design and synthesis of
novel SLC-0111 thiazole and thiadiazole analogues (Fig. 1), with the prime aim of
developing potent and selective hCA IX and hCA XII inhibitors. A bioisosteric
replacement approach was adopted to replace the 4-fluorophenyl tail of SLC-0111 with
thiazole and thiadiazole ones while keeping the ureido linker reported to induce
selectivity towards hCA IX and XII. Further structure extension with un/substituted
phenyl groups at 4- or 5-positions of thiazole and thiadiazole tails, respectively, was
applied to ensure different lipophilic environments that might be harmonious with the
hydrophobic nature of the CA IX active site, and to implement further elaboration of the
target analogues to explore a valuable SAR. All the newly synthesized SLC-0111
analogues were evaluated in vitro for their inhibitory activity towards a panel of hCA
isoforms (hCA I, hCA II, hCA IX and hCA XII), using the stopped-flow CO2 hydrase
assay. Finally, a molecular docking study was carried out for compound 12d within the
hCA IX (PDB 3IAI) active site, to justify its potent inhibitory activity.
Fig. 1. Structures of SLC-0111 and its analogues II-IX, and structures of the target
sulfonamides 11a-d, 12a-d, 16a-c and 17a-d.
4

2.
2.1. Chemistry
The synthesis of the novel sulfonamides in this study (11a-d, 12a-d, 16a-c and 17a-
Results and Discussion
d) is outlined in Schemes 1& 2. Chlorination of 4-sulfamoylbenzoic acid 1 to give 4-
sulfamoylbenzoyl chloride 2 was achieved, in 55% yield, by heating with thionyl chloride
under reflux temperature for 12 hrs. Then aroyl chloride 2 was reacted with sodium azide
at 0°C to afford aroyl azide 3 which subjected to Curtius rearrangement via heating in dry
toluene to furnish the key intermediate 4-isocyanatobenzenesulfonamide (4).
On the other hand, aminothiazole derivatives 7a-d were prepared via the reaction of
thiourea 5 with different α-halogenated carbonyl compounds 6a-d in refluxing absolute
ethanol. Whereas, thiosemicarbazide 8 was reacted with formic acid 9a in HCl to furnish
2-amino-1,3,4-thiadiazole 10a, or with aldehydes 9b-d to afford 2-benzylidenehydrazine-
1-carbothioamide intermediates which cyclized into 2-amino-5-aryl-1,3,4-thiadiazoles
10b-d via refluxing in aqueous FeCl3 solution. Finally, the key intermediates 4 was
refluxed in dry toluene with aminothiazoles 7a-d and aminothiadiazoles 10a-d to furnish
the primary sulfonamide-based ureides 11a-d and 12a-d (Scheme 1).
5

Scheme 1. Synthesis of aminothiazoles 7a-d and aminothiadiazoles 10a-d and target SLC-0111
analogues 11a-d and 12a-d; Reagents and conditions: (i) SOCl2, reflux 12 hrs, (ii) NaN3 / Ice bath
/ stirring 2 hrs, (iii) Dry toluene / reflux 1 hr, (iv) Dry toluene / reflux 4 hrs, (v) Absolute ethanol /
reflux 8 hrs, (vi) HCl / reflux 8 hrs (for 10a), or Glacial acetic acid / reflux 3 hrs (for 10b-d), (vii)
FeCl3 solution / reflux 5 hrs.
In our attempt to improve the yield of chlorination reaction for 4-sulfamoylbenzoic
acid 1 and shorten the reaction time, we used DMF as a catalyst. Interestingly, upon its
addition to the chlorination reaction mixture, DMF condensed with the amino function of
4-sulfamoylbenzoic
acid
1
to
furnish
4-(N-
((dimethylamino)methylene)sulfamoyl)benzoyl chloride 13 in 70 % yield, which
subsequently converted to the key intermediate N'-((4-isocyanatophenyl)sulfonyl)-N,N-
dimethylformimidamide (15) as described above in Scheme 1. The tertiary sulfonamide-
based ureides 16a-c and 17a-d were prepared through reaction of the key intermediate 15
with aminothiazoles 7c-e and aminothiadiazoles 10a-d in refluxing toluene (Scheme 2).
Scheme 2. Synthesis of the tertiary sulfonamide-based ureides 16a-c and 17a-d; Reagents
and conditions: (i) SOCl2/ DMF/reflux 5 hrs (ii) NaN3 / Ice bath / stirring 2 hrs, (iii) Dry toluene /
reflux 1 hr, (iv) Dry toluene / reflux 4 hrs.
6

The elemental and spectral data supported the structures of the newly synthesized
ureas 11a-d, 12a-d, 16a-c and 17a-d. IR spectra of the latter products showed the
absorption bands due to the (C=O) group in the region 1701-1720 cm-1, in addition to two
bands of (SO2) at 1053-1140 and 1327-1338 cm-1.
Furthermore, 1H NMR spectra of compounds 11a-d, 12a-d, 16a-c and 17a-d revealed
the presence of two D2O exchangeable singlet signals assigned to the NH groups of urea
1
function at δ 8.45-9.67 and 9.47-11.96 ppm. While, H NMR spectra of primary
sulfonamides 11a-d and 12a-d displayed an additional D2O exchangeable singlet signal
1
at δ 7.26-7.29 ppm attributable for (NH2) of sulfamoyl group, the H NMR spectra of
tertiary sulfonamides 16a-c and 17a-d revealed the absence of such signal and the
presence of a non-D2O exchangeable singlet signal at δ 7.41-8.27 ppm assigned to the (-
1
N=CH-) olefinic proton. Moreover, H NMR spectra of 16a-c and 17a-d showed two
characteristic signals resonating in the aliphatic range δ 2.90-2.97 and 3.14-3.20 ppm
attributable for the two methyl groups of N-(CH3)2.
13
On the other hand, the C NMR spectra of 11a-d, 12a-d, 16a-c and 17a-d revealed
the presence of the signal of the carbonyl carbon (C=O) in the range δ 159.21-166.94
ppm, whereas the signals due to the carbon of the two methyl groups of N-(CH3)2 in
compounds 16a-c and 17a-d appeared at δ 35.40-35.51 and 41.29-42.09 ppm.
2.2. Biological Evaluation
2.2.1. Carbonic anhydrase inhibition
All the newly prepared ureas (11a-d, 12a-d, 16a-c and 17a-d) were evaluated for
their ability to inhibit the physiologically relevant hCA isoforms, hCA I and II (cytosolic)
as well as hCA IX and XII (trans membrane, tumor associated isoforms) via a stopped
flow CO2 hydrase assay [22] using SLC-0111 and acetazolamide (AAZ) as standard
inhibitors. The following structure–activity relationship (SAR) can be deduced from the
inhibition data displayed in Table 1:
7

Table 1: Inhibition data of hCA isoforms I, II, IX and XII with SLC-0111 and ureides
(11a-d, 12a-d, 16a-c and 17a-d), determined by stopped-flow CO2 hydrase assay, using
SLC-0111 and AAZ as a standard inhibitors.
KI (nM)
Comp.
R
hCA I
1005.1
270.1
hCA II
833.6
17.2
hCA IX
153.0
64.7
hCA XII
94.0
H
11a
C6H5
71.3
11b
11c
4-FC6H4
4-ClC6H4
H
191.4
9.2
13.8
27.6
282.0
24.1
53.9
57.4
11d
12a
7136.0
162.6
671.7
26.7
143.4
8.3
86.9
C6H5
9.4
12b
12c
4-FC6H4
4-ClC6H4
4-FC6H4
4-ClC6H4
4-OCH3C6H4
H
397.2
9.0
12.4
26.2
623.7
45.0
7.9
9.9
12d
16a
>10000
>10000
>10000
>10000
>10000
>10000
>10000
5080.0
250.0
>10000
>10000
>10000
6815.2
>10000
>10000
>10000
960.0
12.5
>10000
>10000
>10000
>10000
>10000
>10000
>10000
45.0
>10000
>10000
>10000
>10000
>10000
>10000
>10000
4.5
16b
16c
17a
C6H5
17b
17c
4-FC6H4
4-ClC6H4
-
17d
SLC-0111
AAZ
-
25.0
5.7
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of
 5-10 % of the reported values).
8

(i) All the tertiary sulfonamides reported here (16a-c and 17a-d) failed to inhibit all
the tested hCAs up to 10 μM, except compound 17a displayed weak inhibitory activity
towards hCA II (KI = 6.815 μM).
(ii) The ubiquitous cytosolic isoform hCA I was moderately inhibited by the prepared
primary sulfonamides (11a-d and 12a-d) with KI values ranging from 162.6 to 1005.1
nM, apart from sulfonamide 12a which exhibited a lower inhibitory efficacy (KI = 7136.0
nM). Noteworthy, grafting un/substituted phenyl groups at thiazole (11b-d) and
thiadiazole (12b-d) rings improved the activity against hCA I.
(iii) The in vitro kinetic data displayed in Table 1 showed that the physiologically
dominant isoform hCA II was efficiently inhibited by the primary sulfonamides (11b-d
and 12b-d) endowed with un/substituted phenyl groups with inhibition constants ranging
in the low nanomolar range, in detail, between 9.0 and 45.0 nM. In particular, the 4-
fluorophenyl-bearing thiazolo (11c) and thiadiazolo (12c) derivatives emerged as the
most potent counterparts against hCA II with single-digit nanomolar inhibitory activities
(KIs = 9.2 and 9.0 nM, respectively). Contrariwise, unsubstituted thiazolo (11a) and
thiadiazolo (12a) derivatives elicited weak activity towards this isoform with KI values of
833.6 and 671.7 nM, respectively.
(vi) The tumor-associated isoform hCA IX was effectively inhibited by all the
prepared primary sulfonamides (11a-d and 12a-d) with KIs spanning in the nanomolar
range: 7.9 – 153 nM. Superiorly, the phenyl and 4-chlorophenyl thiadiazole derivatives
12b and 12d emerged as a single-digit nanomolar hCA IX inhibitors with KIs values of
8.3 and 7.9 nM, respectively, which are 5.5-fold more potent than SLC-0111 (KI = 45.0
nM against hCA IX) and 3-fold more potent than the standard drug AAZ (KI = 25.0 nM
against hCA IX). Also, compounds 11c (KI = 13.8 nM) and 12c (KI = 12.4 nM) were 3.6
times more potent than SLC-0111 and 2 times more active than AAZ with.
Noteworthy, the SAR outcomes highlighted that incorporation of thiadiazole moiety
(compounds 12a-d; KIs: 7.9 – 143.4 nM) is more advantageous for activity towards hCA
IX than the thiazole moiety (compounds 11a-d; KIs: 13.8 – 153.0 nM). In addition,
grafting 4-aryl or 5-aryl substituents in the first (11b-d; KIs: 13.8 – 64.7 nM) and second
(12b-d; KIs: 7.9 – 12.4 nM) series resulted in about 11-2.3 and 18-11.5 fold efficacy
9

enhancement in comparison to their unsubstituted analogues 11a (KI = 153.0 nM) and
12a (KI = 143.4 nM), respectively. Regarding, the impact of substitution with aryl
moieties within the first series 11; the inhibitory activities were decreased in the order of
4-F-C6H4 > 4-Cl-C6H4 > C6H5, whereas the decreasing order for the second series 12 was
4-Cl-C6H4 > C6H5 > 4-F-C6H4.
(v) The second tumor-associated isoform examined here was hCA XII [23]. It was
apparent from the obtained results, Table 1, that the newly prepared sulfonamides 11a-d
and 12a-d exhibited high to excellent inhibitory activities towards hCA XII with KIs in
the range of 94 – 9.4 nM. In particular, thiadiazolo derivatives 12b and 12d were found
to be the most potent hCA XII inhibitors with KI values equal to 9.4 and 9.9 nM,
respectively. Additionally, compounds 11c and 12c showed high inhibitory activity with
KI values of 27.6 and 26.2 nM, respectively. Similar to the SAR for hCA IX inhibition;
both incorporation of thiadiazole moiety rather than thiazole moiety, and grafting an aryl
substituent within the first and second series are more beneficial for inhibitory activity
against hCA XII.
Table 2. Selectivity ratios for the inhibition of hCA IX and XII over hCA I and II for
ureides (11a-d and 12a-d), SLC-0111 and acetazolamide.
Cmpd
I/IX
II/IX
I/XII
II/XII
6.6
4.2
5.4
0.3
0.7
0.4
4.7
3.2
0.7
5.7
21.3
0.5
10.7
3.8
8.9
0.2
11a
11b
13.9
5.2
6.9
0.3
11c
4.9
0.4
11d
49.8
19.6
32.0
78.9
112.9
10.0
82.1
17.3
15.2
63.0
1128.9
43.9
7.7
12a
2.8
12b
0.3
12c
4.5
12d
213.3
2.2
SLC-0111
AAZ
10

(vi) As a result of the inhibitory profiles of the prepared ureides listed in Table 1, the
selectivity index (SI) for each of the primary sulfonamides (11a-d and 12a-d) herein
reported was calculated and displayed in Table 2. Regarding selectivity towards hCA IX
and XII over hCA I, all the examined primary sulfonamides exhibited good SIs spanning
in the range 4.2 – 78.9 and 3.8 – 82.1, respectively. Meanwhile, only compounds 11a,
12a, 12b and 12d were found to be selective towards hCA IX and XII over hCA II with
SIs in the range of 3.2 – 5.7 and 2.8 – 8.9, respectively.
In summary, the adopted bioisosteric replacement approach in this study to replace
the 4-fluorophenyl tail of SLC-0111 with different thiazole and thiadiazole tails,
successfully improved the inhibitory activity towards hCA IX, such as ureas 12b and 12d
that possessed 5.5-fold more potent activity than SLC-0111 towards hCA IX.
Unfortunately, such improved activity against hCA IX was accompanied with an
enhanced activity towards hCA II, too. This trend resulted in a decreasing of the hCA
IX/II selectivity for both compounds 12b and 12d by 6.6- and 3.7-fold in comparison to
SLC-0111, respectively.
In terms of both hCA IX inhibitory activity and selectivity, compound 12d would be
considered as a promising lead molecule for the development of further effective CA
inhibitors candidates targeting the tumor-associated isoforms hCA IX.
3. Molecular modeling studies
Molecular docking simulations were performed in order to justify the inhibitory
activity of compound 12d by investigating its interactions with the key amino acids
within the active site of hCA IX isoform (PDB 3IAI). As shown in Fig. 2, the interaction
of 12d was found to be analogous to SLC-0111 in many points. The primary sulfonamide
moiety make its usual role as zinc-binding group with the additional hydrogen bonds with
Thr199 (2.63 Å) and Thr200 (3.14 Å). Also, the carbonyl moiety of the ureido group of
both 12d and SLC-0111 can interact with Gln92 by forming a hydrogen bond (2.86 and
2.88 Å, respectively) in the middle of the pocket. Moreover, the thiadiazole tail possessed
hydrophobic interactions with Val131, Gln92 and Leu91 which is similar to 4-
fluourophenyl in SLC-0111. Pose analysis for the alignment of both 12d and SLC-0111
11

(as shown in Fig. 2B), unveiled the ability of 12d to establish an extra hydrophobic
interaction between the terminal p-chlorophenyl moiety and Leu91, that could account
for the enhancement of its hCA XI inhibition (Table 1).
(A)
(B)
Fig. 2. (A) Docking pose of compound 12d within the active site of hCA IX (PDB 3IAI) showing
the favorable interactions with key amino acids and Zn metal. (B) Structural superposition of both
compounds 12d and SLC-0111 within the active site of hCA IX (PDB 3IAI) illustrating the extra
hydrophobic interactions of (4-chlorophenyl) ring. Protein unit was illustrated as grey ribbon
except the amino acids of active site were drawn as lines with orange carbon atoms. Hydrogen
bonds were shown as yellow dots, metallic interaction were shown as violet dots and polar
hydrogens only were visible for clarity.
4. Conclusion
In summary, we report here the synthesis of novel series of SLC-0111 analogues via
replacement of 4-fluorophenyl tail of SLC-0111 with different un/substituted thiazole and
thiadiazole tails. The structure of the novel analogues was confirmed by the different
spectral and elemental analyses methods. The inhibition profiles of the newly synthetized
compounds were determined against hCA I, II, IX and XII using the stopped-flow CO2
hydrase assay. All the tertiary sulfonamides (16a-c and 17a-d) failed to inhibit all the
tested hCAs up to 10 μM, except 17a that displayed weak inhibitory activity towards
hCA II (KI = 6.815 μM). Whereas, the tested isoforms were inhibited by the synthesized
primary sulfonamides (11a-d and 12a-d) in variable degrees with KIs range: 162.6–7136
nM for hCA I, 9.0–833.6 nM for hCA II, 7.9–153.0 nM for hCA XI, and 9.4–94.0 nM for
hCA XII. In particular, compounds 11c and 12b-d showed superior inhibitory activity
against hCA IX with 3.3–5.7-fold enhanced potency than SLC-0111. The weightiness of
incorporation of thiadiazole tail, rather than a thiazole one, and grafting an aryl
12

substituent within the first and second series for the inhibitory activity against hCA XI
and XII, was explored via the SAR study. Moreover, the molecular docking for 12d
unveiled its ability to establish an extra hydrophobic interaction between the terminal p-
chlorophenyl moiety and Leu91 that could account for the enhancement of its hCA XI
inhibition.
5. Experimental
5.1. Chemistry
5.1.1. General
Melting points were measured with a Stuart melting point apparatus and were
uncorrected. Infrared (IR) Spectra were recorded as KBr disks using Schimadzu FT-IR
8400S spectrophotometer. Mass spectral data are given by GCMS-QP1000 EX
1
spectrometer at 70 e.V. NMR Spectra were recorded on Bruker spectrometer. H
spectrum was run at 400 MHz and 13C spectrum was run at 100 MHz in deuterated
dimethylsulfoxide (DMSO-d6). Chemical shifts are expressed in values (ppm) using the
solvent peak as internal standard. All coupling constant (J) values are given in hertz. The
abbreviations used are as follows: s, singlet; d, doublet; m, multiplet. Elemental analyses
were carried out at the Regional Center for Microbiology and Biotechnology, Al-Azhar
University, Cairo, Egypt. High-resolution mass spectra were recorded using a Bruker
MicroTOF spectrometer. Analytical thin layer chromatography (TLC) on silica gel plates
containing UV indicator was employed routinely to follow the course of reactions and to
check the purity of products.
5.1.2. Synthesis of compounds 7a-e and 10a-d
Compounds 7a-e and 10a-d were prepared according to the literature procedures [24-26].
5.1.3. Synthesis of isocyanate derivatives 4,15.
A stirred suspension of 4-sulfamoylbenzoic acid 1 (1 gm, 5 mmol) in SOCl2 (30 mL)
was refluxed for 12 hrs in the absence of DMF, or refluxed for 5 hrs in the presence of
DMF (0.73 gm, 10 mmol). The excess of thionyl chloride was removed by repeated
evaporation with dry toluene in vacuo. The obtained crude products of aroyl chlorides 2
13

and 13, respectively, are used in the next step without further purification. Then, an
aqueous solution of sodium azide (0.65 gm, 10 mmole) was added to the solution of
compounds 2 and 13 (5 mmol) in dry acetone (10 mL) at 0oC. The reaction mixture was
stirred for 2 hrs at room temperature. The obtained solid was filtered off and washed with
cold water then petroleum ether to give aroyl azides 3 and 14.
5.1.4. Synthesis of target compounds 11a-d, 12a-d, 16a-c and 17a-d.
Both of azides 3 and 14 (1 mmol) are subjected to Curtius Rearrangement to afford
the isocyanate derivatives 4 and 15, respectively, via heating under reflux temperature in
dry toluene (10 mL) for 1 hr. To this hot solution of 4 and 15 in toluene, the appropriate
aminothiazole (7a-e) and aminothiadiazole (10a-d) derivatives (1 mmol) were added.
This mixture was further heated under reflux for 4 hrs. The formed precipitate was
filtered off while hot and washed with ethanol and petroleum ether, then recrystallized
from DMF/ethanol to obtain ureides 11a-d, 12a-d, 16a-c and 17a-d.
5.1.4.1. 4-(3-(Thiazol-2-yl)ureido)benzenesulfonamide 11a.
Yellow crystal (yield 55%), m.p. 228-230 °C; IR (KBr, ν cm-1): 3313, 3332, 3375 (NH2,
1
2NH), 1712 (C=O) and 1149, 1315 (SO2); H NMR (DMSO-d6, 400 MHz) δ ppm: 7.14
(d, 1H, H-5 of thiazole ring, J = 8.0 Hz), 7.26 (s, 2H, NH2, D2O exchangeable), 7.39 (d,
1H, H-4 of thiazole ring, J = 8.0 Hz), 7.65 (d, 2H, H-3 and H-5 of -C6H4-SO2NH2, J = 8.0
Hz), 7.75 (d, 2H, H-2 and H-6 of -C6H4-SO2NH2, J = 8.0 Hz), 9.34 (s, 1H, NH urea, D2O
13
exchangeable), 10.05 (s, 1H, NH urea, D2O exchangeable); C NMR (DMSO-d6, 100
MHz) δ ppm: 118.36, 126.14, 127.35, 128.14, 38.11, 142.36, 146.64, 159.21 (C=O); MS
m/z [%]: 298.80 [M+, 13.95], 48.26 [100]; Anal. Calcd. for C10H10N4O3S2 (298.02): C,
40.26; H, 3.38; N, 18.78; found C, 40.01; H, 3.41; N, 18.81.
5.1.4.2. 4-(3-(4-Phenylthiazol-2-yl)ureido)benzenesulfonamide 11b.
Buff crystal (yield 51%), m.p. 270-271 °C; IR (KBr, ν cm-1): 3259, 3348, 3498 (NH2,
1
2NH), 1701 (C=O) and 1153, 1319 (SO2); H NMR (DMSO-d6, 400 MHz) δ ppm: 7.29
(s, 2H, NH2, D2O exchangeable), 7.42-7.45 (m, 3H, H-3, H-4 and H-5 of -C6H5), 7.59 (s,
1H, H-5 of thiazole ring), 7.67 (d, 2H, H-3 and H-5 of -C6H4-SO2NH2, J = 8.0 Hz), 7.78
(d, 2H, H-2 and H-6 of -C6H4-SO2NH2, J = 8.0 Hz), 7.89 (d, 2H, H-2 and H-6 of -C6H5, J
14

= 8.0 Hz), 9.35 (s, 1H, NH urea, D2O exchangeable), 10.91 (s, 1H, NH urea, D2O
exchangeable); 13C NMR (DMSO-d6, 100 MHz) δ ppm: 107.91, 118.51, 126.08, 127.39,
128.22, 129.19, 134.67, 138.30, 142.14, 149.23, 151.93, 159.25 (C=O); Anal. Calcd. for
C16H14N4O3S2 (374.05): C, 51.32; H, 3.77; N, 14.96; found C, 51.22; H, 3.75; N, 14.92.
5.1.4.3. 4-(3-(4-(4-Fluorophenyl)thiazol-2-yl)ureido)benzenesulfonamide 11c.
Buff crystal (yield 60%), m.p. 240-242 °C; IR (KBr, ν cm-1): 3390, 3417, 3444 (NH2,
2NH), 1712 (C=O) and 1145, 1327 (SO2); 1H NMR (DMSO-d6, 400 MHz) δ ppm: 7.25-
7.29 (m, 4H; 2H of SO2NH2, H-3 and H-5 of 4-F-C6H4), 7.56 (s, 1H, H-5 of thiazole
ring), 7.68 (d, 2H, H-3 and H-5 of -C6H4-SO2NH2, J = 8.0 Hz), 7.77 (d, 2H, H-2 and H-6
of -C6H4-SO2NH2, J = 8.0 Hz), 7.92 (t, 2H, H-2 and H-6 of 4-F-C6H4, J = 8.0 Hz), 9.67
13
(s, 1H, NH urea, D2O exchangeable), 11.10 (s, 1H, NH urea, D2O exchangeable); C
NMR (DMSO-d6, 100 MHz) δ ppm: 107.62, 115.91, 116.12, 118.45, 120.33, 126.12,
127.36, 128.05, 128.13, 131.37, 138.21, 142.29, 152.10, 159.49 (C=O); Anal. Calcd. for
C16H13FN4O3S2 (392.04): C, 48.97; H, 3.34; N, 14.28; found C, 48.90; H, 3.38; N, 14.22.
5.1.4.4. 4-(3-(4-(4-Chlorophenyl)thiazol-2-yl)ureido)benzenesulfonamide 11d.
Red crystal (yield 49%), m.p. 239-240 °C; IR (KBr, ν cm-1): 3375, 3387, 3390 (NH2,
1
2NH), 1712 (C=O) and 1145, 1327 (SO2); H NMR (DMSO-d6, 400 MHz) δ ppm: 7.28
(s, 2H, NH2, D2O exchangeable), 7.48-7.51 (m, 3H; H-5 of thiazole ring, H-3 and H-5 of
4-Cl-C6H4), 765 (d, 2H, H-3 and H-5 of -C6H4-SO2NH2, J = 8.0 Hz), 7.78 (d, 2H, H-2
and H-6 of -C6H4-SO2NH2, J = 8.0 Hz), 7.91 (d, 2H, H-2 and H-6 of 4-Cl-C6H4, J = 8.0
Hz), 9.39 (s, H, NH urea, D2O exchangeable), 10.95 (s, 2H, NH urea, D2O
exchangeable); 13C NMR (DMSO-d6, 100 MHz) δ ppm: 108.71, 117.95, 118.52, 126.12,
127.39, 127.79, 128.11, 129.21, 132.63, 133.55, 138.33, 142.10, 146.58, 147.98, 151.93,
159.44 (C=O); HRMS (ESI) for C16H14ClN4O3S2, calcd 409.01904, found 409.01901
[M+H]+; Anal. Calcd. for C16H13ClN4O3S2 (408.01): C, 47.00; H, 3.20; N, 13.70; found
C, 47.07; H, 3.15; N, 13.68.
5.1.4.5. 4-(3-(1,3,4-Thiadiazol-2-yl)ureido)benzenesulfonamide 12a.
White crystal (yield 62%), m.p. 240-242 °C; IR (KBr, ν cm-1): 3345, 3380, 3391 (NH2,
1
2NH), 1711 (C=O) and 1149, 1315 (SO2); H NMR (DMSO-d6, 400 MHz) δ ppm: 7.27
(s, 2H, NH2, D2O exchangeable), 7.67 (d, 2H, H-3 and H-5 of -C6H4-SO2NH2, J = 8.0
15

Hz), 7.76 (d, 3H; H-5 of thiadiazole ring, H-2 and H-6 of -C6H4-SO2NH2, J = 8.0 Hz),
9.13 (s, 1H, NH urea, D2O exchangeable), 9.47 (s, 1H, NH urea, D2O exchangeable); 13C
NMR (DMSO-d6, 100 MHz) δ ppm: 118.65, 127.33, 138.45, 140.48, 143.74, 148.50,
162.83 (C=O); Anal. Calcd. for C9H9N5O3S2 (299.01): C, 36.11; H, 3.03; N, 23.40; found
C, 36.01; H, 3.10; N, 23.31.
5.1.4.6. 4-(3-(5-Phenyl-1,3,4-thiadiazol-2-yl)ureido)benzenesulfonamide 12b.
Yellow crystal (yield 50%), m.p. 277-279 °C; IR (KBr, ν cm-1): 3371, 3417, 3470 (NH2,
2NH), 1708 (C=O) and 1150, 1318 (SO2); 1H NMR (DMSO-d6, 400 MHz) δ ppm: 7.46-
7.59 (m, 5H; 2H of SO2NH2, H-3, H-4 and H-5 of -C6H5), 7.70 (d, 2H, H-3 and H-5 of -
C6H4-SO2NH2, J = 8.0 Hz), 7.78 (d, 2H, H-2 and H-6 of -C6H4-SO2NH2, J = 8.0 Hz),
7.91 (d, 2H, H-2 and H-6 of -C6H5, J = 8.0 Hz), 9.20 (s, 1H, NH urea, D2O
13
exchangeable), 9.55 (s, 1H, NH urea, D2O exchangeable); C NMR (DMSO-d6, 100
MHz) δ ppm: 118.17, 118.71, 126.86, 126.92, 127.12, 127.33, 129.72, 129.85, 130.65,
131.06, 138.51, 142.22, 152.87, 161.16 (C=O); MS m/z [%]:375.27 [M+, 74.13], 75.34
[100]; Anal. Calcd. for C15H13N5O3S2 (375.05): C, 47.99; H, 3.49; N, 18.66; found C,
47.90; H, 3.44; N, 18.60.
5.1.4.7. 4-(3-(5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl)ureido)benzenesulfonamide 12c.
Buff crystal (yield 48%), m.p. 255-258 °C; IR (KBr, ν cm-1): 3380, 3407, 3414 (NH2,
2NH), 1710 (C=O) and 1147, 1325 (SO2); 1H NMR (DMSO-d6, 400 MHz) δ ppm: 7.28-
7.40 (m, 4H; 2H of SO2NH2, H-3 and H-5 of 4-F-C6H4), 7.69 (d, 2H, H-3 and H-5 of -
C6H4-SO2NH2, J = 8.0 Hz), 7.77 (d, 2H, H-2 and H-6 of -C6H4-SO2NH2, J = 8.0 Hz),
7.79 (br s, 2H, H-2 and H-6 of 4-F-C6H4), 9.21 (s, 1H, NH urea, D2O exchangeable), 9.56
13
(s, 1H, NH urea, D2O exchangeable); C NMR (DMSO-d6, 100 MHz) δ ppm: 116.80,
117.02, 118.21, 118.71, 126.90, 127.33, 129.41, 138.47, 142.18, 145.52, 154.10, 162.52
(C=O); MS m/z [%]: 393.14 [M+, 43.52], 122.48 [100]; Anal. Calcd. for C15H12FN5O3S2
(393.04): C, 45.80; H, 3.07; N, 17.80; found C, 45.70; H, 3.10; N, 17.83.
5.1.4.8. 4-(3-(5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl)ureido)benzenesulfonamide 12d.
Buff crystal (yield 45%), m.p. 265-267 °C; IR (KBr, ν cm-1): 3370, 3385, 3390 (NH2,
2NH), 1714 (C=O) and 1145, 1327 (SO2); 1H NMR (DMSO-d6, 400 MHz) δ ppm: 7.48-
7.60 (m, 4H; 2H of SO2NH2, H-3 and H-5 of 4-Cl-C6H4), 7.69 (d, 2H, H-3 and H-5 of -
16

C6H4-SO2NH2, J = 8.0 Hz), 7.77 (d, 2H, H-2 and H-6 of -C6H4-SO2NH2, J = 8.0 Hz),
7.93 (d, 2H, H-2 and H-6 of 4-Cl-C6H4, J = 8.0 Hz), 9.22 (s, 1H, NH urea, D2O
13
exchangeable), 9.57 (s, 1H, NH urea, D2O exchangeable); C NMR (DMSO-d6, 100
MHz) δ ppm: 118.74, 12.33, 128.48, 128.81, 129.53, 129.70, 129.89, 135.56, 138.50,
142.13, 147.69, 152.80, 162.34 (C=O); Anal. Calcd. for C15H12ClN5O3S2 (409.01): C,
43.96; H, 2.95; N, 17.09; found C, 43.87; H, 2.99; N, 17.13.
5.1.4.9.
N'-((4-(3-(4-(4-Fluorophenyl)thiazol-2-yl)ureido)phenyl)sulfonyl)-N,N-
dimethylformimidamide 16a.
White crystal (yield 79%), m.p. 232-234 °C; IR (KBr, ν cm-1): 3383, 3401 (2NH), 1711
1
(C=O) and 1140, 1355 (SO2); H NMR (DMSO-d6, 400 MHz) δ ppm: 2.91 (s, 3H, N-
(CH3)2), 3.15 (s, 3H, N-(CH3)2), 7.22 (t, 2H, H-3, H-5 of 4-F-C6H4, J = 8.0 Hz), 7.51 (s,
1H, =CH), 7.61 (d, 2H, H-3, H-5 of -C6H4-SO2N J = 8.0 Hz), 7.69 (d, 2H, H-2, H-6 of -
C6H4-SO2N, J = 8.0 Hz), 7.91 (t, 2H, H-2, H-6 of 4-F-C6H4, J = 8.0 Hz), 8.19 (s, 1H, H-5
of thiazole ring), 9.85 (s, 1H, NH urea, D2O exchangeable), 11.09 (s, 1H, NH urea, D2O
exchangeable); Anal. Calcd. for C19H18FN5O3S2 (447.08): C, 51.00; H, 4.05; N, 15.65;
found C, 51.10; H, 4.00; N, 15.60.
5.1.4.10.
N'-((4-(3-(4-(4-Chlorophenyl)thiazol-2-yl)ureido)phenyl)sulfonyl)-N,N-
dimethylformimidamide 16b.
White crystal (yield 79%), m.p. 250-253 °C; IR (KBr, ν cm-1): 3363, 3444 (2NH), 1701
1
(C=O) and 1141, 1338 (SO2); H NMR (DMSO-d6, 400 MHz) δ ppm: 2.91 (s, 3H, N-
(CH3)2), 3.15 (s, 3H, N-(CH3)2), 7.46 (d, 2H, H-3, H-5 of 4-Cl-C6H4, J = 8.0 Hz), 7.60-
7.62 (m, 3H; H-3, H-5 of -C6H4-SO2N, 1H of =CH), 7.71 (d, 2H, H-2, H-6 of -C6H4-
SO2N, J = 8.0 Hz), 7.91 (d, 2H, H-2, H-6 of 4-Cl-C6H4, J = 8.0 Hz), 8.20 (s, 1H, H-5 of
thiazole ring), 9.83 (s, 1H, NH urea, D2O exchangeable), 11.09 (s, 1H, NH urea, D2O
exchangeable); 13C NMR (DMSO-d6, 100 MHz) δ ppm: 35.47, 41.31 (N-(CH3)2), 108.51,
118.40, 127.71, 127.83, 129.15, 132.55, 133.62, 136.94, 142.30, 147.95, 152.34, 159.80,
160.01 (C=O of uera); HRMS (ESI) for C19H19O3N5ClS2, calcd 464.06124, found
464.06145 [M+H]+; Anal. Calcd. for C19H18ClN5O3S2 (463.05): C, 49.19; H, 3.91; N,
15.10; found C, 49.10; H, 3.96; N, 15.01.
17

5.1.4.11.
N'-((4-(3-(4-(4-Methoxyphenyl)thiazol-2-yl)ureido)phenyl)sulfonyl)-N,N-
dimethylformimidamide 16c.
Yellow crystal (yield 79%), m.p. 223-225 °C; IR (KBr, ν cm-1): 3360, 3390 (2NH), 1708
1
(C=O) and 1153, 1342 (SO2); H NMR (DMSO-d6, 400 MHz) δ ppm: 2.91 (s, 3H, N-
(CH3)2), 3.15 (s, 3H, N-(CH3)2), 3.80 (s, 3H, OCH3), 6.98 (d, 2H, H-3, H-5 of 4-CH3O-
C6H4, J = 8.0 Hz), 7.41 (s, 1H, =CH), 7.62 (d, 2H, H-2, H-6 of 4-CH3O-C6H4, J = 8.0
Hz), 7.72 (d, 2H, H-3, H-5 of -C6H4-SO2N, J = 8.0 Hz), 7.81 (d, 2H, H-2, H-6 of -C6H4-
SO2N, J = 8.0 Hz), 8.21 (s, 1H, H-5 of thiazole ring), 9.27 (s, 1H, NH urea, D2O
13
exchangeable), 10.78 (s, 1H, NH urea, D2O exchangeable); C NMR (DMSO-d6, 100
MHz) δ ppm: 35.48, 41.32 (N-(CH3)2), 55.61 (OCH3), 105.79, 114.54, 118.60, 125.77,
127.43, 127.69, 128.66, 129.36, 137.15, 142.13, 151.88, 159.43, 160.02 (C=O of uera);
Anal. Calcd. for C20H21N5O4S2 (459.10): C, 52.27; H, 4.61; N, 15.24; found C, 52.11; H,
4.67; N, 15.27.
5.1.4.12.
N'-((4-(3-(1,3,4-Thiadiazol-2-yl)ureido)phenyl)sulfonyl)-N,N-
dimethylformimidamide 17a.
White crystal (yield 79%), m.p. 230-231 °C; IR (KBr, ν cm-1): 3373, 3435 (2NH), 1709
1
(C=O) and 1141, 1338 (SO2); H NMR (DMSO-d6, 400 MHz) δ ppm: 2.91 (s, 3H, N-
(CH3)2), 3.15 (s, 3H, N-(CH3)2), 7.66 (d, 2H, H-3, H-5 of -C6H4-SO2N, J = 8.0 Hz), 7.95
(d, 2H, H-2, H-6 of -C6H4-SO2N, J = 8.0 Hz), 8.19 (s, 1H, =CH), 8.94 (s, 1H, H-5 of
thiazole ring), 9.20 (s, 1H, NH urea, D2O exchangeable), 11.96 (s, 1H, NH urea, D2O
exchangeable); 13C NMR (DMSO-d6,100 MHz) δ ppm: 35.44, 41.29 (N-(CH3)2), 117.82,
127.45, 135.37, 144.51, 146.58, 156.99, 160.00, 166.94 (C=O); Anal. Calcd. for
C12H14N6O3S2 (354.06): C, 40.67; H, 3.98; N, 23.71; found C, 40.72; H, 3.91; N, 23.65.
5.1.4.13.
N,N-Dimethyl-N'-((4-(3-(5-phenyl-1,3,4-thiadiazol-2-
yl)ureido)phenyl)sulfonyl)formimidamide 17b.
White crystal (yield 79%), m.p. 240-241 °C; IR (KBr, ν cm-1): 3383, 3401 (2NH), 1711
1
(C=O) and 1147, 1345 (SO2); H NMR (DMSO-d6, 400 MHz) δ ppm: 2.94 (s, 3H, N-
(CH3)2), 3.18 (s, 3H, N-(CH3)2), 7.42-7.56 (m, 3H, H-3, H-4, H-5 of -C6H5), 7.71-7.77
(m, 2H, H-3, H-5 of -C6H4-SO2N), 7.87-8.02 (m, 4H; H-2, H-6 of -C6H4-SO2N, H-2, H-6
of -C6H5), 8.23 (s, 1H, =CH), 8.50 (s, 1H, NH urea, D2O exchangeable), 11.62 (s, 1H,
18

13
NH urea, D2O exchangeable); C NMR (DMSO-d6, 100 MHz) δ ppm: 35.45, 41.30 (N-
(CH3)2), 117.92, 126.87, 127.48, 127.99, 129.66, 130.05, 130.38, 131.92, 135.49, 144.38,
156.07, 159.46, 159.92, 165.15 (C=O); Anal. Calcd. for C18H18N6O3S2 (430.09): C,
50.22; H, 4.21; N, 19.52; found C, 50.15; H, 4.23; N, 19.54.
5.1.4.14.
N'-((4-(3-(5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl)ureido)phenyl)sulfonyl)-
N,N-dimethylformimidamide 17c.
Buff crystal (yield 79%), m.p. 249-250 °C; IR (KBr, ν cm-1): 3375, 3395 (2NH), 1720
1
(C=O) and 1150, 1348 (SO2); H NMR (DMSO-d6, 400 MHz) δ ppm: 2.97 (s, 3H, N-
(CH3)2), 3.20 (s, 3H, N-(CH3)2), 7.37-7.69 (m, 4H, 2H, H-3, H-5 of 4-F-C6H4, 2H, H-3,
H-5 of -C6H4-SO2N), 7.73-7.86 (m, 2H, H-2, H-6 of -C6H4-SO2N), 8.0-8.10 (m, 2H, H-2,
H-6 of 4-F-C6H4), 8.27 (s, 1H, =CH), 8.65 (s, 1H, NH urea, D2O exchangeable), 11.48 (s,
1H, NH urea, D2O exchangeable); 13C NMR (DMSO-d6, 100 MHz) δ ppm: 35.51, 42.09
(N-(CH3)2), 116.91, 118.08, 123.68, 125.69, 127.67, 129.16, 131.63, 133.16, 136.15,
144.17, 152.49, 158.64, 159.95, 162.15 (C=O); Anal. Calcd. for C18H17FN6O3S2
(448.08): C, 48.21; H, 3.82; N, 18.74; found C, 48.10; H, 3.84; N, 18.71.
5.1.4.15.
N'-((4-(3-(5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl)ureido)phenyl)sulfonyl)-
N,N-dimethylformimidamide 17d.
White crystal (yield 79%), m.p. 225-228 °C; IR (KBr, ν cm-1): 3388, 3441 (2NH), 1717
1
(C=O) and 1149, 1342 (SO2); H NMR (DMSO-d6, 400 MHz) δ ppm: 2.91 (s, 3H, N-
(CH3)2), 3.14 (s, 3H, N-(CH3)2), 7.55 (d, 2H, H-3, H-5 of 4-Cl-C6H4, J = 8.0 Hz), 7.64 (d,
2H, H-3, H-5 of -C6H4-SO2N, J = 8.0 Hz), 7.81 (d, 2H, H-2, H-6 of -C6H4-SO2N, J = 8.0
Hz), 7.90 (d, 2H, H-2, H-6 of 4-Cl-C6H4, J = 8.0 Hz), 8.18 (s, 1H, =CH), 8.45 (s, 1H, NH
urea, D2O exchangeable), 11.36 (s, 1H, NH urea, D2O exchangeable); 13C NMR (DMSO-
d6, 100 MHz) δ ppm: 35.45, 41.29 (N-(CH3)2), 117.80, 124.03, 127.42, 128.30, 129.64,
130.96, 131.86, 134.19, 135.20, 144.67, 156.00, 157.83, 159.89, 161.21 (C=O); Anal.
Calcd. for C18H17ClN6O3S2 (464.05): C, 46.50; H, 3.69; N, 18.08; found C, 46.40; H,
3.64; N, 18.01.
4.2. Biological Evaluation
4.2.1. CA inhibitory assay
19

An Applied Photophysics stopped-flow instrument has been used for assaying the CA
catalysed CO2 hydration activity [22]. 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
uncatalyzed rates were determined in the same manner and subtracted from the total
observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized
water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor
and enzyme solutions were preincubated together for 15 min at room temperature prior to
assay, in order to allow for the formation of the E-I complex. The inhibition constants
were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-
Prusoff equation, as reported earlier [27-31], and represent the mean from at least three
different determinations. The four tested CA isofoms were recombinant ones obtained in-
house as reported earlier [27-31].
4.3. Molecular docking simulation
The crystal structure of hCA IX (PDB 3IAI [32]) was downloaded from protein
databank for docking calculations using the Protein Preparation wizard in Maestro -
Schrödinger suite 2017-1, using the default values in the preprocess. The water molecules
were deleted and the default input parameters and Prime were used for adding missing
side chains or loops, also, optimization of the hydrogen bonds was done with refinement
process by running a restrained minimization (OPLS3 force field) which was controlled
by cutoff RMSD value of heavy atoms reached 0.30 Å. The 3D ligand structures were
generated with Maestro (Schrödinger suite). LigPrep was used to prophesy ionization
form of the ligands using a pH of 7.0 ± 0.5 with Epik. In docking simulations using
Glide, the protein was treated as rigid, compounds were flexibly docked, and scoring was
assigned according to the extra precision (XP) mode. Before docking process, the grid
20

box was prepared according to the default size. The constrained were assigned using the
metallic bond for Zn metal [33-35].
References
[1] (a) A. Innocenti, I. Gülçin, S.B.Ö. Sarıkaya, C.T. Supuran, Carbonic anhydrase
inhibitors. Inhibition of mammalian isoforms I–XIV with a series of natural product
polyphenols and phenolic acids, Bioorg. Med. Chem. 18 (2010) 2159–2164. (b) A.
Innocenti, I. Gülçin, A. Scozzafava, C.T.Supuran, Carbonic anhydrase inhibitors.
Antioxidant polyphenols effectively inhibit mammalian isoforms I–XV, Bioorg. Med.
Chem. Lett. 20 (2010) 5050–5053.
[2] (a) F. Turkan, A. Cetin, P. Taslimi, M. Karaman, İ. Gulçin, Synthesis, biological
evaluation and molecular docking of novel pyrazole derivatives as potent carbonic
anhydrase and acetylcholinesterase inhibitors, Bioorg. Chem. 86 (2019) 420-427. (b) K.
Kucukoglu, H. InciGul, P. Taslimi, İ. Gulçin, C.T.Supuran, Investigation of inhibitory
properties of some hydrazone compounds on hCA I, hCA II and AChE enzymes, Bioorg.
Chem. 86 (2019) 316-321.
[3] C. T. Supuran, Carbonic anhydrases: novel therapeutic applications for inhibitors and
activators. Nat. Rev. Drug Discovery 7 (2008) 168-181.
[4] V. Alterio, A. Di Fiore, K. D’Ambrosio, C.T. Supuran, G. De Simone, Multiple
binding modes of inhibitors to carbonic anhydrases: how to design specific drugs
targeting 15 different isoforms? Chem. Rev. 112 (2012) 4421-4468.
[5] D. Neri, C.T. Supuran, Interfering with pH regulation in tumours as a therapeutic
strategy. Nat. Rev. Drug Discov. 10 (2011) 767-777.
[6] G. De Simone, C.T. Supuran, Carbonic anhydrase IX: Biochemical and
crystallographic characterization of a novel antitumor target. Biochim. Biophys. Acta
1804 (2010) 404-409.
21

[7] Y. Lou, P.C. McDonald, A. Oloumi, S. Chia, C. Ostlund, A. Ahmadi, A. Kyle, U. Auf
dem Keller, S. Leung, D. Huntsman, B. Clarke, B.W. Sutherland, D. Waterhouse, M.
Bally, C. Roskelley, C.M. Overall, A. Minchinton, F. Pacchiano, F. Carta, A. Scozzafava,
N. Touisni, J.Y. Winum, C.T. Supuran, S. Dedhar, Targeting tumor hypoxia: suppression
of breast tumor growth and metastasis by novel carbonic anhydrase IX inhibitors, Cancer
Res. 71 (2011) 3364-3376.
[8] F. Pacchiano, M. Aggarwal, B.S. Avvaru, A.H. Robbins, A. Scozzafava, R.
McKenna, C.T. Supuran, Selective hydrophobic pocket binding observed within the
carbonic anhydrase II active site accommodate different 4-substituted-ureido-
benzenesulfonamides and correlate to inhibitor potency, Chem. Commun. (Camb) 46
(2010) 8371–8373.
[9] F. Pacchiano, F. Carta, P.C. McDonald, Y. Lou, D. Vullo, A. Scozzafava, S. Dedhar,
C.T. Supuran, Ureido-substituted benzenesulfonamides potently inhibit carbonic
anhydrase IX and show antimetastatic activity in a model of breast cancer metastasis, J.
Med. Chem. 54 (2011) 1896–1902.
[10] F. Pacchiano, M. Aggarwal, B.S. Avvaru, A.H. Robbins, A. Scozzafava,R.
McKenna, C.T. Supuran, Selective hydrophobic pocket binding observed within the
carbonic anhydrase II active site accommodate different 4-substituted-ureido-
benzenesulfonamides and correlate to inhibitor potency,Chem. Commun. Camb. U. K.)
46 (2010) 8371-8373.
[11] F. Pacchiano, F. Carta, P.C. McDonald, Y. Lou, D. Vullo, A. Scozzafava, S. Dedhar,
C.T. Supuran, Ureido-substituted benzenesulfonamides potently inhibit carbonic
anhydrase IX and show antimetastatic activity in a model of breast cancer metastasis, J.
Med. Chem. 54 (2011) 1896-1902.
[12] Y. Lou, P.C. McDonald, A. Oloumi, S.K. Chia, C. Ostlund, A. Ahmadi, A. Kyle,
U.A. Keller, S. Leung, D.G. Huntsman, B. Clarke, B.W. Sutherland, D. Waterhouse,
M.B. Bally, C.D. Roskelley, C.M. Overall, A. Minchinton, F. Pacchiano, F. Carta, A.
Scozzafava, N. Touisni, J.Y. Winum, C.T. Supuran, S. Dedhar, Targeting tumor hypoxia:
suppression of breast tumor growth and metastasis by novel carbonic anhydrase IX
inhibitors, Cancer Res. 71 (2011) 3364-3376.
22

[13] S.M. Monti, C.T. Supuran, G. De Simone, Anticancer carbonic anhydrase inhibitors:
a patent review (2008 e 2013), Expert Opin. Ther. Pat. 23 (2013) 737-749.
[14] P.C. McDonald, J.Y. Winum, C.T. Supuran, S. Dedhar, Recent developments in
targeting carbonic anhydrase IX for cancer therapeutics, Oncotarget 3 (2012) 84-97.
[15] C.T. Supuran, Carbonic anhydrases: from biomedical applications of the inhibitors
and activators to biotechnological use for CO2 capture, J. Enzyme Inhib. Med. Chem. 28
(2013) 229-230.
[16] W.M. Eldehna, M.F. Abo-Ashour, E. Berrino, D. Vullo, H.A. Ghabbour, S.T. Al-
Rashood, G.S. Hassan, H.M. Alkahtani, A.A. Almehizia, A. Alharbi, SLC-0111
enaminone analogues, 3/4-(3-aryl-3-oxopropenyl) aminobenzenesulfonamides, as novel
selective subnanomolar inhibitors of the tumor-associated carbonic anhydrase isoform
IX, Bioorg. Chem., 83 (2019) 549-558.
[17] W.M. Eldehna, M.F. Abo-Ashour, A. Nocentini, R.S. El-Haggar, S. Bua, A.
Bonardi, S.T. Al-Rashood, G.S. Hassan, P. Gratteri, H.A. Abdel-Aziz, Enhancement of
the tail hydrophobic interactions within the carbonic anhydrase IX active site via
structural extension: Design and synthesis of novel N-substituted isatins-SLC-0111
hybrids as carbonic anhydrase inhibitors and antitumor agents, Eur. J. Med. Chem., 162
(2019) 147-160.
[18] A. Angeli, D. Tanini, T.S. Peat, L. Di Cesare Mannelli, G. Bartolucci, A.
Capperucci, C. Ghelardini, C.T. Supuran, F. Carta, Discovery of new selenoureido
analogues of 4-(4-fluorophenylureido) benzenesulfonamide as carbonic anhydrase
inhibitors, ACS medicinal chemistry letters, 8 (2017) 963-968.
[19] N. Lolak, S. Akocak, S. Bua, M. Koca, C.T. Supuran, Design and synthesis of novel
1, 3-diaryltriazene-substituted sulfonamides as potent and selective carbonic anhydrase II
inhibitors, Bioorganic chemistry, 77 (2018) 542-547.
[20] S. Akocak, N. Lolak, S. Bua, I. Turel, C.T. Supuran, Synthesis and biological
evaluation of novel N, N′-diaryl cyanoguanidines acting as potent and selective carbonic
anhydrase II inhibitors, Bioorganic chemistry, 77 (2018) 245-251.
[21] F. Pacchiano, F. Carta, P.C. McDonald, Y. Lou, D. Vullo, A. Scozzafava, S. Dedhar,
C.T. Supuran, Ureido-substituted benzenesulfonamides potently inhibit carbonic
anhydrase IX and show antimetastatic activity in a model of breast cancer metastasis, J.
Med. Chem. 54 (2011) 1896–1902.
23

[22] R.G. Khalifah, The carbon dioxide hydration activity of carbonic anhydrase I.
Stopflow kinetic studies on the native human isoenzymes B and C, J. Biol. Chem. 246
(1971) 2561–2573.
[23] M. Bo tas, . Cetinkaya, M. Topal, İ. Gülçin, A. Menzek, E. Sahin, M. Tanc, C.T.
Supuran, Synthesis and carbonic anhydrase isoenzymes I, II, IX, and XII inhibitory
effects of dimethoxybromophenol derivatives incorporating cyclopropane moieties, J.
Med. Chem 58 (2014) 640-650.
[24] J. Ramprasad, N. Nayak, U. Dalimba, P. Yogeeswari, D. Sriram, S. Peethambar, R.
Achur, H.S. Kumar, Synthesis and biological evaluation of new imidazo [2, 1-b][1, 3, 4]
thiadiazole-benzimidazole derivatives, European journal of medicinal chemistry, 95
(2015) 49-63.
[25] A. Geronikaki, G. Theophilidis, Synthesis of 2-(aminoacetylamino) thiazole
derivatives and comparison of their local anaesthetic activity by the method of action
potential, European journal of medicinal chemistry, 27 (1992) 709-716.
[26] J. Banothu, K. Vaarla, R. Bavantula, P.A. Crooks, Sodium fluoride as an efficient
catalyst for the synthesis of 2, 4-disubstituted-1, 3-thiazoles and selenazoles at ambient
temperature, Chinese chemical letters, 25 (2014) 172-175.
[27] W.M. Eldehna, M. Fares, M. Ceruso, H.A. Ghabbour, S.M. Abou-Seri, H.A. Abdel-
Aziz, D.A. El Ella, C.T. Supuran, Amido/ureidosubstituted benzenesulfonamides-isatin
conjugates as low nanomolar/subnanomolar inhibitors of the tumor-associated carbonic
anhydrase isoform XII, Eur. J. Med. Chem. 110 (2016) 259-266.
[28] W.M. Eldehna, M.F. Abo-Ashour, A.Nocentini, P.Gratteri, H.I. Eissa, M. Fares,
O.E.Ismael, H.A. Ghabbour, M.M. Elaasser, H.A. Abdel-Aziz, C.T Supuran, Novel 4/3-
((4-oxo-5-(2-oxoindolin-3-ylidene) thiazolidin-2-ylidene) amino) benzenesulfonamides:
Synthesis, carbonic anhydrase inhibitory activity, anticancer activity and molecular
modelling studies, Eur. J. Med. Chem. 139 (2017) 250-262.
[29] W.M. Eldehna, G.H. Al-Ansary, S. Bua, A. Nocentini, P. Gratteri, A. Altoukhy, H.
Ghabbour, H.Y. Ahmed, C.T. Supuran, Novel indolin-2-one-based sulfonamides as
carbonic anhydrase inhibitors: synthesis, in vitro biological evaluation against carbonic
anhydrases isoforms I, II, IV and VII and molecular docking studies, Eur. J. Med. Chem.
127 (2017) 521-530.
24

[30] M.F. Abo-Ashour, W.M. Eldehna, A. Nocentini, H.S. Ibrahim, S. Bua, S.M. Abou-
Seri, C.T. Supuran, Novel hydrazido benzenesulfonamides-isatin conjugates: Synthesis,
carbonic anhydrase inhibitory activity and molecular modeling studies, Eur. J. Med.
Chem. 157 (2018) 28-36.
[31] M. Fares, R.A. Eladwy, A. Nocentini, S.R.A. El Hadi, H.A. Ghabbour, A. Abdel-
Megeed, W.M. Eldehna, H.A. Abdel-Aziz, C.T. Supuran, Synthesis of bulky-tailed
sulfonamides incorporating pyrido [2, 3-d][1, 2, 4] triazolo [4, 3-a] pyrimidin-1 (5H)-yl)
moieties and evaluation of their carbonic anhydrases I, II, IV and IX inhibitory effects,
Bioorg. Med. Chem. 25 (2017) 2210-2217.
[32] V. Alterio, M. Hilvo, A. Di Fiore, C.T. Supuran, P. Pan, S. Parkkila, A. Scaloni, J.
Pastorek, S. Pastorekova, C. Pedone, Crystal structure of the catalytic domain of the
tumor-associated human carbonic anhydrase IX, Proceedings of the National Academy of
Sciences, 106 (2009) 16233-16238.
[33] L. Schrödinger, Schrödinger, LLC; New York, NY: 2015, PyMOL Molecular
Graphics System, Version, 1 (2016).
[34] Schrödinger Release 2018-1: Maestro, Schrödinger, LLC, New York, NY, 2018.
[35] Schrödinger Release 2018-1: LigPrep, Schrödinger, LLC, New York, NY, 2018.
25

Highlights
- New series of thiazole and thiadiazole derivatives were synthesized as SLC-0111
congeners.
- Inhibitory activities of such analogues were evaluated towards hCA I, II, IX and XII
isoforms.
- Compounds 11c and 12b-d showed superior inhibitory activity against hCA IX than
SLC-0111.
- 11a, 12a, 12b and 12d were found to be selective towards hCAIX and XII over hCA II.
- A docking study was done for 12d within the hCA IX active site to justify its inhibitory
activity.
26

Graphical Abstract
Novel synthesized SLC-0111 thiazole and thiadiazole analogues:
Determination of their carbonic anhydrase inhibitory activity and
molecular modeling studies
Mahmoud F. Abo-Ashour, Wagdy M. Eldehna*, Alessio Nocentini, Hany S. Ibrahim,
Silvia Bua, Hatem A. Abdel-Aziz, Sahar M. Abou-Seri*, Claudiu T. Supuran*
A bioisosteric replacement approach was adopted to replace the 4-fluorophenyl tail of
SLC-0111 with thiazole and thiadiazole ones, which were thereafter extended by
lipophilic un/substituted phenyl moieties.
27