Synthesis, biological and molecular dynamics investigations with a series of triazolopyrimidine/triazole-based benzenesulfonamides as novel carbonic anhydrase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.111843

In the presented work, we report the design and synthesis of different new sets of triazolopyrimidine-based (9a-d) and triazole-based (11a-h, 13a-c, 15a,b, 17a,b and 21a-g) benzenesulfonamides. The newly synthesized sulfonamides were assessed for their inhibitory activities toward four human (h) metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) isoforms; hCA I, II, IX and XII. The four examined isoforms were inhibited by the prepared sulfonamides (9a-d, 11a-h, 13a-c, 15a,b, 17a,b and 21a-g) in variable degrees with K_Is ranges: 94.4–4953.5 nM for hCA I, 6.9–837.6 nM for hCA II, 3.3–85.0 nM for hCA XI, and 4.4–105.0 nM for hCA XII. In particular, sulfonamides 11e, 21a and 21e emerged as single-digit nanomolar hCA IX and hCA XII inhibitors. Interestingly, triazolopyrimidine-based sulfonamide 9d and triazole-based sulfonamide 21e were found to be the most selective hCA IX inhibitors over hCA I (SI = 100.85 and 210.58, respectively) and hCA II (SI = 18.54 and 38.36, respectively). Thereafter, sulfonamides 9d and 21e were docked into the active site of CAs II, IX and XII, then poses showing the best scoring values and favorable binding interactions were subjected to a MM-GBSA based refinement and, limited to CA IX and XII, to a cycle of 100 ns molecular dynamics.

Full text of DOI:10.1016/j.ejmech.2019.111843

Journal Pre-proof
Synthesis, biological and molecular dynamics investigations with a series of
triazolopyrimidine/triazole-based benzenesulfonamides as novel carbonic anhydrase
inhibitors
Mohamed A. Said, Wagdy M. Eldehna, Alessio Nocentini, Alessandro Bonardi, Samar
H. Fahim, Silvia Bua, Dalia H. Soliman, Hatem A. Abdel-Aziz, Paola Gratteri, Sahar
M. Abou-Seri, Claudiu T. Supuran
PII:
S0223-5234(19)30995-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.111843
EJMECH 111843
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 October 2019
Revised Date: 31 October 2019
Accepted Date: 31 October 2019
Please cite this article as: M.A. Said, W.M. Eldehna, A. Nocentini, A. Bonardi, S.H. Fahim, S.
Bua, D.H. Soliman, H.A. Abdel-Aziz, P. Gratteri, S.M. Abou-Seri, C.T. Supuran, Synthesis,
biological and molecular dynamics investigations with a series of triazolopyrimidine/triazole-based
benzenesulfonamides as novel carbonic anhydrase inhibitors, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.111843.
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2019 Published by Elsevier Masson SAS.

Graphical Abstract
Synthesis, biological and molecular dynamics investigations with a series of
triazolopyrimidine/triazole-based benzenesulfonamides as novel carbonic
anhydrase inhibitors
New sets of triazolopyrimidine-based (9a-d) and triazole-based (11a-h, 13a-c, 15a,b, 17a,b and
2
1a-g) benzenesulfonamides were synthesized and evaluated for their inhibitory activity toward a
panel of carbonic anhydrase isoforms hCA I, II, IX and XII. hCA IX and XII isoforms were
efficiently inhibited with K s range of 3.3-85 nM and 4.4-105 nM. Compounds 9d and 21e were
I
found to be the most selective hCA IX inhibitors over hCA I and hCA II.

Synthesis, biological and molecular dynamics investigations with a series of
triazolopyrimidine/triazole-based benzenesulfonamides as novel carbonic
anhydrase inhibitors
a
b,*
c,d
c,d
Mohamed A. Said , Wagdy M. Eldehna , Alessio Nocentini , Alessandro Bonardi , Samar H.
e
c
a,f
g
d
Fahim , Silvia Bua , Dalia H. Soliman , Hatem A. Abdel-Aziz , Paola Gratteri , Sahar M.
Abou-Seri , Claudiu T. Supuran
e
c,*
a
Department of Pharmaceutical Chemistry, College of Pharmacy, Egyptian Russian University, Badr
City, Cairo, P.O. Box 11829, Egypt
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh,
P.O. Box 33516, Egypt
c
Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of
Florence, Polo Scientifico, Via U. Schiff 6, 50019, Sesto Fiorentino, Firenze, Italy
d
Department of NEUROFARBA – Pharmaceutical and nutraceutical section; Laboratory of Molecular
Modeling Cheminformatics & QSAR, University of Firenze, Via U. Schiff 6, 50019 Sesto Fiorentino,
Firenze, Italy
e
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street,
Cairo, P.O. Box 11562, Egypt
f
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al Azhar University, Cairo, P.O. Box
1
1471, Egypt
g
Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622, Egypt
ABSTRACT
In the presented work, we report the design and synthesis of different new sets of
triazolopyrimidine-based (9a-d) and triazole-based (11a-h, 13a-c, 15a,b, 17a,b and 21a-g)
benzenesulfonamides. The newly synthesized sulfonamides were assessed for their inhibitory
activities toward four human (h) metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) isoforms;
hCA I, II, IX and XII. The four examined isoforms were inhibited by the prepared sulfonamides
I
(9a-d, 11a-h, 13a-c, 15a,b, 17a,b and 21a-g) in variable degrees with K s ranges: 94.4-4953.5
nM for hCA I, 6.9-837.6 nM for hCA II, 3.3-85.0 nM for hCA XI, and 4.4-105.0 nM for hCA
XII. In particular, sulfonamides 11e, 21a and 21e emerged as single-digit nanomolar hCA IX and
hCA XII inhibitors. Interestingly, triazolopyrimidine-based sulfonamide 9d and triazole-based
sulfonamide 21e were found to be the most selective hCA IX inhibitors over hCA I (SI = 100.85
and 210.58, respectively) and hCA II (SI = 18.54 and 38.36, respectively). Thereafter,
1

sulfonamides 9d and 21e were docked into the active site of CAs II, IX and XII, then poses
showing the best scoring values and favorable binding interactions were subjected to a MM-
GBSA based refinement and, limited to CA IX and XII, to a cycle of 100 ns molecular dynamics.
_
_________
Keywords: Carbonic anhydrase inhibitors; Molecular dynamics; Triazoles; Triazolopyrimidines;
Synthesis.
*
Corresponding authors. E-mail addresses: wagdy2000@gmail.com (W.M. Eldehna),
claudiu.supuran@unifi.it (C.T. Supuran).
2

1
. Introduction
-class carbonic anhydrases (CA) are a family of zinc-containing metallo-enzymes that
The
α
is composed of sixteen isoforms which differ in tissue expression patterns, function and kinetic
properties.[1, 2] The sluggishness and overexpression of various CA isoforms are responsible for
many diseases in human beings.[1] Involvement of CA I and CA II in many important normal
physiological processes such as regulation of the acid–base homeostasis made them act as
worthy drug targets in glaucoma, cerebral edema and epilepsy.[3] On the other hand, the
expression of CA IX and CA XII is limited in normal tissues, but their induction by the hypoxic
conditions in several cancer cell lines made them valuable targets as anticancer agents. CA IX
-
and CA XII have a role in the conversion of CO to HCO and a proton, then facilitating the
2
3
diffusion of that proton through the entire solid tumor leading to low extracellular pH that
expedite matrix breakdown, invasion, and multi-drug resistance.[1, 4] hCA IX isoform is highly
observed in many aggressive tumors including breast, ovarian, bladder, cervix, colorectal,
kidney, lung, pancreatic, head and neck, stomach and oral cavity cancers.[5] Whereas, hCA XII
isoform was robustly expressed in glioblastomas, lung carcinomas, breast carcinoma, urinary
bladder cell carcinomas and T-cell lymphomas.[5]
The design of compounds exhibiting both great affinity and high CA isoform-selectivity for
the specific disease treatment without causing side effects due to off-target CA modulation is a
challenging task because of the high structural homology among human CAs, especially within
their active sites.[6] The CA active site is located in cone-shaped cavity (approximately 15Å
deep). The catalytic zinc ion is situated near the bottom of the cavity and is coordinated to the
side chains of three conserved histidine residues and a water molecule/hydroxide ion. The cavity
extends to the center of the molecule with hydrophobic residues forming one side of the cavity
and hydrophilic residues for the other side. Although, the core of active site is highly conserved
among human CAs, residues of its periphery (10 Å –15 Å from the zinc) are variable in terms of
the polarity and hydrophobicity.[7] This part of the active site is defined as the selectivity pocket
and is used to design ligands targeting the specific CA isoform
.
The most successful strategies of modulating CA IX/XII catalytic activities to date include
the application of sulfonamide function as zinc anchoring group, however only few
3

sulfonamide derivatives have progressed to clinical trials due to lack of selectivity. Among the
different approaches used for development of isoform selective carbonic anhydrase inhibitors
(
CAIs) the tail approach is most widely employed. In the tail approach, the aromatic
sulfonamide ring, zinc anchoring group, is appended with tail moieties through flexible
functionalized linkers. The tails and linkers are selected to modulate interactions with amino acid
residues from the middle and edge of the enzyme active site seeking for isoform-selective
inhibition profiles and /or enhancing the physicochemical properties of the molecule. A flexible
hydrophobic tail group will generally adopt a conformation to interact with the hydrophobic half
of the CA active site; similarly, a flexible hydrophilic tail substituent will generally interact with
the hydrophilic half of the CA active site. A successful example for this approach is the novel
CA IX inhibitor SLC-0111 I (Fig. 1). This novel drug is now in Phase 1b/II clinical trials for the
treatment of advanced and metastatic solid tumors [8]
Literature survey regarding several human carbonic anhydrase inhibitors revealed that,
triazole and fused triazole-linked benzenesulfonamides (as compounds II-VI) have been emerged
as important classes of potent CAIs with much higher selectivity towards the tumor-associated
isoforms; CA IX and XII than on the ubiquitously expressed isoforms CA I and II (Fig. 1). [9-12]
Furthermore, the triazolo moiety enhanced hydro-solubility of the target molecules such as
compound IV (Fig. 1).[11] While fused triazoles revealed better hydrophilic/hydrophobic ratio so
as to improve solubility and CA receptor fitting in both hydrophilic and hydrophobic pockets as
seen in compounds V and VI .[10, 12].
In continuation to our previous work in the search for potent and selective hCA IX and hCA
XII inhibitors [13-16], we report herein the design and synthesis of new sets of
triazolopyrimidine- and triazole-based benzenesulfonamides (9a-d, 11a-h, 13a-c, 15a,b, 17a,b
and 21a-g), as potential CA IX and hCA XII inhibitors. The design of the new compounds relies
on applying the tail approach. The triazole/triazolopyrimidine benzenesulfonamide scaffold was
connected to an aryl tail through different functionalized linker as hydrazide-hydrazone (9a-d,
1
1a-h and 13a-c), semicarbazide/thiosemicarbazide (15a,b and 17a,b) or ureido (21a-g). Such
linkers are selected to donate different levels of flexibility to the aryl tail and impart the
possibility for the new compounds to adopt a diversity of orientations that may permit the
specific interactions between the tail and amino acid residues at the entrance of carbonic
4

anhydrase active site (selectivity pocket). Furthermore, the aryl was designed to ensure different
electronic, hydrophilic and lipophilic environments to explore a valuable SAR.
The newly synthesized sulfonamides were assessed for their inhibitory activities against four
CA isoforms (hCA I, hCA II, hCA IX and hCA XII). In addition, sulfonamides 9d and 21e were
docked into the active site of CAs II, IX and XII, then poses showing the best scoring values and
favorable binding interactions were subjected to a MM-GBSA based refinement and, limited to
CA IX and XII, to a cycle of 100 ns molecular dynamics.
Fig. 1. Structures of certain triazolopyrimidine/triazole-based CAIs I-VI, and structures of the
target sulfonamides 9a-d, 11a-h, 13a-c, 15a,b, 17a,b and 21a-g
5

2
. Results and Discussion
2
.1. Chemistry
The synthetic pathways employed to prepare the key intermediates and the new targeted
derivatives are depicted in Schemes 1-4. Synthesis of 6-methyl-2-thiouracil 2 was achieved by
refluxing ethyl acetoacetate 1 and thiourea in sodium ethoxide through hetero-cyclocondensation
reaction.[17] Subsequently, preparation of the key intermediate ethyl [1,2,4]triazolo[4,3-
a]pyrimidine-3-carboxylate 5 was carried out through refluxing 6-methyl-2-thiouracil 2 with the
hydrazonoyl chloride 3 in dioxane in the presence of a catalytic amount of triethylamine with
4
1% yield. The reaction mechanism proceeds through S-alkylation reaction to give intermediate
1
4
followed by the Smiles rearrangement (Scheme 1).[18] H NMR spectrum of 5 showed a D
2
O-
exchangeable singlet signal attributable to the (NH ) protons of sulfonamido group at δ 7.47 ppm,
2
whereas the unique H-6 triazolopyrimidine proton appeared as a singlet signal at δ 6.02 ppm, in
addition to the singlet corresponding to the 7-methyl triazolopyrimidine protons which resonated
at δ 2.34 ppm. Finally, the terminal ethyl group of the ester moiety appeared as a triplet signal for
2
methyl protons at δ 1.32 ppm and a quartet signal for CH protons at δ 4.45 ppm.
Scheme 1. Reagents and conditions: i. Thiourea, NaOEt, reflux, 7h; ii. Dioxan, TEA, reflux 12h.
Interestingly, hydrazinolysis of the ester derivative 5 yielded two different hydrazide
products (6 and 7) depending on the molar ratio of hydrazine hydrate and the reaction time.
6

Applying two equivalents of hydrazine hydrate in refluxing ethanol for two hours afforded the
expected fused triazolo[4,3-a]pyrimidine hydrazide 6, whereas, refluxing ester 5 with ten
equivalents of hydrazine hydrate for eight hours resulted in hydrazinolysis of not only the ester
group, but also the triazolopyrimidine ring affording the monocyclic 4-(5-amino-3-
(hydrazinecarbonyl)-1H-1,2,4-triazol-1-yl)benzenesulfonamide 7. The former bicyclic hydrazide
6
was heated with different aldehydes in ethanol in the presence of catalytic amount of glacial
1
acetic acid to afford target compounds 9a-d (Scheme 2).[19] H NMR spectrum of 6 revealed
the presence of three D O exchangeable singlet signals assigned to the NH and NH groups of
2
2
2
the hydrazide function at δ 4.87 and 10.52 ppm as well as the sulfonamide NH at 7.52 ppm.
Also, the spectrum showed two singlets at δ 2.39 and 6.09 ppm corresponding to the methyl
1
protons and the H-6 triazolopyrimidine proton, respectively. On the other hand, H NMR
spectrum of hydrazide 7 revealed the absence of C-7 methyl protons and C-6 proton of the
triazolopyrimidine nucleus, concurrently with the appearance of new D O exchangeable singlet
2
2
signal at δ 6.82 ppm attributable for NH group at position 5 of the triazole ring, in addition to the
presence of the D O exchangeable protons of the hydrazide function (CONHNH ) and
2
2
sulfonamido (SO
2
NH
2
) group at δ 4.51, 9.44 and 7.51 ppm, respectively. Moreover, the HRMS
data supported the structure of hydrazide 7 by a molecular ion peak at m/z 298.07171.
Scheme 2. Reagents and conditions: i. NH NH .H O (2 eq.), EtOH, reflux 2h; ii. Xss
2
2
2
NH NH .H O (10 eq.), EtOH, reflux 8h; iii. EtOH, AcOH, reflux 2h.
2
2
2
7

In Scheme 3, hydrazide 7 was reacted with different aromatic aldehydes 10a-h and
acetophenones 12a-c in glacial acetic acid to furnish the target sulfonamides 11a-h or 13a-c,
respectively. Alternatively, refluxing the key intermediate 7 with aryl isocyanates 14a,b or aryl
isothiocynates 16a,b in dry toluene afforded the target sulfonamides 15a,b and 17a,b,
respectively (Scheme 3).
Scheme 3. Reagents and conditions: i. AcOH, reflux 2h; ii. Toluene, reflux 3h.
Finally, the target ureides 21a-g were synthesized through adopting the chemical pathway
outlined in Scheme 4. Hydrazide 7 was stirred with sodium nitrite in cold glacial acetic acid to
8

furnish the azide intermediate 18, which subsequently subjected to Curtius rearrangement
through heating in dry xylene to produce the corresponding isocyanate derivative 19. The target
ureido triazole-based benzenesulfonamides 21a-g were obtained by reaction of isocyanate 19
with different substituted anilines 20a-g in xylene with 44–60% yield (Scheme 4).
Scheme 4. Reagents and conditions: i. NaNO , AcOH, stirring 2 h 0 °C; ii. Xylene, reflux 1 h;
2
iii. Xylene, reflux 3 h.
The structures of the newly synthesized sets of triazolopyrimidine-based (9a-d) and triazole-
based benzenesulfonamides (11a-h, 13a-c, 15a,b, 17a,b and 21a-g) were confirmed under the
basis of spectral and elemental analyses which were in full agreement with the postulated
structures.
1
It is noteworthy that, the H NMR spectra of target ureides 21a-g indicated their presence in
two geometrical conformations (Z,Z, 21) and (E,Z, 21’), where the first geometrical isomer is the
well-known stable form of the ureide derivatives and the other isomer is assumed to be stabilized
9

by intramolecular hydrogen bonding between one of the ureide hydrogens and N-2 atom of the
triazole ring (Scheme 4).[20-22] The spectra revealed the presence of two exchangeable singlets
corresponding to the NH protons of triazole moiety at δ 6.86- 6.88 ppm and 6.99-7.01 ppm,
2
2
beside two other exchangeable singlets corresponding to the NH protons of the sulfonamide
group at δ 7.34-7.47 and 7.44-7.53 ppm. Compounds 21d and 21e spectra showed the presence
of two singlets for the methyl protons at δ 2.25 and 2.27 ppm, as well as two singlet signals for
the methoxy protons at δ 3.73 and 3.75 ppm, respectively.
2
2
.2. Biological Evaluation
.2.1. Carbonic anhydrase inhibition
Six newly prepared triazolopyrimidine-based benzenesulfonamides (5, 6 and 9a-d) as well as
twenty three triazole-based benzenesulfonamides (7, 11a-h, 13a-c, 15a,b, 17a,b and 21a-g) were
examined for their ability to inhibit the physiologically relevant hCA isoforms, hCA I and II
(cytosolic) as well as the tumor associated hCA isoforms hCA IX and XII (trans membrane)
through a stopped flow CO hydrase assay [23] using acetazolamide (AAZ) as a standard
2
inhibitor. The following structure–activity relationship (SAR) can be concluded from the
inhibition data presented in Table 1.
(i) All the tested sulfonamides showed weak inhibitory effect on the slow cytosolic
isoform, hCA I, where the binding affinity constant (K ) values of twenty-eight compounds out
I
of the twenty-nine tested compounds are fluctuating in the hundreds and thousands nM range
(K
I
295.2-4953.5 nM). Only one compound 21b showed moderate inhibition with K
I
value of
9
4.4 nM.
(
ii) The physiologically relevant isoform, hCA II, was much more inhibited by most of the
tested compounds (K s: 6.9-837.6 nM). It is clearly observed that, the triazole-based
compounds (7, 11a-h, 13a-c, 15a,b, 17a,b and 21a-g) were more potent hCA II inhibitors (K s:
I
I
6
I
.9-126.6 nM) than the triazolopyrimidine-based drivatives (5, 6 and 9a-d) (K s: 74.4-837.6
nM). This difference in the activities of the bicyclic and monocyclic structures is markedly
detected by comparing the activity of the triazolopyrimidine hydrazide 6 (K = 74.7 nM) with
I
that of its triazole analogue 7 (K
I
= 19.9 nM), and by comparing the binding affinities (K
I
s:
3
24.6-837.6) of triazolopyrimidines 9a-d with the binding affinities (K s: 6.6-95.4 nM) of
I
triazoles 11a-h.
1
0

Table 1: Inhibition data of CA isoforms I, II, IX and XII with sulfonamides (5, 6, 7, 9a-d, 11a-h, 13a-c,
5a,b, 17a,b and 21a-g), using AAZ as a standard inhibitor.
1
K (nM)
I
Comp.
R
hCA I
697.7
hCA II
85.6
hCA IX
hCA XII
103.9
64.3
-
-
-
5
6
7
35.9
45.4
22.8
63.2
1676.8
522.7
74.7
19.9
29.4
9
9
a
4- OCH
3
-C
6
H
4
2381.9
324.6
38.3
4-(4-methylpiperazin-1-yl)-
b
4953.5
837.6
85.0
26.2
6 4
C H
9
9
1
1
1
1
1
1
1
1
1
1
1
1
1
c
2-pyridyl
3721.8
2481.0
771.4
494.9
702.7
1536.5
334.6
824.6
987.7
476.5
633.1
295.2
793.4
306.9
537.1
536.9
456.2
86.4
65.2
6.9
47.9
24.6
41.1
31.5
73.2
34.4
8.3
59.3
45.3
105.0
29.5
8.2
d
4-pyridyl
1a
1b
1c
1d
1e
1f
C H
6 5
4-F-C
4-Cl-C
4-NO
4-OH-C
4-piperidinyl-C
6
H
4
H
6
4
2
-C
6
H
4
24.7
8.8
10.2
7.8
H
4
6
H
95.4
64.2
74.2
8.9
33.0
19.7
6.6
17.6
49.7
34.4
23.8
26.0
9.9
6
4
1g
1h
3a
3b
3c
5a
5b
4-morpholinyl-C
3-pyridyl
6
H
4
C
6
H
5
41.3
5.8
4-pyridyl
8.5
4-morpholinyl-C
H
53.9
70.5
10.9
31.7
4.9
6
4
C
6
H
5
16.2
48.2
4-Cl-C
H
16.4
6
4
1
1

1
1
2
2
2
2
2
2
2
7a
7b
1a
1b
1c
1d
1e
1f
C
6
H
5
368.2
597.5
442.3
94.4
9.7
11.1
9.8
40.2
16.7
8.2
31.0
21.9
8.1
4-Cl-C H
6
4
C H
6 5
4-F-C
4-Cl-C
4-CH
4-OCH
4-SO NH
4-COOH-C
6
H
4
65.5
9.5
7.8
17.6
73.6
23.8
9.8
H
4
403.3
312.6
694.9
676.6
507.4
250.0
43.3
3.9
6
3
-C
6
H
4
42.8
126.6
78.7
9.6
-C
H
3.3
3
6
4
-C
H
28.6
15.3
25.0
4.4
2
2
6
4
1g
6
H
4
19.9
5.7
AAZ
12.5
*
%
Mean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5-10
of the reported values).
(iii) The tumor associated isoform hCA IX was effectively inhibited by all the tested
triazolopyrimidine/triazole-based sulfonamides with K s ranging from 3.3 nM to 85.0 nM.
I
Superiorly, eight triazole-based tested sulfonamides emerged as the most potent counterparts
against hCA IX isoform in this study with single-digit nanomolar inhibitory activities; 11e (8.3
nM), 11h (6.6 nM), 13b (5.8 nM), 15a (4.9 nM), 21a (8.2 nM), 21b (7.8 nM), 21d (3.9 nM)
and 21e (3.3 nM). Moreover, sulfonamides 11g, 15b, 17b and 21g showed better binding
I I
affinities (K s: 15.3-19.7 nM) than the standard inhibitor AAZ (K = 25.0 nM). In addition,
sulfonamides 7, 9d and 21f showed comparable inhibitory activity (K = 22.8, 24.6 and 28.6
I
nM, respectively) to AAZ toward hCA IX. Notably, among the tested triazole-based
sulfonamides, the ureides series 21 stood out as the most promising one, with binding affinities
better than (K s: 3.3-15.3 nM) or comparable to (K = 28.6 nM) the standard inhibitor AAZ (K
I
I
I
I
=
=
25.0 nM), except sulfonamide 21c whose efficacy raised at slightly higher concentration (K
43.3 nM).
It is noteworthy, that bioisosteric replacement of the terminal un/substituted phenyl ring in
triazolopyrimidine series 9 (K s = 63.2 and 85.0 nM) and triazole series 11 (K s: 8.3-73.2 nM)
I
I
and 13 (K s = 41.3 and 31.7 nM) with the hetero pyridyl moiety enhanced the inhibitory
I
activity toward hCA IX isoform; (K s = 47.9 and 24.6 nM, for series 9), (K = 6.6 nM, for
I
I
series 11) and (K = 5.8 nM, for series 13). In addition, substitution of the terminal phenyl ring
I
in triazole series 11 with electron-withdrawing groups (11b and 11d; K s = 31.5 and 34.4 nM,
I
1
2

I
respectively), or electron-donating groups (11e-11g; K s = 8.3, 33.0 and 19.7 nM, respectively)
improved hCA IX inhibitory activity in comparison to unsubstituted analogue 11a (K = 41.1
I
nM), except 4-Cl derivative 11c which displayed higher K value (73.2 nM). Furthermore,
I
3 3 I
grafting an electron-donating group (4-CH 21d and 4-OCH 21e; K s = 3.9 and 3.3 nM,
respectively) within the phenyl group of the ureido triazole-based sulfonamides 21 resulted in
an enhancement of activity against hCA IX isoform compared to unsubstituted congener 21a
(
K = 8.2 nM).
I
iv) The other tumor associated isoform hCA XII is also effectively inhibited by all the
I
tested triazolopyrimidine-based sulfonamides (K s range: 26.2-103.9 nM) and triazole-based
sulfonamides (K s range: 4.4-105 nM). In particular, six triazole-based tested sulfonamides
I
showed potent binding affinities with single-digit nanomolar K s values, 11c (8.2 nM), 11e
I
(
7.8 nM), 13c (9.9 nM), 21a (8.1 nM), 21e (9.8 nM) and 21f (4.4 nM). Furthermore,
sulfonamides 7, 9b, 11b, 11f, 13a, 13b, 15a, 17b, 21b, 21d and 21g displayed potent hCA XII
inhibitory activity with K values ranging from 16.2 to 29.5 nM.
I
Concerning hCA XII inhibitory activity of the triazole schiff’s bases series 11, we can
conclude that grafting a para-substituent within the benzylidene moiety, as in compounds 11b-
g, is more beneficial for the activity (K
I
s: 7.8-49.7 nM) than unsubstituted congener 11a (K
05.0 nM). In addition, bioisosteric replacement of the terminal phenyl ring in sulfonamide
1a (K = 105.0 nM) with the hetero 3-pyridyl moiety, sulfonamide 11h, led to a 3-fold
I
=
1
1
I
efficacy enhancement (K
through the heterocyclic nitrogen atom within the active site. Whereas, similar bioisosteric
replacement of the phenyl ring in compound 13a (K = 23.8 nM) with the 4-pyridyl ring led to
I
= 34.4 nM) that may be attributed to an extra hydrogen bonding
I
compound 13b whose efficacy rose to a slightly higher concentration (K = 26 nM), unlike
I
what arose for hCA IX.
It is worth highlighting that substitution of the terminal phenyl ring in ureides series 21
elicits a 1.2-fold to 9.1-fold efficacy worsening against hCA XII, except for compound 21f
with incorporated p-sulfamoyl group that displays about 2-fold enhanced activity (K = 4.4
I
I
nM) than unsubstituted analogue 21a (K = 8.1 nM).
1
3

Table 2. Selectivity index (SI) calculated for hCA IX and XII over off-targets isoforms (hCA I and II) for
target sulfonamides (9a-d, 11a-h, 13a-c, 15a,b, 17a,b and 21a-g) reported in the paper, and AAZ.
Cmpd
I/IX
I/XII
62.19
189.06
62.76
54.77
7.35
II/IX
5.14
9.85
11.21
18.54
2.10
2.07
0.09
0.72
1.06
2.89
3.26
11.24
0.22
1.47
1.70
14.39
0.66
0.24
0.66
1.20
8.40
0.22
10.97
38.36
2.75
0.63
0.50
II/XII
8.48
31.97
9.05
10.07
0.82
2.21
0.84
2.42
1.13
5.42
1.29
2.16
0.37
0.33
5.44
4.35
0.23
0.31
0.51
1.21
3.72
0.13
1.80
12.92
17.89
0.48
2.19
9
9
9
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
a
37.69
58.28
77.70
100.85
18.77
15.71
9.60
b
c
d
1a
1b
1c
1d
1e
1f
16.78
85.70
150.64
42.90
46.85
19.87
13.85
26.60
11.35
80.14
18.94
11.14
11.88
27.28
54.60
5.36
44.67
40.31
24.99
50.14
72.20
15.33
50.90
25.03
62.63
32.75
9.16
1g
1h
3a
3b
3c
5a
5b
7a
7b
1a
1b
1c
1d
1e
1f
35.78
53.94
12.10
9.31
5.48
80.15
210.58
23.66
33.16
10.00
13.13
70.91
153.77
25.50
43.86
1g
AAZ
(v) Concerning the selectivity of action of the newly herein reported sulfonamides, the
selectivity index (SI) for hCA IX and XII over the off-targets isoforms hCA I and II are
calculated and listed in Table 2. Regarding selectivity towards hCA IX and XII over hCA I, all
the target sulfonamides (9a-d, 11a-h, 13a-c, 15a,b, 17a,b and 21a-g) exhibited moderate to
excellent SIs spanning in the range 9.16 – 210.58 and 5.36 – 189.06, respectively.
1
4

On the other hand, the triazolopyrimidine-based benzenesulfonamide series 9a-d displayed
good selectivity towards hCA IX and XII over hCA II with SIs spanning in the range 5.14 –
1
8.54 and 8.48 – 31.97, respectively. Whereas, only triazole-based benzenesulfonamide
derivatives 11h, 15a, 21b, 21d and 21e were found to be selective toward hCA IX over hCA II
with SIs equal 11.24, 14.39, 8.40, 10.97 and 38.36, respectively. In addition, triazole-based
benzenesulfonamide derivatives 13c, 15a, 21e and 21f exhibited good selectivity towards hCA
XII over the off-target isoform hCA II with SIs equal 5.44, 4.35, 12.92 and 17.89, respectively.
2
.2.2. Computational studies
Triazolopyrimidine-based benzenesulfonamide 9d and triazole-based benzenesulfonamide
1e were selected as the most selective for the cancer-associated CAs over the ubiquitous
2
isozymes and were docked into the active site of CAs II, IX and XII. Poses showing the best
scoring values and favorable binding interactions were subjected to a MM-GBSA based
refinement (Fig. 2) and, limited to CA IX and XII, to a cycle of 100 ns molecular dynamics
(MD, Fig. 3-5).
Fig. 2. Docked poses of 9d (green) and 21e (magenta) in the active site of CA II. H-Bonds and π-π
interactions are depicted as black and red dashed lines, respectively.
In accordance with the X-ray crystallographic reports [24], the benzenesulfonamide fragment
of 9d and 21e adopted superimposable binding orientations within the three active sites (Fig. 2, 4
1
5

and 5). As a result, the observed specificity of action of the selected compounds needed to be
sought in the interactions established by the tails of 9d and 21e with the enzymatic counterparts.
While the triazolotriazolopyrimidine of 9d occupied a central position within the CA II
binding pocket, its arylidenehydrazide tail accommodated into the cleft formed by Trp5, His64
and His4, establishing a π-π contact with the latter (Fig. 2). Also the 5-amino-1,2,4-triazole
portion of 21e allocated in a middle position of the CA II active site, whereas the 4-
methoxyphenylureido tail accommodated in the pocket lined by Leu204, Val135, Phe131
receiving a H-bond by the Gln136 side chain.
The MD simulation undertaken for the two compounds bound to CA IX and XII showed the
overall stability of the ligand/target adducts over the tested 100 ns, with the interactions
involving the benzenesulfonamide core being steady during the computations (Fig. 4 and 5). In
contrast, movements of the tails of 9d and 21e in relation to the active site residues produced
root-mean-square deviation (RMSD), plotted for the ligands heavy atoms as a function of time,
which settled in the 2.5-3
Å range for CA IX, and reached 4-6 Å in the case of CA XII (Fig. 3).
Figure 4 depicts the main binding orientations reported by 9d (panel A) and 21e (panel B)
within the binding site of CA IX. The arylidenehydrazide tail of 9d was found to stably occupy
the pocket formed by Val20, Trp5, Pro201 and Pro202 where intense hydrophobic contacts
persisted over the MD course (Fig. 4A). Additionally, interesting water bridges connecting the
pyridine N atom and Val20 carbonyl group and the inner hydrazide N atom and Pro201 C=O,
were found for approximately the 20% of the simulation time.
The 4-methoxyphenylureido pendant of 21e oriented towards a small cleft lined by Leu91,
Gln92 and Thr73 and was stabilized by H-bonds persisting for over the 90% of time between
Gln92 side chain and the N2 atom of the triazole and the ureido C=O (Fig. 4B). Further, two
water molecules bridged the oxygen atom of the methoxy group and the Ala90 and Leu74
residues. This network of interaction is present for the 30% of the MD.
1
6

Fig. 3. RMSD analysis of the ligands heavy atoms over the 100 ns MD in CA IX and XII.
Figure 4. Principal predicted orientation produced over a cycle of 100 ns MD for A) 9d and B) 21e bound
in the CA IX active site. H-bonds are depicted as black dashed lines. The bond occupancies over 100 ns
are reported as percentage.
The binding orientations of the molecular tails of 9d and 21e within the CA XII active site
were predicted to be less stable in comparison to the CA IX / ligand interactions, as shown in
Figure 4. The arylidenehydrazide tail of 9d established VdW contacts with Tyr20, Pro201 and
Pro202, but it did not firmly position into the pocket because of the Val20 (CA IX) / Tyr20 (CA
XII) mutation which increases the steric hindrance on the enzymatic counterpart (Fig. 5A). As a
result, during the MD simulation the arylidenehydrazide tail was found to move within the area
comprised from Gln136 to Trp5. The position of the triazolopyrimidine ring was stabilized by π-
cation interactions with the ammonium group of Lys67 (30% of the simulation time) whereas a
water-mediated interaction between the hydrazido moiety and Gln136 side chain persisted for the
2
0% of the MD time (Fig. 5A).
1
7

As far as the adduct of 21e with CA XII is concerned, the RMSD value plotted as a function
of time (Fig. 3) indicated that important changes in the ligand binding orientation occurred at
approximately 35 and 85 ns of MD (Fig. 5B). Indeed, while initially the phenylureido pendant
showed to form a H-bond through its ureido C=O with Gln92 (32% of MD time) and a water
bridge with Thr91 (with an occasional π-cation interaction with Lys67), after 35 ns the molecular
tail shifted toward the α-helix 131-135 only maintaining the water-mediated interaction with
Thr91. Approaching to the end of the MD, the phenylureido tail significantly moved away from
the original position likely because of H-bonds established by the urea with Ser135 side chain
and by the methoxy group with Tyr20 (Fig. 5B).
The different binding orientations predicted for both ligands within CA II, IX and XII, allow
their selectivity values reported in Table 2 to be argued. The greater efficacy of 9d against CA
IX than the other isozymes is likely related to the accommodation and strong interaction of the
arylidenehydrazide moiety with the aforesaid hydrophobic pocket (Val20, Trp5, Pro201 and
Pro202). In fact, the Val20/Phe20 (CA IX/CA II) and the Val20/Tyr20 (CA IX/CA XII)
mutations reduced such an intense binding. Nonetheless, in CA XII π-cation interactions of
Lys67 with both the benzene and pyridinone rings, not present in CA II, likely led to the increase
of K values up to a low nanomolar range (Table 1).
I
Fig. 5. A) Principal predicted orientation produced over a cycle of 100 ns MD for 9d bound in the CA XII
active site. B) Principal predicted orientations extrapolated from the 100 ns MD of 21e bound in the CA
XII active site. H-Bonds and π-cation interactions are depicted as black and red dashed lines, respectively.
The bond occupancies over 100 ns are reported as percentage. The latter are below 10% when not
indicated.
1
8

In the case of 21e, the phenylureido tail occupied a different binding pocket in the CA II
compared to CA IX and XII (Fig. 2, 4, and 5), presumably owing to the presence of a Phe
residue in position 131 which sterically prevent the binding nearby the region where the ligand,
in the tumor-associated CAs, bound to Gln92 and Leu91 (CA IX) and Thr91 and Lys67 (CA
XII).
3
. Conclusion
In summary, we described here the design and synthesis of different new sets of
triazolopyrimidine-based (9a-d) and triazole-based (11a-h, 13a-c, 15a,b, 17a,b and 21a-g)
benzenesulfonamides as potential CA inhibitors. All the newly synthesized sulfonamides were
examined for their inhibitory activities toward hCA I, II, IX and XII. The four examined
isoforms were inhibited by the prepared sulfonamides (9a-d, 11a-h, 13a-c, 15a,b, 17a,b and
2
1a-g) in variable degrees with K
I
s ranges: 94.4-4953.5 nM for hCA I, 6.9-837.6 nM for hCA II,
3
.3-85.0 nM for hCA XI, and 4.4-105.0 nM for hCA XII. In particular, sulfonamides 11e, 21a
and 21e emerged as single-digit nanomolar hCA IX and hCA XII inhibitors. Interestingly,
triazolopyrimidine-based sulfonamide 9d and triazole-based sulfonamide 21e were found to be
the most selective hCA IX inhibitors over hCA I (SI = 100.85 and 210.58, respectively) and hCA
II (SI = 18.54 and 38.36, respectively). Docking and molecular dynamics simulations shed light
on the ligands selectivity for the cancer-associated CAs over CA II. The great inhibition activity
of 9d against CA IX was attributed to strong interactions the arylidenehydrazide moiety forms
with the hydrophobic pocket lined by Val20, Trp5, Pro201 and Pro202, which are absent in CAs
II and XII. Nonetheless, π-cation interactions of Lys67 with both the benzene and pyridinone
I
rings of the ligand occur in CA XII (and not in CA II) which increase the K values up to a low
nanomolar range against the second cancer-related CA.
4
4
4
. Experimental
.1. Chemistry
.1.1. General
1
9

Melting points were measured with a Stuart melting point apparatus and were uncorrected.
Infrared spectra were recorded as KBr disks on Schimadzu FT-IR 8400S spectrophotometer and
-1
1
expressed in wave number (cm ). The NMR spectra were recorded by Bruker spectrometer. H
1
3
NMR spectra were run at 400 MHz, while C NMR spectra were run at 100 MHz in deuterated
dimethylsulfoxide (DMSO-d6). All coupling constant (J) values are given in hertz. Chemical
H C
shifts (δ ) and (δ ) are reported relative to DMSO-d6 as internal standards High-resolution mass
spectra were recorded using a Bruker MicroTOF spectrometer. Elemental analyses were carried
out at the Regional Center for Microbiology and Biotechnology, Al-Azhar University, Cairo,
Egypt.
4
.1.1.1. Synthesis of 6-methyl-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (
Compound (2) was synthesized as reported previously [17]. Yield (88%); white powder; m.p.
87–290 °C as reported [17, 25]
2).
2
4
.1.1.2.
Synthesis
of
ethyl
7-methyl-5-oxo-1-(4-sulfamoylphenyl)-1,5-dihydro-
).
[
1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate (
5
The mixture of 6-methyl-2-thiouracil 2 (1.42 g, 0.01 mol), hydrazonoyl chloride 3 (3 g, 0.01
mol) and triethylamine (1.4 mL, 0.01 mol) in dioxane (30 mL) was heated under reflux for 12h.
After cooling, the precipitated solid was collected by filtration, dried and recrystallized from
acetonitrile to afford ester 5. Yield (41%); yellow powder; m.p. 216-218 °C; IR: 3410, 3348
1
(
NH ), 1735, 1705 (2C=O), 1342, 1165 (SO ); H NMR
δ
ppm: 1.32 (t, 3H, CH , J = 7.2 Hz),
2
2
3
2
2
.34 (s, 3H, CH ), 4.45 (q, 2H, CH , J = 7.2 Hz), 6.02 (s, 1H, H-6 of triazolopyrimidine), 7.47 (s,
3 2
2
H, NH ), 7.99 (d, 2H, Ar-H, J = 8.8 Hz), 8.28 (d, 2H, Ar-H, J = 8.8 Hz); Anal. Calcd. for
C H N O S (377.38): C, 47.74; H, 4.01; N, 18.56; found C, 47.34; H, 3.94; N, 18.66.
15
15
5
5
4
.1.1.3.
a]pyrimidin-1(5H)-yl)benzenesulfonamide (
To a suspension of ester 5 (3.77 g, 0.01 mol) in ethanol (20 mL), 99% hydrazine hydrate (1 mL,
.02 mol) was added. The reaction mixture was heated under reflux for 2h, then left to cool. The
Synthesis
of
4-(3-(hydrazinecarbonyl)-7-methyl-5-oxo-[1,2,4]triazolo[4,3-
6
)
0
precipitated product was filtered, washed with water, dried and recrystallized from ethanol to
furnish hydrazide 6. Yield (30%); white powder; m.p. 260-262 °C; IR: 3363, 3309, 3213 (NH,
2
0

1
2
NH
2 2 3
), 1716, 1689 (2C=O), 1342, 1157 (SO ); H NMR δ ppm: 2.39 (s, 3H, CH ), 4.87 (s, 2H, -
NH ), 6.09 (s, 1H, H-6 of triazolopyrimidine), 7.52 (s, 2H, -SO NH ), 8.05 (d, 2H, Ar-H, J = 8.8
2
2
2
Hz), 8.35 (d, 2H, Ar-H, J = 8.8 Hz), 10.52 (s, 1H, -NH); Anal. Calcd. for C H N O S (363.35):
1
3
13
7
4
C, 42.97; H, 3.61; N, 26.98; found C, 43.09; H, 3.54; N, 27.08.
4
.1.1.4.
yl)benzenesulfonamide (
Excess 99% hydrazine hydrate (5 mL, 0.10 mol) was added to a suspension of ester 5 (3.77 g,
.01mol) in ethanol (10 mL). The reaction mixture was heated under reflux for 8h. The solid
Synthesis
of
4-(5-amino-3-(hydrazinecarbonyl)-1H-[1,2,4]triazol-1-
7
)
0
product obtained upon cooling was filtered off, washed several times with water, dried and
recrystallized from dioxane to give hydrazide 7. Yield (33%); white powder; m.p. 254-256 °C;
1
IR: 3464, 3363, 3298, 3251, 3174 (NH, 3NH ), 1681 (C=O), 1338, 1161 (SO ); H NMR
δ
ppm:
2
2
4
2
.51 (s, 2H, -NH
2
of hydrazide), 6.82 (s, 2H, -NH
2
of triazole), 7.51 (s, 2H, -SO
2
NH
2
), 7.79 (d,
H, Ar-H, J = 8.8 Hz), 7.96 (d, 2H, Ar-H, J = 8.8 Hz), 9.44 (s, 1H, -NH of hydrazide); HRMS
+
(ESI) for C
9
H₁
2
O
3
N
7
³²S, calcd 298.07168, found 298.07171 [M+H] .
4
.1.1.5. Synthesis of 4-(3-(2-(aryl)hydrazine-1-carbonyl)-7-methyl-5-oxo-[1,2,4]triazolo[4,3-
a]pyrimidin-1(5H)-yl)benzenesulfonamide (9a-d
)
A mixture of equimolar quantities of the previously prepared hydrazide 6 (0.726 g, 0.002 mol)
and the appropriate aldehyde 8a-d (0.002 mol) in absolute ethyl alcohol (10 mL) containing
catalytic amount of glacial acetic acid was heated under reflux for 2h, and then cooled to room
temperature. The precipitates formed were collected by filtration, dried and crystallized from
EtOH/DMF to afford compounds 9a-d.
4
.1.1.5.1.
4-(3-(2-(4-Methoxybenzylidene)hydrazine-1-carbonyl)-7-methyl-5-oxo-
[
1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl)benzenesulfonamide (9a
)
2
Yield (61%); white powder; m.p. 284–286 °C; IR: 3410, 3332, 3232 (NH, NH ), 1685, 1604
1
(
2C=O), 1340, 1165 (SO ); H NMR
δ
ppm: 2.42 (s, 3H, CH of triazolopyrimidine), 3.73, 3.83
2
3
(
2s, 3H, OCH ), 6.03, 6.13 (2s, 1H, H-6 of triazolopyrimidine), 6.86, 7.05 (2d, 2H, Ar-H, J = 8.4
3
Hz), 7.32, 7.76 (2d, 2H, Ar-H, J = 8.4 Hz), 7.53, 7.55 (2s, 2H, NH
, 8.06 (t, 2H, Ar-H, J = 8.4 Hz), 8.40 (t, 2H, Ar-H, J = 8.8 Hz), 12.61, 12.68 (2s, 1H, NH); C
NMR ppm: 24.67, 24.82 (CH ), 55.72, 55.83 (OCH ), 101.23, 101.59, 114.78, 114.89, 120.76,
2
), 8.04, 8.28 (2s, 1H, CH=N-
1
3
)
δ
3
3
2
1

1
1
1
3
21.25, 126.31, 126.49, 127.25, 127.64, 129.02, 129.76, 138.55, 138.99, 139.22, 142.77, 143.10,
43.43, 146.46, 147.48, 147.94, 150.43, 151.79, 155.15, 155.93, 158.45, 161.44, 161.88, 167.56,
67.58; Anal. Calcd. for C H N O S (481.49): C, 52.39; H, 3.98; N, 20.36; found C, 52.49; H,
21
19
7
5
.93; N, 20.27.
4
.1.1.5.2. 4-(7-Methyl-3-(2-(4-(4-methylpiperazin-1-yl)benzylidene)hydrazine-1-carbonyl)-5-
oxo-[1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl)benzenesulfonamide (9b
)
2
Yield (64%); yellow powder; m.p. 288–290 °C; IR: 3367, 3298, 3194 (NH, NH ), 1695, 1685
1
(
2C=O), 1346, 1165 (SO ); H NMR
δ
ppm: 1.92 (s, 3H, CH of methyl piprazine), 2.18, 2.23 (2
2
3
br.s, 4H, H-3, H-5 of methyl piprazine), 2.38, 2.45 (2s, 3H, CH of triazolopyrimidine), 3.16,
3
3
6
.27 (2 br.s, 4H, H-2, H-6 of methyl piprazine), 6.02, 6.12 (2s, 1H, H-6 of triazolopyrimidine),
.81, 7.00 (2d, 2H, Ar-H, J = 8.4 Hz), 7.18, 7.63 (2d, 2H, Ar-H, J = 8.4 Hz), 7.54 (br s, 2H,
NH
2.60 (br s, 1H, NH); C NMR
methyl piprazine), 46.06, 46.11, 47.29, 47.31, 47.45, 54.67, 54.77 (piprazine carbons), 101.26,
2
), 7.96, 8.18 (2s, 1H, CH=N-), 8.05 (t, 2H, Ar-H, J = 8.0 Hz), 8.39 (t, 2H, Ar-H, J = 9.6Hz),
1
3
1
δ ppm: 21.58 (CH of triazolopyrimidine), 24.66, 24.81 (CH of
3 3
1
1
1
4
01.58, 114.78, 120.72, 121.17, 123.37, 123.79, 127.59, 128.57, 129.38, 138.63, 139.00, 139.08,
39.20, 142.75, 143.06, 146.86, 147.44, 147.92, 150.82, 151.57, 152.58, 152.96, 155.14, 155.96,
58.23, 167.57, 167.72, 172.62, 172.76; Anal. Calcd. for C25
.95; N, 22.94; found C, 54.93; H, 4.88; N, 22.84.
27 9 4
H N O S (549.61): C, 54.63; H,
4
.1.1.5.3.
4-(7-Methyl-5-oxo-3-(2-(pyridin-2-ylmethylene)hydrazine-1-carbonyl)-
[
1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl)benzenesulfonamide (9c)
2
Yield (60%); white powder; m.p. 290–292 °C; IR: 3498, 3356, 3236 (NH, NH ), 1695, 1670
1
(
2C=O), 1330, 1165 (SO ); H NMR
δ
ppm: 2.40, 2.41 (2s, 3H, CH of triazolopyrimidine), 6.02,
2
3
6
7
.13 (2s, 1H, H-6 of triazolopyrimidine), 7.33-7.40, 7.48-7.54 (2m, 4H, 2H Ar-H and 2H, NH
2
),
.67, 7.93 (2t, 1H, Ar-H, J = 7.6 Hz), 8.07 (t, 2 H, Ar-H, J = 7.8 Hz), 8.13, 8.33 (2s, 1H, CH=N-
), 8.39 (t, 2H, Ar-H, J = 9.4 Hz), 8.55, 8.66 (2d, 1H, Ar-H, J = 4.8 Hz), 12.92 (s, 1H, NH); Anal.
Calcd. for C19
H
16
N
8
O
4
S (452.45): C, 50.44; H, 3.56; N, 24.77; found C, 50.14; H, 3.59; N,
2
4.87.
4
.1.1.5.4.
4-(7-Methyl-5-oxo-3-(2-(pyridin-4-ylmethylene)hydrazine-1-carbonyl)-
[
1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl)benzenesulfonamide (9d)
2
2

2
Yield (57%); white powder; m.p. 289–291°C; IR: 3421, 3305, 3221 (NH, NH ), 1689, 1670
1
(
2C=O), 1350, 1165 (SO ); H NMR
δ
ppm: 2.42 (s, 3H, CH of triazolopyrimidine), 6.03, 6.15
2
3
(
2s, 1H, H-6 of triazolopyrimidine 7.34, 7.75 (2d, 2H, Ar-H, J = 5.6 Hz), 7.52, 7.54 (2s, 2H,
), 8.05 (t, 2H, Ar-H, J = 9.2 Hz), 8.36 (s, 1H, CH=N-), 8.39 (t, 2H, Ar-H, J = 9.2 Hz), 8.51,
.71 (2d, 2H, Ar-H, J = 5.2 Hz), 13.01, 13.03 (2s, 1H, NH); Anal. Calcd. for C H N O S
NH
2
8
1
9
16
8
4
(452.45): C, 50.44; H, 3.56; N, 24.77; found C, 50.64; H, 3.58; N, 24.67.
4
.1.1.6. Synthesis of target sulfonamides (11a-h and 13a-c)
A mixture of equimolar quantities of triazole-based hydrazide 7 (0.59 g, 0.002 mol) and the
appropriate aldehyde 10a-h (0.002 mol) or the appropriate acetophenone 12a-c (0.002 mol) in
glacial acetic acid (5 mL) was refluxed for 2h, and then cooled to room temperature. The formed
precipitate was collected by filtration, dried and recrystallized from EtOH/DMF to afford target
compounds 11a-h and 13a-c, respectively.
4
.1.1.6.1.
4-(5-Amino-3-(2-benzylidenehydrazine-1-carbonyl)-1H-1,2,4-triazol-1-
yl)benzenesulfonamide (11a
)
Yield (66%); white powder; m.p. 283–285 °C; IR: 3479, 3421, 3275, 3186, 3140 (NH, 2NH2),
1
1
685 (C=O), 1327, 1153 (SO
2
); H NMR
δ
ppm: 6.94 (s, 2H, NH
2
), 7.47-7.74 (m, 7H, 2H of -
SO NH and 5H, Ar-H), 7.87 (d, 2H, Ar-H, J = 8 Hz), 8.03 (d, 2H, Ar-H, J = 8 Hz), 8.57 (s, 1H,
2
2
1
3
CH=N-), 11.80 (s, 1H, NH); C NMR
δ
ppm: 123.56, 127.59, 127.66, 129.33, 130.70, 134.69,
1
4
39.59, 143.46, 149.17, 154.35, 156.04, 156.18; Anal. Calcd. for C H N O S (385.40): C,
16
15
7
3
9.86; H, 3.92; N, 25.44; found C, 50.13; H, 3.89; N, 25.35.
4
.1.1.6.2. 4-(5-Amino-3-(2-(4-fluorobenzylidene)hydrazine-1-carbonyl)-1H-1,2,4-triazol-1-
yl)benzenesulfonamide (11b
)
Yield (64%); white powder; m.p. 293–295 °C; IR: 3475, 3421, 3278, 3120 (NH, 2NH ), 1689
2
1
(
C=O), 1327, 1153 (SO ); H NMR
δ
ppm: 6.90 (s, 2H, NH ), 7.29 (t, 2H, Ar-H, J = 8.8 Hz),
2
2
7
.54 (s, 2H, -SO NH ), 7.76 (dd, 2H, Ar-H, J = 8.0 Hz, J = 5.6 Hz), 7.84 (d, 2H, Ar-H, J = 8.4
2 2
Hz), 7.80 (d, 2H, Ar-H, J = 8.4 Hz), 8.54 (s, 1H, CH=N-), 11.78 (s, 1H, NH); Anal. Calcd. for
S (403.39): C, 47.64; H, 3.50; N, 24.31; found C, 47.29; H, 3.47; N, 24.39.
16 7 3
C H14FN O
2
3

4
.1.1.6.3.
4-(5-Amino-3-(2-(4-chlorobenzylidene)hydrazine-1-carbonyl)-1H-1,2,4-triazol-1-
yl)benzenesulfonamide (11c
)
Yield (70%); white powder; m.p. 285-287 °C; IR: 3410, 3367, 3298, 3259 (NH, 2NH ), 1678
2
1
(C=O), 1338, 1157 (SO
2
); H NMR
δ
ppm: 6.91 (s, 2H, NH
2
), 7.53-7.55 (m, 4H, 2H, Ar-H and
2
8
H, SO NH , D O exchangeable), 7.73 (d, 2H, Ar-H, J = 8 Hz), 7.85 (d, 2H, Ar-H, J = 8 Hz),
2 2 2
.00 (d, 2H, Ar-H, J = 8.4 Hz), 8.54 (s, 1H, CH=N-), 11.84 (s, 1H, NH); HRMS (ESI)
+
for C
₁
6
H
₁
5
O
3
N
7
³⁵
Cl³²S, calcd 420.06401, found 420.06390 [M+H] .
4
.1.1.6.4.
4-(5-Amino-3-(2-(4-nitrobenzylidene)hydrazine-1-carbonyl)-1H-1,2,4-triazol-1-
yl)benzenesulfonamide (11d
)
Yield (69%); buff powder; m.p. 296–298 °C; IR: 3471, 3379, 3221, 3194 (NH, 2NH ), 1693
2
1
(
C=O), 1338, 1165 (SO ); H NMR
δ
ppm: 6.94 (s, 2H, NH ), 7.56 (s, 2H, -SO NH ), 7.86 (d,
2
2
2
2
2
8
1
H, Ar-H, J = 7.2 Hz), 7.97, 8.03 (2d, 4H, Ar-H, J = 7.6 Hz), 8.31 (d, 2H, Ar-H, J = 7.2 Hz),
13
.66 (s, 1H, CH=N-), 12.11 (s, 1H, NH); C NMR
δ ppm: 123.52, 124.54, 125.78, 127.57,
39.51, 141.01, 143.54, 146.60, 148.38, 154.10, 156.09, 156.36; Anal. Calcd. for C H N O S
1
6
14
8
5
(430.40): C, 44.65; H, 3.28; N, 26.04; found C, 44.83; H, 3.34; N, 26.09.
4
.1.1.6.5.
4-(5-Amino-3-(2-(4-hydroxybenzylidene)hydrazine-1-carbonyl)-1H-1,2,4-triazol-1-
yl)benzenesulfonamide (11e
)
Yield (70%); buff powder; m.p. >300 °C; IR: 3433, 3271, 3217, 3155, 3136, 3116 (OH, NH,
1
2
NH
2
), 1681 (C=O), 1334, 1165 (SO
2
); H NMR
δ
ppm: 6.91 (br s, 4H, 2H, Ar-H and 2H of
NH ), 7.58-8.04 (m, 8H, 2H of SO NH and 6H, Ar-H), 8.46 (s, 1H, CH=N-), 10.00 (s, 1H, OH),
2
2
2
1
3
1
1
3
1.59 (s, 1H, NH); C NMR δ ppm: 116.20, 123.81, 125.69, 127.54, 129.44, 139.60, 143.39,
49.34, 154.51, 155.85, 155.95, 159.98; Anal. Calcd. for C16
.77; N, 24.43; found C, 48.18; H, 3.83; N, 24.53.
15 7 4
H N O S (401.40): C, 47.88; H,
4
.1.1.6.6.
4-(5-Amino-3-(2-(4-(piperidin-1-yl)benzylidene)hydrazine-1-carbonyl)-1H-1,2,4-
triazol-1-yl)benzenesulfonamide (11f
)
Yield (66%); buff powder; m.p. 298–300 °C; IR: 3460, 3417, 3367, 3278, 3259 (NH, 2NH2),
1
1
678 (C=O), 1330, 1161 (SO ); H NMR
δ
ppm: 1.578 (br s, 6H, H-3, H-4 and H-5 of
), 6.96 (d, 2H, Ar-H, J = 8.4
2
piperidine), 3.27 (br s, 4H, H-2, H-6 of piperidine), 6.90 (s, 2H, NH
2
2
4

Hz), 7.53-7.56 (m, 4H, 2H Ar-H and 2H SO
2 2 2
NH , D O exchangeable), 7.86 (d, 2H, Ar-H, J =
1
3
8
.4 Hz), 8.01 (d, 2H, Ar-H, J = 8.4 Hz), 8.40 (s, 1H, CH=N-), 11.49 (s, 1H, NH); C NMR
δ
ppm: 24.39 (C-4 of piperidine), 25.43 (C-3 and C-5 of piperidine), 48.84 (C-2 and C-6 of
piperidine), 115.00, 123.48, 123.76, 127.56, 128.71, 139.63, 143.16, 149.53, 152.91, 154.53,
1
5
55.94, 159.10; Anal. Calcd. for C H N O S (468.54): C, 53.83; H, 5.16; N, 23.92; found C,
21 24 8 3
3.55; H, 5.11; N, 23.82.
4
.1.1.6.7. 4-(5-Amino-3-(2-(4-morpholinobenzylidene)hydrazine-1-carbonyl)-1H-1,2,4-triazol-1-
yl)benzenesulfonamide (11g
)
Yield (58%); buff powder; m.p. 297–300 °C; IR: 3452, 3309, 3259, 3186, 3143 (NH, 2NH2),
1
1
685 (C=O), 1323, 1153 (SO
2
); H NMR
δ
ppm: 3.22 (br.s, 4H, H-2, H-6 of morpholine), 3.75
(
br.s, 4H, H-3, H-5 of morpholine), 6.89 (s, 2H, NH ), 7.00 (d, 2H, Ar-H, J = 8.4 Hz), 7.54 (s,
2
2
2 2
H, -SO NH ), 7.56 (d, 2H, Ar-H, J = 8.4 Hz), 7.84 (d, 2H, Ar-H, J = 8.4 Hz), 7.99 (d, 2H, Ar-
H, J = 8.4 Hz), 8.41 (s, 1H, CH=N-), 11.52 (s, 1H, NH); Anal. Calcd. for C H N O S (470.51):
2
0
22
8
4
C, 51.06; H, 4.71; N, 23.82; found C, 50.74; H, 4.74; N, 23.92.
4
.1.1.6.8.
4-(5-Amino-3-(2-(pyridin-3-ylmethylene)hydrazine-1-carbonyl)-1H-1,2,4-triazol-1-
yl)benzenesulfonamide (11h
)
Yield (67%); white powder; m.p. 283–285 °C; IR: 3479, 3417, 3294, 3194, 3101 (NH, 2NH2),
1
1
678 (C=O), 1334, 1165 (SO
2
); H NMR
δ
ppm: 6.93 (s, 2H, NH
2
), 7.49-7.56 (m, 3H, H-5 of
pyridine and 2H, -SO NH ), 7.86 (d, 2H, Ar-H, J = 8.4 Hz), 8.01 (d, 2H, Ar-H, J = 8 Hz), 8.13
2
2
(d, 1H, H-4 of pyridine, J = 7.2 Hz), 8.61-8.64 (m, 2H, 1H of CH=N- and 1H of H-6 of
1
3
pyridine), 8.84 (s, 1H, H-2 of pyridine), 11.97 (s, 1H, NH); C NMR
27.57, 130.64, 134.08, 139.54, 143.16, 146.40, 149.21, 151.26, 154.20, 155.77, 156.24; Anal.
Calcd. for C15 S (386.39): C, 46.63; H, 3.65; N, 29.00; found C, 46.33; H, 3.61; N,
δ ppm: 123.52, 124.52,
1
14 8 3
H N O
2
9.10.
4
.1.1.6.9.
4-(5-Amino-3-(2-(1-phenylethylidene)hydrazine-1-carbonyl)-1H-1,2,4-triazol-1-
yl)benzenesulfonamide (13a
)
2
Yield (55%); white powder; m.p. 292–294 °C; IR: 3329, 3259, 3228, 3194, 3155 (NH, 2NH ),
1
1
701 (C=O), 1346, 1168 (SO ); H NMR
δ
ppm: 2.33 (s, 3H, CH ), 6.98 (s, 2H, -NH ), 7.42-7.44
2
3
2
2
5

(m, 3H, Ar-H), 7.50 (s, 2H, -SO
2
2
NH ), 7.81-7.85 (m, 4H, Ar-H), 7.95 (d, 2H, Ar-H, J = 8 Hz),
13
1
0.39 (s, 1H, -NH); C NMR
δ ppm: 14.08 (CH ), 123.76, 126.92, 127.56, 128.87, 130.10,
3
1
38.17, 139.54, 143.42, 154.28, 154.66, 155.89, 172.45; Anal. Calcd. for C H N O S (399.43):
17
17
7
3
C, 51.12; H, 4.29; N, 24.55; found C, 51.31; H, 4.22; N, 24.63.
4
1
.1.1.6.10. 4-(5-Amino-3-(2-(1-(pyridin-4-yl)ethylidene)hydrazine-1-carbonyl)-1H-1,2,4-triazol-
-yl)benzenesulfonamide (13b
)
2
Yield (59%); white powder; m.p. 295–297 °C; IR: 3406, 3329, 3271, 3197, 3163 (NH, 2NH ),
1
1
2
697 (C=O), 1350, 1168 (SO ); H NMR
δ
ppm: 2.35 (s, 3H, CH ), 7.01 (s, 2H, -NH ), 7.52 (s,
2
3
2
H, -SO NH ), 7.77 (d, 2H, Ar-H, J = 5.6 Hz), 7.82 (d, 2H, Ar-H, J = 8.8 Hz), 7.96 (d, 2H, Ar-
2
2
1
3
H, J = 8.8 Hz), 8.64 (d, 2H, Ar-H, J = 6.0 Hz), 10.56 (s, 1H, -NH); C NMR
δ ppm: 13.59
(
CH ), 121.00, 123.83, 127.58, 139.50, 143.49, 145.22, 150.52, 152.11, 154.08, 155.95, 172.47;
3
+
6 7 3 8
HRMS (ESI) for C₁ H₁ O N ³²S, calcd 401.11388, found 401.11300 [M+H] .
4
1
.1.1.6.11.
4-(5-Amino-3-(2-(1-(4-morpholinophenyl)ethylidene)hydrazine-1-carbonyl)-1H-
,2,4-triazol-1-yl)benzenesulfonamide (13c
)
Yield (52%); buff powder; m.p. 299–300 °C; IR: 3456, 3352, 3267 (NH, 2NH ), 1681 (C=O),
2
1
1
4
323, 1153 (SO
2
); H NMR
δ
ppm: 2.27 (s, 3H, CH
3
), 3.18 (t, 4H, H-3, H-5 of morpholine, J =
.6 Hz), 3.73 (t, 4H, H-2, H-6 of morpholine, J = 4.8 Hz), 6.97-7.00 (m, 4H, 2H, Ar-H and 2H, -
NH ), 7.52 (s, 2H, -SO NH ), 7.73 (d, 2H, Ar-H, J = 8.8 Hz), 7.81 (d, 2H, Ar-H, J = 8.8 Hz),
2
2
2
1
3
7
.96 (d, 2H, Ar-H, J = 8.8 Hz), 10.29 (s, 1H, -NH); C NMR
C-5 of morpholine), 66.43 (C-2, C-6 of morpholine), 114.42, 123.73, 127.56, 127.99, 128.16,
39.59, 143.38, 152.37, 154.43, 154. 84, 155.44, 155.84; Anal. Calcd. for C21
484.54): C, 52.06; H, 4.99; N, 23.13; found C, 52.28; H, 4.96; N, 23.22.
3
δ ppm: 13.69 (CH ), 48.03 (C-3,
1
24 8 4
H N O S
(
4
.1.1.7. Synthesis of 2-(5-amino-1-(4-sulfamoylphenyl)-1H-1,2,4-triazole-3-carbonyl)-N-(4-
substituted)hydrazine-1-carboxamide (15a, b) and carbothioamide (17a, b
)
To a solution of the appropriate phenyl isocynate 14a,b or phenyl isothiocynate 16a,b
0.001mol) in dry toluene (10 mL), hydrazide 7 (0.3 g, 0.001 mol) was added, and then the
(
reaction mixture was heated under reflux for 3h. The formed precipitate was filtered while hot,
and recrystallized from dioxane to afford target sulfonamides 15a, b and 17a, b respectively.
2
6

4
1
.1.1.7.1. 2-(5-Amino-1-(4-sulfamoylphenyl)-1H-1,2,4-triazole-3-carbonyl)-N-phenylhydrazine-
-carboxamide (15a
)
Yield (69%); white powder; m.p. 279–281°C; IR: 3406, 3332, 3282, 3178 (3NH, 2NH ), 1643,
2
1
1
562 (2C=O), 1315, 1157 (SO
2
); H NMR
δ
ppm: 6.86 (s, 2H, -NH
2
), 6.92 (t, 1H, Ar-H, J = 8.0
Hz), 7.22 (t, 2H, Ar-H, J = 8.0 Hz), 7.43 (d, 2H, Ar-H, J = 8.0 Hz), 7.49 (s, 2H, -SO NH ), 7.79
2
2
(d, 2H, Ar-H, J = 8.4 Hz), 7.95 (d, 2H, Ar-H, J = 8.8 Hz), 8.18 (s, 1H, -NH), 8.76 (s, 1H, -NH),
1
3
9
1
3
.93 (s, 1H, -NH); C NMR
δ ppm: 118.88, 122.31, 123.63, 127.53, 129.09, 139.62, 140.11,
43.30, 153.97, 155.59, 155.91, 159.69; Anal. Calcd. for C H N O S (416.42): C, 46.15; H,
1
6
16
8
4
.87; N, 26.91; found C, 45.92; H, 3.84; N, 26.81.
4
.1.1.7.2. 2-(5-Amino-1-(4-sulfamoylphenyl)-1H-1,2,4-triazole-3-carbonyl)-N-(4-
chlorophenyl)hydrazine-1-carboxamide (15b
)
Yield (70%); white powder; m.p. 252–254 °C; IR: 3363, 3302, 3278 3194, 3101 (3NH, 2NH2),
1
1
681, 1647 (2C=O), 1307, 1161 (SO
2
); H NMR
δ
ppm: 6.88 (s, 2H, -NH
2
), 7.30 (d, 2H, Ar-H, J
=
8.8 Hz), 7.51-7.52 (m, 4H, 2H, Ar-H and 2H, -SO NH ), 7.82 (d, 2H, Ar-H, J = 8.4 Hz), 7.98
2 2
(d, 2H, Ar-H, J = 8.4 Hz), 8.30 (s, 1H, -NH), 8.95 (s, 1H, -NH), 9.98 (s, 1H, -NH); Anal. Calcd.
for C H ClN O S (450.86): C, 42.62; H, 3.35; N, 24.85; found C, 42.98; H, 3.37; N, 24.76.
1
6
15
8
4
4
1
.1.1.7.3. 2-(5-Amino-1-(4-sulfamoylphenyl)-1H-1,2,4-triazole-3-carbonyl)-N-phenylhydrazine-
-carbothioamide (17a
)
2
Yield (72%); white powder; m.p. 248–250 °C; IR: 3367, 3298, 3251, 3178 (3NH, 2NH ), 1678
1
(
C=O), 1338, 1161 (SO ); H NMR
δ
ppm: 6.78 (s, 2H, -NH ), 7.10 (t, 1H, Ar-H, J = 7.2 Hz),
2
2
7
.28 (t, 2H, Ar-H, J = 8.0 Hz), 7.44-7.53 (m, 4H, 2H, Ar-H and 2H, -SO NH ), 7.75 (t, 2H, Ar-
2 2
H, J = 8.4 Hz), 7.92 (t, 2H, Ar-H, J = 8.4 Hz), 9.40 (s, 1H, -NH), 9.72 (s, 1H, -NH), 10.25 (s, 1H,
1
3
-
NH); C NMR
δ
ppm: 123.44, 123.57, 127.49, 127.56, 139.61, 139.69, 143.10, 143.31, 154.56,
³²S , calcd 433.08595, found 433.08584
1
55.71, 155.86, 159.09; HRMS (ESI) for C
₁
6
H₁
7
O
3
N
8
2
+
[
M+H] .
4
.1.1.7.4.
2-(5-Amino-1-(4-sulfamoylphenyl)-1H-1,2,4-triazole-3-carbonyl)-N-(4-
chlorophenyl)hydrazine-1-carbothioamide (17b
)
2
7

2
Yield (68%); white powder; m.p. 244–246 °C; IR: 3363, 3298, 3251, 3209, 3140 (3NH, 2NH ),
1
1
658 (C=O), 1338, 1161 (SO ); H NMR
δ
ppm: 6.81 (s, 2H, -NH ), 7.36 (d, 2H, Ar-H, J = 8.4
2
2
Hz), 7.51-7.52 (m, 4H, 2H, Ar-H and 2H, -SO NH ), 7.79 (t, 2H, Ar-H, J = 8.8 Hz), 7.95 (t, 2H,
2
2
Ar-H, J = 8.4 Hz), 9.43 (s, 1H, -NH), 9.79 (s, 1H, -NH), 10.31 (s, 1H, -NH); Anal. Calcd. for
C H ClN O S (466.92): C, 41.16; H, 3.24; N, 24.00; found C, 41.45; H, 3.29; N, 23.92.
16
15
8
3 2
4
.1.1.8. Synthesis of 4-(5-amino-3-(3-(4-(un)substituted phenyl)ureido)-1H-1,2,4-triazol-1-
yl)benzenesulfonamide (21a–g
)
In an ice bath, a mixture of the hydrazide 7 (1.49 g, 0.005mol) and sodium nitrite (0.5 g,
.007mol) was stirred in glacial acetic acid for 1h, then stirring was continued at room
0
temperature for another 1h. The obtained solid was collected by filtration, washed with cold
water and air-dried to furnish 5-amino-1-(4-sulfamoylphenyl)-1H-1,2,4-triazole-3-carbonyl azide
1
8, which used in the next reaction without further purification. Then, the appropriate azide was
heated under reflux in dry xylene for 1h before addition of anilines 20a-g. This reaction mixture
was refluxed for 3h then allowed to cool to room temperature. The formed precipitate was
filtered off, washed with cold acetone, dried and recrystallized from dioxane to furnish the target
triazole-based ureides 21a–g.
4
.1.1.8.1. 4-(5-Amino-3-(3-phenylureido)-1H-1,2,4-triazol-1-yl)benzenesulfonamide (21a
)
Yield (48%); white powder; m.p. 251–253 °C; IR: 3379, 3321, 3221 (2NH, 2NH
2
), 1685 (C=O),
1
1
7
334, 1161 (SO ); H NMR
δ
ppm: 6.87-7.02 (m, 3H, 1H Ar -H and 2H, -NH of triazole), 7.28-
2
2
2 2
.44 (m, 3H, 1 H Ar-H and 2H, -SO NH ), 7.50 (d, 2H, Ar-H, J = 7.2 Hz), 7.63-7.76 (m, 2H, Ar-
H), 7.82, 7.91 (2d, 2H, Ar-H, J = 8.8 Hz), 7.98, 9.74 (2s, 1H, -NH), 10.08, 10.38 (2s, 1H, -NH);
Anal. Calcd. for C H N O S (373.39): C, 48.25; H, 4.05; N, 26.26; found C, 48.03; H, 3.99; N,
1
5
15
7
3
2
6.35.
4
.1.1.8.2.
4-(5-Amino-3-(3-(4-fluorophenyl)ureido)-1H-1,2,4-triazol-1-yl)benzenesulfonamide
(21b)
Yield (44%); buff powder; m.p. 261–263 °C; IR: 3475, 3363, 3278, 3170 (2NH, 2NH ), 1695
2
1
(
C=O), 1334, 1157 (SO ); H NMR
δ
ppm: 6.87, 6.99 (2s, 2H, -NH of triazole), 7.11- 7.17 (m,
2
2
2
8
H, Ar-H), 7.34, 7.45 (2s, 2H, -SO NH ), 7.48-7.54 (m, 2H, Ar-H), 7.63, 7.73 (2d, 2H, Ar-H, J =
2 2
.8 Hz), 7.78, 7.90 (2d, 2H, Ar-H, J = 8.8 Hz), 7.98, 9.81 (2s, 1H, -NH), 10.22, 10.37 (2s, 1H, -
2
8