Sulfonamide-based ring-fused analogues for CAN508 as novel carbonic anhydrase inhibitors endowed with antitumor activity: Design, synthesis, and in vitro biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2019.112019

In the present study, we report the design and synthesis of novel CAN508 sulfonamide-based analogues (4, 8a-e, 9a-h and 10a-e) as novel carbonic anhydrase (CA) inhibitors with potential CDK inhibitory activity. A bioisosteric replacement approach was adop

Full text of DOI:10.1016/j.ejmech.2019.112019

Journal Pre-proof
Sulfonamide-based ring-fused analogues for CAN508 as novel carbonic anhydrase
inhibitors endowed with antitumor activity: Design, synthesis, and in vitro biological
evaluation
Mohamed A. Said, Wagdy M. Eldehna, Alessio Nocentini, Samar H. Fahim,
Alessandro Bonardi, Abdullah A. Elgazar, Vladimír Kryštof, Dalia H. Soliman, Hatem
A. Abdel-Aziz, Paola Gratteri, Sahar M. Abou-Seri, Claudiu T. Supuran
PII:
S0223-5234(19)31177-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.112019
EJMECH 112019
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 November 2019
Revised Date: 27 December 2019
Accepted Date: 27 December 2019
Please cite this article as: M.A. Said, W.M. Eldehna, A. Nocentini, S.H. Fahim, A. Bonardi, A.A.
Elgazar, Vladimí. Kryštof, D.H. Soliman, H.A. Abdel-Aziz, P. Gratteri, S.M. Abou-Seri, C.T. Supuran,
Sulfonamide-based ring-fused analogues for CAN508 as novel carbonic anhydrase inhibitors endowed
with antitumor activity: Design, synthesis, and in vitro biological evaluation, European Journal of
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2019.112019.
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Graphical Abstract
Sulfonamide-based Ring-fused analogues for CAN508 as novel carbonic
anhydrase inhibitors endowed with antitumor activity: Design, synthesis, and
in vitro biological evaluation
New sets of novel CAN508 sulfonamide-based analogues (4, 8a-e, 9a-h and 10a-e) were
synthesized and evaluated for their inhibitory activity toward a panel of carbonic anhydrase
isoforms hCA I, II, IX and XII, and toward CDK2 and 9. hCA IX and XII isoforms were
efficiently inhibited with KIs range of 6–67.6 and 10.1–88.6 nM, respectively.
Pyrazolopyrimidines 9d, 9e and 10b were found to be the most potent CDK2 inhibitors with IC50
= 6.4, 8.0 and 11.6 µM, respectively.

Sulfonamide-based Ring-fused analogues for CAN508 as novel carbonic
anhydrase inhibitors endowed with antitumor activity: Design, synthesis, and
in vitro biological evaluation
Mohamed A. Saida, Wagdy M. Eldehnab,*, Alessio Nocentinic,d, Samar H. Fahime, Alessandro
Bonardic,d, Abdullah A. Elgazarf, Vladimír Kryštofg, Dalia H. Solimana,h, Hatem A. Abdel-Azizi,
Paola Gratterid, Sahar M. Abou-Serie,*, Claudiu T. Supuranc,*
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 Pharmacognosy, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, P.O. Box
33516, Egypt
gLaboratory of Growth Regulators, Palacky University & Institute of Experimental Botany, The Czech
Academy of Sciences, Slechtitelu 27, 78371 Olomouc, Czech Republic
h Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al Azhar University, Cairo, P.O. Box
11471, Egypt
i Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622, Egypt
* Corresponding authors. E-mail addresses: wagdy2000@gmail.com (W.M. Eldehna),
sahar.shaarawy@pharma.cu.edu.eg (S.M. Abou-Seri), claudiu.supuran@unifi.it (C.T. Supuran).
1

ABSTRACT
In the present study, we report the design and synthesis of novel CAN508 sulfonamide-based
analogues (4, 8a-e, 9a-h and 10a-e) as novel carbonic anhydrase (CA) inhibitors with potential
CDK inhibitory activity. A bioisosteric replacement approach was adopted to replace the
phenolic OH of CAN508 with a sulfamoyl group to afford compound 4. Thereafter, a ring-fusion
approach was utilized to furnish the 5/5 fused imidazopyrazoles 8a-e which were subsequently
expanded to 6/5 pyrazolopyrimidines 9a-h and 10a-e. All the synthesized analogues were
evaluated for their inhibitory activity toward isoforms hCA I, II, IX and XII. The target tumor-
associated isoforms hCA IX and XII were effectively inhibited with KIs ranges 6–67.6 and 10.1–
88.6 nM, respectively. Furthermore, all compounds were evaluated for their potential CDK2 and
9 inhibitory activities. Pyrazolopyrimidines 9d, 9e and 10b displayed weak CDK2 inhibitory
activity (IC50 = 6.4, 8.0 and 11.6 µM, respectively), along with abolished CDK9 inhibitory
activity. This trend suggested that pyrazolopyrimidine derivatives merit further optimization to
furnish more effective CDK2 inhibitor lead. On account of their excellent activity and selectivity
towards hCA IX and XII, pyrazolopyrimidines 10 were evaluated for their anti-proliferative
activity toward breast cancer MCF-7 and MDA-MB-468 cell lines under normoxic and hypoxic
conditions. The most potent anti-proliferative agents 10a, 10c and 10d significantly increased
cell percentage at sub-G1 and G2-M phases with concomitant decrease in the S phase population
in MCF-7 treated. Finally, a docking study was undertaken to investigate the binding mode for
the most selective hCA IX and XII inhibitors 10a-e, within hCA II, IX and XII active sites.
__________
Keywords: Carbonic anhydrase inhibitors; CDK inhibitors; Molecular docking;
Pyrazolopyrimidines; Imidazopyrazoles; CAN508.
2

1. Introduction
In spite of the significant improvement in survival among cancer patients over the last three
decades [1], cancer, as described by the world health organization (WHO), is considered the
second leading cause of death. It accounted for 9.6 million deaths in 2015[2]. Studies also
estimate that this number is expected to double by 2030, and despite the extensive efforts and
investments in this research field, it seems that the management of these human malignancies
still constitute a major challenge for diagnosis and therapy. Thus, there is an ongoing need to
develop new chemotherapeutic anticancer agents that may act by different mechanisms of action.
It was established that the different carbonic anhydrase (CA) isoforms are involved in
numerous physiological and pathological processes all associated with CO2 hydration reaction,
such as bone resorption, calcification, electrolyte secretion, respiration, lipogenesis,
gluconeogenesis, tumorigenicity and many others.[3, 4] Moreover, the hypoxia-induced hCA IX
and XII isoforms are transmembrane tumor-associated members of the CA family. They are
expressed in a limited number of normal tissues, whereas they are overexpressed in a diversity of
invasive tumors such as colon, pancreas, breast, ovaries, cervix, brain, head and neck, kidney as
well as lung cancers, that are associated with the hypoxic phenotype, so they are capable of
affecting cell proliferation, cell adhesion and malignant cell invasion.[5, 6] Accordingly, CA
isoforms IX and XII represent effective validated targets in cancer treatment strategies.
Superiorly, the zinc anchoring sulfonamides represent the most important class of the CA
inhibitors, where their deprotonated form (SO2NH-) is coordinated to the positively charged
Zn(II) ion in the enzyme active site. The “tail approach” represents the most successful approach
that could be adopted to develop isoform selective CA inhibitors (CAIs). In this approach, the
aromatic/heterocyclic ring possessing the sulfonamide, zinc binding group (ZBG), is appended
with tail moieties through diverse functionalized linkers.[4]
Cyclin-dependent kinases (CDKs) play various essential roles in different key transitions of
the cell cycle as well as in the regulation of apoptosis, neuronal functions, transcription and
exocytosis.[7] Deregulation of cell cycle is accompanied by altered CDK activity in many
cancers, either by activation of CDKs and/or their interacting proteins. For these reasons,
3

therapeutic approaches based on CDK inhibition represent a unique opportunity for drug
discover and a promising strategy for treatment of human malignancies.[8, 9]
Recently, the CDK4/CDk6 inhibitors palbociclib, ribociclib and abemaciclib have received
approval from the US Food and Drug Administration for the treatment of certain breast cancers
and revolutionized the treatment. However, primary and acquired resistance to these drugs
somehow limits the therapy and implies the necessity of development of novel therapeutic
approaches [10]. Other promising CDK inhibitors in the pre/clinical development display
wider kinase selectivity and anticancer potency [11]. Amongst them, purines and related
heterocycles have stood out as the most investigated scaffolds for development of CDK
inhibitors. Seliciclib (Roscovitine or CYC202) (Fig. 1) is a purine-based CDK2/ CDK7/CDK 9
inhibitor that exerts diverse effects on cell proliferation and apoptosis induction in different
cancer cells. [12, 13] The success of roscovitine paved the way for further development of
other CDK inhibitors, either through redistribution of nitrogen atoms of the purine nucleus
such as Dinaciclib (Fig. 1) [14], or via appending different substituents to the purine scaffold
such as purine-based benzenesulfonamide I (Fig. 1).[15] During our past projects, we have
explored various other CDK inhibitors, where the 4-arylazo-3,5-diaminopyrazole derivatives
CAN508 and AAP1742 were identified as efficient CDK2 and/ or CDK 9 inhibitors (Fig. 1).
[9, 16, 17]
Fig. 1. Structures of some reported CDK inhibitors.
In view of the aforementioned findings and in connection with our research program in the
search for effective anticancer candidates that target enzymes/proteins overexpressed in
different tumors such as the tumor-associated hCA IX isoform [18-23], herein we report design
and synthesis for new sets of CAN508 sulfonamide-based analogues (4, 8a-e, 9a-h and 10a-e)
4

with the prime aim of developing novel carbonic anhydrase inhibitors with potential CDK
inhibitory activity, endowed with antitumor activity.
(5/5) Imidazopyrazole
Ar
HN N
N
N
N
HN N
N
Tail
HN
H2N
H2N
NH2
NH2
NH2
N
Spacer
N
N
N
Bioisosteric
Replacement
Ring-Fusion
HBD group
ZBG
OH
O S
O
O S
O
NH2
NH2
CAN508
CDK Inhibitor
8a-e
4
Ring Expansion
(6/5) Pyrazolopyrimidine
NH2
N N
Ar
N
NC
N
Ar
N
N
NH2
NH2
N
N
N
N
O S
O
O S
O
NH2
NH2
9a-h
10a-e
Fig. 2. Design of target sulfonamide-based ring-fused analogues for CAN508 (4, 8a-e, 9a-h and
10a-e).
Initially, the sulfamoyl group was selected to substitute the phenolic OH group of CAN508,
which can interact as a H-bond donor with the side chain carboxylate of Asp145 in the CDK2
active site, in addition to the coordination of its deprotonated form (SO2NH-) to the positively
charged Zn(II) ion in the CA active site. Thereafter, a ring-fusion approach was adopted for the
pyrazole ring to furnish the 5/5 fused imidazo[1,2-b]pyrazole system; compounds 8a-e.
Furthermore, the imidazo[1,2-b]pyrazole nucleus in compounds 8 was expanded to the 6/5
fused pyrazolo[1,5-a]pyrimidine system to afford compounds 9a-h and 10a-e. Both
5

imidazo[1,2-b]pyrazole and pyrazolo[1,5-a]pyrimidine ring systems represent the tail for the
target CAIs (8a-e, 9a-h and 10a-e). Finally, the substitution pattern on both imidazo[1,2-
b]pyrazole and pyrazolo[1,5-a]pyrimidine tails was selected so as to ensure different electronic
and lipophilic environments that could manipulate the potency of the target CAN508
sulfonamide-based analogues (Fig. 2).
All the synthesized sulfonamides were tested for their inhibitory activities toward four CA
isoforms (hCA I, hCA II, hCA IX and hCA XII). Also, their inhibitory activities against CDK2
and CDK9 were assayed. In addition, compounds 8 and 10 were further evaluated against a
panel of 57 human cell lines at National Cancer Institute (NCI, Bethesda, MD). Thereafter, the
most selective hCA IX and XII inhibitors were examined for their anti-proliferative activity
against MCF-7 and MDA-MB-468 breast cancer cell lines under normoxic and hypoxic
conditions. Moreover, sulfonamides 10a, 10c and 10d were assessed for cell cycle disturbance
and apoptosis induction in MCF-7 cells.
2. Results and Discussion
2.1. Chemistry
The synthetic pathways employed to prepare the target CAN508 sulfonamide-based
analogues (4, 8a-e, 9a-h and 10a-e) are depicted in Schemes 1 and 2.
Scheme 1. Reagents and conditions: i. NaNO2, HCl, 0-5 0C; ii. EtOH, CH3COONa, 0-5 0C; iii.
NH2NH2.H2O, EtOH, reflux.
The synthesis of N-(4-sulfamoylphenyl)carbonohydrazonoyl dicyanide 3 was achieved by
diazotization of the amino group of sulfanilamide 1, followed by coupling the obtained
diazonium with malononitrile in ethanolic sodium acetate solution at 0-5 0C as reported. [24, 25]
Subsequently, preparation of (E)-4-((3,5-diamino-1H-pyrazol-4-yl)diazenyl)benzenesulfonamide
6

4 was carried out through refluxing the dicyanide 3 with equimolar amount of hydrazine in
ethanol as described by Hassanien et al. [24, 26] (Scheme 1).
Scheme 2. Reagents and conditions: i. EtOH, DMF, reflux 12h; ii. AcOH, reflux 1h; iii. DMF,
reflux 8-10h.
7

The diazenylbenzenesulfonamide 4 was utilized as a precursor for the synthesis of the
imidazo[1,2-b]pyrazoles 8a-e and the pyrazolo[1,5-a]pyrimidines 9a-h and 10a-e as shown in
Scheme 2 where three different series of reagents were required. The first series included
different phenacyl bromides 5a-e that were obtained by refluxing of appropriate acetophenone
with copper (II) bromide in ethyl acetate as reported.[27, 28] The second series involved 3-
(dimethylamino)-1-(aryl)prop-2-en-1-ones 6a-h and were prepared by refluxing the
corresponding acetophenone with DMF-DMA in xylene following the reported procedure.[29,
30] The third series included different benzylidenemalononitrile derivatives 7a-e and were
synthesized by heating under reflux the appropriate aldehyde with malononitrile in ethanol in
presence of catalytic amount of piperidine.[31]
The target 4-((6-amino-2-(4-substitutedphenyl)-1H-imidazo[1,2-b]pyrazol-7-yl)diazenyl)
benzenesulfonamides 8a-e were obtained by reacting the key intermediate 4 with the phenacyl
bromides 5a-e in refluxing ethanol containing few drops of DMF. Alternatively,
cyclocondensation of diazenylbenzenesulfonamide 4 with the appropriate arylpropinone
derivative 6a-h in glacial acetic acid or the appropriate benzylidenemalononitrile 7a-e in DMF
furnished the corresponding pyrazolo[2,3-a]pyrimidine 9a-h or 10a-e, respectively.
The IR spectra of imidazo[1,2-b]pyrazoles 8a-e showed absorption bands at range of 3448-
3086 cm-1 for the NH and two NH2 groups, in addition to the absorption bands of SO2 of the
sulfamido group at range of 1338-1153 cm-1. The 1H NMR spectra of these compounds revealed
three D2O-exchangeable signals in the regions δ 7.34-7.41 ppm, 7.75-7.79 ppm and 11.86-11.97
ppm corresponding to sulfamido NH2, 6-NH2 and imidazole NH protons, respectively. In
addition to a singlet signal in the region δ 8.44- 8.51 ppm assigned to the imidazo[1,2-
b]pyrazoles H-3 proton. Compound 8b showed additional aliphatic singlet signal corresponding
13
to the 4-methyl substituent at δ 2.36 ppm. Their C NMR spectra showed signals resonating at
110.85-164.02 ppm corresponding to their aromatic carbons. Compound 8b revealed a specific
signal at 21.74 ppm characteristic to the aliphatic carbon of its methyl group. (Scheme 2)
The IR spectra of pyrazolo[1,5-a]pyrimidines 9a-h showed absorption bands in the range of
1
3437-3101 and 1334-1145 cm-1 due to the two NH2 and SO2 of the sulfamido group. The H
NMR spectra of 9a-h revealed the appearance of two characteristic D2O-exchangeable signals
representing the protons of pyrazole NH2 and sulfamido NH2 at δ 7.40-7.57 ppm. Also,
compounds 9b and 9f showed specific signals for protons of methyl and methoxy protons at δ
8

2.43 and 3.87 ppm, respectively. Moreover, 13C NMR spectrum of compounds 9b and 9f
revealed signals resonating at δ 21.57 and 55.98 ppm attributed to the methyl and methoxy
carbons, respectively. Additionally, a high resolution mass analysis showed a molecular ion peak
at 408.12357 and 428.06914 representing [M+H]+ peaks of compounds 9b and 9d, respectively.
(Scheme 2)
Finally, IR spectra of pyrazolo[1,5-a]pyrimidines 10a-e revealed absorption bands at ranges
of 3483-3066 cm-1 for 3NH2, 2210-2222 cm-1 for CN and 1361-1149 cm-1 for SO2 of the
1
sulfamido substituents. The H NMR spectra of these compounds revealed the appearance of
three D2O-exchangeable signals assigned to the protons of the two amino and sulfamido NH2
groups in the ranges of δ 7.24-7.25 ppm, 7.41-7.38 ppm and 8.64-8.71 ppm. Also, compound 10b
showed characteristic singlet signal at δ 2.43 ppm for protons of the methyl substituent, while its
13C NMR spectrum revealed a signal resonating at δ 21.48 ppm due to the methyl carbon. HRMS
spectrum of compound 10c showed a [M+H]+ peak at 452.13999 conforming its molecular
weight. (Scheme 2)
2.2. Biological Evaluation
2.2.1. Carbonic anhydrase inhibition
All the target CAN508 sulfonamide-based analogues (4, 8a-e, 9a-h and 10a-e) were
evaluated for their efficacy to inhibit the physiologically relevant hCA isoforms, hCA I and II
(cytosolic) as well as hCA IX and XII (trans membrane, tumor associated isoforms) through a
stopped flow CO2 hydrase assay [32] using acetazolamide (AAZ) as a standard inhibitor. The
following structure–activity relationship (SAR) can be concluded from the inhibition data
presented in Table 1.
(i) The in vitro kinetic data listed in Table 1 revealed that the ubiquitous cytosolic hCA I isoform
was weakly inhibited by all the CAN508 ring-fused analogues reported in this study (8a-e, 9a-h
and 10a-e) with inhibition constants (KIs) ranging from high nanomolar to low micromolar
concentration; between 442.1 nM and 4.16 µM, apart from the non-fused analogue 4 which
displayed a higher inhibitory efficacy (KI = 270.6 nM). Noteworthy, the expanded
pyrazolopyrimidine series 10 arose as the weakest hCA I inhibitors in this study with KIs in the
micromolar range (2.25 – 4.16 µM).
9

Table 1: Inhibition data of hCA isoforms I, II, IX and XII with CAN508 analogues (4, 8a-e, 9a-
h and 10a-e), using AAZ as a standard inhibitor.
KI (nM)
Comp.
Ar
hCA I
270.6
957.3
1203
769.3
651.3
1589
457.9
1331
681.3
543.7
767.7
442.1
2122
1154
2683
3534
2435
2251
4162
250
hCA II
38.1
75.4
99.7
33.8
22.1
49.3
27.6
6.2
hCA IX
22.0
38.1
9.4
hCA XII
10.1
30.6
27.7
18.6
36.5
52.8
25.4
60.7
31.4
44.3
54.3
88.6
14.4
59.0
39.4
48.8
10.9
14.2
43.2
5.7
4
-
8a
C6H5
8b
8c
4-CH3-C6H4
4-F-C6H4
4-Cl-C6H4
4-Br-C6H4
C6H5
28.4
16.6
44.9
31.8
45.7
6.0
8d
8e
9a
9b
9c
4-CH3-C6H4
4-F-C6H4
4-Cl-C6H4
4-Br-C6H4
4-OCH3-C6H4
4-NO2-C6H4
2-thienyl
C6H5
14.5
23.4
38.7
5.5
9d
9e
56.3
65.0
29.3
67.6
40.1
33.5
44.3
8.8
9f
9g
35.6
7.1
9h
10a
10b
10c
10d
10e
AAZ
921.7
638.4
398.8
496.1
885.2
12
4-CH3-C6H4
4-F-C6H4
4-Cl-C6H4
4-Br-C6H4
31.3
65.7
25
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5-10 % of the
reported values).
10

(ii) The examined sulfonamides displayed weak to potent inhibitory activity toward the
physiologically dominant off-target isoform hCA II (KI values ranging between 5.5 and 921.7
nM, Table 1). The pyrazolopyrimidines 9b, 9f and 9h emerged as the most efficient hCA II
inhibitors with single-digit nanomolar inhibition constants (KIs = 6.2, 5.5 and 7.1 nM,
respectively). Generally, pyrazolopyrimidines 9 showed improved inhibitory profile against hCA
II than their corresponding analogues in both imidazopyrazole 8 and pyrazolopyrimidine 10
series, except 9d which exhibited comparable activity (KI = 23.4 nM) to its imidazopyrazole
analogue 8d (KI = 22.1 nM), whereas pyrazolopyrimidines 10 displayed the weakest hCA II
inhibitory activity with KIs in the high nanomolar range (398.8 – 921.7 µM), similar to the
inhibition profile for hCA I.
The SAR outcomes pointed out that substitution of the pendant phenyl ring in series 8 (KIs:
22.1 – 49.3 nM), 9 (KIs: 5.5 – 23.4 nM) and 10 (KIs: 398.8 – 885.2 nM) elicited an enhancement
of effectiveness against hCA II in comparison to the unsubstituted analogues 8a (KI = 75.4 nM),
9a (KI = 27.6 nM) and 10a (KI = 921.7 nM), except for imidazopyrazole 8b (KI = 99.7 nM), and
pyrazolopyrimidines 9e and 9g (KIs = 38.7 and 35.6 nM, respectively) which displayed an
elevated KI values than their unsubstituted analogues. Moreover, bioisosteric replacement of the
pendant phenyl ring in the pyrazolopyrimidine 9a (KI = 27.6 nM) with the heterocyclic 2-thienyl
ring led to about four-fold enhanced hCA II inhibition potency (9h; KI = 7.1 nM).
(iii) The in vitro kinetic data displayed in Table 1 showed that the target tumor-associated hCA
IX isoform was efficiently inhibited by all the CAN508 analogues herein reported. The inhibition
profiles toward hCA IX isoform were found to be rather flat, as the measured KI values ranged
between 6.0 and 67.6 nM. Superiorly, sulfonamides 8b, 9c and 10c were the most potent hCA IX
inhibitors in this study with KIs in a single-digit nanomolar range; 9.4, 6 and 8.8 nM,
respectively.
Further analysis of the results revealed that incorporation of a small lipophilic electron-
donating group on the pendant phenyl ring of imidazopyrazole series 8 (p-tolyl derivative 8b; KI
= 9.4 nM) resulted in 4-fold enhanced activity compare to the unsubstituted analogues 8a (KI =
38.1 nM), whereas grafting the small lipophilic electron-withdrawing p-fluoro substituent within
the two expanded pyrazolopyrimidine series 9 and 10 led to the best hCA IX inhibitors in this
study; compounds 9c and 10c (KIs = 6.0 and 8.8 nM, respectively). In contrast, incorporation of
11

large electron-withdrawing groups such as p-bromo or p-nitro substituents (compounds 8e, 9e,
9g and 10e; KIs = 44.9, 65.0, 67.6 and 65.7 nM, respectively) elicited a worsening of
effectiveness toward hCA IX in comparison to their unsubstituted counterparts 8a, 9a and 10a
(KIs = 38.1, 31.8 and 33.5 nM, respectively). It is worth stressing that ring-expansion approach
was more beneficial for hCA IX inhibitory activity in case of incorporation of unsubstituted or p-
fluoro substituted phenyl moieties in comparison to the fused imidazopyrazole series 8.
(iv) All the investigated CAN508 analogues emerged as potent inhibitors for the target tumor-
associated hCA XII isoform (KI values ranging between 10.1 and 88.6 nM). Interestingly, the
non-fused pyrazole-based sulphonamide 4 was the most potent hCA XII inhibitor in this study
with KI value equals 10.1 nM. Besides, compounds 8c, 9g, 10c and 10d displayed excellent
inhibitory activity toward hCA XII isoform (KIs = 18.6, 14.4, 10.9 and 14.2 nM, respectively).
Notably, appending the small lipophilic electron-withdrawing p-fluoro substituent within series 8
and 10 led to the best hCA XII inhibitor in each series, (compounds 8c and 10c; KIs = 18.6 and
10.9 nM, respectively).
(v) Taking into account that hCA IX and XII isoforms are validated targets for treatment of
human malignancies, development of selective hCA IX and XII inhibitors stands out as a crucial
element to unveil an effective tumor therapy devoid of the classical side effects attributable for
inhibition of the physiologically relevant off-target isoforms hCA I and II.
The calculated selectivity indexes (SIs) displayed in Table 2 showed that all the tested
compounds possessed good selectivity towards hCA IX and XII over hCA I. The results also
undeniably ascribed to the pyrazolopyrimidine series 10 excellent selectivity towards hCA IX
and XII over hCA I with SIs spanning in the range 63.35 – 276.70 and 68.10 – 223.39,
respectively. In addition, series 10 possessed remarkable selectivity towards hCA IX and XII
over hCA II with SIs ranges 13.47 – 45.32 and 13.08 – 36.59, respectively. In contrast, both
series 8 and 9 failed to display a promising selectivity profile towards hCA IX and XII over
hCA II regardless of their efficient inhibition of hCA IX and XII isoforms, except compound
8b which was capable of achieving moderate selectivity over hCA II isoform.
12

Table 2. Selectivity index (SI) calculated for hCA IX and XII over off-targets isoforms (hCA I and II) for
target sulfonamides (8a-e, 9a-h and 10a-e) and AAZ.
Selectivity indexes
Compound
I\IX
I\XII
II\IX
II\XII
25.13
31.28
1.98
2.46
8a
8b
127.98
27.09
39.23
35.39
14.40
29.12
113.55
9.66
43.43
41.36
17.84
30.09
18.03
21.93
21.70
12.27
14.14
4.99
10.61
1.19
1.33
1.10
0.87
0.14
2.42
0.42
0.60
0.19
0.53
0.18
27.51
14.41
45.32
15.85
13.47
0.48
3.60
1.82
0.61
0.93
1.09
0.10
0.46
0.53
0.71
0.06
2.47
0.12
23.39
13.08
36.59
34.94
20.49
2.11
8c
8d
8e
9a
9b
9c
9d
9e
11.81
15.09
31.39
28.78
80.09
79.77
276.70
71.92
63.35
10.00
9f
9g
147.36
19.56
68.10
72.42
223.39
158.52
96.34
43.86
9h
10a
10b
10c
10d
10e
AAZ
2.2.2. CDK2 and 9 inhibitory activities
The newly prepared sulfonamides (4, 8a-e, 9a-h and 10a-e) were further evaluated for their
potential CDK2 and CDK9 inhibitory activity. Roscovitine and CAN508 were used as
reference drugs. The results were reported as a 50% inhibition concentration values (IC50),
determined from triplicate dose-response curves and are listed in Table 3.
Results in Table 3 revealed that only sulfonamides 9d, 9e and 10b possessed weak
inhibitory activity toward CDK2 (IC50 = 6.4, 8.0 and 11.6 µM, respectively), that is better than
CAN508 (IC50 = 20 µM). In addition, compound 8b displayed comparable activity (IC50 = 20.3
µM) to the lead CAN508 toward CDK2. In contrast, the non-fused compound 4 showed 2-fold
decreased activity (IC50 = 41.7 µM) than CAN508 against CDK2. Unfortunately, all the
13

examined sulfonamides displayed non-significant inhibitory activity towards CDK9 (IC50
>
12.5 µM).
In summary, the adopted bioisosteric replacement of the phenolic group of CAN508 with a
sulfamoyl group resulted in 2-fold decreased potency toward CDK2 (compound 4; IC50 = 41.7
µM), whereas, the ring fusion approach just maintained the CDK2 inhibitory activity
(imidazopyrazole 8b; IC50 = 20.3 µM). On the other hand, the ring expansion approach
successfully enhanced the inhibitory activity towards CDK2, such pyrazolopyrimidines 9d, 9e
and 10b (IC50 = 6.4, 8.0 and 11.6 µM, respectively). Interestingly, increased potency against
CDK2 was accompanied with an abolished activity towards CDK9. Accordingly, this trend of
activity pointed out that the herein reported pyrazolopyrimidine-based CDKIs merit further
optimization and development to furnish more promising selective CDK2 inhibitors.
Table 3. IC50 values for the inhibitory activity of 4, 8a-e, 9a-h and 10a-e against CDK2 and 9.
IC50 (µM)
Compound
CDK2
41.7
CDK9
>12.5
>25
4
>25
8a
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
0.35
8b
8c
8d
8e
9a
9b
9c
9d
9e
9f
9g
9h
10a
10b
10c
10d
10e
20.3
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
6.4
8.0
>12.5
>12.5
>12.5
>12.5
11.6
>12.5
>12.5
>12.5
20
CAN508
Roscovitine
0.1
1.8
14

2.2.3. Anticancer Activity
2.2.3.1. NCI antitumor activity screening
Sulfonamides (8a-e and 10a-e) were selected to be examined for their antitumor activity at
the NCI-Developmental Therapeutic Program (www.dtp.nci.nih.gov). They were screened in a
primary one dose (10 µM) anticancer assay against a panel of 57 cancer cell lines, in accordance
with the protocol of the Drug Evaluation Branch, NCI, Bethesda [33]. The data are reported as
mean-graph of the percent growth of the treated cancer cells and presented, in Table 4, as
percentage growth inhibition (GI%) caused by the examined sulfonamides.
Investigation of the obtained data in Table 4 revealed that the examined CAN508
sulfonamide-based analogues exhibited distinctive patterns of sensitivity and selectivity toward
the different cancer cell panels. Inspecting the GI% values, pointed out that Imidazopyrazole
derivative 8a stood out as the most potent CAN508 analogue in this study with mean GI = 68%.
It exhibited broad spectrum anticancer activity against all the tested cancer cell lines with an
exception to ovarian (NCI/ADR-RES) cancer cell line. Superiorly, sulfonamide 8a displayed an
excellent growth inhibitory activity against Leukemia (MOLT-4, RPMI-8226 and SR), CNS
(SNB-75), ovarian (IGROV1 and SK-OV-3), Renal (A498, ACHN, CAKI-1 and RXF 393), and
breast cancer (HS 578T) cell lines with GI% range = 85- 96. In addition, sulfonamide 8a
displayed potent GI more than 75% toward Leukemia (CCRF-CEM), Non-small cell lung cancer
(NCI-H460), colon cancer (HCT-116 and HCT-15), CNS (SF-539), Melanoma (LOX IMVI),
Renal (SN12C and UO-31), and breast cancer (MDA-MB-231/ATCC) cell lines, Table 4.
Moreover, pyrazolopyrimidine 10b was found to be the second most potent analogue (mean
GI% = 61) with broad spectrum activity against all the tested cancer cell lines, except Ovarian
(NCI/ADR-RES) cancer cell line. 10b exerted outstanding anti-proliferative activity against
Leukemia (CCRF-CEM, HL-60TB, K-562 and MOLT-4), Non-small cell lung cancer (HOP-92),
Colon cancer (COLO 205, HCT-116 and SW-620), Renal (ACHN) and Breast (MDA-MB-468)
cell lines with GI% ranging from 80 to 100 %, Table 4. Furthermore, sulfonamides 8b, 8e and
10e (mean GI = 50, 46 and 50%, respectively) displayed promising broad spectrum growth
inhibitory activity towards most of the tested cancer cell lines.
15

Table 4. Percentage growth inhibition (GI %) of in vitro subpanel tumor cell lines at 10 µM
concentration for sulfonamides 8a-e and 10a-e.
Compound a
Subpanel/ Cell line
8a
80
72
67
86
94
87
67
45
58
74
47
54
63
80
69
55
54
77
77
61
67
74
8b
54
47
48
55
60
63
62
28
39
35
27
34
69
67
57
24
40
62
65
37
46
60
8c
33
8d
----
----
----
----
----
28
8e
45
36
38
42
55
54
51
21
37
29
----
30
65
62
55
28
33
62
63
34
39
48
10a 10b
10c
51
10d 10e
CCRF-CEM
HL-60(TB)
K-562
21
----
----
----
----
----
----
----
----
27
88
97
80
96
116
71
37
30
57
80
35
56
34
72
71
86
48
86
18
64
71
83
65
----
28
104
62
64
98
114
59
32
29
48
69
34
36
25
63
61
74
38
75
----
57
48
56
----
----
----
27
----
33
MOLT-4
RPMI-8226
SR
A549/ATCC
EKVX
62
82
60
58
33
----
----
----
21
----
----
----
24
----
----
----
25
----
----
----
----
----
----
25
HOP-62
HOP-92
58
29
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
----
----
58
24
25
25
----
21
----
21
----
----
21
41
25
----
54
----
32
45
30
29
----
----
37
----
----
----
----
----
----
----
----
----
----
----
----
50
----
----
24
----
----
34
40
----
----
53
----
----
75
HT29
----
27
KM12
SW-620
23
----
----
----
SF-268
63
49
23
----
42
----
65
----
23
44
SF-295
SF-539
52
80
40
99
61
83
74
69
71
29
56
24
66
44
70
63
60
52
----
27
----
44
24
36
42
35
22
----
----
----
23
----
42
26
64
38
64
64
61
46
----
----
----
----
----
----
----
----
----
51
69
42
51
68
78
51
54
48
----
28
----
25
38
61
38
43
59
71
42
59
44
SNB-19
----
33
----
----
30
SNB-75
U251
LOX IMVI
MALME-3M
M14
----
21
28
28
----
----
----
----
----
21
20
----
----
MDA-MB-435
SK-MEL-2
SK-MEL-28
----
68
55
57
38
30
----
----
53
32
----
----
73
47
25
24
57
37
----
----
----
16

SK-MEL-5
UACC-257
UACC-62
73
31
69
85
53
63
35
63
----
87
72
92
91
96
93
80
52
81
51
20
44
80
36
46
23
47
----
77
60
87
79
86
56
63
53
77
23
----
29
38
21
----
----
21
----
26
32
60
48
53
40
31
----
42
----
----
----
21
45
21
47
72
27
43
24
37
----
82
56
83
76
82
55
58
50
74
----
----
----
35
152
28
47
63
76
48
21
56
----
36
35
41
100
59
72
67
34
31
31
----
----
36
31
----
----
----
25
54
----
28
59
45
39
28
53
----
----
30
35
29
47
58
54
27
41
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
----
----
----
----
----
21
----
----
----
----
----
----
----
30
20
----
----
25
----
----
25
----
----
----
----
----
----
59
----
----
----
----
----
26
786-0
A498
28
65
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
PC-3
29
----
----
42
35
29
29
----
27
24
24
27
----
25
----
38
----
36
22
68
43
----
----
39
----
68
29
38
62
57
54
52
30
----
----
47
33
----
60
65
72
----
59
40
51
67
68
DU-145
MCF7
----
----
MDA-MB-
231/ATCC
HS 578T
80
42
25
----
43
----
60
37
52
59
95
41
57
67
68
59
64
33
39
48
50
58
33
26
22
----
25
38
21
34
54
25
46
51
46
57
----
----
26
61
44
66
94
61
59
23
----
48
71
22
30
30
35
56
56
22
30
55
26
68
76
50
56
BT-549
T-47D
----
----
14
MDA-MB-468
Mean growth, %
Sensitive cell lines no.
58
12
18
14
a Only GI % higher than 20% are shown.
Regarding sensitivity of individual cell lines (Table 4), only Non-small cell lung cancer (NCI-
H522) and Renal (RXF 393 and UO-31) cancer cell lines were susceptible to all the examined
CAN508 analogues (8a-e and 10a-e), whereas, the Ovarian NCI/ADR-RES cancer cell line
uniquely hasn’t been affected by any of the examined compounds. Notably, sulfonamides 10b
and 10e exerted cytotoxic effect with GI more than 100% toward certain cancer cell lines.
17

Sulfonamide 10b was proved to be lethal to Leukemia RPMI-8226 and Melanoma SK-MEL-5
cell lines (GI % = 116 and 152, respectively), whereas, sulfonamide 10e exerted its lethal activity
toward Leukemia CCRF-CEM cell line with GI %=114, Table 4.
2.2.3.2. In vitro anti-proliferative activity
While, both imidazopyrazoles 8 and pyrazolopyrimidines 10 exhibited significant growth
inhibitory activities in 57 cancer cell lines-NCI screening assay, only the pyrazolopyrimidine
derivatives 10a-e displayed good selectivity towards tumor associated isoform hCA IX over
hCA I (SI: 63.35 – 276.70) and hCA II (SI: 13.47 – 45.31), in addition to their good inhibitory
activities towards both hCA IX and XII isoforms (KIs: of 8.8 – 65.7 and 10.9 – 48.8 nM,
respectively). Accordingly, compounds 10a-e were selected to be evaluated for their anti-
proliferative activity against two breast cancer cell lines overexpressing CA IX, namely MCF-7
and MDA-MB-468 cell lines [34], using MTT assay [35]. Notably, both breast cancer cell lines
were proved to be susceptible to the influence of compounds 10a-e in the NCI screening assay.
In this assay, cobalt (II) chloride (CoCl2) was utilized as an inducer of HIF-1α to furnish a
chemically-induced hypoxia [36] in order to further evaluate the antiproliferative activity of
10a-e under hypoxic conditions. Staurosporine, a clinically used anticancer drug, was co-
assayed as a reference drug. The results are expressed as IC50 values and displayed in Table 5.
Table 5. In vitro anti-proliferative activity of pyrazolopyrimidines 10a-e against breast MCF-7 and
MDA-MB-468 cancer cell lines under normoxic and hypoxic conditions.
IC50 (µM)a
Compound
MCF-7
MDA-MB-468
Normoxia
Normoxia
0.66±0.03
6.80±0.17
2.96±0.06
2.07±0.08
4.77±0.08
3.39±0.13
Hypoxia
0.96±0.04
15.10±0.82
7.73±0.26
10.30±0.75
7.56±0.33
9.20±0.41
Hypoxia
1.07±0.05
3.03±0.11
11.30±0.74
19.61±1.42
10.02±0.84
6.46±0.26
1.89±0.04
1.37±0.07
5.38±0.16
11.44±0.76
6.81±0.28
4.37±0.17
10a
10b
10c
10d
10e
Staurosporine
a. IC50 values are the mean ± S.D. of three separate experiments.
18

Exploring the results of the MTT assay highlighted that the tested sulfonamides 10a-e
showed potent to good inhibitory activity against both cell lines under the tested normoxic and
hypoxic conditions. The tested compounds were more effective toward MCF-7 cells than MDA-
MB-468 cells, except sulfonamide 10b which displayed much enhanced growth inhibitory
activity, under normoxic and hypoxic conditions, against MDA-MB-468 (IC50 = 1.37±0.07 and
3.03±0.11 µM) than MCF-7 cells (IC50 = 6.80±0.17 and 15.10±0.82 µM). Concerning the anti-
proliferative activity towards MCF-7 cells, sulfonamide 10a (IC50 = 0.66±0.03 and 0.96±0.04
µM, under normoxic and hypoxic conditions, respectively) was found to be the most active
analogue in this study, being 5- and 9-times more potent than reference drug staurosporine (IC50
= 3.39±0.13 and 9.20±0.41 µM, under normoxic and hypoxic conditions, respectively). Also,
compounds 10c and 10d displayed higher or comparable anti-proliferative activities to
staurosporine, with IC50 values equal 2.96±0.06 and 2.07±0.08 µM, respectively, for normoxic
condition, and 7.73±0.26 and 10.30±0.75 µM, respectively, for hypoxic condition.
On the other hand, exploring the growth inhibitory activity of sulfonamides 10a-e against
MDA-MB-468 cells revealed that sulfonamides 10a and 10b were the most effective
counterparts (IC50 = 1.89±0.04 and 1.37±0.07 µM, respectively for normoxic condition, and
1.07±0.05 and 3.03±0.11 µM, respectively, for hypoxic condition), with 2.3- and 3.1-fold
enhanced efficiency under normoxic condition, and 6- and 2.1-fold enhanced efficiency under
hypoxic condition, than staurosporine (IC50 = 4.37±0.17 and 6.46±0.26 µM, under normoxic and
hypoxic conditions, respectively).
2.2.3.3. Cell Cycle Analysis
To acquire mechanistic insights into the anti-proliferative activity of the target
sulfonamides, compounds 10a, 10c and 10d were assessed for their impact on the cell cycle
distribution in MCF-7 cells through a DNA flow cytometric assay (Fig. 3). The outcomes of
this flow cytometric assay highlighted that treatment of MCF-7 cells with sulfonamides 10a,
10c and 10d at their IC50 concentrations (0.66, 2.96 and 2.07 µM) induced a significant cell
cycle arrest at G2-M phase and decreased the S-phase populations with subsequent increase in
the percentages apoptotic cells at Sub-G1 phase by 7, 11 and 8-fold compared to the control,
respectively.
19

Figure 3. Effect of sulfonamides 10a, 10c and 10d on the phases of cell cycle of MCF-7 cells.
2.2.3.4. Annexin V-FITC Apoptosis Assay
To investigate whether the anti-proliferative activities of target sulfonamides 10 is consistent
with the apoptosis induction within the breast cancer MCF-7 cells suggested by the presence of
sub-G1 population of treated cells (Fig. 3), Annexin V-FITC/propidium iodide dual staining
assay (AV/PI) was carried out by flow cytometry (Fig. 4, Table 6).
The obtained results suggested that sulfonamides 10a, 10c and 10d have the ability to
persuade the apoptosis in MCF-7 cells as evidenced by the significant increase in the percent of
annexin V-FITC-positive apoptotic cells, including both the early (from 0.47% to 8.46% (10a),
6.58% (10c), and 5.88% (10d)) and late apoptosis (from 0.29% to 16.94% (10a), 10.89% (10c),
and 8.2% (10d)) phases, which represents about 26-, 23- and 18-fold total increase as compared
with the untreated control (Fig. 4, Table 6).
Control
10a
10c
10d
20

Fig. 4. Effect of sulfonamides 10a, 10c and 10d on the percentage of annexin V-FITC-positive
staining in breast cancer MCF-7 cells. The experiments were done in triplicates. The four quadrants
identified as: LL, viable; LR, early apoptotic; UR, late apoptotic; UL, necrotic.
2.2.4. Computational studies
Docking studies were undertaken to investigate the binding mode and point out the
relationships between the inhibition profile and structural features for the most selective
compounds of the series, 10a-e, within hCA II (pdb 5LJT [37]), IX (pdb 5FL4 [38]) and XII (pdb
JLD0 [39]). In all docking solutions, the benzenesulfonamide moiety accommodates deeply into
the active site of the three isozymes (Fig. 5). In particular, the sulfonamide coordinates to the zinc
ion through the negatively charged nitrogen and also engages two H-bonds, occurring between
the NH− and the S=O groups of the ligand and Thr199 (N-H٠٠٠OG1 and S=O٠٠٠H-N). The
placement of phenyl ring is stabilized by hydrophobic interactions with residues Val121, Val143,
and Leu198.
Within the binding site of the tumor-associated CAs, the 5-phenylpyrazolopyrimidine moiety of
the ligands adopts an orientation whereby intense interactions of the hydrophobic type occur
with the pocket lined by Leu91, Gln92, Val121 and Val131 in CA IX (Fig. 5
B) and Thr91,
Gln92, Val121 and Ala131 in CA XII (Fig. 5 ).
C
As it often occurs with bulky and rigid pendants at the benzenesulfonamide scaffold, the
Phe131/Val131 (CA II/IX) and Phe131/Ala131 (CA II/XII) induces a different placement for the
ligand tail within the active site of the ubiquitous isozyme, which prevents strong contacts with
the hydrophobic cleft of the binding cavity and drives the terminal phenyl moiety toward the
hydrophilic area (Fig. 5A). This could explain the 10a-e better efficacy against the cancer-related
CAs over CA II. The 4-substitution of the outer phenyl ring did not influence the heterocycle
binding orientation within the three active sites (Fig. 5).
21

Figure 5: Binding mode of 10a (cyano) and 10c (magenta) within A) hCA II, B) hCA IX and C) hCA XII
active sites.
3. Conclusion
In summary, herein we described the design and synthesis of new sets of novel CAN508
sulfonamide-based analogues (
potential CDK inhibitory activity. A bioisosteric replacement approach was adopted to replace
the H-bond donor phenolic OH of CAN508 with a sulfamoyl group to afford compound
4, 8a-e, 9a-h and 10a-e) as novel carbonic anhydrase (CA) with
4
.
Thereafter, a ring-fusion approach was utilized to furnish the 5/5 fused imidazopyrazoles 8a-e
which subsequently expanded to 6/5 pyrazolopyrimidines 9a-h and 10a-e. All the synthesized
analogues were evaluated for their inhibitory activity towards a panel of CA isoforms hCA I, II,
IX and XII. The target tumor-associated isoforms hCA IX and XII were effectively inhibited
with KIs ranges 6–67.6 and 10.1–88.6 nM, respectively. In particular, sulfonamides 8b
, 9c and
10c were the most potent CA IX inhibitors in this study with KIs in a single-digit nanomolar
h
range; 9.4, 6 and 8.8 nM, respectively. The obtained inhibition profiles ascribed to
pyrazolopyrimidine series 10 excellent selectivity towards hCA IX and XII over hCA I (SIs:
63.35 – 276.70 and 68.10 – 223.39, respectively) and over hCA II (SIs: 13.47 – 45.32 and 13.08
– 36.59, respectively). Furthermore, all compounds were evaluated for their potential CDK2 and
9 inhibitory activities. Pyrazolopyrimidines 9d, 9e and 10b displayed weak CDK2 inhibitory
activity (IC50 = 6.4, 8.0 and 11.6 M, respectively), along with abolished CDK9 inhibitory
µ
activity. On account of their excellent activity and selectivity towards hCA IX and XII,
pyrazolopyrimidines 10 were evaluated for their anti-proliferative activity toward breast cancer
MCF-7 and MDA-MB-468 cell lines under normoxic and hypoxic conditions. As compounds
10a
,
10c and 10d emerged as the most potent anti-proliferative agents against MCF-7 cells, they
22

were selected to be assessed for their impact on the cell cycle distribution and apoptotic potential
in MCF-7 cells. They disrupted the MCF-7 cell cycle via alteration of the Sub-G1 phase and
arrest of G2-M stage, and displayed significant increase in the percent of annexinV-FITC positive
apoptotic cells. Finally, a docking study was carried out to investigate the binding modes and to
justify the selectivity of series 10 toward isoforms hCA IX and XII over hCA II. It was suggested
that the Phe131/Val131 (CA II/IX) and Phe131/Ala131 (CA II/XII) induced a different
placement for the bulky and rigid pyrazolopyrimidine tail within the active site of the ubiquitous
isozyme, which could prevent strong contacts with the hydrophobic cleft of the binding cavity,
and drives the terminal phenyl moiety toward the hydrophilic area.
4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were measured with a Stuart melting point apparatus and were uncorrected.
Infrared spectra were recorded on Schimadzu FT-IR 8400S spectrophotometer. The NMR
spectra were recorded by Bruker spectrometer at 400 MHz. 13C NMR spectra were run at 100
MHz in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts (δH) are reported relative to
the solvent (DMSO-d6). All coupling constant (J) values are given in hertz. Chemical shifts (δC)
are reported relative to the solvent (DMSO-d6). 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. Unless
otherwise noted, all solvents and reagents were commercially available and used without further
purification. Compounds (3) [24, 25], (4) [25, 26], (5a-e) [27, 28], (6a-h) [29, 30] and (7a-e)
[31] were synthesized as reported previously.
4.1.2.
yl)diazenyl)benzenesulfonamide (8a-e
The key intermediate (7 g, 0.02 mol) was added to a preheated, stirred suspension of 5a-e
Synthesis
of
4-((6-amino-2-(4-substitutedphenyl)-1H-imidazo[1,2-b]pyrazol-7-
)
4
(0.05 mol) in absolute ethanol (25 mL) and few drops of DMF. The resulting reaction mixture
was heated under reflux for 12h with stirring. Then reaction mixture was filtered while hot, and
23

the obtained solid was washed with hot ethanol and recrystallized from DMF/ethanol to afford
the target compounds 8a-e
.
4.1.2.1.
8a
4-((6-Amino-2-phenyl-1
H
-imidazo[1,2-b]pyrazol-7-yl)diazenyl)benzenesulfonamide
(
)
∘
Yield (45%); brick red powder; m.p. >300 C; IR: 3402, 3313, 3240, 3174, 3155 (NH, 2NH2),
1
1338, 1153 (SO2); H NMR
δ
ppm: 7.37 (br s, 2H, -SO2NH2), 7.43 (t, 1H, Ar-H,
= 7.6 Hz), 7.76 (br s, 2H, -NH2 of pyrazole), 7.88 (d, 2H, Ar-H,
= 8.0 Hz), 8.01 (d, 2H, Ar-H,
ppm: 110.85, 115.51, 120.29, 125.06, 127.85, 129.21, 129.73, 130.78,
J
= 7.2 Hz),
7.54 (t, 2H, Ar-H,
7.95 (d, 2H, Ar-H,
1H, -NH); 13C NMR
J
J = 8.8 Hz),
J
J = 8.8 Hz), 8.51 (s, 1H, Ar-H), 11.97 (br s,
δ
138.45, 138.76, 145.32, 153.86, 162.14; MS m/z %: 381.4 (M+, 19.12); Anal. Calcd. for
C17H15N7O2S: C, 53.53; H, 3.96; N, 25.71; found C, 53.71; H, 3.92; N, 25.75.
4.1.2.2.
yl)diazenyl)benzenesulfonamide (8b
Yield (39%); brick red powder; m.p. >300 C; IR: 3390, 3309, 3240, 3174 (NH, 2NH2), 1334,
1153 (SO2); 1H NMR
ppm: 2.36 (s, 3H, CH3), 7.34-7.36 (m, 4H, 2H, -SO2NH2 and 2H, Ar-H),
7.75 (br s, 2H, -NH2 of pyrazole), 7.82 (d, 2H, Ar-H, = 8.0 Hz), 7.88 (d, 2H, Ar-H, = 8.4 Hz),
8.01 (d, 2H, Ar-H, ppm
= 8.4 Hz), 8.44 (s, 1H, Ar-H), 11.86 (br s, 1H, -NH); 13C NMR
4-((6-Amino-2-(4-methylphenyl)-1H-imidazo[1,2-b]pyrazol-7-
)
∘
δ
J
J
J
δ
:
21.74 (CH3), 110.67, 115.24, 121.11, 124.98, 127.09, 128.65, 129.94, 132.39, 142.14, 145.05,
146.15, 155.66, 162.80; MS m/z %: 395.3 (M+, 26.57); Anal. Calcd. for C18H17N7O2S: C, 54.67;
H, 4.33; N, 24.79; found C, 55.01; H, 4.29; N, 24.88.
4.1.2.3.
4-((6-Amino-2-(4-fluorophenyl)-1H-imidazo[1,2-b]pyrazol-7-
yl)diazenyl)benzenesulfonamide (8c
)
∘
Yield (40%); brick red powder; m.p. >300 C; IR: 3417, 3305, 3217, 3174 (NH, 2NH2), 1323,
1
1153 (SO2); H NMR
δ
ppm: 7.39-7.41 (m, 4H, 2H, -SO2NH2 and 2H, Ar-H), 7.75 (br s, 2H, -
= 7.6 Hz), 7.97-8.05 (m, 4H, Ar-H), 8.47 (s, 1H, Ar-H),
ppm: 111.39, 116.05, 116.81 (2JF-C = 21 Hz), 119.12, 125.15,
NH2 of pyrazole), 7.88 (d, 2H, Ar-H,
11.95 (br s, 1H, -NH); 13C NMR
J
δ
127.65, 127.69 (3JF-C = 8 Hz), 128.86, 135.33, 140.02, 145.36, 162.71, 162.79 (1JF-C = 246 Hz);
MS m/z %: 399.7 (M+, 29.37); Anal. Calcd. for C17H14FN7O2S: C, 51.12; H, 3.53; N, 24.55;
found C, 50.84; H, 3.48; N, 24.65.
24

4.1.2.4.
4-((6-Amino-2-(4-chlorophenyl)-1H-imidazo[1,2-b]pyrazol-7-
yl)diazenyl)benzenesulfonamide (8d
)
∘
Yield (43%); brick red powder; m.p. >300 C; IR: 3448, 3410, 3352, 3259, 3170 (NH, 2NH2),
1
1319, 1153 (SO2); H NMR
δ
ppm: 7.36 (br s, 2H, -SO2NH2), 7.60-7.74 (m, 4H, 2H, Ar-H and
= 8.4 Hz), 7.95-8.00 (m, 4H, Ar-H), 8.48 (s, 1H, Ar-
2H, -NH2 of pyrazole), 7.87 (d, 2H, Ar-H,
J
H), 11.89 (br s, 1H, -NH); MS m/z %: 417.8 (M++2, 13.88), 415.8 (M+, 39.41); Anal. Calcd. for
C17H14ClN7O2S: C, 49.10; H, 3.39; N, 23.58; found C, 48.91; H, 3.41; N, 23.54.
4.1.2.5.
4-((6-Amino-2-(4-bromophenyl)-1H-imidazo[1,2-b]pyrazol-7-
yl)diazenyl)benzenesulfonamide (8e
)
∘
Yield (40%); brick red powder; m.p. >300 C; IR: 3344, 3255, 3167, 3086 (NH, 2NH2), 1330,
1
1153 (SO2); H NMR
δ
ppm: 7.36 (br s, 2H, -SO2NH2), 7.73-7.79 (m, 4H, 2H, Ar-H and 2H, -
= 8.0 Hz), 8.50 (s, 1H, Ar-H),
NH2 of pyrazole), 7.87-7.91 (m, 4H, Ar-H), 7.99 (d, 2H, Ar-H,
J
11.88 (br s, 1H, -NH); MS m/z %: 462.3 (M++2, 34.01), 460.3 (M+, 31.26); Anal. Calcd. for
C17H14BrN7O2S: C, 44.36; H, 3.07; N, 21.30; found C, 44.65; H, 3.11; N, 21.20.
4.1.3.
Synthesis
of4-((2-amino-7-(4-substitutedphenyl)pyrazolo[1,5-a]pyrimidin-3-
yl)diazenyl)benzenesulfonamide (9a-h)
A mixture of
heated under reflux for 1h. The formed precipitate was filtered while hot, washed with cold
ethanol and water, and recrystallized from dioxane to furnish target sulfonamides 9a-h
4 (1.4 g, 0.005 mol) and 6a-h (0.005mol) in glacial acetic acid (5 mL) was
.
4.1.3.1. 4-((2-Amino-7-phenylpyrazolo[1,5-a]pyrimidin-3-yl)diazenyl)benzenesulfonamide (9a)
Yield (58%); yellow powder; m.p. 298-300 ∘C; IR: 3437, 3352, 3224, 3101 (2NH2), 1300, 1149
1
(SO2); H NMR
δ
ppm: 7.31 (d, 1H, Ar-H,
J
= 4.8 Hz), 7.42-7.43 (m, 4H, 2H, -NH2 and 2H, -
= 8.0 Hz),
= 4.8 Hz); MS m/z %: 393.3 (M+, 43.43); Anal. Calcd. for C18H15N7O2S: C,
54.95; H, 3.84; N, 24.92; found C, 55.16; H, 3.81; N, 24.84.
SO2NH2), 7.60-7.63 (m, 3H, Ar-H), 7.95-8.00 (m, 4H, Ar-H), 8.07 (d, 2H, Ar-H,
8.63 (d, 1H, Ar-H,
J
J
4.1.3.2.
4-((2-Amino-7-(4-methylphenyl)pyrazolo[1,5-a]pyrimidin-3-
yl)diazenyl)benzenesulfonamide (9b
)
Yield (69%); yellow powder; m.p. 297-299 ∘C; IR: 3417, 3255, 3186, 3143 (2NH2), 1330, 1149
(SO2); 1H NMR
δ
ppm: 2.43 (s, 3H, CH3), 7.32 (d, 1H, Ar-H, J = 4.8 Hz), 7.41-7.43 (m, 6H, 2H,
25

Ar-H, 2H, -NH2 and 2H, -SO2NH2), 7.93-7.99 (m, 4H, Ar-H), 8.02 (d, 2H, Ar-H,
J = 8.0 Hz),
13
8.62 (d, 1H, Ar-H,
J
= 4.8 Hz); C NMR
δ
ppm: 21.57 (CH3), 110.18, 116.13, 121.69, 127.32,
127.73, 129.50, 130.02, 141.82, 143.34, 145.89, 148.66, 151.40, 152.33, 155.41; HRMS (ESI)
for C 9H
8O2N7³²S, calcd 408.12372, found 408.12357 [M+H]+.
₁
₁
4.1.3.3.
yl)diazenyl)benzenesulfonamide (9c
4-((2-Amino-7-(4-fluorophenyl)pyrazolo[1,5-a]pyrimidin-3-
)
∘
Yield (55%); yellow powder; m.p. >300 C; IR: 3437, 3379, 3259, 3186 (2NH2), 1334, 1149
1
(SO2); H NMR
δ
ppm: 7.32 (d, 1H, Ar-H,
J
= 4.8 Hz), 7.42-7.49 (m, 6H, 2H, Ar-H, 2H, -NH2
= 8.0 Hz, = 4.0 Hz), 8.63
ppm: 110.40, 116.04 (2JF-C = 22 Hz), 121.72, 127.04,
127.33, 132.79 (3JF-C = 9 Hz), 143.38, 144.84, 148.57, 151.43, 152.36, 155.37, 164.01 (1JF-C
and 2H, -SO2NH2), 7.94-8.00 (m, 4H, Ar-H), 8.17 (dd, 2H, Ar-H,
(d, 1H, Ar-H,
= 4.4 Hz); 13C NMR
J
J
J
δ
=
248 Hz), 172.49; MS m/z %: 411.4 (M+, 14.04); Anal. Calcd. for C18H14FN7O2S: C, 52.55; H,
3.43; N, 23.83; found C, 52.37; H, 3.49; N, 23.76.
4.1.3.4.
4-((2-Amino-7-(4-chlorophenyl)pyrazolo[1,5-a]pyrimidin-3-
yl)diazenyl)benzenesulfonamide (9d
)
∘
Yield (64%); yellow powder; m.p. >300 C; IR: 3417, 3336, 3259, 3186 (2NH2), 1330, 1153
1
(SO2); H NMR
δ
ppm: 7.36 (d, 1H, Ar-H,
= 8.4 Hz), 7.93 (d, 2H, Ar-H,
= 8.8 Hz), 8.65 (d, 1H, Ar-H,
= 4.8 Hz); 13C NMR
116.12, 121.73, 127.33, 129.05, 129.46, 131.98, 136.43, 143.43, 144.67, 148.52, 151.50, 152.37,
J
= 4.8 Hz), 7.40-7.42 (m, 4H, 2H, -NH2 and 2H, -
= 8.0 Hz), 7.97 (d, 2H, Ar-H,
ppm: 110.46,
SO2NH2), 7.70 (d, 2H, Ar-H,
8.0 Hz), 8.13 (d, 2H, Ar-H,
J
J
J =
J
J
δ
155.35; HRMS (ESI) for C₁8H₁
5O2N7ClS, calcd 428.06910, found 428.06914 [M+H]+.
4.1.3.5.
4-((2-Amino-7-(4-bromophenyl)pyrazolo[1,5-a]pyrimidin-3-
yl)diazenyl)benzenesulfonamide (9e
)
∘
Yield (61%); yellow powder; m.p. >300 C; IR: 3417, 3340, 3255, 3186 (2NH2), 1330, 1153
1
(SO2); H NMR
δ
ppm: 7.33 (d, 1H, Ar-H,
= 8.4 Hz), 7.94-8.00 (m, 4H, Ar-H), 8.04 (d, 2H, Ar-H,
= 4.4 Hz); MS m/z %: 474.2 (M++2, 21.56), 472.2 (M+, 20.94); Anal.
J
= 4.8 Hz), 7.42 (br s, 4H, 2H, -NH2 and 2H, -
SO2NH2), 7.82 (d, 2H, Ar-H,
Hz), 8.64 (d, 1H, Ar-H,
J
J
= 8.8
J
Calcd. for C18H14BrN7O2S: C, 45.77; H, 2.99; N, 20.76; found C, 45.55; H, 2.97; N, 20.78.
26

4.1.3.6.
yl)diazenyl)benzenesulfonamide (9f
Yield (65%); yellow powder; m.p. >300 C; IR: 3425, 3305, 3244, 3186 (2NH2), 1327, 1149
(SO2); 1H NMR
ppm: 3.87 (s, 3H, OCH3), 7.13 (d, 2H, Ar-H, = 8.8 Hz), 7.30 (d, 1H, Ar-H,
= 4.8 Hz), 7.43 (br s, 4H, 2H, -NH2 and 2H, -SO2NH2), 7.94-7.99 (m, 4H, Ar-H), 8.15 (d, 2H,
Ar-H, = 8.8 Hz), 8.58 (d, 1H, Ar-H, ppm: 55.98 (OCH3), 109.75,
= 4.4 Hz); 13C NMR
4-((2-Amino-7-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-
)
∘
δ
J
J
J
J
δ
114.36, 116.11, 121.68, 122.51, 127.32, 131.98, 143.27, 145.53, 148.76, 151.21, 152.29, 155.43,
162.01; MS m/z %: 423.3 (M+, 15.67); Anal. Calcd. for C19H17N7O3S: C, 53.89; H, 4.05; N,
23.15; found C, 54.21; H, 4.12; N, 23.05.
4.1.3.7.
4-((2-Amino-7-(4-nitrophenyl)pyrazolo[1,5-a]pyrimidin-3-
yl)diazenyl)benzenesulfonamide (9g
)
∘
Yield (63%); orange powder; m.p. >300 C; IR : 3417, 3383, 3271, 3190 (2NH2), 1330, 1149
(SO2); 1H NMR
δ ppm: 7.41-7.45 (m, 5H, 1H, Ar-H, 2H, -NH2 and 2H, -SO2NH2), 7.93 (d, 2H,
Ar-H,
Ar-H,
J
J
= 8.4 Hz), 7.98 (d, 2H, Ar-H,
= 8.8 Hz), 8.70 (d, 1H, Ar-H,
J
J
= 8.8 Hz), 8.32 (d, 2H, Ar-H,
= 4.8 Hz); MS m/z %: 438.0 (M+, 30.37); Anal. Calcd.
J = 8.8 Hz), 8.44 (d, 2H,
for C18H14N8O4S: C, 49.31; H, 3.22; N, 25.56; found C, 49.71; H, 3.26; N, 25.65.
4.1.3.8.
4-((2-Amino-7-(thiophen-2-yl)pyrazolo[1,5-a]pyrimidin-3-
yl)diazenyl)benzenesulfonamide (9h
)
∘
Yield (60%); orange powder; m.p. >300 C; IR: 3417, 3321, 3236, 3116 (2NH2), 1319, 1145
(SO2); 1H NMR
1H, Ar-H, = 5.2 Hz), 7.95-8.00 (m, 4H, Ar-H), 8.17 (d, 1H, Ar-H,
δ
ppm: 7.39-7.43 (m, 3H, 1H, Ar-H, 2H, -NH2), 7.57 (s, 2H, -SO2NH2), 7.81 (d,
J
J
= 4.4 Hz), 8.54-8.58 (m,
2H, Ar-H); MS m/z %: 399.4 (M+, 13.60); Anal. Calcd. for C16H13N7O2S2: C, 48.11; H, 3.28; N,
24.55; found C, 48.48; H, 3.24; N, 24.64.
4.1.4. Synthesis of 4-((2,7-diamino-5-(4-substitutedphenyl)-6-cyanopyrazolo[1,5-a]pyrimidin-3-
yl)diazenyl)benzenesulfonamide (10a-e
)
A suspension of the key intermediate
4
(1.4 g, 0.005 mol) in dry DMF (30 mL) was added to
7a-e (0.005 mol), then heated under reflux for 8-10h. Then, the solvent was evaporated under
vacuum and the residue was collected, washed with water, dried and crystallized from
acetonitrile to afford the target sulfonamides 10a-e
.
27

4.1.4.1.
4-((2,7-Diamino-6-cyano-5-phenylpyrazolo[1,5-a]pyrimidin-3-
yl)diazenyl)benzenesulfonamide (10a
)
0
∘
Yield ( 65%); yellow powder, m.p. 298-300 C (reported m.p: 298 C, [26]); IR: 3444, 3394,
1
3332, 3240, 3066 (3NH2), 2214 (CN), 1361, 1149 (SO2); H NMR
δ ppm: 7.25 (s, 2H, -NH2),
7.44 (s, 2H, -SO2NH2), 7.60 (br s, 3H, Ar-H), 7.89-7.98 (m, 6H, Ar-H), 8.68 (br s, 2H, -NH2);
Anal. Calcd. for C19H15N9O2S: C, 52.65; H, 3.49; N, 29.08; found C, 53.00; H, 3.52; N, 29.18.
4.1.4.2.
4-((2,7-Diamino-5-(4-methylphenyl)-6-cyanopyrazolo[1,5-a]pyrimidin-3-
yl)diazenyl)benzenesulfonamide (10b
)
Yield (67%); yellow powder; m.p. 295-297 ∘C; IR: 3429, 3406, 3298, 3224, 3167, (3NH2), 2222
1
(CN), 1334, 1153 (SO2); H NMR
δ
ppm: 2.43 (s, 3H, CH3), 7.24 (s, 2H, -NH2), 7.38-7.43 (m,
= 8.0 Hz), 7.95 (br s, 4H, Ar-H), 8.64 (br s,
ppm: 21.48 (CH3), 116.53, 117.90, 121.88, 127.33, 129.13, 129.37,
4H, 2H, Ar-H, 2H, -SO2NH2), 7.80 (d, 2H, Ar-H,
2H, -NH2); 13C NMR
J
δ
134.76, 140.81, 143.70, 147.13, 149.38, 152.52, 155.11, 161.79, 162.79; MS m/z %: 447.66 (M+,
57.3); Anal. Calcd. for C20H17N9O2S: C, 53.68; H, 3.83; N, 28.17; found C, 53.98; H, 3.77; N,
28.27.
4.1.4.3.
4-((2,7-Diamino-5-(4-fluorophenyl)-6-cyanopyrazolo[1,5-a]pyrimidin-3-
yl)diazenyl)benzenesulfonamide (10c
)
∘
Yield (59%); yellow powder; m.p. >300 C; IR: 3444, 3332, 3236 (3NH2), 2214 (CN), 1338,
1
1149 (SO2); H NMR
δ ppm: 7.24 (s, 2H, -NH2), 7.41 (s, 2H, -SO2NH2), 7.94-7.97 (m, 8H, Ar-
H), 8.68 (br s, 2H, -NH2); HRMS (ESI) for C₁9H₁5O2N9F³²S, calcd 452.10480, found 452.10482
[M+H]+.
4.1.4.4.
4-((2,7-Diamino-5-(4-chlorophenyl)-6-cyanopyrazolo[1,5-a]pyrimidin-3-
yl)diazenyl)benzenesulfonamide (10d
)
Yield (63 %); yellow powder; m.p. >300 ∘C (reported m.p: >3000C [26]); IR: 3433, 3406, 3352,
1
3298, 3228, 3174 (3NH2), 2222 (CN), 1338, 1153 (SO2); H NMR
δ ppm: 7.25 (s, 2H, -NH2),
7.44 (s, 2H, -SO2NH2), 7.65 (d, 2H, Ar-H,
J = 8.4 Hz), 7.91-7.96 (m, 6H, Ar-H), 8.71 (br s, 2H, -
NH2); 13C NMR
δ
ppm: 116.32, 117.95, 121.92, 127.33, 128.95, 131.03, 135.74, 136.36, 143.79,
147.94, 149.28, 152.54, 155.06, 160.59, 162.79; MS m/z %: 469.20 (M++2, 17.85), 467.2 (M+,
55.65); Anal. Calcd. for C19H14ClN9O2S: C, 48.77; H, 3.02; N, 26.94; found C, 49.11; H, 2.96;
N, 26.86.
28