Synthesis, biological evaluation, and docking studies of novel pyrrolo[2,3-b]pyridine derivatives as both ectonucleotide pyrophosphatase/phosphodiesterase inhibitors and antiproliferative agents

Article abstract of DOI:10.1016/j.ejmech.2021.113339

Ecto-nucleotide pyrophosphatases/phosphodiesterases (NPPs) together with nucleoside triphosphate diphosphohydrolases (NTPDases) and alkaline phosphatases (APs) are nucleotidases located at the surface of the cells. NPP1 and NPP3 are important members of NPP family that are known as druggable targets for a number of disorders such as impaired calcification, type 2 diabetes, and cancer. Sulfonylurea derivatives have been reported as antidiabetic and anticancer agents, therefore, we synthesized and investigated series of sulfonylurea derivatives 1a-m possessing pyrrolo[2,3-b]pyridine core as inhibitors of NPP1 and NPP3 isozymes that are over-expressed in cancer and diabetes. The enzymatic evaluation highlighted compound 1a as selective NPP1 inhibitor, however, 1c was observed as the most potent inhibitor of NPP1 with an IC₅₀ value of 0.80 ± 0.04 μM. Compound 1l was found to be the most potent and moderately selective inhibitor of NPP3 (IC₅₀ = 0.55 ± 0.01 μM). Furthermore, in vitro cytotoxicity assays of compounds 1a-m against MCF-7 and HT-29 cancer cell lines exhibited compound 1c (IC₅₀ = 4.70 ± 0.67 μM), and 1h (IC₅₀ = 1.58 ± 0.20 μM) as the most cytotoxic compounds against MCF-7 and HT-29 cancer cell lines, respectively. Both of the investigated compounds showed high degree of selectivity towards cancer cells than normal cells (WI-38). Molecular docking studies of selective and potent enzyme inhibitors revealed promising mode of interactions with important binding sites residues of both isozymes i.e., Thr256, His380, Lys255, Asn277 residues of NPP1 and His329, Thr205, and Leu239 residues of NPP3. In addition, the most potent antiproliferative agent, compound 1h, doesn't produce hypoglycemia as a side effect when injected to mice. This is an additional merit of the promising compound 1h.

Full text of DOI:10.1016/j.ejmech.2021.113339

European Journal of Medicinal Chemistry 217 (2021) 113339
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis, biological evaluation, and docking studies of novel pyrrolo
[2,3-b]pyridine derivatives as both ectonucleotide pyrophosphatase/
phosphodiesterase inhibitors and antiproliferative agents
a
b
,
c
,
d
,
1
e
f
Saif Ullah , Mohammed I. El-Gamal
, Randa El-Gamal , Julie Pelletier ,
Jean S eꢀ vigny , Mahmoud K. Shehata , Hanan S. Anbar , Jamshed Iqbal a
Centre for Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus, Abbottabad, 22060, Pakistan
Department of Medicinal Chemistry, College of Pharmacy, University of Sharjah, Sharjah, 27272, United Arab Emirates
Sharjah Institute for Medical Research, University of Sharjah, Sharjah, 27272, United Arab Emirates
Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura, 35516, Egypt
Department of Medical Biochemistry, Faculty of Medicine, University of Mansoura, Mansoura, 35516, Egypt
Centre de Recherche du CHU de Qu ꢀe bec e Universit ꢀe Laval, Qu ꢀe bec, QC, G1V 4G2, Canada
D ꢀe partement de microbiologie-infectiologie et d’immunologie, Facult ꢀe de M ꢀe decine, Universit ꢀe Laval, Qu ꢀe bec, QC, G1V 0A6, Canada
f
,
g
c
h
,
*
, **
a
b
c
d
e
f
g
h
Department of Clinical Pharmacy and Pharmacotherapeutics, Dubai Pharmacy College for Girls, Dubai, 19099, United Arab Emirates
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 January 2021
Received in revised form
Ecto-nucleotide pyrophosphatases/phosphodiesterases (NPPs) together with nucleoside triphosphate
diphosphohydrolases (NTPDases) and alkaline phosphatases (APs) are nucleotidases located at the sur-
face of the cells. NPP1 and NPP3 are important members of NPP family that are known as druggable
targets for a number of disorders such as impaired calcification, type 2 diabetes, and cancer. Sulfonylurea
derivatives have been reported as antidiabetic and anticancer agents, therefore, we synthesized and
investigated series of sulfonylurea derivatives 1a-m possessing pyrrolo[2,3-b]pyridine core as inhibitors
of NPP1 and NPP3 isozymes that are over-expressed in cancer and diabetes. The enzymatic evaluation
highlighted compound 1a as selective NPP1 inhibitor, however, 1c was observed as the most potent
25 February 2021
Accepted 26 February 2021
Available online 10 March 2021
Keywords:
Antiproliferative activity
Molecular docking studies
NPP1 and NPP3
Pyrrolo[2,3-b]pyridine scaffold
Sulfonylurea
inhibitor of NPP1 with an IC50 value of 0.80 ± 0.04
and moderately selective inhibitor of NPP3 (IC50 ¼ 0.55 ± 0.01
assays of compounds 1a-m against MCF-7 and HT-29 cancer cell lines exhibited compound 1c
(IC50 ¼ 4.70 ± 0.67 M), and 1h (IC50 ¼ 1.58 ± 0.20 M) as the most cytotoxic compounds against MCF-7
m
M. Compound 1l was found to be the most potent
m
M). Furthermore, in vitro cytotoxicity
m
m
and HT-29 cancer cell lines, respectively. Both of the investigated compounds showed high degree of
selectivity towards cancer cells than normal cells (WI-38). Molecular docking studies of selective and
potent enzyme inhibitors revealed promising mode of interactions with important binding sites residues
of both isozymes i.e., Thr256, His380, Lys255, Asn277 residues of NPP1 and His329, Thr205, and Leu239
residues of NPP3. In addition, the most potent antiproliferative agent, compound 1h, doesn’t produce
hypoglycemia as a side effect when injected to mice. This is an additional merit of the promising
compound 1h.
©
2021 Elsevier Masson SAS. All rights reserved.
1
. Introduction
extracellularly located nucleotidases are ectonucleotide pyrophos-
phatase/phosphodiesterases (NPPs), and nucleoside triphosphate
diphosphohydrolases (NTPDases) [1]. NPPs exist as membrane-
bound glycoproteins (NPP1 & NPP3) and as soluble proteins
The prominent ectonucleotidases involved in the hydrolysis of
(NPP2). The plasma membrane-bound proteins hydrolyze the ter-
*
Corresponding author.
minal phosphate of nucleotides through an extracellularly located
active site [2]. Both of NPP1 and NPP3 act on a wide range of sub-
strates, involving adenosine triphosphate (ATP), adenosine
diphosphate (ADP), phosphoadenylate sulfate, nucleotide-based
** Corresponding author.
E-mail addresses: dr.hanan@dpc.edu (H.S. Anbar), drjamshed@cuiatd.edu.pk
(
J. Iqbal).
1
Co-first author.
https://doi.org/10.1016/j.ejmech.2021.113339
223-5234/© 2021 Elsevier Masson SAS. All rights reserved.
0

S. Ullah, M.I. El-Gamal, R. El-Gamal et al.
European Journal of Medicinal Chemistry 217 (2021) 113339
sugars (UDP-glucoside) and dinucleoside polyphosphates (AP3A,
AP4A), to their respective nucleoside monophosphates [3,4].
Expression of NPP1 is found in adipose tissues, bone, heart,
placenta, testes, and liver [5]. The physiological role of NPP1 ap-
pears in the mineralization of bone, and cell calcification of vascular
smooth muscles [6]. Over-expression of NPP1 leads to hyper-
mineralization disorders such as deposition of calcium pyrophos-
phate dihydrate (CPPD), and calcific aortic valve disease [7e10]. The
overexpression of NPP1 leads to insulin resistance because of in-
agent for the treatment or prevention of the diseases associated
with undesirable cytokine signal transduction [19]. Another
important derivative of pyrrolopyridine bearing pyradazinone core
(N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)1-(4-chloro-3-
(trifluoromethyl)phenyl)-4-oxo-1,4-dihydropyridazine-3-
carboxamide) was reported as a potent inhibitor of c-Met kinase
inhibitor (compound B, Fig. 1) [20]. A series of pyrrolopyridine
derivatives showed significant affinity for cannabinoid receptors
CB
quinazoline-4-piperidine sulfamide analogues inhibited the hy-
drolytic activity of NPP1 with K value of 105 nM (compound D,
1 2
/CB (compound C, Fig. 1) [21]. Moreover, a novel series of
hibition of tyrosine kinase activity of
11]. Elevated expression of NPP1 is reported in rat C6 glioma cells,
human stem-like cells glioblastoma, and astrocytic brain tumor
12,13]. In a recent study on small hairpin RNA knockdown of NPP1
expression decreases the cell proliferation and forces the cell death
14].
The second important isozyme NPP3 is mainly expressed on
b-subunit of insulin receptor
[
i
Fig. 1) [22]. Also in our very recent study, we reported pyridine-
pyrazole thioureas and pyridine-pyrazole sulfonamides as highly
[
potent inhibitors of NPP1 and NPP3 with IC50 values of 0.18
mM, and
[
0.21 M, respectively (compounds E and F, Fig. 1). These inhibitors
m
of NPP1 and NPP3 also exhibited antiproliferative activity against
different cancer cell lines [23].
basophils, mast cells, mucosal, and epithelial surfaces. The NPP3
expression is found to be up-regulated due to release of inflam-
matory mediators as a result of basophils activation by antigen-
bound IgE antibody, therefore, NPP3 can act as diagnostic marker
of allergen sensitivity [15,16]. Moreover, NPP3 is reported as tumor
marker because its overexpression is involved in carcinogenesis
and metastasis of tumor cells [8,17].
Pyrrolopyridine-based derivatives have widely been reported
for their medicinal and therapeutic effects. A pyrrolopyridine de-
rivative (BMS-911543) has been reported as potent inhibitor of Ja-
nus kinase 2 (JAK2) (compound A, Fig. 1) [18]. A condensed
structure of pyrrolopyridine derivative was reported to be associ-
ated with lowering the level of JAK3 protein, acting as an important
Another well-known drug bearing the similar pharmacophore is
Sulofenur (diarylsulfonylurea) that was reported as anti-
proliferative agent. However, it suffers from two drawbacks, first, it
induces hypoglycemia as a side effect due to the presence of dia-
rylurea scaffold. The second drawback is it causes hematological
side effects due to formation of aniline derivative as a metabolite
[24e26]. In our target compounds, they were designed to possess
pyrrolopyridine nucleus as an attempt to avoid formation of aniline
metabolite. In addition, the sulfonylurea linker was retained but
embedded into the bicyclic ring (Fig. 1). The inhibitory effects of
sulfonyl-possessing compounds (such as compounds D-F, Fig. 1)
against NPP1 or NPP3 enzymes encouraged us to test our target
Fig. 1. Structures of reported therapeutically active pyrrolopyridine derivatives, sulfonyl/thiourea-based NPP inhibitors, and the target compounds 1a-m.
2

S. Ullah, M.I. El-Gamal, R. El-Gamal et al.
European Journal of Medicinal Chemistry 217 (2021) 113339
compounds against NPP1 and NPP3. Moreover, investigation of the
effect on blood glucose level was a part of our plans, hopefully, we
can avoid this hypoglycemia issue that occurs with sulofenur.
Thus, considering the medicinal importance of fused pyrrolo-
pyridine scaffold and sulfonamide (part of sulfonylurea moiety), we
designed a series of novel structures possessing both pyrrolopyr-
idine nucleus and sulfonylurea groups. We evaluated the newly
synthesized compounds against NPP1 and NPP3 activity and their
chemotherapeutic effects against cancer cell lines (MCF-7 and HT-
2.2. Biological evaluation
2.2.1. Enzymatic assay for NPP1 and NPP3
The inhibitory effect of the target compounds on the activity of
NPP1 and NPP3 was examined by using the already reported
method with minor modifications [23,29]. The inhibitory potential
of newly synthesized derivatives were measured by the decrease of
the hydrolysis of an artificial substrate (thymidine 5ʹ-mono-
phosphate para-nitrophenyl ester (pNP-TMP)), as shown in Table 1.
2
9). Moreover, with the help of enzyme kinetics study we deter-
mined the mode of inhibition for compounds 1c and 1l.The mo-
lecular docking studies were performed to justify the putative
binding mode of inhibitorswith amino acids residues for the most
promising compounds. Furthermore, the most potent anti-
proliferative agent was tested for possible effect on blood sugar
level in mice.
2
.2.1.1. Structure-activity relationship. Comparison of structure-
activity relationship (SAR) of 1a, 1f, 1j, and 1l revealed that struc-
tural variation at position 4 at the pyrrolopyridine core with chlo-
rine was much favorable for inhibition of isoenzyme NPP1 with an
IC50 value of 1.08 ± 0.01
dimethylamino group yielded compound 1l that was considered
among the active inhibitors of NPP3 (IC50 ¼ 0.55 ± 0.01 M). The
simplest structure 1a selectively inhibited the activity of NPP1 to an
IC50 value of 4.50 ± 0.16 M, and moderately affected the activity of
mM whereas, introduction of 4-
m
2
. Results and discussion
m
NPP3 to only 37.5%. The SAR of 1a and 1l assumed that non-
substituted or chloro-substituted structures were much tolerable
for NPP1 inhibition. Introduction of chlorine at para position on
benzenesulfonylurea moiety retained the activity towards NPP3
2
.1. Chemistry
We could successfully synthesize the target molecules 1a-m via
the synthetic pathway shown in Scheme 1. Upon reaction of aryl-
sulfonyl chloride reagents 2a-e with ammonium hydroxide (3), the
corresponding aryl sulfonamides 4a-e were formed [27]. On
another side, fusion of 4-chloro-7-azaindole (5c) with dimethy-
that was IC50 ¼ 0.88 ± 0.01
mM, suggested that for inhibition of
NPP3, chloro substitution was favorable at benzenesulfonylurea
moiety instead of pyrrolopyridine core. SAR study of compound 1c
showed that presence of strong electron-withdrawing group
ꢀ
lammonium chloride at 180 C for 5 h led to formation of 4-
(-NO
(IC50 ¼ 0.80 ± 0.04
with an IC50 value of 1.17 ± 0.07
2
) rendered the selectivity of 1c and activity of NPP1
M) vs. NPP3 which was moderately inhibited
M. The inhibitor 1g with bromine
(
dimethylamino)-7-azaindole (7) [28]. Interaction of compounds
a-c or 7 with phenyl chloroformate (8) in presence of triethyl-
amine (TEA) produced the corresponding carbamate intermediates
a-d. In the final step, reaction of the synthesized sulfonamide
m
5
m
at position 5 of pyrrolopyridine core, proved as dual inhibitor of
both isoenzymes with moderate activity as compared to compound
1c. On the contrary to 1b, presence of electron-donating group
9
reagents 4a-e with carbamates 9a-d in presence of potassium
carbonate yielded the target sulfonylurea products 1a-m.
(-CH
3
) at para position of benzenesulfonylurea moiety (compound
ꢀ
ꢀ
Scheme 1. Reagents and reaction conditions: (i) MeOH, rt, 5e10 min; (ii) 180 C (fusion), 5 h; (iii) TEA, THF, 0 C, 2 h; (iv) K
2
CO
3
, CH
2
Cl
2
, rt, overnight.
3

S. Ullah, M.I. El-Gamal, R. El-Gamal et al.
European Journal of Medicinal Chemistry 217 (2021) 113339
Table 1
Table 1 (continued )
Illustration of inhibitory potential of the compounds 1a-m and suramin against
NPP1 and NPP3 isozymes.
Compound No.
Chemical structure
IC50 ± S.E.M. in mM or %
inhibition at 100
mM
Compound No.
Chemical structure
IC50 ± S.E.M. in mM or %
NPP1
NPP3
inhibition at 100
mM
1
1
l
2.77 ± 0.09
0.55 ± 0.01
NPP1
NPP3
37.5%
1
1
1
1
1
a
b
c
4.50 ± 0.16
m
41.5%
45.7%
10.36 ± 0.03
0.80 ± 0.04
47.9%
0.88 ± 0.01
1.17 ± 0.07
3.72 ± 0.17
44.1%
Suramin
7.77 ± 0.02
0.89 ± 0.16
The results are expressed as means of triplicate assays ± standard error of mean
S.E.M.).
(
d
e
1d) decreased the activity of both isoenzymes to a considerable
level i.e., only 47.9% for NPP1 and IC50 ¼ 3.72 ± 0.17 M for NPP3.
m
Although minor structural variation of 1d at pyrrolopyridine core
with bromine (1h), re-established the inhibitory potential of both
isoenzymes but substitution of dimethylamino group at pyrrolo-
pyridine scaffold (1m) showed limited effect on NPP1 and NPP3
activity merely by 41.5% and 45.7%, respectively. The presence of
naphthalenesulfonylurea motif in compounds 1e, 1i, and 1k pro-
duced interesting results. Compound 1e with simple unsubstituted
pyrrolopyridine core was devoid of inhibitory potential on both
isoenzymes i.e., below 50% inhibition, whereas introduction of
bromine at position 5 to pyrrolopyridine scaffold (1i) moderately
increased the activity and this inhibitory effect was boosted up in
the presence of chlorine. Comparison of SAR of structurally related
compounds 1e, 1i, 1d provoked that moving to substitution of more
electronegative atom in the presence of naphthalenesulfonylurea
moiety was much tolerated for inhibition of both NPP1 and NPP3
activity as presented in Table 1. The SAR of investigated series of
compounds against NPP1 and NPP3 is summarized in Fig. 2.
48.6%
1
1
1
1
f
8.30 ± 0.02
1.15 ± 0.06
1.55 ± 0.01
177.50 ± 4.66
2.01 ± 0.01
2.13 ± 0.05
0.59 ± 0.01
25.31 ± 0.66
g
h
i
2
.2.1.2. Enzymes kinetics studies. Enzyme kinetic studies were car-
ried out to interpret mode of inhibition for compounds 1c and 1l
against NPP1 and NPP3 isozymes, respectively (Fig. 3). The plots
indicate that compound 1c inhibited the NPP1 activity in non-
competitive manner whereas, the compound 1l showed
a
competitive mode of inhibition for isozyme NPP3.
2.3. Effect of compounds on MCF-7 and HT-29 cancer cells viability
1
1
j
1.08 ± 0.01
3.29 ± 0.02
26.09 ± 0.01
The cytotoxic potential of the synthesized derivatives in com-
parison with doxorubicin was determined by using already re-
ported method with slight modifications [30]. Initially, the
compounds were screened at 10
mM concentration against MCF-7
(
breast cancer cell line) and HT-29 (colon cancer cell line). The
percentage effect of compounds on cancer cells viability is shown in
Table 2. The tested compounds showed minor to excellent cytotoxic
potential to both cancer cell lines. The compound 1c reduced the
cells viability of MCF-7 cell lines to 70.66% whereas, the most
effective compound against HT-29 was 1h which caused the cell
death of HT-29 cell lines to 82.43%. The two most cytotoxic com-
pounds were subjected to serial dose-dependent dilutions and IC50
k
1.31 ± 0.01
(half of maximal inhibitory concentration) values were calculated
and compared with standard anticancer drug doxorubicin. The
compound 1c manifested 5.11-folds low cytotoxic effect
4

S. Ullah, M.I. El-Gamal, R. El-Gamal et al.
European Journal of Medicinal Chemistry 217 (2021) 113339
Fig. 2. A summary of SAR of the target compounds against NPP1 and NPP3.
Fig. 3. Lineweaver-Burk plots of the inhibition kinetics against NPP1 by compound 1c (left) showing uncompetitive inhibition and by compound 1l (right) against NPP3 indicating
competitive mode of inhibition.
Table 2
Compound 1l was almost equipotent to 1h against NPP3
enzyme in cell-free assay. However, compound 1l was much less
active as antiproliferative agent against MCF-7 cell line. This can be
attributed to its less hydrophobicity and ability to cross the cell
membrane in whole-cell assays. Compound 1h possessed two hy-
drophobic substituents (methyl and bromo) while compound 1l
lacks both groups and possesses dimethylamino group that con-
fered more polarity.
It is noteworthy that although compounds E and F (Fig. 1) are
more potent inhibitors of NPP1 and NPP3 enzymes, respectively,
they exerted modest activity against MCF-7 cell line compared to
compound 1c. The inhibition percentage values of compounds E
One-dose antiproliferative activity of the target compounds 1a-m.
Compound No.
% inhibition of cell viability at 10
concentration
mM
MCF-7
HT-29
1
1
1
1
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
g
h
i
18.95% ± 1.80%
33.45% ± 2.34%
70.66% ± 0.41%
20.30% ± 2.10%
1.90% ± 0.83%
16.75% ± 0.70%
43.05% ± 0.38%
27.96% ± 0.61%
28.75% ± 1.51%
3.32% ± 0.82%
28.55% ± 0.60%
26.90% ± 0.62%
5.60% ± 0.50%
96.80% ± 0.70%
23.56% ± 2.36%
54.36% ± 0.80%
18.92% ± 0.34%
33.40% ± 2.00%
16.20% ± 0.31%
14.56% ± 1.40%
58.46% ± 1.20%
82.43% ± 1.29%
60.14% ± 1.90%
47.57% ± 0.47%
58.20% ± 0.40%
17.57% ± 0.38%
21.45% ± 2.56%
94.30% ± 1.10%
and F at 100
5.6%, respectively [23].
Overexpression of NPP1 and NPP3 has been reported to be
mM concentration against MCF-7 cell line are 40.3% and
j
k
l
3
involved in propagation and metastasis of various cancers including
breast, colon, lung and brain cancer cell lines. Activation of NPP1
has been found in the generation of breast cancer stem cells
m
Doxorubicin
The results are expressed as means of triplicate assays ± S.E.M.
[
31e33]. Overexpression of NPP1 was found in human brain as-
trocytes tumor and glioblastoma cells [13,14]. Elevated level of
NPP1 was found in the HCC827, A549 lung cancer cell lines [34].
Overexpression of NPP1 and NPP3 was reported to be linked with
metastasis in human colon, and bile duct carcinoma [35,36].
The exact known mechanism by which suppression of NPP1 or
NPP3 leads to inhibition of metastasis and propagation of cancer
cell lines is yet not known. However, there are two possibilities; one
is activation and stimulation of Stimulator of Interferon Genes, also
known as STING pathway and second is preservation or elevation of
extracellular adenosine triphosphate (ATP) level. Activation of
STING pathway leads to initiation of durable and acquired immune
activity for the cure of solid tumors in vivo [37]. One known agonist
that activates the STING receptors, present on the surface of
endoplasmic reticulum is 2 , 3 -cyclic GMP-AMP (cGAMP). cGAMP
is an immunotransmitter that is regularly produced by cancerous
cells and exported to the extracellular surface and level of cGAMP is
regulated by hydrolase enzyme (NPP1) [38]. cGAMP contribute as a
second messenger for the stimulation of STING pathway that
facilitate the production of type 1 interferon and cytolytic T-cells
priming. These cytokines cause the infiltration of immune cells that
(
IC50
¼
4.70
±
0.67
mM) as compared to doxorubicin
(
0.92 ± 0.10
m
M), whereas 1h was found with almost equally
effective as doxorubicin against HT-29 cancer cell lines with an IC50
concentrations of 1.58 ± 0.20 M. Most important, the compounds
c and 1h showed less toxicity and higher selectivity towards
m
1
normal cell lines WI-38 than doxorubicin as shown in Table 3.
Table 3
IC50 ± SEM values (
m
M) of compounds 1c, 1h, and doxorubicin.
Cell line
Compound No.
IC50 value
0
0
MCF-7
1c
4.70 ± 0.67
0.92 ± 0.10
1.58 ± 0.20
1.40 ± 0.23
m
m
m
m
M
M
M
M
Doxorubicin
1h
Doxorubicin
HT-29
WI-38 (normal cells)
1c
>20
18.40 ± 1.25
1.96 ± 0.13
mM
1h
m
M
Doxorubicin
m
M
The results are expressed as means of triplicate assays ± S.E.M.
5

S. Ullah, M.I. El-Gamal, R. El-Gamal et al.
European Journal of Medicinal Chemistry 217 (2021) 113339
facilitate to cure the cancer [37]. NPP1 hydrolyzes the cGAMP
extracellularly and reduces the level of cGAMP required for the
activation of STING pathway, so NPP1 is indirectly involved in
propagation of cancer. Thus, inhibition or reduction of NPP1 activity
by potent inhbitors might lead to cure the slighter immune asso-
ciated cancers due to enhanced level of cGAMP [39]. Adenosine
triphosphate plays a vital role in the suppression of transformed
cancer associated fibroblasts [40], ureter cancerous cells, mast tu-
mor cells [41], thymocytes [42], lymphocytes [43], and melanocytes
stacked linkage with Ser377 and through p-p T-shaped connection
with Tyr382 [48]. Compound 1a formed hydrogen bonds with
important amino acids residues including Thr256, His380 and
Asn277. Moreover, 1a was connected via one
p-cation interaction
with Gly536, one -sulfur interaction with His424, one
p
p-p
T-
shaped linkage with Lys255. Most important, compounds 1a
showed zinc metal chelation with oxygen atoms of phenyl sulfonyl
and carboxamide moiety. It might be suggested that allocation of
compound 1a inside the active pocket site and important in-
teractions such as hydrogen bonding and metal chelation contrib-
uted towards selectivity of inhibitor 1a. Docking studies of most
potent and dual inhibitor 1h with NPP1 bindings site showed five
H-bonds interactions such as Asn277 ꢁ 2, and His380 ꢁ 2 and
Thr256 (Fig. 4c and d). The pyrrolo[2,3-b]pyridine scaffold was
[
44]. ATP suppresses the propagation of human breast cancer [44],
human pancreatic carcinoma, human colon cancer [45], and human
prostate adenocarcinoma [46]. Moreover, hydrolysis of ATP results
in elevated level of adenosine as a secondary product. Adenosine
suppresses the immune response to cancer cells and facilitates
cancer cells proliferation, and angiogenesis [47].
connected via three
p-alkyl interactions with Lys278, similarly
methyl group of para-methylbenzenesulfonamide ring formed two
alkyl bridges with Pro453 and Phe534. Again, more importantly
sulfonyl group of the 1h showed zinc metal chelation.
2
2
.4. Molecular docking studies
.4.1. Docking studies at NPP1 homology model
Docking studies of selective inhibitor of NPP1 (1a) revealed a
2.4.2. Docking studies at NPP3 crystallographic structure
wide range of interactions with amino acids residues of NPP1 ho-
mology model (Fig. 4). The binding interactions of the compounds
were correlated with the already reported molecular binding study
of suramin, used as standard. The complex structure of the suramin
was hydrogen bonded with eight amino acid residues of NPP1
homology model that include Asn277, Ser381, Ser378, His380,
Ser386, Tyr382, two H-bonds with Lys391, and two H-bond in-
The study of putative mode of binding behavior of positive
control (suramin) revealed a wide range of interactions with resi-
dues of NPP3 crystallographic structure (PDB ID ¼ 6C01). Suramin
revealed a number of hydrogen bondings with amino acids
including His329, Asn290, Glu322, Leu239, Thr205, Ser326, Tyr289,
Asn245, Glu275, and Glu322. Moreover, suramin was connected
with Tyr289 through
p-sulfur bonding; Pro288 via p-alkyl linkage;
teractions with Ser387. Moreover, suramin was linked via amide-
p
and zinc metal ion chelation [48]. To get insight into plausible
Fig. 4. 2D and 3D binding interactions and conformations of NPP1 inhibitors 1a (Fig. 3a, b) and 1h (Fig. 3c and d).
6

S. Ullah, M.I. El-Gamal, R. El-Gamal et al.
European Journal of Medicinal Chemistry 217 (2021) 113339
binding modes of interactions of selected inhibitors inside the
active site of NPP3, we docked three compounds; 1b (the most
potent inhibitor), 1d (a selective inhibitor), and 1h (a dual inhibitor)
as shown in Fig. 5. The carboxamide and sulfonyl groups of com-
pound 1b were connected via hydrogen bonds with Asn226,
Thr205, and His483 (Fig. 5a - b). The pyrrolo[2,3-b]pyridine scaffold
selectivity of compound 1b. The docking studies of selective in-
hibitor of NPP3 (1d) demonstrated almost same binding in-
teractions as the most potent inhibitor 1b. The selectivity of 1d vs
1b might be attributed to the presence of tolyl ring instead of
chlorophenyl ring in 1b. The binary inhibitor 1h showed a wide
range of interactions with residues of pocket site. Compound 1h
was stabilized through H-bonds with Asn226, Lys204, and His483.
In addition, 1h was surrounded by His329, Thr205, Phe206, and
Leu239. Most important, both rings of the pyrrolo[2,3-b]pyridine
core were chelated with zinc cation.
showed two
chlorophenyl moiety was connected through
p
-
p
stacked linkages with His329 and also para-
and -alkyl inter-
p
p
action with Leu239. Again, oxygen atom of sulfonyl group was
chelated with zinc-metal ion, contributing to the potency and
Fig. 5. 2D and 3D binding interactions and conformations of NPP3 inhibitors; 1b (Fig. 5a, b), 1d (Fig. 5c, d), and 1h (Fig. 5e and f).
7

S. Ullah, M.I. El-Gamal, R. El-Gamal et al.
European Journal of Medicinal Chemistry 217 (2021) 113339
2.5. In vivo effect of compound 1h on blood glucose level in mice
4. Experimental
Sulfonylurea derivatives are known as hypoglycemic agents
4.1. General
[
49,50]. Due to the presence of sulfonylurea moiety in the target
compound structures, we decided to investigate the effect of
compound 1h, the most potent antiproliferative compound, on
blood glucose level in mice. The compound was injected intra-
peritoneally to male BALB/c mice at a dose of 10 or 20 mg/kg
body weight. The blood glucose level was measured for each
mouse at 30-min time intervals over 2 h, and the results are
illustrated in Table 4. It is obvious that compound 1h did not
produce significant hypoglycemic effect, which is an important
merit.
The melting points were measured on a Stuart melting point
apparatus (Staffordshire, UK), and are uncorrected. Bruker Avance
1
(400 MHz spectrometer) was used to analyze the compounds by H
13
and C NMR. LC-MS analysis was carried out by LC-MS analyzer
(Waters Corporation, MA, USA). All the solvents and reagents were
purchased from commercial companies and used as such. The final
and intermediate compounds were purified by flash column
chromatography (silica gel, pore size 0.040e0.063 mm, 230e400
mesh) using laboratory reagent grade solvents.
4
4
.2. Chemical synthesis of compounds
3
. Conclusion
.2.1. Synthesis of the aryl sulfonamide reagents 4a-e
To a solution of arylsulfonyl chloride 2a-e (0.1 mmol) in meth-
The target sulfonylurea-bearing pyrrolo[2,3-b]pyridine de-
anol (5 mL), ammonium hydroxide (33% solution, 5 mL) was added.
The reaction mixture was stirred at room temperature for 5e10.
Reaction completion was confirmed by TLC then LC-MS. After that,
the reaction mixture was evaporated under reduced pressure to
dryness. Water (10 mL) and ethyl acetate (10 mL) were added to the
residue for extraction. The ethyl acetate layer was separated,
washed with saturated saline (3 ꢁ 5 mL), dried using anhydrous
rivatives were synthesized and evaluated for their potential to
inhibit NPP1 and NPP3 enzymatic activity. The calculated inhibitory
effect was compared with the reference standard inhibitor, sur-
amin. We discovered selective, dual, and even more active in-
hibitors of both NPP1 and NPP3 than suramin (standard drug).
Compound 1a was identified as selective inhibitor of NPP1 with an
IC50 value of 4.50 ± 0.16
selective inhibitor of NPP3 with an IC50 value of 3.72 ± 0.17
Similarly, compound 1c was identified as the most potent inhibitor
of NPP1 (0.80 ± 0.04 M), and 1l had strongest inhibitory activity on
NPP3 (IC50 ¼ 0.55 ± 0.01 M). The kinetic studies showed that
mM, whereas compound 1d was found as
Na
2 4
SO , and evaporated to dryness to obtain the title compounds as
solid products. They were used in the next step as such.
mM.
m
4
.2.2. Synthesis of 4-(dimethylamino)-7-azaindole (7)
A mixture of compound 5c (152 mg, 1 mmol) was fused with
m
compound 1c was an uncompetitive inhibitor of isoenzyme NPP1
and the compound 1l was a competitive inhibitor of isozyme NPP3.
Molecular docking studies of the strongest inhibitors either for
NPP1 (1a and 1h) or NPP3 (1b, 1d, and 1h) showed promising
binding interactions with active site residues of both isozymes,
where the sulfonamide group formed zinc metal ion chelation.
Also, cytotoxic examination of these compounds against MCF-7 and
HT-29 cancer cell lines revealed 1c as most chemoselective against
dimethylammonium chloride (405 mg, 5 mmol) in oil bath without
ꢀ
solvent at 180 C for 5 h. The reaction mixture was cooled, extracted
with ethyl acetate (20 mL), and washed with saline (20 mL). The
organic layer was separated, dried using anhydrous Na SO ,
2 4
filtered, and evaporated to dryness. The product was purified by
column chromatography (silica gel, dichloromethane:methanol
1
0:1 v/v) to obtain the title product (yield: 30%, R
f
¼ 0.54 dichlor-
omethane:methanol 10:1 v/v).
MCF-7 cell lines with an IC50 value of 4.70 ± 0.67
as the most cytotoxic agent against HT-29 cancer cell line with an
IC50 value of 1.58 ± 0.20 M. The IC50 values revealed that com-
mM, and 1h proved
1
H NMR (CDCl
3
, 400 MHz)
d
11.20 (brs, 1H), 7.92 (d, J ¼ 5.6 Hz,
1
H), 7.04 (d, J ¼ 3.6 Hz, 1H), 6.51 (d, J ¼ 3.6 Hz, 1H), 6.06 (d,
m
J ¼ 5.6 Hz, 1H), 3.11 (s, 6H).
pounds 1c and 1h were more selective and less toxic to WI-38
normal cell line as compared to standard doxorubicin for MCF-7
and HT-29 cancer cell lines, respectively. This study led to discov-
ery of a new sulfonylurea-based antiproliferative agent 1h that
does not produce hypoglycemia as a side effect. It is almost equi-
potent to doxorubicin against HT-29 colon cancer cell line with the
merit of higher selectivity index towards cancer cells than normal
cells than doxorubicin. Its molecular mechanism of action could be,
at least in part, inhibition of NPP1 and NPP3 isozymes. Considering
the high potency of compounds E and F (Fig. 1) against NPP1 and
NPP3, respectively, we recommend investigating the impact of
sulfonamide or thiourea spacers on activity in the future.
4
.2.3. Synthesis of carbamate intermediates 9a-d
A mixture of compound 5a-c or 7 (1 mmol) and TEA (2 mL) in
ꢀ
anhydrous THF (5 mL) was cooled to 0 C using ice bath. A solution
of phenyl chloroformate (222 mg, 2 mmol) in anhydrous THF (5 mL)
was added slowly thereto. The mixture was further stirred at the
same temperature for 2 h. The reaction mixture was evaporated in
vacuo, extracted with ethyl acetate (20 mL), and washed with saline
(
20 mL). The organic layer was separated, dried using anhydrous
Na SO , filtered, and evaporated to dryness. It was used in the next
reaction with no further purification.
2
4
4.2.4. Synthesis of the target sulfonyl urea products 1a-m
A mixture of compound 9a-d (0.1 mmol), 4a-e (0.12 mmol), and
anhydrous potassium carbonate (69 mg, 0.5 mmol) in dry
dichloromethane (5 mL) was allowed to stir at room temperature
overnight. After reaction completion, the mixture was washed with
water (5 mL) and the organic layer was separated, dried with
Table 4
Mean blood glucose levels (mg/dL) in mice after i.p. injection of compound 1h at
doses of 10 or 20 mg/kg.
Time after injection (min)
Control
10 mg/kg
20 mg/kg
2 4
anhydrous Na SO , filtered, and evaporated to dryness. The
remaining residue was purified by column chromatography.
0
124 ± 9
106 ± 5
141 ± 6
115 ± 7
120 ± 4
108 ± 5
103 ± 7
122 ± 2
124 ± 12
121 ± 9
114 ± 7
113 ± 8
114 ± 14
128 ± 6
121 ± 7
3
6
9
1
0
0
0
20
4.2.4.1. N-(Pyrrolo[2,3-b]pyridine-1-carbonyl)benzenesulfonamide
(1a). Purification was carried out by flash column chromatography
The results are expressed as means ± standard deviation (S.D.), n ¼ 5 in all groups.
(silica gel, hexane:ethyl acetate 3:1 v/v then switching to ethyl
8

S. Ullah, M.I. El-Gamal, R. El-Gamal et al.
European Journal of Medicinal Chemistry 217 (2021) 113339
acetate); yield: 51%; mp: 130e132 C; 1H NMR (CDCl
d
ꢀ
, 400 MHz)
8.33 (brs, 1H), 7.88 (d, J ¼ 8.0 Hz, 2H), 7.72e7.67 (m, 1H), 7.55e7.43
4.2.4.7. N-(5-Bromopyrrolo[2,3-b]pyridine-1-carbonyl)-3-
nitrobenzenesulfonamide (1g). Purification was carried out by flash
column chromatography (silica gel, hexane:ethyl acetate 2:1 v/v
3
(
2
m, 2H), 7.19 (d, J ¼ 7.6 Hz, 1H), 7.04e7.00 (m, 2H), 6.90e6.82 (m,
13
ꢀ
1
H); C NMR (CDCl
3
, 100 MHz)
d
155.8, 142.0, 132.7, 129.6, 129.1,
then switching to ethyl acetate); yield: 75%; mp: 227e230 C;
NMR (CDCl , 400 MHz) 8.59e8.28 (m, 2H), 8.15e7.93 (m, 3H), 7.71
(brs, 1H), 7.47e7.35 (m, 1H), 6.94 (d, J ¼ 12.8 Hz, 1H), 6.76e6.72 (m,
H
þ
1
28.3, 127.8, 126.4, 125.5, 121.9, 115.4; LC/MS m/z: 302.0 (M þ 1);
3
d
CHN analysis: calculated C:55.80%, H:3.68%, N:13.95%; found:
C:55.70%, H:3.74%, N:13.89%.
13
1H); C NMR (CDCl
3
, 100 MHz) d 145.3, 132.8, 132.6, 131.9, 131.7,
1
29.6, 129.1, 128.6, 126.5, 115.5, 112.7, 102.3; LC/MS m/z: 425.9
þ
þ
(M
þ 2), 424.9 (M þ 1); CHN analysis: calculated C:39.54%,
4
. 2 . 4 . 2 . N - ( P y r r o l o [ 2 , 3 - b ] p y r i d i n e - 1 - c a r b o n y l ) - 4 -
H:2.13%, N:13.18%; found: C:39.40%, H:2.08%, N:13.30%.
chlorobenzenesulfonamide (1b). Purification was carried out by
flash column chromatography (silica gel, hexane:ethyl acetate 3:1
ꢀ
1
4.2.4.8. N-(5-Bromopyrrolo[2,3-b]pyridine-1-carbonyl)-4-
methylbenzenesulfonamide (1h). Purification was carried out by
flash column chromatography (silica gel, hexane:ethyl acetate 4:1
v/v then switching to ethyl acetate); yield: 52%; mp: >280 C; H
NMR (DMSO‑d , 400 MHz)
8.29 (d, J ¼ 8.5 Hz, 1H), 7.95e7.89 (m,
6
d
5
H), 7.50 (d, J ¼ 8.5 Hz, 2H), 7.14e7.12 (m, 1H), 6.46e6.44 (m, 1H);
13
v/v then switching to hexane:ethyl acetate 1:1 v/v); yield: 78%; mp:
C NMR (CDCl
3
, 100 MHz)
d
155.9, 140.6, 137.7, 132.8, 130.1, 128.2,
ꢀ
1
þ
130e132
C; H NMR (CDCl , 400 MHz)
d
12.54 (brs, 1H), 8.43 (s,
127.2, 124.2, 118.9, 117.9, 104.8, 103.6; LC/MS m/z: 336.0 (M þ 1);
3
1
H), 8.08 (t, J ¼ 7.6 Hz, 2H), 7.91 (d, J ¼ 7.2 Hz, 1H), 7.82 (s, 1H),
CHN analysis: calculated C:50.08%, H:3.00%, N:12.51%; found:
C:50.34%, H:2.89%, N:12.45%.
13
7
1
1
.37e7.29 (m, 2H), 6.56 (s, 1H), 2.44 (s, 3H); C NMR (DMSO‑d
00 MHz) 143.2, 142.4, 141.9, 141.4, 130.1, 129.7, 129.4, 129.1, 128.1,
27.7, 127.5, 125.7, 99.7, 21.0; LC/MS m/z: 395.0 (M þ 2), 394.0
6
,
d
þ
4
. 2 . 4 . 3 . N - ( P y r r o l o [ 2 , 3 - b ] p y r i d i n e - 1 - c a r b o n y l ) - 3 -
þ
(M
þ 1); CHN analysis: calculated C:45.70%, H:3.07%, N:10.66%;
nitrobenzenesulfonamide (1c). Purification was carried out by flash
found: C:45.52%, H:3.02%, N:10.74%.
column chromatography (silica gel, hexane:ethyl acetate 3:1 v/v
ꢀ
1
then switching to ethyl acetate); yield: 45%; mp: 171e172 C;
NMR (CD OD, 400 MHz) 8.61 (brs, 2H), 8.36e8.34 (m, 2H),
.18e8.16 (m, 2H), 7.74e7.70 (m, 3H); C NMR (CD
H
4
.2.4.9. N-(5-Bromopyrrolo[2,3-b]pyridine-1-carbonyl)-2-
3
d
13
naphthalenesulfonamide (1i). Purification was carried out by flash
column chromatography (silica gel, hexane:ethyl acetate 5:1 v/v
then switching to hexane:ethyl acetate 2:1 v/v); yield: 30%; mp:
8
3
OD, 100 MHz)
d
1
155.9, 146.2, 143.2, 140.7, 139.4, 139.4, 132.6, 130.1, 128.7, 126.5,
23.9, 118.9, 118.0, 103.9; LC/MS m/z: 347.0 (M þ 1); CHN analysis:
þ
ꢀ
1
2
7
(
1
22e224 C; H NMR (CDCl
.95e7.93 (m, 4H), 7.70e7.56 (m, 4H), 7.43e7.29 (m, 3H); C NMR
CDCl , 100 MHz) 143.3, 140.7, 134.8, 132.4, 129.7, 129.0, 128.8,
28.4, 128.1, 127.8, 127.5, 127.3, 126.7, 126.4, 123.1, 121.8, 102.7; LC/
3
, 400 MHz)
d
8.51 (d, J ¼ 6.4 Hz, 1H),
calculated C:48.55%, H:2.91%, N:16.18%; found: C:48.64%, H:2.83%,
N:16.15%.
13
3
d
4
. 2 . 4 . 4 . N - ( P y r r o l o [ 2 , 3 - b ] p y r i d i n e - 1 - c a r b o n y l ) - 4 -
þ
þ
MS m/z: 431.0 (M þ 2), 430.0 (M þ 1); CHN analysis: calculated
methylbenzenesulfonamide (1d). Purification was carried out by
flash column chromatography (silica gel, hexane:ethyl acetate 3:1
C:50.25%, H:2.81%, N:9.77%; found: C:50.13%, H:2.98%, N:9.66%.
ꢀ
v/v then switching to ethyl acetate); yield: 75%; mp: 150e151 C;
4
.2.4.10. N-(4-Chloropyrrolo[2,3-b]pyridine-1-carbonyl)benzene-
1
H NMR (CDCl
.87e7.68 (m, 2H), 7.53 (d, J ¼ 8.0 Hz, 1H), 7.09 (d, J ¼ 8.0 Hz, 1H),
.81 (d, J ¼ 5.2 Hz,1H), 6.78e6.50 (m, 1H), 6.23 (brs, 1H), 2.25 (s, 3H,
3
, 400 MHz) d 8.28 (brs, 1H), 8.19e7.91 (m, 2H),
sulfonamide (1j). Purification was carried out by flash column
7
chromatography (silica gel, hexane:ethyl acetate 3:1 v/v then
6
ꢀ
1
switching to ethyl acetate); yield: 68%; mp: 225e228 C (dec.); H
NMR (CDCl , 400 MHz) 8.40e8.21 (m, 1H), 7.94e7.85 (m, 2H),
.66e7.46 (m, 3H), 6.92 (s, 2H), 6.73 (d, J ¼ 4.0 Hz, 1H), 6.65e6.57
13
Me); C NMR (DMSO‑d
6
, 100 MHz) d 143.2, 142.5, 142.0, 141.4,
3
d
1
29.4, 128.4, 128.1, 127.3, 126.0, 125.7, 115.5, 99.8, 21.0; LC/MS m/z:
7
þ
316.0 (M þ 1); CHN analysis: calculated C:57.13%, H:4.16%,
13
3
(m, 1H); C NMR (CDCl , 100 MHz) d 145.1, 132.5, 132.3, 131.7, 131.5,
N:13.33%; found: C:57.02%, H:4.08%, N:13.43%.
1
29.4, 128.9, 128.2, 126.2, 115.3, 112.4, 102.3; LC/MS m/z: 337.0
þ
þ
(M
þ 2), 336.0 (M þ 1); CHN analysis: calculated C:50.08%,
4
. 2 . 4 . 5 . N - ( P y r r o l o [ 2 , 3 - b ] p y r i d i n e - 1 - c a r b o n y l ) - 2 -
H:3.00%, N:12.51%; found: C:49.96%, H:2.88%, N:12.60%.
naphthalenesulfonamide (1e). Purification was carried out by flash
column chromatography (silica gel, hexane:ethyl acetate 3:1 v/v
then switching to hexane:ethyl acetate 1:1 v/v); yield: 36%; mp:
4
.2.4.11. N-(4-Chloropyrrolo[2,3-b]pyridine-1-carbonyl)-2-
naphthalenesulfonamide (1k). Purification was carried out by flash
column chromatography (silica gel, hexane:ethyl acetate 2:1 v/v
then switching to ethyl acetate); yield: 56%; mp: 152e153 C;
NMR (CDCl , 400 MHz) 8.53 (brs, 1H), 7.97e7.87 (m, 4H),
ꢀ
1
2
29e231 C (dec.); H NMR (CD
3
OD, 400 MHz) d 8.40 (brs, 1H), 8.34
(
7
d
1
s, 1H), 8.03e7.89 (m, 4H), 7.85e7.79 (m, 2H), 7.75e7.69 (m, 2H),
ꢀ
1
H
13
.55e7.48 (m, 2H), 7.42e7.37 (m, 1H); C NMR (CD
3
OD, 100 MHz)
143.1, 140.6, 134.6, 132.2, 129.6, 128.8, 128.7, 128.2, 128.0, 127.6,
27.4, 127.1, 126.6, 126.2, 122.9, 121.7, 102.5; LC/MS m/z: 352.0
3
d
13
7.67e7.59 (m, 4H), 7.52e7.43 (m, 3H); C NMR (CDCl
3
, 100 MHz)
d
143.4, 140.8, 134.9, 132.6, 129.8, 129.2, 128.9, 128.6, 128.2, 128.0,
þ
(
M
þ 1); CHN analysis: calculated C:61.53%, H:3.73%, N:11.96%;
127.6, 127.5, 126.9, 126.5, 123.2, 121.9, 102.9; LC/MS m/z: 387.0
found: C:61.45%, H:3.58%, N:12.06%.
þ
þ
(M
þ 2), 386.0 (M þ 1); CHN analysis: calculated C:56.03%,
H:3.13%, N:10.89%; found: C:56.09%, H:3.05%, N:10.96%.
4.2.4.6. N-(5-Bromopyrrolo[2,3-b]pyridine-1-carbonyl)benzenesulfo-
namide (1f). Purification was carried out by flash column chro-
4.2.4.12. N-[4-(Dimethylamino)pyrrolo[2,3-b]pyridine-1-carbonyl]
benzenesulfonamide (1l). Purification was carried out by flash col-
umn chromatography (silica gel, ethyl acetate then switching to
matography (silica gel, hexane:ethyl acetate 4:1 v/v then switching
ꢀ
1
to hexane:ethyl acetate 1:1 v/v); yield: 44%; mp: 141e143 C;
H
ꢀ
1
NMR (CDCl , 400 MHz)
3
d
7.80e7.70 (m, 4H), 7.55 (d, J ¼ 8.0 Hz, 1H),
ethyl acetate:methanol 5:1 v/v); yield: 50%; mp: >280 C; H NMR
(CDCl , 400 MHz)
8.13e7.88 (m, 3H), 7.68 (d, J ¼ 8.0 Hz, 2H), 7.46
(brs, 3H), 6.88e6.66 (m, 2H), 3.41 (s, 6H); C NMR (CDCl
156.7,153.5,147.5,142.0,137.7,137.0,129.0,128.4,127.3,105.3,101.9,
13
7
.44 (t, J ¼ 7.2 Hz, 1H), 7.19e7.10 (m, 2H), 6.95e6.73 (m, 2H);
C
3
d
13
NMR (CDCl
3
, 100 MHz)
d
145.2, 132.6, 132.5, 131.8, 131.7, 129.5,
3
,100 MHz)
þ
1
29.0, 128.4, 126.3, 115.4, 112.5, 102.2; LC/MS m/z: 380.9 (M þ 2),
d
þ
þ
379.9 (M þ 1); CHN analysis: calculated C:44.22%, H:2.65%,
100.7, 41.9; LC/MS m/z: 345.1 (M þ 1); CHN analysis: calculated
N:11.05%; found: C:44.40%, H:2.55%, N:10.97%.
C:55.80%, H:4.68%, N:16.27%; found: C:55.85%, H:4.80%, N:16.15%.
9

S. Ullah, M.I. El-Gamal, R. El-Gamal et al.
European Journal of Medicinal Chemistry 217 (2021) 113339
4
4
.2.4.13. N-[4-(Dimethylamino)pyrrolo[2,3-b]pyridine-1-carbonyl]-
-methylbenzenesulfonamide (1m). Purification was carried out by
Burk plots for the both compounds were plotted with the help of
linear regression analysis of PRISM 5.0 (Graph Pad, San Diego,
California, USA).
flash column chromatography (silica gel, ethyl acetate then
switching to ethyl acetate:methanol 6:1 v/v); yield: 68%; mp:
ꢀ
1
2
20e223 C (dec.); H NMR (CDCl
3
, 400 MHz)
d
8.02e7.99 (m, 3H),
4.4. Antiproliferative activity
7
.64 (d, J ¼ 8.0 Hz, 2H), 7.21 (brs, 1H), 6.75 (d, J ¼ 8.0 Hz,1H), 6.69 (d,
13
J ¼ 4.0 Hz, 1H), 6.41 (d, J ¼ 6.4 Hz, 1H), 3.73 (s, 6H), 2.34 (s, 3H);
C
4.4.1. Cell culture
NMR (CDCl
3
, 100 MHz)
d
156.6, 153.4, 147.3, 141.9, 137.6, 136.8,
The breast cancer cell line (MCF-7), and colorectal adenocarci-
noma cell line (HT-29) were collected from the European Collection
of Cell Cultures (ECACC, UK) and later on, maintained in Roswell
Park Memorial Institute medium (RPMI, Sigma-Aldrich, St. Louis,
MO, USA). The normal diploid human fibroblasts (WI-38), were
kindly gifted by Prof. Ekkehard Dikomey (University Cancer Center,
Hamburg University, Hamburg, Germany), that were preserved by
using Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich)
supplemented with 10% concentration of fetal bovine serum and
antibiotics 1% of penicillin/streptomycin, obtained from Sigma-
Aldrich.
1
28.9, 128.3, 127.1, 105.2, 101.8, 100.6, 41.8, 21.7; LC/MS m/z: 359.1
þ
(
M
þ 1); CHN analysis: calculated C:56.97%, H:5.06%, N:15.63%;
found: C:56.80%, H:4.98%, N:15.74%.
4
4
.3. Enzyme testing
.3.1. Transfection of COS-7 cells
COS-7 cells were transfected by using human NPP1 and NPP3
expressing plasmids. For this purpose, 10 cm plates were used and
the Dulbecco’s Modified Eagle Medium/F-12 (DMEM/F-12) was
used for the incubation of confluent cells with plasmid DNA (6 mg)
and Lipofectamine (without fetal bovine serum). The process of
transfection was stopped by adding an equal volume of DMEM/F-12
4.4.2. MTT assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide assay (MTT) was carried out to examine the chemothera-
peutic effect of the tested compounds on cancer cells viability. The
assay was performed according to already reported method with
few modifications [54]. The tested compounds were dissolved in
DMSO to prepare 10 mM stock solution then diluted by dissolution
in fetal bovine serum which is used as an incubation medium. The
(
[
with 20% FBS). The transfected cells were harvested 48e72 h later
51].
4.3.2. Collection of membrane fractions
The harvested transfected cells were subjected to washing by
ꢀ
using Tris-saline buffer at 4 C. The scraping and collection of the
cells were carried out in harvesting buffer (95 mM NaCl, 0.1 mM
phenylmethylsulfonyl fluoride, and 45 mM Tris) at pH 7.5. The cells
4
experimental cell lines were seeded at a density of 4 ꢁ 10 per well
in tissue culturing plate and placed for overnight incubation. The
incubated plates were treated with target compounds, positive
control (doxorubicin), and negative control dimethyl sulfoxide 24 h.
After 24 h, the cell media was separated and again cells were
treated with approximately 200 mL of media with MTT tetrazolium
ꢀ
were centrifuged twice 5 min each at 300 g and 4 C. After that, the
cells were resuspended in harvesting buffer with aprotinin 10 mg/
mL and subjected to sonication. Again, cells were centrifuged at
3
00 g for 10 min and cellular debris was discarded. In the super-
ꢀ
ꢀ
natant, 7.5% glycerol was added and preserved at ꢂ80 C. The
Bradford microplate assay was used for the estimation of protein
contents, using Bovine serum albumin as a standard [52].
dye (0.5 mg/mL) and incubated at 37 C for 2 h. Again, media were
removed and the violet crystals were solubilized by adding 200 mL
of DMSO. Finally, the microplate reader (Thermo Scientific, Mas-
sachusetts, USA) was used for the measurement of wavelength at
570 nm.
4.3.3. NPP1 and NPP3 isoenzymes inhibition assay
The inhibitory effect of compounds 1a-m was determined by
using already reported method with slight modifications [53]. The
assay buffer was composed of Tris. HCl (50 mM), MgCl (5 mM) and
ZnCl (0.1 mM) with final pH 9.5. The assay was performed in 96-
well plate with each well containing assay buffer, 100 M of test
4.5. Molecular docking studies
2
2
For the purpose of molecular docking studies, a previously
generated homology model of NPP1 (PDB ID 4GTW) was used [55].
However, for NPP3, a crystallographic structure with PDB ID 6C01
was downloaded from Protein Data Bank [56]. For the preparation
of database of selected inhibitors and energy minimization, Mo-
lecular Operating Environment (MOE 2014e0901) was used [57].
Structures of both proteins NPP1 and NPP3 were prepared and
docked by using LeadIT (BioSolveIT GmbH, Germany) [58]. The
poses possessing high affinity and low binding energy were
selected for visualization by using Discovery Studio Visualizer DS
[59].
m
compound, enzymes NPP1 (27 ng), NPP3 (35 ng) and substrate p-
0
nitrophenyl 5 -thymidine monophosphate. The plate was placed for
3
4
5 min in an incubator and read was taken at the wavelength of
05 nm by using microplate reader (BioTek FLx800, Instruments,
Inc. USA). The compounds showing more than 50% inhibition of
either of isozyme (NPP1 or NPP3) were subjected to serial dilutions
for the determination of IC50 values. The data was analyzed by using
non-linear regression analysis curve fitting program PRISM 5.0
(
Graph Pad, San Diego, California, USA).
4.3.4. Enzyme kinetics studies
4.6. In vivo effect of blood glucose level in mice
For the determination of kinetic studies, the used substrate
concentration were 0.0 mM, 1.25 mM, 2.50 mM, 5.0 mM, 7.5 mM,
and 10.0 mM. The final assay concentrations for the compound 1c
The protocol was approved by Research Ethics Committee at
Dubai Pharmacy College for Girls, Dubai, United Arab Emirates
(Reference #REC/FD/2020/03). Compound 1h was dissolved in a
were 0.0
l, we used the assay concentrations 0.0
The substrate and the compounds (1c, 1l) were incubated with
assay buffer that was comprised of Tris. HCl (50 mM), MgCl (5 mM)
and ZnCl (0.1 mM) with final pH 9.5. The reaction mixture was
incubated at 37 C, and the reading was taken for 35 min with
5 min intervals. The microplate reader (BioTek FLx800, In-
struments Inc., USA) was used for reading at 405 nm. Lineweaver-
mM, 0.5
mM, 1.0
m
M, 1.5
mM and similarly for the compound
1
m
M, 0.1 M, 0.5 M, 1.0 M.
m
m
m
solution of (2-hydroxypropyl)-b-cyclodextrin in distilled water
(40% w/v) to make a 1 mg/mL solution. The resultant solution was
injected i.p. to male BALB/c mice (30e35 g body weight) in a dose of
10 or 20 mg/kg. A control group was injected with placebo, (2-
hydroxypropyl)-b-cyclodextrin solution devoid of compound 1h.
The number of mice is five per group. The blood glucose level was
2
2
ꢀ
0
measured at 0, 30, 60, 90, and 120 min intervals for each animal as
10

S. Ullah, M.I. El-Gamal, R. El-Gamal et al.
European Journal of Medicinal Chemistry 217 (2021) 113339
per the kit’s instructions. The mice were deprived from any access
to diet during the 2-h measurement period following the injection.
pyrrolopyridine derivative, U.S. Patent Application 13/264 (2012) 8.
20] L.X. Wang, X. Liu, S. Xu, Q. Tang, Y. Duan, Z. Xiao, J. Zhi, L. Jiang, P. Zheng,
W. Zhu, Discovery of novel pyrrolo-pyridine/pyrimidine derivatives bearing
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538e551.
Declaration of competing interest
21] A.R. Blaazer, J.H. Lange, M.A. van der Neut, A. Mulder, F.S. den Boon,
T.R. Werkman, C.G. Kruse, W.J. Wadman, Novel indole and azaindole (pyrro-
lopyridine) cannabinoid (CB) receptor agonists: design, synthesis,
structureeactivity relationships, physicochemical properties and biological
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None.
Acknowledgment
[22] E. Forcellini, S. Boutin, C.A. Lefebvre, E.E. Shayhidin, M.C. Boulanger,
G. Rh eꢀ aume, X. Barbeau, P. Lagüe, P. Mathieu, J.F. Paquin, Synthesis and bio-
logical evaluation of novel quinazoline-4-piperidinesulfamide derivatives as
inhibitors of NPP1, Eur. J. Med. Chem. 147 (2018) 130e149.
This work was funded by University of Sharjah, Dubai Pharmacy
College for Girls, United Arab Emirates as well as by the German
Academic Exchange Service (DAAD) “German-Pakistani Research
Collaboration Programme”. J.S. received support from the Natural
Sciences and Engineering Research Council of Canada (NSERC;
RGPIN-2016-05867).
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23] S. Ullah, M.I. El-Gamal, S. Zaib, H.S. Anbar, S.O. Zaraei, R.M. Sbenati, J. Pelletier,
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studies of new pyrazole-based thiourea and sulfonamide derivatives as in-
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