Synthesis, In-vitro evaluation and molecular docking studies of oxoindolin phenylhydrazine carboxamides as potent and selective inhibitors of ectonucleoside triphosphate diphosphohydrolase (NTPDase)

Article abstract of DOI:10.1016/j.bioorg.2021.104957

Members of the ectonucleoside triphosphate diphosphohydrolases (NTPDases) constitute the major family of enzymes responsible for the maintenance of extracellular levels of nucleotides and nucleosides by catalyzing the hydrolysis of nucleoside triphosphate

Full text of DOI:10.1016/j.bioorg.2021.104957

Bioorganic Chemistry 112 (2021) 104957
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, In-vitro evaluation and molecular docking studies of oxoindolin
phenylhydrazine carboxamides as potent and selective inhibitors of
ectonucleoside triphosphate diphosphohydrolase (NTPDase)
a
b
a
c
c,d
´
Saira Afzal , Mariya al-Rashida , Abdul Hameed , Julie Pelletier , Jean Sevigny
,
Jamshed Iqbala
,
*
a Centre for Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus, Abbottabad, Pakistan
b Department of Chemistry, Forman Christian College (A Chartered University), Lahore, Pakistan
c
´
´
´
Centre de recherche du CHU de Quebec, Universite Laval, Quebec City, QC, Canada
d
´
´
´
´
´
Departement de Microbiologie-Infectiologie et d’Immunologie, Faculte de Medecine, Universite Laval, Quebec City, QC, Canada
A R T I C L E I N F O
A B S T R A C T
Keywords:
Members of the ectonucleoside triphosphate diphosphohydrolases (NTPDases) constitute the major family of
enzymes responsible for the maintenance of extracellular levels of nucleotides and nucleosides by catalyzing the
hydrolysis of nucleoside triphosphate (NTP) and nucleoside diphosphates (NDP) to nucleoside monophosphate
(NMP). Although, NTPDase inhibitors can act as potential drug candidates for the treatment of various diseases,
there is lack of potent as well as selective inhibitors of NTPDases. The current study describes the synthesis of a
number of carboxamide derivatives that were tested on recombinant human (h) NTPDases. The most promising
inhibitors were 2h (h-NTPDase1, IC50: 0.12 ± 0.03 µM), 2d (h-NTPDase2, IC50: 0.15 ± 0.01 µM) and 2a (h-
NTPDase3, IC50: 0.30 ± 0.04 µM; h-NTPDase8, IC50: 0.16 ± 0.02 µM). Four compounds (2e, 2f, 2g and 2h) were
associated with the selective inhibition of h-NTPDase1 while 2b was identified as a selective h-NTPDase3 in-
hibitor. Considering the importance of NTPDase3 in the regulation of insulin release, the NTPDase3 inhibitors
were further investigated to elucidate their role in the insulin release. The obtained data suggested that com-
pound 2a was actively participating in regulating the insulin release without producing any effect on NTPDase3
mRNA. Moreover, the most potent inhibitors were docked within the active site of respective enzyme and the
observed interactions were in compliance with in vitro results. Hence, these compounds can be used as phar-
macological tool to further investigate the role of NTPDase3 coupled to insulin release.
Oxoindolin
Carboxamide
Isatin
Molecular docking
Nucleoside triphosphate diphosphohydrolase
inhibitors
1. Introduction
coupled (P2Y) receptors [6–9]. P2X receptors are ATP-gated ion chan-
nels which are subdivided into seven types (P2X1-7) and associated with
Adenosine triphosphate (ATP) is a versatile signalling molecule that
is present in extracellular space and plays an important role in cell to cell
communication [1–2]. Almost all the cells release ATP that in turn ac-
tivates the purinergic receptors (P2X and P2Y receptors) and partici-
pates in different physiological functions such as hormone secretion,
neurotransmission, sensory transmission, glial-neuron interaction and
specific organ associated functions [3–5]. In pancreatic beta cells, ATP is
co-released with insulin secretory granules and acts in an autocrine
manner to regulate the insulin secretion. This regulation of insulin
secretion is achieved by activating two subfamilies of cell membrane
receptors: ligand gated ion channel (P2X) receptors and G-protein
the exchange of cation across the cell membrane [10–12]. Activation of
P2X receptors by ATP induces an elevated intracellular Ca+2 that
eventually leads to insulin secretion [8,13–14]. In contrast, P2Y re-
ceptors are coupled to G-protein and are comprised of eight members
(P2Y1,2,4,6,11,12,13,14). They signal through intracellular second messen-
gers such as cyclic AMP (cAMP), inositol phosphate and Ca+2 [15–16].
Activation of P2Y receptors leads to an induction of inositol triphosphate
coupled with the transient increase in intracellular Ca+2 level that ul-
timately results in insulin secretion. Hence, ATP operates through P2
receptor activation and amplifies the insulin secretion [9,17].
However, extracellular ATP is progressively metabolized by the
* Corresponding author.
E-mail address: drjamshed@cuiatd.edu.pk (J. Iqbal).
https://doi.org/10.1016/j.bioorg.2021.104957
Received 23 February 2021; Received in revised form 13 April 2021; Accepted 28 April 2021
Available online 30 April 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

S. Afzal et al.
Bioorganic Chemistry 112 (2021) 104957
coordinated action of a network of ectoenzymes known as ecto-
nucleotidases. This chain of ecto-nucleotidases is composed of four
main enzyme families i.e. nucleotide pyrophosphatases/phosphodies-
terases (NPPs), ectonucleoside triphosphate diphosphohydrolases
(NTPDases), alkaline phosphatases (APs or ALPs), and ecto-5′-nucleo-
tidase (e-5′-N) [18–22]. The ecto-nucleotidases catalyze the hydrolysis
of nucleoside triphosphates, diphosphates, monophosphates and dinu-
cleoside polyphosphate leading to formation of nucleoside diphosphate,
nucleoside monophosphate, nucleoside, phosphate and inorganic pyro-
phosphate, respectively. Thus, these ecto-nucleotidases not only regu-
late the concentration of extracellular ATP and related nucleotides but
also provide additional ligands for P2 and P1 receptors [3,23].
and then refluxed for 6–7 h. The precipitated solid was filtered, washed
and dried.
2.2.1. N-(4-fluorophenyl)-2-(2-oxoindolin-3-ylidene)hydrazine-1-
carboxamide (2a)
Yellow solid; Melting point = 223–225 ◦C; Yield: 79%; IR(KBr):
–
–
–
–
3349–3117(>NH stretch), 1701(>C O), 1572(>C
N ), 1524(>NH
–
bend), cmꢀ 1; 1HNMR (300 MHz, DMSO‑d6): δ (ppm) 10.77 (s, 1H, NH),
10.43 (s, 1H, NH), 9.56 (s, 1H, NH), 8.08 (d, 1H, J = 7.8 Hz), 7.61 (dd,
2H, J = 9.0, 5.1 Hz), 7.38 (t, 1H, J = 7.8 Hz), 7.21–7.05 (m, 3H), 6.91 (d,
1H, J = 7.8 Hz); 13C NMR (75 MHz, DMSO‑d6) δ (ppm): 172.49, 165.17,
152.91, 143.60, 135.38, 132.45, 125.72, 122.07, 121.70, 121.60,
116.03, 115.73, 110.99 and positive mode m/z (%): 299 (97%)
NTPDases is the principal enzyme family responsible for degradation
of adenosine triphosphate and adenosine diphosphate (ADP). They are
capable of hydrolyzing ATP to AMP (adenosine monophosphate) via
ADP production [24–25]. So far, eight members (NTPDase1-8) of this
family have been identified, each possessing distinct catalytic proper-
ties, substrate preference and tissue specific distribution [26–27]. Four
members of this family (NTPDase1, ꢀ 2, ꢀ 3, ꢀ 8) are ecto enzymes as
their catalytic site is present in extracellular space [28–29] whereas, the
remaining members have been designated as intracellular proteins and
include NTPDase4, ꢀ 5, ꢀ 6 and ꢀ 7 [27,30].
2.2.2. N-(3-nitrophenyl)-2-(2-oxoindolin-3-ylidene)hydrazine-1-
carboxamide (2b)
Yellow solid; Melting point = 225–228 ◦C; Yield: 74%; IR(KBr):
–
–
–
–
3343–3114(>NH stretch), 1694(>C O), 1597(>C
N ), 1522(>NH
–
bend), cmꢀ 1; 1H NMR (300 MHz, DMSO‑d6): δ (ppm) 10.80 (s, 1H, NH),
10.57 (s, 1H, NH), 10.03 (s, 1H, NH), 8.64 (t, 1H, J = 2.1 Hz), 8.09 (d,
1H, J = 7.8 Hz), 7.99–7.90 (m, 2H), 7.63 (t, 1H, J = 8.1 Hz), 7.39 (td,
1H, J = 7.5, 0.75 Hz), 7.09 (t, 1H, J = 7.65 Hz), 6.92 (d, 1H, J = 7.8 Hz);
13C NMR (75 MHz, DMSO‑d6) δ (ppm): 165.07, 153.17, 148.53, 143.77,
140.45, 136.32, 132.70, 130.66, 125.92, 122.11, 117.91, 115.99,
113.88, 11.05 and positive mode m/z (%): 326 (97%)
NTPDase3 is highly expressed in pancreatic β-cells where it has been
described to regulate the insulin release [31,32]. Lavoie and co-workers
used a commercially available non-selective NTPDase inhibitors termed
as BG0136 and NF279. These studies revealed that the inhibition of
NTPDase3 activity by BG0136 or NF279 affected insulin secretion [32].
Similarly, in another study treatment of pancreatic islets with ARL67156
(a non-selective NTPDase inhibitors) has been shown to induce insulin
secretion [33]. Thus it can be suggested that blocking the ATP hydrolysis
(by inhibiting NTPDase3) might represent a therapeutic strategy to treat
type 2 diabetes. At present, there is lack of data addressing this partic-
ular role of NTPDase3 and only few studies have been undertaken to
illustrate the involvement of NTPDase3 in insulin release. This scarcity
of data encouraged us to synthesize a set of oxoindolin phenylhydrazine
carboxamides that were likely to inhibit NTPDases. The sifted inhibitors
(of NTPDase3) were proceeded towards the insulin secretion study.
2.2.3. N-(4-methoxyphenyl)-2-(2-oxoindolin-3-ylidene)hydrazine-1-
carboxamide (2c)
Yellow solid; Melting point = 205–209 ◦C; Yield: 72%; IR(KBr):
–
–
–
–
3358–3218(>NH stretch), 1690(>C O), 1589(>C
N ), 1511(>NH
–
bend), cmꢀ 1; 1H NMR (300 MHz, DMSO‑d6): δ (ppm) 10.77 (s, 1H, NH),
10.36 (s, 1H, NH), 9.34 (s, 1H, NH), 8.08 (d, 1H, J = 7.5 Hz), 7.51–7.46
(m, 2H), 7.37 (t, 1H, J = 7.65 Hz,), 7.08 (t, 1H, J = 7.35 Hz), 6.95–6.90
(m, 3H), 3.74 (s, 3H; Alkyl-H); 13C NMR (75 MHz, DMSO) δ (ppm):
172.52, 165.20, 155.68, 152.77, 143.49, 134.93, 132.12, 125.61,
122.05, 121.48, 116.09, 114.51, 110.96, 55.65 and positive mode m/z
(%): 311 (69%)
2. Material and methods
2.2.4. N-(4-chlorophenyl)-2-(2-oxoindolin-3-ylidene)hydrazine-1-
carboxamide (2d)
All the chemicals and solvents were procured from commercial
suppliers and were not subjected to any additional purification, unless
specified otherwise. All reactions were checked by thin layer chroma-
tography (TLC), performed on 200 µm thick aluminum sheets laminated
with silica gel 60 F254 (Merck, Germany). Melting points were measured
using a digital Gallenkamp melting point apparatus (UK) and were un-
corrected. The IR spectra were determined using a Perkin Elmer BX-II
spectrometer (USA). Mass spectra were obtained using an API 2000
mass spectrometer (Applied Biosystems, Germany). 1H and 13C spectra
were recorded on a 300 MHz spectrophotometer (Bruker AM-300, USA)
while using DMSO–d6 as a solvent.
Yellow solid; Melting point = 215–217 ◦C; Yield: 74%; IR(KBr):
–
–
–
–
3345–3179(>NH stretch), 1690(>C O), 1595(>C
N ), 1537(>NH
–
bend), cmꢀ 1; 1H NMR (300 MHz, DMSO‑d6): δ (ppm) 10.78 (s, 1H, NH),
10.44 (s, 1H, NH), 9.65 (s, 1H, NH), 8.08 (d, 1H, J = 7.5 Hz), 7.65–7.60
(m, 2H), 7.39 (m, 3H), 7.08 (t, 1H, J = 7.5 Hz), 6.91 (d, 1H, J = 7.5 Hz);
13C NMR (75 MHz, DMSO‑d6) δ (ppm): 165.15, 152.75, 143.64, 138.05,
135.65, 132.52, 129.20, 127.06, 125.75, 122.08, 121.27, 116.02,
111.02 and positive mode m/z (%): 315 (99%)
2.2.5. N-(2,6-dimethylphenyl)-2-(2-oxoindolin-3-ylidene)hydrazine-1-
carboxamide (2e)
Yellow solid; Melting point = 186–189 ◦C; Yield: 75%; IR(KBr):
–
–
–
–
3355–3274(>NH stretch), 1703(>C O), 1580(>C
N ), 1520(>NH
–
2.1. General procedure for the synthesis of substituted phenylhydrazine
bend), cmꢀ 1; 1H NMR (300 MHz, DMSO‑d6): δ (ppm) 10.77 (s, 1H, NH),
10.50 (s, 1H, NH), 10.34 (s, 1H, NH), 8.09 (t, 1H, J = 10.0 Hz), 7.37 (t,
1H, J = 7.65 Hz), 7.12–7.04 (m, 4H), 6.91 (d, 1H, J = 7.8 Hz), 2.19 (d,
6H, J = 9.6 Hz, Alkyl-H); 13C NMR (75 MHz, DMSO) δ (ppm): 167.11,
153.16, 143.15, 136.21, 133.12, 132.51, 130.12, 128.27, 127.82,
122.63, 121.74, 115.45,18.62 and positive mode m/z (%): 309 (98%)
carboxamides (1a–j)
To a solution of hydrazine hydrate (in excess) in acetonitrile, cor-
responding isocyanates (1 mmol) were added drop wise. This reaction
was carried out in an ice bath and allowed to stir for half an hour. The
precipitated product was filtered off, washed and finally dried at room
temperature.
2.2.6. N-(2,6-dichlorophenyl)-2-(2-oxoindolin-3-ylidene)hydrazine-1-
carboxamide (2f)
2.2. General procedure for the synthesis of Oxoindolin-3-ylidene
Yellow solid; Melting point = 198–200 ◦C; Yield: 70%; IR(KBr):
phenylhydrazine carboxamides (2a–j)
–
–
–
–
3345–3205(>NH stretch), 1694(>C O), 1595(>C
N ), 1530(>NH
–
bend), cmꢀ 1; 1H NMR (300 MHz, DMSO‑d6): δ (ppm) 10.77 (s, 1H, NH),
10.35 (s, 1H, NH), 9.09 (s, 1H, NH), 8.03 (d, 1H, J = 7.5 Hz), 7.51 (d, 2H,
A solution consisting of isatin (1 mmol), an appropriate intermediate
(1a-j, 1 mmol) and few drops of acetic acid was prepared in acetonitrile
2

S. Afzal et al.
Bioorganic Chemistry 112 (2021) 104957
J = 8.1 Hz), 7.40–7.27 (m, 2H), 7.06 (t, 1H, J = 7.5 Hz), 6.91 (d, 1H, J =
7.8 Hz); 13C NMR (75 MHz, DMSO‑d6) δ (ppm): 166.15, 152.20, 145.13,
138.32, 136.12, 133.01, 130.24, 128.87, 127.24, 122.90, 121.14,
120.50, 115.92 and positive mode m/z (%): 350 (88%)
washing by centrifugation (300 × g, 4 ◦C) for 5 min. The washed cells
were resuspended in harvesting buffer incorporating aprotinin (10 µg/
mL) and then sonicated. Finally, nuclei and cellular debris were dis-
carded by another centrifugation (300 × g, 4 ◦C, 10 min) and resulting
protein extracts were aliquoted and stored at ꢀ 80 ◦C. Protein concen-
tration of these protein extracts was estimated by Bradford microplate
assay while using bovine serum albumin as a standard [36].
2.2.7. N-(2,4-dimethoxyphenyl)-2-(2-oxoindolin-3-ylidene)hydrazine-1-
carboxamide (2g)
Yellow solid; Melting point = 200–203 ◦C; Yield: 75%; IR(KBr):
–
–
–
–
3367–3022(>NH stretch), 1692(>C O), 1604(>C
N
), 1533(>NH
2.3.2. NTPDase activity assay
–
bend), cmꢀ 1; 1H NMR (300 MHz, DMSO‑d6): δ (ppm) 10.78 (s, 2H, NH),
9.02 (s, 1H, NH), 8.17 (d, 1H, J = 7.8 Hz), 7.95 (d, 1H, J = 8.7 Hz,), 7.36
(t, 1H, J = 7.5 Hz), 7.06 (t, 1H, J = 7.65 Hz), 6.90 (d, 1H, J = 7.5 Hz),
6.67 (d, 1H, J = 2.4 Hz), 6.54 (dd, 1H, J = 8.85, 2.55 Hz), 3.88 (s, 3H;
Alkyl-H), 3.75 (s, 3H, Alkyl-H); 13C NMR (75 MHz, DMSO) δ (ppm):
172.53, 165.14, 156.36, 152.62, 150.35, 143.61, 134.51, 132.33,
125.81, 122.00, 120.87, 116.01, 110.90, 104.73, 99.28, 56.39, 55.78
and positive mode m/z (%): 341 (89%)
NTPDase activity was assayed using malachite green reagent as
previously described, with slight modifications [37]. The assay was
performed in 50 mM tris HCl buffer (pH 7.4) containing 5 mM CaCl2.
Solutions of synthesized products were prepared in 10% DMSO solution
and were tested at 100 µM concentration. Firstly, assay buffer (55 µL)
and test compound solution (10 µL) were incubated with 10 µL of
enzyme solution for 10 min at 37 ◦C. The concentration of enzyme (per
well) used was as follows; h-NTPDase1 (59 ng), h-NTPDase2 (43 ng), h-
NTPDase3 (105 ng), h-NTPDase8 (89 ng). The reaction was started by
adding 10 µL of substrate solution (i.e. ATP) with a final concentration of
2.2.8. N-(4-nitrophenyl)-2-(2-oxoindolin-3-ylidene)hydrazine-1-
carboxamide (2h)
◦
100 µM. Then, the reaction was again incubated at 37 C for 15 min.
Yellow solid; Melting point = 255–257 ◦C; Yield: 80%; IR(KBr):
Finally, malachite green reagent (15 µL) was added to terminate the
reaction and a room temperature incubation was carried out. After 4–6
min, the absorbance of reaction mixture was measured on Omega
FLUOstar microplate reader (BMG Labtech, Germany) using a wave-
length of 630 nm. Percent inhibitions were computed for each com-
pound and all those compounds exhibiting > 50% inhibition of any
enzyme were further diluted to determine the IC50 values. Three inde-
pendent inhibition experiments were conducted in triplicate, dose
response curves were fitted and IC50 values were calculated using PRISM
5.0 (GraphPad, San Diego, USA).
–
–
–
–
3328–3175(>NH stretch), 1697(>C O), 1593(>C
N ), 1526(>NH
–
bend), cmꢀ 1; 1H NMR (300 MHz, DMSO‑d6): δ (ppm) 10.82 (s, 1H, NH),
10.57 (s, 1H, NH), 110.20 (s, 1H, NH), 8.25 (d, 2H, J = 9.0 Hz,), 8.08 (d,
1H, J = 7.5 Hz), 7.85 (d, 2H, J = 9.3 Hz), 7.40 (t, 1H, J = 7.8 Hz), 7.10 (t,
1H, J = 7.65 Hz), 6.92 (d, 1H, J = 7.8 Hz,); 13C NMR (75 MHz,
DMSO‑d6) δ (ppm): 165.07, 152.64, 145.66, 143.83, 142.36, 136.66,
132.79, 125. 92, 123.57, 122.12, 119.09, 115.92, 111.10 and positive
mode m/z (%): 326 (96%)
2.2.9. 2-(2-oxoindolin-3-ylidene)-N-(3-(trifluoromethyl)phenyl)
hydrazine-1-carboxamide (2i)
2.3.3. Islets isolation
Yellow solid; Melting point = 157–160 ◦C; Yield: 76%; IR(KBr):
In this study, BALB/c mice (30–40 g in weight) were used at 6–8
weeks of age to isolate the pancreatic islets. The animals were kept in an
environmentally controlled room at a temperature of 25 ± 2 ◦C with a
12-h light/12-h dark cycle. All procedures involving animal surgery
were in compliance with internationally accepted protocols. In addition,
all the protocols were approved by institutional committee on animal
care and use. (Protocol: PHM.Eth./CS-M01-020-1609)
–
–
–
–
3324–3218(>NH stretch), 1688(>C O), 1600(>C
N ), 1537(>NH
–
bend), cmꢀ 1; 1HNMR (300 MHz, DMSO‑d6): δ (ppm) 11.06 (s, 1H, NH),
9.66 (s, 1H, NH), 9.35 (s, 1H, NH), 7.62–7.49 (m, 4H), 7.32 (d, 1H, J =
7.8 Hz), 7.06 (t, 1H, J = 7.5 Hz), 6.91 (d, 1H, J = 7.8 Hz); 13C NMR (75
MHz, DMSO‑d6) δ (ppm): 184.87, 182.74, 159.83, 153.10, 151.17,
143.81, 142.24, 140.85, 138.82, 130.43, 125.15, 123.21, 122.34,
118.51, 114.55, 112.65 and positive mode m/z (%): 349 (96%)
Isolation of mice islets was performed according to previously
described protocol [38]. Following a general anesthesia, mice were
killed by cervical dislocation and placed under dissection microscope.
After identifying the ampulla, it was clamped and pancreas was dis-
tended with collagenase solution (3 mL), prepared at a concentration of
1 mg/mL. The distended pancreas was excised from the underlying
connective tissue, placed in a 50 mL tube containing collagenase solu-
tion and digested at 37 ◦C for 15 min. In order to stop the digestion, tube
was placed on ice and 20 mL of HBSS was added to wash the digested
islets by centrifugation at 4 ◦C for 1 min at 1000 rpm. This washing was
performed for 2–3 times and then islets were strained through a cell
strainer (70 µm). Finally, the islets were hand-picked under microscope.
All the steps involving isolation and purification of islets were per-
formed in HBSS, in the absence of calcium, magnesium and phenol red.
2.2.10. N-benzyl-2-(2-oxoindolin-3-ylidene)hydrazine-1-carboxamide (2j)
Yellow solid; Melting point = 193–196 ◦C; Yield: 83%; IR(KBr):
–
–
–
–
3350–3125(>NH stretch), 1682(>C O), 1599(>C
N ), 1526(>NH
–
bend), cmꢀ 1; 1H NMR (300 MHz, DMSO‑d6): δ (ppm) 11.99 (s, 1H, NH),
10.72 (s, 1H, NH), 10.35 (s, 1H, NH), 8.06 (d, 1H, J = 7.5 Hz), 7.80 (t,
1H, J = 6 Hz), 7.36–7.23 (m, 5H), 7.02 (t, 1H, J = 7.35 Hz), 6.88 (d, 1H,
J = 7.5 Hz), 4.41 (d, 2H, J = 6 Hz, CH2); 13C NMR (75 MHz, DMSO‑d6) δ
(ppm): 172.53, 165.16, 155.54, 143.38, 140.26, 134.14, 132.15,
128.77, 127.79, 125.73, 122.03, 116.10, 110.81, 43.36 and positive
mode m/z (%): 295 (95%)
2.3. Biological protocols
2.3.4. Insulin secretion by isolated islets
2.3.1. Enzyme preparations of NTPDases
Isolated islets were maintained in Krebs-Ringer bicarbonate buffer
(KRBB, pH 7.4) that was comprised of 118 mM NaCl, 1.9 mM CaCl2, 4.7
mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 10 mM HEPES and 0.1%
bovine serum albumin. During pre-incubation, islets (3 islets/vial) were
suspended in KRBB containing low glucose concentration (3 mM) at
Recombinant h-NTPDases were expressed by transfecting the COS-7
cells in 15 cm dishes using Lipofectamine as transfection reagent as
previously described [34–35]. In short, confluent cells were incubated in
Dulbecco’s Modified Eagle’s Medium (serum free) along with plasmid
◦
(6 µg) and Lipofectamine (24 µL) at 37 C (5 h). Following the termi-
◦
37 C for 45 min. Then, KRBB was substituted with fresh media sup-
nation of transfection, cells were collected by harvesting 40–72 h later.
plemented with high glucose concentration (16.7 mM) and islets were
exposed to test compounds for 1 h at 37 ◦C. Following incubation, su-
pernatant of each sample was removed and an aliquot of this sample was
diluted to determine the amount of secreted insulin. The insulin secre-
tion was quantified using mouse specific insulin ELIZA kit obtained from
Subsequently, the transfected cells were washed with tris-saline
◦
buffer (at 4 C) and used to prepare the protein extracts. Afterwards,
the cells were harvested by scrapping and transferred to harvesting
medium comprised of NaCl (95 mM), tris (45 mM) and phenyl-
methylsulfonyl fluoride (0.1 mM), pH 7.5 and passed through another
3

S. Afzal et al.
Bioorganic Chemistry 112 (2021) 104957
Crystal Chem Inc. (USA), in compliance with manufacturer’s directions.
The optical density was recorded at a wavelength of 450 nm and the
amount of insulin release was normalized to number of islets [38].
2.3.6.2. RNA extraction and real time qPCR. Total RNA extraction from
mice islets was carried out using the TRIzol reagent, following the
manufacturer’s instructions. After quantifying the RNA, cDNA was
generated by reverse transcribing 1 µg of this RNA. Briefly, a reaction
mixture (12 µL) composed of 1 µg RNA and 20 µM oligo-dT (1 µL) was
prepared, incubated for 5 min. at 65 ◦C and instantly placed on ice.
Then, an 8 µL aliquot of master mix consisting of buffer (5X, 4 µL),
dNTPs mix (10 mM, 2 µL), RNase inhibitor (1 µL) and reverse tran-
scriptase (1 µL) was added to above mentioned reaction mixture. The
resulting sample was mixed by gentle pipetting and run under following
temperature profile: 5 min at 25 ◦C, 70 min at 42 ◦C and 5 min at 70 ◦C.
One microliter of cDNA was used for PCR in a total volume of 20 µL.
Hence, PCR reaction mixture consisting of cDNA (1 µL), forward and
revers primers (0.25 µL each) and SYBR Green master mix (10 µL) was
formulated and PCR protocol was set as follows: one cycle of 95 ◦C for
10 min followed by 40 cycles of 95 ◦C for 15 sec and 55 ◦C for 60 sec.
Reactions were carried out with PikoReal 96 Real-Time PCR system
(Thermo Scientific, Vantaa, Finland). PCR of β actin was carried out as a
control for cDNA synthesis and relative expression levels were deter-
mined using ΔΔCT method.
2.3.5. Ectonucleotidase activity of islets
2.3.5.1. Islets’ homogenate preparation. Freshly isolated islets were
transformed into a homogenate in compliance with a previously
described procedure [39]. Briefly, islets after washing with HBSS were
added to an ice-cold buffer composed of 0.25 M sucrose, 1 mM EDTA
and 5 mM tris-HCl, pH 7.0. As a result, a suspension was formed and an
appropriate amount of tris buffer (pH 7.4) was added to dilute it. The
suspension was then sonicated for 30 sec resulting in the preparation of a
homogenate. Finally, nucleus and cellular debris was separated by a
centrifugation (15,000 rpm, 4 ◦C, 10 min) and supernatant was removed
and kept at ꢀ 20 ◦C till used in activity experiments. Bradford method
was used to estimate the protein concentration while using bovine
serum albumin as reference standard [36].
2.3.5.2. Ectonucleotidase activity of islet homogenate. After preparing the
islet homogenate, the ectonucleotidase activity was determined by
quantifying the liberated Pi using a colorimetric assay where KH2PO4
was utilized as a standard [32]. In order to derive a standard curve,
KH2PO4 was diluted and incubated with malachite green reagent at
room temperature. At the end, absorbance was recorded at 630 nm and
plotted against concentrations to construct a standard curve.
2.4. Molecular docking studies
The protein data bank does not contain the crystal structures of
human NTPDases, therefore, their homology models were built in
accordance with our previously described procedure [40]. MOE was
used for the preparation and energy minimization of selected com-
pounds whereas, structures of enzymes were prepared using the Bio-
SolveIT’s LeadIT software [41]. Finally, docking studies were carried
out with the help of BioSolveIT’s LeadIT software and the most favorable
pose with highest affinity and lowest binding energy was selected to
study the binding interactions.
The reaction medium was comprised of tris-HCl (50 mM, pH = 7.4)
supplemented with 5 mM CaCl2. The synthesized molecules were
screened at 100 µM concentrations and the working solution of these
inhibitors were formulated in 10% DMSO. Tris buffer (56 µL) containing
test compound (10 µL) was incubated with 6 µL of islet homogenate
◦
(containing 6 µg of protein) at 37 C. Following a 10 min incubation,
reaction was initiated by adding 10 µL of ATP (100 µM) and incubated
for 15 min at 37 ◦C. Afterwards, an aliquot of trichloroacetic acid (10%,
100 µL) was introduced to terminate the reaction followed by an incu-
bation (15 min.) of samples on ice. At the end, each sample was mixed
with an appropriate quantity of malachite green reagent and liberated
inorganic phosphate (Pi) was measured using standard curve. In order to
generate the dose response curve, each compound was tested at different
concentrations. Ectonucleotidase activity was described in the form
of nm of Pi/min/mg of protein. All the experiments were performed in
triplicate.
2.5. Statistical analysis
Data was shown as mean ± S.E.M. One-way ANOVA and student t
test were performed by the PRISM 5.0 software.
3. Results and discussion
3.1. Chemistry
A series of 10 oxoindolin phenylhydrazine carboxamide derivatives
(2a-j) was synthesized as potential inhibitors of NTPDases. It started
with the synthesis of substituted phenylhydrazine carboxamide (1a–j)
(Scheme 1).
2.3.6. Real time qPCR
2.3.6.1. Incubation of islets with test compounds. After washing the iso-
◦
lated islets, they were maintained at 37 C for 45 min in KRB buffer
The synthesized derivatives were exploited as starting material for
the synthesis of target compounds and refluxed with isatin for 5–6 h
(Table 1). Finally, precipitates of target compounds were obtained by
evaporating the solvent at room temperature. The obtained precipitates
were filtered, washed and characterized using IR, 1H NMR and 13C NMR
and LC/ESI-MS.
containing low glucose concentration (3 mM). The islets were then
exposed to test compounds for 3 h (37 ◦C) whereas, KRBB (supple-
mented with 16.7 mM glucose) was used as incubation medium. At the
end, supernatant was separated while islets were used to extract the
RNA.
Scheme 1. Synthesis of substituted N-phenylhydrazine carboxamides (1a–j).
4

S. Afzal et al.
Bioorganic Chemistry 112 (2021) 104957
Table 1
Synthesis of target compounds (2a–j).
The identity of these products was established by IR, 1H NMR, 13
C
3.2. Inhibitory activity of compounds on recombinant h-NTPDases
and mass spectra. Several peaks indicating the presence of specific
functional groups were observed in IR spectra of these molecules. In this
regard, absorption bands observed within the range of 3367–3302 cmꢀ 1
were corresponding to the NH stretching of amide and indole group. The
This series of carboxamides (2a–j) was evaluated to determine
inhibitory activity of these molecules against h-NTPDases. Four recom-
binant isozymes (h-NTPDase1, ꢀ 2, ꢀ 3 and ꢀ 8) were used and test
compounds were screened at an initial concentration of 0.1 mM. The
results are presented in Table 2.
–
–
–
peaks for C O and C N were observed in the region of 1703–1682
–
(cmꢀ 1) and 1604–1572 (cmꢀ 1), respectively. Similarly, the peaks in the
region of 1537–1511 (cmꢀ 1) were due to the presence of N H. The H
1
–
NMR spectra of the compounds showed three characteristic singlets in
3.2.1. Structure activity relationships
the region of δ (ppm) 9.02–10.35, 9.02–10.72, and 10.77–11.99, which
It was observed that h-NTPDase1 was the most receptive enzyme and
activity of many compounds was in micro molar range against h-
NTPDase1. Interestingly, four compounds (2e, 2f, 2g, 2h) were inhib-
iting the h-NTPDase1 selectively, having micro-molar IC50 values.
However, these compounds affected other isozymes to a lesser extent.
Except 2b, 2c and 2j, all the compounds inhibited h-NTPDase1
(>50%) and their dose response curves were generated. Among them,
IC50s of five compounds were < 1 µM and they included 2h, 2d, 2i, 2a
and 2f. From a structure–activity viewpoint, it was observed that h-
NTPDase1 inhibitory activity was strongly enhanced by introducing
electron withdrawing moiety (F, Cl, CF3 and NO2) at phenyl ring as
compared to electron donating moiety (CH3, OCH3). It can be
–
– –
N NH, respectively.
appeared due to CO NH, NH of indole and
–
Presence of these three NH peaks clearly indicated the formation of
target molecules. As per our anticipation, aromatic protons showed their
signals in the range of δ (ppm) 8.64–6.54. The 13C spectra revealed
carbonyl peaks in the range of δ (ppm) 184.87–165.07, while amide and
imine carbon signals were observed in the range δ (ppm) 182.74–152.20
and 159.83–143.15, respectively. Mass spectra of these compounds were
obtained (in positive mode) and molecular ion peak for each compound
was observed at respective m/z.
Table 2
NTPDase Inhibitory Activity of Compounds (2a–j).
Code
h-NTPDase1
IC50 (µM)a SEM/%inhibitionb
h-NTPDase2
h-NTPDase3
h-NTPDase8
2a
0.37 ± 0.02 a
45.9 ± 1.31b
47.5 ± 0.58b
0.16 ± 0.01a
2.30 ± 0.04a
0.70 ± 0.01a
1.09 ± 0.05a
0.12 ± 0.03a
0.29 ± 0.01a
45.2 ± 1.27b
16.1 ± 1.02a
0.27 ± 0.02a
44.8 ± 0.93b
27.1 ± 0.71b
0.15 ± 0.01a
25.8 ± 1.34b
32.1 ± 0.66b
35.0 ± 2.41b
41.1 ± 1.52b
30.9 ± 0.62b
42.1 ± 0.98b
24.1 ± 3.01a
0.30 ± 0.04a
2.82 ± 0.10a
48.8 ± 0.87b
40.0 ± 0.59b
32.4 ± 1.18b
41.1 ± 2.21b
29.3 ± 0.49b
27.5 ± 1.5b
0.16 ± 0.02a
42.2 ± 0.46b
36.4 ± 0.38b
45.7 ± 1.22b
37.1 ± 0.48b
27.9 ± 0.75b
24.7 ± 1.31b
27.6 ± 0.87b
28.7 ± 0.39b
41.4 ± 1.50b
101.1 ± 2.34a
2b
2c
2d
2e
2f
2g
2h
2i
1.46 ± 0.08 a
20.5 ± 1.14b
4.31 ± 0.41a
2j
Suramin
a
b
IC50 values are presented as mean ± SEM of three independent experiments.
Percent inhibition determined at 100 µM.
5

S. Afzal et al.
Bioorganic Chemistry 112 (2021) 104957
exemplified by making a comparison of 2e with 2f. Similarly, a com-
parison between 2c and 2d also makes the electron withdrawing group
more favorable
Likewise, the most active inhibitor against h-NTPDase1 (2h, IC50
=
0.12 ± 0.03 µM) also incorporated NO2 substituted phenyl ring which is
an electron withdrawing group. However, this activity seemed to be
position dependent because incorporating the same NO2 group at meta
position resulted in <50% inhibition. It seems that activity due to NO2
group is also position dependent.
Another trend observed here was the number of substitution and it
was observed that compounds mono substituted with electron with-
drawing scaffold were more active than those of di substituted. For
instance, 2d (containing 4-Cl phenyl ring) was 4 times more potent than
2f (possessing 2,6-dichloro phenyl ring) in terms of IC50 value.
In case of h-NTPDase2, only two compounds (2a and 2d) were
recognized as potent inhibitors as indicated by their IC50 values (<1
µM). Structures of both the compounds possessed a F- or Cl-substituted
phenyl ring, thus implying the importance of electron withdrawing
group. Moreover, compound 2d showed a dual inhibitory profile against
h-NTPDase1 and h-NTPDase2 sharing almost an equal IC50 value.
However, rest of the compounds showed < 50% inhibition of h-
NTPDase2.
Fig. 2. Mode of inhibition of h-NTPDase2 inhibitor (2d) indicated by
Lineweaver-Burk Plot. Inhibitor (2d) was used at following concentrations
(μM): 0, 0.07, 0.15 and 0.22.
Moving towards next isozyme, three compounds appeared to be
potential h-NTPDase3 inhibitors with their IC50s ranging between 0.30
± 0.04 µM to 2.82 ± 0.10 µM. Compound 2a was the most active h-
NTPDase3 inhibitor (IC50 = 0.30 ± 0.04 µM) whereas, IC50 of compound
2b and 2i was 2.82 ± 0.10 µM and 1.46 ± 0.08 µM, respectively. Here
compound 2b emerged as an important compound since it selectively
inhibited the h-NTPDase3.
Except compound 2a, all the compounds exhibited < 50% inhibition
of h-NTPDase8. Thus, this isoform was least affected by these
compounds.
Fig. 3. Mode of inhibition of h-NTPDase3 inhibitor (2a) indicated by
Lineweaver-Burk Plot. Inhibitor (2a) was used at following concentrations
(μM): 0, 0.15, 0.30 and 0.45.
3.2.2. Enzyme kinetics studies of carboxamide derivatives
Kinetic studies were performed for compounds 2h and 2d as the most
active inhibitors of h-NTPDase1 and h-NTPDase2. Since compound 2a
showed the dual inhibition of h-NTPDase3 and h-NTPDase8, it was
selected to perform kinetics studies against both enzymes. Hence
mechanism of action was demonstrated by generating Lineweaver Burk
plot for each compound. It was observed that compound 2h was an
uncompetitive inhibitor of h-NTPDase1 (Fig. 1) whereas, 2d inhibited
the h-NTPDase2 competitively (Fig. 2). Likewise, compound 2a was also
a competitive inhibitor of h-NTPDase3 and h-NTPDase8 (Figs. 3, 4).
3.3. Effect of NTPDase3 inhibitors on insulin secretion
In order to determine the effects of NTPDase3 inhibitors on insulin
secretion, compound 2a, 2b and 2j were incubated with mice pancreatic
Fig. 4. Mode of inhibition of h-NTPDase8 inhibitor (2a) indicated by
Lineweaver-Burk Plot. Inhibitor (2a) was used at following concentrations
(μM): 0, 0.08, 0.16 and 0.24.
islets under low and high glucose condition. Negative control (con-
taining no drug) and positive control (containing IBMX) were also
included in the study. As shown in Fig. 5, compound 2a elicited an
outstanding surge in insulin release in comparison to control that was
16.7 mM glucose alone. The compound 2a was evaluated at various
concentrations (10 µM to 200 µM); it demonstrated a dose-dependent
behavior indicating a maximum effect on insulin secretion at 200 µM
(Fig. 6). The compound 2a had no prominent effect under low glucose
concentration since glucose is regarded as the bona fide initiator of in-
sulin secretion, while ATP promotes this insulin secretion in the pres-
ence of elevated glucose levels [8].
Fig. 1. Mode of inhibition of h-NTPDase1 inhibitor (2h) indicated by
Lineweaver-Burk Plot. Inhibitor (2h) was used at following concentrations
In contrast, compound 2b and 2i did not show any significant
(μM): 0, 0.05, 0.10 and 0.20.
6

S. Afzal et al.
Bioorganic Chemistry 112 (2021) 104957
Fig. 5. The effect of compounds 2a, 2b, 2i, suramin (Sur) and IBMX on insulin
secretion in mice islets under stimulation of 16.7 mM glucose. Data are shown
as mean ± S.E.M. from 2 to 3 separate experiments. Insulin release resulting
from 16.7 mM glucose (alone) was assumed to be 100% and set as control. ***P
< 0.001 vs. none.
Fig. 7. Test compound 2a and suramin significantly reduced the ectonucleo-
tidase activity in mice islets’ homogenate. A complete enzymatic reaction in the
absence of inhibitor was assumed to be 100% and set as control. Data are shown
as means ± SEM from three separate experiments. ***P < 0.0001.
exploited the ARL67156 as a non-selective inhibitor of NTPDase to
establish that it was the suppression of NTPDase3 activity that was
culminating itself in the stimulation of insulin release. Moreover, they
carried out the siRNA studies to confirm these results [34].
3.4. Effect of compound 2a on ectonucleotidase activity in isolated mice
islets
Since compound 2a appeared to be a potent NTPDase3 inhibitor that
was also involed in the modulation of insulin release, we intended to
determine how this compound (2a) was affecting the ectonucleotidase
activity. Hence, freshly isolated islets were transformed into an ho-
mogenate which was then incubated with compound 2a (100 µM) and
then malachite green assay was used to establish the ectonucleotidase
activity. The results indicated that treating the islets with 2a sub-
stantialy reduced the ectonucleotidase activity and this compound 2a
(in terms of activity) was equivalent to that of suramin (Fig. 7). Subse-
quently, a concentration–response curve was constructed and com-
pound was observed to reduce the ectonucleotidase activity in a dose-
dependent manner (Fig. 8).
Fig. 6. Effect of different doses of test compound (2a) at high glucose con-
centration (16.7 mM) on insulin secretion. The compound (2a) was added at the
concentrations of 10 µM, 50 µM, 100 µM and 200 µM. Data are shown as mean
± S.E.M. from 2 to 3 separate experiments. ***P < 0.001, **P < 0.01.
3.5. Effect of compound 2a on expression of NTPDase3 mRNA
activity, although both of these compounds showed good activity
against h-NTPDase3. Suramin, used as positive control throughout
enzyme inhibition experiments, also had no significant activity. Thus by
blocking the ATP hydrolysis, NTPDase3 inhibitor (2a) is participating in
the regulation of insulin secretion. Since NTPDase3 has been reported to
be the only ectonucleotidase abundantly expressed in pancreatic islets of
human and mice, it is the NTPDase3 inhibition that provokes the insulin
release. Within this framework, Syed et al., performed a study where
they identified the NTPDase3 as the most dominant isoform and
To further explore the action of compound 2a on mRNA of
NTPDase3, mice pancreatic islets were isolated and treated with com-
pound 2a for 3 h. Subsequently, total RNA was isolated from these islets
and analyzed by quantitative real time PCR. The results indicated that
NTPDase3 mRNA level was not significantly affected by NTPDase3 in-
hibitor (2a) exposure. Hence, this inhibition of NTPDase3 did not pro-
duced any change at gene level and only inhibition of protein led to
regulation of insulin secretion (Fig. 9).
7

S. Afzal et al.
Bioorganic Chemistry 112 (2021) 104957
70
60
50
40
30
20
-4
-2
0
2
4
Concentration, Log (µM)
Fig. 8. Different concentrations of compound 2a inhibited the ectonucleotidase
activity (in islets’ homogenate) that was measured in the form of Pi liberated
from externally introduced ATP (100 µM). Data points indicate the mean ± SEM
from three separate experiments.
Fig. 10. Three dimensional interaction pose of 2d docked in human
NTPDase2 model.
Fig. 9. Effect of compound 2a on NTPDase3 mRNA by qPCR. The compound 2a
was incubated with islets for 3 h at 37 ◦C in KRB buffer in the presence of 16.7
mM. RNA was isolated for RT-qPCR. Data was normalized to β-actin. Values are
mean ± S.E.M. from 2 to 3 independent experiments. Control, gene expression
of the group treated with 16.7 mM glucose only. ns: none significant.
Fig. 11. Three dimensional interaction pose of 2a docked in human
3.6. Molecular docking studies of carboxamide derivatives
NTPDase3 model.
In order to determine the putative binding mode, molecular docking
studies of the most potent inhibitors were performed for respective
isozyme. Considering the mode of inhibition, compounds possessing a
competitive mechanism of inhibition were selected for docking studies.
Thus compound 2a and 2d were docked within the active site of
respective enzyme.
were making hydrogen bond with Ala63 whereas, another hydrogen
bond was formed between NH group and Asp62. The indole ring was
experiencing several pi-anion interactions with Asp62, Asp219 and
Glu182. Moreover, a pi-alkyl interaction was also noticed between
Ala223 and 4-fluoro phenyl ring.
The amino acid residues lining the catalytic site of h-NTPDase8 were
comprised of His53, Asp48, Tyr59, Gly42, Trp440, Val436, Thr441 and
Leu442. The compound was attached to active site via non-covalent
bond formation, including hydrogen bond, pi-pi stacking and pi-anion
While docking the compound 2d within the active pocket of
modelled h-NTPDase2, three hydrogen bonds were formed with Arg392
through imine and amide functionalities of the compound. The side
chain of Ala393 was involved in pi-alkyl interaction with 4-chloro
phenyl ring whereas, indole ring appeared to be stabilized by
involving in a pi-pi stacked interaction with Tyr350 and His50 (Fig. 10).
The docking analysis of compound 2a displayed a number of in-
teractions with different amino acids inside the active pocket of
–
interactions. In this regard, imine group (C N) was making hydrogen
–
bond with Trp440 and Thr441 whereas, another hydrogen bond was
formed between fluorine and His53. Similarly, Asp48 was also observed
to have a hydrogen bond interaction with NH of amide functionality.
The alkyl groups in Val436 and Leu442 were observed to have pi-alkyl
interaction with indole phenyl ring, while Tyr59 was found to be
involved in the pi-pi (T-shaped) interaction with the same ring (Fig. 12).
modelled h-NTPDase3. As shown in Fig. 11, the indole ring of compound
–
2a was making a hydrogen bond with Arg67 through its C O group.
–
The amine functionality was also involved in hydrogen bond interaction
with aromatic side chain of Trp549. Similarly, both NH of amide group
8

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In conclusion, oxoindolin phenylhydrazine carboxamide derivatives
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Declaration of Competing Interest
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The authors declare that they have no known competing financial
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the work reported in this paper.
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Acknowledgements
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This work was financially supported by the German.
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Appendix A. Supplementary material
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