Functionalized Oxoindolin Hydrazine Carbothioamide Derivatives as Highly Potent Inhibitors of Nucleoside Triphosphate Diphosphohydrolases

Article abstract of DOI:10.3389/fphar.2020.585876

Ectonucleoside triphosphate diphosphohydrolases (NTPDases) are ectoenzymes that play an important role in the hydrolysis of nucleoside triphosphate and diphosphate to nucleoside monophosphate. NTPDase1, -2, -3 and -8 are the membrane bound members of this enzyme family that are responsible for regulating the levels of nucleotides in extracellular environment. However, the pathophysiological functions of these enzymes are not fully understood due to lack of potent and selective NTPDase inhibitors. Herein, a series of oxoindolin hydrazine carbothioamide derivatives is synthesized and screened for NTPDase inhibitory activity. Four compounds were identified as selective inhibitors of h-NTPDase1 having IC₅₀ values in lower micromolar range, these include compounds 8b (IC₅₀ = 0.29 ± 0.02 μM), 8e (IC₅₀ = 0.15 ± 0.009 μM), 8f (IC₅₀ = 0.24 ± 0.01 μM) and 8l (IC₅₀ = 0.30 ± 0.03 μM). Similarly, compound 8k (IC₅₀ = 0.16 ± 0.01 μM) was found to be a selective h-NTPDase2 inhibitor. In case of h-NTPDase3, most potent inhibitors were compounds 8c (IC₅₀ = 0.19 ± 0.02 μM) and 8m (IC₅₀ = 0.38 ± 0.03 μM). Since NTPDase3 has been reported to be associated with the regulation of insulin secretion, we evaluated our synthesized NTPDase3 inhibitors for their ability to stimulate insulin secretion in isolated mice islets. Promising results were obtained showing that compound 8m potently stimulated insulin secretion without affecting the NTPDase3 gene expression. Molecular docking studies of the most potent compounds were also carried out to rationalize binding site interactions. Hence, these compounds are useful tools to study the role of NTPDase3 in insulin secretion.

Full text of DOI:10.3389/fphar.2020.585876

ORIGINAL RESEARCH
published: 30 November 2020
doi: 10.3389/fphar.2020.585876
Functionalized Oxoindolin Hydrazine
Carbothioamide Derivatives as Highly
Potent Inhibitors of Nucleoside
Triphosphate Diphosphohydrolases
,4
Saira Afzal1, Mariya al-Rashida2, Abdul Hameed1, Julie Pelletier3, Jean Sévigny3 and
Jamshed Iqbal1*
1Centre for Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus, Abbottabad, Pakistan,
2Department of Chemistry, Forman Christian College (A Chartered University), Lahore, Pakistan, 3Centre de Recherche du CHU
de Québec–Université Laval, Québec City, QC, Canada, 4Département de Microbiologie-Infectiologie et d’Immunologie, Faculté
de Médecine, Université Laval, Québec City, QC, Canada
Ectonucleoside triphosphate diphosphohydrolases (NTPDases) are ectoenzymes that play
an important role in the hydrolysis of nucleoside triphosphate and diphosphate to nucleoside
monophosphate. NTPDase1, -2, -3 and -8 are the membrane bound members of this
enzyme family that are responsible for regulating the levels of nucleotides in extracellular
environment. However, the pathophysiological functions of these enzymes are not fully
understood due to lack of potent and selective NTPDase inhibitors. Herein, a series of
oxoindolin hydrazine carbothioamide derivatives is synthesized and screened for NTPDase
inhibitory activity. Four compounds were identified as selective inhibitors of h-NTPDase1
having IC50 values in lower micromolar range, these include compounds 8b (IC50 ꢀ 0.29
0.02 µM), 8e (IC50 ꢀ 0.15 0.009 µM), 8f (IC50 ꢀ 0.24 0.01 µM) and 8l (IC50 ꢀ 0.30
0.03 µM). Similarly, compound 8k (IC50 ꢀ 0.16 0.01 µM) was found to be a selective
h-NTPDase2 inhibitor. In case of h-NTPDase3, most potent inhibitors were compounds 8c
(IC50 ꢀ 0.19 0.02 µM) and 8m (IC50 ꢀ 0.38 0.03 µM). Since NTPDase3 has been reported
to be associated with the regulation of insulin secretion, we evaluated our synthesized
NTPDase3 inhibitors for their ability to stimulate insulin secretion in isolated mice islets.
Promising results were obtained showing that compound 8m potently stimulated insulin
secretion without affecting the NTPDase3 gene expression. Molecular docking studies of the
most potent compounds were also carried out to rationalize binding site interactions. Hence,
these compounds are useful tools to study the role of NTPDase3 in insulin secretion.
Edited by:
Salvatore Salomone,
University of Catania, Italy
Reviewed by:
Dunja Drakulic,
University of Belgrade, Serbia
Terence L. Kirley,
University of Cincinnati, United States
Andreia Machado Cardoso,
Universidade Federal Da Fronteira Sul,
Brazil
*Correspondence:
Jamshed Iqbal
jamshediqb@googlemail.com
Specialty section:
This article was submitted to
Experimental Pharmacology and
Drug Discovery,
a section of the journal
Frontiers in Pharmacology
Received: 21 July 2020
Accepted: 13 October 2020
Published: 30 November 2020
Keywords: carbothioamide, ectonucleotidases, nucleoside triphosphate diphosphohydrolase-3, oxoindolin
hydrazine, molecular docking
Citation:
Afzal S, al-Rashida M, Hameed A,
Pelletier J, Sévigny J and Iqbal J (2020)
Functionalized Oxoindolin Hydrazine
Carbothioamide Derivatives as Highly
Potent Inhibitors of Nucleoside
INTRODUCTION
Glucose acts as a natural insulin secretagogue and maintains the blood glucose level. In response to
an increase in blood glucose level, pancreatic beta cells release insulin to maintain glucose levels
(Chen et al., 2018; Stuhlmann et al., 2018). However, this insulin secretion is not an isolated event,
and is followed by release of other important components like peptides, Zn+2, adenosine
triphosphate (ATP) and other related nucleotides (Tozzi et al., 2018; Vakilian et al., 2019).
Triphosphate Diphosphohydrolases.
Front. Phar. 11:585876.
doi: 10.3389/fphar.2020.585876
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Oxoindolin Hydrazine Carbothioamides as NTPDases Inhibitors
Among them, ATP is an important signaling molecule that
amplifies the glucose stimulated insulin secretion by activating
purinergic (P2X and P2Y) receptors, present on the surface of
β-cell membrane (Cie´slak and Cie´slak, 2017). P2X receptors are
ionotropic receptors that are subdivided into seven types (P2X1
to P2X7). They have been associated with the transmembrane
flow of cations such as Na+, Ca+2, and K+ ions (Kasuya et al.,
2017a; Kasuya et al., 2017b; Giuliani et al., 2019). On the other
hand, P2Y receptors are metabotropic receptors that are
subdivided into eight types (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11,
P2Y12, P2Y13, and P2Y14) and have been linked to Ca+2
mobilization and generation or inhibition of cyclic adenosine
monophosphate (cAMP) (Dou et al., 2018; Lu et al., 2019). In
pancreatic β-cells, both receptor types have been reported to be
involved in the regulation of insulin secretion; however, their
mode of action is slightly different (Galicia-Garcia et al., 2020). In
this regard, P2X receptor activation by ATP leads to Ca+2 flux that
is believed to be responsible for P2X-mediated insulin secretion
(Solini and Novak, 2019). Whereas, activation of P2Y receptors
stimulates the inositol triphosphate, which in turn results in
transient elevation of intracellular concentration of Ca+2
(Bartley et al., 2019).
the need for further detailed investigations to support these claims.
However, to the best of our knowledge, there is still a lack of studies
focusing on this aspect of NTPDase3. Keeping in view this scarcity
of data, we synthesized a class of oxoindolin hydrazine
carbothioamide derivatives as NTPDase inhibitors. The
NTPDase3 inhibitors were further analyzed for their effects on
insulin secretion in mice pancreatic islets.
MATERIALS AND METHODS
Chemical Synthesis
General Comments
All reagents were obtained from commercial sources and used
without further purification, unless stated otherwise. Reaction
progress was monitored by thin layer chromatography, using
aluminum sheets precoated with silica gel 60 F254 (200 μm,
Merck, Darmstadt, Germany). Melting points were
determined on Weiss Gallenkamp melting point apparatus
(Loughborough, England) and are uncorrected. IR spectra
were recorded on Perkin Elmer BX-II spectrometer
1
(Waltham, United States). H and 13C spectra were obtained
The extracellular levels of ATP and related nucleotides are
controlled by a cascade of enzymes known as ectonucleotidases.
This enzymatic cascade is composed of four main families,
using Bruker AM-300 spectrophotometer (Billerica, United
States). Chemical shifts were described in parts per million.
Mass spectra were recorded on an Applied Biosystems API 2000
mass spectrometer (Darmstadt, Germany).
including
NTPDases
(ectonucleoside
triphosphate
diphosphohydrolases), APs or ALPs (alkaline phosphatases),
NPPs (nucleotide pyrophosphatases/phosphodiesterases) and
e-5′-NT (ecto-5′-nucleotidase) (Lee et al., 2017; Lee and
General Procedure for the Synthesis of Substituted
Phenylhydrazine Carbothioamides (7a-m)
´
Excess of hydrazine hydrate was dissolved in ethanol and a
solution of appropriate isothiocyanate (1 mMol) was prepared
in ethanol. The isothiocyanate solution was added drop wise to
hydrazine solution. The reaction was conducted on ice bath and
stirred for half an hour. During the course of reaction, precipitates
were formed which were filtered, washed and then dried.
Müller, 2017; Tozzi and Novak, 2017; Grkovic et al., 2019;
Nabinger et al., 2020). These are plasma membrane bound
ecto-enzymes and regulate the availability of ligands
(nucleotides and hydrolysis products thereof) for P1 and P2
receptors. Among them, NTPDase is an important member
contributing to the hydrolysis of nucleoside tri- and
diphosphates (Longhi et al., 2017; Bagatini et al., 2018). The
NTPDase family comprises of eight members or isozymes
(NTPDase1-8), these isozymes differ in their catalytic
properties and show varying substrate preference (Allard et al.,
2017; Zhong et al., 2017). NTPDase1, -2, -3, and -8 are membrane
bound isoforms that play an important role in purinergic
signaling (Castilhos et al., 2018; Bertoni et al., 2020) whereas
NTPDase4, -5, -6, and -7 are expressed within intracellular
organelles (Zhong et al., 2017; Paes-Vieira et al., 2018).
NTPDase3 is abundantly present in the pancreatic β-cells
where it has been reported to play an important role in the
regulation of insulin secretion (Bartley et al., 2019; Saunders et al.,
2019). In this context, a study was conducted where 1-naphthol-
3, 6-disulfonic acid (BG0136) and 8,8′-[Carbonylbis(imino-
4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,
3,5-naphthalenetrisulfonic acid (NF279) were used to explore
the role of NTPDase3 in insulin secretion (Lavoie et al., 2010).
Similarly, in another study 6-N,N-Diethyl-D-β-c-dibromomethylene
adenosine triphosphate (ARL67156) was used for this purpose and
it was found that NTPDase3 was involved in the modulation of
insulin release (Syed et al., 2013). These data highlight the potential
(important) role of NTPDase3 in insulin secretion and necessitate
General Procedure for the Synthesis of
Oxoindolin-Ylidene Phenylhydrazine
Carbothioamides (8a-m)
A solution of isatin (1 mMol, 0.147 g) was prepared in ethanol
and few drops of acetic acid were added. Resulting solution was
refluxed for 4 to 5 h with equimolar amount of an appropriate
intermediate (7a-m), synthesized during last step. Progress of
reaction was monitored by thin layer chromatography. At the end
of reaction, the precipitated solid was filtered and washed with
ethanol (Pervez et al., 2009).
(Z)-2-(2-oxoindolin-3-ylidene)-N-phenylhydrazine-1-
carbothioamide (8a)
Obtained as yellow solid, Yield ꢀ 78%, mp ꢀ 245–247°C; IR
(KBR): 3,280–3,172 (NH stretching), 1,691 (C ꢀ O), 1,603 (C ꢀ
N), 1,560 (NH bending), 1,161 (C ꢀ S), cm−1; 1HNMR (300 MHz,
DMSO-d6): δ (ppm) 12.80 (s, 1H, N-H), 11.26 (s, 1H, N-H), 10.82
(s, 1H, N-H), 7.78 (d, J ꢀ 7.2, 1H, Ar-H), 7.61 (d, J ꢀ 7.8, 2H, Ar-
H), 7.39 (m, 3H, Ar-H), 7.27 (t, J ꢀ 7.35, 1H, Ar-H) 7.11 (t, J ꢀ 7.5,
1H, Ar-H), 6.95 (d, J ꢀ 7.8, 1H, Ar-H); 13C-NMR (75 MHz,
DMSO-d6) δ (ppm): 175.60, 163.10, 146.23, 135.16, 132.88,
131.10, 128.06, 126.23, 125.45, 123.12, 122.18, 120.05, 111.67;
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Oxoindolin Hydrazine Carbothioamides as NTPDases Inhibitors
LC-MS (m/z): positive mode 297 [M + H]+. Purity determined by
HPLC-UV (254 nm)-ESI-MS: 97.26%
(Z)-N-(4-nitrophenyl)-2-(2-oxoindolin-3-ylidene)hydrazine-
1-carbothioamide (8b)
N), 1,535 (NH bending), 1,170 (C ꢀ S), cm−1; 1HNMR (300 MHz,
DMSO-d6): δ (ppm) 12.66 (s, 1H, N-H), 11.22 (s, 1H, N-H), 9.82
(s,1H, N-H), 7.65 (d, J ꢀ 7.5, 1H, Ar-H), 7.35 (m, 5H, Ar-H), 7.26
(m, 1H, Ar-H), 7.08 (d, J ꢀ 7.5, 1H, Ar-H), 6.93 (d, J ꢀ 7.8, 1H, Ar-
H), 4.88 (d, J ꢀ 6, 2H, Alkyl H); 13C-NMR (75 MHz, DMSO-d6) δ
(ppm): 178.17, 163.11, 142.82, 138.87, 132.53, 131.72, 128.75,
127.81, 127.48, 122.77, 121.37, 120.42, 111.55, 47.67; LC-MS (m/
z): positive mode 311 [M + H]+. Purity determined by HPLC-UV
(254 nm)-ESI-MS: 97.81%
Obtained as yellow solid, Yield ꢀ 86%, mp ꢀ 271–272°C; IR
(KBR): 3,315–3,222 (NH stretching), 1,694 (C ꢀ O), 1,600 (C ꢀ
N), 1,556 (NH bending), 1,168 (C ꢀ S), cm−1; 1HNMR (300 MHz,
DMSO-d6): δ (ppm) 13.01 (s, 1H, N-H), 11.31 (s, 1H, N-H), 11.12
(s, 1H, N-H), 8.29 (m, 2H, Ar-H), 8.09 (m, 2H, Ar-H), 7.78 (d, J ꢀ
7.2, 1H, Ar-H), 7.40 (td, J ꢀ 7.8, 1.2 Hz, 1H, Ar-H), 7.13 (t, J ꢀ 7.5,
1H, Ar-H), 6.96 (d, J ꢀ 7.8, 1H, Ar-H); 13C-NMR (75 MHz,
DMSO-d6) δ (ppm): 176.53, 163.20, 145.12, 144.60, 143.24,
133.79, 132.30, 125.24, 124.47, 122.93, 122.07, 120.15, 111.69;
LC-MS (m/z): positive mode 342 [M + H]+. Purity determined by
HPLC-UV (254 nm)-ESI-MS: 97.18%
(Z)-N-(3-chlorophenyl)-2-(2-oxoindolin-3-ylidene)hydrazine-1-
carbothioamide (8g)
Obtained as yellow solid, Yield ꢀ 74%, mp ꢀ 235–236°C; IR
(KBR): 3,340–3,192 (NH stretching), 1,694 (CꢀO), 1,594 (CꢀN),
1,535 (NH bending), 1,170 (CꢀS), cm−1; 1HNMR (300 MHz,
DMSO-d6): δ (ppm) 12.86 (s, 1H, N-H), 11.28 (s, 1H, N-H), 10.87
(s, 1H, N-H), 7.79 (t, J ꢀ 2.1, 1H, Ar-H), 7.76 (s, 1H, Ar-H), 7.65
(dq, J ꢀ 8.1, 1.2, 1H, Ar-H), 7.45 (t, J ꢀ 7.95, 1H, Ar-H), 7.35 (m,
2H, Ar-H), 7.12 (t, J ꢀ 7.35, 1H, Ar-H), 6.95 (d, J ꢀ 7.8, 1H, Ar-H);
13C-NMR (75 MHz, DMSO-d6) δ (ppm); 176.77, 163.18, 143.09,
140.40, 133.19, 132.91, 132.08, 130.44, 126.28, 125.53, 124.52,
122.87, 121.93, 120.28, 111.62; LC-MS (m/z): positive mode 331
[M + H]+. Purity determined by HPLC-UV (254 nm)-ESI-MS:
98.38%
(Z)-N-(4-chlorophenyl)-2-(2-oxoindolin-3-ylidene)hydrazine-1-
carbothioamide (8c)
Obtained as yellow solid, Yield ꢀ 85% mp ꢀ 240–244°C; IR (KBR):
3,315–3,210 (NH stretching), 1,692 (C ꢀ O), 1,591 (C ꢀ N), 1,527
(NH bending), 1,164 (C ꢀ S), cm−1; 1HNMR (300 MHz, DMSO-d6):
δ (ppm) 12.84 (s, 1H, N-H), 11.27 (s, 1H, N-H), 10.86 (s, 1H, N-H),
7.76 (d, J ꢀ 7.2, 1H, Ar-H), 7.66 (m, 2H, Ar-H), 7.49 (m, 2H, Ar-H),
7.37 (td, J ꢀ 7.65, 1.1, 1H, Ar-H), 7.11 (td, J ꢀ 7.5, 0.7, 1H, Ar-H), 6.94
(d, J ꢀ 7.8, 1H, Ar-H); 13C-NMR (75 MHz, DMSO-d6) δ (ppm):
176.86, 163.17, 143.04, 137.92, 133.06, 132.01, 130.56, 128.79, 127.79,
122.86, 121.88, 120.30, 111.60; LC-MS (m/z): positive mode 331 [M +
H]+. Purity determined by HPLC-UV (254 nm)-ESI-MS: 95.63%
(Z)-N-(4-fluorophenyl)-2-(2-oxoindolin-3-ylidene)hydrazine-1-
carbothioamide (8d)
(Z)-2-(2-oxoindolin-3-ylidene)-N-(p-tolyl)hydrazine-1-
carbothioamide (8h)
Obtained as yellow solid, Yield ꢀ 79%, mp ꢀ 240–242°C; IR
(KBR): 3,260–3,130 (NH stretching), 1,698 (C ꢀ O), 1,608 (C ꢀ
N), 1,530 (NH bending), 1,158 (C ꢀ S), cm−1; 1HNMR (300 MHz,
DMSO-d6): δ (ppm) 12.77 (s, 1H, N-H), 11.25 (s, 1H, N-H), 10.75
(s, 1H, N-H), 7.77 (d, J ꢀ 7.2, 1H, Ar-H), 7.47 (d, J ꢀ 8.1, 2H, Ar-
H), 7.37 (td, J ꢀ 7.8, 1.2, 1H, Ar-H), 7.22 (d, J ꢀ 8.4, 2H, Ar-H),
7.10 (dt, J ꢀ 7.65, 0.7, 1H, Ar-H), 6.94 (d, J ꢀ 7.8, 1H, Ar-H), 2.47
(s, 2H, Alkyl H); 13C-NMR (75 MHz, DMSO-d6) δ (ppm);
176.79, 163.17, 142.92, 136.36, 135.83, 132.61, 131.85, 129.31,
126.04, 122.82, 121.84, 120.41, 111.54, 21.0.
Obtained as yellow solid, Yield ꢀ 87%, mp ꢀ 255–256°C; IR
(KBR): 3,290–3,185 (NH stretching), 1,694 (C ꢀ O), 1,610 (C ꢀ
N), 1,560 (NH bending), 1,166 (C ꢀ S), cm−1; 1HNMR (300 MHz,
DMSO-d6): δ (ppm) 12.81 (s, 1H, N-H), 11.27 (s, 1H, N-H), 10.83
(s, 1H, N-H), 7.75 (d, J ꢀ 7.5, 1H, Ar-H), 7.60 (dd, J ꢀ 8.7, 5.1, 2H,
Ar-H), 7.37 (t, J ꢀ 7.35, 1H, Ar-H), 7.26 (t, J ꢀ 8.85, 2H, Ar-H),
7.11 (t, J ꢀ 7.5, 1H, Ar-H), 6.94 (d, J ꢀ 7.8, 1H, Ar-H); 13C-NMR
(75 MHz, DMSO-d6) δ (ppm): 177.20, 162.10, 158.88, 142.99,
135.26, 132.89, 131.95, 128.52, 122.86, 121.82, 120.35, 115.74,
111.58; LC-MS (m/z): positive mode 315 [M + H]+. Purity
determined by HPLC-UV (254 nm)-ESI-MS: 98.93%
(Z)-N-(2,6-dimethylphenyl)-2-(2-oxoindolin-3-ylidene)
hydrazine-1-carbothioamide (8i)
Obtained as yellow solid, Yield ꢀ 83%, mp ꢀ 250-252°C; IR
(KBR): 3,250–3,155 (NH stretching), 1,688 (C ꢀ O), 1,619 (C ꢀ
N), 1,520 (NH bending), 1,174 (C ꢀ S), cm−1; 1HNMR (300 MHz,
DMSO-d6): δ (ppm) 12.76 (s, 1H, N-H), 11.23 (s, 1H, N-H), 10.61
(s, 1H, N-H), 7.73 (d, J ꢀ 7.5, 1H, Ar-H), 7.36 (t, J ꢀ 7.65, 1H, Ar-
H), 7.17 (m, 3H, Ar-H), 7.09 (t, J ꢀ 7.5, 1H, Ar-H), 6.94 (d, J ꢀ 7.8,
1H, Ar-H), 2.20 (s, 6H, Alkyl H); 13C-NMR (75 MHz, DMSO-d6)
δ (ppm); 177.43, 163.12, 142.92, 136.82, 136.52, 132.58, 131.77,
128.31, 127.89, 122.81, 121.71, 120.49, 111.51, 18.32; LC-MS (m/
z): positive mode 325 [M + H]+. Purity determined by HPLC-UV
(254 nm)-ESI-MS: 98.27%
(Z)-N-(3-methoxyphenyl)-2-(2-oxoindolin-3-ylidene)hydrazine-
1-carbothioamide (8e)
Obtained as yellow solid, Yield ꢀ 80%, mp ꢀ 220–224°C; IR
(KBR): 3,240–3,189 (NH stretching), 1,690 (C ꢀ O), 1,595 (C ꢀ
N), 1,526 (NH bending), 1,161 (C ꢀ S), cm−1; 1HNMR (300 MHz,
DMSO-d6): δ (ppm) 12.80 (s, 1H, N-H), 11.27 (s, 1H, N-H), 10.77
(s, 1H, N-H), 7.79 (d, J ꢀ 7.5, 1H, Ar-H), 7.35 (m, 3H, Ar-H), 7.24
(d, J ꢀ 7.2, 1H, Ar-H), 7.11 (t, J ꢀ 7.5, 2H, Ar-H), 6.94 (d, J ꢀ 7.8,
1H, Ar-H), 6.85 (dd, J ꢀ 8.1, 2.4, 1H, Ar-H); 13C-NMR (75 MHz,
DMSO-d6) δ (ppm): 176.49, 163.17, 159.64, 142.97, 139.99,
132.77, 131.92, 129.59, 127.55, 122.83, 121.92, 120.35, 118.0,
112.0, 111.55, 55.69; LC-MS (m/z): positive mode 327 [M +
H]+. Purity determined by HPLC-UV (254 nm)-ESI-MS: 99.02%
(Z)-N-benzyl-2-(2-oxoindolin-3-ylidene)hydrazine-1-
carbothioamide (8f)
(Z)-N-(2,5-dimethoxyphenyl)-2-(2-oxoindolin-3-ylidene)
hydrazine-1-carbothioamide (8j)
Obtained as yellow solid, Yield ꢀ 74%, mp ꢀ 245–248°C; IR
(KBR): 3,285–3,170 (NH stretching), 1,690 (C ꢀ O), 1,606 (C ꢀ
N), 1,545 (NH bending), 1,164 (C ꢀ S), cm−1; 1HNMR (300 MHz,
DMSO-d6): δ (ppm) 12.81 (s, 1H, N-H), 11.27 (s, 1H, N-H), 10.43
(s, 1H, N-H), 7.77 (d, J ꢀ 2.7, 1H, Ar-H), 7.65 (d, J ꢀ 7.2, 1H, Ar-
H), 7.38 (td, J ꢀ 7.65, 1.1, 1H, Ar-H), 7.09 (m, 2H, Ar-H), 6.95 (d,
J ꢀ 7.8, 1H, Ar-H), 6.82 (dd, J ꢀ 9.0, 3.0, 1H, Ar-H), 3.84 (s, 3H,
Obtained as yellow solid, Yield ꢀ 84%, mp ꢀ 206–208°C; IR
(KBR): 3,271–3,142 (NH stretching), 1,694 (C ꢀ O), 1,594 (C ꢀ
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Oxoindolin Hydrazine Carbothioamides as NTPDases Inhibitors
Alkyl H), 3.72 (s, 3H, Ar-H); 13C-NMR (75 MHz, DMSO-d6) δ
(ppm): 175.64, 163.17, 153.06, 146.68, 143.09, 132.87, 132.05,
128.06, 123.00, 121.41, 120.15, 112.78, 111.64, 111.53, 56.84,
55.95; LC-MS (m/z): positive mode 357 [M + H]+. Purity
determined by HPLC-UV (254 nm)-ESI-MS: 95.39%
(Z)-N-(4-methoxyphenyl)-2-(2-oxoindolin-3-ylidene)hydrazine-
1-carbothioamide (8k)
Obtained as yellow solid, Yield ꢀ 80%, mp ꢀ 255–260°C; IR
(KBR): 3,270–3,160 (NH stretching), 1,692 (C ꢀ O), 1,623 (C ꢀ
N), 1,546 (NH bending), 1,162 (C ꢀ S), cm−1; 1HNMR
(300 MHz, DMSO-d6): δ (ppm) 12.75 (s, 1H, N-H), 11.25 (s,
1H, N-H), 10.73 (s, 1H, N-H), 7.76 (d, J ꢀ 7.5, 1H, Ar-H), 7.46
(d, J ꢀ 8.4, 2H, Ar-H), 7.37 (td, J ꢀ 7.8, 1.0, 1H, Ar-H), 7.10 (t, J ꢀ
7.2, 1H, Ar-H), 6.97 (t, J ꢀ 9.75, 3H, Ar-H), 3.78 (s, 3H, Alkyl-
H); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 177.0, 163.16,
157.85, 142.89, 132.52, 131.80, 131.74, 127.73, 122.82, 121.79,
120.44, 114.03, 111.54, 55.75; LC-MS (m/z): positive mode 327
[M + H]+. Purity determined by HPLC-UV (254 nm)-ESI-MS:
98.73%
(supplemented with 20% FBS) and cells were collected by
harvesting after 40–72 h.
To prepare protein extracts, transfected COS-7 cells were
washed three times with tris-saline buffer while temperature
was maintained at 4°C. Then cells were scraped and
resuspended in harvesting buffer containing 95 mM NaCl,
45 mM tris and 0.1 mM phenylmethylsulfonyl fluoride (pH
7.5). Cells were again washed twice by centrifugation for 5 min
at 300 × g at 4°C and then resuspended in harvesting buffer
(supplemented with 10 μg ml aprotinin). Following sonication,
cellular debris was isolated by 10 min centrifugation at 300 g at
4°C. Subsequently, supernatant was removed very carefully and
protein concentration was determined by using Bradford
microplate assay while using the bovine serum albumin as a
standard (Bradford, 1976).
Nnucleoside Ttriphosphate Ddiphosphohydrolases
Activity Assay
The inhibitory effects of synthesized compounds on
h-NTPDase1, -2, -3, and -8 were determined as described
previously, although with slight modifications (Martín-Satué
et al., 2009). Assay was conducted in a reaction medium
containing 50 mM tris-HCl and 5 mM CaCl2 (pH 7.4). All the
test compounds were dissolved in dimethyl sulfoxide (DMSO,
10%) and initially screened at a concentration of 100 µM. A
reaction mixture containing 55 µl of assay buffer, 10 µl of test
compound solution and 10 µl of h-NTPDase1 (58 ng/well) or
h-NTPDase2 (79 ng/well) or h-NTPDase3 (163 ng/well) or
h-NTPDase8 (66 ng/well) was incubated at 37°C for 10 min.
Following the incubation, a 10 µl solution of adenosine
triphosphate (ATP, 100 µm) was added as substrate and
reaction mixture was again incubated for 15 min at 37°C. The
reaction was stopped by adding malachite green reagent (15 µl)
and placed at room temperature for 4 to 6 min. Finally,
absorbance was measured at 630 nm by using Omega
FLUOstar microplate reader (BMG Labtech, Ortenberg,
Germany) and % inhibitions were calculated. For compounds
showing more than 50% inhibition of any isozyme, dose response
curves were generated and their IC50 values were determined.
Three separate enzyme inhibition assays were carried out in
triplicate and dose-response curve were generated by using
PRISM 5.0 (GraphPad, San Diego, United States).
(Z)-N-(2,6-difluorophenyl)-2-(2-oxoindolin-3-ylidene)hydrazine-
1-carbothioamide (8l)
Obtained as yellow solid, Yield ꢀ 75%, mp ꢀ 243–244°C; IR
(KBR, cm−1): 3,264–3,170 (NH stretching), 1,692 (C ꢀ O), 1,598
1
(C ꢀ N), 1,555 (NH bending), 1,170 (C ꢀ S), cm−1; HNMR
(300 MHz, DMSO-d6) δ (ppm): 12.96 (s, 1H, N-H), 11.28 (s, 1H,
N-H), 10.57 (s, 1H, N-H), 7.69 (d, J ꢀ 7.5, 1H, Ar-H), 7.49 (m, 1H,
Ar-H), 7.39 (td, J ꢀ 7.65, 1.1, 1H, Ar-H), 7.26 (t, J ꢀ 9.9, 2H, Ar-
H), 7.12 (t, J ꢀ 7.65, 1H, Ar-H), 6.95 (d, J ꢀ 7.5, 1H, Ar-H);
13C-NMR (75 MHz, DMSO-d6) δ (ppm): 179.09, 163.11, 157.42,
143.27, 133.83, 132.18, 130.15, 122.94, 121.72, 120.26, 116.56,
112.49, 111.67; LC-MS (m/z): positive mode 333 [M + H]+. Purity
determined by HPLC-UV (254 nm)-ESI-MS: 97%
(Z)-N-ethyl-2-(2-oxoindolin-3-ylidene)hydrazine-1-
carbothioamide (8m)
Obtained as yellow solid, Yield ꢀ 84%, mp ꢀ 190–194°C; IR
(KBR): 3,315–3,281 (NH stretching), 1,686 (C ꢀ O), 1,619 (C ꢀ
N), 1,541 (NH bending), 1,161 (C ꢀ S), cm−1; 1HNMR (300 MHz,
DMSO-d6) δ (ppm): 12.96 (s, 1H, N-H), 11.28 (s, 1H, N-H), 10.57
(s, 1H, N-H), 7.65 (d, J ꢀ 7.2, 1H, Ar-H), 7.35 (td, J ꢀ 7.72, 1.1, 1H,
Ar-H), 7.10 (td, J ꢀ 7.57, 0.7, 1H, Ar-H), 6.93 (d, J ꢀ 7.8, 1H, Ar-
H), 3.64 (m, 2H, CH2), 1.19 (t, J ꢀ 7.05, 3H, CH3); 13C-NMR
(75 MHz, DMSO-d6) δ (ppm): 177.08, 163.09, 142.71, 132.11,
131.61, 122.77, 121.27, 120.44, 111.52, 39.51, 14.52; LC-MS (m/z):
positive mode 249 [M + H]+. Purity determined by HPLC-UV
(254 nm)-ESI-MS: 98.56%
Same procedure was adopted to determine the inhibitory
effects of compounds on mouse NTPDases (m-NTPDases).
However, the amount of protein used was as follows:
m-NTPDase1
(82 ng/well),
m-NTPDase2
(57 ng/well),
m-NTPDase3 (110 ng/well) and m-NTPDase8 (79 ng/well)
Biological Protocols
Cell Transfection and Protein Preparation
Islets Isolation
COS-7 cells were transfected with plasmids expressing human or
mouse NTPDases1, -2, -3, and -8 as previously described
(Levesque et al., 2007; Lecka et al., 2014) Briefly, the confluent
cells were transferred to Dulbecco’s Modified Eagle’s Medium
containing no fetal bovine serum. Then, these cells were
incubated with plasmid DNA (6 µg) and Lipofectamine
reagent (24 µl) at 37°C. After 5 h, transfection was terminated
with the addition of an equal volume of DMEM/F-12
Six to eight week old BALB/c mice, weighing 30–40 g, were
used in the study. These mice were obtained from the animal
house of COMSATS University Islamabad, Abbottabad
Campus and placed under standard temperature (25
2°C) and humidity (50–55%) conditions with an alternate
12-h light/dark cycle. All the experimental animals were
provided with a continuous supply of food and water. The
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Oxoindolin Hydrazine Carbothioamides as NTPDases Inhibitors
animals were handled according to internationally accepted
guidelines of animal care and use. Moreover, this study was
reviewed and approved by Research Ethics Committee,
with ultra-sensitive mouse insulin ELISA kit (Crystal Chem Inc.,
Downers Grove, United States) according to the manufacturer’s
instructions. The absorbance was measured at 450 nm and insulin
secretion was normalized for number of islets used. (Hameed et al.,
2018).
Department
of
Pharmacy,
COMSATS
University
Islamabad, Abbottabad Campus. (Protocol number:
PHM.Eth./CS-M01-020-1609)
The islets were isolated from mice pancreas as previously
described, by a process involving collagenase digestion
(Hameed et al., 2018). Briefly, mice were anesthetized with
sodium thiopental (30 mg kg) and sacrificed by cervical
dislocation to block the flow of blood. Immediately, mice
were transferred to biological hood and their abdominal
cavity was opened completely by making an incision on
abdomen. In order to stop the bile flow toward duodenum,
ampulla was located and clamped under dissection
microscope. Then, distention of pancreas was carried out by
injecting 3 ml of collagenase solution (1 mg/ml) through
common bile duct. The distended pancreas was removed as
a whole, transferred to a 50 ml tube and digested in collagenase
solution at 37°C. After 15 min, digestion was stopped by
placing the tube on ice and 20 ml of Hank’s Balanced Salt
Solution (HBSS) was added to it. Thus the digested islets were
washed with HBSS (two to three times) by centrifugation
(1,000 rpm) for 1 min at 4°C. Finally, islets were purified by
passing through a cell strainer (70 µm) and isolated manually
(by hand picking) under stereomicroscope (Euromex,
Arnhem, Netherlands). All the purification and isolation
procedures were carried out in HBSS, containing no
magnesium, calcium and phenol red.
Ectonucleotidase Activity of Islets
Preparation of Islet Homogenate
Islets were isolated and homogenized, according to the
previously reported method (Hedeskov and Capito, 1974).
After isolation, islets were washed thrice with HBSS and
suspended in an ice-cold buffer containing sucrose (0.25 M),
ethylenediaminetetraacetic acid (EDTA,1 mM) and tris-HCl
(5 mM), maintained at pH 7.0. The resulting suspension was
diluted with assay buffer, composed of tris-HCl (50 mM) and
CaCl2 (5 mM). Afterward, these islets were sonicated for 30 s
leading to disruption of islets and formation of a homogenate.
Cellular debris was removed by centrifugation at 15,000 rpm
for 10 min (4°C). The supernatant was collected and stored on
ice. Protein concentration was estimated by Bradford method,
using bovine serum albumin as reference standard (Bradford,
1976).
Ectonucleotidase Activity of Islet Homogenate
In order to determine the effect of compounds on
ectonucleotidase activity in mice islets, standard curve based
procedure was used where KH2PO4 was used as a standard. A
stock solution of KH2PO4 (1 mM) was prepared in assay buffer
that was used to prepare the working solutions of lower
concentrations (0–100 µm). A standard curve was generated
by incubating increasing concentration of KH2PO4 with buffer
and malachite green reagent.
The assay was carried out in tris-buffer (pH 7.4) containing tris-
HCl (50 mM) and CaCl2 (5 mM), as previously described (Lavoie
et al., 2010). Compounds were dissolved in DMSO (10%) and tested
at a concentration of 100 µM. The assay was started by adding 10 µl
of test compound solution to 56 µl of assay buffer, followed by the
addition of 6 µl of homogenate (i.e., 3 µg of protein). The mixture
was incubated at 37°C for 10 min and then 10 µl of ATP (100 µM)
was added to start the reaction. Reaction mixture was again placed
in incubator at 37°C for 15 min and then reaction was stopped by
adding 100 µl of trichloroacetic acid (10%). These samples were
placed on ice for 15 min. Finally, samples were mixed with
malachite green reagent in an appropriate proportion and
Measurement of Insulin Secretion
Krebs-Ringer bicarbonate buffer (KRBB) was used as an incubation
medium for islets and was composed of NaCl (118 mM), KCl
(4.7 mM), CaCl2 (1.9 mM), MgSO4 (1.2 mM), NaHCO3 (25 mM),
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES,
10 mM) and bovine serum albumin (0.1%). The pH of KRBB
was maintained at 7.4. The isolated islets were size-matched and
pre-incubated (3 islets/tube) for 45 min at 37°C in KRBB,
supplemented with 3 mM glucose. Thereafter, incubation
medium was replaced with fresh KRBB (containing 16.7 mM
glucose) and islets were incubated for 60 min at 37°C with or
without test substance(s). After incubation, supernatant was
collected and stored at −40°C until further use. Finally, collected
samples were properly diluted and insulin secretion was estimated
SCHEME 1 | Synthesis of substituted N-phenylhydrazine carbothioamides (7a-m).
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Oxoindolin Hydrazine Carbothioamides as NTPDases Inhibitors
TABLE 1 | Synthesis of compounds (8a-pm).
released Pi was determined by using standard curve. Moreover, test
compounds were tested at different concentrations and dose
response curves were generated indicating the amount of Pi
released at corresponding dose. All the experiments were carried
out in triplicate and enzyme activity was expressed as nm of Pi/min/
mg of protein.
Real Time Quantitative Polymerase Chain Reaction
Treatment of Islets With Test Compound
Freshly isolated mice islets were incubated in KRBB at 37°C with
glucose (3 mM). After 45 min, these islets were treated with test
compounds in KRBB at 37°C for 3 h, in the presence of
stimulatory glucose concentration. Once the incubation was
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Oxoindolin Hydrazine Carbothioamides as NTPDases Inhibitors
TABLE 2 | 1H NMR data of compound 8i.
Atom No (N or C)
δH
δC
HSQC
HBMC
NH
NˊH
NH
12.77 (brs, 1H)
11.24 (brs, 1H)
10.62 (brs, 1H)
—
—
—
—
—
—
—
—
—
CH-4
CH-6
CH-3ˊ/4ˊ/5ˊ
CH-5
CH-7
Ar(CH3)2
C-1ˊ
C-2ˊ
C-5ˊ
C-2
C-3
7.48 (d, J ꢀ 7.5 Hz, 1H)
7.37 (t, J ꢀ 7.5 Hz, 1H)
7.20–7.46 (m, 3H)
7.09 (t, J ꢀ 7.5 Hz)
6.94 (d, J ꢀ 7.8 Hz)
2.21 (s)
121.7
131.8
128.3/127.9
122.8
111.5
18.3
136.8
136.5
136.5
163.1
132.6
142.9
120.5
171.4
C-4
C-6
C-3ˊ/4ˊ/5ˊ
C-5
C-7
Ar(CH3)2
—
C-6, C-8
C-4, C-8
C-1ˊ/5ˊ, C-2ˊ/6ˊ, C-1ˊ/3ˊ
C-7, C-9
C-5, C-9
C-1ˊ, C-3ˊ, C-5ˊ
—
—
—
—
—
—
—
—
—
—
—
—
C-8
C-9
CꢀS
—
—
—
complete, supernatant was removed carefully, and RNA was
extracted from these islets.
was quantified by using Lvis plate method (FLUOstar Omega,
BMG Labtech, Ortenberg, Germany). This RNA was then reverse
transcribed to cDNA in a total reaction volume of 20 µl. Briefly, a
reaction mixture containing RNA (1 µg) and oligo-dT (20 μM, 1 µl)
was prepared and volume was adjusted to 12 µl with nuclease free
water. The prepared mixture was incubated at 65°C for 5 min and
then immediately chilled on ice. Afterward, 8 µl of master mix
Total RNA Extraction and Real Time Quantitative
Polymerase Chain Reaction
Total RNA was extracted using TRIzol reagent, according to
manufacturer’s instruction. The concentration of isolated RNA
TABLE 3 | Human NTPDase inhibitory data for compounds (8a-m).
Code
NTPDase1
NTPDase2
NTPDase3
NTPDase8
NTPDase1
NTPDase2
NTPDase3
NTPDase8
IC50 (µM) SEMa or %inhibition at 100 µMb
Ki (µM)
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
45%b
0.29 0.02a
0.23 0.01a
49%b
24%b
19%b
20%b
20%b
24%b
24%b
—
—
—
—
—
—
—
0.11
—
—
—
—
—
—
—
—
—
—
—
0.04
0.03
—
46%b
0.19 0.02a
46%b
0.24 0.02a
33%b
—
0.08
—
48%b
—
0.15 0.009a
0.24 0.01a
28%b
34%b
26%b
21%b
0.15
0.03
—
—
—
38%b
27%b
31%b
—
—
29%b
17%b
7%b
—
—
49%b
17%b
26%b
22%b
—
—
—
1.63 0.15a
0.15 0.01a
44%b
0.41 0.03a
0.11 0.08a
0.16 0.01a
45%b
25%b
17%b
0.24
0.02
—
0.17
0.04
0.07
—
—
34%b
19%b
—
19%b
46%b
—
0.30 0.03a
2.60 0.02a
16.1 1.02a
22%b
32%b
0.04
0.38
2.33
—
8m
42%b
24.1 3.01a
0.38 0.03a
4.31 0.41a
38%b
—
0.16
1.85
Suramin3
>100a
9.92
aIC50 values are presented as mean SEM of three independent experiments.
bPercent inhibition determined at 100 μM.
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Oxoindolin Hydrazine Carbothioamides as NTPDases Inhibitors
TABLE 4 | Mouse NTPDase inhibitory data for compounds (8a-m).
Code
NTPDase1
NTPDase2
NTPDase3
NTPDase8
NTPDase1
NTPDase2
NTPDase3
NTPDase8
IC50 (µM) SEMa or %inhibition at 100 µMb
Ki (µM)
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
38%b
14%b
42%b
31%b
31%b
28%b
28%b
30%b
—
—
—
—
—
—
—
—
28%b
36%b
27%b
36%b
—
—
—
—
41%b
27%b
29%b
1.45 0.12a
3.80 0.21a
38%b
—
—
—
0.17
0.44
—
0.60 0.03a
4.14 0.13a
48%b
29%b
0.70 0.05a
32%b
0.006
0.04
—
—
0.07
—
34%b
—
36%b
27%b
36%b
—
—
—
29%b
31%b
31%b
28%b
—
—
—
—
20%a
0.15 0.01a
27%b
32%b
26%b
—
0.04
—
—
—
24%a
30%b
32%b
—
—
—
42%b
29%b
36%b
24%b
—
—
—
—
3.22 0.19a
3.60 0.02a
>100a
0.20 0.01a
1.50 0.11a
21.0 2.0a
0.32 0.04a
2.41 0.15a
31.0 2.0a
13%b
0.03
0.04
—
0.05
0.41
5.67
0.03
0.24
3.07
—
8m
35%b
—
Suramin4
>100a
—
aIC50 values are presented as mean SEM of three independent experiments.
bPercent inhibition determined at 100 μM
containing 5X reaction buffer (4 µl), rnase inhibitor (1 µl), dNTPs mix
(10 mM, 2 µl) and reverse transcriptase (1 µl) was added to above
sample. After gentle pipetting, sample was run under following
conditions: 25°C for 5 min, 42°C for 70 min and 70°C for 5 min.
Once the cDNA was synthesized, real time quantitative PCR was
conducted by using PikoReal 96 Real-Time PCR system (Thermo
Scientific, Vantaa, Finland). For this purpose, a master mixture
containing cDNA (1 µl), forward primer (0.25 µl), reverse primer
(0.25 µl) and SYBER green master mix (10 µl) was prepared and total
volume was adjusted to 20 µl with nuclease free water. Subsequently,
thermal cycling conditions were set as follows: 95°C for 10 min and
then 40 cycles at 95°C for 15 s and 55°C for 1 min. Relative expression
was determined by normalization to β actin mRNA by ΔΔCT method.
mode of inhibition. Compound 8e revealed a non-competitive
mechanism of inhibition against h-NTPDase1, therefore its
docking was not performed. Since the crystal structures of
human NTPDases are not yet available from the Protein Data
Bank, their homology models were built according to our
previously reported method (Iqbal and Shah, 2018). Docking
studies were carried out using BioSolveIT’s LeadIT software1. Out
of the top ten docked conformations obtained for each inhibitor,
the one with the most favorable binding free energy was selected,
using HYDE functionality of software. SeeSAR analysis of the
inhibitors was also performed by BioSolveIT’s SeeSAR2.
Statistical Analysis
The data was presented as mean SEM and statistical analysis
was performed using PRISM 5.0 (GraphPad, San Diego, United
States). Statistical tests were performed by using student t test and
one-way ANOVA. A p value < 0.05 was considered significant.
Molecular Docking Studies
Molecular docking analysis was carried out on the basis of results
obtained in enzyme kinetics. Hence molecular docking was
carried out for the most potent inhibitor of h-NTPDase2 (8j),
h-NTPDase3 (8c) and h-NTPDase8 which showed a competitive
FIGURE 1 | Lineweaver-Burk Plot for h-NTPDase1 inhibitor (8e). S
represents the substrate concentration (μM) and concentration of inhibitor
(8e) are 0, 0.10, 0.15, and 0.20 μM.
FIGURE 2 | Lineweaver-Burk Plot for h-NTPDase2 inhibitor (8j) S
represents the substrate concentration (μM) and concentration of inhibitor (8j)
are 0, 0.05, 0.10, and 0.15 μM.
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Oxoindolin Hydrazine Carbothioamides as NTPDases Inhibitors
FIGURE 3 | Lineweaver-Burk Plot for h-NTPDase3 inhibitor (8m). S
represents the substrate concentration (μM) and concentration of inhibitor
(8m) are 0, 0.20, 0.4, and 0.6 μM.
RESULTS
Chemistry
A series of 13 hydrazine carbothioamide derivatives (8a-m) was
FIGURE 5 | Effect of compound 8c, 8m, suramin (SUR), isatin (IST) and
isobutyl methyl xanthine (IBMX) on glucose stimulated insulin secretion in mice
pancreatic islets at 200 µM. Group of size-matched islets were incubated at
37°C for 1 h in KRB buffer with 16.7 mM glucose supplemented with or
without test compounds. Values are mean S.E.M. from two to three
independent experiments. Insulin secretion induced by 16.7 mM Glucose was
considered 100%. ***p < 0.0001 vs. none. Cnt, insulin secretion induced by
16.7 mM glucose only.
synthesized as potential NTPDase inhibitors. Initially, substituted
phenylhydrazine carbothioamides (7a-m) were prepared by
dropwise addition of appropriate isothiocyanate to excess of
hydrazine hydrate in ethanol as shown in Scheme 1. The
target compounds (8a-m) were synthesized by refluxing the
synthesized intermediates (7a-m) with isatin for 4 to 5 h
(Table 1). After the reaction was completed, gradual
evaporation of solvent at room temperature resulted in
precipitate formation. These precipitates were filtered, washed
with ethanol and then dried. The synthesized compounds (8a-m)
were identified by different spectroscopic techniques including
IR, LC/ESI-MS,1H-NMR and 13C-NMR.
The IR spectra of synthesized compounds showed absorption
bands in the region of 3,315–3,130 cm−1, resulting from NH
FIGURE 6 | Dose-dependent effect of compound 8m on insulin
secretion. Compound 8m was employed at doses of 0, 10, 50, 100, and
200 µM concentration supplemented with 16.7 mM glucose. Group of size-
matched islets were incubated at 37°C for 1 h in KRB buffer with
16.7 mM glucose supplemented with or without test compounds. Values are
mean S.E.M. from two to three independent experiments. **p < 0.001.
FIGURE 4 | Lineweaver-Burk Plot for h-NTPDase8 inhibitor (8c). S
represents the substrate concentration (μM) and concentration of inhibitor
(8c) are 0, 0.10, 0.20, and 0.30 μM.
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Oxoindolin Hydrazine Carbothioamides as NTPDases Inhibitors
Effect of Compounds on Recombinant
h-NTPDases
The synthesized compounds (8a-m) were investigated for their
ability to inhibit h-NTPDases using the malachite green assay.
Recombinant h-NTPDase1, -2, -3 and -8 were employed and initial
screening was carried out at an initial concentration of 100 µM. In
order to compare species difference, compounds were also tested
against mouse NTPDases (m-NTPDases), including m-NTPDase1,
-2, -3 and -8. Dose response curves were generated for compounds
showing >50% inhibition of any isoform of enzyme. The inhibitory
activity of these compounds against human and mouse NTPDases
is presented in Tables 3, 4, respectively.
Structure Activity Relationship
Among thirteen derivatives, eight compounds inhibited
h-NTPDases to a variable extent, however h-NTPDase1 was
more susceptible as compared to other isozymes. Compound
8e (IC50 ꢀ 0.15 0.009 µM) and 8j (IC50 ꢀ 0.15 0.01 µM)
revealed the most promising activity against h-NTPDase1. A
structural comparison of these inhibitors showed that both
compounds contained –OCH3 group attached to phenyl ring,
however, 8e was mono substituted with –OCH3 attached to
phenyl ring whereas 8j was substituted with two –OCH3
groups. Hence, excellent activity of 8e could be due this
–OCH3 group and incorporation of two –OCH3 groups (8j)
resulted in retention of activity.
FIGURE 7 | Effect of compound 8m and suramin on ectonucleotidase
activity in islets homogenate using 100 µM ATP as substrate. Enzyme activity
is expressed in nmol Pi/min/mg of protein. Enzyme activity without any
inhibitor was considered 100%. Treatment of homogenate with
compound 8m and suramin significantly reduced the enzyme activity. Bars
represent means SEM for three independent experiments. ***p < 0.0001.
Cnt (control), enzyme activity without addition of compound, Sur, enzyme
activity by suramin.
Likewise, compounds containing –Cl (8c, IC50 ꢀ 0.23
0.01 µM), –NO2 (8b, IC50 ꢀ 0.29
0.02 µM) and –(F)2 (8l,
IC50 ꢀ 0.30 0.03 µM) also showed promising activities. The
lowest activity in the series was shown by compounds substituted
with an alkyl group as indicated by IC50 values of 8i (IC50 ꢀ 1.63
0.15 µM) and 8m (IC50 ꢀ 2.60 0.02 µM), respectively.
The data suggest that position of substitution also plays an
important role. For example, compound 8c containing 4-Cl
stretching of indole and thioamide function. The absorption bands in
the region of 1,698–1,686 (cm−1) and 1,623–1,591 (cm−1) can be
assigned to the presence of C ꢀ O and C ꢀ N, respectively. Likewise,
bands in the range of 1,560–1,541 (cm−1) and 1,174–1,158 (cm−1)
reflect the presence of N-H and C ꢀ S, respectively. In case of
1H-NMR, spectra showed three characteristic peaks (singlet) of
NH in the range of
δ
9.82–11.12 ppm, 11.22–11.31 ppm,
13.01–12.66 ppm, assigned to CS-NH, indole NH and –N-NH,
respectively, confirming the synthesis of desired compounds. All
the aromatic protons appeared (as expected) in the region of δ
8.29–6.82 ppm. The 13C spectra showed thioamide carbon peak in
the range δ 179.09–175.64 ppm, while carbonyl and imine carbon
appeared in the range
δ
163.20–163.09 ppm and
δ
145.12–142.71 ppm, respectively (Pervez et al., 2009; Ali et al.,
2014). Mass spectra of synthesized compound was recorded in
positive mode, compounds showed molecular ions of varying
intensity indicating molecular weights of the compounds. The
information regarding 1H and 13C spectra of synthesized
compounds was presented in Supplementary Material.
A typical structure of a hydrazine carbothioamide showed
characteristic signals in ID and 2D NMR spectra. The broad signals
for NH groups appeared at
δ
12.77, 11.24, and 10.62 ppm,
H
respectively. The doublets of H-4 and H-7 appeared at δ 7.48
H
and 6.94 ppm, respectively. The carbon signals for δ C ꢀ O and C-S
C
FIGURE 8 | Compound 8m inhibits the ectonucleotidase activity in islet
homogenate, measured as release of Pi from exogenously added ATP at a
concentration of 100 µM (n ꢀ 3).
appeared at 163.1 and 171.4 ppm. The other characteristics signals of
CH, C and CH3 groups confirm the structure of compound 8i and
are presented in Table 2.
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FIGURE 9 | Overlap of docked conformations of compounds 8i (gray), 8j (purple) and 8k (pink) with standard inhibitor suramin (black), the protein backbone is
represented in lined ribbon, the calcium ion is shown as green sphere.
substitution showed remarkable activity (IC50 ꢀ 0.23
0.01 µM); whereas, introduction of 3-Cl resulted in reduced
activity of 8g (28%). Similarly, position of substituent also
contributed toward the activity of 8e (containing 4-OCH3
group) since introduction of 3- OCH3 group lead to
decreased activity of 8k (44%).
Moreover, mono-substitution and di-substitution was another
factor responsible for improved activity of derivatives. In this
case, a comparison of 8d (49.1%) vs. 8l (IC50 ꢀ 0.30 0.03 µM)
showed that di substitution was more favored than mono
substitution. Same pattern of activity was observed for 8h vs.
8i. Above all, four compounds i.e. 8b, 8e, 8f and 8l selectively
FIGURE 10 | 2D (left) and 3D (right) representation of docked conformations of h-NTPDase2 inhibitor 8j.
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Oxoindolin Hydrazine Carbothioamides as NTPDases Inhibitors
FIGURE 11 | See SAR Analysis (visual representation of contribution of each atom to overall binding affinity) of h-NTPDase2 inhibitors 8i (left) and 8j (right). The
structural elements that are contributing favorably to the overall binding affinity are represented with green colored coronas, structural elements that with unfavorable
contribution are represented with red colored coronas, neutral elements are not colored. Unoccupied space in the binding site is in gray color.
inhibited h-NTPDase1and their %inhibition values for other
isozymes was <50%.
respective isozymes. In this regard, compound 8e revealed
a non-competitive mode of inhibition for h-NTPDase1
(Figure 1). However, compound 8j was found to be a
competitive inhibitor of h-NTPDase2 since all the four
lines (indicating different concentrations of inhibitor) were
intersecting at y-axis (Figure 2). Compound 8m showed a
non-competitive mode of inhibition for h-NTPDase3
(Figure 3) whereas the Lineweaver-Burk plot for h-
NTPDase8 with compound 8c exhibited a competitive
mechanism of inhibition (Figure 4).
In case of h-NTPDase2, three compounds could inhibit this
isoform and IC50 values of these compounds were in sub-micro
molar range. Compounds 8j (IC50 ꢀ 0.11 0.08 µM) and 8k
(IC50 ꢀ 0.16
0.01 µM) shared almost comparable activity,
indicating substitution with –OCH3 was well tolerated, whether
it was a mono substitution or a di substitution.
Compound 8i (IC50 ꢀ 0.41 0.03 µM) also possessed good
inhibitory activity and it incorporated two –CH3 groups as a part of its
structure. However, the activity of remaining derivatives was <50%.
There were only two hit compounds (8c, 8m) which showed
excellent inhibition of h-NTPDase3. A comparison of IC50s
showed that 8c was 22 times more active than standard
inhibitor i.e. suramin. Structure of 8c contained 4-Cl attached
to it and this activity appeared position dependent since
introduction of 3-Cl resulted in loss of activity. Another
inhibitor of h-NTPDase3 was 8m and it also showed excellent
Effect of NTPDase3 Inhibitors on Insulin
Secretion
The ability of h-NTPDase3 inhibitors (8c and 8m) to
stimulate insulin secretion was determined by using freshly
isolated mice islets. It was observed that compound 8m
produced
a significant increase in glucose stimulated
inhibitory activity (IC50 ꢀ 0.38
0.03 µM). This compound
insulin secretion as compared to control and activity of
this compound (8m) was comparable to that of isobutyl
methyl xanthine (IBMX), used as positive control. The
results are shown in Figure 5.
The compound 8m was tested at different doses (10–200 µM)
and it induced a dose dependent increase in insulin secretion
(Figure 6). However, compound 8m did not produce any
significant effect at basal glucose level (3 mM).
Although compound 8c was the most potent h-NTPDase3
inhibitor but it did not show any significant effect on insulin
secretion. This discrepancy in the behavior of compound 8c
might be attributed to the species difference. Therefore, we
tested all the compound on mouse NTPDases (m-NTPDases)
contained an ethyl chain attached to –NH of thioamide chain,
indicating that alkyl chain was well tolerated. However, presence
of other substituents like –F, –CH3 and –OCH3 did not show any
remarkable activity toward h-NTPDase3.
Only one compound effectively inhibited h-NTPDase8 and
that was 8c. The IC50 of 8c (0.24 0.02 µM) was 400 times than
that of suramin. However, no other compound could inhibit
h-NTPDase8 to an appreciable extent.
Mechanism of Inhibition of Candidate Compounds
In order to determine the mechanism of inhibition, kinetic
studies of most potent inhibitors were performed against
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Oxoindolin Hydrazine Carbothioamides as NTPDases Inhibitors
FIGURE 12 | Overlap of docked conformations of h-NTPDase3 inhibitor 8c (gray) and standard inhibitor suramin (black), the protein backbone is represented in
lined ribbon, the magnesium ion is shown as a green sphere.
and we found that compound 8c was not an effective inhibitor of
m-NTPDase3 (Table 4).
Effect of 8m on NTPDase3 Activity in Mice
Pancreatic Islets
Suramin, used as positive control during enzyme inhibition
studies, was tested for its insulin secretory activity. Isatin, used as
a reactant during synthesis of target compounds, was also
investigated for its ability to stimulate insulin secretion.
Neither suramin nor isatin had any significant effect on
insulin release (Figure 5).
Given that compound 8m was identified as an effective inhibitor
of h-NTPDase3 as well as a regulator of the glucose-induced
insulin secretion, we decided to determine the compound’s effect
on ectonucleotidase activity in isolated mice islets.
A
homogenate was prepared from the isolated islets which was
treated with compound and then ectonucleotidase activity was
FIGURE 13 | 2D (left) and 3D (right) representation of docked conformations of h-NTPDase3 inhibitor 8c.
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Oxoindolin Hydrazine Carbothioamides as NTPDases Inhibitors
FIGURE 14 | 2D (left) and 3D (right) representation of docked conformations of h-NTPDase8 inhibitor 8c.
determined by malachite green assay. At a concentration of
100 μM, compound 8m significantly decreased the
ectonucleotidase activity as compared to control. The activity
of test compound (8m) was comparable to that of suramin, a
standard inhibitor of NTPDase (Figure 7). Moreover, a dose
response curve was generated for compound 8m and it was
found that compound 8m produced a dose dependent decrease
in enzyme activity (Figure 8).
FIGURE 15 | See SAR Analysis (visual representation of contribution of each atom to overall binding affinity) of h-NTPDase8 inhibitor 8c. The structural elements
that are contributing favorably to the overall binding affinity are represented with green colored coronas, structural elements that with unfavorable contribution are
represented with red colored coronas, neutral elements are not colored. Unoccupied space in the binding site is in gray color.
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TABLE 5 | Summary of amino acids interacting with NTPDase inhibitors.
NTPDase inhibitors
Binding energy (kJ/mol)
Interacting amino acid residues
H-bond acceptors
H-bond donors
8i (NTPDase2)
8j (NTPDase2)
8k (NTPDase2)
8c (NTPDase3)
8c (NTPDase8)
−26
−29
−29
−27
−27
Nil
Nil
Leu202 (CꢀO), Ser346 (−OH), Ser52 (−OH)
Tyr350 (−OH), Ser346 (−OH), Leu202 (CꢀO)
Ser346 (−OH), Ser52 (−OH)
Arg392 (NH(NH2)+NH2)
Ser65 (−NH), Ser66 (−NH)
Ala63 (CꢀO), Ser66 (−OH)
His53 (CꢀO), Ser52 (−OH)
Ser51 (−NH), Ser52 (−NH), His53 (−NH)
BioSolveIT’s SeeSAR is a tool that provides visual display of
binding affinity. The information thus provided can be very
helpful in logically modulating the structures of lead
inhibitors, to synthesize even more potent inhibitors. The
SeeSAR analysis of most active NTPDase inhibitors 8i and 8j
is given in Figure 11. The structural features of the compound
that are contributing positively to the overall binding affinity are
indicated with green coronas; greater the contribution, larger is
the size of the corona. Similarly, the structural elements that are
not contributing favorably to the overall binding are indicated
with red coronas, whereas the structural features with no
contribution are not colored. As can be seen, most of the
atoms in the molecule 8i are contributing favorably to overall
binding (indicated by green colored coronas), except two
structural elements, i) one of the methyl carbon atoms of the
2,6-dimethyl substituted phenyl ring (labeled C23 in Figure 11,
9.8 kJ/mol), and ii) the sulfur atom (S15 in Figure 11, 1.2 kJ/mol)
of thiourea (C ꢀ S) moiety. These unfavorable contributions are
because of high desolvation energy which has not been
compensated by any (favorable) non-bonded interaction.
Based on the unoccupied space in the binding pocket
(indicated in gray), the structure of the molecule can be
changed/extended further to provide a more snug fit into the
binding pocket. Similarly, for compound 8j, unfavorable
contribution to binding free energy is because of somewhat
high desolvation energy of methoxy oxygen atoms (O22 and
O24, 3.1 and 3.7 kJ/mol, respectively) and one of the phenyl ring
carbon atoms (C18, 1.4 kJ/mol). Replacement of these atoms with
some other suitable atoms/group is expected to result in even
more active inhibitors.
Effect of 8m on NTPDase3 mRNA by Real
Time Quantitative Polymerase Chain
Reaction
To determine if the reduction in ectonucleotidase activity by
compound 8m in mice pancreatic islets was an outcome of
downregulation of NTPDase3 mRNA, we analyzed the effect
of this compound (8m) on NTPDase3 gene expression. Freshly
isolated mice islets were incubated with test compound, total
RNA was extracted and relative fold change in the NTPDase3
mRNA expression was determined by real time qPCR
experiments. The results showed that test compound (8m) had
no significant effect (p > 0.05) on the NTPDase3 gene expression
in mice pancreatic islets as compared to control. Moreover,
expression level of β-actin (housekeeping gene) was also not
affected by compound 8m.
Molecular Docking
Compounds 8i, 8j and 8k were highly active against h-NTPDase2
and were therefore docked against this enzyme. For reference,
docking of standard inhibitor suramin was also carried out. All
compounds were found to bind in the same area of the active site,
in the region that is also shared by suramin. Figure 9 shows an
overlap of docked conformations of inhibitors 8i, 8j, 8k with
suramin.
The most probable docked conformation of compound 8j, the
most active h-NTPDase2 inhibitor, is shown in Figure 10. As can
be seen, the NH of the indole ring was making a hydrogen bond
with Leu202. Likewise, one of the NH groups of thiourea moiety
was making hydrogen bond with Ser346 whereas the other NH
group was making hydrogen bond with Tyr350. The 2,5-
dimethoxy phenyl ring was making a pi-cation interaction
with Ala433, pi-sigma and pi-alkyl interactions were observed
between the indole ring and the amino acids Ala347 and Arg245,
respectively.
Compound 8c was the most active inhibitor of h-NTPDase3
and showed competitive mode of inhibition, hence its docking
studies were carried out. For reference, suramin the standard
inhibitor, was also docked. Figure 12 shows overlap of docked
conformation of 8c with that of suramin. Docking studies of
To further highlight the role of each structural element toward
the overall binding affinity SeeSAR analysis was carried out.
compound 8c revealed
a number of hydrogen bonded
interactions, the indole NH was making a hydrogen bond with
Ser66, while the carbonyl oxygen (of indole ring) was within
hydrogen bond distance of Ser65 and Ser66. The NH group of
thiourea moiety was making hydrogen bond with Ala63
(Figure 13).
TABLE 6 | Comparison of IC50, Ki and binding energy of the most potent
NTPDase inhibitors.
h-NTPDase inhibitor
IC50 (µM)
Ki (µM)
Binding energy (KJ/mol)
Compound 8c was the only inhibitor of h-NTPDase8 and
showed competitive mode of inhibition, hence its docking studies
were carried out. As shown in Figure 14, compound 8c revealed a
number of hydrogen bonded interactions, the indole NH was
making a hydrogen bond with His53, while the carbonyl oxygen
8i (NTPDase2)
8j (NTPDase2)
8k (NTPDase2)
8c (NTPDase3)
8c (NTPDase8)
0.41 0.03
0.11 0.08
0.16 0.01
0.19 0.02
0.24 0.02
0.17
0.04
0.07
0.08
0.11
−26
−29
−29
−27
−27
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Oxoindolin Hydrazine Carbothioamides as NTPDases Inhibitors
(of indole ring) was within hydrogen bond distance of Ser52,
Ser51 and His53. The NH group of thiourea moiety was making
hydrogen bond with Ser52. Additionally, a pi-pi stacked
interaction was also observed between the indole ring and
His53. A pi-anion interaction was observed between indole
phenyl ring and Asp48.
derivatives. Among them, POMs showed best inhibitory
activity with Ki value as low as 0.0038 µM (Lee et al., 2015)
whereas 8-Bus-ATP (nucleotide analogue) was found to be a
selective inhibitor of h-NTPDase1 (Ki ꢀ 0.8 0.2 µM) (Lecka
et al., 2013). The Ki values of our compounds against
h-NTPDases were found to be in the range of 0.02–0.38 µM.
Thus, our compounds tend to be more potent than 8-Bus-ATP
but their activity is low as compared to POMs. A recent study
(Baqi et al., 2020) reported synthesis of anthraquinone derivatives
as potent h-NTPDase inhibitors, showing selectivity against
h-NTPDase2 and -3. The IC50 values of these compounds
were found to be within the range of 0.39–15.3 µM, while the
IC50 values of our compounds were within the range of
0.11–2.60 µM, indicating that our compounds are more active
h-NTPDase inhibitors than anthraquinone derivatives.
NTPDase3 is the most prominent isoform in pancreatic islets
where it has been reported to regulate the insulin secretion (Bartley
et al., 2019; Saunders et al., 2019). Therefore, NTPDase3 inhibitors
i.e. 8c and 8m were evaluated for their effects on insulin secretion
in mice islets. Compound 8m produced a dose dependent increase
in insulin release, in the presence of high glucose concentration. At
basal glucose level, compound 8m has no effect on insulin secretion
since glucose is the actual initiator of insulin release, whereas ATP
can stimulate the insulin secretion only in the presence of high
glucose concentration (Cie´slak and Cie´slak, 2017). On the other
hand, compound 8c had no significant effect on insulin secretion
due to species difference, as the screening of compounds on
m-NTPDases showed that compound 8c exhibited < 50%
inhibition against m-NTPDase3. Although compound 8m was
showing good inhibitory activity against m-NTPDase1, -2 and -3, it
was the m-NTPDase3 inhibition that potentiated the insulin
secretion since NTPDase3 is the predominant isoform expressed
in mice islets. In this context, a study had reported that NTPDase3
was the most abundant isoform in mouse and human islets
whereas least amounts of NTPDase1 and -2 were detected.
They used a non-selective NTPDase inhibitor (ARL67156) and
established that it was the NTPDase3 inhibition that potentiated
insulin secretion. They also performed siRNA experiments and
observed that the knockdown of NTPDase3 expression was
producing the same effects on insulin secretion as those
obtained with ARL67156, thus suggesting that it is NTPDase3
inhibition that is contributing to insulin secretion (Syed et al.,
2013).
SeeSAR analysis of h-NTPDase8 inhibitor 8c was also carried
out as depicted in Figure 15. The structural elements with
unfavorable contribution to the overall binding free energy are
indicated in red coronas. These include the nitrogen atoms (N14,
1.3 kJ/mol; N12, 2.2 kJ/mol) and the sulfur atom (S15, 2.4 kJ/mol)
of the thiourea moiety. This is because of high desolvation energy.
The oxygen atom of the carbonyl group (O10 4.1 kJ/mol) also had
high desolvation energy, although it was making a hydrogen
bond, yet the compensation is not enough to fully compensate the
penalty of (high) desolvation energy. It is suggested that
replacement of these atoms with some other suitable atoms/
group is expected to result in even more active inhibitors.
A summary of amino acids interacting with NTPDase
inhibitors as well as binding energy associated with these
inhibitors is presented in Table 5. These computed binding
energies were compared with biological data in Table 6, where
biological data is presented in the form of IC50 and Ki values. The
binding energy values were in close agreement with the
experimentally determined IC50/Ki values. For instance, the
most potent h-NTPDase2 inhibitor (8j, IC50 ꢀ 0.11 0.08 µM,
Ki ꢀ 0.04 µM) had the best binding energy of −29 kJ/mol.
Similarly, compounds 8c with the binding energy of −27 kJ/
mol was found to be the most potent inhibitor of
h-NTPDase3 (IC50 ꢀ 0.19
0.02 µM, Ki ꢀ 0.08) and
h-NTPDase8 (IC50 ꢀ 0.24 0.02 µM, Ki ꢀ 0.11 µM).
DISCUSSION
In this study, we have synthesized and evaluated a series of
oxoindolin hydrazine carbothioamide derivatives as potential
NTPDase inhibitors. Most of the compounds appeared to be
more potent inhibitors of h-NTPDase1 than other NTPDases.
Compound 8e and 8j were the most potent h-NTPDase1
inhibitors whereas compound 8j was also the most potent
inhibitor of h-NTPDase2. Both of these compounds contained an
–OCH3 group as a part of their structure, thus implying the
importance of this –OCH3 for h-NTPDase1 and -2 inhibitors.
On the other hand, compound 8c was found to be potent dual
inhibitor of h-NTPDase3 and -8. Interestingly, compound 8b, 8e, 8f,
and 8l were identified as selective inhibitors of h-NTPDase1 whereas
compound 8k selectively inhibited the h-NTPDase2. Furthermore,
compound 8e, 8j, 8c, and 8m were studied in detail to establish their
mechanism of inhibition. Compound 8e inhibited h-NTPDase1 in a
non-competitive manner whereas compound 8j and 8c revealed a
competitive mode of inhibition against h-NTPDase2 and -8,
respectively. Kinetics studies of compound 8m revealed that it
was inhibiting the h-NTPDase3 in a non-competitive manner.
In literature, different classes of compounds have been
synthesized as NTPDase inhibitors, including nucleotide
analogues, polyoxometalates (POMs) and anthraquinone
Furthermore, we also determined the inhibitory effect of
compound (8m) in mice islets and it produced a significant
reduction in ectonucleotidase activity, suggesting that
compound (8m) is stimulating the insulin secretion via
NTPDase3 inhibition. However, inhibition of NTPDase3 (by
compound 8m) was not occurring due to the downregulation
of NTPDase3 mRNA since compound (8m) had not suppressed
the gene expression of NTPDase3. Finally, molecular docking
studies and SeeSAR analysis of the most potent inhibitors were
also carried out and the calculated binding energies were in
accordance with our experimental data. Taken together, our
study demonstrates that 8m is a potent inhibitor of NTPDase3
which is involved in the stimulation of glucose induced insulin
secretion without suppressing the NTPDase3 gene.
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Oxoindolin Hydrazine Carbothioamides as NTPDases Inhibitors
CONCLUSION
ETHICS STATEMENT
In a series of oxoindolin hydrazine carbothioamide derivatives, only
This study was reviewed and approved by Research Ethics
Committee, Department of Pharmacy, COMSATS University
Islamabad, Abbottabad Campus.
two compounds i.e. 8c (IC50 ꢀ 0.19 µM 0.02) and 8m (IC50
ꢀ
0.38 0.03) showed excellent inhibition of h-NTPDase3. These
inhibitors of h-NTPDase3 were investigated for their effects on
insulin secretion and only compound 8m was found as a lead
regulator of insulin secretion. Further studies revealed that
compound 8m significantly reduced ectonucleotidase activity in
mice pancreatic islets. In consistence with our in vitro data, docking
studies displayed strong binding interaction of potent inhibitors
within the active site of respective enzyme. In conclusion, we report
compound 8m as a potent inhibitor of h-NTPDase3, stimulating
the glucose induced insulin secretion. Further study is needed to
investigate 8m as a potential drug candidate.
AUTHOR CONTRIBUTIONS
SA carried out synthesis and enzyme assays, Mal-R Designed
Synthesis of the derivatives, AH supervised the effect on glucose
stimulated insulin secretion, JP and JS expressed the enzymes
used in the study. JI designed the project and supervised the
biological experiments.
DATA AVAILABILITY STATEMENT
SUPPLEMENTARY MATERIAL
The raw data supporting the conclusions of this article will be made
available by the authors, without undue reservation, to any qualified
researcher.
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fphar.2020.585876/
full#supplementary-material
Dou, L., Chen, Y. F., Cowan, P. J., and Chen, X. P. (2018). Extracellular ATP
signaling and clinical relevance. Clin. Immunol. 188, 67–73. doi:10.1016/j.clim.
2017.12.006
REFERENCES
Ali, A. Q., Teoh, S. G., Salhin, A., Eltayeb, N. E., Ahamed, M. B., and Majid, A. A.
(2014). Synthesis of isatin thiosemicarbazones derivatives: in vitro anti-cancer,
DNA binding and cleavage activities. Spectrochim. Acta Mol. Biomol. Spectrosc.
125, 440–448. doi:10.1016/j.saa.2014.01.086
Allard, B., Longhi, M. S., Robson, S. C., and Stagg, J. (2017). The ectonucleotidases
CD 39 and CD 73: novel checkpoint inhibitor targets. Immunol. Rev. 276,
121–144. doi:10.1111/imr.12528
Bagatini, M. D., dos Santos, A. A., Cardoso, A. M., Maˆnica, A., Reschke, C. R., and
Carvalho, F. B. (2018). The impact of purinergic system enzymes on
noncommunicable, neurological, and degenerative diseases. J. Immunol. Res.
2018, 4892473. doi:10.1155/2018/4892473
Baqi, Y., Rashed, M., Schäkel, L., Malik, E. M., Pelletier, J., Sévigny, J., et al. (2020).
Development of anthraquinone derivatives as ectonucleoside triphosphate
diphosphohydrolase (NTPDase) inhibitors with selectivity for NTPDase2
and NTPDase3. Front. Pharmacol. 11, 1282. doi:10.3389/fphar.2020.01282
Bartley, C., Brun, T., Oberhauser, L., Grimaldi, M., Molica, F., Kwak, B. R., et al.
(2019). Chronic fructose renders pancreatic β-cells hyper-responsive to
glucose-stimulated insulin secretion through extracellular ATP signaling.
Am. J. Physiol. Endocrinol. Metab. 317, E25–E41. doi:10.1152/ajpendo.00456.
2018
Bertoni, A. P., de Campos, R. P., Tamajusuku, A. S., Stefani, G. P., Braganhol, E.,
Battastini, A. M., et al. (2020). Biochemical analysis of ectonucleotidases on
primary rat vascular smooth muscle cells and in silico investigation of their role
in vascular diseases. Life Sci. 256, 117862. doi:10.1016/j.lfs.2020.117862
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem. 72, 248–254. doi:10.1016/0003-2697(76)90527-3
Castilhos, L. G., Adefegha, S. A., Doleski, P. H., Bertoldo, T. M., Moritz, C. E.,
Casali, E. A., et al. (2018). NTPDase, 5′-nucleotidase and adenosine deaminase
activities and purine levels in serum of sickle cell anemia patients. J. Appl.
Biomed. 16, 208–213. doi:10.1016/j.jab.2017.12.004
Galicia-Garcia, U., Benito-Vicente, A., Jebari, S., Larrea-Sebal, A., Siddiqi, H.,
Uribe, K. B., et al. (2020). Pathophysiology of type 2 diabetes mellitus. Int.
J. Mol. Sci. 21, 6275. doi:10.3390/ijms21176275
Giuliani, A. L., Sarti, A. C., and Di Virgilio, F. (2019). Extracellular nucleotides and
nucleosides as signalling molecules. Immunol. Lett. 205, 16–24. doi:10.1016/j.
imlet.2018.11.006
Grkovic´, I., Drakulic´, D., Martinovic´, J., and Mitrovic´, N. (2019). Role of
ectonucleotidases in synapse formation during brain development:
physiological and pathological implications. Curr. Neuropharmacol. 17,
84–98. doi:10.2174/1570159X15666170518151541
Hameed, A., Hafizur, R. M., Hussain, N., Raza, S. A., Rehman, M., Ashraf, S., et al.
(2018). Eriodictyol stimulates insulin secretion through cAMP/PKA signaling
pathway in mice islets. Eur. J. Pharmacol. 820, 245–255. doi:10.1016/j.ejphar.
2017.12.015
Hedeskov, C. J., and Capito, K. (1974). The effect of starvation on insulin secretion
and glucose metabolism in mouse pancreatic islets. Biochem. J. 140, 423–433.
doi:10.1042/bj1400423
Iqbal, J., and Shah, S. J. A. (2018). Molecular dynamic simulations reveal structural
insights into substrate and inhibitor binding modes and functionality of Ecto-
Nucleoside Triphosphate Diphosphohydrolases. Sci. Rep. 8, 1–11. doi:10.1038/
s41598-018-20971-4
Kasuya, G., Fujiwara, Y., Tsukamoto, H., Morinaga, S., Ryu, S., Touhara, K., et al.
(2017a). Structural insights into the nucleotide base specificity of P2X receptors.
Sci. Rep. 7, 45208. doi:10.1038/srep45208
Kasuya, G., Yamaura, T., Ma, X. B., Nakamura, R., Takemoto, M., Nagumo, H., et al.
(2017b). Structural insights into the competitive inhibition of the ATP-gated
P2X receptor channel. Nat. Commun. 8, 876. doi:10.1038/s41467-017-00887-9
Lavoie, E. G., Fausther, M., Kauffenstein, G., Kukulski, F., Künzli, B. M., Friess, H.,
et al. (2010). Identification of the ectonucleotidases expressed in mouse, rat, and
human Langerhans islets: potential role of NTPDase3 in insulin secretion. Am.
J. Physiol. Endocrinol. Metab. 299, E647–E656. doi:10.1152/ajpendo.00126.2010
Lecka, J., Fausther, M., Künzli, B., and Sévigny, J. (2014). Ticlopidine in its prodrug
form is a selective inhibitor of human NTPDase1. Mediat. Inflamm. 2014,
547480. doi:10.1155/2014/547480
Chen, Z., Wang, J., Sun, W., Archibong, E., Kahkoska, A. R., Zhang, X., et al. (2018).
Synthetic beta cells for fusion-mediated dynamic insulin secretion. Nat. Chem.
Biol. 14, 86–93. doi:10.1038/nchembio.2511
Cie´slak, M., and Cie´slak, M. (2017). Role of purinergic signalling and
proinflammatory cytokines in diabetes. Clin. Diabetol. 6, 90–100. doi:10.
5603/DK.2017.0015
Lecka, J., Gillerman, I., Fausther, M., Salem, M., Munkonda, M. N., Brosseau, J. P.,
et al. (2013). 8-BuS-ATP derivatives as specific NTPD ase1 inhibitors. Br.
J. Pharmacol. 169, 179–196. doi:10.1111/bph.12135
Frontiers in Pharmacology | www.frontiersin.org
17
November 2020 | Volume 11 | Article 585876

Afzal et al.
Oxoindolin Hydrazine Carbothioamides as NTPDases Inhibitors
Lee, S. Y., Fiene, A., Li, W., Hanck, T., Brylev, K. A., Fedorov, V. E., et al. (2015).
Polyoxometalates—potent and selective ecto-nucleotidase inhibitors. Biochem.
Pharmacol. 93, 171–181. doi:10.1016/j.bcp.2014.11.002
Lee, S. Y., and Müller, C. E. (2017). Nucleotide pyrophosphatase/
phosphodiesterase 1 (NPP1) and its inhibitors. MedChemComm 8, 823–840.
doi:10.1039/C7MD00015D
Lee, S. Y., Sarkar, S., Bhattarai, S., Namasivayam, V., De Jonghe, S., Stephan, H.,
et al. (2017). Substrate-dependence of competitive nucleotide pyrophosphatase/
phosphodiesterase1 (NPP1) inhibitors. Front. Pharmacol. 8, 54. doi:10.3389/
fphar.2017.00054
Levesque, S. A., Lavoie, É. G., Lecka, J., Bigonnesse, F., and Sévigny, J. (2007).
Specificity of the ecto-ATPase inhibitor ARL 67156 on human and mouse
ectonucleotidases. Br. J. Pharmacol. 152, 141–150. doi:10.1038/sj.bjp.0707361
Longhi, M. S., Moss, A., Jiang, Z. G., and Robson, S. C. (2017). Purinergic signaling
during intestinal inflammation. J. Mol. Med. 95, 915–925. doi:10.1007/s00109-
017-1545-1
Lu, R., Zhang, Z., and Jiang, C. (2019). Recent progress on the discovery of P2Y14
receptor antagonists. Eur. J. Med. Chem. 175, 34–39. doi:10.1016/j.ejmech.2019.
04.068
Martín-Satué, M., Lavoie, E. G., Pelletier, J., Fausther, M., Csizmadia, E.,
Guckelberger, O., et al. (2009). Localization of plasma membrane bound
NTPDases in the murine reproductive tract. Histochem. Cell Biol. 131,
615–628. doi:10.1007/s00418-008-0551-3
Nabinger, D. D., Altenhofen, S., and Bonan, C. D. (2020). Zebrafish models:
gaining insight into purinergic signaling and neurological disorders. Prog.
Neurosychopharmacol. Biol. Psychiatry. 98, 109770. doi:10.1016/j.pnpbp.
2019.109770
Paes-Vieira, L., Gomes-Vieira, A. L., and Meyer-Fernandes, J. R. (2018). NTPDase
activities: possible roles on Leishmania spp infectivity and virulence. Cell Biol.
Int. 42, 670–682. doi:10.1002/cbin.10944
targets adult human pancreatic β cells for in vitro and in vivo analysis. Cell
Metabol. 29, 745–754. doi:10.1016/j.cmet.2018.10.007
Solini, A., and Novak, I. (2019). Role of the P2X7 receptor in the pathogenesis of
type 2 diabetes and its microvascular complications. Curr. Opin. Pharmacol. 47,
75–81. doi:10.1016/j.coph.2019.02.009
Stuhlmann, T., Planells-Cases, R., and Jentsch, T. J. (2018). LRRC8/VRAC anion
channels enhance β-cell glucose sensing and insulin secretion. Nat. Commun. 9,
1974. doi:10.1038/s41467-018-04353-y
Syed, S. K., Kauffman, A. L., Beavers, L. S., Alston, J. T., Farb, T. B., Ficorilli, J., et al.
(2013). Ectonucleotidase NTPDase3 is abundant in pancreatic β-cells and
regulates glucose-induced insulin secretion. Am. J. Physiol. Endocrinol.
Metab. 305, E1319–E1326. doi:10.1152/ajpendo.00328.2013
Tozzi, M., Larsen, A. T., Lange, S. C., Giannuzzo, A., Andersen, M. N., and Novak, I.
(2018). The P2X7 receptor and pannexin-1 are involved in glucose-induced
autocrine regulation in β-cells. Sci. Rep. 8, 8926. doi:10.1038/s41598-018-27281-9
Tozzi, M., and Novak, I. (2017). Purinergic receptors in adipose tissue as potential
targets in metabolic disorders. Front. Pharmacol. 8, 878. doi:10.3389/fphar.
2017.00878
Vakilian, M., Tahamtani, Y., and Ghaedi, K. (2019). A review on insulin trafficking
and exocytosis. Gene 706, 52–61. doi:10.1016/j.gene.2019.04.063
Zhong, A. H., Jiang, Z. G., Cummings, R. D., and Robson, S. C. (2017). Various
N-glycoforms differentially upregulate E-NTPDase activity of the NTPDase3/
CD39L3 ecto-enzymatic domain. Purinergic Signal. 13, 601–609. doi:10.1007/
s11302-017-9587-y
Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
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Pervez, H., Chohan, Z. H., Ramzan, M., Nasim, F. U. H., and Khan, K. M. (2009).
Synthesis and biological evaluation of some new N4-substituted isatin-3-
thiosemicarbazones. J. Enzym. Inhib. Med. Chem. 24, 437–446. doi:10.1080/
14756360802188420
Saunders, D. C., Brissova, M., Phillips, N., Shrestha, S., Walker, J. T., Aramandla, R.,
et al. (2019). Ectonucleoside triphosphate diphosphohydrolase-3 antibody
Frontiers in Pharmacology | www.frontiersin.org
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