Medicinal Chemistry Research (2023) 32:314–325 MEDICINAL

https://doi.org/10.1007/s00044-022-03000-y
CHEMISTRY
RESEARCH
ORIGINAL RESEARCH

Synthesis, biological evaluation, and molecular modeling studies of

a new series of imidazothiazole or imidazooxazole derivatives as
inhibitors of ectonucleoside triphosphate diphosphohydrolases
(NTPDases)
Mahmoud K. Shehata1 Muhammad Uzair2 Seyed–Omar Zaraei1 Afnan I. Shahin1 Syed J. A. Shah2 Saif Ullah2
● ● ● ● ● ●

Jamshed Iqbal2 Mohammed I. El–Gamal 1,3,4

●

Received: 4 September 2022 / Accepted: 5 December 2022 / Published online: 27 December 2022
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022

Abstract
NTPDases are a group of enzymes belonging to ecto–nucleotidases family. NTPDases have a vital role in the regulation of
ATP levels via the catalysis of ATP hydrolysis. Particularly, NTPDases are considered as interesting drug targets due to their
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involvement in pathological conditions, inflammation, infection, and cancer. In this article, we are reporting new
imidazothiazole and imidazooxazole derivatives possessing sulfonate or sulfamate moiety as potential inhibitors of human
NTPDase (1, 2, 3, and 8). Compound 1q is a selective inhibitor of NTPDase1 with IC50 value of 0.36 µM. Compound 1c is a
selective inhibitor of NTPDase2 with IC50 value of 0.29 µM. Compound 1p is a selective inhibitor of NTPDase3 with IC50
value of 0.37 µM. Lastly, compound 1u is a selective inhibitor of NTPDase8 with IC50 value of 0.36 µM. Molecular docking
of compounds 1q, 1c, 1p, and 1u within the active sites of NTPDase1, 2, 3, and 8, respectively, was carried out to investigate
the putative binding interaction. The contribution of each structural moiety to the binding with the active site was studied.
The detailed binding interactions are provided and illustrated with putative binding figures.
Keywords Ecto–nucleotidases h–NTPDase1, 2, 3, and 8 Imidazo[2,1–b]oxazole Imidazo[2,1–b]thiazole Sulfamate
● ● ● ● ●

Sulfonate

Introduction

These authors contributed equally: Mahmoud K. Shehata, Adenosine triphosphate (ATP) is an important nucleotide that
Muhammad Uzair plays a crucial role in communication between cells. Every cell
Supplementary information The online version contains in our body generates ATP which in turn acts as an agonist for
supplementary material available at https://doi.org/10.1007/s00044- P2 receptors (P2X and P2Y) [1, 2]. P2 receptors are the
022-03000-y. membrane-bound extracellular receptors that are involved in
various physiological functions of our body e.g. sensory
* Jamshed Iqbal
drjamshed@cuiatd.edu.pk transmission, hormone secretion, glial–neuron interaction and
* Mohammed I. El–Gamal neurotransmission [3, 4]. However, cascades of
drmelgamal2002@gmail.com ecto–nucleotidases metabolize ATP. This chain of ectoen-
zymes is composed of four enzyme families such as ectonu-
1
Sharjah Institute for Medical Research, University of Sharjah, cleoside triphosphate diphosphohydrolases (NTPDases),
Sharjah 27272, United Arab Emirates nucleotide pyrophosphatases/phosphodiesterases (NPPs),
2
Centre for Advanced Drug Research, COMSATS University ecto–5′–nucleotidase (e–5′–N), and alkaline phosphatases
Islamabad, Abbottabad Campus, Abbottabad 22060, Pakistan (APs or ALPs). These ecto–nucleotidases catalyze hydrolysis
3
Department of Medicinal Chemistry, College of Pharmacy, of extracellular nucleotides to produce inorganic end product
University of Sharjah, Sharjah 27272, United Arab Emirates (pyrophosphate) and adenosine [4, 5]. Therefore, these cas-
4
Department of Medicinal Chemistry, Faculty of Pharmacy, cades of ecto–nucleotidases are well known for their role in
Mansoura University, Mansoura 35516, Egypt regulating extracellular ATP concentration and generating
Medicinal Chemistry Research (2023) 32:314–325
additional ligands for P2 (P2X and P2Y) receptors. Fur-
thermore, NTPDases family of enzymes is the most
dominant for formation of adenosine monophosphate
(AMP) through degradation of adenosine triphosphate and
adenosine diphosphate (ADP). This family possesses eight
members such as NTPDase1–8. NTPDase1, 2, 3 and 8 have
been identified as extracellular membrane–bound enzymes
because they are responsible for catalyzing the hydrolysis
of extracellular ATP whereas, NTPDase4, 5 and 6 are
reported as intracellular ectoenzymes [6]. The first isozyme
of NTPDases family named CD39 was expressed in B
lymphocytes [7], which was later identified in T lympho-
cytes and renamed NTPDase1 [8] while the other members
of this family began to be uncovered. Therefore,
ecto–nucleotidases play a role in controlling the bioavail-
ability of purines and purinergic signals through P2X and
P2Y receptors in the extracellular domain [9].
NTPDases have five active regions (ACR1–ACR5)
known as apyrase-conserved regions [10]. Extracellular
NTPDases have two transmembrane domains, which are
responsible to control the specificity of substrate for binding
and hydrolysis [11]. Moreover, this enzyme family requires
Ca2+ and Mg2+ ions (as cofactors) for their catalytic
activity, any alteration in these ions leads to a change in the
enzymatic activity [12]. NTPDases are expressed in all
tissues, where they are responsible for various physiological
functions such as development, blood flow, hormone
secretion, and neurotransmitter release [11]. In addition,
extracellular NTPDases family is also involved in patho-
logical functions such as injury, inflammation, infection,
and cancer [9].
Several studies reported that NTPDase2 is localized in
specialized astrocytes in rodent brain, such as laminar
astrocytes associated with fiber tracts in the brain stem and
cerebrum [13, 14], tanycytes, non–stellate astrocytes in the
gray matter of discrete regions like habenula, satellite
astrocytes in the dorsal root ganglion [13], and
astrocyte–like progenitor cells of the subventricular zone
(SVZ) of the lateral ventricle. NTPDase3 is localized in the
midline regions in the thalamus, hypothalamus, and the
medulla oblongata [15, 16]. NTPDase2, and to a lesser
extent NTPDase3, preferentially catalyze the depho-
sphorylation of ATP to ADP, generating the physiological
ligand for P2Y1, P2Y12, and P2Y13 receptors [17–20].
Therefore, NTPDase2 and –3 may modulate inflammatory
reactions within the CNS and could represent useful ther-
apeutic targets in neuroinflammatory and neurodegenerative
diseases.
Inhibitors of NTPDases can be categorized into
nucleotide–based and nonnucleotide–based inhibitors.
ARL67156, 8–BuS–ATP, and PSB–6426 (Fig. 1) are
nucleotide–based inhibitors of NTPDases with different
profiles against different isomers. Nonnucleotide–based
315
inhibitors such as suramin, Reactive blue–2, and PPADS
(Fig. 1) were discovered, yet some proved to be
non–selective, moderately active, or had limited stability
[21]. Quinoline–based derivatives containing ethers and/or
esters (Fig. 1) were also developed as inhibitors of
NTPDases, where varying selectivity profiles were dis-
covered as inhibitors of a single isozyme or pan inhibitors
against all 4 isozymes [22, 23]. Other classes of
nonnucleotide–based inhibitors include polyoxometalates
and antibodies. Both suffer from liabilities such as stability
and/or incomplete inhibition [21]. To our knowledge, no
inhibitors containing sulfonate warheads have been reported
in the literature. Such development provides an advantage
in investigating new modalities and strategies to develop
NTPDases inhibitors.
Results and discussion
Chemistry
Synthesis of the methoxy intermediates 5a and 5b was
achieved using the reported procedure in [24, 25], where
2–bromo–1–(4–methoxyphenyl)ethan–1–one (3) and
2–aminothiazole (4a)/2–aminooxazole (4b) were refluxed
in ethanol to yield methoxy derivatives 5a and 5b,
respectively. The yield percentages of compounds 5a and
5b were 90 and 85%, respectively. The demethylation of
5a and 5b proceeded using boron trifluoride methylsulfide
complex in dichloromethane to yield intermediates 6a and
6b similar to the reported procedure [26]. Compounds 6a
and 6b were obtained in yields of 80 and 75%, respec-
tively. The target sulfonate compounds 1a–1v were syn-
thesized via reacting the hydroxyl intermediates 6a,b with
the appropriate sulfonyl chloride in the presence of trie-
thylamine (Scheme 1) [27]. The yields of compounds
1a–1v ranged from 11 to 97%. Most of the compounds
were obtained in high yield, while few of them were
isolated in rather lower yields. Sulfamoyl derivatives
2a–2f were synthesized by the treatment of the hydroxyl
intermediates 6a and 6b with sodium hydride in dry DMF
at 0 °C, followed by an interval of 15 min before the
addition of appropriate sulfamoyl chloride reagent to the
reaction mixture (Scheme 1) [27]. Compounds 2a–2f were
isolated in low-to-moderate yields (9–59%). The identity
of the target compounds 1a–1v and 2a–2f was confirmed
by 1H NMR, 13C NMR, and LC–MS. The signals of the
attached sulfonate and sulfamate moieties were confirmed
by NMR, and the molecular weight increased than the
intermediate hydroxyl intermediates and confirmed by
LC–MS. The detailed spectral data are provided in the
Experimental section, and the charts are provided in the
Supplementary File.
316 Medicinal Chemistry Research (2023) 32:314–325

Fig. 1 Structures of known N NH2 O

NTPDases inhibitors N N
NH
N N N N N
O
N N O O
S OH
O O
NH
OH OH HO
HN CHO O
O ONa
O O OH O O OH HO P
P P O ONa
O O
O O O O
P P N
Br O O O
O N
O P Br O P P
O O O O O O
NaO3S SO3Na

ARL67156 8-BuS-ATP PSB-6426 PPADS

O NH2
SO3Na
NO2
Cl
O HN N
N N
O
N N N MeO
H H O
SO3Na SO3Na

Reactive blue-2 Qunoline based inhibitor

O
H
NaO3S O N
O N N
H H
NH O
N
H
NaO3S SO3Na O NH SO3Na

SO3Na
SO3Na
Suramin

Biological activity and structure–activity
relationship
The target compounds were tested against four recombinant
isozymes of h–NTPDase, i.e., h–NTPDase1, 2, 3 and 8. All
the compounds were initially screened at 0.1 mM con-
centration, then the active derivatives were further tested at
other concentrations to calculate their IC50 values. Their
results are shown in Table 1. Suramin was utilized as a
reference standard in this assay.
Imidazothiazole-possessing sulfonate and sulfamate
derivatives (compounds 1a–k and 2a–2c)
The aliphatic sulfonate derivatives 1a–1d did not show any
significant inhibitory activity against NTPDase1, 3, and 8
isozymes at 0.1 mM. Compound 1c bearing n–propylsulfonyl
side chain showed promising activity against NTPDase2 with
an IC50 of 0.29 µM. The aromatic derivatives showed any
significant inhibitory activity against NTPDase2 and 3.
Interestingly, compound 1i bearing m–CF3 phenylsulfonyl
side chain was the only derivative among the imidazothiazole
series that showed an inhibitory effect against NTPDase8
(IC50 = 6.10 µM). Furthermore, four aromatic sulfonyl deri-
vatives exhibited inhibitory activity against NTPDase1. The
unsubstituted phenylsulfonate derivative 1e was the most
potent among the aromatic sulfonyl derivatives with an IC50
of 2.83 µM. Introducing para substitution (e.g. 4–F and
4–Me, 1f (IC50 = 4.02 µM) and 1g (IC50 = 4.25 µM),
respectively) reduced the potency in comparison to the
unsubstituted derivative 1e. Compound 1j bearing 3–CF3,
4–Cl substitution on the phenylsulfonate side chain dramati-
cally reduced the activity by 2.5 folds in comparison to
unsubstituted phenyl analog 1e. It is apparent that substitution
on the phenyl ring is detrimental to activity against
NTPDase1. Compounds 1e and 1i were the most active
toward NTPDase1 and 8, respectively among the aromatic
sulfonate derivatives.
Furthermore, the sulfamate derivatives 2a–2c lacked
inhibitory activity against NTPDase3 and 8. Compounds 2a
(free sulfamate) and 2b (N-methylsulfamate) showed inhi-
bitory effect against NTPDase1 (IC50 = 2.51 µM) and
NTPDase2 (IC50 = 2.63 µM), respectively. However, com-
pound 2c bearing a piperidinylsulfonyl side chain exhibited
Medicinal Chemistry Research (2023) 32:314–325 317

O O
NH2 N X N X S N X
O i ii iii R
+ O HO O N
O N X N N
Br
4a, X = S 5a, X = S 6a, X = S 1a-v
3 4b, X = O 5b, X = O 6b, X = O

iv

O O O
O N X
R S N X S
N N O
H O N N

2a,b,d,e 2c,f

Scheme 1 Reagents and reaction conditions: (i) EtOH, reflux, 16 h, THF, 0 °C then rt, overnight (sulfonate derivatives), 11–97%; (iv)
90% (5a), 85% (5b); (ii) BF3.Me2S, DCM, rt, overnight, 80% (6a), NaH, appropriate sulfamoyl chloride derivative, DMAC, 0 °C then rt,
75% (6b); (iii) triethylamine, appropriate sulfonyl chloride derivative, overnight (sulfamate derivatives), 9–59%

Table 1 Structures of the target

compounds 1a–1v and 2a–2f
and their inhibitory effects O O
O O O O
together with suramin against S N X R S N X S N X
R N N
NTPD–1, –2, –3, and –8 O N H O N
O N

1a-v 2a,b,d,e 2c,f

Compound no. R X NTPDase1 NTPDase2 NTPDase3 NTPDase8

IC50 (µM) ± SEM/% Inhibition at 100 µM

1a Me S 41.27% 30% 12.19% 31%

1b Et S 13.42% 22% 18.27% 26%
1c n–Pr S 30.48% 0.29 ± 0.01 14.44% 32%
1d Cyclohexyl S 30.40% 19.35% 20% 44%
1e Ph S 2.83 ± 0.19 33% 10.06% 37.22%
1f 4–F(C6H4) S 4.02 ± 0.02 27% 30% 35%
1g 4–Me(C6H4) S 4.25 ± 0.28 24% 13% 40%
1h 3,4–diCl(C6H3) S 24% 40% 16% 31%
1i 3–CF3(C6H4) S 36% 38.23% 24.36% 6.1 ± 0.13
1j 3– CF3,4–Cl(C6H3) S 7.15 ± 0.18 20.05% 23% 21.29%
1k 3,5–bisCF3(C6H3) S 2.68% 29.37% 20.34% 29%
1l Me O 10% 47.2% 24.24% 1.15 ± 0.02
1m Et O 29.13% 46% 20.08% 26%
1n n–Pr O 30.43% 12.35% 24% 17.23%
1o Cyclohexyl O 10.34% 6.50 ± 0.04 22% 1.15 ± 0.18
1p Ph O 23.18% 3.06 ± 0.04 0.37 ± 0.02 22%
1q 4–F(C6H4) O 0.36 ± 0.02 6.92 ± 0.05 32.32% 37%
1r 4–Me(C6H4) O 5.72 ± 0.40 6.35 ± 0.36 31.31% 1.40 ± 0.06
1s 3,4–diCl(C6H3) O 29% 0.36 ± 0.04 32.20% 12.32%
1t 3–CF3(C6H4) O 38.15% 2.00 ± 0.20 36% 13%
1u 3– CF3,4–Cl(C6H3) O 28.01% 0.79 ± 0.02 33.33% 0.36 ± 0.02
1v 3,5–bisCF3(C6H3) O 28% 1.43 ± 0.14 21% 37.22%
2a H S 2.51 ± 0.19 37.30% 11.40% 34.40%
2b Me S 37% 2.63 ± 0.19 14.08% 34.00%
2c – S 7.32 ± 0.48 1.43 ± 0.02 27.33% 33.20%
2d H O 45.40% 0.70 ± 0.02 41.16% 38%
2e Me O 2.92 ± 0.14 47% 33.33% 7.34%
2f – O 47.31% 0.96 ± 0.08 13% 11.66 ± 0.73
Suramin 16.1 ± 1.08 24.1 ± 0.15 4.30 ± 0.84 101.1 ± 2.34
318
an inhibitory activity toward two isoenzymes, NTPDase2
(IC50 = 1.43 µM) with a higher sensitivity by 5 folds com-
pared to NTPDase1 (IC50 = 7.32 µM).
Imidazooxazole-possessing sulfonate and sulfamate
derivatives (compounds 1l–1v, and 2d–2f)
Upon comparing the imidazooxazole derivatives and the
isosteric imidazothiazole analogs, imidazooxazole deriva-
tives 1l–1v and 2d–2f showed dramatic rise in the potency
against most of the NTPDases isoforms, especially against
NTPDase2 isoform. The aliphatic sulfonates 1l–1o revealed
weak inhibitory effects against isoenzymes 1 and 3, similar
to the imidazothiazole aliphatic analogs. Astonishingly, the
methyl sulfonate 1l and the cyclohexyl sulfonate 1o
exhibited activity against NTPDase8 with an IC50 value of
1.15 µM for both of them. (Substituted) phenyl derivatives
1p–1v were all sensitive mainly toward NTPDase2, being
more potent than suramin, suggesting a synergistic effect of
the aryl sulfonate moiety together with the imidazooxazole
nucleus to target NTPDase2, since the imidazothiazole
analogs lacked sensitivity to it. Evidently, substitution on
phenylsulfonate side chain impacted activity based on the
position of the substituent on the phenyl ring, as well as the
number of substitutions. A reduction in potency was
experienced when a substituent was introduced at para
position on the terminal phenyl ring (1q, 4–F,
IC50 = 6.92 µM) and (1r, 4–Me, IC50 = 6.35 µM) compared
to unsubstituted phenylsulfonate derivative (1p,
IC50 = 3.06 µM). However, meta substitution (1t, 3–CF3,
IC50 = 2.00 µM) led to an improved potency. Disubstitution
on the phenyl ring improved the activity such as derivative
1v (3,5–bisCF3, IC50 = 1.43 µM), while meta, para-dis-
ubstituted phenyl ring potentiated the inhibition of
NTPDase2 (1s, 3,4–diCl, IC50 = 0.36 µM) and (1u, 3–CF3,
4–Cl, IC50 = 0.79 µM). It is possible that the aromatic side
chain has a positive effect on inhibitory activity since the
aliphatic cyclohexyl ring was less potent, also increasing
hydrophobicity on the terminal aryl group with halogens
substituted on positions 3 and 4 exhibited positive impact
on the potency. Surprisingly, compound 1p (unsubstituted
phenylsulfonate) was the only derivative among both series
that revealed an activity against NTPDase3
(IC50 = 0.37 µM) with 12–fold higher potency than suramin
(IC50 = 4.30 µM). Compounds 1r (p–methyl) and 1u
(3–CF3, 4–Cl) displayed activity against NTPDase8
(IC50 = 1.40 µM and 0.36 µM, respectively). Interestingly,
1u is more potent toward isoenzyme 8 by 280–folds com-
pared to Suramin. Compounds 1q (4–fluoro) and 1r
(4–methyl) showed inhibitory activity toward NTPDase1
(IC50 = 0.36 µM and 5.72 µM, respectively), where com-
pound 1q showed the highest potency among both series by
45–folds compared to Suramin (IC50 = 16.1 µM). Fluoro is
Medicinal Chemistry Research (2023) 32:314–325
a small substituent, maybe bigger substituents or more
number of substituents are not tolerated in case of
NTPDase1.
Moreover, the sulfamate derivatives 2d–2f had no pro-
mising inhibitory effect toward NTPDase3. Compounds 2e
and 2f were the only derivatives among the imidazooxazole
sulfamates which exhibited inhibitory activity against
NTPDase1 and NTPDase8, respectively. Derivatives 2d
and 2f revealed activity against NTPDase2 with IC50 values
of 0.70 and 0.96 µM, respectively.
Molecular docking studies
Molecular docking studies were conducted to study the
putative binding modes of the most potent inhibitors inside
the h–NTPDases crystal structures. Compounds 1q, 1c, 1p,
and 1u were docked within the active sites of h–NTPDase1,
2, 3, and 8, respectively. It has been noticed that the sul-
fonate group of all compounds was actively participating in
both hydrophilic and hydrophobic interactions with amino
acid residues of modeled proteins.
With h–NTPDase1, compound 1q was found to be lined
with active pocket residues Asp54, Gly56, Ser57, Ser58,
His59, and Thr131 amino acid residues as shown in Fig. 2.
Compound 1q bound within h–NTPDase1 active site with
binding energy of –22 KJmol–1 via different hydrogen and
hydrophobic bonds. In this regard, sulfonate oxygen atom
formed hydrogen bond with His59 and Ser57. Furthermore,
the same moiety interacts with Thr131 and Gly56 amino
acid residues.
The amino acid residues lining the h–NTPDase2 active
site around compound 1c were Gly47, Ser48, Ser49, His50,
Ala123, Gly204, and Tyr350. The inhibitor has a binding
energy of –14 KJmol–1 with the active site. In Fig. 3, the
sulfonate moiety of compound 1c formed a network of
hydrogen bonds with Ser48, Ser49 and His50 residues.
Fig. 2 Putative binding mode of inhibitor 1q within the h–NTPDase1
active site
Medicinal Chemistry Research (2023) 32:314–325
Fig. 3 Putative binding mode of inhibitor 1c within the h–NTPDase2
active site
Fig. 4 Putative binding mode of inhibitor 1p within the h–NTPDase3
active site
Hydrophobic interactions were monitored with Gly204 and
Gly47. A pi–pi stacked interaction was found between the
residue His50 and imidazo[2,1–b]thiazole ring. However,
sulfur atom of the imidazothiazole nucleus was noticed to
form pi–sulfur interaction with residue Tyr350.
Compound 1p adjusted itself in the active site of mod-
eled h–NTPDase3, as illustrated in Fig. 4. The most
important amino acid residues that were involved in the
binding interactions with the inhibitor were Gly64, Ser65,
Ser66, Arg67, Lys95, Gly221, and Asn265. Compound 1p
bound with binding energy of –9.0 KJmol–1 inside the
h–NTPDase3 active site. It formed hydrogen bonding
interactions with Ser65, Ser66, and Arg67 through its sul-
fonate oxygen. Furthermore, pi–alkyl hydrophobic interac-
tion was observed between Arg67 residue and the central
phenyl ring of compound 1p, while pi–cation interaction
was noticed between imidazo[2,1–b]oxazole ring of com-
pound 1p with Lys95.
319
Fig. 5 Putative binding mode of inhibitor 1u within the h–NTPDase8
active site
In the active site of modeled h–NTPDase8 (Fig. 5),
compound 1u bound with binding energy of –17 KJmol–1.
The amino acid residues lining the active site of
h–NTPDase8 surrounding compound 1u were Asp48,
Gly50, Ser51, Ser52, His53, Phe57, Asp205, Met206,
Ser353, Asn354, and Asp437. The benzenesulfonate scaf-
fold was showing both hydrophilic and hydrophobic inter-
actions such as three hydrogen bonds formed with Ser51,
Ser52, and His53. The fluorine atom formed halogen bonds
with residues Met206 and Asp205. Furthermore, the central
phenyl ring of compound 1u was stabilized by forming
pi–anion interaction with Asp48. A pi–pi stacked interac-
tion was noticed between residue Phe57 and imidazo[2,1–b]
oxazole ring of compound 1u.
Molecular dynamic simulation
Molecular dynamic simulations of the most potent
h–NTPDase1 inhibitor 1q were carried out using GRO-
MACS software package. Simulation of the inhibitor was
carried out in the presence of Ca2+ and Mg2+ divalent ions
in the active site. Simulation of the apo–enzyme and with
inhibitor was carried out for 50 ns.
Root mean square deviation (RMSD) profile of the
h–NTPDase1 with and without the inhibitor is given in
Fig. 6. The h–NTPDase1 apo–enzyme and h–NTPDase1
with inhibitor 1q were found to have a similar root mean
square deviation during the entire molecular dynamic
simulations and no abrupt fluctuations in the RMSD value
of the enzyme–inhibitor complex was observed.
The root means square fluctuation (RMSF) profile of the
h–NTPDase1 apo–enzyme and with compound 1q is given
in Fig. 7. The RMSF profile of apo–enzyme and with
320
Fig. 6 RMSD profile of h–NTPDase1 apo–enzyme and with inhibitor
1q. The apo–enzyme’s profile is shown in black while that of the
complex in red color
Fig. 7 RMSF profile of h–NTPDase1 apo–enzyme and with inhibitor
1q. The apo–enzyme’s profile is shown in black while that of the
complex in red color
inhibitor bound were determined with an aim to determine
the fluctuations of key residues that were affected due to the
attachment of the inhibitor. As shown in Fig. 6, the residues
180–220 were found to be the most flexible followed by
residues around 140–170 and around 360–485 of
h–NTPDase1. The h–NTPDase1 in complex with inhibitor
1q however, did not change the RMSF profile significantly.
The fluctuation in RMS values for h–NTPDase1 in complex
with compound 1q is much similar to that of the
h–NTPDase1 apo–enzyme.
Conclusion
This study delivered new series of imidazothiazole- and
imidazooxazole-based sulfonates and sulfamates. This
series was screened for their inhibitory activity toward
four isozymes of human NTPDases, 1, 2, 3, and 8. The
SAR of these derivatives has shown variability in the
Medicinal Chemistry Research (2023) 32:314–325
inhibition potencies against different isoenzymes, speci-
fically the effect of replacing sulfur atom of imida-
zothiazole core with oxygen of imidazooxazole core had
an improved sensitivity toward NTPDase2. The activity
of the reported compounds was compared to the standard
inhibitor, Suramin. Some derivatives have shown selec-
tive inhibition, while others as multi–targeted inhibitor.
The results suggested that some derivatives were tre-
mendously more potent compared to the standard, such as
compounds 1q, 1c, 1p, and 1u, exhibiting more potent
activity against certain NTPDase isozymes in comparison
to suramin. Compounds 1q, 1c, 1p, and 1u showed
stronger potency compared to Suramin, with IC50 values
of 0.36 µM (against NTPDase1), 0.29 µM (against
NTPDase2), 0.37 µM (against NTPDase3), and 0.36 µM
(against NTPDase8), respectively. Molecular docking
studies were performed for the most promising com-
pounds against their particularly inhibited isoenzyme.
The sulfonate/sulfamate moiety was introduced to form
hydrogen bonds by oxygen atoms, hydrophobic interac-
tions by sulfur atom, and to chelate the metal cation in the
enzymes. Moreover, the imidazothiazole/imidazooxazole
nucleus was utilized to form pi–pi and arene–cation
interactions.
Experimental
General
Bruker Avance (500 MHz spectrometer) was used to
analyze the compounds by 1H and 13C NMR. LC–MS
analysis was carried out by LC–MS analyzer (Waters
Corporation, MA, USA). All the solvents and reagents
were purchased from commercial companies and used as
such. The final and intermediate compounds were pur-
ified by flash column chromatography (silica gel, pore
size 0.040~0.063 mm, 230–400 mesh) using laboratory
reagent grade solvents.
Chemical synthesis
Synthesis of 6–(4–methoxyphenyl)imidazo[2,1–b]thiazole
(5a) and 6–(4–methoxyphenyl)imidazo[2,1–b]oxazole (5b)
The synthesis of fused heterocyclic rings was accomplished
using the reported procedure in [24, 25].
Synthesis of 4–(imidazo[2,1–b]thiazol-6–yl)phenol (6a) and
4–(imidazo[2,1–b]oxazol-6–yl)phenol (6b)
The demethylation of methoxy intermediates was accom-
plished using the procedure reported in [26]. The methoxy
Medicinal Chemistry Research (2023) 32:314–325
intermediates (2.17 mmol) were dissolved and stirred in
anhydrous dichloromethane (5 mL), BF3.MeS (3.43 mL,
32.6 mmol) was added under N2 (g) at room temperature
overnight. The completion of reaction was confirmed via
TLC. The mixture was basified with excess saturated aqu-
eous K2CO3 solution and extracted with ethyl acetate
(10 mL). The organic layer was dried over anhydrous
Na2SO4, concentrated in vacuo, and was purified using
column chromatography. The product was eluted using 1:1
ethyl acetate/hexane.
Synthesis of sulfonate target compounds 1a–1v
The synthesis of sulfonate derivatives was accomplished
using a similar reported procedure [27]. Appropriate phenol
(0.28 mmol) was dissolved in anhydrous THF (2 mL) and
was stirred under N2 (g). Triethylamine (0.15 mL, 1 mmol)
was then added at 0 °C and was further stirred for 15 min.
Appropriate sulfonyl chloride (0.83 mmol) dissolved in
anhydrous THF (2 mL) was then added dropwise to the
reaction mixture at 0 °C and then left to stir at rt overnight.
The completion of reaction was confirmed via TLC. THF
was removed in vacuo and the resultant crude was extracted
using ethyl acetate (5 mL) and water (5 mL). Aqueous layer
was reextracted with ethyl acetate (5 mL). The organic
layers were collected, dried over anhydrous Na2SO4, con-
centrated in vacuo, and the crude product was purified by
flash column chromatography by using ethyl acetate:hexane
(1:3 v/v) as an eluent.
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl methanesulfonate
1
(1a) Yield: 61%; mp: 202–205 °C; H NMR (MeOD-d4,
500 MHz) δ 8.12 (s, 1H), 7.88 (d, 2H, J = 8.5 Hz), 7.81 (d,
1H, J = 4.0 Hz), 7.28 (d, 2H, J = 8.5 Hz), 7.20 (d, 1H,
J = 4.0 Hz), 3.25 (s, 3H); 13C NMR (MeOD-d4, 125 MHz)
δ 151.9, 150.3, 127.8, 123.7, 121.0, 114.8, 110.9, 37.5; LC/
MS m/z: 295.1 (M++1).
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl ethanesulfonate (1b)
Yield: 96%; mp: 158–160 °C; 1H NMR (DMSO-d6,
500 MHz) δ 8.27 (s, 1H), 7.96 (d, 1H, J = 4.5 Hz), 7.92 (d,
2H, J = 8.5 Hz), 7.35 (d, 2H, J = 8.5 Hz), 7.29 (d, 1H,
J = 4.0 Hz), 3.53 (q, 2H, J = 7.0 Hz), 1.39 (t, 3H,
J = 7.0 Hz); 13C NMR (DMSO-d6, 125 MHz) δ 149.4,
147.8, 145.1, 133.3, 126.2, 122.4, 120.1, 113.4, 109.9,
44.5, 8.1; LC/MS m/z: 309.1 (M++1).
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl propane-1-sulfonate
(1c) Yield: 41%; mp: 108–110 °C; 1H NMR (CDCl3,
500 MHz) δ 7.85 (dd, 2H, J = 2.0, 7.0 Hz), 7.74 (s, 1H),
7.45 (d, 1H, J = 4.5 Hz), 7.30 (dd, 2H, J = 1.5, 6.5 Hz),
6.86 (d, 1H, J = 4.5 Hz), 3.51–3.22 (m, 2H), 2.05–1.99 (m,
2H), 1.12 (t, 3H, J = 7.5 Hz); 13C NMR (CDCl3, 125 MHz)
321
δ 150.5, 148.5, 146.4, 133.2, 126.8, 122.4, 118.7, 113.2,
108.4, 52.2, 17.5, 13.0; LC/MS m/z: 322.9 (M++1).
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl cyclohexanesulfonate
(1d) Yield: 21%; mp: 156–158 °C; 1H NMR (CDCl3,
500 MHz) δ 7.83 (d, 2H, J = 8.5 Hz), 7.73 (s, 1H), 7.44 (d,
1H, J = 4.5 Hz), 7.29 (d, 2H, J = 8.5 Hz), 6.85 (d, 1H,
J = 4.0 Hz), 3.23–3.18 (m, 1H), 2.35 (d, 2H, J = 11.5 Hz),
1.97–1.94 (m, 2H), 1.78– 1.70 (m, 3H), 1.34–1.25 (m, 3H);
13
C NMR (CDCl3, 125 MHz) δ 150.5, 148.4, 146.6, 133.0,
126.8, 122.5, 122.5,118.7, 113.1, 108.4, 60.3, 26.7, 25.1,
25.1; LC/MS m/z: 363.1 (M++1).
4–(Imidazo[2,1–b]thiazol-6–yl)phenyl benzenesulfonate
(1e) Yield: 70%; mp: >230 °C; 1H NMR (DMSO-d6,
500 MHz) δ 8.22 (s, 1H), 7.94 (d, 1H, J = 4.5 Hz),
7.89–7.87 (m, 2H), 7.84–7.80 (m, 3H), 7.68 (t, 2H,
J = 7.5 Hz), 7.27 (d, 1H, J = 4.0 Hz), 7.03 (d, 2H,
J = 8.5 Hz); 13C NMR (DMSO-d6, 125 MHz) δ 149.5,
147.8, 144.9, 135.0, 134.3, 133.5, 129.8, 128.2, 126.1,
122.3, 120.1, 113.4, 110.0; LC/MS m/z: 357.1 (M++1).
4–(Imidazo[2,1–b]thiazol-6–yl)phenyl
4-fluorobenzenesulfonate (1f) Yield: 14%; mp:
148–150 °C; 1H NMR (CDCl3, 500 MHz) δ 7.86–7.83 (m,
2H), 7.75–7.72 (m, 3H), 7.45 (d, 1H, J = 4.0 Hz),
7.21–7.17 (m, 2H), 7.00 (d, 2H, J = 9.0 Hz), 6.87 (d, 1H,
J = 4.5 Hz); 13C NMR (CDCl3, 125 MHz) δ 166.2 (d, 1C,
J = 256.1 Hz), 150.5, 148.8, 133.2, 131.6 (d, 1C,
J = 9.7 Hz), 131.4 (d, 1C, J = 11.5 Hz), 126.6, 122.8,
118.7, 116.7 (d, 1C, J = 17.6 Hz), 113.3, 108.5; LC/MS
m/z: 375.1 (M++1).
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl
4-methylbenzenesulfonate (1g) Yield: 63%; mp:
168–170 °C; 1H NMR (CDCl3, 500 MHz) δ 7.73–7.69 (m,
5H), 7.44 (d, 1H, J = 4.5 Hz), 7.30 (d, 2H, J = 8.0 Hz), 7.00
(dd, 2H, J = 2.0, 7.0 Hz), 6.86 (d, 1H, J = 4.5 Hz), 2.44 (s,
3H); 13C NMR (CDCl3, 125 MHz) δ 150.4, 149.0, 145.5,
132.4, 129.9, 128.7, 126.5, 122.9, 118.7, 113.3, 108.4,
21.8; LC/MS m/z: 371.2 (M++1).
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl 3,4-dichlorobenzene-
sulfonate (1h) Yield: 63%; 1H NMR (CDCl3, 500 MHz)
δ 7.96 (d, 1H, J = 2.0 Hz), 7.77 (d, 2H, J = 8.5 Hz), 7.72 (s,
1H), 7.63–7.58 (m, 2H), 7.44 (d, 1H, J = 4.0 Hz), 7.03 (d,
2H, J = 8.5 Hz), 6.86 (d, 1H, J = 4.5 Hz); 13C NMR
(CDCl3, 125 MHz) δ 150.6, 148.5, 146.3, 139.6, 135.1,
134.2, 133.6, 131.3, 130.4, 127.7, 126.7, 122.6, 118.6,
113.2, 108.5; LC/MS m/z: 425.0 (M++1).
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl 3-(trifluoromethyl)ben-
zenesulfonate (1i) Yield: 28%; mp: 112–114 °C; 1H NMR
322
(CDCl3, 500 MHz) δ 8.15 (s, 1H), 8.01 (d, 1H, J = 8.0 Hz),
7.93 (d, 1H, J = 8.0 Hz), 7.77–7.66 (m, 4H), 7.45 (d, 1H,
J = 4.5 Hz), 7.03–7.00 (m, 2H), 6.87 (d, 1H, J = 4.5 Hz);
13
C NMR (CDCl3, 125 MHz) δ 149.6 (d, 1C, J = 242.5 Hz),
146.1, 136.7, 133.4, 131.9, 131.0 (d, 1C, J = 3.5 Hz),
130.1, 126.7, 125.7 (d, 1C, J = 4.0 Hz), 122.7, 118.6,
113.3, 108.5; LC/MS m/z: 425.0 (M++1).
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl 4-chloro-3-(trifluoro-
methyl)benzenesulfonate (1j) Yield: 81%; 1H NMR
(CDCl3, 500 MHz) δ 8.19 (d, 1H, J = 1.5 Hz), 7.89 (dd, 1H,
J = 1.0, 8.0 Hz), 7.78–7.77 (m, 2H), 7.72 (m, 1H),
7.68–7.66 (m, 1H), 7.45–7.38 (m, 1H), 7.04–7.02 (m, 2H),
6.86–6.86 (m, 1H); 13C NMR (CDCl3, 125 MHz) δ 149.5
(d, 1C, J = 268.7 Hz), 146.1, 139.3, 134.6, 133.7, 132.7 (d,
1C, J = 8.6 Hz), 130.2 (d, 1C, J = 32.6 Hz), 129.7 (d, 1C,
J = 32.9 Hz), 128.0 (d, 1C, J = 5.4 Hz), 127.9 (d, 1C,
J = 5.7 Hz), 126.8, 123.0, 122.6, 120.8, 118.6, 113.3,
108.5; LC/MS m/z: 459.1 (M++1).
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl 3,5-bis(trifluoromethyl)
benzenesulfonate (1k) Yield: 74%; mp: 169–172 °C; 1H
NMR (CDCl3, 500 MHz) δ 8.30 (s, 2H), 8.17 (s, 1H), 7.79
(d, 2H, J = 9.0 Hz), 7.73 (s, 1H), 7.44 (d, 1H, J = 4.5 Hz),
7.04 (d, 2H, J = 8.5 Hz), 6.87 (d, 1H, J = 4.5 Hz); 13C
NMR (CDCl3, 125 MHz) δ 150.6, 147.1 (d, 1C,
J = 266.9 Hz), 138.4, 134.0, 133.3 (d, 1C, J = 34.5 Hz),
128.9 (d, 1C, J = 3.2 Hz), 127.9, 127.9 (d, 1C, J = 3.5 Hz),
126.9, 123.4, 122.4, 121.2, 118.6, 113.3, 108.6; LC/MS m/
z: 493.1 (M++1).
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl methanesulfonate
(1l) Yield: 78%; mp: 172–175 °C; 1H NMR (DMSO-d6,
500 MHz) δ 7.98 (d, 1H, J = 2.0 Hz), 7.94 (d, 1H,
J = 1.5 Hz), 7.87 (d, 2H, J = 8.5 Hz), 7.82 (s, 1H), 7.35 (d,
2H, J = 9.0 Hz), 3.38 (s, 3H); 13C NMR (DMSO-d6,
125 MHz) δ 155.6, 147.8, 141.5, 139.1, 133.8, 125.9,
122.5, 112.8, 104.7, 37.3; LC/MS m/z: 279.0 (M++1).
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl ethanesulfonate
1
(1m) Yield: 97%; mp: 134–136 °C; H NMR (CDCl3,
500 MHz) δ 7.81 (d, 2H, J = 8.5 Hz), 7.40 (d, 1H,
J = 1.5 Hz), 7.34 (d, 1H, J = 1.5 Hz), 7.29–7.28 (m, 3H),
3.29 (q, 2H, J = 7.5 Hz), 1.55 (t, 3H, J = 7.5 Hz); 13C NMR
(CDCl3, 125 MHz) δ 156.2, 148.3, 143.1, 138.0, 133.6,
126.6, 122.3, 111.7, 103.2, 45.1, 8.4; LC/MS m/z: 293.0
(M++1).
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl propane-1-sulfonate
(1n) Yield: 73%; mp: 122–125 °C; 1H NMR (CDCl3,
500 MHz) δ 7.81 (d, 2H, J = 8.5 Hz), 7.39 (d, 1H,
J = 1.5 Hz), 7.33 (d, 1H, J = 1.5 Hz), 7.29–7.27 (m, 3H),
3.24–3.21 (m, 2H), 2.05–2.00 (m, 2H), 1.12 (t, 3H,
Medicinal Chemistry Research (2023) 32:314–325
J = 7.5 Hz); 13C NMR (CDCl3, 125 MHz) δ 156.1, 148.3,
143.0, 138.0, 133.4, 126.6, 122.3, 111.7, 103.2, 52.1, 17.5,
13.0; LC/MS m/z: 307.0 (M++1).
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl cyclohexanesulfonate
(1o) Yield: 75%; mp: 160–163 °C; 1H NMR (CDCl3,
500 MHz) δ 7.80 (dd, 2H, J = 2.0, 6.5 Hz), 7.40 (d, 1H,
J = 1.5 Hz), 7.34 (d, 1H, J = 1.5 Hz), 7.29–7.26 (m, 3H),
3.23–3.18 (m, 1H), 2.35 (dd, 2H, J = 2.0, 13.5 Hz),
1.97–1.94 (m, 2H), 1.78– 1.70 (m, 3H), 1.38–1.23 (m, 3H);
13
C NMR (CDCl3, 125 MHz) δ 155.8, 148.4, 142.8, 138.2,
132.8, 126.6, 122.4, 111.8, 103.2, 108.4, 60.3, 26.7, 25.1,
25.1; LC/MS m/z: 693.0 (M++1).
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl benzenesulfonate
(1p) Yield: 50%; mp: 168–171 °C; 1H NMR (DMSO-d6,
500 MHz) δ 7.97 (d, 1H, J = 2.0 Hz), 7.93 (d, 1H,
J = 1.5 Hz), 7.87 (d, 2H, J = 7.0 Hz), 7.82 (t, 1H,
J = 7.5 Hz), 7.77–7.40 (m, 3H), 7.67 (t, 2H, J = 7.5 Hz), 7.00
(d, 2H, J = 9.0 Hz); 13C NMR (DMSO-d6, 125 MHz) δ
155.5, 147.6, 141.2, 139.1, 135.0, 134.3, 133.8, 129.8, 128.3,
125.7, 122.2, 112.8, 104.8; LC/MS m/z: 341.0 (M++1).
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl
4-fluorobenzenesulfonate (1q) Yield: 25%; mp:
183–185 °C; 1H NMR (CDCl3, 500 MHz) δ 7.84 (s, 2H),
7.69 (d, J = 5.5 Hz, 2H), 7.38–7.19 (m, 5H), 6.97 (d,
J = 5.5 Hz, 2H); 13C NMR (CDCl3, 125 MHz) δ 166.1 (d,
1C, J = 255.7 Hz), 156.3, 148.6, 143.1, 138.0, 133.8, 131.6
(d, 1C, J = 9.5 Hz), 131.4 (d, 1C, J = 3.0 Hz), 126.4, 122.7,
116.7 (d, 1C, J = 22.7 Hz), 111.9, 103.3; LC/MS m/z: 359.1
(M++1).
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl
4-methylbenzenesulfonate (1r) Yield: 11%; mp:
180–183 °C; 1H NMR (CDCl3, 500 MHz) δ 7.71–7.67 (m,
4H), 7.39–7.29 (m, 6H), 6.98 (d, 2H, J = 8.5 Hz), 2.44 (s,
3H); 13C NMR (CDCl3, 125 MHz) δ 155.9, 148.9, 145.5,
142.9, 138.3, 133.0, 132.4, 129.9, 128.7, 126.4, 122.8,
112.0, 103.3, 29.8; LC/MS m/z: 355.0 (M++1).
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl 3,4-dichlorobenzene-
sulfonate (1s) Yield: 30%; mp: 159–162 °C; 1H NMR
(CDCl3, 500 MHz) δ 7.96 (d, 1H, J = 1.0 Hz), 7.72 (d, 2H,
J = 7.5 Hz), 7.61 (q, 2H, J = 8.0 Hz), 7.40–7.29 (m, 3H),
7.02 (d, 2H, J = 7.0 Hz); 13C NMR (CDCl3, 125 MHz) δ
156.0, 148.5, 142.8, 139.6, 138.4, 135.1, 134.2, 133.7,
131.4, 130.5, 127.7, 126.7, 122.6, 112.1, 103.5; LC/MS
m/z: 409.0 (M++1).
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl 3-(trifluoromethyl)ben-
zenesulfonate (1t) Yield: 96%; mp: 132–135 °C; 1H NMR
(CDCl3, 500 MHz) δ 8.16 (s, 1H), 8.03 (d, 1H, J = 8.0 Hz),
Medicinal Chemistry Research (2023) 32:314–325
7.95 (d, 1H, J = 8.0 Hz), 7.74–7.68 (m, 3H), 7.44 (d, 1H,
J = 1.0 Hz), 7.36 (d, 1H, J = 1.0 Hz), 7.30 (s, 1H), 7.02 (d,
2H, J = 8.5 Hz); 13C NMR (CDCl3, 125 MHz) δ 155.9,
148.5, 142.6, 138.3, 136.7, 133.5, 131.9, 131.0 (d, 1C,
J = 3.5 Hz), 130.1, 126.5, 125.7 (d, 1C, J = 3.6 Hz), 122.7,
111.8, 103.4; LC/MS m/z: 409.0 (M++1).
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl 4-chloro-3-(trifluoro-
methyl)benzenesulfonate (1u) Yield: 95%; mp:
138–141 °C; 1H NMR (CDCl3, 500 MHz) δ 8.19 (d, 1H,
J = 2.0 Hz), 7.89 (dd, 1H, J = 2.0, 8.5 Hz), 7.74 (d, 2H,
J = 8.5 Hz), 7.67 (d, 1H, J = 8.5 Hz), 7.41 (s, 1H), 7.34 (s,
1H), 7.29 (s, 1H), 7.01 (d, 2H, J = 8.5 Hz); 13C NMR (CDCl3,
125 MHz) δ 156.0, 148.3, 142.7, 139.3, 138.3, 134.7, 133.8,
132.8 (d, 1C, J = 8.9 Hz), 130.0 (d, 1C, J = 32.9 Hz), 128.0
(d, 1C, J = 5.2 Hz), 127.9 (d, 1C, J = 5.4 Hz), 126.6, 123.0,
122.5, 120.8, 111.8, 103.4; LC/MS m/z: 443.0 (M++1).
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl 3,5-bis(trifluoromethyl)
benzenesulfonate (1v) Yield: 96%; mp: 166–169 °C; 1H
NMR (CDCl3, 500 MHz) δ 8.30 (s, 2H), 8.17 (s, 1H), 7.77
(d, 2H, J = 8.5 Hz), 7.43 (d, 1H, J = 1.5 Hz), 7.36 (d, 1H,
J = 1.5 Hz), 7.30 (s, 1H), 7.03 (d, 2H, J = 8.5 Hz); 13C
NMR (CDCl3, 125 MHz) δ 148.3, 138.6, 138.3, 133.5,
133.2, 128.9, 127.9, 127.9, 126.8, 123.4, 122.5, 121.2,
111.9, 103.5; LC/MS m/z: 477.0 (M++1).
Synthesis of sulfamate target compounds 2a–2f
The synthesis of sulfamate derivatives was accomplished
using the procedure in [26, 27]. Appropriate phenol
(0.28 mmol) was dissolved in anhydrous DMAc (0.5 mL) and
was stirred under N2 (g). NaH (66 mg, 0.28 mmol) was then
added at 0 °C and was further stirred for 15 min. Appropriate
sulfamoyl chloride (1.39 mmol) dissolved in anhydrous
DMAc (0.5 mL) was then added dropwise to the reaction
mixture at 0 °C and then left to stir at rt for 1–2 h. The
completion of reaction was confirmed via TLC. The reaction
was quenched with ice–water (5 mL) and extracted using
ethyl acetate (2 × 5 mL). The organic layers were collected,
dried over anhydrous Na2SO4, concentrated in vacuo, and the
crude product was purified by flash column chromatography
by using ethyl acetate:hexane (1:1 v/v) as an eluent.
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl sulfamate (2a) Yield:
21%; 1H NMR (DMSO-d6, 500 MHz) δ 8.25 (s, 1H), 8.00
(s, 2H), 7.95 (d, 1H, J = 4.5 Hz), 7.90 (d, 2H, J = 8.5 Hz)
7.31–7.27 (m, 3H); 13C NMR (DMSO-d6, 125 MHz) δ
149.4, 149.1, 145.3, 132.7, 125.9, 122.5, 120.1, 113.3,
109.6; LC/MS m/z: 296.1 (M++1).
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl methylsulfamate (2b)
Yield: 59%; mp: 172–175 °C; 1H NMR (DMSO-d6,
323
500 MHz) δ 8.25 (s, 1H), 8.22 (d, 1H, J = 4.5 Hz), 7.94 (d,
1H, J = 4.5 Hz), 7.90 (d, 2H, J = 9.0 Hz) 7.31–7.27 (m, 3H),
2.73 (d, 3H, J = 4.5 Hz); 13C NMR (DMSO-d6, 125 MHz) δ
149.4, 148.8, 145.3, 132.9, 126.1, 122.3, 120.1, 113.3,
109.7, 29.2; LC/MS m/z: 310.0 (M++1).
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl piperidine-1-sulfonate
(2c) Yield: 26%; mp: 176–178 °C; 1H NMR (CDCl3,
500 MHz) δ 7.82 (d, 2H, J = 8.5 Hz), 7.73 (s, 1H), 7.44 (d,
1H, J = 4.0 Hz), 7.31 (d, 2H, J = 8.5 Hz) 6.85 (d, 1H,
J = 4.5 Hz), 3.37 (t, 4H, J = 5.0 Hz), 1.65 (d, 4H,
J = 4.5 Hz), 1.57 (d, 2H, J = 4.0 Hz); 13C NMR (CDCl3,
125 MHz) δ 150.5, 149.7, 146.7, 132.6, 126.6, 122.2,
118.7, 113.1, 108.3, 48.1, 25.2, 23.6; LC/MS m/z: 364.0
(M++1).
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl sulfamate (2d) Yield:
27%; mp: >230 °C; 1H NMR (DMSO-d6, 500 MHz) δ
8.00–7.97 (m, 3H), 7.93 (d, 1H, J = 2.0 Hz), 7.84 (d, 2H,
J = 9.0 Hz) 7.79 (s, 1H), 7.27 (d, 2H, J = 8.5 Hz); 13C NMR
(DMSO-d6, 125 MHz) δ 155.5, 148.9, 141.7, 139.0, 133.0,
125.6, 122.4, 112.8, 104.4; LC/MS m/z: 280.0 (M++1).
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl methylsulfamate (2e)
Yield: 25%; mp: 182–185 °C; 1H NMR (DMSO-d6,
500 MHz) δ 8.20 (s, 1H), 7.97 (d, 1H, J = 2.0 Hz), 7.93 (d,
1H, J = 2.0 Hz), 7.84 (dd, 2H, J = 2.0, 7.0 Hz) 7.79 (s, 1H),
7.28 (dd, 2H, J = 2.0, 7.0 Hz), 2.72 (s, 3H); 13C NMR
(DMSO-d6, 125 MHz) δ 155.6, 148.6, 141.6, 139.0, 133.2,
125.8, 122.2, 112.8, 104.5, 29.2; LC/MS m/z: 294.0 (M++1).
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl piperidine-1-sulfonate
(2f) Yield: 9%; mp: 190–193 °C; 1H NMR (CDCl3,
500 MHz) δ 7.79 (d, 2H, J = 8.5 Hz), 7.39 (d, 1H,
J = 2.0 Hz), 7.33 (s, 1H), 7.29 (d, 3H, J = 8.5 Hz), 3.37 (t,
4H, J = 5.5 Hz), 1.66 (pentet, 4H, J = 5.5 Hz), 1.59–1.56
(m, 2H); 13C NMR (CDCl3, 125 MHz) δ 156.1, 149.6,
143.3, 138.0, 132.8, 126.4, 122.2, 111.7, 103.1, 48.1, 29.8,
25.2; LC/MS m/z: 348.1 (M++1).
NTPDase activity assay
According to the previously reported method [28], anti-
NTPDase assay was performed with little modification.
This assay was carried out with the help of malachite green
reagent. Assay buffer was prepared by mixing 5 mM CaCl2
with 50 mM tric HCl (pH 7.4). The compounds that would
be tested against h–NTPDases were prepared in 10%
DMSO and tested at 0.1 mM concentration in assay. The
assay was performed in 96-well plate and keeping total
assay volume 100 µL/well. Firstly, 55 µL assay buffer and
10 µL of prepared compound solution were added in each
well and incubated at 37 °C for 10 min after adding 10 µL of
324
enzyme solution. The end concentration of enzyme per well
was h–NTPDase1 (12 ng), h–NTPDase2 (41 ng),
h–NTPDase3 (40 ng) and h–NTPDase8 (63 ng). After
incubation, absorbance of the reaction mixture was mea-
sured at 630 nm of wavelength via Omega FLUOstar
microplate reader (BMG Labtech, Germany) as pre-read
and then substrate (ATP) solution was added with 0.1 mM
(10 µL) final concentration to start the reaction. The reaction
plate was again kept at 37 °C for 15 min. Finally, malachite
green reagent (15 µL) was added to make up the 100 µL
volume and to terminate the reaction. After that, incubation
at room temperature was carried out for 4–6 min, then the
absorbance was measured at the same wavelength as after-
read to determine the change in absorbance. Percent inhi-
bition of all tested compounds was calculated against iso-
zymes of h–NTPDase while those compounds showing
more than 50% inhibition were further diluted in triplicate
to determine their IC50 values. For IC50 value determination,
PRISM 5.0 (GraphPad, San Diego, California, USA)
was used.
Molecular docking studies
As the crystal structures for h–NTPdases are not available in
the Protein Data Bank (PDB), we built the homology
models of h–NTPDase1, 2, 3, and 8 according to our pre-
viously reported method [29]. Molecular Operating Envir-
onment (MOE) software was used to prepare the potent
inhibitors and to minimize their energy. For protein struc-
ture preparation, the BioSolveIT’s LeadIT was used [30].
The selected compounds were docked with modeled
NTPDases by using BioSolveIT’s LeadIT and only the pose
having the highest binding affinity was selected to evaluate
the compound-protein binding interactions.
Molecular dynamics simulation
Molecular dynamic simulation of compound 1q was car-
ried out inside the h–NTPDase1 using GROMACS
[31, 32]. As an explicit water model TIP3P with the latest
CHARMM36 forcefield was employed [33]. The topology
and parameter data were obtained using the CHARMM
General Force Field (CGENFF) web-based server (https://
cgenff.umaryland.edu), with the docked pose of inhibitors
serving as the initial coordinate. The protein-inhibitor
complex was generated and wrapped in a TIP3P water box
before being neutralized with counter sodium and chloride
ions. Using the steepest descent and conjugate gradient
methods, the complex system was reduced until the max-
imum force experienced by the system was less than 103
KJmol–1nm–1. Using NVT (isothermal–isochoric) and NPT
(isothermal–isobaric) ensembles, the system was allowed
to equilibrate for 100 ps. Prior to running the production
Medicinal Chemistry Research (2023) 32:314–325
run, the complex system was observed to achieve a tem-
perature of 300 K and a pressure of roughly 1 atm. A 50 ns
MD simulation was performed. VMD v9.13 and
XMGRACE v5.1.19 were used for the visualization and
plotting of graphs [34, 35].
Acknowledgements The authors are thankful to the University of
Sharjah, United Arab Emirates for funding this project (grant No.
2201110159). The authors gratefully acknowledge also the financial
support for this research provided by the Higher Education Commis-
sion of Pakistan (HEC) via NRPU project No. 20-15846/NRPU/R&D/
HEC/2021, German-Pakistani Research Collaboration Programme and
Equipment Grant funded by DAAD, Germany.
Funding This study was funded by the University of Sharjah, United
Arab Emirates (grant No. 2201110159) & the Higher Education
Commission of Pakistan.
Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
Informed consent All the authors have read the article content and agree
on it. They also agree on submitting it to Medicinal Chemistry Research.
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