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) 32:314–325 315

additional ligands for P2 (P2X and P2Y) receptors. Fur- inhibitors such as suramin, Reactive blue–2, and PPADS
thermore, NTPDases family of enzymes is the most (Fig. 1) were discovered, yet some proved to be
dominant for formation of adenosine monophosphate non–selective, moderately active, or had limited stability
(AMP) through degradation of adenosine triphosphate and [21]. Quinoline–based derivatives containing ethers and/or
adenosine diphosphate (ADP). This family possesses eight esters (Fig. 1) were also developed as inhibitors of
members such as NTPDase1–8. NTPDase1, 2, 3 and 8 have NTPDases, where varying selectivity profiles were dis-
been identified as extracellular membrane–bound enzymes covered as inhibitors of a single isozyme or pan inhibitors
because they are responsible for catalyzing the hydrolysis against all 4 isozymes [22, 23]. Other classes of
of extracellular ATP whereas, NTPDase4, 5 and 6 are nonnucleotide–based inhibitors include polyoxometalates
reported as intracellular ectoenzymes [6]. The first isozyme and antibodies. Both suffer from liabilities such as stability
of NTPDases family named CD39 was expressed in B and/or incomplete inhibition [21]. To our knowledge, no
lymphocytes [7], which was later identified in T lympho- inhibitors containing sulfonate warheads have been reported
cytes and renamed NTPDase1 [8] while the other members in the literature. Such development provides an advantage
of this family began to be uncovered. Therefore, in investigating new modalities and strategies to develop
ecto–nucleotidases play a role in controlling the bioavail- NTPDases inhibitors.
ability of purines and purinergic signals through P2X and
P2Y receptors in the extracellular domain [9].
NTPDases have five active regions (ACR1–ACR5) Results and discussion
known as apyrase-conserved regions [10]. Extracellular
NTPDases have two transmembrane domains, which are Chemistry
responsible to control the specificity of substrate for binding
and hydrolysis [11]. Moreover, this enzyme family requires Synthesis of the methoxy intermediates 5a and 5b was
Ca2+ and Mg2+ ions (as cofactors) for their catalytic achieved using the reported procedure in [24, 25], where
activity, any alteration in these ions leads to a change in the 2–bromo–1–(4–methoxyphenyl)ethan–1–one (3) and
enzymatic activity [12]. NTPDases are expressed in all 2–aminothiazole (4a)/2–aminooxazole (4b) were refluxed
tissues, where they are responsible for various physiological in ethanol to yield methoxy derivatives 5a and 5b,
functions such as development, blood flow, hormone respectively. The yield percentages of compounds 5a and
secretion, and neurotransmitter release [11]. In addition, 5b were 90 and 85%, respectively. The demethylation of
extracellular NTPDases family is also involved in patho- 5a and 5b proceeded using boron trifluoride methylsulfide
logical functions such as injury, inflammation, infection, complex in dichloromethane to yield intermediates 6a and
and cancer [9]. 6b similar to the reported procedure [26]. Compounds 6a
Several studies reported that NTPDase2 is localized in and 6b were obtained in yields of 80 and 75%, respec-
specialized astrocytes in rodent brain, such as laminar tively. The target sulfonate compounds 1a–1v were syn-
astrocytes associated with fiber tracts in the brain stem and thesized via reacting the hydroxyl intermediates 6a,b with
cerebrum [13, 14], tanycytes, non–stellate astrocytes in the the appropriate sulfonyl chloride in the presence of trie-
gray matter of discrete regions like habenula, satellite thylamine (Scheme 1) [27]. The yields of compounds
astrocytes in the dorsal root ganglion [13], and 1a–1v ranged from 11 to 97%. Most of the compounds
astrocyte–like progenitor cells of the subventricular zone were obtained in high yield, while few of them were
(SVZ) of the lateral ventricle. NTPDase3 is localized in the isolated in rather lower yields. Sulfamoyl derivatives
midline regions in the thalamus, hypothalamus, and the 2a–2f were synthesized by the treatment of the hydroxyl
medulla oblongata [15, 16]. NTPDase2, and to a lesser intermediates 6a and 6b with sodium hydride in dry DMF
extent NTPDase3, preferentially catalyze the depho- at 0 °C, followed by an interval of 15 min before the
sphorylation of ATP to ADP, generating the physiological addition of appropriate sulfamoyl chloride reagent to the
ligand for P2Y1, P2Y12, and P2Y13 receptors [17–20]. reaction mixture (Scheme 1) [27]. Compounds 2a–2f were
Therefore, NTPDase2 and –3 may modulate inflammatory isolated in low-to-moderate yields (9–59%). The identity
reactions within the CNS and could represent useful ther- of the target compounds 1a–1v and 2a–2f was confirmed
apeutic targets in neuroinflammatory and neurodegenerative by 1H NMR, 13C NMR, and LC–MS. The signals of the
diseases. attached sulfonate and sulfamate moieties were confirmed
Inhibitors of NTPDases can be categorized into by NMR, and the molecular weight increased than the
nucleotide–based and nonnucleotide–based inhibitors. intermediate hydroxyl intermediates and confirmed by
ARL67156, 8–BuS–ATP, and PSB–6426 (Fig. 1) are LC–MS. The detailed spectral data are provided in the
nucleotide–based inhibitors of NTPDases with different Experimental section, and the charts are provided in the
profiles against different isomers. Nonnucleotide–based 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 series that showed an inhibitory effect against NTPDase8
relationship (IC50 = 6.10 µM). Furthermore, four aromatic sulfonyl deri-
vatives exhibited inhibitory activity against NTPDase1. The
The target compounds were tested against four recombinant unsubstituted phenylsulfonate derivative 1e was the most
isozymes of h–NTPDase, i.e., h–NTPDase1, 2, 3 and 8. All potent among the aromatic sulfonyl derivatives with an IC50
the compounds were initially screened at 0.1 mM con- of 2.83 µM. Introducing para substitution (e.g. 4–F and
centration, then the active derivatives were further tested at 4–Me, 1f (IC50 = 4.02 µM) and 1g (IC50 = 4.25 µM),
other concentrations to calculate their IC50 values. Their respectively) reduced the potency in comparison to the
results are shown in Table 1. Suramin was utilized as a unsubstituted derivative 1e. Compound 1j bearing 3–CF3,
reference standard in this assay. 4–Cl substitution on the phenylsulfonate side chain dramati-
cally reduced the activity by 2.5 folds in comparison to
Imidazothiazole-possessing sulfonate and sulfamate unsubstituted phenyl analog 1e. It is apparent that substitution
derivatives (compounds 1a–k and 2a–2c) on the phenyl ring is detrimental to activity against
NTPDase1. Compounds 1e and 1i were the most active
The aliphatic sulfonate derivatives 1a–1d did not show any toward NTPDase1 and 8, respectively among the aromatic
significant inhibitory activity against NTPDase1, 3, and 8 sulfonate derivatives.
isozymes at 0.1 mM. Compound 1c bearing n–propylsulfonyl Furthermore, the sulfamate derivatives 2a–2c lacked
side chain showed promising activity against NTPDase2 with inhibitory activity against NTPDase3 and 8. Compounds 2a
an IC50 of 0.29 µM. The aromatic derivatives showed any (free sulfamate) and 2b (N-methylsulfamate) showed inhi-
significant inhibitory activity against NTPDase2 and 3. bitory effect against NTPDase1 (IC50 = 2.51 µM) and
Interestingly, compound 1i bearing m–CF3 phenylsulfonyl NTPDase2 (IC50 = 2.63 µM), respectively. However, com-
side chain was the only derivative among the imidazothiazole 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 Medicinal Chemistry Research (2023) 32:314–325

an inhibitory activity toward two isoenzymes, NTPDase2 a small substituent, maybe bigger substituents or more
(IC50 = 1.43 µM) with a higher sensitivity by 5 folds com- number of substituents are not tolerated in case of
pared to NTPDase1 (IC50 = 7.32 µM). NTPDase1.
Moreover, the sulfamate derivatives 2d–2f had no pro-
Imidazooxazole-possessing sulfonate and sulfamate mising inhibitory effect toward NTPDase3. Compounds 2e
derivatives (compounds 1l–1v, and 2d–2f) and 2f were the only derivatives among the imidazooxazole
sulfamates which exhibited inhibitory activity against
Upon comparing the imidazooxazole derivatives and the NTPDase1 and NTPDase8, respectively. Derivatives 2d
isosteric imidazothiazole analogs, imidazooxazole deriva- and 2f revealed activity against NTPDase2 with IC50 values
tives 1l–1v and 2d–2f showed dramatic rise in the potency of 0.70 and 0.96 µM, respectively.
against most of the NTPDases isoforms, especially against
NTPDase2 isoform. The aliphatic sulfonates 1l–1o revealed Molecular docking studies
weak inhibitory effects against isoenzymes 1 and 3, similar
to the imidazothiazole aliphatic analogs. Astonishingly, the Molecular docking studies were conducted to study the
methyl sulfonate 1l and the cyclohexyl sulfonate 1o putative binding modes of the most potent inhibitors inside
exhibited activity against NTPDase8 with an IC50 value of the h–NTPDases crystal structures. Compounds 1q, 1c, 1p,
1.15 µM for both of them. (Substituted) phenyl derivatives and 1u were docked within the active sites of h–NTPDase1,
1p–1v were all sensitive mainly toward NTPDase2, being 2, 3, and 8, respectively. It has been noticed that the sul-
more potent than suramin, suggesting a synergistic effect of fonate group of all compounds was actively participating in
the aryl sulfonate moiety together with the imidazooxazole both hydrophilic and hydrophobic interactions with amino
nucleus to target NTPDase2, since the imidazothiazole acid residues of modeled proteins.
analogs lacked sensitivity to it. Evidently, substitution on With h–NTPDase1, compound 1q was found to be lined
phenylsulfonate side chain impacted activity based on the with active pocket residues Asp54, Gly56, Ser57, Ser58,
position of the substituent on the phenyl ring, as well as the His59, and Thr131 amino acid residues as shown in Fig. 2.
number of substitutions. A reduction in potency was Compound 1q bound within h–NTPDase1 active site with
experienced when a substituent was introduced at para binding energy of –22 KJmol–1 via different hydrogen and
position on the terminal phenyl ring (1q, 4–F, hydrophobic bonds. In this regard, sulfonate oxygen atom
IC50 = 6.92 µM) and (1r, 4–Me, IC50 = 6.35 µM) compared formed hydrogen bond with His59 and Ser57. Furthermore,
to unsubstituted phenylsulfonate derivative (1p, the same moiety interacts with Thr131 and Gly56 amino
IC50 = 3.06 µM). However, meta substitution (1t, 3–CF3, acid residues.
IC50 = 2.00 µM) led to an improved potency. Disubstitution The amino acid residues lining the h–NTPDase2 active
on the phenyl ring improved the activity such as derivative site around compound 1c were Gly47, Ser48, Ser49, His50,
1v (3,5–bisCF3, IC50 = 1.43 µM), while meta, para-dis- Ala123, Gly204, and Tyr350. The inhibitor has a binding
ubstituted phenyl ring potentiated the inhibition of energy of –14 KJmol–1 with the active site. In Fig. 3, the
NTPDase2 (1s, 3,4–diCl, IC50 = 0.36 µM) and (1u, 3–CF3, sulfonate moiety of compound 1c formed a network of
4–Cl, IC50 = 0.79 µM). It is possible that the aromatic side hydrogen bonds with Ser48, Ser49 and His50 residues.
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 Fig. 2 Putative binding mode of inhibitor 1q within the h–NTPDase1
45–folds compared to Suramin (IC50 = 16.1 µM). Fluoro is active site
Medicinal Chemistry Research (2023) 32:314–325 319

Fig. 3 Putative binding mode of inhibitor 1c within the h–NTPDase2

active site

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-
Fig. 4 Putative binding mode of inhibitor 1p within the h–NTPDase3
active site tion was noticed between residue Phe57 and imidazo[2,1–b]
oxazole ring of compound 1u.

Hydrophobic interactions were monitored with Gly204 and Molecular dynamic simulation
Gly47. A pi–pi stacked interaction was found between the
residue His50 and imidazo[2,1–b]thiazole ring. However, Molecular dynamic simulations of the most potent
sulfur atom of the imidazothiazole nucleus was noticed to h–NTPDase1 inhibitor 1q were carried out using GRO-
form pi–sulfur interaction with residue Tyr350. MACS software package. Simulation of the inhibitor was
Compound 1p adjusted itself in the active site of mod- carried out in the presence of Ca2+ and Mg2+ divalent ions
eled h–NTPDase3, as illustrated in Fig. 4. The most in the active site. Simulation of the apo–enzyme and with
important amino acid residues that were involved in the inhibitor was carried out for 50 ns.
binding interactions with the inhibitor were Gly64, Ser65, Root mean square deviation (RMSD) profile of the
Ser66, Arg67, Lys95, Gly221, and Asn265. Compound 1p h–NTPDase1 with and without the inhibitor is given in
bound with binding energy of –9.0 KJmol–1 inside the Fig. 6. The h–NTPDase1 apo–enzyme and h–NTPDase1
h–NTPDase3 active site. It formed hydrogen bonding with inhibitor 1q were found to have a similar root mean
interactions with Ser65, Ser66, and Arg67 through its sul- square deviation during the entire molecular dynamic
fonate oxygen. Furthermore, pi–alkyl hydrophobic interac- simulations and no abrupt fluctuations in the RMSD value
tion was observed between Arg67 residue and the central of the enzyme–inhibitor complex was observed.
phenyl ring of compound 1p, while pi–cation interaction The root means square fluctuation (RMSF) profile of the
was noticed between imidazo[2,1–b]oxazole ring of com- h–NTPDase1 apo–enzyme and with compound 1q is given
pound 1p with Lys95. in Fig. 7. The RMSF profile of apo–enzyme and with
320 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
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 NTPDase2), 0.37 µM (against NTPDase3), and 0.36 µM
complex in red color (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

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 analyze the compounds by 1H and 13C NMR. LC–MS
complex in red color analysis was carried out by LC–MS analyzer (Waters
Corporation, MA, USA). All the solvents and reagents
inhibitor bound were determined with an aim to determine were purchased from commercial companies and used as
the fluctuations of key residues that were affected due to the such. The final and intermediate compounds were pur-
attachment of the inhibitor. As shown in Fig. 6, the residues ified by flash column chromatography (silica gel, pore
180–220 were found to be the most flexible followed by size 0.040~0.063 mm, 230–400 mesh) using laboratory
residues around 140–170 and around 360–485 of reagent grade solvents.
h–NTPDase1. The h–NTPDase1 in complex with inhibitor
1q however, did not change the RMSF profile significantly. Chemical synthesis
The fluctuation in RMS values for h–NTPDase1 in complex
with compound 1q is much similar to that of the Synthesis of 6–(4–methoxyphenyl)imidazo[2,1–b]thiazole
h–NTPDase1 apo–enzyme. (5a) and 6–(4–methoxyphenyl)imidazo[2,1–b]oxazole (5b)

The synthesis of fused heterocyclic rings was accomplished

Conclusion using the reported procedure in [24, 25].

This study delivered new series of imidazothiazole- and Synthesis of 4–(imidazo[2,1–b]thiazol-6–yl)phenol (6a) and
imidazooxazole-based sulfonates and sulfamates. This 4–(imidazo[2,1–b]oxazol-6–yl)phenol (6b)
series was screened for their inhibitory activity toward
four isozymes of human NTPDases, 1, 2, 3, and 8. The The demethylation of methoxy intermediates was accom-
SAR of these derivatives has shown variability in the plished using the procedure reported in [26]. The methoxy
Medicinal Chemistry Research (2023) 32:314–325 321

intermediates (2.17 mmol) were dissolved and stirred in δ 150.5, 148.5, 146.4, 133.2, 126.8, 122.4, 118.7, 113.2,
anhydrous dichloromethane (5 mL), BF3.MeS (3.43 mL, 108.4, 52.2, 17.5, 13.0; LC/MS m/z: 322.9 (M++1).
32.6 mmol) was added under N2 (g) at room temperature
overnight. The completion of reaction was confirmed via 4-(Imidazo[2,1-b]thiazol-6-yl)phenyl cyclohexanesulfonate
TLC. The mixture was basified with excess saturated aqu- (1d) Yield: 21%; mp: 156–158 °C; 1H NMR (CDCl3,
eous K2CO3 solution and extracted with ethyl acetate 500 MHz) δ 7.83 (d, 2H, J = 8.5 Hz), 7.73 (s, 1H), 7.44 (d,
(10 mL). The organic layer was dried over anhydrous 1H, J = 4.5 Hz), 7.29 (d, 2H, J = 8.5 Hz), 6.85 (d, 1H,
Na2SO4, concentrated in vacuo, and was purified using J = 4.0 Hz), 3.23–3.18 (m, 1H), 2.35 (d, 2H, J = 11.5 Hz),
column chromatography. The product was eluted using 1:1 1.97–1.94 (m, 2H), 1.78– 1.70 (m, 3H), 1.34–1.25 (m, 3H);
ethyl acetate/hexane. 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,
Synthesis of sulfonate target compounds 1a–1v 25.1; LC/MS m/z: 363.1 (M++1).

The synthesis of sulfonate derivatives was accomplished 4–(Imidazo[2,1–b]thiazol-6–yl)phenyl benzenesulfonate

using a similar reported procedure [27]. Appropriate phenol (1e) Yield: 70%; mp: >230 °C; 1H NMR (DMSO-d6,
(0.28 mmol) was dissolved in anhydrous THF (2 mL) and 500 MHz) δ 8.22 (s, 1H), 7.94 (d, 1H, J = 4.5 Hz),
was stirred under N2 (g). Triethylamine (0.15 mL, 1 mmol) 7.89–7.87 (m, 2H), 7.84–7.80 (m, 3H), 7.68 (t, 2H,
was then added at 0 °C and was further stirred for 15 min. J = 7.5 Hz), 7.27 (d, 1H, J = 4.0 Hz), 7.03 (d, 2H,
Appropriate sulfonyl chloride (0.83 mmol) dissolved in J = 8.5 Hz); 13C NMR (DMSO-d6, 125 MHz) δ 149.5,
anhydrous THF (2 mL) was then added dropwise to the 147.8, 144.9, 135.0, 134.3, 133.5, 129.8, 128.2, 126.1,
reaction mixture at 0 °C and then left to stir at rt overnight. 122.3, 120.1, 113.4, 110.0; LC/MS m/z: 357.1 (M++1).
The completion of reaction was confirmed via TLC. THF
was removed in vacuo and the resultant crude was extracted 4–(Imidazo[2,1–b]thiazol-6–yl)phenyl
using ethyl acetate (5 mL) and water (5 mL). Aqueous layer 4-fluorobenzenesulfonate (1f) Yield: 14%; mp:
was reextracted with ethyl acetate (5 mL). The organic 148–150 °C; 1H NMR (CDCl3, 500 MHz) δ 7.86–7.83 (m,
layers were collected, dried over anhydrous Na2SO4, con- 2H), 7.75–7.72 (m, 3H), 7.45 (d, 1H, J = 4.0 Hz),
centrated in vacuo, and the crude product was purified by 7.21–7.17 (m, 2H), 7.00 (d, 2H, J = 9.0 Hz), 6.87 (d, 1H,
flash column chromatography by using ethyl acetate:hexane J = 4.5 Hz); 13C NMR (CDCl3, 125 MHz) δ 166.2 (d, 1C,
(1:3 v/v) as an eluent. 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,
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl methanesulfonate 118.7, 116.7 (d, 1C, J = 17.6 Hz), 113.3, 108.5; LC/MS
1
(1a) Yield: 61%; mp: 202–205 °C; H NMR (MeOD-d4, m/z: 375.1 (M++1).
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, 4-(Imidazo[2,1-b]thiazol-6-yl)phenyl
J = 4.0 Hz), 3.25 (s, 3H); 13C NMR (MeOD-d4, 125 MHz) 4-methylbenzenesulfonate (1g) Yield: 63%; mp:
δ 151.9, 150.3, 127.8, 123.7, 121.0, 114.8, 110.9, 37.5; LC/ 168–170 °C; 1H NMR (CDCl3, 500 MHz) δ 7.73–7.69 (m,
MS m/z: 295.1 (M++1). 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,
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl ethanesulfonate (1b) 3H); 13C NMR (CDCl3, 125 MHz) δ 150.4, 149.0, 145.5,
Yield: 96%; mp: 158–160 °C; 1H NMR (DMSO-d6, 132.4, 129.9, 128.7, 126.5, 122.9, 118.7, 113.3, 108.4,
500 MHz) δ 8.27 (s, 1H), 7.96 (d, 1H, J = 4.5 Hz), 7.92 (d, 21.8; LC/MS m/z: 371.2 (M++1).
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, 4-(Imidazo[2,1-b]thiazol-6-yl)phenyl 3,4-dichlorobenzene-
J = 7.0 Hz); 13C NMR (DMSO-d6, 125 MHz) δ 149.4, sulfonate (1h) Yield: 63%; 1H NMR (CDCl3, 500 MHz)
147.8, 145.1, 133.3, 126.2, 122.4, 120.1, 113.4, 109.9, δ 7.96 (d, 1H, J = 2.0 Hz), 7.77 (d, 2H, J = 8.5 Hz), 7.72 (s,
44.5, 8.1; LC/MS m/z: 309.1 (M++1). 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
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl propane-1-sulfonate (CDCl3, 125 MHz) δ 150.6, 148.5, 146.3, 139.6, 135.1,
(1c) Yield: 41%; mp: 108–110 °C; 1H NMR (CDCl3, 134.2, 133.6, 131.3, 130.4, 127.7, 126.7, 122.6, 118.6,
500 MHz) δ 7.85 (dd, 2H, J = 2.0, 7.0 Hz), 7.74 (s, 1H), 113.2, 108.5; LC/MS m/z: 425.0 (M++1).
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, 4-(Imidazo[2,1-b]thiazol-6-yl)phenyl 3-(trifluoromethyl)ben-
2H), 1.12 (t, 3H, J = 7.5 Hz); 13C NMR (CDCl3, 125 MHz) zenesulfonate (1i) Yield: 28%; mp: 112–114 °C; 1H NMR
322 Medicinal Chemistry Research (2023) 32:314–325

(CDCl3, 500 MHz) δ 8.15 (s, 1H), 8.01 (d, 1H, J = 8.0 Hz), J = 7.5 Hz); 13C NMR (CDCl3, 125 MHz) δ 156.1, 148.3,
7.93 (d, 1H, J = 8.0 Hz), 7.77–7.66 (m, 4H), 7.45 (d, 1H, 143.0, 138.0, 133.4, 126.6, 122.3, 111.7, 103.2, 52.1, 17.5,
J = 4.5 Hz), 7.03–7.00 (m, 2H), 6.87 (d, 1H, J = 4.5 Hz); 13.0; LC/MS m/z: 307.0 (M++1).
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), 4-(Imidazo[2,1-b]oxazol-6-yl)phenyl cyclohexanesulfonate
130.1, 126.7, 125.7 (d, 1C, J = 4.0 Hz), 122.7, 118.6, (1o) Yield: 75%; mp: 160–163 °C; 1H NMR (CDCl3,
113.3, 108.5; LC/MS m/z: 425.0 (M++1). 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),
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl 4-chloro-3-(trifluoro- 3.23–3.18 (m, 1H), 2.35 (dd, 2H, J = 2.0, 13.5 Hz),
methyl)benzenesulfonate (1j) Yield: 81%; 1H NMR 1.97–1.94 (m, 2H), 1.78– 1.70 (m, 3H), 1.38–1.23 (m, 3H);
(CDCl3, 500 MHz) δ 8.19 (d, 1H, J = 1.5 Hz), 7.89 (dd, 1H, 13
C NMR (CDCl3, 125 MHz) δ 155.8, 148.4, 142.8, 138.2,
J = 1.0, 8.0 Hz), 7.78–7.77 (m, 2H), 7.72 (m, 1H), 132.8, 126.6, 122.4, 111.8, 103.2, 108.4, 60.3, 26.7, 25.1,
7.68–7.66 (m, 1H), 7.45–7.38 (m, 1H), 7.04–7.02 (m, 2H), 25.1; LC/MS m/z: 693.0 (M++1).
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, 4-(Imidazo[2,1-b]oxazol-6-yl)phenyl benzenesulfonate
1C, J = 8.6 Hz), 130.2 (d, 1C, J = 32.6 Hz), 129.7 (d, 1C, (1p) Yield: 50%; mp: 168–171 °C; 1H NMR (DMSO-d6,
J = 32.9 Hz), 128.0 (d, 1C, J = 5.4 Hz), 127.9 (d, 1C, 500 MHz) δ 7.97 (d, 1H, J = 2.0 Hz), 7.93 (d, 1H,
J = 5.7 Hz), 126.8, 123.0, 122.6, 120.8, 118.6, 113.3, J = 1.5 Hz), 7.87 (d, 2H, J = 7.0 Hz), 7.82 (t, 1H,
108.5; LC/MS m/z: 459.1 (M++1). 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
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl 3,5-bis(trifluoromethyl) 155.5, 147.6, 141.2, 139.1, 135.0, 134.3, 133.8, 129.8, 128.3,
benzenesulfonate (1k) Yield: 74%; mp: 169–172 °C; 1H 125.7, 122.2, 112.8, 104.8; LC/MS m/z: 341.0 (M++1).
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), 4-(Imidazo[2,1-b]oxazol-6-yl)phenyl
7.04 (d, 2H, J = 8.5 Hz), 6.87 (d, 1H, J = 4.5 Hz); 13C 4-fluorobenzenesulfonate (1q) Yield: 25%; mp:
NMR (CDCl3, 125 MHz) δ 150.6, 147.1 (d, 1C, 183–185 °C; 1H NMR (CDCl3, 500 MHz) δ 7.84 (s, 2H),
J = 266.9 Hz), 138.4, 134.0, 133.3 (d, 1C, J = 34.5 Hz), 7.69 (d, J = 5.5 Hz, 2H), 7.38–7.19 (m, 5H), 6.97 (d,
128.9 (d, 1C, J = 3.2 Hz), 127.9, 127.9 (d, 1C, J = 3.5 Hz), J = 5.5 Hz, 2H); 13C NMR (CDCl3, 125 MHz) δ 166.1 (d,
126.9, 123.4, 122.4, 121.2, 118.6, 113.3, 108.6; LC/MS m/ 1C, J = 255.7 Hz), 156.3, 148.6, 143.1, 138.0, 133.8, 131.6
z: 493.1 (M++1). (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
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl methanesulfonate (M++1).
(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, 4-(Imidazo[2,1-b]oxazol-6-yl)phenyl
J = 1.5 Hz), 7.87 (d, 2H, J = 8.5 Hz), 7.82 (s, 1H), 7.35 (d, 4-methylbenzenesulfonate (1r) Yield: 11%; mp:
2H, J = 9.0 Hz), 3.38 (s, 3H); 13C NMR (DMSO-d6, 180–183 °C; 1H NMR (CDCl3, 500 MHz) δ 7.71–7.67 (m,
125 MHz) δ 155.6, 147.8, 141.5, 139.1, 133.8, 125.9, 4H), 7.39–7.29 (m, 6H), 6.98 (d, 2H, J = 8.5 Hz), 2.44 (s,
122.5, 112.8, 104.7, 37.3; LC/MS m/z: 279.0 (M++1). 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,
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl ethanesulfonate 112.0, 103.3, 29.8; LC/MS m/z: 355.0 (M++1).
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, 4-(Imidazo[2,1-b]oxazol-6-yl)phenyl 3,4-dichlorobenzene-
J = 1.5 Hz), 7.34 (d, 1H, J = 1.5 Hz), 7.29–7.28 (m, 3H), sulfonate (1s) Yield: 30%; mp: 159–162 °C; 1H NMR
3.29 (q, 2H, J = 7.5 Hz), 1.55 (t, 3H, J = 7.5 Hz); 13C NMR (CDCl3, 500 MHz) δ 7.96 (d, 1H, J = 1.0 Hz), 7.72 (d, 2H,
(CDCl3, 125 MHz) δ 156.2, 148.3, 143.1, 138.0, 133.6, J = 7.5 Hz), 7.61 (q, 2H, J = 8.0 Hz), 7.40–7.29 (m, 3H),
126.6, 122.3, 111.7, 103.2, 45.1, 8.4; LC/MS m/z: 293.0 7.02 (d, 2H, J = 7.0 Hz); 13C NMR (CDCl3, 125 MHz) δ
(M++1). 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
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl propane-1-sulfonate m/z: 409.0 (M++1).
(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, 4-(Imidazo[2,1-b]oxazol-6-yl)phenyl 3-(trifluoromethyl)ben-
J = 1.5 Hz), 7.33 (d, 1H, J = 1.5 Hz), 7.29–7.27 (m, 3H), zenesulfonate (1t) Yield: 96%; mp: 132–135 °C; 1H NMR
3.24–3.21 (m, 2H), 2.05–2.00 (m, 2H), 1.12 (t, 3H, (CDCl3, 500 MHz) δ 8.16 (s, 1H), 8.03 (d, 1H, J = 8.0 Hz),
Medicinal Chemistry Research (2023) 32:314–325 323

7.95 (d, 1H, J = 8.0 Hz), 7.74–7.68 (m, 3H), 7.44 (d, 1H, 500 MHz) δ 8.25 (s, 1H), 8.22 (d, 1H, J = 4.5 Hz), 7.94 (d,
J = 1.0 Hz), 7.36 (d, 1H, J = 1.0 Hz), 7.30 (s, 1H), 7.02 (d, 1H, J = 4.5 Hz), 7.90 (d, 2H, J = 9.0 Hz) 7.31–7.27 (m, 3H),
2H, J = 8.5 Hz); 13C NMR (CDCl3, 125 MHz) δ 155.9, 2.73 (d, 3H, J = 4.5 Hz); 13C NMR (DMSO-d6, 125 MHz) δ
148.5, 142.6, 138.3, 136.7, 133.5, 131.9, 131.0 (d, 1C, 149.4, 148.8, 145.3, 132.9, 126.1, 122.3, 120.1, 113.3,
J = 3.5 Hz), 130.1, 126.5, 125.7 (d, 1C, J = 3.6 Hz), 122.7, 109.7, 29.2; LC/MS m/z: 310.0 (M++1).
111.8, 103.4; LC/MS m/z: 409.0 (M++1).
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl piperidine-1-sulfonate
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl 4-chloro-3-(trifluoro- (2c) Yield: 26%; mp: 176–178 °C; 1H NMR (CDCl3,
methyl)benzenesulfonate (1u) Yield: 95%; mp: 500 MHz) δ 7.82 (d, 2H, J = 8.5 Hz), 7.73 (s, 1H), 7.44 (d,
138–141 °C; 1H NMR (CDCl3, 500 MHz) δ 8.19 (d, 1H, 1H, J = 4.0 Hz), 7.31 (d, 2H, J = 8.5 Hz) 6.85 (d, 1H,
J = 2.0 Hz), 7.89 (dd, 1H, J = 2.0, 8.5 Hz), 7.74 (d, 2H, J = 4.5 Hz), 3.37 (t, 4H, J = 5.0 Hz), 1.65 (d, 4H,
J = 8.5 Hz), 7.67 (d, 1H, J = 8.5 Hz), 7.41 (s, 1H), 7.34 (s, J = 4.5 Hz), 1.57 (d, 2H, J = 4.0 Hz); 13C NMR (CDCl3,
1H), 7.29 (s, 1H), 7.01 (d, 2H, J = 8.5 Hz); 13C NMR (CDCl3, 125 MHz) δ 150.5, 149.7, 146.7, 132.6, 126.6, 122.2,
125 MHz) δ 156.0, 148.3, 142.7, 139.3, 138.3, 134.7, 133.8, 118.7, 113.1, 108.3, 48.1, 25.2, 23.6; LC/MS m/z: 364.0
132.8 (d, 1C, J = 8.9 Hz), 130.0 (d, 1C, J = 32.9 Hz), 128.0 (M++1).
(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 sulfamate (2d) Yield:
27%; mp: >230 °C; 1H NMR (DMSO-d6, 500 MHz) δ
4-(Imidazo[2,1-b]oxazol-6-yl)phenyl 3,5-bis(trifluoromethyl) 8.00–7.97 (m, 3H), 7.93 (d, 1H, J = 2.0 Hz), 7.84 (d, 2H,
benzenesulfonate (1v) Yield: 96%; mp: 166–169 °C; 1H J = 9.0 Hz) 7.79 (s, 1H), 7.27 (d, 2H, J = 8.5 Hz); 13C NMR
NMR (CDCl3, 500 MHz) δ 8.30 (s, 2H), 8.17 (s, 1H), 7.77 (DMSO-d6, 125 MHz) δ 155.5, 148.9, 141.7, 139.0, 133.0,
(d, 2H, J = 8.5 Hz), 7.43 (d, 1H, J = 1.5 Hz), 7.36 (d, 1H, 125.6, 122.4, 112.8, 104.4; LC/MS m/z: 280.0 (M++1).
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, 4-(Imidazo[2,1-b]oxazol-6-yl)phenyl methylsulfamate (2e)
133.2, 128.9, 127.9, 127.9, 126.8, 123.4, 122.5, 121.2, Yield: 25%; mp: 182–185 °C; 1H NMR (DMSO-d6,
111.9, 103.5; LC/MS m/z: 477.0 (M++1). 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),
Synthesis of sulfamate target compounds 2a–2f 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,
The synthesis of sulfamate derivatives was accomplished 125.8, 122.2, 112.8, 104.5, 29.2; LC/MS m/z: 294.0 (M++1).
using the procedure in [26, 27]. Appropriate phenol
(0.28 mmol) was dissolved in anhydrous DMAc (0.5 mL) and 4-(Imidazo[2,1-b]oxazol-6-yl)phenyl piperidine-1-sulfonate
was stirred under N2 (g). NaH (66 mg, 0.28 mmol) was then (2f) Yield: 9%; mp: 190–193 °C; 1H NMR (CDCl3,
added at 0 °C and was further stirred for 15 min. Appropriate 500 MHz) δ 7.79 (d, 2H, J = 8.5 Hz), 7.39 (d, 1H,
sulfamoyl chloride (1.39 mmol) dissolved in anhydrous J = 2.0 Hz), 7.33 (s, 1H), 7.29 (d, 3H, J = 8.5 Hz), 3.37 (t,
DMAc (0.5 mL) was then added dropwise to the reaction 4H, J = 5.5 Hz), 1.66 (pentet, 4H, J = 5.5 Hz), 1.59–1.56
mixture at 0 °C and then left to stir at rt for 1–2 h. The (m, 2H); 13C NMR (CDCl3, 125 MHz) δ 156.1, 149.6,
completion of reaction was confirmed via TLC. The reaction 143.3, 138.0, 132.8, 126.4, 122.2, 111.7, 103.1, 48.1, 29.8,
was quenched with ice–water (5 mL) and extracted using 25.2; LC/MS m/z: 348.1 (M++1).
ethyl acetate (2 × 5 mL). The organic layers were collected,
dried over anhydrous Na2SO4, concentrated in vacuo, and the NTPDase activity assay
crude product was purified by flash column chromatography
by using ethyl acetate:hexane (1:1 v/v) as an eluent. According to the previously reported method [28], anti-
NTPDase assay was performed with little modification.
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl sulfamate (2a) Yield: This assay was carried out with the help of malachite green
21%; 1H NMR (DMSO-d6, 500 MHz) δ 8.25 (s, 1H), 8.00 reagent. Assay buffer was prepared by mixing 5 mM CaCl2
(s, 2H), 7.95 (d, 1H, J = 4.5 Hz), 7.90 (d, 2H, J = 8.5 Hz) with 50 mM tric HCl (pH 7.4). The compounds that would
7.31–7.27 (m, 3H); 13C NMR (DMSO-d6, 125 MHz) δ be tested against h–NTPDases were prepared in 10%
149.4, 149.1, 145.3, 132.7, 125.9, 122.5, 120.1, 113.3, DMSO and tested at 0.1 mM concentration in assay. The
109.6; LC/MS m/z: 296.1 (M++1). assay was performed in 96-well plate and keeping total
assay volume 100 µL/well. Firstly, 55 µL assay buffer and
4-(Imidazo[2,1-b]thiazol-6-yl)phenyl methylsulfamate (2b) 10 µL of prepared compound solution were added in each
Yield: 59%; mp: 172–175 °C; 1H NMR (DMSO-d6, well and incubated at 37 °C for 10 min after adding 10 µL of
324 Medicinal Chemistry Research (2023) 32:314–325

enzyme solution. The end concentration of enzyme per well run, the complex system was observed to achieve a tem-
was h–NTPDase1 (12 ng), h–NTPDase2 (41 ng), perature of 300 K and a pressure of roughly 1 atm. A 50 ns
h–NTPDase3 (40 ng) and h–NTPDase8 (63 ng). After MD simulation was performed. VMD v9.13 and
incubation, absorbance of the reaction mixture was mea- XMGRACE v5.1.19 were used for the visualization and
sured at 630 nm of wavelength via Omega FLUOstar plotting of graphs [34, 35].
microplate reader (BMG Labtech, Germany) as pre-read
and then substrate (ATP) solution was added with 0.1 mM Acknowledgements The authors are thankful to the University of
Sharjah, United Arab Emirates for funding this project (grant No.
(10 µL) final concentration to start the reaction. The reaction
2201110159). The authors gratefully acknowledge also the financial
plate was again kept at 37 °C for 15 min. Finally, malachite support for this research provided by the Higher Education Commis-
green reagent (15 µL) was added to make up the 100 µL sion of Pakistan (HEC) via NRPU project No. 20-15846/NRPU/R&D/
volume and to terminate the reaction. After that, incubation HEC/2021, German-Pakistani Research Collaboration Programme and
Equipment Grant funded by DAAD, Germany.
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- Funding This study was funded by the University of Sharjah, United
Arab Emirates (grant No. 2201110159) & the Higher Education
bition of all tested compounds was calculated against iso-
Commission of Pakistan.
zymes of h–NTPDase while those compounds showing
more than 50% inhibition were further diluted in triplicate
Compliance with ethical standards
to determine their IC50 values. For IC50 value determination,
PRISM 5.0 (GraphPad, San Diego, California, USA) Conflict of interest The authors declare no competing interests.
was used.
Informed consent All the authors have read the article content and agree
Molecular docking studies on it. They also agree on submitting it to Medicinal Chemistry Research.

As the crystal structures for h–NTPdases are not available in References

the Protein Data Bank (PDB), we built the homology
1. Wei L, Mousawi F, Li D, Roger S, Li J, Yang X, et al. Adenosine
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inhibitors of NTPDases: synthesis, SAR analysis and molecular
docking studies. Bioorg Chem. 2019;87:218–26. Springer Nature or its licensor (e.g. a society or other partner) holds
23. Murtaza A, Afzal S, Zaman G, Saeed A, Pelletier J, Sévigny J, exclusive rights to this article under a publishing agreement with the
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inhibitor. Bioorg Chem. 2021;115:105240. such publishing agreement and applicable law.