Synthesis, biological evaluation, and molecular docking study of sulfonate derivatives as nucleotide pyrophosphatase/phosphodiesterase (NPP) inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.04.031

A new series of sulfonate derivatives 1a–zk were synthesized and evaluated as inhibitors of nucleotide pyrophosphatases. Most of the compounds exhibited good to moderate inhibition towards NPP1, NPP2, and NPP3 isozymes. Compound 1m was a potent and selective inhibitor of NPP1 with an IC₅₀ value of 0.387 ± 0.007 μM. However, the most potent inhibitor of NPP3 was found as 1x with an IC₅₀ value of 0.214 ± 0.012 μM. In addition, compound 1e was the most active inhibitor of NPP2 with an IC₅₀ value of 0.659 ± 0.007 μM. Docking studies of the most potent compounds were carried out, and the computational results supported the in vitro results.

Full text of DOI:10.1016/j.bmc.2019.04.031

Accepted Manuscript
Synthesis, biological evaluation, and molecular docking study of sulfonate de-
rivatives as nucleotide pyrophosphatase (NPPs) inhibitors
Mohammad H. Semreen, Mohammed I. El-Gamal, Saif Ullah, Saquib Jalil,
Sumera Zaib, Hanan S. Anbar, Joanna Lecka, Jean Sévigny, Jamshed Iqbal
PII:
S0968-0896(19)30571-1
https://doi.org/10.1016/j.bmc.2019.04.031
BMC 14885
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 April 2019
23 April 2019
25 April 2019
Please cite this article as: Semreen, M.H., El-Gamal, M.I., Ullah, S., Jalil, S., Zaib, S., Anbar, H.S., Lecka, J.,
Sévigny, J., Iqbal, J., Synthesis, biological evaluation, and molecular docking study of sulfonate derivatives as
nucleotide pyrophosphatase (NPPs) inhibitors, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/
10.1016/j.bmc.2019.04.031
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Synthesis, biological evaluation, and molecular docking study of sulfonate
derivatives as nucleotide pyrophosphatase (NPPs) inhibitors
Mohammad H. Semreen 1,2, Mohammed I. El-Gamal 1,2,3,*, Saif Ullah4, Saquib Jalil 4, Sumera
Zaib 4, Hanan S. Anbar 3, Joanna Lecka 5,6, Jean Sévigny 5,6, and Jamshed Iqbal 4,*
1
Department of Medicinal Chemistry, College of Pharmacy, University of Sharjah, Sharjah
27272, United Arab Emirates.
2
Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab
Emirates.
3 Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt.
4 Centre for Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus,
Abbottabad-22060, Pakistan.
5 Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université
Laval, Québec, QC, G1V 0A6, Canada.
6 Centre de Recherche du CHU de Québec – Université Laval, Québec, QC, G1V 4G2, Canada.
*Corresponding authors: e-mail address: drmelgamal2002@gmail.com ; address: Department
of Medicinal Chemistry, College of Pharmacy, University of Sharjah, Sharjah 27272, United
Arab Emirates [M.I. El-Gamal] & e-mail address: drjamshed@cuiatd.edu.pk; address: Centre for
Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus, Abbottabad-
22060, Pakistan [J. Iqbal].
Page 1 of 36

Abstract
A new series of sulfonate derivatives 1a-zk were synthesized and evaluated as inhibitors of
nucleotide pyrophosphatases. Most of the compounds exhibited good to moderate inhibition towards
NPP1, NPP2, and NPP3 isozymes. Compound 1m was a potent and selective inhibitor of NPP1 with
an IC50 value of 0.387 ± 0.007 µM. However, the most potent inhibitor of NPP3 was found as 1x with
an IC50 value of 0.214 ± 0.012 µM. In addition, compound 1e was the most active inhibitor of NPP2
with an IC50 value of 0.659 ± 0.007 µM. Docking studies of the most potent compounds were carried
out, and the computational results supported the in vitro results.
Keywords: Homology modeling; Immune modulation; Molecular docking; Nucleotide
pyrophosphatase; Sulfonate.
1. Introduction
Cells communicate via a route of purinergic signaling that is arbitrated by a number of
extracellularly positioned nucleotides accompanying nucleosides. This purine nucleotide and
nucleoside signaling play a role in the synaptic transmission, determination of cell fate, and
immunologic response. Secreted nucleotides are capable of switching receptors on cell surface
named P1, P2X and P2Y.1 A number of extracellular nucleotide-hydrolyzing enzyme classes are
engaged in terminating purinergic signaling. The classes of cell surface-located proteins include
ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases), alkaline phosphatases (APs),
and the ectonucleotide pyrophosphatases/phosphodiesterases (ENPPs).2 ENPPs deal with
multiple nucleotide substrates, in particular nucleotide triphosphate, nucleotide diphosphates,
nucleotide sugars, and dinucleotide polyphosphates to generate nucleotide monophosphate
(NMP) as product. NMP is hydrolyzed by APs and ecto-5'-nucleotidases (eNs) and release
nucleoside as a final product.1-3
In accordance with relation to substrate preference, seven members of human ENPPs can be
grouped into two categories. ENPP1, 3, 4 occupy first category for nucleotide degrading
potency, and second category may include ENPP2, 6, 7 which have affinity for phospholipid-
Page 2 of 36

based substrates. ENPP1 early represented as PC1, is a homodimer (linked with disulfide bonds)
glycoprotein.4 Inorganic pyrophosphate generated as a result of nucleotide hydrolysis by ENPP1,
selectively binds to hydroxyapatite crystals and inhibits the bone mineralization.5 In contrast to
PPi, produced by tissue non-specific alkaline phosphatase (TNAP), which facilitates the bone
formation.6,7 Reduced level of ENPP1 is associated with atherosclerosis,8 hypophosphatemic
rickets9 and general arterial calcification of infancy (GACI).10 On the other hand, over
expression of ENPP1 leads to the release of calcium pyrophosphate dihydrate (CPPD) that
accumulate in the joints resulting in disorder chondrocalcinosis.11 Increased level of PPi causes
chondrocytes apoptosis, facilitating the disorder osteoarthritis12 and aortic valve calcification.13
ENPP2 is a distinctive among the other members, capable of producing lysophosphatidic
acids (LPA), in particular from lysophosphatidylcholine (LPC),14 by hydrolyzing the
phosphodiester bonds of substrate. Non-nucleotide substrates for ENPP2 include lipid
compounds and phosphate esters of choline, however, it has also affinity for nucleotide
substrates to some extent. The crystallographic structure of ENPP2 is much related in sequence
identity (about 45%) to ENPP1, in which catalytic domain phosphodiesterase (PDE) is
surrounded at N-terminal by somatomedin B-like domain and nuclease like domain via C-
terminal.15 ENPP2 crystal structure16,17 also revealed a rigid hydrophobic pocket capable for
binding of acyl chain of lysophospholipid, whereas deep hydrophilic region coordinate the
glycerol moiety of lysophospholipid and nucleotide substrate as well. The N-terminal of
hydrophobic pocket acts like a signal peptide, explaining the statement that EPP2 is a secreted
protein.18 The somatomedin B (SMB)-like domains in contact with catalytic region contribute to
the genesis of open tunnel for LPA product, transport it to its associated G protein coupled
receptors19 and binds the ENPP2 with the integrin’s on cell surface as well.20,21 Different
signaling pathways are activated by ENPP2 (autotaxin) and formed LPA product that leads to
invigoration of cell survival, proliferation and migration.22 Pathophysiological contribution of
LPA is reported in a number of illness including cancer,23 fibrotic upset (pulmonary fibrosis),24
neuropathic pain,25 inflammatory affliction,26 as well as cardiovascular27 and cholestatic
pruritus.28
ENPP3 (CD203c) is expressed in different body organs29 on surfaces such as epithelial,
mucosal,30 especially on mast cells and basophils.31 The crystal structure of ENPP3 revealed the
Page 3 of 36

presence of parallel β-sheets based eight stranded catalytic phosphodiesterase (PDE) region
holding five active N-glycosylation places and on each side, eight α-helices, representing a
characteristic feature of alkaline phosphatase superfamily.32,33 This PDE domain is connected to
nuclease-like (NUC) region via a linker L2, two somatomedin B like domains SMB1 and SMB2,
attached to NUC with linker L1.34 Catalytic regions occupy two zinc ions (Zn1 and Zn2)
surrounded by seven amino acids side chains. Zn1 is linked by Asp-325, His-483, and His-329,
and facilitates the escape of leaving group, whereas Zn2 allows nucleophilic attacks coordinated
by Asp-167, Asp 372, His-373, and nucleophilic residue Thr-205.35 Upregulation of ENPP3 was
reported on cell surface in addition to other inflammatory mediators as a result basophils
activation with antigen bounded IgE.36 As a response to this activation, ATP releases which is
degraded by ENPP3, thus, ENPP3 act as a marker for recognizing allergen responsiveness on
basophils of patients.37 In rats, this enzyme was reported in intestine possibly facilitating the
absorption and digestion of nucleotides in diet.38 ENPP3 is actively engaged in fluid
homeostasis, modulating bile formation and secretions of cerebral spinal fluid.39 ENPP3 is
incriminated in synchronizing the glycosylation of nucleotide sugars of brain specific proteins40
and hydrolyzing the intracellular and extracellular dipolyphosphate.41,42 Thus the involvement of
these three isozymes in various biological and pathophysiological processes made them
attractive target for development of therapeutic leads. Up to now, very few inhibitors of ENPP1,
3 have been identified (Fig. 1) with different potential nucleus. These compounds are non-
selective, unstable and not easy to synthesize due to bulk structure. Whereas, in case of ENPP2,
large number of lipid based compounds were reported, capable of targeting lysophospholipase D
activity but a very small number of compounds were available against phosphodiesterase
inhibition (Fig. 1). Here, we developed a new class of sulfonate derivatives and evaluated their
phosphodiesterase inhibitory potential against all the three isozymes.
Page 4 of 36

O
O
NH2
NH
O
SO3H
O
O
O
N
N
O
O
N
N
H
H
NH
HN
N
O
NH SO3H
NH SO3H
SO3H
Cl
HN
N
O
N
N
SO3H
SO3H
HN
S
SO3H
SO3H
NH2
O
N
H
(c)
HO3S
(m or p)
(a)
(b)
S
HN
N
N
OH
Cl
Cl
Cl
O
O
Cl
S
OH
CHO
N
O
O
Cl
O
O
O
S
N
NH
(e)
(d)
(g)
(f)
Figure 1. Structures of previously published NPP1 inhibitors. (a) Suramin,43 (b) Reactive blue
2,43 (c) Quinazoline-4-piperidine-4-ethylsulfamide derivative,44 (d) Oxadiazole derivative. Non-
lipid NPP2 inhibitors45-47 (e) Hexachlorophene, (f) Damnacanthal (g) Vinpocetine.
2. Results and discussion
2.1.
Chemistry
The target sulfonate compounds 1a-zk were synthesized through the 2-step synthetic
pathway illustrated in Scheme 1. Reaction of p-aminophenol (2) with the appropriate acid
chloride 3a-f to produce the corresponding phenolic intermediates 4a-f. In order to keep the
hydroxyl group free and avoid diacylation, the acid chloride was diluted in acetone and
added dropwise slowly to a mixture of 4-aminophenol and potassium carbonate in acetone
while stirring. The target sulfonate derivatives 1a-zk were obtained through treatment of the
phenolic intermediates 4a-f with the appropriate sulfonyl chloride reagent in presence of
Page 5 of 36

triethylamine. The target compounds were purified by column chromatography and their
identity and purity were confirmed by spectral analysis.
H
N
H
N
R1
R1
NH2
ii
i
Cl
R1
O
R2
O
+
O
S
O
HO
HO
O
O
3a-f
2
4a-f
1a-zk
R2 = alkyl, aryl
4a: R1 = cyclopentyl
4b: R1 = cyclohexyl
4c: R1 = cycloheptyl
4d: R1 = cyclooctyl
4e: R1 = p-chlorophenyl
4f: R1 = 2-naphthyl
Scheme 1. Reagents and conditions: (i) anhydrous potassium carbonate, acetone, 0 ºC, rt, 4 h,
65-82%; (ii) appropriate sulfonyl chloride derivative, Et3N, anhydrous THF, 0 ºC, rt, 2 h, 43-
91%.
2.2.
Structure-activity relationship
The target compounds 1a-zk were tested for inhibitory effects against NPP1, 2, and 3
isozymes. These compounds differed in substitution at linkers R1 and R2 (Table 1). Six series of
compounds were synthesized with R1 equals cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, p-
chlorophenyl, or 2-naphthyl moiety, diverging with reference to terminal R2 that was subjected
to variable aliphatic or aromatic substituents keeping R1 with constant structural moiety. These
derivatives were analyzed against three forms of NPPs by using colorimetric method with an
artificial substrate p-Nph-5´-TMP.48,49
Page 6 of 36

Table 1. Structures of the target sulfonate derivatives 1a~zk and their inhibitory activities (IC50,
µM) or inhibition% at 100 µM concentration against NPP1, NPP2, and NPP3.
H
N
R1
O
R2
O
S
O
O
Codes
R1
R2
NPP1
NPP2
NPP3
IC50 ± SEM (µM)/% inhibition
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cycloheptyl
Cycloheptyl
Cycloheptyl
Cycloheptyl
Ph
46.67 ± 0.026
8.137 ± 0.003
1.648 ± 0.032
5.76 ± 0.004
1.134 ± 0.002
1.918 ± 0.004
1.629 ± 0.006
3.461 ± 0.030
11.85 ± 0.011
43%
38%
13.57 ± 0.001
42%
1.724 ± 0.002
0.856 ± 0.003
2.675 ± 0.001
1.014 ± 0.008
3.963 ± 0.023
1.354 ± 0.005
0.369 ± 0.004
2.409 ± 0.011
1.079 ± 0.002
39%
1a
1b
1c
1d
1e
1f
4-Me(C6H4)
4-tert-butyl(C6H4)
4-F(C6H4)
4-CF3(C6H4)
Me
1.473 ± 0.003
0.659 ± 0.007
33%
Et
1.824 ± 0.008
26.21 ± 0.002
23.76 ± 0.68
27%
1g
1h
1i
n-Pr
Ph
4-Me(C6H4)
4-tert-butyl(C6H4)
4-F(C6H4)
4-CF3(C6H4)
Ph
1j
2.55 ± 0.079
0.564 ± 0.008
0.387 ± 0.007
13.84 ± 0.005
0.422 ± 0.008
1.493 ± 0.003
29%
39%
0.807 ± 0.001
0.254 ± 0.004
1.79 ± 0.005
6.602 ± 0.001
0.255 ± 0.007
47.01 ± 0.15
37%
1k
1l
37%
1.6 ± 0.46
28%
1m
1n
1o
1p
1q
4-Me(C6H4)
4-tert-butyl(C6H4)
24%
3.908 ± 0.003
23%
2,4,6-
triisopropylphenyl
Cycloheptyl
Cycloheptyl
Cyclooctyl
Cyclooctyl
Cyclooctyl
Cyclooctyl
4-F(C6H4)
4-CF3(C6H4)
Ph
39.3%
0.431 ± 0.007
45%
4.944 ± 0.004
34%
2.267 ± 0.001
2.578 ± 0.006
47%
1r
1s
65.36 ± 1.26
80.25 ± 2.12
49%
1t
4-Me(C6H4)
4-tert-butyl(C6H4)
43%
35%
1u
1v
1w
40%
46%
2,4,6-
triisopropylphenyl
46%
30%
30%
Page 7 of 36

Cyclooctyl
4-Cl(C6H4)
4-Cl(C6H4)
4-Cl(C6H4)
4-Cl(C6H4)
4-CF3(C6H4)
Ph
29%
0.679 ± 0.007
> 100
0.214 ± 0.012
2.772 ± 0.005
1.624 ± 0.017
1.525 ± 0.006
1.203 ± 0.004
1x
1y
9.614 ± 0.003
1.437 ± 0.006
1.799 ± 0.001
0.975 ± 0.001
4-Me(C6H4)
4-tert-butyl(C6H4)
7.693 ± 0.635
31%
1z
1za
1zb
2,4,6-
24%
triisopropylphenyl
4-Cl(C6H4)
4-F(C6H4)
2.897 ± 0.007
19.91 ± 1.7
46%
1zc
4-Cl(C6H4)
2-naphthyl
2-naphthyl
2-naphthyl
2-naphthyl
4-CF3(C6H4)
Ph
21.57 ± 0.001
9.296 ± 0.016
24%
1.07 ± 0.28
27.97 ± 0.935
7.995 ± 0.009
38%
3.424 ± 0.004
20.51 ± 0.002
23%
1zd
1ze
1zf
4-Me(C6H4)
4-tert-butyl(C6H4)
2.195 ± 0.004
3.429 ± 0.002
49%
1zg
1zh
2,4,6-
42%
2.892 ± 0.008
triisopropylphenyl
2-naphthyl
4-F(C6H4)
1.487 ± 0.002
1.118 ± 0.007
37%
33%
22%
13.52 ± 0.025
5.426 ± 0.113
42%
1zi
1zj
2-naphthyl
4-CF3(C6H4)
2-naphthyl
8-quinolinyl
2.461 ± 0.004
-
1zk
Suramin48
LPA49
-
-
-
-
8.670 ± 1.030
-
1.270 ± 0.080
-
0.06 ± 0.01
The cyclopentyl derivatives 1a-e
Introduction of cyclopentyl at R1 and substitution of sulfonate with phenyl ring at position R2
of compound 1a, resulted in promising inhibitory effect on NPP3. Optimal substitution of phenyl
at para position with electron-donating group, methyl for compound 1b, enhanced the selectivity
for both NPP3 and NPP1, in contrast to compound 1a, with an IC50 values of 0.856 ± 0.003 and
8.137 ± 0.003 µM, respectively, resulting in the most potent derivative against NPP3 among the
cyclopentyl analogues. Compound 1b also showed inhibition against NPP2, however not
selective and less potent, with inhibitory potency of 13.57 ± 0.001 µM. It was suggested that
replacement of methyl with bulky and kinetic-stabilizing group, tert-butyl, will enhance the
activity of the compound 1c, surprisingly, there was weaker potency against NPP3, with almost 5
times greater active against NPP1, demonstrating that a compound with bulky substituent has
more affinity for NPP1 in contrast to NPP3. Replacement of tert-butyl with electron-
Page 8 of 36

withdrawing moiety, fluorine atom, for compound 1d, retrieved the potency towards NPP2 with
an IC50 ± SEM = 1.473 ± 0.003 µM, however, declined the affinity for NPP1 by 3 folds with
little effect on NPP3. Substitution of fluorine element with less electronegative group
trifluoromethyl (–CF3) in compound 1e exhibited 5 folds and 2.3 folds enhanced inhibition
towards NPP1 and NPP2, respectively, in contrast to NPP3 showing 3.9 times decreased
potency. This compound was the most potent against NPP2 with auspicious IC50 ± SEM values
of 0.659 ± 0.007 µM. In case of compounds 1d and 1e, it can be assumed that substitution with
electronegative atom increases the activity against NPP2.
The cyclohexyl derivatives 1f-m
Inhibitory potential of the sulfonate derivatives was further evaluated by modification of
terminal R1 with cyclohexyl cyclic ring instead of cyclopentyl. Insertion of methyl group at
position R2 afforded compound 1f, found favorable inhibitory potency with respect to NPP1 and
NPP3 with an IC50 ± SEM values of 1.918 ± 0.004 and 1.354 ± 0.005 µM, respectively.
However, it showed only 33% inhibition against NPP2. Elongation of aliphatic side chain to
ethyl moiety developed compound 1g, resulted in greater inhibitory potential in respect to NPP3
with an IC50 ± SEM values of 0.369 ± 0.004 µM. The compound 1g recovered the potency
against NPP2 exhibiting an IC50 ± SEM equals 1.824 ± 0.008 µM, however it retained the
potency towards NPP1. Further elongation of the aliphatic side chain length to n-propyl
(compound 1h) exerted declined the activity towards all of three isoenzymes to greater extent.
This chain was replaced with aromatic component, phenyl in compound 1i, which showed less
activity to NPP1, NPP2 only 11.85 ± 0.011 µM and 23.76 ± 0.68 µM, respectively but promising
effect towards NPP3 representing an IC50 values of 1.079 ± 0.002 µM. Further substitution of
phenyl at position 4 with electropositive element, methyl for compound 1j, declined the activity
against all the three isoenzymes with less than 50% inhibition, in contrast to compound 1b that
was active against all of three isozymes. This difference might be due to the increase in carbon in
the cyclic ring at R1 which produced steric hindrance and decreased the affinity of compound
towards the enzymes. However, replacement of methyl with 4-(tert-butyl) in 1k stabilized the
compound and recovered the inhibitory activity against NPP1 and NPP3 with an IC50 ± SEM of
2.55 ± 0.079 and 0.807 ± 0.001 µM, respectively. Further replacement of tert-butyl with
Page 9 of 36

electronegative atom fluorine, enhanced the inhibitory ability of compound 1l for NPP1 and
NPP3 up to 4.5 folds. Introduction of slightly less electronegative group trifluoromethyl, in place
of fluorine, in structure 1m, resulted in stronger potency against NPP1 and NPP2 but weaker
activity against NPP3. It is noteworthy that compounds 1b and 1f-m were previously tested
against steroid sulfatase enzyme but showed no promising inhibitory effect.50
The cycloheptyl derivatives 1n-s
Substitution at place R1, was moved to greater carbon atoms, cycloheptyl and R2 with
aromatic ring phenyl, gave rise to compound 1n that was evaluated against NPP1, NPP2 and
NPP3, resulted in weaker potency against all three isozymes than compound 1i with similar side
moiety, phenyl at R2. It might be assumed that increase in the carbon atoms in cyclic ring at R1
did not contribute towards the affinity to greater extent. The substitution of phenyl at p-position
with methyl in compound 1o gave the outstanding results for NPP1 with promising IC50 value of
0.422 ± 0.008 µM. It could be proposed that para substitution of phenyl ring with some
electropositive radical such as methyl, as in compounds 1b, 1j, and 1o, may produce
considerable potential for NPPs 1, 2, 3 by balancing the inductive electron-withdrawing effect of
the phenyl ring because of sp2 hybridization of carbon atoms and stabilizing the compounds.
Replacement of methyl substituent with a bulky group, tert-butyl in compound 1p, revealed
increased activity against NPP2 to the IC50 values of 3.908 ± 0.003 µM, adding to the concept
that more bulky substitution was also effective against NPP2 and not only against NPP1 and
NPP3 in accordance with 1c and 1k. It was evaluated by replacing it with more spacious group 2,
4, 6-triisopropyl for a compound 1q. Unexpectedly, activity against all the isoenzymes was less
than 50%. It may be suggested that the increase in proportion of electron-donating groups and/or
the bulkiness was detrimental for the activity. Substitution of the phenyl ring with electron-
withdrawing element in compound 1r, supported the suggestion to boost the affinity same like
1d and 1l, with considerably potency against NPP2 and NPP3 yielding IC50 ± SEM value 4.944 ±
0.004 and 2.267 ± 0.001 µM, respectively, with a much lower activity against NPP1 that was
only 39.3%. Further changes in substitution at this position with less effective electron-
withdrawing group (–CF3) at para position of phenyl ring, yielded the compound 1s, which
exhibited remarkable activity against NPP1 with IC50 value of 0.431 ± 0.007 µM. However, no
Page 10 of 36

change in activity against NPP3 in contrast to 1r and revealed just 34% inhibition in case of
NPP2.
The cyclooctyl derivatives 1t-x
The compounds 1t-x of this series were synthesized with increasing carbon atoms in
cyclic ring at position R1 to 8 (cyclooctyl) and substituting the same groups at position R2 as for
earlier in particular phenyl, p-methylphenyl, p-(tert-butyl)phenyl, 2,4,6-triisopropylphenyl and p-
(trifluoromethyl)phenyl. These compounds did not manifest describable potential for either of
three isoenzymes except compound 1x. Compound 1x possessing p-(trifluoromethyl)phenyl was
found the most potent and active against NPP2 and NPP3 among these synthesized sulfonate
derivatives. For NPP2, it exhibited IC50 ± SEM value of 0.679 ± 0.007 µM. It can be suggested
that presence of 8-carbon saturated ring contributed to the hydrophobicity of the compound 1x,
maked it more selective for NPP2. The compound, 1x was the most favorable for NPP3 showing
an IC50 ± SEM value of 0.214 ± 0.012 µM that was ~6 fold more effective as compared to
reference inhibitor, suramin. It was assumed that substitution of electronegative electron-
withdrawing group on para position of phenyl ring, make the unsaturated ring more accepted for
π-π interactions inside the active pocket of isozymes NPP3.
The p-chlorophenyl derivatives 1y-zd
Compounds 1y-zd were synthesized by substituting the p-chlorophenyl at position R1 and
different groups at place R2 in particular phenyl, p-methylphenyl, p-(tert-butyl)phenyl, 2,4,6-
triisopropylphenyl, p-fluorophenyl and p-(trifluoromethyl)phenyl, respectively. All these
compounds demonstrated encouraging results suggesting that placement of electron-withdrawing
substituent at para position of cyclic ring has greater influence towards the activity. The
compounds 1z, 1za, 1zb revealed favorable potential for NPP1 with an IC50 ± SEM = 1.437 ±
0.006, 1.799 ± 0.001, and 0.975 ± 0.001 µM, respectively and at the same time these compounds
were active against NPP3 with an IC50 ± SEM values of 1.624 ± 0.017, 1.525 ± 0.006, and 1.203
± 0.004 µM, respectively. Among these three only compound 1z exhibited affinity for NPP2
Page 11 of 36

with an IC50 ± SEM values of 7.693 ± 0.635 µM. Involvement of electronegative substitutes on
both sides R1, R2 as for compounds 1zc and 1zd resulted in selective potency. Differentiation of
results among 4-chlorobenzene substituted sulfonate derivative 1zc and cycloheptyl substituted
compound 1r with similar attachment at sulfonate group 4-fluorophenyl, delineated contrary
effects against all the three isozymes. Compound 1zc depicted considerable affinity for NPP1
facing the IC50 ± SEM = 2.897 ± 0.007 µM, while compound 1r showed only 39% inhibition. On
the other hand, compound 1r represented potential against NPP2 and NPP3 with an IC50 ± SEM
= 4.944 ± 0.004 and 2.267 ± 0.001 µM, respectively, while compound 1zc was less active
towards NPP2 and NPP3. This might be due to presence of 4-chloro attachment on phenyl ring,
induced the negative inductive property in compound 1zc, making it more stable and favorable
for NPP1 as compared to NPP2. The compound 1zd was found among the most potent structures
against NPP2 with IC50 ± SEM = 1.07 ± 0.28 µM.
The 2-naphthyl derivatives 1ze-zk
A new combination of sulfonate derivatives were synthesized by adding bicyclic weak
electron donating group, 2-naphthyl, at position R1 and different types of groups in place of R2.
2-Naphthyl was considered as privileged unit for NPPs inhibition. Placement of phenyl linker at
point R2 in compound 1ze, manifested weak potency. Further substitution of phenyl with
introduction of electropositive methyl group in compound 1zf, re-attained the inhibitory potential
for NPP2 with an IC50 ± SEM values of 7.995 ± 0.001 µM, however, there was further decrease
in potency against NPP1 and NPP3. Surrogating the R2 with divergent groups as an illustration
p-(tert-butyl)phenyl, 2,4,6-triisopropylphenyl, p-fluorophenyl, p-(trifluoromethyl)phenyl
produced the compounds 1zg-1zj, respectively. These compounds were less than 50% inhibitor
against NPP2, although endowed favorable results against NPP1 and NPP3. The compounds 1zi
and 1zj substituted with electron withdrawing groups, were more potent against NPP1 in contrast
to NPP3 with an IC50 ± SEM = 1.487 ± 0.002 and 1.118 ± 0.007 µM, respectively. The most
feasible inhibition against NPP2 of these naphthyl derivatives was attained when R2 was
positioned with 8-quinolinyl group in compound 1zk. Its inhibitory potency was found among
the most selective and potent compounds for NPP2 with an IC50 ± SEM value of 2.461 ± 0.004
µM.
Page 12 of 36

2.3.
Molecular docking studies
The experimental results suggested that few of the compounds were selective inhibitors of
NPP1, 2 and 3, however, some of the compounds exhibited dual inhibition for NPP1 and NPP2,
or NPP1 and NPP3 and some also were found active against NPP2 and NPP3. Therefore,
docking studies were carried out for all these potent inhibitors having selectivity towards
respective isozyme or showing dual inhibition. As the crystal structure of the human nucleotide
pyrophosphatases (NPP1) is not available at protein data bank, the docking studies were
performed in the homology model generated by our group using mouse ENPP1 with PDB ID:
4B56 as template.51-53 However, the human crystal structures of NPP2 and NPP3 were
downloaded from protein data bank with the PDB IDs 4ZG654 and 6C01.55 In order to validate
the docking studies, the reference compound suramin was docked inside the homology model of
human NPP1 and crystal structure of human NPP3. However, the co-crystallized ligand 4NY
(Figure 3a) was extracted and docked inside the crystal structure of NPP2 (4ZG6). The
validation was carried out before performing the docking studies of selected compounds for each
isozyme. After docking, the root mean square deviation of 0.71 Å was found for co-crystallized
ligand, 4NY of NPP2. While for NPP1 (homology model) and NPP3 (6C01), the positive
compound suramin was docked and the binding interactions were compared with selected
compounds. After the validation process, the docking studies were carried out.
2.3.1. Docking studies of hNPP1 inhibitors
The docking studies of NPP1 were carried out for compounds 1e, 1l, 1m, 1o, 1zd and 1zj in
addition to suramin. The 3D binding poses for all the selected compounds are shown in Figure 2.
The interaction diagram of suramin showed that the amino acids involved in the hydrogen
bonding were Lys295, Lys255, Trp307, Lys338, Tyr340, Lys291, Asn277, Lys528 and Thr256
with the oxygen atoms of suramin. However, the π-π interactions were noticed between the
phenyl group of suramin and His424, Pro323, Trp322, Ser325, and Asp218. In addition to these
interactions, the metal interactions were noticed between two zinc atoms of the active site within
the NPP1 and the oxygen atom of suramin. When the detailed investigation of the compound 1e
Page 13 of 36

was carried out, the most noticeable interactions were hydrogen bonding and metal interactions
within the active pocket of NPP1. The trifluoromethyl group was inclined more towards outer
region of active pocket, however, the carboxamido group was found to involve in metal
interactions with zinc atoms in addition to π-π interaction with His380. The cyclopentyl moiety
was found to make π-π interactions with Tyr451 and Lys528. The carboxamido group was
located in the middle of the active site of NPP1. When the interactions of the compound 1l
(Figure 2c) were examined, it was found that phenyl ring was interacting with one zinc atom,
whereas, oxygen atom of carboxamide part has showed metal interactions with second zinc
atom. The fluorine atom at phenyl ring was showing hydrogen bonds with Lys255, Thr254,
Gly536 and in addition to hydrogen bond, π-π interactions were observed between His424 and
fluorophenyl ring. Compound 1m (Figure 2d) showed metal interactions with both the zinc
atoms present in the active site of NPP1. However, hydrogen bonding were noticed between
Gly452, Tyr451, Phe534 and 4-(trifluoromethyl)benzenesulfonate moiety. Moreover, the Asn277
was also involved in hydrogen bonding with carboxamide part of the compound 1m. The π-π
interactions were observed between the cyclohexyl group and Lys278. Upon investigating the
docking pose of compound 1o (Figure 2e), the important interactions found were the metal
interactions of zinc ion with central phenyl moiety in addition to oxygen of carboxamide part.
Methylbenzenesulfonate moiety showed hydrogen bonding with Lys255 along with π-π
interactions with Asn277. When the other compound 1zd (Figure 2f) was taken into account, the
noticeable interactions were shown by important amino acids of the active site and 4-
(trifluoromethyl)benzenesulfonate moiety of the compound. The hydrogen bonding were
important with residues Tyr451 and Lys255. Moreover, π-π interactions were found Lys255.
His380 and Tyr451 were involved in π-phenyl interactions. In case of compound 1zj (Figure 2g),
the 2-naphthamido part was oriented towards the Glu373 and Ser377, whereas, the 4-
(trifluoromethyl)benzenesulfonate moiety was found deeper inside the active pocket near the
amino acids, Tyr451, Lys255, Thr256 and Asn277. Moreover, the phenyl group in the middle of
the compound exhibited arene-cation interaction with zinc present inside the active site of NPP1.
Page 14 of 36

(a)
(b)
(d)
(c)
(e)
(f)
Page 15 of 36

(g)
Figure 2. 3D interaction poses (with hydrogen bond surface) of the selective inhibitors of human
NPP1 (inside the homology model) (a): Suramin; (b): 1e; (c): 1l; (d): 1m; (e): 1o; (f): 1zd and
(g): 1zj
2.3.2. Docking studies of hNPP2 inhibitors
The docking studies of NPP2 were carried out for 1e, 1g, 1x and 1zk in addition to co-
crystallized ligand 4NY. The 3D binding poses for all the selected compounds are illustrated in
Figure 3. The cognate ligand 4NY (Figure 3a) of the crystal structure of NPP2 (4ZG6) showed
that the important amino acid residues involved in the formation of interactions were Leu214,
Phe211, Phe274, Tyr307, Leu217 and Ala218.54 Among them, Phe274 and Leu217 were
showing hydrogen bonding with cognate ligand. However, the amino acid residues involved in
the π-π interactions were Trp276, Tyr307, Ala305, Leu217, Leu214, Phe211 and Phe274. When
the compound 1e was examined for the favorable interactions within the active site (Figure 3b),
similar types of interactions were observed as found with the cognate ligand inside the active
pocket. The 4-(trifluoromethyl)benzenesulfonate moiety was in π-π interactions with the His316,
His475, Asp474, and interactions with zinc atoms. The hydrogen bonding was noticed with
His475 and 4-(trifluoromethyl)benzenesulfonate. Another hydrogen bond was noticed between
the carboxamido moiety of the compound and His316. The compound 1g, after docking inside
the active pocket, showed the interaction pose depicted in Figure 3c. The oxygen atoms of the
compound was found in hydrogen bonding with Asn231. Whereas, π-π interactions of compound
were noticed by Leu244, His475, His316, Tyr307 and Val278. The ethanesulfonate group of the
Page 16 of 36

compound 1g was found towards the zinc atoms inside the active pocket. However, the 4-
cyclohexanecarboxamido moiety was oriented towards the Tyr307 and Val278. The compound
1x (Figure 3d) showed hydrogen bonding with Phe211 and Lys249 by its trifluoromethyl moiety,
while with the same phenyl group of 1x, Phe275 and Phe250 were making the π-π interactions.
The cyclooctanecarboxamido moiety was only involved in the formation of π-π interactions with
Ala271. When the binding mode of compound 1zk was examined (Figure 3e), the most
noticeable interactions were hydrogen binding with Trp276, in addition to several π-π
interactions with Trp255, Phe274, Tyr307 and Phe275. The zinc atoms did not show any
interactions in case of compound 1zk.
O
(b)
HO
N
Cl
N
F
(a)
Page 17 of 36

(c)
(d)
(e)
Figure 3. 3D interaction poses (with hydrogen bond surface) of the selective inhibitors of
human NPP2 (inside the PDB ID: 4ZG6) (a): 4NY and its structure; (b): 1e; (c): 1g; (d): 1x
and (e): 1zk
2.3.3. Docking studies of hNPP3 inhibitors
The docking studies of NPP3 were carried out for 1d, 1g, 1l, 1r, 1x, 1zd, and 1zh in addition
to suramin. The 3D binding poses for all the selected compounds are shown in Figure 4. The
docking studies of suramin (Figure 4a) inside the active pocket of NPP3 (6C01) revealed that the
most important amino acid residues involved in the interactions were His483, His329 and
Asp325 coordinated to one of zinc atom, while, Asp372, His373, Thr205 and Asp167 were
found in coordination to second zinc atom.55 Due to the bulkier structure of suramin, the
interactions are noticed in wide active site of the enzyme’s pocket. Similarly, many interactions
are noticed such as hydrogen bonding, π-π interactions, metal interactions, and π-alkyl
Page 18 of 36

interactions. Compound 1d (Figure 4b) showed hydrogen bonding with Asn477, Asn475,
Glu400, Lys204, Gly401 and His483. However, the π-π interactions were found between Glu400
and 4-fluorobenzenesulfonate group. When the interactions of compound 1g (Figure 4c) were
taken into account, the 3D diagram suggested that the cyclohexanecarboxamide was found more
towards the middle of active pocket towards the zinc atoms and showed metal interactions.
Hydrogen bonding were observed by the ethanesulfonate group with Asn482, Asn477, Gly401
and Pro402. The 4-fluorobenzenesulfonate moiety of compound 1l (Figure 4d), more like the
compound 1d, was found deep inside the active pocket exerting similar interactions with side
residues including Asp167, Thr205 and Asn226 giving rise to important π-π interactions and
hydrogen bonding. Moreover, the cyclohexanecarboxamido group was found profoundly located
near amino acids Leu468, Asn477 and Asn482. The phenyl ring located in the center of the
compound and the enzyme pocket and was located parallel to the amino acid residues His483
and Asn482. Compound 1r (Figure 4e) possessing cyclohexanecarboxamide ring showed more
or less similar pattern of binding inside the active pocket of NPP3 to that of compound 1d.
However, the cyclopentane ring was replaced by cyclohexane ring and therefore, the 4-
fluorobenzenesulfonate was found deeper inside the active pocket towards the important amino
acids and showing π-π interactions with Gly401. Moreover, hydrogen bonds were noted by
fluorine atom with Glu400 and Asn475. However, the oxygen was making metal interaction with
one of the zinc atom inside the active pocket. The compound 1x (Figure 4f) was monitored, the
notable interactions were hydrogen bonding of triflouromethyl group with Asn475, Glu400 and
Pro402. However, the Gly401 was involved in π-π interactions with the compound. Oxygen
atom showed metal interactions with both the zinc atoms in addition to Thr205. Moreover,
compound 1zd (Figure 4g) having 4-(trifluoromethyl)benzenesulfonate group was investigated
after docking inside the active pocket of NPP3 and the noticeable interactions were hydrogen
bonding with Asn477, Asn475, Glu400, Asn482 and Pro402. The 4-chlorobenzamido group of
the compound showed π-π interactions with Tyr320, Tyr289, Asp167 and hydrogen bond with
Lys204 and Leu239. When compound 1zh (Figure 4h) from the 2-naphthyl series was taken into
account, the 2,4,6-triisopropylbenzenesulfonate moiety was inclined within the pocket of enzyme
and showing several π-π interactions with Asn477 and Asn482. However, the important part of
the compound, 2-naphthamido moiety exhibited π-π interactions with Thr205 and therefore both
sides of this compound exhibited several interactions with the amino acids of active pocket.
Page 19 of 36

The overall docking studies were in accordance with the results of in vitro studies and the
binding affinities and interactions were following the experimental results. Therefore, both the
results are well matched, and justification by docking studies have been provided for the in vitro
experiments.
(a)
(b)
(d)
(c)
Page 20 of 36

(f)
(e)
(h)
(g)
Figure 4. 3D interaction poses (with hydrogen bond surface) of the selective inhibitors of human
NPP3 (inside the PDB ID: 6C01) (a): Suramin; (b): 1d; (c): 1g; (d): 1l; (e): 1r; (f): 1x; (g): 1zd
and (h): 1zh
HYDE assessment of selective and potent compounds against h-NPP1, h-NPP2 and h-NPP3
The HYDE affinity assessment was done by using LeadIT for the first 30 top ranking docked
conformations within the active sites of the homology models of human NPP1, crystal structure
of NPP2 and NPP3 and it helped in the selection of correct binding mode as well as the
selectivity towards the most potent compound. The FlexX docking score for the selective
Page 21 of 36

derivatives and their binding free energy ΔG were given in Table 2. The FlexX docking score
presented the lower energy scores. Moreover, the binding free energies ΔG given in Table 2
showed that the potent inhibitors exhibited higher affinity towards their respective target.
Table 2. Docking and Hyde scores and their corresponding ranks by Hyde affinity assessment
(h-NPP1, h-NPP2 and h-NPP3)
Code
FlexX score of the top Binding free energy
ranking pose
h-NPP1
ΔG (kJ mol‒1)
-18.28
-20.79
-20.09
-19.15
-19.37
-22.67
-23
-21
-30
-30
-26
-10
1e
1l
1m
1o
1zd
1zj
h-NPP2
-20.65
-20.17
-20.48
-34.97
-36
-35
-33
-38
1e
1g
1x
1zk
h-NPP3
-18.25
-16.88
-19.23
-20.04
-17.27
-16.46
-24.13
-12
-10
-3
-18
-17
-39
-19
1d
1g
1l
1r
1x
1zd
1zh
3. Conclusion
The target sulfonate derivatives were screened for their inhibitory activity against NPP1~3. The
results suggested some selective and potent inhibitors of NPP1, 2, and 3 isozymes. However, few
compounds were found as dual inhibitors and exhibited significant inhibition against more than one
isozyme. The docking investigation resulted in putative binding modes of compounds within each
receptor, which further strengthen the in vitro results. The most promising compounds as well as other
Page 22 of 36

derivatives that will be designed after lead optimization may further be tested in animal models to get
insight of the pharmacological investigation.
4. Experimental
4.1.
General
The chemicals and solvents were purchased from Sigma-Aldrich or Alfa Aesar. All the
chemicals were used without purification. The target compounds were purified by column
chromatography using silica gel (0.040-0.063 mm, 230-400 mesh) and technical grade solvents.
1H NMR and 13C NMR spectra were recorded on a Bruker Avance 400 or 500 MHz
spectrometers using tetramethylsilane as an internal standard. IR spectra (KBr disks) were
recorded with a Bruker FT-IR instrument (Bruker Bioscience, Billerica, MA, USA). Mass
spectra (MS) were taken in ESI mode on a Waters 3100 Mass Detecter (Waters, Milford, MA,
USA).
4.2.
Synthesis of phenolic intermediates 4a-f
To a solution of p-aminophenol (500 mg, 4.58 mmol) in acetone (60 mL), anhydrous
potassium carbonate (760 mg, 5.5 mmol), was added. The reaction mixture was stirred at room
temperature for 15 minutes, then cooled to 0 °C. A solution of the appropriate acid chloride
(4.165 mmol) in acetone (30 mL) was added dropwise to the reaction mixture at 0 °C with
continuous stirring. After complete addition, the reaction mixture was stirred at room
temperature for 4 h. The reaction mixture was filtered and the filtered solid was washed with
acetone (3 x 10 mL). The combined filtrate and wash were evaporated to dryness. The residue
was dissolved in ethyl acetate (50 mL) and extracted with dilute HCl. The organic layer was then
washed with saline (2 x 30 mL) and dried with anhydrous Na2SO4. The organic solvent was
evaporated under reduced pressure to get the intermediate title compounds. They were used in
the next steps as such without further purification.
Page 23 of 36

4.3.
General procedure for synthesis of the target sulfonate compounds 1a-zk
The appropriate phenolic intermediate 4a-f (0.456 mmol) was dissolved in dry THF (10 mL),
and the mixture was cooled to 0 °C. Triethylamine (0.25 mL, 2.47 mmol) was added thereto. A
solution of appropriate sulfonyl chloride (0.912 mmol) in dry THF (2 mL) was added dropwise
to the reaction mixture at 0 °C. The reaction mixture was stirred at room temperature for 2 h until
reaction completion. The mixture was quenched with ethyl acetate (10 mL) and water (10 mL).
The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3 x 5
mL). The combined organic layer extract was washed with saline (3 x 10 mL) and dried over
anhydrous Na2SO4. The organic solvent was evaporated under reduced pressure and the crude
residue was purified by column chromatography (silica gel, appropriate ratio of hexane: ethyl
acetate) to obtain the pure product. The spectral data of compounds 1b and 1f-m have been
reported in our previous report50 and those for the other target compounds are reported herein in
details.
1
Compound 1a: Yield: 86%; H NMR (400 MHz, CDCl3) δ 7.67-7.62 (m, 4H), 7.48 (d, 2H, J =
8.0 Hz), 7.32 (d, 2H, J = 8.0 Hz), 6.91 (d, 2H, J = 8.0 Hz), 2.72-2.64 (m, 1H), 2.46 (s, 3H), 1.94-
13
1.75 (m, 6H), 1.62-1.59 (m, 2H); C NMR (100 MHz, CDCl3) δ 175.1, 145.6, 145.3, 137.4,
132.2, 129.9, 128.6, 122.9, 120.6, 46.5, 30.6, 26.0; LC-MS: m/z: 346.1 [M+ + 1].
Compound 1c: Yield: 90%; 1H NMR (400 MHz, CDCl3) δ 7.67 (d, 2H, J = 12.0 Hz), 7.61 (brs,
1H), 7.45 (d, 2H, J = 8.0 Hz), 7.30 (d, 2H, J = 8.0 Hz), 6.87 (d, 2H, J = 8.0 Hz), 2.70-2.62 (m,
13
1H), 1.91-1.73 (m, 6H), 1.60-1.56 (m, 2H), 1.34 (s, 9H); C NMR (100 MHz, CDCl3) δ 175.1,
145.4, 145.1, 137.2, 132.0, 129.7, 128.4, 122.7, 120.4, 46.4, 30.5, 29.6, 26.0, 25.6; LC-MS: m/z:
401.82 [M+ + 1].
Compound 1d: Yield: 82%; 1H NMR (500 MHz, CDCl3) δ 7.84-7.81 (m, 2H), 7.47 (d, 2H, J =
9.0 Hz), 7.22-7.18 (m, 3H), 6.93-6.90 (m, 2H), 2.67-2.64 (m, 1H), 1.94-1.75 (m, 6H), 1.64-1.42
13
(m, 2H); C NMR (125 MHz, CDCl3) δ 174.8, 167.2, 165.1, 145.3, 137.4, 131.6, 123.0, 120.6,
116.7 (d, J = 22.6 Hz), 47.0, 30.6, 26.1; LC-MS: m/z: 363.66 [ M+ + 1].
1
Compound 1e: Yield: 85%; H NMR (400 MHz, CDCl3) δ 7.84-7.81 (m, 2H), 7.46 (d, 2H, J =
9.0 Hz), 7.23-7.17 (m, 3H), 6.94-6.90 (m, 2H), 2.69-2.65 (m, 1H), 1.94-1.76 (m, 6H), 1.64-1.45
Page 24 of 36

13
(m, 2H); C NMR (100 MHz, CDCl3) δ 174.9, 167.3, 165.2, 145.4, 137.4, 131.8, 123.1, 120.7,
116.8, 47.0, 30.5, 26.1; LC-MS: m/z: 413.73 [M+ + 1].
1
Compound 1n: Yield: 78%; H NMR (500 MHz, CDCl3) δ 7.80 (d, 2H, J = 2.0Hz), 7.70-7.68
(m, 1H), 7.54-7.50 (m, 2H),7.45 (d, 2H, J = 9.0Hz), 7.38 (brs, 1H), 6.87 (d, 2H, J = 9.0Hz),
2.37-2.33 (m, 1H), 1.95-1.90 (m, 2H), 1.80-1.68 (m, 4H), 1.59-1.51 (m, 4H), 1.49-1.42 (m, 2H);
13C NMR (125 MHz, CDCl3) δ 175.8, 137.4, 135.3, 134.7, 133.0, 129.4, 123.2, 122.9, 120.6,
48.4, 31.7, 29.8, 28.2; LC-MS: m/z: 374.13 [M+ +1].
1
Compound 1o: Yield: 85%; H NMR (500 MHz, CDCl3) δ 7.67 (d, 2H, J = 8.0 Hz), 7.44 (d,
2H, J = 9.0 Hz), 7.30-7.28 (m, 3H), 6.88 (d, 2H, J = 9.0 Hz), 2.43 (s, 3H), 2.37-2.33 (m, 1H),
13
1.95-1.90 (m, 2H), 1.80-1.68 (m, 4H), 1.60-1.50 (m, 48), 1.49-1.42 (m, 2H); C NMR (125
MHz, CDCl3) δ 175.8, 145.6, 145.4, 137.3, 132.3, 130.0, 129.8, 123.0 120.6, 48.4, 31.7, 28.3,
26.6; LC-MS: m/z: 388.18 [M+ + 1].
Compound 1p: Yield: 76%; 1H NMR (500 MHz, CDCl3) δ 7.71 (brs, 1H), 7.65 (d, 2H, J = 8.5
Hz), 7.45-7.39 (m, 4H), 6.81 (d, 2H, J = 9.0 Hz), 2.32-2.27 (m, 1H), 1.86-1.80 (m, 2H), 1.69-
13
1.59 (m, 4H), 1.49-1.31 (m, 6H), 1.25 (s, 9H); C NMR (125 MHz, CDCl3) δ 176.1, 158.5,
145.2, 137.4, 132.1, 128.3, 126.2, 122.8, 120.6, 48.1, 35.4, 31.5, 31.0, 28.1, 26.5; LC-MS: m/z:
430.35 [M+ +1].
1
Compound 1q: Yield: 72%; H NMR (500 MHz, CDCl3) δ 7.61 (s, 1H), 7.40 (d, 2H, J = 9.0
Hz), 6.82 (d, 2H, J = 8.5 Hz), 4.01-3.95 (m, 2H), 2.88-2.82 (m, 1H), 2.31-2.27 (m, 1H), 1.85-
13
1.81 (m, 2H), 1.69-1.64 (m, 4H), 1.44-1.38 (m, 7H), 1.23-1.07 (m, 19H); C NMR (125 MHz,
CDCl3) δ 175.9, 154.4, 151.2, 145.1, 173.3, 129.6, 124.0, 122.8, 120.6, 48.1, 34.3, 31.5, 29.8,
28.1, 26.5, 24.6, 23.5; LC-MS: m/z: 430.35 [M+ + 1].
1
Compound 1r: Yield: 76%; H NMR (500 MHz, CDCl3) δ 7.83-7.79 (m, 2H), 7.50 (brs, 1H),
7.46 (d, 2H, J = 9.0 Hz), 7.21-7.16 (m, 2H), 6.88 (d, 2H, J = 5.0), 2.38-2.34 (m, 1H), 1.95-1.90
(m, 2H), 1.79-1.68 (m, 4H), 1.59-1.50 (m, 4H), 1.49-1.38 (m, 2H); 13C NMR (125 MHz, CDCl3)
δ 175.9, 145.1, 137.5, 131.5, 131.5, 122.9, 120.7, 116.8, 116.6, 48.3, 31.6, 28.2, 26.6; LC-MS:
m/z: 392.31 [M+ + 1].
Page 25 of 36

1
Compound 1s: Yield: 83%; H NMR (500 MHz, CDCl3) δ 7.98 (d, 2H, J = 8.0 Hz), 7.82 (d,
2H, J = 8.0 Hz), 7.50 (d, 2H, J = 9.0 Hz), 7.28 (brs, 1H), 6.94 (d, 2H, J = 8.5 Hz), 2.41-2.36 (m,
1H), 1.99-1.94 (m, 2H), 1.84-1.49 (m, 10H); 13C NMR (125 MHz, CDCl3) δ 175.6, 144.9, 138.8,
137.5, 136.0, 129.1, 126.3, 126.3, 124.0, 121.9; LC-MS: m/z: 442.11 [M+ +1].
Compound 1t: Yield: 68%; 1H NMR (500 MHz, CDCl3) δ 7.82 (d, 2H, J = 7.0 Hz), 7.65 (t, 1H,
J = 7.5 Hz), 7.51 (t, 2H, J = 8.0 Hz), 7.44 (d, 2H, J = 9.0 Hz), 7.39 (brs, 1H), 6.88 (d, 2H, J =
9.0 Hz), 2.41-2.38 (m, 1H), 1.90-1.87 (m, 2H), 1.86-1.75 (m, 4H), 1.61-1.53 (m, 8H); 13C NMR
(125 MHz, CDCl3) δ 176.1, 145.3, 137.4, 135.3, 134.4, 129.3, 128.6, 123.0, 120.7, 46.8, 29.8,
26.7, 26.3, 22.8; LC-MS: m/z: 388.0 [M+ +1].
1
Compound 1u: Yield: 78%; H NMR (500 MHz, CDCl3) δ 7.68 (d, 2H, J = 8.5 Hz), 7.44 (d,
2H, J = 9.0 Hz), 7.29 (d, 2H, J = 8.0 Hz), 7.24 (brs, 1H), 6.90 (d, 2H, J = 7.5 Hz), 2.44 (s, 3H),
13
2.41-2.38 (m, 1H), 1.92-1.87 (m, 2H), 1.81-1.78 (m, 4H), 1.64-1.57 (m, 8H); C NMR (125
MHz, CDCl3) δ 175.0, 144.5, 144.4, 136.3, 131.3, 128.9, 127.7, 122.1, 119.6, 45.9, 28.8, 25.8,
25.3, 24.6, 20.9; LC-MS: m/z: 402.0 [M+ +1].
1
Compound 1v: Yield: 64%; H NMR (500 MHz, CDCl3) δ 7.73 (d, 2H, J = 9.0 Hz), 7.52 (d,
2H, J = 7.0 Hz), 7.45 (d, 2H, J = 9.0 Hz), 7.16 (brs, 1H), 6.93 (d, 2H, J = 8.5 Hz), 2.42-2.37 (m,
13
1H), 1.91-1.88 (m, 2H), 1.82-1.75 (m, 4H), 1.63-1.53 (m, 8H), 1.35 (s, 9H); C NMR (125
MHz, CDCl3) δ 176.0, 158.5, 145.5, 137.2, 132.4, 128.5, 126.3, 123.1, 120.6, 120.5, 46.9, 35.5,
31.1, 29.8, 26.7, 26.3, 25.5; LC-MS: m/z: 444.23 [M+ +1].
1
Compound 1w: Yield: 70%; H NMR (500 MHz, CDCl3) δ 7.38 (d, 2H, J = 9.0 Hz), 7.12 (s,
2H), 7.03 (brs, 1H), 6.86 (d, 2H, J = 9.0 Hz), 2.36-2.29 (m, 1H), 1.84-1.81 (m, 2H), 1.74-1.68
(m, 4H), 1.55-1.47 (m, 8H), 1.21-1.18 (m, 9H), 1.12 (d, 12H, J = 7.0 Hz); 13C NMR (125 MHz,
CDCl3) δ 175.9, 154.4, 151.4, 145.5, 137.1, 129.8, 124.0, 123.1, 120.6, 47.0, 34.4, 29.9, 29.8,
26.8, 26.3, 25.6, 24.7, 23.7; LC-MS: m/z: 514.2 [M+ +1].
1
Compound 1x: Yield: 74%; H NMR (500 MHz, CDCl3) δ 7.96 (d, 2H, J = 8.0 Hz), 7.80 (d,
2H, J = 8.5 Hz), 7.48 (d, 2H, J = 9.0 Hz), 7.10 (brs, 1H), 6.93 (d, 2H, J = 8.5 Hz), 2.41-2.34 (m,
13
1H), 1.80-1.78 (m, 6H), 1.71-1.53 (m, 8H); C NMR (125 MHz, CDCl3) δ 175.1, 144.6, 144.3,
133.4, 129.3, 126.5, 122.9, 120.8, 45.9, 29.8, 26.8, 25.5, 22.8, 14.3; LC-MS: m/z: 456.0 [M+ +1].
Page 26 of 36

Compound 1y: Yield: 81%; 1H NMR (500 MHz, DMSO-d6) δ 10.42 (brs, 1H), 7.95 (d, 2H, J =
8.5 Hz), 7.87-7.86 (m, 3H), 7.74 (d, 2H, J = 9.0 Hz), 7.68 (t, 2H, J = 8.0 Hz), 7.61 (d, 2H, J = 7.0
Hz), 7.02 (d, 2H, J = 7.0 Hz); 13C NMR (125 MHz, CDCl3) δ 164.6, 144.6, 138.1, 136.6, 135.0,
134.2, 133.3, 129.8, 129.7, 128.5, 128.3, 122.4, 121.5; LC-MS: m/z: 388.0 [M+ +1].
Compound 1z: Yield: 78%; 1H NMR (500 MHz, DMSO-d6) δ 10.42 (brs, 1H), 7.96 (d, 2H, J =
8.5 Hz), 7.74 (m, 4H), 7.61 (d, 2H, J = 9.0 Hz), 7.48 (d, 2H, J = 8.0 Hz), 7.01 (d, 2H, J = 9.0
Hz), 2.43 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 164.5, 145.8, 144.6, 138.0, 136.6, 133.3,
131.4, 130.2, 129.6, 128.5, 128.3, 122.4, 121.5, 21.2; LC-MS: m/z: 402.0 [M+ +1].
1
Compound 1za: Yield: 76%; H NMR (500 MHz, DMSO-d6) δ 10.42 (brs, 1H), 7.98-7.95 (m,
2H), 7.81-7.79 (m, 4H), 7.76 (d, 2H, J = 5.0 Hz), 7.61 (d, 2H, J = 9.0 Hz), 7.04 (d, 2H, J = 10.0
13
Hz), 1.34 (s, 9H); C NMR (125 MHz, DMSO-d6) δ 164.5, 158.2, 144.6, 138.0, 136.6, 133.3,
131.5, 129.6, 128.5, 128.1, 126.7, 122.3, 121.4, 35.2, 30.7; LC-MS: m/z: 444.1 [M+ +1].
Compound 1zb: Yield: 85%; 1H NMR (500 MHz, DMSO-d6) δ 10.43 (brs, 1H), 7.96 (d, 2H, J =
8.5 Hz), 7.77 (d, 2H, J = 9.0 Hz), 7.60 (d, 2H, J = 8.5 Hz), 7.36 (s, 2H), 6.99 (d, 2H, J = 9.0 Hz),
13
3.96-3.90 (m, 2H), 3.01-2.96 (m, 1H), 1.23 (d, 6H, J = 7.0 Hz), 1.15 (d, 12H, J = 7.0 Hz); C
NMR (125 MHz, DMSO-d6) δ 164.5, 154.6, 150.7, 144.4, 138.0, 136.6, 133.3, 129.7, 128.8,
128.5, 124.1, 122.4, 121.6, 33.5, 29.4, 24.2, 23.3; LC-MS: m/z: 514.15 [M+ +1].
Compound 1zc: Yield: 88%; 1H NMR (500 MHz, DMSO-d6) δ 10.44 (brs, 1H), 8.09 (q, 4H, J =
8.5 Hz), 7.96 (d, 2H, J = 9.0 Hz), 7.77 (d, 2H, J = 9.0 Hz), 7.61 (d, 2H, J = 9.0 Hz), 7.07 (d, 2H,
13
J = 9.0 Hz); C NMR (125 MHz, DMSO-d6) δ 164.6, 144.3, 138.4, 138.2, 136.6, 134.5, 133.3,
129.7, 128.5, 127.1, 124.3, 122.4, 121.6; LC-MS: m/z: 406.0 [M+ +1].
1
Compound 1zd: Yield: 90%; H NMR (500 MHz, DMSO-d6) δ 10.43 (brs, 1H), 7.97-7.93 (m,
4H), 7.77 (d, 2H, J = 7.0 Hz), 7.61 (d, 2H, J = 5.0 Hz), 7.53 (t, 2H, J = 8.5 Hz), 7.04 (d, 2H, J =
13
7.0 Hz); C NMR (125 MHz, DMSO-d6) δ 166.6, 164.5, 144.4, 138.2, 136.6, 133.3, 131.7,
130.5, 129.6, 128.5, 122.4, 121.5, 117.3, 117.1; LC-MS: m/z: 456.0 [M+ +1].
1
Compound 1ze: Yield: 74%; H NMR (500 MHz, CDCl3) δ 8.38 (brs, 1H), 8.11 (s, 1H), 7.95-
7.88 (m, 4H), 7.78 (dd, 2H, J = 4.5 Hz, J = 6.5 Hz), 7.65-7.50 (m, 7H), 7.03 (dd, 2H, J = 2.0 Hz,
13
J = 7.0 Hz); C NMR (125 MHz, CDCl3) δ 165.9, 158.5, 145.9, 137.1, 135.1, 132.7, 132.3,
Page 27 of 36

131.9, 129.1, 129.0, 128.5, 128.2, 128.0, 127.8, 127.2, 126.3, 123.6, 123.2, 121.2; LC-MS: m/z:
404.97 [M+ +1].
1
Compound 1zf: Yield: 81%; H NMR (500 MHz, CDCl3) δ 8.39 (brs, 1H), 8.11 (s, 1H), 7.94-
7.87 (m, 4H), 7.78 (dd, 2H, J = 4.5 Hz, J = 6.5 Hz), 7.67-7.51 (m, 6H), 7.04 (dd, 2H, J = 2.0 Hz,
13
J = 7.0 Hz), 2.43 (s, 3H); C NMR (125 MHz, CDCl3) δ 165.9, 158.5, 145.9, 137.1, 135.1,
132.7, 132.3, 131.9, 129.1, 129.0, 128.5, 128.2, 128.0, 127.8, 127.2, 126.3, 123.6, 123.2, 121.2,
21.2; LC-MS: m/z: 418.08 [M+ +1].
1
Compound 1zg: Yield: 85%; H NMR (500 MHz, CDCl3) δ 8.35 (brs, 1H), 8.10 (s, 1H), 7.93-
7.87 (m, 4H), 7.76 (dd, 2H, J = 4.5 Hz, J = 6.5 Hz), 7.65-7.51 (m, 6H), 7.01 (dd, 2H, J = 2.0 Hz,
13
J = 7.0 Hz), 1.35 (s, 9H); C NMR (125 MHz, CDCl3) δ 165.9, 158.5, 145.9, 137.1, 135.1,
132.7, 132.3, 131.9, 129.1, 129.0, 128.5, 128.2, 128.0, 127.8, 127.2, 126.3, 123.6, 123.2, 121.2,
35.5, 31.2; LC-MS: m/z: 460.19 [M+ +1].
1
Compound 1zh: Yield: 78%; H NMR (500 MHz, CDCl3) δ 8.35 (brs, 1H), 8.00 (s, 1H), 7.94
(dd, 2H, J = 5.5 Hz, J = 7.0 Hz), 7.93-7.88 (m, 2H), 7.65 (d, 2H, J = 10.5 Hz), 7.63-7.55 (m,
2H), 7.21 (s, 2H), 7.04-7.01 (m, 2H), 4.12-4.07 (m, 2H), 2.97-2.92 (m, 1H), 1.28 (d, 6H, J = 7.0
Hz), 1.21 (d, 12H, J = 7.0 Hz); 13C NMR (125 MHz, CDCl3) δ 165.8, 154.5, 151.4, 145.9, 136.9,
135.1, 132.7, 131.9, 129.8, 129.1, 129.0, 128.2, 128.0, 127.7, 127.2, 124.1, 123.5, 123.3, 121.2,
34.4, 30.0, 24.8, 23.7; LC-MS: m/z: 530.19 [M+ +1].
1
Compound 1zi: Yield: 87%; H NMR (500 MHz, DMSO-d6) δ 10.60 (brs, 1H), 8.56 (s, 1H),
13
8.12-8.02 (m, 8H), 7.84 (d, 2H, J = 9.0 Hz), 7.67-7.63 (m, 2H), 7.13 (d, 2H, J = 9.0 Hz); C
NMR (125 MHz, DMSO-d6) δ 165.8, 144.3, 138.5, 138.1, 134.3, 134.2, 134.1, 132.0, 131.8,
129.2, 129.0, 128.0, 127.7, 127.5, 127.0, 126.6, 124.3; LC-MS: m/z: 422.03 [M+ +1].
1
Compound 1zj: Yield: 91%; H NMR (500 MHz, DMSO-d6) δ 10.58 (brs, 1H), 8.57 (s, 1H),
13
8.13-8.00 (m, 8H), 7.86 (d, 2H, J = 9.0 Hz), 7.65-7.62 (m, 2H), 7.11 (d, 2H, J = 9.0 Hz); C
NMR (125 MHz, DMSO-d6) δ 165.7, 144.2, 138.6, 138.2, 134.4, 134.3, 134.2, 132.0, 131.9,
129.4, 129.0, 128.1, 127.9, 127.7, 127.1, 127.0, 126.9, 124.4; LC-MS: m/z: 472.14 [M+ +1].
1
Compound 1zk: Yield: 43%; H NMR (500 MHz, DMSO-d6) δ 8.41 (brs, 1H), 8.14 (s, 1H),
7.98-7.89 (m, 7H), 7.82 (dd, 2H, J = 4.5 Hz, J = 6.5 Hz), 7.68-7.53 (m, 7H), 7.06 (dd, 2H, J =
Page 28 of 36

2.0 Hz, J = 7.0 Hz); 13C NMR (125 MHz, DMSO-d6) δ 166.0, 158.5, 145.9, 137.1, 135.1, 133.6,
134.4, 132.7, 132.4, 131.9, 129.2, 129.0, 128.8, 128.5, 128.2, 128.0, 127.9, 127.3, 126.4, 123.7,
123.3, 121.3; LC-MS: m/z: 455.24 [M+ +1].
4.4.
Nucleotide pyrophosphatase/ phosphodiesterase inhibition assays
The inhibition potential of all the sulfonate derivatives against NPP1, 2, and 3 was
determined by considering already reported colorimetric method54,55 with minor modifications.
The reaction buffer was comprised of 50 mM Tris-hydrochloric acid, 5 mM magnesium chloride
(MgCl2) and 0.1 mM zinc chloride (ZnCl2) with final pH 9.5. For initial screening of
compounds, enzyme and substrate parameters (concentration, temperature, time) were first
optimized. The reaction assay was performed at 100 µL well volume containing assay buffer, 10
µL of 1 mM test compound prepared in 10% DMSO, enzymes NPP1 (35 ng), NPP2 (338 ng), or
o
NPP3 (37 ng) and assay buffer. The reaction mixture was incubated at 37 C, 10 minutes for
NPP1 and 3 and 5 minutes for NPP2. Absorbance was measured at wavelength of 405 nm as pre-
read by using microplate reader (BioTek FLx800, Instruments, Inc. USA). After pre-read
artificial substrate p-nitrophenyl 5'-thymidine monophosphate (pNP-TMP), 400 µM for NPP1,
500 µM for NPP2, or 600 µM in case of NPP3, was added followed by second incubation at 37
oC, 15 minutes for NPP1, 3 and 35 minutes for NPP2. Absorbance was measured as after read.
All the experiments were performed in triplicates. Compounds exhibiting more than 50%
inhibition against either of NPP1, NPP2, and NPP3 were further subjected to serial dilutions for
the determination of IC50 values, using non-linear curve fitting program PRISM 5.0 (Graph Pad,
San Diego, California, USA).
4.5.
Molecular docking studies
4.5.1. Selection of protein structure
Due to the unavailability of crystal structure of the human nucleotide pyrophosphatases
(NPP1), the docking studies were performed in the homology model generated by our group
using mouse ENPP1 with PDB ID: 4B56 as template.48,50 However, the human crystal structures
of NPP2 and NPP3 were downloaded from protein data bank with the PDB IDs 4ZG651 and
Page 29 of 36