Coumarin sulfonates: New alkaline phosphatase inhibitors; in vitro and in silico studies

Article abstract of DOI:10.1016/j.ejmech.2017.03.003

A library of coumarin derived sulfonyl esters (1–38) was synthesized by reacting various hydroxy coumarins with different alkyl and aryl sulfonyl chlorides. All compounds were evaluated for their potential to inhibit alkaline phosphatases (hTNAP and hIAP)

Full text of DOI:10.1016/j.ejmech.2017.03.003

Accepted Manuscript
Coumarin sulfonates: New alkaline phosphatase inhibitors; in vitro and in silico
studies
Uzma Salar, Khalid Mohammed Khan, Jamshed Iqbal, Syeda Abida Ejaz, Abdul
Hameed, Mariya al-Rashida, Shahnaz Perveen, Muhammad Nawaz Tahir
PII:
S0223-5234(17)30152-6
10.1016/j.ejmech.2017.03.003
EJMECH 9263
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 October 2016
Revised Date: 1 March 2017
Accepted Date: 3 March 2017
Please cite this article as: U. Salar, K.M. Khan, J. Iqbal, S.A. Ejaz, A. Hameed, M. al-Rashida, S.
Perveen, M.N. Tahir, Coumarin sulfonates: New alkaline phosphatase inhibitors; in vitro and in silico
studies, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.03.003.
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ACCEPTED MANUSCRIPT

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Coumarin Sulfonates: New Alkaline Phosphatase Inhibitors; In Vitro and In
Silico Studies
a
*a
*b
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Uzma Salar, Khalid Mohammed Khan, Jamshed Iqbal, Syeda Abida Ejaz, Abdul Hameed,
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Mariya al-Rashida, Shahnaz Perveen and Muhammad Nawaz Tahir
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H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
b
Centre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabad 22060, Pakistan
Department of Chemistry, Forman Christian College (A Chartered University), Ferozepur
Road, Lahore-54600, Pakistan
c
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PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280,
Pakistan
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Department of Physics, University of Sargodha, Sargodha, Pakistan
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Abstract: A library of coumarin derived sulfonyl esters (1-38) was synthesized by reacting
various hydroxy coumarins with different alkyl and aryl sulfonyl chlorides. All compounds were
evaluated for their potential to inhibit alkaline phosphatases (hTNAP and hIAP). Most of the
compounds were found to be inhibitors of APs. Compound 20 was found to be the most active
hIAP inhibitor (IC = 1.11 ± 0.15 µM), whereas, compound 13 was found to be the most active
5
0
hTNAP inhibitor (IC = 0.58 ± 0.17 µM). Detailed structure activity relationship studies (SAR),
5
0
and molecular docking studies were carried out to identify structural elements necessary for AP
inhibition, in addition to rationalize most probable binding site interaction of the inhibitors with
the AP enzymes.
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Keywords: Coumarins, Sulfonates, Inhibitory activity, Alkaline Phosphatase, SAR, Docking
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studies
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*
Corresponding Authors: K. M. Khan, khalid.khan@iccs.edu, drkhalidhej@gmail.com; Tel. 00922134824910; Fax.
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0922134819018 / J. Iqbal, drjamshed@ciit.net.pk, jamshediqb@googlemail.com; Tel. 00923365101651, 0092-
0
992-383591-5; Fax. 0092-0992-383441
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Introduction
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Alkaline phosphatases (APs, E.C. 3.1.3.1) belong to a broad family of ectonucleotidase enzymes
that are responsible for catalytic dephosphorylation of nucleoside phosphates [1]. However, APs
are promiscuous enzymes and are capable of hydrolyzing a variety of phosphomonoesters along
with other phosphate containing compounds, such as, inorganic pyrophosphate (PPi) and
polyphosphates, glucose phosphate, and phosphatidates [2,3]. APs are also known to catalyze
certain transphosphorylation reactions, and exhibit optimum function at an alkaline pH.
Adenosine monophosphate (AMP) is an important substrate of AP, its dephosphorylation results
in the release of adenosine, an important signaling molecule that is also implicated in purinergic
cell-signaling. Adenosine modulates a variety of immunological, cardiovascular, inflammatory
and angiogenic responses [4-7]. In APs ectoenzymes catalytic active site is located on the
external cell surface via a glycosylphosphatidyl inositol (GPI) anchor. This makes them ideal
drug targets, since potential drug molecules do not necessarily have to be cell-permeable in order
to elicit the desired physiological effect via AP inhibition [4,8]
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There are two types of APs, tissue non-specific alkaline phosphatase (TNAP), and tissue specific
alkaline phosphatase which are further divided in three types; germ-cell alkaline phosphatase
(GCAP), intestinal alkaline phosphatase (IAP), and placental alkaline phosphatase (PLAP) [1,9].
Tissue-nonspecific alkaline phosphatase (TNAP) has been found in variety of tissues such as
kidney, liver, bone, and brain [10]. High concentrations of TNAP have also been found in
mineralizing tissues such as bones and teeth, where role of TNAP is essential for normal bone
formation [11]. Over-expression of TNAP has been known to cause abnormal calcification
including vascular calcification [12,13] and leads to increased deposition of mineral
hydroxyapatite causing hydroxyapatite deposition disorder (HADD) [14,15]. A decrease in
concentration of extracellular ATP, via TNAP inhibition has been demonstrated to be essential
for development of neuritic processes of the neurons [16]. An important role for TNAP has been
suggested in neuronal development and synaptic function [17]. A number of Alzheimer’s disease
patients have indicated considerable elevated TNAP activity in the hippocampus section of brain
[18]. TNAP can therefore be explored as a potential target for treatment of neurological
disorders.
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The role of coumarin scaffold in medicinal chemistry is well established due to its broad range of
therapeutic activities. Over the years a number of different coumarin based molecules, both
natural and synthetic, have been reported for their different bioactivities [19]. Warfarin is a
coumarin based drug which prolonged the survival of cancer patients [20]. The strategy to link,
or fuse other heterocyclic moieties with coumarin template has also been widely explored in
search of new lead molecules as potential drug candidates [19,21]. Sulfonyl group, either as
sulfonamide (-SO NH-R) or sulfonate (-OSO -R), is a biologically active pharmacophore that
2
2
has been a part of many clinically used drugs [22,23]. Although, less commonly encountered in
natural products isolated from plant sources, the sulfonyl moiety is an integral part of a fairly
large number of synthetic drugs commercially available, for example COX-2 inhibitor,
®
ampiroxicam (Flucam ) (Figure 1). Our research group has already reported sulfonamide
derivatives as potent and selective inhibitors of APs [24,25]. We wanted to investigate if
sulfonate containing compounds also be able to inhibit AP. Accordingly a library of thirty eight
compounds has been prepared and evaluated as alkaline phosphate (hIAP, hTNAP) inhibitors.
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9
0
7
Figure 1: Coumarin (Warfarin) and sulfonyl group (Ampiroxicam) based drugs
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A simple and less expensive method was adopted in which different substituted hydroxyl
coumarins were reacted with various substituted phenyl or alkyl sulfonyl chlorides to get the
desired sulfonates (1-38) (Scheme 1).
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4
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Results and Discussion
Chemistry
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Different sulfonyl chlorides underwent nucleophilic substitution reactions with a variety of
hydroxy substituted coumarin derivatives, such as 7-hydroxy-4-methylcoumarin, 7-hydroxy-4-
methyl-8-nitrocoumarin,
3-chloro-7-hydroxy-4-methylcoumarin,
3-cyano-7-hydroxy-4-
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methylcoumarin, and 7,8-dihydroxy-4-methylcoumarin in tetrahydrofuran (THF) in the presence
of triethyl amine (Scheme 1). Reaction mixture was stirred at room temperature for 4 h. Progress
of reaction was monitored via TLC. On completion of reaction, THF was evaporated to obtain
the solid product which was extensively washed with distilled water to remove triethyl
ammonium chloride salt to afford pure desired coumarin sulfonate esters 1-38 in good yields
(Table 1). The structures of the synthetic compounds 1-38 were confirmed with the help of
1
13
different spectroscopic techniques such as EI-MS, HREI-MS, H-NMR and C-NMR. Crystal
structure of compound 31 also confirmed the formation of the desired compounds (Figure 2).
The 7-hydroxy-4-methyl-2H-chromen-2-one group A (C1-C10/O1/O2/O5) and n-butyl group B
(C11-C14) are planar with r. m. s. deviation of 0.0626 and 0.0039 Å, respectively. The nitro
group C (N1/O3/O4) and sulfonyl group D (S1/O5/O6) is of course planar. The dihedral angle
between A/B, A/C, A/D, B/C, B/D and C/D is 48.19 (14)°, 63.13 (15)°, 70.43 (9)°, 69.04 (26)°,
71.06 (20)°, and 81.92 (23)°, respectively. Their exist four C-H…O interactions of each
molecule with three H-atoms from two adjacent CH
2
groups of n-butyl and one from aromatic
1
CH group. The carbonyl group of heterocyclic ring completes R (8) ring as shown in Figure 2a.
2
The molecules are overall stabilized in the form infinite 3-dimensional network with these H-
interactions.
R5 R4
R5 R4
O
O O
S
R6
R3
O
R6
R3
O O
S
R S
THF, TEA, r.t.
O
O
R
+
R
Cl
R7
O
O
R7
O
O
R8
R8
(
1-38)
7
0-90% Yields
9
6
7
9
Scheme 1: Synthesis of coumarin based sulfonate esters (1-38)
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Figure 2: Crystal structure of compound 31.
1
1
Note: The thermal ellipsoids are drawn at 50% probability level. The H-atoms shown as small circles of arbitrary
radii.
1
02
1
1
03
04
Figure 2a: Three dimensional network of compound 3I with hydrogen bond interactions
Note: The H-atoms not involved in H-interactions are omitted for clarity.
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Characteristic Spectral Feature of Representative Compound 20
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13
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1
1
1
1
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H- and C-NMR spectra of compound 20 were recorded in deuterated DMSO-d
6
at 300 MHz
1
machine. In H-NMR spectrum, H-3ʹ/5ʹ and H-2ʹ/6ʹ were resonated at δH 8.47 (d, J3',2'/5',6' = 9.0
Hz) and δH 8.22 (d, J2',3'/6',5' = 9.0 Hz), respectively. H-5 and H-8 were appeared as doublet at δH
8.04 (d, J5,6 = 8.7 Hz) and δH 7.41 (d, J8,6 = 2.1 Hz), respectively. H-6 resonated as double
doublet at δH 7.25 (dd, J = 2.1 Hz, J = 8.7 Hz) (Figure 3).
6,8
6,5
1
1
11
12
1
Figure 3: H-NMR chemical shifts of compound 20
1
3
1
1
13
14
C-NMR broad-band decoupled spectrum (DMSO-d ) showed total 15 carbon signals including
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one methyl, seven methine and nine quarternary carbons (Figure 4).
1
1
15
16
1
3
Figure 4: C-NMR chemical shifts of compound 20
+
1
1
1
1
1
1
1
17
18
19
20
21
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23
EI-MS of compound 20 displayed the molecular ion peak [M] at m/z 386.1. HREI-MS showed
+
the [M] at 386.0221, corresponding to the formula C H N O S calcd (386.0209). However,
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7
10
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the fragmentation pattern observed in the EI-MS spectrum confirmed the structure of the desired
compound. First molecular ion observed at m/z 386.1 undergoes the homolytic cleavage to afford
a radical cation at m/z 201.1 by the neutral loss of 2-nitrocyclohexa-1,3-dien-5-yne molecule.
Then radical cation m/z 201.1 further cleaves to afford another radical cation observed at m/z 173
by another neutral loss of carbon monoxide. In other way, molecular ion undergoes a heterolytic
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cleavage to give a 4-nitrobenzene sulfonium cation at m/z 186 by the loss of a radical. Then 4-
nitrobenzene sulfonium cation m/z 186 cleaves to afford 4-nitrobenzene-1-ylium cation at m/z
122.0 (Figure 5).
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1
27
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Figure 5: Key EI-MS fragmentation of compound 20.
Alkaline Phosphatase Inhibition and Structure-Activity Relationship (SAR)
hIAP Inhibition
1
1
29
30
1
1
1
1
1
1
31
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34
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All synthesized compounds 1-38 were investigated for their potential to inhibit human intestinal
alkaline phosphatase (hIAP) and human tissue non-specific alkaline phosphatase (hTNAP).
Levamisole (IC50 = 20.21 ± 1.9 µM) and L-phenylalanine (IC50 = 100.1 ± 3.15 µM) were used as
standards for human intestinal alkaline phosphatase (hIAP) and human tissue non-specific
alkaline phosphatase (hTNAP), respectively. Except few derivatives, all compounds showed
promising and potent hIAP and hTNAP inhibitory activity as compared to standards Table 1.
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Table 1: In vitro alkaline phosphatase (hIAP and hTNAP) inhibition activities of coumarin sulfonate esters 1-38
1
38
hIAP
IC (µM) ± SEM )
hTNAP
IC (µM) ± SEM )
Comp. No.
R
R
3
R
4
R
5
R
6
R
7
R
8
a
a
50
50
1
2
3
4
5
6
7
8
9
Ph
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
5.52 ± 0.67
10.3 ± 1.26
1.42 ± 0.02
9.01 ± 0.94
1.25 ± 0.07
6.38 ± 0.78
17.6 ± 2.06
1.64 ± 0.12
2.56 ± 0.46
2.51 ± 0.26
1.83 ± 0.11
9.37 ± 0.69
3.53 ± 0.22
0.58 ± 0.17
1.07 ± 0.04
2.52 ± 0.31
2.42 ± 0.32
9.23 ± 0.46
0.85±0.03
4'-Cl-3'-NO
Ph
4.06 ± 0.12
2.13 ± 0.17
5.49 ± 0.59
13.1 ± 1.43
-
3.22 ± 0.43
3.12 ± 0.76
3.58 ± 0.77
-
4.18 ± 0.14
-
3.59 ± 0.76
-
7.03 ± 0.99
5.16 ± 0.36
4.63 ± 0.27
6.29 ± 0.33
2.81 ± 0.11
1.11 ± 0.15
-
2
4'-OMePh
2'-NO Ph
4'-ClPh
4'-nPrPh
n-Bu
n-Oct
Me
2',5'-diClPh
Et
2
1
1
1
1
1
1
1
1
1
1
2
2
0
1
2
3
4
5
6
7
8
9
0
1
Cl
Cl
Ph
4'-OMePh
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
2'-NO
4'-ClPh
3'-NO Ph
4'-nPrPh
n-Bu
n-Oct
4'-NO
Me
Ph
2
2
0.92±0.04
1.33±0.23
1.99 ± 0.56
2
Ph
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3
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3
3
3
3
3
3
3
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
2',5'-diClPh
CN
CN
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
OH
H
OH
OH
OH
H
2.26 ± 0.19
4.06 ± 0.27
-
2.72 ± 0.19
-
4.57 ± 0.59
20.6 ± 2.67
3.91 ± 0.11
5.95 ± 0.48
-
2.47 ± 0.23
-
Et
Ph
7.36 ± 0.56
2.31 ± 0.12
2.59 ± 0.23
1.17 ± 0.14
3.69 ± 0.16
6.59 ± 0.87
-
4'-Cl-3'-NO
4'-OMePh
4'-NO Ph
Ph
2
2
4'-ClPh
2'-Me-5'-NO
4'-nPrPh
n-Bu
Ph
2
OH
NO
NO
-
2
n-Oct
Me
2
3.06 ± 0.47
-
2.54 ± 0.17
2.16 ± 0.14
7.71 ± 1.09
1.33 ± 0.04
8.37 ± 0.45
0.92 ± 0.04
1.94 ± 0.13
OH
4'-Cl-3'-NO
2
Ph
NO
OH
H
OH
OH
2
4.65 ± 0.58
3.21 ± 0.21
1.33 ± 0.06
10.3 ± 0.99
2.87 ± 0.37
20.21 ± 1.9
2',5'-diClPh
2',4',5'-triClPh
n-Oct
bEt
Levamisole
c
L-Phenylalanine
100.1 ± 3.15
a
b
c
1
1
39
40
SEM (Standard Error Mean); Levamisole (Standard Inhibitor for hIAP); L-Phenylalanine (Standard Inhibitor for hTNAP)
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Detailed structure-activity relationship (SAR) was rationalized in order to identify structural
elements necessary for inhibition. Although all structural features are cordially taking part in the
inhibitory activity but variation of “R” on main scaffold is responsible for variation in inhibitory
potential. Since, all compounds have a common 4-methyl coumarin scaffold in their structures,
the observed SAR is mainly due to the variation of other functionalities on this scaffold.
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In compounds containing 3-CN and 7-sulfonate (-OSO -R) substituents on the coumarin ring,
2
compound 20 (-OSO
2
-4'-NO
2
Ph) having a 4'-NO
2
substituent on phenyl ring “R”, was found to
M). Compound 20 was eighteen fold more
active than the standard inhibitor levamisole (IC50 = 20.21 ± 1.9 M). Upon changing the
be the most active hIAP inhibitor (IC = 1.11 ± 0.15
µ
5
0
µ
position of NO group from position 4' to 3', as in compound 16 (-OSO -3'-NO Ph), almost five
2
2
2
times decreased hIAP inhibition was observed (IC = 5.16 ± 0.36
µM). Similarly, when the nitro
50
group was switched to position 2', as in compound 14 (-OSO
2
-2'-NO Ph), the hIAP inhibitory
2
activity was completely lost. Comparison of docked conformations of most active compound 20,
and inactive compound 14 revealed different mode of binding for compound 14 as compared to
compound 20 which did not involve a direct binding of sulfonate group with the Zn metal ion,
instead the coumarin ring was oriented towards the Zn metallic center with significantly weak
binding affinity (Figure S1 in SI). The hIAP inhibition was restored in compound 4 (IC50 = 5.49
± 0.59
molecule 14. This indicates that chloro group plays a major part in reinstating the inhibitory
activity. 7,8-Disulfonated compound 27 (IC50 = 4.57 ± 0.59 M) having 4'- and 4''-NO2
µM) having a 3-Cl substituent instead of 3-CN group as compared to its closely related
µ
substituents on both phenyl rings, also showed potent hIAP inhibitory activity (Figure 6).
According to the molecular docking studies, the bulky nature of this compound hindered access
of sulfonate group to the catalytic Zn center, instead the more easily accessible terminal nitro
group was oriented toward the catalytic metal center (Figure S2 in SI).
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Figure 6: Structure-activity relationship of compound 20 with 16, 14, 4, and 27
1
1
1
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1
1
1
1
1
1
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Compound 25 (-OSO -4'-Cl-3'NO Ph; IC = 2.72 ± 0.19 µM) with 4'-Cl-3'-NO groups on ring
2 2 50 2
“R”, showed good inhibitory potential as compared to standard levamisole. Structurally similar
compound 2 (-OSO -4'-Cl-3'-NO Ph; IC50 = 4.06 ± 0.12 M) with an additional 3-Cl group
showed slightly diminished inhibitory activity. Similarly another compound 34 (-OSO -4'-Cl-3'-
2
2
µ
2
2 2
NO Ph; IC50 = 4.65 ± 0.58 µM) with an additional 8-NO group at coumarin scaffold showed
almost similar activity as 2 (Figure 7). Figures S3-S5 in SI showed most probable docked
conformations of compounds 2, 25 and 34, respectively. All three compounds have inhibitory
activities that are not very different from one another, as can be seen from their similar binding
modes. The most important interactions were found to be hydrogen bonded interaction between
the sulfonate group and Arg185, while the terminal nitro group was in direct contact with the
catalytic Zn ion.
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Figure 7: Structure-activity relationship of compound 25 with 2 and 34
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1
1
1
1
1
1
1
1
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Compounds having 3-CN and 7-sulfonate moieties such as 15 (-OSO
0.99 M), with 4'-Cl substitution on phenyl ring “R” was also found to be a good hIAP inhibitor.
Upon replacement of 3-CN with 4-Cl, as in compound 5 (IC50 = 13.1 ± 1.43 M), a diminished
activity was observed. However, disulfonated compound 28 having 4',4''-Cl substitution (IC =
2
-4'-ClPh; IC50 = 7.03 ±
µ
µ
5
0
20.6 ± 2.64
µM), displayed inhibition comparable to the standard levamisole (IC = 20.21 ± 1.9
50
µ
M) (Figure 8). Molecular docking analysis of compound 15 revealed favorable interaction of
CN group with amino acid residue Thr450 which may explain its good inhibitory activity as
compared to 5 and 28, where in the absence of CN group this interaction was lost (Figures S6-
S8).
1
1
90
91
Figure 8: Structure-activity relationship of compound 15 with 5 and 28
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1
1
1
1
1
1
1
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95
96
97
98
99
Compound 36 (-OSO
was found to be one of the most potent hIAP inhibitor. Absence of 4'-Cl substitution on ring “R”,
in case of compound 22 (-OSO -2',5'-diClPh; IC = 2.26 ± 0.19 M), resulted in almost half of
2
-2',4',5'-triClPh; IC50 = 1.33 ± 0.06 µM) having 3-Cl, 7-sulfonate groups,
µ
2
50
the hIAP inhibitory activity. Its structurally similar analog 10 which has 3-Cl instead of 3-CN
was found to be inactive, possibly due to change in conformation of 10 which did not fit
favorably in the active site of enzyme as shown in Figure S9. However, disulfonated compound
35 (IC50 = 3.21 ± 0.21
µM) having 2',5'- and 2'',5''-di-Cl substitutions was found to be a good
hIAP inhibitor (Figure 9).
12

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2
00
01
Figure 9: Structure-activity relationship of compound 36 with 22, 10, and 35
2
2
2
2
2
2
2
02
03
04
05
06
07
08
Compound 3 (-OSO -4'-OMePh; IC = 2.13 ± 0.17
µ
M), having 4'-methoxy on phenyl ring (R),
also exhibited good inhibitory activity. Upon replacement of 3-Cl substituent with 3-CN as in
compound 13 (IC50 = 3.59 ± 0.76 M), a slight decline in the hIAP inhibitory activity was
2
50
µ
observed. However, among the 7,8-disulfonated compounds, compound 26 with 4'- and 4''-OMe
substituents on phenyl ring was found to be inactive (Figure 10). Due to the bulky conformation
of this molecule, it does not fit favorably inside the active site of the enzyme, instead it was
found to bind slightly away from the active site (Figure S10).
2
2
09
10
Figure 10: Structure-activity relationship of compound 3 with 26 and 13
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2
2
2
2
2
2
2
2
2
2
11
12
13
14
15
16
17
18
19
20
21
Among the 7-alkyl sulfonates (-OSO
2
-R), derivative 19 with 3-CN group and n-octyl substitution
M), was found to be seven-fold more active than the
M). Inhibitory activity was slightly decrease on
shortening of chain length, as compound 23 having ethyl group (-OSO -Et; IC50 = 4.06 ± 0.27
M) followed by n-butyl substituted compound 18 (-OSO -But; IC = 6.29 ± 0.33 M). On
-Me), complete loss of activity was
at ring “R” (-OSO -Oct; IC = 2.81 ± 0.11
µ
2
50
standard levamisole (IC50 = 20.21 ± 1.9
µ
2
µ
µ
2
50
further shortening of chain length as in compound 21 (-OSO
2
observed (Figure 11). Molecular docking analysis revealed that smaller methyl group next to the
sulfonate group makes the sulfonate oxygen more accessible for binding via hydrogen bond to
amino acid residues Thr450 and His339, these interactions preventing this molecule for the same
binding conformation showed by compounds 18, 19, and 23 where these molecules fit tightly
into the active site (Figure S11).
2
2
22
23
Figure 11: Structure-activity relationship of compound 19 with 18, 23, and 21
2
2
2
2
2
2
24
25
26
27
28
29
Compounds with 3-Cl and 7-alkyl sulfonate (-OSO
2
-R) groups on coumarin ring also exhibited
M) was
M), followed
M), 9 (-OSO -Me; IC = 3.58 ± 0.77 M) and
µM) (Figure 12). All these compounds had almost comparable
good hIAP inhibitory activity. Such as compound 8 (-OSO -Oct; IC = 3.12 ± 0.76
µ
2
50
observed to be six-fold more active than standard levamisole (IC50 = 20.21 ± 1.9
by compounds 7 (-OSO -Bu; IC = 3.22 ± 0.43
µ
µ
µ
2
50
2
50
11 (-OSO -Et; IC = 4.18 ± 0.14
2
50
binding affinities.
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2
30
31
Figure 12: Structure-activity relationship of compound 8 with 7, 11, and 9
2
2
2
2
2
2
32
33
34
35
36
37
In compounds containing 8-nitro and 7-sulfonate substituents on the coumarin ring, compound
32, containing n-octyl group (-OSO -Oct; IC = 3.06 ± 0.47 µM), was identified as a much
2
50
better inhibitor compared to levamisole. Whereas, compound 31 with n-butyl group (-OSO -n-
2
But) was found to be inactive (Figure 13). Molecular docking analysis revealed that inactive
compound 31 does not fit tightly into the active site and a large portion of the active site cavity
remains empty (Figure S12).
2
2
38
39
Figure 13: Structure-activity relationship of compound 32 with 31
2
2
2
40
41
42
Among the 7,8-disulfonated compounds such as 33, 37 and 38, compound 38 having ethyl group
as R (-OSO
2
-Et; IC50 = 2.87 ± 0.37
µ
M) was found to be a good hIAP inhibitor. Replacement of
M),
ethyl group with n-octyl residue, as in compound 37 (-OSO -Oct; IC = 10.3 ± 0.99
µ
2
50
15

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2
43
44
resulted in more than four-fold decrease in hIAP inhibitory activity, as compared to 38.
Similarly, compound 33 having methyl as “R”, was found to be completely inactive (Figure 14).
2
2
45
46
Figure 14: Structure-activity relationship of compound 38 with 37 and 33.
hTNAP Inhibition
2
47
2
2
2
2
2
2
2
2
2
2
48
49
50
51
52
53
54
55
56
57
Except compounds 23, 30 and 31, all derivatives were found to have many fold better inhibitory
activity for hTNAP as compared to the standard L-phenylalanine (IC50 = 100.1 ± 3.15
Compound 13 (-OSO -4'-OMePh; IC = 0.58 ± 0.17 M) with 3-CN and 4'-OMe substitutions,
was found to be more than hundred-fold better hTNAP inhibitor than L-phenylalanine.
Structurally similar compound 3 (-OSO -4'-OMePh; IC = 9.01 ± 0.94 M) having a 3-Cl
instead of a 3-CN group, was found to have decreased activity than 13, but was still ten times
better inhibitor than standard. 4'- and 4''-di-OMe substituted disulfonate derivative 26 (IC50
2.59 ± 0.23 M) was also found to possess good hTNAP inhibitory activity (Figure 15). Figures
µM).
µ
2
50
µ
2
50
=
µ
S13-S15 give comparison of most probable docked conformations of compounds 13, 26, and 3,
respectively.
2
58
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59
2
2
60
61
Figure 15: Structure-activity relationship of compound 13 with 3 and 26
2
2
2
2
2
2
2
2
2
2
2
2
2
62
63
64
65
66
67
68
69
70
71
72
73
74
Alkyl sulfonate substituted coumarins having 3-CN substitution also showed potent inhibitory
activity. The second most active hTNAP inhibitor was an n-butyl substituted derivative 18 (-
OSO
activity than standard L-phenylalanine. It was observed that extension of chain length as in case
of 19 (-OSO -Oct; IC = 0.92 ± 0.04 M) brings slight decline in the activity. Compound 21 (-
M) with 7-methyl sulfonate substitution showed good hTNAP
2
-n-But; IC50 = 0.85 ± 0.03 µM), which also demonstrated more than hundred-fold better
µ
2
50
2
OSO -Me; IC50 = 1.99 ± 0.56 µ
inhibitory activity too, however replacement of methyl sulfonate with ethyl sulfonate as in
compound 23 makes compound inactive (Figure 16). Comparison of docked conformations of
these compounds revealed different orientation for compound 23 as compared to compounds 19,
18, and 21 (Figures S16-S19). The common binding features observed for compounds 19, 18,
2
+
and 21 were the binding of sulfonate oxygen to Zn ion and hydrogen bonded interactions of
remaining sulfonate oxygens to Ser93 and Arg167. These interactions were missing for
compound 23.
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2
75
76
Figure 16: Structure-activity relationship of compound 18 with 19, 21, and 23
2
2
2
2
2
2
2
2
2
2
77
78
79
80
81
82
83
84
85
86
Another, alkyl sulfonate derivative 7 (-OSO -n-But; IC = 1.64 ± 0.12 µM) has similar structure
2 50
as compound 18 but have 3-Cl instead of 3-CN group; this compound 18 demonstrated sixty-fold
enhanced activity as compare to the standard L-phenylalanine. Extension of chain length, slightly
decreases the hTNAP inhibitory potential as observed in case of compound 8 (-OSO -Oct; IC =
2
50
2.56 ± 0.46
potential as 8. However, replacement of methyl sulfonate with ethyl sulfonate as in compound 11
(-OSO -Et; IC = 9.37 ± 0.69 M) showed decreased activity, however, it is worth noting that
µM). Compound 9 (-OSO -Me; IC = 2.51 ± 0.26 µM) also showed almost similar
2 50
µ
2
50
all compounds are still much more potent than standard L-phenylalanine (Figure 17).
Comparison of docked conformations of compounds 7, 8, 9, and 11 are given in Figures S20-
S23.
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2
87
88
Figure 17: Structure-activity relationship of compound 7 with 8, 11, and 9
2
2
2
2
2
2
89
90
91
92
93
94
Among the disulfonate derivatives, 4'- and 4''-di-Oct substituted derivative 37 (IC50 = 2.59 ± 0.23
µ
M) was found to be the third most potent derivative. Replacement of octyl substitution with
ethyl sulfonate derivative 38 (-OSO -Et; IC = 1.94 ± 0.13
µ
M) and methyl sulfonate derivative
2
50
33 (-OSO -Me; IC = 2.16 ± 0.14
µ
M) showed almost twice decrease in the inhibitory activity
2
50
of hTNAP (Figure 18). Comparison of docked conformations of compounds 38, 3, and 33 are
given in Figures S23-S26. All compounds showed similar binding orientation.
2
2
95
96
Figure 18: Structure-activity relationship of compound 37 with 38 and 33
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3
3
3
3
3
3
3
97
98
99
00
01
02
03
04
05
06
07
08
2 2
Among the 3-cyano and 7-sulfonate substituted coumarins, compound 14 (-OSO -2'-NO Ph; IC50
= 1.07 ± 0.04
µM) with 2'-NO group showed hundred-fold enhanced activity as compared to L-
2
phenylalanine (IC = 100.1 ± 3.15
µ
M). Switching of NO group from position-2' to 4' and 3',
2
5
0
showed slight decline in the activity, as in compounds 20 (-OSO
2
-4'-NO
M), respectively. However, compound 4 (-
M) having a 3-Cl instead of a 3-CN group, showed almost
2
Ph; IC50 = 1.33 ± 0.23
µ
M) and 16 (-OSO -3'-NO Ph; IC = 2.42 ± 0.32 µ
2 2 50
OSO
2
-2'-NO
2
Ph; IC50 = 1.25 ± 0.07
µ
similar activity as 14. Similarly, disulfonate derivative with 4'- and 4''-NO such as 27 (IC
=
50
2
1.17 ± 0.14 µM) also showed potent activity (Figure 19). Detailed molecular docking analysis of
these compounds (Figures S27-S31) revealed similar binding orientations, the most characteristic
binding features observed for these compounds were the binding of oxygen atom of nitro group
2
+
to the Zn ion and hydrogen bonded interactions with catalytically important amino acid Ser93,
these interactions were found to be conserved in all these compounds.
3
3
09
10
Figure 19: Structure-activity relationship of compound 14 with 20, 16, 4, and 27
3
3
3
3
11
12
13
14
Comparison of activity of compound 20 (IC = 1.33 ± 0.23 µM) with 15 (-OSO -4'-ClPh; IC =
50 2 50
2.52 ± 0.31
2
µM) revealed that replacement of 4'-NO with 4'-Cl resulted in decreased inhibitory
activity to almost half. Similarly, replacement of 3-Cl with 3-CN as in compound 5 (-OSO -4'-
2
ClPh; IC50 = 6.38 ± 0.78
µM), demonstrated three-fold lesser hTNAP inhibitory activity.
20

ACCEPTED MANUSCRIPT
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3
15
16
However, disulfonate derivative 28 (IC50 = 3.69 ± 0.16
µ
M) showed better activity (Figure 20).
Most probable docked conformations of these compounds are given in Figures S32-S34.
3
3
17
18
Figure 20: Structure-activity relationship of compound 15 with 5 and 28
3
3
3
3
3
3
3
3
3
19
20
21
22
23
24
25
26
27
Among compounds having 4
= 1.42 ± 0.02 M) showed potent inhibition. Absence of 3-Cl substitution as in compound 25
(IC = 2.31 ± 0.12 M), showed a decrease in activity which indicates that 3-Cl substitution is
playing an important role in the inhibitory activity. Similarly, addition of 8-NO group brings out
ʹ-Cl-3ʹ-NO substitutions, compound 2 (-OSO -4'-Cl-3'NO Ph; IC
2 2 2 50
µ
µ
5
0
2
the notable decreased activity as in case of compound 34 (IC50 = 7.71 ± 1.09 µM) (Figure 21).
Figures S35-S37 showed most probable docked conformations of compounds 2, 35, and 34. The
characteristic binding features conserved in all of these compounds as expected from the similar
2
+
nitro group containing compounds i.e. binding of oxygen atom of the nitro group to the Zn ion
and interactions with amino acids Ser93 and Arg167 (Figures S35-S37).
3
3
28
29
Figure 21: Structure-activity relationship of compound 2 with 25 and 34.
3
3
3
3
3
30
31
32
33
34
Compounds with di and tri-Cl substituted phenyl ring “R” demonstrated a noticeable hTNAP
inhibitory activity. Out of these, compound 35 (IC = 1.33 ± 0.04
µ
M) with 2',5'- and 2'',5''-di-Cl
substitutions, showed good hTNAP inhibition. Its activity can be compared with compounds 10
(-OSO -2',5'-di-ClPh; IC50 = 1.83 ± 0.11 M) and 22 (-OSO -2',5'-di-ClPh; IC50 = 2.47 ± 0.23
M), that are mono-sulfonated derivatives with 2',5'-di-Cl groups, exhibiting slightly lower
5
0
2
µ
2
µ
21

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3
3
3
3
3
35
36
37
38
39
40
2
activity. Similarly, activity of compound 10 can also be compared with compound 36 (-OSO -
2',4',5'-tri-ClPh; IC = 8.37 ± 0.45
µM) which has similar structure but has an additional chloro
5
0
group at position-4ʹ, this derivative exhibited more than four-fold decline in the activity (Figure
22). Docked conformations of compounds 10, 22, 35, and 36 are given in Figures S38-S41. The
common binding features observed for these compounds were the binding of sulfonate oxygen to
2
+
the Zn ion and interactions with amino acids Ser93, Arg167, His437, and His154.
3
3
41
42
Figure 22: Structure-activity relationship of compound 35 with 22, 10, and 36
3
3
3
3
43
44
45
46
A crucial investigation of the excellent inhibitory potential of compounds against hIAP and
hTNAP, leads to the perception that all structural features of the synthetic compounds are taking
part in the inhibition. However, the comparatively low activity or inactivity of compounds might
be due to attaining the conformation which is less favorable to bind with the active site.
3
47
Homology Modeling and Molecular Docking Studies
3
3
3
3
3
48
49
50
51
52
Since the crystal structures of hTNAP and hIAP are not available from the PDB, homology
models for these enzymes were built and validated according to our already reported method
+
2
[26], and used for docking studies. APs are metalloenzymes, containing two Zn ions and one
2
+
Mg ion in the active site. Our research group has already reported sulfonamides as competitive
inhibitors of APs [24-26]. Herein, we investigated bioisosteres of sulfonamides, that is,
22

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3
3
3
3
3
3
3
3
53
54
55
56
57
58
59
60
61
sulfonates as potent inhibitors of APs. All compounds were found to bind in the same region of
the active site. From the results of molecular docking studies of hIAP inhibitors, the most
important binding features common to all inhibitors were identified as, 1) interaction of sulfonate
+
2
oxygen atoms with the Zn ion of the active site, this interaction is analogous to the interaction
of sulfonamides with APs [24,25]; 2) interaction of sulfonate oxygen atoms with Arg185. These
two interactions were found in all hIAP inhibitors studied and can be deemed important for hIAP
inhibition. Where nitro group was present, particularly at position 2 to the sulfonate group on the
+
2
phenyl ring, the oxygen atoms of the nitro group were in interaction with the Zn ion of the
active site.
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
Among all the compounds screened, derivatives 20 containing a 4-nitro phenyl group was most
active hIAP inhibitor and was selected for detailed analysis via molecular docking studies.
Figures 23 and 24 show the most probable docked configuration for compound 20. The sulfonate
oxygen atom (-O-SO ) was found to be involved in a hydrogen bond interaction with the amino
2
+2
acid Arg185 (2.55 Å), the same oxygen atom was also in direct contact with the Zn ion (2.29
Å) of the active site. The coumarin ring oxygen was acting as a hydrogen bond acceptor toward
NH of His172 (2.02 Å). One of the oxygen atoms of the nitro group was making a hydrogen
bond with His451 (2.12 Å), both oxygen atoms of the nitro group were making a bridging
hydrogen bond contact with Thr450 (1.40 Å and 2.12 Å). Figure 22 shows the surface drawn
around the active site with purple color indicating H-bond donor region/features of the active,
and the green color indicating the H-bond acceptor region/feature of the active site. As can be
seen the structure of the most active inhibitor 20, in its most probable docked conformation, is
complementary to the H-bond acceptor/donor regions of the active site, such that the structural
elements of compound 20, capable of acting as H-bond acceptor, such as the coumarin ring
2
oxygen and the oxygen atom of the sulfonate group (-OSO ), are bound in a complementary
region of the enzyme containing amino acid residues with H-bond donor features.
3
78
23

ACCEPTED MANUSCRIPT
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79
3
3
3
3
80
81
82
83
Figure 23. 3D interactions of most active inhibitor 20 inside the active site of hIAP. Surface
is colored according to hydrogen bond donor (purple) and acceptor (green) features of the
enzyme
24

ACCEPTED MANUSCRIPT
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3
84
85
Figure 24. 2D interactions of most active inhibitor 20 inside the active site of hIAP
3
3
3
3
3
3
3
3
86
87
88
89
90
91
92
93
Similar to hIAP inhibition, the common feature observed for all hTNAP inhibitors was the
interaction of the sulfonate group to the zinc ion of the active site. Indeed compounds containing
structural features that act as metal (here Zn) binding function, are deemed necessary for the
design of inhibitors of metalloenzymes. Figures 25 and 26 show detailed interaction of most
active hTNAP inhibitor 13, in its most probable docked conformation inside binding site of
+
2
hTNAP. One of the sulfonate group oxygen was in direct contact with the Zn ion (1.99 Å), the
other oxygen atom of the sulfonate group was acting as a hydrogen bond acceptor towards Ser93
(2.26 Å) and Arg167 (1.57 Å). The other amino group of Arg167 was acting as a hydrogen bond
25

ACCEPTED MANUSCRIPT
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3
94
95
donor towards the oxygen atom of the sulfonate group (1.53 Å). The coumarin ring oxygen was
making a hydrogen bond with His154 (2.22 Å).
3
96
3
3
3
97
98
99
Figure 25. 3D interactions of most active inhibitor 13 inside the active site of hTNAP.
Surface is colored according to hydrogen bond donor (purple) and acceptor (green)
features of the enzyme
26

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4
00
01
Figure 26. 2D interactions of most active inhibitor 13 inside the active site of hTNAP.
4
4
4
4
4
4
4
4
4
4
4
4
02
03
04
05
06
07
08
09
10
11
12
13
The binding efficiency of the selected docked conformation of hIAP inhibitor 20 and hTNAP
inhibitor 13 was further confirmed via Hyde affinity utility of LeadIT software, whereby a
favorable binding free energy of -11 kJ/mol was calculated for hIAP inhibitor 20, and -13 kJ/mol
for hTNAP inhibitor 13. The Hyde utility not only assesses the binding efficiency/affinity but
also shows which regions or structural elements of the compound contributes favorably and
unfavorably to the overall binding. Figures 27 and 28 show the Hyde-assessment of docked
conformation of compounds 20, and 13, respectively. Regions or structural features that
contribute favorably are colored in green, white for neutral (that is neither favors nor un-favors),
and red for unfavorable contribution to binding. As can be seen, the most favorable structural
element contributing favorably to the binding is the sulfonate group oxygen (colored green) that
+
2
directly binds the Zn ion of the active site. This supports our hypothesis and study that
sulfonate group, which is analogous to the sulfonamide group, is the main structural elements
27

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4
4
4
4
4
14
15
16
17
18
19
required for efficient binding/inhibition to the metallic center of APs. Previously, we have
reported many sulfonamides to be potent inhibitors of APs and other ectonucleotidases as well
[24-26]. For compound 13, the only structural element that contributes un-favorably (colored
red) toward the binding is the C7 carbon atom. Detailed analysis of binding contributions due to
this atom indicated that this is due to the high desolvation cost; introducing polar substituents
near the vicinity of this atom may lead to even better inhibition.
4
20
4
4
4
4
4
4
21
22
23
24
25
26
Figure 27. Hyde assessment of most favorable docked conformation of hIAP inhibitor 20
inside active site of hIAP. Part of the ligand structure contributing favorably to the overall
binding is indicated in green, neutral in white, and the part contributing unfavorably is
colored in red. Surface is colored according to formal charges, blue for positive charge and
red for negative
28