2-Alkoxy-3-(sulfonylarylaminomethylene)-chroman-4-ones as potent and selective inhibitors of ectonucleotidases

Article abstract of DOI:10.1016/j.ejmech.2016.02.073

A facile method for the modulation of 2-alkoxy side chain of 3-formylchromone enamines has been exploited for the synthesis of a series of 2-alkoxy-3-(sulfonylarylaminomethylene)-chroman-4-ones. This modulation was achieved by simply changing the alcoholi

Full text of DOI:10.1016/j.ejmech.2016.02.073

Accepted Manuscript
2-Alkoxy-3-(sulfonylarylaminomethylene)-chroman-4-ones as potent and selective
inhibitors of ectonucleotidases
Mariya al-Rashida, Gazala Batool, Almas Sattar, Syeda Abida Ejaz, Samiullah Khan,
Joanna Lecka, Jean Sévigny, Abdul Hameed, Jamshed Iqbal
PII:
S0223-5234(16)30173-8
10.1016/j.ejmech.2016.02.073
EJMECH 8424
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 October 2015
Revised Date: 26 February 2016
Accepted Date: 28 February 2016
Please cite this article as: Mariya al-Rashida, G. Batool, A. Sattar, S.A. Ejaz, S. Khan, J. Lecka, J.
Sévigny, A. Hameed, J. Iqbal, 2-Alkoxy-3-(sulfonylarylaminomethylene)-chroman-4-ones as potent and
selective inhibitors of ectonucleotidases, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.02.073.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
2-Alkoxy-3-(sulfonylarylaminomethylene)-chroman-4-ones as potent and selective inhibitors of
ectonucleotidases
Mariya al-Rashida1*, Gazala Batool1, Almas Sattar1, Syeda Abida Ejaz2, Samiullah Khan2, Joanna
Lecka3,4, Jean Sévigny3,4, Abdul Hameed5, Jamshed Iqbal2*
1Department of Chemistry, Forman Christian College (A Chartered University), Ferozepur Road-54600,
Lahore, Pakistan
2Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad-
22060, Pakistan
3 Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université Laval,
Québec, QC, G1V 0A6, Canada
4 Centre de Recherche du CHU de Québec – Université Laval, Québec, QC, G1V 4G2, Canada
5H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan.
Corresponding Author
Tel: +923324213592; Email (M. al-Rashida): maria_al_rashida@hotmail.com, mariyara-
shida@fccollege.edu.pk; (J. Iqbal): drjamshed@ciit.net.pk; jamshediqb@gmail.com.
KEYWORDS: Chromones, sulfonamides, ectonucleotidase inhibitors, ecto-5′-nucleotidase, tissue non-
specific alkaline phosphatase, intestinal alkaline phosphatase.
ABSTRACT: A facile method for the modulation of 2-alkoxy side chain of 3-formylchromone
enamines has been exploited for the synthesis of a series of 2-alkoxy-3-(sulfonylarylaminomethylene)-
chroman-4-ones. This modulation was achieved by simply changing the alcoholic reaction media from
methanol to ethanol, iso-propanol and n-butanol while reacting various 3-formylchromones with amino-
benzenesulfonamides. Alcohols are sufficiently nucleophilic and add into the C2-C3 olefinic bond of 3-
formylchromones
without
causing
any
ring cleavage.
The
resulting
2-alkoxy-3-
(sulfonylarylaminomethylene)-chroman-4-ones were found to be potent and selective inhibitors of ecto-
5′-nucleotidase and alkaline phosphatases (TNAP and IAP). Detailed enzyme kinetics studies revealed
competitive inhibition against alkaline phosphatases and un-competitive inhibition against rat and hu-

ACCEPTED MANUSCRIPT
man ecto-5′-nucleotidase. The most active TNAP inhibitor 23 (Ki = 0.078±0.001µM), exhibited 28 times
more selectivity for TNAP over IAP (Ki = 2.18±0.12 µM). Compound 9 was most active IAP inhibitor
(Ki = 0.24±0.01 µM), and was 300 times more selective towards IAP than TNAP (Ki = 72.9±1.68 µM).
Compound 40 was most active human ecto-5′-nucleotidase inhibitor exhibiting inhibition in low nano-
molar
range
(Ki
=
14
nM).
Introduction
Ectonucleotidases are a group of cell-surface located ecto-enzymes. The members of ectonucleotidase
family include ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs), ecto-nucleoside triphos-
phate diphosphohydrolases (E-NTPDases), ecto-5ʹ-nucleotidase (e5NT, CD73) and alkaline phosphatase
(AP) [1, 2]. These enzymes hydrolyze a variety of extracellular nucleotides to nucleosides including
adenosine [3]. Extracellular nucleotides are important signaling molecules that trigger cellular responses
by acting on their respective receptors (adenosine activates P1 and nucleotides activate P2 receptors) [3,
4]. Adenosine 5'-monophosphate (AMP) is a major substrate of e5NT that is hydrolyzed to adenosine
(ado). Adenosine is ubiquitously present in the body and is an important regulator of purinergic cell sig-
naling that is vital for many physiological and patho-physiological processes [5-9]. However it has been
found that e5NT is up-regulated in a variety of tumors and its expression has been known to be associat-
ed with tumor neovascularization, invasiveness and metastasis [10-11]. Extracellular adenosine is also
implicated in regulating adaptive responses to hypoxia [12] hence inhibitors of e5NT are being keenly
explored for their therapeutic potential against cancer [13].
Another major source of adenosine production is alkaline phosphatase (AP) mediated hydrolysis of
AMP to adenosine either directly, AMP ꢀ Ado or indirectly, ATP ꢀ ADP ꢀ AMP ꢀ Ado. AP is sub-
strate non-specific and hydrolyzes a variety of substrates other than nucleotides, such as inorganic poly-
phosphates, bis(p-nitrophenyl)phosphate, glucose-phosphates, and phosphatidates [14]. APs are also
responsible for catalyzing certain transphosphorylation reactions [15, 16]. APs have been divided into
two major groups, tissue non-specific alkaline phosphatase (TNAP) and tissue specific alkaline phos-
phatase, consisting of germ-cell alkaline phosphatase (GAP), placental alkaline phosphatase (PAP) and
intestinal alkaline phosphatase (IAP) [17]. PAP is mainly expressed during embryonic stages and is an
important tumor marker [18]. GAP is polymorphic and is known to be expressed in seminoma and car-
cinoma [19]. IAP is mainly present in intestinal brush border membrane and is believed to be vital for
pH regulation, lipid accumulation and for detoxification of bacterial lipopolysaccharides [20]. High
concentrations of TNAP are found in mineralizing tissues such as teeth and bones. TNAP aids in bone
2

ACCEPTED MANUSCRIPT
formation by providing a phosphate pool, which is necessary for mineralization, and by hydrolyzing in-
organic pyrophosphate (PPi) which is a potent inhibitor of mineralization [21-22]. Increased levels of
TNAP are responsible for ectopic calcification including hydroxyapatite deposition disorder (HADD)
[23] and vascular calcification [24]. In continuation of our research on discovery and development of
inhibitors of ectonucleotidases, herein we report synthesis of a series of 2-alkoxy-3-
(sulfonylarylaminomethylene)-chroman-4-ones and their ectonucleotidase inhibition. In order to carry
out detailed structure activity relationship analysis, a diverse series of compounds were synthesized by
reacting variously 6-substituted and 6,8-disubstituted 3-formylchromones with various aminobenzene-
sulfonamides (Scheme 1).
Results and Discussion
Chemistry
3-Formylchromone (4-oxo-4H-chromene-3-carbaldehyde), is a fairly versatile molecule that is easily
synthesized and frequently used for the incorporation of chromone moiety into other aro-
matic/heterocyclic molecules, or to construct new scaffolds based on this molecule. Much of the syn-
thetic utility of 3-formylchromones is due to the presence of three electron deficient centers, the carbon-
yl carbon atoms C-4 and C-3’of the keto and formyl groups respectively, and carbon atom C-2 of the
chromone ring [25]. The reaction of 3-formylchromones with primary amines results in the initial for-
mation of a 3-(aryliminomethyl)chromone, this product makes the chromone nucleus susceptible to-
wards nucleophilic addition reactions at 2 position of chromone ring. Alcohols are sufficiently nucleo-
philic and add into the 2 position of chromone ring without causing any ring cleavage [26]. Previously,
we reported that when 3-formylchromones are reacted with aminobenzenesulfonamides using ethanol as
a solvent, an enamine product containing a 2-ethoxy side chain is obtained [27, 28]. Hence, 2-ethoxy-3-
(sulfonylarylaminomethylene)-chroman-4-ones (24-38, 43-48), were synthesized and reported as potent
and selective inhibitors of alkaline phosphatase [29]. This prompted us to explore whether it was possi-
ble to modulate the 2-alkoxy side chain by simply changing the alcoholic reaction medium. We found,
that indeed, the 2-alkoxy side chain of 2-alkoxy-3-(sulfonylarylaminomethylene)-chroman-4-one was
very easily modulated by simply changing the alcoholic reaction medium from methanol to ethanol, iso-
propanol, and n-butanol. Subsequently 2-alkoxy-3-(sulfonylarylaminomethylene)-chroman-4-ones (9-
23,
39-44)
were
synthesized
according
to
Scheme
1.
3

ACCEPTED MANUSCRIPT
Scheme 1. Scheme for the synthesis of 2-alkoxy-3-(sulfonylarylaminomethylene)-chroman-4-ones.
It is interesting to note that when 3-formylchromones are reacted with ortho-
aminobenzenesulfonamide in methanol, an enamine product containing a 2-methoxy group (11, 14, 17,
20, 23) is obtained, however when the same reaction is carried out in higher alcohols, that is, ethanol,
iso-propanol and n-butanol, a benzothiadiazine ring (26) is obtained every time. By carrying similar re-
actions using a 3-formylchromone, where C-2 position of chromone ring is already substituted, we
hoped to obtain an imine (Schiff base) product. However when 2-amino-6-chloro-3-formylchromone
was condensed with 4-, 3-, and 2-aminobenzenesulfonamide (4-, 3- and 2-ABS), the product obtained in
small amounts (< 20 % yield) was identified as an unexpected self-condensation product of 2-amino-6-
chloro-3-formylchromone (49).
4

ACCEPTED MANUSCRIPT
Figure 1. 1H NMR spectrum of the un-expected condensation product, compound 49.
1
The structures of synthesized compounds were elucidated using H and 13C-NMR spectroscopy. All
compounds (except 49, which is achiral) existed as racemic mixtures (as evidenced by optical activity,
data not presented). Serendipitously we were able to isolate single crystals of two different enantiomers
of compound 42, R-isomer, compound 42(a), and S-isomer, compound 42(b) (CCDC No. 1059015 and
1059016 respectively, Figure 2).
Figure 2. ORTEP plot of compound 42(a), R isomer (left), and compound 42(b), S-isomer (right).
Thermal ellipsoids are drawn at 50% probability level.
5

ACCEPTED MANUSCRIPT
In our view, there is much ambiguous data present in literature regarding the structures of reaction
products obtained by condensing 3-formylchromones and primary amines in alcohols, many of such re-
action products are claimed to be Schiff bases synthesized in ethanol or methanol, we believe Schiff ba-
ses of 3-formylchromone cannot be formed in alcoholic reaction media, this calls for re-investigation
into the structure of such compounds. However studies also need to take into account the effect of na-
ture of amines on the formation of an enamine product. In all the literature available, the chirality of the-
se compounds has never been reported or investigated. We have reported here, for the first time, crystal
structures of the two enantiomers.
Ectonucleotidase Inhibition Studies
Tissue Non Specific Alkaline Phosphatase (TNAP) and Intestinal Inhibition Alkaline Phosphatase
(IAP) Inhibition Studies
We have previously reported 2-ethoxy-3-(sulfonylarylaminomethylene)-chroman-4-ones (24
48), as potent and selective inhibitors of alkaline phosphatase [29] the newly synthesized compounds (
23 39 44) were also assayed as inhibitors of bovine tissue non-specific alkaline phosphatase (TNAP)
and calf intestinal alkaline phosphatase (IAP). Most of the compounds in the series were found to be
potent selective inhibitors of IAP (Table 1). The most potent TNAP inhibitor (23), and IAP inhibitor (
-38, 43-
9
-
,
-
9
)
were selected for the kinetics study in order to determine their type of inhibition. Changes in the initial
velocities of the reaction were measured at different concentrations of inhibitor (0 µM, 0.5 µM, 1.00
µM, 2.00 µM) using substrate (CDP-star) concentrations of 55µM, 110 µM, 165 µM and 220 µM. A
double-reciprocal plot of the inhibition kinetics of TNAP by 23 and IAP by inhibitor
9 was measured by
using PRISM 5.0 (GraphPad, San Diego, California, USA), both compounds exhibited competitive
mode of inhibition (Figure 3).
6

ACCEPTED MANUSCRIPT
0.00015
0.00010
0.00005
0 µM
0.50 µM
1.00 µM
2.00 µM
-0.06
-0.04
0.00
0.02
1/[S](µM)
-0.00005
Figure 3. Double-reciprocal plot of the inhibition kinetics of bovine tissue non-specific alkaline phos-
phatase (TNAP) by compound 23 (left), and double-reciprocal plot of the inhibition kinetics of bovine
intestinal alkaline phosphatase (IAP) by compound
9 (right), both indicating competitive inhibition.
Changes in the initial velocities of the reaction were measured at different concentrations of the inhibi-
tors using substrate CDP-Star (disodium 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2′-(5-
chlorotricyclo[3.3.1.13.7]decan])-4-yl]-1-phenyl
phosphate).
Table 1. Bovine tissue non-specific alkaline phosphatase (TNAP) and bovine intestinal alkaline phos-
phatase (IAP) inhibition activities of compounds.
TNAP
IAP
R1, R2, R3
Position of
sulfonamide
group
Code
IC50±SEM
Ki±SEM
(µM)
IC50±SEM
(µM)
Ki±SEM
(µM)
(µM)
9
H, H, CH3
H, H, CH3
p
-SO2NH2
170.9±2.41
32.12%
72.9±1.68
-
0.565±0.01 0.24±0.01
39.8±0.21 16.9±0.14
10
m-SO2NH2
7

ACCEPTED MANUSCRIPT
11
12
13
14
15
16
17
18
19
20
H, H, CH3
F, H, CH3
o
p
-SO2NH2
-SO2NH2
-SO2NH2
714.5±3.11 305.15±1.11 7.81±1.11
3.34±1.11
29.1±1.21
5.66±0.91
4.26±0.06
1.89±0.06
13.6±0.11
17.6±0.02
1.46±0.33
17.1±0.09
20.87%
7.51±0.21
0.623±0.01
7.29±0.06
-
68.6±0.04
13.5±1.12
10.2±0.34
4.43±0.32
31.9±0.98
F, H, CH3
m
F, H, CH3
o
p
-SO2NH2
-SO2NH2
Cl, H, CH3
Cl, H, CH3
Cl, H, CH3
Cl, Cl, CH3
Cl, Cl, CH3
Cl, Cl, CH3
m
-SO2NH2
34.25%
-
o
p
-SO2NH2
-SO2NH2
29.92%
-
2.32±0.99 0.992±0.12
16.2±0.56
3.52±0.18
5.36±0.03
8.17±0.98
6.19±0.12
1.49±0.13
2.28±0.03
3.49±0.91
3.25±0.05
8.34±0.16
9.87±0.11
3.13±0.76
1.38±0.11
3.59±0.42
4.21±0.16
1.34±0.14
m
-SO2NH2
o
p
-SO2NH2
-SO2NH2
CH3, H,
CH3
21
22
CH3, H,
CH3
m
-SO2NH2
110±1.19
47.2±2.06
6.56±0.26
2.77±0.64
2.18±0.12
CH3, H,
CH3
o
p
-SO2NH2
-SO2NH2
0.18±0.02 0.078±0.001 5.06±1.21
23
39
40
41
42
H, H,
CH(CH3)2
47.06±2.88 20.09±1.54 0.527±0.08 0.22±0.01
H, H,
CH(CH3)2
m
-SO2NH2
-SO2NH2
-SO2NH2
111.3±1.82
4.57±0.01
77.8±2.67
47.5±1.06
1.95±0.001
33.21±1.67
17.9±2.13
48.3±1.3
7.66±0.13
20.6±1.5
H, H,
(CH2)3CH3
p
H, H,
(CH2)3CH3
m
2.29±0.07 0.978±0.07
8

ACCEPTED MANUSCRIPT
For 3-formylchromone derived compounds (without having any substituent on 6 position of chromone
ring), as the position of sulfonamide group on phenyl ring was changed from para to meta, the TNAP
and IAP inhibition activity was found to decrease. The only exception was 2-butoxy containing com-
pound, where the meta isomer 42 (Ki = 0.978±0.07 µM) was more active than its para isomer 41 (Ki =
20.6±1.5 µM). However, when the 6-position of chromone ring is substituted by highly electronegative
halogen groups as in 6-fluoro, 6-chloro and 6,8-dichloro substituted compounds, the TNAP inhibition
activity of
= 1.49±0.13 µM) increases as compared to their
7.51±0.21 µM), 15 (21%) and 18
halogen substituent in increasing TNAP inhibition activity. However, no such trend was found for IAP
inhibitors, all 6-halogen substituted compounds containing -SO2NH2 group except 12 were better IAP
inhibitors as compared to the corresponding -SO2NH2 containing compounds. The 6-methyl substitut-
ed compound 23, containing -SO2NH2 group was the most active TNAP inhibitor having Ki value of
0.078±0.001µM and 28 times selectivity for TNAP over IAP. Among all the compounds assayed, com-
pound was identified as a potent lead compound that was most active IAP inhibitor and exhibited most
selectivity for IAP as compared to TNAP. Compound was 300 times more active inhibitor of IAP (
Ki = 0.24±0.01 µM) over TNAP ( Ki = 72.9±1.68 µM), closely followed by 39 which was 95 times
m
-SO2NH2 containing compounds 13
(
Ki = 0.623±0.01 µM), 16 (34% inhibition) and 19
(Ki
p
-SO2NH2 group containing isomers 12 Ki
(
=
(Ki = 6.19±0.12 µM). This trend seems to suggest significance of 6-
p
m
o
9
9
9;
9;
more active IAP inhibitor (Ki = 0.22±0.01 µM) as compared to TNAP (Ki = 20.09±1.54 µM).
Ecto-5′-Nucleotidase (e5NT) Inhibition Studies
Encouraged by excellent AP inhibitory activity of these compounds, we decided to test their inhibitory
potential against another AMP hydrolyzing enzyme, ecto-5’-nucleotidase. Accordingly the compounds
were assayed against human ecto-5′-nucleotidase (he5NT), and rat ecto-5′-nucleotidase (re5NT) en-
zymes (Table 2). 2-Ethoxy substituted compounds (24-38, 43-48) were previously assayed against
he5NT and did not exhibit any significant activities [29], however when assayed against re5NT in this
study, some of the compounds from this series were found to be active inhibitors (Table 3). The inhibi-
tors (40, 35) with potent IC50 values were then selected for the kinetics study in order to determine their
type of inhibition. Changes in the initial velocities of the reaction were measured at different concentra-
tions of the inhibitor (0 µM, 0.5 µM, 1.00 µM, 2.00 µM) using substrate (AMP) at concentrations of 4
mM, 5 mM, 6 mMM and 8 mM. A double-reciprocal plot of the inhibition kinetics of he5NT by 40
and re5NT by inhibitor 35 was measured by using PRISM 5.0 (GraphPad, San Diego, California, USA).
9

ACCEPTED MANUSCRIPT
Both compounds (35
,
40) indicated uncompetitive inhibition (Figure 4) suggesting that the inhibitors
bind to the enzyme only after the enzyme-substrate complex has been formed. This suggests existence
of binding pocket other than the active site for these inhibitors. Since this enzyme has been known to
undergo significant domain movement upon substrate binding, it seems quite likely that this binding site
is made available as a result of conformational changes following the domain movement.
Figure 4. Double-reciprocal plot of the inhibition kinetics of human ecto-5′-nucleotidase (he5NT) by
compound 40 (left), and double-reciprocal plot of the inhibition kinetics of rat ecto-5′-nucleotidase
(re5NT) by compound 37 (right), indicating uncompetitive inhibition. Changes in the initial velocities of
the reaction were measured at different concentrations of the inhibitors using substrate AMP (adenosine
monophosphate). The experiments were carried out in triplicate (n=3).
Compounds 35 and 39 were most active re5NT inhibitor (Ki = 0.0047±0.01 µM and 0.026±0.08 µM
respectively). All newly synthesized compounds were assayed against both human and rat e5NT. In
general the compounds containing
sponding -SO2NH2 compounds. Compounds containing
SO2NH2 compounds, but less active than -SO2NH2 compounds. The inhibition activity against both
m
-SO2NH2 group were more active against he5NT than the corre-
p
o
-SO2NH2 group are more active than
p-
m
human and rat e5NT was found to increase upon substitution of halogen groups at 6 position of chro-
mone ring. For 4-ABS derived compounds, as the number of carbon atoms in the 2-alkoxy side chain
increases, that is from 2-methoxy to 2-butoxy, the ectonucleotidase inhibition activity also increases. For
3-ABS derived compounds there was no significant effect of change of 2-alkoxy side chain on ectonu-
cleotidase inhibition activity. The most active he5NT inhibitors were 40 and 42 with Ki values of
10

ACCEPTED MANUSCRIPT
0.014±0.08 and 0.018±0.06 µM. Compounds
e5NT inhibitors.
9, 40 and 41 were found to be selective human versus rat
Table 2. Human and rat ecto-5′-nucleotidase (he5′NT and re5′NT) inhibition activities of compounds.
Ecto-5′-NT (Human)
Ecto-5′-NT (Rat)
R1, R2, R3
Position of
sulfonamide
group
Code
Ki±SEM
IC50±SEM
(µM)
Ki±SEM
(µM)
IC50±SEM
(µM)
(µM)
9
H, H, CH3
H, H, CH3
H, H, CH3
F, H, CH3
p
-SO2NH2
7.11±0.66
0.38±0.11
1.91±0.033
46.79%
0.338±0.01
27.71%
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
m
-SO2NH2
-SO2NH2
-SO2NH2
0.018±0.01
2.81±0.99
2.39±0.04
46.44%
0.134±0.011
o
p
0.091±0.03
0.114±0.02
-
-
F, H, CH3
m
-SO2NH2
44.39%
-
46.05%
-
F, H, CH3
o
p
-SO2NH2
-SO2NH2
48.22%
-
45.41%
-
Cl, H, CH3
Cl, H, CH3
Cl, H, CH3
Cl, Cl, CH3
Cl, Cl, CH3
Cl, Cl, CH3
CH3, H, CH3
CH3, H, CH3
CH3, H, CH3
1.66±0.12
0.56±0.21
1.35±0.16
2.92±0.02
0.97±0.03
0.88±0.01
47.01%
0.079±0.02
0.027±0.01
0.064±0.02
0.138±0.2
0.046±0.01
0.042±0.02
-
1.71±0.11
2.38±0.06
1.31±0.77
10.1±0.98
4.81±0.1
1.84±0.01
42.86%
0.081±0.04
0.113±0.01
0.062±0.04
0.472±0.1
0.229±0.2
0.087±0.4
-
m
-SO2NH2
o
p
-SO2NH2
-SO2NH2
m
-SO2NH2
o
p
-SO2NH2
-SO2NH2
m
-SO2NH2
44.49%
-
47.48%
-
o
-SO2NH2
39.13%
-
47.76%
-
11

ACCEPTED MANUSCRIPT
H, H,
CH(CH3)2
p
-SO2NH2
1.17±0.02
0.287±0.04
1.32±0.03
0.377±0.05
0.053±0.01
0.014±0.08
0.063±0.01
0.018±0.06
0.54±0.98
25.71%
0.026±0.08
39
40
41
42
H, H,
CH(CH3)2
m
-SO2NH2
-
H, H,
(CH2)3CH3
p
-SO2NH2
6.66%
-
H, H,
(CH2)3CH3
m
-SO2NH2
3.61±0.02
0.172±0.05
Table 3. Rat ecto-5′-nucleotidase (re5NT) inhibition activities of compounds (24-38, 43-48).
24-38
26, 29, 32
43-48
Ecto-5′-NT (Rat)
IC50±SEM Ki±SEM
R1, R2, R3, R4
Position of terminal
sulfonamide group
Code
(µM)
(µM)
24
25
26
27
28
29
30
31
32
33
34
35
37
H, H, C2H5,
H, H, C2H5
H, H
p
-SO2NH2
39.53%
30.08%
-
m
-SO2NH2
-
-
3.81±0.16
2.13±0.04
0.99±0.11
2.71±0.34
29.73%
0.181±0.2
0.101±0.2
0.047±0.09
0.129±0.07
-
F, H, C2H5,
F, H, C2H5,
F, H
p-SO2NH2
m
-SO2NH2
-
Br, H, C2H5,
Br, H, C2H5,
Br, H
p-SO2NH2
m
-SO2NH2
6.13±0.98
20.31%
0.292±0.01
-
-
C2H5, H, C2H5,
C2H5, H, C2H5,
C2H5, H, C2H5,
Br, Br, C2H5,
p
-SO2NH2
-SO2NH2
-SO2NH2
-SO2NH2
3.73±0.26
40.48%
0.178±0.04
-
m
o
0.101±0.05
0.583±0.11
0.0047±0.01
0.027±0.01
m
12

ACCEPTED MANUSCRIPT
38
43
Br, Br, C2H5
H, H, C2H5, 5-methyl-1,3,4-
thiadiazol-2-yl
o
-SO2NH2
-
40.1±1.21
48.02%
1.91±0.07
-
H, H, C2H5, 2-pyridyl
-
-
-
45
46
15.18±0.98
23.57%
0.712±0.2
-
Br, H, C2H5, 2-pyridyl
H, H, C2H5, 6-
methoxypyridazin-3-yl
H, H, C2H5, 2,6-
1.09±0.11
0.0517±0.08
47
48
-
28.21%
-
dimethoxypyrimidin-4-yl
Molecular Modelling Studies
Molecular docking studies were carried out to, (1) investigate if any of the stereoisomer was more active
over the other, and (2) to identify possible structural elements that render selectivity (if any) to one par-
ticular stereoisomer over the other. Since crystal structures of bovine APs are not known, homology
models of bIAP and bTNAP were made according to our previously reported method [30]. APs are
metalloenzymes, the active site of both bIAP and bTNAP contains two Zn+2 ions and one Mg+2 ion.
Compounds 13 and 23, being most active bTNAP inhibitors were selected for docking studies. Similarly
compounds 9 and 39 were most active bIAP inhibitors and were accordingly selected for docking stud-
ies. The R and S isomers of these compounds were constructed in HyperChem 8.0 [31], the initial coor-
dinates for the construction of these stereoisomers were obtained from the experimentally determined
crystal structures of 42(a) and 42(b). Charges were added on ligands using ANTECHAMBER [32] utili-
ty of Chimera [33]. The geometries of the ligands were optimized and energies minimized using Chime-
ra [33] via steepest descent minimization through 100 steps followed by a more refined conjugate gradi-
ent minimization, using steepest descent step size and conjugate gradient step size of 0.02 each. Molecu-
lar docking studies were carried out using BiosolveIT’s LeadIT software [34], which makes use of Flex
X docking method. Docked poses were rescored and binding free energies ꢀG (kJ/mol) were calculated
using BioSolveIT’s Hyde utility. Since these compounds were experimentally found to be competitive
inhibitors via enzyme kinetics studies, a 10 Å area around the active site was selected for docking.
For both bIAP inhibitors 9 and 39, no significant difference in binding efficiency of two enantiomers
was observed (Table S3, in SI). Detailed analysis of binding site interactions reveal a similar binding
pattern for both enantiomers of compounds 9 as seen in Figure 5 (2D interactions are given in Figure S1
of SI). The most significant binding patterns observed for both enantiomers of compound 9 are binding
of sulfonamide nitrogen to Zn ion, and binding of sulfonamide oxygens to His336, His172 and Arg185.
13

ACCEPTED MANUSCRIPT
No noteworthy interactions could be observed for the chiral methoxy chain. Similar interactions were
observed for enantiomers of compounds 39 (Figure S2).
Figure 5. 3D interactions of most probable docked conformation of bIAP inhibitor 9(R) (left), and 9(S)
(right).
Most active bTNAP inhibitors, 13 and 23 were selected for molecular docking studies. Similar to bIAP
inhibitors, for studied bTNAP inhibitors (13, 23) no evidence could be put forward that indicates prefer-
ential binding of any one particular isomer over the other. This can be seen from similar biding patterns
observed for both enantiomers as well as comparable binding free energies (Table S3). The most signifi-
cant binding patterns observed for both enantiomers of compound 13 are binding of sulfonamide nitro-
gen to Zn ion, and binding of sulfonamide oxygens to His171, Ser110, Arg184. The oxygen atom of the
chromone ring as well as oxygen atom of the chiral methoxy chain were found to act as hydrogen bond
acceptors towards Thr453 (Figure 6; Figure S2 for 2D interactions). It is interesting to note here that the
only indication of involvement of chiral methoxy chain as a hydrogen bond donor (via oxygen atom of
methoxy group) could be seen in compound 13, containing
m-sulfonamide group. For compound 23
(Figure S3), the chromone ring oxygen of both enantiomers was found to act as a hydrogen bond accep-
tor toward Arg168. The sulfonamide oxygen atoms were acting as hydrogen bond acceptors toward
His171 in addition to being in contact with Zn ion. The sulfonamide oxygen of 23(S) was found to make
an additional hydrogen bond contact with Arg184. The sulfonamide nitrogen of both enantiomers was
found to interact with Arg184 and Ser110 as a hydrogen bond donor, for 23(S) sulfonamide nitrogen
was also found to be in contact with Zn ion (2.14 Å).
14

ACCEPTED MANUSCRIPT
Figure 6. 3D interactions of most probable docked conformation of bTNAP inhibitor 13(R) (left), and
13(S) (right).
From docking studies of most active bIAP (9, 39), and bTNAP inhibitors (13, 23), containing a chiral 2-
methoxy side chain, no evidence could be put forward that unequivocally establishes preferential bindꢁ
ing of any one particular stereoisomer over the other. The reason may be attributed to the similar alignꢁ
ment of zinc biding function that is the sulfonamide group, of both enantiomers to the Zn ion, as shown
in Figure 7, indicating overlap of most favorable docked orientation of both enantiomers of 13.
Figure 7. Overlap of most probable docked conformations of bTNAP inhibitor 13(R) (blue) and 13(S)
(purple), indicating similar alignment of Zn binding function, the sulfonamide group.
15

ACCEPTED MANUSCRIPT
Conclusion
In summary, we have described here a simple method for modulation of 2-alkoxy side chain in 2-
alkoxy-3-(sulfonylarylaminomethylene)-chroman-4-ones. All synthesized compounds were assayed as
inhibitors of human and rat e5NT, and bovine TNAP and IAP. Most of the compounds were found to be
potent and selective inhibitors of ectonucleotidases. Changing the 2-alkoxy side chain was found to
have a profound effect on enzyme inhibition activities. Some lead compounds have been identified that
have exhibited promising activities and can be developed further. Molecular modelling studies were car-
ried out on selective, most active AP inhibitors. No evidence could be put forward that unequivocally
establishes preferential binding of any one particular stereoisomer over the other, this primarily seems to
be attributed to the similar alignment of zinc binding function, that is, the sulfonamide group, in both
enantiomers. However, this statement is based only on molecular modelling study of selective 2-
methoxy containing compounds, as the length of 2-alkoxy group varies, the preferential binding of any
one particular stereoisomer over the other may appear. Indeed, detailed study of such system will pro-
vide useful information that my help in development of even more potent and selective ectonucleotidase
inhibitors from this class of compounds.
Experimental Section
General Comments
All reagents were purchased from either Sigma or Aldrich and were used as such. Commercially availa-
ble analytical grade solvents were used. Progress of the reaction was monitored by TLC silica gel plates
(0.2 mm, 60 HF254, Merck). TLC spots were analyzed under short and long wavelength UV radiation.
Melting points were taken on a Gallenkamp melting point apparatus and remain uncorrected. Purity of
compounds was established by using HPLC (Shimadzu Liquid Chromatograph LC-10 AS with UV
Spectrophotometric Detector, Shimadzu SPD-6A) on C18 column (M2 Analytical column 250 x 4.6
mm; 5 µm). All the chromatograms obtained were very similar to one another due to chemical similarity
of the compounds. Mass spectra (EIMS) were recorded on FinniganMAT- 321A, Germany. IR spectra
1
were recorded on Perkin Elmer Spectrum BX-II. H-NMR and 13C-NMR spectra were recorded on
Bruker (300, 400 and 500 MHz) AMX Spectrometer. Chemical shift values were referenced against tri-
methylsilane (TMS). DMSO-d6 was used as solvent for NMR spectroscopy. Calf intestine alkaline
phosphatase IALP (1000 U), bovine kidney alkaline phosphatase TNALP (100 U), Tris HCl, MgCl2,
ZnCl2 and phenyl phosphate disodium salt were purchased from Sigma, Germany. For the spectropho-
tometric determination of p-nitrophenol, catalytic product of ALP, a 96 well plate microplate reader
from Bio-TekELx 800TM, Instruments, Inc. USA was used.
16

ACCEPTED MANUSCRIPT
General Method for the synthesis of 2-alkoxy-3-(sulfonylarylaminomethylene)-chroman-4-one de-
rivatives
These compounds were synthesized by modification of our already reported methods [29]. A solution of
0.001 mol 6-(un)substituted 3-formylchromone (1-8, scheme 1) in 15-20 mL appropriate alcohol (meth-
anol, ethanol, iso-propanol, n-butanol) was stirred with heating until dissolved. An equimolar amount of
appropriate sulfonamide was added to this solution. The reaction mixture was refluxed for 3 hrs and
kept overnight. Solid product was obtained by filtration and purified by recrystallization from a mixture
of hot ethanol and acetone (1:1) or acetonitrile.
4-{[(Z)-(2-methoxy-4-oxo-2H-chromen-3(4H)-ylidene)methyl]amino}benzenesulfonamide (9)
Yield 76 %. m.p 187-189 °C. MS-EI m/z 360 (M+). Anal. Calcd for C17H16N2O5S: C(56.66) H(4.47)
1
3
N(7.77); found: C(56.46) H(4.43) N(7.50). H NMR (600MHz, DMSO) δ: 11.87 (1H, d, J = 12.3 Hz,
3
3
4
3
NH), 8.16 (1H, d, J = 12.3 Hz, CH enamine), 7.84 (1H, dd, J = 7.9 Hz, J = 1.6 Hz), 7.8 (2H, d, J =
8.8 Hz), 7.53 (2H, d, 3J = 8.8 Hz), 7.27 (2H, br s, SO2NH2), 7.14 (1H, t, J = 8x(2) Hz), 7.11 (1H, dd, 3J =
8.4 Hz, 4J = 0.8 Hz), 5.84 (1H, s, H-2), 3.39 (3H, s, OCH3). 13C NMR (150.92 MHz, DMSO)
δ: 179.36
(C-4), 156.52 (C-9), 151.87 (C-1’), 145.79 (C-11), 145.55 (C-3’), 140.24 (C-6), 130.61 (C-7), 123.58
(C-10),121.10 (C-5), 120.34 (C-5’), 101.28 (C-2’), 113.76 (C-8), 111.05 (C-6’), 110.90 (C-4’), 103.98
(C-3), 97.44 (C-2).
3-{[(Z)-(2-methoxy-4-oxo-2H-chromen-3(4H)-ylidene)methyl]amino}benzenesulfonamide (10)
Yield 87 %. m.p 185-187 °C. MS-EI m/z 360 (M+). Anal. Calcd for C17H16N2O5S: C(56.66) H(4.47)
1
3
N(7.77); found: C(56.81) H(4.39) N(7.58). H NMR (600MHz, DMSO) δ: 11.87 (1H, d, J = 12.5 Hz,
NH), 8.19 (1H, d, 3J = 12.5 Hz, CH enamine), 5.88 (1H, s, H-2), 7.38 (2H, br s, SO2NH2), 7.82 (1H, t, 4J
= 1.7 Hz, H-2’), 3.38 (3H, s, OCH3), 7.41 (1H, dt, 3J = 6 Hz, 4J = 3.3 Hz, H-5’), 7.51-7.61 (5H, m, ArH).
13C NMR (150.92 MHz, DMSO)
δ: 179.36 (C-4), 156.52 (C-9), 151.87 (C-1’), 145.79 (C-11), 145.55
(C-3’), 140.24 (C-6), 130.61 (C-7), 123.58 (C-10),121.10 (C-5), 120.34 (C-5’), 101.28 (C-2’), 113.76
(C-8), 111.05 (C-6’), 110.90 (C-4’), 103.98 (C-3), 97.44 (C-2).
2-{[(Z)-(2-methoxy-4-oxo-2H-chromen-3(4H)-ylidene)methyl]amino}benzenesulfonamide (11)
17

ACCEPTED MANUSCRIPT
Yield 71 %, m.p 183-185 °C. MS-EI m/z 360 (M+). Anal. Calcd for C17H16N2O5S: C(56.66) H(4.47)
3
N(7.77); found: C(56.73) H(4.50) N(7.81).1H NMR (600MHz, DMSO)
δ
: 12.19 (1H, d, J = 12.0 Hz,
3
NH), 8.01 (1H, d, J = 12.0 Hz, CH enamine), 7.86-7.83 (2H, m, ArH), 7.64-7.63 (2H, m, ArH), 7.61
(2H, br s, SO2NH2), 7.53 (1H, ddd, J = 8.4 Hz, J = 7.1 Hz, J = 1.6 Hz), 7.26 (1H, ddd, 8.0 Hz, 6.0 Hz,
2.4 Hz), 7.13 (1H, td, J = 7.5x(2) Hz, J = 1.1 Hz), 7.1 (1H, d, J = 8.2 Hz), 5.87 (1H, s, H-2), 3.39 (3H, s,
OCH3).13C NMR (150.92 MHz, DMSO)
δ: 180.01 (C-4), 155.77 (C-9), 143.74 (C-1’), 137.68 (C-11),
134.70 (C-2’), 133.66 (C-7), 131.27 (C-5’), 128.45 (C-6), 125.94 (C-3’), 123.43 (C-5), 122.79 (C-8),
122.09 (C-10), 118.17 (C-4’), 117.60 (C-6’), 105.88 (C-3), 101.63 (C-2), 55.00 (OCH3).
4-{[(Z)-(6-fluoro-2-methoxy-4-oxo-2H-chromen-3(4H)-ylidene)methyl]amino}benzenesulfonamide
(12)
Yield 80 %, m.p 190-192 °C. MS-EI m/z 378 (M+). Anal. Calcd for C17H15FN2O5S: C(53.96) H(4.00)
1
3
N(7.40); found: C(54.54) H(4.33) N(7.47). H NMR (500MHz, DMSO)
δ
: 11.78 (1H, d, J = 12.8 Hz,
3
NH), 8.21 (1H, d, 3J = 12.8 Hz, CH enamine),7.8 (2H, d, J = 8.8 Hz, H-3’, H-5’), 7.55 (2H, d, 3J = 8.8
Hz, H-2’, H-6’), 7.51 (1H, dd, 3J = 8.4, 4J = 3.3 Hz, H-5), 7.43 (1H, dt, 3J = 8.5 Hz, 4J = 4.4x(2) Hz, H-
7), 7.29 (2H, br s, SO2NH2), 7.17 (1H, dd, 3J = 9.0 Hz, 4J = 4.2 Hz, H-8), 5.85 (1H, s, H-2), 3.38 (3H, s,
13
OCH3). C NMR (150.92 MHz, DMSO)
δ: 188.38 (C-4), 163.92 (C-9), 156.52 (C-1’), 151.88 (C-4’),
139.35 (C-11), 145.18 (C-7), 139.30 (C-6), 127.58 (C-3’, C-5’), 123.22(C-5), 121.96 (C-10), 120.27(C-
8), 116.83 (C-2’, C-6’), 112.5724 (C-3), 101.32(C-2), 55.17 (OCH3).
3-{[(Z)-(6-fluoro-2-methoxy-4-oxo-2H-chromen-3(4H)-ylidene)methyl]amino}benzenesulfonamide
(13)
Yield 79 %, m.p 179-181 °C. MS-EI m/z 378 (M+). Anal. Calcd for C17H15FN2O5S: C(53.96) H(4.00)
1
3
N(7.40); found: C(54.21) H(4.41) N(7.52). H NMR (500MHz, DMSO) δ: 11.87 (1H, d, J = 12.5 Hz,
NH), 8.19 (1H, d, 3J = 12.5 Hz, CH enamine), 7.82 (1H, t, 4J = 1.7 Hz, H-2’), 7.52 (1H, dd, 3J = 8.4 Hz,
, 4J = 3.3 Hz, H-5),7.43-7.39 (m, 1H, H-7), 7.17 (1H, dd, 3J = 9.0 Hz, 4J = 4.2 Hz, H-8), 7.38 (2H, br s,
SO2NH2), 7.61-7.56 (m, 3H, ArHbenzene), 5.88 (1H, s, H-2), 3.38 (3H, s, OCH3). 13C NMR (150.92 MHz,
DMSO) δ: 179.36 (C-4), 156.52 (C-9), 152.87 (C-1’), 1495. (C-11), 145.55 (C-3’), 141.35 (C-7), 140.24
(C-6), 130.61 (C-5’), 123.58 (C-8), 121.10 (C-5), 120.34 (C-6’), 113.78 (C-4’), 111.05 (C-2’), 103.98
(C-10), 101.28 (C-3), 97.44 (C-2), 55.14 (OCH3).
2-{[(Z)-(6-fluoro-2-methoxy-4-oxo-2H-chromen-3(4H)-ylidene)methyl]amino}benzenesulfonamide
(14)
18

ACCEPTED MANUSCRIPT
Yield 87 %, m.p 172-174 °C. MS-EI m/z 378 (M+); MS (FAB+) 379.2. Anal. Calcd for C17H15FN2O5S:
1
C(53.96) H(4.00) N(7.40); found: C(54.23) H(4.13) N(7.49). H NMR (500MHz, DMSO)
δ: 12.2 (1H,
d, 3J = 12.1 Hz, NH), 8.05 (1H, d, 3J = 12.1 Hz, CH enamine), 7.85 (1H, dd, 3J = 7.5 Hz, 4J = 1.3 Hz, H-
3
3
3
3’), 7.64-7.65 (m, 3H, H-5, SO2NH2), 7.53 (dd, 1H, J = 8.4 Hz, J = 3.3 Hz, H-6’), 7.4 (1H, td, J =
8.6x(2) Hz, 4J = 3.3 Hz, H-7), 7.28 (1H, ddd, J = 8.1, 6.2, 1.8 Hz, H-5’), 7.16 (1H, dd, 3J = 9.0 Hz, 4J =
4.2 Hz, H-8), 5.87 (1H, s, H-2), 3.38 (3H, s, OCH3). 7.64-7.65 (m, 4H, H-4’, H-5’, SO2NH2).13C NMR
(150.92 MHz, DMSO) δ: 178.98 (C-4), 158.08 (C-6), 156.51 (C-9), 151.96 (C-1’), 144.54 (C-11),
137.52 (C-7), 133.74 (C-8), 131.49 (C-2’), 128.48 (C-5), 123.79 (C-5’), 121.83 (C-3’), 120.23 (C-4’),
117.85 (C-6’), 111.09 (C-10), 105.43 (C-3), 101.68 (C-2), 55.14 (OCH3).
4-{[(Z)-(6-chloro-2-methoxy-4-oxo-2H-chromen-3(4H)-ylidene)methyl]amino}benzenesulfonamide
(15)
Yield 76 %, m.p 164-166 °C. MS-EI m/z 394 (M+). Anal. Calcd for C17H15ClN2O5S: C(51.71) H(3.83)
1
3
N(7.10); found: C(52.27) H(3.87) N(7.14). H NMR (500MHz, DMSO)
δ
: 11.76 (1H, d, J = 12.6 Hz,
3
NH), 8.21 (1H, d, 3J = 12.6 Hz, CH enamine),7.8 (2H, d, J = 8.8 Hz, H-3’, H-5’), 7.76 (1H, d, 4J = 2.8
3
4
3
Hz, H-5), 7.59 (1H, dd, J = 8.7 Hz, J = 2.7 Hz, H-7), 7.55 (2H, d, J = 8.8 Hz, H-2’, H-6’), 7.28 (2H,
br s, SO2NH2), 7.18 (1H, d, 3J = 8.8 Hz, H-8), 5.88 (1H, s, H-2), 3.39 (3H, s, OCH3). 13C NMR (150.92
MHz, DMSO) δ: 179.04 (C-4), 154.35 (C-9), 145.33 (C-1’), 142.45 (C-4’), 139.35 (C-11), 134.39 (C-
7), 127.54 (C-3’, C-5’), 125.27 (C-6), 126.35 (C-5), 124.85 (C-10), 120.56 (C-8), 116.88 (C-2’, C-6’),
104.24 (C-3), 101.44 (C-2), 55.22 (OCH3).
3-{[(Z)-(6-chloro-2-methoxy-4-oxo-2H-chromen-3(4H)-ylidene)methyl]amino}benzenesulfonamide
(16)
Yield 83 %, m.p 177-179 °C. MS-EI m/z 360 (M+). Anal. Calcd for C17H15ClN2O5S: C(51.71) H(3.83)
1
3
N(7.10); found: C(51.82) H(3.85) N(7.13). H NMR (500MHz, DMSO)
δ
: 11.85 (1H, d, J = 12.9 Hz,
3
NH), 8.19 (1H, d, J = 12.6 Hz, CH enamine), 5.91 (1H, s, H-2), 7.37 (2H, br s, SO2NH2), 7.76 (1H, d,
3
4J = 2.8 Hz, H-5), 7.18 (1H, d, J = 8.8 H-8), 7.56-7.61 (4H, m, ArH benzene, H-7), 7.82 (1H, s, H-2’),
3.39 (3H, s, OCH3).13C NMR (150.92 MHz, DMSO)
δ: 178.90 (C-4), 154.32 (C-9), 145.77 (C-1’),
145.69 (C-11), 140.18 (C-3’), 134.28 (C-3’), 130.56 (C-6), 126.30 (C-5’), 124.82 (C-7), 123.91 (C-4’),
120.53 (C-8), 113.81 (C-2’), 103.84 (C-3), 101.39 (C-2), 55.17 (OCH3), 120.35 (C-10), 121.12 (C-5).
2-{[(Z)-(6-chloro-2-methoxy-4-oxo-2H-chromen-3(4H)-ylidene)methyl]amino}benzenesulfonamide
(17)
19

ACCEPTED MANUSCRIPT
Yield 87 %, m.p 188-190 °C. MS-EI m/z 360 (M+). Anal. Calcd for C17H15ClN2O5S: C(51.71) H(3.83)
1
3
N(7.10); found: C(51.79) H(3.88) N(7.16). H NMR (500MHz, DMSO)
δ
: 12.21 (1H, d, J = 12.3 Hz,
3
NH), 8.06 (1H, d, J = 12.3 Hz, CH enamine), 7.86 (1H, d, 3J = 7.3 Hz, H-7), 7.77 (1H, d, 4J = 2.5 Hz,
3
H-5), 7.64-7.65 (2H, m, ArHbenzene),7.63 (2H, br s, SO2NH2), 7.57 (1H, dd, J = 8.8 Hz, 4J = 2.8 Hz, H-
6’), 7.29 (1H, ddd, , J = 8.0 Hz, 5.7 Hz, 2.7 Hz, H-5’), 7.16 (1H, d , 3J = 8.5 Hz, H-8), 5.90 (1H, s, H-2),
3.39 (3H, s, OCH3). 13C NMR (150.92 MHz, DMSO)
δ: 178.56 (C-4), 154.42 (C-9), 144.70 (C-1’),
137.45 (C-11), 134.22 (C-2’), 133.70 (C-7), 131.52 (C-5’), 128.46 (C-6), 126.28 (C-3’), 125.06 (C-5),
124.02 (C-8), 123.83 (C-10), 120.48 (C-4’), 117.87 (C-6’), 105.29 (C-3), 101.80 (C-2), 55.19 (OCH3).
m/z (%): 359.9 (M+, 16), 297 (100), 280.9 (22), 269.0 (13), 206.0 (28), 181.9 (25.6), 172.0 (50), 154.9
(69), 125.9 (13.5), 108 (14.1), 91 (), 65 (31), 52 (12).
4-{[(Z)-(6,8-dichloro-2-methoxy-4-oxo-2H-chromen-3(4H)-
ylidene)methyl]amino}benzenesulfonamide (18)
Yield 74 %, m.p 179-181 °C. MS-EI m/z 429 (M+). Anal. Calcd for C17H14Cl2N2O5S: C(47.56) H(3.29)
1
3
N(6.53); found: C(47.61) H(3.31) N(6.57). H NMR (600MHz, DMSO)
δ: 11.77 (1H, d, J = 12.8 Hz,
NH), 8.28 (1H, d, 3J = 12.8 Hz, CHenamine), 7.88 (1H, d, 4J = 2.6 Hz, H-5), 7.81 (2H, d, J = 8.8 Hz, H-3’,
4
3
H-5’), 7.73 (1H, d, J = 2.6 Hz, H-7), 7.58 (2H, d, J = 8.8 Hz, H-2’, H-6’), 7.30 (2H, br s, SO2NH2),
13
6.01 (1H, s, H-2), 3.42 (3H, s, CH3). C NMR (150.92 MHz, DMSO)
δ: 177.93 (C-4), 150.12 (C-9),
146.17 (C-1’), 142.30 (C-4’), 139.65 (C-11), 133.73 (C-7), 127.55 (C-3’, C-5’), 126.35 (C-6), 124.93
(C-5), 124.03 (C-10), 123.73 (C-8), 117.17 (C-2’, C-6’), 103.68 (C-3), 102.07 (C-2), 55.48 (OCH3).
3-{[(Z)-(6,8-dichloro-2-methoxy-4-oxo-2H-chromen-3(4H)-
ylidene)methyl]amino}benzenesulfonamide (19)
Yield 80 %, m.p 175-177 °C. MS-EI m/z 429 (M+). Anal. Calcd for C17H14Cl2N2O5S: C(47.56) H(3.29)
1
N(6.53); found: C(47.59) H(3.32) N(6.55). H NMR (600MHz, DMSO)
δ
: 11.85 (1H, d, 3J = 12.5 Hz,
3
NH), 8.26 (1H, d, J = 12.8 Hz, CHenamine), 6.06 (1H, s, H-2), 7.39 (2H, br s, SO2NH2), 3.42 (3H, s,
CH3), 7.84 (1H, s, H-2’), 7.63-7.64 (1H, m, H-5’), 7.57-7.59 (2H, m, H-4’ benzene, H-6’benzene), 7.88 (1H,
4
4
d, J = 2.6 Hz, H-5), 7.73 (1H, d, J = 2.6 Hz, H-7).13C NMR (150.92 MHz, DMSO)
δ: 177.78 (C-4),
55.44 (OCH3), 150.09 (C-9), 146.52 (C-1’), 140.05 (C-11), 133.64 (C-3’), 130.60 (C-7), 126.32 (C-6),
124.98 (C-5’), 124.01 (C-8), 123.70 (C-5), 121.45 (C-6’), 120.66 (C-4’), 114.06 (C-2’), 105.24 (C-10),
103.30 (C-3), 102.03 (C-2).
2-{[(Z)-(6,8-dichloro-2-methoxy-4-oxo-2H-chromen-3(4H)-
ylidene)methyl]amino}benzenesulfonamide (20)
20

ACCEPTED MANUSCRIPT
Yield 87 %, m.p 189-191 °C. MS-EI m/z 429 (M+). Anal. Calcd for C17H14Cl2N2O5S: C(47.56) H(3.29)
1
3
N(6.53); found: C(47.59) H(3.34) N(6.55). H NMR (600MHz, DMSO) δ: 12.25 (1H, d, J = 12.5 Hz,
NH), 8.14 (1H, d, 3J = 12.5 Hz, CHenamine), 7.86-7.87 (2H, m, H-5, H-3’), 7.87 (1H, d, 4J = 2.6 Hz, H-5),
7.86 (1H, d, 4J = 1.1 Hz, H3’), 7.75 (1H, d, 4J =2.6 Hz, H-7), 7.66-7.67 (4H, m, ArHbenzene,(H-4’, H-5’),
13
SO2NH2), 7.31 (1H, ddd, J = 8.2 Hz, 5.6 Hz, 2.8 Hz, H-6’), 6.04 (1H, s, H-2), 3.42 (3H, s, CH3). C
NMR (150.92 MHz, DMSO) δ: 177.53 (C-4), 55.48 (OCH3), 150.18 (C-9), 145.81 (C-1’), 137.25 (C-
11), 133.76 (C-7), 131.73 (C-2’), 128.47 (C-5’), 126.31 (C-6), 125.08 (C-5), 124.26 (C-5’), 124.20 (C-
8), 123.84 (C-3’), 118.12 (C-10), 104.70 (C-3), 102.40 (C-2), 55.46 (OCH3).
4-{[(Z)-(2-methoxy-6-methyl-4-oxo-2H-chromen-3(4H)-
ylidene)methyl]amino}benzenesulfonamide (21)
Yield 80 %, m.p 180-182°C. MS-EI m/z 374 (M+). Anal. Calcd for C18H18N2O5S: C(57.74) H(4.85)
1
3
N(7.48); found: C(57.78) H(4.90) N(7.51). H NMR (500MHz, DMSO) δ: 11.77 (1H, d, J = 12.5 Hz,
NH), 8.13 (1H, d, 3J = 12.1 Hz, CH enamine), 7.79 (2H, d, 3J = 8.8 Hz, H-3’, H-5’), 7.62 (1H, d, 4J = 1.8
Hz, H-5), 7.52 (2H, d, 3J = 8.8 Hz, H-2’, H-6’), 7.35 (1H, dd, 3J = 5.1 Hz, 4J = 2.1 Hz, H-7), 7.27 (2H,
3
br s, SO2NH2), 7.0 (1H, d, J = 8.1 Hz, H-8), 5.80 (1H, s, H-2), 3.36 (3H, s, OCH3).13C NMR (150.92
MHz, DMSO) δ: 180.84 (C-4), 153.70 (C-9), 144.26 (C-1’), 142.70 (C-4’), 138.97 (C-11), 135.70 (C-
7), 131.20 (C-6), 127.61 (C-3’, C-5’), 125.57 (C-5), 122.35 (C-10), 118.82 (C-8), 116.56 (C-2’, C-6’),
105.04 (C-3), 101.17 (C-2), 54.98 (OCH3), 20.37 (ArCH3).
3-{[(Z)-(2-methoxy-6-methyl-4-oxo-2H-chromen-3(4H)-
ylidene)methyl]amino}benzenesulfonamide (22)
Yield 77 %, m.p 183-185 °C. MS-EI m/z 374 (M+). Anal. Calcd for C18H18N2O5S: C(57.74) H(4.85)
1
3
N(7.48); found: C(57.80) H(4.92) N(7.51). H NMR (500MHz, DMSO)
δ
: 11.88 (1H, d, J = 12.6 Hz,
3
NH), 8.12 (1H, d, J = 12.6 Hz, CH enamine), 5.83 (1H, s, H-2), 7.36 (2H, br s, SO2NH2), 7.78 (1H, s,
H-2’), 3.36 (3H, s, OCH3), 2.31 (3H, s, CH3), 7.62 (1H, d, 4J = 1.9 Hz, H-5), 7.35 (1H, dd, 3J = 6 Hz, 4J
= 2.5 Hz, H-7), 7.0 (1H, d, 3J = 8.2 Hz, H-8), 7.53-7.58 (3H, m, ArH benzene). 13C NMR (150.92 MHz,
DMSO) δ: 180.70 (C-4), 54.90 (OCH3), 20.35 (ArCH3), 153.65 (C-9), 145.76 (C-1’), 144.60 (C-11),
140.38 (C-3’), 135.54 (C-7), 131.11 (C-6), 130.55 (C-5), 125.53 (C-5’), 122.35 (C-2’), 120.72 (C-10),
120 (C-8), 118.05 (C-6’), 113.50 (C-4’), 104.58 (C-3), 101.10 (C-2).
2-{[(Z)-(2-methoxy-6-methyl-4-oxo-2H-chromen-3(4H)-
ylidene)methyl]amino}benzenesulfonamide (23)
21

ACCEPTED MANUSCRIPT
Yield 87 %, m.p 169-171 °C. MS-EI m/z 374 (M+). Anal. Calcd for C18H18N2O5S: C(57.74) H(4.85)
1
3
N(7.48); found: C(57.81) H(4.91) N(7.53). H NMR (500MHz, DMSO) δ: 12.19 (1H, d, J = 12.1 Hz,
3
3
NH), 7.97 (1H, d, J = 12.1 Hz, CH enamine), 7.85 (1H, d, J = 7.3 Hz, H-3’), 7.61-7.64 (4H, m, H-5,
ArHbenzene, SO2NH2), 7.34 (1H, dd, 3J = 8.4 Hz, 4J = 2.6 Hz, H-7), 7.26 (1H, ddd, J = 8.2 Hz, 6.0 Hz, 2.4
3
Hz, H-6’), 6.99 (1H, d, J = 8.1 Hz, H-8), 5.82 (1H, s, H-2), 3.36 (3H, s, OCH3). 13C NMR (150.92
MHz, DMSO) δ: 180.23 (C-4), 153.76 (C-9), 143.57 (C-11), 137.75 (C-1’), 135.46 (C-7), 133.70 (C-
2’), 131.26 (C-6), 131.09 (C-5), 128.48 (C-5’), 125.81 (C-3’), 123.39 (C-4’), 122.48 (C-6’), 123.39 (C-
10), 122.48 (C-8), 118.02, 117.57, 106.10 (C-3), 101.55 (C-2), 54.96(OCH3), 20.34 (ArCH3).
4-({(Z)-[4-oxo-2-(propan-2-yloxy)-2H-chromen-3(4H)-ylidene]methyl}amino)benzenesulfonamide
(39)
Yield 77 %, m.p 191-193 °C. MS-EI m/z 388 (M+). Anal. Calcd for C19H20N2O5S: C(58.75) H(5.19)
1
3
N(7.21); found: C(58.79) H(5.23) N(7.27). H NMR (300MHz, DMSO) δ: 11.81 (1H, d, J = 12.3 Hz,
NH), 8.12 (1H, d, 3J = 12.3 Hz, CH enamine), 6.03 (1H, s, H-2), 7.27 (2H, br s, SO2NH2), 7.85 (1H, dd,
3J = 7.8 Hz, 4J = 1.8 Hz, H-5), 7.81 (2H, d, 3J = 9.0 Hz, H-3’, H-5’), 7.56 (1H, m, H-7), 7.54 (2H, d, 3J =
3
3
9 Hz, H-2’, H-6’), 7.14 (1H, t, J = 6 Hz, H-6), 7.04 (1H, d, J = 8.1 Hz, H-8), 4.13 (1H, m, CH), 1.19
3
3
13
(3H, d, J = 6.3 Hz, CH3), 1.06 (3H, d, J = 6.3 Hz, CH3). C NMR (150.92 MHz, DMSO) δ: 180.77
(C-4), 155.85 (C-1’), 148.34 (C-9), 105.30 (C-3), 122.20 (10), 138.76 (C-4’), 127.44 (C-3’, C-5’),
116.39 (C-2’, C-6’), 135.21 (C-7), 118.11 (C-8), 121.82 (C-6), 125.69 (C-5), 69.76 (OCH), 23.16 (CH3),
22.03 (CH3), 98.67 (C-2), 143.74 (C-11).
3-({(Z)-[4-oxo-2-(propan-2-yloxy)-2H-chromen-3(4H)-ylidene]methyl}amino)benzenesulfonamide
(40)
Yield 68 %, m.p 189-191°C. MS-EI m/z 388 (M+). Anal. Calcd for C19H20N2O5S: C(58.75) H(5.19)
1
3
N(7.21); found: C(58.80) H(5.26) N(7.30). H NMR (500MHz, DMSO) δ: 11.90 (1H, d, J = 12.0 Hz,
NH), 8.11 (1H, d, 3J = 12.5 Hz, CH enamine), 6.07 (1H, s, H-2), 7.38 (2H, br s, SO2NH2), 7.84 (1H, d,
3J = 7.5 Hz, H-5), 7.78 (1H, s, H-2’), 7.58-7.51 (4H, m, ArH), 7.13 (1H, t, 3J = 7.5 Hz, H-6), 7.05 (1H,
3
3
3
d, J = 8.0 Hz, H-8), 4.11 (1H, m, CH), 1.18 (3H, d, J = 5.5 Hz, CH3), 1.02 (3H, d, J = 5.5 Hz,
CH3).13C NMR (150.92 MHz, DMSO)
: 180.65 (C-4), 155.81 (C-1’), 145.61 (C-3’), 140.26 (C-9),
δ
122.47 (C-10), 104.88 (C-3), 134.63 (C-2’), 130.43 (C-7), 125.66 (C-5), 121.78 (C-5’), 120.57 (C-6),
119.83 (C-6’), 118.09 (C-4’), 1313.36 (C-8), 69.73 (OCH), 23.19 (CH3), 22.10 (CH3), 144.12 (C-11),
98.67 (C-2).
22

ACCEPTED MANUSCRIPT
4-{[(Z)-(2-butoxy-4-oxo-2H-chromen-3(4H)-ylidene)methyl]amino}benzenesulfonamide
(41)
Yield 87 %, m.p 169-171°C. MS-EI m/z 402 (M+). Anal. Calcd for C20H22N2O5S: C(59.69) H(5.51)
1
3
N(6.96); found: C(59.73) H(5.55) N(6.71). H NMR (500MHz, DMSO) δ: 11.87 (1H, d, J = 12.5 Hz,
NH), 8.22 (1H, d, 3J = 12.0 Hz, CH enamine), 5.75 (1H, s, H-2), 7.26 (2H, br s, SO2NH2), 7.87 (1H, dd,
3J = 7.5 Hz, 4J = 1.8 Hz, H-5), 7.61 (2H, d, 3J = 8.5 Hz, H-3’, H-5’), 7.56 (1H, m, H-7), 7.53 (2H, d, 3J =
3
3
13
9.0 Hz, H-2’, H-6’), 7.45 (1H, t, J = 7.5 Hz, H-6), 7.38 (1H, d, J = 8.5 Hz, H-8). C NMR (150.92
MHz, DMSO) : 180.04 (C-4), 156.33 (C-1’), 148.29 (C-9), 104.53 (C-3), 122.80 (C-10), 138.65 (C-
δ
4’), 135.14 (C-7), 112.63 (C-8), 121.57 (C-6), 125.24 (C-5), 100 (C-2), 143.33 (C-11), 127.33 (C-3’, C-
5’), 112.36 (C-2’, C-6’), 60.30 (OCH2), 34.61 (CH2), 18.51 (CH2), 13.75 (CH3).
3-{[(Z)-(2-butoxy-4-oxo-2H-chromen-3(4H)-ylidene)methyl]amino}benzenesulfonamide
(42)
Yield 86 %, m.p 168-170 °C. MS-EI m/z 402 (M+). Anal. Calcd for C20H22N2O5S: C(59.69) H(5.51)
1
3
N(6.96); found: C(59.77) H(5.56) N(6.70). H NMR (500MHz, DMSO) δ: 11.89 (1H, d, J = 12.5 Hz,
NH), 8.14 (1H, d, 3J = 12.0 Hz, CH enamine), 5.96 (1H, s, H-2), 7.38 (2H, br s, SO2NH2), 7.84 (1H, dd,
3J = 7.7 Hz, 4J = 2.0 Hz, H-5), 7.78 (1H, s, H-2’), 7.58-7.49 (4H, m, ArH), 7.14 (1H, t, 3J = 7.0 Hz, H-6),
7.08 (1H, d, 3J = 8.5 Hz, H-8), 3.7-3.5 (2H, m, OCH2), 1.46-1.40 (2H, m, CH2), 1.20-1.15 (2H, m, CH2),
0.79 (3H, t, 3J = 7.5 Hz, CH3). 13C NMR (150.92 MHz, DMSO)
δ: 180.47 (C-4), 155.63 (C-1’), 145.57
(C-3’), 140.19 (C-9), 122.42 (C-10), 104.48 (C-3), 100.02 (C-2), 144.32 (C-11), 134.57 (57), 130.38 (C-
7), 125.60 (C-5), 121.74 (C-5’), 120.56 (C-6), 119.32 (C-6’), 118.0 (C-4’), 113.52 (C-8).
Bis-9-chloro-7H-chromeno[2,3-b][1,5]diazocin-7-one (49)
Yield 71 %. Anal. Calcd for C20H10N2O4: C(70.18) H(2.94) N(8.18); found: C(70.21) H(2.30) N(8.21).
δ
1H NMR (500MHz, DMSO) : 10.06 (2H, s, CHazomethine), 7.93 (2H, d, 4J = 2.5 Hz, H5), 7.75 (dd, 2H, 3J
4
3
13
= 8.8 Hz, J = 2.5 Hz, H7), 7.46 (2H, d, J = 8.8 Hz, H8). C NMR (150.92 MHz, DMSO)
δ: 187.95
(C4), 174.13 (C2), 165.51 (CH=N azomethine), 151.84 (C9), 133.88 (C7), 129.83 (C6), 124.42 (C5),
123.85 (C10), 119.45 (C8), 98.74 (C3).
Biochemical Assays
Alkaline Phosphatase Assay
A chemiluminescent substrate, CDP-star, was used for the determination of alkaline phosphatase activi-
ty of a compound against bovine kidney alkaline phosphatase (TNAP) enzyme and calf intestine alka-
23

ACCEPTED MANUSCRIPT
line phosphatase (IAP). The conditions for the assay were optimized with the slight modifications in
previously used spectrophotometric method [35]. The assay buffer which contained 8 M DEA (pH 9.8),
2.5 mM MgCl2 and 0.05 mM ZnCl2 was used.Initial screening was performed at a concentration of 0.2
mM of the tested compounds. The total volume of 50 µL contained 10 µL of tested compound (0.2 mM
with final DMSO 1% (v/v)), followed by the addition of 20 µL of TNAP (1:800 times diluted (0.8
units/mL) enzyme in assay buffer) or 20 µL of IAP (1:800 times diluted (1 unit/mL) enzyme in assay
buffer). The mixture was pre-incubated for 3-5 minutes at 37 oC and luminescence was measured as pre-
read using microplate reader (BioTek FLx800, Instruments, Inc. USA).Then, 20 µL of CDP-star (final
concentration of 110 µM) was added to initiate the reaction and the assay mixture was incubated again
o
for 15 min at 37 C. The change in the luminescence was measured as after-read. The activity of each
compound was compared with total activity control (without any inhibitor). Levamisole (2 mM per
well) and L-phenylalanine (4 mM per well) were used as a positive control against tissue-nonspecific
alkaline phosphatase (TNAP) and calf intestinal alkaline phosphatase (IAP), respectively. For potential-
ly active compounds, full concentration inhibition curves were produced. The compounds which exhib-
ited over 50% inhibition of either the tissue-nonspecific alkaline phosphatase (TNAP) activity or calf
intestinal alkaline phosphatase (IAP) activity were further evaluated for determination of IC50 values.
For this purpose 6 to 8 serial dilutions of each compound (2mM to 20 nM) were prepared in assay buff-
er and their dose response curves were obtained by assaying each inhibitor concentration against both
ALPs using the above mentioned reaction conditions. All experiments were repeated in triplicate. The
Cheng Prusoff equation was used to calculate the IC50values, determined by the non-linear curve fitting
program PRISM 5.0 (GraphPad, San Diego, California, USA).
Kinetics Study of Alkaline Phosphatase Inhibitors
Changes in the initial velocities of the reaction were measured at different concentrations of inhibitor (0
µM, 0.5 µM, 1.00 µM, 2.00 µM) using substrate (CDP-star) concentrations of 55µM, 110 µM, 165 µM
and 220 µM. A double-reciprocal plot of the inhibition kinetics of TNAP by 23 and IAP by inhibitor 9
was measured by using PRISM 5.0 (GraphPad, San Diego, California, USA),
Ecto-5′-nucleotidase Assay
At the beginning of each day, the capillary was conditioned with the following sequence to obtain the
highly reproducible migration times and good separation peaks: (i) rinse with 0.1 N NaOH for 5 min (ii)
rinse with distilled water for 5 min and (iii) rinse with running buffer (sodium tetra borate 20mM, pH
9.00) for 5 min. The bioactivity assay of both rat and human Ecto-5′-Nucleotidase was performed with
the slight modifications in the method as described previously [36, 37]. Stock solutions of 10 mM con-
24

ACCEPTED MANUSCRIPT
centration of each compound were prepared in DMSO. Working solutions were prepared by appropri-
ately diluting the stock solutions to 1mM concentration in assay buffer (2 mM MgCl2, 10 mM Tris HCl
and 1 mM CaCl2, pH 7.4). The volume of the final assay was kept 100 µL. A sample of 10 µL of each
tested compound was pre-incubated with 10 µL of human ecto-5′-nucleotidase (6.94 µg/mL) protein ex-
tract or rat ecto-5′-nucleotidase (7.17 µg/mL) for 10 min in the presence of 70 µL assay buffer (2 mM
MgCl2, 10 mM Tris HCl and 1 mM CaCl2, pH 7.4). Enzymatic reaction was initiated by the addition of
10 µL of AMP (adenosine monophosphate) as the substrate at a final concentration of 500 µM. The re-
action mixture was allowed to incubate for 10 min at 370C. To stop the enzymatic reaction, the reaction
mixture was quenched by thermal denaturation at 990C for 20 min. Aliquots of 50 µL of each reaction
mixture were transferred to CE minivial and injected into the CE instrument for data collection and
analysis. Prior to each injection, the capillary was rinsed with 0.1 N NaOH for 2 min, distilled water for
2 min, and running buffer (sodium tetra borate 20 mM, pH 9.00) for 2 min. The enzyme reaction mix-
ture was hydrodynamically injected into the capillary by applying pressure of 0.5 psi for 5s, followed by
the application of 15 kV voltage for separation of peaks of substrate and product. The concentration of
product i.e. adenosine was determined by calculating the area under its absorbance peak at 260 nm. For
active compounds, full concentration inhibition curves were produced. The compounds which exhibited
over 50% inhibition of either the rat ecto-5′-nucleotidase activity or human ecto-5′-nucleotidase activity
were further evaluated for determination of IC50 values. For this purpose serial dilutions of each com-
pound were prepared in assay buffer and their dose response curves were obtained by assaying each
inhibitor concentration against both nucleotidases using the above mentioned reaction conditions. All
experiments were performed in triplicate. The Cheng Prusoff equation was used to calculate the IC50
values, determined by the non-linear curve fitting program PRISM 5.0 (GraphPad, San Diego, Califor-
nia, USA).
Kinetics Study of ecto-5′-Nucleotidase Inhibitors
In order to determine the type of inhibition, kinetics experiments were carried out. For this purpose
changes in the initial velocities of the reaction were measured at different concentrations of the inhibitor
(0 µM, 0.5 µM, 1.00 µM, 2.00 µM) using substrate (AMP) at concentrations of 400 µM, 500 µM, 600
µM and 800 µM. A double-reciprocal plot of the inhibition kinetics of he5NT by 40 and re5NT by in-
hibitor 35 was measured by using PRISM 5.0 (GraphPad, San Diego, California, USA).
25

ACCEPTED MANUSCRIPT
Supporting Information. NMR spectra and summary of single crystal XRD data collection and re-
finement parameters for compound 42(a), R-isomer and compound 42(b), S-isomer are given in sup-
porting information. Crystallographic data for compounds 42(a) and 42(b) reported in this paper has
been deposited with the Cambridge Crystallographic Data Center, CCDC No. 1059015 and 1059016
respectively. Copies of the data can be obtained free of charge on application to The Director, CCDC, 12
Union Road, Cambridge, UK, fax: +44 1223 336033, e-mail: deposit@ccdc.cam.ac.uk or http:
www.ccdc.cam.ac.uk.
ACKNOWLEDGMENT
M. al-Rashida is grateful to HEC for providing funds to support this research via HEC NRPU Project
No. 20-3133/NRPU/R&D/HEC/13/675. J. Iqbal is thankful to the Organization for the Prohibition of
Chemical Weapons (OPCW), The Hague, The Netherlands and Higher Education Commission of Paki-
stan for the financial support through Project No. 20-3733/NRPU/R&D/ 14/520 for the financial sup-
port. JS received support from the Canadian Institutes of Health Research (CIHR) and was also a recipi-
ent of a “Chercheur National” research award from the Fonds de recherche du Québec – Santé (FRQS).
ABBREVIATIONS
AP, alkaline phosphatase; TNAP, tissue non-specific alkaline phosphatase, IAP, intestinal alkaline phos-
phatase, he5NT, human ecto-5′-nucleotidase; r5NT, rat ecto-5′-nucleotidase; 4-, 3-, 2-ABS, 4-, 3-, 2-
aminobezenesulfonamide; E-NPPs, pyrophosphatase/phosphodiesterases; E-NTPDases, ecto-nucleoside
triphosphate diphosphohydrolases.
REFERENCES
[1] G.G. Yegutkin. Biochim. Biophys. Acta. 1783 (2008) 673-94.
[2] M. al-Rashida, J. Iqbal. Med. Res. Rev. 34 (2014) 703-43.
[3] H. Zimmermann, M. Zebisch, N. Sträter. Purinergic Signal. 8 (2012) 437-502.
[4] G. Burnstock. Cell. Mol. Life Sci. 64, (2007), 1471–1483.
[5] R. Resta, Y. Yamashita, L. F. Thompson. Immunol. Rev. 161 (1998) 95-109.
[6] B. P. Klyuch, N. Dale, M. J. Wall. J. Neurosci. 32 (2012) 3842-7.
[7] C. D. Bonan. CNS Neurol. Disord. Drug Targets. 11 (2012) 739-50.
26

ACCEPTED MANUSCRIPT
[8] Z. Peng, P. Fernandez, T. Wilder, H. Yee, L. Chiriboga, E. S. Chan, B. N. Cronstein. FASEB J.
22 (2008) 2263-72.
[9] P. Koszalka, B. Ozüyaman, Y. Huo, A. Zernecke, U. Flögel, N. Braun, A. Buchheiser, U. K.
Decking, M. L. Smith, J. Sévigny, A. Gear, A. A. Weber, A. Molojavyi, Z. Ding, C. Weber, K. Ley,
H. Zimmermann, A. Gödecke, J. Schrader. Circ. Res. 95 (2004) 814-21.
[10] L. Wang, J. Fan, L. F. Thompson, Y. Zhang, T. Shin, T. J. Curiel, B. Zhang. J. Clin. Invest. 121
(2011) 2371-82.
[11] L. Wang, S. Tang, Y. Wang, S. Xu, J. Yu, X. Zhi, Z. Ou, J. Yang, P. Zhou, Z. Shao. Clin. Exp.
Metastasis. 30 (2013) 671-80.
[12] L. F. Thompson, H. K. Eltzschig, J. C. Ibla, C. J. Van De Wiele, R. Resta, J. C. Morote-Garcia,
S. P. Colgan. J. Exp. Med. 200 (2004) 1395-405.
[13] B. Allard, M. Turcotte, K. Spring, S. Pommey, I. Royal, J. Stagg. Int. J. Cancer. 134 (2014)
1466-73.
[14] J. L. Millán. Purinergic Signal., 2 (2006) 335-41.
[15] R. Han, J. E. Coleman. Biochemistry. 34 (1995) 4238-45.
[16] M. G. Roig, F. J. Burguillo, A. Del Arco, J. L. Usero, C. Izquierdo, M. A. Herraez. Int. J. Bio-
chem, 14 (1982) 655-66.
[17] J. E. Coleman. Annu. Rev. Biophys. Biomol. Struct. 21 (1992) 441-83.
[18] C. E. Ovitt, A. W. Strauss, D. Alpers, J. Y. Chou, I. Boime. Proc. Natl. Acad. Sci. U S A. 83
(1986) 3781–3785.
[19] M. C. Hofmann, J. L. Millán. Eur. Urol. 23 (1993) 38-44.
[20] J. P. Lallès. Nutr. Rev. 72 (2014) 82–94.
[21] H. Orimo. J. Nippon Med. Sch. 77 (2010) 4-12.
[22] B. L. Foster, K. J. Nagatomo, H. W. Tso, A. B. Tran, F. H. Nociti Jr, S. Narisawa, M. C.
Yadav, M. D. McKee, J. I. Millán, M. J. Somerman. J. Bone Miner. Res. 28 (2013) 271-82.
[23] C. W. Hayes, W. F. Conway. Radiographics. 10 (1990) 1031-48.
[24] M. Schoppet, C. M. Shanahan. Kidney Int. 73 (2008) 989-91.
[25] M. A. Ibrahim, T. El
311 328.
‐Sayed Ali, N. M. El‐Gohary, A. M. El‐Kazak. Eur. J. Chem. 4 (2013)
‐
[26] H. M. el-Shaaer, P. Foltínová, M. Lácová, J. Chovancová, H. Stankovicová. Farmaco. 53
(1998) 224-32.
[27] M. al-Rashida, M. Ashraf, B. Hussain, S. A. Nagra, G. Abbas. Bioorg. Med. Chem. 19 (2011)
3367-3371.
[28] M. al-Rashida, M.N Tahir, S.A. Nagra, M. Imran, J. Iqbal. Acta Crystallogr. Sect. E. Struct.
Rep. Online. 2009, E65, o1818-o1819.
27

ACCEPTED MANUSCRIPT
[29] M. al-Rashida, R. Raza, G. Abbas, M. S. Shah, G. E. Kostakis, J. Lecka, J. Sévigny, M.
Muddassar, C. Papatriantafyllopoulou, J. Iqbal. Eur. J. Med. Chem. 66 (2013) 438-449.
[30] M. al-Rashida, S.A. Ejaz, S. Ali, A. Shaukat, M. Hamayoun, M. Ahmed, J. Iqbal. Bioorg Med
Chem. 23 (2015) 2435-44.
[31] HyperChem 8.0, Hypecube Inc., Gainesville, FL, USA. [http://www.hyper.com/]
[32] J. Wang, W. Wang, P. A. Kollman, D. A. Case. J. Mol. Graph Model. 25 (2006) 247260.
[33] E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng, T. E.
Ferrin, J. Comput. Chem., 25 (2004) 1605.
[34] http://www.biosolveit.de/LeadIT/.
[35] S. Narisawa, D. Harmey, M. C. Yadav, W. C. O’Neill, M. F. Hoylaerts, J. L. Millán. J. Bone
Miner. Res.22 ( 2007) 1700–1710.
[36] J. Iqbal, D. Jirovsky, S. Y. Lee, H. Zimmermann, C. E. Müller. Anal. Biochem. 373 (2008)
129-140.
[37] R. Raza, A. Saeed, J. Lecka, J. Sevigny, J. Iqbal. Med. Chem. 8 (2012) 1573-4064.
28