Tricyclic coumarin sulphonate derivatives with alkaline phosphatase inhibitory effects: in vitro and docking studies

Article abstract of DOI:10.1080/14756366.2018.1428193

Tissue-nonspecific alkaline phosphatase (TNAP) is an important isozyme of alkaline phosphatases, which plays different pivotal roles within the human body. Most importantly, it is responsible for maintaining the balanced ratio of phosphate and inorganic pyrophosphate, thus regulates the extracellular matrix calcification during bone formation and growth. The elevated level of TNAP has been linked to vascular calcification and end-stage renal diseases. Consequently, there is a need to search for highly potent and selective inhibitors of alkaline phosphatases (APs) for treatment of disorders associated with the over-expression of APs. Herein, a series of tricyclic coumarin sulphonate 1a-za with known antiproliferative activity, was evaluated for AP inhibition against human tissue nonspecific alkaline phosphatase (h-TNAP) and human intestinal alkaline phosphatase (h-IAP). The methylbenzenesulphonate derivative 1f (IC₅₀ = 0.38 ± 0.01 μM) was found to be the most active h-TNAP inhibitor. Another 4-fluorobenzenesulphonate derivative 1i (IC₅₀ = 0.45 ± 0.02 μM) was found as the strongest inhibitor of h-IAP. Some of the derivatives were also identified as highly selective inhibitors of APs. Detailed structure-activity relationship (SAR) was investigated to identify the functional groups responsible for the effective inhibition of AP isozymes. The study was also supported by the docking studies to rationalise the most possible binding site interactions of the identified inhibitors with the targeted enzymes.

Full text of DOI:10.1080/14756366.2018.1428193

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Tricyclic coumarin sulphonate derivatives with
alkaline phosphatase inhibitory effects: in vitro
and docking studies
Jamshed Iqbal, Mohammed I. El-Gamal, Syeda Abida Ejaz, Joanna Lecka, Jean
Sévigny & Chang-Hyun Oh
To cite this article: Jamshed Iqbal, Mohammed I. El-Gamal, Syeda Abida Ejaz, Joanna Lecka,
Jean Sévigny & Chang-Hyun Oh (2018) Tricyclic coumarin sulphonate derivatives with alkaline
phosphatase inhibitory effects: in vitro and docking studies, Journal of Enzyme Inhibition and
Medicinal Chemistry, 33:1, 479-484, DOI: 10.1080/14756366.2018.1428193
To link to this article: https://doi.org/10.1080/14756366.2018.1428193
© 2018 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
View supplementary material
Submit your article to this journal
View related articles
Published online: 02 Feb 2018.
Article views: 19
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2018
VOL. 33, NO. 1, 479–484
https://doi.org/10.1080/14756366.2018.1428193
RESEARCH PAPER
Tricyclic coumarin sulphonate derivatives with alkaline phosphatase inhibitory
effects: in vitro and docking studies
a
b,c,d
a
e,f
e,f
ꢀ
ꢀ
ꢀ
Jamshed Iqbal , Mohammed I. El-Gamal
, Syeda Abida Ejaz , Joanna Lecka , Jean Sevigny and
Chang-Hyun Ohg,h
b
aCentre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad, Pakistan; Department of Medicinal Chemistry,
c
College of Pharmacy, University of Sharjah, Sharjah, United Arab Emirates; Sharjah Institute for Medical Research, University of Sharjah, Sharjah,
d
e
ꢀ
United Arab Emirates; Department of Medicinal Chemistry, University of Mansoura, Mansoura, Egypt; Departement de microbiologie-
f
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
infectiologie et d'immunologie, Faculte de Medecine, Universite Laval, Quebec, Canada; Centre de Recherche du CHU de Quebec, Universite
Laval, Quebec, Canada; Center for Biomaterials, Korea Institute of Science and Technology, Seoul, Republic of Korea; Department of
Biomolecular Science, University of Science and Technology, Daejeon, Republic of Korea
g
h
ꢀ
ABSTRACT
ARTICLE HISTORY
Received 12 December 2017
Revised 11 January 2018
Accepted 11 January 2018
Tissue-nonspecific alkaline phosphatase (TNAP) is an important isozyme of alkaline phosphatases, which
plays different pivotal roles within the human body. Most importantly, it is responsible for maintaining the
balanced ratio of phosphate and inorganic pyrophosphate, thus regulates the extracellular matrix calcifica-
tion during bone formation and growth. The elevated level of TNAP has been linked to vascular calcifica-
tion and end-stage renal diseases. Consequently, there is a need to search for highly potent and selective
inhibitors of alkaline phosphatases (APs) for treatment of disorders associated with the over-expression of
APs. Herein, a series of tricyclic coumarin sulphonate 1a-za with known antiproliferative activity, was eval-
uated for AP inhibition against human tissue nonspecific alkaline phosphatase (h-TNAP) and human intes-
tinal alkaline phosphatase (h-IAP). The methylbenzenesulphonate derivative 1f (IC50 ¼ 0.38 0.01 lM) was
found to be the most active h-TNAP inhibitor. Another 4-fluorobenzenesulphonate derivative 1i
(IC50 ¼ 0.45 0.02 lM) was found as the strongest inhibitor of h-IAP. Some of the derivatives were also
identified as highly selective inhibitors of APs. Detailed structure-activity relationship (SAR) was investi-
gated to identify the functional groups responsible for the effective inhibition of AP isozymes. The study
was also supported by the docking studies to rationalise the most possible binding site interactions of the
identified inhibitors with the targeted enzymes.
KEYWORDS
Alkaline phosphatase
inhibitor; coumarin;
molecular docking;
structure-activity
relationship; Tricyclic
coumarin sulfonate
Introduction
by detoxifying the bacterial toxins7. TNAP is widely distributed
almost in each body part but is abundantly present in central ner-
vous tissues, mineralising tissues and also in kidney8. As compared
to the other body tissues, high concentration of TNAP is present
in the mineralising tissues such as teeth and bones for the normal
teeth and bone formation. But too high concentrations of TNAP in
mineralising tissue results in abnormal calcification and mineral
deposition9. The identification of effective inhibitors of AP iso-
zymes becomes an emerging drug target for the disorders related
to the hyper-activity of TNAP and IAP. To date, different inhibitors
of APs based on triazole, pyrazoles, coumarin sulphonates, diaryl
sulphonamides, and chromones have been identified as effective
Alkaline phosphatases (APs, E.C. 3.1.3.1) are membrane-bound
ecto-enzymes with an extracellular-oriented active site1. They
belong to the large family of ecto-enzyme known as ecto-nucleoti-
dases, and are involved in various physiological functions within
the human body, most importantly in phosphorylation and
dephosphorylation reactions2. They are also responsible for the
hydrolysis and breakdown of wide variety of nucleoside tri- and
di-phosphates substrates to their respective monophosphates3,4
.
They also hydrolyse many other phosphate-containing substrates,
such as inorganic pyrophosphate (PPi), glucose phosphate, poly-
phosphates, phosphomonoesters, and phosphatidates5. APs are
further categorised into two types, the tissue-specific and tissue-
nonspecific alkaline phosphatases (TNAPs). From the tissue-specific
AP type, intestinal alkaline phosphatase (IAP) plays a pivotal role
in the maintenance of physiological environment of the intestine6.
Moreover, this isozyme is important for the regulation of bicarbon-
ate secretion balance, duodenal luminal pH, dephosphorylation
inhibitors of TNAP and IAP (Figure 1)10–13
.
Recently, we have reported coumarinyl sulphonates derivatives
as novel and potent inhibitors of h-TNAP and h-IAP13. In continu-
ation of our efforts for searching the effective and selective inhibi-
tors, herein we evaluated a series of 27 tricyclic-fused coumarin
sulphonate derivatives 1a-za as inhibitors of APs which were
already tested for their antiproliferative and anti-inflammatory activ-
reaction, and helps in the maintenance of normal gut environment ities14–17. We assumed that the fused cycloheptane or cyclooctane
CONTACT Chang-Hyun Oh
choh@kist.re.kr
Center for Biomaterials, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650,
Republic of Korea
ꢀ
These authors contributed equally to this work.
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

480
J. IQBAL ET AL.
Figure 1. APs inhibitors; (I) Levamisole (II) Theophylline (III) Aryl sulphonamide-based inhibitors (IV) trizole-based inhibitors (V) Pyrazole-based Inhibitors, (VI) coumarinyl
sulphonates.
Figure 2. 3D binding interactions of most potent inhibitor 1f within the active site of h-TNAP. Hydrogen bonds are shown in green dotted line while p–p interactions
are shown in purple dotted line.
ring might contribute to stronger affinity than the previously
reported bicyclic coumarin sulphonate derivatives through forma-
tion of additional hydrophobic interaction(s). Our assumption suc-
ceeded as shown in Figure 2. Except two derivatives 1w and 1 y, all
the other compounds were found as effective AP inhibitors with
sub-micromolar or very low micro-molar range IC50 values. Further,
the most effective inhibitors of either h-TNAP or h-IAP were
selected for the molecular docking studies to rationalise the pos-
sible binding interactions with the respective targeted enzymes.
Bioactivity protocol
Alkaline phosphatase inhibition assay (h-TNAP and h-IAP)
The inhibition assay was performed by following the already
reported method18 and the inhibitory activity was measured at
the final concentration of 200 mM in diethanolamine (DEA) buffer
as discussed in our published article19. The first step of the
inhibition assay was the treatment of 10 ml of tested derivatives
with 20 ml of h-TNAP (46 ng/well) or of h-IAP (57 ng/well). The
plates were incubated at 37 ꢁC for 5–10 min, and luminescence
Experimental
signals were recorded by microplate reader (BioTek FLx800,
R
Instruments, Inc., Winooski, VT). Then, 20 ml of CDP-starV sub-
Synthesis of the target molecules 1a-za
strate was added to each reaction mixture and signals were
again measured as after-read, after 15–20 min of incubation. The
data were scrutinised by PRISM 5.0 (GraphPad, San Diego, CA)
software and the inhibitory concentration values (IC50 values)
were attained as discussed earlier.
The synthetic procedures, purification methods, and the spectral
analysis data have been reported14–17. The experimental details as
well as representative analysis charts are provided in the supple-
mentary file.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
481
Molecular docking studies
potential in the range of IC50 ¼ 0.38 0.01 to 8.57 0.73 lM as
compared to standard levamisole (IC50 ¼ 20.21 1.9 lM), and h-IAP
inhibitory activity in the range of IC50 ¼ 0.45 0.09 to
11.8 1.91 lM as compared to standard L-phenylalanine
(IC50 ¼ 100.1 3.15 lM) (Table 1).
Molecular docking studies of compounds 1f and 1i, potent inhibi-
tors of h-TNAP and h-IAP, respectively, were conducted to investi-
gate the probable binding interactions of these compounds within
the active site of the respective target enzyme. Our previously
reported models of h-TNAP and h-IAP were used for docking cal-
culation20. Before performing docking studies, protonation and
energy minimisation of all enzyme structures were carried out
Structure-activity relationship (SAR)
In order to find out the comprehensive structure-activity
relationship for AP inhibition, all tricyclic-fused coumarin sulpho-
nates 1a-za were divided into two categories, on the basis of
fused cycloheptane and cyclooctane ring. Moreover, the detailed
structure-selectivity relationship was also rationalised on the basis
of effect of unsubstituted, and chloro- and methoxy-substituted
derivatives on the inhibition of both APs. Compounds 1q and 1z
were found as selective inhibitors of h-TNAP while none of the
derivative was found as a selective inhibitor of IAP.
using molecular operating environment (MOE) 2014, 09 software21
.
Structure generation as well as energy minimisation of the effect-
ive inhibitors were also carried out via MOE software. After prepar-
ation of all the prerequisite compounds and enzymes structures,
total 50 independent docking runs for each ligand were executed
by MOE docking program. The best scored pose of potent com-
pounds in each target enzyme was selected for evaluation of bind-
ing interactions. Binding free energy for each pose was also
determined and pose with the lowest free binding energy was
selected for further visualisation studies. 3D visualisation of bind-
ing interaction between potent compound and respective target
was carried out using Discovery studio visualizer v422.
Among the tested derivatives, compound 1f expressed max-
imum inhibitory effects on h-TNAP and compound 1i depicted the
strongest potency against h-IAP. From the unsubstituted deriva-
tives, except compound 1c, 1g, and 1m, the other target deriva-
tives expressed varying inhibitory effects on h-TNAP. Compounds
1f, 1a, and 1d possessing p-toluene sulphonate, methane sul-
phonate, and cyclopropane substitution, respectively, depicted
higher inhibition of h-TNAP than compounds 1c, 1g, and 1j-m.
Among the cyclooctane-fused derivatives, compound 1l with ben-
zenesulphonate substitution demonstrated the strongest inhibitory
potential on h-TNAP (IC50 SEM ¼ 1.13 0.21 mM). Compounds 1c
with the fused cycloheptane ring and compound 1j with the fused
cyclooctane ring were found as stronger inhibitors of h-IAP with
the inhibitory values of IC50 SEM ¼ 0.91 0.06 and 0.84 0.09 mM,
respectively. Among the cyclooctane-fused derivatives, compound
1j with ethanesulphonate substitution demonstrated the strongest
inhibitory effect on h-IAP (IC50 SEM ¼ 0.84 0.09 mM). From the
cycloheptane-fused derivatives, compound 1c with propanesulph-
onate substitution demonstrated the strongest inhibitory potential
on h-IAP (IC50 SEM ¼ 0.91 0.06 mM). Although more lipophilic
side chain was substituted in 1c, the inhibitory effect of this
derivative was found less because of the presence of seven-mem-
bered fused ring. Moreover, from the obtained results this may be
rationalised that the presence of more lipophilic side chain in
compound 1c may form stronger hydrophobic interactions with
the target enzyme.
Results and discussion
Chemistry
Scheme 1 illustrates the synthetic pathway via which the target
compounds 1a-za were prepared. Cycloheptanone (2a) or cyclooc-
tanone (2b) was reacted with diethyl carbonate in the presence of
NaH in benzene at the reflux temperature to get the ester inter-
mediates 3a,b that exist in keto-enol tautomers23. Subsequent
reaction with (substituted) resorcinol in the presence of trifluoro-
acetic acid and concentrated sulphuric acid led to the production
of the tricyclic phenolic intermediates 4a-f24. They were used in
the next step as such with no prior purification. In the last step,
those phenolic intermediates were treated with the appropriate
sulphonyl chloride reagent in the presence of triethylamine as a
catalyst to get the target sulphonate products 1a-za14–17. Table 1
illustrates the structure of each target compound.
Alkaline phosphatase inhibition
All synthesised tricyclic coumarin sulphonate derivatives 1a-za
were tested for their potential to inhibit h-TNAP and h-IAP. Except
two compounds (1w and 1 y), all compounds were found to be
active against both isozymes of AP with IC50 values in the lower
The inhibitory effect by other derivatives; 1h and 1n with tert-butyl-
benzene suphonate moiety expressed the dual inhibition of both iso-
zymes of APs. The derivative 1n depicted more inhibitory potential
micro-molar range. Compounds showed their h-TNAP inhibitory because of the presence of fused eight membered ring (h-TNAP;
R1
R1
n
n
ii
i
iii
O
S
O
O
R2
O
n
n
n
HO
O
O
O
O
O
O
O
O
HO
O
4a-e
1a-za
2a,b
3a,b
a: n = 1
b: n = 2
4a: R1 = H, n = 1
4b: R1 = H, n = 2
4c: R1 = OMe, n = 1
4d: R1 = OMe, n = 2
4e: R1 = Cl, n = 1
n = 1, 2
R1 = H, OMe, Cl
R2 = Me, Et, n-Pr, c-Pr, Ph, p-tolyl, p-(F)C6H4, p-(CF3)C6H4, p-(tert-butyl)C6H4
Scheme 1. Reagents and conditions: (i) diethyl carbonate, NaH, benzene, reflux, 90% (3a, n ¼ 1), 85% (3 b, n ¼ 2); (ii) (substituted) resorcinol, CF3COOH, conc. H2SO4,
0 ꢁC; rt, 3 h; (iii) appropriate sulphonyl chloride derivative, triethylamine, CH2Cl2, 0 ꢁC; rt, 1 h.

482
J. IQBAL ET AL.
Table 1. Structures of the target compounds 1a-za along with their yield percentages and in vitro inhibitory effects (IC50, lM) against h-TNAP and h-IAP.
R1
n
O
S
O
R2
O
O
O
IC50 (mM) SEMa
Compound No.
R1
R2
n
Yield%
h-TNAP
h-IAP
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
1s
1t
1u
1v
1w
1x
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
Cl
Me
Et
n-Pr
Cyclo-Pr
Ph
p-Tolyl
p-(CF3)C6H4
p-(tert-butyl)C6H4
p-(F)C6H4
Et
n-Pr
1
1
1
1
1
1
1
1
1
2
2
2
2
2
1
2
1
2
1
2
1
2
1
2
1
95
92
93
85
93
95
90
95
82
90
88
92
95
94
85
85
95
91
92
87
96
90
87
85
85
0.48 0.01
2.13 0.19
1.52 0.17
0.98 0.13
8.57 0.73
0.38 0.01
1.59 0.12
6.33 0.99
1.06 0.11
1.74 0.14
1.91 0.11
1.13 0.21
1.59 0.16
1.85 0.13
1.21 0.11
1.49 0.13
4.31 0.99
2.60 0.49
1.26 0.07
2.68 0.18
1.65 0.13
0.64 0.02
>100
1.98 0.19
2.71 0.23
0.91 0.06
0.96 0.08
1.84 0.19
1.94 0.11
2.64 0.45
3.92 0.47
0.45 0.09
0.84 0.09
3.37 0.79
2.82 0.82
2.12 0.23
1.28 0.13
11.80 1.91
1.51 0.17
>100
0.75 0.07
3.88 0.32
0.83 0.06
0.79 0.05
1.45 0.04
>100
Ph
p-Tolyl
p-(tert-butyl)C6H4
Me
Me
n-Pr
n-Pr
Ph
Ph
p-Tolyl
p-Tolyl
p-(CF3)C6H4
p-(CF3)C6H4
Me
1.29 0.12
>100
1.63 0.19
>100
1y
1z
1za
Cl
Cl
——
——
Ph
p-Tolyl
——
1
1
85
86
——
——
5.73 1.06
2.22 0.27
20.2 1.90
——
>100
1.11 0.01
——
Levamisole
L-Phenylalanine
——
——
——
100 3.15
aIC50 is the concentration at which the 50% of the enzyme activity was inhibited. All the values were expressed as IC50 SEM (standard error mean), n ¼ 3.
IC50 SEM ¼ 1.85 0.13 mM, h-IAP; IC50 SEM ¼ 1.28 0.13 mM). In com- i.e. 1t having the same substitution of benzenesulphonate as in
parison to 1h, the replacement of tert-butylbenzene with flouoroben- compound 1s, but with the cyclooctane ring, a reduced inhibitory
zenesulphonate results in the most potent inhibitor of h-IAP i.e. 1i. effect on TNAP was observed with an increased inhibitory effect
Compound 1i was identified as the strongest inhibitor of h-IAP with on IAP isozymes (h-TNAP; (IC50 SEM ¼ 2.68 0.18 mM, h-IAP;
and inhibitory value of IC50 SEM ¼ 0.45 0.09 mM. The increased IC50 SEM ¼ 0.83 0.06 mM). The derivatives 1w and 1x having the
potency might be due to the presence of most electronegative same substitution at R1 and R2 positions but with cycloheptane
halogen.
On the other hand, the chloro- and methoxy-substituted deriv- these two derivatives was considered, it was observed that cyclo-
atives were found more active against both APs than the unsubsti-
and cyclooctane ring in 1w and 1x, respectively. When the SAR of
heptane derivative 1w was found less reactive than that of the
tuted analogues. With reference to h-TNAP, compound 1o cyclooctane derivative 1x which inhibited both isozymes effect-
(IC50 SEM ¼ 1.21 0.11 mM) was found more potent as compared ively. This inhibitory effect confirms that like 1p, the presence of
to compound 1q (IC50 SEM ¼ 4.31 0.99 mM), 1p (IC50 SEM ¼
more carbon number in the derivative is responsible for the more
1.49 0.13 mM), and 1r (IC50 SEM ¼ 2.60 0.49 mM) but with refer- inhibition.
ence to h-IAP, 1p (IC50 SEM ¼ 1.51 0.17 mM) was found more
better than 1o (IC50 SEM ¼ 11.8 1.91 mM) and 1r (IC50 SEM ¼
An interesting inhibition of IAP was also exhibited by the aro-
matic derivative 1u and 1za processing cycloheptane ring along
0.75 0.07 mM) was found better than 1q (IC50 SEM ¼ >100 mM). with p-tosylate substitution at R2 position but different substitu-
It means that the presence of functional group is playing more tion at R1 position. The derivative with electron-donating group
important role on the inhibitory effects. The derivatives with less i.e. OMe in case of 1u (IC50 SEM ¼ 0.79 0.05 mM) was found more
carbon number were found more potent that the propane deriva- active inhibitor as compared to the derivative 1za (IC50 SEM ¼
tive. But, with detailed SAR it was found that the heptane ring 1.11 0.01 mM) with electron-withdrawing substitution. These two
was favourable for TNAP, while octanone derivative was found derivative also exert some inhibitory potential on h-TNAP, but less
potent towards IAP. When the activity of 1o was compared with (1u; IC50 SEM ¼ 1.65 0.13 mM, 1za; IC50 SEM ¼ 2.22 0.27 mM) in
1r and 1p, it was observed that the reduced inhibitory effect of comparison to h-IAP. The other derivatives 1 y and 1z with metha-
both the derivatives on TNAP was due to the presence of n-pro- nesulphonate and benzenesulphonate substitution were found
pane substitution in case of 1r, and the presence of fused cyclooc- inactive towards h-IAP. Moreover, it was found that the aromatic
tane ring in case of 1p.
sulphonate derivatives 1s, 1u, and 1za depicted stronger inhibi-
In comparison to 1o, compound 1s having benzenesulphonate tory effects on h-IAP than h-TNAP.
substitution was found almost as an equipotent inhibitor of TNAP
The results obtained here are in consistent with our previously
with the inhibitory value of 1.26 0.07 mM. The other derivative reported work. The 2-methoxy- or 2-chloro- substituted derivatives

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
483
Figure 3. 3D binding interactions of most potent inhibitor 1i within the active site of h-IAP. Hydrogen bonds are shown in green dotted line while p–p interactions
are shown in purple dotted line.
possessing the fused cycloheptane ring were found stronger inhib- h-IAP is shown in Figure 3. In comparison to docking interaction
itors of h-TNAP than the corresponding analogues having fused of compound 1f with h-TNAP, compound 1i formed four hydro-
cyclooctane ring. Similarly, an inverse effect was observed towards gen bonds with different amino acids of h-IAP. Oxygen atom in
h-IAP. It can be suggested from the obtained data that the deriva- sulphonate group of compound 1i also formed two hydrogen and
tives with more lipophilic side chains led to the more inhibition of one metal interaction. These two hydrogen bonds were formed by
h-IAP and vice versa in case of h-TNAP. This lipophilic side chains one oxygen atom in sulphonate group with amino acid residue of
might be involved in some hydrophobic interactions within the Arg166 and His143. While other oxygen of sulphonate group
pocket of the receptor site and increase the affinity of such deriva- formed interaction with Znþ2 ion. Carbonyl oxygen in coumarin
tives for the targeted enzyme and ultimately accounts for the ring and fluorine atom in compound 1i also formed hydrogen
enhanced h-IAP inhibition.
bond with amino acid residues Arg150 and Gln108 as shown in
Figure 3. Additional to hydrogen bonding, two p–p interaction
were also formed by benzene ring of coumarin ring with
amino acid residue of His317 and His320. Metal interaction of sul-
phonate group with metal ion is analogous to previously reported
interaction of sulphonamides with different APs13. p-alkyl inter-
action between seven-membered ring adjacent to coumarin ring
was not shown as in 1i. Docking of the inactive derivatives 1q
and 1w showed weaker affinity and fewer bonds formed,
which supports the SAR discussion. The figures are given in the
supplementary file.
Molecular docking studies
Molecular docking studies of the most potent derivatives i.e. 1f
and 1i were carried out within the respective target enzyme and
their most probable binding interactions within the modelled
enzymes are depicted in Figures 2 and 3. Detailed analysis of
binding interaction of compound 1f within h-TNAP revealed that
oxygen atom in both sulphonate group and carbonyl group are
responsible for making three strong hydrogen bonds with amino
acid residues. Two hydrogen bonds were formed by oxygen of sul-
phonate group with amino groups of Arg167 while the third
hydrogen bond was formed by carbonyl oxygen in coumarin ring
Conclusions
with amino acid Thr436. Additional to hydrogen bonding, second Tricyclic-fused coumarin sulphonates 1a-za were evaluated for
oxygen atom of the sulphonate group also formed direct inter- their effective inhibitory potential against h-TNAP and h-IAP.
action with the Znþ2 ion within the active site of h-TNAP. The Except 1w and 1 y derivatives, all the other compounds exhibited
excellent h-TNAP inhibition activity of compound 1f can be related excellent inhibitory potential against both h-TNAP and h-IAP.
with this direct binding interaction of sulphonate oxygen with the Methylbenzenesulphonate derivative 1f (IC50 ¼ 0.38 0.01 lM) was
Znþ2 ion of the active site. Furthermore, two p–p interactions found to be the most active h-TNAP inhibitor. Another 4-fluoro-
were formed by benzene ring adjacent to sulphonate group with benzenesulphonate derivative 1i (IC50 ¼ 0.45 0.02 lM) was found
amino acid residues His154 and His321. One p-alkyl interaction as the strongest inhibitor of h-IAP. Detailed SAR studies and
was also exhibited by the seven-membered ring fused with the molecular docking studies were performed to identify the import-
coumarin ring and His434 as shown in Figure 2. This interaction ance of functional groups within the structures and to explore the
may account for the stronger affinity and potency of this tricyclic possible binding site interactions, respectively. Moreover, previ-
compound compared to the previously reported bicyclic coumarin ously these derivatives have been reported for their antiprolifera-
sulphonate derivatives13. Detailed binding interaction of 1i with tive effects against different cell lines including, leukaemia, CNS,

484
J. IQBAL ET AL.
prostate, renal, colon, lung cancer, melanoma, ovarian, and breast
cancers. From that data we got 1i and 1 m as the strongest anti-
proliferative derivatives against colon cancer cell line along with
the maximum anti-inflammatory effects. Here in this study, we
have identified these compounds as stronger inhibitors of h-IAP
isozymes. This can be rationalised that the IAP, responsible for the
maintenance of intestinal homeostasis, might be over-expressed in
the colon cancer cell lines. Moreover, the over-expression of h-
TNAP can be associated with the lung cancer, melanoma, ovarian,
breast cancers, and renal cancer. Therefore, the obtained results
synergise with the previously reported data. It can be suggested
that the potent inhibitors of both h-TNAP and h-IAP can be
selected for further exploration at molecular level.
and alkaline phosphatase function: a unified model of the
mechanisms of initiation of skeletal calcification. J Bone
Miner Res 2011;26:286–97.
ꢀ
10. Chung TD, Sergienko E, Millan JL. Assay format as a critical
success factor for identification of novel inhibitor chemo-
types of tissue-nonspecific alkaline phosphatase from high-
throughput screening. Molecules 2010;15:3010–37.
11. Dahl R, Sergienko EA, Su Y, et al. Discovery and validation of
a series of aryl sulfonamides as selective inhibitors of tissue-
nonspecific alkaline phosphatase (TNAP).
J Med Chem
2009;52:6919–25.
12. Al-Rashida M, Raza R, Abbas G, et al. Identification of novel
chromone based sulfonamides as highly potent and select-
ive inhibitors of alkaline phosphatases. Eur J Med Chem
2013;66:438–49.
13. Salar U, Khan KM, Iqbal J, et al. Coumarin sulfonates: new
alkaline phosphatase inhibitors; in vitro and in silico studies.
Eur J Med Chem 2017;131:29–47.
Disclosure statement
The authors have declared no conflict of interest.
14. Jang HL, El-Gamal MI, Choi HE, et al. Synthesis of tricyc-
lic fused coumarin sulfonates and their inhibitory effects
on LPS-induced nitric oxide and PGE2 productions in
RAW 264.7 macrophages. Bioorg Med Chem Lett
2014;24:571–5.
15. El-Gamal MI, Oh C-H. Synthesis, in vitro antiproliferative
activity, and in silico studies of fused tricyclic coumarin sul-
fonate derivatives. Eur J Med Chem 2014;84:68–76.
16. El-Gamal MI, Baek D, Oh C-H. A new series of cycloalk-
ane-fused coumarin sulfonates: synthesis and in vitro anti-
proliferative screening. Bull Korean Chem Soc 2016;37:
184–91.
17. El-Gamal MI, Lee WS, Shin JS, et al. Synthesis of new tricyclic
and tetracyclic fused coumarin sulfonate derivatives and
their inhibitory effects on LPS-Induced Nitric Oxide and
PGE2 Productions in RAW 264.7 Macrophages: part 2. Archiv
Der Pharmazie 2016;349:853–63.
Funding
J. Iqbal is thankful to the Organization for the Prohibition of
Chemical Weapons (OPCW), The Hague, The Netherlands and
Higher Education Commission, Pakistan for the financial support
through Project No. 20–3733/NRPU/R&D/14/520. This work was
also supported by Korea Institute of Science and Technology
(KIST), KIST Project (E26900).
ORCID
Mohammed I. El-Gamal
http://orcid.org/0000-0002-4269-5264
References
ꢀ
18. Sergienko EA, Millan JL. High-throughput screening of tis-
€
1. Zimmermann H, Zebisch M, Strater N. Cellular function and
molecular structure of ecto-nucleotidases. Purinergic Signal
2012;8:437–502.
sue-nonspecific alkaline phosphatase for identification of
effectors with diverse modes of action. Nat Protoc
2010;5:1431–9.
ꢀ
2. Millan JL. Mammalian alkaline phosphatases: from biology to
19. Khan I, Shah SJA, Ejaz SA, et al. Investigation of quinoline-
applications in medicine and biotechnology. Hoboken, NJ,
USA: John Wiley & Sons; 2006.
4-carboxylic acid as
a highly potent scaffold for the
development of alkaline phosphatase inhibitors: synthesis,
SAR analysis and molecular modelling studies. RSC Advances
2015;5:64404–13.
ꢀ
3. Millan JL. Alkaline phosphatases: structure, substrate specifi-
city and functional relatedness to other members of a large
superfamily of enzymes. Purinergic Signal 2006;2:335–41.
20. Khan I, Ibrar A, Ejaz SA, et al. Influence of the diversified
structural variations at the imine functionality of 4-bromo-
phenylacetic acid derived hydrazones on alkaline phosphat-
ase inhibition: synthesis and molecular modelling studies.
RSC Advances 2015;5:90806–18.
ꢀ
4. Millan JL. The role of phosphatases in the initiation of skel-
etal mineralization. Calcif Tissue Int 2013;93:299–306.
5. Al-Rashida M, Iqbal J. Inhibition of alkaline phosphatase: an
emerging new drug target. Mini Rev Med Chem
2015;15:41–51.
21. Molecular Operating Environment (MOE), 2014.09; Chemical
Computing Group Inc., 1010 Sherbooke St. West, Suite #910,
Montreal, QC, Canada, H3A 2R7, 2015.
22. Accelrys Software Inc., Discovery Studio Modeling
Environment, Release 4.0, Accelrys Software Inc., San Diego,
CA; 2013.
23. Li C-J, Chen D-L, Lu Y-Q, et al. Metal-mediated two-atom car-
bocycle enlargement in aqueous medium. Tetrahedron
1998;54:2347–64.
6. Kiffer-Moreira T, Sheen CR, da Silva Gasque KC, et al.
Catalytic signature of a heat-stable, chimeric human alkaline
phosphatase with therapeutic potential. PLoS One
2014;9:e89374.
7. Guerville M, Boudry G. Gastrointestinal and hepatic mecha-
nisms limiting entry and dissemination of lipopolysaccharide
into the systemic circulation. Am J Physiol Gastrointest Liver
Physiol 2016;311:G1–G15.
ꢀ
8. Buchet R, Millan JL, Magne D. Multisystemic functions of
24. Woo LWL, Purohit A, Malini B, et al. Potent active site-
directed inhibition of steroid sulphatase by tricyclic couma-
rin-based sulphamates. Chem Biol 2000;7:773–91.
alkaline phosphatases. Methods Mol Biol 2013;1053:27–51.
9. Yadav MC, Simao AMS, Narisawa S, et al. Loss of skeletal
mineralization by the simultaneous ablation of PHOSPHO1