3-(5-(Benzylideneamino)thiazol-3-yl)-2H-chromen-2-ones: A new class of alkaline phosphatase and ecto-5′-nucleotidase inhibitors

Article abstract of DOI:10.1039/c5ra24684a

A new series of 3-(2-(benzylideneamino)thiazol-4-yl)-2H-chromen-2-ones has been synthesized. The structures of the compounds were established by means of ¹H and ¹³C NMR spectroscopy. All compounds were evaluated for their potential t

Full text of DOI:10.1039/c5ra24684a

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S. Ejaz, M. Shehzad, S. Hassan, M. al-Rashida, J. Sevigny and J. Lecka, RSC Adv., 2016, DOI:
1
0.1039/C5RA24684A.
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3
ꢀ(5ꢀ(Benzylideneamino)thiozolꢀ3ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀones: A new class of alkaline
phosphatase and ectoꢀ5′ꢀnucleotidase inhibitors
Aamer Saeed, Syeda Abida Ejaz, Muddasar Shehzad, Sidra Hassan, Mariya alꢀRashida, c
*
a
b
a
b
d,e
d,e
*b
Joanna Lecka, Jean Sévigny, Jamshed Iqbal
a
Department of Chemistry, Quaid-I-Azam University, 45320, Islamabad, Pakistan
Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad 22060,
b
Pakistan
c
Department of Chemistry, Forman Christian College (A Chartered University), Ferozepur Road-54600,
Lahore, Pakistan
d
Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université Laval,
Québec, QC, Canada
e
Centre de Recherche du CHU de Québec – Université Laval, Québec, QC, Canada
*
Address correspondence to:
Prof. Dr. Jamshed Iqbal
Centre for Advanced Drug Research,
COMSATS Institute of Information Technology,
Abbottabad, Postal Code 22060, Pakistan
Tel: +92ꢀ992ꢀ383591ꢀ96
Fax: +92ꢀ992ꢀ383441
EꢀMail: drjamshed@ciit.net.pk / jamshediqb@googlemail.com
Abstract
A new series of 3ꢀ(2ꢀ(benzylideneamino)thiazolꢀ4ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀones has been
1
13
synthesized. The structures of compounds were established by means of H and C NMR
spectroscopy. All compounds were evaluated for their potential to inhibit human recombinant
ectoꢀnucleotidases, including human tissueꢀnonspecific alkaline phosphatase (hꢀTNAP), tissue
specific human intestinal alkaline phosphatase (hꢀIAP), human and rat ectoꢀ5′ꢀnucleotidase (h-
e5′NT & r-e5′NT). All compounds were found to be potent and selective inhibitors of hꢀe5′NT,
the most active hꢀe5′NT inhibitors were compounds 7a (IC = 0.25±0.07 µM) and 7g (IC =
5
0
50
0.28±0.05 µM). Most of the compounds were found to selectively inhibit hꢀTNAP over hꢀIAP,
1

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with inhibitory activities in lower micromolar range. The most active hꢀTNAP inhibition was
exhibited by compounds 7h and 7c (IC = 0.21±0.04 µM and 0.22±0.03, respectively), which is
5
0
≈
91 times greater than the inhibitory activity of standard inhibitor levamisole. Compound 7i was
found to be most potent hꢀIAP inhibitor (IC = 0.05±0.001 µM), exhibiting ≈11 times greater
5
0
selectivity for hꢀIAP over hꢀTNAP. Homology modeling and molecular docking studies were
carried out to determine the most plausible binding site interactions of potent inhibitors with the
ectoꢀnucletidases.
Keywords: human recombinant ectoꢀnucleotidases, alkaline phosphatase,levamisole, homology
modeling, molecular docking
Introduction
The membrane bound ectoꢀnucleotidases are subdivided into four major families, the alkaline
phosphatases (APs), ectoꢀnucleoside triphosphate diphosphohydrolases (NTPDases),
ectoꢀnucleotide
pyrophosphatase/phosphodiesterases
(NPPs)
and
ectoꢀ5′ꢀ
1
2
nucleotidase (e5′NT) , . These ectoꢀnucleotidase enzymes act as important regulators of
purinerguc cell signaling by carrying out catalytic degradation of various nucleotides, which
subsequently act on their respective receptors (P1, adenosine activated; or P2, nucleotide
3
activated) to affect a certain physiological process . Ectoꢀ5′ꢀnucleotidase (e5′NT) is a
glycosyl phoshatidylinositol (GPI) anchored membrane protein responsible for the
hydrolysis of extracelluar adenosine monophosphate (AMP) to adenosine, which
4
acts on purinergic (P1) receptors . This enzyme regulates various physiological
processes that are modulated by adenosine, including vasodilation, stimulation of
angiogenesis, cytoprotection, immunosuppression and control of cell growth, maturation and
5
differentiation . In addition to enzymatic hydrolysis, e5′NT acts as an adhesion
2

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6
molecule, is also involved in cell–cell and cell–matrix interactions . There are two
+
2
domains in e5′NT, an Nꢀterminal domain containing a bimetallic Zn center, and a
Cꢀterminal domain containing nucleotide (substrate) binding site. The two domains
+
2
are linked via an alphaꢀhelix chain. The active site (consisting of bimetallic Zn
center) is located at the junction of these two domain, therefore substrate binding
and subsequesnt product release involves considerable domain movement and
3
conformation . Overexpression of e5′NT is associated with various physiological disorders
and malignancies, as progressive tumor growth depends on vasodilation, angiogenesis,
7
cytoprotective and immunosuppressive activities . Moreover, the levels of e5′NT exhibit a
8
significant correlation with tumor grade and localization . Therefore the inhibitors of e5′NT are
highly speculated to be important pharmacological agents for cancer therapy as they reduce
3
extracellular level of adenosine which leads to the reduction of purinergic receptor expression ,
9
.
Alkaline phosphatases (APs), also members of ectoꢀnucleotidase family, are responsible for the
production of adenosine directly by hydrolyzing AMP. APs can also hydrolyse extracellular
1
0
adenosine triphosphate (ATP) via adenosine diphosphate (ADP) and AMP to adenosine
.
However their activities in relation to their substrate is nonꢀspecific, they can hydrolyze a
number of substrates other than nucleotides such as bis(pꢀnitrophenyl)phosphate, inorganic
1
1
polyphosphates,
phosphatidates and glucoseꢀphosphates . In the presence of large
concentration of phosphate acceptors, these APs are also responsible for catalyzing various
transphosphorylation reactions. In humans, there are four AP isozymes, three of these are tissue
specific enzymes, i.e., intestinal AP (IAP), germ cell AP (GCAP) and placental AP (PLAP). The
2
+
2+
catalytic site of all APs contains two Zn ions and a Mg ion. The crystal structure of hPLAP
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reveals important information abobut amino acid residues coordinating metal ions. One of the
2
+
2+
Zn ions is coordinated to three amino acid residues and a water molecule. The other Zn ion is
pentaꢀcoordinated to four amino acid residues and a water molecule which can be replaced upon
2
+
substrate binding. The Mg ion is hexaꢀcoordinated to three amino acid residues and three water
molecules that can be replaced upon substrate binding. In addition to these catalytic metal ions,
2
+
the presence of a Ca ion as been proven mandatory for normal functioninig of these enzymes,
1
2
although exact mechanism of it’s involvement remains somewhat vague . Tissue nonꢀspecific
enzyme (TNAP) is 50% identical to other isozymes and is located in liver, kidney and
1
2
excessively in bones . This enzyme plays an important role in regulating normal bone
mineralization. TNAP is located at the surface of osteoblasts and chrondrocytes, and produces
inorganic phosphate for the crystallization of hydroxyapatite. It is also involved in the
hydrolysis of inorganic pyrophosphate (PPi) to inorganic phosphate in bone tissues. The
1
3
overexpression of TNAP leads to impaired mineralization .
The tissue specific intestinal alkaline phosphatase (IAP) is found at the brush border membrane
of the intestinal epithelium and is enriched in surfactantꢀlike particles (SLP). It is likely to be
involved in intestinal absorption of lipid/nutrients across the cell membrane via its association
1
4
15ꢀ19
with SLP . Although a number of inhibitors of AP are known
. But because of high
homology between TNAP and tissueꢀspecific IAP, very few selective inhibitors of IAP have
been reported. Previously we reported chromone containing sulfonamides as potent and selective
2
0
inhibitors of APs . Herein, we report a convenient method for the synthesis of 3ꢀ(5ꢀ
benzylideneamino)thiazolꢀ3ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone derivatives (7aꢀj) and their inhibitory
(
potential against human tissueꢀnonspecific alkaline phosphatase (hꢀTNAP) and human intestinal
alkaline phosphatase (hꢀIAP). The inhibitory activities of these molecules were also evaluated
4

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against human and rat ectoꢀ5′ꢀnucleotidases (hꢀe5′NT and rꢀe5′NT). Homology modeling and
molecular docking studies were carried out to predict the most plausible binding site interactions
of potent inhibitors.
Experimental
General procedure for the synthesis of 3ꢀ(5ꢀ(benzylideneamino)thiazolꢀ3ꢀyl)ꢀ2Hꢀchromenꢀ
2ꢀones
2.2.1 Synthesis of 3ꢀacetylꢀ2Hꢀchromenꢀ2ꢀone (3)
To a cold mixture of salicylaldehyde (0.1 mol, 10.64 mL) and ethylacetoacetate (0.1 mol,
2.63 mL), was added 1 g (1.12 mL) of pipredine with rapid shaking. The solid separated was
1
filtered and washed with ethanol. Crystallization of the solid from pure water gave pure 3ꢀacetylꢀ
Hꢀchromeneꢀ2ꢀone.
2
ꢀ1
White solid (83%): m.p.: 123ꢀ124°C; R : 0.48 (a). IR (pure cm ): 3029 (C ꢀH), 1738 (C=O
f
sp2
1
ketone), 1673 (C=O lactone), 1555 (C=C aromatic), H NMR (300 MHz, CDCl ): δ 8.51 (s, 1H,
3
13
Hꢀ4), 7.68ꢀ7.63 (m, 2H, ArꢀH), 7.38ꢀ7.28 (m, 2H, ArꢀH), 2.72 (s, 3H, CH ), C NMR: (75 MHz,
3
CDCl ): δ 195.5, 159.2, 155.3, 147.5, 134.4, 130.2, 125.0, 124.4, 118.2, 116.6, 30.6. ESI LC/MS
3
DAD (80% H O, 20% CH CN): R.T 3.6 min, m/z M+1 189, 171, 143, 115. λ 305 nm.
2
3
max
2
.2.2 Synthesis of 3ꢀ(2ꢀbromoacetyl)ꢀ2Hꢀchromeneꢀ2ꢀone (4)
A solution of bromine (0.01 mol, 1.72g) in chloroform was added with rapid shaking to a
solution 3ꢀacetylꢀ2Hꢀchromeneꢀ2ꢀone (0.01 mol, 2.0g) in chloroform. The mixture was heated
under reflux for 2ꢀ3 hours and cooled. The solid separated was crystallized from chloroformꢀ
ethanol (2:1) to get pure 3ꢀ(2ꢀbromoacetyl)ꢀ2Hꢀchromeneꢀ2ꢀone.
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ꢀ1
Off white solid (85.7%): m.p. 162ꢀ165°C; R : 0.51 (a) IR (pure cm ): 3024 (C ꢀH), 1728 (C=O
f
sp2
1
ketone), 1684 (C=O lactone), 1554 (C=C aromatic), H NMR (300MHz, C D O): δ 8.78 (s, 1H,
3
6
1H), 7.83 (dd 1H , J= 1.5 Hz, 7.8, Hꢀ2 ), 8.02ꢀ7.92 (m, 1H, Hꢀ4), 7.41ꢀ7.41 (m, 2H, ArꢀꢀH) 4.87
1
3
(
s, 2H, Hꢀ6), C NMR: (75MHz, C D O): δ 188.2, 158.5, 148.5, 134.9, 130.8, 125.1, 122.5,
3 6
118.3, 117.4, 116.3, 36.2.
2.2.3 Synthesis of 3ꢀ(2ꢀaminoꢀ1,3ꢀthiazolꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone (5)
To a hot suspension of compound 4 (0.00857 mol) in 200 mL ethanol, thiourea
(0.00857mol) was added and the resulting mixture heated under reflux for three hours. The
precipitates that separated out were filtered, washed with ethanol and boiled with an aqueous
solution of sodium acetate yielding compound 5. The product was recrystallized from ethanol.
ꢀ1
Pale yellow (89.67%): m.p. 223ꢀ225°C; R : 0.11 (a) IR (pure cm ): 3379, 3313 (NH ), 3153
f
2
1
(
Csp2ꢀH), 1694 (C=O lactone), 1537 (C=C aromatic), 1178 (C–O), H NMR (300MHz, DMSOꢀ
d6): δ 8.49 (s, 1H, Hꢀ1), 7.8 (dd 1H , J= 1.2 Hz, 7.8 Hz, Hꢀ2 ), 7.58 (dt, 1H, J=1.5 Hz, 8.4 Hz, Hꢀ
), 7.507 (s, 1H, Hꢀ6), 7.41 (d, 1H, J=8.1 Hz, Hꢀ5), 7.34 (at, 1H, J=7.8 Hz, Hꢀ3), 7.183 (s, 2H,
4
1
3
NH ) C NMR: (75MHz, DMSOꢀd ): δ 167.88, 159.23, 152.61, 143.73, 138.54, 131.89, 129.12,
2
6
1
25.11, 120.90, 119.74, 116.25, 109.19. ESI LC/MS DAD (80% H O, 20% CH CN): R.T 3.8
2 3
mint, m/z M+1 245, λmax 355 nm.
2.2.4 Synthesis of 3ꢀ(5ꢀ(benzylideneamino)thiazolꢀ3ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀones (7aꢀj)
1
'
5'
S
2'
6
"
5"
5
8
4
4
'
N
6
7
1"
N
4
"
3
3
'
O
2
O
2"
3"
R
1
6

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General procedure
A mixture of 2ꢀaminoꢀ4ꢀ(coumarrinꢀ4ꢀyl) thiazole (5) (0.001 mole, 0.244g) and
substituted benzaldehyde (0.001mole) was dissolved in 20 mL of ethanol taken in a round
bottom flask fitted with a reflux condenser. Then the mixture was refluxed for 5ꢀ6 h. The solvent
was removed by vacuum distillation after the completion of the reaction. The product was
filtered, dried and reꢀcrystallized from absolute alcohol.
3
ꢀ(2ꢀ(3ꢀNitrobenzylideneamino)thiazolꢀ4ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone (7a)
ꢀ1
Yellow (69.35%): m.p. 158ꢀ160°C; R : 0.37(a) IR (pure cm ): 3105 (Csp2ꢀH), 1698 (C=O
f
1
lactone), 1605 (C=N), 1520 (C=C aromatic), 1161 (C–O), H NMR (300 MHz, DMSOꢀd ): δ
6
8.161 (s, 1H, Hꢀ2″), 8.1 (d 1H ,
J= 8.4 Hz, Hꢀ4″), 8.0 (s, 2H, Hꢀ3, Hꢀ5″), 7.8 (d, 1H, J=7.8 Hz,
Hꢀ6″), 7.68ꢀ7.5 (m, 3H, Hꢀ7, Hꢀ6, Hꢀ5), 7.312ꢀ7.21 (m, 2H, Hꢀ5″, Hꢀ8), 6.13 (s, 1H, –N=CH–),
1
3
C NMR: (75MHz, DMSOꢀd ): δ 167.7, 158.9, 153.5, 148.1, 145.4, 143.0, 134.9, 133.4, 130.8,
6
129.4, 125.3, 124.1, 123.1, 118.6, 118.5, 116.5
3
ꢀ(2ꢀ(4ꢀNitrobenzylideneamino)thiazolꢀ4ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone (7b)
S
N
N
NO2
O
O
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ꢀ1
Orange yellow (62.45%): m.p. 149ꢀ151°C; R : 0.36(a) IR (pure cm ): 3105 (C ꢀH), 1697 (C=O
f
sp2
1
lactone), 1607 (C=N), 1535 (C=C aromatic), 1169 (C–O), H NMR (300MHz, DMSOꢀd ): δ 8.2
6
(d,
J= 7.9 Hz, 2H, Hꢀ3″), 8.12 (d 2H ,
J
= 7.9 Hz, Hꢀ2″), 7.9 (s, 1H, Hꢀ4), 7.8 (d, 1H,
J
=7.8 Hz,
1
3
Hꢀ8), 7.7ꢀ7.45 (m, 3H, Hꢀ7, Hꢀ6, Hꢀ5), 7.03(s, 1H, Hꢀ5′), 6.4 (s, 1H, –N=CH–), C NMR:
(
75MHz, DMSOꢀd ): δ 167, 160, 155.1, 147.8, 145, 143, 134.9, 134, 131.8, 129.4, 127.3, 125.1,
6
123.1, 119.6, 119.5, 116.5, 115.
3
ꢀ(2ꢀ(4ꢀFluorobenzylideneamino)thiazolꢀ4ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone (7c)
S
N
N
F
O
O
ꢀ1
Light yellow (70.6%): m.p. 148ꢀ150°C; R : 0.40 (a) IR (pure cm ): 3126(C ꢀH), 1692 (C=O
f
sp2
1
lactone), 1610 (C=N), 1565 (C=C aromatic), 1156 (C–O), H NMR (300MHz, DMSOꢀd ): δ
6
8
.1(s, 1H, Hꢀ4), 8.05 (d,
Hz, Hꢀ8), 7.6ꢀ7.36 (m, 3H, Hꢀ7, Hꢀ6, Hꢀ5), 6.95 (s, 1H, Hꢀ5′), 6.45 (s, 1H, –N=CH–), C NMR:
75MHz, DMSOꢀd ): δ 167.9, 158.3, 151.5, 147.1, 144.4, 143.5, 133.4, 132.1, 131.2, 128.5,
J= 7.9 Hz, 2H, Hꢀ2″), 7.96 (d 2H , J= 8.4 Hz, Hꢀ3″), 7.79 (d, 1H, J=8
1
3
(
6
124.3, 124.1, 123.1, 118.3, 117.2, 115.6
3
ꢀ(2ꢀ(4ꢀBromobenzylideneamino)thiazolꢀ4ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone (7d)
S
N
N
Br
O
O
ꢀ1
Pale yellow (62.86%): m.p. 141ꢀ142°C; R : 0.41 (a) IR (pure cm ): 3105 (Csp2ꢀH), 1696 (C=O
f
1
lactone), 1609 (C=N), 1529 (C=C aromatic), 1159 (C–O), H NMR (300MHz, DMSOꢀd ): δ
6
8

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8.16 (s, 1H, Hꢀ4), 8 (d,
J= 7.8 Hz, 2H, Hꢀ2″), 7.9 (d 2H , J= 8.4 Hz, Hꢀ3″), 7.6 (d, 1H, J= 8 Hz,
1
3
Hꢀ8), 7.5ꢀ7.35 (m, 3H, Hꢀ7, Hꢀ6, Hꢀ5), 6.86 (s, 1H, Hꢀ5′), 6.32 (s, 1H, –N=CH–), C NMR:
75MHz, DMSOꢀd ): δ 167.1, 157.4, 152.4, 146.9, 145, 144.3, 132.5, 131.5, 131, 127.6, 124.3,
(
6
124.1, 123.1, 118.3, 116.2, 114.3
3
ꢀ(2ꢀ(4ꢀMethoxyꢀ3ꢀnitrobenzylideneamino)thiazolꢀ4ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone (7e)
S
N
N
OCH3
O
O
NO2
ꢀ1
Off white (65.9%): m.p. 152ꢀ155°C; R : 0.41 (a) IR (pure cm ): 3126 (C ꢀH), 1688 (C=O
f
sp2
1
lactone), 1611 (C=N), 1537 (C=C aromatic), 1149 (C–O), H NMR (300MHz, DMSOꢀd ): δ
6
8.19 (s, 1H, Hꢀ4), 8.18 (d,
J= 7.7 Hz, 1H, Hꢀ2″), 7.76 (d 1H , J= 7.7 Hz, Hꢀ6″), 7.59 (d, 1H, J=8
Hz, Hꢀ8), 7.47ꢀ7.2 (m, 4H, Hꢀ7, Hꢀ6, Hꢀ5, Hꢀ5″), 6.62 (s, 1H, Hꢀ5′), 6.15 (s, 1H, –N=CH–), 3.68
1
3
(
s, 3H, ꢀOCH ), C NMR: (75MHz, DMSOꢀd ): δ 167.5, 156.9, 153.7, 147.3, 146.4, 140.3,
3
6
132.5, 131.2, 129.5, 127.2, 125.3, 124.7, 123.8, 116.5, 114.2, 112.3, 18.7
3
ꢀ(2ꢀ(3ꢀMethoxyꢀ4ꢀphenoxybenzylideneamino)thiazolꢀ4ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone (7f)
S
N
N
OPh
O
O
OCH3
9

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ꢀ1
Light yellow (58.3%): m.p. 160ꢀ162°C; R : 0.38 (a) IR (pure cm ): 3126(C ꢀH), 1692 (C=O
f
sp2
1
lactone), 1610 (C=N), 1535 (C=C aromatic), 1156 (C–O), H NMR (300MHz, DMSOꢀd ): δ
6
8
.1(s, 1H, Hꢀ4), 8.05 (d,
J= 7.9 Hz, 1H, Hꢀ5), 7.96 (d 2H , J= 8.4 Hz, Hꢀ7), 7.79 (d, 1H, J=8 Hz,
Hꢀ8), 7.6ꢀ7.36 (m, 8H, ArꢀH), 6.95 (s, 1H, Hꢀ5′), 6.43 (s, 1H, –N=CH–), 3.64 (s, 1H, ꢀOCH3),
1
3
C NMR: (75MHz, DMSOꢀd ): δ 167.9, 160.2, 157.3, 156.4, 154.3, 150.2, 151.5, 147.1, 144.4,
6
1
43.5, 133.4, 132.1, 131.2, 128.5, 124.3, 124.1, 123.1, 118.3, 117.2, 115.6, 17.9.
3
ꢀ(2ꢀ(4ꢀChlorobenzylideneamino)thiazolꢀ4ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone (7g)
S
N
N
Cl
O
O
ꢀ1
Green (60.32%): m.p. 147ꢀ148°C; R : 0.413 (a) IR (pure cm ): 3134 (C ꢀH), 1687 (C=O
f
sp2
1
lactone), 1604 (C=N), 1530 (C=C aromatic), 1130 (C–O), H NMR (300MHz, DMSOꢀd ): δ
6
8
.13 (s, 1H, Hꢀ4), 8.09 (d,
Hz, Hꢀ8), 7.6ꢀ7.28 (m, 3H, Hꢀ7, Hꢀ6, Hꢀ5), 6.78 (s, 1H, Hꢀ5′), 6.20 (s, 1H, –N=CH–), C NMR:
75MHz, DMSOꢀd ): δ 167.8, 158.6, 153.5, 149.3, 147.3, 145.6, 138.2, 134.5, 132.7, 129.4,
J= 7.8 Hz, 2H, Hꢀ2″), 7.95 (d 2H , J= 8.4 Hz, Hꢀ3″), 7.65 (d, 1H, J= 8
1
3
(
6
1
28.2, 125.7, 124.6, 119.7, 117.3, 115.2.
3
ꢀ(2ꢀ(3ꢀChlorobenzylideneamino)thiazolꢀ4ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone (7h)
S
N
N
O
O
Cl
ꢀ1
Greenish yellow (65.17%): m.p. 138ꢀ140°C; R : 0.42(a) IR (pure cm ): 3141 (C ꢀH), 1691
f
sp2
1
(
C=O lactone), 1607 (C=N), 1543 (C=C aromatic), 1137 (C–O), H NMR (300MHz, DMSOꢀd ):
6
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δ 8.197 (s, 1H, Hꢀ4), 8.09 (d, 2H, Hꢀ2″), 7.89 (m, 1H, Hꢀ8, Hꢀ6″), 7.65 (d, 1H, J=7.8 Hz, Hꢀ8),
13
7
.7ꢀ7.3 (m, 5H, Hꢀ7, Hꢀ6, Hꢀ5, Hꢀ4″, Hꢀ5″), 6.65 (s, 1H, Hꢀ5′), 6.17 (s, 1H, –N=CH–),
C
NMR: (75MHz, DMSOꢀd ): δ 167.8, 157.8, 155.5, 148.5, 147.3, 145.6, 138.2, 134.5, 132.7,
6
1
29.4, 128.2, 125.7, 124.6, 119.7, 117.3, 115.2.
3
ꢀ(2ꢀ(3,4,5ꢀTrimethoxybenzylideneamino)thiazolꢀ4ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone (7i)
S
OCH3
OCH3
OCH3
N
N
O
O
ꢀ1
Pale yellow (59.8%): m.p. 142ꢀ144°C; R : 0.42 (a) IR (pure cm ): 3126(C ꢀH), 1692 (C=O
f
sp2
1
lactone), 1610 (C=N), 1547 (C=C aromatic), 1156 (C–O), H NMR (300MHz, DMSOꢀd ): δ
6
8
.1(s, 1H, Hꢀ4), 7.96 (d, J= 7.9 Hz, 2H, Hꢀ2″), 7.79 (d, 1H, J= 8 Hz, Hꢀ8), 7.6ꢀ7.36 (m, 3H, Hꢀ7,
1
3
Hꢀ6, Hꢀ5), 6.91 (s, 1H, Hꢀ5′), 6.4 (s, 1H, –N=CH–), 3.23 (s, 9H, ꢀOCH ), C NMR: (75MHz,
3
DMSOꢀd ): δ 167.2, 157.3, 154.5, 146.8, 144.9, 144, 133.8, 133, 132, 128.9, 124.3, 124.1, 123.7,
6
1
18.2, 117.2, 115.6.18.2. 17.6.
3
ꢀ(2ꢀ(Furanꢀ2ꢀylmethyleneamino)thiazolꢀ4ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone (7j)
S
N
O
N
O
O
ꢀ1
Greenish yellow (64.87%): m.p. 140ꢀ141°C; R : 0.456 (a) IR (pure cm ): 3124 (C ꢀH), 1697
f
sp2
1
(
C=O lactone), 1612 (C=N), 1534 (C=C aromatic), 1148 (C–O), H NMR (300MHz, DMSOꢀd ):
6
δ 8.09 (s, 1H, Hꢀ4), 7.88 (d,
J= 7.8 Hz, 2H, Hꢀ5), 7.65 (d 2H ,
J= 8.4 Hz, Hꢀ7), 7.43 (d, 1H,
J=8
Hz, Hꢀ8), 7.28 (td, = 7.8 Hz, 3.4, 1H, Hꢀ6), 6.51 (s, 1H, Hꢀ5'), 7.88 (d,
J
J= 7.1 Hz, 2H, Hꢀ2"),
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1
3
7
.88 (d,
J= 7.1 Hz, 2H, Hꢀ4"), 7.88 (t, J= 7.8 Hz, 2H, Hꢀ3"), 5.86 (s, 1H, –N=CH–), C NMR:
(
75MHz, DMSOꢀd ): δ 167.8, 158.9, 152.8, 149.3, 147.3, 146.2, 145.6, 138.2, 134.5, 132.7,
6
1
29.4, 128.2, 125.7, 124.6, 119.7, 109.3, 109.1.
Biochemical Assays
Cell Transfection with Human APs and eꢀ5′ꢀNT
2
2
The plasmid expressing human APs: TNAP and IAP 21 or ectoꢀ5′ꢀnucleotidase , was
transfected into the COSꢀ7 cells by utilizing lipofectamine. The confluent cells were again
incubated in DMEM/Fꢀ12 (without fetal bovine serum) having 24 µL of Lipofectamine reagent
0
and 6 µg of plasmid DNA for 5 h at 37 C. DMEM/Fꢀ12 solution containing 20% FBS was used
to cease the transfection and cells were allowed to harvest for 48ꢀ72 h.
Preparation of membrane fractions
0
These cells were washed with trisꢀsaline buffer at 4 C and finally collected by scraping in the
harvesting buffer (95 mM NaCl, 0.1 mM PMSF, and 45 mM Tris buffer, pH 7.5). These cells
16
were centrifuged at 300×
harvesting buffer containing 10 µg/mL aprotinin and sonicated. Cellular and nuclear debris were
removed by centrifugation (300× at 4 °C) for 10 min. Glycerol (final concentration of 7.5%. )
was added to the resulting supernatant and all the samples were stored at ꢀ80 °C until used.
g twice for 5 min at 4 °C . Later the cells were resuspended in the
g
2
3
Bradford microplate assay was used for the estimation of protein concentration. Bovine serum
albumin was used as a reference standard.
Alkaline Phosphatase Inhibition Assay (hꢀTNAP & hꢀIAP)
The alkaline phosphatase (
hꢀTNAP and hꢀIAP) activity of compounds was determined by using a
chemiluminescent substrate, CDPꢀstar (disodium 2ꢀchloroꢀ5ꢀ(4ꢀmethoxyspiro[1,2ꢀdioxetaneꢀ
1
2

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3
,2′ꢀ(5ꢀchlorotricyclo[3.3.1.13.7]decan])ꢀ4ꢀyl]ꢀ1ꢀphenyl phosphate). The assay was carried out
2
4
and optimized with slight modifications in previously reported spectrophotometric method . The
activity was measured in 8 M DEA buffer (pH 9.8), containing 2.5 mM MgCl and 0.05 mM
2
ZnCl in the presence of 10 µL potential inhibitors (0.2 mM concentration) and 20 µL of hꢀ
2
,
TNAP (from 2.29µg of protein/mL in assay buffer) or of hꢀIAP (from 2.84 µg protein/mL in
assay buffer). The luminescence signals were measured as preꢀread after 10 minutes of
o
incubation at 37 C at microplate reader (BioTek FLx800, Instruments, Inc. USA). Then 20 µL
of CDPꢀstar (final concentration of 110 µM) was added at last ten minutes to initiate the reaction.
The change in the luminescence was measured as afterꢀread. The activity of each compound was
compared with total activity control (without any inhibitor). For the compounds which exhibited
over 50% inhibition of either hꢀTNAP activity or hꢀIAP activity, full concentration inhibition
curves were produced to evaluateIC50 values. All experiments were repeated three times in
triplicate. A nonꢀlinear regression analysis of the program PRISM 5.0 (GraphPad, San Diego,
California, USA). was used to fit the doseꢀresponse curves and derive IC values.
5
0
Ectoꢀ5′ꢀNucleotidase Inhibition Assay
Capillary electrophoresis P/ACE MDQ system (Beckman Instruments, Fullerton, CA) having a
UV detection system coupled with diode array detector (DAD) was used. The data analysis
were performed by using the P/ACEMDQsoftware 32 KARAT, obtained from Beckman Coulter.
o
The sample storing unit of CE were maintained at temperature of 25 C. The eCAP fusedꢀsilica
capillary (30 cm) was used for eletrophoretic separation of analyte. The current of 60 ꢁA was
applied and data was recorded at a acquisition rate of 4Hz. Analytes were detected at 260 nm.
Prior to each injection, the capillary was rinsed with 0.1 N NaOH for 2 min, distilled water for 2
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min, and running buffer (sodium tetra borate 20 mM, pH 9.0) for 2 min. Sample injections were
made by using the forward phase analysis.
The bioactivity assay of both rat and human ectoꢀ5′ꢀnucleotidase was performed with the slight
2
5
modifications in the method as described previously . Stock solutions of 10 mM concentration
of each compound were prepared in DMSO. Working solutions were prepared by appropriately
diluting the stock solutions to 1mM concentration in assay buffer (2 mM MgCl , 1 mM CaCl2
2
and 10 mM Tris HCl (pH 7.4). The volume of the final assay was kept 100 µL. A 10 µL of each
tested compound was preꢀincubated with 10 µL of human e5′NT (6.94 µg/mL) protein extract or
rat e5′NT (7.17 µg/mL) for 10 min in the presence of 70 µL assay buffer (2 mM MgCl , 1 mM
2
CaCl and 10 mM Tris HCl, pH 7.4). Afterwards, 10 µL of AMP substrate (final concentration of
2
5
00 µM) was added to initiate the enzymatic reaction. The reaction mixture was allowed to
o
incubate for 10 min at 37 C. To stop the enzymatic reaction, the reaction mixture was quenched
o
by thermal denaturation at 99 C for 20 min. Aliquots of 50 µL of each reaction mixture were
transferred to CE minivial and injected into the CE instrument. The enzyme reaction mixture 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. The compounds which exhibited over 50% inhibition of either the
rꢀ
e5′NT or ꢀe5′NT activity were further evaluated for determination of IC50 values. For this
h
purpose serial dilutions of each compound 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. A nonꢀ
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4

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linear regression analysis of the program PRISM 5.0 (GraphPad, San Diego, California, USA).
was used to fit the doseꢀresponse curves and derive IC values.
5
0
Homology modelling of hꢀTNAP and hꢀIAP
Since crystal structure of human TNAP and IAP are not available from the Protein Data Bank
2
6
(PDB), homology models of
h
ꢀTNAP and
h
ꢀIAP were generated using Chimera and Modeller
2
7
10
according to our already reported method . The sequence of target protein
h
ꢀTNAP (uniprot
id P05186) and hꢀIAP (P09923) was fetched in Chimera. BLAST (Basic Local Alignment Search
2
8
Tool) protein database was used to search for sequence similarity with target proteins, human
placental alkaline phosphatase (hPALP, PDB id 1ZED and 1EW2) was identified among the top
five matches and wasaccordingly selected as a template for modelling of
respectively. The sequence of template protein ꢀPALP, was added to the sequence of target
protein using Needleman Wunschglobal alignment algorithm embedded in Chimera.
hꢀTNAP and hꢀIAP
h
2
7
Comparative modelling was run using Modeller via Chimera. The overall quality of the protein
2
9
was evaluated, and Ramachandran plot generated using Molprobity . In generated
Ramachandran plot of ꢀTNAP, 95.2% of the residues lied in favored region, and for ꢀIAP,
6.9% of residues lied in favored region .
h
h
1
0
9
Molecular Docking and Simulation Studies
In order to rationalize most plausible binding site interactions of inhibitors, molecular docking
studies were carried out against homology built models of human alkaline phosphatases (
TNAP and ꢀIAP). Crystal structure of closed (active) form human ectoꢀ5′ꢀnucleotidase (
h
ꢀ
ꢀ
h
h
e5′NT) in complex with inhibitor adenosine 5′ꢀ(α,βꢀmethylene)diphosphate (AMPCP) was
3
0
downloaded from the Protein Data Bank (PDB id 4H2I) . Prior to docking the enzymes
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(receptors) were prepared for docking using DockPrep utility of Chimera, whereby all hetero and
solvent molecules are removed, hydrogen atoms and Gasteiger chargers are added and
incomplete side chains (if any) are repaired using dunbrackrotamer library. Alkaline
phosphatases
sites, charges of +2 were assigned on each metallic center. Human ectoꢀ5′ꢀnucleotidase (
e5′NT) contains only two zinc ions in its active site, charge of +2 was assigned to each zinc ion.
hꢀTNAP and hꢀIAP, contain two zinc ions and one magnesium ion intheir active
h
ꢀ
3
1
Structures of all ligands were sketched in ChemSketch , the geometries of all ligands were
3
2
optimized using semiꢀempirical PM3 method inArgusLab 4.0.1 . Molecular docking studies
3
3
were carried out using BiosolveIT’sLeadIT software version 2.1.8 . Docked conformations were
3
4
analyzed using LeadIT and Discovery Studio Visualizer 4.0 .
For
h
ꢀe5′NT,method validation was carried out by reꢀdocking the ligand (AMPCP) extracted
ꢀe5′NT in complex with AMPCP (PDB id 4H2I), the docking
from the crystal structure of
h
method was successfully able to reproduce the experimentally observed bound conformation of
AMPCP with an rmsd of 1.7 Å.
Molecular dynamics simulations were carried out using MOE software, whrereby most favorable
doked conformation of ligand inside the enzyme was imported into MOE. The enzymeꢀinhibitor
complex was solvated in a periodic cell using “solvate” option of MOE, default settings were
kept. MD simulations were carried out using NPA ensemble, the system was equilibrated for 10
ps followed by an MD run of 100 ps at a physiological temperature of 300 K using MMFF94x
forcefield. Analysis of MD trajectories and distances of selected hydrogen bonds was carried out
in MOE, the data obtained was copied to MS Excel where appropriate graphs were plotted.
Results and Discussion
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6

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Chemistry
General Procedure for the synthesis of 3ꢀ(5ꢀ(benzylideneamino)thiozolꢀ3ꢀyl)ꢀ2Hꢀchromenꢀ
ꢀones
2
The synthetic strategy adopted for 3ꢀ(5ꢀ(benzylideneamino)thiozolꢀ3ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀones
(7aꢀj) is shown in schemeꢀ1.
Scheme 1. The synthetic route to 3ꢀ(5ꢀbenzylideneamino)thiazoleꢀ3ꢀyl)ꢀ2Hꢀchromenꢀ
ꢀones (7aꢀj).
2
The acetylcoumarin (3) was prepared by the solvent free reaction of salicaldehyde with ethyl
1
acetoacetate in the presence of a catalytic amount of piperidine. The H NMR spectrum of (3)
displayed the characteristic signal for Hꢀ4 of the coumarin ring at 8.51 ppm and those of three
1
3
methyl protons at 2.72 ppm. In the C NMR spectrum, the characteristic signal for the ketonic
and lactonic carbon atoms appeared at 195.5 and 159.2 ppm respectively. Bromination of
acetylcoumarin (3) was carried out by refluxing equimolar amounts of acetylcoumarin and
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1
bromine in chloroform to afford αꢀbromoacetylcoumarin (4). The H NMR spectrum displayed
methylene protons at 4.8 ppm. Due to the attachment of electronegative bromine, the signal for
CH carbon appeared at 36.2 ppm more downfield than the methyl carbon of acetyl coumarin.
2
Synthesis of coumarinyl aminothiazole (5) was carried out by the heterocyclization of the αꢀ
bromoacetylcoumarin (4) with thiourea in ethanol under reflux for three hours. In FTIR
ꢀ1
spectrum, the appearance of absorption bands for NH at 3379, 3313cm due to symmetric and
2
ꢀ1
asymmetric stretchings, 1694 cm (C=O lactone), as well as the disappearance of absorption
1
band for C=O (ketone) indicated successful synthesis of (5). In the H NMR spectrum of (5),
signal for –NH at 7.183 ppm appeared as a singlet, and the signal for two methylene protons
2
disappeared due to cyclization with thiourea. The characteristic signal for Hꢀ5' of the thiazole
ring appeared at 7.05 ppm.
1
3
In the C NMR spectrum of the compound (5), disappearance of the characteristic C=O
ketone carbon was observed. The signal for highly deshielded thiazole ring carbon surrounded by
three heteroatoms appeared at 167.8 ppm, and the signal for lactonic carbon appeared at 159.23
ppm. The signal for the (CH ) carbon appeared at 36.2 ppm confirming the formation of the
2
product (5)
.
Finally, the synthesis of target secondary aldimines (7aꢀj) was achieved by refluxing an
equimolar mixture of coumariyl aminothiazole (5) with suitably substituted benzaldehydes (6aꢀj)
in ethanol in the presence of catalytic amount of conc. H SO . The reaction progress was
2
4
monitored with the help of TLC, the secondary aldimines exhibited higher R values as compared
f
to that of coumarinyl aminothiazole. Secondary aldimines, that precipitated out were filtered and
recrystallized from aqueous ethanol to obtain pure products. The FTIR data showed the
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8

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ꢀ1
ꢀ1
disappearance of absorption peaks for (NH ) at 3379 cm , 3313 cm and the appearance of
2
ꢀ1
ꢀ1
(
C=O lactone) in the region of 1687ꢀ1698 cm , for C=N in the region of 1604ꢀ1612 cm , and
ꢀ1
1
C=C aromatic in the region of 1520ꢀ1595 cm was observed. The H NMR spectra showed the
disappearance of signal for ꢀNH2 protons confirming the successful completion of reaction. The
appearance of characteristic signal of a proton corresponding to –N=CH– functionality, also
confirmed the formation of the secondary aldimines. The characteristic signal of Hꢀ4 proton of
the coumarin resides in the range 8.0ꢀ8.2 ppm, the signal for methyl protons was observed in the
range 3.23ꢀ3.68 ppm.
1
3
The
C
NMR spectra of all the synthesized derivatives of 3ꢀ(5ꢀ
ꢀchromenꢀ2ꢀone (7aꢀj) showed the characteristic signal for
(
benzylideneamino)thiazolꢀ3ꢀyl)ꢀ2
H
the thiazole carbon in the range 167ꢀ168 ppm, for C=O (lactone) carbon atom the signal
appeared in the range 157ꢀ158 ppm. The characteristic signal for –N=CH– carbon atom appeared
in the range 152ꢀ153 ppm confirming the formation of secondary aldimines (7aꢀj). Rest of the
signal for the aromatic and thiazole carbons appeared in the aromatic region.
Alkaline Phosphatase and Ectoꢀ5′ꢀNucleotidase Inhibition Activities and SAR
The synthesized derivatives of 3ꢀ(5ꢀ(benzylideneamino)thiazolꢀ3ꢀyl)ꢀ2
evaluated for their inhibitory potential against human alkaline phosphatases (
TNAP), and human and rat ectoꢀ5′ꢀnucleotidases ( ꢀe5′NT and rꢀe5′NT). All derivatives were
tested at final concentration of 200 µM against alkaline phosphatases, and at 100 µM against
ectoꢀ5′ꢀnucleotidases. The inhibition activities of compounds 7aꢀj against ꢀIAP and ꢀTNAP, is
given in Table 1. Compounds 7a 7i and 7j exhibited non selective inhibition against both
isozymes ꢀTNAP and h-IAP, whereas all other compounds 7c, 7d, 7e, 7f, 7g and 7h were found
Hꢀchromenꢀ2ꢀone were
h
ꢀIAP and
hꢀ
h
h
h
,
h
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to be potent and selective inhibitors of only hꢀTNAP. Compound 7i was found to be most potent
hꢀIAP inhibitor (IC50 = 0.05±0.001 µM), possessing ≈1500 fold greater inhibition than the
standard inhibitor Lꢀphenylalanine (IC50 = 80.2 ± 0.001 µM). Compound 7i is the only
compound in this series containing a triꢀmethoxy substituted benzene ring attached to the
azomethine group, this triꢀmethoxy substituted pattern seems to be the reason for increased hꢀ
IAP inhibition exhibited by this compound. Compounds 7a (IC = 0.39±0.09 µM) and 7j (IC =
5
0
50
0
.31±0.04 µM) exhibited almost comparable inhibtion.
For inhibition against
h
ꢀTNAP, the inhibitory activities of compounds 7aꢀ7j vary only slightly
(the difference in inhibition activities of most and least active hꢀTNAP inhibitors is only 0.31
µM) as the nature of substituents on the benzene ring attached to the azomethine group is varied.
Compounds 7h and 7c were found to be most potent inhibitors, possessing inhibitory activities in
lower micromolar range (IC50 = 0.21±0.04 µM and 0.22±0.03, respectively). Their inhibitory
potential is ≈ 91 fold greater than the inhibitory activity of standard inhibitor levamisole (IC =
5
0
1
0
9.2±0.001 µM). This is closely followed by compound 7f, exhibiting an IC50 value of
.25±0.01 µM and ≈77 fold greater inhibition than levamisole. In this series, compounds 7a 7d
ꢀTNAP inhibition (IC = 0.51±0.01 µM, 0.51±0.01 µM and 0.52±0.02
,
and 7i exhibited least
h
5
0
µM respectively), though least in the series, their inhibition is still ≈37 fold greater than the
standard inhibitor levamisole.
Compounds 7aꢀ7j were also evaluated for their inhibitory potential against human and rat ectoꢀ
5
′ꢀnucleotidase enzymes (
h
ꢀe5′NT and rꢀe5′NT). All compounds were found to selectively inhibit
ꢀe5′NT (Table 1). Compounds 7a and 7g were found to be most active
ꢀe5′NT inhibitors with IC values of 0.25±0.07 µM and 0.28±0.05 µM respectively, which is
only the human enzyme,
h
h
5
0
≈
150ꢀ168 fold greater than inhibition of reference standard sulfamic acid. The 3ꢀmethoxyꢀ4ꢀ
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phenoxy substituted compound 7f also exhibited good inhibitory activity (IC = 0.41±0.03 µM)
5
0
followed by the 2ꢀfurfuryl substituted compound 7j (IC = 0.92±0.09 µM). The 3ꢀCl substituted
5
0
compound 7h had IC value of 2.23±0.68 µM. Compounds 7d (4ꢀBr substituted) and 7c (4ꢀF
5
0
substituted) had comparable inhibition activities (IC = 4.38±0.79 µM and 5.15±0.18 µM). The
5
0
triꢀOMe substituted compound 7i (IC = 10.2±1.36 µM) exhibited least inhibition activity in the
5
0
series, which is still ≈4 fold greater than the inhibition exhibited by standard sulfamic acid.
Table1. Alkaline phosphatase (
h
ꢀTNAP &
h
ꢀIAP) and ectoꢀ5′ꢀnucleotidase (
h
ꢀe5′NT &
r
ꢀe5′NT)
inhibition activities of compounds 7aꢀ7j
.
Alkaline Phosphatase
Ectoꢀ5′ꢀNucleotidase
hꢀTNAP
hꢀIAP
hꢀe5′NT
IC50 (µM) ±SEM
0.25±0.07
rꢀe5′NT
Code
IC50 (µM) ±SEM
IC50 (µM) ±SEM
IC50 (µM) ±SEM
7
7
7
7
7
7
7
7
a
c
d
f
0.51±0.01
0.22±0.03
0.51±0.01
0.25±0.01
0.33±0.01
0.21±0.04
0.52±0.02
0.41±0.06
19.2 ±.001
ꢀ
0.39±0.09
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
5.15±0.18
ꢀꢀ
4.38±0.79
ꢀꢀ
0.41±0.03
g
h
i
ꢀꢀ
ꢀꢀ
0.28±0.05
2.23±0.68
0.05±0.001
0.31±0.04
ꢀ
10.2±1.36
j
0.92±0.09
Levamisole
LꢀPhenylalanine
Sulfamic acid
80.2 ± 0.001
42.1±7.8
77.3±7.0
The IC50 is the concentration at which 50% of the enzyme activity is inhibited. Alkaline phosphatase (hꢀTNAP & hꢀ
IAP) activity was performed at final concentration of 200 µM. Ectoꢀ5′ꢀnucleotidase (hꢀe5′NT & rꢀe5′NT ) activity
was carried out at final concentration of 100 µM.
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Molecular Docking
Molecular Docking Studies against h-TNAP and hꢀIAP
In order to rationalize the inhibition caused by inhibitors, most plausible binding site interactions
were generated using molecular docking studies of most active inhibitors. Compound 7h was
found to be the most active hꢀTNAP inhibitor and was therefore selected for the molecular
docking studies. Molecular docking interactions (Figure 1) indicated that compound 7h binds
near the active site, but does not interact directly with the metallic center. The coumarin ring was
found to be oriented towards amino acid residues His321 and His154, whereas the other end of
the molecule, 3ꢀchloro phenyl ring was in close contact with amino acid residues Arg167,
His437, Asp92, and Val90 as shown in Figure below. The oxygen atom of the coumarin ring was
acting as hydrogen bond acceptor and making a hydrogen bond (1.85 Å) with amino group of
Arg167. The carbonyl oxygen atom was also found to act as a hydrogen bond acceptor while
making a bridging hydrogen bond contact (2.65 Å and 1.86 Å) with amino group of Arg167 and
Ser93 respectively.
(a)
(b)
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Figure 1. (a) Most plausible binding mode of most active inhibitor 7h against
h
ꢀTNAP (b) 2D
binding site interactions of 7h against hꢀTNAP
Compound 7i was found to be the most active hꢀIAP inhibitor, and was accordingly selected for
molecular docking studies (Figure 2). The coumarin ring was found to be oriented towards the amino
acid residues Thr450 and Val108. The thiazole ring and azomethine moiety were surrounded by
His451, Phe126 and His339. The trimethoxy phenyl ring was making extensive hydrogen bond
networks with the surrounding amino acid residues. The oxygen atom of one of the methoxy groups
acted as a hydrogen bond acceptor towards NH group of His172 (2.12 Å), while the oxygen atom of an
adjacent methoxy group was found to be involved in making a bridging hydrogen bond with NH of
His172 (1.90 Å) and His336 (2.18 Å). This extensive hydrogen bond network due to triꢀmethoxy
groups, may explain the excellent inhibitory activity of compound 7i towards hꢀIAP.
(a)
(b)
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Figure 2. (a) Most plausible binding mode of most active inhibitor 7i against
hꢀIAP (b) 2D
binding site interactions of 7i against hꢀIAP
In order to have a further confirmation regarding the stability of hydrogenꢀbonded interactions
3
6
indicated by the docking studies, molecular dynamics studies were carried out. MOE software
was used to carry out MD simulations of 100 ps. Figures 3 and 4 indicate stability of Hꢀbonded
interactions for inhibitors 7h and 7i againt ꢀTNAP and ꢀIAP respectively.
h
h
Figure 3. Stability of Hꢀbond interaction between oxygen atom of chromone ring of hꢀTNAP
inhibitor 7h and amino group of Arg167 over 100ps MD run.
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Figure 4. Stability of Hꢀbond interaction between oxygen atom of methoxy group of hꢀIAP
inhibitor 7i and nitrogen atom of His336 over 100ps MD run.
Molecular Docking Studies against Ectoꢀ5′ꢀNucleotidase (hꢀe5′NT)
Active site residues of closed (active) form of hꢀe5′NT are Phe500, Phe417, Arg395, Asn390,
Arg354, Asn245, His243, His220, Asp121, His118, Asn117, Asp85, His38 and Asp3635.
Compound 7a was found to be most active hꢀe5′NT inhibitor and was therefore selected for the
molecular docking stdies (Figure 5). The thiazole ring and associated azomethine moiety of
compound 7a were found to be surrounded by amino residues His118, Phe500, and Phe417,
while the phenyl ring containing nitro substituent, was surrounded by His243 and Leu184. One
of the negatively charged oxygen atom of the nitro group was making a direct contact (2.31 Å)
+
2
with the Zn ion of the active site, the same oxygen atom was also making a hydrogen bond
contact with amino group of Asn117 (1.77 Å). The other oxygen atom the nitro group was
making a hydrogen bond with amino group of Arg395 (1.78 Å).This extensive hydrogen bond
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network due to the presence of nitro group on phenyl ring may be responsible for excellent
inhibition exhibited by this compound.
(a)
(b)
Figure 5. (a) Most plausible binding mode of most active inhibitor 7a against hꢀ eꢀ5′NT (b) 2D
binding site interactions of 7a against hꢀ eꢀ5′NT.
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Figure 6. Stability of ZnꢀO interaction between oxygen atom of nitro group of hꢀ eꢀ5′NT
inhibitor 7a and zinc ion over 100ps MD run.
Inꢀsilico ADME Properties
Evaluation of ADME properties of any molecule is an important step in drug discovery process
as the ADME profile has a major impact on the likelihood of success of a molecule as a drug.
Keeping this in mind, inꢀsilico evaluation of ADME properties of the synthesized compounds
was carried out. Table 2 shows the calculated ADME parameters such as octanolꢀwater partition
coefficient (S+logP and MlogP), the pHꢀdependent octanolꢀwater distribution coefficient
(S+logD), number of hydrogen bond donors (HBDH), hydrogen bond acceptor (sum of nitrogen
and oxygen atoms MNO) and topological polar surface area (TPSA). Generally a compound is
more likely to be membrane permeable and hence easily absorbed by the body if it has a
molecular weight (MWt) less than 500, the logP value, which is a measure of lipophilicity,
should be less than 5, number of hydrogen bond donors (HBDH) is less than 5 and the number of
hydrogen bond acceptors (MNO) is less than 10. The estimation of topological polar surface area
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(TPSA) is often used as a model for estimation of ability of compounds to cross the blood–brain
2
barrier (BBB). Molecules with TPSA values less than 60 Å are usually expected to be
2
completely absorbed while those having TPSA values greater than 140 Å are considered
3
7
undesirable and cannot be expected to possess sufficient bioavailability . All compounds
indicated favorable ADME profile.
Table 2. In-silico evaluation of ADME properties.
Compound S + logP S + logD M logP
MWt
HBDH
0.0
MNO
7.0
7.0
4.0
4.0
8.0
6.0
4.0
4.0
7.0
5.0
TPSA
101.28
101.28
55.46
55.46
110.51
73.92
55.46
55.46
83.15
68.60
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
3.165
3.251
3.529
3.919
2.953
4.55
3.165
3.251
3.529
3.919
2.953
4.55
2.839
2.839
3.174
3.40
377.38
377.38
350.37
411.28
407.40
454.50
366.82
366.82
422.46
322.34
0.0
0.0
0.0
2.318
2.804
3.288
3.288
1.411
1.872
0.0
0.0
g
h
i
3.924
3.924
3.025
2.369
3.924
3.924
3.024
2.369
0.0
0.0
0.0
j
0.0
Conclusion
Series of 3ꢀ(5ꢀ(benzylideneamino)thiazolꢀ3ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone derivatives were synthesized and
characterized. When evaluated for their inhibitory potential against human alkaline phosphatases (hꢀ
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TNAP and hꢀIAP), and human and rat ectoꢀ5′ꢀnucleotidases (hꢀe5′NT and rꢀe5′NT), all compounds were
found to have a selective inhibition against human e5′NT over the corresponding rat enzyme (rꢀe5′NT).
Compounds 7a (IC = 0.25±0.07 µM) and 7g (IC = 0.28±0.05 µM) were most active e5′NT inhibitors.
5
0
50
With the exception of only three compounds (7a, 7i and 7j), all other compounds were found to act as
selective inhibitors of hꢀTNAP over the hꢀIAP isozyme. The most active ꢀTNAP inhibition was
exhibited by compounds 7h and 7c (IC = 0.21±0.04 µM and 0.22±0.03, respectively), which is
h
5
0
≈
91 times greater than the inhibitory activity of standard inhibitor levamisole. Compound 7i was
found to be most potent ꢀIAP inhibitor (IC = 0.05±0.001 µM), exhibiting ≈11 times greater
selectivity for ꢀIAP over
interactions, molecular docking studies were carried out against homology built models of ectoꢀ
nucleotidases ( ꢀTNAP and ꢀIAP). From AP docking studies, no evidence of direct ligand
h
5
0
h
hꢀTNAP. In order to rationalize the most plausible binding site
h
h
interaction with the active site metallic center was observed. However, in case of most active
he5NT inhibitor, the oxygen atom of the nitro group was found to be bound to the catalytic zinc
ion (2.31 Å). In order to further ascertain the stability of predicted interactions, molecular
dynamics simulations were carried out that confirmed stable interactions.
Acknowledgment
AK and MAH acknowledge the funding from Higher Education Commission (HEC) of Pakistan
for NRPU project (No. 1691) and PhD studies, respectively. J. Iqbal is thankful to the
Organization for the Prohibition of Chemical Weapons (OPCW), The Hague, The Netherlands
and Higher Education Commission of Pakistan for the financial support through Project No. 20ꢀ
3
733/NRPU/R&D/ 14/520 for the financial support." JS received support from the Canadian
Institutes of Health Research (CIHR) and was also a recipient of a “Chercheur National”
research award from the Fonds de recherche du Québec – Santé (FRQS).
References
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