Synthesis, characterization and biological evaluation of N-(2,3-dimethyl-5-oxo-1-phenyl-2,5-dihydro-1H-pyrazol-4-yl)benzamides

Article abstract of DOI:10.1039/c5ra17568b

We report the synthesis of a series of different substituted N-(2,3-dimethyl-5-oxo-1-phenyl-2,5-dihydro-1H-pyrazol-4-yl)benzamides by making use of a non-steroidal anti-inflammatory drug known as 4-aminophenazone also called as antipyrine, a compound of great interest in drug chemistry. These compounds possess potential biological applications and were screened against human recombinant alkaline phosphatase including human tissue-nonspecific alkaline phosphatase (h-TNAP), tissue specific human intestinal alkaline phosphatase (h-IAP), human placental alkaline phosphatase (h-PLAP) and human germ cell alkaline phosphatase (h-GCAP). These compounds were also tested for their inhibitory potential against recombinant human and rat ecto-5′-nucleotidases (h-e5-NT & r-e5-NT, respectively). All benzamide derivatives inhibited APs to a lesser degree than e5-NT. The reported compounds are of considerable interest for further applications in the field of medicinal chemistry as these compounds have potential to bind nucleotide protein targets.

Full text of DOI:10.1039/c5ra17568b

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10.1039/C5RA17568B.
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Synthesis, characterization and biological evaluation of Nꢀ(2,3ꢀdimethylꢀ5ꢀoxoꢀ1ꢀphenylꢀ
2,5ꢀdihydroꢀ1Hꢀpyrazolꢀ4ꢀyl)benzamides
Aamer Saeeda*, Syeda Abida Ejazb, Asma Khurshida, Sidra Hassanb, Mariya alꢀRashidac,
Muhammad Latifd, Joanna Leckae,f, Jean Sévignye,f & Jamshed Iqbalb*
a Department of Chemistry, Quaid-I-Azam University, 45320, Islamabad, Pakistan
bCentre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabad 22060, Pakistan
cDepartment of Chemistry, Forman Christian College (A Chartered University), Ferozepur
Road-54600, Lahore, Pakistan
dMedicinal/Pharmaceutical Chemistry Laboratory,Korea Research Institute of Chemical
Technology 141-Gajeongro, Yuseong, Daejeon 305-600, South Korea.
e Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université
Laval, Québec, QC, Canada
f Centre de Recherche du CHU de Québec – Université Laval, Québec, QC, Canada
To whom Correspondence should address:
Prof. Dr. Jamshed Iqbal
Centre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabad 22060, Pakistan.
Tel.: +92 992 383591 96; Fax: +92 992 383441. Eꢀmail addresses: drjamshed@ciit.net.pk
∗ Email: aamersaeed@yahoo.com, Tel +92ꢀ51ꢀ9064ꢀ2128; Fax: +92ꢀ51ꢀ9064ꢀ2241
Abstract:
We report the synthesis of a series of different substituted Nꢀ(2,3ꢀdimethylꢀ5ꢀoxoꢀ1ꢀphenylꢀ2,5ꢀ
dihydroꢀ1Hꢀpyrazolꢀ4ꢀyl)benzamides by making use of a nonꢀsteroidal antiꢀinflammatory drug

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known as 4ꢀaminophenazone also called as antipyrine, a compound of great interest in drug
chemistry. These compounds possess potential biological applications and were screened against
human recombinant alkaline phosphatase including human tissueꢀnonspecific alkaline
phosphatase (hꢀTNAP), tissue specific human intestinal alkaline phosphatase (hꢀIAP), human
placental alkaline phosphatase (hꢀPLAP) and human germ cell alkaline phosphatase (hꢀGCAP).
These compounds were also tested for their inhibitory potential against recombinant human and
rat ectoꢀ5’ꢀnucleotidases (hꢀe5ꢀNT & rꢀe5ꢀNT, respectively). All benzamide derivatives inhibited
APs to a lesser degree than e5ꢀNT. The reported compounds are of considerable interest for
further applications in the field of medicinal chemistry as these compounds have potential to
bind nucleotide protein targets.
Keywords: alkaline phosphatase, antipyrine, human recombinant, human Germ cell, intestinal
alkaline phosphatase
Introduction
Ectoꢀnucleotidases enzymes are subdivided into four major families: the ectoꢀnucleoside 5′ꢀ
triphosphate diphosphohydrolases (NTPDases), the alkaline phosphatases (APs), the
ectoꢀnucleotide pyrophosphatases (NPPs) and the ectoꢀ5′ꢀnucleotidases (e5NT).1
These enzymes are responsible for the hydrolysis of a wide range of extracellular nucleotides to
nucleosides and adenosine.2 Adenosine monophosphate (AMP) is a significant biological
substrate of ectoꢀ5'ꢀnucleotidase as its hydrolysis produces adenosine, which acts on P1
receptors.3 AMP is produced by the degradation of P2 receptor agonists ATP and ADP by the
other ectonucleotidases mentioned above. Among the different nucleotidases, ectoꢀ5'ꢀ
nucleotidase is expressed by several cells including by those localized on the outer surface of
endothelial cells of large and small arteries. Hypoxia increases the expression of ectoꢀ5'ꢀ
nucleotidase and it is generally upꢀregulated at the site of inflammation.4
Alkaline phosphatases are the other important enzymes for the production of adenosine either
directily by the conversion of adenosine monophosphate (AMP) into adenosine (Ado) or
indirectly by the conversion of ATP ꢀ ADP which is then converted to AMP and finally to
Ado.5 AP hydrolyzes a variety of substrates other than nucleotides, such as bis(pꢀ
nitrophenyl)phosphate, inorganic polyphosphates, phosphatidates and glucoseꢀphosphates.6 In

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the presence of large concentrations 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. germ cell AP (GCAP), placental AP (PLAP) and
intestinal AP (IAP). They have relative homologies in the range of 90–98% and their genes are
clustered on chromosome 2. The fourth (tissueꢀnonspecific AP; TNAP) is found in liver, kidney
and most abundantly in bones.7 The gene of TNAP is located on chromosome 1 and it is only
50% identical to the other three isozymes. TNAP has various functions depending of the tissue
where it is expressed. An important role demonstrated for this enzyme is to hydrolyze a potent
calcification inhibitor, inorganic pyrophosphate.8 The intestinal alkaline phosphatase is found at
the brush boarder of the intestinal epithelium and enriched in surfactantꢀlike particles (SLP).
They are likely to be involved in intestinal absorption of the lipid/nutrients across the cell
membrane via its association with SLP. Because of a high homology between TNAP and tissueꢀ
specific IAP, very few selective inhibitors of IAP have been reported.9 The tissueꢀspecific
placental alkaline phosphatase (PLAP) is highly expressed in placental tissue and its biological
function is still unknown.10 The PLAPꢀlike enzymes are observed in serum of patients with
primary testicular tumors, in particular seminoma11 and other cancers.12 PLAP and GCAP are
often coꢀexpressed in ovarian cancers and it has been reported that transformation from normal
to malignant trophoblast might be associated with a switch from PLAP to GCAP expression.3
We report here various benzamide derivatives containing pyrazole as excellent inhibitors of e5ꢀ
NT and alkaline phosphatases (hꢀTNAP,hꢀ IAP, hꢀPLAP and hꢀGCAP). Some of the compounds
were found to be more selective as inhibitors of e5ꢀNT even at lower concentrations and can be
developed further to synthesize even more potent and selective compounds lead derivatives.
Aminophenazone is one of the antipyrine derivatives which was first synthesized by Knorr in
1833 and ever since the antipyrine derivatives (APDs) have been studied. The antipyrine
derivatives have been investigated in a variety of fields.13 The nonlinear optical and UVꢀvis
studies indicate that these antipyrine derivatives are the promising photo electronic materials.
Aminophenazone derivatives have been shown to possess a number of potent biological
applications including their use as antipyretic, analgesic and antiꢀinflammatory agents.14 The
presence of amide linkage imparts useful biological applications to aminophenazone moiety.
Since amides are an important class of organic compounds and can be synthesized in a number
of ways either by direct coupling of carboxylic acids and amines at high temperatures or through

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microwave assisted methodologies and even through organo catalysts. The exploration of amidic
linkage in various drugs has far more enhanced the importance of amides and has broadened the
scope of their biological applications like anthelmintic, antihistamine, antifungal, and
antibacterial etc.15 Both alkaline phosphatase (AP) and ectoꢀ5′ꢀnucleotidase (e5NT) are
responsible for hydrolyzing nucleotides. AP is substrate nonꢀspecific and is a major enzyme
responsible for AMP hydrolysis (although it hydrolysis other nucleotides as well), on the other
hand ectoꢀ5ꢀnucleotidase is a substrate specific enzyme and only hydrolyzes AMP to adenosine
with the release of phosphate group16,17. Pyrazole derivatives have previously been identified as
inhibitors of AP18, keeping this in mind our research group focused on design of new pyrazole
derivatives containing amide linkages as potential inhibitors of AP, since e5NT is also a major
AMP hydrolyzing enzyme, therefore activity against this enzyme was also evaluated.
Experimental Section
Melting points were determined using Gallenkamp melting point apparatus (MPꢀD) and are
1
uncorrected. Infrared spectra were recorded using a Shimadzu IR 460 as KBr pellets. HꢀNMR
spectra were obtained using a Bruker 300 NMR MHz spectrometer in CDCl3 solution using TMS
as an internal reference. 13CNMR spectra were obtained by (75 MHz) NMR spectrometer in
deuterated solvents. Thin layer chromatography was performed on preꢀcoated silica gel
aluminum plates (layer thickness 0.2 mm, HF 254, ReidalꢀdeꢀHaen from Merck). Chromatogram
was detected by using ultraviolet light (254 and 260nm). MS were recorded using an EI source
of (70 EV) on Agilent Technologies 6890N (GC) and an inert mass selective detector 5973 mass
spectrometer.
General procedure for the synthesis of Nꢀ(1,5ꢀDimethylꢀ3ꢀoxoꢀ2ꢀphenylꢀ2,3ꢀdihydroꢀ1Hꢀ
pryazoleꢀ4ꢀyl)benzamide (4a):
Brownish black amorphous solid; Yield; 89%, m.p: 133ꢀ134 °C; IR (neat): 3140 (CONH), 2967,
1
2829, (Csp3ꢀH), 1677 (CONH), 1421, 1340 (Csp3ꢀH bending): HꢀNMR (CDCL3, 300MHz): δ
8.09 (m, 5H, ArꢀH, s, 1H, NꢀH), 7.84 (m, 4H, ArꢀH), 3.07 (s, 3H), 2.10 (s, 3H); 13CꢀNMR
(CDCL3, 75MHz): δ 173.02 (CONH), 170.89 (ꢀC=CꢀCOꢀNꢀ), 161.45, 152.28, 134.67, 133.46,
132.33, 130.10, 129.72, 129.31, 128.46, 127.47, 124.77,35.36 (CH3ꢀNꢀ), 10.62 (CH3ꢀCSP2ꢀ),
12.78 (CH3ꢀArꢀC).LCꢀMS (m/z) %: M+ 308, 214, 203. Elemental analysis found C18H17N3O2: C
69.00, H 6.92, N 12.05.

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Nꢀ(1,5ꢀDimethylꢀ3ꢀoxoꢀ2ꢀphenylꢀ2,3ꢀdihydroꢀ1Hꢀpryazoleꢀ4ꢀyl)ꢀ2ꢀmethylbenzamide (4b):
Brown amorphous solid; Yield; 86%, m.p: 125ꢀ126 °C; IR (neat): 3150 (CONH), 2997, 2809,
(Csp3ꢀH), 1670 (CONH), 1428, 1342 (Csp3ꢀH bending): 1HꢀNMR (CDCL3, 300MHz): δ 7.65 (s,
13
1H, NꢀH), 7.58ꢀ7.21 (m, 9H, ArꢀH), 3.12 (s, 3H), 2.53 (s, 3H), 2.37 (s, 3H); CꢀNMR (CDCL3,
75MHz): δ 168.63 (CONH), 161.50 (ꢀC=CꢀCOꢀNꢀ), 149.05, 136.81, 134.54,131.24, 131.00,
130.80, 130.41, 129.28, 124.21,36.27 (CH3ꢀNꢀ), 20.16 (CH3ꢀCSP2ꢀ), 12.78 (CH3ꢀArꢀC). GCꢀMS
(m/z): 321 M+, 245, 187, 119 (100%), 91, 56. Elemental analysis found C19H19N3O2: C 72.32, H
5.25, N 13.05.
Nꢀ(1,5ꢀDimethylꢀ3ꢀoxoꢀ2ꢀphenylꢀ2,3ꢀdihydroꢀ1Hꢀpryazoleꢀ4ꢀyl)ꢀ4ꢀmethylbenzamide (4c):
Yellow crystalline solid; Yield; 85%, m.p: 183ꢀ184 °C; IR (neat): 3145 (CONH), 2954, 2891,
(Csp3ꢀH), 1637 (CONH), 1425, 1347 (Csp3ꢀH bending): 1HꢀNMR (CDCL3, 300MHz): δ 8.04 (s,
1H, NꢀH), 7.78 (d, 2H, ArꢀH, J = 6Hz), 7.47ꢀ7.40 (m, 4H, ArꢀH), 7.33ꢀ7.21 (m, 3H, ArꢀH), 3.10
(s, 3H), 2.39 (s, 3H), 2.32 (s, 3H); 13CꢀNMR (CDCL3, 75MHz): δ 166.09 (CONH), 161.86 (ꢀ
C=CꢀCOꢀNꢀ), 149.31, 142.27, 134.66, 130.87, 129.27, 129.19, 127.50, 126.98, 124.33, 109.07,
36.23 (CH3ꢀNꢀ), 21.49 (CH3ꢀCSP2ꢀ), 12.68 (CH3ꢀArꢀC).LCꢀMS (m/z) %: M+322. Elemental
analysis found C19H19N3O2: C 72.32, H 5.25, N 13.05.
2ꢀChloro-Nꢀ(1,5ꢀdimethylꢀ3ꢀoxoꢀ2ꢀphenylꢀ2,3ꢀdihydroꢀ1Hꢀpryazoleꢀ4ꢀyl)benzamide (4d):
Yellow crystalline solid; Yield; 82%, m.p: 103ꢀ104 °C; IR (neat): 3150 (CONH), 2991, 2865,
1
(Csp3ꢀH), 1675 (CONH), 1435, 1346 (Csp3ꢀH bending): HꢀNMR (CDCL3, 300MHz): δ 7.64ꢀ
7.60 (m, 3H, ArꢀH), 7.45 (m, 2H, ArꢀH), 7.40 (m, 3H, ArꢀH), 7.37ꢀ7.26 (m, 2H, ArꢀH, s, 1H, Nꢀ
13
H), 3.13 (s, 3H), 2.41 (s, 3H); CꢀNMR (CDCL3, 75MHz): δ 166.25 (CONH), 161.36 (ꢀC=Cꢀ
COꢀNꢀ), 149.45, 137.40, 134.55,133.40, 131.45, 129.63, 129.24, 127.48, 126.98, 124.21, 119.70,
108.31, 36.17 (CH3ꢀNꢀ), 12.79 (CH3ꢀCSP2ꢀH). LCꢀMS (m/z) %: M+342 (1:3), 214. Elemental
analysis found C18H16ClN3O2: C 61.25, H 6.72, N 14.42.
4ꢀChloro-Nꢀ(1,5ꢀdimethylꢀ3ꢀoxoꢀ2ꢀphenylꢀ2,3ꢀdihydroꢀ1Hꢀpryazoleꢀ4ꢀyl)benzamide (4e):
Yellow crystalline solid; Yield; 86%, m.p: 113ꢀ114°C; IR (neat): 3151 (CONH), 2993, 2876,
1
(Csp3ꢀH), 1674 (CONH), 1425, 1341 (Csp3ꢀH bending): HꢀNMR (CDCL3, 300MHz): δ 7.97ꢀ
13
7.94 (m, 2H, ArꢀH), 7.55ꢀ7.41 (m, 7H, ArꢀH, s, 1H, NꢀH), 3.31 (s, 3H), 2.26 (s, 3H); CꢀNMR

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(CDCL3, 75MHz): δ 164.66 (CONH), 161.86 (ꢀC=CꢀCOꢀNꢀ), 152.91, 136.44, 135.04, 132.49,
129.56, 129.07, 128.44, 126.24, 123.51, 35.94 (CH3ꢀNꢀ), 10.97 (CH3ꢀCSP2ꢀ).LCꢀMS (m/z) %:
M+342 (1:3), 278, 214. Elemental analysis found C18H16ClN3O2: C 61.25, H 6.72 , N 14.42.
2ꢀBromoꢀNꢀ(1,5ꢀdimethylꢀ3ꢀoxoꢀ2ꢀphenylꢀ2,3ꢀdihydroꢀ1Hꢀpryazoleꢀ4ꢀyl)benzamide (4f):
Brown crystals, Yield; 87%, m.p: 108ꢀ109 °C ; IR (neat): 3151 (CONH), 2990, 2832, (Csp3ꢀH),
1678 (CONH), 1423, 1332 (Csp3ꢀH bending): 1HꢀNMR (CDCL3, 300MHz): δ 7.81 (s, 1H, NꢀH),
7.66ꢀ7.28 (m, 9H, ArꢀH), 3.15 (s, 3H), 2.48 (s, 3H) ; 13CꢀNMR (CDCL3, 75MHz): δ 166.31
(CONH), 161.19 (ꢀC=CꢀCOꢀNꢀ), 36.09 (CH3ꢀNꢀ), 12.81 (CH3ꢀCSP2ꢀ), 149.37, 137.29, 134.37
,133.46, 131.56, 129.63, 129.33, 127.56, 124.40. GCꢀMS (m/z): 387 (M+), 341, 321 (100%),
202, 119, 91, 56.LCꢀMS (m/z) %: M+386 (1:1), 368, 214, 203. Elemental analysis found
C18H16BrN3O2: C 59.47, H 4.06, N 9.19.
Nꢀ(1,5ꢀDimethylꢀ3ꢀoxoꢀ2ꢀphenylꢀ2,3ꢀdihydroꢀ1Hꢀpryazoleꢀ4ꢀyl)ꢀ3,5ꢀdihydroxybenzamide
(4g):
Brownish black amorphous solid; Yield; 82%, m.p: 163ꢀ164 °C; IR (neat): 3159 (CONH), 2927,
1
2815, (Csp3ꢀH), 1679 (CONH), 1422, 1332 (Csp3ꢀH bending): HꢀNMR (CDCL3, 300MHz): δ
8.79 (s, 1H, NꢀH), 7.21 (m, 2H, ArꢀH), 6.92 (m, 3H, ArꢀH), 6.58 (m, 2H, ArꢀH), 3.27 (s, 3H),
2.37 (s, 3H); 13CꢀNMR (CDCL3, 75MHz): δ 165.62 (CONH), 162.53 (ꢀC=CꢀCOꢀNꢀ), 158.73.
158.28, 144.74, 142.74, 129.32, 129.08, 126.04, 124.67, 123.41, 123.27, 108.12, 106.36,
36.12(CH3ꢀNꢀ), 11.03 (CH3ꢀCSP2ꢀ).LCꢀMS (m/z) %: M+340, 322, 214, 135. Elemental analysis
found C18H17N3O4: C 62.00, H 6.76, N 11.01.
Nꢀ(1,5ꢀDimethylꢀ3ꢀoxoꢀ2ꢀphenylꢀ2,3ꢀdihydroꢀ1Hꢀpryazoleꢀ4ꢀyl)ꢀ4ꢀphenylbutanamide (4h):
Yellow solid; Yield; 84%, m.p: 137ꢀ138 °C; IR (neat): 3256 (CONH), 2923, 2827, (Csp3ꢀH),
1
1659 (CONH), 1433, 1352 (Csp3ꢀH bending): HꢀNMR (CDCL3, 300MHz): δ 8.80 (d, 2H, Arꢀ
H), 8.37 (d, 1H, ArꢀH), 8.20 (s, 1H, NꢀH), 3.55 (2H, t, 9Hz),1.81 (m, 2H),3.08 (s, 3H); 13CꢀNMR
(CDCL3, 75MHz): δ 177.30 (CONH), 173.29 (ꢀC=CꢀCOꢀNꢀ), 161.08, 149.27, 144.94, 141.78,
140.25, 134.07, 128.54, 128.47, 127.44, 126.83, 123.26, 54.22 (ꢀCOꢀCH2ꢀCꢀ), 35.83 (CH3ꢀNꢀ),
26.56(CH3ꢀCSP2ꢀ), 12.32 (ꢀCH2ꢀCH2ꢀArꢀ), 12.20 (ꢀCH2ꢀCH2ꢀCH2ꢀ).LCꢀMS (m/z) %: M+349,
348, 232, 214.Elemental analysis found C21H23N3O2: C 70.18, H 8.83, N 10.00.

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Nꢀ(1,5ꢀDimethylꢀ3ꢀoxoꢀ2ꢀphenylꢀ2,3ꢀdihydroꢀ1Hꢀpryazoleꢀ4ꢀyl)furanꢀ2ꢀcarboxamide (4i):
Red crystalline solid, Yield; 83%, m.p: 143ꢀ144 °C; IR (neat): 3146 (CONH), 2953, 2896,
(Csp3ꢀH), 1634 (CONH), 1455, 1327 (Csp3ꢀH bending): 1HꢀNMR (CDCL3, 300MHz): δ 8.52 (s,
1H, NꢀH), 7.60ꢀ7.52 (m, 4H, ArꢀH), 7.41ꢀ7.36 (m, 3H, ArꢀH,), 6.49 (d, 1H, Cꢀ4ꢀFuroic ring, J =
1.8Hz), 6.49 (s, 1H), 3.42 (s, 3H), 2.96 (s, 3H) ; 13CꢀNMR (CDCL3, 75MHz): δ 161.91 (CONH),
156.27 (ꢀC=CꢀCOꢀNꢀ), 155.55, 148.70, 145.83, 132.26, 130.10, 130.01, 116.97, 112.29,107.56,
33.60 (CH3ꢀNꢀ), 14.80 (CH3ꢀCSP2ꢀ). GCꢀMS (m/z): 387 (M+), 341, 321 (100%), 202, 119, 91,
56. LCꢀMS (m/z) %: M+322, 233, 215, 214. Elemental analysis found C16H15N3O3: C 63.56, H
5.12, N 15.08.
Nꢀ(1,5ꢀDimethylꢀ3ꢀoxoꢀ2ꢀphenylꢀ2,3ꢀdihydroꢀ1Hꢀpryazoleꢀ4ꢀyl)pivalamide (4j):
Yellow amorphous solid, Yield; 86%, m.p: 127ꢀ128 °C ; IR (neat): 3250 (CONH), 2978, 2932,
1
(Csp3ꢀH), 1644 (CONH), 1456, 1337 (Csp3ꢀH bending): HꢀNMR (CDCL3, 300MHz): δ 7.45ꢀ
7.28 (m, 4H, ArꢀH), 7.26 (m, 1H, ArꢀH), 7.07 (s, 1H), 3.05 (s, 3H), 2.26 (s, 3H), 1.31 (s, 9H,
CH3) ; 13CꢀNMR (CDCL3, 75MHz): δ 177.47 (CONH), 161.68 (ꢀC=CꢀCOꢀNꢀ), 148.92, 134.81,
129.17, 126.66, 123.80, 39.28 (CH3ꢀNꢀ), 36.49 (CH3ꢀCSP2ꢀH), 27.67 (ꢀCOꢀCꢀCH3). GCꢀMS
(m/z): 387 (M+), 341, 321 (100%), 202, 119, 91, 56.LCꢀMS (m/z) %: M+288, 214, 135.
Elemental analysis found C16H21N3O2: C65.82, H7.43, N 14.67.
Biochemical Assays
Cell Transfection with Human APs and e5ꢀNT
The COSꢀ7 cells were transfected with plasmids expressing human APs: TNAP, IAP, PLAP &
GCAP 19 or ectoꢀ5’ꢀnucleotidase either human or rat5 in 10ꢀcm plates, by using Lipofectamine.
The confluent cells were incubated for 5 h at 37 °C in DMEM/Fꢀ12 in the absence of fetal bovine
serum and with 6 µg of plasmid DNA and 24 µL of Lipofectamine reagent. The same volume of
DMEM/Fꢀ12 containing 20% FBS was added to stop the transfection and cells were harvested
48–72 h later.
Preparation of membrane fractions
These transfected cells were washed three times with Tris–saline buffer at 4 °C and then the cells
were collected by scraping in the harvesting buffer (95 mM NaCl, 0.1 mM PMSF, and 45 mM

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Tris buffer, pH 7.5). The cells were washed twice by centrifugation at 300×g for 5 min at 4 °C.5
Later the cells were resuspended in the harvesting buffer containing 10 µg/mL aprotinin and then
sonicated. Cellular and nuclear debris were discarded by 10 min centrifugation (300×g at 4 °C).
Glycerol (final concentration of 7.5%) was added to the resulting supernatant and all the samples
were kept at ꢀ80 °C until used. Bradford microplate assay20 was used for the estimation of
protein concentration. Bovine serum albumin was used as a reference standard.
Protocol of Alkaline Phosphatase Assay (hꢀTNAP, hꢀIAP, hꢀPLAP and hꢀGCAP)
The determination of phosphatase activity with hꢀTNAP, hꢀIAP, hꢀPLAP and hꢀGCAP in
presence or absence of the tested compounds, was performed with a chemiluminescent substrate,
CDPꢀstar. The conditions for the assay were optimized with the slight modifications in
previously used spectrophotometric method.3 The composition of assay buffer was: 2.5 mM
MgCl2, 0.05 mM ZnCl2 and 8 M DEA (pH 9.8). Initial screening of the tested compounds was
performed at a concentration of 0.2 mM. The total volume of 50 µL contained 10 µL of tested
compound (0.2 mM with final DMSO 1% (v/v)), 20 µL of hꢀTNAP (46 ng of protein from COS
cell lysate in assay buffer) or of hꢀIAP (57 ng protein in assay buffer) or of hꢀPLAP (55 ng
protein in assay buffer) or of hꢀGCAP (51 ng protein in assay buffer), depending on assay. The
o
mixture was preꢀincubated for 5 to 7 minutes at 37 C and luminescence was measured as preꢀ
read using microplate reader (BioTek FLx800, Instruments, Inc. USA). Then, 20 µL of CDPꢀstar
(final concentration of 110 µM) was added to initiate the reaction and the assay mixture was
o
allowed to incubate for 15 min more at 37 C. The change in the luminescence was measured as
afterꢀread. The activity of each compound was compared with total activity control (without any
inhibitor). Levamisole (2 mM per well) and Lꢀphenylalanine (4 mM per well) were used as a
positive control for the inhibition of hꢀTNAP and hꢀIAP, respectively. For the compounds which
exhibited over 50% inhibition of either enzyme activity, full concentration inhibition curves were
produced to evaluate IC50 values. All experiments were performed in triplicate. The IC50 values
were calculated by using using a nonꢀlinear curve fitting program PRISM 5.0 (GraphPad, San
Diego, California, USA).
Ectoꢀ5ʹꢀnucleotidase Inhibition Assay

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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 20 mM, pH 9.0) for 5 min.
The bioactivity assay of both rat and human Ectoꢀ5′ꢀnucleotidase was performed with the slight
modifications in the method as described previously.21 Stock solutions of 10 mM concentration
of each compound were prepared in DMSO. Working solutions were prepared by appropriately
diluting the stock solutions to 1 mM concentration in assay buffer (2 mM MgCl2, 1 mM CaCl2
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 e5NT (6.94 µg/mL) protein extract or
rat e5NT (7.17 µg/mL) for 10 min in the presence of 70 µL assay buffer (2 mM MgCl2, 1 mM
CaCl2 and 10 mM Tris HCl, pH 7.4). Afterwards, 10 µL of AMP substrate (final concentration of
500 µM) was added to initiate the enzymatic reaction. The reaction mixture was allowed to
incubate for 10 min at 37 oC. 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 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
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
e5NT or hꢀe5NT activity were further evaluated for determination of IC50 values. For this
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. The IC50
values were calculated by using a nonꢀlinear curve fitting program PRISM 5.0 (GraphPad, San
Diego, California, USA).

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Kinetics Study (Alkaline Phosphatase)
The inhibitors (compounds) 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 concentrations of the inhibitor (0 µM, 0.5 µM, 1.00 µM, 2.00 µM) using
substrate (CDPꢀstar) concentrations of 55µM, 110 µM, 165 µM & 220 µM. A doubleꢀreciprocal
plot of the inhibition kinetics of hꢀTNAP by 4i & 4h by hꢀIAP and 4f by hꢀPLAP by inhibitor
was measured by using PRISM 5.0 (GraphPad, San Diego, California, USA).
4i (TNAP)
0.00016
0 ꢀM
0.50 ꢀM
1.00 ꢀM
2.00 ꢀM
0.00015
0.00014
0.00013
ꢀ0.04
ꢀ0.03
ꢀ0.02
ꢀ0.01
0.00012
0.00
0.01
0.02
1/[S](ꢀM)
Figure 1. Doubleꢀreciprocal plots of the inhibition kinetics of bovine tissue nonꢀspecific alkaline
phosphatase (TNAP) indicating competitive inhibition by compound 4i. Changes in the initial
velocities of the reaction were measured at different concentrations of the inhibitor 4i using
substrate
CDPꢀStar
(disodium
2ꢀchloroꢀ5ꢀ(4ꢀmethoxyspiro[1,2ꢀdioxetaneꢀ3,2′ꢀ(5ꢀ
chlorotricyclo[3.3.1.13.7]decan])ꢀ4ꢀyl]ꢀ1ꢀphenyl phosphate).
4h (IAP)

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0.00017
0.00016
0.00015
0.00014
0.00013
0.00012
0 ꢀM
0.50 ꢀM
1.00 ꢀM
2.00 ꢀM
ꢀ0.03
ꢀ0.02
ꢀ0.01
0.00
1/[S](ꢀM)
0.01
0.02
Figure 2. Doubleꢀreciprocal plots of the inhibition kinetics of tissue specific alkaline
phosphatase (IAP) indicating competitive inhibition by compound 4h. Changes in the initial
velocities of the reaction were measured at different concentrations of the inhibitor 4h using
substrate
CDPꢀStar
(disodium
2ꢀchloroꢀ5ꢀ(4ꢀmethoxyspiro[1,2ꢀdioxetaneꢀ3,2′ꢀ(5ꢀ
chlorotricyclo[3.3.1.13.7]decan])ꢀ4ꢀyl]ꢀ1ꢀphenyl phosphate).
4f (hꢀPLAP)

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0.00015
0.00014
0.00013
0.00012
0.00011
0 ꢀM
0.50 ꢀM
1.00 ꢀM
2.00 ꢀM
ꢀ0.05
ꢀ0.04
ꢀ0.03
ꢀ0.02
ꢀ0.01
0.00
0.01
0.02
1/[S](ꢀM)
Figure 3. Doubleꢀreciprocal plots of the inhibition kinetics of tissue specific alkaline
phosphatase (hꢀPlAP) indicating competitive inhibition by compound 4f. Changes in the initial
velocities of the reaction were measured at different concentrations of the inhibitor 4f using
substrate
CDPꢀStar
(disodium
2ꢀchloroꢀ5ꢀ(4ꢀmethoxyspiro[1,2ꢀdioxetaneꢀ3,2′ꢀ(5ꢀ
chlorotricyclo[3.3.1.13.7]decan])ꢀ4ꢀyl]ꢀ1ꢀphenyl phosphate).
Homology modelling of Human Alkaline Phosphatases (h-TNAP, h-IAP, hꢀGCAP)
Since crystal structures of human tissue nonꢀspecific alkaline phosphatase (h-TNAP), intestinal
alkaline phosphatase (hꢀIAP) and germꢀcell alkaline phosphatase (hꢀGCAP) have not yet been
determined experimentally, therefore homology models of h-TNAP, hꢀIAP and hꢀGCAP were
generated using Chimera22 and Modeller.23 Sequences of all three human AP enzymes, h-TNAP
(uniprot id: P05186), hꢀIAP (uniprot id: P09923), and hꢀGCAP (uniprot id: P10696) were
fetched in Chimera. A search on BLAST database24 returned h-PLAP (PDB id: 1ZED; uniprot
id: P05187) among top five searched results for all APs, therefore h-PLAP was selected to be
used as a template protein. The sequences of target proteins were added to that of template

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protein via Needleman Wunsch global alignment algorithm25 embedded in Chimera. The
structures of all target proteins h-TNAP, hꢀIAP and hꢀGCAP were modelled using Modelle23.
Ramachandran plots were generated using Molprobity26. Comparative sequence alignments and
Ramachandran plots are given in supporting information, Figures S6ꢀS8 and Figures S2ꢀS4,
respectively. All active site residues among modelled proteins (h-TNAP, hꢀIAP, hꢀGCAP), and
h-PLAP were found to be highly conserved (Table 1). Amino acid residues Phe107, Gln108,
Ser155 and Glu429 in h-PLAP have been replaced by amino acid residues Glu108, Gly109,
Thr156 and His434 respectively in hꢀTNAP. Glu429 of h-PLAP, was found to be the only active
site amino acid residue that was not conserved in any of the hꢀAPs, in hꢀIAP it is replaced by a
Ser448 residue, whereas in hꢀGCAP, it has been replaced by Gly448 residue. Figures 1ꢀ3 show
comparison of active site residues of modelled proteins h-TNAP, hꢀIAP and hꢀGCAP against the
template protein h-PLAP.
Homology modelling of Rat ectoꢀ5′ꢀNucleotidase (rꢀe5NT)
Crystal structure of rat ectoꢀ5′ꢀnucleotidase (r-e5NT), is not available from the Protein Data
Bank (PDB), therefore its homology model was generated using Chimera22 and Modeller23. The
protein sequence of rꢀe5NT (uniprot id: Q66HL0) was fetched in Chimera. A search on BLAST
database24 indicated human ectoꢀ5′ꢀnucleotidase (hꢀe5NT), (PDB id: 4H2I; uniprot id: P21589)
among top five results (sharing 88% sequence identity), and was therefore selected to be used as
a template protein for modelling of rꢀe5NT. The sequences of target protein (r-e5NT) was added
to that of template protein (hꢀe5NT) via Needleman Wunsch global alignment algorithm25
embedded in Chimera. The structure of target protein, rꢀe5NT, was modelled using Modeller23
Ramachandran plots were generated using Molprobity26. Five models were generated, each
model was analyzed individually and the most favorable model, having 87.97% identity and
0.141 rmsd was selected. Comparative sequence alignment and Ramachandran plot are given in
supporting information, Figures S1 and Figures S5, respectively. Active site residues among
modelled proteins (rꢀe5NT), and target protein (hꢀ5T) were found to be highly conserved.
Comparison of active site residues of rꢀe5NT and hꢀe5NT are given in Figure 4.
Molecular Docking

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In order to rationalize most plausible binding site interactions of inhibitors, molecular docking
studies were carried out against three homology built models of alkaline phosphatases (h-TNAP,
hꢀIAP and hꢀGCAP), and one experimentally determined h-PLAP (PDB id: 1ZED). Crystal
structure of closed (active) form human ectoꢀ5′ꢀnucleotidase (hꢀe5NT) in complex with inhibitor
adenosine 5′ꢀ(α,βꢀmethylene)diphosphate (AMPCP) was downloaded from the Protein Data
Bank (PDB id 4H2I).27 Prior to docking the enzymes (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 Dunbrack Rotamer library. All alkaline phosphatases, contain two zinc ions and
one magnesium ion intheir active sites, charges of +2 were assigned on each metallic center.
Human ectoꢀ5′ꢀnucleotidase (hꢀe5NT) contains only two zinc ions in its active site, charge of +2
was assigned to each zinc ion. Structures of all ligands were sketched in ChemSketch,28 the
geometries of all ligands were optimized using semiꢀempirical PM3 method in ArgusLab 4.0.1.29
Molecular docking studies were carried out using BiosolveIT’s LeadIT software version 2.1.8.30
Docked conformations were analyzed using LeadIT and Discovery Studio Visualizer 4.0.31 For
hꢀe5NT method validation was carried out by reꢀdocking the ligand (AMPCP) extracted from the
crystal structure of hꢀe5NT in complex with AMPCP (PDB id 4H2I), the docking method was
successfully able to reproduce the experimentally observed bound conformation of AMPCP with
an rmsd of 1.7 Å
Results and Discussion
General procedure for the synthesis ofNꢀ(2,3ꢀdimethylꢀ5ꢀoxoꢀ1ꢀphenylꢀ2,5ꢀdihydroꢀ1Hꢀ
pyrazolꢀ4ꢀyl)benzamides (4aꢀj)
A calculated amount of suitably substituted aromatic acids 1aꢀj (0.5 mmol) was set to reflux with
thionyl chloride (0.03 mL, 0.5 mmol) for the fresh preparation of respective acid halides (2aꢀj).
The acid chlorides (2aꢀj) were added to the solution of 4ꢀaminophenazone 3 (0.1g, 0.5 mmol),
dissolved in pyridine (0.03 mL, 0.5 mmol) and the reaction mixture was refluxed for 3 hours.
The completion of the reaction was confirmed by TLC, the solids appeared were filtered off and
recrystallized by aqueous ethanol to afford the purified products (4aꢀj).

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Chemistry
The synthetic pathway adopted for syntheses of title compounds is depicted in Scheme 1. The
benzamides are easily accessible by reaction of suitably substituted acid chlorides to the solution
of 4ꢀaminophenazone in dry pyridine. The solids products obtained were recrystallized by
aqueous ethanol to afford the purified products (4aꢀj). The spectral and microelemental data
confirmed the structural assignments. The compound 4i has already been reported.32
Alkaline Phosphatase and Ectoꢀ5’ꢀnucleotidase Activity and SAR:

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Different benzamide derivatives of phenazone were synthesized and further evaluated for their
anticancer properties. For this purpose inꢀvitro activity against membrane bounded nucleotidases
including alkaline phosphatases and ectoꢀnucleotidase were performed. These compounds were
found to inhibit all APs at the concentration of 200 µM. However, at the lower concentration of
100 µM these compounds significantly inhibited both human and rat e5ꢀNT
.
As regards to AP activity, the compounds were tested against four isozymes of human
recombinant APs: hꢀTNAP, h-IAP, hꢀPLAP and hꢀGCAP. It was found that all the derivatives
exhibited significant inhibitory potential against hꢀTNAP within the IC50 ± SEM values of
0.337±0.08 µM to 5.91±0.99 µM. Among ten derivatives, compound 4i was the most potent
inhibitor of hꢀTNAP with an IC50 value ±SEM of 0.337±0.08 µM which is 70 fold lower than
the reference standard inhibitor Levamisole (IC50 ± SEM = 20.2 ± 1.9 µM). The detailed study of
the structure showed that in this compound the benzene ring attached to acetamide group is
replaced with the furan ring. Thus, the name benzamide is replaced with carboxamide. The
hydrophilic character of the compound was increased as furan ring is electron donating and it
increases the electron density of the compound by donating its lone pair of electrons to it, so
increasing its hydrophilicity property. It can be suggested that the activity of this compound
might be due to the presence of furan ring. Furthermore, this statement was proved by observing
inhibitory values of the other compounds. The compounds having less carbon number are more
active against hꢀTNAP than the compounds containing higher carbon number. The presence of
halogens also affects the property of the compound. The compounds halogens are more
lipophilic in character thus exhibiting lower IC50 values against hꢀTNAP as mentioned in the
Table 1. On the other hand the inhibition of hꢀIAP by this compound increased with the
hydrophobicity or lipophilicity character of the compound. In this case the maximum inhibition
was shown by 4h with an IC50 value of ± SEM = 2.83±0.03 µM which is 35 fold lower than the
known reference standard inhibitor i.e. LꢀPhenylalanine (IC50 ± SEM = 100 ± 3 µM). With the
decrease in lipophilic character other compounds inhibited more modestly as reported in Table 1.
When these compounds were tested for their inhibition potential against hꢀPLAP we observed
that with the exception of compounds 4i and 4j, all the other derivatives were strong inhibitors of
hꢀPLAP. In comparison to hꢀTNAP the inhibition of hꢀPLAP by these compounds was weaker
but stronger than with reference inhibitor i.e Levamisole (IC50 ± SEM = 120.0 ± 2.51 µM). The

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maximum inhibition was obtained in presence of compound 4c and 4f with IC50 value of
4.97±0.66 µM and 4.63±0.11 µM, respectively. Only few derivatives inhibited hꢀGCAP. The
compound 4e and 4g inhibited this enzyme at a concentration of 180 and 300 fold less than the
standard inhibitor LꢀPhenylalanine. The structures of these compounds showed that substitution
at para position is important for inhibition of hꢀGCAP. However the presence of bulky groups
and substitution at other sites will result in loss of hꢀGCAP inhibition by the compound.
When the benzamide derivatives were tested for inhibition of human and rat e5ꢀNT it was
observed that these compounds exhibited more inhibitory potential against e5ꢀNT rather than the
same member of the family i.e. APs (Table 1). The compounds 4c and 4i displayed strongest
inhibition of hꢀe5ꢀNT with IC50 value of 0.78±0.26 µM and 0.77±0.42 µM, respectively. The
structures of these compounds showed electron donating groups i.e. methyl and furan
respectively, which are responsible for their inhibitory behavior against human e5ꢀNT. All
derivatives inhibited rat e5ꢀNT. The structure activity relationship of all derivatives displayed
that the compound 4h having substitution of large carbon chain showed the strongest inhibition
of rat e5ꢀNT with IC50 value 0.17±0.02 µM which is a concentration 450 fold lower than its
previously mentioned standard inhibitor sulfamic acid. The compound 4g was found to be the
selective inhibitor of rat e5ꢀNT with IC50 value 1.38±0.29 µM which is the concentration 55 fold
lower than its previously mentioned standard inhibitor sulfamic acid. The reported compounds
herein are of considerable interest for further applications in the field of medicinal chemistry as
these compounds have potential to bind nucleotide protein targets.

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Table 1. Alkaline phosphatase and Ectoꢀ5ꢀNucleotidase inhibition in presence of the synthesized
compounds
Alkaline Phosphatase
Ectoꢀ5’ꢀnucleotidase
Code
hꢀTNAP
hꢀIAP
hꢀPLAP
hꢀGCAP
hꢀe5ꢀNT
rꢀe5ꢀNT
IC50 (µM) ±SEM IC50 (µM) ±SEM IC50 (µM) ±SEM
IC50 (µM) ±SEM
IC50 (µM) ±SEM
IC50 (µM) ±SEM
4a
4.22±0.34
3.32±0.98
5.91±0.99
2.19±0.87
0.691±0.11
0.408±0.09
5.15±1.01
0.917±0.02
0.337±0.08
4.36±0.71
19.2 ± 0.01
12.3±1.08
8.58±0.91
6.67±0.87
4.97±0.66
9.21±1.11
10.5±1.09
4.63±0.11
10.9±1.22
6.57±1 .02
ꢀ
4.94±0.23
1.49±0.86
5.59±0.94
0.78±0.26
172.62±3.34
1.25±0.38
10.5±1.46
ꢀ
23.5±0.87
7.41±0.64
0.83±0.41
1.92±0.61
1.05±0.46
0.961±0.21
1.38±0.29
0.174±0.02
0.224±0.05
0.625±0.18
4b
ꢀ
ꢀ
4c
3.27±0.67
7.63±0.99
4d
ꢀ
ꢀ
4e
ꢀ
1.65±0.22
4f
6.29±0.99
ꢀ
4g
15.1±1.06
1.03±0.11
4h
2.83±0.03
ꢀ
ꢀ
ꢀ
ꢀ
17.9±1.87
0.769±0.42
7.36±0.41
4i
ꢀ
ꢀ
ꢀ
4j
ꢀ
Levamisole
120.0 ± 2.51
Lꢀ
ꢀ
82.1 ± 0.01
ꢀ
301 ± 5.1
Phenylalanine
Sulfamic acid
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, h-PLAP and hꢀGAP) activity was performed at the final concentration of 200 µM. Ectoꢀ5’ꢀnucleotidase
(human and rat) activity was done at final concentration of 100 µM.
Homology Modelling of h-TNAP, hꢀIAP and hꢀGCAP
Since crystal structures of humantissue nonꢀspecific alkaline phosphatase (h-TNAP), intestinal
alkaline phosphatase (hꢀIAP) and germꢀcell alkaline phosphatase (hꢀGCAP) are not available

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from the Protein Data Bank (PDB), homology models of h-TNAP, hꢀIAP and hꢀGCAP were
generated using Chimera22 and Modeller.23 The sequence of target proteins h-TNAP (uniprot id:
P05186), hꢀIAP (uniprot id: P09923), and hꢀGCAP (uniprot id: P10696) were fetched in
Chimera. BLAST (Basic Local Alignment Search Tool) protein database24 was used to search for
sequence similarity with target proteins, human placental alkaline phosphatase (h-PLAP, PDB
id: 1ZED) was identified among the top five matches in all cases, and was accordingly selected
to be used as a template. The sequence of template protein was added to the sequence of target
protein using Needleman Wunsch global alignment algorithm25 embedded in Chimera.
Comparative modelling was run using Modeller via Chimera. The overall quality of the protein
was evaluated, and Ramachandran plots were generated using Molprobity.26 Comparative
sequence alignments and Ramachandran plots are given in supporting information, Figures S6ꢀ
S8 and Figures S2ꢀS4, respectively. All active site residues among modelled proteins (hTNAP,
hIALP, hGCALP), and hPALP were found to be highly conserved (Table 1). Amino acid
residues Phe107, Gln108, Ser155 and Glu429 in hPALP have been replaced by amino acid
residues Glu108, Gly109, Thr156 and His434 respectively in hTNALP. Glu429 of hPALP, was
found to be the only active site amino acid residue that was not conserved in any of the hAPs, in
hIALP it is replaced by a Ser448 residue, whereas in hGCALP, it has been replaced by Gly448
residue. Figures 1ꢀ3 show comparison of active site residues of modelled proteins hTNALP,
hIALP and hGCALP against the template protein hPALP.

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Figure 1. Comparison of active site residues of modelled h-TNAP (purple, A) with template h-
PLAP (light brown, B).
Figure 2. Comparison of active site residues of modelled hꢀIAP (green, A) with template h-
PLAP (light brown, B).

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Figure 3. Comparison of active site residues of modelled hꢀGCAP (blue, A) with template h-
PLAP (light brown, B).
Homology Modelling of Rat ectoꢀ5′ꢀNucleotidase (rꢀe5NT)
Homology model of rꢀe5NT was generated using chimera22 and Modeller.23 The sequence of
target protein rꢀe5NT (uniprot id: Q66HL0), was opened in Chimera. BLAST (Basic Local
Alignment Search Tool) protein database24 was used to search for sequence similarity with target
proteins, human ectoꢀ5′ꢀnucleotidase (hꢀe5NT, PDB id: 4H2I) was identified among the top five
matches, and was accordingly selected to be used as a template for modeling of rꢀe5NT. The
sequence of template protein was added to the sequence of target protein using Needleman
Wunsch global alignment algorithm25 embedded in Chimera. Comparative modelling was run
using Modeller.23 The overall quality of the protein was evaluated, and Ramachandran plots were
generated using Molprobity.26
Table 2. Comparison of active site residues of hꢀe5NT (PDB id: 4H2I) with rꢀe5NT. Conserved
residues (with respect to hꢀe5NT) are in purple font color, the residues which are not conserved
are in red font color.

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h-e5NT
Asp36
rꢀe5NT
Asp38
His38
His40
Asp85
Asp87
Asn117
His118
His220
His243
Asn245
Arg354
Arg395
Phe417
Asp506
Phe500
Asn119
His120
His222
His245
Asn247
Arg356
Arg397
Phe419
Asp508
Tyr502
Figure 4. Comparison of active site residues of modelled rꢀe5NT (violet, A) with template hꢀ
e5NT (grey, B).

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Molecular Docking
Molecular Docking Studies against ectoꢀ5′ꢀNucleotidase (CD73)
Human ectoꢀ5′ꢀNucleotidase (hꢀe5NT) Docking
Table 2 shows comparison of active site residues of human and rat e5NT, the differences in the
amino acids that are not conserved, for example Phe500 in he5NT has been replaced by Tyr502
in re5NT, may contribute towards selectivity in inhibition exhibited by these compounds.
Compound 4i was found to be the most active hꢀe5NT inhibitor, and was therefore selected for
molecular docking studies against hꢀe5NT (PDB id: 4H2I). No direct binding with the zinc ions
was found. Detailed analysis of the docked mode of 4i indicated presence of three hydrogen
bonds, the oxygen atom of furan ring was found to act as a hydrogen bond acceptor towards
amino group of Phe500 (2.34 Å). The amino group of amino acid Asn390 was making a bridging
hydrogen bond with oxygen atom of carbonyl group of pyrazole ring (2.04 Å), and with oxygen
atom of carboxamide group (2.14 Å). The 3D and 2D binding site interactions of 4i are given in
Figure 5.

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Figure 5. 3D and 2D binding site interactions of 4i against hꢀe5NT.
Rat ectoꢀ5′ꢀNucleotidase (rꢀe5NT) Docking
Compound 4h was found to be the most active rꢀe5NT inhibitor, and was therefore selected for
molecular docking studies against homology modelled rꢀe5NT. No direct binding with the zinc
ions was found, the most hydrophobic part of the molecule, that is, ethylphenyl group was found
to have a snug fit in the binding cavity of enzyme, surrounded by hydrophobic residues Phe419
and Leu186. The carboxamide group and the pyrazole ring were found to be surrounded by
amino acid residues His120, Asn119 and His245. The NH of the carboxamide group was making
a hydrogen bond with carbonyl oxygen atom of amino acid residue Leu186 (1.95 Å). The 3D and
2D binding site interactions of 4h are given in Figure 6.

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Figure 6. 3D and 2D binding site interactions of 4h against homology modelled rꢀe5NT.
Molecular Docking Studies against Alkaline Phosphatases (APs)
Human Tissue NonꢀSpecific Alkaline Phosphatase(h-TNAP) Docking
Detailed comparison of active site residues of different APs has been given in Table S1 of the
supporting information, the differences in the amino acids that are not conserved may contribute
towards selectivity in inhibition exhibited by these compounds. The most active h-TNAP

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inhibitor 4i, was subjected to molecular docking studies. Detailed analysis of binding site
interactions reveal a directing binding of oxygen atom of furan ring with the catalytic zinc ion
with a distance of 2.12 Å. The furan ring is surrounded by amino acid residues His321 and
His324. The imidazole ring nitrogen atom the amino acid residue His437, was found to be
involved in a hydrogen bond contact with the NH of carboxamide group of 4i at a distance of
2.08 Å. The NH group of same amino acid His437 was making another hydrogen bond (2.31 Å)
with oxygen atom of carbonyl group of 4i. The 3D and 2D binding site interactions of 4i are
given in Figure 7.

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Figure 7. 3D and 2D binding site interactions of 4i.
Human Intestinal Alkaline Phosphatase (hꢀIAP) Docking
The hydrophobic part, ethylphenyl, of the most active inhibitor 4h was found to be surrounded
by hydrophobic amino acid residues Phe126 and Val108, whereas the rest of the ligand was lying
in close vicinity of the hydrophilic residues His339 and His336. The oxygen atom of the
carboxamide group was making a hydrogen bond (2.12 Å) with NH group of His451 amino acid.
A direct binding of carbonyl oxygen of pyrazole ring with the catalytic zinc ion was observed
having a distance of 2.24 Å. Putative binding site interactions are given in figures below. The 3D
and 2D binding site interactions of 4h are given in Figure 8.

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Figure 8. 3D and 2D binding site interactions of 4h.
Human Germ Cell Alkaline Phosphatase (hꢀGCAP) Docking
Detailed molecular docking studies for compound 4g, the most active hꢀGCAP were carried out
using the homology modelled structure of hꢀGCAP. The docking studies revealed putative
binding site interactions, which are given in figures below. Compound 4g was found to bind near
the active site such that the phenyl pyrazole moiety was surrounded by amino acids Phe126,
Thr450, Val108, His339 and His451. NH of carboxamide group was making a hydrogen bond
with His451 at a distance of 2.21 Å.One of the hydroxyl substituent on the phenyl group was

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making bridging hydrogen bonds with His172 (2.07 Å) and Asp335 (2.39 Å). The 3D and 2D
binding site interactions of 4i are given in Figure 9.
Figure 9. 3D and 2D binding site interactions of 4g.
Human Placental Alkaline Phosphatase (h-PLAP) Docking