Exploration of carboxy pyrazole derivatives: Synthesis, alkaline phosphatase, nucleotide pyrophosphatase/phosphodiesterase and nucleoside triphosphate diphosphohydrolase inhibition studies with potential anticancer profile

Article abstract of DOI:10.1016/j.ejmech.2018.07.002

In the present work we report the synthesis of new aryl pyrazole derivatives using 1,3-dicarbonyl motifs. The reaction was proceeded by the cyclization of pentane-2,4-dione (1a), 3-chloropentane-2,4-dione (1b) or ethyl 3-oxobutanoate (1c) with different a

Full text of DOI:10.1016/j.ejmech.2018.07.002

Accepted Manuscript
Exploration of Carboxy pyrazole derivatives: Synthesis, alkaline Phosphatase,
nucleotide pyrophosphatase/phosphodiesterase and nucleoside triphosphate
diphosphohydrolase inhibition studies with potential anticancer profile
Pervaiz Ali Channar, Saira Afzal, Syeda Abida Ejaz, Aamer Saeed, Fayaz Ali Larik,
Parvez Ali Mahesar, Joanna Lecka, Jean Sévigny, Mauricio F. Erben, Jamshed Iqbal
PII:
S0223-5234(18)30558-0
10.1016/j.ejmech.2018.07.002
EJMECH 10537
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 February 2018
Revised Date: 15 June 2018
Accepted Date: 2 July 2018
Please cite this article as: P.A. Channar, S. Afzal, S.A. Ejaz, A. Saeed, F.A. Larik, P.A. Mahesar,
J. Lecka, J. Sévigny, M.F. Erben, J. Iqbal, Exploration of Carboxy pyrazole derivatives: Synthesis,
alkaline Phosphatase, nucleotide pyrophosphatase/phosphodiesterase and nucleoside triphosphate
diphosphohydrolase inhibition studies with potential anticancer profile, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.07.002.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Exploration of Carboxy Pyrazole Derivatives: Synthesis, Alkaline Phosphatase, Nucleotide
Pyrophosphatase/Phosphodiesterase and Nucleoside Triphosphate Diphosphohydrolase
Inhibition Studies with Potential Anticancer Profile
1
ǂ
2ǂ
2
∗
1
1
Pervaiz Ali Channar, Saira Afzal, Syeda Abida Ejaz, Aamer Saeed, Fayaz Ali Larik, Parvez
1
,3
4,5
4,5,
6
2
Ali Mahesar, Joanna Lecka, Jean Sévigny, Mauricio F. Erben , Jamshed Iqbal *
O
CH3
O
CH3
H C
3
N
N
N
N
3
a
H N
2
3
c
CH3
CH3
h-TNAP= 3.58±0.67 µM
h-IAP= 0.39±0.08 µM
h-TNAP=4.58±0.23 µM
O
CH3
O
O
CH3
CH3
CH3
N
N
N
N
N
N
N
R
3d
3
b
CH3
Cl
CH3
h-NPP3=0.35±0.02 µM
h-NPP3=1.15±0.13 µM
O
C H
3
CH3
O
C H 3
N
N
N
N
3
f
3
e
CH3
C H 3
h-NPP3=1.81±0.17 µM
h-TNAP=8.24±0.78 µM
h-IAP= 0.81±0.01 µM
Structure-activity relationship of compounds

ACCEPTED MANUSCRIPT
Exploration of Carboxy Pyrazole Derivatives: Synthesis, Alkaline Phosphatase, Nucleotide
Pyrophosphatase/Phosphodiesterase and Nucleoside Triphosphate Diphosphohydrolase
Inhibition Studies with Potential Anticancer Profile
1
2
2
∗
1
1
Pervaiz Ali Channarǂ, Saira Afzalǂ, Syeda Abida Ejaz, Aamer Saeed, Fayaz Ali Larik,
1
,3
4,5
4,5,
6
2
Parvez Ali Mahesar, Joanna Lecka, Jean Sévigny, Mauricio F. Erben , Jamshed Iqbal *
1
2
Department of Chemistry, Quaid-I-Azam University, 45320, Islamabad, Pakistan
Centre for Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus, Abbottabad-
2
2060, Pakistan
3
Institute of Chemistry, Shah Abdul Latif University, Khairpur-66020, Pakistan
Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université Laval, Québec,
4
QC, G1V 0A6, Canada
5
Centre de Recherche du CHU de Québec – Université Laval, Québec, QC, G1V 4G2, Canada
CEQUINOR (UNLP, CONICET-CCT La Plata), Departamento de Química, Facultad de Ciencias Exactas,
6
Universidad Nacional de La Plata, C.C. 962 (1900). La Plata, Argentina.
ǂ These authors contributed equally to the manuscript.
Abstract:
In the present work we report the synthesis of new aryl pyrazole derivatives using 1,3-dicarbonyl
motifs. The reaction was proceeded by the cyclization of pentane-2,4-dione (1a), 3-
chloropentane-2,4-dione (1b) or ethyl 3-oxobutanoate (1c) with different aryl hydrazines. The
products, which can be regarded as 1H-pyrazol-1-yl-one analogues (3a-f, 3g-o, 4a-c, 5a-b) and
represent drug like molecules along with well-developed structure–activity relationships, were
obtained in good to excellent yield. The structures of synthesized compounds were charcterized
1
13
on the basis of FT-IR, H NMR, C NMR and mass spectroscopic data. Considering alkaline
phosphatases (APs), nucleotide pyrophosphatases/phosphodiesterases (NPPs) and nucleoside
triphosphate diphosphohydrolase as the molecular targets, the effects of these synthesized
compounds were investigated on different isozymes of APs, NPPs and NTPDases. The data
revealed that the synthesized compounds inhibited both enzymes but most of them inhibited
tissue non-specific alkaline phosphatase (TNAP) more selectively. The antitumor activity results
indicated that the synthesized derivatives have strong inhibitory effects on the growth of selected
cell lines from different tissues such as breast, bone marrow and cervix (MCF-7, K-562 and
Hela) but with varying intensities. Moreover the binding mode of interactions were explained on
the basis of molecular docking and in-silico studies.
Keywords: Hydrazides, pyrazoles, alkaline phosphatases (APs), nucleotide pyrophosphatases
(NPPs), antitumor activity.
*
Corresponding Authors:
Aamer Saeed; aamersaeed@yahoo.com,
Jamshed Iqbal; drjamshed@ciit.net.pk
1

ACCEPTED MANUSCRIPT
1
. INTRODUCTION
Phosphatases and kinases are the group of enzymes which play an important role in the
regulation of phosphorylation and dephosphorylation reactions and help in the transfer of
phosphoryl groups from one entity to another, inside as well as outside the cell [1, 2]. These
reactions act as a key mechanism and control many pathways of the cells including; regulation of
gene expression, cell proliferation and cell differentiation activities [3, 4]. They also control
several metabolic processes like biosynthesis of nucleic acid building blocks and
synthetase/phosphorylase pathways [5, 6]. The two classes of phosphatase enzyme which are
involved in the hydrolysis of phosphate-ester bonds include alkaline phosphatase (AP) and
nucleotide pyrophosphatase/phospdiesterase (NPP) [7]. Alkaline phosphatase exists in two major
isoforms i.e., tissue specific alkaline phosphatase (intestinal, placental and germ-cell types) and
tissue non-specific alkaline phosphatase (TNAP) [8]. They all act as membrane-bounded
enzymes and are widely distributed in nature [9]. Moreover, they are also known as homodimeric
+
2
+2
enzymes having three metal ions i.e., two Zn and one Mg , necessary for enzymatic activity.
The physiological role of alkaline phosphatases have been reported in various biological
processes like cell adhesion, bone mineralization and cell signaling pathways; majorly in
purinergic signaling pathways [10]. Elevated level of APs is commonly associated to the diseases
of liver and bone such as obstructive jaundice, hepatitis, Paget's disease, osteomalacia,
osteosarcoma, fractured bone, cirrhosis, liver tumor, liver metastasis, bone metastasis, prostatic
cancer and others such as renal diseases and diabetes [11-13]. Nucleotide
pyrophosphatase/phosphodiesterase (NPP), another important phosphatase, involved in the
hydrolysis of phosphodiester or pyrophosphate bond in a variety of substrates such as nucleoside
triphosphate, choline phosphate esters and lysophospholipids [14]. Among the seven different
types (NPP1-7), only three members (NPP1-3) have been well characterized. Numerous
physiological functions of NPP1-3 have been reported such as regulation of extracellular
pyrophosphate levels, stimulation of cell motility, nucleotide recycling and modulation of
purinergic receptor signaling [15-16]. Elevated levels of NPP1-3 is associated with bone
diseases; chondrocalcinosis, diabetes and malignant cancers [17-18]. Interestingly, TNAP from
alkaline phosphatase and NPP1 from nucleotide pyrophosphatase/phosphodiesterase synergize in
their functions during bone mineralization process; TNAP is associated with the hydrolysis of
2

ACCEPTED MANUSCRIPT
pyrophosphate and NPP1 is associated with the regulation of extracellular pyrophosphate levels
[
19]. Thus the optimum level of TNAP and NPP1 is associated with the normal bone functioning
and other cellular pathways [20-21]. Similarly, ecto-nucleoside triphosphate diphosphohydrolase
E-NTPDase) represent a predominant family of eukaryotic enzymes characterized by the
(
presence of five conserved domains known as “apyrase conserved region”. These domains,
consisting of small stretches of amino acids containing residues, are vital for the proper
functioning of enzyme. The enzyme activity also strictly depends on the presence of divalent
cations such as calcium and magnesium [22]. In mammals, E-NTPDases consist of eight
members and they are named as NTPDase1-8. These enzymes have varying preference for
nucleotides. NTPDase 1, 3 and 8 hydrolyze nucleoside tri and diphosphate at an equal rate
whereas NTPDase 2 strongly prefers nucleoside triphosphate [23].
Considering the role of TNAP and NPP1 in various pathophysiological processes along with
their extracellular actions makes them an attractive target for some specific therapeutic
interventions. Therefore, potent and selective inhibitors of TNAP and NPP1 might be useful
candidates for the drug used in treatment or prevention of some of these diseases especially;
bone mineralization dysfunctions, cancer (hepatic, breast) and its associated malignancies. A
large number of compounds bearing nitrogen-containing fused heterocyclic skeletons have been
reported with excellent inhibitory values and most of them also exhibited anticancer activity [24-
2
8]. Encouraged by those results, aromatic heterocycles containing two nitrogen atoms in their
five-membered rings have attracted considerable attention in recent studies due to their wide
spectrum of biological activities as such as anticancer, analgesic, anti-inflammatory,
antimicrobial, antiviral, anticonvulsant, antihistaminic and anti-HIV [29-32]. The recent success
of pyrazole as COX-2 inhibitors has further highlighted the importance of these heterocycles in
medicinal chemistry [33].
Pyrazole is five member cyclic compound with two adjacent nitrogen atoms. Pyrazole unit
possess significance importance in synthetic chemistry, as it can be used as useful reagent in
multicomponent reactions and designing of chiral catalyst [34]. Pyrazole derivatives exhibit
extensive spectrum of biological activities such as anti-inflammatory, antiproliferative, anti-
hepatotoxicity, antileishmanial, analgesic, anticancer, insecticidal, antimicrobial and antioxidant.
Furthermore, pyrazole nucleus has been widely studied as an entry to new class of several
3

ACCEPTED MANUSCRIPT
enzyme inhibitors such as, monoamide oxidase inhibitors, BRAF (BRAF is a human gene that
makes a protein called B-Raf, formally known as serine/threonine protein kinase B-Raf)
inhibitors, DNA gyrase inhibitors, cyclin dependant kinase inhibitors, Interleukin receptor-
associated kinase 4 (IRAK4) inhibitors, 5α-Reductase inhibitor and tissue-non-specific alkaline
phosphatase (TNAP) inhibitors [35].
In recent years, several drugs have been developed from pyrazole derivatives. For example,
celecoxib demonstrates anti-inflammatory effects and inhibits COX-2; rimonabant functions as a
cannabinoid receptor and is utilized to treat obesity; fomepizole inhibits alcohol dehydrogenase;
and sildenafil inhibits phosphodiesterase [35]. The examples of few drug molecules containing
pyrazole scaffolds (Figure 1).
<
Figure 1>
Pyrazole moiety is considered as privileged unit for the designing of new enzyme inhibitors and
drugs because it possess two nitrogen atoms in a five membered ring, the one nitrogen atom act
as hydrogen bond donor, while the other nitrogen atom can coordinate with metal atoms. Methyl
groups attached to pyrazole ring provide necessary free bond rotation (flexibility). Polarized pi
ring of pyrazole sets the ground for aromatic interactions. These intriguing structural features
assist to incorporate various pharmacophores to furnish potent and safe drugs. Moreover,
compounds having Nitrogen as heteroatom in heterocyclic moieties display enhanced solubility
and salt-formation properties that enable their oral absorption and bioavailability.
Motivated by the aforesaid bioactivities concerning the scarcity of oxygen containing pyrazole
systems [36] herein, we report a mechanochemical method for synthesis of oxygen containing
pyrazole derivatives. The grinding-induced method (green synthesis) not only reduces the time
of reaction from several hours to a few minutes but also improves the yield compared with that
of conventional conditions. Moreover, the biological data of synthesized derivatives revealed a
non-selective mode of inhibition within the same group i.e., h-TNAP and h-IAP. But when the
results were compared with reference to the family; APs, NPPs; except four compounds (3g, 3h,
3
j and 4a) rest of the compounds showed selectivity towards h-TNAP rather than h-NPP1. The
anticancer assay findings may provide a support and idea to the researcher to synthesize more
analogues of effective derivatives along with the exploration of more molecular pathways.
4

ACCEPTED MANUSCRIPT
2
2
.1. RESULTS AND DISCUSSION
.1.1. Chemistry
The cyclocondensation of pentane-2,4-dione (1a) or 3-chloropentane-2,4-dione (1b) with
different aryl hydrazides (2a-o) was successfully achieved in the presence of piperidine as
catalyst to affords (3,5-dimethyl-1H-pyrazol-1-yl)(phenyl)methanone (3a-f) and (4-chloro-3,5-
dimethyl-1H-pyrazol-1-yl)(phenyl)methanone derivatives (3g-o) respectively (Table 1 and table-
2
) [37-38]. The progress of the reaction was monitored by TLC using n-hexane and ethyl acetate
in the ratio of 8:2. On completion, the reaction mixture was diluted with ethyl acetate and
filtered; the filtrate was concentrated and recrystallized from ethanol to afford pure products in
good yield (3a-o).
<
Table 1>
Table 2>
<
For the second series initially, 2,4-diones 3-(phenoxy)-pentane were prepared, following a
known procedure [37], by potassium carbonate mediated reaction of phenols (2) with 3-
(chloro)pentane-2,4-dione (1b). The corresponding 3-(phenoxy) pentane-2,4-diones which were
obtained in good yield (intermediate) were further undergone cyclization reaction with different
aryl hydrazides and led to the formation of 3,5-dimethyl-1H-pyrazol-4-ol derivatives (4a-c) as
shown in table 3. The reaction was carried out in the presence of piperidine under the inert
atmospheric conditions. Except 4a (70%), the % yield of the other two derivative was very low
i.e., 30%.
<Table 3>
The reaction of ethyl 3-oxobutanoate 1c with arylhydrazide containing electron donating
substituents i.e., NH and CH groups at position 4 of the aryl ring, was equally successful as that
2
3
of the other reactions (Table 1, 2 and 3) and afforded products 5a and 5b in 72% yield,
respectively (Table 4).
<Table 4>
1
The products were characterized on the basis of their FTIR, H NMR, mass spectroscopic and
micro elemental analytical data. The stretching at 3160 (N-H), 1528 (C=N), 1597 (C=C), and
5

ACCEPTED MANUSCRIPT
-1
1
7
80 (C–S) cm were noted in the FTIR spectra of pyrazoles. In H NMR spectra, the typical
sharp singlet’s were noticed at δ 9.34 for NH and at δ 2.42, 2.32 ppm because of CH protons''.
3
Those in the range of δ 7.31–7.83 for the aromatic protons, indicating the successful linkage of
1
3
aryl through sulfur bridge. C NMR spectra displayed the signal at δ 156.5 (C=N), 136.1 (C=C),
0.6 (C–O), and 9.6 (CH ) ppm, in addition to those for aromatic carbons. Mechanistically, it is
6
3
likely that first 3-aryloxy pentane-2,4-dione is formed as an intermediate, and then 3-aryloxy
pentane-2,4-dione acetyl acetone, 3-chloro-2,4-pentanedione, compounds were then condensed
with an equimolar amount of hydrazine hydrate to afford the target pyrazoles.
2
2
.1.2. Biological Results:
.1.2.1. Alkaline
Phosphatases
(h-TNAP
&
h-IAP),
Nucleotidase
Pyrophosphatase/Phosphodiesterase (h-NPP1 & h-NPP3) and Nucleoside Triphosphate
Diphosphohydrolase (r-NTPDase1, 2, 3 and 8) Inhibition Studies:
All synthesized pyrazole derivatives (3a-3o, 4a-c & 5a-b) were tested for their potential to
inhibit AP (h-TNAP and h-IAP), NPPs (h-NPP1 and h-NPP3) and nucleoside triphosphate
diphosphohydrolase (r-NTPDase1, r-NTPDase2, r-NTPDase3 and r-NTPDase8) isozymes.
Although these derivatives revealed a non-selective mode of inhibition within the same group
i.e., APs but when the results were compared with reference to the family, except four
compounds (3g, 3h, 3j and 4a) rest of the compounds showed selectivity towards h-TNAP rather
than h-NPP1. The synthesized derivatives showed h-TNAP inhibition in the range of (IC50
±
SEM) 0.91±0.04 to 42.0±2.62 µM, h-IAP inhibition in the range of (IC ± SEM) 0.39±0.08 to
5
0
2
2.3±1.31 µM, h-NPP1 inhibition in the range of (IC ± SEM) 2.53±0.42 to 8.04±1.01 µM and
50
h-NPP3 inhibition in the range of (IC ± SEM) 0.23±0.05 to 5.98±0.76 µM as shown in Table 6.
5
0
The synthesized derivatives showed 20-49% inhibitory activity, when tested against r-
NTPDase2, 3 and 8. However, for NTPDase1, most of the derivatives exhibited IC50 ± SEM in
lower micromolar range. For example, compound 3f, having 2-methyl group as substituent
possessed 6.21±0.61 µM. Among the derivatives, (3g-o), 3i showed IC ± SEM = 2.30±0.77
5
0
µM, while, 3n and 3o presented IC50 ± SEM = 1.05±1.09 and 1.10±0.33 µM, respectively.
Among series 4a-c, the compound showing inhibition was 4a with an IC value of 9.02±0.12
5
0
6

ACCEPTED MANUSCRIPT
µM, and among 5a-b, 5b was active derivative having inhibitory concentration of 10.10±0.04
µM.
<
Table 5>
2
.1.2.2. Structure Activity and Structure Selectivity Relationship of (3,5-dimethyl-1H-
pyrazol-1-yl)(phenyl)methanone Derivatives (3a-3f, 3g-3o):
In scheme 1, total fifteen derivatives of (3,5-dimethyl-1H-pyrazol-1-yl)(phenyl)methanone were
prepared (3a-o), possessing different aryl hydrazide substituents on phenyl ring (R). Briefly these
fifteen derivatives were divided into two groups, the first group of six derivatives (3a-f) was
synthesized by keeping di-methyl substituted pyrazole ring along with variably substituted R
position on phenyl ring. While the second group of nine derivatives (3g-o) was synthesized by
substituting the pyrazol ring at 3 position with electronegative atom such as chlorine atom and R
position was substituted further with different groups. In this case, the possible effect of chlorine
atom on the pyrazole ring and the substitutional effect (R) at phenyl ring were examined on the
basis of their inhibitory effects on alkaline phosphatase (AP) isozymes and nucleotide
pyrophosphatase/phosphodiesterase (NPPs) isozymes. The overall inhibitory effect of these
derivatives (3a-3f and 3g-o) was found to be increased on both APs and NPPs isozymes (Table
6
).
<Table 6>
It was observed that, from first group of derivatives i.e., 3a-f, derivative 3a revealed maximum
inhibitory effects with IC ± SEM= 3.58±0.67 and 0.39±0.08 µM on both h-TNAP and h-IAP
50
isozymes, respectively. The detailed study of this derivative suggested that the significant
inhibition might be due to the presence of strong electron donating group i.e., NH at position 4
2
of phenyl ring. The strong electron donating group increases ~ 9 fold inhibitory effects on h-IAP
isozyme as compared to h-TNAP. In case of other derivatives, the presence of weak electron
donating group i.e., CH , pyridine or naphthyl ring, resulted in the reduced inhibitory effects on
3
both enzymes as compared to 3a. When the activity of 3c and 3f was compared, it was observed
that substitution of CH group at position 3 of phenyl ring (3c) resulted in higher inhibitory
3
3
effects on h-TNAP as compared to derivative 3f having the same CH group substitution but at
7

ACCEPTED MANUSCRIPT
position 2. An inverse effect was observed by these two derivatives against h–IAP isozymes,
derivative 3f showed greater inhibition of h–IAP with selectivity of ~ 37 fold more than
derivative 3c. This showed that the stearic hindrance reduces the inhibitory behavior of these
derivatives against h-TNAP. Other derivatives (3d, 3e, 3b) with weak electronegative
substitution or presence of pyridine or naphthyl ring in place of phenyl ring, showed
comparatively less inhibitory effects on both isozymes as compared to 3a. Presence of pyridal
group makes the molecule relatively electron deficient. Moreover, in contrast to benzene, the
electron density is not evenly distributed over the ring, reflecting the negative inductive effect of
the nitrogen atom. This makes compound less stable and therefore resulted in the reduced
activity. Similarly, presence of bulky naphthyl group makes the compound less hydrophilic and
resulted in less inhibitory effects of TNAP.
The inhibitory effect of these six derivatives on h-NPP1 and h-NPP3 isozymes was found very
interesting. All of these derivatives remained inactive against h-NPP1 while possessed effective
inhibitory activity against h-NPP3. Compound 3b depicted maximum inhibition of h-NPP3 with
IC ± SEM= 0.35±0.02 µM. The structural elucidations of derivative 3b suggested that the
5
0
substitution of pyridine ring in place of substituted aryl ring possibly responsible for the
enhanced inhibitory effects on h-NPP3. In case of compound 3e, the attachment of napthyl ring
in place of aryl ring resulted in increased inhibitory effects on h-NPP3 with IC50
±
SEM=1.81±0.17 µM but less as compared to derivative 3b. The derivative 3d also showed
significant h-NPP3 inhibition with IC value of IC ± SEM=1.15±0.13 µM (Figure 2).
5
0
50
<
Figure 2>
The nine derivatives from the other group also influenced the activity of both enzymes to some
extent depending on the type and position of substitutional functional group. The reverse effect
was observed by the derivatives from both groups having similar substitution at R position but
replacement of H atom with chloride group on pyrazole ring. For example the substitution of
chloro group both at position 3 of pyrazol ring and position 4 (R) of phenyl ring (3g) resulted in
enhanced inhibitory effects on h-TNAP, h-NPP1 and h-NPP3 as compared to derivative 3d
having no chloro group at pyrazole ring but only one chloro group at position 4 of the phenyl
ring. Similarly another chloro substituted derivative 3h possessing 4-NH group substitution at R
2
8

ACCEPTED MANUSCRIPT
position also exhibited higher inhibitory effects on NPPs isozymes in comparison to 3a having
same R substitution. The derivative 3n exhibited the same inhibitory behavior with relation to h-
NPP3 isozyme. From these results, it was observed that the substitution of chlorine atom at
pyrazole ring resulted in increased inhibitory effects on NPPs.
From this group, except three derivatives i.e., 3g, 3h and 3j, the other six derivatives remained
inactive against h-NPP1. The introduction of t-butyl group substitution at 4 position of phenyl
ring (R) (3o) resulted in maximum inhibition of h-IAP as well as h-TNAP with the inhibitory
value of IC ± SEM = 1.01±0.01 and 1.07±0.01 µM, respectively. Moreover, the substitution of
5
0
4
3
-CH group at phenyl ring in addition to chloride substitution at pyrazole ring (3l) resulted in
the maximum inhibitory effects on h-NPP3 (IC ±SEM = 0.23±0.05 µM) while revealed least
5
0
inhibitory effects on the other isozymes (Figure 3).
<
Figure 3>
2
.1.2.3. Structure Activity and Structure Selectivity Relationship of 3,5 dimethyl-1H-
pyrazol-4-ol Derivatives:
In Scheme 2, three derivatives (4a-c) of 3,5-dimethyl-1H-pyrazol-4-ol were synthesized
with varying substitutions at position 2 and 4 on pyrazol ring. The derivative 4a depicted least
inhibitory effects on h-TNAP, h-IAP and h-NPP1 while it revealed significant inhibitory effects
on h-NPP3. The structure activity relationship of derivative 4a suggested that the substitution of
naphthyl ring at R1 might be responsible for its inhibitory activity. Another derivative 4b having
substitution of p-tolyl group at R1 and aniline group at R2 ended up with increased inhibitory
activity on h-TNAP and h-NPP3 but with abolished inhibitory effects on h-NPP1. Similarly
another derivative (4c) was synthesized by keeping the same substitution at R1 like 4b but
different R2 substitution i.e., with p-chlorophenyl ring. The obtained results revealed that R2
position might be responsible for maximum inhibitory effects on h-IAP and h-NPP1. In both
derivatives 4b and 4c, the substitution at R2 position determined the inhibitory effects on
different isozyme. Moreover, the presence of more nucleophilic substitution at R2 position (4b)
might be responsible for potent activity against h-TNAP and h-NPP3 (Figure 4).
<
Figure 4>
9

ACCEPTED MANUSCRIPT
2
.1.2.4. Structure Activity and Structure Selectivity Relationship of 2-benzoyl-5-methyl-1H-
pyrazol-3(2H)-one Derivatives
In Scheme 3, two more derivatives (5a-b) were synthesized by changing the R group on phenyl
ring of 2-benzoyl-5-methyl-1H-pyrazol-3(2H)-one. It was observed from their structure activity
relationship that the substitution of less electron donating group (4-CH ) resulted in greater
3
inhibitory effects on h-TNAP and h-IAP while the substitution of strong electron donating group
2
(4-NH ) resulted in significant inhibition of h-NPP3. Both these derivatives did not show any
promising effects against h-NPP1.
It can be observed from the structure activity and structure selectivity relationship of 3a-o, 4a-c
3 ,
and 5a-b that in comparison to CH group presence of more strong electron donating group i.e.,
NH greatly enhanced the inhibitory effects of most of the derivatives on the isozymes of both
2
selected enzyme families i.e., h-TNAP and h-IAP and h-NPP3 (Figure 5).
<
Figure 5>
2
.1.3. Molecular Docking Discussion
In order to identify the most possible binding interactions of the most effective derivatives within
active site of target enzymes, molecular docking studies were carried out by using molecular
operating environment (MOE) and LeadIT software. The most probable binding interactions of
the most effective inhibitors of h-TNAP, h-IAP, h-NPP1 and h-NPP3 are shown in Figure 5-8,
respectively. The detailed 3D and 2D binding interactions of derivative 3f within active site of h-
TNAP is shown in Figure 6. The amino acid residues which were involved in bonding and non-
bonding interactions with 3g included; Arg167, Ser93, Asp92, His154, His321, His324 and
His437, which played a very significant role in ligand-protein binding. Briefly, compound 3g
formed two hydrogen bonds with side chain of Ser93 and Arg167 amino acid groups. These two
hydrogen bonds were formed by carbonyl group of compound 3g. Moreover, two different π-π
interactions were also formed by compound 3g with His324 and His437. The amino acid His324
was further involved in making π-π stacked interaction with pyrazole ring while amino acid
His437 was involved in forming π-π T shaped interaction with pyrazole ring in compound 3g.
<
Figure 6>
1
0

ACCEPTED MANUSCRIPT
The detailed 3D and 2D binding interactions of effective inhibitor of h-IAP i.e., 3a within active
site of h-IAP is shown in Figure 7. The amino acid residues which were involved in bonding and
non-bonding interactions with 3a included; Arg166, Ser92, Gly313, His317, Asp316, His153,
His432 andAsp42 which played a very significant role in ligand-protein binding as compared to
two hydrogen bonds formed by compound 3f, compound 3a formed three hydrogen bonds with
the side chain of Ser92, Arg166 and Gly313 amino acid residues. Among these interactions, two
hydrogen bonds were formed by carbonyl group of compound 3a with amino acid residue Ser92
and Arg166 while one hydrogen bond was formed by amine moiety with side chain of
Gly313.Three different π interactions were also formed by compound 3a with amino acid groups
of His153, Arg166 and Zinc. Moreover, amino acid Arg166 was also involved in making one π-
cation interaction with pyrazole ring while amino acid His153 was involved in forming one π-π
T shaped interaction with benzene moiety (Figure 7).
<
Figure 7>
To check the selectivity of potent compounds against selective isozyme, the cross docking
studies were carried out, i-e., potent inhibitor of h-TNAP (3g) was docked in h-IAP and potent
inhibitor of h-IAP (3a) was docked against h-TNAP. The resultant results were reported in
Figure S10-S11. The Figure suggested that no strong hydrogen bond and π-π interactions were
noted. However, unfavorable (bump, red color lines) interactions were observed. It therefore
suggested that which is in line with the observations in the enzyme inhibition studies and
supports the high selectivity of 3a for h-IAP over h-TNAP and 3g for h-TNAP over h-IAP.
The detailed 3D and 2D binding interactions of the most effective inhibitor 4c within active site
of h-NPP1 is shown in Figure 8. The amino acid residues which were involved in bonding and
non-bonding interactions with compound 4c included; His380. His535, Asn277, The256,
Phe257, Leu290 and Lys528 which played a very significant role in ligand-protein binding.
Analysis of binding interactions revealed that five different strong binding interactions were
formed between compound 4c and amino acid residue of h-NPP1. Among these bonding
interactions, three strong hydrogen bonds were formed between compound 4c and the side chain
of Asn277, His380 and His535 amino acids. Carbonyl group was also involved in making two
hydrogen bonds with amino acid residue of His380 and His53. In addition to hydrogen bonds
1
1

ACCEPTED MANUSCRIPT
formation, carbonyl group was also involved in making a metal interaction with zinc atom within
the active site of h-NPP1. Furthermore one more hydrogen bond was also formed by nitrogen in
pyrazole ring and the side chain of Asn277. Two π-π T shaped interactions were also formed by
benzene ring of methylbenzene moiety and amino acid His535 as well as Tyr451 as depicted in
Figure 8.
<
Figure 8>
The detailed 3D and 2D binding interactions of the most effective inhibitor of h-NPP3; 3l within
active site of h-NPP3 is shown in Figure 9. The amino acid residues which were involved in
bonding and non-bonding interactions with compound 3l included; THr205, His483, Leu239,
Tyr289, Phe206 and His329 which played a very significant role in ligand binding. Analysis of
binding interactions as depicted in Figure 9 revealed that four different strong binding
interactions were formed between compound 3l and amino acid residues of h-NPP3. Among
these binding interactions, two strong hydrogen bonds were formed between compound 3l and
side chain of Thr205 and His483 amino acids. Carbonyl group of compound 3l was involved in
making one hydrogen bond with Thr205 and two metal acceptor interactions with zinc atoms
present within the active site of h-NPP3. Addition to hydrogen bond formed by carbonyl group,
one hydrogen bond was also formed by nitrogen of the pyrazole ring and side chain of Asn277.
Moreover, one π-π T shaped interaction was also formed by pyrazole ring and amino acid residue
of His329 as shown in Figure 9.
<
Figure 9>
Similarly, the selectivity of potent compounds against selective isozymes NPP1 and NPP3 was
evaluated by cross docking studies. For this purpose, the potent inhibitor of h-NPP1 (4c) was
docked in h-NPP3 and potent inhibitor of h-NPP3 (3l) was docked against h-NPP1. The resultant
results were reported in Figure S12-S13. The Figure suggested that no strong hydrogen bond and
π-π interactions were noted. However, many unfavorable (bump, red color lines) interactions
were observed. It therefore suggested that which is in line with the observations in the enzyme
inhibition studies and supports the high selectivity of 4c for h-NPP1 over h-NPP3 and 3l for h-
NPP3 over h-NPP1.
1
2

ACCEPTED MANUSCRIPT
2
.1.4. Cytotoxic Potential by MTT Assay
The synthesized pyrazole moieties i.e., 3a-o, 4a-c and 5a-b were subjected to in vitro MTT assay
against cancer cell lines taken from different tissues (breast, bone marrow and cervix) in
comparison to the normal cells taken from the baby hamster kidney. In order to rationalize the
effect of identified inhibitors of AP and NPP isozymes, above mentioned cancer cell lines were
selected having maximum expression of these ecto-nucleotidases [39,40]. All the synthesized
derivatives were tested at the single-dose concentration of 100 µM, and the percentages of the
growth reduction (inhibition) over the four tested cell lines were determined. The mean growth
reduction percentages for each of the tested derivatives are illustrated in Table 7.
<Table 7>
From Scheme 1 (3a-f and 3g-o), 4-chloro substituted derivative (3d) exhibited stronger growth
reduction of breast cancer cell line than the other derivatives of the same group containing
variably substituent. Upon comparing the activities of (3,5-dimethyl-1H-pyrazol-1-
yl)(phenyl)methanone derivatives (3d) and chloro substituted derivatives (at 3 position of the
phenyl ring) it was observed that the substitution of chloro group both at position 3 of pyrazole
ring and position 4 (R) of phenyl ring (3g) resulted in enhanced growth inhibitory effects on
breast cancer cell line (MCF-7). These two derivatives also exhibit promising growth reduction
effect on the other cell lines derived from the bone-marrow (K-562) and cervix (HeLa) but to the
lesser extent than the breast cancer cells. Although the two substituted chlorine derivative
exhibited much stronger effects than the derivative possessing 4-chloro phenyl substitution.
From the methyl substituted derivatives, it was observed that 4-position was found more
favorable for MCF-7 cells as compared to the K-562 and HeLa cell line. Among 3a-o
derivatives, the chloro substituted derivative (at pyrazole) showed stronger reduction as
compared to non-chloro derivatives. The derivatives 3c and 3i having the electron donating
methyl group at position 3 were found equi-effective, but less as compared to 4-position
derivatives i.e., 3l. Moreover, the effect of 2-substituted derivatives were also observed.
Compound 3f showed higher mean growth reduction value than the corresponding derivative 3n.
Similarly from compounds 3a and 3h with amine substitution at 4 position, derivative 3h was
1
3

ACCEPTED MANUSCRIPT
found more effective than 3a. When the 4 position substitution was occupied with napthyl (3e
and 3m) and pyridal group (3b and 3j) less growth reduction was observed. The reduction effect
was more promising on breast cancer cell line (MCF-7) than that of the cells obtained from bone-
marrow i.e., (K-562). When the obtained mean growth inhibition values were compared with the
results obtained during the enzyme inhibition assay, it was interestingly observed that derivatives
from both groups of Scheme 1 having substitution at position 4 of the phenyl ring, exhibited
stronger enzyme inhibition effects as well as maximum growth reduction effects on cancer cell
lines. It can be concluded from the enzyme inhibition data as well as anticancer potential, that the
activity 4-position substituted derivatives was because of the presence of electron-withdrawing
groups such as chloro.
Similarly from 3,5 dimethyl-1H-pyrazol-4-ol (4a-c) derivatives, the chloro substituted derivative
(4c) exhibited stronger growth inhibition effects than the other two derivatives. The growth
inhibitory effects 4c were found more on K-562 cell line along with the moderate inhibitory
effects on the other selected cell lines. Moreover, amine substituted derivative (4b) showed
higher inhibition than the naphthyl substituted derivative (4a), but less in comparison to the
chloro substituted derivative (4c). Both derivatives of 2-benzoyl-5-methyl-1H-pyrazol-3(2H)-one
(5a-b) showed more promising growth reduction effects on K-562 and HeLa cells as compared
to MCF-7 cells. Here, amino substituted derivatives showed much growth reduction than the
methyl substituted derivative.
2
.1.5. Drug Likeness Evaluation and ADME Properties
Now-a-days, evaluation of ADME-Tox (Adsorption, Distribution, Metabolism, and Excretion)
properties and drug likeliness properties of the synthesized compounds has become an important
factor for the designing of new drugs. The initial findings of ADME properties can help to cut
down the total R&D cost by screening out the unwanted molecules in the initial stages of drug
discovery process. For the estimation of drug likeness properties of compounds, physicochemical
parameters were calculated using MedChem Designer [39]. The calculated ADME-Tox
parameters of compounds 3a-o, 4a-c & 5a-b are given in Table-8. The logP value (octanol-water
partition coefficient) serves as a measure of lipophilicity. Preferably the logP value should not be
greater than five and it can be seen from Table-8 that all the derivatives had logP values in the
1
4

ACCEPTED MANUSCRIPT
range 1.49-4.258. LogD (pH dependent octanol-water partition coefficient) was also calculated
for all the derivatives and it was observed that all the compounds had favorable logD values in
the range 0.161-4.984. Moreover, logSw (Logarithm of water solubility) of compound was also
calculated and values for compounds 3a-o, 4a-c & 5a-b were in the range 0.624 to 4.984.
Furthermore, the most important aspect of these derivatives i.e., topological Polar Surface Area
(
TPSA) values give an estimation of surface contributions of polar moieties of the compounds.
It is also useful for the determination of the ability of molecules to cross the blood brain barrier
BBB). The values are given in Table 8. Most of the obtained values were <40, indicating it’s
(
potential to reach the brain quickly, bypassing the BBA cut off filter <60. According to
Lipinski’s rule of 5, total number of hydrogen bond donors (HBD) should not be more than 5,
and total number of hydrogen bond acceptors (HBA) should not be greater than 10. Except few
derivative i.e., 3a, 3h, 4a, 4b, 5a & 5b, rest of the derivatives did not contain any hydrogen bond
donar moieties. The number of hydrogen bond acceptors was less than 5 for all the derivatives. It
was observed from the computed values that all the compounds have <500 molecular weight, <5
HBD number, <10 HBA number and also <5 logP values. Therefore these derivatives can be
considered to have good oral-bioavailability as well as ADME profile [40].
<Table 8>
3
.0. Conclusion
It was concluded from the above discussion that the inhibitory effects of synthesized pyrazole
moieties were found dependent on both type and position of the functional group. Moreover,
these derivatives were found least effective against h-NPP1 and in comparison to h-NPP1, except
3
g, 3h, 3j, 4a and 4c, we got rest of the derivatives as selective inhibitors of h-TNAP with
effective inhibitory values. These derivatives might be useful for the exploration of their
mechanism of inhibition at the molecular level where the lead molecules are required for the
treatment of health disorders associated with the over-expression of h-TNAP. From the
anticancer profile, it can be concluded that from Scheme 1, compounds 3g and 3d having
electronegative substitution were found more effective against h-TNAP as well as on the breast
cancer cell line. Similarly, the compounds having 4-substituted methyl group (3l) were found
less effective on the selected targets also showed less response on the selected cell lines. The
1
5

ACCEPTED MANUSCRIPT
chloro derivative from Scheme 2 was found most effective against h-NPP1 as well as on cancer
cells. Moreover, the derivative from Scheme 3 showed moderate effects but strong electron
donating group showed higher inhibitory effects than the less strong electron donating methyl
group.
4
4
.0 Experimental:
.1. Chemistry:
Melting points were recorded using a digital Gallenkamp (SANYO, Moriguchi, Japan) model
1
MPD BM 3.5 apparatus and were uncorrected. H NMR spectra were determined as CDCl
3
solutions at 300 MHz using a Bruker AM-300 spectrophotometer (Billerica, MA, USA). FTIR
spectra were recorded using Bio-Rad Excalibur FTS 3000 MX spectrophotometer (Madison,
WI), Mass Spectra (EI, 70 eV) on a GC-MS instrument. The reactions were carried out in glass
mortar and pestle
.
4
.1.2. General procedure for the preparation of (substituted phenyl)(3,5-dimethyl-1H-
pyrazol-1-yl) methanone derivatives (3a-3o), (4a-c) and (5a-b)
A mixture containing acetyl acetone, 3-chloro-2,4-pentanedione, 3-aryloxy-2,4-pentanediones
and ethyl acetoacetate was ground in the presence of piperidine as a catalyst. The reaction was
carried out in a glass mortar and pestle at room temperature. After grinding for 5–10 min,
hydrazine hydrate or suitable aryl hydrazines (1 mmol) was added, and the grinding was
continued for further 10 min. The progress of the reaction was monitored by TLC using n-
hexane/ethyl acetate (8:2). On completion, the reaction mixture was diluted with ethyl acetate
and filtered to remove salts, and the filtrate was concentrated. Recrystallization from ethanol
afforded pure products (3a–3o), (4a-c) [38] and (5a-b).
(4-Aminophenyl)(3,5-dimethyl-1H-pyrazol-1-yl)methanone (3a)
1
Orange Solid (710 mg, 71%), m.p; 147-151 °C, R
f
; 0.42 (Pet Ether : Ethyl Acetate; 8:2), H
NMR; (300 MHz, CDCl ): δ= 7.95 (d, 2H, J = 8.0 Hz, ArH), 6.68 (d, 2H, J = 7.5 Hz, ArH),
3
1
3
6
.042 (s, 1H, CH), 2.6(s, 3H, CH
3 3 2
), 2.28 (s, 3H, CH ), 4.10 (s, 2H, NH ); C NMR; (75 MHz,
CDCl ): δ = 167.5 (C=O), 151.2 (Ar-C), 151.0 (Ar-C), 144.7 (Ar-C), 134.3 (Ar-C), 122.0 (Ar-
3
1
6

ACCEPTED MANUSCRIPT
-1
C), 113.4 (pyrazole-C), , 110.3 (pyrazole-C) 14.1 (CH
3
), 13.9 (CH
437(NH); 3131 (sp CH), 2923 (sp CH), 1661(C=O), 1513 (C=N), 1597-1479 (C=C), GC-MS;
EI, 70 eV): m/z (%): 216 M+1, 215(10), 136(12), 120 (100), 95 (8), 92 (60), 82 (51); Elemental
3
). IR; (KBr, cm ):
2
3
3
(
Analysis: calcuated C, 66.96; H, 6.09; N, 19.52; Found C, 66.93; H, 6.07; N, 19.50; accurate
mass 215.1.
(3,5-Dimethyl-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (3b)
1
Light brown (750 mg, 75%), m.p; 237-240 °C, R
f
; 0.45 (Pet Ether : Ethyl Acetate; 8:2),
H
NMR; (300 MHz, CDCl ): δ = 7.79-8.79 (m, 4H, pyridine), 5.80 (s, 1H, CH), 2.35 (s, 3H, CH ),
3
3
13
2
.10 (s, 3H, CH3), C NMR; (75 MHz, CDCl ): δ = δ 166.7 (C=O), 151.4 (Pyr-C), 151.4 (Pyr-
3
C), 149.5 (Pyr-C), 140.5 (Pyr-C), 138.7 (Pyr-C), 124.7 (Pyr-C), 124.7 (Pyrazole-C) , 113.4
-
1
2
3
(
(
(
Pyrazole-C), 13.8 (CH ), 12.7 (CH ). IR; (KBr, cm ): 3200 (sp CH), 2954 (sp CH), 1730
3 3
C=O), 1561 (C=N), 1580-1476 (C=C), GC-MS; (EI, 70 eV): m/z (%):202 M+1; 201 (12), 122
13), 106 (100), 95 (8), 82 (51); Elemental Analysis: calculated; C, 65.66; H, 5.51; N, 20.88;
Found C, 65.64; H, 5.54; N, 20.83, accurate mass: 201.2
(3,5-Dimethyl-1H-pyrazol-1-yl)(m-tolyl)methanone (3c)
1
Yellow Oil (84%), R
f
; 0.60 (Pet Ether : Ethyl Acetate; 8:2), H NMR; (300 MHz, CDCl
3
): δ=
7
3
.72 (s, 1H, N-H), 7.68 – 7.54 (m, 1H, Ar-H), 7.42 – 7.30 (m, 2H, Ar-H), 6.09 (s, 1H, CH), 2.5(s,
1
3
3 3 3 3
H, CH ), 2.35 (s, 3H,CH ), 2.30 (s, 3H, CH ), C NMR; (75 MHz, CDCl ); δ = δ 166.1
(C=O), 149.8 (Ar-C) , 140.1 (Ar-C), 138.8 (Ar-C), 134.1 (Ar-C), 131.8 (Ar-C), 129.5 (Ar-C),
1
29.1 (Ar-C), 127.9 (pyrazol-C), 113.4 (pyrazole-C), 21.2 (CH ), 13.1 (CH ), 12.2 (CH ).. IR;
3
3
3
-1
2
3
(
KBr, cm ): 3135 (sp CH), 2955 (sp CH), 1668 (C=O), 1540 (C=N), 1598-1480 (C=C); GC-
MS; (EI, 70 eV); m/z (%): 215 M+1; 214 (13), 135 (16), 119 (100), 95 (11), 91 (68), 82 (57);
Elemental Analysis: calculated; C, 72.87; H, 6.59; N, 13.07; Found; C, 72.85; H, 6.53; N, 13.04
accurate mass: 214.1
(4-Chlorophenyl)(3,5-dimethyl-1H-pyrazol-1-yl)methanone (3d)
1
Light yellow solid (800 mg, 80%), m.p; 138 °C, R ; 0.71 (Pet Ether : Ethyl Acetate; 8:2), H
f
NMR; (300 MHz, CDCl ): δ= 7.73 (d, 2H, J = 7.8 Hz, ArH), 7.47 (d, 2H, J = 7.8 Hz, ArH),
3
1
3
6
.07 (s, 1H, CH), 2.5 (s, 3H, CH ), 2.27 (s, 3H, CH ); C NMR; (75 MHz, CDCl ); δ = 166.7
3 3 3
1
7

ACCEPTED MANUSCRIPT
(C=O), 148.5 (Ar-C), 138.7 (Ar-C), 138.7 (Ar-C), 132.8 (Ar-C), 129.8 (Ar-C), 129.8 (Ar-C),
-
1
1
3
29.2 (Ar-C), 129.2 (Pyrazole-C), 113.4 (Pyrazole-C), 12.7 (CH ), 12.3 (CH ). IR; (KBr, cm ):
3 3
2
3
110 (sp CH), 2962 (sp CH), 1700 (C=O), 1600-1559 (C=C); 1490 (C=N), GC-MS; (EI, 70
+
37
+ 35
eV); m/z (%): 235 M+1; 234 (M , Cl, 24), 232 (M , Cl, 74),135(12), 139.5 (100), 95 (8), 112
60), 82(51); Elemental Analysis: Calculated; C, 61.41; H, 4.72; Cl, 15.11; N, 11.94; Found C,
1.39; H, 4.69; N, 11.90, accurate mass 234.2.
3,5-Dimethyl-1H-pyrazol-1-yl)(naphthalen-2-yl)methanone (3e)
(
6
(
1
Light brown (650 mg, 65%), m.p; 215 °C, R ; 0.63 (Pet Ether : Ethyl Acetate; 8:2), HNMR;
f
(300 MHz, CDCl
3
): δ= 9.08 (m, 1H, N-H), 9.08 – 7.59 (m, 4H, Ar-H), 8.19 (d, J = 7.5 Hz, 1H,
1
3
Ar-H) 7.51 (d, J 7.5, 1H, Ar-H), 6.22 (s, 1H, CH), 2.5 (s, 3H, CH ), 2.4 (s, 3H, CH ).
C
3
3
NMR; (75 MHz, CDCl ); δ 166.0 (C=O), 150.5 (Ar-C), 139.7 (Ar-C), 137.0 (Ar-C), 134.5 (Ar-
3
C), 134.0 (Ar-C), 130.3 (Ar-C), 129.7 (Ar-C), 128.7 (Ar-C), 128.2 (Ar-C), 127.0 (Ar-C), 126.5
-1
(
Ar-C), 125.5 (Pyrazole-C), 113.4 (Pyrazole-C), 12.8 (CH ), 11.3 (CH ); IR; (KBr, cm ):
3 3
2
3
3
140(sp CH), 2980(sp CH), 1681 (C=O),1605-1550 (C=C); 1510(C=N), GC-MS; (EI, 70 eV):
m/z (%):251 M+1; 250 (14), 171 (12), 155 (100), 95 (9); Elemental Analysis: Calculated; C,
6.78; H, 5.64; N, 11.19; Found; C, 76.75; H, 5.61; N, 11.15 accurate mass: 250.1
7
(3,5-dimethyl-1H-pyrazol-1-yl)(o-tolyl)methanone (3f)
1
Yellow Oil (84%), R ; 0.60 (Pet Ether : Ethyl Acetate; 8:2), H NMR; (300 MHz, CDCl ): δ=
f
3
7
3
.68 – 7.32 (m, 4H, Ar-H), 6.09 (s, 1H, CH), 2.51 (s, 3H, CH
3
), 2.45 (s, 3H, CH
3
), CH
3
), 2.60 (s,
1
3
H, CH ), C NMR; (75 MHz, CDCl ): δ = δ 167.1 (C=O), 148.8 (Ar-C), 142.1 (Ar-C), 139.8
3
3
(Ar-C), 135.1 (Ar-C), 131.7 (Ar-C), 129.6 (Ar-C), 129.3 (Ar-C), 126.9 (Pyrazole-C), 114.5
-1
2
3
(
pyrazole-C), 21.3 (CH ), 13.2 (CH ), 12.7 (CH ). IR; (KBr, cm ): 3134 (sp CH), 2956 (sp CH),
3 3 3
1
1
666 (C=O), 1542 (C=N), 1598-1482 (C=C); GC-MS; (EI, 70 eV): m/z (%):215 M+1; 212 (13),
36 (16), 118 (100), 98 (11), 95 (68), 82 (57); Elemental Analysis: Calculated; C, 72.87; H, 6.59;
N, 13.07, Found; C, 72.84; H, 6.55; N, 13.04 accurate mass: 214.2.
(4-Chloro-3,5-dimethyl-1H-pyrazol-1-yl)(4-chlorophenyl)methanone (3g)
1
8

ACCEPTED MANUSCRIPT
o
1
White Solid (600 mg, 60%), m.p,155 C, R
f
; 0.55 (Pet Ether : Ethyl Acetate; 8:2), H NMR;
(
300 MHz, CDCl ): δ= 7.90 (d, 2H, J = 7.5 Hz, ArH), 7.42 (d, 2H, J = 7.5 Hz, ArH), 2.6 (s, 3H,
3
13
CH ), 2.24 (s, 3H, CH ), C NMR; (75 MHz, CDCl ): δ = 166.3 (C=O), 152.4 (Ar-C), 139.7
3
3
3
(Ar-C), 134.0 (Ar-C), 132.8 (Ar-C), 129.8 (Ar-C), 129.8 (Ar-C), 129.2 (Ar-C), 129.2 (Ar-C),
-1
2
1
19.4 (Pyrazole-C), 11.7 (Pyrazole-C), 9.8 (CH3). IR; (KBr, cm ): 3100 (sp CH), 2972
3
(
sp CH), 1700 (C=O), 1582 (C=N), 1590-1487 (C=C), 614-732 (C-Cl); GC-MS; (EI, 70 eV):
3
7
37
35
m/z (%):270 M+2, 269 M+1; 269 ([M]+, [2× Cl], 3), 266 ([M]+, [ Cl],[ Cl], 15) , 156 ([M]+,
35
[
2× Cl], 23), 140 (100), 130 (8), 112 (60), 82 (51); Elemental Analysis: calculated; C, 53.55; H,
3
.75; N, 10.41; Found C, 53.52; H, 3.71; N, 10.39 accurate mass: 268.1.
(4-aminophenyl)(4-chloro-3,5-dimethyl-1H-pyrazol-1-yl)methanone (3h)
o
1
Orange Solid (600 mg, 60%), m.p; 149 C, R ; 0.41, (Pet Ether : Ethyl Acetate; 8:2), H NMR;
f
(300 MHz, CDCl
3
):δ= 7.55 (d, 2H, J = 7.5 Hz, ArH), 6.73 (d, 2H, J = 7.5 Hz, ArH), 4.14 (s, 2H,
1
3
NH ), 2.6 (s, 3H, CH ), ), 2.31 (s, 3H, CH ), C NMR; (75 MHz, CDCl ): δ =166.3 (C=O),
2
3
3
3
1
52.7 (Ar-C) , 150.4 (Ar-C), 134.0 (Ar-C), 131.8 (Ar-C), 131.8 (Ar-C), 122.8 (Ar-C), 119.4 (Ar-
-1
3 3
C), 112.3 (Pyrazole-C), 112.3 (Pyrazole-C), 12.7 (CH ), 10.9 (CH ). IR; (KBr, cm ): 3147
2
3
(
(
(
sp CH), 2933 (sp CH), 1673 (C=O), 1560 (C=N), 1597-1489(C=C), 695-570(C-Cl); GC-MS;
+
37
+ 35
EI, 70 eV): m/z (%):251 M+2, 250M+1; 249 (M , Cl, 17), 247 (M , Cl, 62), 136(12), 130
8), 120 (100), 92 (60), 82 (51); Elemental Analysis: calculated, C, 57.72; H, 4.84; N, 16.83;
Found C, 57.70; H, 4.80; N, 16.80; accurate mass: 249.1
(4 -Chloro-3,5-dimethyl-1H-pyrazol-1-yl)(m-tolyl)methanone (3i)
1
Yellow Oil (62%), R
f
; 0.47 (Pet Ether : Ethyl Acetate; 8:2), H NMR; (300 MHz, CDCl
3
): δ =
7
3
1
.70 (d, J = 1.2 Hz, 1H, Ar-H), 7.68 – 7.54 (m, 1H, Ar-H), 7.42 – 7.30 (m, 2H, Ar-H), 2.50 (s,
1
3
H, CH
3 3 3 3
), 2.36 (s, 3H, CH ), 2.29 (s, 3H, CH ); C NMR; (75 MHz, CDCl ): δ = 165.6 (C=O),
50.4 (Ar-C), 140.1 (Ar-C), 134.0 (Ar-C), 133.1 (Ar-C), 131.8 (Ar-C), 129.5 (Ar-C), 129.1 (Ar-
-1
C), 127.9 (Pyrazole-C), 119.4 (Pyrazole-C), 21.2 (CH ), 10.1 (CH ), 9.3 (CH ). IR; (KBr, cm ):
3
3
3
2
3
3
117 (sp CH), 2967 (sp CH), 1675 (C=O), 1565 (C=N), 1595-1480 (C=C), 601-735 (C-Cl); GC-
+
37
+ 35
MS; (EI, 70 eV): m/z (%):250 M+2, 249 M+1; 248 (M , Cl, 18), 246 (M , Cl, 65), 135 (14),
1
9

ACCEPTED MANUSCRIPT
1
1
19 (100), 130 (8), 91 (60), 82 (51); Elemental Analysis: calculated, C, 62.78; H, 5.27; N,
1.26; Found C, 62.75; H, 5.24; N, 11.22 accurate mass: 248.1.
(4-Chloro-3,5-dimethyl-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (3j)
o
1
Light brown Solid (500 mg, 50%), m.p; 215 C, R ; 0.40 (Pet Ether : Ethyl Acetate; 8:2),
H
f
NMR; (300 MHz, CDCl
3
): δ= 8.79-7.79 (m, 4H, pyridine), 2.77 (s, 3H, CH
3
), ), 2.10 (s, 3H,
1
3
CH ), C NMR; (75 MHz, CDCl ): δ =166.3 (C=O), 151.4 (Ar-C), 150.4 (Ar-C), 140.5 (Ar-C),
3
3
1
34.0 (Ar-C), 124.7 (Ar-C), 124.7 (Pyrazole-C), 119.4 (Pyrazol-C), 10.8 (CH
3
), 9.9 (CH
3
). IR;
-
1
2
3
(
(
KBr, cm ): 3147 (sp CH), 2933 (sp CH), 1673 (C=O),1560 (C=N), 1597-1489 (C=C), 695-570
+
37
+ 35
C-Cl); GC-MS; (EI, 70 eV): m/z (%):237 M+2, 236 M+1; 237 (M , Cl, 19), 235 (M , Cl,
5
1
5), 130 (8), 122 (13), 106 (100), 82 (51); Elemental Analysis: calculated C, 56.06; H, 4.28; C N,
7.83; Found C, 56.04; H, 4.27; N, 17.81 accurate mass: 235.2
(E)-1-(4-Chloro-3,5-dimethyl-1H-pyrazol-1-yl)-3-phenylprop-2-en-1-one (3k)
1
Orange Oil (70%), R
f
; 0.58 (Pet Ether : Ethyl Acetate; 8:2), H NMR; (300MHz, CDCl
3
): δ =
7
.68 (d, J = 15.2 Hz, 1H, Ar-H), 7.60 – 7.50 (m, 2H, Ar-H), 7.50 – 7.32 (m, 3H, Ar-H), 6.79 (d,
13
J = 15.2 Hz, 1H, Ar-H), 6.17 (s, 1H, CH), 2.5 (s, 3H, CH ),2.3 (s, 3H, CH ), C NMR; (75
3
3
MHz, CDCl
3
): δ = 160.2 (C=O), 150.6 (Ar-C), 150.1 (Ar-C), 135.8 (Ar-C), 135.0 (Ar-C), 129.4
(
(
Ar-C), 129.0 (Ar-C), 129.0 (Ar-C), 128.0 (Ar-C), 128.0 (Ar-C), 119.6 (Pyrazole-C), 117.6
-
1
2
3
Pyrazole-C), 10.7 (CH
3 3
), 9.9 (CH ). IR; (KBr,cm ): 3122 (sp CH), 2856 (sp CH), 1677 (C=O),
1
583 (C=N), 1555-1478 (C=C), 700-627 (C-Cl), GC-MS; (EI, 70 eV): m/z (%): 262 M+2, 261
+
37
+ 35
M+1; 262 (M , Cl, 23), 260 (M , Cl, 67), 147 (12), 131 (100), 130 (8); Elemental Analysis:
calculated, C, 64.49; H, 5.03; N, 10.74; Found C, 64.47; H, 5.01; N, 10.72; accurate mass:
2
60.1
(4-Chloro-3,5-dimethyl-1H-pyrazol-1-yl)(p-tolyl)methanone (3l)
o
1
Yellow Solid (630 mg, 63%), m.p; 122 C, R ; 0.48 (Pet Ether : Ethyl Acetate; 8:2), H NMR;
f
(
300 MHz, CDCl ): δ= 7.85 (d, 2H, J = 7.5 Hz, ArH), 7.25 (d, 2H, J = 7.5 Hz, ArH), 2.60 (s,
3
1
3
3
1
1
H, CH ), 2.4 (s, 3H, CH ), 2.24 (s, 3H, CH ), C NMR; (75 MHz, CDCl ): δ = 167.7 (C=O),
3 3 3 3
49.2 (Ar-C), 143.6 (Ar-C), 140.2 (Ar-C), 131.5 (Ar-C), 129.5 (Ar-C), 128.6 (Pyrazole-C),
-1
2
14.62 (Pyrazole-C), 21.6 (CH ), 12.27 (CH ), 11.76 (CH ); IR; (KBr, cm ): 3057 (sp CH),
3
3
3
2
0

ACCEPTED MANUSCRIPT
3
2
2
6
922 (sp CH), 1701 (C=O), 1683 (C=C), 1509 (C=N), GC-MS; (EI, 70 eV): m/z (%):250 M+2,
+
37
49 M+1; 248(M , Cl, 24), 119 (100), 91 (99), 65 (80); Elemental Analysis: calculated, C,
2.78; H, 5.27; N, 11.26; Found; C, 62.75; H, 5.25; N, 11.24 accurate mass: 248.1
(4-Chloro-3,5-dimethyl-1H-pyrazol-1-yl)(naphthalen-2-yl)methanone (3m)
1
Light brown 720 mg, 72%), m.p; 205 °C, R
f
; 0.57 (Pet Ether : Ethyl Acetate; 8:2), H NMR;
(
(
(
300 MHz, CDCl ): δ= δ 8.14 (d, J = 15.6 Hz, 2H, Ar-H), 7.86 (d, J = 16.8 Hz, 2H, Ar-H), 7.56
3
1
3
s, 1H, Ar-H), 7.49 (d, J = 4.7 Hz, 2H, Ar-H) 2.62 (s, 3H, CH
3 3
), 2.29 (s, 3H, CH ), C NMR;
75 MHz, CDCl ): δ = 165.7 (C=O), 150.8 (Ar-C), 137.0 (Ar-C), 134.5 (Ar-C), 134.0 (Ar-C),
3
1
34.0 (Ar-C), 130.3 (Ar-C), 129.7 (Ar-C), 128.7 (Ar-C), 128.2 (Ar-C), 127.0 (Ar-C), 126.5 (Ar-
-1
3 3
C), 125.5 (Pyrazole-C), 119.4 (Pyrazole-C), 12.7 (CH ), 10.9 (CH ). IR; (KBr, cm ): 3134
2
3
(
(
(
sp CH), 2969 (sp CH), 1679 (C=O), 1578 (C=N), 1602-1483 (C=C), 595-725 (C-Cl); GC-MS;
+
37
+ 35
EI, 70 eV): m/z (%):286 M+2, 285 M+1; 286 (M , Cl, 22), 284 (M , Cl, 70), 171 (12), 155
100), 130 (9); Elemental Analysis: calculated; C, 67.49; H, 4.60; N, 9.84; Found C, 67.47; H,
4
.58; N, 9.81 accurate mass: 284.3.
(4-Chloro-3,5-dimethyl-1H-pyrazol-1-yl)(o-tolyl)methanone (3n)
1
Yellow Oil (59%), R ; 0.45 (Pet Ether : Ethyl Acetate; 8:2), H NMR; (300MHz, CDCl ): δ=
f
3
7
.67 – 7.61 (m, 1H, Ar-H), 7.45 (td, J = 7.5, 1.5 Hz, 1H, Ar-H), 7.36 – 7.27 (m, 2H, Ar-H); 2.5
1
3
(
s, 3H, Pyrazole CH
3 3 3
), 2.4 (s, 3H, Toluene CH ), 2.37 (s, 3H, Pyrazole CH ), C NMR; (75
MHz, CDCl ): δ = 163.6 (C=O), 151.4 (Ar-C), 137.8 (Ar-C), 134.0 (Ar-C), 133.6 (Ar-C), 132.3
3
3 3
(Ar-C), 130.8 (Ar-C), 130.1 (Ar-C), 126.7 (Ar-C), 119.4 (Ar-C), 20.2 (CH ), 10.7 (CH ), 9.9
-1
2
3
(
CH ); IR; (KBr, cm ): 3140 (sp CH), 2995 (sp CH), 1662 (C=O), 1552 (C=N), 1595-1485
3
(C=C), 605-745 (C-Cl); 1567-1488 (C=C), 1070 (C-O); GC-MS; (EI, 70 eV): m/z (%):250 M+2,
+
37
+ 35
2
49 M+1, 248(M , Cl, 22), 246 (M , Cl, 74), 135 (14), 119 (100), 130 (8), 91 (60), 82 (51).
Elemental Analysis: Calculated, C, 62.78; H, 5.27; N, 11.26; Found C, 62.75; H, 5.24; N, 11.23
accurate mass: 248.3
(R)-1-(4-chloro-3,5-dimethyl-1H-pyrazol-1-yl)-2-(4-isobutylphenyl)propan-1-one (3o)
2
1

ACCEPTED MANUSCRIPT
1
Orange Oil (59%), R
f
; 0.48 (Pet Ether : Ethyl Acetate; 8:2), H NMR; (300 MHz, CDCl
3
): δ=
7
.85 (d, 2H, J = 7.5 Hz, ArH), 7.25 (d, 2H, J = 7.5 Hz, ArH), 4.64 (1H, q, J 6.99 Hz, CHCH3),
.41 (3H, d, J 7.10 Hz,CHCH ), 2.40 (s, 3H, CH ), 2.47 (s, 3H, CH ), 1.72 {1.85 (1H, m,
2
3
3
3
1
3
CH(CH
3
)
2
) , 1.39 (3H, d, CHCH
3
3 2
) , 0.85 (6H, d, J 6.60 Hz, CH(CH ) ), C NMR; (75 MHz,
CDCl ): δ = 198.6 (C=O), 146.4 (Ar-C), 139.83 (Ar-C), 137.8 (Ar-C), 133.6 (Ar-C), 130.8 (Ar-
3
C), 130.11 (Ar-C), 109.5 (Ar-C), 45.24 (CH
2
), 39.78 (CH), 29.78 (CH
3
3
), 22.78 (CH ), 17.78
-1
2
3
(
CH ), 10.78 (CH ), 8.78 (CH ), IR; (KBr, cm ): 3140 (sp CH), 2995 (sp CH), 1662 (C=O),
3
3
3
1
552 (C=N), 1595-1485 (C=C), 605-745 (C-Cl); GC-MS; (EI, 70 eV): m/z (%):320 M+2, 319
+
37
+
35
M+1;318(M , Cl, 22), 316 (M , Cl, 74), 161 (14), 157 (100), 129 (8), 91 (60), 189 (51);
Elemental Analysis: calculated, C, 67.81; H, 7.27; N, 8.79; Found C, 67.79; H, 7.24; N, 8.76
accurate mass: 318.2
3
,5-Dimethyl-4-(naphthalen-2-yloxy)-1H-pyrazole (4a)
Starting with 3-(naphthalen-2-yloxy)pentane-2,4-dione (478 mg, 2.0 mmol) and hydrazine
hydrate (100 mg, 2.0 mmol), 4a was isolated as a pink solid (700mg, 70%), mp.120 °C;
1
HNMR; (300MHz, CDCl
.4 Hz, 2H, Ar-H), 7.46 – 7.43 (m, 3H, Ar-H), 2.23 (d, J = 11.5 Hz, 6H); CNMR (75 MHz,
CDCl ): δ =156.5 (Ar-C), 136.1 (Ar-C), 134.2 (Ar-C), 129.8 (Ar-C), 129.6 (Ar-C), 127.7 (Ar-C),
3
): δ = 9.32 (s, 1H, NH), 7.84 – 7.81 (m, 2H, Ar-H), 7.69 (qd, J = 7.5,
1
3
1
3
1
1
1
26.9 (Ar-C), 126.6 (Ar-C), 124.2 (Ar-C), 117.6 (Ar-C), 108.9 (Pyrazole-C), 108.6 (Pyrazole-C),
-1
2
3
06.2 (C-O), 19.6 (CH ); IR; (KBr, cm ): 3159 (sp CH), 2917 (sp CH), 1629 (C=N), 1597-
3
508(C=C), 1053(C-O); GC-MS; (EI, 70 eV): m/z (%):239 M+1; 238 (M+, 100), 209 (10), 155
(15), 127 (30), 115 (11) , 77 (10); Elemental Analysis: calculated C, 75.61; H, 5.92; N, 11.76
Found C, 75.59; H, 5.89; N, 11.73 accurate mass: 238.1
(4-Aminophenyl)(3,5-dimethyl-4-(p-tolyloxy)-1H-pyrazol-1-yl)methanone (4b):
Starting with 3-(p-tolyloxy)pentane-2,4-dione (412 mg, 2.0 mmol) and 4-aminobenzohydrazide
(312 mg, 2.0 mmol), 4b was isolated as a red solid (300 mg, 30%). mp.160 °C;
1
HNMR;(300MHz, CDCl
3
): δ = 7.57 (d, J = 7.5 Hz, 2H, ArH), 6.97 (d, J = 7.5 Hz, 2H, ArH ),
6
.76 – 6.67 (m, 4H), 3.82 (s, 2H, NH2), 2.52 (s, 1H, CH ), 2.43 (s, 3H, CH ), 2.32 (s, 3H, CH ),
3
3
3
1
3
CNMR; (75 MHz, CDCl ): δ = 166.3 (C=O), 153.7 (Ar-C), 152.7 (Ar-C), 141.9 (Ar-C), 140.6
3
2
2

ACCEPTED MANUSCRIPT
(Ar-C), 134.2 (Ar-C), 131.8 (Ar-C), 131.2 (Ar-C), 128.6 (Ar-C), 128.6 (Ar-C), 125.0 (Ar-C),
1
1
1
1
7
22.8 (Ar-C), 119.9 (Ar-C), 117.4 (Ar-C), 112.3 (Pyrazole-C), 110.5 (Pyrazole-C), 21.1 (CH3),
-1
2
3
9.5 (CH ), 18.3 (CH ) IR; (KBr, cm ): 3127(sp CH), 2952(sp CH), 1670 (C=O), 1573(C=N),
3
3
592-1498(C=C), 1060(C-O); GC-MS (EI, 70 eV): m/z (%):322 M+1; 321 (M+, 100), 209 (7),
19 (35), 91(30), 65 (25) , 51 (10); Elemental Analysis: C, 71.01; H, 5.96; N, 13.08 Found C,
1.68; H, 5.94; N, 13.05 accurate mass: 321.1
(4-Chlorophenyl)(3,5-dimethyl-4-(p-tolyloxy)-1H-pyrazol-1-yl)methanone (4c):
Starting with 3-(p-tolyloxy)pentane-2,4-dione (412 mg, 2.0 mmol) and 4-chlorobenzo-hydrazide
1
(
140 mg, 2.0 mmol), 4c was isolated as a yellow solid.(300 mg, 30%), m.p;155 °C; HNMR;
3
(300 MHz, CDCl ) : δ = 7.71 (d, J = 7.5 Hz, 2H, ArH), 7.44 (d, J = 7.5 Hz, 2H, ArH ), 6.97 (d, J
=
7.5 Hz, 2H, ArH), 6.72(d, J = 7.5 Hz, 2H, ArH), 2.52 (s, 1H, CH ), 2.42 (s, 1H, CH ), 2.32 (s,
3
3
1
3
3
H, CH
3 3
), CNMR; (75 MHz, CDCl ): δ = 166.3 (C=O), 153.7 (Ar-C), 141.9 (Ar-C), 140.6
(Ar-C), 139.7 (Ar-C), 134.1 (Ar-C), 132.8 (Ar-C), 129.8 (Ar-C), 129.8 (Ar-C), 129.2 (Ar-C),
1
2
28.8 (Ar-C), 128.6 (Ar-C), 127.9 (Ar-C), 125.0 (Ar-C), 119.9 (Pyrazole-C), 117.1 (Pyrazole-C),
-
1
2
3
3 3 3
1.1 (CH ), 18.3 (CH ), 17.6 (CH ) IR; (KBr, cm ): 3095 (sp CH), 2919 (sp CH), 1640 (C=O).
+
37
)
;
1586 (C=N), GC-MS (EI, 70 eV): m/z (%):342 M+2, 341 M+1; 340 (M , Cl, 24), 238
+
35
(
M , Cl, 54), 207 (26), 164 (16), 139 (100), 111 (44), 91 (15); Elemental Analysis: calulated C,
6
6.96; H, 5.03; N, 8.22; Found C, 66.93; H, 5.01; N, 8.20 accurate mass: 340.1
1
-(4-aminobenzoyl)-5-methyl-1H-pyrazol-3(2H)-one (5a)
Starting with ethyl 3-oxobutanoate (200mg, 2.0 mmol), 4-amino benzohydrazide (302 mg, 2.0
mmol), and added a drop of concentrated sulphuric acid. 5a was isolated as a pink Solid (714
1
mg, 72%), m.p; 158 °C, R ; 0.62 (Pet Ether : Ethyl Acetate; 8:2), HNMR; (300 MHz, CDCl ):
f
3
δ= 10.24 (s, 1H, NH), 7.95 (d, 2H, J = 8.0 Hz, ArH), 6.58 (d, 2H, J = 7.5 Hz, ArH), 6.07 (s, 1H,
1
3
CH), 2.6 (s, 3H, CH ), 2.29 (s, 3H, CH ), 4.10 (s, 2H, NH ); C NMR; (75 MHz, CDCl ): δ =
3
3
2
3
1
92.5 (C=O), 166.7 (C=O), 152.7 (Ar-C), 138.7 (Ar-C), 131.8 (Ar-C), 130.5 (Ar-C), 122.8 (Ar-
C), 113.4 (Ar-C), 112.3 (Pyrazole-C), 110.5 (Pyrazole-C), 13.67 (CH ), 10.8 (CH ). IR; (KBr,
3
3
-1
2
3
cm ): 3131 (sp CH), 2923 (sp CH), 1513 (C=N), 1661 (C=O), 1597-1479 (C=C), 3437 (NH);
2
3

ACCEPTED MANUSCRIPT
GC-MS; (EI, 70 eV): m/z (%):218 M+1; 217(10), 97(12), 120 (100), 92 (60); Elemental
Analysis: calculated, C, 60.82; H, 5.10; N, 19.34; Found; C, 60.80; H, 5.08; N, 19.31 accurate
mass: 217.1
3
-methyl-2-(4-methylbenzoyl)-1,2-dihydropyrazol-5-one (5b)
Starting with ethyl 3-oxobutanoate (204 mg, 2.0 mmol), 4-methyl benzohydrazide (301 mg, 2.0
mmol), and added a drop of concentrated sulfuric acid. 5b was isolated as a pink Solid (714 mg,
1
7
1
2%), m.p; 158 °C, R
f
; 0.62 (Pet Ether : Ethyl Acetate; 8:2), HNMR; (300 MHz, CDCl
3
): δ=
0.21 (s, 1H, NH), 7.85 (d, 2H, J = 8.0 Hz, ArH), 6.78 (d, 2H, J = 7.5 Hz, ArH), 6.06 (s, 1H,
1
3
CH), 2.71(s, 3H, CH ), 2.39 (s, 3H, CH ), 4.12 (s, 2H, NH ); C NMR; (75 MHz, CDCl ): δ =
3
3
2
3
1
1
82.57 (C=O), 167.7 (C=O), 151.71 (Ar-C), 138.73 (Ar-C), 131.71 (Ar-C), 131.61 (Ar-C),
22.77 (Ar-C), 113.49 (Ar-C), 112.34 (Pyrazole-C), 112.34 (Pyrazole-C), 12.67 (CH ), 10.19
3
-1
2
3
(
CH
3
) ; IR; (KBr, cm ): 3131(sp CH), 2923(sp CH), 1513(C=N), 1661(C=O), 1597-1479(C=C),
3
437(NH); GC-MS; (EI, 70 eV): m/z (%): 217 M+1; 216(10), 97(12), 109 (100), 91 (60);
Elemental Analysis: calculated C, 66.65; H, 5.59; N, 12.96; Found C, 66.61; H, 5.55; N, 12.93,
accurate mass: 216.1
4
4
.2. Biological Protocols:
.2.1 Cell transfection and preparation of membrane fractions
Initially, the plasmids expressing alkaline phosphatase (h-TNAP & h-IAP) [41] or human
nucleotide pyrophosphatase/phosphodiesterase (h-NPP1 & h-NPP3) [42, 43] were transfected
into the COS-7 cells, using lipofectamine. Next day, serum free dulbecco’s modified eagle’s
medium (DMEM); containing plasmid DNA (6 µg) and lipofectamine reagent (24 µL) was
added to the transfected cells and the plates were kept under incubation at 37 °C for 5 hr. After
4
8 hr, the transfection reaction was stopped by replacing the serum free media with the media
containing 20% FBS. After incubation of 48-72 hr, the transfected cells were washed with tris-
saline buffer and further processed as reported earlier [44]. For the transfection of NTPDases,
chinese hamster ovary (CHO) cells were transfected by using electroporation method as
described previously [45]. Briefly, CHO cells were transfected with plasmid expressing rat
NTPDase1 [46] or NTPDase2 [47] or rat NTPDase3 [45] by using a BTX Electrocell
2
4

ACCEPTED MANUSCRIPT
manipulator 600. NTPDase was transfected and purified by previously used protocol [39]. The
total protein estimation was carried out by Bradford method and and the aliquotes of protein
were kept at -80 °C after the addition of 7.5% glycerol [48].
4
.2.2. Alkaline phosphatase inhibition assay (h-TNAP & h-IAP)
The alkaline phosphatase (h-TNAP and h-IAP) inhibition assay was carried out as discussed in
our previously published article [49]. For the inhibition assay, 10 µL of each tested compound
was pre-treated with 20 µL of h-TNAP (46 ng/well) or h-IAP (57 ng/well) isozyme. The
o
reaction mixture was kept at 37 C for 5-10 min. and luminescence signals were recorded by
TM
using FLx800 microplate reader (BioTek , Instruments, Inc. USA). Finally, 20 µL of CDP-
®
star substrate was added to each reaction well and after 20 min. of incubation, the signals were
again measured. The data was analyzed by using PRISM 5.0 (GraphPad, San Diego, California,
USA) software and the inhibitory concentration values (IC50 values) of the most effective
derivatives were determined as discussed earlier [49].
4
.2.3. Nucleotide pyrophosphatase/phosphodiesterase (h-NPP1 & h-NPP3) inhibition assay:
The nucleotide pyrophosphatase (h-NPP1 & h-NPP3) inhibitory assay was performed as reported
earlier [50]. Intially, to the each well, 10 µL of tested compound, 10 µL of h-NPP1 (final conc.
of 27 ng) or h-NPP3 (final conc. of 25 ng) and 70 µL of 50 mM tris- HCl (pH 9.5) buffer were
o
added and kept at 37 C for 5-10 min. After incubation, absorbance was determined at 405 nm
TM
using ELx800 microplate reader (BioTek,
Instruments, Inc. USA). Finally, 10 µL of
substrate p-nitrophenyl-5'-thymidine monophosphate (p-Nph-5'-TMP, 0.5 mM) was added to
each reaction mixture and change in absorbance was evaluated after 35 min. of incubation. The
PRISM 5.0 (GraphPad, San Diego, California, USA) software was used for data analysis and the
inhibitory concentration values (IC values) were determined as mentioned in previous
5
0
publication [50].
4
.2.4. Nucleoside Triphosphate Diphosphohydrolase (r-NTPDase1, 2, 3 & 8) Inhibition
Assay
2
5

ACCEPTED MANUSCRIPT
All the synthesized derivatives were tested for their r-NTPDase activity by slightly modifying
the previously reported method [51]. The assay buffer containing 50 mM tis-HCl supplemented
with 5 mM CaCl (pH 7.4) was used. The total assay volume of 100 µL contained 59 µL of assay
2
buffer, 10 µL of tested compound and 6 µL of r-NTPDase1 (final conc. of 0.99 µg) or r-
NTPDase2 (final conc. of 1.21 µg) or r-NTPdase3 (final conc. of 1.04 µg) or r-NTPdase8 (final
conc. of 1.85 µg). The reaction mixture was allowed to incubate at 37 ˚C for 10 minutes and
absorbance was measured at 630 nm using microplate reader (BioTek ELx800, Instruments, Inc.
USA). Reaction was initiated by the addition of 10 µL of adenosine triphosphate (0.5 mM) and
the reaction mixture was again incubated at 37 ˚C for 10 minutes followed by the addition of 15
µL of malachite green reagent. The change in absorbance was measured within 3-5 minutes of
incubation. The compounds having more than 50% inhibition of any isoform of r-NTPDase were
further evaluated to determine the IC values. The IC values were determined by using non-
5
0
50
linear curve fitting program PRISM 5.0 (GraphPad, San Diego, California, USA).
4
.2.5. Molecular docking methodology
The molecular docking studies of the most effective inhibitors i.e., 3g, 3a, 4c and 3l were
performed to find out the most possible binding interactions of the identified effective
derivatives within the selected targeted enzymes i.e., h-TNAP, h-IAP, h-NPP1 and h-NPP3,
respectively. The x-ray crystallographic structures of h-TNAP, h-IAP, h-NPP1 and h-NPP3 are
not reported in Protein Data Bank (PDB) till to-date so already published homology models from
our research groups were used for docking studies [52, 53]. Prior to the docking studies, the
homology models were refined and protonated using AMBER99 force field and energy was
minimized with a RMSD gradient of < 0.05 kcal/mol/Å using Molecular Operating Environment
(MOE 2014.09) [54]. The structures of all effective inhibitors were generated using the builder
tool within MOE software. Then hydrogen atoms were added to all compounds and charges were
assigned to each atom. Finally the energy minimization of compounds structure was carried
using force field MMFF94x with RMSD gradient of <0.01 kcal/mol/Å. After preparation of
protein structures and compounds, MOE Site finder utility was used to identify the binding sites
within all target enzymes. MOE Site finder uses a geometric approach to find the possible
binding sites in a protein structure starting from its 3D atomic coordinates. For docking
2
6

ACCEPTED MANUSCRIPT
calculation default triangle matcher placement method and default scoring function GBVI/WSA
∆G were used which estimates free binding energy of bound ligand within active site. After
completion of docking calculation, those possess which were with the lowest S score and free
binding energies were selected further for visualizing the putative binding interactions within the
active site of all selected enzymes. Discovery studio 4.0 and PoseView BioSolveIT GmbH,
Germany were used for 3D and 2D visualization purpose, respectively [55,56].
4
.2.6. Cell viability assays (MTT assay)
The cytotoxic potential of the synthesized pyrazole derivatives was scrutinized against three
different cancer cell lines i.e., breast cancer cells (MCF-7), cervical cancer cells (HeLa) and bone
marrow cells i.e., Lymphoblast cells (k-562). The assay was performed by using MTT
(Dimethyl–2–thiazolyl–2,5–diphenyl–2H–tetrazolium bromide)–based cell viability assay [57]
and [58] as discussed earlier [59].
Acknowledgement
The authors are grateful 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. 20-3733/NRPU/R&D/ 14/520.
References
[1] Johnson, L.N., Lewis, R.J., 2001. Structural basis for control by phosphorylation. Chem.
Rev.101, 2209-2242.
[2] Adams, J.A., 2001. Kinetic and catalytic mechanisms of protein kinases. Chem. Rev.101,
2
271-2290.
[3] Krol, J., Loedige, I. and Filipowicz, W., 2010. The widespread regulation of microRNA
biogenesis, function and decay. Nature Rev Genet. 11, 597-610.
[4] Keyse, S.M., 2000. Protein phosphatases and the regulation of mitogen-activated protein
kinase signalling. Curr. Opin. Cell Biol. 12,186-192.
2
7

ACCEPTED MANUSCRIPT
[5] Romero, P., Wagg, J., Green, M.L., Kaiser, D., Krummenacker, M., Karp, P.D., 2005.
Computational prediction of human metabolic pathways from the complete human
genome. Genome Biol. 6, R2-R2.
[6] Munoz-Pinedo, C., Ricci, J.E. and El Mjiyad, N., 2012. Cancer metabolism: current
perspectives and future directions. Cell death & disease. 3(1), p.e248.
[7] Guanhua, H., Cui, Q., 2011.QM/MM analysis suggests that alkaline phosphatase (AP) and
nucleotide pyrophosphatase/phosphodiesterase slightly tighten the transition state for phosphate
diester hydrolysis relative to solution: implication for catalytic promiscuity in the AP
superfamily. J. Am. Chem. Soc. 134, 229-246.
[8] Hoylaerts, M.F., Ding, L., Narisawa, S., Van Kerckhoven, S., Millán, J.L., 2006. Mammalian
alkaline phosphatase catalysis requires active site structure stabilization via the N-terminal amino
acid microenvironment. Biochem. 45, 9756-9766.
[9] Jangra, S., Raghav, N., 2012. Inhibitors of alkaline phosphatase: a review. Nat. J. Basic. App.
Sci. 1, 91-97.
[
10] Hui, M., Hu, M., Tenenbaum, H. C., 1993. Changes in cell adhesion and cell proliferation
are associated with expression of tissue non-specific alkaline phosphatase. Cell Tissue Res. 274,
29-437.
4
[11] Giannini, E.G., Testa, R., Savarino, V., 2005. Liver enzyme alteration: a guide for
clinicians. CMAJ. 172, 367-379.
[12] Coleman, R.E., 2006. Clinical features of metastatic bone disease and risk of skeletal
morbidity. Clin. Cancer Res. 12, 6243s-6249s.
[13] Blayney, M.J., Pisoni, R.L., Bragg-Gresham, J.L., Bommer, J., Piera, L., Saito, A., Akiba,
T., Keen, M.L., Young, E.W., Port, F.K., 2008. High alkaline phosphatase levels in hemodialysis
patients are associated with higher risk of hospitalization and death. Kidney int. 74, 655-663.
[14] Stefan, C., Jansen, S., Bollen, M., 2005. NPP-type ectophosphodiesterases: unity in
diversity. Trends Biochem. Sci. 30, 542-550.
[15] Stella, J., Bavaresco, L., Braganhol, E., Rockenbach, L., Farias, P.F., Wink, M.R.,
Azambuja, A.A., Barrios, C.H., Morrone, F.B., Battastini, A.M.O., 2010. Differential
ectonucleotidase expression in human bladder cancer cell lines. Urol Oncol. Seminars and
Original Invest.28, 260-267.
[
16] al‐Rashida, M., Iqbal, J., 2014. Therapeutic Potentials of Ecto‐Nucleoside Triphosphate
Diphosphohydrolase, Ecto‐Nucleotide Pyrophosphatase/Phosphodiesterase,
Ecto‐5′‐Nucleotidase, and Alkaline Phosphatase Inhibitors. Med. Res. Rev. 34, 703-743.
[17] Lecka, J., Ben-David, G., Simhaev, L., Eliahu, S., Oscar Jr, J., Luyindula, P., Pelletier, J.,
Fischer, B., Senderowitz, H., Sévigny, J., 2013. Nonhydrolyzable ATP analogues as selective
2
8