Design, synthesis and biological evaluation of certain CDK2 inhibitors based on pyrazole and pyrazolo[1,5-a] pyrimidine scaffold with apoptotic activity

Article abstract of DOI:10.1016/j.bioorg.2019.01.008

Different series of novel pyrazole and pyrazolo[1,5-a] pyrimidine derivatives (2a-g), (3a-c), (7a-d) and (10a-e) were designed, synthesized and evaluated for their ability to inhibit CDK2/cyclin A2 enzyme in vitro. In addition, the cytotoxicity of the newly synthesized compounds was screened against four different human cancer cell lines. The CDK2/cyclin A2 enzyme inhibitory activity revealed that compounds (2d) and (2 g) are among the most active with inhibitory activity values of 60% and 40%, respectively, while compounds (7d) and (10b) exhibited the highest activity among the newly synthesized derivatives against four tumor cell lines (HepG2, MCF-7, A549 and Caco2) with IC50 values 24.24, 14.12, 30.03 and 29.27 μM and 17.12, 10.05, 29.95 and 25.24 μM, respectively. Flow cytometry cell cycle assay was carried for compounds (7d) and (10b) to investigate their apoptotic activity. The obtained results revealed that they induced cell-cycle arrest in the G0-G1phase and reinforced apoptotic DNA fragmentation. Molecular modeling studies have been carried out to gain further understanding the binding mode of the target compounds together with field alignment to define the similar field properties.

Full text of DOI:10.1016/j.bioorg.2019.01.008

Accepted Manuscript
Design, synthesis and biological evaluation of certain CDK2 inhibitors based
on pyrazole and pyrazolo[1,5-a] pyrimidine scaffold with apoptotic activity
Ghada M.E. Ali, Diaa A. Ibrahim, Amira M. Elmetwali, Nasser S.M. Ismail
PII:
S0045-2068(18)31342-7
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.008
YBIOO 2712
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
18 November 2018
31 December 2018
6 January 2019
Please cite this article as: G.M.E. Ali, D.A. Ibrahim, A.M. Elmetwali, N.S.M. Ismail, Design, synthesis and
biological evaluation of certain CDK2 inhibitors based on pyrazole and pyrazolo[1,5-a] pyrimidine scaffold with
apoptotic activity, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.01.008
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Design, synthesis and biological evaluation of certain CDK2
inhibitors based on pyrazole and pyrazolo[1,5-a] pyrimidine scaffold
with apoptotic activity.
a
a
a
b *
Ghada M.E. Ali , Diaa A. Ibrahim , Amira M. Elmetwali , Nasser S.M. Ismail
a
National Organization for Drug Control & Research, P.O. Box: 29, Cairo, Egypt
b
Pharmaceutical Chemistry Department, Faculty of Pharmaceutical Sciences and Pharmaceutical
Industries, Future University in Egypt, Cairo 12311, Egypt.
*Corresponding authors email: saadnasser2003@yahoo.com
Abstract
Different series of novel pyrazole and pyrazolo[1,5-a] pyrimidine derivatives (2a-g), (3a-c), (7a-
d) and (10a-e) were designed, synthesized and evaluated for their ability to inhibit CDK2/cyclin
A2 enzyme in vitro. In addition, the cytotoxicity of the newly synthesized compounds was
screened against four different human cancer cell lines. The CDK2/cyclin A2 enzyme inhibitory
activity revealed that compounds (2d) and (2g) are among the most active with inhibitory
activity values of 60% and 40%, respectively, while compounds (7d) and (10b) exhibited the
highest activity among the newly synthesized derivatives against four tumor cell lines (HepG2,
MCF-7, A549 and Caco2) with IC50 values 24.24, 14.12, 30.03 and 29.27 μM and 17.12, 10.05,
2
9.95 and 25.24 μM, respectively. Flow cytometry cell cycle assay was carried for compounds
(
7d) and (10b) to investigate their apoptotic activity. The obtained results revealed that they
induced cell-cycle arrest in the G0-G1phase and reinforced apoptotic DNA fragmentation.
Molecular modeling studies have been carried out to gain further understanding the binding
mode of the target compounds together with field alignment to define the similar field properties.
Keywords:
Pyrazolo[1,5-a]pyrimidine, Anticancer, CDK2, Pyrazole, Apoptosis, Molecular Modeling, Cell
cycle.
1
. Introduction
Protein kinases constitute a large group of evolutionary and structurally related enzymes that
regulate the function of other proteins through the catalysis of the transfer of the γ phosphate
group from a nucleoside triphosphate (ATP), to the side chain of an amino acid residue in the

substrate protein; serine, threonine, histidine or tyrosine residues [1]. Protein phosphorylation
plays a critical role in the regulation of cellular function [2].
Based upon their catalytic specificity, kinases can be categorized into: (I) Tyrosine kinases
(
TK) [3], (II) Serine/Threonine kinases [4] and (III) Histidine Kinases [5]. The cyclin dependent
kinases (CDK) (a serine/threonine kinase family) [6] are responsible for the initiation and the
succession of each cell cycle phase; CDK2 activity is required for progression through G1 to the
S phase [7].CDK2 hyper activation in human cancers is often associated with amplification
and/or overexpression of its partner cyclins A and E in a diverse of human cancers particularly in
breast, ovarian, endometrial, lung and thyroid carcinomas, melanoma and osteosarcoma
[
8].Pyrazoles and pyrazolo pyrimidines, especially pyrazolo[1,5-a]pyrimidines, represent
common nucleus in many pharmaceutical active compounds that have a wide range of significant
pharmacological activities especially the inhibition of CDK2/Cyclin A [9-11] as shown in Fig.1.
Fig.1. The Chemical structure for clinically approved CDK2 inhibitors (I-V).
In this research, the design of the target compounds with the pyrazole core (2a-g) and (3a-c)
was derived from the structure optimization of the reference compound AT7519 (IV) based on
its reported structure activity relationship SAR [14,15] as following: Fig.2&3.


 The core pyrazole nucleus of the lead compound AT7519 (IV) that occupy the adenine region
of ATP binding pocket is maintained in our target compounds.
AT7519 (IV) is anchored to the hinge region of CDK2 through a donor-acceptor donor H-
bonding with the backbone GLU81 and LEU83. This interaction is retained in compounds (2a-g)
via the pyrazole ring (N & CO) and the NH of the amino methylene group at position 4 of
pyrazole ring. While compounds (3a-c) exhibit donor- acceptor H-bond interaction only.
The amino piperidine amide group of AT7519 (IV) that improved the hydrophobic filling of

2

the region bounded by the backbone of the, was replaced at position 4 of the pyrazole ring by; a)
substituted phenyl amino methylene groups in compounds (2a-g), b) substituted phenyl furanyl
methylene groups in compounds (3a-b) and c) substituted benzylidene group in compound (3c).

The 2, 6-dichlorobenzamide of AT7519 (IV) with the two chlorine atoms efficiently filling
small hydrophobic pockets, was swapped with phenyl amino group at position 5 of the pyrazole
ring to occupy this hydrophobic pocket.
Fig.2. Schematic illustration of the binding mode of the reference compound
AT7519 (IV) to the active site of CDK2 (2H-bond with LEU 83).

R
H
N
NH
NH
N
O
Cl
(2a-g)
N
O
H
NH
NH
Cl
O
N
N
H
AT7519
(IV)
NH
N
R
O
N
(3a-c)
H
Fig.3. Similarities of pharmacophoric features between the reference compound AT7519 (IV) and the
target compounds (2a-g) and (3a-c).
The design the pyrazolo[1,5-a]pyrimidine scaffold ((7a-d) and (10a-e)) was based on some
structural modifications of the lead compounds Roscovitine (II) and 4k (BS-194) (III) [8,13] as
shown in Fig.4&5.

Purine of Roscovitine (II) that occupies approximately the same region as the purine ring of
ATP was bioisosterically replaced by pyrazolo[1,5-a]pyrimidine nucleus in the reference
compounds 4k (BS-194) (III), so it becomes an elegant nucleus of choice.
2

The hydroxylated ethylamino and propylamino substitutions at C of Roscovitine (II) and the
three side chain hydroxyl groups of 4k (BS-194) (III) are essential for hydrophilic interaction in
the ribose binding site. This substitutions were changed to carboxylic acid ethyl ester group at
position 6 of the pyrazolo[1,5-a]pyrimidine nucleus.
6

The N benzyl amino substituent of Roscovitine (II) provides π-π interaction between with the
7
amino acid residues ILE10, PHE82 and HIS84.While the N benzyl amino substituent of 4k (BS-
1
94) (III) occupies a hydrophobic pocket outside the ATP binding site. In the newly designed
compounds, this substitution was replaced by phenyl amino substituent but at position 2 of the
pyrazolo[1,5-a]pyrimidine nucleus.
9

The isopropyl substituent at N of Roscovitine (II) or at position 3 of 4k (BS-194) (III) was
replaced at position 3 of the pyrazolo[1,5-a]pyrimidine nucleus with hydrophilic carbamide or

cyanide moiety in compounds (7a-d) and (10a-e) , respectively, to provide extra H-bonding
interaction.

The introduction of hydrophobic group as substituted phenyl group at position 5 of the
pyrazolo[1,5-a]pyrimidine nucleus aiming to provide additional hydrophobic interaction.
(
a)
Fig.4. Schematic illustration of the binding modes of the reference compounds (a) Roscovitine (II) and
b) 4k (BS-194) (III) to the active site of CDK2 (2H-bond with LEU 83).
(b)
(
N
NH
N
N
(7a-d)
N
NH
Roscovitine
II)
(
HO
N
OH
NH
N
N
NH
4
k (BS-194)
III)
(10a-e)
(
Fig.5. Similarities of the pharmacophoric features among the reference compounds Roscovitine (II), 4k
BS-194) (III) and the target compounds (7a-d) and (10a-e).
(

2
. Results and discussion
2.1. Chemistry
Final compounds incorporating substituted methylene and amino methylene moieties were
obtained utilizing 5-phenylamino-2,4-dihydro-pyrazol-3-one (1) [17], which was synthesized
according to the route outlined in (Scheme 1a) via nucleophilic addition reaction of the highly
2
reactive C of ethyl acetoacetate to phenyl isothiocyanate, to give the sodium salt of 2-
(
Mercapto-phenylamino-methylene)-3-oxo-butyric acid ethyl ester. Cyclization took place
through the addition of hydrazine hydrate to the previous alcoholic reaction mixture.
1
Scheme 1a Reagents and conditions: (a) Na, CH
3
OH, phenyl isothiocyanate, reflux /
2
hr, cool (b)
Hydrazine hydrate, reflux 1 hr, 72-75%.
The synthesis of the target compounds (2a-g) was achievable via the fusion of the 5-
phenylamino-2,4-dihydro-pyrazol-3-one (1) with the appropriate aryl amine and triethyl
orthoformate in DMSO for 2.5-4.5hrs, afforded the corresponding 4-[(4-subistituted-
phenylamino)-methylene]-5-phenylamino-2,4-dihydro-pyrazol-3-one (2a-g) with a good yield
(
Scheme 1b). According to Knoevenagel condensation reaction [18,19], the nucleophilic
4
addition reaction of C of 5-phenylamino-2,4-dihydro-pyrazol-3-one (1) to the corresponding
aldehyde, followed by the dehydration reaction affording the corresponding (4-subistituted)
methylene-5-phenylamino-2,4-dihydro-pyrazol-3-one (3a-c) (Scheme 1b).

a
b
Scheme 1b Reagents and conditions: (a) Aryl amine, triethyl orthoformate, DMSO, Oil Bath, 2.5-4.5 hrs,
81-85 % (b) Aromatic aldehyde, sodium acetate, AcOH, reflux 6 hrs, 78-81%.
The synthesis of the pyrazolo[1,5-a]pyrimidine target compounds (7a-d) was initiated via the
2
preparation of the key cyanoacrylamide intermediate (5), by nucleophilic addition reaction of C
of cyanoacetamide to phenyl isothiocyanate followed by the methylation reaction using dimethyl
sulfate [20-22]. The later was then cyclized directly upon the fusion with hydrazine hydrate [23]
to afford 5-amino-3-phenylamino-1H-pyrazole-4-carboxylic acid amide (6), which in turn was
refluxed with different aromatic aldehydes and a β-ketoester (ethyl acetoacetate) in DMF to
afford the final target compounds (7a-d) through biginelli reaction [24,25] (Scheme 2).

Scheme 2 Reagents and conditions: (a) KOH, DMF, phenyl isothiocyanate, stirring, RT, 24 hrs (b)
Dimethyl sulphate, stirring, RT, 8 hrs, 80% (c) Hydrazine hydrate, W. B., 3-4 hrs, 64% (d) Hydrochloric
acid, aromatic aldehyde, ethyl acetoacetate, ethanol, reflux, 4-8 hrs 80-83%.
Whilst similar synthetic procedures were adopted for the synthesis of the pyrazolo[1,5-
a]pyrimidine target compounds (10a-e). Malononitrile was our key intermediate instead of the
cyanoacrylamide and gave 2-(Methylsulfanyl-phenylamino-methylene)-malononitrile (8). The
later was then cyclized directly upon the fusion with equimolar amount of hydrazine hydrate to
afford 5-Amino-3-phenylamino-1H-pyrazole-4-carbonitrile (9), which in turn was refluxed with
different aromatic aldehydes and a β-ketoester (ethyl acetoacetate) in DMF to afford the final
target compounds (10a-e).

Scheme 3 Reagents and conditions: (a) KOH, DMF, phenyl isothiocyanate, stirring, RT, 24 hrs (b)
Dimethyl sulphate, stirring, RT, 8 hrs, 82% (c) Hydrazine hydrate, W. B., 3-4 hrs, 80% (d) Hydrochloric
acid, aromatic aldehyde, ethyl acetoacetate, ethanol, reflux, 4-8 hrs 80-83%.
The newly synthesized derivatives (2a-g) revealed spectral analysis data matched with their
structures. The IR spectra of compounds (2a-g) showed the appearance of extra absorption band
-1
of NH around 3100 cm , while compound (2e) illustrated the existence of two extra absorption
-1
-1
bands; one for NH at 3873 cm and the other for CO at 1743 cm .Whereas compound (2f)
-1
demonstrated one extra absorption band of CO at 1743 cm . Compound (2g) proved the
-1
appearance of two absorption bands; one for NH at 3032 cm and for the other for SO -N at
2
-1
1
377 cm .
1
The H-NMR (300 MHz, DMSO-d
6
) spectra of the synthesized compounds (2a-g)
exemplified the presence of extra aromatic protons at the range of δ 7.4 to 7.6 ppm and an extra
olefinic singlet proton around δ 8.7 ppm. While compounds (2d) , (2e) & (2g) illustrated the
appearance of an extra singlet exchangeable proton by D
at δ 10.49 ppm and Ar-NH at δ 9.79 ppm , respectively. The spectra of the produced compounds
2c), (2e) & (2f) exhibited the existence of one singlet peak around δ 2.5 ppm for the CH group
and compound (2g) showed the appearance of one singlet overlapped peak at δ 2.6 ppm of the
2
O of Ar-OH at δ 9.47 ppm, Amide-NH
(
3

1
3
two CH
3
groups. The C-NMR (DMSO-d
6
) spectrum of the produced compound (2c) confirmed
group.
the appearance of an absorption peak at δ 30.61 ppm for the added CH
3
For compounds (7a-d), their IR spectra displayed the disappearance of one absorption band
of NH that was present in the intermediate (6) and the existence of extra CO band around 1700
-1
-1
cm .While, compound (7c) illustrated additional absorption band of OH at 3425 cm .
1
Even as the H-NMR (300 MHz, DMSO-d
6
) spectra of the synthesized compounds (7a-d)
, CH CH and CH CH
demonstrated the presence of extra aliphatic protons of Pyrimidinyl-CH
3
3
2
3
2
groups at the range of δ 2.5 to 4.14 ppm and the appearance of singlet proton of Pyrimidinyl-H
around δ 5.5 ppm. Compound (7b) showed the presence of extra aliphatic singlet proton of
OCH
exchangeable proton by D
produced compound (7b) showed the appearance of absorption peak at δ 55.59 ppm of the added
OCH group.
3
group at δ 3.85 ppm. Where, compound (7c) proved the presence of extra singlet
1
3
2
O of OH at δ 9.58 ppm. The C-NMR (DMSO-d ) spectrum of the
6
3
The structure of compounds (10a-e) was elucidated on the basis of spectral data. The IR
spectra showed the disappearance of one absorption band of NH was present in the intermediate
-1
(
9) and the appearance of CO band around 1680 cm . Compound (10c) illustrated the
-1
appearance of OH band at 3429 cm .
While the synthesized compounds (10a-e) showed the presence of extra aliphatic protons of
Pyrimidinyl-CH
3
, CH
3
CH
2
and CH
3
CH
2
groups at the range of δ 2.4 to 4.1 ppm and the
1
appearance of singlet proton of Pyrimidinyl-H around δ 5.4 ppm in their H-NMR (300 MHz,
DMSO-d
OCH group at δ 3.68 ppm. While, compound (10e) showed the presence of one singlet aliphatic
proton of N(CH at δ 2.82 ppm. The C-NMR (DMSO-d ) spectrum of the produced
6
) spectra. Compound (10b) showed the presence of extra aliphatic singlet proton of
3
1
3
3
)
2
6
compound (10b) showed the appearance of absorption peak at δ 55.6 ppm the added OCH
3
group. The mass analysis of all compounds was consistent.
3
. Biological Evaluation
3
.1. In vitro CDK2/cyclin A2 activity
The CDK2/cyclin A2 protein kinase assays were performed at BPS Bioscience, San Diego,
CA, USA (www.bpsbioscience.com) using Kinase-Glo Plus luminescence kinase assay kit
Promega). It measures kinase activity by quantitating the amount of ATP remaining in solution
(

following a kinase reaction. The luminescent signal from the assay is correlated with the amount
of ATP present and is inversely correlated with the amount of kinase activity. The percent
inhibition of the tested compounds against CDK2/cyclin A2 protein kinase was compared to
reference kinase inhibitor dinaciclib at a single concentration of 10 µM. The computer software
Graphpad Prism was used for analyzing the luminescence data. The difference between
luminescence intensities in the absence of CDK2/cyclin A2 protein kinase (Lu
presence of kinase (Lu ) was defined as 100 % activity (Lu – Lu ). Using luminescence signal
Lu) in the presence of the compound, % activity was calculated as:
activity = {(Lu - Lu)/(Lu - Lu )}×100%, where Lu= the luminescence intensity in the
t
) and in the
c
t
c
(
%
t
t
c
presence of the compound. % Inhibition was calculated as: % inhibition = 100 (%) _ % activities.
An initial screening; all synthesized final compounds were evaluated for their inhibitory
activity against CDK2/cyclin A2 protein kinase at a single dose concentration of 10 μM. At this
concentration, two compounds (2d) and (2g) of the pyrazole series had the highest competitive
inhibitory effect among the newly synthesized derivatives with % inhibitory values 60% and
4
4%, respectively. Nevertheless, the pyrazole derivatives (3a) & (3c) and the pyrazolo[1,5-
a]pyrimidine derivatives (7c) , (10a), (10b), (10c) & (10d) showed low to moderate competitive
inhibitory effect with % activity changes values ranging from 10% to 28%. The mean percent
CDK2/cyclin A2 protein kinase inhibition of the investigated compounds at 10 μM concentration
are shown in (Table 1). (Dinaciclib [12] as a reference compound showed 100% CDK2/cyclin
A2 protein kinase inhibition).

Table 1: inhibition activity results (% Inhibition) of the synthesized compounds against
CDK2/cyclin A2.
Cpd ID
R
%Inhibition
(2a)
(2b)
(2c)
(2d)
(2e)
(2f)
Cl
6
Br
CH
4
3
8
OH
60
8
NHCOCH
3
COCH
3
9
N
S
N
(
2g)
3a)
O
44
11
N
Cl
(
O
O
Cl
(
3b)
3c)
8
O
(
11
(
(
(
(
(
(
(
(
(
7a)
Cl
5
7b)
OCH
OH
F
3
3
7c)
28
6
7d)
10a)
10b)
10c)
10d)
10e)
Cl
12
10
11
13
9
OCH
OH
F
3
N(CH
3 2
)
3
.2. In vitro antiproliferative activity.
Cytotoxicity assays were conducted to determine the level of sensitivity as well as selectivity of
cancer and normal cells to the targeted compounds. All the newly synthesized compounds (2a-g),
3a-c), (7a-d) and (10a-e) were screened for their anticancer activity against four different human
(

tumor cell lines including human liver cancer cell line (HepG2), human breast cancer cell line
MCF-7), human lung cancer cell line (A549) and human colon cancer cell line (Caco2) at
(
pharmacology department of Al- Azhar University, Egypt, using MTT colorimetric assay according
to Mosmann [26]. This test is based on the selective ability of living cells, not dead cells, to reduce
the yellow soluble salt of MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to
a purple-blue insoluble formazan precipitate, this dye is accomplished by the mitochondrial
dehydrogenases of viable cells. The viable cell number is proportional to the production of
formazan salts.
The experiments were performed in six replicates for each compound and the results were
normalized to the control value and expressed as percentage of control. The compound
concentrations which give 50% growth inhibition are referred to as the IC50. The anticancer
activity was determined using Doxorubicin [27] as a reference (Table 2).
Table 2
In vitro cytotoxic activity of the newly synthesized compounds and doxorubicin against MCF-7, HepG2,
A549 and Caco-2 tumor cell lines.
MCF-7
IC50 μM
HepG2
IC50 μM
A549
IC50 μM
Caco-2
IC50 μM
Cpd
Doxorubicin
0.44
0.977
>500
76.34
34.92
37.24
79.02
123
5.842
>500
112.11
61.34
41.42
90.35
120
8.508
>500
79.78
58.22
50.3
(
(
(
(
(
(
(
(
(
(
(
(
2a)
2b)
2c)
2d)
2e)
2f)
346.01
97.64
30.96
23.32
42.19
92.72
334.47
133.1
100.1
30.06
132.5
39.09
88.26
146
2g)
3a)
3b)
3c)
7a)
7b)
>500
184.7
120.92
37.91
160.4
72.52
>500
192.1
160.5
42.90
185.8
111.22
265.35
160.2
208.1
52.12
352
88.14

(
(
(
(
(
(
(
7c)
30.09
14.12
35.53
10.05
30.06
30.47
86.23
66.12
24.24
62.35
17.12
69.44
69.35
125.24
58.54
30.03
71.23
29.95
82.15
81.44
150.4
99.91
29.27
90.35
25.24
41.07
59.96
157.9
7d)
10a)
10b)
10c)
10d)
10e)
The analysis of the IC50 results in (Table 2) disclosed that:
Compounds (7d) and (10b) revealed the highest activity against the four tumor cell lines
[
(HepG2), (MCF-7), (A549) and (Caco2)] among the newly synthesized derivatives with IC50
values for (7d) are 24.24, 14.12, 30.03 and 29.27 μM and for (10b) are 17.12, 10.05, 29.95 and
5.24 μM , respectively. Whereas, compounds (2c) , (2d) , (7c), (10c) and (10d) divulged
comparable anticancer activity against (MCF-7) cancer cell line with IC50 values 30.96, 23.32,
2
3
0.09, 30.06 and 47μM , respectively.
Derivatives (2c), (2d) and (3c) showed moderate antiproliferative activity against three tumor
cell lines [(HepG2), (A549) and (Caco2)] with IC50 values ranging from 34.92 to 61.34 μM.
Rather though this moderate activity has been also displayed against (MCF-7) cancer cell line by
compounds (2e), (7b) and (10a) with IC50 values ranging from 35.53 to 42.19.34 μM. Where,
compounds (10c) and (10d) exhibited well to moderate antiproliferative activity against (Caco2)
cancer cell line with IC50 values 41.07and 59.96 μM, respectively.
3
.3. Flow cytometry cell cycle analysis
Cell cycle analysis is a method in cell biology that employs flow cytometry to distinguish cells
in different phases of the cell cycle. Before analysis, the cells are permeabilised and treated with a
fluorescent dye that stains DNA quantitatively, as Propidium Iodide (PI). The fluorescence intensity
of the stained cells will therefore correlate with the amount of DNA they contain. As the DNA
content of cells duplicates during the S phase of the cell cycle, the relative amount of cells in the
G
0
phase and G
1
phase (before S phase), in the S phase, and in the G phase and M phase (after S
2

phase) can be determined, since the fluorescence of G
cells.
2
/M cells is twice as high as that of G
0
/G
1
Cell staining with PI is considered as univariate measurement of the cellular DNA content
alone. This approach reveals the distribution of cells in three major phases of the cycle (G1 vs S
vs G2/M) and makes it possible to detect apoptotic cells with fractional DNA content. It utilizes
Propidium Iodide (PI) as the DNA fluorochrome and requires blue light as the excitation source
(
e.g., 488 nm argon ion laser). Because PI also stains double-stranded RNA, the latter is removed
by the addition of RNase A to the staining solution [28].
Since compounds (7d) and (10b) showed the highest anticancer activity against the four
tumor cell lines and represent different series that were synthesized in this work, so they were
selected to test their effect on cell cycle of HCT-116 cells. The results are presented in the
following (Table 3).
Table 3
The percentage of HCT-116 cells in G0-G1 vs. S vs. G2/M after being treated with compounds (7d) and
(10b) in comparison to control (untreated cells)
%
G0-G1
%S
%G2-M
Sample
Control
47.91
67.37
65.43
20.85
18.6
31.24
14.03
9.89
(7d)
(10b)
24.68
As shown in Fig.6., the assay outcomes proved that upon exposure HCT-116 cells to
compounds (7d) and (10b), a significant increase in the percentage of the sub-G0/G1 cells by
7.37% and 65.43% , respectively occurred when they were compared to the cells of control
6
sample with 47.91%. Moreover, (10b) causes cell cycle arrest at S phase by 24.68% in
comparison to control sample with 20.85%. A sub-G0/G1 peak represents the relative amount of
apoptotic cells in the sample. These results demonstrated that compounds (7d) and (10b) inhibit
the proliferation of HCT-116 cells and cause apoptotic DNA fragmentation.

Control (untreated cells)
(7d) treated cells
(10b) treated cells
Fig.6. Cell cycle assay and effect of compounds (7d) and (10b) on HCT-116 cells in comparison to
control (untreated cells) evaluated by flow cytometry.
4
. Molecular modeling studies
4
.1. Docking study
Molecular modeling simulation study was performed through docking of the target
compounds in the binding site of CDK2 enzyme using C-Docker protocol in Discovery Studio 2.5
Software. In this investigation, docking study on the target compounds and analysis of their
binding modes was performed to interpret the biological results and to gain further insight into

binding orientations and interactions. The selected docking pose among the 10 retrieved possible
ones was chosen based on the similarity of its binding mode to that of the co-crystallized ligand.
It is worth noting that both the X-ray crystallographic enzyme (CDK2) substrate complex
with AT7519 (IV) (PDB code 2VU3) and with Roscovitine (II) (PDB code 2A4L) revealed the
formation the essential two H-bonds with LEU83.Validation of C-Docker protocol used in this
study was performed by re-docking the lead compounds in the CDK2 kinase active site. This was
followed by the alignment of the X-ray bioactive conformer of the lead compound with the best
fitted pose achieved from the docking run. The alignment showed good coincidence between
them with RMSD=0.5 A°, indicating the ability of the used docking protocol to retrieve valid
docking poses.
The docking study of the synthesized compounds into CDK2 active site revealed comparable
binding modes of the docked molecules to the lead compound. The binding interactions of the
docked compounds together with their binding energies are presented below in Fig.7. By studying
the CDOCKER interaction and the binding mode of the biologically active compounds (2d), (2g),
(7d) and (10b), we figured out that:
Compound (2d) conserved the formation of the two H bonds with LEU83 of the reference
compound AT7519 (IV) and exhibited high antitumor activity against (MCF-7) cancer cell line
with high % of inhibition against CDK2/A2 which proves that it’s antitumor activity due to the
inhibition of CDK2 enzyme and can be used as a lead for further studies.
Whereas compound (2g) formed only one H bond with LEU83 and showed different binding
mode other than the reference compound AT7519 (IV). Compounds (7d) and (10b) didn’t form
this essential H-bonding with LEU83 other than the reference compounds Roscovitine (II) and
(
4k) (BS-194) (III), but they exhibited new binding mode to the active site relative to these
reference compounds.

(a)
(b)
(c)
(d)
Fig.7. (a) Retrieved docking pose of the pyrazole-based inhibitor (IV) (PDB code 2VU3) showing the same
key interactions as reported. (b) Docking pose of the target compounds (2a) to the ATP binding pocket of
CDK2 in its inactive conformation. (c) Retrieved docking pose of the pyrazolo[1,5-a]pyrimidine based
inhibitor (II) (PDB code 2A4L) showing the same key interactions as reported.(d) Docking pose of the
target compounds (10b) to the ATP binding pocket of CDK2 in its inactive conformation.

4
.2. Field alignment study methodology.
2
D structure of the reference compounds [AT7519 (IV) and (4k) (BS-194) (III)] and the target
®
compounds were constructed using ChemDraw Ultra . The 2D structures were subjected a
conformational search MMFF94x forcefield applying the default settings. The energy minimized
structures were saved in files with the appropriate extensions. FieldAlign was used to align the
structures of the target compounds to the reference compounds. The most accurate mode was used
in the settings. FieldAlign, conformation hunter algorithm was used to generate representative sets
of conformations corresponding to local minima of energy calculated within the extended electron
distribution force field. Superimpositions of the conformations to the template were refined by the
simplex optimization algorithm incorporated in FieldAlign Fig.8.
(
a)
(b)
(
c)
(d)

Fig.8. (a& b) the best aligned poses of compounds (2d) & (2g) (grey) overlaid with the reference ligand
AT7519 (IV) (light green) , respectively. (c & d) The best aligned poses of compounds (7d) & (10e) (grey)
overlaid with the reference ligand (4k) (BS-194) (III) (light green) , respectively.
Compound (2d) showed good alignment with approximately the same field points of the
reference compound AT7519 (19), whereas compound (2g) illustrated different alignment other
than the reference compound AT7519 (IV) due to the steric sulfonamide moiety. Compounds (7d)
and (10b) didn’t exhibit good alignment with the reference compound (4k) (BS-194) (III), but they
showed different field points in the active site relative to the reference compound, therefore they
displayed lower CDK2/cyclin A2 affinity.
5
. Conclusion
A new series of pyrazole and pyrazolo[1,5-a]pyrimidine derivatives (2a-g), (3a-c), (7a-d) and
(
10a-e) were designed to act as anticancer via CDK2 inhibition. The promising candidates were
synthesized and evaluated as inhibitors of CDK2/cyclin A2. Compounds (2d) and (2g) with the
highest inhibitory activity against CDK2/cyclin A2 with % activity values 60% and 44% ,
respectively which showed high docking scores and low binding energy values and their binding
mode was compatible with the reported binding mode of reference compound AT7519 (IV).
Two compounds (7d) and (10b) exhibited high anticancer activity against four tumor cell lines
[
(HepG2), (MCF-7), (A549) and (Caco2)] among the newly synthesized series with IC50 values
for (7d) are 24.24, 14.12, 30.03 and 29.27 μM and for (10b) are 17.12, 10.05, 29.95 and 25.24 μM
respectively, but they showed different binding mode and field points rather than the reference
,
compounds in the docking and field alignment study .
Compounds (7d) and (10b) were selected to test their effect on cell cycle of HCT-116 cells by
Propidium Iodide (PI) flow cytometric assay; they cause a significant increase in the percentage of
the sub-G0/G1 cells by 67.37% and 65.43%, respectively when they were compared to the cells of
control sample with 47.91%. This peak reveals that compounds (7d) and (10b) inhibit the
proliferation of HCT-116 cells and cause apoptotic DNA fragmentation.
6
. Experimental
Starting materials, reagents and solvents were purchased from Sigma-Aldrich (USA) or Alfa-
Aesar Organics and used without further purification. Reactions were monitored by analytical thin

layer chromatography (TLC) , performed on silica GF254 plates packed on Aluminium sheets,
purchased from (E. Merck, Germany) with visualization under U.V. light (254 nm).Melting points
(
°C) were determined by open capillary tube method using (XT4 MP) apparatus and they are
uncorrected. Mass spectrum was carried out on Direct Inlet part to mass analyzer in Thermo
Scientific GCMS model ISQ at the Regional Center for Mycology and Biotechnology (RCMB),
1
13
Al-Azhar University, Nasr City, Cairo. The H NMR and C NMR spectra were recorded on a
Varian spectrometer 300MHz, at the Micro-analytical Data Center of Cairo University, or 500
MHz, at National Research Center, at 25°C with TMS and solvent signals allotted as internal
standards. All coupling constant ( ) values are given in Hertz. Chemical shifts (ꢀC) are reported
relative to DMSO-ꢁ as internal standards. The abbreviations used are as follows: s: singlet; d:
6
doublet; t: triplet; q: quartet and m: multiplet. Analytical data (IR) was performed at NODCAR,
the Micro-analytical Data Center of Cairo University and National Research Center. Elemental
analyses were performed on a CHNS-O-Rapid instrument in the Micro-analytical Data Center at
Cairo University.
Thin layer chromatography was performed on pre-coated (0.25 mm) silica gel GF254 plates (E.
Merck, Germany), compounds were detected with 254 nm UV lamp. Silica gel (60–230 mesh) was
employed for routine column chromatography separations.
6
.1. Chemistry
The synthesis of the reported intermediates is presented in Supplementary Materials.
.1.1. General procedure for preparation of 5-Phenylamino-2,4-dihydro-pyrazol-3-one (1) [17].
6
To a solution of sodium methylate (8.8 gm of sodium in 120 ml of methanol), (50 gm, 0.38 mole)
of ethyl acetoacetate was added and adjoined with (52 gm, 0.38 mole) of phenyl isothiocyanate in
1
a thin stream at room temperature. The mixture was refluxed for /
2
hr, cooled to about 50 ˚C, so it
was ready for hydrazine hydrate addition in the next step. Hydrazine hydrate 85% (11.9 ml, 0.38
moles) added in a thin stream with stirring to the previous reaction mixture. After refluxing for 1
hr, the mixture was cooled, filtered and the product was washed and crystallized from ethanol and
gave white crystals of the title compound (72%).
6
.1.1.1. 5-Phenylamino-2,4-dihydro-pyrazol-3-one (1).White powder; yield (72%); m.p.: 268-270
1
˚
C; H-NMR (300 MHz)(DMSO-d
6
) δ: 3.41 (s, 2H, Pyrazole-CH ), 6.74 (d, 2H, J=8.4HZ, Ar-H),
2

7
1
.15 (t, 1H, Ar-H), 7.43 (d, 2H, overlapped, Ar-H), 9.16 (s, 1H, Ar-NH, exchangeable by D O),
2
+
·
0.35 (s, 1H, Pyrazole-NH, exchangeable by D O); MS: (Mwt.: 175.19) :m/z, 175.07 (M ,
2
[
100%]).
6
.1.2. General procedure for preparation of 4-[(4-subistituted-phenylamino)-methylene]-5-
phenylamino-2,4-dihydro-pyrazol-3-one (2a-g) [29,30]. A mixture of 5-phenylamino-2,4-dihydro-
pyrazol-3-one (1) (0.8 gm, 0.005mole) and appropriate aryl amine (0.005mole) was fused with
triethyl orthoformate (0.66 gm, 0.005mole) in oil bath at 140-160˚C for 2.5-4.5 hrs. The resulting
precipitate was washed well via diethyl ether, filtered and crystallized from ethanol to give to the
tilted compounds (2a-g).
6
.1.2.1.4-[(4-Chloro-phenylamino)methylene]-5-phenylamino-2,4-dihydro-pyrazol-3-one
(2a).
-
1
Brown powder; (85%); m.p.: ˃ 300 ˚C; IR: (ύ max, cm ): 3421 (NH), 3232 (NH), 3140 (NH),
1
1
7
670 (CO); H-NMR (300 MHz)(DMSO-d
6
) δ: 6.84 (t, 1H, Ar-H), 7.26 (d, 2H, J=8.4HZ, Ar-H),
.43 (d, 2H, J=8.7HZ, Ar-H), 7.51 (d, 2H, overlapped, Ar-H), 7.61 (d, 2H, J=8.5HZ, Ar-H), 8.55
O), 8.71( s, 1H, Olefinic-CH), 10.54 (s, 1H, Ar-NH,
(
s, 1H, Ar-NH, exchangeable by D
2
exchangeable by D
2
O), 11.1 (s, 1H, Pyrazole-NH, exchangeable by D
2
O); MS: (Mwt.: 312.75)
O; C, 61.44; H,
+
·
+2.
:
m/z, 312.11 (M , [100%]), 314.09 (M , [39.77%]); Anal. Calcd. for C16
H
13ClN
4
4
.19; N, 17.91; Found: C, 61.73; H, 4.21; N, 18.11.
6
.1.2.2.4-[(4-Bromo-phenylamino)methylene]-5-phenylamino-2,4-dihydro-pyrazol-3-one
(2b).
-
1
Orange powder; yield (82%); m.p.: 285-287 ˚C; IR: (ύ max, cm ): 3440 (NH), 3238 (NH), 3143
1
(
NH), 1671 (CO); H-NMR (300 MHz)(DMSO-d
6
) δ: 6.82 (t, 1H, Ar-H), 7.23 (d, 2H, J=8.4HZ,
Ar-H), 7.34 (d, 2H, J=8.6HZ, Ar-H), 7.56 (d, 2H, J=8.7HZ, Ar-H), 7.61 (d, 2H, J=8.4HZ, Ar-H),
.51 (s, 1H, Ar-NH, exchangeable by D O), 8.68 ( s, 1H, Olefinic -CH), 10.50 (s, 1H, Ar-NH,
8
2
exchangeable by D
2
O), 11.1 (s, 1H, Pyrazole-NH, exchangeable by D O); MS: (Mwt.: 357.20):
2
+
·
+2.
m/z, 356.07 (M , [100%]), 358.08(M , [95.67%]); Anal. Calcd., for C16
H13BrN
4
O; C, 53.80; H,
3
.67; N, 15.68; Found: C, 54.11; H, 3.85; N, 15.91.
6
.1.2.3. 5-Phenylamino-4-(4-tolylamino-methylene)-2,4-dihydro-pyrazol-3-one
(2c).
-1
Brownish orange powder; yield (84%); m.p.: 293-295˚C; IR: (ύ max, cm ): 3427 (NH),
1
3
242(NH), 3145 (NH), 1671 (CO); H-NMR (300 MHz)(DMSO-d
6
) δ: 2.46 (s, 3H, Ar-CH ), 6.81
3
(
t, 1H, Ar-H), 7.21 (d, 2H, J=8.4HZ, Ar-H), 7.27 (d, 2H, J=8.7HZ, Ar-H), 7.4 (d, 2H, J=8.6HZ,

Ar-H), 7.56 (d, 2H, J=8.5HZ, Ar-H), 8.49 (s, 1H, Ar-NH, exchangeable by D
Olefinic-CH), 10.46 (s, 1H, Ar-NH, exchangeable by D O), 11.11 (s, 1H, Pyrazole-NH,
) δ: 30.61, 107.35, 115.18, 115.31, 115.54,
15.65, 118.59, 128.66, 129.22, 129.5, 130.31, 130.55, 142.75, 146.61, 149.16, 159.55, 167.20;
2
O), 8.71 (s, 1H,
2
1
3
exchangeable by D
2
O); C-NMR (75 MHz, DMSO-d
6
1
+
·
MS: (Mwt.: 292.34): m/z, 292.17 (M ,[100%]); Anal.Calcd., for C17
H
16
4
N O; C, 69.85; H, 5.52;
N, 19.17; Found: C, 70.11; H, 5.75; N, 19.33.
6
.1.2.4. 4-[(4-Hydroxy-phenylamino)-methylene]-5-phenylamino-2,4-dihydro-pyrazol-3-one (2d).
-1
Brown powder; yield (85%); m.p.: 210-215 ˚C; IR: (ύ max, cm ): 3426 (OH), 3392 (NH), 3240
1
(
NH), 3145 (NH), 1665 (CO); H-NMR (300 MHz)(DMSO-d
6
) δ: 6.8 (t, 1H, Ar-H), 7.18 (d, 2H,
J=8.7HZ, Ar-H), 7.24 (d, 2H, J=8.5HZ, Ar-H), 7.43 (d, 2H, J=8.4HZ, Ar-H), 7.55 (d, 2H,
J=8.4HZ, Ar-H), 8.44 (s, 1H, Ar-NH, exchangeable by D O), 8.59 (s, 1H, Olefinic-CH), 10.4 (s,
2
1
H, Ar-NH, exchangeable by D
Pyrazole-NH, exchangeable by D
Calcd.,for C16
2
O), 9.47 (s, 1H, Ar-OH, exchangeable by D
2
·
O), 11.09 (s, 1H,
+
2
O); MS: (Mwt.: 294.31): m/z, 294.17 (M ,[14.77%]); Anal.
H
14
N
4
O
2
; C, 65.3; H, 4.79; N, 19.04; Found: C, 65.44; H, 4.95; N, 19.21.
6
.1.2.5. N-{4-[(5-Oxo-3-phenylamino-1,5-dihydro-pyrazol-4-ylidenemethyl)-amino]
-phenyl}
-1
acetamide (2e) . Brown powder; yield (81%); m.p.: ˃ 300 ˚C; IR: (ύ max, cm ): 3873 (NH), 3248
1
(
NH), 3140 (NH), 3062 (NH), 1743(CO), 1666 (CO); H-NMR (300 MHz)(DMSO-d
6
) δ: 2.5 (s,
3
H, Amide-CH
.47 (d, 2H, J=8.6HZ, Ar-H), 7.63 (d, 2H, J=8.7HZ, Ar-H), 8.53 (s, 1H, Ar-NH, exchangeable by
O), 8.7 (s, 1H, Olefinic-CH), 9.8 (s, 1H, Ar-NH, exchangeable by D O), 10.01 (s, 1H, Pyrazole-
NH, exchangeable by D O), 10.49 (s, 1H, Amide-NH, exchangeable by D O); MS: (Mwt.:
; C, 64.47; H, 5.11; N, 20.88;
3
), 6.9 (t, 1H, Ar-H), 7.24 (d, 2H, J=8.4HZ, Ar-H), 7.32 (d, 2H, J=8.5HZ, Ar-H),
7
D
2
2
2
2
+
·
3
35.36): m/z, 335.2 (M ,[8.35%]); Anal.Calcd.,for C18
H
17
N
5
O
2
Found: C, 64.61; H, 5.39; N, 20.97.
6
.1.2.6. 4-[(4-Acetyl-phenylamino)-methylene]-5-phenylamino-2,4-dihydro-pyrazol-3-one (2f).
-1
Brown powder; yield (85%); m.p.: ˃ 300 ˚C; IR: (ύ max, cm ): 3325 (NH), 3209 (NH), 3093
1
(
NH), 1743 (CO), 1640 (CO); H-NMR (300 MHz)(DMSO-d
6
) δ: 2.5 (s, 3H, Acetyl-CH ), 6.84 (t,
3
1
H, Ar-H), 7.26 (d, 2H, , J=8.5HZ, Ar-H), 7.43 (d, 2H, , J=8.4HZ, Ar-H), 7.51 (d, 2H,
overlapped, Ar-H), 7.61 (d, 2H, , J=8.6 HZ, Ar-H), 8.55 (s, 1H, Ar-NH, exchangeable by D O),
.71 (s, 1H, Olefinic-CH), 10.54 (s, 1H, Ar-NH, exchangeable by D O), 11.16 (s, 1H, Pyrazole-
2
8
2

+
·
NH, exchangeable by D
2
O); MS: (Mwt.: 320.35): m/z, 320.15 (M ,[1.95%]); Anal.Calcd.,for
C
18
H
16
N
4
O
2
; C, 67.49; H, 5.03; N, 17.49; Found: C, 67.61; H, 5.21; N, 17.65.
6
.1.2.7.N-(4,6-Dimethyl-pyrimidin-2-yl)
-4-[(5-oxo-3-phenylamino
-1,5-dihydro-pyrazol-4-
ylidenemethyl)-amino]-benzene sulfonamide (2g). Black powder; yield (82%); m.p.: ˃ 300 ˚C; IR:
-
1
1
(
ύ max, cm ): 3367 (NH), 3232 (NH), 3055 (NH), 3032 (NH), 1620 (CO), 1377 (SO
2
-N); H-
NMR (300 MHz) (DMSO-d ) δ: 2.6 (s, 6H, 2Ar-CH ), 6.28 (s, 1H, Pyrimidinyl-CH), 6.53 (t, 1H,
6
3
Ar-H), 7.15 (d, 2H, J=8.4HZ, Ar-H), 7.41 (d, 2H, J=8.5HZ, Ar-H), 7.58 (d, 2H, J=8.6HZ, Ar-
H), 7.64 (d, 2H, J=8.7HZ, Ar-H), 7.66 (s, 1H, Olefinic-CH), 8.03 (s, 1H, Ar-NH, exchangeable by
D
2
O), 9.79 (s, 1H, Ar-NH, exchangeable by D
2
O), 10.26 (s, 1H, Ar-NH, exchangeable by D
O); MS: (Mwt.: 463.51): m/z, 463.17
S; C, 57.01; H, 4.57; N, 21.15; Found: C, 57.21; H,
2
O),
1
2.85 (s, 1H, Pyrazole-NH, exchangeable by D
2
+
·
(
M ,[2.47%]); Anal. Calcd., for C22
H
21
N
7
O
3
4
.75; N, 21.31.
6
.1.3. General procedure for preparation of (4-subistituted) methylene-5-phenylamino-2,4-
dihydro-pyrazol-3-one (3a-c) [18,19]. A mixture of 5-phenylamino-2, 4-dihydro-pyrazol-3-one 1
4gm, 0.025mol) with appropriate aromatic aldehyde (0.025mol) was refluxed for 6 hrs with
(
freshly fused sodium acetate (7gm) in glacial acetic acid (80ml). After that, the reaction mixture
was cooled and poured on ice where the precipitate occurred. The later was filtered and washed
well with water and crystallized from ethanol to give the title compound (3a-c).
6
.1.3.1. 4-[5-(4-Chloro-phenyl)-furan-2-ylmethylene]-5-phenylamino-2,4-dihydro pyrazol-3-one
-1
(
3a). Olive green powder; yield (80%); m.p.:128-130˚C; IR: (ύ max, cm ): 3550 (NH), 3478
1
(
NH), 1617(CO); H-NMR (300 MHz)(DMSO-d
6
) δ: 6.91 (d, 2H, J=8.7HZ, Ar-H), 7.3 (t, 1H, Ar-
H), 7.48 (d, 1H, J=8.4HZ, furan-H), 7.59 (d, 2H, J=8.6HZ, Ar-H), 7.68 (d,1H, J=8.5HZ, furan-H),
7
1
.9 (d, 2H, J=8.5HZ, Ar-H), 8.07 (d, 2H, overlapped, Ar-H), 8.64 (s, 1H, Olefinic-CH), 8.8 (s,
H, Ar-NH, exchangeable by D
2
O), 10.72 (s, 1H, Pyrazole -NH, exchangeable by D O); MS:
2
+
·
+2·
(
;
Mwt.:363.8): m/z, 363.12 (M ,[29.04%]), 365.11 (M ,[14.1%]); Anal. Calcd., for C20
H14ClN
3
O
2
C, 66.03; H, 3.88; N, 11.55; Found: C, 66.21; H, 4.11; N, 11.75.
6
.1.3.2. 4-[5-(3-Chloro-phenyl)-furan-2-ylmethylene]-5-phenylamino-2,4-dihydro pyrazol-3-one
-1
(
3b). Dark green powder; yield (78%); m.p.: 198-200˚C; IR: (ύ max, cm ): 3547 (NH), 3413
1
(
NH), 1680 (CO); H-NMR (300 MHz)(DMSO

-
d
6
) δ: 6.89 (d, 2H, J=8.7HZ, Ar-H), 7.15 (t, 1H, Ar-H), 7.28 (d, 1H, J=8.5HZ, furan-H), 7.44 (d,
H, J=8.5HZ, furan-H), 7.53 (d, 2H, J=8.4HZ, Ar-H), 7.66 (d, 2H, J=8.5HZ, Ar-H), 7.73 (t, 1H,
Ar-H), 7.89 (d, 2H, J=8.7HZ, Ar-H), 8.1 (s, 1H, Ar-H), 8.63 (s, 1H, Olefinic-CH), 9.11 (s, 1H, Ar-
1
NH, exchangeable by D
2
O), 10.5 (s, 1H, Pyrazole -NH, exchangeable by D
2
O); MS: (Mwt.:
; C,
+
·
+2·
3
6
63.80): m/z, 363.1 (M ,[19.01%]), 365.1 (M ,[7.59%]); Anal. Calcd., for C20
H
14ClN
3
O
2
6.03; H, 3.88; N, 11.55; Found: C, 66.21; H, 3.81; N, 11.74.
6
.1.3.3. 4-(4-Methoxy-benzylidene)-5-phenylamino-2,4-dihydro pyrazol-3-one (3c). Dark red
-1
1
powder; yield (81%); m.p.: 184-186˚C; IR: (ύ max, cm ): 3525 (NH), 3387 (NH), 1678 (CO); H-
NMR (300 MHz)(DMSO-d ) δ: 3.86 (s, 3H, OCH ), 6.91 (d, 2H, overlapped, Ar-H), 7.12 (t, 1H,
Ar-H), 7.27 (d, 2H, J=8.6HZ, Ar-H), 7.66 (d, 2H, J=8.7HZ, Ar-H), 7.9 (d, 2H, J=8.5HZ, Ar-H),
6
3
8
.63 (s, 1H, Olefinic-CH), 9.8 (s, 1H, Ar-NH, exchangeable by D
2
O), 10.68 (s, 1H, Pyrazole -NH,
1
3
exchangeable by D
2
O); C-NMR (75 MHz, DMSO-d
6
) δ: 55.59, 114.35, 114.66, 118.6, 126.26,
1
27.89, 128.3, 128.69, 129.22, 129.55, 140.45, 142.75, 146.7, 149.16, 155.75, 165.20, 169.78;
+
·
MS: (Mwt.: 293.32): m/z, 293.13 (M ,[31.33%]); Anal. Calcd., for C17
H
15
N
3
2
O ; C, 69.61; H,
5
.15; N, 14.33; Found: C, 69.89; H, 5.31; N, 14.55.
6
.1.4. General procedure for preparation of Cyanoacetamide (4) [31]. Ethyl 2-cyanoacetate (52
ml, 0.442mole) was poured on ammonium hydroxide (40 ml, 1.14mole), then stirred until a turbid
solution formed. The mixture was gently heated until it turned clear. The mixture has been kept in
an ice bath for 1 hr upon which a crystalline white precipitate formed. The precipitate was filtered
and crystallized from ethanol to give the title compound as crystalline white powder (70%).
6
.1.5. General procedure for preparation of 2-Cyano-3-methylsulfanyl-3-phenylamino-acrylamide
(
5) [20,21]. To a cooled solution of finely divided KOH (1.65gm, 0.025mol) in DMF,
cyanoacetamide (4) (2.1gm, 0.025mol) was added then followed by the addition of phenyl
isothiocyanate (3.37gm, 0.025mol). Reaction mixture was stirred at room temperature for 24 hrs
then cooled again with addition of water (30ml). Dimethyl sulphate (3.15gm, 0.025mol) was added
drop wisely and stirred continuously for extra 8 hrs to give a crystalline yellow precipitate. The
precipitate was filtered and washed well with ether then crystallized from ethanol to give the title
compound (5) (80%).

6
.1.5.1. 2-Cyano-3-methylsulfanyl-3-phenylamino-acrylamide (5). Pale yellow powder; yield
-1
1
(
80%); m.p.: 147-149˚C; IR: (ύ max, cm ): 3382 (NH
NMR (300 MHz)(DMSO-d ) δ: 2.51 (s, 3H, CH ), 7.14 (d, 2H, J=8.4HZ, Ar-H), 7.29 (t, 1H,
J=8.7HZ, Ar-H), 7.42 (d, 2H, J=8.6HZ, Ar-H), 12.11 (s, 1H, NH, exchangeable by D O), 12.46 (s,
2
), 3329 (NH), 2225 (CN), 1652 (CO); H-
6
3
2
+
·
2
H, NH
2
, exchangeable by D
2
O); MS: (MWt.: 233.1): m/z, 233.05 (M , [0.78%]).
6
.1.6. General procedure for preparation of 5-Amino-3-phenylamino-1H-pyrazole-4-carboxylic
acid amide (6) [23]. 2-Cyano-3-methylsulfanyl-3-phenylamino-acrylamide (5) (8gm, 0.034mol)
1
was refluxed with methanol (250ml) for /
2
hr till completely dissolved then cooled. Hydrazine
1
hydrate (1.6ml, 0.034mol) was added drop wisely on cold with continuous stirring for extra /
2
hr,
then followed with water bath for 3-4 hrs till precipitate occurred. The later was filtered and
washed well with ether then crystallized from ethanol to give the title compound (6) as crystalline
whitish grey powder (64%).
6
.1.6.1. 5-Amino-3-phenylamino-1H-pyrazole-4-carboxylic acid amide (6). Whitish grey powder;
-
1
yield (64%); m.p.: 178-179˚C; IR: (ύ max, cm ): 3441 (NH
2
), 3379 (NH
2
), 3275 (NH), 3197
, exchangeable by
1
(
NH), 1654 (CO); H-NMR (300 MHz)(DMSO-d
6
) δ: 5.82 (s, 2H, Pyrazole-NH
2
D
2
O), 6.74 (d, 2H, J=8.7HZ, Ar-H), 7.17 (t, 1H, Ar-H), 7.2 (d, 2H, J=8.6HZ, Ar-H), 7.33 (s, 1H,
Ar-NH, exchangeable by D O), 8.97 (s, 1H, Pyrazole -NH, exchangeable by D O), 11 (s, 2H,
Amide-NH , exchangeable by D O); MS: (Mwt.: 217.2): m/z, 217.05 (M ,[47.43%]).
2
2
+
·
2
2
6
.1.7. General procedure for preparation of 3-Carbamoyl-5-(4-subistituted phenyl)-7-methyl-2-
phenylamino-4,5-dihydro pyrazolo[1,5-a]pyrimidine-6-carboxylic acid ethyl ester (7a-d) [24,25].
Hydrochloric acid (0.1ml) was added to a mixture of aldehyde derivative (0.01mol), ethyl
acetoacetate (1.43gm, 0.01mol) and 5-amino-3-phenyl-amino-1H-pyrazole-4-carboxylic acid
amide (6) (2.17gm, 0.01mol) in ethanol (40ml) and then refluxed for 4-8 hrs. After that, the
reaction mixture was poured upon ice and the resulting precipitate was filtered, washed by diethyl
ether and crystallized from ethanol to give the titled compounds (7a-d).
6
.1.7.1. 3-Carbamoyl-5-(4-chloro-phenyl)-7-methyl-2-phenylamino-4,5-dihydro pyrazolo[1,5-a]
pyrimidine-6-carboxylic acid ethyl ester (7a). Dark yellow powder; yield (80%); m.p.: 96-98 ˚C;
-
1
1
IR: (ύ max, cm ): 3480 (NH
2
), 3325 (NH), 3093 (NH), 1720 (CO), 1658 (CO); H-NMR (300
-CH ), 2.73 (s, 3H, Pyrimidinyl-CH ), 4.13 (dd, 2H, J=8.6HZ,
MHz)(DMSO-d ) δ: 2.5 (t, 3H, CH
6
3
2
3

CH
H, J=8.6HZ, Ar-H), 7.4 (d, 2H, J=8.6HZ, Ar-H), 7.51 (d, 2H, J=8.7HZ, Ar-H), 8.26 (s, 1H, Ar-
NH, exchangeable by D O), 9.52 (s, 1H, Pyrimidinyl-NH, exchangeable by D O), 10 (s, 2H,
Amide-NH , exchangeable by D O); MS: (Mwt.: 467.95): m/z, 467.1 (M ,[1.03%]), 469.32
M ,[0.91%]); Anal.Calcd.,for C24 ; C, 61.6; H, 5.6; N, 14.97; Found: C, 61.86; H,
3
-CH
2
), 5.52 (s, 1H, Pyrimidinyl-H), 6.87 (d, 2H, J=8.5HZ, Ar-H), 7.03 (t, 1H, Ar-H), 7.27 (d,
2
2
2
+
·
2
2
+
2·
(
H26ClN
5
O
3
5
.89; N, 15.11.
6
.1.7.2. 3-Carbamoyl-5-(4-methoxy-phenyl)-7-methyl-2-phenylamino-4,5-dihydro pyrazolo[1,5-a]
pyrimidine-6-carboxylic acid ethyl ester (7b). Grayish green powder; yield (81%); m.p.: 76-78˚C;
-
1
1
IR: (ύ max, cm ): 3479 (NH
MHz)(DMSO-d ) δ: 2.5 (t, 3H, CH
.14 (dd, 2H, J=8.5HZ, CH -CH ), 5.47 (s, 1H, Pyrimidinyl-H), 6.87 (d, 2H, J=8.7HZ, Ar-H), 6.97
t, 1H, Ar-H), 7.13 (d, 2H, J=8.6HZ, Ar-H), 7.44 (d, 2H, J=8.4HZ, Ar-H), 7.73 (d, 2H, J=8.5HZ,
Ar-H), 8.17 (s, 1H, Ar-NH, exchangeable by D O), 9.55 (s, 1H, Pyrimidinyl-NH, exchangeable by
O), 9.71 (s, 2H, Amide-NH , exchangeable by D
2
), 3332 (NH), 3271 (NH), 1708 (CO), 1658 (CO); H-NMR (300
6
3
-CH ), 2.72 (s, 3H, Pyrimidinyl-CH ), 2.85 (s, 3H, OCH ),
2
3
3
4
3
2
(
2
1
3
D
2
2
2
O); C-NMR (75 MHz, DMSO-d ) δ: 15.5,
6
1
1
4
6
5.9, 55.59, 56.4, 60.1, 78.8, 110.66, 114.12, 114.56, 115.38, 115.9, 119.7, 128.5,128.9, 129.6,
30.55, 135.32, 138.6, 148.6, 155.5, 156.9, 161.2, 164.5, 170.32 ; MS: (Mwt.: 447.49): m/z,
+
·
47.21 (M ,[21.99%]); Anal.Calcd.,for C24
H
25
N
5
4
O ; C, 64.42; H, 5.63; N, 15.65; Found: C,
4.63; H, 5.85; N, 15.84.
6
.1.7.3. 3-Carbamoyl-5-(4-hydroxy-phenyl)-7-methyl-2-phenylamino-4,5-dihydro pyrazolo[1,5-a]
pyrimidine-6-carboxylic acid ethyl ester (7c). Off-white powder; yield (83%); m.p.: 90-92˚C; IR:
-
1
1
(
ύ max, cm ): 3425 (OH), 3309 (NH
300 MHz)(DMSO-d ) δ: 2.5 (t, 3H, CH
-CH ), 5.39 (s, 1H, Pyrimidinyl-H), 6.66 (d, 2H, J=8.5HZ, Ar-H), 6.86 (t, 1H,
Ar-H), 7.12 (d, 2H, J=8.5HZ, Ar-H), 7.42 (d, 2H, J=8.4HZ, Ar-H), 7.84 (d, 2H, J=8.7HZ, Ar-H),
2
), 3209 (NH), 2978 (NH), 1685 (CO), 1651 (CO); H-NMR
(
6
3
-CH ), 2.7 (s, 3H, Pyrimidinyl-CH ), 4.02 (dd,
2
3
overlapped 2H, CH
3
2
8
9
.22 (s, 1H, Ar-NH, exchangeable by D
2
O), 9.39 (s, 1H, Pyrimidinyl-NH, exchangeable by D
2
O),
O);
.58 (s, 1H, Ar-OH, exchangeable by D
2
O), 11.55 (s, 2H, Amide-NH , exchangeable by D
2
2
+
·
MS: (Mwt.: 433.46): m/z, 433.15 (M ,[18.8%]); Anal.Calcd.,for C23
H
23
N
5
4
O ; C, 63.73; H, 5.35;
N, 16.16; Found : C, 63.98; H, 5.59; N, 16.34.
6
.1.7.4. 3-Carbamoyl-5-(4-fluoro-phenyl)-7-methyl-2-phenylamino-4,5-dihydro pyrazolo[1,5-a]
pyrimidine-6-carboxylic acid ethyl ester (7d). grey powder; yield (80%); m.p.: 121-125˚C; IR: (ύ

-
1
1
max, cm ): 3460 (NH
MHz)(DMSO-d ) δ: 2.7 (t, 3H, CH
overlapped, CH -CH ), 5.52 (s, 1H, Pyrimidinyl -H), 6.68 (d, 2H, J=8.5HZ, Ar-H), 6.92 (t, 1H, Ar-
H), 7.21 (d, 2H, J=8.7HZ, Ar-H), 7.49 (d, 2H, J=8.5HZ, Ar-H), 7.71 (d, 2H, J=8.6HZ, Ar-H), 8.22
2
), 3336 (NH), 3217 (NH), 1697 (CO), 1662 (CO); H-NMR (300
6
3
-CH ), 2.88 (s, 3H, Pyrimidinyl-CH ), 4.12 (dd, 2H,
2
3
3
2
(
(
s, 1H, Ar-NH, exchangeable by D
s, 2H, Amide-NH , exchangeable by D
; C, 63.44; H, 5.09; N, 16.08; Found: C, 63.64; H, 5.21; N, 16.25.
2
O), 9.51 (s, 1H, Pyrimidinyl-NH, exchangeable by D
2
O), 9.73
+
·
2
2
O); MS: (Mwt.: 435.45): m/z, 435.18 (M ,[78.62%]);
Anal.Calcd.,for C23
H
22FN
5
O
3
6
.1.8. General procedure for preparation of 2-(Methylsulfanyl-phenylamino-methylene)-
malononitrile (8) [21,22]. To a cooled solution of finely divided KOH (1.65gm, 0.025mol),
malononitrile (1.65gm, 0.025mol) was added then followed by the addition of phenyl
isothiocyanate (3.37gm, 0.025mol). Reaction mixture was stirred at room temperature for 24 hrs
then cooled again with addition of water (30ml). Dimethyl sulphate (3.15gm, 0.025mol) was added
drop wisely then stirred continuously for extra 8 hrs to give a crystalline yellow precipitate. The
precipitate was filtered and washed well with ether then crystallized from ethanol to give the title
compound (8) (82%).
6
.1.8.1.2-(Methylsulfanyl-phenylamino-methylene)-malononitrile (8). Yellow powder; yield
-
1
1
(
82%); m.p.: 166-168˚C; IR: (ύ max, cm ): 3320 (NH), 2219 (CN), 2125 (CN); H-NMR (300
MHz)(DMSO-d ) δ: 3.9 (s, 3H, CH ), 7.28 (d, 2H, J =8.4HZ, Ar-H), 7.4 (t, 1H, J=8.5HZ, Ar-H),
.53 (d, 2H, overlapped, Ar-H), 10.53 (s, 1H, NH, exchangeable by D O); MS: (MWt.: 215.27):
6
3
7
2
+
·
m/z, 215.05 (M , [100%]).
6
.1.9. General procedure for preparation of 5-Amino-3-phenylamino-1H-pyrazole-4-carbonitrile
(
9) [23]. 2-(Methylsulfanyl-phenylamino-methylene)-malononitrile (8) (7.3gm, 0.034mol) was
1
refluxed with methanol (250ml) for /
2
hr till completely dissolved then cooled. Hydrazine hydrate
1
(
1.6ml, 0.034mol) was added drop wisely on cold with continuous stirring for extra /
2
hr, then
followed with water bath for 3-4 hrs till precipitate occurred. The later was filtered and washed
well with ether then crystallized from ethanol to give the title compound (9) as crystalline whitish
grey powder as reported (80%).
6
.1.9.1. 5-Amino-3-phenylamino-1H-pyrazole-4-carbonitrile (9). Whitish grey powder; yield
-1
1
(
80%); m.p.: 204-205˚C; IR: (ύ max, cm ): 3468 (NH ), 3371 (NH), 3305 (NH), 2214 (CN), H-
2

NMR (300 MHz)(DMSO-d
J=8.7HZ, Ar-H), 7.16 (t, 1H, Ar-H), 7.45 (d, 2H, J=8.5HZ, Ar-H), 8.27 (s, 1H, Ar-NH,
exchangeable by D O), 11.11(s, 1H, Pyrazole-NH, exchangeable by D O) ; MS: (Mwt.: 199.21):
m/z, 199.09 (M ,[100%]).
6
) δ: 6.23 (s, 2H, Pyrazole-NH
2
, exchangeable by D
2
O), 6.75 (d, 2H,
2
2
+
·
6
.1.10. General procedure for preparation of 5-(4-subistituted-phenyl)-3-cyano-7-methyl-2-
phenylamino-4,5-dihydro-pyrazolo[1,5-a]pyrimidine-6-carboxylic acid ethyl ester (10a-e) [24,25].
Hydrochloric acid (0.1ml) was added to a mixture of aldehyde derivative (0.01mol), ethyl
acetoacetate (1.43gm, 0.01mol) and 5-amino-3-phenylamino-1H-pyrazole-4-carbonitrile (9)
(
1.99gm, 0.01mol) in ethanol (40ml) and then refluxed for 4-8 hrs. After that, the reaction mixture
was poured onto ice and the resulting precipitate was filtered, washed well by diethyl ether and
crystallized from ethanol to give the titled compounds (10a-e).
6
.1.10.1. 5-(4-Chloro-phenyl)-3-cyano-7-methyl-2-phenylamino-4,5-dihydro pyrazolo[1,5-a]
pyrimidine -6-carboxylic acid ethyl ester (10a). Dark yellow powder; yield (81%); m.p.: 177-
-
1
1
1
79˚C; IR: (ύ max, cm ): 3421 (NH), 3317 (NH), 2210 (CN), 1685 (CO); H-NMR (300
MHz)(DMSO-d ) δ: 2.69 (t, 3H, CH -CH ), 2.83 (s, 3H, Pyrimidinyl-CH ), 4.12 (dd, 2H,
overlapped, CH -CH ), 5.48 (s, 1H, Pyrimidinyl-H), 6.88 (d, 2H, J=8.5HZ, Ar-H), 7.24 (t, 1H, Ar-
H), 7.34 (d, 2H, J=8.7HZ, Ar-H), 7.63 (d, 2H, J=8.5HZ, Ar-H), 7.76 (d, 2H, overlapped, Ar-H),
.91 (s, 1H, Ar-NH, exchangeable by D O), 9.15 (s, 1H, Pyrimidinyl-NH, exchangeable by D O);
MS: (Mwt.: 433.89): m/z, 433.13 (M ,[100%]), 435.12 (M ,[29.27%]); Anal.Calcd.,for
; C, 63.67; H, 4.65; N, 16.14; Found: C, 63.91; H, 4.86; N, 16.35.
6
3
2
3
3
2
8
2
2
+
·
+2·
C
23
H20ClN
5
O
2
6
.1.10.2. 3-Cyano-5-(4-methoxy-phenyl)-7-methyl-2-phenylamino-4,5-dihydro pyrazolo[1,5-a]
pyrimidine-6-carboxylic acid ethyl ester (10b). Pale green powder; yield (82%); m.p.: 178-180˚C;
-1
1
IR: (ύ max, cm ): 3363 (NH), 3325 (NH), 2218 (CN), 1674 (CO); H-NMR (300 MHz)(DMSO-
) δ: 2.48 (t, 3H, CH -CH ), 2.72 (s, 3H, Pyrimidinyl-CH ), 3.68 (s, 3H, OCH ), 4.07 (dd, 2H,
J=8.5HZ, CH -CH ), 5.4 (s, 1H, Pyrimidinyl-H), 6.84 (d, 2H, overlapped, Ar-H), 6.88 (t, 1H, Ar-
H), 7.14 (d, 2H, J=8.5HZ, Ar-H), 7.22 (d, 2H, J=8.5HZ, Ar-H), 7.59 (d, 2H, J=8.6HZ, Ar-H), 8.87
d
6
3
2
3
3
3
2
1
3
(
s, 1H, Ar-NH, exchangeable by D
NMR (75 MHz, DMSO-d ) δ: 16.5, 16.9, 55.6, 56.5, 60.2, 75.8, 108.66, 113.12, 113.56, 115.5,
15.9, 118, 118.8, 128.25, 128.7, 129.66, 131.58, 134.12, 139.76, 150.32, 154.5, 155.9, 161.2,
2
O), 9.05 (s, 1H, Pyrimidinyl-NH, exchangeable by D O); C-
2
6
1