Synthesis and in-vitro anti-proliferative evaluation of some pyrazolo[1,5-a]pyrimidines as novel larotrectinib analogs

Article abstract of DOI:10.1016/j.bioorg.2019.103458

A series of 2-phenyl-7-(aryl)pyrazolo[1,5-a]pyrimidine-3-carbonitriles 11a–j and 2-phenyl-7-(aryl)pyrazolo[1,5-a]pyrimidine-3,6-dicarbonitriles 16a–c was synthesized by the reaction of 5-amino-3-phenyl-1H-pyrazole-4-carbonitrile (5) with 3-(dimethylamino)

Full text of DOI:10.1016/j.bioorg.2019.103458

Journal Pre-proofs
Synthesis and in-vitro anti-proliferative evaluation of some pyrazolo[1,5-
a]pyrimidines as novel larotrectinib analogs
Mohamed H. Attia, Eman Z. Elrazaz, Soad Z. El-Emam, Azza T. Taher,
Hatem A. Abdel-Aziz, Khaled A.M. Abouzid
PII:
S0045-2068(19)31392-6
DOI:
https://doi.org/10.1016/j.bioorg.2019.103458
YBIOO 103458
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
26 August 2019
12 November 2019
19 November 2019
Please cite this article as: M.H. Attia, E.Z. Elrazaz, S.Z. El-Emam, A.T. Taher, H.A. Abdel-Aziz, K.A.M.
Abouzid, Synthesis and in-vitro anti-proliferative evaluation of some pyrazolo[1,5-a]pyrimidines as novel
larotrectinib analogs, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103458
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Synthesis and in-vitro anti-proliferative evaluation of some
pyrazolo[1,5-a]pyrimidines as novel larotrectinib analogs
Mohamed H. Attia1, Eman Z. Elrazaz2, Soad Z. El-Emam3, Azza T. Taher4, Hatem
A. Abdel-Aziz5* and Khaled A.M. Abouzid2,6*
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, October 6 University, 6 of
October City, Giza, Egypt
2Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia
11566, Cairo, Egypt
3Department of Pharmacology and Toxicology, Faculty of Pharmacy, October 6 University, 6 of
October City, Giza, Egypt
4Cairo University, Faculty of Pharmacy, Pharmaceutical Organic Chemistry Department, Cairo,
Egypt
5Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622,
Egypt
6Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat City,
Sadat City, Egypt
ABSTRACT
A series of 2-phenyl-7-(aryl)pyrazolo[1,5-a]pyrimidine-3-carbonitriles 11a-j and 2-
phenyl-7-(aryl)pyrazolo[1,5-a]pyrimidine-3,6-dicarbonitriles 16a-c was synthesized by the
reaction of 5-amino-3-phenyl-1H-pyrazole-4-carbonitrile (5) with 3-(dimethylamino)-1-
arylprop-2-en-1-ones 6a-j or 2-aryl-3-(dimethylamino)acrylonitriles 12a-c, respectively. In
addition, 7-amino-5-oxo-2-phenyl-4,5-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (22) was
prepared from the reaction of compound 5 with ethyl cyanoacetate. The anticancer activity of the
newly synthesized compounds against Huh-7, HeLa, MCF-7 and MDA-MB231 cell lines
showed moderate activity of compound 11f as anti-proliferative agent against Huh-7 cell line
with IC50 = 6.3 µM when compared with doxorubicin (IC50 = 3.2 µM). On the other hand,
compound 16b revealed potent anti-proliferative activity against HeLa cell line with IC50 = 7.8
1

µM when compared with doxorubicin (IC50 = 8.1 µM). Also compound 11i exhibited a
promising anti-proliferative activity against MCF-7 cell line (IC50 = 3.0 µM) whereas IC50 of
doxorubicin = 5.9 µM, finally compounds 11i and 16b have potent activity as anti-proliferative
agents against MDA-MB231 cell line with IC50 = 4.32 and 5.74 µM, respectively when
compared with doxorubicin (IC50 = 6.0 µM).
____________
Keywords: Synthesis, Pyrazolo[1,5-a]pyrimidine, Anticancer activity, MDA-MB231 cell line
*Corresponding authors: Khaled A.M. Abouzid, E-mail: khaled.abouzid@pharma.asu.edu.eg
and Hatem A. Abdel-Aziz, E-mail: hatem_741@yahoo.com
1-Introduction
Pyrazolo[1,5-a]pyrimidine scaffold is an important block in the constructions of several
anticancer agents [1-3]. For instance, the anticancer drug VITRAKVI® (Larotrectinib, i) (Fig 1),
was innovated by medicinal scientists using pyrazolo[1,5-a]pyrimidine as core moiety [4].
VITRAKVI®, generated by Loxo Oncology Incorporation with Bayer and got FDA approval on
November, 2018, is useful for treatments of solid tumors with gene fusion [5]. It showed potent
anticancer activity as TRKA-C kinases inhibitor with IC50 < 1 nM and it revealed activity against
TRKA G595R, TRKC G623R, and TRKA G667C cell lines with IC50 = 2.0-9.8 nM [6]. In
addition, LOXO-195 (ii, Fig 1) is 2nd generation TRK inhibitor, it exhibited activity against TRK
fusion- KM12, CUTO-3, and MO-91 cell line with IC50 ≤ 5 nM [6]. Furthermore, pyrazolo[1,5-
a]pyrimidines iii (Fig 1) are useful for treatment of diseases associated with Interleukin-1
Receptor Associated Kinase (IRAK). These compounds modulate the function of IRAK-1 and/or
IRAK-4 which represent an attractive approach to the development of therapeutic agents for the
treatment of cancer, inflammatory and immune-related diseases [7].
On the other side, 3-(thiophene-2-carbonyl)pyrazolo[1,5-a]pyrimidine derivatives iv with
aryl substitution in position 7 (Fig 1) showed potent activity as proliferative agents against
HCT116 and 80S14 cell lines with IC50 = 11 and 0.45 µM respectively [8] and they revealed a
significant anticancer activity against different six colon cell lines with IC50 = 0.012-2.3 µM [9].
2

pyrazolo[1,5-a]pyrimidines v (Fig 1), with phenyl moiety in position 2, have been reported as
CDK inhibitors. Their anticancer activity against A549, DU-145, ACHN, MCF-7 and Hela cell
lines with IC50 in the range of 1.94-7.07 µM [10].
Pyrazolo[1,5-a]pyrimidine vi (Fig 1), with carbonitrile function in position 3, has been
reported as potent anticancer agents against Bel-7402 and HT-1080 cell lines [11], whereas
pyrazolo[1,5-a]pyrimidine-3-carbonitrile vii (Fig 1) showed potent anticancer activity (IC50 =
0.002 µM) against HCT116 cell line [12]. Furthermore, 7-amino-pyrazolo[1,5-a]pyrimidines viii
and ix (Fig 1) have been reported as potent Lck and CHK1 inhibitors, respectively [13][14].
HO
O
N
R1
NH
R
O
N
O
N
O
N
NH
N
S
N
N
N
N
N
N
N
N
N
N
N
F
Ar
F
Ar
N
F
i, VITRAKVI® (Larotrectinib)
ii, LOXO-195
iv
iii
Ar
CN
CN
S
Ph
N
N
N
R
N
N
N
N
R
N
N
N
N
N
H2N
Ph
R
Ar
R
Ar
viii, Ar =
3-substituted phenyl
vii
v
vi
ix, Ar =
substituted pyrazol-3-yl
CN
Ph
N
CN
Ph
CN
Ph
N
N
N
N
N
N
NH
O
N
Ar
Ar
H2N
CN
11a-j
16a-c
22
3

Figure 1. Structure of i (Larotrectinib), ii (LOXO-195), iii-ix and the target compounds 11a-j,
16a-c, and 22.
In view of the previous reports [4-15], we aim to report herein the synthesis of 7-aryl-2-
phenylpyrazolo[1,5-a]pyrimidine-3-carbonitriles 11a-j, 7-aryl-2-phenylpyrazolo[1,5-
a]pyrimidine-3,6-dicarbonitriles 16a-c and 7-amino-2-phenylpyrazolo[1,5-a]pyrimidine-3-
carbonitrile 22 as larotrectinib analogs to evaluation their anticancer properties against four
human cancer cell lines namely; Huh-7, HeLa, MCF-7 and MDA-MB231 cell lines.
2-Results and Discussion
2.1. Chemistry
The key starting material 5-amino-3-phenyl-1H-pyrazole-4-carbonitrile (3) was prepared
by the reaction of 3-oxo-3-phenylpropanenitrile (1) with trichloroacetonitrile to yield 3-amino-2-
benzoyl-4,4,4-trichlorobut-2-enenitrile (2) which then reacted with hydrazine hydrate (Scheme
1). There are two alternative methods have been reported for the synthesis of 3 by reacting
arylmethylenemalononitrile with hydrazine [16-17] or via transformation of isoxazoles using
hydrazine [18]. On the other hand, enaminones 3-(dimethylamino)-1-arylprop-2-en-1-ones 6a-j
are versatile substrates in the synthesis of several biologically active heterocyclic compounds and
drug intermediates, were chosen in this study for the synthesis of pyrazolo[1,5-a]pyrimidines
11a-j. Enaminones 6a-j are generally prepared by the condensation of methyl aryl ketones 4a-j
with dimethylformamide dimethylacetal (5) in refluxing xylene (Scheme 1) [19-22]. Next, the
reaction of 5-amino-3-phenyl-1H-pyrazole-4-carbonitrile (3) with enaminones 6a-j in refluxing
acetic acid resulted in the formation of 2-phenyl-7-(aryl)pyrazolo[1,5-a]pyrimidine-3-
carbonitriles 11a-j, respectively. The latter reaction was assumed to proceed through an initial
Michael type addition of the amino group in pyrazole 3 to the double bond in enaminones 6a-j to
give intermediate 7a-j followed by the elimination of dimethylamine to afford the non-isolable
intermediates 8a-j which cyclized by losing one mole of H2O to give pyrazolo[1,5-a]pyrimidines
11a-j as final isolable products (Scheme 1). The 1H NMR spectra of 11a-j showed the down-
field doublet signal of pyrimidine H2 at δ 8.78–8.96 with J = 4.4–6.0 Hz, whereas the up-field
doublet signal of pyrimidine H3 appeared at δ around 7.61 with J around 5.8 Hz whereas the
signal of amino group not appeared in their NMR spectra. In addition, the mass spectra of 11a-j
exhibited, in each case, a peak corresponding to their molecular ions. The IR spectra of 11a-j
4

revealed the absence of the characteristic absorption band in the region 3150-3400 cm-1 due to
amino group.
O
O
O
N
+
Ar
4a-j
5
iii
O
O
Ar
N
CN
Ph
O
CN
NH2
Ph
CN
6a-j
iv
N
ii
i
Ph
H2N
O
CN
Ph
N
N
N
N
CCl3
1
N
H
Ar
H
H
2
H
3
7a-j
- NH(Me)2
CN
N
Ph
CN
Ph
CN
NH
Ph
CN
Ph
O
- H2O
N
N
N
N
N
N
N
Ar
N
N
H
N
Ar
Ar
HO
9a-j
H
Ar
HO
8a-j
H
11a-j
10a-j
11
a
b
c
d
e
f
Ar
Ph
4-CH3-C6H4
4-CH3O-C6H5
3,4-di-MeO-C6H4
4-F-C6H4
4-Cl-C6H4
g
h
i
4-Br-C6H4
4-NO2-C6H4
Naphthalen-2-yl
Furan-2-yl
j
Scheme 1. Reagents and conditions: i) Cl3C-CN/anhydrous CH3COONa/abs. EtOH/stirring 48
h; ii) NH2-NH2.H2O/abs. EtOH/reflux 4 h; iii) dry xylene/reflux 8h; iv) glacial AcOH/reflux 3 h.
5

Furthermore, the reaction of pyrazole 3 with 2-aryl-3-(dimethylamino)acrylonitrile 12a-c
in refluxing acetic acid resulted in the formation of the corresponding pyrazolopyrimidines 16a-
c. Their 1H NMR spectra revealed the down-field singlet signal of H2 proton of pyrimidine
moiety in the region δ 9.04–9.25 and the signal of amino group not appeared in their NMR
spectra, whereas their mass spectra showed a peak corresponding to their molecular ion values.
The IR spectra of 16a-c revealed the characteristic sharp absorption band of two carbonitrile
groups in the region 2230-2240 cm-1 and the absence of amino group absorption band in the
region 3150-3400 cm-1. This reaction was proposed to proceed through the same reaction
pathway of compounds 16a-c (Scheme 1). However, the possible structure 18a-c is easily
excluded on the basis of the spectral data of the isolated products (Scheme 2).
6

O
CN
1a-c
Ar
i
5
O
Ar
N
CN
NH2
Ph
CN
12a-c
CN
Ph
O
N
ii
N
Ar
N
N
N
H
H
H
CN
3
13a-c
X
CN
NH
Ph
CN
NH
Ph
N
N
N
N
Ar
HO
HN
CN
14a-c
Ar
O
17a-c
CN
Ph
Ar
N
N
N
CN
Ph
CN
HO
N
H
N
N
15a-c
H2N
- H2O
Ar
O
18a-c
CN
N
Ph
N
N
16
a
b
Ar
Ph
Ar
4-CH3O-C6H5
Furany-2-yl
CN
16a-c
c
Scheme 2. Reagents and conditions: i) dry xylene/reflux 1 h; ii) glacial AcOH/reflux 3 h.
7

Next, the neat reaction of compound 3 with ethyl cyanoacetate (19) gave 7-amino-5-oxo-
2-phenyl-4,5-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (22) (Scheme 3). The IR spectrum
of 22 showed absorption band due to the carbonyl group at 1685 cm-1. 1H NMR spectrum of 22
revealed two D2O exchangeable signals at δ 7.77 and 12.07 which were assigned to NH2 and NH
protons, respectively. Furthermore, the acetylation of compound 3 with glacial acetic acid gave
N-(4-cyano-3-phenyl-1H-pyrazol-5-yl)acetamide (23). The IR spectrum of 23 showed a carbonyl
band at 1685 cm-1. 1H NMR spectrum of 23 revealed two D2O exchangeable signals at δ 10.73
and 13.67 which were assigned to NH of COCH3 and NH of pyrazole protons, respectively.
Finally, diazotization of compound 3 yielded the corresponding diazonium chloride 24 which
was coupled with 3-oxo-3-phenylpropanenitrile (1) to give the corresponding hydrazone 25
(Scheme 3). The IR spectrum of 25 showed a carbonyl absorption band at 1650 cm-1. 1H NMR
spectrum of 25 revealed two D2O exchangeable signals at δ 9.61 and 9.91 which were assigned
to the protons of hydrazone NH and pyrazole NH, respectively.
8

O
CN
CN
NH2
Ph
O
CN
Ph
CN
N N Cl
24
Ph
O
19
i
iii
N
N
N
N
N
H
3
N
N
H
N
H
H
20
ii
3a
iv
CN
Ph
CN
Ph
CN
Ph
O
N
NH
N
N
N
N
NH
N
N
N
H
O
H
H
HN
23
O
21
CN
Ph
25
CN
NH
Ph
N
N
O
H2N
22
Scheme 3. Reagents and conditions: i) fusion, 45 min; ii) AcOH/reflux 2 h; iii)
AcOH/HCl/NaNO2/0-5oC; iv) CH3COONa.3H2O/abs. EtOH/stirring 4 h, 0-5oC.
2.2. Anticancer Activity
2.2.1. In vitro anti-proliferative activity against Huh-7, HeLa and MCF-7 cell lines
All synthesized pyrazolo[1,5-a]pyrimidines 11a-j, 16a-c, 22 in addition to N-(4-cyano-3-
phenyl-1H-pyrazol-5-yl)acetamide 23 and N-(4-cyano-3-phenyl-1H-pyrazol-5-yl)-2-oxo-2-
phenylacetohydrazonoyl cyanide 25 were examined for their antiproliferative activity towards
three cancer cell lines: hepatocellular carcinoma Huh-7, cervical adenocarcinoma HeLa and
breast adenocarcinoma MCF-7 cell lines. The MTT colorimetric assay was adopted to assess the
anti-proliferative activity as described by Mosmann [23]. Doxorubicin was used as a control in
this assay. The results were expressed as median growth inhibitory concentration (IC50) values
that represent the compound concentration required to produce a 50% inhibition of cell growth
after 24 h of incubation (Table 1). The results of the MTT assay listed in Table 1 suggested that
9

the examined compounds 11a-j, 16a-c, 22, 23 and 25 exhibited growth inhibitory activity against
the tested Huh-7, HeLa and MCF-7 cancer cell lines. Huh-7 and HeLa cells were found to be
more sensitive to the impact of the tested compounds than MCF-7 cells, except compound 11i
which is more effective towards MCF-7 cells. Compounds 11f, 16b and 11i emerged as the most
active compounds towards Huh-7, HeLa and MCF-7 cell lines with IC50 values equal 6.3, 7.8
and 3.0 µM, respectively, which are comparable to those of Doxorubicin (IC50 = 3.2, 8.1 and 5.9
µM, respectively). Regarding the activity of tested compounds against Huh-7 cells, compounds
16a and 23 displayed moderate antitumor activity with IC50 values in the range of 10.2–29.1 µM,
whereas the compounds 16b and 25 exhibited a significant potency towards Huh-7 cell line with
IC50 values 53.7 and 54.1 µM, respectively. On the other hand, investigation of the anti-
proliferative activity against HeLa cell line elucidated that 11c, 11i, 16a and 16c had moderate
anti-proliferative activity with IC50 values equal 25.3, 21.6, 15.5 and 26.2 µM, respectively.
Furthermore, compounds 11d, 11e, 11f, 11h, 11j, 22, 23 and 25 were significantly active
towards Huh-7 cells with IC50 values of 56.4, 39.6, 48.8, 60.2, 55.6, 62.6, 44.6 and 56.6 µM,
respectively. Finally, the anti-proliferative activity against MCF-7 cell line elucidated that 11a
and 11e were significantly active towards MCF-7 cells with IC50 values of 39.3 and 49.0 µM,
respectively.
10

Table 1. In vitro anti-proliferative activity of the synthesized compounds against Huh-7, HeLa and
MCF-7 cell lines
IC50 (μM)
Compound
Huh-7
77.5
109.5
123.6
75.2
75.2
6.3
88.4
70.9
95.0
82.6
29.1
54.1
65.6
83.1
10.2
53.7
3.2
HeLa
245.2
155.1
25.3
56.4
39.6
48.8
79.1
60.2
21.6
55.6
15.5
7.8
MCF-7
39.3
507.4
194.9
171.4
49.0
118.4
84.2
87.8
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
16a
16b
16c
22
3.0
100.5
133.8
106
26.2
62.6
44.6
56.6
8.1
70.1
189.3
247.0
71.2
23
25
DOX
5.9
2.2.2. In vitro anti-proliferative activity of 11f, 11i and 16b against MDA-MB231 cell lines
The promising results of compounds 11f, 11i and 16b against the tested cell lines
stimulate our interest to evaluate their antiproliferative potency against the MDA-MB231 cell
lines. Interestingly, compounds 11i and 16b showed potent antiproliferative activity with IC50
4.32 µM and 5.74 µM, respectively, when compared with that of Doxorubicin with IC50 = 6 µM
(Table 2).
Table 2. In vitro anti-proliferative activity of compounds 11f, 11i and 16b against MDA-MB231
cell lines.
Compound
11f
IC50 (μM)
74.25
4.32
11i
16b
5.74
DOX
6.0
11

2.2.3. Structure activity relationship (SAR)
Observing the results, we could deduce valuable data about the structure activity
relationship (SAR). Firstly, Huh-7 cell line on investigation of the impact of the substitution on
the pendant phenyl group in compounds 11a revealed that grafting a chloro group (11f, IC50 =
6.3 µM) is more beneficial for activity than fluoro and bromo group (11e, IC50 = 75.2 µM and
11g, IC50 = 88.4 µM, respectively) independent on the electronic displacement effect of Cl atom.
The effect of replacement of pyrimidine ring in pyrazolopyrimidine 11f (IC50 = 6.3 µ M) with N-
acetyl group in compound 23 (IC50 = 10.2 µM), resulted in remarkable decrease in the activity.
Also, introduce of cyano group in position 4 of pyrimdine ring in compounds 11a and 11c (IC50
= 77.5 and 25.3 µM, respectively) resulted in an increase of the anti-proliferative activity of
compounds 16a and 16b (IC50 = 29.1 and 6.8 µM, respectively) against Huh-7 and HeLa cell
lines respectively. In HeLa cell lines, the substitution on the pendant phenyl group with methoxy
function in compound 16b (IC50 = 6.8 µM) is more beneficial for activity than unsubstituted
phenyl group in compound 16a (IC50 15.5 = µM). In MCF-7 cell lines, the effect of replacement
of phenyl ring in compound 11a (IC50 = 39.3 µM) with more lipophilic naphthalene group
increase the activity in compound 11i, (IC50 = 3.0 µM) resulted in remarkable increase in the
activity. In MDA-MB231 cell lines, compound 11i with lipophilic naphthyl substitution and
compound 16b with 4-methoxyphenyl substitution showed potent antiproliferative activity (IC50
= 4.32 and 5.74 µM, respectively) whereas compound 11f with 4-chlorophenyl substitution
showed very low antiproliferative activity (IC50 = 74.25 µM) when compared with 11f or 16b.
2.2.4. Effect of compound 11f, 11i and 16b on cell cycle distribution of Huh-7, HeLa, MCF-
7 and MDA-MB231 cell lines after 48 h incubation
The ability of compounds 11f, 11i and 16b to induce cell cycle growth arrest was assessed
by using the PI/RNase staining solution for quantification of DNA content under each phase of the
cell cycle and subsequent analysis by flow cytometry. As shown in Figure 2, treatment of MCF-7
cells with compound 11i for 48 h induced a marked reduction in G1 phase, with significant arrest
in S phase as well as G2/M phase when compared to that of control untreated cells (P < 0.05). In
Figure 3, treatment of MDA-MB-231 cells with compound 11i for 48 h induced a significant arrest
in G1 phase, with marked reduction in S phase when compared to that of control untreated cells
(P < 0.05). Also, treatment of Hela cells with compound 16b for 48 h induced a marked reduction
12

in G1 phase, with significant arrest in G2/M phase when compared to that of control untreated
cells (Fig 4). Finally, treatment of Huh-7 cells with compound 11f for 48 h showed no change
when compared with that of control untreated cells (P < 0.05).
A
B
C
D
Figure 2. Cell cycle analysis in MCF-7 cells.Cells were subjected to cell cycle analysis using flow
cytometry. Representative histogram of the gated cells in the G0/G1, S, and G2/M phases for A:
control; B: Compound 11i; C: PTX. D: Quantitative analysis of distribution or proportion of the
cells in each phase was performed from at least 10,000 cells per sample. Each bar represents mean
± SEM of the data obtained from three independent experiments. *p < 0.05 vs. control
13

A
B
C
Figure 3. Cell cycle analysis in MDA-Mb-231 cells. Cells were subjected to cell cycle analysis
using flow cytometry. Representative histogram of the gated cells in the G0/G1, S, and G2/M
phases for A: control; B: Compound 11i. C: Quantitative analysis of distribution or proportion
of the cells in each phase was performed from at least 10,000 cells per sample. Each bar
represents mean ± SEM of the data obtained from three independent experiments. *p < 0.05 vs.
control.
14

A
B
C
Figure 4. Cell cycle analysis in Hela cells. Cells were subjected to cell cycle analysis using flow
cytometry. Representative histogram of the gated cells in the G0/G1, S, and G2/M phases for A:
control; B: Compound 16b. C: Quantitative analysis of distribution or proportion of the cells in
each phase was performed from at least 10,000 cells per sample. Each bar represents mean ±
SEM of the data obtained from three independent experiments. *p < 0.05 vs. control.
2.2.5. In-silico ADME study
Computer aided ADME study was performed by using the software: Swiss ADME tool.
These studies are based on the chemical structure of the molecule and involves the calculation of
certain parameters including; human gastrointestinal absorption (HIA) and blood-brain barrier
(BBB) permeation both consist in the readout of the BOILED-Egg model substrate or non-
15

substrate of the permeability glycoprotein (P-gp), interaction of molecules with cytochromes P450
(CYP) and bioavailability score also calculate the molecular properties of the ligands following
the Lipinski rule of five (5), molar refractivity, The total polar surface area (TPSA) and The
partition coefficient between n-octanol and water (log Po/w).
2.2.5.1. Results of the ADME study
The results of the ADME study are presented as BOILIED-EGG, which is a 2D plot drawn
using calculated TPSA and A WLOGP properties. Blood Brain Barrier (BBB) and Human
Intestinal Absorption (HIA) plots were drawn using all the compounds. (Fig 5).
In BBB plot, showed nine of the newly synthesized compounds fell inside the yolk which
may be able to penetrate the blood brain barrier and seven of these compounds fell outside the yolk
which may not be able to penetrate the blood brain barrier which indicate that the chances of CNS
side effects in these seven compounds are predicted to be low. In HIA plot, all the synthesized
compounds fell inside the white, which indicate all compounds have excellent human intestinal
absorption.
ADME Aqueous solubility level of most of the compounds was found to be moderate
solubility.
The CYP2D6 score predicts the inhibitory and non-inhibitory character of the given chemical
structure on Cytochrome P450 2D6 enzyme. Most of the compounds are predicted as inhibitors of
CYP2D6. The calculated parameters from the ADMET study are tabulated in (Table 3).
16

Figure 5. The BOILED-Egg chart for the synthesized compounds developed by swiss ADME.
Table 3. Computer aided ADME screening of the synthesized compounds:
Parameters
CPD
ID
GI Absorption
BBB
permea substra
tion te
Pg-p
CYP1
A2
inhibit
or
CYP2
C19
inhibit
or
CYP2
C9
inhibit
or
CYP2
D6
inhibit
or
CYP3
A4
inhibit permeatio
or n)
Logkp
(Skin
Bioavailability
score
11a
-5.71
High
High
High
High
High
High
High
High
High
High
High
High
High
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
11b
11c
11d
11e
11f
11g
11h
11i
-5.54
-5.92
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
No
No
No
No
No
No
Yes
Yes
No
No
No
Yes
Yes
Yes
Yes
Yes
No
-6.12
-5.75
-5.48
-5.71
-6.11
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
-5.13
-6.29
-6.07
-6.27
-6.64
11j
16a
16b
16c
Yes
Yes
Yes
Yes
No
No
No
17

22
23
25
High
High
High
No
No
No
No
No
No
Yes
Yes
Yes
No
No
Yes
No
No
Yes
No
No
No
No
No
No
-7.39
-6.5
0.55
0.55
0.55
-5.45
2.2.5.2. Prediction of toxicity
Protox-II server [24] was used to predict the organ toxicities and toxicological end points of
the ligands and their LD50. The integrated PubChem search (https:/pubchem.ncbi.nlm.nih.gov/)
was used to search for chemical structures using the compound names. The models to be used were
selected and the webserver computed the acute toxicity and toxicity targets selected.
The prediction of toxicity was based on 6 different targets linked to adverse drug-reactions.
The hepatotoxicity, carcinogenicity, immunotoxicity, mutagenicity and cytotoxicity of the
compounds were determined. All compounds are found to have hepatotoxicity except compound
11f. Compounds 11d, 11h, 22, 23 and 25 are found to be carcinogenic, also compounds 11h and
25 are found to have mutagenicity. The acute toxicity of the ligands was checked using PROTOX
-II server. All compounds with LD50 of 300 mg/kg have class 3 except compound (23 and 25) have
class 4 with LD50 of 600 mg/kg and 2000 mg/kg, respectively (Table 4).
Table 4. Toxicity prediction of the synthesized compounds
Predicted target
Compound
Hepatotoxicity Carcinogenicity Immunotoxicity Mutagenicity Cytotoxicity
Acute
toxicity
Class 3
Class 3
Class 3
Class 3
Class 3
Class 3
Class 3
Class 3
Class 3
Class 3
Class 3
11a
11b
11c
11d
11e
11f
11g
11h
11i
Active
Active
Active
Active
Active
Inactive
Active
Active
Active
Active
Active
Inactive
Inactive
Inactive
Active
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Active
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Active
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
11j
16a
18

16b
16c
22
Active
Active
Active
Active
Active
Inactive
Inactive
Active
Active
Active
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Active
Inactive
Inactive
Inactive
Inactive
Inactive
Class 3
Class 3
Class 3
Class 4
Class 4
23
25
3. Conclusion
A series of pyrazolo[1,5-a]pyrimidines 11a-j, 16a-c, 22 in addition to acetamide 23 and
hydrazone 25 were synthesized. Compounds 11f, 16b and 11i emerged as the most active
compounds towards Huh-7, HeLa and MCF-7 cell lines with IC50 = 6.3, 7.8 and 3.0 µM,
respectively. Compounds 11i and 16b showed potent antiproliferative activity with IC50 = 4.32 µM
and 5.74 µM, respectively, against the MDA-MB231 cell lines. Cell cycle analysis of 11i induced
a reduction in G1 phase with arrest in S phase and G2/M phase when treated with MCF-7 cells.
Compound 16b induced a reduction in G1 phase and arrest in G2/M phase when treated with Hela
cells. Compound 11i induced arrest in G1 phase and reduction in S phase when treated with MDA-
MB-231 cells.Taken together, these compounds may act as promising hits for further development
of new antiproliferative agents.
4. Experimental
4.1. Chemistry
Melting points (°C, uncorrected) were determined using a Stuart melting point apparatus.
The IR spectra (KBr) were recorded on a SHIMADZU FT/IR spectrometer. The NMR spectra
recorded by BRUKER 400 MHz NMR spectrometers use DMSO-d6 as solvent. Chemical shifts
were reported in parts per million (δ), and coupling constants (J) expressed in Hertz. TMS was
used as an internal standard and chemical shifts were measured in  ppm. 1H and 13C spectra were
run at 400 and 100 MHz, respectively. Electron impact mass spectra were measured on Direct
probe controller inlet part to single quadropole mass analyzer in THERMO SCIENTIFIC GCMS
model (ISQ LT) using THERMO X-CALIBUR SOFTWARE at the regional center for mycology
and biotechnology (RCMB) Al-azhar University, Naser city, Cairo.
19

4.1.1. Synthesis of 3-amino-2-benzoyl-4,4,4-trichlorobut-2-enenitrile (2)
To a stirred solution of 3-oxo-3-phenylpropanenitrile (1) (1.45 g, 10 mmol) in EtOH (20
mL) and in the presence of anhydrous AcONa (1.0 g), 3,3,3-trichloropropanenitrile (10 mmol,
1.57 g), was added. The resulting mixture was stirred for 6 h at rt., and then the solvent was
evaporated under vacuum to half of its volume. The reaction mixture was then poured into water.
The solid product obtained was collected by filtration and recrystallization from ethanol to give
compound 2 as pale yellow crystals in 80 % yield, mp 182oC (Let. mp 182-184oC, [25]).
4.1.2. 5-Amino-3-phenyl-1H-pyrazole-4-carbonitrile (3)
''A mixture of compound 2 (2.90 g, 10 mmol) and hydrazine hydrate (80%, 3 mL) in
dioxane (20 mL) was refluxed for 30 min. The reaction mixture is then allowed to cool at
ambient temperature. The precipitate was filtered off and recrystallized from ethanol from
ethanol to give compound 3. The physical properties of 3 were identical to those reported for
compound 3 as buff crystals in 86 % yield, mp 200oC (Let. mp 200-202oC, [25]).
4.1.3. Synthesis of 3-(dimethylamino)-1-arylprop-2-en-1-ones 6a-j
To a solution of aryl ethenone 4a-j (20 mmol) in xylene (50 mL), dimethylformamide-
dimethylacetal (5) (2.38 g, 20 mmol), was added and then the reaction was refluxed for 7 h. The
xylene was distilled off and the product was triturated with diethyl ether (5 mL). The resulting
solid was filtered and washed with cold petroleum ether to afford the pure compounds 6a-j,
respectively. The physical properties of 6a-j were identical to those reported [19-22].
4.1.4. Synthesis of 7-(aryl)-2-phenylpyrazolo[1,5-a]pyrimidine-3-carbonitrile 11a-j
A mixture of the appropriate enaminone 6a-j (10 mmol) and 5-amino-3-phenyl-1H-
pyrazole-4-carbonitrile (3) (1.84 g, 10 mmol) in acetic acid (25 mL) was refluxed for 3 h, then
left to cool. The solid product filtered off, washed with ethanol, dried and finally recrystallized
from dimethylformamide/H2O to afford the corresponding 11a-j, respectively.
4.1.4.1. 2,7-Diphenylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (11a)
20

White powder, 55% yield; mp 210-211oC; IR (KBr) νmax/cm-1 3150 (Aromatic CH), 2225
(C≡N), 1610 (C=N); 1H NMR  7.63-7.64 (m, 4H, H3, H3`, H4 of phenyl of pyrazole and H3 of
pyrimidine), 7.65-7.71 (m, 3H, H3, H3` and H4 of phenyl of pyrimidine), 8.04-8.07 (m, 2H, H2
and H2` of phenyl of pyrazole), 8.17-8.20 (m, 2H, H2 and H2` of phenyl of pyrimidine), 8.88 (d,
13
J = 5.6 Hz, 1H, H2 of pyrimidine); C NMR δ 79.02 (C4 of pyrazole), 111.59, 114.75, 127.56
(2C), 129.14 (2C), 129.72 (2C), 129.91 (2C), 130.27, 130.60, 131.08, 132.22, 147.67, 153.25,
154.10, 156.09. MS m/z (%) 297.24 (M++1, 22.21), 296.22 (M+, 100); Anal. Calcd. For: C19H12N4
(296.33): C, 77.01; H, 4.08; N, 18.91; Found: C, 76.89; H, 4.25; N, 19.17.
4.1.4.2. 2-Phenyl-7-(p-tolyl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile (11b)
White powder, 60% yield; mp 220-222oC; IR (KBr) νmax/cm-1 3100 (Aromatic CH), 2900
(Aliphatic CH), 2225 (C≡N), 1625 (C=N); 1H NMR (DMSO-d6)  2.44 (s, 3H, CH3), 7.44 (d, J =
8.8 Hz, 2H, H3, H3` of Aryl), 7.54-7.61 (m, 4H, H3, H3`, H4 of phenyl and H3 pyrimidine), 8.04-
8.05 (m, 2H, H2 and H2` of phenyl), 8.08 (d, J = 9.6 Hz, 2H, H2 and H2` of aryl), 8.83 (d, J = 4.4
Hz, 1H, H2 of pyrimidine); 13C NMR (DMSO-d6) δ 21.59 (-CH3), 78.87 (C4 of pyrazole), 111.11,
114.77, 126.95, 127.52 (2C), 129.69 (3C), 130.19 (2C), 130.62 (2C), 131.04, 142.52, 147.65,
153.29, 153.93, 155.97. MS m/z (%) 311.31 (M++1, 9.73), 310.27 (M+, 58.26), 77.19 (100), 51.16
(44.52); Anal. Calcd. For: C20H14N4 (310.35): C, 77.40; H, 4.55; N, 18.05; Found: C, 77.68; H,
4.69; N, 18.31.
4.1.4.3. 7-(4-Methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (11c)
Buff powder, 58% yield; mp 184-185oC; IR (KBr) νmax/cm-1 3100 (Aromatic CH), 2850
1
(Aliphatic CH), 2224 (C≡N), 1620 (C=N); H NMR  3.88 (s, 3H, OCH3), 7.16 (d, J = 6.8 Hz,
2H, H3, H3`of aryl), 7.52 (d, J = 6.8 Hz, 1H, H3 of pyrimidine), 7.55-7.60 (m, 3H, H3, H3` and
H4 of phenyl), 8.04-8.05 (m, 2H, H2 and H2` of phenyl), 8.22 (d, J = 9.6 Hz, 2H, H2, H2` of aryl),
13
8.78 (d, J = 5.6 Hz, 1H, H2 of pyrimidine); C NMR δ 56.01 (OCH3), 78.70 (C4 of pyrazole),
110.46, 114.57 (2C), 114.81, 121.68, 127.49 (2C), 129.64 (2C), 130.63, 131.01, 132.18 (2C),
147.22, 153.39, 153.67, 155.86, 162.48. MS m/z (%) 327.27 (M++1, 21.01), 326.27 (M+, 100),
77.14 (93.68); Anal. Calcd. For: C20H12N4O (326.35): C, 73.61; H, 4.32; N, 17.17; Found: C,
73.89; H, 4.60; N, 17.40.
21

4.1.4.4. 7-(3,4-Dimethoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (11d)
Buff powder, 62% yield; mp 220-221oC; IR (KBr) νmax/cm-1 3100 (Aromatic CH), 2850
1
(Aliphatic CH), 2226 (C≡N), 1620 (C=N); H NMR  3.88 (s, 3H, OCH3), 3.90 (s, 3H, OCH3),
7.21 (d, J = 6.8 Hz, 1H, H5 of aryl), 7.55-7.64 (m, 4H, H3, H3`, H4 of phenyl and H3 of
pyrimidine), 7.89 (s, 1H, H2 of aryl), 7.92 (d, J = 8.4 Hz, 1H, H6 of aryl), 8.07 (d, J = 8.4 Hz, 2H,
H2, H2` of phenyl), 8.81 (d, J = 6 Hz, 1H, H2 of pyrimidine); 13C NMR δ 56.19 (OCH3), 56.23
(OCH3), 78.66 (C4 of pyrazole), 110.64, 111.90, 113.50, 114.86, 121.66, 124.15, 127.44 (2C),
129.74 (2C), 130.69, 131.07, 147.27, 148.77, 152.31, 153.49, 153.69, 155.83. MS m/z (%) 357.30
(M++1, 25.77), 356.28 (M+, 100); Anal. Calcd. For: C21H16N4O2 (356.38): C, 70.77; H, 4.53; N,
15.72; Found: C, 70.85; H, 4.76; N, 15.94.
4.1.4.5. 7-(4-Fluorophenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (11e)
White powder, 65% yield; mp 250-252oC; IR (KBr) νmax/cm-1 3100 (Aromatic CH), 2230
(C≡N), 1600 (C=N); 1H NMR  7.49-7.54 (m, 2H, H3, H3` of aryl), 7.57-7.63 (m, 4H, H3, H3`,H4
of phenyl and H3 of pyrimidine), 8.05-8.07 (m, 2H, H2 and H2` of phenyl), 8.27-8.31 (m, 2H, H2
and H2` of aryl), 8.89 (d, J = 4.4 Hz, 1H, H2 of pyrimidine); 13C NMR δ 79.04 (C4 of pyrazole),
111.51, 114.72, 116.19, 116.41, 126.34, 127.56 (3C), 129.72 (3C), 130.57, 131.11, 133.03, 133.12,
146.67, 153.24, 154.06, 156.06. MS m/z (%) 315.22 (M++1, 20.92), 314.20 (M+, 100), 77.13
(95.33); Anal. Calcd. For: C19H11N4 (314.32): C, 72.60; H, 3.53; N, 17.83; Found: C, 72.84; H,
3.67; N, 18.19.
4.1.4.6. 7-(4-Chlorophenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (11f)
Buff powder, 78% yield; mp 271-273oC; IR (KBr) νmax/cm-1 3100 (Aromatic CH), 2225
(C≡N), 1625 (C=N); 1H NMR  7.59-7.62 (m, 4H, H3, H3`, H4 of phenyl and H3 of pyrimidine),
7.73 (d, , J = 9.2 Hz, 2H, H3, H3` of aryl), 8.04 (d, J = 8.8 Hz, 2H, H2 and H2` of phenyl), 8.21
(d, J = 8.8 Hz, 2H, H2 and H2` of aryl), 8.89 (d, J = 5.2 Hz, 1H, H2 of pyrimidine); 13C NMR δ
79.13 (C4 of pyrazole), 111.63, 115.01, 127.61 (3C), 129.29 (3C), 129.76 (3C), 131.16, 132.20
(3C), 137.04, 154.15, 187.96. MS m/z (%) 331.21 (M++1, 35.70), 330.18 (M+, 100);Anal. Calcd.
For: C19H11N4 (330.77): C, 68.99; H, 3.35; N, 16.94; Found: C, 69.21; H, 3.59; N, 17.12.
22

4.1.4.7. 7-(4-Bromophenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (11g)
Off white powder, 70% yield; mp 260-261oC; IR (KBr) νmax/cm-1 3100 (Aromatic CH),
1
2900 (Aliphatic CH), 2227 (C≡N), 1610 (C=N); H NMR  7.54-7.62 (m, 4H, H3, H3`, H4 of
phenyl and H3 of pyrimidine), 7.86 (d, J = 8 Hz, 2H, H3 and H3` of aryl), 8.05-8.06 (m, 2H, H2
and H2` of phenyl), 8.13 (d, J = 9.6 Hz, 2H, H2 and H2` of aryl), 8.88 (d, J = 6 Hz, 1H, H2 of
13
pyrimidine); C NMR δ 79.10 (C4 of pyrazole), 111.56, 114.69, 125.98, 127.56 (2C), 129.04,
129.72 (2C), 130.52, 131.13, 132.20 (2C), 132.28 (2C), 146.60, 153.20, 154.10, 156.05. MS m/z
(%) 376.19 (M++2, 93.50), 375.22 (M++1, 54.67), 374.21 (M+, 94.46), 77.13 (100);Anal. Calcd.
For: C19H11N4 (375.22): C, 60.82; H, 2.95; N, 14.93; Found: C, 61.06; H, 3.11; N, 15.20.
4.1.4.8. 7-(4-Nitrophenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (11h)
Pale yellow, 68% yield; mp 290-291oC; IR (KBr) νmax/cm-1 3100 (Aromatic CH), 2220
(C≡N), 1600 (C=N), 1350-1360 (NO2); 1H NMR  7.59-7.60 (m, 3H, H3, H3`and H4 of phenyl),
7.70 (d, J = 4.8 Hz, 1H, H3 of pyrimidine), 8.06-8.07 (m, 2H, H2 and H2` of phenyl), 8.42 (d, J
= 9.6 Hz, 2H, H2 and H2` of aryl), 8.47 (d, J = 8.8 Hz, 2H, H3 and H3` of aryl), 8.96 (d, J = 5.6
13
Hz, 1H, H2 of pyrimidine); C NMR δ 73.52, 79.35 (C4 of pyrazole), 112.40, 114.59, 124.08
(2C), 127.58, 129.74 (2C), 130.42, 131.20, 131.87 (C2), 135.89, 145.58, 149.42, 153.11, 154.26,
156.16. MS m/z (%) 342.33 (M++1, 16.29), 341.28 (M+, 100); Anal. Calcd. For: C19H11N5O2
(341.32): C, 66.86; H, 3.25; N, 20.52; Found: C, 67.12; H, 3.49; N, 20.36.
4.1.4.9. 7-(Naphthalen-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (11i)
Buff powder, 70% yield; mp 226-228oC; IR (KBr) νmax/cm-1 3100 (Aromatic CH), 2223
(C≡N), 1620 (C=N); 1H NMR  7.60-7.62 (m, 3H, H3, H3`and H4 of phenyl), 7.65-7.71 (m, 3H,
naphthalene Hs and H3 of pyrimidine), 8.03-8.07 (m, 3H, H2, H2` of phenyl and 1H of
naphthalene), 8.10-8.14 (m, 2H, naphthalene Hs), 8.20-8.23 (m, 1H, naphthalene H), 8.80 (s, 1H,
H1 of naphthalene), 8.90 (d, J = 5.6 Hz, 1H, H2 of pyrimidine); 13C NMR δ 79.05 (C4 of pyrazole),
111.84, 114.77, 126.33, 127.33, 127.56 (3C), 128.15, 128.46, 128.84, 129.53 (2C), 129.71, 130.60,
131.07, 131.11, 132.65, 134.50, 147.58, 153.29, 154.02, 156.10. MS m/z (%) 347.27 (M++1,
25.61), 346.25 (M+, 100), 345.25 (60.43); Anal. Calcd. For: C23H14N4 (346.38): C, 79.75; H, 4.07;
N, 16.17; Found: C, 79.91; H, 4.23; N, 16.45.
23

4.1.4.10. 7-(Furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidine-3-carbonitrile (11j)
Brown powder, 62% yield; mp 204-206oC; IR (KBr) νmax/cm-1 3150 (Aromatic CH), 2225
(2C≡N), 1575 (C=N); 1H NMR  6.97 (s, 1H, H4 of furan), 7.58-7.65 (m, 3H, H3, H3` and H4 of
phenyl), 7.68 (d, J = 6.4 Hz, 1H, H3 of pyrimidine), 8.18 (d, J = 5.6 Hz, 2H, H2 and H2` of
phenyl), 8.26 (s, 2H, H3 and H5 of furan), 8.82 (d, J = 4.4 Hz, 1H, H2 of pyrimidine); 13C NMR
δ 78.83 (C4 of pyrazole), 106.13, 114.38, 114.71, 121.84, 127.57 (2C), 129.79 (2C), 130.47,
131.25, 136.46, 142.71, 148.90, 152.83, 153.27, 156.32. MS m/z (%) 287.21 (M++1, 14.93),
286.21 (M+, 66.07), 77.14 (100); Anal. Calcd. For: C17H10N4O (286.29): C, 71.32; H, 3.52; N,
19.57; Found: C, 71.08; H, 3.80; N, 19.71.
4.1.5. Synthesis of 2-Aryl-3-(dimethylamino)acrylonitrile 12a-c
To a mixture of 3-(aryl)-3-oxopropanenitrile 3a-c (20 mmol), in xylene (50 mL), was added
dimethylformamide-dimethylacetal (2.38 g, 20 mmol) and the reaction refluxed for 1 h. The xylene
was distilled off and the product was triturated with petroleum ether. The resulting solid was
filtered and washed with cold petroleum ether to afford the pure compounds 12a-c, respectively.
The physical properties of 12a-c were identical to those reported [25-26]).
4.1.6. Synthesis of 2-Phenyl-7-(aryl)pyrazolo[1,5-a]pyrimidine-3,6-dicarbonitrile 16a-c
A mixture of the appropriate enaminone 12a-c (10 mmol) and 5-amino-3-phenyl-1H-
pyrazole-4-carbonitrile (3) (1.84 g, 10 mmol) in acetic acid (25 mL) was refluxed for 3 h, then
left to cool. The solid product filtered off, washed with ethanol, dried and finally recrystallized
from dimethylformamide/H2O to give the corresponding 2-phenyl-7-(aryl)pyrazolo[1,5-
a]pyrimidine-3,6-dicarbonitriles 16a-c, respectively.
4.1.6.1. 2,7-Diphenylpyrazolo[1,5-a]pyrimidine-3,6-dicarbonitrile (16a)
Yellow powder, 42% yield; mp 229-230oC; IR (KBr) νmax/cm-1 3100 (Aromatic CH), 2240
1
(2C≡N), 1610 (C=N); H NMR  7.59-7.64 (m, 3H, H3, H3`and H4 of phenyl of pyrimidine),
7.71-7.80 (m, 3H, H3, H3` and H4 of phenyl of pyrazole), 7.99-8.01 (m, 4H, H2, H2` of phenyl
13
of pyrazole, H2 and H2` of phenyl of pyrimidine), 9.25 (s, 1H, H2 of pyrimidine); C NMR δ
81.30 (C4 of pyrazole), 98.47 (C3 of pyrimidine), 113.79, 115.49, 127.27, 127.65 (2C), 129.17,
24

129.36 (2C), 129.50, 129.74, 129.88, 130.73, 131.70, 133.14, 153.09, 153.29, 154.87, 157.98;
Anal. Calcd. For: C20H11N5 (321.33): C, 74.76; H, 3.45; N, 21.79; Found: C, 74.90; H, 3.66; N,
21.57.
4.1.6.2. 7-(4-Methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-3,6-dicarbonitrile (16b)
Yellow powder, 40% yield; mp > 300oC; IR (KBr) νmax/cm-1 3400 (Aromatic CH), 2900
(Aliphatic CH), 2230 (2C≡N), 1520 (C=N); 1H NMR  3.92 (s, 3H, OCH3), 7.24 (d, J = 8.8 Hz,
2H, H3 and H3` of aryl), 7.63-7.66 (m, 3H, H3, H3` and H4 of phenyl), 7.87-7.89 (m, 2H, H2 and
H2` of phenyl), 8.15 (d, J = 7.4 Hz, 2H, H2 and H2` of aryl), 9.04 (s, 1H, H2 of pyrimidine). MS
m/z (%) 351.12 (M+, 7.60), 77.07 (100); Anal. Calcd. For: C21H13N5O (351.36): C, 71.79; H, 3.73;
N, 19.93; Found: C, 72.04; H, 3.87; N, 20.16.
4.1.6.3. 7-(Furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidine-3,6-dicarbonitrile (16c)
Brown powder, 69% yield; mp 265-267oC; IR (KBr) νmax/cm-1 3200 (Aromatic CH), 2230
(2C≡N), 1610 (C=N); 1H NMR  7.11 (d, J = 4 Hz, 1H, H4 of furan), 7.60-7.66 (m, 3H, H3, H3`
and H4 of phenyl), 8.18-8.19 (m, 2H, H2 and H2` of phenyl), 8.49 (s, 1H, H3 of furan), 8.55-8.57
(d, J = 4.4 Hz, 1H, H5 of furan), 9.11 (s, 1H, H2 of pyrimidine); 13C NMR δ 80.93 (C4 of pyrazole),
92.26 (C3 of pyrimidine), 113.81, 115.30, 115.92, 127.10, 127.69 (2C), 129.68, 129.95 (2C),
131.84, 139.09, 141.31, 150.76, 153.03, 155.43, 157.61. MS m/z (%) 312.17 (M++1, 22.68),
311.15 (M+, 100); Anal. Calcd. For: C18H9N5O (311.30): C, 69.45; H, 2.91; N, 22.50; Found: C,
69.18; H, 3.24; N, 22.37.
4.1.7. Synthesis of 7-Amino-5-oxo-2-phenyl-4,5-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile
(22)
A mixture of compound 3 (0.18 g, 1 mmol) and ethyl cyanoacetate (19) (0.113 g, 1 mmol)
was heated to reflux for 45 min. and then left to cool at r.t. Ethanol (5 mL) was added, the solid
separated was collected by filtration, washed with ethanol, and finally recrystallized from
EtOH/DMF to afford compound 22 as light brown powder, 75% yield; mp > 300oC; IR (KBr)
νmax/cm-1 3150-3400 (NH, NH2), 3100 (Aromatic CH), 2220 (C≡N), 1680 (C=O), 1630 (C=N); 1H
NMR  5.41 (s, 1H, CH pyrimidine), 7.51-7.59 (m, 3H, H3, H3` and H4 of phenyl), 7.77 (s, D2O
exchangeable, 2H, NH2), 8.01-8.03 (m, 2H, H2 and H2` of phenyl), 12.07 (br. s, D2O
25

13
exchangeable, 1H, NH); C NMR δ 77.82 (C4 of pyrazole), 115.46, 127.27 (3C), 129.42 (3C),
130.53, 131.09, 150.21, 154.20, 164.90. MS m/z (%) 252.18 (M++1, 2.45), 251.19 (M+, 10.87),
68.11 (100); Anal. Calcd. For: C13H9N5O (251.24): C, 62.15; H, 3.61; N, 27.87; Found: C, 62.37;
H, 3.89; N, 27.61.
4.1.8. Synthesis of N-(4-cyano-3-phenyl-1H-pyrazol-5-yl)acetamide (23)
A solution of compound 3 (1.84 g, 10 mmol) in acetic acid (25 mL) was refluxed for 2 h, and
then left to cool. The solid product filtered off, washed with ethanol, dried and finally recrystallized
from DMF/H2O to gave the corresponding N-(4-cyano-3-phenyl-1H-pyrazol-5-yl)acetamide (23)
as buff powder in 40% yield; mp 290-293oC; IR (KBr) νmax/cm-1 3225, 3235 (2NH), 3150
(Aromatic CH), 2900 (Aliphatic CH), 2228 (C≡N), 1700 (C=O), 1620 (C=N); 1H NMR  2.10 (s,
3H, CH3), 7.50-7.59 (m, 3H, H3, H3`and H4 of phenyl), 7.82-7.84 (m, 2H, H2 and H2` of phenyl),
10.73 (br. s, D2O exchangeable, 1H, NHCOMe), 13.67 (br. s, D2O exchangeable, 1H, NH of
13
pyrazole); C NMR δ 23.15 (CH3), 114.79, 126.75 (3C), 129.67 (3C), 130.31, 169.42, 196.70.
MS m/z (%) 227.21 (M++1, 1.07), 226.18 (M+, 21.04), 77.15 (100); Anal. Calcd. For: C12H10N4O
(226.23): C, 63.71; H, 4.46; N, 24.76; Found: C, 63.54; H, 4.70; N, 24.89.
4.1.9. Synthesis of N-(4-cyano-3-phenyl-1H-pyrazol-5-yl)-2-oxo-2-phenylacetohydrazonoyl
cyanide (25)
A solution of 5-amino-3-phenyl-1H-pyrazole-4-carbonitrile (3) (1.84 g, 10 mmol) in glacial
acetic acid (10 mL) was cooled to 0-5ºC, then hydrochloric acid (10 mL) was added. A solution
of sodium nitrite (0.6 g, 10 mmol) in water (5 ml) was then gradually added with stirring. The
reaction mixture was left in an ice chest for 15 min. The latter solution of dizonium chloride (24)
is used in the next coupling reactions directly. To a cold solution of 2-benzoylacetonitrile (1) (1.45
g 10 mmol) and sodium acetate trihydrate (3.0 g) in ethanol (50 mL), the diazonium salt 24 was
added. The addition was carried out portion-wise with stirring at 0–5°C over a period of 30 min.
After complete addition, the reaction mixture was stirred for further 4 h, then kept in an ice-chest
for 12 h and finally diluted with water. The product was filtered off, washed with water, dried and
recrystallised from EtOH/DMF to give the corresponding hydrazone 25 as buff powder in 80%
yield; mp 240-243oC; IR (KBr) νmax/cm-1 3200, 3300 (2NH), 3100 (Aromatic CH), 2225, 2235
1
(2C≡N), 1650 (C=O), 1560 (C=N); H NMR  7.55-7.64 (m, 2H, H4 of phenyl of pyrazole and
26

H4 of ph-CO), 7.65-7.70 (m, 4H, H3, H3` of phenyl of pyrazole, H3 and H3` of phenyl of
hydrazone), 7.95-7.97 (m, 2H, H2 and H2` of phenyl of pyrazole), 8.20-8.22 (m, 2H, H2 and H2`
of phenyl of hydrazone), 9.61 (s, 1H, NH of hydrazone), 9.91 (s, 1H, NH of pyrazole); 13C NMR
δ 78.97 (C4 of pyrazole), 114.08, 127.84 (2C), 128.40, 129.72 (2C), 129.80, 129.89 (2C), 130.95
(2C), 131.73, 132.76, 138.22, 142.38, 151.56, 157.24, 193.44. MS m/z (%) 341.23 (M++1, 2.90),
340.24 (M+, 12.56), 77.14 (100); Anal. Calcd. For: C19H12N6O (340.35): C, 67.05; H, 3.55; N,
24.69; Found: C, 67.31; H, 3.73; N, 24.41.
4.2. Biological Evaluation
4.2.1. In vitro anti-proliferative activity against Huh-7, HeLa and MCF-7 cell lines
Huh-7 (Hepatocellular Carcinoma) cells, HeLa (cervical adenocarcinoma) cells and MCF-
7 (breast adenocarcinoma) cells were used to evaluate the potential cytotoxicity of the tested
compounds using the method described by Tim Mosmann [23]. Briefly, in flat bottom 96 well-
microplates, cells (0.5×105) were cultured in 180 μl/well RPMI media supplemented with 10%
fetal bovine serum, 2μmol/ml L-glutamine, 250 ng/ml fungizone, 100 units/ml penicillin
streptomycin solutions at 37°C in a CO2 incubator. The plates were incubated for 24 h at 37°C in
a humidified 5% CO2 atmosphere to allow cells to settle down. After incubation, cells were then
treated with different concentrations of tested compounds or doxorubicin (DOX) as a standard
drug (100 – 0.8 μM) and incubated for 24 h at 37 °C, in atmosphere of 5% CO2. After incubation,
the media were removed and MTT solution 40 µl/well was added and incubated for additional 4
h. MTT crystals were solubilized by adding acidified isopropanol (160 µl/well) and the plate was
shaken at room temperature. This was followed by photometric determination of the absorbance
at 570 nm using the microplate ELISA reader (FLUOstar Omega, BMG, Labtech, Germany). The
IC50 was determined by using a program Graph-Pad PRISM version 5.
4.2.2. In vitro anti-proliferative activity of 11f, 11i and 16b against MDA-MB231 cell lines
MDA-MB231 cells were cultured in Medium Human breast cancer MCF-7 and colon
cancer Caco-2 cell lines were maintained in RPMI-1640 supplemented with 100 µg/mL
27

streptomycin, 100 units/mL penicillin and 10% heat-inactivated fetal bovine serum in a
humidified, 5% (v/v) CO2 atmosphere at 37 ºC.
The tested agent was dissolved in DMSO and kept at a stock concentration of 100 mM.
Cell seeding was done at a density of 2000 cells/ well in 96-well plates. Cells were exposed to
different treatments for 48 h during which five different drug concentrations were tested.
Cytotoxicity was assessed at the end of drug exposure using SRB assay as previously
described[27]. Absorbance was measured at 545 nm using microplate reader (BioTek
instruments, Vermont, USA). Results were expressed as the relative percentage of absorbance
compared to control. Experiments were done in triplicates. Half-maximal inhibitory
concentration (IC50), the drug concentration at which 50% growth inhibition is achieved, was
calculated using GraphPad Prism software, version 5.00 (GraphPad Software, Inc. La Jolla, CA,
USA).
4.2.3. In vitro Cell cycle distribution against Huh-7, HeLa and MCF-7 cell lines
After treatment with test compounds for 48 h and paclitaxel (PTX) 1 µM for 24 h as positive
control, cells (105 cells) are collected by trypsinization and washed twice with ice-cold PBS (pH
7.4). Cells are re-suspended in two milliliters of 60% ice-cold ethanol and incubated at 4ºC for 1
h for fixation. Fixed cells are washed twice again with PBS (pH 7.4) and resuspended in 1 mL of
PBS containing 50 µg/mL RNAase A and 10 µg/mL propidium iodide (PI). After 20 min of
incubation in dark at 37 C, cells are analyzed for DNA contents using flow cytometry analysis
using FL2 (λex/em 535/617 nm) signal detector (ACEA Novocyte™ flowcytometer, ACEA
Biosciences Inc., San Diego, CA, USA). For each sample, 12,000 events are acquired. Cell cycle
distribution is calculated using ACEA NovoExpress™ software (ACEA Biosciences Inc., San
Diego, CA, USA).
28

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