An Efficient Synthesis of Novel Pyrazole-Based Heterocycles as Potential Antitumor Agents

Abstract

A new series of pyrazolylpyridines was prepared by reaction of ethyl-3-acetyl-1,5-diphenyl-1H-pyrazole-4-carboxylate with the appropriate aldehyde, malononitrile, or ethyl acetoacetate and an excess of ammonium acetate under reflux in acetic acid. Similarly, two novel bipyridine derivatives were prepared by the above reaction using terephthaldehyde in lieu of benzaldehyde derivatives. In addition, a series of 1,2,4-triazolo[4,3-a]pyrimidines was synthesized by a reaction of 6-(pyrazol-3-yl)pyrimidine-2-thione with a number of hydrazonoyl chlorides in dioxane and in the presence of triethylamine. The structure of the produced compounds was established by elemental analyses and spectral methods, and the mechanisms of their formation was discussed. Furthermore, the pyrazolyl-pyridine derivatives were tested as anticancer agents and the results obtained showed that some of them revealed high activity against human hepatocellular carcinoma (HEPG2) cell lines.

Full text

applied
sciences
Article
An Efficient Synthesis of Novel Pyrazole-Based
Heterocycles as Potential Antitumor Agents
Magda A. Abdallah 1, Sobhi M. Gomha 1, *ID , Ikhlass M. Abbas 1, Mariam S. H. Kazem 2,
Seham S. Alterary 3and Yahia N. Mabkhot 3,*ID
1Department of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt;
mkamalh2009@yahoo.com (M.A.A.); son2karim@gmail.com (I.M.A.)
2Department of Chemistry, Faculty of Dentistry, October University for Modern Science & Arts,
Giza 12613, Egypt; mariamkazem@hotmail.com
3Department of Chemistry, College of Science, King Saud University, P.O. Box 2455,
Riyadh 11451, Saudi Arabia; salterary@ksu.edu.sa
*Correspondence: s.m.gomha@gmail.com (S.M.G.); yahia@ksu.edu.sa (Y.N.M.);
Tel.: +20-237-400-304 (S.M.G.); +966-11-467-5898 (Y.N.M.);
Fax: +20-025-685-799 (S.M.G.); +966-11-467-5992 (Y.N.M.)
Received: 22 July 2017; Accepted: 31 July 2017; Published: 3 August 2017
Abstract:
A new series of pyrazolylpyridines was prepared by reaction of ethyl-3-acetyl-1,5-diphenyl-
1H-pyrazole-4-carboxylate with the appropriate aldehyde, malononitrile, or ethyl acetoacetate
and an excess of ammonium acetate under reflux in acetic acid. Similarly, two novel bipyridine
derivatives were prepared by the above reaction using terephthaldehyde in lieu of benzaldehyde
derivatives. In addition, a series of 1,2,4-triazolo[4,3-a]pyrimidines was synthesized by a reaction of
6-(pyrazol-3-yl)pyrimidine-2-thione with a number of hydrazonoyl chlorides in dioxane and in the
presence of triethylamine. The structure of the produced compounds was established by elemental
analyses and spectral methods, and the mechanisms of their formation was discussed. Furthermore,
the pyrazolyl-pyridine derivatives were tested as anticancer agents and the results obtained showed
that some of them revealed high activity against human hepatocellular carcinoma (HEPG2) cell lines.
Keywords:
pyrazoles; pyridines; multicomponent reactions; pyrazolyl-pyridines; antitumor activity
1. Introduction
A literature survey revealed that compounds, including the pyrazole nucleus, are extensively
used as a precursor for the synthesis of compounds presenting many applications, such as electrolyte
additives in batteries [
1
], catalysis [
2
], photographic materials [
3
], agrochemicals [
4
], and dyes [
5
].
The chemical versatility of the pyrazole and its analogues has attracted interest because it allows a
range of applications in the pharmaceutical industry. Many pyrazole-derived compounds are known
to exhibit anticancer [
6
–
10
], antimicrobial [
11
,
12
], antiviral [
13
], antiparasitic [
14
], anti-inflammatory [
15
,
16
],
antipyretic [
17
], analgesic [
18
], anticoagulant [
19
], and anti-obesity [
20
] biological activities. The
pyridine nucleus is a key constituent, present in a range of bioactive compounds, occurring both
synthetically and naturally with wide range of biological applications [
21
,
22
]. Among the successful
examples as drug candidates possessing pyridine nuclei are streptonigrin, streptonigrone, and
lavendamycin, which are described in the literature as anticancer drugs. Some pyridine derivatives
were studied for their topoisomerase inhibitory activity and cytotoxicity against several human cancer
cell lines for the development of novel anticancer agents. As a result, it has been reported that various
pyridine derivatives, as bioisosteres of
α
-terthiophene (potent protein kinase C inhibitor) [
23
], have
significant topoisomerase I and/or II inhibitory activity, and cytotoxicity against several human cancer
cell lines [
24
–
28
]. Early reports on the ability of
α
-terpyridine to form metal complexes [
29
] and to
Appl. Sci. 2017,7, 785; doi:10.3390/app7080785 www.mdpi.com/journal/applsci

Appl. Sci. 2017,7, 785 2 of 13
bind with DNA/RNA [
30
] have been the base for the study on pyridine derivatives as antitumor
agents. On the other hand, multicomponent reactions (MCRs) are powerful tools in modern medicinal
chemistry, facilitating the lead generation by providing access to drug-like compounds, helping in
drug discovery [
31
–
33
]. Additionally, the utility of MCR under microwave irradiation in the synthesis
of heterocyclic compounds enhanced the reaction rates and improved the regioselectivity [
34
,
35
].
Over the last decade, several research groups adopted a hybridization approach for the design of
pyrazole-pyridine hybrid analogs and illuminated their synthetic and medicinal importance [36–42].
In light of the above findings and in continuation of our efforts to synthesize new anticancer
compounds [
43
–
52
], the aim of presented report is to synthesize a new series of pyrazolyl-pyridines
via multicomponent reactions which are expected to be active as antitumor agents.
2. Results and Discussion
2.1. Chemistry
Ethyl 3-acetyl-1,5-diphenyl-1H-pyrazole-4-carboxylate (
1
) [
53
] was used as the starting compound
for the preparation of a number of novel pyrazolyl-pyridine derivatives via one-pot multicomponent
reactions. For example, a series of novel 2-amino-3-cyano-6-(pyrazol-3-yl)-pyridines
4a
–
f
was prepared
by a one-pot reaction of 3-acetylpyrazole derivative
1
with the appropriate aldehyde
2
, malononitrile
3
, and ammonium acetate under reflux in acetic acid (Scheme 1). Both elemental analyses and spectral
data were used to elucidate the structures of the products
4a
–
f
. The IR spectra of compounds
4a
–
f
revealed in each case three absorption bands in the regions
υ
3431–3211, 2218–2210, 1715–1709 cm
−1
attributed to the NH
2
, CN and C=O groups. The
1
HNMR spectrum of compound
4a
taken as a
typical example of the products
4
, revealed two signals at
δ
= 6.93 (brs, 2H, NH
2
) and 8.11 (s, 1H,
pyridine-H5), in addition to the expected signals for the aryl and ester protons. Moreover, the mass
spectra of product
4
showed in each case the respective molecular ion peak which is consistent with
the assigned structure.
Appl. Sci. 2017, 7, 785 2 of 13
and to bind with DNA/RNA [30] have been the base for the study on pyridine derivatives as
antitumor agents. On the other hand, multicomponent reactions (MCRs) are powerful tools in
modern medicinal chemistry, facilitating the lead generation by providing access to drug-like
compounds, helping in drug discovery [31–33]. Additionally, the utility of MCR under microwave
irradiation in the synthesis of heterocyclic compounds enhanced the reaction rates and improved the
regioselectivity [34,35]. Over the last decade, several research groups adopted a hybridization
approach for the design of pyrazole-pyridine hybrid analogs and illuminated their synthetic and
medicinal importance [36–42].
In light of the above findings and in continuation of our efforts to synthesize new anticancer
compounds [43–52], the aim of presented report is to synthesize a new series of pyrazolyl-pyridines
via multicomponent reactions which are expected to be active as antitumor agents.
2. Results and Discussion
2.1. Chemistry
Ethyl 3-acetyl-1,5-diphenyl-1H-pyrazole-4-carboxylate (1) [53] was used as the starting compound
for the preparation of a number of novel pyrazolyl-pyridine derivatives via one-pot multicomponent
reactions. For example, a series of novel 2-amino-3-cyano-6-(pyrazol-3-yl)-pyridines 4a–f was prepared
by a one-pot reaction of 3-acetylpyrazole derivative 1 with the appropriate aldehyde 2, malononitrile 3,
and ammonium acetate under reflux in acetic acid (Scheme 1). Both elemental analyses and spectral
data were used to elucidate the structures of the products 4a–f. The IR spectra of compounds 4a–f
revealed in each case three absorption bands in the regions υ 3431–3211, 2218–2210, 1715–1709 cm−1
attributed to the NH2, CN and C=O groups. The 1HNMR spectrum of compound 4a taken as a typical
example of the products 4, revealed two signals at δ = 6.93 (brs, 2H, NH2) and 8.11 (s, 1H, pyridine-H5),
in addition to the expected signals for the aryl and ester protons. Moreover, the mass spectra of
product 4 showed in each case the respective molecular ion peak which is consistent with the
assigned structure.
Scheme 1. Synthesis of pyridine derivatives 4a–f.
In a similar manner, another series of pyrazolylpyridines 6a–f was synthesized using ethyl
acetoacetate in lieu of malononitrile. Thus, the reaction of 3-acetylpyrazole derivative 1 with the
appropriate aldehyde 2, ethyl acetoacetate 5, and ammonium acetate in refluxing acetic acid afforded
the corresponding products 6a–f (Scheme 2). The structure 6 assigned for the obtained products was
established by elemental analyses and spectral (IR, 1HNMR, and MS) data. For example, the IR
spectra of products 6a–f revealed, in each case, four absorption bands assigned for the three carbonyl
groups and the -NH group of the pyridinone ring (see Section 3). The 1HNMR spectra displayed three
singlet signals near δ 2.58, 9.80 and 7.79 ppm attributed to the acetyl, NH and pyridinyl-5H protons,
in addition to the expected signals due to the ester and aryl protons (see Section 3).
Scheme 1. Synthesis of pyridine derivatives 4a–f.
In a similar manner, another series of pyrazolylpyridines
6a
–
f
was synthesized using ethyl
acetoacetate in lieu of malononitrile. Thus, the reaction of 3-acetylpyrazole derivative
1
with the
appropriate aldehyde
2
, ethyl acetoacetate
5
, and ammonium acetate in refluxing acetic acid afforded
the corresponding products
6a
–
f
(Scheme 2). The structure
6
assigned for the obtained products
was established by elemental analyses and spectral (IR,
1
HNMR, and MS) data. For example, the IR
spectra of products
6a
–
f
revealed, in each case, four absorption bands assigned for the three carbonyl
groups and the -NH group of the pyridinone ring (see Section 3). The
1
HNMR spectra displayed three
singlet signals near
δ
2.58, 9.80 and 7.79 ppm attributed to the acetyl, NH and pyridinyl-5H protons,
in addition to the expected signals due to the ester and aryl protons (see Section 3).

Appl. Sci. 2017,7, 785 3 of 13
Appl. Sci. 2017, 7, 785 3 of 13
Scheme 2. Synthesis of pyridine derivatives 6a–f.
To account for the formation of products 4 and 6, it was suggested that the reaction proceeds by
condensation of the acetyl group of Compound 1 with the aldehyde to give the corresponding
chalcone which reacts with ammonium acetate to give the imino derivative, followed by tandem Michael
addition of the active methylene group of 3 (or 5) to afford the non-isolable tetrahydropyridine
intermediates A (or B). The latter undergo in situ auto-oxidation (followed by tautomerization in case
of A) and formation of the final products 4 (or 6) (Scheme 3).
Scheme 3. Mechanism of the synthesis of pyridine derivatives 4a–f and 6a–f.
Our study was extended to prepare another new bipyridine derivatives including the pyrazole
moiety via multi-component reaction. Thus, the reaction of 3-acetylpyrazole derivative 1 with
terephthaldehyde 7, malononitrile 3, and ammonium acetate in acetic acid under reflux furnished the
bipyridine derivative 8 (Scheme 4).
Similarly, the reaction of compound 1 with terephthaldehyde, ethyl acetoacetate 5, and ammonium
acetate in acetic acid under reflux gave the respective bipyridinone 9 (Scheme 4). The structure
of products 8 and 9 were confirmed by elemental analyses and spectral data (IR, 1HNMR, and MS)
(see Section 3).
Scheme 2. Synthesis of pyridine derivatives 6a–f.
To account for the formation of products
4
and
6
, it was suggested that the reaction proceeds
by condensation of the acetyl group of Compound
1
with the aldehyde to give the corresponding
chalcone which reacts with ammonium acetate to give the imino derivative, followed by tandem
Michael addition of the active methylene group of
3
(or
5
) to afford the non-isolable tetrahydropyridine
intermediates
A
(or
B
). The latter undergo in situ auto-oxidation (followed by tautomerization in case
of A) and formation of the final products 4(or 6) (Scheme 3).
Appl. Sci. 2017, 7, 785 3 of 13
Scheme 2. Synthesis of pyridine derivatives 6a–f.
To account for the formation of products 4 and 6, it was suggested that the reaction proceeds by
condensation of the acetyl group of Compound 1 with the aldehyde to give the corresponding
chalcone which reacts with ammonium acetate to give the imino derivative, followed by tandem Michael
addition of the active methylene group of 3 (or 5) to afford the non-isolable tetrahydropyridine
intermediates A (or B). The latter undergo in situ auto-oxidation (followed by tautomerization in case
of A) and formation of the final products 4 (or 6) (Scheme 3).
Scheme 3. Mechanism of the synthesis of pyridine derivatives 4a–f and 6a–f.
Our study was extended to prepare another new bipyridine derivatives including the pyrazole
moiety via multi-component reaction. Thus, the reaction of 3-acetylpyrazole derivative 1 with
terephthaldehyde 7, malononitrile 3, and ammonium acetate in acetic acid under reflux furnished the
bipyridine derivative 8 (Scheme 4).
Similarly, the reaction of compound 1 with terephthaldehyde, ethyl acetoacetate 5, and ammonium
acetate in acetic acid under reflux gave the respective bipyridinone 9 (Scheme 4). The structure
of products 8 and 9 were confirmed by elemental analyses and spectral data (IR, 1HNMR, and MS)
(see Section 3).
Scheme 3. Mechanism of the synthesis of pyridine derivatives 4a–fand 6a–f.
Our study was extended to prepare another new bipyridine derivatives including the pyrazole
moiety via multi-component reaction. Thus, the reaction of 3-acetylpyrazole derivative
1
with
terephthaldehyde
7
, malononitrile
3
, and ammonium acetate in acetic acid under reflux furnished the
bipyridine derivative 8(Scheme 4).
Similarly, the reaction of compound
1
with terephthaldehyde, ethyl acetoacetate
5
, and ammonium
acetate in acetic acid under reflux gave the respective bipyridinone
9
(Scheme 4). The structure of
products
8
and
9
were confirmed by elemental analyses and spectral data (IR,
1
HNMR, and MS)
(see Section 3).

Appl. Sci. 2017,7, 785 4 of 13
Appl. Sci. 2017, 7, 785 4 of 13
Scheme 4. Synthesis of bipyridine derivatives 8 and 9.
On the other hand, chalcone 10, prepared by the reaction of 1 with benzaldehyde in ethanol
containing catalytic amounts of NaOH [54], was used for preparation of 6-(pyrazol-3-yl) pyrimidine-
2-thione derivative 11 via its reaction with thiourea in ethanol containing a catalytic amount of
sodium hydroxide [54]. Reaction of the latter compound 11 with a number of hydrazonoyl chlorides
12a–h [55] in dioxane in the presence of triethylamine afforded the respective products 15a–h through
the non-isolated intermediates 13 and 14 (Scheme 5). The structure assigned for the products 15 was
established via microanalytical and spectral data (see Section 3). For example, the IR spectra of
product 15 revealed the absence of the pyrimidinyl-NH groups, and instead showed two absorption
bands near υ 1706 and 1649 cm−1 assigned for the two carbonyl groups. Additionally, 1HNMR spectra
of product 15 showed the absence of the signals attributed to the pyrimidinyl-NH protons and,
instead, revealed the signals assigned for the acetyl protons (for 15a–d) or the ethoxycarbonyl protons
(for 15e–h), in addition to the characteristic signals due to the ester and aromatic protons (see Section 3).
The mass spectra of product 15 showed, in each case, the respective molecular ion peak, which is
consistent with the assigned structure.
Scheme 5. Synthesis of 1,2,4-triazolo[4,3-a]pyrimidines 15a–h.
2.2. Antitumor Activity
The cytotoxicity of the synthesized pyridines 4a,b,e and 6a,b,e was evaluated against the human
liver carcinoma cell line (HepG2-1) using doxorubicin as a reference drug (IC50 value of doxorubicin
Scheme 4. Synthesis of bipyridine derivatives 8and 9.
On the other hand, chalcone
10
, prepared by the reaction of
1
with benzaldehyde in ethanol containing
catalytic amounts of NaOH [
54
], was used for preparation of 6-(pyrazol-3-yl) pyrimidine-2-thione
derivative
11
via its reaction with thiourea in ethanol containing a catalytic amount of sodium
hydroxide [
54
]. Reaction of the latter compound
11
with a number of hydrazonoyl chlorides
12a
–
h
[
55
]
in dioxane in the presence of triethylamine afforded the respective products
15a
–
h
through the
non-isolated intermediates
13
and
14
(Scheme 5). The structure assigned for the products
15
was
established via microanalytical and spectral data (see Section 3). For example, the IR spectra of product
15
revealed the absence of the pyrimidinyl-NH groups, and instead showed two absorption bands
near
υ
1706 and 1649 cm
−1
assigned for the two carbonyl groups. Additionally,
1
HNMR spectra of
product
15
showed the absence of the signals attributed to the pyrimidinyl-NH protons and, instead,
revealed the signals assigned for the acetyl protons (for
15a
–
d
) or the ethoxycarbonyl protons (for
15e
–
h
), in addition to the characteristic signals due to the ester and aromatic protons (see Section 3).
The mass spectra of product
15
showed, in each case, the respective molecular ion peak, which is
consistent with the assigned structure.
Appl. Sci. 2017, 7, 785 4 of 13
Scheme 4. Synthesis of bipyridine derivatives 8 and 9.
On the other hand, chalcone 10, prepared by the reaction of 1 with benzaldehyde in ethanol
containing catalytic amounts of NaOH [54], was used for preparation of 6-(pyrazol-3-yl) pyrimidine-
2-thione derivative 11 via its reaction with thiourea in ethanol containing a catalytic amount of
sodium hydroxide [54]. Reaction of the latter compound 11 with a number of hydrazonoyl chlorides
12a–h [55] in dioxane in the presence of triethylamine afforded the respective products 15a–h through
the non-isolated intermediates 13 and 14 (Scheme 5). The structure assigned for the products 15 was
established via microanalytical and spectral data (see Section 3). For example, the IR spectra of
product 15 revealed the absence of the pyrimidinyl-NH groups, and instead showed two absorption
bands near υ 1706 and 1649 cm−1 assigned for the two carbonyl groups. Additionally, 1HNMR spectra
of product 15 showed the absence of the signals attributed to the pyrimidinyl-NH protons and,
instead, revealed the signals assigned for the acetyl protons (for 15a–d) or the ethoxycarbonyl protons
(for 15e–h), in addition to the characteristic signals due to the ester and aromatic protons (see Section 3).
The mass spectra of product 15 showed, in each case, the respective molecular ion peak, which is
consistent with the assigned structure.
Scheme 5. Synthesis of 1,2,4-triazolo[4,3-a]pyrimidines 15a–h.
2.2. Antitumor Activity
The cytotoxicity of the synthesized pyridines 4a,b,e and 6a,b,e was evaluated against the human
liver carcinoma cell line (HepG2-1) using doxorubicin as a reference drug (IC50 value of doxorubicin
Scheme 5. Synthesis of 1,2,4-triazolo[4,3-a]pyrimidines 15a–h.

Appl. Sci. 2017,7, 785 5 of 13
2.2. Antitumor Activity
The cytotoxicity of the synthesized pyridines
4a
,
b
,
e
and
6a
,
b
,
e
was evaluated against the
human liver carcinoma cell line (HepG2-1) using doxorubicin as a reference drug (IC
50
value of
doxorubicin = 0.08
±
0.07 nM) and MTT assay. The data generated were used to plot a dose response
curve of which the concentration of the tested compounds required to kill 50% of cell population (IC
50
)
was determined. Cytotoxic activity was expressed as the mean IC
50
of three independent experiments.
The results are depicted in Table 1and Figure 1.
Table 1. IC50 values of tested compounds 4and 6±standard deviation against HEPG2-1.
Compound No. X Y Z IC50 (nM) General Structure
Doxorubicin - - - 0.08 ±0.07
Appl. Sci. 2017, 7, 785 5 of 13
= 0.08 ± 0.07 nM) and MTT assay. The data generated were used to plot a dose response curve of
which the concentration of the tested compounds required to kill 50% of cell population (IC
50
) was
determined. Cytotoxic activity was expressed as the mean IC
50
of three independent experiments.
The results are depicted in Table 1 and Figure 1.
Table 1. IC
50
values of tested compounds 4 and 6 ± standard deviation against HEPG2-1.
Compound No. X Y Z IC
50
(nM) General Structure
Doxorubicin - - - 0.08 ± 0.07
4a H CN NH
2
9.7 ± 0.85
4b Me CN NH
2
1.9 ± 0.16
4e Cl CN NH
2
17.2 ± 0.83
6a H MeCO OH 12.3 ± 0.37
6b Me MeCO OH 2.4 ± 0.29
6e Cl MeCO OH 22.3 ± 0.36
The results revealed that the descending order of the antitumor activity of the tested compounds
against HEPG2-1cell line is as follow: 4b > 6b > 4a > 6a > 4e > 6e.
The pyridine derivatives 4b and 6b (IC
50
= 1.9 ± 0.16 and 2.4 ± 0.29 nM, respectively) have
promising antitumor activity against HEPG2-1. On the other hand, pyridine derivatives 4e and 6e
have poor inhibitory activity (IC
50
> 17 nM) compared with doxorubicin which used as reference drug.
Figure 1. Cytotoxic activities of tested compounds against HEPG2-1.
Structural Activity Relationship SAR
Examination of the SAR led to the following conclusions:
The activity of the synthesized compounds 4 and 6 against hepatocellular carcinoma depends
on the structural skeleton and electronic environment of the molecules. For example, the activity of
the tested compounds 4a,b,e and 6a,b,e were found to be highly related to their structures since
replacement of electron-donating groups in the two aryl groups in compounds 4b and 6b with
electron-withdrawing groups in compounds 4e and 6e dramatically decreases their cytotoxicity
against HEPG2-1. On the other hand, the cytotoxicity of compounds 4a and 6a whose structures
contain two phenyl groups (no substituent), is intermediate between the highly-potent and the
weakly-potent compounds (See Table 1).
0
5
10
15
20
25
IC 50 (nM)
4a H CN NH29.7 ±0.85
4b Me CN NH21.9 ±0.16
4e Cl CN NH217.2 ±0.83
6a H
MeCO
OH 12.3 ±0.37
6b Me
MeCO
OH 2.4 ±0.29
6e Cl
MeCO
OH 22.3 ±0.36
Appl. Sci. 2017, 7, 785 5 of 13
= 0.08 ± 0.07 nM) and MTT assay. The data generated were used to plot a dose response curve of
which the concentration of the tested compounds required to kill 50% of cell population (IC
50
) was
determined. Cytotoxic activity was expressed as the mean IC
50
of three independent experiments.
The results are depicted in Table 1 and Figure 1.
Table 1. IC
50
values of tested compounds 4 and 6 ± standard deviation against HEPG2-1.
Compound No. X Y Z IC
50
(nM) General Structure
Doxorubicin - - - 0.08 ± 0.07
4a H CN NH
2
9.7 ± 0.85
4b Me CN NH
2
1.9 ± 0.16
4e Cl CN NH
2
17.2 ± 0.83
6a H MeCO OH 12.3 ± 0.37
6b Me MeCO OH 2.4 ± 0.29
6e Cl MeCO OH 22.3 ± 0.36
The results revealed that the descending order of the antitumor activity of the tested compounds
against HEPG2-1cell line is as follow: 4b > 6b > 4a > 6a > 4e > 6e.
The pyridine derivatives 4b and 6b (IC
50
= 1.9 ± 0.16 and 2.4 ± 0.29 nM, respectively) have
promising antitumor activity against HEPG2-1. On the other hand, pyridine derivatives 4e and 6e
have poor inhibitory activity (IC
50
> 17 nM) compared with doxorubicin which used as reference drug.
Figure 1. Cytotoxic activities of tested compounds against HEPG2-1.
Structural Activity Relationship SAR
Examination of the SAR led to the following conclusions:
The activity of the synthesized compounds 4 and 6 against hepatocellular carcinoma depends
on the structural skeleton and electronic environment of the molecules. For example, the activity of
the tested compounds 4a,b,e and 6a,b,e were found to be highly related to their structures since
replacement of electron-donating groups in the two aryl groups in compounds 4b and 6b with
electron-withdrawing groups in compounds 4e and 6e dramatically decreases their cytotoxicity
against HEPG2-1. On the other hand, the cytotoxicity of compounds 4a and 6a whose structures
contain two phenyl groups (no substituent), is intermediate between the highly-potent and the
weakly-potent compounds (See Table 1).
0
5
10
15
20
25
IC 50 (nM)
Figure 1. Cytotoxic activities of tested compounds against HEPG2-1.
The results revealed that the descending order of the antitumor activity of the tested compounds
against HEPG2-1cell line is as follow: 4b >6b >4a >6a >4e >6e.
The pyridine derivatives
4b
and
6b
(IC
50
= 1.9
±
0.16 and 2.4
±
0.29 nM, respectively) have
promising antitumor activity against HEPG2-1. On the other hand, pyridine derivatives
4e
and
6e
have poor inhibitory activity (IC
50
> 17 nM) compared with doxorubicin which used as reference drug.
Structural Activity Relationship SAR
Examination of the SAR led to the following conclusions:
The activity of the synthesized compounds
4
and
6
against hepatocellular carcinoma depends
on the structural skeleton and electronic environment of the molecules. For example, the activity
of the tested compounds
4a
,
b
,
e
and
6a
,
b
,
e
were found to be highly related to their structures since
replacement of electron-donating groups in the two aryl groups in compounds
4b
and
6b
with
electron-withdrawing groups in compounds
4e
and
6e
dramatically decreases their cytotoxicity against
HEPG2-1. On the other hand, the cytotoxicity of compounds
4a
and
6a
whose structures contain
two phenyl groups (no substituent), is intermediate between the highly-potent and the weakly-potent
compounds (See Table 1).

Appl. Sci. 2017,7, 785 6 of 13
3. Experimental
3.1. Chemistry
Melting points were measured on an Electrothermal IA 9000 series (Bibby Sci. Lim. Stone, Staffordshire,
UK) digital melting point apparatus. The IR spectra were recorded in potassium bromide discs on a Pye
Unicam SP 3300 (Cambridge, UK) and a Shimadzu FT IR 8101 PC infrared (Shimadzu, Tokyo, Japan)
spectrophotometer.
1
H-NMR spectra were recorded in deuterated dimethyl sulfoxide (DMSO-d
6
)
using a Varian Gemini 300 NMR spectrometer (Varian, Inc., Karlsruhe, Germany). Mass spectra were
recorded on a Shimadzu GCMS-QP1000 EX mass spectrometer (Tokyo, Japan) at 70 eV. Elemental
analysis was carried out at the Microanalytical Centre of Cairo University, Giza, Egypt. All reactions
were followed by TLC (Silica gel, Merck, Darmstadt, Germany).
3.1.1. Synthesis of Tetra-Substituted Pyridine Derivatives (4a–fand 6a–f)
General procedure: A mixture of ethyl 3-acetyl-1,5-diphenyl-1H-pyrazole-4-carboxylate (
1
)
(0.334 g, 1 mmol), the appropriate aldehyde
2a
–
f
(1 mmol) and malononitrile (
3
), or ethyl acetoacetate
(
5
) (1 mmol) in glacial acetic acid (20 mL) containing ammonium acetate (0.616 g, 8 mmol) was refluxed
for 6–8 h (monitored by TLC). After complete reaction, the mixture was cooled and the precipitated
products were filtered, washed with water, dried, and crystallized from ethanol to give the pyridine
derivatives
4a
–
f
and
6a
–
f
, respectively
.
Compounds
4a
–
f
and
6a
–
f
together with their physical and
spectral data are listed below:
Ethyl 3-(6-amino-5-cyano-4-phenylpyridin-2-yl)-1,5-diphenyl-1H-pyrazole-4-carboxylate (
4a
). Brown solid,
(70% yield), mp 169–171
◦
C; IR (KBr)
νmax
3364, 3208 (NH
2
), 2218 (CN), 1715 (C=O) cm
−1
;
1
H NMR
(DMSO-d
6
)
δ
1.02 (t, J= 7.2 Hz, 3H, CH
3
), 4.13 (q, J= 7.2 Hz, 2H, CH
2
), 6.93 (s, br, 2H, NH
2
), 7.18–7.90
(m, 15H, Ar-H), 8.11 (s, 1H, Pyridine-H5); MS m/z(%) 485 (M
+
, 14), 322 (47), 252 (29), 167 (38), 77 (52),
43 (100). Anal. Calcd. for C
30
H
23
N
5
O
2
(485.55): C, 74.21; H, 4.77; N, 14.42. Found: C, 74.05; H, 4.52;
N, 14.26%.
Ethyl 3-(6-amino-5-cyano-4-(p-tolyl)pyridin-2-yl)-1,5-diphenyl-1H-pyrazole-4-carboxylate (
4b
). Brown solid,
(72% yield), mp 180–182
◦
C; IR (KBr)
νmax
3379, 3211 (NH
2
), 2210 (CN), 1712 (C=O) cm
−1
;
1
H NMR
(DMSO-d
6
)
δ
1.01 (t, J= 7.2 Hz, 3H, CH
3
), 2.36 (s, 3H, CH
3
), 4.12 (q, J= 7.2 Hz, 2H, CH
2
), 6.92 (s, br, 2H,
NH
2
), 7.14–7.94 (m, 14H, Ar-H), 8.15 (s, 1H, Pyridine-H5); MS m/z(%) 499 (M
+
, 15), 468 (32), 364 (39),
209 (42), 104 (38), 78 (72), 43 (100). Anal. Calcd. for C
31
H
25
N
5
O
2
(499.57): C, 74.53; H, 5.04; N, 14.02.
Found: C, 74.37; H, 5.00; N, 13.85%.
Ethyl 3-(6-amino-5-cyano-4-(4-methoxyphenyl)pyridin-2-yl)-1,5-diphenyl-1H-pyrazole-4-carboxylate (
4c
).
Pale green solid, (68% yield), mp 154–156
◦
C; IR (KBr)
νmax
3367, 3219 (NH
2
), 2210 (CN), 1714
(C=O) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.02 (t, J= 7.2 Hz, 3H, CH
3
), 3.78 (s, 3H, OCH
3
), 4.15 (q, J= 7.2 Hz,
2H, CH
2
), 6.93 (s, br, 2H, NH
2
), 7.18–7.80 (m, 14H, Ar-H), 8.12 (s, 1H, Pyridine-H5); MS m/z(%) 515
(M
+
, 9), 452 (42), 316 (100), 234 (51), 182 (37), 118 (50), 76 (66). Anal. Calcd. for C
31
H
25
N
5
O
3
(515.57): C,
72.22; H, 4.89; N, 13.58. Found: C, 72.01; H, 4.77; N, 13.30%.
Ethyl 3-(6-amino-5-cyano-4-(4-(dimethylamino)phenyl)pyridin-2-yl)-1,5-diphenyl-1H-pyrazole-4-carboxylate
(
4d
). Dark yellow solid, (73% yield), mp 150–152
◦
C; IR (KBr)
νmax
3431, 3212 (NH
2
), 2210 (CN), 1709
(C=O) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.01 (t, J= 7.2 Hz, 3H, CH
3
), 2.97 (s, 6H, 2CH
3
), 4.11 (q, J= 7.2 Hz,
2H, CH
2
), 6.82 (s, br, 2H, NH
2
), 7.14–7.82 (m, 14H, Ar-H), 8.10 (s, 1H, Pyridine-H5); MS m/z(%) 528
(M
+
, 14), 416 (80), 212 (100), 170 (27), 105 (48), 76 (63). Anal. Calcd. for C
32
H
28
N
6
O
2
(528.62): C, 72.71;
H, 5.34; N, 15.90. Found: C, 72.59; H, 5.30; N, 15.73%.
Ethyl 3-(6-amino-4-(4-chlorophenyl)-5-cyanopyridin-2-yl)-1,5-diphenyl-1H-pyrazole-4-carboxylate (
4e
). Dark
yellow solid, (76% yield), mp 181–183
◦
C; IR (KBr)
νmax
3362, 3218 (NH
2
), 2213 (CN), 1712 (C=O) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.02 (t, J= 7.2 Hz, 3H, CH
3
), 4.14 (q, J= 7.2 Hz, 2H, CH
2
), 6.98 (s, br, 2H, NH
2
),
7.17–7.84 (m, 14H, Ar-H), 8.17 (s, 1H, Pyridine-H5); MS m/z(%) 521 (M
+
, 23), 519 (M
+
, 8), 397 (32), 316

Appl. Sci. 2017,7, 785 7 of 13
(60), 191 (55), 127 (51), 85 (47), 57 (100). Anal. Calcd. for C
30
H
22
ClN
5
O
2
(519.99): C, 69.30; H, 4.26; N,
13.47. Found: C, 69.16; H, 4.18; N, 13.28%.
Ethyl 3-(6-amino-5-cyano-4-(2,4-dichlorophenyl)pyridin-2-yl)-1,5-diphenyl-1H-pyrazole-4-carboxylate (
4f
).
Yellow solid, (75% yield), mp 197–199
◦
C; IR (KBr)
νmax
3367, 3215 (NH
2
), 2214 (CN), 1714 (C=O)
cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.04 (t, J= 7.2 Hz, 3H, CH
3
), 4.15 (q, J= 7.2 Hz, 2H, CH
2
), 7.06 (s, br, 2H,
NH
2
), 7.28–7.85 (m, 13H, Ar-H), 8.14 (s, 1H, Pyridine-H5); MS m/z(%) 554 (M
+
, 100), 316 (77), 281 (41),
193 (71), 105 (33), 58 (72). Anal. Calcd. for C
30
H
21
Cl
2
N
5
O
2
(554.43): C, 64.99; H, 3.82; N, 12.63. Found:
C, 64.80; H, 3.61; N, 12.44%.
Ethyl 3-(5-acetyl-6-oxo-4-phenyl-1,6-dihydropyridin-2-yl)-1,5-diphenyl-1H-pyrazole-4-carboxylate (
6a
).
Brown solid, (68% yield), mp 186–188
◦
C; IR (KBr)
νmax
3367 (NH), 1722, 1690, 1657 (3C=O) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.03 (t, J= 7.2 Hz, 3H, CH
3
), 2.58 (s, 3H, CH
3
), 4.12 (q, J= 7.2 Hz, 2H, CH
2
),
7.24–7.49 (m, 15H, Ar-H), ), 7.77 (s, 1H, Pyridine-H5), 9.63 (s, br, 1H, NH); MS m/z(%) 503 (M
+
, 48),
458 (27), 334 (52), 232 (46), 99 (54), 57 (68), 43 (100). Anal. Calcd. for C
31
H
25
N
3
O
4
(503.56): C, 73.94; H,
5.00; N, 8.34. Found: C, 73.73; H, 4.86; N, 8.17%.
Ethyl 3-(5-acetyl-6-oxo-4-(p-tolyl)-1,6-dihydropyridin-2-yl)-1,5-diphenyl-1H-pyrazole-4-carboxylate (
6b
).
Brown solid, (66% yield), mp 134–136
◦
C; IR (KBr)
νmax
3409 (NH), 1718, 1681, 1662 (3C=O) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.02 (t, J= 7.2 Hz, 3H, CH
3
), 2.35 (s, 3H, CH
3
), 2.56 (s, 3H, CH
3
), 4.11 (q,
J= 7.2 Hz, 2H, CH
2
), 7.19–7.49 (m, 14H, Ar-H), ), 7.79 (s, 1H, Pyridine-H5), 9.81 (s, br, 1H, NH); MS m/z
(%) 517(M
+
, 23), 385 (33), 294 (38), 147 (50), 120 (100), 76 (62). Anal. Calcd. for C
32
H
27
N
3
O
4
(517.59): C,
74.26; H, 5.26; N, 8.12. Found: C, 74.20; H, 5.14; N, 8.03%.
Ethyl 3-(5-acetyl-4-(4-methoxyphenyl)-6-oxo-1,6-dihydropyridin-2-yl)-1,5-diphenyl-1H-pyrazole-4-carboxylate
(
6c
). Pale brown solid, (67% yield), mp 141–143
◦
C; IR (KBr)
νmax
3423 (NH), 1715, 1687, 1660 (3C=O)
cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.00 (t, J= 7.2 Hz, 3H, CH
3
), 2.57 (s, 3H, CH
3
), 3.77 (s, 3H, OCH
3
), 4.01
(q, J= 7.2 Hz, 2H, CH
2
), 7.16–7.54 (m, 14H, Ar-H), ), 7.74 (s, 1H, Pyridine-H5), 9.80 (s, br, 1H, NH);
MS m/z(%) 533 (M
+
, 14), 423 (37), 313 (51), 279 (100), 105 (36), 76 (43). Anal. Calcd. for C
32
H
27
N
3
O
5
(533.58): C, 72.03; H, 5.10; N, 7.88. Found: C, 71.85; H, 5.02; N, 7.63%.
Ethyl 3-(5-acetyl-4-(4-(dimethylamino)phenyl)-6-oxo-1,6-dihydropyridin-2-yl)-1,5-diphenyl-1H-pyrazole-4-
carboxylate (
6d
). Brown solid, (69% yield), mp 141–143
◦
C; IR (KBr)
νmax
3425 (NH), 1721, 1682, 1657
(3C=O) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.00 (t, J= 7.2 Hz, 3H, CH
3
), 2.58 (s, 3H, CH
3
), 2.99 (s, 6H, 2CH
3
),
4.11 (q, J= 7.2 Hz, 2H, CH
2
), 6.78–7.39 (m, 14H, Ar-H), 7.72 (s, 1H, Pyridine-H5), 9.73 (s, br, 1H, NH);
MS m/z(%) 546 (M
+
, 14), 406 (36), 349 (55), 241 (49), 121 (36), 76 (30), 43 (100). Anal. Calcd. for
C33H30 N4O4(546.63): C, 72.51; H, 5.53; N, 10.25. Found: C, 72.39; H, 5.38; N, 10.02%.
Ethyl 3-(5-acetyl-4-(4-chlorophenyl)-6-oxo-1,6-dihydropyridin-2-yl)-1,5-diphenyl-1H-pyrazole-4-carboxylate
(
6e
). Brown solid, (68% yield), mp 170–172
◦
C; IR (KBr)
νmax
3366 (NH), 1720, 1680, 1663 (3C=O) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.06 (t, J= 7.2 Hz, 3H, CH
3
), 2.58 (s, 3H, CH
3
), 4.14 (q, J= 7.2 Hz, 2H, CH
2
),
7.24–7.59 (m, 14H, Ar-H), 7.78 (s, 1H, Pyridine-H5), 10.06 (s, br, 1H, NH); MS m/z (%) 540 (M
+
+ 2, 1),
538 (M
+
, 3), 368 (53), 214 (100), 120 (55), 40 (79). Anal. Calcd. for C
31
H
24
ClN
3
O
4
(538.00): C, 69.21; H,
4.50; N, 7.81. Found: C, 69.46; H, 4.35; N, 7.66%.
Ethyl 3-(5-acetyl-4-(2,4-dichlorophenyl)-6-oxo-1,6-dihydropyridin-2-yl)-1,5-diphenyl-1H-pyrazole-4-carboxylate
(
6f
). Brown solid, (69% yield), mp 197–199
◦
C; IR (KBr)
νmax
3414 (NH), 1720, 1683, 1659 (3C=O) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.09 (t, J= 7.2 Hz, 3H, CH
3
), 2.61 (s, 3H, CH
3
), 4.15 (q, J= 7.2 Hz, 2H, CH
2
),
7.26–7.52 (m, 13H, Ar-H), 7.76 (s, 1H, Pyridine-H5), 10.24 (s, br, 1H, NH); MS m/z(%) 572 (M
+
, 12), 388
(64), 256 (44), 207 (67), 125 (50), 83 (42), 55 (100). Anal. Calcd. for C
31
H
23
Cl
2
N
3
O
4
(572.44): C, 65.04; H,
4.05; N, 7.34. Found: C, 65.24; H, 4.02; N, 7.16%.

Appl. Sci. 2017,7, 785 8 of 13
3.1.2. Synthesis of Bipyridine Derivatives 8and 9
A mixture of 3-acetylpyrazole derivative
1
(0.668 g, 2 mmol), terephthalaldehyde
7
(0.134 g,
1 mmol), and malononitrile
3
or ethyl acetoacetate
5
(2 mmol) in acetic acid (30 mL) containing
ammonium acetate (1.232 g, 16 mmol) was refluxed for 8 h. After cooling the reaction mixture it
was poured into an ice-water mixture, the formed a precipitate that was collected by filtration, then
crystallized from dioxane to give the bipyridine products 8and 9, respectively.
Diethyl 3,3
0
-(1,4-phenylenebis(6-amino-5-cyanopyridine-4,2-diyl))bis(1,5-diphenyl-1H-pyrazole-4-carboxylate)
(
8
). Brown solid, (68% yield), mp 187–189
◦
C; IR (KBr)
νmax
3378, 3201 (NH
2
), 2211 (CN), 1709 (C=O)
cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.03 (t, J= 7.2 Hz, 6H, 2CH
3
), 4.14 (q, J= 7.2 Hz, 4H, 2CH
2
), 6.93 (s, br,
4H, 2NH
2
), 7.18–7.49 (m, 20H, Ar-H), 7.85 (s, 4H, Ar-H), 8.10 (s, 2H, 2Pyridine-H3); MS m/z(%) 892
(M
+
, 39), 724 (48), 622 (63), 368 (39), 82 (60), 76 (57), 43 (100). Anal. Calcd. for C
54
H
40
N
10
O
4
(892.98): C,
72.63; H, 4.52; N, 15.69. Found: C, 72.69; H, 4.36; N, 15.47%.
Diethyl 3,3
0
-(1,4-phenylenebis(5-acetyl-6-oxo-1,6-dihydropyridine-4,2-diyl))bis(1,5-diphenyl-1H-pyrazole-4-
carboxylate) (
9
). Brown solid, (66% yield), mp 207–209
◦
C; IR (KBr)
νmax
3423 (NH), 1723, 1677, 1653
(3C=O) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.11 (t, J= 7.2 Hz, 6H, 2CH
3
), 2.58 (s, 6H, 2CH
3
), 4.14 (q, J= 7.2 Hz,
4H, 2CH
2
), 7.24–7.48 (m, 20H, Ar-H), ), 7.77 (s, 2H, 2Pyridine-H3), 7.81 (s, 4H, Ar-H), 10.06 (s, br, 2H,
2NH); MS m/z(%) 929 (M
+
, 17), 776 (41), 509 (37), 386 (55), 267 (40), 148 (32), 77 (100), 43 (68). Anal.
Calcd. for C56 H44N6O8(929.00): C, 72.40; H, 4.77; N, 9.05. Found: C, 72.17; H, 4.62; N, 9.01%.
3.1.3. Synthesis of 1,5-Diphenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine derivatives 15a–h
General procedure: Triethylamine (0.14 mL, 1 mmol) was added to a mixture of equimolar
amounts of thione
11
(0.480 g, 1 mmol) and the appropriate hydrazonoyl halides
12a
–
h
(1 mmol) in
dioxane (20 mL) at room temperature. The reaction mixture was then refluxed for 10–15 h until all
hydrogen sulfide gas stopped evolving. The solid that formed after concentration of the reaction
mixture was filtered and crystallized from the proper solvent to give the products
15a
–
h
, respectively.
Ethyl 3-(3-acetyl-1,5-diphenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidin-7-yl)-1,5-diphenyl-1H-pyrazole-4-
carboxylate (
15a
).Yellow solid, (74% yield), mp 233–235
◦
C (DMF); IR (KBr)
νmax
3026, 2956 (C-H),
1706, 1649 (2C=O), 1595 (C=N) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.15 (t, J= 7.2 Hz, 3H, CH
3
), 2.43 (s, 3H,
CH
3
), 4.19 (q, J= 7.2 Hz, 2H, CH
2
), 5.33 (d, J= 4 Hz, 1H, CH), 6.62 (d, J= 4Hz, 1H, CH), 7.03–7.80 (m,
20H, Ar-H); MS m/z(%) 606 (M
+
, 5),406 (36), 287 (29), 247 (75), 194 (37), 92 (71), 65 (60), 43 (100). Anal.
Calcd. for C37 H30N6O3(606.69): C, 73.25; H, 4.98; N, 13.85. Found: C, 73.07; H, 4.84; N, 13.67%.
Ethyl 3-(3-acetyl-5-phenyl-1-(p-tolyl)-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidin-7-yl)-1,5-diphenyl-1H-
pyrazole-4-carboxylate (
15b
). Yellow solid, (72% yield), mp 211–213
◦
C (DMF); IR (KBr)
νmax
3030, 2951
(C-H), 1697, 1642 (2C=O), 1597 (C=N) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.04 (t, J= 7.2 Hz, 3H, CH
3
), 2.24
(s, 3H, CH
3
), 2.44 (s, 3H, CH
3
), 4.16 (q, J= 7.2 Hz, 2H, CH
2
), 5.32 (d, J= 4 Hz, 1H, CH), 6.61 (d, J= 4Hz,
1H, CH), 7.05–7.73 (m, 19H, Ar-H); MS m/z(%) 620 (M
+
, 7), 498 (27), 390 (35), 285 (60), 105 (41), 77
(100), 43 (92). Anal. Calcd. for C
38
H
32
N
6
O
3
(620.71): C, 73.53; H, 5.20; N, 13.54. Found: C, 73.39; H,
5.38; N, 13.36%.
Ethyl 3-(3-acetyl-1-(4-chlorophenyl)-5-phenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidin-7-yl)-1,5-diphenyl-
1H-pyrazole-4-carboxylate (
15c
). Yellow solid, (74% yield), mp 242–244
◦
C (DMF/EtOH); IR (KBr)
νmax
3028, 2963 (C-H), 1707, 1641 (2C=O), 1597 (C=N) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.02 (t, J= 7.2 Hz, 3H,
CH
3
), 2.44 (s, 3H, CH
3
), 4.15 (q, J= 7.2 Hz, 2H, CH
2
), 5.36 (d, J= 4 Hz, 1H, CH), 6.69 (d, J= 4Hz, 1H,
CH), 7.27–7.70 (m, 19H, Ar-H); MS m/z(%) 643 (M
+
+ 2, 4), 641 (M
+
, 13), 499 (57), 322 (39), 180 (28),
105 (35), 77 (100). Anal. Calcd. for C
37
H
29
ClN
6
O
3
(641.13): C, 69.32; H, 4.56; N, 13.11. Found: C, 69.19;
H, 4.51; N, 13.00%.
Ethyl 3-(3-acetyl-1-(4-nitrophenyl)-5-phenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidin-7-yl)-1,5-diphenyl-1H-
pyrazole-4-carboxylate (
15d
). Yellow solid, (75% yield), mp 204–206
◦
C (EtOH); IR (KBr)
νmax
3031, 2950

Appl. Sci. 2017,7, 785 9 of 13
(C-H), 1712, 1656 (2C=O), 1598 (C=N) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.02 (t, J= 7.2 Hz, 3H, CH
3
), 2.47 (s,
3H, CH
3
), 4.15 (q, J= 7.2 Hz, 2H, CH
2
), 5.39 (d, J= 4 Hz, 1H, CH), 6.72 (d, J= 4Hz, 1H, CH), 7.24–8.52
(m, 19H, Ar-H); MS m/z(%) 651 (M+, 26), 484 (48), 400 (71), 252 (39), 179 (42), 105 (100), 57 (83). Anal.
Calcd. for C37 H29N7O5(651.68): C, 68.19; H, 4.49; N, 15.05. Found: C, 68.04; H, 4.33; N, 14.92%.
Ethyl 7-(4-(ethoxycarbonyl)-1,5-diphenyl-1H-pyrazol-3-yl)-1,5-diphenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]
pyrimidine-3-carboxylate (
15e
). Yellow solid, (72% yield), mp 180–182
◦
C (DMF/EtOH); IR (KBr)
νmax
3056, 2973 (C-H), 1713, 1679 (2C=O), 1596 (C=N) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.04 (t, J= 7.2 Hz, 3H,
CH
3
), 1.26 (t, J= 7.6 Hz, 3H, CH
3
), 4.14 (q, J= 7.2 Hz, 2H, CH
2
), 4.26 (q, J= 7.6 Hz, 2H, CH
2
), 5.46 (d,
J= 4 Hz, 1H, CH), 6.47 (d, J= 4Hz, 1H, CH), 6.96–7.79 (m, 20H, Ar-H); MS m/z(%) 636 (M
+
, 9), 394
(51), 283 (33), 235 (49), 194 (62), 83 (53), 57 (100). Anal. Calcd. for C
38
H
32
N
6
O
4
(636.71): C, 71.68; H,
5.07; N, 13.20. Found: C, 71.62; H, 5.01; N, 13.03%.
Ethyl 7-(4-(ethoxycarbonyl)-1,5-diphenyl-1H-pyrazol-3-yl)-5-phenyl-1-(p-tolyl)-1,5-dihydro-[1,2,4]triazolo
[4,3-a]pyrimidine-3-carboxylate (
15f
). Yellow solid, (73% yield), mp 172–174
◦
C (EtOH); IR (KBr)
νmax
3052, 2955 (C-H), 1710, 1699 (2C=O), 1595 (C=N) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.02 (t, J= 7.2 Hz, 3H,
CH
3
), 1.23 (t, J= 7.6 Hz, 3H, CH
3
), 2.30 (s, 3H, CH
3
), 4.10 (q, J= 7.2 Hz, 2H, CH
2
), 4.26 (q, J= 7.6 Hz,
2H, CH
2
), 5.39 (d, J= 4 Hz, 1H, CH), 6.45 (d, J= 4Hz, 1H, CH), 7.12–7.76 (m, 19H, Ar-H); MS m/z(%)
650 (M
+
, 6), 439 (44), 361 (30), 244 (57), 104 (100), 91 (48), 43 (60). Anal. Calcd. for C
39
H
34
N
6
O
4
(650.74):
C, 71.98; H, 5.27; N, 12.91. Found: C, 71.75; H, 5.19; N, 12.74%.
Ethyl 1-(4-chlorophenyl)-7-(4-(ethoxycarbonyl)-1,5-diphenyl-1H-pyrazol-3-yl)-5-phenyl-1,5-dihydro-[1,2,4]
triazolo[4,3-a]pyrimidine-3-carboxylate (
15g
). Yellow solid, (75% yield), mp 188–190
◦
C (DMF/EtOH);
IR (KBr)
νmax
3037, 2966 (C-H), 1713, 1667 (2C=O), 1597 (C=N) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.05 (t,
J= 7.2 Hz, 3H, CH
3
), 1.19 (t, J= 7.6 Hz, 3H, CH
3
), 4.13 (q, J= 7.2 Hz, 2H, CH
2
), 4.24 (q, J= 7.6 Hz, 2H,
CH
2
), 5.42 (d, J= 4 Hz, 1H, CH), 6.47 (d, J= 4Hz, 1H, CH), 7.25–7.79 (m, 19H, Ar-H); MS m/z(%) 673
(M
+
+ 2, 11), 671 (M
+
, 36), 387 (100), 324 (68), 278 (50), 105 (42), 78 (83). Anal. Calcd. for C
38
H
31
ClN
6
O
4
(671.15): C, 68.00; H, 4.66; N, 12.52. Found: C, 68.25; H, 4.40; N, 12.46%.
Ethyl 7-(4-(ethoxycarbonyl)-1,5-diphenyl-1H-pyrazol-3-yl)-1-(4-nitrophenyl)-5-phenyl-1,5-dihydro-[1,2,4]
triazolo[4,3-a]pyrimidine-3-carboxylate (
15h
). Brown solid, (71% yield), mp 206–208
◦
C (EtOH); IR
(KBr)
νmax
3030, 2948 (C-H), 1713, 1644 (2C=O), 1593 (C=N) cm
−1
;
1
H NMR (DMSO-d
6
)
δ
1.07 (t,
J= 7.2 Hz, 3H, CH
3
), 1.25 (t, J= 7.6 Hz, 3H, CH
3
), 4.12 (q, J= 7.2 Hz, 2H, CH
2
), 4.27 (q, J= 7.6 Hz, 2H,
CH2), 5.46 (d, J= 4 Hz, 1H, CH), 6.49 (d, J= 4Hz, 1H, CH), 7.25–8.42 (m, 19H, Ar-H); MS m/z(%) 681
(M
+
, 31), 577 (73), 390 (66), 327 (95), 115 (100), 83 (52). Anal. Calcd. for C
38
H
31
N
7
O
6
(681.71): C, 66.95;
H, 4.58; N, 14.38. Found: C, 66.77; H, 4.42; N, 14.23%.
3.2. Cytotoxic Activity
The cytotoxic evaluation of the synthesized compounds was carried out at the Regional Center
for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt according to the reported
method [56].
4. Conclusions
Two series of functionalized pyrazolyl-pyridines were prepared by multi-component reaction of
3-acetylpyrazole derivative with the appropriate aldehyde, malononitrile (or ethyl acetoacetate) in
acetic acid in the presence of excess ammonium acetate. The mechanism of formation of the novel
products was also discussed. Additionally, two novel bipyridine derivatives were synthesized by
the above described reaction and under the same reaction conditions using terephthaldehyde in lieu
of benzaldeyde derivatives. Another series of 1,2,4-triazole[4,3-a]pyrimidines, including a pyrazole
moiety, was prepared by the reaction of a pyrazolylpyrimidine-2-thione derivative with a variety of
hydrazonoyl chlorides under reflux in dioxane in the presence of triethylamine. The assigned structure
for the products was elucidated based on elemental analyses and spectral data (IR,
1
HNMR, MS).

Appl. Sci. 2017,7, 785 10 of 13
Moreover, the novel pyrazolyl-pyridines were tested for their reactivity as antitumor agents and the
results obtained revealed high potency of some of them against HEPG2-1 compared with doxorubicin
used as the reference drug.
Acknowledgments:
The authors extend their sincere appreciation to the Deanship of Scientific Research at the
King Saud University for its funding this Prolific Research group (PRG-1437-29).
Author Contributions:
Magda A. Abdallah, Sobhi M. Gomha, and Ikhlass M. Abbas designed research;
Mariam S. H. Kazem, Seham S. Alterary and Yahia N. Mabkhot performed the research, analyzed the data,
wrote the paper, and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interests.
References
1.
Kam, D.; Kim, K.; Kim, H.-S.; Liu, H.K. Studies on film formation on cathodes using pyrazole derivatives as
electrolyte additives in the Li-ion battery. Electrochem. Commun. 2009,11, 1657–1660. [CrossRef]
2.
Keter, F.K.; Darkwa, J. Perspective: The potential of pyrazole-based compounds in medicine. Biometals
2012
,
25, 9–21. [CrossRef] [PubMed]
3.
Yutilov, Y.M.; Smolyar, N.N.; Minkina, L.V. Synthesis of 3-(3
00
-Aminobenzoylamido)-1-(2
0
,4
0
,6
0
-trichlorophenyl)
pyrazol-5-one, an intermediate in the synthesis of the purple component of color photographic materials.
Russ. J. Appl. Chem. 2005,78, 278–280. [CrossRef]
4.
Tingle, C.C.; Rother, J.A.; Dewhurst, C.F.; Lauer, S.; King, W.J. Fipronil: Environmental fate, ecotoxicology,
and human health concerns. Rev. Environ. Contam. Toxicol. 2003,176, 1–66. [PubMed]
5.
Metwally, M.A.; Abdel-Galil, E.; Metwally, A.; Amer, F.A. New azodisperse dyes with thiazole, thiophene,
pyridone and pyrazolone moiety for dyeing polyester fabrics. Dyes Pigment. 2012,92, 902–908. [CrossRef]
6.
Aly, H.M.; El-Gazzar, M.G. Novel pyrazole derivatives as anticancer and radiosensitizing agents.
Arzneimittelforschung 2012,62, 105–112. [CrossRef] [PubMed]
7.
Puthiyapurayil, P.; Poojary, B.; Chikkanna, C.; Buridipad, S.K. Design, synthesis and biological evaluation of
a novel series of 1,3,4-oxadiazole bearing N-methyl-4-(trifluoromethyl) phenyl pyrazole moiety as cytotoxic
agents. Eur. J. Med. Chem. 2012,53, 203–210. [CrossRef] [PubMed]
8.
Hassan, G.S.; Kadry, H.H.; Abou-Seri, S.M.; Ali, M.M.; Mahmoud, A.E. Synthesis and
in vitro
cytotoxic
activity of novel pyrazolo[3,4-d]pyrimidines and related pyrazole hydrazones toward breast adenocarcinoma
MCF-7 cell line. Bioorg. Med. Chem. 2011,19, 6808–6817. [CrossRef] [PubMed]
9.
El-Zahar, M.I.; El-Karim, S.S.A.; Haiba, M.E.; Khedr, M.A. Synthesis, antitumor activity and molecular
docking study of novel benzofuran-2-yl pyrazole pyrimidine derivatives. Acta Pol. Pharm.
2011
,68, 357–373.
[PubMed]
10.
El-Gamal, M.I.; Sim, T.B.; Hong, J.H.; Cho, J.-H.; Yoo, K.H.; Oh, C.-H. Synthesis of 1H-pyrazole-1-carboxamide
derivatives and their antiproliferative activity against melanoma cell line. Arch. Pharm.
2011
,344, 197–204.
[CrossRef] [PubMed]
11.
Sharshira, E.M.; Hamada, N.M.M. Synthesis and antimicrobial evaluation of some pyrazole derivatives.
Molecules 2012,17, 4962–4971. [CrossRef] [PubMed]
12.
Ragavan, R.V.; Vijayakumar, V.; Kumari, N.S. Synthesis and antimicrobial activities of novel 1,5-diaryl
pyrazoles. Eur. J. Med. Chem. 2010,45, 1173–1180. [CrossRef] [PubMed]
13.
Ouyang, G.; Chen, Z.; Cai, X.-J.; Song, B.-A.; Bhadury, P.S.; Yang, S.; Jin, L.-H.; Xue, W.; Hu, D.-Y.; Zeng, S.
Synthesis and antiviral activity of novel pyrazole derivatives containing oxime esters group. Bioorg. Med.
Chem. 2008,16, 9699–9707. [CrossRef] [PubMed]
14.
Sanchez-Moreno, M.; Gomez-Contreras, F.; Navarro, P.; Marin, C.; Ramirez-Macias, I.; Olmo, F.; Sanz, A.M.;
Campayo, L.; Cano, C.; Yunta, M.J.
In vitro
leishmanicidal activity of imidazole- or pyrazole-based
benzo[g]phthalazine derivatives against Leishmania infantum and Leishmania braziliensis species.
J. Antimicrob. Chemoth. 2012,67, 387–397. [CrossRef] [PubMed]
15.
El-Din, M.M.M.; Senbel, A.M.; Bistawroos, A.A.; El-Mallah, A.; El-Din, N.A.N.; Bekhit, A.A.; El Razik, H.A.A.
A novel COX-2 inhibitor pyrazole derivative proven effective as an anti-inflammatory and analgesic drug.
Basic Clin. Pharmacol. 2011,108, 263–273. [CrossRef] [PubMed]

Appl. Sci. 2017,7, 785 11 of 13
16.
Raffa, D.; Migliara, O.; Maggio, B.; Plescia, F.; Cascioferro, S.; Cusimano, M.G.; Tringali, G.; Cannizzaro, C.;
Plescia, F. Pyrazolobenzotriazinone derivatives as COX inhibitors: Synthesis, biological activity, and
molecularmodeling studies. Arch. Pharm. Chem. Life Sci. 2010,10, 631–638. [CrossRef] [PubMed]
17.
Tomazetti, J.; Avila, D.S.; Ferreira, A.P.; Martins, J.S.; Souza, F.R.; Royer, C. Baker yeast-induced fever in
young rats: Characterization and validation of an animal model for antipyretics screening. J. Neurosci.
Methods 2005,147, 29–35. [CrossRef] [PubMed]
18.
Dekhane, D.V.; Pawar, S.S.; Gupta, S.; Shingare, M.S.; Patil, C.R.; Thore, S.N. Synthesis and anti-inflammatory
activity of some new 4,5-dihydro-1,5-diaryl-1H-pyrazole-3-substituted-heteroazole derivatives. Bioorg. Med.
Chem. Lett. 2011,21, 6527–6532. [CrossRef] [PubMed]
19.
Agrawal, R.; Jain, P.; Dikshit, S.N. Apixaban: A new player in the anticoagulant class. Curr. Drug Targets
2012,13, 863–875. [CrossRef] [PubMed]
20.
Backhouse, K.; Sarac, I.; Shojaee-Moradie, F.; Stolinski, M.; Robertson, M.D.; Frost, G.S.; Bell, J.D.;
Thomas, E.L.; Wright, J.; Russell-Jones, D.; et al. Fatty acid flux and oxidation are increased by rimonabant in
obese women. Metabolis 2012,61, 1220–1223. [CrossRef] [PubMed]
21.
Ranu, B.C.; Jana, R.; Sowmiah, S. An improved procedure for the three-component synthesis of highly
substituted pyridines using ionic liquid. J. Org. Chem. 2007,72, 3152–3154. [CrossRef] [PubMed]
22.
Abbas, I.M.; Gomha, S.M.; Elaasser, M.M.; Bauomi, M.A. Synthesis and biological evaluation of new
pyridines containing imidazole moiety as antimicrobial and anticancer agents. Turk. J. Chem.
2015
,39,
334–346. [CrossRef]
23.
Xu, W.C.; Zhou, Q.; Ashendel, C.L.; Chang, C.T.; Chang, C.J. Novel protein kinase C inhibitors: Synthesis
and PKC inhibition of
β
-substituted polythiophene derivatives. Bioorg. Med. Chem. Lett.
1999
,9, 2279–2282.
[CrossRef]
24.
Zhao, L.X.; Moon, Y.S.; Basnet, A.; Kim, E.K.; Jahng, Y.; Park, J.G.; Jeong, T.C.; Cho, W.J.; Choi, S.U.;
Lee, C.O.; et al. The discovery and synthesis of novel adenosine receptor (A2A) antagonists. Bioorg. Med.
Chem. Lett. 2004,14, 1333–1336. [CrossRef] [PubMed]
25.
Zhao, L.X.; Sherchan, J.; Park, J.K.; Jahng, Y.; Jeong, B.S.; Jeong, T.C.; Lee, C.S.; Lee, E.S. Synthesis, cytotoxicity
and structure-activity relationship study of terpyridines. Arch. Pharm. Res.
2006
,29, 1091–1095. [CrossRef]
[PubMed]
26.
Son, J.K.; Zhao, L.X.; Basnet, A.; Thapa, P.; Karki, R.; Na, Y.; Jahng, Y.; Jeong, T.C.; Jeong, B.S.; Lee, C.S.; et al.
Synthesis of 2,6-diaryl-substituted pyridines and their antitumor activities. Eur. J. Med. Chem.
2008
,43,
675–682. [CrossRef] [PubMed]
27.
Thapa, P.; Karki, R.; Choi, H.Y.; Choi, J.H.; Yun, M.; Jeong, B.S.; Jung, M.J.; Nam, J.M.; Na, Y.; Cho, W.J.; et al.
Synthesis of 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine derivatives and evaluation of their topoisomerase
I and II inhibitory activity, cytotoxicity, and structure–activity relationship. Bioorg. Med. Chem.
2010
,18,
2245–2254. [CrossRef] [PubMed]
28.
Jeong, B.S.; Choi, H.Y.; Thapa, P.; Karki, R.; Lee, E.; Nam, J.M.; Na, Y.; Ha, E.-M.; Kwon, Y.; Lee, E.S. Synthesis,
Topoisomerase I and II Inhibitory activity, cytotoxicity, and structure-activity relationship study of rigid
analogues of 2,4,6-trisubstituted pyridine containing 5,6-dihydrobenzo[h]quinoline moiety. Bull. Korean
Chem. Soc. 2011,32, 303–306. [CrossRef]
29.
Eryazici, I.; Moorefield, C.N.; Newkome, G.R. Square-Planar Pd(II), Pt(II), and Au(III) Terpyridine Complexes:
Their Syntheses, Physical Properties, Supramolecular Constructs, and Biomedical Activities. Chem. Rev.
2008,108, 1834–1895. [CrossRef] [PubMed]
30.
Patel, K.K.; Plummer, E.A.; Darwish, M.; Rodger, A.; Hannon, M.J. Aryl substituted ruthenium
bis-terpyridine complexes: Intercalation and groove binding with DNA. J. Inorg. Biochem.
2002
,91, 220–229.
[CrossRef]
31.
Weber, L. The application of multi-component reactions in drug discovery. Curr. Med. Chem.
2002
,9,
2085–2093. [CrossRef] [PubMed]
32.
Hulme, C.; Gore, V. Multi-component reactions: Emerging chemistry in drug discovery from xylocain to
crixivan. Curr. Med. Chem. 2003,10, 51–80. [CrossRef] [PubMed]
33.
Nair, V.; Rajesh, C.; Vinod, A.U.; Bindu, S.; Sreekanth, A.R.; Mathen, J.S.; Balagopal, L. Strategies for
heterocyclic construction via novel multicomponent reactions based on isocyanides and nucleophilic
carbenes. Acc. Chem. Res. 2003,36, 899–907. [CrossRef] [PubMed]

Appl. Sci. 2017,7, 785 12 of 13
34.
Andrade, C.K.Z.; Barreto, A.S.; Silva, W.A. Microwave assisted solvent-, support- and catalyst-free synthesis
of enaminones. Ark. Arch. Org. Chem. 2008,xii, 226–232.
35.
Bortolini, O.; D’Agostino, M.; De Nino, A.; Maiuolo, L.; Nardi, M.; Sindona, G. Solvent-free, microwave
assisted 1,3-cycloaddition of nitrones with vinyl nucleobases for the synthesis of N,O-nucleosides. Tetrahedron
2008,64, 8078–8081. [CrossRef]
36.
Ganesan, S.; Muthuraaman, B.; Madhavan, J.; Mathew, V.; Maruthamuthu, P.; Suthanthiraraj, S.A. The use
of 2,6-bis (N-pyrazolyl) pyridine as an efficient dopant in conjugation with poly(ethylene oxide) for
nanocrystalline dye-sensitized solar cells. Electrochim. Acta 2008,53, 7903–7907. [CrossRef]
37.
Manikandan, P.; Padmakumar, K.; Justin Thomas, K.R.; Varghese, B.; Onodera, H.; Manoharan, P.T.
Lattice-Dictated Conformers in Bis(pyrazolyl)pyridine-Based Iron(II) Complexes: Mössbauer, NMR, and
Magnetic Studies. Inorg. Chem. 2001,40, 6930–6939. [CrossRef] [PubMed]
38.
Jameson, D.L.; Goldsby, K.A. 2,6-bis(N-pyrazolyl)pyridines: The convenient synthesis of a family of planar
tridentate N3 ligands that are terpyridine analogs. J. Org. Chem. 1990,55, 4992–4994. [CrossRef]
39.
Zoppellaro, G.; Enkelmann, V.; Geies, A.; Baumgarten, M. A Multifunctional High-Spin Biradical
Pyrazolylbipyridine-bisnitronylnitroxide. Org. Lett. 2004,6, 4929–4932. [CrossRef] [PubMed]
40.
Prakash, O.; Hussain, K.; Kumar, R.; Wadhwa, D.; Sharma, C.; Aneja, K.R. Synthesis and antimicrobial
evaluation of new1,4-dihydro-4-pyrazolylpyridines and 4-Pyrazolylpyridines. Org. Med. Chem. Lett.
2011
,1,
1–7. [CrossRef] [PubMed]
41.
Motaung, M.P.; Ajibade, P.A. Ru(II) and Co(II) Complexes of bis(pyrazolyl)pyridine and pyridine-2,6-
dicarboxylic Acid: Synthesis, Photo Physical Studies and Evaluation of Solar Cell Conversion Efficiencies.
Int. J. Electrochem. Sci. 2015,10, 8087–8096.
42.
Zoppellaro, G.; Geies, G.; Enkelmann, V.; Baumgarten, M. 2,6-Bis(pyrazolyl)pyridine Functionalised with
Two Nitronylnitroxide and Iminonitroxide Radicals. Eur. J. Org. Chem. 2004,2004, 2367–2374. [CrossRef]
43.
Gomha, S.M.; Salah, T.A.; Abdelhamid, A.O. Synthesis, characterization and pharmacological evaluation
of some novel thiadiazoles and thiazoles incorporating pyrazole moiety as potent anticancer agents.
Monatsh. Chem. 2015,146, 149–158. [CrossRef]
44.
Gomha, S.M.; Abdel-aziz, H.M. Synthesis and antitumor activity of 1,3,4-thiadiazole derivatives bearing
coumarine ring. Heterocycles 2015,91, 583–592. [CrossRef]
45.
Dawood, K.M.; Gomha, S.M. Synthesis and anti-cancer activity of 1,3,4-thiadiazole and 1,3-thiazole
derivatives having 1,3,4-oxadiazole moiety. J. Heterocycl. Chem. 2015,52, 1400–1405. [CrossRef]
46.
Abbas, I.M.; Gomha, S.M.; Elneairy, M.A.A.; Elaasser, M.M.; Mabrouk, B.K.A. Antimicrobial and anticancer
evaluation of novel synthetic tetracyclic system through Dimroth rearrangement. J. Serb. Chem. Soc.
2015
,80,
1251–1264.
47.
Gomha, S.M.; Abbas, I.M.; Elneairy, M.A.A.; Elaasser, M.M.; Mabrouk, B.K.A. Synthesis and biological
evaluation of novel fused triazolo[4,3-a] pyrimidinones. Turk. J. Chem. 2015,39, 510–531.
48. Gomha, S.M.; Zaki, Y.H.; Abdelhamid, A.O. Utility of 3-acetyl-6-bromo-2H-chromen-2-one for synthesis of
new heterocycles as potential anticancer agents. Molecules 2015,20, 21826–21839. [CrossRef] [PubMed]
49.
Gomha, S.M.; Riyadh, S.M.; Mahmmoud, E.A.; Elaasser, M.M. Chitosan-grafted-poly(4-vinylpyridine) as a
novel copolymer basic catalyst for synthesis of arylazothiazoles and 1,3,4-thiadiazoles under microwave
irradiation. Chem. Heterocycl. Comp. 2015,51, 1030–1038. [CrossRef]
50.
Gomha, S.M.; Salah, T.A.; Hassaneen, H.M.E.; Abdel-aziz, H.; Khedr, M.A. Synthesis, characterization and
molecular docking of novel bioactive thiazolyl-thiazole derivatives as promising cytotoxic antitumor drug.
Molecules 2016,21, 3. [CrossRef] [PubMed]
51.
Gomha, S.M.; Badrey, M.G.; Edrees, M.M. Heterocyclization of a bis-thiosemicarbazone of 2,5-
diacetyl-3,4-disubstituted-thieno[2,3-b]thiophene bis-thiosemicarbazones leading to bis-thiazoles and
bis-1,3,4-thiadiazoles as anti-breast cancer agents. J. Chem. Res. 2016,40, 120–125. [CrossRef]
52.
Gomha, S.M.; Abdallah, M.A.; Mourad, M.A.; Elaasser, M.M. Application of Mannich and Michael reactions
in synthesis of pyridopyrimido[2,1-b][1,3,5]thiadiazinones and pyridopyrimido[2,1-b][1,3]thiazinones and
their biological activity. Heterocycles 2016,92, 688–699. [CrossRef]
53.
Ibrahim, M.K.; El-Ghandour, A.H.H.; Abou-hadeed, K.; Abdelhafiz, I.S. Utility of hydzidoyl chlorides:
Synthesis of new pyrazoles, pyrazolo[3,4-d]pyridazines, pyrazolo[4,5-b]pyridines and pyrazolo[4,5-d]
pyrimidine derivatives. J. Ind. Chem. Soc. 1992,69, 378–380.

Appl. Sci. 2017,7, 785 13 of 13
54.
Gomha, S.M.; Abdalla, M.A.; Abdelaziz, M.; Serag, N. Eco-friendly one-pot synthesis and antiviral evaluation
of pyrazolyl chalcones and pyrazolines of medicinal interest. Turk. J. Chem. 2016,40, 484–498. [CrossRef]
55.
Shawali, A.S.; Gomha, S.M. A new entry for short and regioselective synthesis of [1,2,4]triazolo[4,3-b][1,2,4]-
triazin-7(1H)-one. Adv. Synth. Catal. 2000,342, 599–604. [CrossRef]
56.
Gomha, S.M.; Riyadh, S.M.; Abbas, I.M.; Bauomi, M.A. Synthetic utility of ethylidenethiosemicarbazide:
Synthesis and and anti-cancer activity of 1,3-thiazines and thiazoles with imidazole moiety. Heterocycles
2012,87, 341–356.
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