Synthesis of some new Pyridine-based Heterocyclic Compounds with Anticipated Antitumor Activity

Article abstract of DOI:10.1002/jhet.3210

A novel class of 5-amino-N′-(1-(pyridin-4-yl)ethylidene)-1H-pyrazole-4-carbohydrazides and 8-(pyridin-4-yl)pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-ones was synthesized from reaction of 2-cyano-N′-(1-(pyridin-4-yl)ethylidene)-acetohydrazide and

Full text of DOI:10.1002/jhet.3210

Month 2018
Synthesis of some new Pyridine-based Heterocyclic Compounds with
Anticipated Antitumor Activity
Sobhi M. Gomha,a
Fathy M. Abdelrazek,a,b
Aly H. Abdelrahman,a and Peter Metzb
*
aChemistry Department, Faculty of Science, Cairo University, Giza 12613, Egypt
bInstitute of Organic Chemistry, TU Dresden, Dresden 01062, Germany
*
E-mail: prof.fmrazek@gmail.com
Received December 17, 2017
DOI 10.1002/jhet.3210
Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com).
A novel class of 5-amino-N0-(1-(pyridin-4-yl)ethylidene)-1H-pyrazole-4-carbohydrazides and 8-(pyridin-
4-yl)pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-ones was synthesized from reaction of 2-cyano-N0-
(1-(pyridin-4-yl)ethylidene)-acetohydrazide
and
7-(pyridin-4-yl)-2-thioxo-2,3-dihydropyrido[2,3-d]
pyrimidin-4(1H)-one with the appropriate hydrazonoyl halides. Moreover, 2-cyano-N0-(1-(pyridin-4-yl)-
ethylidene)-acetohydrazide was used for the synthesis of 2-cyano-N0-(1-(pyridin-4-yl)ethylidene)-
acrylohydrazides and 2-oxo-2-(2-(1-(pyridin-4-yl)ethylidene)-hydrazinyl)-acetohydrazonoyl cyanides. The
structures of the newly prepared compounds were confirmed by both elemental and spectral analyses as well
as by alternate synthesis. The anticancer activities of the prepared compounds were screened against the he-
patocellular carcinoma (HepG2) cell line, and the results showed that most of the compounds exhibit consid-
erable activities.
J. Heterocyclic Chem., 00, 00 (2018).
INTRODUCTION
As a result, it has been reported that various pyridine
derivatives, as bioisosteres of α-terthiophene that are
potent protein kinase C inhibitor [4,5], has significant
Identification of novel structure leads that may be of
use in designing new, potent, selective, and less toxic
topoisomerase
I
and/or II inhibitory activity,
anticancer agents remains
a
major challenge for
and cytotoxicity against several human cancer cell lines
[6–16]. Early reports on the ability of α-terpyridine to
form metal complex [17,18] and to bind with DNA/RNA
[19,20] has been the base for the study on pyridine
derivatives as antitumor agents.
In the last decade, many researchers have reported a
large series of pyrazole derivatives having promising
anticancer activities [21–30], indicating the use of
medicinal chemistry researchers. 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 [1–3].
Among the successful examples as drug, candidates
possessing
pyridine
nucleus
are
streptonigrin,
streptonigrone, and lavendamycin that 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.
pyrazole motif as
a
powerful tool for novel
anticancer drug development. Moreover, the 1,2,4-
triazolopyrimidines have also attracted growing
interest due to their important pharmacological activities,
such as antitumor potency, antimalarial, antimicrobial,
© 2018 Wiley Periodicals, Inc.

S. M. Gomha, F. M. Abdelrazek, A. H. Abdelrahman, and P. Metz
Vol 000
anti-inflammatory, antifungal, and macrophage activation
[31–35].
υmax = 1708 and 1663 cmꢀ1 due to the two carbonyl
groups, in addition to the presence of a broad absorption
band at υmax = 3430–3223 cmꢀ1 attributed to the
In the course of our recent investigations aiming at the
synthesis of a variety of heterocyclic ring systems with
remarkable biological importance [36–38], it was
envisioned that the combination of a pyridine ring with
other heterocyclic rings in one entity may lead to
enhanced anticancer activity due to the synergistic effect
of both rings. 4-Acetylpyridine scaffold, as a highly
versatile building block for the synthesis of a wide
variety of several new pyridine-based heterocyclic
derivatives, seemed a suitable starting material to fulfill
this objective.
1
amide-NH and the NH2 groups. The HNMR spectrum of
products 6 revealed the absence of the signal at δ
2.98 ppm of the CH2 group and instead appeared
one broad singlet for two NH2 protons, in addition to the
expected signals of the methyl, amide NH, and aryl
protons. The mass spectra of products 6 showed
in each case a molecular ion peak at the correct
molecular weight (Experimental). The obtained data
gave a firm support for the aminopyrazole structure
6
and excluded the other possible cyanopyrazole
derivative 7.
A further confirmation of structures 6a–h was deduced
via the alternative synthesis of 6a. Thus, reaction of 4-
acetylpyridine 1 with 5-amino-1,3-diphenyl-1H-pyrazole-
4-carbohydrazide 8 [40] in ethanol under reflux afforded
a product that is identical in all respects with 6a (m.p.,
mixed m.p., and IR spectra) obtained from the reaction of
3 with 4a (Scheme 1).
It is assumed that 3 reacts with 4a–h via elimination of
HCl molecule to give the non-isolable intermediates
5, which undergo in situ cyclization via nucleophilic
addition of the hydrazone-NH to the cyano group to
give the pyrazole derivative 6a–h as outlined in
Scheme 1.
RESULTS AND DISCUSSION
2-Cyano-N0-(1-(pyridin-4-yl)-ethylidene)-acetohydrazide
was obtained from the condensation of 2-
cyanoacetohydrazide 2 with 4-acetylpyridine 1 in refluxing
ethanol as reported by EL-Hawash et al. as outlined in
Scheme 1 [39].
3
The reaction of the pyridylhydrazide derivative 3 with
hydrazonoyl chlorides 4a–h in ethanolic sodium ethoxide
solution [prepared from sodium metal and absolute
ethanol] at room temperature afforded in each case one
isolable product (TLC analysis). The aminopyrazole
structures 6a–h, rather than the other possible structure 7
(Scheme 1) were assigned for these products on the basis
Moreover, compound 3 was allowed to condense with
a number of benzaldehyde derivatives 9a–d in EtOH in
the presence of piperidine under reflux for 6 h
1
of their microanalyses and spectral (IR, H NMR, Mass)
data. For example, the IR spectra of products 6 revealed
(monitored by TLC) to afford the corresponding
in each case three characteristic absorption bands at
arylidene derivative 10a–d (Scheme 2). The structures
Scheme 1. Synthesis of pyrazole derivatives 6a–h.
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Pyridine-based Heterocyclic Compounds
Scheme 2. Synthesis of compounds 10a–d and 12a–d.
of the products 10a–d were established via elemental
analysis and spectral (IR, 1HNMR, Mass) data. For
example, the IR spectra of products 10 revealed in each
case the characteristic absorption bands as in compound
3, except υmax value of the nitrile group decreased due
to its conjugation with the arylmethylene double bond
(Experimental). The mass spectrum of 18 exhibited a
molecular ion peak at m/z = 332 that is consistent with
the assigned structure.
Reaction of compound 18 with each of the
hydrazonoyl chlorides 4a–e in dioxane, in the presence
of triethylamine under reflux, gave in each case a single
product consistent with structure 21 (Scheme 2) based
on spectral data (IR, 1HNMR, and MS) and elemental
analyses (Experimental). As depicted in Scheme 3, the
reaction is assumed to proceed through S-alkylation to
give the non-isolable S-alkylated intermediates 19,
which undergo in situ tandem rearrangement into 20
followed by cyclization via loss of H2S to give the
final products 21.
1
(ArCH¼). The HNMR spectra showed the absence of
the singlet signal of the methylene protons, and instead
revealed the characteristic signal assigned for the
olefinic proton (ArCH¼) near δ 8.8 ppm, in addition to
the other expected signals for the aromatic, NH, and
Me protons.
The azo coupling reaction of the pyridinylacetohydrazide
derivative 3 with arenediazonium chloride derivatives
11a–d in ethanol in the presence of pyridine as basic
catalyst at low temperature (0–5°C) afforded the hydrazo
products 12a–d (Scheme 2). The structures of 12a–d were
deduced from their elemental and spectral data (IR,
1HNMR, Mass) (Experimental).
ANTICANCER ACTIVITY
The anticancer activity of some newly synthesized
compounds was determined against a liver carcinoma
cell line HEPG2, using doxorubicin as a reference drug.
The data generated were used to plot a dose–response
curve of which the concentration (μM) of the test
compounds required to kill 50% of cell population
(IC50) was determined. The cytotoxic activity was
expressed as the mean IC50 of three independent
experiments (Table 1) and the results revealed that all
the tested compounds showed inhibitory activity to the
tumor cell lines in a concentration dependent manner.
The small values of IC50 for the selected compounds
indicate that for more anticancer effect, higher
concentrations should be used.
We have also investigated reaction of the chalcone 13
[41] with 6-amino-2-thioxo-(1H)-pyrimidin-4-one 14
that produced 5-phenyl-7-(pyridin-4-yl)-2-thioxo-2,3-
dihydropyrido[2,3-d]pyrimidin-4(1H)-one 18 as shown in
Scheme 3. Structure 18 was assigned on the basis of its
1
analytical and spectral data (IR, HNMR, Mass). Thus,
the IR spectrum of thione 18 revealed three characteristic
absorption bands at υmax = 3346, 3217, and 1678 cmꢀ1
for the two NH groups and the carbonyl group. The
1HNMR spectrum of 18 revealed three singlet signals at
δ = 8.06, 11.73, and 12.42 assignable for the pyridine–
CH5 and two NH protons, respectively, in addition to the
expected signals attributed to the nine aromatic protons
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S. M. Gomha, F. M. Abdelrazek, A. H. Abdelrahman, and P. Metz
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Scheme 3. Synthesis of triazolopyridopyrimidines 21a–e.
Table 1
The in vitro inhibitory activity of tested compounds against a liver carcinoma cell line HEPG2 expressed as IC50 values (μM). [Color table can be viewed
at wileyonlinelibrary.com]
Compound No.
Doxorubicin
6a
6b
6c
6d
6e
6f
6 g
R
-
X
-
H
IC50 (μM)
0.74
35.04
5.33
2.12
10.9
17.9
1.05
0.97
7.64
5.31
2.48
4.9
Ph
CH3CO
CH3CO
CH3CO
CH3CO
CH3CH2COO
CH3CH2COO
CH3CH2COO
H
4-Me
4-Cl
4-NO2
H
4-Me
4-Cl
H
4-OMe
4-Cl
4-NO2
H
4-Me
4-Cl
4-NO2
6 h
10a
10b
10c
10d
12a
12b
12c
12d
-
-
-
-
-
-
-
-
21.3
20.3
10.3
17.9
19.0
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Pyridine-based Heterocyclic Compounds
The results represented in Table 1 showed that
NH2), 3063, 2927 (C–H), 1664 (C¼O), 1599 (C¼N)
cmꢀ1 1H NMR (DMSO-d6): δ 2.46 (s, 3H, CH3),
;
• The in vitro inhibitory activities of tested compounds
against the human liver carcinoma (HEPG2) have the
descending order as follows: 6g > 6f > 6c > 10b >
10c > 10a > 6b > 6h > 6d > 12b > 6e > 12c >
12d > 12a > 10d > 6a.
• The aminopyrazole derivatives that containing ester group
havein vitro inhibitory activity more than the aminopyrazole
derivatives containing acetyl group (6f > 6b, 6f > 6c, 6h
> 6d).
6.98–8.11 (m, 14H, Ar–H), 8.65 (s, br, 2H, NH2), 10.79 (s,
br, 1H, NH); 13C-NMR (DMSO-d6): δ 22.3 (q, CH3), 99.6
(s), 123.2 (d), 124.3 (d), 126.3 (d), 127.4 (d), 128.9 (d),
129.1 (d), 129.3 (d), 132.9 (s), 137.1 (s), 138.5 (s), 147.8
(s), 149.2 (s), 149.4 (d), 151.5 (s), 163.4 (s, C¼O) ppm.
MS m/z (%): 396 (M+, 9), 271 (49), 189 (47), 105 (52), 77
(100). Anal. Calcd for C23H20N6O (396.17): C, 69.68; H,
5.08; N, 21.20. Found C, 69.49; H, 5.01; N, 21.07%.
3-Acetyl-5-amino-1-phenyl-N0-(1-(pyridin-4-yl)ethylidene)-
• The introduction of electron-withdrawing group (chlorine
atom and nitro group) at the 4-position of phenyl group at
the pyrazole ring decreases the antitumor activity. In
contrast, introduction of electron-donating group (methyl
group) enhances the antitumoractivity (6c > 6b > 6d> 6e
and 6g > 6f > 6h).
1H-pyrazole-4-carbohydrazide (6b).
Yield 73%; yellow
solid; mp 203–205°C (EtOH). IR (KBr): v 3434–3223
(NH and NH2), 3030, 2923 (C–H), 1708, 1654 (2C¼O),
1
1599 (C¼N) cmꢀ1; H NMR (DMSO-d6): δ 2.34 (s, 3H,
CH3), 2.49 (s, 3H, CH3), 6.90–8.07 (m, 9H, Ar–H), 8.69
(s, br, 2H, NH2), 10.81 (s, br, 1H, NH); MS m/z (%): 362
(M+, 18), 297 (24), 189 (36), 109 (73), 77 (100). Anal.
Calcd for C19H18N6O2 (362.15): C, 62.97; H, 5.01; N,
23.19. Found C, 62.88; H, 4.87; N, 23.12%.
EXPERIMENTAL
3-Acetyl-5-amino-N0-(1-(pyridin-4-yl)ethylidene)-1-(p-tolyl)-
All melting points were measured on a Gallenkamp
melting point apparatus. The infrared spectra were
recorded in potassium bromide disks on a Pye Unicam
SP 3300 and Shimadzu FT IR 8101 PC infrared
1H-pyrazole-4-carbohydrazide (6c).
solid; mp 219–221°C (DMF/EtOH); IR (KBr): v 3436–
3216 (NH and NH2), 3030, 2923 (C–H), 1703, 1647
Yield 76%; yellow
(2C¼O), 1599 (C¼N) cmꢀ1
;
1H NMR (DMSO-d6): δ
1
spectrophotometers. The H NMR & 13C NMR spectra
2.24 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.49 (s, 3H, CH3),
6.79–7.73 (m, 8H, Ar–H), 8.68 (s, br, 2H, NH2), 10.95
(s, br, 1H, NH); MS m/z (%): 376 (M+, 31), 233 (73),
218 (100), 190 (58), 71 (39). Anal. Calcd for
C20H20N6O2 (376.16): C, 63.82; H, 5.36; N, 22.33.
were recorded on a Varian Mercury VX-300 NMR
spectrometer (1H NMR: 300 MHz & 13C NMR:
75 MHz) in DMSO-d6. Chemical shifts were related to
that of the solvent. 13C multiplicities were determined by
using DEPT and off-resonance pulse sequence. Mass
spectra were recorded on a Shimadzu GCMS-QP 1000
EX mass spectrometer at 70 e.V. Elemental analyses were
carried out at the Microanalytical Center of Cairo
University, Giza, Egypt. Antitumor activity was evaluated
at the Regional Center for Mycology and Biotechnology
at Al-Azhar University, Cairo, Egypt. Hydrazonoyl halides
4a–h [42] were prepared as previously reported in the re-
spective literature.
Found C, 63.64; H, 5.31; N, 22.16%.
3-Acetyl-5-amino-1-(4-chlorophenyl)-N0-(1-(pyridin-4-yl)
ethylidene)-1H-pyrazole-4-carbohydrazide (6d). Yield 73%;
brown solid; mp 230–232°C (DMF/EtOH); IR (KBr): v
3433–3216 (NH and NH2), 3039, 2925 (C–H), 1707,
1655 (2C¼O), 1601 (C¼N) cmꢀ1; H NMR (DMSO-d6):
1
δ 2.39 (s, 3H, CH3), 2.49 (s, 3H, CH3), 7.00–8.10 (m,
8H, Ar–H), 8.77 (s, br, 2H, NH2), 10.74 (s, br, 1H, NH);
MS m/z (%): 396 (M+, 12), 316 (38), 230 (62), 127 (83),
81 (100), 57 (73). Anal. Calcd for C19H17ClN6O2
(396.11): C, 57.51; H, 4.32; N, 21.18. Found C, 57.46;
Reaction of
3 with hydrazonoyl chlorides 4a–h.
Pyridinylacetohydrazide derivative 3 (0.202 g, 1 mmol)
was added while stirring to an ethanolic sodium ethoxide
solution [prepared from sodium metal (0.23 g, 1 mmol)
and absolute ethanol (15 mL)]. The resulting solution
was stirred for 15 min, then the appropriate hydrazonoyl
halide 4a–h (1 mmol) was added portionwise and stirring
of the reaction mixture was continued for further 6–10 h
at room temperature. The solid that formed in each case
was filtered off, washed with water, and dried.
Recrystallization from DMF or DMF\EtOH afforded the
corresponding pyrazole derivatives 6a–h in 66–80%
H, 4.27; N, 21.10%.
3-Acetyl-5-amino-1-(4-nitrophenyl)-N0-(1-(pyridin-4-yl)
ethylidene)-1H-pyrazole-4-carbohydrazide (6e). Yield 77%;
brown solid; mp 193–195°C (EtOH); IR (KBr): v 3432–
3216 (NH and NH2), 3053, 2926 (C–H), 1716, 1664
(2C¼O), 1597 (C¼N) cmꢀ1;1H NMR (DMSO-d6): δ
2.37 (s, 3H, CH3), 2.50 (s, 3H, CH3), 7.31–8.38 (m, 8H,
Ar–H), 8.78 (s, br, 2H, NH2), 10.48 (s, br, 1H, NH); MS
m/z (%): 407 (M+, 20), 366 (46), 323 (37), 228 (92), 121
(100), 42 (84). Anal. Calcd for C19H17N7O4 (407.13): C,
56.02; H, 4.21; N, 24.07. Found C, 55.94; H, 4.14; N,
23.93%.
yields.
5-Amino-1,3-diphenyl-N0-(1-(pyridin-4-yl)ethylidene)-1H-
pyrazole-4-carbohydrazide (6a). Yield 70%; yellow solid;
Ethyl
5-amino-1-phenyl-4-(2-(1-(pyridin-4-yl)ethylidene)
mp 247–249°C (DMF). IR (KBr): v 3426–3210 (NH and
hydrazinecarbonyl)-1H-pyrazole-3-carboxylate (6f).
Yield
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S. M. Gomha, F. M. Abdelrazek, A. H. Abdelrahman, and P. Metz
Vol 000
73%; yellow solid; mp 242–244°C (DMF/EtOH); IR
(KBr): v 3444–3190 (NH and NH2), 3041, 2924 (C–
213–215°C (EtOH); IR (KBr): v 3429 (NH), 3056, 2930
1
(C–H), 2207 (CN), 1662 (C¼O), 1599 (C¼N) cmꢀ1; H
1
H), 1737, 1634 (2C¼O), 1599 (C¼N) cmꢀ1; H NMR
NMR (DMSO-d6): δ 2.29 (s, 3H, CH3), 6.73–7.90 (m,
9H, Ar–H), 8.84 (s, 1H, CH¼), 10.02 (s, br, 1H, NH);
13C-NMR (DMSO-d6): δ 22.5 (q, CH3), 106.6 (s), 115.6
(s, CN), 124.0 (d), 127.6 (d), 128.2 (d), 131.3 (d), 132.4
(s), 138.2 (s), 147.4 (s), 149.5 (d), 150.5 (d), 168.9 (s,
C¼O) ppm. MS m/z (%): 290 (M+, 18), 217 (40), 178
(49), 129 (66), 93 (100), 71 (60). Anal. Calcd for
C17H14N4O (290.12): C, 70.33; H, 4.86; N, 19.30. Found
(DMSO-d6): δ 1.23 (t, J = 7.2 Hz, 3H, CH3), 2.41 (s,
3H, CH3), 4.25 (q, J = 7.2 Hz, 2H, CH2), 7.20–7.74
(m, 9H, Ar–H), 8.62 (s, br, 2H, NH2), 10.45 (s, br,
1H, NH); MS m/z (%): 392 (M+, 9), 284 (51), 202
(61), 77 (100). Anal. Calcd for C20H20N6O3 (392.16):
C, 61.21; H, 5.14; N, 21.42. Found C, 61.09; H, 5.10;
N, 21.31%.
Ethyl
5-amino-4-(2-(1-(pyridin-4-yl)ethylidene)
C, 70.21; H, 4.75; N, 19.00%.
2-Cyano-3-(4-methoxyphenyl)-N0-(1-(pyridin-4-yl)
ethylidene)acrylohydrazide (10b). Yield 77%; yellow solid;
hydrazinecarbonyl)-1-(p-tolyl)-1H-pyrazole-3-carboxylate
(6g). Yield 74%; yellow solid; mp 220–222°C (DMF/
EtOH); IR (KBr): v 3425–3193 (NH and NH2), 3041,
mp 177–179°C; IR (KBr): v 3429 (NH), 3042, 2930
1
2920 (C–H), 1737, 1634 (2C¼O), 159 (C¼N) cmꢀ1; H
1
(C–H), 2199 (CN), 1660 (C¼O), 1599 (C¼N) cmꢀ1; H
NMR (DMSO-d6): δ 1.05 (t, J = 7.2 Hz, 3H, CH3), 2.34
(s, 3H, CH3), 2.42 (s, 3H, CH3), 4.43 (q, J = 7.2 Hz, 2H,
CH2), 7.13–8.31 (m, 8H, Ar–H), 8.68 (s, br, 2H, NH2),
10.37 (s, br, 1H, NH); MS m/z (%): 406 (M+, 24), 316
(100), 249 (44), 127 (72), 81 (65), 57 (85). Anal. Calcd
for C21H22N6O3 (406.18): C, 62.06; H, 5.46; N, 20.68.
Found C, 61.92; H, 5.41; N, 20.57%.
NMR (DMSO-d6): δ 2.28 (s, 3H, CH3), 3.79 (s, 3H,
OCH3), 7.25–8.58 (m, 8H, Ar–H), 8.74 (s, 1H, CH¼),
10.00 (s, br, 1H, NH); MS m/z (%): 320 (M+, 17), 203
(73), 160 (62), 119 (54), 78 (100). Anal. Calcd for
C18H16N4O2 (320.13): C, 67.49; H, 5.03; N, 17.49.
Found C, 67.37; H, 5.01; N, 17.35%.
3-(4-Chlorophenyl)-2-cyano-N0-(1-(pyridin-4-yl)ethylidene)
Ethyl
5-amino-1-(4-chlorophenyl)-4-(2-(1-(pyridin-4-yl)
acrylohydrazide (10c).
Yield 74%; yellow solid; mp
ethylidene)hydrazinecarbonyl)-1H-pyrazole-3-carboxylate
(6h). Yield 76%; yellow solid; mp 251–253°C (DMF/
EtOH); IR (KBr): v 3438–3173 (NH and NH2), 3032,
210–212°C; IR (KBr): v 3435 (NH), 3034, 2929 (C–H),
1
2199 (CN), 1663 (C¼O), 1596 (C¼N) cmꢀ1; H NMR
(DMSO-d6): δ 2.33 (s, 3H, CH3), 7.32–8.56 (m, 8H,
Ar–H), 8.72 (s, 1H, CH¼), 10.03 (s, br, 1H, NH); MS
m/z (%): 324 (M+, 14), 229 (63), 177 (49), 125 (68), 78
(100). Anal. Calcd for C17H13ClN4O (324.08): C, 62.87;
1
2926 (C–H), 1739, 1644 (2C¼O), 1599 (C¼N) cmꢀ1; H
NMR (DMSO-d6): δ 1.16 (t, J = 7.2 Hz, 3H, CH3), 2.42
(s, 3H, CH3), 4.46 (q, J = 7.2 Hz, 2H, CH2), 7.19–8.27
(m, 8H, Ar–H), 8.66 (s, br, 2H, NH2), 10.39 (s, br, 1H,
NH); MS m/z (%): 426 (M+, 9), 330 (100), 272 (30), 194
(63), 57 (81). Anal. Calcd for C20H19ClN6O3 (426.12):
C, 56.28; H, 4.49; N, 19.69. Found C, 56.16; H, 4.40; N,
19.43%.
H, 4.03; N, 17.25. Found C, 62.69; H, 3.80; N, 17.13%.
2-Cyano-3-(4-nitrophenyl)-N0-(1-(pyridin-4-yl)ethylidene)
acrylohydrazide (10d).
Yield 74%; yellow solid; mp
241–243°C (DMF/EtOH); IR (KBr): v 3431 (NH), 3046,
2929 (C–H), 2199 (CN), 1682 (C¼O), 1596 (C¼N)
1
cmꢀ1; H NMR (DMSO-d6): δ 2.38 (s, 3H, CH3), 7.27–
Alternate synthesis of 6a.
A mixture of equimolar
amounts of the 4-acetylpyridine (1) (0.121 g, 1 mmol)
and 5-amino-1,3-diphenyl-1H-pyrazole-4-carbohydrazide
(8) (0.293 g, 1 mmol) in 20 mL of ethanol was refluxed
for 6 h (monitored by TLC). The solvent was evaporated
and the residue was triturated with methanol. The solid
that formed was filtered and recrystallized from DMF to
give compound 6a that was identical in all respects (m.p.,
mixed m.p., and IR spectra) with that obtained from
reaction of 3 with 4a.
Reaction of 3 with aromatic aldehydes 9a–d. General
procedure: To a mixture of pyridinylacetohydrazide
derivative 3 (0.202 g, 1 mmol) and the appropriate
benzaldehyde derivative 9 (1 mmol) in 20 mL absolute
EtOH was added 0.5 mL piperidine. The reaction mixture
was refluxed for 4–6 h (monitored by TLC), left to cool
to room temperature, and the formed solid was filtered
off, washed with water, dried, and recrystallized from
8.59 (m, 8H, Ar–H), 8.77 (s, 1H, CH¼), 10.03 (s, br, 1H,
NH); MS m/z (%): 335 (M+, 19), 216 (100), 183 (100),
125 (47), 81 (70). Anal. Calcd for C17H13N5O3 (335.10):
C, 60.89; H, 3.91; N, 20.89. Found C, 60.81; H, 3.77; N,
20.69%.
Coupling of compound 3 with arenediazonium chlorides
11a–d.
A cold solution of arenediazonium chloride
[prepared by diazotizing substituted aniline derivatives
(1 mmol) dissolved in hydrochloric acid (6 M, 1 mL)
with a solution of sodium nitrite (0.14 g, 1 mmol) in
water (3 mL)] was added portionwise onto a cold
solution of 3 (0.202 g, 1 mmol) in pyridine (20 mL, 0–
5°C/ice bath). After complete addition, the reaction
mixture was stirred for a further 30 min in the ice bath.
The solid that separated was filtered off, washed with
water, and finally recrystallized from proper solvent to
give the corresponding products 12a–d.
2-Oxo-N0-phenyl-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)
acetohydrazonoylcyanide (12a). Yield 72%; yellow solid;
DMF to give 10a–d.
2-Cyano-3-phenyl-N0-(1-(pyridin-4-yl)ethylidene)
acrylohydrazide (10a).
Yield 76%; yellow solid; mp
mp 213–215°C (EtOH); IR (KBr): v 3426, 3212 (2NH),
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Pyridine-based Heterocyclic Compounds
2198 (CN), 1681 (C¼O), 1596 (C¼N) cmꢀ1; H NMR
(DMSO-d6): δ 2.37 (s, 3H, CH3), 7.40–7.85 (m, 9H,
Ar–H), 8.73 (s, br, 1H, NH), 12.72 (s, br, 1H, NH); MS
m/z (%): 306 (M+, 12), 236 (42), 119 (62), 78 (86), 57
(100). Anal. Calcd for C16H14N6O (306.12): C, 62.74; H,
4.61; N, 27.44. Found C, 62.68; H, 4.46; N, 27.30%.
2-Oxo-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)-N0-(p-
C18H12N4OS (332.07): C, 65.04; H, 3.64; N, 16.86.
1
Found: C, 65.00; H, 3.62; N, 16.76%.
Synthesis of pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-
5(1H)-one derivatives (21a–e). General procedure: To a
mixture of equimolar amounts of the thione 18 (0.332 g,
1 mmol) and the appropriate hydrazonoyl halides 4a–e
(1 mmol) in dioxane (10 mL) was added triethylamine
(0.14 mL, 1 mmol). The reaction mixture was refluxed
till all of the starting materials disappeared and hydrogen
sulfide gas ceased to evolve (6–10 h, monitored by TLC).
The solvent was evaporated and the residue was triturated
with MeOH. The solid that formed was filtered off and
recrystallized from the proper solvent to give the products
21a–e, respectively.
tolyl)acetohydrazonoylcyanide (12b).
Yield 74%; yellow
solid; mp 184–186°C (DMF/EtOH); IR(KBr) υ (cmꢀ1
)
3412, 3182 (2NH), 2218 (CN), 1679 (C¼O), 1599
1
(C¼N) cmꢀ1; H NMR (DMSO-d6): δ 2.26 (s, 3H, CH3),
2.37 (s, 3H, CH3), 7.36–7.84 (m, 8H, Ar–H), 10.15 (s,
br, 1H, NH), 12.83 (s, br, 1H, NH); MS m/z (%): 320
(M+, 29), 265 (52), 106 (84), 97 (100), 56 (96). Anal.
Calcd for C17H16N6O (320.14): C, 63.74; H, 5.03; N,
26.23. Found C, 63.58; H, 5.00; N, 26.15%.
3-Acetyl-1,6-diphenyl-8-(pyridin-4-yl)pyrido[2,3-d][1,2,4]
triazolo[4,3-a]pyrimidin-5(1H)-one (21a).
Brown solid,
yield 72%; mp 233–235°C (DMF); IR (KBr) υ: 3041,
N0-(4-Chlorophenyl)-2-oxo-2-(2-(1-(pyridin-4-yl)ethylidene)
2930 (C–H), 1698, 1652 (2C¼O), 1600 (C¼N) cmꢀ1; H
1
hydrazinyl)acetohydrazonoyl cyanide (12c).
Yield 77%;
NMR (DMSO-d6) δ: 2.62 (s, 3H, CH3), 7.02–8.65 (m,
15H, Ar¼H) ppm; MS m/z (%): 458 (M+, 15), 395 (50),
295 (37), 168 (78), 96 (100), 78 (73). Anal. Calcd for
C27H18N6O2 (458.15): C, 70.73; H, 3.96; N, 18.33.
yellow solid; mp 203–205°C (DMF); IR(KBr) υ (cmꢀ1
)
3437, 3195 (2NH), 2219 (CN), 1679 (C¼O), 1599
1
(C¼N) cmꢀ1; H NMR (DMSO-d6): δ 2.39 (s, 3H, CH3),
7.60–8.26 (m, 8H, Ar–H), 10.72 (s, br, 1H, NH), 12.96
(s, br, 1H, NH); 13C-NMR (DMSO-d6): δ 22.6 (q, CH3),
106.3 (s), 112.5 (s, CN), 117.6 (d), 124.2 (d), 127.8 (s),
129.5 (d), 138.5 (s), 141.2 (s), 147.9 (s), 149.5 (d), 165.5
(s, C¼O) ppm. MS m/z (%): 340 (M+, 16), 218 (100),
137 (73), 79 (94), 44 (69). Anal. Calcd for C16H13ClN6O
(340.08): C, 56.39; H, 3.85; N, 24.66. Found C, 56.28;
H, 3.77; N, 24.52%.
Found: C, 70.66; H, 3.79; N, 18.26%.
3-Acetyl-1-(4-nitrophenyl)-6-phenyl-8-(pyridin-4-yl)
pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one
(21b). Brown solid, yield 74%; mp 192–194°C (DMF/
EtOH); IR (KBr) υ: 3037, 2928 (C–H), 1699, 1671
(2C¼O), 1593 (C¼N) cmꢀ1
;
1H NMR (DMSO-d6) δ:
2.59 (s, 3H, CH3), 6.91–8.39 (m, 14H, Ar–H) ppm; MS
m/z (%): 503 (M+, 13), 383 (31), 267 (39), 192 (64), 123
(84), 83 (100). Anal. Calcd for C27H17N7O4 (503.13): C,
64.41; H, 3.40; N, 19.47. Found: C, 64.34; H, 3.31; N,
19.35%.
N0-(4-Nitrophenyl)-2-oxo-2-(2-(1-(pyridin-4-yl)ethylidene)
hydrazinyl)acetohydrazonoyl cyanide (12d).
Yield 73%;
yellow solid; mp 227–229°C (DMF); IR(KBr) υ (cmꢀ1
)
3431, 3199 (2NH), 2221 (CN), 1683 (C¼O), 1597
3-Acetyl-1-(4-chlorophenyl)-6-phenyl-8-(pyridin-4-yl)
pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one (21c).
1
(C¼N) cmꢀ1; H NMR (DMSO-d6): δ 2.41 (s, 3H, CH3),
7.63–8.29 (m, 8H, Ar–H), 10.79 (s, br, 1H, NH), 12.90
(s, br, 1H, NH); MS m/z (%): 351 (M+, 18), 239 (70),
154 (100), 81 (46), 55 (68). Anal. Calcd for C16H13N7O3
(351.11): C, 54.70; H, 3.73; N, 27.91. Found C, 54.63;
H, 3.68; N, 27.76%.
Brown solid, yield 76%; mp 231–233°C (DMF); IR
(KBr) υ: 3053, 2925 (C–H), 1689, 1671 (2C¼O), 1593
(C¼N) cmꢀ1
;
1H NMR (DMSO-d6) δ: 2.66 (s, 3H,
CH3), 7.01–8.12 (m, 14H, Ar–H) ppm; 13C-NMR
(DMSO-d6):
δ
23.9 (q, CH3), 117.7 (s), 121.0
Synthesis
of
5-phenyl-7-(pyridin-4-yl)-2-thioxo-2,3-
(d), 121.4 (d), 121.5 (d), 127.3 (d), 127.5 (s), 129.1
(d), 129.3 (d) 129.4 (d), 138.3 (s), 140.7 (s), 142.6 (s),
145.8 (s), 149.8 (d), 152.0 (s), 152.2 (s), 153.8
(s), 154.7 (s), 170.5 (s, ring C¼O), 194.0(s, acetyl
C¼O) ppm. MS m/z (%): 492 (M+, 19), 335 (68), 280
(57), 126 (98), 67 (90), 44 (100). Anal. Calcd for
C27H17ClN6O2 (492.11): C, 65.79; H, 3.48; N, 17.05.
dihydropyrido[2,3-d]pyrimidin-4(1H)-one (18).
A mixture
of 3-phenyl-1-(pyridin-4-yl)prop-2-en-1-one (13) (2.09 g,
10 mmol) and 6-amino-2-thioxo-2,3-dihydropyrimidin-
4(1H)-one (14) (1.43 g, 10 mmol) in acetic acid (30 mL)
was refluxed for 8 h. The reaction mixture was cooled
and diluted with MeOH and the solid product was
collected by filtration and recrystallized from DMF to
give thione 18 as brown solid in yield 76%, mp 305–
307°C (DMF); IR (KBr) υ: 3346, 3217 (2 NH), 3032,
Found: C, 65.59; H, 3.29; N, 17.01%.
Ethyl 5-oxo-1,6-diphenyl-8-(pyridin-4-yl)-1,5-dihydropyrido[2,3-
d][1,2,4]triazolo[4,3-a]pyrim-idine-3-carboxylate (21d). Yellow
2923 (C–H), 1678 (C¼O) cmꢀ1; H NMR (DMSO-d6) δ:
1
solid, yield 70%; mp 217–219°C (DMF/EtOH); IR (KBr) υ:
3032, 2934 (C–H), 1737, 1685 (2C¼O), 1597 (C¼N) cmꢀ1
;
7.29–7.53 (m, 5H, Ar–H), 8.06 (s, 1H, pyridine–H), 8.12
(d, 2H, J = 7.5 Hz, pyridine–H), 8.70 (d, 2H, J = 7.5 Hz,
pyridine–H), 11.73 (s, br, 1H, NH), 12.42 (s, br, 1H, NH)
ppm; MS m/z (%): 332 (M+). Anal. Calcd for
1H NMR (DMSO-d6) δ: 1.27 (t, 3H, CH3, J = 7.2 Hz), 4.28
(q, 2H, CH2, J = 7.2 Hz), 6.85–8.42 (m, 15H, Ar–H) ppm;
MS m/z (%): 488 (M+, 32), 329 (41), 247 (74), 178 (53), 65
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

S. M. Gomha, F. M. Abdelrazek, A. H. Abdelrahman, and P. Metz
Vol 000
(100). Anal. Calcd for C28H20N6O3 (488.16): C, 68.84; H,
4.13; N, 17.20. Found: C, 68.79; H, 4.04; N, 17.13%.
Ethyl 1-(4-nitrophenyl)-5-oxo-6-phenyl-8-(pyridin-4-yl)-1,5-
synthesis of a new series of aminopyrazole derivatives
via its reaction with hydrazonoyl chlorides. The
mechanism that account for formation of products was
discussed. The structures of the newly synthesized
products were elucidated based on elemental analysis,
spectral data, and by alternative methods. The anticancer
activities of the synthesized compounds were screened
for their activity against hepatocellular carcinoma
(HepG2) cell line comparable with doxorubicin using
MTT assay and the results showed that most of such
compounds exhibit considerable activities.
dihydropyrido[2,3-d]
[1,2,4]
triazolo-[4,3-a]pyrimidine-3-
carboxylate (21e).
Light yellow solid, yield 69%; mp
200–202°C (EtOH); IR (KBr) υ: 3036, 2926 (C–H), 1739,
1
1677 (2C¼O), 1594 (C¼N) cmꢀ1; H NMR (DMSO-d6)
δ: 1.24 (t, 3H, CH3, J = 7.2 Hz), 4.35 (q, 2H, CH2,
J = 7.2 Hz), 6.89–8.40 (m, 14H, Ar–H) ppm; MS m/z (%):
533 (M+, 20), 447 (40), 313 (36), 213 (52), 77 (100), 41
(74). Anal. Calcd for C28H19N7O5 (533.14): C, 63.04; H,
3.59; N, 18.38. Found: C, 63.01; H, 3.47; N, 18.30%.
Antitumor activity. Human liver carcinoma (HepG-2)
cell lines were obtained from the American Type Culture
Collection (ATCC, Rockville, MD). The cells were grown
on RPMI-1640 medium supplemented with 10%
inactivated fetal calf serum and 50 μg/mL entamycin. The
cells were maintained at 37°C in a humidified atmosphere
with 5% CO2 and were subcultured two to three times/week.
For antitumor assays, the tumor cell lines were
suspended in medium at concentration 5 × 104 cell/well
in corning® 96-well plates (six replicates) to achieve
eight concentrations for each compound. Six vehicle
controls with media or 0.5% DMSO were run for each
96-well plate as a control. After incubating for 24 h, the
numbers of viable cells were determined by the MTT
test. Briefly, the media was removed from the 96-well
plates and replaced with 100 μL of fresh culture RPMI
1640 medium without phenol red then 10 μL of the
12 Mm MTT stock solution (5 mg of MTT in 1 mL of
PBS) to each well including the untreated controls. The
96-well plates were then incubated at 37°C and 5% CO2
for 4 h. An 85 μL aliquot of the media was removed
from the wells, and 50 μL of DMSO was added to each
well and mixed thoroughly with the pipette and incubated
at 37°C for 10 min. Then, the optical density was
measured at 590 nm with the microplate reader (SunRise,
TECAN, Inc., USA) to determine the number of viable
cells and the percentage of viability was calculated as
(1-(ODt/ODc)) × 100% where ODt is the mean optical
density of untreated cells. The relation between surviving
cells and drug concentration is plotted to get the survival
curve of each tumor cell line after treatment with the
specified compound. The 50% inhibitory concentration
(IC50), the concentration required to cause toxic effects
in 50% of intact cells, was estimated from graphic plots
of the dose response curve for each conc. Using Graphad
Prism software (San Diego, CA. USA) [43–46].
Acknowledgments. We thank the Alexander von Humboldt-
Foundation (Germany) for granting short research fellowships,
continued help and support to F. M. Abdelrazek.
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