Synthesis and anticancer properties of novel hydrazone derivatives incorporating pyridine and isatin moieties

Article abstract of DOI:10.1002/ardp.202000377

Nine novel hydrazone derivatives (4a–i) incorporating pyridine and isatin moieties were synthesized through one-pot, four-component heterocyclic condensation reactions. The structures of all new compounds (2a–e, 3a, 3c–e, and 4a–e) were identified by

Full text of DOI:10.1002/ardp.202000377

Received: 6 October 2020
Revised: 20 November 2020
Accepted: 27 November 2020
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DOI: 10.1002/ardp.202000377
F U L L P A P E R
Synthesis and anticancer properties of novel hydrazone
derivatives incorporating pyridine and isatin moieties
Zineddine Zebbiche1,2 | Suat Tekin3 | Hasan Küçükbay1
| Furkan Yüksel3
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Boudjemaa Boumoud2
1Department of Chemistry, Faculty of Arts
and Sciences, İnönü University, Malatya,
Abstract
Turkey
Nine novel hydrazone derivatives (4a–i) incorporating pyridine and isatin moieties
were synthesized through one‐pot, four‐component heterocyclic condensation re-
actions. The structures of all new compounds (2a–e, 3a, 3c–e, and 4a–e) were
identified by 1H nuclear magnetic resonance (NMR), 13C NMR, and Fourier‐
transform infrared spectroscopic techniques and elemental analysis. Cell viability
assays for the tested hydrazone derivatives were performed and the log IC50 values
of the compounds were calculated after a 24‐h treatment. All hydrazide derivatives
tested showed a promising antitumor activity against A‐2780 cells as compared with
the standard drug docetaxel with a log IC50 value of 0.2200 μM (p < .05). Seven of
the examined compounds (4b–e, 4g–i) showed high cytotoxic activity against
A‐2780 cells as compared with the standard drug docetaxel. Whereas the log IC50 of
docetaxel was 0.2200 μM for A‐2780 cells at 24 h, the IC50 values of these com-
pounds were −0.4987, −0.4044, −0.8138, −0.3868, −0.6954, −0.4751, and 0.1809
μM, respectively. Three of the compounds, 4b, 4d, and 4i, showed high cytotoxic
activity against MCF‐7 cells as compared with docetaxel (p < .05). Whereas the
log IC50 of docetaxel was 0.2400 μM for MCF‐7 cells at 24 h, the log IC50 values of
compounds 4b, 4d, and 4i were −0.1293, −0.1700, and 0.2459 μM, respectively.
2Laboratory of Synthesis of Molecules with
Biological Interest, Mentouri Constantine
University, Constantine, Algeria
3Department of Physiology, Faculty of
Medicine, İnönü University, Malatya, Turkey
Correspondence
Hasan Küçükbay, Department of Chemistry,
Faculty of Arts and Sciences, İnönü University,
44280 Malatya, Turkey.
Email: hasan.kucukbay@inonu.edu.tr
K E Y W O R D S
anticancer activity, hydrazone derivatives, isatin derivatives, pyridine derivatives
most important groups in medicinal chemistry,[2–5] and they are
present in a large number of compounds due to their diverse bio-
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1
INTRODUCTION
Due to the increase in cancer incidence, scientific research on cancer
morbidity and mortality and improvement in the quality of life of
cancer patients is increasing rapidly. Cancer is a serious health
concern in all societies, regardless of wealth or social status.
In 2018, 18.1 million people worldwide suffered from can-
cer and 9.6 million patients died from the disease. By 2040, these
figures will almost double, and the largest increase will be noticed
in low‐ and middle‐income countries where more than two‐thirds of
world cancer cases occur.[1] Over the last two decades,
hydrazide–hydrazone derivatives have been known as one of the
logical properties such as anti‐inflammatory,[6] antimicrobial,[7,8]
analgesic,[9] anti‐HIV,[10] anticonvulsant,[11,12] antileishmanial,[13,14]
antimalarial,[15] antitumor,[16–19] and antitubercular activities.[20]
Furthermore, hydrazide–hydrazones are increasingly considered to
be a valuable core in medicinal chemistry.[21–24] However, isatin is a
natural product discovered in the early 19th century in the genus
Isatis and in Couroupita guianensis Aubl. plants,[25,26] and it is known
as oxidized indole that attracts much attention as a building block in
the design of countless compounds. Isatin has a wide variety of
biological and pharmacological activities such as antifungal and
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Arch Pharm. 2021;354:e2000377.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
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antibacterial properties[27] and potent caspase inhibitory[28,29] and
anticancer activities.[30] Schiff bases of isatin have a wide range of
pharmacological activities including antismallpox,[31] anti‐
inflammatory,[32] antibacterial,[33] antiviral,[34] and antitubercular
activities[35]; they also act as a GAL3 receptor antagonist.[36]
However, 3‐cyano‐2‐pyridones show remarkable biological and phar-
macological properties, particularly antidepressant,[37] anticancer,[38,39]
antimicrobial activity,[40] and p38 MAP kinase inhibitory capabilities.[41]
Although the developments in chemotherapy increase the fight
against cancer, numerous side effects of the drugs used pose
serious problems in cancer treatment. A great number of drugs
have been used to treat cancer, such as cis‐platinum and ruthe-
nium compounds, but good treatable values have not been
achieved with existing drugs. To eliminate these disadvantages,
increasing numbers of studies are continuously being carried out in this
area. We have also synthesized and tested anticancer activities of many
heterocyclic compounds against several cell lines such as A‐2780
(human ovarian),[42,43] PC‐3 (human prostate),[43] DU‐145 (human
prostate),[42] A549 (human lung cancer),[44] and BEAS‐2B (human lung
cancer).[44] As a part of these research works, we planned to synthesize
a number of new hydrazone derivatives incorporating pyridine and
isatin moieties and examine their anticancer activities against A‐2780
and MCF‐7 cell lines.
2
RESULTS AND DISCUSSION
Chemistry
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2.1
The synthetic pathways utilized to prepare the new hydrazone de-
rivatives are depicted in Scheme 1.
Different cyanopyridinones 1a–e were prepared as a starting
material via one‐pot, four‐component heterocyclic condensation
process, as reported in the literature.[39] The reaction of 1a–e with
methyl bromoacetate in the presence of anhydrous K2CO3 in
dimethylformamide (DMF) yielded the corresponding methyl ester
derivatives 2a–e. In infrared data, the presence of υ(COOR) at around
1750 cm−1 and the disappearance of the amide carbonyl band at
position 2 of the pyridine core prove that the desired ester deriva-
tives (2a–e) are synthesized. The singlet peaks around 5.00 ppm for
compounds 2a–e were assigned to the OCH2 protons of the acetyl
group, whereas the singlet signals around 3.70 ppm were assigned to
the methyl protons of the ester group for compounds 2a–e. The 13
C
nuclear magnetic resonance (NMR) spectra of 2a–e showed three
signals around δ 168.9, 55.47, and 52.24 ppm, corresponding to
(C═O, ester), (OCH3), and CH2O, respectively. The reaction of esters
(2a–e) with hydrazine monohydrate in boiling THF yielded the cor-
responding acetohydrazides 3a–e. The optimization studies were
SCHEME 1 Synthetic pathway for compounds 1–4. Reagents and conditions: (I) CNCH2COOEt/CH3COONH4/EtOH/reflux; (II) methyl
bromoacetate/anhydrous K2CO3/dimethylformamide/RT; (III) N2H4 100%/tetrahydrofuran/reflux; (IV) appropriate isatin derivative/1,4‐
dioxane/10% p‐toluenesulfonic acid/RT

ZEBBICHE ET AL.
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cis/trans amide conformers (Scheme 3). In this respect, the 1H NMR
spectra of 4a–i in dimethyl sulfoxide (DMSO)‐d6 showed only the
presence of E conformers; however, the spectra for compounds 4i
and 4g revealed two sets of protons at 13.49–12.61 and
5.80–5.61 ppm, which are attributed to CO–NH amide and OCH2
due to cis and trans conformers. According to the literature, the cis
conformers of amide were assigned to upfield signals of CO–NH
and OCH2 protons, whereas the downfield peaks were caused by
the trans conformer.
The N‐benzylisatin derivatives used in this study were prepared
from isatin and an appropriate benzyl halide in the presence of an-
hydrous K2CO3 and KI in DMF, according to the literature.[46,47] The
structures of all isatin derivatives were confirmed by 1H NMR
and 13C NMR, and melting point data and all spectral and analytical
data were in accordance with the literature values.[46,47]
FIGURE 1 The effect of solvent with time in the reaction. THF,
tetrahydrofuran
accomplished for the current reaction using 2a (1.0 eq) and hy-
drazine monohydrate (4 eq). The use of THF has proven to be es-
sential to achieve maximum conversion for this reaction. Attempts to
use other solvents (such as ethanol, methanol, propanol, dioxane,
CH3CN, and benzene) resulted in lower yields or no reaction
(Figure 1). The use of protic solvents such as ethanol, methanol, and
propanol generated a side product, as indicated in Scheme 2. The
infrared spectra of 3a–e show an absorption peak at around 3333.42
and 1675.80 cm−1 for NH/NH2 and the amide carbonyl groups, re-
spectively, which confirms the presence of the hydrazide group. The
1H NMR data confirm the presence of the NH2 and NH peaks in the
regions 4.33–4.37 and 9.43–9.45 ppm, respectively, and the dis-
appearance of OCH3 protons. The 13C NMR spectra of compounds
3a–e confirmed the absence of the methoxy carbon atom.
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2.2
Cytotoxicity study
Cytotoxic activities of nine new hydrazone derivatives including pyr-
idine and isatin moieties were determined using human ovarian
(A‐2780) and human breast cancer (MCF‐7) cell lines. To obtain the
cytotoxic properties of the newly synthesized hydrazone derivatives on
A‐2780 and MCF‐7 cells, the respective cell lines were incubated with
increasing concentrations (0–100 µg/ml) of the compounds for 48 h
and then subjected to an MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐
diphenyltetrazolium bromide) assay. Among the tested nine hydrazone
derivatives, those bearing hydrogen atoms for R, R1, and R2 sub-
stituents and with 4‐methoxy substituent for R, R1, and hydrogen for R2
at position 1 of the isatin moiety showed a lower cytotoxicity on
A‐2780 cells than the other compounds and the standard drug
docetaxel at 0.1 µM. A nearly similar result was obtained at 1 µM
concentration for the A‐2780 cell line. The cell viability results of
A‐2780 and MCF‐7 cells after a 48‐h treatment with the nine
hydrazone derivatives are shown in Tables 1 and 2, respectively. As
shown in Table 1, all tested hydrazone derivatives demonstrated an
anticancer activity against the A‐2780 cell line at 0.1, 1, 10, and 100 µM
concentrations with p < .05, except compounds 4a and 4f at 0.1 µM
concentration. Similar to A‐2780 cell line results, all tested compounds
show a cytotoxic activity against MCF‐7 cell line at 0.1, 1, 10, and 100
µM concentrations with p < .05, except compounds 4a, 4f, and 4h at 0.1
µM concentration. Among the tested nine hydrazone derivatives,
The condensation of hydrazides 3a–e with different isatins in
dioxane containing 10% PTSOH (p‐toluenesulfonic acid) at room
temperature afforded the corresponding isatin Schiff base deriva-
tives 4a–i. The infrared spectra of these compounds showed the
appearance of NH stretching bands in the range of 3301–3089 cm−1
.
The 1H NMR spectra of 4a–i also showed two D2O exchangeable
signals due to the NH group of isatin (4a, 4d, 4f, 4h, and 4i) in the
range of 10.90–10.93 ppm and NH of the hydrazide linker in the
range of 11.66–11.80 ppm. All other spectral data were in ac-
cordance with the assumed structures.
According to the literature,[45] the N‐substituted hydrazones may
exist as Z/E geometrical isomers about C═N double bonds and as
SCHEME 2 The condensation of ester with hydrazine under protic solvents conditions

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SCHEME 3 E/Z geometrical isomers about
the C═N double bond and cis/trans amide
conformers of the N‐substituted hydrazones
compounds 4b and 4d showed a better cytotoxicity against MCF‐7 cells
as compared with the standard drug docetaxel with an inhibitory
logarithmic 50 (log IC50) value of 0.24 µM.
tested nine hydrazone derivatives, compounds 4b and 4d showed
better cytotoxicity against MCF‐7 cells as compared with the standard
drug docetaxel with a log IC50 value of 0.24 µM.
A time‐dependent cell viability assay for the tested hydrazone
derivatives was performed, and the log IC50 values of the compounds
were calculated after a 24‐h treatment. Log IC50 values calculated for
the A‐2780 and MCF‐7 cells, based on the results of MTT assays for a
24‐h interaction of hydrazone derivatives, are presented in Table 3. As
can be seen in Table 3, all tested hydrazone derivatives showed pro-
mising antitumor activity against A‐2780 cells as compared with the
standard drug docetaxel with a log IC50 value of 0.22 µM. Among the
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3
CONCLUSIONS
Nine new hydrazone derivatives were synthesized successfully and
their anticancer activities were evaluated against human cancer cell
lines (A‐2780 and MCF‐7). Seven out of the nine compounds (4b, 4c,
4d, 4e, 4g, 4h, and 4i) showed better anticancer activity than the
TABLE 1 The cell viability results of A‐2780 cells after a 48‐h treatment with nine hydrazone derivativesa
A2780 cell viability (%)
Compound no.
Control
Solvent (DMSO)
94.72 2.39
94.72 2.39
94.72 2.39
94.72 2.39
94.72 2.39
94.72 2.39
94.72 2.39
94.72 2.39
94.72 2.39
94.72 2.39
0.1 μM
1 μM
10 μM
100 μM
4a
100.00 3.90
100.00 3.90
100.00 3.90
100.00 3.90
100.00 3.90
100.00 3.90
100.00 3.90
100.00 3.90
100.00 3.90
100.00 3.90
83.60 6.70
55.11 8.36*
44.80 1.48*
21.45 5.13*
50.24 7.65*
103.44 7.64*
31.21 7.96*
50.34 9.10*
58.50 5.06*
60.21 5.97
64.85 5.21*
23.48 4.43*
39.28 5.30*
21.23 1.10*
38.11 3.64*
67.46 5.78*
25.77 4.95*
27.44 3.50*
61.93 3.23*
40.13 6.58
42.47 3.98*
18.53 2.70*
30.10 2.49*
18.08 5.14*
19.50 3.29*
27.82 3.84*
23.87 7.15
38.35 2.09*
25.76 3.30*
14.12 2.73
37.76 5.44*
17.59 3.08*
9.74 1.08*
13.45 1.34*
12.64 3.05*
11.78 0.79*
20.97 5.44
13.03 3.67*
12.27 2.00*
0.48 0.02
4b
4c
4d
4e
4f
4g
4h
4i
Docetaxel (ref. drug)
Abbreviation: DMSO, dimethyl sulfoxide.
aThe changes in cell viability caused by hydrazone derivatives are compared with the control data. Each data point is an average of eight viability
measurements.
*p < .05.

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TABLE 2 The cell viability results of MCF‐7 cells after a 48‐h treatment with nine hydrazone derivativesa
MCF‐7 cell viability (%)
Compound no.
Control
Solvent (DMSO)
95.79 2.47
95.79 2.47
95.79 2.47
95.79 2.47
95.79 2.47
95.79 2.47
95.79 2.47
95.79 2.47
95.79 2.47
95.79 2.47
0.1 μM
1 μM
10 μM
100 μM
4a
100.00 5.32
100.00 5.32
100.00 5.32
100.00 5.32
100.00 5.32
100.00 5.32
100.00 5.32
100.00 5.32
100.00 5.32
100.00 5.32
76.51 9.13
53.98 6.15*
70.19 6.91*
50.95 2.55*
69.37 3.95*
84.42 4.70
63.21 6.60*
80.37 2.73
58.96 4.60*
64.56 6.14
65.60 6.61*
44.46 4.47*
68.06 2.10*
43.11 4.16*
68.48 5.68*
71.87 6.84*
54.36 3.57*
57.02 6.97*
51.89 4.92*
36.85 4.92
64.37 3.77*
39.14 3.09*
51.57 3.50*
42.22 1.96*
57.96 4.97*
53.51 7.44*
50.44 1.93*
56.52 5.12*
44.88 2.13*
20.01 3.09
46.87 2.59*
34.32 3.30*
53.38 3.55*
44.05 3.32*
49.57 5.74*
51.73 7.16*
53.13 3.75*
45.03 1.79*
42.56 2.11*
0.88 0.04
4b
4c
4d
4e
4f
4g
4h
4i
Docetaxel (ref. drug)
Abbreviation: DMSO, dimethyl sulfoxide.
aThe changes in cell viability caused by hydrazone derivatives are compared with the control data. Each data point is an average of eight viability measurements.
*p < .05.
standard drug docetaxel at a concentration of 0.1 μM against the A‐
2780 cell line. The log IC50 of docetaxel was 0.2200 μM for A‐2780
cells at 24 h, whereas the IC50 values of compounds 4b, 4c, 4d, 4e,
4g, 4h, and 4i were −0.4987, −0.4044, −0.8138, −0.3868, −0.6954,
−0.4751, and 0.1809 μM, respectively. Three out of the nine com-
pounds (4b, 4d, and 4i) at a concentration of 0.1 µM showed better
anticancer activity against the MCF‐7 cancer cell line than the
standard drug. The log IC50 of docetaxel was 0.2400 μM for MCF‐7
cells at 24 h, whereas the log IC50 values of compounds 4b, 4d, and 4i
were −0.1293, −0.1700, and 0.2459 μM, respectively. The outcomes
of this study indicate that the newly studied hydrazone derivatives
may act as potential drug candidates for cancer treatment, and the
results of the present study are encouraging us to continue our an-
ticancer activity screening with further modification in their struc-
ture in the future.
by the addition of D2O. Elemental analyses were performed by a LECO
CHNS 932 Elemental Analyzer. The infrared spectra were recorded with
ATR equipment in the range of 4000–650 cm−1 on
a Perkin
Elmer Spectrum One Fourier‐transform infrared spectrophotometer.
Melting points (mp) were measured in open capillary tubes and
were uncorrected, using a Gallenkamp MPD350.BM3.5 apparatus. 1‐
Benzylindoline‐2,3‐dione,[46,47] 1‐(4‐bromobenzyl)indoline‐2,3‐dione,[46,47]
and compound 3b[48] were prepared according to the published
procedures.
The InChI codes of the investigated compounds, together with some
biological activity data, are provided as Supporting Information.
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4.1.2
General procedure for the synthesis of the
products 2a–e
A mixture of an appropriate pyridin‐2(1H)‐one (1a–e; 0.01 mol) and
anhydrous K2CO3 (0.015 mol) was stirred at room temperature in
DMF (10 ml) for 1 h, and then 0.011 mol of methyl bromoacetate was
added. The reaction mixture was stirred for an additional 3 h and
poured into ice‐cooled water. The obtained product was filtered off,
dried, and crystallized from ethanol/acetone (2:1).
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4
EXPERIMENTAL
Chemistry
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4.1
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4.1.1
General
The starting materials and reagents used in the reactions were supplied
commercially by Aldrich, Acros, ABCR, and Merck. The human breast
(MCF‐7) cancer cell line and female ovarian (A‐2780) cancer cell line
were retrieved from the American Type Culture Collection (ATCC). The
nuclear magnetic resonance (1H NMR, 13C NMR) spectra (see the
Supporting Information) were recorded using a Bruker Advance III
400 MHz spectrometer in DMSO‐d6. Chemical shifts are reported in
parts per million (ppm) and the coupling constants (J) are expressed in
Hertz (Hz). The assignment of exchangeable protons (NH) was confirmed
Methyl 2‐[(3‐cyano‐4,6‐diphenylpyridin‐2‐yl)oxy]acetate (2a)
White crystals, yield 89%, mp: 157–159°C, 1H NMR (CDCl3,
400 MHz) δ 8.01 (dd, J = 6.8, 3.0 Hz, 2H, Ar–H), 7.70–7.64 (m, 2H,
Ar–H), 7.58–7.45 (m, 7H, Ar–H, pyridine C5–H), 5.11 (s, 2H, OCH2),
3.81 (s, 3H, COOCH3). 13C NMR (CDCl3, 400 MHz) δ 164.14
(COOCH3), 158.67, 152.85, 152.37, 132.05, 131.37, 125.95, 125.42,
124.31, 124.22, 123.68, 122.50, 110.28, 109.70 (Ar–C), 88.54 (CN),
58.85 (OCH2), 47.51 (COOCH3). ν(C–O–C) aliphatic ether:
1145.43 cm−1 ν(C═O) ester: 1733.12 cm−1 ν(CN): 2226.56 cm−1
, , ,

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TABLE 3 Log IC50 (µM) concentrations calculated for A‐2780 and MCF‐7 cells in the GraphPad Prism 6 program of hydrazone derivatives
log IC50 calculation
Compound
4a
A‐2780
MCF‐7
0.7816
1.660
4b
4c
−0.4987
−0.4044
−0.1293
1.465
4d
4e
4f
−0.8138
−0.3868
0.4636
−0.1700
1.598
1.551
4g
−0.6954
1.085
4h
−0.4751
0.1809
0.2200
1.267
4i
0.2459
0.2400
Docetaxel

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ν(C–H) aromatic: 2959.37 cm−1. Anal. calcd. for C21H16N2O3: C,
8.26 (d, J = 8.2 Hz, 1H, Ar–H), 8.20 (dd, J = 6.7, 3.0 Hz, 2H, Ar–H),
8.05 (s, 1H, pyridine C5–H), 7.92 (t, J = 8.0 Hz, 1H, Ar–H), 7.59–7.52
(m, 3H, Ar–H), 5.23 (s, 2H, OCH2), 3.75 (s, 3H, COOCH3). 13C NMR
(CDCl3, 400 MHz) δ 169.23 (COOCH3), 163.25, 157.75, 154.76,
148.42, 137.44, 136.50, 135.86, 131.56, 131.00, 129.47, 127.90,
125.31, 124.18, 115.23, 115.12 (Ar–C), 92.96 (CN), 64.18 (OCH2),
73.24; H, 4.68; N, 8.13. Found: C, 74.10; H, 4.95; N, 8.21.
Methyl 2‐[(3‐cyano‐4,6‐bis(4‐methoxyphenyl)pyridin‐2‐yl)oxy]‐
acetate (2b)
White solid, yield 75%, mp: 130–132°C, 1H NMR (CDCl3, 400 MHz) δ
7.96 (d, J = 9.0 Hz, 2H, Ar–H), 7.63 (d, J = 8.8 Hz, 2H, Ar–H), 7.43 (s,
1H, pyridine C5–H), 7.04 (d, J = 8.8 Hz, 2H, Ar–H), 6.98 (d, J = 9.0 Hz,
2H, Ar–H), 5.07 (s, 2H, OCH2), 3.87 (s, 6H, 2OCH3), 3.79 (s, 3H,
COOCH3). 13C NMR (CDCl3, 400 MHz) δ 169.04 (COOCH3), 163.47,
161.73, 161.16, 157.07, 156.45, 129.90, 129.42, 128.78, 128.51,
115.61, 114.45, 114.29, 113.15 (Ar–C), 91.75 (CN), 63.55 (OCH2),
55.46 (2OCH3), 52.21 (COOCH3). ν(C–O–C) aliphatic ether: 1140.53,
52.44 (COOCH3). ν(C–O–C) aliphatic ether: 1155.06 cm−1
ν(N–O) nitro: 1528.48 cm−1 ν(C═O) ester: 1754.76 cm−1
ν(CN):
2225.46 cm−1 ν(C–H) aromatic: 3092.87 cm−1
Anal. calcd. for
21H15N3O5: C, 64.78; H, 3.88; N, 10.79. Found: C, 65.04; H, 4.02;
N, 10.69.
,
,
,
,
.
C
1170.69, 1182.51 cm−1
2220.54 cm−1 ν(C–H) aromatic: 2838.91, 2949.49 cm−1. Anal. calcd.
,
ν(C═O) ester: 1761.85 cm−1
,
ν(CN):
4.1.3
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General procedure for the synthesis of the
products 3a–e
,
for C23H20N2O5: C, 68.31; H, 4.98; N, 6.93. Found: C, 68.98; H, 5.02;
N, 6.94.
A mixture of 2a–e (0.01 mol) and hydrazine monohydrate (0.04 mol,
1 ml) was boiled in tetrahydrofuran (10 ml) for 2.5–3 h. After cooling,
the formed precipitate was filtered off, dried, and crystallized from
ethanol/DMF (2:1) to give the target compounds 3a–e.
Methyl 2‐{[3‐cyano‐6‐(4‐methoxyphenyl)‐4‐phenylpyridin‐2‐yl]oxy}‐
acetate (2c)
White crystals, yield 92%, mp: 148–150°C, 1H NMR (CDCl3,
400 MHz) δ 7.97 (d, J = 9.0 Hz, 2H, Ar–H), 7.67–7.62 (m, 2H, Ar–H),
7.55–7.49 (m, 3H, Ar–H), 7.45 (s, 1H, pyridine C5–H), 6.98 (d,
J = 9.0 Hz, 2H, Ar–H), 5.08 (s, 2H, OCH2), 3.87 (s, 3H, OCH3), 3.79 (s,
3H, COOCH3). 13C NMR (CDCl3, 400 MHz) δ 168.99 (COOCH3),
163.36, 161.83, 157.28, 156.87, 136.28, 130.06, 129.27, 129.00,
128.83, 128.40, 115.26, 114.33, 113.45 (Ar–C), 92.15 (CN), 63.58
(OCH2), 55.47 (OCH3), 52.24 (COOCH3). ν(C–O–C) aliphatic ether:
2‐[(3‐Cyano‐4,6‐diphenylpyridin‐2‐yl)oxy]acetohydrazide (3a)
Yellowish white crystals, yield 80%, mp: 227–228°C, 1H NMR
(DMSO‐d6, 400 MHz) δ 9.45 (s, 1H, NH), 8.21 (dd, J = 6.7 Hz, 3.3 Hz,
2H, Ar–H), 7.85 (s, 1H, pyridine C5–H), 7.77–7.75 (m, 2H, Ar–H),
7.61–7.52 (m, 6H, Ar–H), 5.04 (s, 2H, OCH2), 4.34 (s, 2H, NH2). 13C
NMR (DMSO‐d6, 400 MHz) δ 167.06 (CONH–NH2), 163.77, 157.49,
156.88, 136.90, 136.25, 131.22, 130.61, 129.40, 129.38, 129.10,
1140.53, 1172.51 cm−1
,
ν(C═O) ester: 1760.38 cm−1
,
ν(CN):
128.03, 115.75, 114.67 (Ar–C), 93.10 (CN), 64.81 (OCH2). (C–O–C)
ν
2223.11 cm−1 ν(C–H) aromatic: 2842.39, 2950.17 cm−1. Anal. calcd.
,
aliphatic ether: 1142.28 cm−1
,
ν(C═O) amide: 1675.80 cm−1
,
ν(CN):
for C22H18N2O4: C, 70.58; H, 4.85; N, 7.48. Found: C, 71.25; H, 4.90;
N, 7.61.
2224.20 cm−1 ν(C–H) aromatic: 2934.87, 3051.01 cm−1
3282.25, 3333.42 cm−1. Anal. calcd. for C20H16N4O2: C, 69.76; H,
,
, ν(N–H):
4.68; N, 16.27. Found: C, 70.29; H, 4.91; N, 16.31.
Methyl 2‐{[3‐cyano‐6‐(4‐methoxyphenyl)‐4‐(3‐nitrophenyl)pyridin‐2‐
yl]oxy}acetate (2d)
2‐{[3‐Cyano‐6‐(4‐methoxyphenyl)‐4‐phenylpyridin‐2‐yl]oxy}‐
acetohydrazide (3c)
Yellow solid, yield 77%, mp: 190–192°C, 1H NMR (DMSO‐d6,
400 MHz) δ 8.60 (t, J = 1.9 Hz, 1H, Ar–H), 8.47–8.40 (m, 1H, Ar–H),
8.26–8.20 (m, 1H, Ar–H), 8.16 (d, J = 8.9 Hz, 2H, Ar–H), 7.94 (s, 1H,
pyridine C5–H), 7.90 (t, J = 8.0 Hz, 1H, Ar–H), 7.09 (d, J = 9.0 Hz, 2H,
Ar–H), 5.19 (s, 2H, OCH2), 3.85 (s, 3H, OCH3), 3.75 (s, 3H, COOCH3).
13C NMR (CDCl3, 400 MHz) δ 169.28 (COOCH3), 163.20, 162.23,
157.61, 154.45, 148.39, 137.58, 135.81, 130.97, 129.65, 128.85,
125.21, 124.09, 115.31, 114.86, 114.17 (Ar–C), 91.63 (CN), 64.10
(OCH2), 55.91 (OCH3), 52.42 (COOCH3). ν(C–O–C) aliphatic ether:
White solid, yield 73%, mp: 209–211°C, 1H NMR (DMSO‐d6,
400 MHz) δ 9.44 (s, 1H, NH), 8.19 (d, J = 8.9 Hz, 2H, Ar–H), 7.80–7.69
(m, 3H, Ar–H, pyridine C5–H), 7.64–7.55 (m, 3H, Ar–H), 7.06 (d,
J = 8.9 Hz, 2H, Ar–H), 5.01 (s, 2H, OCH2), 4.33 (s, 2H, NH2), 3.85 (s,
3H, OCH3). 13C NMR (DMSO‐d6, 400 MHz) δ 167.14 (CONH–NH2),
163.71, 161.94, 157.31, 156.64, 136.41, 130.51, 129.76, 129.35,
129.30, 129.06, 115.94, 114.76, 113.66 (Ar–C), 91.89 (CN), 64.72
(OCH2), 55.87 (OCH3). ν(C–O–C) aliphatic ether: 1141.80,
1149.23, 1176.64 cm−1
,
ν(N–O) nitro: 1531.41 cm−1
ν(C–H) aromatic: 2837.24,
,
ν(C═O) ester:
1171.45 cm−1 ν(C═O) amide: 1665.12 cm−1 ν(CN): 2217.43 cm−1
, , ,
ν(N–H): 3281.79 cm−1. Anal. calcd. for C21H18N4O3: C, 67.37; H, 4.85;
1787.33 cm−1 ν(CN): 2217.02 cm−1
,
,
2950.66 cm−1. Anal. calcd. for C22H17N3O6: C, 63.01; H, 4.09; N,
N, 14.96. Found: C, 68.15; H, 4.91; N, 14.90.
10.02. Found: C, 63.69; H, 4.09; N, 10.02.
2‐{[3‐Cyano‐6‐(4‐methoxyphenyl)‐4‐(3‐nitrophenyl)pyridin‐2‐
yl]oxy} acetohydrazide (3d)
Yellow solid, yield 89%, mp: 236–238°C, 1H NMR (DMSO‐d6,
400 MHz) δ 9.44 (s, 1H, NH), 8.59 (t, J = 2 Hz, 1H, Ar–H), 8.46–8.39
(m, 1H, Ar–H), 8.23–8.18 (m, 3H, Ar–H, pyridine C5–H), 7.92
Methyl 2‐{[3‐cyano‐4‐(3‐nitrophenyl)‐6‐phenylpyridin‐2‐yl]oxy}‐
acetate (2e)
Yellow solid, yield 72%, mp: 170–172°C, 1H NMR (DMSO‐d6,
400 MHz) δ 8.64 (t, J = 1.9 Hz, 1H, Ar–H), 8.47–8.41 (m, 1H, Ar–H),

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(t, J = 7.9 Hz, 2H, Ar–H), 7.08 (d, J = 9.0 Hz, 2H, Ar–H), 5.03 (s, 2H,
OCH2), 4.34 (s, 2H, NH2), 3.85 (s, 3H, OCH3). 13C NMR (DMSO‐d6,
400 MHz) δ 167.03 (CONH–NH2), 163.60, 162.09, 157.70, 154.15,
148.38, 137.74, 135.76, 131.03, 129.87, 129.10, 125.17, 124.00,
115.61, 114.78, 113.84 (Ar–C), 92.05 (CN), 64.79 (OCH2), 55.90
(E)‐Nʹ‐(1‐Benzyl‐2‐oxoindolin‐3‐ylidene)‐2‐[(3‐cyano‐4,6‐
diphenylpyridin‐2‐yl)oxy]acetohydrazide (4b)
Yellow solid, yield 68%, mp: 217–219°C, 1H NMR (DMSO‐d6,
400 MHz) δ 11.79 (s, 1H, NH), 8.27 (d, J = 7.6 Hz, 1H, Ar–H), 8.16 (d,
J = 7.2 Hz, 2H, Ar–H), 7.89 (s, 1H, pyridine C5–H), 7.84–7.77 (m, 2H,
Ar–H), 7.67–7.60 (m, 3H, Ar–H), 7.48–7.26 (m, 9H, Ar–H), 7.14
(t, J = 7.6 Hz, 1H, Ar–H), 7.09 (d, J = 8.0 Hz, 1H, Ar–H), 5.70 (s, 2H,
OCH2), 5.01 (s, 2H, NCH2). 13C NMR (DMSO‐d6, 400 MHz) δ 163.80
(CONHN), 163.64 (CONCH2 isatin), 157.46, 157.15, 144.40, 136.79,
136.61, 136.18 (Ar–C), 133.18 (C═Nimine), 131.21, 130.68, 129.40,
129.22, 129.20, 129.18, 128.02, 127.92, 127.73, 126.65, 122.95,
115.67, 115.21, 114.87, 110.44 (Ar–C), 92.73 (CN), 43.20 (NCH2).
(OCH3). ν(C–O–C) aliphatic ether: 1150.79, 1171.99 cm−1
nitro: 1516.22 cm−1 ν(C═O) amide: 1665.56 cm−1
2218.28 cm−1 ν(C–H) aromatic: 2836.60, 3016.19 cm−1
,
ν(N–O)
, ν(CN):
,
,
,
ν(N–H):
3290.90 cm−1. Anal. calcd. for C21H17N5O5: C, 60.14; H, 4.09; N,
16.70. Found: C, 59.85; H, 4.16; N, 16.18.
2‐{[3‐Cyano‐4‐(3‐nitrophenyl)‐6‐phenylpyridin‐2‐yl]oxy}‐
acetohydrazide (3e)
ν(C–O–C) aliphatic ether: 1143.54 cm−1, ν(C═N) imine: 1675.58 cm−1
ν(C═O) amide: 1694.48, 1724.92 cm−1, (CN): 2225.44 cm−1, (N–H):
,
Yellow solid, yield 69%, mp: 219–221°C, 1H NMR (DMSO‐d6,
400 MHz) δ 9.45 (s, 1H, NH), 8.61 (t, J = 1.8 Hz, 1H, Ar–H), 8.45
(dd, J = 8.3, 1.5 Hz, 1H, Ar–H), 8.26–8.21 (m, 3H, Ar–H), 8.00 (s,
1H, pyridine C5–H), 7.96–7.88 (m, 1H, Ar–H), 7.56–7.53 (m, 3H,
Ar–H), 5.06 (s, 2H, OCH2), 4.34 (s, 2H, NH2). 13C NMR (DMSO‐d6,
400 MHz) δ 166.98 (CONH–NH2), 163.65, 157.86, 154.41, 148.41,
137.59, 136.70, 135.78, 131.41, 131.06, 129.42, 128.11, 125.25,
124.06, 115.41, 114.85 (Ar–C), 93.34 (CN), 64.89 (OCH2).
ν
ν
3198.56 cm−1. Anal. calcd. for C35H25N5O3: C, 74.59; H, 4.47; N,
12.43. Found: C, 74.47; H, 4.46; N, 12.48.
(E)‐Nʹ‐[1‐(4‐Bromobenzyl)‐2‐oxoindolin‐3‐ylidene]‐2‐[(3‐cyano‐4,6‐
diphenylpyridin‐2‐yl)oxy]acetohydrazide (4c)
Yellow solid, yield 71%, mp: 228–230°C, 1H NMR (DMSO‐d6,
400 MHz) δ 11.81 (s, 1H, NH), 8.27 (d, J = 7.6 Hz, 1H, Ar–H),
8.19–8.11 (m, 2H, Ar–H), 7.89 (s, 1H, pyridine C5–H), 7.83–7.77 (m,
2H, Ar–H), 7.65–7.60 (m, 3H, Ar–H), 7.55 (d, J = 8.4 Hz, 2H, Ar–H),
7.49–7.27 (m, 6H, Ar–H), 7.15 (t, J = 7.6 Hz, 1H, Ar–H), 7.08 (d,
J = 8.0 Hz, 1H, Ar–H), 5.73 (s, 2H, OCH2), 4.98 (s, 2H, NCH2).
ν(C–O–C) aliphatic ether: 1158.46 cm−1
1542.40 cm−1 ν(C═O) amide: 1665.97 cm−1 ν(CN): 2219.83 cm−1
,
,
ν(N–O) nitro:
,
,
ν(C–H) aromatic: 2954.09 cm−1, ν(N–H): 3299.03 cm−1. Anal. calcd.
for C20H15N5O4: C, 61.69; H, 3.88; N, 17.99. Found: C, 59.83; H,
4.04; N, 17.35.
ν(C–O–C) aliphatic ether: 1143.67 cm−1, ν(C═N) imine: 1672.08 cm−1
ν(C═O) amide: 1690.59, 1725.73 cm−1 ν(CN): 2226.51 cm−1
ν(C–H)
aromatic: 2982.40 cm−1 ν(N–H): 3220.55 cm−1
Anal. calcd. for
35H24BrN5O3: C, 65.43; H, 3.77; N, 12.44. Found: C, 65.42; H, 4.46;
N, 12.48.
,
,
,
,
.
|
4.1.4
General procedure for the synthesis of the
C
products 4a–j
A mixture of isatin derivative (0.01 mol) and hydrazides 3a‐e (0.01
mol) in dioxane (10 ml) containing 10% PTSOH as catalyst was
stirred at room temperature for 3 h. Then the resulting solid was
filtered, washed with ethanol, filtered and recrystallized from etha-
nol/DMF.
(E)‐2‐{[3‐Cyano‐6‐(4‐methoxyphenyl)‐4‐phenylpyridin‐2‐yl]oxy}‐Nʹ‐
[(2‐oxoindolin‐3‐ylidene)acetohydrazide] (4d)
Yellow solid, yield 80%, mp: 230–232°C, 1H NMR (DMSO‐d6,
400 MHz) δ 11.67 (s, 1H, NH), 10.92 (s, 1H, NHisatin), 8.21 (d,
J = 7.7 Hz, 1H, Ar–H), 8.15–8.05 (m, 2H, Ar–H), 7.82–7.72 (m, 3H,
Ar–H, pyridine C5–H), 7.65–7.57 (m, 3H, Ar–H), 7.44 (t, J = 7.7 Hz,
1H, Ar–H), 7.10 (t, J = 7.2 Hz, 1H, Ar–H), 6.97 (d, J = 7.8 Hz, 1H,
Ar–H), 6.79 (d, J = 8.8 Hz, 2H, Ar–H), 5.67 (s, 2H, OCH2), 3.71 (s, 3H,
OCH3). 13C NMR (DMSO‐d6, 400 MHz) δ 164.95 (CONHN), 163.56
(CONHisatin), 161.85, 157.24, 156.87, 144.47, 136.33 (Ar–C), 133.38
(C═Nimine), 130.56, 129.59, 129.35, 129.11, 126.80, 122.27, 115.85,
115.66, 114.54, 113.80, 111.21 (Ar–C), 91.51 (CN), 66.82 (OCH2),
(E)‐2‐[(3‐Cyano‐4,6‐diphenylpyridin‐2‐yl)oxy]‐Nʹ‐[(2‐oxoindolin‐3‐
ylidene)acetohydrazide] (4a)
Yellow solid, yield 70%, mp: 243–245°C, 1H NMR (DMSO‐d6,
400 MHz) δ 11.67 (s, 1H, NH), 10.90 (s, 1H, NHisatin), 8.22–8.10 (m,
3H, Ar–H), 7.88 (s, 1H, pyridine C5–H), 7.83–7.77 (m, 2H, Ar–H),
7.67–7.58 (m, 3H, Ar–H), 7.48–7.38 (m, 2H, Ar–H), 7.32 (t, J = 7.6 Hz,
2H, Ar–H), 7.08 (t, J = 7.5 Hz, 1H, Ar–H), 6.96 (d, J = 7.8 Hz, 1H,
Ar–H), 5.70 (s, 2H, OCH2). 13C NMR (DMSO‐d6, 400 MHz) δ 164.93
(CONHN), 163.65 (CONHisatin), 157.44, 157.14, 144.45, 136.78,
136.18 (Ar–C), 133.38 (C═Nimine), 131.23, 130.67, 129.39, 129.22,
129.17, 127.91, 126.78, 122.25, 115.67, 115.63, 114.83, 111.20
(Ar–C), 92.72 (CN), 66.83 (OCH2). ν(C–O–C) aliphatic ether:
55.73 (OCH3). ν(C–O–C) aliphatic ether: 1146.56, 1170.17 cm−1
ν(C═N) imine: 1584.37 cm−1 ν(C═O) amide: 1715.81 cm−1
ν(CN):
2222.83 cm−1, ν(C–H) aromatic: 2839.13 cm−1, ν(N–H): 3170.35 cm−1
,
,
,
.
Anal. calcd. for C29H21N5O4: C, 69.18; H, 4.20; N, 13.91. Found: C,
68.95; H, 4.21; N, 14.03.
1146.83 cm−1
1717.75 cm−1
,
,
ν(C═N) imine: 1608.97 cm−1
ν(CN): 2228.78 cm−1 ν(C–H) aromatic: 2837.66 cm−1
,
ν(C═O) amide:
(E)‐Nʹ‐[1‐(4‐Bromobenzyl)‐2‐oxoindolin‐3‐ylidene]‐2‐{[3‐cyano‐6‐
(4‐methoxyphenyl)‐4‐phenylpyridin‐2‐yl]oxy}acetohydrazide (4e)
Yellow solid, yield 71%, mp: 212–214°C, 1H NMR (DMSO‐d6,
400 MHz) δ 11.80 (s, 1H, NH), 8.28 (d, J = 7.7 Hz, 1H, Ar–H),
,
,
ν(N–H): 3147.71 cm−1. Anal. calcd. for C28H19N5O3: C, 71.03; H, 4.04;
N, 14.79. Found: C, 70.87; H, 4.04; N, 14.82.

ZEBBICHE ET AL.
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|
8.14–8.06 (m, 2H, Ar–H), 7.82–7.74 (m, 3H, Ar–H, pyridine C5–H),
7.64–7.59 (m, 3H, Ar–H), 7.53 (d, J = 8.3 Hz, 2H, Ar–H), 7.46 (t,
J = 7.8 Hz, 1H, Ar–H), 7.33 (d, J = 8.3 Hz, 2H, Ar–H), 7.16 (t, J = 7.6 Hz,
1H, Ar–H), 7.09 (d, J = 8.0 Hz, 1H, Ar–H), 6.77 (d, J = 8.8 Hz, 2H,
Ar–H), 5.67 (s, 2H, OCH2), 4.98 (s, 2H, NCH2), 3.66 (s, 3H, OCH3).
(E)‐2‐{[(3‐Cyano‐6‐(4‐methoxyphenyl)]‐4‐[(3‐nitrophenyl)pyridin‐2‐
yl]oxy}‐Nʹ‐(2‐oxoindolin‐3‐ylidene)acetohydrazide (4h)
Yellow solid, yield 66%, mp: 224–226°C, 1H NMR (DMSO‐d6,
400 MHz) δ 11.66 (s, 1H, NH), 10.90 (s, 1H, NHisatin), 8.62 (m, 1H,
Ar–H), 8.47–8.40 (m, 1H, Ar–H), 8.29–8.17 (m, 2H, Ar–H), 8.16–8.07
(m, 2H, Ar–H), 7.96–7.86 (m, 2H, Ar–H, pyridine C5–H), 7.44 (t,
J = 7.7 Hz, 1H, Ar–H), 7.10 (t, J = 7.5 Hz, 1H, Ar–H), 6.96 (d, J = 7.9 Hz,
1H, Ar–H), 6.81 (d, J = 8.8 Hz, 2H, Ar–H), 5.66 (s, 2H, OCH2), 3.72 (s,
3H, OCH3). 13C NMR (DMSO‐d6, 400 MHz) δ 164.95 (CONHN),
163.48 (CONHisatin), 162.01, 157.63, 154.42, 148.41, 144.48, 137.69,
135.82 (Ar–C), 133.39 (C═Nimine), 130.99, 129.71, 128.95, 126.79,
125.19, 124.09, 122.27, 115.65, 115.51, 114.58, 114.01, 111.21
13C NMR (DMSO‐d6, 400 MHz)
δ 163.82 (CONHN), 163.57
(CONCH2 isatin), 161.84, 157.27, 156.91, 144.20, 136.33, 136.09
(Ar–C), 133.20 (C═Nimine), 132.09, 130.57, 130.02, 129.60, 129.36,
129.14, 129.11, 126.70, 123.06, 121.15, 115.84, 115.27, 114.55,
113.86, 110.39 (Ar–C), 91.55 (CN), 55.69 (OCH3), 42.61 (NCH2).
ν(C–O–C) aliphatic ether: 1142.76, 1175.30 cm−1
1675.11 cm−1 ν(C═O) amide: 1693.27, 1724.24 cm−1
2224.31 cm−1, ν(C–H) aromatic: 2974.03 cm−1, ν(N–H): 3143.95 cm−1
,
ν(C═N) imine:
ν(CN):
,
,
.
(Ar–C), 91.70 (CN), 64.33 (OCH2), 55.76 (OCH3). (C–O–C) aliphatic
ν
Anal. calcd. for C36H26BrN5O4: C, 64.29; H, 3.90; N, 10.41. Found: C,
64.21; H, 3.89; N, 10.53.
ether: 1148.39, 1173.92 cm−1
imine: 1606.06 cm−1 ν(C═O) amide: 1669.94, 1722.80 cm−1
2225.58 cm−1, ν(C–H) aromatic: 2967.74 cm−1, ν(N–H): 3089.46 cm−1
,
ν(N–O) nitro: 1536.99 cm−1
,
ν(C═N)
, ν(CN):
,
.
(E)‐2‐{[3‐Cyano‐4,6‐bis(4‐methoxyphenyl)pyridin‐2‐yl]oxy}‐Nʹ‐[(2‐
oxoindolin‐3‐ylidene)acetohydrazide] (4f)
Anal. calcd. for C29H20N6O6: C, 63.50; H, 3.68; N, 15.32. Found: C,
63.43; H, 3.67; N, 15.35.
Yellow solid, yield 70%, mp: 240–242°C, 1H NMR (DMSO‐d6,
400 MHz) δ 11.68 (s, 1H, NH), 10.93 (s, 1H, NHisatin), 8.21 (d,
J = 7.7 Hz, 1H, Ar–H), 8.14–8.03 (m, 2H, Ar–H), 7.80–7.70 (m, 3H,
Ar–H, pyridine C5–H), 7.44 (t, J = 7.8 Hz, 1H, Ar–H), 7.16 (d,
J = 8.8 Hz, 2H, Ar–H), 7.10 (d, J = 7.3 Hz, 1H, Ar–H), 6.96 (d,
J = 7.8 Hz, 1H, Ar–H), 6.78 (d, J = 8.9 Hz, 2H, Ar–H), 5.66 (s, 2H,
OCH2), 3.86 (s, 3H, OCH3), 3.70 (s, 3H, OCH3). 13C NMR (DMSO‐d6,
400 MHz) δ 164.96 (CONHN), 163.67 (CONHisatin), 161.76, 161.30,
157.00, 156.44, 144.46 (Ar–C), 133.36 (C═Nimine), 130.72, 129.52,
129.23, 128.53, 128.38, 126.79, 125.98, 122.72, 116.15, 115.65,
114.79, 114.50, 113.49, 111.21 (Ar–C), 91.04 (CN), 67.49 (OCH2),
55.89 (OCH3), 55.71 (OCH3). ν(C–O–C) aliphatic ether: 1141.86,
2‐{[3‐Cyano‐4‐(3‐nitrophenyl)‐6‐phenylpyridin‐2‐yl]oxy}‐Nʹ‐(2‐
oxoindolin‐3‐ylidene)acetohydrazide (4i)
Yellow solid, yield 69%, mp: 280–282°C, 1H NMR (DMSO‐d6, 600 MHz) δ
13.49 (s, 1H, NHcis conformer), 12.70 (s, 1H, NHtrans conformer), 11.35 (s, 1H,
NHisatin), 8.65–8.61 (m, 1H, Ar–H), 8.44 (d, J = 8.0 Hz, 1H, Ar–H), 8.26 (d,
J = 7.8 Hz, 1H, Ar–H), 8.22–7.95 (m, 3H, Ar–H, pyridine C5–H), 7.92 (t,
J = 7.9 Hz, 1H, Ar–H), 7.64–7.32 (m, 5H, Ar–H), 6.99 (m, 2H, Ar–H), 5.80
(s, 2H, OCH2 trans conformer), 5.39 (s, 2H, OCH2 cis conformer). 13C NMR
(DMSO‐d6, 400 MHz) δ 163.41 (CONHN), 163.02 (CONHisatin), 157.83,
154.72, 148.43, 143.08, 137.46, 136.54, 135.85 (Ar–C), 132.42 (C═Ni-
mine), 131.48, 131.02, 129.30, 128.00, 125.31, 124.15, 123.16, 121.41,
120.00, 115.26, 115.21, 115.18, 111.72 (Ar–C), 93.06 (CN), 63.67
1171.08, 1189.61 cm−1
1721.22, 1750.23 cm−1
2946.79 cm−1 ν(N–H): 3301.03, 3393.82 cm−1
, , ν(C═O) amide:
ν(C═N) imine: 1603.46 cm−1
,
ν(CN): 2226.39 cm−1
,
ν(C–H) aromatic:
(OCH2). ν(C–O–C) aliphatic ether: 1141.09 cm−1
1526.53 cm−1 ν(C═N) imine: 1617.90 cm−1
ν(C═O) amide: 1691.35,
1729.31 cm−1 ν(CN): 2226.86 cm−1 ν(N–H): 3204.30 cm−1. Anal. calcd.
, ν(N–O) nitro:
,
.
Anal. calcd. for
,
,
C
30H23N5O5: C, 67.54; H, 4.35; N, 13.13. Found: C, 67.47; H, 4.35;
,
,
N, 13.19.
for C28H18N6O5: C, 64.86; H, 3.50; N, 16.21. Found: C, 64.77; H, 3.48;
N, 16.26.
Nʹ‐(1‐Benzyl‐2‐oxoindolin‐3‐ylidene)‐2‐{[3‐cyano‐4,6‐bis(4‐
methoxyphenyl)pyridin‐2‐yl]oxy}acetohydrazide (4g)
Yellow solid, yield, mp: 168–170°C, 1H NMR (DMSO‐d6, 600 MHz) δ
13.38 (s, 1H, NHcis conformer), 12.61 (s, 1H, NHtrans conformer), 8.20–8.04
(m, 2H, Ar–H), 7.79–7.57 (m, 4H, Ar–H, pyridine C5–H), 7.46–7.24
(m, 6H, Ar–H), 7.21–7.02 (m, 4H, Ar–H), 7.02–6.77 (m, 2H, Ar–H),
5.78 (s, 2H, OCH2 trans conformer), 5.35 (s, 2H, OCH2 cis conformer), 5.00 (s,
2H, NCH2), 3.86 (s, 3H, OCH3), 3.57 (s, 3H, OCH3). 13C NMR (DMSO‐d6,
400 MHz) δ 161.86 (CONHN), 161.33 (CONCH2 isatin), 161.14, 157.06,
156.50, 143.26, 136.06 (Ar–C), 132.21 (C═Nimine), 130.71, 129.57,
129.20, 128.33, 128.15, 127.88, 123.85, 121.26, 119.57, 115.96,
114.79, 114.61, 113.75, 111.02 (Ar–C), 91.21 (CN), 66.83 (OCH2),
4.2
Cytotoxicity study
|
|
4.2.1
Cell lines and culture conditions
The human cancer lines, ovarian cancer (A‐2780) and prostate
cancer (MCF‐7), were used for in vitro screening experiments.
Both cell lines were both purchased from the ATCC. All cells were
fed in 25‐ and 75‐cm2 flasks with the RPMI‐1640 medium
(containing 10% fetal bovine serum, 100 U/ml penicillin, and
0.1 mg/ml streptomycin) 2 days apart. In cells with a carbon di-
oxide (5% CO2) incubator (Panasonic), the cells maintained at
37°C and in a humid environment were separated from the flasks
using a solution of trypsin–EDTA (Sigma‐Aldrich) when confluent.
The viability of the cells was determined using 0.4% trypan blue
and experiments were started when the viability was above 90%.
55.90 (OCH3), 55.72 (OCH3), 43.06 (NCH2). (C–O–C) aliphatic ether:
ν
1142.92, 1170.32 cm−1
1698.07, 1729.42 cm−1
2963.10 cm−1 ν(N–H): 3225.70 cm−1. Anal. calcd. for C37H29N5O5: C,
71.26; H, 4.69; N, 11.23. Found: C, 71.34; H, 4.70; N, 11.39.
,
ν(C═N) imine: 1610.41 cm−1
, ν(C═O) amide:
,
ν(CN): 2215.51 cm−1
,
ν(C–H) aromatic:
,

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4.2.2
MTT assay
[2] J. S. da Silva, D. Gabriel‐Costa, R. T. Sudo, H. Wang, L. Groban,
E. B. Ferraz, J. H. M. Nascimento, C. A. M. Fraga, E. J. Barreiro,
G. Zapata‐Sudo, Drug Des., Dev. Ther. 2017, 11, 553. https://doi.org/
10.2147/DDDT.S113289
Antitumor activities of these substances were evaluated by MTT
assay.[30] Cells were removed using a trypsin–EDTA solution from
flasks and counted by a hemocytometer to determine cytotoxic ef-
fects. Furthermore, 15 × 103 cells per well were plated in 96‐well
plates including 200 µl of the RPMI‐1640 medium. Cells were in-
cubated at 37°C in a CO2 incubator for 24 h to adhere to a 96‐well
plate base. When the incubation ended, concentrations of 0.1, 1, 10,
and 100 µM of the hydrazone derivatives were added to the wells in
which the cells were contained. Incubation with cancer cells for 24 h
at 37°C in a CO2 incubator was performed to determine the effects
of different concentrations of hydrazone derivatives on cell viability
for 24 h. When the incubation was over, 0.5 mg/ml of MTT solution
in sterile phosphate‐buffered solution was prepared and added to
96‐well plates. After the addition of MTT, the plates were incubated
again for 3 h. After this time, incubation was stopped by adding
DMSO to the wells, and the optical densities of the cells in the plates
were read on a spectrophotometer (Synergy HTX) at a wavelength of
550 nm.[31] The cell viability percentage was calculated by pro-
portioning the absorbance values obtained from hydrazone
derivative‐applied wells to that of control group. MTT trials were
performed 10 times in triplicate on different days, and the log IC50
values of the applied compounds were calculated on the basis of
MTT results using the GraphPad Prism 6 program on a computer.
[3] D.G. Costa, J. S. Da Silva, A. E. Kümmerle, R. T. Sudo, S. S. Landgraf,
C. Caruso‐Neves, C. A. M. Fraga, E. J. De Lacerda Barreiro,
G. Zapata‐Sudo, Am. J. Hypertens. 2010, 23, 1220. https://doi.org/
10.1038/ajh.2010.157
[4] E. J. Barreioro, C. A. M. Fraga, A. L. P. Miranda, C. R. Rodrigues, Quim.
Nova 2002, 25, 129. https://doi.org/10.2174/092986706775197881
[5] R.T. Sudo, G. Zapata‐Sudo, E. J. Barreiro, Br. J. Pharmacol. 2001,
134, 603. https://doi.org/10.1038/sj.bjp.0704291
[6] U. Salgin‐Gökşen, N. Gökhan‐Kelekçi, Ö. Göktaş, Y. Köysal, E. Kiliç,
Ş. Işik, G. Aktay, M. Özalp, Bioorg. Med. Chem. 2007, 15, 5738.
https://doi.org/10.1016/j.bmc.2007.06.006
[7] S. Rollas, N. Gulerman, H. Erdeniz, Farmaco 2002, 57, 171. https://
doi.org/10.1016/S0014-827X(01)01192-2
[8] Z. Lian, J. Sun, H. Zhu, J. Mol. Struct. 2016, 1117, 8. https://doi.org/
10.1016/j.molstruc.2016.03.036
[9] P. C. Lima, L. M. Lima, K. C. M. Da Silva, P. H. O. Léda,
A. L. P. De Miranda, C. A. M. Fraga, E. J. Barreiro, Eur. J. Med. Chem.
2000, 35,187. https://doi.org/10.1016/S0223-5234(00)00120-3
[10] P. Vicini, M. Incerti, P. La Colla, R. Loddo, Eur. J. Med. Chem. 2009,
44, 1801. https://doi.org/10.1016/j.ejmech.2008.05.030
[11] A. P. Nikalje, A. Ansari, S. Bari, V. Ugale, Arch. Pharm. 2015, 348,
435. https://doi.org/10.1002/ardp.201500020
[12] J. R. Dimmock, S. C. Vashishtha, J. P. Stables, Eur. J. Med. Chem.
2000, 35, 241. https://doi.org/10.1016/S0223-5234(00)00123-9
[13] Z. Garkani‐Nejad, B. Ahmadi‐Roudi, Eur. J. Med. Chem. 2010, 45,
719. https://doi.org/10.1016/j.ejmech.2009.11.019
[14] A. M. R. Bernardino, A. O. Gomes, K. S. Charret, A. C. C. Freitas,
G. M. C. Machado, M. M. Canto‐Cavalheiro, L. L. Leon, V. F. Amaral,
Eur. J. Med. Chem. 2006, 41, 80. https://doi.org/10.1016/j.ejmech.
2005.10.007
|
4.3
Statistical analysis
[15] A. Walcourt, M. Loyevsky, D. B. Lovejoy, V. R. Gordeuk, D. R.
Richardson, Int. J. Biochem. Cell Biol. 2004, 36, 401. https://doi.org/
10.1016/S1357-2725(03)00248-6
The IBM SPSS Statistics 24.0 (Windows) package program was used
in the analysis. Conformity to normal distribution was evaluated by
the Shapiro–Wilk test. Intergroup comparisons of quantitative vari-
ables were measured by Kruskal–Wallis H test. When significant
statistical differences were determined between groups, multiple
comparisons were made with Bonferroni correction Mann–Whitney
U test. All p values <.05 were considered statistically significant.
Log IC50 values of melatonin and agomelatine were calculated using
GraphPad Prism 6 program on a computer based on the MTT results
obtained from the experiments.
[16] N. Terzioglu, A. Gu, Eur. J. Med. Chem. 2003, 38, 781. https://doi.
org/10.1016/S0223-5234(03)00138-7
[17] L. Savini, L. Chiasserini, V. Travagli, C. Pellerano, E. Novellino,
S. Cosentino, M. B. Pisano, Eur. J. Med. Chem. 2004, 39, 113. https://
doi.org/10.1016/j.ejmech.2003.09.012
[18] A. H. A. Badi, A. Abdel, H. E. Issa, G. S. H. Assan, Chem. Pharm. Bull.
2003, 51, 838.
[19] H. Zhang, J. Drewe, B. Tseng, S. Kasibhatla, S. X. Cai, Med. Chem.
2004, 12, 3649. https://doi.org/10.1016/j.bmc.2004.04.017
[20] Z. Rec, J. Kaustova, Bioorg. Med. Chem. 2007, 15, 2551. https://doi.
org/10.1016/j.bmc.2007.01.051
[21] W. Wahba, W. Nahed, N. El‐Sayed, R. Mohareb, Acta Pharm. 2013,
63, 45. https://doi.org/10.2478/acph-2013-0004
ACKNOWLEDGMENTS
[22] H. Maniak, M. Talma, K. Matyja, A. Trusek, M. Giurg, Molecules
2020, 25. https://doi.org/10.3390/molecules25051255
[23] M. Bingul, S. Ercan, M. Boga, J. Mol. Struct. 2020, 1213, 128202.
https://doi.org/10.1016/j.molstruc.2020.128202
The authors thank İnönü University, Malatya Turkey, and Mentouri
Constantine University, Constantine, Algeria, for financial support.
[24] Ł. Popiołek, B. Rysz, A. Biernasiuk, M. Wujec, Chem. Biol. Drug Des.
2020, 95, 260. https://doi.org/10.1111/cbdd.13639
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
[25] J. F. M. Da Silva, S. J. Garden, A. C. Pinto, J. Braz. Chem. Soc. 2001,
12, 273. https://doi.org/10.1590/s0103-50532001000300002
[26] U. T. J. Bergman, J. O. Lindström, Tetrahedron 1985, 41, 2879.
[27] Z. H. Chohan, H. Pervez, A. Rauf, K. M. Khan, C. T. Supuran,
J. Enzyme Inhib. Med. Chem. 2004, 19, 417. https://doi.org/10.1080/
14756360410001710383.
ORCID
Hasan Küçükbay
http://orcid.org/0000-0002-7180-9486
REFERENCES
[28] W. Chu, J. Zhang, C. Zeng, J. Rothfuss, Z. Tu, Y. Chu, D. E. Reichert,
M. J. Welch, R. H. Mach, J. Med. Chem. 2005, 48, 7637. https://doi.
org/10.1021/jm0506625
[1] WHO, WHO report on cancer: Setting priorities investing wisely
and providing care for all, 2020.

ZEBBICHE ET AL.
11 of 11
|
[29] W. Chu, J. Rothfuss, Y. Chu, D. Zhou, R. H. Mach, J. Med. Chem.
2009, 52, 2188. https://doi.org/10.1021/jm900135r
[43] H. Küçükbay, A. Mumcu, S. Tekin, S. Sandal, Turkish J. Chem. 2016,
40, 1510. https://doi.org/10.3906/kim-1510-15
[30] Y.‐O. Teng, H.‐Y. Zhao, J. Wang, H. Liu, M.‐L. Gao, Y. Zhau, K.‐L.
Han, Z.‐C. Fan, Y.‐M. Zhang, H. Sun, P. Yu, Eur. J. Med. Chem 2016,
112, 145. https://doi.org/10.1016/j.ejmech.2015.12.050
[44] E. Apohan, U. Yilmaz, O. Yilmaz, A. Serindag, H. Küçükbay,
O. Yesilada, Y. Baran, J. Organomet. Chem. 2017, 828, 52. https://
doi.org/10.1016/j.jorganchem.2016.11.020
[31] M. C. Pirrung, S. V. Pansare, K. Das Sarma, K. A. Keith, E. R. Kern,
J. Med. Chem. 2005, 48, 3045. https://doi.org/10.1021/jm049147h.
[32] V. Alagarsamy, S. Meena, S. Vijayakumar, K. V. Ramseshu,
R. Revathi, Pharmazie 2003, 4, 233.
[45] E. Wyrzykiewicz, D. Prukała, J. Heterocycl. Chem. 1998, 35, 381.
[46] S. J. Garden, J. C. Torres, L. E. Da Silva, A. C. Pinto, Synth. Commun.
1998, 28, 1679. https://doi.org/10.1080/00397919808006872
[47] J. C. Torres, A. C. Pinto, S. J. Garden, Tetrahedron 2004, 60, 9889.
https://doi.org/10.1016/j.tet.2004.08.030
[33] V. A. M. S. George, Arkivoc 2008, 2008, 187.
[34] A. S. P. Singh, S. K. Shukla, L. P. Awasthi, Curr. Sci. 1983, 52, 766.
[35] T. Aboul‐Fadl, F. A. S. Bin‐Jubair, Int. J. Res. Pharm. Sci. 2010, 1, 113.
[36] M. J. Konkel, B. Lagu, L. W. Boteju, H. Jimenez, S. Noble,
M. W. Walker, G. Chandrasena, T. P. Blackburn, S. S. Nikam,
J. L. Wright, B. E. Kornberg, T. Gregory, T. A. Pugsley, H. Akunne,
K. Zoski, L. D. Wise, V. De, P. Road, A. Arbor, R. V. January, J. Med.
Chem. 2006, 49, 3757.
[48] M. S. A. E. A. Sammour, A. F. Fahmy, S. A. El‐Rahman, S. A. Aknookh,
Chem. Informationsdienst 1973, 52, 143.
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
supporting information tab for this article.
[37] N. A. Abdel‐Latif, Sci. Pharm. 2005, 74, 195.
[38] A. H. Abadi, D. A. Abouel‐Ella, J. Lehmann, H. N. Tinsley, B. D. Gary,
G. A. Piazza, M. A. O. Abdel‐Fattah, Eur. J. Med. Chem. 2010, 45, 90.
https://doi.org/10.1016/j.ejmech.2009.09.029.
How to cite this article: Zebbiche Z, Tekin S, Küçükbay H,
Yüksel F, Boumoud B. Synthesis and anticancer properties of
novel hydrazone derivatives incorporating pyridine and isatin
moieties. Arch Pharm. 2021;354:e2000377.
[39] A. H. Abadi, T. M. Ibrahim, K. M. Abouzid, J. Lehmann, H. N. Tinsley,
B. D. Gary, G. A. Piazza, Bioorg. Med. Chem. 2009, 17, 5974. https://
doi.org/10.1016/j.bmc.2009.06.063
[40] S. A. Malladi, A. M. Isloor, Pharm. Chem. 2012, 4, 43.
[41] A. M. Serry, S. Luik, S. Laufer, A. H. Abadi, J. Comb. Chem. 2010, 12, 559.
[42] Ü. Yılmaz, S. Tekin, N. Buğday, K. Yavuz, H. Küçükbay, S. Sandal, Inorg.
Chim. Acta 2019, 495. https://doi.org/10.1016/j.ica.2019.118977
https://doi.org/10.1002/ardp.202000377