Synthesis, cytotoxicity and in vitro antibacterial screening of novel hydrazones bearing thienopyridine moiety as potent COX-2 inhibitors

Article abstract of DOI:10.1007/s13738-020-01987-y

Abstract: The starting precursors 2-carbohydrazides, bearing thienopyridine moiety, were prepared using 2-thioxopyridine-3-carbonitriles as key synthons. Next, 2-carbohydrazides were reacted with a variety of 4-substituted benzylidinemalononitriles or 4-substituted benzaldehydes to afford a new series of the target hydrazones incorporating thienopyridine moiety. The elemental analyses and spectral data were used to demonstrate the structures of new hydrazones series. The in vitro antibacterial activities of the target hydrazones were evaluated against different strains of Gram-positive and Gram-negative bacteria. In comparison with chloramphenicol as a reference drug, hydrazones 13b, 13c and 13d, linked to p-Cl, p-Br and p-Me moiety, respectively, exhibited the strongest activities against all tested bacteria with MIC values in the range of 6.2–12.5?μg/mL. In addition, several new hydrazones were tested as in vitro cytotoxic agents against each of human breast carcinoma MCF-7 cell line, colon cancer Caco2 cell line and liver hepatocellular carcinoma HEPG2 cell line. The hydrazones 13b, 13c and 13d demonstrated the best cytotoxicity against the tested eukaryotic cells. Furthermore, both experimental and docking studies could predict the promising inhibitory activities of hydrazones 13c and 13d against COX-2 enzyme with IC₅₀ of 0.110 and 0.104?μM, respectively, when compared with Celecoxib (IC₅₀ of 0.115?μM). Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s13738-020-01987-y

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-020-01987-y
ORIGINAL PAPER
Synthesis, cytotoxicity and in vitro antibacterial screening of novel
hydrazones bearing thienopyridine moiety as potent COX‑2 inhibitors
Sherif M. H. Sanad1 · Ahmed E. M. Mekky1
Received: 24 March 2020 / Accepted: 26 June 2020
© Iranian Chemical Society 2020
Abstract
The starting precursors 2-carbohydrazides, bearing thienopyridine moiety, were prepared using 2-thioxopyridine-3-carbon-
itriles as key synthons. Next, 2-carbohydrazides were reacted with a variety of 4-substituted benzylidinemalononitriles or
4-substituted benzaldehydes to aford a new series of the target hydrazones incorporating thienopyridine moiety. The elemen-
tal analyses and spectral data were used to demonstrate the structures of new hydrazones series. The in vitro antibacterial
activities of the target hydrazones were evaluated against diferent strains of Gram-positive and Gram-negative bacteria. In
comparison with chloramphenicol as a reference drug, hydrazones 13b, 13c and 13d, linked to p-Cl, p-Br and p-Me moiety,
respectively, exhibited the strongest activities against all tested bacteria with MIC values in the range of 6.2–12.5 μg/mL.
In addition, several new hydrazones were tested as in vitro cytotoxic agents against each of human breast carcinoma MCF-7
cell line, colon cancer Caco2 cell line and liver hepatocellular carcinoma HEPG2 cell line. The hydrazones 13b, 13c and 13d
demonstrated the best cytotoxicity against the tested eukaryotic cells. Furthermore, both experimental and docking studies
could predict the promising inhibitory activities of hydrazones 13c and 13d against COX-2 enzyme with IC50 of 0.110 and
0.104 µM, respectively, when compared with Celecoxib (IC50 of 0.115 µM).
Graphic abstract
Keywords Hydrazones · Thieno[2,3-b]pyridine · In vitro antibacterial screening · COX-2 inhibitors · In silico study
Introduction
In recent years, design of synthetic routes for preparation
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s13738-020-01987-y) contains
supplementary material, which is available to authorized users.
of new thieno[2,3-b]pyridines has attracted considerable
attention because of their important medicinal activities
as antimicrobial [1, 2], antiviral [3, 4] and anti-inflamma-
tory agents [5–7]. Also, these derivatives exhibit interest-
ing antiproliferative and anticancer activities [8–11] as
well as their capability for treatment of disorders of cen-
tral nervous system [12–14]. Moreover, thienopyridines
* Sherif M. H. Sanad
sherif_hamed1980@yahoo.com
1
Chemistry Department, Faculty of Science, Cairo University,
Giza 12613, Egypt
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
exhibit broad spectrum of bioactivities as antidiabetic
[15, 16], antihypertensive [17, 18] and osteogenic [19,
20] activities.
Results and discussion
Chemistry
On the other hand, many publications reported the
remarkable biological and pharmacological properties of
several hydrazone containing derivatives as antituberculo-
sis [21], antibacterial, antifungal [22–24], anticonvulsant
[25], antimalarial [26] and anti-HIV [27] agents.
In connection with our efforts in designing facile pro-
cedures for the preparation of thienopyridines and their
related fused heterocyclic derivatives [28–36], the goal
of this work was aimed to construct an efficient pro-
cedure for the synthesis of a new series of hydrazones
incorporating thienopyridine moieties. Evaluation of the
in vitro antimicrobial potencies of the target hydrazones
was performed against various Gram-positive and Gram-
negative bacteria. The in vitro cytotoxicity of some new
hydrazones was evaluated against several eukaryotic cell
lines. In addition, in vitro and in silico studies were per-
formed to predict the capability of new hydrazones as
potent COX-2 enzyme inhibitors using Celecoxib as a
reference drug.
Initially, carbonyl derivatives 3a,b were prepared by the
reaction of thiophene-2-carboxaldehyde 1 with 4-substituted
acetophenones 2a,b [37]. Compounds 3a,b were reacted
with 2-cyanothoacetamide 4 in ethanol in the presence of
piperidine at refux to give the starting precursors 2-thiox-
opyridine-3-carbonitriles 5a,b (see Scheme 1 and “Experi-
mental” section) [38].
Then, 2-thioxopyridine-3-carbonitriles 5a,b were reacted
with ethyl 2-chloroacetate 6 in ethanol in the presence of
sodium acetate under constant stirring at room temperature
to give ethyl 2-((pyridin-2-yl)thio)acetates 7a,b. The reac-
tion most probably proceeded via a simple nucleophilic
substitution reaction (Scheme 2). Both elemental analyses
and spectral data were performed to confrm the chemical
structures of 7a,b. The IR spectrum of 7a, as a representa-
tive example, showed both nitrile and carbonyl functions
1
at 2214 and 1729 cm−1. Its H-NMR spectrum showed a
triplet, a quartet and a singlet signals at δ 1.14, 4.10 and
4.24 corresponding to OCH2CH3, OCH2CH3 and SCH2
Scheme 1 Synthesis of 2-thioxopyridine-3-carbonitriles 5
Scheme 2 Synthesis of nicotinonitriles 7 and carbohydrazides 9
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Journal of the Iranian Chemical Society
protons, respectively. Its 13C-NMR spectrum revealed fve
signals at δ 14.4, 33.1, 61.6, 116.2 and 169.0 correspond-
ing to CH2CH3, CH2CH3, SCH2, CN and CO carbons (see
Experimental section).
with subsequent removal of one molecule of malononitrile
(Scheme 4) [30, 39].
In the same way, 9a,b were reacted with a variety of
4-substituted benzylidinemalononitrile 11b–11k to give
the corresponding hydrazones 13b–13k and 14a–14f
(Scheme 4). TLC analyses were performed to determine the
reaction times. The 1H-NMR spectrum of 13f, as a repre-
sentative example, revealed fve singlet signals at δ 3.83,
6.71, 7.86, 8.04 and 11.43 attributed to OCH3, NH2, pyr-
idine-H5, methine-H and NH protons (see Experimental
section).
The isolation of nicotinonitriles 7a,b with ethoxycarbonyl
group stimulated our interest to investigate their reaction
with nucleophiles. Therefore, the reaction between 7a and
hydrazine hydrate in pyridine at refux was investigated. The
IR spectrum of the above product showed the absence of
cyano group and the presence of NH and NH2 groups at
3396, 3344, 3188 cm−1. Its 1H-NMR spectrum showed the
absence of any COOCH2CH3 or SCH2 protons. Instead, it
showed three singlet signals at δ 4.51, 6.11 and 9.15 corre-
sponding to NHNH2, thiophene-NH2 and NH protons (see
Experimental section). Based on these fndings, the product
was assigned as 3-aminothieno[2,3-b]pyridine-2-carbohy-
drazide 9a. Likewise, the reaction of 7b with hydrazine
hydrate in pyridine aforded the corresponding 2-carbohy-
drazide 9b. The reaction probably proceeded by the initial
nucleophilic acyl substitution reaction to give 2-((pyridin-
2-yl)thio)acetohydrazide [8] followed by intramolecular
cyclization via addition of methylene group to cyano group
to give carbohydrazide 9, bearing thienopyridine moiety
(Scheme 2).
It was reported the presence of hydrazones prepared from
substituted hydrazides in the E-confguration at N=CH bond
[40, 41]. So, the confguration of the target hydrazones was
assigned as (E)-13(14)A and not the (Z)-isomers 13(14)B
(Scheme 4).
The structure of target hydrazones 13 was confrmed
chemically via their synthesis by another route. So, hydra-
zones 13a, 13b and 13d were prepared by the direct con-
densation of carbohydrazide 9 with the appropriate aromatic
aldehydes 15a–15c in pyridine (Scheme 5).
Biology
The structure of 9a was more confrmed via its independ-
ent synthesis by another two-step synthetic route. Thus,
nicotinonitrile derivative 7a reacted with ethanolic sodium
ethoxide solution at reflux to afford ethyl thieno[2,3-b]
pyridine-2-carboxylate 10 via intramolecular cyclization.
Then, 2-carboxylate 10 reacted with hydrazine hydrate to
aford 2-carbohydrazide 9a (see Scheme 3 and Experimental
section).
Antibacterial screening
The in vitro antibacterial activities were estimated for the
starting precursors 7 and 9 as well as the new series of hydra-
zones 13 and 14, bearing thienopyridine moiety, using agar
well difusion method [42]. For this purpose, three Gram-
positive bacterial strains (Staphylococcus aureus, Strepto-
coccus mutans and Enterococcus faecalis) were selected
as well as two Gram-negative bacterial strains (Klebsiella
pneumoniae and Escherichia coli). The tested derivatives
inhibition zones are listed in the supplementary fle, while
their MIC values are listed in Table 1.
Next, carbohydrazides 9a,b were taken as key synthons to
prepare the target hydrazones, incorporating thienopyridine
moiety. Therefore, 9a was reacted with benzylidinemalon-
onitrile 11a in pyridine at refux. TLC analysis was used to
follow up the reaction. After 4 h of reaction, TLC showed
the separation of one product with some slight impurities.
After recrystallization, a sole product was isolated which
formulated as the corresponding N’-benzylidenethieno-
pyridine-2-carbohydrazide 13a. The reaction most prob-
ably proceeded via an initial hetero-Michael addition of
N-NH2 group to the C=C bond in 11a to give [12] followed
by an intramolecular nucleophilic attack of the NH group
When compared with chloramphenicol as a reference
drug (MIC values of 12.5 μg/mL against all the tested
strains), the starting precursors 2-carbohydrazide 9a, bear-
ing 6-(4-chlorophenyl)thienopyridine moiety, exhibited
more efective antibacterial activities than nicotinoni-
trile 7a. Hence, 9a showed MIC values in the range of
12.5–25 μg/mL, while 7a showed MIC values in the range
of 25–50 μg/mL against all the tested strains. Likewise,
Scheme 3 Synthesis of 2-carbohydrazide 9a and 2-carboxylate 10
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Journal of the Iranian Chemical Society
Scheme 4 Synthesis of N’-substituted benzaldehyde hydrazones 13 and 14
Scheme 5 Direct synthesis of hydrazones 13
2-carbohydrazide 9b, bearing 6-(4-nitrophenyl)thieno-
pyridine moiety, showed more potent antibacterial activi-
ties (MIC values of 25–50 μg/mL) than nicotinonitrile 7b
(MIC values of 50–100 μg/mL).
On the other hand, hydrazones 14, bearing 6-(4-nitrophe-
nyl)thienopyridine moiety, showed less efective antibacte-
rial activities than hydrazones 13, bearing 6-(4-chlorophe-
nyl)thienopyridine moiety. Hence, hydrazones 14b, 14c and
14d, with p-Cl, p-Br and p-Me, respectively, exhibited MIC
values of the range 25–50 μg/mL, while hydrazones 14a,
with p–H, exhibited MIC values of 100–200 μg/mL, when
compared with the reference drug. In addition, 14e, and 14f,
with p-OH, and p-MeO, respectively, exhibited poor anti-
bacterial activities.
The new series of hydrazones 13, bearing 6-(4-chlorophe-
nyl)thienopyridine moiety, showed a diverse antibacterial
activity. Hence, hydrazones 13b, 13c and 13d, with p-Cl,
p-Br and p-Me, respectively, exhibited the best antibacterial
activities. These derivatives exhibited more potent or equal
antibacterial activities when compared with chlorampheni-
col. Their MIC values were in the range of 6.2–12.5 μg/mL.
The hydrazones 13a, 13g and 13k, with p–H, p-PhCOO
and p-OCH2O, respectively, showed decreased antibacterial
activities. Hence, 13g exhibited MIC values of the range
25–50 μg/mL, while hydrazones 13a and 13k exhibited MIC
values of the range 50–100 μg/mL.
The substitution pattern of 13, bearing 6-(4-chlorophenyl)
moiety, was carefully chosen to provide diverse electronic
environment to the target derivatives. Therefore, electron
releasing substituents such as p-Me, p-MeO, p-OH and p-
NMe2 groups and electron-withdrawing substituents, such
as p-Cl, p-Br, p-NO2, p-PhCOO and p-EtOCO groups,
were selected as substituents on the skeleton of hydrazones
13a–13k. Experimental results revealed diferences in the
in vitro antibacterial activities between p-substituted benza-
ldehyde hydrazones 13 indicating the efect of substituents
on the resulting activity.
Moreover, hydrazones 13i and 13j, with p-NO2 and
p-EtOCO, as well as 13e, 13f and 13h, with p-OH, p-MeO
and p-NMe2, respectively, exhibited mild or poor antibacte-
rial activities with MIC values of 100–200 μg/mL, when
compared with the reference drug.
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Journal of the Iranian Chemical Society
Table 1 MIC values of
new hydrazones, bearing
6-(4-chlorophenyl)
Compound
MIC (µg/mL)
Gram-positive bacteria
Gram-negative bacteria
thienopyridine moiety
S. aureus
S. mutans
E. faecalis
K. pneumoniae
E. coli
7a
25
50
12.5
25
50
6.2
12.5
6.2
−ve
200
25
−ve
100
100
50
50
100
25
50
50
12.5
12.5
6.2
−ve
−ve
25
200
200
100
50
25
50
12.5
50
25
50
12.5
25
50
100
25
50
100
6.2
12.5
12.5
−ve
200
50
200
200
200
100
200
25
7b
9a
9b
13a
100
6.2
6.2
12.5
200
−ve
50
−ve
200
100
100
200
25
100
12.5
6.2
12.5
200
−ve
25
−ve
100
100
50
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
14a
100
25
50
100
50
50
200
50
25
14b
14c
25
25
−ve
−ve
12.5
25
50
−ve
−ve
12.5
14d
25
25
50
14e
−ve
−ve
12.5
−ve
−ve
12.5
−ve
−ve
12.5
14f
Chloramphenicol
In order to elucidate the relationship between electronic
properties of the hydrazones 13 and their antibacterial
activities, we compared the values of electronic substituent
constant (σp), feld constant (F), resonance efect constant
(R) [43] with the MIC values against both Staphylococcus
aureus (bacteria) and Klebsiella pneumoniae as examples of
Gram-positive and Gram-negative bacterial strains, respec-
tively (Table 2). After examining Table 2, no relation was
observed between values of MIC and σp or F. On the other
hand, a clear relation was observed between the values of
R and MIC. It is obvious that resonance efect constant (R)
values of electron releasing Cl (R=−0.19), Br (R=−0.22)
and Me (R=−0.18) groups are very close. The hydrazones
13b, 13c and 13d with these substituents are the most potent
antibacterial agents with lower or equal MIC values when
compared with the reference drug (Table 2).
Table 2 The relation between
Compound
Substituent
σp
F
R
MIC (µg/mL)
electronic constants of new
hydrazones 13 and their MIC
values
S. aureus
K. pneumoniae
13a
13b
13c
13d
13e
13f
13g
13h
13i
H
Cl
Br
Me
0.00
0.23
0.23
−0.17
−0.37
−0.27
0.13
−0.83
0.78
0.45
0.03
0.44
0.45
0.01
0.33
0.29
0.26
0.15
0.65
0.34
0.00
50
100
12.5
6.2
12.5
200
−ve
25
−ve
100
100
50
−0.19
−0.22
−0.18
−0.70
−0.56
−0.13
−0.98
0.13
6.2
12.5
6.2
−ve
200
25
−ve
100
100
50
OH
MeO
PhCOO
NMe2
NO2
EtOCO
OCH2O
13j
13k
0.11
−0.16
−0.11
−0.05
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Journal of the Iranian Chemical Society
Table 2 reveals that as the resonance efect constant (R)
for the substituent attached to hydrazone become more
+ve, the antibacterial activity decreased. This may be
attributed to the electron density of the hydrazone. Since,
electron releasing group has increased the electron den-
sity making the target hydrazone efective against diferent
bacterial strains [44].
Cytotoxicity against eukaryotic cells
Several hydrazones, carrying thienopyridine moiety, 13a,
13b, 13c, 13d, 13g, 13k, 14b, 14c and 14d have been
reported for in vitro cytotoxic activities against each human
breast carcinoma cell line (MCF-7), colon cancer cell
line (Caco2) and liver hepatocellular carcinoma cell line
(HEPG2) (Table 4). The cytotoxicity fndings are expressed
as the concentration of the tested hydrazone 13 needed to
inhibit 50 percent of cell growth (IC50, μg/mL).
However, Table 2 also reveals that hydrazones attached
to strong electron substituents exhibited decreased anti-
bacterial potency. Therefore, hydrazones 13e, 13f and 13h
that attached to OH (R = − 0.70), MeO (R = − 0.56) and
NMe2 (R=−0.98) groups exhibited poor or fair antibacte-
rial activities. This may be attributed to too high electron
density resulted in more difcult difusion through the bac-
teria cells, and so, substantial activity loss may occur [45].
On the other hand, experimental fndings showed the
reliance of the antibacterial activities on the substitution at
para-position of 6-aryl group on the skeleton of thienopyr-
idine. Therefore, hydrazones 14a–14f, linked to more elec-
tron-withdrawing 6-(4-nitrophenyl) group, demonstrated
less efective antibacterial activity than their analogues
13a–13f, linked to more electron-donating 6-(4-chloro-
phenyl) group.
The hydrazones 14b, 14c and 14d were found to be non-
cytotoxic to all of the cell lines tested. The cell viability
percentage of diferent cell lines was evaluated after treat-
ment with hydrazones 14 and the reference Doxorubicin for
24 h (see the supplementary fle). The viability of all the
tested cells was > 70 percent when the cells were treated
with concentrations of up to 75 μg/mL of these hydrazones
(lowest viability value defned by ISO 10993-5 [47] to fnd
a substance as non-cytotoxic).
Among the hydrazones 13, compound 13d exhib-
ited the strongest cytotoxic effect against MCF-7 with
IC50 =18.03 0.5 µg/mL (Fig. 1). In addition, hydrazones
13b, 13c and 13g exhibited decreased cytotoxic activities
with IC50 =23.67 0.3, 25.51 0.4 and 29.37 0.3 µg/mL,
respectively, while the remaining hydrazones 13 exhibited
mild cytotoxic activities when compared with Doxorubicin
(IC50 =16.39 0.3 µg/mL) (Table 4).
Likewise, we observed the dependence of antibacte-
rial activities of hydrazones 14 on the values of resonance
efect constant (R). The relation between R and the MIC
values of hydrazones 14a–14f against both Staphylococcus
aureus and Klebsiella pneumoniae is described in Table 3.
Among the new series of hydrazones 14, compounds
14b, 14c and 14d (R values from − 0.18 to − 0.22) had
the best antibacterial potencies. Such derivatives showed
MIC values of 25–50 μg/mL (Table 3) [41]. In addition,
hydrazones 14e and 14f linked to OH (R = − 0.70) and
MeO (R = − 0.56) moieties exhibited fair antibacterial
activities [45].
With regard to the cytotoxicity against colon cancer
Caco2 cell line, hydrazone 13d exhibited strong cytotoxic
activity with IC50 =25.99 0.6 µg/mL (Fig. 2). Moreover,
hydrazones 13b and 13c exhibited moderate activities with
IC50 =31.09 0.4 and 32.64 0.4 µg/mL, respectively. Rest
of hydrazones 13 exhibited mild cytotoxic activities when
compared with Doxorubicin (IC50 = 25.30 0.4 µg/mL)
(Table 4).
As a summary, synthesis of effective antibacterial
hydrazones, bearing thienopyridine moiety, could be
accomplished by optimizing the electron density of the
substituents on their structure [46].
With regard to the cytotoxicity against liver hepatocellu-
lar carcinoma HEPG2 cell line, both hydrazones 13b and 13c
exhibited strong cytotoxic activity with IC50 =19.48 0.5
Table 4 The cell inhibition percentage and IC50 values of some novel
hydrazones against each of MCF-7, Caco2 and HEPG2, and using
Doxorubicin as a reference drug
Table 3 The relation between resonance efect constant (R) of new
hydrazones 14 and their MIC values
Compound
IC50 (µg/mL)
MCF-7
Compound
Substituent
R
MIC (µg/mL)
Caco2
HEPG2
S. aureus
K. pneumoniae
13a
48.02 0.4
23.67 0.3
25.51 0.4
18.03 0.5
29.37 0.3
46.47 0.4
16.39 0.3
68.14 0.5
31.09 0.4
32.64 0.4
25.99 0.6
42.56 0.6
58.51 0.3
25.30 0.4
64.15 0.5
19.48 0.5
19.30 0.3
25.85 0.4
31.00 0.3
53.26 0.4
18.89 0.3
13b
14a
14b
14c
14d
14e
14f
H
Cl
Br
Me
OH
MeO
0.00
100
25
50
25
−ve
−ve
200
50
25
50
−ve
−ve
13c
−0.19
−0.22
−0.18
−0.70
−0.56
13d
13g
13k
Doxorubicin
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Journal of the Iranian Chemical Society
Fig. 1 Cell viability percentage
of MCF-7 after treatment with
some hydrazones 13 for 24 h
16.39 0.3
25.30 0.4 18.89 0.3
Doxorubicin
120
100
80
60
40
20
0
0
5
25
50
75
29.4
6.5
13a
100
100
100
100
100
100
100
75.3
74.4
69.8
64.1
74.4
80.2
66.9
62.9
47.3
55.6
47.1
54.7
69.5
36.6
55.4
10.7
15.7
7.7
13b
13c
13d
13g
13k
Dox
6.5
5.3
25.4
49.3
14.4
7.3
25.4
10.8
Concentraꢀon (μg/mL)
Fig. 2 Cell viability percentage
of Caco2 after treatment with
some hydrazones 13 for 24 h
120
100
80
60
40
20
0
0
5
25
50
75
13a
100
100
100
100
100
100
100
84.4
80.3
82.3
79.6
81.6
80.2
76.8
67.8
55.6
50.4
47.3
60.3
60.5
49.9
53.7
22.2
29.1
11.4
44.6
48.8
9.8
50.8
9.5
13b
13c
13d
13g
13k
Dox
15.4
7.3
25.4
47.1
6.6
Concentraꢀon (μg/mL)
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Journal of the Iranian Chemical Society
Table 5 The IC50 values in µM
of some hydrazones against
COX-2 enzyme. Celecoxib was
used as the standard drug
and 19.30 0.3 µg/mL, respectively (Fig. 3). Moreover,
hydrazones 13d and 13g exhibited moderate activities with
IC50 =25.85 0.4 and 31.00 0.3 µg/mL, respectively. Rest
of hydrazones 13 exhibited mild cytotoxic activities when
compared with Doxorubicin (IC50 = 18.89 0.3 µg/mL)
(Table 4).
IC50 (µM)
against
COX-2
13a
0.182
0.121
0.110
0.104
0.133
0.165
0.115
13b
13c
13d
The in vitro evaluation of cyclooxygenase‑2 inhibition
13g
13k
The hydrazones 13a, 13b, 13c, 13d, 13g and 13k were tested
for inhibiting cyclooxygenase-2 enzyme (COX-2) [48]. The
concentration required to inhibit 50% of COX-2 enzyme
(IC50) is expressed in µM. Celecoxib was used as a refer-
ence drug with IC50 of 0.115 µM (see Table 5).
Celecoxib
13c and 13d as well as Celecoxib were docked as ligand
molecules with COX-2 (PDB ID: 4cox) as the target pro-
tein to achieve their optimum conformation, with reduced
free energy [49, 50]. Computational docking studies have
been used to estimate the binding energies of the interac-
tions between each ligand and the amino acid residues in
the target enzyme [51]. The fndings are shown in Table 6.
Figure 4 displays 2D and 3D ligand interactions of
Celecoxib with the target 4cox. It showed a large network
of hydrogen bonding interactions with residues of GLU
46, CYS 47, TYR 136 and LYS 137 amino acids in the
target enzyme with binding distances and energies varying
from − 0.6 to 5.6 kcal/mol and from 2.92 to 3.36 Å.
Hydrazones 13c and 13d revealed more inhibitory activ-
ity than Celecoxib with IC50 of 0.110 and 0.104 µM, respec-
tively. Hydrazone 13b revealed nearly inhibition activity
equipotent to Celecoxib with IC50 of 0.121 µM. On the other
hand, hydrazones 13a, 13g and 13k exhibited decreased
inhibition activity against COX-2 enzyme with IC50 in the
range of 0.133–0.182 µM.
The in silico study: Molecular modeling
In comparison with Celecoxib, molecular docking was
performed to test the capability of some new hydrazones
13 as potential COX-2 inhibitors. Thus, compounds 13b,
Fig. 3 Cell viability percentage
of HEPG2 after treatment with
some hydrazones 13 for 24 h
120
100
80
60
40
20
0
0
5
25
50
75
13a
100
100
100
100
100
100
100
79.8
67.6
62.3
69
66.5
44.1
49.2
52.4
50.2
63.5
39.6
50.2
11.6
15.4
20.3
37.6
54.5
11.5
49.7
7.3
13b
13c
13d
13g
13k
Dox
6.1
17.7
26.6
37.3
10.1
67.8
74.6
69.8
Concentraꢀon (µg/mL)
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Journal of the Iranian Chemical Society
Table 6 Energy values obtained
in docking calculations of new
hydrazones 13 with COX-2
(PDB ID: 4cox)
Compound
Ligand moiety
Site
Interaction
Distance (Å)
E (kcal/mol)
13b
N 48
O 12
6-ring
5-ring
S 10
O CYS 47 (A)
N CYS 47 (A)
CA GLN 42 (A)
CA MET 48 (A)
O CYS 41 (A)
O ALA 151 (A)
N CYS 47 (A)
CG ARG 44 (A)
CA GLU 46 (A)
O ARG 44 (A)
O ARG 44 (A)
OH TYR 130 (A)
OH TYR 130 (A)
CA GLU 46 (A)
N CYS 47 (A)
OE1 GLU 46 (A)
O CYS 47 (A)
CA TYR 136 (A)
NZ LYS 137 (A)
H-donor
H-acceptor
Pi-H
2.80
2.72
3.56
3.61
2.84
2.67
2.59
3.34
2.98
3.15
2.84
2.42
3.11
3.85
3.09
3.11
2.92
3.28
3.36
−1.5
−5.4
−3.2
−1.1
−0.7
−1.7
−5.7
−1.4
−3.7
−0.7
−4.2
−6.2
−0.7
−0.8
−3.8
−5.6
−2.5
−0.6
−0.8
Pi-H
13c
13d
H-donor
H-donor
H-acceptor
Pi-H
S 23
O 12
5-ring
6-ring
S 10
N 13
O 12
N 15
6-ring
6-ring
N 19
N 19
O 18
O 18
Pi-H
H-donor
H-donor
H-acceptor
H-acceptor
Pi-H
Pi-H
Celecoxib
H-donor
H-donor
H-acceptor
H-acceptor
Fig. 4 The 2D and 3D ligand interactions of Celecoxib with COX-2 (4cox)
Experimental results demonstrated capability of hydra-
zone 13d as a potent COX-2 inhibitor. Docking study of
compound 13d revealed more strong binding interactions
with COX-2 than the reference Celecoxib (see Fig. 5).
The fgure revealed a strong network of hydrogen bonding
interactions of both hydrazone-NH and thienopyridine-S
with residue of ARG 44 (2.84 Å, − 4.2 kcal/mol; 3.15 Å,
− 0.7 kcal/mol, respectively) as well as between both
carbonyl-O and hydrazone-N with residue of TYR 130
(2.42 Å, − 6.2 kcal/mol; 3.11 Å, − 0.7 kcal/mol, respec-
tively). In addition, it showed strong π–H interactions
between hydrazone-aryl moieties with residues of both CYS
47 and GLU 46 (3.09 Å, −3.8 kcal/mol; 3.85 Å, −0.8 kcal/
mol, respectively).
Hydrazone 13c showed good binding interactions
with COX-2 (see supplementary fle). Its docking poses
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Journal of the Iranian Chemical Society
Fig. 5 The 2D and 3D ligand interactions of hydrazone 13d with COX-2 (4cox)
revealed a network of hydrogen bonding interactions of
carbonyl-O with residue of CYS 47 (2.59 Å, − 5.7 kcal/
mol), thienopyridine-S with residue of CYS 41 (2.84 Å,
−0.7 kcal/mol) and thiophene–S with residue of ALA 151
(2.67 Å, −1.7 kcal/mol). Also, it showed strong π–H inter-
actions between hydrazone-aryl moiety with residue of
GLU 46 (2.98 Å, − 3.7 kcal/mol) and thiophene ring with
residue of ARG 44 (3.34 Å, − 1.4 kcal/mol).
Conclusion
The starting intermediate carbohydrazide was prepared
and used to prepare a series the target hydrazones incor-
porating thienopyridine moiety. The in vitro antibacterial
potency of the target hydrazones was evaluated against
different strains of Gram-positive and Gram-negative
bacteria. The hydrazones 13b, 13c and 13d, with p-Cl,
p-Br and p-Me, respectively, exhibited the best antibacte-
rial potencies against all the tested bacteria. The in vitro
cytotoxicity of some novel hydrazones was tested against
several eukaryotic cell lines. The hydrazones 13b, 13c and
13d exhibited the best cytotoxicity results against all the
tested cells. Furthermore, both experimental and docking
studies could predict the promising inhibitory activities
of hydrazones 13c and 13d against COX-2 enzyme, when
compared with Celecoxib.
Furthermore, hydrazone 13b showed decreased binding
interactions with COX-2 when compared with Celecoxib
(see supplementary file). Its docking poses demon-
strated a network of hydrogen bonding interactions of
both carbonyl-O and amino-N with residue of CYS 47
(2.72 Å, − 5.4 kcal/mol; 2.80 Å, − 1.5 kcal/mol, respec-
tively). Also, it showed strong π–H interactions between
hydrazone-aryl moiety with residue of GLN 42 (3.56 Å,
− 3.2 kcal/mol) and thiophene ring with residue of MET
48 (3.61 Å, − 1.1 kcal/mol).
As a summary, both experimental and docking studies
could predict the promising potential of the hydrazones
being tested as efective COX-2 inhibitors. Docking poses
of these hydrazones showed the relation between the elec-
tronic properties of the hydrazones and their inhibitory
activities. Therefore, hydrazones 13b, 13c, and 13d, linked
to aryl group with p-Cl, p-Br and p-Me, respectively,
revealed favorable hydrophobic interactions between
hydrazone-aryl moieties with amino acids residues of
COX-2 (energy of − 3.2 to − 3.8 kcal/mol). In addition,
docking poses of these hydrazones demonstrated strong
networks of hydrogen bonding interactions of carbonyl-
O with COX-2 amino acids residues (energy of − 5.4 to
− 6.2 kcal/mol).
Experimental
Introduction
All solvents were acquired from commercial sources and
used as received unless otherwise stated. All other chemi-
cals were acquired from Merck or Aldrich. These chemi-
cals were used without further purifcation. The melting
points were measured on a Stuart melting point appara-
tus and are uncorrected. IR spectra were recorded on a
Smart iTR, which is an ultra-high-performance, versatile
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Journal of the Iranian Chemical Society
attenuated total refectance (ATR) sampling accessory on
the Nicolet iS10 FT-IR spectrometer manufactured. NMR
spectra were recorded on Bruker Avance III 400 MHz
product that formed was collected by fltration, washed with
water, dried and then recrystallized from the proper solvent.
1
spectrophotometer (400 MHz for H and 100 MHz for
Ethyl
2‑((6‑(4‑chlorophenyl)‑3‑cyano‑4‑(thiophen‑2‑yl)
13C) using TMS as an internal standard and DMSO-d6 as
solvent, and chemical shifts were expressed as δ ppm units.
Mass spectra were recorded on a GC–MS-QP1000EX
spectrometer using inlet type at 70 eV. Elemental analy-
ses were carried out on a EuroVector instrument C, H, N
analyzer EA3000 Series.
pyridin‑2‑yl)thio)acetate (7a) Colorless solid (ethanol/diox-
ane mixture, 95%); m.p. 200–202 °C;; IR (υ cm−1): 2214
1
(CN), 1729 (CO); H-NMR (DMSO-d6): δ 1.14 (t, 3H,
CH2CH3), 4.10 (q, 2H, CH2CH3), 4.24 (s, 2H, CH2), 7.34
(t, 1H, thiophene-H4), 7.60 (d, 2H, Ar–H’s), 7.97–8.02 (m,
3H, pyridine-H5, thiophene-H5 and H3), 8.24 (d, 2H, Ar–
H’s); 13C-NMR (DMSO-d6): δ 14.4 (CH3), 33.1 (SCH2),
61.6 (OCH2), 100.2, 114.3 (pyridine-C3, C5), 116.2 (CN),
127.8, 129.1, 129.9, 130.2, 130.9, 135.0, 135.8, 136.7 (Ar–
C’s and thiophene-C’s), 146.1, 158.2, 162.7 (pyridine-C4,
C6, C2), 169.0 (CO); MS m/z (%): 414 (M+, 40.9), 416
(M++2, 14.0); Anal. for C20H15ClN2O2S2: C, 57.90; H,
3.64; N, 6.75; found: C, 58.02; H, 3.53; N, 6.81%.
Methods and spectral data
Synthesis of pyridine‑2(1H)‑thiones 5
A binary mixture of carbonyl derivative 3 (5 mmol) and
2-cyanothioacetamide 4 (5 mmol) in ethanol (15 mL) in the
presence of piperidine (0.3 mL) was boiled at refux for 4 h.
The reaction mixture was cooled, fltered of, washed with
ethanol, and then, the product was recrystallized from the
proper solvent.
Ethyl 2‑((3‑cyano‑6‑(4‑nitrophenyl)‑4‑(thiophen‑2‑yl)pyri‑
din‑2‑yl)thio)acetate (7b) Colorless solid (dioxane, 90%);
m.p. 224–225 °C;; IR (υ cm−1): 2214 (CN), 1728 (CO);
1H-NMR (DMSO-d6): δ 1.13 (t, 3H, CH2CH3), 4.10 (q, 2H,
CH2CH3), 4.21 (s, 2H, CH2), 7.37 (t, 1H, thiophene-H4),
7.95 (s, 1H, pyridine-H5), 7.99 (d, 1H, thiophene-H5), 8.06
(d, 1H, thiophene-H3), 8.21 (d, 2H, Ar–H’s), 8.33 (d, 2H,
Ar–H’s); 13C-NMR (DMSO-d6): δ 14.3 (CH3), 33.2 (SCH2),
61.6 (OCH2), 100.1, 114.2 (pyridine-C3, C5), 116.3 (CN),
122.2, 124.5, 127.7, 129.9, 130.8, 136.5, 138.8, 144.8 (Ar–
C’s and thiophene-C’s), 146.0, 157.7, 162.5 (pyridine-C4,
C6, C2), 169.1 (CO); Anal. for C20H15N3O4S2 (425.4): C,
56.46; H, 3.55; N, 9.88; found: C, 56.31; H, 3.42; N, 10.06%.
6‑(4‑Chlorophenyl)‑4‑(thiophen‑2‑yl)‑2‑thioxo‑1,2‑dihy‑
dropyridine‑3‑carbonitrile (5a) Pale orange solid (ethanol,
71%); m.p. 286–287 °C; IR (υ cm−1): 3155 (NH), 2218
(CN);1H-NMR (DMSO-d6): δ 7.38 (t, 1H, thiophene-H4),
7.43 (d, 2H, Ar–H’s), 8.04 (d, 1H, thiophene-H5), 8.08–8.11
(m, 3H, pyridine-H5, 2 Ar–H’s), 8.17 (d, 1H, thiophene-
H3), 14.08 (s, 1H, NH); 13C-NMR (DMSO-d6): 109.6, 111.7
(pyridine-C3, C5), 117.6 (CN), 128.7, 128.9, 129.0, 130.4,
133.1, 135.1, 135.7, 136.4 (Ar–C’s and thiophene-C’s),
147.8, 152.4, 180.2 (pyridine-C4, C6, C2); MS m/z (%): 329
(M+, 28.5), 331 (M++2, 9.9); Anal. for C16H9ClN2S2: C,
58.44; H, 2.76; N, 8.52; found: C, 58.30; H, 2.89; N, 8.64%.
Synthesis of thieno[2,3‑b]pyridine‑2‑carbohydrazides 9
A mixture of nicotinonitrile 7 or thienopyridine 10 (5 mmol)
and hydrazine hydrate (5 mL, 80%) in pyridine (15 mL) was
boiled at refux for 5–8 h. The product that formed was col-
lected by fltration, washed with ethanol, dried and then
recrystallized from the proper solvent.
6‑(4‑Nitrophenyl)‑4‑(thiophen‑2‑yl)‑2‑thioxo‑1,2‑dihydro‑
pyridine‑3‑carbonitrile (5b) Pale orange solid (dioxane/
ethanol mixture, 74%); m.p. above 300 °C; IR (υ cm−1):
3158 (NH), 2219 (CN);1H-NMR (DMSO-d6): δ 7.32 (t, 1H,
thiophene-H4), 7.98 (d, 1H, thiophene-H5), 8.04 (d, 2H,
Ar–H’s), 8.10 (s, 1H, pyridine-H5), 8.15 (d, 1H, thiophene-
H3), 8.41 (d, 2H, Ar–H’s), 14.22 (s, 1H, NH); 13C-NMR
(DMSO-d6): 109.2, 111.6 (pyridine-C3, C5), 117.2 (CN),
121.9, 124.2, 128.4, 128.7, 132.8, 136.2, 138.6, 144.5 (Ar–
C’s and thiophene-C’s), 146.8, 152.7, 180.6 (pyridine-C4,
C6, C2); Anal. for C16H9N3O2S2 (339.3): C, 56.62; H, 2.67;
N, 12.38; found: C, 56.51; H, 2.54; N, 12.48%.
3‑Amino‑6‑(4‑chlorophenyl)‑4‑(thiophen‑2‑yl)thieno[2,3‑b]
pyridine‑2‑carbohydrazide (9a) Yellow solid (ethanol/
dioxane mixture, 77%); m.p. 250–251 °C; IR (υ cm−1):
3396, 3344, 3188 (NH, NH2); 1H-NMR (DMSO-d6): δ
4.51 (br s, 2H, NHNH2), 6.11 (br s, 2H, NH2), 7.28 (t, 1H,
thiophene-H4), 7.42 (d, 1H, thiophene-H5), 7.53 (d, 2H,
Ar–H’s), 7.72 (s, 1H, pyridine-H5), 7.84 (d, 1H, thiophene-
H3), 8.22 (d, 2H, Ar–H’s), 9.15 (br s, 1H, NH); 13C-NMR
(DMSO-d6): δ 94.2, 118.9, 121.8 (thienopyridine-C2, C5,
C3a), 127.9, 128.5, 128.9, 129.4, 130.6, 134.7, 135.9, 136.4
(Ar–C’s and thiophene-C’s), 139.9, 145.1 (2C), 153.8
(thienopyridine-C3, C4, C6, C7a), 164.4 (thienopyridine-
CO); MS m/z (%): 400 (M+, 20.4), 402 (M++2, 8.3); Anal.
Synthesis of nicotinonitriles 7
A mixture of 2-thioxopyridine 5 (5 mmol) and ethyl chlo-
roacetate 6 (5 mmol) in ethanol (15 mL) in the presence
of sodium acetate (5 mmol) was stirred at rt for 1 h. The
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Journal of the Iranian Chemical Society
for C18H13ClN4OS2: C, 53.93; H, 3.27; N, 13.98; found: C,
53.84; H, 3.41; N, 14.09%.
thiophene-H3), 8.12 (s, 1H, methine-H), 8.28 (d, 2H, Ar–
H’s), 11.49 (s, 1H, NH); 13C-NMR (DMSO-d6): δ 94.6,
119.5, 122.2 (thienopyridine-C2, C5, C3a), 127.5, 127.9,
128.5, 128.9, 129.2, 129.4, 130.5, 130.7, 134.3, 134.7,
135.9, 136.5 (Ar–C’s and thiophene-C’s), 139.9 (thienopyr-
idine-C3), 144.8 (methine-C), 145.5, 145.8, 154.4 (thieno-
pyridine-C4, C6, C7a), 168.6 (thienopyridine-CO); Anal.
for C25H17ClN4OS2 (489.0): C, 61.40; H, 3.50; N, 11.46;
found: C, 61.23; H, 3.36; N, 11.62%.
3‑Amino‑6‑(4‑nitrophenyl)‑4‑(thiophen‑2‑yl)thieno[2,3‑b]
pyridine‑2‑carbohydrazide (9b) Yellow solid (dioxane,
81%); m.p. 259–262 °C; IR (υ cm−1): 3468, 3313, 3255 (NH,
1
NH2); H-NMR (DMSO-d6): δ 4.42 (br s, 2H, NHNH2),
6.14 (br s, 2H, NH2), 7.30 (t, 1H, thiophene-H4), 7.40 (d,
1H, thiophene-H5), 7.69 (s, 1H, pyridine-H5), 7.82 (d, 1H,
thiophene-H3), 8.14 (d, 2H, Ar–H’s), 8.31 (d, 2H, Ar–H’s),
9.20 (br s, 1H, NH); 13C-NMR (DMSO-d6): δ 94.5, 118.5,
121.5 (thienopyridine-C2, C5, C3a), 122.1, 124.0, 127.7,
128.6, 130.7, 136.3, 138.5 (Ar–C’s and thiophene-C’s),
139.5 (thienopyridine-C3), 144.4 (Ar–C), 144.9, 145.2,
153.4 (thienopyridine-C4, C6, C7a), 164.2 (thienopyridine-
CO); Anal. for C18H13N5O3S2 (411.4): C, 52.54; H, 3.18; N,
17.02; found: C, 52.62; H, 3.22; N, 16.87%.
(E)‑3‑Amino‑N’‑(4‑chlorobenzylidene)‑6‑(4‑chlorophenyl)
‑4‑(thiophen‑2‑yl)thieno[2,3‑b]pyridine‑2‑carbohydrazide
(13b) Yellow solid (ethanol/dioxane mixture); m.p. above
1
300 °C; IR (υ cm−1): 3322, 3240 (NH, NH2); H-NMR
(DMSO-d6): δ 6.64 (br s, 2H, NH2), 7.29 (t, 1H, thiophene-
H4), 7.43 (d, 1H, thiophene-H5), 7.54 (d, 2H, Ar–H’s),
7.60 (d, 2H, Ar–H’s), 7.84–7.86 (m, 3H, 2 Ar–H’s and
pyridine-H5), 7.94 (d, 1H, thiophene-H3), 8.11 (s, 1H,
methine-H), 8.27 (d, 2H, Ar–H’s), 11.55 (s, 1H, NH); 13C-
NMR (DMSO-d6): δ 94.3, 119.0, 121.9 (thienopyridine-C2,
C5, C3a), 127.8, 128.4, 128.6, 128.8, 128.9, 129.4, 130.6,
132.8, 134.5, 134.8, 135.9, 136.5 (Ar–C’s and thiophene-
C’s), 140.2 (thienopyridine-C3), 142.9 (methine-C), 145.3,
146.2, 154.5 (thienopyridine-C4, C6, C7a), 167.9 (thieno-
pyridine-CO); Anal. for C25H16Cl2N4OS2 (523.4): C, 57.36;
H, 3.08; N, 10.70; found: C, 57.51; H, 3.00; N, 10.58%.
Synthesis of ethyl 3‑amino‑6‑(4‑chlorophenyl)‑4‑(thiophen‑
2‑yl)thieno[2,3‑b]pyridine‑2‑carboxylate (10)
A mixture of nicotinonitrile 7 (5 mmol) and ethyl chloro-
acetate 6 (5 mmol) in ethanol (20 mL) in the presence of
sodium metal (5 mmol) was heated at refux for 2 h. The
products that formed were collected by fltration, washed
with ethanol, dried and then recrystallized from dioxane as
yellow solid (93%); m.p. 160–161 °C; IR (υ cm-1): 3462,
1
3333 (NH2), 1664 (CO); H-NMR (DMSO-d6): δ 1.30 (t,
(E)‑3‑Amino‑N’‑(4‑bromobenzylidene)‑6‑(4‑chlorophenyl)
‑4‑(thiophen‑2‑yl)thieno[2,3‑b]pyridine‑2‑carbohydrazide
(13c) Yellow solid (ethanol/dioxane mixture); m.p. above
3H, CH2CH3), 4.28 (q, 2H, CH2CH3), 6.11 (s, 2H, NH2),
7.31 (t, 1H, thiophene H-4), 7.46 (d, 1H, thiophene-H5),
7.56 (d, 2H, Ar–H’s), 7.89 (s, 1H, pyridine-H5), 7.93 (d, 1H,
thiophene-H3), 8.23 (d, 2H, Ar–H’s); MS m/z (%): 414 (M+,
76.4), 416 (M++2, 28.7); Anal. for C20H15ClN2O2S2: C,
57.90; H, 3.64; N, 6.75; found: C, 57.74; H, 3.77; N, 6.92%.
1
300 °C; IR (υ cm−1): 3322, 3240 (NH, NH2); H-NMR
(DMSO-d6): δ 6.66 (br s, 2H, NH2), 7.31 (t, 1H, thiophene-
H4), 7.44 (d, 1H, thiophene-H5), 7.57–7.60 (m, 4H, Ar–
H’s), 7.85–7.87 (m, 3H, 2 Ar–H’s and pyridine-H5), 7.93
(d, 1H, thiophene-H3), 8.10 (s, 1H, methine-H), 8.27 (d, 2H,
Ar–H’s), 11.52 (s, 1H, NH); Anal. for C25H16BrClN4OS2
(567.9): C, 52.87; H, 2.84; N, 9.87; found: C, 52.99; H,
2.97; N, 9.66%.
General procedure for synthesis of hydrazones 13 and 14
A mixture of each of 2-carbohydrazides 9a,b (5 mmol)
and the appropriate arylidenemalononitrile 11a–11k or the
appropriate aromatic aldehydes 15a–15c (5 mmol) in pyri-
dine (15 mL) was boiled at refux. The reaction was followed
by TLC analyses to determine the reaction time. Then, the
solvent was evaporated to its half volume and cooled. The
solid product was collected by fltration, washed with etha-
nol, dried and then recrystallized from the proper solvent.
(E)‑3‑Amino‑6‑(4‑chlorophenyl)‑N’‑(4‑methylbenzylidene)
‑4‑(thiophen‑2‑yl)thieno[2,3‑b]pyridine‑2‑carbohydrazide
(13d) Yellow solid (dioxane); m.p. 280–281 °C; IR (υ
cm−1): 3319, 3250 (NH, NH2); 1H-NMR (DMSO-d6):
δ 2.35 (s, 3H, CH3), 6.73 (br s, 2H, NH2), 7.29–7.32 (m,
3H, 2 Ar–H’s and thiophene-H4), 7.47 (d, 1H, thiophene-
H5), 7.55 (d, 2H, Ar–H’s), 7.63 (d, 2H, Ar–H’s), 7.82 (s,
1H, pyridine-H5), 7.93 (d, 1H, thiophene-H3), 8.10 (s, 1H,
methine-H), 8.25 (d, 2H, Ar–H’s), 11.41 (s, 1H, NH); Anal.
for C26H19ClN4OS2 (503.0): C, 62.08; H, 3.81; N, 11.14;
found: C, 61.95; H, 3.74; N, 11.26%.
(E)‑3‑amino‑N’‑benzylidene‑6‑(4‑chlorophenyl)‑4‑(thiophe
n‑2‑yl)thieno[2,3‑b]pyridine‑2‑carbohydrazide (13a) Yel-
low solid (ethanol/dioxane mixture); m.p. 240–242 °C; IR
1
(υ cm−1): 3329, 3244 (NH, NH2); H-NMR (DMSO-d6): δ
6.63 (br s, 2H, NH2), 7.32 (t, 1H, thiophene-H4), 7.46–7.50
(m, 4H, 3 Ar–H’s and thiophene-H5), 7.55 (d, 2H, Ar–H’s),
7.84–7.86 (m, 3H, 2 Ar–H’s and pyridine-H5), 7.90 (d, 1H,
(E)‑3‑Amino‑6‑(4‑chlorophenyl)‑N’‑(4‑hydroxybenzylidene
)‑4‑(thiophen‑2‑yl)thieno[2,3‑b]pyridine‑2‑carbohydrazide
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Journal of the Iranian Chemical Society
(13e) Yellow solid (dioxane); m.p. above 300 °C; IR
1H, thiophene-H5), 7.63–7.66 (m, 4H, Ar–H’s), 7.85 (s,
1H, pyridine-H5), 7.91 (d, 1H, thiophene-H3), 8.07 (s,
1H, methine-H), 8.25 (d, 2H, Ar–H’s), 11.49 (s, 1H, NH);
13C-NMR (DMSO-d6): δ 39.8 (N(CH3)2), 94.7 (thienopyr-
idine-C2), 111.7 (Ar–C), 118.2 (thienopyridine-C5), 121.1
(Ar–C), 121.6 (thienopyridine-C3a), 127.7, 128.2, 128.6,
129.1, 129.5, 130.4, 134.6, 135.8, 136.5 (Ar–C’s and thio-
phene-C’s), 140.3 (thienopyridine-C3), 145.2 (methine-C),
145.4, 145.9 (thienopyridine-C4, C6), 149.3 (Ar–C), 155.4
(thienopyridine-C7a), 167.5 (thienopyridine-CO); Anal. for
C27H22ClN5OS2 (532.0): C, 60.95; H, 4.17; N, 13.16; found:
C, 60.95; H, 4.17; N, 13.16%.
1
(υ cm−1): 3445 (OH), 3318, 3252 (NH, NH2); H-NMR
(DMSO-d6): δ 6.72 (br s, 2H, NH2), 6.91 (d, 2H, Ar–H’s),
7.33 (t, 1H, thiophene-H4), 7.45 (d, 1H, thiophene-H5), 7.57
(d, 2H, Ar–H’s), 7.66 (d, 2H, Ar–H’s), 7.83 (s, 1H, pyridine-
H5), 7.93 (d, 1H, thiophene-H3), 8.06 (s, 1H, methine-H),
8.27 (d, 2H, Ar–H’s), 10.02 (br s, 1H, OH), 11.44 (s, 1H,
NH); 13C-NMR (DMSO-d6): δ 95.0 (thienopyridine-C2),
115.7 (Ar–C), 118.2, 121.5 (thienopyridine-C5, C3a), 124.9,
127.8, 128.5, 128.7, 129.1, 129.4, 130.5, 134.8, 135.8, 136.6
(Ar–C’s and thiophene-C’s), 140.3 (thienopyridine-C3),
144.6 (methine-C), 145.1, 145.9, 155.2 (thienopyridine-C4,
C6, C7a), 159.4 (Ar–C), 167.8 (thienopyridine-CO); Anal.
for C25H17ClN4O2S2 (505.0): C, 59.46; H, 3.39; N, 11.09;
found: C, 59.31; H, 3.53; N, 10.87%.
(E)‑3‑Amino‑6‑(4‑chlorophenyl)‑N’‑(4‑nitrobenzylidene)‑
4‑(thiophen‑2‑yl)thieno[2,3‑b]pyridine‑2‑carbohydrazide
(13i) Orange solid (dioxane); m.p. 184–187 °C; IR (υ
1
(E)‑3‑Amino‑6‑(4‑chlorophenyl)‑N’‑(4‑methoxybenzylidene
)‑4‑(thiophen‑2‑yl)thieno[2,3‑b]pyridine‑2‑carbohydrazide
(13f) Yellow solid (ethanol/dioxane mixture); m.p. 284 °C;
IR (υ cm−1): 3310, 3251 (NH, NH2); 1H-NMR (DMSO-d6):
δ 3.83 (s, 3H, OCH3), 6.71 (br s, 2H, NH2), 7.11 (d, 2H, Ar–
H’s), 7.31 (t, 1H, thiophene-H4), 7.46 (d, 1H, thiophene-
H5), 7.57 (d, 2H, Ar–H’s), 7.77 (d, 2H, Ar–H’s), 7.86 (s,
1H, pyridine-H5), 7.91 (d, 1H, thiophene-H3), 8.04 (s, 1H,
methine-H), 8.27 (d, 2H, Ar–H’s), 11.43 (s, 1H, NH); 13C-
NMR (DMSO-d6): δ 55.2 (OCH3), 94.8 (thienopyridine-
C2), 114.2 (Ar–C), 118.3, 121.8 (thienopyridine-C5, C3a),
126.5, 127.8, 128.5, 128.6, 129.1, 129.5, 130.6, 134.8,
135.7, 136.4 (Ar–C’s and thiophene-C’s), 140.5 (thienopyr-
idine-C3), 144.2 (methine-C), 145.3, 146.2, 155.5 (thien-
opyridine-C4, C6, C7a), 160.8 (Ar–C), 167.9 (thienopyri-
dine-CO); Anal. for C26H19ClN4O2S2 (519.0): C, 60.17; H,
3.69; N, 10.79; found: C, 60.29; H, 3.75; N, 10.95%.
cm−1): 3329, 3237 (NH, NH2); H-NMR (DMSO-d6): δ
6.68 (br s, 2H, NH2), 7.31 (t, 1H, thiophene-H4), 7.47 (d,
1H, thiophene-H5), 7.59 (d, 2H, Ar–H’s), 7.82 (s, 1H, pyri-
dine-H5), 7.93 (d, 1H, thiophene-H3), 8.05 (d, 2H, Ar–H’s),
8.12 (s, 1H, methine-H), 8.26 (d, 2H, Ar–H’s), 8.38 (d, 2H,
Ar–H’s), 11.52 (s, 1H, NH); Anal. for C25H16ClN5O3S2
(534.0): C, 56.23; H, 3.02; N, 13.12; found: C, 56.08; H,
3.14; N, 13.00%.
Ethyl (E)‑4‑((2‑(3‑amino‑6‑(4‑chlorophenyl)‑4‑(thiophen‑
2‑yl)thieno[2,3‑b]pyridine‑2‑carbonyl)hydrazineylidene)
methyl)benzoate (13j) Orange solid (dioxane); m.p. 242–
1
243 °C; IR (υ cm−1): 3329, 3237 (NH, NH2); H-NMR
(DMSO-d6): δ 1.30 (t, 3H, CH2CH3), 4.28 (q, 2H, CH2CH3),
6.74 (br s, 2H, NH2), 7.33 (t, 1H, thiophene-H4), 7.47 (d,
1H, thiophene-H5), 7.57–7.60 (m, 4H, Ar–H’s), 7.82 (s, 1H,
pyridine-H5), 7.90 (d, 1H, thiophene-H3), 8.00 (d, 2H, Ar–
H’s), 8.09 (s, 1H, methine-H), 8.24 (d, 2H, Ar–H’s), 11.44
(s, 1H, NH); Anal. for C28H21ClN4O3S2 (561.0): C, 59.94;
H, 3.77; N, 9.99; found: C, 60.13; H, 3.81; N, 9.85%.
(E)‑4‑((2‑(3‑Amino‑6‑(4‑chlorophenyl)‑4‑(thiophen‑2
‑yl)thieno[2,3‑b]pyridine‑2‑carbonyl)hydrazineylidene)
methyl)phenyl benzoate (13g) Brown solid (dioxane);
m.p. above 300 °C; IR (υ cm−1): 3327, 3243 (NH, NH2),
(E)‑3‑Amino‑N’‑(benzo[d] [1, 3] dioxol‑5‑ylmethylene)‑6‑(4‑c
hlorophenyl)‑4‑(thiophen‑2‑yl)thieno[2,3‑b]pyridine‑2‑car‑
bohydrazide (13k) Yellow solid (ethanol/dioxane mix-
ture); m.p. above 300 °C; IR (υ cm−1): 3322, 3253 (NH,
1
1739 (CO); H-NMR (DMSO-d6): δ 6.68 (br s, 2H, NH2),
7.30–7.33 (m, 3H, 2 Ar–H’s and thiophene-H4), 7.44–7.48
(m, 3H, 2 Ar–H’s and thiophene-H5), 7.55–7.58 (m, 4H,
Ar–H’s), 7.65 (t, 1H, Ar–H’s), 7.87 (s, 1H, pyridine-H5),
7.92 (d, 1H, thiophene-H3), 8.01 (s, 1H, methine-H), 8.11
(d, 2H, Ar–H’s), 8.28 (d, 2H, Ar–H’s), 11.48 (s, 1H, NH);
Anal. for C32H21ClN4O3S2 (609.1): C, 63.10; H, 3.48; N,
9.20; found: C, 63.26; H, 3.53; N, 9.06%.
1
NH2); H-NMR (DMSO-d6): δ 6.12 (s, 3H, OCH2O), 6.62
(br s, 2H, NH2), 7.06 (d, 1H, piperonal-H7), 7.22 (d, 1H,
piperonal-H6), 7.31 (t, 1H, thiophene-H4), 7.44–7.46 (m,
2H, piperonal-H4 and thiophene-H5), 7.55 (d, 2H, Ar–H’s),
7.87 (s, 1H, pyridine-H5), 7.92 (d, 1H, thiophene-H3), 8.01
(s, 1H, methine-H), 8.25 (d, 2H, Ar–H’s), 11.45 (s, 1H,
NH); 13C-NMR (DMSO-d6): δ 94.5 (thienopyridine-C2),
101.2 (OCH2O), 105.2, 108.3 (Ar–C), 118.1, 122.4 (thien-
opyridine-C5, C3a), 123.1, 127.6, 128.3, 128.6, 128.9,
129.4, 130.6, 134.7, 135.7, 136.5 (Ar–C’s and thiophene-
C’s), 140.3 (thienopyridine-C3), 143.9 (methine-C), 145.2,
146.1 (thienopyridine-C4, C6), 147.9, 149.3 (Ar–C), 154.8
(E)‑3‑Amino‑6‑(4‑chlorophenyl)‑N’‑(4‑(dimethylamino)
benzylidene)‑4‑(thiophen‑2‑yl)thieno[2,3‑b]pyridine‑2‑car‑
bohydrazide (13h) Brown solid (dioxane); m.p. 297–
1
299 °C; IR (υ cm−1): 3326, 3241 (NH, NH2); H-NMR
(DMSO-d6): δ 2.97 (s, 6H, N(CH3)2), 6.65 (br s, 2H, NH2),
6.84 (d, 2H, Ar–H’s), 7.31 (t, 1H, thiophene-H4), 7.44 (d,
1 3

Journal of the Iranian Chemical Society
(thienopyridine-C7a), 168.0 (thienopyridine-CO); Anal.
for C26H17ClN4O3S2 (533.0): C, 58.59; H, 3.21; N, 10.51;
found: C, 58.67; H, 3.12; N, 10.72%.
(thienopyridine-C4, C6, C7a), 168.2 (thienopyridine-CO);
Anal. for C25H16BrN5O3S2 (578.4): C, 51.91; H, 2.79; N,
12.11; found: C, 52.08; H, 2.67; N, 12.22%.
(E)‑3‑Amino‑N’‑benzylidene‑6‑(4‑nitrophenyl)‑4‑(thiophen‑
2‑yl)thieno[2,3‑b]pyridine‑2‑carbohydrazide (14a) Orange
solid (dioxane); m.p. 266–267 °C; IR (υ cm−1): 3464, 3271
(E)‑3‑Amino‑N’‑(4‑methylbenzylidene)‑6‑(4‑nitrophenyl)‑
4‑(thiophen‑2‑yl)thieno[2,3‑b]pyridine‑2‑carbohydrazide
(14d) Yellow solid (dioxane); m.p. 294–297 °C; IR (υ
cm−1): 3464, 3290, 3109 (NH, NH2); 1H-NMR (DMSO-d6):
δ 2.34 (s, 3H, CH3), 6.68 (br s, 2H, NH2), 7.31 (d, 2H, Ar–
H’s), 7.37 (t, 1H, thiophene-H4), 7.45 (d, 1H, thiophene-
H5), 7.60 (d, 2H, Ar–H’s), 7.79 (s, 1H, pyridine-H5), 7.92
(d, 1H, thiophene-H3), 8.08 (s, 1H, methine-H), 8.18 (d,
2H, Ar–H’s), 8.37 (d, 2H, Ar–H’s), 11.52 (s, 1H, NH); 13C-
NMR (DMSO-d6): δ 19.8 (CH3), 94.3, 119.6, 121.7 (thien-
opyridine-C2, C5, C3a), 122.4, 124.1, 127.3, 127.5, 128.8,
128.9, 130.6, 131.6, 136.6, 136.9, 138.7 (Ar–C’s and thio-
phene-C’s), 141.7 (thienopyridine-C3), 143.5 (methine-C),
144.4 (Ar–C), 145.1, 145.9, 154.3 (thienopyridine-C4, C6,
C7a), 168.0 (thienopyridine-CO); Anal. for C26H19N5O3S2
(513.5): C, 60.80; H, 3.73; N, 13.64; found: C, 60.68; H,
3.81; N, 13.47%.
1
(NH, NH2); H-NMR (DMSO-d6): δ 6.68 (br s, 2H, NH2),
7.27 (t, 1H, thiophene-H4), 7.40 (d, 1H, thiophene-H5),
7.47–7.54 (m, 3H, Ar–H’s), 7.84 (s, 1H, pyridine-H5), 7.88
(d, 2H, Ar–H’s), 7.96 (d, 1H, thiophene-H3), 8.10 (s, 1H,
methine-H), 8.17 (d, 2H, Ar–H’s), 8.35 (d, 2H, Ar–H’s),
11.44 (s, 1H, NH); 13C-NMR (DMSO-d6): δ 95.2, 119.2
(thienopyridine-C2, C5), 122.3 (Ar–C), 122.7 (thienopyri-
dine-C3a), 124.1, 127.4, 127.7, 128.3, 129.2, 130.3, 130.6,
134.5, 136.4, 138.3 (Ar–C’s and thiophene-C’s), 139.6
(thienopyridine-C3), 144.2 (Ar–C), 144.9 (methine-C),
145.4, 145.7, 153.8 (thienopyridine-C4, C6, C7a), 168.2
(thienopyridine-CO); Anal. for C25H17N5O3S2 (499.5):
C, 60.11; H, 3.43; N, 14.02; found: C, 60.03; H, 3.30; N,
14.28%.
(E)‑3‑Amino‑N’‑(4‑chlorobenzylidene)‑6‑(4‑nitrophenyl)‑
4‑(thiophen‑2‑yl)thieno[2,3‑b]pyridine‑2‑carbohydrazide
(14b) Yellow solid (dioxane); m.p. above 300 °C; IR (υ
(E)‑3‑Amino‑N’‑(4‑hydroxybenzylidene)‑6‑(4‑nitrophenyl)
‑4‑(thiophen‑2‑yl)thieno[2,3‑b]pyridine‑2‑carbohydrazide
(14e) Orange solid (dioxane); m.p. above 300 °C; IR
1
1
cm−1): 3336, 3242 (NH, NH2); H-NMR (DMSO-d6): δ
(υ cm−1): 3443 (OH), 3338, 3244 (NH, NH2); H-NMR
6.58 (br s, 2H, NH2), 7.29 (t, 1H, thiophene-H4), 7.42
(d, 1H, thiophene-H5), 7.58 (d, 2H, Ar–H’s), 7.81 (s, 1H,
pyridine-H5), 7.87 (d, 2H, Ar–H’s), 7.95 (d, 1H, thiophene-
H3), 8.09 (s, 1H, methine-H), 8.16 (d, 2H, Ar–H’s), 8.36 (d,
2H, Ar–H’s), 11.47 (s, 1H, NH); 13C-NMR (DMSO-d6): δ
94.7, 119.4 (thienopyridine-C2, C5), 122.3 (Ar–C), 122.8
(thienopyridine-C3a), 124.2, 127.6, 128.5, 128.7, 128.9,
130.4, 132.6, 134.2, 136.3, 138.4 (Ar–C’s and thiophene-
C’s), 141.3 (thienopyridine-C3), 143.6 (methine-C), 144.2
(Ar–C), 145.4, 145.9, 154.1 (thienopyridine-C4, C6, C7a),
168.1 (thienopyridine-CO); Anal. for C25H16ClN5O3S2
(534.0): C, 56.23; H, 3.02; N, 13.12; found: C, 56.08; H,
3.14; N, 12.99%.
(DMSO-d6): δ 6.65 (br s, 2H, NH2), 6.94 (d, 2H, Ar–H’s),
7.36 (t, 1H, thiophene-H4), 7.47 (d, 1H, thiophene-H5),
7.63 (d, 2H, Ar–H’s), 7.80 (s, 1H, pyridine-H5), 7.95 (d,
1H, thiophene-H3), 8.10 (s, 1H, methine-H), 8.18 (d, 2H,
Ar–H’s), 8.37 (d, 2H, Ar–H’s), 10.05 (br s, 1H, OH), 11.50
(s, 1H, NH); 13C-NMR (DMSO-d6): δ 95.2 (thienopyridine-
C2), 115.6 (Ar–C), 117.9, 121.8 (thienopyridine-C5, C3a),
122.3, 124.1, 124.9, 127.8, 128.5, 128.7, 130.5, 136.6,
138.2 (Ar–C’s and thiophene-C’s), 140.3 (thienopyridine-
C3), 144.2 (Ar–C), 144.6 (methine-C), 145.3, 145.6, 155.4
(thienopyridine-C4, C6, C7a), 159.5 (Ar–C), 167.9 (thien-
opyridine-CO); Anal. for C25H17N5O4S2 (515.5): C, 58.24;
H, 3.32; N, 13.58; found: C, 58.24; H, 3.32; N, 13.58%.
(E)‑3‑Amino‑N’‑(4‑bromobenzylidene)‑6‑(4‑nitrophenyl)‑
4‑(thiophen‑2‑yl)thieno[2,3‑b]pyridine‑2‑carbohydrazide
(14c) Orange solid (dioxane); m.p. above 300 °C; IR (υ
(E)‑3‑Amino‑N’‑(4‑methoxybenzylidene)‑6‑(4‑nitrophe
nyl)‑4‑(thiophen‑2‑yl)thieno[2,3‑b]pyridine‑2‑carbohy‑
drazide (14f) Yellow solid (dioxane); m.p. above 300 °C;
1
1
cm−1): 3327, 3243 (NH, NH2); H-NMR (DMSO-d6): δ
IR (υ cm−1): 3347, 3246 (NH, NH2); H-NMR (DMSO-
6.60 (br s, 2H, NH2), 7.29 (t, 1H, thiophene-H4), 7.42 (d,
1H, thiophene-H5), 7.57 (d, 2H, Ar–H’s), 7.79 (s, 1H, pyri-
dine-H5), 7.84 (d, 2H, Ar–H’s), 7.94 (d, 1H, thiophene-H3),
8.12 (s, 1H, methine-H), 8.17 (d, 2H, Ar–H’s), 8.38 (d, 2H,
Ar–H’s), 11.56 (s, 1H, NH); 13C-NMR (DMSO-d6): δ 94.2,
118.9, 121.9 (thienopyridine-C2, C5, C3a), 122.2, 123.8,
124.3, 127.7, 128.3, 128.7, 130.5, 131.3, 132.5, 136.4,
138.6 (Ar–C’s and thiophene-C’s), 141.6 (thienopyridine-
C3), 143.2 (methine-C), 144.3 (Ar–C), 145.4, 146.2, 154.4
d6): δ 3.82 (s, 3H, OCH3), 6.68 (br s, 2H, NH2), 7.08
(d, 2H, Ar–H’s), 7.36 (t, 1H, thiophene-H4), 7.44 (d,
1H, thiophene-H5), 7.74 (d, 2H, Ar–H’s), 7.83 (s, 1H,
pyridine-H5), 7.93 (d, 1H, thiophene-H3), 8.08 (s, 1H,
methine-H), 8.18 (d, 2H, Ar–H’s), 8.39 (d, 2H, Ar–H’s),
11.51 (s, 1H, NH); 13C-NMR (DMSO-d6): δ 55.3 (OCH3),
94.5 (thienopyridine-C2), 114.4 (Ar–C), 118.6, 121.9
(thienopyridine-C5, C3a), 122.2, 123.9, 126.6, 127.8,
128.4, 128.7, 130.5, 136.6, 138.4 (Ar–C’s and thiophene-
1 3

Journal of the Iranian Chemical Society
C’s), 140.8 (thienopyridine-C3), 144.1 (Ar–C), 144.3
(methine-C), 145.6, 146.3, 155.8 (thienopyridine-C4, C6,
C7a), 160.6 (Ar–C), 167.7 (thienopyridine-CO); Anal.
for C26H19N5O4S2 (529.5): C, 58.97; H, 3.62; N, 13.22;
found: C, 59.12; H, 3.48; N, 13.07%.
Cytotoxicity
Cell line, culture conditions and preparation of compounds
The human breast carcinoma cell line (MCF-7), human
colon cancer cell line (Caco2) and human liver hepatocellu-
lar carcinoma cell line (HEPG2) were obtained from ATCC
via Holding company for biological products and vaccines
(VACSERA), Cairo, Egypt.
The in vitro antibacterial assay
The agar well difusion method was used to evaluate the
in vitro antibacterial activity of the new derivatives [42,
52] against each of Escherichia coli (ATCC:9637), Kleb-
siella pneumoniae (ATCC:10031), Staphylococcus aureus
(ATCC:6538), Streptococcus mutans (ATCC:25175) and
Enterococcus faecalis (ATCC:29212). The assay involved
using of nutrient agar medium, Chloramphenicol (30 µg
susceptibility disk, Unipath, Basingstoke, UK) as a refer-
ence drug and DMSO as a solvent and negative control.
The hydrazones were tested using 15 mg/mL against each
strain. Interpretation of results was done using of Chloram-
phenicol breakpoint for each strain. Petri dishes containing
the sterilized media were prepared and allowed to solidify
at rt. Microbial suspension in sterilized saline equivalent
to McFarland 0.5 standard solution (1.5 × 105 CFU mL−1)
was prepared, whose turbidity modifed to OD=0.13 using
spectrophotometer at 625 nm. In the solidifed media,
6 mm diameter wells were made and 100 μL of each of
the tested hydrazones was added. Then, the plates were
left to stand for 24 h at 37 °C, and then, inhibition zones
in mm were observed.
The selected cell lines were cultivated in Dulbecco’s
Modified Eagle’s Medium (DMEM). All of the growth
media were supplemented with 10% Fetal Bovine Serum
(FBS) and antibiotics (100 U/mL penicillin and 100 mg/mL
streptomycin) at 37 °C in a humidifed atmosphere contain-
ing 5% CO2. Compounds 13a, 13b, 13c, 13d, 13g, 13k, 14b,
14c and 14d as well as Doxorubicin, as a positive control,
were dissolved in DMSO, and fnal concentrations were
diluted in culture medium.
Neutral red uptake assay (NRU assay)
The NRU assay depends on the ability of living cells to bind
neutral red, in lysosomes [54]. The cytotoxicity of hydra-
zones 13a, 13b, 13c, 13d, 13g, 13k, 14b, 14c and 14d was
evaluated against each of MCF-7, Caco2 and HEPG2 cell
lines using Doxorubicin as a standard drug. Exponentially
growing cells were collected using 0.25% Trypsin–EDTA;
then, the cell suspension was counted using hemocytometer,
and cell viability was checked by trypan blue (100% viabil-
ity). Then, the cells suspension was diluted with complete
medium to have an approximately 1.0×105 cell/mL; then,
200 µL of the cell suspension was dispensed by multichannel
pipette into the inner 60 wells of the 96 well plate, and the
peripheral wells were flled with PBS; then, the plate incu-
bated for 24 h before treatment with the tested compounds
to allow attachment of cells to the wall of the plate. Diferent
concentrations of the tested hydrazones (5, 25, 50 and 75 µg/
mL) were prepared using DMEM media. 200 μL of treat-
ment media was dispensed into 4 replicates for each concen-
tration, and other wells were flled with untreated cells only
(as a negative control) and wells flled with media containing
Doxorubicin HCL as a positive control. The 96-well plate
incubated at 37° C for 48 h. Then, the medium and extracts
were discarded and replaced with 100 µL of neutral red solu-
tion (50 mg/mL) and centrifuged at 1800 rpm for ten min
to remove any crystals of dye. After incubation at 37 °C for
3 h, the dye medium was discarded and the microplate was
washed twice with 150 µL PBS to remove the unabsorbed
neutral red dye contained in the wells. The cellular morphol-
ogy of each of the treated MCF-7, Caco2 and HEPG2 cell
lines with the tested hydrazones was observed using Inverted
Microscope Leica DMI3000B. The absorbance of acidifed
ethanol solution containing extracted neutral red dye was
Minimum inhibitory concentration (MIC)
The twofold serial dilution method was used to evaluate
the MIC values [53]. MIC value is the minimum concen-
tration of the tested compound required to stop bacterial
growth. For each strain of bacteria, 3–5 isolated colonies
were transferred into 3–4 mL of sterile broth medium. The
bacterial suspension was allowed to incubate for 2–6 h at
35–37 °C until its turbidity become equal to or greater
than the turbidity of a McFarland Standard 0.5. Then, a
stock solution of 1 mg/mL was prepared using DMSO as
a solvent. The tested hydrazones were subjected to further
progressive dilutions with the broth medium. Addition of
a fxed volume of the prepared bacterial inoculum to each
concentration of the tested hydrazones was done and then
allowed to incubation for 16–20 h at 37 °C. The bacterial
growth in the tested tubes was observed by comparing the
resulted turbidity with the growth in the original inocu-
lum without any of the tested hydrazones. For comparison,
Chloramphenicol was tested in the same assay.
1 3

Journal of the Iranian Chemical Society
2. A.M. Bernardino, L.C. da Silva Pinheiro, C.R. Rodrigues, N.I.
Loureiro, H.C. Castro, A. Lanfredi-Rangel, J. Sabatini-Lopes, J.C.
Borges, J.M. Carvalho, G.A. Romeiro, V.F. Ferreira, I.C. Frugul-
hetti, M. Vannier-Santos, a. Bioorg. Med. Chem. 14, 5765 (2006)
3. D. Shuck-Lee, F.F. Chen, R. Willard, S. Raman, R. Ptak, Antimi-
crob. Agents Chemother. 52, 3169 (2008)
determined using microplate reader (BioTek, ELX808) at
540 nm to estimate the optical density, and the cell viability
percentage was measured.
COX‑2 inhibition assay
4. M.E. Schnute, D.J. Anderson, R.J. Brideau, F.L. Ciske, S.A. Col-
lier, M.M. Cudahy, M. Eggen, M.J. Genin, T.A. Hopkins, T.M.
Judge, E.J. Kim, Bioorg. Med. Chem. Lett. 17, 3349 (2007)
5. K. Madhusudana, B. Shireesha, V.G. Naidu, S. Ramakrishna,
B. Narsaiah, A.R. Rao, P.V. Diwan, Eur. J. Pharmacol. 678, 48
(2012)
The ability of the tested derivatives to inhibit COX-2 enzyme
was estimated [55, 56]. Briefy, COX-2 (purchased from
Sigma-Aldrich) was added in 180 µL of the assay bufer con-
taining 100 mM Tris–HCl bufer of pH 8.0, 1.0 mM hema-
tin, 2.0 mM phenol and 5 mM EDTA. Each sample of the
tested compounds and positive control, Celecoxib, was dis-
solved in DMSO and tested at three diferent concentrations
(5, 10 and 25 µM). After addition of the tested derivatives or
positive control (10 µL), the reaction mixture was allowed
to incubate for 5 min at rt. The reaction was started by addi-
tion of 5 µL of arachidonic acid solution (5.0 mM) dissolved
in methanol and N,N,N′,N′-tetramethylpphenylenediamine
dihydrochloride and allowed to incubate at 37 °C for 1 h.
The reaction was quenched by adding 50 mL of 1 M HCl.
The absorbance was measured using spectrophotometer at
610 nm. The inhibition percentage was estimated by com-
paring the test compounds with the blank and calculated
using the equation:
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(
)
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