Syntheses, crystal structures, in vitro antitumor and free radical scavenging activity evaluation of a series of 2-substituted thiophenes

Article abstract of DOI:10.1007/s00044-015-1353-5

A series of thiazole, pyridine and benzylidene derivatives derived from thiophene scaffold have been synthesized. The antitumor evaluation of the newly synthesized products against four cancer cell lines, namely breast carcinoma (MCF-7), liver carcinoma (HepG2), colon carcinoma (HCT-116) and prostate carcinoma (PC3), indicated that the thiazole derivative 11b showed remarkable activity against all cell lines with LC₅₀ values of 18.3, 2.5, 7.5 and 7.6 μM, respectively. Cytotoxicity toward normal cell lines was also investigated and indicated that compound 21c displayed remarkable potency against PC3 with LC₅₀ values of 7.1 μM and showed weak inhibition of normal cell lines at (GI %) of 41.2 % and thus could be considered as an important lead compound for potential application in anticancer chemotherapy. Brine shrimp lethality assay of the most active compounds was carried out to detect possible cytotoxicity effects and indicated that highly active compound, 21c, is not harmful. The X-ray crystallographic analysis of compounds 3 and 11b was obtained thus establishing with certainty the proposed structures in this work. The synthesized compounds were also screened for their free radical scavenging activity. Hydrazino-thiazole derivatives 15 and 16 showed remarkable antioxidant activity with IC₅₀ values of 60.9 and 61.9 μg ml^-1, respectively.

Full text of DOI:10.1007/s00044-015-1353-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1353-5
ORIGINAL RESEARCH
Syntheses, crystal structures, in vitro antitumor and free radical
scavenging activity evaluation of a series of 2-substituted
thiophenes
•
Yara E. El-Shorafa Daisy H. Fleita Ola K. Sakka
•
•
•
William T. A. Harrison Khaled Mahmoud
•
Rafat M. Mohareb
Received: 9 June 2014 / Accepted: 27 February 2015
Ó Springer Science+Business Media New York 2015
Abstract A series of thiazole, pyridine and benzylidene
derivatives derived from thiophene scaffold have been syn-
thesized. The antitumor evaluation of the newly synthesized
products against four cancer cell lines, namely breast carci-
noma (MCF-7), liver carcinoma (HepG2), colon carcinoma
(HCT-116) and prostate carcinoma (PC3), indicated that the
thiazole derivative 11b showed remarkable activity against
all cell lines with LC50 values of 18.3, 2.5, 7.5 and 7.6 lM,
respectively. Cytotoxicity toward normal cell lines was also
investigated and indicated that compound 21c displayed
remarkable potency against PC3 with LC50 values of 7.1 lM
and showed weak inhibition of normal cell lines at (GI %) of
41.2 % and thus could be considered as an important lead
compound for potential application in anticancer che-
motherapy. Brine shrimp lethality assay of the most active
compounds was carried out to detect possible cytotoxicity
effects and indicated that highly active compound, 21c, is not
harmful. The X-ray crystallographic analysis of compounds
3 and 11b was obtained thus establishing with certainty the
proposed structures in this work. The synthesized
compounds were also screened for their free radical scav-
enging activity. Hydrazino–thiazole derivatives 15 and 16
showed remarkable antioxidant activity with IC50 values of
60.9 and 61.9 lg ml-1, respectively.
Keywords Thiophene ꢀ Antitumor activity ꢀ
Free radical scavenging activity ꢀ
Brine shrimp lethality assay ꢀ X-ray
Introduction
Hydrazines have traditionally been employed as reagents
for the derivatization and characterization of carbonyl
compounds, and in recent years, the N–N linkage has been
used as a key structural motif in various bioactive agents.
Recently, hydrazide–hydrazones have gained great impor-
tance as antitumor agents (Terzioglu and Gu¨rsoy, 2003;
Pandey et al., 2002; Abadi et al., 2003). A number of
hydrazones have been reported to induce apoptosis. Vogel
et al. reported the synthesis of aroyl hydrazones of
2-phenylindole-3-carbaldehydes and their ability to inhibit
the growth of MDA-MB 231 and MCF-7 breast cancer
cells (Vogel et al., 2008). In addition, 4-amino-6-ary-
lamino-pyrimidine-5-carbaldehyde hydrazones were iden-
tified as potent dual ErbB-2/EGFR kinase inhibitors,
resulting in growth inhibition of ErbB-2 over-expressing
human cancer cell lines (Xu et al., 2008). Thiophenes
represent a class of heterocyclic compounds of great im-
portance in biological chemistry, especially for their anti-
tumor activities (Shaaban et al., 2010; Jin et al., 2008;
Pinkerton et al., 2007; Garton et al., 2006). For example,
OSI-930 has shown a broad range of antitumor activity
through inhibition of its target proteins in vitro and in
preclinical models in vivo. In addition, it is currently
Y. E. El-Shorafa ꢀ D. H. Fleita ꢀ O. K. Sakka (&)
Department of Chemistry, American University in Cairo,
P.O. Box 74, New Cairo 11835, Egypt
e-mail: Olasakka@aucegypt.edu
W. T. A. Harrison
Department of Chemistry, University of Aberdeen, Meston
Walk, Aberdeen AB24 3UE, Scotland, UK
K. Mahmoud
Department of Pharmacognosy, National Research Center,
Cairo 12622, Egypt
R. M. Mohareb
Department of Chemistry, Faculty of Science, Cairo University,
Giza, A.R., Egypt
123

Med Chem Res
evaluated in clinical studies (Garton et al., 2006; Yap et al.,
2013). Raloxifen, currently marketed as EvistaÓ, has
shown to be effective in invasive breast cancer in post-
menopausal women at increased risk (Vogel et al., 2006;
Barrett-Connor et al., 2006; Fig. 1).
pyridine and furan pharmacophore with promising antitumor
activities (Mohareb et al., 2009, 2011). In continuation to
this program, we utilized the reaction of hydrazide–hydra-
zone derivative 3 in the synthesis of a series of thiazole,
pyridine and benzylidene derivatives derived from the
thiophene scaffold. Even though compound 3 was previ-
ously reported (Steiner, 1960; Scheme 1), the structure of 3
was further confirmed by X-ray crystallographic analysis
(Figs. 2, 3, Table 1). Thus, the reaction of 3 with ben-
zaldehyde, 4-methoxybenzaldehyde, 4-chlorobenzaldehyde,
4-methylbenzaldehyde and 3-nitrobenzaldehyde afforded
the corresponding arylidene derivatives 5a–e, respectively.
Moreover, condensation of 3 with salicylaldehyde afforded
the chromen-2-imine derivative 7 (Scheme 2). The reaction
of compound 3 with cyclopentanone in the presence of
ammonium acetate in an oil bath at 140 °C gave the Kno-
evenagel condensation product 9 (Scheme 3). The structure
of compound 9 was confirmed based on its analytical and
Antioxidants are chemical entities that are capable of pre-
venting cell death that occurs due to the release of free radi-
cals. Many forms of cancer are thought to be the result of
reactions between free radicals and DNA, resulting in muta-
tions that can adversely affect the cell cycle, potentially
leading to malignancy (Halliwell and Gutteridge, 1985;
Cooke et al., 2003). Hydrazide–hydrazone derivatives have
been shown recently to possess radical scavenging ability
(Shih and SuY, 2007; Yilmaz et al., 2012; Gwaram et al.,
2012). In view of the aforementioned facts, our efforts were
directed toward combining the hydrazone and thiophene
scaffolds together in a molecular framework to study their
additiveeffecttowardthe antioxidantand antitumoractivities.
The present article embodies the synthesis and single-crystal
characterization of a family of hydrazide–hydrazone deriva-
tives incorporating the thiophene pharmacophore. In addition,
their antioxidant and cytotoxic abilities have been explored.
1
spectroscopic data. Thus, the H-NMR spectrum showed a
multiplet at d 0.76–1.23 for the cyclopentanone-H, a singlet
at d 2.27 for the CH3 group, a multiplet at d 7.06–7.60 for
thiophene-H and a singlet at d 10.6 for the NH group.
Moreover, the 13C-NMR spectrum showed peaks at d: 14.7
(CH3), 25.1, 25.2, 146.4 (cyclopentanone C), 143.1 (C=C),
116.5 (C:N), 128.2, 128.4, 129.1, 131.6 (thiophene C),
159.6 (C=N) and 165.7 (C=O). The reaction of compound 3
with ethyl cyanoacetate and elemental sulfur in the presence
of triethylamine was studied as an application of Gewald’s
thiophene synthesis and afforded the 2,4-diaminothiophene-
3-carboxylate derivative 10 (Scheme 3). The structure of
Results and discussion
Synthesis of compounds 3–21
Recently, our research group has reported the synthesis of a
series of hydrazide–hydrazone derivatives containing the
Fig. 1 Chemical structures of
OSI-930 and raloxifen
HO
O
S
HN
S
N
OH
O
F
F
F
O
O
N
H
N
OSI-930
Raloxifen
H2
C
Scheme 1 Synthesis of the
hydrazide–hydrazone
derivative 3
H
N
NC
CH2
O
CH3
NC
NH2
NH
CH3
S
O
N
O
2
1
S
3
123

Med Chem Res
The reactivity of the active methylene group in 3 was
studied by coupling it with either benzene diazonium
chloride, 4-bromobenzene diazonium chloride or 2-methyl-
benzene diazonium chloride in ethanol to yield the aryl
hydrazones 11a–c (Scheme 3). The E conformation of the
arylhydrazones was established via the X-ray structure
determination of 11b (Figs. 4, 5, Table 2). Moreover, the
reaction of 3 with acetyl acetone afforded the oxo-pyridine
1
derivative 13 whose structure was confirmed from its H-
NMR spectrum, which showed the presence of three CH3
signals at d 2.17, 2.23 and 2.37, a multiplet at d 7.23–7.90
due to thiophene-H and a singlet at d 6.44 owing to the
presence of the pyridine-H (Scheme 3). The 13C-NMR
spectrum is consistent with this structure due to the pres-
ence of three CH3 signals at d 17.2, 19.6, 21.0, a signal at d
116.5 for the nitrile carbon atom (C:N), four signals at d
99.7, 108.5, 150.6, 157.7 for the pyridine C, four signals at
d 128.8, 133.4, 133.6, 139.8 for the thiophene C, a signal at
d 155.9 (C=N) and a signal at d 172 (C=O).
Fig. 2 Molecular structure of
ellipsoids
3 showing 50 % displacement
Next, we studied the reactivity of 3 toward phenyl isothio-
cyanate in basic dimethylformamide solution followed by
heterocyclization with active methylene reagents like a-
haloketones. Thus, compound 3 reacted with phenylisothio-
cyanate in DMF/KOH solution at room temperature to give the
intermediate potassium sulfide salt 14. Heterocyclization of 14
with a-haloketones like ethyl chloroacetate gave the thiazole
derivative 15. The 1H-NMR showed a singlet at d 2.23 for the
CH3 protons and another singlet at d 4.34 for thiazole protons, a
singlet at d 11.02 owing to NH proton and a multiplet at d
7.05–7.60 owing to thiophene and benzene ring hydrogens. In a
similar way, the reaction of 14 with chloroacetonitrile afforded
the thiazole derivative 16 (Scheme 4).
Finally, compound 3 underwent a one-pot three-com-
ponent reaction with malononitrile and salicylaldehyde in
the presence of ethanol and a few drops of piperidine to
afford compound 19. Formation of compound 19 could be
explained via the initial condensation of salicylaldehyde
with malononitrile to afford the arylidene derivative 17,
followed by the Michael addition of the active methylene
group of 3 to the ß carbon atom of 17 to afford the acyclic
intermediate 18. The attack of the amidic nitrogen on the
cyano group along with the attack of the hydroxyl group on
the second cyano group affords derivative 19. In a similar
manner, the one-pot reaction of compound 3 with mal-
ononitrile and benzaldehyde or p-methoxybenzaldehyde or
p-tolualdehyde afforded derivatives 21a–c (Scheme 5).
Fig. 3 Dimer formation in the crystal of 3, with hydrogen bonds
shown as double-dashed lines. See Table 1 for symmetry codes
˚
Table 1 Hydrogen bonds (A,°) in the structure of compound 3
Bond
Bond lengths/
˚
angles (A,°)
N2–H1ꢀꢀꢀO1iii
0.87 (3)
0.98
2.02 (3)
2.36
2.881 (3)
3.337 (3)
169 (2)
174
C6–H6AꢀꢀꢀO1iii
Symmetry code: (iii) 2 - x, -y, 1 - z
compound 10 was consistent with its spectroscopic data.
Thus, the 1H-NMR spectrum showed a triplet at d 1.056 for
the CH3 group in the ethyl side chain, a singlet at d 2.27 for
the second CH3 group, a quartet at d 3.454 belonging to the
CH2 of the ethyl group and two singlets at d 3.85, 4.09
belonging to the two amino groups. A multiplet at d
7.05–7.60 was also observed due to the presence of the
thiophene ring hydrogens. Finally, a singlet at d 10.63 was
observed due to the presence of the NH group.
X-ray structure determination
Crystal structures of 3 and 11b
Figure 2 shows the molecular structure of 3. The dihedral angle
between the aromatic ring and the carbohydrazide grouping
123

Med Chem Res
Scheme 2 Reaction of
R
H
hydrazide–hydrazone derivative
3 with different aldehydes to
afford derivatives 5a–e and 7
dioxane/piperidine
3
R
CHO
CH3
C
H
N
NC
N
S
O
4
5
5
a
b
c
d
e
R
H
4-OCH3
4-Cl
4-CH3
3-NO2
OH
O
O
NH
NH
dioxane/piperidine
3
CHO
CH3
N
6
7
S
˚
(r.m.s. deviation = 0.036 A) is 4.41 (14)°. The C=O and cya-
nide groups are approximately syn but distinctly twisted [O=C–
C–Cc (c = cyanide) torsion angle = -15.7 (3)°]. The con-
formations of the C=N–N–C grouping is pseudo-trans [torsion
angle = -173.1 (2)°]. In the crystal, pairs of N–HꢀꢀꢀO hydro-
gen bonds (Table 1) link the molecules into inversion dimers
with an R22 (8) loop graph set motif. The dimer linkages are
reinforced by a pair of weak C–HꢀꢀꢀO interactions (Fig. 3).
Figure 4 shows the molecular structure of 11b. The dihedral
angle between the aromatic rings is 24.85 (10)°, and the central
fragment of the molecule is approximately planar (r.m.s. de-
compared with the reference drug doxorubicin. The human
tumor cell line panel consisted of breast carcinoma (MCF-
7), liver carcinoma (HepG2), colon carcinoma (HCT-116)
and prostate carcinoma (PC3) using MTT assay. Tumor
cells were incubated either alone (negative control) or with
different concentrations of the test compounds (100–50–
25–12.5–6.25–3.125–0.78 and 1.56 lM). The data in
Table 3 are reported as percentage growth inhibition (GI
%). With regard to sensitivity against individual cell lines,
compounds 7 and 19 showed selective potency against
HepG2 cell line with increasing concentration with (GI %)
of 65.3 and 56.7 at 100 lM concentration, respectively.
However, compound 11b showed effectiveness against all
cell lines with GI % of 100, 96.5, 100 and 100 % for
HepG2, MCF-7, HCT-116 and PC3, respectively (Fig. 6).
In addition, compounds 21b and 21c displayed selective
potency against PC3 cell line with GI % of 70.8 and 100 %
at 100 lM concentration, respectively. Moreover, com-
pound 15 displayed moderate potency against PC3 cell line
with GI % of 51.8 %. Consequently, two response pa-
rameters, LC50 and LC90, were calculated for compounds
7, 11b, 15, 19, 21b, 21c and indicated that compounds 7
and 19 possessed moderate activity compared to positive
control doxorubicin with LC50 values of 73.9 and 87 lM,
respectively, toward HepG2 cell line. On the other hand,
compound 11b was almost 15-fold more active toward
HepG2 cell line (LC50 2.5 lM), twofold more active to-
ward MCF-7 cell line (LC50 18.3 lM), eightfold more
active toward HCT-116 cell line (LC50 7.5 lM) and five-
fold more active toward PC3 cell line (LC50 7.6 lM) than
the positive control doxorubicin (Table 4). Compound 21c
˚
viation for the 11 non-hydrogen atoms = 0.030 A). The C=O
and cyanide groups are syn and statistically coplanar [O=C–C–
Cc (c = cyanide) torsion angle = -0.3 (3)°], and the confor-
mations of both the C=N–N–C groupings are pseudo-trans
[torsion angles = -177.02 (18)° and –172.80 (19)°]. A pos-
sible intramolecular N–HꢀꢀꢀN(\ = 112°) interaction generates
an S(5) ring. In the crystal, pairs of N–HꢀꢀꢀNc (c = cyanide)
hydrogen bonds (Table 2) link the molecules into inversion
dimers with an R22 (12) loop graph set motif: The dimer linkages
are reinforced by pairs of weak C–HꢀꢀꢀN interactions (Fig. 5).
Finally, C–HꢀꢀꢀO interactions link the dimers into [001] chains.
Pharmacology
In vitro antitumor screening against MCF-7, HepG2,
HCT-116 and PC3 cell lines
The cytotoxic potencies of compounds 3–21 against a
panel of four human tumor cell lines were investigated and
123

Med Chem Res
O
oil bath/NH4OAc
NC
3
CH3
C
H
N
N
8
9
S
O
COOEt
H2N
NH2
CN
EtOH/Et3N
3
S
S
COOEt
NH
CH3
O
N
10
S
S
EtOH/AcONa
3
R
N
NCl
N
O
C
H3C
HN
CN
11
11
N
R
R
a
b
c
H
HN
4-Br
2-CH3
H3C
CN
O
O
EtOH/Et3N
O
3
H3C
CH3
N
CH3
12
H3C
N
S
13
Scheme 3 Synthesis of derivatives 9, 10, 11 and 13
displayed a sixfold higher potency than the standard dox-
orubicin with LC50 value of 7.1 for PC3 cell line.
(WISH; Table 3, Fig. 7). Compounds 7, 19, 21b and 21c
displayed weak inhibition of normal cells with GI % of 6.9,
33.4, 30.5 and 41.2 %, respectively, suggesting that these
molecules can selectively inhibit the growth of cancer
cells. Compound 21c displayed remarkable potency against
prostate carcinoma (PC3) (LC50 7.1 lM) and showed weak
inhibition of normal cell lines at (GI %) of 41.2 % and thus
could be considered as an important lead compound for
potential application in anticancer chemotherapy.
Cytotoxicity toward normal cell lines (WISH)
Specific killing of cancer cells without affecting normal
cell growth is a key safety feature of cancer chemotherapy.
Therefore, the most active compounds 7, 11b, 19, 21b and
21c were evaluated for possible cytotoxicity to normal cells
123

Med Chem Res
in the absorbance produced in this reaction has been widely
applied to test the capacity of numerous molecules to act as
free radical scavengers. The effective concentration for the
antioxidant activity of compounds (3–21) was evaluated
using the DPPH system. The samples were prepared at
concentrations of (100–0.39 lg ml-1), and both a synthetic
antioxidant (butylated hydroxy anisole, BHA) and the nat-
ural antioxidant ascorbic acid (vitamin C) were used as
reference standards. The DPPH radical assay indicated that
the radical scavenging activity of compounds 15, 16 and
11b was dose dependent and increased with the concen-
tration of the tested compounds (Fig. 8). Using the DPPH
assay, compounds 15, 16 and 11b showed approximately
80.7, 80.9 and 51.4 % antioxidant activity, respectively, at
100 lg ml-1. Compound 11b displayed moderate an-
tioxidant activity with an IC50 value of 91 lg ml-1. The
hydrazino–thiazole derivatives 15 and 16 showed remark-
able antioxidant activity compared to butylated hydroxy
Fig. 4 Molecular structure of 11b showing 50 % displacement
ellipsoids
anisole, BHA with IC50 values of 60.9 and 61.9 lg ml-1
,
respectively. Thus, the hydrazine–thiazole moiety might be
one of the factors behind the exclusive antioxidant activity
of compounds 15 and 16. The remaining compounds did not
display any antioxidant activity compared with butylated
hydroxyanisole (BHA; Table 5).
Determination of in vivo toxicity by brine shrimp
lethality assay (BSLA)
Fig. 5 Dimer formation in the crystal of 11b, with hydrogen bonds
shown as double-dashed lines. See Table 2 for symmetry codes
Bioactive compounds are often toxic to shrimp larvae.
Thus, in order to monitor these chemicals’ in vivo lethality
to shrimp larvae (Artemia salina), brine shrimp lethality
assay (Rahman et al., 2001) was used. The shrimp lethality
assay is considered as a useful tool for preliminary
assessment of toxicity, and it has been used for the de-
tection of fungal toxins, plant extract toxicity, heavy
metals, cyanobacteria toxins, pesticides and cytotoxicity
testing of dental materials (Carballo et al., 2002), natural
and synthetic organic compounds (Rahman et al., 2001). It
has also been shown that A. salina toxicity test results have
a correlation with rodent and human acute oral toxicity
data. Thus, the highest bioactive compounds, 7, 11b, 15,
16, 19, 21b and 21c, that exhibited the highest cytotoxicity
against cancer cell lines were subjected to a shrimp leth-
ality assay to investigate their toxicity. Results were ana-
lyzed with LC50 program to determine LC50 values (Brayn
et al., 1993; Table 6).
˚
Table 2 Hydrogen bonds (A,°) in the structure of compound 11b
Bond
Bond lengths/
˚
angles (A,°)
N2–H1NꢀꢀꢀN3
N4–H2 NꢀꢀꢀN5i
C2–H2ꢀꢀꢀO1ii
C15–H15ꢀꢀꢀN5i
0.80 (3)
0.85 (2)
0.95
2.24 (2)
2.28 (2)
2.56
2.645 (2)
3.090 (3)
3.404 (3)
3.372 (3)
112 (2)
159 (2)
148
0.95
2.62
137
Symmetry codes: (i) -x, 2 - y, -z; (ii) 1 ? x, 3/2 - y, ‘ ? z
Antioxidant activity
The in vitro method of the scavenging of the stable DPPH
radical is extensively used to evaluate antioxidant activities
in less time than other methods (Marxen et al., 2007). DPPH
is a stable free radical molecule that can accept an electron
or hydrogen radical and thus be converted into a stable,
diamagnetic molecule. DPPH has an odd electron and so
has a strong absorption band at 518 nm. When this electron
is paired, the absorption decreases stoichiometrically with
respect to the number of electrons taken up. Such a change
Inorder topreventpossiblefalsetoxicityresultsoriginating
from solubility of compounds and DMSO’s possible toxicity
effect, compounds were prepared by dissolving in DMSO in
the suggested DMSO volume ranges. Results indicated that
compounds 15, 19, 21c showed non-toxicity against the tested
organisms. The lethality concentrations of compounds 15, 19
123

Med Chem Res
Scheme 4 Synthesis of
derivatives 15 and 16
O
Ph
N
O
O
S
Cl
OEt
NC
NH
CH3
Ph
NH
O
N
SK
NH
S
NC
KOH/DMF
15
3
Ph
N
C
S
CH3
N
H2N
N
S
CN
Ph
Cl
14
S
NC
NH
CH3
O
N
S
16
CHO
OH
CN
O
CN
OH
NH
EtOH/piperidine
3
NC
O
NC
O
CN
CN HN
CH3
N
CH3
N
H2N
N
S
S
19
18
OH
CN
O
NC
HN
CH3
N
CN
S
3
17
R
R
CHO
CN
CN
CN
EtOH/piperidine
NC
O
3
NC
O
N
CH3
CN
CN HN
CH3
H2N
N
R
N
S
S
20
21
21
R
a
b
c
H
4-OCH3
4-CH3
Scheme 5 Reaction of hydrazide–hydrazone derivative 3 with malononitrile and different aldehydes to afford derivatives 19 and 21a–c
123

Med Chem Res
Table 3 Percentage growth inhibition (GI %) at 100 lM of HepG2, MCF-7, HCT-116, PC-3 and normal WISH cell lines of test compounds
3–21
Compound no.
GI (%)
Liver cancer
HepG2
Breast cancer
MCF-7
Colon cancer
HCT-116
Prostate cancer
PC3
Normal cells
WISH
3
47.9 4.2
0
-24.2 2.4
2.1 0.7
22.1 4.1
-44.5 6.4
-40.4 8.5
0
-14.5 5.6
-17.5 2.8
-18.2 8.5
0
22.4 3.1
9.1 2.9
7.1 1.5
22.5 5.2
-7.8 2.5
-11.6 2.7
29.4 4.1
0
-11 1.2
35.5 4.1
19.6 2.8
50.4 3.9
40.8 1.8
23.1 3.9
6.9 1.5
11.1 2.5
9.6 1.2
27.4 1.5
90.7 2.1
6.1 0.9
17.3 1.3
28.8 1.8
29.5 1.1
33.4 2.7
8.3 1.4
30.5 3.7
41.2 2.4
100
5a
5b
-77 10.5
50.7 4.6
13.8 2.1
33.7 0.41
65.3 5.1
33.9 3.5
36.8 3.8
45.80 0.9
5c
5d
6.4 1.2
5e
12.9
0
3
7
50.7 3.8
-30.4 4.3
13.2 1.8
15.6 1.8
96.5 0.5
28.8 1.2
33.2 2.3
-34.7 3.4
-70.2 5.4
42.8 2.1
9.4 0.5
39.5 0.3
37.2 3.7
100
9
14.5 1.9
-14 3.4
11.5 1.9
100
10
23.6 2.9
20.1 0.9
100
11a
11b
100
1
11c
27.6 3.1
29.2 1.5
46.8 4.2
44.8 2.4
56.7 3.5
35.5 3.1
33.7 5.1
18.5 2.4
100
23.4 0.2
13.8 3.1
4.3 0.4
0
30.6 0.3
21.4 0.4
51.8 2.8
15.7 1.2
25.3 1.6
35.2 6.1
70.8 4.6
100
13
15
16
19
15.2 1.5
38.9 2.1
22.7 1.0
17.3 0.7
99.5
21a
21b
21c
Doxorubicin
100
Structure activity relationships
Regarding the structure activity relationships of the syn-
thesized thiophene derivatives 3–21 against all tested cell
lines, it was found that transformation of the hydrazide–
hydrazone derivative 3 into the corresponding arylidene
derivatives 5a–e did not cause any considerable enhance-
ment in antitumor activity. However, close inspection of
the substituents attached to the benzene ring reveals that
the 4-chloro-substituted derivative was the most active
compared to the 4-methoxy-, 4-methyl- and 3-nitro-sub-
stituted derivatives. Close examination of the structures of
compounds 7 and 19 reveals that their enhanced cyto-
toxicity toward HepG2 cell line may be due to the fact that
they both possess the 2-imino-chromene ring system. On
the other hand, upon inspecting the 2-phenylhydrazono-
acetohydrazide derivatives 11a–c, it was obvious that the
addition of a 4-bromo phenyl hydrazono moiety in 11b
greatly enhanced the cytotoxicity with reported GI % val-
ues of 100 % for liver, colon and prostate cancer cell lines
and 96 % growth inhibition for breast cancer cell line. It
was evident that addition of an electron-withdrawing bro-
mine group at the p-position of the benzene ring greatly
favored the cytotoxicity of compound 11b toward all cell
Fig. 6 Effects of compound 11b on cell death in HepG2, HCT-116,
MCF-7 and PC3 cell lines
and 21c were 880.42, 999.33 and 982.15, respectively. On the
other hand, compounds 16 and 21b were very toxic, with
lethality concentrations of 18.38 and 14.88, respectively. On
the other hand, compounds 7 and 11b were harmful and dis-
played lethal concentrations of 420.28 and 22.70, respec-
tively. The degree of lethality was directly proportional to the
concentration of all tested compounds.
123

Med Chem Res
Table 4 Compounds 7, 11b, 15, 19, 21b and 21c median lethal (LC50, lM) and (LC90, lM) concentrations of in vitro tumor cell lines HepG2,
MCF-7, HCT-116, PC3
Compound no.
Activity
Tumor cell lines
Liver cancer
HepG2
Breast cancer
MCF-7
Colon cancer
HCT-116
Prostate cancer
PC3
7
LC50
LC90
LC50
LC90
LC50
LC90
LC50
LC90
LC50
LC90
LC50
LC90
LC50
73.9 4.6
126.9 10.6
2.5 6.5
5.2 3.4
ND
NDa
ND
ND
ND
ND
ND
11b
18.3 3.3
33.1 6.2
ND
7.5 2.4
15.1 2.1
ND
7.6 1.7
14.9 1.9
90.5 3.4
144.0 4.1
ND
15
ND
ND
ND
19
87 3.5
138.5 6.7
ND
ND
ND
ND
ND
ND
21b
ND
ND
70.6 3.7
121.3 6.7
7.1 1.8
12.9 0.5
41.1 3.1
ND
ND
ND
21c
ND
ND
ND
ND
ND
ND
Doxorubicin
37.8 2.4
45.02 5.1
65.1 0.9
Cytotoxicity as LC50 for each cell line is the concentration of compound which caused 50 % cell growth inhibition of treated cells with respect to untreated cells
after continuous exposure of 48 h using MTT assay
Cytotoxicity as LC90 for each cell line is the concentration of compound which caused 90 % cell growth inhibition of treated cells with respect to untreated cells
after continuous exposure of 48 h using MTT assay
a
ND; IC50 not determined because less than 50 % inhibition was observed at the highest tested concentration (100 lM)
Fig. 7 Effects of compounds
3–21c on cell death in normal
cell line (WISH)
lines as compared to its inactive analog 11c which pos-
sessed an electron-releasing methyl group at the o-position
of the benzene ring. The introduction of a pyridine or
thiazole moiety in compounds 13, 15 and 16 did not induce
a remarkable enhancement in cytotoxicity. On the other
hand, the addition of an electron-releasing methyl group in
21c favored the cytotoxicity as compared to an electron-
withdrawing methoxy group in compound 21b, with a GI
% value of 100 % for PC3 cell line.
In vivo toxicity of the most antitumor bioactive com-
pound 21c revealed that it is not toxic to shrimp larvae.
Thus, it can be concluded that the substitution of the p-
methoxy group in 21b by the p-methyl group enhanced the
antitumor activity of 21c against PC3 cell line and sig-
nificantly decreased its toxicity.
Fig. 8 Free radical scavenging activity of synthesized compounds
11b, 15 and 16
123

Med Chem Res
116) and prostate carcinoma (PC3) were investigated. In
addition, their cytotoxic activities against normal cell line
(WISH) were studied. Results indicated that compound 11b
was almost 15-fold more active toward HepG2 cell line
(LC50 2.5 lM), twofold more active toward MCF-7 cell line
(LC50 18.3 lM), eightfold more active toward HCT-116 cell
line (LC50 7.5 lM) and fivefold more active toward PC3 cell
line (LC50 7.6 lM) than the positive control doxorubicin.
Compound 21c displayed a sixfold higher potency than the
standard doxorubicin with LC50 value of 7.1 for PC3 cell line
and showed weak inhibition of normal cell lines at (GI %) of
41.2 %. In addition, the toxicity of compound 21c toward
brine shrimp larvae was investigated and the results revealed
that the compound was non-toxic with LC50 value corre-
sponding to 982 lg ml-1. Thus, compound 21c could be
considered as an important lead compound for potential
application in anticancer chemotherapy. In addition, the
antioxidant activity of compounds (3–21) was evaluated
using the DPPH system. The hydrazino–thiazole derivatives
15 and 16 showed remarkable antioxidant activity with IC50
Table 5 Compounds 15, 16 and 11b free radical scavenging activity
Compound no.
ICa50 (lg ml-1
)
15
60.9
61.9
91
16
11b
Butylated hydroxyanisole (BHA)
53
Vitamin C
12
a
The IC50 value for each sample, defined as the concentration of the
test sample leading to 50 % reduction of the initial DPPH concen-
tration, was calculated from the nonlinear regression curve of log
concentration of the test extract (lg ml-1) against the mean per-
centage of the radical scavenging activity
Table 6 Brine shrimp lethality assay for compounds 7, 11b, 15, 16,
19, 21b and 21c
Compound Concentration Mortalitya Toxicity
LC50
(lg ml-1
)
no.
(lg ml-1
)
7
10
100
1000
10
0
5
Harmful
420.28
values of 60.9 and 61.9 lg ml-1
.
10
0
11b
15
Harmful
22.70
100
1000
10
6
Experimental
8
0
Non-toxic
Very toxic
Non-toxic
Very toxic
880.42
18.38
General
100
1000
10
1
4
Melting points were determined on an Electrothermal 9100
melting point apparatus and are uncorrected. IR spectra
were recorded for KBr disks on a ThermoScientific, Ni-
colet 380 FT-IR spectrophotometer. Mass spectra were
16
1
100
1000
10
6
10
0
1
recorded on a GCMS-Shimadzu Qp-2010 Plus. H-NMR
19
999.33
14.88
and 13C-NMR spectra were measured on a Varian EM-
390–300 MHz in DMSO-d6 as solvent using TMS as in-
ternal standard, and chemical shifts are expressed as d. The
products of the reactions were monitored by thin-layer
chromatography (TLC) on pre-coated plates (Merck TLC
aluminum sheets silica 60 F254) with detection by UV
light.
100
1000
10
0
5
21b
2
100
1000
100
1000
10
8
10
8
10
0
21c
Non-toxic
982.15
Cytotoxicity assays
100
1000
0
2
Cell viability was assessed by the mitochondrial-dependent
reduction of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide) to purple formazan (Mos-
mann, 1983). All the following procedures were done in a
sterile area using a laminar flow cabinet biosafety class II
level (Baker, SG403INT, Sanford, ME, USA). Cells were
suspended in RPMI 1640 medium, 1 % antibiotic–antimy-
cotic mixture (10,000 lg ml-1 potassium penicillin,
10,000 lg ml-1 streptomycin sulfate and 25 lg ml-1 am-
photericin B) and 1 % L-glutamine at 37 °C under 5 % CO2.
Cells were batch cultured for 10 days and then seeded at a
a
Ten organisms (A. salina) tested for each concentration
Conclusion
This work reports the synthesis and single-crystal charac-
terization of a family of hydrazide–hydrazone derivatives
incorporating the thiophene pharmacophore. The cytotoxic
potencies of all compounds 3–21 against breast carcinoma
(MCF-7), liver carcinoma (HepG2), colon carcinoma (HCT-
123

Med Chem Res
concentration of 10 9 103 cells per well in fresh complete
growth medium in 96-well microtiter plastic plates at 37 °C
for 24 h under 5 % CO2 using a water-jacketed carbon
dioxide incubator (Sheldon, TC2323, Cornelius, OR, USA).
The media were aspirated, fresh medium (without serum)
was added, and cells were incubated either alone (negative
control) or with different concentrations of sample to give a
final concentration (100–50–25–12.5–6.25–3.125–0.78 and
1.56 lM). After 48 h of incubation, the medium was aspi-
rated, 40 ll MTT salt (2.5 lg ml-1) was added to each well
and incubated for further 4 h at 37 °C under 5 % CO2. To
stop the reaction and to dissolve the formed crystals, 200 ll
of 10 % sodium dodecyl sulfate (SDS) in deionized water
was added to each well and incubated overnight at 37 °C. A
positive control composed of 100 lg ml-1 was used as a
known cytotoxic natural agent, which gives 100 % lethality
under the same conditions (Thabrew et al., 1997). The ab-
sorbance was then measured using a microplate multi-well
reader (Bio-Rad Laboratories Inc., model 3350, Hercules,
California, USA) at 595 nm and a reference wavelength of
620 nm. A statistical significance was tested between sam-
ples and negative control (cells with vehicle) using an in-
dependent t test with the SPSS 11 program. DMSO is the
vehicle used for dissolution of compounds, and its final
concentration on the cells was less than 0.2 %. The per-
centage of change in viability was calculated accordingto the
formula: (Reading of extract/Reading of negative con-
trol) - 1 9 100 %. A probit analysis was carried for IC50
and IC90 determination using the SPSS 11 program.
extract (lg ml-1) against the mean percentage of the
radical scavenging activity.
Brine shrimp lethality assay (BSLA)
The brine shrimp lethality bioassay was used to predict the
toxicity of the synthesized compounds. For the experiment,
4 mg of each compound was dissolved in dimethylsul-
foxide (DMSO) and solutions of varying concentrations
(10, 100, 1000) lg ml-1 were obtained by the serial dilu-
tion technique using simulated seawater. The solutions
were then added to the pre-marked vials containing ten live
brine shrimp nauplii in 5 ml simulated seawater. After
24 h, the vials were inspected using a magnifying glass and
the number of survived nauplii in each vial was counted.
The mortality endpoint of this bioassay was defined as the
absence of controlled forward motion during 30 s of ob-
servation. From these data, the percent of lethality LC50 of
the brine shrimp nauplii for each concentration and control
was calculated.
Crystal structure determinations
Intensity data for each crystal were collected at 100 (2) K
on a Rigaku RX CCD diffractometer (Mo Ka radiation,
˚
k = 0.71073 A). The structures were routinely solved by
direct methods with SHELXS-97, and the structural models
refined and completed by full-matrix least squares refine-
ment against |F|2 with SHELXL-97 (Sheldrick, 2008). The
N-bound H atoms were located in difference maps, and
their positions were freely refined. The C-bound H atoms
were geometrically placed and refined as riding atoms with
Free radical scavenging activity of (DPPH)
˚
C–H = 0.95–0.99 A depending on the assumed C-atom
The free radical scavenging activity of all compounds was
measured by 1,1-diphenyl-2-picrylhydrazyl (DPPHꢀ) using
the method of Shimada et al. (1992). All compounds were
screened at 100 lg ml-1, while the most potent com-
pounds (gave more 90 %) were assayed at
0.39–50 lg ml-1. Briefly, 0.1 mM solution of DPPHꢀ in
methanol was prepared. Then, 1 ml of this solution was
added to 3 ml of each compound at different concentra-
tions. The mixture was shaken vigorously and allowed to
stand at room temperature for 30 min. Then, the ab-
sorbance was measured at 517 nm in Asys microplate
reader. Lower absorbance of the reaction mixture indicated
higher free radical scavenging activity. DPPH scavenging
effect (%) = 100 - [((A0 - A1)/A0) 9 100] where A0
was the absorbance of the control reaction and A1 was the
absorbance in the presence of the sample (Oktay et al.,
2003).
hybridization. The constraint Uiso (H) = 1.2Ueq (carrier) or
1.5Ueq (methyl carrier) was applied in all cases.
2-Cyano-N0-(1-(thiophen-2-yl)ethylidene)acetohydrazide
(3)
Pale yellow crystals (from ethanol). To a solution of
cyanoacetylhydrazine (0.99 g, 0.01 mol) in 1,4-dioxane
(40 ml), 2-acetyl thiophene (1.26 g, 0.01 mol) was added.
The reaction mixture was heated under reflux for 3 h and
then poured onto a beaker containing an ice/water mixture.
The formed solid product was collected by filtration and
dried to give pale yellow crystals (from ethanol). Yield:
1.50 g, 72 %, m.p. 152 °C; IR (KBr) t/cm-1: 3331 (NH),
3167 (CH-aromatic), 2966 (CH3), 2259 (CN), 1680 (C=O),
1
1640 (C=N); MS m/z (%) 207 [M?, 100 %]; H-NMR d:
The IC50 value for each sample, defined as the con-
centration of the test sample leading to 50 % reduction of
the initial DPPH concentration, was calculated from the
nonlinear regression curve of log concentration of the test
2.27 (s, 3H, CH3), 4.09 (s, 2H, CH2), 7.05–7.60 (m, 3H,
thiophene), 11.02 (s, 1H, NH); 13C-NMR d: 15.1 (CH3),
25.3 (CH2), 116.6 (CN), 128.1, 128.3, 128.9, 129.1 (thio-
phene C), 165.8 (C=N), 172.2 (C=O).
123

Med Chem Res
Crystal data for 3
(thiophene C), 114.9, 116.5, 116.7, 161.4 (benzene C),
155.8 (C=N), 165.7 (C=O).
C9H9N3OS, Mr = 207.25, monoclinic, P21/n, a = 4.5453
˚
3-(4-Chlorophenyl)-2-cyano-N0-(1-(thiophen-2-
˚
˚
(2) A, b = 22.6817 (16) A, c = 10.1007 (7) A, b =
3
˚
102.289 (5)°, V = 1017.47 (11) A , Z = 4, F(000) = 432,
yl)ethylidene)acrylohydrazide (5c)
qcalc = 1.353 g cm-3, l = 0.288 mm-1, 8879 data scan-
ned, RInt = 0.099, 2288 data merged [1386 with I [ 2r(I)],
R(F) = 0.045, wR(F2) = 0.096. Full details of the crystal
structure determination are available from the Cambridge
Crystallographic Data Centre (CCDC-960261).
Dark orange crystals (from acetic acid). This compound
was prepared by the following method: To a solution of 3
(2.07 g, 0.01 mol) in 1,4-dioxane (40 ml), p-chloroben-
zaldehyde (1.12 g, 0.01 mol) was added. The reaction
mixture was heated under reflux for 6 h in the presence of a
few drops of piperidine and then poured onto a beaker
containing an ice/water mixture. The formed solid product
was collected by filtration and dried. It was recrystallized
from acetic acid. Yield: 0.43 g, 13.1 %, m.p. 140 °C; IR
(KBr) t/cm-1: 3445 (NH), 3098 (CH-aromatic), 2941
(CH3), 2260 (CN), 1680 (C=O), 1629 (C=N); MS m/z (%)
329 [M?, 10.3 %]; 1H-NMR (DMSO-d6, 300 MHz) d: 2.39
(s, 3H, CH3), 7.96 (s, 1H, olefinic CH), 7.12–7.67 (m, 7H,
C6H4, thiophene-H), 12.80 (s, 1H, NH); 13C-NMR
(DMSO-d6) d: 15.3 (CH3), 146.2 (C=CH), 165.8 (C=CH),
114.4 (C:N), 129.2, 130.2, 130.3, 131.6 (thiophene C),
132.3, 132.6, 132.9, 133.1 (benzene C), 153.9 (C=N), 167.1
(C=O).
2-Cyano-3-phenyl-N0-(1-(thiophen-2-yl)ethylidene)
acrylohydrazide (5a)
Yellow crystals (from ethanol). This compound was pre-
pared by the following method: To a solution of 3 (2.07 g,
0.01 mol) in 1,4-dioxane (40 ml), benzaldehyde (1.06 g,
0.01 mol) was added. The reaction mixture was heated
under reflux for 6 h in the presence of a few drops of
piperidine and then poured onto a beaker containing an
ice/water mixture. The formed solid product was collected
by filtration and dried. It was recrystallized from ethanol.
Yield: 1.50 g, 50.8 %, m.p. 118 °C; IR (KBr) t/cm-1
:
3440 (NH), 3090 (CH-aromatic), 2929 (CH3), 2200 (CN),
1686 (C=O), 1629 (C=N); MS m/z (%) 295 [M?, 0.8 %];
1H-NMR (DMSO-d6, 300 MHz) d: 2.38 (s, 3H, CH3),
7.90 (s, 1H, olefinic CH), 7.12–7.66 (m, 8H, C6H5,
thiophene-H), 10.50 (s, 1H, NH); 13C-NMR (DMSO-d6)
d: 14.7 (CH3), 146.6 (C=CH), 165.1 (C=CH), 114.6
(C:N), 126.7, 128.2, 128.4, 129.2 (thiophene C), 114.7,
114.8, 116.5, 116.9 (benzene C), 155.1 (C=N), 165.7
(C=O).
2-Cyano-N0-(1-(thiophen-2-yl)ethylidene)-3-p-
tolylacrylohydrazide (5d)
Red crystals (from ethanol). This compound was prepared
by the following method: To a solution of 3 (2.07 g,
0.01 mol) in 1,4-dioxane (40 ml), p-tolualdehyde (1.20 g,
0.01 mol) was added. The reaction mixture was heated
under reflux for 6 h in the presence of a few drops of
piperidine and then poured onto a beaker containing an ice/
water mixture. The formed solid product was collected by
filtration and dried. It was recrystallized from ethanol.
2-Cyano-3-(4-methoxyphenyl)-N0-(1-(thiophen-2-yl)
ethylidene)acrylohydrazide (5b)
Yield: 1.95 g, 63.1 %, m.p. 120 °C; IR (KBr) t/cm-1
:
Pale yellow crystals (from ethanol). This compound was
prepared by the following method: To a solution of 3
(2.07 g, 0.01 mol) in 1,4-dioxane (40 ml), p-methoxyben-
zaldehyde (1.08 g, 0.01 mol) was added. The reaction
mixture was heated under reflux for 6 h in the presence of a
few drops of piperidine and then poured onto a beaker
containing an ice/water mixture. The formed solid product
was collected by filtration and dried. It was recrystallized
from ethanol. Yield: 1.99 g, 61.4 %, m.p. 167 °C; IR
(KBr) t/cm-1: 3435 (NH), 3092 (CH-aromatic), 2928
(CH3), 2258 (CN), 1680 (C=O), 1607 (C=N); MS m/z (%)
3447 (NH), 3024 (CH-aromatic), 2933 (CH3), 2260 (CN),
1685 (C=O), 1610 (C=N); MS m/z (%) 309 [M?, 4.5 %];
1H-NMR (DMSO-d6, 300 MHz) d: 2.40 (s, 3H, CH3), 3.62
(s, 3H, Ph-CH3), 7.81 (s, 1H, olefinic CH), 7.03–7.42 (m,
7H, C6H4, thiophene-H), 9.95 (s, 1H, NH); 13C-NMR
(DMSO-d6) d: 15.6 (CH3),21.4 (Ph-CH3), 144.7 (C=CH),
145.7 (C=CH), 116.5 (CN), 128.1, 129.1, 129.3,129.7
(thiophene C), 130.1, 130.2, 135.2, 143.7 (benzene C),
155.6 (C=N), 165.7 (C=O).
2-Cyano-3-(3-nitrophenyl)-N0-(1-(thiophen-2-
1
324 [M?, 22 %]; H-NMR (DMSO-d6, 300 MHz) d: 2.29
yl)ethylidene)acrylohydrazide (5e)
(s, 3H, CH3),3.79 (s, 3H, OCH3), 8.10 (s, 1H, olefinic CH),
6.97–7.94 (m, 7H, C6H4, thiophene-H), 11.64 (s, 1H, NH);
13C-NMR (DMSO-d6) d: 14.7, 55.8 (CH3), 145.1 (C=CH),
165.1 (C=CH), 114.8 (C:N), 126.7, 128.2, 128.4, 129.2
Yellow crystals (from ethanol). This compound was pre-
pared by the following method: To a solution of 3 (2.07 g,
0.01 mol) in 1,4-dioxane (40 ml), m-nitrobenzaldehyde
123

Med Chem Res
(1.51 g, 0.01 mol) was added. The reaction mixture was
heated under reflux for 6 h in the presence of a few drops
of piperidine and then poured onto a beaker containing an
ice/water mixture. The formed solid product was collected
by filtration and dried. It was recrystallized from ethanol.
cyclopentanone), 2.27 (s, 3H, CH3), 7.06–7.60 (m, 3H,
thiophene-H), 10.6 (s, 1H, NH); 13C-NMR (DMSO-d6) d:
14.7 (CH3), 25.1, 25.2, 146.4 (cyclopentanone C), 143.1
(C=C), 116.5 (C:N), 128.2, 128.4, 129.1,131.6 (thiophene
C), 159.6 (C=N), 165.7 (C=O).
Yield: 1.05 g, 30.9 %, m.p. 130 °C; IR (KBr) t/cm-1
:
3427 (NH), 3050 (CH-aromatic), 2937 (CH3), 2212 (CN),
1650 (C=O), 1630 (C=N); MS m/z (%) 340 [M?, 20.2 %];
1H-NMR (DMSO-d6, 300 MHz) d: 2.39 (s, 3H, CH3), 7.96
(s, 1H, olefinic CH), 7.12–7.67 (m, 7H, C6H4, thiophene-
H), 12.8 (s, 1H, NH); 13C-NMR (DMSO-d6) d: 14.2 (CH3),
146.2 (C=CH), 153.5 (C=CH), 115.9 (C:N), 126, 127.1,
127.4, 129 (thiophene C), 120.3, 121.3, 129.6, 132.5,
136.1, 148.3 (benzene C), 155.6 (C=N), 168 (C=O).
Ethyl 5-(1-(thiophen-2-yl)ethylideneaminocarbamoyl)-2,4-
diaminothiophene-3-carboxylate (10)
Pink crystals (from ethanol). This compound was prepared
by the following method: To a solution of 3 (2.07 g,
0.01 mol) in ethanol (20 ml), ethylcyanoacetate (1.13 g,
0.01 mol) and sulfur (0.32 g, 0.01 mol) were added. The
reaction mixture was heated under reflux for 3 h in the
presence of a few drops of triethylamine and then poured
onto a beaker containing an ice/water mixture. The formed
solid product was collected by filtration and dried to give
pink crystals (from ethanol). Yield: 2.26 g, 64.2 %, m.p.
165 °C; IR (KBr) t/cm-1: 3400–3337 (NH, NH2), 3101
(CH-aromatic), 2928 (CH3), 2258 (CN), 1690, 1812 (2
C=O), 1609 (C=N), 1119 (CO); MS m/z (%) 352 [M?,
2-Imino-N0-(1-(thiophen-2-yl)ethylidene)-2H-chromene-3-
carbohydrazide (7)
White crystals (from ethanol). This compound was pre-
pared by the following method: To a solution of 3 (2.07 g,
0.01 mol) in 1, 4-dioxane (20 ml), salicylaldehyde (1.22 g,
0.01 mol) was added. The reaction mixture was heated
under reflux for 3 h in the presence of a few drops of
piperidine and then poured onto a beaker containing an ice/
water mixture. The formed solid product was collected by
filtration and dried to give white crystals (from ethanol).
1
10.9 %]; H-NMR (DMSO-d6, 300 MHz) d: 1.056 (3H, t,
J = 6.9 Hz, CH3), 2.27 (s, 3H, CH3), 3.454 (2H, q,
J = 7 Hz, CH2), 3.85, 4.09 (2 NH2), 7.05–7.60 (m, 3H,
thiophene-H), 10.63 (s, 1H, NH); 13C-NMR (DMSO-d6) d:
17.6, 21.6 (2 CH3), 55.8 (CH2), 127.0, 128.3, 130.5, 130.7,
133.4, 133.6, 140.3, 161.3 (thiophene C), 159.6 (C=N),
172.6, 175.4 (2 C=O).
Yield: 1.24 g, 39.8 %, m.p. 210 °C IR (KBr) t/cm-1
:
3435, 3207 (2 NH), 3059 (CH-aromatic), 2900 (CH3), 2212
(CN), 1700 (C=O), 1677 (C=N); MS m/z (%) 311 [M?,
1
20.5 %]; H-NMR (DMSO-d6, 300 MHz) d: 2.34 (s, 3H,
2-(2-Phenylhydrazono)-2-cyano-N0-(1-(thiophen-2-
CH3), 8.91 (s, 1H, coumarin H-4), 6.93–8.70 (m, C6H4,
thiophene-H), 11.86 (s, 1H, NH); 13C-NMR (DMSO-d6) d:
17.6 (CH3), 116.6 (C:N), 130.7, 133.5, 133.7, 135.2,
140.2, 146.4, 152.9, 153.9 (chromene C), 128.4, 128.6,
128.8, 129.1 (thiophene C), 155.6 (C=N), 160.3 (C=NH),
175.4 (C=O).
yl)ethylidene)acetohydrazide (11a)
Brown crystals (from EtOH). This compound was pre-
pared by the following method: A cold solution of ben-
zenediazonium chloride (0.01 mol) was prepared by
adding a solution of sodium nitrite (1.4 g dissolved in
10 ml water) to cold solution of aniline hydrochloride
(0.93 g of aniline in 10 ml 6 M HCl). The resulting so-
lution of benzenediazonium chloride was then added to a
cold solution of 3 (2.70 g, 0.01 mol) in ethanol (50 ml) in
the presence of sodium acetate (1.64 g, 0.02 mol). The
reaction mixture was stirred at room temperature for 1 h.
The mixture was poured into ice-cold water, and the
formed solid product was collected by filtration and
crystallized from ethanol.
2-Cyano-2-cyclopentylidene-N0-(1-(thiophen-2-
yl)ethylidene)acetohydrazide (9)
Gray crystals (from ethanol). This compound was prepared
by the following method: Equimolar amounts of compound
3 (2.07 g, 0.01 mol) and cyclopentanone 10 (0.84 g,
0.01 mol) were heated in an oil bath at 140 °C for 1 h in
the presence of ammonium acetate. After cooling the re-
action mixture, it was heated in ethanol and then poured
into ice/water mixture and the formed solid product was
collected by filtration and dried to give gray crystals (from
ethanol).
Yield: 0.84 g, 27 %, m.p. 156 °C; IR (KBr) t/cm-1
:
3400, 3336 (2 NH), 3100 (CH-aromatic), 2928 (CH3), 2258
(CN), 1685 (C=O), 1609 (C=N); MS m/z (%) 311 [M?,
1
1.2 %]; H-NMR (DMSO-d6, 300 MHz) d: 2.27 (s, 3H,
Yield: 1.24 g, 45.4 %, m.p. 160 °C; IR (KBr) t/cm-1
:
CH3), 7.1–7.6 (m, 8H, thiophene-H, C6H5), 10.63, 11.01 (2
NH); 13C-NMR (DMSO-d6) d: 15.3 (CH3), 114.9 (CN),
125.8, 126, 127.1, 127.4 (thiophene C), 116.3, 118.8, 129.6,
143.1 (benzene C), 106.1, 155.6 (2 C=N), 165 (C=O).
3400 (NH), 3100 (CH-aromatic), 2853 (CH3), 2258 (CN),
1690 (C=O), 1609 (C=N); MS m/z (%) 273 [M?, 13.2 %];
1H-NMR (DMSO-d6, 300 MHz) d: 0.76–1.23 (m, 8H,
123

Med Chem Res
2-(2-(4-Bromophenyl)hydrazono)-2-cyano-N0-(1-
2258 (CN), 1672 (C=O), 1659 (C=N); MS m/z (%) 325
1
[M?, 0.6 %]; H-NMR(DMSO-d6, 300 MHz) d: 2.27 (s,
(thiophen-2-yl) ethylidene) acetohydrazide (11b)
3H, CH3), 3.86 (s, 3H, CH3), 7.05–7.60 (m, 7H, thio-
phene-H, C6H4), 10.64, 11.01 (2 NH); 13C-NMR (DMSO-
d6) d: 15.2, 16.3 (2 CH3), 114.9 (C:N), 131.4, 132.6,
132.9, 133.2 (thiophene C), 116.2, 118.7, 129.0, 129.6,
129.9, 141.6 (benzene C), 118.9, 163.1 (2 C=N), 167.5
(C=O).
Orange crystals (from EtOH). This compound was prepared
by the following method: A cold solution of benzenediazo-
niumchloride(0.01 mol)waspreparedbyaddingasolutionof
sodiumnitrite(1.4 gdissolvedin10 mlwater)tocoldsolution
of aniline hydrochloride (p-bromoaniline) (1.70 g in 10 ml
6 M HCl). The resulting solution of benzenediazonium
chloride was then added to a cold solution of 3 (2.70 g,
0.01 mol)inethanol (50 ml)inthe presenceofsodiumacetate
(1.64 g, 0.02 mol). The reaction mixture was stirred at room
temperature for 1 h. The mixture was poured into ice-cold
water, and the formed solid product was collected by filtration
and crystallized from ethanol.
Yield: 0.53 g, 14 %, m.p. 137 °C; IR (KBr) t/cm-1: 3634,
3361 (2 NH), 3184 (CH-aromatic), 2928 (CH3), 2258 (CN),
1672 (C=O), 1659 (C=N); MS m/z (%) 390 [M?, 42 %]; 1H-
NMR (DMSO-d6, 300 MHz) d: 2.37 (s, 3H, CH3), 7.10–7.84
(m, 7H, thiophene-H, C6H4), 10.51, 12.02 (2 NH); 13C-NMR
(DMSO-d6) d: 15.3(CH3), 116.7 (C:N), 132.4, 132.6, 132.9,
133.1 (thiophene C), 111.6, 120.9, 133.3, 138.1 (benzene C),
118.9, 163.1 (2 C=N), 167.5 (C=O).
1-(1-(Thiophen-2-yl)ethylideneamino)-1,2-dihydro-4,6-
dimethyl-2-oxopyridine-3-carbonitrile (13)
White crystals (from ethanol). This compound was pre-
pared by the following method: To a solution of 3 (2.07 g,
0.01 mol) in ethanol (20 ml), acetylacetone (0.10 g,
0.01 mol) was added. The reaction mixture was heated
under reflux for 3 h in the presence of a few drops of
triethylamine and then poured onto a beaker containing an
ice/water mixture. The formed solid product was collected
by filtration and dried to give white crystals (from ethanol).
Yield: 0.92 g, 33.5 %, m.p. 180 °C; IR (KBr) t/cm-1
:
3106 (CH-aromatic), 2927 (CH3), 2218 (CN), 1640 (C=O),
1
1592 (C=N); MS m/z (%) 243 [M?, 15.06 %]; H-NMR
(DMSO-d6, 300 MHz) d: 2.17 (s, 3H, CH3), 2.23 (s, 3H,
CH3), 2.37 (s, 3H, CH3), 7.23–7.90 (m, 3H, thiophene-H),
6.44 (s, pyridine-H); 13C-NMR (DMSO-d6) d: 17.2, 19.6,
21.0 (3CH3), 116.5 (CN), 99.7, 108.5, 150.6, 157.7 (pyr-
idine C), 128.8, 133.4, 133.6, 139.8 (thiophene C), 155.9
(C=N), 172 (C=O).
Crystal data for 11b
C15H12BrN5OS, Mr = 390.27, monoclinic,P21/c, a =6.1486
˚
˚
˚
(4) A, b = 23.1125 (16) A, c = 11.2442 (8) A, b = 90.505
3
˚
(2)°, V = 1597.85 (19) A , Z = 4, F(000) = 784,
qcalc = 1.622 g cm-3, l = 2.714 mm-1, 22,664 data scan-
ned, RInt = 0.043, 3643 data merged [2894 with I[ 2r(I)],
R(F) = 0.034, wR(F2) = 0.073. Full details of the crystal
structure determination are available from the Cambridge
Crystallographic Data Centre (CCDC-960260).
Ethyl 4-(1-(thiophen-2-yl)ethylideneaminocarbamoyl)-3-
amino-5-(phenylamino)thiophene-2-carboxylate (15)
Pale yellow crystals (from EtOH). This compound was
prepared by the following method: Equimolecular amounts
of 3 (2.07 g, 0.01 mol) and phenylisothiocyanate (1.35 g,
0.01 mol) and potassium hydroxide (0.56 g, 0.01 mol)
were stirred for 24 h. Next, ethyl chloroacetate (1.22 g,
0.01 mol) was added and the reaction mixture was stirred
for another 24 h. The reaction mixture was then poured
onto an ice/water mixture containing few drops of hy-
drochloric acid, and the formed solid product was collected
by filtration and crystallized from ethanol.
2-(2-o-Tolylhydrazono)-2-cyano-N0-(1-(thiophen-2-
yl)ethylidene)acetohydrazide (11c)
Orange crystals (from EtOH). This compound was prepared
by the following method: A cold solution of benzene-
diazonium chloride (0.01 mol) was prepared by adding a
solution of sodium nitrite (1.4 g dissolved in 10 ml water) to
cold solution of aniline hydrochloride [o-toluidine (1.07 g in
10 ml 6 M HCl)]. The resulting solution of benzenediazo-
nium chloride was then added to a cold solution of 3 (2.70 g,
0.01 mol) in ethanol (50 ml) in the presence of sodium ac-
etate (1.64 g, 0.02 mol). The reaction mixture was stirred at
room temperature for 1 h. The mixture was poured into ice-
cold water, and the formed solid product was collected by
filtration and crystallized from ethanol.
Yield: 2.1 g, 54.9 %, m.p. 155 °C; IR (KBr) t/cm-1: 3434
(NH), 3101 (CH-aromatic), 2928 (CH3), 2215 (CN), 1690
1
(C=O), 1596 (C=N); MS m/z (%) 382 [M?, 21.3 %]; H-
NMR (DMSO-d6, 300 MHz)d: 2.23(s, 3H, CH3), 4.34(s, 2H,
thiazole), 7.05–7.60 (m, 8H, thiophene-H, C6H5), 11.02 (s,
1H, NH); 13C-NMR (DMSO-d6) d: 17.6 (CH3),87.6 (C=C),
116.9 (C:N), 128.4, 128.6, 128.8, 129.1 (thiophene C),
130.7, 133.5, 133.7, 140.2 (benzene C), 75.5, 160.2 (thiazole
C), 155.6 (C=N), 175.4 (C=O), 188.4 (thiazole C=O).
Yield: 1.12 g, 34.4 %, m.p. 152 °C; IR (KBr) t/cm-1
3435, 3300 (2 NH), 3184 (CH-aromatic), 2928 (CH3),
:
123

Med Chem Res
2-(4-Amino-3-phenylthiazol-2(3H)-ylidene)-2-cyano-N0-(1-
0.01 mol) and benzaldehyde (1.06 g, 0.01 mol) were
added. The reaction mixture was heated under reflux for
3 h in the presence of a few drops of piperidine and then
poured onto a beaker containing an ice/water mixture fil-
tration and crystallized from ethanol.
(thiophen-2-yl)ethylidene) acetohydrazide (16)
Gray crystals (from EtOH). This compound was prepared
by the following method: Equimolecular amounts of 3
(2.07 g, 0.01 mol) and phenylisothiocyanate (1.35 g,
0.01 mol) and potassium hydroxide (0.56 g, 0.01 mol)
were stirred for 24 h. Next, chloroacetonitrile (0.75 g,
0.01 mol) was added and the reaction mixture was stirred
for another 24 h. The reaction mixture was then poured
onto an ice/water mixture containing few drops of hy-
drochloric acid, and the formed solid product was collected
by filtration and crystallized from ethanol.
Yield: 0.84 g, 23 %, m.p. 278 °C; IR (KBr) t/cm-1
:
3456 (NH2), 3097 (CH-aromatic), 2980 (CH3), 2216 (CN),
1654 (C=O), 1610 (C=N); MS m/z (%) 359 [M?, 23.2 %];
1H-NMR (DMSO-d6, 300 MHz) d: 2.26 (s, 3H, CH3),
7.24–8.26 (m, 8H, thiophene-H, C6H5), 3.34 (NH2). 13C-
NMR (DMSO-d6) d: 17.6 (CH3), 114.6, 116.3 (2 C:N),
75.5, 140.2, 161.3, 175.4 (pyridine C), 130.5, 130.7, 133.5,
133.6 (thiophene C), 114.4, 127.1, 128.4, 159.9 (benzene
C), 155.6 (C=N), 172.6 (C=O).
Yield: 1.92 g, 50.4 %, m.p. 180 °C; IR (KBr) t/cm-1
:
3434–3300 (NH2, NH), 3101 (CH-aromatic), 2928 (CH3),
2218 (CN), 1640 (C=O), 1592 (C=N); MS m/z (%) 381
1
1-(1-(Thiophen-2-yl)ethylideneamino)-6-amino-1,2-
dihydro-4-(4-methoxyphenyl)-2-oxopyridine-3,5-
dicarbonitrile (21b)
[M?, 17.1 %]; H-NMR (DMSO-d6, 300 MHz) d: 2.27 (s,
3H, CH3), 3.86 (s, 1H, thiazole-H), 4.09 (s, NH2),
7.06–7.60 (m, 8H, thiophene-H, C6H5), 11.02 (s, 1H, NH);
13C-NMR (DMSO-d6) d: 17.6 (CH3), 75.5 (C=C), 116.6
(C:N), 81.8, 152.9, 160.3 (thiazole C), 128.4, 128.6,
128.8, 129.1 (thiophene C),130.7, 133.5, 133.7, 135.1
(benzene C), 155.6 (C=N), 175.4 (C=O).
Brown crystals (from EtOH). This compound was prepared
by the following method: To a solution of 3 (2.07 g,
0.01 mol) in ethanol (20 ml), malononitrile (0.66 g,
0.01 mol) and anisaldehyde (1.36 g, 0.01 mol) were added.
The reaction mixture was heated under reflux for 3 h in the
presence of a few drops of piperidine and then poured onto
a beaker containing an ice/water mixture filtration and
crystallized from ethanol.
3-(1-(Thiophen-2-yl)ethylideneamino)-2-amino-4,5-
dihydro-5-imino-4-oxo-3H-chromeno[3,4-c] pyridine-1-
carbonitrile (19)
Yield: 0.79 g, 23 %, m.p. 110 °C; IR (KBr) t/cm-1
:
Dark orange crystals (from EtOH). This compound was
prepared by the following method: To a solution of 3
(2.07 g, 0.01 mol) in ethanol (20 ml), malononitrile
(0.66 g, 0.01 mol) and salicylaldehyde (1.22 g, 0.01 mol)
were added. The reaction mixture was heated under reflux
for 3 h in the presence of a few drops of piperidine and
then poured onto a beaker containing an ice/water mixture
filtration and crystallized from ethanol.
3324 (NH2), 3080 (CH-aromatic), 2935 (CH3), 2214 (CN),
1670 (C=O), 1607 (C=N); MS m/z (%) 389 [M?, 10.2 %];
1H-NMR (DMSO-d6, 300 MHz) d: 2.25 (s, 3H, CH3),
6.87–8.20 (m, 8H, thiophene-H, C6H4), 3.86 (s, NH2), 3.87
(s, 3H, CH3); 13C-NMR (DMSO-d6) d: 17.6,55.8 (2CH3),
114.6, 116.3 (2 C:N), 75.5, 140.2, 161.3, 175.4 (pyridine
C), 130.5, 130.7, 133.5, 133.6 (thiophene C), 114.4, 127.1,
128.4, 159.9 (benzene C), 155.6 (C=N), 172.6 (C=O).
Yield: 1.84 g, 49 %, m.p. 120 °C IR (KBr) t/cm-1
:
3100–3441 (NH, NH2), 3097 (CH-aromatic), 2933 (CH3),
1-(1-(Thiophen-2-yl)ethylideneamino)-6-amino-1,2-
2204 (CN), 1600 (C=O), 1560 (C=N); MS m/z (%) 375
1
dihydro-2-oxo-4-p-tolylpyridine-3,5-dicarbonitrile (21c)
[M?, 21.2 %]; H-NMR (DMSO-d6, 300 MHz) d: 2.35 (s,
3H, CH3), 6.8–8.28 (m, 8H, thiophene-H, C6H4), 4.00
(NH2),13.26 (NH); 13C-NMR (DMSO-d6) d: 17.6 (CH3),
116.9 (C:N), 75.5, 116.08, 153.9, 160.2 (pyridine C),
109.7, 130.7, 133.5, 133.7,140.2, 152.9 (benzene C), 128.4,
128.6, 128.8, 129.1 (thiophene C), 155.5 (C=N),175.4
(C=NH), 167.1 (C=O).
Dark yellow crystals (from EtOH). This compound was
prepared by the following method: To a solution of 3
(2.07 g, 0.01 mol) in ethanol (20 ml), malononitrile
(0.66 g, 0.01 mol) and tolualdehyde (1.2 g, 0.01 mol) were
added. The reaction mixture was heated under reflux for
3 h in the presence of a few drops of piperidine and then
poured onto a beaker containing an ice/water mixture fil-
tration and crystallized from ethanol.
1-(1-(Thiophen-2-yl)ethylideneamino)-6-amino-1,2-
Yield: 0.89 g, 24 %, m.p. 120 °C; IR (KBr) t/cm-1
:
dihydro-2-oxo-4-phenylpyridine-3,5-dicarbonitrile (21a)
3434 (NH2), 3080 (CH-aromatic), 2921 (CH3), 2212, 2149
(CN), 1610 (C=O), 1578 (C=N); MS m/z (%) 373 [M?,
Yellow crystals (from EtOH). This compound was pre-
pared by the following method: To a solution of 3 (2.07 g,
0.01 mol) in ethanol (20 ml), malononitrile (0.66 g,
1
10.2 %]; H-NMR (DMSO-d6, 300 MHz) d: 2.25 (s, 3H,
CH3), 2.38 (s, 3H, CH3), 7.13–8.2 (m, 8H, thiophene-H,
123

Med Chem Res
C6H4), 3.39 (NH2); 13C-NMR (DMSO-d6) d: 13C-NMR d:
15.3, 16.3 (CH3), 116.7, 117.8 (2C:N), 73.3, 108.5, 160.3,
163.8 (pyridine C), 118.6, 119.2, 120.5, 122.4 (benzene C),
125.0, 128.6, 129.5, 133.2 (thiophene C), 161.7 (C=N),
164.7 (C=O).
˘
Oktay M, Gu¨lc¸in I, Ku¨frevioglu II (2003) Determination of invitro
antioxidant activity of fennel (Foeniculum vulgare) seed
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