Synthesis and biological evaluation of new curcumin analogues as antioxidant and antitumor agents: Molecular modeling study

Article abstract of DOI:10.1016/j.ejmech.2015.07.014

New curcumin analogues have been synthesized and their antioxidant activities were investigated by measuring their free radical scavenging capacities. The in vitro and in vivo antitumor activities of the synthesized compounds on Ehrlich ascites carcinoma (EAC) cell line were evaluated. 4-(4-Chlorophenyl)-2-(5-ethyl-7-(4-methoxybenzylidene)-3-(4-methoxyphenyl)-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c] pyridin-2-yl)thiazole 7h showed excellent antineoplastic activity in both in vitro and in vivo studies more than that of tested compounds and reference drug, cisplatin. Different molecular modeling studies were performed, where docking of compound 7h into telomerase active site suggested that it could exert its antitumor potential by telomerase inhibition.

Full text of DOI:10.1016/j.ejmech.2015.07.014

European Journal of Medicinal Chemistry 101 (2015) 584e594
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and biological evaluation of new curcumin analogues as
antioxidant and antitumor agents: Molecular modeling study
Said M. Bayomi a, Hassan A. El-Kashef b, Mahmoud B. El-Ashmawy a, Magda N.A. Nasr c,
Magda A. El-Sherbeny a, Naglaa I. Abdel-Aziz a, Magda A.-A. El-Sayed c,
,
*
Ghada M. Suddek b, Shahenda M. El-Messery c, Mariam A. Ghaly a
a Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
b Department of Pharmacology, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
c Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
New curcumin analogues have been synthesized and their antioxidant activities were investigated by
measuring their free radical scavenging capacities. The in vitro and in vivo antitumor activities of the
synthesized compounds on Ehrlich ascites carcinoma (EAC) cell line were evaluated. 4-(4-Chlorophenyl)-
2-(5-ethyl-7-(4-methoxybenzylidene)-3-(4-methoxyphenyl)-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]
pyridin-2-yl)thiazole 7h showed excellent antineoplastic activity in both in vitro and in vivo studies more
than that of tested compounds and reference drug, cisplatin. Different molecular modeling studies were
performed, where docking of compound 7h into telomerase active site suggested that it could exert its
antitumor potential by telomerase inhibition.
Received 8 December 2014
Received in revised form
6 July 2015
Accepted 7 July 2015
Available online 14 July 2015
Keywords:
Curcumin analogues
Synthesis
© 2015 Elsevier Masson SAS. All rights reserved.
Antioxidant effect
Antitumor effect
Molecular modeling studies
1. Introduction
progress of many chronic diseases, including cancer [1].
Curcumin (A) is one of the most potent and multi-targeting
Despite significant progress achieved in anticancer therapy, high
systemic toxicity and drug resistance remain a major challenge for
contemporary medicine in the management of cancers. Chemo-
therapy causes severe side-effects, which may be due to its cyto-
toxic effect on normal cells [1]. Therefore, it is important that
anticancer drugs display antiproliferative and cytotoxic activity in
tumor cells without affecting normal tissues. Reactive oxygen
species (ROS) have been linked with cancer, coronary heart disease,
and neurodegenerative diseases. Furthermore, their presence in the
body causes damage to the DNA of cells. Antioxidants exert their
effects by scavenging or preventing the generation of ROS, thus can
protect against the formation of free radicals and retard the
phytochemicals against a variety of cancers. The cancer preven-
tive capability of curcumin is linked to its direct antioxidant ability
to eliminate free radicals and to reduce oxidative stress [2]. How-
ever, the clinical application of curcumin has been significantly
limited by its instability and poor metabolic property. A number of
synthetic modifications of curcumin have been studied intensively
in order to develop a molecule with enhanced bioactivities. A
number of curcumin analogues have partial structural resemblance
to curcumin motif but are appended with different pharmaco-
phores for targeting proteins that are crucial for survival of tumor
cells in question. For example, monocarbonyl 5-carbon spacer,
curcumin analogues having cycloalkanone or piperidone central
motifs (B) were reported to exert good antioxidant and antitumor
activities [3,4].
* Corresponding author.
E-mail address: mariamaghaly2@yahoo.com (M.A. Ghaly).
http://dx.doi.org/10.1016/j.ejmech.2015.07.014
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

S.M. Bayomi et al. / European Journal of Medicinal Chemistry 101 (2015) 584e594
585
Our previous study also showed the incorporation of the
shortened carbon linker in heterocyclic parts as hexahydro-2H-
indazole ring (C) and tetrahydro-4H-chromene ring (D) and re-
ported their promising activities [4].
polarized carbonyl group, followed by intramolecular cyclization.
The spectral and microanalytical data of the formed products were
consistent with their structures. The amino functional group was
observed at 3280e3463 cmꢀ1 in the IR spectra. The evidence for the
Telomere is a non-coding DNA sequence, repeating (50-TTAGGG-
30), located at the ends of the chromosomes. It functions by pre-
venting chromosomes from losing base pair sequences at their
ends. Telomerase, a reverse transcriptase, has been found to be
activated in more than 80% of human cancers and, therefore, can be
considered as a potential marker for tumorigenesis [5]. Several
studies comprising a variety of mechanistic approaches, like mu-
tations and RNA interference of the telomerase enzyme activity,
provided the proof of concept for this form of cancer treatment
[6e8]. Telomerase provides a good target not only for cancer
diagnosis, but also for the development of novel therapeutic agents
[9]. Regulation of telomerase activity of cancerous cells by curcu-
min was estimated and found beneficial to human beings [10,11].
Drug design in the area of cancer therapeutics has used curcu-
min and its analogues for developing a trend toward more precise
mechanisms of cancer cell destruction [12]. So in our research
program for novel antitumor agents, we designed and synthesized
a series of curcumin analogues based on chalcone scaffold. We
chose chalcone moiety as it represents an important pharmaco-
phore of both natural products and synthetic precursors that were
found to possess our beseeched biological activity, including anti-
oxidants, and antitumor activities. Recently there is a strong link
between curcumin cancer potential and telomerase inhibition
[11e18]. So it was quite interesting to make in silico study on the
most active antitumor curcumin analogue to investigate the
mechanistic way for its antiumor potential, and compare it against
the least active one.
formation of 4,5-dihydropyrazole ring was obtained from 1H NMR
spectrum which provided the diagnostic tool for the positional
elucidation of protons. The geminal pyrazoline protons at C4
appeared in the region of 3.20e4.10 ppm as doublet of doublets in
all compounds. In addition, CH proton at C5 also appeared as
doublet of doublets in the region of 6.10e6.23 ppm due to vicinal
coupling with the two non-magnetically equivalent geminal pro-
tons of C4. 13C NMR spectrum of compound 3a confirmed its pro-
posed structure, since the C4 and C5 of pyrazoline ring resonated at
45.11 and 70.20 ppm, respectively. Moreover, the aforementioned
compounds 3a,b were cyclized to pyrazolothiazole derivatives 4a,b
through reaction with phenacyl bromide in ethanol. Analytical and
spectral data of the prepared compounds 4a,b were in agreement
with the proposed structure.
2.1.2. Synthesis of compounds 5e8 (Scheme 2)
The key chalcone intermediates 5aed were synthesized through
condensation of p-methoxybenzaldehyde with different cyclic ke-
tones in accordance with the method described in the literature [3].
Heating at reflux equimolar amounts of thiosemicarbazide and the
key chalcones 5aed in ethanol in the presence of NaOH afforded
the corresponding 1-thiocarbamoyl pyrazole derivatives 6aed. The
thiocarbamoyl moiety in the latter compounds was cyclized to 4-
(substituted phenyl)thiazole ring, affording compounds 7aeh in a
good yield. The structures of the new compounds 7aeh were
confirmed using 1H NMR spectra which revealed the presence of 5-
H of thiazole in the aromatic region. Moreover, 13C NMR for com-
pound 7c confirmed the proposed structure due to the appearance
2. Results and discussion
2.1. Chemistry
of a characteristic peak at d169.20 corresponding to C2 of thiazole.
In addition, compounds 8aed were obtained by the reaction of 1-
thiocarbamoyl pyrazole derivative 6b with chloroacetic acid and
different aromatic aldehydes in ethanol. The spectral and micro-
analytical data of compounds 8aed were consistent with their
structures. For example, IR spectrum of compound 8a showed a
strong absorption band at 1696 cmꢀ1 due to carbonyl group. In
addition, 13C NMR confirmed the proposed structure due to the
appearance of a signal at 165.11 ppm for the carbonyl group as well
as a signal assignable to C2 of thiazole at 160.21 ppm.
The reaction sequences employed for synthesis of the target
derivatives are illustrated in Schemes 1e3.
2.1.1. Synthesis of compounds 3 and 4 (Scheme 1)
Thiocarbamoyl pyrazole derivatives 3a,b were obtained by
refluxing equimolar amounts of thiosemicarbazide and the corre-
sponding chalcone 2a,b in hot ethanolic NaOH solution. The reac-
tion involved nucleophilic attack of the amino group on the

586
S.M. Bayomi et al. / European Journal of Medicinal Chemistry 101 (2015) 584e594
Scheme 1. Synthesis of the designed 1-thiocarbamoyl pyrazole and pyrazolylthiazole derivatives.
Scheme 2. Synthesis of the designed pyrazolylthiazole and indazolylthiazole derivatives.
2.1.3. Synthesis of compounds 11 (Scheme 3)
2-amino-3-cyanopyridine derivatives. IR spectra of compounds
11bed were characterized by the presence of absorption bands
assignable to both NH2 and CN groups in the expected regions of
the spectra.
Construction of a cyanoaminopyridine ring could be achieved by
refluxing a mixture of 2,6-bis(substituted benzylidene)cyclohexa-
nones 10aed with malononitrile in the presence of ammonium
acetate in n-butanol to afford the corresponding cyanoaminote-
trahydroquinolines 11aed. The reaction may proceed via a,b-un-
2.2. Biological evaluation
saturated imino intermediate formed by reaction of ketonic group
and ammonium acetate, and stimultaneous Michael addition of
2.2.1. Assay for 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) radical (ABTSꢁþ) scavenging activity
malononitrile on
a,b-unsaturated moiety to give an adduct, fol-
lowed by cycloaddition, isomerization, and aromatization to afford
The antioxidant activity assay employed here is one of the

S.M. Bayomi et al. / European Journal of Medicinal Chemistry 101 (2015) 584e594
587
Scheme 3. Synthesis of the designed cyanoaminopyridine derivatives.
several assays that depend on measuring the consumption of stable
free radicals, to evaluate the free radical scavenging activity of the
investigated compound. The methodology assumes that con-
sumption of the stable free radical (Xꢁ) by hydrogen-donating an-
tioxidants (YH) will be determined by reaction as follows:
Xꢁ þ YH/XH þ Yꢁ
The rate and/or the extent of the process measured in terms of
the decrease in Xꢁ concentration, would be related to the ability of
the added compounds (YH) to trap free radicals. The decrease in
colour intensity of the free radical solution due to scavenging of the
free radical by the antioxidant material is measured colourimetri-
cally at a specific wavelength. The assay employs the radical cation
derived from 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS) as stable free radical. The advantage of ABTSederived free
radical method over other methods is that the produced colour
remains stable for more than 1 h and the reaction is stoichiometric.
In this study, thirteen of the synthesized compounds were sub-
jected to ABTS test and their percentage inhibition of the ABTS
radical cation were listed in Table 1. From these results, it is
concluded that compounds that exhibited more than 50% inhibition
of the ABTS radical cation were in the order of: ascorbic acid
> 6d > curcumin > 3a > 7h > 4a > 7f > 7g as presented in Fig. 1.
Fig. 1. The percentage inhibition of the ABTS radical cation by the tested compounds.
Table 1
Compounds 3a and 6d, with a thiocarbamoyl substituent at N1 of
the pyrazoline ring, showed the highest free radical scavenging
activities among the tested compounds with percentage inhibition
84.52 and 88.17, respectively. However, cyclization of the thio-
carbamoyl moiety in the latter compounds to a phenylthiazole ring
slightly decreased the activity as in compounds 4a, 7g and 7h. The
rest of the examined compounds showed weak antioxidant activity
except a tetrahydroquinoline derivative, 11b, that showed per-
centage inhibition of 46.60.
The percentage inhibition of the ABTS radical cation by the tested compounds.
Compound
Absorbance
%Inhibition
Control
Ascorbic acid
Curcumin
3a
4a
6d
7b
7f
7g
7h
8a
8b
8d
11a
11b
11c
0.575
0.067
0.077
0.089
0.138
0.068
0.417
0.175
0.227
0.100
0.427
0.545
0.465
0.525
0.307
0.519
0
88.34
86.60
84.52
76.00
88.17
27.47
69.56
60.52
82.60
25.73
5.21
2.2.2. Antitumor testing
This research aimed to evaluate the in vitro and in vivo antitumor
activity of the synthesized compounds on Ehrlich ascites carcinoma
(EAC) cell line and this method was proven to give accurate and
reliable results as corroborated by the literature [19e21].
19.13
8.69
46.60
9.73

588
S.M. Bayomi et al. / European Journal of Medicinal Chemistry 101 (2015) 584e594
Table 3
2.2.2.1. Antitumor activity using in vitro Ehrlich ascites assay.
The antitumor activity of thirteen of the synthesized compounds
against EAC cell line was determined using trypan blue assay and
cisplatin as a reference drug control. The cells were incubated with
Effect of tested compounds on tumor size in mice after 21 days
treatment.
Compound
% Tumor growth inhibition
three different concentrations (25,50 and100 mM) for each com-
Control
Cisplatin
3a
4a
6d
7f
7h
11b
0
80
76
65
77
70
82
80
pound and cisplatin. The trypan blue dye exclusion test was used
for assessment of cell viability. It is based on the principle that live
cells possess intact cell membranes that exclude trypan, whereas
dead cells do not and so a viable cell will have a clear cytoplasm and
the dead cell will have a blue cytoplasm. Control experiment was
also conducted in which EAC cells were incubated in a drug-free
medium and the viability of the cells used in control experiments
exceeded 95%. Percent survival of cells ¼ (T/C) X 100 was calculated
where T and C represent the number of viable cells in a unit volume
of the test drug tube and the control tube, respectively (Table 2).
It is evident from the results in Table 2 that the mortality of EAC
cells increased with the concentration of the tested compounds as
there was an increase in the number of cells stained with trypan
blue dye. All the tested compounds showed % dead of cultured
(80%), while compound 11b showed equal activity as cisplatin.
However, the rest of the screened compounds exhibited reasonable
antitumor activities against (EAC) cell line in the following order:
6d > 3a > 7f > 4a with percentage tumor growth inhibition 77, 76,
70 and 65, respectively.
EAC cells more than 75% at a concentration of 100 mM in compar-
2.3. Molecular modeling studies
ison to that of the standard cisplatin (100%). Generally, the
most active compounds in this assay were in the order of:
7h > 11b > 6d > 4a > 7f > 3a. Compound 7h was proved to be the
most active member among the tested compound and it appeared
promising with antineoplastic activity higher than that of the
positive control, cisplatin. Moreover, compound 11b exhibited
equipotent antitumor activity comparable to that of cisplatin.
2.3.1. Docking study
It was considered interesting to investigate actions of curcumin
analogues on signaling molecule antitelomeric activity. The struc-
tures of the ligands, the most active compound 7h and the least
active one 8d, were energy minimized to an RMSD of 0.01 kcal/mol
using MMFF94X forcefield. They were docked into telomerase
active site and the best and the most energetically favorable
conformation of each compound were selected. Minimization
methods were used for predicting relative binding affinities of the
ligands using energies obtained both in solvent as well as complex
phases of each ligand. Molecular docking of the lowest energy
conformer (Fig. 3) into ATP binding site of telomerase was per-
formed on the binding model based on the telomerase structure
3DU6 pdb. The binding model of 7h into telomerase is depicted in
(Fig. 4), where it is nicely bound to telomerase with one methoxy
group project toward Gln B367, and the methoxy group on the
other side forming a more optimal dual interaction, with Lys B169
and Asn B303 through a side chain acceptor binding which might
make the 2D structure more stable. These residues influenced the
accessibility of the hydrophobic pocket that flanks the active
binding site, and their size could be important in controlling telo-
merase selectivity. Meanwhile, the 2D interaction diagram of the
lowest energy conformer of compound 8d (Fig. 5) showed that the
oxygen atom of both methoxy groups didn't show any interactions
with any amino acid in telomerase enzyme, and it only shows a
single interaction with telomerase through its thiazol-4-one ring
(Fig. 6). Hence, This could support the hypothesis that the most
active antitumor synthesized compound 7h could have its both
in vitro and in vivo antitumor activity through telomerase inhibi-
tion, in contrary to the least active antitumor synthesized deriva-
tive 8d. Our docking studies provide a good explanation with our
preliminary pharmacological experimental data and support
further telomerase binding assay methods.
2.2.2.2. In vivo antitumor study in tumor-bearing mice. To confirm
the in vitro results, an in vivo study was carried out using tumor-
bearing mice. Implantation of EAC cells into mice resulted in a
solid palpable tumor mass that appeared after 5 days from inocu-
lation (day 0). The size of tumor progressively increased with time
and reached about 6-folds its initial mass after additional 21 days
(day 21) that considered 100% tumor growth (i.e. 0% tumor growth
inhibition). Treatment with cisplatin significantly decreased the
relative tumor size compared to the control group, showing 20%
tumor growth (i.e. 80% tumor growth inhibition). The synthesized
compounds that showed the highest activity in the in vitro anti-
tumor assay, 3a, 4a, 6d, 7f, 7h and 11b, were administered to
tumour-bearing mice. Antitumor activity was calculated by the
determination of
and C (change of tumor size in the control). The degree of tumor
growth inhibition can be obtained from T/
DT (change of tumor size in the treatment group)
D
D
D
C ꢂ 100 (Table 3 and
Fig. 2). Compound 7h showed a promising degree of tumor growth
inhibition (82%) being more active than the reference drug cisplatin
Table 2
Effect of synthesized compounds on viability of cultured EAC cells in vitro.
Compound
% Dead of cultured EAC cells
25
m
M
50
m
M
100 mM
Cisplatin
3a
4a
6d
7b
7f
7g
7h
8a
8b
8d
11a
11b
11c
60
75
100
98
98.3
99
93.5
97.9
95.4
28.7
30.4
31.7
25
29.2
27.8
98.3
23.8
22
20.4
21.7
60.2
24.9
57.5
61.6
66.5
47.1
58
50.2
100
46
43.8
40
2.3.2. Flexible alignment
Ligand-based active site alignment is a widely adopted tech-
nique for the structural analysis of proteineligand complexes. An
alignment is good if a) the strain energy of each molecule is small;
b) they have a similar shape; c) their aromatic atoms overlap [22].
Our objective when computing a multiple ligand alignment of a set
of input ligands is to maximize the similarity between these li-
gands, while keeping them in sound conformations. So to probe
similarity between the 3D structures of the lowest energy
100
84
81.6
75.8
81
99.7
84.2
43.4
84
45.1

S.M. Bayomi et al. / European Journal of Medicinal Chemistry 101 (2015) 584e594
589
Fig. 2. Percentage tumor growth inhibition by the tested compounds.
Fig. 3. Lowest energy conformer of most active compound 7h.
Fig. 5. Lowest energy conformer of least active compound 8d.
conformers of the most and least active compounds, 7h and 8d
respectively, flexible alignment was employed. Our initial approach
was to employ MOE/MMFF94 flexible alignment to automatically
generate superposition of the compounds under investigation with
minimal user bias. 200 conformers of each compound were
generated and minimized with a distance-dependant dielectric
model. A low energy set of 100 was selected for further analysis.
The top scoring alignment with the least strain energy is shown in
(Fig. 7) where there is a sharp difference in the alignment profile
between compound 7h, and 8d which is consistent with our
experimental data.
2.3.3. Hydrophopic surface mapping
In a deep research for reasons behind the diminished activity of
compound 8d, in comparison to the most active one 7h, hydrophobic
surface mapping study was conducted for both compounds in which
compound 7h, pointed out hydrophobic regions (Fig. 8a) more than
compound 8d (Fig. 8b). That could be attributed to the cyclic piper-
idine structure core in addition to the chlorophenyl substitution on
the thiazole ring in compound 7h so it could be deduced that the
lipophilicity represents important factor for activity.
Fig. 4. The 2D binding mode and residues involved in the recognition for compound 7h, in the telomerase binding pocket.

590
S.M. Bayomi et al. / European Journal of Medicinal Chemistry 101 (2015) 584e594
Fig. 6. The 2D binding mode and residues involved in the recognition for compound 8d, in the telomerase binding pocket.
further application of telomerase binding assay methods.
4. Experimental protocols
4.1. Chemistry
Melting points (ºC) were recorded using a FishereJohns melting
point apparatus and are uncorrected. Microanalyses were per-
formed in the Microanalytical Unit, Cairo University. IR spectra
(KBr) were recorded on Mattson 5000 FT-IR spectrometer (
n
Fig. 7. Flexible alignment of the most active compound 7h (pink) and the least active
compound 8d (brown). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
in cmꢀ1). 1H NMR spectra were obtained on FT-NMR spectrometer
(200 MHz) Varian Gemini using TMS as internal standard (chemical
shifts in ppm,
d units). MS analyses were performed on JEOL JMS-
600H spectrometer. Thin layer chromatography (TLC) was per-
formed on silica gel G for TLC (Merck) and spots were visualized by
irradiation with ultraviolet light (UV; 254 nm). The syntheses of
compounds 2a [3], 2b [3], 5a [3], 5b [23], 5c [3], 5d [3], 10a [24], 10b
[24], 10c [25] and 10d [4] have been previously reported.
3. Conclusion
In this study, a series of chalcone-type newly synthesized cur-
cumin analogues were subjected to antioxidant and antitumor
screening. The obtained data revealed that compounds 3a, 4a, 6d,
7f, 7g, 7h and 11b exhibited significantly higher antioxidant and
antitumor activities than other derivatives. Among the latter active
compounds, compound 7h showed excellent in vitro and in vivo
antitumor activities higher than that of cisplatin. In addition,
compound 11b possessed equivalent activity to the standard drug.
In silico modeling studies were performed. Docking simulation of
compound 7h into the telomerase 3DU6 active site suggested that
compound 7h could exert its antitumor potential by telomerase
inhibition. Moreover, conformational analyses, flexible alignment,
and hydrophobic mappings of the most active (7h) and the least
active (8d) compounds were applied showing completely different
profiles explaining their sharp difference in activity though having
similar structure. Modeling studies showed a fairly good agreement
with preliminary pharmacological experimental data and support
4.1.1. General method for the synthesis of 3-(substituted
benzylidene)-5-(substituted phenyl)-1-thiocarbamoyl-4,5-
dihydro-1H-pyrazole (3a,b)
A mixture of the appropriate chalcone 2a,b (0.01 mol), thio-
semicarbazide (0.9 g, 0.01 mol) and NaOH (1 g, 0.025 mol) in
ethanol (50 mL) was refluxed for 8 h. The progress of reaction was
monitored by TLC. After the completion of reaction, the reaction
mixture was poured into acidic ice water (~pH 2, adjusted by HCl).
The separated products was filtered, dried and crystallized from
aqueous ethanol.
4.1.1.1. 3-(4-Methoxybenzylidene)-5-(4-methoxyphenyl)-1-
thiocarbamoyl-4,5-dihydro-1H-pyrazole (3a). Yield, 40%; mp
135e137 ꢃC; IR (KBr) nmax in cmꢀ1 3440 and 3280 (NH2), 1579 (C]
Fig. 8. (a) Surface map for the most active compound 7h (element) in pocket side. (b) Surface map for the least active compound 8d (cyan) in pocket side pink: hydrogen bond, blue:
mild polar, green: hydrophobic. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

S.M. Bayomi et al. / European Journal of Medicinal Chemistry 101 (2015) 584e594
591
N), 1334 (C]S). 1H NMR (CDCl3);
d
3.20 (dd, J ¼ 5.2, 17,8 Hz, 1H, 4-H
ylidene CH and NH2, D2O exchangeable). Anal. Calcd for
C22H23N3O2S (%): C, 67.15; H, 5.89; N,10.68. Found: C, 67.55; H, 6.21;
N, 10.95.
of pyrazole), 3.78 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.90 (dd, J ¼ 11.9,
17.8 Hz, 1H, 4-H of pyrazole), 6.10 (dd, J ¼ 11.9, 5.2 Hz, 1H, 5-H of
pyrazole), 6.71 (br s, 2H, NH2), 7.10e8.07 (m, 10H, AreH and CH]
CH). 13C NMR (CDCl3):
d
176.80,159.21, 159.01, 153.22, 135.11, 134.21,
4.1.3.2. (E)-7-(4-Methoxybenzylidene)-3-(4-methoxyphenyl)-
133.21, 132.11, 129.11, 129.00, 129.23, 115.11, 114.81, 70.21, 54.22,
45.11. Anal. Calcd for C20H21N3O2S (%):C, 65.37; H, 5.76; N, 11.44.
Found: C, 65.85; H, 6.00; N, 11.94.
3,3a,4,5,6,7-hexahydro-2H-indazole-2-carbothioamide
(6b).
Yield, 64%; mp 218e219 ꢃC. IR (KBr) nmax in cmꢀ1 3400 and 3389
(NH2), 2190 (C^N), 1672, 1635, 1600 (C]C), and 1291 (CeN).1H
NMR (CDCl3); d 1.30e1.45 (m, 4H, 2CH2), 1.98e2.26 (m, 3H, CH2 and
4.1.1.2. 3-(4,5-Dimethoxybenzylidene)-5-(4,5-dimethoxyphenyl)-1-
thiocarbamoyl-4,5-dihydro-1H-pyrazole (3b). Yield, 50%; mp
121e123 ꢃC; IR (KBr) nmax in cmꢀ1 3463 and 3301 (NH2), 1577 (C]
CH), 3.70 (s, 3H, OCH3); 3.75 (s, 3H, OCH3), 5.32 (d, J ¼ 12.5 Hz, 1H,
NCH), 6.45e6.98 (m, 10H, 8ArH and NH2, D2O exchangeable), 7.29
(s, 1H, ylidene CH). 13C NMR (CDCl3):
d 174.70, 158.22, 150.22,
N), 1336 (C]S).1H NMR (CDCl3);
d
3.34 (dd, J ¼ 5.2, 17.8 Hz, 1H, 4-H
134.21, 133.00, 132.50, 132.21, 128.21, 128.11, 127.55, 116.12, 115.54,
71.11, 53.22, 43.21, 26.55, 25.21, 25.00. Anal. Calcd for C23H25N3O2S
(%): C, 67.79; H, 6.18; N, 10.31. Found: C, 68.10; H, 6.51; N, 10.65.
of pyrazole), 3.80 (s, 6H, 2OCH3), 3.88 (s, 6H, 2OCH3), 4.10 (dd,
J ¼ 11.8,17.2 Hz,1H, 4-H of pyrazole), 6.23 (dd, J ¼ 11.8, 5.2 Hz,1H, 5-
H of pyrazole), 6.89e8.00 (m, 10H, AreH, NH2 and CH]CH). Anal.
Calcd for C22H25N3O4S (%):C, 61.81; H, 5.89; N, 9.83. Found: C, 62.10;
H, 6.10; N, 10.30.
4.1.3.3. (E)-7-(4-Methoxybenzylidene)-3-(4-methoxyphenyl)-5-
methyl-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridine-2-
carbothioamide (6c). Yield, 50%; mp 130e131 ꢃC. 1H NMR (CDCl3);
4.1.2. General method for synthesis of 2-(3-(substituted benzylidene)-
5-(substituted phenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-phenylthiazole
(4a,b)
A mixture of compound 3a,b (0.01 mol), phenacyl bromide
(1.9 g, 0.01 mol) in ethanol (50 mL) was heated at reflux for 2 h.
After cooling, the separated products was filtered, dried and crys-
tallized from ethanol.
d 2.23 (s, 3H, NCH3), 2.30e2.45 (m, 3H, NCH2 and CH), 2.90 (s, 2H,
NCH2), 3.70 (s, 6H, 2OCH3), 5.32 (d, J ¼ 12.0 Hz, 1H, NCH), 6.55e7.18
(m, 10H, 8ArH and NH2, D2O exchangeable), 7.29 (s, 1H, ylidene CH).
Anal. Calcd for C23H26N4O2S (%): C, 65.38; H, 6.20; N, 13.26. Found:
C, 65.90; H, 6.60; N, 13.70.
4.1. 3. 4. (E) -5-Ethyl-7 -(4-m ethoxybenzylidene) -3-(4-
methoxyphenyl)-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridine-
2-carbothioamide (6d). Yield, 50%; mp 187e188 ꢃC. 1H NMR
4.1.2.1. 2-(3-(4-Methoxybenzylidene)-5-(4-methoxyphenyl))-4,5-
dihydro-1H-pyrazol-1-yl)-4-phenylthiazole (4a). Yield, 45%; mp
(CDCl3); d 1.03e1.15 (t, 3H, CH2CH3), 2.40e2.50 (m, 3H, NCH2 and
155e157 ꢃC; 1H NMR (CDCl3);
d
3.0 (dd, J ¼ 4.5, 18.0 Hz, 1H, 4-H of
CH), 2.80 (s, 2H, NCH2), 3.52e3.70 (q, 2H, CH2CH3), 3.80 (s, 6H,
2OCH3), 5.00 (d, J ¼ 12.1 Hz, 1H, NCH), 6.60e7.30 (m, 11H, 8ArH and
NH2, D2O exchangeable, ylidene CH). MS m/z (%); 436, Mþ (19.63);
437, Mþþ1 (20.24); 75 (100.00). Anal. Calcd for C24H28N4O2S (%): C,
66.03; H, 6.46; N, 12.83. Found: C, 66.43; H, 6.90; N, 13.20.
pyrazole), 3.78 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.88 (dd, J ¼ 11.7,
18.0 Hz, 1H, 4-H of pyrazole), 6.0 (dd, J ¼ 11.7, 4.5 Hz, 1H, 5-H of
pyrazole), 6.76 (s, 1H, 5-H of thiazole), 7.10e8.27 (m, 15H, AreH and
CH]CH). 13C NMR (CDCl3):
d 168.10, 159.20, 154.10, 150.20, 135.01,
135.20, 135.11, 130.45, 130.33, 130,25, 130.22, 129.21, 129.11, 128.55,
128.34, 128.00, 127.21, 120.22, 114.22, 114.00, 105.55, 60.22, 55.8,
40.34. Anal. Calcd for C28H25N3O2S (%):C, 71.92; H, 5.39; N, 8.99.
Found: C, 72.20; H, 5.50; N, 9.20.
4.1.4. General method for the synthesis of 2-(pyrazolyl)-4-
(substituted phenyl)thiazole derivatives (7a-h)
A mixture of the appropriate 1-thiocarbamoyl pyrazole deriva-
tive 6aed (0.01 mol) and phenacyl bromide derivative (0.01 mol) in
ethanol (50 mL) was heated at reflux for 6 h. The reaction mixture
was then evaporated, and the residue obtained was washed with
water, collected by filtration, dried, and recrystallized from ethyl
acetate to yield the title compounds.
4.1.2.2. 2-(3-(4-Dimethoxybenzylidene)-5-(4-dimethoxyphenyl))-
4,5-dihydro-1H-pyrazol-1-yl)-4-phenylthiazole (4b). Yield, 60%; mp
145e147 ꢃC; 1H NMR (CDCl3);
d
3.0 (dd, J ¼ 5.2, 18.0 Hz, 1H, 4-H of
pyrazole), 3.70 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.80 (s, 6H, 2OCH3),
3.9 (dd, J ¼ 12.0, 18.0 Hz, 1H, 4-H of pyrazole), 6.20 (dd, J ¼ 12.0,
5.4 Hz, 1H, 5-H of pyrazole), 6.80e8.27 (m, 14H, AreH, 5-H of
thiazole and CH]CH). MS m/z (%); 527.00, Mþ (9.14); 55 (100.00).
Anal. Calcd for C30H29N3O4S (%):C, 68.29; H, 5.54; N, 7.96. Found: C,
68.54; H, 5.90; N, 8,25.
4.1.4.1. (E)-2-(6-(4-Methoxybenzylidene)-3-(4-methoxyphenyl)-
3,3a,4,5,6-pentahydrocyclopenta[c]pyrazol-2-yl)-4-phenylthiazole
(7a). Yield, 75%; mp 179e180 ꢃC. 1H NMR (CDCl3);
d 1.29 (m, 2H,
CH2), 2.00e2.21 (m, 3H, CH2 and CH), 3.89 (s, 3H, OCH3); 3.80 (s, 3H,
OCH3), 5.55 (d, J ¼ 11.5 Hz, 1H, NCH), 6.65e7.79 (m, 15H, 13ArH,
ylidene CH and 5-H of thiazole). Anal. Calcd for C30H27N3O2S (%): C,
73.00; H, 5.51; N, 8.51. Found: C, 73.50; H, 5.90; N, 9.00.
4.1.3. General method for the synthesis of (E)-7(6)-(4-
methoxybenzylidene)-3-(4-methoxyphenyl)-2-
carbothioamide (6aed)
A mixture of the appropriate chalcone 5aed (0.01 mol), thio-
semicarbazide (0.9 g, 0.01 mol) and NaOH (1 g, 0.025 mol) in
ethanol (50 mL) was refluxed for 10 h. The progress of reaction was
monitored by TLC. After the completion of reaction, the reaction
mixture was poured into acidic ice water (~pH 2, adjusted by HCl).
The separated product was filtered, dried and crystallized from
ethyl acetate.
4.1.4.2. (E)-4-(4-Chlorophenyl)-2-(6-(4-methoxybenzylidene)-3-(4-
methoxyphenyl)-3,3a,4,5,6-pentahydrocyclopenta[c]pyrazol-2-yl)
thiazole (7b). Yield, 80%; mp 150e151 ꢃC. 1H NMR (CDCl3);
d 1.30
(m, 2H, CH2), 2.10e2.21 (m, 3H, CH2 and CH), 3.80 (s, 6H, 2OCH3),
5.60 (d, J ¼ 11.3 Hz, 1H, NCH), 6.64e7.80 (m, 14H, 12ArH, ylidene CH
and 5-H of thiazole). Anal. Calcd for C30H26ClN3O2S (%): C, 68.23; H,
4.96; N, 7.96. Found: C, 68.55; H, 5.20; N, 8.50.
4.1.3.1. (E)-6-(4-Methoxybenzylidene)-3-(4-methoxyphenyl)-
3,3a,4,5,6-pentahydro-2H-cyclopenta[c]pyrazole-2-carbothioamide
4.1.4.3. (E)-2-(7-(4-Methoxybenzylidene)-3-(4-methoxyphenyl)-
3,3a,4,5,6,7-hexahydro-2H-indazol-2-yl)-4-phenylthiazole
(7c).
(6a). Yield, 55%; mp 135e136 ꢃC. 1H NMR (CDCl3);
d
1.30 (m, 2H,
Yield, 75%; mp 179e180 ꢃC. 1H NMR (CDCl3);
d
1.32e1.46 (m, 4H,
CH2), 1.99e2.27 (m, 3H, CH2 and CH), 3.80 (s, 3H, OCH3); 3.82 (s, 3H,
OCH3), 5.52 (d, J ¼ 12.0 Hz, 1H, NCH), 6.55e7.20 (m, 11H, 8ArH,
2CH2), 1.88e2.19 (m, 3H, CH2 and CH), 3.75 (s, 3H, OCH3); 3.79 (s,
3H, OCH3), 5.22 (d, J ¼ 11.0 Hz, 1H, NCH), 6.65e6.78 (m, 15H, 13ArH,

592
S.M. Bayomi et al. / European Journal of Medicinal Chemistry 101 (2015) 584e594
ylidene CH and 5-H of thiazole). 13C NMR (CDCl3):
155.10, 149.50, 136.00, 135.21, 135.01, 129.45, 129.25, 128.25, 128.22,
127.52, 127.11, 126.55, 126.22, 125.20, 125.00, 120.00, 113.22, 112.10,
103.55, 62.32, 51.8, 40.00, 26.21, 24.50, 24.00. Anal. Calcd for
d
169.20, 158.20,
2H-indazol-2-yl)thiazol-4(5H)-one (8a). Yield, 65%; mp 167e168 ꢃC.
IR (KBr) nmax in cmꢀ1 1696 (C]O), 2190 (C]N).1H NMR (CDCl3);
d
1.33e1.46 (m, 4H, 2CH2), 1.78e2.25 (m, 3H, CH2 and CH), 3.69 (s,
3H, OCH3); 3.74 (s, 3H, OCH3), 5.22 (d, J ¼ 12.0 Hz, 1H, NCH),
C
31H29N3O2S (%): C, 73.34; H, 5.76; N, 8.28. Found: C, 73.40; H, 5.96;
6.43e6.99 (m, 14H, 12ArH and 2 ylidene CH). 13C NMR (CDCl3):
N, 8.60.
d 165.11,160.21,158.80,158.10,153.11,135.02,134.50,134.23,134.00,
133.12, 133.00, 130.21, 129.51, 129.41, 127.11, 127.01, 126.20, 126.11,
114.50, 114.20, 114.12, 72.11, 55.8, 40.12, 27.51, 24.23, 24.21. Anal.
Calcd for C32H28ClN3O3S (%): C, 67.42; H, 4.95; N, 7.37. Found: C,
67.92; H, 5.21; N, 7.00.
4.1.4.4. (E)-4-(4-Chlorophenyl)-2-(7-(4-methoxybenzylidene)-3-(4-
methoxyphenyl)-3,3a,4,5,6,7-hexahydro-2H-indazol-2-yl)thiazole
(7d). Yield, 83%; mp 169e170 ꢃC. 1H NMR (CDCl3);
d 1.42e1.46 (m,
4H, 2CH2), 1.78e2.00 (m, 3H, CH2 and CH), 3.8 (s, 6H, 2OCH3), 5.00
(d, J ¼ 12.0 Hz, 1H, NCH), 6.61e6.77 (m, 14H, 12ArH, ylidene CH and
5-H of thiazole). Anal. Calcd for C31H28ClN3O2S (%): C, 68.68; H,
5.21; N, 7.75. Found: C, 69.00; H, 5.60; N, 7.85.
4 .1. 5 . 2 . ( Z ) - 5 - ( 4 - B r o m o b e n z y l i d e n e ) - 2 - ( ( E ) - 7 - ( 4 -
methoxybenzylidene)-3-(4-methoxyphenyl)-3,3a,4,5,6,7-hexahydro-
2H-indazol-2-yl)thiazol-4(5H)-one (8b). Yield, 60%; mp 230e
231 ꢃC. 1H NMR (CDCl3);
d 1.32e1.46 (m, 4H, 2CH2), 1.76e2.26 (m,
4.1.4.5. (E)-2-(7-(4-Methoxybenzylidene)-3-(4-methoxyphenyl)-5-
methyl-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridin-2-yl)-4-
phenylthiazole (7e). Yield, 78%; mp 179e180 ꢃC. 1H NMR (CDCl3);
3H, CH2 and CH), 3.90 (s, 6H, 2OCH3), 6.00 (d, J ¼ 12.0 Hz, 1H, NCH),
6.55e7.12 (m, 14H, 12ArH and 2 ylidene CH). Anal. Calcd for
C32H28BrN3O3S (%): C, 62.54; H, 4.59; N, 6.84. Found: C, 63.04; H,
d
2.13 (s, 3H, NCH3), 2.31e2.44 (m, 3H, NCH2 and CH), 3.00 (s, 2H,
5.11; N, 6.34.
NCH2), 3.80 (s, 6H, 2OCH3), 5.12 (d, J ¼ 11.5 Hz, 1H, NCH), 6.55e7.58
(m, 15H, 13ArH, ylidene CH and 5-H of thiazole). MS m/z (%); 522,
Mþ (25.37), 85, (100.00). Anal. Calcd for C31H30N4O2S (%): C, 71.24;
H, 5.79; N, 10.72. Found: C, 71.44; H, 6.11; N, 11.00.
4 .1. 5 . 3 . ( Z ) - 5 - ( 4 - M e t h o x y b e n z yl i d e n e ) - 2 - ( ( E ) - 7 - ( 4 -
methoxybenzylidene)-3-(4-methoxyphenyl)-3,3a,4,5,6,7-hexahydro-
2H-indazol-2-yl)thiazol-4(5H)-one (8c). Yield, 70%; mp 199e
200 ꢃC. 1H NMR (CDCl3);
d 1.30e1.45 (m, 4H, 2CH2), 1.77e2.27 (m,
4.1.4.6. (E)-4-(4-Chlorophenyl)-2-(7-(4-methoxybenzylidene)-3-(4-
methoxyphenyl)-5-methyl-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3-
c]pyridin-2-yl)thiazole (7f). Yield, 80%; mp 170e171 ꢃC. 1H NMR
3H, CH2 and CH), 3.90 (s, 6H, 2OCH3), 3.96 (s, 3H, OCH3), 5.90 (d,
J ¼ 12.2 Hz, 1H, NCH), 6.77e7.40 (m, 14H, 12ArH and 2 ylidene CH).
Anal. Calcd for C33H31N3O4S (%): C, 70.07; H, 5.52; N, 7.43. Found: C,
70.37; H, 5.42; N, 7.03.
(CDCl3); d 2.33 (s, 3H, NCH3), 2.36e2.45 (m, 3H, NCH2 and CH), 3.10
(s, 2H, NCH2), 3.88 (s, 6H, 2OCH3), 5.14 (d, J ¼ 11.9 Hz, 1H, NCH),
6.45e7.88 (m, 14H, 12ArH, ylidene CH and 5-H of thiazole). Anal.
Calcd for C31H29ClN4O2S (%): C, 66.83; H, 5.25; N, 10.06. Found: C,
67.05; H, 5.45; N, 10.12.
4.1.5.4. (Z)-5-(3,4-Dimethoxybenzylidene)-2-((E)-7-(4-
methoxybenzylidene)-3-(4-methoxyphenyl)-3,3a,4,5,6,7-hexahydro-
2H-indazol-2-yl)thiazol-4(5H)-one (8d). Yield, 65%; mp 255e
256 ꢃC. 1H NMR (CDCl3);
d 1.22e1.37 (m, 4H, 2CH2), 1.79e2.27 (m,
4.1.4.7. (E)-2-(5-Ethyl-7-(4-methoxybenzylidene)-3-(4-
methoxyphenyl)-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridin-
2-yl)-4-phenylthiazole (7g). Yield, 80%; mp 220e221 ꢃC. 1H NMR
3H, CH2 and CH), 3.91 (s, 6H, 2OCH3), 3.97 (s, 6H, 2OCH3), 6.00 (d,
J ¼ 12.5 Hz, 1H, NCH), 6.87e7.50 (m, 13H, 11ArH and 2 ylidene CH).
MS m/z (%); 595.50, Mþ (20.10); 596.12, Mþþ1 (20.00); 151
(100.00).Anal. Calcd for C34H33N3O5S (%): C, 68.55; H, 5.58; N, 7.05.
Found: C, 68.11; H, 5.28; N, 7.55.
(CDCl3);
d 1.02e1.16 (t, 3H, CH2CH3), 2.30e2.55 (m, 3H, NCH2 and
CH), 2.90 (s, 2H, NCH2), 3.50e3.80 (q, 2H, CH2CH3), 3.80 (s, 6H,
2OCH3), 5.22 (d, J ¼ 12.0 Hz, 1H, NCH), 6.65e7.90 (m, 15H, 13ArH,
ylidene CH and 5-H of thiazole). MS m/z (%); 536, Mþ (23.17);
537.12, Mþþ1 (30.00); 75 (100.00). Anal. Calcd for C32H32N4O2S (%):
C, 71.61; H, 6.01; N, 10.44. Found: C, 71.78; H, 6.50; N, 10.89.
4.1.6. General method for the synthesis of 2-amino-8-(substituted
benzylidene)-4-(substituted phenyl)-5,6,7,8-tetrahydroquinoline-3-
carbonitriles (11aed)
A mixture of the appropriate 2,6-bis(substituted benzylidine)
cyclohexanone 10aed (0.001 mol), malononitrile (0.07 g,
0.001 mol), and ammonium acetate (1.542 g, 0.02 mol) in n-butanol
(10 mL) was heated under reflux for 10 h. The reaction mixture was
concentrated. The separated crystalline solid was collected by
filtration, air dried, and recrystallized from ethanol.
4 . 1. 4 . 8 . ( E ) - 4 - ( 4 - C h l o r o p h e n y l ) - 2 - ( 5 - e t h y l - 7 - ( 4 -
methoxybenzylidene)-3-(4-methoxyphenyl)-3,3a,4,5,6,7-hexahydro-
2H-pyrazolo[4,3-c]pyridin-2-yl)thiazole (7h). Yield, 80%; mp
212e213 ꢃC. 1H NMR (CDCl3);
d 1.12e1.17 (t, 3H, CH2CH3), 2.40e2.60
(m, 3H, NCH2 and CH), 3.00 (s, 2H, NCH2), 3.40e3.70 (q, 2H,
CH2CH3), 3.89 (s, 6H, 2OCH3), 6.00 (d, J ¼ 11.0 Hz, 1H, NCH),
6.66e7.80 (m, 14H, 12ArH, ylidene CH and 5-H of thiazole). Anal.
Calcd for C32H31ClN4O2S (%): C, 67.29; H, 5.47; N, 9.81. Found: C,
67.70; H, 5.40; N, 10.05.
4.1. 6.1. (E)-2-Amino-8-(4-hydroxybenzylidene)-4-(4-
hydroxyphenyl)-5,6,7,8-tetrahydroquinoline-3-carbonitrile
Yield, 50%; mp 250e252 ꢃC. MS m/z (%); 369 (Mþ, 12.79), 177
(100.00). 13C NMR (CDCl3):
161.22, 160.10, 159.23, 157.01, 151.23,
(11a).
d
4.1.5. (Z)-5-(4-Substitutedbenzylidene)-2-((E)-7-(4-
140.11, 131.10, 130.91, 130.21, 129.51, 129.30, 129.01, 123.20, 115.50,
115.11, 114.50, 113.40, 90.12, 35.00, 23.50, 24.30. Anal. Calcd for
methoxybenzylidene)-3-(4-methoxyphenyl)-3,3a,4,5,6,7-
hexahydro-2H-indazol-2-yl)thiazol-4(5H)-ones (8aed)
A mixture of the appropriate1-thiocarbamoyl pyrazole deriva-
tive 6aed (0.01 mol), chloroacetic acid (0.94 gm, 0.01 mol), aro-
matic aldehyde (0.01 mol) in ethanol (50 mL) was heated at reflux
for 16 h. The separated solid was collected by filtration, washed
with water, dried and recrystallized from acetonitrile to yield the
title compounds.
C23H19N3O2 (%): C, 74.78; H, 5.18; N, 11.37. Found: C, 75.00; H, 5.48;
N, 11.47.
4.1.6.2. (E)-2-Amino-8-(4-hydroxy-3-methoxybenzylidene)-4-(4-
hydroxy-3-methoxyphenyl)-5,6,7,8-tetrahydroquinoline-3-
carbonitrile (11b). Yield, 50%; mp 247e249 ꢃC. IR (KBr) nmax in cmꢀ1
3416 and 3333 (NH2), 2202 (C^N), 1256 (CeN).1H NMR (CDCl3);
d
1.52e2.96 (m, 6H, 3CH2), 3.79 (s, 6H, 2OCH3), 6.45e6.98 (m, 8H,
4 .1. 5 .1. ( Z ) - 5 - ( 4 - C h l o r o b e n z y l i d e n e ) - 2 - ( ( E ) - 7 - ( 4 -
methoxybenzylidene)-3-(4-methoxyphenyl)-3,3a,4,5,6,7-hexahydro-
6ArH and NH2, D2O exchangeable), 7.89 (s, 1H, benzylidene-H), 9.36
(br s, 2H, 2OH, D2O exchangeable). MS m/z (%); 429, Mþ (100.00).

S.M. Bayomi et al. / European Journal of Medicinal Chemistry 101 (2015) 584e594
593
Anal. Calcd for C25H23N3O4 (%): C, 69.92; H, 5.40; N, 9.78. Found: C,
69.40; H, 5.90; N, 10.11.
washed by phosphate buffered saline and resuspended in a drug-
free medium. The percent survival of EAC cells was determined
by trypan blue dye exclusion method in which the dye stained the
dead cells only [29]. Cytotoxicity was determined 3 times and the
mean was recorded. Control experiments in which EAC cells were
incubated in a drug-free medium were also conducted. Percent
survival of cells ¼ (T/C) X 100 was calculated where T and C
represent the number of viable cells in a unit volume of the test
drug tube and the control tube, respectively.
4.1.6.3. (E)-2-Amino-8-(3,4-dimethoxybenzylidene)-4-(3,4-
dimethoxyphenyl)-5,6,7,8-tetrahydroquinoline-3-carbonitrile (11c).
Yield, 50%; mp 175e177 ꢃC. IR (KBr) nmax in cmꢀ1 3438 and 3348
(NH2), 2205 (C^N), 1253 (CeN).1H NMR (CDCl3);
d 1.59e2.78 (m,
6H, 3CH2), 3.78 (s, 12H, 4OCH3), 6.49 (s, 2H, NH2, D2O exchange-
able), 6.84e7.07 (m, 6H, 6ArH), 7.91 (s, 1H, benzylidene-H). MS m/z
(%); 457, Mþ (0.83), 45 (100.00). Anal. Calcd for C27H27N3O4 (%):C,
70.88; H, 5.95; N, 9.18. Found: 70.44; H, 5.22; N, 9.00.
4.2.2.2. In vivo experiment. Ascitic fluid was withdrawn under
aseptic conditions from tumor-bearing mice by needle aspiration
from the peritoneal cavity, 7e8 days after EAC cells inoculation, and
washed three times with normal saline by centrifugation at 67 ꢂ g.
EAC cells that obtained after washing were tested for viability using
trypan blue. The cells were examined microscopically using a
haemocytometer, suspended in normal saline so that each 0.1 ml
contained 5 ꢂ 105 viable EAC cells. The cells were counted under
the microscope. Solid tumors were induced in mice by S.C. inocu-
lation of 0.1 ml containing 5 ꢂ 105 viable tumor cells on the left
flank anterior to the hind leg. [30] Tumor growth was determined
by caliper measurement of the largest diameter and its
perpendicular.
4.1.6.4. (E)-2-Amino-8-(2,5-dimethoxybenzylidene)-4-(2,5-
dimethoxyphenyl)-5,6,7,8-tetrahydroquinoline-3-carbonitrile (11d).
Yield, 50%; mp 48e50 ꢃC. IR (KBr) nmax in cmꢀ1 3438 and 3357
(NH2), 2209 (C^N), 1223 (CeN). 1H NMR (CDCl3);
d 1.51e2.97 (m,
6H, 3CH2), 3.66 (s, 12H, 4OCH3), 5.90 (s, 2H, NH2, D2O exchange-
able), 6.49e7.08 (m, 6H, 6ArH), 7.85 (s, 1H, benzylidene-H). Anal.
Calcd for C27H27N3O4 (%):C, 70.88; H, 5.95; N, 9.18. Found: C, 71.00;
H, 6.00; N, 8.89.
4.2. Biological testing
4.2.1. ABTS test
ꢀ
ꢁ
Tumor size mm3 ¼ 0:5Xa Xb2:
A mixture of solution of ABTS (2 ml, 0.1 g/100 ml, Sigma-
eAldrich, St. Louis, Mo, USA) and solution of MnO2 (3 ml, 25 mg/
ml), prepared in phosphate buffer (pH 7, 0.1 M, BP 1998) was
shaken, centrifuged, and decanted. The absorbance (Acontrol) of
the resulting green-blue solution (ABTS þ radical solution) was
recorded at lmax 734 nm. The absorbance (Atest) was measured
where a is the largest diameter and b is its perpendicular.
When the primary tumor reached a size of 50e100 mm3, mice
were grouped into 14 groups (10 mice each). Group (1) received
normal saline (EAC-bearing control, 5 ml/kg). Group (2) received
cisplatin (2 mg/kg from 0.1% solution) I.P. Group (3e14) received
tested compounds in a dose of 75 mg/kg orally suspended in CMC.
Treatment continued for 21 consecutive days. Tumor size at day
0 (five days after tumor inoculation) and after treatment (day 21)
was measured. Antitumor activity was calculated by the determi-
upon the addition of 20 ml of 1 mg/ml solution of the test sample in
spectroscopic grade MeOH/buffer (1:1 v/v) to the ABTS solution.
The decrease in absorbance is expressed as % inhibition which is
calculated from the equation [26,27].
Acontrol ꢀ Atest
% inhibition ¼
ꢂ 100
nation of
(change of tumor size in the control). The degree of tumor growth
inhibition can be obtained from T/
C ꢂ 100 [31].
DT (change of tumor size in the treatment group) and DC
Acontrol
D
D
Ascorbic acid 20 ml (2 mM) solution, and curcumin 20 ml (1 mg/
ml) solution were used as standard antioxidants (positive controls).
Blank sample was run using solvent without ABTS.
4.3. Molecular modeling methodology
4.2.2. Antitumor testing
4.3.1. Docking study
EAC cells were established in the Netherlands Cancer Institute.
The Ehrlich tumor line was maintained in the laboratory of Faculty
of Pharmacy, Mansoura University in female Swiss albino mice by
serial intraperitoneal passage at 7e10 day intervals.
The three-dimensional structures of compounds 7h and 8d
which presented best and worst biological profiles, respectively,
were constructed using ‘Molecular Operating Environment’ soft-
ware (MOE of Chemical Computing Group Inc., 2009.10 on a Core 2
duo 2.3 GHz workstation). Lowest energy conformer of each new
analogue ‘global-minima’ was docked into the telomerase enzyme-
binding domain. Starting coordinate of telomerase enzyme, code ID
3DU6, was obtained from the Protein Data Bank of Brookhaven
National Laboratory [32]. All the hydrogens were added and
enzyme structure was subjected to a refinement protocol in which
the constraints on the enzyme were gradually removed and mini-
mized until the RMS gradient was 0.01 kcal/mol A. The energy
minimization was carried out using the molecular mechanics force
field ‘AMBER.’ The energy-minimized structure was used for mo-
lecular modeling studies keeping all the heavy atoms fixed until a
RMSD gradient of 0.05 kcal mol 1 Å1 was reached. Ligand structures
were built with MOE and minimized using the MMFF94x forcefield
until a RMSD gradient of 0.05 kcal mol 1Å 1 was reached. For both
compounds 7h and 8d, energy minimizations (EM) were per-
formed using 1000 steps of steepest descent, followed by conjugate
gradient minimization to a RMS energy gradient of 0.01 kcal/mol Å
[33,34]. The docking was performed using the Alpha Triangle
4.2.2.1. In vitro cytotoxic assay. Ascitic fluid from the intraperito-
neal cavity of the donor animal was aseptically aspirated, 7e8 days
after EAC cells inoculation, and washed three times with N-2-
hydroxyethyl-piperazine-N-2-ethanesulfonic acid buffered Hanks’
balanced salt solution. EAC cells were counted under the micro-
scope using a haemocytometer and resuspended in normal saline
so that each 0.1 ml contained 2 ꢂ 105 cells [28]. EAC cells were
incubated in RPMI 1640 medium supplemented with 10% fetal calf
serum for 24 h in cell culture tubes in a humidified atmosphere
containing 5% CO2 at 37 ꢃC. Each tube contained 0.1 ml cells and
0.9 ml medium (final concentration of the cells ¼ 2 ꢂ 105 cells/ml).
After 24 h incubation, the tubes were centrifuged at 67 ꢂ g and cells
were separated. EAC cells were resuspended in RPMI 1640 medium
and drugs were added so that the content of each tube was 0.8 ml
medium, 0.1 ml cells and 0.1 ml drug. The final concentrations of
tested compounds and cisplatin were 25, 50, and 100
mM. After
incubation of cells with drugs for 24 h, cells were separated,

594
S.M. Bayomi et al. / European Journal of Medicinal Chemistry 101 (2015) 584e594
76e86.
placement method and the London dG scoring method. 300 results
for each ligand were generated, discarding the results with a RMSD
value> 3 Å 0.19 The best scored result of the remaining confor-
mations for each ligand was further analyzed. The protein/ligand
complexes were minimized using the MMFF94x force field, until an
RMSD gradient o 0.1 kcal mol 1 Å 1 was reached.
[13] Z. Xiao, A. Zhang, J. Lin, Z. Zheng, X. Shi, W. Di, W. Qi, Y. Zhu, G. Zhou, Y. Fang,
Telomerase: a target for therapeutic effects of curcumin and a curcumin de-
rivative in Ab1-42 insult in vitro, PLoS One 9 (2014) e101251.
[14] M. Mollazade, K. Nejati-Koshki, A. Akbarzadeh, N. Zarghami, M. Nasiri,
R. Jahanban-Esfahlan, A. Alibakhshi, PAMAM dendrimers augment inhibitory
effects of curcumin on cancer cell proliferation: possible inhibition of telo-
merase, Asian Pac. J. Cancer Prev. 14 (2013) 6925e6928.
[15] M. Nasiri, N. Zarghami, K.N. Koshki, M. Mollazadeh, M.P. Moghaddam,
M.R. Yamchi, R.J. Esfahlan, A. Barkhordari, A. Alibakhshi, Curcumin and sili-
binin inhibit telomerase expression in T47D human breast cancer cells, Asian
Pac. J. Cancer Prev. 14 (2013) 3449e3453.
[16] F. Kazemi-Lomedasht, A. Rami, N. Zarghami, Comparison of inhibitory effect of
curcumin nanoparticles and free curcumin in human telomerase reverse
transcriptase gene expression in breast cancer, Adv. Pharm. Bull. 3 (2013)
127e130.
[17] A.K. Khaw, M.P. Hande, G. Kalthur, Curcumin inhibits telomerase and induces
telomere shortening and apoptosis in brain tumour cells, J. Cell. Biochem. 114
(2013) 1257e1270.
[18] I.L. Hsin, G.T. Sheu, H.H. Chen, L.Y. Chiu, H.D. Wang, H.W. Chan, C.P. Hsu,
J.L. Ko, N-acetyl cysteine mitigates curcumin-mediated telomerase inhibition
through rescuing of Sp1 reduction in A549 cells, Mutat. Res. 688 (2010)
72e77.
4.3.2. Flexible alignment
The investigated compounds were subjected to flexible align-
ment and superposition experiments using the MOE of Chemical
Computing Group Inc. software, 2009.10 on Core 2 duo 2.3 GHz
workstation. The molecules were built using the Builder module of
MOE. Lowest energy aligned conformation(s) were identified. Their
geometry was optimized by using the MMFF94 forcefield followed
by a flexible alignment using systematic conformational search
[35].
Acknowledgment
[19] M. Gupta, U.K. Mazumder, R.S. Kumar, T. Sivakumar, M.L. Vamsi, Antitumor
activity and antioxidant status of Caesalpinia bonducella against Ehrlich ascites
carcinoma in Swiss albino mice, J. Pharmacol. Sci. 94 (2004) 177e184.
[20] M.E. Zumrutdal, M. Ozaslan, M. Tuzcu, M.E. Kalender, K. Daglioglu, A. Akova,
I.D. Karagoz, H. Kilic, O. Colak, F. Koksal, Effect of Lawsonia inermis treatment
on mice with sarcoma, Afr. J. Biotechnol. 7 (2008) 2781e2786.
[21] N. Bromberg, J.L. Dreyfuss, C.V. Regatieri, M.V. Palladino, N. Duran, H.B. Nader,
M. Haun, G.Z. Justo, Growth inhibition and pro-apoptotic activity of violacein
in Ehrlich ascites tumor, Chem. Biol. Interact. 186 (2010) 43e52.
[22] P. Labute, C. Williams, M. Feher, E. Sourial, J.M. Schmidt, Flexible alignment of
small molecules, J. Med. Chem. 44 (2001) 1483e1490.
The authors extend their appreciation to Mansoura University
research center, Egypt for funding this work.
References
[1] A. Joseph, C.S. Shah, S.S. Kumar, A.T. Alex, N. Maliyakkal, S. Moorkoth,
J.E. Mathew, Synthesis, in vitro anticancer and antioxidant activity of thia-
diazole substituted thiazolidin-4-ones, Acta. Pharm. 63 (2013) 397e408.
[2] A.T. Dinkova-Kostova, P. Talalay, Direct and indirect antioxidant properties of
inducers of cytoprotective proteins, Mol. Nutr. Food Res. 52 (2008)
S128eS138.
[3] K.M. Youssef, M.A. El-Sherbeny, F.S. El-Shafie, H.A. Farag, O.A. Al-Deeb,
S.A. Awadalla, Synthesis of curcumin analogues as potential antioxidant,
cancer chemopreventive agents, Arch. Pharm. Weinh. 337 (2004) 42e54.
[4] S.M. Bayomi, H.A. El-Kashef, M.B. El-Ashmawy, M.N.A. Nasr, M.A. El-Sherbeny,
F.A. Badria, L.A. Abou-zeid, M.A. Ghaly, N.I. Abdel-Aziz, Synthesis and biolog-
ical evaluation of new curcumin derivatives as antioxidant and antitumor
agents, Med. Chem. Res. 22 (2013) 1147e1162.
[5] N. Watanabe, Down regulation of telomerase activity may enhanced by
nanoparticle mediated curcumin delivery, Hokkaido Igaku Zasshi 76 (2001)
127e133.
[6] P. Gandellini, M. Folini, R. Bandiera, M. De Cesare, M. Binda, S. Veronese,
M.G. Daidone, F. Zunino, N. Zaffaroni, Down-regulation of human telomerase
reverse transcriptase through specific activation of RNAi pathway quickly
results in cancer cell growth impairment, Biochem. Pharmacol. 73 (2007)
1703e1714.
[7] L. Zou, P. Zhang, C. Luo, Z. Tu, shRNA-targeted hTERT suppress cell prolifera-
tion of bladder cancer by inhibiting telomerase activity, Cancer Chemother.
Pharmacol. 57 (2006) 328e334.
[8] M. Folini, C. Brambilla, R. Villa, P. Gandellini, S. Vignati, F. Paduano,
M.G. Daidone, N. Zaffaroni, Antisense oligonucleotide-mediated inhibition of
hTERT, but not hTERC, induces rapid cell growth decline and apoptosis in the
absence of telomere shortening in human prostate cancer cells, Eur. J. Cancer
41 (2005) 624e634.
[9] X.D. Gao, Y.R. Chen, Inhibition of telomerase with human telomerase reverse
transcriptase antisense enhances tumor necrosis factor-alpha-induced
apoptosis in bladder cancer cells, Chin. Med. J. 120 (2007) 755e760.
[10] C. Ramachandran, H.B. Fonseca, P. Jhabvala, E.A. Escalon, S.J. Melnick, Curcu-
min inhibits telomerase activity through human telomerase reverse tran-
scritpase in MCF-7 breast cancer cell line, Cancer Lett. 184 (2002) 1e6.
[11] N.C.S. Mukherjee, U. Ghosh, N.P. Bhattacharyya, R.K. Bhattacharya, S. Dey,
M. Roy, Curcumin-induced apoptosis in human leukemia cell HL-60 is asso-
ciated with inhibition of telomerase activity, Mol. Cell. Biochem. 297 (2007)
31e39.
[23] L. Wang, J. Sheng, H. Tian, J. Han, Z. Fan, C. Qian, A convenient synthesis of a, -
a0
bis(substituted benzylidene)cycloalkanones catalyzed by Yb(OTf)3 under
solvent-free conditions, Synthesis 18 (2004) 3060e3064.
[24] Z. Du, R. Liu, W. Shao, X. Mao, L. Ma, L. Gu, Z. Huang, A.S.C. Chan, a-Glucosidase
inhibition of natural curcuminoids and curcumin analogs, Eur. J. Med. Chem.
41 (2006) 213e218.
[25] N. Singh, J. Pandey, A. Yadav, V. Chaturvedi, S. Bhatnagar, A.N. Gaikwad,
S.K. Sinha, A. Kumar, P.K. Shukla, R.P. Tripathi, A facile synthesis of a,a
0-(EE)-
bis(benzylidene)-cycloalkanones and their antitubercular evaluations, Eur. J.
Med. Chem. 44 (2009) 1705e1709.
[26] C.F. Hsu, H. Peng, C. Basle, J. Travas-Sejdic, P.A. Kilmartin, ABTSþ scavenging
activity of polypyrrole, polyaniline and poly(3,4-ethylenedioxythiophene),
Polym. Int. 60 (2011) 69e77.
[27] E.A. Lissi, B. Modak, R. Torres, J. Escobar, A. Urzua, Total antioxidant potential
of resinous exudates from Heliotropium species, and a comparison of ABTS and
DPPH methods, Free Radi. Res. 30 (1999) 471e477.
[28] O.A. Badary, S.A. Sharaby, S.A. Kenawy, E.E. El-Denshary, F.M. Hamada, Eval-
uation of cisplatin combined with ondansetron in Ehrlich ascites carcinoma
in vitro and in vivo, Tumori 86 (2000) 153e156.
[29] L.M. Weisenthal, P.L. Dill, N.B. Kurnick, M.E. Lippman, Comparison of dye
exclusion assays with
a clonogenic assay in the determination of drug-
induced cytotoxicity, Cancer Res. 43 (1983) 258e264.
[30] R.R.A. Raja, N. Ndupa, D.P. Uma, Effect of macrophage activation on niosome
encapsulated bleomycin in tumor-bearing mice, Ind. J. Pharmacol. 28 (1996)
175e180.
[31] M. Schirner, J. Hoffmann, A. Menrad, M.R. Schneider, Antiangiogenic chemo-
therapeutic agents: characterization in comparison to their tumor growth
inhibition in human renal cell carcinoma models, Clin. Cancer Res. 4 (1998)
1331e1336.
[32] A.J. Gillis, A.P. Schuller, E. Skordalakes, Structure of the Tribolium castaneum
telomerase catalytic subunit TERT, Nature 455 (2008) 633e637.
[33] S. Profeta, N.L. Allinger, Molecular mechanics calculations on aliphatic amines,
J. Am. Chem. Soc. 107 (1985) 1907e1918.
[34] N.L. Allinger, Conformational analysis. 130. MM2. A hydrocarbon force field
utilizing V1 and V2 torsional terms, J. Am. Chem. Soc. 99 (1977) 8127e8134.
[35] S. Kearsley, G.M. Smith, An alternative method for the alignment of molecular
structures: maximizing electrostatic and steric overlap, Tetrahedron Comput.
Methodol. 3 (1990) 615e633.
[12] J.H. Lee, I.K. Chung, Curcumin inhibits nuclear localization of telomerase by
dissociating the Hsp90 co-chaperone p23 from hTERT, Cancer Lett. 290 (2010)