Synthesis, biological evaluation, drug-likeness, and in silico screening of novel benzylidene-hydrazone analogues as small molecule anticancer agents

Article abstract of DOI:10.1007/s12272-015-0699-z

A series of fifteen benzylidene-hydrazone analogues (3a-o), including eight new compounds, were synthesized and evaluated for their cytotoxic activities in four human cancer cell lines and for their antioxidant activities using DPPH. Of the tested compounds 3e, which possesses two methoxy substituents in its benzylidene phenyl ring, was found to be potently cytotoxic to all cancer cell lines tested with IC₅₀ values of 0.12 (lung), 0.024 (ovarian), 0.097 (melanoma), and 0.05 μM (colon), and these IC₅₀ values were comparable to those of the doxorubicin standard (IC₅₀ = 0.021, 0.074, 0.001, and 0.872 μM, respectively). DPPH assay showed compounds 3f, 3i, and 3g had IC₅₀ values of 0.60, 0.99, and 1.30 μM, respectively, which were comparable to that of ascorbic acid (IC₅₀ = 0.87 μM). Computational parameters such as, drug-likeness, ADME properties, toxicity effects, and drug scores were evaluated, and none of the fifteen compounds violated Lipinski's rule of five or Veber's rule, and thus they demonstrated good drug-likeness properties. In addition, all fifteen compounds had a higher drug score than the doxorubicin and BIBR1532. In silico screening was also conducted by docking of the active compounds on the active site of telomerase reverse transcriptase catalytic subunit, an important therapeutic target of anticancer agents, to determine the probable binding properties. The total binding energies of docked compounds are correlated well with cytotoxic potencies (pIC₅₀) against lung, ovarian, melanoma, and colon cancer cell lines indicating that the benzylidene-hydrazones could use for the development of new anticancer agents as a telomerase inhibitor.

Full text of DOI:10.1007/s12272-015-0699-z

Arch. Pharm. Res.
DOI 10.1007/s12272-015-0699-z
RESEARCH ARTICLE
Synthesis, biological evaluation, drug-likeness, and in silico
screening of novel benzylidene-hydrazone analogues as small
molecule anticancer agents
Mohammad Sayed Alam1,2 Dong-Ung Lee1
•
Received: 3 October 2015 / Accepted: 12 December 2015
Ó The Pharmaceutical Society of Korea 2015
Abstract A series of fifteen benzylidene-hydrazone
analogues (3a–o), including eight new compounds, were
synthesized and evaluated for their cytotoxic activities in
four human cancer cell lines and for their antioxidant
activities using DPPH. Of the tested compounds 3e, which
possesses two methoxy substituents in its benzylidene
phenyl ring, was found to be potently cytotoxic to all
cancer cell lines tested with IC50 values of 0.12 (lung),
0.024 (ovarian), 0.097 (melanoma), and 0.05 lM (colon),
and these IC50 values were comparable to those of the
doxorubicin standard (IC50 = 0.021, 0.074, 0.001, and
0.872 lM, respectively). DPPH assay showed compounds
3f, 3i, and 3g had IC50 values of 0.60, 0.99, and 1.30 lM,
respectively, which were comparable to that of ascorbic
acid (IC50 = 0.87 lM). Computational parameters such as,
drug-likeness, ADME properties, toxicity effects, and drug
scores were evaluated, and none of the fifteen compounds
violated Lipinski’s rule of five or Veber’s rule, and thus
they demonstrated good drug-likeness properties. In addi-
tion, all fifteen compounds had a higher drug score than the
doxorubicin and BIBR1532. In silico screening was also
conducted by docking of the active compounds on the
active site of telomerase reverse transcriptase catalytic
subunit, an important therapeutic target of anticancer
agents, to determine the probable binding properties. The
total binding energies of docked compounds are correlated
well with cytotoxic potencies (pIC50) against lung, ovarian,
melanoma, and colon cancer cell lines indicating that the
benzylidene-hydrazones could use for the development of
new anticancer agents as a telomerase inhibitor.
Keywords Benzylidene-hydrazones Á Antitumor agents Á
Antioxidant Á Drug-likeness Á In silico screening Á
Molecular modelling
Introduction
The most common cancers, such as, those of the lung, breast,
colorectum, stomach, liver, and skin, are a major global health
concern (Seigel et al. 2015). Chemotherapy is most widely
used cancer treatment, but is associated with serious side
effects and other problems, for example, the drugs used
interact with unnecessary binding sites, their selectivities and
efficacies are limited, and their effectiveness are sometimes
undermined by the development of drug resistance. Further-
more, cancer growth and metastasis are driven by multiple
pathways. For these reasons, the discovery of new, multi-
targeting anticancer agents is of critical importance.
In vivo oxidative stress produces free radical species
(ROS), which can oxidize many biomolecules, and are
causally associated with several diseases, such as, cancer,
Alzheimer’s disease (AD), metabolic disorders, cellular
aging, reperfusion damage, and inflammatory diseases.
Anti-oxidants are administered to prevent the formation or
to neutralize free radicals and to enable repair of the
damage caused (Sun-Waterhouse et al. 2009), and thus,
importantly provide protection from the developments of
such diseases.
Electronic supplementary material The online version of this
article (doi:10.1007/s12272-015-0699-z) contains supplementary
material, which is available to authorized users.
& Dong-Ung Lee
dulee@dongguk.ac.kr
1
Division of Bioscience, Dongguk University,
Gyeongju 780-714, Republic of Korea
2
Department of Chemistry, Jagannath University, Dhaka 1100,
Bangladesh
123

M. S. Alam, D.-U. Lee
Hydrazones are used as building blocks for the synthe-
ses of a variety of useful bioactive compounds, and are
characterized by the presence of a open chain –RR1-
NN=CR2R3-group. Furthermore, hydrazones exhibit a
broad range of biological activities, such as, anticancer
(Zheng et al. 2009), antibacterial (Rasras et al. 2010), anti-
tuberculosis (Hearn et al. 2009), anti-convalescent (Jain
et al. 2007), anti-oxidant (Anouar 2014), and diuretic
properties (Mishra et al. 1995; Supuran et al. 1996), and
have been used as herbicides, insecticides, nematocides,
rodenticides, plant growth regulators, and as housefly
sterilants (Mohan et al. 1988; Akelah et al. 1993). The wide
ranging biochemical activities and uses of hydrazone ana-
logues have attracted considerable research attention. As
part of our continued search for novel bioactive compounds
(Alam et al. 2011, 2012, 2013) and the results of literature
survey, we synthesized a series of small molecule ben-
zylidene-hydrazone analogues with pharmacodynamic
scaffolds and anticancer properties similar to those of
telomerase inhibitors (Fig. 1). Telomerase, a unique
ribonucleoprotein enzyme, is a potential therapeutic target
for several structurally diverse anticancer agents (Tian
et al. 2010; Read et al. 2001; Moorthy et al. 2012; Luo
et al. 2013; Xing et al. 2014). Literature survey revealed
that some telomerase inhibitors possessing amide
(–NHCO–) and azomethine (–C=N–) functional group to
exhibit telomerase inhibitor activity, and they are also
abundantly observed in pharmaceutically active com-
pounds as a prominent structural motif. For instance,
BIBR1532 (Fig. 1a), an amide derivative of 3-(naph-
thalene-2-yl)but-2-enoic acid, is a promising human
telomerase reverse transcriptase (hTERT) inhibitor so far
(Tian et al. 2010). It exerts its anticancer activity through
the specific blocking of DNA elongation and thereby, acts
as a non-competitive telomerase inhibitor (Pascolo et al.
2002). Benzylidene-hydrazone analogues in the present
study also possess amide and azomethine structural motif
including polar and non-polar substituents in the phenyl
rings, and they might involve in hydrogen bond formation
and hydrophobic interactions with targeting protein or
DNA to exert cell proliferation inhibition activity (Tian
et al. 2010). Moreover, hydrazone derivatives have excel-
lent coordination properties to form complex with metal
ions, especially Fe3? and Ni2?, leading to block the ion
catalyzed physiological reaction or promote the absorption,
transportation, distribution, and metabolism of drugs
(Bernhardt et al. 2007). The cytotoxicities of synthesized
benzylidene-hydrazone analogues were evaluated for
against four human cancer cell lines, and their antioxidant
effects using a DPPH assay. A computational study was
also performed to predict the pharmacokinetic properties
(ADME), MTIR (mutagenic, tumorigenic, irritant, and
reproductive) toxicity profiles, and drug scores to identify
the molecular features responsible for the cytotoxic
Fig. 1 Structures of several
reported anticancer agents as
telomerase inhibitors with
pharmacodynamic scaffolds
similar to the proposed
benzylidene-hydrazone
analogues
123

Synthesis, biological evaluation, drug-likeness, and in silico screening of novel…
properties of these compounds. Furthermore, in silico
screening was performed using the X-ray crystallographic
structure of the catalytic subunit of telomerase to investi-
gate compound binding affinity and modes at its active site.
Exploiting the result of the present study, design and syn-
thesis of more benzylidene-hydrazone derivatives are in
progress to invent a potent anticancer agent as a telomerase
inhibitor.
which were crystallized from ethanol yielding the pure
compounds. All compounds except 3a, 3c, 3g, 3h, and 3m–o
are new (Alam and Lee 2015b) and their spectral and char-
acterization data are included in the supplementary material.
(E)-N0-(4-hydroxybenzylidene)benzohydrazide (3b) Yield:
85 %, m.p. 267–268 °C (brown powder). IR (KBr) mmax
3609 (OH), 3203 (NH), 2919 (CH), 1676 (C=O) cm-1; 1H
NMR (DMSO-d6, 400 MHz), d: 7.02 (d, 2H, J = 7.6, Ar–
H), 7.70–8.08 (m, 5H, Ar–H), 8.53 (s, 1H, CH), 8.67 (d, 2H,
J = 7.8, Ar–H), 11.86 (s, 1H, NH); 13C NMR (DMSO-d6,
100 MHz), d: 116.58 (C 9 2), 126.17, 128.42 (C 9 2),
129.30 (C 9 2), 129.72 (C 9 2), 132.45, 134.55, 148.99
(C=N), 160.31, 163.71 (C=O); EIMS m/z (%) 240 (M?, 30),
110 (100); Anal. calcd. for C14H12N2O2: C, 69.99; H, 5.03;
N, 11.66. Found: C, 70.08; H, 5.09; N, 11.71.
Materials and methods
General
The melting points were determined using a Stuart SMP3
apparatus, and were uncorrected. FT-IR spectra were
obtained using a Bruker Tensor 37 spectrometer (Billerica,
MA, USA) using KBr disc. NMR spectra were recorded
using a Bruker 400 MHz (Billerica, MA, USA) spectrom-
eter and TMS was used as the internal standard. Mass
spectra were acquired using a Jeol JMS700 high-resolution
mass spectrometer at the Korea Basic Science Center
(Daegu, Korea). Elemental analyses (C, H, N) were per-
formed on a Perkin Elmer 2400 II CHN elemental
analyzer.
(E)-N0-(2,4-dihydroxybenzylidene)benzohydrazide (3d)
Yield: 87 %, m.p. 246–247 °C (brown powder). IR (KBr)
mmax 3399 (OH), 3252 (NH), 2919 (CH), 1662 (C=O)
1
cm-1; H NMR (DMSO-d6, 400 MHz), d: 6.32–6.39 (m,
2H, Ar–H), 7.28–7.33 (m, 1H, Ar–H), 7.49–7.60 (m, 3H,
Ar–H), 7.90–7.94 (m, 2H, Ar–H), 8.51 (s, 1H, CH), 11.49
(s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz), d: 109.12,
112.37, 122.74, 127.93, 128.45 (C 9 2), 129.33 (C 9 2),
132.52, 134.49, 148.91 (C=N), 150.97, 152.64, 163.87
(C=O); EIMS m/z (%) 256 (M?, 40), 137 (100); Anal.
calcd. for C14H12N2O3: C, 65.62; H, 4.72; N, 10.93. Found:
C, 65.69; H, 4.77; N, 11.01.
Chemistry
General procedure for preparation of benzohydrazides (2)
Benzohydrazide and 2-hydroxybenzohydrazide were pre-
pared as we previously described (Alam and Lee 2015a).
Briefly, methyl benzoate (13.6 g, 0.1 M) and methyl sali-
cylate (15.2 g, 0.1 M), respectively were refluxed with
hydrazine hydrate (12.5 g, 0.25 M) in approximately
100 cm3 of ethanol for 3 h. The progress of the reaction
was monitored by TLC (Silica gel 60F254, Merck, Ger-
many). Precipitated benzohydrazides were filtered and
recrystallized from an ethanol–water mixture. Mps and
spectral data were compared with previously reported
values to confirm structures.
(E)-N0-(2,4-dimethoxybenzylidene)benzohydrazide (3e)
Yield: 88.5 %, m.p. 178–179 °C (white powder). IR (KBr)
1
mmax 3183 (NH), 3008 (CH), 1725 (C=O) cm-1; H NMR
(DMSO-d6, 400 MHz), d: 3.80 (s, 3H, –OCH3), 3.88 (s,
3H, –OCH3), 6.38–6.47 (m, 2H, Ar–H), 7.40–7.87 (m, 6H,
Ar–H), 8.54 (s, 1H, CH), 10.91 (s, 1H, NH); 13C NMR
(DMSO-d6, 100 MHz), d: 56.31 (CH3), 56.65 (CH3),
99.16, 107.26, 116.04, 127.57, 128.42 (C 9 2), 129.27
(C 9 2), 132.42, 134.46, 144.20 (C=N), 160.04, 161.31,
163.59 (C=O); EIMS m/z (%) 284 (M?, 15), 163 (100);
Anal. calcd. for C16H16N2O3: C, 67.59; H, 5.67; N, 9.85.
Found: C, 67.68; H, 5.72; N, 9.89.
General procedure for the preparation of (E)-N0-
benzylidene-hydrazide analogues (3a–o)
(E)-N0-(3,4-dihydroxybenzylidene)benzohydrazide (3f)
Yield: 91 %, m.p. 208–209 °C (white powder). IR (KBr)
mmax 3660 (OH), 3309 (NH), 2904 (CH), 1720 (C = O)
Compounds 3a–o were prepared as previously described
(Alam and Lee 2015b). Briefly, benzohydrazides (2, 10 mM)
in 30 cm3 ethanol were added drop-wise into 20 mL of
suitably substituted benzaldehydes (10 mM) in ethanol. The
mixture was stirred and refluxed for 1.5–2.5 h. Reaction
progress was monitored by TLC (Silica gel 60F254, Merck,
Germany). After cooling reaction mixtures to ambient tem-
perature, mixtures were filtered to give solid crude products,
1
cm-1; H NMR (DMSO-d6, 400 MHz), d: 6.82–7.01 (m,
2H, Ar–H), 7.31 (s, 1H, Ar–H), 7.57–7.61 (m, 3H, Ar–H),
7.93-7.98 (d, 2H, J = 7.6 Hz, Ar–H), 8.32 (s, 1H, CH),
11.68 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz), d:
113.55, 116.43, 121.47, 126.63, 128.39 (C 9 2), 129.30
(C 9 2), 132.42, 134.55, 146.60 (C=N), 148.88, 149.21,
123

M. S. Alam, D.-U. Lee
163.68 (C=O); EIMS m/z (%) 256 (M?, 35), 105 (100);
Anal. calcd. for: C14H12N2O3: C, 65.62; H, 4.72; N, 10.93.
Found: C, 65.71; H, 4.78; N, 11.00.
Biological evaluation
The DPPH free radical-scavenging activities and cytotox-
icities were assayed using the Blois (Blois 1958) and the
SRB (sulforhodamine-B) method (Skehan et al. 1990),
respectively and description of the assays are included in
the supplementary material.
(E)-N0-(4-Hydroxy-3,5-dimethoxybenzylidene)benzohy-
drazide (3i) Yield: 90 %, m.p. 227–228 °C (white
powder). IR (KBr) mmax 3536 (OH), 3224 (NH), 2904
(CH), 1710 (C=O) cm-1; 1H NMR (DMSO-d6, 400 MHz),
d: 3.83 (s, 6H, OCH3), 6.99 (s, 2H, Ar–H), 7.49–7.60 (m,
3H, Ar–H), 7.88–7.93 (m, 2H, Ar–H), 8.34 (s, 1H, CH),
11.73 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz), d:
56.89 (CH3 9 2), 105.51 (C 9 2), 125.42, 128.42 (C 9 2),
129.36 (C 9 2), 132.52, 134.40, 138.80, 148.99 (C 9 2),
149.40 (C=N), 163.99 (C=O); EIMS m/z (%) 300 (M?, 22),
105 (100); Anal. calcd. for C16H16N2O4: C, 63.99; H, 5.37;
N, 9.33. Found: C, 64.10; H, 5.42; N, 9.39.
Computational studies
The details of the computational studies e.g. drug-likeness
and ADME properties, in silico screening and conforma-
tional analysis are described as supplementary material.
Results and discussion
Synthesis and characterisation
(E)-N0-(2,4,6-trihydroxybenzylidene)benzohydrazide (3j)
Yield: 81 %, mp 260–261 °C (deep-red powder). IR (KBr)
mmax 3610 (OH), 3182 (NH), 2912 (CH), 1710 (C=O)
cm-1; 1H NMR (DMSO-d6, 400 MHz), d: 5.85 (s, 2H, Ar–
H), 7.53 (d, 2H, J = 7.6 Hz, Ar–H), 7.92 (d, 2H,
J = 7.6 Hz, Ar–H), 8.81 (s, 1H, Ar–H), 8.83 (s, 1H, CH),
11.10 (s, OH), 11.90 (s, 1H, NH); 13C NMR (DMSO-d6,
100 MHz), d: 95.25 (C 9 2), 99.92, 128.39 (C 9 2),
129.39 (C 9 2), 132.63, 133.76, 147.72 (C=N), 160.61
(C 9 2), 162.44, 163.15 (C=O); EIMS m/z (%) 272 (M?,
20), 105 (100); Anal. calcd. for C14H12N2O4: C, 61.76; H,
4.44; N, 10.29. Found: C, 61.83; H, 4.50; N, 10.37.
The desired benzylidene-hydrazone analogues (3a–o) were
prepared using a straightforward two step reactions, as
presented in Scheme 1. The structures of the synthesized
compounds were elucidated by FT-IR, 1H NMR, 13C
NMR, mass spectroscopy, and elemental analyses. The FT-
IR spectra of compounds 3a–o showed absorption bands in
the 3430–3660 and 3174–3309 cm-1 regions resulting
from –OH and –NH– stretch, respectively, the [C=O
stretch absorption band was observed at 1648–1667 cm-1
.
1
The H NMR spectra of compounds 3a–o showed singlet
protons at 8.13–8.70 and 10.27–11.94 ppm corresponding
to the –CH=N– and =N–NH– protons, respectively. Aro-
matic protons and the protons of other functional group
produced bands in accord with substitution patterns. In the
13C NMR spectra, azomethine (C=N) and carbonyl (C=O)
carbon showed two characteristics peaks around
144.20–149.78 and 163.15–165.78 ppm, respectively. The
aromatic and methoxyl functional group carbons were
assigned in the usual way. The previously reported crystal
structure of compounds 3g and 3m (Alam and Lee 2015a,
b) confirmed the E-configuration around the azomethine
functional group (–CH=N–NHCO–) of benzylidene
hydrazone analogues. The EI-MS spectra of 3a–o showed
molecular ion peaks with intensities of 22–35 %. FT-IR,
1H-NMR, 13C-NMR and mass spectrometric data together
with elemental analyses data of newly synthesized com-
pounds are summarized in ‘‘Experimental’’ part.
(E)-2-Hydroxy-N0-(4-hydroxybenzylidene)benzohydrazide
(3k) Yield 86 %, m.p. 269–270 °C (white powder). IR
(KBr) mmax 3490 (OH), 3212 (NH), 2909 (CH), 1718 (C=O)
cm-1; 1H NMR (DMSO-d6, 400 MHz), d: 6.45–7.84 (m, 8H,
Ar–H), 8.45 (1H, s, N=CH), 11.25 (s, 1H, NH); 13C NMR
(DMSO-d6, 100 MHz), d: 116.61 (C 9 2), 117.81, 118.37,
125.96, 127.51, 129.27, 130.97 (C 9 2), 131.75, 146.60
(C=N), 160.18, 161.13, 165.78 (C=O); EIMS m/z (%) 256
(M?, 35), 137 (100); Anal. calcd. for C14H12N2O3: C, 65.62;
H, 4.72; N, 10.93. Found: C, 65.66; H, 4.62; N, 10.95.
(E)-2-hydroxy-N0-(2-hydroxybenzylidene)benzohydrazide
(3l) Yield 89 %, m.p. 274–275 °C (brown powder). IR
(KBr) mmax 3569 (OH), 3192 (NH), 2949 (CH), 1680
(C=O) cm-1
;
1H NMR (DMSO-d6, 400 MHz), d:
6.35–7.90 (m, 8H, Ar–H), 8.70 (s, 1H, =CH–), 11.27, (s,
1H, NH); 13C NMR (DMSO-d6, 100 MHz), d: 116.52,
117.31, 118.16, 119.50, 119.89, 120.29, 129.45, 130.30,
132.52, 134.85, 149.78 (C=N), 158.37, 159.86, 165.38
(C=O); EIMS m/z (%) 256 (M?, 40), 105 (100); Anal.
calcd. for C14H12N2O3: C, 65.62; H, 4.72; N, 10.93. Found:
C, 65.68; H, 4.73; N, 10.89.
Anticancer activity
The in vitro cytotoxicities of benzylidene-hydrazone ana-
logues (3a–o) were examined in four human cancer cell
lines, namely, a lung cancer (A549), an ovarian cancer
(SK-OV-3), a skin cancer (SK-MEL-2), and a colon cancer
123

Synthesis, biological evaluation, drug-likeness, and in silico screening of novel…
Scheme 1 The synthesis of
novel (E)-N0-
benzylidenebenzohydrazide
analogues
R1
Comp.
3a
3b
3c
3d
3e
3f
R2
H
H
R3
H
H
H
H
H
OH
OCH3
R4
H
OH
OCH3
OH
OCH3
OH
R5
H
H
H
H
H
H
H
R6
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OCH3
H
H
H
3g
H
OH
3h
3i
3j
3k
3l
H
H
H
OH
H
H
OH
H
OCH3 OCH3
H
OCH3
H
H
H
H
H
OH
H
H
OCH3
H
OH
OH
OH
H
H
OH OH
H
3m
H
H
H
H
H
H
Cl
H
H
3n
3o
H
H
N(CH3)2
H
H
H
H
H
CHO
cell line (HCT15). Cytotoxic potencies were evaluated by
measuring inhibitions of net cell proliferation (measured as
percentages of controls) after incubation for 48 h using the
SRB (sulforhodamine-B) method. Doxorubicin, a positive
control is well-known telomerase inhibitor (Lanvers-
Kaminsky et al. 2005; Yoon et al. 2003). As presented in
Table 1, of the fifteen compounds, compound 3e showed
greatest cytotoxic activity against all four cancer cell lines,
followed by compounds 3l, 3d, and 3n. In particular,
compound 3e exhibited [ 17 and 3 times greater activity
against HCT15 (IC50 0.05 lM) and SK-OV-3 (IC50
0.024 lM), but less activity against A549 (IC50 0.12 lM)
and SK-MEL-2 (IC50 0.097 lM) than doxorubicin (IC50
0.872, 0.074, 0.021 and 0.001 lM, respectively), a potent
anticancer drug.
Table 1 In vitro cytotoxicity data of the 15 benzylidene-hydrazone
analogues (3a–o) against selected human cancer cell lines
Comp.
IC50 values/lMa
A549b
SK-OV-3c
SK-MEL-2d
HCT15e
3a
470.0
[500.0
498.0
125.0
145.0
76.74
4.26
176.0
128.0
110.0
6.84
402.0
[500.0
193.0
7.03
3b
3c
3d
5.71
3e
0.12
62.19
435.0
167.0
[500.0
498.0
37.51
2.25
0.024
31.57
0.097
38.16
235.0
51.44
270.0
325.0
36.14
2.41
0.05
3f
57.34
395.0
59.76
[500.0
333.0
47.33
1.96
3g
[500.0
3h
63.25
[500.0
[500.0
34.46
1.50
3i
3j
Our structure–activity relationship study revealed that
the cytotoxic activities of the fifteen benzylidene hydra-
zone analogues were significantly influenced by the posi-
tions and natures of groups on the phenyl rings. Compound
3a, which has no substituent on its phenyl rings, had weak
or very weak activity against the four human cancer lines
as determined by the SRB method. However, introducing a
methoxy group at the ortho- and para-positions of the
benzylidene phenyl ring of 3a to produce 3e drastically
increased cytotoxicity. In particular, the cytotoxicities of 3e
against ovarian and colon cancer cells were higher than
those of doxorubicin. However, a shift in the position of the
methoxy group or OH group at the ortho-position of the
benzylidene phenyl ring in 3e or 3d to the meta-position in
3h or 3f, respectively, appreciably reduced cytotoxic
effects, and 3i possessing methoxy groups at both meta-
positions led to least activity against all cell lines. These
3k
3l
3m
93.51
17.14
[500.0
0.021
40.34
8.27
36.17
8.12
58.96
7.86
3n
3o
480.0
0.074
320.0
0.001
[500.0
0.872
Doxorubicin
a
IC50 values were obtained using dose response curves by nonlinear
regression using a curve fitting program, OriginPro 7.5
b
Human lung cancer
c
Human ovarian cancer
d
Human skin cancer
e
Human colon cancer
results suggest that the ortho- and para-positions in the
benzylidene phenyl ring importantly determine cytotoxic-
ity. When both methoxy groups in 3e were replaced by OH
123

M. S. Alam, D.-U. Lee
to produce 3d, cytotoxicities against all cell lines sub-
stantially were decreased, although 3d was also potent.
Whereas, cytotoxicity of 3l with an OH group at the ortho-
positions of both phenyl rings was much greater than that
of 3d against all cancer cells. Furthermore, an OH group at
the meta-position (as in 3f) significantly reduced activity
versus 3d or 3l. The above findings indicate that the sub-
stituent position in the phenyl ring plays more crucial role
than the kinds of the substituent, that is, the ortho-substi-
tution led to potent activity (cf. 3e [ 3l [ 3d), whereas the
meta-substitution decreased activity greatly (cf. 3f–i). But a
methoxy substituent with moderate electron-donating
ability more enhances cytotoxicity than an OH group,
which has a stronger electron-donating effect (cf. 3e [ 3d).
Notably, when an electron-withdrawing group was present
on the benzylidene phenyl ring (e.g. 3o), the cytotoxic
effect almost disappeared. These structure–activity rela-
tionships led us to speculate that an optimum electron
density on the benzylidene phenyl ring of hydrazone ana-
logues might determine their cytotoxic activities.
However, compounds having OH group at the ortho posi-
tion (3j [ 3d [ 3l) showed much weaker activity than the
potent compounds (e.g. 3f [ 3i [ 3g [ 3b). Furthermore,
compounds possessing an electron-withdrawing group (3o)
or a strong electronegative group (e.g. 3m) exhibited poor
activity. The above structure–activity relationship study led
us to hypothesize that the position of substituents (partic-
ularly the OH group) on the benzylidene phenyl ring of
hydrazone analogues plays a key role in determining
antioxidant activity.
ADME and drug-likeness properties
Absorption, distribution, metabolism, and excretion
(ADME) and drug-likeness properties of compounds 3a–
o have been calculated and are presented in Table 3.
Moreover, the calculated toxicity effects and drug scores of
the synthesized compounds and of doxorubicin and
BIBR1532 are also presented in Fig. 2. The discussion of
these results has been provided as a ‘‘supplementary
material’’. The results of ADME and drug-likeness prop-
erties together with the calculated toxicity effects and drug
scores of synthesized compounds suggest that benzylidene-
hydrazone analogues (3a–o) have good drug-likeness
properties.
Antioxidant activity
The synthesized benzylidene-hydrazone analogues (3a–o)
were also evaluated for their free radical-scavenging
activity using DPPH. As listed in Table 2, all compounds
exhibited antioxidant activity with a wide range of IC50
values (0.60–264 lM). Compound 3f with two OH groups
at the meta- and para-positions of the benzylidene ring
displayed greatest free radical-scavenging activity
(IC50 = 0.60 lM), and this was higher than that of ascor-
bic acid (IC50 = 0.87 lM). Other potent compounds
(3i [ 3g) possessed OH group at the para-position and a
methoxy group at the meta-position. Methoxy group sub-
stitution in 3f, instead of OH substitution, decreased
activity drastically (cf. 3h). These results suggest that the
polar OH group at the para-position of the benzylidene
phenyl ring is essential for DPPH scavenging activity.
In silico screening
To predict the binding affinity and mode of the benzyli-
dene-hydrazone analogues, the most active (3d, 3e, 3l and
3n) and moderately active (3f, 3h, 3k and 3m) compounds
were docked into the catalytic subunit of telomerase (Gillis
et al. 2008) using AutoDock tools (Morris et al. 2009) 1.5.6
and AutoDock Vina in PyRx 0.8 software (Trott and Olson
2010). Telomerase is an enzyme, and its active site is
composed of two major components, that is, the human
telomerase RNA (hTR) and the human telomerase reverse
transcriptase (hTERT) catalytic subunit, and also includes
many associated protein that are important for proper
function. It is revealed that hTERT is the attractive target
for the discovery of telomerase inhibitors (Gillis et al.
2008; Levelle et al. 2000). The crystal structure of telom-
erase reverse transcriptase (TERT) catalytic subunit was
retrieved from the Protein Data Bank (PDB ID: 3DU6). In
silico screening results are summarized in Table 4. These
studies revealed that the benzylidene-hydrazones (3d-f, 3h
and 3k–n) bind to the active site of telomerase (Luo et al.
2013; Xing et al. 2014) by various hydrophobic, electro-
static, covalent, and Van der Wall’s interactions. Binding
modes and the different types of interactions found for
most active compound 3e is shown in Fig. 3, and docking
models for compounds 3d, 3f, 3h, and 3k–n have been
provided as supporting data (Figs. S1–S7, respectively).
Table 2 DPPH radical scavenging activities of the benzylidene-hy-
drazone analogues (3a–o)
Comp. no
IC50 values/lM
Comp. no
IC50 values/lM
3a
3b
3c
3d
3e
3f
3g
3h
a
360a
2.49
3i
0.99
3.67
36.58
9.55
264a
11.0a
114a
0.87
3j
133.71
8.19
3k
3l
12.31
0.60
3m
3n
1.30
164a
3o
Ascorbic acid
Alam and Lee (2015a)
123

Synthesis, biological evaluation, drug-likeness, and in silico screening of novel…
Table 3 Calculated Lipinski’s rule of five, Veber’s rule, and solubility and absorption parameters of the benzylidene-hydrazone analogues (3a–
o) and doxorubicin
Comp.
Lipinski’s violations
(B1)
Based on Lipinski rule
Based on Veber rule
LogSg %ABSh
HBAa
(B10)
HBDb
(B5)
ClogPc
(B5)
MWd
(B500)
NROTBe
(B10)
TPSAf
(B140 A )
2
˚
3a
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
3
4
1
2
1
3
1
3
2
1
2
4
3
3
1
1
1
7
3.099
2.619
3.155
2.536
3.14
224.263
240.262
254.289
256.261
284.315
256.261
270.288
284.315
300.314
272.26
3
3
4
3
5
3
4
5
5
3
3
3
3
4
4
5
41.462
61.69
-3.72 94.70
-3.42 87.72
-3.74 91.51
-3.13 80.74
-3.76 88.32
-3.13 80.74
-3.44 84.53
-3.76 88.32
-3.46 81.35
-2.83 73.76
-3.13 80.74
-3.13 80.74
-4.46 94.70
-3.76 93.58
-4.04 88.81
-5.32 41.09
3b
3c
4
50.696
81.918
59.93
3d
5
3e
5
3f
5
2.13
81.918
70.924
59.93
3g
5
2.438
2.745
2.454
2.452
2.56
3h
5
3i
6
80.158
102.146
81.918
81.918
41.462
44.70
3j
6
3k
5
256.261
256.261
258.708
267.332
252.273
541.553
3l
5
3.528
3.777
3.201
2.841
0.879
3m
3
3n
4
3o
4
58.533
196.844
Doxorubicin
11
a
Number of hydrogen-bond acceptors
Number of hydrogen-bond donors
b
c
d
e
f
Calculated octanol/water partition coefficient
Molecular weight
Number of rotatable bonds
Molecular polar surface area
Solubility parameter
g
h
Percentage absorption
Toxicity effects
Fig. 2 Toxicity profiles (left)
and drug scores (right) of the
benzylidene-hydrazone
analogues (3a–o), and of the
anticancer agents doxorubicin
and BIBR1532 (M mutagenic,
T tumorigenic, I irritant,
R reproductive)
Comp.
M
T
Low
Low
Low
Low
Low
Low
Medium
Low
Low
Low
Low
Low
Low
High
Low
Low
Low
I
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Medium
High
R
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
High
Low
0
0.2
0.4
0.6
0.8
0.81
0.84
0.81
0.87
0.79
0.87
1
3a
3b
3c
3d
3e
3f
3g
0.67
0.83
0.83
0.89
3h
3i
3j
0.87
0.87
3k
3l
3m
3n
3o
0.70
0.48
0.32
0.29
0.20
Doxorubicin
BIBR1532
Doxorubicin
BIBR1532
Drug score
Medium Medium
123

M. S. Alam, D.-U. Lee
Table 4 Docking results of the synthesized benzylidene-hydrazone analogues
Binding energy (E/kJ mol-1
)
H-bonding residues (ligands functional group)
No of H-bonds
˚
Bond distance (A)
Comp.
3d
3e
3f
-32.24
-47.31
225.96
-24.28
-29.31
-37.26
-24.70
-26.38
ILE338 ([C=O), PHE329 (–OH)
2.72, 2.26
2.25, 2.14
1.95, 2.12
2.26
2
2
2
1
0
1
0
1
LEU419([C=O), ARG592(–OCH3)
LYS392([C=O), PHE443(–OH)
ARG592(–OCH3)
3h
3k
3l
–
–
GLU386(–OH)
–
2.13
3m
3n
–
GLU386 (–NH)
2.83
a
b
c
d
ARG592
4.03
4.27
ARG592
ARG592
MET483
MET483
ARG414
MET483
2.14
ARG414
4.35
ARG414
PHE417
4.50
3.43
GLU84
PHE417
GLU84
PHE417
LEU419
GLU84
SER82
2.25
SER82
SER82
LEU419
LEU419
Fig. 3 a Binding of compound 3e into telomerase (Protein Data Bank entry: 3DU6). The green dotted lines show the hydrogen bond, the blue
dotted lines show p-alkyl interactions, and the yellow dotted lines show p-cation, p-pi, and p-sulfur interactions. b 2D ligand interaction diagram
with telomerase using Discovery Studio program with the essential amino acid residues at the binding site are tagged in circles. The purple
circles show the amino acids which involve in electrostatic and covalent interactions and the green circles show the amino acids which
participate in the van der Waals interactions. c Hydrogen bond interaction showing donor (pink) and acceptor surfaces (green). d Hydrophobic
interaction showing hydrophobic surface (brown)
˚
˚
The most active compounds, 3e and 3l, had low binding
energies of -47.31 and -37.26 kJ mol-1, respectively,
which were lower than those of the moderately active
(distance: 4.44 A) and TYR577 (distance: 5.31 A),
respectively involving benzoyl- or benzylidene-phenyl
rings. The following van der Waal’s interactions were also
observed between: 3d and THR232, GLU236, PHE237,
LEU332, PHE337, HIS339, and ARG340; 3e and SER81,
SER82, PHE83, CYS85, HIS144, ARG414, HIS415,
LYS418, PHE434, MET483, ALA484, THR487, LYS576,
ARG547, ILE590, and ASN591; 3f and CYS390, THR403,
LEU404, TRY405, and PHE417; 3h and LYS76, GLU84,
LEU419, TRP420, ALA484, MET483, and THR487; 3k
and SER81, ARG414, PHE417 and LEU419; 3l and ILE93,
ALA465, VAL573, ASP574, TYR577, PHE578 and
LYS584; 3m and PRO582, PRO388, GLU386, ILE387,
ILE 393, ARG402, LEU404, and TYR577; 3n and
GLN388, ILE387, PRO388, ILE393, LEU404, ALA465,
ASP574, LYS584, and TYR577 amino acid residues. The
higher binding affinities of the most active compounds (3d,
3e, 3l and 3n) could have been caused by additional p-
sulfur and p-pi interactions with amino acid residues of the
telomerase active site.
compounds
3h
(-24.28 kJ mol-1
)
and
3m
(-24.70 kJ mol-1). This result indicates that compounds
3e and 3l bind more tightly (Figs. 3 and S5, respectively) to
the active site of telomerase to that of 3h and 3m (Figs. S3
and S6, respectively) due to hydrogen bonding,
hydrophobic, and van der Waal’s interactions. In addition
to hydrogen bonding (Table 4), 3d–f, 3h and 3k–n exhib-
ited electrostatic p-cation interactions with LYS225 (dis-
˚
˚
tance: 3.84 A), ARG 592 (distance: 4.35 A), LYS416
˚
(distance: 3.73 A), ARG592 (distance: 5.09 A), GLU84
˚
˚
(distance: 3.74 A), TYR577 (distance: 5.31 A), LYS 584
˚
˚
(distance: 3.90 A), and ARG402 (distance: 5.33 A),
˚
respectively, and 3d, 3e, 3f, and 3k exhibited p-sulfur
˚
interactions with CYS233 (distance: 2.46 A), MET483
˚
(distance: 4.27 A), CYS473 (distance: 5.95 A), and
˚
˚
MET483 (distance: 5.30 A), respectively, and 3k and 3l
exhibited p-sigma and p-pi interactions with THR487
123

Synthesis, biological evaluation, drug-likeness, and in silico screening of novel…
Fig. 4 Correlation between the
total binding energy (E/
8
7
6
5
4
3
2
8
b
a
R² = 0.93
R² = 0.95
7
6
5
4
3
2
kJ mol-1) with TERT protein
catalytic subunit (PDB ID:
3DU6) determined by docking
studies and the experimentally
determined cytotoxic activity
(pIC50) of benzylidene-
hydrazone analogues (3d–f, 3h
and 3k–n) against a human lung
cancer (A549), b human ovarian
cancer (SK-OV-3), c human
skin cancer (SK-MEL-2), and
d human colon cancer (HCT15)
cell lines
-50
-45
-40
-35
-30
-25
-20
-50
-45
-40
-35
-30
-25
-20
E / kJ mol-1
E / kJ mol-1
8
7
6
5
4
3
2
8
7
6
5
4
3
2
c
d
R² = 0.91
R² = 0.91
-50
-45
-40
-35
-30
-25
-20
-50
-45
-40
-35
-30
-25
-20
E / kJ mol-1
E / kJ mol-1
Finally, to validate the in silico screening results of
benzylidene-hydrazone analogues into the TERT catalytic
subunit, we examined the relationship between the total
binding energies calculated by the docking studies and the
experimentally determined cytotoxic potencies, pIC50 (-
log10IC50) values. A good correlations were observed
between the binding energies of compounds 3d-f, 3h and
3k–n and their corresponding pIC50 values against A549
(lung), SK-OV-3 (ovarian), SK-MEL-2 (skin), and HCT15
(colon) cancer cell lines (Fig. 4) with correlation coeffi-
cients (r2) of 0.95, 0.93, 0.91, and 0.91, respectively. These
findings suggest compounds 3e, 3l, and 3d be considered
leads to the development of anticancer agents as a telom-
erase inhibitor.
docked conformation of 3n deviated more (69.738), while
the benzoyl phenyl ring showed minor deviation (5.318)
from its lowest energy optimized conformation. In contrast,
the docked conformation of lower active compounds e.g.
3f, 3h, 3k and 3m (Fig. 5c–e, and g, respectively) are
conformationally and energetically distinct to their corre-
sponding lowest energy conformation. This result indicates
that compounds 3f, 3h, 3k, and 3m require more energy to
adopt docked conformation compare to that of compounds
3d, 3e, 3l, and 3n. Therefore, we could anticipate that
compounds 3d, 3e, 3l, and 3n could bind more efficiently
to the telomerase RT catalytic subunit to exert more
potency compare to 3f, 3h, 3k, and 3m. The above con-
sequence is consistent with the result of docking studies of
benzylidene-hydrazone analogues.
Conformational analysis
To gain a better understanding about the bioactive con-
formations of benzylidene-hydrazone analogues, we com-
pared by the superimposition of the lowest energy
optimized conformation and the docked conformation of
the compounds 3d–f, 3h, and 3k–n and their superimpo-
sitions are presented in Fig. 5. The lowest energy con-
former was obtained by the optimization with MM2 semi-
empirical molecular orbital calculation (Allinger 1977).
Analysis of these conformations indicates that the docked
conformation of the higher active compounds e.g. 3d, 3e,
and 3l (Fig. 5a,b,f, respectively) are conformationally and
energetically (data not shown) closer to their corresponding
lowest energy optimized conformation except compound
3n. As shown in Fig. 5h, the benzylidene phenyl ring of
Conclusion
We report the synthesis of novel benzylidene-hydrazone
analogues as potential anticancer, and antioxidant agents.
Using simple condensation reactions, fifteen benzylidene-
hydrazone analogues were prepared and evaluated for
cytotoxicity in four cancer cell lines and for DPPH free
radical scavenging activity. Compound 3e with two
methoxy groups at ortho- and para-positions of the ben-
zylidene phenyl ring was found to be most cytotoxic to all
four cancer cell lines, and its activity against colon- and
ovarian cancer cells was greater than that of doxorubicin.
Furthermore, compound 3f with two hydroxyl substituents
at meta- and para-positions of the benzylidene phenyl ring
123

M. S. Alam, D.-U. Lee
Fig. 5 Comparison between
lowest energy conformation
(green color) calculated by the
semi-empirical molecular
orbital MM2 method and
docked conformation (blue
color) of 3d (a), 3e (b), 3f (c),
3h (d), 3k (e), 3l (f), 3m (g) and
3n (h)
a
c
e
b
d
f
15.51º
23.21º
22.05º
18.19º
28.83º
34.66º
18.01º
72.64º
68.29º
26.97º
106.63º
12.07º
5.31º
h
g
28.15º
69.73º
79.81º
base analogs: synthesis, molecular structure, hirshfeld and
density functional theory analyses. Bull Korean Chem Soc 36:
682–691
had greater DPPH radical scavenging activity than ascorbic
acid. In silico screening studies were used to examine the
probability of binding between these benzylidene-hydra-
zone analogues and the active site of telomerase, a unique
ribonucleoprotein enzyme, and an important therapeutic
target for anticancer agents. A good correlations were
observed between the total binding energies of docked
compounds and their corresponding cytotoxic potencies
against A549 (lung), SK-OV-3 (ovarian), SK-MEL-2
(skin), and HCT15 (colon) cancer cell lines, which indi-
cated benzylidene-hydrazones could be used for the
development of new anticancer agents as a telomerase
inhibitor. None of the fifteen analogues violated Lipniski’s
rule of five (ROF) or Veber’s rule, and thus exhibited good
drug-likeness and ADME properties and high drug scores.
The above findings provided the theoretical basis for
design and synthesis of novel benzylidene-hydrazone
analogues as potential telomerase inhibitors.
Alam MS, Lee DU (2015b) Syntheses, crystal structure, Hirshfeld
surfaces, fluorescence properties, and DFT analysis of benzoic
acid hydrazone Schiff bases. Spectrochim Acta, Part A 145:563–
574
Alam MS, Liu L, Lee DU (2011) Cytotoxicity of new 5-phenyl-4,5-
dihydro-1,3,4-thiadiazole analogues. Chem Pharm Bull 59:
1413–1416
Alam MS, Choi JH, Lee DU (2012) Synthesis of novel Schiff base
analogues of 4-amino-1,5-dimethyl-2-phenylpyrazol-3-one and
their evaluation for antioxidant and anti-inflammatory activity.
Bioorg Med Chem 20:4103–4108
Alam MS, Nam YJ, Lee DU (2013) Synthesis and evaluation of (Z)-
2,3-diphenylacrylonitrile analogs as anti-cancer and anti-micro-
bial agents. Eur J Med Chem 69:790–797
Allinger NL (1977) A hydrocarbon force field utilizing V1 and V2
torsional terms. J Am Chem Soc 99:8127–8134
Anouar EH (2014) A quantum chemical and statistical study of
phenolic Schiff bases with antioxidant activity against DPPH
free radical. Antioxidants 3:309–322
Bernhardt PV, Chin P, Sharpe PC, Richardson DR (2007) Hydrazone
chelators for the treatment of iron overload disorders: iron
coordination chemistry and biological activity. Dalton Trans
30:3232–3244
Blois MS (1958) Antioxidant determinations by the use of a
stable free radical. Nature 455:1199–1200
References
Gillis AJ, Schuller AP, Skordalakes E (2008) Structure of the
Tribolium castaneum telomerase catalytic subunit TERT. Nature
455:633–637
Hearn MJ, Cynamon MH, Chen MF, Coppins R, Davis J, Joo-On
Kang H, Noble A, Tu-Sekine B, Terrot MS, Trombino D, Thai
Akelah A, Kenawy ER, Sherrington DC (1993) Agricultural polymers
with herbicide/fertilizer function-III. Polyureas and poly (Schiff
base)s based systems. Eur Polymer J 29:1041–1045
Alam MS, Lee DU (2015a) Quantum-chemical studies to approach
the antioxidant mechanism of nonphenolic hydrazone Schiff
123

Synthesis, biological evaluation, drug-likeness, and in silico screening of novel…
M (2009) Preparation and antitubercular activities in vitro and
in vivo of novel Schiff bases of isoniazid. Eur J Med Chem
44:4169–4178
Read M, Harrison RJ, Romagnoli B, Tanious FA, Gowan SH, Reszka
AP, Wilson WD, Kelland LR, Neidle S (2001) Structure-based
design of selective and potent G quadruplex-mediated telom-
erase inhibitors. Proc Natl Acad Sci 98:4844–4849
Jain JS, Srivastava RS, Aggrawal N, Sinha R (2007) Synthesis and
evaluation of schiff bases for anticonvulsant and behavioral
depressant properties. Cent Nerv Syst Agents Med Chem
7:200–204
Lanvers-Kaminsky C, Winter B, Koling S, Frodermann B, Braun Y,
Schaefer KL, Diallo R, Koenemann S, Wai D, Willich N,
Poremba C (2005) Doxorubicin modulates telomerase activity in
Ewing’s sarcoma in vitro and in vivo. Oncol Rep 14:751–758
Levelle F, Riou JF, Laoui A, Mailliet P (2000) Telomerase: a
therapeutic target for the third milleneum? Crit Rev Oncol
Hematol 34:111–126
Luo Y, Zhang S, Qiu KM, Liu ZJ, Yang YS, Fu J, Zhong WQ, Zhu
HL (2013) Synthesis, biological evaluation, 3D-QSAR studies of
novel aryl-2H-pyrazole derivatives as telomerase inhibitors.
Bioorg Med Chem Lett 23:1091–1095
Mishra P, Gupta PN, Shakya AK, Shukla R, Srimal RC (1995) Anti-
inflammatory and diuretic activity of a new class of compounds-
Schiff bases of 3-amino-2-methylquinazolin 4(3H)-ones. Indian
J Physiol Pharmacol 39:169–172
Mohan M, Gupta MP, Chandra L, Jha NK (1988) Synthesis,
characterization and antitumour properties of some metal(II)
complexes of 2-pyridinecarboxaldehyde 20-pyridyl hydrazone
and related compounds. Inorg Chim Acta 151:61–68
Moorthy NSHN, Nuno SC, Maria JR, Pedro AF (2012) QSAR
analysis of 2-benzoxazolyl hydrazone derivatives for anticancer
activity and its possible target prediction. Med Chem Res 21:
133–144
Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell
DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: auto-
mated docking with selective receptor flexibility. J Comput
Chem 30:2785–2791
Seigel RL, Miller KD, Jemal A (2015) Cancer statistics, 2015. CA
Cancer J Clin 65:5–29
Skehan P, Streng R, Scudiero D, Monks A, McMahon J, Vistica D,
Warren JT, Bokesch H, Kenney S, Boyd MR (1990) New
colorimetric cytotoxicity assay for anticancer-drug screening.
J Natl Cancer Inst 82:1107–1112
Sun-Waterhouse D, Chen J, Chuah C, Wibisono R, Melton LD, Laing
W, Ferguson LR, Skinner MA (2009) Kiwifruit-based polyphe-
nols and related antioxidants for functional foods: kiwifruit
extract-enhanced gluten-free bread. Int
60:251–264
J Food Sci Nutr
Supuran CT, Barboiu M, Luca C, Pop E, Brewster ME, Dinculescu A
(1996) Carbonic anhydrase activators. Part 14. Syntheses of
mono and bis pyridinium salt derivatives of 2-amino-5-(2-
aminoethyl)- and 2-amino-5-(3-aminopropyl)-1,3,4-thiadiazole
and their interaction with isozyme II. Eur J Med Chem
31:597–606
Tian FF, Jiang FL, Han XL, Xiang C, Ge YS, Li JH, Zhang Y, Li R,
Ding XL, Liu Y (2010) Synthesis of a novel hydrazone
derivative and biophysical studies of its interactions with bovine
serum albumin by spectroscopic, electrochemical, and molecular
docking methods. J Phys Chem B 114:14842–14853
Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and
accuracy of docking with a new scoring function, efficient
optimization and multithreading. J Comput Chem 31:455–461
Xing M, Zhao TT, Ren YJ, Peng NN, Yang XH, Li X, Zhang H, Liu
GQ, Zhang LR, Zhu HL (2014) Synthesis, biological evaluation,
and molecular docking studies of pyrazolyl-acylhydrazone
derivatives as novel anticancer agents. Med Chem Res
23:3274–3286
Pascolo E, Wenz C, Lingner J, Hauel N, Priepke H, Kauffmann I
(2002) Mechanism of human telomerase inhibition by
BIBR1532, a synthetic, non-nucleosidic drug candidate. J Biol
Chem 77:15566–15572
Rasras AJM, Al-Tel TH, Amal AF, Al-Qawasmeh RA (2010)
Synthesis and antimicrobial activity of cholic acid hydrazone
analogues. Eur J Med Chem 45:2307–2313
Yoon KA, Ku JL, Yang JO, Park JG (2003) Telomerase activity,
expression of Bcl-2 and cell cycle regulation in doxorubicin
resistant gastric carcinoma cell lines. Int J Mol Med 11:343–348
Zheng LW, Wu LL, Zhao BX, Dong WL, Miao JY (2009) Synthesis
of novel substituted pyrazole-5-carbohydrazide hydrazone
derivatives and discovery of a potent apoptosis inducer in
A549 lung cancer cells. Bioorg Med Chem 17:1957–1962
123