Synthesis, biological activities and molecular docking simulation of hydrazone scaffolds of carvacrol, thymol and eugenol

Article abstract of DOI:10.1007/s11164-017-3007-3

In present work, we report the synthesis of 12 new hydrazones and sulfonyl hydrazones linkage containing carvacrol, thymol and eugenol derivatives by simple condensation reactions. Synthesized derivatives have been characterized by ¹H NMR, ¹³C NMR, LC–MS, and X-ray single crystallography techniques, and these derivatives were screened for anticancer testing by using sulforhodamine B assay and anti-oxidant testing by using DPPH assay. Docking studies of all the derivatives against the active site of human heme oxygenase-1 indicated that interaction with the maximum site of the amino acid residue of human heme oxygenase-1 was crucial for anti-oxidant activity. The results show that all derivatives possess interesting biological activities.

Full text of DOI:10.1007/s11164-017-3007-3

Res Chem Intermed
DOI 10.1007/s11164-017-3007-3
Synthesis, biological activities and molecular docking
simulation of hydrazone scaffolds of carvacrol, thymol
and eugenol
1
1
•
Jamatsing D. Rajput
1
S. Bendre
•
Suresh D. Bagul
Ratnamala
Received: 16 March 2017 / Accepted: 7 June 2017
Ó Springer Science+Business Media B.V. 2017
Abstract In present work, we report the synthesis of 12 new hydrazones and sul-
fonyl hydrazones linkage containing carvacrol, thymol and eugenol derivatives by
simple condensation reactions. Synthesized derivatives have been characterized by
1
13
H NMR, C NMR, LC–MS, and X-ray single crystallography techniques, and
these derivatives were screened for anticancer testing by using sulforhodamine B
assay and anti-oxidant testing by using DPPH assay. Docking studies of all the
derivatives against the active site of human heme oxygenase-1 indicated that
interaction with the maximum site of the amino acid residue of human heme
oxygenase-1 was crucial for anti-oxidant activity. The results show that all
derivatives possess interesting biological activities.
Keywords Hydrazones Á Sulfonyl hydrazones Á SRB Á DPPH Á Carvacrol Á Thymol Á
Eugenol
Introduction
Hydrazones linkage constitutes an imperative class of biologically active drug
molecules which have attracted the attention of pharmaceutical chemists due to their
huge range of medicinal properties [1, 2]. These compounds are being synthesized
as drugs by many researchers in order to combat diseases with minimal toxicity and
Electronic supplementary material The online version of this article (doi:10.1007/s11164-017-3007-3)
contains supplementary material, which is available to authorized users.
&
Ratnamala S. Bendre
bendrers@gmail.com
1
School of Chemical Sciences, North Maharashtra University Jalgaon, Jalgaon,
Maharashtra 425 001, India
123

J. D. Rajput et al.
maximal effects [3]. These predictions have provided a therapeutic pathway to
develop new effective biologically active hydrazones [4]. Many hydrazone
derivatives has been reported to exert notably biological activities [5]. Throughout
the world, medicinal chemists have carried out vast research work on hydrazones
and established new scaffolds with better activity and low toxicity profile;, Fig. 1
shows hydrazone linkage-based bioactive molecules [6, 7]. Numerous synthetic
methodologies have been developed for the synthesis of hydrazone derivatives and
they have been found to be active against diverse pharmacological targets [8]. They
are known to possess antimicrobial, anticancer, anti-inflammatory and antimalarial
activities [9–11]. These observations have been key for the development of new
hydrazones that possess diverse biological activities [12].
It is well known that the essential oils obtained from plants, a traditional
Mediterranean spice, possess strong biological activity due to their very high
contents of monoterpenes and oxygenated compounds [13]. Carvacrol, thymol and
eugenol are present in essential oils of many plants. These three naturally occurring
phenolic monoterpenoids are interesting resourceful molecules incorporated as
useful constituents in several products [14, 15]. The wide range of pharmacological
activities, including analgesic, antimicrobial, anti-inflammatory, anti-oxidant and
anticancer, etc., have been well researched [16–18].
This has created an interest among researchers who have synthesized a variety of
hydrazones and screened them for their various biological activities. In the present
study, we report on 12 new hydrazone and sulfonyl hydrazone derivatives of
carvacrol, thymol, and eugenol. All the synthesized compounds show excellent anti-
oxidant and good anticancer activity.
Fig. 1 Some biological active hydrazone linkage-based biological active molecules
1
23

Synthesis, biological activities and molecular docking…
Experimental
Chemicals, characterizations and procedures
All the chemicals and reagents necessary for the reactions were procured from
Sigma-Aldrich and Fisher Scientific with purity 98% and used without further
1
13
purification. The products were characterized using H NMR, C NMR, IR spectra
and LC–MS. IR spectra were recorded with a Shimadzu FTIR spectrometer model
using KBr disks. The NMR spectra (CDCl and DMSO d6) were recorded on a
3
1
13
Bruker AC-400 MHz spectrometer (for H NMR use AC-400 MHz and NMR use
AC-100 MHz) with TMS as an internal standard. The crystal structure was
examined using a Bruker APEX-II CCD diffractometer.
1
.
General procedure for the synthesis of ortho formyl phenolic monoterpenoids
29].
[
Synthesis of ortho formyl phenolic monoterpenoids was carried out using a
previously reported method.
2
.
General procedure for the synthesis of substituted hydrazine [30].
Hydrazine hydrate (0.25 mol, 2.5 equiv) was added dropwise to a solution of
4
,6 dichloro pyrimidine 3,6 dichloro pyridazine (0.1 mol, 1 equiv) in ethanol
(100 mL) for 30 °C at room temperature. After stirring at 30 °C for 1 h, the
reaction mixture after 1 h produced a creamy precipitate. After filtering the
product (78% yield), it was used for the next step in the synthesis.
3
.
General procedure for the synthesis of substituted sulfonyl hydrazides [30].
Hydrazine hydrate (0.25 mol, 2.5 equiv) was added dropwise to a solution of
p-toluene sulfonyl chloride or 2, 4, 6 trimethyl benzene sulfonyl chloride
(0.1 mol, 1 equiv) in THF (100 mL) at 0 °C under an inert atmosphere. After
stirring for 0 to 5 °C for 30 min, ice-cold ethyl acetate (200 mL) was added to
the cooled reaction mixture and the mixture was washed repeatedly with ice-
cold 10% aqueous sodium chloride (5 9 150 mL). The organic layer was dried
over sodium sulfate at 0 °C, and was then added slowly to a stirring solution of
hexane (1.2 L) during 5 min. Substituted benzene sulfonyl hydrazide precip-
itated as a white solid and was collected by vacuum filtration. The filter cake
was washed with hexanes (2 9 50 mL) and then dried in vacuo yielding the
title compound as a white solid (17.61 g, 81%).
4
.
General procedure for the synthesis of phenolic monoterpenoid-based hydra-
zones [31].
The hydrazones were prepared by reaction of equimolar quantities of
substituted hydrazine (A or B) and ortho formyl thymol (or ortho formyl
carvacrol or ortho formyl eugenol). Each reactant was dissolved in a minimum
amount of ethanol, and then they were mixed together, adding 2–3 drops of
acetic acid. The reaction mixture was refluxed for 2 h, then cooled to room
temperature and poured into ice-cold water. The solid product obtained was
collected by filtration and then dried using a drying oven at 70 °C. The product
was crystallized from ethanol and dried to obtain the pure product (yield 80%).
123

J. D. Rajput et al.
5
.
General procedure for the synthesis of phenolic monoterpenoid-based sulfonyl
hydrazones [31].
The hydrazone were prepared by the reaction of equimolar quantities of
substituted benzene sulfonyl hydrazide and ortho formyl thymol (or ortho
formyl carvacrol or ortho formyl eugenol). Each reactant was dissolved in a
minimum amount of ethanol, and then they were mixed together, adding 2–3
drops of acetic acid. The reaction mixture was refluxed for 2 h, then cooled to
room temperature and poured into ice-cold water. The solid product obtained
was collected by filtration and then dried using a drying oven at 70 °C. The
product was crystallized from ethanol and dried to obtain the pure product
(yield 80%).
Spectroscopic characterizations and physical properties of synthesized
derivatives
(
(
E)-2-((2-(6-chloropyridazine-3-yl)hydrazone)methyl)-3-isopropyl-6-methylphenol,
1
IIA), Color—white, mp- 110 °C. H NMR (DMSO) d 1.24–1.25 (d, J = 4.0 Hz,
6
H), 2.19 (s, 3H), 3.21–3.34 (m, J = 4.0 Hz, 1H), 6.75 (d, J = 8.0 Hz, 1H), 6.78 (s,
H), 7.10–7.12 (d, 1H), 8.44 (s, 1H) 8.80 (s, 1H for imine), 11.30 (s, 1H for OH D O
1
2
1
3
exchangeable), 11.79 (s, 1H for NH D O exchangeable) C NMR (DMSO) d
2
1
5.39, 23.60, 27.96, 113.64, 115.30, 122.15, 132.22, 145.70, 156.09, LC–MS
?
methanol), m/z: [M ? 1] 305.50, [M ? 3] 307.51
?
(
(
(
E)-2-((2-(6-chloropyridazine-3-yl)hydrazone)methyl)-6-isopropyl-3-methylphenol,
1
IA), Color—white, mp- 110 °C. H NMR (DMSO) d 1.33–1.35 (d, J = 4.0 Hz,
6
1
H), 2.8 (s, 3H), 3.63–3.70 (m, J = 4.0 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 6.91 (s,
H), 7.12–7.18 (d, 1H), 8.48 (s, 1H) 8.92 (s, 1H for imine), 11.34 (s, 1H for OH D O
2
1
3
exchangeable), 11.69 (s, 1H for NH D O exchangeable) C NMR (DMSO) d
2
1
5.40, 23.63, 27.90, 114.22, 116.31, 126.20, 138.48, 146.10, 157.16, LC–MS
?
methanol), m/z: [M ? 1] 305.50, [M ?3] 307.52
?
(
(
(
E)-4-allyl-2-((2-(6-chloropyridazine-3-yl)hydrazone)methyl)-6-methoxyphenol,
1
IIIA), Color—yellow, mp- 108 °C. H NMR (CDCl ) d 3.40 (d, 2H), 3.92 (s,
3
1
1
H), 5.0–5.13 (t, 2H), 5.91–6.01 (m, 1H), 6.80–6.83 (s, 2H), 7.21 (s, 2H), 8.63 (s,
H for imine), 9.72 (s, 1H for OH D O exchangeable), 11.43 (s, 1H for NH D O
2
2
1
3
exchangeable) C NMR (CDCl ) d 39.55, 56.22, 115.81, 116.21, 117.04, 123.40,
3
1
31.14, 137.13, 148.01, 148.24, 164.77, LC–MS (methanol), m/z: [M ? 1] ?
19.47, [M ? 3] ? 321.33
3
(
(
E)-2-((2-(6-chloropyrimidin-4-yl)hydrazone)methyl)-3-isopropyl-6-methylphenol,
1
IIB), Color—white, mp- 112 °C. H NMR (DMSO) d 1.24–1.25 (d, J = 4.0 Hz,
6
H), 2.19 (s, 3H), 3.21–3.34 (m, J = 4.0 Hz, 1H), 6.75 (d, J = 8.0 Hz, 1H), 6.78 (s,
H), 7.10–7.12 (d, 1H), 8.44 (s, 1H) 8.80 (s, 1H for imine), 11.30 (s, 1H for OH D O
1
2
1
23

Synthesis, biological activities and molecular docking…
1
3
exchangeable), 11.79 (s, 1H for NH D O exchangeable) C NMR (DMSO) d
2
1
5.39, 23.60, 27.96, 113.64, 115.30, 122.15, 132.22, 145.70, 156.09, LC–MS
?
methanol), m/z: [M ? 1] 305.50, [M ? 3] 307.51.
?
(
(
(
E)-2-((2-(6-chloropyrimidin-4-yl)hydrazone)methyl)-6-isopropyl-3-methylphenol
1
IIB), Color—white; mp- 110 °C. H NMR (DMSO) d 1.17–1.19 (d, J = 4.0 Hz,
6
H), 2.35 (s, 3H), 3.26–3.35 (m, J = 4.0 Hz, 1H), 6.66 (d, J = 8.0 Hz, 1H), 6.77 (s,
H), 7.07–7.09 (d, 1H), 8.43 (s, 1H), 8.65 (s, 1H for imine), 11.30 (s, 1H for OH
1
1
3
D O exchangeable), 11.79 (s, 1H for NH D O exchangeable) C NMR (DMSO) d
2
2
1
MS (methanol), m/z: [M ? 1] 350.50, [M ? 3] 307.52
8.76, 22.28, 25.78, 115.16, 211.07, 127.58, 133.16, 135.06, 155.18, 158.38, LC–
?
?
(E)-4-allyl-2-((2-(6-chloropyrimidin-3-yl)hydrazone)methyl)-6-methoxyphenol
1
(IIIB), Color—white; mp- 100 °C. H NMR (DMSO) d 3.28–3.30 (d, 2H), 3.80
(s, 1H), 5.0–5.08 (t, 2H), 5.87–5.98(m, 1H), 6.72–6.73 (s, 1H), 7.00 (s, 1H), 7.08 (s,
1
1
1
1
H), 8.33 (s, 1H), 8.41 (s, 1H for imine), 9.25 (s, 1H for OH D O exchangeable),
2
1
3
1.67 (s, 1H for NH D O exchangeable) C NMR (DMSO) d 55.64, 101.30,
2
13.05, 155.34, 117.69, 119.74, 130.34, 137.54, 142.74, 144.74, 147.65, 157.79,
?
59.31, 161.75, LC–MS (methanol), m/z: [M ? 1] 319.47, [M ?3] 321.34.34
?
(
E)-N-(2-hydroxy-6-isopropyl-3-methylbenzylidene)-4-methylbenezenesulfonohy-
1
drazide (IID), Color—white; mp- 98 °C. H NMR (CDCl ) d 1.18–1.20 (d,
J = 4.0 Hz, 6H), 2.19 (s, 3H), 2.40–2.41 (s, 3H) 3.09–3.16 (m, J = 4.0 Hz, 1H),
3
6
.69–6.71 (d, J = 8.0 Hz, 1H), 7.11–7.13 (d, J = 8.0 Hz, 1H), 7.32–7.34 (d,
J = 8.0 Hz, 2H), 7.83–7.85 (d, J = 8.0 Hz, 2H), 8.00 (s, 1H for imine), 8.49 (s, 1H
1
for OH D O exchangeable), 11.05 (s, 1H for NH D O exchangeable) C NMR
3
2
2
(
CDCl ) d 15.68, 21.66, 23.94 28.40, 112.93, 115.50, 123.67, 127.90, 128.39,
3
1
29.66, 130.07, 133.72, 134.52, 144.80, 146.82, 151.93, 157.40, LC–MS
?
? ?
methanol), m/z: [M ? 1] 347.49, [M ? Na ] 369.47
(
(
E)-N-(2-hydroxy-3-isopropyl-6-methylbenzylidene)-4-methylbenezenesulfonohy-
1
drazide (ID), Color—white; mp- 104 °C. H NMR (DMSO) d 1.11–1.13 (d,
J = 4.0 Hz, 6H), 2.26 (s, 3H), 2.34 (s, 3H) 3.19–3.22 (m, J = 4.0 Hz, 1H),
6
J = 8.0 Hz, 2H), 7.76–7.80 (d, J = 8.0 Hz, 2H), 8.39 (s, 1H for imine), 11.31 (s,
.63–6.65 (d, J = 8.0 Hz, 1H), 7.06–7.08 (d, J = 8.0 Hz, 1H), 7.41–7.43 (d,
1
3
1
H for OH D O exchangeable), 11.74 (s, 1H for NH D O exchangeable) C NMR
2 2
(
DMSO) d 18.53, 20.92, 22.21, 25.70, 114.70, 121.09, 127.07, 128.03, 129.89,
?
1
3
33.16, 135.35, 135.95, 143.90, 148.99, 155.22, LC–MS (methanol), m/z: [M ? 1]
?
.47.50, [M ? Na ] 369.47
?
(
E)-N-(5-allyl-2-hydroxy-3-methoxybenzylidene)-4-methylbenzenesulfonohy-
1
drazide (IIID), Color—yellow; mp- 114 °C. H NMR (CDCl ) d 2.39 (S, 3H),
3
3
.27–3.29 (d, 2H), 3.86 (s, 1H), 5.02–5.07 y7.82 (d, 2H), 7.93 (s, 1H for imine), 8.04
1
3
C
(
s, s, 1H for OH D O exchangeable),10.01 (s, 1H for NH D O exchangeable)
2
2
NMR (CDCl ) d 21.64, 39.50, 56.12, 114.61, 116.76, 121.95, 127.93, 130.10,
3
123

J. D. Rajput et al.
1
31.12, 134.36, 137.11, 144.88, 146.01, 148.06, 152.34, LC–MS (methanol), m/z:
? ? ?
M ? 1] 361.46, [M ? Na ] 383.42
[
(
E)-N-(2-hydroxy-6-isopropyl-3-methylbenzylidene)-2,4,6-trimethylbenezenesul-
1
fonohydrazide (IIC), Color—white; mp- 114 °C. H NMR (CDCl ) d 1.16–1.18
(
J = 4.0 Hz, 1H), 6.67–6.69 (d, J = 8.0 Hz, 1H), 6.99 (s, 2H), 7.07–7.09 (d,
3
d, J = 4.0 Hz, 6H), 2.15 (s, 3H), 2.27–2.29 (s 3H), 2.70 (s, 6H), 3.08–3.19 (m,
J = 8.0 Hz, 1H), 8.30 (s, 1H for imine), 8.44 (s, 1H for OH D O exchangeable),
2
1
3
1
2
1
0.82 (s, 1H for NH D O exchangeable) C NMR (CDCl ) d 15.66, 21.06, 23.13,
2
3
3.91, 28.38, 112.93, 113.02, 115.40, 123.54, 131.39, 132.38, 133.40, 140.22,
?
43.62, 146.56, 149.82, 157.13, LC–MS (methanol), m/z: [M ? 1] 375.51,
?
?
[
M ? Na ] 397.47
(
E)-N-(2-hydroxy-3-isopropyl-6-methylbenzylidene)-2,4,6-trimethylbenezenesul-
1
fonohydrazide (IC), Color—white; mp- 108 °C. H NMR (DMSO) d 1.09–1.11
(
d, J = 4.0 Hz, 6H), 2.23–2.26 (s, 6H), 2.62 (s, 6H), 3.13–3.20 (m, d, J = 4.0 Hz
1
1
H), 6.63–6.65 (d, J = 8.0 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 7.06 (s, 2H), 8.43 (s,
H for imine), 11.10 (s, 1H for OH D O exchangeable), 12.47 (s, 1H for NH D O
2
2
1
3
exchangeable) C NMR (DMSO) d 18.56, 20.35, 22.17, 22.21, 22.48, 25.65,
1
14.88, 121.12, 127.18, 131.81, 131.39, 132.51, 133.04, 165.27, 139.13, 142.65,
? ? ?
47.36, 154.90, LC–MS (methanol), m/z: [M ? 1] 375.51, [M ? Na ] 397.48
1
(
E)-N-(5-allyl-2-hydroxy-3-methoxybenzylidene)-2,4,6-trimethylbenzenesulfono-
1
hydrazide (IIID), Color—yellow; mp- 114 °C. H NMR (CDCl ) d 2.26 (S, 3H),
2
3
.68 (s, 6H) 3.25–3.27 (d, 2H), 3.84 (s, 3H), 5.01–5.02 (s, 1H), 5.03–5.06 (t, 1H),
.85–5.92 (m, 1H), 6.56 (s, 1H), 6.69–6.70 (s, 1H), 6.95 (s, 2H), 7.90 (s, 1H for
5
imine), 8.49 (s, 1H for OH D O exchangeable), 9.74 (s, 1H for NH D O
2
2
1
3
exchangeable) C NMR (CDCl ) d 21.03, 23.15, 39.49, 56.08, 114.31, 116.07,
3
1
16.97, 121.92, 131.06, 131.17, 132.27, 137.16, 140.25, 143.61, 145.64, 147.92,
? ? ?
50.44, LC–MS (methanol), m/z: [M ? 1] 389.48, [M ? Na ] 411.44
1
Bioassay
Anticancer activity
In vitro anticancer activity of synthesized derivatives was performed using
sulforhodamine B (SRB) assay on a panel of human cancer cell lines HCT-12
(
colon cancer) and MIPaCa-2 (pancreatic cancer) [32]. The results are expressed as
GI50 (growth inhibitory concentration at 50%), i.e. the concentration of the
compounds which inhibits the tumor cell growth by 50%.
Anti-oxidant activity
DPPH radical-scavenging activity was performed by the reported method [33]. The
concentrations of the sample necessary to decrease the DPPH concentration by 50%
was obtained by interpolation from linear regression analysis and denoted as the
1
23

Synthesis, biological activities and molecular docking…
EC50 value (lg/mL). All determinations were carried out in triplicate. Butylated
hydroxy toluene (BHT) was used as a reference compound.
Molecular docking studies
The docking study was performed by FRED (Open Eye) and used to determine the
orientation of molecules bound in the active site of human heme oxygenase-1 (heme
oxy-1, PDB code: 3CZY). The PDB files were downloaded from the Protein Data
Bank (www.rsc.org). The ligands were drawn in Chem Draw Ultra 12.0 and the
(
Freeware) FRED docking programme was used to perform molecular docking. It is
a shape-based docking approach. The steps performed by FRED along with the used
parameters are as follows. Mostly, default parameters are used unless specified. Site
˚
box volume was 4818 A. All possible poses of the ligand around the active site were
enumerated by rigidly rotating and translating each conformer within the site. The
resulting pose ensemble was filtered by rejecting poses that do not fit within the
larger of the two volumes specified by the receptor file’s shape potential grid and a
˚
contour level (referred to as the outer contour, volume = 1020 A). The resulting
pose ensemble was filtered by rejecting poses that do not have a least one heavy
atom within the smaller of the two volumes specified by the receptor file’s shape
potential grid and a contour level (referred to as the inner contour, vol-
˚
ume = 209 A). The pose ensemble was filtered by rejecting poses that do not match
any user-defined docking constraints [34].
Results and discussion
Chemistry of hydrazones
Hydrazones, possessing an azomethine –NHN=CH– group, constitute an important
class of compounds as target structures for their biological activities [19]. These
molecules are easily synthesized by the reaction of hydrazine or hydrazide or
sometimes sulfonyl hydrazide with aldehydes and ketones. They are a class of
organic compounds and widely used in organic synthesis [20]. They are not only
intermediates but also very effective organic compounds when they are used as
intermediates and coupling products which can be synthesized by using the active
hydrogen component of the –CONHN=CH– azomethine group [21]. Many effective
compounds, such as iproniazid and isocarboxazid, are synthesized by the reduction
of hydrazide–hydrazones [22].
The targeted hydrazones and sulfonyl hydrazones were synthesized in three steps.
The first step involved the synthesis of ortho formyl derivatives of natural phenolic
monoterpenes (Part-I) via Reimer Tiemann reaction. The second step involved
hydrazino and sulfonyl hydrazino analogs (A, B, C and D) of 4,6-dichloropyrim-
idine, 3,6-dichloropyridazine, p-toluene sulfonyl chloride and 2,4,6, trimethyl
123

J. D. Rajput et al.
benzene sulfonyl chloride (Part-II), which was further reacted with ortho formyl
derivative of natural phenolic monoterpenes (Part-I) gave substituted hydrazones
and sulfonyl hydrazones. (Part-III and Part-IV).
Reaction scheme
(
Part-I) Synthesis of ortho formyl phenolic monoterpenoids
3
A = CHCl , NaOH in water reflux
(
Part-II) Synthesis of substituted hydrazine and sulfonyl hydrazides
O
B = NH
2
NH
2
:H
2
O in ethanol at RT for 2h. C = NH
2
NH
2
:H
2
O in THF at 0 to 5 C for 40 min.
(
Part-III) Synthesis of phenolic monoterpenoid-based hydrazones
1
23

Synthesis, biological activities and molecular docking…
o
D= substituted hydrazine (A or B) in ethanol reflux at 80 C for 2h.
(
Part-IV) Synthesis of phenolic monoterpenoid-based sulfonyl hydrazones
o
E= substituted sulfonyl hydrazine (C or D) in ethanol reflux at 80 C for 2h.
123

J. D. Rajput et al.
Biological evaluation
Cell growth inhibition was investigated by SRB assay and the results show that the
some of compounds show an inhibitory effect on the proliferation of HCT15
(
human colon cancer cell line) and MIAPaCa-2 (human pancreatic cancer cell line)
in a dose-dependent manner (Table 1). The cytotoxic effect of IB, IIIB, IIC and
IID derivatives against pancreatic cancer was excellent as compared to colon
cancer: these six compounds were found to exhibit excellent cytotoxic potency
(
10 lg/mL) which is comparable with of Adriamycin (10 lg/mL) against pancreatic
cancer on the MIAPaCa-2 cell line. Similarly, the compounds IB, IIIB and IID
were found to possess moderate anticancer activity compared with that of
Adriamycin (10 lg/mL) against colon cancer on the HCT-15 cell line. Of note is
that the phenolic monoterpenoid-based derivatives show better activity against
pancreatic cancer on the MIAPaCa-2 cell line as compared to colon cancer with the
HCT-15 cell line. The total results suggested that, of all the compounds, there are
six which possess excellent anticancer activity against pancreatic cancer. The
theoretical reason behind the elevation in activity may be due to multiple influences
involved in chemo-therapeutic mechanisms (Figs. 2, 3).
The DPPH radical scavenging activity of the compounds was found to be
outstanding as compared to the standard BHT. In particular, the eugenol derivatives,
i.e., IIIA, IIIB, IIIC and IIID, showed outstanding EC50 values which are
comparable with STD, which may be due tothe free phenolic group and the allyl
Table 1 EC50 and GI50 values in anti-oxidant and anticancer activities of hydrazones and sulfonyl
hydrazones
Sample
no.
Compound
code
Anti-oxidant test
EC50 in lg/mL
Anticancer test
Colon cancer
GI50 in lg/mL
Pancreatic cancer
GI50 in lg/mL
1
2
3
4
5
6
7
8
9
IA
25.12 ± 0.123
22.54 ± 0.224
0.3608 – 0.154
26.08 ± 0.145
18.02 ± 0.245
0.3883 – 0.247
18.25 ± 0.115
20.8 ± 0.117
[80
[80
[80
69.2
[80
19.2
[80
[80
[80
[80
66.2
[80
10
[80
[80
[80
10
IIA
IIIA
IB
IIB
IIIB
IC
[80
10
[80
10
IIC
IIIC
ID
0.3212 – 0.138
23.25 ± 0.123
19.06 ± 0.349
0.3502 – 0.350
0.1203 – 0.213
Butylated hydroxy toluene
54.74
10
1
1
1
1
0
1
2
3
IID
IIID
STD
10
10
10
Adriamycin
(ADR)
Adriamycin
(ADR)
(
BHT)
1
23

Synthesis, biological activities and molecular docking…
Fig. 2 Cytotoxicity effects of hydrazones and sulfonyl hydrazones against measured against colon on the
HCT-15 cell line
Fig. 3 Cytotoxicity effects of hydrazones and sulfonyl hydrazones against measured against pancreatic
on the MIAPaCa-2 cell line
group present at the 5-position [23], while thymol derivatives, i.e., IA, IIA, IC and
ID, demonstrated a decrease in % anti-oxidant activity with higher EC50 values. The
lowest activity was noted for thymol derivatives at every concentration, perhaps due
to the phenolic OH present in the steric crow [24]. The anti-oxidant activity of the
compounds relates to their electron or hydrogen radical releasing abilities to DPPH
so that they become stable diamagnetic molecules. This might be the reason for the
higher or lower anti-oxidant activity [25], while carvacrol derivatives also showed
inaudible anti-oxidant activity with high EC50 values, which is comparatively lower
123

J. D. Rajput et al.
than eugenol derivatives. All EC50 values for synthesizing derivatives are
mentioned in Table 1.
Molecular docking outcomes
The docking of ligand molecules with human heme oxygenase-1 (3CZY) indicated
that all the potent compounds exhibited the bonding with more than one amino acids
in the active pocket (Fig. 4), and they may be considered as good inhibitors of heme
oxygenase-1 (3CZY). The study also showed that the phenolic monoterpenoid-
based hydrazone scaffolds are attached to the key residues, i.e., GLY143, ARG136,
SER142, HEM140, ASP140 and GLN38 of the active pocket of oxygenase-1.
Moreover, some of the compounds have minimum binding energy and docking
scores comparable with 1(adamantan1yl) 2(1Himidazol1yl) ethanone from Tables 2
and 3, and hence could be considered to have a good affinity with heme oxygenase-
1
(3CZY). The theoretical outcome highlighted that the minimum binding energy of
the molecules with the targeted enzyme, which is lower than 1(adamantan1yl)
(1Himidazol1yl) ethanone, suggests that the synthesized compounds IA, IIA, IB
2
and IC are good inhibitors of heme oxygenase-1 (3CZY). Therefore, it is striking to
state that the docking studies have extended the scope of developing phenolic
monoterpenoid-based scaffolds as promising anti-oxidant and anticancer agents.
X-ray single crystallographic analysis
Appropriate crystals were selected and mounted on a Bruker APEX-II CCD
diffractometer. During data collection, the crystal was kept at 296.15 K. The
structure was solved using Olex2 [26] with the ShelXS [27] structure solution
program by Direct Methods, and refined with the ShelXL [28] refinement package
using least squares minimisation. The crystal structures of IIC and IIA with atomic
Fig. 4 Anti-oxidant activity of hydrazones and sulfonyl hydrazones determined by the DPPH free radical
method at various concentrations
1
23

Synthesis, biological activities and molecular docking…
123

J. D. Rajput et al.
Table 3 Docking score, steric score and structural parameter of synthesized derivatives
Sample
no.
Name
Docking
score
Steric
score
Desolvation
Hydrogen bond
acceptor
Hydrogen
bond donor
1
2
3
4
5
6
7
8
9
IA
-86.19
-78.81
-84.30
-82.44
-82.80
-81.93
-92.77
-72.55
-83.62
-82.44
-82.80
-81.93
-82.20
-91.13
-86.44
-87.58
-91.14
-77.56
-92.65
-103.05
-79.07
-90.65
-91.14
-77.56
-92.65
-88.20
8.01
13.39
13.54
8.87
0.00
-3.36
-6.76
-0.17
-5.13
-0.47
0.00
-3.07
-2.41
-3.50
0.00
IIA
IIIA
IB
IIB
10.53
11.29
10.28
11.41
10.87
8.87
-10.64
-0.10
0.00
IIIB
IC
IIC
-3.57
-3.81
-0.17
-5.13
-0.47
-0.22
-1.31
-0.02
0.00
IIIC
10
11
12
13
ID
IID
10.53
11.29
9.47
-10.64
-0.10
-8.4
IIID
1(adamantan1yl)
2(1Himidazol1yl)
ethanone (Std)
Fig. 5 2D binding interactions of a IB with structural amino acid residue, b IC with structural amino
acid residue, c 3D binding interactions IC with structural amino acid residue and d 3D image of IC in the
active site of heme oxygenase-1 (3CZY)
1
23

Synthesis, biological activities and molecular docking…
Fig. 6 ORTEPs representing 50% probability ellipsoids of a IIC and b IIA
numbering are presented in Fig. 5. The IIC crystal structures show a monoclinic
crystal system and IIA crystal structures show a triclinic crystal system. The 3D
representation diagrams of anhydrous IIC and IIA are shown in Fig. 6. The crystal
structures indicate that structures of hydrazone and sulfonyl hydrazone scaffolds of
phenolic monoterpenoids are connected through the substituted hydrazine and
formyl functionality of phenolic monoterpenoids. The CCDC No. for IIC is
1
515851 and for and IIA is 1501299.
Conclusion
In conclusion, novel derivatives of carvacrol- and thymol-containing hydrazone and
sulfonyl hydrazone linkage have been synthesized by a multistep pathway with
good practical yields. Anticancer and anti-oxidant activities were tested in vitro.
The obtained results indicated that the free hydroxy as well as hydrazone and
sulfonyl hydrazone groups in given scaffolds have significantly remarkable
anticancer and anti-oxidant potency. All synthesized compounds displayed exten-
sively in vitro anti-oxidant activities by DPPH assay. In an anticancer test by using a
SRB assay against pancreatic cancer with the MIAPaCa-2 cell line and colon cancer
with the HCT-15 cell line, the GI50 values of six compounds indicated their good
anticancer efficacy which is comparable with that of STD (Adriamycin). All these
results will be useful in the future to guide the design and modification of new
natural phenolic monoterpenoid-based analogues as biologically active agents.
Acknowledgements RSB thanks UGC, New Delhi, for SAP (DSA-I) (Grant No. (No. F. 4-6/2015/DSA-I
(SAP-II)) and Jamatsing D. Rajput gratefully thanks UGC, New Delhi, for a BSR Fellowship of PhD
Candidates (Grant No. F. 25- l/2014-15 (BSR/7-136/2007(BSR), dated 13 March, 2015). We are also
grateful to Dr. Davinder Tuli for molecular docking studies.
Compliance with ethical standards
Conflict of interest The authors declare no competing financial interests.
123

J. D. Rajput et al.
References
1
2
3
4
5
6
. S. Rollas, N. Gulerman, H. Erdeniz, II Farmaco 57, 171–174 (2002)
¨
. S¸ .G. K u¨ c¸ u¨ kg u¨ zel, E.E. Oru c¸ , S. Rollas, F. S¸ ahin, A. Ozbek, Eur. J. Med. Chem. 37, 197–206 (2002)
. M. Singh, N. Raghav, Int. J. Pharm. Pharm. Sci. 3, 26–32 (2011)
. T. Scior, S.J. Garces-Eisele, A critical review. Curr. Med. Chem. 13, 2205–2219 (2006)
. N. Terzioglu, A. G u¨ rsoy, Eur. J. Med. Chem. 38, 781–786 (2003)
. P.F.M. Oliveira, M. Baron, A. Chamayou, C. Andre-Barres, B. Guidetti, M. Baltas, RSC Adv. 4,
56736–56742 (2014)
7
. S. Rollas, S.G. K u¨ c¸ u¨ kg u¨ zel, Molecules 12, 1910–1939 (2007)
. M.R. Cunha, M.T. Tavares, C.F. Carvalho, N.A. Silva, A.D. Souza, G.J. Pereira, R. Parise-Filho,
ACS Sustain. Chem. Eng. 4, 1899–1903 (2016)
8
9. M. Sharma, K. Chauhan, R.K. Srivastava, S.V. Singh, K. Srivastava, J.K. Saxena, P. Chauhan, Chem.
Biol. Drug Des. 84, 175–181 (2014)
1
0. J.D. Rajput, S.D. Bagul, R.S. Bendre, Res. Chem. Intermed. (2016). doi:10.1007/s11164-017-2919-2
1. S. Sarkar, A.A. Siddiqui, S.J. Saha, R. De, S. Mazumder, C. Banerjee, U. Bandyopadhyay,
Antimicrob. Agents Chemother. 60, 4217–4228 (2016)
1
1
2. W. Dhifi, S. Bellili, S. Jazi, N. Bahloul, W. Mnif, Crit. Rev. Med. 3, 25 (2016)
3. R.S. Bendre, J.D. Rajput, S.D. Bagul, P.S. Kar, Nat. Prod. Chem. Res. (2016). doi:10.4172/2329-
1
6836.1000212
1
1
4. C.S. Mathela, K.K. Singh, V.K. Gupta, Acta Pol. Pharm. 67, 375–380 (2010)
5. J.D. Rajput, S.D. Bagul, S. Tadavi, R.S. Bendre, Med. Aromat. Plants (Los Angel) 5, 279 (2016).
doi:10.4172/2167-0412.1000279
1
1
6. D. Kumar, D.S. Rawat, Bio. Med. Chem. Lett. 23, 641–645 (2013)
7. D.B. Suresh, D.R. Jamatsing, S.K. Pravin, S.B. Ratnamala, Mod. Chem. Appl. 4, 193 (2016). doi:10.
4172/2329-6798.1000193
1
1
2
2
2
2
2
8. L. Rubi o´ , M.J. Motilva, M.P. Romero, Crit. Rev. Food Sci. Nutr. 53, 943–953 (2013)
9. H. Am aˆ l, N. Ergen c¸ , IU Fen Fak. Mecm. 22, 390–392 (1957)
˙ ¨
0. S. Tyagarajan, P.K. Chakravarty, Tetrahedron Lett. 46, 7889–7891 (2005)
1. R.M. Mohareb, D.H. Fleita, O.K. Sakka, Molecules 16, 16–27 (2010)
2. I. Fagervall, S.B. Ross, Biochem. Pharmacol. 35, 1381–1387 (1986)
3. E. Nagababu, J.M. Rifkind, S. Boindala, L. Nakka, Free Radic. Antioxid. Protoc. 165, 180 (2010)
4. J. Mastelic, I. Jerkovic, I. Bla zˇ evi c´ , M. Poljak-Bla zˇ i, S. Borovi c´ , I. Ivan cˇ i c´ -Ba c´ e, N. M u¨ ller, J. Agric.
Food Chem. 56, 3989–3996 (2008)
2
5. E. Bendary, R.R. Francis, H.M.G. Ali, M.I. Sarwat, S. El Hady, Ann. Agric. Sci. 58, 173–183 (2013)
6. O.V. Dolomanov, L.J. Bourhis, R.J. Gildea, J.A. Howard, H. Puschmann, J. Appl. Crystallogr. 42,
2
339–341 (2009)
2
2
2
7. C.B. H u¨ bschle, G.M. Sheldrick, B. Dittrich, J. Appl. Crystallogr. 44, 1281–1284 (2011)
8. G.M. Sheldrick, Acta Crystallogr. Sect. A 71, 3–8 (2015)
9. J.D. Rajput, S.D. Bagul, S.K. Tadavi, P.S. Karandikar, R.S. Bendre, Med. Chem. (Los Angeles) 6,
123 (2016). doi:10.4172/2161-0444.1000336
3
3
0. S.D. Bagul, J.D. Rajput, S.K. Tadavi, R.S. Bendre, Res. Chem. Intermed. (2016). doi:10.1007/
s11164-016-2759-5
1. J.D. Rajput, S.D. Bagul, A.A. Hosamani, M.M. Patil, R.S. Bendre, Res. Chem. Intermed. (2017).
doi:10.1007/s11164-017-2933-4
2. V. Vichai, K. Kirtikara, Nat. Protoc. 1, 1112–1116 (2006)
3
3
3
3. S. Son, B.A. Lewis, J. Agric. Food Chem. 50, 468–472 (2002)
4. M.N. Rahman, J.Z. Vlahakis, W.A. Szarek, K. Nakatsu, Z. Jia, J. Med. Chem. 51, 5943–5952 (2008)
1
23