Synthesis, characterizations, biological activities and docking studies of novel dihydroxy derivatives of natural phenolic monoterpenoids containing azomethine linkage

Article abstract of DOI:10.1007/s11164-017-2933-4

Abstract: In the present work, we report the synthesis of six new azomethine linkage containing dihydroxy derivatives of carvacrol, thymol, and eugenol. All the synthesized derivatives have been characterized by spectroscopic techniques and their structures were confirmed by X-ray single crystallography. Synthesized derivatives were screened for anti-oxidant activity using DPPH radical scavenging assay, and anticancer activity by using SRB assay against pancreatic cancer with MIAPaCa-2 and colon cancer with HCT-15 cell lines. The molecular docking studies of all the synthesized derivatives were performed on cyclooxygenases (COX-2) protein enzyme. In the anti-oxidant test, the values of EC₅₀ indicated that all the compounds show excellent anti-oxidant potency, and similarly the GI₅₀ values in anticancer tests indicated that most of the compounds possess good anticancer efficacy. The overall docking score suggested that all the synthesized compounds exhibit good binding affinity towards cyclooxygenases (COX-2) protein enzyme.

Full text of DOI:10.1007/s11164-017-2933-4

Res Chem Intermed
DOI 10.1007/s11164-017-2933-4
Synthesis, characterizations, biological activities
and docking studies of novel dihydroxy derivatives
of natural phenolic monoterpenoids containing
azomethine linkage
Jamatsing D. Rajput1 Suresh D. Bagul1
•
•
Amar A. Hosamani2 Manohar M. Patil1
•
•
Ratnamala S. Bendre1
Received: 26 December 2016 / Accepted: 4 March 2017
Ó Springer Science+Business Media Dordrecht 2017
Abstract In the present work, we report the synthesis of six new azomethine linkage
containing dihydroxy derivatives of carvacrol, thymol, and eugenol. All the synthesized
derivatives have been characterized by spectroscopic techniques and their structures
were confirmed by X-ray single crystallography. Synthesized derivatives were screened
for anti-oxidant activity using DPPH radical scavenging assay, and anticancer activity by
using SRB assay against pancreatic cancer with MIAPaCa-2 and colon cancer with
HCT-15 cell lines. The molecular docking studies of all the synthesized derivatives were
performed on cyclooxygenases (COX-2) protein enzyme. In the anti-oxidant test, the
values of EC50 indicated that all the compounds show excellent anti-oxidant potency,
and similarly the GI50 values in anticancer tests indicated that most of the compounds
possess good anticancer efficacy. The overall docking score suggested that all the
synthesized compounds exhibit good binding affinity towards cyclooxygenases (COX-2)
protein enzyme.
Electronic supplementary material The online version of this article (doi:10.1007/s11164-017-2933-
4) contains supplementary material, which is available to authorized users.
&
Ratnamala S. Bendre
bendrers@gmail.com
1
2
School of Chemical Sciences, North Maharashtra University, Jalgaon, Maharashtra 425001,
India
Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560 012, India
123

J. D. Rajput et al.
Graphical Abstract
Keywords Phenolic monoterpenoids Á COX-2 Á Molecular docking Á Biologically
active scaffolds Á Anticancer Á Anti-oxidant
Introduction
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved in
a number of biological processes, such as degenerative, carcinogenesis, mutation
and other diseases, inflammation, development, and aging [1–5]. Both ROS and
RNS are well documented for performing a double role as both injurious and
beneficial species [6, 7]. Free radicals can destroy DNA and cause mutagenicity and
cytotoxicity, and thus perform a significant role in carcinogenesis. It is believed that
ROS can induce mutations and inhibit the DNA repair process that results in the
activation of certain tumor repressors, leading to cancer [8, 9]. To counterbalance
such adverse effects, there is a demand for natural and synthetic anti-oxidants to
support the endogenous defenses [10]. Anti-oxidants must react with radicals and
other reactive species more quickly than the biological entity, thus protecting
biological targets from oxidative injury [11, 12]. In addition, the ensuing anti-
oxidant radical must possess excessive stability, that is, the anti-oxidant radical must
disturb a chain reaction [13, 14]. Recently, research has shown that cancer cells
123

Synthesis, characterizations, biological activities and…
produce higher levels of ROS to help feed their abnormal growth [15]. In laboratory
and animal studies, the presence of increased levels of exogenous anti-oxidants has
been shown to prevent the types of free radical damage that have been associated
with cancer development [16, 17].
The literature has suggested that phenolic compounds usually have probable anti-
oxidant and anticancer activity due to phenolic hydroxyl groups which act as a
proton donor and are responsible for a redox-type mechanism [18, 19]. Mastelic
et al. reported anti-oxidant activity of four dihydroxy derivatives of carvacrol,
thymol and eugenol for the first time [1–4], concluding that the results of DPPH and
Rancimat assay demonstrated that all synthesized derivatives possess remarkable
anti-oxidative properties as well as antiproliferative effects against human uterine
carcinoma cells (HeLa) [20].
Carvacrol, thymol and eugenol are present in the essential oils of several plants
[21, 22]. These naturally occurring phenolic monoterpenoids are outstanding
creative molecules incorporated as useful constituents in various products and have
found wide applications in pharmaceutical, agricultural, fragrance, flavor, cosmetic
and various other industries [23, 24]. Their vast range of pharmacological actions
including antimicrobial, anti-inflammatory, analgesic, anti-oxidant and anticancer
activities have been well researched [25, 26] (Fig. 1).
In the present study, we report six new dihydroxy derivatives of carvacrol,
thymol, and eugenol containing azomethine functionality which possess excellent
anti-oxidant and good anticancer activities. Docking simulations of all the
derivatives was performed on the COX-2 protein enzyme which is responsible for
undeclared changes in biochemical processes.
Experimental
All the chemicals and reagents necessary for the reactions were procured from
Sigma-Aldrich and Fisher scientific chemicals with purity 98% and used without
further purification. The products were characterized using 1H NMR, 13C NMR, IR,
LC–MS spectra and X-ray single crystallography. NMR spectra of the products
were obtained using Bruker AC-400 MHz spectrometers with TMS as the internal
standard. Mass spectra of the products were obtained using Waters 2795 micromass
Q-TOF micro LC–MS.
HO
HO
HO
OH
OH
OH
HO
HO
O
2
1
3
4
Fig. 1 Reported dihydroxy derivatives of carvacrol, thymol and eugenol
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J. D. Rajput et al.
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 [32] with the ShelXS [33] structure solution
program by Direct Methods and refined with the ShelXL [34] refinement package
using Least Squares minimization. The crystal structures of IIA and IIB with atomic
numbering are presented in Fig. 2. Both the crystal structures show monoclinic
crystal system. The 3D packing diagram of anhydrous carvacryl thymol (IIA) and
carvacryl carvacrol (IIB) are shown in Fig. 3. Hydrogen bonding present in all the
derivatives are observable in NMR which has been confirmed by X-ray single
crystallography. Figure 4 showing the intermolecular and intramolecular hydrogen
bonding in IIA and IIB. The presented structures illustrate the formation of
carvacryl thymol (IIA) and carvacryl carvacrol (IIB) upon the condensation of
respective ortho formyl phenolic monoterpenoids with para amino phenolic
monoterpenoids. The crystal structures indicated that the two-fold dimeric structures
of azomethine scaffolds of phenolic monoterpenoids are connected through the
nitrogens of para amino phenolic monoterpenoids and formyl functionality of
phenolic monoterpenoids. The CCDC No. for carvacryl carvacrol (CC) is 1452108
and for carvacryl thymol (CT) is 1482770.
Synthesis of ortho formylation on phenolic monoterpenes [35]
NaOH (6.66 g, 0.16 M) in (7 ml) distilled water was placed in a round-bottom flask
fitted with a water reflux condenser and a thermometer, to which phenolic
monoterpene (0.02 mol) was added. The temperature inside the flask was adjusted
to 60–65 °C. Chloroform (0.04 mol) was introduced in small portions with stirring
by maintaining the temperature at 60–65 °C during addition. The mixture was
stirred for 2 h. Excess of chloroform was removed by steam distillation from the
alkaline solution. The solution was allowed to cool and was then acidified with
dilute HCl (1%). The acidified solution was steam-distilled until no more oily drops
Fig. 2 ORTEPs representing 50% probability ellipsoids of a CT (IIA) and b CC (IIB)
123

Synthesis, characterizations, biological activities and…
Fig. 3 Packing diagram of a form IIA, space group P21/n, and b form IIB, space group Cc
Fig. 4 Existence of intermolecular and intramolecular hydrogen bonding in IIA and IIB
were collected. The distillate was extracted with ether (3 9 30 ml). Most of the
ether was removed by distillation. The residue, which contains unreacted phenolic
monoterpene and ortho formylated phenolic monoterpene, was transferred to a small
glass-stoppered flask to which about twice the volume of saturated sodium
metabisulfite solution was added. The solution was stirred vigoursly for 30 min and
allowed to stand for 1 h. The paste of the bisulfite compound was filtered and
washed with a little ethanol and finally with a little ether. The bisulfite compound
was decomposed by warming in a round-bottom flask on a water bath with dilute
H2SO4. The mixture was allowed to cool and then the ortho formylated phenolic
monoterpene was extracted with ether (3 9 30 ml). Removal of the ether gave pure
ortho formylated phenolic monoterpene. Finally, all three ortho formyl phenolic
monoterpenes were characterized by spectroscopic methods with yields of carvacrol
aldehyde 70%, thymol aldehyde 70% and eugenol aldehyde 75%.
Synthesis of 4-nitroso thymol and 4-nitroso carvacrol [36]
To a solution of carvacrol or thymol (3.00 g, 0.02 mol) in 95% ethyl alcohol
(20 ml), concentrated hydrochloric acid (20 ml) was added. The mixture was cooled
to 0–5 °C and sodium nitrite (1.08 g, 0.015 mol) was continuously added slowly in
small lots in 1 h. The mixture was stirred well after each addition. It became brown
in colour and a green precipitate soon began to form. After 0.5 g of nitrite had been
123

J. D. Rajput et al.
added, the mixture became pasty, so the intervals between the additions were
lengthened and the stirring was made more vigorous. When all the nitrite had been
added, the bulk of the product was transferred to a 250-ml beaker containing 120 ml
of cold water and the remainder was washed with water. After agitation with water,
the product became a light yellow, fluffy solid. It was filtered off by suction, washed
well with water and crystallized from ethanol with yields for both 4-nitroso
carvacrol and thymol of 75%.
Synthesis of 4-amino thymol and 4-amino carvacrol [37]
The crude wet 4-nitrosocarvacrol (4-nitrosothymol) (3.1 g, 0.0174 mol) from the
second step was dissolved in a mixture of 28% aqueous ammonia (30 ml) and water
(50 ml). The brown solution thus obtained was filtered to remove resinous matter
and hydrogen sulfide was passed into it. The brown colour disappeared and a white
precipitate of 4-aminocarvacrol (4-aminothymol) was obtained, with a yield of 82%.
Due to less stability, the product was used for the next step without purification.
Synthesis of Azomethine Scaffolds of thymol, carvacrol and eugenol [38]
The Schiff bases were prepared by the reaction of equimolar quantities of 4-amino
thymol (or 4-amino carvacrol) and ortho formyl thymol (or ortho formyl carvacrol
or ortho formyl eugenol). Each reactant was dissolved in a minimum amount of
ethanol, and they were mixed together. The reaction mixture was refluxed for 4 h,
then cooled to room temperature and poured into ice-cold water. The solid product
obtained was collected by filtration and dried in an oven at 70 °C. The product was
recrystallized from ethanol and dried to obtain pure product.
Spectroscopic characterizations and physical properties of synthesized
derivatives
(E)-4-(2-hydroxy-6-isopropyl-3-methylbenzylideneamino)-5-isopropyl-2-methylphenol
(CC) Color light red mp-118 °C, Yield: 68%, 1H NMR (DMSO) d 1.21 (d,
J = 4.0 Hz, 6H), d 1.27 (d, J = 4.0 Hz, 6H), 2.15–2.18 (s, 6H), 3.33–3.34 (m,
J = 4.0 Hz, 1H), 3.58–3.62 (m, J = 4.0 Hz, 1H), 6.71–6.73 (d, J = 8.0 Hz, 1H),
6.79 (s, 1H), 7.04 (s, 1H), 7.15–7.17 (d, J = 8.0 Hz, 1H), 9.03 (s, 1H for OH), 9.26
(s, 1H for imine), 14.98 (s, 1H for OH hydrogen bonded with nitrogen of imine), 13
C
NMR (DMSO) d 15.30, 15.57, 23.01,23.90, 27.12, 27.50, 78.4, 78.76, 79.43,
111.51, 114.30, 114.83, 120.09, 122.34, 122.53, 133.47, 137.13, 140.47, 147.28,
154.98, 157.13, 159.84. LC–MS (methanol), m/z: [M ? 1]? 326.57, [M ? 2]?
327.37.
(E)-4-(2-hydroxy-3-isopropyl-6-methylbenzylideneamino)-5-isopropyl-2-methylphenol
(CT) Color light red, mp-112 °C, Yield: 66%, 1H NMR (DMSO) d 1.21 (d,
J = 4.0 Hz, 6H), d 1.24 (d, J = 4.0 Hz, 6H), 2.16–2.26 (s, 6H), 3.61–3.62 (m,
J = 4.0 Hz, 1H), 3.65–3.67 (m, J = 4.0 Hz, 1H), 6.71–6.74 (d, Hz, 2H), 7.17–7.19
(d, Hz, 2H), 9.07 (s, 1H for OH), 9.43 (s, 1H for imine), 15.22 (s, 1H for OH
123

Synthesis, characterizations, biological activities and…
hydrogen bonded with nitrogen of imine), 13C NMR (DMSO) d 15.33, 17.62, 22.36,
23.88, 26.73, 27.03, 27.50, 114.36, 114.85, 115.60, 116.76, 122.59, 130.16, 133.04,
138.00, 147.42, 153.77, 153.77, 156.68, 160.09. LC–MS (methanol), m/z: [M ? 1]?
326.57, [M ? 2]? 327.37.
(E)-4-(5-allyl-2-hydroxy-3-methoxybenzylideneamino)-5-isopropyl-2-methylphe-
1
nol (CE) Color orange, mp-118 °C, Yield: 70%, H NMR (DMSO) d 1.21 (d,
J = 4.0 Hz, 6H), 2.27 (s, 3H), 3.39-3.40 (m, J = 4.0 Hz, 1H), 6.71–6.73 (d,
J = 8.0 Hz, 1H), 3.33–3.35 (d, 2H), 3.84 (s, 3H), 5.05 (dd 2H), 5.92–5.98 (m 1H),
6.71 (s, 1H), 7.84 (s, 1H), 6.98 (s, 1H), 7.17 (s, 1H), 8.77 (s, 1H for OH), 9.26 (s, 1H
for imine), 14.04 (s, 1H for OH hydrogen bonded with nitrogen of imine), 13C NMR
(DMSO) d 17.49, 22.10, 26.38, 55.67, 78.35, 78.68, 76.01, 114.70, 114.95, 152.52,
116.66, 118.91, 122.79, 129.29, 130.35, 133.09, 137.31, 137.39, 153.78, 158.65.
LC–MS (methanol), m/z: [M ? 1]? 340.55, [M ? 2]? 341.58.
(E)-4-(2-hydroxy-3-isopropyl-6-methylbenzylideneamino)-2-isopropyl-5-methylphenol
(TT) Color red, mp-116 °C, Yield: 70%, 1H NMR (DMSO) d 1.18 (d, J = 4.0 Hz,
6H), d 1.22 (d, J = 4.0 Hz, 6H), 2.27 (s, 3H), 2.51 (s, 3H), 3.20–3.22 (m,
J = 4.0 Hz, 1H), 3.31–3.32 (m, J = 4.0 Hz, 1H), 6.63–6.65 (d, J = 8.0 Hz, 1H),
6.72 (s, 1H), 7.11 (s, 1H), 7.12 (d, J = 8.0 Hz, 1H), 8.92 (s, 1H for OH), 9.30 (s, 1H
for imine), 14.98 (s, 1H for OH hydrogen bonded with nitrogen of imine), 13C NMR
(DMSO) d 17.15, 18.50, 22.28, 22.36, 25.67, 26.64, 78.49, 78.82, 79.15, 115.42,
116.42, 116.70, 120.01, 128.75, 130.04, 133.01, 133.46, 136.54, 138.11, 153.68,
157.14, 159.14. LC–MS (methanol), m/z: [M ? 1]? 326.57, [M ? 2]? 327.37.
(E)-4-(2-hydroxy-6-isopropyl-3-methylbenzylideneamino)-2-isopropyl-5-methylphenol
(TC) Color red, mp-112 °C, Yield: 68%, 1H NMR (DMSO) d 1.21 (d, J = 4.0 Hz,
6H), d 1.24 (d, J = 4.0 Hz, 6H), 2.16–2.26 (s, 6H), 3.61–3.62 (m, J = 4.0 Hz, 1H),
3.65–3.67 (m, J = 4.0 Hz, 1H), 6.71–6.74 (d,8 Hz, 2H), 7.17–7.19 (d, 8 Hz, 2H),
9.07 (s, 1H for OH), 9.43 (s, 1H for imine), 15.22 (s, 1H for OH hydrogen bonded
with nitrogen of imine), 13C NMR (DMSO) d 15.33, 17.62, 22.36, 23.88, 26.73,
27.03, 27.50, 114.36, 114.85, 115.60, 116.76, 122.59, 130.16, 133.04, 138, 147.42,
153.77, 153.77, 156.68, 160.09. LC–MS (methanol), m/z: [M ? 1]? 326.57,
[M ? 2]? 327.37.
(E)-4-(5-allyl-2-hydroxy-3-methoxybenzylideneamino)-2-isopropyl-5-methylphenol
1
(TE) Color light orange, mp-120 °C, Yield: 70%, H NMR (DMSO) d 1.21 (d,
J = 4.0 Hz, 6H), 2.27 (s, 3H), 3.39–3.40 (m, J = 4.0 Hz, 1H), 3.33 (d, 2H), 3.84 (s,
3H), 5.05 (dd 2H), 5.92–5.98 (m 1H), 6.71 (s, 1H), 7.84 (s, 1H), 6.98 (s, 1H), 7.17
(s, 1H), 8.77 (s, 1H for OH), 9.26 (s, 1H for imine), 14.04 (s, 1H for OH hydrogen
bonded with nitrogen of imine), 13C NMR (DMSO) d 17.49, 22.10, 26.38, 55.67,
78.35, 78.68, 76.01, 114.70, 114.95, 152.52, 116.66, 118.91, 122.79, 129.29,
130.35, 133.09, 137.31, 137.39, 153.78, 158.65. LC–MS (methanol), m/z: [M ? 1]?
340.55, [M ? 2]? 341.58.
123

J. D. Rajput et al.
Results and discussion
Reaction schemes
The synthesis of compounds in Schemes 1 and 2 involved a multi-step pathway
leading to the formation of a variety of novel dihydroxy phenolic monoterpenoid-
based azomethine scaffolds, IA–IIIB in excellent yields. The structures of the
compounds obtained are shown in Scheme 3. Their analytical and spectroscopic
data are in agreement with the predicted structures. Recently, we have reported
number of derivatives of phenolic monoterpenes and their biological activities
[27, 28]. Syntheses of ortho formyl derivative of (I, II and III) and 4-amino analogs
(A and B) of phenolic monoterpenoids were carried out by reported methods. The
reaction of ortho formyl phenolic monoterpenoids (I, II and III) and 4-amino
phenolic monoterpenoids (A and B) in ethanol at 80 °C for 5 h resulted in the
coupling products (IA–IIIB) in exceptional yields.
OH
OH
HO
HO
A
A
CHO
OHC
I
II
HO
O
A
OHC
HO
O
III
Reagents & Reaction conditions: A = CHCl3, NaOH in water, reflux at 60-65 oC for 2h.
Scheme 1 Synthesis of ortho formyl phenolic monoterpenoids
HO
HO
HO
OH
OH
C
B
OH
B
C
NO
NH2
ON
H2N
B
A
Reagents & reaction conditions: B= Ethanol, NaNO2 + HCl at 0-5 oC for 4h. C= H2O, 28% NH3 + H2S for 3h.
Scheme 2 Synthesis of 4-amino phenolic monoterpenoids
123

Synthesis, characterizations, biological activities and…
OH
N
OH
N
HO
HO
D
IIB (CC)
D
IA (TT)
OH
HO
D
OH
OH
D
N
N
CHO
OHC
HO
OHC
HO
HO
IB (TC)
IIA (CT)
O
D
D
O
OH
O
OH
N
N
HO
IIIA (TE)
HO
IIIB (CE)
Reaction Conditions: D= A or B in Ethanol, Reflux at 80 oC for 2h.
Scheme 3 Synthesis of phenolic monoterpenoids based azomethine scaffolds
Bioassay
Anticancer activity
In vitro anticancer activities of synthesized derivatives were performed using
sulforhodamine B (SRB) assay on a panel of human cancer cell lines HCT-12 (colon
cancer) and MIAPaCa-2 (pancreatic cancer) [29]. The cell lines were grown in
RPMI 1640 medium containing 10% fetal bovine serum and 2 mM L-glutamine. For
the present screening experiment, cells were inoculated into 96-well microtiter
plates in 90 ll at 5000 cells per well. After cell inoculation, the microtiter plates
were incubated at 37 °C, 5% CO2, 95% air and 100% relative humidity for 24 h
prior to the addition of the experimental drugs, which were solubilized in the
appropriate solvent to prepare a stock of 10-2 concentration. At the time of the
experiment, four tenfold serial dilutions were made using complete medium.
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J. D. Rajput et al.
Aliquots of 10 ll of these different drug dilutions were added to the appropriate
micro-titer wells already containing 90 ll of medium, resulting in the required final
drug concentrations. After addition of the compound, plates were incubated at
standard conditions for 48 h and the assay was terminated by the addition of cold
TCA. Cells were fixed in situ by the gentle addition of 50 ll of cold 30% (w/v) TCA
(final concentration, 10% TCA) and incubated for 60 min at 4 °C. The supernatant
was discarded, and the plates were washed five times with tap water and air-dried.
Sulforhodamine B (SRB) solution (50 ll) at 0.4% (w/v) in 1% acetic acid was
added to each of the wells, and the plates were incubated for 20 min at room
temperature. After staining, unbound dye was recovered and the residual dye was
removed by washing five times with 1% acetic acid. The plates were air-dried.
Bound stain was subsequently eluted with 10 mM trizma base, and the absorbance
was read on an Elisa plate reader at a wavelength of 540 nm with 690-nm reference
wavelength. The GI50 values, defined as the drug concentration required for
inhibiting the growth of cell proliferation by 50%, were calculated from percent
growth and were expressed as the ratio of average absorbance of the test well to the
average absorbance of the control well 9100. Using the six absorbance measure-
ments [time zero (Tz), control growth (C), and test growth in the presence of drug at
the four concentration levels (Ti)], the percentage growth was calculated at each of
the drug concentration level and the data are presented in Figs. 5 and 6, and the
results are expressed as GI50 (growth inhibitory concentration at 50%) in Table 1.
Cell growth inhibition was analyzed by SRB assay and the results show that the
compounds IA–IIIA and IB–IIIB exhibit an inhibitory effect on the proliferation of
HCT-15 and MIAPaCa-2 cells in a dose-dependent manner (Table 1). Compounds
IA and IIIA were found to exhibit moderate cytotoxic potency (22.1 and 59.2 lg/
ml) compared with that of Doxorubicin (10 lg/ml) against the HCT-15 cell.
Similarly, the compounds IA and IIA displayed excellent cytotoxic potency (10.77
and 5.2 lg/ml) copmared with that of Doxorubicin (10 lg/ml) against MIAPaCa-2.
Of note is that the amino carvacrol derivatives (IB–IIIB) presented better activity.
Fig. 5 Cytotoxicity effects of IA–IIIA and IB–IIIB against measured against Colon on HCT-15 cell line
123

Synthesis, characterizations, biological activities and…
Fig. 6 Cytotoxicity effects of IIIA and IB–IIIB measured against Pancreatic on MIAPaCa-2 cell line
Table 1 EC50 and GI50 values in anti-oxidant and anticancer activities of compounds IA–IIIA and IB–
IIIB
Sample
no.
Name of derivatives
Anti-oxidant test
EC50 in lg/ml
Anticancer test
Colon cancer GI50 Pancreatic
in lg/ml
cancer GI50 in
lg/ml
1.
2.
3.
4.
5.
6.
7.
Carvacryl carvacrol CC (IIB) 0.3906 0.350
Carvacryl thymol CT (IIA) 0.5188 0.349
Carvacryl eugenol CE (IIIB) 0.2316 0.331
80
16.9
80
10.77
16.5
80
Thymyl thymol TT (IA)
Thymyl carvacrol TC (IB)
Thymyl eugenol TE (IIIA)
STD
18.74 0.214
0.1171 0.542
0.4136 0.113
0.1203 0.213
22.1
5.8
80
82.5
59.2
43.4
10
10
Butylated
hydroxy toluene
(BHT)
Doxorubicin
Doxorubicin
Anti-oxidant activity
DPPH radical-scavenging activity was performed by the reported method [30]. For
each determination, the stock solution (1 mg/ml) was diluted by serial dilution
(25–500 lg/ml) with 60% (v/v) ethanol. An aliquot of each dilution (0.5 ml) was
mixed with a methanolic solution of DPPH (5 ml, 0.06 mM). The mixtures were
shaken vigorously and incubated at 37 °C in dthe ark for 30 min. At the same time,
123

J. D. Rajput et al.
a control containing 60% (v/v) ethanol (0.5 ml) and the methanolic solution of
DPPH (5 ml, 0.06 mM) was run. The absorbance was measured at 517 nm. The
percentage of DPPH scavenging versus concentration of samples was plotted. The
concentration of the sample necessary to decrease the DPPH concentration by 50%
was obtained by interpolation from linear regression analysis and denoted as the
EC50 value (lg/ml). All determinations were carried out in triplicate. Butylated
hydroxy toluene (BHT) was used as the reference compound. The % radical
scavenging against concentration of entity has been represented in Fig. 7.
DPPH radical scavenging activity of the compounds was found to be good to
outstanding as compared to the standard BHT. Derivatives IB, IIIB, IIB, IIIA and
IIA showed outstanding EC50 values which are comparable with STD, while IA
showed a decrease in % anti-oxidant activity with a higher EC50 value. The lowest
activity was noted for IA at every concentration, perhaps due to the phenolic OH
Fig. 7 Anti-oxidant activity of azomethine scaffolds (IA–IIIB) determined by the DPPH free radical
method at various concentrations
Fig. 8 Binding mode of inhibitors in active site of cyclooxygenases (4PH9). a 2D interaction of IB with
4PH9. b 3D binding of IIA with amino acids of cyclooxygenases (4PH9)
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Synthesis, characterizations, biological activities and…
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J. D. Rajput et al.
Table 3 Docking score, steric score and structural parameter of synthesized derivatives
Sample Name
no.
Docking
score
Steric
score
Desolvation Hydrogen bonding
acceptor
Hydrogen bonding
donor
1.
2.
3.
4.
5.
6.
7.
TE
CT
CE
TT
CC
TC
-65.09
-63.07
-62.62
-61.56
-60.89
-56.69
-66.29
-67.19
-69.62
-62.90
-62.91
-60.05
-82.91
8.79
5.66
8.20
6.50
6.18
6.30
10.35
-7.55
-0.30
-0.17
-3.89
-1.82
-0.25
-8.72
-0.03
-1.23
-1.03
-1.26
-2.34
-2.70
0.00
Ibuprofen -81.28
present in the steric crowd. The anti-oxidant activity of the compounds is related to
their electron or hydrogen radical releasing abilities towards DPPH, so that they
become stable diamagnetic molecules. Among the series, IB showed the highest
anti-oxidant activity with a remarkable EC50 value particularly lower in comparison
with STD. All EC50 values are listed in Table 1.
Molecular docking studies
The docking study was performed by FRED (Open Eye.lnc.) and has been used to
determine the orientation of inhibitors bound in the active site of cyclooxygenases
(COX-2, PDB code: 4PH9). The PDB files were downloaded from the Protein Data
Bank (www.rsc.org). The ligands were drawn in Chem Draw Ultra 12.0. The FRED
docking programme was used to perform molecular docking [31]. The docking of
ligand molecules with COX-2 indicated that all the inhibitor compounds exhibit
bonding with more than one amino acids in the active pocket as shown in Fig. 8, and
may be considered as good inhibitors of cyclooxygenases (COX-2). The study also
revealed that the phenolic monoterpenoid-based scaffolds are attached to key
residues, i.e. TYR386, ARG121, ARG514 and SER354 of the active site of
cyclooxygenases (COX-2). Moreover, all the compounds possess the lowest binding
energy and docking score as presented in Tables 2 and 3, and hence could be
considered as a good inhibitors of cyclooxygenases (COX-2). The theoretical out-
come highlighted that the minimum binding energy of the molecules with the
targeted protein may make these newly synthesized phenolic monoterpenoid-based
scaffolds good inhibitors of cyclooxygenases (COX-2). Therefore, it is striking that
the docking studies have extended the scope of developing phenolic monoterpenoid
derivatives as promising anti-oxidant and anticancer agents.
Conclusion
In conclusion, we have prepared novel dihydroxy analogues of phenolic monoter-
penoids (IA–IIIB) using simple chemistry, and synthesized structures were
confirmed by X-ray single crystallography. Anticancer and anti-oxidant activities
123

Synthesis, characterizations, biological activities and…
were tested in vitro. The obtained results indicated that the introduction of another
hydroxy group in a given scaffold has significantly improved its anti-oxidant
potency. All synthesized compounds displayed extensive in vitro anti-oxidant
activities by DPPH assay. Anticancer tests were performed 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 value in the anticancer test of some of the compounds
showed good anticancer efficacy. The molecular docking studies of all the
synthesized derivatives were performed on COX-2 enzyme. Overall docking scores
suggested that the synthesized derivatives will lead to new cyclooxygenases (COX-
2) inhibitors.
Supplementary data (ESI)
1
Complete H, 13C NMR, LC–MS, IR and crystal data e included, CIF files. Also
contain 3D model of molecular docking of IA to IIIC.
Acknowledgements RSB thanks UGC, New Delhi, for SAP (DSA-I) and Jamatsing D. Rajput gratefully
thanks UGC, New Delhi, for BSR Fellowship of PhD Candidates. We are also grateful to Prof. T. N. Guru
Row, IISC, Bangalore, for help in solving the crystal structures. Finally, we are grateful to Dr. Davinder
Tuli for the molecular docking studies.
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