Synthesis and evaluation of anti-oxidant and cytotoxic activities of novel 10-undecenoic acid methyl ester based lipoconjugates of phenolic acids

Article abstract of DOI:10.3762/bjoc.13.4

The synthesis of five novel methyl 10-undecenoate-based lipoconjugates of phenolic acids from undecenoic acid was carried out. Undecenoic acid was methylated to methyl 10-undecenoate which was subjected to a thiol-ene reaction with cysteamine hydrochloride. Further amidation of the amine was carried out with different phenolic acids such as caffeic, ferulic, sinapic, coumaric and cinnamic acid. All synthesized compounds were fully characterized and their structures were confirmed by spectral data. The antioxidant activity of the synthesized lipoconjugates of phenolic acids was studied by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and also by the inhibition of linoleic acid oxidation in micellar medium by differential scanning calorimetry (DSC). The prepared compounds were also screened for their cytotoxic activity against five cell lines. It was observed that the lipoconjugates of caffeic acid, sinapic acid, ferulic acid, and coumaric acid displayed anticancer and anti-oxidant properties. The anticancer properties of these derivatives have been assessed by their IC₅₀ inhibitory values in the proliferation of MDA-MB231, SKOV3, MCF7, DU 145 and HepG2 cancer cell lines.

Full text of DOI:10.3762/bjoc.13.4

Synthesis and evaluation of anti-oxidant and cytotoxic
activities of novel 10-undecenoic acid methyl ester
based lipoconjugates of phenolic acids
Naganna Narra1,2, Shiva Shanker Kaki*1,2,§, Rachapudi Badari Narayana Prasad1,2,
Sunil Misra2,3, Koude Dhevendar2,3, Venkateshwarlu Kontham1,2
and Padmaja V. Korlipara*1,2,§
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2017, 13, 26–32.
1Centre for Lipid Research, CSIR-Indian Institute of Chemical
Technology, Uppal Road, Hyderabad 500007, India, 2Academy of
Scientific and Innovative Research, New Delhi, India and 3Biology
Division, CSIR-Indian Institute of Chemical Technology, Uppal Road,
Hyderabad 500007, India
doi:10.3762/bjoc.13.4
Received: 24 October 2016
Accepted: 19 December 2016
Published: 04 January 2017
Email:
This article is part of the Thematic Series "Lipids: fatty acids and
derivatives, polyketides and isoprenoids".
Shiva Shanker Kaki* - shivashanker.kaki@iict.res.in;
Padmaja V. Korlipara* - kvpadmaja@iict.res.in
Guest Editor: J. S. Dickschat
* Corresponding author
§ Tel/Fax +91-40-27193370
© 2017 Narra et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
anticancer; anti-oxidants; caffeic acid; coumaric acid; ferulic acid;
phenolic lipids; sinapic acid; undecenoic acid
Abstract
The synthesis of five novel methyl 10-undecenoate-based lipoconjugates of phenolic acids from undecenoic acid was carried out.
Undecenoic acid was methylated to methyl 10-undecenoate which was subjected to a thiol–ene reaction with cysteamine hydro-
chloride. Further amidation of the amine was carried out with different phenolic acids such as caffeic, ferulic, sinapic, coumaric and
cinnamic acid. All synthesized compounds were fully characterized and their structures were confirmed by spectral data. The anti-
oxidant activity of the synthesized lipoconjugates of phenolic acids was studied by the 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical scavenging assay and also by the inhibition of linoleic acid oxidation in micellar medium by differential scanning calorime-
try (DSC). The prepared compounds were also screened for their cytotoxic activity against five cell lines. It was observed that the
lipoconjugates of caffeic acid, sinapic acid, ferulic acid, and coumaric acid displayed anticancer and anti-oxidant properties. The
anticancer properties of these derivatives have been assessed by their IC50 inhibitory values in the proliferation of MDA-MB231,
SKOV3, MCF7, DU 145 and HepG2 cancer cell lines.
Introduction
Phenolic compounds are a class of natural compounds which oxidant, antimicrobial, anti-inflammatory, anticarcinogenic and
are found ubiquitously in the plant kingdom. They are reported antiviral activities [1]. The phenolic acids are also reported to
to possess a wide range of biological properties like anti- show in vitro anti-oxidant activity against many reactive oxygen
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Beilstein J. Org. Chem. 2017, 13, 26–32.
species and to protect neuronal cells against various types of ox- where the phenolic acids were linked to the olefinic group of
idative damage [2,3]. To increase the effectiveness of phenolic undecenoic acid via a thioamide spacer. Among the various
compounds, their lipophilization has been the choice of deriva- fatty acids reported, 10-undecenoic acid is unique due to its
tization as it provides beneficial effects of both the phenolics bifunctional nature with an odd-numbered carbon atom chain
and the lipid involved in one chemical entity [4,5]. Lipids, espe- length derived from castor oil. There have been several reports
cially fatty acids and their derivatives are known for their broad on the synthesis and evaluation of undecenoic acid-based deriv-
spectrum of activity which expands their application in devel- atives due to its wide applicability ranging from biological ac-
oping new hybrid biomolecules which help in host defenses tivity, natural products and polymer applications [10,11]. This
against potential pathogenic microbes. Research interest in pro- type of compounds could be useful as potential novel lipid de-
ducing new phenolipids has been increasing due to the poten- rivatives because of the presence of lipophilic chain and the
tial applications of such products in biomedical fields. Earlier phenolic amide conjugate.
reports on the production of phenolipids were focused on the in-
Results and Discussion
corporation of phenolic compounds into triglycerides where a
number of phenolic acids were transesterified with different oils Synthesis
or triglycerides [6]. Apart from these structured phenolipids, 10-Undecenoic acid was chosen as the lipid part as the deriva-
different fatty acids were esterified with phenolic compounds to tives of undecenoic acid have been reported to be potent bioac-
produce novel esters which were evaluated for anti-oxidant and tive compounds [12,13]. Additionally the terminal double bond
antimicrobial activities [7,8]. However, there are very few of undecenoic acid provides a reactive group for further deriva-
reports where the phenolic acids have been derivatized with tization for producing potential functional derivatives. The syn-
other functionalities apart from esters. The reported compounds thetic route followed for the synthesis of the phenolipids is
other than esters were amides where bioconjugates of fatty shown in Scheme 1. Initially, undecenoic acid was treated with
acids and amino acids were prepared and evaluated for their sulfuric acid in methanol to obtain methyl undecenoate (1) in
anti-oxidant activity by a DPPH radical assay [9]. In view of quantitative yield. Next, ester 1 was treated with 1,1′-
developing new conjugates of phenolic lipids, we have synthe- azobis(cyclohexanecarbonitrile) (ABCN) and 2-mercaptoethyl-
sized novel derivatives of phenolic lipids from undecenoic acid amine hydrochloride in dioxane/ethanol 70:30 (v:v) to obtain
Scheme 1: Synthetic procedure for the preparation of 10-undecenoic acid methyl ester-based lipoconjugates of phenolic acids.
27

Beilstein J. Org. Chem. 2017, 13, 26–32.
methyl 11-(2-aminoethylthio)undecanoate (2) in 89% yield. The acid containing the synthesized compounds were subjected to
structures of compounds 1 and 2 were in agreement with the re- DSC analysis. The results of the assay are shown in Table 2 and
ported literature data [14,15].
α-TP and TBHQ were included as standard anti-oxidants.
Finally, amine 2 was reacted with different phenolic acids in the
presence of EDC·HCl and HOBt to give amides 3a–3e with
reproducible yields in the range of 84–86%.
Table 2: DSC study of the synthesized 10-undecenoic acid methyl
ester–based lipoconjugates 3a–e.
Compounda
OITb (°C)
Anti-oxidant activity
LA + 3a
LA + 3b
LA + 3c
LA + 3d
LA + 3e
LA + α-TP
LA +TBHQ
LA
116
130
136
141
142
130
126
116
The anti-oxidant activities of the prepared derivatives were
studied by the well-established DPPH radical scavenging assay
and also by studying the oxidation of linoleic acid using DSC.
The DPPH radical is a commercially available stable free
radical which is widely used to preliminarily determine the
radical scavenging potential of compounds. The results for the
ability of the prepared compounds to scavenge the DPPH
radical are shown in Table 1 along with reference anti-oxidants
α-tocopherol (α-TP) and tert-butylhydroquinone (TBHQ). As
can be seen, all synthesized derivatives exhibit radical scav-
enging ability except the cinnamic acid derivative 3a. This
aLA: linoleic acid, bOIT: oxidative induction temperature.
could be due to the absence of a phenolic hydroxy group which Pure linoleic acid showed an oxidative induction temperature
is responsible for the anti-oxidant activity of most of the natural (OIT) of 116 °C which was found to increase when anti-
phytochemicals. Among all compounds, 3c was found to be the oxidants were added. It can be observed that the prepared deriv-
most efficient free radical scavenger which showed a value atives had a positive influence on the oxidation of linoleic acid
closest to the standard anti-oxidant, α-TP. Compounds 3d and except for derivative 3a which did not show any anti-oxidant
3e also showed free radical scavenging activity (FRSA) of 68% activity (see Supporting Information File 1, Figure S20 for DSC
and 67%, respectively, whereas compound 3b showed only curves). All other derivatives were found to exhibit very good
moderate activity with 30% FRSA.
protective activity against oxidation of linoleic acid and the
OITs were found to be similar or in case of compounds 3c, 3d
and 3e even higher compared to the reference anti-oxidants
TBHQ and α-TP. The OIT for TBHQ and α-TP were observed
to be 126 and 130 °C, respectively, whereas compound 3b
showed an OIT of 130 °C. As the anti-oxidant activity has been
reported [17] to depend on several factors such as the medium
of an assay, number and position of phenolic hydroxy groups,
etc. the differences in the anti-oxidant potentials of the pre-
pared phenolipids could be due to different media used for the
assays; the DPPH assay is conducted in a polar medium but the
linoleic acid oxidation study is conducted in a non-polar envi-
ronment.
Table 1: DPPH radical scavenging activity of the synthesized
10-undecenoic acid methyl ester-based lipoconjugates.
Compound
FRSA (%) at 1.0 mM concentration
3a
3b
–a
30.23
87.05
67.68
66.57
90.23
92.34
3c
3d
3e
α-TP
TBHQ
ano activity.
Cytotoxic activity
As there were studies reported on the cytotoxicity of phenolic
lipids, we have further screened the prepared compounds for
In another study, the ability of the prepared derivatives in inhib- their anticancer activity [18]. The anticancer activity of com-
iting the oxidation of linoleic acid was studied by differential pounds 3a–e was tested against five cell lines along with
scanning calorimetry (DSC). DSC is a sensitive technique and doxorubicin as positive control and all of them showed moder-
has been used for investigating the thermotropic properties of ate to good anticancer effects. The results are collected in
several compounds including biological macromolecules, drugs Table 3. The compounds whose IC50 values are observed to be
and lipid-based materials for their stability and other character- lower and closer to the reference drug are considered as having
istics [16]. In the present study, pure linoleic acid and linoleic good anticancer potential.
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Beilstein J. Org. Chem. 2017, 13, 26–32.
Table 3: Anticancer activity of 10-undecenoic acid methyl ester–based lipoconjugates.a
Entry
Compound
IC50 values (μM)
MDA-MB-231
SKOV3
MCF7
DU145
HepG2
1
2
3
4
5
3a
21.2 ± 0.31
14.5 ± 0.26
12.0 ± 0.28
29.0 ± 0.36
12.5 ± 0.25
0.8 ± 0.14
99.2 ± 0.79
31.5 ± 0.41
38.9 ± 0.37
32.2 ± 0.32
38.3 ± 0.40
0.7 ± 0.16
17.2 ± 0.23
39.2 ± 0.45
10.55 ± 0.27
28.8 ± 0.36
13.9 ± 0.22
0.8 ± 0.12
25.4 ± 0.31
81.6 ± 0.77
13.0 ± 0.26
56.7 ± 0.62
28.8 ± 0.39
0.8 ± 0.15
38.2 ± 0.42
58.3 ± 0.61
67.4 ± 0.59
93.9 ± 0.85
141.4 ± 0.98
0.7 ± 0.14
3b
3c
3d
3e
doxorubicin
aCell lines: MDA-MB-231, breast cancer (ATCC® HTB-26™); SKOV3, ovarian cancer (ATCC® HTB-77™); MCF7, breast cancer (ATCC® HTB-22™);
DU 145, prostate cancer (ATCC® HTB-81™); HepG2, liver hepatocellular carcinoma (ATCC® HB-8065™).
Specifically compounds 3c, 3b and 3e were found to show Sigma–Aldrich (St.Louis, USA), and pre-coated TLC plates
promising anticancer activity according to their IC50 values, (silica gel 60 F254) were purchased from Merck (Darmstadt,
whereas 3d and 3a exhibited only moderate activity. Among all Germany). All solvents were purchased from Sd Fine Chemi-
tested derivatives, compound 3c was found to exhibit best anti- cals (Mumbai, India) and were of the highest grade of purity
cancer activity against MCF7, DU145 and MDA-MB-231 cell available.
lines with IC50 values of 10.55, 13.0 and 12.0 µM, respectively.
Instruments
It was found that the anticancer activity against some cell lines
was much better compared to our previous reports on phenolic
lipids [19]. However, all prepared derivatives were observed to
exhibit lower anticancer activity when compared to the refer-
ence drug doxorubicin which showed IC50 values in the range
of 0.7 to 0.8 µM against the tested cell lines.
1H and 13C NMR spectra were recorded on a Bruker Avance
operating at 700/500 MHz and 175/125 MHz. The NMR spec-
tra were referenced to δ 7.26 ppm and δ 77.0 ppm in CDCl3 sol-
vent for 1H and 13C, respectively. Coupling constants (J)
patterns in the 1H NMR spectra are given as follows:
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet
protons. Mass spectra were recorded using electron spray
Conclusion
In conclusion, the synthesis of five novel methyl 10-unde- ionization (ESI) on a Waters e2695 Separators module (Waters,
cenoate-based lipoconjugates of phenolic acids is reported. The Milford, MA, USA) mass spectrometer. FTIR spectra were re-
synthetic route was simple with product yields in the range of corded in chloroform on a Perkin–Elmer Fourier Transform
84–86% over three steps. The lipid part, methyl 10-unde- (FTIR) spectrum BX instrument (Model: Spectrum BX;
cenoate was subjected to a thiol–ene reaction with cysteamine Connecticut, USA). HRMS spectra were obtained from an
hydrochloride and the resulting intermediate was conjugated Exactive Orbitrap mass spectrometer (Thermo Scientific,
with the phenolic acid via amidation. The evaluation of the five Waltham, MA, USA). Melting points of lipoconjugates of
novel phenolic lipids as anti-oxidants was studied using the phenolic molecules were determined with a capillary tube
DPPH radical scavenging assay and DSC studies where some melting point apparatus. Gas chromatography (GC) was per-
compounds showed excellent anti-oxidant activity. Finally the formed on an Agilent 6890N gas chromatograph (Delaware,
compounds were further screened for anticancer activity where USA) equipped with a flame ionization detector using a
a few derivatives showed interesting activity.
HP-1 capillary column (30 m × 0.25 mm × 0.25 μm). The
injector and detector temperatures were set at 280 and 300 °C,
respectively. The oven temperature was programmed at 150 °C
for 2 min and then increased to 300 °C at 10 °C/min and final
Experimental
Materials
10-Undecenoic acid, 1,1’-azobis(cyclohexanecarbonitrile) temperature hold for 20 min. The carrier gas used was nitrogen
(ABCN), hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3’- at a flow rate of 1.0 mL/min.
dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl),
cysteamine hydrochloride, cinnamic acid, sinapic acid, ferulic Methods
acid, p-coumaric acid, caffeic acid, α-tocopherol (α-TP), tert- Synthesis of methyl undec-10-enoate (1): 10-Undecenoic acid
butylhydroquinone (TBHQ), linoleic acid (LA) and 2,2- (10 g, 54.34 mmol), was added to methanol (17.6 mL) and
diphenyl-1-picrylhydrazyl (DPPH) radical were purchased from sulfuric acid (0.1 mL, 2 wt % 10-undecenoic acid) and stirred at
29

Beilstein J. Org. Chem. 2017, 13, 26–32.
refluxing temperature of methanol for 6 h. After completion of The product was characterized by 1H and 13C NMR, IR, ESIMS
the reaction as shown by TLC (hexane/ethyl acetate 80:20, v/v), and HRMS spectral studies. Mp 55–56 °C; 1H NMR (500 MHz,
excess methanol was removed under reduced pressure and the CDCl3) δ 7.64 (d, J = 15.6 Hz, 1H), 7.54–7.46 (m, 5H), 6.24 (d,
product was diluted with ethyl acetate (30 mL), washed with J = 15.6 Hz, 1H), 3.66 (s, 3H), 3.56 (q, 2H), 2.73 (t, 2H), 2.58
5% aqueous NaHCO3 solution (3 × 30 mL), and dried over an- (t, 2H), 2.30 (t, 2H), 1.24–1.62 (m, 12H, CH2); 13C NMR
hydrous Na2SO4. The organic solvent was removed under (75 MHz, CDCl3) δ 174.37 (-C(O)-OCH3), 165.50
reduced pressure to afford crude methyl ester of 10-undecenoic (-NH-C(O)-), 141.25 (-NH-C(O)-CH=CH-),134.81–127.83,
acid. The product was purified by column chromatography with 120.52 (-NH-C(O)-CH=CH-), 51.47 (-C(O)-OCH3-), 38.49
basic alumina and hexane as the eluent to get 99% pure methyl (-CH2-NH-), 29.44 (-S-CH2-), 29.36 (-CH2-S-), 29.22 (-CH2-
undec-10-enoate (1) as indicated by GC. The product was CH2-S-), 29.19–24.96 (-CH2-CH2-); IR (cm−1, KBr): 2853,
analyzed by 1H NMR, 13C NMR, ESIMS, and FTIR and the 2853, 1720, 1654, 1599, 1527, 1441, 1365; ESIMS (m/z): 406
structure was confirmed by comparing the data with those re- [M + H]+, 428 [M + Na]+; HRMS (m/z): [M + H]+ calcd for
ported in the literature [14].
C23H36O3NS, 406.24104; found, 406.24077.
Synthesis of methyl 11-(2-aminoethylthio) undecanoate (2): Synthesis of methyl 11-((2-((E)-3-(4-hydroxyphenyl)acryl-
For the synthesis of compound 2, a reported protocol was fol- amido)ethyl)sulfanyl)undecanoate (3b): Similarly, methyl
lowed with slight modifications [20]. Briefly, methyl unde- 11-((2-((E)-3-(4-hydroxyphenyl)acrylamido)ethyl)sulfanyl)un-
cenoate (1, 6 g, 30.3 mmol) and ABCN (0.18 g, 3 wt % of 1) decanoate (3b) was prepared from 2 (0.6 g, 2.1 mmol) and
were dissolved in 40 mL chloroform. Then, 2-mercaptoethyl- coumaric acid (0.5 g, 3.2 mmol) in 85% yield (0.78 g) and the
amine hydrochloride (6.8 g, 60 mmol) and 40 mL of 1,4- product was characterized by 1H and 13C NMR, IR, ESIMS and
dioxane/ethanol (70:30; v/v) were added and the mixture was HRMS spectral studies. Mp 64–65 °C; 1H NMR (500 MHz,
stirred at 85 °C for 48 h. The progress of the reaction was moni- CDCl3) δ 7.57 (d, J = 15.6 Hz, 1H), 7.39 (d, J = 8.6 Hz, 1H),
tored by TLC (hexane/ethyl acetate 80:20, v/v). After maximum 6.85 (d, J = 8.6 Hz, 1H), 6.26 (d, J = 15.6 Hz, 1H), 3.67 (s, 3H),
conversion, the reaction mixture was extracted with dichloro- 3.58 (q, 2H), 2.73 (t, 2H), 2.58 (t, 2H), 2.30 (t, 2H), 1.24–1.62
methane (2 × 40 mL) and the combined organic phases were (m, 12H, CH2); 13C NMR (75 MHz, CDCl3) δ 174.71 (-C(O)-
washed with saturated K2CO3, brine and finally with water and OCH3), 166. 84 (-NH-C(O)-), 158.33, 141.51 (-NH-C(O)-
dried over anhydrous Na2SO4. This crude product mixture was CH=CH-),129.64, 128.99, 117.32 (-NH-C(O)-CH=CH-),
concentrated and purified by column chromatography with 115.98, 51.58 (-C(O)-OCH3-), 38.63 (-CH2-NH-), 34.16 (-S-
hexane/ethyl acetate (92:8, v/v) to obtain pure methyl 11-(2- CH2-), 31.89 (-CH2-S-), 31.84 (-CH2-CH2-S-), 29.69–24.96
aminoethylthio)undecanoate (2) in 89% yield (7.41 g). The (-CH2-CH2-); IR (cm−1, KBr): 3409, 2923, 2853, 1729, 1652,
purified product was characterized by 1H and 13C NMR, IR and 1595, 1519, 1452, 1373; ESIMS (m/z): 422 [M + H]+, 444
ESIMS spectral studies and the structure was confirmed by [M + Na]+; HRMS (m/z): [M + H]+ calcd for C26H36O4NS,
comparing the data with those reported in the literature [15]. 422.23596; found, 422.23491.
Synthesis of methyl 11-((2-(cinnamamido)ethyl)sulfanyl)un- Synthesis of methyl 11-((2-((E)-3-(3,4-dihydroxyphenyl)-
decanoate (3a): The amidation reaction was performed acrylamido)ethyl)sulfanyl)undecanoate (3c): Similarly,
following a reported protocol with slight modifications [21]. methyl 11-((2-((E)-3-(3,4-dihydroxyphenyl)acrylamido)-
Briefly, compound 2 (0.58 g, 2.1 mmol) and cinnamic acid ethyl)sulfanyl)undecanoate was prepared from 2 (0.6 g,
(0.4 g, 3.1 mmol) were dissolved in dichloromethane (30 mL) 2.1 mmol) and caffeic acid (0.5 g, 3.2 mmol) in 85% yield
and the mixture was stirred at 0–5 °C under a nitrogen atmo- (0.81 g) and the product was characterized by 1H and
sphere. EDC·HCl (0.4 g, 2.52 mmol) and HOBt (0.3 g, 13C NMR, IR, ESIMS and HRMS spectral studies. 1H NMR
3.1 mmol) were added and the contents were stirred at 0–5 °C (500 MHz, CDCl3) δ 7.55 (d, J = 15.6 Hz, 1H), 7.12 (d, J = 8.6
for 10 min. After the addition, the mixture was stirred for 12 h Hz, 1H), 6.95 (s, 1H) 6.87 (d, J = 8.6 Hz, 1H), 6.24 (d, J = 15.6
at rt under a nitrogen atmosphere and the progress of reaction Hz, 1H), 3.68 (s, 3H), 3.56 (q, 2H), 2.73 (t, 2H), 2.58 (t, 2H),
was monitored by TLC using the solvent system chloroform/ 2.30 (t, 2H), 1.24–1.62 (m, 12H, CH2); 13C NMR (75 MHz,
methanol (80:20, v/v). After maximum conversion, the reaction CDCl3) δ 174.74 (-C(O)-OCH3), 167. 38 (-NH-C(O)-), 146.87,
mixture was extracted with dichloromethane, washed with 144.53, 142.53 (-NH-C(O)-CH=CH-), 127.07, 121.33, 117.12
water and dried over anhydrous Na2SO4 and concentrated to (-NH-C(O)-CH=CH-), 115.46, 114.71, 51.60 (-C(O)-OCH3-),
obtain the crude product. The crude product was purified by 39.05 (-CH2-NH-), 38.88 (-S-CH2-), 38.76 (-CH2-S-), 34.16
column chromatography (chloroform/methanol 90:10, v/v) to (-CH2-CH2-S-), 29.59–24.96 (-CH2-CH2-); IR (cm−1, KBr):
obtain the thioamide of cinnamic acid in 86% yield (0.73 g). 3359, 2953, 2854, 1721, 1654, 1599, 1527, 1441, 1365; ESIMS
30

Beilstein J. Org. Chem. 2017, 13, 26–32.
(m/z): 438 [M + H]+, 460 [M + Na]+; HRMS (m/z): thesized phenolic lipoconjugates (1 mM concentrations) were
[M + H]+ calcd for C23H36O5NS, 438.23087; found, added to 2 mL of a methanolic solution of the DPPH radical
438.23023.
(1 mM concentration) and the total volume was made up to
3 mL with methanol. After 40 min of standing, the absorbance
Synthesis of methyl 11-((2-((E)-3-(4-hydroxy-3-methoxy- of the mixture was measured at 517 nm against methanol as
phenyl)acrylamido)ethyl)sulfanyl)undecanoate (3d): Simi- blank sample. TBHQ and α-TP (1 mM concentration) were
larly, methyl 11-((2-((E)-3-(4-hydroxy-3-methoxyphenyl)acryl- used as a positive control. The radical-scavenging activities (%)
amido)ethyl)sulfanyl)undecanoate was prepared from 2 (0.6 g, of the tested samples were evaluated by comparison with the
2.1 mmol) and ferulic acid (0.6 g, 3.2 mmol) in 84% yield control (2 mL DPPH radical solution and 1 mL methanol). Each
(0.82 g) and the product was characterized by 1H and sample was measured in triplicate and averaged. The free-
13C NMR, IR, ESIMS and HRMS spectral studies. 1H NMR radical scavenging activity (FRSA) was calculated using the
(500 MHz, CDCl3) δ 7.55 (d, J = 15.5 Hz, 1H), 7.06 (dd, J = following formula: FRSA = [(Ac − As)/Ac] × 100 where Ac is
8.2, 1.5 Hz, 1H), 7.00 (d, J = 1.6 Hz, 1H), 6.91 (d, J = 8.2 Hz, the absorbance of the control and As is the absorbance of the
1H), 6.27 (d, J = 15.5 Hz, 1H), 3.92 (s, 3H), 3.67 (s, 3H), 3.58 tested sample after 40 min.
(q, 2H), 2.73 (t, 2H), 2.58 (t, 2H), 2.30 (t, 2H), 1.24–1.62 (m,
12H, CH2); 13C NMR (75 MHz, CDCl3) δ 174.36 (-C(O)- DSC measurements
OCH3), 166. 21 (-NH-C(O)-), 147.48, 146.76, 141.26 (-NH- The anti-oxidant activity was also evaluated by differential
C(O)-CH=CH-), 127.33, 122.22, 118.06 (-NH-C(O)-CH=CH-), scanning calorimetry using pure linoleic acid as a lipid model
114.76, 109.64, 55.95 (-OCH3-), 51.45 (-C(O)-OCH3-), 38.42 system [23,24]. All studied anti-oxidants were dissolved in
(-CH2-NH-), 34.11 (-S-CH2-), 31.97 (-CH2-S), 31.74 (-CH2- methanol to prepare 1 mM solutions. Samples of linoleic acid
CH2-S-), 29.47–24.95 (-CH2-CH2-); IR (cm−1, KBr): 3375, (2.5–3.0 mg) were placed in standard aluminum pans
2926, 2853, 1730, 1656, 1596, 1516, 1433, 1273; ESIMS (m/z): and spiked with 10 µL of the anti-oxidant solution.
452 [M + H]+, 474 [M + Na]+; HRMS (m/z): [M + H]+ calcd A blank run of linoleic acid, spiked with 10 µL of
for C24H38O5NS, 452.24652; found, 452.24475.
methanol was also carried out simultaneously to find the
oxidative induction temperature (OIT) of linoleic acid. OIT is
Synthesis of methyl-11-((2-((E)-3-(4-hydroxy-3,5-dimethoxy- determined from the first exothermal peak of the plot of heat
phenyl)acrylamido)ethyl)sulfanyl)undecanoate (3e): Simi- flow (mW/g) vs temperature. All measurements for
larly, methyl 11-((2-((E)-3-(4-hydroxy-3,5-dimethoxyphenyl)- each compound were run in triplicate and the results were aver-
acrylamido)ethyl)sulfanyl)undecanoate was prepared from 2 aged.
(0.6 g, 2.1 mmol) and sinapic acid (0.7 g, 3.2 mmol) in 85%
yield (0.89 g) and the product was characterized by 1H and Cytotoxicity test (MTT assay)
13C NMR, IR, ESIMS and HRMS spectral studies. The cytotoxicity assay (MTT) was evaluated for all test com-
Mp 69–70 °C; 1H NMR (500 MHz, CDCl3) δ 7.53 (d, J = 15.5 pounds as described in our earlier work [25]. Five different
Hz, 1H), 6.76 (s, J = 8.2, 1.5 Hz, 2H), 6.28 (d, J = 15.5 Hz, 1H), cancer cell lines viz., MDA-MB-231, breast cancer (ATCC®
3.93 (s, 6H), 3.67 (s, 3H), 3.58 (q, 2H), 2.73 (t, 2H), 2.58 (t, HTB-26™); SKOV3, ovarian cancer (ATCC® HTB-77™);
2H), 2.30 (t, 2H), 1.24–1.62 (m, 12H, CH2); 13C NMR MCF7, breast cancer (ATCC® HTB-22™); DU 145, prostate
(75 MHz, CDCl3) δ 174.37 (-C(O)-OCH3), 166.07 cancer (ATCC® HTB-81™); HepG2, liver hepatocellular carci-
(-NH-C(O)-), 147.22, 141.43 (-NH-C(O)-CH=CH-),136.64, noma (ATCC® HB-8065™) were obtained from the ATCC
126.29, 118.44 (-NH-C(O)-CH=CH-), 104.82, 56.34 (-OCH3-), (Bethesda, MD, USA) and maintained in DMEM supplemented
51.48 (-C(O)-OCH3-), 38.39 (-CH2-NH-), 34.11 (-S-CH2-), with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and
31.96 (-CH2-S-), 31.69 (-CH2-CH2-S-), 29.65–24.95 (-CH2- 100 μg/mL streptomycin at 37 °C in a 5% CO2 incubator.
CH2-); IR (cm−1, KBr): 3371, 2926, 2852, 1730, 1658, 1612, After seeding of cells in 96 well culture plates, they were
1514, 1455 1285; ESIMS (m/z): 482 [M + H]+, 504 [M + Na]+; allowed to attach properly. Test compounds of different concen-
HRMS (m/z): [M + H]+ calcd for C25H40O6NS, 482.25709; trations ranging from 1 to 50 µM were added in triplicates
found, 482.25532.
and incubated for 24 h. The cells were then incubated
with MTT (0.5 mg/mL) for 3 h and, to dissolve the insoluble
formazan crystals, 100 µL DMSO was added to
each well. Finally the absorbance of the plates was
Anti-oxidant activity
DPPH radical scavenging assay
The anti-oxidant activity was determined by the radical scav- measured using a Synergy H1 multi-mode plate reader
enging ability using the stable DPPH radical method as re- (USA). Doxorubicin was used as the positive control for com-
ported [22]. Briefly, 200 µL of a methanolic solution of the syn- parison.
31

Beilstein J. Org. Chem. 2017, 13, 26–32.
20.Sammaiah, A.; Padmaja, K. V.; Prasad, R. B. N.
Supporting Information
Eur. J. Lipid Sci. Technol. 2016, 118, 495. doi:10.1002/ejlt.201500119
21.Kaki, S. S.; Arukali, S.; Padmaja, K. V.; Prasad, R. B. N.; Yedla, P.;
Kumar, C. G. Bioorg. Med. Chem. Lett. 2016, 26, 209.
doi:10.1016/j.bmcl.2015.10.086
Supporting Information File 1
Copies of 1H NMR, 13C NMR, HRMS and DSC spectra.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-13-4-S1.pdf]
22.Akowuah, G. A.; Zhari, I.; Norhayati, I.; Mariam, A.
J. Food Compos. Anal. 2006, 19, 118. doi:10.1016/j.jfca.2005.04.007
23.Gaspar, A.; Martins, M.; Silva, P.; Garrido, E. M.; Garrido, J.; Firuzi, O.;
Miri, R.; Saso, L.; Borges, F. J. Agric. Food Chem. 2010, 58, 11273.
doi:10.1021/jf103075r
Acknowledgements
Naganna Narra thanks the University Grants Commission
(UGC), New Delhi, India, for financial support through a Senior
Research Fellowship (UGC-SRF).
24.Reis, B.; Martins, M.; Barreto, B.; Milhazes, N.; Garrido, E. M.;
Silva, P.; Garrido, J.; Borges, F. J. Agric. Food Chem. 2010, 58, 6986.
doi:10.1021/jf100569j
25.Bollu, V. S.; Nethi, S. K.; Dasari, R. K.; Shiva Nageshwara Rao, S.;
Misra, S.; Patra, C. R. Nanotoxicology 2015, 10, 413.
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