Synthesis and cytotoxic activities of hexyl-esters derivatives of gallic acid against MCF-7 cell line

Article abstract of DOI:10.13005/ojc/340132

Gallic acid is found in many plants, fruits, and foods where the anti-cancer activity is found. However, gallic acid has a problem on the high polarity and low bio availability. So, it takes molecular modifications in order to increase its lipophilicity, which is expected to increase bio availability and cytotoxic activity of gallic acid. Hexyl esters derivatives of gallic acid were synthesized and characterized by spectrometer ¹H-NMR, ¹³C-NMR, mass spectrometry and infrared spectrophotometer (FTIR). All compounds were then evaluated for cytotoxic activity on MCF-7 cell line using MTT method. Compound cis-2′-hexenyl-3,4,5-trimethoxygallate (19) had the lowest IC50 value compared with gallic acid and other derivatives hexyl esters. IC50 value of cis-2′-hexenyl-3,4,5-trimethoxygallate (19) is 14.48 μg/ml. Compound (19) also has approached with IC50 values of gossypol as a positive control. Compound (19) is a potential compound to inhibit growth of MCF-7 cell line.

Full text of DOI:10.13005/ojc/340132

ISSN: 0970-020 X
CODEN: OJCHEG
2018, Vol. 34, No.(1):
Pg. 295-300
ORIENTAL JOURNAL OF CHEMISTRY
An International Open Free Access, Peer Reviewed Research Journal
www.orientjchem.org
Synthesis and Cytotoxic Activities of Hexyl-esters
Derivatives of Gallic Acid Against MCF-7 Cell Line
RAFIKA INDAH PARAMITA1, ADE ARSIANTI1* and MAKSUM RADJI2
1Department of Medical Chemistry and Drug Development Research Cluster,
Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia.
2Faculty of Pharmacy, Universitas Indonesia, Depok, Indonesia.
*Corresponding author E-mail: arsi_ade2002@yahoo.com
http://dx.doi.org/10.13005/ojc/340132
(Received: August 06, 2017; Accepted: December 20, 2017)
ABSTRACT
Gallic acid is found in many plants, fruits, and foods where the anti-cancer activity is found.
However, gallic acid has a problem on the high polarity and low bio availability. So, it takes
molecular modifications in order to increase its lipophilicity, which is expected to increase bio
availability and cytotoxic activity of gallic acid. Hexyl esters derivatives of gallic acid were
synthesized and characterized by spectrometer 1H-NMR, 13C-NMR, mass spectrometry and
infrared spectrophotometer (FTIR). All compounds were then evaluated for cytotoxic activity on
MCF-7 cell line using MTT method. Compound cis-2’-hexenyl-3,4,5-trimethoxygallate (19) had
the lowest IC50 value compared with gallic acid and other derivatives hexyl esters. IC50 value of
cis-2’-hexenyl-3,4,5-trimethoxygallate (19) is 14.48 µg/ml. Compound (19) also has approached
with IC50 values of gossypol as a positive control. Compound (19) is a potential compound to
inhibit growth of MCF-7 cell line.
Keywords: Gallic acid, MCF-7, Cytotoxicity assay, Synthesis of hexyl esters gallate derivative.
INTRODUCTION
synthesis new potential anticancer drugs. Although
many potential anticancer drugs have been made
but the medical need is still largely unmet due to
many factors among which the lack of selectivity of
conventional drugs leading to toxicity, the
metastatic spreading, and multi-drug resistance;
(MDR).Therefore, the search for novel and selective
anticancer agents is highly required due to currently
available anticancer drugs problems.2,3
Breast cancer is the second most common
cancer in the world and the most frequent cancer
among women with an estimated 1.67 million new
cancer cases diagnosed in 2012 (25% of all
cancers). Breast cancer ranks as the fifth cause of
death from cancer overall1. A lot of research to
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In recent decades, the development of acid. Hexyl esters of trimethoxygallic acid showed
novel and advanced cancer therapies common use
natural plants, fruits, or foods as a valuable resource.
Gallic acid (3,4,5-trihydroxyl-benzoic acid) is
compound which is widely distributed in various
plants, fruits, and foods. Gallic acid was
demonstrated to have various biological
activities including antibacterial, antiviral, and
anti-inflammatory, in which the antitumor activity is
most striking.3 Growth of human breast cancer cell,
MCF-7, is significantly reduced by treatment with
gallic acid. Gallic acid causes apoptosis induction
and cells treated with gallic acid showed
significantly downregulated of Bcl-xL protein and
upregulation of Bak and Bad proteins.4
more interaction of hydrophobic amino acid than
gallic acid.5 This leads to increased bond stability
with the receptor. Compound cis-2’-hexenyl-3,4,5-
trimethoxygallate serve as potential inhibitor of anti-
apoptotic Bcl-xL protein because had the lowest
ΔG dan Ki. There is also pi-pi interaction with
TYR105 which adds stability to protein-inhibitor
interactions and protein complexes.6
To prove the docking result, we had
synthesized the hexyl esters derivative of gallic acid
(Fig. 1) and further testing the cytotoxic activity
against MCF-7 cell line. The in vitro result then
compared to in silico docking result to get a potent
anticancer drug for breast cancer.
In previous study, we were done with in
silico study about hexyl esters derivatives of gallic
Fig. 1. Scheme of synthesis of gallic acid derivative
magnetic resonance) was measured using JEOL
JNM-ECP (125 Hz) in Acetone-d6 as solvent. The
chemical shifts were reported in ppm and coupling
constants were measured in Hertz (Hz). Reactions
were monitored with thin layer chromatography
using aluminum sheets silica gel 60 F254 2x5 cm
( Merck & Co.). Ultra-violet light (254 nm) and iodine
vapour were used to visualized the chromatograms.
EXPERIMENTAL
Synthesis reaction
Chemicals, reagents and solvents were
purchased from Merck & Co. and Sigma-Aldrich.
1H-NMR (proton nuclear magnetic resonance) was
measured using JEOL JNM-ECP (500 Hz) in
Acetone-d6 as solvent. 13C-NMR (carbon nuclear

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ARSIANTI et al., Orient. J. Chem., Vol. 34(1), 295-300 (2018)
Mass spectra (MS) were recorded on Shimadzu Cytotoxicity effects
GCMS QP-5000, IR spectrum were recorded using
IR (JASCO FT/IR-420 spectrophotometer.
The inhibitory effect of synthesized
compounds against colorectal adenocarcinoma
cell line, HT-29, was determined using MTT (3-(4,5-
dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium
bromide) assay. First, cells were seeded in 96-well
plates at 5000 cells/well and allowed to attach in
24 h Media was renewed and the cells were
added with several concentrations of the extract
(3.125–100 ppm) incubated for an additional 24 h.
After 24 h, 20 µM of MTT solution (0.5 mg/ml MTT
solution in media) was added to each well and
Methylation reaction
A solution of gallic acid (17.65 mmol), and
K2CO3 (52,83 mmol) in DMF (25 mL) were added
Methyl iodide (52.83 mmol) and stirred at room
temperature for 48 hours. The reaction mixture was
then added 75 ml ethyl acetate and then washed
with 3x25 ml saturated NaHCO3 and 2x25 ml
saturated NaCl, sequently. The organic phase was
then added Mg2SO4 anhydrate to take out the water
residue. The filtrate was then drying to obtain the
crude product. The product was purified using
chromatography colomn with chloroform as mobile
phase and give 3 products : 4-monomethoxymethyl
gallate, 4,5-dimethoxymethyl gallate, 3,4,5-
trimethoxymethyl gallate.
0
incubated for 4 h at 37 C. The supernatant was
aspirated and the MTT-formazan crystals formed
by metabolically viable cells were dissolved in 100
µl of dimethyl sulphoxide (DMSO). Finally, the
absorbance was monitored by a microplate reader
at a wavelength of 570 nm. The percentage of viable
cells was plotted versus the concentration of the
test compound. The concentration by which to
mediate 50% cytotoxicity (IC50) was determined by
linear regression analysis.
Hidrolysis reaction
A solution of each methylation product and
LiOH monohydrate 0.5 M in mixture THF:Methanol
(3:1) were stirred at room temperature for 24 hours.
The reaction mixture was then acidified using
KHSO4 1M until pH-3, then added 15 mL of
aquadest, and extracted with 3x15 ml ethyl acetate.
The organic phase was washed with saturated
NaCl and then added Mg2SO4 anhydrate to take
out the water residue. The filtrate was then drying
to obtain the product and give 3 products:
4-monomethoxygallic acid, 4,5-dimethoxygallic
acid, 3,4,5-trimethoxygallic acid, respectively.
RESULT AND DISCUSSION
Synthesis of the desired compound
The compounds were synthesized as per
Fig. 1 and the structure was elucidated by
1
spectroscopic data, i.e IR, H-NMR, 13C-NMR and
LCMS are presented as follows:
Hexylgallate (2)
IR (KBr): v cm-1, 3326.61 (OH), 2927.41
(Al. C–H), 1685.48 (C=O), 1535.06 (Ar C=C),
1
1222.65 (C–O); H NMR (Acetone-d6) d: 7.11 (s,
Esterification reaction
2H, Ar-CH), 4.19 (t, 2H, OCH2-CH2), 1.74 – 1.66 (m,
2H, CH2CH2-CH2), 1.47 – 1.39 (m, 2H, CH2CH2-
CH2), 1.36 – 1.30 (m, 4H, CH2CH2-CH2), 0.89 (t, 3H,
CH2CH3). 13C NMR (Acetone-d6) d: 166.72, 146.02
(2C), 138.48, 122.03 (2C), 109.64, 64.91, 42.04,
32.18, 26.41, 23.59, 14.24. LCMS m/z: [M+]
254.1154 (C13H18O5).
A solution of gallic acid or 3,4,5-
trimethoxygallic acid (1 mmol), alcohols (2 mmol)
and 1.5 mmol diisopropyl carbadiimide (DIC) as
catalisator in tetrahydofurane (10 mL) were stirred
in 0 °C for 30 min. then added solution of 0,1 mmol
N,N-Dimethylaminopiridine (DMAP) in 1 ml THF.
This mixture was stirred at room temperature for
24 hours. The reaction mixture was then added
aquadest and extracted with chloroform. The
organic phase was then added Mg2SO4 anhydrate
to take out the water residue. The filtrate was then
drying to obtain the crude product. The product was
purified using chromatography colomn with
appropriate mobile phase.
Trans-2’-hexenylgallate (3)
IR (KBr): v cm-1, 3272.61 (OH), 2927.41
(Al. C–H), 1685.48 (C=O), 1535.06 (Ar C=C), 1226.5
(C–O); 1H NMR (Acetone-d6) d: 7.08 (s, 2H, Ar-CH),
5.84 – 5.76 (m, 1H, CH2CH=CH2), 5.67 – 5.59 (m,
1H, CH=CHCH2), 4.61 (dd, 2H, OCH2-CH2), 1.41 –
1.31 (q, 2H, CH2CH2-CH2), 1.10 – 1.02 (s, 2H,

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ARSIANTI et al., Orient. J. Chem., Vol. 34(1), 295-300 (2018)
CH2CH2CH3) 0.86 (t, 3H, CH2CH3).13C NMR 105.49, 60.96, 60.59, 58.21, 29.65, 23.06, 13.72.
(Acetone-d6) d: 166.50, 146.08 (2C), 136.03, 131.22, LCMS m/z: [M+]280.1311 (C15H20O5).
125.56 (2C), 109.66 (2C), 65.46, 42.18, 23.53,
13.82. LCMS m/z: [M+] 252.0998 (C13H16O5).
Hexyl-3,4,5-trimethoxygallate (17)
IR (KBr): v cm-1, 2935.13 (Al. C–H),
1714.41 (C=O), 1589.06 (Ar C=C), 1224.58 (C–O);
1H NMR (Acetone-d6) d: 7.30 (s, 2H, Ar-CH), 4.29
(t, 2H, OCH2-CH2), 3.88 (s, 6H, OCH3), 3.79 (s, 3H,
OCH3), 1.79 – 1.72 (m, 2H, CH2CH2-CH2), 1.49 –
1.42 (m, 2H, CH2CH2-CH2), 1.38 – 1.33 (m, 4H,
CH2CH2-CH2), 0.90 (t, 3H, CH2CH3). 13C NMR
(Acetone-d6) d: 166.69, 154.45 (2C), 143.63, 126.73,
107.86 (2C), 65.91 60.94, 56.79 (2C), 32.49, 26.71,
23.52 (2), 14.57. LCMS m/z: [M+] 296.1624
(C16H24O5).
Cis-2’-hexenylgallate (4)
IR (KBr): v cm-1, 3326.61 (OH), 2927.41
(Al. C–H), 1685.48 (C=O), 1535.06 (Ar C=C),
1
1222.65 (C–O); H NMR (Acetone-d6) d: 7.01 (s,
2H, Ar-CH), 5.65 – 5.54 (m, 2H, CH2CH=CH), 4.72
(d, 2H, OCH2-CH2), 2.14 – 2.06 (q, 2H, CH2CH2-
CH2), 1.42 – 1.32 (m, 2H, CH2CH2CH3) 0.92 (t, 3H,
CH2CH3). 13C NMR (Acetone-d6) d: 166.52, 157.91,
146.77 (2C), 135.11, 125.14 (2C), 109.59 (2C),
60.37, 23.13, 21.43, 13.85. LCMS m/z: [M+]252.0997
(C13H16O5).
Trans-2’-hexenyl-3,4,5-trimethoxygallate (18)
IR (KBr): v cm-1, 2958.27 (Al. C–H), 1714.41
(C=O), 1589.06 (Ar C=C), 1222.65 (C–O); 1H NMR
Cis-2’-hexenyl-3,4-dimethoxygallate (16)
IR (KBr): v cm-1, 3403.74 (OH), 2956.34 (Acetone-d6) d: 7.24 (s, 2H, Ar-CH), 5.87 – 5.77 (m,
(Al. C–H), 1683.35 (C=O), 1587.13 (Ar C=C), 1H, CH2CH=CH), 5.70 – 5.58 (m, 1H, CH=CHCH2),
1324.86 (C–O); H NMR (Acetone-d6) d: 7.20 (s, 4.69 (dd, 2H, OCH2-CH2), 3.82 (s, 6H, OCH3), 3.74
1
1H, Ar-CH), 7.16 (s, 1H, Ar-CH), 5.73 – 5.64 (m, 2H, (s, 3H, OCH3), 2.04 – 2.02 (q, 2H, CH2CH2-CH2),
CH2CH=CH), 4.83 (d, 2H, OCH2-CH2), 3.89 (s, 3H, 1.42 – 1.32 (m, 2H, CH2CH2CH3) 0.85 (t, 3H,
OCH3), 3.83 (s, 3H, OCH3), 2.20 – 2.15 (q, 2H, CH2CH3). 13C NMR (Acetone-d6) d: 166.47, 154.46
CH2CH2-CH2), 1.47 – 1.39 (s, 2H, CH2CH2CH3) 0.93 (2C), 143.69, 136.91, 126.59, 125.65, 107.91 (2C),
(t, 3H, CH2CH3). 13C NMR (Acetone-d6) d: 166.10, 64.45, 60.94, 56.81 (2C), 35.27, 23.09, 14.17. LCMS
163.15, 153.75, 135.56, 132.24, 126.31, 124.68, m/z: [M+] 294.1467 (C16H22O5).
Table. 1 : Physicochemical data of title compounds
Compound
Molecular
formula
R
R1
R2
R3
MW
Yield(%)
Rf
2
3
4
16
17
18
19
C13H18O5
C13H16O5
C13H16O5
C15H20O5
C16H24O5
C16H22O5
C16H22O5
-C6H13
-C6H12
-C6H12
–OH
–OH
–OH
–OH
–OH
–OH
–OH
–OH
–OH
–OH
254.11
252.10
252.10
280.13
296.16
294.15
294.15
72
63
66
67
56
53
54
0.70a
0.65a
0.64a
0.32b
0.75b
0.65b
0.64b
-C6H12 –OCH3 –OCH3
-C6H13 –OCH3 –OCH3 –OCH3
-C6H12 –OCH3 –OCH3 –OCH3
-C6H12 –OCH3 –OCH3 –OCH3
a Mobile phase : chloroform : methanol (5:1)
b Mobile phase : chloroform

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ARSIANTI et al., Orient. J. Chem., Vol. 34(1), 295-300 (2018)
Cis-2’-hexenyl-3,4,5-trimethoxygallate (19)
IR (KBr): v cm-1, 2958.27 (Al. C–H),
that alkyl esters of gallic acid had better cytotoxic
effects than gallic acid. This is due to an increase in
lipophilicity that contributes in increasing affinity for
cell membrane and permeability.8 IC50 values of
hexylgallate 2, trans-2'-hexenylgallate 3, and
cis-2'-hexenylgallate 4 are 24.38 μg / ml, 21.28 μg
/ ml, 20.80 μg / ml, respectively, where the value is
lower than the gallic acid.
1714.41 (C=O), 1589.06 (Ar C=C), 1220.72 (C–O);
1H NMR (Acetone-d6) d: 7.29 (s, 2H, Ar-CH), 5.73 –
5.61 (m, 2H, CH2CH=CH), 4.85 (d, 2H, OCH2-CH2),
3.86 (s, 6H, OCH3), 3.78 (s, 3H, OCH3), 2.20 – 2.15
(q, 2H, CH2CH2-CH2), 1.47 – 1.38 (m, 2H,
CH2CH2CH3), 0.92 (t, 3H, CH2CH3). 13C NMR
(Acetone-d6) d: 166.24, 154.14 (2C), 143.38,
135.77, 126.25, 124.85, 107.60 (2C), 61.29, 60.64,
56.49 (2C), 29.84, 23.24, 13.92. LCMS m/z: [M+]
294.1465 (C16H22O5).
In the double-bonded hexenyl ester
compound, both the cis 4 and trans 3 positions
exhibit lower IC50 than the hexyl ester compound
without the double bond 2. This suggests that the
presence of double bonds may increase the
cytotoxic activity of the compound. In addition,
double bond positions with cis configurations in
compounds 4 and 19 exhibit slightly better activity
than trans configurations in compounds 3 and 18.
In ester compounds without methoxy groups,
ie hexylgallate 2, trans-2'-hexenylgallate 3, and
cis-2'-hexenylgallate 4, showed less cytotoxic
activity than those with methoxy groups. In
compounds with the same alkyl ester, cis-2'-hexenyl
ester, in compounds 2, 16, 19, with differences in
the number of methoxy groups, also shows
increased cytotoxic activity as the number of
methoxy groups increases. The bonding sites of
the Bcl-xL protein are mostly composed of non-
polar amino acids.9 Thus the methoxy group has a
more stable bond on the protein binding site of Bcl-
xL compared with the hydroxy group.
After completion of the synthesis, cytotoxic
activity of gallic acid, target derivative 2, 3, 4, 16,
17, 18, and 19 were determined MTT cell
proliferation assay against MCF-7 cell line, cytotoxic
activity represented by IC50. The smaller IC50 value,
the higher cytotoxic activity. From the MTT assay
result, compound 19 showed the best cytotoxic
activity than other compound, and the IC—50 value
of compound 19 similar to IC50 value of Gossypol
as positive control. This result was agreevwith in
silico study in previous research by Paramita, 2017.
Cytotoxicity assay of gallic acid derivative are
summarized in Table. 2 as follow.
Table. 2 : Cytotoxic activity of hexyl
esters derivatives of gallic acid
Compound
IC50 (µg/ml)
Gallic acid
32.25 0.014
24.38 0.014
21.28 0.030
20.80 0.087
18.93 0.062
16.54 0.012
14.93 0.000
14.48 0.087
10.90 0.000
2
3
4
16
17
From the MTT test results, it is known that
the cis-2'-hexenyl-3,4,5-trimethoxygallate 19
compound exhibited the best activity with the lowest
IC50 value compared to the other gallic acid
derivative compounds and the results obtained
supported the previous results of in silico study by
Paramita et al., 2017. Compound 19 also had an
IC50 value close to the IC50 value of gossypol as a
positive control. Compound 19 is a potential
compound in inhibiting growth of MCF-7 cell line
because has the best in vitro activity between
synthesized gallic acid derivatives.
18
19
Gossypol
As shown in Table. 2, seven derived
compounds had greater inhibitory activity against
MCF-7 cell line than gallic acid. Gallic acid has an
IC50 value of 32.25 μg / ml. The results are similar to
IC50 of gallic acid in previous studies, i.e. 36 μg /
ml.7 Increased lipophilicity of the gallic acid
derivative (alkyl ester derivative) will increase the
cytotoxic activity. In the previous study, it was shown
CONCLUSION
We described synthesis of hexyl esters
derivatives of gallic acid for in vivo cytotoxic activities

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ACKNOWLEDGEMENT
against MCF-7 cell line. Among all the compounds
synthesized, compounds 19 exhibited most potent
cytotoxic activity and had an IC50 value close to the
IC50 value of gossypol. Based on the findings of
these in vitro results, further studies need to be
carried out to investigate in vivo assays and
toxicological studies.
This article does not contain any studies
with human and animal subjects performed by any
of the authors. We thank to Faculty of Medicine,
Universitas Indonesia for the Cluster Research
Grant 2017.
REFERENCES
5.
6.
7.
Paramita, R.I.; Arsianti, A.; Radji, M. Intl. J.
ChemTech Res. 2017, 1, 385.
1.
2.
Ferlay, J.; Soerjomataram, Dikshit, R.; Eser,
S.; Mathers, C.; Rebelo, M. Intl. J. of Cancer.
2015,136 (5).
Shipra, G.; Gauri, M.;Chandra, P.M.;Kishore,
S.P. Prot. & Pept. Let.. 2012, 19,1302–17.
Mukherjee, P.; Desai, P.; Zhou, Y. D.; Avery,
M. J. Chem. Inform. Modeling. 2010, 50(5),
906–923.
Tor, Y. S., PLoS ONE. 2015, 10(6), 1–25.
Locatelli, C.; Filippin-Monteiro, F. B.;
Creczynski-Pasa, T. B. Euro. J. Med. Chem.
2013, 60, 233–239.
Shukla, S.; Srivastava, R.S.; Shrivastava, S.
K.; Sodhi, A.; Kumar, P. Asian Pac. J. Trop.
Biomed. 2012, 5, 1040–46.
3.
4.
Jaikumar, B. & Jasmine, R. Int. J. PharmTech
Res. 2016, 9, 333–365.
8.
9.
Wang, K.;Zhu, X.;Zhang, K.;Zhu, L.;Zhou, F.
J. Biochem. Mol. Tox. 2014, 28(9), 387–93.