Antioxidant, cytotoxic activities, and structure-activity relationship of gallic acid-based indole derivatives

Article abstract of DOI:10.1002/ardp.201000223

A new series of gallic hydrazones containing an indole moiety was synthesized through the reaction of gallic hydrazide and different indole carboxaldehydes. Their antioxidant activities were determined on DPPH radical scavenging and inhibition of lipid peroxidation. The in-vitro cytotoxic activities of the compounds were evaluated against HCT-116 (human colon cancer cell line) and MCF-7 (estrogen-dependent human breast cancer cell line) by the MTT method. An attempt to correlate the biological results with their structural characteristics has been done. A limited positive structure activity relationship was found between cytotoxic and antioxidant activities. Gallic acid derivatives are naturally occurring compounds with interesting biological properties. Similarly, indolic compounds represent an important class of therapeutical agents in medicinal chemistry. A series of hybrid molecules incorporating these two pharmacophores has been synthesized and assessed for their antioxidant and cytotoxic activities. Copyright

Full text of DOI:10.1002/ardp.201000223

Arch. Pharm. Chem. Life Sci. 2011, 344, 703–709
703
Full Paper
Antioxidant, Cytotoxic Activities, and Structure–Activity
Relationship of Gallic Acid-based Indole Derivatives
Hamid Khaledi1, Abeer A. Alhadi1, Wagee A. Yehye1, Hapipah Mohd Ali1, Mahmood A. Abdulla2, and
Pouya Hassandarvish2
1 Department of Chemistry, University of Malaya, Kuala Lumpur, Malaysia
2 Department of Molecular Medicine, University of Malaya, Kuala Lumpur, Malaysia
A new series of gallic hydrazones containing an indole moiety was synthesized through the reaction of
gallic hydrazide and different indole carboxaldehydes. Their antioxidant activities were determined
on DPPH radical scavenging and inhibition of lipid peroxidation. The in-vitro cytotoxic activities of the
compounds were evaluated against HCT-116 (human colon cancer cell line) and MCF-7 (estrogen-
dependent human breast cancer cell line) by the MTT method. An attempt to correlate the biological
results with their structural characteristics has been done. A limited positive structure activity
relationship was found between cytotoxic and antioxidant activities.
Keywords: Antioxidant activity / Cytotoxic activity / Gallic hydrazone / Indole
Received: July 30, 2010; Revised: September 18, 2010; Accepted: September 24, 2010
DOI 10.1002/ardp.201000223
Introduction
tives [3–11]. These compounds have been proved to have
various biological properties including neuroprotective, anti-
oxidant [12] and anticancer activities [13]. Several studies
have demonstrated that gallic acid and its derivatives can
selectively induce cancer cell death by apoptosis without
harming healthy cells [14–16].
The generation of reactive oxygen species (ROS) and
reactive nitrogen species (RNS) has been considered to be a
possible mediator of cellular damage in many diseases [1].
Human bodies are constantly exposed to ROS and RNS
generated from endogenous and some exogenous sources.
Antioxidants, both enzymatic and non-enzymatic, prevent
oxidative damage to biological molecules by various mech-
anisms. Polyphenols constitute an important class of chemo-
preventive agents because they can quench or prevent the
formation of ROS and RNS [2]. The ability of phenolic com-
pounds to quench free radicals arises because of both their
acidity (ability to donate protons) and their delocalized
p-electrons (ability to transfer electrons while remaining
relatively stable) characteristic of benzene rings. Among
polyphenols, gallic acid (3,4,5-trihydroxybenzoic acid) deriva-
tives are a well-known group of naturally occurring com-
pounds which have been found in many phytomedicines.
In recent years, a number of their biological and pharmaco-
logical activities have been described resulting in much
attention in the development of synthetic gallic acid deriva-
Indole derivatives are biologically important chemicals
with a wide range of therapeutic properties. Antibacterial
[17], antifungal [18], antiviral [19], antimalarial [20], anti-HIV
[21], anticancer [22–24], and antioxidant properties [25, 26]
have been reported to be associated with the indolic nucleus.
Indolic compounds are very efficient antioxidants, protect-
ing both lipids and proteins from peroxidation, and it is
known that the indole structure influences the antioxidant
efficacy in biological systems [27].
In the design of new drugs, the development of hybrid
molecules through the combination of different pharmaco-
phores in one frame may lead to compounds with interesting
pharmacological properties. The co-administration of the
moieties, acting by different mechanisms may have a syner-
gistic effect, resulting in a higher activity than each of the
components. In the light of interesting biological activities
associated with gallic acid derivatives and indolic com-
pounds, it was considered worthwhile to synthesize some
hybrid molecules incorporating both indole and 3,4,5-trihy-
droxy benzoyl nucleus. The synthesized compounds were
investigated for their antioxidant and cytotoxic activities.
Correspondence: Hamid Khaledi, Department of Chemistry, University of
Malaya, 50603 Kuala Lumpur, Malaysia.
E-mail: khaledi@siswa.um.edu.my, hamid.khaledi@gmail.com
Fax: þ603-79674193
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Arch. Pharm. Chem. Life Sci. 2011, 344, 703–709
Table 1. Structure of the synthesized indole gallic hydrazones.
Scheme 1. All the compounds were characterized by elemen-
tal analysis and spectroscopic methods.
O
In vitro antioxidant activities
HN
3
The synthesized compounds were subjected to DPPH radical
scavenging and lipid peroxidation inhibitory assays to deter-
mine their antioxidant activity. The results were compared
with Ascorbic acid and a-tocopherol. All the tested com-
pounds showed noticeable DPPH radical scavenging activity,
with IC50 values of 0.17–0.37mM, when compared to the
reference compounds, ascorbic acid and a-tocopherol with
IC50 values of 0.98 and 0.91 mM, respectively (Table 2).
Among them, compounds 3e and 3f with a halogen group
in position 5 of the indole ring showed the highest activity.
All the compounds under study also possess inhibitory effect
on lipid peroxidation. The effect of the test compounds and
a-tocopherol (positive control) on their reaction with lipid is
shown in Table 2. The results show a very similar trend to the
one of DPPH radical scavenging assay. The halogenated com-
pounds, 3e and 3f, showed strong inhibitory activity, with
the lowest IC50 values, and comparable to a-tocopherol.
N
X
5
OH
R
2
N
R'
1
7
HO
OH
Compound Position of the gallic
hydrazide moiety
X
R
R0
2a
3a
3b
3c
3d
3e
3f
2
3
3
3
3
3
3
7
H
H
H
-
H
H
CH3
H
H
H
H
CH3
H
H
H
H
CH3
Cl
Br
H
H
H
H
H
7a
Results
Cytotoxic activities
Chemistry
The synthesized compounds were tested for their cytotoxic
activity on HCT-116 and MCF-7 cell lines by the MTT assay.
As shown in Table 2, all compounds have comparable activities
with the positive control, curcumin. The highest activities
against MCF-7 cell line were found for the halogenated com-
pounds 3e and 3f with IC50 values of 19.2 mM and 13.3 mM,
respectively. A similar trend was observed with HCT-116 cell
line, with the halogenated compounds 3e and 3f yielding the
lowest IC50 values, 6.7 mM. Figure 1 displays the effect of increas-
ing concentrations of the compounds on the cancer lines. It is
apparent that the cell viability decreased almost linearly as a
function of the log concentration of the compounds.
The structures of the synthesized compounds are presented
in Table 1. The gallic hydrazide moiety is connected via an
imine link on the indole nucleus, at different position (2, 3
or 7). Compounds differentiate also in substitution at
position 1, 2 and 5 of the indole nucleus. Among those,
the crystal structures of compounds 3a [28], 3e [29], and 3f
[30] have been reported previously by us. The compounds
were synthesized upon the reaction of the appropriate indole
carboxaldehydes with gallic hydrazide which had itself been
prepared from the reaction between methyl 3,4,5-trihydroxy-
benzoate and hydrazine. The synthetic route is depicted in
Scheme 1. Synthetic route for the preparation of compounds 2a–7a.
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Arch. Pharm. Chem. Life Sci. 2011, 344, 703–709
Gallic Acid-based Indole Derivatives
705
Table 2. DPPH radical scavenging, anti-lipid peroxidation (LP) and cytotoxic activities of 2a–7a1.
Compound
DPPH IC50 (mM)
LP IC50 (nM)
Cytotoxicity IC50 (mM)
MCF-7
HCT-116
2a
3a
3b
3c
3d
3e
3f
7a
357 ꢀ 12a,b
259 ꢀ 4c,d
303 ꢀ 10a,d
216 ꢀ 9c,e
358 ꢀ 10a,b
189 ꢀ 8e
172 ꢀ 9e
373 ꢀ 12b
981 ꢀ 7f
911 ꢀ 19g
–
403 ꢀ 8a
344 ꢀ 9b
621 ꢀ 4c
179 ꢀ 4d
713 ꢀ 18e
29 ꢀ 3f
50 ꢀ 3f
657 ꢀ 16c
–
40.0 ꢀ 2.5a,b
46.9 ꢀ 3.1b
35.1 ꢀ 2.8a,b
27.9 ꢀ 3.1a,c
39.0 ꢀ 2.1a,b
19.2 ꢀ 1.1c,d
13.3 ꢀ 0.9d
44.4 ꢀ 1.6b
–
29.4 ꢀ 1.6a
29.2 ꢀ 2.8a
30.0 ꢀ 3.7a
21.0 ꢀ 1.5a,b
29.4 ꢀ 3.0a
6.7 ꢀ 0.1c
6.7 ꢀ 0.2c
29.9 ꢀ 0.1a
–
Ascorbic acid
a-Tocopherol
Curcumin
38 ꢀ 6f
–
–
–
34.7 ꢀ 3.5a,b
13.7 ꢀ 0.9b,c
The values represent the mean ꢀ S.E.M. of three determinations.a–g Values in a column with the same superscript letter are not
1
significantly different, P < 0.05 (ANOVA and Tukey’s test).
Discussion
indolyl cation radical intermediate by the conjugated imine
side chain without disruption of the benzene aromaticity.
Introduction of a chlorine or bromine atom in position 5 of
the indole moiety significantly enhances the antioxidant
activity. Thus, compounds 3e and 3f show the highest activi-
ties among all the target compounds. This might be ascribed
to the electron donating properties of the halogens by reso-
nance, making the lone pair electron on the indole nitrogen
atom more available to a plausible electron transfer.
However, some other factors may be involved, as there are
some reports which show different influences by halogens in
the 5-position of indole [25, 33]. The effect of a methyl sub-
stituent depends on its attached position. Compound 3c,
with a methyl group in 2-position, has a higher activity than
the unsubstituted compound 3a, whereas introduction of a
methyl group on the indole nitrogen or at the 5-position
diminishes the antioxidant activity.
The present study was conducted to investigate structure–
activity relationship for the antioxidant and cytotoxic activi-
ties of hybrid molecules containing indole and gallic hydra-
zone moieties. Eight different indole gallic hydrazones were
synthesized and tested for their antioxidant potency by well
known in-vitro antioxidant assays, DPPH radical scavenging
ability and lipid peroxidation inhibitory. It is well established
that gallic acid derivatives possess antioxidant properties.
The antioxidant activity of these phenolic compounds is
mainly due to their redox properties, which can play an
important role in the absorption and neutralization of free
radicals, the quenching of singlet and triplet oxygen, or the
decomposition of peroxides [31]. Likewise, indolic com-
pounds are known to have antioxidant properties. The active
redox center of indoles is the nitrogen atom. Replacement of
the nitrogen by oxygen significantly reduces antioxidant
activity [32]. Delocalization of the nitrogen electron pair over
the aromatic system seems to be of great importance for
antioxidant activity of indole derivatives. It is reasonable
to conclude that multiple mechanisms regulate antioxidant
action of the hybrid molecules, although they may contrib-
ute to the antioxidant activity to different degrees.
For further assessment, the compounds were subjected to
cytotoxicity assay against HCT-116 and MCF-7 cell lines. In a
previously reported study on cytotoxic and antioxidant
activities of some phenolic systems (hydroxycinnamic acid
derivatives), a positive structure-activity-property relation-
ship between the activities was observed. It was suggested
that the antitumor activity of phenolic derivatives is highly
dependent upon their conformational characteristics, which,
in turn determine their antioxidant properties [14]. The con-
siderable antioxidant activities of our synthesized molecules
intrigued us to know how relevant these results are to their
cytotoxic activity. According to the in-vitro results, the target
compounds have comparable activities with the positive
control, curcumin. In excellent agreement with antioxidant
efficiency, the halogenated compounds, 3e and 3f, showed
the highest activities against both cell lines with IC50 values
lower than curcumin. Furthermore, consistent with the anti-
All the tested compounds showed significant DPPH radical
scavenging activity and with a very similar trend, inhibitory
effect on lipid peroxidation. Comparison of the activity of
compounds 2a, 3a, and 7a shows the importance of the
attachment position of the imine link, while the activities
of the other compounds reveal the effect of the substitution
patterns of the halogen or methyl group. The higher anti-
oxidant activity of compound 3a compared to 2a and 7a can
be explained by the fact that the attachment of the gallic
hydrazone at the 3-position permits stabilization of the
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Arch. Pharm. Chem. Life Sci. 2011, 344, 703–709
Figure 1. Dose-response curves of MCF-7 and HCT-116 cells to varying concentrations of compounds 2a–7a.
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Arch. Pharm. Chem. Life Sci. 2011, 344, 703–709
Gallic Acid-based Indole Derivatives
707
oxidant activity results, the replacement of the hydrogen in
position 2 by a methyl group enhanced the cytotoxicity, thus,
compound 3c is more active than 3a. To a lesser extent,
introduction of a methyl group on the indole nitrogen (3b)
or at position 5 of the indole moiety (3d) also increased the
activity against MCF-7 cells, while it did not show any
obvious effects on HCT-116 cell line (3a ꢁ 3b ꢁ 3d). A dis-
crepancy was also observed with the effect of the position of
the imine link on the indole system. While the 3-imineindole
3a showed higher antioxidant activity compared to the 2-
imineindole, 2a, and the 7-imineindole, 7a, the cytotoxicity
effectiveness against MCF-7 cell line followed the order of
2a > 7a > 3a. These three compounds showed almost the
same effect on HCT-116 cells. Overall, the results obtained
from this study showed a limited correlation between the
antioxidant and cytotoxic properties of the synthesized
compounds.
General procedure for the synthesis of compounds 2a–7a
An equimolar (5 mmol) mixture of indole carboxaldehyde and
3,4,5 trihydroxybenzoylhydrazine in the presence of acetic acid
(1 mL) was heated in ethanol (70 mL) for 6 h. The solution was
then cooled and filtered to remove the unreacted hydrazide. The
hydrazide was washed with ethanol and the filtrates added
together, evaporated partially, and poured to water (400 mL).
The solid product formed was filtered off, washed with diethyl
ether and dried over silica gel to give the related indole gallic
hydrazone with 57–77% yield.
3,4,5-Trihydroxy-N 0-[(1H-indol-2-yl)methylidene]-
benzohydrazide (2a)
Yield 68%, mp 261–2638C; 1H-NMR (DMSO-d6): d 6.77 (s, 1H, Ar-H),
6.93 (s, 2H, Ar-H), 6.99 (t, 1H, Ar-H), 7.13 (t, 1H, Ar-H), 7.42 (d, 1H,
Ar-H), 7.54 (d, 1H, Ar-H), 8.43 (s, 1H, CHN), 9.13 (br, 3H, OH), 11.50
(s, 1H, NH), 11.53 (s, 1H, NH); 13C-NMR (DMSO-d6): d 106.26, 107.17,
111.92, 120.66, 123.16, 123.36, 127.68, 133.50, 136.96, 137.72,
139.34 (CHN), 145.57, 163.16 (CONH); Anal. calcd. for C16H13N3O4:
C, 61.73; H, 4.21; N, 13.50%. Found: C, 61.20; H, 4.98; N, 13.10%.
Conclusions
3,4,5-Trihydroxy-N 0-[(1H-indol-3-yl)methylidene]-
benzohydrazide (3a)
Yield 73%, mp 238–2408C; 1H-NMR (DMSO-d6): d 6.92 (s, 2H, Ar-H),
7.16 (t, 1H, Ar-H), 7.20 (t, 1H, Ar-H), 7.43 (d, 1H, Ar-H), 7.76 (s, 1H,
Ar-H), 8.28 (d, 1H, Ar-H), 8.57 (s, 1H, Ar-H), 8.75 (s, 1H, OH), 9.13
(s, 2H, OH), 11.21 (s, 1H, indole NH), 11.53 (s, 1H, CONH); 13C-NMR
(DMSO-d6): d 107.45, 112.22, 112.33, 120.97, 122.41, 123.22,
124.34, 124.76, 130.39, 137.14, 137.41, 144.92 (CHN), 145.95,
163.57 (CONH); Anal. calcd. for C16H13N3O4: C, 61.73; H, 4.21;
N, 13.50%. Found: C, 61.44; H, 4.75; N, 13.17%.
In conclusion, we have designed a series of hybrid molecules
on the basis of the biological significance of indole and gallic
acid. With a high degree of consistency, the compounds
showed significant antioxidant activities in DPPH radical
scavenging and inhibitory of lipid peroxidation assays. The
compounds also exhibited noticeable cytotoxicity against
HCT-116 and MCF-7 cell lines. A limited degree of agreement
was observed between cytotoxic and antioxidant activities.
The activities are dependent on the position of the imine link
and different substituents on indole moiety. Among all, the
halogenated compounds, 3e and 3f, are the most efficient
compounds. Further study is required to understand the
mechanisms of action of this class of compounds.
3,4,5-Trihydroxy-N 0-[(1-methyl-1H-indol-3-yl)-
methylidene]benzohydrazide (3b)
Yield 65%, mp 256–2588C; 1H-NMR (DMSO-d6): d 3.81 (s, 3H, CH3),
6.92 (s, 2H, Ar-H), 7.17 (t, 1H, Ar-H), 7.26 (t, 1H, Ar-H), 7.48 (d, 1H,
Ar-H), 7.75 (s, 1H, Ar-H), 8.28 (d, 1H, Ar-H), 8.54 (s, 1H, Ar-H), 8.72
(s, 1H, OH), 9.10 (s, 2H, OH), 11.19 (s, 1H, CONH); 13C-NMR (DMSO-
d6): d 32.76 (CH3), 107.04, 110.14, 110.99, 120.55, 122.18, 122.67,
124.11, 124.79, 133.48, 136.59, 137.54, 143.38 (CHN), 145.56,
162.70 (CONH); Anal. calcd. for C17H15N3O4: C, 62.76; H, 4.65;
N, 12.92%. Found: C, 63.01; 4.89; N, 12.57%.
Experimental
Chemistry
Melting points were determined using a MEL-TEMP II melting
point instrument and were not corrected. Microanalyses were
carried out on a Perkin-Elmer 2400 elemental analyzer. 1H-NMR
and 13C-NMR spectra were determined with a Lambda JEOL
400 MHz FT-NMR (1H-NMR: 400 MHz and 13C-NMR: 100.4 MHz)
spectrometer. Chemical shifts are given in d values (ppm) using
TMS as the internal standard.
3,4,5-Trihydroxy-N 0-[(2-methyl-1H-indol-3-yl)-
methylidene]benzohydrazide (3c)
Yield 77%, mp 284–2868C; 1H-NMR (DMSO-d6): d 2.50 (s, 3H, CH3),
6.69 (s, 2H, Ar-H), 7.10 (m, 2H, Ar-H), 7.32 (d, 1H, Ar-H), 8.21 (d, 1H,
Ar-H), 8.66 (s, 1H, Ar-H), 8.76 (s, 1H, OH), 9.12 (s, 2H, OH), 11.10
(s, 1H, indole NH), 11.41 (s, 1H, CONH); 13C-NMR (DMSO-d6):
d 11.50 (CH3), 106.98, 107.78, 110.79, 120.13, 121.27, 121.75,
124.20, 125.47, 135.72, 136.53, 139.54, 143.57 (CHN), 145.59,
162.46 (CONH); Anal. calcd. for C17H15N3O4: C, 62.76; H, 4.65;
N, 12.92. Found: C, 62.51; H, 5.05; N, 13.00%.
Synthesis of 3,4,5-trihydroxybenzoic hydrazide (gallic
hydrazide)
A mixture of methyl 3,4,5-trihydroxybenzoate (9.2 g, 50 mmol)
and hydrazine hydrate (45 mL) was stirred at room temperature
for 30 min until the ester was dissolved completely. Ethanol
(250 mL) was added to the mixture and it was stirred under
reflux for 6 h and then at room temperature overnight. The
white solid was filtered, washed with ethanol and dried over
silica gel to give 5.13 g, 56% gallic hydrazide.
3,4,5-Trihydroxy-N 0-[(5-methyl-1H-indol-3-yl)-
methylidene]benzohydrazide (3d)
Yield 65%, mp 259–2608C; 1H-NMR (DMSO-d6): d 2.41 (s, 3H, CH3),
6.91 (s, 2H, Ar-H), 7.01 (d, 1H, Ar-H), 7.30 (d, 1H, Ar-H), 7.69 (s, 1H,
Ar-H), 8.05 (s, 1H, Ar-H), 8.54 (s, 1H, Ar-H), 8.70–9.10 (br, 3H, OH),
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Arch. Pharm. Chem. Life Sci. 2011, 344, 703–709
11.15 (s, 1H, indole NH), 11.41 (s, 1H, CONH); 13C-NMR (DMSO-d6):
d 21.58 (CH3), 107.28, 111.65, 111.76, 121.85, 124.29, 124.48,
124.86, 129.41, 130.33, 135.63, 136.90, 144.88 (CHN), 145.79,
163.18 (CONH); Anal. calcd. for C17H15N3O4: C, 62.76; H, 4.65;
N, 12.92. Found: C, 62.35; H, 4.98; N, 13.06%.
Lipid Peroxidation Assay
The lipid peroxidation assay was carried out following the
previously described method [35]. Fowl egg yolk, comprising
mainly of phospholipids, triacyglycerols and proteins, was used
as an alternative to rat liver microsomes and linoleic acid. The
reactive mixture for inducing lipid peroxidation contained
1 mL egg yolk emulsified with phosphate buffer saline
(0.1 M, pH 7.4), to obtain a final concentration of 12.5 g/L
and 200 mL of 3000 mM FeSO4. 40–43 mg (1 ꢄ 10ꢂ4 M) of each
test compound were dissolved in 1 mL DMSO (100%). This stock
solution was then diluted to final extraction concentration
3,4,5-Trihydroxy-N 0-[(5-chloro-1H-indol-3-yl)-
methylidene]benzohydrazide (3e)
Yield 67%, mp 274–2768C; 1H-NMR (DMSO-d6): d 6.89 (s, 2H, Ar-H),
7.18 (dd, 1H, Ar-H), 7.42 (d, 1H, Ar-H), 7.82 (d, 1H, Ar-H), 8.28 (s, 1H,
Ar-H), 8.52 (s, 1H, Ar-H), 8.77 (s, 1H, OH), 9.13 (s, 2H, OH), 11.26
(s, 1H, indole NH), 11.69 (s, 1H, CONH); 13C-NMR (DMSO-d6):
1 ꢄ 10ꢂ4, 1 ꢄ 10ꢂ5, 1 ꢄ 10ꢂ6, 1 ꢄ 10ꢂ7, 1 ꢄ 10ꢂ8, 1 ꢄ 10ꢂ9
,
and 1 ꢄ 10ꢂ10 M. Each assay was carried out in triplicates.
The mixture was incubated at 378C for 1 h, after which it
was treated with 0.5 mL of freshly prepared 15% TCA and
1.0 mL of 1% TBA. The reaction tubes were kept in boiling water
bath for 10 min. Upon cooling, the tubes were centrifuged at
3500 ꢄ g for 10 min to remove precipitated protein. LP was
measured spectrometrically by estimation of thiobarbituric
reactive substances (TBARS). The formation of TBARS was
measured by removing 100 mL of supernatant and measuring
the absorbance at 532 nm. a-Tocopherol ranging from 1 ꢄ 10ꢂ4
to 1 ꢄ 10ꢂ10 M was used as the standard reference. The control
was buffered egg with FeSO4 only. The percentage of inhibition
was calculated from the following equation:
d
107.52, 112.22, 113.90, 121.71, 123.08, 124.44, 125.46,
125.87, 131.93, 136.02, 137.14, 143.84 (CHN), 146.05, 163.25
(CONH); Anal. calcd. for C16H12ClN3O4: C, 55.58; H, 3.50;
N, 12.15. Found: C, 55.76; H, 3.89; N, 11.96%.
3,4,5-Trihydroxy-N 0-[(5-bromo-1H-indol-3-yl)-
methylidene]benzohydrazide (3f)
Yield 71%, mp 273–2758C; 1H-NMR (DMSO-d6): d 6.89 (s, 2H, Ar-H),
7.29 (dd, 1H, Ar-H), 7.38 (d, 1H, Ar-H), 7.81 (d, 1H, Ar-H), 8.42 (s, 1H,
Ar-H), 8.51 (s, 1H, Ar-H), 8.77 (s, 1H, OH), 9.13 (s, 2H, OH), 11.26
(s, 1H, indole NH), 11.70 (s, 1H, CONH); 13C-NMR (DMSO-d6):
d
107.51, 112.11, 113.51, 114.37, 124.43, 124.72, 125.63,
126.51, 131.79, 136.27, 137.14, 143.88 (CHN), 146.05, 163.45
(CONH); Anal. calcd. for C16H12BrN3O4: C, 49.25; H, 3.10;
N, 10.77. Found: C, 49.70; H, 3.43; N, 10.70%.
% Inhibition ¼ ½1ꢂðAs=A0Þꢃ ꢄ 100
where A0 refers to the absorbance of the control and As is the
absorbance of the sample. To determine the concentration
required to achieve 50% inhibition (IC50) of phospholipid oxi-
dation in egg yolk, the percentage of lipid peroxidation inhi-
bition was plotted against extract concentration.
3,4,5-Trihydroxy-N 0-[(1H-indol-7-yl)methylidene]-
benzohydrazide (7a)
Yield 57%, mp 237–2408C; 1H-NMR (DMSO-d6): d 6.56 (m, 1H, Ar-H),
6.98 (s, 2H, Ar-H), 7.12 (t, 1H, Ar-H), 7.31 (d, 1H, Ar-H), 7.53 (s, 1H,
Ar-H), 7.68 (d, 1H, Ar-H), 8.63 (s, 1H, CHN), 8.88 (s, 1H, OH), 9.22
(s, 2H, OH), 10.91 (s, 1H, indole NH), 11.81 (s, 1H, CONH); 13C-NMR
(DMSO-d6): d 102.3, 107.30, 118.10, 119.23, 122.73, 123.07,
124.04, 126.21, 128.23, 131.89, 137.26, 145.72, 147.55 (CHN),
163.44 (CONH); Anal. calcd. for C16H13N3O4: C, 61.73; H, 4.21;
N, 13.50%. Found: C, 62.2; H, 5.11; N, 13.77%.
Cell culture
The human colon carcinoma HCT-116 and hormone-dependent
breast carcinoma MCF7 were purchased from the American Type
Culture Collection (ATCC, USA). HCT 116 and MCF 7 cells in RPMI
1640 Medium (Sigma), supplemented with 10% (v/v) heat-inacti-
vated fetal bovine serum (FBS, PAA Lab, Austria), 100 mg/mL
streptomycin and 100 U/ml penicillin (PAA Lab, Austria) and
50 mg/mL fungizone (PAA Lab, Austria). The media were filter
sterilized using a 0.22 mm filter membrane (Minisart, Sartorius
stedim). The cells were cultured in 5% CO2 incubator at 378C in a
humidified atmosphere. The culture was subcultured every 2 or
3 days and routinely checked under an inverted microscope
(Motic) for any contamination. Cells in the exponential growth
phase were used for all experiments. The cells were harvested
from culture flasks by accutase (Innovative Cell Technologies)
and the viable cell count was determined using trypan blue
exclusion assay with a hemocytometer.
DPPH free radical scavenging activity
Radical scavenging activity was determined by a spectrophoto-
metric method based on the reduction of an ethanol solution of
1,1-diphenyl-2-picrylhydrazyl (DPPH) [34]. Briefly, to a solution of
DPPH (200 mM) in absolute ethanol, the compound dissolved in
DMSO was added at various concentrations. All the samples were
incubated in dark for 30 min at room temperature. When DPPH
reacts with an antioxidant, it will be reduced. The change in color
(from purple to yellow) was measured spectrophotometrically at
517 nm for each sample after incubation. Ascorbic acid and a-
tocopherol were used as positive controls. The free radical scav-
enging activity of the compounds was calculated as a percentage
of radical reduction. Each experiment was carried out in tripli-
cates and each experiment was performed three times. IC50 values
were determined from a calibration curve for each compound.
The radical scavenging activity was obtained from the equation:
MTT cytotoxicity assay
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay was modified from Mossmann [36]. Cells were seeded
at density of 5000 cells/well into a 96-well flat-bottomed culture
plates (Orange Scientific) and allowed to adhere overnight. The
experimental compounds were predissolved in dimethyl sulph-
oxide (DMSO) and diluted to the required concentration (five
concentrations, 0.1–100 mg/mL), such that the total DMSO con-
centration did not exceed 0.5%. At this concentration, DMSO was
Percentage of scavenging activity ð%Þ¼½1ꢂðAs=AcÞꢃꢄ100
where Ac is the absorbance of control (DPPH solution þ DMSO)
and As is the absorbance of compound/positive control.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 344, 703–709
Gallic Acid-based Indole Derivatives
709
found to be nontoxic to the cells tested. Vehicle DMSO was used
as a control. Cells were incubated with the samples (three wells
on a plate for each concentration) for 72 h. Treated and
untreated cells were inspected qualitatively using an inverted
light microscope (20 ꢄ). Then, 20 ml of MTT (5 mg/mL) (Sigma)
was added to each well and the plates were incubated at 378C for
4 h. The media was then gently aspirated, and 150 mL DMSO was
added to dissolve the formazan crystals. The amount of formazan
product was measured spectrophotometrically at 570 nm and
650nm as a background using a microplate reader (Oasys
UVM340).
Negi, S. P. S. Khanuja, Bioorg. Med. Chem. Lett. 2008, 18,
3914–3918.
[14] M. Esteves, C. Siquet, A. Gaspar, V. Rio, J. B. Sousa, S. Reis,
M. P. M. Marques, F. Borges, Arch. Pharm. Chem. Life. Sci. 2008,
341, 164–173.
[15] M. Inoue, R. Suzuki, T. Koide, N. Sakaguchi, Y. Ogihara,
Y. Yabu, Biochem. Biophys. Res. Commun. 1994, 204, 898–904.
[16] H. M. Chen, Y. C. Wu, Y. C. Chia, F. R. Chang, H. K. Hsu, Y. C.
Hsieh, C. C. Chen, S. S. Yuan, Cancer Lett. 2009, 286, 161–171.
[17] F. Palluotto, A. Carotti, G. Casini, M. Ferappi, A. Rosato,
C. Vitali, F. Campagna, Farmaco 1999, 54, 191–194.
The percentage of viability ¼ (absorbance of treated cells/
absorbance of untreated cells) ꢄ 100.
[18] C. K. Ryu, J. Y. Lee, R. E. Park, M. Y. Ma, J. H. Nho, Bioorg. Med.
Chem. Lett. 2007, 17, 127–131.
[19] J. J. Chen, Y. Wei, J. C. Drach, L. B. Townsend, J. Med. Chem.
2000, 43, 2449–2456.
We thank the staff members of the Institute of Biological Science, University
of Malaya for carrying out the biological tests. The authors thank the
University of Malaya for funding this study (FRGS grant No. FP009/
2008 C).
[20] A. Agarwal, K. Srivastava, S. K. Puri, P. M. S. Chauhan, Bioorg.
Med. Chem. Lett. 2005, 15, 3133–3136.
[21] R. Ragno, A. Coluccia, G. La Regina, G. de Martino,
F. Piscitelli, A. Lavecchia, E. Novellino, A. Bergamini,
C. Ciaprini, A. Sinistro, G. Maga, E. Crespan, M. Artico,
R. Silvestri, J. Med. Chem. 2006, 49, 3172–3184.
The authors have declared no conflict of interest.
[22] P. Singh, M. Kaur, P. Verma, Bioorg. Med. Chem. Lett. 2009, 19,
3054–3058.
References
[23] J. J. Wang, Y. K. Shen, W. P. Hu, M. C. Hsieh, F. L. Lin, M. K.
Hsu, M. H. Hsu, J. Med. Chem. 2006, 49, 1442–1449.
[1] D. V. Ratnam, D. D. Ankola, V. Bhardwaj, D. K. Sahana, M. N.
Kumar, J. Controlled Release 2006, 113, 189–207.
[24] R. Kumar, D. Rai, S. C. C. Ko, J. W. Lown, Heterocycl. Commun.
2002, 8, 521–530.
[2] S. D’Angelo, A. Morana, A. Salvatore, V. Zappia, P. Galletti,
J. Med. Food. 2009, 12, 1326–1333.
[25] G. Gurkok, T. Coban, S. Suzen, J. Enz. Inhib. Med. Chem. 2009,
24, 506–515.
[3] S. Kanai, H. Okano, Am. J. Chin. Med. 1998, 26, 333–341.
[4] P. Dwibedy, G. R. Dey, D. B. Naik, K. Kishore, P. N. Moorthy,
Phys. Chem. Chem. Phys. 1999, 1, 1915–1918 .
[26] Z. Ates-Alagoz, C. Kus, T. Coban, J. Enzyme Inhib. Med. Chem.
2005, 20, 325–331.
[5] K. Saeki, A. You, M. Isemura, I. Abe, T. Seki, H. Noguchi, Biol.
Pharm. Bull. 2000, 23, 1391–1394.
[27] J. Antosiewicz, E. Damiani, W. Jassem, M. Wozniak,
M. Orena, L. Greci, Free Radical Biol. Med. 1997, 22, 249–255.
[6] H. Sakagami, K. Satoh, T. Hatano, T. Yoshida, T. Okuda,
Anticancer Res. 1997, 17, 377–380.
[28] H. Khaledi, H. Mohd Ali, S. W. Ng, Acta Crystallogr., Sect. E:
Struct. Rep. Online 2008, 64, o2481.
[7] A. Serrano, C. Papacios, G. Roy, C. Cespon, M. L. Villar, M.
Nocito, P. Gonzalez-Porque, Arch. Biochem. Biophys. 1998, 350,
49–54.
[29] H. Khaledi, H. Mohd Ali, S. W. Ng, Acta Crystallogr., Sect. E:
Struct. Rep. Online 2009, 65, o169.
[30] H. Khaledi, H. Mohd Ali, S. W. Ng, Acta Crystallogr., Sect. E:
Struct. Rep. Online 2008, 64, o2108.
[8] I. Abe, T. Seki, H. Noguchi, Biochem. Biophys. Res. Commun.
2000, 270, 137–140.
[31] B. G. Heo, Y. S. Park, S. U. Chon, S. Y. Lee, J. Y. Cho,
S. Gorinstein, Biofactors 2007, 30, 79–89.
[9] N. Sakaguchi, M. Inoue, Y. Ogihara, Biochem. Pharm. 1998, 55,
1973–1981 .
[32] A. Gozzo, D. Lesieur, P. Duriez, J. C. Fruchart, E. Teissier, Free
Radical Biol. Med. 1999, 26, 1538–1543.
[10] M. Inoue, N. Sakaguchi, K. Isuzugawa, H. Tani, Y. Ogihara,
Biol. Pharm. Bull. 2000, 23, 1153–1157.
[33] I. Andreadou, A. Tsantili-Kakoulidou, E. Spyropoulou,
T. Siatra, Chem. Pharm. Bull. 2003, 51, 1128–1131.
[11] K. K. Sohi, N. Mittal, M. K. Hundal, K. L. Khanduja, J. Nutr. Sci.
Vitaminol. 2003, 49, 221–227.
[34] M. S. Blois, Nature 1958, 181, 1199–1200.
[12] Z. Lu, G. Nie, P. Belton, H. Tang, B. Zhao, Neurochem. Int. 2006,
48, 263–274.
[35] M. Daker, N. Abdullah, S. Vikineswary, P. C. Goh, U. R.
Kuppusamy, Food Chem. 2008, 107, 1092–1098.
[13] H. O. Saxena, U. Faridi, S. Srivastava, J. K. Kumar, M. P.
Darokar, S. Luqman, C. S. Chanotiya, V. Krishna, A. S.
[36] T. Mosmann, J. Immunol. Methods 1983, 65, 55–63.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com