Synthesis and biological evaluation of novel hybrid compounds derived from gallic acid and the 2-aminothiophene derivatives

Article abstract of DOI:10.1007/s13738-019-01813-0

Abstract: Gallic acid (GA) and its benzamide derivatives have a wide variety of biological activities, such as antimicrobial, antioxidant, anticancer. In this study, we have reported the synthesis of some new hybrid compounds comprised of the 2-aminothiophene and GA moieties and evaluation of their cytotoxic activities against HeLa (cervical cancer), HCT116 (human colon cancer), and FT (fibroblast) cell lines as well as antimicrobial activities against some Gram-positive and Gram-negative bacteria. The reaction of some 2-aminothiophene derivatives (previously prepared from the Gewald reaction) with galloyl chloride having the acetylated hydroxyl groups and the subsequent deprotection of the hydroxyl groups gave the desired hybrid compounds. Then, the antimicrobial activity of the compounds was evaluated using disc diffusion and minimum inhibitory concentration assays. Finally, the MTT assay was carried out to evaluate the cytotoxicity of the synthesized compounds on the mentioned cell lines. The structure of the synthesized compounds was elucidated by conventional spectroscopic methods such as NMR, FT-IR, and UV–Vis spectroscopy. All compounds prevented the growth of Staphylococcus coagulase more than the positive control of chloramphenicol, and one compound was more sensitive to the growth of Klebsiella pneumonia compared to the standard antibiotic. All compounds showed acceptable activity against cancer cells. The highest activity was observed against HeLa with an IC₅₀ value of 3.2 μg/mL for compound 3d and against HCT116 with IC₅₀ of 59.4 μg/mL for 3b. The high anticancer activity of compound 3d against HeLa allows us to consider it as a good lead compound for the development of new potent anticancer agents for the treatment of cervical cancer. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s13738-019-01813-0

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-019-01813-0
ORIGINAL PAPER
Synthesis and biological evaluation of novel hybrid compounds
derived from gallic acid and the 2‑aminothiophene derivatives
1
1
1
2
Behnam Mahdavi · Seyed Mahmood Hosseyni‑Tabar · Esmail Rezaei‑Seresht · Hasan Rezaei‑Seresht ·
Farahnaz Falanji3
Received: 21 July 2019 / Accepted: 11 November 2019
©
Iranian Chemical Society 2019
Abstract
Gallic acid (GA) and its benzamide derivatives have a wide variety of biological activities, such as antimicrobial, antioxidant,
anticancer. In this study, we have reported the synthesis of some new hybrid compounds comprised of the 2-aminothiophene
and GA moieties and evaluation of their cytotoxic activities against HeLa (cervical cancer), HCT116 (human colon cancer),
and FT (ꢀbroblast) cell lines as well as antimicrobial activities against some Gram-positive and Gram-negative bacteria. The
reaction of some 2-aminothiophene derivatives (previously prepared from the Gewald reaction) with galloyl chloride having
the acetylated hydroxyl groups and the subsequent deprotection of the hydroxyl groups gave the desired hybrid compounds.
Then, the antimicrobial activity of the compounds was evaluated using disc diꢁusion and minimum inhibitory concentration
assays. Finally, the MTT assay was carried out to evaluate the cytotoxicity of the synthesized compounds on the mentioned
cell lines. The structure of the synthesized compounds was elucidated by conventional spectroscopic methods such as NMR,
FT-IR, and UV–Vis spectroscopy. All compounds prevented the growth of Staphylococcus coagulase more than the posi-
tive control of chloramphenicol, and one compound was more sensitive to the growth of Klebsiella pneumonia compared to
the standard antibiotic. All compounds showed acceptable activity against cancer cells. The highest activity was observed
against HeLa with an IC value of 3.2 μg/mL for compound 3d and against HCT116 with IC of 59.4 μg/mL for 3b. The
5
0
50
high anticancer activity of compound 3d against HeLa allows us to consider it as a good lead compound for the development
of new potent anticancer agents for the treatment of cervical cancer.
Graphic abstract
Keywords Synthesis · Biological evaluation · Anticancer activity · Antimicrobial activity · 2-Aminothiophenes · Gallic acid
Introduction
3
,4,5-trihydroxybenzoic acid or gallic acid is a polyhydroxy
*
Esmail Rezaei-Seresht
e.rezaei@hsu.ac.ir
phenolic compound that can be found in various plants such
as gallnuts, sumac, tea leaves, oak bark, green tea, apple
Extended author information available on the last page of the article
Vol.:(0
1
123456789)
3

Journal of the Iranian Chemical Society
peels, grapes, strawberries, pineapples, bananas, lemons [1,
spectroscopy was determined using a Shimadzu UV-1601
PC instrument. Elemental analyses were performed on a
Costech ECS 4010 CHNS analyser.
2
]. Gallic acid and its esters, in turn, are hydroxybenzoic
derivatives used as antioxidant additives in both food and
pharmaceutical industry, E-310 (propyl gallate) and E-311
(
octyl gallate), which are known to protect against oxida-
General procedure for the preparation of the substi‑
tuted 2‑aminothiophene‑3‑carbonitriles (1a‑d)
tive damage induced by reactive oxygen species (ROS), as
hydroxyl radicals or hydrogen peroxide, and reactive sulphur
species (RSS) [3, 4]. Moreover, gallic acid derivatives are
known to cause apoptosis in tumour cell lines and to inhibit
lymphocyte proliferation [5]. However, the mechanism by
which gallic acid analogues induce apoptosis in some cell
lines is not yet completely understood, probably involving
the paradoxical generation of ROS, which interferes with
the homoeostatic redox balance of the cell [6–8]. It is also a
fact that these compounds are excellent inhibitors of protein
tyrosinase kinases (PTKs) [9, 10].
These compounds were prepared according to literature
procedures with slight modiꢀcation [19, 20]. Brieꢂy, mor-
pholine (5 mL, 60 mmol) was slowly added to a mixture of
ketone (50 mmol), malonodinitrile (3.3 g, 50 mmol), and
sulphur (1.6 g, 50 mmol) in ethanol (75 mL) at 30 °C. Then,
the mixture was stirred for 8 h at this temperature. After
completion, the reaction mixture was ꢀltered, and the result-
ing ꢀltrate was poured into cold water, and allowed to stand
for 1 h. Finally, the precipitate was ꢀltered, washed with cold
aqueous ethanol (50%), and recrystallized from ethanol to
give the desired Gewald product.
The chemistry of 2-aminothiophenes has received much
attention upon their convenient availability through their
most versatile synthetic method, namely the Gewald reac-
tion [11]. An important feature of 2-aminothiophenes is that
they can serve as good precursors for the synthesis of bio-
logically active thiophene-derived heterocycles, conjugates,
and hybrids [12]. For example, olanzapine is a 2-aminothio-
phene-derived medication that is used as antipsychotic for
the treatment of schizophrenia and bipolar disorder [13].
Moreover, the 2-aminothiophene scaꢁolds exhibit diverse
pharmacological activities in medicinal chemistry, including
anticancer [14, 15], antimicrobial [14, 16, 17], and antiviral
activities [15, 18].
2‑Amino‑4,5‑dimethylthiophene‑3‑carbonitrile (1a)
Yield: 45%; brown crystals; M.p. 136–138 °C (Lit. [21]
141-142 °C).
2‑Amino‑5,6‑dihydro‑4H‑cyclopentathiophene‑3‑carboni‑
trile (1b)
Yield: 90%; brown crystals; M.p. 146–148 °C (Lit. [22]
144-145 °C).
Herein, we report the synthesis and biological evaluation
of four novel hybrid compounds comprised of one gallic acid
moiety and one 2-aminothiophene nucleus. Their antimicro-
bial activities against three Gram-positive and four Gram-
negative bacteria were studied. Also, cytotoxicity activities
of the hybrid compounds on the HCT116, HeLa, and FT
cell lines were carried out and determined by the MTT assay
method.
2‑Amino‑4,5,6,7‑tetrahydrobenzo[b]thiophene‑3‑carboni‑
trile (1c)
Yield: 97%; cream crystals; M.p. 145–146 °C (Lit. [20]
147-148 °C).
2‑Amino‑5,6,7,8‑tetrahydro‑4H‑cyclohepta[b]thio‑
phene‑3‑carbonitrile (1d)
Materials and methods
Yield: 95%; light brown needles; M.p. 120–122 °C (Lit. [22]
1
17-119 °C).
General
3,4,5‑Triacetoxybenzoic acid (2a)
All chemicals were used as received without further puriꢀ-
cation. Solvents were redistilled prior to use. All reactions
were monitored by TLC on commercially available pre-
coated glass plates (silica gel 60 F , Merck), and the plates
A solution of gallic acid (5.1 g, 30 mmol) in acetic anhy-
dride (20 mL) was reꢂuxed for 3 h. The solution was then
poured into hot water (100 mL). During cooling time, com-
pound 2a was crystallized. After ꢀltering and washing with
water, the white crystals were dried in vacuo. Yield: 95%;
2
54
were visualized in UV light (254 nm). Melting points were
recorded on an Electrothermal-9200 melting point apparatus
and are uncorrected. FT-IR spectra were measured using a
M.p. 173–174 °C; FT-IR (KBr disc) ν :2400–3200 (OH),
max
1
13
−1 1
Shimadzu 8400 FT-IR instrument. H NMR and C NMR
spectra were obtained using a Bruker 300 MHz NMR spec-
trophotometer with TMS as the internal reference. UV–Vis
1782 and 1699 (CO), 1605 (CC), 1178 (CO) cm ; H NMR
(DMSO-d , 300 MHz) δ 2.30 (s, 6H), 2.33 (s, 3H), 7.75 (s,
6
1
3
2H), 13.38 (s, 1H) ppm; C NMR (DMSO-d , 100 MHz):
6
1
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Journal of the Iranian Chemical Society
δ 19.8, 20.3, 121.9, 128.9, 138.2, 143.2, 165.3, 166.9,
2H), 6.97 (s, 2H), 9.04 (s, 1H), 9.36 (s, 2H), 11.31 (s, 1H)
1
3
1
68.0 ppm.
ppm; C NMR (DMSO-d , 75 MHz) δ 27.9, 28.1, 29.6,
6
9
1.8, 108.2, 115.2, 122.5, 134.9, 138.8, 141.7, 146.1, 152.1,
3,4,5‑Triacetoxybenzoyl chloride (2b)
165.4 ppm; UV–Vis (DMSO): λ 462 nm; Anal. Calcd for
max
C H N O S: C, 56.95; H, 3.82; N, 8.86; S, 10.13. Found:
1
5
12
2
4
Acid 2a (5.9 g, 20 mmol) was reꢂuxed with thionyl chloride
C, 57.28; H, 3.68; N, 8.51; S, 9.76.
(
6 mL) for 1 h. The excess of thionyl chloride was removed
by evaporating under reduced pressure. After drying, the
pale yellow residue was washed with the dried diethyl ether.
The raw product 2b was recrystallized in xylene. Yield: 91%;
N‑(3‑cyano‑4,5,6,7‑tetrahydrobenzo[b]
thiophen‑2‑yl)‑3,4,5‑trihydroxybenzamide (3c)
white crystals; M.p. 102–104 °C; FT-IR (KBr disc) ν
:
Yield: 45%; pale yellow precipitate; M.p. 267–268 °C;
max
3
092 and 2939 (CH), 1784 and 1762 (CO), 1605 (CC), 1180
FT-IR (KBr disc) ṽ : 3379 (NH), 3286 (OH), 2849 (CH),
max
−
1 1
(
CO), 740 (CCl) cm ; H NMR (DMSO-d , 300 MHz) δ
2216 (CN), 1662 (CO), 1612 (CC), 1207 (CO), 1036 (CN)
6
−
1 1
2
.31 (s, 6H), 2.32 (s, 3H), 7.89 (s, 2H) ppm.
cm ; H NMR (DMSO-d , 300 MHz) δ 1.76 (s, 4H), 2.58
6
(
s, 4H), 7.00 (s, 2H), 9.06 (s, 1H), 9.40 (s, 2H), 11.34 (s,
13
General procedure for the synthesis of the hybrid
compounds (3a‑d)
1H) ppm; C NMR (DMSO-d , 75 MHz) δ 22.4, 23.3, 24.0,
6
24.1, 94.5, 108.2, 115.8, 124.0, 127.1, 130.9, 137.9, 145.9,
1
50.6, 165.9 ppm; UV–Vis (DMSO): λ
462 nm; Anal.
max
A solution of compound 1 (8 mmol) and triethylamine
Calcd for C H N O S: C, 58.17; H, 4.27; N, 8.48; S, 9.70.
16 14 2 4
(
1.6 mL) in the dried dichloromethane (40 mL) was slowly
Found: C, 58.64; H, 4.12; N, 8.18; S, 9.36.
added to a solution of 2b (2.5 g, 8 mmol) in dried dichlo-
romethane (40 mL), and the mixture was stirred at 0 °C for
N‑(3‑cyano‑5,6,7,8‑tetrahydro‑4H‑cyclohepta[b]
thiophen‑2‑yl)‑3,4,5‑trihydroxybenzamide (3d)
2
h. Then, the mixture was washed with water (3×40 mL),
dried over anhydrous sodium sulphate, evaporated to dry-
ness. The obtained residue was re-dissolved in dichlo-
romethane (40 mL), and hydrazine hydrate (1.3 mL) was
slowly added to the solution and stirred for 1 h. The mixture
was washed with water (3×40 mL), dried over anhydrous
sodium sulphate, and evaporated to dryness. Finally, the
recrystallization using water–methanol yielded compound 3.
Yield: 40%; cream precipitate; M.p. 262–264 °C; FT-IR
(KBr disc) ṽ : 3345 (NH), 3290 (OH), 2847 (CH), 2214
max
−
1
(CN), 1626 (CO), 1608 (CC), 1207 (CO), 1036 (CN) cm ;
1
H NMR (DMSO-d , 300 MHz) δ 1.60 (qui, J = 6.0 Hz,
6
4H), 1.78–1.81 (m, 2H), 2.65–2.70 (m, 4H), 6.99 (s, 2H),
1
3
9.01 (s, 1H), 9.34 (s, 2H), 11.30 (s, 1H) ppm; C NMR
(
DMSO-d , 75 MHz) δ 27.5, 28.2, 28.9, 29.0, 31.9, 97.1,
6
N‑(3‑cyano‑4,5‑dimethylthiophen‑2‑yl)‑3,4,5‑trihydroxy‑
benzamide (3a)
108.1, 116.3, 124.0, 130.8, 135.7, 138.0, 145.9, 148.8,
166.0 ppm; UV–Vis (DMSO): λ 457 nm; Anal. Calcd
max
for C H N O S: C, 59.29; H, 4.68; N, 8.13; S, 9.31. Found:
17
16
2
4
Yield: 48%;brown precipitate; M.p. 292–294 °C; FT-IR
C, 59.71; H, 4.47; N, 7.86; S, 8.93.
(
KBr disc) ṽ : 3311 (NH), 3242 (OH), 2984 (CH), 2222
max
−
1
(
CN), 1620 (CO), 1610 (CC), 1219 (CO), 1036 (CN) cm ;
Antimicrobial activity
1
H NMR (DMSO-d , 300 MHz) δ 2.12 (s, 3H), 2.25 (s, 3H),
6
1
3
6
.96 (s, 2H), 9.32 (br. s, 3H), 11.25 (s, 1H) ppm; C NMR
Three Gram-positive bacteria of Staphylococcus aureus
ATCC25923, Enterococcus faecalis ATCC14506, Staphy-
lococcus coagulase, and four Gram-negative bacteria includ-
ing Escherichia coli ATCC25922, Klebsiella pneumonia
ATCC13883, Proteus vulgaris ATCC33420, Pseudomonas
aeruginosa ATCC27853 were used as the tested strains. All
the microorganisms were obtained from the microbiology
laboratory culture collection of Sabzevar Medical Science
University.
(
DMSO-d , 75 MHz) δ 12.6, 12.6, 97.6, 99.9, 108.1, 115.3,
6
1
22.5, 125.6, 129.2, 138.4, 146.0, 146.1165.3 ppm; UV–Vis
(
DMSO): λ
461 nm; Anal. Calcd for C H N O S: C,
max
14 12
2
4
5
5.26; H, 3.97; N, 9.21; S, 10.53. Found: C, 55.61; H, 3.80;
N, 8.86; S, 10.17.
N‑(3‑cyano‑5,6‑dihydro‑4H‑cyclopenta[b]
thiophen‑2‑yl)‑3,4,5‑trihydroxybenzamide (3b)
Yield: 43%; mustard-yellow precipitate; M.p. 262–264 °C;
Disc diꢀusion assay
FT-IR (KBr disc) ṽ : 3298 (OH), 3296 (NH), 2922
max
(
(
CH), 2220 (CN), 1652 (CO), 1606 (CC), 1207 (CO), 1032
The assay was carried out according to a previous study [23];
ꢀrst, a plate of Mueller–Hinton agar medium was inocu-
−
1 1
CN) cm ; H NMR (DMSO-d , 300 MHz) δ 2.36 (qui,
6
8
J=6.0 Hz, 2H), 2.74 (t, J=6.0 Hz, 2H), 2.85 (t, J=6.0 Hz,
lated by the microorganism suspension (1×10 CFU/mL) of
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Journal of the Iranian Chemical Society
microorganism. Then, a paper disc with a diameter of 6 mm,
which had been impregnated with 30 µg of the compounds
were kept in medium supplemented with 10% FBS and anti-
biotics (100 U/mL penicillin G, 100 μg/mL streptomycin) at
(
3×10 µL of the solution of the compound with a concentra-
37 °C in a humidiꢀed incubator with 5% CO .
2
tion of 1 mg/mL), was placed on the inoculated agar. The
plates were incubated at 37 °C for 24 h. The diameter of
inhibition zones against the microorganisms was measured
and reported as the antibacterial activity of the synthesized
compounds. The assay was carried out in triplicate for each
strain. Chloramphenicol 30 µg was used as a positive con-
trol. The values were reported in terms of inhibition levels
percentage (%). The inhibition level was measured by divid-
ing the inhibition zone diameter of the samples with that of
the antibiotic (positive control) as following:
Cytotoxicity assay
3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) method was used to determine the eꢁects of
the synthesized compounds on the cytotoxicity of HeLa,
3
HCT116, and FT cell lines. Brieꢂy, 5×10 cells/well were
evenly distributed and incubated in 96-well plates (SPL Life
Sciences) overnight. The cells were treated with the synthe-
sized compounds at concentrations of 5, 10, 30, 50, 75,100,
Inhibition level (%) = (Inhibition zone diameter of the sample)∕(Inhibition zone diameter of the antibiotic) × 100
Antimicrobial activity was categorized as strong for inhi-
bition level ≥ 70%, moderate for inhibition level 50–70%,
and weak for inhibition level<50% [24].
130, 170, 200 µg/mL and negative control and then incu-
bated for 24 h. Subsequently, the medium in each well was
replaced with 20 µL MTT (5 mg/mL in PBS) and incubated
at 37 °C for 4 h. The purple-blue formazan precipitate was
dissolved in 100 µL DMSO, and the optical density was
measured at a wavelength of 492 nm and a reference wave-
length of 630 nm on a 96-well plate reader. The IC values
Minimum inhibitory concentration assay
The minimum inhibitory concentration (MIC) assay was
run on the bacteria strains that showed sensitivity to the
compounds in the disc diꢁusion method. For this purpose,
a 96-well plate (8×12 wells) was ꢀlled with 100 μL of the
culture media Mueller–Hinton broth (MHB); the ꢀrst well
was charged with 100 μL of DMSO solution of the synthe-
sized compounds; then, 100 μL from each of their serial
dilutions was transferred into consecutive wells; each well
was charged with 50 μL of the MHB and 50 μL of the bac-
teria inoculums described earlier. The ꢀnal volume in each
well was 200 μL with concentrations of 1.00, 0.50, 0.25,
5
0
were calculated as the concentration of compounds 3a-d that
caused a 50% inhibition of cell viability. Data were analysed
using a GraphPad prism program (Graphpad Software Inc.,
San Diego, CA, USA) to determine the IC values of each
5
0
compound independently.
Results and discussion
Synthesis
0
.125, 0.062, 0.0312, 0.0156, and 0.0078 mg/mL for each
compound and positive control of chloramphenicol. Each
plate was used for one microorganism, respectively. Three
wells in the last line of the plate were used as growth con-
trols by ꢀlling the wells with 50 μL of inoculums and 150 μL
of MHB; also, three wells were used as a negative control by
During our previous studies on the Gewald synthesis of
2-aminothiophenes [21, 25], we found numerous reports
regarding the biological activities of the 2-aminothiophene
derivatives in the literature such as anticancer, antimicrobial,
antiviral, anti-tubercular, anti-parasitic activity, and also a
prior molecule in modern drug design [26–28]. On the other
hand, the various biological activities of gallic acid and its
derivatives including anti-tumourigenic, anti-inꢂamma-
tory, antioxidant, anticancer, antiviral, and antimutagenic
have been extensively reported in the recent years [29–32].
These studies motivated us to design and synthesize new
compounds consisting of a galloyl group and a 2-aminothio-
phene unit joined by an amide linkage and then examine
whether the connection of these active moieties together
would cause a synergic eꢁect on their biological activities.
Therefore, 2-aminothiophene-3-carbonitrile derivatives 1a-
d as one component of the desired hybrid compounds were
synthesized through the Gewald three-component reaction
ꢀ
lling them with 200 μL of MHB. The covered plates were
incubated at 37.5 °C for 24 h in an incubator. The turbidity
of each well was then observed and recorded. The minimum
inhibitory concentration was assessed as the minimum con-
centration that resulted in no visible growth. All tests were
carried out in triplicate [23].
Cells culture and treatment
Human cervical carcinoma cell line (HeLa), human colon
cancer cell line (HCT116), and human embryonic kidney
(
HEK293-FT) cells were purchased from Pasteur Institute,
Iran. The cell lines were cultured in DMEM (GIBCO) and
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Journal of the Iranian Chemical Society
of 2-butanone and three cyclic ketones, and malononitrile
(
Scheme 1).
To ꢀt the other component (galloyl group) for the linkage,
the phenolic hydroxyl groups of gallic acid were acetylated
to prevent them from participating in the linkage step. The
fully acetylated acid was then activated with thionyl chlo-
ride to obtain the corresponding acid chloride (Scheme 2).
Finally, the two components were linked together via a
nucleophilic substitution reaction in the presence of trieth-
ylamine, and in situ deprotection of the hydroxyl groups
with hydrazine hydrate yielded the desired products 3a-d
Scheme 1 Synthesis of 2-aminothiophene-3-carbonitriles 1a-d
(
Scheme 3).
Antibacterial activity
Scheme 2 Synthesis of compounds 2a and 2b
The antibacterial activity of compounds 3a-d is shown in
Table 1. According to the results, the compounds prevented
the growth of all Gram-positive bacteria including S. aureus,
S. coagulase, and E. faecalis. In disc diꢁusion assay, the
compounds were recognized as a weak antibacterial agent
against S. aureus and E. faecalis due to their inhibition level
values (less than 50%); however, the compounds inhibited
the growth of S. coagulase more than positive control
(
chloramphenicol) and they were known as strong agent
against the bacterium. The maximum activity was obtained
for 3b with inhibition level of 120.8± 2.3 followed by 3a
(
104.3± 2.0), 3c (103.9± 2.6), and 3d (101.3± 2.6). Simi-
lar results were collected for the MIC assay. 3b inhibited
the strain of S. coagulase with the lowest concentration of
Scheme 3 Synthesis of the hybrid compounds 3a-d
Table 1 Antimicrobial activity
Positive controla
Microorganisms Test
3a
3b
3c
3d
of hybrid compounds using
disc diꢁusion (DD test)
and minimum inhibition
concentration (MIC) assays
Gram-positive
S. aureus
26.7± 0.4b 31.2± 1.5
23.2± 0.3c
DD %
35.1± 1.1
35.3± 1.1
MIC mg/mL 0.41± 0.14 0.21± 0.07 0.21± 0.07 0.21± 0.07 0.03± 0.01
DD % 104.3± 2.0 120.8± 2.3 103.9± 2.6 101.3± 2.6 7.7± 0.3
MIC mg/mL 0.15± 0.01 0.10± 0.03 0.15± 0.01 0.15± 0.01 0.41± 0.14
DD % 33.8± 0.7 33.2± 0.9 42.8± 1.2 38.0± 0.7 21.4± 0.4
S. coagulase
E. faecalis
MIC mg/mL 0.25± 0.00 0.25± 0.00 0.17± 0.07 0.25± 0.00 0.05± 0.01
Gram-negative
E. coli
d
DD %
MIC mg/mL NT
K. pneumoniae DD % 51.1± 1.0
NA
NA
NT
36.3± 0.6
NA
NT
30.5± 0.9
NA
NT
65.2± 0.8
19.8± 0.7
0.05± 0.01
20.3± 0.6
e
MIC mg/mL 0.25± 0.00 0.17± 0.07 0.41± 0.14 0.17± 0.07 0.03± 0.01
DD % 59.9± 1.2 82.7± 1.6 109.3± 1.6 91.7± 1.2 12.9± 0.4
MIC mg/mL 0.17± 0.07 0.17± 0.07 0.10± 0.03 0.15± 0.01 0.03± 0.01
P. vulgaris
P. aeruginosa
DD %
NA
NA
NT
NA
NT
NA
NT
24.7± 0.8
0.05± 0.01
MIC mg/mL NT
a
b
c
d
e
Chloramphenicol
Values are presented as means± SD (n=3)
The results of positive control in disc diꢁusion assay were reported in terms of mm
Non-active
Not tested
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Journal of the Iranian Chemical Society
0
.10 ± 0.03 mg/mL. The compounds inhibited two strains
3c
3d
3a
GA
3b
out of 4 Gram-negative microorganisms including K. pneu-
monia and P. vulgaris. The samples were inactive against
E. coli and P. aeruginosa. Maximum inhibition level was
obtained for 3c against P. vulgaris (109.3 ± 1.6) followed
by 3d (91.7 ± 1.2), 3b (82.7 ± 1.6), and 3a (59.9 ± 1.2). A
similarity in the results was observed for MIC assay; 3c rep-
resented the highest activity (0.10± 0.03 mg/mL) followed
by 3d (0.15± 0.01 mg/mL), 3b, and 3a (0.17± 0.07 mg/mL).
1
00
50
0
0
50
100
150
200
250
-
50
Concentration (μg/ml)
a)
Cytotoxicity
(
The cytotoxicity evaluation of the synthesized compounds
was measured by the MTT assay method on three cell lines
including HeLa, HCT116, and FT cells. The MTT results
showed that the cytotoxicity of the compounds signiꢀcantly
increases in a dose-dependent manner (Fig. 1). The IC
3
c
3d
3a
GA
3b
1
-
00
5
0
values are shown in Table 2. Compound 3d with a cyclo-
heptane ring incorporated into its structure exhibited excel-
lent anticancer activity against HeLa cell line, while it had
weak cytotoxicity on FT cells. Recently, Schiavon et al. have
reported high anticancer activities against the HeLa cell line
for a thiophene derivative which is structurally similar to
compounds 3d with respect to the thiophene moiety [33].
Therefore, it seems that the fusion of the cycloheptane ring
to the 2-aminothiophene ring gives a nucleus that would
have strong anticancer activity against HeLa cells.
5
0
0
0
50
100
150
200
250
50
Concentration (μg/ml)
b)
(
3c
3d
3a
GA
3b
1
-
00
Recently in a review work by Subramanian et al., molecu-
lar mechanisms of the anticancer activity of gallic acid and
its derivatives have been comprehensively surveyed. Accord-
ing to the report, the activity of the compounds is related
to the induction apoptosis through diꢁerent mechanisms
including generation of reactive oxygen species (ROS),
suppression, and promotion of oncogenes, regulation of
apoptotic and anti-apoptotic proteins, cell cycle arrest, and
inhibition of matrix metalloproteinases (MMPs), depend-
ing upon the type of cancer studied [34]. As reported there,
gallic acid induces cell lysis in the HeLa cells. Cell death is
accompanied by the loss of mitochondrial membrane poten-
tial in the HeLa cells. Also, an increase in the ROS genera-
tion and GSH depletion in the HeLa cells treated with gallic
acid has been observed [35].
5
0
0
0
50
100
150
200
250
50
Concentration (μg/ml)
c)
(
Fig. 1 Cytotoxicity activity of compounds 3a-d and gallic acid (GA)
on the HCT116 (a), HeLa (b), and FT (c) cell lines
Table 2 In vitro anticancer activity data (μg/mL) of compounds 3a-d
and gallic acid
Compound
HeLa (IC50)
HCT116 (IC50)
FT (IC50)
3
3
3
3
a
b
c
120.1
100.8
165.4
3.2
162.7
59.4
102.2
68.9
107.4
132.6
10.7
Conclusion
d
74.5
Gallic acid
26.3
41.8
NAa
Four novel hybrid compounds consist of gallic acid, and
some 2-aminothiophene derivatives were synthesized,
and their biological activities evaluated. The compounds
inhibited the growth of S. coagulase more than the posi-
tive control (chloramphenicol). The maximum activity was
obtained for 3b with an inhibition level of 120.8± 2.3 and
aNon-active
respectively; followed by 3c against P. vulgaris (109.3± 1.6
for inhibition level and 0.10± 0.03 mg/mL for MIC). Moreo-
ver, the anticancer activities of compounds 3a-d and gallic
0
.10 ± 0.03 mg/mL in the disc diꢁusion and MIC assays,
1
3

Journal of the Iranian Chemical Society
acid (for comparison purpose), on the HCT116, HeLa, and
FT cell lines, were determined. The results showed that
compound 3d with a seven-membered ring fused to the
thiophene ring had an excellent activity (IC =3.2 μg/mL)
14. K. Bozorov, H.R. Ma, J.Y. Zhao, H.Q. Zhao, H. Chen, K. Boba-
kulov, X.L. Xin, B. Elmuradov, K. Shakhidoyatov, H.A. Aisa, Eur.
J. Med. Chem. 84, 739 (2014)
1
5. K. Bozorov, L.F. Zhao Jy Nie, H.R. Ma, K. Bobakulov, R. Hu, N.
Rustamova, G. Huang, T. Eꢁerth, H.A. Aisa, RSC Adv. 7, 31417
(2017)
5
0
against HeLa cell line while 3c having a six-membered ring
1
6. S. Al-Mousawi, M. El-Apasery, H. Mahmoud, Molecules 18, 7081
exhibited the lowest activity (IC =165.4 μg/mL). There-
5
0
(2013)
fore, it seems that compound 3d could be considered as a
good lead for designing of the anticancer agents targeting
cervical cancer.
1
7. L. Scotti, M.T. Scotti, E.D.O. Lima, M.S.D. Silva, M.D.C.A.D.
Lima, I.D.R. Pitta, R.O.D. Moura, J.G.B.D. Oliveira, R.M.D.D.
Cruz, F.J.B. Mendonça-Junior, Molecules 17, 2298 (2012)
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1
Acknowledgements We are grateful to Hakim Sabzevari University
and Sabzevar University of Medical Sciences for the support of the
present study.
1
2
2
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Compliance with ethical standards
22. F. Javadi, R. Tayebee, Microporous Mesoporous Mater. 231, 100
Conflict of interest The authors declare that they have no conꢂict of
(2016)
interest.
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(
Aꢀliations
1
1
1
2
Behnam Mahdavi · Seyed Mahmood Hosseyni‑Tabar · Esmail Rezaei‑Seresht · Hasan Rezaei‑Seresht ·
Farahnaz Falanji3
1
3
Department of Chemistry, School of Sciences, Hakim
Cellular and Molecular Research Center, Sabzevar University
of Medical Sciences, Sabzevar, Iran
Sabzevari University, P.O. Box 96179-76487, Sabzevar, Iran
2
Traditional and Complementary Medicine Research Center,
Sabzevar University of Medical Sciences, Sabzevar, Iran
1
3