Synthesis, antiproliferative activity and molecular properties predictions of galloyl derivatives

Article abstract of DOI:10.3390/molecules20045360

The present study was designed to investigate the in vitro antiproliferative activity against ten human cancer cell lines of a series of galloyl derivatives bearing substituted-1,3,4-oxadiazole and carbohydrazide moieties. The compounds were also assessed in an in silico study of the absorption, distribution, metabolism and excretion (ADME) in the human body using Lipinski's parameters, the topological polar surface area (TPSA) and percentage of absorption (%ABS). In general, the introduction of N'-(substituted)-arylidene galloyl hydrazides 4-8 showed a moderate antitumor activity, while the 2-methylthio- and 2-thioxo-1,3,4-oxadiazol-5-yl derivatives 9 and 10 led to increased inhibition of cancer cell proliferation. The precursor compound methyl gallate 2 and the intermediary galloyl hydrazide 3 showed greater antiproliferative activity with GI₅₀ values < 5.54 μM against all human tumor cell lines tested. A higher inhibition effect against ovarian cancer (OVCAR-3) (GI₅₀ = 0.05-5.98 μM) was also shown, with compounds 2, 3, 9 and 10 with GI₅₀ ≤ 0.89 μM standing out in this respect. The in silico study revealed that the compounds showed good intestinal absorption.

Full text of DOI:10.3390/molecules20045360

Molecules 2015, 20, 5360-5373; doi:10.3390/molecules20045360
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Antiproliferative Activity and Molecular Properties
Predictions of Galloyl Derivatives
1
2
2
3
Marciane Maximo da Silva , Marina Comin , Thiago Santos Duarte , Mary Ann Foglio ,
3
4
4,
João Ernesto de Carvalho , Maria do Carmo Vieira and Anelise Samara Nazari Formagio *
1
Faculdade de Ciências Biológicas e Ambientais, Universidade Federal da Grande Dourados,
Rodovia Dourados—Itahum, Km 12, Dourados, 79.804-970 MS, Brazil;
E-Mail: marcianemaximo@hotmail.com
2
Faculdade de Ciências Exatas e Tecnologia, Universidade Federal da Grande Dourados,
Rodovia Dourados—Itahum, Km 12, Dourados, 79.804-970 MS, Brazil;
E-Mails: marina_comin@hotmail.com (M.C.); thiagosantos948@gmail.com (T.S.D.)
3
Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas,
Universidade Estadual de Campinas, 6171, Campinas, 13083-970 SP, Brazil;
E-Mails: foglioma@cpqba.unicamp.br (M.A.F.); carvalho_je@yahoo.com.br (J.E.C.)
4
Faculdade de Ciências Agrárias, Universidade Federal da Grande Dourados,
Rodovia Dourados—Itahum, Km 12, Dourados, 79.804-970 MS, Brazil;
E-Mail: mariavieira@ufgd.edu.br
*
Author to whom correspondence should be addressed; E-Mail: aneliseformagio@ufgd.edu.br;
Tel.: +55-67-3410-2426.
Academic Editor: Panayiotis A. Koutentis
Received: 11 November 2014 / Accepted: 9 March 2015 / Published: 25 March 2015
Abstract: The present study was designed to investigate the in vitro antiproliferative
activity against ten human cancer cell lines of a series of galloyl derivatives bearing
substituted-1,3,4-oxadiazole and carbohydrazide moieties. The compounds were also
assessed in an in silico study of the absorption, distribution, metabolism and excretion
(
ADME) in the human body using Lipinski’s parameters, the topological polar surface area
TPSA) and percentage of absorption (%ABS). In general, the introduction of
(
N'-(substituted)-arylidene galloyl hydrazides 4–8 showed a moderate antitumor activity,
while the 2-methylthio- and 2-thioxo-1,3,4-oxadiazol-5-yl derivatives 9 and 10 led to
increased inhibition of cancer cell proliferation. The precursor compound methyl gallate 2
and the intermediary galloyl hydrazide 3 showed greater antiproliferative activity with GI50

Molecules 2015, 20
values < 5.54 µM against all human tumor cell lines tested. A higher inhibition effect
5361
against ovarian cancer (OVCAR-3) (GI50 = 0.05–5.98 µM) was also shown, with
compounds 2, 3, 9 and 10 with GI50 ≤ 0.89 µM standing out in this respect. The in silico
study revealed that the compounds showed good intestinal absorption.
Keywords: galloyl; hydrazide; oxadiazole; antiproliferative activity; in silico study
1
. Introduction
Gallic acid (3,4,5-trihydroxybenzoic acid) is a polyphenol that possesses a wide spectrum of
important pharmacological properties. In particular, gallic acid affects several pharmacological and
biochemical pathways and has strong antioxidant [1,2], anti-inflammatory [3], antimutagenic and
anticancer properties [4–6], showing selective cytotoxicity against a variety of tumor cells and much
less toxicity against normal cells [7–15].
Studies with a variety of synthetic galloyl derivatives have demonstrated the influence of
substituents on cytotoxic activity, e.g., digalloylresveratrol shows induction of apoptosis and necrosis
in HT-29 colon cancer cells and HL-60 human promyelocytic leukemia cells, and gallic acid esters
show inhibition of cancer cell proliferation [14,16–23]. Appropriate substituents in the galloyl group
positions could lead to more potent compounds.
The isolation of methyl gallate from the methanol extract of Schinus terebinthifolius leaves by our
research group (data not shown) and the biological potential of the galloyl group directed us to develop
derivatives as antitumor agents. Mannich bases of oxadiazoles and carbohydrazides possess important
activities, including anticancer functions [24–27] and our prior studies showed that introducing
1
,3,4-oxadiazole and carbohydrazide units at the C-3 position of the β-carboline derivatives led to
significant antitumor action [28–30]. As such, we proposed the synthesis and in vitro antiproliferative
activity evaluation of a series of galloyl derivatives bearing the substituted-1,3,4-oxadiazole and
carbohydrazide moiety at position C-1, expecting that the incorporation of these substituent may
improve the antitumor activities of the galloyl group. Herein, the synthesis of 2-methylthio- and
2
-thioxo-1,3,4-oxadiazol-5-yl derivatives is reported for the first time. Additionally, an in silico study
of the absorption, distribution, metabolism, and excretion (ADME) properties of the compounds was
performed by investigating their match of Lipinski’s rules, topological polar surface area (TPSA) and
percentage of absorption (%ABS). In silico ADME is currently used widely to determine whether it is
possible for a drug candidate to reach its site of action.
2
. Results and Discussion
2
.1. Chemistry
The synthetic route for the preparation of the galloyl derivatives is presented in Scheme 1. Methyl
gallate (2) was prepared by esterification of the corresponding carboxylic acid 1 with methanol and
sulfuric acid [31]. The reaction of methyl gallate with hydrazine hydrate yielded the galloyl hydrazide 3.
Condensation of the hydrazide 3 with aromatic aldehydes (benzaldehyde, 4-dimethylaminobenzaldehyde,

Molecules 2015, 20
5362
4
-nitrobenzaldehyde, 4-methoxybenzaldehyde and 4-hydroxybenzaldehyde), in ethanol heated at
1
reflux, yielded the N'-(substituted)-arylidenegalloyl hydrazides 4–8, respectively. The IR and H-NMR
spectroscopic data of these compounds were consistent with the related literature and the experimental
conditions and yields for the reaction were satisfactory [32].
O
O
O
HO
HO
O
R
OH
HO
HO
HO
HO
OCH3
NHNH2
HO
HO
N
a
b
c
N
H
OH
1)
OH
2)
OH
OH
(
(4) R= H
(
(3)
(5) R= 4-N(CH3)2
(6) R= 4-NO2
(7) R= 4-OCH3
(8) R= 4-OH
d
N NH
O
N
N
HO
HO
S
e
HO
HO
SCH3
O
OH
OH
(10)
(9)
Reagents and conditions: (a) MeOH, H
c) RCHO, EtOH, H SO (cat), reflux, 36 h; 74%–80%; (d) CS
HCl; (e) MeI, K CO , THF, rt, 48 h.
2
SO
4
, reflux, 48 h; 85%; (b) NH
2 2 2
NH ·H O, EtOH, reflux, 48 h; 74%;
(
2
4
2
/KOH, EtOH, reflux, 48 h, acidified dilute
2
3
Scheme 1. Synthetic route for the preparation of the galloyl derivatives.
For preparation of new derivatives of galloyl-2-thioxo-1,3,4-oxadiazole (9), the key intermediate 3
was subjected to a reaction with carbon disulfide in the presence of KOH and ethanol heated at reflux.
The 1,3,4-oxadiazolyl 9 was subsequently S-methylated with methyl iodide in the presence of K
2
CO
3
at room temperature to yield afford the galloyl-2-methylthio-1,3,4-oxadiazole 10.
1
13
The chemical structures of all compounds were confirmed by spectral data ( H- and C-NMR, IR,
1
and MS) (see Section 3). The H-NMR spectra of N'-(substituted)-arylidene galloyl hydrazides 4–8
displayed signals at δ
the imine and aromatic hydrogens, respectively, of the (substituted-benzylidene) galloyl hydrazide
group. The presence of this group was confirmed by the signals at δ 147–150 (C=N), 158–162 (C=O),
H
8.40 ppm integrating for one proton and δ
H
6.70–9.00 ppm, corresponding to
C
1
3
and 110–135 ppm (aromatic carbons of R group) in the C-NMR spectra.
The galloyl-2-thioxo-1,3,4-oxadiazole 9 may exist in a thione-thiol tautomeric structure, as
1
indicated by the –NH and –SH proton signals at 13.7–14.1 ppm in the H-NMR spectrum, as a broad
singlet integrating for one hydrogen each. In the solid state, these compounds are present in the thione
(
C=S) form, as indicated in their IR spectrum by the absence of the ν
500 cm−1 and the presence of two ν(C=S) absorption bands at 1230–1339 cm , which is
s
(S–H) absorption band at
−1
2
characteristic for this class of compounds. The S-methylated derivative 10 was characterized by the
presence of an addition signal at δ
H
/δ 2.84/14.7 ppm, corresponding to the S-methyl group attached to
C
the oxadiazole nucleus.

Molecules 2015, 20
.2. Antiproliferative Activity
5363
2
Three response parameters (GI50, TGI, and LC50) were calculated for each compound and cell line,
and the results are summarized in Tables 1 and 2.
Table 1. GI50 values (µM) for compounds 2–10 in ten tumor cell lines.
Cancer Cell Lines
U251 MCF-7 NCI/ADR-RES 786-0 NCI-H460 PC-3 OVCAR-3 HT-29 K-562 HaCaT
2
3
4
5
6
7
8
9
0.16
1.11
7.80
24.10
5.12
9.14
6.60
4.20
5.20
0.16
0.85
5.55
8.61
-
0.12
0.91
7.16
4.68
8.46
11.34
-
0.65
1.56
9.06
8.93
7.60
18.85
26.80
8.90
9.20
0.04
0.13
5.54
11.20
21.64
-
0.57
3.53
5.07
-
0.05
0.88
1.24
2.93
5.91
5.98
1.35
0.89
0.14
0.02
0.49
1.21
7.53
16.24
-
0.31
1.17
9.96
2.05
-
0.26
2.50
8.29
4.05
31.55
18.08
8.72
2.25
2.50
0.01
13.99
19.64
-
15.66
-
41.05
9.38
8.77
9.44
0.01
25.12
-
8.93
9.17
1.72
0.60
0.03
1.08
0.34
0.01
2.83
0.17
0.20
6.05
7.43
0.18
2.61
2.77
0.09
1
0
Dox 0.02
GI50: (growth inhibitory activity) the drug concentration that reduces cellular growth by 50%; Dox:
Doxorubicin; (-): GI50 value not presented.
Table 2. TGI and LC50 (values in parentheses) in µg/mL for compounds 2–10 in ten tumor
cell lines.
Cancer Cell Lines
U251
.10
29.19)
.13
MCF-7 NCI/ADR-RES
786-0
8.62
NCI-H460
7.97
PC-3
13.10
(-)
OVCAR-3 HT-29
K-562 HaCaT
4
5.12
(-)
3.62
(30.26)
2.56
1.59
(33.69)
9.77
(-)
8.52
(30.60)
3.27
5.67
16.74
(-)
2
3
4
5
6
7
8
9
(
(
(31.36)
17.98
(-)
(34.92)
17.35
(-)
(-)
-
6
2.36
(-)
18.31
(-)
29.02
>100
31.36)
(-)
(26.21)
-
2
3.54
-)
0.26
29.61
(-)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(
2
83.07
(-)
41.52
(-)
48.50
(-)
8.98
(-)
18.41
-
-
(
47.93)
(55.32)
8
4.67
-
-
-
-
-
-
-
-
-
-
-
(
-)
6
2
1
4.22
-)
6.07
-)
4.58
66.56
(-)
41.76
(-)
-
-
-
(
-
-
-
-
(
5.06
(-)
15.86
(-)
24.68
(-)
19.43
(42.67)
29.52
(65.78)
3.73
18.09
(56.99)
28.45
(69.30)
1.03
13.62
(58.99)
2.38
13.74
11.35
16.53
(-)
(
41.26)
6.40
(58.99) (58.99)
17.66 3.70
(60.32) (20.20)
1
3.20
2.33
(19.65)
22.42
(-)
29.40
(-)
18.00
(-)
1
0
(
30.43) (20.24)
(17.34)
1.47
2
.68
-)
2.52
(-)
0.51
(17.82)
26.03
(-)
2.60
0.77
(-)
Dox
(
(-)
(7.95)
(-)
(20.09)
TGI: the drug concentration required for total growth inhibition; LC50: the drug concentration required for
killing 50% of cells; Dox: Doxorubicin; (-): not presented.

Molecules 2015, 20
5364
The GI50 values (growth inhibitory activity) (Table 1) refer to the drug concentration that produces a
0% reduction of cellular growth compared with the untreated control cells. The TGI (cytostatic
5
activity) and LC50 (cytotoxic activity) values (Table 2) refer to the drug concentration required for total
growth inhibition and killing 50% of the cells, respectively. GI50 values were used to classify a
compound’s activity as follows: inactive, >100 µM; moderate, between >10 and <100 µM; and active,
<
10 µM.
The N'-(substituted)-arylidene galloyl hydrazide 4, which has a phenyl group attached to the galloyl
hydrazide moiety, possesses antiproliferative activity with GI50 values ranging from 1.24 to 9.96 µM
for nine human tumor cell lines. In particular, the compound displayed activity against the ovarian cell
line OVCAR-3 with a GI50 value of 1.24 µM (Table 1). Derivative 5, which has a dimethylaminophenyl
group attached to the galloyl hydrazide moiety, displayed cytotoxic efficacy against OVCAR-3
(GI50 = 2.93 µM; TGI = 41.52 µM) and leukemia K-562 (GI50 = 2.05 µM; TGI = 8.98 µM) cell lines
(
Tables 1 and 2). Compounds 6 and 7 did not demonstrate cytotoxic efficacy in these cell lines. The
activity against the OVCAR-3 cell line was also observed for compound 8 (GI50 = 1.35 µM),
containing the p-hydroxyphenyl group to the galloyl hydrazide moiety.
The introduction of the 2-thioxo-1,3,4-oxadiazol-5-yl group 9 at C-1 of the galloyl group led to
significant cytotoxicity, with GI50 less than 8.90 µM towards all cell lines, and displayed significant
activity against OVCAR-3 (GI50 = 0.89 µM), MCF-7 (GI50 = 1.08 µM), K-562 (GI50 = 1.72 µM),
HaCaT (GI50 = 2.25 µM), HT-29 (GI50 = 2.61 µM), and NCI/ADR/RES (GI50 = 2.83 µM) cell lines.
The 2-methylthio analogue 10 showed an increase in antiproliferative activity, with particular effectiveness
against OVCAR-3 (GI50 = 0.14 µM, TGI = 2.38 µM, LC50 = 17.34 µM), MCF-7 (GI50 = 0.34 µM,
TGI = 3.20 µM, LC50 = 20.24 µM), K-562 (GI50 = 0.60 µM, TGI = 3.70 µM, LC50 = 20.20 µM) and
NCI/ADR/RES (GI50 = 0.17 µM, TGI = 2.33 µM, LC50 = 19.65 µM) cell line (Tables 1 and 2).
In addition, the precursor compound methyl gallate 2 exhibited potent, broad-spectrum antitumor
activity with GI50 values < 1.0 µM against all human tumor cell lines tested, with GI50 (TGI) values
ranging from 0.05 to 0.65 µM (1.59–16.74 µM) (Tables 1 and 2). The intermediary, galloyl hydrazide
3
, also showed significant activity, with GI50 values in the range of 0.85–5.54 µM for all cell lines
tested, and displayed cytotoxic efficacy against MCF-7 breast (GI50 = 0.85 µM, TGI = 2.36 µM),
NCI/ADR-RES multiple drug resistant breast (GI50 = 0.91 µM, TGI = 2.56 µM) and OVCAR-3
ovarian (GI50 = 0.88 µM, TGI = 9.77 µM) cells (Tables 1 and 2). Methyl gallate is a derivative of
gallic acid that has been extensively investigated for important biological properties such as
antioxidant, anti-inflammatory, antimicrobial and antitumor activities by inhibiting tumor infiltration
+
+
of CD4 CD25 regulatory T cell and glioma cells [19,33–37]. However, the antitumor activity of this
compound has not been extensively examined in other cancer cells. We examined the effect of methyl
gallate in ten human tumor cell lines and observed selective cytotoxicity in all cell lines tested.
An analysis of the relationship between the GI50 data and the effects of the substitutions in the
aromatic ring at the N'-(substituted)-arylidene galloyl hydrazide with electron-withdrawing and
electron-donating substituents 5–8 did not display enhanced activity compared with the phenyl
substituent 4, which demonstrated a pronounced effect on cell line proliferation. These results suggest
that the electronic features of the various substituents affect how easily the drug can interact with
biological molecules. Regarding the relationship between the 1,3,4-oxadiazolyl derivatives, compound
1
0 displayed the highest antiproliferative activity compared to the corresponding precursor 9,

Molecules 2015, 20
5365
exhibiting selectivity for four cell lines. Substitution by ester, hydrazine and 1,3,4-oxadiazolyl moiety
side chains in compounds 2, 3, 9 and 10 resulted in a reduction in the percentage of growth inhibition,
thereby indicating the importance of these side chain groups.
The anthracyclins doxorubicin (DOX) and daunorubicin (DNR) are among the most useful
antibiotic, antitumor natural isolates from a species of the bacteria Streptomyces for the treatment of
breast cancer, solid tumors and leukemia [38–42]. The mechanism of action is complex. Briefly,
doxorubicin interferes with DNA replication by intercalation into DNA molecules, thus inhibiting the
biosynthesis of DNA, RNA and protein as well as the induction of the free radicals inside the cell,
leading to senescence and cell death by apoptosis or necrosis [43,44]. The compounds synthesized
have functional groups similar to DOX, which suggests that they may act in a similar manner. Thus,
the development of new molecular targets for antitumor therapies is directly correlated to many tumors
that are resistant to therapeutic strategies and side effects. The main side effect of DOX is cardiac
insufficiency, which is caused by the production of free radicals in the myocardium. In clinical
research, chemotherapy resistance has been identified as a serious problem when the concentrations of
chemotherapy drugs reach toxic and harmful doses for killing tumors.
2
.3. Lipinski’s Rule of Five
The present study focused on the antiproliferative activity and synthesis of structurally modified
galloyl derivatives, but we also explored the bioavailability of the synthesized derivatives, which is of
importance for further development of drugs based upon these substances and their analogues.
Drug-likeness is a promising paradigm to identify a balance that influences the pharmacodynamic and
pharmacokinetic properties of a compound that ultimately optimizes its absorption, distribution,
metabolism and excretion (ADME) in the human body [45].
These parameters were tentatively assessed using theoretical calculations following Lipinski’s rule
of five, which establishes that the absorption or permeation of an orally administered compound is
more likely to be efficient if the drug satisfies the established criteria: molecular weight (MW) ≤ 500 Da,
log P ≤ 5, H-bond donors (HBD) ≤ 5 and H-bond acceptors (HBA).
Our results (Table 3) revealed that derivatives 2, 4–10, presented lipophilicities less than 5, with
values between 0.34 and 1.94. Galloyl hydrazide (3) showed lipophilicity, Log P = −0.94, which is not
in violation of Lipinski’s rules. To further evaluate the drug-likeness, the rules have spawned many
extensions, such as the partition coefficient, Log P in −0.4 to +5.6 ranges [46]. All derivatives have a
number of hydrogen bond acceptors (HBA) (n-ON = 5–9), and their molecular weights were smaller
than 500 (184.15 > MW < 317.26), which is in agreement with Lipinski’s rules. With the exception of
the hydrazide 3, they all have a number of hydrogen bond donors (HBD) (n-OHNH = 6), violating one
of the Lipinski’s rules. The calculated percent absorption (%ABS) of all derivatives ranged between
5
7.95% and 78.98%, indicating that these compounds have good permeability in the cellular
membrane. Other rules include the number of rotatable bonds, indicating the flexibility of the
molecule, the volume and the polar surface area. The topological polar surface area (TPSA) is
recognized as a good indicator of drug absorption in the intestine (TPSA less than 140 Angstroms
2
2
squared [Å ]) and blood-brain barrier penetration (TPSA less than 60 Å ). The compounds exhibit
2
computational TPSA values between 86.99 and 122.37 Å and have good intestinal absorption except

Molecules 2015, 20
5366
2
6
(147.97 Å ). However, the derivatives do not have adequate blood-brain barrier penetration, as the
TPSA values are more than 60 Å2.
The empirical conditions to satisfy Lipinski’s rule and to manifest a good oral bioavailability
involve a balance between the solubility of a compound and its ability to diffuse passively through the
different biological barriers. Compounds with high solubility are more easily metabolized and
eliminated from the organism, thus leading to a lower probability of adverse effects and
bioaccumulation. The compound 2 (Log S = −0.87) presented good solubility, thus encouraging its use
as a precursor to produce new drugs that have better absorption.
Table 3. Lipinski’s rule and %ABS, TPSA, Log S for compounds 2–10.
Lipinski’s Parameters
TPSA a
Å2)
nHBA a
nHBD a
Comp. %ABS
Log P a MW a n violations a Log S b
(
(nON) (nOHNH)
2
3
4
5
6
7
8
9
78.98
69.04
73.75
72.64
57.95
70.57
66.78
73.63
74.63
86.99
115.80
102.15
105.38
147.97
111.38
122.37
102.51
99.61
5
6
6
7
9
7
7
6
6
3
6
4
4
4
4
5
4
3
0.85
−0.94
1.84
1.94
1.80
1.89
1.36
0.34
1.27
184.15
184.15
272.26
315.33
317.26
302.29
288.26
226.21
240.24
0
1
0
0
0
0
0
0
0
−0.87
−1.09
−2.83
−2.87
−3.47
−2.85
−2.54
−2.10
−3.07
1
0
a
b
www.molinspiration.com [47]; www.organic-chemistry.org/prog/peo [48]; %ABS = 109 − 0.345 × TPSA;
Number hydrogen bond acceptor (NO) = nHBA ≤ 10; Number hydrogen bond donors (OHNH) = nHBD ≤ 5;
MW ≤ 500; Octanol-water partition coefficient = Log P < 5; Solubility = Log S between −1 and −5.
3
3
3
. Experimental Section
.1. Chemistry
.1.1. General
1
13
H- and C-NMR spectra were recorded at 300 MHz and 75 MHz, respectively, using a model
Mercury plus BB spectrometer (Varian, Palo Alto, CA, USA). Mass spectra (MS) were recorded in a
Focus-DSQ II spectrometer (Thermoelectron Corporation, Austin, TX, USA). IR spectra were
recorded on a model MB-100 spectrometer (BOMEM, Quebec, CA, USA). For TLC, precoated plates
(
silica gel 60 G254, Macherey-Nagel, Duren, GER) were used. All reagents were purchased from
commercial suppliers.
3.1.2. Galloyl hydrazide (3)
Briefly, 80% hydrazine hydrate (1.8 mL, 48.2 mmol) was added to a solution of methyl gallate (2)
(
2.5 g, 2.97 mmol) that was previously prepared by esterification of commercial gallic acid (1) in
ethanol (30 mL). This mixture was heated at reflux for 60 h, when TLC analysis indicated the end of
the reaction. Then, the media was poured on ice, and the resulting precipitate was filtered, affording

Molecules 2015, 20
5367
the title compound 3 (1.85 g, 74%) as pale white powder, mp 164–167 °C (MeOH); mp 167 °C [32];
−
1
1
IR (KBr) νmax (cm ): 3365–3284 (O-H, N-H), 1623 (C=O), 1509–1303 (C=C), 1260 (C-O); H-NMR
1
3
(
DMSO-d
6
): δ
H
6.77 (s, 2H); 8.59 (NH); 9.32 (OH). C-NMR (DMSO-d
6
): δ 106.5, 123.9, 136.2,
C
+
1
45.4, 166.4. MS m/z (%): 185 (MH , 12), 153 (100), 126 (42).
3
.1.3. General Procedure for the Preparation of N'-(Substituted)-arylidene galloyl hydrazides 4–8
A solution of galloyl hydrazide (3) (0.28 g, 1 mmol) in water (10 mL) containing two drops of conc.
H
2
SO
benzaldehyde, 4-N-dimethylaminobenzaldehyde, 4-nitrobenzaldehyde, 4-methoxybenzaldehyde and
-hydroxybenzaldehyde, 1.5 mmol) in EtOH (3 mL) were added to the solution, which was then
heated at reflux for 48 h. The mixtures were poured into cold water and neutralized with 10% aqueous
NaHCO . The precipitates were collected by filtration, recrystallized and dried, yielding the
4
was heated at reflux for 0.5 h until complete dissolution. Then, aromatic aldehydes
(
4
3
compounds 4–8 in 74%–83% yields.
N'-(Benzylidene)galloyl hydrazide (4). White powder (0.20 g, 71%), mp 170–173 °C (MeOH); mp
−
1
1
70–172 °C [32]; IR (KBr) νmax (cm ): 3362–3270 (O-H, N-H), 1630 (C=O), 1518 (C=C), 1264 (C-O);
1H-NMR (CD
OD): δ
7.30–7.68 (5H, m), 7.70 (2H, s), 8.65 (1H, s, N'=CH). C-NMR (CD
13
OD): δ
3
H
3
C
+
1
15.4, 126.3, 128.7, 128.8, 131.0, 133.9, 140.4, 145.2, 148.7, 162.5. MS m/z (%): 272 (MH , 8), 153
(
100), 126 (30).
N'-(4-Dimethylaminobenzylidene)galloyl hydrazide (5). White powder, (0.22 g, 78%) mp 174–178 °C
−
1
(
MeOH); mp 175–178 °C [32]; IR (KBr) νmax (cm ): 3325 (O-H, N-H), 1661 (C=O), 1520 (C=C),
1
1
264 (C-O); H-NMR (CD
3
OD): δ 3.03 (6H, s), 6.93 (2H, d, J = 8.7 Hz), 7.68 (2H, s), 7.78 (2H, d,
H
1
3
J = 8.7 Hz), 8.36 (1H, s, N'=CH). C-NMR (CD
3
OD): δ
C
40.3, 111.9, 114.1, 125.9, 129.8, 130.4,
+
1
41.4, 146.2, 149.4, 151.2, 162.6. MS m/z (%): 315 (MH , 12), 153 (100), 126 (33).
N'-(4-Nitrobenzylidene)galloyl hydrazide (6). Pale yellow powder (0.24 g, 83%); mp 171–174 °C
−
1
(
(
MeOH); mp 171–172 °C [32]; IR (KBr) νmax (cm ): 3422–3280 (O-H, N-H), 1665 (C=O), 1520
1
C=C), 1264 (C-O); H-NMR (CD
3
OD): δ
H
6.98 (2H, s), 7.94 (2H, d, J = 8.5 Hz), 7.28 (2H, d, J = 8.5 Hz),
OD): δ 116.3, 122.8, 124.1, 127.8, 137.3, 141.0,
43.9, 145.6, 147.6, 163.4; MS m/z (%): 317 (MH , 10), 153 (100), 126 (21).
1
3
8
1
.49 (1H, s, N'=CH), 9.19 (OH, NH). C-NMR (CD
3
C
+
N'-(4-Methoxybenzylidene)galloyl hydrazide (7). Pale white powder (0,23 g, 80%), mp 170–172 °C
−
1
(
(
MeOH); mp 171–173 °C [32]; IR (KBr) νmax (cm ): 3410–3260 (O-H, N-H), 1668 (C=O), 1520
1
C=C), 1262 (C-O); H-NMR (CD
3
OD): δ
H
3.87 (3H, s, OCH ), 7.00 (2H, s), 7.70 (2H, d, J = 8.3 Hz),
3
1
3
6
1
.80 (2H, d, J = 8.3 Hz), 8.58 (1H, s, N'=CH). C-NMR (CD
3
OD): δ
C
55.6, 110.2, 114.4, 126.5,
+
28.6, 130.0, 136.8, 145.5, 146.6, 160.5, 161.6; MS m/z (%): 302 (MH , 31), 153 (100), 126 (17).
N'-(4-Hydroxybenzylidene)galloyl hydrazide (8). White powder (0,20 g, 78%), mp 170–173 °C
−
1
(
(
MeOH); mp 172–174 °C [32]; IR (KBr) νmax (cm ): 3418–3266 (O-H, N-H), 1668 (C=O), 1571
1
C=C), 1260 (C-O); H-NMR (CD
3
OD): δ
H
7.12 (2H, s), 7.34 (2H, d, J = 7.8 Hz), 6.60 (2H, d, J = 7.8
OD): δ 115.6, 114.8, 125.5, 128.7, 132.0, 136.8, 147.5,
47.6, 158.5, 160.6; MS m/z (%): 288 (MH , 18), 153 (100), 126 (44).
1
3
Hz), 8.66 (1H, s, N'=CH). C-NMR (CD
3
C
+
1

Molecules 2015, 20
.1.4. Galloyl-2-thioxo-1,3,4-oxadiazole (9)
Carbon disulfide (0.8 mL, 5 mmol) and potassium hydroxide (0,213 g, 1 mmol) were added to a
5368
3
solution of galloyl hydrazide (3) (0.456 g, 1 mmol) in EtOH (10 mL) at 0 °C. The resulting solution
was heated at reflux for 48 h. The solvent was evaporated, and the residue was dissolved in water and
acidified with a dilute solution of HCl. The solid obtained was filtered and recrystallized to give the
−1
title compound 9 (0.36 g, 80%) as a white powder, mp 168–170 °C (MeOH); IR (KBr) νmax (cm ):
1
3
9
370–3280 (O-H), 1595–1430 (C=C), 1245 (C-S), 1168 (C-O-C); H-NMR (CD
3
OD): δ 6.82 (2H, s),
H
1
3
.05 (OH, NH); C-NMR (CD
3
OD): δ 105.0, 112.0, 137.6, 146.5, 161.1, 176.9. MS m/z (%): 226
C
+
(
MH , 12), 150 (100), 77 (28).
3
.1.5. Galloyl-2-methylthio-1,3,4-oxadiazole (10)
To a solution of galloyl-2-thioxo-1,3,4-oxadiazole (9) (0.20 g, 0.6 mmol) in anhydrous THF (10 mL)
was to added K
2
CO (0.14 g, 0.6 mmol). The resulting solution was stirred at room temperature for 1 h.
3
Methyl iodide (0.19 mL, 0.8 mmol) was then added, and the mixture was stirred for an additional 48 h.
The solvent was removed, and the residue obtained was crystallized to give the title compound 10
−
1
(
0.1192 g, 60%), as a brown powder, mp 166–168 °C (MeOH); IR (KBr) νmax (cm ): 3376 (O-H,
1
N-H), 1668 (C=O), 1565–1430 (C=C), 1170 (C-O-C), 720 (C-S-C); H-NMR (CD
3
OD): δ 2.80 (3H,
H
1
3
s, SCH
3
); 7.00 (2H, s); 9.62 (OH). C-NMR (CD
3
OD): δ 16.2, 110.2, 112.4, 136.6, 146.8, 162.2,
C
+
1
60.4; MS m/z (%): 240 (MH , 8), 150 (100), 91 (28).
3.2. Anticancer Assay in Vitro
The synthesized compounds were assessed in the following ten human tumor cell lines from various
tissues kindly provide by the National Cancer Institute (Frederick, MD, USA): U251 (glioma, CNS),
MCF-7 (breast), NCI-ADR/RES (breast expressing the multiple drug resistance phenotype), 786-0
(
renal), NCI-H460 (lung, non-small cells), PC-3 (prostate), OVCAR-3 (ovarian), HT-29 (colon),
K-562 (leukemia) and HaCaT (keratinocytes). Stock cultures were grown in media containing 5 mL of
RPMI 1640 (Gibco BRL) supplemented with 5% fetal bovine serum. Gentamicin (50 mg/mL) was
added to the experimental cultures. Cells in 96-well plates (100 µL cells/well) were exposed to sample
concentrations in DMSO/RPMI (0.25, 2.5, 25, and 250 µg/mL) at 37 °C and 5% CO for 48 h. The
2
final DMSO concentration did not affect cell viability. Next, cells were fixed with 50% trichloroacetic
acid, and cell proliferation was determined by spectrophotometric quantification (540 nm) of cellular
protein content employing the sulforhodamine B assay [49]. Doxorubicin (0.025–25 µg/mL) was used
as a positive control. Three measurements were obtained: first at time point zero (T
of incubation) and then 48 h post-incubation for both the compound-free (C) and tested (T) cells. Cell
proliferation was determined using the equation 100 × [(T − T )/C − T ]. A cytostatic effect was
, while a cytocidal effect occurred when T < T . The experiments were
0
, at the beginning
0
0
observed when T ≥ T
0
0
performed in triplicate.

Molecules 2015, 20
.3. In Silico Study
An in silico computational study of the synthesized compounds 2–10 was performed to determine
5369
3
Lipinski’s rules, topological polar surface area (TPSA) and percentage of absorption (%ABS).
Calculations were performed using Molinspiration online property calculation toolkit software [47]
and OSIRIS property explorer software [48]. The percentage of absorption was estimated using the
following equation: %ABS = 109 − [0.345 × TPSA]. Lipinski’s rule states that an orally active drug
generally has no more than one violation of the following criteria [50]:
(
(
(
(
I) hydrogen bond donors ≤ 5 (OH and NH groups);
II) hydrogen bond acceptors ≤ 10 (N and O atoms);
III) molecular weight < 500;
IV) calculated logP < 5.
4
. Conclusions
The results suggest that synthetic derivatives of N'-(substituted)-arylidenegalloyl hydrazide do not
have a very strong synergistic effect in the inhibition of cancer cell proliferation compared with methyl
gallate (2), galloyl hydrazide (3) and 1,3,4-oxadiazolyl derivatives 9 and 10. These compounds showed
higher inhibition effect against ovarian (OVCAR-3) cells. Computational drug-likeness and TPSA
calculations revealed that the compounds show good intestinal absorption. Finally, the in silico study
identified these derivatives as potential new drug candidates. More detailed studies with other models,
such in vivo assays, are essential for the characterization of these derivatives as anticancer agents.
Acknowledgments
We are grateful to Fundect and CAPES fellowships for financial support (ASNF and MMS). The
authors are grateful Maria Helena Sarragiotto (Departamento de Química—UEM, Maringá-PR) for
technical support. Maria do Carmo Vieira in acquisition materials.
Author Contributions
ASNF, MMS, TSD and MC designed the study, synthesis of compounds and in silico study. MAF
and JEC participated in the antiproliferative assay. MCV helped conduct the literature review and in
implementation of the study.
Conflicts of Interest
The authors declare that they have no competing interests.
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©
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http://creativecommons.org/licenses/by/4.0/).
(