Synthesis, biological evaluation and in silico study of bis-thiourea derivatives as anticancer, antimalarial and antimicrobial agents

Article abstract of DOI:10.1007/s00044-017-2008-5

Two sets of bis-thioureas including a para series (8–14) and a meta series (4, 5, 15–19), were synthesized and evaluated for their anticancer, antimalarial and antimicrobial activities. Most of the synthesized bis-thioureas, except for analogs 8–11, displayed cytotoxicity against MOLT-3 cell line (IC₅₀ = 1.55–32.32 μM). Derivatives 5, 14, 18 and 19 showed a broad spectrum of anticancer activity. Analogs (4, 5, 8, 13, 14, 18 and 19) exhibited higher inhibitory efficacy in HepG2 cells than the control drug, etoposide. Significantly, bis-trifluoromethyl analog 19 was the promising potent cytotoxic agent (IC₅₀ = 1.50–18.82 μM) with the best safety index (1.64–20.60). Antimalarial activity results showed that trifluoromethyl derivative 18 was the most potent compound (IC₅₀ = 1.92 μM, selective index = 6.86). Antimicrobial activity revealed that bis-thioureas 12, 18 and 19 exhibited selective activity against Gram-positive bacteria and fungi. Promisingly, the bis-trifluoromethyl derivative 19 was the most potent compound in the series and displayed higher potency, against most of the Gram-positive bacteria and fungi, than that of ampicillin, the reference drug. Among the tested strains of microorganisms, compound 19 inhibited the growth of Staphylococcus epidermidis ATCC 12228 and Micrococcus luteus ATCC 10240 with the lowest MIC of 1.47 μM. The findings demonstrated that trifluoromethyl group plays a crucial role in their biological activities. Furthermore, the molecular docking was performed to reveal possible binding modes of the compounds against target proteins.

Full text of DOI:10.1007/s00044-017-2008-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2017) 26:3136–3148
DOI 10.1007/s00044-017-2008-5
ORIGINAL RESEARCH
Synthesis, biological evaluation and in silico study of bis-thiourea
derivatives as anticancer, antimalarial and antimicrobial agents
Ratchanok Pingaew1 Nujarin Sinthupoom2 Prasit Mandi3 Veda Prachayasittikul4
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Rungrot Cherdtrakulkiat2 Supaluk Prachayasittikul4 Somsak Ruchirawat5,6,7
Virapong Prachayasittikul2
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Received: 29 January 2017 / Accepted: 27 July 2017 / Published online: 5 September 2017
© Springer Science+Business Media, LLC 2017
Abstract Two sets of bis-thioureas including a para series
(8–14) and a meta series (4, 5, 15–19), were synthesized
and evaluated for their anticancer, antimalarial and anti-
microbial activities. Most of the synthesized bis-thioureas,
except for analogs 8–11, displayed cytotoxicity against
MOLT-3 cell line (IC50 = 1.55–32.32 µM). Derivatives 5,
14, 18 and 19 showed a broad spectrum of anticancer
activity. Analogs (4, 5, 8, 13, 14, 18 and 19) exhibited
higher inhibitory efficacy in HepG2 cells than the control
drug, etoposide. Significantly, bis-trifluoromethyl analog
19 was the promising potent cytotoxic agent (IC50
=
1.50–18.82 µM) with the best safety index (1.64–20.60).
Antimalarial activity results showed that trifluoromethyl
derivative 18 was the most potent compound (IC50 = 1.92
µM, selective index = 6.86). Antimicrobial activity revealed
that bis-thioureas 12, 18 and 19 exhibited selective activity
against Gram-positive bacteria and fungi. Promisingly, the
bis-trifluoromethyl derivative 19 was the most potent
compound in the series and displayed higher potency,
against most of the Gram-positive bacteria and fungi, than
that of ampicillin, the reference drug. Among the tested
strains of microorganisms, compound 19 inhibited the
growth of Staphylococcus epidermidis ATCC 12228 and
Micrococcus luteus ATCC 10240 with the lowest MIC of
1.47 µM. The findings demonstrated that trifluoromethyl
group plays a crucial role in their biological activities.
Furthermore, the molecular docking was performed to
reveal possible binding modes of the compounds against
target proteins.
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-2008-5) contains supplementary material,
which is available to authorized users.
* Ratchanok Pingaew
ratchanok@swu.ac.th
* Supaluk Prachayasittikul
supaluk@g.swu.ac.th
1
Department of Chemistry, Faculty of Science, Srinakharinwirot
University, Bangkok 10110, Thailand
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Keywords Thiourea Trifluoromethyl group Anticancer
2
Department of Clinical Microbiology and Applied Technology,
Faculty of Medical Technology, Mahidol University, Bangkok
10700, Thailand
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activity Antimalarial activity Antimicrobial activity
Molecular docking
3
Department of Community Medical Technology, Faculty of
Medical Technology, Mahidol University, Bangkok 10700,
Thailand
4
Center of Data Mining and Biomedical Informatics, Faculty of
Medical Technology, Mahidol University, Bangkok 10700,
Thailand
Introduction
5
Chulabhorn Research Institute, Bangkok 10210, Thailand
Thiourea derivatives have drawn considerable interest in the
field of medicine, agriculture and analytical chemistry.
These compounds display a diverse range of pharmacolo-
gical activities including anticancer (Kumar and Chimni
2015; Pingaew et al. 2013; Liu et al. 2015; Vega-Pérez et al.
6
Program in Chemical Biology, Chulabhorn Graduate Institute,
Bangkok 10210, Thailand
7
Center of Excellence on Environmental Health and Toxicology
(EHT), CHE, Ministry of Education, Bangkok 10400, Thailand

Med Chem Res (2017) 26:3136–3148
Fig. 1 Bioactive thioureas
3137
2012; Tahir et al. 2015; Bielenica et al. 2015), antimalarial
(Mishra and Batra 2013) and antimicrobial (Vega-Pérez
et al. 2012; Tahir et al. 2015; Bielenica et al. 2015; Mishra
and Batra 2013; Saeed et al. 2009; Suresha et al. 2011;
Stefanska et al. 2015; Cunha et al. 2007) activities. Bioac-
tive thioureas possess specific binding sites known as
hydrogen binding area (NH), complementary area (S) and
auxiliary binding area (1,3-substituents) (Mishra and Batra
2013). Thiourea has more lipophilicity than urea congener
thus rendering its easy penetration to cells. Various struc-
tural modifications of thioureas by introducing halogen and
trifluoromethyl moieties can improve biological effects of
the compounds (Vega-Pérez et al. 2012; Tahir et al. 2015;
Bielenica et al. 2015; Suresha et al. 2011; Stefanska et al.
2015). Several studies (Bielenica et al. 2015; Azam et al.
2015; Ehmann and Lahiti 2014; Grillot et al. 2014; Basarab
et al. 2013; Charifson et al. 2008) indicated that potent
antibacterial activity of halogenated urea/thiourea deriva-
tives such as compound (1) (Bielenica et al. 2015) was
elucidated by an inhibition of topoisomerase IV (Fig. 1).
Bis-thioureas have been denoted as compounds with two
thiourea units which showed significant pharmacological
activities. For example, phenyl-bis(phenylthiourea) 2 dis-
played cytotoxic activity toward multiple cancer cell lines at
nanomolar concentrations and its action involved the inhi-
bition of tubulin polymerization (Shing et al. 2014) (Fig. 1).
Many alkylated (bis)thiourea polyamine analogs (3) showed
antitumor activity by acting as a lysine-specific demethylase
1 inhibitor (Sharma et al. 2010; Nowotarski et al. 2015) as
well as antimalarial activity against Plasmodium falciparum
(Verlinden et al. 2015, 2011). In our group, bis-thioureas (4
and 5) derived from m-xylylenediamine have been revealed
to exert potent anticancer activity against HuCCA-1,
HepG2 and MOLT-3 cancer cell lines (Pingaew et al.
2013).
Fig. 2 Designed bis-thiourea derivatives
The importance of our work lies in the possibility that the
obtained synthetic bis-thioureas may serve as a useful lead
compound for the discovery of novel bioactive agents by
using a simple chemical synthetic procedure. Herein, a
variety of known bis-thioureas (4, 5, 15 and 19) and novel
bis-thiourea derivatives (8–14, 16–18) were synthesized and
evaluated for their anticancer, antimalarial and antimicrobial
activities. Furthermore, the molecular docking was per-
formed to reveal possible binding modalities of the inves-
tigated compounds against target proteins accounting for
anticancer (i.e., tubulin) and antimicrobial (i.e., topoi-
somerase IV) activities. Two-dimensional ligand-protein
interaction diagrams also were generated to elucidate crucial
binding moieties and interactions.
Materials and methods
Chemistry
Column chromatography was carried out using silica gel 60
(70–230 mesh ASTM). Analytical thin-layer chromato-
graphy (TLC) was performed on silica gel 60 F254 alumi-
1
num sheets. H- and13C-NMR spectra were recorded on a
In view of the existing reports and continuation of our
interest in the search for novel bis-thioureas endowed with
potential chemotherapeutic activities, the synthetic bis-
thiourea targets have been designed as xylylene-bis(phe-
nylthiourea) (Fig. 2). The core structure of target com-
pounds bears phenylthioureas at para and meta positions on
a xylene ring A, and different substituted groups (R = CH3,
OCH3, Cl, Br, NO2, CF3 and 2 × CF3) on phenyl rings B.
Bruker AVANCE 300 NMR spectrometer (operating at
1
300 MHz for H and 75 MHz for 13C). FTIR spectra were
obtained using a universal attenuated total reflectance
attached on a Perkin–Elmer Spectrum One spectrometer.
High resolution mass spectra (HRMS) were recorded on a
Bruker Daltonics (microTOF). Melting points were deter-
mined using a Griffin melting point apparatus and were
uncorrected.

3138
Med Chem Res (2017) 26:3136–3148
General procedure for the synthesis of bis-thioureas (4,
5, 8–19)
NMR (300 MHz, DMSO-d6): δ (ppm) 4.71 (s, 4H, 2 ×
CH2), 7.31 (s, 4H, ArH), 7.37 (d, J = 8.7 Hz, 4H, ArH),
7.48 (d, J = 8.7 Hz, 4H, ArH), 8.27 (br.s, 2H, NH), 9.69 (br.
s, 2H, NH). 13C NMR (75 MHz, DMSO-d6) δ 47.4 (2 ×
CH2), 125.3 (2 × C-3′, 2 × C-5′), 127.9 (C-2, C-3, C-5, C-
6), 128.4 (2 × C-4′), 128.9 (2 × C-2′, 2 × C-6′), 138.0 (C-1,
C-4), 138.8 (2 × C-1′), 181.3 (C = S). HRMS-TOF: m/z [M
+ H]+ 475.0581 (calcd for C22H21Cl2N4S2: 475.0579).
A mixture of xylylenediamine (2 mmol) and appropriate
phenylisothiocyanate (4 mmol) in dichloromethane (20 mL)
was stirred at room temperature for 3–16 h (monitored by
TLC). The solid product formed was filtered, and recrys-
tallized from methanol to give the bis-thiourea derivatives
(4, 5, 8–19).
1H NMR data of 1,1′-(1,3-phenylenebis(methylene))bis
(3-(4-chlorophenyl)thiourea) (4) (Pingaew et al. 2013), 1,1′-
(1,3-phenylenebis(methylene))bis(3-(4-nitrophenyl)thiourea)
(5) (Pingaew et al. 2013), 1,1′-(1,3-phenylenebis(methy-
lene))bis(3-(4-methylphenyl)thiourea) (15) (Gulgas and
Reineke 2008) and 1,1′-(1,3-phenylenebis(methylene))bis
(3-(3,5-ditrifluoromethylphenyl)thiourea) (19) (Jones et al.
2008) are consistent with that reported in the literature.
1,1′-(1,4-phenylenebis(methylene))bis(3-(4-bromophenyl)
thiourea) (11)
White solid. 81%. Mp 208–209 °C. IR (UATR) cm−1
:
3271, 3200, 1588, 1537, 1524, 1485, 1334, 1284, 1070,
822. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 4.69 (d, J =
5.4 Hz, 4H, 2 × CH2), 7.28 (s, 4H, ArH), 7.41 (d, J = 8.8
Hz, 4H, ArH), 7.48 (d, J = 8.8 Hz, 4H, ArH), 8.24 (br.s,
2H, NH), 9.66 (br.s, 2H, NH). 13C NMR (75 MHz, DMSO-
d6) δ 47.4 (2 × CH2), 116.6 (2 × C-4′), 125.6 (2 × C-2′,
2 × C-6′), 127.9 (C-2, C-3, C-5, C-6), 131.8 (2 × C-3′, 2 ×
C-5′), 137.9 (C-1, C-4), 139.2 (2 × C-1′), 181.3 (C = S).
HRMS-TOF: m/z [M + Na]+ 584.9368 (calcd for
C22H20Br2N4NaS2: 584.9388).
1,1′-(1,4-phenylenebis(methylene))bis(3-(4-methylphenyl)
thiourea) (8)
White solid. 80%. Mp 209–210 °C. IR (UATR) cm−1
:
1
3276, 1585, 1540, 1516, 1310, 1189, 956, 788, 662. H
NMR (300 MHz, DMSO-d6): δ (ppm) 2.25 (s, 6H, 2 ×
CH3), 4.68 (s, 4H, 2 × CH2), 7.11 (d, J = 8.2 Hz, 4H, ArH),
7.25 (d, J = 8.2 Hz, 4H, ArH), 7.27 (s, 4H, ArH), 8.04 (br.s,
2H, NH), 9.51 (br.s, 2H, NH). 13C NMR (75 MHz, DMSO-
d6) δ 20.9 (2 × CH2), 47.4 (2 × CH3), 124.3 (2 × C-2′, 2 ×
C-6′), 127.8 (C-2, C-3, C-5, C-6), 129.6 (2 × C-3′, 2 × C-
5′), 134.2 (2 × C-4′), 136.9 (2 × C-1′), 138.1 (C-1, C-4),
181.3 (C = S). HRMS-TOF: m/z [M + H]+435.1675 (calcd
for C24H27N4S2: 435.1672).
1,1′-(1,4-phenylenebis(methylene))bis(3-(4-nitrophenyl)
thiourea) (12)
Yellow solid. 76%. mp 254–255 °C. IR (UATR) cm−1
:
1
3310, 1596, 1504, 1328, 1302, 1257, 1112, 850, 698. H
NMR (300 MHz, DMSO-d6): δ (ppm) 4.75 (s, 4 H, 2 ×
CH2), 7.35 (s, 4H, ArH), 7.86 (d, J = 8.8 Hz, 4H, ArH),
8.18 (d, J = 8.8 Hz, 4H, ArH), 8.80 (br.s, 2H, NH). 13C
NMR (75 MHz, DMSO-d6) δ 47.1 (2 × CH2), 121.1 (2 ×
C-3′, 2 × C-5′), 124.9 (2 × C-2′, 2 × C-6′), 128.0 (C-2, C-3,
C-5, C-6), 137.6 (C-1, C-4), 142.4 (2 × C-4′), 146.9 (2 × C-
1′), 180.8 (C = S). HRMS-TOF: m/z [M + H]+ 497.1076
(calcd for C22H21N6O4S2: 497.1060).
1,1′-(1,4-phenylenebis(methylene))bis(3-(4-methoxyphenyl)
thiourea) (9)
White solid. 79%. mp 209–210 °C. IR (UATR) cm−1: 3245,
1565, 1542, 1509, 1298, 1243, 1032, 970, 831, 727, 679.
1H NMR (300 MHz, DMSO-d6): δ (ppm) 3.72 (s, 6H, 2 ×
OCH3), 4.67 (d, J = 5.4 Hz, 4H, 2 × CH2), 6.90 (d, J = 8.8
Hz, 4H, ArH), 7.23 (d, J = 8.8 Hz, 4H, ArH), 7.26 (s, 4H,
ArH), 7.91 (br.s, 2H, NH), 9.39 (br.s, 2H, NH). 13C NMR
(75 MHz, DMSO-d6) δ 47.5 (2 × CH2), 55.7 (2 × OCH3),
114.5 (2 × C-3′, 2 × C-5′), 126.6 (2 × C-2′, 2 × C-6′),
127.7 (C-2, C-3, C-5, C-6), 132.1 (2 × C-1′), 138.2 (C-1, C-
4), 157.1 (2 × C-4′), 181.6 (C = S). HRMS-TOF: m/z [M +
H]+ 467.1581 (calcd for C24H27N4O2S2: 467.1570).
1,1′-(1,4-phenylenebis(methylene))bis(3-(4-trifluoromethyl-
phenyl)thiourea) (13)
White solid. 74%. Mp 215–216 °C. IR (UATR) cm−1
:
1
3230, 1560, 1527, 1515, 1328, 1135, 841, 706. H NMR
(300 MHz, DMSO-d6): δ (ppm) 4.72 (s, 4H, 2 × CH2), 7.32
(s, 4H, ArH), 7.65 (d, J = 8.6 Hz, 4H, ArH), 7.73 (d, J =
8.6 Hz, 4H, ArH), 8.46 (br.s, 2H, NH), 9.94 (br.s, 2H, NH).
13C NMR (75 MHz, DMSO-d6) δ 47.4 (2 × CH2), 122.6
2
1,1′-(1,4-phenylenebis(methylene))bis(3-(4-chlorophenyl)
thiourea) (10)
(2 × C-3′, 2 × C-5′), 124.0 (q, JCF = 32 Hz, 2 × C-4′),
1
125.2 (q, JCF = 269 Hz, CF3), 126.1 (2 × C-2′, 2 × C-6′),
128.0 (C-2, C-3, C-5, C-6), 137.8 (C-1, C-4), 143.8 (2 × C-
1′), 181.2 (C = S). HRMS-TOF: m/z [M + H]+ 543.1122
(calcd for C24H21F6N4S2: 543.1106).
White solid. 75%. mp 233–234 °C. IR (UATR) cm−1: 3271,
3202, 1590, 1524, 1489, 1332, 1286, 1091, 825, 728. H
1

Med Chem Res (2017) 26:3136–3148
3139
1,1′-(1,4-phenylenebis(methylene))bis(3-(3,5-ditrifluoro-
methylphenyl)thiourea) (14)
s, 2H, NH). 13C NMR (75 MHz, DMSO-d6) δ 47.2 (2 ×
2
CH2), 122.3 (2 × C-3′, 2 × C-5′), 123.7 (q, JCF = 32 Hz,
1
2 × C-4′), 124.4 (q, JCF = 270 Hz, CF3), 125.7 (2 × C-2′,
White solid. 70%. Mp 234–235 °C. IR (UATR) cm−1
:
2 × C-6′), 126.3 (C-4, C-6), 126.4 (C-2), 128.4 (C-5), 138.8
(C-1, C-3), 143.3 (2 × C-1′), 180.8 (C = S). HRMS-TOF:
m/z [M + H]+ 543.1086 (calcd for C24H21F6N4S2:
543.1106).
1
3230, 1545, 1382, 1275, 1181, 1140, 955, 893. H NMR
(300 MHz, DMSO-d6): δ (ppm) 4.74 (s, 4H, 2 × CH2), 7.32
(s, 4H, ArH), 7.72 (s, 2H, ArH), 8.24 (s, 4H, ArH), 8.69 (br.
s, 2H, NH), 10.00 (br.s, 2H, NH). 13C NMR (75 MHz,
DMSO-d6) δ 47.3 (2 × CH2), 116.7 (2 × C-4′), 122.7 (2 ×
Cytotoxic assay: cancer cell lines
1
C-2′, 2 × C-6′), 123.7 (q, JCF = 271 Hz, CF3), 128.0 (C-2,
C-3, C-5, C-6), 130.6 (q, 2JCF = 32 Hz, 2 × C-3′, 2 × C-5′),
137.6 (C-1, C-4), 142.4 (2 × C-1′), 181.4 (C = S). HRMS-
TOF: m/z [M + H]+ 679.0871 (calcd for C26H19F12N4S2:
679.0854).
Cells suspended in the corresponding culture medium were
inoculated in 96-well microtiter plates (Corning Inc., NY,
USA) at a density of 10,000–20,000 cells per well, and
incubated for 24 h at 37 °C in a humidified atmosphere with
95% air and 5% CO2. An equal volume of additional
medium containing either serial dilutions of the test com-
pounds, positive control (etoposide and/or doxorubicin), or
negative control [dimethyl sulfoxide (DMSO)] was added to
the desired final concentrations, the microtiter plates were
incubated for an additional 48 h. The number of surviving
cells in each well was determined using MTT assay (Car-
michael et al. 1987; Mosmann 1983) (for adherent cells:
HuCCA-1, HepG2, and A549 cells) and XTT assay (Doyle
and Griffiths 1997) (for suspended cells: MOLT-3 cells).
The IC50 value is defined as the drug (or compound) con-
centration that inhibits cell growth by 50% (relative to
negative control).
1,1’-(1,3-phenylenebis(methylene))bis(3-(4-methoxyphenyl)
thiourea) (16)
White solid. 79%. Mp 169–170 °C. IR (UATR) cm−1
:
1
3256, 3219, 1542, 1511, 1299, 1221, 1032, 974, 830. H
NMR (300 MHz, DMSO-d6): δ (ppm) 3.71 (s, 6H, 2 ×
OCH3), 4.70 (d, J = 6.0 Hz, 4H, 2 × CH2), 6.89 (d, J = 8.8
Hz, 4H, ArH), 7.15–7.31 (m, 8H, ArH), 7.93 (s, 2H, NH),
9.42 (s, 2H, NH).13C NMR (75 MHz, DMSO-d6): δ (ppm)
47.7 (2 × CH2), 55.7 (2 × OCH3), 114.5 (2 × C-3′, 2 × C-
5′), 126.3 (C-4, C-6), 126.6 (C-2, 2 × C-2′, 2 × C-6′), 128.6
(C-5), 132.1 (2 × C-1′), 139.7 (C-1, C-3), 157.1 (2 × C-4′),
181.6 (C = S). HRMS-TOF: m/z [M + H]+ 467.1564 (calcd
for C24H27N4O2S2: 467.1570).
Antimalarial assay: radioisotope techniques
1,1′-(1,3-phenylenebis(methylene))bis(3-(4-bromophenyl)
thiourea) (17)
In vitro culture of P. falciparum (K1, multidrug resistant
strain) was performed, according to (Trager and Jensen
1976), in RPMI 1640 medium containing 20 mM HEPES
(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), 32
mM NaHCO3 and 10% heat activated human serum with
3% erythrocytes, in humidified 37 °C incubator with 3%
CO2. The culture was passaged with fresh mixture of ery-
throcytes and medium every day to maintain cell growth.
Antimalarial activity was determined by microculture
radioisotope techniques as described by Desjardins et al.
(1979) (Desjardins et al. 1979). Briefly, a mixture of 200 μL
of 1.5% erythrocytes with 1% parasitemia at the early ring
stage was pre-exposed to 25 μL of the medium containing a
test sample dissolved in 1% DMSO (0.1% final con-
centration) for 24 h. Subsequently, 25 μL of [3H] hypox-
anthine (Amersham, USA) in culture medium (0.5 μCi) was
added to each well and the plates were incubated for an
additional 24 h. Levels of incorporated radioactive labeled
hypoxanthine, indicating parasite growth, were determined
using the Top Count microplate scintillation counter
(Packard, USA). The percentage of parasite growth was
calculated using the signal count per minute of treated
White solid. 80%. Mp 135–136 °C. IR (UATR) cm−1
:
1
3311, 3210, 1540, 1526, 1344, 1068, 825. H NMR (300
MHz, DMSO-d6): δ (ppm) 4.71 (s, 4H, CH2), 7.17–7.33 (m,
4H, ArH), 7.40 (d, J = 8.8 Hz, 4H, ArH), 7.46 (d, J = 8.8
Hz, 4H, ArH), 8.28 (s, 2H, NH), 9.70 (s, 2H, NH).13C NMR
(75 MHz, DMSO-d6): δ (ppm) 47.2 (2 × CH2), 116.2 (2 ×
C-4′), 125.3 (2 × C-2′, 2 × C-6′), 126.1 (C-4, C-6), 126.3
(C-2), 128.4 (C-5), 131.4 (2 × C-3′, 2 × C-5′), 138.7 (2 ×
C-1′), 139.0 (C-1, C-3), 180.9 (C = S). HRMS-TOF: m/z
[M + Na]+ 584.9371 (calcd for C22H20Br2N4NaS2:
584.9388).
1,1′-(1,3-phenylenebis(methylene))bis(3-(4-trifluoromethyl-
phenyl)thiourea) (18)
White solid. 73%. Mp 193–194 °C. IR (UATR) cm−1
:
1
3245, 1542, 1324, 1119, 1067, 840. H NMR (300 MHz,
DMSO-d6): δ (ppm) 4.75 (d, J = 4.8 Hz, 4H, 2 × CH2),
7.20–7.35 (m, 4H, ArH), 7.61 (d, J= 8.6 Hz, 4H, ArH),
7.70 (d, J = 8.6 Hz, 4H, ArH), 8.47 (br.s, 2H, NH), 9.95 (br.

3140
Med Chem Res (2017) 26:3136–3148
CPMT and untreated conditions (CPMU) as shown by the
following equation;
Salmonella choleraesuis ATCC 10708, Shewanella putre-
faciens ATCC 8071, Achromobacter xylosoxidans ATCC
2706, Pseudomonas aeruginosa ATCC 15442, Pseudomo-
nas aeruginosa ATCC 27853, Pseudomonas stutzeri ATCC
17587, Shigella dysenteriae, Salmonella enteritidis, Mor-
ganella morganii, Aeromonas hydrophila, Citrobacter
freundii, Plesiomonas shigelloides; ten Gram-positive-
bacteria: Staphylococcus aureus ATCC 29213, Staphylo-
coccus aureus ATCC 25923, Staphylococcus epidermidis
ATCC 12228, Enterococcus faecalis ATCC 29212, Enter-
ococcus faecalis ATCC 33186, Micrococcus luteus ATCC
10240, Corynebacterium diphtheriae NCTC 10356, Bacil-
lus subtilis ATCC 6633, Listeria monocytogenes, Bacillus
cereus, and two diploid fungi (yeast): Candida albicans
ATCC 90028, and Saccharomyces cerevisiae ATCC 2601.
% Parasite growth ¼ CPMT=CPMU Â 100:
Cytotoxicity assay: normal cell line (Vero)
Cytotoxicity assay was performed using the green fluor-
escent protein (GFP) detection method (Hunt et al. 1999).
African green monkey kidney (Vero, ATCC CCL-81) cell
line was stably transfected with pEGFP-N1 plasmid
(Clontech) for constructing GFP-expressing Vero cell. The
cell line was propagated in a medium containing 10% fetal
bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine,
1.5 g/L sodium bicarbonate and 0.8 mg/mL geneticin, and
maintained at 37 °C in a humidified incubator with 5% CO2.
In brief, a 45 µL of cell suspension at 3.3 × 104 cells/mL
was added to each well of 384 well plates containing 5 µL
of tested compounds diluted in 0.5% DMSO. The plate was
further incubated under the same condition for 4 days.
Fluorescence signals were measured at excitation and
emission wavelengths of 485 and 535 nm, respectively by
using SpectraMax M5 microplate reader (Molecular Devi-
ces, USA). Fluorescence signal at day 4 was substrated with
background fluorescence at day 0. IC50 values were deter-
mined from dose-response curves. Ellipticine and 0.5%
DMSO were used as a positive and a negative control,
respectively.
Molecular docking
Molecular docking was performed to investigate possible
binding modalities of the synthesized ligands toward their
possible targets. Crystal structures of the target proteins
were downloaded from RSCB protein data bank (http://
www.rcsb.org/). The studied targets for anticancer and
antimicrobial activities include α-tubulin and β-tubulin
complex (PDB id 1SA0) from Bos Taurus in co-
crystallized form with colchicines and DNA topoisome-
rase IV from Staphylococcus aureus in co-crystallized form
with novobiocin (PDB id 4URN), respectively. The target
protein structures were prepared by adding essential
hydrogen atoms and repairing missing side chains using the
WHAT IF web server version 10.1 (Vriend 1990). Non-
polar hydrogen atoms were merged, Gasteiger atomic
charges were assigned, and atom type of the protein struc-
tures were specified using AutoDock Tools version 1.5.6.
(Morris et al. 2009; Sanner 1999). The investigated bis-
thiourea ligands were constructed using Marvin Sketch
version 6.1.4 (ChemAxon 2013) and were geometrically
optimized by Gaussian 09 (Frisch et al. 2009) using Becke’s
three-parameter hybrid method with the Lee–Yang–Parr
correlation functional (B3LYP) together with the 6-31 g(d)
basis set. All ligands were prepared by merging non-polar
hydrogen atoms, defining rotatable bonds and assigning
partial atomic charge using AutoDock Tools version 1.5.6
(Morris et al. 2009; Sanner 1999). Grid boxes for tubulin
(25 Å × 25 Å × 25 Å) and topoisomerase (25 Å × 25 Å ×
25 Å) were generated to intend searching modality. Center
of the receptor binding site on grid boxes were allocated
using x, y and z coordinates of 118.36, 90.56, and 5.25 for
tubulin, and −50.33, −8.80 and −29.79 for topoisomerase
IV. AutoDock Vina, as a part of the PyRx 0.8 software
(Dallakyan 2013), was used to perform the molecular
docking simulation. The co-crystallized ligands were re-
docked to their target proteins to validate docking protocol
Antimicrobial assay: agar dilution
Agar dilution method was employed to investigate anti-
microbial properties of the tested compounds as described
previously (Prachayasittikul et al. 2011). Briefly, tested
compounds dissolved in DMSO were mixed with Müller
Hinton (MH) broth and added to MH agar for preparing
their serial dilutions at the final concentrations of 0.5–256
µg/mL. The DMSO without the tested compounds was
tested in parallel with the compounds. The MH broth was
the negative control, whereas ampicillin and ciprofloxacin
were used as reference drugs. Twenty-eight strains of
microorganisms as shown below were cultured in MH broth
at 37 °C for 24 h. The microbial cultures were individually
adjusted the cell suspension to 0.5 McFarland standards
(1.5 × 108 CFU/mL) using 0.9% normal saline solution.
The inoculums of microorganisms were dropped onto the
prepared plate using multipoint inoculator and incubated at
37 °C for 18–48 h. After the incubation, the lowest con-
centration of each tested compound which inhibited the
visible growth of microorganisms was reported as MIC.
Twenty-eight strains of tested microorganisms were sixteen
Gram-negative bacteria: Escherichia coli ATCC 25922,
Klebsiella pneumoniae ATCC 700603, Serratia marcescens
ATCC 8100, Salmonella typhimurium ATCC 13311,

Med Chem Res (2017) 26:3136–3148
3141
Scheme 1 Synthesis of bis-
thioureas (4, 5, 8–19) from
xylylenediamines 6 and
isothiocyanates 7
(i.e., colchicine for α-tubulin and β-tubulin complexes and
novobiocin for DNA topoisomerase IV). Re-docked results
were evaluated by calculation of root mean standard
deviation (RMSD) using Chimera software (Pettersen et al.
2004). Docking poses of the investigated compounds were
visualized using PyMOL (Delano 2002) and two-
dimensional ligand-protein interaction diagrams were gen-
erated using Discovery Studio Visualization version
16.1.0.15350 (BioVia 2017).
the distinctive peaks as two quartets with coupling constant
of 269–272 Hz (1JCF) and 32 Hz (2JCF), which proved the
presence of CF3 moiety and the carbon adjacent to CF3
group, respectively.
Biological activities
Cytotoxic activity
Cytotoxicity of the synthesized bis-thiourea derivatives was
examined against human cancer cell lines; HuCCA-1
(cholangiocarcinoma), HepG2 (hepatocellular carcinoma),
A549 (lung carcinoma) and MOLT-3 (lymphoblastic leu-
kemia), as summarized in Table 1. These compounds were
also evaluated toward non-cancerous, Vero cell line derived
from African green monkey kidney (Table 1), and a selec-
tive index was calculated as provided in Table S1.
In the para derivatives (8–14), compound 8 with methyl
substituent (R = CH3) displayed cytotoxic activity against
HuCCA-1 and HepG2 cells with IC50 values of 82.83 and
21.67 μM, respectively. Replacement of the methyl group
with methoxy, chloro and bromo (R = OCH3, Cl, Br) sub-
stituents led to compounds (9–11) with a total loss of
cytotoxicity. However, compounds 12 and 13 obtained by
the replacement of methyl group of compound 8 with nitro
(R = NO2) and trifluoromethyl (R = CF3) moieties showed
inhibitory potencies against HepG2 and MOLT-3 cell lines.
Significantly, an introduction of two trifluoromethyl groups
(R = 3,5-diCF3) on the phenyl ring provided the analog 14
which displayed a broad spectrum of anticancer activity
toward all of the tested cells with IC50 values in the range of
2.49–30.95 μM, and had a selective index in the range of
Results and discussion
Chemistry
Two sets of bis-thiourea derivatives constituting various
substitutuents (R = CH3, OCH3, Cl, Br, NO2, CF3) at
position-4 and position-3,5 (R = diCF3) on phenyl ring, a
para series (8–14) and a meta series (4, 5 and 15–19), were
readily synthesized by treatment of (p- or m-) xylylenedia-
mines 6 with the corresponding isothiocyanates 7 in
dichloromethane (Scheme 1). Structures of all obtained bis-
1
thioureas (4, 5 and 8–19) were characterized by H NMR,
13C NMR, IR, and HRMS. Typically, infrared spectra of the
synthesized compounds had strong N–H absorptions
(3202–3375 cm−1), and thiocarbonyl groups (C = S)
showed stretching absorption band values in the range of
1050–1250 cm−1. 1H NMR spectra exhibited two singlets at
δ 7–11 ppm, which were assigned to the N–H protons. 13C
NMR spectral data analysis of thioureas showed a typical
signal for thiocarbonyl carbon in the range of δ 180–182
ppm. In13C NMR, compounds 13, 14, 18 and 19 revealed

3142
Med Chem Res (2017) 26:3136–3148
Table 1 Cytotoxic and antimalarial activities (IC50, µM) of bis-thiourea derivatives (4, 5, 8–19)
Compound
Cancer cell linesa
Antimalarialb
Vero cell linec
HuCCA-1
HepG2
A549
MOLT-3
8
82.83 8.48
Non-cytotoxic
Non-cytotoxic
30.95 4.58
Non-cytotoxic
Non-cytotoxic
30.22 1.25
non-cytotoxic
14.84 0.50
14.47 1.38
18.82 2.34
ND
21.67 2.24
61.02 2.56
15.52 1.12
8.81 0.71
Non-cytotoxic
Non-cytotoxic
13.29 1.05
41.30 0.03
10.53 0.58
8.40 0.43
1.50 0.23
26.05 0.50
0.57 0.05
ND
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
26.16 0.35
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
44.71 3.49
17.97 2.96
16.67 1.11
ND
Non-cytotoxic
11.40 0.45
6.06 0.79
2.49 0.19
13.90 1.09
32.32 6.92
2.23 0.26
2.62 0.28
3.40 0.44
1.55 0.17
3.63 0.46
0.041 0.003
ND
Inactive
Inactive
17.95
Inactive
Inactive
Inactive
5.09
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
70.81
12
13
14
15
33.50
16
Non-cytotoxic
2.84
4
17
2.80
3.28
5
4.29
11.24
18
1.92
13.18
19
3.33
30.90
Etoposided
Doxorubicind
Mefloquined
Dihydroartemisinined
Ellipticined
ND
ND
0.42 0.02
ND
0.37 0.02
ND
ND
ND
ND
0.0247
0.0019
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2.16
IC50 > 50 μg/mL denoted as non-cytotoxic. IC50 > 10 μg/mL denoted as inactive. ND = not determined
a
Cancer cell lines comprise the following: HuCCA-1 human cholangiocarcinoma cancer cell line, HepG2 human hepatocellular carcinoma cell
line, A549 human lung carcinoma cell line, MOLT-3 human lymphoblastic leukemia cell line
b
Antimalarial against Plasmodium falciparum
c
Vero cell line was African green monkey kidney cell line
d
Etoposide, doxorubicin, mefloquine, dihydroartemisinine and ellipticine were used as reference drugs
2.29–28.44. Notably, the derivatives 8, 13 and 14 were
shown to be more potent anticancer agents against HepG2
cell compared with etoposide, the reference drug.
In the meta derivatives (4, 5, 15-19), results showed that
all of the tested molecules exerted significant cytotoxicity,
was the promising compound showing the best safety pro-
file with selective index (SI) values of 1.64–20.60. Inter-
estingly, derivatives 4, 5, 18 and 19 exhibited higher
anticancer activity against HepG2 cell than that of the eto-
poside. Among these, compound 19 (SI = 20.60) was the
most potent cytotoxic agent (HepG2) with 17.4-fold stron-
ger potency than the etoposide, but with 2.6-fold weaker
potency than the doxorubicin. Clearly, most of the meta-
analogs with R = NO2, CF3 and diCF3 were more active
than the corresponding para-counterparts as seen in com-
pounds 5 > 12, 18 > 13 and 19 > 14. Considering isomeric
and substituent effects, it could be presumed that an
orientation of bis-thiourea groups in meta-position on phe-
nyl ring A afforded the compounds that better occupy at the
target sites of action compared with para-isomers. Along
this line, electron withdrawing groups can create ionic
charge on N-atom of the thiourea moiety that may be
required for interacting with the sites of action.
especially against MOLT-3 cancer cell line (IC50
=
1.55–32.22 µM). Methyl compound 15 and methoxy analog
16 selectively inhibited MOLT-3 cell line with IC50 values
of 13.90 and 32.32 μM, respectively. Improvement of
cytotoxic activity against HepG2 and MOLT-3 cells was
observed when the molecules contained halogen sub-
stituents (R = Cl and Br) as seen in compounds 4 and 17.
Furthermore, the chloro analog 4 also inhibited HuCCA-1
cell line with IC50 of 30.22 μM. It should be noted that the
substitution with strong electron withdrawing groups (R =
NO2 and CF3) led to the derivatives (5, 18 and 19) which
enhanced inhibitory potency with a wide range of antic-
ancer activity against all tested cells. Apparently, tri-
fluoromethyl analog 18 was shown to be the most potent
cytotoxic compound against HuCCA-1 and MOLT-3 cells
with IC50 of 14.47 and 1.55 μM, respectively. Moreover,
bis-trifluoromethyl compound 19 displayed the most potent
cytotoxic activity against HepG2 and A549 cells with IC50
values of 1.50 and 16.67 μM, respectively. Derivative 19
Antimalarial activity
Antimalarial activity of the bis-thioureas (4, 5, 8-19) was
assayed against Plasmodium falciparum (K1, multidrug
resistant strain) as shown in Table 1.

Med Chem Res (2017) 26:3136–3148
3143
Trifluoromethyl derivative (13) was the only compound
in the para series which exhibited antimalarial activity with
an IC50 of 17.95 μM. It was found that meta-thioureas (15
and 16)-bearing electron donating groups (R = CH3 and
OCH3) were shown to be inactive compounds, while the
meta-thioureas (4, 5, 17–19) containing electron with-
drawing groups (R = Cl, Br, NO2, CF3, diCF3) displayed
significant antimalarial activity with IC50 values in the range
of 1.92–5.09 μM. The order of decreasing inhibitory
potency of these thioureas based on the substituent effects
(R) as shown by the following trend: CF3 > Br > 2 × CF3 >
NO2 > Cl. Notably, compounds 18 and 19 were found to be
the most promising compounds that exerted antimalarial
activity with the best safety index (SI = 6.86 and 9.28,
respectively). The meta-compound 18 with R = CF3
showed 9-fold more active than the corresponding para-
compound 13 (R = CF3). At this point, it is reasonable to
conclude that the presence of lipophilic trifluoromethyl
moiety at the 4-position on the phenyl rings B of thiourea
groups plays an important role in the inhibition of P. fal-
ciparum as indicated by the most potent compounds 13 and
18 in the para-series and meta-series, respectively.
potent antimicrobial activity than that of compound 18.
Similarly, compound 19 displayed higher activity against
Gram-negative bacteria including S. dysenteriae and P. shi-
gelloides. However, compound 19 did not show growth
inhibition against A. xylosoxidans ATCC 2706 while com-
pound 18 exerted the activity with MIC value of 471.84 µM.
Promisingly, bis-trifluoromethyl derivative 19 was the
most potent compound in the meta-series. Compound 19
had 1.8 and 3.7-fold higher potency against Gram-negative
bacteria (S. dysenteriae and P. shigelloides) than that of the
reference drug, ampicillin. Mostly, compound 19 inhibited
the growth of Gram-positive bacteria and fungi better than
the ampicillin. Moreover, compound 19 possessed the
lowest MIC value of 1.47 µM against S. epidermidis ATCC
2228 and M. luteus ATCC 10240, which were 1.8 and 7.3
fold more potent than the ampicillin, respectively. Inter-
estingly, compound 19 showed comparable activity to
ciprofloxacin against S. dysenteriae, P. shigelloides, M.
luteus ATCC 10240 and L. monocytogenes.
The antimicrobial activity revealed that bis-thioureas 12,
18 and 19 exhibited selective activity against Gram-positive
bacteria and fungi. Results are in accordance with the pre-
vious report of thiourea derivatives (Cunha et al. 2007). It
should be noted that the position of substituents (R) and
electronegativity of fluorine atom play an important role in
the observed antimicrobial activity. There are many CF3-
containing drugs have been approved by the food and drug
administration (Zhu et al. 2014; Wang et al. 2014). The role
of trifluoromethyl function is recognized in medicinal
chemistry as a substituent of distinctive properties such as
enhancing efficacy by promoting electrostatic interactions
with targets, improving cellular membrane permeability due
to its lipophilicity and increasing strength toward oxidative
metabolism of the drug (Müller et al. 2007; Purser et al.
2008; Böhm et al. 2004; Smart 2001).
Antimicrobial activities
All bis-thiourea derivatives (4, 5, 8-19) were tested for
antimicrobial activity, using the agar dilution method,
against 28 strains of microorganisms including 16 Gram-
negative bacterial, 10 Gram-positive bacterial and 2 fungal
strains. Results (Table 2) showed that most of active anti-
microbial compounds were Cl, NO2, CF3 and diCF3 deri-
vatives (4, 5, 18, 19) in the meta-series, except for
compounds 10 and 12 (para-series).
The chloro derivatives in both meta-series and para-
series (4 and 10) showed selective complete growth inhi-
bition against Gram-positive bacteria, M. luteus ATCC
10240 with a MIC of 538.43 µM. The nitro analog 12 dis-
played antigrowth activity against Gram-negative bacteria,
P. shigelloides and A. xylosoxidans ATCC 2706 with MIC
values of 257.77 and 515.55 µM, respectively. However, A.
xylosoxidans ATCC 2706 was partially inhibited (25%) by
the nitro compound 5 (meta-series) at 515.55 µM. Mostly,
the para-analog with NO2 substituent (12) exerted complete
inhibition against Gram-positive bacteria such as S. aureus
ATCC 29213, S. aureus ATCC 25923, M. luteus ATCC
10240, B. subtilis ATCC 6633, L. monocytogenes and B.
cereus with the MIC value of 257.77 µM. However, S.
epidermidis ATCC 12228 and fungus, S. cerevisiae ATCC
2601 were inhibited by the analog 12 with the MIC of
515.55 µM. Trifluoromethyl (18) and bis-trifluoromethyl
(19) derivatives displayed a broad spectrum of antimicrobial
activity against the same strains of Gram-positive bacteria
and fungi. It was observed that compound 19 showed more
Molecular docking
As previously mentioned, the anticancer and antimicrobial
activities of the lead thioureas 2 (Shing et al. 2014) and 1
(Bielenica et al. 2015) are associated with their abilities to
inhibit tubulin and topoisomerase IV, respectively. Fur-
thermore, many (thio)urea derivatives are known inhibitors
of the two putative targets, which have been supported by
the literatures (Li et al. 2009; Shing et al. 2014; Bielenica
et al. 2015; Azam et al. 2015; Ehmann and Lahiti 2014;
Grillot et al. 2014; Basarab et al. 2013; Charifson et al.
2008). Molecular docking was performed to investigate
binding modalities of the synthesized bis-thiourea deriva-
tives against target proteins, i.e., tubulin (anticancer activ-
ity) and topoisomerase IV (antimicrobial activity). Due to
the lack of literature specifying the target proteins for
antimalarial activity of the compounds in this class, thus,

3144
Med Chem Res (2017) 26:3136–3148
Table 2 Antimicrobial activity (MIC) of the bis-thiourea derivatives (4, 5, 8–19)
Microorganisms
MIC (μM)
10
12
4
5
18
19
Ampicillina
Ciprofloxacina
Gram-negative bacteria
E. coli ATCC 25922
K. pneumoniae ATCC 700603
S. marcescens ATCC 8100
S. typhimurium ATCC 13311
S. choleraesuis ATCC 10708
S. putrefaciens ATCC 8071
A. xylosoxidans ATCC 2706
P. aeruginosa ATCC 15442
P. aeruginosa ATCC 27853
P. stutzeri ATCC 17587
S. dysenteriae
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
21.54
<0.32
0.32
I
I
I
I
I
I
I
I
I
I
<0.32
<0.32
0.32
I
I
I
I
1.35
I
I
I
I
I
I
I
I
I
1.35
I
<0.32
10.37
1.30
515.55
515.55b
471.84
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
2.59
I
I
I
0.67
5.39
2.69
I
<0.32
2.59
I
14.74
2.95
S. enteritidis
I
I
I
<0.32
<0.32
<0.32
<0.32
2.59
M. morganii
I
I
I
A. hydrophila
I
I
I
10.77
21.54
10.77
C. freundii
I
I
I
P. shigelloides
257.77
3.69
2.95
Gram-positive bacteria
S. aureus ATCC 29213
S. aureus ATCC 25923
S. epidermidis ATCC 12228
E. faecalis ATCC 29212
E. faecalis ATCC 33186
M. luteus ATCC 10240
C. diphtheriae NCTC 10356
B. subtilis ATCC 6633
L. monocytogenes
I
257.77
257.77
515.55 257.77c
515.55c
515.55c
257.77
I
I
I
I
I
I
I
I
I
I
I
I
3.69
3.69
3.69
29.49
29.49
3.69
14.74
3.69
3.69
3.69
2.95
0.34
1.30
1.30
<0.32
1.30
2.59
1.30
2.59
<0.32
2.59
0.32
I
I
2.95
0.34
I
I
1.47
2.69
I
I
5.89
21.54
21.54
10.77
10.77
1.35
I
I
5.89
538.43
538.43
1.47
I
I
I
I
I
I
I
I
5.89
257.77
257.77
257.77
2.95
2.95 1.47d
<0.34
43.08
B. cereus
2.95
Fungi
C. albicans ATCC 90028
S. cerevisiae ATCC 2601
I
I
I
I
I
I
I
7.37
2.95
2.95
5.39
5.39
1.30
1.30
515.55
14.74
Minimum inhibitory concentration (MIC) was the lowest concentration that inhibited the growth of microorganisms
MIC > 256 µg/mL was indicated as an inactive (I)
a
Ampicillin and ciprofloxacin were used as reference drugs
b
At 515.55 µM, compound 5 showed 25% inhibition against A. xylosoxidans ATCC 2706
c
At 515.55 µM and 257.77 µM, compound 12 showed 25% inhibition against E. faecalis ATCC 29212 and E. faecalis ATCC 33186 and 75 %
inhibition against S. epidermidis ATCC 12228, respectively
d
At 1.47 µM, compound 19 showed 75% inhibition against L. monocytogenes
molecular docking against antimalarial target was excluded
from this study. Co-crystallized ligands of tubulin (i.e.,
colchicine) and topoisomerase IV (i.e., novobiocin) were re-
docked to their target protein structures to validate the
docking protocols. Re-docking poses of colchicine to
tubulin (RMSD = 0.614 A°), and novobiocin to topoi-
somerase IV (RMSD = 0.388 A°) are shown in Fig. S1A,
B, respectively. The calculated RMSD values indicated that
the docking protocols are acceptable for the analysis of
possible binding modes of the investigated compounds. All
bis-thiourea derivatives (4, 5, 8–19) were docked to tubulin
and topoisomerase IV structures (Fig. S2A, S2B, respec-
tively). Results suggested that all bis-thiourea analogs
possibly bind to the target proteins via the same binding

Med Chem Res (2017) 26:3136–3148
3145
Fig. 3 Two-dimensional
ligand–protein interaction
diagrams of GTP (a) colchicine
(b) and compound 19 (c) with
tubulin
sites of the co-crystallized ligands i.e., colchicine binding
site on the β-tubulin (Fig. S2A) and novobiocin binding site
on the topoisomerase IV (Fig. S2B). Binding energy of the
investigated compounds against the target proteins, tubulin
and topoisomerase IV, are in the range of −7.7 to −10.5
and −6.6 to −7.7 kcal/mol, respectively (Table S2).
Docking results of the topoisomerase IV demonstrated that
all of the investigated bis-thioureas exhibited weaker
binding affinities than the co-crystallized ligand, novobicin
(−9.8 kcal/mol, Table S2). In contrast, a promising poten-
tial of the compounds in this class was highlighted against
tubulin target as the stronger binding affinities observed for
three bis-thioureas 14 (−10.5 kcal/mol), 18 (−9.8 kcal/mol)
and 19 (−10.3 kcal/mol), compared with the co-crystallized
ligand, colchicine (−9.5 kcal/mol, Table S2). Furthermore,
the promising compound 19 with highly potent cytotoxic
activity (Table 1) and selectivity index (Table S1) was
selected as a model for elucidating the ligand-protein
binding interactions. Two-dimensional binding interaction
diagrams of tubulin to co-crystalize ligands, i.e., guanosine-
5′-triphosphate (GTP, Fig. 3a) and colchicine (Fig. 3b) as
well as compound 19 (Fig. 3c) are shown in Fig. 3. Con-
sidering the tubulin binding, fluorine (F) and sulfur (S)
atoms play role in the formation of hydrogen bonds, using F
atom of CF3 group with THR145, GLY146 and SER178
residues on the α-chain as well as using S atom of thiourea
moiety with LEU248 residue on the β-chain of tubulin
complex. Hydrophobic interactions (Fig. 3c) also take part
in the binding between phenyl rings and CF3 moieties of the
compound 19 via several residues of both α-chain and β-
chain (i.e., ALA 180 of α-chain as well as LYS254,
MET259 and LYS352 of β-chain). Moreover, the docking
post of compound 19 suggested that this compound has
several common binding sites with co-crystallized ligands
found in the tubulin. GTP found in the binding pocket of α-
chain tubulin also formed hydrogen bondings with
THR145, GLY146 and SER178 (Fig. 3a) as observed in the
docking pose of compound 19. Furthermore, colchicine that
occupied the binding site of β-chain also shared the com-
mon binding residues with compound 19. Colchicine
interacted with β-chain tubulin via attractive charge and
hydrophobic interaction with MET259 and LYS352,
respectively (Fig. 3b) whereas compound 19 formed
hydrophobic interaction with these mentioned residues.
This circumstance suggested that compound 19 might pre-
vent multimerization of tubulin by acting as the dual site
blocker to the binding sites of GTP and colchicine. Addi-
tionally, two-dimensional binding interaction diagrams of

3146
Med Chem Res (2017) 26:3136–3148
13, 14, 18 and 19) displayed higher cytotoxic potency
against HepG2 cells than the control drug, etoposide. In
particular, the meta-thiourea containing bis-trifluoromethyl
moiety (19) was the promising potent cytotoxic agent (IC50
= 1.50–18.82 μM) with the best safety index (1.64–20.60).
Antimalarial activity results fulfilled that the trifluoromethyl
derivative 18 was shown to be the most potent compound
(IC50 = 1.92 μM, SI = 6.86). Significantly, the bis-
trifluoromethyl derivative 19 was also shown to be the
most potent antimicrobial agent. The compound 19 dis-
played higher potency than that of the ampicillin against
Gram-negative bacteria: S. dysenteriae and P. shigelloides;
Gram-positive bacteria: S. epidermidis ATCC 12228, E.
faecalis ATCC 29212, E. faecalis ATCC 33186, M. luteus
ATCC 10240, C. diphtheriae NCTC 10356, B. cereus and
fungi: C. albicans ATCC 90028 and S. cerevisiae ATCC
2601. Promisingly, the potency of compound 19 against
S. dysenteriae, P. shigelloides, M. luteus ATCC 10240 and
L. monocytogenes was comparable to that of ciprofloxacin.
The molecular docking revealed that trifluoromethyl moiety
and thiourea group of the investigated compounds play
roles in binding interaction with the target proteins. It
should be noted that the high electronegativity atoms, a
relatively small size and tightly bound of trifluoromethyl
moiety are essential features for anticancer, antimalarial and
antimicrobial activities of the compounds. In conclusion,
the findings suggested that the bis-thioureas with tri-
fluoromethyl group(s) (18 and 19) are promising com-
pounds to be further developed as novel anticancer,
antimalarial and antimicrobial agents.
Fig. 4 Two-dimensional ligand–protein interaction diagrams of
novobiocin (a) and compound 19 (b) with topoisomerase IV
topoisomerase IV to novobiocin and compound 19 are
shown in Fig. 4. Likewise, the compound 19 binds to
topoisomerase IV (Fig. 4b) via the formation of hydrogen
bonds (ASN49, GLY121, ALA122 and THR168 residues)
and hydrophobic interactions (MET81 and ILE96 residues).
In addition, attractive charge interaction and amide-pi
stacking may be essential for the binding of compound 19
with topoisomerase IV as noted for the charge interaction of
positively charged nitrogen (N+) atoms with ASP52 and
GLU53 residues including the amide-pi stacking with
GLY80 residue (Fig. 4b). Interestingly, the common char-
acteristics of compound 19 and novobiocin bindings to
topoisomerase IV were observed. Both compounds can
form hydrogen bond with ASN49 as well as hydrophobic
interactions with the pockets of ILE96 and MET81. This
suggested that the compound 19 could possibly inhibit
topoisomerase IV by the similar mechanism of novobiocin.
Acknowledgements This project is financially supported by Srina-
kharinwirot University (grant no. 497/2559). Great supports from the
office of the Higher Education Commission and Mahidol University
under the National Research Universities Initiative are appreciated. We
are also indebted to Chulabhorn Research Institute for recording mass
spectra and bioactivity testing.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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