Synthesis and molecular docking of N,N′-disubstituted thiourea derivatives as novel aromatase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2018.05.002

A three series of thioureas, monothiourea type I (4a–g), 1,4-bisthiourea type II (5a–h) and 1,3-bisthiourea type III (6a–h) were synthesized. Their aromatase inhibitory activities have been evaluated. Interestingly, eight thiourea derivatives (4e, 5f–h, 6d, 6f–h) exhibited the aromatase inhibitory activities with IC₅₀ range of 0.6–10.2 μM. The meta-bisthiourea bearing 4-NO₂ group (6f) and 3,5-diCF₃ groups (6h) were shown to be the most potent compounds with sub-micromolar IC₅₀ values of 0.8 and 0.6 μM, respectively. Molecular docking also revealed that one of the thiourea moieties of these two compounds could mimic steroidal backbone of the natural androstenedione (ASD) via hydrophobic interactions with enzyme residues (Val370, Leu477, Thr310, and Phe221 for 6f, Val370, Leu477, Ser478, and Ile133 for 6h). This is the first time that the bisthioureas have been reported for their potential to be developed as aromatase inhibitors, in which the 4-NO₂ and 3,5-diCF₃ analogs have been highlighted as promising candidates.

Full text of DOI:10.1016/j.bioorg.2018.05.002

Accepted Manuscript
Synthesis and molecular docking of N,N'-disubstituted thiourea derivatives as
novel aromatase inhibitors
Ratchanok Pingaew, Veda Prachayasittikul, Nuttapat Anuwongcharoen,
Supaluk Prachayasittikul, Somsak Ruchirawat, Virapong Prachayasittikul
PII:
S0045-2068(18)30273-6
DOI:
https://doi.org/10.1016/j.bioorg.2018.05.002
YBIOO 2355
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 March 2018
1 May 2018
3 May 2018
Please cite this article as: R. Pingaew, V. Prachayasittikul, N. Anuwongcharoen, S. Prachayasittikul, S. Ruchirawat,
V. Prachayasittikul, Synthesis and molecular docking of N,N'-disubstituted thiourea derivatives as novel aromatase
inhibitors, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.05.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1
Synthesis and molecular docking of N,N'-disubstituted thiourea
derivatives as novel aromatase inhibitors
Ratchanok Pingaew a,*, Veda Prachayasittikul b,*, Nuttapat Anuwongcharoen b,
Supaluk Prachayasittikul b, Somsak Ruchirawat c,d,e, Virapong Prachayasittikulf
aDepartment of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110,
Thailand
bCenter of Data Mining and Biomedical Informatics, Faculty of Medical Technology,
Mahidol University, Bangkok 10700, Thailand
cChulabhorn Research Institute, Bangkok 10210,Thailand
dProgram in Chemical Biology, Chulabhorn Graduate Institute, Bangkok 10210,Thailand
eCenter of Excellence on Environmental Health and Toxicology (EHT), CHE, Ministry
of Education, Thailand
fDepartment of Clinical Microbiology and Applied Technology, Faculty of Medical
Technology, Mahidol University, Bangkok 10700, Thailand
*Corresponding authors:
E-mail: ratchanok@swu.ac.th (R. Pingaew)
Tel: 662-649-5000 ext 18253; Fax: 662-260-0128
E-mail: veda.pra@mahidol.ac.th (V. Prachayasittikul)
Tel: 662-441-4376; Fax: 662-441-4380

2
Abstract
A three series of thioureas, monothiourea type I (4a-g), para-1,4-bisthiourea type II
(5a-h) and meta-1,3-bisthiourea type III (6a-h) were synthesized. Their aromatase inhibitory
activities have been evaluated. Interestingly, eight thiourea derivatives (4e, 5f-h, 6d, 6f-h)
exhibited the aromatase inhibitory activities with IC50 range of 0.6-10.2 μM. The meta-
bisthiourea bearing 4-NO2 group (6f) and 3,5-diCF3 groups (6h) were shown to be the most
potent compounds with sub-micromolar IC50 values of 0.8 and 0.6 μM, respectively.
Molecular docking also revealed that one of the thiourea moieties of these two compounds
could mimic steroidal backbone of the natural androstenedione (ASD) via hydrophobic
interactions with enzyme residues (Val370, Leu477, Thr310, and Phe221 for 6f, Val370,
Leu477, Ser478, and Ile133 for 6h). This is the first time that the bisthioureas have been
reported for their potential to be developed as aromatase inhibitors, in which the 4-NO2 and
3,5-diCF3 analogs have been highlighted as promising candidates.
Keywords: Thiourea; Bisthiourea; Breast cancer; Aromatase inhibitor; Molecular docking

3
1. Introduction
Breast cancer is one of the most common cancers found in women worldwide.
Estrogens have been noted for their roles in pathogenesis and progress of the hormone-
dependent (estrogen-receptor positive or ER+) breast cancer [1]. The amount of
biosynthesized estrogen products involves in the final step conversion of androgens to
estrogens, which is catalyzed by the key rate-limiting enzyme namely aromatase cytochrome
P450 [1-6]. Currently, there are two main types of drugs that are used for the treatment of
hormone-dependent breast cancer i.e., the drugs acting on the estrogen receptor (selective
estrogen receptor modulators) and the drugs that inhibit activity of the aromatase enzyme
(aromatase inhibitors or AIs) [1]. The inhibition of aromatase enzyme activity is considered
to be a selective strategy to decrease the amount of estrogen production without affecting the
production of other steroids in the estrogen biosynthetic pathway [1]. AIs can be classified
based on their mechanisms of action into two types i.e., steroidal and nonsteroidal aromatase
inhibitors. The steroidal AIs are androstenedione (ASD) analogs which interact irreversibly to
the active site of the aromatase either by a mechanism-based or by a competitive manner via
covalent interactions, whereas the nonsteroidal AIs bind to the aromatase active site by
reversible process through noncovalent interaction [3, 6]. Although the clinically available
drugs for treatment of ER+ breast cancer have been proven as highly effective, their side
effects and resistance in prolonged treatment persist [7-10]. Thus, the improvement of the
hormone-dependent breast cancer drugs still remains an energetic field of study as established
in a number of recent publications [11-14].
Thiourea and its derivatives have a wide range of versatile applications in many fields
including medicine, agriculture and analytical chemistry. Compounds bearing thiourea
scaffold hold a broad spectrum of biological activities such as anticancer [15, 16],
antimalarial, antiviral and antimicrobial activities [17]. The thiourea pharmacophore
possesses specific binding sites known as hydrogen binding area (NH), complementary area
(S) and auxiliary binding area (1,3-substituents) [17]. Many thiourea analogs have been
shown to exert potent anti-breast cancer activity [18-25]. For example, 1-(chrysen-6-yl)-3-(4-
nitrophenyl)thiourea (SL-1-18) 1 (Fig. 1) has been reported to inhibit proliferation of two
ER+ breast cancer cell lines MCF-7 and T-47D with GI50 values of 3.72 and 2.52 µM,
respectively. The growth inhibitory effect of the SL-1-18 was due to its ability to promote
downregulation and degradation of the ERα as well as to block the cancer cell cycle [25].

4
Although an aromatase inhibitory effect of thiourea derivatives has not been reported in the
literature, many compounds bearing N, O and S electron donor atoms have been reported to
display aromatase inhibitory effect [1, 2]. It could be hypothesized that thiourea bearing N
and S electron donors might exert aromatase inhibitory activity.
As aforementioned, this study aimed to design, synthesize and investigate aromatase
inhibitory and anti-breast cancer activities of N,N'-disubstituted thiourea derivatives. Based
on the attractive phenotypic molecule 1, the synthetic N,N'-disubstituted thioureas have been
designed including monothiourea type I (4), para-bisthiourea type II (5) and meta-bisthiourea
type III (6) (Fig. 1). In addition, a molecular docking was carried out to reveal possible
binding modes and the crucial protein-ligand interactions toward the aromatase.
2. Results and discussion
2.1 Chemistry
A three series (I, II, III) of N,N'-disubstituted thioureas (4-6) constituting various
substitutuents (R = CH3, OCH3, F, Cl, Br, NO2, CF3) on phenyl rings (B) were readily
synthesized by treatment of benzylamine 2a or xylylenediamines (para-2b and meta-2c) with
the corresponding phenylisothiocyanates 3 in dichloromethane [26, 27] (Scheme 1).
Chemical structures of all desired bisthioureas were confirmed by HRMS, 1H NMR,
13C NMR, and IR. All thioureas displayed molecular ion peaks corresponding to their
molecular formula. Infrared spectra of the synthesized compounds exhibited strong N–H
stretching absorption values in the range of 3190–3380 cm-1. 1H NMR spectral data analysis
13
showed the N-H protons as two singlets at  7.5–10.5 ppm. C NMR spectra had a typical
signal for thiocarbonyl carbon in the range of  180–183 ppm. Spectroscopic data of the
known thioureas 5a-b, 5d-h, 6a-b and 6d-h have been reported in the literature [26, 27].
2.2 Aromatase inhibitory activity
Three types of thioureas including monothiourea type I (4a-g), para-bisthiourea type
II (5a-h) and meta-bisthiourea type III (6a-h) were evaluated for their aromatase inhibitory
activities using letrozole (IC50 = 1.9 nM) as the reference drug summarized in Table 1.
Apparently, eight thiourea derivatives (4e, 5f-5h, 6d, and 6f-6h) inhibited the
aromatase activity with IC50 range of 0.6-10.2 μM. To the best of our knowledge, this is the
first time that aromatase inhibitory activity of thiourea derivatives has been reported. Results

5
showed that the number and position of thiourea moieties (X) as well as substituents (R) on
the rings B play important roles in governing their aromatase inhibitory activities. In
monothioureas type I, only compound 4e (X = H) with nitro substituent (R = 4-NO2)
displayed the aromatase inhibitory activity with IC50 of 4.2 µM. The inhibition of aromatase
was significantly improved when the thiourea groups (X = para- and meta-thioureas) were
introduced as seen in bisthioureas types II and III. In type II, there are three active derivatives
(5f-h) with R = 4-NO2, 4-CF3 and 3,5-diCF3 substituents. Apparently, 1.5-fold improvement
of the activity was noted when compound 4e was introduced by N-methylene,N'4-
nitrophenylthiourea on para-position as observed in compound 5f (IC50 = 2.8 µM).
Replacement of 4-NO2 group of compound 5f with 4-CF3 moiety provided analog 5g (IC50 =
10.2 µM) with decreasing the aromatase inhibitory potency; however, insertion of CF3 groups
on 3- and 5- positions led to compound 5h (IC50 = 1.2 µM) with 2.3-fold and 8.5-fold
increasing of inhibition potency compared with the compounds 5f and 5g, respectively.
The meta-bisthiourea type III seemed to be the most potent series of compounds.
Chloro derivative 6d (R = 4-Cl) exhibited the aromatase inhibitory activity with IC50 of 5.5
μM whereas 4-Cl derivatives of monothiourea 4c and para-bisthiourea 5d were shown to be
inactive. Obviously, meta-bisthiourea 6f (R = 4-NO2, IC50 = 0.8 µM) displayed 3.5-fold
higher activity compared with para-analog 5f (R = 4-NO2). Analogously, 4-NO2 group of
compound 6f was replaced with 4-CF3 group to give compound 6g with decreasing in 3.5-
fold inhibitory potency. It should be noted that compound 6h (IC50 0.6 μM) bearing two CF3
groups (R) on 3- and 5- positions of the rings B exerted comparable activity with the
compound 6f (R = 4-NO2), in which both of them were shown to be the most potent
derivatives. At this point, it is noted that the enhanced aromatase inhibitory potency is
dependent on the increased number of thiourea groups, in which the meta-thioureas displayed
better activity than that of the para-counterpart, and the substituent (R) effect was shown in
the following trend: 3,5-diCF3 > 4-NO2 > 4-CF3 > 4-Cl.
Besides the potency, the selectivity of the compounds towards the intended
cytochrome (CYP), aromatase enzyme CYP450, is necessary for minimizing the undesirable
side effects and toxicities [28-30]. Accordingly, potent and selective inhibitors of CYPs (e.g.,
CYP17, CYP19, CYP11B1 and CYP11B2) have been extensively studied [28-32]. Therefore,
it is suggested that further investigations on the selectivity of these thiourea inhibitors over
other CYPs is essential. Regardingly, the structural modification of the compounds is one of

6
the most effective strategies to improve both potency and selectivity of the bioactive
compounds towards the intended targets.
2.3. Anti-breast cancer activity
These active thioureas (4e, 5f-h, 6d and 6f-h) were further tested for their cytotoxic
effects against the breast cancer cell line (MCF-7) using Resazurin microplate assay (Table
1). In overview, the variation of cytotoxic effect of the para- and meta-bisthiourea
compounds against MCF-7 cell line was observed. The para-bisthioureas (5f-5h) showed low
cytotoxicity whereas those of meta-bisthioureas, except for compound 6h, exhibited moderate
cytotoxic effect (IC50 25.01-54.19 µM). The meta-thiourea 6f exhibited the most potent
cytotoxic activity (IC50 = 25.01 µM); however, it is not the one possessing the highest
aromatase inhibitory effect (IC50 = 0.8 μM). In contrast, the most potent aromatase inhibitor
6h (IC50 = 0.6 μM) was shown to be non-cytotoxic compound (IC50 > 73.69 μM) against
MCF-7. This indicated that the aromatase inhibitory effect of the compound is not always in
parallel with its cytotoxic potency [11, 33-35]. It should be noted that the compounds bearing
R = 4-NO2 exerted the highest cytotoxic effect in both series of para-isomer (5f, IC50 = 99.63
μM) and meta-isomer (6f, IC50 = 25.01 μM). This might be due to the NO2 substituent that
may produce reactive nitrogen species (RNS) in the cell. Ultimately, the RNS may initiate a
series of redox reactions resulting in the formation of reactive oxygen sepecies (ROS) leading
to antiangiogenesis and cell growth inhibition [36, 37]. These could be a possible explanation
for the potent cytotoxic effect of compound 6f, which may be responsible by different
mechanisms and/or targets. Currently, many receptors and enzymes such as estrogen receptor
[38], aldo-keto reductase (AKR1C3)[39, 40] and steroid sulfatase [41, 42] have been
proposed as targets for breast cancer. The concurrent preferable aromatase inhibitory and
cytotoxic effects of compound 6f suggested its synergistic potential for management of
estrogen-dependent breast cancer. In contrast, the highest potent aromatase inhibitor 6h with
non-cytotoxic effect against MCF-7 may be suitable for management of other estrogen-
related cancers as well as conditions when the decrease of estrogen products is desirable.
The bisthioureas 6f and 6h have been reported to display cytotoxicity against cancer cell
lines (HuCCA, A549, HepG2 and MOLT-3), antimalarial and antimicrobial activities [26,
27]. Thus, both thioureas are noted as potential candidates to be further developed as multi-
target compounds corresponding to the hot issue in current medicinal chemistry [43-45].

7
2.4. Molecular docking
Molecular docking was performed to reveal possible binding modes of the active bis-
thiourea analogs (i.e., 4e, 5f-5h, 6d, and 6f-6h) toward the target protein, aromatase enzyme.
Redocking of the co-crystallized ligand (ASD) to the aromatase protein was performed to
validate the docking protocol. The redocking provided an acceptable accuracy as indicated by
the root mean standard deviation (RMSD) value of 0.722 A° (Fig. 2A). The results indicated
that all active bisthiourea analogs could occupy within the same binding site of the natural
ASD substrate on the aromatase enzyme, the docking poses of the active thiourea analogs are
shown in Fig. 2B. Additionally, the binding modalities of the ASD and the most potent
compound 6h are comparatively illustrated in alternative view (Fig. 2C). The docking poses
showed that one of the thiourea moieties substituted on the meta-xylenyl ring of compound
6h could occupy within the same binding pocket of the ASD. The two dimensional ligand-
protein interactions also revealed that the right thiourea part of compound 6h could mimic
steroidal backbone of the ASD (Fig. 3A-3B) through the formations of hydrophobic
interactions between the meta-xylenyl ring and the residues Val370, Leu477, and Ser478 as
well as between two trifluoromethyl groups substituted on the terminal benzene ring of
thiourea and the residue Ile133. In addition, the trifluoromethyl group substituted on the
benzene ring of another (on the left) thiourea moiety of 6h could form hydrophobic
interaction with the residue Phe221 where the benzene ring itself takes part for π-π interaction
with Phe221 (Fig. 3B). The ligand-protein interactions of the second most (6f) and the third
most potent (5h) compounds are also provided in Fig. 3C-3D.
For meta-xylenyl bisthiourea 6f (R = 4-NO2), one thiourea moeity (on the left) could
mimic steroidal backbone of the ASD (Fig. 3C) through the hydrophobic interaction between
the meta-xylenyl ring and the Val370, Leu477, Thr310 and Phe221 residues including the π-π
interaction of Phe221 with benzene ring containing 4-NO2 group. Another thiourea (on the
right) displayed Fe3+-π interaction with the benzene ring substituted with 4-NO2 group. H-
bond formations of compound 6f were noted for both terminal 4-NO2 groups with Gln218
and Thr310 residues, and for thiol group with Ser478.
Bisthiourea 5h of para-xylenyl ring could bind to the pocket of enzyme (Fig. 3D)
through hydrophobic interactions with Thr310, Trp224, Phe221, and Asp309 residues along
with π-π interactions of 3,5-diCF3 substituted benzene and para-xylenyl rings with Phe221 as
well as π-charge interaction of 3,5- diCF3 phenyl group with Arg192.

8
3. Conclusions
Twenty-three thiourea derivatives (4-6) have been achieved by a simple one step
synthesis using commercially available and inexpensive starting materials. Eight thiourea
derivatives displayed aromatase inhibitory activity. In particular, the meta-bisthiourea bearing
NO2 (6f) and 3,5-diCF3 (6h) were shown to be the promising potent agents with IC50 values
of 0.8 and 0.6 μM, respectively. The molecular docking revealed that one of the thiourea
moieties of these two compounds (6f and 6h) could form hydrophobic interactions with the
enzyme residues (Val370, Leu477, Thr310, and Phe221 for 6f, Val370, Leu477, Ser478, and
Ile133 for 6h) to mimic steroidal backbone of the ASD substrate, whereas another substituted
thiourea moiety interacted with the enzyme via the formation of π-π interaction with Phe221
(6h) and cation-π interaction with Fe3+ (6f). H-bonding interactions with the enzyme residues
(Gln218, Thr310, and Ser478) were only observed for the nitro derivative 6f. The present
work provides bisthiourea as a novel skeleton with potentially useful for the developement of
aromatase inhibitors.
4. Experimental section
4.1 Chemistry
Column chromatography was carried out using silica gel 60 (70–230 mesh ASTM).
Analytical thin-layer chromatography (TLC) was performed using silica gel 60 F254
aluminum sheets. 1H- and 13C- NMR spectra were recorded on a Bruker AVANCE 300 NMR
1
spectrometer (operating at 300 MHz for H and 75 MHz for 13C). The following standard
abbreviations were used for signal multiplicities: singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m), and broad (br). Mass spectra were recorded on a Bruker Daltonics
(microTOF). IR spectra were obtained using a universal attenuated total reflectance attached
on a Perkin–Elmer Spectrum One spectrometer. Melting points were determined using a
Griffin melting point apparatus and were uncorrected.
4.2 General procedure for the synthesis of thioureas (4-6)
A mixture of amine (4 mmol) or diamine (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 recrystallized from methanol to give the
thiourea derivatives (4-6).

9
4.2.1 1-Benzyl-3-(4-methoxyphenyl)thiourea (4a)
o
Off-white solid. 69%. Mp 100-102 C. IR (UATR) cm-1: 3196, 1607, 1530, 1508, 1298,
1
1239, 1029, 964, 833, 736, 697. H NMR (300 MHz, DMSO-d6): δ (ppm) 3.73 (s, 3H,
OCH3), 4.70 (d, J=5.7 Hz, 2H, CH2), 6.89 (d, J=8.8 Hz, 2H, ArH), 7.23 (d, J=8.8 Hz, 2H,
13
ArH), 7.20-7.35 (m, 5H, ArH), 7.93 (s, 1H, NH), 9.40 (s, 1H, NH). C NMR (75 MHz,
DMSO-d6) δ 48.0, 56.1, 115.2, 100.7, 128.0, 128.6, 129.5, 132.9, 140.6, 158.1, 182.8.
HRMS-TOF: m/z [M + H]+ 273.1064 (Calcd for C15H17N2OS: 273.1056).
4.2.2 1-Benzyl-3-(4-fluorophenyl)thiourea (4b)
o
White solid. 72%. Mp 106-108 C. IR (UATR) cm-1: 3200, 1603, 1539, 1505, 1309, 1213,
1
968, 840, 735, 697. H NMR (300 MHz, DMSO-d6): δ (ppm) 4.72 (d, J=5.7 Hz, 2H, CH2),
7.15 (t, J=8.8 Hz, 2H, ArH), 7.21-7.46 (m, 7H, ArH), 8.18 (s, 1H, NH), 9.62 (s, 1H, NH). 13C
2
NMR (75 MHz, DMSO-d6) δ 47.6, 115.6 (d, JCF=22.5), 126.4, 127.3, 127.9, 128.7, 136.0,
1
139.5, 159.2 (d, JCF=240), 181.7. HRMS-TOF: m/z [M + H]+ 261.0856 (Calcd for
C14H14FN2S: 261.0856).
4.2.3 1-Benzyl-3-(4-chlorophenyl)thiourea (4c)
o
White solid. 70%. Mp 111-112 C. IR (UATR) cm-1: 3228, 1533, 1490, 1338, 1307, 1234,
1089, 1014, 966, 828, 735, 697. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 4.72 (d, J=4.4 Hz,
2H, CH2), 7.21-7.39 (m, 7H, ArH), 7.47 (d, J=8.8 Hz, 2H, ArH), 8.24 (s, 1H, NH), 9.66 (s,
1H, NH). 13C NMR (75 MHz, DMSO-d6) δ 47.6, 125.3, 127.4, 127.9, 128.5, 128.8, 128.9,
138.8, 139.3, 181.4. HRMS-TOF: m/z [M + H]+ 277.0563 (Calcd for C14H14ClN2S:
277.0561).
4.2.4 1-Benzyl-3-(4-bromophenyl)thiourea (4d)
o
White solid. 84%. Mp 128-129 C. IR (UATR) cm-1: 3221, 1587, 1529, 1487, 1306, 1071,
1
1010, 823, 696, 735. H NMR (300 MHz, DMSO-d6): δ (ppm) 4.72 (s, 2H, CH2), 7.18-7.35
(m, 5H, ArH), 7.42 (d, J=8.9 Hz, 2H, ArH), 7.48 (d, J=8.7 Hz, 2H, ArH), 8.27 (s, 1H, NH),
9.66 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6) δ 47.6, 116.5, 125.6, 127.4, 127.9, 128.8,
131.8, 139.2, 139.3, 181.4. HRMS-TOF: m/z [M + H]+ 321.0050 (Calcd for C14H14BrN2S:
321.0056).
4.2.5 1-Benzyl-3-(4-nitrophenyl)thiourea (4e)
o
Yellow solid. 80%. Mp 178-179 C. IR (UATR) cm-1: 3336, 1596, 1495, 1324, 1300, 1255,
1177, 1111, 966, 850, 749, 697. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 4.75 (s, 2H, CH2),

10
7.20-7.40 (m, 5H, ArH), 7.85 (d, J=9.1 Hz, 2H, ArH), 8.17 (d, J=9.1 Hz, 2H, ArH), 8.66 (s,
13
1H, NH), 10.21 (s, 1H, NH). C NMR (75 MHz, DMSO-d6) δ 40.7, 121.2, 124.9, 127.6,
128.1, 128.8, 138.7, 142.5, 146.8, 180.9. HRMS-TOF: m/z [M + H]+ 288.0799 (Calcd for
C14H14N3O2S: 288.0801).
4.2.6 1-Benzyl-3-(4-trifluoromethylphenyl)thiourea (4f)
o
White solid. 76%. Mp 132-134 C. IR (UATR) cm-1: 3274, 1620, 1556, 1335, 1115, 1071,
1
969, 839, 734, 696. H NMR (300 MHz, DMSO-d6): δ (ppm) 4.74 (s, 2H, CH2), 7.20-7.40
(m, 5H, ArH), 7.64 (d, J=8.6 Hz, 2H, ArH), 7.73 (d, J=8.8 Hz, 2H, ArH), 8.43 (s, 1H, NH),
2
9.91 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6) δ 47.6, 122.7, 124.1 (q, JCF=32), 124.8 (q,
1JCF=269), 126.1 (q, 3JCF=3.8), 127.5, 128.0, 128.8, 139.0, 143.8, 181.2. HRMS-TOF: m/z [M
+ H]+ 311.0828 (Calcd for C15H14F3N2S: 311.0824).
4.2.7 1-Benzyl-3-(3,5-ditrifluoromethylphenyl)thiourea (4g)
o
Off-white solid. 71%. Mp 139-140 C. IR (UATR) cm-1: 3222, 1550, 1385, 1276, 1176,
1
1129, 957, 892, 705, 681. H NMR (300 MHz, DMSO-d6): δ (ppm) 4.77 (d, J=3.4 Hz, 2H,
CH2), 7.23-7.39 (m, 5H, ArH), 7.73 (s, 1H, ArH), 8.25 (s, 2H, ArH), 8.67 (s, 1H, NH), 10.14
(s, 1H, NH). 13C NMR (75 MHz, DMSO-d6) δ 47.7, 116.6, 122.8, 123.7 (q, 1JCF=272), 127.5,
2
128.0, 128.8, 130.7 (q, JCF=33), 138.7, 142.5, 181.6. HRMS-TOF: m/z [M + H]+ 379.0696
(Calcd for C16H13F6N2S: 379.0698).
4.2.8 1,1'-(1,4-phenylenebis(methylene))bis(3-(4-fluorophenyl)thiourea) (5c)
o
White solid. 78%. Mp 218-219 C. IR (UATR) cm-1: 3247, 1603, 1542, 1508, 1337, 1219,
971, 842, 800, 724, 685. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 4.69 (d, J=5.5 Hz, 4H, 2 ×
CH2), 7.15 (t, J=8.8 Hz, 4H, ArH), 7.28 (s, 4H, ArH), 7.40 (dd, J=9.0, 5.0 Hz, 4H, ArH), 8.13
2
(s, 2H, NH), 9.56 (s, 2H, NH). 13C NMR (75 MHz, DMSO-d6) δ 47.4, 115.7 (d, JCF=22.5
1
Hz), 126.5, 127.8, 135.9, 138.1, 159.6 (d, JCF=239 Hz), 181.6. HRMS-TOF: m/z [M + H]+
443.1166 (Calcd for C22H12F2N4S2: 443.1170).
4.2.9 1,1'-(1,3-phenylenebis(methylene))bis(3-(4-fluorophenyl)thiourea) (6c)
o
White solid. 71%. Mp 174 C. IR (UATR) cm-1: 3261, 3225, 1542, 1508, 1339, 1216, 974,
1
842, 726, 682. H NMR (300 MHz, DMSO-d6): δ (ppm) 4.72 (d, J=5.4 Hz, 4H, 2 × CH2),
7.14 (t, J=8.8 Hz, 4H, ArH), 7.17-7.33 (m, 4H, ArH), 7.40 (dd, J=8.9, 5.1 Hz, 4H, ArH), 8.15
2
(s, 2H, NH), 9.58 (s, 2H, NH). 13C NMR (75 MHz, DMSO-d6) δ 47.7, 115.6 (d, JCF=21.8
3
1
Hz), 126.4 (d, JCF=8.25 Hz), 126.5, 126.9, 128.7, 136.1, 139.5, 159.6 (d, JCF=240 Hz),
181.9. HRMS-TOF: m/z [M + Na]+ 465.0981 (Calcd for C22H10F2N4NaS2: 465.0990).

11
4.3 Aromatase inhibition assay
Aromatase inhibitory effect was evaluated using the modified method described by
Stressor et al. [46]. The assay was carried out according to the Gentest kit using CYP19
enzyme and O-benzyl fluorescein benzyl ester (DBF) as a fluorometric substrate. The DBF
substrate was dealkylated by the aromatase and then hydrolyzed to give the fluorescein
product.
In brief, 100 µL of cofactor, containing 78.4 µL of 50 mM phosphate buffer (pH 7.4);
20 µL of 20 × NADPH-generating system (26 mM NADP+, 66 mM glucose-6-phosphate, and
66 mM MgCl2); and 1.6 µL of 100 U/mL glucose-6-phosphate dehydrogenase, were added
o
into a 96-well black plate and preincubated in water bath (37 C) for 10 min. The reaction
was initiated by adding 100 µL of enzyme/substrate mixture containing 77.3 µL of 50 mM
phosphate buffer (pH 7.4); 12.5 µL of 16 pmol/mL CYP19; 0.2 µL of 0.2 mM DBF, and 10
µL of tested sample or 10% DMSO as a negative control or letrozole as a positive control.
Fluorescence signal was recorded using an excitation wavelength of 490 nm and emission
wavelength of 530 nm with cutoff 515 nm. Percentage (%) of inhibition was calculated using
equation (1). Samples with % inhibition greater than 50 were further determined to obtain
their IC50 values.
(1)
4.4 Cytotoxicity assay
Cytotoxic activity was performed using resazurin microplate assay (REMA)[47].
MCF-7 cell line (ATCC HTB-22) was derived from breast adenocarcinoma. Cells were
grown and maintained in a complete medium (minimal essential medium (MEM)
supplemented with 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 0.01
mg/mL insulin, 1X non-essential amino acid and 1.5 g/L sodium bicarbonate), and incubated
at 37 °C in a humidified incubator with 5% CO2. Cells at a logarithmic growth were prepared
to 3.3 × 104 cells/mL in complete medium prior to assay. This assay was performed in 384-
well plate in triplicate. Each well was added with 5 μL of the tested compound and 45 μL of
cell suspension. Plate was then incubated at 37 °C in the humidified incubator with 5% CO2
for 3 days. After that, 12.5 μL of 62.5 μg/mL resazurin solution was added to each well, and

12
the plate was further incubated at 37 °C for 4 hours. Fluorescence was measured at 530 nm
excitation and 590 nm emission wavelengths by using the bottom-reading mode of
fluorometer. The signal was subtracted with the blank before calculation. The percentage of
cytotoxicity is calculated by the following equation:
% Cytotoxicity = [1-(FUT/ FUC)] x100
(2)
Whereas FUT and FUC are the mean fluorescent unit from cells treated with the tested
compound and that treated with 0.5% DMSO, respectively.
A threshold of 50% cytotoxicity is used as a cut off for cytotoxic activity of compound,
which can be classified by these criteria:
If % cytotoxicity is < 50%, the activity is reported as 'Non-cytotoxic'.
If % cytotoxicity is ≥ 50%, the activity is reported as 'Cytotoxic'.
The IC50 value is derived from dose-response-curve that is plotted between %
cytotoxicity versus the sample concentrations by using SOFTMax Pro software (Molecular
Devices, USA).
4.5 Molecular docking
Crystal structure of the target protein, human placental aromatase co-crystallized with
natural substrate ASD, was retrieved from RSCB protein data bank (PDB ID: 3EQM,
http://www.rcsb.org/). The preparation of the protein was initially performed by adding
essential hydrogen atoms and repairing missing side chains, using the WHAT IF web server
version 10.1 [48]. Subsequently, non-polar hydrogen atoms were merged, Gasteiger atomic
charges were assigned, and atom type of the protein structures were specified using
AutoDock Tools version 1.5.6. [49, 50]. The chemical structures of the investigated ligands
(1-23) were drawn using Marvin Sketch version 6.1.4 [51], and were geometrically optimized
by Gaussian 09 [52] using Becke’s three-parameter hybrid method with the Lee-Yang-Parr
correlation functional (B3LYP) together with the 6-31g(d) basis set. The geometrically
optimized compounds then were prepared by merging non-polar hydrogen atoms, assigning
partial atomic charge, and defining rotatable bonds using AutoDock Tools version 1.5.6 [49,
50]. Molecular docking simulation was conducted using AutoDock Vina, as a part of the
PyRx 0.8 software [53]. The grid boxes sizing 25.00 × 25.00 × 25.00° were generated and the
center of binding cavity was allocated using x, y and z coordinates of 85.5392, 54.3040, and

13
46.3803, respectively. The validation of docking protocol was performed by redocking of the
co-crystallized ligand ASD to the aromatase protein. The accuracy of the docking protocol
was evaluated by RMSD value, which was calculated by the difference between original and
redocking poses using the Chimera software [54]. Docking poses of the investigated
compounds were visualized using PyMOL [55], and two-dimensional ligand-protein
interaction diagrams were generated using PoseView (http://proteins.plus) [56].
Acknowledgments
Financial support for this work was provided by Srinakharinwirot University (grant no.
039/2561). We are grateful for the support from the Annual Government Grant under
Mahidol University (2557-2559 B.E.). We would like to thank Chulabhorn Research Institute
for recording mass spectra and bioactivity testing.
References
[1] I. Ahmad, Shagufta, Recent developments in steroidal and nonsteroidal aromatase
inhibitors for the chemoprevention of estrogen-dependent breast cancer, Eur. J. Med.
Chem. 102 (2015) 375-386.
[2] D. Ghosh, J. Lo, C. Egbuta, Recent progress in the discovery of next generation inhibitors
of aromatase from the structure−function perspective, J. Med. Chem. 59 (2016) 5131-
5148.
[3] M.R. Yadav, M.A. Barmade, R.S. Tamboli, P.R. Murumkar, Developing steroidal
aromatase inhibitors-an effective armament to win the battle against breast cancer, Eur. J.
Med. Chem. 105 (2015) 1-38.
[4] C. Palmieri, D.K. Patten, A. Januszewski, G. Zucchini, S.J. Howell, Breast cancer: current
and future endocrine therapies, Mol. Cell. Endocrinol. 382 (2014) 695-723.
[5] R. Sainsbury, The development of endocrine therapy for women with breast cancer,
Cancer Treat. Rev. 39 (2013) 507-517.
[6] N. Adhikari, S.A. Amin, A. Saha, T. Jha, Combating breast cancer with non-steroidal
aromatase inhibitors (NSAIs): Understanding the chemico-biological interactions
through comparative SAR/QSAR study, Eur. J. Med. Chem. 137 (2017) 365-438.
[7] P.E. Lonning, H.P. Eikesdal, Aromatase inhibition 2013: clinical state of the art and
questions that remain to be solved, Endocr. Relat. Cancer, 20 (2013), R183-201

14
[8] A.F. Sobral, C. Amaral, G. Correia-da-Silva, N. Teixeira, Unravelling exemestane: from
biology to clinical prospects, J. Steroid Biochem. Mol. Biol. 163 (2016), 1-11.
[9] R.J. Santen, E.K. Lobenhofer, C.A. Afshari, Y. Bao, R.X. Song, Adaptation of estrogen-
regulated genes in long-term estradiol deprived MCF-7 breast cancer cells, Breast Cancer
Res. Treat. 94 (2005) 213-223.
[10] G.J. Sabnis, D. Jevolac, B. Lomg, A. Brodie, The role of growth factor receptor
pathways in human breast cancer cells adapted to long-term estrogen deprivation, Cancer
Res. 65 (2005) 3903-3910.
[11] H. Kang, C., Xiao, C., Huang, Y. Yuan, D. Tang, X. Dai, X. Zeng, Potent aromatase
inhibitors and molecular mechanism of inhibitory action, Eur. J. Med. Chem. 143 (1028)
416-437.
[12] M. Di Matteo, A. Ammazzalorso, F. Andreoli, I. Caffa, B. De Filippis, M. Fantacuzzi, L.
Giampietro, C. Maccallini, A. Nencioni, M.D. Parenti, D. Soncini, A. Del Rio, R.
Amoroso, Synthesis and biological characterization of 3-(imidazol-2-ylmethyl)piperidine
sulfonamides as aromatase inhibitors, Bioorg. Med. Chem. Lett. 26 (1026) 3291-3294.
[13] Y. Yamaguchi, N. Nishizono, D. Kobayashi, T. Yoshimura, K. Wada, K. Oda,
Evaluation of synthesized coumarin derivatives on aromatase inhibitory activity, Bioorg.
Med. Chem. Lett. 27 (1027) 1645-1649.
[14] R. Pingaew, P. Mandi, V. Prachayasittikul, S. Prachayasittikul, S. Ruchirawat, V.
Prachayasittikul, Synthesis, molecular docking, and QSAR study of sulfonamide-based
indoles as aromatase inhibitors. Eur. J. Med. Chem. 143 (2018) 1604-1615.
[15] V. Kumar, S.S. Chimni, Recent developments on thiourea based anticancer
chemotherapeutics, Anticancer agents Med. Chem. 15 (2015) 163-175.
[16] S. Prachayasittikul, R. Pingaew, A. Worachartcheewan, N. Sinthupoom, V.
Prachayasittikul, S. Ruchirawat, V. Prachayasittikul, Roles of pyridine and pyrimidine
derivatives as privileged scaffolds in anticancer agents, Mini Rev. Med. Chem. 17 (2017)
869-901.
[17] A. Mishra, S. Batra, Thiourea and Guanidine derivatives as antimalarial and
antimicrobial agents, Curr. Top. Med. Chem. 23 (1023) 1022-1015.
[18] S. Madabhushi, K.K.R. Mallu, V.S. Vangipuram, S. Kurva, Y. Poornachandra, C.G.
Kumar, Synthesis of novel benzimidazole functionalized chiral thioureas and evaluation

15
of their antibacterial and anticancer activities, Bioorg. Med. Chem. Lett. 24 (2014) 4822-
4825.
[19] S. Saeed, N. Rashid, P.G. Jones, M. Ali, R. Hussain, Synthesis, characterization and
biological evaluation of some thiourea derivatives bearing benzothiazole moiety as
potential antimicrobial and anticancer agents. Eur. J. Med. Chem. 45 (2010) 1323-1331.
[20] S.M. Sondhi, J. Singh, R. Rani, P.P. Gupta, S.K. Agrawal, A.K. Saxena, Synthesis, anti-
inflammatory and anticancer activity evaluation of some novel acridine derivatives. Eur.
J. Med. Chem. 45 (2010) 555-563.
[21] İ. Koca, A. Özgür, M. Er, M. Gümüş, K.A. Coşkun, Y. Tutar, Design and synthesis of
pyrimidinyl acyl thioureas as novel Hsp90 inhibitors in invasive ductal breast cancer and
its bone metastasis. Eur. J. Med. Chem. 122 (2016) 280-290.
[22] X. Huang, R. Huang, Z. Liao, Y. Pan, S. Gou, H. Wang, Synthesis and pharmacological
evaluation of dehydroabietic acid thiourea derivatives containing bisphosphonate moiety
as an inducer of apoptosis, Eur. J. Med. Chem. 108 (2016) 381-391.
[23] W. Liu, J. Zhou, T. Zhang, H. Zhu, H. Qian, H. Zhang, W. Huang, G. Gust, Design and
synthesis of thiourea derivatives containing a benzo[5,6]cyclohepta[1,2-b]pyridine
moiety as potential antitumor and anti-inflammatory agents, Bioorg. Med. Chem. Lett.
22 (2012) 2701-2704.
[24] S. Liu, M.C. Louie, V. Rajagopalan, G. Zhou, E. Ponce, T. Nguyen, L. Green, Synthesis
and evaluation of the diarylthiourea analogs as novel anti-cancer agents, Bioorg. Med.
Chem. Lett. 25 (2015) 1301-1305.
[25] M.M. Fallatah, S. Liu, M.B. Sevigny, H. Zou, M.C. Louie, Novel flexible
heteroarotinoid, SL-1-18, promotes ERα degradation to inhibit breast cancer cell growth,
Cancer letters. 408 (2017) 82-91.
[26] R. Pingaew, P. Tongraung, A. Worachartcheewan,C. Nantasenamat, S. Prachayasittikul,
S. Ruchirawat, V. Prachayasittikul, Cytotoxicity and QSAR study of (thio)ureas derived
from phenylalkylamines and pyridylalkylamines, Med. Chem. Res. 22 (2013) 4026-
4019.
[27]R. Pingaew, N. Sinthupoom, P. Mandi, V. Prachayasittikul, R. Cherdtrakulkiat, S.
Prachayasittikul, S. Ruchirawat, V. Prachayasittikul, Synthesis, biological evaluation and
in silico study of bis-thiourea derivatives as anticancer, antimalarial and antimicrobial
agents, Med. Chem. Res. 26 (2017) 3136–3148.

16
[28] M.G. Ferlin, D. Carta, R. Bortolozzi, R. Ghodsi, A. Chimento, V. Pezzi, S. Moro,
N. Hanke, R.W. Hartmann, G. Basso, G. Viola, Design, synthesis, and structure-
activity relationships of azolylmethylpyrroloquinolines as nonsteroidal aromatase
inhibitors, J. Med. Chem. 56 (2013) 7536-7551.
[29] A. Stefanachi, A.D. Favia, O. Nicolotti, F. Leonetti, L. Pisani, M. Catto, C. Zimmer,
R.W. Hartmann, A. Carotti, Design, synthesis, and biological evaluation of imidazolyl
derivatives of 4,7-disubstituted coumarins as aromatase inhibitors selective over 17-α-
hydroxylase/C17-20 lyase, J. Med. Chem. 54 (2011) 1613-1625.
[30] F. Leonetti, A. Favia, A. Rao, R. Aliano, A. Paluszcak, R.W. Hartmann, A. Carotti,
Design, synthesis, and 3D QSAR of novel potent and selective aromatase inhibitors, J.
Med. Chem. 47 (2004) 6792-6803.
[31] A. Stefanachi, N. Hanke, L. Pisani, F. Leonetti, O. Nicolotti, M. Catto, SCellamare,
R.W. Hartmann, A. Carotti, Discovery of new 7-substituted-4-imidazolylmethyl
coumarins and 4′-substituted-2-imidazolyl acetophenones open analogues as potent and
selective inhibitors of steroid-11β-hydroxylase, Eur. J. Med. Chem. 134 (2017) 304-315.
[32] A.H. Abadi, S.M. Abou-Seri, Q. Hu, M. Negri, R.W. Hartmann, Synthesis and
biological evaluation of imidazolylmethylacridones as cytochrome P-450 enzymes
inhibitors, MedChemComm 3 (2012) 663-666.
[33] M.M. Ghorab, M.S. Alsaid, N. Samir, G.A. Abdel-Latif, A.M. Soliman, F.A. Ragab,
D.A. Abou El Ella, Aromatase inhibitors and apoptotic inducers: design, synthesis,
anticancer activity and molecular modeling studies of novel phenothiazine derivatives
carrying sulfonamide moiety as hybrid molecules, Eur. J. Med. Chem. 134 (2017) 304-
315.
[34] E. Amato, T. Bankemper, R. Kidney, T. Do, A. Onate, F.S. Thowfeik, E.J. Merino, S.
Paula, L. Ma, Investigation of fluorinated and bifunctionalized 3-phenylchroman-4-one
(isoflavanone) aromatase inhibitors, Bioorg. Med. Chem. 22 (2014) 126-134.
[35] R. Ghodsi, E. Azizi, M.G. Ferlin, V. Pezzi, A. Zarghi, Design, synthesis and biological
evaluation of 4-(imidazolylmethyl)-2-aryl-quinoline derivatives as aromatase inhibitors
and anti-breast cancer agents, Lett. Drug Design Discov.13 (2016), 89-97.
[36] H. Jiang, J.E. Taggart, X. Zhang, D.M. Benbrook, S.E. Lind, W.Q. Ding, Nitroxoline (8-
hydroxy-5-nitroquinoline) is more a potent anti-cancer agent than clioquinol (5-chloro-7-
iodo-8-quinoline), Cancer Lett., 312 (2011) 11-17.

17
[37] V. Prachayasittikul, S. Prachayasittikul, S. Ruchirawat, 8-Hydroxyquinolines: A review
of their metal chelating properties and medicinal applications, Drug Des. Devel. Ther., 7
(2013) 1157-1178.
[38] G.P. Skliris, E. Leygue, P.H. Watson, L.C. Murphy, Estrogen receptor alpha negative
breast cancer patients: Estrogen receptor beta as a therapeutic target, J. Steroid Biochem.
Mol. Biol. 109 (2008) 1-10.
[39] T.M. Penning, M.C. Byrns, Steroid hormone transforming aldo-keto reductases and
cancer, Ann. N. Y. Acad. Sci. 1155 (2009) 33-42.
[40] S. Endo, D. Hu, T. Matsunaga, Y. Otsuji, O. El-Kabbani, M. Kandeel, A. Ikari, A. Hara,
Y. Kitade, N. Toyooka, Synthesis of non-prenyl analogues of baccharin as selective and
potent inhibitors for aldo-keto reductase 1C3, Bioorg. Med. Chem. 22 (2014) 5220-5233.
[41] M.P. Thomas, B.V.L Potter, Discovery and development of the aryl O-sulfamate
pharmacophore for oncology and womens health, J. Med. Chem. 58 (2015) 7634-7658.
[42] K.M. McNamara, H. Sasano, The intracrinology of breast cancer J. Steroid Biochem.
Mol. Biol. 145 (2015) 172-178.
[43] N. M. Raghavendra, D. Pingili, S. Kadasi, A. Mettu, S.V.U.M. Prasad, Dual or multi-
targeting inhibitors: The next generation anticancer agents, Eur. J. Med. Chem. 143
(2018) 1277-1300.
[44] A. Abdolmaleki, J.B. Ghasemi, F. Ghasemi, Computer aided drug design for multi-target
drug design: SAR /QSAR, molecular docking and pharmacophore methods, Curr. Drug
Targets 18(2017) 556-575.
[45] P. Csermely, V. Agoston, S. Pongor, The efficiency of multi-target drugs: the network
approach might help drug design, Trends Pharmacol. Sci. 26 (2005) 178-182.
[46] D.M. Stresser, S.D. Turner, J. McNamara, P. Stocker, V.P. Miller, C.L. Crespi, C.J.
Patten, A high-throughput screen to identify inhibitors of aromatase (CYP19), Anal.
Biochem. 284 (2000) 427-430.
[47] J.O. Brien, I. Wilson, T. Orton, F. Pognan, Investigation of the alamar blue (resazurin)
fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267
(2000) 5421-5426.
[48] G. Vriend, WHAT IF: A molecular modeling and drug design program, J. Mol. Graph. 8
(1990) 52-56.

18
[49] M.F. Sanner, Python: a programming language for software integration and
development, J. Mol. Graph. Model. 17 (1999) 57-61.
[50] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J.
Olson, AutoDock4 and AutoDockTools4: Automated docking with selective receptor
flexibility, J. Comput. Chem. 30 (2009) 2785-2791.
[51] ChemAxon, Marvin Sketch Version 6.0, in, Budapest, Hungary, 2013.
[52] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G.
Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li,
H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M.
Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
H. Nakai, T. Vreven, J.A.M. Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E.
Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A.
Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene,
J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann,
O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K.
Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich,
A.D. Daniels, Ö. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, in, Gaussian
Inc., Wallingford CT, 2009.
[53] S. Dallakyan, PyRx Version 0.8, in, http://pyrx.scripps.edu, 2013.
[54] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng,
T.E. Ferrin, UCSF Chimera--a visualization system for exploratory research and
analysis, J. Comput. Chem. 25 (2004) 1605-1612.
[55] W. Delano, PyMOL Release 0.99, DeLano Scientific LLC, Pala Alto, Ca, 2002.
[56] R. Fährrolfes, S. Bietz, F. Flachsenberg, A. Meyder, E. Nittinger, T. Otto, A. Volkamer,
M. Rarey, ProteinsPlus: a web portal for structure analysis of macromolecules, Nucleic
Acids Res. 45 (2017) W337-W343.

19
Figure legends
Fig. 1. Structure of SL-1-18 and our designed thiourea derivatives.
Scheme 1. Synthesis of thiourea derivatives (4-6).
Fig. 2. Molecular docking of the investigated N,N'-disubstituted thiourea derivatives.
Heme and Fe atom are shown in orange and red, respectively. (A) The redocking
provided acceptable RMSD value of 0.722 A°, the crucial enzyme residues for the
binding of the natural substrate are shown in pale pink. (B) Docking poses of active
thiourea derivatives. Active compounds with IC50 values (µM ) < 1, 1 < IC50 (µM ) <
5, and IC50 (µM ) > 5 are shown in purple, green, and yellow, respectively. The crucial
enzyme residues for the binding of the compounds are shown in pale blue. (C) The
binding modes of natural substrate and the most potent compound (6h) are
comparatively illustrated in alternative view. The natural substrate ASD and 6h are
shown in yellow and purple, respectively. The residues played role for the binding of
ASD and 6h are shown in pale pink and green, respectively.
Fig. 3. The 2D ligand-protein interactions. (A) Natural substrate ASD, (B) The most potent
compound 6h, (C) compound 6f, and (D) compound 5h.

20
Fig. 1. Structure of SL-1-18 and our designed thiourea derivatives.
Scheme 1. Synthesis of thiourea derivatives (4-6).

21
B
A
C
Fig. 2. Molecular docking of the investigated N,N'-disubstituted thiourea derivatives. Heme and
Fe atom are shown in orange and red, respectively. (A) The redocking provided acceptable
RMSD value of 0.722 A°, the crucial enzyme residues for the binding of the natural substrate
are shown in pale pink. (B) Docking poses of active thiourea derivatives. Active compounds
with IC50 values (µM ) < 1, 1 < IC50 (µM ) < 5, and IC50 (µM ) > 5 are shown in purple, green,
and yellow, respectively. The crucial enzyme residues for the binding of the compounds are
shown in pale blue. (C) The binding modes of natural substrate and the most potent
compound (6h) are comparatively illustrated in alternative view. The natural substrate ASD
and 6h are shown in yellow and purple, respectively. The residues played role for the binding
of ASD and 6h are shown in pale pink and green, respectively.

22
A
B
C
D
Fig. 3. The 2D ligand-protein interactions. (A) Natural substrate ASD, (B) The most potent
compound 6h, (C) compound 6f, and (D) compound 5h.

23
Table 1
Aromatase inhibitory and cytotoxic activities of thioureas 4e, 5f-h, 6d and 6f-h
IC50 against MCF-7a (µM)
Compound
IC50 of aromatase (µM)
4.2 ± 0.9
>174.01b
99.63
>92.16b
>73.69b
38.11
25.01
54.19
>73.69b
ND
4e
2.8 ± 0.4
5f
10.2 ± 0.1
1.2 ± 0.3
5g
5h
5.5 ± 0.2
6d
0.8 ± 0.2
6f
2.8 ± 0.2
6g
0.6 ± 0.1
6h
Letrozolec
Doxorubicinc
0.0019 ± 0.0002
ND
15.24
ND = Not determine
a MCF-7 is breast cancer cell line.
b IC50 > 50 μg/mL denoted as non-cytotoxic.
c Letrozole and doxorubicin were used as reference drugs.
Highlights:

24
 Twenty-three thiourea derivatives were synthesized.
 The meta-bisthiourea analogs 6f and 6h were shown to be the most potent compounds.
 4-NO2 and 3,5-diCF3 substituents were crucial moieties for aromatase inhibitory potency.
 Molecular docking of the thioureas with aromatase has been studied.

Compound 6f
Compound 6h
aromatase inhibitory activity IC50 0.6 μM
aromatase inhibitory activity IC50 0.8 μM