Design, synthesis, and biological evaluation of novel substituted thiourea derivatives as potential anticancer agents for NSCLC by blocking K-Ras protein-effectors interactions

Article abstract of DOI:10.1080/14756366.2019.1702653

Mutation of the proto-oncogene K-Ras is one of the most common molecular mechanisms in non-small cell lung cancer. Many drugs for treating lung cancer have been developed, however, due to clinical observed K-Ras mutations, corresponding chemotherapy and targeted therapy for such mutation are not efficient enough. In this study, on the basis of the crystal structure of K-Ras, 21 analogues (TKR01–TKR21) containing urea or thiourea were rationally designed, which can effectively inhibit the lung cancer cell A549 growth. The designing of these compounds was based on the structure of K-Ras protein, and the related groups were replaced by bioisosteres to improve the affinity and selectivity. Biological testing revealed that compound TKR15 could significantly inhibit the proliferation of A549 cell with IC₅₀ of 0.21 μM. Docking analysis showed that the TKR15 can effectively bind to the hydrophobic cavity and form a hydrogen bond with the Glu37. In addition, through flow apoptosis assay and immunofluorescence staining assay, it confirmed that this compound can inhibit A549 cell proliferation with the mechanism of blocking K-Ras^G12V protein and effector proteins interactions through the apoptotic pathway. In conclusion, our studies in finding novel potent compound (TKR15) with confirmed mechanism showed great potential for further optimisation and other medicinal chemistry relevant studies.

Full text of DOI:10.1080/14756366.2019.1702653

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis, and biological evaluation of
novel substituted thiourea derivatives as potential
anticancer agents for NSCLC by blocking K-Ras
protein-effectors interactions
Yuan Zhang, Xin Meng, Haikang Tang, Minghui Cheng, Fujun Yang & Wenqing
Xu
To cite this article: Yuan Zhang, Xin Meng, Haikang Tang, Minghui Cheng, Fujun Yang &
Wenqing Xu (2020) Design, synthesis, and biological evaluation of novel substituted thiourea
derivatives as potential anticancer agents for NSCLC by blocking K-Ras protein-effectors
interactions, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 344-353, DOI:
10.1080/14756366.2019.1702653
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 344–353
https://doi.org/10.1080/14756366.2019.1702653
RESEARCH PAPER
Design, synthesis, and biological evaluation of novel substituted thiourea
derivatives as potential anticancer agents for NSCLC by blocking K-Ras
protein-effectors interactions
Yuan Zhang, Xin Meng, Haikang Tang, Minghui Cheng, Fujun Yang and Wenqing Xu
Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical
Sciences and Peking Union Medical College, Tianjin, China
ABSTRACT
ARTICLE HISTORY
Received 8 July 2019
Revised 27 November 2019
Accepted 2 December 2019
Mutation of the proto-oncogene K-Ras is one of the most common molecular mechanisms in non-small
cell lung cancer. Many drugs for treating lung cancer have been developed, however, due to clinical
observed K-Ras mutations, corresponding chemotherapy and targeted therapy for such mutation are not
efficient enough. In this study, on the basis of the crystal structure of K-Ras, 21 analogues (TKR01–TKR21)
containing urea or thiourea were rationally designed, which can effectively inhibit the lung cancer cell
A549 growth. The designing of these compounds was based on the structure of K-Ras protein, and the
related groups were replaced by bioisosteres to improve the affinity and selectivity. Biological testing
revealed that compound TKR15 could significantly inhibit the proliferation of A549 cell with IC50 of
0.21 mM. Docking analysis showed that the TKR15 can effectively bind to the hydrophobic cavity and form
a hydrogen bond with the Glu37. In addition, through flow apoptosis assay and immunofluorescence
staining assay, it confirmed that this compound can inhibit A549 cell proliferation with the mechanism of
blocking K-RasG12V protein and effector proteins interactions through the apoptotic pathway. In conclu-
sion, our studies in finding novel potent compound (TKR15) with confirmed mechanism showed great
potential for further optimisation and other medicinal chemistry relevant studies.
KEYWORDS
K-Ras mutations; malignant
carcinoma; anticancer drug;
non-small cell lung cancer;
A459 cell
Introduction
also lead to different tumours3. H-Ras protein mutations mainly
occur in the cancer of brain and neck; K-Ras protein mutations
mainly occur in pancreatic cancer, colon cancer and lung cancer;
N-Ras mutations are mainly found in skin cancer and blood dis-
eases3–5. The Ras protein mutations are often key mutations, with
approximately 99% of the mutations at positions 12 and 13 of gly-
cine and 61 of glutamine4. These mutations will result in the
decrease of the catalytic hydrolysis activity of GAP, a hindrance of
the GAP-mediated GTP hydrolysis process, and finally a significant
increase in the level of activation of Ras-GTP binding.
Consequently, these processes sustained of the downstream
effector proteins activation6–8, which finally contribute to the
forming of malignant tumour.
Ras proteins are small and membrane-bound guanine nucleotide-
binding proteins; they serve as molecular switches between active
guanine triphosphate (GTP)-bound and inactive guanine diphos-
phate-bound conformations. The Ras proteins usually consist of
188 or 189 amino acids. The 1–165 amino acids of each subtype
have around 92–98% identical sequences, they are highly con-
served during evolution and called G regions, which are respon-
sible for the passage of guanine nucleotides1. The remaining
amino acids vary widely and are known as the hyper variable
region, and the Ras proteins are located in the cytoplasmic mem-
brane binding to the lipid1. Ras proteins have a crucial role in the
regulation of cell proliferation, differentiation and survival through
some critical pathways, such as the Raf–MAPK/ERK kinase–extra-
cellular signal-regulated kinase (Raf–MEK–ERK), phosphoinositide
3-kinase–AKT–mechanistic target of rapamycin (PI3K–AKT–mTOR),
K-Ras protein, like other members of the Ras proteins family, is
a switch comprising of GTP-binding complex which regulates sig-
nalling pathways related to cell growth, differentiation, and apop-
tosis by interacting with multiple effector proteins9. Currently,
there are not existing therapies specifically for targeting mutant K-
and
Ral
guanine
nucleotide-dissociation
stimulator–RAL
(RalGDS–Ral) pathways.
Ras for the treatment of non-small cell lung cancer (NSCLC)10,11
.
Ras proto-oncogene and proteins dysfunction exist in a variety
of malignant tumours. The Ras proto-oncogene encodes four sub-
types of Ras proteins including H-Ras, N-Ras, K-Ras4A, and K-
Ras4B, of which K-Ras4A and K-Ras4B are different splice variants
of the K-Ras gene2. And because of different Ras proto-oncogene
But potential alternative therapeutic strategies in this direction are
always being pursued, with particular efforts directed towards the
inhibition of interactions between K-Ras protein and downstream
effector proteins12,13. The development of small molecule inhibi-
subtypes presenting in different cells, Ras protein mutations will tors targeting mutant K-Ras and inhibiting K-Ras-effector proteins
CONTACT Wenqing Xu
xuwenqing@irm-cams.ac.cn
Institute of Radiation Medicine, Chinese Academy of Medical Science, 238 Baidi Road, Nankai District,
Tianjin, 300192, China
Supplemental data for this article is available online at here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
345
Scheme 1. (a) MeONa, MeOH, rt; (b) NH2NH2, MeOH; (c) CS2, TEA, THF, rt; (d)
Boc2O, DMAP, 5 ꢀC to rt; (e) C, CH3CN, TEA, rt. R1 ¼ (o-tolyl)/(p-tolyl)/(3-chloro-
phenyl)/(2-chlorophenyl)/(4-(trifluoromethyl)phenyl)/(naphthalen-1-yl)/(3,5-dime-
thylphenyl); R2 ¼ (2,6-dinitro-4-(trifluoromethyl)phenyl).
goat were obtained from Abcam (Shanghai, China). Sorafenib
Tosylate was purchased from MCE (Shanghai, China).
Figure 1. Three reported compounds targeting K-RasG12V protein.
All melting points were measured by a Melting Point YRT-
3apparatus (Tianjin precision apparatus factory, China) and were
corrected. NMR spectra were performed using 300 MHz spectrom-
eters (Bruker, USA) with TMS as an internal standard. High reso-
lution mass spectra were determined by Thermo Scientific
Exactive Plus mass spectrometry with ESI method (Thermo, USA).
The purity of all these synthesised compounds was determined by
HPLC analysis (Waters, USA). TLC analysis was carried out on silica
gel plates GF254 (Yantai chemical research institute, China).
Column chromatography was performed on silica gel (200–300
mush; Qingdao Marine Chemical Inc.).
interactions in NSCLC will provide very important preclinical
research information.
In 2012, Stephen W. Fesik’s group14 and Guowei Fang’s
group15, through fragment-based virtual screening and high-reso-
lution crystallographic studies, respectively, found DCAI and com-
pound 1 (Figure 1) which could efficiently bind to K-Ras protein.
In 2013, Tohru Kataoka’s group16 discovered bioactive small com-
pound Kobe0065 (Figure 1), through virtual screening and bio-
activity evaluation, that effectively binded K-Ras protein and
significantly inhibited the K-Ras protein-effector proteins interac-
tions. By the analysis of the binding cavity, it was found that
although the protein crystal models used in molecular docking
and the novel binding fragments were different, but the binding
sites were basically comprised of the same amino acid residues
containing Lys5, Glu37, Aap54, Leu56, Met67, Gln70, Tyr71, and
Thr74. Meanwhile, the structure-activity relationship (SAR) of com-
pound Kobe0065 was not discussed by Tohru Kataoka. In addition,
the compound Kobe0065 was just used to inhibit the growth and
proliferation of pancreatic carcinoma and colorectal carcinoma,
with a slightly higher IC50 value compared with marked antitumor
agent Sorafenib, both of which leave space to further optimise
this small molecule.
General procedures for synthesis
Compounds TKR01-TKR21 were synthesised as described methods
in previous literatures (all the 1H NMR Spectra of Compounds
TKR01-21 in Supporting Information)17–22
.
Synthetic routes of target compounds TKR01–TKR07 were outlined
in Scheme 1
Compound
A (1.0 equiv.) dissolved in anhydrous methanol
(MeOH) was added to sodium methylate (1.0 equiv.) in small por-
tions under magnetic stirring, and continued stirring at room tem-
perature for 1–2 h. The mixture quickly turned yellow and then
gradually changed into red. After the complete depletion of A,
the reaction mixture was extracted with dichloromethane and
washed with distilled water to obtain the product B without fur-
ther purification.
A solution of hydrazine monohydrate (1.1 equiv.) in anhydrous
ethanol was added dropwise to a solution of B (1.0 equiv.) in
anhydrous ethanol under an argon atmosphere. The reaction mix-
ture was stirred at 0 ꢀC for 0.5 h and then stirred at room tempera-
ture, monitored by TLC. After the complete depletion of B, the
solvent was removed under vacuum, and crude product C was
purified by column chromatography on silica gel in hexane/ethyla-
cetate (4:1).
Encouraged by these results, in this study we designed and
synthesised a series of compounds with structure of urea or thio-
urea on the basis of the lead compound Kobe0065, and examined
their cytotoxic effects on NSCLC A549 cell, in order to find small
molecule that could target K-Ras protein, block K-Ras-effector pro-
teins interactions and significantly inhibit the growth of NSCLC. As
a result, the structure of compound Kobe0065 was optimised and
the SAR was discussed. For the most desirable compound TKR15
with IC50 of 0.21 mM, we have carried out molecular docking and
cell-based experiments to gain the mechanism of function. The
results of this study will help to develop and design future new
generations of small molecule compounds that could further
improve on targeting mutant K-Ras protein and cytotoxic efficacy
for NSCLC.
An amine D (1.0 equiv.) was dissolved in THF. While stirring,
CS2 (10 equiv.) and triethylamine (1.1 equiv.) was added. After the
complete conversion to dithiocarbamic acid salt (monitored via
TLC, generally within 30–60 min), the reaction mixture was cooled
in an ice bath with immediate addition of Boc2O (1.0 equiv.) and
DMAP (0.05 equiv.) in THF. Complete consumption of dithiocarba-
mic acid salt proceeded within 15–60 min. Solvent and other vola-
tiles were removed under reduced pressure yielding
Materials and methods
Materials and instruments
The commercial reagents and materials were purchased from
commercial suppliers such as Sigma-Aldrich (Shanghai, China) or
TCI (Shanghai, China), and used without purification. K-Ras
Antibody (Rabbit Polyclonal) was purchased from Proteintech
Group Inc (Wuhan, China). Raf-1 Antibody (Rabbit Polyclonal), the isothiocyanates
anti-mouse IgG and anti-rabbit secondary antibodies raised from purification.
E and used in next step without further

346
Y. ZHANG ET AL.
J ¼ 8.3 Hz, 2H).13C NMR (75 MHz, DMSO) d 143.21, 138.10, 127.54,
126.19, 124.83, 122.95, 121.23. HRMS (ESI): calculated for
C15H9F6N5O4S [M þ H]þ: 470.0313; found:470.0352.
Scheme 2. (f) BTC, TEA, EA, 50 ꢀC; (g) C, CH3CN, TEA, rt. R1 ¼ 4-(trifluoromethyl)-
phenyl)/(3-chloro-4-methylphenyl); R2 ¼ (2,6-dinitro-4-(trifluoromethyl)phenyl).
2–(2,6-Dinitro-4-(trifluoromethyl)phenyl)-N-(naphthalen-1-yl)hy-
drazine-1-carbothioamide (TKR06)
This compound was obtained as light yellow solid in 43% yield;
1
Melting point: 204–205 ꢀC. H NMR (300 MHz, CDCl3) d 9.67 (s, 1H),
To a stirred solution of substituted hydrazine C (1.1 equiv.) in
enough anhydrous acetonitrile, then triethylamine (3.0 equiv.) was
added to the reaction mixture and appropriately substituted iso-
thiocyanate E (1.0 equiv.) was added at room temperature. There
sultant mixture was heated at reflux with magnetic stirring for 3 h
or stirred at room temperature for 4 h. Precipitate was formed
immediately. The mixture was filtered, and the precipitate was
washed with acetonitrile or dichloromethane three times to give
the desired product thioureas F.
8.38 (s, 2H), 8.05 – 7.85 (m, 4H), 7.75 – 7.53 (m, 3H), 7.45 (d,
J ¼ 7.2 Hz, 1H).13C NMR (75 MHz, DMSO) d 134.18, 128.34, 127.47,
126.45, 125.91, 124.92, 121.32. HRMS (ESI): calculated for
C18H12F3N5O4S [M þ H]þ: 452.0596; found:452.0628.
N-(3,5-Dimethylphenyl)-2–(2,6-dinitro-4-(trifluoromethyl)phenyl)-
hydrazine-1-carbothioamide (TKR07)
This compound was obtained as light yellow solid in 40% yield;
1
Melting point: 194–195 ꢀC. H NMR (300 MHz, CDCl3) d 9.70 (s, 1H),
2–(2,6-Dinitro-4-(trifluoromethyl)phenyl)-N-(o-tolyl)hydrazine-1-
carbothioamide (TKR01)
8.43 (s, 2H), 7.85 (s, 1H), 7.37 (s, 1H), 7.07 (s, 1H), 6.82 (s, 2H), 2.42
(s, 5H).13C NMR (75 MHz, DMSO) d 138.42, 127.37, 124.86, 121.26,
117.67. HRMS (ESI): calculated for C16H14F3N5O4S [M þ H]þ:
430.0752; found:430.0777.
This compound was obtained as claybank solid in 39% yield;
Melting point: 181–182 ꢀC.1H NMR (300 MHz, CDCl3) d 9.67 (s, 1H),
8.39 (s, 2H), 7.71 (s, 1H), 7.37 (d, J ¼ 2.8 Hz, 3H), 7.20 (s, 2H), 2.30
(s, 3H).13C NMR (75 MHz, DMSO) d 137.96, 130.62, 127.29, 126.35,
124.89, 121.29, 17.99. HRMS (ESI): calculated for C15H12F3N5O4S
[M þ H]þ: 416.0596; found:416.0628.
Synthetic routes of target compounds TKR08-TKR09 were outlined
in Scheme 2
A solution of amine D (1.0 equiv.) dissolved in dichloromethane
was added dropwise to a solution of triphosgene (1.5 equiv.) in
dry dichloromethane with continuous stirring under nitrogen
atmosphere in an ice bath. Catalytic amount of triethylamine in
dichloromethane was added dropwise to the mixture. The reac-
tion mixture was stirred for 0.5 h at room temperature and for
another 3 h at 50–60 ꢀC. The solvent was removed under vacuum
to obtain product substituted isocyanates G.
An appropriate substituted hydrazine C (1.1 equiv.) was dis-
solved in dichloromethane, and then triethylamine (3.0 equiv.)
was added to the reaction mixture. A solution of substituted iso-
cyanate G (1.0 equiv.) in dichloromethane was added dropwise
under stirring. The reaction mixture was stirred at room tempera-
ture for 2 h. The solvent was removed under reduced pressure
and the residue was recrystallised from EtOH or hexane to obtain
pure product ureas H.
2–(2,6-Dinitro-4-(trifluoromethyl)phenyl)-N-(p-tolyl)hydrazine-1-
carbothioamide (TKR02)
This compound was obtained as claybank solid in 38% yield;
1
Melting point: 179–180 ꢀC. H NMR (300 MHz, CDCl3) d 9.67 (s, 1H),
8.42 (s, 2H), 7.90 (s, 1H), 7.33 (t, J ¼ 7.2 Hz, 3H), 7.12 (d, J ¼ 8.2 Hz,
2H), 2.42 (d, J ¼ 8.3 Hz, 3H).13C NMR (75 MHz, DMSO) d 138.06,
136.72, 135.16, 129.22, 127.44, 124.86, 121.26, 117.66. HRMS (ESI):
calculated for C15H12F3N5O4S [M þ H]þ: 416.0596; found:416.0628.
N-(3-Chlorophenyl)-2–(2,6-dinitro-4-(trifluoromethyl)phenyl)hy-
drazine-1-carbothioamide (TKR03)
This compound was obtained as yellow solid in 36% yield; Melting
1
point: 174–175 ꢀC. H NMR (300 MHz, CDCl3) d 9.70 (s, 1H), 8.42 (s,
2H), 8.05 (s, 1H), 7.55 – 7.30 (m, 4H), 7.21 (d, J ¼ 7.7 Hz, 1H).13C
NMR (75 MHz, DMSO) d 148.46 – 145.99, 140.90, 138.02, 132.80,
130.33, 127.51, 125.17, 121.23 (s), 119.08 – 115.80. HRMS (ESI): cal-
culated for C14H9ClF3N5O4S [M þ H]þ: 436.0049; found:436.0083.
N-(3-Chloro-4-methylphenyl)-2–(2,6-dinitro-4-(trifluoromethyl)-
phenyl)hydrazine-1-carboxamide (TKR08)
This compound was obtained as kelly solid in 43% yield; Melting
point: 204–206 ꢀC.1H NMR (300 MHz, DMSO) d 9.80 (s, 1H), 9.01 (s,
1H), 8.51 (d, J ¼ 22.1 Hz, 3H), 7.65 (d, J ¼ 19.6 Hz, 1H), 7.23 (s, 3H),
2.25 (s, 4H). 13 C NMR (75 MHz, DMSO) d 155.90, 141.96, 140.11,
137.30, 133.54, 131.55, 129.14, 127.57, 124.85, 121.25, 118.33.
HRMS (ESI): calculated for C15H11ClF3N5O5 [M þ H]þ: 434.0434;
found:434.0478.
N-(2-Chlorophenyl)-2–(2,6-dinitro-4-(trifluoromethyl)phenyl)hy-
drazine-1-carbothioamide (TKR04)
This compound was obtained as yellow solid in 37% yield; Melting
1
point: 171–172 ꢀC. H NMR (300 MHz, CDCl3) d 9.93 – 9.62 (m, 1H),
8.64 – 8.21 (m, 2H), 7.97 (s, 1H), 7.62 – 7.32 (m, 5H).13C NMR
(75 MHz, DMSO) d 138.28, 129.85, 127.56, 124.85, 121.25. HRMS
(ESI): calculated for C14H9ClF3N5O4S [M þ H]þ: 436.0049;
found: 436.0074.
2–(2,6-Dinitro-4-(trifluoromethyl)phenyl)-N-(4-(trifluoromethyl)-
phenyl)hydrazine-1-carboxamide (TKR09)
2–(2,6-Dinitro-4-(trifluoromethyl)phenyl)-N-(4-(trifluoromethyl)-
phenyl)hydrazine-1-carbothioamide (TKR05)
This compound was obtained as yellow solid in 41% yield; Melting
1
point: 202–203 ꢀC. H NMR (300 MHz, DMSO) d 9.83 (s, 1H), 9.33 (s,
This compound was obtained as yellow solid in 41% yield; Melting
1H), 8.58 (s, 1H), 8.56 (s, 2H), 7.64 (d, J ¼ 2.4 Hz, 5H).13C NMR
1
point: 171–173 ꢀC. H NMR (300 MHz, CDCl3) d 9.69 (s, 1H), 8.41 (s,
2H), 8.21 (s, 1H), 7.75 (d, J ¼ 8.4 Hz, 2H), 7.58 (s, 1H), 7.47 (d, (75 MHz, DMSO) d 155.82, 143.26, 138.03, 127.64, 126.53, 124.83,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
347
1-Mesityl-3–(4-(trifluoromethyl)phenyl)thiourea (TKR14)
This compound was obtained as grey solid in 45% yield; Melting
point: 204–205 ꢀC. 1H NMR (300 MHz, CDCl3) d 7.70 (s, 1H), 7.58
(q, J ¼ 8.4 Hz, 4H), 7.10 (s, 1H), 6.99 (d, J ¼ 13.4 Hz, 2H), 2.31
(d, J ¼ 6.5 Hz, 9H).13C NMR (75 MHz, DMSO) d 180.96, 143.99,
136.38, 128.81, 126.12, 122.47. HRMS (ESI): calculated for
C17H17F3N2S [M þ H]þ: 339.1098; found: 339.1141.
Scheme 3. (h) G, CH3CN, TEA, rt; (i) E, CH3CN, TEA, rt. R1 ¼ 4-(trifluoromethyl)-
(1,10-biphenyl)/(3s,5s,7s)-adamantan-1-yl)/(4-phenoxyphenyl).
1,3-bis(4-(Trifluoromethyl)phenyl)thiourea (TKR15)
phenyl)/(3-chloro-4-methylphenyl); R2
¼
mesitylene/4-(trifluoromethyl)phenyl)/
This compound was obtained as grey solid in 49% yield; Melting
1
point: 179–181 ꢀC. H NMR (300 MHz, CDCl3) d 8.15 (s, 1H), 7.70 (d,
J ¼ 8.5 Hz, 3H), 7.56 (d, J ¼ 8.4 Hz, 3H).13C NMR (75 MHz, DMSO) d
180.17 (s), 143.54 (s), 126.29 – 125.92 (m), 123.49 (s). HRMS (ESI):
calculated for C15H10F6N2S [M þ H]þ: 365.0502; found: 365.0553.
123.13, 121.23, 118.74, 113.45. HRMS (ESI): calculated for
C15H9F6N5O5 [M þ H]þ: 454.0541; found:454.0586.
Synthetic routes of target compounds TKR10–TKR18 were outlined
in Scheme 3
1-([1,10-Biphenyl]-4-yl)-3–(4-(trifluoromethyl)phenyl)th-
iourea (TKR16)
To a solution of amine I (1.1 equiv.) in anhydrous acetonitrile was
added triethylamine (3.0 equiv.), and then the resulted solution
was added isocyanate G or isothiocyanate E (1.0 equiv.). The reac-
tion mixture was then stirred until complete conversion of the
starting material monitored by TLC. The solvent was removed
under reduced pressure and the residue was recrystallised from
EtOH or hexane to get the desired ureas J or thioureas K.
This compound was obtained as grey solid in 45% yield; Melting
point: 201–203 ꢀC. 1H NMR (300 MHz, DMSO) d 10.25 (s, 1H), 7.79
(d, J ¼ 8.6 Hz, 1H), 7.66 (ddd, J ¼ 19.9, 12.2, 6.8 Hz, 4H), 7.47 (dd,
J ¼ 10.3, 4.8 Hz, 1H), 7.39 – 7.32 (m, 1H).13C NMR (75 MHz, DMSO)
d 179.79, 144.01, 140.13, 139.20, 136.81, 129.40, 127.20, 126.87,
124.25, 123.14. HRMS (ESI): calculated for C20H15F3N2S [M þ H]þ:
373.0942; found: 373.0958.
1–(3-Chloro-4-methylphenyl)-3-mesitylurea (TKR10)
1-((3s,5s,7s)-Adamantan-1-yl)-3–(4-(trifluoromethyl)phenyl)th-
iourea (TKR17)
This compound was obtained as white solid in 46% yield; Melting
This compound was obtained as light yellow solid in 45% yield;
Melting point: 261–262 ꢀC. 1H NMR (300 MHz, DMSO) d 8.78 (s,
1H), 7.69 (d, J ¼ 1.8 Hz, 1H), 7.66 (s, 1H), 7.27 – 7.12 (m, 2H), 6.88
(s, 2H), 2.24 (d, J ¼ 4.4 Hz, 6H), 2.15 (s, 6H).13C NMR (75 MHz, point: 176–177 ꢀC. 1H NMR (300 MHz, DMSO) d 9.63 (s, 1H), 7.75
DMSO) d 153.72, 140.05, 135.83, 135.49, 133.53, 132.95, 131.48,
(d, J ¼ 8.5 Hz, 2H), 7.62 (d, J ¼ 8.7 Hz, 2H), 7.57 (s, 1H), 2.25 (d,
J ¼ 2.5 Hz, 6H), 2.07 (s, 3H), 1.65 (s, 6H).13C NMR (75 MHz, DMSO) d
128.79, 128.08, 118.37, 117.14. HRMS (ESI): calculated for
C17H19ClN2O [M þ H]þ: 303.1219; found: 303.1262.
178.93, 144.08, 125.77, 122.22, 54.06, 36.44, 29.51. HRMS (ESI): cal-
culated for C18H21F3N2S [M þ H]þ: 355.1411; found: 355.1421.
1–(3-Chloro-4-methylphenyl)-3–(4-(trifluoromethyl)pheny-
l)urea (TKR11)
1–(4-Phenoxyphenyl)-3–(4-(trifluoromethyl)phenyl)urea (TKR18)
This compound was obtained as light yellow solid in 46% yield;
Melting point: 235–236 ꢀC. 1H NMR (300 MHz, DMSO) d 9.15 (s,
1H), 8.91 (s, 1H), 7.69 (t, J ¼ 2.2 Hz, 1H), 7.64 (s, 4H), 7.22 (dt,
J ¼ 8.3, 5.2 Hz, 2H), 2.26 (s, 4H). 13 C NMR (75 MHz, DMSO) d
152.66, 143.77, 139.08, 133.64, 131.55, 129.10, 126.89, 125.99,
123.19, 122.64, 122.21, 118.89, 118.40, 117.56. HRMS (ESI): calcu-
lated for C15H12ClF3N2O [M þ H]þ: 329.0624; found: 329.0676.
This compound was obtained as white solid in 45% yield; Melting
1
point: 203–204 ꢀC. H NMR (300 MHz, DMSO) d 9.09 (s, 1H), 8.82 (s,
1H), 7.65 (q, J ¼ 9.1 Hz, 5H), 7.49 (t, J ¼ 6.2 Hz, 2H), 7.38 (dd,
J ¼ 11.2, 4.7 Hz, 2H), 7.10 (t, J ¼ 7.4 Hz, 1H), 6.99 (dd, J ¼ 11.6,
6.5 Hz, 5H).13C NMR (75 MHz, DMSO) d 158.07, 152.86, 151.53,
144.00, 135.74, 130.32, 126.47, 123.24, 120.73, 120.18, 118.23.
HRMS (ESI): calculated for C20H15F3N2O2 [M þ H]þ: 373.1119; found:
373.1182.
1-Mesityl-3–(4-(trifluoromethyl)phenyl)urea (TKR12)
This compound was obtained as light yellow solid in 44% yield;
Melting point: 207–209 ꢀC. 1H NMR (300 MHz, DMSO) d 9.15 (s,
1H), 7.78 (s, 1H), 7.63 (q, J ¼ 8.9 Hz, 5H), 6.90 (s, 2H), 2.24 (s, 4H),
2.16 (s, 7H).13C NMR (75 MHz, DMSO) d 153.51, 144.58, 135.73,
132.78, 128.80, 126.38, 118.01. HRMS (ESI): calculated for
C17H17F3N2O [M þ H]þ: 323.1327; found: 323.1367.
Synthetic routes of target compounds TKR19–TKR21 were outlined
in Scheme 4
The substituted urea or thiourea (1.0 equiv.) was dissolved in gla-
cial acetic acid with gentle heating, and then appropriate of conc.
HCl and distilled water were added. The mixture was cooled to
0–5 ꢀC, stirring constantly. A separate solution of NaNO2 (1.0
equiv.) in distilled water was then prepared and cooled to 0–5 ꢀC.
With efficient stirring, this solution was then added in small ali-
quots to the solution of urea or thiourea, maintaining the tem-
perature at less than 10 ꢀC throughout. After 5 min of further
stirring at or less than 5 ꢀC, the product was vacuum filtered,
washed with cold distilled water, and dried to constant weight in
an oven at 50–60 ꢀC to obtain product L.
1,3-bis(4-(Trifluoromethyl)phenyl)urea (TKR13)
This compound was obtained as light grey solid in 49% yield;
Melting point: 237–238 ꢀC. 1H NMR (300 MHz, DMSO) d 9.26 (s,
2H), 7.72 – 7.63 (m, 8H).13C NMR (75 MHz, DMSO) d 152.55, 143.54,
126.73 – 125.87, 123.13, 122.92, 122.50, 118.51. HRMS (ESI): calcu-
lated for C15H10F6N2O [M þ H]þ: 349.0731; found: 349.0779.

348
Y. ZHANG ET AL.
Scheme 4. (j) DCM, TEA, 1–5 ꢀC; (k) HCl, H2O, CH3COOH, 1–5 ꢀC; (l) NaNO2/H2O, 1–5 ꢀC. R3 ¼ 4-(trifluoromethyl)phenyl)/(3-chloro-4-methylphenyl).
Table 1. Compounds TKR01-TKR07 and their anti-tumour activity.
N-(3-Chloro-4-methylphenyl)-5-(trifluoromethyl)-1H-ben-
zo[d][1,2,3]triazole-1-carboxamide (TKR19)
This compound was obtained as white solid in 39% yield; Melting
1
point: 180–181 ꢀC. H NMR (300 MHz, DMSO) d 8.83 (s, 1H), 8.46 (s,
1H), 8.12 (d, J ¼ 8.7 Hz, 1H), 7.77 (dd, J ¼ 8.7, 1.4 Hz, 1H), 7.68 (d,
J ¼ 2.1 Hz, 1H), 7.20 (dt, J ¼ 8.3, 5.2 Hz, 2H), 2.26 (s, 4H).13C NMR
(75 MHz, DMSO) d 152.78, 140.24, 139.07, 133.57, 131.47, 128.70,
122.76, 118.63, 117.33, 115.78, 115.07. HRMS (ESI): calculated for
C15H10ClF3N4O [M þ H]þ: 355.0529; found:355.0541.
Compound
R1
R2
R3
R4
IC50 (lM)
Sorafenib
TKR01
TKR02
–
–CH3
–H
–H
–
–H
–H
–Cl
–
–H
–CH3
–H
–
–H
–H
8.50
101.7
>100
2.72
5-(Trifluoromethyl)-N-(4-(trifluoromethyl)phenyl)-1H-ben-
zo[d][1,2,3]triazole-1-carboxamide (TKR20)
TKR03
–H
This compound was obtained as white solid in 40% yield; Melting TKR04
–Cl
–H
–H
–H
–H
–CF3
–H
–H
–H
–H
–H
–CH3
>100
1.25
>100
>100
point: 219.3 ꢀC. 1H NMR (300 MHz, DMSO) d 9.62 (d, J ¼ 14.1 Hz,
TKR05
TKR06
1H), 8.43 (d, J ¼ 32.7 Hz, 1H), 7.98 (dd, J ¼ 9.7, 5.0 Hz, 1H), 7.77 –
TKR07
Naphthalene
–H
Naphthalene
–CH3
7.60 (m, 4H), 7.49 (dd, J ¼ 8.6, 1.6 Hz, 1H).13C NMR (75 MHz, DMSO)
d 153.44, 153.04, 143.73, 135.68, 131.15, 126.55, 118.48. HRMS
(ESI):
found:375.0649.
calculated
for
C15H8F6N4O
[M þ H]þ:
375.0636;
added to each well. The cells were incubated for 3 h, and the
absorbance was measured at 450 nm using Spectra Max i3.
5-(Trifluoromethyl)-N-(4-(trifluoromethyl)phenyl)-1H-ben-
zo[d][1,2,3]triazole-1-carbothioamide (TKR21)
This compound was obtained as white solid in 39% yield; Melting
Molecular docking
1
point: 135–136 ꢀC. H NMR (300 MHz, DMSO) d 9.62 (d, J ¼ 14.1 Hz,
TKR15 was docked into the binding pocket of K-Ras protein (PDB
1H), 8.43 (d, J ¼ 32.7 Hz, 1H), 7.98 (dd, J ¼ 9.7, 5.0 Hz, 1H), 7.76 –
7.60 (m, 4H), 7.49 (dd, J ¼ 8.6, 1.6 Hz, 1H).13C NMR (75 MHz, DMSO) code: 4DSO) using docking software Autodock Vina (Designed by
d 153.44, 153.04, 143.73, 135.68, 131.15, 126.66, 123.98, 118.48.13C
NMR (75 MHz, DMSO) d 178.91, 140.21, 138.92, 125.78, 122.56,
122.21, 115.76, 115.04, 54.03, 36.35, 29.43. HRMS (ESI): calculated
for C15H8F6N4S [M þ H]þ: 391.0417; found:391.0407.
Dr. Oleg Trott in the Molecular Graphics Lab at The Scripps
Research Institute). All required were the structures of the mole-
cules being docked and the specification of the search space
including the binding site. Calculating grid maps and assigning
atom charges was not needed. The binding pocket was searched
by Autodock 4.2 (Autodock suite-4.2.6.i86Windows) according to
the literature14–16. Water molecules were deleted and hydrogen
atoms were added. The structure of compound 10 was drawn on
Cell culture
The human NSCLC cell line A549 were purchased from Institute of
Basic Medical Science of Peking Union Medical College (IBMS, ChemDraw and copied to Autodock 4.2. The active site of K-
PUMC, China) and maintained according to the provided proto- RasG12V was defined as the collection of amino acids enclosed
cols. Briefly, A549 cells were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% foetal bovine serum
(Gibbco, Gland Island, NY, USA) and 1% penicillin/streptomycin
(Gibbco, Gland Island, NY, USA) at 37 ꢀC with 5% CO2.
within a 6.5 Å radius of compound TKR15.
Cell apoptosis assay
In order to investigate the mechanism of compound TKR15 in
inhibiting A549 growth and proliferation, the cell apoptosis assay
analysed by flow cytometry was conducted. Cells were treated
with 0.1% DMSO, 24 mM Sorafenib, and 1.0 mM and 2.0 mM TKR15
Cell proliferation assay
To measure cell viability and proliferation, A549 cells were seeded
at a final concentration of 5 ꢁ 103 cells/mL, respectively, in a 96-
well plate. Compounds were dissolved in DMSO, and the cells
were treated with different concentrations (0.2, 1, 5, 25, and
75 mM) of compounds. Cell viability and cell proliferation were
determined after 48 h of incubation, respectively. After the treat-
R
in 2.5% FBS-DMEM for 48 h and stained using the Alexa FluorV
488 Annexin V/Dead Cell Apoptosis Kit according to the manufac-
turer’s protocol (Molecular Probes). Annexin V and PI were ana-
lysed by flow cytometry. Annexin V-positive cells were counted as
ment, cell counting kit-8 reagent (MCE, Shanghai, China) was Apoptosis cells in total 5 ꢁ 104 cells per each sample.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
349
Immunofluorescence staining assay
fixated with 4% Paraformaldehyde for 5 min. After an additional
three times PBS washing, cells were permeabilised with 0.1%
Triton-X for 10 min and washed with PBS three times before with
1% BSA blocking buffer at a period of 30 min. The anti-K-RAS
mouse monoclonal antibody was incubated in blocking buffer
overnight at 4 ꢀC. Afterwards, the cells were washed with PBS
three times. A secondary antibody, goat anti-Mouse antibody, was
incubated for 2 h in PBS. Then, cells were washed with PBS for
three times again and incubated with the anti-Raf-1 antibody in
blocking buffer overnight at 4 ꢀC, subsequently incubated with a
secondary antibody, anti-goat antibody for 2 h in PBS. The cells
were washed three times with PBS and stored in PBS
for microscopy.
Immunofluorescence Staining Assay was conducted in the study
to demonstrate the effects of blocking K-Ras-Raf-1 proteins inter-
actions23. After 2 h incubation with 1% DMSO and compound
TKR15 (20 mM), A549 cells were washed with PBS three times and
Table 2. Compounds TKR08-TKR09 and their anti-tumour activity.
Compound
R4
R5
IC50 (lM)
Statistics
TKR08
TKR09
–Cl
–H
–CH3
–CF3
125.9
81.9
All of the experiments were repeated a minimum of three times.
The data are expressed as the means (SD). Statistical significance
Table 3. Compounds TKR10–TKR18 and their anti-tumour activity.
Compound
TKR10
R7
X
O
R8
IC50(µM)
46.07
TKR11
TKR12
O
O
48.44
10.37
TKR13
TKR14
O
S
22.81
>100
TKR15
TKR16
S
S
0.21
>100
TKR17
TKR18
S
>100
5.37
O

350
Y. ZHANG ET AL.
was analysed using either Unpaired-Student’s t-test (two-tailed) or can markedly inhibit Raf-1 kinase protein. Followed by 48 h incu-
analysis of variance test. The p values of less than 0.05 was con- bation with these compounds, cells were treated with cell count-
ing kit-8reagent to measure their growth/viability (% of the
untreated control) by Spectra Max i3. The 50% inhibitory concen-
tration (IC50) for each derivative was calculated according to the
equation of Boltzmann sigmoidal concentration-response curve
using Graph Pad Prism 8, which represented the concentration
that resulted in a 50% decrease in cell growth after 48 h of incu-
bation. As shown in the Tables 1–4, several compounds out of
TKR01–TKR21 can effectively inhibit A549cells growth in a dose-
dependent manner. Especially for compound TKR15, it could sig-
nificantly inhibit the growth of A549 cell with IC50 of 0.21 mM.
Initially, as shown in Table 1, we attempted to increase the
affinity binding to K-Ras protein by changing the hydrophobic
groups of phenyl, such as methyl, naphthyl, and trifluoromethyl.
And we finally found that only TKR03 and TKR05 can significantly
inhibit A549 cells proliferation compared with Sorafenib. It dem-
onstrates that the substitutions (2-chlorophenyl and p-trifluoro-
methyl) can markedly contribute to the hydrophobic interactions
between small molecule and receptor protein in these hydropho-
bic groups, especially for p-trifluoromethyl which lead compound
TKR05 inhibit A549 cell proliferation with IC50 of 1.25 mM. The
stronger contributions of p-trifluoromethyl to the hydrophobic
interactions may lead these observations of anti-proliferation.
As shown in Table 2, we wanted to learn about the impacts of
urea and thiourea for the affinity. And we found that urea struc-
ture molecule has higher IC50 value compared with thiourea and
Sorafenib (Table 1), in other words, the urea structure cannot
effectively promote the binding between small molecule and
receptor protein. This phenomenon may be owing to the different
sidered statistically significant.
Results and discussion
Antiproliferative effects of compounds TKR01–TKR21 on NSCLC
A549 cells
The newly synthesised urea or thiourea compounds
(TKR01–TKR21) were evaluated for their antiproliferative effects
using NSCLC A549 cell lines, for which these compounds were
diluted to achieve five different concentrations ranging from 0.2
to 75 mM. And we choose Sorafenib as the controlled agent, which
Table 4. Compounds TKR19–TKR21 and their anti-tumour activity.
Compound
R9
X
R10
IC50 (lM)
TKR19
TKR20
TKR21
–CH3
–CF3
–CF3
O
O
S
–Cl
–H
–H
>100
22.75
18.57
Figure 2. (A) Cartoon structure and (B) Surface structure of molecular docking analysis of small molecule TKR15 and K-RasG12V protein.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
351
(A)
(B) 100
1.0 (µM)
2.0 (µM)
24 (µM)
80
60
40
20
0
5
5
ib
1
1
nk
n
a
R
R
e
f
Bl
a
TK
TK
r
So
Compounds
Figure 3. (A) Flow cytometric analysis of compound TKR-15 induced apoptosis on A549 cells; (B) Cell apoptosis amount of A549 after treating TKR15 and Sorafenib
(the results were significant, p < 0.05, n ¼ 3).
electronic effect lead by oxygen and sulphur in the structures of Binding mode of small molecule TKR15 with K-RasG12V protein
urea and thiourea.
Molecular docking was performed using TKR15 and the active site
In addition, we intended to shorten the linker of the two phe-
of K-Ras protein (PDB ID: 4DSO). In order to rationalise the bio-
nyls and investigate the impacts of volume of group on hydrazine.
logical results and to gain insight into the SAR of the most potent
As shown in Table 3, we found that small molecule TKR15 and
compound, an attempt to interpret the observed anti-proliferation
TKR18 can significantly inhibit A549 cells growth compared with
TKR05 and Sorafenib (Table 1), with IC50 of 0.21 mM and 5.37 mM,
activity of the tested compound TKR15 on the basis of the ligand-
protein interactions was done. The anti-proliferation activity of K-
respectively. This demonstrates that decreasing of hydrazine nitro-
Ras-effector protein interactions inhibitors depends on the ability
gen can markedly in inhibit A549 cells and the thiourea structure
of the compound to properly binding into the allosteric site.
is needed for the activity. At the same time, volume increasing of
As shown in the Figure 2(A,B), the benzotrifluoride moiety of
group on hydrazine will not contribute to inhibition, and hydro-
TKR15 was located in the active site consisting of Lys5, Glu37,
phobic group p-trifluoromethyl can further contribute to affinity
of binding. This may be owing to that the all binding pocket
need appropriately size of small molecule to interact with.
Finally, we attempted to construct a five-member cycle in the
compound, because there may form hydrogen bond between
nitryl and hydrazine in the interactions. But this change cannot
markedly contribute to the inhibitions of compounds against
A549 cells compared with TKR05 and Sorafenib (Table 1). That is
to say, the rigid rings of compounds TKR19–TKR21 were not the
practical conformations in the interactions with active site.
Ile55, Aap54, Leu56, Met67, Gln70, Tyr71, and Thr74 residues of K-
RasG12V. These Lys5, Glu37, Ile55, Aap54, and Leu56 residues
formed a hydrophobic surface pocket in the neighbourhood of
switch, indicating that the benzotrifluoride part was inserted into
the pocket through hydrophobic interaction. In addition, the resi-
due of Glu37 can form a hydrogen bond with hydrogen of nitro-
gen. And the other moiety of benzotrifluoride of TKR15 was
located binding site forming by Met67, Gln70, Tyr71, and Thr74.
Thus, this binding mode between small molecule and receptor
protein provided the binding affinity and anti-proliferation activity.

352
Y. ZHANG ET AL.
Figure 4. Immunostaining of A549 after treatment with compound TKR15 for 2 h (20lM). The vehicle control for the above experiments was 0.1% DMSO.
Flow cytometric analysis of compound TKR15 induced apoptosis compound TKR15 can significantly target K-Ras protein and inhibit
on A549 cells
K-Ras protein-Raf-1 protein interaction.
DNA mutations and protein mutations of malignant carcinoma
cells can continue to multiply. The K-Ras protein mutations of
NSCLC A549 also can contribute to persistent proliferation. The
role of PPI inhibitors, which can target K-Ras protein and block
PPI, is to inhibit the over activity of K-Ras downstream effector
proteins by blocking PPI, such as Raf-1 and PI3K. And that will
further induce carcinoma cells apoptosis. In other words, treat-
ment with these anticancer agents can lead malignant carcinoma
cells apoptosis by blocking K-Ras PPI. In this study, A549 cells
were treated with compound TKR15 at 1.0 and 2.0 mM and con-
trolled agent Sorafenib at 24 mM. As shown in Figure 3(A,B), com-
pound TKR15 resulted in apoptosis of A549 cells (31.1% and
82.3%, respectively; Figure 3(A)), compared with blank group,
Conclusion
K-Ras protein is an attractive and effective therapeutic target
because of such crucial role in the cell signalling paths. However,
most of the attempts to develop targeting at K-Ras protein inhibi-
tors have faced huge challenges due to their low druggability. At
the same time, protein-protein interactions inhibitors offer another
strategy to study targeting K-Ras protein with the role of inducing
apoptosis of carcinoma cell.
In this study, we have designed a series of compounds on the
basis of Kobe0065, one of Kobe0065-family compounds with con-
firmed mechanism action against K-Ras which further showed
antitumor activity by blocking the Ras-effctor interaction. Under
these backgrounds, more than 20 novel inhibitors were designed,
synthesised and biologically tested using the lead compound
(Kobe0065) as template. Among these inhibitors, a promising ana-
logue TKR15 could inhibit A549 cell growth in a dose dependent
manner. Moreover, by flow cytometry assay, compound TKR15 sig-
nificantly induced the apoptosis of A549 cells with the percentage
of apoptosis cells ranging from around 30% to around 80% com-
pared with the control group. Immunofluorescence staining assay
demonstrates that TKR15 can significantly inhibit K-Ras-effector
proteins interactions. And molecular docking demonstrates that
small molecule TKR15 bind to the active pocket through the
strong hydrophobic interactions between benzotrifluoride moiety
and binding site forming by Gln70, Tyr71, and Thr74 and hydro-
gen bond between Glu37 and hydrogen of nitrogen.
and the controlled group in
a dose-dependent manner.
Moreover, when improved the concentration of TKR15, the pro-
portion of apoptosis cells was significantly increasing, while
Sorafenib resulted in apoptosis of A549 cells around 50% (Figure
3(B)). These results demonstrate that compound TKR15 antitumor
activity is better than Sorafenib by targeting K-Ras protein and
blocking PPI.
Effects of compound TKR15Blocking K-Ras protein-Raf-1 protein
interaction
If compound can significantly block K-Ras-effector proteins interac-
tions, K-Ras protein and effector proteins cannot be close to each
other in the space. In order to directly observe the effects of
blocking PPI of compound TKR15, we conducted an immunofluor-
escence staining experiment. In this experiment, after treatment
compound TKR15, the K-Ras protein was stained into red colour,
Taken together, compound TKR15 performed stronger anti-
and Raf-1 protein was stained green colour. And then, these cells cancer activity towards A549 cells. It inhibited cells proliferation
were observed under laser scanning confocal microscope, and we and induced apoptosis of A549 cells via targeting the K-RasG12V
obviously found that there were several red colour dots in the protein. In conclusion, urea and thiourea derivatives should be
TKR15 group compared with blank (DMSO) group after being served as a promising antitumor leads with good promising to be
merged (Figure 4). This result can directly demonstrate that further exploited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
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Disclosure statement
We declare that there are no financial and personal relationships
with other people or organisations that could inappropriately
influence this study. In other words, there is no commercial or
associative interest that represents a conflict of interest in connec-
tion with the work submitted.
Funding
This work was supported by a grant from Medical and Health
Innovation Project of Chinese Academy of Medical Sciences
(CIFMS) [2017-I2M-3–019].
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