Design and synthesis of novel 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thiadiazol- 2-yl)urea derivatives with potent anti-CML activity throughout PI3K/AKT signaling pathway

Article abstract of DOI:10.1016/j.bmcl.2019.05.005

In this investigation, a series of 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4- thiadiazol-2-yl)urea receptor tyrosine kinase inhibitors were synthesized by a simple and efficient structure-based design. Structure-activity relationship (SAR) analysis of these compounds based on cellular assays led to the discovery of a number of compounds that showed potent activity against human chronic myeloid leukemia (CML) cell line K562, but very weak or no cellular toxicity through monitoring the growth kinetics of K562 cell during a period of 72 h using the real-time live-cell imaging. Among these compounds, 1-(5-((6-((3-morpholinopropyl) amino)pyrimidin-4-yl)thio)-1,3,4-thiadiazol-2-yl)-3-(4-(trifluoromethyl)phenyl)urea (7) exhibited the least cellular toxicity and better biological activity in cellular assays (K562, IC₅₀: 0.038 μM). Compound 7 also displayed very good induced-apoptosis effect for human CML cell line K562 and exerted its effect via a significantly reduced protein phosphorylation of PI3K/Akt signal pathway by Human phospho-kinase array analysis. In vitro results indicate that 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4- thiadiazol-2-yl)urea derivatives are lead molecules for further development as treatment of chronic myeloid leukemia and cancer.

Full text of DOI:10.1016/j.bmcl.2019.05.005

Accepted Manuscript
Design and synthesis of novel 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thia-
diazol- 2-yl)urea derivatives with potent anti-CML activity throughout PI3K/
AKT signaling pathway
Weiwei Li, Jianjie Chu, Tingting Fan, Wei Zhang, Minna Yao, Zeqiong Ning,
Mingming Wang, Jin Sun, Xian Zhao, Aidong Wen
PII:
S0960-894X(19)30290-2
https://doi.org/10.1016/j.bmcl.2019.05.005
BMCL 26425
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 January 2019
20 April 2019
5 May 2019
Please cite this article as: Li, W., Chu, J., Fan, T., Zhang, W., Yao, M., Ning, Z., Wang, M., Sun, J., Zhao, X., Wen,
A., Design and synthesis of novel 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thiadiazol- 2-yl)urea derivatives with
potent anti-CML activity throughout PI3K/AKT signaling pathway, Bioorganic & Medicinal Chemistry Letters
(2019), doi: https://doi.org/10.1016/j.bmcl.2019.05.005
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.

Design and synthesis of novel 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thiadiazol- 2-
yl)urea derivatives with potent anti-CML activity throughout PI3K/AKT signaling
pathway
Weiwei Li a, Jianjie Chu a, Tingting Fan a, Wei Zhang a, Minna Yao a, Zeqiong Ning a,
Mingming Wang a, Jin Sun a, Xian Zhao a, Aidong Wen a*
a
Department of Pharmacy, Xijing Hospital, The Fourth Military Medical University,
127 Changle Western Road, Xi'an, Shaanxi Province, 710032, China.
* Corresponding author. Aidong Wen. E-mail: wwzoe.1@163.com
Abstract: In this investigation, a series of 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-
thiadiazol-2-yl)urea receptor tyrosine kinase inhibitors were synthesized by a simple
and efficient structure-based design. Structure-activity relationship (SAR) analysis of
these compounds based on cellular assays led to the discovery of a number of
compounds that showed potent activity against human chronic myeloid leukemia
(CML) cell line K562, but very weak or no cellular toxicity through monitoring the
growth kinetics of K562 cell during a period of 72 hours using the real-time live-
cell imaging.
Among
these
compounds,
1-(5-((6-((3-morpholinopropyl)
amino)pyrimidin-4-yl)thio)-1,3,4-thiadiazol-2-yl)-3-(4-(trifluoromethyl)phenyl)urea
(7) exhibited the least cellular toxicity and better biological activity in cellular assays
(K562, IC50: 0.038 μM). Compound 7 also displayed very good induced-apoptosis
effect for human CML cell line K562 and exerted its effect via a significantly reduced
protein phosphorylation of PI3K/Akt signal pathway by Human phospho-kinase array
analysis. In vitro results indicate that 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-
thiadiazol-2-yl)urea derivatives are lead molecules for further development as
treatment of chronic myeloid leukemia and cancer.
Keywords: 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thiadiazol-2-yl)urea derivatives;
chronic myeloid leukemia (CML); cellular toxicity; K562 cell; PI3K/Akt signal
pathway
1

Chronic myeloid leukemia (CML) is a malignant disease of hematopoietic stem cell
caused by the t(9;22) translocation generated fusion gene BCR-ABL, a constitutively
active tyrosine kinase (TK) that drives the leukemia. The t(9; 22) chromosome can
occur in more than 95% of CML patients, or 5% of adult acute lymphoblastic
leukemia (ALL) patients.1-2 CML is characterised by prolonged asymptomatic period
with gradual development of splenomegaly, culminating in acute leukaemia amongst
untreated patients. According to the course of disease progression, clinical CML can
be divided into three phases: chronic phase, acceleration phase and blast crisis phase.
Most patients are in the chronic phase, and if the chronic phase is not treated timely or
resistant, it will progress to an accelerated or blast phase.3-5 Currently, specific Bcr-
Abl tyrosine kinase inhibitor, Imatinib, Dasatinib or Nilotinib, is highly effective and
considered standard therapy as first-line treatment for CML.6-8 However, resistance or
intolerance to these tyrosine kinase inhibitors (TKI), Heavy economic burden and
reduced quality of life are often encountered that ultimately leads to disease
progression.9-10 Therefore, the development of other strategies and alterative targets
for CML patients is of intense interest.
Phosphatidylinositol 3-kinase (PI3K) signaling is involved in the regulation of various
cellular functions such as proliferation, differentiation, apoptosis and glucose
transport. In recent years, the signaling pathway composed of PI3K and its
downstream molecular Akt is closely related to the occurrence and development of
human tumors.11-13 This pathway regulates the proliferation and survival of tumor
cells. Its abnormal activity can not only lead to malignant transformation of cells, but
also related to tumor cell migration, adhesion, tumor angiogenesis and degradation of
extracellular matrix. Currently, the tumor therapy strategy targeting the key molecules
of PI3K-Akt signaling pathway is developing.14-15 Activation of PI3K leads to
activation of Akt, which activates or inhibits its downstream target proteins and then
regulates cell proliferation, differentiation, apoptosis and migration.
Recently, several multi-target TKIs such as Sorafenib and Linifanib have been
approved or investigated in clinical trials. Sorafenib is a potent inhibitor of several
RTKs (VEGFR/PDGFR/ERK/Raf), and effectively inhibits Raf/MEK/ERK signaling
2

pathway.16 Linifanib is an active multi-target RTK inhibitor of KDR and PDGFR,
VEGFR.17 The diarylurea scaffold of these compounds is thought to form multiple
important hydrogen bonds with the hydrophobic pocket. Numbers of reports have
recently highlighted diarylureas as potential antiproliferative agents for cancer.18-19
Therefore, diarylurea scaffold has been identified as an effective scaffold for
multikinase inhibitors, and a promising anti-cancer agents. In the present investigation,
considering that the N and S atom on the thiazole ring of Dasatinib may form critical
interactions with hydrophobic pocket residues, so we utilized thiadiazole ring instead
of thiazole ring of Dasatinib. And then the diarylurea fragments were incorporated
with the modified Dasatinib framework to synthesize a series of 1-phenyl-3-(5-
(pyrimidin-4-ylthio)-1,3,4-thiadiazol-2-yl)urea derivatives, which was a potentially
interesting scaffold for design of multi-target RTK inhibitors and downregulated the
phosphorylation of kinases within the PI3K/Akt signaling pathway. The SAR analysis
will focus on two regions: the 3/4-position substituent of phenyl ring (ring A) and 6-
position substituent of pyrimidin (Figure 1).
Figure 1. Design strategy based on Dasatinib and structural modifications of novel 1-
phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thiadiazol-2-yl)urea derivatives.
The synthetic route for preparation of the title compounds was depicted in Scheme 1.
Briefly, the key intermediates 2a-f were achieved by reaction of commercially
available 5-amino-1,3,4-thiadiazole-2-thiol with substituent phenyl isocyanates or
diverse aromatic amines and triphosgene 20 under reflux for 3~5h using acetonitrile as
a solvent in high yield (85%~95%). Then intermediates 2a-f were reacted with
commercially available 4,6-dichloropyrimidine in the presence of potassium
3

carbonate in acetonitrile under reflux for 8~12h to provide the 1-(5-((6-chloro-
pyrimidin-4-yl)thio)-1,3,4-thiadiazol-2-yl)-3-phenylurea analogues 3a-f in high yield
(90%~96%). Finally, 3a-f were substituted with 3-morpholinopropan-1-amine, 3-
morpholinopropan-1-ol and N1,N1-diethylpropane-1,3-diamine in ethanol under reflux
for 8-12 h to afford the 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thiadiazol- 2-yl)urea
derivatives 4-9. A total of 10 compounds were synthesized and characterized by
hydrogen nuclear magnetic resonance spectra and electrospray ionization mass
spectra (see the Supporting Information).
Cl
Cl
H
H
N
N
NCO
N
N
N
S
N
a
SH
+
HS
b
NH2
O
N
N
S
R1
R1
1a-f
2a-f
R1
R1
O
N
N
X
R2
N
H
O
N
N
N
N
N
S
H
NH
S
N
NH
c
R2
X
S
S
N
4-9
3a-f
Cl
4a:R1=-4-CH3, R2=-morpholine, X=N
4b:R1=-4-CH3, R2=-morpholine, X=O
4c:R1=-4-CH3, R2=-diethylamine, X=N
5a:R1=-3-CF3-4-Cl, R2=-morpholine, X=N
5b:R1=-3-CF3-4-Cl, R2=-morpholine, X=O
5c:R1=-3-CF3-4-Cl, R2=-diethylamine, X=N
6:R1=-4-Cl, R2=-morpholine, X=N
3a:R1=-4-CH3
3b:R1=-3-CF3-4-Cl
3c:R1=-4-Cl
3d:R1=-4-CF3
3e:R1=-3-Cl-4-CH3
3f:R1=-3-OCH3
7:R1=-4-CF3, R2=-morpholine, X=N
8:R1=-3-Cl-4-CH3, R2=-morpholine, X=N
9:R1=-3-OCH3, R2=-morpholine, X=N
Scheme 1. Synthesis of 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thiadiazol-2-yl)urea
derivatives. Reagents and conditions: (a) acetonitrile, reflux, 3~5h, 85%~95%; (b)
potassium carbonate, acetonitrile, reflux, 8~12h, 90%~96%; (c) ethanol, reflux, 8~12h,
35%~60%.
The results of the antiproliferative activities of these compounds and Imatinib (a
standard drug approved by FDA) were summarized in Table 1. Figure 2 showed
cellular toxicity comparison between standard drug Imatinib and compounds 4-9 on
4

K562 cells. Cell proliferation and cell killing experiments were tested with the
method of the real-time live-cell imaging, and kinetics of cell growth was monitored
and recorded by an InCucyteZOOM device during a period of 72 hours.
Firstly, we changed the substituent of 6-position pyrimidin with different groups and
fixed the substituent of phenyl ring (4a vs 4b vs 4c, 5a vs 5b vs 5c). Obviously, there
is no significant difference in the biological activities of compounds 4a-c or 5a-c.
However, the cellular toxicity of compounds 4b and 5b was significantly higher than
that of 4a or 4c and 5a or 5c, respectively (shown in Figure 2A and 2B). The cellular
toxicity and antiproliferative activities of compounds 4a and 5a are similar to those of
4c and 5c, respectively. These results imply that the substituent of 6-position
pyrimidin does not affect the bioactivities but change the cellular toxicity of
compounds to K562 cells.
We next fixed 6-(3-morpholinopropyl)amino substituent of pyrimidin and varied the
3/4-position substituent of phenyl ring (4a, 5a, 6, 7, 8, 9). Among these compounds,
4a (4-methyl phenyl, IC50 = 0.056 μM), 7 (4-trifluoromethyl phenyl, IC50 = 0.038 μM),
8 (3-chloro-4-methyl phenyl, IC50 = 0.035 μM) showed excellent activities compared
to standard drug Imatinib (IC50 = 0.14 μM). The most potent compound corresponds
to 7 displayed a very weak or no cellular toxicity against K562 cells compared to 8
(shown in Figure 2C). The results indicate that the antiproliferative activity of
compound 4a and 7 is superior to that of Imatinib, and the cellular toxicity is also
slightly superior or similar to Imatinib, which indicating that the anti-viability
activities of compounds 4a and 7 against K562 cells were not due to cellular toxicity
(shown in Figure 2D). Considering that compound 7 has the least cellular toxicity and
better biological in cellular assays, further studies were performed just on compound
7.
Table 1. Anti-proliferation activities of 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-
thiadiazol-2-yl)urea derivatives 4-9 against K562 cells
Comp.
K562 (IC50, μM)
Comp.
K562 (IC50, μM)
5

4a
4b
4c
5a
5b
5c
0.056±0.0028
0.078±0.0044
0.014±0.001
0.12±0.025
0.15±0.026
0.28±0.064
6
0.11±0.013
0.038±0.0013
0.035±0.0025
0.69±0.081
0.14±0.010
7
8
9
Imatinib
Figure 2. Cellular toxicity of compounds 4-9 on K562 cells (see the Supporting
Information for the experimental operation). In this experiment, kinetics of growth
was monitored and recorded by an InCucyteZOOM device during a period of 72
hours. All the conditions were in triplicates and the assay was repeated twice. (A)
Cellular toxicity of compounds 4a, 4b and 4c against K562 cells. (B) Cellular toxicity
of compounds 5a, 5b and 5c against K562 cells. (C) Cellular toxicity of compounds 6,
7, 8, 9 against K562 cells. (D) Cellular toxicity of compounds 4a, 6, 7, 9 and Imatinib
against K562 cells.
To confirm compound 7 could induce apoptosis, we further performed cell apoptosis
6

assays using the CellEvent® Caspase-3/7 Green ReadyProbes® reagent. The total
apoptotic cell count was monitored and recorded by an InCucyteZOOM device during
a period of 48 hours. As shown in Figure 3, compound 7 at 10 μM significantly
induced apoptosis. Apoptotic cell count significantly increased from 24 h to 48 h after
administration, and was different from the vehicle control (P< 0.0001). Both
compound 7 and Imatinib inhibited K562 cell proliferation, but Imatinib had stronger
apoptosis effect on K562 cells than compound 7 at the same concentration of 10 μM.
These results revealed that compound 7 inhibited proliferation and induced apoptosis
in K562 cells, which was the same to most of the clinical anticancer chemotherapy
agents.
Figure 3. K562 cells were loaded with CellEvent® Caspase-3/7 Green ReadyProbes®
reagent, then treated with 10 μM compound 7 and Imatinib, respectively, compared to
the vehicle control for 48 hours. Untreated cells (CTRL) have minimal fluorescence,
while treated cells (7 and Imatinib) have a significant increase in fluorescence (shown
in green) indicating apoptosis (see the Supporting Information for the experimental
operation).
We next performed a human phospho-kinase array, which simultaneously determine
relative phosphorylation levels in over 40 different kinases, and disclosed the effects
of the treatment with compound 7 on activation of the AKT downstream signaling
7

pathways in K562 cells (Figure 4A). In the human phospho-kinase array, treatment
with compound 7 clearly downregulated the phosphorylation of kinases within the
PI3K/Akt signaling pathway, AKT1/2/3 (S473, T308) and its downstream kinases
p70S6K (T421/S424), TOR (S2448) and GSK-3α/β(S21/S9). Phosphorylation of the
kinases RSK1/2/3 (S380/S386/S377) and ERK1/2 (T202/Y204, T185/Y187) involved
in the Ras/Raf/MEK/ERK pathway was decreased after addition of compound 7 in
K562 cells. Whereas the phosphorylation of Src kinases (Src, Lck, PYK2) signaling
pathways was slightly reduced upon treatment with compound 7. We slao detected
significant changes in the phosphorylation of P53 (S392, S46, S15), p27 (T198).
Phosphorylation of kinases that belong to the MAPK pathway (p38, HSP27, JNK) and
STAT pathway was differentially effected upon compound 7. Together, these data
suggest that a major mechanism by which compound 7 mediates K562 cells is
through down-regulation of AKT signaling pathway, all resulting in lower cell
survival, proliferation and apoptosis. In addition, the array results further revealed that
the compound 7 could inhibit the effect of the phosphorylation of WNK1 (T60) and
PRAS40 (T246) kinases.21 The down-regulation of WNK1 activation may indicate
that other AKT-independent pathways could also play roles in the observed effects of
cell growth inhibition and cell apoptosis. 22-23
8

Figure 4. Compound 7 activate PI3K/Akt signaling pathway (see the Supporting
Information for the experimental operation). (A) Representative images showing the
changes of kinase phosphorylation in the Human Phospho-kinase Array on K562 cells
treated with compound 7 and Imatinib. Select phosphorylated proteins on the kinase
array membrane are indicated. (B) Relative expression of indicated protein in K562
cells treated for 48 hours.
In this study, a series of 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thiadiazol-2-yl)urea
derivatives were synthesized and evaluated for antiproliferative activity against
human chronic myeloid leukemia cell line K562. As expected, these compounds
9

exhibited remarkable effects on human chronic myeloid leukemia cell line K562.
Especially, compounds 4a (IC50 = 0.056 μM) and 7 (IC50 = 0.038 μM) showed much
better inhibitory activity than standard drug Imatinib (IC50 = 0.14 μM) against human
chronic myeloid leukemia cell line K562, and the cellular toxicities of compounds 4a
and 7 were also slightly superior or similar to Imatinib. In conclusion, SAR studies of
1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thiadiazol-2-yl)urea derivatives based on
structure design combined with cellular assays led to the discovery of a number of
potent TKI inhibitors. Compound 7 was the most active one and lower cellular
toxicity in cellular levels. It also displayed very good induced-apoptosis effect for
human CML cell line K562. Human phospho-kinase array analysis showed that
compound 7 exerted its effect via a significantly reduced phosphorylation of
downstream signal proteins, mainly via the PI3K/Akt kinase. Overall, compound 7
could be taken as a good lead compound for further lead optimization for the
treatment of CML. The further structure expansion of these new series of compounds
and evaluation of their pharmacological profile, as well as improvement of inhibition
ability are still on-going.
Acknowledgements
The research was financially supported by the National Natural Science Foundation of
China (Nos. 81502903 and 81503285).
Appendix A. Supplementary data
Supplementary data associated with this article can be found online version, at
References and Notes
Design and synthesis of novel 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thiadiazol-2-yl) urea
derivatives with potent anti-CML activity throughout PI3K/AKT signaling pathway
Weiwei Li, Jianjie Chu, Tingting Fan, Wei Zhang, Minna Yao, Zeqiong Ning, Mingming Wang,
Jin Sun, Xian Zhao, Aidong Wen*
10

Department of Pharmacy, Xijing Hospital, The Fourth Military Medical University, 127 Changle
Western Road, Xi'an, Shaanxi Province, 710032, China.
* Corresponding author. E-mail addresses: wwzoe.1@163.com
N
N
N
N
skeleton
HN
O
N
N
N
S
S
N
CF3
S
N
N
simplification
H
Cl
H
N
HO
Dasatinib
O
N
N
incorporation
N
N
NH
NH
S
N
N
S
F
H
H
H
H
H
O
NH2
N
N
CF3
Cl
N
N
N
N
HN
K562 IC50=0.038μ M
K562 IC50=0.14μ M
H
N
O
O
O
Imatinib
O
Sorafenib
Linifanib
diarylureas is potential antiproliferative agent for cancer
1 Lugo TG, Pendergast AM, Muller AJ, et al. Tyrosine kinase activity and transformation potency
of bcr-abl oncogene products. Science (New York, N.Y.). 1990;247(4946):1079-1082.
https://doi.org/10.1126/science.2408149.
2 Sawyers CL. Chronic myeloid leukemia. N Engl J Med. 1999;340(17):1330-1340.
https://doi.org/10.1056/NEJM199904293401706.
3 Clarkson B, Strife A, Wisniewski D, et al. Chronic myelogenous leukemia as a paradigm of
early cancer and possible curative strategies. Leukemia. 2003;17(7):1211-1262.
https://doi.org/10.1038/sj.leu.2402912.
4 Mauro MJ, Deininger MW. Chronic myeloid leukemia in 2006: a perspective. Heamatologica.
2006;91(2):152-158. http://www.haematologica.org/content/91/2/152.full.pdf+html.
5 National Comprehensive Cancer Network (NCCN). NCCN Clinical Practice Guidelines in
Oncology:
Chronic
Myeloid
Leukemia
(Version
3.2018).
https://www.nccn.org/professionals/physician_gls/default.aspx. 2019 Accessed 15 January 2019.
6 Buchdunger E, Matter A, Druker BJ. Bcr-Abl inhibition as a modality of CML therapeutics.
Biochim Biophys Acta. 2001;1551(1):M11-8. https://doi.org/10.1016/S0304-419X(01)00022-1.
7 Weisberg E, Manley PW, Breitenstein W, et al. Characterization of AMN107, a selective
inhibitor
of
native
and
mutant
Bcr-Abl.
Cancer
Cell.
2005;7(2):129-141.
https://doi.org/10.1016/j.ccr.2005.01.007.
11

8 Bjorkholm M, Ohm L, Eloranta S, et al. Success story of targeted therapy in chronic myeloid
leukemia: a population-based study of patients diagnosed in Sweden from 1973 to 2008. J Clin
Oncol. 2011;29(18):2514-2520. https://doi.org/10.1200/JCO.2011.34.7146.
9 Pellicano F, Šimara P, Sinclair A, et al. The MEK inhibitor PD184352 enhances BMS-214662-
induced apoptosis in CD34+ CML stem/progenitor cells. Leukemia. 2011;25(7):1159-1167.
https://doi.org/10.1038/leu.2011.67.
10 Gora-Tybor J. Emerging therapies in chronic myeloid leukemia. Curr Cancer Drug Targets.
2012;12(5):458-470. https://doi.org/10.2174/156800912800673202.
11 Brugge J, Hung MC, Mills GB. A new mutational AKTivation in the PI3K pathway. Cancer
Cell. 2007;12(2):104-107. https://doi.org/10.1016/j.ccr.2007.07.014.
12 Hers I, Vincent EE, Tavars JM. Akt signalling in health and disease. Cell Signal. 2011;23(10):
1515-1527. https://doi.org/10.1016/j.cellsig.2011.05.004.
13 Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell. 2007;129(7):
1261-1274. https://doi.org/10.1016/j.cell.2017.04.001.
14 Liu P, Cheng H, Roberts TM, et al. Targeting the phosphoinositide 3-kinase pathway in
cancer. Nat Rev Drug Discov. 2009;8(8):627-644. https://doi.org/10.1038/nrd2926.
15 Carnero A, Blanco-Aparicio C, Renner O, et al. The PTEN/PI3K/AKT signalling pathway in
cancer, therapeutic implications. Curr Cancer Drug Targets. 2008;8(3):187-198.
https://doi.org/10.2174/156800908784293659.
16 Liu L, Cao Y, Chen C, et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor
angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5.
Cancer Res. 2006;66(24):11851-11858. https://doi.org/10.1158/0008-5472.CAN-06-1377.
17 Albert DH, Tapang P, Magoc TJ, et al. Preclinical activity of ABT-869, a multitargeted
receptor
tyrosine
kinase
inhibitor.
Mol
Cancer
Ther.
2006;5(4):995-1006.
https://doi.org/10.1158/1535-7163.MCT-05-0410.
18 Li Y, Tan C, Gao C, et al. Discovery of benzimidazole derivatives as novel multi-target EGFR,
VEGFR-2 and PDGFR kinase inhibitors. Bioorg Med Chem. 2011;19(15):4529-4535.
https://doi.org/10.1016/j.bmc.2011.06.022.
12

19 Choi WK, El-Gamal MI, Choi HS, et al. New diarylureas and diarylamides containing 1,3,4-
triarylpyrazole scaffold: Synthesis, antiproliferative evaluation against melanoma cell lines, ERK
kinase inhibition, and molecular docking studies. Eur J Med Chem. 2011;46(12):5754- 5762.
https://doi.org/10.1016/j.ejmech.2011.08.013.
20 (a) Almajan GL, Innocenti A, Puccetti L, et al. Carbonic anhydrase inhibitors. Inhibition of the
cytosolic and tumor-associated carbonic anhydrase isozymes I, II, and IX with a series of 1,3,4-
thiadiazole- and 1,2,4-triazole-thiols. Bioorg Med Chem Lett. 2005;15(9):2347-2352.
https://doi.org/10.1016/j.bmcl.2005.02.088. (b) Kim KS, Kimball SD, Misra RN, et al. Discovery
of aminothiazole inhibitors of cyclin-dependent kinase 2: synthesis, X-ray crystallographic
analysis,
and
biological
activities.
J
Med
Chem.
2002;45(18):3905-3927.
https://doi.org/10.1021/jm0201520.
21 Wang H, Zhang Q, Wen Q, et al. Proline-rich Akt substrate of 40kDa (PRAS40): a novel
downstream target of PI3k/Akt signaling pathway. Cell Signal. 2012;24(1):17-24.
https://doi.org/10.1016/j.cellsig.2011.08.010.
22 Moniz S, Jordan P. Emerging roles for WNK kinases in cancer. Cell Mol Life Sci. 2010;67(8):
1265-1276. https://doi.org/10.1007/s00018-010-0261-6.
23 Lenertz LY, Lee BH, Min X, et al. Properties of WNK1 and implications for other family
members. J Biol Chem. 2005;280(29):26653-6658. https://doi.org/10.1074/jbc.M502598200.
13