Design and synthesis of novel orally selective and type II pan-TRK inhibitors to overcome mutations by property-driven optimization

Article abstract of DOI:10.1016/j.ejmech.2021.113673

Rare oncogenic NTRK gene fusions result in uncontrolled TRK signaling leading to various adult and pediatric solid tumors. Based on the architecture of our multi-targeted clinical candidate BPR1K871 (10), we designed and synthesized a series of quinazoline compounds as selective and orally bioavailable type II TRK inhibitors. Property-driven and lead optimization strategies informed by structure-activity relationship studies led to the identification of 39, which showed higher (about 15-fold) selectivity for TRKA over AURA and AURB, as well as potent cellular activity (IC₅₀ = 56.4 nM) against the KM12 human colorectal cancer cell line. 39 also displayed good AUC and oral bioavailability (F = 27%), excellent in vivo efficacy (TGI = 64%) in a KM12 xenograft model, and broad-spectrum anti-TRK mutant potency (IC₅₀ = 3.74–151.4 nM), especially in the double-mutant TRKA enzymatic assays. 39 is therefore proposed for further development as a next-generation, selective, and orally-administered type II TRK inhibitor.

Full text of DOI:10.1016/j.ejmech.2021.113673

European Journal of Medicinal Chemistry 224 (2021) 113673
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design and synthesis of novel orally selective and type II pan-TRK
inhibitors to overcome mutations by property-driven optimization
,
b
,
,
,
Mu-Chun Li a c, Wen-Hsing Lin a, Pei-Chen Wang a 1, Yu-Chieh Su a 1, Pei-Yi Chen a,
Chu-Min Fan a, Ching-Ping Chen a, Chen-Lung Huang a, Chun-Hsien Chiu a, Ling Chang a,
Chiung-Tong Chen a, Teng-Kuang Yeh a, Hsing-Pang Hsieh a
, b, c, *
a Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 35, Keyan Road, Zhunan Town, Miaoli County, 350401,
Taiwan, ROC
b Department of Chemistry, National Tsing Hua University, No. 101, Sec. 2, Kuang-Fu Road, Hsinchu, 300044, Taiwan, ROC
c Biomedical Translation Research Center, Academia Sinica, No. 99, Ln. 130, Sec. 1, Academia Road, Taipei City, 115202, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
Rare oncogenic NTRK gene fusions result in uncontrolled TRK signaling leading to various adult and
pediatric solid tumors. Based on the architecture of our multi-targeted clinical candidate BPR1K871 (10),
we designed and synthesized a series of quinazoline compounds as selective and orally bioavailable type
II TRK inhibitors. Property-driven and lead optimization strategies informed by structure-activity rela-
tionship studies led to the identification of 39, which showed higher (about 15-fold) selectivity for TRKA
over AURA and AURB, as well as potent cellular activity (IC50 ¼ 56.4 nM) against the KM12 human
colorectal cancer cell line. 39 also displayed good AUC and oral bioavailability (F ¼ 27%), excellent in vivo
efficacy (TGI ¼ 64%) in a KM12 xenograft model, and broad-spectrum anti-TRK mutant potency
(IC50 ¼ 3.74e151.4 nM), especially in the double-mutant TRKA enzymatic assays. 39 is therefore proposed
for further development as a next-generation, selective, and orally-administered type II TRK inhibitor.
© 2021 Elsevier Masson SAS. All rights reserved.
Received 12 May 2021
Received in revised form
22 June 2021
Accepted 24 June 2021
Available online 29 June 2021
Keywords:
Tropomyosin receptor kinase (TRK)
Kinase inhibitor
Fusion
Mutation
Property-driven optimization
1. Introduction
fusion, such as TPM3-NTRK1 fusion gene found in a colorectal
cancer (CRC) patient in 1982 (the first NTRK gene abnormality to be
In the tropomyosin receptor kinase (TRK) family, TRKA, TRKB,
and TRKC are the members of tyrosine receptor kinase encoded by
the NTRK1, NTRK2, and NTRK3 genes, respectively. These tyrosine
kinases are highly expressed in neuronal tissues and play crucial
roles in the development and function of neurons by neurotrophin
(NT) activation [1]. The corresponding neurotrophins of TRK ki-
nases include nerve growth factor (NGF) for TRKA, both brain-
derived growth factor and neurotrophin 4 (NT4) for TRKB, and
neurotrophin 3 (NT3) for TRKC [2]. Thus, TRK kinases are involved
in the regulation of synaptic strength and plasticity in the
mammalian nervous system [3]. A variety of NTRK gene alterations
have been diagnosed in various tumor types [4], including gene
identified [5]); and mutant TRKA or TRKB proteins (i.e. TRKAR577G
and TRKBP507L), NTRK1 deletion, and overexpressed TRK proteins
have been observed in lung adenocarcinoma, melanoma, acute
myeloid leukemia, neuroblastoma, etc. in the last decade [6e11].
NTRK gene fusions occur when a NTRK gene becomes abnormally
connected to another unrelated gene, such as ETV6, LMNA, TPM3,
etc. [12], causing uncontrolled TRK signaling leading to various
adult and pediatric solid tumors. NTRK-fusions are rare (< 1%) in
common cancer types but common (up to > 90%) in rare cancer
types [13], and therefore the discovery of TRK inhibitors is an
important therapeutic strategy.
Several TRK inhibitors have been evaluated for the treatment of
a variety of cancers (Fig.1) [14,15] including larotrectinib (Vitrakvi®,
1) [16] and entrectinib (Rozlytrek®, 2) [17] which were approved by
U.S. Food and Drug Administration (FDA) as tumor-agnostic cancer
treatment in 2018 and 2019, respectively. Larotrectinib (1) and
entrectinib (2) are both proved as type I TRK inhibitors according to
their binding modes with TRKA protein, in which the compounds
occupy the ATP-binding site and present the “DFG-in”
* Corresponding author. Institute of Biotechnology and Pharmaceutical Research,
National Health Research Institutes, No. 35, Keyan Road, Zhunan Town, Miaoli
County, 350401, Taiwan, ROC.
E-mail
addresses:
hphshieh@nhri.edu.tw,
alexhsieh@gate.sinica.edu.tw
(H.-P. Hsieh).
1
These authors contributed equally.
https://doi.org/10.1016/j.ejmech.2021.113673
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

M.-C. Li, W.-H. Lin, P.-C. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113673
Fig. 1. Chemical structures of representative TRK inhibitors.
conformation [14]. The kinase activity is regulated by the Asp-Phe-
Gly (DFG) motif significantly. Milciclib (3) [18] and belizatinib (4)
[19] are two other potent type I TRK inhibitors, though neither has
been approved for clinical use. Type II TRK inhibitors include GNF-
5837 (5) [20], cabozantinib (6) [21], sitravatinib (7) [22], etc., all of
which exhibit potent inhibitory activity against TRK kinases pos-
sessing “DFG-out” conformation, and some of which are under
clinical investigation for the treatment of NTRK-fusion cancers.
Apart from these abovementioned clinical candidates, there are
numerous developing compounds under various clinical trials at
different stages in the past ten years [23e25].
Despite the success of these anti-TRK drugs as tumor-agnostic
treatment, acquired drug resistance mediated by somatic muta-
tions in the TRK fusion is inevitable [26]. For example, the muta-
tions TRKAG595R and TRKAG667C significantly reduce the binding
affinity of larotrectinib (1) [27]. TRKAG595R is located in the solvent
front region of the kinase domain, whereas TRKAG667C is in the
xDFG position [28]. Several second-generation TRK inhibitors are in
development to address these specific mutations, such as seli-
trectinib (LOXO-195, 8) [29] and repotrectinib (TPX-0005, 9) [30],
whose macrocyclic structures artfully mitigate the influence of the
mutant amino acids (Fig. 1). However, in clinical trials, some pa-
tients who acquired the TRKA solvent front mutation and were
treated with the abovementioned compounds eventually became
non-responsive, due to mutations at the xDFG position,[31] which
may diminish the type I binding affinity of second-generation TRK
inhibitors. Consequently, the discovery of novel selective type II
TRK inhibitors to overcome the resistance is extremely important
for patients who are suffering from rare cancers harboring NTRK-
fusion genes. Interestingly, cabozantinib (6) and other type II ki-
nase inhibitors preferentially bind to suppress xDFG TRK muta-
tions, and showed strong inhibitory ability against double mutants
in both in vitro and in vivo models [32].
(10) exhibited potent inhibitory activity against TRK kinases with
IC50 values of 2.23 nM, 1.78 nM and 0.21 nM for TRKA/B/C,
respectively (Fig. 2); and 6 and 13 nM against aurora kinases A
(AURA) and B (AURB). However, the poor selectivity of BPR1K871
(10) may cause unwanted side effects [34]; and several AURA/B
inhibitors are known to be associated with common adverse effects
including febrile neutropenia, stomatitis, hematological toxicities,
etc. [35,36] In addition, the low oral absorption of BPR1K871 (10)
necessitated its intravenous administration, and raised questions
regarding how frequently the patient would have to be dosed. In
this study, inspired by the architecture of BPR1K871 (10), we
designed and synthesized a series of novel TRK inhibitors display-
ing excellent selectivity, significantly improved oral bioavailability,
and double-digit nanomolar potency against a panel of TRK family
enzymes, including some drug-resistant mutants. Structure-
activity relationship (SAR) studies were accomplished using the
TRKA enzymatic assay and KM12 human CRC cell line, which ex-
presses the chimeric TPM3-TRKA protein and is hypersensitive to
TRKA kinase inhibition. In addition, our novel inhibitors were
counter-screened against AURA and AURB kinases, to assess their
selectivity.
2. Design and chemistry
Our previous efforts to develop dual AURA and FLT3 inhibitors
resulted in the identification of BPR1K871 (10), a promising
candidate for multiple cancer treatments (Fig. 2). However, its
bioavailability was poor e perhaps due to its relatively large
number of free rotatable bonds (NRB) and/or flexible dimethyla-
minopropoxy side chain on the quinazoline scaffold. BPR1K871
(10) did incorporate a thiazole moiety on the ethylene linker, a
common structural characteristic of small-molecule drugs that
might be key to the poor selectivity of BPR1K871 (10) [37]. To
discover desired TRK inhibitors inspired by BPR1K871 (10) based
on property-driven strategies, we initially screened different side
chains on the quinazoline scaffold, and compared the inhibitory
Previously, we developed BPR1K871 (10) [33], a multi-kinase
inhibitor (MKI) for which IND approval was obtained for the
treatment of solid tumors. In a kinase profiling study, BPR1K871
2

M.-C. Li, W.-H. Lin, P.-C. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113673
Fig. 2. Design strategy to improve the oral bioavailability and selectivity of BPR1K871 (10).
activity of analogues incorporating either an ethylene thiazole or
ethylene benzene linker. We also experimented with shorter
linkers, to reduce the flexibility of the compounds. A selection of
derivatives incorporating various functional groups were also
synthesized, to screen for their effect on both TRKA and ARUA
inhibitory ability.
Our synthesis started with quinazolines 11aef, to which were
attached an ethylene thiazole ring (to give 12aed) or an ethylene
benzene ring group (to give 19aef), respectively, as depicted in
Schemes 1 and 2. The core skeletons, 4-chloroquinazoline de-
rivatives 11aef, were either commercially available or prepared
based on our previous reports [33,38]. For the ureas 14e17, 4-
chloroquinazoline derivatives 11aed were treated with tert-butyl
[5-(2-aminoethyl)-1,3-thiazol-2-yl]carbamate under basic condi-
tions to give 12aed. Then the acid-deprotected products 13aed
was treated with either phenyl isocyanate or 3-chlorophenyl iso-
cyanate to yield the target ureas 14e17. Finally, the chloride moiety
of 14 was substituted by N,N-dimethylamino group to obtain urea
18.
Compounds incorporating an ethylene benzene linker were
analogously synthesized. The core skeleton 4-chloroquinazoline
derivatives 11aef were reacted with 4-(2-aminoethyl)aniline, 4-
(aminomethyl)aniline, or benzene-1,4-diamine and trimethyl-
amine in N,N-dimethylformamide solution to give 19aef. Then, the
amine derivatives 19aef were coupled with appropriate iso-
cyanates to give ureas 20e23 and 25e40, respectively.
3. Results and discussion
3.1. Biological evaluation of AURA and TRKA and SAR analysis
Our initial results are presented in Table 1. Based on our
knowledge and experience [39], number of rotatable bonds (NRB)
and value of polar surface area (PSA) were hypothesized to influ-
ence the proportion to oral absorption, for each compound.
Therefore, to improve the oral bioavailability of BPR1K871 (10),
several analogues bearing shorter side chains on the C-6 or C-7
positions of the quinazoline scaffold were screened. Varying the R1
Scheme 1. Synthesis of analogues 15e18.
Reagents and conditions: (i) tert-butyl [5-(2-aminoethyl)-1,3-thiazol-2-yl]carbamate, Et3N, EtOH, reflux, 15 h; (ii) 6.0 N HCl(aq), rt, 10 h or TFA, CH2Cl2, rt, 12 h; (iii) PhNCO, MeOH,
CH2Cl2, rt, 16 h; (iv) 3-Cl-PhNCO, MeOH, CH2Cl2, rt, 16 h; (v) N,N-dimethylamine, DMF, 100 ꢀC, 6 h.
3

M.-C. Li, W.-H. Lin, P.-C. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113673
Scheme 2. Synthesis of TRK analogues 20e39.
Reagents and conditions: (i) (a) 4-(2-aminoethyl) aniline, nBuOH, reflux, 1 h; or (b) 4-(2-aminoethyl)aniline, EtOH, Et3N, reflux, 1 h; or (c) 4-(aminomethyl)aniline, EtOH, reflux, 12 h;
or (d) benzene-1,4-diamine, propan-2-ol, reflux, 1 h; (ii) R3-PhNCO, MeOH, CH2Cl2, rt, 1e16 h; (iii) N,N-dimethylamine, DMF, 120 ꢀC, 100 min, MW.
Table 1
SAR investigation by side chain modification.a
Compound
R1
R2
R3
Ringb
AURA
AURB
TRKA
NRB
PSA
IC50 (nM)
Inhibition
Inhibition
Inhibition
@ 0.5
m
M
@ 10
m
M
@ 1 mM
BPR1K871 (10)
H
H
OCH3
F
H
H
OCH3
F
O(CH2)3N(CH3)2
H
OCH3
OCH2CH3
O(CH2)3N(CH3)2
H
OCH3
OCH2CH3
O(CH2)3N(CH3)2
3-Cl
H
H
3-Cl
H
H
H
3-Cl
H
T
T
T
T
T
B
B
B
B
5.6
13c
100%
71%
98%
98%
99%
18%
65%
62%
91%
99%
1%
80%
96%
95%
1%
6%
51%
3%
11
6
8
8
11
6
8
8
11
132.54
120.07
138.53
129.29
132.54
78.94
97.40
88.17
91.41
15
16
17
18
21
22
23
24
11.1
10.0
8.4
85%
86%
64%
76%
85%
81%
75%
77%
6.0
140.0
23.0
26.9
59.0
H
a
The IC50 and inhibition values of AURA, AURB and TRKA were performed using in-house Kinase-Glo® assay; all data are expressed as the mean of at least two independent
experiments and are mostly within 15% error margins.
b
The “T” represents a thiazole ring, and the “B” represents a benzene ring.
IC50 value.
c
and R2 substituents of 15e17 not only conserved the AURA, AURB
and TRKA activities but also decreased the number of rotatable
bonds. Next, replacement of the thiazole ring of ureas 15e18 with a
benzene ring gave ureas 21e24 which had lower PSA values and
AURA activities by 2.3- to 12.7-fold (Table 1). Compared with
thiazole-containing analogue 17, benzene-containing analogue 23
exhibited improved selectivity when profiled using the KINO-
MEscan™ technology against a panel of 456 kinases at a concen-
tration of 100 nM (Fig. 3, Table S1, Table S2). Taken together, 23 not
only showed lower values of NRB and PSA but also satisfied
selectivity than 17, which increases the potential to obtain selective
and orally efficacious compounds. These results convinced us to
further study the series of compounds incorporating a benzene ring
in the joint linker moiety, between the scaffold and
pharmacophore.
Of urea derivatives 21e24 (all of which incorporated a benzene
ring in the linker), 21 without any substituents on the quinazoline
scaffold and a terminal phenyl ring showed the weakest TRKA
ability. Urea 24 incorporating a solubilizing dimethylaminopropoxy
moiety at the C-7 position was a more potent inhibitor of TRKA
kinase than 21 at a concentration of 10 mM. However, the accom-
panying increases in both flexibility and AURA kinase inhibition
were undesirable. Introduction of electron-donating methoxy
groups onto both the C-6 and C-7 position of 21 to yield 22 [40]
improved the TRKA inhibitory ability by a factor of approximately
three times at a concentration of 10 mM; 22 was also a more potent
4

M.-C. Li, W.-H. Lin, P.-C. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113673
Fig. 3. Kinase profiling of 17 and 23 using KINOMEscan™ technology. The S(35) of 17 and 23 were 0.022 and 0.012, respectively.
inhibitor of AURA, compared to 21. However, of ureas 21e24, 23
BPR1K871 (10) such that 23 is lacking the p-p interaction as well.
was the most potent inhibitor of TRKA, exhibiting 51% inhibition at
a concentration of 1 mM, and having moderate AURA and AURB
kinase activities. Additionally, the NRB and PSA were both relatively
low. Consequently, compound 23 was regarded as the potential hit
compound and selected for further studies.
These observations could account for the decreased AURA inhibi-
tory ability of BPR1K871 (10) compared to hit compound 23.
Both BPR1K871 (10) and hit 23 anchor to the ATP-binding site of
TRKA kinase (PDB: 6PL1) [41] via hinge binding with Met592,
adopting a type II binding mode with TRKA (Fig. 4b). The urea
moiety in these two compounds generates a strong hydrogen bond
interaction with the Asp668 backbone amide NH of the DFG motif,
and bidentate hydrogen bonds with the Glu560 sidechain carbox-
3.2. Binding mode analysis of BPR1K871 (10) and 23
ylate of the
a
-helix. Also, the thiazole ring of BPR1K871 (10) and
interaction
Molecular docking studies were undertaken to better under-
stand the structural basis for the differences in the inhibitory po-
tencies of BPR1K871 (10) and 23 against AURA and TRKA kinases.
The X-ray co-crystal structure of BPR1K871-AURA complex (pro-
tein data bank (PDB): 4JBO) was utilized as the basis for the study of
hit compound 23 with AURA kinase. As depicted in Fig. 4a, both
BPR1K871 (10) and hit 23 adopt a type II binding mode with ARUA;
both compounds overlap extensively in the ATP binding site.
BPR1K871 (10) formed seven hydrogen bond interactions with
Arg137, Lys162, Glu181 and Ala213 via either nitrogen or oxygen
atoms. The unique hydrogen bond interaction between the sulfur of
the thiazole ring of BPR1K871 (10) and the Asp274 backbone amide
the benzene ring of urea 23 both form the crucial
p-p
with the Phe669 of the DFG motif, as well as the Phe589 of the
gatekeeper, leading to the DFG-out conformation. Due to its strong
interactions with TRKA kinase, 23 was selected for further hit-to-
lead optimization.
3.3. Biological evaluation of TRKA and SAR analysis
Knowing the binding modes between hit 23 and the AURA ki-
nase, we attempted to decreased inhibition of AURA by 23 by
redesigning the ethylene linker connecting the quinazoline scaffold
of 23 with the benzene ring. We hypothesized that shortening the
linker might decrease binding activity with AURA/AURB by
reducing the number of possible degrees of freedom e thereby
impeding its interaction with the back pocket. As shown in Table 2,
ureas 25 and 26 incorporating a methylene linker (n ¼ 1) had very
low TRKA and AURA/AURB inhibiting activities, as did ureas 27 and
28 (lacking a linker entirely, n ¼ 0). The importance of the ethylene
NH of the DFG motif, as well as the crucial p-p interaction between
the terminal phenyl ring of BPR1K871 (10) and the Phe144 in the
AURA back pocket could account for the nanomolar-potency of
BPR1K871 (10) against AURA kinase. However, urea 23 which in-
corporates a benzene ring instead of a thiazole ring cannot form the
specific hydrogen bond interaction with the sulfur. Additionally, the
terminal phenyl ring of 23 is flipped relative to its orientation in
5

M.-C. Li, W.-H. Lin, P.-C. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113673
Fig. 4. Characterization of inhibitor-binding modes: (a) Binding site analysis for BPR1K871 (10, cyan) and hit 23 (rose-pink) docking with AURA X-ray protein (PDB: 4JBO, green);
(b) Binding site analysis for BPR1K871 (10, cyan) and hit 23 (rose-pink) docking with TRKA X-ray protein (PDB: 6PL1, orange).
Table 2
In Vitro TRKA kinase inhibitory activities of inhibitors 23e34.a
Compound
n
R1
R2
R3
TRKA
KM12
AURA
AURB
Inhibition
Inhibition
@ 1
IC50 (nM)
IC50 (nM)
IC50 (nM)
Inhibition
@ 0.5 mM
@ 10
mM
m
M
23
25
26
27
28
29
30
31
2
1
1
0
0
2
2
2
2
2
F
F
F
F
F
F
F
OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
F
F
F
3-Cl
H
3-Cl
H
3-Cl
H
3-Cl
H
61.6%
14.7%
18.6%
23.1%
30.2%
17.6%
46.1%
21.1%
68.3%
7.9%
50.7%
3.1%
3.1%
1.5%
7.6%
ꢁ0.2%
0.7%
4.4%
15.3%
0.5%
99%
91.4
N.D.
N.D.
N.D.
7410
N.D.
3590
N.D.
611
86.6
26.9
8%a
75%
2%
0%
>10000
>10000
2400
1270
2022
283.3
1523
492.7
323.7
42.0
2%a
337.8
155.7
N.D.
N.D.
65.6
107.8
29.2
6.0
73%
76%
75%
75%
50%
66%
101%
75%
OCH2CH3
OCH2CH3
F
32
33
F
3-Cl
H
OCH2CH3
>9000
36.8
BPR1K871 (10)
100%
a
The IC50 values of AURA and inhibition values of TRKA and AURB were performed using in-house Kinase-Glo® assay; the IC50 values of TRKA were performed using
HotSpot™ kinase assay according to the manufacturer's instructions [42]; all data are expressed as the mean of at least two independent experiments and are mostly within
15% error margins.
linker was also evident in a docking study of 23, 26 and 28. The
distance between hinge Met592 and N1 on the quinazoline scaffold
increased with decreasing linker length (Fig. 5). Accordingly, the
ethylene linker (n ¼ 2) was concluded to be essential for TRKA
activity, and was conserved in further investigations.
Next, we further explored the influence of the substituents on
the quinazoline scaffold. Replacing an electron-donating group
with an electron-withdrawing group at the R2 position (for
example 23 to 30, and 33 to 29) resulted in loss of TRKA inhibitory
activity. In addition, 31 and 32 e both substituent-swapped ana-
logues of 23 e showed weaker inhibition in the TRKA enzymatic
and KM12 cellular assays. These results indicated that inhibition of
TRKA and KM12 cells may be influenced by the electronic charac-
teristics of the inhibitor. We also verified the importance of the
substituent on the terminal phenyl ring. Urea analogues bearing a
chloro-substituent (cf. 23, 30, 32) were more potent inhibitors of
KM12 cells than their unsubstituted counterparts (cf. 33, 29, 31) by
Fig. 5. Binding site analysis for hit 23 (pink), 26 (green) and 28 (yellow) docking with
TRKA X-ray protein (PDB: 6PL1, orange).
approximately 3e8-fold. Thus, the substituent on the distal phenyl
ring was concluded to exhibit an inhibitory effect.
6

M.-C. Li, W.-H. Lin, P.-C. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113673
The effect of the substituent at the R3 position is outlined in
Table 3. When the 3-chloro substituent was replaced by a hydrogen
or fluoro group to give 33 and 34, inhibition of TRKA and KM12 cells
significantly decreased. Interestingly, 35 bearing a trifluoromethyl
moiety exhibited a significantly enhanced IC50 value as compared
to 33 and 34. Moreover, compound 35 was 2.5-fold less effective
against AURA than hit 23. Next, we further examined the effect of
di-substituted functional groups on the terminal phenyl ring.
Incorporation of a phenyl ring bearing two election-withdrawing
substituents (36 (3-Cl, 4-F) and 37 (3-CF3, 4-Cl)) decreased inhibi-
tory TRKA potency. Based on our preceding docking studies [43],
the back pocket of AURA is cramped and unable to accommodate
too large of an ortho-substituent. Therefore, we artfully introduced
a methoxyl group into 23 (to give 38) and 35 (to give 39), in order to
exclude the undesired AURA activity. The results revealed that both
38 and 39 demonstrated excellent inhibitory potency against TRKA,
and improved selectivity for TKRA vs. AURA; the profile of 39 was
considered to be better than that of 38, based on its improved KM12
potency and weaker AURA/AURB activities. Remarkably, the dock-
ing study of compound 39 exhibited an additional hydrogen bond
interaction between His648 and the fluorine atom on the distal
phenyl ring (Fig. 6). Interaction with His648 has been proposed to
stabilize TRKA protein in the inactive state (DFG-out conformation)
[31]. More importantly, 39 not only exhibited similar KM12 activity
to BPR1K871 (10), but also significantly decreased 50-fold IC50
value against AURA and AURB compared to BPR1K871 (10). Hence,
39 was further pursued as a promising TRKA inhibitor.
Fig. 6. Binding site analysis for lead 39 (purple) docking with TRKA X-ray protein
(PDB: 6PL1, orange).
(Table S4). It is noteworthy that 39 also showed powerful inhibitory
ability against DDR1 e the only potential off-target kinase. These
data emphasize the high selectivity of lead 39 and its suitability for
further in vivo studies and development.
3.5. Biological evaluation of mutant forms of TRK family
3.4. Biological evaluation of wild-type TRKs and selectivity profile
There are several known NTRK gene fusions, of which TRKAG595R
and TRKAG667C are the two major mutant forms of the TRKA pro-
tein; TRKCG623R and TRKCG696A are the paralogous mutant TRKC
proteins. TRKAF589L is another amino acid substitution that has also
been identified in patients who have progressed on current ther-
apies including larotrectinib (1) and entrectinib (2) [5]. Since lead
39 exhibited promising performance in both enzymatic and cellular
assays, we were interested in its ability to suppress mutant TRKA
and TRKC proteins. In addition to the aforementioned NTRK gene
fusions, IC50 values against TRKAL657M and TRKCL686M were also
ascertained. Lead 39 proved to be a potent inhibitor of seven
Given the high sequence identity and similarity of the three TRK
members, we further examined the enzymatic and cellular activ-
ities of lead 39 with view to better understanding its selectivity. As
shown in Fig. 7, 39 demonstrated inhibitory activity against wild-
type (WT) TRKA/B/C in enzymatic assays with IC50 values of 21.4,
29.2, 4.55 nM, respectively. Profiling of a panel of selected wild-
type kinases was conducted at a screening concentration of 1 mM
(Fig. 7, Table S3); the results showed that 39 is highly selective, with
standard selectivity scores 0.086 (for S (35)) and 0.057 (S (10))
Table 3
In Vitro TRKA kinase inhibitory activities of inhibitors 33e39.a
Compound
R3
TRKA
KM12
AURA
AURB
Inhibition
Inhibition
@ 1
IC50 (nM)
IC50 (nM)
IC50 (nM)
Inhibition
@ 0.5 mM
@ 10
m
M
m
M
23
33
34
35
36
37
38
39
3-Cl
H
3-F
3-CF3
3-Cl, 4-F
3-CF3, 4-Cl
3-Cl, 6-OCH3
3-CF3, 6-OCH3
61.6%
7.9%
50.7%
0.5%
4.9%
88.5%
0.6%
40.9%
92.1%
93.7%
99%
91.4
86.6
26.8
29.2
26.2
63.2
44.0
349.4
351.6
454.2
6.0
75%
101%
95%
62%
63%
62%
> 9000
4500
26.7
2630
2120
23.0
323.7
411.8
70.2
125.2
227.9
95.5
17.4%
91.4%
32.0%
80.7%
98.0%
98.9%
100%
511.8a
548.4a
75%
21.4
36.8
56.4
42.0
BPR1K871 (10)
a
The IC50 values of AURA and inhibition values of TRKA and AURB were performed using in-house Kinase-Glo® assay; the IC50 values of TRKA were performed using
HotSpot™ kinase assay according to the manufacturer's instructions [42]; all data are expressed as the mean of at least two independent experiments and are mostly within
15% error margins.
7

M.-C. Li, W.-H. Lin, P.-C. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113673
against double-mutant TRKs such as TRKAG595R/G667C, which has
been associated with selitrectinib-treated patients [28].
On the other hand, the selectivity indices (SI; IC50 of TRKAWT
/
mutant-TRKA) of 39 and representative compounds were evaluated
and listed inTable 5. The first-generationTRKA drug, larotrectinib (1),
is potent against wild-type TRKA proteinwith an IC50 value of 3.9 nM,
but it is not capable to overcome the inevitable drug-acquired resis-
tance with poor selectivity indices between wild-type and mutant-
TRKA proteins. Two second-generation type I TRKA inhibitors, seli-
trectinib (8) and repotrectinib (9) exhibited extremely strong inhib-
itory ability against TRKAWT, but 30-fold and 100e400-fold reduced
potency against TRKAG595R and TRKAG667C enzymes, the two typical
single-mutant TRKA proteins. More interestingly, both selitrectinib
(8) and repotrectinib (9) are much less potent against the double-
mutant TRKAG595R/G667C enzyme. Instead, cabozantinib (6) and fore-
tinib show good ability of overcoming resistance against TRKAG667C
and TRKAG595R/G667C enzymes. Being a type II TRKA inhibitor, 39 has
similar performance as cabozantinib (6) and foretinib did. The IC50
values of 39 against TRKAWT, TRKAG667C, and TRKAG595R/G667C en-
zymes are 21.4 nM, 13.3 nM, and 19.6 nM, respectively. Differ from
most of type II TRK inhibitors with multi-targeted characters, 39 is a
highly selective pan-TRK inhibitor which could prevent from unex-
pected side effects due to off-target issues.
3.6. Western blotting analysis
The ability of lead 39 to inhibit activation of TRKA was examined
by western blotting analysis, Fig. 8. KM12 cells were treated with
increasing concentrations (0e500 nM) of 39; after 40 minutes, the
autophosphorylation sites of TRKA and phospho-TRKA (Tyr490)
were analyzed. The TRKA protein was found to be predominantly
phosphorylated when the KM12 cells were not treated with 39, and
the amount of phospho-TRKA decreased with increasing concen-
trations of 39. A concentration of about 50 nM of 39 was sufficient
to suppress TRKA autophosphorylation, and 39 reduced the phos-
phorylation of KM12 cells in a dose-dependent manner. These data
were also consistent with the antiproliferative function in
KM12 cells (IC50 ¼ 56.4 nM). Therefore, lead 39 was concluded to
target the TRKA enzyme inside KM12 cells.
Fig. 7. Kinome-tree representation of the selectivity profile of 39 as measured by ki-
nase inhibition assay [42]. Red dots indicate the key target TRK proteins in which the
remained enzyme activity is less than 10%. A garnet dot indicates the remained
enzyme activity is less than 10%. Gold dots indicate the remained enzyme activity are
between 30 and 50%. Gray dots indicate the remained enzyme activity are over 50%.
3.7. Pharmacokinetic study
mutant TRKAs and TRKCs (Table 4). Compared with larotrectinib
(1), 39 was approximately 3e18 fold more potent an inhibitor of six
mutant TRKAs and TRKCs (though not TRKCG696A). In particular, 39
showed promising inhibitory activity against the major TRK protein
mutations TRKAG667C and TRKCG696A and the gatekeeper F589L
substitution of TRKA, with IC50 values of 13.3, 22.5, and 10.4 nM,
respectively. Lead 39 even exhibited promising inhibitory activity
The in vivo pharmacokinetic (PK) profile of 39 was next inves-
tigated in different species, including mice and rats (Table 6). The
half-life, AUC, etc. of 39 were monitored after i.v. dosages of 1 mg/
kg and 5 mg/kg, and p.o. dosages of 10 mg/kg and 20 mg/kg in mice
and rats, respectively. 39 was found to exhibit acceptable drug
exposure (AUC(0-inf)) and reasonable PK profiles in both species,
with good oral absorption (based on AUC values) and moderate oral
bioavailability of 27.9% (mice) and 27.2% (rats). These results sug-
gest the suitability of 39 for oral administration, and spurred our
evaluation of its in vivo efficacy.
Table 4
Inhibitory ability of 39 against mutant TRKs.a
Kinase (IC50, nM)
39
Larotrectinib (1)
3.8. Evaluation of in vivo efficacy of 39
TRKAF589L
10.4
151.4
13.3
3.74
75.4
22.5
48.4
5.22
19.6
37.6
52.1
TRKAG595R
1125
237.2
20.7
208.7
3.28
149.7
80.2
> 10000
2510
The antitumor efficacy of lead 39 was evaluated in a KM12 nude
mice xenograft model with a five-days-on, two-days-off (FOTO)
treatment schedule. 39 was orally administered to the mice daily
and the marketed TRK inhibitor larotrectinib (1) was utilized as a
positive control. The experiment was progressed for three weeks,
as shown in Fig. 9, 39 demonstrated excellent in vivo efficacy with
the tumor growth inhibition (TGI) value of 64% at day 18. Compared
with larotrectinib (1), lead 39 exhibited identical efficacy in the
KM12 mice model. These data indicated that lead 39 is an orally
efficacious TRK-inhibiting agent by delaying tumor growth.
TRKAG667C
TRKAL657M
TRKCG623R
TRKCG696A
TRKCL686M
TRKAG595R/A608D
TRKAG595R/G667C
TRKAG595R/L657M
a
The experiments were performed using HotSpotTM Kinase Assay according to
the manufacturer's instructions; also, the control compound, staurosporine, was
tested in 10-dose IC50 mode with 4-fold serial dilution starting at 20 mM [42].
8

M.-C. Li, W.-H. Lin, P.-C. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113673
Table 5
TRKAWT and mutant-TRKA selectivity comparison between 39 and TRKA inhibitors.a
39
Larotrectinib (1)
Selitrectinib (8)
Repotrectinib (9)
Cabozantinib (6)
Foretinib
TRKAWT IC50 (nM)
21.4
151.4
13.3
19.6
0.14
1.61
1.09
3.9
2,203
961
> 1000
0.002
0.004
0.004
0.07
2.5
30.8
596
0.028
0.002
0.0001
0.11
3.1
10.1
205
0.035
0.011
0.0005
72.3
305.6
1.0
2.97
143.2
0.44
2.4
0.021
6.75
1.24
TRKAG595R IC50 (nM)
TRKAG667C IC50 (nM)
TRKAG595R/G667C IC50 (nM)
Selectivity (TRKAWT/TRKAG595R
Selectivity (TRKAWT/TRKAG667C
4.3
)
)
0.237
72.3
16.8
Selectivity (TRKAWT/TRKAG595R/G667C
)
a
The data of TRKA inhibitors except 39 are received from literature [31].
Fig. 8. Western blot analysis for cellular target modulation by 39. Phospho-TRKA
(Y490) was inhibited by 39 in KM12 cells after 40 minutes.
4. Conclusion
A series of quinazoline derivatives of varying polar surface areas
and incorporating variable numbers of rotatable bonds was syn-
thesized and evaluated in a TRKA enzymatic assay. Of these, 39
showed high enzymatic potency against TRKA, approximately 15-
fold selectivity over AURA and AURB, and potent cellular activity
with an IC50 value of 56.4 nM against the KM12 human CRC cell line
expressing the chimeric TPM3-TRKA protein. Molecular docking
studies revealed that 39 conducted a type II binding mode, which
especially interacted with the His648 in the back pockets of TRKA
kinase e a crucial interaction stabilizes the DFG-out conformation.
More importantly, 39 exhibited significantly improved drug-like
properties (NRB and PSA) compared to our previously reported
candidate BPR1K871 (10), as well as good AUC and oral bioavail-
ability (F ¼ 27.9% for mice and 27.2% for rats), and excellent in vivo
efficacy (TGI of 64% at day 18) in a KM12 mice xenograft model.
Fig. 9. In vivo antitumor efficacy of compound 39 and larotrectinib (1) in the KM12
xenograft mouse model. Compound 39 was orally administered to the mice at a dosage
of 100 mg/kg once a day for three days (days 1e3), whereupon the dosage was reduced
to 75 mg/kg due to observed body weight loss (days 4e5); then the optimum dosage
and dosing schedule were finally determined to be three times per week at 75 mg/kg.
Larotrectinib (1) at a dosage of 75 mg/kg dosage was utilized as a positive control.
Finally,
39
demonstrated
broad-spectrum
potency
reactions were carried out under dry nitrogen or argon atmosphere
and were monitored for completion by TLC using Merck 60 F254
silica gel glass-backed plates; zones were detected visually under
UV irradiation (254 nm) or by spraying with potassium perman-
ganate reagent (Aldrich) followed by heating at 80 ꢀC. Flash column
chromatography was carried out using silica gel (Silicycle Silia-
Flash® P60, R12030B, 230e400 mesh). 1H and 13C NMR spectra
were recorded with Varian Mercury-300 or Varian Mercury-400
spectrometers or Bruker 400 MHz or 600 MHz AVANCE III spec-
trometers and the chemical shifts were recorded in parts per
(IC50 ¼ 3.74e151.4 nM) against mutant TRKAs and TRKCs, and, in
particular, was 3e65 fold more potent an inhibitor than laro-
trectinib (1), especially of double-mutant TRKAs. Accordingly, 39 is
considered a next-generation orally selective type II TRK inhibitor
as a potential candidate for the treatment of rare cancers incor-
porating NTRK gene fusions.
5. Experimental section
million (ppm, d) and reported relative to the solvent peak. Data
5.1. General methods for chemistry
analysis was done using Mnova software. Low-resolution mass
spectra (LRMS) data were measured with Agilent MSD-1100 ESI-
MS/MS system. High-resolution mass spectra (HRMS) data were
All commercial chemicals and solvents are of reagent grade and
were used without further purification unless otherwise stated. All
Table 6
Pharmacokinetic profile of compound 39 in mice and rats.
Species
i.v.
p.o.
t1/2 (h)
CL (ml/min/kg)
Vss (l/kg)
AUC(0-inf)
t1/2 (h)
Cmax (ng/ml)
Tmax (h)
AUC(0-inf)
F (%)
(ng/mL ꢂ hr)
(ng/mL ꢂ hr)
micea
ratb
2.9
5.3
7.4
12.2
0.6
2.3
5081
2772
2.2
5.1
2406
454.7
1.7
4.3
5793
3012
27.9
27.2
a
The dosage of i.v. is 1 mg/kg and the dosage of p.o. is 10 mg/kg.
The dosage of i.v. is 5 mg/kg and the dosage of p.o. is 20 mg/kg.
b
9

M.-C. Li, W.-H. Lin, P.-C. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113673
measured with Varian 901-MS FT-ICR HPLC/MS-MS system. Purity
of the final compounds were determined with a Hitachi 2000 series
HPLC system using C-18 column (Agilent ZORBAX Eclipse XDB-C18
yield as pale orange solid. 1H NMR (300 MHz, DMSO‑d6)
d 8.41 (s,
1H), 8.13e8.00 (m, 2H), 7.24 (d, J ¼ 8.4 Hz, 1H), 6.90 (d, J ¼ 8.3 Hz,
2H), 6.49 (d, J ¼ 8.3 Hz, 2H), 4.86 (s, 2H), 4.24 (q, J ¼ 7.1 Hz, 2H), 3.62
(td, J ¼ 8.1, 6.3 Hz, 2H), 2.74 (t, J ¼ 8.1 Hz, 2H), 1.41 (t, J ¼ 7.1 Hz, 3H).
LC-MS (ESI) m/z: 327.2 [MþH]þ.
5
m
m. 4.6 mm ꢂ 150 mm) operating at 25 ꢀC. The elution was
carried out using acetonitrile as mobile phase A, and water con-
taining 0.1% formic acid þ 2 mmol NH4OAc as mobile phase B.
Elution conditions: at 0 min, phase A 10% þ phase B 90%; at 25 min,
phase A 90% þ phase B 10%; at 30 min, phase A 90% þ phase B 10%;
at 30.5 min, phase A 10% þ phase B 90%; at 37 min, phase A
10% þ phase B 90%. The flow-rate of the mobile phase was 0.5 mL/
N-(4-Aminobenzyl)-7-ethoxy-6-fluoroquinazolin-4-amine (19d0).
The title compound was synthesized following the general pro-
cedure A and using the reactants/reagents 11d (150 mg, 0.66 mmol,
1.0 equiv.), 4-(aminomethyl)aniline (86 mg, 0.80 mmol, 1.2 equiv.)
and ethanol (3.0 mL). After stirred at reflux for 12 h and work-up,
the title compound 19d’ was obtained in 73% yield as khaki solid.
min and the injection volume of the sample was 10 or 20 mL. Peaks
were detected at 254 nm. IUPAC nomenclature of compounds were
obtained with the software ACD/Name Pro.
1H NMR (400 MHz, DMSO‑d6)
d 8.43e8.32 (m, 2H), 8.15 (d,
J ¼ 12.4 Hz, 1H), 7.25 (d, J ¼ 8.4 Hz, 1H), 7.02 (d, J ¼ 8.2 Hz, 2H), 6.50
(d, J ¼ 8.2 Hz, 2H), 4.97 (br s, 2H), 4.56 (d, J ¼ 5.6 Hz, 2H), 4.24 (q,
J ¼ 7.0 Hz, 2H), 1.40 (t, J ¼ 7.0 Hz, 3H). LC-MS (ESI) m/z: 313.3
[MþH]þ.
General Procedure A. To a solution of the 4-chloropyrimidine
derivatives 11aef (1.0 equiv.) in appropriate alcohol type solution
(1.0e20.0 mL) was added appropriate aniline (1.1e3.0 equiv.) and
triethylamine (0e1.9 equiv.) then the reaction mixture was stirred
at reflux. After stirred for 1e12 h, the reaction mixture was cooled
down to room temperature, concentrated in vacuo and purified by
silica gel column chromatography using the appropriate mobile
phase to yield the title compounds 19aef.
General Procedure B. To a solution of amine derivatives 19aef
(1.0 equiv.) in dichloromethane (0.5e30 mL) or N,N-dime-
thylformamide (1 mL) and was added appropriate isocyanate
(1.0e2.1 equiv.) and methanol (0e0.20 mL) then the reaction
mixture was stirred at room temperature. After stirred for 1e16 h,
the resulting precipitate was collected, washed with dichloro-
methane (5e20 mL) and dried in vacuo to yield the title compounds
20e23, 25e39 in 28e98% yield.
N-(7-Ethoxy-6-fluoroquinazolin-4-yl)benzene-1,4-diamine
(19d’’). The title compound was synthesized following the general
procedure A and using the reactants/reagents 11d (150 mg,
0.66 mmol, 1.0 equiv.), benzene-1,4-diamine (79 mg, 0.73 mmol, 1.1
equiv.) and propan-2-ol (1.0 mL). After stirred at reflux for 1 h and
work-up, the title compound 19d’’ was obtained in 97% yield as
khaki solid. 1H NMR (400 MHz, DMSO‑d6)
d 8.68 (s, 1H), 8.52 (d,
J ¼ 11.6 Hz, 1H), 7.43 (d, J ¼ 8.0 Hz, 2H), 7.35 (d, J ¼ 8.0 Hz, 1H), 6.78
(d, J ¼ 8.4 Hz, 2H), 4.31 (q, J ¼ 7.0 Hz, 2H), 1.45 (t, J ¼ 7.0 Hz, 3H). LC-
MS (ESI) m/z: 299.1 [MþH]þ.
N-[2-(4-Aminophenyl)ethyl]-6,7-difluoroquinazolin-4-amine
(19d’’). The title compound was synthesized following the general
procedure A and using the reactants/reagents 11e (310 mg,
1.55 mmol, 1.0 equiv.), 4-(2-aminoethyl)aniline (634 mg,
4.66 mmol, 3.0 equiv.), triethylamine (305 mg, 3.01 mmol, 1.9
equiv.) and propan-2-ol (1.3 mL). After stirred at reflux for 1 h and
work-up, the title compound 19e was obtained in 85% yield as
1-(5-{2-[(6,7-Dimethoxyquinazolin-4-yl)amino]ethyl}-1,3-
thiazol-2-yl)-3-phenylurea (16). To a solution of 13c (208 mg,
0.63 mmol, 1.0 equiv.) in dichloromethane (3.0 mL) was added
phenyl isocyanate (97 mg, 0.82 mmol, 1.3 equiv.) and methanol
(0.3 mL) then the reaction mixture was stirred at room tempera-
ture. After stirred for 4 h, the reaction mixture was concentrated in
vacuo and purified by column chromatography (2e5% methanol/
dichloromethane) to yield the title compound 16 (165 mg,
0.37 mmol, 58%) as white solid. 1H NMR (400 MHz, DMSO‑d6)
white solid. 1H NMR (400 MHz, DMSO‑d6)
d 8.48 (s, 1H), 8.38e8.29
(m, 2H), 7.69 (dd, J ¼ 12.0, 8.0 Hz, 1H), 6.90 (d, J ¼ 8.2 Hz, 2H), 6.49
(d, J ¼ 8.2 Hz, 2H), 4.87 (s, 2H), 3.64 (td, J ¼ 8.2, 6.0 Hz, 2H), 2.76 (t,
J ¼ 8.2 Hz, 2H). LC-MS (ESI) m/z: 301.1 [MþH]þ.
N-[2-(4-Aminophenyl)ethyl]-6-ethoxy-7-fluoroquinazolin-4-
amine (19f). The title compound was synthesized following the
general procedure A and using the reactants/reagents 11f (165 mg,
0.73 mmol, 1.0 equiv.), 4-(2-aminoethyl)aniline (119 mg,
0.87 mmol, 1.2 equiv.) and ethanol (3.6 mL). After stirred at reflux
for 1 h and work-up, the title compound 19f was obtained in 72%
d
10.34 (br s,1H), 8.97 (s,1H), 8.37 (s,1H), 8.11 (t, J ¼ 5.6 Hz,1H), 7.59
(s, 1H), 7.45 (d, J ¼ 8.0 Hz, 1H), 7.31 (dd, J ¼ 8.0, 7.4 Hz, 2H), 7.13 (s,
1H), 7.10 (s, 1H), 7.02 (t, J ¼ 7.4 Hz, 1H), 3.90 (s, 3H), 3.89 (s, 3H), 3.41
(td, J ¼ 7.0, 5.6 Hz, 2H), 3.08 (t, J ¼ 7.0 Hz, 2H). 13C NMR (150 MHz,
DMSO‑d6)
d 158.4, 154.2, 152.9, 151.9, 148.7, 144.3, 138.9, 134.0,
129.0,127.7,122.7,118.6,108.4,106.0,102.2, 56.1, 55.8, 42.0, 26.0. LC-
yield as pale yellow solid. 1H NMR (400 MHz, DMSO‑d6)
d 8.39 (s,
MS (ESI) m/z: 451.2 [MþH]þ. HRMS (ESI) m/z: calcd for
1H), 8.22 (t, J ¼ 5.2 Hz, 1H), 7.82 (d, J ¼ 9.2 Hz, 1H), 7.45 (d,
J ¼ 12.4 Hz, 1H), 6.91 (d, J ¼ 8.0 Hz, 2H), 6.50 (d, J ¼ 8.0 Hz, 2H), 4.87
(s, 2H), 4.21 (q, J ¼ 6.8 Hz, 2H), 3.64 (td, J ¼ 8.2, 6.0 Hz, 2H), 2.77 (t,
J ¼ 8.2 Hz, 2H), 1.43 (t, J ¼ 6.8 Hz, 3H). LC-MS (ESI) m/z: 327.2
[MþH]þ.
C
22H23N6O3S, 451.1552 [MþH]þ, found 451.1560. HPLC purity:
99.5%, tR ¼ 13.97 min.
N-[2-(4-Aminophenyl)ethyl]-7-(3-chloropropoxy)quinazolin-4-
amine (19a). The title compound was synthesized following the
general procedure A and using the reactants/reagents 11a (200 mg,
0.78 mmol, 1.0 equiv.), 4-(2-aminoethyl)aniline (127 mg,
0.93 mmol, 1.2 equiv.), triethylamine (118 mg, 1.17 mmol, 1.5 equiv.)
and ethanol (5.0 mL). After stirred at reflux for 1 h and work-up, the
title compound 19a was obtained in 40% yield as light yellow solid.
1-[4-(2-{[7-(3-Chloropropoxy)quinazolin-4-yl]amino}ethyl)
phenyl]-3-phenylurea (20). The title compound was synthesized
following the general procedure B and using the reactants/reagents
19a (2.00 g, 5.60 mmol, 1.0 equiv.), phenyl isocyanate (1.33 g,
11.2 mmol, 2.0 equiv.) and dichloromethane (30 mL). After stirred at
room temperature overnight and work-up, the title compound 20
was obtained in 71% yield as light yellow solid. 1H NMR (300 MHz,
1H NMR (300 MHz, CD3OD-d4)
d
8.37 (s,1H), 7.95 (dd, J ¼ 9.0, 0.6 Hz,
1H), 7.11 (dd, J ¼ 9.0, 2.6 Hz, 1H), 7.07 (d, J ¼ 2.6 Hz, 1H), 7.01 (d,
J ¼ 8.6 Hz, 2H), 6.68 (d, J ¼ 8.6 Hz, 2H), 4.25 (t, J ¼ 6.0 Hz, 2H),
3.84e3.69 (m, 4H), 2.87 (t, J ¼ 7.5 Hz, 2H), 2.29 (tt, J ¼ 6.0, 6.0 Hz,
2H). LC-MS (ESI) m/z: 357.1 [MþH]þ.
DMSO‑d6)
d
8.66 (s, 1H), 8.62 (s, 1H), 8.42 (s, 1H), 8.20 (t, J ¼ 6.0 Hz,
1H), 8.13 (d, J ¼ 8.4 Hz, 1H), 7.44 (d, J ¼ 8.6, 1.1 Hz, 2H), 7.37 (d,
J ¼ 8.6 Hz, 2H), 7.27 (dd, J ¼ 8.6, 7.5 Hz, 2H), 7.16 (d, J ¼ 8.6 Hz, 2H),
7.14 (d, J ¼ 2.7 Hz, 1H), 7.10 (dd, J ¼ 8.4, 2.7 Hz, 1H), 6.95 (tt, J ¼ 7.5,
1.1 Hz, 1H), 4.23 (t, J ¼ 6.2 Hz, 2H), 3.82 (t, J ¼ 6.5 Hz, 2H), 3.70 (td,
J ¼ 7.8, 6.0 Hz, 2H), 2.89 (t, J ¼ 7.8 Hz, 2H), 2.22 (tt, J ¼ 6.5, 6.2 Hz,
2H). LC-MS (ESI) m/z: 476.2 [MþH]þ.
N-[2-(4-Aminophenyl)ethyl]-7-ethoxy-6-fluoroquinazolin-4-
amine (19d). The title compound was synthesized following the
general procedure A and using the reactants/reagents 11d (453 mg,
2.00 mmol, 1.0 equiv.), 4-(2-aminoethyl)aniline (331 mg,
2.43 mmol, 1.2 equiv.) and ethanol (10.0 mL). After stirred at reflux
for 1 h and work-up, the title compound 19d was obtained in 60%
1-Phenyl-3-{4-[2-(quinazolin-4-ylamino)ethyl]phenyl}urea (21).
The title compound was synthesized following the general
10

M.-C. Li, W.-H. Lin, P.-C. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113673
procedure B and using the reactants/reagents 19b (200 mg,
0.76 mmol, 1.0 equiv.), isocyanatobenzene (135 mg, 1.13 mmol, 1.5
equiv.) and dichloromethane (20 mL). After stirred for 8 h at room
temperature and work-up, the title compound 21 was obtained in
2H), 1.41 (t, J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6)
d 158.7 (d,
JC-F ¼ 4.3 Hz), 155.1, 152.5, 151.1 (d, JC-F ¼ 12.9 Hz), 150.2 (d, JC-
¼ 245.5 Hz), 148.0, 139.7, 138.5, 132.7, 128.7, 127.9, 121.7, 118.2,
F
118.1, 109.7, 107.9 (d, JC-F ¼ 6.3 Hz), 107.8, 64.6, 43.2, 14.3. LC-MS
(ESI) m/z: 432.4 [MþH]þ. HRMS (ESI) m/z: calcd for C24H23FN5O2,
432.1836 [MþH]þ, found 432.1835. HPLC purity: 90.9%,
tR ¼ 16.57 min.
98% yield as pale yellow solid. 1H NMR (400 MHz, DMSO‑d6)
d 8.61
(s, 1H), 8.58 (s, 1H), 8.49 (s, 1H), 8.38 (t, J ¼ 6.0 Hz, 1H), 8.21 (d,
J ¼ 8.0 Hz, 1H), 7.78 (ddd, J ¼ 8.0, 6.8, 1.2 Hz, 1H), 7.67 (dd, J ¼ 8.4,
1.2 Hz, 1H), 7.50 (ddd, J ¼ 8.4, 6.8, 1.2 Hz, 1H), 7.44 (d, J ¼ 7.8 Hz, 2H),
7.37 (d, J ¼ 8.4 Hz, 2H), 7.27 (dd, J ¼ 7.8, 7.6 Hz, 2H), 7.18 (d,
J ¼ 8.4 Hz, 2H), 6.95 (t, J ¼ 7.6 Hz, 1H), 3.73 (td, J ¼ 7.6, 6.0 Hz, 2H),
1-(3-Chlorophenyl)-3-(4-{[(7-ethoxy-6-fluoroquinazolin-4-yl)
amino]methyl}phenyl)urea (26). The title compound was synthe-
sized following the general procedure B and using the reactants/
reagents 19d’ (50 mg, 0.16 mmol, 1.0 equiv.), 1-chloro-3-
isocyanatobenzene (25 mg, 0.16 mmol, 1.0 equiv.) and dichloro-
methane (1 mL). After stirred for 16 h at room temperature and
work-up, the title compound 26 was obtained in 90% yield as pale
2.91 (t, J ¼ 7.6 Hz, 2H). 13C NMR (150 MHz, DMSO‑d6)
d 159.3, 155.2,
152.6, 149.1, 139.8, 137.8, 132.9, 132.4, 129.0, 128.8, 127.5, 125.5,
122.6, 121.7, 118.4, 118.2, 115.0, 42.3, 33.9. LC-MS (ESI) m/z: 384.3
[MþH]þ. HRMS (ESI) m/z: calcd for C23H14N1O5, 384.0872 [MþH]þ,
found 384.0871. HPLC purity: 97.9%, tR ¼ 20.82 min.
yellow solid. 1H NMR (400 MHz, DMSO‑d6)
d 8.87 (s, 1H), 8.75 (s,
1-(3-Chlorophenyl)-3-(4-{2-[(7-ethoxy-6-fluoroquinazolin-4-yl)
amino]ethyl}phenyl)urea (23). The title compound was synthesized
following the general procedure B and using the reactants/reagents
19d (130 mg, 0.40 mmol, 1.0 equiv.), 1-chloro-3-isocyanatobenzene
(88 mg, 0.58 mmol, 1.4 equiv.), methanol (0.13 mL) and dichloro-
methane (13 mL). After work-up, the title compound 23 was ob-
tained in 46% yield as white solid. 1H NMR (400 MHz, DMSO‑d6)
1H), 8.52 (t, J ¼ 5.8 Hz, 1H), 8.41 (s, 1H), 8.15 (d, J ¼ 12.4 Hz, 1H), 7.71
(dd, J ¼ 2.0, 1.6 Hz, 1H), 7.41 (d, J ¼ 8.8 Hz, 2H), 7.33e7.20 (m, 5H),
6.82 (ddd, J ¼ 6.8, 2.0, 2.0 Hz, 1H), 4.70 (d, J ¼ 5.8 Hz, 2H), 4.22 (q,
J ¼ 7.0 Hz, 2H), 1.40 (t, J ¼ 7.0 Hz, 3H). 13C NMR (150 MHz, DMSO‑d6)
d
158.7 (d, JC-F ¼ 2.1 Hz), 155.0, 152.4, 151.2 (d, JC-F ¼ 12.8 Hz), 150.2
(d, JC-F ¼ 245.4 Hz), 148.0, 141.3, 138.2, 133.2, 133.0, 130.3, 127.9,
121.4, 118.4, 117.5, 116.6, 109.7, 108.0 (d, JC-F ¼ 8.4 Hz), 107.9 (d, JC-
d
8.87 (s, 1H), 8.70 (s, 1H), 8.43 (s, 1H), 8.14e8.02 (m, 2H), 7.71 (dd,
¼ 20.6 Hz), 64.6, 43.2, 14.3. LC-MS (ESI) m/z: 466.4 [MþH]þ. HRMS
F
J ¼ 2.4, 1.6 Hz, 1H) 7.38 (d, J ¼ 8.6 Hz, 2H), 7.34e7.24 (m, 3H), 7.18 (d,
J ¼ 8.6 Hz, 1H), 7.00 (ddd, J ¼ 8.0, 1.6, 1.6 Hz, 1H), 4.24 (q, J ¼ 7.0 Hz,
2H), 3.70 (td, J ¼ 7.8, 6.0 Hz, 2H), 2.89 (t, J ¼ 7.8 Hz, 2H), 1.41 (t,
(ESI) m/z: calcd for C24H22ClFN5O2, 466.1446 [MþH]þ, found
466.1446. HPLC purity: 95.2%, tR ¼ 18.60 min.
1-{4-[(7-Ethoxy-6-fluoroquinazolin-4-yl)amino]phenyl}-3-
J ¼ 7.0 Hz, 3H). 13C NMR (150 MHz, DMSO‑d6)
d
158.7 (d, JC-
phenylurea (27). The title compound was synthesized following the
general procedure B and using the reactants/reagents 19d’’ (33 mg,
0.11 mmol, 1.0 equiv.), isocyanatobenzene (27 mg, 0.23 mmol, 2.1
equiv.) and dichloromethane (0.5 mL). After stirred for 16 h at room
temperature and work-up, the title compound 27 was obtained in
¼ 3.5 Hz), 155.1, 152.4, 151.0 (d, JC-F ¼ 12.8 Hz), 150.1 (d, JC-
¼ 245.1 Hz), 147.8, 141.3, 137.5, 133.18, 133.16, 130.3, 129.0, 121.3,
F
F
118.5, 117.5, 116.5, 109.7, 108.0 (d, JC-F ¼ 7.4 Hz), 107.7 (d, JC-
¼ 20.0 Hz), 64.5, 42.2, 33.9, 14.3. LC-MS (ESI) m/z: 480.2 [MþH]þ.
F
HRMS (ESI) m/z: calcd for C25H24ClFN5O2, 480.1603 [MþH]þ, found
480.1600. HPLC purity: 99.3%, tR ¼ 18.95 min.
28% yield as pale yellow solid. 1H NMR (400 MHz, DMSO‑d6)
d 9.51
(s, 1H), 8.66 (s, 1H), 8.65 (s, 1H), 8.49 (s, 1H), 8.40 (d, J ¼ 12.8 Hz,1H),
7.69 (d, J ¼ 8.8 Hz, 2H), 7.50e7.42 (m, 4H), 7.34 (d, J ¼ 8.4 Hz, 1H),
7.28 (dd, J ¼ 8.0, 7.6 Hz, 2H), 6.97 (t, J ¼ 7.6 Hz, 1H), 4.28 (q,
J ¼ 7.0 Hz, 2H), 1.43 (t, J ¼ 7.0 Hz, 3H). 13C NMR (150 MHz, DMSO‑d6)
1-{4-[2-({7-[3-(Dimethylamino)propoxy]quinazolin-4-yl}amino)
ethyl]phenyl}-3-phenylurea (24). To
a solution of 20 (1.46 g,
3.07 mmol, 1.0 equiv.) in N,N-dimethylacetamide (10 mL) was
added a solution of dimethylamine in water (6.91 mg, 61.34 mmol,
20.0 equiv.) then the reaction was heated at 120 ꢀC under micro-
wave irradiation. After stirred for 100 min, the reaction mixture
was cooled down and poured into an ice bath. The resulting pre-
cipitate was collected, washed with water (50 mL) and acetone
(20 mL). Then the crude product was purified by silica gel column
chromatography to yield the title compound 24 in 64% yield as
d
157.1, 154.6, 152.6, 151.4 (d, JC-F ¼ 13.1 Hz), 150.5 (d, JC-
¼ 245.6 Hz), 148.5, 139.8, 135.7, 133.2, 128.8, 123.0, 121.7, 118.3,
F
118.1, 109.9, 108.2 (d, JC-F ¼ 7.7 Hz), 108.0 (d, JC-F ¼ 20.3 Hz), 64.7,
14.3. LC-MS (ESI) m/z: 418.4 [MþH]þ. HRMS (ESI) m/z: calcd for
C
23H21FN5O2, 418.1679 [MþH]þ, found 418.1786. HPLC purity:
98.9%, tR ¼ 16.57 min.
1-(3-Chlorophenyl)-3-{4-[(7-ethoxy-6-fluoroquinazolin-4-yl)
amino]phenyl}urea (28). The title compound was synthesized
following the general procedure B and using the reactants/reagents
19d’’ (100 mg, 0.34 mmol, 1.0 equiv.), 1-chloro-3-
isocyanatobenzene (63 mg, 0.41 mmol, 1.2 equiv.) and dichloro-
methane (1 mL). After stirred for 2 h at room temperature and
work-up, the title compound 28 was obtained in 46% yield as pale
white solid. 1H NMR (400 MHz, DMSO‑d6)
d 8.69 (s, 1H), 8.65 (s, 1H),
8.41 (s, 1H), 8.19 (t, J ¼ 5.8 Hz, 1H), 8.11 (d, J ¼ 9.0 Hz, 1H), 7.44 (dd,
J ¼ 8.6, 1.2 Hz, 2H), 7.37 (d, J ¼ 8.4 Hz, 2H), 7.27 (dd, J ¼ 8.6, 7.2 Hz,
2H), 7.17 (d, J ¼ 8.4 Hz, 2H), 7.10 (dd, J ¼ 9.0, 2.8 Hz, 1H), 7.04 (d,
J ¼ 2.8 Hz, 1H), 6.95 (tt, J ¼ 7.2, 1.2 Hz, 1H), 4.12 (t, J ¼ 6.6 Hz, 2H),
3.70 (td, J ¼ 8.0, 5.8 Hz, 2H), 2.88 (t, J ¼ 8.0 Hz, 2H), 2.38 (t, J ¼ 7.0 Hz,
2H), 2.16 (s, 6H), 1.89 (tt, J ¼ 7.0, 6.6 Hz, 2H). 13C NMR (150 MHz,
yellow solid. 1H NMR (400 MHz, DMSO‑d6)
d 9.56 (s, 1H), 8.94 (s,
DMSO‑d6)
d
161.6, 159.0, 155.7, 152.6,151.4, 139.8, 137.8, 132.9,129.0,
1H), 8.80 (s, 1H), 8.51 (s, 1H), 8.42 (d, J ¼ 12.8 Hz, 2H), 7.75e7.67 (m,
3H), 7.47 (d, J ¼ 8.8 Hz, 2H), 7.38e7.24 (m, 3H), 7.01 (ddd, J ¼ 7.2, 2.0,
2.0 Hz, 1H), 4.29 (q, J ¼ 6.8 Hz, 2H), 1.43 (t, J ¼ 6.8 Hz, 3H). 13C NMR
128.7, 124.2, 121.7, 118.3, 118.1, 116.8, 109.1, 107.5, 66.0, 55.5, 45.0,
42.2, 34.0, 26.6. LC-MS (ESI) m/z: 485.3 [MþH]þ. HRMS (ESI) m/z:
calcd for C28H33N6O2, 485.2665 [MþH]þ, found 485.2663. HPLC
purity: 98.2%, tR ¼ 17.35 min.
(150 MHz, DMSO‑d6)
d
157.1, 154.4, 152.5, 151.4 (d, JC-F ¼ 13.1 Hz),
150.5 (d, JC-F ¼ 245.9 Hz), 148.3, 141.4, 135.4, 133.4, 133.2, 123.0,
121.3, 118.5, 117.4, 116.5, 109.7, 108.2, 108.1 (d, JC-F ¼ 20.6 Hz), 64.7,
14.3. LC-MS (ESI) m/z: 452.3 [MþH]þ. HRMS (ESI) m/z: calcd for
1-(4-{[(7-Ethoxy-6-fluoroquinazolin-4-yl)amino]methyl}phenyl)-
3-phenylurea (25). The title compound was synthesized following
the general procedure B and using the reactants/reagents 19d’
(70 mg, 0.22 mmol, 1.0 equiv.), isocyanatobenzene (55 mg,
0.46 mmol, 2.1 equiv.) and dichloromethane (1 mL). After stirred for
16 h at room temperature and work-up, the title compound 25 was
obtained in 43% yield as creamy-white solid. 1H NMR (400 MHz,
C
23H20ClFN5O2, 452.1290 [MþH]þ, found 452.1323. HPLC purity:
97.1%, tR ¼ 19.59 min.
1-(3-Chlorophenyl)-3-(4-{2-[(6,7-difluoroquinazolin-4-yl)amino]
ethyl}phenyl)urea (29). The title compound was synthesized
following the general procedure B and using the reactants/reagents
19e (60 mg, 0.20 mmol, 1.0 equiv.), isocyanatobenzene (29 mg,
0.24 mmol, 1.2 equiv.) and dichloromethane (5.0 mL). After stirred
for 90 min at room temperature and work-up, the title compound
DMSO‑d6)
d
8.65 (s, 1H), 8.61 (s, 1H), 8.51 (t, J ¼ 5.6 Hz, 1H), 8.40 (s,
1H), 8.16 (d, J ¼ 12.4 Hz, 1H), 7.46e7.36 (m, 4H), 7.31e7.22 (m, 5H),
6.95 (t, J ¼ 7.2 Hz, 1H), 4.69 (d, J ¼ 5.6 Hz, 2H), 4.25 (q, J ¼ 6.8 Hz,
11

M.-C. Li, W.-H. Lin, P.-C. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113673
29 was obtained in 88% yield as white solid. 1H NMR (400 MHz,
HPLC purity: 98.3%, tR ¼ 19.37 min.
DMSO‑d6)
d
8.70 (s, 1H), 8.67 (s, 1H), 8.50 (s, 1H), 8.41e8.29 (m, 2H),
1-(4-{2-[(7-Ethoxy-6-fluoroquinazolin-4-yl)amino]ethyl}phenyl)-
3-phenylurea (33). The title compound was synthesized following
the general procedure B and using the reactants/reagents 19d
(98 mg, 0.30 mmol, 1.0 equiv.), isocyanatobenzene (55 mg,
0.46 mmol, 1.5 equiv.), methanol (0.09 mL) and dichloromethane
(9 mL). After stirred for 2 h at room temperature and work-up, the
title compound 33 was obtained in 97% yield as white solid. 1H NMR
7.70 (dd, J ¼ 11.6, 8.0 Hz, 1H) 7.44 (d, J ¼ 7.6 Hz, 2H), 7.38 (d,
J ¼ 8.4 Hz, 2H), 7.26 (dd, J ¼ 7.8, 7.6 Hz, 2H), 7.17 (d, J ¼ 8.4 Hz, 2H),
6.95 (t, J ¼ 7.8 Hz,1H), 3.72 (td, J ¼ 7.8, 6.4 Hz, 2H), 2.89 (t, J ¼ 7.8 Hz,
2H). 13C NMR (150 MHz, DMSO‑d6)
d 158.8, 155.8, 152.9 (dd, JC-
¼ 252.0,15.2 Hz),152.5,147.8 (dd, JC-F ¼ 246.0,14.2 Hz),147.3 (d, JC-
F
¼ 11.3 Hz), 139.8, 137.9, 132.6, 129.0, 128.7, 121.7, 118.3, 118.1, 114.4
F
(d, JC-F ¼ 16.1 Hz), 111.6 (d, JC-F ¼ 6.6 Hz), 110.1 (d, JC-F ¼ 18.6 Hz),
42.3, 33.7. LC-MS (ESI) m/z: 420.2 [MþH]þ. HRMS (ESI) m/z: calcd
for C23H20F2N5O, 420.1636 [MþH]þ, found 420.1622. HPLC purity:
95.9%, tR ¼ 17.77 min.
(400 MHz, DMSO‑d6) d 8.61 (s, 1H), 8.58 (s, 1H), 8.43 (s, 1H),
8.13e8.04 (m, 2H), 7.44 (dd, J ¼ 8.8, 1.4 Hz, 2H), 7.37 (d, J ¼ 8.4 Hz,
2H), 7.30e7.23 (m, 3H), 7.17 (d, J ¼ 8.4 Hz, 2H), 6.95 (tt, J ¼ 7.6,
1.2 Hz, 1H), 4.24 (q, J ¼ 7.2 Hz, 2H), 3.70 (td, J ¼ 7.8, 6.4 Hz, 2H), 2.88
(t, J ¼ 7.8 Hz, 2H), 1.41 (t, J ¼ 7.2 Hz, 3H). 13C NMR (100 MHz,
1-(4-{2-[(6,7-Difluoroquinazolin-4-yl)amino]ethyl}phenyl)-3-
phenylurea (30). The title compound was synthesized following the
general procedure B and using the reactants/reagents 19e (60 mg,
0.20 mmol, 1.0 equiv.), 1-chloro-3-isocyanatobenzene (37 mg,
0.24 mmol, 1.2 equiv.) and dichloromethane (6 mL). After stirred for
90 min at room temperature and work-up, the title compound 30
was obtained in 99% yield as white solid. 1H NMR (400 MHz,
DMSO‑d6)
d
158.8 (d, JC-F ¼ 3.8 Hz), 155.1, 152.6, 151.1 (d, JC-
¼ 12.9 Hz), 150.2 (d, JC-F ¼ 245.2 Hz), 147.9, 139.8, 137.8, 132.9,
F
129.0, 128.8, 121.8, 118.4, 118.2, 109.7, 108.0 (d, JC-F ¼ 6.5 Hz), 107.8
(d, JC-F ¼ 20.1 Hz), 64.6, 42.3, 34.0, 14.3. LC-MS (ESI) m/z: 446.2
[MþH]þ. HRMS (ESI) m/z: calcd for C25H25FN5O2, 446.1992 [MþH]þ,
found 446.1986. HPLC purity: 99.4%, tR ¼ 17.08 min.
DMSO‑d6)
d
8.89 (s, 1H), 8.72 (s, 1H), 8.50 (s, 1H), 8.41e8.29 (m, 2H),
1-(4-{2-[(7-Ethoxy-6-fluoroquinazolin-4-yl)amino]ethyl}phenyl)-
3-(3-fluorophenyl)urea (34). The title compound was synthesized
following the general procedure B and using the reactants/reagents
19d (98 mg, 0.30 mmol, 1.0 equiv.), 1-fluoro-3-isocyanatobenzene
(60 mg, 0.44 mmol, 1.5 equiv.), methanol (0.09 mL) and dichloro-
methane (9 mL). After stirred for 2 h at room temperature and
work-up, the title compound 34 was obtained in 84% yield as white
7.74e7.66 (m, 2H), 7.38 (d, J ¼ 8.4 Hz, 2H) 7.32e7.22 (m, 2H), 7.18 (d,
J ¼ 8.4 Hz, 2H), 7.00 (ddd, J ¼ 7.6, 2.0, 2.0 Hz, 1H), 3.72 (td, J ¼ 7.6,
6.0 Hz, 2H), 2.90 (t, J ¼ 7.6 Hz, 2H). 13C NMR (150 MHz, DMSO‑d6)
d
158.8, 155.8, 152.9 (dd, JC-F ¼ 251.8, 15.2 Hz), 152.4, 147.8 (dd, JC-
¼ 254.2, 14.5 Hz), 147.3 (d, JC-F ¼ 11.0 Hz), 141.3, 137.6, 133.2, 133.0,
F
130.3, 129.0, 121.3, 118.5, 117.4, 116.5, 114.4 (d, JC-F ¼ 15.9 Hz), 111.6
(d, JC-F ¼ 6.0 Hz),110.1 (d, JC-F ¼ 18.8 Hz), 42.3, 33.7. LC-MS (ESI) m/z:
454.2 [MþH]þ. HRMS (ESI) m/z: calcd for C23H19ClF2N5O, 454.1246
[MþH]þ, found 454.1254. HPLC purity: 94.2%, tR ¼ 20.77 min.
1-(4-{2-[(6-Ethoxy-7-fluoroquinazolin-4-yl)amino]ethyl}phenyl)-
3-phenylurea (31). The title compound was synthesized following
the general procedure B and using the reactants/reagents 19f
(114 mg, 0.35 mmol, 1.0 equiv.), phenyl isocyanate (55 mg,
0.46 mmol, 1.3 equiv.), methanol (0.1 mL) and dichloromethane
(10 mL). After stirred at room temperature for 1 h and work-up, the
title compound 31 was obtained in 87% yield as white solid. 1H NMR
solid. 1H NMR (400 MHz, DMSO‑d6)
d 8.87 (s, 1H), 8.67 (s, 1H), 8.43
(s, 1H), 8.16e8.02 (m, 2H), 7.48 (ddd, J ¼ 12.0, 2.6, 2.2 Hz, 1H), 7.37
(d, J ¼ 8.4 Hz, 1H), 7.34e7.22 (m, 2H), 7.18 (d, J ¼ 8.4 Hz, 2H), 7.10
(ddd, J ¼ 8.4, 2.2, 2.2 Hz,1H), 6.77 (dddd, J ¼ 8.8, 8.4, 2.6, 2.2 Hz,1H),
4.24 (q, J ¼ 7.0 Hz, 2H), 3.70 (td, J ¼ 8.0, 6.0 Hz, 2H), 2.88 (t,
J ¼ 8.0 Hz, 2H), 1.41 (t, J ¼ 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6)
d
162.5 (d, JC-F ¼ 239.6 Hz), 158.8, 155.1, 152.4, 151.1 (d, JC-
¼ 12.8 Hz), 150.2 (d, JC-F ¼ 145.1 Hz), 147.9, 141.7 (d, JC-F ¼ 11.5 Hz),
F
137.6,133.2,130.3 (d, JC-F ¼ 9.7 Hz),129.1,118.6,113.9,109.7,108.1 (d,
JC-F ¼ 21.6 Hz),107.8 (d, JC-F ¼ 23.7 Hz),104.8 (d, JC-F ¼ 25.9 Hz), 64.6,
42.3, 34.0, 14.3. LC-MS (ESI) m/z: 464.2 [MþH]þ. HRMS (ESI) m/z:
calcd for C25H24F2N5O2, 464.1898 [MþH]þ, found 464.1860. HPLC
purity: 98.8%, tR ¼ 17.90 min.
(400 MHz, DMSO‑d6) d 8.61 (s, 1H), 8.58 (s, 1H), 8.41 (s, 1H), 8.26 (t,
J ¼ 5.6 Hz, 1H), 7.83 (d, J ¼ 8.8 Hz, 1H), 7.50e7.41 (m, 3H), 7.38 (d,
J ¼ 8.2 Hz, 2H), 7.27 (dd, J ¼ 8.2, 7.4 Hz, 2H), 7.18 (d, J ¼ 8.2 Hz, 2H),
6.96 (t, J ¼ 7.4 Hz,1H), 4.17 (q, J ¼ 7.0 Hz, 2H), 3.72 (td, J ¼ 7.8, 5.6 Hz,
2H), 2.90 (t, J ¼ 7.8 Hz, 2H), 1.43 (t, J ¼ 7.0 Hz, 3H). 13C NMR
1-(4-{2-[(7-Ethoxy-6-fluoroquinazolin-4-yl)amino]ethyl}phenyl)-
3-[3-(trifluoromethyl)phenyl]urea (35). The title compound was
synthesized following the general procedure B and using the re-
actants/reagents 19d (196 mg, 0.60 mmol, 1.0 equiv.), 1-isocyanato-
3-(trifluoromethyl)benzene (163 mg, 0.87 mmol, 1.5 equiv.),
methanol (0.2 mL) and dichloromethane (20 mL). After work-up,
the title compound 35 was obtained in 89% yield as white solid.
(100 MHz, DMSO‑d6)
d
158.4, 155.3 (d, JC-F ¼ 251.7 Hz), 154.2, 152.6,
145.7 (d, JC-F ¼ 12.4 Hz), 144.9 (d, JC-F ¼ 11.8 Hz), 139.8, 137.9, 132.9,
129.0, 128.8, 121.8, 118.4, 118.2, 112.6 (d, JC-F ¼ 17.3 Hz), 111.9, 105.3
(d, JC-F ¼ 2.4 Hz), 64.9, 42.4, 34.1, 14.4. LC-MS (ESI) m/z: 446.3
[MþH]þ. HRMS (ESI) m/z: calcd for C25H25FN5O2, 446.1992 [MþH]þ,
found 446.1986. HPLC purity: 95.5%, tR ¼ 17.35 min.
1H NMR (400 MHz, DMSO‑d6)
d 9.03 (s,1H), 8.75 (s,1H), 8.43 (s,1H),
1-(3-Chlorophenyl)-3-(4-{2-[(6-ethoxy-7-fluoroquinazolin-4-yl)
amino]ethyl}phenyl)urea (32). The title compound was synthesized
following the general procedure B and using the reactants/reagents
19f (94 mg, 0.29 mmol, 1.0 equiv.), 1-chloro-3-isocyanatobenzene
(53 mg, 0.35 mmol, 1.2 equiv.) and N,N-dimethylacetamide
(1 mL). After stirred at room temperature for 2 h and work-up, the
title compound 32 was obtained in 87% yield as white solid. 1H
8.16e7.98 (m, 3H), 7.56 (d, J ¼ 8.0 Hz, 1H) 7.50 (dd, J ¼ 8.0, 7.6 Hz,
2H), 7.39 (d, J ¼ 8.4 Hz, 2H), 7.30 (d, J ¼ 7.2 Hz, 1H), 7.26 (d,
J ¼ 8.4 Hz, 1H), 7.18 (d, J ¼ 8.4 Hz, 2H), 4.24 (q, J ¼ 7.0 Hz, 2H), 3.70
(td, J ¼ 7.2, 6.0 Hz, 2H), 2.89 (t, J ¼ 7.2 Hz, 2H), 1.41 (t, J ¼ 7.0 Hz, 3H).
13C NMR (150 MHz, DMSO‑d6)
d 158.7, 155.1, 152.5, 151.1 (d, JC-
¼ 12.9 Hz), 150.1 (d, JC-F ¼ 245.4 Hz), 147.8, 140.7, 137.5, 133.3,
F
129.8, 129.5 (q, JC-F ¼ 31.4 Hz), 129.0, 124.2 (q, JC-F ¼ 270.6 Hz), 121.7,
118.6, 117.9, 114.0, 109.7, 108.0 (d, JC-F ¼ 5.7 Hz), 107.8 (d, JC-
NMR (400 MHz, DMSO‑d6)
d 8.84 (s, 1H), 8.68 (s, 1H), 8.41 (s, 1H),
8.26 (t, J ¼ 5.6 Hz, 1H), 7.83 (d, J ¼ 9.2 Hz, 1H), 7.71 (dd, J ¼ 2.0,
1.6 Hz, 1H), 7.47 (d, J ¼ 12.4 Hz, 1H), 7.38 (d, J ¼ 8.6 Hz, 2H),
7.32e7.22 (m, 2H), 7.19 (d, J ¼ 8.6 Hz, 2H), 7.00 (ddd, J ¼ 7.6, 2.4,
2.0 Hz, 1H), 4.21 (q, J ¼ 6.8 Hz, 2H), 3.72 (td, J ¼ 7.6, 5.6 Hz, 2H), 2.91
(t, J ¼ 7.6 Hz, 2H), 1.43 (t, J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz,
¼ 20.0 Hz), 64.5, 42.2, 33.9, 14.3. LC-MS (ESI) m/z: 514.3 [MþH]þ.
F
HRMS (ESI) m/z: calcd for C26H24F4N5O2, 514.1866 [MþH]þ, found
514.1863. HPLC purity: 98.6%, tR ¼ 19.61 min.
1-(3-Chloro-4-fluorophenyl)-3-(4-{2-[(7-ethoxy-6-
fluoroquinazolin-4-yl)amino]ethyl}phenyl)urea (36). The title com-
pound was synthesized following the general procedure B and
using the reactants/reagents 19d (40 mg, 0.12 mmol, 1.0 equiv.), 2-
chloro-1-fluoro-4-isocyanatobenzene (81 mg, 0.30 mmol, 2.5
equiv.), methanol (0.2 mL) and dichloromethane (2 mL). After
work-up, the title compound 36 was obtained in 73% yield as white
DMSO‑d6)
d
158.4, 155.3 (d, JC-F ¼ 251.7 Hz), 154.2, 152.4, 145.7 (d, JC-
¼ 12.4 Hz), 144.8 (d, JC-F ¼ 12.1 Hz), 141.4, 137.6, 133.3, 133.2, 130.4,
F
129.1, 121.4, 118.6, 117.5, 116.6, 112.5 (d, JC-F ¼ 17.3 Hz), 111.9, 105.4,
64.9, 42.4, 34.1,14.4. LC-MS (ESI) m/z: 480.2 [MþH]þ. HRMS (ESI) m/
z: calcd for C25H24ClFN5O2, 480.1603 [MþH]þ, found 480.1601.
12

M.-C. Li, W.-H. Lin, P.-C. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113673
solid. 1H NMR (400 MHz, DMSO‑d6)
d
8.83 (s, 1H), 8.68 (s, 1H), 8.42
J ¼ 7.2 Hz, 2H), 1.41 (t, J ¼ 6.8 Hz, 3H). 13C NMR (150 MHz, DMSO‑d6)
(s, 1H), 8.13e8.04 (m, 2H), 7.79 (dd, J ¼ 4.8, 2.4 Hz, 1H) 7.37 (d,
J ¼ 8.4 Hz, 2H), 7.34e7.27 (m, 2H), 7.24 (d, J ¼ 8.4 Hz, 1H), 7.17 (d,
J ¼ 8.4 Hz, 2H), 4.23 (q, J ¼ 7.0 Hz, 2H), 3.69 (td, J ¼ 7.6, 6.4 Hz, 2H),
2.88 (t, J ¼ 7.6 Hz, 2H), 1.40 (t, J ¼ 7.0 Hz, 3H). 13C NMR (150 MHz,
d
158.7 (d, JC-F ¼ 3.0 Hz), 155.1, 152.4, 151.1 (d, JC-F ¼ 12.9 Hz), 150.1
(d, JC-F ¼ 245.3 Hz),150.0,147.9,137.6,133.2,129.5,129.1,124.6 (q, JC-
¼ 269.6 Hz), 121.1 (q, JC-F ¼ 31.5 Hz), 118.6 (q, JC-F ¼ 3.0 Hz), 118.2,
F
114.0 (q, JC-F ¼ 31.5 Hz), 110.7, 109.7, 108.0 (d, JC-F ¼ 7.2 Hz), 107.8 (d,
JC-F ¼ 20.1 Hz), 64.5, 56.2, 42.2, 34.0, 14.3. LC-MS (ESI) m/z: 544.2
[MþH]þ. HRMS (ESI) m/z: calcd for C27H26F4N5O3, 544.1972
[MþH]þ, found 544.1966. HPLC purity: 98.8%, tR ¼ 20.18 min.
Docking Analysis of BPR1K871 and Compound 23 with AURA and
TRKA proteins. The protein structures of AURA (PDB: 4JBO) and
TRKA (PDB: 6PL1) were utilized for this study. The docking analysis
was conducted using the Discovery Studio 2020//LigandFit pro-
gram (BIOVIA Inc., San Diego, CA, USA) with the CHARMm force
field [44]. The number of docking poses was set as 10 with default
parameters. The decision of the best pose was according to the
lowest binding energy of the compound as well as the nitrogen
atom of the compound formed a hydrogen bond with the back bone
amide nitrogen of Ala213 in AURA and Met592 in TRKA, respec-
tively. This was imposed based on the observation of such both
hydrogen bonds in the crystal structures of the AURA kinase
complexed to BPR1K871 (PDB: 4JBO), and in the crystal structures
of the TRKA kinase complexed to entrectinib (PDB: 5KVT).
DMSO‑d6)
d
158.7 (d, JC-F ¼ 3.5 Hz), 155.1, 152.3 (d, JC-F ¼ 239.4 Hz),
152.2, 151.0 (d, JC-F ¼ 12.9 Hz), 150.1 (d, JC-F ¼ 245.3 Hz), 147.8, 137.5,
137.1 (d, JC-F ¼ 2.1 Hz), 133.1, 129.0, 119.4, 119.1 (d, JC-F ¼ 18.2 Hz),
118.6, 118.4 (d, JC-F ¼ 6.5 Hz), 116.8 (d, JC-F ¼ 21.6 Hz), 109.7, 108.0 (d,
JC-F ¼ 7.4 Hz), 107.7 (d, JC-F ¼ 20.1 Hz), 64.5, 42.1, 33.9, 14.3. LC-MS
(ESI) m/z 498.1 [MþH]þ. HRMS (ESI) m/z: calcd for
C
25H23ClF2N5O2, 498.1508 [MþH]þ, found 498.1506. HPLC purity:
96.4% (tR ¼ 21.57 min).
1-[4-Chloro-3-(trifluoromethyl)phenyl]-3-(4-{2-[(7-ethoxy-6-
fluoroquinazolin-4-yl)amino]ethyl}phenyl)urea (37). The title com-
pound was synthesized following the general procedure B and
using the reactants/reagents 19d (40 mg, 0.12 mmol, 1.0 equiv.), 1-
chloro-4-isocyanato-2-(trifluoromethyl)benzene
0.31 mmol, 2.5 equiv.), methanol (0.2 mL) and dichloromethane
(97
mg,
(2 mL). After work-up, the title compound 37 was obtained in 37%
yield as white solid. 1H NMR (400 MHz, DMSO‑d6)
d 9.15 (s, 1H),
8.79 (s, 1H), 8.42 (s, 1H), 8.14e8.02 (m, 3H), 7.62 (dd, J ¼ 8.8, 2.4 Hz,
1H), 7.58 (d, J ¼ 8.8 Hz, 1H), 7.38 (d, J ¼ 8.4 Hz, 2H), 7.24 (d,
J ¼ 8.4 Hz, 1H), 7.18 (d, J ¼ 8.4 Hz, 2H), 4.24 (q, J ¼ 6.8 Hz, 2H), 3.69
(td, J ¼ 7.4, 6.4 Hz, 2H), 2.89 (t, J ¼ 7.4 Hz, 2H), 1.40 (t, J ¼ 6.8 Hz, 3H).
Purified Kinase Confirmatory Activity Assay for TRKA, AURA and
AURB. The recombinant GST-TRKA (residues G443~G796,
NP_002520.2) containing kinase domain (KD) expressed in Sf9 in-
sect cells. The TRKA ADP-Kinase-Glo assay was carried out in 96-
13C NMR (150 MHz, DMSO‑d6)
d
158.7 (d, JC-F ¼ 3.0 Hz), 155.1, 152.4,
151.1 (d, JC-F ¼ 12.8 Hz), 150.1 (d, JC-F ¼ 245.2 Hz), 147.9, 139.4, 137.3,
well plates at 30 ꢀC for 180 min in a final volume of 10
mL
133.4, 131.9, 129.0, 126.7 (q, JC-F ¼ 30.3 Hz), 122.9, 122.8 (q, JC-
including 200 ng GST-TRKA proteins, 25 mM TriseHCl, pH 7.4,
2 mM DTT, 10 mM MgCl2, 0.01% bovine serum albumin, 40 M poly
(Glu,Tyr) 4:1 peptide, 0.02% Triton X-100, 0.5 mM Na3VO4, and
40 M ATP. Following incubation, 5 L ADP-Glo™ Reagent (Prom-
¼ 271.4 Hz), 122.1, 118.8, 116.6 (d, JC-F ¼ 5.4 Hz), 109.7, 108.0 (d, JC-
m
F
¼ 7.1 Hz),107.7 (d, JC-F ¼ 20.0 Hz), 64.5, 42.2, 33.9,14.3. LC-MS (ESI)
F
m/z: 548.1 [MþH]þ. HRMS (ESI) m/z: calcd for C26H23ClF4N5O2,
548.1476 [MþH]þ, found 548.1454. HPLC purity: 98.1%
(tR ¼ 24.11 min).
m
m
ega, Madison, WI, USA) was added and the mixture was incubated
at 25 ꢀC for 40 min. The KDR (Kinase Detection Reagent) reagent
1-(5-Chloro-2-methoxyphenyl)-3-(4-{2-[(7-ethoxy-6-
10
After incubation, 15
and mixed well. Then 30
m
L was added and the mixture was incubated 25 ꢀC for 30 min.
fluoroquinazolin-4-yl)amino]ethyl}phenyl)urea (38). The title com-
pound was synthesized following the general procedure B and
using the reactants/reagents 19d (82 mg, 0.25 mmol, 1.0 equiv.), 4-
chloro-2-isocyanato-1-methoxybenzene (69 mg, 0.38 mmol, 1.5
equiv.), methanol (0.2 mL) and dichloromethane (2 mL). After
stirred for 2 h at room temperature and work-up, the title com-
pound 38 was obtained in 80% yield as white solid. 1H NMR
mL buffer (25 mM Tris-HCl, pH 7.4) was added
m
L aliquot of each reaction mixture was
transferred to 96-well black microtiter plate (237108, NUNC) and
the luminescence was measured on Wallac Vector 1420 multi-label
counter (PerkinElmer, Shelton, CT, USA). The AURKA ATP-Kinase-
Glo and AURKB ATP-Kinase-Glo assays for the compounds were
carried out as described in our previous studies [33,45].
(400 MHz, DMSO‑d6)
d
9.34 (s, 1H), 8.43 (s, 1H), 8.37 (s, 1H), 8.23 (d,
Cellular Proliferation Assays. To determine cell viability after drug
treatment, assays were performed by seeding 5000 KM12 cells per
well in a 96-well culture plate. After 16 h, cells were then treated
with vehicle or various concentrations of compound in medium for
72 h. The viable cells were quantitated using the MTS method
(Promega, Madison, WI, USA) according to the manufacturer's
recommended protocol. The results were determined by measuring
the absorbance at 490 nm using a plate reader (Victor 2; Perki-
nElmer, Shelton, CT, USA). The IC50 value was defined as the amount
of compound that caused 50% reduction in cell viability in com-
parison with DMSO-treated (vehicle) control and was calculated
using Prism version 8 software (GraphPad, San Diego, CA, USA).
Western Blot Analysis. KM12 cells (1 ꢂ 106) were seeded in each
well of a six-well plate for overnight in culture medium. After
40 min of compound treatment, cells were harvested by washing
twice with cold PBS and lysed in RIPA buffer (20 mM Tris, pH 7.5,
150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium
J ¼ 2.6 Hz, 1H), 8.13e8.04 (m, 2H), 7.37 (d, J ¼ 8.4 Hz, 2H), 7.25 (d,
J ¼ 8.4 Hz, 1H), 7.18 (d, J ¼ 8.4 Hz, 2H), 7.02 (d, J ¼ 8.8 Hz, 1H), 6.97
(dd, J ¼ 8.8, 2.6 Hz, 1H), 4.24 (q, J ¼ 7.0 Hz, 2H), 3.88 (s, 3H), 3.70 (td,
J ¼ 7.8, 6.4 Hz, 2H), 2.89 (t, J ¼ 7.8 Hz, 2H), 1.41 (t, J ¼ 7.0 Hz, 3H). 13
C
NMR (100 MHz, DMSO‑d6)
d
158.7 (d, JC-F ¼ 3.8 Hz), 155.1, 152.2,
151.0 (d, JC-F ¼ 13.0 Hz), 150.1 (d, JC-F ¼ 245.2 Hz), 147.9, 146.3, 137.6,
133.1, 130.1, 129.1, 124.4, 120.7, 118.2, 117.3, 111.8, 109.7, 108.0 (d, JC-
¼ 7.4 Hz),107.8 (d, JC-F ¼ 20.2 Hz), 64.5, 56.1, 42.2, 33.9,14.3. LC-MS
F
(ESI) m/z: 510.1 [MþH]þ. HRMS (ESI) m/z: calcd for C26H26ClFN5O3,
510.1708 [MþH]þ, found 510.1725. HPLC purity: 98.4%,
tR ¼ 20.75 min.
1-(4-{2-[(7-Ethoxy-6-fluoroquinazolin-4-yl)amino]ethyl}phenyl)-
3-[2-methoxy-5-(trifluoromethyl)phenyl]urea (39). The title com-
pound was synthesized following the general procedure B and
using the reactants/reagents 19d (82 mg, 0.25 mmol, 1.0 equiv.), 2-
isocyanato-1-methoxy-4-(trifluoromethyl)benzene
(82
mg,
0.38 mmol, 1.5 equiv.), methanol (0.2 mL) and dichloromethane
(2 mL). After stirred for 2 h at room temperature and work-up, the
title compound 39 was obtained in 76% yield as white solid. 1H NMR
deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM
b-glycer-
ophosphate, 1 mM Na3VO4, 1 g/mL leupeptin, 1 mM PMSF, phos-
m
phatase inhibitor cocktail (Bimake, B15001), and protease inhibitor
(Thermo Fisher, A32955). After addition of SDS sample buffer, the
(400 MHz, DMSO‑d6)
d
9.38 (s, 1H), 8.55 (d, J ¼ 2.4 Hz, 1H), 8.49 (s,
1H), 8.43 (s,1H), 8.20e8.02 (m, 2H), 7.39 (d, J ¼ 8.4 Hz, 2H), 7.30 (dd,
J ¼ 8.8, 2.4 Hz, 1H), 7.25 (d, J ¼ 8.4 Hz, 1H), 7.22e7.15 (m, 3H), 4.24
(q, J ¼ 6.8 Hz, 2H), 3.96 (s, 3H), 3.70 (td, J ¼ 7.2, 6.8 Hz, 2H), 2.89 (t,
lysate was heated at 95 ꢀC for 10 min and an amount of 50
mg of
samples were resolved by SDS-PAGE and transferred onto a poly-
vinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA,
13

M.-C. Li, W.-H. Lin, P.-C. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113673
USA). The membranes were immunoblotted with appropriate an-
tibodies (TRKA Antibody (Cell Signaling Technology, #2505),
Phospho-TrkA (Tyr490) Antibody (Cell Signaling Technology,
#9141), and b-ACTIN antibody (Thermo Fisher, MA5-15739)) and
detected using the SuperSignal reagent (Pierce, Rockford, IL, USA)
followed by exposure to X-ray film.
Cell Culture. Human cancer cell line KM12 cells were cultured
and maintained in flasks with RPMI-1640 Media. The culture me-
dium was supplemented with 10% fetal bovine serum. The cells
were incubated at 37 ꢀC in a humidified atmosphere containing 5%
CO2.
After all this 39 group was treated at 75 mg/kg three time per week
for 3 weeks. Vehicle (P.O.) 10 mL/kg QD for three continuous weeks
the treatment schedule was 5 days on, 2 days off. Larotrectinib (1)
at 75 mg/kg (P.O.), QD, the treatment schedule was 5 days on, 2 days
off, and continued for 4 weeks. Tumor dimensions were measured
with a digital caliper, and the tumor volume in mm3 was calculated
by the formula: Volume ¼ (length x width^2)/2. Tumor-bearing
mice were randomized when the mean tumor volume was
approximately to 200 mm3. All mice were monitored daily for signs
of toxicity. Body weight and tumor size were measured twice a
week.
Pharmacokinetics. Male ICR mice (25e35 g) and male Sprague
Dawley rats (300e400 g) were obtained from BioLASCO (Taiwan
Co., Ltd, Ilan, Taiwan). The animal studies were performed ac-
cording to NHRI institutional animal care and committee-approved
procedures. Rats were surgically prepared with a jugular-vein
cannula 1 day before dosing. Rats and mice were fasted overnight
(for approximately 18e20 h) before dosing. Water was available ad
libitum throughout the experiment. Food was provided at 4 h after
dosing. A single 2.0 and 10 mg/kg dose of compound, as a PEG400/
DMA (80/20, v/v) for both IV and oral and DMSO/CrEL/D5W (5%/5%/
90%,v/v/v) and 1%CMCþ0.5%Tween 80 (oral) solution, was sepa-
rately administered to rats and mice. The groups consisted of three
rats each route and a total of 33 and 27 mice for intravenously (IV)
and oral gavage (PO), respectively. Each rat received 2 or 10 mL of
the dosing solution/kg of body weight and each mouse was given
Data Analysis and Statistics. Tumor growth inhibition (TGI) in
percentage was determined by the formula: TGI (%) ¼ (1-T/C) x 100,
where T indicates the mean tumor volume of compound-treated
group and C indicates the mean tumor volume of vehicle-treated
control. The significant difference between drug treatment and
vehicle control were performed with statistical analysis (p-
value < 0.05 vs. control).
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgement
100 and 200 mL of dosing solution by intravenous injection and by
gavage, respectively. At 0 (before dosing), 2, 5 (IV only), 15, and
30 min and at 1, 2, 4, 6, 8, 16 (mice only), and 24 h after dosing, a
blood sample (0.15 mL) was collected from each rat through the
Support from the National Health Research Institutes and Min-
istry of Science and Technology, Taiwan (MOST 109-2113-M-400-
003 and MOST 109-3114-Y-001-001 for H.-P. H.) is acknowledged.
This work is also financially supported by the Center of Applied
Nanomedicine, National Cheng Kung University from The Featured
Areas Research Center Program within the framework of the Higher
Education Sprout Project by the Ministry of Education (MOE) in
Taiwan. M.-C. L. is supported by the Ministry of Science and Tech-
nology, Taiwan (MOST 109-2113-M-400-003 and MOST 109-3114-
Y-001-001), National Health Research Institutes, Taiwan, and
Biomedical Translation Research Center, Academia Sinica, Taiwan.
We thank Dr. Hsin-Ru Wu, Instruments Center at National Tsing
Hua University, for her help in obtaining mass spectral data (MOST
110-2731-M-007-001) and Mr. Richard Guy for his help with En-
glish editing.
jugular-vein cannula and ~500
mL was collected from groups of
three mice at each time point by cardiac puncture and stored in ice
(0e4 ꢀC). Immediately after collecting the blood sample, 150 mL of
physiological saline (containing 30 Units of heparin per ml) was
injected into the rat through the jugular-vein cannula. Plasma was
separated from the blood by centrifugation (14,000 g for 15 min at
4 ꢀC in a Beckman Model AllegraTM 6R centrifuge) and stored in a
freezer (ꢁ80 ꢀC). All samples were analyzed for the parent drug by
LCꢁMS/MS. Data were acquired through selected reaction ion
monitoring. Plasma concentration data were analyzed with a
noncompartmental method.
Animal. The animal use protocol was approved by The Institu-
tional Care and Use Committee of the National Health Research
Institutes (animal use protocol number: NHRI-IACUC-106076-A).
Briefly, Nude mice (Nu-Fox 1nu) of six weeks old were purchased
from BioLasco (Ilan, Taiwan) and housed in sterilized cages equip-
ped with air filter and sterile beddings at the AAALAC accredited
facility, Laboratory Animal Center of the National Health Research
Institutes. Animals were fed standard mice chow with water
available ad libitum. The animals were housed in group cages under
a 12 h light/dark cycle.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113673.
Abbreviations
TRK
NTRK
NT
tropomyosin receptor kinase
neurotrophic tyrosine receptor kinase
neurotrophin
In Vivo Antitumor Efficacy Studies in the KM12 Human Colorectal
Cancer Xenograft Model (Subcutaneous inoculation of tumor cells in
mice) KM12 cells were suspended in phenol red free RPMI-1640
medium and mixed with Matrigel™ (356237, BD Bioscience, San
Jose CA, USA) in 1:1 ratio. The cells were subcutaneously inoculated
into the left flanks of mice using a 1 mL syringe (needle 24G x 1 in.,
0.25 mm; TERUMO). Cells were suspended in a volume so that
NGF
CRC
ETV6
FDA
DFG
MKI
IND
AUR
SAR
FLT3
NRB
PSA
nerve growth factor
colorectal cancer
ETS variant transcription factor 6
food and drug administration
Asp-Phe-Gly
multi-kinase inhibitor
investigational new drug
aurora kinase
structure-activity relationship
FMS-like tyrosine kinase 3
number of free rotatable bond
polar surface area
100
m
L contains 1 ꢂ 106 KM12 cells per s. c. inoculation injection.
The mice were randomly divided into 3 groups of 7e8 animals
each, and the treatment was initiated. The animals will be grouped
and treated with 39 at various doses for an optimal treatment
period. Animals were treated with 39 at 100 mg/kg (P.O.) at day 1, 2,
3 after treatment, while the animal body weight loss<ꢁ10% on day
4. The dosage was reduced to 75 mg/kg and treated on day 4 and 5.
14

M.-C. Li, W.-H. Lin, P.-C. Wang et al.
European Journal of Medicinal Chemistry 224 (2021) 113673
F. Bentzien, B. Cancilla, J. Orf, A. You, A.D. Laird, S. Engst, L. Lee, J. Lesch,
Y.C. Chou, A.H. Joly, Cabozantinib (XL184), a novel MET and VEGFR2 inhibitor,
simultaneously suppresses metastasis, angiogenesis, and tumor growth, Mol.
Canc. Therapeut. 10 (2011) 2298e2308.
ATP
WT
DDR1
PK
AUC
FOTO
TGI
adenosine triphosphate
wild-type
discoidin domain receptor 1
pharmacokinetic
area under curve
[22] P.P. Patwardhan, K.S. Ivy, E. Musi, E. de Stanchina, G.K. Schwartz, Significant
blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a
novel small molecule inhibitor, shows potent anti-tumor activity in preclinical
models of sarcoma, Oncotarget 7 (2016) 4093e4109.
[23] J.J. Bailey, R. Schirrmacher, K. Farrell, V. Bernard-Gauthier, Tropomyosin re-
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Expert Opin. Ther. Pat. 27 (2017) 733e751.
[24] J.J. Bailey, R. Schirrmacher, K. Farrell, V. Bernard-Gauthier, Tropomyosin re-
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five-days-on, two-days-off
tumor growth inhibition
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