Design, synthesis and biological activity of bicyclic carboxamide derivatives as TRK inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115811

‘precision medicine’ is characterized by the selection of targeted drugs based on genetic characteristics of tumor from patients, and no longer selected basis on the type of cancer tissue. Among them, clinical trials on neurotrophin receptor tyrosine kinase genes (NTRK) have proven that great anti-cancer effects can be achieved in different cancer patients. In this paper, a novel total of twenty compounds in two categories have been designed and synthesized. Results of Kinase activity tests showed that I-9 (TRKA IC₅₀ = 1.3 nM, TRKA^G595R IC₅₀ = 6.1 nM), and I-10 (TRKA IC₅₀ = 1.1 nM, TRKA^G595R IC₅₀ = 5.3 nM) have significant inhibitory activity, and results of cell viability tests showed that I-9 and I-10 can maintain a great inhibitory effect in the Ba/F3-LMNA-NTRK1 cell line(IC₅₀ = 81.1 nM and 41.7 nM, respectively), and in Ba/F3-LMNA-NTRK1-G595R cell line, I-9 and I-10 have better cell activity (IC₅₀ was 495.3 nM, 336.6 nM, respectively) compared with the positive control drug LOXO-101. These results indicate that I-9 and I-10 are potential TRK inhibitors that can overcome drug resistance for further investigation.

Full text of DOI:10.1016/j.bmc.2020.115811

Journal Pre-proofs
Design, synthesis and biological activity of bicyclic carboxamide derivatives
as TRK inhibitors
Minghao Sun, Shi Cai, Pei Li, Fangqing Zhang, Huibin Zhang, Jinpei Zhou
PII:
S0968-0896(20)30641-6
https://doi.org/10.1016/j.bmc.2020.115811
BMC 115811
DOI:
Reference:
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Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
15 August 2020
27 September 2020
3 October 2020
Please cite this article as: M. Sun, S. Cai, P. Li, F. Zhang, H. Zhang, J. Zhou, Design, synthesis and biological
activity of bicyclic carboxamide derivatives as TRK inhibitors, Bioorganic & Medicinal Chemistry (2020), doi:
https://doi.org/10.1016/j.bmc.2020.115811
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Design，synthesis and biological activity of bicyclic carboxamide derivatives as
TRK inhibitors
Minghao Suna,Shi Caib,Pei Lia, Fangqing Zhangb, Huibin Zhangb,Jinpei Zhoua,*
a Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
b Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang,
Nanjing 210009, China
* Corresponding authors.
E-mail addresses: jpzhou668@163.com (J. Zhou)
Abstract
‘precision medicine’ is characterized by the selection of targeted drugs based on genetic
characteristics of tumor from patients, and no longer selected basis on the type of cancer tissue.
Among them, clinical trials on neurotrophin receptor tyrosine kinase genes (NTRK) have proven
that great anti-cancer effects can be achieved in different cancer patients. In this paper, a novel total
of twenty compounds in two categories have been designed and synthesized. Results of Kinase
activity tests showed that I-9 (TRKA IC50 = 1.3 nM, TRKAG595R IC50 = 6.1 nM), and I-10 (TRKA
IC50 = 1.1 nM, TRKAG595R IC50 = 5.3 nM) have significant inhibitory activity, and results of cell
viability tests showed that I-9 and I-10 can maintain a great inhibitory effect in the Ba/F3-LMNA-
NTRK1 cell line(IC50 = 81.1 nM and 41.7 nM, respectively), and in Ba/F3-LMNA-NTRK1-G595R
cell line, I-9 and I-10 have better cell activity (IC50 was 495.3 nM, 336.6 nM, respectively)
compared with the positive control drug LOXO-101. These results indicate that I-9 and I-10 are
potential TRK inhibitors that can overcome drug resistance for further investigation.
Keywords: Tropomyosin Receptor Kinases; TRK inhibitors; Molecular docking; Synthesis
1. Introduction
In the 21st century, the emerging field of "precision medicine" is gradually developing, which
is mainly carried out by detecting relevant genes that lead to the development of diseases from
patients. Then, specific drugs are given for targeted therapy to achieve precise treatment for
individual patients. In 2018, the US Food and Drug Administration (FDA) approved Larotectinib
(LOXO-101) (Table.1), the first TRK inhibitor, for the treatment of adult and paediatric patients
with solid tumors that have a NTRK gene fusion1, which is the second broad-spectrum anti-cancer
drug after the PD1 monoclonal antibody drug Keytruda. In the next year, Entrectinib (RXDX-101)
(Table.1) was approved by Japan for the treatment of patients with solid tumors that have
NTRK/ROS1/ALK gene fusion2. Although Entrectinib is a multi-target inhibitor, it has an excellent
therapeutic effect on patients and can also achieve precise treatment. In addition, the successful
listing of TRK inhibitors marked the great success of precision medicine in cancer patients and laid
the foundation for the rapid development of precision medicine. Annually, it is estimated that
approximately 20000 new cases of TRK cancer have been diagnosed, with the majority of cases
exhibiting a TRK genomic rearrangement3. TRK protein is a single transmembrane receptor tyrosine
kinase, and there are three members of the TRK family: TRKA (encoded by NTRK1 gene), TRKB
(NTRK2 gene), and TRKC (NTRK3 gene), which have all played an important role in the initiation
and progression of malignancies3, 4. Although studies have shown that the three subtypes of TRK

protein are highly homologous, the ligands that activate these three subtypes are indeed different:
TRKA (activated by nerve growth factor (NGF))5, 6, TRKB (brain-derived neurotrophic factor
(BDNF) and neurotrophin-4 (NT-4)), and TRKC (neurotrophin-3 (NT-3)) 7-9. There are five key
amino acid residues which are closely related to the activation of the entire TRK protein kinase in
the intracellular domain of TRK protein10.
Table 1. representative TRK inhibitors under clinical research or on the market
IC50(nM)
Compound
Company
Structure
Phase
TRKA
TRKAG595R
F
OH
N
Larotinib
Loxo
N
F
9.7
>2000
Launched
N
N
(LOXO-101)
Oncology
N
HN
O
O
O
HN
Entrectinib
HN
Roche
1
>700
Launched
F
(RXDX-101)
N
N
N
N
H
F
N
N
N
N
Selitrectinib
(LOXO-195)
Loxo
O
< 1
2
I/II
Oncology
N
H
N
F
N
N
HN
Repotrectinib
(TPX-0005)
TP
N
O
< 1
0.2
I/II
O
Therapeutics
N
H
F
The TRK protein is initially activated by dimerizing when the ligand binds to the extracellular
region of the TRK protein11. Then, it can cause phosphorylation of tyrosine residues in the
intracellular kinase region (take TRKA as an example, such as Y676, Y680, Y681) and activate the
entire TRK protein12. After that, phosphorylated Y496 can directly bind to and activate SRC
homology 2 domain containing(SHC), fibroblast growth factor receptor substrate 2(FRS2) proteins,
while phosphorylated Y791 interacts with Phospholipase C-γ(PLCγ)13, 14. The activation of SHC
protein can activate RAS and AKT signaling pathways15, 16. It recruits growth factor receptor-bound
protein 2(GRB2), and promotes the activation of RAS through GRB2-associated-binding protein 1
(GAB1) and son-of-sevenless homologue (SOS) factors. Afterwards, activates the mitogen-
activated protein kinase (MAPK) cascade, including RAF/MEK/ERK. Eventually, ERK kinase
enters the nucleus to activate target genes related to cell survival and differentiation by penetrating
the nuclear membrane17, 18. The activation of the AKT pathway is mainly accomplished by recruiting
SHC and GRB2 proteins to the NPXY motif of TRK19. Signal transmission mediated by GAB1
protein activates PI3K and phosphorylates PI4,5 lipids at the 3'position20. There is a conserved PH
domain in AKT that can interact with lipids phosphorylated at the 3'position to promote the

activation of AKT/mTORC, thereby increasing the transcriptional expression of genes related to
cell proliferation and survival21, 22
.
Mutations in NTRK induce cancer due to abnormal expression of TRK protein. Currently,
studies have reported that NTRK mutations in a variety of cancer types, including mammary
analogue secretory carcinoma(MASC), soft tissue sarcoma, glioma, thyroid cancer, salivary gland
cancer and so on23, 24. The mutation types of NTRK mainly include: NTRK fusion, NTRK point
mutation, TRK protein overexpression and TRK protein resistance mutation. Although TRK
inhibitors Larotinib and Entrectinib have shown excellent therapeutic effects in clinical studies,
TRK kinase also inevitably appears drug-resistant mutations with the prolonged treatment time.
Research on the mechanism of clinical drug resistance found that the secondary mutations of TRK
kinase mainly include: the front of the solvent area of TRKA and TRKC (TRKAG595R,TRKCG623R),
xDFG area (TRKAG667C,TRKCG696A) and gatekeeper area (TRKAF589L)13. In addition, biochemical
mechanism studies have revealed that the mutant TRK protein will also increase the kinase activity
through the improvement of its binding affinity to ATP, thereby further weakening the effect of
inhibitor25.
In order to solve the problem of drug resistance, the second generation of TRK inhibitors has
been developed. LOXO-195 and TPX-0005, from Loxo Oncology and TP Therapeutics respectively
(Table.1), have shown great inhibitory effects on drug-resistant mutant TRK kinases in vitro tests.
Since Loxo oncology dominates the development of TRK inhibitors, we used LOXO-101 as the
lead compound. Due to drug resistance, a new type of small molecule TRK inhibitor was designed
and synthesized, which was expected to have a certain inhibitory effect on mutant TRK while
maintaining its inhibitory effect on wild-type TRK.
The structure of LOXO-101 can be divided into three parts: phenyl-1-pyrrolidinyl,
pyrazolopyrimidine core and ureido moiety. Up to now, Loxo Oncology has not announced the co-
crystal structure of the compound and TRK protein. We docked LOXO-101 to explore its binding
conformation with TRKA protein by using Schrodinger software and the existing eutectic complex
(PDB ID: 4YNE). Through the docking test, it can be concluded that the nitrogen atom on the
pyrazole ring of the nucleus can form a hydrogen bond with Met592 in the hinge area, and have a
hydrophobic interaction with the surrounding Phe589, Glu590, and Tyr591(Fig.1). The hydroxypyr-
rolidine part of the compound extends to the front of the solvent region, the difluorophenyl moiety
that well integrated in the hydrophobic pocket maintains a vertical conformation with the entire
skeleton, and has hydrophobic interactions with surrounding Phe521, Val524, Arg654, Asn655,
Cys656, Gly667 and Asp668. The whole protein is in the DFG-in conformation. As a result, we
found that the hydrogen bond is essential for LOXO-101 to bind with the hinge region. Based on
this finding, we retained the core of the lead compound and simplified the structure of this part to
investigate its influence on the hydrophobic effect of surrounding amino acids by using the ring-
opening strategy to replace the pyrrolidinyl group with aliphatic groups. The hydroxypyrrolidinyl
part is located in the front of the solvent zone. Since this part is sterically hindered with the 595th
arginine of the mutant TRKAG595R, we designed and synthesized compounds I to shorten the side

chain by converting urea groups to amide groups on the basis of bioelectronics, so as to study
whether it will reduce steric hindrance (Fig.1).
N
R2
N
F
N
N
R3
NH
O
F
R1
I-1~10
N
N
F
OH
N
N
N
HN
O
N
LOXO-101
N
F
N
N
R2
H
N
O
R1
II-1~10
Fig.1. Docking of compound LOXO-101 and TRKA; Design ideas of compounds I and compounds II.
The design of compounds II was based on compounds I. Based on the skeleton transition
principle, the pyrazolopyrimidine core was replaced by imidazopyridazine to explore whether
hydrogen bonds in the hinge region could be maintained while other groups remained basically
unchanged (Fig.1).
2. Results and discussion
2.1. Chemistry
The synthesis of compounds I-1~I-10 is shown in Scheme 1. Briefly, ethyl 3-amino-4-
pyrazolecarboxylate (1) was heated with (E)-ethyl-3-ethoxyacrylate and cesium carbonate in N,N-
dimethylformide (DMF) to generate the intermediate 2, which chlorinated by phosphorus
oxychloride to obtain 3. Then, starting from compound 4a-b, the key intermediate 5a-c were
obtained with sodium cyanoborohydride by reductive amination reaction, which were reacted with
3 to yield 6a-c. Afterwards, treatment of 6a-c with lithium hydroxide produced 7a-c by hydrolysis

reaction. Finally, 7a-c were further converted to the target compounds I-1~I-10 by condensation
with differently substituted amines.
N
N
N
HN
H2N
N
N
a
b
HO
N
Cl
N
N
N
R2
O
O
O
O
O
O
F
d
N
N
1
2
3
O
R3
O
O
R2
c
6a~c
F
F
R3
R2
N
H
4a~b
5a~c
4a~7a
4b~7b
R2=H,R3=H
R2=CH3,R3=H
N
N
N
N
R2
R2
f
e
F
F
N
N
N
N
R3
NH
R1
OH
R3
O
O
=
=
R2 H,R3 CH3
5c~7c
I-1~10
7a~c
I-3
I-4
I-5
I-6
I-7
I-8
I-9
I-10
I-2
I-1
OH
O
OH
CH3
N
O
OH
CH3
O
O
O
N
R1
H
H
H
R2
R3
CH3
H
CH3
H
CH3
H
H
H
H
H
H
H
H
CH3
CH3
CH3
H
Scheme 1. General synthesis of compounds I-1~I-10. Reagents and conditions: (a) (E)-Ethyl-3-ethoxyacrylate,
Cs2CO3, DMF, 110 °C, 12 h, 94.9%; (b) POCl3, DMF, 100 °C, 1 h, 68.9%; (c) NaBH3CN, NH4OAc, MeOH, 60 °C,
3 h, 59.6-90.5%; (d) DIPEA, DMSO, 90 °C, 1 h., 59.8-79.0%; (e) LiOH, THF/MeOH/H2O, 60 °C, 12 h, 84.5-93.3%;
(f) R1NH2, HATU, DIPEA, DMF, rt, 3 h, 30.9-93.0%.
Target compounds II-1~II-10 were synthesized according to the route shown in Scheme 2.
Starting from 3-amino-6-chloropyridazine (8), which was transformed to the intermediate 9 with
N,N-dimethylformamide dimethyl acetal. After that, the intermediate 10 was synthesized by the
ring closure reaction. Next, the obtained 10 was treated with 3-fluorobenzylamine to afford 11,
which was further converted to 12 by hydrolysis reactions. Then condensation of 12 with different
substituted amines resulted in target compounds II-1~II-10.

N
N
NH2
N
a
b
c
N
F
N
N
N
N
N
N
Cl
N
Cl
N
Cl
N
H
O
O
O
9
8
O
10
11
N
N
d
e
F
N
F
N
N
H
N
N
H
N
R2
OH
N
O
O
R1
12
II-1~10
NR1R2=
N
O
HN
N
CH3
HN
N
OH
HN
O
HN
N
O
II-1
II-2
II-3
II-4
II-5
HN
N
HN
HN
N
O
II-9
II-10
II-8
II-6
II-7
Scheme 2. General synthesis of compounds II-1~II-10. Reagents and conditions: (a) N,N-dimethylformamide
dimethyl acetal, reflux, 2 h, 87.7%; (b) Ethyl bromoacetate, MeCN, 80 °C, 3 h, 61.4%; (c) 3-fluorobenzylamine,
KF·2H2O, DMSO, 120 °C, 12 h , 61.0%; (d) LiOH, THF/MeOH/H2O, rt, 3 h, 83.7%; (e) R1R2NH, HATU, DIPEA,
DMF, rt, 4 h, 22.5-94.9%.
2.2. Biological evaluation
In this paper, the caliper mobility shift assay based on microfluidic chip technology was used.
First, most target compounds were screened for TRKA wild-type and TRKAG595R mutant kinases.
Besides, LOXO-101 was evaluated as the control compound. Afterwards, the compounds with
good inhibition rate were selected for the IC50 test of the kinase, and finally the compounds with
the best activity were selected for the anti-proliferative activity test of BA/F3 cells.
2.2.1. In vitro inhibitory activity and SAR study of compounds I and II
First, we tested the inhibitory rate of the compound against TRKA and TRKAG595R at a
concentration of 500 nM, as shown in Table.2. The results show that the inhibition rate of most
compounds is about 100%, and only compounds I-1, I-5 and II-1 have poor inhibitory effects on
TRKAG595R. We found that the activity declined when N-methylpiperidinyl of the R1 substituent
was introduced. In compounds I, we investigated the effects of the R2, R3 substituents on the activity
and compared the IC50 kinase test results of compounds I-2, I-6 and I-8 on TRKA and TRKAG595R
(3.6, 32.7 nM; 2.1, 10.8 nM; 3.2, 22.9 nM, respectively), indicating that the inhibitory activity of
these compounds on TRKA is equivalent, and the inhibitory effect on TRKAG595R is: I-6> I-8> I-2.
It can be concluded that when R2 or R3 is substituted by a methyl group, its activity is better than
that without a substituent, and the methyl substitution on R3 is inferior to R2. For the substituent on
the R1 position, compared the test results of compounds I-1, I-2, I-3 and I-4, indicating that the
activity is better than tetrahydropyranyl when trans cyclohexanol substitution or trans p-
methoxycyclohexyl was introduced, and the activity is weakest as R1 substituent became N-
methylpiperidinyl.
Later, compounds II were synthesized to preliminarily explore the effect of changes in the
nucleus on the activity. We found that although compounds II maintianed great inhibitory activity

on TRKA, the inhibitory activity on TRKAG595R had been declined ( IC50 is around 20~60 nM)
through the test results. It is inferior to compounds I, indicating that the pyrazolopyrimidin is
assuredly vital for the activity of such compounds.
Table.2. Kinase activity of most compounds I and II
R2
H
Ar
F
N
N
R1
R3
O
N
N
N
Ar=
N
N
N
II-1~II-4
I-1~I-10
Inhibitory% (500nM)
IC50 (nM)
Compound
R1
R2
H
R3
H
TRKA
TRKAG595R
TRKA
TRKAG595R
I-1
I-2
99.8 ± 0.3
12.7 ± 1.6
NTa
3.6
NT
N
CH3
O
H
H
108.3 ± 1.2
108.0 ± 2.5
100.4 ± 0.1
97.8 ± 1.1
99.0 ± 1.6
98.8 ± 0.3
100.6 ± 1.5
100.9 ± 0.4
101.5 ± 0.1
88.5 ± 1.8
99.7 ± 5.4
107.1 ± 1.5
99.3 ± 1.2
94.6 ± 0.5
96.8 ± 1.8
103.0 ± 1.0
16.4 ± 2.1
93.6 ± 1.7
93.3 ± 1.2
99.5 ± 2.2
100.7 ± 0.2
103.2 ± 0.2
-2.4 ± 0.3
87.1 ± 6.4
97.8 ± 2.0
94.6 ± 0.1
32.7
OH
I-3
I-4
H
H
1.6
1.5
NT
2.1
3.0
3.2
1.3
1.1
NT
8.4
2.8
3.0
1.2
10.5
15.7
NT
O
H
H
N
CH3
I-5
CH3
CH3
CH3
H
H
O
I-6
H
10.8
29.4
22.9
6.1
OH
I-7
H
O
I-8
CH3
CH3
CH3
——
——
——
——
OH
O
I-9
H
I-10
II-1
H
5.3
N
CH3
——
——
——
——
NT
O
II-2
59.9
33.0
24.9
237.4
OH
O
II-3
II-4
LOXO-101

a: NT: no test
2.2.2. Anti-proliferative effects against cancer cell lines
We selected two cell lines, Ba/F3-LMNA-NTRK1 and Ba/F3-LMNA-NTRK1-G595R, to test
the IC50 of the compounds. Among compounds I-9 and I-10 have better inhibitory activity against
Ba/F3-LMNA-NTRK1 cells (IC50 of 81.1 nM and 41.7 nM, respectively), and the activity of
compound I-10 is closer to that of the positive drug LOXO-101 (IC50 = 18.4 nM). Although the
inhibitory activity was decreased against Ba/F3-LMNA-NTRK1-G595R cells (IC50 is 0.495 μM and
0.337 μM, respectively), the activity is better than LOXO-101 in Ba/F3-LMNA-NTRK1-G595R
cells.
This result indicates that the compound has a better inhibitory activity against wild-type TRKA
at the cellular level, but the inhibitory effect against mutant TRKA is not as high as the enzymatic
level. It is speculated that the possible cause is the compound with poor membrane permeability in
the Ba/F3-LMNA-NTRK1-G595R cell line lead to low intracellular concentration. It needs to be
further modified in subsequent work to obtain compounds with better cell activity.
Table.3. Cellular activity of main compounds
Ba/F3-LMNA-
NTRK1-G595R
IC50 (nM)
IC50 (nM)
Ba/F3-LMNA-
Compound
Structure
TRKAG595R
NTRK1 IC50(nM)
TRKA
N
N
F
N
H
N
N
N
NH
NH
NH
O
I-3
I-9
1.6
1.3
1.1
1.2
10.5
316.4
81.1
41.7
18.4
NTa
495.3
336.6
2504.7
OH
N
N
F
N
O
6.1
5.3
OH
N
N
F
N
O
I-10
O
F
N
N
F
O
LOXO-101
237.4
N
N
N
HN
O
a: NT: no test
2.3. Molecular docking study of compounds
Among compounds I, we selected compound I-10 with the best activity for molecular docking.
Analysis of the docking conformation of compound I-10 and LOXO-101 with TRKA revealed that
the ligand-protein interactions between these two molecules and TRKA were similar (Fig.2). The
nitrogen atom on the core pyrazole ring can form a hydrogen bond with Met592 in the hinge area,

thus maintaining the hydrophobic interaction with the surrounding Phe589, Glu590 and Tyr591.
The p-methoxycyclohexane part extends to the front of the solvent region, and the benzylamine part
maintains a vertical conformation with the entire skeleton, forming hydrophobic interactions with
the surrounding Gly517, Glu518, Phe521, Val524, Asp668. The benzene ring can facilitate its
anchoring in the hydrophobic pocket via a π-π stacking effect with Phe521. Besides, the whole
protein is in the DFG-in conformation.
Fig.2. The docking result of compound I-10 (yellow) and TRKA (left). The overlap result of compound I-10
(yellow) and LOXO-101 (pink) (right).
In addition, the docking result of this compound with TRKAG595R shows that it can maintain
the hydrophobic interaction with the surrounding amino acids (Fig.3) and form hydrogen bonds
with Met592. However, the p-methoxycyclohexane part of the compound is not sterically hindered
by the mutation of 595 glycine in front of the solvent region to giant arginine. The possible reason
is that the urea group is substituted by the amide moiety, which can change the spatial conformation
of the substituents on the amino group and avoid the mutant arginine. The hydroxypyrrolidine of
LOXO-101 is closer to Arg595, which will cause a certain steric hindrance. This can also explain
the poor inhibitory effect of LOXO-101 on TRKAG595R, while Compound I-10 has a good inhibitory
effect on TRKA and TRKAG595R
.
Fig.3. The docking result of compound I-10 (yellow) and TRKAG595R (left). The overlap result of compound I-10
(yellow) and LOXO-101 (pink) (right).

3. Conclusion
In summary, we used LOXO-101 as the lead compound and utilized molecular docking
technology to obtain their binding mode with TRKA and TRKAG595R protein. On this basis, the
design and synthesis of second-generation TRK inhibitors were carried out. Most target compounds
were initially screened for TRKA wild-type and TRKAG595R. The inhibition rate test results at a
concentration of 500 nM found that most of the above compounds have good inhibitory effect on
TRKA. However, for TRKAG595R, only compounds I can maintain high inhibition rates. Therefore,
we selected compounds I with good inhibition rates for the kinase IC50 test. We found that I-9
(TRKA IC50 = 1.3 nM, TRKG595R IC50 = 6.1 nM) and I-10 (TRKA IC50 = 1.1 nM, TRKG595R IC50 =
5.3 nM) have excellent activity, both reaching the nanomolar level and better than positive medicine
LOXO-101. Then, compounds with excellent kinase inhibitory activity were tested for the anti-
proliferative activity of BA/F3 cells. It was found that the inhibitory activities of I-9 and I-10 were
good with IC50 of 81.1 nM and 41.7 nM in Ba/F3-LMNA-NTRK1 cells, while the IC50 was 495.3
nM and 336.6 nM in Ba/F3-LMNA-NTRK1-G595R cells. The cell test results show that the above
compounds have better cell activity to overcome resistance compared with the positive control drug
LOXO-101 in Ba/F3-LMNA-NTRK1-G595R cells. In the next study, we will do further structural
optimization, and strive to obtain a second-generation TRK inhibitor with better activity.
4. Experimental section
4.1. Chemistry
All chemical reagents were commercially available and used without further purification unless
expressly stated. Reaction progress was monitored by TLC using HSGF 254 (150–200 μm thickness;
Yantai Jiangyou Co., China) with detection by UV. Silica gel 200–300 was used for column
chromatography. Melting points of the synthesized compounds were determined with capillary
apparatus and were not corrected. IR spectra (in KBr pellets) were taken using Shimadzu FTIR-
1
8400S spectrophotometer. H NMR and 13C NMR spectra were measured on Bruker ARX-300
apparatus at 300 MHz or 400 MHz with TMS as the internal standard in CDCl3 or DMSO-d6 unless
otherwise indicated. Mass spectrometry (MS) was performed using a Hewlett-Packard 1100
LC/MSD spectrometer.
4.1.1. Synthesis of ethyl 5-hydroxypyrazolo[1,5-a]pyrimidine-3-carboxylate (2).
To a solution of Ethyl 3-amino-4-pyrazolecarboxylate (3.0 g, 19.34 mmol) in DMF (8ml) was
added (E)-Ethyl-3-ethoxyacrylate (3.35 mL, 23.20 mmol) and cesium carbonate (9.45 g, 29.00
mmol). The mixture was stirred at 110 °C for 12 h. After the reaction was completed, the mixture
was cooled to room temperature and the pH value was adjusted to 4 with acetic acid (20 ml) solution.
Then added with water (40 mL), the mixture was filtered and the precipitate was washed with water
(20 ml × 3) and EtOAc (20 ml × 3). After dried, the precipitate was collected to afford ethyl 5-
hydroxypyrazolo[1,5-a]pyrimidine-3-carboxylate (2) as a light yellow solid (3.8 g, 94.9% yield ).
1H NMR (300 MHz, DMSO-d6) δ 8.48 (d, J = 7.8 Hz, 1H), 8.08 (s, 1H), 6.08 (d, J = 8.1 Hz, 1H),
4.32 – 4.18 (m, 2H), 1.27 (td, J = 7.1, 2.4 Hz, 3H).
4.1.2. Synthesis of ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (3).

In a round-bottom flask, the intermediate 2 (2.8 g, 13.51 mmol) was added to N,N-
dimethylformamide (10 mL), the mixture was stirred in an ice bath. And phosphorus oxychloride
(2.52 mL, 27.03 mmol) was added dropwise at 0 °C. Then the reaction solution was stirred at 100 °C
for 1 h. After the reaction was completed, the reaction solution was cooled to room temperature and
diluted with water (50 mL), the reaction mixture was stirred at room temperature for 0.5 h. The solid
precipitated out, and dried after filtration to obtain ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-
carboxylate (3) as a light yellow solid(2.1 g, 68.9% yield). 1H NMR (300 MHz, DMSO-d6) δ 8.66
(dd, J = 7.3, 1.8 Hz, 1H), 8.57 (d, J = 1.8 Hz, 1H), 7.02 (dd, J = 7.4, 1.8 Hz, 1H), 4.43 (qd, J = 7.0,
1.8 Hz, 2H), 1.43 (td, J = 7.0, 1.7 Hz, 3H).
4.1.3.1. Synthesis of 1-(3-fluorophenyl)ethan-1-amine (5b).
To a solution of 3-fluoroacetophenone (4b, 2.0 g, 14.48 mmol) in methanol (10ml) was added
ammonium acetate (1.82 g, 28.96 mmol) followed by the addition of sodium cyanoborohydride
(11.16 g, 144.78 mmol). The mixture was heated at 60 °C for 3 h. The reaction solution was cooled
to room temperature and was concentrated in vacuo, then diluted with water (30 mL). The mixture
was extracted with EtOAc (50 mL × 3), and the combined organic phase was washed with saturated
sodium chloride solution (40 mL ×2), dried over anhydrous sodium sulfate, filtered and concentrated
in vacuo. The crude residue was purified by flash column chromatography (petroleum ether/ EtOAc
= 1:1)) to afford 1-(3-fluorophenyl)ethan-1-amine (5b) as colorless liquid (1.2 g, 59.6% yield). 1H
NMR (300 MHz, DMSO-d6) δ 7.40 – 7.37 (m, 1H), 7.23 – 7.19 (m, 1H), 7.14 (dt, J = 9.6, 2.2 Hz,
1H), 7.06 (tdd, J = 8.3, 2.6, 1.0 Hz, 1H), 4.40 (q, J = 6.5 Hz, 1H), 1.63 (dd, J = 7.0, 1.7 Hz, 3H).
4.1.3.2. Synthesis of 1-(3-fluorophenyl)-N-methylmethanamine (5c)
In a round-bottom flask, to a solution of methylamine hydrochloride (1.63 g, 24.17 mmol) in
methanol (10ml) was added potassium carbonate (1.67 g, 12.09 mmol). The mixture was stirred at
room temperature for 1 h. To the mixture was then added 3-fluorobenzaldehyde (4c, 2.0 g, 16.11
mmol) and continued to stir at room temperature for 2 h. The reaction solution was cooled to room
temperature, then sodium borohydride (914 mg, 24.17 mmol) was added slowly in an ice bath and
the mixture was continually stirred at 0 °C for 1 h. After the reaction was completed, the mixture
was diluted with water (30 mL) and extracted with EtOAc (30 mL  3). The combined organic phase
was washed with saturated sodium chloride solution (30 mL× 3), dried over anhydrous sodium
sulfate, filtered, the filtrate was evaporated to give 1-(3-fluorophenyl)-N-methylmethanamine (5c)
as colorless liquid (2.03 g, 90.5% yield). 1H NMR (300 MHz, CDCl3) δ 7.35 – 7.29 (m, 1H), 7.16
– 7.02 (m, 2H), 6.97 (td, J = 8.5, 2.7 Hz, 1H), 3.77 (s, 2H), 2.47 (s, 3H).
4.1.4 General procedure for the preparation of 6a-c..
To a solution of the intermediate 3 (4.43 mmol) in dimethyl sulfoxide (5ml) was added 5a-c
(5a, available for purchase)(5.32 mmol) followed by the addition of N,N- diisopropylethylamine
(DIPEA, 8.86 mmol), the reaction was heated to 120 °C and stirred for 1 h. After the reaction was
completed, the reaction solution was cooled to room temperature and was added to water (30 ml).
The mixture was filtered and the precipitate was dried to afford compound 6a-c.
4.1.4.1. Ethyl 5-((3-fluorobenzyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (6a).White solid,
79.0% yield; 1H NMR (300 MHz, DMSO-d6) δ 8.57 (d, J = 7.6 Hz, 1H), 8.43 (s, 1H), 8.16 (s, 1H),

7.43 – 7.23 (m, 3H), 7.09 (t, J = 8.5 Hz, 1H), 6.43 (d, J = 7.6 Hz, 1H), 4.63 (d, J = 6.3 Hz, 2H), 4.20
(q, J = 7.1 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H).
4.1.4.2. Ethyl 5-((1-(3-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (6b).
Light yellow solid, 62.8% yield; 1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 7.6 Hz, 1H), 8.37
(d, J = 7.6 Hz, 1H), 8.14 (s, 1H), 7.38 (td, J = 7.9, 7.5, 5.5 Hz, 1H), 7.34 – 7.23 (m, 2H), 7.11 – 7.01
(m, 1H), 6.42 (d, J = 7.6 Hz, 1H), 5.35 – 5.27 (m, 1H), 4.26 – 4.16 (m, 2H), 1.52 (d, J = 7.0 Hz,
3H), 1.30 (t, J = 7.1 Hz, 3H).
4.1.4.3. Ethyl 5-((3-fluorobenzyl)(methyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (6c).
White solid, 59.8% yield;1H NMR (300 MHz, DMSO-d6) δ 8.76 (d, J = 7.7 Hz, 1H), 8.24 (s, 1H),
7.47 – 7.19 (m, 3H), 7.12 (t, J = 8.4 Hz, 1H), 6.74 (d, J = 7.9 Hz, 1H), 4.94 (s, 2H), 4.21 (q, J = 7.1
Hz, 2H), 3.23 (s, 3H), 1.26 (t, J = 7.2 Hz, 3H).
4.1.5. General procedure for the preparation of 7a-c.
The intermediate 6a-c (3.18 mmol) was dissolved in a mixed solvent of tetrahydrofuran (8 mL)
and methanol (4 mL), then was added 2 mL lithium hydroxide aqueous solution (15.91 mmol). The
mixture was stirred at 70 °C for 12 h and was diluted with water (5 mL). The pH was adjusted to 6
with 1 mol/L dilute hydrochloric acid and then dried after filtration to afford compound 7a-c.
4.1.5.1. 5-((3-fluorobenzyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (7a). White solid,
1
93.3% yield; H NMR (300 MHz, DMSO-d6) δ 11.65 (s, 1H), 8.57 (s, 1H), 8.40 (s, 1H), 8.14 (s,
1H), 7.31 (d, J = 21.5 Hz, 3H), 7.09 (s, 1H), 6.43 (s, 1H), 4.62 (s, 2H).
4.1.5.2. 5-((1-(3-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (7b).
White solid, 85.8% yield; 1H NMR (400 MHz, DMSO-d6) δ 11.50 (s, 1H), 8.55 (d, J = 7.5 Hz, 1H),
8.38 (d, J = 7.7 Hz, 1H), 8.12 (s, 1H), 7.43 – 7.32 (m, 1H), 7.32 – 7.23 (m, 2H), 7.11 – 6.98 (m,
1H), 6.42 (d, J = 7.6 Hz, 1H), 5.30 (s, 1H), 1.52 (d, J = 7.0 Hz, 3H).
4.1.5.3. 5-((3-fluorobenzyl)(methyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (7c).
White solid, 84.5% yield; 1H NMR (300 MHz, DMSO-d6) δ 11.60 (s, 1H), 8.73 (d, J = 7.9 Hz, 1H),
8.20 (s, 1H), 7.38 (q, J = 7.3 Hz, 1H), 7.23 (s, 2H), 7.10 (t, J = 8.6 Hz, 1H), 6.72 (d, J = 7.9 Hz, 1H),
4.92 (s, 2H).
4.1.6. General procedure for the preparation of I-1~ I-10.
In a round-bottom flask, HATU (0.262 mmol) was added to a mixture solution of 7a-c (0.175
mmol), differently substituted amines (0.21 mmol) and DIPEA (0.349 mmol) in DMF (2 mL). The
reaction solution was stirred at room temperature for 4 h. After the reaction was completed, the
mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL  2). The organic
phase was washed with saturated sodium chloride (30 mL × 2), dried over Na2SO4, filtered and
concentrated in vacuo. The crude residue was purified by flash column chromatography
(CH2Cl2/CH3OH = 20:1) to give compounds I-1~ I-10.

4.1.6.1. 5-((3-fluorobenzyl)amino)-N-(1-methylpiperidin-4-yl)pyrazolo[1,5-a]Pyrimidine-3-carb-
oxamide (I-1). This compound was prepared from 7a and 4-amino-1-methylpiperidine as a white
solid, 46.4% yield; m.p.: >250 °C; IR (KBr, cm-1): 3428, 2775, 1638, 1576, 1499, 1487, 840, 773,
559; 1H NMR (300 MHz, DMSO-d6) δ 8.64 (d, J = 7.8 Hz, 2H), 8.12 (s, 1H), 7.74 (d, J = 7.2 Hz,
1H), 7.43 (d, J = 7.8 Hz, 1H), 7.29 – 7.09 (m, 3H), 6.48 (d, J = 7.6 Hz, 1H), 4.64 (d, J = 5.4 Hz,
2H), 3.90 (s, 1H), 3.20 (s, 2H), 2.91 (s, 2H), 2.63 (s, 3H), 1.95 (d, J = 13.6 Hz, 2H), 1.57 (s, 2H).
13C NMR (75 MHz, DMSO-d6) δ 163.98, 161.58, 156.51, 146.10, 144.15, 141.34, 136.29, 130.60,
123.59, 114.21, 114.00, 100.96, 99.45, 61.13, 51.58, 49.32, 43.58, 29.24. MS (ESI) m/z calculated
for C20H23FN6O [M+H]+: 383.2; found: 383.2.
4.1.6.2.
5-((3-fluorobenzyl)amino)-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[1,5-a]pyrimidine-3-
carboxamide (I-2). This compound was prepared from 7a and 4-aminotetrahydropyran as a white
solid, 93% yield; m.p: >250 °C; IR (KBr, cm-1): 3472, 3260, 3126, 3028, 1624, 1595, 1552, 1504,
1443, 1355, 1252, 915, 824, 775; 1H NMR (300 MHz, DMSO-d6) δ 8.63 (d, J = 7.7 Hz, 2H), 8.10
(s, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.40 (q, J = 7.7 Hz, 1H), 7.19 (t, J = 7.4 Hz, 2H), 7.10 (t, J = 8.8
Hz, 1H), 6.49 (d, J = 7.6 Hz, 1H), 4.65 (d, J = 5.7 Hz, 2H), 3.90 (s, 1H), 3.76 (d, J = 11.4 Hz, 2H),
1.71 (d, J = 12.7 Hz, 2H), 1.23 (d, J = 9.7 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 164.00, 161.17,
156.59, 145.99, 144.13, 141.55, 136.22, 130.54, 122.98, 113.87, 113.58, 101.20, 100.84, 72.90,
65.79, 44.13, 32.98. MS (ESI) m/z calculated for C19H20FN5O2 [M+Na]+: 392.2; found: 392.2.
4.1.6.3. 5-((3-fluorobenzyl)amino)-N-((1r,4r)-4-hydroxycyclohexyl)pyrazolo[1,5-a]pyrimidine-3-
carboxamide (I-3). This compound was prepared from 7a and trans-4-aminocyclohexanol as a white
solid, 67.2% yield; m.p: >250 °C; IR (KBr, cm-1): 3393, 3267, 1633, 1590, 1505, 1440, 1415, 1085,
912, 773; 1H NMR (300 MHz, DMSO-d6) δ 8.62 (d, J = 8.0 Hz, 2H), 8.07 (s, 1H), 7.54 (d, J = 8.0
Hz, 1H), 7.43 – 7.33 (m, 1H), 7.12 (dd, J = 23.6, 9.0 Hz, 3H), 6.48 (d, J = 7.7 Hz, 1H), 4.63 (d, J =
5.7 Hz, 2H), 4.56 (d, J = 4.2 Hz, 1H), 3.60 (s, 1H), 1.73 (d, J = 11.0 Hz, 4H), 1.20 (d, J = 12.0 Hz,
2H), 0.93 (d, J = 12.1 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 164.01, 161.16, 156.59, 145.91,
144.10, 141.65, 136.22, 130.50, 122.64, 113.77, 113.39, 101.34, 100.80, 67.92, 46.18, 43.59, 33.75,
30.67. MS (ESI) m/z calculated for C20H22FN5O2 [M+H]-: 384.2; found: 384.1836.
4.1.6.4. 5-((3-fluorobenzyl)amino)-N-((1r,4r)-4-methoxycyclohexyl)pyrazolo[1,5-a]pyrimidine-3-
carboxamide (I-4). This compound was prepared from 7a and trans-4-methoxycyclohexanamine as
a light yellow solid, 30.9% yield; m.p: 235-243 °C; IR (KBr, cm-1): 3449, 3261, 3128, 1627, 1560,
1508, 1442, 1423, 1099, 774; 1H NMR (300 MHz, DMSO-d6) δ 8.64 (d, J = 7.5 Hz, 2H), 8.09 (s,
1H), 7.59 (d, J = 8.0 Hz, 1H), 7.42 (q, J = 7.3 Hz, 1H), 7.15 (dd, J = 22.7, 8.7 Hz, 3H), 6.51 (d, J =
7.6 Hz, 1H), 4.65 (d, J = 5.7 Hz, 2H), 3.70 (d, J = 15.1 Hz, 1H), 3.24 (s, 3H), 3.05 (d, J = 11.1 Hz,
1H), 1.90 (d, J = 12.2 Hz, 2H), 1.78 (d, J = 12.6 Hz, 2H), 1.24 (t, J = 8.7 Hz, 2H), 0.95 (q, J = 12.1
Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 164.00, 161.13, 156.60, 145.92, 144.07, 141.60, 136.22,
130.52, 122.66, 113.79, 113.53, 101.31, 100.80, 77.46, 55.11, 46.01, 43.59, 30.24, 29.70. MS (ESI)
m/z calculated for C21H24FN5O2 [M+H]+: 398.2; found: 398.2.
4.1.6.5. 5-((1-(3-fluorophenyl)ethyl)amino)-N-(1-methylpiperidin-4-yl)pyrazolo[1,5-a]pyrimidine-
3-carboxamide (I-5). This compound was prepared from 7b and 4-amino-1-methylpiperidine as a
white solid, 45.5% yield; m.p: 215-219 °C; IR (KBr, cm-1): 3443, 1638, 1573, 1491, 1432, 1238,

840, 555; 1H NMR (400 MHz, DMSO-d6) δ 8.61 (d, J = 7.5 Hz, 1H), 8.54 (d, J = 7.1 Hz, 1H), 8.08
(s, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.42 (q, J = 7.4 Hz, 1H), 7.24 (dd, J = 15.4, 8.8 Hz, 2H), 7.11 (d,
J = 8.6 Hz, 1H), 6.49 (d, J = 7.6 Hz, 1H), 5.25 – 5.14 (m, 1H), 3.82 (s, 1H), 3.03 (s, 2H), 1.98 (d, J
= 12.5 Hz, 2H), 1.56 (d, J = 7.2 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 164.04, 161.40, 155.75,
147.02, 145.91, 144.22, 136.24, 130.68, 122.17, 113.99, 113.72, 101.09, 100.97, 61.12, 53.18, 49.70,
43.72, 30.55, 22.40. MS (ESI) m/z calculated for C21H25FN6O [M+H]+: 397.2; found: 397.2.
4.1.6.6.
5-((1-(3-fluorophenyl)ethyl)amino)-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[1,5-
a]pyrimidine-3-carboxamide (I-6). This compound was prepared from 7b and 4-
aminotetrahydropyran as a white solid, 62.7% yield; m.p: 177-180 °C; IR (KBr, cm-1): 3421, 2933,
1643, 1582, 1499, 1437, 1231, 1089, 849, 775; 1H NMR (400 MHz, DMSO-d6) δ 8.61 (d, J = 7.3
Hz, 1H), 8.53 (d, J = 6.9 Hz, 1H), 8.05 (d, J = 4.6 Hz, 1H), 7.36 (d, J = 9.5 Hz, 2H), 7.25 – 7.17 (m,
2H), 7.09 (d, J = 10.9 Hz, 1H), 6.51 (d, J = 7.5 Hz, 1H), 5.10 (s, 1H), 4.59 (d, J = 5.4 Hz, 1H), 3.56
(d, J = 68.1 Hz, 2H), 1.85 (d, J = 27.6 Hz, 4H), 1.64 (s, 2H), 1.55 (d, J = 6.9 Hz, 3H). 13C NMR (75
MHz, DMSO-d6) δ 164.01, 161.12, 155.75, 147.26, 145.78, 144.17, 136.16, 130.54, 121.49, 113.73,
112.62, 101.32, 100.91, 68.11, 49.88, 46.32, 34.13, 34.05, 22.95. MS (ESI) m/z calculated for
C20H22FN5O2 [M+Na]+: 406.2; found: 406.2.
4.1.6.7.
5-((1-(3-fluorophenyl)ethyl)amino)-N-((1r,4r)-4-hydroxycyclohexyl)pyrazolo[1,5-
a]pyrimidine-3-carboxamide (I-7). This compound was prepared from 7b and trans-4-
aminocyclohexanol as a white solid, 65% yield; m.p: 226-227 °C; IR (KBr, cm-1): 3449, 3283, 1630,
1591, 1500, 1439, 1231, 769; 1H NMR (400 MHz, DMSO-d6) δ 8.61 (s, 1H), 8.53 (s, 1H), 8.13 –
8.02 (m, 1H), 7.49 (s, 1H), 7.40 (s, 1H), 7.22 (d, J = 11.2 Hz, 2H), 7.08 (s, 1H), 6.50 (s, 1H), 5.17
(s, 1H), 4.09 – 3.69 (m, 4H), 3.45 (s, 2H), 1.85 (d, J = 12.3 Hz, 1H), 1.64 (d, J = 10.4 Hz, 1H), 1.56
(s, 3H), 1.48 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 164.02, 161.13, 155.77, 147.17, 145.86,
144.22, 136.19, 130.48, 121.77, 113.53, 112.52, 101.20, 100.94, 66.02, 49.78, 44.32, 33.23, 33.12,
22.73. MS (ESI) m/z calculated for C21H24FN5O2 [M+Na]+: 420.2; found: 420.2.
4.1.6.8.
5-((3-fluorobenzyl)(methyl)amino)-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[1,5-
a]pyrimidine-3-carboxamide (I-8). This compound was prepared from 7c and 4-
aminotetrahydropyran as a white solid, 70.5% yield; m.p: 219-220 °C; IR (KBr, cm-1): 3456, 1632,
1
1594, 1571, 1505, 1406, 1239, 776; H NMR (300 MHz, DMSO-d6) δ 8.80 (s, 1H), 8.18 (s, 1H),
7.74 (d, J = 77.8 Hz, 1H), 7.42 (dt, J = 9.0, 7.0 Hz, 1H), 7.20 – 7.07 (m, 3H), 6.80 (d, J = 7.9 Hz,
1H), 4.95 (s, 2H), 3.92 (s, 1H), 3.76 (s, 2H), 3.27 (s, 3H), 1.71 (s, 2H), 1.28 – 1.21 (m, 1H), 1.16 (s,
1H). 13C NMR (75 MHz, DMSO-d6) δ 164.09, 161.17, 156.45, 145.68, 144.69, 140.18, 136.84,
130.77, 122.70, 113.68, 111.23, 100.84, 99.44, 65.77, 49.32, 44.18, 36.87, 33.02. MS (ESI) m/z
calculated for C20H22FN5O2 [M+H]+: 384.2; found: 384.1.
4.1.6.9.
5-((3-fluorobenzyl)(methyl)amino)-N-((1r,4r)-4-hydroxycyclohexyl)pyrazolo[1,5-
a]pyrimidine-3-carboxamide (I-9). This compound was prepared from 7c and trans-4-
aminocyclohexanol as a white solid, 72.5% yield; m.p: 235-237 °C; IR (KBr, cm-1): 3421, 3302,
1
2925, 1638, 1572, 1508, 1419, 1223, 1090, 774; H NMR (300 MHz, DMSO-d6) δ 8.80 (s, 1H),
8.15 (s, 1H), 7.61 – 7.32 (m, 2H), 7.17 – 7.07 (m, 3H), 6.80 (d, J = 7.9 Hz, 1H), 4.93 (s, 2H), 4.58
(d, J = 4.2 Hz, 1H), 3.61 (s, 1H), 3.26 (s, 3H), 1.72 (s, 4H), 1.28 – 1.11 (m, 4H). 13C NMR (75 MHz,

DMSO-d6) δ 164.11, 161.14, 156.41, 145.59, 144.63, 140.05, 136.82, 130.72, 122.50, 113.59,
111.23, 100.97, 97.62, 67.93, 54.96, 46.17, 36.92, 33.73, 30.68. MS (ESI) m/z calculated for
C21H24FN5O2 [M+H]+: 398.2; found: 398.1992.
4.1.6.10.
5-((3-fluorobenzyl)(methyl)amino)-N-((1r,4r)-4-methoxycyclohexyl)pyrazolo[1,5-
a]pyrimidine-3-carboxamide (I-10). This compound was prepared from 7c and trans-4-
methoxycyclohexanamine as a white solid, 39.4% yield; m.p: 232-236 °C; IR (KBr, cm-1): 3449,
1
3315, 2931, 1635, 1572, 1507, 1418, 1101, 901, 773; H NMR (300 MHz, DMSO-d6) δ 8.80 (s,
1H), 8.16 (s, 1H), 7.42 (q, J = 7.7 Hz, 2H), 7.13 (d, J = 9.2 Hz, 3H), 6.80 (d, J = 7.9 Hz, 1H), 4.94
(s, 2H), 3.67 (s, 1H), 3.27 (s, 3H), 3.23 (s, 3H), 2.96 (d, J = 24.7 Hz, 1H), 1.87 (s, 4H), 1.26 (d, J =
8.8 Hz, 4H). 13C NMR (75 MHz, DMSO-d6) δ 164.12, 161.15, 156.46, 145.62, 144.64, 140.61,
136.85, 130.65, 122.48, 113.60, 111.22, 100.95, 97.68, 77.46, 55.11, 46.11, 43.43, 36.95, 30.27,
29.67. MS (ESI) m/z calculated for C22H26FN5O2 [M+H]+: 412.2; found: 412.2147.
4.1.7. Synthesis of (E)-N'-(6-chloropyridazin-3-yl)-N,N-dimethylformamidine (9).
To a solution of 3-amino-6-chloropyridazine (8, 2.0 g,15.44 mmol) in N,N-dimethylformamide
dimethyl acetal (4.13 mL, 30.88 mmol). The mixture was heated at 110 °C for 2 h. After the reaction
was completed, the reaction solution was cooled to room temperature. The solid precipitated out,
washed with ethyl acetate (20 mL × 2) and dried after filtration to obtain (E)-N-(6-chloropyridazin-
3-yl)-N,N-dimethylformamidine (9) as an off-white solid (2.5 g, 87.7% yield). 1H NMR (300 MHz,
DMSO-d6) δ 8.47 (s, 1H), 7.57 (d, J = 9.1 Hz, 1H), 7.14 (d, J = 9.1 Hz, 1H), 3.12 (s, 3H), 3.01 (s,
3H).
4.1.8. Synthesis of ethyl 6-chloroimidazo[1,2-b]pyridazine-3-carboxylate (10).
To a solution of the intermediate 9 (2.0 g, 10.83 mmol) in acetonitrile (10 mL) was added ethyl
bromoacetate (3 mL, 27.08 mmol), the reaction solution was stirred at 80 °C for 3 h. After cooling
to room temperature, a large amount of solids precipitated, which was filtered off with suction,
washed with diethyl ether (20 mL × 2) to afford yellow solid. The solid was dissolved in a solution
of N,N-diisopropylethylamine (2.27 mL, 13.00 mmol) in acetonitrile (10 mL). The reaction mixture
was stirred at room temperature for 3 h. The solvent was removed in vacuo and the residue was
washed with water to give ethyl 6- chloroimidazo[1,2-b]pyridazine-3-carboxylate (10) as a yellow
solid (1.5g, 61.4% yield). 1H NMR (300 MHz, DMSO-d6) δ 8.37 (d, J = 9.7 Hz, 2H), 7.62 (d, J =
9.5 Hz, 1H), 4.35 (q, J = 7.1 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H).
4.1.9. Synthesis of ethyl 6-((3-fluorobenzyl)amino)imidazo[1,2-b]pyridazine-3-carboxylate (11).
To a solution of the intermediate 10 (1.0 g, 4.43 mmol) in dimethyl sulfoxide (6ml) was added
3-fluorobenzylamine (666 mg, 5.32 mmol) followed by the addition of potassium fluoride dihydrate
(4.17 g, 44.32 mmol), the reaction was heated to 120 °C and stirred for 12 h. After the reaction was
completed, the reaction solution was cooled to room temperature. The residue was added water
(30ml) and extraction with EtOAc (20 mL × 3), the combined organic phase was washed with
saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated in
vacuo. The residue was purified by flash column chromatography (petroleum ether/EtOAc = 1:1)
to afford ethyl 6-((3-fluorobenzyl)amino)imidazo[1,2-b]pyridazine-3-carboxylate (11) as a light
yellow solid (850mg, 61.0% yield). 1H NMR (300 MHz, CDCl3) δ 8.14 (s, 1H), 7.71 (d, J = 9.5 Hz,

1H), 7.37 – 7.25 (m, 2H), 7.20 (d, J = 9.4 Hz, 1H), 6.99 (t, J = 8.4 Hz, 1H), 6.64 (d, J = 9.3 Hz, 1H),
5.07 (s, 1H), 4.64 (d, J = 5.6 Hz, 2H), 4.41 (t, J = 7.2 Hz, 2H), 1.46 – 1.34 (m, 3H).
4.1.10. Synthesis of 6-((3-fluorobenzyl)amino)imidazo[1,2-b]pyridazine-3-carboxylic acid (12).
The intermediate 11 (840 mg, 2.67 mmol) was dissolved in a mixed solvent of tetrahydrofuran
(8 mL) and methanol (4 mL), then was added 2 mL lithium hydroxide aqueous solution (192 mg，
8.02 mmol). The mixture was stirred at room temperature for 3 h and was diluted with water (5 mL).
The pH was adjusted to 6 with 1mol/L dilute hydrochloric acid and then dried after filtration to
obtain 6-((3-fluorobenzyl)amino)imidazo[1,2-b]pyridazine-3-carboxylic acid (12) as a light yellow
solid (640mg, 83.7% yield). 1H NMR (300 MHz, DMSO-d6) δ 12.56 (s, 1H), 7.98 (s, 1H), 7.86 (q,
J = 12.3, 9.4 Hz, 2H), 7.47 – 7.26 (m, 3H), 7.07 (s, 1H), 6.88 (d, J = 9.7 Hz, 1H), 4.48 (d, J = 5.5
Hz, 2H).
4.1.11. General procedure for the preparation of II-1~II-10.
A solution of differently substituted amines (0.210 mmol), the intermediate 12(0.175 mmol),
HATU (0.262 mmol) and N,N-diisopropylethylamine (0.349 mmol) in DMF (2 mL) for 1 h at 60 °C.
The reaction was extracted with ethyl acetate (20ml × 3), washed with water (20ml × 2) and
saturated brine (30ml × 2). The organic phase was dried over anhydrous sodium sulfate, filtered,
and concentrated. The residue was purified by flash column chromatography (CH2Cl2 :CH3OH=
30:1) to afford compounds II-1~II-10.
4.1.11.1.
6-((3-fluorobenzyl)amino)-N-(1-methylpiperidin-4-yl)imidazo[1,2-b]pyridazine-3-
carboxamide (II-1). This compound was prepared from 4-amino-1-methylpiperidine as a light
yellow solid. 22.5% yield; m.p: 195-198°C; IR (KBr, cm-1): 3651, 3418, 1638, 1491, 1400, 841,
555; 1H NMR (300 MHz, DMSO-d6) δ 8.61 (d, J = 7.3 Hz, 1H), 8.04 (t, J = 5.5 Hz, 1H), 7.97 – 7.90
(m, 2H), 7.44 (td, J = 8.0, 6.0 Hz, 1H), 7.30 – 7.22 (m, 2H), 7.14 (td, J = 8.6, 2.7 Hz, 1H), 6.95 (d,
J = 9.7 Hz, 1H), 4.53 (d, J = 5.4 Hz, 2H), 3.90 (d, J = 11.1 Hz, 1H), 3.13 (s, 2H), 2.74 (s, 2H), 2.58
– 2.53 (m, 2H), 1.93 (d, J = 9.5 Hz, 3H), 1.55 (d, J = 12.5 Hz, 2H). 13C NMR (75 MHz, DMSO-d6)
δ 164.02, 160.79, 157.61, 153.58, 141.10, 137.76, 135.37, 130.60, 126.74, 123.37, 122.01, 114.23,
113.94, 53.73, 45.51, 45.00, 44.29, 31.31. MS (ESI) m/z calculated for C20H23FN6O [M+H]+: 383.2;
found: 383.2.
4.1.11.2.
6-((3-fluorobenzyl)amino)-N-(tetrahydro-2H-pyran-4-yl)imidazo[1,2-b]pyridazine-3-
carboxamide (II-2). This compound was prepared from 4-aminotetrahydropyran as a white solid.
94.9% yield; m.p: >250 °C; IR (KBr, cm-1): 3568, 3421, 3276, 1626, 1593, 1492, 1421, 1291, 819;
1H NMR (300 MHz, DMSO-d6) δ 8.52 (d, J = 7.6 Hz, 1H), 8.07 (s, 1H), 7.98 – 7.86 (m, 2H), 7.46
– 7.35 (m, 1H), 7.23 (s, 2H), 7.11 (t, J = 8.8 Hz, 1H), 7.00 – 6.89 (m, 1H), 4.55 (d, J = 5.5 Hz, 2H),
3.93 (s, 1H), 3.77 (d, J = 11.4 Hz, 2H), 1.70 (d, J = 12.5 Hz, 2H), 1.19 (d, J = 13.3 Hz, 2H). 13C
NMR (75 MHz, DMSO-d6) δ 164.10, 160.88, 157.50, 153.66, 141.16, 137.81, 135.43, 130.52,
126.76, 123.02, 121.96, 114.00, 113.73, 65.88, 44.64, 44.21, 32.62. MS (ESI) m/z calculated for
C19H20FN5O2 [M+H]+: 370.2; found: 370.2.
4.1.11.3. 6-((3-fluorobenzyl)amino)-N-((1r,4r)-4-hydroxycyclohexyl)imidazo[1,2-b]pyridazine-3-
carboxamide (II-3). This compound was prepared from trans-4-aminocyclohexanol as a white solid.

81.2% yield; m.p: >250 °C; IR (KBr, cm-1): 3289, 1623, 1589, 1564, 1490, 1293, 1084;1H NMR
(300 MHz, DMSO-d6) δ 8.45 – 8.33 (m, 1H), 8.10 (s, 1H), 8.00 – 7.83 (m, 2H), 7.40 (d, J = 6.9 Hz,
1H), 7.18 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 9.9 Hz, 1H), 4.57 (d, J = 12.7 Hz, 2H), 3.63 (s, 1H), 1.72
(s, 4H), 1.18 (d, J = 12.2 Hz, 2H), 0.84 (d, J = 12.6 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ
164.11, 160.88, 157.42, 153.66, 141.36, 137.75, 135.31, 130.58, 126.76, 122.59, 122.01, 113.90,
113.65, 67.93, 46.52, 44.15, 33.88, 30.45. MS (ESI) m/z calculated for C20H22FN5O2 [M-H]-: 382.2;
found: 382.2.
4.1.11.4. 6-((3-fluorobenzyl)amino)-N-((1r,4r)-4-methoxycyclohexyl)imidazo[1,2-b]pyridazine-3-
carboxamide (II-4). This compound was prepared from trans-4-methoxycyclohexanamine as a
white solid. 88.5% yield; m.p: >250 °C; IR (KBr, cm-1): 3456, 3274, 3108, 1638, 1617, 1595, 1567,
1490, 1291, 1099, 685; 1H NMR (300 MHz, DMSO-d6) δ 8.42 (d, J = 8.0 Hz, 1H), 8.12 (t, J = 5.8
Hz, 1H), 8.01 – 7.88 (m, 2H), 7.45 (q, J = 7.5 Hz, 1H), 7.19 (dt, J = 17.2, 6.9 Hz, 3H), 6.98 (d, J =
9.7 Hz, 1H), 4.58 (d, J = 5.6 Hz, 2H), 3.70 (d, J = 7.5 Hz, 1H), 3.00 (t, J = 9.6 Hz, 1H), 1.92 (d, J =
12.4 Hz, 2H), 1.78 (d, J = 12.6 Hz, 2H), 1.18 (q, J = 12.9, 12.1 Hz, 2H), 0.86 (td, J = 13.5, 13.1, 6.7
Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 164.09, 160.87, 157.40, 153.65, 141.32, 137.77, 135.30,
130.61, 126.75, 122.58, 121.99, 113.89, 113.69, 77.52, 55.12, 46.38, 44.17, 30.11, 29.90. MS (ESI)
m/z calculated for C21H24FN5O2 [M+H]+: 398.2; found: 398.8.
4.1.11.5. (6-((3-fluorobenzyl)amino)imidazo[1,2-b]pyridazin-3-yl)(morpholino)methanone (II-5).
This compound was prepared from morpholine as a yellow solid. 95.6% yield; m.p: 158-160°C; IR
(KBr, cm-1): 3416, 1653, 1618, 1496, 1114, 437, 426, 409 ; 1H NMR (300 MHz, DMSO-d6) δ 7.78
(t, J = 8.7 Hz, 1H), 7.72 (t, J = 5.5 Hz, 1H), 7.59 (s, 1H), 7.37 (dd, J = 14.1, 7.8 Hz, 1H), 7.22 (d, J
= 7.6 Hz, 2H), 7.07 (t, J = 8.1 Hz, 1H), 6.82 (d, J = 9.7 Hz, 1H), 4.46 (d, J = 5.5 Hz, 2H), 3.47 (d, J
= 9.5 Hz, 4H). 13C NMR (75 MHz, DMSO-d6) δ 163.97, 160.75, 159.59, 153.63, 142.41, 142.32,
131.89, 130.36, 125.84, 123.46, 122.71, 114.37, 113.55, 66.20, 44.07, 41.60. MS (ESI) m/z
calculated for C18H18FN5O [M+H]+: 356.1; found: 356.1.
4.1.11.6. N-cyclohexyl-6-((3-fluorobenzyl)amino)imidazo[1,2-b]pyridazine-3-carboxamide (II-6).
This compound was prepared from cyclohexanamine as a white solid. 89.6% yield; m.p: >250 °C;
IR (KBr, cm-1): 3474, 3279, 2929, 1616, 1594, 1489, 428, 421, 410 ; 1H NMR (300 MHz, DMSO-d6)
δ 8.42 (d, J = 8.0 Hz, 1H), 8.09 (t, J = 5.6 Hz, 1H), 7.95 – 7.85 (m, 2H), 7.43 – 7.37 (m, 1H), 7.18
(d, J = 7.8 Hz, 2H), 7.10 (t, J = 8.4 Hz, 1H), 6.95 (d, J = 9.8 Hz, 1H), 4.56 (d, J = 5.6 Hz, 2H), 3.75
– 3.62 (m, 1H), 1.70 (d, J = 11.8 Hz, 2H), 1.56 (t, J = 15.6 Hz, 4H), 1.21 (s, 2H), 1.11 (d, J = 17.0
Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 164.08, 160.86, 157.17, 153.68, 141.22, 137.73, 135.29,
130.60, 126.77, 122.62, 122.06, 113.85, 113.38, 46.77, 44.14, 32.49, 24.91, 24.54. MS (ESI) m/z
calculated for C20H22FN5O [M+H]+: 368.2; found: 368.2.
4.1.11.7.
(6-((3-fluorobenzyl)amino)imidazo[1,2-b]pyridazin-3-yl)(4-morpholinopiperidin-1-
yl)methanone (II-7). This compound was prepared from 4-(piperidin-4-yl)morpholine as a light
yellow solid. 91.39% yield; m.p: 198-202 °C; IR (KBr, cm-1): 3451, 1618, 1560, 847, 420; 1H NMR
(300 MHz, DMSO-d6) δ 7.79 (d, J = 9.7 Hz, 1H), 7.71 (t, J = 5.8 Hz, 1H), 7.55 (s, 1H), 7.42 – 7.33
(m, 1H), 7.24 – 7.14 (m, 2H), 7.07 (td, J = 8.2, 1.8 Hz, 1H), 6.81 (d, J = 9.7 Hz, 1H), 4.44 (d, J =
5.7 Hz, 2H), 3.52 (d, J = 4.1 Hz, 4H), 3.41 (s, 4H), 3.17 (s, 1H), 2.70 (t, J = 11.5 Hz, 2H), 2.36 (d,

J = 28.7 Hz, 4H), 1.22 (dd, J = 12.5, 7.7 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 163.98, 160.76,
159.25, 153.56, 142.55, 136.06, 131.49, 130.40, 125.86, 123.40, 123.28, 114.31, 113.54, 66.47,
60.98, 49.29, 48.66, 44.05, 29.07. MS (ESI) m/z calculated for C23H27FN6O2 [M+H]+: 439.2; found:
439.2.
4.1.11.8.
[1,4'-bipiperidin]-1'-yl(6-((3-fluorobenzyl)amino)imidazo[1,2-b]pyridazin-3-
yl)methanone (II-8). This compound was prepared from 1,4'-bipiperidine as a light yellow solid.
95.1% yield; m.p: >250 °C; IR (KBr, cm-1): 3454, 1618, 1400, 472, 457, 445, 421, 405; 1H NMR
(300 MHz, DMSO-d6) δ 7.78 (d, J = 9.7 Hz, 1H), 7.71 (t, J = 5.7 Hz, 1H), 7.54 (s, 1H), 7.42 – 7.33
(m, 1H), 7.22 – 7.16 (m, 2H), 7.10 – 7.02 (m, 1H), 6.81 (d, J = 9.7 Hz, 1H), 4.45 (d, J = 5.6 Hz,
2H), 3.51 (s, 4H), 2.65 (d, J = 7.9 Hz, 1H), 2.42 (s, 4H), 1.43 (d, J = 3.8 Hz, 4H), 1.38 – 1.29 (m,
4H), 1.17 (dd, J = 8.9, 5.1 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 163.96, 160.73, 159.23, 153.55,
142.40, 136.01, 131.46, 130.36, 125.83, 123.40, 123.30, 114.31, 113.50, 61.62, 59.81, 49.53, 44.04,
25.80, 24.29, 20.80. MS (ESI) m/z calculated for C24H29FN6O [M+H]+: 437.2; found: 437.2.
4.1.11.9. N-cyclopropyl-6-((3-fluorobenzyl)amino)imidazo[1,2-b]pyridazine-3-carboxamide (II-9).
This compound was prepared from cyclopropanamine as a white solid. 82.5% yield; m.p: 238-
242 °C; IR (KBr, cm-1): 3474, 3225, 1618, 1560, 1491, 1329, 1294, 413, 404; 1H NMR (300 MHz,
DMSO-d6) δ 8.50 (s, 1H), 8.08 (s, 1H), 7.88 (s, 2H), 7.40 (d, J = 5.8 Hz, 1H), 7.16 (d, J = 33.3 Hz,
3H), 6.88 (t, J = 21.0 Hz, 1H), 4.50 (s, 2H), 2.73 (s, 1H), 0.67 (s, 2H), 0.30 (s, 2H). 13C NMR (75
MHz, DMSO-d6) δ 164.03, 160.79, 159.27, 153.41, 141.44, 137.68, 135.19, 130.59, 126.67, 122.85,
121.96, 113.93, 113.48, 44.18, 21.85, 6.38. MS (ESI) m/z calculated for C17H16FN5O [M+H]+:
326.1; found: 326.2.
4.1.11.10. N-cyclopentyl-6-((3-fluorobenzyl)amino)imidazo[1,2-b]pyridazine-3-carboxamide (II-
10). This compound was prepared from cyclopentanamine as a white solid. 93.1% yield; m.p: 239-
1
242 °C; IR (KBr, cm-1): 3474, 3275, 1618, 1596, 1492, 473, 421, 416, 410, 401; H NMR (300
MHz, DMSO-d6) δ 8.51 (d, J = 7.5 Hz, 1H), 8.05 (t, J = 5.4 Hz, 1H), 7.90 (d, J = 11.6 Hz, 2H), 7.40
(dd, J = 14.2, 7.7 Hz, 1H), 7.25 – 7.14 (m, 2H), 7.10 (t, J = 8.2 Hz, 1H), 6.94 (d, J = 9.7 Hz, 1H),
4.54 (d, J = 5.5 Hz, 2H), 4.17 (h, J = 6.9 Hz, 1H), 1.81 (dd, J = 11.6, 5.8 Hz, 2H), 1.48 (s, 4H), 1.16
– 1.09 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ 164.06, 160.84, 157.70, 153.65, 141.25, 137.73,
135.22, 130.59, 126.73, 122.83, 122.11, 113.96, 113.49, 49.74, 44.10, 32.64, 23.31. MS (ESI) m/z
calculated for C19H20FN5O [M+H]+: 354.2; found: 354.2.
4.2. Enzyme activity using caliper mobility shift assay.
The compound was dissolved in 100% DMSO to prepare a 10 mM stock solution. 1×Kinase
buffer and compound concentration gradient were prepared. Next, 2.5 times the final concentration
of kinase solution with 1×Kinase buffer was prepared. Added 10 μL of 2.5 times the final
concentration of kinase solution to the compound wells and the positive control well respectively,
and added 10 μL of 1×Kinase buffer to the negative control well. After that, the mixture plate was
centrifuged at 1000 rpm for 30 seconds, shook the reaction plate and incubated at room temperature
for 10 minutes, and used 1×Kinase buffer to prepare a mixed solution of 5/3 times the final
concentration of ATP and Kinase substrate 22. Started the reaction by adding 15 μL of a mixed
solution of 5/3 times the final concentration of ATP and substrate. The reaction was centrifuged in

a centrifuge at 1000 rpm for 30 seconds, shook and mixed, and incubated at room temperature for
30 minutes. Finally, added 30 μL stop detection solution to stop the kinase reaction, centrifuged at
1000 rpm for 30 seconds, shook and mixed well. Used Caliper EZ Reader to read the conversion
rate and calculate the inhibition rate by formula. Calculated the inhibitory rate of the compound on
the kinase at each concentration, and then used the GraphPad Prism 5 software to fit the dose-effect
curve to obtain the IC50 value of the compound on the kinase activity.
4.3. Cell viability assay
Resuspended the cells and counted them with an automatic cell counter. According to the
seeding density of BaF3 cells, 2000 cells per well, the cell suspension was diluted to the required
density. Then spread 95 μL of cells in each well and cultivated stable equilibrium at 37 °C. The
compound was formulated into a diluted solution with a final concentration of 1000 times. The
compound was diluted with culture medium to prepare a compound with a final concentration of 20
times. Added 5 μL of compound to each well. Used the same volume of DMSO as the control. Next,
incubated for 72 hours at 37 °C and 5% CO2. Equilibrated the cell plate to room temperature. Added
40 μL of CellTiter-Glo reagent to each well, shook for 2 minutes, and let stand for 10 minutes. Used
SpectraMax Paradigm to detect and GraphPad Prism5 to calculate IC50.
4.4. Docking study.
Docking studies were processed with Glide docking in Schrodinger 2018-1. The TRKA crystal
complex structure (PDB ID: 4YME, 6DKB) was obtained from the RSCB protein database, then
opened the Maestro 11.2 module, imported the obtained TRKA crystal complex into this module,
and used the Protein Preparation Wizard module to pretreat and optimize the protein (on this basis,
the Residue Loop Mutation module can induce the G595R mutation of the protein to obtain the
TRKA protein with site-directed mutation). Hydrogen atoms were added to the structure and the
water was removed. All ligand molecules were drawn in ChemDraw 14.0, and saved as sdf style.
Then ligand was processed at a simulated pH of 7.4 ± 1.0 to generate all possible tautomers,
stereoisomers, and protonation states and were finally minimized at the OPLS force field with
Ligand preparation protocol of Maestro 10.6. After docking finished, only one docking
conformation was saved for every compound. All the graphical images were produced using Pymol.
Acknowledgement
This study was supported by the Natural Science Foundation of Jiangsu Province (No. BK
20141349) and the China National Key HiTech Innovation Project for the R&D of Novel Drugs
(No. 2013ZX09301303-002).
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Highlights
1. A new class of TRK inhibitors were designed and synthesized.
2. Synthesized compounds were evaluated for their ability to inhibit TRKA and TRKAG595R
.
3. Cell proliferation inhibition was assessed in Ba/F3-LMNA-NTRK1 and Ba/F3-LMNA-
NTRK1-G595R cell lines.
4. I-10 have better cell activity to overcome drug resistance compared with the positive control
drug LOXO-101 in Ba/F3-LMNA-NTRK1-G595R cell line.
Declarations of interest: the authors have no relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial conflict with the subject matter or
materials discussed in the manuscript.