Design, synthesis and biological evaluation of 3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-2-methylbenzamides as potent and selective pan-tropomyosin receptor kinase (TRK) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.06.064

A series of 3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-2-methylbenzamides was designed and synthesized as new tropomyosin receptor kinases (Trks) inhibitors by utilizing a structure-guided optimization strategy. One of the most potent compounds 9o suppressed TrkA/B/C with IC₅₀ values of 2.65, 10.47 and 2.95 nM, respectively. The compound dose-dependently inhibited brain-derived neurotrophic factor (BDNF)-mediated TrkB activation and suppressed migration and invasion of SH-SY5Y-TrkB neuroblastoma cells expressing high level of TrkB. Inhibitor 9o also inhibited the proliferation of SH-SY5Y-TrkB cells with an IC₅₀ value of 58 nM, which was comparable to that of an US FDA recently approved drug LOXO-101. Compound 9o may serve as a new lead compound for further anti-cancer drug discovery.

Full text of DOI:10.1016/j.ejmech.2019.06.064

Accepted Manuscript
Design, synthesis and biological evaluation of 3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-2-
methylbenzamides as potent and selective pan-tropomyosin receptor kinase (TRK)
inhibitors
Shenyang Cui, Yongjin Wang, Yuting Wang, Xia Tang, Xiaomei Ren, Lei Zhang,
Yong Xu, Zhang Zhang, Zhimin Zhang, Xiaoyun Lu, Ke Ding
PII:
S0223-5234(19)30591-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.064
EJMECH 11467
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 February 2019
Revised Date: 21 June 2019
Accepted Date: 21 June 2019
Please cite this article as: S. Cui, Y. Wang, Y. Wang, X. Tang, X. Ren, L. Zhang, Y. Xu, Z. Zhang,
Z. Zhang, X. Lu, K. Ding, Design, synthesis and biological evaluation of 3-(imidazo[1,2-a]pyrazin-3-
ylethynyl)-2-methylbenzamides as potent and selective pan-tropomyosin receptor kinase
(TRK) inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.06.064.
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ACCEPTED MANUSCRIPT
GA
Design, Synthesis and Biological Evaluation of
3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-2-methylbenzamides as Potent
and Selective pan-Tropomyosin receptor kinase (TRK) Inhibitors
Shenyang Cui,a,b# Yongjin Wang,c# Yuting Wang,c# Xia Tang,c Xiaomei Ren,c Lei Zhang,d Yong Xu,a,b
Zhang Zhang,c* Zhimin Zhang,c* Xiaoyun Lu,c* Ke Dingc*
A series of 3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-2-methylbenzamides was designed and synthesized
as new Trk inhibitors. 9o suppressed TrkA/B/C with IC50 values of 2.65, 10.47 and 2.95 nM,
respectively. 9o also inhibited the proliferation of SH-SY5Y-TrkB cells with an IC50 value of 58 nM,
and dose-dependently inhibited the BDNF-mediated TrkB activation and suppressed migration and
invasion of SH-SY5Y-TrkB neuroblastoma.

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of
3-(Imidazo[1,2-a]pyrazin-3-ylethynyl)-2-methylbenzamides as New
Tropomyosin receptor kinase (Trk) Inhibitors
Shenyang Cui,a,b# Yongjin Wang,c# Yuting Wang,c# Xia Tang,c Xiaomei Ren,c Lei Zhang,d Yong Xu,a,b
Zhang Zhang,c* Zhimin Zhang,c* Xiaoyun Lu,c* Ke Dingc*
a Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, No. 190 Kaiyuan
Avenue, Guangzhou 510530, China
b University of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing 100049, China
c
International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative
Drug Development of Chinese Ministry of Education (MOE), Guangzhou City Key Laboratory of
Precision Chemical Drug Development, School of pharmacy, Jinan University, No. 601 Huangpu
Avenue West, Guangzhou 510632, China.
d National Facility for Protein Science in Shanghai, Zhangjiang Lab, Shanghai, 201210, China.
# These authors contributed equally.
*Corresponding authors: Tel: +86-20-85223764. E-mail: dingke@jnu.edu.cn.
Abstract
A
series of 3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-2-methylbenzamides was designed and
synthesized as new tropomyosin receptor kinases (Trks) inhibitors by utilizing a structure-guided
optimization strategy. One of the most potent compounds 9o suppressed TrkA/B/C with IC50 values of
2.65, 10.47 and 2.95 nM, respectively. The compound dose-dependently inhibited brain-derived
neurotrophic factor (BDNF)-mediated TrkB activation and suppressed migration and invasion of
SH-SY5Y-TrkB neuroblastoma cells expressing high level of TrkB. Inhibitor 9o also inhibited the
proliferation of SH-SY5Y-TrkB cells with an IC50 value of 58 nM, which was comparable to that of a
US FDA recently approved drug LOXO-101. Compound 9o may serve as a new lead compound for
further anti-cancer drug discovery.
Introduction
Tropomyosin receptor kinases (i.e. TrkA, TrkB and TrkC) are encoded by NTRK1, NTRK2 and
NTRK3 genes, respectively, belong to the tyrosine receptor kinase superfamily. Trks are activated by
neurotrophin family of growth factors including nerve growth factor (NGF), brain-derived neurotrophic
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factor (BDNF), Neurotrophin-4 (NT-4) and Neurotrophin-3 (NT-3) [1]. Activation of Trks importantly
affects differentiation, growth and survival of neurons. Particularly, Trks are pivotally involved in the
synaptic strength and plasticity of the mammalian central system and peripheral nervous system [1-3].
Lack of Trks would result in incapacitation and deficiencies of neurons. For instance, the population of
corneal sensory neurons is markedly depleted in TrkA (–/–) mice [4], animals lacking TrkB in
parvalbumin-positive cells displayed sexually dimorphic behavioral phenotypes [5], while TrkC
knockout mice exhibited profound deficiencies in CNS glial cells [6]. In tumor tissues, upon binding
with the ligands, Trks are activated and downstream signals involving Ras/Erk, PI3K/Akt, and
PLC-γ/PKC transduction pathways are triggered to stimulate cancer cell differentiation, proliferation,
survival, angiogenesis, migration and metastasis [7, 8]. NTRKs gene fusion has been detected in
multiple human cancers to express chimeric Trk proteins and leads to spontaneous ligand-independent
dimerization and subsequent pathological activation of transduction pathways [7-9].
Neuroblastoma (NB) is one of most common and lethal solid tumors in children, for which less than
50% of patients classified as high-risk achieved long-term survival [10, 11]. Although TrkA and TrkC
are commonly expressed in low-stage neuroblastomas, and are considered as predictive markers of a
favorable outcome, TrkB and its ligand BDNF are highly expressed in more aggressive and fatal
neuroblastomas [12, 13]. Collective investigations suggested that TrkB was heavily involved in
proliferation and migration of NB cells and BDNF was able to enhance survival of TrkB-expressing
neuroblastoma cell line SMS-KCM in serum-free media [12, 14-16]. TrkB expression in
neuroblastomas is also associated with drug resistance and expression of angiogenic factors [17, 18].
Pan-Trk inhibitors, e.g. Entrectinib (1) and AZ623, had been demonstrated to strongly suppress
BDNF-mediated proliferation of SH-SY5Y-TrkB neuroblastoma cells stably expressing high level of
TrkB [15, 16]. Our previous investigation also showed that a novel Trk inhibitor GZD2202 effectively
suppressed BDNF-mediated proliferation and metastasis in neuroblastoma models [19]. Moreover,
studies also showed that TrkB contributes greatly to the poor prognosis of NB patients [8, 12]. These
results collectively suggested Trks as highly promising molecular targets for new anti-NB drug
discovery [8, 20].
A number of small molecule Trk inhibitors have been reported to date [20-23] (Figure 1). Among
them, a type-I Trk inhibitor Larotrectinib (LOXO-101, 2) was recently approved by U.S. Food and
Drug Administration (FDA) for treatment of multiple cancers containing NTRKs fusions [24-26].
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Entrectinib (1) represents another promising type-I Trk inhibitor which is currently in phase II clinical
trials and has demonstrated robust therapeutic efficacy in patients with NTRKs, ROS1, and ALK
fusions, regardless of cancer types [27-30]. Most recently, RIPMICF16 (3) and TRACK (4) were also
reported as novel Type I Trk inhibitors [31, 32]. Several other type-II multi-target kinase inhibitors,
e.g. cabozantinib (5), sitravatinib (6) and altiratinib (7), are also in different stages of clinical
investigation for treatment of cancer patients with NTRKs fusions [33-36]. Nevertheless, most of the
Trk inhibitors are still suffering from relatively low target specificity. Moreover, acquired drug
resistance against the available Trk inhibitor drugs was reported [37, 38]. It is highly desirable to
discover new selective Trk inhibitors with distinct chemical scaffolds as new potential
anti-neuroblastoma (NB) drugs.
Figure 1. Chemical structures of representative Trk inhibitors.
3-(Imidazo[1,2-a]pyrazin-3-ylethynyl)-4-methyl-N-(3-((4-methylpiperazin-1-yl)methyl)-5-(trifluoro
methyl)phenyl)benzamide (8) is a highly potent Abelson tyrosine kinase (Abl) inhibitor recently
discovered in our laboratory [39]. Interestingly, this compound also exhibited strong suppressive effect
against TrkA/B/C with IC50 values of 8.2, 9.4 and 4.4 nM, respectively. Aiming to eliminate the Abl
inhibitory potency which may cause potential cardiotoxicity by inducing mitochondrial dysfunction [40,
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41], a series of 3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-2-methylbenzamide derivatives were designed
and synthesized as new selective Trk inhibitors.
Figure 2. Structure-based optimization of 3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-2-methylbenzamides
as new selective Trk inhibitors.
Chemistry
The designed molecules were readily prepared using palladium-catalyzed Sonogashira coupling
reactions [42] as the key steps (Scheme 1). Briefly, commercially available 3-bromobenzoic acid or
3-iodobenzoic acid (10) were treated with sulfurous dichloride in methanol to yield the methyl
benzoates (11). The methyl benzoates (11) were reacted with excessive amounts of
ethynyltrimethylsilane under palladium catalysis to afford the Sonogashira coupling products, which
were further deprotected with K2CO3 to produce the terminal alkynes (12). The coupling of compounds
12 with commercially available or self-prepared heterocyclic bromides 13 (Scheme S1) under
Sonogashira conditions afforded the intermediate methyl benzoates 14. Methyl benzoates 14 were
treated with 3-((4-methylpiperazin-1-yl)methyl)-5-(trifluoromethyl)aniline which were prepared based
on the previously reported protocols [43] under basic conditions to produce designed inhibitors 9a-9i,
9k and 9l. Intermediates 14 were hydrolyzed to generate benzoic acids 15. The condensation of benzoic
acids 15 with a series of substituted 3-((4-methylpiperazin-1-yl)methyl) anilines which were prepared
based on the previously reported protocols [43] afforded the corresponding amides, which were further
deprotected with trifluoroacetic acid to produce the designed inhibitors 9j, 9m-9q.
Scheme 1. Syntheses of the designed molecules 9a-9q. a
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a Reagents and conditions: (a) sulfurous dichloride, methanol (MeOH), 60 , 6 h, ~95%. (b) (i)
trimethylsilyl acetylene, CuI, Bis(triphenylphosphine)palladium(II) chloride (PdCl2(PPh3)2),
triethylamine (Et3N), acetonitrile (MeCN), 80 , 12 h; (ii) K2CO3, MeOH, rt, 2 h, 50−92% (two steps).
(c) CuI, PdCl2(PPh3)2, N-ethyl-N-isopropylpropan-2-amine (DIPEA), N, N-dimethylformamide (DMF),
80 , 18 h, 40~88%. (d) 3-((4-Methylpiperazin-1-yl)methyl) -5- (trifluoromethyl)aniline, potassium
tert-butoxide (t-BuOK), tetrahydrofuran (THF), −20 °C to rt, 1 h, 40~70%. (e) Lithium hydroxide
(LiOH), THF: H2O = 2 : 1, rt, 4 h, 50%~70%. (f) 1-[Bis(dimethylamino)methylene]-1H-1, 2, 3
-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), DIPEA, DMF, rt, 6 h, 50~65%. (g)
trifluoroacetic acid (CF3COOH), dichloromethane (DCM), rt, 6 h, 80~90%.
Results and Discussion
Sequence analysis of TrkA/B/C and Abl kinase domains revealed that TrkA/B/C share 34%, 36%
and 37% sequence identity with Abl in their kinase domains, respectively (Figure S1). A preliminary
docking investigation suggested that 8 could bind with the inactive configurations of TrkA/B/C and
Abl with similar type-II binding modes (data not shown). The investigation also suggested that the
middle methylphenyl moiety of 8 adopted a different orientation in Trks (e.g. TrkC) comparing with it
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in Abl (Figure 3A and 3B). This variation could be explained by the fact that methylphenyl moiety of 8
might form a potential π−π stacking interaction with Phe617 in TrkC, while the corresponding residue
in Abl is Thr315 (Figure 3B). Investigation further implied that switching methyl group from 4’-
position to other positions of the phenyl group could be a feasible strategy to improve the target
selectivity against Trks over Abl. Based on this hypothesis, 9a, 9b and 9c, in which a methyl was
introduced at 5’-, 6’- or 2’- position of the corresponding phenyl moiety, respectively, were first
designed and synthesized (Scheme 1). It was shown that the modification indeed significantly
decreased inhibitory potencies against Abl (Table 1). However, the compounds also displayed
obviously less Trks suppressive activities comparing to the lead molecule 8. Significantly,
6’-methaylted molecule 9b totally abolished inhibitory function against all of 3 isoforms of Trks.
Although the 2’-methyl analogue 9c exhibited 3~5-fold less potencies with IC50 values of 39.78, 38.35
and 11.46 nM, against TrkA, B and C, respectively, its inhibition against Abl decreased more than 500
folds. Further investigation also revealed that removal of 2’- methyl group in 9c barely affected its
potency against Trks (9d), but the modification obviously rescued Abl inhibition with an IC50 value of
33.31 nM. These data collectively suggested that 2’- position might be an optimal position for further
structural modification to increase target selectivity of the compounds. However, structure activity
relationship (SAR) exploration suggested that this position was not tolerated to a variety of substituents
such as chloride (9e), methoxyl (9f), ethyl (9g), n-propyl (9h) and i-propyl (9i).
Figure 3. A) Docking studies of TrkC (PDB: 3V5Q) and compound 8; the key residues of TrkC kinase
is shown in blue stick and ribbon representation. Compound 8 is shown in orange stick structure.
Hydrogen bonds are indicated by yellow dashed lines to key amino acids and π−π stacking interaction
is indicated by black dashed lines. B) Superposition of the docking models of TrkC-8 and Abl (PDB:
3OXZ) -8 complexes. The residue thr315 and 8 in Abl are shown in green stick.
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Table 1. In vitro inhibitory activities of compounds 9a-9i against TrkA, TrkB, TrkC and Abl kinases. a
R2
N
R3
N
N
O
N
2'
N
N
H
6'
4'
R1
5'
TrkA
TrkB
TrkC
Abl
Compound
R1
R2
R3
IC50(nM)
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
8.24±1.4
9.37±2.76
4.35±0.08
2.14±0.73
CH3(4’-)
CH3(5’-)
CH3(6’-)
CH3(2’-)
H
H
H
8
9a
9b
9c
9d
9e
9f
132.25±1.6 178.45±23. 66.98±20.9 449.1±56.9
H
H
H
H
H
H
H
H
>1µM
>1µM
934.9±261.
11.46±1.03
>1µM
>1µM
39.78±8.2
38.35±9.3
41.61±0.65 43.92±3.74 11.48±3.36 33.31±14.0
226.55±34. 253.65±20. 44.58±5.31
>1µM
>1µM
>1µM
>1µM
>1µM
>1µM
>1µM
>1µM
>1µM
>1µM
>1µM
>1µM
>1µM
>1µM
Cl(2’-)
>1µM
186±18.82
>1µM
>1µM
315.13±6.0
>1µM
692.5±229.
129±27.35
>1µM
OCH3(2’-)
Et(2’-)
9g
9h
9i
n-Pr(2’-)
>1µM
>1µM
>1µM
i-Pr(2’-)
CH3(2’-)
7.75±2.56
29.2±6.25
28.79±2.3
13.11±3.75
5.71±1.79
9j
CH3(2’-)
CH3(2’-)
CH3(2’-)
CH3(2’-)
CH3(2’-)
CH3(2’-)
CH3(2’-)
22.73±8.69 16.8±10.52
43.47±31.9 8.84±0.76
9k
9l
17.43±3.97 41.34±19.9 14.55±5.52
Me
Et
9m
9n
9o
9p
3.56±0.44
2.65±0.26
6.4±1.46
9.3±1.3
4.1±1.16
2.95±0.21
6.1±2.37
6.52±0.07
3.49±0.25
10.47±2.58
15.59±3.93
24.19±7.1
5.21±1.41
i-Pr
t-Bu
Ph
7.34±0.48
3.34±0.15
9q
LOXO-101
a Trks and Abl activity experiments were performed using the FRET-based Z′-Lyte assay according to
the manufacturer's instructions [44]. The data are mean values from at least three independent
experiments.
We were able to solve a 2.0 Å X-ray co-crystal structure of 9c-TrkC complex to elucidate the detail
interaction of 9c with Trks (Figure 4, Table S1). It was shown that 9c fitted nicely into the ATP binding
site of TrkC with a similar type-II binding mode to that was predicted (Figure 3A). The
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imidazo[1,2-a]pyrazine moiety of 9c formed an essential hydrogen bond with Met620 in the hinge
region. Two additional hydrogen bonds were also formed between the amide and Glu588, and Asp697
from DFG-motif, respectively, while the methyl fitted nicely into a small hydrophobic pocket formed
by the residues Val601, Leu686, and Phe698. In addition, the methylphenyl of 9c indeed formed a π−π
stacking interaction with Phe617.
Figure 4. Co-crystal structure of TrkC and 9c (PDB: ***). The key residues of TrkC kinase are shown
in blue stick and ribbon representation. Compound 9c is shown in yellow stick structure. Hydrogen
bonds are indicated by yellow dashed lines to key amino acids and π−π stacking interaction is indicated
by black dashed lines.
Analysis of TrkC-9c structure suggested that a polar recess formed by Asp624, Arg683, Leu686 and
Phe698 could be accessible by the imidazo[1,2-a]pyrazine moiety of 9c (Figure 5A). A hydrophilic
substituent at R2 might capture additional interaction with this pocket to achieve improved potency.
Indeed, the 4-aniline substituted compound (9j) exhibited approximately a 3-fold improved potency
with IC50 values of 7.75, 13.11 and 5.71 nM against TrkA, B and C, respectively, while the phenyl (9k)
and pyridine (9l) derivatives were equally potent to 9c (Table 1). Structural analysis also showed that
the CF3 group in 9c bound deeply into a hydrophobic pocket formed by the DFG-out conformation.
Consequently, SAR investigation was conducted to introduce various hydrophobic groups at R3
position. It was found that this position was well tolerated to a variety of hydrophobic substituents with
different sizes. Among all of molecules designed and synthesized, the i-Pr substituted derivative (9o)
displayed the most potent inhibitory activities against TrkA, B and C with IC50 values of 2.65, 10.47
and 2.95 nM, respectively, which was equally potent to the US FDA recently approved drug
LOXO-101.
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A 1.7 Å resolution X-ray crystallographic structure of TrkC-9o complex was further determined to
elucidate detail interactions between 9o and Trks (Figure 5B, Table S1). It was shown that 9o bound to
TrkC with a similar type-II binding mode to that of 9c. In accordance with the prediction, 4-aniline
moiety indeed extended to the polar groove and the amino group formed hydrogen bonds network with
Asp624, Lys627 and Arg683 mediated by waters (Figure 5B). Whereas, the i-Pr group fitted nicely into
the hydrophobic pocket formed by the DFG-out conformation.
Figure 5. A) A small polar recess formed by Asp624, Arg683, Leu686 and phe698 in the crystal
structure of TrkC-9c. The key residues of TrkC kinase are shown in gray stick and ribbon
representation. Compound 9c is shown in yellow stick structure. B) The crystal structure of TrkC and
9o (PDB: ***). The key residues of TrkC kinase are shown in gray stick and ribbon representation.
Compound 9o is shown in yellow stick structure. Hydrogen bonds are indicated by yellow dashed lines
to key amino acids and π−π stacking interaction is indicated by black dashed lines.
Inhibitory effects of compound 9o on activation of TrkB and its downstream signals, e.g. Akt, Erk
and PLC-γ, were also investigated in SH-SY5Y-TrkB cells stably expressing high level of TrkB
(Figure 6). It was shown that 9o dose-dependently inhibited the phosphorylation of TrkB and
downstream signaling proteins Akt, Erk, PLC-γ. Substantial inhibition of TrkB activation by 9o was
observed at a concentration as low as 0.64 nM, and almost complete inhibition could be achieved at 16
nM or higher concentrations (Figure 6). However, no obvious effect was observed on Akt, Erk or
PLC-γ signals by BDNF or 9o in SH-SY5Y parental cells with low level of TrkB (Supporting
Information). These data collectively suggested the selective inhibition of 9o on the target.
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Figure 6. 9o inhibited TrkB activation induced by BDNF in SH-SY5Y-TrkB cells. SH-SY5Y-TrkB
cells were pretreated with 0 to 2.0 µmol/L of 9o for 6 h followed by BDNF (10 ng/mL) treatment for 30
min or not, then harvested and lysed. Cell lysates were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analysed by Western blot for
phosphorylated TrkB, TrkB, phosphorylated PLC-γ1, PLC-γ1, phosphorylated Akt (Ser473, Thr308),
Akt, phosphorylated Erk and Erk. GAPDH was used as a control.
The migration inhibitory effect of 9o on SH-SY5Y-TrkB neuroblastoma cells was initially
investigated with a well-established wound healing assay [45]. It was shown that treatment of 9o
effectively inhibited migrating process in SH-SY5Y-TrkB cells, suppressing wound closure induced by
BDNF (10 ng/mL) by 50.1%, 76.6% and 77.5% at concentrations of 8, 40 and 200 nM, respectively,
compared to the untreated control (Figure 7A and 7B). Migration and invasion inhibitory potency of 9o
against SH-SY5Y-TrkB neuroblastoma cells was further validated by a standard transwell assay, which
showed that 9o dose-dependently inhibited migration and invasiveness of SH-SY5Y-TrkB
neuroblastoma cells. Treatment with 9o at 8, 40 or 200 nM for 24 h inhibited cancer cell migration by
22.7%, 53.3%, 82.0%; and inhibited invasion by 9.1%, 61.9% and 85.5% respectively (p < 0.05),
compared to the BDNF (10 ng/mL) treatment; (Figure 7C and 7D). Collectively, these results suggest
the promising potential of 9o to produce an anti-metastasis effect in SH-SY5Y-TrkB neuroblastoma
cells.
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Figure 7. 9o suppresses wound healing, migration and invasion induced by BDNF in SH-SY5Y-TrkB
neuroblastoma cells. A) Effect of 9o on the wound healing induced by BDNF in SH-SY5Y-TrkB
neuroblastoma cells. B) Quantitative analysis of wound healing. C) Compound 9o suppresses migration
and invasion induced by BDNF (10 ng/mL) of neuroblastoma cells. D) Quantitative analysis of
migration and invasion. The results are presented as the mean ± standard deviation (* P < 0.05, ** P <
0.01, compare with BDNF treatment by two-tailed Student’s t test).
The antiproliferative activity of 9o was also examined against SH-SY5Y-TrkB cell lines. It was
shown that 9o inhibited the proliferation of SH-SY5Y-TrkB cells with an IC50 value of 58 nM, which
was comparable to that of LOXO-101 (Figure 8). However, both 9o and LOXO-101exhibited
significantly less potency in SH-SY5Y parental cells with low level of TrkB. Further pharmacokinetics
investigation also revealed the compound exhibited low blood-brain-barrier (BBB) penetration ability,
which may avoid the potential neurotoxicity issue of a pan-Trk inhibitor (Supporting Information).
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Figure 8. Anti-proliferative activities of compound 9o and LOXO-101 in SH-SY5Y-TrkB cells. The
data were means from at least three independent experiments.
Conclusion
A
series of 3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-2-methylbenzamides were designed and
synthesized as new selective Trks inhibitors. One of the most promising compounds 9o exhibited IC50
values of 2.65, 10.47, 2.95 nM against TrkA, B and C, respectively. Compound 9o dose-dependently
inhibited the phosphorylation of TrkB and downstream signaling proteins in the SH-SY5Y-TrkB
neuroblastoma cells. In addition, 9o also dose-dependently inhibited the migration and invasiveness of
SH-SY5Y-TrkB neuroblastoma cells as well as cellular proliferation. 9o exhibited the promising
anti-proliferative activity against SH-SY5Y-TrkB cells with an IC50 of 58 nM. Further
pharmacokinetics investigation also suggested that compound 9o was peripherally restricted,
avoiding the potential neurotoxic issue of a pan-Trk inhibitor. Further structural optimization and
extensive biological investigation are undergoing and the results will be disclosed in due course.
Experimental section
General Methods for Chemistry
Reagents and solvents were obtained from commercial suppliers and used without further
purification. Flash chromatography was performed using silica gel (200-300 mesh or 300-400 mesh).
All reactions were monitored by (TLC), and silica gel plates with fluorescence F254 were used and
visualized with UV light. 1H and 13C NMR spectra were recorded on a Bruker AV-400 spectrometer at
400 MHz and Bruker AV-500 spectrometer at 125 MHz, respectively. Coupling constants (J) are
expressed in hertz (Hz). Chemical shifts (δ) of NMR are reported in parts per million (ppm) units
relative to internal control (TMS). The low resolution of ESI-MS was recorded on an Agilent 1200
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HPLC-MSD mass spectrometer, and high resolution of ESI-MS was recorded on an Applied
Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer. The purity of compounds was
determined to be over 95% (>95%) by reverse-phase high performance liquid chromatography (HPLC)
analysis. HPLC instrument: DIONEX SUMMIT HPLC (reverse-phase column, Diamonsil C18, 5.0 µm,
4.6 mm × 250 mm (Dikma Technologies); detector, PDA-100 photodiode array; injector, ASI-100
autoinjector; pump: P-680A). Elution: 85% MeOH in water (0.1% ammonia water); flow rate, 1.0
mL/min.
Methyl 3-bromo-5-methylbenzoate (11a). 3-bromo-5-methylbenzoic acid (5.0 g, 23.3 mmol) was
dissolved in dry methanol (150 mL) and cooled to 0 °C. Thionyl chloride (5.0 ml, 68.8 mmol) was
added dropwise over 10 min. The solution allowed to reach room temperature and then heated to 60
for 6 h. Removal of MeOH under reduced pressure resulted in crude product. The crude product was
treated with 100 mL water, and a saturated solution of NaHCO3 (50 mL) was added slowly. The
aqueous solution was extracted ethyl acetate (3 × 100 mL). The combined organic layers were washed
with brine (100 mL), dried over by magnesium sulfate and the solvent was removed in vacuo to get
product as yellow oil (5.0 g, 21.8 mmol), yielded 93.6%. 1H NMR (400 MHz, DMSO-d6) δ 7.84 (s, 1H),
7.76 (s, 1H), 7.71 (s, 1H), 3.85 (s, 3H), 2.37 (s, 3H). LC-MS (ESI) m/z 229 [M + H] +.
Methyl 3-ethynyl-5-methylbenzoate (12a). Step (I): To a solution of 11a (500 mg, 1.81 mmol) and
DIPEA (468 mg, 3.62 mmol) in 100 mL dry MeCN, CuI (34 mg, 0.18 mmol) and Pd(PPh3)2Cl2 (70 mg,
0.1 mmol) was added. The solution was then degassed with argon for 5 min. Ethynyltrimethylsilane
(267 mg, 2.72 mmol) was added dropwise via syringe under argon and the solution was stirred at 80
for 12 h. The reaction suspension was cooled to room temperature and filtered through Celite. The
solvent was removed by rotary evaporation to get crude mixture. Step (II): The crude mixture was
dissolved in 100 mL MeOH, and K2CO3 (500 mg, 3.62 mmol) was added to the solution. The
suspension was stirred at room temperature for 2 h. The reaction suspension was filtered through Celite,
and the solvent was removed by rotary evaporation. The residue was treated with water and extracted
with ethyl ether. The combined organic layer was washed with brine and dried by magnesium sulfate.
After the removal of solvent via rotary evaporation, the crude product was purified with silica gel
column chromatography (ethyl acetate/petroleum ether, v/v
=
1/100) to provide methyl
3-ethynyl-5-methylbenzoate (230 mg 1.32 mmol) as light red oil, yield 73%. 1H NMR (400 MHz,
DMSO-d6) δ 7.80 (s, 1H), 7.78 (s, 1H), 7.58 (s, 1H), 4.27 (s, 1H), 3.85 (s, 3H), 2.37 (s, 3H). LC-MS
13

ACCEPTED MANUSCRIPT
(ESI) m/z 175 [M + H] +.
Methyl 3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-5-methylbenzoate (14a). A round bottom flask was
charged with 12a (250 mg, 0.86 mg), 3-bromoimidazo[1,2-a]pyrazine (255 mg, 1.29 mmol),
Pd(PPh3)2Cl2 (30 mg, 0.043 mmol), CuI (16 mg, 0.086 mmol). 80 mL dry DMF and DIPEA (333 mg,
2.58 mmol) were added to the flask and the solution was degassed with argon for 5 min. The solution
was stirred under argon atmosphere at 80 for 18 h. The reaction solution was filtered through Celite
and the solvent was removed by rotary evaporation to get crude mixture. The crude mixture was
purified with silica gel column chromatography (MeOH/DCM, v/v = 1/100) to provide Mthyl
3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-2-methylbenzoate (160 mg, 0.55 mmol) as light yellow solid,
1
yielded 64%. H NMR (400 MHz, DMSO-d6) δ 9.20 (s, 1H), 8.81 (dd, J 1 = 4.4 Hz, J 2 = 1.2 Hz), 8.23
(s, 1H), 8.12 (d, J = 4.4 Hz), 8.06 (s, 1H), 7.85 (s, 1H), 7.82 (s, 1H). LC-MS (ESI) m/z 292 [M + H] +.
3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-5-methyl-N-(3-((4-methylpiperazin-1-yl)methyl)-5-
(trifluoromethyl)phenyl)benzamide (9a). To a solution of 14a (160 mg, 0.55 mmol) in anhydrous
THF (30 mL), 3-((4-methylpiperazin-1-yl)methyl)-5-(trifluoromethyl)aniline (136 mg, 0.5 mmol) was
added. The solution was solution was stirred at -20 under argon for 30 minutes, and then t-BuOK
solid (185 mg, 1.65 mmol) was added. The temperature allowed to increase to room temperature and
the reaction mixture stirred at room temperature for 1 h. The solvent was removed by rotary
evaporation. The residue was treated with water and extracted with ethyl ether, then washed with brine,
dried over by magnesium sulfate. The organic layer was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (MeOH/DCM, v/v = 1/40) to afford the
1
target compound (130 mg, 0.24 mmol), light yellow solid, yielded 44.4%. H NMR (400 MHz,
DMSO-d6) δ 10.62 (s, 1H), 9.21 (s, 1H), 8.79 (d, J = 4.4 Hz, 1H), 8.25 (s, 1H), 8.19 (s, 1H), 8.14 (d, J
= 4.8 Hz, 1H), 8.12 (s, 1H), 8.03 (s, 1H), 7.88 (s, 1H), 7.80 (s, 1H), 7.37 (s, 1H), 3.56 (s, 2H), 2.46 (s,
3H), 2.42 (br, 8H), 2.21 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 164.9, 143.2, 140.7, 140.3, 139.8,
139.4, 138.8, 135.0, 134.6, 130.7, 129.2, 129.2 (q, J = 31.5 Hz), 127.5, 124.1 (q, J = 270.5 Hz), 123.8,
121.4, 120.1, 119.1, 115.1, 108.9, 99.3, 75.8, 61.3, 54.5, 52.2, 45.4, 20.7. HRMS (ESI) calcd for
C29H27F3N6O [M + H] + 533.2271, found 533.2258. Purity 96.6% (tR = 13.83 min).
5-(imidazo[1,2-a]pyrazin-3-ylethynyl)-2-methyl-N-(3-((4-methylpiperazin-1-yl)methyl)-5-
(trifluoromethyl)phenyl)benzamide (9b). Compound 9b was prepared by following a similar
procedure to that for 9a. 1H NMR (400 MHz, DMSO-d6) δ 10.75 (s, 1H), 9.20 (s, 1H), 8.79 (d, J = 4.0
14

ACCEPTED MANUSCRIPT
Hz, 1H), 8.22 (s, 1H), 8.16 (s, 1H), 8.11 (d, J = 4.4 Hz), 7.93 (d, J = 3.2 Hz, 2H), 7.74 (d, J = 7.6 Hz,
1H), 7.45 (d, J = 8.0 Hz, 1H), 7.37 (s, 1H), 3.55 (s, 1H), 2.45 (s, 3H), 2.40 (br, 8H), 2.18 (s, 3H). 13C
NMR (125 MHz, DMSO-d6) δ 167.0, 143.1, 140.8, 140.2, 139.8, 139.2, 137.2, 136.9, 132.2, 131.2,
130.6, 130.2, 129.3 (q, J = 31.0 Hz), 124.1 (q, J = 270.6 Hz), 123.2, 119.9, 119.1, 118.7, 114.5, 109.0,
99.3, 75.6, 61.2, 54.5, 52.3, 45.5, 19.4. HRMS (ESI) calcd for C29H27F3N6O [M + H] + 533.2271, found
533.2253. Purity 98.1% (tR = 10.96 min).
3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-2-methyl-N-(3-((4-methylpiperazin-1-yl)methyl)-5-
(trifluoromethyl)phenyl)benzamide (9c). Compound 9c was prepared by following a similar
procedure to that for 9a. 1H NMR (400 MHz, DMSO-d6) δ 10.76 (s, 1H), 9.21 (s, 1H), 8.67 (d, J = 4.4
Hz, 1H), 8.27 (s, 1H), 8.15 (s, 1H), 8.13 (d, J = 4.4 Hz, 1H), 7.92 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.59
(d, J = 7.6 Hz, 1H), 7.44 (t, J = 7.7 Hz, 1H), 7.37 (s, 1H), 3.54 (s, 2H), 2.61 (s, 3H), 2.40 (br, 8H), 2.16
(s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 167.5, 143.2, 141.0, 140.3, 139.8, 139.5, 137.7, 136.7,
133.1, 130.8, 129.3 (q, J = 31.2 Hz), 128.2, 126.1, 124.1 (q, J = 270.7 Hz), 123.1, 122.5, 120.0, 119.0,
+
114.4, 109.0, 98.2, 79.8, 61.2, 54.6, 52.4, 45.6, 17.9. HRMS (ESI) calcd for C29H27F3N6O [M + H]
533.2271, found 533.2257. Purity 99.3% (tR = 11.45 min).
3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-N-(3-((4-methylpiperazin-1-yl)methyl)-5-
(trifluoromethyl)phenyl)benzamide (9d). Compound 9d was prepared by following a similar
procedure to that for 9a. 1H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 9.22 (d, J = 1.3 Hz, 1H), 8.82
(dd, J 1 = 4.5 Hz, J 2 = 1.3 Hz, 1H), 8.35 (s, 1H), 8.26 (s, 1H), 8.20 (s, 1H), 8.14 (d, J = 4.4 Hz, 1H),
8.05 (d, J = 8.0 Hz, 2H), 7.95 (d, J = 7.7 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.38 (s, 1H), 3.57 (s, 2H),
2.43 (br, 8H), 2.22 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 164.9, 143.2, 140.6, 140.3, 139.8, 139.5,
134.9, 134.2, 130.7, 130.3, 129.2 (q, J = 31.3 Hz), 129.1, 128.7, 124.1 (q, J = 270.7 Hz), 123.9, 121.5,
120.1, 119.2, 115.2, 115.1, 108.8, 99.1, 76.2, 61.3, 54.4, 52.1, 45.4. HRMS (ESI) calcd for
C28H25F3N6O [M + H] + 519.2115, found 519.2100. Purity 99.5% (tR = 11.82 min).
2-chloro-3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-N-(3-((4-methylpiperazin-1-yl)methyl)-5-
(trifluoromethyl)phenyl)benzamide (9e). Compound 9e was prepared by following a similar
procedure to that for 9a. 1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 9.24 (d, J = 1.2 Hz, 1H), 8.67
(dd, J 1 = 4.4 Hz, J 2 = 1.2 Hz), 8.31 (s, 1H), 8.17 (d, J = 4.8 Hz, 1H), 8.12 (s, 1H), 7.99 (dd, J1 = 8.0
Hz, J 2 = 1.6 Hz, 1H), 7.88 (s, 1H), 7.72 (dd, J 1 = 7.6 Hz, J 2 = 1.2 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H),
7.39 (s, 1H), 3.55 (s, 2H), 2.40 (br, 4H), 2.33 (br, 4H), 2.15 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ
15

ACCEPTED MANUSCRIPT
164.7, 143.3, 141.2, 140.5, 139.9, 139.5, 137.5, 134.0, 131.1, 130.7, 129.4 (d, J = 30.5 Hz), 129.3,
127.6, 124.1 (q, J = 269.9 Hz), 123.0, 122.2, 120.2, 118.9, 114.3, 108.5, 96.2, 81.1, 61.2, 54.7, 52.5,
45.7, 29.0. HRMS (ESI) calcd for C28H24ClF3N6O [M + H] + 553.1725, found 553.1726. Purity 97.8%
(tR = 11.64 min).
3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-2-methoxy-N-(3-((4-methylpiperazin-1-yl)methyl)-5-
(trifluoromethyl)phenyl)benzamide (9f). Compound 9f was prepared by following a similar
procedure to that for 9a. 1H NMR (400 MHz, DMSO-d6) δ 10.75 (s, 1H), 9.22 (d, J = 1.2 Hz, 1H), 8.64
(dd, J 1 = 4.4 Hz, J 2 = 1.2 Hz, 1H), 8.27 (s, 1H), 8.16 (d, J = 4.4 Hz, 2H), 7.91 (s, 1H), 7.87 (dd, J1 =
7.6 Hz, J 2 = 1.6 Hz, 1H), 7.66 (dd, J 1 = 7.6 Hz, J 2 = 1.4 Hz, 1H), 7.38 (s, 1H), 7.34 (t, J = 7.6 Hz, 1H),
4.03 (s, 3H), 3.54 (s, 2H), 2.39 (br, 4H), 2.33 (br, 4H), 2.15 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ
164.9, 157.6, 143.3, 141.0, 140.3, 139.7, 139.5, 135.0, 131.1, 130.9, 130.2, 129.4 (d, J = 31.1 Hz),
124.1 (q, J = 270.8 Hz), 124.1, 123.1, 120.0, 118.8, 115.9, 114.4, 108.9, 95.8, 80.0, 62.3, 61.2, 54.7,
+
52.5, 45.7. HRMS (ESI) calcd for C29H27F3N6O2 [M + H] 533.1725, found 533.1726. Purity 95.8%
(tR = 11.30 min).
2-ethyl-3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-N-(3-((4-methylpiperazin-1-yl)methyl)-5-
(trifluoromethyl)phenyl)benzamide (9g). Compound 9g was prepared by following a similar
1
procedure to that for 9a. H NMR (400 MHz, DMSO-d6) δ 10.80 (s, 1H), 9.21 (s, 1H), 8.62 (dd, J 1 =
4.6 Hz, J 2 = 1.4 Hz, 1H), 8.26 (s, 1H), 8.15 (d, J = 4.4 Hz, 2H), 7.91 (s, 1H), 7.85 (dd, J 1 = 7.6 Hz, J2
= 0.8 Hz, 1H), 7.59 (d, J = 6.8 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.37 (s, 1H), 3.54 (s, 2H), 3.01 (q, J =
7.3 Hz, 2H), 2.39 (br, 4H), 2.15 (br, 4H), 1.99 (s, 3H), 1.30 (t, J = 7.4 Hz, 3H). 13C NMR (125 MHz,
DMSO-d6) δ 167.5, 143.1, 143.0, 140.9, 140.2, 139.7, 139.4, 137.2, 133.5, 130.7, 129.2 (q, J = 31.2
Hz), 128.2, 126.1, 124.0 (q, J = 270.6 Hz), 123.0, 121.7, 119.8, 118.7, 114.3, 108.9, 97.8, 78.9, 61.2,
54.6, 52.4, 45.6, 24.9, 15.3. HRMS (ESI) calcd for C30H29F3N6O [M + H] + 547.2428, found 547.2418.
Purity 99.8% (tR = 11.82 min).
3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-N-(3-((4-methylpiperazin-1-yl)methyl)-5-
(trifluoromethyl)phenyl)-2-propylbenzamide (9h). Compound 9h was prepared by following a
similar procedure to that for 9a. 1H NMR (400 MHz, DMSO-d6) δ 10.80 (s, 1H), 9.21 (s, 1H), 8.62 (d, J
= 4.4 Hz, 1H), 8.25 (s, 1H), 8.15 (d, J = 4.4 Hz, 1H), 8.12 (s, 1H), 7.91 (s, 1H), 7.85 (d, J = 7.7 Hz, 1H),
7.59 (d, J = 7.2 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.37 (s, 1H), 3.54 (s, 2H), 3.00 (t, J = 6.8 Hz, 2H),
2.39 (br, 4H), 2.33 (br, 4H), 2.15 (s, 3H), 1.71 (q, J = 6.8 Hz, 2H), 0.94 (t, J = 7.0 Hz, 3H). 13C NMR
16

ACCEPTED MANUSCRIPT
(125 MHz, DMSO-d6) δ 167.7, 143.3, 141.6, 141.1, 140.3, 139.9, 139.6, 137.5, 133.6, 130.8, 129.4 (d,
J = 31.2 Hz), 128.4, 126.3, 124.2 (q, J = 268.6 Hz), 123.3, 122.2, 120.1, 118.8, 114.5, 109.1, 98.3, 79.0,
61.3, 54.7, 52.5, 45.7, 33.4, 23.9, 14.2. HRMS (ESI) calcd for C31H31F3N6O [M + H] + 561.2584, found
561.2577. Purity 96.5% (tR = 11.60 min).
3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-2-isopropyl-N-(3-((4-methylpiperazin-1-yl)methyl)-5-(tri
fluoromethyl)phenyl)benzamide (9i). Compound 9i was prepared by following a similar procedure to
that for 9a. 1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 9.21 (s, 1H), 8.62 (d, J = 4.4 Hz, 1H), 8.25
(s, 1H). 8.15 (d, J = 4.8 Hz, 2H), 7.90 (s, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.51 (d, J =7.6 Hz, 1H), 7.42 (t,
J = 7.6 Hz, 1H), 7.37 (s, 1H), 3.54 (s, 2H), 3.50-3.45 (m, 1H), 2.39 (br, 4H), 2.33 (br, 4H), 2.15 (s, 3H),
1.50 (d, J = 7.0 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 168.4, 145.5, 143.3, 141.1, 140.3, 139.8,
139.4, 138.0, 134.9, 130.9, 129.4 (d, J = 31.1 Hz), 128.3, 126.2, 124.1 (q, J = 269.3Hz), 123.1, 120.6,
120.0, 118.9, 114.3, 109.1, 99.1, 80.4, 61.2, 54.7, 52.5, 45.7, 32.0, 21.4. HRMS (ESI) calcd for
C31H31F3N6O [M + H] + 561.2584, found 561.2584. Purity 95.5% (tR = 11.60 min).
3-((6-(4-((tert-butoxycarbonyl)amino)phenyl)imidazo[1,2-a]pyrazin-3-yl)ethynyl)-2-methylben
zoic
acid
(15).
A
solution
of
methyl
3-((6-(4-((tert-butoxycarbonyl)amino)
similar
phenyl)imidazo[1,2-a]pyrazin-3-yl)ethynyl)-2-methylbenzoate (prepared by following
a
procedure to that for 14a; 1.5 g, 3.11 mmol) in 200 ml of THF/H2O (2:1) was treated with LiOH (372
mg, 15.5 mmol) in 10 ml water. The mixture was stirred at 25 °C for 4 h. The solvent was removed
under reduced pressure and the resulting solution was diluted with 100 ml water, then acidified with 1
M
HCl to pH
=
5-7. The resulting solid
was
filtered
to afford of
3-((6-(4-((tert-butoxycarbonyl)amino)phenyl)imidazo[1,2-a]
pyrazin-3-yl)ethynyl)-2-methylbenzoic
acid (1.1 g, 2.35 mmol) as white solid, yielded 75.5%. 1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H),
9.24 (d, J = 1.2 Hz, 1H), 8.86 (d, J = 1.6 Hz, 1H), 8.22 (s, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.81 (d, J =
7.2 Hz, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.33 (s, J = 8.0 Hz, 1H), 2.76 (s, 3H), 1.49 (s, 9H). LC-MS (ESI)
m/z 467 [M - H] -.
3-((6-(4-aminophenyl)imidazo[1,2-a]pyrazin-3-yl)ethynyl)-2-methyl-N-(3-((4-methylpiperazin-
1-yl)methyl)-5-(trifluoromethyl)phenyl)benzamide (9j). Step (I): To a solution of 15 (250 mg, 0.53
mmol) and 3-((4-methylpiperazin-1-yl)methyl)-5-(trifluoromethyl)aniline (136 mg, 0.5 mmol) in
anhydrous DMF (10 mL), was added HATU (380 mg, 1.0 mmol) and 0.3 ml DIPEA (235 mg, 1.82
mmol). The solution was stirred for 6 h at room temperature. The solution was diluted with 20 ml water,
17

ACCEPTED MANUSCRIPT
then the resulting solid was filtered to afford crude mixture. The crude mixture was purified with silica
gel
column
chromatography
(MeOH/DCM,
v/v
=
1/30)
to
provide
tert-butyl(4-(3-((2-methyl-3-((3-((4-methylpiperazin-1-yl)methyl)-5-(trifluoromethyl)phenyl)carbamoy
l)phenyl)ethynyl)imidazo[1,2-a]pyrazin-6-yl)phenyl)carbamate (207 mg, 0.28 mmol) as yellow solid,
yield 56.0%. LC-MS (ESI) m/z 724 [M + H] +. Step (II): The product of last step (207 mg, 0.28 mmol)
was dissolved in 50mL of CF3COOH/DCM (1:1). The solution was stirred for 6 h at room temperature.
The solvent was removed by rotary evaporation, and then diluted with 10 mL MeOH. The MeOH
solution was alkalified with 1 M KOH to pH = 9-11. The solution was treated with water and extracted
with ethyl ether, then washed with brine, dried over by anhydrous sodium sulfate. The organic layer
was concentrated under reduced pressure. The residue was purified by silica gel column
chromatography (MeOH/DCM, v/v = 1:30) to afford the target compound (129 mg, 0.21 mmol), white
1
solid, yielded 75.0%. H NMR (400 MHz, DMSO-d6) δ 10.79 (s, 1H), 9.20 (s, 1H), 8.71 (s, 1H), 8.21
(d, J = 2.8 Hz, 1H), 8.13 (s, 1H), 7.97 (s, 1H), 7.90 (d, J = 3.6 Hz, 1H), 7.81 (d, J = 5.7 Hz, 2H), 7.59 (s,
1H), 7.45 (s, 1H), 7.40 (s, 1H), 6.68 (d, J = 5.7 Hz, 2H), 5.45 (s, 2H), 3.60 (s, 2H), 2.74 (br, 4H), 2.64
(s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 167.6, 158.1, 157.9, 149.8, 142.1, 140.8, 139.9, 139.8,
139.3, 137.6, 136.8, 133.2, 129.5 (d, J = 31.8 Hz), 128.1, 127.2, 126.1, 124.1 (q, J = 272.9 Hz), 123.3,
122.8, 120.1, 118.4, 116.0, 114.7, 113.9, 111.7, 109.2, 98.5, 80.2, 60.8, 53.7, 50.9, 44.0, 17.9. HRMS
(ESI) calcd for C35H32F3N7O [M + H] + 624.2693, found 624.2666. Purity 97.0% (tR = 13.31 min).
2-methyl-N-(3-((4-methylpiperazin-1-yl)methyl)-5-(trifluoromethyl)phenyl)-3-((6-phenylimidaz
o[1,2-a]pyrazin-3-yl)ethynyl)benzamide (9k). Compound 9k was prepared by following a similar
procedure to that for 9a. 1H NMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H), 9.31 (d, J = 0.8 Hz, 1H), 8.98
(s, 1H), 8.29 (s, 1H), 8.17 (s, 1H), 8.14 (d, J = 7.5 Hz, 2H), 7.90 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 7.6 Hz,
1H), 7.54 (t, J = 7.4 Hz, 2H), 7.45 (t, J = 7.4 Hz, 2H), 7.37 (s, 1H), 3.54 (s, 2H), 2.65 (s, 3H), 2.40 (br,
4H), 2.33 (br, 4H), 2.15 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 167.6, 142.5, 140.9, 140.1, 140.0,
139.6 (d, J = 4.8 Hz), 137.7, 136.9, 135.8, 133.2, 129.3 (q, J = 31.1 Hz), 128.8, 128.2, 126.3, 126.0,
124.1 (q, J = 270.8 Hz), 123.2, 122.6, 119.9, 114.8, 114.4, 109.8, 98.6, 79.9, 61.3, 54.7, 52.5, 45.7, 17.9.
+
HRMS (ESI) calcd for C35H31F3N6O [M + H] 609.2584, found 609.2580. Purity 98.4% (tR = 20.22
min).
2-methyl-N-(3-((4-methylpiperazin-1-yl)methyl)-5-(trifluoromethyl)phenyl)-3-((6-(pyridin-4-yl)
imidazo[1,2-a]pyrazin-3-yl)ethynyl)benzamide (9l). Compound 9l was prepared by following a
18

ACCEPTED MANUSCRIPT
1
similar procedure to that for 9a. H NMR (400 MHz, DMSO-d6) δ 10.80 (s, 1H), 9.35 (d, J = 1.2 Hz,
1H), 9.22 (s, 1H), 8.73 (d, J = 5.6 Hz, 2H), 8.34 (s, 1H), 8.18 (s, 1H), 8.15 (d, J = 6.0 Hz, 2H), 7.93 (d,
J = 7.4 Hz, 1H), 7.92 (s, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.37 (s, 1H), 3.54 (s,
2H), 2.66 (s, 3H), 2.39 (br, 4H), 2.33 (br, 4H), 2.15 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 167.6,
150.4, 143.1, 142.9, 141.0, 140.6, 139.9, 137.7, 137.1, 136.8, 133.4, 129.4 (q, J = 30.9 Hz), 128.3,
126.1, 124.2 (q, J = 260.0 Hz), 122.6, 120.5, 117.0, 114.4, 110.3, 98.7, 79.7, 61.3, 54.7, 52.5, 45.8, 29.0,
+
17.9. HRMS (ESI) calcd for C34H30F3N7O [M + H] 610.2537, found 610.2512. Purity 98.1% (tR =
13.88 min).
3-((6-(4-aminophenyl)imidazo[1,2-a]pyrazin-3-yl)ethynyl)-2-methyl-N-(3-methyl-5-((4-methylp
iperazin-1-yl)methyl)phenyl)benzamide (9m). Compound 9m was prepared by following a similar
1
procedure to that for 9j. H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.20 (s, 1H), 8.70 (s, 1H),
8.20 (s, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.81 (d, J = 8.3 Hz, 2H), 7.53 (d, J = 8.8 Hz, 1H), 7.50 (d, J =
9.4 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 6.85 (s, 1H), 6.68 (d, J = 8.3 Hz, 2H), 5.44 (s, 2H), 3.39 (s, 2H),
2.63 (s, 3H), 2.44-2.21 (br, 8H), 2.29 (s, 3H), 2.14 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 167.1,
149.8, 142.1, 140.8, 139.7, 139.3, 138.9, 138.8, 138.3, 137.6, 136.6, 132.8, 128.0, 127.9, 127.2, 126.0,
124.9, 122.8, 122.6, 118.8, 117.3, 113.9, 111.7, 109.2, 98.6, 80.0, 79.1, 62.2, 54.7, 52.6, 45.7, 21.2,
17.9. HRMS (ESI) calcd for C35H35N7O [M + H] + 570.2976, found 570.2957. Purity 99.8% (tR = 8.91
min).
3-((6-(4-aminophenyl)imidazo[1,2-a]pyrazin-3-yl)ethynyl)-N-(3-ethyl-5-((4-methylpiperazin-1-
yl)methyl)phenyl)-2-methylbenzamide (9n). Compound 9n was prepared by following a similar
1
procedure to that for 9j. H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.19 (s, 1H), 8.70 (s, 1H),
8.20 (s, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.81 (d, J = 7.6 Hz, 2H), 7.53 (d, J = 5.8 Hz, 3H), 7.42 (t, J = 7.2
Hz, 1H), 6.88 (s, 1H), 6.68 (d, J = 7.9 Hz, 2H), 5.43 (s, 2H), 3.41 (s, 2H), 2.63 (s, 2H), 2.59 (q, J = 7.6
Hz, 2H), 2.37 (br, 4H), 2.33 (br, 4H), 2.15 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 167.1, 149.8,
144.0, 142.1, 140.8, 139.7, 139.3, 139.0, 138.9, 138.3, 136.6, 132.8, 128.0, 127.2, 126.0, 123.7, 122.8,
122.6, 117.7, 117.5, 113.9, 111.7, 109.2, 98.6, 80.0, 62.2, 54.7, 52.6, 45.7, 28.2, 17.9, 15.5. HRMS (ESI)
calcd for C36H37N7O [M + H] + 584.3132, found 584.3115. Purity 99.6% (tR = 10.23 min).
3-((6-(4-aminophenyl)imidazo[1,2-a]pyrazin-3-yl)ethynyl)-N-(3-isopropyl-5-((4-methylpiperazi
n-1-yl)methyl)phenyl)-2-methylbenzamide (9o). Compound 9o was prepared by following a similar
procedure to that for 9j. 1H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 9.19 (d, J = 1.6 Hz, 1H), 8.70
19

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(d, J = 1.2 Hz, 1H), 8.20 (s, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.81 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 4.0 Hz,
2H), 7.53 (s, 1H), 7.42 (t, J = 8.0 Hz, 1H), 6.91 (s, 1H), 6.68 (d, J = 8.4 Hz, 2H), 5.42 (s, 2H), 3.42 (s,
2H), 2.90-2.83 (m, 1H), 2.64 (s, 3H), 2.37-2.33 (br, 8H), 2.15 (s, 3H), 1.21 (d, J = 7.6 Hz, 2H). 13C
NMR (125 MHz, DMSO-d6) δ 167.1, 149.7, 148.7, 142.1, 140.8, 139.7, 139.3, 139.0, 138.8, 138.3,
136.6, 132.8, 128.0, 127.2, 125.9, 122.8, 122.6, 122.3, 117.6, 116.2, 113.9, 111.7, 109.2, 98.6, 80.0,
+
62.3, 54.7, 52.6, 45.7, 33.4, 23.9, 17.9. HRMS (ESI) calcd for C37H39N7O [M + H] 598.3289, found
598.3276. Purity 95.1% (tR = 12.93 min).
3-((6-(4-aminophenyl)imidazo[1,2-a]pyrazin-3-yl)ethynyl)-N-(3-(tert-butyl)-5-((4-methylpipera
zin-1-yl)methyl)phenyl)-2-methylbenzamide (9p). Compound 9p was prepared by following a
similar procedure to that for 9j. 1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.19 (d, J = 1.2 Hz,
1H), 8.70 (d, J = 1.2 Hz, 1H), 8.20 (s, 1H), 7.86 (d, J = 6.8 Hz, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.66 (s,
1H), 7.63 (s, 1H), 7.54 (d, J = 7.2 Hz, 2H), 7.42 (t, J = 7.6 Hz, 1H), 6.69 (s, 1H), 6.68 (d, J = 8.4 Hz,
2H), 5.44 (s, 2H), 3.45 (s, 2H), 2.64 (s, 3H), 2.40 (br, 8H), 2.19 (s, 3H), 2.03-1.95 (m, 1H), 1.28 (s, 9H).
13C NMR (125 MHz, DMSO-d6) δ 167.1, 151.0, 149.7, 142.1, 140.8, 139.7, 138.8, 138.3, 136.6, 132.8,
129.6, 128.0, 127.2, 125.9, 122.8, 122.6, 121.1, 117.2, 115.3, 113.9, 111.7, 98.6, 80.0, 62.3, 54.6, 52.3,
45.5, 34.4, 31.1, 17.9. HRMS (ESI) calcd for C38H41N7O [M + H] + 612.3445, found 612.3435. Purity
98.0% (tR = 13.13 min).
3-((6-(4-aminophenyl)imidazo[1,2-a]pyrazin-3-yl)ethynyl)-2-methyl-N-(5-((4-methylpiperazin-
1-yl)methyl)-[1,1'-biphenyl]-3-yl)benzamide (9q). Compound 9q was prepared by following a
1
similar procedure to that for 9j. H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 9.20 (s, 1H), 8.71 (s,
1H), 8.32 (s, 1H), 8.21 (s, 1H), 8.00 (s, 1H), 7.88 (d, J = 7.5 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.72 (s,
1H), 7.62 (d, J = 7.6 Hz, 2H), 7.59 (d, J = 7.6 Hz, 1H), 7.51-7.37 (m, 4H), 7.31 (s, 1H), 6.68 (d, J = 8.5
Hz, 2H), 5.44 (s, 2H), 3.52 (s, 2H), 2.66 (s, 3H), 2.42 (br, 4H), 2.34 (br, 4H), 2.15 (s, 3H). 13C NMR
(125 MHz, DMSO-d6) δ 167.3, 149.7, 142.1, 140.8, 140.6, 140.2, 139.7, 139.6, 139.3, 138.2, 136.7,
132.9, 129.0, 128.0, 127.5, 127.2, 126.6, 126.0, 122.8, 122.7, 122.5, 119.0, 116.7, 113.9, 111.7, 109.2,
+
98.6, 80.0, 79.1, 62.1, 54.7, 52.6, 45.7, 17.9. HRMS (ESI) calcd for C40H37N7O [M + H] 632.3132,
found 632.3111. Purity 97.0% (tR = 11.73 min).
Computational Study
The crystal structures of TrkC and Abl were taken from PDB ID 3V5Q and 3OXZ. Hydrogen
atoms and charges were added during a brief relaxation performed using the “Protein Preparation
20

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Wizard” workflow in Maestro 11.7 (Schrodinger LLC). All HETATM residues and crystal water
molecules were removed. After the hydrogen bond network was optimized, the crystal structures
were minimized until the RMSD between the minimized structure and the starting structure
reached 0.3 Å with OPLS 2005 force field. The grid-enclosing box was placed on the centroid of
the binding ligand in the optimized crystal structure as described above, and accept the remaining
default values. The structure of compound 8 was optimized with Lig-prep module in Maestro to
afford multi conformations. Standard precision (SP) approach of Glide was adopted with the
default parameters, and the top-ranking pose was retained as the reasonable binding pose.
Crystallization and Structure Determination.
The kinase domain of human TrkC (residues 526-839) was expressed as an N-terminal
hexahistidine-SUMO fusion in E. Coli cells and purified by Nickel affinity chromatography. The
tag was cleaved with ULP1 protease. The proteins were further fractioned by ion exchange
chromatography on a Q column (GE Healthcare) and size exclusion chromatography on a
Superdex-75 column (GE Healthcare). To form complex, TrkC in 25mM Tris pH 8.0, 100 mM
NaCl, 5 mM DTT was incubated with 1 mM compound 9c and 9o, respectively. Hanging drops
were set up with diffracting crystals obtained from a 1:1 ratio of protein to mother liquor (0.1 M
MES, pH 6.0, 0.15 M (NH4)2SO4, 15% PEG 4000). Prior to flash frozen in liquid nitrogen,
crystals were equilibrated in mother liquor supplemented with 25%~30% glycerol. All data sets
were collected on beam-line BL17U1 at Shanghai Synchrotron Radiation Facility. Data were
indexed, integrated and scaled using HKL2000 package [46]. Initial phases were identified using
molecular replacement in Phaser [47]. Iterative cycles of model building and refinement were
carried out using COOT [48] and PHENIX [49].
In vitro enzymatic activity assay
The kinases and Z’-Lyte Kinase Assay Kit were commercially purchased from Invitrogen.
Firstly, TrkA/B/C and the Z’-Lyte Kinase Assay Kit were purchased from Invitrogen. The assays were
performed according to the manufacturer’s instructions. The concentrations of kinases were determined
by optimization experiments and the concentrations used were 1.22 ng/µL, 0.056 ng/µL and 1.73 ng/µL.
First, the solutions of the compounds were diluted from 1x10-10M to 1x10-4M in DMSO and a 400 µM
compound solution was prepared (4 µL compound dissolved in 96 µL water). Second, a 100 µM ATP
solution in 1.33 X kinase buffer was prepared. Third, a kinase/peptide mixture containing 2 X kinase
21

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and 4 µM Tyr1 peptide (PV3190; Invitrogen, Waltham, MA, USA) was prepared immediately before
use. A kinase/peptide mixture was prepared by diluting Z’-Lyte Tyr1 peptide (PV3190; Invitrogen) and
kinase in 1 X Kinase Buffer, and an 0.2 µM Tyr1 phospho-peptide solution was made by adding
Z’-Lyte Tyr1 phosphopeptide to 1 X Kinase Buffer. The final 10 µL reaction solution consists of 12.2
ng TrkA/0.56 ng TrkB/17.3 ng TrkC, 2 µM Tyr1 peptide in 1 X kinase buffer. For each assay, 10 µL
kinase reactions, including 2.5 µL compound solution, 5 µL Kinase/Peptide Mixture, and 2.5 µL ATP
solution were made at first. The plate wells were mixed thoroughly and incubated for 1 h at room
temperature. Then 5 µL development solution was added to each well and the plate was incubated for 1
h at room temperature; the phosphopeptides were cleaved at this time. Finally, 5 µL of stop reagent was
added to stop the reaction. For the control setting, 5 µL phospho-peptide solution instead of the
kinase/peptide mixture was used as a 100% phosphorylation control. 2.5 µL 1.33 X kinase buffer
instead of ATP solution was used as a 100% inhibition control, and 2.5 µL 4% DMSO instead of
compound solution was used as the 0% inhibitor control. The plate was measured on an EnVision
Multilabel Reader (Perkin-Elmer). Curve fitting and data presentation was performed using GraphPad
Prism, version 5.0. Every experiment was repeated at least three times.
Secondly, the kinase Abl1 was P3049, and it was full-length human recombinant protein expressed
in insect cells and histidine-tagged. Inhibition activities of inhibitors against Abl1 were performed in
384-well plates using the FRET-based Z′-Lyte assay system according to the manufacturer’s
instructions (Invitrogen). Briefly, the kinase substrate was diluted into 5 µL of 1X kinase reaction
buffer; and the kinase was added. Compounds (10 nL) with indicated concentrations were then
delivered to the reaction by using Echo liquid handler, and the mixture was incubated for 30 min at
room temperature. Then 5 µL of 2X ATP solution was added to initiate the reaction, and the mixture
was further incubated for 2 h at room temperature. The resulting reactions contained 10 µM of ATP, 2
µM of Tyr2 Peptide substrate in 50 mM HEPES (PH 7.5), 0.01% BRIJ-35, 10 mM MgCl2, 1 mM
EGTA, 0.0247 µg/mL Abl1, and inhibitors as appropriate. Then, 5 µL of development reagent was
added, and the mixture was incubated for 2 h at room temperature before 5 µL of stop solution was
added. Fluorescence signal ratio of 445 nm (Coumarin)/ 520 nm (Fluorescein) was examined on an
EnVision Multilabel Reader (PerkinElmer, Inc.). The data were analyzed using Graphpad Prism5
(Graphpad Software, Inc.). The data were the mean value of three experiments.
Cell lines
22

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SH-SY5Y cells were obtained from Shanghai Cell Bank (Type Culture Collection (TCC), Chinese
Academy of Sciences) and cultured in a 1:1 mixture of MEM (01-040-1 A, BI) and F-12
(C11765500BT, Gibco) supplemented with 10% foetal bovine serum (04-001-1 A, BI), 1%
Penicillin/Streptomycin (03-031-1B, BI), 1% Gluta-max (25030-081, Gibco), 1% sodium pyruvate
(11360-070, Gibco) and 1% NEAA (11140050, life). SH-SY5Y cells were stably transfected with
pRP-TrkB plasmids using Lipofectamine 2000 (11668019, Invitrogen) according to the manufacturer’s
protocol. SH-SY5Y-TrkB stable cell lines were selected in the mixed medium added with 0.5 µg/ml
Puromycin (HY-B1743, MedChemExpress).
Agents
Primary antibodies against TrkB (4603), PLC-γ (5690), phosphor-PLC-γ (14008), Erk1/2 (4695),
phosphor-Erk1/2 (4370), phosphor-Akt (13038, 4060), GAPDH (2118) and anti-rabbit or anti-mouse
IgG horseradish peroxidase (HRP)-linked secondary antibodies were purchased from Cell Signaling
Technology (Boston, MA, USA). Primary antibodies against Akt (SC8312), phosphor-TrkB
(SC-135645), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Larotrectinib
(LOXO-101) were purchased from the Selleckchem Company (Houston, TX, USA). These compounds
were dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 10 mmol/L and
the solution was stored at -20
.
Western blot analysis (WB)
Cells were treated with various concentrations of 9o with or without BDNF (10 ng/ml) for a fixed
time. Then cells were lysed using 1 X SDS sample lysis buffer (CST recommended) with protease and
phosphatase inhibitors. Cell lysates were loaded and electrophoresed onto 8–12% SDS-PAGE gel, and
then the separated proteins were transferred to a PVDF film. The film was blocked with 5% BSA
(Sigma-Aldrich, St. Louis, MO, USA) in TBS solution containing 0.5% Tween-20 for 4 h at room
temperature, then incubated with corresponding primary antibody (1:1000–1:200) overnight at 4
.
After washing with TBST, HRP-conjugated secondary antibody was incubated for 2 h. The protein
signals were visualized by ECL Western Blotting Detection Kit (Thermo Scientific, Grand Island, NY,
USA), and detected with Amersham Imager 600 system (GE, Boston, MA, USA).
Anti-proliferation assay in vitro
Cells were placed in 96-well plates (1500-3000/well) in complete medium. After incubation
overnight, the cells were exposed to various concentrations (0.000457~ 3 µM) of compounds with or
23

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without BDNF (10 ng/ml) for further 72 h. Cell proliferation was evaluated by Cell Counting Kit 8
(CCK8, CK04, Dojindo Laboratories, Kumamoto, Japan). IC50 values were calculated by
concentration-response curve fitting using GraphPad Prism 5.0 software. Each IC50 value is expressed
as mean ± SD.
Wound healing, migration and invasion assay
Cells (2 X 106/well) were seeded in a 6 well plate and allowed to grow to nearly 100% confluence in
a culture medium. Subsequently, a cell free line was manually created by scratching the confluent cell
monolayers with a 200 µL pipette tip. The wounded cell monolayers were washed three times with
PBS and incubated in mixed medium of MEM and F-12 (containing 2% FBS) with or without BDNF at
a concentration of 10 ng/mL and different concentrations of 9o for 24 h. Three scratched fields were
randomly chosen and the images were captured by a bright field microscope (CKX41; Olympus). The
percentage of wound closure was measured using Adobe Photoshop 7.0.1 (Adobe Systems Inc., San
Jose, CA). The experiment was performed three times, each in triplicate.
Cell migration assays were evaluated in Transwell chambers (Corning Costar). Cell invasion assays
were evaluated in Magrigel invasion chambers (Corning Costar). 1.5 X 106 tumor cells in 200 µL FBS
free medium of different doses of test compound (0, 8, 40, 200 nM) were plated in the top chamber,
800 µL complete medium with or without BDNF (10 ng/mL) was added to the bottom chamber. After
incubation for 24 h at 37 , the cells were fixed in 100% methanol and stained with 0.25% crystal
violet; the cells that had not migrated from the top surface of the filters were removed with cotton.
Migrated cells were quantitated by counting cells in six randomly selected fields on each filter under a
microscope at 200X magnification and graphed as the mean of three independent experiments.
Acknowledgements
The authors appreciate the financial support from National Natural Science Foundation of
China (81820108029, 21572230, 81874285, 81673285, 81425021 and 31800638), Key
Laboratory of Precision Chemical Drug Development (201805010007), Natural Science
Foundation of Guangdong Province (2018A030313003, 2015A030312014, 2015A030306042 and
2016A050502041 and Jinan University for their financial support.
Abbreviations
Trk, tropomyosin receptor kinases; IC50, half-maximal (50%) inhibitory concentration of a substance;
Abl, Abelson tyrosine kinase; BDNF, brain-derived neurotrophic factor; NGF, nerve growth factor;
24

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NT-4, Neurotrophin-4; NT-3, Neurotrophin-3; Ras, rat sarcoma protein; Erk, extracellular regulated
protein kinases; PI3K, phosphoinositide 3-kinases; Akt, protein kinase B; PLC-γ, phospholipase C
isotype γ; PKC, protein kinase C; NB, neuroblastoma; FDA, Food and Drug Administration; ROS1,
proto-oncogene tyrosine-protein kinase 1; ALK, anaplastic lymphoma kinase; ER, endoplasmic
reticulum; Bcr, breakpoint cluster region protein; MeOH, methanol; PdCl2(PPh3)2,
Bis(triphenylphosphine)palladium(II) chloride; Et3N, triethylamine; MeCN, acetonitrile; rt, room
temperature; DIPEA, N,N-Diisopropylethylamine; DMF, N,N-Dimethylformamide; t-BuOK,
potassium tert-butoxide; THF, tetrahydrofuran; LiOH, Lithium hydroxide; HATU,
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate;
CF3COOH, trifluoroacetic acid; DCM, dichloromethane; DFG, Asp-Phe-Gly; SAR, structure activity
relationship; ATP, Adenosine Triphosphate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel
electrophoresis; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; BBB, blood-brain-barrier; TLC,
thin-layer chromatography; NMR, nuclear magnetic resonance; TMS, tetramethylsilane; ESI-MS,
electrospray ionization mass spectrometry; HPLC, high-performance liquid chromatography; LC-MS,
liquid chromatography–mass spectrometry; HRMS, high-resolution mass spectrometry; DMSO,
dimethylsulfoxide;
Tris,
tris(hydroxymethyl)aminomethane;
DTT,
dithiothreitol;
MES,
2-(N-morpholino)ethanesulfonic acid; PEG, polyethylene glycol; FRET, fluorescence resonance
energy transfer; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EGTA, ethylene
glycol-bis( β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; MEM, minimum essential medium;
NEAA, non-essential amino acids; PVDF, polyvinylidene fluoride; BSA, albumin from bovine serum;
TBS, tris buffered saline. HRP, horseradish peroxidase; FBS, fetal bovine serum.
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