The discovery of novel 3-aryl-indazole derivatives as peripherally restricted pan-Trk inhibitors for the treatment of pain

Article abstract of DOI:10.1016/j.bmcl.2019.06.018

The design, synthesis, and biological evaluation of novel 3-aryl-indazole derivatives as peripherally selective pan-Trk inhibitors are described. Three strategies were used to obtain a potent compound exhibiting low central nervous system (CNS) penetratio

Full text of DOI:10.1016/j.bmcl.2019.06.018

Accepted Manuscript
The Discovery of Novel 3-Aryl-Indazole Derivatives as Peripherally Restricted
Pan-Trk Inhibitors for the Treatment of Pain
Hiromitsu Shirahashi, Eisuke Toriihara, Yoshihito Suenaga, Hideyuki Yoshida,
Kensuke Akaogi, Yukiko Endou, Makoto Wakabayashi, Misato Takashima
PII:
S0960-894X(19)30398-1
https://doi.org/10.1016/j.bmcl.2019.06.018
BMCL 26496
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 April 2019
24 May 2019
15 June 2019
Please cite this article as: Shirahashi, H., Toriihara, E., Suenaga, Y., Yoshida, H., Akaogi, K., Endou, Y.,
Wakabayashi, M., Takashima, M., The Discovery of Novel 3-Aryl-Indazole Derivatives as Peripherally Restricted
Pan-Trk Inhibitors for the Treatment of Pain, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/
10.1016/j.bmcl.2019.06.018
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The Discovery of Novel 3-Aryl-Indazole Derivatives as Peripherally Restricted Pan-Trk
Inhibitors for the Treatment of Pain
Hiromitsu Shirahashi*, Eisuke Toriihara, Yoshihito Suenaga, Hideyuki Yoshida, Kensuke Akaogi,
Yukiko Endou, Makoto Wakabayashi, Misato Takashima*
Pharmaceuticals Research Center, Asahi Kasei Pharma Corporation, 632-1 Mifuku, Izunokuni,
Shizuoka 410-2321, Japan
*Correspondence:
Hiromitsu Shirahashi, E-mail address: shirahashi.hd@om.asahi-kasei.co.jp
Misato Takashima, E-mail address: takashima.mc@om.asahi-kasei.co.jp
Abstract
The design, synthesis, and biological evaluation of novel 3-aryl-indazole derivatives as peripherally
selective pan-Trk inhibitors are described. Three strategies were used to obtain a potent compound
exhibiting low central nervous system (CNS) penetration and high plasma exposure: 1) a structure-
based drug design (SBDD) approach was used to improve potency; 2) a substrate for an efflux
transporter for lowering brain penetration was explored; and 3) the most basic pKa (pKa–MB) value
was used as an indicator to identify compounds with good membrane permeability. This enabled the
identification of the peripherally targeted 17c with the potency, kinase-selectivity, and plasma
exposure required to demonstrate in vivo efficacy in a Complete Freund's adjuvant (CFA)-induced
thermal hypersensitivity model.
Keywords: pan-Trk inhibitor; peripherally restricted; 3-aryl-indazole derivative; efflux transporter;
pKa-MB
Tropomyosin-related kinase (Trk) is a family of receptor tyrosine kinases highly expressed in
neurons, comprised of three isomers TrkA, TrkB, and TrkC. Each isomer is activated by the binding
of a complementary neutrophin, such as nerve growth factor (NGF) for TrkA, brain-derived
neurotrophic factor (BDNF) for TrkB, and neurotrophin-3 for TrkC.1 The activation of Trk causes
dimerization and autophosphorylation, followed by initiation of the intracellular signaling
pathway.1,2 Trk signaling pathways are implicated in various cancers and pain.3,4,5 We have a
particular interest in the NGF/TrkA signaling pathway because preclinical studies on antagonists of
NGF and clinical research on a humanized anti-NGF monoclonal antibody (tanezumab) suggest that
NGF plays a crucial role in pain.6,7,8

Trk inhibitors can cause CNS side effects. The inhibition of NGF/TrkA signaling in the CNS may
induce dysfunction in the cholinergic circuitry9 and BDNF/TrkB signaling in the CNS appears to
regulate hippocampal long-term potentiation and eating behavior.10,11 The highly homologous nature
of the ATP binding sites in TrkA, B and C severely hampers the design of small molecules with high
selectivity towards Trk family members.12 We considered the CNS-related risk and high similarity of
the ATP binding sites of the Trk family proteins and explored a peripherally selective small
molecule as a pan-Trk inhibitor for the treatment of chronic pain.
We embarked on the design and synthesis of a substrate for efflux transporters to obtain a safe orally
administrable compound with restricted CNS penetration. Whether a compound is an efflux
transporter substrate or not can be assessed by the efflux ratio (ER) value obtained by in vitro assays.
In our Trk inhibitor program, the Caco-2 assay was used to calculate ER values. Compounds having
ER values over 2 were evaluated as efflux transporter substrates.13
Screening of our in-house compound library identified hit 1 exhibiting moderate potency (Figure 1).
We therefore optimized hit 1 by focusing on the A part (substituted pyridine moiety) and the B part
(substituted benzylamine moiety), while maintaining the 3-aryl-indazole structure as our original
hinge binder. This structure is not found in known pan-Trk inhibitors such as 2 (AZ-23) and 4
(Figure 2).14a,15,16 While compound 3 (Entrectinib) has an indazole structure, the nitrogen atom at the
3-position is important for binding with the hinge.
O
H
S
N
O
NH
B
N
N
H
O
A
N
1
TrkA IC50= 7 nM
Cell IC50 = 53 nM
Figure 1. The structure of hit 1.
Cl
H
H
N
N
N
N
NH
O
F
N
N
F
HN
HN
O
F
NH
N
N
N
2
3,
Entrectinib
, AZ-23
O
O
N
N
Cl
N
N
O
H
N
H2N
N
OH
4

Figure 2. Trk inhibitors described in the literature.
First, we optimized the A part with the aim of improving the potency and metabolic stability. The
structure activity relationship (SAR) of the A part is summarized in Table 1. Potency and metabolic
stability appeared to be affected by the position of the nitrogen atom and the orientation of the
substituent group in the pyridine moiety (1, 10a and 10b). Regarding the methoxypyridine
derivatives, 10b exhibited the highest potency and moderate metabolic stability. Substitution of the
methoxy group in 10b by 4-hydroxypiperidine or 4-aminopiperidine maintained the potency in an
enzyme assay and metabolic stability but decreased the potency in a cell-based assay (10c and 10d).
These results suggested that the larger substituents were tolerated at R1 and that the low membrane
permeability of these compounds probably decreased their potency in the cell-based assay (the
Madine−Darby canine kidney cell (MDCK) apparent permeability coefficients (Papp) at pH 7.4 of
10c and 10d were both 0.3 × 10−6 cm/sec). Indeed, 10e possessing a larger substituent than methoxy
group at R1, which has a higher membrane permeability (MDCK Papp at pH 7.4 of 1.2 × 10−6 cm/sec)
than 10c or 10d, showed excellent potency in both the enzyme and cell-based assays as well as good
metabolic stability.
The binding mode of 10e was confirmed via X-ray crystallography using a 2.3 Å complex structure
with TrkA protein (Figure 3). Compound 10e was found to be a Type I inhibitor that binds to the
DFG-in conformation of TrkA. Figure 3 shows that the indazole ring of 10e interacts with the kinase
hinge through two key hydrogen bonds between Glu590 and Met592, and that the piperazine and
benzylamine moieties (in the B part) do not appear to form hydrogen bonds with TrkA. The
observation that the piperazine group is located at the entrance to solvent region, is considered to be
the reason why the substituent larger than methoxy group was allowed at R1.
As mentioned above, 10e exhibited high potency, good metabolic stability, and high ER value
(Caco-2 ER = 7.8), and the interaction with TrkA, However, we considered that the membrane
permeability of 10e was still not high enough to be administered orally. We therefore conducted
further SAR development of the B part in 10e to improve the permeability.
Table 1. SAR of the A part of hit 1
O
H
S
N
O
NH
N
N
H
X
Y
R2
R1
TrkA IC50
(nM)
Cell IC50
(nM)
hCLinta
ID
R1
R2
X
Y
(mL/min/kg)

1
H
H
-OMe
-OMe
H
C
N
C
N
C
N
7.0
7.8
2.9
53
64
35
217
833
359
10a
10b
-OMe
O
NH
NH
10c
10d
10e
H
H
H
C
C
C
N
N
N
2.3
0.9
1.6
175
224
27
323
184
70
H
N
N
NH
ahCLint was determined from human liver microsome incubations; ND = not determined.
Figure 3. Co-crystal structure of 10e bound to TrkA protein in a DFG-in conformation. Two key
hydrogen bonds are highlighted with blue dashed lines. Nitrogen atoms are shown in blue, oxygen
atoms in red, and the sulfur atom in yellow. Some protein residues were omitted for clarity. The
PDB code for 10e is 6J5L.
We first studied the SAR of the linker structure using a compound with a simple phenyl group to
facilitate synthesis of the derivatives (Table 2). The results suggested that a non-branched nitrogen
linker is the most suitable structure in terms of potency, metabolic stability and membrane
permeability (13c, 22a, 22b, 22c and 22d). The potency reduction of 22c and 22d indicated that the
space around the linker was not enough to introduce a substituent. The decreased metabolic stability
of 22a and 22d was considered to be due to the increased lipophilicity. Compound 22a has two
benzyl positions that might be metabolized.

Next, we explored the SAR of substituents on the phenyl group (Table 3) and found that the 2-
substituted derivative was more potent than the 3-or 4-substituted derivatives (10e, 13a and 13b).
Furthermore, replacement of the methanesulfonamide group with the methanesulfonyl group
improved potency in the cell-based assay and retained potency in the enzyme assay (13d). In
addition, 13d exhibited higher membrane permeability and ER than 10e while maintaining metabolic
stability. The improvement of membrane permeability might be achieved by the decrease of the
hydrogen bond donors (HBD). The increase of the ER value implied that the methanesulfonyl group
had a higher affinity for the efflux transporter than the methanesulfonamide group.
Figure 4 shows an overlay of 10e and AZ-23 bound to TrkA protein in which the para-position of
the methanesulfonyl group in 10e corresponds to the space occupied by the fluorine atom in the
fluoropyridine ring of AZ-23. The fluorine atom in AZ-23 was reported to contact the backbone
carbonyl atom of Asn 665 and the Cα of Gly 667 N-terminal to the DFG sequence of the activation
loop,14a and to be important for the high potency against TrkA.14b Thus, we predicted that the
introduction of a fluorine atom at the para-position of the methanesulfonyl group in 13d could
improve the potency, and as expected, the para-fluorinated analogue 13e exhibited higher potency
than 13d. In addition, the fluorine substitution improved the ER value but decreased the membrane
permeability. The reason for the increase of ER value can be considered that the efflux transporter
affinity was enhanced by introducing fluorine atom. On the other hand, there is the report that the
increase in molecular weight due to the insertion of fluorine does not lead to increased ER value,
although high molecular weight tends to show high ER value.17 Therefore, the cause of the increase
of ER value with the fluorine introduction was considered to be increase of molecular weight, which
may be exceptionally cased only in our compounds.
Table 2. SAR of the linker structure of the B part of 10e
H
N
N
R3
R4
L
R5
N
N
HN
b
TrkA IC50 Cell IC50
hCLinta
(mL/min/kg)
12
MDCK Papp
(cm/sec)
ID
L
R3
R4
R5
(nM)
12.3
18.1
11.1
27.6
(nM)
73
13c
22a
22b
22c
N
CH
O
H
H
H
H
H
H
H
H
H
H
13.0× 10−6
3.1× 10−6
9.6× 10−6
7.3× 10−6
>300
127
264
2726
-
694
N
Me
417

22d
N
Me
Me
H
58.6
253
1737
11.0× 10−6
ahCLint was determined from human liver microsome incubations; bMDCK Papp was evaluated at pH
7.4.
Table 3. SAR of the substituted phenyl ring of the B part of 10e
H
N
N
2
N
H
3
N
N
4
5
R6
HN
b
TrkA IC50
(nM)
1.6
Cell IC50
(nM)
27
hCLinta
(mL/min/kg)
69.5
MDCK Papp
(cm/sec)
1.2× 10−6
ND
ID
R6
Caco-2 ER
10e
13a
13b
13d
13e
2-NH-SO2Me
3-NH-SO2Me
4-NH-SO2Me
2-SO2Me
7.8
ND
ND
13.6
71.1
143.2
766.2
1.4
ND
ND
16
ND
ND
ND
74
12.5× 10−6
0.4× 10−6
2-SO2Me-5-F
0.3
2
176
ahCLint was determined from human liver microsome incubations; bMDCK Papp was evaluated at pH
7.4; ND = not determined.
Figure 4. Overlay of 10e and AZ-23 bound to TrkA protein. The PDB code for AZ-23 is 4AOJ.14a
The in vivo PK profiles of 13d and 13e were determined but the concentration of these compounds
in plasma was below the detection limit when administered orally at a dose of 10 mg/kg to SD rats.
Thus, we investigated the underlying cause of the poor plasma exposure of these compounds (Table
4). Although 13d showed good membrane permeability at pH 7.4 (Table 3), we considered that the
membrane permeability was the reason for their poor plasma exposure, because both 13d and 13e

had the piperazine group that is likely ionizable in the small intestine, and the membrane
permeability of ionized compounds is typically lower than that of non-ionized compounds. We
therefore confirmed whether 13d and 13e would be ionized in the small intestine by calculating the
most basic pKa (pKa-MB) of these compounds with ACD/Percepta, since pKa determines the degree
of ionization. As a result, more than 99% of both 13d and 13e was estimated to be ionized at pH 6.5,
the pH of the small intestine,18 and the terminal amino group in the piperazine moiety was found to
be the most basic site in these compounds. Moreover, the membrane permeability of 13d at pH 6.5
dramatically decreased from the membrane permeability at pH 7.4, possibly due to increased
ionization of the compound. This result indicated that the membrane permeability at pH 6.5 was
important to impart good plasma exposure to a compound. These findings suggest that the
membrane permeability of 13d and 13e at pH 6.5 might be too low, resulting in poor plasma
exposure and the piperazine group might cause low membrane permeability.
Table 4. pKa-MB value, ionized portion, and membrane permeability of 13e at pH 6.5
Ionized portion Ionized portion at MDCK Papp at pH MDCK Papp at pH
ID
pKa-MB
at pH 7.4a
pH 6.5a
> 99.1%
> 99.1%
7.4 (cm/sec)
12.5×10-6
0.4×10-6
6.5 (cm/sec)
0.4×10-6
13d
13e
8.55
8.55
73.7 %
73.7 %
0.3×10-6
b
aIonized portion was determined from the Henderson-Hasselbach equation; MDCK Papp was
evaluated at both pH 6.5 and pH 7.4.
Consequently, we identified an alternative structure to the piperazine group by conducting another
SAR exploration of the A part of 13e while retaining the structure of the B part (predicted to
contribute to high potency) (Table 5). The pKa-MB and membrane permeability at pH 6.5 were also
examined as indices for compound selection for an in vivo pharmacokinetics (PK) study.
The results shown in Table 5 suggested that removal of the piperazine group would yield the desired
pKa-MB values and increase the membrane permeability significantly at pH 6.5. Unfortunately, the
pyridine derivatives 17a and 17b exhibited decreased metabolic stability; however, the pyrazole
derivatives 17c and 13f showed metabolic stability comparable to 13e. 17c exhibited the same
potency as 13e, providing a possible substrate for an efflux transporter (Caco-2 ER = 12.6), and
exhibited the best balance of potency, metabolic stability, and membrane permeability of the
compounds synthesized. We determined the in vivo PK characteristics of 17c by administering it
orally at a dose of 5 mg/kg to SD rats and found that the Cmax of 17c was moderate (161.0 nmol/L)
and there was very low CNS penetration (brain/plasma concentration ratio (B/P) = 0.03) (Table 6).
Table 5. SAR of the A part of 13e

H
N
N
F
N
H
R7
S
O
O
TrkA
IC50
Cell IC50
hCLinta
MDCK Papp Solubility pKa- Caco-2
ID
R7
(nM) (mL/min/kg) (cm/sec)b
(µM)
38.7
23.0
15.2
140.7
80.1
MBc
8.5
4.8
3.9
2.6
2.3
ER
71.1
ND
ND
12.6
ND
(nM)
N
13e
17a
17b
17c
13f
0.3
0.2
2.1
0.2
4.5
2.0
2.4
177.5
793.4
442.7
127.4
136.8
0.3×10-6
61.5×10-6
73.2×10-6
15.2×10-6
34.0×10-6
N
NH
N
N
18.0
1.7
N
N
N
N
104.8
aCLint determined from human liver microsome incubations; b MDCK Papp was evaluated at pH 6.5;
cpKa-MB determined by ACD calculation; ND = not determined.
Table 6. Summary of the PK properties of 17c
Cmax (nmol/L)a
CL (L/h/kg)b
161.0
4.4
T1/2 (h)c
1.46
86.6
96.2
0.03
plasma protein binding (%)
brain protein binding (%)
B/Pd
b
c
aCmax was determined by the oral dosing of SD rats at 5 mg/kg; CL and T1/2 were determined by
intravenous administration to SD rats at 1 mg/kg; dB/P was determined by intraperitoneal
administration to SD rats at 2.5 mg/kg.
We investigated the kinase selectivity of 17c at 20 nM against a panel of 49 kinases and found that
no kinase was inhibited by more than 50%, excluding TrkA, and this compound exhibited no
selectivity against TrkB (TrkB IC50 = 0.2 nM). The selectivity of 17c against TrkC was not
determined. We concluded that 17c had sufficient selectivity as a pan-Trk inhibitor to warrant
evaluating its in vivo efficacy.

We investigated the effect of 17c in an inflammatory pain model using a CFA-induced thermal
hypersensitivity assay. Compound 17c was orally administered to rats at increasing doses once daily
after administration of CFA. Table 7 shows the plasma and brain levels of 17c at 1 h post-dose. The
concentration of 17c in the brain was too low to detect at doses of 0.8 mg/kg and 4.0 mg/kg.
Correction for plasma protein binding revealed that even at a dose of 0.8 mg/kg, there was a higher
concentration of unbound 17c in plasma than the IC50 value obtained by the cell-based assay. Figure
5 shows the rate of weight gain of rats to which 17c was administered. The weight gain was
established as 100 mg/kg. This is considered to be caused by the inhibition of BDNF/TrkB signaling
in the CNS by increased brain penetration of 17c.11 Therefore, as a peripherally selective pan-Trk
inhibitor, the acceptable dosing amount could be up to 20 mpk because no weight gain was observed.
Figure 6 demonstrates the results of the CFA-induced thermal hypersensitivity assay using 17c. A
dose of 0.8 mg/kg seemed to provide weak efficacy and doses of 4.0, 20, and 100 mg/kg had a
tendency for reversed thermal hyperalgesia comparable to a dose of 100 mg/kg ibuprofen. Although
no dose dependency was observed, we considered the reason the maximum effect was achieved at
4.0 mg/kg (p-value was 0.0522) was because there was an individual difference in rats (p-values
were 0.2531 at 20 mg/kg and 0.2875 at 100 mg/kg).
Table 7. Plasma and brain concentrations of 17c 1 h post-dose
a
b
Dose Cu,plasma
(mg/kg) (nM)
Cu,brain
(nM)
Cu,plasma/IC50
Cu,brain/IC50
0.8
4.0
4.7
2.8
8.8
BLQc
BLQc
1.5
-
14.9
70.9
-
20.0
41.7
156.8
0.9
2.1
100.0 266.5
3.5
aCu,plasma and bCu,brain were determined by oral administration to CFA rats; cBLQ means “below limit
of quantification.”

Figure 5. Weight gain rate of rat to which 17c was administered
Figure 6. Results of CFA-induced thermal hypersensitivity assay using 17c.
The indazole derivatives listed in Tables 1, 2, 3 and 5 were synthesized as outlined in Schemes 1 and
2. Scheme 1 starts with the hydrogenation of 5 to form 6, followed by reductive amination to afford
7 and 14. Suzuki-Miyaura coupling of 7 with the corresponding boronic acid or pinacol boronate
gave 8a-b. 9a-e were prepared via a nucleophilic aromatic substitution reaction in which 8a-b were
reacted with the corresponding alcohol or amine. Deprotection of the THP group in 9a-e provided
10a-e. 14 was reacted with an organotin reagent to obtain 15, which was converted into 16a-c by
Stille coupling. The deprotection of 16a-c furnished 17a-c. 6 was reacted with the corresponding
pinacol boronate to form 11a-b. The reductive amination of 11a-b with the corresponding aldehyde
afforded 12a-f, which were converted to 13a-f by deprotection of the THP group. The synthetic
route to 22a-d is shown in Scheme 2. Commercially available 18 was protected using the THP group
to obtain 19, which was converted to 20 by Suzuki-Miyaura coupling. A second Suzuki-Miyaura
coupling and Buchwald–Hartwig aryl amination of 20 provided 21a and 21b-c, respectively. 21d
was prepared by reaction with benzyl bromide and 24, which was synthesized from commercially
available 23 by Suzuki-Miyaura coupling. The hydrogenation of 21a afforded 21e and deprotection
of 21b-e provided 22a-d.
In conclusion, we identified the novel 3-aryl-indazole derivative 17c as a low-CNS-penetrating
targeted small molecule pan-Trk inhibitor. Three methodologies contributed to the design of this
compound: 1) employing the SBDD approach; 2) exploring a substrate as an efflux transporter; and
3) calculating the value of pKa–MB. Compound 17c exhibited sub-nanomolar potency, high
selectivity, and satisfactorily low brain penetration, and in addition demonstrated dose-independent
in vivo efficacy in a CFA-induced thermal hypersensitivity model.

O
S
O
S
THP
THP
THP
THP
N
N
N
N
a
b
c or d
O
NH
O
NH
N
N
N
N
NO2
THP
NH2
N
N
R8
H
H
Br
Br
Br
5
6
7
8 a-b
8a: R8 = 6-fluoropyridin-2-yl
8b: R8 = 6-fluoropyridin-3-yl
O
O
H
S
S
N
N
N
O
NH
O
NH
e or f or g or h
N
i
N
H
N
H
X
X
Y
Y
9 a-e
R2
R2
10 a-e
R1
R1
9a, 10a: X = N, Y = C, R1 = H, R2 = methoxy
9b, 10b: X = C, Y = N, R1 = methoxy, R2 = H
9d, 10d: X = C, Y = N, R1 = piperidin-4-amino, R2 = H
9e: X = C, Y = N, R1 = 4-[(tert-butoxy)carbonyl]piperazin-1-yl, R2 = H
9c, 10c: X = C, Y = N, R1 = tetrahydro-2H-pyran-4-amino, R2 = H 10e: X = C, Y = N, R1 = 1-piperazinyl, R2 = H
THP
THP
N
N
NH
N
N
N
d
b
i or j or k
N
N
N
N
N
N
NH
HN
R9
NH2
NH
N
N
R9
Boc
Boc
12 a-e
13 a-e
11 a
12a, 13a: R9 = 3-[(methylsulfonyl)amino]benzyl 12d, 13d: R9 = 2-(methansulfonyl)benzyl
12b, 13b: R9 = 4-[(methylsulfonyl)amino]benzyl 12e, 13e: R9 = 2-(methansulfonyl-4-fluoro)benzyl
12c, 13c: R9 = phenyl
THP
THP
O
S
O
S
H
O
O
N
N
N
THP
N
N
N
N
b
i or j or k
c
N
NH2
N
N
H
H
NH2
N
N
N
Br
6
N
N
N
F
F
12 f
13 f
11 b
THP
THP
THP
O
O
S
O
N
N
N
S
F
S
b
l
m or n
N
N
O
N
O
O
N
H
N
N
R7
H
H
Br
Me3Sn
14
15
16 a-c
k
F
F
S
H
N
O
N
O
N
H
16a, 17a: R7 = pyridin-3-yl
16b, 17b: R7 = pyridin-2-yl
16c, 17c: R7 = 1-methyl-1H-pyrazol-4-yl
R7
17 a-c
F
Scheme 1. Synthesis of 10a-e, 13a-f and 17a–c. Reagents and conditions: (a) H2 gas, Pt(S)/C, THF,
30 °C, 70%; (b) corresponding aldehyde, NaBH3CN, AcOH, MeOH-THF, rt; (c) corresponding
pinacol boronate, PdCl2(dppf)·CH2Cl2, 2 M Na2CO3 in H2O, DME, 90 °C or 95 °C, 39%-quant; (d)
corresponding boronic acid or pinacol boronate, Pd(PPh3)4, Na2CO3 or K2CO3, DME-H2O, 80 °C or
85 °C, 64-74%; (e) NaOMe, MeOH, 60 °C; (f) 1-(tert-butoxycarbonyl)-4-hydroxypiperidine, tBuOK,
DMSO, rt; (g) 1-(tert-butoxycarbonyl)-4-aminopiperidine, DMSO, 80 °C to 100 °C; (h)1-(tert-
butylcarbonyl)piperazine, DMSO, 80 °C; (i) TFA, CH2Cl2, rt, 9-63% (two steps); (j) 2 M HCl in
MeOH, rt, 17-70% (two steps); (k) 4 M HCl in 1,4-dioxane, 1,4-dioxane, rt, 2-23% (two steps); (l)

hexamethylditin, Pd(PPh3)4, 1,4-dioxane, 80 °C, 38%; (m) corresponding bromide, Pd(PPh3)4, LiCl,
DMF, 60 °C; (n) 4-bromo-1-methylpyrazole, Pd(PPh3)4, DMF, 90 °C;
THP
R10
N
H
N
N
N
N
N
THP
H
N
N
a
b
c or d
g or h
N
N
N
N
R10
N
R10
Br
Br
Br
I
N
N
I
N
N
N
18
19
20
Boc
21 a-d
21 e
22 a-d
HN
Boc
N
Boc
f
21a, R10 = styryl
21b, 22c, R10 = N-benzylmethylamino
21c, 22d, R10 = 1-methyl-1-phenylethylamino
21d, 22b, R10 = benzyloxy
21e, 22a, R10 = phenethyl
N
N
Boc
b
e
N
N
OTBS
N
I
OH
N
Boc
23
24
Scheme 2. Synthesis of 22a-d. Reagents and conditions: (a) DHP, TsOH-H2O, toluene, 60 °C, 89%;
(b) 2-(4-tert-Butoxycarbonylpiperazin-1-yl)pyridine-5-boronic acid, pinacol ester, Pd(PPh3)4, K2CO3,
DME-H2O, 80 °C, 68%-quant; (c) trans-2-phenylvinylboronic acid, Pd(PPh3)4, K2CO3, DME-H2O,
100 °C; (d) corresponding benzylamine, Pd(crotyl)Q-PhosCl, tBuONa, toluene, 35 °C or 100 °C; (e)
benzyl bromide, K2CO3, acetone, rt, 61% (two steps); (f) H2 gas, Pd/C, MeOH-EtOAc, rt, 89% (two
steps); (g) TFA, CH2Cl2, rt, 51%; (h) 4 M HCl in 1,4-dioxane, rt, 44-51%.
Abbreviations
ATP, adenosine triphosphate; BDNF, brain-derived neurotrophic factor; B/P, brain/plasma
concentration ratio; CFA, Complete Freund's adjuvant; CL, clearance; T1/2, half-life; CLint, intrinsic
clearance; CNS, central nervous system; Cu,brain, unbound brain concentration; Cu,plasma, unbound
plasma concentration; ER, efflux ratio; HBD, hydrogen bond donor; IC50, half-maximum inhibitory
concentration; MDCK, Madine−Darby canine kidney; ND, not determined; NGF, nerve growth
factor; Papp, apparent permeability; PDB, Protein Data Bank; PK, pharmacokinetics; pKa–MB,
most basic pKa; SBDD, structure based drug design; Trk, tropomyosin-related kinase
Acknowledgments
We are grateful to Shinichi Oguma, Tsuyoshi Arai and Naomi Shinotsuka for conducting the
biological evaluations, and to Tokuhito Goto and Tomohisa Toyama for their insightful chemistry
support.
Notes
The authors declare no competing financial interest.
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Highlights:




New 3-Aryl-indazole derivatives as pan-Trk inhibitors were designed and synthesized.
Substrates for efflux transporter were explored by the Caco-2 assay.
Membrane permeability was increased by lowing basicity.
17c demonstrated restricted brain penetration and good in vivo efficacy in a CFA-induced
thermal hypersensitivity model.