Discovery of (E)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-yl)thiazol-2-yl)-2-(4-methylpiperazin-1-yl)acetamide (IHMT-TRK-284) as a novel orally available type II TRK kinase inhibitor capable of overcoming multiple resistant mutants

^aInstitute of Health & Medical Technology, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science,

Chinese Academy of Sciences, Hefei, Anhui, 230031, PR China

^bUniversity of Science and Technology of China, Hefei, Anhui 230026, PR China

^cPrecision Medicine Research Laboratory of Anhui Province, Hefei, Anhui 230088, PR China

^dInstitute of Physical Science and Information Technology, Anhui University, Hefei, Anhui 230601, PR China

a r t i c l e i n f o

a b s t r a c t

Article history:

Due to the critical tumorigenic role of fused NTRK genes in multiple cancers, TRK kinases have attracted

extensive attention as a drug discovery target. Starting from an indazole based scaffold, through the type

II kinase inhibitor fragments hybrid design approach with a ring closure strategy, we discovered a novel

potent type II TRK kinase inhibitor compound 34 (IHMT-TRK-284), which exhibited IC₅₀values of

10.5 nM, 0.7 nM and 2.6 nM to TRKA, B, and C respectively. In addition, it displayed great selectivity

Received 16 May 2020

Received in revised form

1 August 2020

Accepted 2 August 2020

Available online 30 August 2020

proﬁle in the kinome when tested among 468 kinases and mutants (S score (1) ¼ 0.02 at 1

mM).

Importantly, 34 could overcome drug resistant mutants including V573M and F589L in the ATP binding

pocket as well as G667C/S in the DFG region. In vivo, 34 exhibited good PK proﬁles in different species

including mice, rats, and dogs. It also displayed good in vivo antitumor efﬁcacies in the TRKA/B/C, TRKA

mutants, and KM-12-LUC cells mediated mouse models. The potent activity against clinically important

TRK mutants combined with the good in vivo PK and efﬁcacy properties of 34 indicated that it might be a

new potential therapeutic candidate for TRK kinase fusion or mutants driven cancers.

Keywords:

NTRK

Kinase inhibitor

TRK mutant

Drug resistance

1. Introduction

The tropomyosin receptor kinases including TRKA, B, and C

regulate synaptic strength and plasticity in the mammalian ner-

vous system [1], which can be activated by speciﬁc neurotrophins

such as nerve growth factors (NGF), brain-derived neurotrophic

factor (BDNF), neurotrophin-4,5 (NT-4,5), and neurotrophin-3 (NT-

3). The binding of TRKA receptor by NGF causes the activation of the

Abbreviations: TRK, tropomyosin receptor kinase; CSF1R, colony stimulating

factor 1 receptor; SAR, structureꢀactivity relationship; THP, tetrahydropyranyl;

DMSO, dimethylsulfoxide; DMF, N,N-dimethylformamide; DCM, dimethyl chloride;

DIPEA, N,N-diisopropylethylamine; HATU, 1-[bis(dimethylamino)methylene]-1H-

1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexaﬂuorophosphate.

* Corresponding author. Institute of Health & Medical Technology, Hefei In-

stitutes of Physical Science, Chinese Academy of Sciences, Science Island, Hefei,

Anhui, 230031, PR China.

MAPK, phospholipase C-

increased proliferation and ERK kinase signaling. TRKB transduces

the BDNF signal via MAPK, PI3K, and PLC pathway as well,

g (PLCg), and PI3K pathway, which leads to

** Corresponding author. Institute of Health & Medical Technology, Hefei In-

stitutes of Physical Science, Chinese Academy of Sciences, Science Island, Hefei,

Anhui, 230031, PR China.

g

resulting in neuronal differentiation and survival. TRKC coupling

with NT-3 causes activation of the PI3K/AKT pathway which pre-

vents apoptosis and increases cell survival [2]. It is well known that

E-mail addresses: qsliu97@hm ﬂ .ac.cn (Q. Liu), jingliu@hm ﬂ .ac.cn (J. Liu).

These authors contribute equally.

1

https://doi.org/10.1016/j.ejmech.2020.1 12744

B. Wang, W. Zhang, X. Liu et al.

European Journal of Medicinal Chemistry 207 (2020) 112744

gene rearrangements which resulted in the aberrant activity of

kinases can drive the oncogenesis in a variety of cancers. NTRK

rearrangements and fusion products (mostly NTRK1 and 3) can lead

to transcription of chimeric TRK proteins with constitutively acti-

vated or over expressed kinase function conferring oncogenic po-

tential, which have been observed in numerous types of cancers

such as thyroid cancer, glioblastoma, colon cancer, breast cancer,

and melanoma etc [2,3]. Therefore, targeting TRK kinases has

emerged as an attractive therapeutic approach for a diverse set of

cancers.

compound 6, through a fragments hybrid type II kinase inhibitor

design approach [15], we designed and obtained compound 34

with a ring-closure strategy in the linker region. 34 was a potent,

selective, and orally available TRK inhibitor. It could not only

potently inhibit the wt TRKA, B, and C kinases, but also overcome

the drug resistant mutants including TRKA F589L and G667C/S

(Fig. 2).

2. Results and discussion

Recently, a number of kinase inhibitors bearing activities against

TRKA, TRKB and/or TRKC have been developed [4,5] (Fig. 1). Laro-

trectinib (LOXO-101, 1), which was the ﬁrst FDA approved type I TRK

inhibitor, has achieved great clinical response [6]. However, muta-

tions in the kinase domain of TRK such as G595R (located adjacent to

the hinge binding area) and G667C/S (located in the DFG motif) were

discovered after chronic treatment. Entrectinib (RXDX-101, 2) is the

second FDA approved type I TRK inhibitor which also has activity

against ROS1 and ALK kinases [7]. Despite the high clinical response,

drug acquired resistance was also observed in the metastatic colo-

rectal cancer patients after 4 months of treatment [8e10]. Belizatinib

(TSR-011, 3), a potent dual TRK/ALK inhibitor under clinical devel-

opment [1 1], also exhibited resistance to G595R and G667C/S mu-

tations [10]. Therefore, the second generation TRK inhibitors that can

overcome some of the acquired resistances such as selitrectinib

(LOXO-195, 4) and repotrectinib (TPX-0005, 5) were developed. The

macrocyclic compound 4 can overcome G595R and G667C/S muta-

tions mediated resistances [12]. Compound 5, a TRK inhibitor which

also inhibits the kinases ROS1/SRC/FAK/ALK, can overcome acquired

drug resistant mutants including G595R and G667C/S [13]. In addi-

tion, several other TRK inhibitors were developed and tried in

different cancers [5].

2.1. Chemistry

As shown in Scheme 1, intermediate 45a-b and 45d-f were

synthesized from 6-bromo-3-iodo-1H-indazole via THP protection

and Suzuki or Heck coupling reaction of 44. Palladium-catalyzed

CeS cross-coupling reaction of 45a-b with methyl 3-

mercaptopropanoate provided methyl ester propanoate 46a-b,

which were then hydrolyzed to give carboxylic acid 47a-b. Finally,

amide coupling reaction and removal of the THP protecting group

afforded the target compounds 11e12. Scheme 2 depicts the syn-

thetic route for preparation of target compounds 17e18. Palladium-

catalyzed CeS cross-coupling of 48 with 3-aminobenzenethiol

provided the intermediate 49. Amide coupling reaction or forma-

tion of urea followed by deprotection of the THP group afforded the

desired compounds. Thiazole analogues 19e43 were prepared as

illustrated in Scheme 3. Amide coupling reaction of 50 with

different carboxylic acids provided the intermediates 51a-t.

Palladium-catalyzed coupling reaction and THP deprotection

offered the target compounds.

2.2. Design rationale and structureeactivity relationship (SAR)

exploration

During the journey of seeking novel TRK kinase inhibitor, we

found that CHMFL-ABL-121 (6), a type II ABL kinase inhibitor pre-

viously reported by our group [14], exhibited potent inhibitory

activities against TRKA, B, and C. However, it also bore activities

against FLT3, c-KIT, and VEGFR2 kinases and displayed poor PK

proﬁles in vivo. Therefore, starting from the indazole scaffold of

Compound 6 is a highly potent type II ABL kinase inhibitor which

is capable of overcoming the T315I gatekeeper mutant [14]. The

primary test with kinase transformed BaF3 cells showed that it could

also inhibit TRKA (GI₅₀¼ 0.063

mM), TRKB/C, VEGFR2, c-KIT, and FLT3

kinases potently. In order to better understand its binding mode to

Fig. 1. Chemical structures of representative TRK kinase inhibitors.

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European Journal of Medicinal Chemistry 207 (2020) 112744

Fig. 2. Schematic illustration of discovery of compound 34 (IHMT-TRK-284) from compound 6.

Scheme 1. Synthesis of Compounds 11e12. Reagents and conditions: (a) THP, CH₃SO₃H, THF, 80 ^ꢁC, overnight; (b) for 45a-b: 4-vinylpyridine or 2-vinylpyrazine, DIPEA, tri-o-

tolyphosphine, PdCl₂, DMF, 100 ^ꢁC, 12 h; for 45d-f: arylboronic acid pinacol ester, Pd(PPh₃)₄, K₂CO₃, DMF, 80 ^ꢁC, overnight; (c) 3-mercaptopropionate, DIPEA, xantphos, Pd(OAc)₂,

DMA, 150 ^ꢁC, 2 h; (d) LiOH, MeOH, H₂O, rt, 6 h; (e) (i) 4-((4-methylpiperazin-1-yl)methyl)-3-(triﬂuoromethyl)aniline, HATU, DIPEA, DMF, rt, 2 h; (ii) CF₃COOH, DCM, rt, 1 h.

Scheme 2. Synthesis of Compounds 17e18. Reagents and conditions: (a) 3-aminobenzenethiol, Pd₂(dba)₃, NaHCO₃, xantphos, DMF, 60 ^ꢁC, overnight; (b) (i) for 17: 4-((4-

methylpiperazin-1-yl)methyl)-3-(triﬂuoromethyl)benzoic acid, HATU, DIPEA, DMF, rt, 1 h; for 18: 1-(4-isocyanato-2-(triﬂuoromethyl)benzyl)-4-methylpiperazine, DCM, rt, 1 h;

(ii) CF₃COOH, DCM, rt, 1 h.

the TRK, we ﬁrst docked compound 6 to TRKA kinase (PDB ID: 6PL1)

(Fig. 3). The docking results demonstrated that 6 preferred to adopt

the canonical type II binding mode with the “DFG-out” inactive

conformation of TRKA. The indazole core formed two hydrogen

bonds with Glu590 and Tyr591 in the hinge binding area (Fig. 3A).

The carbonyl and NH of the linkage moiety formed two canonical

hydrogen bonds with Asp668 of the DFG motif and Glu560 of the C-

helix, respectively. Interestingly, the calculation also showed that the

carbonyl formed a weak aromatic hydrogen bond with the DFG motif

(Fig. 3B). In addition, the pyridine in the “head” moiety formed two

non-classical hydrogen bonds with Met592 and Arg593 [16]

(Fig. 3C). The “tail” moiety occupied the hydrophobic pocket formed

by the “DFG-out” shift which was comprised of four hydrophobic

residues Leu563, Leu567, Ile572, and Ile667 (Fig. 3D). In addition, the

nitrogen atom of the piperazine in the “tail” moiety formed a

hydrogen bond with Asp668 of the DFG motif. Based on this analysis,

we envisioned that the ﬂexible “linker” moiety might be the key part

that could improve the activity to TRK kinases, and meanwhile

achieve selectivity over VEGFR2, c-KIT, and FLT3 kinases. Therefore,

we launched the medicinal chemistry campaign with the type II

kinase inhibitor binding elements hybrid design approach [15,1 7]

and explored the SAR by varying the “head”, “linker”, and “tail”

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European Journal of Medicinal Chemistry 207 (2020) 112744

Scheme 3. Synthesis of Compounds 19e43. Reagents and conditions: (a) R²COOH, HATU, K₂CO₃, DMF, rt, overnight; (b) (i) for 19e38: 48, 1.5e2 h₂, tri-tert-butylphosphinete-

traﬂuoroborate, Cs₂CO₃, DMSO, 135 ^ꢁC, 1.5e2 h; for 39e43: 45c-g, Pd(OAc)₂, tri-tert-butylphosphinetetraﬂuoroborate, Cs₂CO₃, DMSO, 135 ^ꢁC, 1.5e2 h; (ii) CF₃COOH, DCM, rt, 1 h.

moieties of 6. We used the TEL fused TRKA kinase transformed BaF3

cells for potency evaluation of the new compounds. Meanwhile,

given the fact that VEGFR2, c-KIT, and FLT3 kinases share high

structural similarity, and considering that VEGFR2 kinase activity

was associated with hypertension mediated cardiovascular toxicities

[18,19], we chose VEGFR2 as primary selectivity readout in the SAR

exploration.

We ﬁrst ﬁxed the hinge binding part as indazole and explored

the “linker” (L) and “head” (R¹) moieties (Table 1). In the beginning

we checked several previously reported derivatives of compound 6

[14] (7e10, 13e15) from in-house compound library for the SAR

exploration. The growth inhibitions of IL-3 independent BaF3 cells

expressing TRKA wt (BaF3-TEL-TRKA) and VEGFR2 (BaF3-TEL-

VEGFR2) were used as potency and selectivity readouts respec-

tively. Replacement of the R¹moiety in 6 with methyl (7), pyridine

(8, 10), and 2-methyl pyridine groups (9) resulted in signiﬁcant

activity loss to TRKA but maintaining the potency to VEGFR2.

Therefore, we synthesized two compounds by changing the “head”

moiety to 4-vinylpyridine (11) and 2-vinylpyrazine (12), which

both showed strong inhibition against VEGFR2. While 11 gained

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European Journal of Medicinal Chemistry 207 (2020) 112744

Fig. 3. Design rationale of new type II TRK kinase inhibitor. (A) Docking model of compound 6 with TRKA (PDB ID: 6PL1). (B) Cartoon view of the interaction between Phe589 and

the “linker” moiety of compound 6. (C) Solid surface view of the “head” moiety adjacent to the hinge binding area. (D) Surface illustration of the pocket created by the “DFG-out” ﬂip

of TRKA kinase.

better potency to TRKA, it also exhibited stronger cytotoxicity in

parental BaF3 cells. The inhibitory activity of 12 to TRKA was not

improved compared with 6. For the “linker” moiety, switching the

direction of the amide bond (13), transforming the amide to urea

(14), and increasing the length (15) all led to dramatic activity

decrease to TRKA. Replacement of the amide linkage with malo-

namide (16) or phenyl thiol ether (17) did not achieve potency to

TRKA either. Interestingly, switching the amide moiety of 17 to urea

(18) led to increase of the activity to TRKA meanwhile retained the

activity to VEGFR2. We hypothesized that increasing the rigidity of

such ﬂexible linker through a ring-closure strategy might allow it to

differentiate conformational ﬂexibility of kinases and thus lead to

improved selectivity [20,21]. In addition, aromatic ring of the

the selectivity to VEGFR2, we reasoned that the hydrophobic ar-

omatic rings at R²might be preferred by VEGFR2. Therefore, we

returned to hydrophilic moieties such as N-methyl piperidine

(33), N-methyl-piperazine (34), piperidine (35e36), more hydro-

philic N-hydroxyl ethyl-piperazine (37), and the PEG like linear

chain (38). In general, all these compounds showed less inhibition

to VEGFR2 and more potent activity to TRKA. Among them, 34

exhibited the most potent inhibition to TRKA and the best selec-

tivity over VEGFR2 kinase.

Finally, we ﬁxed the “linker” (L) and “tail” (R²) moieties as in 34,

and further explored the “head” (R¹) moiety (Table 3). Unfortu-

nately, simply removing the vinyl group (39) signiﬁcantly

decreased the activity to TRKA. Addition of a hydrophobic F-sub-

stituent to the pyridine ring (40) started to gain the activity to TRKA

but still not as good as 34. Changing the pyridine to phenyl ring (41)

lost the activity to TRKA completely. Other aromatic heterocycles

such as amino-pyrimidine (42) and N-methyl pyrazole (43) didn’t

increase the activity to TRKA either relative to 34.

linkage moiety may form p-p interaction with Phe589 and Phe669

residues, which might help to retain the potency (Fig. 3). As ex-

pected, introduction of thiazole ring (19) resulted in decreased

activity to VEGFR2 (GI₅₀¼ 0.38

m

M) and meanwhile achieved

strong inhibition to TRKA (GI₅₀¼ 0.044

mM).

Since 19 started to gain potency to TRKA and selectivity over

VEGFR2, we then explored the “linker” (L) and “tail” (R²) moieties

by ﬁxing R¹as vinyl pyridine (Table 2). When the linkage part was

the same with 19, changing R²to methyl (20) and isopropyl groups

(21) both led to activity loss. Switching R²to longer alkyl chain 1-

amino-3-methylthio propyl (22) caused higher cytotoxicity in

parental BaF3 cells. Introduction of the hydrophilic six-member

ring piperidine (23) and N-methyl piperidine (24) both gained

selectivity over VEGFR2 but slightly decreased the activity to

TRKA. Amino-phenethyl moiety at R²(25) again increased cyto-

toxicity in parental BaF3 cells. 4-(N-Methyl)piperazine (26) and 3-

CF₃substituted phenyl groups (27) resulted in lower selectivity

over VEGFR2. The amide group in the “linker” moiety was then

changed to urea (28), which resulted in higher cytotoxicity in

parental BaF3 cells. It was further changed to acetamide, and

meanwhile R²was switched to different aromatic rings (29e32),

which did not achieve selectivity over VEGFR2 kinase though

potent activity to TRKA was retained. Since 23 and 24 improved

2.3. Selectivity proﬁling of compound 34

Since 34 displayed the best potency against TRKA and selectivity

against VEGFR2, we next evaluated its activity to TRKB, TRKC, and a

panel of mutants of TRKA. As expected, 34 also displayed potent

inhibitory activity to the TEL fused TRKB and TRKC transformed

BaF3 cells (GI₅₀s ¼ 0.008

mM and 0.027 mM) (Fig. 4C). In order to

examine the antiproliferative effect of 34 against TRKA mutants, we

then generated the clinically relevant LMNA fused TRKA trans-

formed BaF3 cells (Table 4). In this system, 34 was still very potent

to TRKA wt (GI₅₀: 0.007 mM). In addition, it was also potent to the

V573M, F589L (gatekeeper mutation) and G667C/S mutants. By

comparison, the clinically used ﬁrst generation TRK inhibitor 1 was

resistant to the F589L and G677C/S mutants. Both 1 and 34 were

resistant to the G595R mutant. The second generation TRK inhibitor

4 could overcome most of these mutants except that it was slightly

resistant to the gatekeeper mutant F589L.

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European Journal of Medicinal Chemistry 207 (2020) 112744

Table 1

SAR exploration focused on the L and R¹Moities^a.

Compd.

R¹

L

BaF3 (GI₅₀

:

m

M)

BaF3-TEL-TRKA (GI₅₀

0.063 0.01

:

m

M)

BaF3-TEL-VEGFR2 (GI₅₀

0.002 0.001

: mM)

6

2.48 0.12

3.9 0.08

2.65 0.32

1.18 0.25

1.06 0.10

7

CH₃-

0.729 0.054

0.736 0.068

0.516 0.031

0.448 0.021

0.009 0.02

0.009 0.004

0.001 0.001

8

9

10

11

12

13

14

15

16

0.543 0.028

1.64 0.011

4.4 0.32

0.018 0.001

0.088 0.003

0.37 0.011

0.646 0.034

0.37 0.031

0.248 0.013

0.002 0.001

0.041 0.009

0.042 0.002

0.001 0.001

0.004 0.001

3.1 0.10

4.4 0.18

2.5 0.087

17

18

1.26 0.15

3.09 0.26

0.92 0.031

0.062 0.003

0.013 0.002

0.015 0.004

19

1.77 0.17

0.044 0.008

0.376 0.054

a

All GI₅₀values were obtained by triplet testing.

We also investigated the selectivity proﬁle of 34 against other

kinases in the kinome. In the DiscoverX’s KINOMEscan assay [22],

34 exhibited good selectivity (S score (1) ¼ 0.02) against a panel of

(IC₅₀

¼

2.62 nM), CSF1R (IC₅₀

¼

1.2 nM), and PDGFR

a

b

(IC₅₀¼ 24.2 nM), and it inhibited ABL1 (IC₅₀¼ 83.6 nM), PDGFR

(IC₅₀¼ 95.7 nM), and c-KIT wt (IC₅₀¼ 2167 nM) much less potently.

In order to further validate these targets in the cellular background,

we then used different kinase transformed BaF3 cells to test 34

468 kinases and mutants at 1

mM concentration (Fig. 4A and

Supplementary Table 1). Besides TRKA, TRKB, and TRKC kinases, 34

also displayed strong binding afﬁnity to ABL1, CSF1R, KIT, PDGFR

and PDGFR kinases (percent activity remaining less than 1% at

M of 34). Given the fact that KINOMEscan is a binding assay and

a

(Fig. 4C). The data showed that TRKA/B/C, PDGFRa/b, and CSF1R

were potently inhibited, which recapitulated the biochemical assay

results.

b

1

m

sometimes it cannot really reﬂect the inhibitory activity of a com-

pound, we then used the Invitrogen’s Z’LYTE and Promega’s ADP-

Glo biochemical assays with the puriﬁed kinase proteins to

conﬁrm the inhibitory activity of 34 against these kinase targets

(Fig. 4B). The results showed that 34 could potently inhibit the

activities of TRKA (IC₅₀¼ 10.5 nM), TRKB (IC₅₀¼ 0.71 nM), TRKC

2.4. Binding mode of compound 34

In order to better understand the binding mode of 34, we then

docked the compound into TRKA wt and different mutants. The

docking results demonstrated that 34 could ﬁt well in the “DFG-

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European Journal of Medicinal Chemistry 207 (2020) 112744

Table 2

SAR exploration focused on the L and R²Moieties^a.

Compd.

L

R²

BaF3 (GI₅₀

:

mM)

BaF3-TEL-TRKA (GI₅₀

:

mM)

BaF3-TEL-VEGFR2 (GI₅₀

0.301 0.081

: mM)

20

CH₃-

5.24 0.18

4.31 0.21

21

22

23

24

25

26

27

28

1.58 0.012

0.251 0.006

0.013 0.001

0.145 0.021

0.089 0.001

0.068 0.002

0.008 0.001

0.003 0.001

0.181 0.009

0.101 0.001

3.05 0.18

0.594 0.011

8.71 0.19

2.51 0.012

0.708 0.058

1.46 0.061

>10

2.04 0.098

0.097 0.015

0.012 0.002

0.005 0.001

0.004 0.001

0.105 0.008

29

30

31

32

33

34

2.31 0.054

4.1 0.11

0.002 0.001

0.04 0.001

0.033 0.001

0.032 0.003

0.009 0.001

0.013 0.001

0.01 0.001

0.019 0.001

0.015 0.002

0.57 0.01

4.87 0.098

3.69 0.18

1.56 0.065

1.4 0.045

1.18 0.016

(continued on next page)

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European Journal of Medicinal Chemistry 207 (2020) 112744

Table 2 (continued)

Compd.

L

R²

BaF3 (GI₅₀

:

mM)

BaF3-TEL-TRKA (GI₅₀

:

mM)

BaF3-TEL-VEGFR2 (GI₅₀

0.982 0.054

: mM)

35

>10

0.116 0.01

36

37

38

8.35 0.27

0.3 0.019

1.93 0.65

0.081 0.009

1.78 0.14

1.02 0.069

1.07 0.098

0.018 0.002

0.035 0.002

a

All GI₅₀values were obtained by triplet testing.

out” inactive conformation of TRKA and adopted a type II binding

mode as 6. The indazole core formed two hydrogen bonds with

Glu590 and Tyr591 in the hinge binding area. The thiazole ring in

2.5. Cellular evaluation of compound 34

In order to conﬁrm the on-target effects of 34, we ﬁrst examined

its inhibition effect against the phosphorylation of TRK kinase in

transformed BaF3 cells (Fig. 6A). The results showed that 34 was

the linkage moiety formed a sandwich

p-p interaction with the

phenyl rings of Phe589 and Phe669. The carbonyl and NH groups

formed two hydrogen bonds with Glu560 and Gly667, respectively.

The pyridine in the “head” moiety formed two non-classical

hydrogen bonds with Met592 and Arg593. The N-methyl-pipera-

zine in the “tail” moiety occupied the small hydrophobic pocket

formed by Leu563, Leu567, Ile572, and Ile667 residues (Fig. 5A). The

3-D models of V573M, F589L, G667C/S mutants were generated

from TRKA wt (PDB code: 6PL1) using homology modeling. The

docking results showed that these four mutants didn’t affect the

binding of 34 with TRKA kinase directly, which is consistent with

the biological assay results (Fig. 5BeE). However, the steric hin-

drance to the pyridine ring in the “head” moiety increased when

Gly595 residue was mutated to arginine, which might help to

explain the resistance of 34 to the G595R mutant (Fig. 5F).

able to inhibit the phosphorylation of TRKA Y490 (EC₅₀¼ 0.026

m

M)

and corresponding tyrosine residues TRKB Y515 (EC₅₀¼ 0.069

and TRKC Y516 (EC₅₀¼ 0.029

mM), which is required for SHC as-

sociation and activation of the RAS-MAP kinase cascades [23,24].

Among the ﬁve tested TRKA mutants, 34 could potently inhibit the

phosphorylation of Y490 in V573M, F589L, and G667C/S mutants

with EC₅₀s of 0.013

mM, 0.021 mM, 0.067 mM, and 0.074 mM

respectively (Fig. 6B). It was less potent to G595R mutant, which is

consistent with the growth inhibition results in BaF3 cells.

Next, we examined the antiproliferative effects of 34 against the

more clinically relevant colon cancer cell line KM-12-LUC which is

TPM3-TRKA fused. 34 exhibited similar growth inhibition activity

(GI₅₀: 0.002

mM) to 1 (GI₅₀: 0.011 mM) and 4 (GI₅₀: 0.005 mM).

Signaling pathway analysis demonstrated that 34 blocked the

Table 3

SAR exploration focused on the R¹Moiety^a.

Compd.

R¹

BaF3

BaF3-TEL-TRKA (GI₅₀

:

mM)

BaF3-TEL-VEGFR2 (GI₅₀

: mM)

39

5.13 0.21

0.251 0.028

2.63 0.21

40

41

1.69 0.036

0.054 0.001

1.8 0.025

1.74 0.025

>10

1.49 0.016

0.752 0.045

0.073 0.002

1.71 0.068

5.63 0.098

0.351 0.012

42

43

a

1.53 0.054

All GI₅₀values were obtained by triplet testing.

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European Journal of Medicinal Chemistry 207 (2020) 112744

Fig. 4. Selectivity proﬁling of compound 34. (A) Treespot demonstration of selectivity proﬁling of 34 with DiscoverX’s KINOMEscan technology. Measurements were performed at a

concentration of 1 M of the inhibitor. The afﬁnity was deﬁned with DMSO as control. (B) Biochemical tests of 34 with Invitrogen’s Z’LYTE assay or Promega’s ADP-Glo assay. (C)

m

Antiproliferative effects of 34 against a panel of kinase transformed BaF3 cells.

Table 4

Antiproliferative Effects of 1, 4, and 34 against a Panel of TRKs wt/Mutants Trans-

formed BaF3 Cells.

following intravenous injection and oral administration (Table 5).

34 exhibited acceptable drug exposure (AUC_0-t) in all these three

species (1431 ng/mL^.h at 10 mg/kg in mice, 952 ng/mL^.h at

10 mg/kg in rats, and 464 ng/mL^.h at 5 mg/kg in dogs). The half-

life (T_1/2) of 34 was different in these three species through oral

administration, i.e., 2.5 h in rats, 3.4 h in mice, and relatively

longer time in dogs (11.8 h), indicating that 34 was eliminated

very slowly in dogs relative to mice and rats. 34 possessed

acceptable bioavailability in mice (F ¼ 20%), rats (F ¼ 26%), and

dogs (F ¼ 35%). These PK properties indicated that 34 would be

suitable for oral administration. We also performed the dose-

dependent pharmacokinetic study of 34 in Sprague Dawlay

rats. The data showed that the AUC and C_maxincreased dose-

dependently (Fig. 8).

We then evaluated the in vivo antitumor efﬁcacy of 34 in

different cells (BaF3-TEL-TRKA, BaF3-TEL-TRKB, BaF3-TEL-TRKC,

BaF3-LMNA-TRKA-F589L, BaF3- LMNA-TRKA-G667S, and cancer

cell line KM-12-LUC) inoculated allograft or xenograft mouse

models by oral administration (Figs. 9 and 10). The two dosages of

34 in the study (40 mg/kg and 80 mg/kg) did not affect the animal

body weights and no abnormal behavior was observed, which

indicated that there was no general cytotoxicity at these dosages

(Fig. 9AeC,10A-C left lane). During 10 days of continuous treatment

with 34 at a dosage of 80 mg/kg/day in transformed BaF3 cells

allograft mouse models, the compound dose-dependently inhibi-

ted the BaF3-TEL-TRKA, BaF3-TEL-TRKB, and BaF3-TEL-TRKC tumor

progression with TGI (tumor growth inhibition) of 68%, 93%, and

58% (Fig. 9). At the same dosage, 1 showed moderate inhibition in

Cells (GI₅₀

BaF3

:

m

M)

Compd. 1

Compd. 4

Compd. 34

>10

3.57 0.14

1.4 0.011

BaF3-LMNA-TRKA

0.029 0.005

0.258 0.01

0.102 0.008

1.19 0.015

0.602 0.013

0.148 0.021

0.006 0.001

0.016 0.001

0.052 0.002

0.005 0.001

0.01 0.01

0.007 0.001

0.003 0.001

0.004 0.001

0.194 0.013

0.034 0.002

0.002 0.001

BaF3-LMNA-TRKA-V573M

BaF3-LMNA-TRKA-F589L

BaF3-LMNA-TRKA-G595R

BaF3-LMNA-TRKA-G667C

BaF3-LMNA-TRKA-G667S

0.029 0.003

^aAll GI₅₀values were obtained by triplet testing.

phosphorylation of TRKA Y490 in KM-12-LUC cells at the concen-

tration of 0.01 M upon 2 h exposure, and it also remarkably

inhibited the phosphorylation of downstream signaling mediators

AKT T308/S473 and ERK1/2 (T202/Y204) (EC₅₀less than 0.03 M)

m

(Fig. 7A). Compounds 1 and 4 displayed similar effects. In addition,

34 could induce dose-dependent cell apoptotic death (by exam-

ining the cleaved PARP) and arrest the cell cycle into G0/G1 phase in

colon cancer cell line KM-12-LUC upon 24 h exposure (Fig. 7A and

B). These data suggested that 34 exerts its inhibitory effect to the

colon cancer cells through on-target inhibition of TRK.

2.6. In vivo PK and efﬁcacy evaluation of compound 34

The in vivo PK properties of 34 were evaluated in different

species including mice, Sprague Dawley rats and Beagle dogs

9

B. Wang, W. Zhang, X. Liu et al.

European Journal of Medicinal Chemistry 207 (2020) 112744

Fig. 5. Molecular modeling analyses of the binding modes of TRKA/mutants with compound 34. (A) Binding mode of 34 with TRKA homology model (PDB code: 6PL1). Binding

modes of 34 with TRKA (B) F589L, (C) V573M, (D) G667C, (E) G667S, and (F) G595R homology models (generated based on PDB code: 6PL1).

BaF3-TEL-TRKA and BaF3-TEL-TRKC cells mediated allograft mouse

models, however it almost couldn’t inhibit the tumor progression

of BaF3-TEL-TRKB. In the KM-12-LUC cells inoculated xenograft

mouse model, 34 dose-dependently inhibited the tumor progres-

sion and exhibited the TGI of 93% at 40 mg/kg/day and 95% at

80 mg/kg/day (Fig. 10). In addition, in the 1-resistant mutants BaF3-

LMNA-TRKA-F589L and BaF3-LMNA-TRKA-G667S cells [25] inocu-

lated allograft mouse models, 34 also potently inhibited the tumor

growth with TGI values of 88% and 89% respectively at 80 mg/kg

dosage. Compared with the positive control groups (4, 80 mg/kg

bid), 34 treatment groups (80 mg/kg, qd or bid) exhibited relatively

better efﬁcacies in these mouse models.

4. Experimental section

4.1. e 4.2. Chemistry

All reagents and solvents were purchased from commercial

sources and used without further puriﬁcation. ¹H NMR and ¹³C

NMR spectra were recorded on a Bruker 400 or Bruker 500 spec-

trometer and referenced to chloroform-d (CDCl₃) or dimethyl

sulfoxide (DMSO)‑d₆. Chemical shifts are expressed in ppm. In the

NMR tabulation, s indicates singlet; d, doublet; t, triplet; q, quartet;

m, multiplet; and br, broad peak. LC/MS were performed on an

Agilent 6224 TOF using an ESI source coupled to an Agilent 1260

Inﬁnity HPLC system operating in reverse mode with an Agilent

Eclipse Plus C18 1.8

chromatography was conducted using silica gel (Silicycle

40e64 m). All the compounds were determined to be >95% pure.

m

m, 3.0 mm ꢂ 50 mm column. Flash column

3. Conclusions

m

The syntheses of compounds 6e10, 13e15, 45c, and 45g were re-

ported in previous work [14].

Starting from the previously developed type II multi-target ki-

nase inhibitor 6, which has good TRK potency but poor selectivity

and PK proﬁle, we have discovered a novel potent TRK kinase in-

hibitor 34 through the type II kinase inhibitor binding elements

hybrid drug design approach. 34 exhibited potent inhibitory effects

against TRKA/B/C wt, and it could overcome drug resistant mutants

including V573M, F589L at the gatekeeper position, and G667C/S in

the DFG region. In addition, from the KINOMEscan binding assay,

in-vitro biochemical assay and antiproliferation assay with trans-

formed BaF3 cell lines, 34 exhibited good selectivity proﬁle in the

kinome and only showed inhibitory activities to TRKs and the type

4.1.1. (E)-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)-3-((3-(2-(pyridin-4-yl)vinyl)-1H-indazol-

6-yl)thio)propanamide (11)

To a solution of 47a (50 mg, 0.12 mmol) in DMF (3 mL) were

added 4-((4-methylpiperazin-1-yl)methyl)-3-(triﬂuoromethyl)an-

iline (30 mg, 0.12 mmol), HATU (90 mg, 0.24 mmol), and DIEPA

(40

mL, 0.24 mmol). The resulting mixture was stirred at room

temperature for 2 h. Then it was diluted with EtOAc (10 mL),

washed with water (10 mL ꢂ 3) and brine (10 mL). The organic

layers were dried over anhydrous sodium sulfate and concentrated.

The residue was diluted with DCM (5 mL) and CF₃COOH (2 mL). The

reaction mixture was stirred at room temperature for 4 h and then

III kinase family including CSF1R and PDGFRa/b kinases but no

other protein kinases. The good in vivo PK and antitumor efﬁcacy

properties suggested that 34 might be a potential drug candidate as

a supplementation for TRK fusionepositive cancers.

10

B. Wang, W. Zhang, X. Liu et al.

European Journal of Medicinal Chemistry 207 (2020) 112744

Fig. 6. (A) Inhibitory effects against the phosphorylation of TRKA, TRKB, and TRKC at different sites in BaF3 isogenic cell lines after treatment of compounds 34, 1 and 4. (B) In-

hibition of the phosphorylation of TRKA at different sites in BaF3 isogenic cell lines harboring mutants in solvent front domain, ATP binding pocket, and xDFG moiety after treatment

of compounds 34, 1 and 4.

the pH value was adjusted to 12 with saturated sodium bicarbon-

ate. The mixture was extracted by EtOAc (10 mL) and washed with

water (10 mL ꢂ 2) followed by brine (10 mL). The organic layers

were dried over sodium sulfate, ﬁltered, concentrated and puriﬁed

by silica gel column chromatography (eluting with 0e5% MeOH in

DCM) to afford 11 (43 mg, 62%) as a yellow solid. ¹H NMR (500 MHz,

DMSO‑d₆) d 169.14, 147.39, 145.93, 141.32, 140.85, 137.94, 133.87,

130.89, 129.66, 126.95 (q, J ¼ 29.6 Hz), 125.92, 125.56, 124.70,

121.82, 121.70, 120.76, 120.62, 118.67, 115.42 (q, J ¼ 6.1 Hz), 108.59,

55.92, 51.81, 48.47, 41.30, 35.30, 27.49; HRMS (ESI) m/z calcd for

C

₃₀H₃₂F₃N₆OS [MþH]^þ581.2310; found, 581.2311.

Compounds 12 was prepared following the synthetic procedure

DMSO‑d₆)

d

13.43 (s, 1H), 10.45 (s, 1H), 8.63 (d, J ¼ 6.1 Hz, 2H), 8.19

of 11.

(d, J ¼ 8.5 Hz, 1H), 8.05 (d, J ¼ 2.0 Hz, 1H), 7.91 (d, J ¼ 16.7 Hz, 1H),

7.85 (d, J ¼ 5.5 Hz, 2H), 7.80 (dd, J ¼ 8.5, 1.8 Hz, 1H), 7.63 (d,

J ¼ 8.5 Hz, 1H), 7.56e7.49 (m, 2H), 7.22 (dd, J ¼ 8.5, 1.4 Hz, 1H), 3.62

(s, 2H), 3.43e3.21 (m, 4H), 3.01e2.97 (m, 2H), 2.89e2.79 (m, 2H),

2.77e2.70 (m, 5H), 2.46e2.41 (m, 2H); ¹³C NMR (126 MHz,

4.1.2. (E)-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)-3-((3-(2-(pyrazin-2-yl)vinyl)-1H-indazol-

6-yl)thio)propanamide (12)

Yield ¼ 64%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.38 (s, 1H), 10.43

11

B. Wang, W. Zhang, X. Liu et al.

European Journal of Medicinal Chemistry 207 (2020) 112744

Fig. 7. Cellular effects of compound 34 in colon cancer cell line KM-12-LUC. (A) Effects of 34 on TRKA mediated signaling and apoptosis pathways. (B) Effects of 34 on cell cycle

progression.

(s, 1H), 8.92 (d, J ¼ 1.2 Hz, 1H), 8.65 (dd, J ¼ 3.8, 1.5 Hz, 1H), 8.52 (d,

J ¼ 2.4 Hz, 1H), 8.16 (d, J ¼ 8.5 Hz, 1H), 8.05 (d, J ¼ 2.0 Hz, 1H), 8.02

(d, J ¼ 16.4 Hz, 1H), 7.79 (dd, J ¼ 8.5, 1.6 Hz, 1H), 7.65e7.59 (m, 2H),

7.52 (s, 1H), 7.22 (dd, J ¼ 8.5, 1.2 Hz, 1H), 3.61 (s, 2H), 3.34e3.37 (m,

4H), 3.07e2.90 (s, 2H), 2.88e2.77 (m, 2H), 2.77e2.67 (m, 5H),

2.46e2.39 (m, 2H); ¹³C NMR (126 MHz, DMSO‑d₆)

d 170.22, 151.14,

144.99, 144.55, 143.60, 142.45, 141.94, 139.01, 134.89, 131.91, 130.83,

127.99 (q, J ¼ 29.6 Hz), 126.51, 125.79, 125.67, 123.61, 122.92, 122.79,

121.60, 119.66, 116.49 (q, J ¼ 6.1 Hz), 109.75, 70.24, 57.05, 52.97,

49.65, 42.49, 36.38, 28.61; HRMS (ESI) m/z calcd for C₂₉H₃₁F₃N₇OS

[MþH]^þ582.2263; found, 582.2263.

4.1.3. (E)-N¹-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)-N³-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-

6-yl)malonamide (16)

Compound 16 was synthesized following previously reported

Fig. 8. Dose-dependent pharmacokinetic study of compound 34 in Sprague Dawley

rats.

method [26]. Yield ¼ 53%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.15 (s,

1H), 10.60 (s,1H),10.48 (s,1H), 8.66e8.57 (m, 1H), 8.19 (d, J ¼ 1.7 Hz,

1H), 8.13 (d, J ¼ 8.7 Hz, 1H), 8.09 (d, J ¼ 2.2 Hz, 1H), 7.91 (d,

J ¼ 16.5 Hz, 1H), 7.81 (dt, J ¼ 7.8, 1.9 Hz, 2H), 7.68 (d, J ¼ 8.5 Hz, 1H),

7.66 (d, J ¼ 7.8 Hz, 1H), 7.54 (d, J ¼ 16.4 Hz, 1H), 7.30e7.21 (m, 2H),

3.58 (s, 2H), 3.57 (s, 2H), 2.78e2.56 (m, 4H), 2.48e2.40 (m, 4H), 2.33

(s, 3H); HRMS (ESI) m/z calcd for C₃₀H₃₁F₃N₇O₂[MþH]^þ578.2491;

found, 578.2488.

Table 5

Pharmacokinetic study of compound 34 in mice, sprague dawley rats, and beagle dogs^a.

34

Mice

Rats

Beagle Dogs

Parameter

i.v. (1 mg/kg)

748

p.o. (10 mg/kg)

1431

i.v. (1 mg/kg)

393

p.o. (10 mg/kg)

952

i.v. (1 mg/kg)

323

p.o. (5 mg/kg)

464

AUC(0-t)

(ng/mL$h)

Tmax (h)

T_1/2(h)

Vz (mL/kg)

CLz (m/h/kg)

Cmax (ng/L)

F (%)

0.033

2.6

4934

1302

1107

e

1.5

3.4

31567

6502

258

20

0.03

2.7

9682

2481

363

e

4.7

2.5

37364

10182

139

0.08

0.03

17511

2861

284

4.3

11.8

149260

8408

131

26

e

35

a

All testing data were obtained from three independent mice.

12

B. Wang, W. Zhang, X. Liu et al.

European Journal of Medicinal Chemistry 207 (2020) 112744

Fig. 9. Antitumor efﬁcacies of 34 in the (A) BaF3-TEL-TRKA, (B) BaF3-TEL-TRKB, and (C) BaF3-TEL-TRKC allograft mouse models.

4.1.4. (E)-4-((4-Methylpiperazin-1-yl)methyl)-N-(3-((3-(2-

(pyridin-2-yl)vinyl)-1H-indazol-6-yl)thio)phenyl)-3-

(triﬂuoromethyl)benzamide (17)

J ¼ 8.2 Hz, 1H), 7.86 (s, 1H), 7.81 (t, J ¼ 6.6 Hz, 2H), 7.66 (d, J ¼ 7.8 Hz,

1H), 7.56 (d, J ¼ 16.4 Hz, 1H), 7.48 (s, 1H), 7.43 (t, J ¼ 8.0 Hz, 1H), 7.28

(dd, J ¼ 6.9, 5.2 Hz, 1H), 7.19 (t, J ¼ 6.6 Hz, 2H), 3.75 (s, 2H), 2.69 (s,

3H), 3.25e2.93 (m, 4H), 2.91e2.56 (m, 7H); HRMS (ESI) m/z calcd

for C₃₄H₃₂F₃N₆OS [MþH]^þ629.2310; found, 629.2305.

To a solution of 49 (30 mg, 0.07 mmol) in DMF (5 mL) were

added

4-((4-methylpiperazin-1-yl)methyl)-3-(triﬂuoromethyl)

benzoic acid (24 mg, 0.08 mmol), HATU (41.8 mg, 0.11 mmol), and

DIPEA (18.1 mg, 0.14 mmol). The resulting mixture was stirred at

room temperature for 1 h. Then it was diluted with EtOAc (50 mL)

and washed with water (100 mL ꢂ 3) followed by brine (100 mL).

The organic layers were dried over anhydrous sodium sulfate and

concentrated. The residue was diluted with DCM (1 mL) and

CF₃COOH (0.5 mL). The reaction mixture was stirred at room tem-

perature for 1 h and then the pH value was adjusted to 12 with

saturated sodium bicarbonate. The mixture was extracted by EtOAc

(50 mL ꢂ 2) and washed with water (50 mL ꢂ 2) followed by brine

(50 mL). The organic layers were dried over anhydrous sodium

sulfate, ﬁltered, and concentrated. The residue was puriﬁed by silica

gel column chromatography (eluting with 0e5% MeOH in DCM) to

afford 17 as a yellow solid (27 mg, 61%). ¹H NMR (500 MHz,

4.1.5. (E)-1-(4-((4-Methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)-3-(3-((3-(2-(pyridin-2-yl)vinyl)-1H-

indazol-6-yl)thio)phenyl)urea (18)

To a solution of 49 (30 mg, 0.07 mmol) in DCM (5 mL) was added

1-(4-isocyanato-2-(triﬂuoromethyl)benzyl)-4-methylpiperazine

(24 mg, 0.08 mmol). The resulting mixture was stirred at room

temperature for 1 h and then concentrated to dryness. The residue

was diluted with DCM (5 mL) and CF₃COOH (0.5 mL). The reaction

mixture was stirred at room temperature for 1 h and then the pH

value was adjusted to 12 with saturated sodium bicarbonate. The

mixture was extracted by EtOAc (50 mL ꢂ 2) and washed with

water (50 mL ꢂ 2) followed by brine (50 mL). The organic layers

were dried over anhydrous sodium sulfate, ﬁltered, and concen-

trated. The residue was puriﬁed by silica gel column chromatog-

raphy (eluting with 0e5% MeOH in DCM) to afford 18 as a yellow

DMSO‑d₆)

d

13.32 (s, 1H), 10.57 (s, 1H), 8.60 (d, J ¼ 4.3 Hz, 1H), 8.25

(s, 1H), 8.24e8.19 (m, 2H), 7.93 (d, J ¼ 16.4 Hz, 1H), 7.90 (d,

13

B. Wang, W. Zhang, X. Liu et al.

European Journal of Medicinal Chemistry 207 (2020) 112744

Fig. 10. Antitumor efﬁcacies of 34 in the (A) KM-12-LUC xenograft mouse model, (B) BaF3-LMNA-TRKA-F589L and (C) BaF3-LMNA-TRKA-G667S allograft mouse models.

solid (34 mg, 75%). ¹H NMR (500 MHz, DMSO‑d₆)

d

13.29 (s, 1H),

solvent was removed under reduced pressure, and the residue was

puriﬁed by silica gel ﬂash chromatography (eluting with 0e5%

MeOH in DCM) to give the title compound 19 as a white solid

9.13 (d, J ¼ 49.0 Hz, 2H), 8.60 (d, J ¼ 4.5 Hz, 1H), 8.21 (d, J ¼ 8.4 Hz,

1H), 7.91 (d, J ¼ 5.3 Hz, 2H), 7.81 (t, J ¼ 7.7 Hz,1H), 7.66 (d, J ¼ 7.8 Hz,

1H), 7.61e7.56 (m, 3H), 7.47e7.41 (m, 2H), 7.36e7.25 (m, 3H), 7.18

(d, J ¼ 8.5 Hz, 1H), 7.02 (d, J ¼ 7.7 Hz, 1H), 3.54 (s, 2H), 2.76e2.28 (m,

11H); HRMS (ESI) m/z calcd for C₃₄H₃₃F₃N₇OS [MþH]^þ644.2419;

found, 644.2416.

(20 mg, 60%). ¹H NMR (500 MHz, DMSO‑d₆)

d 13.46 (s, 1H), 13.02 (s,

1H), 8.62 (d, J ¼ 4.4 Hz, 1H), 8.51 (s, 1H), 8.41 (d, J ¼ 8.2 Hz, 1H), 8.29

(d, J ¼ 8.5 Hz, 1H), 7.98 (dd, J ¼ 12.1, 8.5 Hz, 2H), 7.83 (t, J ¼ 7.1 Hz,

1H), 7.70 (d, J ¼ 7.8 Hz, 1H), 7.67e7.58 (m, 2H), 7.37 (d, J ¼ 8.4 Hz,

1H), 7.31e7.28 (m, 1H), 3.80 (s, 2H), 3.15e2.80 (m, 4H), 2.73 (s, 3H),

2.67e2.22 (m, 4H), 2.49 (s, 3H); HRMS (ESI) m/z calcd for

4.1.6. (E)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-

yl)thiazol-2-yl)-4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)benzamide (19)

C

₃₂H₃₁F₃N₇OS [MþH]^þ618.2263; found, 618.2268.

Compounds 20e43 were prepared following the synthetic

procedure of 19.

To a solution of 51a (30 mg, 0.08 mmol) in DMSO (10 mL) were

added 48 (39 mg, 0.09 mmol), Pd(OAc)₂(5 mg, 0.02 mmol), tri-tert-

butylphosphinetetraﬂuoroborate (13 mg, 0.05 mmol), and Cs₂CO₃

(49 mg,0.15 mmol). The resulting mixture was stirred at 135 ^ꢁC in

N₂atmosphere for 1.5 h, and then the mixture was cooled to rt

before poured into water (100 mL). The mixture was extracted with

EtOAc (30 mL ꢂ 3). The organic layers were washed with saturated

aqueous NaCl solution and then dried over anhydrous Na₂SO₄. The

4.1.7. (E)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-

yl)thiazol-2-yl)acetamide (20)

Yield ¼ 62%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.39 (s, 1H), 12.17

(s, 1H), 8.61 (d, J ¼ 4.4 Hz, 1H), 8.25 (d, J ¼ 8.5 Hz, 1H), 7.97 (d,

J ¼ 16.4 Hz, 1H), 7.82 (t, J ¼ 7.6 Hz, 1H), 7.70 (t, J ¼ 9.9 Hz, 1H),

7.64e7.55 (m, 2H), 7.35e7.24 (m, 2H), 2.41 (s, 3H), 2.16 (s, 3H). ¹³

C

14

B. Wang, W. Zhang, X. Liu et al.

European Journal of Medicinal Chemistry 207 (2020) 112744

NMR (126 MHz, DMSO‑d₆)

142.28, 142.14, 137.33, 130.65, 129.49,124.50, 124.40,123.06, 122.90,

d

168.92, 155.77, 155.45, 150.02, 142.74,

2.48 (s, 3H), 2.28 (d, J ¼ 83.4 Hz, 8H), 2.17 (s, 3H); HRMS (ESI) m/z

calcd for C₃₁H₃₂N₇OS [MþH]^þ550.2389; found, 550.2384.

122.84, 121.64, 120.19, 110.47, 29.09, 16.64; HRMS (ESI) m/z calcd for

C

₂₀H₁₈N₅OS [MþH]^þ376.1232; found, 376.1230.

4.1.14. (E)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-

yl)thiazol-2-yl)-3-(triﬂuoromethyl)benzamide (27)

4.1.8. (E)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-

Yield ¼ 59%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.43 (s, 1H), 13.05

yl)thiazol-2-yl)isobutyramide (21)

(s, 1H), 8.65 (d, J ¼ 4.2 Hz, 1H), 8.52 (s, 1H), 8.41 (d, J ¼ 7.8 Hz, 1H),

8.28 (t, J ¼ 12.3 Hz, 1H), 8.02 (dd, J ¼ 12.4, 3.8 Hz, 2H), 7.90 (t,

J ¼ 7.4 Hz, 1H), 7.86e7.74 (m, 2H), 7.70e7.57 (m, 2H), 7.44e7.28 (m,

Yield ¼ 61%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.34 (s, 1H), 12.12

(s, 1H), 8.73e8.49 (m, 1H), 8.26 (d, J ¼ 8.4 Hz, 1H), 7.97 (d,

J ¼ 16.4 Hz, 1H), 7.83 (td, J ¼ 7.7, 1.8 Hz, 1H), 7.70 (d, J ¼ 7.8 Hz, 1H),

7.60 (d, J ¼ 16.5 Hz, 2H), 7.36e7.23 (m, 2H), 2.75 (hept, J ¼ 6.8 Hz,

1H), 2.42 (s, 3H), 1.13 (d, J ¼ 6.9 Hz, 6H). ¹³C NMR (126 MHz,

2H), 2.49 (s, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 155.35, 149.90,

142.41, 142.12, 137.46, 132.74, 130.44, 130.40, 129.98, 129.72, 129.46,

127.63, 125.47, 125.37, 125.34, 125.31, 125.28, 124.44, 123.30, 123.10,

122.95, 122.89, 121.76, 121.13, 120.38, 110.56, 16.22; HRMS (ESI) m/z

calcd for C₂₆H₁₉F₃N₅OS [MþH]^þ506.1262; found, 506.1263.

DMSO‑d₆)

d 175.68, 155.92, 155.38, 149.93, 142.80, 142.39, 142.13,

137.43,130.76, 129.46, 124.56,124.45, 123.09, 122.93, 121.68, 120.25,

110.40, 34.29, 19.57, 16.59; HRMS (ESI) m/z calcd for C₂₂H₂₂N₅OS

[MþH]^þ404.1545; found, 404.1550.

4.1.15. (E)-1-(4-Methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-

yl)thiazol-2-yl)-3-(3-(triﬂuoromethyl)phenyl)urea (28)

4.1.9. (E)-2-Amino-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-

Yield ¼ 65%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.36 (s, 1H), 10.78

indazol-6-yl)thiazol-2-yl)-4-(methylthio)butanamide (22)

(s, 1H), 9.40 (s, 1H), 8.62 (d, J ¼ 4.1 Hz, 1H), 8.27 (d, J ¼ 8.4 Hz, 1H),

8.07 (s, 1H), 7.98 (d, J ¼ 16.4 Hz, 1H), 7.83 (td, J ¼ 7.7, 1.6 Hz, 1H), 7.69

(d, J ¼ 7.7 Hz, 2H), 7.64e7.50 (m, 3H), 7.38 (d, J ¼ 7.4 Hz, 1H),

7.35e7.24 (m, 2H), 2.41 (s, 3H); HRMS (ESI) m/z calcd for

Yield ¼ 58%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.36 (s, 1H),

8.63e8.59 (m, 1H), 8.26 (d, J ¼ 8.4 Hz, 1H), 8.02e7.91 (m, 1H),

7.86e7.78 (m, 1H), 7.69 (d, J ¼ 7.8 Hz, 1H), 7.64e7.57 (m, 2H),

7.35e7.25 (m, 2H), 7.03 (s, 1H), 3.52e3.38 (m, 1H), 2.64e2.53 (m,

2H), 2.43 (s, 3H), 2.07 (s, 3H), 2.05e1.94 (m, 1H),1.85 (m, 1H); HRMS

(ESI) m/z calcd for C₂₃H₂₅N₆OS₂[MþH]^þ465.1531; found, 465.1535.

C

₂₆H₂₀F₃N₆OS [MþH]^þ521.1371; found, 521.1374.

4.1.16. (E)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-

yl)thiazol-2-yl)-2-(3-(triﬂuoromethyl)phenyl)acetamide (29)

4.1.10. (E)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-

Yield ¼ 63%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.35 (s, 1H), 12.50

yl)thiazol-2-yl)piperidine-4-carboxamide (23)

(s, 1H), 8.61 (d, J ¼ 4.2 Hz, 1H), 8.25 (d, J ¼ 8.5 Hz, 1H), 7.97 (d,

J ¼ 16.4 Hz, 1H), 7.82 (td, J ¼ 7.7, 1.6 Hz, 1H), 7.74 (s, 1H), 7.71e7.63

(m, 3H), 7.63e7.56 (m, 3H), 7.33e7.25 (m, 2H), 3.93 (s, 2H), 2.43 (s,

3H); HRMS (ESI) m/z calcd for C₂₇H₂₁F₃N₅OS [MþH]^þ520.1419;

found, 520.1421.

Yield ¼ 62%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.37 (s, 1H), 8.62

(d, J ¼ 5.0, 1H), 8.26 (d, J ¼ 8.4 Hz, 1H), 7.97 (d, J ¼ 16.4 Hz, 1H), 7.82

(td, J ¼ 7.7, 1.9 Hz, 1H), 7.68 (d, J ¼ 7.8 Hz, 1H), 7.67e7.55 (m, 2H),

7.36e7.22 (m, 2H), 3.25e3.14 (m, 2H), 2.85e2.66 (m, 3H), 2.42 (s,

3H), 1.95e1.85 (m, 2H), 1.78e1.61 (m, 2H); HRMS (ESI) m/z calcd for

C

₂₄H₂₅N₆OS [MþH]^þ445.1811; found, 445.1813.

4.1.17. (E)-2-(3-Chlorophenyl)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)

vinyl)-1H-indazol-6-yl)thiazol-2-yl)acetamide (30)

4.1.11. (E)-1-Methyl-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-

Yield ¼ 61%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.37 (s, 1H), 12.46

indazol-6-yl)thiazol-2-yl)piperidine-4-carboxamide (24)

(s, 1H), 8.62 (s, 1H), 8.26 (d, J ¼ 8.3 Hz, 1H), 7.97 (d, J ¼ 16.8 Hz, 1H),

7.83 (t, J ¼ 7.5 Hz, 1H), 7.69 (d, J ¼ 7.8 Hz, 1H), 7.60 (d, J ¼ 10.8 Hz,

2H), 7.44 (s, 1H), 7.42e7.34 (m, 2H), 7.30 (t, J ¼ 9.8 Hz, 3H), 3.82 (s,

Yield ¼ 64%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.34 (s, 1H), 12.18

(s, 1H), 8.61 (d, J ¼ 3.8 Hz, 1H), 8.25 (d, J ¼ 8.5 Hz, 1H), 7.97 (d,

J ¼ 16.4 Hz, 1H), 7.82 (td, J ¼ 7.7, 1.8 Hz, 1H), 7.68 (d, J ¼ 7.8 Hz, 1H),

7.60 (d, J ¼ 16.4 Hz, 2H), 7.34e7.25 (m, 2H), 2.63e2.53 (m, 2H), 2.42

(s, 3H), 2.27 (s, 3H), 2.16e1.94 (m, 2H), 1.83 (d, J ¼ 12.3 Hz, 2H), 1.69

2H), 2.43 (s, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 169.19, 155.62,

155.37, 149.98, 142.96, 142.38, 142.08, 137.75, 137.39, 133.40, 130.73,

130.57, 129.68, 129.50, 128.53, 127.35, 124.84, 124.37, 123.12, 122.94,

122.88, 121.73, 120.27, 110.42, 41.59, 16.62; HRMS (ESI) m/z calcd for

(d, J ¼ 11.3 Hz, 2H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 173.68, 155.76,

155.41, 150.03, 142.82, 142.28, 137.35, 130.69, 129.57, 124.64, 124.27,

123.09, 122.94, 122.91, 121.70, 120.23, 110.50, 54.40, 45.64, 27.89,

16.59; HRMS (ESI) m/z calcd for C₂₅H₂₇N₆OS [MþH]^þ459.1967;

found, 459.1971.

C

₂₆H₂₁ClN₅OS [MþH]^þ486.1155; found, 486.1157.

4.1.18. (E)-2-(3,4-Dimethoxyphenyl)-N-(4-methyl-5-(3-(2-

(pyridin-2-yl)vinyl)-1H-indazol-6-yl)thiazol-2-yl)acetamide (31)

Yield ¼ 62%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.35 (s, 1H), 12.36

4.1.12. (E)-2-Amino-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-

(s, 1H), 8.61 (d, J ¼ 4.3 Hz, 1H), 8.25 (d, J ¼ 8.4 Hz, 1H), 7.96 (d,

J ¼ 16.4 Hz, 1H), 7.82 (td, J ¼ 7.7, 1.6 Hz, 1H), 7.68 (d, J ¼ 7.8 Hz, 1H),

7.59 (d, J ¼ 16.5 Hz, 2H), 7.33e7.26 (m, 2H), 6.97 (d, J ¼ 1.6 Hz, 1H),

6.92 (d, J ¼ 8.3 Hz,1H), 6.86 (dd, J ¼ 8.2,1.5 Hz,1H), 3.76 (s, 3H), 3.73

(s, 3H), 3.69 (s, 2H), 2.43 (s, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

indazol-6-yl)thiazol-2-yl)-3-phenylpropanamide (25)

Yield ¼ 60%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.33 (s, 1H),

8.71e8.53 (m, 1H), 8.35e8.17 (m, 1H), 7.97 (d, J ¼ 16.4 Hz, 1H), 7.82

(td, J ¼ 7.7, 1.8 Hz, 1H), 7.69 (d, J ¼ 7.9 Hz, 1H), 7.64e7.55 (m, 2H),

7.37e7.17 (m, 8H), 5.6 (br, 2H), 3.75 (dd, J ¼ 7.7, 6.0 Hz,1H), 3.01 (dd,

J ¼ 13.4, 7.7 Hz, 1H), 2.78 (dd, J ¼ 13.4, 7.7 Hz, 1H), 2.42 (s, 3H);

HRMS (ESI) m/z calcd for C₂₇H₂₅N₆OS [MþH]^þ481.1811; found,

481.1810.

d

169.93, 155.77, 155.43, 150.02, 149.11, 148.34, 142.90, 142.40,

142.11, 137.32, 130.65, 129.58, 127.72, 124.72, 124.33, 123.07, 122.91,

122.87, 121.73, 121.69, 120.27, 113.69, 112.45, 110.40, 56.05, 55.98,

41.81, 16.61; HRMS (ESI) m/z calcd for C₂₈H₂₆N₅O₃S [MþH]^þ

512.1756; found, 512.1757.

4.1.13. (E)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-

yl)thiazol-2-yl)-4-((4-methylpiperazin-1-yl)methyl)benzamide (26)

4.1.19. (E)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-

Yield ¼ 62%. ¹H NMR (500 MHz, DMSO‑d₆)

d

13.38 (s, 1H), 12.68

yl)thiazol-2-yl)-2-(naphthalen-1-yl)acetamide (32)

(s, 1H), 8.62 (d, J ¼ 4.1 Hz, 1H), 8.28 (d, J ¼ 8.4 Hz, 1H), 8.09 (d,

J ¼ 8.2 Hz, 2H), 7.98 (d, J ¼ 16.4 Hz, 1H), 7.83 (td, J ¼ 7.7, 1.7 Hz, 1H),

7.69 (d, J ¼ 7.8 Hz, 1H), 7.66e7.57 (m, 2H), 7.47 (d, J ¼ 8.2 Hz, 2H),

7.37 (dd, J ¼ 8.4,1.1 Hz,1H), 7.29 (dd, J ¼ 6.6, 4.9 Hz,1H), 3.55 (s, 2H),

Yield ¼ 64%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.35 (s, 1H), 12.59

(s, 1H), 8.61 (d, J ¼ 4.5 Hz, 1H), 8.24 (d, J ¼ 8.5 Hz, 1H), 8.11 (d,

J ¼ 8.3 Hz, 1H), 7.93e7.98 (m, 2H), 7.89 (d, J ¼ 8.0 Hz, 1H), 7.82 (td,

J ¼ 7.7, 1.7 Hz, 1H), 7.67 (d, J ¼ 7.8 Hz, 1H), 7.62e7.48 (m, 6H),

15

B. Wang, W. Zhang, X. Liu et al.

European Journal of Medicinal Chemistry 207 (2020) 112744

7.31e7.26 (m, 2H), 4.30 (s, 2H), 2.44 (s, 3H). ¹³C NMR (126 MHz,

4.1.25. (E)-2-(2-Methoxyethoxy)-N-(4-methyl-5-(3-(2-(pyridin-2-

DMSO‑d₆)

d

169.67, 155.71, 155.42, 150.02, 142.96, 142.40, 142.09,

yl)vinyl)-1H-indazol-6-yl)thiazol-2-yl)acetamide (38)

137.32, 133.88, 132.34, 131.90, 130.60, 129.57, 128.97, 128.62, 128.05,

126.76,126.27,126.05,124.79, 124.54, 124.32, 123.09, 122.91, 122.86,

121.70, 120.27, 110.40, 16.62; HRMS (ESI) m/z calcd for C₃₀H₂₄N₅OS

[MþH]^þ502.1702; found, 502.1697.

Yield ¼ 62%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.50 (s, 1H), 11.87

(s, 1H), 9.29e9.15 (m, 1H), 8.63 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.49e8.29

(m, 1H), 8.17 (d, J ¼ 8.3 Hz, 1H), 7.72e7.49 (m, 2H), 7.33 (dd, J ¼ 8.5,

1.5 Hz, 1H), 3.28 (s, 2H), 2.62e2.51 (m, 4H), 2.43 (s, 3H), 2.40e2.25

(m, 4H), 2.16 (s, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 169.11, 155.41,

155.13, 149.99, 142.94, 142.40, 142.12, 137.36, 130.56, 129.55, 124.94,

124.37, 123.07, 122.92, 121.70, 120.31, 110.49, 71.60, 70.84, 69.80,

58.57, 16.60; HRMS (ESI) m/z calcd for C₂₃H₂₄N₅O₃S [MþH]^þ

450.1600; found, 450.1606.

4.1.20. (E)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-

yl)thiazol-2-yl)-2-(1-methylpiperidin-4-yl)acetamide (33)

Yield ¼ 64%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.36 (s, 1H), 12.16

(s, 1H), 8.73e8.50 (m, 1H), 8.25 (d, J ¼ 8.4 Hz, 1H), 7.97 (d,

J ¼ 16.4 Hz, 1H), 7.82 (td, J ¼ 7.7, 1.8 Hz, 1H), 7.69 (d, J ¼ 7.9 Hz, 1H),

7.64e7.57 (m, 2H), 7.31 (dd, J ¼ 8.4,1.4 Hz,1H), 7.28 (ddd, J ¼ 7.5, 4.8,

1.0 Hz, 1H), 2.91e2.82 (m, 2H), 2.42 (s, 3H), 2.38 (d, J ¼ 7.1 Hz, 2H),

2.26 (s 3H), 2.13e1.98 (m, 2H), 1.83e1.72 (m, 1H), 1.68e1.65 (d,

J ¼ 12.1 Hz, 2H), 1.34e1.21 (m, 2H). ¹³C NMR (126 MHz, DMSO‑d₆)

4.1.26. N-(4-methyl-5-(3-(pyridin-3-yl)-1H-indazol-6-yl)thiazol-2-

yl)-2-(4-methylpiperazin-1-yl)acetamide (39)

Yield ¼ 63%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.50 (s, 1H), 11.87

(s, 1H), 9.29e9.15 (m, 1H), 8.63 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.49e8.29

(m, 1H), 8.17 (d, J ¼ 8.3 Hz, 1H), 7.72e7.49 (m, 2H), 7.33 (dd, J ¼ 8.5,

1.5 Hz,1H), 3.28 (s, 2H), 2.43 (s, 3H), 2.16 (s, 3H). ¹³C NMR (126 MHz,

d

170.73, 155.61, 155.43, 150.02, 142.82, 142.26, 142.22, 137.33,

130.70, 129.56, 124.56, 124.27, 123.07, 122.91, 121.68, 120.23, 110.52,

DMSO‑d₆)

d 168.95, 155.29, 149.23, 147.92, 142.98, 142.35, 141.13,

55.14, 45.82, 41.96, 32.40, 31.35, 16.60; HRMS (ESI) m/z calcd for

C

₂₆H₂₉N₆OS [MþH]^þ473.2124; found, 473.2120.

134.41, 130.67, 129.87, 124.73, 124.53, 123.19, 121.55, 119.60, 110.58,

60.61, 55.07, 52.95, 46.21, 16.59; HRMS (ESI) m/z calcd for

C

₂₃H₂₆N₇OS [MþH]^þ448.1920; found, 448.1920.

4.1.21. (E)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-

yl)thiazol-2-yl)-2-(4-methylpiperazin-1-yl)acetamide (34)

4.1.27. N-(5-(3-(5-ﬂuoropyridin-3-yl)-1H-indazol-6-yl)-4-

methylthiazol-2-yl)-2-(4-methylpiperazin-1-yl)acetamide (40)

Yield ¼ 63%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.36 (s, 1H), 11.88

(s, 1H), 8.62 (d, J ¼ 4.0 Hz, 1H), 8.26 (d, J ¼ 8.4 Hz, 1H), 7.97 (d,

J ¼ 16.4 Hz, 1H), 7.82 (td, J ¼ 7.7, 1.7 Hz, 1H), 7.69 (d, J ¼ 7.8 Hz, 1H),

7.64e7.57 (m, 2H), 7.35e7.30 (m, 1H), 7.29 (dd, J ¼ 7.0, 5.2 Hz, 1H),

3.28 (s, 2H), 2.53e2.51 (m, 4H), 2.43 (s, 3H), 2.40e2.24 (m, 4H), 2.16

Yield ¼ 60%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.66 (s,1H), 9.07 (t,

J ¼ 1.8 Hz, 1H), 8.60 (d, J ¼ 2.7 Hz, 1H), 8.22 (dt, J ¼ 9.9, 2.3 Hz, 1H),

8.19 (d, J ¼ 8.5 Hz, 1H), 7.66 (s, 1H), 7.34 (dd, J ¼ 8.5, 1.3 Hz, 2H), 3.32

(s, 3H), 2.62 (s, 14H), 2.40 (s, 3H), 2.35 (s, 4H); HRMS (ESI) m/z calcd

for C₂₃H₂₅FN₇OS [MþH]^þ466.1825; found, 466.1830.

(s, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 168.97, 155.36, 155.25,

150.01, 142.90, 142.29, 142.19, 137.38, 130.61, 129.57, 124.82, 124.25,

123.08, 122.96, 122.93, 121.70, 120.24, 110.55, 60.59, 55.01, 52.92,

46.16, 16.56; HRMS (ESI) m/z calcd for C₂₅H₂₈N₇OS [MþH]^þ

474.2076; found, 474.2071.

4.1.28. N-(5-(3-(3-ﬂuorophenyl)-1H-indazol-6-yl)-4-

methylthiazol-2-yl)-2-(4-methylpiperazin-1-yl)acetamide (41)

Yield ¼ 64%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.46 (s, 1H), 8.15

(d, J ¼ 8.5 Hz,1H), 7.86 (d, J ¼ 7.8 Hz,1H), 7.74 (d, J ¼ 9.9 Hz,1H), 7.64

(s, 1H), 7.58 (dd, J ¼ 14.3, 8.0 Hz, 1H), 7.32 (dd, J ¼ 8.5, 1.3 Hz, 1H),

7.24 (td, J ¼ 8.6, 2.5 Hz, 1H), 3.30 (s, 2H), 2.62e2.52 (m, 4H), 2.40 (s,

3H), 2.38e2.25 (m, 4), 2.26 (s, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

4.1.22. (E)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-

yl)thiazol-2-yl)-2-(piperidin-4-yl)acetamide (35)

Yield ¼ 60%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.36 (s, 1H), 12.16

(s, 1H), 8.61 (d, J ¼ 4.0 Hz, 1H), 8.25 (d, J ¼ 8.5 Hz, 1H), 8.01e7.92 (m,

1H), 7.82 (t, J ¼ 7.7 Hz, 1H), 7.68 (d, J ¼ 7.8 Hz, 1H), 7.62e7.55 (m,

2H), 7.34e7.24 (m, 2H), 3.24 (d, J ¼ 12.6 Hz, 2H), 2.95e2.80 (m, 2H),

2.45e2.39 (m, 5H), 2.12e2.01 (m, 1H), 1.84e1.76 (m, 2H), 1.70e1.58

(m, 1H), 1.44e1.32 (m, 2H); HRMS (ESI) m/z calcd for C₂₅H₂₇N₆OS

[MþH]^þ459.1967; found, 459.1962.

d

168.90, 164.01, 162.07, 155.25, 142.92, 142.45, 136.38, 136.32,

131.50, 131.43, 130.48, 124.75, 123.24, 123.22, 123.10, 121.57, 119.41,

115.09, 114.92, 113.58, 113.40, 110.64, 60.35, 54.65, 52.30, 45.51,

16.58; HRMS (ESI) m/z calcd for C₂₄H₂₆FN₆OS [MþH]^þ465.1873;

found, 465.1870.

4.1.29. N-(5-(3-(2-aminopyrimidin-5-yl)-1H-indazol-6-yl)-4-

methylthiazol-2-yl)-2-(4-methylpiperazin-1-yl)acetamide (42)

4.1.23. (E)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-

yl)thiazol-2-yl)-2-(piperidin-3-yl)acetamide (36)

Yield ¼ 59%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.24 (s, 1H), 11.85

Yield ¼ 61%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.34 (s, 1H), 12.18

(s, 1H), 8.83 (s, 2H), 8.07 (d, J ¼ 8.5 Hz, 1H), 7.57 (d, J ¼ 15.2 Hz, 1H),

7.25 (dt, J ¼ 26.4, 13.2 Hz, 1H), 6.89 (s, 2H), 3.28 (s, 2H), 2.61e2.55

(m, 8H), 2.42 (s, 3H), 2.16 (s, 3H); HRMS (ESI) m/z calcd for

(s, 1H), 8.61 (d, J ¼ 3.8 Hz, 1H), 8.25 (d, J ¼ 8.5 Hz, 1H), 7.97 (d,

J ¼ 16.4 Hz, 1H), 7.82 (td, J ¼ 7.7, 1.8 Hz, 1H), 7.68 (d, J ¼ 7.8 Hz, 1H),

7.60 (d, J ¼ 16.4 Hz, 2H), 7.34e7.25 (m, 2H), 3.15e3.06 (m, 2H),

2.71e2.61 (m, 1H), 2.52 (s, 2H), 2.45e2.38 (m, 5H), 2.16e2.07 (m,

1H), 1.78e1.66 (m, 2H), 1.61e1.48 (m, 1H); HRMS (ESI) m/z calcd for

C

₂₂H₂₆N₉OS [MþH]^þ464.1981; found, 464.1980.

4.1.30. N-(4-methyl-5-(3-(1-methyl-1H-pyrazol-4-yl)-1H-indazol-

6-yl)thiazol-2-yl)-2-(4-methylpiperazin-1-yl)acetamide (43)

C

₂₅H₂₇N₆OS [MþH]^þ459.1967; found, 459.1966.

Yield ¼ 64%. ¹H NMR (500 MHz, DMSO‑d₆)

d 12.93 (d,

4.1.24. (E)-2-(4-(2-Hydroxyethyl)piperazin-1-yl)-N-(4-methyl-5-

(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-yl)thiazol-2-yl)acetamide

(37)

J ¼ 66.6 Hz, 1H), 11.87 (s, 1H), 8.36 (d, J ¼ 17.3 Hz, 1H), 8.06 (d,

J ¼ 8.4 Hz, 1H), 8.00 (s, 1H), 7.54 (s, 1H), 7.23 (dd, J ¼ 8.4, 1.4 Hz, 1H),

3.94 (s, 3H), 3.30 (s, 2H), 2.64e2.52 (m, 6H), 2.48e2.42 (m, 2H), 2.41

(s, 3H), 2.29e2.19 (s, 3H); HRMS (ESI) m/z calcd for C₂₂H₂₇N₈OS

[MþH]^þ451.2029; found, 451.2028.

Yield ¼ 61%. ¹H NMR (500 MHz, DMSO‑d₆)

d 13.36 (s, 1H), 11.95

(s, 1H), 8.62 (d, J ¼ 4.0 Hz, 1H), 8.26 (d, J ¼ 8.5 Hz, 1H), 7.97 (d,

J ¼ 16.4 Hz, 1H), 7.82 (td, J ¼ 7.7, 1.8 Hz, 1H), 7.69 (d, J ¼ 7.8 Hz, 1H),

7.62e7.56 (m, 2H), 7.30 (ddd, J ¼ 12.9, 7.9, 3.4 Hz, 2H), 4.81 (s, 1H),

3.69e3.54 (m, 2), 3.36 (s, 2H), 2.90e2.58 (m, 8H), 2.50e2.49 (m,

2H), 2.43 (s, 3H); HRMS (ESI) m/z calcd for C₂₆H₃₀N₇O₂S [MþH]^þ

504.2182; found, 504.2182.

4.1.31. 6-Bromo-3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole

(44)

To a solution of 6-bromo-3-iodo-1H-indazole (5 g, 15.5 mmol) in

THF (200 mL) were added THP (6.5 g, 77.4 mmol) and CH₃SO₃H

16

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European Journal of Medicinal Chemistry 207 (2020) 112744

(225 mg, 2.4 mmol). The resulting mixture was stirred at 80 ^ꢁC

overnight, and then it was cooled to room temperature before

water was added. The mixture was extracted with EtOAc

(3 ꢂ 200 mL). The organic layers were washed with brine and dried

over anhydrous sodium sulfate. The solvent was removed by

evaporation under reduced pressure, and the residue was puriﬁed

by silica gel ﬂash chromatography (eluting with 0e10% EtOAc in

heptane) to afford 44 as a white solid (5.5 g, 88%). ¹H NMR

3.93e3.78 (m, 1H), 2.36 (s, 2H), 2.11e2.02 (m, 2H), 1.83e1.57 (m,

3H); HRMS (ESI) m/z calcd for C₁₇H₁₆BrFN₃O [MþH]^þ376.0461;

found, 376.0460.

Compounds 45e-f were prepared following the synthetic pro-

cedure of 45d.

4.1.35. 6-Bromo-3-(3-ﬂuorophenyl)-1-(tetrahydro-2H-pyran-2-

yl)-1H-indazole (45e)

(500 MHz, DMSO‑d₆)

d

8.11 (dd, J ¼ 2.8, 1.5 Hz, 1H), 7.51e7.32 (m,

¹H NMR (500 MHz, DMSO‑d₆)

d

8.15 (s, 1H), 8.07 (d, J ¼ 8.6 Hz,

2H), 5.96e5.76 (m, 1H), 4.00e3.66 (m, 2H), 2.42e2.26 (m, 1H),

1H), 7.83 (dt, J ¼ 7.8, 1.2 Hz, 1H), 7.72 (ddd, J ¼ 10.2, 2.6, 1.5 Hz, 1H),

7.59 (td, J ¼ 8.1, 6.2 Hz, 1H), 7.45e7.41 (m, 1H), 7.30 (td, J ¼ 8.7,

2.4 Hz, 1H), 6.03e5.95 (m, 1H), 3.95e3.78 (m, 2H), 2.48e2.40 (m,

1H), 2.11e1.92 (m, 2H), 1.82e1.55 (m, 3H); HRMS (ESI) m/z calcd for

2.14e1.83 (m, 2H), 1.83e1.45 (m, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d

141.03, 127.69, 125.77, 123.24, 122.05, 113.90, 95.29, 84.47, 67.10,

29.30, 25.12, 22.59; HRMS (ESI) m/z calcd for C₁₂H₁₃BrIN₂O [MþH]^þ

406.9256; found, 406.9254.

C

₁₈H₁₇BrFN₂O [MþH]^þ375.0508; found, 375.0505.

4.1.32. (E)-6-Bromo-3-(2-(pyridin-4-yl)vinyl)-1-(tetrahydro-2H-

pyran-2-yl)-1H-indazole (45a)

4.1.36. 5-(6-Bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-

yl)pyrimidin-2-amine (45f)

Compound 44 (2 g, 4.9 mmol) was dissolved in DMF (50 mL). 4-

Vinylpyridine (2.6 mL, 24.6 mmol), DIPEA (1.6 mL, 10.0 mmol),

Palladium chloride (177 mg, 1.0 mmol), and o-tritolylphosphine

(595 mg, 2.0 mmol) were added, and then stirred for 12 h at 95 ^ꢁC

under an argon atmosphere. After the starting materials were

consumed, it was cooled to room temperature before water was

added. The mixture was extracted with EtOAc (3 ꢂ 50 mL). The

organic layers were washed with brine and dried over anhydrous

sodium sulfate. The solvent was removed by evaporation under

reduced pressure, and the residue was puriﬁed by silica gel ﬂash

chromatography (eluting with 0e25% EtOAc in heptane) to afford

45a as a white solid (1.15 g, 60%). ¹H NMR (500 MHz, DMSO‑d₆)

¹H NMR (500 MHz, DMSO‑d₆)

d

8.78 (s, 1H), 8.10 (d, J ¼ 1.6 Hz,

1H), 7.98 (d, J ¼ 8.7 Hz, 1H), 7.37 (dd, J ¼ 8.6, 1.6 Hz, 1H), 6.99 (s, 1H),

5.92 (dd, J ¼ 9.8, 2.4 Hz, 1H), 3.95e3.73 (m, 2H), 2.48e2.38 (m, 1H),

2.13e1.94 (m, 2H), 1.83e1.53 (m, 3H); HRMS (ESI) m/z calcd for

C

₁₆H₁₇BrN₅O [MþH]^þ374.0616; found, 374.0612.

4.1.37. (E)-methyl3-((3-(2-(pyridin-4-yl)vinyl)-1-(tetrahydro-2H-

pyran-2-yl)-1H-indazol-6-yl)thio)propanoate (46a)

To a mixture of 45a (1 g, 3.5 mmol) and DMA (20 mL) were

added methyl 3-mercaptopropionate (781

mL, 7.1 mmol), DIPEA

(1.2 mL, 7.1 mmol), xantphos (404 mg, 0.7 mmol), and Pd(OAc)₂

(62 mg, 0.35 mmol). The reaction mixture was stirred at 150 ^ꢁC for

2 h under argon. Then the reaction mixture was cooled to rt before

poured into water (50 mL). The mixture was extracted by EtOAc

(50 mL) and washed with water (50 mL ꢂ 2) followed by brine

(50 mL). The organic layers were dried over sodium sulfate, ﬁltered,

concentrated, and puriﬁed by silica gel column chromatography

(eluting with 0e30% EtOAc in heptane) to afford 46a (650 mg, 59%)

d

8.58 (dd, J ¼ 4.6, 1.4 Hz, 2H), 8.22 (d, J ¼ 8.6 Hz, 1H), 8.11 (d,

J ¼ 1.4 Hz, 1H), 7.83 (d, J ¼ 16.7 Hz, 1H), 7.72 (dd, J ¼ 4.7, 1.4 Hz, 2H),

7.53 (d, J ¼ 16.7 Hz,1H), 7.45 (dd, J ¼ 8.6,1.6 Hz,1H), 5.94 (dd, J ¼ 9.7,

2.4 Hz, 1H), 3.94e3.87 (m, 1H), 3.84e3.76 (m, 1H), 2.47e2.36 (m,

1H), 2.10e1.95 (m, 2H), 1.82e1.68 (m, 1H), 1.64e1.54 (m, 2H); HRMS

(ESI) m/z calcd for C₁₉H₁₉BrN₃O [MþH]^þ384.0711; found, 384.0710.

Compound 45b were prepared following the synthetic proced-

ure of 45a [14].

as a white solid. ¹H NMR (500 MHz, DMSO‑d₆)

d

8.57 (dd, J ¼ 4.6,

1.4 Hz, 2H), 8.19 (d, J ¼ 8.5 Hz, 1H), 7.81 (d, J ¼ 16.7 Hz, 1H), 7.73 (d,

J ¼ 1.4 Hz, 1H), 7.71 (dd, J ¼ 4.6, 1.4 Hz, 2H), 7.51 (d, J ¼ 16.7 Hz, 1H),

7.26 (dd, J ¼ 8.5, 1.4 Hz, 1H), 5.95 (dd, J ¼ 9.7, 2.4 Hz, 1H), 3.94e3.87

(m, 1H), 3.83e3.74 (m, 1H), 3.61 (s, 3H), 3.30 (t, J ¼ 7.0 Hz, 2H), 2.70

(t, J ¼ 7.0 Hz, 2H), 2.47e2.35 (m, 1H), 2.12e1.94 (m, 2H), 1.83e1.68

(m, 1H), 1.64e1.55 (m, 2H); HRMS (ESI) m/z calcd for C₂₃H₂₆N₃O₃S

[MþH]^þ424.1695; found, 424.1692.

4.1.33. (E)-6-Bromo-3-(2-(pyrazin-2-yl)vinyl)-1-(tetrahydro-2H-

pyran-2-yl)-1H-indazole (45b)

Yield ¼ 64%. ¹H NMR (500 MHz, DMSO‑d₆)

d

8.95 (d, J ¼ 1.4 Hz,

1H), 8.67 (dd, J ¼ 2.3, 1.5 Hz, 1H), 8.54 (d, J ¼ 2.5 Hz, 1H), 8.21 (d,

J ¼ 8.6 Hz, 1H), 8.13 (d, J ¼ 1.3 Hz, 1H), 8.01 (d, J ¼ 16.4 Hz, 1H), 7.68

(d, J ¼ 16.4 Hz, 1H), 7.46 (dd, J ¼ 8.6, 1.6 Hz, 1H), 5.95 (dd, J ¼ 9.7,

2.4 Hz, 1H), 3.95e3.87 (m, 1H), 3.84e3.75 (m, 1H), 2.46e2.35 (m,

1H), 2.11e1.96 (m, 2H), 1.82e1.69 (m, 1H), 1.67e1.55 (m, 2H); HRMS

Compounds 46b were prepared following the synthetic pro-

cedure of 46a.

(ESI) m/z calcd for

C

₁₈H₁₈BrN₄O [MþH]^þ385.0664; found,

4.1.38. (E)-Methyl 3-((3-(2-(pyrazin-2-yl)vinyl)-1-(tetrahydro-2H-

385.0666.

pyran-2-yl)-1H-indazol-6-yl)thio)propanoate(46b)

Yield ¼ 62%. ¹H NMR (500 MHz, DMSO‑d₆)

d

8.95 (d, J ¼ 1.5 Hz,

4.1.34. 6-Bromo-3-(5-ﬂuoropyridin-3-yl)-1-(tetrahydro-2H-pyran-

2-yl)-1H-indazole (45d)

1H), 8.66 (dd, J ¼ 2.5, 1.5 Hz, 1H), 8.53 (d, J ¼ 2.5 Hz, 1H), 8.17 (d,

J ¼ 8.5 Hz, 1H), 8.00 (d, J ¼ 16.4 Hz, 1H), 7.74 (d, J ¼ 1.4 Hz, 1H), 7.66

(d, J ¼ 16.4 Hz, 1H), 7.27 (dd, J ¼ 8.5, 1.5 Hz, 1H), 5.96 (dd, J ¼ 9.8,

2.6 Hz, 1H), 3.95e3.86 (m, 1H), 3.85e3.74 (m, 1H), 3.61 (s, 3H), 3.34

(t, J ¼ 7.0 Hz, 2H), 2.70 (t, J ¼ 7.0 Hz, 2H), 2.47e2.37 (m, 1H),

2.11e1.95 (m, 2H),1.83e1.70 (m,1H),1.66e1.56 (m, 2H); HRMS (ESI)

m/z calcd for C₂₂H₂₅N₄O₃S [MþH]^þ425.1647; found, 425.1644.

To a solution of 44 (150 mg, 0.35 mmol) in DMF (15 mL) were

added

3-ﬂuoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)

pyridine (100 mg, 0.45 mmol), Pd(PPh₃)₄(45 mg, 0.035 mmol), and

K₂CO₃(100 mg, 0.75 mmol). The resulting mixture was stirred at

80 ^ꢁC overnight under argon, and then it was cooled to room

temperature before water was added. The mixture was extracted

with EtOAc (3 ꢂ 50 mL). The organic layers were washed with brine

and dried over anhydrous sodium sulfate. The solvent was removed

by evaporation under reduced pressure, and the residue was pu-

riﬁed by silica gel ﬂash chromatography (eluting with 0e5% MeOH

in DCM) to afford 45d as a yellow solid (85 mg, 60%). ¹H NMR

4.1.39. (E)-3-((3-(2-(pyridin-4-yl)vinyl)-1-(tetrahydro-2H-pyran-

2-yl)-1H-indazol-6-yl)thio)propanoic acid (47a)

To a solution of 46a (200 mg, 0.47 mmol) in methanol (5 mL)

were added LiOH (113 mg, 4.7 mmol) and water (5 mL). The reac-

tion mixture was stirred at room temperature for 6 h under argon

protection. The mixture was then acidiﬁed with 1 N hydrochloric

acid to pH ¼ 3. The white precipitate was ﬁltered and dried to

(500 MHz, DMSO‑d₆)

d

8.16 (d, J ¼ 8.5 Hz, 1H), 7.86 (s, 1H), 7.76 (dt,

J ¼ 10.2, 2.0 Hz, 1H), 7.60 (td, J ¼ 8.0, 6.2 Hz, 1H), 7.38 (dd, J ¼ 8.5,

1.5 Hz, 1H), 7.29 (td, J ¼ 8.6, 2.6 Hz, 1H), 6.04 (dd, J ¼ 9.4, 2.3 Hz, 1H),

provide 47a (160 mg, 83%). ¹H NMR (500 MHz, DMSO‑d₆)

d 8.94 (dd,

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European Journal of Medicinal Chemistry 207 (2020) 112744

J ¼ 11.3, 1.5 Hz, 1H), 8.66 (dt, J ¼ 6.4, 1.9 Hz, 1H), 8.52 (dd, J ¼ 6.6,

2.5 Hz, 1H), 8.17 (d, J ¼ 8.5 Hz, 1H), 7.98 (dd, J ¼ 18.3, 16.3 Hz, 1H),

7.73 (d, J ¼ 1.5 Hz, 1H), 7.64 (dd, J ¼ 19.2, 16.3 Hz, 1H), 7.26 (dd,

J ¼ 8.5, 1.5 Hz, 1H), 5.96 (dd, J ¼ 9.7, 2.6 Hz, 1H), 3.97e3.66 (m, 3H),

3.29 (t, J ¼ 7.0 Hz, 2H), 2.61 (t, J ¼ 7.0 Hz, 2H), 2.41 (dddd, J ¼ 29.7,

16.5, 10.0, 4.0 Hz, 2H), 2.01 (dddd, J ¼ 31.0, 17.7, 13.3, 5.7 Hz, 2H),

1.82e1.68 (m, 2H); HRMS (ESI) m/z calcd for C₂₂H₂₄N₃O₃S [MþH]þ

410.1538; found, 410.1540.

4.1.44. N-(4-methylthiazol-2-yl)isobutyramide (51c)

Yield ¼ 84%. ¹H NMR (500 MHz, CDCl₃)

d 9.48 (s,1H), 6.46 (s,1H),

2.52 (hept, J ¼ 6.9 Hz, 1H), 2.27 (s, 3H), 1.20 (s, 3H), 1.18 (s, 3H);

HRMS (ESI) m/z calcd for C₈H₁₃N₂OS [MþH]^þ185.0749; found,

185.0747.

4.1.45. 2-Amino-N-(4-methylthiazol-2-yl)-4-(methylthio)

butanamide (51d)

Compounds 47b were prepared following the synthetic pro-

cedure of 47a.

Yield ¼ 85%. ¹H NMR (500 MHz, CDCl₃)

d 6.53 (s, 1H), 3.79 (dd,

J ¼ 8.5, 4.3 Hz, 1H), 2.75e2.58 (m, 2H), 2.36 (s, 3H), 2.35e2.29 (m,

1H), 2.12 (s, 3H), 1.86 (dt, J ¼ 14.2, 7.3 Hz, 1H); HRMS (ESI) m/z calcd

for C₉H₁₆N₃OS₂[MþH]^þ246.0735; found, 246.0730.

4.1.40. (E)-3-((3-(2-(pyrazin-2-yl)vinyl)-1-(tetrahydro-2H-pyran-

2-yl)-1H-indazol-6-yl)thio)propanoic acid(47b)

Yield ¼ 82%. ¹H NMR (500 MHz, DMSO‑d₆)

d 12.42 (s, 1H), 8.62

4.1.46. tert-Butyl 4-((4-methylthiazol-2-yl)carbamoyl)piperidine-

1-carboxylate (51e)

(d, J ¼ 1.7 Hz, 1H), 8.21 (d, J ¼ 8.5 Hz, 1H), 7.87 (s, 1H), 7.84 (s, 1H),

7.73 (d, J ¼ 1.5 Hz,1H), 7.54 (dd, J ¼ 16.7, 8.6 Hz,1H), 7.26 (dd, J ¼ 8.5,

1.5 Hz, 1H), 5.96 (dd, J ¼ 9.7, 2.6 Hz, 1H), 3.96e3.70 (m, 3H), 3.29 (t,

J ¼ 7.0 Hz, 2H), 2.61 (t, J ¼ 7.0 Hz, 2H), 2.48e2.35 (m, 1H), 2.10e1.95

(m, 2H), 1.82e1.70 (m, 1H), 1.65e1.56 (m, 2H); HRMS (ESI) m/z calcd

for C₂₁H₂₃N₄O₃S [MþH]^þ411.1491; found, 411.1494.

Yield ¼ 81%. ¹H NMR (500 MHz, DMSO‑d₆)

d 12.04 (s,1H), 6.73 (s,

1H), 4.05e3.85 (m, 2H), 2.91e2.67 (m, 2H), 2.63 (tt, J ¼ 11.4, 3.6 Hz,

1H), 2.25 (s, 3H), 1.85e1.73 (m, 2H), 1.47 (td, J ¼ 12.4, 3.9 Hz, 2H),

1.40 (s, 9H); HRMS (ESI) m/z calcd for C₁₅H₂₄N₃O₃S [MþH]^þ

326.1538; found, 326.1538.

4.1.41. (E)-3-((3-(2-(Pyridin-2-yl)vinyl)-1-(tetrahydro-2H-pyran-

2-yl)-1H-indazol-6-yl)thio)aniline (49)

4.1.47. 1-Methyl-N-(4-methylthiazol-2-yl)piperidine-4-

To a solution of 48 (1.29 g, 3.0 mmol) in DMF (20 mL) were

added 3-aminobenzenethiol (0.45 g, 3.6 mmol), NaHCO₃(1.16 g,

9.0 mmol), xantphos (0.35 g, 0.6 mmol), and Pd(dba)₂(0.07 g,

0.3 mmol). The reaction mixture was placed into an oil bath pre-

heated to 60 ^ꢁC and stirred at this temperature for 2 h under argon.

Then the reaction mixture was cooled to rt before poured into

water (100 mL). The mixture was extracted by EtOAc (100 mL) and

washed with water (100 mL ꢂ 2) followed by brine (100 mL). The

organic layers were dried over sodium sulfate, ﬁltered, concen-

trated and puriﬁed by silica gel column chromatography (eluting

with 0e30% EtOAc in heptane) to afford 49 as a white solid (0.90 g,

carboxamide (51f)

Yield ¼ 82% ¹H NMR (500 MHz, DMSO‑d₆)

d 12.00 (s,1H), 6.73 (d,

J ¼ 0.8 Hz, 1H), 2.89e2.81 (m, 2H), 2.42 (tt, J ¼ 11.5, 3.8 Hz, 1H), 2.25

(d, J ¼ 0.6 Hz, 3H), 2.21 (s, 3H), 1.95 (t, J ¼ 11.2 Hz, 2H), 1.77 (d,

J ¼ 11.3 Hz, 2H), 1.70e1.61 (m, 2H). ¹³C NMR (126 MHz, DMSO‑d₆)

d

173.78, 157.78, 146.98, 107.95, 54.90, 46.38, 41.29, 28.46, 17.33;

HRMS (ESI) m/z calcd for C₁₁H₁₈N₃OS [MþH]^þ240.1171; found,

240.1170.

4.1.48. 2-Amino-N-(4-methylthiazol-2-yl)-3-phenylpropanamide

79%). ¹H NMR (500 MHz, DMSO‑d₆)

d

8.61 (d, J ¼ 4.3 Hz, 1H), 8.17 (d,

(51g)

Yield ¼ 83%. ¹H NMR (500 MHz, CDCl₃)

d 10.59 (s, 1H), 7.30e7.13

J ¼ 8.5 Hz, 1H), 7.91 (d, J ¼ 16.3 Hz, 1H), 7.85e7.80 (m, 1H), 7.79 (s,

1H), 7.68 (d, J ¼ 7.8 Hz, 1H), 7.59 (d, J ¼ 16.3 Hz, 1H), 7.29 (dd, J ¼ 6.9,

5.2 Hz, 1H), 7.15 (d, J ¼ 8.5 Hz, 1H), 7.04 (t, J ¼ 7.8 Hz, 1H), 6.57 (s,

1H), 6.52 (d, J ¼ 7.9 Hz, 2H), 5.87 (dd, J ¼ 9.7, 2.4 Hz, 1H), 5.29 (br,

2H), 3.92e3.70 (m, 2H), 2.46e2.30 (m, 1H), 2.10e1.91 (m, 2H),

1.84e1.68 (m, 1H), 1.58 (hept, J ¼ 3.9 Hz, 2H); HRMS (ESI) m/z calcd

for C₂₅H₂₅N₄OS [MþH]^þ429.1749; found, 429.1750.

(m, 5H), 6.48 (s, 1H), 3.75 (dd, J ¼ 9.5, 3.8 Hz, 1H), 3.31 (dd, J ¼ 13.9,

3.7 Hz, 1H), 2.73 (dd, J ¼ 13.7, 9.7 Hz, 1H), 2.28 (s, 3H), 1.52 (s,

2H)$¹³C NMR (126 MHz, DMSO‑d₆)

d 172.57, 157.44, 147.12, 137.93,

129.80, 128.64, 126.87, 108.33, 56.14, 40.25, 17.30; HRMS (ESI) m/z

calcd for C₁₃H₁₆N₃OS [MþH]^þ262.1014; found, 262.1018.

4.1.49. 4-((4-Methylpiperazin-1-yl)methyl)-N-(4-methylthiazol-2-

4.1.42. 4-((4-Methylpiperazin-1-yl)methyl)-N-(4-methylthiazol-2-

yl)-3-(triﬂuoromethyl)benzamide (51a)

yl)benzamide (51h)

Yield ¼ 84% ¹H NMR (500 MHz, CDCl₃)

7.81 (d, J ¼ 8.2 Hz, 2H),

d

To a solution of 50 (1 g, 2.3 mmol) in DMF (20 mL) were added

K₂CO₃(3.2 g, 23 mmol), 4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)benzoic acid (0.84 g, 2.8 mmol) and HATU (1.1 g,

3 mmol) in batches, and the reaction was allowed to be stirred at rt

for 8h. The mixture was extracted by DCM (100 mL ꢂ 3) and

washed with water (100 mL ꢂ 2) followed by brine (100 mL). The

organic layers were dried over sodium sulfate, ﬁltered, concen-

trated and puriﬁed by silica gel column chromatography (eluting

with 0e30% EtOAc in heptane) to afford 51a as a white solid (0.72 g,

7.41 (d, J ¼ 8.1 Hz, 2H), 6.51 (s, 1H), 3.55 (s, 2H), 2.80e2.51 (m, 8H),

2.44 (s, 3H), 2.25 (s, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 165.21,

158.64, 146.94, 142.83, 131.60, 129.38, 128.59, 108.53, 61.07, 53.55,

50.49, 43.48, 17.24; HRMS (ESI) m/z calcd for C₁₇H₂₃N₄OS [MþH]^þ

331.1593; found, 331.1591.

4.1.50. N-(4-methylthiazol-2-yl)-3-(triﬂuoromethyl)benzamide

(51i)

Yield ¼ 80%. ¹H NMR (500 MHz, DMSO‑d₆)

d 12.89 (s,1H), 8.47 (s,

72%). ¹H NMR (500 MHz, CDCl₃)

d

8.19 (s, 1H), 8.05 (d, J ¼ 8.0 Hz,

1H), 8.37 (d, J ¼ 7.8 Hz,1H), 8.00 (d, J ¼ 7.8 Hz,1H), 7.79 (t, J ¼ 7.8 Hz,

1H), 6.85 (s, 1H), 2.32 (s, 3H); HRMS (ESI) m/z calcd for

1H), 7.89 (d, J ¼ 8.1 Hz, 1H), 6.60 (s, 1H), 3.77 (s, 2H), 2.91e2.68 (m,

8H), 2.56 (s, 3H), 2.22 (s, 3H); HRMS (ESI) m/z calcd for

C

₁₂H₁₀F₃N₂OS [MþH]^þ287.0466; found, 287.0461.

C

₁₈H₂₂F₃N₄OS [MþH]^þ399.1466; found, 399.1469.

Compounds 51b-t were prepared following the synthetic pro-

cedure of 51a.

4.1.51. 1-(4-Methylthiazol-2-yl)-3-(3-(triﬂuoromethyl)phenyl)urea

(51j)

4.1.43. N-(4-methylthiazol-2-yl)acetamide (51b)

Yield ¼ 82%. ¹H NMR (500 MHz, CDCl₃)

d 10.24 (br, 1H), 7.87 (s,

Yield ¼ 82%. ¹H NMR (500 MHz, CDCl₃)

d

10.66 (s, 1H), 6.54 (s,

1H), 7.63 (d, J ¼ 8.0 Hz,1H), 7.44 (t, J ¼ 7.9 Hz,1H), 7.36 (d, J ¼ 7.7 Hz,

1H), 6.35 (s, 1H), 2.31 (s, 3H); HRMS (ESI) m/z calcd for

1H), 2.36 (s, 3H), 2.23 (s, 3H); HRMS (ESI) m/z calcd for C₆H₉N₂OS

[MþH]^þ157.0436; found, 157.0432.

C

₁₂H₁₁F₃N₃OS [MþH]^þ302.0575; found, 302.0580.

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European Journal of Medicinal Chemistry 207 (2020) 112744

4.1.52. N-(4-methylthiazol-2-yl)-2-(3-(triﬂuoromethyl)phenyl)

acetamide (51k)

4.1.60. 2-(4-(2-Hydroxyethyl)piperazin-1-yl)-N-(4-methylthiazol-

2-yl)acetamide (51s)

Yield ¼ 81%. ¹H NMR (500 MHz, DMSO‑d₆)

d

12.32 (s,1H), 7.71 (s,

Yield ¼ 80%. ¹H NMR (500 MHz, DMSO‑d₆)

d 11.80 (s, 1H), 6.77 (s,

1H), 7.67e7.55 (m, 3H), 6.75 (s, 1H), 3.88 (s, 2H), 2.26 (s, 3H); HRMS

1H), 4.99 (br, 1H), 3.65 (s, 2H), 3.12e2.61 (m, 12H), 2.26 (s, 3H);

HRMS (ESI) m/z calcd for C₁₂H₂₁N₄O₂S [MþH]^þ285.1385; found,

285.1382.

(ESI) m/z calcd for

301.0620.

C

₁₃H₁₂F₃N₂OS [MþH]^þ301.0622; found,

4.1.61. 2-(2-Methoxyethoxy)-N-(4-methylthiazol-2-yl)acetamide

4.1.53. 2-(3-Chlorophenyl)-N-(4-methylthiazol-2-yl)acetamide

(51t)

(51l)

Yield ¼ 81%. ¹H NMR (500 MHz, DMSO‑d₆)

d 11.78 (s, 1H), 6.77 (s,

Yield ¼ 83%. ¹H NMR (500 MHz, DMSO‑d₆)

d 12.29 (s, 1H), 7.41 (s,

1H), 4.19 (s, 2H), 3.65 (dd, J ¼ 5.5, 3.7 Hz, 2H), 3.50 (dd, J ¼ 5.5,

3.7 Hz, 2H), 3.28 (s, 3H), 2.26 (s, 3H); HRMS (ESI) m/z calcd for

C₉H₁₅N₂O₃S [MþH]^þ231.0803; found, 231.0806.

1H), 7.39e7.24 (m, 3H), 6.75 (s, 1H), 3.77 (s, 2H), 2.26 (s, 3H). ¹³C

NMR (126 MHz, DMSO‑d₆)

d 168.94, 157.61, 147.16, 137.84, 133.37,

130.67, 129.64, 128.49, 127.27, 108.24, 41.62, 17.31; HRMS (ESI) m/z

calcd for C₁₂H₁₂ClN₂OS [MþH]^þ267.0359; found, 267.0362.

4.2. Biology

4.1.54. 2-(3,4-Dimethoxyphenyl)-N-(4-methylthiazol-2-yl)

4.2.1. Cell lines, cell culture, antibodies, and chemicals

acetamide (51m)

Cellular assays using human colon cancer cells KM-12-LUC were

performed at PreceDo Pharmaceuticals Co., Ltd. (Hefei, China). TEL-

and LMNA-isogenic BaF3 cell lines were established in our lab

following previously reported procedure²⁷. All the isogenic BaF3

cells were grown in RPMI 1640 (Gibco, USA) medium supported

with 10% FBS and 1% penicillin/streptomycin. All the cells were

grown in a humidiﬁed incubator (Thermo, USA) at 37 ^ꢁC under 5%

CO₂. Adherent cells were grown in tissue culture ﬂasks until they

were 85e95% conﬂuent prior to use. For suspension cells, cells were

collected by spinning down at 800 rpm/min for 4 min before use.

The following antibodies were purchased from Cell Signaling

Technology (Danvers, MA): Phospho-TRKA (Tyr490) Antibody (no.

9141S), TRK (pan) (A7H6R) Rabbit mAb (no. 92991S), AKT (pan)

(C67E7) rabbit mAb (no. 4691), Phospho-AKT (Thr308) (244F9)

rabbit mAb (no. 4056), Phospho-AKT (Ser473) (D9E) XP rabbit mAb

(no. 4060), PARP (46D11) rabbit mAb (no. 9532), Phospho-p44/42

MAPK (ERK1/2) (Thr202/Tyr204) (197G2) rabbit mAb (no. 4377),

p44/42 MAPK (ERK1/2) (137F5) rabbit mAb (no. 4695). Phospho-

TRKB (Tyr515) (no, abs131087) was purchased from Absin. Anti-

phospho-TRKC (pTyr516) antibody SAB4504648-100UG was pur-

chased from Merck. Monoclonal anti-FLAG M2 (no. F3165) were

purchased from Sigma Aldrich.

Yield ¼ 84%. ¹H NMR (500 MHz, DMSO‑d₆)

d 12.19 (s, 1H), 6.94 (s,

1H), 6.89 (d, J ¼ 8.2 Hz, 1H), 6.83 (dd, J ¼ 8.2,1.7 Hz, 1H), 6.73 (s, 1H),

3.74 (s, 3H), 3.72 (s, 3H), 3.63 (s, 2H), 2.25 (s, 3H). ¹³C NMR

(126 MHz, DMSO‑d₆) d 169.70, 157.77, 149.08, 148.30, 147.09, 127.82,

121.70, 113.68, 112.42, 108.08, 56.03, 55.95, 41.83, 17.31; HRMS (ESI)

m/z calcd for C₁₄H₁₇N₂O₃S [MþH]^þ293.0960; found, 293.0960.

4.1.55. N-(4-methylthiazol-2-yl)-2-(naphthalen-1-yl)acetamide

(51n)

Yield ¼ 85%. ¹H NMR (500 MHz, CDCl₃)

d 8.48 (s, 1H), 7.89e7.78

(m, 3H), 7.47 (pd, J ¼ 6.9, 3.5 Hz, 2H), 7.43e7.38 (m, 2H), 6.40 (s, 1H),

4.18 (s, 2H), 2.15 (s, 3H); HRMS (ESI) m/z calcd for C₁₆H₁₅N₂OS

[MþH]^þ283.0905; found, 283.0909.

4.1.56. 2-(1-Methylpiperidin-4-yl)-N-(4-methylthiazol-2-yl)

acetamide (51o)

Yield ¼ 80%. ¹H NMR (500 MHz, CDCl₃)

d 8.48 (s,1H), 6.52 (s,1H),

2.86 (d, J ¼ 11.7 Hz, 2H), 2.33 (s, 3H), 2.27 (d, J ¼ 11.7 Hz, 3H),

2.01e1.92 (m, 2H), 1.92e1.85 (m, 1H), 1.80e1.73 (m, 2H), 1.45e1.21

(m, 4H); HRMS (ESI) m/z calcd for C₁₂H₂₀N₃OS [MþH]^þ254.1327;

found, 254.1324.

Compounds 1 and 4 were purchased from MedChemExpress

(Shanghai, China).

4.1.57. 2-(4-Methylpiperazin-1-yl)-N-(4-methylthiazol-2-yl)

acetamide (51p)

4.2.2. General procedure for antiproliferation assays

For the non-adherent BaF3 cells, a density of 1.5 ꢂ 10⁴cells/mL

Yield ¼ 87%. ¹H NMR (500 MHz, CDCl₃)

d 10.12 (s, 1H), 6.48 (s,

cells were mixed with various concentrations of compounds, then

100 mL suspension was added to each well and then incubated at

1H), 3.19 (s, 2H), 2.59 (d, J ¼ 42.5 Hz, 8H), 2.33 (s, 3H), 2.30 (d,

J ¼ 0.8 Hz,3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d

168.69, 157.21,

37 ^ꢁC with 5% CO₂for 72 h. For the KM-12-LUC cells, a density of

3 ꢂ 10⁴cells/mL were added to 96-well plate, incubated at 37 ^ꢁC

with 5% CO₂overnight. The next day the supernatant was changed

with fresh medium containing different concentrations of com-

pounds and then incubated for 72 h. Cell viability was determined

using CCK-8 assay (MedChemExpress, China) according to the in-

struction manual. The absorbance was measured in a multimode

plate reader (EnVision, PerkinElmer, USA) at 450 nm. Data were

normalized to control group (DMSO). GI₅₀values were calculated

using Prism 7.0 (GraphPad Software, San Diego, CA).

147.10, 108.22, 60.62, 55.07, 52.96, 46.19, 17.30; HRMS (ESI) m/z

calcd for C₁₁H₁₉N₄OS [MþH]^þ255.1280; found, 255.1279.

4.1.58. tert-Butyl 4-(2-((4-methylthiazol-2-yl)amino)-2-oxoethyl)

piperidine-1-carboxylate (51q)

Yield ¼ 82%. ¹H NMR (500 MHz, CDCl₃)

d 9.87 (s,1H), 6.46 (s,1H),

4.32e3.75 (m, 2H), 2.73e2.55 (m, 2H), 2.28 (s, 2H), 2.27 (s, 3H),

2.04e1.92 (m, 1H), 1.68e1.60 (m, 2H), 1.38 (s, 9H), 1.15e1.03 (m,

2H); HRMS (ESI) m/z calcd for C₁₆H₂₆N₃O₃S [MþH]^þ340.1695;

found, 340.1698.

4.2.3. Signaling pathway study

4.1.59. tert-Butyl 3-(2-((4-methylthiazol-2-yl)amino)-2-oxoethyl)

piperidine-1-carboxylate (51r)

BaF3-TEL-TRKA, BaF3-TEL-TRKB, BaF3-TEL-TRKC, BaF3-LMNA-

TRKA-V573M, BaF3-LMNA-TRKA-F589L, BaF3-LMNA-TRKA-G595R,

BaF3-LMNA-TRKA-G667C/S, and KM-12-LUC cells were cultured in

RPMI 1640 medium (Gibco, USA) supported with 10% FBS and 1%

Yield ¼ 84%. ¹H NMR (500 MHz, CDCl₃)

d 10.29 (br, 1H), 6.44 (s,

1H), 3.92e3.51 (d, J ¼ 69.2, 2H), 3.22e2.63 (m, 2H), 2.46e2.31 (m,

1H), 2.27 (s, 3H), 2.23e2.14 (m, 1H), 2.11e2.01 (m, 1H), 1.88e1.73

(m, 1H), 1.59e1.48 (m, 1H), 1.39 (s, 9H), 1.22e1.16 (m, 1H); HRMS

(ESI) m/z calcd for C₁₆H₂₆N₃O₃S [MþH]^þ340.1695; found, 340.1697.

penicillin/streptomycin. Serially diluted compound 34, 0.5

compound 1 and 0.1 M compound 4 were added to cells for 2 h.

The cells were collected and lysed. The cell lysates were analyzed by

mM

m

19

B. Wang, W. Zhang, X. Liu et al.

European Journal of Medicinal Chemistry 207 (2020) 112744

western blotting. Western blotting was performed following stan-

dard method as previously described [27].

Declaration of competing interest

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

4.2.4. Apoptosis effect examination

KM-12-LUC cells were cultured in RPMI 1640 medium (Gibco,

USA) supported with 10% FBS and 1% penicillin/streptomycin.

Serially diluted compound 34, 0.5

m

M compound 1 and 0.1

mM

Acknowledgments

compound 4 were added to cells for 24 h. Apoptosis of KM-12-LUC

cells was then detected by western blotting using PARP and

antibodies (Cell Signaling Technology).

b

-actin

This work was supported by the National Natural Science

Foundation of China (Grant Nos. 81773777, 81872748, 81903659,

81903650), the “Personalized Medicines-Molecular Signature-

Based Drug Discovery and Development”, Strategic Priority

Research Program of the Chinese Academy of Sciences (Grant No.

XDA12020224), the National Science & Technology Major Project

“Key New Drug Creation and Manufacturing Program” of China

(Grant No. 2018ZX09711002), the Natural Science Foundation of

Anhui Province (Grant Nos. 1808085MH268, 1908085QH348,

1908085MH259), the China Postdoctoral Science Foundation

(Grants Nos. 2019M652057, 2020M671916), the Postdoctoral Sci-

ence Foundation of Anhui Province (Grant Nos. 2019B326,

2019B300), the Frontier Science Key Research Program of CAS

(Grant No. QYZDB-SSW-SLH037), the CASHIPS Director’s Fund

(Grant No. BJPY2019A03), and the University Synergy Innovation

Program of Anhui Province(Grant No. GXXT-2019-045). We are also

grateful for the support of Hefei leading talent for F.Z.

4.2.5. Cell cycle analysis

KM-12-LUC cells were treated with DMSO, serially diluted

compound 34, 0.5 M compound 1 and 0.1 M compound 4 for

m

indicated periods. The cells were ﬁxed in 70% cold ethanol and

incubated at ꢀ20 ^ꢁC overnight and then stained with PI/RNase

staining buffer (BD Pharmingen). Flow cytometry was performed

using a FACS Calibur (BD), and the results were analyzed by ModFit

software.

4.3. Xenograft and allograft tumor models

The mouse models were constructed using the modiﬁed

method according to previous work [28]. Four-week old female nu/

nu mice were purchased from Beijing Vital River Laboratory Animal

Technology Co., Ltd (Beijing, China). All animals were housed in a

speciﬁc pathogen-free facility and used according to the animal

care regulations of Hefei Institutes of Physical Science, Chinese

Academy of Sciences (Hefei, China). Prior to implantation, cells

were harvested during exponential growth. One million BaF3-TEL-

TRKA, BaF3-TEL-TRKB, BaF3-TEL-TRKC, BaF3-LMNA-TRKA-F589L,

BaF3-LMNA-TRKA-G667S, and ﬁve million KM-12-LUC cells in

DMEM medium were formulated as a 1:1 mixture with matrigel

(BD Biosciences) and injected into the subcutaneous space on the

right ﬂank of nu/nu mice. Daily oral administration was initiated

when tumors had reached a size of 150e200 mm³for the xenograft

model or 200e400 mm³for the allograft models. Animals were

then randomized into treatment groups of 5 mice each for efﬁcacy

study. 34 was delivered daily in an HKI solution (0.5% Methylcel-

lulose/0.4% Tween 80 in ddH₂O) by oral gavage. A range of doses of

34 or its vehicle were administered, as indicated in Figs. 9 and 10.

Body weight and tumor growth were measured daily after 34

treatment. Tumor volumes were calculated as follows: tumor vol-

ume (mm³) ¼ [(W²ꢂ L)/2] in which width (W) is deﬁned as the

smaller of the two measurements and length (L) is deﬁned as the

larger of the two measurements.

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2020.1 12744.

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Article Doi

Discovery of (E)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-yl)thiazol-2-yl)-2-(4-methylpiperazin-1-yl)acetamide (IHMT-TRK-284) as a novel orally available type II TRK kinase inhibitor capable of overcoming multiple resistant mutants

DOI: 10.1016/j.ejmech.2020.112744

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Full text of DOI:10.1016/j.ejmech.2020.112744

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