Discovery of Conformational Control Inhibitors Switching off the Activated c-KIT and Targeting a Broad Range of Clinically Relevant c-KIT Mutants

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Cite This: J. Med. Chem. XXXX, XXX, XXX −XXX

Discovery of Conformational Control Inhibitors Switching oﬀ the

Activated c‑KIT and Targeting a Broad Range of Clinically Relevant

c‑KIT Mutants

Tsung-Sheng Wu,^†Wen-Hsing Lin,^†Hui-Jen Tsai,^‡,§Ching-Cheng Hsueh,^†Tsu Hsu,^†Pei-Chen Wang,^†

Hui-You Lin,^‡Yi-Hui Peng,^†Cheng-Tai Lu,^†Lung-Chun Lee,^†Chih-Hsiang Tu,^†Fang-Chun Kung,^†

Hui-Yi Shiao,^†Teng-Kuang Yeh,^†Jen-Shin Song,^†Jia-Yu Chang,^†Yu-Chieh Su,^†Li-Tzong Chen,^‡,∥

Chiung-Tong Chen,^†Weir-Torn Jiaang,*^,†and Su-Ying Wu*^,†

^†Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, 35, Keyan Road, Zhunan Town,

Miaoli County 350, Taiwan R.O.C.

^‡National Institute of Cancer Research, National Health Research Institutes, Tainan City 704, Taiwan R.O.C.

^§Division of Hematology/Oncology, Department of Internal Medicine, National Cheng Kung University Hospital, Tainan City 704,

Taiwan R.O.C

^∥Institute of Molecular Medicine, National Cheng Kung University, Tainan City 704, Taiwan R.O.C.

S

* Supporting Information

ABSTRACT: Drug resistance due to acquired mutations that constitutively activate c-KIT is a signiﬁcant challenge in the

treatment of patients with gastrointestinal stromal tumors (GISTs). Herein, we identiﬁed 1-(5-ethyl-isoxazol-3-yl)-3-(4-{2-[6-

(4-ethylpiperazin-1-yl)pyrimidin-4-ylamino]-thiazol-5-yl}phenyl)urea (10a) as a potent inhibitor against unactivated and

activated c-KIT. The binding of 10a induced rearrangements of the DFG motif, αC-helix, juxtamembrane domain, and the

activation loop to switch the activated c-KIT back to its structurally inactive state. To the best of our knowledge, it is the ﬁrst

structural evidence demonstrating how a compound can inhibit the activated c-KIT by switching back to its inactive state

through a sequence of conformational changes. Moreover, 10a can eﬀectively inhibit various c-KIT mutants and the

proliferation of several GIST cell lines. The distinct binding features and superior inhibitory potency of 10a, together with its

excellent eﬃcacy in the xenograft model, establish 10a as worthy of further clinical evaluation in the advanced GISTs.

each of the c-KIT monomers in close proximity and

rearranging them in a proper orientation critical for the

following intracellular activation events.⁵While how the

extracellular conformational changes transmit and initiate

intracellular activation of c-KIT remains unresolved, it is

clear that upon SCF stimulation, the JM domain is relieved

from an autoinhibited position that impedes the activation

loop (A-loop) for adopting the activated conformation.⁶The

A-loop in turn switches from an inactive position that sterically

hinders the ATP-binding cleft to a proper activated position to

allow a γ-phosphate of ATP to be transferred to tyrosine

residues of protein substrates.^4,7While these two major

conformational changes initiate the catalytic activity of the c-

INTRODUCTION

■

Receptor tyrosine kinases (RTKs) are cell membrane receptors

conducting signaling of growth factors, cytokines, and

hormones to orchestrate normal cell development and

behavior in a highly regulated manner.¹Perturbation of

RTKs-mediated signaling is characteristic in many disorders,

including a signiﬁcant proportion of human cancers.²On the

basis of sequence similarity and structural homology, the

proto-oncogenic protein c-KIT is categorized as a type III RTK

comprised of ﬁve IgG-like extracellular domains, a trans-

membrane helix followed by an autoinhibitory juxtamembrane

(JM) domain, and a cytoplasmic kinase domain.³

The catalytic activity of c-KIT remains autoinhibited⁴until

its cognate ligand, stem cell factor (SCF), binds to and initiates

a series of activation processes. Binding of SCF initiates

homodimerization of c-KIT in a divalent manner, bringing

Received: November 26, 2018

A

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Table 1. Inhibition of c-KIT Enzymatic Activity and GIST-T1 Cell Proliferation by 10a and the Analogues

B

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Table 1. continued

a

b

Values of IC₅₀and GI₅₀are expressed as the mean of three independent experiments and are within 15%. GIST cell line: heterozygous deletion

of V560-Y578.

C

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a

Scheme 1

a

Reagents and conditions: (a) NaH, THF, 0 °C; (b) 2° amines, pyridine, 80 °C; (c) pyridine, (5-ethylisoxazol-3-yl)carbamic acid 4-nitro-phenyl

ester for 10a,b, 10g−k, and 11, phenyl isocyanate for 10c, benzenesulfonyl chloride for 10e, acetyl chloride for 10f, rt. KOCN, AcOH, H₂O for

10d.

KIT, a series of autophosphorylation of tyrosine residues at JM

domain and A-loop are considered as the necessity for full

activation and as a stabilizer for the active conformation of c-

KIT.⁸

of Val654 in the ATP binding pocket to alanine may result in

loss of c-KIT-drug interaction,²¹whereas substitutions of

gatekeeper Thr670 to residues with a bulkier side chain, such

as isoleucine, may induce steric clashes,²²and its hydro-

phobicity may stabilize c-KIT in an activated conformation.²³

In contrast, mutations in the A-loop, such as D816H, shift the

c-KIT activation equilibrium from inactive to the active state,

rendering it insensitive to many inhibitors.^24,25Although the

second line sunitinib²⁶and the third line regoraﬁnib²⁷have

been approved and implemented for treatment of the

refractory GISTs, sunitinib was shown to be ineﬀective against

c-KIT A-loop mutants,²⁴and regoraﬁnib treatment was found

to be only modestly beneﬁcial.²⁷Accordingly, there is an

urgent need for novel c-KIT inhibitors that eﬀectively target a

broad spectrum of c-KIT mutants and improve the survival of

patients with advanced GISTs.²⁸

The concept of designing conformational control inhibitors

to target the activated form of kinases and enable the inhibition

of a wide range of kinase mutants has been proposed. Indeed, a

class of inhibitors targeting the “switch control pocket”²⁹to

force the activated p38-MAP kinase into a conformation that

resembled its type II inactive state was discovered.^30,31In the

case of BCR-ABL tyrosine kinase, a switch control inhibitor

showed potency against several clinically relevant chronic

myeloid leukemia (CML) related mutants.³²In the case of c-

KIT, although several inhibitors with diﬀerent chemical

scaﬀolds have been discovered,²⁸a mechanistic understanding

of how the activated c-KIT and various c-KIT mutants can be

inhibited is still unclear. Therefore, discovery of novel

Aberrant activation of c-KIT activity due to naturally

occurring or drug-induced mutations plays an important role

in etiology and drug resistance in gastrointestinal stromal

tumors (GISTs).⁹The most common primary mutations in

GISTs occur in the exon 11 of c-KIT.¹⁰Given that exon 11

encodes the JM domain of c-KIT, these mutations may alter

the autoinhibitory properties of the JM domain and/or

compromise the binding of negative regulators that dephos-

phorylate and inactivate the c-KIT kinase activity,¹¹either of

which contributes to the transformation of c-KIT to its active

form.¹⁰Mutations in cKIT exon 9, which encodes the IgG-like

extracellular domain D5, account for the second most common

primary c-KIT mutations in GISTs.^12,13These mutations may

enhance the interaction between D5 domains and promote c-

KIT dimerization and activation.⁵

Imatinib was approved and implemented as the ﬁrst line

treatment¹⁴for patients with resectable primary GISTs¹⁵as

well as those with unresectable or advanced metastatic

GISTs¹⁶caused by mutations at exon 11 of c-KIT.¹⁷This

ﬁrst line treatment, however, induces mutations in c-KIT gene

that decrease c-KIT sensitivity to imatinib.^18,19In contrast to

the primary c-KIT mutations, these acquired secondary

mutations are mostly found in exon 13/14 and exon 17 of c-

KIT, which encode the kinase catalytic pocket and A-loop,

respectively, and are responsible for the failure in the treatment

of GISTs by diﬀerent mechanisms.²⁰For example, substitution

D

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a

Scheme 2

a

Reagents and conditions: (a) NaH, THF, rt; (b) 1-ethylpiperazine, pyridine, 65 °C; (c) NIS, CHCl₃, rt; (d) Pd(dppf)Cl₂, Na₂CO₃, 1,4-dioxane,

80 °C; (e) TFA, CH₂Cl₂, rt; (f) pyridine, (5-ethylisoxazol-3-yl)carbamic acid 4-nitro-phenyl ester, rt; (g) Pd(PPh₃)₄, CuI, NEt₃, THF, rt; (h) H₂N-

NH₂, THF, rt; (i) H₂, Pd/BaSO₄, MeOH, rt.

conformational control inhibitors against c-KIT with support

of structural evidence is needed.

tion of the activated c-KIT and revealed the therapeutic

potential of 10a in treatment of advanced GISTs.

In this study, we synthesized a series of 5-phenyl-thiazol-2-

ylamine based compounds bearing a urea substituent, and

identiﬁed 10a as a potent c-KIT inhibitor on the basis of its

enzymatic and cellular activities. The crystal structure of c-KIT

in complex with 10a revealed 10a as a type II inhibitor, in

which 10a binds to c-KIT in a “DFG-out” inactive

conformation. More importantly, binding of 10a shuts oﬀ

the activities of the activated c-KIT by inducing dramatic

conformational changes that switch the activated c-KIT back to

its inactive state. Moreover, 10a was found to eﬀectively inhibit

activities of a broad spectrum of c-KIT mutants, including

those bearing mutations in the A-loop, as well as the

proliferation of several GIST cell lines and the growth of

tumors in a xenograft mouse model. These results provided

novel structural insights into the compound-induced inactiva-

RESULTS

■

Identiﬁcation of 10a and Its Analogues as c-KIT

Inhibitors. A class of 5-phenyl-thiazol-2-ylamine pyrimidine

derivatives³³has been utilized as a versatile template for the

development of kinase inhibitors. On the basis of this scaﬀold,

urea-substituted series of compounds were synthesized and

they were identiﬁed as c-KIT inhibitors by our in-house kinase

screening programs. Among these derivatives, 10a exhibited

high potency against c-KIT enzymatic (IC₅₀= 82 nM) and

cellular (GI₅₀= 2.2 nM) activities (Table 1).

The general synthetic route for 10a and its analogues is

shown in Scheme 1 and 2. Compounds 10a−k and 11 were

synthesized based on our previous publication with mod-

iﬁcation.³³The synthesis began with the preparation of 4- or 5-

E

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aromatic ring substituted thiazol-2-ylamines 1−2,³⁴3,³⁵or 4³⁶

according to previously published protocols. 4,6-Disubstituted

pyrimidine 8 was coupled with (5-ethylisoxazol-3-yl)carbamic

acid 4-nitrophenyl ester in pyridine to aﬀord ureas 10a−c and

10g−k. The treatment of 4,6-disubstituted pyrimidine 8 with

potassium cyanate in acetic acid/water, benzenesulfonyl

chloride in pyridine, and acetyl chloride in pyridine aﬀorded

the ﬁnal urea 10d, sulfonamide 10e,³³and amide 10f,

respectively. The synthetic route to 5-substituted thiazol-2-

ylamine pyrimidines 18, 22, and 24 (Table 1) is shown in

Scheme 2. Treatment of thiazol-2-ylamine 12 with 4,6-

dichloropyrimidines 5 in the presence of NaH and THF at

room temperature aﬀorded 4-monosubstituted pyrimidine

derivatives 13. The treatment of derivatives 13 with 1-

ethylpiperazine in pyridine at 80 °C yielded 4,6-disubstituted

pyrimidines 14. Electrophilic iodination of thiazole using N-

iodosuccinimide (NIS) in CHCl₃aﬀorded 5-iodothiazol-2-

ylamine 15, which was coupled with N-Boc protected 1,2,3,6-

tetrahydropyridyl borane 16 or phthalimide-protected prop-2-

ynylamine 19 to yield N-Boc protected 5-(1,2,3,6-tetrahy-

dropyridin-4-yl)thiazol-2-ylamine pyrimidine 17 or phthali-

mide-protected 5-(3-aminoprop-1-ynyl)thiazol-2-ylamine pyr-

imidine 20, respectively. Deprotection of Boc-17 or

phthalimide-protected 20 using TFA or hydrazine followed

by coupling with (5-ethylisoxazol-3-yl)carbamic acid 4-nitro-

phenyl ester aﬀorded the ﬁnal pyrimidine urea 18 or 22,

respectively. Unsaturated prop-2-ynylamine 21 reacted with

hydrogen in the presence of Pd catalyst in MeOH to provide

butylamine 23, which was coupled with (5-ethylisoxazol-3-

yl)carbamic acid 4-nitro-phenyl ester to give ﬁnal pyrimidine

urea 24.

Compound 10a and the analogues were evaluated in kinase

activity and cell proliferation assays to facilitate the elucidation

of structure−activity relationships. As shown in Table 1, 2-

methylpyrimidine 10b³³exhibited similar inhibitory activities

against wild type c-KIT kinase and GIST-T1 cells as 10a.

Phenyl-substituted urea 10c³³showed potent inhibition

against c-KIT. Removing the 5-ethylisoxazole moiety of 10a

(10d), replacing the urea moiety of 10a with sulfonamide

(10e)³³or amide moiety (10f) did not signiﬁcantly aﬀect the

inhibition of c-KIT kinase activity but did result in reduced

cellular potency. With maintenance of the urea moiety,

replacement of 10a’s phenyl ring by a pyridin-2-yl ring

(10g), a 1,2,3,6-tetrahydropyridine ring (18), or linear alkyl

groups, such as propynyl (22) and propyl (24), did not

signiﬁcantly aﬀect the inhibitory potency against c-KIT kinase

and GIST-T1 cells.

pyrrolidin-3-ylamine group increased the enzymatic inhibition

against c-KIT but slightly decreased the cellular activity as

compared to 10a.

Among the analogues listed in Table 1, only 10g showed

show slightly better activities than 10a both in enzymatic and

cellular assays. However, 10g has low synthetic yield as

compared to 10a. Moreover, 10c and 10k have poor PK

proﬁles³³although they exhibited potent inhibition against c-

KIT. Therefore, compound 10a was selected for further

biological activity and in vivo eﬃcacy studies.

Crystal Structure of c-KIT Kinase in Complex with

10a. The excellent enzymatic and cellular potency of 10a

against c-KIT and GIST-T1 cells prompted us to further

investigate the molecular mechanisms underlying this activity.

After expression and puriﬁcation, the recombinant c-KIT

proteins were found to be unphosphorylated and in their

unactivated form (Supporting Information, Figure S1, lane 1).

The crystal structure of the unactivated c-KIT kinase domain

in complex with 10a (hereafter referred to as unactivated c-

KIT/10a) was determined in a resolution of 2.10 Å. Data

collection and reﬁnement statistics are summarized in Table 2.

Table 2. Statistics of X-ray Diﬀraction Data and Structure

Reﬁnement for c-KIT Complex Structure

unactivated c-KIT/10a

activated c-KIT/10a

(6ITT)

(6ITV)

resolution (Å)

space group

unit cell

28.43−2.10

P2₁2₁2₁

26.77−1.88

P4₃2₁2

a, b, c (Å)

62.63, 63.09, 196.81

90, 90, 90

45999 (4548)

21.77 (2.48)

5.7 (59.6)

99.5 (100.0)

36.66

59.86, 59.86, 197.98

90, 90, 90

30279 (2935)

25.14 (2.45)

5.5 (65.4)

99.7 (100.0)

30.42

α, β, γ (deg)

unique reﬂections

I/σ

R_merge(%)

completeness (%)

Wilson B-factor

R

_work/R_free

0.1862/0.2346

0.008

0.1798/0.2306

0.006

rmsd (bond) (Å)

rmsd (angle) (deg)

0.976

0.828

Ramachandran favored

(%)

97.63

98.29

MolProbility clash

score

MolProbility overall

score

6.05

1.41

2.08

0.98

The overall structure shows that 10a binds to the unactivated

c-KIT at the ATP-binding pocket located between the N- and

C-lobe (Figure 1B). In addition, 10a stabilizes c-KIT in an

inactive conformation, wherein the A-loop is retained in a

DFG-out inactive conformation (Figure 1B, magenta),

resembling the autoinhibited conformation of the native

form of unactivated c-KIT⁴(Figure 1A, magenta, hereafter

referred to as unactivated c-KIT; PDB 1T45). Instead of an

ordered and compact hairpin loop conformation observed in

the unactivated c-KIT (Figure 1A, blue), the N-terminal

segment (Tyr547-Glu562) of the JM domain of unactivated c-

KIT/10a is exposed to the solvent region and is too ﬂexible to

be detected in the crystal structure (Figure 1B, blue).

Next, we examined eﬀects of substitutions on the thiazole

ring and compared the potencies of compounds 10h and 11

with 10a. Compound 10h, with a methyl substituent at the

fourth position of the thiazole ring, exhibited a slightly reduced

and comparable potency against c-KIT kinase and GIST-T1

cells, respectively. By contrast, a signiﬁcant decrease in both

enzymatic and cellular potency was observed when the

phenylurea tail of 10a was moved from the ﬁfth position to

the fourth position (11) of the thiazole ring. The importance

of the pyrimidine moiety of 10a was revealed by the

observation that changing of pyrimidine moiety of 10a to 2-

methyl-1,3,5-triazine (10i) led to 6-fold decrease of potency

against GIST-T1 cells. Finally, the eﬀects of water-solubilizing

groups were investigated. N-(2-Hydroxyethyl)piperazine 10j

exhibited a potency that was similar to 10a in the enzymatic

and cellular assays. Compound 10k³³bearing a primary

The pharmacophore of 10a can be divided into a solvent-

exposed headgroup (ethylpiperazine), a hinge scaﬀold

(thiazolylamine), a middle linker (phenyl), and a hydrogen-

bonding moiety (urea) along with a tail group (5-ethyl-

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Our previous SAR analysis revealed that the inhibitory

potency signiﬁcantly decreased when the phenylurea group of

10a was switched from the ﬁfth to the fourth position on the

thiazole ring (11) (Table 1). According to our crystal

structure, the distance between the fourth carbon of the

thiazole ring of 10a with the surrounding residues ranges from

3.3 to 4.7 Å (Supporting Information, Figure S2A). To

maintain the critical H-bond between the nitrogen on the

thiazole ring and Cys673 in the hinge region, the thiazole ring

must remain in the original position with its nitrogen facing to

the backbone of Cys673. Accordingly, if this conserved H-

bond has to be maintained between the nitrogen atom of

thiazole ring in 11 and Cys673, the linker and tail groups on

the fourth position of the thiazole ring of 11 would cause

serious clashes with the surrounding residues (Supporting

Information, Figure S2B). To avoid this, the thiazole ring of 11

would need to ﬂip into a new orientation that allows the

following linker and tail groups to properly ﬁt into the binding

site, but this results in a loss of the critical H-bond with

Cys673 (Supporting Information, Figure S2C). Either of the

above scenarios are expected to diminish binding aﬃnity of 11

to c-KIT, which rationalizes the 10 times lower inhibitory

potency of 11 to c-KIT compared to 10a (Table 1).

On the basis of the observation of short distances between

the fourth carbon of the thiazole ring of 10a with the

surrounding residues (Supporting Information, Figure S2A),

addition of a methyl group on the fourth position of the

thiazole ring (10h) is also expected to cause steric eﬀects with

the surrounding residues, such as Glu671. To avoid the steric

eﬀects, 10h would adopt a diﬀerent conformation, as

supported by our molecular docking analysis (Figure S3).

Although 10h was docked into c-KIT in a highly similar

orientation as 10a, the thiazole ring moved away from the

hinge region to accommodate the additional methyl group of

10h (Supporting Information, Figure S3A, red arrow),

resulting in a weaker H-bond between thiazolylamine N3

and Cys673/N. Moreover, the additional methyl group of 10h

induced a rotation of the phenyl linker, consequently directing

the tail group away from the αC-helix and leading to loss of

one of the H-bonds with Glu640 (compare Supporting

Information, Figure S3C with S3B). The steric hindrance

induced by the additional methyl group of 10h, together with

its possible adjustments indicated by molecular docking

analysis, are plausible explanations for the reduced inhibitory

potency of 10h to c-KIT when compared to 10a (Table 1).

Substitution of the phenyl linker of 10a with six-membered

carbon ring (10g and 18) or three-carbon alkyl group (22 and

24) did not reduce the inhibitory potency (Table 1), indicating

that replacement of the linker with a moiety of optimum length

size to properly position the hinge scaﬀold and the urea group

for hydrogen bond formation to be practical. Indeed,

cocrystallized structures revealed that many type II inhibitors

bear linker moieties comprising an aliphatic chain or aromatic

ring with 3−5 chemical bonds in length.³⁸

Figure 1. Overall structure of the (A) apo form (PDB 1T45) and (B)

10a-bound unactivated c-KIT (PDB 6ITT). The JM domain and A-

loop are shown in blue and magenta, respectively. (C) Pharmaco-

phore of 10a: solvent-exposed headgroup (yellow), hinge scaﬀold

(light blue), linker (light salmon), and tail group (light green). (D)

The structure of the unactivated c-KIT kinase domain in complex

with 10a. Compound 10a is shown in orange, with its electron density

map (1.0 sigma). The H-bonds between c-KIT and 10a are shown in

red dash lines. Residue Asp810 and Phe811 of the apo form of

unactivated c-KIT are shown in gray.

isoxazole) (Figure 1C). The binding features of 10a to the

unactivated c-KIT are shown in Figure 1D. The thiazolylamine

scaﬀold of 10a forms two H-bonds with the backbone of

Cys673 in the hinge region of c-KIT. In addition, the tail group

of 10a occupies the back pocket of the ATP-binding site by

forming three more H-bonds between the nitrogen and oxygen

of the urea group and the side chain of Glu640 and the

backbone of Asp810, respectively. Upon binding of 10a, the

rearranged Phe811 forms a perpendicular “edge-to-face”

aromatic interaction with the phenyl ring of 10a (Figure

1D). The ethylpiperazine group of 10a is exposed in the

solvent region in the crystal structure, which is usually utilized

to improve the physiochemical properties of compounds.³⁷

The above features, especially the interaction network of 10a

and Glu640 at the αC-helix and Asp810 of the DFG motif,

have characterized 10a as a type II kinase inhibitor.³⁸

The crystal structure and the SAR study consistently

revealed the importance of the tail group in the activity of

10a (Figure 1D and Table 1). Removal of the ethylisoxazole

group of 10a to give 10d had little eﬀect on the inhibitory

activity against c-KIT, indicating the intact urea group but not

the ethylisoxazole group to be important for maintaining the

H-bond network and contributing to the potency. In contrast,

substitution of the urea group of 10d with an amide group to

give 10f reduced the inhibitory potency against c-KIT activity,

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Figure 2. (A) Comparison of the structure of c-KIT/10a and c-KIT/imatinib. (B) Comparison of the structure of c-KIT/10a and c-KIT/sunitinib.

(C) Comparison of the structure of c-KIT/10a and c-KIT/ponatinib. (D) Chemical structures of 10a, imatinib, sunitinib. and ponatinib. Hydrogen

bonds between c-KIT and 10a are shown with red dashed lines. Hydrogen bonds between c-KIT and imatinib (or sunitinib or ponatinib) are

shown with green dashed lines.

Figure 3. Determination of IC₅₀of inhibitors to the unactivated and activated c-KIT. IC₅₀of inhibitors to c-KIT were determined as the

concentrations (nM) of inhibitors required to inhibit 50% of c-KIT kinase activity. Values are expressed as means SEM of three independent

assays.

suggesting that the intact urea group may provide optimal

interactions of 10a to c-KIT.

hydrogen bond with Glu640, while the interaction network of

10a with Glu640 and Asp810 was more extensive with three

hydrogen bonds. Moreover, unlike 10a, instead of exposing the

headgroup to the solvent region, imatinib extended deeply into

the binding pocket and formed interactions with Ile789 and

His790. The structure of c-KIT/10a was also compared with

that of c-KIT/sunitinib (PDB 3G0E). The pyrrole and indole

groups of sunitinib overlapped with the pyrimidine and

thiazolylamine groups of 10a, respectively (Figure 2B). The

(2-diethylaminoethyl)amine group of sunitinib was exposed to

Comparison with the Structures of Other Inhibitors

Bound to c-KIT. First, the structure of c-KIT/10a was

compared with the structures of c-KIT/imatinib (PDB 1T46).

Superimposing of these two structures revealed that the

pyridine and pyrimidin-2-ylamine groups of imatinib occupied

a similar position as that of the thiazolylamine and phenyl

groups of 10a and interacted with the hinge region of c-KIT

(Figure 2A). The amide group of imatinib formed one

H

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Figure 4. Overall structure of the (A) native form (PDB 1PKG) and (B) 10a-bound (PDB 6ITV) activated c-KIT. The JM domain and A-loop are

shown in blue and magenta, respectively. (C) The structure of the activated c-KIT in complex with 10a. Compound 10a is shown in orange with its

electron density map (1.0σ). The H-bonds between c-KIT and 10a are shown in red dash lines. (D) Structural comparison of the N-terminal JM

domain of the unactivated c-KIT/10a (light teal) and activated c-KIT/10a (pink). (E) Structural diﬀerences with electron density maps (0.9σ) of

the Tyr570 and phospho-Tyr570 in the unactivated and activated c-KIT.

the solvent region and was not observable in the crystal

structure. Sunitinib interacted with the hinge region and the

DFG-motif of c-KIT and kept c-KIT in a DFG-out

conformation. However, the interactions of sunitinib with

Glu640 were absent. Moreover, the structure of c-KIT/10a

was compared with that of c-KIT/ponatinib (PDB 4U0I). The

imidazo[1,2-b]pyridazine group of ponatinib occupied a

similar position as that of the thiazolylamine groups of 10a

and interacted with the hinge region of c-KIT (Figure 2C).

The amide group of ponatinib also formed interactions with

Glu640. The piperazine group of ponatinib extended deeply

into the binding pocket while the ethylpiperazine group of 10a

exposed toward the solvent region.

the activated form of kinases often also show high potency

against many drug-induced kinase mutants.³²This prompted

us to further investigate whether compound 10a could inhibit

the activated c-KIT with a similar potency as to the unactivated

c-KIT. To obtain the activated c-KIT, the puriﬁed unactivated

c-KIT recombinant proteins were incubated with an excess

amount of ATP, and their phosphorylation was conﬁrmed by

Western blot analysis (Supporting Information, Figure S1). In

addition to phosphorylation, the activation of c-KIT was

further conﬁrmed by the observation that imatinib and

sunitinib inhibited the activated c-KIT with a signiﬁcantly

lower potency compared to that of the unactivated c-KIT

(Figure 3), which is consistent with the previous reports.²⁴In

contrast to imatinib and sunitinib, compound 10a was found to

inhibit the enzymatic activities of both unactivated and

Compound 10a Potently Inhibits the Activated c-KIT.

Studies have revealed that inhibitors with high activities against

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Figure 5. Binding of 10a to the activated c-KIT induced a sequence of conformational changes in the DFG motif, αC-helix and JM domain. (A)

Interactions of A-loop with αC-helix stabilized A-loop in a DFG-in conformation in the activated c-KIT structure (PDB 1PKG). (B) Interaction

network between αC-helix, 10a, and DFG motif upon binding of 10a to the activated c-KIT (PDB 6ITV). (C) In addition to the interactions with

αC-helix, Lys818, and Asn819 of the activated A-loop were stabilized by forming strong interactions with p-Tyr570 of the activated JM domain.

(D) Binding of 10a moved αC-helix (red arrow), which caused a steric eﬀect toward JM domain (red explosion drawing). The JM domain was

then rearranged and stabilized in a new position (pink) by forming H-bond (red dash line) and hydrophobic (gray dash line) interactions with

residues at the C-lobe.

activated c-KIT with similarly high potencies (Figure 3). The

ability of 10a to potently inhibit the activated c-KIT suggested

a possibility that 10a would also inhibit the constitutively

active c-KIT mutants.

PDB 1PKG). Instead, the overall structure closely resembles

that of the unactivated c-KIT/10a (Figure 1B). Moreover, the

binding mode of 10a to the activated c-KIT was almost

identical to that observed in the unactivated c-KIT/10a

structure (Figure 1D), wherein the interactions between the

thiazolylamine scaﬀold of 10a and the Cys673 of the hinge

region, as well as those of the conventional Glu640−10a−

Asp810 interaction network, were properly formed (Figure

4C). However, the most signiﬁcant diﬀerence between the

activated c-KIT/10a and unactivated c-KIT/10a structures

was found in the JM domain (Figure 4D), wherein the Ile571,

p-Tyr570, and Val569 residues were orientated diﬀerently and

the N-terminal segment (Tyr547-Asn566) was absent in the

activated c-KIT/10a crystal structure. In addition, the electron

Compound 10a Induces the Conformational Changes

That Switch the Activated c-KIT to Its Inactive State. On

the basis of the enzymatic results that 10a potently inhibits the

activated c-KIT, we next investigated the structural mecha-

nisms underlying this important observation. The activated c-

KIT proteins were cocrystallized with 10a and a high-quality

crystal structure (hereafter referred to as activated c-KIT/10a)

with a resolution of 1.88 Å was obtained (Table 2).

Surprisingly, the activated c-KIT/10a (Figure 4B) was found

to no longer adopt the activated conformation⁷(Figure 4A;

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density map of the phosphate group of the p-Tyr570 can be

clearly seen (Figure 4E, pink), which provided direct evidence

that the proper phosphorylation and activation of the c-KIT

proteins had taken place.

line). These strong H-bond interactions and extensive

hydrophobic interactions stabilized JM domain in the space

that was previously occupied by the activated A-loop (Figure 6,

To elucidate the mechanism underlying the 10a-induced on-

to-oﬀ switch of the activated c-KIT, the structure of activated

c-KIT/10a was compared with the native form of the activated

c-KIT⁷(PDB 1PKG) in detail. In the native form of the

activated c-KIT, the hydrolyzed ADP after trans-autophos-

phorylation is retained in the ATP binding pocket (Figure 4A).

The JM domain is phosphorylated and a large part of it is

exposed to the solvent region (Figure 4A, blue). With a DFG-

in conformation,⁷the activated A-loop (Figure 4A, magenta)

extends into a space that was occupied by the autoinhibited JM

domain of the unactivated c-KIT (Figure 1A, blue) and is

stabilized by αC-helix by forming the H-bond network and

hydrophobic interactions (Figure 5A). The main chains of

Phe811 and Gly812 of the DFG motif on the A-loop form H-

bonds with the side chains of Glu640 and Ser639 in αC-helix,

respectively, while the side chain of Phe811 forms hydrophobic

interactions with Val643, Lue644, and Leu647 in αC-helix

(Figure 5A). In comparison, binding of 10a ﬂipped the DFG

motif to an out conformation and also induced a conforma-

tional change of A-loop (Figure 4B), resulting in a loss of the

interactions with αC-helix (Figure 5B). Instead of stabilizing

the A-loop, the Glu640 of the αC-helix formed extensive H-

bond interactions with the urea moiety of 10a. In addition, the

Phe811 of the A-loop moved away from the αC-helix and

formed strong “edge-to-face” aromatic interactions with the

phenyl ring of 10a (Figure 5B).

Figure 6. Compound 10a switched the activated c-KIT back to the

inactive state. Upon binding of 10a (orange) to the activated c-KIT

(green) (PDB 1PKG), the αC-helix shifted (red arrow) and the JM

domain (pink) repositioned (blue arrow) (PDB 6ITV) to the space

that was previously occupied by the activated A-loop (green). The A-

loop (pink), therefore, was detached and switched back to the inactive

conformation (yellow arrow).

Signiﬁcant structural diﬀerences between the activated c-

KIT and activated c-KIT/10a were observed in the JM

domain. Residue Tyr568 and Tyr570 in the JM domain are

two important phosphorylation sites at the early stage of c-KIT

activation.⁸Upon phosphorylation of Tyr570, critical inter-

actions are formed between the JM domain and the activated

A-loop (Figure 5C). In addition to an H-bond interaction

between Asn819 in the A-loop and main chain of p-Tyr570,

the salt bridge between the ε-NH₃⁺group of Lys818 in the A-

loop and the phosphate group of p-Tyr570, as well as

hydrophobic interaction between these two lateral side chains,

provide a strong and speciﬁc force tethering these two domains

(Figure 5C). However, binding of 10a induced a movement of

αC-helix and concurrently resulted in the rearrangement of the

JM domain (Figure 5D). The movement of αC-helix shifted

the Ser639 and consequently deprived the interaction network

between Ser639 and the DFG motif (Figure 5D). More

importantly, due to the movement of the αC-helix, Met638

was observed to cause a steric clash with the Tyr578 of the JM

domain (Figure 5D). In addition, the side chain of Lys642 on

the αC-helix shifted to a new orientation, which consequently

disrupted its original H-bond interaction with Pro577 (Figure

5C) and formed a new interaction with Thr574 of the JM

domain (Figure 5D). These initiated conformational changes

of the JM domain, and the p-Tyr570 of the redirected JM

domain no longer interacted with and stabilized the A-loop.

The movement of JM domain was further accomplished by

formation of new interaction network (Figure 5D). The p-

Tyr570 and Ile571 formed new H-bond interactions with

residues Phe848, Glu849 and Asn787 at the C-lobe (Figure

5D, red dash line). In addition to H-bonds, the p-Tyr570 and

Ile571 also formed hydrophobic interactions with Phe848 and

Ile789, and with Cys788, respectively (Figure 5D, gray dash

blue arrow), which led to a full detachment of the A-loop from

its activated composition. Consequently, the destabilized and

detached A-loop can be leveraged back and stabilized by 10a in

a DFG-out inactive conformation (Figure 6, yellow arrow).

Taken together, our results constituted the ﬁrst structural

insights into the mechanism underlying actions of a small

molecule inhibiting the activated c-KIT.

Compound 10a Inhibits Enzymatic Activities of a

Broad Spectrum of c-KIT Mutants. The ability of 10a to

potently inhibit activated c-KIT suggested to us that 10a would

also inhibit c-KIT mutants that are constitutively activated.

Accordingly, various single- or double-mutant isoforms of c-

KIT were evaluated for their susceptibility to inhibition by 10a.

As shown in Table 3, compound 10a more eﬀectively inhibited

a broad spectrum of c-KIT mutants bearing single mutations in

the JM domain, ATP-binding pocket and the A-loop than

sunitinib. In addition to single c-KIT mutants, 10a also

potently inhibited c-KIT double mutants containing primary

V560G and secondary mutations at the A-loop (D816V and

N822K), although reduced potency was observed in the

V559D/V654A double mutant (Table 3). The reduced

potency observed in the V559D/V654A double mutant was

investigated by the modeling study. Unlike the extensive

hydrophobic network formed between Val654, 10a, and the

surrounding residues Cys809 and Leu799 (Supporting

Information, Figure S4A), mutation of Val654 to Ala654

loses the hydrophobic interactions with 10a as well as with the

surrounding residues Cys809 and Leu799 (Supporting

Information, Figure S4B). As for Val559, the other mutated

residue in the c-KIT double mutant (V559D/V654A), the

structural explanation of how it aﬀects the aﬃnity of 10a is

uncertain because it locates in a ﬂexible region that is

unobservable in our crystal structure.

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Table 3. Inhibition of Enzymatic Activities of c-KIT Mutants by 10a or Sunitinib (by HotSpot Kinase Proﬁling, Reaction

Biology Corporation)

% inhibition of enzymatic activities

10a sunitinib

IC₅₀(nM)

mutant forms of c-KIT

Exon 11

10a

100 nM

99

10 nM

95

100 nM

41

10 nM

8

JM domain

V560G

0.98

ATP binding pocket

Exon 13

Exon 14

V654A

K642E

T670I

75

99

83

43

82

55

73

80

93

48

60

43

4.14

1.37

1.66

A-loop

Exon 17

Exon 18

D816H

D816V

D820E

D820Y

Y823D

A829P

96

73

100

99

73

37

83

99

97

90

56

47

65

81

85

43

12

5

16

36

28

6

0.67

8.56

0.81

0.02

0.12

0.89

double mutant

Exon 11/13

Exon 11/17

V559D/V654A

V560G/D816V

V560G/N822K

43

63

99

10

22

94

82

52

51

32

9

7

102.0

9.27

0.18

In conclusion, 10a inhibits not only the activated form of c-

KIT, but also a broad range of c-KIT mutants including those

notoriously resistant to sunitinib treatment at the A-loop.^39,24

In addition to c-KIT, the ability of 10a to inhibit other

kinases was also investigated. 10a was proﬁled over a panel of

468 kinases. The TREEsport interactions maps and the

selectivity score of 10a are shown in Supporting Information,

Figure S5.

Antiproliferative Eﬀects of 10a on c-KIT Mutant GIST

Cell Lines. To investigate the inhibitory properties of 10a at a

cellular level, its antiproliferative activities against GIST cell

lines were evaluated. Three GIST cell lines, GIST882 cells

carrying a c-KIT exon 13 mutation encoding K642E, GIST430

cells harboring a primary c-KIT exon 11 in-frame deletion and

a heterozygous secondary exon 13 missense mutation encoding

V654A, and GIST48 cells containing a homozygous exon 11

missense mutation encoding V560D and a heterozygous

secondary exon 17 mutation encoding D820A were inves-

tigated. Compound 10a showed superior antiproliferative

activities against all the three GIST cell lines, GIST882,

GIST430, and GIST48, with GI₅₀values of 3, 1, and 2 nM,

respectively (Table 4). Consistent with the previous studies,⁴⁰

imatinib showed activity in GIST882 cells but was ineﬀective

in GIST430 and GIST48 cells, whereas treatment with

sunitinib had an antiproliferative eﬀect on GIST882 and

GIST430 cells but not GIST48 cells (Table 4).

(Supporting Information, Figure S6). The GI₅₀value of 10a

is >100 nM in both c-KIT independent GIST cell lines,

suggesting that 10a exhibited inhibition selectivity between the

c-KIT driven GIST cell lines and c-KIT independent GIST cell

lines.

In addition, the inhibition eﬀect of 10a on other cancer cell

lines and normal cell line was also evaluated (Supporting

Information, Figure S7). The GI₅₀of 10a in PC3 (human

prostate cancer cell line) and A549 (human nonsmall-cell lung

cancer cell line) are >20 and 14.6 μM, respectively. Moreover,

10a had no inhibition eﬀect on Detroit 551 (normal human

skin ﬁbroblast cell), with GI₅₀> 10 μM.

Furthermore, the eﬀect of 10a on the c-KIT-mediated

signaling pathway was investigated. As shown in Figure 7, 10a

inhibits c-KIT phosphorylation and the phosphorylation of its

downstream signaling molecules, such as AKT, MEK, and

MAPK, conﬁrming the on-target eﬀect of 10a.

Moreover, the inhibition eﬀect of 10a on c-KIT independent

GIST cell lines, GIST48B and GIST62, was evaluated

Table 4. Inhibitory Activity of Imatinib, Sunitiniband 10a

against GIST Cell Lines

a

GI₅₀(nM)

cell line

primary/secondary mutation

imatinib

sunitinib

10a

GIST882

GIST430

GIST48

K642E/none

V560-L576del/V654A

V560D/D820A

195

929

625

64

47

2000

3

1

2

a

Values of GI₅₀are expressed as the mean of three independent

experiments.

Figure 7. Inhibition eﬀect of compound 10a on the phosphorylation

of c-KIT and its downstream signaling molecules in GIST430.

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Pharmacokinetics, Pharmacodynamics, and in Vivo

Eﬃcacy. The pharmacokinetic properties of 10a were

evaluated using a mouse model. In ICR mice, intravenous

administration of 10a at a dosage of 2 mg/kg gave rise to high

volume of distribution (V_ss= 7.1 L/kg) and plasma clearance

rate (Cl = 20.2 mL/min/kg). Oral administration of 10a in

water solution containing 20% hydroxypropyl-β-cyclodextrin

with dosage of 10 mg/kg revealed a short half-life (t_1/2= 2.8

h), moderate C_max(562 ng/mL) and AUC (2796 ng/mL·h),

and 38% of bioavailability.

The pharmacodynamic study of 10a was also performed.

Pharmacodynamic analysis of tumor in GIST430 xenografts

harvested 8 h after 10a administration revealed that decreased

c-KIT phosphorylation (p-c-KIT) in a dose-dependent

response was observed in mice treated with 10a compared

with those in the controls, demonstrating the on-target eﬀect

of 10a on c-KIT inhibition in vivo. 10a at a 20 mg/kg dose

resulted in 85% p-c-KIT inhibition. while 10a at a 80 mg/kg

dose resulted in 95.9% p-c-KIT inhibition (Supporting

Information, Figure S8).

On the basis of the excellent cellular potency and these

favorable pharmacokinetic properties and pharmacodynamic

results, the antitumor eﬃcacy of 10a was further investigated in

the GIST430 tumor xenograft model (Figure 8). NOD-SCID

mice were implanted subcutaneously (SC) with GIST430

tumor cells, and treatment with either sunitinib or 10a was

initiated when tumors reached an average volume of 155 mm³

at an oral dose of 40 or 25 mg/kg, respectively, on days 1−5

and 8−12. The antitumor growth eﬀect of 10a is superior to

sunitinib (Figure 8). Compound 10a not only inhibited tumor

growth throughout the dosing period, but also reduced tumor

size by 59% on day 14. In comparison, treatment of sunitinib

only inhibited tumor growth, but no obvious tumor regression

was observed.

DISCUSSION AND CONCLUSIONS

■

Tumor recurrence due to emergence of imatinib-resistant

secondary mutations in c-KIT is an ongoing challenge in the

treatment of GISTs. The aim of this study is to discover a more

promising inhibitor to ﬁll the gap that current inhibitors leave

in full coverage of known c-KIT mutants, especially those

arising at the A-loop region, in hopes to reach better clinical

beneﬁts. Using 5-phenyl-thiazol-2-ylamine as a core structure,

10a was identiﬁed as a potent inhibitor that eﬀectively inhibits

c-KIT (Table 1) and a broad spectrum of clinically relevant c-

KIT mutants (Table 3). The inhibitory potency of 10a against

c-KIT A-loop mutants is signiﬁcantly greater than that of

sunitinib (Table 3). Consistent with the enzymatic results, 10a

had a promising antiproliferative eﬀect on GIST cell lines

(Table 4) and induced tumor regression in the xenograft

mouse model (Figure 8). More importantly, our crystal

structure results demonstrated that 10a not only tightly

binds to the unactivated c-KIT as a type II inhibitor (Figure 1),

it also induces a serial conformational changes that switch the

activated c-KIT back to its inactive state (Figures 4−6). Upon

binding of 10a, the urea moiety forms new interactions with

αC-helix and the DFG motif, and rearranges them to

compositions that facilitate the movement of the JM domain

and the ﬁnal switch of the A-loop into the inactive

conformation. The ability of 10a to switch oﬀ the active

conformation of c-KIT is consistent with the observation of

high potency of 10a against the activated c-KIT in the

enzymatic activity (Figure 3). The structure insights of the

conformational changes upon binding of 10a to the activated

c-KIT also rationalize the crosstalk between the JM domain

and A-loop in regulation of c-KIT activation. Perturbation of

the interaction between the activated JM domain and A-loop,

as well as induction of the JM domain into a position that

displaces the A-loop in its active conformation, switch c-KIT

from active to inactive state in the c-KIT activation

equilibrium. Taken together, the excellent biological activities

of 10a and the accompanying structural rationale establish 10a

as a potential drug candidate worthy of clinical evaluation in

patients with advanced GISTs.

EXPERIMENTAL SECTION

■

Chemistry Methods. All commercial chemicals and solvents are

reagent grade and were used without further treatment unless

otherwise noted. ¹H NMR spectra were obtained with a Varian

Mercury-300 or a Varian Mercury-400 spectrometer. Chemical shifts

were recorded in parts per million (ppm, δ) and were reported

relative to the solvent peak or TMS. LC/MS data were measured on

an Agilent MSD-1100 ESI-MS/MS system. High-resolution mass

spectra (HRMS) were measured with a Thermo Finnigan (TSQ

Quantum) electrospray ionization (ESI) mass spectrometer. Flash

column chromatography was done using silica gel (Merck Kieselgel

60, no. 9385, 230−400 mesh ASTM). Reactions were monitored by

TLC using Merck 60 F₂₅₄silica gel glass backed plates (5 cm × 10

cm); zones were detected visually under ultraviolet irradiation (254

nm) or by spraying with phosphomolybdic acid reagent (Aldrich),

followed by heating at 80 °C. All starting materials and amines were

commercially available unless otherwise indicated. The purity of target

compounds was determined with a Hitachi 2000 series HPLC system

based on reverse phase C₁₈column (Agilent ZORBAX Eclipse XDB-

Figure 8. Comparison of antitumor eﬃcacy of 10a and sunitinib in

GIST430 xenograft mouse models. After the average size of the

tumors reached 155 nm³, mice were dosed with 10a orsunitinib (5

days on/2 days oﬀ schedule for 2 weeks) by oral gavage. Tumor sizes

are expressed as the mean SEM (n = 6/group). Mean diﬀerences

between 10a and sunitinib treatments at day 14 and 21 were analyzed

using Wilcoxon rank sum test. *, p < 0.05

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C18 5 μm, 4.6 mm × 150 mm) under the following gradient elution

condition: Mobile phase A-acetonitrile (10% to 90%, 0 to 45 min)

and mobile phase B-10 mM NH₄OAc aqueous solution containing

0.1% formic acid (90% to 10%, 0−45 min). The ﬂow-rate was 0.5

mL/min and the injection volume was 20 μL. The system operated at

25 °C. Peaks were detected at λ = 254 nm. The purity of all tested

compounds is ≥95% purity except for compounds 10f (93.8%), 10j

(94.0%), and 11 (93.9%).

NMR (300 MHz, DMSO-d₆): δ 11.13 (s, 1H), 10.35 (s, 1H), 7.79−

7.76 (d, J = 8.1 Hz, 2H), 7.63−7.53 (m, 4H), 7.44 (d, J = 8.7 Hz,

2H), 7.11 (d, J = 8.7 Hz, 2H), 5.76 (s, 1H), 4.08 (br s, 1H), 3.33 (s,

1H), 3.16 (d, J = 5.4 Hz, 2H), 2.41−2.32 (m, 9H), 1.02 (t, J = 7.2 Hz,

3H). MS (ES⁺) m/z calcd for C₂₆H₂₉N₇O₂S₂535.68, found 536.2 (M

+ H⁺). HRMS (ESI) calcd for C₂₆H₃₀N₇O₂S₂536.1902, found

536.1902 (M + H⁺).

N-(4-{2-[6-(4-Ethylpiperazin-1-yl)pyrimidin-4-ylamino]thiazol-5-

1

yl}phenyl)acetamide HCl Salt (10f). Melting point 312−313 °C. H

Compounds 10−11 were prepared following the previous

publication with some modiﬁcations.³³

NMR (400 MHz, D₂O): δ 8.15 (s, 1H), 7.36 (s, 1H), 7.27 (d, J = 8.4

Hz, 2H), 7.20 (d, J = 8.4 Hz, 2H), 5.92 (s, 1H), 4.28 (d, J = 13.6 Hz,

2H), 3.61 (d, J = 11.6 Hz, 2H), 3.18 (q, J = 7.3 Hz, 4H), 2.89 (d, J =

10.8 Hz, 2H), 2.05 (s, 3H), 1.30 (t, J = 7.4 Hz, 3H). MS (ES⁺) m/z

calcd for C₂₁H₂₅N₇OS 423.18, found 424.4 (M + H⁺). HRMS (ESI)

calcd for C₂₁H₂₆N₇OS 424.1920, found 424.1916 (M + H⁺). HPLC t_R

= 11.81 min, 93.8%.

1-(5-Ethylisoxazol-3-yl)-3-(4-{2-[6-(4-ethylpiperazin-1-yl)-

pyrimidin-4-ylamino]thiazol-5-yl}phenyl)urea HCl Salt (10a). Melt-

1

ing point 276−277 °C. H NMR (400 MHz, DDMSO-d₆): δ 11.24

(bs, 1H), 9.81 (s, 1H), 9.71 (s, 1H), 8.50 (s, 1H), 7.76 (s, 1H), 7.55−

7.49 (m,4H), 6.56 (s, 1H), 6.42 (s, 1H), 4.36 (d, J = 13.2 Hz, 2H),

3.57 (d, J = 11.6 Hz, 2H), 3.45 (t, J = 12.4 Hz, 2H), 3.17−3.01 (m,

4H), 2.71 (q, J = 7.6 Hz, 2H), 1.28 (t, J = 7.2 Hz, 3H), 1.21 (t, J = 7.6

Hz, 3H). MS (ES⁺) m/z calcd for C₂₅H₂₉N₉O₂S 519.22, found 520.2

(M + H⁺). HRMS (ESI) calcd for C₂₅H₃₀N₉O₂S 520.2243, found

520.2241 (M + H⁺).

1-(5-Ethylisoxazol-3-yl)-3-(5-{2-[6-(4-ethylpiperazin-1-yl)-

pyrimidin-4-ylamino]thiazol-5-yl}pyridin-2-yl)urea HCl Salt (10g).

Melting point 243−244 °C. ¹H NMR (400 MHz, DMSO-d₆): δ 11.26

(bs, 1H), 10.76 (bs, 1H), 9.91 (s, 1H), 8.55 (s, 1H), 8.48 (s, 1H),

8.03 (d, J = 8.8 Hz, 1H), 7.56 (s, 1H), 7.70 (d, J = 8.8 Hz, 1H), 6.60

(s, 1H), 6.39 (s, 1H), 4.37 (d, 2H, overing water peak), 3.56 (d, J =

12.0 Hz, 2H), 3.44 (t, J = 12.4 Hz, 2H), 3.13 (t, J = 6.2 Hz, 2H), 3.02

(q, J = 10.0 Hz, 2H), 2.73 (q, J = 7.6 Hz, 2H), 1.27 (t, J = 7.2 Hz,

3H), 1.22 (t, J = 7.6 Hz, 3H), MS (ES⁺) m/z calcd for C₂₄H₂₈N₁₀O₂S

520.21, found 521.2 (M + H⁺). HRMS (ESI) calcd for C₂₄H₂₉N₁₀O₂S

521.2196, found 521.2189 (M + H⁺).

1-(5-Ethyl-3-isoxazolyl)-3-[4-(2-[6-(4-ethylpiperazino)-2-methyl-

4-pyrimidinyl]amino-1,3-thiazol-5-yl)phenyl]urea HCl Salt (10b).

Melting point 259−260 °C. ¹H NMR (400 MHz, DMSO-d₆): δ 11.12

(s, 1H), 9.48 (s, 1H), 8.95 (s, 1H), 7.66 (s, 1H), 7.48 (ABq, Δν_AB

=

16.4 Hz, J_AB= 8.8 Hz, 4H), 6.55 (s, 1H), 6.05 (s, 1H), 3.50 (br s,

4H), 2.70 (q, J = 7.6 Hz, 2H), 2.41−2.32 (m, 9H), 1.22 (t, J = 7.6 Hz,

3H), 1.03 (t, J = 7.2 Hz, 3H). MS (ES+) m/z calcd for C₂₆H₃₁N₉O₂S:

533.23, found 534.2 (M + H⁺). HRMS (ESI) calcd for C₂₆H₃₂N₉O₂S

534.2400, found 534.2390 (M + H⁺).

1-(5-Ethylisoxazol-3-yl)-3-(4-{2-[6-(4-ethylpiperazin-1-yl)-

pyrimidin-4-ylamino]-4-methylthiazol-5-yl}phenyl)urea HCl Salt

1

(10h). Melting point 275−276 °C. H NMR (400 MHz, DMSO-

1-[4-(2-{[2-Methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl]-

amino}-1,3-thiazol-5-yl)phenyl]-3-phenylurea (10c). Melting point

252−253 °C (HCl salt). ¹H NMR (300 MHz, DMSO-d₆): δ 11.11 (s,

1H), 8.77 (s, 1H), 8.67 (s, 1H), 7.64 (s, 1H), 7.49−7.44 (m, 6H),

7.28 (t, J = 7.8 Hz, 2H), 6.97 (t, J = 7.2 Hz, 1H), 6.05 (s, 1H), 3.50

(br s, 4H), 2.41−2.34 (m,7H), 2.22 (s, 3H). MS (ES⁺) m/z calcd for

C₂₆H₂₈N₈OS 500.63, found 501.47 (M + H⁺). HRMS (ESI) calcd for

C₂₆H₂₉N₈OS 501.2185, found 501.2178 (M + H⁺).

d₆): δ 11.08 (bs, 1H), 9.76 (s, 1H), 9.58 (s, 1H), 8.47 (s, 1H), 7.54

(d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 6.56 (s, 1H), 6.38 (s,

1H), 4.36 (d, J = 10.8 Hz, 2H), 3.56 (d, J = 11.6 Hz, 2H), 3.42 (t, J =

12.4 Hz, 2H), 3.13−3.01 (m, 4H), 2.71 (q, J = 7.2 Hz, 4H), 2.34 (s,

3H), 1.28 (t, J = 6.4 Hz, 3H), 1.21 (t, J = 7.2 Hz, 3H). MS (ES⁺) m/z

calcd for C₂₆H₃₁N₉O₂S 533.23, found 534.2 (M + H⁺). HRMS (ESI)

calcd for C₂₆H₃₂N₉O₂S 534.2400, found 534.2383 (M + H⁺).

1-(5-Ethylisoxazol-3-yl)-3-(4-{2-[4-(4-ethylpiperazin-1-yl)-6-

methyl-[1,3,5]triazin-2-ylamino]thiazol-5-yl}phenyl)urea HCl Salt

(10i). Melting point 277−278 °C. ¹H NMR (400 MHz, DMSO-d₆): δ

11.32 (br, 1H), 9.77 (d, J = 8.8 Hz, 1H), 9.63 (d, J = 19.6 Hz, 1H),

8.13 (d, J = 10.0 Hz, 1H), 7.58 (dd, J = 29.2, 7.6 Hz, 4H), 6.55 (s,

1H), 4.83 (d, J = 14.0 Hz, 1H), 3.80 (br, 2H), 3.59 (d, J = 10.0 Hz,

2H), 3.16−3.08 (m, 4H), 2.72 (t, J = 15.2, 7.6 Hz, 3H), 2.39 (s, 2H),

1.30 (t, J = 7.2 Hz, 3H), 1.21 (t, J = 7.6 Hz, 3H). MS (ES⁺) m/z calcd

for C₂₅H₃₀N₁₀O₂S 534.2, found 535.2 (M + H⁺). HRMS (ESI) calcd

for C₂₅H₃₁N₁₀O₂S 535.2352, found 535.2341 (M + H⁺).

(4-{2-[6-(4-Ethylpiperazin-1-yl)pyrimidin-4-ylamino]thiazol-5-

ylphenyl)urea HCl Salt (10d). A solution of potassium cyanate (41

mg, 0.5 mmol) in water (1 mL) was added dropwise to a solution of 8

(95 mg, 0.25 mmol) in glacial acetic acid (4 mL) at room

temperature. After the reaction mixture was stirred for 30 min,

most of solvent was removed under reduced pressure, and the residue

was washed with saturated NaHCO₃solution (5 mL). The precipitate

was collected by ﬁltration, washed with acetic acid (2 mL), and dried

in vacuo. The residue was puriﬁed by chromatography on silica gel

(10% MeOH/CH₂Cl₂) to give the desired product 10d as an oﬀ-

white solid (87 mg, 82%). The above white solid 10d was added to a

stirred solution of 6 N HCl (1 mL) at 0 °C. The solution was ﬁltered

through a 0.45 μm PVDF membrane, and the ﬁltrate was collected.

Acetone (10 mL) was added to the stirred ﬁltrate dropwise over 1 h.

Stirring was continued for an additional 1 h at 0 °C. The precipitate

was collected by ﬁltration, washed with acetone (10 mL), and dried in

vacuo to give the desired 10d HCl salt as a yellow solid (102 mg,

1-(5-Ethylisoxazol-3-yl)-3-[4-(2-{6-[4-(2-hydroxyethyl)piperazin-

1-yl]-pyrimidin-4-ylamino}thiazol-5-yl)phenyl]urea Dihydrochlor-

ide (10j). Melting point 272−273 °C. ¹H NMR (400 MHz,

DMSO-d₆): δ 10.77 (bs, 1H), 9.79 (s, 1H), 9.66 (s, 1H), 8.49 (s,

1H), 7.74 (s, 1H), 7.55−7.49 (m, 4H), 6.56 (s, 1H), 6.40 (s, 1H),

4.34 (d, J = 13.2 Hz, 2H), 3.81 (t, J = 5.0 Hz, 2H), 3.62 (d, J = 12.0

Hz, 2H), 3.45 (t, J = 12.4 Hz, 2H), 3.22−3.13 (m, 4H), 2.71 (q, J =

7.6 Hz, 4H), 1.21 (t, J = 7.6 Hz, 3H). MS (ES⁺) m/z calcd for

C₂₅H₂₉N₉O₃S 535.21, found 536.2 (M + H⁺). HRMS (ESI) calcd for

C₂₅H₃₀N₉O₃S 536.2192, found 536.2170 (M + H⁺). HPLC t_R= 14.61

min, 94.0%.

1

99%). H NMR (400 MHz, DMSO-d₆): δ 11.05 (s, 1H), 8.89 (s,

1H), 8.46 (s, 1H), 7.67 (s, 1H), 7.43−7.40 (m, 4H), 6.36 (s, 1H),

4.34 (d, J = 12.8 Hz, 2H), 3.55 (d, J = 11.6 Hz, 2H), 3.39 (t, J = 12.6

Hz, 2H), 3.15−3.08 (m, 2H), 3.01 (t, J = 10.8 Hz, 2H), 1.25 (t, J =

7.2 Hz, 3H). MS (ES⁺) m/z calcd for C₂₀H₂₄N₈OS 424.18, found

425.4 (M + H⁺). HRMS (ESI) calcd for C₂₀H₂₅N₈OS 425.1872,

found 425.1867 (M + H⁺).

N-[4-(2-[6-(3-Aminotetrahydro-1H-1-pyrrolyl)-2-methyl-4-

pyrimidinyl]amino-1,3-thiazol-5-yl)phenyl]-N′-(5-ethyl-3-

isoxazolyl)urea Dihydrochloride (10k). Melting point 267−268 °C.

¹H NMR (400 MHz, DMSO-d₆): δ 8.94 (s, 1H), 7.63 (s, 1H), 7.48

N-[4-(2-{[6-(4-Ethylpiperazin-1-yl)-2-methylpyrimidin-4-yl]-

amino}-1,3-thiazol-5-yl)phenyl]benzenesulfonamide (10e). To a

solution of 8 (1.0 equiv) in pyridine at room temperature was added

benzenesulfonyl chloride (1.1 equiv). After 2 h of stirring at room

temperature, the reaction mixture was evaporated to dryness. The

residue was suspended in saturated NaHCO₃solution, and the

resulting suspension was vigorously stirred at room temperature for 1

h. The resultant solid was collected by ﬁltration and recrystallized

(ABq, Δν_AB= 14.0 Hz, J_AB= 8.4 Hz, 4H), 6.53 (s, 1H), 5.73 (s, 1H),

3.37 (br s, 7H, overlapping with water peak), 2.69 (q, J = 7.2 Hz, 2H),

2.37 (s, 3H), 1.99 (br s, 1H), 1.67 (br s, 1H), 1.19 (t, J = 7.6 Hz, 3H).

MS (ES⁺) m/z calcd for C₂₄H₂₇N₉O₂S 505.20, found 506.2 (M +

H⁺). HRMS (ESI) calcd for C₂₄H₂₈N₉O₂S 506.2087, found 506.2083

(M + H⁺).

1-(5-Ethylisoxazol-3-yl)-3-(4-{2-[6-(4-ethylpiperazin-1-yl)-2-

methylpyrimidin-4-ylamino]thiazol-4-yl}phenyl)urea HCl Salt (11).

Melting point 258−259 °C. ¹H NMR (400 MHz, DMSO-d₆): δ 11.21

1

from EtOAc/CH₃OH to yield 10e. Melting point 281−282 °C. H

N

DOI: 10.1021/acs.jmedchem.8b01845

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Journal of Medicinal Chemistry

Article

(bs, 1H), 9.83 (s, 1H), 9.72 (s, 1H), 7.81 (d, J = 8.4, 2H), 7.51 (d, J =

8.4, 2H), 7.43 (s, 1H), 6.55 (s, 1H), 6.34 (bs, 1H), 4.37 (d, J = 13.6

Hz, 2H), 3.57−3.41 (m, 4H), 3.14−3.01 (m, 4H), 2.70 (q, J = 7.6 Hz,

2H), 2.45 (s, 3H), 1.27 (t, J = 7.2 Hz, 3H), 1.20 (t, J = 7.6 Hz, 3H).

MS (ES⁺) m/z calcd for C₂₇H₃₃N₉O₂S 533.23, found 534.1 (M +

H⁺). HRMS (ESI) calcd for C₂₇H₃₄N₉O₂S 534.2400, found 534.2350

(M + H⁺). HPLC t_R= 22.74 min, 93.9%.

Synthetic Procedure for the Preparation of Urea Analogues 18,

22, and 24. Sodium hydride (1.6 g, 60% in oil, 39.94 mmol) was

added to a solution of 2-aminothiazole 12 (2.0 g, 19.97 mmol) in

anhydrous THF (100 mL) at room temperature. After the reaction

mixture was stirred for 10 min, a solution of 4,6-dichloro-2-

methylpyrimidine 5b (R = CH₃, 4.9 g, 29.96 mmol) in anhydrous

THF (50 mL) was added dropwise and the mixture was stirred

overnight. The reaction was quenched by the addition of saturated

NH₄Cl solution, and most of solvent was removed under reduced

pressure. The residue was stirred vigorously in saturated NaHCO₃

solution (250 mL) for 1 h. The precipitate was collected by ﬁltration,

washed with water (2 × 30 mL), and puriﬁed by chromatography on

silica gel (5% MeOH/CH₂Cl₂) to give the desired product 13b as an

oﬀ-white solid (3.5 g, 78%).

(6-Chloro-2-methylpyrimidin-4-yl)thiazol-2-yl-amine (13b, R =

CH₃). ¹H NMR (400 MHz, DMSO-d₆): δ 11.87 (s, 1H), 7.48 (d, J =

3.6 Hz, 1H), 7.23 (d, J = 3.6 Hz, 1H), 6.92 (s, 1H), 2.55 (s, 3H). MS

(ES⁺) m/z calcd for C₈H₇ClN₄S 226.01, found 227.0 (M + H⁺). A

mixture of 13b (2.0 g, 8.82 mmol) and 1-ethylpiperazine (2.0 g, 17.51

mmol) in pyridine (15 mL) was heated at 65 °C overnight. After

cooling to room temperature, most of solvent was removed under

reduced pressure and the residue was puriﬁed by chromatography on

silica gel (5% MeOH/CH₂Cl₂) to give the desired product 14b as a

brown solid (2.4 g, 89%).

[6-(4-Ethylpiperazin-1-yl)-2-methylpyrimidin-4-yl]thiazol-2-yl-

amine (14b, R = CH₃). ¹H NMR (400 MHz, DMSO-d₆): δ 11.02 (s,

1H), 7.33 (d, J = 3.6 Hz, 1H), 7.01 (d, J = 3.6 Hz, 1H), 6.03 (s, 1H),

3.50−3.42 (m, 4H), 2.45−2.28 (m, 9H), 1.01 (t, J = 7.2 Hz, 3H). MS

(ES⁺) m/z calcd for C₁₄H₂₀N₆S 304.15, found 305.1 (M + H⁺). A

mixture of 14b (745 mg, 2.45 mmol) and N-iodosuccinimide (580

mg, 2.58 mmol) in CHCl₃(20 mL) was stirred at room temperature

for 2 h. The reaction was quenched by the addition of saturated

sodium thiosulfate solution (5 mL), and the product was extracted

with CH₂Cl₂(2 × 20 mL). The combined organic extract was dried

over MgSO₄and concentrated under reduced pressure. The residue

was puriﬁed by chromatography on silica gel (7% MeOH/CH₂Cl₂) to

give the desired product 15b as a white solid (843 mg, 80%).

[6-(4-Ethylpiperazin-1-yl)-2-methylpyrimidin-4-yl]-(5-iodothia-

zol-2-yl)amine (15b, R = CH₃). ¹H NMR (400 MHz, CDCl₃): δ 8.90

(bs, 1H), 7.39 (s, 1H), 5.67 (s, 1H), 3.68−3.62 (m, 4H), 2.68−2.55

(m, 4H), 2.50 (s, 3H), 2.47 (q, J = 6.8 Hz, 2H), 1.13 (t, J = 7.0 Hz,

3H). MS (ES⁺) m/z calcd for C₁₄H₁₉IN₆S 430.04, found 431.0 (M +

H⁺). A mixture of 15b (120 mg, 0.28 mmol), N-boc-1,2,3,6-

tetrahydropyridine-4-boronic acid pinacol ester 16 (172 mg, 0.56

mmol), PdCl₂(dppf) (80 mg, 0.11 mmol), and Na₂CO₃(1.0 mmol in

0.5 mL of water) in 1,4-dioxane (4 mL) was heated at 80 °C for 2 h.

After cooling to room temperature, the reaction mixture was ﬁltered

through a short pad of Celite and most of solvent was removed under

reduced pressure. The residue was puriﬁed by chromatography on

silica gel (10% MeOH/CH₂Cl₂) to aﬀord crude Boc-17 as a brown

solid (100 mg). A mixture of Boc-17 (100 mg, 0.21 mmol) and TFA

(1 mL) in CH₂Cl₂(4 mL) was stirred at room temperature overnight.

Most of solvent was removed under reduced pressure, and the residue

was puriﬁed by chromatography on silica gel (15% MeOH/CH₂Cl₂)

to give the desired product 17 as a brown solid (30 mg, 38%).

[6-(4-Ethylpiperazin-1-yl)-2-methylpyrimidin-4-yl]-[5-(1,2,3,6-

tetrahydropyridin-4-yl)thiazol-2-yl]amine (17). ¹H NMR (300

MHz, DMSO-d₆): δ 7.24 (s, 1H), 6.03 (s, 1H), 5.89 (s, 1H),

3.55−3.36 (m, 6H, overlapping with water), 2.91 (t, J = 5.6 Hz, 2H),

2.49−2.31 (m, 11H), 0.98 (t, J = 7.1 Hz, 3H). MS (ES⁺) m/z calcd

for C₁₉H₂₇N₇S 385.20, found 386.2 (M + H⁺). 5-Ethylisoxazol-3-

yl)carbamic acid 4-nitro-phenyl ester (45 mg, 0.16 mmol) was added

to a solution of 17 (30 mg, 0.08 mmol) in pyridine (1 mL) at room

temperature, and the mixture was stirred overnight. Most of solvent

was removed under reduced pressure, and the residue was puriﬁed by

chromatography on silica gel (10% MeOH/CH₂Cl₂) to give the

desired product 18 as a yellow solid (27 mg, 65%).

4-{2-[6-(4-Ethylpiperazin-1-yl)-2-methylpyrimidin-4-ylamino]-

thiazol-5-yl}-3,6-dihydro-2H-pyridine-1-carboxylic Acid (5-Ethyl-

isoxazol-3-yl)amide (18). Melting point 245−246 °C. ¹H NMR

(300 MHz, DMSO-d₆): δ 11.08 (bs, 1H), 9.68 (s, 1H), 7.32 (s, 1H),

6.49 (s, 1H), 6.04 (s, 1H), 5.90 (bs, 1H), 4.10 (bs, 2H), 3.65 (bs,

2H), 3.55−3.40 (m, 4H), 2.68 (q, J = 7.5 Hz, 2H), 2.41−2.34 (m,

11H, overlapping with DMSO), 1.19 (t, J = 7.5 Hz, 3H), 1.02 (t, J =

7.2 Hz 3H). MS (ES⁺) m/z calcd for C₂₅H₃₃N₉O₂S 523.25, found

524.3 (M + H⁺). HRMS (ESI) calcd for C₂₅H₃₄N₉O₂S 524.2556,

found 524.2552 (M + H⁺). A mixture of 15a (R = H, 200 mg, 0.48

mmol), N-propargylphthalimide 19 (178 mg, 0.96 mmol), Pd(PPh₃)₄

(58 mg, 0.05 mmol), copper(I) iodide (19 mg, 0.10 mmol), and

triethylamine (0.20 mL, 1.44 mmol) in dry THF (5 mL) was stirred

at room temperature overnight. The reaction mixture was ﬁltered

through a short pad of Celite, and most of the solvent was removed

under reduced pressure. The residue was partitioned between water

(20 mL) and CH₂Cl₂(20 mL). The organic layer was separated, and

the aqueous layer was extracted with CH₂Cl₂(2 × 10 mL). The

combined organic layer was dried over MgSO₄and concentrated to

aﬀord 20a (R = H) as a brown solid which was used in the next step

without further puriﬁcation (43 mg, 19%). Hydrazinium hydroxide

(0.25 mL, 4.8 mmol) was added dropwise to a solution of crude 20a

(43 mg, 0.09 mmol) in THF (5 mL) at 0 °C, and the mixture was

stirred at room temperature overnight. The volatiles were removed

under reduced pressure, and the residue was partitioned between

water (10 mL) and CH₂Cl₂(20 mL). The organic layer was

separated, and the aqueous layer was extracted with CH₂Cl₂(2 × 20

mL). The combined organic layer was dried over MgSO₄and

concentrated. The residue was puriﬁed by chromatography on silica

gel (10% MeOH/CH₂Cl₂) to give the desired product 21a (R = H)

as a white solid (26 mg, 85%). The same synthetic procedure as 21a

yielded 21b (R = CH₃) as a white solid (90%).

[5-(3-Aminoprop-1-ynyl)thiazol-2-yl]-[6-(4-ethylpiperazin-1-yl)-

pyrimidin-4-yl]amine (21a, R = H). ¹H NMR (400 MHz, CDCl₃): δ

8.42 (s, 1H), 7.44 (s, 1H), 5.97 (s, 1H), 3.68−3.65 (m, 6H), 2.54 (t, J

= 5.0 Hz, 4H), 2.48 (q, J = 7.2 Hz, 2H), 1.14 (t, J = 7.2 Hz, 3H).

[5-(3-Aminoprop-1-ynyl)thiazol-2-yl]-[6-(4-ethylpiperazin-1-yl)-

2-methylpyrimidin-4-yl]amine (21b, R = CH₃). ¹H NMR (400 MHz,

DMSO-d₆): δ 7.47 (s, 1H), 5.97 (s, 1H), 3.51 (s, 2H), 3.50−3.44 (m,

4H), 2.42−2.35 (m, 4H), 2.36 (s, 3H), 2.33 (q, J = 7.2 Hz, 2H), 1.01

(t, J = 7.2 Hz, 3H). MS (ES⁺) m/z calcd for C₁₇H₂₃N₇S 357.17, found

358.1 (M + H⁺). The synthesis of ﬁnal products 22 from amine 21a

(yield 80%) was the same as that of urea 18 from amine 17.

1-(5-Ethylisoxazol-3-yl)-3-(3-{2-[6-(4-ethylpiperazin-1-yl)-

pyrimidin-4-ylamino]thiazol-5-yl}prop-2-ynyl)urea (22). ¹H NMR

(300 MHz, DMSO-d₆): δ 11.48 (s, 1H), 9.50 (s, 1H), 8.34 (s, 1H),

7.56 (s, 1H), 6.91 (t, J = 5.4 Hz, 1H), 6.42 (s, 1H), 6.18 (s, 1H), 4.19

(d, J = 5.4 Hz, 2H), 3.52−3.42 (m, 4H), 2.69 (q, J = 7.2 Hz, 2H),

2.50−2.35 (m, 6H, overlapping with DMSO), 1.19 (t, J = 7.5 Hz,

3H), 1.06 (t, J = 6.9 Hz, 3H). MS (ES⁺) m/z calcd for C₂₂H₂₇N₉O₂S

481.2, found 482.2 (M + H⁺). HRMS (ESI) calcd for C₂₂H₂₈N₉O₂S

481.2087, found 482.2085 (M + H⁺). A mixture of 21b (50 mg, 0.14

mmol) and Pd/BaSO₄(30 mg)in MeOH (2 mL) was stirred under a

hydrogen atmosphere at room temperature overnight. The reaction

mixture was ﬁltered through a short pad of Celite, and most of the

solvent was removed under reduced pressure. The residue was

puriﬁed by chromatography on silica gel (15% MeOH/CH₂Cl₂) to

give the desired product 23 as a brown solid (29 mg, 57%).

[5-(3-Aminopropyl)-thiazol-2-yl]-[6-(4-ethylpiperazin-1-yl)-2-

1

methylpyrimidin-4-yl]amine (23). H NMR (300 MHz, CDCl₃): δ

7.02 (s, 1H), 5.88 (s, 1H), 3.78−3.57 (m, 4H), 2.82−2.74 (m, 4H),

2.54−2.41 (m, 9H), 1.90−1.75 (m, 2H), 1.12 (t, J = 7.2 Hz, 3H). MS

(ES⁺) m/z calcd for C₁₇H₂₇N₇S 361.2, found 362.2 (M + H⁺). The

synthesis of ﬁnal product 24 from amine 23 (yield 90%) was the same

as that of urea 18 from amine 17.

O

DOI: 10.1021/acs.jmedchem.8b01845

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Journal of Medicinal Chemistry

Article

1-(5-Ethylisoxazol-3-yl)-3-(3-{2-[6-(4-ethylpiperazin-1-yl)-2-

methylpyrimidin-4-ylamino]thiazol-5-yl}propyl)urea (24). ¹H NMR

(300 MHz, DMSO-d₆): δ 10.88 (bs, 1H), 9.28 (s, 1H), 7.06 (s, 1H),

6.59 (t, J = 5.4 Hz, 1H), 6.38 (s, 1H), 6.04 (s, 1H), 3.58−3.42 (m,

4H), 3.15 (td, J = 6.3, 5.4 Hz, 2H), 2.73−2.63 (m, 4H), 2.55−2.32

(m, 9H, overlapping with DMSO), 1.82−1.71 (m, 2H), 1.18 (t, J =

7.5 Hz, 3H), 1.03 (t, J = 7.1 Hz, 3H). MS (ES⁺) m/z calcd for

C₂₃H₃₃N₉O₂S 499.25, found 500.2 (M + H⁺). HRMS (ESI) calcd for

C₂₃H₃₄N₉O₂: 500.2556, found 500.2551 (M + H⁺). ¹³C NMR

spectrum of 10a and HPLC traces of 10a−10k, 11, 18, 22, and 24 are

shown in Supporting Information, Figure S9.

Kinase Expression, Puriﬁcation, and Activation. Human c-

KIT kinase was expressed using a baculovirus expression system.

Brieﬂy, cDNA encoding c-KIT residues 547−935, with the kinase

insertion domain (residues 694−753) being deleted and replaced by a

6-nucleotide fragment encoding Thr-Ser, was cloned. A 6x-histidine

tag followed by a thrombin cleavage site was added in-frame to the N-

terminus of the cloned c-KIT, and the whole fragment was inserted

into the pBacPAK8 vector. Recombinant c-KIT was expressed in Sf9

insect cells grown in Grace’s insect medium supplemented with 10%

fetal bovine serum (FBS) and antibiotics (100 U penicillin/mL and

100 μg streptomycin/mL). Cell pellets were lysed in lysis buﬀer (25

mM Tris-HCl, pH7.6, 250 mM NaCl, and 0.5 mM TCEP) by

sonication. His-tagged c-KIT proteins were aﬃnity puriﬁed with

HisTrap HP column (GE Life Sciences). Proteins were eluted by a

liner imidazole gradient, and the buﬀer was exchanged to thrombin

buﬀer (20 mM Tris-HCl, pH8.4, 150 mM NaCl, and 2.5 mM CaCl₂)

for overnight thrombin cleavage at 4 °C. After removal of the histidine

tag, the c-KIT proteins were subjected to a buﬀer exchange to the

ﬁnal buﬀer containing 25 mM Tris-HCl, pH 7.6, 250 mM NaCl, and

5 mM DTT.

To obtain the activated c-KIT proteins, 10 times in molar ratio of

ATP was incubated with c-KIT at room temperature for 2 h, followed

by a ﬁnal concentrating step at 4 °C. To verify the phosphorylation

status of the activated c-KIT, Western blotting assays using

antiphospho-tyrosine antibodies and Coomassie Brilliant Blue staining

of total proteins were performed. As shown in Supporting

Information, Figure S1, the puriﬁed c-KIT proteins were unphos-

phorylated (lane 1), indicating that the proteins are in the unactivated

state before ATP activation. In contrast, the c-KIT proteins were

predominantly phosphorylated in the ﬁrst hour of ATP incubation

(lane 2), and the activation reached to a level that almost all c-KIT

proteins were phosphorylated after 2 h of ATP incubation (lane 3).

Crystallization of c-KIT with Compound 10a. Crystallization

of the c-KIT kinase domain with 10a was performed by the hanging

drop method. A solution of the unactivated c-KIT (5.0 mg/mL) was

preincubated with 10a (0.6 mM) on ice for 20 min and mixed with an

equal volume of reservoir solution (15% PEG3350, 0.1 M sodium

citrate tribasic dihydrate, pH 5.6, and 3% ethylene glycol) plus 2%

benzamidine hydrochloride as an additive. The activated c-KIT was

cocrystallized with 10a in a similar way except for an extended

incubation time up to 1 h on ice and using 3% 1,6-hexanediol as an

additive. Crystals were grown at 18 °C and immersed quickly in

cryoprotectant (reservoir solutions with addition of 9% glycerol and a

mixture of 3% glycerol and 7.5% ethylene glycol for the unactivated

and activated c-KIT crystals, respectively) before being ﬂash-frozen in

liquid nitrogen. The X-ray diﬀraction data sets were collected at

beamline 15A1 (NSRRC, Taiwan) and beamline 44XU (Spring-8,

Japan) and processed using the software program HKL2000 (HKL

Research, Inc.). The initial phases of c-KIT kinase domain structures

were obtained by molecular replacement in the PHENIX package

using the previously reported c-KIT structure (PDB 1T46) as a

template. Structure reﬁnement and model building were performed

using PHENIX and COOT, and structure results were depicted using

Tris−HCl, pH 7.4, 2 mM MnCl₂, 2 mM DTT, 20 mM MgCl₂, 0.1

mg/mL bovine serum albumin, 1 mM Na₃VO₄) containing 20 μM

ATP and 40 μM poly(Glu₄-Tyr₁) as substrates at 30 °C for 150 min.

The ADP-Glo reagent was then added, and the mixture was incubated

at 25 °C for 40 min. The kinase detection reagent was added for

another 30 min at 25 °C. The converted ADP was determined by

luminescence measurement using the Wallac Vector 1420 multilabel

counter (PerkinElmer).

To determine the IC₅₀values of imatinib, sunitinib, or 10a to the

unactivated and activated c-KIT (Figure 3), kinase assays were

performed using the Kinase-GloPlus reagents (Promega). Brieﬂy, 250

nM of the unactivated or activated c-KIT proteins were incubated

with imatinib, sunitinib, or 10a in the reaction buﬀer (40 mM Tris-

HCl, pH 7.4, 2 mM MnCl₂, 2 mM DTT, 20 mM MgCl₂, 0.1 mg/mL

bovine serum albumin, 1 mM Na₃VO₄) containing 10 μM poly(Glu₄-

Tyr₁) as substrates at room temperature for 30 min. After incubation,

5 μM ATP was added to the unactivated c-KIT or activated c-KIT

mixtures and incubated at 37 °C for 120 or 30 min, respectively. The

Kinase-Glo Plus reagent was then added, and the amount of remained

ATP was determined by luminescence measurement using the Wallac

Vector 1420 multilabel counter (PerkinElmer).

ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.8b01845.

Material and methods of antiproliferation cellular assays;

Western blot analysis, pharmacokinetic studies, in vivo

pharmacodynamics, and eﬃcacy studies in GIST430

xenografts model; Coomassie Brilliant Blue staining and

Western blot analysis of the unactivated and activated c-

KIT; Illustration of possible binding modes of 11 with c-

KIT; molecular docking of 10f with c-KIT; modeling

study of 10a bound to c-KIT V654A; the TREEspot

interaction maps and selectivity score of 10a; cell

viability of GIST48B and GIST62 cell lines treated with

10a; cell viability of PC3, A549, and Detroit 551 cell

lines treated with 10a; pharmacodynamic study of 10a;

¹³C NMR spectrum of 10a and HPLC traces of 10a−

10k, 11, 18, 22, and 24 (PDF)

Molecular formula strings (CSV)

Accession Codes

6ITT (unactivated c-KIT/10a); 6ITV (activated c-KIT/10a).

Authors will release the atomic coordinates and experimental

data upon article publication.

AUTHOR INFORMATION

Corresponding Authors

*For S.Y.W.: phone, +886-37-246-166 ext 35713; fax, + 886-

37-586-456; E-mail, suying@nhri.org.tw.

■

*For W.T.J.: phone, +886-37-246-166 ext 35712; fax, + 886-

37-586-456; E-mail, wtjiaang@nhri.org.tw.

ORCID

Su-Ying Wu: 0000-0003-2816-6961

Notes

The authors declare no competing ﬁnancial interest.

̈

PyMOL (Schrodinger, LLC).

ACKNOWLEDGMENTS

■

Biochemical Kinase Assays. For SAR analysis (Table 1), the

enzymatic activities of c-KIT were determined by in vitro kinase

assays using the ADP-Glo reagent (Promega) according to

manufacturer’s instructions. Brieﬂy, 250 ng of c-KIT proteins were

incubated with compounds in the 10 μL of reaction buﬀer (40 mM

We thank the staﬀ of beamlines TPS05A, BL15A1, and

BL13B1 at the National Synchrotron Radiation Research

Center (NSRRC), Taiwan, and SP44XU at the Spring-8,

Japan, for technical assistance. This work was supported by

P

DOI: 10.1021/acs.jmedchem.8b01845

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

H.; Bono, P.; Kallio, R.; Junnila, J.; Alvegard, T.; Reichardt, P.

Adjuvant imatinib for high-risk GI stromal tumor: analysis of a

randomized trial. J. Clin. Oncol. 2016, 34, 244−250.

National Health Research Institute, and Ministry of Economic

Aﬀairs, Taiwan, R.O.C.

(16) Casali, P. G.; Zalcberg, J.; Le Cesne, A.; Reichardt, P.; Blay, J.

Y.; Lindner, L. H.; Judson, I. R.; Schoffski, P.; Leyvraz, S.; Italiano, A.;

Grunwald, V.; Pousa, A. L.; Kotasek, D.; Sleijfer, S.; Kerst, J. M.;

Rutkowski, P.; Fumagalli, E.; Hogendoorn, P.; Litiere, S.; Marreaud,

S.; van der Graaf, W.; Gronchi, A.; Verweij, J. Ten-year progression-

free and overall survival in patients with unresectable or metastatic GI

stromal tumors: long-term analysis of the European organisation for

research and treatment of cancer, Italian sarcoma group, and

Australasian gastrointestinal trials group intergroup phase III

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2017, 35, 1713−1720.

ABBREVIATIONS USED

■

GIST, gastrointestinal stromal tumor; RTK, receptor tyrosine

kinase; JM, juxtamembrane; A-loop, activation loop; SCF, stem

cell factor; SAR, structure−activity relationships

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DOI: 10.1021/acs.jmedchem.8b01845

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Discovery of Conformational Control Inhibitors Switching off the Activated c-KIT and Targeting a Broad Range of Clinically Relevant c-KIT Mutants

DOI: 10.1021/acs.jmedchem.8b01845

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