Identification of a potent 5-phenyl-thiazol-2-ylamine-based inhibitor of FLT3 with activity against drug resistance-conferring point mutations

Article abstract of DOI:10.1016/j.ejmech.2015.05.008

Abstract Numerous FLT3 inhibitors have been explored as a viable therapy for the treatment of acute myeloid leukemia (AML). However, clinical data have been underwhelming due to incomplete inhibition of FLT3 or the emergence of resistant mutations treated

Full text of DOI:10.1016/j.ejmech.2015.05.008

Accepted Manuscript
Identification of a potent 5-phenyl-thiazol-2-ylamine-based inhibitor of FLT3 with
activity against drug resistance-conferring point mutations
Chiung-Tong Chen, John T.-A. Hsu, Wen-Hsing Lin, Cheng-Tai Lu, Shih-Chieh Yen,
Tsu Hsu, Yu-Ling Huang, Jen-Shin Song, Chun-Hwa Chen, Ling-Hui Chou, Kuei-Jung
Yen, Ching-Ping Chen, Po-Chu Kuo, Chen-Lung Huang, H. Eugene Liu, Yu-Sheng
Chao, Teng-Kuang Yeh, Weir-Torn Jiaang
PII:
S0223-5234(15)30034-9
10.1016/j.ejmech.2015.05.008
EJMECH 7887
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 March 2015
Revised Date: 30 April 2015
Accepted Date: 5 May 2015
Please cite this article as: C.-T. Chen, J.T.-A. Hsu, W.-H. Lin, C.-T. Lu, S.-C. Yen, T. Hsu, Y.-L. Huang,
J.-S. Song, C.-H. Chen, L.-H. Chou, K.-J. Yen, C.-P. Chen, P.-C. Kuo, C.-L. Huang, H. Eugene Liu, Y.-
S. Chao, T.-K. Yeh, W.-T. Jiaang, Identification of a potent 5-phenyl-thiazol-2-ylamine-based inhibitor
of FLT3 with activity against drug resistance-conferring point mutations, European Journal of Medicinal
Chemistry (2015), doi: 10.1016/j.ejmech.2015.05.008.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Identification of a potent 5-phenyl-thiazol-2-ylamine-based inhibitor of FLT3 with activity against drug
resistance-conferring point mutations
Chiung-Tong Chen, John T.-A. Hsu, Wen-Hsing Lin, Cheng-Tai Lu, Shih-Chieh Yen, Tsu Hsu, Yu-Ling Huang, Jen-Shin
Song, Chun-Hwa Chen, Ling-Hui Chou, Kuei-Jung Yen, Ching-Ping Chen, Po-Chu Kuo, Chen-Lung Huang, H. Eugene Liu,
Yu-Sheng Chao, Teng-Kuang Yeh, and Weir-Torn Jiaang
FLT3-ITD/D835Y tumor xenografts
3
2
2
1
000
500
000
500
Vehicle
10 mg/kg
7h 10 mg/kg
3
N
N
NH
HN
S
O
N
HN
N
1000
5
00
O
N
0
N
0
5
10
15
20
25
30
7h
Days after treatment
1

ACCEPTED MANUSCRIPT
Identification of a potent 5-phenyl-thiazol-2-ylamine-based inhibitor of
FLT3 with activity against drug resistance-conferring point mutations
1
,#
1,#
1,#
1
1
Chiung-Tong Chen, John T.-A. Hsu, Wen-Hsing Lin, Cheng-Tai Lu, Shih-Chieh Yen,
1
1
1
1
1
Tsu Hsu, Yu-Ling Huang, Jen-Shin Song, Chun-Hwa Chen, Ling-Hui Chou, Kuei-Jung
1
1
1
1
2
Yen, Ching-Ping Chen, Po-Chu Kuo, Chen-Lung Huang, H. Eugene Liu, Yu-Sheng
1
1,
1,
Chao, Teng-Kuang Yeh, * and Weir-Torn Jiaang *
1
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes,
No. 35, Keyan Rd., Zhunan Town, Miaoli Country 350, Taiwan, R.O.C.
2
Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan, R.O.C.
Division of Hematology and Oncology, Department of Medicine, Wan Fang Hospital, Taipei
Medical University, Taipei, Taiwan, R.O.C.
#
Chiung-Tong Chen, John T.-A. Hsu and Wen-Hsing Lin contributed equally.
*
Corresponding authors
Please send all correspondence to
Dr. Weir-Tong Jiaang (email: wtjiaang@nhri.org.tw)
Institute of Biotechnology and Pharmaceutical Research
National Health Research Institutes, Taiwan, R.O.C.
TEL: 886-37-246166 ext. 35712; FAX: 886-37-586456
Abstract
Numerous FLT3 inhibitors have been explored as a viable therapy for the treatment of
acute myeloid leukemia (AML). However, clinical data have been underwhelming due to
incomplete inhibition of FLT3 or the emergence of resistant mutations treated with these older
1

ACCEPTED MANUSCRIPT
agents. We previously developed a series of 3-phenyl-1H-5-pyrazolylamine derivatives as
highly potent and selective FLT3 inhibitors with good in vivo efficacy using an intravenous
(
IV) route. However, the poor bioavailability of these pyrazole compounds limits the
development of these promising antileukemic compounds for clinical use. Herein, we
describe a novel class of 5-phenyl-thiazol-2-ylamine compounds that are multi-targeted FLT3
inhibitors. From this class of compounds, compound 7h was very potent against AML cell
lines and exhibited excellent oral efficacy in AML xenograft models. In addition, further
studies demonstrated that compound 7h exhibited potent in vitro and in vivo activities against
clinically relevant AC220 (3)-resistant kinase domain mutants of FLT3-ITD.
Keywords: FLT3 inhibitor; Acute myeloid leukemia; Receptor tyrosine kinase; ITD/D835Y;
Xenograft model.
Abbreviations: FLT3, FMS-like Tyrosine Kinase-3; AML, acute myeloid leukemia; IV,
intravenous; RTK, receptor tyrosine kinase; ITDs, internal tandem duplications; AL,
activation loop; KD, kinase domain; TKIs, tyrosine-kinase inhibitors; SAR, structure-activity
relationships; VEGFR2, vascular endothelial growth factor receptor 2; CSF1R,
macrophage-colony stimulating factor receptor; PDGFR, platelet-derived growth factor
receptor; RET, proto-oncogene tyrosine-protein kinase receptor; ALL, acute lymphoblastic
leukemia; CML, chronic myelogenous leukemia; CR, complete regression; SC, subcutaneous;
PO, per oral.
2

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1
.
Introduction
Acute myeloid leukemia (AML) is an aggressive disease in which the rapid growth of
abnormal leukemic cells in bone marrow inhibits the production of normal blood cells.
Despite the success of conventional chemotherapy, the rate of relapse among AML patients is
relatively high. The failure of the initial chemotherapy treatment and the refractory nature of
AML have been associated with mutations that activate signal transduction pathways,
resulting in enhanced proliferation and survival of leukemia cells [1,2]. The FMS-like
tyrosine kinase-3 (FLT3) is a member of the class III membrane-bound receptor tyrosine
kinase (RTK) family, along with c-KIT, FMS, and PDGFR [3,4]. The most frequent mutations
among AML are FLT3 mutations, which account for approximately 30% of the genetic
mutations that are predictive markers for a poor prognosis [4-6]. These mutations are typically
internal tandem duplications (ITDs) in the juxtamembrane domain of the receptor and the
missense point, or short-length mutations in the activation loop (AL) of the tyrosine kinase
domain (KD) [7-9]. In addition to mutant forms of FLT3, wt-FLT3 is highly expressed in most
cases of acute leukemia, and FLT3 over-expression is an unfavorable prognostic factor for
overall survival with AML. In addition, over-expressed wt-FLT3 has the same sensitivity to
FLT3 inhibitors as the FLT3/ITD mutants [10,11].
Due to the prevalence and refractory nature of the FLT3 mutations as well as the poor
prognosis for those affected by it, numerous agents have been developed to directly inhibit
wild-type and mutant FLT3. Several of these agents, such as midostaurin (PKC412, 1) [12],
3

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sorafenib (2) [13] and quizartinib (AC220, 3) [14] (Figure 1), have been investigated as
potential agents for the treatment of AML [15]. However, clinical responses to several of the
early multi-targeted agents, such as lestaurtinib and 1, have been underwhelming with limited
reduction in bone marrow blasts and weak short-term responses. These underwhelming results
may be primarily due to low potency and/or inadequate toleration of the inhibitors at effective
doses, which result in failure to achieve complete and sustained inhibition of FLT3 in the
patients’ leukemic blast cells [15]. Newer, more potent and selective FLT3 inhibitors, such as
2
and 3, possess the ability to achieve more sustained in vivo inhibition of FLT3 and have
exhibited highly promising activity in early clinical studies [16]. Nevertheless, K. W. Pratz et
al. reported that the inhibition of FLT3 autophosphorylation in a FLT3/ITD specimen does not
always induce cell death, suggesting that some FLT3/ITD AML may not be addicted to FLT3
signaling. In addition, the response rate for diagnostic specimens is clearly higher for the least
selective of the drugs due to the cytotoxic effect. These results indicate the potential
therapeutic use of FLT3 inhibitors in patients with newly diagnosed FLT3-mutant AML might
be less likely to respond clinically to highly selective FLT3inhibition [17].
4

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Cl
H
N
HN
HN
CF
3
O
O
N
N
O
N
O
O
N
HN
O
O
Midostaurin (PKC412, 1)
Sorafenib (2)
O
N
N
N
O
S
O
N
O
N
N
H
H
Quizartinib (AC220, 3)
Figure 1. Structures of FLT3 inhibitors in the clinic.
Another significant result was that patients achieving a response to 3 often developed
resistance-conferring point mutations (i.e., most commonly at D835 and less frequently at a
“
gatekeeper” residue, phenylalanine 691 (F691)) [18,19]. In addition, the emergence of
resistant mutations has been reported in relapsed AML patients with FLT3-ITD treated with 1
20] and 2 [21,22]. These clinical results suggest that secondary mutations conferring
[
resistance were clinically problematic, which has spurred the development of new
tyrosine-kinase inhibitors (TKIs) with activity against drug-resistant FLT3-ITD mutations
[
23].
Using structure-based design, we previously developed a novel class of
-phenyl-1H-5-pyrazolylamine derivatives (Figure 2) as a versatile template for the
development of specific kinase inhibitors and successfully identified
3
a
sulfonamide-substituted series of benzamides (Structure I) that inhibit both FLT3 and cellular
proliferation [24,25]. Using rational design, we identified a second sulfonamide-substituted
5

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series of pyrimidines (Structure L) [25] and two carbamate-substituted series of benzamides
and pyrimidines (Structures J and M, respectively) [26] as potent inhibitors of FLT3. Recently,
we developed another series of 3-phenyl-1H-5-pyrazolylamine-based inhibitors capable of
greater inhibition of in vitro AML MOLM-13 cell growth and with a prolonged duration of in
vivo action. In the previous study, two urea-substituted series of benzamides (Structure K) and
pyrimidines (Structure N) were reported (Figure 2) [27,28]. However, these
pyrazolylamine-based inhibitors were not orally active and exhibited moderate inhibitory
activity toward drug-resistant FLT3-ITD mutations [24,28]. Both drawbacks could limit the
future development of this pyrazole class of inhibitors for the treatment of AML patients in
clinical trials.
NH
N
NH
N
NH
R
2
NH
R
2
O
H
O
N
O
O
n
HN
R
S
XR
O
J
X = O
n = 0 or 1
I
K X = NR3
NH
N
N
NH
N
NH
N
Rational design
S
O
3
R2
R
R
2
N
N
R1
N
H
H
N
O
1
R3
NHC(O)OR3
2
L
R = 3-NHSO
2
1
M R = 4-CH
1
3 4
N R = 4-(CH ) NHC(O)NR R
2
n
(n = 0 or 1)
Figure 2. Identification of 5-phenyl-thiazol-2-ylamine derivatives as FLT3 inhibitors.
We supposed that little or no oral absorption for this class of pyrazole compounds may
be due to the presence of more hydrogen-bond donors in the molecules. To continue to
develop potent and orally active FLT3 inhibitors, we rationally designed a second class of
6

ACCEPTED MANUSCRIPT
5
-phenyl-thiazol-2-ylamine-based inhibitors (Structure O, Figure 2). The approach can
remove a hydrogen-bond donor in the pyrazole ring. In this study, the structure-activity
relationships (SAR), in vitro properties and pharmacokinetics of a series of pyrimidine
derivatives are reported. This study has led to the discovery of urea 7h, which is a
multi-targeted kinase inhibitor with significant in vitro potency and in vivo efficacy against
FLT3-ITD–expressing MOLM-13 and MV4;11 cell lines and FLT3-ITD/D835Y–expressing
3
2D cell line [29]. These promising results demonstrate the potential of 7h as a drug
candidate for further preclinical and clinical development.
2
.
Results and discussion
.1. Chemistry
The general synthetic route to 5-phenyl-thiazol-2-ylamine pyrimidines 7-10 (Table 1) is
2
shown in Scheme 1. The synthesis began with the preparation of 5-phenyl-thiazol-2-ylamine 4
according to a previously published protocol [30]. Treatment of 5-phenyl-thiazol-2-ylamine 4
with 4,6-dichloropyrimidine in the presence of NaH and THF at 0 ° C afforded
o
4
-monosubstituted pyrimidine derivatives 5. The treatment of these derivatives with 2 amines
in pyridine at 80 °C yielded 4,6-disubstituted pyrimidines 6, which were coupled with phenyl
isocyanates (or heteroaryl carbamic acid 4-nitro-phenyl esters), benzenesulfonyl chloride,
benzyl chloroformate, or benzoyl chloride to afford the final pyrimidine ureas 7 (except for
Boc-7b, -7d and -7i), sulfonamide 8, carbamate 9 or benzoyl 10, respectively. Deprotection of
7

ACCEPTED MANUSCRIPT
Boc-7 using TFA yielded final products 7b, 7d and 7i.
The synthetic route to 5-phenyl-thiazol-2-ylamine benzamide 12 (Table 1) is shown in
Scheme 1. The benzoyl chloride derivative with a water-solubilizing substituent was prepared
according to a modified literature procedure [31]. The benzoyl chloride with a morphine
group was employed to acylate amine 4 in pyridine to yield amide 11. Intermediate 11 was
coupled with (5-methyl-isoxazol-3-yl)-carbamic acid 4-nitro-phenyl ester to afford urea 12.
N
S
NH
2
NH
2
Cl
N
N
N
N
N
NH
2
a
b
S
+
N
S
HN
N
HN
N
H
2
N
Cl
4
R1
Cl
5
6
e
c, d
N
O
N
O
H
c
NH
N
N
R2
NH
S
N
O
N
S
O
O
O
O
N
S
O
N
N
HN
2
H N
H
H
1
1
12
N
R1
N
2
3
7
8
9
R = -C(O)NHR
2
R = -SO
2
R3
2
3
R = -C(O)OR
R = -C(O)R3
2
10
o
Scheme 1. Reagents and conditions: (a) NaH, THF, 0 °C; (b) 2 amines, pyridine, 80 °C; (c) pyridine, phenyl
isocyanate or (5-alkyl-isoxazol-3-yl)-carbamic acid 4-nitro-phenyl ester for 7 or 12, benzenesulfonyl chloride for
8
, benzyl chloroformate for 9, benzoyl chloride for 10, rt; (d) CF COOH, rt for 7b, 7d and 7i; (e) pyridine,
3
benzoyl chloride with a morphine group, rt.
2
.2. In vitro pharmacology and rat oral exposure assay
As shown in Table 1, the target compounds were tested against FLT3, vascular
endothelial growth factor receptor 2 (VEGFR2), and Aurora A kinase (in-house kinas assays).
In addition, these compounds were further evaluated against the mutant FLT3 AML cell line
MOLM-13 and oral exposure in rats. The first compound 7a, which had a 4-methylpiperazine
1
2
at R and a phenyl substituent at R , moderately inhibited FLT3 (IC = 0.079 µM) and
5
0
8

ACCEPTED MANUSCRIPT
VEGFR2 (IC = 0.16 µM) but not Aurora A (IC > 10 µM). Lead 7a potently inhibited the
5
0
50
growth of MOLM-13 cells (GI = 0.024 µM) but suffered from undetectable drug levels in
5
0
rat plasma after oral dosing (10 mg/kg). Conserving the phenyl urea moiety, hydrogen (7b),
para-chlorination (7c) and meta-chlorination (7d) bearing different water-solubilizing
substituents increased the inhibitory activity against VEGFR2 (IC = 0.031 to 0.053 µM) and
5
0
Aurora A (IC = 0.095 to 0.27 µM) but resulted in no improvement in the oral absorption of
5
0
the compounds.
Table 1. Inhibition of enzymes and cell proliferation with thiazole analogues.
H
H
N
H
N
N
R2
N
O
N
N
N
O
S
HN
O
S
HN
N
O
N
O
R1
1
2
2
3
7
R = -C(O)NHR
2
3
8
9
1
R = -SO R
2
2
3
R = -C(O)OR
2
3
0 R = -C(O)R
*
IC (µM)
GI (µM)
Rats, PO 10 mg/kg
5
0
50
1
3
Compd
R
R
＊
wt-FLT3 VEGFR2 Aurora A MOLM-13
AUC(0-inf) ng/mL hr
7
7
a
N
N
-C
6
6
H
H
5
0.079
0.073
0.32
0.16
0.053
0.043
>10
0.15
0.024
0.016
0.009
undetectable
undetectable
undetectable
b
N
NH
2
-C
5
7
c
N
N
4-Cl-C
6
6
H
5
5
-
-
0.095
N
NH
3-Cl-C
H
0.020
0.010
0.031
0.015
0.27
0.024
0.001
undetectable
313
7
d
N
O
O
O
O
O
7
e
N
N
N
N
0.056
CH
3
N
7
f
N
N
0.049
0.022
0.038
0.014
0.01
0.055
0.025
0.041
0.038
0.032
0.038
0.003
0.014
0.002
0.003
632
—
2 5
C H
N
N
N
7
g
4 9
C H
7
h
N
N
1075
C
C
2
2
H
H
5
N
NH2
0.007
9
undetectable
7
i
5

ACCEPTED MANUSCRIPT
N
O
7
j
N
N
N
N
0.033
0.015
0.036
0.002
0.14
—
—
8
-C H
6 5
0.52
0.17
0.043
9
N
N
N
N
-CH
2
C
6
H
5
0.083
0.85
0.12
>1.0
0.37
0.25
0.042
0.43
0.32
0.14
0.025
0.080
3.8
0.003
0.019
0.21
66
—
—
—
—
—
1
0
2
-C
H
6 5
1
0.58
1
0.037
0.044
0.069
0.055
0.056
0.004
2
3
>10
*
IC and GI values are the means of at least two independent experiments.
5
0
50
2
Compounds 7a-d, which contain a phenyl ring at R , exhibit potent inhibition against
AML cell line MOLM-13. However, these compounds are not comparable to FLT3 inhibitor 3
2
(
MOLM-13 GI = 4 nM) and cannot be orally absorbed. To overcome these problems, the R
50
substituent of 7a was changed from a phenyl group to a 5-alkyl-isoxazole group. According to
the previous results from the development of 3-phenyl-1H-5-pyrazolylamine-based inhibitors,
the terminal isoxazole ring of the urea maintains or increases the potency in enzymatic and
cellular assays and improves the drug-like physical properties, such as solubility and
2
7
lipophilicity (calculated LogP and CLogP) compared to the terminal phenyl ring of the urea.
As expected, 5-methyl-isoxazole 7e increased the inhibitory activity against wt-FLT3 (IC =
5
0
0
.010), VEGFR2 (IC = 0.015), Aurora A (IC = 0.056) and MOLM-13 cells (GI = 0.001
50 50 50
µM) and exhibited a considerable increase in oral exposure and oral bioavailability (F% =10).
-Ethyl-isoxazole 7f was 5 times less potent against FLT3 (IC = 0.049 µM) but maintained
5
5
0
its potency against VEGFR2 and Aurora A kinases relative to 5-methyl-isoxazole 7e. In
1
0

ACCEPTED MANUSCRIPT
addition, inhibitor 7f potently inhibited the growth of MOLM-13 cells (IC = 0.003 µM) and
5
0
had a positive effect on the pharmacokinetic profile (F% = 18). The bulky
-tert-butyl-isoxazole (7g) had a detrimental effect on the MOLM-13 inhibitory potency (GI50
5
=
0,014 µM) compared to methyl analogue 7e and ethyl analogue 7f.
Next, we examined the effects of water-solubilizing substituents on the 4-position of the
pyrimidine ring and compared the potencies of compounds 7h-i with N-methylpiperazine
analogue 7f. N-ethylpiperazine 7h exhibited a potency that was similar to N-methylpiperazine
7
f in the enzymatic and cellular assays with slight improvements in the oral exposure and F%
20. In contrast, 7i bearing a primary pyrrolidin-3-ylamine group produced extremely low
=
circulating plasma levels but maintained its potency in the enzymatic and cellular assays. The
hydrogen-bond donor number may partially account for the difference in the oral absorption
between N-ethylpiperazine 7h and pyrrolidin-3-ylamine 7i. Although the more bulky
5
-cyclopropyl-isoxazole 7j was approximately as potent as 5-ethyl-isoxazole group 7h in all
of the in vitro assays, the preliminary toxicity study in mice indicated that compound 7j was
more toxic than 7h. Therefore, the pharmacokinetic profile of 7j was not further studied in
rats. The preliminary toxicity study for compound testing was performed as previously
2
7
described.
Previous
sulfonamide-substituted series of pyrimidines (Structure L) and the carbamate-substituted
studies
indicated
that
the
3-phenyl-1H-5-pyrazolylamine-based
2
5,26
series of pyrimidines (Structure M) were potent and selective FLT3 inhibitors (Figure 2).
11

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An extensive SAR study was carried out by replacing the urea moiety of 7 with
phenyl-substituted sulfonamide, carbamate and amide moieties to yield analogues 8, 9 and 10,
respectively. Sulfonamide 8 was significantly less potent against the FLT3 and MOLM-13
cells as well as Aurora A (IC = 0.043 µM). Carbamate 9 exhibited only moderate inhibition
5
0
of FLT3, VEGFR2 and Aurora A kinases (IC = 0.083 to 0.32 µM) and suffered from low
5
0
oral exposure in rats. However, 9 still exhibited a GI value of 0.003 µM against MOLM-13
5
0
cells. Amide 10 moderately inhibited Aurora A, weakly inhibited FLT3 and VEGFR2 (IC >
5
0
0
.80 µM), and potently inhibited MOLM-13 cell growth (GI = 0.019 µM). The ability of 8
50
and 10 to inhibit the growth of MOLM-13 cells was not comparable to FLT3 inhibitor 3.
Therefore, the pharmacokinetic profiles of these compounds were not determined.
After optimizing the urea-substituted series of pyrimidines (compounds 7, Structure O),
we investigated the effect of the urea-substituted series of benzamides. Benzamide 12
effectively inhibited Aurora A (IC = 0.025 µΜ) but not FLT3, VEGFR2 and MOLM-13 cell
5
0
growth. Therefore, further modification of lead 12 was not carried out. In comparison to FLT3
inhibitors 1, 2 and 3, which are currently in clinical development (Table 1), the inhibition of
FLT3-mutant MOLM-13 cell growth by some of the potent urea compounds (7) is comparable
to 3 (GI = 4 nM) and greater than 1 and 3 (GI = 55 and 56 nM, respectively). Among these
5
0
50
potent compounds (7), compound 7h exhibited better oral exposure and oral bioavailability in
rats, and therefore, this compound was chosen for more extensive in vitro and in vivo
pharmacokinetic and antitumor efficacy studies.
1
2

ACCEPTED MANUSCRIPT
2
.3. Kinase inhibition profiles of 7h
To better understand the enzyme inhibition by the potent compound, 7h was assayed
against 73 protein kinases covering the major oncogenic kinases of the human protein kinome
at a concentration of 0.1 µM. As shown in Table 2, 7h inhibited 28 kinases by more than 50%
at 0.1 µM out of a panel of 73 tested kinases. Among these kinases, 18 kinases were potently
inhibited by more than 80% including FLT3 (98%), PDGFRα (98%), SRC (97%), LYN A
(96%), Aurora A (96%), RET (95%), CSF1R (94%), LCK (92%), and PDGFRβ (91%).
Therefore, this kinase inhibition profile indicated that compound 7h is a multi-targeted kinase
inhibitor that inhibits certain receptor tyrosine kinases, which include Aurora kinase A and B,
macrophage-colony stimulating factor receptor (CSF1R), FMS-like tyrosine kinase-3 (FLT3),
platelet-derived growth factor receptors (PDGFRα and PDGFRβ), proto-oncogene
tyrosine-protein kinase receptor (RET), Src-family non-receptor tyrosine kinases and the
receptors of vascular endothelial growth factor (VEGFR type 2 and 3).
Table 2. Kinase inhibition profile of compounds 7h.
0
.1 µM
0.1 µM
% inhibition
Kinase
Kinase
%
inhibition
FLT3
PDGFRα
SRC
98
VEGFR2
AURKB
TRKA
VEGFR3
BLK
85
85
82
80
78
72
66
98
97
96
96
95
94
LYN A
AURKA
RET
YES1
CSF1R
ABL1
RET
V804L
LCK
92
65
1
3

ACCEPTED MANUSCRIPT
PDGFRβ
91
KIT
64
RET
V804L
PDGFRα
V561D
8
9
63
FGR
89
88
MEK1
IRAK4
62
61
LYN B
FLT3
D835Y
RET
8
8
7
6
Tie2
61
51
ABL1
T315I
Y791F
2
.4. Antiproliferative effects of 7h on leukemia cell lines
The activity of 7h was evaluated in a panel of leukemic cell lines (Table 3). Potent
anti-proliferative activity of 7h was observed in FLT3-driven MOLM-13 and MV4;11 AML
cell lines containing the FLT-ITD-activating mutation. The IC50 values of 7h for the
MOLM-13 and MV4;11 cells were 0.002 and 0.001 nM, respectively. Compound 7h was
weakly cytotoxic against U937 human leukemic monocyte lymphoma cells (not expressing
FLT3) [32] (GI = 1.1 µM). In contrast, the IC values for the inhibition of RS4;11 AML
5
0
50
cells (expressing native FLT3) [33] and K562 human chronic myeloid leukemia cells
expressing wt-BCR/ABL) [11] were between 0.1 and 0.2 µM. Among the FLT3 inhibitors
(
shown in Table 3, the growth inhibitory activity of 7h against the MOLM-13 and MV4;11
AML cell lines was comparable to that of 3, which is a specific FLT3 inhibitor.
Multi-targeted kinase inhibitors 1 and 7h appeared to induce higher growth inhibition against
U937 and RS4;11.
Table 3. Inhibition of leukemia cell growth by 7h and 1-3.
GI (µM)
50
*
Cell line
Characterization
7
h
1
2
3
MOLM-13 AML-FLT3-ITD (heterozygous)
0.002
0.055
0.056
0.004
1
4

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MV4;11
U937
AML-FLT3-ITD (homozygous)
AML-FLT3-negative
0.001
1.1
0.038
1.4
0.043
3.4
0.003
>20
2.2
RS4;11
ALL-wt-FLT3 (homozygous)
CML-Bcr-Abl FLT3-negative
0.11
0.19
0.40
>20
9.3
K562
7.3
>20
*
AML, acute myelocytic leukemia; ALL, acute lymphoblastic leukemia; CML, chronic myelogenous leukemia
2
.5 In vitro activity of 7h against clinically relevant drug resistance caused by FLT3-ITD KD
mutants
Due to its promising potency toward the FLT3-driven AML cell lines, the activity of 7h
against four different KD mutants of FLT3-ITD, which confer resistance to 3 [20-23]. In this
study, we first examined the cellular GI values of 3 and 7h against 32D cells expressing
5
0
FLT3-ITD and its KD mutants D835V/Y/F and F691L [29]. As shown in Table 4, compounds
and 7h potently inhibited proliferation of native FLT3-ITD with a GI value of 10 pM. The
3
5
0
cell proliferation–based assays indicated that the sensitivity toward 7h differed significantly
for inhibiting FLT3-ITD D835Y (GI = 0.006 µM), D835V (GI = 0.004 µM), D835F (GI
5
0
50
50
=
0.038 µM) and F691L (GI 50 = 0.071 µM). These results are similar to the sensitivity
profiles of 3 against FLT3-ITD D835Y/V/F and F691L, which exhibited GI values of 0.016,
5
0
0
.027, 0.15 and 0.22 µM, respectively, for the four mutants. Along with the results from Table
, 7h exhibits very potent in vitro growth inhibitory effects (GI < 10 nM) against FLT3-ITD
4
5
0
D835Y/V and is 3- to 6-fold more potent than 3 at inhibiting proliferation of these FLT3-ITD
mutants, as shown in Table 4.
Table 4. Activity of 3 and 7h against 32D cells expressing native and drug-resistant mutants of FLT3-ITD.
GI (µM)*
5
0
Cell line
7
h
3
1
5

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ITD alone
0.00001
0.006
0.004
0.038
0.070
0.00001
ITD-D835Y
ITD-D835V
ITD-D835F
ITD-F691L
0.016
0.027
0.15
0.22
*
GI values are the means of three independent experiments.
50
Because inhibitor of 7h exhibits cytotoxicity in 32D cells expressing FLT3-ITD KD
mutants, it was important to verify that the mechanism of cell death was directly linked to the
inhibition of FLT3. Next, we analyzed the activity of 7h against wild-type, ITD, ITD D835Y
or F691L mutants of FLT3 in a biochemical assay for the phosphorylation of FLT3 (Figure 3).
A western blot was performed using HEK293T cells engineered to express FLT3-ITD/D835Y
or FLT3-ITD/F691L [27]. According to the cell-based proliferation assay, the phosphorylation
of all of the FLT3-WT, FLT3-ITD, FLT3-ITD/D835 and FLT3-ITD/F691L was inhibited by
compound 7h with IC values of approximately 10 nM.
5
0
FLT3-WT
FLT3-ITD/F691L
0
0.1
1
10 100 1000 (nM)
pFLT3
0
0.1
1
10 100 1000 (nM)
pFLT3
FLT3
FLT3
ß-actin
ß-actin
FLT3-ITD
FLT3-ITD/D835Y
0
0.1
1
10 100 1000 (nM)
pFLT3
0
0.1
1
10 100 1000 (nM)
pFLT3
FLT3
FLT3
ß-actin
ß-actin
Figure 3. HEK293T cells engineered to express FLT3-WT, FLT3-ITD, FLT3-ITD/D835Y or FLT3-ITD/F691L
were analyzed. The HEK293T-FLT3 cells were treated with compound 7h at various concentrations for 1 hr, and
the FLT3 ligand (50 ng/mL) was added for 5 min to prepare the cell lysate for detection of changes in FLT3
phosphorylation by western blot analysis.
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6

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2
.6. Pharmacokinetic and in vivo efficacy studies of 7h
Table 5 shows the PK data obtained from the evaluation of 7h in male Sprague–Dawley
rats and ICR mice. In both species, 7h exhibited high volumes of distribution (V = 8.2 L/kg
ss
in rats and 21 L/kg in mice) and plasma clearances (Cl = 31.6 mL/min/kg in rats and 82
mL/min/kg in mice) after intravenous (IV, 2 mg/kg) administration. 10 mg/kg was orally
administered as a solution containing 22% hydroxypropyl-β-cyclodextrin in water and
absorbed with a moderate half-life (t1/2 = 4.3 hr) in rats and a shorter half-life (t1/2 = 1.8 hr) in
mice. Both the Cmax (122 ng/mL) and AUC (1075 ng/mL*hr) in rats were similar to those in
mice, and the oral bioavailability ranged from 20% in rats to 35% in mice. With favorable
pharmacokinetic properties and excellent cellular potency against AML cell lines, 7h was
appropriate for continued investigation in in vivo studies including the MV4;11 tumor
xenograft model, MOLM-13 myeloproliferative mouse model and FLT3-ITD/D835Y tumor
xenograft model (Figure 4).
Table 5. Pharmacokinetic profile of compound 7h.
IV (dose: 2 mg/kg)
CL Vss
Species (mL/min/kg) (L/kg)
PO (dose: 10 mg/kg)
Cmax AUC(0-inf)
(hr) (ng/mL) (ng/mL*hr) (%)
T1/2
F
Rats
31.6
82
8.2
21
4.3
1.8
122
168
1,075
942
20
35
Mice
In a MV4;11 tumor xenograft model (Figure 4a), compound 7h was administered orally
at doses of 10 and 25 mg/kg qd on days 1-5 and 8-12. By the end of this period, the MV4;11
3
tumors reached an average volume of 200 mm . At 10 or 25 mg/kg, the tumor growth was
1
7

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inhibited for the first 5 days. After this period, complete disappearance of the tumor mass was
observed after dosing was halted. Treatment with 10 or 25 mg/kg resulted in 6 of 7 and 7 of 7
treated animals with complete regression (CR), respectively, during the 48 days of
post-treatment observation. Treatment with 3, which is a clinically active FLT3 inhibitor, also
exhibited substantial activity with a once daily oral dosing at 10 mg/kg, and complete
regression was observed in 6 of the 7 treated animals, which comparable to the efficacy of 7h
at the same dose.
To determine whether 7h is also efficacious in a MOLM-13 xenograft model, we used a
myeloproliferative mouse model where the MOLM-13 cells were IV inoculated into SCID
mice and allowed to proliferate for one day prior to treatment with either the vehicle, 3 or 7h
(Figure 4b). The median survival for the mice treated with the vehicle was 20 days. At a dose
of 10 mg/kg once a day using 3 and 7h, the median survival was extended to 33 and 29 days,
respectively. The median survival in the group treated with 7h at a daily dose of 25 mg/kg
was extended to 38 days, which is an increase of 90% relative to the vehicle control and twice
the percentage relative to treatment with 7h at a low dose (Figure 4b). FLT3 inhibitor 3
exhibited better efficacy than 7h in this animal model.
1
8

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A
C
3
2
2
1
1
000
500
000
500
000
3000
Vehicle
10 mg/kg
7h 25 mg/kg
h 10 mg/kg
Vehicle
3 10 mg/kg
3
2500
2000
1500
1000
500
7h 10 mg/kg
7
5
00
0
0
0
10
20
30
40
50
0
5
10
15
20
25
30
Days after treatment
Days after treatment
B
Vehicle
3
7
7
10 mg/kg
h 25 mg/kg
h 10 mg/kg
1
1
0
0
0
0
0
.2
.0
.8
.6
.4
.2
.0
percentage increase in
Regimen
Vehicle
median survival (days)
survival
20
33
38
29
0
3
7
, 10mg/kg
h, 25mg/kg
65
90
45
7h, 10mg/kg
0
10
20
30
40
50
60
Days after treatment
Figure 4. Comparison of antitumor efficacy of compounds
3
and 7h in xenograft mouse models. (A) Human
3
MV4;11 cells were implanted SC in nude mice. After the average size of the tumors reached to 200 nm , mice
were dosed with
n = 6-7/group). (B) MOLM-13 cells were IV inoculated into SCID mice. From the 2nd day after inoculation,
mice were administered 7h at 10 or 20 mg/kg or at 10 mg/kg or vehicle (qd, days 2-5 and 8-12) by oral gavage
n = 6/group). (C) Mice were implanted subcutaneously (SC) with 32D/FLT3-ITD-D835Y cells. When the
3 or 7h (qd, days 1-5 and 8-12) by oral gavage. The tumor size is expressed as the mean ± SEM
(
3
(
3
average tumor volume reached 200 mm in size,
3 or 7h was administered orally at a dose of 10 mg/kg with a 5
days on/2 days off schedule for 4 weeks. The tumor size is expressed as the mean ± SEM (n = 9/group).
Compound 7h was very active against FLT3-ITD AL mutant D835Y in both the
cell-based proliferation and biochemical assay for the phosphorylation of FLT3 (Table 4 and
Figure 3). To determine if the in vitro effects of 7h correlate with in vivo tumor growth
inhibition, the efficacy of 7h was examined against FLT3-ITD/D835Y tumor xenografts in
1
9

ACCEPTED MANUSCRIPT
nude mice (Figure 4c). Mice were implanted subcutaneously (SC) with
3
2D/FLT3-ITD-D835Y tumor cells, and treatment with compound 3 or 7h was initiated when
3
tumors reached an average volume of 200 mm . Compound 3 or 7h was administered orally at
a dose of 10 mg/kg with a 5 days on/2 days off schedule for 4 weeks. Compound 7h exhibited
a statistically significant TGI (68%) [34] in tumor-bearing mice treated with the drug
compared with vehicle-treated control mice. However, treatment with 3 at the same dose did
not yield a biologically significant TGI (32%). The drug-resistant xenograft model
demonstrates that 7h effectively reduced the tumor size with 4 weeks of treatment at an oral
dose of 10 mg/kg/day. 7h is well tolerated in mice at the effective dose.
3
. Conclusion
A rational design approach successfully identified a potent and orally bioavailable
compound (7h), which is a multi-targeted FLT3 inhibitor. In AML cells, 7h exhibited similar
in vitro potency and in vivo efficacy to known FLT3 inhibitor 3. Importantly, 7h appears to be
highly active against clinically relevant FLT3-ITD AL mutations, especially at the D835
residue (D835Y/V), and demonstrates in vivo efficacy in the FLT3-ITD/D835Y xenograft
model. These promising results indicate that 7h could be an effective first-line TKI therapy in
AML patients or second-line therapy for the treatment of clinical drug resistance.
4
. Experimental
2
0

ACCEPTED MANUSCRIPT
4
4
.1. Chemistry
.1.1. General methods
All commercial chemicals and solvents are reagent grade and were used without further
1
treatment unless otherwise noted. H 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 F254
silica gel glass backed plates (5 × 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 compounds was determined by a Hitachi 2000 series HPLC
system. Except for compound 12, purity of target compounds 7, 8, 9 and 10 were over 95%
based on reverse phase C column (Agilent ZORBAX Eclipse XDB-C18 5 µm, 4.6 mm ×
18
1
50 mm, Condition A) and reverse phase phenyl column (Waters XBridge Phenly 5 υ m, 4.6
mm × 150 mm, Condition B) under the following gradient elution condition: Mobile phase
A-acetonitrile (10% to 90%, 0 to 45 min) and mobile phase B-10 mM NH OAc aqueous
4
solution containing 0.1% formic acid (90% to 10%, 0 to 45 min). The flow-rate was 0.5
2
1

ACCEPTED MANUSCRIPT
ml/min and the injection volume was 20 µl. The system operated at 25 °C. Peaks were
detected at λ = 254 nm.
4
.1.2. N-[5-(4-aminophenyl)-1,3-thiazol-2-yl]-6-chloro-2-methylpyrimidin-4-amine (5)
To a solution of 5-(4-aminophenyl)-1,3-thiazol-2-amine 4 (1.0 equiv) and
,6-dichloro-2-methylpyrimidine (1.0 equiv) in THF at 0 °C was added NaH (60% in oil, 2.0
4
equiv) and the resultant mixture was stirred at room temperature for 4 h. The reaction was
o
cooled to 0 C and quenched with 2 N HCl to pH = 3. The mixture was partitioned between
THF and saturated NaHCO solution. The organic layer was separated, dried over MgSO and
3
4
concentrated in vacuo. Purification by flash column chromatography (eluted with
1
CH Cl /EtOAc/THF) yielded 5. H NMR (300 MHz, DMSO-d6): δ 11.77 (s, 1H), 7.54 (s,
2
2
+
1
H), 7.28-7.24 (m, 2H), 6.89 (s, 1H), 6.59-6.56 (m, 2H), 5.31 (s, 2H), 2.55 (s, 3H); MS (ES )
+
m/z calcd. for C H ClN S: 317.05; found: 318.1 (M+H ).
1
4
12
5
4
.1.3. General procedure for the preparation of compounds 6
To a solution of 5 (1.0 equiv) in pyridine at room temperature was added 2 amines (2.0
o
equiv) and the resultant mixture was stirred at 80 °C for 2 h. After cooling to room
temperature, the reaction mixture was evaporated to dryness and the residue suspended in
saturated NaHCO solution. The suspension was vigorously stirred at room temperature for 1
3
h and the resultant solid was collected by filtration. The crude product was used for the next
step without further purification.
2
2

ACCEPTED MANUSCRIPT
4
.1.4. General procedure for the preparation of urea analogues 7
To a solution of 6 (1.0 equiv) in pyridine at room temperature was added phenyl
isocyanate (1.1 equiv) or (5-alkyl-isoxazol-3-yl)-carbamic acid 4-nitro-phenyl ester (1.5
equiv). After 2 h of stirring at room temperature, the reaction was worked up as described for
compounds 6 to yield crude products 7, N-Boc 7b, N-Boc 7d and N-Boc 7i. The crude
products 7 were further purified by recrystallization with CH CN/CH OH to yield 7.
3
3
To a solution of N-Boc 7b N-Boc 7d or N-Boc 7i (1.0 equiv) in CH Cl at room
2
2
temperature was added trifluoroacetic acid (10 equiv). After 0.5 h of stirring at room
temperature, the reaction mixture was evaporated to dryness. The residue was suspended in
saturated NaHCO solution and the resulting suspension was vigorously stirred at room
3
temperature for 1 h. The resultant solid was collected by filtration and recrystallized from
CH CN/CH OH to yield 7b, 7d or 7i.
3
3
4
1
.1.4.1.
-[4-(2-{[2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl]amino}-1,3-thiazol-5-yl)phenyl]
-3-phenylurea (7a)
°
1
Mp 252–253 C (HCl salt). H NMR (300 MHz, DMSO-d6): δ 11.11 (s, 1H), 8.77 (s,
1
1
H), 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,
1
3
H), 6.05 (s, 1H), 3.50 (br s, 4H), 2.41-2.34 (m,7H), 2.22 (s, 3H); C NMR (100 MHz,
DMSO-d ) δ 26.1, 43.9, 46.2, 54.6, 82.6, 118.7, 119.1, 122.3, 126.2, 126.3, 129.3, 130.7,
6
2
3

ACCEPTED MANUSCRIPT
+
1
5
5
9
33.0, 139.2, 140.1, 152.9, 157.7, 158.5, 162.9, 165.6; MS (ES ) m/z calcd. for C H N OS:
26 28 8
+
00.63; found: 501.47 (M+H ); HRMS (ESI) calcd. for C H N OS: 501.2185; found:
2
6
29
8
+
01.2178 (M+H ); HPLC (condition A) t = 22.41 min, 99.5%, (condition B) t = 24.49 min,
R
R
9.1%.
4
.1.4.2.
N-[4-(2-[6-(4-aminopiperidino)-2-methyl-4-pyrimidinyl]amino-1,3-thiazol-5-yl)phenyl]-N'-ph
enylurea (7b)
°
1
Mp 269–270 C. H NMR (400 MHz, DMSO-d6): δ 8.80 (s, 1H), 8.70 (s, 1H), 7.64 (s,
1
H), 7.49-7.45 (m, 6H), 7.28 (t, J = 7.6 Hz, 2H), 6.97 (t, J = 7.2 Hz, 1H), 6.06 (s, 1H), 4.12 (d,
J = 12.0 Hz, 2H), 3.33 (br s, 2H, overlapping with water peak), 2.94 (t, J = 11.6 Hz, 4H), 2.41
1
3
(
s, 3H), 1.76 (d, J = 10.0 Hz, 2H), 1.16 (d, J = 10.4 Hz, 2H); C NMR (100 MHz, DMSO-d )
6
δ 19.1, 26.1, 32.3, 42.9, 56.5, 82.4, 118.7, 119.0, 122.1, 126.0, 126.2, 129.2, 130.7, 132.9,
1
39.5, 140.4, 153.1, 157.7, 158.5, 162.5, 165.7; HRMS (ESI) calcd. for C H N OS:
26 29 8
+
5
01.2185; found: 501.2184 (M+H ); HPLC (condition A) t = 21.51 min, 99.6%, (condition
R
B) t = 23.57 min, 98.1%.
R
4
.1.4.3.
N-(4-chlorophenyl)-N'-[4-(2-[6-(4-ethylpiperazino)-2-methyl-4-pyrimidinyl]amino-1,3-thiazol
-5-yl)phenyl]urea (7c)
°
1
Mp 256–257 C (HCl salt). H NMR (300 MHz, DMSO-d6): δ 11.10 (s, 1H), 8.84-8.81
(m, 2H), 7.63 (s, 1H), 7.49-7.43 (m, 6H), 7.32 (s, 1H), 7.29 (s, 1H), 6.03 (s, 1H), 3.48 (br s,
1
3
4
H), 2.40-2.30 (m, 9H), 1.01 (t, J = 7.2 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 12.4, 26.1,
6
2
4

ACCEPTED MANUSCRIPT
4
1
5
4.0, 52.1, 52.4, 82.6, 110.0, 119.2, 120.2, 125.3, 125.8, 129.1, 130.6, 133.1, 139.0, 139.1,
+
52.8, 157.7, 158.6, 162.9, 165.6; MS (ES ) m/z calcd. for C H ClN OS: 548.19; found:
2
7
29
8
+
+
49.2 (M+H ); HRMS (ESI) calcd. for C H ClN OS: 549.1952; found: 549.1948 (M+H );
2
7
30
8
HPLC (condition A) t = 25.92 min, 97.4%, (condition B) t = 28.18 min, 97.5%.
R
R
4
.1.4.4.
N-(3-chlorophenyl)-N'-(4-2-[(2-methyl-6-piperazino-4-pyrimidinyl)amino]-1,3-thiazol-5-ylph
enyl)urea (7d)
°
1
Mp 345–346 C. H NMR (400 MHz, DMSO-d6): δ 11.07 (s, 1Η), 9.01 (s, 1H), 8.99 (s,
1
1
H), 7.72-7.65 (m, 2H), 7.57-7.50 (m, 4H), 7.32-7.27 (m, 2H), 7.04-7.00 (m, 2H), 6.06 (s,
1
3
H), 3.43 (br s, 4H, overlapping with water peak), 2.76 (t, J = 4.8 Hz, 4H), 2.41 (s, 3H); C
NMR (100 MHz, DMSO-d ) δ 26.1, 45.2, 45.7, 82.3, 117.1, 117.9, 119.2, 121.6, 125.4, 126.2,
6
+
1
30.7, 130.8, 133.0, 133.6, 139.3, 142.1, 153.0, 157.7, 158.5, 162.9, 165.6; MS (ES ) m/z
+
calcd. for C H ClN OS: 520.16; found: 521.1 (M+H ); HRMS (ESI) calcd. for
2
5
25
8
+
C H ClN OS: 521.1639; found: 521.1636 (M+H ); HPLC (condition A) t = 24.80 min,
25
26
8
R
9
7.6%, (condition B) t = 27.09 min, 99.0%.
R
4
.1.4.5.
N-(5-methyl-3-isoxazolyl)-N'-[4-(2-[2-methyl-6-(4-methylpiperazino)-4-pyrimidinyl]amino-1,
-thiazol-5-yl)phenyl]urea(7e)
3
2
5

ACCEPTED MANUSCRIPT
Mp 344–345 C. H NMR (400 MHz, DMSO-d6): δ 11.15 (s, 1H), 9.46 (s, 1H), 8.92 (s, 1H),
°
1
7
.67(s, 1H), 7.50 (ABq, ꢀνAB = 13.4 Hz, J = 8.8 Hz, 4H), 6.55 (s, 1H), 6.04 (s, 1H), 3.50 (t,
AB
13
J = 4.8 Hz, 4H), 2.41 (s, 3H), 2.37 (m, 7H), 2.21 (s, 3H); C NMR (75 MHz, DMSO-d ) δ
6
1
1
5
2.6, 26.0, 43.9, 46.2, 54.6, 82.6, 96.0, 119.5, 126.3, 126.9, 130.5, 133.3, 138.3, 151.7, 157.7,
+
58.6, 159.1, 162.9, 165.6, 169.7; MS (ES ) m/z calcd. for C H N O S: 505.20; found:
2
4
27
9
2
+
+
06.2 (M+H ); HRMS (ESI) calcd. for C H N O S: 506.2087; found: 506.2082 (M+H );
2
4
28
9
2
HPLC (condition A) t = 19.85 min, 100%, (condition B) t = 21.31 min, 98.3%.
R
R
4
1
.1.4.6.
-(5-ethyl-1,2-oxazol-3-yl)-3-[4-(2-{[2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl]amin
o}-1,3-thiazol-5-yl)phenyl]urea Diformate (7f)
°
1
Mp 326–327 C. H NMR (400 MHz, DMSO-d6): δ 9.83 (s, 1H), 9.29 (s, 1H), 8.29 (s,
2
H), 7.75 (s, 1H), 7.58 (s, 4H), 6.63 (s, 1H), 6.14 (s, 1H), 5.53 (br s, 1H), 3.59 (s, 4H), 2.79 (d,
1
3
J = 6.0 Hz, 2H), 2.58 (s, 3H), 2.33 (s, 3H), 1.29 (s, 3H); C NMR (75 MHz, DMSO-d ) δ
6
11.9, 20.2, 26.1, 43.9, 46.14, 54.6, 82.6, 94.7, 119.5, 126.3, 127.0, 130.5, 133.3, 138.3, 151.7,
+
1
57.7, 158.7, 156.0, 162.9, 165.6, 174.7; MS (ES ) m/z calcd. for C H N O S: 519.62;
25 29 9 2
+
found: 520.2 (M+H ); HRMS (ESI) calcd. for C H N O S: 520.2243; found: 520.2239
2
5
30
9
2
+
(
M+H ); HPLC (condition A) t = 21.89 min, 98.7%, (condition B) t = 23.25 min, 98.9%.
R R
4
.1.4.7.
N-[5-(tert-butyl)-3-isoxazolyl]-N'-[4-(2-[2-methyl-6-(4-methylpiperazino)-4-pyrimidinyl]amin
o-1,3-thiazol-4-yl)phenyl]urea (7g)
°
1
Mp 330–331 C. H NMR (400 MHz, DMSO-d6): δ 11.13 (s, 1H), 9.53 (s, 1H), 8.93 (s,
1
H), 7.67 (s, 1H), 7.53-7.47 (m, 4H), 6.51 (s, 1H), 6.04 (s, 1H), 3.50 (br s, 4H), 2.41 (s, 3H),
2
6

ACCEPTED MANUSCRIPT
1
3
2
2
1
5
.36 (t, J = 4.8 Hz, 4H), 2.21 (s, 3H), 1.30 (s, 9H); C NMR (100 MHz, DMSO-d ) δ 26.1,
6
8.8, 32.9, 43.7, 46.2, 54.6, 82.6, 92.9, 119.4, 126.3, 126.9, 130.4, 133.3, 138.3, 151.7, 157. 7,
+
58.6, 158.8, 162.8, 165.6, 180.7; MS (ES ) m/z Calcd for C H N O S: 547.25; found:
2
7
33
9
2
+
+
48.3 (M+H ); HRMS (ESI) calcd. for C H N O S: 548.2556; found: 548.2553(M+H );
2
7
34
9
2
HPLC (condition A) t = 25.28 min, 95.6%, (condition B) t = 26.66 min, 95.5%.
R
R
4
.1.4.8.
N-(5-ethyl-3-isoxazolyl)-N'-[4-(2-[6-(4-ethylpiperazino)-2-methyl-4-pyrimidinyl]amino-1,3-th
iazol-5-yl)phenyl]urea Dihydrochloride (7h)
°
1
Mp 259–260 C. H NMR (400 MHz, DMSO-d6): δ 11.12 (s, 1H), 9.48 (s, 1H), 8.95 (s,
H), 7.66 (s, 1H), 7.48 (ABq, ꢀνAB = 16.4 Hz, JAB = 8.8 Hz, 4H), 6.55 (s, 1H), 6.05 (s, 1H),
.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
1
3
=
1
3
7.2 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 11.9, 12.4, 20.2, 26.1, 44.0, 52.1, 52.4, 82.6,
6
9
4.7, 119.5, 126.3, 127.0, 130.4, 133.3, 138.3, 151.7, 157.7, 158.6, 159.0, 162.9, 165.6, 174.7;
+
+
MS (ES ) m/z calcd. for C H N O S: 533.23; found: 534.2 (M+H ); HRMS (ESI) calcd. for
26
31
9
2
+
C H N O S: 534.2400; found: 534.2390 (M+H ); HPLC (condition A) t = 22.75 min,
26
32
9
2
R
1
00%, (condition B) t = 24.41 min, 98.9%.
R
4
.1.4.9.
N-[4-(2-[6-(3-aminotetrahydro-1H-1-pyrrolyl)-2-methyl-4-pyrimidinyl]amino-1,3-thiazol-5-y
l)phenyl]-N'-(5-ethyl-3-isoxazolyl)urea Dihydrochloride (7i)
2
7

ACCEPTED MANUSCRIPT
°
1
Mp 267–268 C. H NMR (400 MHz, DMSO-d6): δ 8.94 (s, 1H), 7.63 (s, 1H), 7.48
(ABq, ꢀνAB = 14.0 Hz, JAB = 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,
1
3
1
5
1
H), 1.19 (t, J = 7.6 Hz, 3H); C NMR (100 MHz, DMSO-d ) δ 11.9, 20.2, 26.0, 45.0, 50.8,
6
4.7, 60.2, 82.3, 94.7, 119.4, 126.2, 126.9, 130.3, 133.2, 138.4, 151.8, 156.7, 158.8, 159.0,
+
+
60.9, 165.4, 174.6; MS (ES ) m/z calcd. for C H N O S: 505.20 ; found: 506.2 (M+H );
2
4
27
9
2
+
HRMS (ESI) calcd. for C H N O S: 506.2087; found: 506.2083 (M+H ); HPLC (condition
24
28
9
2
A) t = 19.72 min, 99.7%, (condition B) t = 21.87 min, 98.3%.
R
R
4
.1.4.10.
N-(5-cyclopropyl-3-isoxazolyl)-N'-[4-(2-[6-(4-ethylpiperazino)-2-methyl-4-pyrimidinyl]amino
-1,3-thiazol-5-yl)phenyl]urea (7j)
°
1
Mp 271–272 C. H NMR (400 MHz, DMSO-d6): δ 11.12 (s, 1Η), 9.44 (s, 1H), 8.88 (s,
H), 7.65 (s, 1H), 7.48 (ABq, ꢀνAB = 18.4 Hz, JAB = 8.8 Hz, 4H), 6.47 (s, 1H), 6.03 (s, 1H),
.48 (br s, 4H), 2.40 (br s, 7H), 2.34 (q, J = 7.2 Hz, 2H), 2.12-2.06 (m, 1H), 1.05-0.99 (m, 5H),
1
3
0
8
1
1
3
.89-0.85 (m, 2H); C NMR (75 MHz, DMSO-d ) δ 8.3, 8.5, 12.4, 26.1, 44.0, 52.0, 52.4,
6
2.6, 93.0, 119.5, 126.3, 126.6, 130.5, 131.9, 138.3, 151.6, 157.7, 158.7, 159.0, 162.9, 165.6,
+
+
74.6; MS (ES ) m/z calcd. for C H N O S: 545.23; found: 546.2 (M+H ); HRMS (ESI)
2
7
31
9
2
+
calcd. for C H N O S: 546.2400; found: 546.2390 (M+H ); HPLC (condition A) t = 22.79
2
7
32
9
2
R
min, 98.4%, (condition B) t = 24.65 min, 95.6%.
R
2
8