Discovery of novel N-(5-(tert-butyl)isoxazol-3-yl)-N′-phenylurea analogs as potent FLT3 inhibitors and evaluation of their activity against acute myeloid leukemia in vitro and in vivo

Article abstract of DOI:10.1016/j.bmc.2015.06.033

FLT3 inhibitors have been explored as a viable therapy for acute myeloid leukemia (AML). However, the clinical outcomes of these FLT3 inhibitors were underwhelming except AC220. Therefore, the development of novel FLT3 inhibitors with high potency against

Full text of DOI:10.1016/j.bmc.2015.06.033

Bioorganic & Medicinal Chemistry 23 (2015) 4333–4343
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel N-(5-(tert-butyl)isoxazol-3-yl)-N0-phenylurea
analogs as potent FLT3 inhibitors and evaluation of their activity
against acute myeloid leukemia in vitro and in vivo
Ying Xu a, , Ning-Yu Wang a, , Xue-Jiao Song a, Qian Lei a, Ting-Hong Ye a, Xin-Yu You a,b, Wei-Qiong Zuo a,
Yong Xia a, Li-Dan Zhang a,b, Luo-Ting Yu a,
⇑
a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu 610041, China
b College of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610065, China
a r t i c l e i n f o
a b s t r a c t
Article history:
FLT3 inhibitors have been explored as a viable therapy for acute myeloid leukemia (AML). However, the
clinical outcomes of these FLT3 inhibitors were underwhelming except AC220. Therefore, the develop-
ment of novel FLT3 inhibitors with high potency against both FLT3-WT and FLT3-ITD mutants are
strongly demanded at the present time. In this study, we designed and synthesized a series of novel
N-(5-(tert-butyl)isoxazol-3-yl)-N0-phenylurea derivatives as FLT3 inhibitors. SAR studies focused on the
fused rings led to the discovery of a series of compounds with high potency against FLT3-ITD-bearing
MV4-11 cells and significantly inhibitory activity toward FLT3. Among these compounds, N-(5-(tert-
butyl)isoxazol-3-yl)-N0-(4-(7-methoxyimidazo[1,2-a]pyridin-2-yl)phenyl)urea (16i), displayed accept-
able aqueous solubility, desirable pharmacokinetic profile and high cytotoxicity selectivity against
MV4-11 cells. This compound can inhibit phosphorylation of FLT3 and induce apoptosis in a concentra-
tion-dependent manner. Further in vivo antitumor studies showed that 16i led to complete tumor regres-
sion in the MV4-11 xenograft model at a dose of 60 mg/kg/d while without observable body weight loss.
This study had provided us a new chemotype of FLT3 inhibitors as novel therapic candidates for AML.
Ó 2015 Published by Elsevier Ltd.
Received 22 March 2015
Revised 10 June 2015
Accepted 11 June 2015
Available online 19 June 2015
Keywords:
FMS-like tyrosine kinase 3 (FLT3)
Inhibitor
Acute myeloid leukemia
N-(5-(tert-Butyl)isoxazol-3-yl)-N0-
phenylurea
1. Introduction
of the tyrosine kinase domain, which are found in approximately
23% and 7% of patients with AML, respectively.7,8
FMS-like tyrosine kinase 3 (FLT3), a member of the class III
receptor tyrosine kinase family, is mainly expressed on hematopoi-
etic system, nervous system, gonads and placenta.1,2 In the
hematopoietic system, FLT3 is a key mediator of early haematopoi-
esis and plays several important roles in the proliferation, differen-
tiation and apoptosis of hematopoietic cells.3,4 Over-expression of
FLT3, or its constitutive activation, appears to play a major role in
the development and progress of leukemia, especially in acute
myeloid leukemia (AML).5 Actually, approximately 90% of AML
patients have been found over-expression of FLT3. While some of
these patient-derived AML cells exhibit over-expression of FLT3-
WT, others are found to harbor activating mutations of FLT3.6
There are two major classes of activating mutations of FLT3, inter-
nal tandem duplications (ITDs) in the juxtamembrane domain of
the receptor and ‘activation loop’ mutations in the activation loop
FLT3-ITD mutations trigger constitutive activation of FLT3,
which activated downstream signaling cascades and resulted in
survival and proliferation of leukemic cells.9 Besides, the over acti-
vation of FLT3 by FLT3-ITD mutants is relevant to a poor prognosis
and the complete remission rate of patients with FLT3-ITD muta-
tions is much lower than that of patients with FLT3-WT when trea-
ted with conventional chemotherapy like cytarabine and
anthracycline.10,11 Therefore, FLT3 presents us an attractive target
for the treatment of AML, especial patients with FLT3-ITD muta-
tions. And FLT3 inhibitors have emerged in a rash in recent years
for the treatment of AML patients.12
The first panel of FLT3 inhibitors nearly all belong to multi-ki-
nase inhibitors. Several of them, such as sunitinib (SU-11248),13
sorafenib (BAY-43-900),14 tandutinib (MLN518),15 lestaurtinib
(CEP-701),16 midostaurin (PKC412)17 and KW-244918 (Fig. 1) have
been advanced to clinical trials as potential drug candidates for
AML patients.19 However, the clinical outcomes of these FLT3 inhi-
bitors is far from being satisfactory, probably due to their inability
of completely and sustained inhibition of FLT3 in vivo.20 Thus, the
⇑
Corresponding author.
E-mail address: yuluot@scu.edu.cn (L.-T. Yu).
This author contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2015.06.033
0968-0896/Ó 2015 Published by Elsevier Ltd.

4334
Y. Xu et al. / Bioorg. Med. Chem. 23 (2015) 4333–4343
F
H
N
H
N
F
F
N
O
N
HN
O
N
H
O
O
N
Cl
N
N
H
F
O
N
NH
H
N
H
KW-2449
O
sunitinib
sorafenib
H
N
O
H
H
N
N
O
O
N
OH
OH
N
N
HN
O
N
N
O
N
H
H
O
O
O
N
O
N
N
O
CEP-701
MLN518
PKC412
Figure 1. Structure of known FLT3 inhibitors.
development of novel FLT3 inhibitors with high potency and selec-
tivity against both FLT3-WT and FLT3-ITD mutants are urgent
needed at the present time.
To date, AC220 (Fig. 2), a second generation selective FLT3 inhi-
bitor, is one of the most potential clinical candidate for AML.
Moreover, the excellent kinase selectivity implied that it repre-
sented an attractive new class of drug candidates for the treatment
of FLT3-ITD patients with relapsed and refractory AML.21 Our
research group aimed at developing novel second generation
O
H
N
H
N
N
N
O
O
N
N
O
S
AC220
H
N
H
N
W1
W6
O
W5
R
N
W3W2
W4
FLT3 inhibitors. In this study,
a series of novel N-(5-(tert-
O
butyl)isoxazol-3-yl)-N0-phenylurea derivatives (Fig. 2) were
designed via the principle of isostere based on AC220. Herein, we
describe the chemical synthesis, structure–activity relationship
(SAR) investigation and biologic evaluation of this new kind of
compounds.
a
Figure 2. Structure of AC220 and a.
position. As show in Table 1, a series of nitrogen-contained bicyclic
derivatives was first synthesized and evaluated for its FLT3 inhibi-
tory activity as well as the in vitro antiproliferative activity. 15a,
which contains an imidazo[1,2-a]pyridine substituent at phenyl,
was found to potently inhibit MV4-11 (IC50 = 3.09 nM) and FLT3
(65.7% inhibition at 100 nM). While 15b–15d, which contain imi-
dazo[1,2-a]pyrimidine substituent, imidazo[1,2-a]pyrazine sub-
stituent and imidazo[1,2-b]pyridazine substituent, respectively,
were about 15–50 fold less potent than 15a in with respect to
antiproliferative activity against MV4-11. This probably due to
the lower electron cloud density of these fused rings compared
with imidazo[1,2-a]pyridine ring. Having demonstrated the impor-
tance of the electron-rich group, we then focused on the oxygen-
contained and sulfur-contained bicyclic derivatives. Likewise, the
cytotoxicity against MV4-11 of 15e (benzo[b]thiophene,
2. Chemistry
The synthesis of our analogs was outlined in Schemes 1 and 2.
As shown in Scheme 1, the intermediates 2, 4, 6, 8 and 11 were pre-
pared through five different routes. Briefly, condensation of 1 with
4-nitrobenzaldehyde in the presence of [bis(trifluoroace-
toxy)iodo]benzene (PIFA) provided 2.22 Benzo[b]thiophene
intermediate 4 was formed using a Suzuki coupling of 3 and
1-bromo-4-nitrobenzene.23 Then benzofuran derivative 6 was
achieved by reacting 5 with 4-nitrobenzyl bromide in refluxing
DMF, while condensation of different heterocyclic 7 with 2-
bromo-40-nitroacetophenone gave various N-contained fused rings
8.24 Finally, coupling of 9 with bis(pinacolato)diboron followed by
H2O2 treatment yielded 10, which was alkylated with iodoalkane
or acylated with acyl chloride under basic condition provided 11.
Scheme 2 depicted the general reaction route of the target prod-
ucts. The key building blocks 12 were first reduced with iron pow-
der and then coupled with 5-tert-butyl-isoxazole-3-isocyanate 14,
which was achieved by reacting of 3-amino-5-tert-butyl-isoxazole
with triphosgene, to yield the ureas 15 and 16.25
IC50 > 900 nM),
15f
(benzofuran,
IC50 > 195.14 nM),
15g
(benzo[d]oxazole, IC50 = 82.57 nM) and 15h (benzo[d]thiazole,
IC50 = 172.31 nM) were dramatic decreased compared with 15a,
indicating that the N4 position of the imidazo[1,2-a]pyridine ring
was important for the activity. In addition, compared with com-
pounds 15f–15h, the potency of 15e was absolutely lost, which
might be due to its shortage of a hydrogen bond acceptor at the
bicyclic fragment.
Since the cellular activity of imidazo[1,2-a]pyridine substituent
15a was the best among the first batch of compounds, our next SAR
study focused on four positions of the imidazo[1,2-a]pyridine ring
2.1. Structure–activity relationship (SAR) analysis
In our initial SAR study, various fused rings were introduced to
the para-position of the phenyl to find a suitable scaffold in this

Y. Xu et al. / Bioorg. Med. Chem. 23 (2015) 4333–4343
4335
NH2
XH
OH
N
b
a
B
NO2
NO2
S
OH
S
X
2a
2b: X=S
1
3
: X=O
4
O
X1
X3
X2 X1
N
d
c
X2
H
R1
NH2
NO2
NO2
N
X3
N
O
R1
OH
7
8a: X1=X2=X3=CH
5
6
8b
: X1=N X2=X3=CH
8c: X2=N X1=X3=CH
8d: X3=N X1=X2=CH
O
N
R2
Br
HO
N
N
e
NO2
f or g
N
NO2
NO2
N
N
11a: R2=CH3
10
9
11b
11c
: R2=CH2CH3
: R2=COCH3
Scheme 1. Reagents and conditions: (a) (i) 4-nitrobenzaldehyde, grinding, rt; (ii) PIFA, grind, rt; (b) 1-bromo-4-nitrobenzene, Na3PO4ꢀ12H2O, 10% Pd/C (1.0 mol %), i-PrOH,
80 °C; (c) 4-nitrobenzyl bromide, K2CO3, DMF, reflux; (d) 2-bromo-40-nitroacetophenone, EtOH, reflux; (e) (i) bis(pinacolato)diboron, CH3COOK, PdCl2(dppf), 1,4-dioxane,
100 °C; (ii) H2O2, H2O; (f) CH3I or CH3CH2I, K2CO3, DMF; (g) DMAP, CH2Cl2, CH3COCl.
W6
W5
W6
W5
W1
W1
NCO
a
O
N
NO2
NH2
14
W
W
3W2
R
W4
3W2
R
W4
b
12
13
15a, 16
: W1=W3=N W2=W4=W5=W6=CR1
W5W6
15b: W1=W3=W6=N W2=W4=W5=CH
15c: W1=W3=W5=N W2=W4=W6=CH
R
W1
W
4W3
W2
15d
: W1=W3=W4=N W2=W5=W6=CH
O
N
O
15e: W2=S W1=W3=W4=W5=W6=CH
15f: W2=O W1=W3=W4=W5=W6=CH
N
N
H
H
15g
: W1=N W2=O W3=W4=W5=W6=CH
15h: W1=N W2=S W3=W4=W5=W6=CH
Scheme 2. Reagents and conditions: (a) Fe, NH4Cl, H2O/EtOH, reflux; (b) CHCl3, 60 °C.
(Fig. 3). Various derivatives of 15a together with their bioactivities
were presented in Table 2.
We then explored the influence of different substituents at C6
position. When this position was substituted with halogen groups,
the resulting compounds 16l (fluorine, IC50 = 20.94 nM), 16m
(chlorine, IC50 = 24.75 nM), 16n (bromine, IC50 = 44.48 nM) and
16o (iodine, IC50 = 98.55 nM) showed a 7–30 fold loss of potency
against MV4-11 compared with the unsubstituted counterpart
15a. And the potency against MV4-11 was decreased along with
the increasing size of the substituent. Furthermore, the antiprolif-
erative activity was reserved as the introduction of a methyl (16p,
IC50 = 4.08 nM) or an ethynyl (16q, IC50 = 8.62 nM) to this position,
while 5 fold loss of potency was observed when a cyclopropane
was introduced (16r, IC50 = 15.83 nM), which indicated that a rela-
tive large cycloalkyl group at this position was disadvantage to the
potency against MV4-11 and FLT3. Interestingly, the cell growth
inhibitory activities against MV4-11 of the electron-donating
derivatives 16p, 16q and 16r were higher than that of the elec-
tron-withdrawing derivatives 16l–16o, which was consistent with
the SAR of C7 position. These results further supported the conclu-
sion that an electron-rich bicyclic motif was important for the
antiproliferative activity as well as FLT3 inhibitory potency.
Lastly, in order to explore the substitution effect of C5 position
of imidazo[1,2-a]pyridine ring, we synthesized compounds 16s–
16t that contained different substituents at this position. The
bioactivity of 16s–16t at the cellular level was clearly decreased
relative to their counterpart 15a (Table 2), which suggested that
substitution at this position was not beneficial for increasing the
bioactivity against MV4-11.
The H atom at C8 position of imidazo[1,2-a]pyridine ring was
replaced by fluorine, chlorine, methyl and trifluoromethyl group,
yielding compounds 16a–16d, respectively. As show in Table 2,
compounds 16a (IC50 = 12.21 nM), 16b (IC50 = 352.76 nM), 16c
(IC50 = 248.42 nM) and 16d (IC50 > 900 nM) exhibited much lower
anti-proliferative and anti-FLT3 activities than their unsubstituted
counterpart 15a and the antiproliferative activity decreased as the
size of the substituent increasing. Moreover, it’s worth noting that
a trifluoromethyl introduced to this position led to an inactive com-
pound (16d IC50 > 900 nM), indicated that a bulky substituent at this
position was detrimental to the potency against MV4-11 and FLT3.
Next, a series of substituents were introduced in C7 position to
study their influence on the potency. The results show that the
introduction of chlorine (16e, IC50 = 5.91 nM), bromine (16f,
IC50 = 9.59 nM), methyl (16g, C50 = 0.79 nM) or ethynyl (16h,
IC50 = 3.46 nM) at this position was well tolerated. Moreover, the
methyl and ethynyl analogs 16g and 16h displayed a better inhibi-
tory activity in the cellular assay than their chlorine and bromine
counterparts 16e and 16f, suggesting that electron-donating sub-
stituent at this position was superior to electron-withdrawing sub-
stituent. Methoxy substituent analog 16i maintained excellent
anti-MV4-11 activity with an IC50 value of 2.84 nM while incorpo-
ration of ethoxy and acetoxy group at this position resulted in 4–10
fold decrease of potency compared with 16i (16j, IC50 = 11.59 nM
and 16k, IC50 = 29.39 nM).

4336
Y. Xu et al. / Bioorg. Med. Chem. 23 (2015) 4333–4343
Table 1
tissue, including leukemia and solid tumor, were treated with 16i
and the results are presented in Table 5. 16i potently inhibited
SAR of derivatives with varied fused rings
the growth of FLT3-ITD-bearing MV4-11 cells, with an IC50 value
R
O
O
N
of 0.003
activity against human acute monocytic leukemia THP-1 cells
and human lung cancer A549 cells (IC50 of 3.37 M and 5.61 M,
lM. While it just exhibited very weak antiproliferative
N
H
N
H
l
l
respectively). For other cancer cell lines, including K562, U937,
Jurkat, MCF-7, PANC-1, U251, HepG2, A875 and HCT116, 16i only
exhibited a negligible growth inhibition effect at the concentration
Compd
R
MV4-11
(IC50, nM)
FLT3
(inhibition at 100 nM, %)
AC220
—
0.68
69.3
of 20 lM (Table 5). Furthermore, the viability of MV4-11 cells was
N
N
attenuated along with increasing concentration of 16i and duration
of exposure (Fig. 4a). These data suggested that 16i was highly
selective for the human AML cell line MV4-11 and inhibited
MV4-11 cells proliferation in a dose- and time-dependent manner.
15a
3.09
65.7
N
N
N
N
15b
15c
170.68
44.83
19.9
35.5
N
N
2.4. Phosphorylation of FLT3 and apoptosis assays in MV4-11
cells
N
N
15d
59.05
30.9
To examine whether 16i inhibited phosphorylation of FLT3 in
intact cells, MV4-11 cells were treatment with increasing concen-
trations of 16i for 2 h, and the level of FLT3 as well as phosphory-
lated FLT3 was determined by Western blot (Fig. 4b). It was found
that 16i inhibited FLT3 phosphorylation in a dose-dependent man-
ner without observable influence on FLT3 expression. Then flow
cytometry assay was further performed to examine the apoptosis
inducing effect upon treatment with 16i for 48 h. As shown in
Figure 4c, 16i increased the percentage of apoptotic cells (positive
Annexin V-FITC and negative PI staining) in a dose-dependent
manner, and at a concentration of 100 nM, the early stage apopto-
sis rate and late stage apoptosis rate were 6.9% and 23%,
respectively.
N
15e
15f
15g
>900
3.8
S
195.14
82.57
11.5
21.8
O
N
O
N
15h
172.31
14.7
S
2.5. In vivo efficacy and immunohistochemical analysis
H
N
H
N
8
N
Finally, in vivo anti-AML activity of 16i was evaluated using the
FLT3-ITD-bearing MV4-11 xenograft model. Oral administration of
16i at dose of 10, 30 and 60 mg/kg once a daily were initiated when
the tumor grew to a volume of 100–200 mm3, and the tumor vol-
umes were measured every 3 days. As shown in Figure 5a, at the
dose of 60 mg/kg/d, 16i was able to yield complete regression of
established tumors during the dosing period. In addition, 16i sub-
stantially suppressed tumor growth in a dose-dependent manner
with inhibition rates of 51% and 87% at 10 mg/kg/d and
30 mg/kg/d, respectively. Moreover, no significant weight loss
(Fig. 5b) or any other obvious signs of toxicity were observed for
all of the 16i-treated mice during the whole study.
The effects of 16i on cancer cell proliferation and apoptosis
were also evaluated using immunohistochemical assays. Mice
bearing MV4-11 xenograft tumors were treated orally once daily
with 60 mg/kg/d of 16i, and tumor tissue were collected and ana-
lyzed 7 days later. Tumor tissues from the vehicle group were
stained strongly with Ki67, indicating a large number of highly
proliferative cells (Fig. 5c). Conversely, tumor tissues from the
16i-treated groups showed significantly fewer Ki67-positive cells.
Furthermore, the TUNEL assay of the 16i-treated groups showed
an obvious increase of apoptotic cells compared with the vehicle
group (Fig. 5c).
O
7
N
N
O
15a
6
5
Figure 3. The structure of compound 15a and the four regions our SAR study
focused on.
Collectively, the structural optimization and SAR studies led to
the discovery of a series of compounds which exhibited superior
potency against human AML MV4-11 cells and delightful inhibition
of FLT3. Then we studied the solubility of the most potent com-
pounds 15a, 16g, 16h and 16i in several organic solvent and water.
As show in Table 3, all of these compounds were well soluble in
organic solvent, while compound 16i was significantly more sol-
uble than compounds 15a, 16g and 16h in water. Thus, 16i was
selected to carry out further PK profile, in-depth in vitro and
in vivo anti-AML studies.
2.2. Pharmacokinetic assays
The preliminary pharmacokinetic characteristic of 16i following
oral administration to rats was analyzed. As shown in Table 4, at an
oral dose of 30 mg/kg, 16i demonstrated desirable maximum
plasma level (Cmax 3.29 mg/L), favorable drug exposure (AUC0–24h
14.32 mg/Lꢀh) and moderate apparent plasma half-life (T1/2
4.86 h), indicating that 16i was orally available for tumor xenograft
studies in vivo.
3. Conclusion
In this paper, we designed and synthesized a series of novel N-
(5-(tert-butyl)isoxazol-3-yl)-N0-phenylurea derivatives based on
the FLT3 inhibitor AC220 through bioisostere principle.
Structure–activity relationship studies demonstrated that an elec-
tron-rich fused ring at the phenyl was beneficial for the
2.3. In vitro cell growth inhibitory activity of 16i
To further study the growth inhibitory potencies of compound
16i, a panel of human cancer cell lines originated from different

Y. Xu et al. / Bioorg. Med. Chem. 23 (2015) 4333–4343
4337
Table 2
SAR of carried substitutions at the imidazo[1,2-a]pyridine
R
O
N
O
N
H
N
H
Compd
15a
R
MV4-11
(IC50, nM)
FLT3
(inhibition of 100 nM, %)
N
N
3.09
65.7
N
N
F
16a
12.21
38.9
Cl
N
N
16b
16c
16d
352.76
248.42
>900
12.5
13.6
2.5
N
N
CF3
N
N
Cl
Br
N
N
16e
16f
16g
5.91
9.59
0.79
44.2
39.5
69.0
N
N
N
N
N
16h
3.46
57.8
N
N
O
O
N
16i
16j
16k
2.84
57.4
44.2
27.4
O
O
N
N
11.59
29.39
N
N
N
16l
20.94
24.75
44.48
31.8
35.7
28.4
N
F
N
16m
16n
N
Cl
Br
I
N
N
N
16o
16p
16q
98.55
4.08
29.3
48.8
39.9
N
N
N
N
8.62
N
N
N
16r
15.83
30.5
(continued on next page)

4338
Y. Xu et al. / Bioorg. Med. Chem. 23 (2015) 4333–4343
Table 2 (continued)
Compd
R
MV4-11
FLT3
(IC50, nM)
(inhibition of 100 nM, %)
N
N
N
N
16s
16t
56.08
20.24
28.5
41.5
Cl
4. Experimental section
4.1. Chemistry methods
Table 3
The solubility of compounds 15a, 16g, 16h and 16i in different solvent
Compd
lg/mL
MeOH
EtOH
H2O
PBS
SPSS
1H and 13C NMR spectra were generated in DMSO-d6 on a
Bruker AVANCEi 400. Chemical shifts (d) were reported in ppm rel-
ative to Me4Si (internal standard), and coupling constants (J) were
reported in Hz. Low-resolution mass spectra (ESI) were performed
on a Bruker Amazon SL ESI mass spectrometer. HPLC conditions
15a
16g
16h
16i
5276
6827
5842
7460
4072
3340
4105
3149
NDa
NDa
NDa
10.2
36.6
NDa
NDa
NDa
1.47
NDa
26.7
303.6
a
ND: not detected.
were as follows: column, Agilent Eclipse Plus C18 3.5
4.6 mm ꢁ 150 mm; solvent system, ACN/H2O; flow rate
1.0 ml/min; UV detection, 270 nM; injection volume, 5 L; temper-
lM,
l
Table 4
Pharmacokinetic parameters of 16i in ratsa
ature, 30 °C. All tested compounds were purified until the purity
was P95%, detected by HPLC, NMR and ESI-MS.
Compd
Parameter
AUC0–24h (mg/
Lꢀh)
14.32
Cmax (mg/
L)
t1/2
(h)
Vdss (L/
kg)
CL (L/h/
kg)
4.1.1. General procedure for the preparation of intermediates
4.1.1.1. General procedure for the preparation of 2.
A mix-
16i
3.29
4.86
14.12
2.01
ture of 1 (1.0 equiv) and 4-nitrobenzaldehyde (1.0 equiv) was
blended thoroughly in pestle with mortar for 2 min. Then we
added [bis(trifluoroacetoxy)iodo]benzene (PIFA, 1.2 equiv) and
ground the reaction mixture for 3 min. After completion of reac-
tion, reaction mixture was triturated with hexane to remove
iodobenzene. Then we added cold saturated NaHCO3 to quench
the reaction and filtered the product. The solid thus obtained
was recrystallized with methanol to afford 2.
a
Sprague–Dawley rats were dosed at 30 mg/kg po (n = 3).
Table 5
Antiproliferative activities of 16i against various cell lines
Cell lines
Tumor type
IC50 (lM)
MV4-11
K562
U937
THP-1
Jurkat
A549
MCF-7
PANC-1
U251
Human acute myeloid leukemia
Human chronic myelogenous leukemia
Human leukemic monocyte lymphoma
Human acute monocytic leukemia
Human T lymphoma
Human lung cancer
Human breast cancer
Human pancreatic carcinoma
Human glioma
Human hepatic cancer
0.003
>20
>20
3.37
>20
5.61
>20
>20
>20
>20
>20
>20
4.1.1.2. General procedure for the preparation of 4.
To a
mixture of benzo[b]thiophen-2-ylboronic acid 3 (1.5 equiv) and
1-bromo-4-nitrobenzene (1.0 equiv) in isopropanol/H2O (v/v:
10:1), Na3PO4ꢀ12H2O (3.5 equiv) and 10% Pd/C (0.05 equiv) were
added, and the mixture was stirred under nitrogen at 80 °C for
4 h. After completion of reaction, the mixture was diluted with
H2O and acetic ether, and filtered through a filter. The filtrate
was separated into two layers and the aqueous layer was extracted
with acetic ether. Then the combined organic layers were dried
over MgSO4 and concentrated under vacuum. The residue was
purified by silica gel chromatography to give 4.
HepG2
A875
HCT116
Human melanoma
Human colorectal carcinoma
antiproliferative activity and the introduction of substituents in C6
and C7 position were more tolerated than in C5 and C8 position.
Four compounds (15a, 16g, 16h and 16i) showed superior potency
against FLT3-ITD-bearing MV4-11 cells (IC50 = 3.09, 0.79, 3.46 and
2.84 nM, respectively) and delightful inhibition of FLT3. Among
these compounds, 16i displayed well aqueous solubility, desirable
pharmacokinetic profile and high selectivity against MV4-11 cells.
Further in vivo anti-AML activity assays showed that 16i led to
complete tumor regression in the MV4-11 xenograft model at a
dose of 60 mg/kg/d. In addition, western blot, apoptosis and
immunohistochemical analysis demonstrated that 16i could
downregulate the phosphorylation of FLT3, induced apoptosis
and block the proliferation of MV4-11 cell in vitro and in tumor tis-
sue. Our preliminary optimization of this new chemotype of FLT3
inhibitors had provided us novel drug candidates for AML, and fur-
ther lead optimization as well as biologic studies are underway.
4.1.1.3. General procedure for the preparation of 6.
A solu-
tion of 2-hydroxybenzaldehyde (1.0 equiv), 4-nitrobenzyl bromide
(1.0 equiv), and K2CO3 (3.0 equiv) in DMF was stirred under reflux
overnight. After completion of reaction, the solvent was removed
and the residue was recrystallized with ethyl acetate to give 6.
4.1.1.4. General procedure for the preparation of 8.
A mix-
ture of pyridine amine 7 (1.0 equiv) and 2-bromo-40-nitroace-
tophenone (1.1 equiv) in ethanol was heated to reflux overnight.
The solution was then allowed to stand at room temperature for
2 h. The precipitate was collected by filtration, washed with
methanol and dried under vacuum to give 8.
4.1.1.5. General procedure for the preparation of 11a and
11b.
7-Bromo-2-(p-tolyl)imidazo[1,2-a]pyridine (9, 1.0 equiv),

Y. Xu et al. / Bioorg. Med. Chem. 23 (2015) 4333–4343
4339
Figure 4. (a) MV4-11 cells were treated with different concentrations of 16i for 24–72 h and cell viability was determined by MTT assay. (b) Western blot analysis was used
to examine the expression and phosphorylation of FLT3 in MV4-11 cells treated with different doses of 16i for 2 h. (c) Flow cytometric analysis of cells stained with Annexin
V-FITC/PI after treatment with various concentrations (0 nM, 1 nM, 10 nM, 100 nM) of 16i for 48 h.
potassium acetate (3.0 equiv), bis(pinacolato)diboron (2.25 equiv)
and PdCl2(dppf) (0.1 equiv) in 1,4-dioxane were heated to 100 °C
for 2 h. The mixture was cooled to room temperature before acetic
acid (2.0 equiv) and water were added. The mixture was stirred for
1 h and 30% hydrogen peroxide (2.0 equiv) was added. After 3 h,
the precipitate was collected by filtration, washed with EtOH and
dried under vacuum to give 10.
NaHCO3 and water, dried over MgSO4 and then purified by silica
gel chromatography to give 11c.
4.1.2. General procedure for the preparation of compounds
15a–15h and 16a–16t
Compound 12 (1.0 equiv) was suspended in EtOH/H2O (v/v:
3:1), and iron powder (10.0 equiv) and NH4Cl (3.0 equiv) were
Then 10 (1.0 equiv), potassium carbonate (2.0 equiv) and
methyl iodide or ethyl iodide (1.0 equiv) in DMF were stirred at
room temperature for 2 h. The mixture was diluted with water,
and the precipitate was collected by filtration, washed with water
and dried under vacuum to give 11a or 11b.
added. The suspension was heated to reflux for 1 h with
vigorous stirring. The precipitate was separated by filtration
and washing with ethanol. The filtrates were evaporated and
the resulting oil was added to cold saturated NaHCO3 fol-
lowed by extracted with acetic ether. The combined organic
layers were dried (MgSO4), filtered and the filtrate was con-
centrated under reduced pressure. The crude amide (13) was
used for the next reaction without further purification. A solu-
tion of 13 (1.0 equiv) and 14 (1.0 equiv) in chloroform was
heated at 60 °C for 2 h. The reaction mixture was purified
by silica gel chromatography to give 15a–15h and 16a–16t
as solid.
4.1.1.6. General procedure for the preparation of 11c.
To a
stirred solution of 10 (1.0 equiv) and 4-dimethylaminopyridine
(DMAP, 0.2 equiv) in CH2Cl2 at 0 °C was added drop-wise acetyl
chloride (1.1 equiv). The resulting suspension was allowed to
warm to ambient temperature and then stirred for 30 min. The
reaction was then diluted with CH2Cl2, washed with saturated

4340
Y. Xu et al. / Bioorg. Med. Chem. 23 (2015) 4333–4343
Figure 5. (a) Antitumor efficacy of 16i at concentrations of 10, 30, and 60 mg/kg/d in an in vivo MV4-11 xenograft model. (b) Average body weights for 16i-treated mice
groups at different doses and vehicle group in an in vivo MV4-11 xenograft model. (c) Ki67 and TUNEL staining of tumor tissues showing the inhibition of cell proliferation
and the induction of apoptosis.
4.1.2.1. N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(imidazo[1,2-a]pyr-
idin-2-yl)phenyl)urea (15a). Overall yield: 14.5%. 1H NMR
142.26, 140.32, 129.11, 127.38, 126.65, 119.92, 118.60, 109.86,
98.36, 92.44, 32.46. 28.22; MS (ES+) m/z calcd for C20H20N6O2:
376.16; found: 399.2 (M+Na+).
(400 MHz, DMSO-d6)
d 9.56 (s, 1H), 8.92 (s, 1H), 8.50 (d,
J = 6.6 Hz, 1H), 8.32 (s, 1H), 7.92 (d, J = 7.8 Hz, 2H), 7.56 (d,
J = 7.5 Hz, 2H), 7.52 (d, J = 6.7 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 6.87
(t, J = 6.5 Hz, 1H), 6.54 (s, 1H), 1.30 (s, 9H); 13C NMR (101 MHz,
4.1.2.4. N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(imidazo[1,2-b]pyr-
idazin-2-yl)phenyl)urea (15d). Overall yield: 12.3%. 1H NMR
(400 MHz, DMSO-d6) d 9.64 (s, 1H), 9.16 (s, 1H), 8.77 (s, 1H), 8.47
(d, J = 8.3 Hz, 1H), 8.09 (d, J = 8.1 Hz, 1H), 7.99 (d, J = 7.1 Hz, 2H),
7.57 (d, J = 7.1 Hz, 2H), 7.21 (t, J = 8.5 Hz, 1H), 6.54 (s, 1H), 1.29
(s, 9H); 13C NMR (101 MHz, DMSO-d6) d 180.16, 158.31, 151.31,
144.55, 143.56, 139.06, 138.83, 127.55, 126.30, 124.68, 118.49,
117.45, 112.39, 92.45, 32.43, 28.31; MS (ES+) m/z calcd for
DMSO-d6)
d 180.16, 158.36, 151.27, 144.74, 144.29, 138.49,
128.29, 126.70, 126.15, 124.68, 118.59, 116.41; MS (ES+) m/z calcd
for C21H21N5O2: 375.17; found: 376.2 (M+H+).
4.1.2.2. N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(imidazo[1,2-a]pyr-
imidin-2-yl)phenyl)urea (15b). Overall yield: 7.2%. 1H NMR
(400 MHz, DMSO-d6) d 10.34 (s, 1H), 10.03 (s, 1H), 9.32 (d,
J = 6.1 Hz, 1H), 8.96 (d, J = 5.8 Hz, 1H), 8.69 (s, 1H), 7.96 (d,
J = 7.9 Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H), 7.64 (t, J = 6.5 Hz, 1H), 6.53
(s, 1H), 1.30 (s, 9H); 13C NMR (101 MHz, DMSO-d6) d 180.10,
158.04, 156.13, 151.45, 144.20, 141.39, 137.41, 136.38, 127.16,
120.00, 118.20, 113.61, 108.36, 92.48, 32.43, 28.34; MS (ES+) m/z
calcd for C20H20N6O2: 376.16; found: 377.2 (M+H+).
C
20H20N6O2: 376.16; found: 377.2 (M+H+).
4.1.2.5. N-(4-(Benzo[b]thiophen-2-yl)phenyl)-N0-(5-(tert-butyl)
isoxazol-3-yl)urea (15e). Overall yield: 8.5%. 1H NMR (400 MHz,
DMSO-d6)
d 9.60 (s, 2H), 7.94 (d, J = 7.7 Hz, 1H), 7.81 (d,
J = 7.6 Hz, 1H), 7.76 (s, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.60 (d,
J = 8.0 Hz, 2H), 7.38 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H),
6.54 (s, 1H), 1.30 (s, 9H); 13C NMR (101 MHz, DMSO-d6) d 180.04,
158.53, 151.51, 143.19, 140.61, 139.63, 138.22, 127.48, 126.63,
124.71, 124.29, 123.42, 122.33, 118.76, 92.57, 32.43, 28.34; MS
(ES+) m/z calcd for C21H20N3O2S: 391.14; found: 414.2 (M+Na+).
4.1.2.3. N-(5-(tert-butyl)Isoxazol-3-yl)-N0-(4-(imidazo[1,2-a]pyr-
azin-2-yl)phenyl)urea (15c). Overall yield: 11.6%. 1H NMR
(400 MHz, DMSO-d6) d 9.58 (s, 1H), 9.05 (s, 1H), 8.99 (s, 1H), 8.58
(d, J = 6.5 Hz, 1H), 8.53 (s, 1H), 7.98 (d, J = 7.9 Hz, 2H), 7.89 (d,
J = 6.3 Hz, 1H), 7.58 (d, J = 8.2 Hz, 2H), 6.53 (s, 1H), 1.31 (s, 9H);
13C NMR (101 MHz, DMSO-d6) d 180.21, 158.31, 151.25, 144.74,
4.1.2.6. N-(4-(Benzofuran-2-yl)phenyl)-N0-(5-(tert-butyl)isoxa-
zol-3-yl)urea (15f). Overall yield: 10.8%. 1H NMR (400 MHz,

Y. Xu et al. / Bioorg. Med. Chem. 23 (2015) 4333–4343
4341
DMSO-d6) d 9.61 (s, 1H), 9.06 (s, 1H), 7.86 (d, J = 6.0 Hz, 2H), 7.62 (s,
4H), 7.29 (d, J = 6.2 Hz, 2H), 7.25 (s, 1H), 6.54 (s, 1H), 1.30 (s, 9H);
13C NMR (101 MHz, DMSO-d6) d 180.23, 158.27, 155.24, 154.02,
151.22, 139.56, 129.00, 125.46, 124.14, 123.91, 123.12, 120.83,
118.62, 110.91, 100.58, 92.46, 32.45, 28.31; MS (ES+) m/z calcd
for C22H21N3O3: 375.16; found: 398.2 (M+Na+).
158.33, 151.23, 145.12, 139.84, 138.98, 130.80, 127.36, 126.50,
123.76 (q, J = 4.2 Hz), 123.09 (q, J = 270.8 Hz), 118.56, 116.33 (q,
J = 32.0 Hz), 110.63, 109.61, 92.44, 32.44, 28.31; MS (ES+) m/z calcd
for C22H20F3N5O2: 443.16; found: 444.2 (M+H+).
4.1.2.13. N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(7-chloroimidazo
[1,2-a]pyridin-2-yl)phenyl)urea (16e).
Overall yield: 13.9%.
4.1.2.7.
N-(4-(Benzo[d]oxazol-2-yl)phenyl)-N0-(5-(tert-butyl)
1H NMR (400 MHz, DMSO-d6) d 9.81 (s, 1H), 9.63 (s, 1H), 8.83 (d,
J = 7.1 Hz, 1H), 8.65 (s, 1H), 8.01 (s, 1H), 7.92 (d, J = 8.2 Hz, 2H),
7.66 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 6.8 Hz, 1H), 6.53 (s, 1H), 1.30
(s, 9H); 13C NMR (101 MHz, DMSO-d6) d 180.21, 158.16, 151.36,
144.49, 141.11, 140.77, 129.53, 127.43, 126.94, 124.37, 118.57,
111.94, 110.13, 108.00, 92.47, 32.46, 28.33; MS (ES+) m/z calcd
for C21H20ClN5O2: 409.13; found: 410.1 (M+H+).
isoxazol-3-yl)urea (15g). Overall yield: 4.8%. 1H NMR (400 MHz,
DMSO-d6) d 9.65 (s, 1H), 9.19 (s, 1H), 8.12 (d, J = 7.7 Hz, 1H), 8.06
(d, J = 7.8 Hz, 2H), 8.02 (d, J = 7.5 Hz, 1H), 7.67 (d, J = 7.9 Hz, 2H),
7.54 (t, J = 7.2 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 6.55 (s, 1H), 1.31 (s,
9H); 13C NMR (101 MHz, DMSO-d6) d 180.31, 166.95, 158.19,
153.62, 151.15, 141.92, 134.20, 128.13, 126.87, 126.54, 125.18,
122.50, 122.21, 118.55, 92.48, 32.47, 28.32; MS (ES+) m/z calcd
for C21H20N4O2: 376.15; found: 377.2 (M+H+).
4.1.2.14. N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(7-bromoimidazo
[1,2-a]pyridin-2-yl)phenyl)urea (16f).
Overall yield: 16.1%. 1H
4.1.2.8.
N-(4-(Benzo[d]thiazol-2-yl)phenyl)-N0-(5-(tert-butyl)
NMR (400 MHz, DMSO-d6) d 10.03 (s, 1H), 9.95 (s, 1H), 8.73 (s,
1H), 8.63 (d, J = 6.3 Hz, 1H), 7.96 (d, J = 8.1 Hz, 2H), 7.66 (s, 1H),
7.64 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 5.8 Hz, 1H), 6.54 (s, 1H), 1.30
(s, 9H); 13C NMR (101 MHz, DMSO-d6) d 180.11, 158.14, 151.46,
144.49, 141.11, 140.59, 129.53, 127.05, 126.94, 124.37, 118.29,
111.13, 110.13, 108.00, 92.47, 32.44, 28.34; MS (ES+) m/z calcd
for C21H20BrN5O2: 453.08; found: 454.1 (M+H+).
isoxazol-3-yl)urea (15h). Overall yield: 5.7%. 1H NMR (400 MHz,
DMSO-d6) d 9.67 (s, 1H), 9.21 (s, 1H), 8.15 (d, J = 7.8 Hz, 2H), 7.77
(d, J = 4.1 Hz, 2H), 7.71 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 4.0 Hz, 2H),
6.55 (s, 1H), 1.31 (s, 9H); 13C NMR (101 MHz, DMSO-d6) d
162.24, 158.17, 151.16, 150.08, 142.40, 141.63, 128.35, 125.09,
124.74, 120.09, 119.46, 118.47, 110.71, 99.49, 92.48, 32.47,
28.32; MS (ES+) m/z calcd for C21H20N4O2S: 392.13; found: 393.1
(M+H+).
4.1.2.15. N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(7-methylimidazo
[1,2-a]pyridin-2-yl)phenyl)urea (16g). Overall yield: 12.8%. 1H
NMR (400 MHz, DMSO-d6) d 9.57 (s, 1H), 8.97 (s, 1H), 8.37 (d,
J = 7.1 Hz, 1H), 8.21 (s, 1H), 7.88 (d, J = 8.2 Hz, 2H), 7.53 (d,
J = 8.2 Hz, 2H), 7.32 (s, 1H), 6.71 (d, J = 6.8 Hz, 1H), 6.53 (s, 1H),
2.34 (s, 3H), 1.29 (s, 9H); 13C NMR (101 MHz, DMSO-d6) d 180.15,
158.35, 151.28, 145.12, 143.96, 138.34, 135.15, 128.39, 126.03,
125.90, 118.54, 114.66, 114.55, 107.70, 92.43, 32.43, 28.31,
20.78; MS (ES+) m/z calcd for C22H23N5O2: 389.19; found: 390.2
(M+H+).
4.1.2.9.
dazo[1,2-a]pyridin-2-yl)phenyl)urea (16a).
N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(8-fluoroimi-
Overall yield:
5.5%. 1H NMR (400 MHz, DMSO-d6) d 9.56 (s, 1H), 8.95 (s, 1H),
8.74 (s, 1H), 8.32 (s, 1H), 7.90 (d, J = 8.2 Hz, 2H), 7.62 (m, 1H),
7.54 (d, J = 8.2 Hz, 2H), 7.31 (t, J = 9.0 Hz, 1H), 6.53 (s, 1H), 1.30
(s, 9H); 13C NMR (100 MHz, DMSO-d6) d 180.17, 158.33, 152.47
(d, J = 230.2 Hz), 151.26, 145.56, 142.65, 138.66, 127.92, 126.16,
118.57, 117.02 (d, J = 9.4 Hz), 116.37 (d, J = 26.5 Hz), 113.42 (d,
J = 41.4 Hz), 109.88, 92.43, 32.44, 28.31; MS (ES+) m/z calcd for
C
21H20FN5O2: 393.16; found: 394.2 (M+H+).
4.1.2.16.
dazo[1,2-a]pyridin-2-yl)phenyl)urea (16h).
N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(7-ethynylimi-
Overall yield:
4.1.2.10. N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(8-chloroimidazo
[1,2-a]pyridin-2-yl)phenyl)urea (16b). Overall yield: 15.1%.
15.5%. 1H NMR (400 MHz, DMSO-d6) d 9.83 (s, 1H), 9.68 (s, 1H),
8.74 (d, J = 6.3 Hz, 1H), 8.64 (s, 1H), 8.13 (s, 1H), 7.93 (d,
J = 7.9 Hz, 2H), 7.66 (d, J = 8.1 Hz, 2H), 7.56 (d, J = 6.1 Hz, 1H),
6.53 (s, 1H), 3.07 (s, 1H), 1.30 (s, 9H); 13C NMR (101 MHz, DMSO-
d6) d 180.20, 179.74, 158.16(s), 152.86, 151.37, 146.41, 140.72,
136.53, 126.94, 122.07, 118.54, 116.87, 110.12, 106.02, 92.47,
80.47, 75.32, 32.45, 28.34; MS (ES+) m/z calcd for C23H21N5O2:
399.17; found: 400.3 (M+H+).
1H NMR (400 MHz, DMSO-d6) d 9.55 (s, 1H), 8.91 (s, 1H), 8.31
(s, 1H), 8.18 (s, 1H), 7.88 (d, J = 8.0 Hz, 2H), 7.53
(d, J = 7.9 Hz, 2H), 7.45 (d, J = 9.2 Hz, 1H), 6.96 (d, J = 9.2 Hz, 1H),
6.53 (s, 1H), 1.30 (s, 9H); 13C NMR (101 MHz, DMSO-d6) d 180.16,
158.36, 151.26, 144.16, 143.90, 138.36, 128.39, 127.43,
126.02, 124.19, 122.67, 118.56, 116.00, 108.00, 92.42, 32.44,
28.31; MS (ES+) m/z calcd for C21H20ClN5O2: 409.13; found: 410.2
(M+H+).
4.1.2.17. N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(7-methoxyimi-
dazo[1,2-a]pyridin-2-yl)phenyl)urea (16i). Overall yield: 9.6%.
1H NMR (400 MHz, DMSO-d6) d 9.54 (s, 1H), 8.91 (s, 1H), 8.36 (d,
J = 7.1 Hz, 1H), 8.11 (s, 1H), 7.85 (d, J = 7.9 Hz, 2H), 7.52 (d,
J = 8.0 Hz, 2H), 6.95 (s, 1H), 6.60 (d, J = 7.0 Hz, 1H), 6.53 (s, 1H),
3.84 (s, 3H), 1.30 (s, 9H); 13C NMR (101 MHz, DMSO-d6) d 180.16,
158.36, 157.34, 151.27, 146.16, 143.85, 138.17, 128.53,
127.24, 125.86, 118.57, 106.98, 106.56, 94.20, 92.42, 55.49, 32.44,
28.32; MS (ES+) m/z calcd for C22H23N5O3: 405.18; found: 406.2
(M+H+).
4.1.2.11. N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(8-methylimidazo
[1,2-a]pyridin-2-yl)phenyl)urea (16c). Overall yield: 11.9%.
1H NMR (400 MHz, DMSO-d6) d 9.55 (s, 1H), 8.95 (s, 1H), 8.33 (d,
J = 6.6 Hz, 1H), 8.29 (s, 1H), 7.92 (d, J = 6.8 Hz, 2H), 7.55 (d,
J = 6.9 Hz, 2H), 7.01 (d, J = 6.7 Hz, 1H), 6.77 (t, J = 7.2 Hz, 1H), 6.54
(s, 1H), 2.53 (s, 3H), 1.30 (s, 9H); 13C NMR (101 MHz, DMSO-d6) d
180.19, 158.36, 151.27, 145.21, 143.63, 138.36, 128.41, 126.13,
125.92, 124.42, 123.21, 118.57, 112.02, 108.79, 92.44, 32.43, 28.30,
16.66; MS (ES+) m/z calcd for C22H23N5O2: 389.19; found: 390.2
(M+H+).
4.1.2.18. N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(7-ethoxyimidazo
[1,2-a]pyridin-2-yl)phenyl)urea (16j).
Overall yield: 7.1%. 1H
4.1.2.12. N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(8-(trifluoromethyl)
NMR (400 MHz, DMSO-d6) d 9.55 (s, 1H), 8.90 (s, 1H), 8.35 (d,
J = 7.2 Hz, 1H), 8.11 (s, 1H), 7.84 (d, J = 8.0 Hz, 2H), 7.51 (d,
J = 8.1 Hz, 2H), 6.92 (s, 1H), 6.59 (d, J = 7.2 Hz, 1H), 6.53 (s, 1H),
4.10 (q, J = 8.1 Hz, 2H), 1.37 (t, J = 6.4 Hz, 3H), 1.30 (s, 9H); 13C
NMR (101 MHz, DMSO-d6) d 180.16, 158.35, 156.51, 151.26,
146.14, 143.76, 138.17, 128.48, 127.27, 125.84, 118.56, 106.95,
imidazo[1,2-a]pyridin-2-yl)phenyl)urea (16d).
Overall yield:
9.9%. 1H NMR (400 MHz, DMSO-d6) d 9.55 (s, 1H), 8.97 (s, 1H),
8.79 (d, J = 5.5 Hz, 1H), 8.51 (s, 1H), 7.94 (d, J = 7.4 Hz, 2H), 7.69
(d, J = 5.8 Hz, 1H), 7.57 (d, J = 7.5 Hz, 2H), 7.02 (t, J = 7.8 Hz, 1H),
6.54 (s, 1H), 1.30 (s, 9H); 13C NMR (101 MHz, DMSO-d6) d 180.19,

4342
Y. Xu et al. / Bioorg. Med. Chem. 23 (2015) 4333–4343
106.76, 94.65, 92.42, 63.55, 32.45, 28.33, 14.28; MS (ES+) m/z calcd
4.1.2.25.
dazo[1,2-a]pyridin-2-yl)phenyl)urea (16q).
N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(6-ethynylimi-
Overall yield:
for C23H22N5O3: 419.20; found: 420.3 (M+H+).
12.1%. 1H NMR (400 MHz, DMSO-d6) d 9.33 (s, 1H), 8.74 (s, 1H),
8.56 (d, J = 6.2 Hz, 1H), 8.05 (d, J = 6.0 Hz, 1H), 7.66 (d, J = 8.2 Hz,
2H), 7.31 (d, J = 8.0 Hz, 2H), 7.27 (s, 1H), 7.00 (s, 1H), 6.29 (s, 1H),
4.02 (s, 1H), 1.05 (s, 9H); 13C NMR (101 MHz, DMSO-d6) d 180.19,
158.33, 151.26, 145.23, 143.66, 138.80, 130.35, 127.66, 127.07,
126.28, 118.59, 116.45, 108.76, 106.75, 92.43, 81.62, 80.21, 32.43,
28.30; MS (ES+) m/z calcd for C23H21N5O2: 399.17; found: 400.2
(M+H+).
4.1.2.19. 2-(4-(3-(5-(tert-Butyl)isoxazol-3-yl)ureido)phenyl)imi-
dazo[1,2-a]pyridin-7-yl acetate (16k).
Overall yield: 6.3%. 1H
NMR (400 MHz, DMSO-d6) d 9.56 (s, 1H), 8.96 (s, 1H), 8.55 (d,
J = 7.9 Hz, 1H), 8.33 (s, 1H), 7.89 (d, J = 7.3 Hz, 2H), 7.54 (d,
J = 7.7 Hz, 2H), 7.35 (s, 1H), 6.81 (d, J = 7.6 Hz, 1H), 6.53 (s, 1H),
2.32 (s, 3H), 1.31 (s, 9H); 13C NMR (101 MHz, DMSO-d6) d 180.18,
169.39, 158.34, 151.25, 145.24, 143.13, 138.79, 138.62, 126.17,
125.85, 121.87, 119.43, 118.58, 116.20, 109.37, 92.43, 32.45,
28.42, 28.33; MS (ES+) m/z calcd for C23H23N5O4: 433.18; found:
434.2 (M+H+).
4.1.2.26. N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(6-cyclopropylim-
idazo[1,2-a]pyridin-2-yl)phenyl)urea (16r).
Overall yield:
12.6% 1H NMR (400 MHz, DMSO-d6) d 9.52 (s, 1H), 8.89 (s, 1H),
8.32 (s, 1H), 8.18 (s, 1H), 7.87 (d, J = 8.1 Hz, 2H), 7.52 (d,
J = 8.1 Hz, 2H), 7.45 (d, J = 9.2 Hz, 1H), 6.97 (d, J = 9.3 Hz, 1H),
6.52 (s, 1H), 2.04–1.88 (m, 1H), 1.30 (s, 9H), 0.94 (m, 2H), 0.71
(m, 2H); 13C NMR (101 MHz, DMSO-d6) d 180.17, 158.34, 151.24,
144.12, 143.89, 138.34, 128.37, 127.45, 126.02, 124.21, 122.67,
118.54, 116.00, 108.02, 92.42, 32.45, 28.32, 12.37, 7.65; MS (ES+)
m/z calcd for C24H25N5O2: 415.20; found: 416.3 (M+H+).
4.1.2.20. N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(6-fluoroimidazo
[1,2-a]pyridin-2-yl)phenyl)urea (16l).
Overall yield: 7.2%. 1H
NMR (400 MHz, DMSO-d6) d 9.61 (s, 1H), 9.08 (s, 1H), 8.52 (d,
J = 6.3 Hz, 1H), 8.45 (s, 1H), 7.93 (d, J = 7.8 Hz, 2H), 7.57 (d,
J = 7.8 Hz, 2H), 7.41 (s, 1H), 6.87 (d, J = 6.6 Hz, 1H), 6.54 (s, 1H),
1.30 (s, 9H); 13C NMR (101 MHz, DMSO-d6) d 180.16, 158.33,
151.28 (d, J = 24.2 Hz), 144.64, 141.86, 138.86, 127.57, 126.33,
125.96, 123.89, 120.91 (d, J = 43.4 Hz), 118.53 (d, J = 240.8 Hz),
111.91 (d, J = 39.8 Hz), 110.32, 92.45, 32.43, 28.31; MS (ES+) m/z
calcd for C21H20FN5O2: 393.16; found: 394.2 (M+H+).
4.1.2.27.
dazo[1,2-a]pyridin-2-yl)phenyl)urea (16s).
N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(5-chloroimi-
Overall yield:
8.5%. 1H NMR (400 MHz, DMSO-d6) d 9.99 (s, 1H), 9.93 (s, 1H),
8.89 (s, 1H), 8.07 (d, J = 8.0 Hz, 2H), 7.94 (d, J = 8.1 Hz, 1H), 7.86
(t, J = 8.0 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H),
6.55 (s, 1H), 1.31 (s, 9H); 13C NMR (101 MHz, DMSO-d6) d 180.15,
158.09, 151.38, 141.63, 141.14, 136.95, 132.48, 127.93, 127.22,
120.30, 118.18, 116.70, 111.05, 108.59, 92.48, 32.44, 28.33; MS
(ES+) m/z calcd for C21H20ClN5O2: 409.13; found: 410.2 (M+H+).
4.1.2.21.
dazo[1,2-a]pyridin-2-yl)phenyl)urea (16m).
N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(6-chloroimi-
Overall yield:
12.6%. 1H NMR (400 MHz, DMSO-d6) d 8.96 (s, 1H), 8.80 (s, 1H),
8.30 (s, 1H), 7.91 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 9.3 Hz, 1H), 7.55
(d, J = 7.5 Hz, 2H), 7.28 (d, J = 9.3 Hz, 1H), 6.54 (s, 1H), 1.31 (s,
9H); 13C NMR (101 MHz, DMSO-d6) d 180.18, 158.33, 151.25,
145.30, 143.21, 138.78, 127.71, 126.26, 125.50, 124.62, 118.79,
118.58, 117.15, 109.00, 92.43, 32.44, 28.32; MS (ES+) m/z calcd
for C21H20ClN5O2: 409.13; found: 410.2 (M+H+).
4.1.2.28.
dazo[1,2-a]pyridin-2-yl)phenyl)urea (16t).
N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(5-methylimi-
Overall yield:
7.1%. 1H NMR (400 MHz, DMSO-d6) d 9.57 (s, 1H), 8.93 (s, 1H),
8.28 (s, 1H), 7.98 (d, J = 6.2 Hz, 2H), 7.54 (d, J = 6.4 Hz, 2H), 7.46
(d, J = 7.8 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 6.76 (d, J = 7.8 Hz, 1H),
6.54 (s, 1H), 2.63 (s, 3H), 1.30 (s, 9H); 13C NMR (101 MHz,
4.1.2.22. N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(6-bromoimidazo
[1,2-a]pyridin-2-yl)phenyl)urea (16n).
Overall yield: 12.8%.
1H NMR (400 MHz, DMSO-d6) d 9.60 (s, 1H), 9.06 (s, 1H), 8.86 (s,
1H), 8.29 (s, 1H), 7.90 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 8.3 Hz, 2H),
7.50 (d, J = 9.0 Hz, 1H), 7.34 (d, J = 9.4 Hz, 1H), 6.53 (s, 1H), 1.30
(s, 9H); 13C NMR (101 MHz, DMSO-d6) d 180.16, 158.32, 151.28,
145.09, 143.25, 138.82, 127.65, 127.58, 126.70, 126.271 118.54,
117.42, 108.78, 105.72, 92.44, 32.44, 28.33; MS (ES+) m/z calcd
for C21H20BrN5O2: 453.08; found: 454.1 (M+H+).
DMSO-d6)
d 180.16, 158.37, 151.27, 145.13, 144.25, 138.49,
135.09, 126.20, 124.83, 118.51, 113.79, 111.10, 106.17, 99.48,
92.45, 32.44, 28.31, 18.29; MS (ES+) m/z calcd for C22H23N5O2:
389.19; found: 390.2 (M+H+).
4.2. Kinase inhibitory assays
The kinase inhibitory assays were performed according to the
KinaseProfiler assay protocols of Upstate Biotechnology
(Millipore).
4.1.2.23.
[1,2-a]pyridin-2-yl)phenyl)urea (16o).
N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(6-iodoimidazo
Overall yield: 9.5%.
1H NMR (400 MHz, DMSO-d6) d 9.92 (s, 1H), 9.88 (s, 1H), 9.21 (s,
1H), 8.55 (s, 1H), 8.04 (d, J = 8.7 Hz, 1H), 7.90 (d, J = 7.4 Hz, 2H),
7.73 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 7.7 Hz, 2H), 6.52 (s, 1H), 1.30
(s, 9H); 13C NMR (101 MHz, DMSO-d6) d 180.15, 158.08, 151.35,
141.12, 139.69, 139.13, 136.00, 132.95, 127.06, 119.96, 118.34,
113.08, 109.47, 92.48, 81.53, 32.44, 28.34; MS (ES+) m/z calcd for
4.3. Cell culture and cell viability assays
All cell lines were obtained from the American Type Culture
Collection (Manassas, VA, USA). Cells were maintained in RPMI
1640 or DMEM medium supplemented with 10% FBS (v/v) in 5%
CO2 at 37 °C, except for MV4-11 cells, which were cultured in
IMDM medium. The leukemia cells were seeded in a 96-well plate
at 1–4 ꢁ 104 cells per well, and an equal volume of medium con-
taining increasing concentrations of inhibitors was added to each
C
21H20IN5O2: 501.07; found: 502.1 (M+H+).
4.1.2.24.
dazo[1,2-a]pyridin-2-yl)phenyl)urea (16p).
N-(5-(tert-Butyl)isoxazol-3-yl)-N0-(4-(6-methylimi-
Overall yield:
18.4%. 1H NMR (400 MHz, DMSO-d6) d 9.88 (s, 1H), 9.82 (s, 1H),
8.68 (s, 1H), 8.63 (s, 1H), 7.93 (d, J = 8.3 Hz, 2H), 7.86 (d,
J = 9.1 Hz, 1H), 7.79 (d, J = 9.1 Hz, 1H), 7.67 (d, J = 8.2 Hz, 2H),
6.54 (s, 1H), 2.42 (s, 3H), 1.31 (s, 9H); 13C NMR (101 MHz,
well. At the end of the incubation period (72 h at 37 °C), 20
5 mg/mL MTT reagent was added per well for 2–4 h, and 50
l
l
L of
L of
20% SDS (w/v) per well was used to dissolve the cells. The other cell
lines were seeded in 96-well plates at a density of 2–5 ꢁ 103
cells/well. After incubation for 24 h in serum-containing media,
the cells were treated with inhibitors diluted with culture medium
for 72 h at 37 °C under a 5% CO2 atmosphere. Thereafter, the MTT
reagent was added for 2–4 h, and DMSO was used to dissolve the
DMSO-d6)
d 180.20, 158.12, 151.38, 143.27, 141.02, 138.73,
135.45, 135.40, 126.96, 126.33, 120.14, 118.45, 111.21, 109.80,
92.47, 32.46, 28.33, 17.37; MS (ES+) m/z calcd for C22H23N5O2:
389.19; found: 390.2 (M+H+).

Y. Xu et al. / Bioorg. Med. Chem. 23 (2015) 4333–4343
4343
cells. Finally, the light absorption (OD) of the dissolved cells was
4.8. Immunohistochemistry
measured at 570 nM using a Multiskan MK3 ELISA photometer
(Thermo Scientific).
NOD-SCID mice bearing tumors were treated with 16i
(60 mg/kg/d) via oral gavage, and the tumors were harvested
7 days later. The tumors were fixed with formalin and embedded
4.4. Pharmacokinetic analysis
in paraffin. Sections measuring 4–8 lm in thickness were prepared
Female Sprague–Dawley rats (200–250 g) were used and ran-
domly divided into two groups (n = 3 in each group). A catheter
was surgically placed into the femoral vein for collection of blood
samples. Rats were fasted overnight before dosing. Compounds
16i was administered orally (po) by gavage at 30 mg/kg in 25%
PEG400, 5% DMSO (Sigma–Aldrich) and 70% distilled water.
Blood was collected into heparin-containing tubes for plasma at
15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 12 h and 24 h postdose, and the
plasma was isolated by centrifugation. Plasma concentrations of
each compound were determined using liquid–liquid extraction
followed by HPLC (an UltiMate 3000 HPLC system, Dionex, USA).
for immunohistochemical analysis. Proliferation was detected
using immunostaining with the Ki67 antibody (Thermo Fisher
Scientific, Fremont, CA). Apoptosis was determined using trans-
ferase-mediated dUTP nick-end labeling (TUNEL) and staining
(Roche Applied Science). Finally, images were captured with an
Olympus digital camera attached to a light microscope.
References and notes
1. Rosnet, O.; Marchetto, S.; DeLapeyriere, O.; Birnbaum, D. Oncogene 1991, 6,
1641.
2. Rosnet, O.; Bühring, H.-J.; Beslu, N.; Lavagna, C.; Marchetto, S.; Rappold, I.;
Drexler, H. G.; Birg, F.; Rottapel, R.; Hannum, C. Acta Haematol. 1996, 95, 218.
3. Rosnet, O.; Schiff, C.; Pebusque, M. J.; Marchetto, S.; Tonnelle, C.; Toiron, Y.;
Birg, F.; Birnbaum, D. Blood 1993, 82, 1110.
4.5. Western blot analysis
4. Mackarehtschian, K.; Hardin, J. D.; Moore, K. A.; Boast, S.; Goff, S. P.; Lemischka,
I. R. Immunity 1995, 3, 147.
5. Stacchini, A.; Fubini, L.; Severino, A.; Sanavio, F.; Aglietta, M.; Piacibello, W.
Leukemia 1996, 10, 1584.
6. Quentmeier, H.; Reinhardt, J.; Zaborski, M.; Drexler, H. G. Leukemia 2003, 17,
120.
7. Yamamoto, Y.; Kiyoi, H.; Nakano, Y.; Suzuki, R.; Kodera, Y.; Miyawaki, S.; Asou,
N.; Kuriyama, K.; Yagasaki, F.; Shimazaki, C.; Akiyama, H.; Saito, K.; Nishimura,
M.; Motoji, T.; Shinagawa, K.; Takeshita, A.; Saito, H.; Ueda, R.; Ohno, R.; Naoe,
T. Blood 2001, 97, 2434.
After treatment with compound 16i for 2 h at 37 °C, MV4-11
cells were harvested, washed with ice-cold PBS, and suspended
in RIPA lysis buffer (Beyotime) including 1% cocktail (Sigma–
Aldrich). Cell lysates were centrifuged for 15 min at 13,000 rpm
and 4 °C to remove any insoluble material. Proteins were separated
by gel electrophoresis on 5–10% polyacrylamide gels and trans-
ferred onto PVDF membranes (Millipore). The PVDF membranes
were incubated with the anti-FLT3, anti-p-FLT3 and anti-b-actin
antibody, washed with TBST buffer and blotted with horseradish
peroxidase-conjugated secondary antibody. Finally, the mem-
branes were developed with SuperSignal reagent (Pierce,
Rockford, IL, USA) upon exposure to X-ray film.
8. Nakao, M.; Yokota, S.; Iwai, T.; Kaneko, H.; Horiike, S.; Kashima, K.; Sonoda, Y.;
Fujimoto, T.; Misawa, S. Leukemia 1996, 10, 1911.
9. Mizuki, M.; Fenski, R.; Halfter, H.; Matsumura, I.; Schmidt, R.; Muller, C.;
Gruning, R.; Kratz-Abers, K.; Serve, S.; Steur, C.; Buchner, T.; Kienast, J.;
Kanakura, Y.; Berdel, W. E.; Serve, H. Blood 2000, 96, 3907.
10. Gale, R. E.; Green, C.; Allen, C.; Mead, A. J.; Burnett, A. K.; Hills, R. K.; Linch, D. C.
Blood 2008, 111, 2776.
11. Kiyoi, H.; Yanada, M.; Ozekia, K. Int. J. Haematol. 2005, 82, 85.
12. Heinrich, M. C. Mini-Rev. Med. Chem. 2004, 4, 255.
4.6. Apoptosis analysis in MV4-11 cells
13. O0Farrell, A.-M.; Abrams, T. J.; Yuen, H. A.; Ngai, T. J.; Louie, S. G.; Yee, K. W.;
Wong, L. M.; Hong, W.; Lee, L. B.; Town, A. Blood 2003, 101, 3597.
14. Zhang, W.; Konopleva, M.; Shi, Y.-X.; McQueen, T.; Harris, D.; Ling, X.; Estrov,
Z.; Quintás-Cardama, A.; Small, D.; Cortes, J. J. Natl. Cancer I 2008, 100, 184.
15. DeAngelo, D. J.; Stone, R. M.; Heaney, M. L.; Nimer, S. D.; Paquette, R. L.;
Klisovic, R. B.; Caligiuri, M. A.; Cooper, M. R.; Lecerf, J.-M.; Karol, M. D. Blood
2006, 108, 3674.
A total of 4 ꢁ 105 MV4-11 cells were seeded in a six-well plate
and treated with compound 16i for 48 h at 37 °C. After incubation,
the cells were harvested and washed with PBS. The apoptosis ratio
was analyzed using an Annexin V-FITC Apoptosis Analysis Kit
(TianJin Suangene Biotech Co.) and BD FACSCalibur.
16. Smith, B. D.; Levis, M.; Beran, M.; Giles, F.; Kantarjian, H.; Berg, K.; Murphy, K.
M.; Dauses, T.; Allebach, J.; Small, D. Blood 2004, 103, 3669.
17. Stone, R.; De Angelo, J.; Galinsky, I.; Estey, E.; Klimek, V.; Grandin, W.; Lebwohl,
D.; Yap, A.; Cohen, P.; Fox, E. Ann. Haematol. 2003, 83, S89.
18. Shiotsu, Y.; Kiyoi, H.; Ishikawa, Y.; Tanizaki, R.; Shimizu, M.; Umehara, H.; Ishii,
K.; Mori, Y.; Ozeki, K.; Minami, Y. Blood 2009, 114, 1607.
19. Fathi, A. T.; Chabner, B. A. Oncologist 2011, 16, 1162.
20. Smith, C. C.; Wang, Q.; Chin, C.-S.; Salerno, S.; Damon, L. E.; Levis, M. J.; Perl, A.
E.; Travers, K. J.; Wang, S.; Hunt, J. P. Nature 2012, 485, 260.
21. Zarrinkar, P. P.; Gunawardane, R. N.; Cramer, M. D.; Gardner, M. F.; Brigham, D.;
Belli, B.; Karaman, M. W.; Pratz, K. W.; Pallares, G.; Chao, Q. Blood 2009, 114,
2984.
22. Kumar, P.; Meenakshi; Kumar, S.; Kumar, A.; Hussain, K.; Kumar, S. J.
Heterocycl. Chem. 2012, 49, 1243.
23. Kitamura, Y.; Sako, S.; Tsutsui, A.; Monguchi, Y.; Maegawa, T.; Kitade, Y.; Sajiki,
H. Adv. Synth. Catal. 2010, 352, 718.
24. Ono, M.; Cheng, Y.; Kimura, H.; Cui, M.; Kagawa, S.; Nishii, R.; Saji, H. J. Med.
Chem. 2011, 54, 2971.
25. Chao, Q.; Sprankle, K. G.; Grotzfeld, R. M.; Lai, A. G.; Carter, T. A.; Velasco, A. M.;
Gunawardane, R. N.; Cramer, M. D.; Gardner, M. F.; James, J. J. Med. Chem. 2009,
52, 7808.
4.7. In vivo model
MV4-11 cells were harvested during the exponential-growth
phase, washed three times with serum-free medium, and
resuspension at a concentration of 1 ꢁ 108 cell/mL. Tumor xeno-
graft models were established by subcutaneously injecting
100 lL of cell suspension into NOD-SCID mice (5–6 weeks).
When the tumors had grown to 100–200 mm3, the mice were ran-
domized into 4 groups (6 mice for each group) and treated with 16i
(10, 30, or 60 mg/kg/d) or vehicle alone (25% PEG400 plus 5%
DMSO in distilled water) via oral gavage. Tumor growth was mea-
sured twice weekly using Vernier calipers and the volume was cal-
culated as follows: tumor size = a ꢁ b2 ꢁ 0.5 (a = long diameter;
b = short diameter).