Discovery of 3-phenyl-1H-5-pyrazolylamine derivatives containing a urea pharmacophore as potent and efficacious inhibitors of FMS-like tyrosine kinase-3 (FLT3)

Article abstract of DOI:10.1016/j.bmc.2013.03.083

Preclinical investigations and early clinical trials suggest that FLT3 inhibitors are a viable therapy for acute myeloid leukemia. However, early clinical data have been underwhelming due to incomplete inhibition of FLT3. We have developed 3-phenyl-1H-5-p

Full text of DOI:10.1016/j.bmc.2013.03.083

Bioorganic & Medicinal Chemistry 21 (2013) 2856–2867
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of 3-phenyl-1H-5-pyrazolylamine derivatives containing a
urea pharmacophore as potent and efficacious inhibitors of FMS-like
tyrosine kinase-3 (FLT3)
Wen-Hsing Lin , John T.-A. Hsu , Shu-Yi Hsieh , Chiung-Tong Chen, Jen-Shin Song, Shih-Chieh Yen,
Tsu Hsu, Cheng-Tai Lu, Chun-Hwa Chen, Ling-Hui Chou, Yung-Ning Yang, Ching-Hui Chiu,
⇑
Ching-Ping Chen, Ya-Ju Tseng, Kuei-Jung Yen, Ching-Fang Yeh, Yu-Sheng Chao, Teng-Kuang Yeh ,
Weir-Torn Jiaang
⇑
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 35, Keyan Rd., Zhunan Town, Miaoli Country 350, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
Preclinical investigations and early clinical trials suggest that FLT3 inhibitors are a viable therapy for
acute myeloid leukemia. However, early clinical data have been underwhelming due to incomplete inhi-
bition of FLT3. We have developed 3-phenyl-1H-5-pyrazolylamine as an efficient template for kinase
inhibitors. Structure–activity relationships led to the discovery of sulfonamide, carbamate and urea series
of FLT3 inhibitors. Previous studies showed that the sulfonamide 4 and carbamate 5 series were potent
and selective FLT3 inhibitors with good in vivo efficacy. Herein, we describe the urea series, which we
found to be potent inhibitors of FLT3 and VEGFR2. Some inhibited growth of FLT3-mutated MOLM-13
cells more strongly than the FLT3 inhibitors sorafenib (2) and ABT-869 (3). In preliminary in vivo toxicity
studies of the four most active compounds, 10f was found to be the least toxic. A further in vivo efficacy
study demonstrated that 10f achieved complete tumor regression in a higher proportion of MOLM-13
xenograft mice than 4 and 5 (70% vs 10% and 40%). These results show that compound 10f possesses
improved pharmacologic and selectivity profiles and could be more effective than previously disclosed
FLT3 inhibitors in the treatment of acute myeloid leukemia.
Received 4 February 2013
Revised 28 March 2013
Accepted 30 March 2013
Available online 6 April 2013
Keywords:
FMS-like tyrosine kinase-3
Inhibitor
Acute myeloid leukemia
Receptor tyrosine kinase
Internal tandem duplications
Ó 2013 Published by Elsevier Ltd.
1. Introduction
These mutations are generally internal tandem duplications (ITDs)
in the juxtamembrane domain of the receptor and the missense
The FMS-like tyrosine kinase-3 (FLT3) is a member of the class
III membrane-bound receptor tyrosine kinase (RTK) family, to-
gether with cKIT, FMS, and PDGFR.1,2 Inhibition of RTK signaling
pathways has become an important area of new cancer drug dis-
covery,3–5 particularly for the treatment of acute myeloid leukemia
(AML), the most common type of leukemia in adults. AML is an
aggressive disease with rapid growth of abnormal leukemic cells
in bone marrow that inhibits production of normal blood cells. De-
spite the success of conventional chemotherapy, the rate of relapse
amongst AML patients is relatively high. The failure of initial che-
motherapy and the refractory nature of AML have been associated
with mutations that activate signal transduction pathways, result-
ing in enhanced proliferation and survival of leukemia cells.6,7 Acti-
vating mutations in FLT3 are present in ꢀ30% of AML patients.2,8
point, or short-length mutations in the activation loop of the tyro-
sine kinase domain (TKD).9–11 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 over-
all survival in AML. Also, over-expressed wt-FLT3 has the same
sensitivity to FLT3 inhibitors as the FLT3/ITD mutants.12,13
Due to the prevalence and refractory nature of 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, such as AC220 (1),14 sorafenib
(BAY-43-9006, 2)15 and ABT-869 (3)16 (Fig. 1), have been investi-
gated as potential agents for the treatment of AML.17 However,
early clinical data for direct FLT3 inhibitors has been underwhelm-
ing, with a limited reduction in bone marrow blasts and weak
short-term responses. It has been suggested that this is mainly
due to low potency and/or inadequate toleration of the inhibitors
at effective doses, which together result in a failure to achieve
complete and sustained inhibition of FLT3 in patients’ leukemic
blast cells. Second-generation FLT3 inhibitors which are highly
⇑
Corresponding authors. Tel.: +886 37 246166x35712; fax: +886 37 586456
(W.-T.J.).
E-mail address: wtjiaang@nhri.org.tw (W.-T. Jiaang).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.03.083

W.-H. Lin et al. / Bioorg. Med. Chem. 21 (2013) 2856–2867
2857
Cl
HN
HN
CF3
O
HN
HN
F
O
O
N
O
O
N
N
S
H2N
N
N
O
O
N
N
HN
O
N
H
N
H
H
AC220 (1)
Sorafenib (2)
ABT-869 (3)
O
HN
S
O
O
O
HN
N
N
HN
HN
HN
HN
O
O
N
O
N
4
5
N
FLT3
IC50 = 11 nM
FLT3
IC50 = 10 nM
MOLM-13 GI50 = 21 nM
MOLM-13 GI50 = 2 nM
Figure 1. Previously reported FLT3 inhibitors.
potent against both wt-FLT3 and mutants, highly selective, and less
toxic than their predecessors, are therefore urgently required.14,17,18
Using structure-based design, we previously developed a novel
class of 3-phenyl-1H-5-pyrazolylamine derivatives (Fig. 2), a versa-
tile template for the development of specific kinase inhibitors, and
successfully identified a sulfonamide-substituted series of benzam-
ides (Structure I) inhibiting both FLT3 and cellular proliferation.19
of action in vivo. In this study, the structure–activity relationships
(SAR) and in vitro properties of two urea-substituted series of benz-
amides (Structure K) and pyrimidines (Structure N) are reported.
Western blots illustrating 10f’s mechanism of action are also pre-
sented. This study led to the discovery of urea 10f with greater
in vivo efficacy than sulfonamide 4 and carbamate 5.
Using rational design, we identified
a second sulfonamide-
2. Chemistry
substituted series of pyrimidines (Structure L)19,20 and two carba-
mate-substituted series of benzamides and pyrimidines (Structures
J and M, respectively)21 as potent inhibitors of FLT3 (Fig. 2).
Representative sulfonamide (4) and carbamate (5) FLT3 inhibitors
are shown in Figure 1. Continuing our efforts to develop potent
FLT3 inhibitors, we searched for another series of 3-phenyl-1H-5-
pyrazolylamine-based inhibitors capable of greater inhibition of
AML MOLM-13 cell growth in vitro and with a prolonged duration
The general synthetic route to the 3-phenyl-1H-5-pyrazolyl-
amine benzamides 9 and 10 (Table 1) is depicted in Scheme 1.
The synthesis began with 4-nitro or 4-CH2NHCbz substituted 3-
phenyl-1H-5-pyrazolylamine 6, prepared according to literature
procedures.22,23 Benzoyl chlorides 7 with water-solubilizing sub-
stituents were prepared from the corresponding acids, which were
either commercially available or prepared according to modified
NH
N
R2
NH
H
N
O
O
n
XR1
NH
N
J
X = O
NH
R'
NH
n = 0 or 1
N
R2
NH
K X = NR3
Kinase
Rational design
O
Counterscreening
NH
N
O
R
HN
R1
NH
N
S
O
3-Phenyl-1H-5-
pyrazolylamine-based
derivatives
R2
I
R1
N
L
R1= 3-NHSO2R3
M R1= 4-CH2NHC(O)OR3
N R1= 4-(CH2)nNHC(O)NR3R4
(n = 0 or 1)
Figure 2. Identification of 3-phenyl-1H-5-pyrazolylamine derivatives as FLT3 inhibitors.

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W.-H. Lin et al. / Bioorg. Med. Chem. 21 (2013) 2856–2867
Table 1
Inhibition of enzymes and cell proliferation by urea analogues K
NH
NH
N
N
3
NH
3
NH
R
R
4
4
O
R
R
H
N
1
O
O
O
R
N
N
H
2
N
R
1
2
R
R
10
9
R1, R2
R3
R4
IC50
(lM)
GI50 (lM)
a
a
Compd
wt-FLT3
VEGFR2
Aurora A
MOLM-13 cell
4
5
0.011
0.010
0.13
0.12
0.34
>1.0
0.021
0.002
9a
9b
9c
9d
C6H5, H
C2H5, H
C3H5, H
H
H
H
H
N
N
N
N
N
N
N
N
N
N
0.013
0.094
0.052
0.070
0.026
0.10
0.30
>1.0
0.33
>1.0
>1.0
>1.0
0.006
0.033
0.018
0.037
N
N
, H
, H
9e
9f
H
H
0.040
0.074
0.073
0.19
0.61
0.30
0.042
0.022
N
N
N
N
N
N
N
, H
, H
9g
9h
9i
H
0.050
0.006
0.030
0.009
0.39
0.035
0.031
0.012
0.038
>1.0
0.065
0.12
0.009
0.003
0.005
0.001
0.007
0.002
0.002
0.001
0.008
0.002
CH3
S
O
O
O
O
O
O
O
O
O
N
N
N
N
N
N
N
N
H
CH
3
N
N
N
N
N
N
N
N
, H
H
0.053
0.25
C4H9
, H
9j
CH3
Cl
CH
CH
3
3
, H
9k
9l
0.60
4
O(CH2)2
O(CH2)2
N
N
, H
, H
, H
, H
, H
H
0.015
0.019
0.008
0.020
0.002
0.012
0.022
0.012
0.027
0.007
0.047
0.16
C4H9
C4H9
C4H9
C4H9
C4H9
4
4
4
4
O
9m
9n
9o
9p
H
CH2
N
N
H
0.16
O(CH2)2
N
N
O
O
CH3
CH3
0.069
0.073
CH2
N
N
N
10a
10b
10c
10d
C6H5, H
C6H5, H
4-F-C6H5,
H
H
H
H
4
4
4
4
O(CH2)2
CH2
0.009
0.022
0.066
0.016
0.054
0.082
0.13
0.76
>1.0
>1.0
>1.0
0.003
0.004
0.005
0.003
N
H
O(CH2)2
O(CH2)2
N
N
O
O
4-Cl-C6H5, H
0.071
O
O
O
N
N
N
N
N
, H
10e
10f
10g
H
H
H
0.013
0.018
0.004
0.050
0.028
0.018
0.94
>1.0
>1.0
0.002
0.003
0.003
CH
3
4
4
O(CH2)2
N
O
, H
, H
C4H9
C4H9
CH2
N
N
a
IC50 and GI50 values are the means of at least two independent experiments.
literature procedures.24 These substituted benzoyl chlorides 7
were employed to acylate amines 6 in pyridine to give amides.
Reduction of the nitro or Cbz groups using a Pd/C catalyst under
H2 yielded the corresponding amines 8. These were coupled with
alkyl or benzyl isocyanates, or heteroaryl carbamic acid 4-nitro-
phenyl esters, to afford the corresponding ureas 9 or 10.

W.-H. Lin et al. / Bioorg. Med. Chem. 21 (2013) 2856–2867
2859
NH
N
H
H
N
1
N
O
R
NH
N
N
R'
N=C=O
a
NH
O
b,
2
or
NH
2
R
R'
R'
+
H
N
R"
Cl
R"
O
N
2
1
R
R
n
R"
1
R
R
R
N
O
7
O
K (9 n = 0, 10 n = 1)
8a (R = 4-NH2, R' = H, CH or Cl )
6a (R = 4-NO , R' = H, CH or Cl)
3
2
2
3
O
8b (R = 4-CH NH )
6b (R = 4-CH NHCbz)
2
2
Cl
NO
2
c
N
Cl
N
O
N
N
HN
n
N
HN
HN
HN
HN
R
1
R
1
N
R
R
N=C=O
b,
or
d
2
HN
R
HN
2
N
R
N
N
1
R
N
Cl
N
R'
N
O
R'
N
O
N (13 n = 0, 14 n = 1)
11a (R = 4-NO )
o
2
2
12a (R = 4-NH , R' = 2 amine)
12b (R = 4-CH NH , R' = 2 amine)
2
11b (R = 4-CH NHCbz)
o
2
2
NO
2
Scheme 1. Reagents and conditions: (a) (i) pyridine, rt, (ii) H2, Pd/C, MeOH/HCl (aq); (b) pyridine, rt or DMSO, NaHCO3, 50 °C; (c) 4 N HCl/1,4-dioxane, DMF, 80 °C; (d) (i) 2°
amines, pyridine, 80 °C, (ii) H2, Pd/C, MeOH/HCl (aq).
The general synthetic route to the 3-phenyl-1H-5-pyrazolyl-
amine pyrimidines 13–14 (Table 2) is shown in Scheme 1. Treatment
of 3-phenyl-1H-5-pyrazolylamine 6 with 4,6-dichloropyrimidine,
in the presence of 4 N HCl in 1,4-dioxane and DMF at 80 °C,
provided 4-monosubstituted pyrimidine derivatives 11. Treatment
of these derivatives with substituted or unsubstituted piperazine
in pyridine at 80 °C gave 4,6-disubstituted pyrimidines, which
were reduced with Pd/C under H2 to yield the corresponding
amines 12. These were coupled with alkyl or benzyl-substituted
isocyanates, or heteroaryl carbamic acid 4-nitro-phenyl esters to
obtain the final pyrimidine ureas 13 or14.
3. Biological evaluation
3.1. In vitro pharmacology
As shown in Table 1, the target compounds were tested against
FLT3, vascular endothelial growth factor receptor 2 (VEGFR2), and
Table 2
Inhibition of enzymes and cell proliferation by urea analogues N
NH
N
NH
N
NH
N
NH
N
O
1
R2
H
N
H
N
R
R2
N
H
N
H
1
N
R
N
O
13
14
a
a
Compd
R1
R2
IC50
(lM)
GI50 (lM)
wt-FLT3
0.28
VEGFR2
Aurora A
0.50
MOLM-13 cell
N
N
N
13
C6H5
0.62
0.16
O
O
O
O
O
N
N
14a
0.021
0.035
0.017
0.095
0.10
0.012
0.12
0.05
0.32
0.12
0.098
0.49
0.009
0.003
0.016
0.005
CH
3
N
N
N
N
N
N
N
N
N
N
14b
14c
14d
C4H9
N
CH
3
OH
N
C4H9
C4H9
OH
NH
NH
14e
14f
0.026
0.026
0.085
0.038
0.71
0.79
0.008
0.018
3-Cl-C6H5
1
2
3
0.069
0.054
0.020
0.43
0.020
0.007
>10
3.8
0.76
0.004
0.056
0.037
a
IC50 and GI50 values are the means of at least two independent experiments.

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W.-H. Lin et al. / Bioorg. Med. Chem. 21 (2013) 2856–2867
aurora A kinase (in-house kinas assays), and further evaluated
against the mutant FLT3 AML cell line MOLM-13. Previous studies
showed that sulfonamides 4 and carbamates 5 were potent and
logue 9n in FLT3 enzyme-inhibition assay. Compounds 9o and 9p
both exhibited potent inhibition against VEGFR2 (IC50 <30 nM)
and aurora A (IC50—70 nM) kinases.
selective FLT3 inhibitors (FLT3 IC50 = 0.01
l
M; VEGFR2 and aurora
Having demonstrated the importance of the urea pharmaco-
phore, SAR studies to investigate the effect of chain length between
the urea moiety and the 4-position of the phenyl ring were carried
out (benzylurea compounds 10a–g, Table 1). When R1 was a phe-
nyl group, compounds 10a and 10b (possessing different water-
solubilizing substituents) inhibited FLT3 and MOLM-13 cell growth
with potencies comparable to homolog 9a. para-Fluorination or
chlorination of the R1 phenyl ring had no impact in the MOLM-
A IC50 >0.1 M, Table 1). An extensive SAR study was firstly carried
l
out, replacing the carbamate moiety of J (n = 0, Fig. 2) with a urea
moiety to give potential K-based FLT3 inhibitors. These have a urea
group at the 4-position of the phenyl ring, connected to pyrazolyl-
amine at the 3-position (phenylurea compounds 9a–p). The first
compound 9a, with a phenyl substituent at R1 and a 4-methylpi-
perazine at R4, was found to potently inhibit FLT3 (IC50 = 0.013
l
M)
M). Compound 9a also weakly inhibited
M) and potently inhibited growth of MOLM-
M). A significant drop in potency was ob-
and VEGFR2 (IC50 = 0.026
aurora A (IC50 = 0.33
13 cells (GI50 = 0.006
l
13 assay (p-fluoro 10c: GI50 = 0.005
GI50 = 0.003 M), but p-fluoro compound 10c was almost sevenfold
less active than the unsubstituted analogue 10a against FLT3 (10c:
FLT3 IC50 = 0.066 M). These phenyl-substituted ureas 10a–d had
lM, and p-chloro 10d:
l
l
l
served in all assays when the terminal phenyl ring of the urea
was replaced by alkyl groups such as ethyl (9b), cyclopropyl (9c),
or amine NR1R2, together yielding a five-membered monocyclic
ring (9d).
l
slightly decreased inhibitory potencies against VEGFR2 and aurora
A compared to homolog 9a. Because the SAR of the terminal het-
eroaryl benzylureas 10 was similar to phenylureas 9, only repre-
sentative isoxazol-3-yl compounds 10e–g are shown in Table 1.
Compounds 10e–g potently inhibited FLT3 (IC50 = 0.004–
Compound 9a, bearing a phenyl substituent at R1, is a potent
FLT3 inhibitor; however, it is poorly soluble in organic solvents.
To overcome this problem, the R1 substituent of 9a was changed
from a phenyl group to heteroaryl groups. As expected, compounds
9e–p were more soluble in organic solvents, and easily purified by
flash column chromatography. In comparison with 9a, pyridin-2-yl
(9e) and pyridin-3-yl rings (9f) decreased inhibitory activity
against wt-FLT3 and VEGFR2, and also against MOLM-13 cells.
With a five-membered heteroaryl ring, thiazol-2-yl compound 9g
0.018
l
M), VEGFR2 (IC50 = 0.018–0.050
lM) and MOLM-13 cells
(GI50 = 0.002–0.003
lM). However, significant loss of potency
against aurora A was observed for these three compounds com-
pared to analogues 9h and 9m–n, respectively.
According to prior reports, the 3-aminopyrazole moiety is
conformationally well suited to form hydrogen bond interactions
with the kinase hinge region of the ATP pocket,25 and 2-methyl-
pyrimidine is a known scaffold for kinase inhibitors.26 Accordingly,
we replaced the benzamide group with a 2-methyl-pyrimidine
group bearing a solubilizing substituent at the 4-position, giving
phenylureas 13 and benzylureas 14a–f (N, Fig. 2). As shown in
Table 2, pyrimidine 13, with a phenyl substituent at R1 and a
water-solubilizing N-methylpiperazine group at R2, was less po-
was 4 times less potent against FLT3 (IC50 = 0.05
tained potency against MOLM-13 cells (IC50 = 0.009
to phenyl compound 9a. 5-Methyl-isoxazole 9h and 5-tert-butyl-
isoxazole 9i potently inhibited growth of MOLM-13 cells
(GI50 = 0.003 and 0.005 lM, respectively); but 5-tert-butyl-isoxaz-
ole 9i was only half as potent as 9a in FLT3 inhibition. Five-mem-
lM), but main-
lM) relative
bered heteroaryl compounds 9g–i were potent VEGFR2 inhibitors
tent against FLT3 (IC50 = 0.28
lM), VEGFR2 (IC50 = 0.62 lM) and
(IC50 = 0.012–0.035
lM), and potent to moderate inhibitors of aur-
MOLM-13 cells (GI50 = 0.16 M) than the related benzamide 9a
l
ora A (IC50 = 0.053–0.12
l
M).
and was, therefore, not optimized further. Nevertheless, benzylu-
rea compounds 14a–f (based upon a 2-methyl-pyrimidine scaf-
fold) were found to have promising potency in vitro. Retaining
the N-methylpiperazine group, isoxazol-3-yl ureas 14a and b
were found to be potent FLT3 inhibitors, with IC50 values of
Next, we examined the effects of the R3 substituent on the phe-
nyl ring connected to the pyrazolylamine group at the 3-position.
Compound 9j, with a methyl substituent at R3, potently inhibited
FLT3 and VEGFR2 (IC50 = 0.009 and 0.038
lM, respectively), and
very
(GI50 = 0.001
potently
inhibited
growth
of
MOLM-13 cells
0.021 and 0.035
growth of MOLM-13 cells with GI50 values of <0.01 l
l
M, respectively. Both of them also inhibited
l
M). Chlorination at R3 (9k) dramatically decreased
M. How-
potency against FLT3 and VEGFR2 (FLT3 IC50 = 0.39
IC50 >1.0 M). Nevertheless, 9k still exhibited a GI50 value of
0.007 M against MOLM-13 cells.
l
M, VEGFR2
ever, 5-tert-butyl-isoxazole 14b was a more potent inhibitor of
VEGFR2, aurora A and MOLM-13 cell growth than 5-methyl-
isoxazole 14a.
l
l
Previous studies have demonstrated the importance of a water-
solubilizing substituent on the benzamide for optimal activity.20,21
The SAR of ureas with various water-solubilizing benzamide sub-
stituents (R4) was similar to that displayed in the carbamate ser-
Next, we investigated the effects of water-solubilizing substit-
uents on the 4-position of the pyrimidine ring and compared the
potencies of compounds 14c–e with N-methylpiperazine ana-
logues 14a and 14b. N-(2-hydroxyethyl)piperazine 14c had sim-
ilar potency to N-methylpiperazine 14a in FLT3, VEGFR2, and
MOLM-13 cell proliferation assays but was threefold more po-
tent against aurora A. In contrast, 5-tert-butyl-isoxazole 14d
ies.21 Conserving the 5-tert-butyl-isoxazole urea moiety,
a
pyrrolidine (9l) or morpholine group (9m) tethered by a two-car-
bon ether or a N-methylpiperazine group (9n) linked by a one-car-
bon tether potently inhibited FLT3 and VEGFR2, comparable in
potency to N-methylpiperazine 9i in both cases (FLT3
bearing
a N-(2-hydroxyethyl)piperazine group was three- to
fourfold weaker in the FLT3, VEGFR2 and aurora A assays, but
maintained potency in the cellular assay compared to 14b. Fur-
thermore, piperazine 14e was about as potent as 14b against
FLT3 and six- to sevenfold less potent than 14b in inhibiting
VEGFR2 and aurora A. Piperazine 14e strongly inhibited growth
IC50 = 0.008–0.019
pounds 9l–n were more potent against MOLM-13 cells
(GI50 = 0.001–0.002 M) than the N-methylpiperazine analogue 9i
(GI50 = 0.005 M). 5-tert-Butyl-isoxazole ureas 9i and 9l–n bearing
different water-solubilizing substituents displayed moderate to
potent inhibition of aurora A (IC50 = 0.047–0.16
M). When R3
lM, VEGFR2 IC50 = 0.012–0.022 lM). Com-
l
l
of MOLM-13 cells, with a GI50 value of 0.008 lM. Replacing
l
the 5-tert-butyl-isoxazole ring of 14e with 3-chloro substituted
benzene ring (14f) had no significant effect on potency in any
assay. In comparison with FLT3 inhibitor ureas 1, 2 and 3 cur-
rently in clinical development (Table 2), the inhibition of FLT3-
mutant MOLM-13 cell growth by some potent urea compounds
(K and N) is comparable to 1 (GI50 = 4 nM) and greater than 2
and 3 (GI50 = 56 and 37 nM, respectively).
was substituted with a methyl group, the resulting compound 9o
showed a fourfold lost of potency compared to the unsubstituted
analogue 9m (R3 = H). Compound 9p bearing an N-methylpipera-
zine group was the most potent FLT3 inhibitor (IC50 = 0.002
in this series. It had a GI50 value of 0.002 M against MOLM-13
cells and was fourfold more potent than the R3 unsubstituted ana-
lM)
l

W.-H. Lin et al. / Bioorg. Med. Chem. 21 (2013) 2856–2867
2861
Table 3
3.2. Kinase inhibition profiles of 9p and 10f
Kinase inhibition profiles of compounds 9p and 10f
To better understand enzyme inhibition by these potent com-
pounds, 9p (a phenylurea) and 10f (a benzylurea) were assayed
against a panel of selected protein tyrosine kinases at a concentra-
Kinase
% Inhibition @ 1 l
Ma
10f
9p
ABL1
59
43
21
tion of 1.0
inhibited CSF1R (P95%), the VEGFR family (VEGFR1, 2 and 3,
P86%), FLT3 (100%), PDGFR- and -b (P85%), TRKA (P95%), RET
lM. As shown in Table 3, both compounds potently
ABL1 G250E
AKT1 (PKB alpha)
AMPK A1/B1/G1
AURKA (aurora A)
AURKB (aurora B)
AURKC (aurora C)
BRAF
NTb
ꢁ17
94
ꢁ20
70
a
66 (97)c
79
(48)
79
(P97%) and Tie2 (P83%). Regarding other, structurally unrelated
tyrosine kinases, phenylurea 9p moderately inhibited AMPK A1/
B1/G1 (70%), aurora B (79%) and SRC (77%), while benzylurea 10f
strongly inhibited AMPK A1/B1/G1 (94%), JAK1 (82%), TYK2 (86%),
aurora B and JAK2 (79% in each case).
16
15
NT
NT
NT
ꢁ6
ꢁ2
5
(2)
(10)
95
BTK
CDC42 BPA (MRCKA)
CDK2/cyclin A
CHEK1 (CHK1)
CHEK2 (CHK2)
CSF1R (FMS)
EPHA1
EPHB2
ERBB2 (HER2)
FGFR1
ꢁ5
16
ꢁ9 (1)
37 (68)
97
3.3. Antiproliferative effects of 9p and 10f on leukemia cell lines
NT
NT
11
NT
ꢁ20
We evaluated the effects of 9p and 10f on growth of leukemia
cells with or without FLT3 mutations (Table 4). In addition to
MOLM-13, both compounds inhibited growth of human AML
19
ꢁ1
48
99
100
(86)
(ꢁ1)
6
MV4;11 cells harboring FLT3/ITD, with a GI50 of 0.002 lM. Com-
FLT1 (VEGFR1)
FLT3
90
pounds 9p and 10f were as potent as FLT3 inhibitor 1, and at least
15-fold more potent than 2 and 3, in MOLM-13 and MV4;11 cells.
In RS4;11 cells (FLT3-wt), the antiproliferative effects of 9p and 10f
100
93 (102)
ꢁ1 (3)
24
FLT4 (VEGFR3)
FRAP1 (mTOR)
GSK3A (GSK3 alpha)
GSK3B (GSK3 beta)
IGF1R
IKBKB (IKK beta)
IKBKE (IKK epsilon)
JAK1
were modest (GI50 = 0.4 and 0.9 lM, respectively). Phenylurea 9p
13
33
NT
12
35
44
NT
2
was weakly cytotoxic against K562 human chronic myeloid leuke-
mia cells (expressing wt-BCR/ABL)13 and U937 human leukemic
monocyte lymphoma cells (not expressing FLT3);27 GI50 = 2.3 and
ꢁ1
ꢁ2
ꢁ5
82
79
27
5.5
lM, respectively. In contrast, benzylurea 10f did not signifi-
JAK2
JAK3
cantly inhibit growth of K562 cells and U937 cells (GI50 >10
lM).
Both the cell lines, K562 and U937, are not FLT3-dependent cells.
The results reveal the specificity of compound 10f on FLT3 inhibi-
tion. Of the urea compounds shown in Table 4, we found that the
growth-inhibitory profile of 10f against various leukemia cell lines
was similar to that of urea 1; 2 appeared to induce lower growth
KDR (VEGFR2)
KIT
97 (88)
71
5
NT
51
2
(28)
6
(99)
68
13
MAP2K1 (MEK1)
MAPK10 (JNK3)
MAPK14 (p38 alpha)
MAPK8 (JNK1)
MET (cMet)
MST1R (RON)
NTRK1 (TRKA)
PDGFRA (PDGFR alpha)
PDGFRB (PDGFR beta)
RAF1 (cRAF) Y340D Y341D
RET
RPS6KB1 (p70S6K)
SRC
TEK (Tie2)
TYK2
TYRO3 (RSE)
ꢁ3
3
ꢁ3
2 (40)
13
inhibition against RS4;11 (GI50 = 9.3 lM).
95
98
3.4. Inhibition of FLT3 and STAT5 phosphorylation in cells
94 (95)
87 (99)
6
99
49
77
90
3
NT
(85)
(97)
NT
97
41
35
83
86
24
Compound 10f showed promising antiproliferative activity
against the human AML cell lines MOLM-13 and MV4;11, with
GI50 values of 3 and 2 nM, respectively. To examine whether 10f
inhibited phosphorylation of FLT3 and downstream signaling path-
ways in FLT3-driven cells, MV4;11 cells were cultured with differ-
ent concentrations of 10f for 1 h; inhibition of FLT3 and STAT5
phosphorylation was then determined by Western blot analysis
(Fig. 3). It was found that compound 10f suppressed the phosphor-
ylation of FLT3 and STAT5 in MV4;11 cells, with IC50 values of 1 nM
against p-FLT3 and p-STAT5, respectively.
a
Percentage inhibition was determined by Invitrogen SelectScreen kinase pro-
filing service.
b
NT = not tested.
Parenthesis means in-house Kinase-Glo assay.
c
3.5. Pharmacokinetics, toxicity and in vivo efficacy
Table 4
Inhibition of leukemia cell growth by 9p and 10f
5-tert-Butyl-isoxazole compounds 9o, 9p, 10f and 10g were se-
lected for pharmacokinetic evaluation after intravenous (2 mg/kg)
or oral (10 mg/kg) administration to rats (Table 5). After iv dosing,
each compound demonstrated moderate to long half-life (5.3–
Cell line
Characterization
GI50
10f
(lM)
9p
1
2
MOLM-13
MV4;11
RS4;11
K562
AML-FLT3-ITD
AML-FLT3-ITD
ALL-wt-FLT3
CML-Bcr-Abl FLT3-negative
AML-FLT3-negative
0.003
0.002
0.4
2.3
5.5
0.003
0.002
0.9
11.8
>20
0.004
0.003
2.2
>20
>20
0.056
0.043
9.3
7.3
3.4
17 h), favorable drug exposure (AUC0–inf.
) (2118–3145 ng/ml),
moderate plasma clearance (10.7–13.2 ml/min/kg) and moderate-
to-high volume of distribution (3.6–10 l/kg), but oral bioavailabil-
ity was less than 5%. Due to this poor oral bioavailability in rats,
these four compounds were administered intravenously for evalu-
ation of efficacy and acute toxicity in mice. Before proceeding, tox-
icity was evaluated to allow selection of the least toxic compound
and to determine the proper dose range for evaluation of efficacy in
the tumor xenograft model. Compounds 9o, 9p, 10f and 10g were
administered intravenously (25 mg/kg) once daily for 5 consecu-
tive days, in normal mice. Compound 10f was the safest of these
U937
AML, acute myelocytic leukemia; ALL, acute lymphoblastic leukemia; CML, chronic
myelogenous leukemia.
compounds. It was well tolerated; no loss of body weight, and no
mortality, were observed. Based on its enzyme-inhibitory and
antiproliferative potency as well as its favorable pharmacokinetic

2862
W.-H. Lin et al. / Bioorg. Med. Chem. 21 (2013) 2856–2867
to be 66% on day 11. At 3 mg/kg, tumor growth was inhibited
throughout the dosing period with 7% tumor regression on day
11; tumor growth resumed soon after dosing was halted. At
10 mg/kg, near-complete tumor regression was observed in all ani-
mals by day 16 (84% tumor regression); complete regression (CR)
occurred in 5 of 7 treated animals during 48 days of post-treatment
observation. No loss of body weight was observed in animals trea-
ted with any dose of 10f. In comparison with sulfonamide 4 (1/8
CR, 25 mg/kg)19 and carbamate 5 (3/7 CR, 25 mg/kg)21 in the same
model, 10f gave a higher CR ratio (70%) at a lower dose (10 mg/kg).
These results demonstrate that 10f effectively reduces tumor size
with 2 weeks of treatment at an intravenous dose of 10 mg/kg/
day; 10f is well tolerated in mice at the effective dose.
Figure 3. Compound 10f inhibits FLT3 phosphorylation in MV4;11 AML cells,
which are homozygous for FLT-ITD. Cells were treated with 10f at the indicated
concentrations for 1 h. Phosphorylation of FLT3 and STAT5 was evaluated by
Western blot analysis.
4. Conclusion
A rational design approach has been used to identify a novel class
of 3-phenyl-1H-5-pyrazolylamine-based urea derivatives, which in-
clude benzamide (K) and pyrimidine (N) FLT3 inhibitors. SAR was
used to optimize these series, yielding potent FLT3 inhibitors, some
of which exhibited comparable antiproliferative potency to the
known FLT3 inhibitor 1, being at least 10-fold more potent than 2
and 3 in MOLM-13 and MV4;11 cells. Broad kinase profiling indi-
cated that phenylurea 9p and benzylurea 10f inhibited VEGFR1-3,
Table 5
Pharmacokinetic parameters of compounds 9o, 9p, 10f and 10g in ratsa
Parameter
Unit
Compound
9o
9p
10f
10g
AUC0–inf.
CL
Vdss
t1/2
F
ng/ml h
ml/min/kg
l/kg
h
%
2856
13.9
4.7
5.3
4
3015
11.3
10
3145
10.7
3.6
6.2
1
2118
17.2
8.0
PDGFRa, PDGFRb, CSF1R and RET by P85% at 1 lM. In addition,
10f downregulated the phosphorylation of FLT3/STAT5 in MV4;11
cells, with an IC50 of 1 nM, and showed high efficacy and tolerability
in the MOLM-13 human AML xenograft model.
17
11
NTb
NDc
a
b
c
Sprague–Dawley rats were dosed at 2 mg/kg iv (n = 3).
NT: not tested.
ND: not detected.
5. Experimental
5.1. Chemistry methods
All commercial chemicals and solvents are reagent grade and
were used without further treatment unless otherwise noted. 1H
NMR spectra were obtained with a Varian Mercury-300 or a Varian
Mercury-400 spectrometer. Chemical shifts were recorded in parts
per million (ppm, d) 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 mea-
sured with a Thermo Finnigan (TSQ Quantum) electrospray ioniza-
tion (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 ꢂ 10 cm); zones were detected visually
under ultraviolet irradiation (254 nm) or by spraying with phos-
phomolybdic acid reagent (Aldrich) followed by heating at 80 °C.
All starting materials and amines were commercially available un-
less otherwise indicated. The purity of compounds was determined
by a Hitachi 2000 series HPLC system. Except for compounds 9k
and 9n, purity of target compounds 9 and 10 were over 95% based
on reverse phase C18 column (Agilent ZORBAX Eclipse XDB-C18
Figure 4. Compound 10f demonstrates antitumor efficacy after intravenous (iv)
administration in the MOLM-13 xenograft model (qd, days 1–5 and 8–12). Tumor
size is expressed as the mean SEM (n = 6–8/group).
profile and low toxicity, compound 10f was chosen for further
in vivo evaluation.
5
l
m, 4.6 mm ꢂ 150 mm) and reverse phase phenyl column
FLT3-ITD-bearing MV4;11 and MOLM-13 xenografts have been
widely used to evaluate the in vivo efficacy of FLT3 inhibitors
against AML.7,13 We19,20 and others7,28 have found the MV4;11
xenograft model to be noticeably more sensitive to FLT3 inhibitors.
To assess the ability of compound 10f to inhibit tumor growth in
an AML xenograft model, efficacy testing of 10f was therefore con-
ducted using a MOLM-13 tumor xenograft model (Fig. 4). Com-
pound 10f was administered intravenously (1, 3 and 10 mg/kg
qd) on days 1–5 and 8–12; by the end of this period the MOLM-
13 tumors reached an average volume of 200 mm3. At 1 mg/kg, tu-
mor growth was inhibited for the first 5 days, after which tumor
growth resumed and tumor growth inhibition (TGI) was observed
(Waters XBridge Phenly 5
t
m, 4.6 mm ꢂ 150 mm) under the fol-
lowing gradient elution condition: Mobile phase A-acetonitrile
(10–90%, 0–45 min) and mobile phase B-10 mM NH4OAc aqueous
solution containing 0.1% formic acid (90–10%, 0–45 min). The
flow-rate was 0.5 ml/min and the injection volume was 20 ll.
The system operated at 25 °C. Peaks were detected at k = 254 nm.
5.2. General procedure for the preparation of compounds 9 and
10
The starting materials 6a and 6b were synthesized according to
the literature report.22,23 Pyrazolylamine 6 (1.0 equiv) in pyridine

W.-H. Lin et al. / Bioorg. Med. Chem. 21 (2013) 2856–2867
2863
at room temperature was added acyl chloride 7 (1.2 equiv). After 1 h
of stirring at room temperature, the reaction mixture was evapo-
rated to dryness and the residue suspended in saturated NaHCO3
solution. The suspension was vigorously stirred at room tempera-
ture for 1 h, and the resultant solid was collected by filtration and
dried in vacuo. The crude amide was used for the next reaction with-
out further purification. To a solution of crude amide prepared from
last step in MeOH was added 6 N HCl (5.0 equiv) and a catalytic
amount of 10% Pd/C at room temperature. The mixture was vigor-
ously stirred at room temperature under an atmospheric pressure
of hydrogen for 12 h. The catalyst was removed by filtration and
the filtrate was concentrated in vacuo. The crude product was puri-
fied by flash column chromatography (eluted with CH2Cl2/CH3OH)
yielded the desired amine 8. Only representative compounds 8a
and one of 8b are selected to show their NMR and mass data.
s, 1H), 3.40–3.20 (m, 4H, overlapping with water peak), 2.64–
2.60 (m, 4H), 2.35 (s, 3H); MS (ES+) m/z calcd for C28H29N7O2:
495.24; found: 496.2 (M+H+); HRMS (ESI) calcd for C28H30N7O2:
496.2455; found: 496.2465 (M+H+).
5.2.6. N1-[3-(4-[(Ethylamino)carbonyl]aminophenyl)-1H-5-
pyrazolyl]-4-(4-methylpiperazino)benzamide (9b)
Mp 322–324 °C. 1H NMR (300 MHz, DMSO-d6): d 12.67 (s, 1H),
10.47 (s, 1H), 8.57 (s, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz,
2H), 7.46 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 6.90 (br s, 1H),
6.17 (t, J = 5.3 Hz, 1H), 3.29–3.26 (m, 4H), 3.16–3.07 (m, 2H), 2.45–
2.42 (m, 4H), 2.22 (s, 3H), 1.06 (t, J = 7.2 Hz, 2H); MS(ES+) m/z calcd
for C24H29N7O2: 447.24; found: 448.1 (M+H+); HRMS (ESI) calcd for
C
24H30N7O2: 448.2455; found: 448.2465 (M+H+).
5.2.7. N1-[3-(4-[(Cyclopropylamino)carbonyl]aminophenyl)-
1H-5-pyrazolyl]-4-(4-methylpiperazino)benzamide (9c)
5.2.1. N1-[3-(4-Aminophenyl)-1H-5-pyrazolyl]-4-(4-
Mp 258–259 °C. 1H NMR (400 MHz, DMSO-d6): d 12.68 (s, 1H),
10.48 (s, 1H), 8.48 (s, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 8.4 Hz,
2H), 7.47 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 8.8 Hz, 2H), 6.90 (br s, 1H),
6.49 (s, 1H), 3.27 (m, 4H), 4.54 (m, 1H), 2.44 (m, 4H), 2.22 (s, 3H),
0.64 (td, J = 3.4 Hz, 6.0 Hz, 2H), 0.41 (m, 2H); MS (ES+) m/z calcd for
methylpiperazino)benzamide (8aa, R0 = H)
Mp 251–252 °C. 1H NMR (400 MHz, DMSO-d6): d 12.41 (s, 1H),
10.43 (s, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 7.6 Hz, 2H), 6.96 (d,
J = 8.8 Hz, 2H), 6.71 (s, 1H), 6.59 (d, J = 8.0 Hz, 2H), 5.30 (s, 2H), 3.26
(t, J = 4.2 Hz, 4H), 2.42 (t, J = 4.4 Hz, 4H), 2.21(s, 3H); MS (ES+) m/z
calcd for C21H24N6O: 376.20; found: 377.2 (M+H+), 399.2 (M+Na+).
C
C
25H29N7O2: 459.24; found: 460.2 (M+H+); HRMS (ESI) calcd for
25H30N7O2: 460.2455; found: 460.2465 (M+H+).
5.2.2. N1-[3-(4-Amino-3-methylphenyl)-1H-5-pyrazolyl]-4-(4-
methylpiperazino)benzamide (8ab, R0 = CH3)
5.2.8. N1-[3-(4-[(Pyrrolid-1-yl)carbonyl]aminophenyl)-1H-5-
pyrazolyl]-4-(4-methylpiperazino)benzamide (9d)
Mp 272–274 °C. 1H NMR (300 MHz, DMSO-d6): d 12.40 (s, 1H),
10.38 (s, 1H), 7.91 (d, J = 8.7 Hz, 2H), 7.31 (s, 1H), 7.27 (d,
J = 8.4 Hz, 1H), 6.96 (d, J = 8.7 Hz, 2H), 6.74 (s, 1H), 6.63 (d,
J = 8.4 Hz, 1H), 5.06 (s, 2H), 3.26 (br s, 4H), 2.42 (br s, 4H), 2.21
(s, 3H), 2.09 (s, 3H); MS (ES+) m/z calcd for C22H26N6O: 390.21;
found: 391.0 (M+H+), 413.0 (M+Na+).
Mp 304–305 °C. 1H NMR (400 MHz, DMSO-d6): d 12.68 (s, 1H),
10.50 (s, 1H), 8.24 (s, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.60 (s, 4H),
6.98 (d, J = 8.4 Hz, 2H), 6.90 (s, 1H), 3.38 (t, J = 6.0 Hz, 4H over-
lapped with water peak), 3.28 (t, J = 6.4 Hz, 4H), 2.46 (t,
J = 6.4 Hz, 4H), 2.23 (s, 3H), 1.86 (t, J = 6.0 Hz, 4H); MS (ES+) m/z
calcd for C26H31N7O2: 473.25; found: 474.3 (M+H+); HRMS (ESI)
calcd for C26H32N7O2: 474.2612; found: 474.2622 (M+H+).
5.2.3. N1-[3-(4-Amino-3-chlorophenyl)-1H-5-pyrazolyl]-4-(4-
methylpiperazino)benzamide (8ac, R0 = Cl)
5.2.9. N1-[3-(4-[(2-Pyridylamino)carbonyl]aminophenyl)-1H-5-
pyrazolyl]-4-(4-methylpiperazino)benzamide (9e)
1H NMR (300 MHz, DMSO-d6): d 12.55 (s, 1H), 10.45 (s, 1H),
7.91 (d, J = 9.0 Hz, 2H), 7.60 (d, J = 1.8 Hz, 1H), 7.40 (dd, J = 8.7,
1.8 Hz, 1H), 6.81 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.4 Hz, 2H), 5.57
(s, 2H), 3.31–3.29 (m, 4H), 2.51–2.49 (m, 4H), 2.26 (s, 3H); MS
(ES+) m/z calcd for C21H23ClN6O: 410.16; found: 411.0 (M+H+).
Mp 300–302 °C. 1H NMR (300 MHz, DMSO-d6): d 10.68 (s, 1H),
10.49 (s, 1H), 9.51 (s, 1H), 8.31 (dd, J = 1.2, 4.8 Hz, 1H), 7.93 (d,
J = 8.4 Hz, 2H), 7.82–7.55 (m, 5H), 7.50 (d, J = 8.4 Hz, 2H), 7.10–
6.90 (m, 3H), 3.28 (t, J = 4.8 Hz, 4H), 2.44 (t, J = 4.8 Hz, 4H), 2.22
(s, 3H); MS (ES+) m/z calcd for C27H28N8O2: 496.23; found: 497.0
(M+H+); HRMS (ESI) calcd for C27H29N8O2: 497.2408; found:
497.2412 (M+H+).
5.2.4. N1–3-[4-(Aminomethyl)phenyl]-1H-5-pyrazolyl-4-(4-
methylpiperazino)benzamide (8b, R0 = H)
Mp 164–165 °C. 1H NMR (400 MHz, DMSO-d6): d 7.89 (d,
J = 8.8 Hz, 2H), 7.61 (d, J = 8.4 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H),
7.12 (d, J = 8.0 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.75 (d, J = 8.8 Hz,
2H), 4.01 (s, 1H), 3.61 (s, 1H), 3.10 (br s, 4H), 2.54 (br s, 4H), 2.22
(s, 3H); MS (ES+) m/z calcd for C22H26N6O: 390.22; found: 391.2
(M+H+).
5.2.10. N1-[3-(4-[(3-Pyridylamino)carbonyl]aminophenyl)-1H-
5-pyrazolyl]-4-(4-methylpiperazino)benzamide (9f)
Mp 191–193 °C. 1H NMR (400 MHz, DMSO-d6): d 12.75 (s, 1H),
10.53 (s, 1H), 9.53 (s, 2H), 8.62 (d, J = 2.4 Hz, 1H), 8.20 (dd, J = 1.2,
4.8 Hz, 1H), 7.96–7.93 (m, 3H), 7.68 (d, J = 8.8 Hz, 2H), 7.54 (d,
J = 8.4 Hz, 2H), 7.32 (dd, J = 8.4, 4.4 Hz, 1H), 6.99 (d, J = 8.8 Hz,
2H), 6.94 (br s, 1H), 3.34 (m, 4H overlapped with water peak),
2.49–2.54 (m, 4H), 2.33 (s, 3H); MS (ES+) m/z calcd for
C27H28N8O2: 496.23; found: 497.0 (M+H+); HRMS (ESI) calcd for
To a solution of 8 (1.0 equiv) in pyridine at room temperature or
in DMSO and NaHCO3 (4.0 equiv) at 50 °C was added alkyl or ben-
zyl isocyanate or heteroaryl carbamic acid 4-nitro-phenyl ester
(1.2 equiv). After 1 h of stirring at room temperature, the reaction
mixture was evaporated to dryness and the residue suspended in
saturated NaHCO3 solution. The suspension was vigorously stirred
at room temperature for 1 h before the resultant solid was col-
lected by filtration and dried in vacuo. The crude product was puri-
fied by flash column chromatography (eluted with CH2Cl2/CH3OH)
yielded the desired phenylurea 9 or benzylurea 10.
C
27H29N8O22: 497.2408; found: 497.2417 (M+H+).
5.2.11. N1-3-[4-([(5-Methyl-1,3-thiazol-2-
yl)amino]carbonylamino)phenyl]-1H-5-pyrazolyl-4-(4-
methylpiperazino)benzamide (9g)
Mp 275–277 °C. 1H NMR (300 MHz, DMSO-d6): d 10.52 (s, 1H),
10.49 (s, 1H), 9.09 (s, 1H), 7.93 (d, J = 9.0 Hz, 2H), 7.89 (d,
J = 9.0 Hz, 1H), 7.81 (d, J = 8.7 Hz, 1H), 7.69 (d, J = 8.7 Hz, 2H),
7.60 (d, J = 8.7 Hz, 1H), 7.55 (d, J = 8.7 Hz, 2H),7.04–6.97 (m, 3H),
3.29 (t, J = 4.8 Hz, 4H), 2.45 (t, J = 4.8 Hz, 4H), 2.32 (s, 3H), 2.23 (s,
3H); MS (ES+) m/z calcd for C26H28N8O2S: 516.21; found: 517.0
(M+H+); HRMS (ESI) calcd for C26H29N8O2S: 517.2129; found:
517.2136 (M+H+).
5.2.5. N1-(3-4-[(Anilinocarbonyl)amino]phenyl-1H-5-
pyrazolyl)-4-(4-methylpiperazino)benzamide (9a)
Mp 337–338 °C. 1H NMR (400 MHz, DMSO-d6): d 12.68 (br s,
1H), 10.54 (s, 1H), 9.22 (s, 1H), 9.11 (s, 1H), 7.93 (d, J = 8.8 Hz,
2H), 7.65 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.8 Hz, 2H), 7.45 (d,
J = 7.6 Hz, 2H), 7.27 (t, J = 7.6 Hz, 2H), 7.02–6.88 (m, 3H), 6.86 (br

2864
W.-H. Lin et al. / Bioorg. Med. Chem. 21 (2013) 2856–2867
5.2.12. N1–3-[4-([(5-Methyl-3-
isoxazolyl)amino]carbonylamino)phenyl]-1H-5-pyrazolyl-4-(4-
methylpiperazino)benzamide HCl salt (9h)
C30H35N7O5: 573.27; found: 574.2 (M+H+); HRMS (ESI) calcd for
C30H36N7O5: 574.2772; found: 574.2786 (M+H+).
Mp 243–245 °C. 1H NMR (400 MHz, DMSO-d6): d 10.64 (s, 1H),
10.42 (br s, 1H), 9.61 (s, 1H), 9.23 (s, 1H), 7.97 (d, J = 8.8 Hz, 2H),
7.67 (d, J = 8.8 Hz, 2H), 7.51 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 8.8 Hz,
2H), 6.88 (s, 1H), 6.54 (s, 1H), 4.08–4.00 (m, 2H), 3.54–3.40 (m,
2H, overlapped with water peak), 3.18–3.06 (m, 4H), 2.81 (d,
J = 4.4 Hz, 3H), 2.35 (s, 3H); MS (ES+) m/z calcd for C26H28N8O3:
500.23; found: 501.1 (M+H+); HRMS (ESI) calcd for C26H29N8O3:
501.2357; found: 501.2368 (M+H+).
5.2.18. N1-(3-4-[([5-(tert-Butyl)-3-
isoxazolyl]aminocarbonyl)amino]phenyl-1H-5-pyrazolyl)-4-
[(4-methylpiperazino)methyl]benzamide (9n)
Mp 247–248 °C. 1H NMR (300 MHz, DMSO-d6): d 10.79 (s, 1H),
9.73 (s, 1H), 9.23 (s, 1H), 7.98 (d, J = 8.1 Hz, 2H), 7.69 (d,
J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H),
6.97 (s, 1H), 6.52 (s, 1H), 3.51 (d, J = 5.1 Hz, 2H), 3.28 (t,
J = 4.8 Hz, 4H), 2.37 (t, J = 4.8 Hz, 4H), 2.16 (s, 3H), 1.30 (s, 9H);
MS (ES+) m/z calcd for C30H36N8O3: 556.29; found: 557.4 (M+H+);
HRMS (ESI) calcd for C30H37N8O3: 557.2983; found: 557.2998
(M+H+). HPLC (C18 column): tR = 22.29 min, 93.3%. HPLC (phenyl
column): tR = 23.54 min, 93.9%.
5.2.13. N1-(3-4-[([5-(tert-Butyl)-3-
isoxazolyl]aminocarbonyl)amino]phenyl-1H-5-pyrazolyl)-4-(4-
methylpiperazino)benzamide (9i)
Mp 332–334 °C. 1H NMR (400 MHz, DMSO-d6): d 12.74 (s, 1H),
10.51 (s, 1H), 9.58 (s, 1H), 8.99 (s, 1H), 7.92 (d, J = 9.2 Hz, 2H), 7.69
(d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H), 6.99–6.92 (m, 3H), 6.53
(s, 1H), 3.29–3.27 (m, 4H), 2.47–2.43 (m, 4H), 2.23 (s, 3H), 1.27 (s,
9H); MS(ES+) m/z calcd for C29H34N8O3: 542.28; found: 543.1
(M+H+); HRMS (ESI) calcd for C29H35N8O3: 543.2827; found:
543.2832 (M+H+).
5.2.19. N1-(3-4-[([5-(tert-Butyl)-3-
isoxazolyl]aminocarbonyl)amino]-3-methylphenyl-1H-5-
pyrazolyl)-4-(2-morpholinoethoxy)benzamide (9o)
Mp 258–260 °C. 1H NMR (300 MHz, DMSO-d6): d 12.77 (s, 1H),
10.69 (s, 1H), 9.98 (s, 1H), 9.41 (s, 1H), 8.38 (s, 1H), 8.01 (t,
J = 9.9 Hz, 3H), 7.57 (t, J = 9.9 Hz, 2H), 7.31 (d, J = 5.4 Hz, 2H), 7.05
(s, 1H), 6.48 (s, 1H), 4.16 (t, J = 5.7 Hz, 2H), 3.58 (t, J = 4.5 Hz, 4H),
2.71 (t, J = 5.7 Hz, 2H), 2.50–2.46 (m, 4H, overlapped with DMSO),
2.30 (s 3H), 1.30 (s, 9H); MS (ES+) m/z calcd for
5.2.14. N1-3-[3-Methyl-4-([(5-methyl-3-
isoxazolyl)amino]carbonylamino)phenyl]-1H-5-pyrazolyl-4-(4-
methylpiperazino)benzamide (9j)
C
C
31H37N7O5:587.28; found: 588.4 (M+H+); HRMS (ESI) calcd for
31H38N7O5: 588.2934; found: 588.2926 (M+H+).
Mp 321–323 °C. 1H NMR (400 MHz, DMSO-d6): d 12.73 (s, 1H),
10.49 (s, 1H), 9.88 (s, 1H), 8.35 (s, 1H), 7.97–7.91 (m, 3H), 7.60 (s,
1H), 7.55 (d, J = 8.4 Hz, 1H), 6.97 (d, J = 8.0 Hz, 3H), 6.51 (s, 1H),
3.27 (t, J = 4.8 Hz, 4H), 2.42 (t, J = 4.8 Hz, 4H), 2.36 (s, 3H), 2.29 (s,
3H), 2.21 (s, 3H); MS (ES+) m/z calcd for C27H30N8O3: 514.24;
found: 515.2 (M+H+); HRMS (ESI) calcd for C27H31N8O3:
515.2514; found: 515.2520 (M+H+).
5.2.20. N1-(3-4-[([5-(tert-Butyl)-3-
isoxazolyl]aminocarbonyl)amino]-3-methylphenyl-1H-5-
pyrazolyl)-4-[(4-methylpiperazino)methyl]benzamide (9p)
Mp 263–264 °C. 1H NMR (300 MHz, DMSO-d6): d 10.78 (s, 1H),
9.95 (s, 1H), 8.35 (s, 1H), 7.99–7.97 (m, 3H), 7.73 (s, 1H), 7.57 (d,
J = 8.4 Hz, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.11 (s, 1H), 6.48 (s, 1H),
3.52 (s, 2H), 3.35 (s, 4H), 2.37 (s, 4H), 2.30 (s, 3H), 2.15 (s, 3H),
1.30 (s, 9H); MS (ES+) m/z calcd for C31H38N8O3: 570.31; found:
571.4 (M+H+); HRMS (ESI) calcd for C31H39N8O3: 571.3145; found:
571.3136 (M+H+).
5.2.15. N1-3-[3-Chloro-4-([(5-methyl-3-
isoxazolyl)amino]carbonylamino)phenyl]-1H-5-pyrazolyl-4-(4-
methylpiperazino)benzamide (9k)
Mp 240–242 °C. 1H NMR (400 MHz, DMSO-d6): d 12.88 (s, 1H),
10.54 (s, 1H), 10.25 (s, 1H), 8.81 (s, 1H), 8.24 (d, J = 8.4 Hz, 1H),
7.94–7.91 (m, 3H), 7.71 (d, J = 8.8 Hz, 1H), 7.05–6.98 (m, 3H),
6.52 (s, 1H), 3.28 (t, J = 4.8 Hz, 4H), 2.44 (t, J = 4.8 Hz, 4H), 2.38 (s,
3H), 2.22 (s, 3H); MS (ES+) m/z calcd for C26H27ClN8O3: 534.19;
found: 535.2 (M+H+); HRMS (ESI) calcd for C26H28ClN8O3:
535.1967; found: 535.1978 (M+H+). HPLC (C18 column):
tR = 19.65 min, 94.0%. HPLC (phenyl column): tR = 20.26 min, 94.0%.
5.2.21. N1-[3-(4-[(Anilinocarbonyl)amino]methylphenyl)-1H-5-
pyrazolyl]-4-(2-morpholinoethoxy)benzamide (10a)
Mp 237–238 °C. 1H NMR (300 MHz, DMSO-d6): d 10.71 (s, 1H),
8.56 (s, 1H), 8.01 (d, J = 8.7 Hz, 2H), 7.71 (d, J = 7.8 Hz, 2H), 7.42–
7.37 (m, 4H), 7.22 (t, J = 7.8 Hz, 2H), 7.04–7.02 (m, 3H), 6.90 (t,
J = 7.2 Hz, 1H), 6.65 (t, J = 5.7 Hz, 1H), 4.32 (d, J = 5.7 Hz, 2H), 4.17
(t, J = 5.7 Hz, 2H), 3.58 (t, J = 4.8 Hz, 4H), 3.38–3.23 (m, 4H), 2.71
(t, J = 5.7 Hz, 2H); MS (ES+) m/z calcd for C30H32N6O4: 540.25;
found: 541.3 (M+H+); HRMS (ESI) calcd for C30H33N6O4:
541.2558; found: 541.2569 (M+H+).
5.2.16. N1-(3-4-[([5-(tert-Butyl)-3-
isoxazolyl]aminocarbonyl)amino]phenyl-1H-5-pyrazolyl)-4-(2-
tetrahydro-1H-1-pyrrolylethoxy)benzamide (9l)
Mp 256–257 °C. 1H NMR (400 MHz, DMSO-d6): d 13.59 (s, 1H),
11.48 (s, 1H), 10.39 (s, 1H), 9.80 (s, 1H), 8.82 (d, J = 8.8 Hz, 2H), 8.50
(d, J = 8.8 Hz, 2H), 8.34 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 8.8 Hz, 2H),
7.77 (br s, 1H), 7.32 (s, 1H), 4.96 (t, J = 5.8 Hz, 2H), 2.11 (s, 9H);
MS (ES+) m/z calcd for C30H35N7O4: 557.64; found: 558.6 (M+H+);
HRMS (ESI) calcd for C30H36N7O4: 558.2823; found: 558.2841
(M+H+).
5.2.22. N1-[3-(4-[(Anilinocarbonyl)amino]methylphenyl)-1H-5-
pyrazolyl]-4-[(4-methylpiperazino)methyl]benzamide (10b)
Mp 235–237 °C. 1H NMR (300 MHz, DMSO-d6): d 12.91 (s, 1H),
10.80 (s, 1H), 8.63 (s, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 8.4 Hz,
2H), 7.43–7.37 (m, 6H), 7.23 (t, J = 7.2 Hz, 2H), 7.03 (s, 1H), 6.90 (d,
J = 7.2 Hz, 1H), 6.68 (t, J = 6.0 Hz, 1H), 4.33 (d, J = 6.0 Hz, 2H), 3.52
(s, 2H), 2.37 (br s, 8H), 2.16 (s, 3H); MS (ES+) m/z calcd for
5.2.17. N1-(3-4-[([5-(tert-Butyl)-3-
isoxazolyl]aminocarbonyl)amino]phenyl-1H-5-pyrazolyl)-4-(2-
morpholinoethoxy)benzamide (9m)
C
C
30H33N7O2: 523.27; found: 524.3 (M+H+); HRMS (ESI) calcd for
30H34N7O2: 524.2768; found: 524.2773 (M+H+).
Mp 270–272 °C. 1H NMR (400 MHz, DMSO-d6): 12.79 (s, 1H),
10.67 (s, 1H), 9.57 (s, 1H), 8.94 (s, 1H), 8.01 (d, J = 8.8 Hz, 2H),
7.69 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H), 7.04 (d, J = 8.8 Hz,
2H), 6.96 (br s, 1H), 6.52 (s, 1H), 4.17 (t, J = 5.8 Hz, 2H), 3.58 (t,
J = 4.6 Hz, 4H), 2.71 (t, J = 5.8 Hz, 2H), 2.48–2.47 (m, 4H, overlap-
pimg with solvent peak), 1.30 (s, 9H); MS (ES+) m/z calcd for
5.2.23. N1-[3-(4-[([4-Fluoro-
phenyl]aminocarbonyl)amino]methylphenyl)-1H-5-pyrazolyl]-
4-(2-morpholinoethoxy)benzamide (10c)
Mp 238–240 °C. 1H NMR (300 MHz, DMSO-d6): d 12.87 (s, 1H),
10.67 (s, 1H), 8.63 (s, 1H), 8.00 (d, J = 8.7 Hz, 2H), 7.71 (d,
J = 8.1 Hz, 2H), 7.44–7.35 (m, 4H), 7.10–7.02 (m, 4H), 6.65 (t,

W.-H. Lin et al. / Bioorg. Med. Chem. 21 (2013) 2856–2867
2865
J = 6.0 Hz, 1H), 4.32 (d, J = 6.0 Hz, 2H), 4.17 (t, J = 5.7 Hz, 2H), 3.58
5.3.1. (4-[5-(6-Chloro-2-methyl-pyrimidin-4-ylamino)-1H-
(t, J = 4.8 Hz, 4H), 2.71 (t, J = 5.7 Hz, 2H), 2.50–2.46 (m, 4H, overlap-
ping with DMSO peak); MS (ES+) m/z calcd for C30H31FN6O4:
558.24; found: 559.2 (M+H+); HRMS (ESI) calcd for C30H32FN6O4:
559.2464; found: 559.2468 (M+H+).
pyrazol-3-yl]benzyl)carbamic acid benzyl ester (11b)
Mp 208–209 °C. 1H NMR (400 MHz, DMSO-d6): d 12.85 (s, 1H),
10.26 (s, 1H), 7.85 (t, J = 6.0 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.20–
7.50 (m, 7H), 6.63 (br s, 1H), 5.04 (s, 2H), 4.22 (d, J = 6.0 Hz, 2H),
2.44 (s, 3H); MS (ES+) m/z calcd for C23H21ClN6O2: 448.14 and
450.14; found: 449.1 and 451.1 (M+H+).
5.2.24. N1-[3-(4-[([4-Chloro-
phenyl]aminocarbonyl)amino]methylphenyl)-1H-5-pyrazolyl]-
4-(2-morpholinoethoxy)benzamide (10d)
To a solution of 11 (1.0 equiv) in pyridine at room temperature
was added 1° or 2° amines (2.0 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 NaHCO3 solution. The suspension was vig-
orously stirred at room temperature for 1 h, and the resultant solid
was collected by filtration and dried in vacuo to yield crude prod-
uct which was used without further purification. To a solution of
the crude product prepared from last step in MeOH was added
12 N HCl (5.0 equiv) and a catalytic amount of 10% Pd/C at room
temperature. The mixture was vigorously stirred at room temper-
ature under an atmospheric pressure of hydrogen for 12 h. The cat-
alyst was removed by filtration and the filtrate was concentrated in
vacuo. The crude amine was used for the next reaction without fur-
ther purification. Starting with the crude amine, the same proce-
dures were followed as described in the synthesis of ureas 9–10
to yield compounds 13–14.
Mp 228–229 °C. 1H NMR (400 MHz, DMSO-d6): d 12.87 (s, 1H),
10.68 (s, 1H), 8.76 (s, 1H), 8.01 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.0 Hz,
2H), 7.45 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 7.6 Hz, 2H), 7.27 (d,
J = 8.4 Hz, 2H), 7.04 (d, J = 8.0 Hz, 2H), 6.72 (t, J = 5.6 Hz, 1H), 4.32
(d, J = 6.0 Hz, 2H), 4.17 (t, J = 5.6 Hz, 2H), 3.58 (t, J = 4.8 Hz, 4H),
2.71 (t, J = 5.6 Hz, 2H), 2.50–2.48 (m, 4H, overlapping with DMSO
peak); MS (ES+) m/z calcd for C30H31ClN6O4: 574.21; found: 575.2
(M+H+); HRMS (ESI) calcd for C30H32ClN6O4: 575.2174; found:
575.2186 (M+H+).
5.2.25. N1-(3-4-[([(5-Methyl-3-
isoxazolyl)amino]carbonylamino)methyl]phenyl-1H-5-
pyrazolyl)-4-(4-methylpiperazino)benzamide (10e)
Mp 259–260 °C. 1H NMR (400 MHz, DMSO-d6): d 12.83 (s, 1H),
10.52 (s, 1H), 9.42 (s, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.69 (d,
J = 7.6 Hz, 2H), 7.33 (d, J = 7.6 Hz, 2H), 7.03–6.88 (m, 4H), 6.39 (s,
1H), 4.32 (d, J = 5.2 Hz, 2H), 3.28–3.18 (m, 4H), 2.48–2.38 (m,
4H), 2.31 (s, 3H), 2.20 (s, 3H); MS (ES+) m/z calcd for C27H30N8O3:
514.24; found: 515.3 (M+H+); HRMS (ESI) calcd for C27H31N8O3:
515.2514; found: 515.2518 (M+H+).
5.3.2. N-[4-(5-[2-Methyl-6-(4-methylpiperazino)-4-
pyrimidinyl]amino-1H-3-pyrazolyl)phenyl]-N0-phenylurea (13)
1H NMR (400 MHz, DMSO-d6): d 12.58 (s, 1H), 9.32 (s, 1H), 8.88
(s, 1H), 8.78 (s, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 4.4 Hz, 1H),
7.52 (d, J = 8.8 Hz, 2H), 7.45–7.27 (m, 3H), 6.96 (t, J = 7.2 Hz, 1H),
6.63 (s, 1H), 6.38 (s, 1H), 3.41 (br s, 6H), 2.37 (br s, 3H), 2.28 (s,
3H), 2.20 (s, 2H); MS (ES+) m/z calcd for C26H29N9O: 483.25; found:
484.1 (M+H+).
5.2.26. N1-[3-(4-[([5-(tert-Butyl)-3-
isoxazolyl]aminocarbonyl)amino]methylphenyl)-1H-5-
pyrazolyl]-4-(2-morpholinoethoxy)benzamide (10f)
Mp 250–252 °C. 1H NMR (400 MHz, DMSO-d6): d 12.87 (s, 1H),
10.68 (s, 1H), 9.48 (s, 1H), 8.00 (d, J = 8.0 Hz, 2H), 7.71 (d,
J = 7.2 Hz, 2H), 7.35 (d, J = 7.6 Hz, 2H), 7.03 (d, J = 8.0 Hz, 2H),
7.00 (s, 1H), 6.38 (s, 1H), 4.33 (t, J = 5.2 Hz, 2H), 4.16 (t, J = 5.6 Hz,
2H), 3.57 (t, J = 4.4 Hz, 4H), 2.70 (t, J = 5.2 Hz, 2H), 2.49–2.47 (m,
4H, overlapped with DMSO), 1.27 (s, 9H); MS (ES+) m/z calcd for
5.3.3. N-(5-Methyl-3-isoxazolyl)-N0-4-(5-[2-methyl-6-(4-
methylpiperazino)-4-pyrimidinylamino]-1H-3-
pyrazolyl)benzylurea (14a)
Mp 154–156 °C. 1H NMR (400 MHz, DMSO-d6): d 12.67 (s, 1H),
9.40 (s, 1H), 9.34 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz,
2H), 7.03 (t, J = 6.0 Hz, 1H), 6.63 (br s, 1H), 6.54 (s, 1H), 6.44 (s, 1H),
4.34 (d, J = 5.6 Hz, 2H), 3.48 (m, 4H), 2.36 (t, J = 4.8 Hz, 4H), 2.33 (s,
3H), 2.29 (s, 3H), 2.21 (s, 3H); MS (ES+) m/z calcd for C258H30N10O2:
502.58; found: 503.1 (M+H+).
C
31H37N7O5: 587.28; found: 588.3 (M+H+); HRMS (ESI) calcd for
C31H38N7O5: 588.2929; found: 588.2934 (M+H+).
5.2.27. N1-[3-(4-[([5-(tert-Butyl)-3-
isoxazolyl]aminocarbonyl)amino]methylphenyl)-1H-5-
pyrazolyl]-4-[(4-methylpiperazino)methyl]benzamide (10g)
Mp 226–227 °C. 1H NMR (300 MHz, DMSO-d6): d 10.82 (s, 1H),
10.71 (s, 1H), 9.50 (s, 1H), 7.98 (d, J = 8.1 Hz, 2H), 7.72 (d,
J = 8.1 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.36 (t, J = 8.4 Hz, 2H), 7.02
(t, J = 5.7 Hz, 2H), 6.38 (s, 1H), 4.35 (d, J = 5.7 Hz, 2H), 3.52 (s,
2H), 3.25 (s, 4H), 2.37 (s, 4H), 2.15 (s, 3H), 1.27 (s, 9H); MS (ES+)
m/z calcd for C31H38N8O3: 570.31; found: 571.4 (M+H+); HRMS
(ESI) calcd for C31H39N8O3: 571.3140; found: 571.3147 (M+H+).
5.3.4. N-[5-(tert-Butyl)-3-isoxazolyl]-N0-4-(5-[2-methyl-6-(4-
methylpiperazino)-4-pyrimidinylamino]-1H-3-
pyrazolyl)benzylurea (14b)
Mp 166–167 °C. 1H NMR (400 MHz, DMSO-d6): d 12.70 (s, 1H),
9.49 (s, 1H), 9.31 (s, 1H), 7.66 (d, J = 7.2 Hz, 2H), 7.34 (d,
J = 8.0 Hz, 2H), 7.01 (t, J = 6.0 Hz, 1H), 6.69 (s, 1H), 6.46 (s, 1H),
6.37 (m, 1H), 4.33 (d, J = 6.0 Hz, 2H), 3.48 (d, J = 4.8 Hz, 4H), 2.36
(t, J = 4.8 Hz, 4H), 2.28 (s, 3H), 2.20 (s, 3H), 1.26 (s, 9H); MS (ES+)
m/z calcd for C28H36N10O2: 544.30; found: 545.3 (M+H+); HRMS
(ESI) calcd for C28H37N10O2: 545.3101; found: 545.3098 (M+H+).
5.3. General procedure for the preparation of compounds 13
and 14
5.3.5. N-(5-Methyl-3-isoxazolyl)-N0-4-[5-(6-[4-(2-
To a solution of 6 (1.0 equiv) and 4,6-dichloro-2-methylpyrim-
idine (1.5 equiv) in DMF at room temperature was added hydrogen
chloride solution (4.0 M in dioxane, 1.6 equiv) and the resultant
mixture was stirred at 80 °C for 4 h. After cooling to room temper-
ature, the solvent was evaporated in vacuo to give a viscous resi-
due, which was partitioned between EtOAc and saturated
NaHCO3 solution. The organic layer was separated, dried over
MgSO4 and concentrated in vacuo. Purification by flash column
chromatography (eluted with CH2Cl2/CH3OH) yielded compound
11. Only 11b is selected to show its NMR and mass data.
hydroxyethyl)piperazino]-2-methyl-4-pyrimidinylamino)-1H-
3-pyrazolyl]benzylurea (14c)
Mp 105–107 °C. 1H NMR (400 MHz, DMSO-d6): d 12.69 (s, 1H),
9.43 (s, 1H), 9.31 (s, 1H), 7.66 (d, J = 7.2 Hz, 2H), 7.33 (d,
J = 7.6 Hz, 2H), 7.03 (t, J = 5.6 Hz, 1H), 6.65 (s, 1H), 6.40–6.60 (m,
1H), 6.39 (s, 1H), 4.46 (s, 1H), 4.33 (d, J = 5.6 Hz, 2H), 3.38–3.60
(m, 8H), 2.40–2.55 (m, 4H overlapped with DMSO peak), 2.36 (s,
3H), 2.28 (s, 3H); MS (ES+) m/z calcd for C26H32N10O3: 532.60;
found: 533.3 (M+H+); HRMS (ESI) calcd for C26H33N10O3:
533.2737; found: 533.2736 (M+H+).

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W.-H. Lin et al. / Bioorg. Med. Chem. 21 (2013) 2856–2867
5.3.6. N-[5-(tert-Butyl)-3-isoxazolyl]-N0-4-[5-(6-[4-(2-
hydroxyethyl)piperazino]-2-methyl-4-pyrimidinylamino)-1H-
3-pyrazolyl]benzylurea (14d)
5.5. Western blot analysis
Cultured cells were suspended in lysis buffer (50 mM Tris, pH
8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate,
0.1% SDS, 1 mM sodium orthovanadate, 1 mM PMSF, and 1 mM
DTT). Cell lysates were kept on ice for 10 min and then cleared
by centrifugation at 14,000ꢂg for 15 min at 4 °C. After adding 5ꢂ
sample buffer, the supernatants were heated at 95 °C for 5 min,
Mp 155–157 °C. 1H NMR (300 MHz, DMSO-d6): d 12.65 (s, 1H),
9.48 (s, 1H), 9.31 (s, 1H), 7.64 (d, J = 8.1 Hz, 2H), 7.32 (d, J = 8.1 Hz,
2H), 7.00 (t, J = 5.7 Hz, 1H), 6.65 (s, 1H), 6.42 (br s, 1H), 6.35 (s, 1H),
4.43 (t, J = 5.4 Hz,1H), 4.31 (d, J = 5.7 Hz, 2H), 3.40–3.60 (m, 8H),
2.30–2.55 (m, 4H overlapped with DMSO peak), 2.27 (s, 3H), 1.25
(s, 9H); MS (ES+) m/z calcd for C29H38N10O3: 574.69; found:
575.3 (M+H+).
and cell extract samples (10 or 20 lg) were separated by 10% or
15% SDS–PAGE and transferred onto a polyvinylidene difluoride
membrane (Millipore, Bedford, MA, USA). The membrane was then
probed using the indicated primary antibodies anti-b-actin (Santa
Cruz Biotechnology, Santa Cruz, CA, USA), and the following anti-
bodies including anti-Stat5, anti-phospho-Stat5 (Tyr694), anti-FLT3,
anti-phospho-FLT3 (Tyr591) from Cell Signaling Technology (Dan-
vers, MA, USA). After the membrane was first incubated with pri-
mary antibody in TBST buffer (20 mM Tris pH 7.5, 135 mM NaCl,
and 0.1% (v/v) Tween 20) with 5% (wt/v) milk powder (or 2% (wt/
v) bovine serum albumin) and washed with TBST buffer, the mem-
brane then blotted with horseradish peroxidase–conjugated sec-
ondary antibody (1:1000 dilution). The membrane was then
developed with SuperSignal reagent (Pierce, Rockford, IL, USA)
upon exposure to X-ray film.
5.3.7. N-[5-(tert-Butyl)-3-isoxazolyl]-N0-4-[5-(2-methyl-6-
piperazino-4-pyrimidinylamino)-1H-3-pyrazolyl]benzylurea
(14e)
Mp 74–75 °C. 1H NMR (400 MHz, DMSO-d6): d 12.69 (s, 1H),
9.52 (s, 1H), 9.33 (s, 1H), 7.65 (d, J = 7.6 Hz, 2H), 7.33 (d,
J = 8.4 Hz, 2H), 7.08 (t, J = 6.0 Hz, 1H), 6.50 (s, 1H), 6.46 (d,
J = 6.4 Hz, 1H), 6.36 (m, 1H), 4.32 (d, J = 5.6 Hz, 2H), 3.53–3.49
(m, 4H, overlapped with water peak), 2.91 (t, J = 4.8 Hz, 4H), 2.28
(s, 3H), 1.25 (s, 9H); MS (ES+) m/z calcd for C27H34N10O2: 530.29;
found: 531.3 (M+H+); HRMS (ESI) calcd for C27H35N10O2:
531.2944; found: 531.2943 (M+H+).
5.3.8. N-(3-Chlorophenyl)-N0-4-[5-(2-methyl-6-piperazino-4-
pyrimidinylamino)-1H-3-pyrazolyl]benzylurea (14f)
5.6. Cell lines and MTS cell viability assays
Mp 96–97 °C. 1H NMR (400 MHz, DMSO-d6): d 10.00 (s, 1H),
9.04 (s, 3H), 7.65–7.75 (m, 3H), 7.36 (d, J = 8.0 Hz, 2H), 7.19–7.23
(m, 2H), 6.99 (t, J = 6.4 Hz, 1H), 6.85–6.88 (m, 1H), 6.57 (s, 1H),
6.50 (s, 1H), 4.30 (d, J = 5.6 Hz, 2H), 3.65–3.83 (m, 4H), 3.12–3.33
(m, 4H), 2.37 (s, 3H); MS (ES+) m/z calcd for C26H28ClN9O:
517.21; found: 518.2 (M+H+); HRMS (ESI) calcd for C26H29ClN9O:
518.2184; found: 518.2182 (M+H+).
The leukemias cell lines MOLM-13, MV4:11, RSV4;11, U937,
and K562 were purchased from American Type Culture Collection
(ATCC, Manassas, VA, USA). All leukemias cell lines were main-
tained in RPMI 1640 medium supplemented with 10% fetal bovine
serum (FBS), 10 U/ml penicillin, and 10 g/ml streptomycin at 37 °C
and 5% CO2. To determine cell viability after drug treatment, assays
were performed by seeding 10,000 cells (leukemias cell lines) per
well in a 96-well culture plate. After 16 h, cells were then treated
with vehicle or various concentrations of compound in medium
for 72 h. Viable cells were quantitated using the MTS method (Pro-
mega, Madison, WI, USA) according to the manufacturer’s recom-
mended protocol. The results were determined by measuring the
absorbance at 490 nm using a plate reader (Victor2; PerkinElmer,
Shelton, CT, USA). The IC50 value was defined as the amount of
compound that caused 50% reduction in cell viability in compari-
son with DMSO-treated (vehicle) control and was calculated using
Prism version 4 software (GraphPad, San Diego, CA, USA).
5.4. Kinase assays
FLT3: GST-FLT3-KDWT containing the FLT3 kinase catalytic do-
main (residues Y567ꢀS993) were expressed in Sf9 insect cells
transfected the baculovirus containing pBac-PAK8-GST-FLT3-KD
plasmid. The FLT3WT Kinase-Glo assays were carried out in 96-
well plates at 30 °C for 4 h and tested compound in a final vol-
ume of 50 ll including the following components: 75 ng GST-
FLT3-KDWT proteins, 25 mM HEPES, pH 7.4, 4 mM MnCl2,
10 mM MgCl2, 2 mM DTT, 0.02% Triton X-100, 0.1 mg/ml bovine
serum albumin, 25
lM Her2 peptide substrate, 0.5 mM Na3VO4,
and 1 M ATP. VEGFR2: The recombinant GST-VEGFR2 (residues
l
5.7. Pharmacokinetics
V789-V1356) containing kinase domain were expressed in Sf9
insect cells. The kinase assay were carried out in 96-well plates
with tested compound in a final volume of 50 ll reaction at
30 °C for 120 min with following components: 25 mM HEPES
pH 7.4, 10 mM MgCl2, 4 mM MnCl2, 0.5 mM Na3VO4, 2 mM
Male Sprague–Dawley rats weighing 300–400 g each (8–
12 weeks old) were obtained from BioLASCO (Taiwan Co., Ltd, Ilan,
Taiwan). The animal studies were performed according to NHRI
institutional animal care and committee-approved procedures.
Animals were surgically prepared with a jugular-vein cannula
1 day before dosing and fasted overnight (for approximately 18–
20 h) before dosing. Water was available ad libitum throughout
the experiment. Food was provided at 4 h after dosing. A single
2.0 mg/kg and 10 mg/kg dose of compound, as a PEG400/DMA
(80/20, v/v) solution, was separately administered to groups of 3
rats each intravenously (iv) and oral gavage, respectively. Each ani-
mal received 2 or 10 ml of the dosing solution per kg of body
weight for iv and po, respectively. At 0 (before dosing), 2, 5 (iv
only), 15, and 30 min and at 1, 2, 4, 6, 8, and 24 h after dosing, a
blood sample (0.15 ml) was collected from each animal through
the jugular-vein cannula and stored in ice (0–4 °C). Immediately
after collecting the blood sample, 150 ml of physiological saline
(containing 30 Units of heparin per ml) was injected into the rat
through the jugular-vein cannula. Plasma was separated from the
DTT, 0.02% Triton X100, 0.01% BSA, 1 lM ATP, 2 lM polyGlu4:Tyr
peptide, 50ꢀ100 ng recombinant VEGFR2. Aurora Kinase A: The
recombinant GST-Aurora A (residues S123-S401) containing ki-
nase domain were expressed in Sf9 insect cells. The kinase assay
were carried out in 96-well plates with tested compound in a fi-
nal volume of 50
components: 50 mM Tris–HCl pH 7.4, 10 mM NaCl, 10 mM
MgCl2, 0.01% BSA, 5 M ATP, 1 mM DTT and 15 M tetra(-
LRRASLG) peptide, and 150 ng recombinant Aurora A.
Following incubation, 50 l Kinase-Glo Plus Reagent (Promega,
Madison, WI, USA) was added and the mixture was incubated at
25 °C for 20 min. A 70- l aliquot of each reaction mixture was
ll reaction at 37 °C for 90 min with following
l
l
l
l
transferred to a black microtiter plate and the luminescence was
measured on Wallac Vector 1420 multilabel counter (PerkinElmer,
Shelton, CT, USA).

W.-H. Lin et al. / Bioorg. Med. Chem. 21 (2013) 2856–2867
2867
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5.8. Efficacy in tumor xenograft model
Nine weeks old athymic nu/nu nude mice were obtained from
BioLASCO, Taiwan Co., Ltd (Ilan, Taiwan). The mice were housed
in sterilized cages equipped with an air filter and sterile bedding
materials at the Laboratory Animal Center. All mice were fed with
sterilized water and chow under 12-h light/dark schedule. 100 ll
suspensions of MOLM-13 cells (final concentration: 1 ꢂ 107 cells/
ml; 1 ꢂ 106/flank) were transplanted sc into the left flank of nu/
nu nude mice. Tumor diameters are measured with digital calipers,
and the tumor volume in mm3 is calculated by the formula: Vol-
ume = (length ꢂ width2)/2. In Figure 4, tumor-bearing mice were
randomized when the mean tumor volume was ꢀ200 mm3. The
mice were randomly divided into 3 groups of 6–8 animals each,
and the treatment was initiated. Compound 10f was dissolved in
vehicle formulation (10% DMSO/20% Cremophor EL/70% saline, v/
v/v) and intravenously administrated at 1, 3 and 10 mg/kg once a
day for two continuous weeks (on days 1–5 and 8–12). A vehicle
control group received intravenously in the same manner. (Animal
Use Protocol for this project has been approved by National Health
Research Institutes, the protocol No: NHRI-IACUC-100023-A.)
Acknowledgments
The National Health Research Institutes and National Science
Council of Taiwan (NSC 100-2325-B-400-002-) financially sup-
ported the study.
24. Grese, T. A.; Cho, S.; Finley, D.; Godfrey, A. G.; Jones, C. D.; Lugar, C. W.; Martin,
M. J.; Matsumoto, K.; Pennington, L. D.; Winter, M. W.; Adrian, M. D.; Cole, H.
W.; Magee, D. E.; Phillips, D. L.; Rowley, E. R.; Short, L. L.; Glasebrook, A. L.;
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