3-Phenyl-1H-5-pyrazolylamine-based derivatives as potent and efficacious inhibitors of FMS-like tyrosine kinase-3 (FLT3)

Article abstract of DOI:10.1016/j.bmcl.2012.05.116

A new class of FLT3 inhibitors has been identified based on the 3-phenyl-1H-5-pyrazolylamine scaffold. The structure-activity relationships led to the discovery of two carbamate series, and some potent compounds within these two series exhibited better gr

Full text of DOI:10.1016/j.bmcl.2012.05.116

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4654–4659
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-Phenyl-1H-5-pyrazolylamine-based derivatives as potent and efficacious
inhibitors of FMS-like tyrosine kinase-3 (FLT3)
John T.-A. Hsu , Teng-Kuang Yeh , Shih-Chieh Yen , Chiung-Tong Chen, Shu-Yi Hsieh, Tsu Hsu,
Cheng-Tai Lu, Chun-Hwa Chen, Ling-Hui Chou, Ching-Hui Chiu, Yun-I Chang, Ya-Ju Tseng,
⇑
⇑
Kuei-Rong Yen, Yu-Sheng Chao, Wen-Hsing Lin , 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:
A new class of FLT3 inhibitors has been identified based on the 3-phenyl-1H-5-pyrazolylamine scaffold.
The structure–activity relationships led to the discovery of two carbamate series, and some potent com-
pounds within these two series exhibited better growth inhibition of FLT3-mutated MOLM-13 cells than
FLT3 inhibitors sorafenib (2) and ABT-869 (3). In particular, compound 8d exhibited the ability to regress
tumors in mouse xenograft model using MOLM-13 cells.
Received 15 March 2012
Revised 18 May 2012
Accepted 24 May 2012
Available online 7 June 2012
Ó 2012 Published by Elsevier Ltd.
Keywords:
FMS-like tyrosine kinase-3
Inhibitor
Acute myeloid leukemia
Receptor tyrosine kinase
Internal tandem duplications
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 Acute myeloid leukemia (AML) is the most common
type of leukemia in adults. AML is an aggressive disease with rapid
growth of abnormal leukemic cells in bone marrow that results in
failure of production of normal blood cells. Activating mutations in
the FLT3 receptor tyrosine kinase are present in ꢀ30% of AML
patients.6,7 These mutations involve internal tandem duplications
(ITDs) in the juxtamembrane domain of the receptor and missense
point or short-length mutations in the activation loop of the tyro-
sine kinase domain (TKD).8–10 In addition to FLT3 mutation,
wt-FLT3 has been found to be highly expressed in most acute leu-
kemia and FLT3 overexpression is an unfavorable prognostic factor
for overall survival in AML; moreover, it has been revealed that
overexpressed FLT3 had the same sensitivity to the FLT3 inhibitor
as FLT3/ITD.11,12 Over the past decade, due to the incidence and
poor prognosis associated with FLT3 and the refractory nature of
this disease associated with the mutant forms, numerous agents
have been developed to directly inhibit the activity of wild type
and mutated FLT3, and several of them, such as AC220 (1),13
sorafenib (BAY-43-9006, 2)14 and ABT-869 (3),15 have been inves-
tigated as potential agents for treating AML.16
We initially developed, through a structure-based design, a novel
class of 3-phenyl-1H-5-pyrazolylamine-based derivatives (Fig. 1) as
a versatile template for developing specific kinase inhibitors. Previ-
ous report from our laboratories described a counterscreen against
in-house kinases to identify a sulfonamide series of benzamide com-
pounds (Structure I) and a rational design strategy to identify a
second sulfonamide series of pyrimidine compounds (Structure K)
as potent inhibitors of FLT3 (Fig. 1).17 In continuation to our efforts
to develop potent FLT3 inhibitors, we searched for other series of
3-phenyl-1H-5-pyrazolylamine-based inhibitors that not only in-
creased the in vitro growth-inhibitory effect on AML MOLM-13 cells
and with prolonged duration of action in vivo. In this letter, the
structure–activity relationships of two carbamate series of benzam-
ide compounds (Structure J) and pyrimidine compounds (Structure
L) as FLT3 inhibitors as well as their in vitro properties are reported.
This study has led to the discovery of carbamate series of FLT3 inhib-
itors with better in vitro potency to other FLT3 inhibitors 2 and 3 in
clinical development. In addition, in vivo efficacy of the selected
compound 8d is also presented.
The general synthetic route for the preparation of 3-phenyl-1H-
5-pyrazolylamine benzamide derivatives 7–8 is illustrated in
Scheme 1 and these derivatives are listed in Table 1. The synthesis
began with prepared 4-nitro or 4-CH2NHCbz substituted 3-phenyl-
⇑
Corresponding authors. Tel.: +886 37 246166x35712; fax: +886 37 586456
(W.-T.J.).
1H-5-pyrazolylamine
4 as a starting material. Compound 4
E-mail address: wtjiaang@nhri.org.tw (W.-T. Jiaang).
These authors contributed equally.
was prepared according to the literature procedures.18,19 Benzoyl
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.05.116

J. T.-A. Hsu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4654–4659
4655
NH
N
NH
O
R1
R2
O
O
N
H
O
H
N
H
N
R2
O
N
N
7
b,
O
Cl
a
NH
O
or
NO2
HN
NH2
R1
+
Cl
R2
O
R1
NH
R
R
O
N
5
6a (R = 4-NH2)
NH
O
4a (R = 4-NO2)
6b (R = 4-CH2NH2)
4b (R = 4-CH2NHCbz)
R1
H
N
O
O
R2
O
Cl
8
c
N
Cl
N
O
HN Cbz
N
NH2
b,
N
O
HN
N
HN
HN
R2
O
R2
O
Cl
or
d
HN
HN
HN
R2
O
N
N
N
O
R1
N
Cl
N
R1
N
O
10
9
11
NO2
Scheme 1. Reagents and conditions: (a) (i) Pyridine, rt; (ii) H2, Pd/C, MeOH/HCl (aq), 75–80% (two steps); (b) pyridine, rt, 80–85%; (c) 4 N HCl/1,4-dioxane, DMF, 80 °C, 40–
45%; (d) (i) 1° or 2° amines, pyridine, 80 °C; (ii) H2, Pd/C, MeOH/HCl (aq), 85–90% (two steps) after recrystallization.
chlorides 5 with different water-solubilizing groups were prepared
from their corresponding acids which are commercially available or
prepared according to the literature procedures with some modifi-
cations.20 Substituted benzoyl chlorides 5 acylated amines 4 in pyr-
idine to form amides, which were subjected to the next reaction
involving reduction of the nitro or Cbz groups using a Pd/C catalyst
under H2 to yield corresponding amines 6. Amines 6 were coupled
with phenyl or benzyl chloroformate or carbonic acid 4-nitro-phe-
nyl ester heteroarylmethyl ester to afford the corresponding carba-
mates 7 or 8.
The general synthetic route for the preparation of 3-phenyl-1H-
5-pyrazolylamine pyrimidines 11 is shown in Scheme 1 and these
derivatives are listed in Table 2. Treatment of 3-phenyl-1H-5-pyraz-
olylamine 4 with 4,6-dichloropyrimidine in the presence of 4 N HCl/
1,4-dioxane and DMF at 80 °C provided 4-monosubstituted pyrimi-
dine derivatives 9. Reaction of 9 with the substituted or unsubstitut-
ed piperazine in pyridine at 80 °C gave 4,6-disubstituted pyrimidine
derivatives. The crude products were subjected to reduction by the
treatment with Pd/C under H2 to yield the corresponding amines
10, which were subsequently coupled with phenyl or benzyl chlor-
formate or carbonic acid 4-nitro-phenyl ester heteroarylmethyl
ester to obtain the final pyrimidine carbamates 11.
Previous report has demonstrated the importance of the water-
solubilizing group and sulfonamide moiety which is located at the
3-position of phenyl ring (Structure I, Fig. 1).17 An extensive SAR
study to identify potent structure J-based FLT3 inhibitors which
have carbamate group at the 4-position of phenyl ring connecting
with pyrazolylamine at the 3-position is shown in Table 1. The tar-
get compounds were tested in the FLT3, vascular endothelial
growth factor receptor (VEGFR) and Aurora A kinase assays, and
were further evaluated the inhibition of proliferation of the FLT3
mutated AML cell line MOLM-13 in a cellular assay. Compounds
7a with benzyl substituent and 7b with phenyl substituent at R1
position was found to show potent inhibition against FLT3
NH
N
R2
NH
H
N
O
O
n
NH
OR1
N
NH
R'
NH
N
R2
NH
J (n = 0 or 1)
Kinase
Rational design
O
Counterscreening
O
R
HN
R1
NH
N
S
O
3-Phenyl-1H-5-
NH
N
pyrazolylamine-based
derivatives
R2
I
R1
N
K R1= 3-NHSO2R3
R1= 4-CH2NHC(O)OR3
L
Figure 1. Identification of 3-phenyl-1H-5-pyrazolylamine-based derivatives as FLT3 inhibitors.

4656
J. T.-A. Hsu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4654–4659
Table 1
Inhibitory activity of structure-J carbamate analogues
NH
N
NH
N
NH
R2
NH
N
N
H
N
O
O
H
O
O
S
O
N
n
n
OR1
7d n = 0, 8n n =1
J (7 n = 0, 8 n =1)
Compd
R1
R2
IC50 (nM)
GI50 (nM)
wt-FLT3
VEGFR1
VEGFR2
Aurora A
MOLM-13 cell
7a
7b
–CH2C6H5
–C6H5
4
N
N
N
N
7
200
320
48
>1000
>1000
16
5
4
4
12
180
O(CH2)2
N
7c
–C6H5
40
>1000
>1000
300
>1000
>1000
47
7d
590
>1000
500
8a
8b
8c
8d
8e
8f
–C6H5
16
17
6
54
>1000
20
92
560
100
120
170
370
240
>1000
>1000
880
20
48
2
4
4
N
N
N
N
N
N
–C2H5
–CH2C6H5
–CH2C6H5
–CH2C6H5
–CH2C6H5
–CH2C6H5
4
4
O(CH2)2
CH2
N
10
7
70
>1000
>1000
590
2
4
N
N
320
370
180
4
17
19
8
4
4
O(CH2)2
O(CH2)3
N
N
O
O
O
8g
>1000
9
N
4
4
N
N
8h
8i
14
35
20
250
>1000
>1000
220
>1000
>1000
>1000
>1000
>1000
2
8
7
N
N
O(CH2)2
O(CH2)2
N
N
4
4
8j
N
N
N
N
N
O
O
O
8k
8l
27
43
350
590
370
480
>1000
>1000
1
2
4
4
O(CH2)2
O(CH2)2
N
N
8m
8n
52
>1000
>1000
>1000
>1000
>1000
1000
4
140
47
(IC50 = 7 and 12 nM, respectively) and had potent growth inhibi-
tory activity against MOLM-13 cells (GI50 = 16 and 5 nM, respec-
tively). A significant drop in both enzymatic and cellular potency
was observed when the water-solubilizing group (R2) on the benz-
amide was changed from 4-methylpiperazine (7b) to a pyrrolidine
group linked by a two-carbon ether tether (7c). The sulfonamide
group at the 4-position of phenyl ring (7d) dramatically decreased
potency with respect to wt-FLT3 and MOLM-13 cell. Compounds
7a–c exhibited modest (IC50 >100 nM) to weak inhibition (IC50
>1000 nM) against VEGFR1, 2 and Aurora A kinases except that
7a is a potent VEGFR2 inhibitor (IC50 = 48 nM).
8a or ethyl 8b, FLT3 potency was comparable to homolog 7b, how-
ever with a significant loss in cellular potency. Next, benzyl substi-
tuted compounds possessing different water-solubilizing groups
(8c–g) were investigated. 4-Methylpiperazine 8c was a potent
FLT3 inhibitor (IC50 = 6 nM) and exhibited extremely potent
growth-inhibitory activity against MOLM-13 cells (GI50 = 2 nM).
Compound 8d with pyrrolidine group linked by a two-carbon ether
tether and compound 8e with piperazine group linked by a one-
carbon tether had minimal effects on FLT3 enzymatic and cellular
potency (FLT3 IC50 = 10 and 7 nM, MOLM-13 GI50 = 2 and 4 nM,
respectively). A slight drop in both the enzymatic and cellular
potency was observed when the pyrrolidine of 8d was replaced
by morpholine (8f: FLT3 IC50 = 17 nM, MOLM-13 GI50 = 8 nM) or
was linked by a three-carbon ether tether (8g: FLT3 IC50 = 19 nM,
MOLM-13 GI50 = 9 nM).
Having demonstrated the importance of the carbamate phar-
macophore, extensive SAR studies were carried out on elongation
of the carbon chain by one carbon between the carbamate moiety
and 4-position of phenyl ring (8a–m, Table 1). When R1 is phenyl

J. T.-A. Hsu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4654–4659
4657
Table 2
Inhibitory activity of structure-L carbamate analogues
NH
N
NH
H
N
R2
O
N
R1
N
O
L
Compd
R1
R2
IC50 (nM)
GI50 (nM)
wt-FLT3
18
VEGFR1
420
VEGFR2
310
Aurora A
430
MOLM-13 cell
11a
11b
–CH2C6H5
N
N
N
N
2
N
4
5
>1000
>1000
>1000
>1000
>1000
>1000
2
4
N
N
N
O
11c
N
N
N
N
N
N
11d
11e
–CH2C6H5
10
21
290
240
170
2
3
OH
N
>1000
>1000
>1000
OH
OH
N
O
11f
72
17
>1000
82
>1000
190
>1000
350
5
6
11g
–CH2C6H5
N
NH
1
2
3
69
54
20
>10,000
165
70
430
20
7
>10,000
3800
760
4
56
37
Due to benzyl substituent at R1 position leading to better po-
tency than that of phenyl substituent (8c vs 8a), we set out to
investigate the effect on varying the R1 substituent of 8c, 8d and
8f from arylalkyl (benzyl) group to heteroarylmethyl group (Table
1). Carbamic acid pyridin-3-ylmethyl esters 8h and 8j displayed
similar enzymatic and cellular activities to that shown by 8c and
8f, respectively. A fourfold loss in both enzyme and cell potency
was observed when 8d was changed from benzyl to pyridylmethyl
ester 8i (FLT3 IC50 = 35 nM, MOLM-13 GI50 = 8 nM). As for five-
membered 5-methyl-isoxazole ring, compounds 8k, 8l and 8m
were 3 to 5-fold less potent than 8c, 8d and 8f, respectively. But
all of these isoxazole analogues had very potent growth inhibitory
activity against MOLM-13 cells with GI50 values of 1–4 nM. Com-
pounds 8k–m were more potent in the cellular assay (MOLM-13)
than in the enzymatic inhibition assay (FLT3). The likely reasons
for this behavior include slight differences in enzyme conformation
between the in vitro system and cellular environment and the
difference in amount of protein present in the two assays13 and/
or molecular differences between wild-type (WT) and mutant
(ITD) FLT3. Replacing the carbamate moiety of 8c with sulfonamide
group (8n) had profound negative effects on the inhibition against
FLT3 and MOLM-13.
As for the selectivity profile of 8a–m shown in Table 1, phenyl
substituted carbamate 8a had the IC50 values of 50 and 100 nM
against VEGFR1 and 2, respectively. Ethyl substituted 8b exhibited
modest to weak inhibition against VEGFR1 and 2 (IC50 >1000 nM
and IC50 = 560 nM, respectively). In general, benzyl substituted
carbamates 8c–g possessing different water-solubilizing group
exhibited potent to modest inhibitory potency against VEGFR1
and 2 (IC50 = 20–370 nM). Notable, carbamate 8c had and IC50
10000
1000
IV
100
10
1
0
6
12
18
24
Time, hr
Figure 2. Compound 8d inhibits FLT3 phosphorylation in MV4;11 AML cells. MV4-
11 cells, which are homozygous for FLT-ITD, were treated with compound 8d at the
indicated concentrations for 2 h. The phosphorylation state of FLT3 was evaluated
by western blot.
Figure 3. Pharmacokinetic profile of 8d in Sprague-Dawley rats (n = 3). Compound
was dosed intravenously with 2 mg/kg formulated in DMA/PEG400 (20/80, v/v).

4658
J. T.-A. Hsu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4654–4659
and improved over compounds 2 and 3 (GI50 = 56 and 37 nM,
respectively). Additionally, representative compound 8d showed
promising antiproliferative activity against the human AML cell
line MV4;11, which is a typical FLT3-ITD-expressing and a FLT3-
driven AML cell line, with a GI50 value of 3 nM. In accordance with
growth inhibitory effect, Western blot analysis showed that com-
pound 8d suppressed the phosphorylations of FLT3 (P-FLT3) in
MV4;11 cells in a dose-dependent manner (Fig. 2). The observed
IC50 value was approximately 1 nM for FLT3-ITD activity.
Due to high cellular potency of carbamate compounds, in vivo
anticancer efficacy of potent compound has to be evaluated. Quick
pharmacokinetics studies screened some potent carbamate com-
pounds and found that 8d possessed higher iv AUC0–4h.
23 Therefore,
compound 8d was further evaluated for its complete pharmacoki-
netic properties in Sprague–Dawley rats. Figure 3 shows the PK
curves for compound 8d dosed intravenously (IV) with 2 mg/kg.
The Cmax at 5 min and AUC for 8d was 2932 ng/mL (5433 nM) and
3126 ng/mL h (5793 nM h), respectively. Plasma concentration of
8d remained at a concentration of 8.9 ng/mL (17 nM) at 24 h. Com-
pound 8d exhibited a high volume of distribution (6.0 L/kg) and a
moderate plasma clearance (16.2 mL/min/kg). Its plasma IV half-
life was 6.2 h in rat, while oral bioavailability was found to be 5%.
Since compound 8d has poor oral bioavailability, the antitumor
efficacy of 8d was performed using the IV route. The efficacy testing
of 8d was conducted in a MOLM-13 tumor xenograft model in male
athymic NU-Fox1nu nude mice (Fig. 4). Compound 8d was dissolved
in 10% (v/v) DMSO, 20% cremphore EL and 70% saline and dosed at
25 mg/kg given iv qd for 1–5 and 8–12 days when the MOLM-13
tumors reached an average of 200 mm3 in volume. A single
25 mg/kg dose of 8d resulted in rapid and complete regression of
tumors in all animals and the tumor volume stayed suppressed
after dosing was halted, complete regressions were observed in 3
of 7 treated animals during the 30-day post-treatment observation
period. No body weight loss was observed in animals treated with
8d at the dose. These results demonstrate that 8d can effectively
reduce tumor size within 2 weeks of treatment and is well tolerated
in mice at the efficacious dose.
In summary, through counterscreening and rational design
approaches, we identified a novel class of 3-phenyl-1H-5-pyrazol-
ylamine-based carbamate derivatives which contains benzamide
series (Structure-J) and pyrimidine series (Structure-L) as FLT3
inhibitors. This report describes the SAR studies of these two series
of compounds to develop potent FLT3 inhibitors. Some of these
potent compounds exhibited similar cellular potency to FLT3
inhibitors 1 and at least 10-fold more potent than 2 and 3 in cellu-
lar assay. In addition, compound 8d showed highly antitumor effi-
cacy and tolerability in the MOLM-13 human AML xenograft
model.
Figure 4. Compound 8d demonstrates antitumor efficacy after intravenous (iv)
treatment in the MOLM-13 xenograft model (qd for 1–5 and 8–12 days). Tumor
sizes are expressed as mean SEM (n = 7/group).
value of 20 nM for VEGFR1. Compared to benzyl carbamates 8c–g,
heteroarylmethyl carbamates 8h–m are less potent VEGFR1-2
inhibitors with the IC50 values of 220 to >1000 nM. In general, com-
pounds 8a–m showed weak inhibition against Aurora A.
According to literature reports, the 3-aminopyrazole moiety is
stereochemically well suited to form hydrogen bonding interac-
tions with the kinase hinge region of the ATP pocket21 and 2-
methyl-pyrimidine is a known scaffold for kinase inhibitors.22
Based on these facts, we replaced the benzamide with 2-methyl-
pyrimidine which had a solubilizing group at 4-position to create
structure-L carbamate analogues 11a–g. As shown in Table 2,
pyrimidine 11a with water-solubilizing N-methylpiperazine group
reduced inhibitory potency toward FLT3 (IC50 = 18 nM) and VEGFR1
(IC50 = 420 nM) and VEGFR2 (IC50 = 310 nM) but maintained
growth inhibitory activity against MOLM-13 (GI50 = 2 nM) com-
pared to the related benzamide 8c. By keeping the N-methylpiper-
azine group, heteroarylmethyl carbamates 11b–c were highly
potent FLT3 inhibitors with the IC50 values of 4–5 nM and highly
selective for FLT3 over VEGFR1-2 and Aurora A. Importantly, com-
pounds 11b–c maintained cellular potency compared to 11a.
Next, we investigated the effects of water solubilizing groups on
the 4-position of pyrimidine ring and compared the potency of
compounds 11d–g with that of N-methylpiperazine analogues
11a–c, respectively. In comparison with N-methylpiperazine 11a,
N-(2-hydroxyethyl)piperazine 11d was approximately equipopo-
tent in the FLT3, VEGFR1-2 enzymatic assays and MOLM-13 cell
proliferation assay, but it was threefold more potent against
Aurora A kinase. On the countrary, heteroarylmethyl analogues
11e–f with N-(2-hydroxyethyl)piperazine were 5- and 14-fold
weaker in FLT3 enzymatic assay, but maintained potency in cellu-
lar assay when compared to related analogues 11b–c, respectively.
Compounds 11e–f exhibited weak inhibition against VEGFR1-2
and Aurora A (IC50 >1000 nM). Furthermore, no alkyl substituted
piperazine 11g was approximately equipotent to 11a in the FLT3
enzymatic potency and threefold less potent than 11a in the cellu-
lar potency. Piperazine 11g showed modest inhibition against
VEGFR1-2 and Aurora A kinases with the IC50 values of 82–
350 nM). According to SAR studied shown in Table 2, we find that
heteroarylmethyl carbamates 11b–c and 11e–f appear to be more
selective than benzyl carbamates 11a, 11d and 11g.
Acknowledgment
National Health Research Institutes and National Science Council
(NSC 100-2325-B-400-002-), Taiwan, financially supported the
study.
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23. Quick PK studies in rats: compounds were administered intravenously in a
dose of 2 mg/kg in DMA/PEG400 (20/80, v/v). Blood was collected at 2 min, 1 h
and 4 h postdose. The plasma exposure (AUC0–4h) was calculated from the
normalized LC–MS/MS peak areas and the unit is ng/ml h; 8d (1380), 8h (272),
8i (266), 8l (448), 8m (1031) and 11f (443).