Arylcarboxyamino-substituted diaryl ureas as potent and selective FLT3 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.07.024

A series of diaryl ureas with an amide substitution at the 4-position was prepared and found to be potent and selective FLT3 inhibitors with good oral bioavailability and efficacy in a tumor xenograft model.

Full text of DOI:10.1016/j.bmcl.2009.07.024

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5182–5185
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Arylcarboxyamino-substituted diaryl ureas as potent and selective FLT3
inhibitors
Hitesh K. Patel, Robert M. Grotzfeld, Andiliy G. Lai, Shamal A. Mehta, Zdravko V. Milanov, Qi Chao,
Kelly G. Sprankle, Todd A. Carter, Anne Marie Velasco, Miles A. Fabian, Joyce James, Daniel K. Treiber,
*
David J. Lockhart, Patrick P. Zarrinkar, Shripad S. Bhagwat
Ambit Biosciences Inc., 4215 Sorrento Valley Boulevard, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of diaryl ureas with an amide substitution at the 4-position was prepared and found to be potent
and selective FLT3 inhibitors with good oral bioavailability and efficacy in a tumor xenograft model.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 8 May 2009
Revised 29 June 2009
Accepted 2 July 2009
Available online 9 July 2009
Keywords:
Potent
Selective
FLT3
Kinase inhibitor
H
N
H
N
FMS-related tyrosine kinase 3 (FLT3) is a transmembrane tyro-
sine kinase that is expressed on dendritic cells, immature hemato-
poietic progenitors and also on some mature myeloid and
lymphoid cells.1 Activation of FLT3 is found to play a significant
role in maturation and proliferation of these cell types. In acute
myeloid leukemia (AML), FLT3 is expressed in myeloid cells in
approximately 90% of the patients and this kinase is responsible
for survival and proliferation of leukemic blasts.2 Activating muta-
tions of FLT3 are present in approximately one-third of all AML pa-
tients and are associated with adverse clinical outcome. FLT3
internal tandem duplications (ITDs) are associated with a poor
prognosis in AML. Inhibition of the kinase activity of FLT3 using
small molecule inhibitors has the potential to reduce leukemic
blasts and hence control the disease in AML patients.3
A number of small molecule inhibitors of FLT3, such as CEP-701,
PKC-412, MLN-518, sunitinib (SU-11248), and sorafenib (BAY-43-
9006) have been evaluated in preclinical as well as clinical setting.4,5
These FLT3 inhibitors are found to be less than optimal because of
lack of sufficient potency or sub-optimal oral bioavailability or lack
of adequate tolerability at efficacious doses. This Letter describes a
novel class of FLT3 inhibitors that are highly potent, significantly
more selective, and orally bioavailable with desirable efficacy and
tolerability in animals.
O
O
N
N
N
N
O
O
N
O
OH
HO
O
CEP-701
CF3
O
Cl
O
PKC412
O
NH
N
N
H
N
H
Sorafenib (BAY-43-9006)
H
N
O
N
O
N
O
N
H
N
N
H
MeO
O
F
N
O
N
H
N
N
Sunitinib
(SU-11248)
MLN-518
* Corresponding author. Tel.: +1 858 334 2180; fax: +1 858 334 2198.
E-mail address: sbhagwat@ambitbio.com (S.S. Bhagwat).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.024

H. K. Patel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5182–5185
5183
Profiling of small kinase focused libraries using KinomeScanTM, a
O
O
i
N
S
novel kinase screening platform to evaluate the binding affinity of
small molecules to the active site of a large number of kinases6,
led us to identify urea derivatives, I, as potent FLT3 inhibitors. Eval-
uation of the effect of X on the FLT3 inhibitory activity indicated
that compounds with an amino-carbonyl moiety at the para-posi-
tion, like in II, have improved cellular activity over the carboxam-
ides III. This Letter describes the SAR generated for the
amide–urea series culminating in the identification of compounds
13 (AB460) and 14 (AB530), which were found to be potent and
selective FLT3 inhibitors with excellent pharmacokinetic properties
and efficacy in a human tumor xenograft model in mice.
N
NH2
+
Br
OEt
OH
N
O
S
ii
1
Scheme 2. Reagents and conditions: (i) EtOH, 90 °C, 28%; (ii) LiOH, THF, water, 80%.
CO2H
NH2
O
i
R
O
N
H
+
NCO
O
O
N
N
N
H
N
H
ii
H
N
R
O
O
III
O
O
O
N
N
H
N
H
Scheme 3. Reagents and conditions: (i) DMA, 90 °C, 84%; (ii) RNH2, HATU, DMA,
II
O
I
10–24%.
X
O
O
N
N
H
N
H
R
N
H
for inhibiting MV4-11 proliferation. Among the three positional
isomers of the pyridyl amides, 6 with the 2-pyridyl substitution
inhibited the cell proliferation significantly more potently than
the other isomers, 7 and 8 (1.6 nM vs 159 nM and 132 nM). Intro-
ducing a hydroxyl group on the pyridine ring gave the two isomers,
9 and 10. Compound 10 was significantly more potent in cells with
sub-nanomolar IC50 for inhibiting cell proliferation. It is not clear if
the slight difference in intramolecular hydrogen bonding pattern
contributes to the unexpected increase in cell penetration and po-
tency of 10 over 9. The 2-substituted benzothiophene analog, 11,
N
N
H
N
H
III
The amide (NHCO)–urea series of compounds, II, were prepared by
condensing 3-amino-5-t-butyl-isoxazole with p-nitrophenyl isocya-
nate, followed by reduction of the nitro group to the corresponding
amine and coupling of the amine with a variety of carboxylic acids
(Scheme 1). The commercially unavailable carboxylic acid 1 was
prepared as shown in Scheme 2. 2-Amino benzothiazole was con-
densed with ethyl bromopyruvate to give ethyl imidazo[2,1-
b][1,3]benzothiazole-2-carboxylate, which was saponified to give
1. The amide (CONH)-urea series of compounds, III, was prepared
using the general synthetic sequence shown in Scheme 3. 5-t-Bu-
tyl-3-isoxazole isocyanate was condensed with 4-aminobenzoic
acid, which was coupled with a number of commercially available
amines to yield III.
The binding affinity was measured for the catalytic domain
(amino acids 592–969) of FLT3. The compounds which had desired
binding affinity for the enzyme were tested for their ability to in-
hibit the proliferation of MV4-11 cells derived from AML patients.
Amide analogs of II derived from alkyl or substituted alkyl car-
boxylic acids were in general less potent in binding to the enzyme
as shown for compounds 2 and 3 in Table 1.
also inhibited MV4-11 cell proliferation with sub-nanomolar IC50
.
Since the 2-pyridyl amide was superior to the corresponding 3-
and 4-isomers, we prepared 12 and 13 (AB460), two isoquinoline
amides and found that, of the two, 13 was significantly more po-
tent than 12 in the cell proliferation assay. Comparison of the cell
potency of 4 vs 5 and 11 and of 6 vs 13 and 12 allows one to point
out that adding hydrophobicity at this position increases cell pen-
etration and that the topology of the added hydrophobicity makes
a significant difference to the cell penetration while having little
effect on enzyme binding. Adding additional hydrophobicity
through the imidazo-benzothiazole ring of 14 (AB530) was not
detrimental and retained the binding and cell potency. The amides
10, 13 and 14 were found to be the most potent FLT3 inhibitors as
measured by their enzyme binding affinity and ability to inhibit
proliferation of MV4-11 cells. A hydrogen bond donor heteroatom
at the ortho-position of the carboxamide linkage appears to impart
better cell penetration (compound 4 vs 6 and 10; compound 5 vs
11, 13 and 14). Compounds 13 and 14 appear to be more potent
in the MV4-11 cell proliferation assay compared to their enzyme
binding affinity. The likely reasons for this observation include
changes in enzyme conformation from in vitro system to cells. It
is also worth noticing that the cell proliferation assay was carried
out in the presence of 0.5% serum protein. The IC50 for the inhibi-
tion of proliferation decreased significantly for all compounds in
the presence of 10% serum protein.
Therefore, the chemistry efforts focused on amide analogs de-
rived from aryl carboxylic acids, which bound to FLT3 with high
potency. A large number of aryl amides were prepared and the
examples in Table 1 highlight the observed SAR. The phenyl and
naphthyl amides, 4 and 5, were potent in binding to the enzyme,
with the naphthyl amide showing nearly 10-fold increased potency
NCO
NO2
i
O
+
O
O
N
N
H
N
H
A number of amide analogs of the type III (compounds 15–18)
were prepared and found to have potent enzyme binding affinity
but significantly lower potency in inhibiting cell proliferation.
N
NH2
NO2
H
N
Every compound was screened initially at 10 lM against a panel
R
of at least 40 kinases7 using the KinomeScanTM platform. A subset of
the compounds was tested further to obtain a dose–response and
Kd for the kinases in the panel. In general, the amides of Type II
were very selective with potent binding affinity restricted to the
Type III RTK family of kinases, that is, FLT3, KIT, CSF1R, RET,
ii
O
O
O
N
N
H
N
H
iii
II
PDGFR
a and PDGFRb. In particular, compounds 13 and 14 were
Scheme 1. Reagents and conditions: (i) toluene, 100 °C, 97%; (ii) Pd/C, THF, water,
>99%; (iii) RCOOH, CDI, DMF, 65–75%.
found to be highly selective in a panel of 402 kinases with Kd of

5184
H. K. Patel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5182–5185
Table 1
The SAR of FLT3 inhibitors
X
O
R
O
N
N
H
N
H
a
a
Compd
X
R
FLT3 Kd (nM)
MV4-11 IC50 (nM)
2
3
4
5
6
7
8
9
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
CONH
CONH
CONH
CONH
Benzyl
4-Piperidyl
Phenyl
2-Naphthyl
2-Pyridyl
3-Pyridyl
4-Pyridyl
6-Hydroxy, 2-pyridyl
3-Hydroxy, 2-pyridyl
5-Methyl, 2-benzo[b]thienyl
1-Isoquinolyl
3-Isoquinolyl
2-Imidazo[2,1-b][1,3]benzothiazolyl
Phenyl
2-Pyridyl
510
240
0.98
0.75
0.76
15
10.1
2.5
0.74
0.77
0.74
1.1
1.6
7.4
8.15
25
38.4
1090
10,500
30
3.1
1.6
159
132
46.7
0.71
0.85
2.8
0.29
0.44
101
104
199
414
10
11
12
13 (AB460)
14 (AB530)
15
16
17
18
3-Pyridyl
4-Pyridyl
a
Each experiment was run in duplicate and the values shown are the average of the two; for 10, 13 and 14, the values are average of three experiments.
Table 2
The selectivity profile of compounds 10, 13 and 14
a
a
a
a
a
a
Compd
FLT3 Kd (nM)
KIT Kd (nM)
RET Kd (nM)
CSF1R Kd (nM)
PDGFR
a
Kd (nM)
PDGFRb Kd (nM)
10
0.74
1.1
1.6
0.96
1.8
7.5
4.7
7.3
3
4.4
8.5
10
1
9.9
4.3
2.6
7
2.2
13 (AB460)
14 (AB530)
a
The values are an average of three experiments.
ꢀ1–2 nM for FLT3, ꢀ1–10 nM for KIT, RET, CSF1R, and PDGFR
a
/b,
are orally available in nude mice for testing their ability to inhibit
tumor growth. Compound 10 was poorly bioavailable with signif-
icantly lower Cmax, AUC and T1/2 values.
The oral efficacy of compounds 13 and 14 was evaluated in a
xenograft model in athymic nude mice with a subcutaneous im-
plant of a pellet of MV4-11 cells (Fig. 2). When the tumor grew
to a size of ꢀ160 mm3, the animals were dosed orally once daily
with 10 mg/kg of 13 and 14 for 28 days. The daily dosing was
stopped and the animals were observed between days 29–56.
>200 nM for VEGFR2 and other kinases (Table 2).
Among the known FLT3 inhibitors, CEP-701, PKC-412 and sun-
itinib were found to bind tightly to a large number of kinases in
this panel, while sorafenib bound tightly to moderate number of
kinases and MLN-518 bound to a small number of kinases like
compounds 13 and 14.8
Compounds 10, 13 and 14, three of the more potent analogs in
inhibiting cell proliferation, were tested for their PK profile upon
oral administration in nude mice, the species used for assessing
in vivo efficacy (Fig. 1). At an oral dose of 10 mg/kg of compounds
13 and 14, a desirable Cmax (1.31
lM and 2.36 lM respectively),
AUC (4.25 M h and 13.20 M h respectively), and T1/2 (2.2 h and
l
l
2.3 h, respectively) were observed, indicating that the compounds
Figure 2. Antitumor activity of 13, 14 and sunitinib (SU-11248) in a xenograft
model in nude mice (n = 10 animals per group).
Figure 1. Pharmacokinetic profile of compounds 10, 13, and 14.

H. K. Patel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5182–5185
5185
Sunitinib, a positive control, was dosed orally at 10 and 30 mg/kg
Acknowledgements
once a day for 28 days. Compounds 13 and 14 were effective in
shrinking the tumor to ꢀ90 mm3 during the dosing period and this
tumor shrinking effect persisted between days 29–36 when the
animals did not receive the drugs. The tumor started growing back
between days 36–56. The tumor grew rapidly in the untreated and
vehicle treated animals. At 10 mg/kg po dose, the efficacy of suni-
tinib in shrinking the tumor in this model was modest, while at
30 mg/kg po dose it was superior to 13 and 14. The effect of 13
and 14 on the body weight of treated animals, a measure of the
compounds’ tolerability, was minimal with <5% change in this
parameter with either compound.
In conclusion, profiling of a focused library of diarylurea-based
kinase inhibitors led us to discover potent and selective FLT3 ki-
nase inhibitors. Optimization of the substituents on one of the aro-
matic rings helped us identify a series of urea–amide compounds
like 13 and 14 that were found to bind to FLT3 kinase with sub-
nanomolar potency and with exquisite selectivity in a panel of
402 kinases when compared to reported potent FLT3 inhibitors,
CEP-701, PKC-412, MLN-518, sunitinib and sorafenib. Compounds
13 (AB460) and 14 (AB530) were found to have good oral availabil-
ity in rats (data not shown) and mice with potent tumor growth
inhibitory activity in a mouse xenograft model.
The authors thank Dr. Robert C. Armstrong for helping analyze
the efficacy data.
References and notes
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7. In the early stages of this lead optimization campaign, the panel consisted of 40
kinases, which grew steadily in size during lead optimization and currently has
402 kinases. Compounds 10, 13 and 14 have been screened against 402 kinases.
8. The kinase binding affinity profile for PKC-412, sunitinib, sorafenib and MLN-
518 have been published in Ref. 6 above. The profile for CEP-701 will be
published in a manuscript submitted for publication.