Identification of N-(5-tert-butyl-isoxazol-3-yl)-N′-{4-[7-(2- morpholin-4-yl-ethoxy)imidazo-[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea dihydrochloride (AC220), a uniquely potent, selective, and efficacious FMS-like tyrosine kinase-3 (FLT3) inhibitor

Article abstract of DOI:10.1021/jm9007533

Treatment of AML patients with small molecule inhibitors of FLT3 kinase has been explored as a viable therapy. However, these agents are found to be less than optimal for the treatment of AML because of lack of sufficient potency or suboptimal oral pharma

Full text of DOI:10.1021/jm9007533

7808 J. Med. Chem. 2009, 52, 7808–7816
DOI: 10.1021/jm9007533
Identification of N-(5-tert-Butyl-isoxazol-3-yl)-N0-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo-
[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea Dihydrochloride (AC220), a Uniquely Potent, Selective,
and Efficacious FMS-Like Tyrosine Kinase-3 (FLT3) Inhibitor
Qi Chao, Kelly G. Sprankle, Robert M. Grotzfeld, Andiliy G. Lai, Todd A. Carter, Anne Marie Velasco,
Ruwanthi N. Gunawardane, Merryl D. Cramer, Michael F. Gardner, Joyce James, Patrick P. Zarrinkar,
Hitesh K. Patel, and Shripad S. Bhagwat*
Ambit Biosciences, 4215 Sorrento Valley Boulevard, San Diego, California 92121
Received May 29, 2009
Treatment of AML patients with small molecule inhibitors of FLT3 kinase has been explored as a viable
therapy. However, these agents are found to be less than optimal for the treatment of AML because of
lack of sufficient potency or suboptimal oral pharmacokinetics (PK) or lack of adequate tolerability at
efficacious doses. We have developed a series of extremely potent and highly selective FLT3 inhibitors
with good oral PK properties. The first series of compounds represented by 1 (AB530) was found to be a
potent and selective FLT3 kinase inhibitor with good PK properties. The aqueous solubility and oral PK
properties at higher doses in rodents were found to be less than optimal for clinical development. A novel
series of compounds were designed lacking the carboxamide group of 1 with an added water solubilizing
group. Compound 7 (AC220) was identified from this series to be the most potent and selective FLT3
inhibitor with good pharmaceutical properties, excellent PK profile, and superior efficacy and
tolerability in tumor xenograft models. Compound 7 has demonstrated a desirable safety and PK
profile in humans and is currently in phase II clinical trials.
Introduction
(CEP-701), midostaurin(PKC-412), tandutinib(MLN-518or
CT 53518), sunitinib (SU-11248), and sorafenib (BAY-
43-9006), shown in Figure 1, have been investigated as
potential therapeutic agents for the treatment of AML.3-6
However, these agents are found to be less than optimal for
the treatment of AML because of lack of sufficient potency,
suboptimal oral PK, or lack of adequate tolerability at
efficacious doses that contribute ultimately to suboptimal
inhibition of FLT3 in AML patients.7
FLT3 is expressed in dendritic cells, immature hematopoie-
tic progenitors, and some mature myeloid and lymphoid cells.
The role of FLT3 in bone marrow derived cells is relatively
more clearly understood than in dendritic cells, the primary
antigen presenting cells for T- cells. Activation of FLT3
induces proliferation and differentiation of dendritic cells,
and this implies a potential role of FLT3 inhibitors in immune
modulation.8,9 A potent FLT3 inhibitor, lestaurtinib, was
found to protect mice from disease progression in an experi-
mental autoimmune encephalomyelitis (EAE) model that
mimics multiple sclerosis (MS) in humans. FLT3 inhibitors,
therefore, are likely to be efficacious in animal models of other
autoimmune disorders. While efficacy of lestaurtinib in psor-
iasis patients is being studied (www.clinicaltrials.gov), it is not
clear if any of the known FLT3 inhibitors in the clinic are
being evaluated in other autoimmune disorders in humans.
We have developed a series of extremely potent and highly
selective FLT3 inhibitors with good oral pharmacokinetic
(PK) properties. One such compound, 1 (AB-530), shown in
Figure 1, was found to regress tumors in a mouse xenograft
model using MV4-11 cells.10 The aqueous solubility and oral
PK properties at higher doses were found to be less than
Acute myeloid leukemia (AMLa) is the most common and
aggressive form of acute leukemias in elderly people. AML
patients treated with frontline chemotherapy of a combina-
tion of an anthracycline derivative such as daunorubicin
or idarubicin and a nucleoside analogue such as cytarabine
(Ara-C) often respond initially but relapse or are refractory to
frontline therapy and ultimately succumb to the disease.
A large majority of myeloid cells in AML patients are found
to express FMS-like tyrosine kinase 3 (FLT3), a trans-mem-
brane tyrosine kinase that belongs to the type III split-kinase
domain family of receptor tyrosine kinases (RTKs).1 Binding
of FLT3 ligand to the receptor triggers receptor dimerization
and autophosphorylation, leading to FLT3 kinase activation
and subsequent signal transduction culminating in survival
and proliferation of leukemic cells. Two types of activating
mutations (internal tandem duplications (ITD) in the juxta-
membrane (JM) domain and point mutations at residue D835
or deletion of I836 in the activation loop) are present in
approximately one-third of all AML patients and are asso-
ciated with poor response to chemotherapy, resulting in
adverse clinical outcome.2
Treatment of AML patients with small molecule inhibitors
of FLT3 has been explored as a viable therapy.3-5 A number
of potent small molecule FLT3 inhibitors such as lestaurtinib
*To whom correspondence should be addressed. Phone: 858-344-
4924. Fax: 858-334-2198. E-mail: sbhagwat@ambitbio.com.
a Abbreviations: FLT3, FMS-like tyrosine kinase 3; AML, acute
myeloid leukemia; RTK, receptor tyrosine kinase; ITD, internal tandem
duplications.
r
pubs.acs.org/jmc
Published on Web 09/16/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7809
Figure 1. Structure of known FLT3 inhibitors.
optimal for clinical development. We reasoned that the
carboxamide group in 1 may be contributing to lower solubi-
lity as well as to the absorption limited PK profile and that
removal of this functionality coupled with addition of a water
solubilizing group at a suitable position could address the
issues in the resulting molecules. We were gratified to find the
des-carboxamido-derivatives of 1 with a solubilizing group
not only retained the potency and selectivity of 1 but also
showed improved solubility and PK profiles. We describe
herein the discovery of a novel series of FLT-3 inhibitors
culminating in the identification and characterization of
compound 7, N-(5-tert-butyl-isoxazol-3-yl)-N0-{4-[7-(2-mor-
pholin-4-yl-ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phe-
nyl}urea dihydrochloride (AC220), as a uniquely potent,
selective, and efficacious compound suitable for clinical
development.
Chemistry
The commercially available 2-aminobenzothiazole (2a),
2-amino-6-hydroxybenzothiazole (2b), and 2c (prepared as
shown in Scheme 2) were condensed with 2-bromo-40-nitroace-
tophenone in refluxing ethanol to yield 3a, 3b, and 3c having the
imidazobenzothiazole ring attached directly to the p-nitrophenyl
ring (Scheme1).11 Reduction of the nitro group using iron and
HCl or stannous chloride gave the corresponding amines, 4,
which were coupled with 5-t-butyl-isoxazole-3-isocyanate, 5, to
yield the ureas 6a, 6b, and 6c. Compound 5 was prepared from
the commercially available 3-amino-5-t-butyl-isoxazole by treat-
ment with triphosgene. Mitsunobu reaction of 6b with various
ω-substituted alcohols gave compounds 7-9 with different
water solubilizing groups. Compound 7 could be prepared using
an alternate method shown in Scheme 4 (vide infra).
Scheme 1. Synthesis of Compounds 6a, 6b, 6c, 7, 8, and 9a
a Reagents and conditions: (a) 2-bromo-40-nitroacetophenone, EtOH, reflux; (b) Fe, iPrOH, HCl, or SnCl2 2H2O, EtOH; (c) toluene or CHCl3,
3
90-110 °C; (d) ROH, Ph3P, diisopropyl azodicarboxylate, THF.

7810 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Chao et al.
Compound 2c was prepared by the cyclization of 1-( p-
aminophenyl)-4- methylpiperazine, 10, with ammonium
thiocyanate in the presence of bromine and acetic acid
(Scheme 2).12
Compounds 14 and 15 were preparedasshownin Scheme3.
The phenol 3b was alkylated using 1-bromo-3-chloropropane
in DMF and potassium carbonate to give 11. Substitution of
the chloro group of 11 using N-methylpiperazine or metha-
nesulfonylpiperazine in the presence of tetra-n-butylammo-
nium iodide gave 12a and 12b, respectively, which were
subjected to the sequence of reactions involving reduction of
the nitro group using iron and HCl followed by coupling with
5 to yield compounds 14 and 15.
Scheme 2. Synthesis of Intermediate 2ca
Two regioisomers of 7 were prepared starting from
the corresponding hydroxy-substituted 2-aminobenzothia-
zoles 16a and 16b as shown in Scheme 4. Condensation of
16a and 16b with 2-bromo-40-nitroacetophenone as before
gave 17a and 17b. Alkylation of the phenolic group with
4-(2-chloroethy)morpholine hydrochloride followed by re-
duction of the nitro group with iron and HCl and coupling
a Reagents and conditions: (a) NH4SCN, AcOH, Br2.
Scheme 3. Synthesis of Compounds 14 and 15a
a Reagents and conditions: (a) Br(CH2)3Cl, K2CO3, DMF, 80 °C; (b) HN(CH2CH2)2NSO2Me, Bu4NI, DMF, 90 °C; (c) Fe, 10% HCl, iPrOH, 90 °C;
(d) 5, CHCl3, reflux.
Scheme 4. Synthesis of Compounds 20a, 20b, and 7a
a Reagents and conditions: (a) 2-bromo-40-nitroacetophenone, EtOH, reflux; (b) O(CH2CH2)2NCH2CH2Cl HCl, K2CO3, DMF, 90 °C; (c) Fe,
3
NH4Cl, EtOH; (d) 5, toluene, 120 °C.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7811
Scheme 5. Synthesis of Compounds 26a, 26b, 27a, 27b, 27c, and 28a
a Reagents and conditions: (a) MeOH, H2SO4. (b) (1) 2-bromo-40-nitroacetophenone, MeOCH2CH2OH, 40 °C, 24 h; (2) 140 °C, 18 h. (c) 2-bromo-40-
nitroacetophenone, EtOH, reflux. (d) SnCl2 2H2O, EtOH, relux. (e) 5, toluene, 110 °C. (f ) LiOH H2O, THF, water. (g) HNRR, EDC, HOBt, DMF.
3
3
(h) BH3 Me2S, THF, 95 °C.
3
Scheme 6. Synthesis of Compound 34a
a Reagents and conditions: (a) (1) 2-Bromo-40-nitroacetophenone, MeOCH2CH2OH, 24 h, 40 °C; (2) 140 °C, 18 h. (b) HN(CH2CH2)2NEt, EDC,
HOBt, DMF. (c) SnCl2 2H2O, EtOH, relux. (d) BH3 Me2S, THF, 95 °C. (e) 5, toluene 110 °C.
3
3
with 5 gave 20a and 20b. Compound 7 could also be
prepared using this method as shown in Scheme 4. The
yield of 7 using this reaction sequence was superior to that
from Scheme 1 and hence the Experimental Section de-
scribes the synthesis of 7 using the reaction sequence in
Scheme 4.

7812 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Chao et al.
Table 1. In Vitro Kinase Binding, Cell Proliferation, and Kinase Selectivity Data
FLT3
Kda,b (nM)
MV4-11
IC50a,c (nM)
selectivity scored
S(10)
compd
R1, R2, R3
1
6a
1.6
1.9
2.7
1.2
1.6
1.5
0.99
1.7
9.7
5.2
2.3
1.6
1.3
0.98
2.0
1.5
1.3
1.1
0.47
0.44
0.35
0.15
0.62
0.56
0.88
0.79
0.78
1.1
0.050
NDe
ND
H, H, H
OH, H, H
6b
6c
4-methylpiperazin-1-yl, H, H
2-morpholinoethoxy, H, H
ND
7
0.079
ND
ND
8
2-(piperidin-1-yl)ethoxy, H, H
3-morpholinopropoxy, H, H
9
14
3-(4-methyl)piperazin-1-yl-propoxy, H, H
3-(4-(methylsulfonyl)piperazin-1-yl)propoxy, H, H
H, H, 2-morpholinoethoxy
0.069
0.045
0.059
0.040
0.064
0.074
0.054
0.059
0.035
0.079
0.064
0.41
15
20a
20b
26a
26b
27a
27b
27c
28
9.3
H, 2-morpholinoethoxy, H
CO2H, H, H
CH2CH2CO2H, H, H
0.56
1.0
0.41
0.41
0.66
0.23
0.53
0.38
4.3
4-ethylpiperazine-1-carbonyl, H, H
3-(4-ethylpiperazin-1-yl)-3-oxopropyl, H, H
3-(morpholin-4-yl)-3-oxo-propyl, H, H
3-morpholinopropyl, H, H
34
sunitinib
(4-ethylpiperazin-1-yl)methyl, H, H
a Each experiment was run in duplicate and the values shown are the average of the two. b Kd values are for binding to FLT3.13 c IC50 values are for
inhibiting the proliferation of MV4-11 cell line. d Selectivity score or S(10), calculated as described in ref 15, is the ratio of kinase targets of the compound to the
total number of kinases screened (202 distinct kinases in a panel of 227) at a 10 μM compound concentration; kinases were defined as targets if the primary screen
showed >90% competition. e ND = not determined.
The compounds described above have the water solubiliz-
ing group attached to a heteroatom such as O or N. The
corresponding analogues with carbon atom were prepared
as shown in Scheme 5. The commercially available ethyl
2-aminobenzothiazole-6-carboxylate (22a) and methyl 3-(2-
aminobenzothiazol-6-yl)propionate (22b), prepared by ester-
ification of the corresponding commercially available car-
boxylic acid (21), were condensed with 2-bromo-40-
nitroacetophenone in 2-methoxyethanol for 22a or in ethanol
for 22b to give the nitro ethyl esters 23a and 23b, respectively.
Reduction of the nitro group followed by coupling with 5 gave
the ureas 25a and 25b. Saponification of the esters with LiOH
followed by coupling with appropriate amines using EDCI
and HOBt yielded the amides 27a, 27b, and 27c. Reduction of
the amide 27c using borane-dimethylsulfide in THF gave the
corresponding amine 28.
Compound 34 with a one carbon tether to the ethylpiper-
azine ring could not be prepared using the reaction sequence
used for 28 because the borane reduction conditions led to
extensive decomposition of the compound. The commercially
available 2-amino-benzothiazole-6-carboxylic acid, 29, was
condensed with 2-bromo-40-nitroacetophenone in ethanol to
give the nitro acid 30 (Scheme 6). Amide coupling with
N-ethylpiperazine gave the amide 31. Reduction of the nitro
group using stannous chloride followed by reduction of the
carboxamide using borane gave the amine 33. Coupling of 33
with the isoxazole 5 gave 34 with an ethylpiperazine group as
the solubilizing moiety.
assay with an ATP-competitive FLT3 inhibitor bound to a
solid surface13 and were found to have high binding affinity
to the kinase (Table 1). The compounds were then evaluated
in a cellular assay measuring the inhibition of proliferation of
the human leukemia cell line MV4-11, which is FLT3
dependent and harbors a homozygous FLT3-ITD mutation.
The first compound in this series, 6a, an analogue of 1 with-
out the carboxamide group, was found to be equipotent
to 1 in the binding as well as cell proliferation assay (Kd of
1.9 nM for 6a vs 1.6 nM for 1; IC50 of 0.35 nM for 6a vs
0.44 nM for 1). This crucial result indicated that the differ-
ence in conformation between 1 and 6a affects neither the
binding of 6a to the enzyme active site nor its cell perme-
ability and reinforced the rationale to optimize the series
further.
The compounds reported here were almost uniformly
found to be more potent in the cell proliferation assay than
in the in vitro binding assay. A number of factors may
contribute to this observed property of the molecules includ-
ing changes in enzyme conformation between the in vitro
system and cells and the difference in amount of protein
present in the two assays. The cell assay was carried out in the
presence of 0.5% serum proteins. The IC50 for the inhibition
of proliferation decreased significantly for all compounds
tested in the presence of 10% serum protein. In contrast
to the series of compounds reported herein, sunitinib has
∼10-fold weaker cellular activity compared to the binding
potency. The likely reasons for this behavior include ineffi-
cient penetration of cell membranes and/or a preference for
the enzyme conformation that is not favored in cells.14
Sorafenib, on the other hand, had a profile similar to the
In Vitro Pharmacology. The compounds described herein
were tested for their binding affinity to the catalytic domain
(amino acids 592-969) of FLT3 in a competition binding

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7813
series of compounds reported here with Kd = 13 nM and cell
IC50 = 0.87 nM (data shown in refs 15 and 16).
additional kinases in the larger panel. All of the compounds
in this series were found to be highly selective compared to
sunitinib, which has a S(10) score of 0.47. These compounds
are also significantly more selective than lestaurtinib and
midostaurin.15,16 The kinase profile of tandutinib is similar
to the series of compounds in Table 1, although the former is
about 100-fold less potent than 7 in the cell proliferation
assay.15,16 The selectivity profile of 7 is typical of this series of
compounds. The binding affinity for every kinase in the
panel was determined for 7,16 and the kinases with binding
affinities within 10-fold of the Kd value for FLT3 (1.6 nM)
were the closely related RTKs, Kit, PDGFRR, PDGFRβ,
RET, and CSF1R (4.8, 11, 7.7, 9.9, and 12 nM respectively).
The additional kinases with Kd values within 100-fold of Kd
for FLT3 were the closely related RTKs, FLT1, FLT4,
DDR1, and VEGFR2 (41, 41, 81, and 87 nM, respectively).
These data suggest that 7 and analogues are among the
most potent inhibitors of FLT3 in binding as well as pro-
liferation assays and also among the most selective in a panel
of 402 kinases. Additional details of the in vitro profile,
kinase selectivity, mouse PK, and in vivo pharmacology of 7
are described in a separate paper.16
Compound 6b and 6c were found to be similar in potency
to 6a. Addition of polar groups either as a solubilizing group
or as a handle to attach one, did not adversely affect the cell
permeability. This encouraging result indicated that further
optimization of the series for an optimal pharmaceutical
profile may be possible with water solubilizing groups on the
imidazobenzothiazole ring. A number of water solubilizing
groups were attached to the hydroxyl group of 6b, a few of
which are shown in Table 1. Compounds 7-9, 14, and 15
were all approximately equipotent in binding as well as cell
proliferation tests, with the exception of the Kd value of 15,
which was about 5-fold weaker than the others. Compound 7
had a binding constant of 1.6 nM, and it inhibited prolifera-
tion of MV4-11 cells with IC50 = 0.56 nM. Replacing the
morpholine ring of 7 with nitrogen containing solubilizing
groups such as piperidine (8) or N-methylpiperazine (14)
yielded compounds with equal potency but inferior PK
properties, probably due to metabolism of the N-alkyl group
(data not shown). Moving the morpholinoethoxy group of 7
to the 6-position (compound 20b) had a minimal effect on
binding and cell potency, while moving that group to the
5-position (compound 20a) had a profound negative effect
on cellular potency (∼17-fold less potent).
An advantage of screening compounds against
a
large panel of kinases is that one has the choice of guiding
the lead optimization based on potency as well as a desired
level of selectivity, and this helps reduce the time required for
lead optimization.17,18 In fact, during the lead optimization
of the current series of compounds culminating in the
identification of 7 and analogues, we identified potent but
less selective compounds (data not shown); however, we
chose to avoid these compounds to maintain a high degree
of selectivity. Our approach of extensive kinase profiling
during lead optimization guided us to focus on the potent
and selective compounds rather than potent but less selective
compounds. It is important to note that while we chose to
optimize compounds for high kinase selectivity, one can
choose to dial in an appropriate level of selectivity with a
desired list of kinases inhibited by the compounds.
Pharmacokinetics. A large number of compounds pre-
pared during this SAR campaign had similar properties in
kinase binding as well as cell proliferation tests. Therefore,
we studied the pharmacokinetic profile of the more potent
compounds in rats and in athymic nude mice. The PK profile
in mice helped us prioritize compounds for the in vivo
efficacy studies in the same species.
Analogues of the compounds above with a carbon atom in
place of the oxygen of the aryloxyalkyl group (26a-b,
27a-c, 28, and 34) were all found to be equipotent to 7 in
binding as well as cellular tests. However, these compounds
had inferior PK properties (vide infra). Analogues of 7 with a
nitrogen atom in place of the oxygen atom of the side chain
were difficult to prepare. Compound 6c with an N-methyl-
piperazine group attached directly to the 7-position of the
imidazobenzothiazole ring was one of the few analogues
prepared with a nitrogen atom as the handle to attach water
solubilizing groups.
Evaluation of SAR of a number of compounds, some of
which are included in Table 1, indicated that the morpholi-
noethoxy group appended at the 7-position of the imidazo-
benzothiazole ring rendered optimal overall properties to the
molecule. Analogues of 7 with shorter or longer linkers to the
basic nitrogen atom or with terminal alkylamino group or
with the solubilizing group attached at 5- or 6-position of the
imidazobenzothiazole ring were found to be inferior in PK
properties.
Kinase Selectivity. The compounds shown in Table 1 were
tested for their kinase selectivity in a panel of 227 kinases
(202 distinct kinases excluding the mutants) using the Kino-
meScan technology.13 The compounds were screened at
10 μM concentration and the kinases for which >90%
competition was observed in the binding assay (measured
as <10% of control experiment) were designated as “kinase
targets of the compound”. A selectivity score, S(10), based
on this single concentration primary screen, was calculated
by dividing the “kinase targets of the compound” by the total
number of kinases in the panel.15 Lower S(10) scores indicate
higher selectivity and vice versa. As shown in Table 1, the
selectivity score of the compounds in this series was <0.1,
indicating that these are highly selective FLT3 inhibitors.
Compound 7, having a selectivity score of 0.079 in a panel
of 227 kinases, was also screened against a kinase panel of
402 kinases (359 distinct kinases excluding the mutants) and
the S(10) score was calculated to be 0.061. This result
indicates that 7 does not bind to a large number of the
The compounds were dosed as a solution in 22% hydro-
xypropyl β-cyclodextrin in water, except for 27a, which was
dosed in 0.5% methylcellulose. The compounds differen-
tiated themselves in the PK profile in mice and rats.
For example, 7, at an oral dose of 10 mg/kg in mice, showed
the best Cmax (3.8 μM) and AUC (35 μM h) in comparison
3
with its analogues shown in Table 2. Compound 28
was the next best compound with Cmax (2.09 μM) and
AUC (21.32 μM h). The morpholinoethoxy and morpholi-
3
nopropyl side chains of 7 and 28 respectively offer the
better in vitro, cellular, and PK profile than the other
substituents. The PK profile of 27a and other analogues
with terminal alkyl amines or N-alkyl piperazines (data
not shown) were found to be inferior to 7. The poor PK
profile of the carboxylic acid 26a is not surprising, and it
is possible that the terminal alkylamine functionality of
15 contributes to its lower Cmax and AUC in mice than 7
(but similar to 28).

7814 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Table 2. Pharmacokinetic Profile of Compounds in Micea
Chao et al.
dose
(mg/kg)
Cmax
(μM)
Tmax
(h)
AUC(0-inf )
(μM h)
oral t1/2
(h)
compd
3
7
10
10
10
10
10
3.8
1.5
1.7
1.7
0.5
2
35.04
11.72
21.32
0.67
3.6
4.6
4.5
6.7
2.3
15
28
26a
27a
1.58
2.09
0.49
2.19
14.58
a Compounds were dosed orally to nude mice (n = 3, semilongitu-
dinal sampling). All compounds dosed as oral gavage in hydroxypropyl
β-cyclodextrin, except 27a, which was dosed in 0.5% methylcellulose.
Figure 3. Efficacy of compound 7 in a mouse tumor xenograft
model. Compounds were dosed orally once a day to nude mice
bearing a tumor with volume of ∼200 mm3, 10 animals per treat-
ment group, drug treatment for 28 days followed by 32 days of
observation, sunitinib used as a positive control, compound 7 was
dosed as oral gavage in 22% hydroxypropyl β-cyclodextrin, suniti-
nib was dosed in an aqueous citric acid vehicle.
efficacious and well tolerated compound in inhibiting
MV4-11 tumor growth in animals.
Figure 2. Pharmacokinetic profile of 7, 15, and 28 in rats. Com-
pounds were dosed orally (10 mg/kg) to Sprague-Dawley rats
(n = 3); all compounds dosed as oral gavage in hydroxypropyl
β-cyclodextrin.
Conclusion
Lead optimization of a potent and selective FLT3 inhibitor,
1, was carried out to improve its pharmaceutical profile.
Removal of the carboxamide group in 1 and adding a water
solubilizing group has resulted in a novel series of highly potent
and selective compounds with a significantly improved pharma-
ceutical as well as PK profile. Compound 7 was identified as one
of the most potent and selective FLT3 kinase inhibitors we have
characterized. The good aqueous solubility and other pharma-
ceutical properties, excellent PK profile in different species, and
superior efficacy and tolerability in tumor xenograft models
have supported the selection of 7 as a clinical candidate.
A number of compounds were also profiled in Sprague-
Dawley rats upon oral and intravenous dosing for their
PK characteristics. Figure 2 shows the PK curves for com-
pounds 7, 15, and 28 dosed orally at 10 mg/kg. The Cmax and
AUC for 7 (2.16 μM and 20.66 μM h, respectively) was
3
clearly superior to 15 and 28. The clearance of 7 in rats was
7.11 mL/min/kg, half-life was 5.5 h, and the oral bioavail-
ability was found to be 48%. The PK profile of 7 was found
to be dose linear in mice and rats between 1 and 100 mg/kg
doses, and its PK profile in dogs and monkeys was also
acceptable (data not shown).
Efficacy in Tumor Xenograft Studies. The antitumor effi-
cacy of 7 was assessed in a subcutaneous flank-tumor
xenograft model in athymic nude mice using the MV4-11
cell line. Once the tumors had grown to about 200 mm3 size,
compounds were dosed orally once a day for 28 days. The
dosing was stopped, and the animals were observed for
32 additional days. Compound 7 was dosed at 1, 3, and
10 mg/kg (Figure 3). Sunitinib, used as a positive control,
was dosed at 10 and 30 mg/kg.19 At 1 mg/kg of 7, tumor
growth was completely inhibited during the dosing period,
after which growth resumed. At 3 and 10 mg/kg of 7, tumors
regressed almost completely and the tumor volume stayed
suppressed after dosing was halted. At 3 mg/kg, tumors
appeared to regrow after day 49 (21 days post last dose),
while there was no sign of tumor regrowth until day 60
(32 days post last dose) in the animals treated with 10 mg/kg
of 7. In contrast, the 10 mg/kg dose of sunitinib inhibited
tumor growth during the first 14 days of dosing, after which
they started regrowing even during the dosing period. The
tumor profile of animals treated with 30 mg/kg of sunitinib
was similar to the animals treated with 7 at 3 mg/kg dose in
terms of regression and regrowth.
Compound 7 has demonstrated an acceptable toxicology
profile in GLP studies and was tested in AML patients in a
phase I study.20 The acceptable tolerability and excellent PK
profile in humans combined with signs of beneficial effect in
AML patients have justified the progression of 7 into phase II
clinical trials.21
Experimental Section
Chemistry: General Methods. Reactions involving air or
moisture sensitive reagents were carried out under an argon
atmosphere. If not otherwise specified, reactions were carried
out at ambient temperature. Organic extracts were usually
dried over anhydrous MgSO4. Proton NMR spectra were
obtained in the deuterated solvents indicated on a Bruker
Avance 300 referencing to tetramethylsilane. LC-MS analyses
No body weight loss was observed in animals treated
with 7 at all doses, indicating it is well tolerated in mice
at efficacious doses. These results indicate 7 is a highly

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7815
were carried out on Shimadzu LC-MS 2010 EV system. Chemi-
cal purity for all target compounds was determined by HPLC
with a C18 column (Phenomenex Luna 5 μ C18(2) 100A,
250 mm ꢀ 4.6 mm), detected by ELSD and MS, confirming
g95% purity.
2-(4-Nitrophenyl)imidazo[2,1-b][1,3]benzothiazol-7-ol (3b):
General Procedure A. A mixture of 2-amino-1,3-benzothia-
zol-6-ol (20.0 g, 0.12 mol) and 2-bromo-40-nitroacetophenone
(29.3 g, 0.12 mol) in 600 mL of ethanol was heated to reflux
overnight. The reaction mixture was cooled to 0 °C in an
ice-water bath. Filtration of the resulting precipitate pro-
vided 2-(4-nitrophenyl)imidazo[2,1-b][1,3]benzothiazol-7-ol
(3b) as a yellow solid (17.0 g, 46% yield). 1H NMR (DMSO-
d6) δ 10 (s, 1H), 8.9 (s, 1H), 8.3 (d, J = 8.6 Hz, 2H), 8.1 (d, J =
8.6 Hz, 2H), 7.8 (d, J = 8.7 Hz, 1H), 7.4 (s, 1H), 6.9 (d, J = 8.7
Hz, 1H). LC-MS (ESI) m/z 312 (M þ H)þ.
General Procedure E for Preparation of Hydrochloride Salt.
The free base was dissolved in a mixture of dichloromethane
(20 mL) and methanol (1 mL). A solution of 1.0 M HCl in ethyl
ether (1.1 equiv for all compounds except 7, for which 2.5 equiv
were used) was added dropwise, followed by addition of ethyl
ether. The precipitate was collected by filtration to give N-(5-
tert-butyl-isoxazol-3-yl)-N0-{4-[7-(2-morpholin-4-yl-ethoxy)-
imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea dihydrochloride
(7) (2.441 g, 98%). 1H NMR (DMSO-d6) δ 11.0 (br, 1H), 9.68 (s,
1H), 9.26 (s, 1H), 8.66 (s, 1H), 7.93 (d, J=8.9 Hz, 1H), 7.78 (m,
3H), 7.53 (d, J=8.7 Hz, 2H), 7.26 (dd, J=2.4 and 8.9 Hz, 1H), 6.53
(s, 1H), 4.50 (t, J=4.1 Hz, 2H), 3.97 (m, 2H), 3.81 (t, J=12.1 Hz,
2H), 3.6 (overlapping, 4H), 3.23 (m, 2H), 1.30 (s, 9H). LC-MS
(ESI) m/z 561 (M þ H)þ. Anal. (C29H32N6O4S 2HCl) C, H, N. C:
3
calcd 54.97; found 54.54. H: calcd 5.22; found 5.87. N: calcd 13.26;
found 13.16.
7-(2-Morpholin-4-yl-ethoxy)-2-(4-nitrophenyl)imidazo[2,1-b][1,3]-
benzothiazole (18c): General Procedure B. To a mixture of 2-(4-
nitrophenyl)imidazo[2,1-b][1,3]benzothiazol-7-ol (3b) (4.67 g,
15.0 mmol) in DMF (100 mL) was added potassium carbonate
(5.52 g, 40 mmol) and 4-(2-chloroethyl)morpholine hydrochloride
(3.72 g, 20 mmol). The mixture was heated to 60 °Cfor6hand70°C
for 12 h. The reaction mixture was poured into water (400 mL),
filtered, and washed with water and ethyl ether to give 7-(2-
morpholin-4-yl-ethoxy)-2-(4-nitrophenyl)imidazo[2,1-b][1,3]-
benzothiazole (18c), which was used in the next reaction without
further purification. 1H NMR (DMSO-d6) δ 9.00 (s, 1H), 8.31 (d,
J=8.1 Hz, 2H), 8.09 (d, J=8.1 Hz, 2H), 7.92 (d, J=8.8 Hz, 1H),
7.72 (d, J=1.5Hz, 1H), 7.2(dd, J=8.8 and 1.5Hz, 1H), 4.17 (t, J=
5.4 Hz, 2H), 3.59 (t, J = 4.2 Hz, 4H), 3.34 (overlapping
with solvent, 4H), 2.73 (t, J =5.4 Hz, 2H). LC-MS (ESI) m/z
425 (M þ H)þ.
Kinase Competition Binding. KinomeScan competition bind-
ing assays (www.kinomescan.com) were performed as described
previously.13,15 Kinases were produced displayed on T7 phage
or by expression in HEK-293 cells and tagged with DNA,
binding reactions were performed at room temperature for
one hour, and the fraction of kinase not bound to test compound
determined by capture with an immobilized affinity ligand and
quantitation by quantitative PCR.
MV4-11 Cell Proliferation. MV4-11 cells were cultured in
Iscove’s media with 10% FBS and RPMI complete with 10%
FBS, respectively. For proliferation assays, cells were cultured
overnight in low serum media (0.5% FBS) and then seeded in a
96-well plate at 40000 cells per well. Inhibitors were added to the
cells and incubated at 37 °C for 72 h. Cell viability was measured
using the Cell Titer-Blue cell viability assay from Promega
(Madison, WI).
2-(4-Aminophenyl)-7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b]-
[1,3]benzothiazole (19c): General Procedure C. A mixture of 7-(2-
morpholin-4-yl-ethoxy)-2-(4-nitrophenyl)imidazo[2,1-b][1,3]-
benzothiazole (18c) (15.0 mmol) and ammoniumchloride(2.14g,
40 mmol) in ethanol was heated to reflux, and then iron powder
(8.378 g, 150 mmol) was added. The mixture was heated to reflux
for 3 h and then immediately filtered through celite and washed
with hot ethanol. The filtrate was concentrated to approximately
1/5 of the original volume, neutralized with saturated NaHCO3
solution, and extracted with dichloromethane. The combined
extracts was dried over MgSO4 and concentrated to give 2-(4-
aminophenyl)-7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b][1,3]-
benzothiazole (19c) as an orange solid (4.067 g, 69% for two
steps). 1H NMR (DMSO-d6) δ 8.40 (s, 1H), 7.86 (d, J=8.3 Hz,
1H), 7.70 (s, 1H), 7.51 (d, J=7.6 Hz, 2H), 7.22 (d, J=8.3 Hz,
1H), 6.61 (d, J=7.6 Hz, 2H), 4.34 (t, J=5.6 Hz, 2H), 3.76 (m,
4H), 2.99 (m, 6H). LC-MS (ESI) m/z 395 (M þ H)þ.
Pharmacokinetics. Precatheterized (jugular vein), male Spra-
gue-Dawley rats (230-300 g; Charles River, Hollister, CA) and
female athymic nude mice (20-35 g; Harlan, Livermore, CA)
were acclimated at the vivarium for at least three days following
delivery and prior to entering a study. Rats were fasted over-
night before dosing. Compounds were administered orally (PO)
by gavage at 10 mg/kg in 22% hydroxypropyl-β-cyclodextrin
(HPBCD) or 0.5% methylcellulose. Blood was collected into
K3EDTA tubes for plasma at 15 min, 30 min, 1 h, 2 h, 4 h, 6 h,
and 24 h postdose. Three rats were used per study, however, for
blood collection in mice 150 μL retro-orbital bleeds were taken
semilongitudinally using nine animals, taking 2-3 time-points
per animal for a total of n = 3 plasma concentration-time
curves. Plasma samples, calibration, and quality control stan-
dards (25 μL) were extracted with four volumes of acetonitrile
containing an internal standard and analyzed using LC-MS/MS
analysis (Sciex 4000 Qtrap). Sample separation was achieved on
a Zorbax SBC8 5 μm column (2.1 or 4.6 mm ꢀ 50 mm) using a
0.8 mL/min flow rate over a 3 min gradient from 5 to 95% ACN
containing 0.05% formic acid. The parent compound to frag-
ment mass transitions were monitored. Pharmacokinetic para-
meters were calculated from the normalized LC-MS/MS peak
areas using a noncompartmental model and the linear trape-
zoidal estimation method with WinNonlin (Pharsight v5.2).
Efficacy in Tumor Xenograft Studies. Xenograft studies were
performed at Piedmont Research Center LLC (Morrisville,
NC). MV4-11 human leukemia cells (1 ꢀ 107) that had been
cultured in Iscove’s Modified Dulbecco’s Medium supplemen-
ted with 10% heat-inactivated fetal bovine serum, 100 units/mL
penicillin G, 100 μg/mL streptomycin sulfate, 0.25 μg/mL
amphotericin B, 2 mM glutamine, 0.075% sodium bicarbonate,
and 25 μg/mL gentamicin were harvested during logarithmic
phase growth and resuspended at a concentration of 5 ꢀ 107
cells/mL in 50% Matrigel matrix (BD Biosciences) and 50%
PBS, and implanted subcutaneously into the right flank of
female athymic nude mice (nu/nu, Harlan). Sixteen days later,
mice were sorted into groups, each consisting of 10 mice with
individual tumor sizes of 126-221 mm3 and group mean tumor
N-(5-tert-Butyl-isoxazol-3-yl)-N0-{4-[7-(2-morpholin-4-yl-
ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea Dihy-
drochloride (7): General Procedure D. A suspension of
2-(4-aminophenyl)-7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b]-
[1,3]benzothiazole (19c) (4.06 g, 10.3 mmol) and 5-tert-butyl-
isoxazole-3-isocyanate (5) (1.994 g, 12 mmol) in toluene (60 mL)
was heated at 120 °C overnight. The reaction was quenched with
a mixture of dichloromethane and water containing a little
methanol, and the mixture was neutralized with saturated aqu-
eous NaHCO3. The aqueous phase was extracted twice with
dichloromethane, and the combined organic extracts were dried
over MgSO4 and filtered. The filtrate was concentrated to a
volume of about 20 mL and ethyl ether was added, resulting in
the formation of a solid. The precipitate was collected by
filtration, washed with ethyl ether, and dried under vacuum to
give the free base of 7 (2.342 g, 41%). 1H NMR (DMSO-d6) δ 9.6
(br, 1H), 8.9 (br, 1H), 8.61 (s, 1H), 7.86 (d, J=8.9 Hz, 1H), 7.76
(d, J=8.0 Hz, 2H), 7.69 (d, J=1.3 Hz, 1H), 7.51 (d, J=8.0 Hz,
2H), 7.18 (dd, J=1.3 and 8.9 Hz, 1H), 6.52 (s, 1H), 4.16 (t, J=5.7
Hz, 2H), 3.59 (t, J=4.2 Hz, 4H), 3.36 (overlapping, 4H), 2.72 (t,
J=5.7 Hz, 2H), 1.30 (s, 9H).

7816 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Chao et al.
sizes of 160 mm3. Tumor dimensions were measured by caliper
and tumor volume was calculated using the formula: tumor
volume (mm3)=w2 ꢀ l/2, where w=width and l=length in mm
of an MV4-11 tumor. Each dose of drug was given in a volume of
0.2 mL per 20 g of body weight (10 mL/kg) and was adjusted for
the body weight of the animal. Each animal was euthanized
when its tumor reached the predetermined end point size of
approximately 1000 mm3 or on the last day of the study (day 60),
whichever came first.
(10) Patel, H. K.; Grotzfeld, R. M.; Lai, A. G.; Mehta, S. A.; Milanov,
Z. V.; Chao, Q.; Sprankle, K. G.; Carter, T. A.; Velasco, A. M.;
Fabian, M. A.; James, J.; Treiber, D. T.; Lockhart, D. J.;
Zarrinkar, P. P.; Bhagwat, S. S. Arylcarboxyamino-Substituted
Diaryl Ureas as Potent and Selective FLT3 Inhibitors. Bioorg.
Med. Chem. Lett. 2009, 19, 5182–5185.
(11) Grin, N. P.; Krasovskii, A. N.; Kochergin, P. M. Investigations
in the Imidazole Series. Chem. Heterocycl. Compd. 1972, 8,
1149–1152.
(12) Kaye, I. A.; Burlant, W. J.; Price, L. Thiocyanation of p-Dialky-
lamino-alkoxyanilines. J. Org. Chem. 1951, 16, 1421–1426.
(13) Fabian, M. A.; Biggs, W. H., III; Treiber, D. K.; Atteridge, C. E.;
Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.;
Edeen, P. T.; Floyd, M.; Ford, J. M; Galvin, M.; Gerlack, J. L.;
Grotzfeld, R. M.; Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai,
A. G.; Lelias, J.-M.; Mehta, S. A.; Milanov, Z. V.; Velasco, A. M.;
Wodicka, L. M.; Patel, H. K.; Zarrinkar, P. P.; Lockhart, D. J. A
small molecule-kinase interaction map for clinical kinase inhibi-
tors. Nat. Biotechnol. 2005, 23, 329–336.
Acknowledgment. We thank Dr. Robert C. Armstrong for
his assistance in the analysis and graphical representation of
the tumor xenograft data. We also thank Paul Gallant, Mazen
W. Karaman, Antonio Torres, and the Ambit High Through-
put Screening team for kinase profiling and Kd measurements.
(14) Knight, Z. A.; Shokat, K. M. Features of selective kinase inhibi-
tors. Chem. Biol. 2005, 12, 621–637.
Supporting Information Available: Experimental data for
compounds 6a, 6b, 6c, 8, 9, 14, 15, 20a, 20b, 26a, 26b, 27a,
27b, 27c, 28, and 34. This material is available free of charge via
the Internet at http://pubs.acs.org.
(15) Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.;
Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis,
M. I.; Edeen, P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart,
D. J.; Milanov, Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.;
Pritchard, L. M.; Wodicka, L. M.; Zarrinkar, P. P. A quantitative
analysis of kinase inhibitor selectivity. Nat. Biotechnol. 2008, 26,
127–132.
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