3-Amino-benzo[d]isoxazoles as novel multitargeted inhibitors of receptor tyrosine kinases

Article abstract of DOI:10.1021/jm701096v

A series of benzoisoxazoles and benzoisothiazoles have been synthesized and evaluated as inhibitors of receptor tyrosine kinases (RTKs). Structure-activity relationship studies led to the identification of 3-amino benzo[d]isoxazoles, incorporating a N,N′-

Full text of DOI:10.1021/jm701096v

J. Med. Chem. 2008, 51, 1231–1241
1231
3-Amino-benzo[d]isoxazoles as Novel Multitargeted Inhibitors of Receptor Tyrosine Kinases
Zhiqin Ji,*,† Asma A. Ahmed,‡ Daniel H. Albert,† Jennifer J. Bouska,† Peter F. Bousquet,‡ George, A. Cunha,‡ Gilbert Diaz,†
Keith B. Glaser,† Jun Guo,† Christopher M. Harris,‡ Junling Li,† Patrick A. Marcotte,† Maria D. Moskey,‡ Tetsuro Oie,†
Lori Pease,† Nirupama B. Soni,† Kent D. Stewart,† Steven K. Davidsen,† and Michael R. Michaelides†
Global Pharmaceutical Research and DeVelopment, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064-6100, and Abbott
Bioresearch Center, 100 Research DriVe, Worcester, Massachusetts 01605-5314
ReceiVed September 4, 2007
A series of benzoisoxazoles and benzoisothiazoles have been synthesized and evaluated as inhibitors of
receptor tyrosine kinases (RTKs). Structure–activity relationship studies led to the identification of 3-amino
benzo[d]isoxazoles, incorporating a N,N′-diphenyl urea moiety at the 4-position that potently inhibited both
the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor families
of RTKs. Within this series, orally bioavailable compounds possessing promising pharmacokinetic profiles
were identified, and a number of compounds demonstrated in vivo efficacy in models of VEGF-stimulated
vascular permeability and tumor growth. In particular, compound 50 exhibited an ED50 of 2.0 mg/kg in the
VEGF-stimulated uterine edema model and 81% inhibition in the human fibrosarcoma (HT1080) tumor
growth model when given orally at a dose of 10 mg/kg/day.
Introduction
important target for therapeutic intervention. Although VEGFR
and PDGFR are compelling cancer targets individually, tumors
are capable of secreting multiple angiogenic factors and they
depend on these factors at different stages of progression.27–29
For example, VEGFs cause a large increase in blood vessel
formation, yet these vessels are immature and leaky. The
formation of thicker, more stable vessels requires encapsulation
by pericytes that are driven by PDGFR-ꢀ signaling. The
multifactorial mechanisms by which cancer cells proliferate
demand the development of multitargeted agents to interfere
with multiple pathways. Simultaneous blockade of all the VEGF
and PDGF receptors is expected to provide a synergistic effect
by impacting multiple stages of tumor formation and, thus, may
offer greater potential for treating a broader range of human
cancers. The recent approval of the multitargeted agents 1
(sunitinib)30 and 2 (sorafenib)31 demonstrates that clinical
benefit with manageable side effects is possible with broad-
acting kinase inhibitors (Figure 1). As part of a research
program at Abbott aiming at the identification of novel
multitargeted RTK inhibitors, we have explored several
chemotypes such as thienopyrimidines,32 isothiazolopyrim-
idines,33 thienopyridines,34 and indazoles35 as potent inhibi-
tors of multitargeted RTKs. Extensive SAR studies in the
aminoindazole series led to the discovery of 3 (ABT-869),35,36
which is currently in phase II clinical trials.
Receptor tyrosine kinases (RTKsa), a subclass of cell surface
growth factor receptors, mediate cellular response to environ-
mental signals and facilitate a broad range of cellular processes
including proliferation, migration, and survival.1,2 RTK signaling
pathways are normally highly regulated. However, aberrant
activation of RTKs, in particular, vascular endothelial growth
factor receptor (VEGFR) and platelet-derived growth factor
receptor (PDGFR), has been linked to the development and
progression of various human cancers.3–5 The VEGFR family,
comprised of FLT1 (VEGFR1), KDR (VEGFR2), and FLT4
(VEGFR3), plays a pivotal role in angiogenesis and contributes
to tumor progression through its ability to mediate tumor
angiogenesis and lymphangiogenesis, and to enhance vascular
permeability.6–10 The PDGFR family, consisting of PDGFR-R,
PDGFR-ꢀ, colony stimulating factor 1 receptor (CSF1R), KIT,
and FLT3, promotes tumor cell growth and metastasis through
modification of the tumor microenvironment.11,12 Additionally,
mutations of FLT3 and KIT are directly associated with
proliferation of acute myeloid leukemia (AML) blast cells and
gastrointestinal stromal tumor (GIST) cells, respectively.13–18
Inhibition of RTK signaling pathways has become an
important area of new cancer drug discovery.19–21 The approval
of bevacizumab,22 an anti-VEGF antibody, and the antitumor
efficacy exhibited by small molecule inhibitors such as
(Z)-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)indolin-2-one
(SU5416),23 N-(4-chlorophenyl)-4-(pyridin-4-ylmethyl)phthalazin-
1-amine (PTK787, vatalanib),24,25 and 3-(4-bromo-2,6-difluo-
robenzyloxy)-5-(3-(4-(pyrrolidin-1-yl)butyl)ureido)isothiazole-
4-carboxamide (CP-547632)26 have established VEGFR as an
A model of 3 docked into the active site of KDR in its inactive
conformation shows that the 3-amino group along with its
adjacent ring nitrogen serves as an anchor binding to the hinge
region of KDR, while the diaryl urea moiety extends into the
hydrophobic back pocket of the kinase domain (Figure 2).
According to this model, the endocyclic indazole N1-H of 3
does not form additional hydrogen bonds with the kinase, and
SAR studies confirm that methylation of the N1-H only results
in a slight loss in KDR potency.35 Based on these observations,
we rationalize that the N1-H of endocyclic indazole can be
replaced by oxygen or sulfur, leading to the novel chemotypes
aminobenzoisoxazole (4) and aminobenzoisothiazole (5; Figure
* To whom correspondence should be addressed. Tel.: (847) 938-9442.
Fax: (847) 935-5165. E-mail: zhiqin.ji@abbott.com.
†
Abbott Laboratories.
Abbott Bioresearch Center.
‡
a Abbreviations: RTK, receptor tyrosine kinase; VEGFR, vascular
endothelial growth factor receptor; PDGFR, platelet-derived growth factor
receptor; FLT, Fms-like tyrosine kinase; KDR, kinase insert domain-
containing receptor tyrosine kinase; CSF1R, colony stimulating factor 1
receptor; AML, acute myeloid leukemia; GIST, gastrointestinal stromal
tumor; ATP, adenosine 5′-triphosphate; FGFR, fibroblast growth factor
receptor; UE, uterine edema.
10.1021/jm701096v CCC: $40.75
2008 American Chemical Society
Published on Web 02/09/2008

1232 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Ji et al.
Figure 1. Advanced multitargeted RTK inhibitors.
Scheme 1. Synthesis of 3-Aminobenoisoxazolesa
a Reagents and conditions: (a) acetohydroxamic acid, t-BuOK, DMF,
rt; (b) 4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenylamine, PdCl2 ·
dppf·CH2Cl2, Na2CO3, DME, H2O, 85 °C; (c) acetic acid, NaOCN; (d)
RCOCl, pyridine, DMF; (e) RNCO, CH2Cl2; (f) 7, PdCl2 ·dppf·CH2Cl2,
Na2CO3, DME, H2O, 85 °C.
Figure 2. Molecular models of inhibitors bound to the inactive
conformation of KDR (based on cKit crystal structure). The inset shows
the previously described interaction of compound 3 with two hydrogen
bonds to the hinge region. The larger picture shows model of amino-
benzisoxazole 14 bound to KDR with two analogous hinge hydrogen
bonds to Glu 917 and Cys 919. The urea of 14 donates a hydrogen
bond to Glu 885.
pound 8 served as a key intermediate for the preparation of a
number of target molecules. For example, urea 9 was prepared
by the treatment of 8 with NaOCN in acetic acid solution; 10
and 11 were synthesized by the reaction of 8 with carboxylic
acid chlorides. Treatment of 8 with the requisite isocyanates
gave rise to the corresponding urea analogs 13 and 14. To
confirm the regioselectivity of the reaction of 8 with isocyanates,
we also prepared 13 and 14 via Suzuki coupling of 7 with urea
boronates 12a,b, which were readily prepared via the treatment
of 4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenylamine
with the appropriate isocyanates. The products obtained from
both methods were identical. Consequently, urea analogs 23–45
were prepared by treatment of 8 with the corresponding
isocyanates.
3-Aminobenzoisothiazoles were synthesized as illustrated in
Scheme 2. Compound 6 was converted to benzylthioether 15
by displacement of the fluoride with phenylmethanethiol.
Sequential treatment of 15 with SO2Cl2 and ammonia generated
the intermediate 16, which then underwent cyclization in the
presence of lithium methoxide to afford 3-aminobenzoisothi-
azole 17. Compounds 18 and 19 were obtained via Suzuki
coupling reactions of 17 with urea boronates 12a,b. 7-Substi-
tuted 3-aminobenzoisoxazoles 49–57 were prepared from the
previously reported intermediates 46a-f,35 following the pro-
cedures described in Scheme 3.
Figure 3. Inhibitor design.
3). We wish to report the effects of these replacements on
structure–activity relationships, pharmacokinetics, and in vivo
efficacy.
Chemistry
The general synthetic route for the preparation of 3-ami-
nobenzoisoxazole derivatives is shown in Scheme 1. Treatment
of commercially available 6 with acetohydroxamic acid and
potassium t-butoxide provided the core 3-aminobenzisoxazole
7, which underwent Suzuki coupling with 4-(4,4,5,5-tetramethyl-
[1,3,2]dioxaborolan-2-yl)-phenylamine to afford aniline 8. Com-
Results and Discussion
Compounds were assayed against a panel of enzymes
including KDR, KIT, and FLT3. The measurements were

3-Amino-benzo[d]isoxazole Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1233
Scheme 2. Synthesis of 3-Aminobenzoisothiazolesa
a Reagents and conditions: (a) t-BuOK, PhCH2SH, DMF; (b) SO2Cl2; (c) 7 M NH3 in MeOH; (d) LiOMe, MeOH; (e) 12, PdCl2 ·dppf·CH2Cl2, Na2CO3,
DME, H2O.
Scheme 3. Synthesis of 7-Substituted 3-Aminobenzoisoxazolesa
a Reagents and conditions: (a) for 47a-e, acetohydroxamic acid, t-BuOK, DMF, rt; (b) for 47f, acetone oxime, t-BuOK, THF; (c) 5% HCl, EtOH; (d)
4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenylamine, PdCl2 ·dppf·CH2Cl2, Na2CO3, DME, H2O, 85 °C; (e) RNCO, CH2Cl2.
conducted in the presence of a physiologically relevant con-
centration of adenosine 5′-triphosphate (ATP, 1.0 mM), as
previously described.32 As KDR plays a central role in angio-
genesis, structural optimization was primarily focused on KDR.
A brief SAR summary is shown in Table 1. As observed in the
3-aminoindazole series,35 the N,N′-diphenyl urea moiety was
found to be essential for potency in the aminobenzoisoxazole
and aminobenzoisothiazole series. This suggests that these
compounds exhibit similar binding modes with the enzyme, with
the amino and its adjacent ring nitrogen forming hydrogen bonds
with KDR hinge region, and the diaryl urea moiety extending
into the hydrophobic back pocket of the kinase domain, as
shown in Figure 2. Replacement of endocyclic indazole NH-1
with oxygen led to a 5-fold loss of potency (13 vs 3), whereas
the replacement of NH-1 with sulfur resulted in a 157-fold loss
of potency (18 vs 3). In general, 3-aminobenzoisoxazole
derivatives exhibited improved potency when compared with
the related 3-aminobenzoisothiazole compounds (13 vs 18 and
14 vs 19).
In an attempt to better understand the SAR trends described
above, we studied the intrinsic properties of the three hetero-
cyclic structures, aminoindazole (20), aminobenzoisoxazole (21),
and aminobenzoisothiazole (22), in greater detail (Table 2). The
binding mode of aminoindazole (20) has previously been
described as possessing a hydrogen bond donation from the
exocyclic N-H(b) to a protein backbone carbonyl and possessing
a hydrogen bond accepting interaction to the ring nitrogen N(a)
from a protein backbone amide N-H (positions identified in
Table 2). Therefore, we calculated the hydrogen bond energies
of these two positions as well as the basicity (pKa) of the ring
nitrogen N(a). Relative to aminoindazole (20), aminobenzoix-
azole (21) shows decreased basicity, lower hydrogen bond
accepting potential at N(a), and increased hydrogen bond
donating potential at N(b). The aminobenzoisothiazole (22)
shows increased basicity, unchanged hydrogen bond accepting
potential, and increased hydrogen bond donation potential. The
higher electronegativity of oxygen and lower electronegativity
of sulfur, relative to nitrogen, provides a rationalization for these
calculated observations. In addition to these electronic effects,
the larger sulfur atom has the potential to perturb the hinge
interactions away from optimal geometry relative to the smaller
oxygen and nitrogen atoms. The significant decrease in potency
of the aminobenzoisothiazole series, relative to the aminoinda-
zole and aminobenzisoxazole series, is likely due to a combina-
tion of the electronic and steric effects noted here. A complete
understanding of the relative contributions of these effects will
require further study.
Having demonstrated the importance of the N,N′-diphenyl
urea moiety, we then focused on optimization of the terminal
phenyl ring of the urea. The effects of ortho-, meta-, and para-
substitution and disubstitution on potency were investigated.
Several trends can be observed from Table 3. ortho-Substitution
(cf. 24–26) is not tolerated; even a small group such as fluoro
causes a large drop in potency. The detrimental effect of ortho-
substitution in this series is more significant than in the
aminoindazole series.35 On the other hand, consistent with the
aminoindazole series, a variety of meta-substituents, including
both electron-donating and electron-withdrawing groups (cf. 14,
27–32) are tolerated, with the m-methyl compound 27 providing
an approximate 3-fold boost in potency. Substitution at the para-

1234 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Ji et al.
Table 3. Inhibitory Activity of Aminobenzoisoxazole Urea Analogs
Table 1. Inhibitory Activity of Aminobenzoisoxazoles and
Aminobenzoisothiazoles
KDR
KIT
FLT3
KDR Cell
b
a
a
a
compds
R
IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM)
23
24
25
26
27
28
14
29
30
31
32
33
34
35
36
37
13
38
39
40
41
42
43
44
45
H
o-F
o-OCF3
o-CF3
m-Me
m-OMe
m-CF3
m-NO2
m-F
m-Br
m-Cl
23
318
806
6161
7
17
37
19
44
10
8
16
32
22
134
42
21
8
104
15
23
59
67
8
18
21
166
23440
12
4
114
14
11
7
10
5
27
5
7
102
23
37
339
12
62
36
14
24
50
7
8
25
497
1000
NA
5
11
65
17
21
5
28
44
173
106
23
123
13
3
80
40
94
19
81
36
32
60
1570
14
NA
64
5
NA
NA
6
p-CH3
p-CF3
p-OMe
p-F
4
NA
NA
4
p-OCF3
2-F-5-Me
3,5-di-Me
2-F-5-CF3
3-F-4-Me
3-CF3-4-F
3-Cl-4-F
3,5-di-F
3-Cl-4-OMe
3-Cl-4-Me
27
22
17
213
NA
28
22
NA
8
8
37
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate. b Each cellular IC50 determination
was performed with five concentrations, and each assay point was determined
in duplicate.
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate.
cellular potency (cf. 13), overcoming the deleterious effect of
ortho-substitution.
Table 2. pKa and Hydrogen Bond Parameters in Three Pharmacophores
As the binding modes of 3-aminobenzoisoxazoles and 3-ami-
noindazoles are similar, substituents at the C7 position in the
3-aminobenzoisoxazole series would be expected to project
toward solvent.35 Thus, we further investigated the effect of
C7 substitution, including groups that might improve aqueous
solubility. As shown in Table 4, electron-donating groups such
as methoxy, methyl, are tolerated (cf. 49-53); in contrast,
electron-withdrawing groups such as fluoro and trifluoromethoxy
are detrimental to potency (cf. 54, 55). Installation of the water
solubilizing morpholine group linked by a two-carbon ether
tether led to retention of both enzymatic and cellular potency
(cf. 57).
Select compounds were further evaluated for their selectivity
against other tyrosine kinases. As shown in Table 5, the
compounds potently inhibited all members of the VEGR and
PDGFR families and exhibited modest inhibitory activity against
Tie-2, but were selective over other nonstructurally related
tyrosine kinases such as FGFR, HCK, SRC, and LYN.
Compounds with promising in vitro enzymatic and cellular
inhibitory activity were further evaluated for their pharmaco-
kinetic properties in mice. Data for 3 are included for compari-
son. As shown in Table 6, the 2-F-5-Me phenyl urea analog 49
had the lowest oral bioavailability (34%), however, all other
analogs incorporating halo or trifluoromethyl substituents had
very high levels of oral bioavailability that are comparable to
H-bond parameter ∆E, kcal/molb
a
compd
X
pKa
acceptor N(a)
donor H(b)
20
21
22
NH
O
S
3.72
2.04
6.04
10.8
9.60
10.6
2.60
3.40
3.30
a pK values were calculated using ACD/pKa software (version 10.05,
Advanced Chemistry Development Inc., Toronto, ON). b Hydrogen bond
energies were calculated by using gas phase energies at the Hartree–Fock
6-31G* level. The energy refers to the energy released upon formation of
a hydrogen bonded complex between the heterocycle and hydrogen fluoride
(HF) by either hydrogen bond donation to the fluoride of HF or hydrogen
bond accepting interaction from the hydrogen of HF. Larger values
correspond to stronger hydrogen bonds.
position does not significantly affect KDR enzymatic inhibitory
activity, however, the KDR cellular activity is diminished (cf.
33–37). Disubstitution is generally well tolerated (cf. 13, 38–45),
with the 3,5-dimethyl compound 38 being the most potent,
exhibiting both enzymatic and cellular KDR inhibitory activity
of less than 10 nM. Interestingly, addition of a methyl on the
o-F compound 24 results in an approximate 40-fold boost in

3-Amino-benzo[d]isoxazole Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1235
Table 4. Inhibitory Activity of 7-Substituted Benzoisoxazole Urea
Analogs
oral bioavailability and improved antitumor efficacy in the
human fibrosarcoma (HT1080) model relative to 3.
Experimental Section
HTRF Assays of Receptor Tyrosine Kinases. Assays were
performed in a total of 40 µL in 96-well Costar black half-volume
plates using HTRF technology. Peptide substrate (Biotin-Ahx-
AEEEYFFLFA-amide) at 4 µM, 1 mM ATP, enzyme, and inhibitors
were incubated for 1 h at ambient temperature in 50 mM Hepes/
NaOH, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTT, 0.1
mM orthovanadate, and 0.01% BSA. Inhibitors were added to the
wells at final concentrations of 3.2 nM to 50 µM with 5% DMSO
added as cosolvent. The reactions were stopped with 10 µL/well
0.5 M EDTA and then 75 µL of buffer containing streptavidin-
allophycocyanin (Prozyme; 1.1 µg/mL) and PT66 antibody Eu-
ropium cryptate (Cis-Bio; 0.1 µg/mL) was added to each well. The
plates were read from 1 to 4 h after addition of the detection reagents
and the time-resolved fluorescence (665 to 615 ratio) measured
using a Packard Discovery instrument. The amount of each tyrosine
kinase added to the wells was calibrated to give a control (no
inhibitor) to background (prequenched with EDTA) ratio of 10–15
and was shown to be in the low nanomolar concentration range
for each kinase. The inhibition of each well was calculated using
the control and background readings for that plate. KDR, CSF1R,
KIT, FLT1, and FLT3 were assayed using the above protocol. The
HTRF detection was done in the same way as for the other kinases.
Inhibition constants are the mean of two determinations performed
with seven concentrations of the test compounds.
Cellular KDR Phosphorylation Assay Determined by ELISA.
NIH3T3 cells stably transfected with full length human KDR
(VEGFR2) were maintained in DMEM medium with 10% fetal
bovine serum and 500 µg/mL geneticin. KDR cells were plated at
20000 cells/well into duplicate 96-well tissue culture plates and
cultured overnight in an incubator at 37 °C with 5% CO2 and 80%
humidity. The growth medium was replaced with serum-free growth
medium for 2 h prior to compound addition. Compounds in DMSO
were diluted in serum-free growth medium (final DMSO concentra-
tion 1%) and added to cells for 20 min prior to stimulation for 10
min with VEGF (50 ng/mL). Cells were lysed by addition of RIPA
buffer (50 mM Tris-HCl (pH 7.4), 1% IGEPAL, 150 mM NaCl, 1
mM EDTA, and 0.25% sodium deoxycholate) containing protease
inhibitors (Sigma cocktail), NaF (1 mM), and Na3VO4 (1 mM) and
placed on a microtiter plate shaker for 10 min. The lysates from
duplicate wells were combined, and 170 µL of the combined lysate
was added to the KDR ELISA plate. The KDR ELISA plate was
prepared by adding anti-VEGFR2 antibody (1 µg/well, R&D
Systems) to an unblocked plate and incubated overnight at 4 °C.
The plate was then blocked for at least 1 h with 200 µL/well of
5% dry milk in PBS. The plate was washed two times with PBS
containing 0.1% Tween 20 (PBST) before addition of the cell
lysates. Cell lysates were incubated in the KDR ELISA plate with
constant shaking on a microtiter plate shaker for 2 h at room
temperature. The cell lysate was then removed and the plate was
washed five times with PBST. Detection of phospho-KDR was
performed using a 1:2000 dilution of biotinylated 4G10 antiphos-
photyrosine (UBI, Lake Placid, NY), incubated with constant
shaking for 1.5 h at room temperature, and washed five times with
PBST, and for detection, a 1:2000 dilution of strepavidin-HRP (UBI,
Lake Placid, NY) was added and incubated with constant shaking
for 1 h at room temperature. The wells were then washed five times
with PBST and K-Blue HRP ELISA substrate (Neogen) was added
to each well. Development time was monitored at 650 nm in a
SprectrMax Plus plate reader until 0.4–0.5 absorbance units were
obtained (approximately 10 min) in the VEGF only wells. Phos-
phoric acid (1 M) was added to stop the reaction, and the plate
was read at 450 nm. Percent inhibition was calculated using the
VEGF only wells as 100% controls and wells containing 5 µM
pan-kinase inhibitor as 0% controls (no VEGF wells were used to
monitor endogenous phosphorylation state of the cells). IC50 values
were calculated by nonlinear regression analysis of the concentra-
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate. b Each cellular IC50 determination
was performed with five concentrations, and each assay point was determined
in duplicate.
3. In general, the compounds were characterized by moderate
volume of distribution (Vd) and low clearance.
Acute in vivo efficacy was assessed using the estradiol-
induced murine uterine edema (UE) assay, an in vivo model of
VEGF-stimulated vascular permeability. As seen in Table 7, a
number of compounds displayed excellent efficacy in this model
after oral adminisration (ED50 < 10 mg/kg). Compounds that
showed efficacy in the uterine edema model and possessed
favorable PK profiles were evaluated in the human fibrosarcoma
HT1080 xenograft tumor growth model. Several compounds
exhibited significant inhibition of tumor growth. In particular,
compound 50 displayed 81% inhibition at 10 mg/kg/day (Figure
4), which is more potent on a dose basis than 3 in this model.37
Furthermore, the cardiac safety of inhibitors 49 and 50 were
evaluated using a [3H] dofetilide binding assay, a predictive
screening tool for hERG blockade. Both 49 and 50 showed low
affinity with an IC50 value greater than 30 uM.
Conclusion
By substituting the NH-1 of the 3-aminoindazole series with
oxygen, we have developed a novel series of 3-aminobenzoisox-
azole N,N′-diphenyl urea derivatives as multitargeted RTK
inhibitors that potently inhibit both VEGFR and PDGFR family
members. Optimization of the urea moiety and substitution at
7-position led to several compounds that displayed high oral
bioavailability and excellent in vivo efficacy in the estradiol-
induced murine uterine edema (UE) assay and HT1080 tumor
growth inhibition model. In particular, compound 50 shows high

1236 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Table 5. Kinase Inhibition Profiles of Selected Compoundsa
Ji et al.
VEGFR IC50 (nM)
PDGFR IC50 (nM)
other kinases IC50 (nM)
compd
KDR
FLT1
FLT3
KIT
CSF1R
PDGFR-ꢀ
TIE-2
FGFR
NA
3000
NA
>12500
HCK
SRC
LYN
49
50
51
3
21
11
41
4
24
16
38
3
36
19
30
4
43
56
72
14
27
30
40
3
96
249
270
1000
170
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
130
NA
66
a Each IC50 determination was performed with seven concentrations, and each assay point was determined in duplicate.
Table 6. Mouse Pharmacokinetic Parameters of Selected Compounds
IV
POa
dose
CL
compd
R′
R″
(mg/kg)
t1/ 2 (h)
Vd (L/Kg)
(L/hr·kg)
F (%)
AUC (µM·h)
41
49
50
51
52
3
H
3-CF3-4-F
2-F-5-Me
3-CF3
3-Cl
3-Br
3
3
1
1
3
3
1.2
0.66
0.9
0.9
1.9
1.7
1.4
0.94
2.2
1.2
3.1
2.7
0.79
1.0
1.7
0.93
1.1
1.1
100
34
100
99
71
100
33.1
8.5
30.6
25.8
13.8
24.5
OMe
OMe
OMe
OMe
a Dose orally at 10 mg/kg.
Table 7. In Vivo Efficacy in Murine Uterine Edema Model and
Antitumor Efficacy in HT1080 Model
compd
UE ED50 (mg/kg)
HT1080 TGI (PI 2g)a
41
49
50
51
52
3
5.4
3.2
2.0
63% @ 30 mg/kg/dayb
55% @ 30 mg/kg/dayb
81% @ 10 mg/kg/dayb
45% @ 30 mg/kg/dayb
ND
90% @ 3 mg/kg
1.0
0.4
69% @ 10 mg/kg/dayb
a PI: percent inhibition of tumor growth relative to control at 2 g.
b Compounds were dosed twice daily (BID).
tion–response curve. Each IC50 determination was performed with
five concentrations and each assay point was determined in
duplicate.
Estradiol-Induced Murine Uterine Edema Assay. Female
Balb/C mice greater than twelve weeks old (Taconic, Germantown,
NY) were pretreated with 10 units of Pregnant Mare’s Serum
Gonadotropin (PMSG; Calbiochem) at 72 and 24 h prior to
compound administration (0.1 mL/mouse IP). Mice were random-
ized the day of the experiment. Test compounds were formulated
in a variety of vehicles and administered p.o. 30 min prior to
stimulation with an i.p. injection of water-soluble 17ꢀ-estradiol
(20–25 µg/mouse). Animals were sacrificed and the uteri were
removed 2.5 h following estradiol stimulation by cutting just
proximal to the cervix and at the fallopian tubes. After removal of
fat and connective tissue, uteri were weighed, squeezed between
filter paper to remove fluid, and weighed again. The difference
between wet and blotted weights represented the fluid content of
the uterus. Compound-treated groups were compared to vehicle-
treated animals after subtracting the background water content of
unstimulated uteri. Experimental group size was 5 or 6.
HT1080 Tumor Growth Inhibition Model. A total of 1080
human fibrosarcoma cells were obtained from the American type
Tissue Culture Collection and maintained in Dulbecco’s Modified
Eagle Medium supplemented with 10% fetal bovine serum and
antibiotics. For tumor xenograft studies, cells were suspended in
PBS, mixed with an equal volume of Matrigel (phenol red free) to
a final concentration of 2 million cells/mL, and inoculated (0.25
Figure 4. Effects of inhibitor 50 on the growth of HT1080 human
xenograft, dosing twice daily (BID) started on day 7. Tumor volumes
are expressed as mean ( SEM, n ) 10 per group. Significant differences
(p > 0.05 vs control) in mean tumor volume were observed for all
treatment groups by day 14. Total daily dose (mg/kg/day) and percent
inhibition of control (2 g) are indicated in the legend.
mL) into the flank of SCID-Beige mice. One week after inoculation,
tumor bearing animals were divided into groups (n ) 10) and
administration of vehicle (2% EtOH, 5% Tween80, 20% PEG400,
73% saline) or inhibitor at the indicated dose was initiated. Tumor
growth was assessed every 2–3 days by measuring tumor size and
calculating tumor volume using the formula [length × width2]/2.
[3H] Dofetilide/HEK-293 Membrane Competition Binding
Assay. The affinity of test drugs for the hERG cardiac K+ channel
was determined by their ability to displace tritiated dofetilide (a
class III antiarrhythmic drug and potent hERG blocker) in
membrane homogenates from HEK-293 cells heterogeneously

3-Amino-benzo[d]isoxazole Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1237
1
expressing the hERG cannel. Drug dilutions were prepared from
10 mM DMSO stocks, and the following were added to a 96-well
polystyrene plate (Perkin-Elmer Optiplate): 20 µL of assay binding
buffer (for total bounds), 1 µM astemizole (for nonspecific bounds),
or test drug, 50 µL of [3H]dofetilide (20 nM, 85 Ci/mmol specific
activity), and 130 µL of membrane homogenate (final protein
concentration of 30 µg per well). This plate was incubated at
ambient temperature for 45 min, aspirated onto GF/B filter plates
(Perkin-Elmer), and washed with 2 mL of cold wash buffer. After
allowing the plates to dry, 50 µL of scintillant (Perkin-Elmer
MicroScint 20) was added to each well, and the radioactivity was
counted in a Perkin-Elmer Topcount NXT scintillant counter. TC50
determinations were calculated from competition curves using six
drug concentrations, half-log apart, starting at a high concentration
of 100 µL (final assay DMSO concentration ) 1%) using a four-
parameter logistic equation.
white solid (42 mg, 64%). H NMR (DMSO-d6) δ 5.22 (s, 2 H),
7.09–7.23 (m, 1 H), 7.44–7.68 (m, 7 H), 7.88–8.06 (m, 4 H),
10.20–10.56 (m, 1 H). MS (ESI) m/z 330 (M + H)+. Anal.
(C20H15N3O2) C, H, N.
N-[4-(3-Aminobenzo[d]isoxazol-4-yl)-phenyl]-3-(3-trifluoro-
methyl-phenyl)-acrylamide (11). Compound 11 was prepared
using the same procedure described for the synthesis of 10,
substituting trans-3-(trifluromethyl)cinnamoyl chloride for benzoyl
chloride. 1H NMR (DMSO-d6) δ 5.22 (s, 2 H), 7.01 (d, J ) 15.94
Hz, 1 H), 7.16 (m, 1 H), 7.45–8.09 (m, 11 H), 10.45 (s, 1 H). MS
(ESI) m/z 424 (M + H)+. Anal.(C23H16F3N3O2) C, H, N.
1-(2-Fluoro-5-methylphenyl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl)urea (12a). To a solution of 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (219 mg, 1mmol) in
dichloromethane (5 mL) was added dropwise 2-fluoro-5-meth-
ylphenyl isocyanate (151 mg, 1 mmol). The mixture was stirred at
room temperature for 10 h. The solid was collected by filtration to
afford the title compound as a white solid (330 mg, 89%). 1H NMR
(DMSO-d6) δ 1.28 (s, 12 H), 2.27 (s, 3 H), 6.49–6.90 (m, 1 H),
7.10 (dd, J ) 11.53, 8.48 Hz, 1 H), 7.47 (d, J ) 8.48 Hz, 2 H),
7.53–7.65 (m, 2 H), 7.98 (dd, J ) 7.97, 1.86 Hz, 1 H), 8.51 (d, J
) 1.70 Hz, 1 H), 9.19 (s, 1 H). MS (ESI) m/z 371 (M + 1)+.
Chemistry. 1H NMR spectra were recorded on a 300 MHz or a
500 MHz (GE QE 300 or QE 500) spectrometer and chemical shifts
are reported in parts per million (ppm, δ) relative to tetramethyl-
silane as an internal standard. Mass spectra were obtained on a
Finnigan MAT SSQ700 instrument. Elemental analysis (C, H, N)
was performed by Robertson Microlit Laboratories, Inc., Madison,
WI. Silica gel 60 (E. Merck, 230–400 mesh) was used for
preparative column chromatography.
1-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-
(3(trifluoromethyl)-phenyl)urea (12b). Compound 12b was pre-
pared using the same procedure described for the synthesis of 12a,
substituting 3-(trifluoromethyl)phenyl isocyanate for 2-fluoro-5-
4-Iodo-benzo[d]isoxazol-3-ylamine (7). t-BuOK (4.72 g, 42.1
mmol) was added to a solution of acetohydroxamic acid (3.16 g,
42.1 mmol) in DMF (50 mL). The mixture was stirred at room
temperature for 30 min, and then 2-fluoro-6-iodo-benzonitrile (5.2
g, 21.05 mmol) was added. The reaction mixture was stirred for
an additional 10 h, then diluted with water (300 mL), and filtered.
The solid was rinsed with water and dried to provide the title
compound as a white solid (3.65 g, 67%). 1H NMR (DMSO-d6) δ
5.92 (s, 2 H), 7.21 – 7.31 (m, 1 H), 7.55 (d, J ) 8.48 Hz, 1 H),
7.70 (d, J ) 7.46 Hz, 1 H). MS (ESI) m/z 261 (M + H)+.
4-(4-Aminophenyl)-benzo[d]isoxazol-3-ylamine (8). A mixture
of compound 7, (585 mg, 2.25 mol), 4-(4,4,5,5-tetramethyl [1,3,2]-
dioxaborolan-2-yl)-phenylamine (591 mg, 2.7 mol), Pd(PPh3)4 (260
mg, 0.225 mol), and Na2CO3 (596 mg, 5.6 mmol) in DME (40
mL) and water (10 mL) was degassed with nitrogen, then heated
to 85 °C for 4 h. The reaction mixture was cooled to room
temperature and partitioned between ethyl acetate and water. The
aqueous layer was extracted with ethyl acetate. The combined
extracts were washed with water and brine, dried (MgSO4), filtered,
and concentrated to dryness. The crude product was purified by
flash column chromatography on silica gel with 0–5% MeOH in
CH2Cl2 to afford the title compound as a light tan solid (300 mg,
59%). 1H NMR (DMSO-d6) δ 5.21 (s, 2 H), 5.39 (s, 2 H), 6.70 (d,
J ) 8.14 Hz, 2 H), 7.05 (d, J ) 6.78 Hz, 1 H), 7.16 (d, J ) 8.14
Hz, 2 H), 7.39 (d, J ) 8.14 Hz, 1 H), 7.52 (t, J ) 7.80 Hz, 1 H).
MS (ESI) m/z 226 (M + H)+. Anal. (C13H11N3O) C, H, N.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)urea (9). Com-
pound 8 (45 mg, 0.2 mmol) and NaOCN (26 mg, 0.4 mmol) were
suspended in acetic acid (1 mL) and water (1 mL). The reaction
mixture was stirred at room temperature for 10 h, then poured into
water (20 mL). The resulting solid was collected by filtration. The
crude product was recrystallized from THF and hexane to afford
1
methylphenyl isocyanate. H NMR (DMSO-d6) δ 1.29 (s, 12 H),
7.32 (d, J ) 7.12 Hz, 1 H), 7.42 – 7.66 (m, 6 H), 8.03 (s, 1 H),
8.94 (s, 1 H), 9.07 (s, 1 H). MS (ESI) m/z 407 (M + 1)+.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(2-fluoro-5-meth-
ylphenyl)urea (13). Method A. A mixture of compound 7 (52 mg,
0.2 mmol), compound 12a (81 mg, 0.22 mmol), Na2CO3 (64 mg,
0.6 mmol) and PdCl2(dppf)·CH2Cl2 (8.2 mg, 0.01mmol) in DME
(4 mL) and water (1 mL) were degassed with nitrogen, then heated
to 85 °C for 4 h. The reaction mixture was cooled to room
temperature and partitioned between ethyl acetate and water. The
aqueous layer was extracted with ethyl acetate. The combined
extracts were washed with water and brine, dried (MgSO4), filtered,
and concentrated to dryness. The crude product was purified by
flash column chromatography on silica gel with 0–5% MeOH in
CH2Cl2 to afford the title compound (38 mg, 50%).
Method B. A solution of compound 8 (45 mg, 0.2 mol) in
CH2Cl2 (2 mL) was cooled to 0 °C, and then 2-fluoro-5-
methylphenyl isocyanate (30 mg, 0.2 mmol) was added dropwise.
The mixture was stirred at 0 °C for 1 h and then at room temperature
for 10 h. The resulting white suspension was filtered, affording the
title compound as a white solid (49 mg, 65%). 1H NMR (DMSO-
d6) δ 2.28 (s, 3 H), 5.22 (s, 2 H), 6.75–6.85 (m, 1 H), 7.06–7.18(m,
2 H), 7.40–7.66 (m, 6 H), 8.00 (dd, J ) 7.97, 1.86 Hz, 1 H), 8.55
(d, J ) 2.37 Hz, 1 H), 9.25 (s, 1 H). MS (ESI) m/z 377 (M + H)+.
Anal. (C21H17FN4O2) C, H, N.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(3-(trifluoro-
methyl)phenyl)urea (14). Compound 14 was prepared using the
same procedure described for the synthesis of 13 (method A) by
1
substituting 12b for 12a. H NMR (DMSO-d6) δ 5.22 (s, 2 H),
1
the title compound as an off-white solid (35 mg, 65%). H NMR
7.14 (d, J ) 6.78 Hz, 1 H), 7.33 (d, J ) 7.12 Hz, 1 H), 7.40–7.75
(m, 8 H), 8.04 (s, 1 H), 9.00 (s, 1 H), 9.12 (s, 1 H). MS (ESI) m/z
413 (M + H)+. Anal. (C21H15F3N4O2) C, H, N.
(DMSO-d6) δ 5.20 (s, 2 H), 5.92 (s, 2 H), 7.11 (d, J ) 7.12 Hz, 1
H), 7.30–7.40 (m, 2 H), 7.42–7.50 (m, 1 H), 7.53–7.64 (m, 3 H),
8.72 (s, 1 H). MS (ESI) m/z 269 (M + H)+. Anal. (C14H12N4O2)
C, H, N.
N-[4-(3-Aminobenzo[d]isoxazol-4-yl)-phenyl]-benzamide (10).
To a solution of compound 8 (45 mg, 0.2 moml) in DMF (2 mL)
was added pyridine (19 µL, 0.24mmol), followed by benzoyl
chloride (23.2 µL, 0.2 mmol). The mixture was stirred at room
temperature for 3 h, then partitioned between water and ethyl
acetate. The aqueous layer was extracted with ethyl acetate, and
the combined extracts were washed with water and brine, dried
(MgSO4), filtered, and concentrated to dryness. The crude product
was purified by flash column chromatography on silica gel, eluting
with 0–4% MeOH in CH2Cl2 to afford the title compound as a
2-(Benzylthio)-6-iodobenzonitrile (15). Benzyl mercaptan (2.36
mL, 20 mmol) was added to a suspension of t-BuOK (2.69 g, 24
mmol) in anhydrous DMF (100 mL). The mixture was stirred at
room temperature for 10 min, and then 2-fluoro-6-iodo-benzonitrile
(4.94 g, 20 momol) was added. Stirring was continued for 3 h and
then the reaction mixture was poured into water (1 L). Upon stirring
a yellow precipitate was formed. This solid was collected by
filtration and rinsed with water. The crude product was stirred in
hot hexane, then cooled to room temperature, and filtered to collect
the title compound as a yellow solid (6.0 g, 85%). 1H NMR
(DMSO-d6) δ 4.40 (s, 2 H), 7.19–7.46 (m, 6 H), 7.61 (d, J ) 7.12
Hz, 1 H) 7.81 (d, J ) 8.81 Hz, 1 H). MS (ESI) m/z 350 (M - H)-.

1238 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Ji et al.
2-(Aminothio)-6-iodobenzonitrile (16). Compound 15 (1.76 g,
5 mmol) was dissolved in CH2Cl2 (10 mL) and SO2Cl2 (0.49 mL,
6 mmol) was added. The mixture was stirred at room temperature
for 10 h and then concentrated to dryness. To the residue was added
7 M NH3 in MeOH (20 mL). The mixture was stirred 2 h at room
temperature, then diluted with water, extracted with ethyl acetate,
washed with brine, dried (MgSO4), and concentrated to dryness.
The residue was dissolved in a minimum amount of ethyl acetate
and reprecipitated with hexane. The resulting solid was collected
by filtration to afford the title compound as a light tan solid. (0.75
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-m-tolylurea (27). 1H
NMR (DMSO-d6) δ 2.29 (s, 3 H), 5.22 (s, 2 H), 6.81 (d, J ) 7.12
Hz, 1 H), 7.10–7.70 (m, 10 H), 8.66 (s, 1 H), 8.85 (s, 1 H). MS
(ESI) m/z 359 (M + H)+. Anal. (C21H18N4O2 ·0.4H2O) C, H, N.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(3-methoxyphe-
nyl)urea (28). 1H NMR (DMSO-d6) δ 3.74 (s, 3 H), 5.22 (s, 2 H),
6.57 (dd, J ) 7.97, 2.20 Hz, 1 H), 6.85–7.03 (m, 1 H), 7.07–7.29
(m, 3 H), 7.37–7.52 (m, 3 H), 7.53–7.71 (m, 3 H), 8.75 (s, 1 H),
8.86 (s,
(C21H18N4O3 ·0.15H2O) C, H, N.
1 H). MS (ESI) m/z 376 (M +
H)+. Anal.
1
g, 54%). H NMR (DMSO-d6) δ 4.43 (s, 2 H), 7.34–7.46 (m, 1
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(3-nitrophenyl)-
urea (29). 1H NMR (DMSO-d6) δ 5.22 (s, 2 H), 7.15 (d, J ) 6.78
Hz, 1 H), 7.48 (t, J ) 8.65 Hz, 3 H), 7.54–7.70 (m, 4 H), 7.75 (dd,
J ) 7.80, 1.70 Hz, 1 H), 7.84 (dd, J ) 8.14, 2.37 Hz, 1 H), 8.59
(t, J ) 2.20 Hz, 1 H), 9.09 (s, 1 H), 9.34 (s, 1 H). MS (ESI) m/z
H), 7.64 (d, J ) 7.12 Hz, 1 H), 7.70 (d, J ) 7.80 Hz, 1 H). MS
(ESI) m/z 277 (M + H)+.
4-Iodobenzo[d]isothiazol-3-amine (17). To a solution of com-
pound 16 (700 mg, 2.54 mmol) in methanol (10 mL) was added 1
M LiOMe methanol solution (3 mL, 3 mmol). The reaction mixture
was heated to 70 °C for 1 h and then cooled to room temperature.
The resulting solid was collected by filtration to afford the title
390 (M + H)+ Anal. (C20H15N5O4) C, H, N.
.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(3-fluorophe-
1
nyl)urea (30). H NMR (DMSO-d6) δ 5.22 (s, 2 H), 6.75–6.86
1
compound as a light tan solid (408 mg, 58%) H NMR (DMSO-
(m, 1 H), 7.10–7.20 (m, 2 H), 7.26–7.36 (m, 1 H), 7.41–7.66 (m,
7 H), 8.94 (s, 1 H), 8.98 (s, 1 H). MS (ESI) m/z 363 (M + H)+.
Anal. (C20H15FN4O2) C, H, N.
d6) δ 6.50 (s, 2 H), 7.12–7.24 (m, 1 H), 7.92 (d, J ) 8.14 Hz, 1
H), 8.03 (d, J ) 8.14 Hz, 1 H). MS (ESI) m/z 277 (M + H)+.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(3-bromophe-
1-(4-(3-Aminobenzo[d]isothiazol-4-yl)phenyl)-3-(2-fluoro-5-
methylphenyl)urea (18). A mixture of compound 17 (55 mg, 0.2
mmol), 12a (81 mg, 0.22 mmol), and PdCl2(dppf)·CH2Cl2 (16 mg,
0.02 mmol) in DME (2 mL) was degassed with nitrogen and then
a solution of Na2CO3 (64 mg, 0.6 mmol) in water (1 mL) was added.
The mixture was heated to 80 °C for 4 h. After cooling to room
temperature, the reaction mixture was extracted with ethyl acetate,
washed with water and brine, dried (MgSO4), filtered, and
concentrated to dryness. The crude product was purified by flash
column chromatography on silica gel with 0–2% MeOH in CH2Cl2
to afford the title compound (40 mg, 51%).1H NMR (DMSO-d6) δ
2.28 (s, 3 H), 5.45 (s, 2 H), 6.69–6.92 (m, 1 H), 7.02–7.24 (m, 2
H), 7.37 (d, J ) 8.48 Hz, 2 H), 7.49–7.59 (m, 1 H), 7.62 (d,
J ) 8.48 Hz, 2 H), 7.88–8.08 (m, 2 H), 8.56 (d, J ) 2.37 Hz,
1 H), 9.27 (s, 1 H). MS (ESI) m/z 393 (M + H)+. Anal.
(C21H17FN4OS·0.4 CH3OH·0.15CH2Cl2).
1
nyl)urea (31). H NMR (DMSO-d6) δ 5.22 (s, 2 H), 7.16 (t, J )
7.29 Hz, 2 H), 7.25 (t, J ) 7.97 Hz, 1 H), 7.30–7.37 (m, 1 H),
7.40–7.52 (m, 3 H), 7.55–7.68 (m, 3 H), 7.88 (t, J ) 1.86 Hz, 1
H), 8.95 (s, 1 H), 8.96 (s, 1 H). MS (ESI) m/z 425 (M + H)+.
Anal. (C20H15BrN4O2) C, H, N.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(3-chlorophe-
1
nyl)urea (32). H NMR (DMSO-d6) δ 5.22 (s, 2 H), 6.96–7.09
(m, 1 H), 7.14 (d, J ) 7.12 Hz, 1 H), 7.24–7.38 (m, 2 H), 7.40–7.52
(m, 3 H), 7.54–7.67 (m, 3 H), 7.70–7.77 (m, 1 H), 8.96 (s, 2 H).
MS (ESI) m/z 379 (M + H)+. Anal. (C20H15ClN4O2 ·0.15H2O) C,
H, N.
1-(4-(3-Aminobenzo[d]Isoxazol-4-yl)phenyl)-3-p-tolylurea (33). 1H
NMR (DMSO-d6) δ 2.25 (s, 3 H), 5.22 (s, 2 H), 6.97–7.20 (m, 3
H), 7.30–7.52 (m, 5 H), 7.53–7.69 (m, 3 H), 8.62 (s, 1 H), 8.83 (s,
1 H). MS (ESI) m/z 359 (M + H)+. Anal. (C21H18N4O2 ·0.15H2O)
C, H, N.
1-(4-(3-Aminobenzo[d]isothiazol-4-yl)phenyl)-3-(3-(trifluo-
romethyl)phenyl)urea (19). Compound 19 was prepared using the
procedure described for the synthesis of compound 18 by substitut-
ing compound 12b for 12a. 1H NMR (DMSO-d6) δ 5.45 (s, 2 H),
7.17 (d, J ) 6.10 Hz, 1 H), 7.28–7.41 (m, 3 H), 7.48–7.73 (m, 5
H), 7.98 (d, J ) 8.14 Hz, 1 H), 8.04 (s, 1 H), 9.02 (s, 1 H),
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(4-(trifluoro-
1
methyl)phenyl)urea (34). H NMR (DMSO-d6) δ 5.23 (s, 2 H),
7.00–7.26 (m, 1 H), 7.39–7.75 (m, 10 H), 9.04 (s, 1 H), 9.21 (s, 1
H). MS (ESI) m/z 413 (M + H)+. Anal. (C21H15F3N4O2) C, H, N.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(4-methoxyphe-
nyl)urea (35). 1H NMR (DMSO-d6) δ 3.72 (s, 3 H), 5.22 (s, 2 H),
6.88 (d, J ) 8.82 Hz, 2 H), 7.13 (d, J ) 6.44 Hz, 1 H), 7.31–7.53
(m, 5 H), 7.54–7.79 (m, 3 H), 8.54 (s, 1 H), 8.79 (s, 1 H). MS
(ESI) m/z 375 (M + H)+. Anal. (C21H18N4O3 ·0.15H2O) C, H, N.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(4-fluorophe-
9.13 (s,
(C21H15F3N4OS·0.1CH2Cl2) C, H, N.
1 H). MS (ESI) m/z 429 (M +
H)+. Anal.
Compounds 23–45 were prepared using the procedure described
for the synthesis of compound 13 (method B), substituting the
appropriate isocyanate for 2-fluoro-5-methylphenyl isocyanate.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-phenylurea
(23). 1H NMR (DMSO-d6) δ 5.24 (s, 2 H), 7.00 (t, J ) 7.17 Hz,
1 H), 7.15 (d, J ) 7.32 Hz, 1 H), 7.31 (t, J ) 7.78 Hz, 2 H),
7.48 (dd, J ) 22.43, 7.78 Hz, 5 H), 7.55–7.71 (m, 3 H), 8.76 (s,
1 H), 8.89 (s, 1 H). MS (ESI) m/z 345 (M + H)+. Anal.
(C20H16N4O2 · 0.3H2O) C, H, N.
1
nyl)urea (36). H NMR (DMSO-d6) δ 5.22 (s, 2 H), 7.01–7.25
(m, 3 H), 7.35–7.53 (m, 5 H), 7.54–7.73 (m, 3 H), 8.80 (s, 1 H),
8.90 (s, 1 H). MS (ESI) m/z 363 (M + H+). Anal.(C20H15FN4O2)
C, H, N.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(4-(trifluo-
1
romethoxy)phenyl)urea (37). H NMR (DMSO-d6) δ 5.22 (s, 2
H), 7.14 (d, J ) 7.12 Hz, 1 H), 7.30 (d, J ) 9.16 Hz, 2 H),
7.40–7.52 (m, 3 H), 7.54–7.68 (m, 5 H), 8.93 (s, 1 H), 8.96 (s, 1
H). MS (ESI) 429 (M + H)+. Anal. (C21H15F3N4O3) C, H, N.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(3,5-dimeth-
ylphenyl)urea (38). 1H NMR (DMSO-d6) δ 2.49 (s, 6 H), 5.47 (s,
2 H), 6.88 (s, 1 H), 7.20–7.46 (m, 3 H), 7.62–7.77 (m, 3 H),
7.78–7.96 (m, 3 H), 8.82 (s, 1 H), 9.08 (s, 1 H). MS (ESI) m/z 373
(M+ H)+. Anal.(C22H20N4O2 ·0.15H2O) C, H, N.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(2-fluorophe-
1
nyl)urea (24). H NMR (DMSO-d6) δ 5.22 (s, 2 H), 6.89–7.08
(m, 1 H), 7.10–7.36 (m, 3 H), 7.39–7.53 (m, 3 H), 7.53–7.72 (m,
3 H), 8.05–8.29 (m, 1 H), 8.62 (d, J ) 2.37 Hz, 1 H), 9.27 (s, 1
H). MS (ESI) m/z 363 (M + H)+. Anal. (C20H15FN4O2 ·0.1CH2Cl2)
C, H, N.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(2-(trifluo-
1
romethoxy)phenyl)urea (25). H NMR (DMSO-d6) δ 5.23 (s, 2
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(2-fluoro-5 (tri-
1
H), 6.96–7.23 (m, 2 H), 7.30–7.53 (m, 5 H), 7.54–7.77 (m, 3 H),
8.28 (dd, J ) 8.31, 1.53 Hz, 1 H), 8.55 (s, 1 H), 9.48 (s, 1 H). MS
(ESI) m/z 429 (M + H)+. Anal. (C21H15F3N4O3) C, H, N.
fluoromethyl)phenyl)urea (39). H NMR (DMSO-d6) δ 5.22 (s,
2 H), 7.15 (d, J ) 7.12 Hz, 1 H), 7.33–7.70 (m, 8 H), 8.64 (dd, J
) 7.29, 2.20 Hz, 1 H), 8.97 (d, J ) 2.71 Hz, 1 H), 9.38 (s, 1 H).
MS (ESI) m/z 431(M + H)+. Anal. (C21H14F4N4O2 ·0.06CH2Cl2)
C, H, N.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(2-(trifluoro-
1
methyl)phenyl)urea (26). H NMR (DMSO-d6) δ 5.22 (s, 2 H),
7.00–7.75 (m, 10 H), 7.95 (d, J ) 7.80 Hz, 1 H), 8.16 (s, 1 H),
9.57 (s, 1 H). MS (ESI) m/z 413 (M + H)+. Anal. (C21H15-
F3N4O2 ·0.35H2O) C, H, N.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(3-fluoro-4-meth-
ylphenyl)urea (40). 1H NMR (DMSO-d6) δ 2.17 (s, 3 H), 5.22 (s,
2 H), 7.05 (dd, J ) 8.31, 2.20 Hz, 1 H), 7.10–7.27 (m, 2 H),

3-Amino-benzo[d]isoxazole Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1239
7.37–7.53 (m, 4 H), 7.53–7.70 (m, 3 H), 8.84 (s, 1 H), 8.90 (s, 1
H). MS (ESI) m/z 377 (M + H)+. Anal. (C21H17FN4O2) C, H, N.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(4-fluoro-3-(tri-
4-Iodo-7-methoxy-benzo[d]isoxazol-3-ylamine (48a). 1H NMR
(DMSO-d6) δ 3.93 (s, 3 H), 5.19 (s, 2 H), 5.31 (s, 2 H), 6.67 (d,
J ) 8.48 Hz, 2 H), 6.94. MS (ESI) m/z 256 (M + H+).
1
fluoromethyl)phenyl)urea (41). H NMR (DMSO-d6) δ 5.22 (s,
4-(4-Amino-phenyl)-7-methyl-benzo[d]isoxazol-3-ylamine
1
2 H), 7.14 (d, J ) 7.12 Hz, 1 H), 7.37–7.52 (m, 4 H), 7.54–7.74
(m, 4 H), 8.03 (dd, J ) 6.44, 2.71 Hz, 1 H), 9.01 (s, 1 H), 9.11 (s,
1 H). MS (ESI) m/z 431 (M + H)+. Anal. (C21H14F4N4O2) C,
H, N.
(48b). H NMR (DMSO-d6) δ 2.35–2.46 (m, 3 H), 5.19 (s, 1 H),
5.35 (s, 2 H), 6.69 (d, J ) 8.14 Hz, 2 H), 6.94 (d, J ) 7.46 Hz, 1
H), 7.13 (d, J ) 8.14 Hz, 2 H), 7.32 (d, J ) 7.46 Hz, 1 H). MS
(ESI) m/z 240 (M + H+).
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(3-chloro-4-
fluorophenyl)urea (42). 1H NMR (DMSO-d6) δ 5.22 (s, 2 H), 7.14
(d, J ) 6.44 Hz, 1 H), 7.30–7.37 (m, 2 H), 7.41–7.67 (m, 6 H),
7.78–7.86 (m, 1 H), 8.95 (s, 1 H), 8.97 (s, 1 H). MS (ESI) m/z 393
(M + H)+. Anal. (C20H14ClFN4O2) C, H, N.
4-(4-Aminophenyl)-7-fluorobenzo[d]isoxazol-3-amine (48c).
1H NMR (DMSO-d6) δ 5.36 (s, 2 H), 5.40 (s, 2 H), 6.69 (d, J )
8.48 Hz, 2 H), 7.01 (dd, J ) 8.14, 4.07 Hz, 1 H), 7.14 (d, J ) 8.48
Hz, 2 H), 7.45 (dd, J ) 10.85, 8.14 Hz, 1 H). MS (ESI) m/z 244
(M + H+).
4-(4-Aminophenyl)-7-(morpholinomethyl)benzo[d]isoxazol-3-
amine (48d). 1H NMR (DMSO-d6) δ 2.30–2.47 (m, 4 H), 3.47–3.64
(m, 4 H), 3.70 (s, 2 H), 5.21 (s, 2 H), 5.38 (s, 2 H), 6.69 (d, J )
8.48 Hz, 2 H), 7.02 (d, J ) 7.46 Hz, 1 H), 7.15 (d, J ) 8.48 Hz,
2 H), 7.45 (d, J ) 7.46 Hz, 1 H). MS (ESI) m/z 325 (M + H)+.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(3,5-difluorophe-
1
nyl)urea (43). H NMR (DMSO-d6) δ 5.21 (s, 2 H), 6.71–6.89
(m, 1 H), 7.14 (d, J ) 7.12 Hz, 1 H), 7.15–7.27 (m, 2 H), 7.39–7.53
(m, 3 H), 7.54–7.70 (m, 3 H), 9.05 (s, 1 H), 9.16 (s, 1 H). MS
(ESI) m/z 381 (M + H)+. Anal. (C20H14F2N4O2) C, H, N.
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(3-chloro-4-
1
4-(4-Aminophenyl)-7-(2-morpholinoethoxy)benzo[d]isoxazol-
methoxyphenyl)urea (44). H NMR (DMSO-d6) δ 3.82 (s, 3 H),
1
3-amine (48e). H NMR (DMSO-d6) δ 2.40–2.56 (m, 4 H), 2.76
5.22 (s, 2 H), 7.02–7.17 (m, 2 H), 7.29 (dd, J ) 8.98, 2.54 Hz, 1
H), 7.38–7.53 (m, 3 H), 7.53–7.78 (m, 4 H), 8.71 (s, 1 H), 8.87 (s,
1 H). MS (ESI) m/z 409 (M + H)+. Anal. (C21H17ClN4-
O3 ·0.12CH2Cl2) C, H, N.
(t, J ) 5.59 Hz, 2 H), 3.52–3.63 (m, 4 H), 4.28 (t, J ) 5.59 Hz, 2
H), 5.19 (s, 2 H), 5.31 (s, 2 H), 6.58–6.75 (m, 2 H), 6.92 (d, J )
7.80 Hz, 1 H), 7.04–7.16 (m, 3 H). MS (ESI) m/z 355 (M + H)+.
4-(4-Aminophenyl)-7-(trifluoromethoxy)benzo[d]isoxazol-3-
1-(4-(3-Aminobenzo[d]isoxazol-4-yl)phenyl)-3-(3-chloro-4-meth-
ylphenyl)urea (45). 1H NMR (DMSO-d6) δ 2.27 (s, 3 H), 5.22 (s,
2 H), 7.14 (d, J ) 6.10 Hz, 1 H), 7.17–7.32 (m, 2 H), 7.38–7.54
(m, 3 H), 7.54–7.68 (m, 3 H), 7.71 (d, J ) 2.03 Hz, 1 H), 8.84 (s,
1 H), 8.91 (s, 1 H). MS (ESI) m/z 393 (M + H)+. Anal.
(C21H17ClN4O2 ·0.1CH2Cl2) C, H, N.
Compounds 47a-e were prepared using the same procedure
described for the synthesis of 7, substituting compounds 46a-e
for compound 6.
1
amine (48f). H NMR (DMSO-d6) δ 5.42 (s, 2 H), 5.46 (s, 2 H),
6.70 (d, J ) 8.48 Hz, 2 H), 7.11 (d, J ) 8.14 Hz, 1 H), 7.14–7.23
(m, 2 H), 7.60 (dd, J ) 8.14, 1.36 Hz, 1 H). MS (ESI) m/z 310 (M
+ H+).
Compounds 49–57 were prepared using the procedure described
for the synthesis of 13 (method B) substituting compounds 48a-f
for compound 8.
1-(4-(3-Amino-7-methoxybenzo[d]isoxazol-4-yl)phenyl)-3-(2-
fluoro-5 methylphenyl)urea (49). 1H NMR (DMSO-d6) δ 2.28
(s, 3 H), 3.96 (s, 3 H), 5.21 (s, 2 H), 6.75–6.87 (m, 1 H), 7.00–7.21
(m, 3 H), 7.30–7.45 (m, 2 H), 7.52–7.69 (m, 2 H), 8.00 (dd, J )
7.63, 1.86 Hz, 1 H), 8.53 (d, J ) 2.71 Hz, 1 H), 9.22 (s, 1 H). MS
(ESI) m/z 407 (M + H)+. Anal. (C22H19FN4O3) C, H, N.
4-Iodo-7-methoxybenzo[d]isoxazol-3-amine (47a). 1H NMR
(DMSO-d6) δ 3.91 (s, 3 H), 5.90 (s, 2 H), 6.91 (d, J ) 8.14 Hz, 1
H), 7.57 (d, J ) 8.14 Hz, 1 H). MS (ESI) m/z 291 (M + H)+.
4-Iodo-7-methylbenzo[d]isoxazol-3-amine (47b). 1H NMR (DM-
SO-d6) δ 2.36 (s, 3 H), 5.89 (s, 2 H), 7.09 (d, J ) 7.46 Hz, 1 H),
7.58 (d, J ) 7.46 Hz, 1 H). MS (ESI) m/z 275 (M + H)+.
7-fluoro-4-iodobenzo[d]isoxazol-3-amine (47c). 1H NMR (DM-
SO-d6) δ 6.09 (s, 2 H), 6.08 (s, 2 H), 7.28 (dd, J ) 10.85, 8.48 Hz,
1 H), 7.67 (dd, J ) 8.31, 3.90 Hz, 1 H). MS (ESI) m/z 262 (M +
H)+.
1-(4-(3-Amino-7-methoxybenzo[d]isoxazol-4-yl)phenyl)-3-(3
1
(trifluoromethyl)phenyl)urea (50). H NMR (DMSO-d6) δ 3.96
(s, 3 H), 5.21 (s, 2 H), 7.05 (d, J ) 7.80 Hz, 1 H), 7.17 (d, J )
8.14 Hz, 1 H), 7.32 (d, J ) 7.46 Hz, 1 H), 7.39 (d, J ) 8.48 Hz,
2 H), 7.53 (t, J ) 7.80 Hz, 1 H), 7.61 (m, 3 H), 8.04 (s, 1 H), 8.96
(s, 1 H), 9.10 (s, 1 H). MS (ESI) m/z 443 (M + H)+. Anal.
(C22H17F3N4O3 ·0.15CH2Cl2) C, H, N.
4-Iodo-7-(morpholinomethyl)benzo[d]isoxazol-3-amine (47d).
1H NMR (DMSO-d6) δ 2.33–2.44 (m, 4 H), 3.52–3.60 (m, 4 H),
3.65 (s, 2 H), 5.92 (s, 2 H), 7.21 (d, J ) 7.46 Hz, 1 H), 7.67 (d, J
) 7.46 Hz, 1 H). MS (ESI) m/z 360 (M + H)+.
1-(4-(3-Amino-7-methoxybenzo[d]isoxazol-4-yl)phenyl)-3-(3-
chlorophenyl)urea (51). 1H NMR (DMSO-d6) δ 3.96 (s, 3 H),
5.20 (s, 2 H), 7.00–7.06 (m, 2 H), 7.16 (d, J ) 8.14 Hz, 1 H),
7.25–7.35 (m, 2 H), 7.39 (d, J ) 8.81 Hz, 2 H), 7.60 (d, J ) 8.48
Hz, 2 H), 7.73 (t, J ) 2.03 Hz, 1 H), 8.92 (s, 1 H), 8.94 (s, 1 H).
MS (ESI) m/z 409 (M + H)+. Anal. (C21H17ClN4O3) C, H, N.
4-Iodo-7-(2-morpholinoethoxy)benzo[d]isoxazol-3-amine (47e). 1H
NMR (DMSO-d6) δ 2.37–2.53 (m, 4 H), 2.74 (t, J ) 5.59 Hz, 2
H), 3.48–3.63 (m, 4 H), 4.25 (t, J ) 5.59 Hz, 2 H), 5.90 (s, 2 H),
6.94 (d, J ) 8.48 Hz, 1 H), 7.55 (d, J ) 8.14 Hz, 1 H). MS (ESI)
m/z 390 (M + H)+.
1-(4-(3-Amino-7-methoxybenzo[d]isoxazol-4-yl)phenyl)-3-(3-
4-Bromo-7-(trifluoromethoxy)benzo[d]isoxazol-3-amine (47f).
To a solution of 6-bromo-2-fluoro-3-(trifluoromethoxy)benzonitrile
(2.84 g, 10 mmol) in THF (50 mL) was added t-BuOK (1.23 g, 11
mmol). The reaction mixture was stirred for 30 min at room
temperature and then acetone oxime (0.80 g, 11mmol) was added.
The reaction mixture was stirred for 10 h, then diluted with water,
and extracted with ethyl acetate. The organic layer was washed
with brine, dried (MgSO4), filtered, and concentrated to dryness.
The residue was dissolved in ethanol (20 mL), and 5% HCl (20
mL) was added. The resulting mixture was refluxed for 2 h, and
then the solvent was reduced to half-volume. The resulting solid
was collected by filtration, and the crude product was purified by
flash column chromatography with 0–10% EtOAc in hexane to
afford the title compound as a light orange flake (0.95 g, 32%). 1H
NMR (DMSO-d6) δ 6.33 (s, 2 H), 7.46–7.64 (m, 2 H). MS (ESI)
m/z 299 (M + H)+.
1
bromophenyl)urea (52). H NMR (DMSO-d6) δ 3.96 (s, 3 H),
5.21 (s, 2 H), 6.96–7.09 (m, 1 H), 7.12–7.47 (m, 6 H), 7.60 (d,
J ) 8.48 Hz, 2 H), 7.88 (t, J ) 2.03 Hz, 1 H), 8.92 (d, J ) 3.05
Hz, 2 H). MS (ESI) m/z 453 (M + H)+. Anal. (C21H17BrN4O3)
C, H, N.
1-(4-(3-Amino-7-methylbenzo[d]isoxazol-4-yl)phenyl)-3-(2-
fluoro-5-methylphenyl)urea (53). 1H NMR (DMSO-d6) δ 2.28 (s,
3 H), 2.45 (s, 3 H), 5.20 (s, 2 H), 6.71–6.90 (m, 1 H), 6.96–7.20
(m, 2 H), 7.31–7.49 (m, 3 H), 7.51–7.74 (m, 2 H), 8.00 (dd, J )
7.63, 1.86 Hz, 1 H), 8.54 (d, J ) 2.37 Hz, 1 H); 9.24 (s, 1 H). MS
(ESI) m/z 391 (M + H)+. Anal. (C22H19FN4O2 ·0.1H2O) C, H, N.
1-(4-(3-Amino-7-fluorobenzo[d]isoxazol-4-yl)phenyl)-3-(2-
fluoro-5-methylphenyl)urea (54). 1H NMR (DMSO-d6) δ 2.28 (s,
3 H), 5.38 (s, 2 H), 6.73–6.89 (m, 1 H), 7.05–7.19 (m, 2 H),
7.39–7.46 (m, 2 H), 7.52 (dd, J ) 10.85, 8.14 Hz, 1 H), 7.62 (d,
J ) 8.81 Hz, 2 H), 8.00 (dd, J ) 7.63, 1.86 Hz, 1 H), 8.55 (d, J )
2.37 Hz, 1 H), 9.25 (s, 1 H). MS (ESI) m/z 395 (M + H)+. Anal.
(C21H16F2N4O2 ·0.1hexane·0.2H2O) C, H, N.
Compounds 48a-f were prepared using the procedure de-
scribed for the synthesis of 8, substituting compounds 47a-f
for compound 7.

1240 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Ji et al.
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1-(4-(3-Amino-7-(trifluoromethoxy)benzo[d]isoxazol-4-yl)phe-
nyl)-3-(2-fluoro-5-methylphenyl)urea (55). 1H NMR (DMSO-d6)
δ 2.28 (s, 3 H), 5.44 (s, 2 H), 6.75–6.92 (m, 1 H), 7.05–7.26 (m,
2 H), 7.47 (d, J ) 8.82 Hz, 2 H), 7.56–7.78 (m, 3 H), 8.00 (dd, J
) 7.97, 1.86 Hz, 1 H), 8.56 (d, J ) 2.71 Hz, 1 H), 9.28 (s, 1 H).
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δ 2.28 (s, 3 H), 2.35–2.48 (m, 4 H), 3.50–3.65 (m, 4 H), 3.74 (s,
2 H), 5.23 (s, 2 H), 6.75–6.85 (m, J ) 2.37 Hz, 1 H), 7.05–7.18
(m, 2 H), 7.44 (d, J ) 8.48 Hz, 2 H), 7.52 (d, J ) 7.80 Hz, 1 H),
7.62 (d, J ) 8.48 Hz, 2 H), 8.00 (dd, J ) 7.80, 1.70 Hz, 1 H), 8.55
(d, J ) 2.37 Hz, 1 H), 9.25 (s, 1 H). MS (ESI) m/z 476 (M + H)+.
Anal. (C26H26FN5O3 ·0.1CH2Cl2) C, H, N.
1-(4-(3-Amino-7-(2-morpholinoethoxy)benzo[d]isoxazol-4-
yl)phenyl)-3-(2-fluoro-5-methylphenyl)urea (57). 1H NMR (DMSO-
d6) δ 2.28 (s, 3 H), 2.45–2.55 (m, 4 H), 2.78 (t, J ) 5.59 Hz, 2 H),
3.50–3.65 (m, 4 H), 4.31 (t, J ) 5.76 Hz, 2 H), 5.21 (s, 2 H),
6.75–6.84 (m, 1 H), 7.03 (d, J ) 8.14 Hz, 1 H), 7.11 (dd, J )
11.36, 8.31 Hz, 1 H), 7.19 (d, J ) 8.14 Hz, 1 H), 7.39 (d, J ) 8.48
Hz, 2 H), 7.59 (d, J ) 8.48 Hz, 2 H), 8.00 (dd, J ) 7.80, 2.03 Hz,
1 H), 8.53 (d, J ) 2.37 Hz, 1 H), 9.22 (s, 1 H). MS (ESI) m/z 506
(M + 1)+. Anal. (C27H28FN5O4) C, H, N.
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