Isothiazolopyrimidines and isoxazolopyrimidines as novel multi-targeted inhibitors of receptor tyrosine kinases

Article abstract of DOI:10.1016/j.bmcl.2006.05.057

A series of isothiazolopyrimidines and isoxazolopyrimidines were synthesized and identified as potent KDR inhibitors. SAR studies led to isothiazolopyrimidine urea analogs that potently inhibit VEGFR tyrosine kinases (KDR enzymatic and cellular IC_50<

Full text of DOI:10.1016/j.bmcl.2006.05.057

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4326–4330
Isothiazolopyrimidines and isoxazolopyrimidines as novel
multi-targeted inhibitors of receptor tyrosine kinases
Zhiqin Ji,a,* Asma A. Ahmed,b Daniel H. Albert,a Jennifer J. Bouska,a Peter F. Bousquet,b
George A. Cunha,b Keith B. Glaser,a Jun Guo,a Junling Li,a Patrick A. Marcotte,a
Maria D. Moskey,b Lori J. Pease,a Kent D. Stewart,a Melinda Yates,a
Steven K. Davidsena and Michael R. Michaelidesa
aGlobal Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6100, USA
bAbbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605-5314, USA
Received 1 May 2006; revised 16 May 2006; accepted 16 May 2006
Available online 2 June 2006
Abstract—A series of isothiazolopyrimidines and isoxazolopyrimidines were synthesized and identified as potent KDR inhibitors.
SAR studies led to isothiazolopyrimidine urea analogs that potently inhibit VEGFR tyrosine kinases (KDR enzymatic and cellular
IC50 values below 10 nM) as well as cKIT and TIE2. The selected compounds 8 and 13 display 56% and 48% oral bioavailability in
mice, respectively.
Ó 2006 Elsevier Ltd. All rights reserved.
Receptor tyrosine kinases (RTKs), a family of trans-
membrane proteins, have been shown to be important
mediators of signal transduction in cells.1–3 Overactiva-
tion of RTKs is associated with various human cancers.4
A major sub-class of the RTKs is the vascular endothe-
lial growth factor receptor (VEGFR) tyrosine kinases
family which includes FLT1 (VEGFR1), KDR (VEG-
FR2), and FLT4 (VEGFR3). VEGF-mediated KDR
signaling in particular plays a central role in angiogene-
sis through induction of proliferation, migration, and
survival of endothelial cells.5–7 TIE2, another endotheli-
um-specific tyrosine kinase, promotes tumor angiogene-
sis through interaction with angiopoietin,8 playing an
important role in stabilizing the immature endothelial
cell network, attracting pericytes, and maintaining bio-
chemical interactions and vessel integrity.9 The
PDGFRs, another sub-class of RTKs consisting of
PDGFRb, cKIT, CSF1R, and FLT3, are believed to
contribute to tumor angiogenesis and tumor growth as
well.10–12
F
HN
HN
HN
HN
O
CF3
O
NH2
N
NH2
N
N
N
S
O
O
I
II
The development of VEGFR inhibitors has been the
subject of intense research.13 The first generation of clin-
ical candidates, such as SU5416,14 PTK787,15 and CP-
54763216 focused on selective KDR inhibition. However
in recent years, the focus has shifted toward multi-tar-
geted inhibitors in an effort to overcome redundancies
in signaling pathways and thus more effectively inhibit
tumor growth. The recent approval of the multi-targeted
TM17
TM18
agents Sutent
and Nexavar
demonstrates that
clinical benefit with manageable side effects is possible
with broad-acting kinase inhibitors.
As part of our continuing efforts to identify novel RTK
inhibitors with improved efficacy and tolerability, we
have identified a series of thienopyrimidine-based ureas,
exemplified by I as potent multi-targeted inhibitors.19
Keywords: Isothiazolopyrimidine; Isoxazolopyrimidine; Multi-targeted
RTK inhibitor.
*
Corresponding author. Fax: +1 847 9355165; e-mail: zhiqin.ji@
abbott.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.057

Z. Ji et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4326–4330
4327
A closely related series of furopyrimidines (e.g., II) have
also been recently reported as dual inhibitors of KDR
and TIE2.20
was converted to 3 via sequential treatment with PCl5
and ammonium hydroxide. Treating 3 with diethyl
dithiophosphate formed the desired intermediate 4,
which was cyclized to 5 with H2O2. Heating 5a with
formamide at 210 °C under microwave conditions led
to isothiazolopyrimidine 6a. Compound 6b was pre-
pared in a stepwise fashion by first refluxing 5b with
(EtO)3CH then treating with ammonia followed by lith-
ium methoxide-mediated cyclization. Reduction of the
nitro group of 6 afforded aniline 7, which was converted
to the ureas 8–15 and benzoxazole 16.
We now wish to report the results of our expanded SAR
studies on five-membered fused pyrimidine heterocycles
covering the related isothiazolopyrimidines and isoxaz-
olopyrimidines (III).
L
Ar
L
Ar
NH2
N
NH2
N
The synthesis of the isoxazolopyrimidines was accom-
plished by the route illustrated in Scheme 2. Treating 17
with sodium hypochlorite gave compound 18, which
was then condensed with malononitrile to form oxazole
19. Refluxing 19 with (EtO)3CH followed by treatment
with ammonia gave the desired isoxazolopyrimidine 20.
Reduction of the nitro group of 20 afforded aniline 21,
which was converted to products 22–27. The correspond-
ing furano-linked compounds 28 and 29 (Table 2) were
prepared by substituting 17 with 5-nitro-furan-2-carbox-
aldehyde oxime in Scheme 2 except using Al–Hg–THF in
place of SnCl2 for the nitro group reduction.
N
N
N
X
X
III X = S, O
The general synthetic strategy for the preparation of iso-
thiazolopyrimidines is described in Scheme 1. Condensa-
tion of 1 with malononitrile in the presence of NaOH
and a phase transfer reagent gave rise to 2, which in turn
CN
Cl
n
Our initial SAR studies focused on KDR potency. The
inhibitory activity was measured in the presence of a
n
NC
b, c
a
O
NO2
1a: n = 0
1b: n = 1
OH
CN
NO2
2a: n = 0
2b: n = 1
NO2
NO2
CN
Cl
a
b
n
H2N
NC
n
N
d
e
N
HO
NH2
HO
NO2
S
NH2
18
17
NO2
3a: n = 0
3b: n = 1
4a: n = 0
4b: n = 1
NO2
NO2
NO2
NO2
f or g,h,i
NH2
NH2
n
NC
NC
H2N
c,d
n
j
e
N
N
N
N
N
O
N
H2N
N
S
O
N
S
5a: n = 0
5b: n = 1
6a: n = 0
6b: n = 1
20
19
H
N
NH2
H
NH2
H
N
H
N
N
R
O
NH2
NH2
N
n
O
NH2
NH2
N
n
f
R
N
N
N
k
N
N
N
S
N
S
N
N
O
O
24: R =m-Me
25: R = m-Et
26: R = m-CF3
7a: n = 0
7b: n = 1
N
14-15: n=1
8-13: n = 0
21
24-27
8: R = m-CH3 14: R= m-CH3
9: R = m-CF3 15: R = m-CF3
10: R = m-Et
11: R = m-Cl
12: R = 2-F-5-CF3
13: R = 2-F-5-CH3
27: R = 2-F-5-CF3
l
h
g
H
N
N
N
NH
NH
O
NH2
N
n
O
O
N
NH2
N
N
NH2
S
n = 0
16
N
N
N
N
O
O
Scheme 1. Reagents and conditions: (a) malononitrile, 10 N NaOH,
PhCH2NEt3Cl, CH2Cl2, 0–5 °C, 76–82%; (b) PCl5, CH2Cl2, reflux
24 h; (c) NH4OH, EtOH, rt, b and c two-step yield 53–58%; (d) diethyl
dithiophosphate, EtOH/H2O, reflux 24 h, 85–95%; (e) H2O2, EtOH,
90–96%; (f) for 6a: HCONH2, microwave, 210 °C, 30 min, 60%; g–i for
6b: (g) (EtO)3CH, (NH4)2SO4, reflux; (h) NH3 in EtOH, 35%; (i)
LiOMe, MeOH, reflux, 40%; (j) Fe/NH4Cl, EtOH/H2O 50–60 °C, 90–
97%; (k) ArNCO, DMF, 0–5 °C, 30–40%; (l) 2,4-dimethyl-6-amino-
phenol, 1,10-thiocarbonyldiimidazole, EDC, DMF, 10%.
N
22
23
Scheme 2. Reagents and conditions: (a) NaOCl, HCl, dioxane, 70%;
(b) malononitrile, NaOMe, THF, 75%; (c) HC(OEt)3, reflux; (d) NH3
in EtOH, rt, c and d two-step yield 54%; (e) SnCl2/HCl, 80%; (f)
RNCO, Py, DMF, 40–55%; (g) benzoyl chloride, pyridine, 40%; (h)
2,4-dimethyl-6-aminophenol, 1,10-thiocarbonyldiimidazole, EDC,
DMF, 8%.

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Z. Ji et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4326–4330
Table 2. KDR inhibitory activity of isothiazolopyrimidines and
isoxazolopyrimidines with varying links between the heterocycle core
and urea moiety
high concentration of ATP (1.0 mM) as described in the
previous paper.19 Table 1 summarizes the SAR of the
northern part of the molecule. In general, the SAR mir-
rors that seen in the thienopyrimidine series19 with the
ureas being most potent. Both the isothiazole 8 and isox-
azole 24 exhibit IC50 values of less than 100 nM,
although the isoxazole was ca. 7-fold less active.
Replacement of the urea with aminobenzoxazole (cf.
16, 23) led to a more dramatic drop in potency than that
observed with the thienopyrimidine template.19 The
amide analog 22 also had drastically diminished inhibi-
tory activity.
H
H
R
N
N
NH2
N
Y
N
O
N
X
a
Compound
X
S
Y
R
KDR IC50 (lM)
9
m-CF3
m-CF3
m-CH3
m-CF3
0.004
0.070
8.6
The linkage between isothiazolopyrimidine or isoxazol-
opyrimidine core and urea moiety also has profound im-
pact on potency as shown in Table 2, with a 1,4-phenyl
group being the optimal linker (9, 26). Replacing the
phenyl linkage with benzyl (14, 15) or furan (28, 29) re-
sults in a markedly reduced potency.
26
14
15
28
29
O
S
S
2.1
A model of 9 in the active site of KDR, constructed
according to our previous protocol,19 is shown in Figure
1. The isothiazolopyrimidine forms the hinge binding
unit and the trifluoromethylphenyl portion occupies a
pocket allosteric to the ATP-binding site and found in
the inactive (DFG-out) conformation of kinases. The
five-membered ring of the furan-linked compounds,
illustrated in Figure 1 with a model of 29, gives a projec-
tion vector of approximately 140°, in contrast with the
180° of the para-substitution of 9, necessitating signifi-
cant re-alignment of the binding interactions. The
skewed fit and sub-optimal hydrogen bonding of 29
O
m-CH3 22
O
O
O
m-CF3
16
a Each IC50 determination was performed with seven concentrations,
and each assay point was determined in duplicate.
within the active site provides a rationale for the de-
crease in inhibition strength. Similarly, the additional
methylene group of the benzyl-linked compounds neces-
sitates a 109° bend in the overall trajectory of com-
pounds 14 and 15, and leads to a binding mode with
less optimal interactions (model not shown).
Table 1. KDR inhibitory activity of isothiazolopyrimidine and isox-
azolopyrimidine analogs
R
The effect of varying the terminal phenyl substitution of
the urea moiety on KDR enzymatic and cellular activity
is shown in Table 3. The isothiazolopyrimidines are con-
sistently more potent than the corresponding isoxazolo-
NH2
N
N
X
N
a
Compound
X
R
KDR IC50 (lM)
6a
7a
S
S
NO2
NH2
>50
46
O
N
8
S
0.013
N
H
N
HN
O
16
22
23
S
43
O
O
O
>50
>50
N
H
N
O
HN
Figure 1. Models of 9 (green, thick) and 29 (magenta, thin) bound to
KDR kinase using a homology model of the active site.19 Hydrogen
bonds in black are shown between the urea and Glu885 carboxylate,
between the exocyclic amine and Glu917 backbone carbonyl, and
between the ring nitrogen and Cys919 N–H. Also in thick bonds are
residues Asp1046 and Phe1047 of the DFG motif in the ‘inactive’
conformation (DFG-out).
O
N
24
O
0.097
N
H
a Each IC50 determination was performed with seven concentrations,
and each assay point was determined in duplicate.

Z. Ji et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4326–4330
Table 5. Mouse PK profiles of 8 and 13a
4329
Table 3. Enzymatic and cellular activities of isothiazolopyrimidines
and isoxazolopyrimidines
Compound
t1/2
Vdss
Cl
(L/hÆkg)
F
(%)
AUC
(po, lMÆh)
(h)
(L/kg)
HN
O
R
8
13
I19
0.6
0.2
0.7
1.36
1.19
1.30
1.6
4.5
1.2
56
48
65
9.1
2.7
HN
14.0
NH2
a Dosed intravenously at 3 mg/kg and orally at 10 mg/kg.
N
N
X
N
In summary, potent VEGFR and PDGFR inhibitory
activity can be obtained with compounds using an iso-
thiazolopyrimidine template, while the corresponding
isoxazolopyrimidines have weaker inhibitory activity. A
comparison of a limited set of compounds showed that
the pharmacokinetic profile of isothiazolopyrimidines is
comparable to the corresponding thienopyrimidines.
Compound
X
R
KDR IC50 (lM)
Enzymatica
Cellularb
8
9
S
m-Me
m-CF3
m-Et
0.012
0.004
0.005
0.019
0.016
0.008
0.097
0.024
0.070
0.225
0.003
0.001
0.005
0.005
0.050
NA
S
10
11
12
13
24
25
26
27
I
S
S
m-Cl
S
2-F-5-CF3
2-F-5-Me
m-Me
S
0.021
0.10
0.25
References and notes
O
O
O
O
m-Et
m-CF3
2-F-5-CF3
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0.17
0.385
0.001
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 concen-
trations and each assay point was determined in duplicate.
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bers of the VEGFR family, FLT1 and FLT4 (Table 4).
The compounds tested had comparable activity against
the VEGFR family and cKIT. Additionally significant
TIE2 activity was observed with the fluoro-containing
analogs 9, 12, and 13, a trend previously seen in the thi-
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compounds display good oral bioavailability (56% and
48%), that is comparable to the thienopyrimidine I.
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2-fluoro-5-methyl analog 13 has a shorter half-life and
considerably higher plasma clearance rate.
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Table 4. Inhibitory activity against a series of kinases
Compound
IC50 (lM)a
KDR
TIE2
FLT1
FLT 4
CKIT
8
9
0.013
0.004
0.001
0.008
0.003
0.415
0.028
0.022
0.019
0.730
0.079
0.039
0.075
0.010
0.002
0.004
<0.003
0.023
0.011
0.018
0.028
0.007
0.006
12
13
I19
0.068
0.013
a Each IC50 determination was performed with seven concentrations,
and each assay point was determined in duplicate.

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Z. Ji et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4326–4330
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