4-Aminopyrimidine-5-carbaldehyde oximes as potent VEGFR-2 inhibitors. Part II

Article abstract of DOI:10.1016/j.bmcl.2011.01.053

A series of 4-aminopyrimidine-5-carbaldehyde oxime was discovered to have potent VEGFR-2 inhibitory activity. Described here are the chemistry for analogue synthesis and SAR study results. The PK properties, kinase profiling, and in vivo efficacy study for compound 4b are also discussed.

Full text of DOI:10.1016/j.bmcl.2011.01.053

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1815–1818
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4-Aminopyrimidine-5-carbaldehyde oximes as potent VEGFR-2
inhibitors. Part II
⇑
Shenlin Huang , Ronghua Li, Kenneth R. LaMontagne, Lee M. Greenberger, Peter J. Connolly
Johnson & Johnson Pharmaceutical Research & Development L.L.C., 1000 Route 202, Raritan, NJ 08869, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-aminopyrimidine-5-carbaldehyde oxime was discovered to have potent VEGFR-2 inhibitory
activity. Described here are the chemistry for analogue synthesis and SAR study results. The PK proper-
ties, kinase profiling, and in vivo efficacy study for compound 4b are also discussed.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 13 December 2010
Revised 11 January 2011
Accepted 13 January 2011
Available online 21 January 2011
Keywords:
VEGFR-2 inhibitor
4
-Aminopyrimidine-5-carbaldehyde oxime
Angiogenesis, the formation of new capillaries from the endo-
thelium of an existing vascular network, plays a crucial role in tu-
mor growth. Solid tumors cannot grow beyond several cubic
H
N
R2
N
X
N
O
N
millimeters until they establish a blood supply because cells must
O
R1
N
H
1
EtO
F
be within 100–200
l
m of a blood vessel to survive. Many factors
N
N
have been shown to affect endothelial cell function resulting in
angiogenesis in vivo, including FGF (fibroblast growth factor),
angiopoietins, PDGF (platelet-derived growth factor), EGF (epider-
mal growth factor), and VEGF (vascular endothelial growth factor).
N
H
H N
H
I
II
2
Figure 1. 4-Aminopyrimidine-5-carbaldehyde.
Of these factors, VEGF is a major initiator of angiogenesis. It acts
as a potent proangiogenic factor by promoting endothelial sprout-
3
ing, migration, proliferation, and survival both in vitro and in vivo.
scaffold (I, shown inFig. 1) is considered as an open form of quinaz-
oline since there is a pseudo six-membered ring formed via the
intramolecular H-bond. We previously reported a related subseries
of antiproliferative VEGFR-2 inhibitors, exemplified by compound
II. Here a new subseries of VEGFR-2 inhibitors with different prop-
These effects are primarily mediated by the receptor tyrosine ki-
nase, VEGF receptor 2 (VEGFR-2).4 There has been a tremendous
interest in pharmacologically targeting VEGF and VEGFR-2 in
5
oncology. Inhibition of VEGF or VEGFR-2 kinase activity has been
shown to suppress tumor angiogenesis and tumor growth in vari-
1
3
erties is described.
ous models. This research has led to the development of an FDA-
approved anti-VEGF antibody, bevacizumab (Avastin),6 as well as
In an effort to modulate biological properties of II, monocyclic
alternatives to the indol-5-yloxy group were investigated, particu-
larly N-substituted 1-amino-4-phenoxy. Initial SAR focused on urea
derivatives where the methyl oxime was maintained and 2-chloro-
three small molecule inhibitors of VEGFR-2 kinase, sorefenib
7
8
9
(
BAY-43-9006), sunitinib (Su-11248) and pazopanib. Numerous
other small molecule targeting agents and monoclonal antibodies
are currently in clinical development with the intention of inhibit-
4
-phenoxy was used as a starting point. Our previous work showed
ing this critical pathway in angiogenesis.1
0,11
that O-alkyl oximes gave optimal potency. It was also reported by
1
4
Kubo et al. that a Cl atom at the 2-position of the phenyl ring was
beneficial for VEGFR-2 potency. The chemistry is shown in Scheme
In our research efforts to develop small molecule kinase
inhibitors for cancer therapy, a novel series of 4-aminopyrimi-
dine-5-carbaldehyde oxime was discovered to be potent not only
1
. Treating 4-amino-6-chloropyrimidine-5-carbaldehyde15 (1) with
1
2
methoxyamine hydrochloride in acetic acid generated oxime 2. Dis-
placing Cl of compound 2 with 4-amino-3-chlorophenol in DMSO in
for VEGFR-2 but also for several other kinase targets. This
the presence of Cs
acted with various isocyanates to afford urea analogues. Alterna-
tively, intermediate was reacted with 4-nitrophenyl
2 3
CO provided intermediate 3, which was then re-
⇑
Corresponding author. Tel.: +1 858 332 4684; fax: +1 858 812 1684.
E-mail address: shenlinhuang@hotmail.com (S. Huang).
3
0
960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.053

1
816
S. Huang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1815–1818
Cl
gave similar potencies. An unsubstituted phenyl group was also
NH2
potent, but adding Cl, F, or MeO to the 2-position of the phenyl ring
had little impact on potency. 2-Me, however, led to a slight de-
crease in potency. Moving 2-F to the 3- or 4-position also barely af-
fected potency. 2,4-Difluorophenyl substitution conferred the
same potency as mono fluorophenyl. Therefore, for aryl ureas, high
potency with single-digit nM IC50s could be achieved with many
variations. This potency was reduced three-fold with insertion of
H
Cl
N
Cl
N
O
O
O
(a)
(b)
O
N
N
N
N
N
H N
H N
H N
N
2
2
2
1
2
3
2
a CH linkage between phenyl ring and urea group. Interestingly,
cyclohexyl was only twofold less potent than phenyl.
Cl
H
H
N
N
R
Next, the urea group was fixed at Me or Et for a follow-up SAR
study due to favorable physiochemical properties, for example,
lower molecular weight and better solubility. Similar chemistry
was used to modify the urea functionality and 2-chlorophenoxy
group. The results are shown in Table 2. Changing urea NHC(O)N-
HEt to amide NHC(O)Me or reversed amide C(O)NHMe resulted in
a loss of potency (amide) or complete loss of activity (reversed
amide). While replacing urea with carbamate NHC(O)OMe only
led to a 10-fold potency loss, the reversed carbamate OC(O)NHEt
O
O
(c) or (d)
O
N
N
H N
N
2
4a-y
Scheme 1. Synthesis of compounds 4a–y. Reagents and conditions: (a) methoxy-
amine hydrochloride, acetic acid, 95%; (b) 4-amino-3-chlorophenol, Cs CO , DMSO,
4%; (c) RNCO, CH Cl , 60–90%; (d) 4-NO OC(O)Cl, THF; then RNH
2
3
9
2
2
2
C
6
H
4
2
2
was inactive. Replacing NH with CH was also highly detrimental
to activity. These data demonstrate that the urea functionality is
crucial for target inhibition. Replacing 2-Cl on the phenoxy ring
with 2-F, 2-Me, 2-Br or H all resulted in potency loss, especially
with 2-Br substitution, which led to complete loss of activity. Shift-
ing 2-Cl to the 3-position gave a less potent analogue. As stated
above, 2-Cl plays a key role for activity.
chloroformate to give the 4-nitrophenyl carbamate, which was then
treated with amines to generate urea compounds.
These compounds were tested for VEGFR-2 kinase activity. The
results are shown in Table 1. For alkyl ureas, Et group gave higher
potency than Me and Pr groups. It was also more potent than i-Pr
and cyclopropyl groups. A hydrophilic group, for example, hydro-
xyl or amino, reduced potency. For the three pyridyl isomers, pyri-
din-2-yl was three-fold less potent than both pyridin-3-yl and
pyridin-4-yl. Ontheotherhand, allthreefive-memberedheteroaryls
To assess kinase selectivity of this series, compound 4b (JNJ-
3
8158471) was selected for screening against a panel of 184 ki-
nases. At a single concentration of 1 M with 2 M of ATP used,
l
l
only a few kinases including VEGFR-2, FMS, KIT, and RET were
inhibited more than 50%. IC50 values for these and several other ki-
nases of interest were further determined in a 10-point titration
IC50 determination assay. The data are shown in Table 3 and reveal
that compound 4b is a highly selective, potent VEGFR-2 inhibitor.
It is interesting to note that 4b did not potently inhibit closely re-
Table 1
VEGFR-2 potencies for compounds 4a–y
lated VEGF receptors (VEGFR-1 = 4.4 lM and VEGFR-3 = 1.1 lM).
When tested in vitro in tumor cell proliferation assays, 4b showed
no antiproliferative effects on human cancer cell lines, A375 and
HCT116, even at the highest concentration (100 lM) assayed. This
Cl
H
N
H
N
R
O
O
N
is in contrast to the previously reported antiproliferative series.
Pharmacokinetic parameters for compound 4b were measured
in both Balb/c mice and Sprague–Dawley rats. As shown in Table
O
N
H
N
2
N
4, 4b exhibited good bioavailability in rats (30%) though its mouse
Compds
R
VEGFR-2 inhibition IC50 (nM)
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
Methyl
Ethyl
Propyl
Isopropyl
Cyclopropyl
2-Hydroxyethyl
3-Hydroxypropyl
N,N-Dimethylaminoethyl
4-(Pyrrolidin-1-yl)-butyl
Pyridin-2-yl
Pyridin-3-yl
Pyridin-4-yl
25
11
28
73
Table 2
VEGFR-2 potencies for compounds 5a–j
R1
29
R2
168
400
285
759
34
O
N
O
N
N
10
11
H2N
Thiazol-2-yl
3.4
2.0
2.0
3.0
2.6
7.9
2.5
3.3
1.7
2.8
3.0
10.3
6.5
4,5-Dimethylthiazol-2-yl
5-Methylisoxazol-3-yl
Phenyl
Compds
R1
R2
VEGFR-2 inhibition IC50 (lM)
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
2-Cl
H
2-Cl
H
NHC(O)Me
C(O)NHMe
2.21
2-Chlorophenyl
2-Methylphenyl
2-Methoxyphenyl
2-Fluorophenyl
3-Fluorophenyl
4-Fluorophenyl
2,4-Difluorophenyl
2,4-Difluorophenylmethyl
Cyclohexyl
>100
0.152
>100
19.7
0.142
0.180
>100
0.179
0.142
NHC(O)OMe
OC(O)NHEt
CH C(O)NHMe
2
NHC(O)NHMe
NHC(O)NHEt
NHC(O)NHMe
NHC(O)NHEt
NHC(O)NHEt
s
t
H
u
v
w
x
y
2-F
2-Me
2-Br
3-Cl
H
j

S. Huang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1815–1818
1817
Table 3
Kinase selectivity profile for compound 4b
Kinase tested
IC50 (nM)
CSF1R (FMS)
FLT1 (VEGFR-1)
FLT3
FLT4(VEGFR-3)
KDR (VEGFR-2)
KIT
622
4420
4800
1100
42
513
PDGFRA (PDGFR alpha)
RET
1110
183
Figure 3. HCT116 tumor xenograft study.
Table 4
Pharmacokinetic properties of compound 4b
Species
Balb/c mouse Rat
Parameters (IV)
Dose (mg/kg)
2
2
C
max (ng/mL)
AUC (ng h/mL)
ss (L/kg)
1/2 (h)
2987
6114
0.7
5401
4799
0.4
V
t
2.8
0.7
Clearance (mL/min/kg) 5.6
7.4
Parameters (PO)
Dose (mg/kg)
max (ng/mL)
AUC (ng h/mL)
1/2 (h)
10
10
C
2786
12305
2.1
1034
7304
10.0
30
t
Oral bioavailability (%) 16
Figure 4. A375 tumor xenograft study.
activity may be correlated with the compound’s ability to inhibit
the dynamics of tumor blood vessel turnover, similar to SU11248
1
6
maintenance therapy.
In summary, compounds belonging to a novel series of 4-
aminopyrimide-5-carbaldehyde oxime were discovered to be po-
tent and selective VEGFR-2 inhibitors. An efficient chemistry was
developed for analogue synthesis. This series showed reasonable
PK properties in both mice and rats. Compound 4b exhibited activ-
ity against a variety of solid tumor types when administered alone.
It represents an exciting new angiogenesis inhibitor for clinical
development in areas in which dysregulated vascularization
occurs, including rheumatoid arthritis, diabetic retinopathy, and
tumor growth.
Figure 2. A431 tumor xenograft study.
bioavailability (16%) was less desirable. Compound 4b showed
relatively slow clearance in both mice and rats. The volume of dis-
tribution in mice was nearly two-fold higher than in rats. In the
mouse PK study, its IV half-life was similar to that for PO dose.
On the other hand, its oral half-life in rats was much longer than
that for IV dose. This difference could be due to a slow absorption.
The in vivo efficacy of compound 4b was evaluated in several
well-established, diverse human tumor xenograft models in nude
mice. The compound was formulated as a suspension in 0.5% meth-
ylcellulose and dosed PO once daily. Before daily treatment, tumor
cells were implanted and allowed to establish growth for 10–
Acknowledgments
We thank Angel Fuentes-Pesquera for conducting the VEGFR-2
kinase assay and Jeannene Butler for performing in vivo efficacy
studies.
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