Thienopyridine urea inhibitors of KDR kinase

Article abstract of DOI:10.1016/j.bmcl.2006.12.015

A series of substituted thienopyridine ureas was prepared and evaluated for enzymatic and cellular inhibition of KDR kinase activity. Several of these analogs, such as 2, are potent inhibitors of KDR (<10 nM) in both enzymatic and cellular assays. Further

Full text of DOI:10.1016/j.bmcl.2006.12.015

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1246–1249
Thienopyridine urea inhibitors of KDR kinase
H. Robin Heyman,a,* Robin R. Frey,a Peter F. Bousquet,b George A. Cunha,b
Maria D. Moskey,b Asma A. Ahmed,b Niru B. Soni,a Patrick A. Marcotte,a Lori J. Pease,a
Keith B. Glaser,a Melinda Yates,a Jennifer J. Bouska,a Daniel H. Albert,a
Candace L. Black-Schaefer,a Peter J. Dandliker,a Kent D. Stewart,a Paul Rafferty,b
Steven K. Davidsen,a Michael R. Michaelidesa and Michael L. Curtina
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 7 November 2006; revised 4 December 2006; accepted 4 December 2006
Available online 9 December 2006
Abstract—A series of substituted thienopyridine ureas was prepared and evaluated for enzymatic and cellular inhibition of KDR
kinase activity. Several of these analogs, such as 2, are potent inhibitors of KDR (<10 nM) in both enzymatic and cellular assays.
Further characterization of inhibitor 2 indicated that this analog possessed excellent in vivo potency (ED50 2.1 mg/kg) as measured
in an estradiol-induced mouse uterine edema model.
Ó 2006 Elsevier Ltd. All rights reserved.
Reversible protein phosphorylation by protein kinases is
one of the primary biochemical mechanisms mediating
eukaryotic cell signaling.1 A subset of these kinases,
the receptor tyrosine kinases (RTKs), possess both
extracellular and intracellular domains and selectively
catalyze the phosphorylation of tyrosine hydroxyl
groups in response to binding of certain extracellular
growth factors.2 RTK signaling pathways are normally
highly regulated, yet their over-activation has been
shown to promote the growth, survival, and metastasis
of cancer cells, and has been associated with the progres-
sion of various human cancers.3 The VEGF receptor
family of RTKs, most notably VEGFR2 or KDR, medi-
ates the biological function of vascular endothelial
growth factor (VEGF) which is a regulator of vascular
permeability and an inducer of endothelial cell prolifer-
ation, migration, and survival.4 Accordingly, interrup-
tion of the KDR-mediated signaling cascade can
provide an anti-angiogenic effect in human cancers as
recently demonstrated by the FDA approval of the
anti-VEGF antibody AvastinTM for the treatment of
colorectal cancer.5 In addition, the small-molecule
KDR kinase inhibitors sorafenib and sunitinib have
both been approved by the FDA for the treatment of
patients with advanced renal cell carcinoma.6
In an ongoing effort at Abbott Laboratories to develop
small-molecule RTK inhibitors, it was recently disclosed
that a series of thienopyrimidine ureas were potent
inhibitors of both the VEGFR and PDGFR families
of RTKs;7 for example, it was shown that compound
1 was a potent inhibitor of both KDR (IC50 = 6 nM)8
and PDGFRb (IC50 = 60 nM) with excellent cellular
and in vivo activity. The ATP-mimic core of 1 comple-
ments other related nuclei including furanopyrimidine,9
pyrrolopyrimidine,10 and pyrazolopyrimidine11 while
the importance of the diphenyl urea moiety for tyrosine
kinase inhibition is consistent with earlier observations
regarding this structural motif.12 In an effort to expand
the scope of this series of inhibitors and potentially com-
plement the kinase activities and ADME properties of 1,
SAR studies have been conducted on a series of thieno-
pyridine 3-ureas. We wish to report that these com-
pounds are potent inhibitors of KDR kinase and that
7-heteroarylation (e.g., 2) provides compounds with
excellent cellular and in vivo activity.13
CF3
H
H
H
N
H
N
N
N
O
NH2
3
O
NH2
S
F
N
N
7
Keywords: KDR kinase; VEGF; Thienopyridine; Urea.
*
1
2
N
S
Corresponding author. Tel.: +1 847 937 1885; fax: +1 847 935
5165; e-mail: robin.heyman@abbott.com
N
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.015

H. R. Heyman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1246–1249
1247
The preparation of the 3-(diphenylurea)thienopyridine
inhibitors began with the three-step conversion of car-
boxylic acid 3, prepared by condensation of 4-bromo-
2-thiophenecarboxaldehyde with malonic acid, to
thienopyridinone 4 as reported previously for the des-
bromo substrate (Scheme 1).14 Compound 4 was con-
verted to aminopyridine 5 by high-pressure aminolysis
of the corresponding chloropyridine intermediate. Suzu-
ki coupling gave aniline 6 which was condensed with
variously substituted phenyl isocyanates to afford thie-
nopyridine ureas 7 in good yield (70–95%). Due to com-
peting acylation of the aminopyridine moiety of 6, the
urea formation was usually conducted at low tempera-
ture (À20 °C) with a small excess (1.05 equiv) of isocya-
nate; the bis-urea by-product could then be easily
removed by flash chromatography.
A series of 7-aryl thienopyridine ureas were made using
the synthetic route in Scheme 3. Bromide 5 was coupled
with N-Boc-protected aniline boronate to afford thieno-
pyridine 10 which could then be selectively iodinated at
the 7-position to give, after TFA deprotection, iodide 11
in 81% yield over three steps. Coupling with aryl or het-
eroaryl boronate/boronic acids using standard Pd0 cou-
pling conditions and urea formation as in Scheme 1
provided ureas 12 in 65–95% yield.
The SAR of the initial thienopyridine compounds is
shown in Table 1 and is quite similar to that observed
for the thienopyrimidine series of inhibitors.7 The
importance of the urea functionality was demonstrated
by the low activity of non-urea analogs 6, 13, and 14
while the 3-methylphenyl urea (7b) was modestly more
potent than phenyl urea 7a. As with the isoindolinone
series of KDR inhibitors,12a the external urea nitrogen
was much more sensitive to modification (i.e., methyla-
tion or replacement with carbon) than the internal urea
nitrogen as can be seen by comparing the potency of
An alternate preparation of the urea analogs utilizes the
commercially available aniline boronate 8 which was
condensed with substituted phenyl isocyanates to give
urea boronates 9 (Scheme 2). These intermediates would
then undergo Suzuki coupling with bromide 5 to give
the desired thienopyridine phenyl ureas (7) in 50–65%
yield.
NHtBoc
NH2
Br
NH2
b
Br
a
NH2
OH
N
Br
Br
N
a
b
S
N
N
HO
10
S
S
S
S
5
O
c
O
3
4
5
NH2
HN
R1
O
HN
NH2
NH2
HN
c,d
NH2
HN
N
R2
NH2
d
12
N
NH2
S
3
N
11
S
N
7
7
I
S
Ar
S
6
t
Scheme 3. Reagents and conditions: (a) Butyl N-[4-(tetramethyldiox-
aborolanyl)phenyl]carbamate, cat. Pd(PPh3)4, Na2CO3, DME/H2O,
90 °C; (b) N-iodosuccinimide, DMF; TFA, 81% (three steps); (c) aryl
boronate/boronic acid, cat. Pd(PPh3)4, Na2CO3, DME/H2O, 90 °C,
45–70%; (d) 3-methylphenyl isocyanate, NMM, DMF, À20 °C,
65–95%.
Scheme 1. Reagents and conditions: (a) SOCl2, CH2Cl2; NaN3,
dioxane/H2O; CH2Cl2, Ph2O, reflux, 70%; (b) POCl3; NH3, dioxane,
290 psi, 160 °C, 61%; (c) 4-(tetramethyldioxaborolanyl)aniline, cat.
Pd(PPh3)4, Na2CO3, DME/H2O, 90 °C, 75%; (d) aryl isocyanate,
NMM, DMF, À20 °C, 70–95%.
R1
O
Table 1. KDR inhibitory potency of thienopyridine analogs
NH2
HN
R
HN
R2
a
NH2
O
B
O
O
B
O
N
8
9
S
8
KDR IC50 (nM)
Compound
R
R1
O
13
6
H
NH2
> 12,500
4900
12,000
56
HN
HN
R2
14
7a
7b
15
16
17
18
19
–NHCOPh
–NHCONHPh
b
NH2
7
–NHCONH(3-MePh)
–NHCON(Me)(3-MePh)
–N(Me)CONH(3-MePh)
–NHCOCH2(3-MePh)
–CH2CONH(3-MePh)
–NHC(S)NH(3-MePh)
9
N
4900
23
S
520
94
Scheme 2. Reagents and condition: (a) aryl isocyanate, NMM, THF,
85–95%; (b) 3-bromothieno[3,2-c]pyridin-4-ylamine (5), cat. Pd(PPh3)4,
Na2CO3, DME/H2O, 90 °C, 50–65%.
410

1248
H. R. Heyman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1246–1249
compounds 15 and 17 versus 16 and 18. The thiourea 19
showed a significant decrease in activity compared to
urea 7b.
were 3,4-disubstitution (7i) and 3,5-disubstitution (7j).
It can be seen in Table 2 that most of these ureas had
only a small difference (2- to 5-fold) between their enzy-
matic and cellular potencies.
Consistent with the hypothesis that the thienopyridine
nucleus is an ATP-mimic and that the aminopyridine
acts as a ‘hinge-binding’ element,15 N-methylation as
in compound 20 gave a complete loss of activity. As ini-
tial modeling had suggested, replacement of the linking
The KDR inhibitory activities of thienopyridine urea
analogs with non-phenyl terminal rings are shown in
Table 3. It can be seen that while terminal alkyl rings
such as cyclopentyl (7k) and cyclohexyl (7l) were not tol-
erated, heterocycles such as naphthyl (7m, 7n) and thie-
nyl (7o, 7p) were potent KDR inhibitors.
1,4-phenyl of 7 with a 1,3-phenyl (21, KDR IC50
=
250 nM) gave a significant loss of potency while analogs
bearing urea substitution at the 2-position of the thieno-
pyridine nucleus as in 22 were inactive against KDR.
However, it was found that 2-methylation of the thieno-
pyridine, as in inhibitor 23 (KDR IC50 = 27 nM), was
well tolerated.
Initial modeling had suggested that substituents at the
thienopyridine 7-position would project into a solvent
accessible region of the active site and should be well tol-
erated. This substitution could potentially be used to
modulate the pharmacodynamic properties of these
inhibitors and offer an advantage over the thienopyrim-
idine series. Consistent with this hypothesis, arylation of
the thienopyridine 7-position with phenyl (12a) or
substituted phenyls such as in 12b and 12c provided ana-
logs which were equipotent with 7b, both in enzymatic
and cellular assays (Table 4). Furthermore, it was found
that heterocycles at this position, including 3-pyridyl (2),
4-pyridyl (12d), and 5-pyrimidyl (12e), gave a significant
increase in cellular potency while the larger heterocycle
4-isoquinolyl (12f) had only modest enzymatic potency.
O
O
HN
NH
HN
NH
NH2
NH
3
N
N
20
2
S
S
21
O
HN
NH2
O
HN
NH
N
Table 3. KDR enzymatic and cellular activity of ureas 7k–p
NH2
NH
S
O
N
23
HN
22
HN
R
S
NH2
N
S
The effect of substitution on the terminal phenyl of the
urea is shown in Table 2. Aside from the modest potency
difference between 7a and 7b, substitution of this ring at
the 3- and 4-positions generally provided compounds
(7c) that were roughly equipotent with 7a, while 2-sub-
stitution (7d) led to a significant loss of enzymatic poten-
cy. A number of 3-substituents were allowed (7e–h) as
8
16
Compound
R
KDR IC50
(nM)
KDRcell IC50
(nM)
7k
7l
cC5H9
cC6H11
420
491
75
ND
ND
895
15
7m
7n
7o
7p
1-Naphthyl
2-Naphthyl
2-Thienyl
21
30
48
61
3-Thienyl
38
Table 2. KDR enzymatic and cellular activity of ureas 7a–j
R1
O
2
3
Table 4. KDR enzymatic and cellular activity of ureas 2 and 12a–f
HN
4
R2
HN
O
HN
NH2
HN
N
NH2
N
S
8
16
Compound
R1
H
R2
KDR IC50
(nM)
KDRcell IC50
(nM)
S
7
Ar
7a
7b
7c
7d
7e
7f
H
H
H
H
H
H
H
H
56
9
72
32
8
16
Compound Ar
KDR IC50
(nM)
KDRcell IC50
(nM)
3-Me
4-Me
2-Me
3-Et
26
152
25
15
22
45
10
16
141
ND
32
12a
12b
12c
2
Ph
13
11
11
9
33
29
56
4
4-(OH)Ph
3-(CONHMe)Ph
3-Pyridyl
3-F
3-Cl
147
56
7g
7h
7i
12d
12e
12f
4-Pyridyl
5-Pyrimidyl
4-Isoquinolyl
5
4
3-CN
3-Me
3-Me
198
41
8
388
1
ND
4-Me
5-Me
7j
48

H. R. Heyman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1246–1249
1249
Table 5. KDR fold-potency versus a series of tyrosine kinases
Compound FLT1 FLT3 KIT PDGFRb TIE2 FGFR SRC
In summary, a series of potent KDR thienopyridine
urea inhibitors has been identified. Optimal urea and
7-substitution afforded analogs (e.g., 2) with significant
enzymatic and cellular potency as well as selectivity
against the non-VEGF tyrosine kinases FGFR and
SRC. Homology modeling suggested that the predomi-
nant interactions include hydrogen-bonds between the
thienopyridine nucleus and the protein backbone as well
as the urea functionality with a glutamate residue and a
hydrophobic pocket.
7b
2
0.6
1
1
0.5
2
2
10
13
40
5
>1000 >1000
>440 >440
The selectivity of analogs 7b and 2 for KDR versus
other tyrosine kinases as a ratio of enzymatic IC50 val-
ues is shown in Table 5. These inhibitors were equipo-
tent against the kinases most homologous to KDR
(FLT1, FLT3, and KIT), modestly potent against
PDGFRb and TIE2, and much less active against
FGFR and the cytosolic kinase SRC.
References and notes
Further characterization of these inhibitors indicated
that, while 7-unsubstituted analogs such as 7b were only
modestly active in vivo (40% inhibition, 30 mg/kg dose,
po), as measured in an estradiol-induced mouse uterine
edema model,7 inhibitors such as 2 possessed excellent
potency (ED50 2.1 mg/kg). However, it was determined
that 2 had a disappointing pharmacokinetic profile in
mouse (t1/2 0.4 h, 3 mg/kg dose, iv) with low exposure
after oral dosing (1.1 lmol h/L, 10 mg/kg dose, po) which
precluded its assessment in a mouse tumor model. Pyridyl
2 was also a potent inhibitor of Cyp3A4 (IC50 ꢀ250 nM).
Homology modeling17 of inhibitor 2 bound to the ATP-
binding site of the ‘inactive’ conformation of KDR is
reminiscent of our earlier analyses of urea KDR inhibi-
tors (Fig. 1).7,12a In this analysis, two hydrogen bonds
were created with the kinase hinge: the exocyclic amino
group of 2 with the backbone carbonyl of Glu917; and
the proximal ring nitrogen of the thienopyridine with
the backbone N–H of Cys919. In this bound conforma-
tion the urea unit accessed the back hydrophobic pocket
adjacent to the ATP-binding site with the urea N–H
bonds interacting with Glu885 of KDR. The terminal
tolyl group rested in a hydrophobic region comprised
of the side chains of Ile888, Leu889, Leu1019, and
Val898. As mentioned, the 7-pyridyl of inhibitor 2 pro-
jects into a solvent-accessible region of the active site
without any specific contact with the protein and is con-
sistent with the fact that most of the 7-substituted ana-
logs (2 and 12a–f) are equipotent with 7b.
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8. KDR and PDGFRb IC50 values were determined using an
ATP concentration of 1 mM as described in Ref. 7.
9. Miyazaki, Y.; Matsunaga, S.; Tang, J.; Maeda, Y.; Nakano,
M.; Philippe, R. J.; Shibahara, M.; Liu, W.; Sato, H.; Wang,
L.; Nolte, R. T. Bioorg. Med. Chem. Lett. 2005, 15, 2203.
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A.; Steinman, D. H.; Holmes, J. H.; Bousquet, P. F.;
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13. A related series of KDR inhibitors have recently been
reported: Miyazaki, Y.; Nakano, M.; Sato, H.; Truesdale,
A. T.; Stuart, J. D.; Nartey, E. N.; Hightower, K. E.;
Kane-Carson, L. Bioorg. Med. Chem. Lett. 2006.
doi:10.1016/j.bmcl.2006.09.050.
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Chem. 1999, 6, 775.
16. The cellular IC50 values represent inhibition of KDR
phosphorylation in NIH3T3 cells stably transfected with
full length human KDR. The procedure was followed as
described in Ref. 12.
17. The computational analysis used to create this homology
model with a recently published crystal structure of KDR-
homolog KIT kinase (PDB entry 1T46) is described in
Ref. 7.
Figure 1. Model of thienopyridine 2 (green) bound to active site of
KDR (model, inactive conformation, in purple) with hinge hydrogen
bonds to Glu917 C@O and Cys919 N–H in black and urea hydrogen
bonds to Glu885 in brown.