Design and synthesis of 1,5-diarylbenzimidazoles as inhibitors of the VEGF-receptor KDR

Article abstract of DOI:10.1016/S0960-894X(03)00485-2

1,5-Diarylbenzimidazoles have been identified as potent inhibitors of KDR kinase activity. The series was developed with a goal of finding compounds with optimal drug-like properties. This communication describes structural modifications in the series that enhance solubility, lower protein binding, and provide compounds with excellent potency and pharmacokinetic profiles.

Full text of DOI:10.1016/S0960-894X(03)00485-2

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2485–2488
Design and Synthesis of 1,5-Diarylbenzimidazoles as Inhibitors of
the VEGF-Receptor KDR
Mark T. Bilodeau,* April M. Cunningham, Timothy J. Koester, Patrice A. Ciecko,
Kathleen E. Coll, William R. Huckle, Randall W. Hungate, Richard L. Kendall,
Rosemary C. McFall, Xianzhi Mao, Ruth Z. Rutledge and Kenneth A. Thomas
Departments of Medicinal Chemistry and Cancer Research, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
Received 5 March 2003; revised 7 May 2003; accepted 8 May 2003
Abstract—1,5-Diarylbenzimidazoles have been identified as potent inhibitors of KDRkinase activity. The series was developed
with a goal of finding compounds with optimal drug-like properties. This communication describes structural modifications in the
series that enhance solubility, lower protein binding, and provide compounds with excellent potency and pharmacokinetic profiles.
# 2003 Elsevier Ltd. All rights reserved.
Angiogenesis, the formation of new capillaries from
established blood vessels, is a normal developmental
process that has also been shown to be operative in the
progression of diseases such as diabetic retinopathy,1
rheumatoid arthritis,2 psoriasis,3 and cancer.4 In parti-
cular, the growth and metastasis of solid tumors has
been shown to be dependent on the angiogenic process.5
These observations have lead to widespread interest in
the inhibition of angiogenesis as an approach for the
treatment of cancer.
different structural classes, including indolin-2-ones,
phthalazines, and quinazolines.
As part of an effort to develop inhibitors of KDR, 3,6-
diarylpyrazolo[1,5-a]pyrimidines such as 1 have been
found to be potent inhibitors of KDRkinase activity
(IC50=71 nM).11,12 Compounds of this series had been
hindered by poor physical properties, including low
solubility, high logP and high protein binding, leading
us to explore other templates to address these limi-
tations. We sought different cores that could present the
same key pharmacophore as these leads but address
the problems of physical properties. Based on modeling,
the core N-1 was important as a hydrogen bond
acceptor and the aryl groups at positions 3- and 6-made
crucial hydrophobic interactions. Thus, we considered
benzimidazoles to be a likely replacement for the pyr-
azolo[1,5-a]pyrimidine core. The direct benzimidazole
analogue of 1 (2, IC50=113 nM, Scheme 1)13,14 main-
tained good intrinsic potency while significantly
improving solubility in organic solvents.15 However, 2
was still quite lipophilic, with poor cell potency.16 In the
3,6-diarylpyrazolo[1,5-a]pyrimidine class, incorporation
of a basic amine substituent modestly improved intrinsic
potency and significantly improved cell potency. This
was also the case for the benzimidazole class (see com-
pound 3) where enhanced activity was observed in an
assay of VEGF-induced mitogenesis of human umbili-
cal vein endothelial cells. Compound 3 showed sig-
nificantly enhanced solubility relative to 2 although it is
Among the molecular mechanisms elucidated in the
angiogenic process, the role of vascular endothelial
growth factor (VEGF) has elicited particular interest.6
VEGF expression has been shown to be up-regulated in
a variety of tumors and is an important factor in tumor
angiogenesis. VEGF is a selective mitogen for endothe-
lial cells and this signaling is mediated through the
receptor tyrosine kinase KDR(VEGFR-2). A variety of
inhibitors of VEGF-induced angiogenesis have been
shown to be efficacious in in vivo tumor xenograft
models. These inhibitors have included antibodies
against VEGF7 and its receptor KDR,8 a soluble VEGF
decoy-receptor9 and small molecule inhibitors of KDR
kinase activity.10 Clinical trials have been underway for
KDRkinase inhibitors derived from a number of
*Corresponding author. Tel.: +1-215-652-5304; fax: +1-215-652-
6345; e-mail: mark_bilodeau@merck.com
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00485-2

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M. T. Bilodeau et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2485–2488
Scheme 1. Initial optimization of the benzimidazole core.
still quite lipophilic (LogP=4.29).17 This compound
continued to possess good organic solubility but poor
aqueous solubility at neutral pH (<0.00001 mg/mL).
However compound 3 had an appreciable solubility at
acidic pH (solubility of 0.120 mg/mL at pH 5.2).
should be open to substitution, hence the addition of
basic amines off of the 5-aryl moiety.
Since the lipophilicity of the lead compound 3 was high,
we began to incorporate more polarity into the core.
This was accomplished by incorporating pyridyl func-
tionality into the benzimidazole core itself as in the
7-azabenzimidazole derivative 4. This compound had
comparable intrinsic and improved cell potency relative
to compound 3. In addition, 4 had a significantly lower
LogP of 3.55 and consequently an improved aqueous
solubility (Table 2).
Substitution of the 3-phenyl substituent (data not
shown) lead to significantly lower activity in the series,
even with relatively conservative fluorine substitutions.
This observation was consistent with docking studies
using a KDRhomology model that depicted the bicyclic
core bound in the adenine region of the ATP active site.
In this model N-1 of the benzimidazole is engaged in a
hydrogen bond with Cys919 of the hinge region, and the
N-3 aryl substituent occupying the sterically confined
hydrophobic region I.18 Thus, all efforts to substitute
this phenyl moiety lead to steric clashes within the
hydrophobic pocket and decreased potency. According
to this postulate the other benzimidazole 5-substituent
Other changes to improve the physical properties of the
series focused on the 5-aryl substituent (Table 1).17
Pyridyl functionality was incorporated into this position
to produce the alkoxypyridine 5 and the aminopyridine
6. While these changes tended to generally improve the
physical properties of the series, these changes resulted
in low cell potency. Additional heterocycles were inves-
tigated at this position, with the most dramatic
improvement occurring upon incorporation of pyridone
functionality. Thus, the synthesis of 7 resulted in a
compound with good intrinsic potency and, in contrast
to the previous compounds of this series good potency
in cells. Most significantly, 7 is more polar with a
LogP=2.25. Compound 7 had minimal effect on the
unstimulated growth of HUVECS at 2.5 mM and a 60-
fold ratio for inhibition of bFGF-stimulated growth of
Table 1. SARof the benzimidazole 5-substituent
Compd
Ar
KDRIC 50 (nM) Cell IC50 (nM)
5
23Æ12
52Æ13
25Æ9
122
213
37
Table 2. Physical properties of selected compounds
6
7
8
Compd
LogP
Prot. Bind.
(% bound)
Solubility
@ pH 5.2
(mg/mL)
Solubility
@ pH 7.4
(mg/mL)
3
4
7
4.29
3.55
2.25
99.4
97.4
80.7
0.120
6.46
33.9
<0.00001
0.021
8.4
37Æ7
152
Table 3. Pharmacokinetic data for 3a
9
88
nd
PK parameter
Ratb
Dogc
t1/2 (h)
6.1
14.4
1.9
5.2
4.6
3.1
nd
Cl (mL/min/kg)
Vdss (L/kg)
F (%)
10
11
270
nd
76
aCompound dosed iv as a solution in DMSO and p.o. as a suspension
in 0.5% methocel.
bAverage of four rats dosed at each of 5 mpk iv and 16 mpk po.
cAverage of two dogs dosed 1 mpk iv.
96
164

M. T. Bilodeau et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2485–2488
2487
Table 4. Pharmacokinetic data for 7a
primarily to the three carbon chain between hetero-
atoms and potential changes in amine pKa as 10 dis-
plays comparable protein binding (85.0%) and
solubility (>1 mg/mL @ pH 7.4) as 7.
PK parameter
Ratb
Dogc
t1/2 (h)
2.9
17.3
3.9
10.4
8.9
7.9
nd
Cl (mL/min/kg)
Vdss (L/kg)
F (%)
Compound
3 displayed excellent pharmacokinetic
73
behavior in rats and dogs (Table 3). It showed a good
half-life with moderate clearance and excellent oral
bioavailability in rats. Also, the iv pharmacokinetics for
3 was excellent in dogs. It exhibited a long half-life and
low clearance.
aCompound dosed iv as a solution in DMSO and po as a suspension
in 0.5% methocel.
bAverage of four rats dosed at each of 5 mpk iv and 20 mpk po.
cAverage of two dogs dosed 1 mpk iv.
Table 5. Fold-selectivity versus a series of kinases
The pharmacokinetics for compound 7 were also good
in both rats and dogs (Table 4). It also had a moderate
clearance and good half life in rats. In dogs, it had a
long half-life with a moderate clearance and higher
volume of distribution relative to compound 3.
Compd Flt-1 Flt-4 PDGFRb FGFR-1 FGFR-2 c-Src
3
4
5
7
5
6
4
11
6
5
3
6
0.5
0.4
0.5
1
60
38
51
97
47
25
42
57
62
63
50
181
We have examined the selectivity of these compounds
for KDRversus other closely related receptor tyrosine
kinases and a non-receptor tyrosine kinase. Fold selec-
tivities for several compounds are shown for this selec-
tion of kinases versus KDRin Table 5. In general the
compounds show low levels of selectivity against the
most KDR-homologous kinases; Flt-1, Flt-4 and
PDGFb. Higher levels of selectivity are observed
against FGF-1, FGF-2 and c-Src.
HUVECs, consistent with specificity of action through
KDRand moderate selectivity against FGFRs ( Table 5).
Changing the basic amine side-chain as in 8 leads to a
moderate loss of intrinsic activity and in this case 9 is
significantly less active in cells. The length of he chain is
also critical for potency as 9 and 10 demonstrate (com-
pare with 7 and 8 respectively). Again, other hetero-
cycles such as the pyrimidone 11 were less active than
the pyridone 7.
Scheme 2 outlines the synthesis of compounds 3 and 7.19
4-Bromo-1-fluoro-2-nitrobenzene was condensed with
aniline and this was followed by reduction and benzi-
midazole formation to provide 5-bromobenzimidazole
12. A Suzuki reaction provided 2 and the methyl ether
was subsequently deprotected to provide 13. Alkylation
of 13 provided 3. For the synthesis of 7, 12 was con-
verted to a pinacol boronate and that intermediate was
coupled with 4-bromopyridine to provide the
pyridylbenzimidazole 14. Following a standard pyridine
N-oxidation and rearrangement sequence the pyridone
15 was obtained. The pyridone was then alkylated to
Table 2 highlights the physical property improvements
achieved in this series.17 Compound 3 is highly lipophilic
with high human plasma protein binding. Introduction
of the basic functionality in 4 lowered LogP and protein
binding while dramatically improving solubility. A fur-
ther significant improvement in all of these parameters
has been observed with the introduction of pyridone
functionality as in 7. These changes are not due
Scheme 2. Synthesis of compounds 3 and 7.

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M. T. Bilodeau et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2485–2488
provide 7. This alkylation provides a 4:1 ratio of N- and
O-alkylation products.
11. Fraley, M. E.; Hoffman, W. F.; Rubino, R. S.; Hungate,
R. W.; Tebben, A. J.; Rutledge, R. Z.; McFall, R. C.; Huckle,
W. R.; Kendall, R. L.; Coll, K. E.; Thomas, K. A. Bioorg.
Med. Chem. Lett. 2002, 12, 2767.
We have described the development of 1,5-diarylbenz-
imidazoles that are potent and selective KDRinhibi-
tors. These compounds significantly improved physical
properties relative to the corresponding 3,6-diarylpyr-
azolo[1,5-a]pyrimidines. In particular, 3 and 7 have
excellent potency and pharmacokinetic profiles. The
pyridone 7 additionally has a significantly improved
LogP and solubility, properties which may confer
advantages in vivo.
12. Fraley, M. E.; Rubino, R. S.; Hoffman, W. F.; Ham-
baugh, S. R.; Arrington, K. L.; Hungate, R. W.; Bilodeau,
M. T.; Tebben, A. J.; Rutledge, R. Z.; Kendall, R. L.; McFall,
R. C.; Huckle, W. R.; Coll, K. E.; Thomas, K. A. Bioorg.
Med. Chem. Lett. 2002, 12, 3537.
13. All target compounds were fully characterized by 1H
NMRand mass spectroscopy.
14. The KDRIC value represents biochemical inhibition of
50
phosphorylation of a poly-Glu/Tyr (4:1) peptide substrate by
isolated KDRkinase (cloned and expressed as a GST-fusion
protein): see, Kendall, R. L.; Rutledge, R. Z.; Mao, X.; Teb-
ben, A. L.; Hungate, R. W.; Thomas, K. A. J. Biol. Chem.
1999, 274, 6453. Values are reported as single determinations
or as the average of at least two determinationsÆstandard
deviation.
15. N-Aryl benzimidazoles as inhibitors of PDGF: Palmer,
P. D.; Smaill, J. B.; Boyd, M.; Boschelli, D. H.; Doherty,
A. M.; Hamby, J. M.; Khatana, S. S.; Kramer, J. B.; Kraker,
A. J.; Panek, R. L.; Lu, G. H.; Dahring, T. K.; Winters, R. T.;
Showalter, H. D. H.; Denny, W. A. J. Med. Chem. 1998, 41,
5457.
16. The Cell IC50 value represents the inhibition of VEGF-
stimulated mitogenesis as determined in human umbilical vein
endothelial cells. For the key compounds 3, 4 and 7 the
averages are based on 3, 34 and 7 determinations respectively.
17. Partition coefficients were determined by adding an ali-
quot of a methanolic sample solution to equal volumes of 1-
octanol and pH 7.4 buffer and measuring the concentration in
each after an equilibration period of 8 h. Buffer solubilities
were determined by comparing the HPLC peak area of a fil-
tered, saturated solution of compound in pH adjusted buffer
to peaks from standard methanolic solutions. Protein binding
to human plasma is determined by equilibrating buffer solu-
tions of test compounds and human plasma and using ultra-
filtration for separation. The free compound concentration is
measured by HPLC.
18. Traxler, P.; Furet, P. Pharmacol. Ther. 1999, 82, 195.
19. Compound 4 was prepared according to the synthesis of 3,
beginning with 5-bromo-2-chloro-3-nitropyridine. Com-
pounds 5, 8 and 10 were derived from alkyation of 5-iodo-
pyridin-2-ol and subsequent coupling with the boronate ester
in Scheme 2. Compound 6 was prepared by coupling of the
boronate ester with 5-bromo-2-fluoropyridine and displace-
ment of the resulting fluoride with (2-morpholin-4-ylethyl)-
amine. Compound 9 was prepared according to the synthesis
of 7, using the homologous alkylating reagent. Compound 11
was prepared by coupling of the boronate ester with 4-chloro-
2-(methylthio)pyrimidine, followed by hydrolysis of the thio-
methyl-functionality and alkylation according to Scheme 2.
Acknowledgements
We thank Matthew M. Zrada and Kenneth D.
Anderson for LogP, solubility and protein binding
determinations.
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