Optimization of a pyrazolo[1,5-a]pyrimidine class of KDR kinase inhibitors: improvements in physical properties enhance cellular activity and pharmacokinetics.

Article abstract of DOI:10.1016/S0960-894X(02)00827-2

We have introduced solubilizing functionality to a 3,6-disubstituted pyrazolo[1,5-a]pyrimidine series of KDR kinase inhibitors to improve the physical properties of these compounds. The addition of a basic side-chain to the 6-aryl ring, introduction of 3-pyridyl groups, and most significantly, incorporation of a 4-pyridinonyl substituent at the 6-position of the core are modifications that maintain and often enhance the intrinsic potency of this class of inhibitors. Moreover, the improvements in physical properties result in marked increases in cellular activity and more favorable pharmacokinetics in rats. The synthesis and SAR of these compounds are described.

Full text of DOI:10.1016/S0960-894X(02)00827-2

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3537–3541
Optimization of a Pyrazolo[1,5-a]pyrimidine Class of KDR Kinase
Inhibitors: Improvements in Physical Properties Enhance Cellular
Activity and Pharmacokinetics
Mark E. Fraley,* Robert S. Rubino, William F. Hoffman, Scott R. Hambaugh,
Kenneth L. Arrington, Randall W. Hungate, Mark T. Bilodeau, Andrew J. Tebben,
Ruth Z. Rutledge, Richard L. Kendall, Rosemary C. McFall, William R. Huckle,
Kathleen E. Coll and Kenneth A. Thomas
Departments of Medicinal Chemistry and Cancer Research, Merck Research Laboratories, West Point, PA 19486, USA
Received 12 June 2002; accepted 17 September 2002
Abstract—We have introduced solubilizing functionality to a 3,6-disubstituted pyrazolo[1,5-a]pyrimidine series of KDR kinase
inhibitors to improve the physical properties of these compounds. The addition of a basic side-chain to the 6-aryl ring, introduction
of 3-pyridyl groups, and most significantly, incorporation of a 4-pyridinonyl substituent at the 6-position of the core are modifi-
cations that maintain and often enhance the intrinsic potency of this class of inhibitors. Moreover, the improvements in physical
properties result in marked increases in cellular activity and more favorable pharmacokinetics in rats. The synthesis and SAR of
these compounds are described.
# 2002 Elsevier Science Ltd. All rights reserved.
Angiogenesis, the formation of new capillaries from
established blood vessels, contributes to the pathogen-
esis of a number of disease states such as diabetic
retinopathy,1 rheumatoid arthritis,2 psoriasis,3 and can-
cer.4 At an early stage in the development of solid
tumors, angiogenesis is triggered when blood perfusion
from the existing vasculature becomes insufficient for
sustaining tumor cell growth.5 Under hypoxic condi-
tions, the tumor cells elicit an angiogenic response by
expressing a variety of growth factors, including vascular
endothelial growth factor (VEGF), whose mitogenic
signaling is mediated through the receptor tyrosine
kinase VEGFR-2 (KDR).6 Several lines of evidence
indicate that expression and signaling of VEGF are cri-
tical for tumor angiogenesis. Among these, antibodies
against VEGF7 and its receptor KDR8 as well as small
molecule inhibitors of the KDR kinase domain9 have
been shown to inhibit angiogenesis in tumor models.
Recently, the therapeutic potential of small molecule
inhibitors of KDR kinase for use as anti-angiogenic
Figure 1. Initial optimization of screening lead 1.
*Corresponding author. Fax: +1-215-652-6345; e-mail: mark_fraley@merck.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00827-2

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M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3537–3541
Scheme 1. Synthesis of compounds 4a–h.14
agents for the treatment of cancer has come under much
attention. Clinical trials have been initiated for KDR
kinase inhibitors derived from a number of different
structural classes, including indolin-2-ones, phthalazines,
and quinazolines.10 This approach to cancer therapy dif-
fers from the traditional use of cytotoxic agents in that the
target tissue is the vasculature encompassing the neoplasm,
not tumor cells, and relies on ‘starvation’ of the tumor for
growth inhibition rather than direct cell-cycle arrest.
midine class of KDR kinase inhibitors. In that work, we
identified a number of replacements for the 4-pyridyl
groups of screening lead 1 which offered marked
improvements in the biochemical activity of this series.
Potency was fully optimized with 4-methoxyphenyl and
3-thienyl groups at the 6- and 3-positions, respectively,
of the pyrazolo[1,5-a]pyrimidine, as illustrated in the
progression of 1 to 3 (Fig. 1).12 Results from a VEGF-
stimulated endothelial cell mitogenesis assay (ECMA)
indicated that compounds 1 and 2 which bear solubiliz-
ing pyridine groups have enhanced cellular inhibitory
activity (a lower ECMA to KDR IC50 ratio) than the
In a previous paper,11 we reported our initial optimiza-
tion studies of a newly discovered pyrazolo[1,5-a]pyri-
Table 1. KDR kinase and cellular activity of 4a–h17
Compound
Side-chain
position
RO
3-Het/Ar
KDR IC50
(nM)
ECMA
IC50 (nM)
4a
4b
para
para
4
9
22
32
4c
4d
4e
4f
meta
para
para
para
para
para
14
10
3
49% inhibition
@ 100 nM
23
18
19
20
11
31% inhibition
@ 100 nM
4g
4h
29
31

M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3537–3541
3539
Scheme 2. Synthesis of compounds 5a and 5b.
less soluble and more lipophilic compound 3.13 These
data suggested that cellular activity in the mitogenesis
assay was improved by increasing the solubility and
polarity of these inhibitors. In this paper, we describe
structural modifications that improved the physical
properties of inhibitors within this series, and thereby
enhanced the cellular activity and improved pharmaco-
kinetics in rats.
pyrazoles.15 Demethylation of the 6-aryl ethers was
accomplished in one of two ways depending on the
nature of the 3-heteroaryl group as depicted.16 Alkyl-
ation of the resulting phenols with a variety of mustard-
like chloroalkyl amines proceeded in moderate yield to
provide analogues 4a–h (Table 1).
The screening data from this set of inhibitors revealed
that addition of the basic side-chain enhanced the
intrinsic potency and produced the predicted increase in
cellular activity. For example, compound 4a was 5-fold
more potent against isolated KDR and nearly 20 times
more active in the mitogenesis assay compared to 3.
Consistent with the structure–activity relationships
(SARs) established in our preliminary work in this ser-
ies, compounds possessing a 3-thienyl group at the
3-position of the pyrazolo[1,5-a]pyrimidine were 2-fold
more potent biochemically than the corresponding
3-phenyl analogues (4a vs 4b). Likewise, compounds
possessing para-substituted basic ethers on the 6-aryl
ring were 3-fold more active than the meta-substituted
analogues (4c vs 4a). In this data set, inhibitory activity
appeared relatively insensitive to the nature of the basic
side-chain (cf., 4a, 4d, and 4e). This observation was
consistent with docking studies using a KDR homology
model that depicted the bicyclic core bound in the ade-
nine region of the ATP active site, with N-1 of the pyr-
azolo[1,5-a]pyrimidine engaged in a hydrogen bond with
Cys 919 of the hinge region, and the side-chain extending
away from the cleft and exposed to solvent.18 Although
less potent, compound 4f showed elevated KDR kinase
specificity (see below). Lastly, 3- and 4-pyridyl substituents
at the 3-position of the nucleus were well-tolerated (4g
and 4h) and provided greater solubility and polarity
(e.g., logP 4g=3.4 vs logP 4e>4.0).
Toward improving the physical properties of our initial
leads, we first introduced solubilizing basic amines to the
meta and para positions of the 6-aryl ring via an ether
linkage utilizing the chemistry outlined in Scheme 1.
Thus, 6-(3- and 4-methoxyphenyl)pyrazolo[1,5-a]pyri-
midines were assembled in good yield via condensation
reactions of the corresponding 2-(3- and 4-methoxy-
phenyl)malondialdehydes and 3-amino-4-heteroaryl-
Table 2. KDR kinase and cellular activity of 5a–c
Compound
R
3-Ar/Het KDR ECMA LogP
IC50
(nM)
IC50
(nM)
5a
5b
14
13
70
80
2.4
2.0
5c
7
20
1.6
We next incorporated a 4-pyridinonyl substituent at the
6-position of the pyrazolo[1,5-a]pyrimidine, a modification
Scheme 3. Synthesis of compound 5c.

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M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3537–3541
that had been shown to greatly enhance physical prop-
erties in a related benzimidazole class of KDR kinase
inhibitors. The chemistry in the 3-phenyl series involved
oxidation of the corresponding 4-pyridyl ring followed
by rearrangement of the resulting pyridine n-oxide
under standard conditions (Scheme 2). Selective alkyl-
ation of the 4-pyridinonyl nitrogen provided com-
pounds 5a and 5b in modest yield (Table 2).
Introduction of the 6-(4-pyridinonyl) group in the more
potent 3-(3-thienyl) series required an alternative synth-
esis due to the oxidatively sensitive 3-thiophene ring
(Scheme 3). Key steps in the synthesis of 5c included the
Suzuki cross-coupling reaction of 6-bromo-3-(3-thio-
phenyl)pyrazolo[1,5-a]pyrimidine19 with the 4-pinacol-
boronic ester of 2-methoxypyridine,20 hydrolysis of the
2-methoxypyridine intermediate,21 and the selective
N-alkylation of the penultimate pyridinone under the
optimized conditions shown.
In conclusion, we have identified a number of modifi-
cations to the pyrazolo[1,5-a]pyrimidine class of KDR
kinase inhibitors that have improved the physical prop-
erties of these compounds over those of the initial leads.
Modifications such as the addition of a basic side-chain
to the 6-aryl ring, introduction of 3-pyridyl groups, and
incorporation of a 4-pyridinonyl substituent at the
6-position of the nucleus afforded enhanced cellular
potency and more favorable pharmacokinetics in rats.
Acknowledgements
We thank Dr. Art Coddington, Dr. Chuck Ross, and
Dr. Harri Ramjit for mass spectral analyses, Matt
Zrada, Ken Anderson, and Patrice Ciecko for logP
determinations, and Dr. George Hartman and Elaine
Walker for editing and assistance in the preparation of
this manuscript.
Biochemical, cellular, and partition coefficient data are
summarized for compounds 5a–c in Table 2. A sig-
nificant increase in polarity and aqueous solubility was
observed with the incorporation of the 4-pyridinonyl
group within 5a (logP=2.4) as compared to the highly
lipophilic and relatively insoluble homologue 4e (logP
>4.0). Physical properties and potency were further
optimized in this series with addition of the 4-methyl-
piperazinyl and 3-thienyl appendages found in 5c.
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4e
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26
52
>1000
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242
86
358
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t1/2 (h)
Vdss (L/kg)
%F
4e
4g
5c
80
23
28
5.1
3.3
3.4
16
6.0
7.9
18
42
56
13. Partition coefficients were determined by HPLC analysis
in octanol/water.

M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3537–3541
14. All target compounds were fully characterized by 1H
NMR and mass spectroscopy.
3541
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23. The doses and vehicles used for intravenous and oral
administration were 5 mg/kg in DMSO and 20 mg/kg in aqu-
eous 0.5% methyl cellulose (pH 5), respectively.