Synthesis and initial SAR studies of 3,6-disubstituted pyrazolo[1,5-a]pyrimidines: A new class of KDR kinase inhibitors

Article abstract of DOI:10.1016/S0960-894X(02)00525-5

We have synthesized and evaluated the activity of 3,6-disubstituted pyrazolo[1,5-a]pyrimidines as a new class of KDR kinase inhibitors. Starting with screening lead 1, potency against isolated KDR was fully optimized with 3-thienyl and 4-methoxyphenyl substituents at the 6- and 3-positions (3g, KDR IC₅₀=19 nM), respectively. The synthesis and SAR of these compounds are described.

Full text of DOI:10.1016/S0960-894X(02)00525-5

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Bioorganic & Medicinal Chemistry Letters 12 (2002) 2767–2770
Synthesis and Initial SAR Studies of 3,6-Disubstituted
Pyrazolo[1,5-a]pyrimidines: A New Class of
KDR Kinase Inhibitors
Mark E. Fraley,* William F. Hoffman, Robert S. Rubino, Randall W. Hungate,
Andrew J. Tebben, Ruth Z. Rutledge, Rosemary C. McFall, William R. Huckle,
Richard L. Kendall, Kathleen E. Coll and Kenneth A. Thomas
Departments of Medicinal Chemistry and Cancer Research, Merck Research Laboratories, West Point, PA 19486, USA
Received 27 March 2002; accepted 10 June 2002
Abstract—We have synthesized and evaluated the activity of 3,6-disubstituted pyrazolo[1,5-a]pyrimidines as a new class of KDR
kinase inhibitors. Starting with screening lead 1, potency against isolated KDR was fully optimized with 3-thienyl and 4-methoxy-
phenyl substituents at the 6- and 3-positions (3g, KDR IC50=19 nM), respectively. 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 reti-
nopathy,1 rheumatoid arthritis,2 psoriasis,3 and cancer.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 conditions, 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 critical for tumor
angiogenesis. Among these, antibodies against VEGF7
and its receptor KDR8 as well as small molecule inhibi-
tors 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 agents for the
treatment of cancer has come under much attention.
Clinical trials have been initiated for KDR kinase inhib-
itors derived from a number of different structural
classes, including indolin-2-ones, phthalazines, and
quinazolines.10 This approach to cancer therapy differs
from the traditional use of cytotoxic agents in that the
target tissue is the vasculature encompassing the neo-
plasm, not tumor cells, and relies on ‘starvation’ of the
tumor for growth inhibition rather than direct cell cycle
arrest.
In our efforts to identify small molecule inhibitors of
KDR kinase, we discovered a new class whose central
core is comprised of a pyrazolo[1,5-a]pyrimidine. Herein
we describe the synthesis and initial structure–activity
relationship (SAR) studies of this novel series.
Our chemistry efforts began with the goal of improving
the activity of screening lead 1 (Fig. 1, KDR IC50=224
nM). Toward optimization of the substituents at the 3- and
6-positions of 1, an initial set of 3,6-diarylpyrazolo[1,5-a]
pyrimidines was prepared in combinatorial fashion
*Corresponding author. Fax: +1-215-652-6345; e-mail: mark_fraley
@merck.com
Figure 1.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00525-5

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
2768
M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2767–2770
Scheme 1. Synthesis of compounds 2a–h.12
through condensation reactions of a 3-amino-4-aryl-
pyrazoles and commercially available 2-arylmalon-
dialdehydes (Scheme 1). The starting 3-amino-4-
arylpyrazoles were prepared in two steps from the
corresponding acetonitriles in 55–75% yield. Heating
solutions of the 3-amino-4-arylpyrazoles and 2-arylma-
londialdehydes in the presence of acetic acid in ethanol
followed by cooling furnished the 3,6-diarylpyr-
azolo[1,5-a]pyrimidines as yellow crystalline solids (40–
60% yield).11 KDR kinase inhibitory activity for a select
set of compounds is presented in Table 1.
The data in Table 1 indicate that substitution at the
para position of the 6-aryl ring enhances inhibitory
activity in the 3-(4-pyridyl) series. In the best case,
potency was optimized 6-fold over lead compound 1 by
the replacement of the 6-pyridyl substituent with a
4-methoxyphenyl group (2c). A number of more subtle
findings are worthy of note. In the 6-methoxyphenyl
series, the 3-(3-pyridyl) moiety provides nearly the same
potency as the 3-(4-pyridyl) group (2d vs 2c), while the
3-(2-pyridyl) arrangement (2e) is detrimental to activity.
In the 3-phenyl series, very little difference in activity
was observed between the 6-(4-methoxyphenyl) and 6-
(4-pyridyl) ligands (2f vs 2g), which is in marked con-
trast to the 3-(4-pyridyl series) as indicated. In addition,
a 2-pyridyl ring at the 6-position of the ring system does
not have the deleterious effect on activity as it did when
placed in the 3-position (compare 2h to 2e).
Table 1. KDR kinase activity of compounds 2a–h13
With the discovery of the 4-methoxy phenyl group as an
advance in the optimization of the 6-position of the
pyrazolo[1,5-a]pyrimidine nucleus, we next examined
the SAR of the 3-position more closely. A convenient
method for varying the substituent at the 3-position,
which avoids repetitious 3-aminopyrazole synthesis, was
employed in the assembly of a second set of analogues
(Scheme 2). 3-Amino-4-bromopyrazole was utilized in
the condensation reaction to provide 3-bromo pyr-
azolo[1,5-a]pyrimidines, which smoothly cross-coupled
with aryl boronic acids under Suzuki conditions to
afford 3,6-diarylpyrazolo[1,5-a]pyrimidines in 40–60%
yield. Biochemical inhibition of KDR kinase activity is
reported in Table 2 for a set of analogues wherein the
3-substituent was varied by this synthetic method.
Compd
Ar00
Ar0
KDR IC50 (nM)
224
1
2a
2b
2c
2d
2e
2f
113
62
37
41
The data in Table 2 indicate that halogen and methoxyl
substitution of the 3-aryl ring are detrimental to activity
with the exception of the incorporation of a 4-fluorine
atom (3a and 3c). However, replacement of the 3-phenyl
ring with a 3-thienyl group improves activity 3-fold in
both the 6-(4-pyridyl) and 6-(4-methoxyphenyl) series
(3f and 3g, respectively). The data in Table 2 are con-
sistent with docking studies using a KDR homology
model that depicts the 3-substituent occupying the small
and narrow hydrophobic region I of the ATP active
site.14 In addition, modeling and supporting SAR (not
shown) suggest the nitrogen at the 1–position of the
pyrazolo[1,5-a]pyrimidine makes in a key hydrogen
bond with Cys 919 of the hinge region.
1000
71
2g
2h
71
88

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2767–2770
2769
Scheme 2. Synthesis of compounds 3a–g.
In subsequent studies, we have found that a number of
modifications to the substitution pattern about the pyr-
azolo[1,5-a]pyrimidine nucleus have deleterious effects
on kinase inhibitory activity. For example, deletion of
the 3- or 6-aryl group from the core leads to complete
loss of activity, as does independently shifting the 6-aryl
group to the 7- or 5-position, or moving the 3-aryl
group to the 2-position. The addition of a methyl group
at the 7- or 2-position of the pyrazolo[1,5-a]pyrimidine
also produces inactive compounds. In addition, sub-
stitution of the 3- or 6-aryl ring at a position ortho to
the carbon appended to the bicyclic core is detrimental
to activity. These latter results suggest a nearly planar
ring system for optimized binding and are consistent
with kinetics studies which indicate that these com-
pounds bind in the narrow ATP binding cleft of the
kinase domain.
Table 2. KDR kinase activity of compounds 3a–g
Compd
Ar00
Ar0
KDR IC50 (nM)
71
2g
3a
3b
3c
3d
3e
3f
63
350
265
We next examined the SAR of fully and partially satu-
rated ring systems at the 6- and 3-positions of the cen-
tral nucleus. Incorporation of a cyclohexyl ring at the
6-position in compound 4 led to a 15-fold loss in activity
(Scheme 3). Activity was mostly maintained, however,
with cyclopentenyl (5) and cyclohexenyl (6) substitution
at the 3-position (Scheme 4). A fully saturated cyclo-
hexyl ring or small alkyl substituents at the 3-position,
such as a 3-ethyl group, were not well-tolerated (KDR
IC50s>5 mM).
510
71% inh. @ 2500 nM
24
19
The selectivity for KDR kinase inhibition was deter-
mined for 1, 2c, 3f, and 3g via screening against a small
panel of highly homologous receptor tyrosine kinases
3g
Scheme 3. Synthesis and KDR kinase activity of compound 4.

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
2770
M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2767–2770
Scheme 4. Synthesis and KDR kinase activity of compounds 5 and 6.
Table 3. KDR kinase selectivity (fold) of compounds 1, 2c, 3f, and 3g
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.
Compd
PDGFRb
FLT-1
FLT-4
FGFR-1
SRC
1
0.4
0.5
11.4
1.8
4.6
5.7
7.1
10.0
2.7
2.2
9.2
10.0
59
54
104
>100
20
27
146
>100
2c
3f
3g
References and Notes
Table 4. ECMA IC50s for compounds 1, 2c, 3f, and 3g
1. Adamis, A. P.; Shima, D. T.; Yeo, K. T.; Yeo, T. K.;
Brown, L. F.; Berse, B.; D’Amore, P. A.; Folkman, J. Bio-
chem. Biophys. Res. Commun. 1993, 193, 631.
2. Giatromanolaki, A.; Sivridis, E.; Athanassou, N.; Zois, E.;
Thorpe, P. E.; Brekken, R. A.; Gatter, K. C.; Harris, A. L.;
Koukourakis, I. M.; Koukourakis, M. I. J. Path. 2001, 194, 101.
3. Detmar, M. Dermatol. Sci. 2000, 24, S78.
Compd
KDR IC50 (nm)
ECMA IC50 (nm)
Log P17
1
224
29
24
19
622
150
127
387
2.5
4.0
—
> >4.0
2c
3f
3g
4. For reviews, see (a) Carmeliet, P.; Jain, R. K. Nature 2000,
407, 249. (b) Folkman, J. Nature Med. 1995, 1, 27.
5. (a) Hanahan, D.; Folkman, J. Cell 1996, 86, 353. (b)
Holmgen, L.; O’Reilly, M. S.; Folkman, J. Nature Med. 1995,
1, 149.
6. (a) Veikkola, T.; Karkkainen, M.; Claesson-Welsh, L.;
Alitalo, K. Cancer Res. 2000, 60, 203. (b) Thomas, K. A.
J. Biol. Chem. 1996, 271, 603.
7. Kim, K. J.; Li, B.; Winer, J.; Armanini, M.; Gillett, N.;
Phillips, H. S.; Ferrara, N. Nature 1993, 362, 841.
8. Witte, L.; Hicklin, D. J.; Zhu, Z.; Pytowski, B.; Kotanides, H.;
Rockwell, P.; Bohlen, P. Cancer Metastasis Rev. 1998, 17, 155.
9. (a) Fong, T. A. T.; Shawyer, L. K.; Sun, L.; Tang, C.; App,
H.; Powell, T. J.; Kim, Y. H.; Schreck, R.; Wang, X.; Risau,
W.; Ullrich, A.; Hirth, K. P.; McMahaon, G. Cancer Res.
1999, 59, 99. (b) Drevs, J.; Hofmann, I.; Hugenschmidt, H.;
Wittig, C.; Madjar, H.; Muller, M.; Wood, J.; Martiny-Baron,
G.; Unger, C.; Marme, D. Cancer Res. 2000, 60, 4819.
10. For a current review, see Sun, L.; McMahon, G. Drug
Discov. Today 2000, 5, 344.
11. (a) Muhmel, G.; Hanke, R.; Breitmaier, E. Synthesis 1982,
8, 673. (b) Reichardt, C.; Ferwanah, A.-R.; Pressler, W.; Yun,
K.-Y. Liebigs Ann. Chem. 1984, 649.
12. All target compounds were fully characterized by 1H
NMR and mass spectroscopy.
13. The KDR IC50 value represents biochemical inhibition of
phosphorylation of a poly-Glu/Tyr (4:1) peptide substrate by
isolated KDR kinase (cloned and expressed as a GST-fusion
protein): see, Kendall, R. L.; Rutledge, R. Z.; Mao, X.;
Tebben, A. J.; Hungate, R. W.; Thomas, K. A. J. Biol. Chem.
1999, 274, 6453.
and SRC kinase (Table 3). The data, expressed as the
biochemical IC50 ratios to KDR kinase, indicate that
this set of inhibitors is relatively non-selective for KDR
versus PDGFRb, FLT-1, and FLT-4 kinases, but mod-
erately KDR selective versus FGFR-1 and SRC kinases.
The data also suggest that the 3-thienyl ring at the
3-position of the pyrazolo[1,5-a]pyrimidine enhances
KDR selectivity in comparison to the 4-pyridyl ligand.
The ability of compounds 1, 2c, 3f, and 3g to inhibit
VEGF-stimulated mitogenesis was determined in
human umbilical vein endothelial cells. The inhibition
constants determined in the biochemical assay are com-
pared to those measured in the endothelial cell mito-
genesis assay (ECMA) in Table 4. Compounds 1, 2c,
and 3f, which bear a solubilizing 4-pyridyl substituent,
demonstrated a lower ratio of cell to biochemical activ-
ity compared to compound 3g, whose activity may be
limited by poor cell penetration due to unfavorable
physical properties.
In conclusion, we have identified a new and syntheti-
cally accessible class of potent KDR kinase inhibitors
comprised of a pyrazolo[1,5-a]pyrimidine core. Within
this series, potent kinase activity is limited to 3,6-dis-
ubstituted analogues that may adopt planar orientations.
14. Traxler, P.; Furet, P. Pharmacol. Ther. 1999, 82, 195.
15. Fiaud, J.; Malleron, J. Tetrahedron Lett. 1980, 21, 4437.
16. Winters, G.; Sala, A.; DePaoli, A.; Conti, M. Synthesis
1984, 1050.
Acknowledgements
We thank Dr. Art Coddington, Dr. Chuck Ross, and
Dr. Harry Ramjit for mass spectral analyses, Matt
17. Partition coefficients were determined in octanol/water.