7-Aminopyrazolo[1,5-a]pyrimidines as potent multitargeted receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1021/jm701397k

7-Aminopyrazolo[1,5-a]pyrimidine urea receptor tyrosine kinase inhibitors have been discovered. Investigation of structure-activity relationships of the pyrazolo[1,5-a]pyrimidine nucleus led to a series of 6-(4-N,N′-diphenyl) ureas that potently inhibited

Full text of DOI:10.1021/jm701397k

J. Med. Chem. 2008, 51, 3777–3787
3777
7-Aminopyrazolo[1,5-a]pyrimidines as Potent Multitargeted Receptor Tyrosine Kinase
Inhibitors
Robin R. Frey,* Michael L. Curtin, Daniel H. Albert, Keith B. Glaser, Lori J. Pease, Niru B. Soni, Jennifer J. Bouska,
David Reuter, Kent D. Stewart, Patrick Marcotte, Gail Bukofzer, Junling Li, Steven K. Davidsen, and Michael R. Michaelides
Global Pharmaceutical Research and DeVelopment, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064-6100
ReceiVed NoVember 7, 2007
7-Aminopyrazolo[1,5-a]pyrimidine urea receptor tyrosine kinase inhibitors have been discovered. Investigation
of structure-activity relationships of the pyrazolo[1,5-a]pyrimidine nucleus led to a series of 6-(4-N,N′-
diphenyl)ureas that potently inhibited a panel of vascular endothelial growth factor receptor (VEGFR) and
platelet-derived growth factor receptor (PDGFR) kinases. Several of these compounds, such as 34a, are
potent inhibitors of kinase insert domain-containing receptor tyrosine kinase (KDR) both enzymatically
(<10 nM) and cellularly (<10 nM). In addition, compound 34a possesses a favorable pharmacokinetic
profile and demonstrates efficacy in the estradiol-induced murine uterine edema (UE) model (ED50 ) 1.4
mg/kg).
Introduction
VEGF promotes angiogenesis through binding to a family of
homologous VEGF receptors, including KDR (VEGFR-2), Flt-1
(VEGFR-1), and Flt-4 (VEGFR-3). Likewise, PDGF binds to
the PDGFR family of receptors, Flt-3, PDGFRꢀ, cKit, and
CSF1R (colony-stimulating factor 1 receptor). The PDGF
receptors not only trigger angiogenesis but also contribute to
tumor growth.12
Protein kinase signaling is a predominant means of signal
transduction in eukaryotic cells and controls processes such as
proliferation, migration, survival, and cell cycle progression.
Misregulation of these tightly controlled processes through
mutation or overexpression of kinases is implicated in a number
of disease states including cancer and immunological disorders.1,2
Early work in the field of receptor tyrosine kinase inhibitors
was directed toward the identification of agents with a high
degree of selectivity for a particular enzyme.13,14 This led to
the discovery of first-generation RTK inhibitors, including
compounds such as SU5416 ((Z)-3-((3,5-dimethyl-1H-pyrrol-2-yl)-
methylene)indolin-2-one),15,16 a small molecule that is selective
for VEGFR. Later research, however, suggests that compounds
targeting a broader range of RTKs may lead to a more robust
antitumor response, as there are a number of redundant RTK-
mediated processes that can promote angiogenesis.17,18 Fur-
thermore, recent data show that tumors treated with selective
agents can develop resistance through the up-regulation of
alternative kinase-mediated pathways.19 This has led to the
development of broader-spectrum RTK inhibitors, including two
recently launched anticancer drugs, SU11248 ((Z)-N-(2-(diethyl-
amino)ethyl)-2,4-dimethyl-5-((5-methyl-2-oxoindolin-3-ylidene)-
methyl)-1H-pyrrole-3-carboxamide),20 an inhibitor of KDR, Flt-
1, PDGFR, and c-Kit, and BAY 43-9006 (N-[4-chloro-3-
(trifluoromethyl)phenyl]-N′-[4-[2-(N-methylcarbamoyl)-4-
pyridyloxy]phenyl]urea),21 an inhibitor of Raf kinase, as well
as of the VEGFR, EGFR, and PDGFR kinases.
Angiogenesis, the process by which new capillaries are
formed from existing blood vessels, is essential for a tumor to
grow beyond about 1-2 mm.3 As a tumor becomes larger, it
becomes increasingly hypoxic, leading to induction of growth
factors, including vascular endothelial growth factor (VEGFa),1
which trigger angiogenesis.4,5 Once angiogenesis has been
induced, the tumor becomes highly vascularized and can grow
at a much more rapid pace.6 Although VEGF-stimulated
angiogenesis is crucial for embryonic neovascularization,7 in
adults its normal physiologic roles are largely restricted to
reproductive function and wound healing,8 suggesting that
inhibition of angiogenesis may present an opportunity for the
selective treatment of cancer.9 Approval by the U.S. Food and
Drug Administration of the anti-VEGF antibody bevacizumab,10
along with the clinical efficacy shown by this agent, has further
heightened interest in identifying small-molecule inhibitors of
angiogenic kinases.
VEGF, as well as other proangiogenic growth factors includ-
ing platelet-derived growth factor (PDGF), acts through binding
to receptor tyrosine kinases (RTKs), a family of transmembrane
proteins. Upon binding the growth factor, RTKs dimerize and
undergo autophosphorylation, triggering a series of downstream
events leading to proliferation, migration, and cell survival.11
Our laboratories have been engaged in research directed
toward multitargeted RTK inhibitors, including thienopyrim-
idines,22 isoindolinone ureas,23 and aminoindazole ureas includ-
ing 1 (Figure 1).24 In light of the efficacy observed for RTK
inhibitors in the clinic,25 we are continuing our efforts to identify
structurally novel kinase inhibitor classes. Rational design efforts
in our laboratories identified aminoquinoxaline 2, which was a
submicromolar inhibitor of KDR (IC50 ) 168 nM). Brief SAR
study based on the quinoxaline scaffold failed to generate
compounds with substantially improved potency, so we sought
other templates that might exploit a binding mode similar to
that proposed for compound 2, in which the biarylurea extends
into the hydrophobic pocket and the bicyclic core forms
* To whom correspondence should be addressed. Phone: (847) 938-2517.
Fax: (847) 935-5165. E-mail: robin.r.frey@abbott.com.
a Abbreviations: ATP, adenosine 5′ triphosphate; CSF1R, colony-
stimulating factor-1; DMF-DMA, dimethylformamide dimethyl acetal;
EDCI, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride;
EGFR, endothelial growth factor receptor; Flt-1, fms-like tyrosine kinase-
1; Flt-3, fms-like tyrosine kinase 3; KDR, kinase insert domain-containing
receptor tyrosine kinase; PDGF, platelet-derived growth factor; PDGFR,
platelet-derived growth factor receptor; RTK, receptor tyrosine kinase; SAR,
structure-activity relationship; UE, estradiol-induced murine uretine edema
assay; VEGF, vascular endothelial growth factor; VEGFR, vascular
endothelial growth factor receptor.
10.1021/jm701397k CCC: $40.75
2008 American Chemical Society
Published on Web 06/17/2008

3778 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Frey et al.
Scheme 1. Synthesis of 7-Aminopyrazolo[1,5-a]pyrimidines 6
and 7 via Nitrationa
Figure 1. Compound 1.
a Conditions: (a) concentrated HCl, EtOH, reflux, 74%; (b) HNO3, H2SO4,
-20 °C to room temp; (c) Fe0, NH4Cl, THF, EtOH, H2O, 80 °C, 29%
(two-step yield); (d) m-tolyl isocyanate, DMF, -20 °C to room temp, 47%;
(e) benzoic acid, EDCI, HOBt, N-methylmorpholine, DMF, 60%.
Figure 2. Lead structure 2 and pyrazolo[1,5-a]pyrimidine 14.
Suzuki or Sonogoshira couplings. A variety of catalysts as well
as high-temperature microwave reaction conditions were em-
ployed,29 but these compounds would not undergo palladium-
catalyzed coupling reactions even following Boc protection of
the amino functionality. Similar couplings have been reported
for 3-bromo pyrazolo[1,5-a]pyrimidines lacking the 7-amino
substituent,30 and we observed that compound 41 (Table 4)
underwent facile Suzuki coupling with thiophene-3-boronic acid.
Upon discovering that bromide 3 could not be elaborated
through palladium-catalyzed cross-coupling reactions, it became
necessary to find an alternative route that would allow for the
substitution of a broad range of substituents on the pyrazole
and that would also incorporate a nitro group on the 6-phenyl
ring prior to the cyclocondensation, thereby alleviating the need
to perform a nitration on a more highly functionalized system.
The optimized synthesis of 7-aminopyrazolo[1,5-a]pyrimidines
incorporating a diphenylurea substituent in the 6-position is
typified by the preparation of compound 14, shown in Scheme
3. 4-Nitrophenylacetonitrile was treated with dimethylforma-
mide-dimethyl acetal (DMF-DMA) in refluxing toluene to give
the dimethyl enamine 11. Acid-catalyzed cyclocondensation of
11 with 3-aminopyrazole led to the formation of pyrazolo-
[1,5-a]pyrimidine 12. Nitro reduction was followed by treatment
of the resulting aniline 13 with m-tolyl isocyanate to complete
the synthesis of urea 14.
hydrogen bonds to the hinge region of the enzyme as shown in
Figure 2.26 Modification of the aminoquinoxaline lead structure
led to the identification of a series of 7-aminopyrazolo[1,5-a]-
pyrimidine inhibitors of KDR such as 14, which demonstrated
improved potency. In this paper, we describe the synthesis and
characterization of this novel series of 7-aminopyrazolo[1,5-a]-
pyrimidines as potent inhibitors of the VEGFR and PDGFR
tyrosine kinases.27
Chemistry
Our initial synthetic route, shown in Scheme 1, began with
the cyclocondensation of 3-amino-4-bromopyrazole with 3-oxo-
2-phenylpropanenitrile under acidic conditions to give the fused
bicyclic aromatic heterocycle 3.28 This compound was then
subjected to electrophilic aromatic nitration yielding a mixture
of nitration products favoring the para isomer, followed by
reduction of the nitro group to give aniline 5, which was treated
with m-tolyl isocyanate to give the urea 6. Similar conditions
were used to prepare urea derivatives 6a-c (Table 3); amide 7
(Table 1) was prepared by an amide coupling of 5 with benzoic
acid. The 7-des-amino analogue 10 was prepared in an
analogous fashion from 2-phenylmalonaldehyde, as shown in
Scheme 2. Compounds 4 and 41 (Tables 1 and 4, respectively),
which bear a 4-methoxy group in place of the urea, were
prepared using cyclocondensation conditions similar to those
described in Schemes 1 and 2, respectively, starting with
commercially available 3-(4-methoxyphenyl)-3-oxopropaneni-
trile.
Compounds bearing a substituent at the 3-position of the
pyrazolo[1,5-a]pyrimidine nucleus were prepared via a similar
route, using 4-substituted 3-aminopyrazoles in place of 3-ami-
nopyrazole in the cyclocondensation step. 3-Aminopyrazole
derivatives that were not available from commercial sources
were prepared through synthetic sequences based on the route
exemplified for compound 19 in Scheme 4. N-Methylpyrazole
was formylated according to the Vilsmaier-Haack protocol, and
the resulting aldehyde 15 was reduced to the alcohol 16 using
sodium borohydride. Conversion to the chloromethyl derivative
It was envisioned that the bromine atom in compound 3 would
serve not only as a blocking group to prevent potential nitration
at the 3-position of the pyrazole but also as a handle allowing
for subsequent functionalization of the molecule using pal-
ladium-catalyzed coupling chemistry. In practice, however,
neither the bromide 3 nor the analogous iodide would undergo

Pyrimidines as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3779
Table 1. KDR Inhibitory Activity of Initial Pyrazolo[1,5-a]pyrimidine
Analogues
Scheme 3. Synthesis of 7-Aminopyrazolo[1,5-a]pyrimidine Urea
14 from 4-Nitrophenylacetonitrilea
a Conditions: (a) CH(OCH3)2N(CH3)2, toluene, reflux, 95%; (b) 3-ami-
nopyrazole, concentrated HCl, EtOH, reflux, 86%; (c) H2 (1 atm), Pd/C,
MeOH, 94%; (d) m-tolyl isocyanate, DMF, -20 °C to room temp, 75%.
Scheme 4. Synthesis of C3-Substituted
7-Aminopyrazolo[1,5-a]pyrimidine Urea 34a
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate.
Scheme 2. Synthesis of 7-des-Aminopyrazolo[1,5-a]pyrimidine
Urea 10a
a Conditions: (a) POCl3, DMF, 0-70 °C, 79%; (b) NaBH4, MeOH, 92%;
(c) SOCl2, CHCl3, reflux; (d) NaCN, DMSO, 43% (two-steps); (e) HCO2Et,
NaH, catalytic EtOH, toluene; (f) H2NNH2 ·H2O, concentrated HCl, EtOH,
reflux, 45% (two steps); (g) 11, concentrated HCl, EtOH, reflux, 79%; (h)
SnCl2, HCl, 0 °C to room temp, 80%; (i) m-tolyl isocyanate, DMF, -20
°C to room temp, 80%.
commercially available acetonitrile derivatives, as shown for
the conversion of 17 to 20 in Scheme 4. Compounds 26 and 27
(Table 2), which contain substituents at the 2-position, were
prepared from 3-amino-5-methylpyrazole and 3-amino-5-phe-
nylpyrazole, and these starting materials were synthesized
according to published procedures.31
a Conditions: (a) concentrated HCl, EtOH, reflux, 93%; (b) HNO3, H2SO4,
-20 °C to room temp; (c) Fe0, NH4Cl, THF, EtOH, H2O, 80 °C, 34%
(two-step yield); (d) m-tolyl isocyanate, DMF, -20 °C to room temp, 51%.
Compound 28 (Table 2), which bears a methyl group at the
5-position of the bicyclic core, was prepared by substituting
dimethylacetamide-dimethyl acetal for DMF-DMA when form-
ing the cyanoenamine (Scheme 5). Compounds 38-40 incor-
porating an amide substituent in the 3-position of the pyrazolo-
[1,5-a]pyrimidine ring were prepared by saponification of ester
36 followed by EDCI-mediated coupling of the resulting acid
37 with various amines (Scheme 6).
by treatment with thionyl chloride was followed by displacement
of the chloride with sodium cyanide to give acetonitrile
derivative 17. This compound was then formylated under basic
conditions, and the resulting oxopropionitrile derivative 18 was
condensed with hydrazine to give 4-substituted 3-aminopyrazole
19. Cyclocondensation with compound 11 as previously de-
scribed gave the 7-aminopyrazolo[1,5-a]pyrimidine bearing the
N-methylpyrazole substituent in the 3-position. Reduction of
the nitro group to the aniline 20 followed by treatment with
isocyanate provided the urea 34. Compounds 29-33 (Table 2)
bearing substituents in the 3-position were prepared from
Biology
Compounds were assayed against a panel of receptor tyrosine
kinases that included a KDR assay run in the presence of 1

3780 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Frey et al.
Table 2. Substitutions at the 2, 3, and 5 Positions of the
7-Aminopyrazolo[1,5-a]pyrimidines
Table 3. Substitutions at the Terminal Benzene Ring of the of
N,N′-Diarylureas
compd
X
R
KDR (IC50, nM)a KDR (cell) (IC50, nM)b
6
6a
6b
6c
Br 3-CH3
Br 2-CH3
Br 4-CH3
72
86
>12500
810
1600
47
9
44
Br
H
H
H
H
H
H
3-CH3
3-CF3
3-Cl
2-F, 5-CH3
2-F, 5-CF3
14
70
35
14a
14b
14c
14d
74
14
70
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate. b Each cellular IC50
determination was performed with five concentrations, and each assay point
was determined in duplicate.
Table 4. Comparison of the SAR of 7-Amino- and
7-des-Aminopyrazolo[1,5-a]pyrimidine KDR Inhibitors
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate. b Compound reported in ref
34.
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate. b Each cellular IC50
determination was performed with five concentrations, and each assay point
was determined in duplicate.
essential for potency in this series, as evidenced by the loss of
potency shown for non-ureas 3-5 when compared to urea 6
(Table 1). Replacement of the diphenylurea of 6 with a
benzamide (7) was also not tolerated, consistent with our model
suggesting that both of the N-H moieties of the urea form
hydrogen bonds to the enzyme (vide infra). Likewise, thiourea
24 demonstrated substantially diminished potency compared to
the analogous urea 14, and the meta-linked diphenylurea (23)
also led to potency loss.
Substitution about the pyrazolopyrimidine core generated a
distinct SAR (Table 2). Addition of a methyl or phenyl group
in the 2-position (26, 27) led to a loss of potency, consistent
with the hypothesis that N-1 is acting as a hinge binder.
Incorporation of a methyl group in the 5-position (28) was
tolerated, although this led to diminished potency in the KDR
cellular assay in comparison with compound 14, which lacks
this substituent. A variety of substitutions at the 3-position were
tolerated, and in many cases these compounds exhibited
mM ATP, which is reportedly a physiologically relevant
concentration.32 Active compounds were then further character-
ized using a cellular KDR assay, and compounds showing
promise in that assay progressed to in vivo studies (vide infra).
Compound 6 was prepared as the initial compound in the
series and was found to be a nanomolar inhibitor of KDR (Table
1). Compound 10, an analogue lacking the 7-amino group, was
inactive, a finding consistent with the participation of this group
in the hinge-binding motif proposed in Figure 2. Removal of
the 3-bromo substituent of 6 to give 14 provided a roughly
equipotent compound and suggested that this position might be
amenable to substitution.
Consistent with the thienopyrimidine and isoindolinone urea
series,22,23 the 4-diphenylurea substituent in the 6-position was

Pyrimidines as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3781
Scheme 5. Synthesis of C5-Substituted
7-Aminopyrazolo[1,5-a]pyrimidine 28a
a Conditions: (a) CH3C(OCH3)2N(CH3)2, toluene, reflux, 76%; (b)
3-aminopyrazole, concentrated HCl, EtOH, reflux, 46%.
Figure 3. Model of compound 6 bound to KDR kinase. Hydrogen
bonds in black are shown between the urea and Glu 885 carboxylate,
between the exocyclic amine and Glu 917 backbone carbonyl, and
between the ring nitrogen and Cys 919 N-H. Also in thick bond are
residues Asp 1046-Phe 1047 of the “DFG” motif in the “inactive”
conformation (“DFG-out”).
Scheme 6. Synthesis of Compounds 36-38a
a Conditions: (a) LiOH, H2O, THF, MeOH, 70 °C 84%; (b) MeNH2 ·HCl,
EDCI, HOBt, NMM, DMF, 56%.
improved potency. In general, aryl, heteroaryl, and alkyl groups
were favored over more polar groups. Both the 2- and
3-thiophene isomers 29 and 30 were roughly equipotent and
were not significantly different from the phenyl analogue 25.
Other hydrophobic groups in the 3-position, including the
smaller cyclopropyl (31) and larger methylenedioxyphenyl (32),
also showed potency in the 15-25 nM range in the KDR
enzyme assay. The most potent compounds in the KDR cellular
assay incorporated nitrogen heterocycles in the 3-position,
including 3-pyridine 33 and N-methylpyrazole 34. These
compounds exhibited cellular IC50 values in the single-digit
nanomolar range. Compounds such as nitrile 35, acid 37, and
amides 38-40, which bear polar substituents in the 3-position,
were considerably weaker inhibitors, although the ester 36
retained potency similar to aryl compounds such as 25,
presumably because of its somewhat more hydrophobic character.
The SAR of substitution about the terminal ring of the urea
was similar to that observed for the thienopyrimidine urea,
isoindolinone urea, and aminoindazole series studied previously.
Substitution at the 3-position was required for optimal potency,
as evidenced by the strongly enhanced potency of the 3-methyl
analogue 6 when compared with the 2- and 4-methyl compounds
6a and 6b and the unsubstituted phenyl analogue 6c (Table 3).
Further exploration of des-bromo compounds incorporating a
3-substituent on the terminal phenyl ring revealed that the
trifluoromethyl group in compounds 14a and 14d was preferred
over the methyl group in compounds 14 and 14c. The 3-chloro
analogue 14b was equipotent with the methyl analogue 14.
Figure 4. Comparison of proposed binding modes for compounds 4334
and 6.
group and the backbone carbonyl of Glu 917 and between the
pyrazolo ring nitrogen and the backbone N-H of Cys 919. This
is supported by the observation that des-amino compound 10,
an analogue of 6 that lacks a 7-amino group, does not inhibit
KDR at measured concentrations. Consistent with the observed
SAR for compounds 26 and 27, there does not appear to be
room for substitution at C-2 of the pyrazolo ring.
Researchers at Merck have also described a series of
pyrazolo[1,5-a]pyrimidines as inhibitors of KDR, including
compound 43 (Table 4).30,34 In order to establish whether these
7-amino pyrazolo[1,5-a]pyrimidines are binding in the same
fashion as the previously reported compounds, hybrid com-
pounds incorporating features of both the Abbott compounds
reported in this paper and compounds from the Merck series
were prepared. As shown in Table 4, it appears that these two
series of compounds must be binding in distinctly different
fashions with the enzyme. Introduction of a 7-amino group onto
compound 43 to give 42 results in a striking loss of potency.
Conversely, removal of the 7-amino group from compound 6
to give compound 10 in the urea series results in loss of
measurable potency. Furthermore, Merck researchers report that
compounds in their series lacking an aryl or heteroaryl group
in the 3-position are inactive, as shown by comparison of
3-thienyl compound 43 to the 3-bromo compound 41. In the
7-amino series, however, 3-bromo compound 6 is roughly
equipotent with 3-thienyl analogue 30. These findings are
consistent with the contrasting binding modes proposed for the
two series based on homology modeling, as shown in Figure 4.
Although N-1 of the pyrazolo[1,5-a]pyrimidine is thought to
form a hydrogen bond with Cys 919 of the hinge region in both
binding modes, the 7-amino inhibitor 6 appears to be “flipped”
in comparison with the des-amino compound 43 and also forms
A plausible model of the binding mode of 6 in the active
site of KDR was prepared using a method previously reported
and is shown in Figure 3.22 The urea portion of the inhibitor
was docked as reported for another urea-based inhibitor33 with
hydrogen bonds between the inhibitor carbonyl and the Asp
1045 backbone N-H and between the inhibitor urea N-H
groups and the side chain carboxylate of Glu 885. The
pyrazolopyrimidine ring system fit well into the canonical
adenosine-binding portion of the ATP-binding site, with hy-
drogen bonding interactions occurring between the 7-amino

3782 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Frey et al.
Table 5. KDR Enzymatic and Cellular Inhibitory Activity and in Vivo
Oral Uterine Edema Inhibitory Activity of 4-Methylpyrazole-Substituted
Pyrazolo[1,5-a]pyrimidine Ureas
The 7-aminopyrazolo[1,5-a]pyrimidines herein described are
potent inhibitors of KDR, and they also display significant
inhibition of other VEGFR and PDGFR kinase family members,
as shown in Table 7. Compound 34a is a single-digit-nanomolar
inhibitor of the VEGFR kinases KDR and Flt-1 and also of the
PDGFR kinases Flt-3, c-Kit, and CSF1R.38
Conclusion
compd
R
KDR (IC50, nM)a KDR (cell) (IC50, nM)b UEc (mg/kg)
49% @ 10d
ED50 ) 1.4e
27% @ 10d
45% @ 10d
inactivef
Novel 7-aminopyrazolo[1,5-a]pyrimidine inhibitors of VEG-
FR and PDGFR kinases have been discovered. These com-
pounds display an SAR profile that is distinct from the
pyrazolo[1,5-a]pyrimidine KDR inhibitors previously reported
by other researchers. Modeling and SAR studies suggest that
these compounds bind in the ATP pocket of the enzyme, with
the 7-aminopyrazolo[1,5-a]pyrimidine core forming a bidentate
hydrogen bonding interaction with the hinge region and the
N,N′-diarylurea occupying the hydrophobic pocket. Optimal
potency was obtained by incorporation of an N,N′-diarylurea
moiety at the 6-position, with the substituents in a para
orientation on the 6-phenyl ring and a small meta substituent
on the terminal phenyl ring of the urea. Additional substitution
at the 3-position of the pyrazolo[1,5-a]pyrimidine core led to
compounds with enhanced activity in the cellular KDR assay,
and the placement of an N-methylpyrazole linked through C-4
in this position provided compounds typified by 34a, which
exhibited single-digit nanomolar inhibition of KDR in both the
enzymatic and cellular assays and was found to have oral
efficacy in the estradiol-induced murine uterine edema assay,
an in vivo model of VEGF-induced vascular permeability.
34 3-CH3
34a 3-CF3
34b 3-Cl
34c 2-F, 5-CF3
34d 3-CF3-4-F
34e 3-F
4
3
4
5
6
1
0.7
8
28
55
29
10
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate. b Each cellular IC50
determination was performed with five concentrations, and each assay point
was determined in duplicate. c Estradiol-induced murine uterine edema assay.
d Values are expressed as percent inhibition @ mg/kg. e ED50 values in
mg/kg. f “Inactive” indicates the compound provided <15% inhibition at
10 mg/kg.
a second hydrogen bond through the 7-amino group. In the
7-amino series, it is proposed that the 6-substituent occupies
the hydrophobic pocket, whereas in the des-amino series, the
3-substituent is thought to occupy this pocket. Modeling studies
and SAR both suggest that the 7-amino series is indeed binding
in a distinct binding mode from the previously reported series.
This result is consistent with previous reports of kinase inhibitors
exhibiting multiple binding modes.35
Compounds showing potency of <100 nM in the KDR
enzymatic and cellular assays were evaluated in the estradiol-
induced murine uterine edema (UE) assay, an in vivo model of
VEGF-induced vascular permeability.36 Mice (six per group),
which had been primed with pregnant mare’s serum 1 and 2
days prior to the assay, were given a KDR inhibitor orally and
challenged with estradiol 30 min later. Edema was assessed 2 h
following estradiol administration, comparing the uterine weights
of the inhibitor-treated animals to those of estradiol-treated and
untreated controls.37 This model requires a minimal amount of
compound and provides a medium-throughput in vivo assess-
ment of KDR inhibition following a single oral dose and was
used as a screening tool to identify compounds that would
progress to in vivo tumor growth inhibition assays.
In the uterine edema assay, the most potent compounds in
this series bear an N-methylpyrazole substituent in the 3-position
(Table 5). A number of compounds incorporating meta substit-
uents on the terminal phenyl ring were single-digit nanomolar
inhibitors of KDR in the enzymatic assay, although in the
cellular KDR assay the methyl and trifluoromethyl compounds
34 and 34a were found to be somewhat more potent than the
chloro and fluoro compounds 34b and 34e or the trifluoromethyl
compounds 34c,d incorporating an additional fluoro substituent
on the ring. Compound 34a, with an ED50 of 1.4 mg/kg in the
uterine edema model, was the most potent compound evaluated
in this series and had efficacy comparable to that observed for
the best compounds in the thienopyrimidine series.22 This
compound also exhibited a favorable pharmacokinetic profile
characterized by low plasma clearance, high exposure, a half-
life of 2.2 h, and oral availablility in the mouse (Table 6).
Compared to m-tolylurea 34, 3-trifluoromethylphenylurea 34a
showed a 6-fold reduction in clearance following iv administra-
tion (3.7 vs 0.57), as well as a dramatic enhancement in exposure
following oral dosing. This improved clearance and the corre-
sponding enhanced exposure may correlate with the boost in
potency observed for compound 34a in the uterine edema model.
Experimental Section
Chemistry. 1H NMR spectra were recorded on a 300 MHz
spectrometer (Nicolet QE-300 or General Electric GN-300) if not
otherwise indicated, and chemical shifts are reported in parts per
million (ppm, δ) relative to tetramethylsilane as an internal standard.
Mass spectra were obtained on a Finnigan MAT SSQ700 instru-
ment. The above structural data were obtained through the
Department of Structural Chemistry, Abbott Laboratories. Elemental
analyses were performed by Robertson Microlit Laboratories, Inc.,
Madison, NJ, or by Quantitative Technologies Inc., Whitehouse,
NJ, and the results indicated by elemental symbols are within
(0.4% of theoretical values unless otherwise indicated. Preparative
column chromatography was performed using silica gel 60 (E.
Merck, 230-400 mesh) or, when indicated, using an Analogix
IntelliFlash 280 chromatographic purification system and Analogix
prefilled silica gel cartridges as described.
3-Bromo-6-phenylpyrazolo[1,5-a]pyrimidin-7-amine (3). 3-
Amino-4-bromopyrazole (4.00 g, 24.7 mmol), 3-oxo-2-phenylpro-
panenitrile (3.60 g, 24.8 mmol), and concentrated HCl (8 mL) were
combined in EtOH (150 mL). The mixture was heated to reflux
for 16 h, then cooled to room temperature. The mixture was
concentrated to dryness on a rotary evaporator, and the residue was
dissolved in H2O (100 mL). An amount of 1 N NaOH was added
until the mixture reached pH 6, and the resulting white solid was
collected by filtration, rinsing with H2O, to give 3 (6.17 g, 86%).
1H NMR (300 MHz, DMSO-d6) δ 8.29 (s, 1H), 8.17 (s, 1H), 7.70
(s, 2H), 7.48-7.54 (m, 4H), 7.37-7.45 (m, 1H); MS (ESI) m/z
288.8, 290.8 [M + H]+. Anal. (C12H9BrN4 ·0.1EtOH) C, H, N.
6-(4-Aminophenyl)-3-bromopyrazolo[1,5-a]pyrimidin-7-amine
(5). Compound 3 (5.75 g, 19.9 mmol) was dissolved in concentrated
H2SO4 (70 mL), and the solution was chilled in a -20 °C cooling
bath. A solution of concentrated HNO3 (1.25 mL, 20 mmol) in
concentrated H2SO4 (5 mL) was added dropwise, and the mixture
was allowed to warm slowly to room temperature and stirred for
2 h. The mixture was then poured carefully over ice (800 g) and
adjusted to pH 6 by the addition of 5 N NaOH. A yellow precipitate
formed and was collected by filtration, providing a crude nitration

Pyrimidines as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3783
Table 6. Pharmacokinetic Data for Pyrazolo[1,5-a]pyrimidine Ureas 34 and 34aa
ivb
poc
compd
t1/2 (h)
Cl (mL/(min ·kg))
AUC (µg·h/mL)
Vd (L/kg)
Cmax (µM)
AUC (µg·h/mL)
F (%)
34
34a
0.25
2.2
3.7
0.57
1.8
10.6
1.4
1.8
0.36
5.7
0.81
41.0
14
∼100
a Mouse pharmacokinetics. b Dosed at 3 mg/kg in a vehicle comprising 2.5% DMSO, 2.5% Tween-80, 25% PEG400 in PBS. c Dosed at 10 mg/kg in a
vehicle comprising 2.5% EtOH, 5% Tween-80, 25% PEG400 in PBS.
Table 7. Kinase Inhibition Profile of Pyrazolo[1,5-a]pyrimidine Urea
(t, J ) 7.5 Hz, 2H), 6.98 (t, J ) 7.3 Hz, 1H); MS (ESI) m/z 423.0,
425.0 [M + H]+. Anal. (C19H15BrN6O) C, H, N.
34aa
VEGFR kinases (IC50, nM)
PDGFR kinases (IC50, nM)
N-(4-(7-Amino-3-bromopyrazolo[1,5-a]pyrimidin-6-yl)phenyl)-
benzamide (7). A solution of 5 (100 mg, 0.33 mmol), benzoic acid
(41 mg, 0.33 mmol), EDCI (78 mg, 0.41 mmol), HOBt (54 mg,
0.40 mmol), and N-methylmorpholine (0.18 mL, 1.5 mmol) in DMF
(2 mL) was stirred overnight at room temperature, then diluted with
H2O (50 mL). The mixture was extracted with EtOAc (3 × 25
mL), and the organic layers were dried (Na2SO4) and concentrated
to dryness. The residue was triturated with CH2Cl2 and the resulting
solid was collected by filtration, rinsing with CH2Cl2, to give 7 as
an off-white solid (80 mg, 60%). 1H NMR (300 MHz, DMSO-d6)
δ 10.38 (s, 1H), 8.28 (s, 1H), 8.18 (s, 1H), 7.91-8.01 (m, 4H),
7.70 (br s, 2H), 7.47-7.63 (m, 5H); MS (ESI) m/z 408.0, 410.0
[M + H]+. Anal. (C19H15BrN6O·0.3H2O) C, H, N.
compd
KDR
3
Flt-1
3
FGFR
Flt-3
5
c-Kit
9
CSF-1R
4
34a
>12500
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate.
product. MS (ESI) m/z 334.0, 335.9 [M + H]+. This material was
taken up in EtOH (200 mL), THF (80 mL), and H2O (40 mL), and
the mixture was heated to 70 °C, whereupon Fe0 (11.1 g, 199 mmol)
and NH4Cl (1.06 g, 19.8 mmol) were added. The mixture was stirred
at 70 °C overnight, then cooled to room temperature and filtered,
rinsing with MeOH. The filtrate was concentrated to dryness, and
the residue was partitioned between H2O (500 mL) and CH2Cl2
(500 mL). The aqueous layer was further extracted with CH2Cl2
(2 × 250 mL), and the combined extracts were dried (Na2SO4)
and concentrated onto silica gel (50 g). The silica was placed atop
a flash chromatography column and eluted with EtOAc/CH2Cl2
(gradient elution 4-10% EtOAc) to give 5 as a light-orange solid
(1.68 g, 28%). 1H NMR (300 MHz, DMSO-d6) δ 8.23 (s, 1H),
8.08 (s, 1H), 7.45-7.41 (br s, 2H), 7.13 (d, J ) 8.5 Hz, 2H), 6.68
(d, J ) 8.5 Hz, 2H), 5.25-5.24 (br s, 2H); MS (ESI) m/z 303.9,
305.9 [M + H]+. Anal. (C12H10BrN5 ·0.2CH2Cl2) C, H, N.
1-(4-(7-Amino-3-bromopyrazolo[1,5-a]pyrimidin-6-yl)phenyl)-
3-m-tolylurea (6). A solution of 5 (100 mg, 0.33 mmol) in DMF (1
mL) was chilled to -20 °C, and m-tolyl isocyanate (0.045 mL,
0.35 mmol) was added dropwise. The mixture was allowed to warm
to room temperature and stirred overnight, and then H2O (20 mL)
was added. The mixture was extracted with EtOAc (3 × 15 mL).
The extracts were dried (Na2SO4) and concentrated. The residue
was triturated with CH2Cl2, and the resulting solid was collected
by filtration to give 6 (67 mg, 47%). 1H NMR (300 MHz, DMSO-
d6) δ 8.81 (s, 1H), 8.62 (s, 1H), 8.27 (s, 1H), 8.15 (s, 1H), 7.65 (s,
2H), 7.59 (d, J ) 8.5 Hz, 2H), 7.41 (d, J ) 8.8 Hz, 2H), 7.32 (s,
1H), 7.25 (d, J ) 8.5 Hz, 1H), 7.17 (t, J ) 7.8 Hz, 1H), 6.80 (d,
J ) 7.5 Hz, 1H), 2.29 (s, 3H); MS (ESI) m/z 436.9, 438.9 [M +
H]+. Anal. (C20H17BrN6O·0.2H2O·0.1CH2Cl2) C, H, N.
3-Bromo-6-phenylpyrazolo[1,5-a]pyrimidine (8). A mixture 2-phe-
nylmalonaldehyde (500 mg, 3.4 mmol), 3-amino-4-bromopyrazole
(544 mg, 3.4 mmol), and concentrated HCl (1 mL, 12 mmol) in
EtOH (20 mL) was heated to reflux for 2 h. The mixture was
concentrated in vacuo to remove the EtOH, and the resulting residue
was dissolved in H2O (100 mL). The solution was adjusted to basic
pH with 1 N NaOH and the resulting solid was collected by filtration
1
to give 8 (860 mg, 93%). H NMR (300 MHz, DMSO-d6) δ 9.52
(d, J ) 2.4 Hz, 1H), 9.03 (d, J ) 2.0 Hz, 1H), 8.42 (s, 1H),
7.84-7.89 (m, 2H), 7.51-7.58 (m, 2H), 7.43-7.50 (m, 1H); MS
(ESI) m/z 273.9, 275.9 [M + H]+.
4-(3-Bromopyrazolo[1,5-a]pyrimidin-6-yl)phenylamine (9). To
a solution of 8 (500 mg, 1.82 mmol) in concentrated H2SO4 (3
mL) at -20 °C was added dropwise concentrated HNO3 (0.1 mL,
1.6 mmol). The mixture was allowed to warm to room temperature
and stirred for 2 h. The mixture was poured over ice (50 g) and
stirred until the ice had melted. The resulting solid was collected
by filtration, then partitioned between 1 N NaOH (100 mL) and
CH2Cl2 (100 mL), and the aqueous layer was extracted with CH2Cl2
(2 × 100 mL). The organic layers were dried (Na2SO4) and
concentrated to give a mixture of nitration products in which the
desired para isomer predominated. This crude solid was taken up
in EtOH (40 mL), THF (16 mL), and H2O (8 mL) and the mixture
was heated to 75 °C. Fe0 (700 mg, 12.5 mmol) and NH4Cl (106
mg, 1.98 mmol) were added, and the mixture was kept at 75 °C
for 16 h. TLC (25% EtOAc/hexanes) shows that the reaction is
complete. The hot mixture was filtered through a pad of Celite,
rinsing with copious amounts of MeOH, and the filtrate was
concentrated in vacuo. The residue was partitioned between CH2Cl2
(100 mL) and H2O (200 mL), and the aqueous layer was extracted
with CH2Cl2 (2 × 100 mL). The organic layers were dried (Na2SO4)
and concentrated to give a crude solid, which was purified by flash
chromatography (4% EtOAc in CH2Cl2) to give 9 as a pale-yellow
1-(4-(7-Amino-3-bromopyrazolo[1,5-a]pyrimidin-6-yl)phe-
nyl)-3-o-tolylurea (6a). Compound 6a was prepared following
the procedure described for 6, substituting o-tolyl isocyanate for
m-tolyl isocyanate. 1H NMR (300 MHz, DMSO-d6) δ 9.16 (s, 1H),
8.28 (s, 1H), 8.15 (s, 1H), 7.95 (s, 1H), 7.86 (dd, J ) 8.0, 1.2 Hz,
1H), 7.65 (s, 2H), 7.60 (d, J ) 8.8 Hz, 2H), 7.42 (d, J ) 8.5 Hz,
2H), 7.07-7.21 (m, 2H), 6.96 (td, J ) 7.4, 1.2 Hz, 1H), 2.26 (s,
3H); MS (ESI) m/z 437.1, 439.1 [M + H]+. Anal. (C20H17BrN6O)
C, H, N.
1-(4-(7-Amino-3-bromopyrazolo[1,5-a]pyrimidin-6-yl)phenyl)-
3-p-tolylurea (6b). Compound 6b was prepared following the
procedure described for 6, substituting p-tolyl isocyanate for m-tolyl
1
solid (180 mg, 34%). H NMR (300 MHz, DMSO-d6) δ 9.27 (d,
J ) 2.0 Hz, 1H), 8.93 (d, J ) 2.0 Hz, 1H), 8.32 (s, 1H), 7.52 (d,
J ) 8.5 Hz, 2H), 6.68 (d, J ) 8.5 Hz, 2H), 5.41 (s, 2H); MS (ESI)
m/z 288.9, 290.8 [M + H]+.
1
isocyanate. H NMR (300 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.58
(s, 1H), 8.27 (s, 1H), 8.15 (s, 1H), 7.65 (s, 2H), 7.58 (d, J ) 8.5
Hz, 2H), 7.40 (d, J ) 8.8 Hz, 2H), 7.35 (d, J ) 8.1 Hz, 2H), 7.09
(d, J ) 8.1 Hz, 2H), 2.25 (s, 3H); MS (ESI) m/z 437.1, 439.1 [M
+ H]+. Anal. (C20H17BrN6O) C, H, N.
1-[4-(3-Bromopyrazolo[1,5-a]pyrimidin-6-yl)phenyl]-3-m-
tolylurea (10). Compound 10 was prepared from 9 following the
1
procedure described for the synthesis of 6. H NMR (300 MHz,
DMSO-d6) δ 9.46 (d, J ) 2.0 Hz, 1H), 9.02 (d, J ) 2.0 Hz, 1H),
8.92 (s, 1H), 8.71 (s, 1H), 8.39 (s, 1H), 7.80 (d, J ) 8.8 Hz, 2H),
7.62 (d, J ) 8.8 Hz, 2H), 7.32 (s, 1H), 7.25 (d, J ) 9.2 Hz, 1H),
7.17 (t, J ) 7.6 Hz, 1H), 6.80 (d, J ) 7.5 Hz, 1H), 2.29 (s, 3H);
MS (ESI) m/z 422.0, 423.7 [M + H]+. Anal. (C20H16BrN5O) C,
H, N.
1-(4-(7-Amino-3-bromopyrazolo[1,5-a]pyrimidin-6-yl)phenyl)-
3-phenylurea (6c). Compound 6c was prepared following the
procedure described for 6, substituting phenyl isocyanate for m-tolyl
isocyanate. H NMR (300 MHz, DMSO-d6) δ 8.82 (s, 1H), 8.69
(s, 1H), 8.27 (s, 1H), 8.15 (s, 1H), 7.65 (s, 2H), 7.59 (d, J ) 8.8
Hz, 2H), 7.47 (d, J ) 7.8 Hz, 2H), 7.41 (d, J ) 8.8 Hz, 2H), 7.29
1

3784 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Frey et al.
3-Dimethylamino-2-(4-nitrophenyl)acrylonitrile (11). To a solu-
tion of 4-nitrophenylacetonitrile (10 g, 62 mmol) in toluene (400
mL) was added dimethylformamide-dimethyl acetal (25.0 mL, 188
mmol). The mixture was heated to reflux for 16 h and then
concentrated to dryness in vacuo. The residue was purified by flash
chromatography, eluting with CH2Cl2 to give 11 as a bright-yellow
tube filled with Drierite. The flask was then cooled in an ice bath,
and POCl3 (17.0 mL, 186 mmol) was added dropwise over 10 min.
The mixture was stirred for 30 min at 0 °C, followed by dropwise
addition of a solution of N-methylpyrazole (5.0 g, 61 mmol) in
DMF (12 mL) over 15 min. The ice bath was removed, and the
mixture was stirred at room temperature for 16 h. The solution
was then chilled to 0 °C, and saturated aqueous NaHCO3 was added
until the reaction was no longer acidic. The resulting mixture was
extracted with EtOAc (3 × 200 mL), and the extracts were dried
over Na2SO4 and concentrated in vacuo to give 15 as a crude amber
oil (5.3 g, 79%), which was judged sufficiently pure to be used in
the next reaction. 1H NMR (300 MHz, DMSO-d6) δ 9.78 (s, 1 H),
8.42 (s, 1 H), 7.96 (s, 1 H), 3.90 (s, 3 H). MS (ESI) m/z 111.1 [M
+ H]+, 258.0.
(1-Methyl-1H-pyrazol-4-yl)methanol (16). A solution of com-
pound 15 (5.6 g, 51 mmol) in MeOH (500 mL) was chilled to 0
°C, and NaBH4 (3.8 g, 100 mmol) was added portionwise over 10
min. The ice bath was removed, and the mixture was stirred for
1 h. TLC (19:1 CH2Cl2/MeOH, KMnO4 stain) shows that the
reaction is complete. The reaction was quenched with 2 N HCl
(300 mL), and the MeOH was removed by concentrating in vacuo.
The resulting aqueous solution was extracted with EtOAc (3 ×
200 mL). The extracts were dried over Na2SO4 and concentrated
in vacuo to give 16 as an amber oil (5.2 g, 92%), which was used
without further purification. 1H NMR (300 MHz, DMSO-d6) δ 7.54
(s, 1H), 7.30 (s, 1H), 4.77 (t, J ) 5.4 Hz, 1H), 4.32 (d, J ) 5.4 Hz,
2H), 3.78 (s, 3H); MS (ESI) m/z 113.1 [M + H]+.
(1-Methyl-1H-pyrazol-4-yl)acetonitrile (17). A solution of com-
pound 16 (614 mg, 5.5 mmol) in CHCl3 (25 mL) was treated with
SOCl2 (0.80 mL, 11 mmol). The mixture was heated to reflux for
3 h, then cooled to room temperature and concentrated in vacuo to
give an oil that solidified upon standing. This crude chloride was
then dissolved in DMSO (30 mL), the solution was treated with
NaCN (1.1 g, 22 mmol), and the mixture was stirred overnight at
room temperature. The mixture was diluted with H2O (150 mL),
and the mixture was extracted with EtOAc (3 × 75 mL). The
extracts were washed with brine and dried (Na2SO4), then concen-
trated in vacuo. The residue was purified by flash chromatography
(Analogix Intelliflash, RS-12 cartridge, flow rate 30 mL/min),
eluting with a gradient of 0-10% EtOAc/CH2Cl2 over 30 min to
provide 17 as an oil (284 mg, 43%). 1H NMR (300 MHz, DMSO-
d6) δ 7.69 (s, 1H), 7.37 (s, 1H), 3.80 (s, 3H), 3.78 (s, 2H); MS
(ESI) m/z 122.1 [M + H]+.
1
solid (12.7 g, 95%). H NMR (300 MHz, DMSO-d6) δ 8.13 (d, J
) 9.2 Hz, 2H), 7.87 (s, 1H), 7.53 (d, J ) 9.2 Hz, 2H), 3.29 (s,
6H); MS (ESI) m/z 218.0 [M + H]+, 124.1 (base).
6-(4-Nitrophenyl)pyrazolo[1,5-a]pyrimidin-7-ylamine (12). A
solution of 11 (500 mg, 2.3 mmol) and 3-aminopyrazole (205 mg,
2.5 mmol) in EtOH (10 mL) was treated with concentrated HCl
(0.5 mL, 6 mmol), and the mixture was heated to reflux for 16 h,
then cooled to room temperature. A yellow precipitate formed and
was collected by filtration, rinsing with EtOH, to give the title
compound as its hydrochloride salt (627 mg, 86%). 1H NMR (300
MHz, DMSO-d6) δ 9.24 (s, 2H), 8.44 (s, 1H), 8.37 (d, J ) 2.4 Hz,
1H), 8.37 (d, J ) 8.8 Hz, 2H), 7.82 (d, J ) 8.8 Hz, 2H), 6.63 (d,
J ) 2.0 Hz, 1H); MS (ESI) m/z 256.0 [M + H]+.
6-(4-Aminophenyl)pyrazolo[1,5-a]pyrimidin-7-ylamine (13). A
suspension of the hydrochloride salt of 12 (0.62 g, 2.1 mmol) in
MeOH (20 mL) was purged with N2, and 10% Pd/C (30 mg) was
added. The mixture was placed under H2 (1 atm) for 3 h. The
mixture was purged with N2 and filtered through a pad of Celite,
rinsing with MeOH. The filtrate was concentrated to give the
hydrochloride salt of 13 as a yellow solid (0.52 g, 94%). 1H NMR
(300 MHz, DMSO-d6) δ 8.66 (s, 2H), 8.28 (d, J ) 2.4 Hz, 1H),
8.21 (s, 1H), 7.23 (d, J ) 8.5 Hz, 2H), 6.83 (d, J ) 8.1 Hz, 2H),
6.53 (d, J ) 2.4 Hz, 1H); MS (ESI) m/z 226.0 [M + H]+.
1-[4-(7-Aminopyrazolo[1,5-a]pyrimidin-6-yl)phenyl]-3-m-
tolylurea (14). Compounds 14 and 14a-d were prepared from
1
13 following the procedure described for the synthesis of 6. H
NMR (300 MHz, DMSO-d6) δ 8.80 (s, 1H), 8.62 (s, 1H), 8.12 (d,
J ) 2.4 Hz, 1H), 8.10 (s, 1H), 7.58 (d, J ) 8.5 Hz, 2H), 7.42 (d,
J ) 8.8 Hz, 2H), 7.37-7.41 (m, 2H), 7.32 (s, 1H), 7.25 (d, J )
8.5 Hz, 1H), 7.17 (t, J ) 7.8 Hz, 1H), 6.80 (d, J ) 7.1 Hz, 1H),
6.44 (d, J ) 2.4 Hz, 1H), 2.29 (s, 3H); MS (ESI) m/z 359.1 [M +
H]+. Anal. (C20H18N6O·0.2H2O) C, H, N.
1-(4-(7-Aminopyrazolo[1,5-a]pyrimidin-6-yl)phenyl)-3-(3-
(trifluoromethyl)phenyl)urea (14a). 1H NMR (300 MHz, DMSO-
d6) δ 9.09 (s, 1H), 8.95 (s, 1H), 8.12 (d, J ) 2.0 Hz, 1H), 8.10 (s,
1H), 8.02-8.04 (m, 1H), 7.58-7.63 (m, 3H), 7.52 (t, J ) 8.0 Hz,
1H), 7.43 (d, J ) 8.8 Hz, 2H), 7.40 (br s, 2H), 7.32 (d, J ) 7.5 Hz,
1H), 6.45 (d, J ) 2.4 Hz, 1H); MS (ESI) m/z 413.1 [M + H]+.
Anal. (C20H15F3N6O·0.5H2O) C, H, N.
1′-Methyl-2H,1′H-[4,4′]bipyrazolyl-3-ylamine (19). To a mixture
of ethyl formate (1.60 mL, 19.8 mmol), NaH (60% dispersion in
mineral oil, 1.16 g, 29 mmol), and EtOH (2 drops) in toluene (35
mL) at room temperature was added dropwise a solution of (1-
methyl-1H-pyrazol-4-yl)acetonitrile) 17 (1.17 g, 9.7 mmol) in
toluene (10 mL) over 30 min. The mixture was stirred at room
temperature for 6 h, then quenched with H2O and brought to pH 4
using 1 N HCl (50 mL). The mixture was extracted with EtOAc (3
× 100 mL). The combined extracts were dried (Na2SO4) and
concentrated to give 18 as a crude offwhite solid, which was carried
forward without purification. MS (ESI) m/z 150.0 [M + H]+. This
material was combined with EtOH (40 mL), hydrazine monohydrate
(0.95 mL, 19.6 mmol), and concentrated HCl (0.9 mL). The mixture
was heated to reflux for 16 h, then cooled to room temperature
and concentrated to dryness. The residue was dissolved in H2O
and neutralized by the addition of saturated aqueous NaHCO3. The
resulting mixture was extracted with EtOAc (3 × 50 mL). The
extracts were dried (Na2SO4) and concentrated in vacuo to provide
1-(4-(7-Aminopyrazolo[1,5-a]pyrimidin-6-yl)phenyl)-3-(3-chlo-
1
rophenyl)urea (14b). H NMR (300 MHz, DMSO-d6) δ 8.94 (s,
1H), 8.92 (s, 1H), 8.13 (d, J ) 2.4 Hz, 1H), 8.10 (s, 1H), 7.72-7.74
(m, 1H), 7.59 (d, J ) 8.8 Hz, 2H), 7.43 (d, J ) 8.5 Hz, 2H), 7.40
(br s, 2H), 7.27-7.32 (m, 2H), 7.03 (ddd, J ) 6.3, 2.4, 2.2 Hz,
1H), 6.45 (d, J ) 2.4 Hz, 1H); MS (ESI) m/z 377.0 [M + H]+.
Anal. (C19H15ClN6O·0.4H2O) C, H, N.
1-(4-(7-Aminopyrazolo[1,5-a]pyrimidin-6-yl)phenyl)-3-(2-fluoro-
5-methylphenyl)urea (14c). 1H NMR (300 MHz, DMSO-d6) δ 9.19
(s, 1H), 8.50 (d, J ) 2.4 Hz, 1H), 8.13 (d, J ) 2.4 Hz, 1H), 8.10
(s, 1H), 8.01 (dd, J ) 8.0, 1.9 Hz, 1H), 7.58 (d, J ) 8.5 Hz, 2H),
7.43 (d, J ) 8.5 Hz, 2H), 7.40 (br s, 2H), 7.12 (dd, J ) 11.4, 8.3
Hz, 1H), 6.78-6.84 (m, 1H), 6.45 (d, J ) 2.4 Hz, 1H), 2.28 (s,
3H); MS (ESI) m/z 377.2 [M + H]+. Anal. (C20H17FN6O·1.0H2O)
C, H, N.
1-(4-(7-Aminopyrazolo[1,5-a]pyrimidin-6-yl)phenyl)-3-(2-fluoro-
5-(trifluoromethyl)phenyl)urea (14d). 1H NMR (300 MHz, DMSO-
d6) δ 9.31 (s, 1H), 8.93 (s, 1H), 8.64 (dd, J ) 7.3, 2.2 Hz, 1H),
8.13 (d, J ) 2.4 Hz, 1H), 8.10 (s, 1H), 7.60 (d, 2H), 7.51 (dd, J )
10.5, 8.5 Hz, 1H), 7.37-7.47 (m, 5H), 6.45 (d, J ) 2.4 Hz, 1H);
MS (ESI) m/z 431.1 [M + H]+. Anal. (C20H14F4N6O·0.1H2O) C,
H, N.
1
19 as a yellow wax (0.71 g, 45%). H NMR (300 MHz, DMSO-
d6) δ 11.42-11.54 (br s, 1H), 7.78 (s, 1H), 7.55 (app s, 2H),
4.36-4.51 (br s, 2H), 3.81 (s, 3H); MS (ESI) m/z 164.1 [M +
H]+.
6-(4-Aminophenyl)-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]-
pyrimidin-7-ylamine (20). 3-(1-Methyl-1H-pyrazol-4-yl)-6-(4-ni-
trophenyl)pyrazolo[1,5-a]pyrimidin-7-amine was prepared via cy-
clocondensation of 11 with 19 following the procedure described
for the synthesis of 12, and the resulting nitro compound was
reduced to 20 as follows: 3-(1-methyl-1H-pyrazol-4-yl)-6-(4-
1-Methyl-1H-pyrazole-4-carbaldehyde (15). A 500 mL, two-
necked round-bottomed flask equipped with an addition funnel, a
water-cooled reflux condenser, and a magnetic stir bar was charged
with DMF (35 mL). The condenser was equipped with a drying

Pyrimidines as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3785
nitrophenyl)pyrazolo[1,5-a]pyrimidin-7-amine (408 mg, 1.2 mmol)
was suspended in concentrated HCl (10 mL), and mixture was
chilled to 0 °C. SnCl2 ·2H2O (824 mg, 3.6 mmol) was added, and
the mixture was stirred at room temperature for 5 h. The precipitate
was collected by filtration and rinsed with a little concentrated HCl,
then dissolved in H2O (1000 mL). The solution was filtered, and
the filtrate was neutralized by the addition of saturated aqueous
NaHCO3. The mixture was extracted with EtOAc (3 × 50 mL).
The extracts were dried (Na2SO4) and concentrated to provide 20
as a yellow solid (300 mg, 80%). 1H NMR (300 MHz, DMSO-d6)
δ 8.37 (s, 1H), 8.13 (s, 1H), 8.08 (s, 1H), 7.91 (s, 1H), 7.21 (s,
2H), 7.16 (d, J ) 8.1 Hz, 2H), 6.69 (d, J ) 8.5 Hz, 2H), 5.22 (s,
2H), 3.88 (s,3 H); MS (ESI) m/z 306.1 [M + H]+.
1-{4-[7-Amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]py-
rimidin-6-yl]phenyl}-3-m-tolyl-urea (34). Compounds 34 and 34a-e
were prepared from 20 following the procedure described for the
synthesis of 6. 1H NMR (300 MHz, DMSO-d6) δ 8.79 (s, 1H),
8.61 (s, 1H), 8.41 (s, 1H), 8.15 (s, 1H), 8.14 (s, 1H), 7.93 (d, J )
0.7 Hz, 1H), 7.59 (d, J ) 8.8 Hz, 2H), 7.41-7.46 (m, 4H), 7.32 (s,
1H), 7.25 (d, J ) 8.1 Hz, 1H), 7.17 (t, J ) 7.8 Hz, 1H), 6.80 (d,
J ) 7.5 Hz, 1H), 3.89 (s, 3H), 2.29 (s, 3H); MS (ESI) m/z 439.1
[M + H]+. Anal. (C24H22N8O·0.2H2O) C, H, N.
7-Amino-6-[4-(3-m-tolylureido)phenyl]pyrazolo[1,5-a]pyrimidine-
3-carboxylic Acid Ethyl Ester (36). Compound 36 was prepared
from 3-aminopyrazole-4-carboxylic acid ethyl ester following the
sequence described for the synthesis of 14 from 3-aminopyrazole,
substituting the nitro reduction protocol described for the synthesis
of 5. 1H NMR (300 MHz, DMSO-d6) δ 8.82 (s, 1H), 8.62 (s, 1H),
8.52 (s, 1H), 8.25 (s, 1H), 7.80 (s, 2H), 7.60 (d, J ) 8.8 Hz, 2H),
7.42 (d, J ) 8.5 Hz, 2H), 7.32 (s, 1H), 7.25 (d, J ) 8.5 Hz, 1H),
7.17 (t, J ) 7.6 Hz, 1H), 6.80 (d, J ) 7.1 Hz, 1H), 4.27 (q, J ) 7.1
Hz, 2H), 2.29 (s, 3H), 1.31 (t, J ) 7.1 Hz, 3H); MS (ESI) m/z
431.2 [M + H]+. Anal. (C23H22N6O3 ·1.0DMF) C, H, N.
7-Amino-6-[4-(3-m-tolylureido)phenyl]pyrazolo[1,5-a]pyrimidine-
3-carboxylic Acid (37). A mixture of compound 36 (113 mg, 0.26
mmol), 2 N aqueous LiOH (0.7 mL, 1.4 mmol), MeOH (0.5 mL),
and THF (1 mL) was heated to 70 °C for 3 h, at which time TLC
(19:1 CH2Cl2/MeOH) showed consumption of starting material. The
mixture was concentrated to dryness, and the residue was taken up
in H2O (25 mL). The solution was adjusted to pH 4 with with glacial
HOAc and the resulting white solid was collected by filtration,
1
rinsing with H2O, to give 37 (88 mg, 84%). H NMR (300 MHz,
DMSO-d6) δ 9.26 (s, 1H), 9.06 (s, 1H), 8.42 (s, 1H), 8.18 (s, 1H),
7.67-7.79 (br s, 2H), 7.63 (d, J ) 8.5 Hz, 2H), 7.42 (d, J ) 8.5
Hz, 2H), 7.36 (s, 1H), 7.29 (d, J ) 8.1 Hz, 1H), 7.16 (t, J ) 7.8
Hz, 1H), 6.79 (d, J ) 7.5 Hz, 1H), 2.29 (s, 3H); MS (ESI) m/z
403.1 [M + H]+. Anal. (C21H18N6O3 ·2.7H2O) C, N. H calcd 5.23,
found 4.66.
7-Amino-6-[4-(3-m-tolylureido)phenyl]pyrazolo[1,5-a]pyrimidine-
3-carboxylic Acid Methylamide (38). A mixture of compound 37
(25 mg, 0.062 mmol), MeNH2 ·HCl (9 mg, 0.13 mmol), EDCI (36
mg, 0.19 mmol), HOBt (25 mg, 0.18 mmol), and N-methylmor-
pholine (0.07 mL, 0.63 mmol) in DMF (0.3 mL) was stirred for
16 h at room temperature. The mixture was diluted with H2O (10
mL) and extracted with EtOAc (3 × 10 mL). The extracts were
dried (Na2SO4) and concentrated in vacuo, and the crude residue
was triturated with CH2Cl2, yielding a solid that was collected by
filtration to provide 38 (14 mg, 56%). 1H NMR (300 MHz, DMSO-
1-{4-[7-Amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]py-
rimidin-6-yl]phenyl}-3-(3-trifluoromethylphenyl)urea (34a). 1H NMR
(300 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.95 (s, 1H), 8.41 (s, 1H),
8.15 (s, 2H), 8.03 (s, 1H), 7.93 (s, 1H), 7.59-7.64 (m, 3H), 7.53
(t, J ) 7.8 Hz, 1H), 7.43-7.48 (m, 4H), 7.32 (d, J ) 7.5 Hz, 1H),
3.89 (s, 3H); MS (ESI) m/z 493.1 [M + H]+. Anal. (C24H19F3N8O)
C, H, N.
1-{4-[7-Amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]py-
rimidin-6-yl]phenyl}-3-(3-chlorophenyl)urea (34b). 1H NMR (300
MHz, DMSO-d6) δ 8.92 (s, 1H), 8.91 (s, 1H), 8.41 (s, 1H), 8.15
(app s, 2H), 7.93 (s, 1H), 7.72-7.75 (m, 1H), 7.60 (d, J ) 8.8 Hz,
2H), 7.42-7.48 (m, 4H), 7.27-7.35 (m, 2H), 7.03 (dt, J ) 6.1,
2.7 Hz, 1H), 3.89 (s, 3H); MS (ESI) m/z 459.1 [M + H]+. Anal.
(C23H19ClN8O·0.1H2O·0.1CH2Cl2) C, H, N.
d6) δ 8.82 (s, 1H), 8.62 (s, 1H), 8.45 (s, 1H), 8.22 (s, 1H), 7.99 (q,
J ) 4.5 Hz, 1H), 7.90 (s, 2H), 7.60 (d, J ) 8.8 Hz, 2H), 7.42 (d,
J ) 8.5 Hz, 2H), 7.31 (s, 1H), 7.25 (d, J ) 8.5 Hz, 1H), 7.17 (t,
J ) 7.8 Hz, 1H), 6.80 (d, J ) 7.1 Hz, 1H), 2.88 (d, J ) 4.7 Hz,
3H), 2.29 (s, 3H); MS (ESI) m/z 416.2 [M + H]+. Anal.
(C22H21N7O2 ·1.5H2O) C, H, N.
1-{4-[7-Amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]-
pyrimidin-6-yl]phenyl}-3-(2-fluoro-5-trifluoromethylphenyl)-
1
urea (34c). H NMR (300 MHz, DMSO-d6) δ 9.32 (s, 1H), 8.94
(s, 1H), 8.65 (dd, J ) 7.1, 2.4 Hz, 1H), 8.41 (s, 1H), 8.15 (s, 2H),
7.93 (s, 1H), 7.58-7.63 (d, J ) 8.8 Hz, 2H), 7.37-7.56 (m, 6H),
3.86-3.92 (s, 3H); MS (ESI) m/z 511.2 [M + H]+. Anal.
(C24H18F4N8O) C, H, N.
Homogeneous Time-Resolved Fluorescence (HTRF) Assays of
Receptor Tyrosine Kinases (KDR, CSR1R, cKIT, FLT1, FLT3).
Assays were performed in a total of 40 µL in 96-well Costar black
half-volume plates using HTRF technology.39 Peptide substrate
(Biotin-Ahx-AEEEYFFLFA-amide) at 4 µM, 1 mM ATP, enzyme,
and inhibitors were incubated for 1 h at ambient temperature in 50
mM Hepes/NaOH, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM
DTT, 0.1 mM orthovanadate, and 0.01% BSA. Inhibitors were
added to the wells at final concentrations of 3.2 nM to 50 µM with
5% DMSO added as cosolvent. The reactions were stopped with
10 µL/well 0.5 M EDTA and then 75 µL buffer containing
streptavidin-allophycocyanin (Prozyme, 1.1 µg/mL), and PT66
antibody europium cryptate (Cis-Bio, 0.1 µg/mL) was added to each
well. The plates were read from 1 to 4 h after addition of the
detection reagents, and the time-resolved fluorescence (665-615
ratio) was measured using a Packard Discovery instrument. The
amount of each tyrosine kinase added to the wells was calibrated
to give a control (no inhibitor) to background (prequenched with
EDTA) ratio of 10-15 and was shown to be in the low nanomolar
concentration range for each kinase. The inhibition of each well
was calculated using the control and background readings for that
plate. Inhibition constants are the mean of two determinations
performed with seven concentrations of the test compounds.
Enzyme-Linked Immunisorbent Assay (ELISA) of KDR Cel-
lular Phosphorylation. NIH3T3 cells stably transfected with full
length human KDR (VEGFR2) were maintained in DMEM medium
with 10% fetal bovine serum and 500 µg/mL Geneticin. KDR cells
were plated at 20 000 cells/well into duplicate 96-well tissue culture
plates and cultured overnight in an incubator at 37 °C with 5%
1-{4-[7-Amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]py-
rimidin-6-yl]phenyl}-3-(4-fluoro-3-trifluoromethylphenyl)urea (34d).
1H NMR (300 MHz, DMSO-d6) δ 9.06 (s, 1H), 8.95 (s, 1H), 8.41
(s, 1H), 8.15 (app s, 2H), 8.02 (dd, J ) 0.6, 2.5 Hz, 1H), 7.93 (s,
1H), 7.64-7.71 (m, 1H), 7.61 (d, J ) 8.5 Hz, 2H), 7.41-7.49 (m,
5H), 3.89 (s, 3H); MS (ESI) m/z 511.2 [M + H]+. Anal.
(C24H18F4N8O·0.2H2O) C, H, N.
1-{4-[7-Amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]py-
rimidin-6-yl]phenyl}-3-(3-fluorophenyl)urea (34e). 1H NMR (300
MHz, DMSO-d6) δ 8.94 (s, 1H), 8.90 (s, 1H), 8.41 (s, 1H), 8.15
(app s, 2H), 7.93 (s, 1H), 7.60 (d, J ) 8.8 Hz, 2H), 7.51 (dt, J )
12.0, 2.3 Hz, 1H), 7.45 (s, 2H), 7.42-7.47 (m, J ) 8.5 Hz, 2H),
7.32 (td, J ) 8.2, 7.0 Hz, 1H), 7.13-7.17 (m, 1H), 6.79 (td, J )
8.4, 2.5 Hz, 1H), 3.89 (s, 3H); MS (ESI) m/z 443.1 [M + H]+.
Anal. (C23H19FN8O·0.1CH2Cl2) C, H, N.
1-[4-(7-Amino-5-methylpyrazolo[1,5-a]pyrimidin-6-yl)phenyl]-
3-m-tolylurea (28). Compound 28 was prepared from 3-aminopy-
razole and dimethylacetamide-dimethyl acetal following the same
sequence described for the synthesis of 14 from 3-aminopyrazole
and dimethylformamide-dimethyl acetal, substituting the nitro
reduction protocol described for the synthesis of compound 20. 1H
NMR (300 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.62 (s, 1H), 8.04 (d,
J ) 2.4 Hz, 1H), 7.59 (d, J ) 8.8 Hz, 2H), 7.32 (s, 1H), 7.21-7.28
(m, 3H), 7.17 (t, J ) 7.6 Hz, 1H), 6.78-6.83 (m, 3H), 6.30 (d, J
) 2.0 Hz, 1H), 2.29 (s, 3H), 2.14 (s, 3H); MS (ESI) m/z 373.1 [M
+ H]+. Anal. (C21H20N6O·0.3H2O) C, H, N.

3786 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Frey et al.
CO2 and 80% humidity. The growth medium was replaced with
serum-free growth medium for 2 h prior to compound addition.
Compounds in DMSO were diluted in serum-free growth medium
(final DMSO concentration of 1%) and added to cells for 20 min
prior to stimulation for 10 min with VEGF (50 ng/mL). Cells were
lysed by addition of RIPA buffer (50 mM Tris-HCl (pH 7.4), 1%
IGEPAL, 150 mM NaCl, 1 mM EDTA, and 0.25% sodium
deoxycholate) containing protease inhibitors (Sigma cocktail), NaF
(1 mM), and Na3VO4 (1 mM) and placed on a microtiter plate
shaker for 10 min. The lysates from duplicate wells were combined,
and 170 µL of the combined lysate was added to the KDR ELISA
plate. The KDR ELISA plate was prepared by adding anti-VEGFR2
antibody (1 µg/well, R&D Systems) to an unblocked plate and
incubated overnight at 4 °C. The plate was then blocked for at least
1 h with 200 µL/well of 5% dry milk in PBS. The plate was washed
two times with PBS containing 0.1% Tween 20 (PBST) before
addition of the cell lysates. Cell lysates were incubated in the KDR
ELISA plate with constant shaking on a microtiter plate shaker for
2 h at room temperature. The cell lysate was then removed and the
plate washed five times with PBST. Detection of phospho-KDR
was performed using a 1:2000 dilution of biotinylated 4G10
antiphosphotyrosine (UBI, Lake Placid, NY), incubated with
constant shaking for 1.5 h at room temperature, and washed five
times with PBST, and for detection a 1:2000 dilution of strepavidin-
HRP (UBI, Lake Placid, NY) was added and incubated with
constant shaking for 1 h at room temperature. The wells were then
washed five times with PBST, and K-Blue HRP ELISA substrate
(Neogen) was added to each well. Development time was monitored
at 650 nm in a SprectrMax Plus plate reader until 0.4-0.5
absorbance units were obtained (approximately 10 min) in the
VEGF only wells. Phosphoric acid (1 M) was added to stop the
reaction, and the plate was read at 450 nm. Percent inhibition was
calculated using the VEGF only wells as 100% controls and wells
containing 5 µM pan-kinase inhibitor as 0% controls (no VEGF
wells were used to monitor endogenous phosphorylation state of
the cells). IC50 values were calculated by nonlinear regression
analysis of the concentration-response curve. Each IC50 determi-
nation was performed with five concentrations and each assay point
determined in duplicate.
Estradiol-Induced Murine UE Assay. The 12 week old balb/c
female mice (Taconic, Germantown, NY) were pretreated with 10
units of pregnant mare’s serum gonadotropin (PMSG, Calbiochem)
intraperitoneally (ip) administered 72 and 24 h prior to estradiol.
Mice were randomized the day of the experiment. Test compounds
were formulated in a variety of vehicles and administered po 30
min prior to stimulation with an ip injection of water soluble 17ꢀ-
estradiol (20-25 µg/mouse). Animals were sacrificed and uteri
removed 2.5 h following estradiol stimulation by cutting just
proximal to the cervix and at the fallopian tubes. After the removal
of fat and connective tissue, uteri were weighed, squeezed between
filter paper to remove fluid, and weighed again. The difference
between wet and blotted weights represented the fluid content of
the uterus. Compound-treated groups were compared to vehicle-
treated groups after subtracting the background water content of
unstimulated uteri. Experimental group size was five or six.
Mouse PK Analysis. Male CD-1 mice weighing 26-30 g
(Charles River Laboratories) were dosed intravenously via the tail
vein or orally by gavage with a metal feeder tube. Dosing solutions
were prepared in 2.5% ethanol, 2.5% DMSO, 5% Tween-80, 25%
PEG 400, and pH 7.4 PBS for a dosing volume of 10 mL/kg. Blood
samples were collected with a heparinized syringe by cardiac
puncture following CO2 asphixiation at specified times. Plasma
samples were aliquoted into 96-well plates, and proteins were
precipitated using acidified methanol. Supernatants were stored at
-20 °C. Sample analyses were performed by LC-MS using a
Shimadzu 10A-VP chromatography system with a Waters YMC-
AQ 5 cm column. The mobile phase consisted of 45% acetonitrile
and 0.1% acetic acid in water, and the flow rate was 0.4 mL/min.
Mass detection was accomplished with an ESI equipped LCQ-Duo
by ThermoFinnegan. External standards were prepared from spiked
control plasma and used to generate a response factor for every
study. Limits of detection were between 20 and 50 nM.
Acknowledgment. We thank the Department of Structural
Chemistry, Abbott Laboratories, for recording the NMR and
MS spectral data.
Supporting Information Available: Synthesis and spectral data
for compounds 2, 4, 23-27, 29-33, 35, 39, 40, and 42. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam,
S. The Protein Kinase Complement of the Human Genome. Science
2002, 298, 1912–1934.
(2) Blume-Jensen, P.; Hunter, T. Oncogenic Kinase Signalling. Nature
2001, 411, 355–365.
(3) Holmgren, L.; O’Reilly, M. S.; Folkman, J. Dormancy of microme-
tastases: Balanced proliferation and apoptosis in the presence of
angiogenesis suppression. Nat. Med. 1995, 1, 149–153.
(4) Fukumura, D.; Xu, L.; Chen, Y.; Gohongi, T.; Seed, B.; Jain, R. K.
Hypoxia and Acidosis Independently Up-Regulate Vascular Endot-
helial Growth Factor Transcription in Brain Tumors in Vivo. Cancer
Res. 2001, 61, 6020–6024.
(5) Dvorak, H. F. Vascular Permeability Factor/Vascular Endothelial
Growth Factor: A Critical Cytokine in Tumor Angiogenesis and a
Potential Target for Diagnosis and Therapy. J. Clin. Oncol. 2002, 20,
4368–4380.
(6) Carmeliet, P. VEGF as a Key Mediator of Angiogenesis in Cancer.
Oncology 2005, 69 (Suppl. 3), 4–10.
(7) Beck, L.; D’Amore, P. A. Vascular development: cellular and
molecular regulation. FASEB J. 1997, 11, 365–373.
(8) Reynolds, L. P.; Killilea, S. D.; Redmer, D. A. Angiogenesis in the
female reproductive system. FASEB J. 1992, 6, 886–892.
(9) Augustin, H. G. Antiangiogenic tumor therapy: will it work? Trends
Pharmacol. Sci. 1998, 19, 216–222.
(10) Ferrara, N.; Hillan, K. J.; Gerber, H.-P.; Novotny, W. Discovery and
Development of Bevacizumab, an Anti-VEGF Antibody for Treating
Cancer. Nat. ReV. Drug DiscoVery 2004, 3, 391–400.
(11) Gschwind, A.; Fischer, O. M.; Ullrich, A. The Discovery of Receptor
Tyrosine Kinases: Targets for Cancer Therapy. Nat. ReV. Cancer 2004,
4, 361–370.
(12) Board, R.; Jayson, G. C. Platelet-Derived Growth Factor Receptor
(PDGFR): A Target for Anticancer Therapeutics. Drug Resist. Updates
2005, 8, 75–83.
(13) Sun, L.; Tran, N.; Tang, F.; App, H.; Hirth, P.; McMahon, G.; Tang,
C. Synthesis and Biological Evaluations of 3-Substituted Indolin-2-
ones: A Novel Class of Tyrosine Kinase Inhibitors That Exhibit
Selectivity toward Particular Receptor Tyrosine Kinases. J. Med. Chem.
1998, 41, 2588–2603.
(14) Laird, A. D.; Vajkoczy, P.; Shawver, L. K.; Thurnher, A.; Liang, C.;
Mohammadi, M.; Schlessinger, J.; Ullrich, A.; Hubbard, S. R.; Blake,
R. A.; Fong, T. A. T.; Strawn, L. M.; Sun, L.; Tang, C.; Hawtin, R.;
Tang, F.; Shenoy, N.; Hirth, K. P.; McMahon, G.; Cherrington, J. M.
SU6668 Is a Potent Antiangiogenic and Antitumor Agent That Induces
Regression of Established Tumors. Cancer Res. 2000, 60, 4152–4160.
(15) Fong, T. A. T.; Shawver, 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.; McMahon, G. SU5416 Is a Potent and Selective Inhibitor
of the Vascular Endothelial Growth Factor Receptor (Flk-1/KDR) That
Inhibits Tyrosine Kinase Catalysis, Tumor Vascularization, and Growth
of Multiple Tumor Types. Cancer Res. 1999, 59, 99–106.
(16) Sun, L.; Tran, N.; Liang, C.; Hubbard, S.; Tang, F.; Lipson, K.;
Schreck, R.; Zhou, Y.; McMahon, G.; Tang, C. Identification of
Substituted 3-[(4,5,6,7-Tetrahydro-1H-indol-2-yl)methylene]-1,3-di-
hydroindol-2-ones as Growth Factor Receptor Inhibitors for VEGF-
R2 (Flk-1/KDR), FGF-R1, and PDGF-Rꢀ Tyrosine Kinases. J. Med.
Chem. 2000, 43, 2655–2663.
(17) Klebl, B. M.; Mu¨ller, G. Second-generation kinase inhibitors. Expert
Opin. Ther. Targets 2005, 9, 975–993.
(18) Hicklin, D. J.; Ellis, L. M. Role of the Vascular Endothelial Growth
Factor Pathway in Tumor Growth and Angiogenesis. J. Clin. Oncol.
2005, 23, 1011–1027.
(19) Casanovas, O.; Hicklin, D. J.; Bergers, G.; Hanahan, D. Drug
Resistance by Evasion of Antiangiogenic Targeting of VEGF Signaling
in Late-Stage Pancreatic Islet Tumors. Cancer Cell 2005, 8, 299–
309.
(20) Sun, L.; Liang, C.; Shirazian, S.; Zhou, Y.; Miller, T.; Cui, J.; Fukuda,
J. Y.; Chu, J.-Y.; Nematalla, A.; Wang, X.; Chen, H.; Sistla, A.; Luu,
T. C.; Tang, F.; Wei, J.; Tang, C. Discovery of 5-[5-Fluoro-2-oxo-

Pyrimidines as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3787
1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-car-
boxylic Acid (2-Dimethylaminoethyl)amide, a Novel Tyrosine Kinase
Inhibitor Targeting Vascular Endothelial and Platelet-Derived Growth
Factor Receptor Tyrosine Kinase. J. Med. Chem. 2003, 46, 1116–
1119.
(26) Traxler, P.; Furet, P. Strategies toward the Design of Novel and
Selective Protein Tyrosine Kinase Inhibitors. Pharmacol. Ther. 1999,
82, 195–206.
(27) Bold, G.; Florsheimer, A.; Furet, P.; Imbach, P.; Masuya, K.;
Schoepfer, R. Scientists at Novartis have independently discovered
7-amino-pyrazolo[1,5-a]pyrimidine ureas as kinase inhibitors: Organic
compounds, US2005/0222171 A1, 2005.
(21) Wilhelm, S. M.; Carter, C.; Tang, L.; Wilkie, D.; McNabola, A.; Rong,
H.; Chen, C.; Zhang, X.; Vincent, P.; McHugh, M.; Cao, Y.; Shujath,
J.; Gawlak, S.; Eveleigh, D.; Rowley, B.; Liu, L.; Adnane, L.; Lynch,
M.; Auclair, D.; Taylor, I.; Gedrich, R.; Voznesensky, A.; Riedl, B.;
Post, L. E.; Bollag, G.; Trail, P. A. BAY 43-9006 Exhibits Broad
Spectrum Oral Antitumor Activity and Targets the RAF/MEK/ERK
Pathway and Receptor Tyrosine Kinases Involved in Tumor Progres-
sion and Angiogenesis. Cancer Res. 2004, 64, 7099–7109.
(28) Alcade, E.; deMendoza, J.; Garcia-Marquina, J. M.; Almera, C.;
Elguero, J. Etude de la Re´action du ꢀ-Aminocrotonitrile et du
R-Formyl Phe´nylace´tonitrile avec l’Hydrazine: Synthe`se d’Amino-7-
pyrazolo[1,5-a]pyrimidines. J. Heterocycl. Chem. 1974, 11, 423–429.
(29) Miyaura, N.; Suzuki, A. Palladium-Catalyzed Cross-Coupling Reac-
tions of Organoboron Compounds. Chem. ReV. 1995, 95, 2457–2483.
(30) 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. Synthesis and Initial SAR Studies of 3,6-
Disubstituted Pyrazolo[1,5-a]pyrimidines: A New Class of KDR
Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2002, 12, 2767–2770.
(31) Nam, N. L.; Grandberg, I. I.; Sorokin, V. I. Pyrazolopyrimidines Based
on 5-Aminopyrazoles Unsubstituted at the Position 1. Chem. Hetero-
cycl. Compd. 2002, 38, 1371–1374.
(32) Gribble, F. M.; Loussouarn, G.; Tucker, S. J.; Zhao, C.; Nichols, C. G.;
Ashcroft, F. M. A Novel Method for Measurement of Submembrane
ATP Concentration. J. Biol. Chem. 2000, 275, 30046–30049.
(33) Miyazaki, Y.; Matsunaga, S.; Tang, J.; Maeda, Y.; Nakano, M.;
Philippe, R. J.; Shibahara, M.; Liu, W.; Sato, H.; Wang, L.; Nolte,
R. T. Novel 4-Amino-furo[2,3-d]pyrimidines as Tie-2 and VEGFR2
Dual Inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 2203–2207.
(34) Fraley, M. E.; Rubino, R. S.; Hoffman, W. F.; Hambaugh, 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. Optimization of a Pyrazolo[1,5-a]pyrimidine
Class of KDR Kinase Inhibitors: Improvements in Physical Properties
Enhance Cellular Activity and Pharmacokinetics. Bioorg. Med. Chem.
Lett. 2002, 12, 3537–3541.
(22) Dai, Y.; Guo, Y.; Frey, R. R.; Ji, Z.; Curtin, M. L.; Ahmed, A. A.;
Albert, D. H.; Arnold, L.; Arries, S. S.; Barlozzari, T.; Bauch, J. L.;
Bouska, J. J.; Bousquet, P. F.; Cunha, G. A.; Glaser, K. B.; Guo, J.;
Li, J.; Marcotte, P. A.; Marsh, K. C.; Moskey, M. D.; Pease, L. J.;
Stewart, K. D.; Stoll, V. S.; Tapang, P.; Wishart, N.; Davidsen, S. K.;
Michaelides, M. R. Thienopyrimidine Ureas as Novel and Potent
Multitargeted Receptor Tyrosine Kinase Inhibitors. J. Med. Chem.
2005, 48, 6066–6083.
(23) Curtin, M. L.; Frey, R. R.; Heyman, H. R.; Sarris, K. A.; Steinman,
D. H.; Holms, J. H.; Bousquet, P. F.; Cunha, G. A.; Moskey, M. D.;
Ahmed, A. A.; Pease, L. J.; Glaser, K. B.; Stewart, K. D.; Davidsen,
S. K.; Michaelides, M. R. Isoindolinone Ureas: A Novel Class of
KDR Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2004, 14, 4505–
4509.
(24) (a) Albert, D. H.; Tapang, P.; Magoc, T. J.; Pease, L. J.; Reuter, D. R.;
Wei, R.-Q.; Li, J.; Guo, J.; Bousquet, P. F.; Ghoreishi-Haack, N. S.;
Wang, B.; Bukofzer, G. T.; Wang, Y.-C.; Stavropoulos, J. A.; Hartandi,
K.; Niquette, A. L.; Soni, N.; Johnson, E. F.; McCall, J. O.; Bouska,
J. J.; Luo, Y.; Donawho, C. K.; Dai, Y.; Marcotte, P. A.; Glaser, K. B.;
Michaelides, M. R.; Davidsen, S. K. Preclinical activity of ABT-869,
a multitargeted receptor tyrosine kinase inhibitor. Mol. Cancer Ther.
2006, 5, 995–1006. (b) Dai, Y.; Hartandi, K.; Ji, Z.; Ahmed, A. A.;
Albert, D. H.; Bauch, J. L.; Bouska, J. J.; Bousquet, P. F.; Cunha,
G. A.; Glaser, K. B.; Harris, C. M.; Hickman, D.; Guo, J.; Li, J.;
Marcotte, P. A.; Marsh, K. C.; Moskey, M. D.; Martin, R. L.; Olson,
A. M.; Osterling, D. J.; Pease, L. J.; Soni, N. B.; Stewart, K. D.; Stoll,
V. S.; Tapang, P.; Reuter, D. R.; Davidsen, S. K.; Michaelides, M. R.
Discovery of N-(4-(3-Amino-1H-indazol-4-yl)phenyl)-N′-(2-fluoro-5-
methylphenyl)urea (ABT-869), a 3-Aminoindazole-Based Orally Ac-
tive Multitargeted Receptor Tyrosine Kinase Inhibitor. J. Med. Chem.
2007, 50, 1584–1597.
(35) Aronov, A. M.; Baker, C.; Bemis, G. W.; Cao, J.; Chen, G.; Ford,
P. J.; Germann, U. A.; Green, J.; Hale, M. R.; Jacobs, M.; Janetka,
J. W.; Maltais, F.; Martinez-Botella, G.; Namchuk, M. N.; Straub, J.;
Tang, Q.; Xie, X. Flipped Out: Structure-Guided Design of Selective
Pyrazolylpyrrole ERK Inhibitors. J. Med. Chem. 2007, 50, 1280–1287.
(36) Ma, W.; Tan, J.; Matsumoto, H.; Robert, B.; Abrahamson, D. R.; Das,
S. K.; Dey, S. K. Adult Tissue Angiogenesis: Evidence for Negative
Regulation by Estrogen in the Uterus. Mol. Endocrinol. 2001, 15,
1983–1992.
(37) Inhibition values exceeding 30% were significantly different (p < 0.05)
from vehicle-treated controls.
(38) Compound 34a was screened against a wider panel of 150 kinases.
At 1 mM ATP concentration, IC50 values of <1000 nM were noted
for TrkA (99 nM) and TrkB (52 nM). Kinases showing IC50 < 1000
nM at lower ATP concentration included Src, Lck, JAK2, Abl, and
Fyn. Without inhibition data at 1 mM ATP, however, it is not possible
to make a direct comparison of these screening data with the data
reported in Table 7.
(39) Kolb, A. J.; Kaplita, P. V.; Hayes, D. J.; Park, Y.-W.; Pernell, C.;
Major, J. S.; Mathis, G. Tyrosine Kinase Assays Adapted to
Homogeneous Time-Resolved Fluorescence. Drug DiscoVery Today
1998, 3, 333–342.
(25) The development of RTK inhibitors in the clinic has been extensively
reviewed. For recent reviews, see the following: (a) Arora, A.; Scholar,
E. M. Role of Tyrosine Kinase Inhibitors in Cancer Therapy.
J. Pharmacol. Exp. Ther. 2005, 315, 971–979. (b) Laird, A. D.;
Cherrington, J. M. Small Molecule Tyrosine Kinase Inhibitors: Clinical
Development of Anticancer Agents. Expert Opin. InVest. Drugs 2003,
12, 51–64. (c) Manley, P. W.; Bold, G.; Br¨uggen, J.; Fendrich, G.;
Furet, P.; Mestan, J.; Schnell, C.; Stolz, B.; Meyer, T.; Meyhack, B.;
Stark, W.; Strauss, A.; Wood, J. Advances in the Structural Biology,
Design and Clinical Development of VEGF-R Kinase Inhibitors for
the Treatment of Angiogenesis. Biochim. Biophys. Acta 2004, 1697,
17–27.
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