Synthesis and discovery of pyrazine-pyridine biheteroaryl as a novel series of potent vascular endothelial growth factor receptor-2 inhibitors

Article abstract of DOI:10.1021/jm040099a

Pathological angiogenesis is associated with disease states such as cancer, diabetic retinopathy, rheumatoid arthritis, endometriosis, and psoriasis. There is much evidence that direct inhibition of the kinase activity of vascular endothelial growth facto

Full text of DOI:10.1021/jm040099a

1886
J. Med. Chem. 2005, 48, 1886-1900
Synthesis and Discovery of Pyrazine-Pyridine Biheteroaryl as a Novel Series
of Potent Vascular Endothelial Growth Factor Receptor-2 Inhibitors
Gee-Hong Kuo,* Aihua Wang, Stuart Emanuel, Alan DeAngelis, Rui Zhang, Peter J. Connolly,
William V. Murray, Robert H. Gruninger, Jan Sechler, Angel Fuentes-Pesquera, Dana Johnson,
Steven A. Middleton, Linda Jolliffe, and Xin Chen
Drug Discovery Division, Johnson & Johnson Pharmaceutical Research and Development, L.L.C.,
1000 Route 202, P.O. Box 300, Raritan, New Jersey 08869
Received May 6, 2004
Pathological angiogenesis is associated with disease states such as cancer, diabetic retinopathy,
rheumatoid arthritis, endometriosis, and psoriasis. There is much evidence that direct inhibition
of the kinase activity of vascular endothelial growth factor receptor-2 (VEGFR-2) will result in
the reduction of angiogenesis and the suppression of tumor growth. Attempts to optimize a
cyclin-dependent kinase-1 (CDK1) inhibitor by using palladium-catalyzed C-C bond, C-N bond
formation reactions to assemble diverse biheteroaryl molecules led to the unexpected discovery
of a pyrazine-pyridine biheteroaryl as a novel series of potent VEGFR-2 inhibitors. Compound
15, which had IC50 ) 0.084 µM at VEGFR-2, showed very modest selectivity against fibroblast
growth factor receptor-2 (IC50 ) 0.21 µM), platelet-derived growth factor receptor (IC50 ) 0.36
µM), and glycogen synthase kinase-3 (IC50 ) 0.478 µM), while it exhibited more than 10-fold
selectivity against epidermal growth factor receptor (IC50 ) 1.36 µM) and insulin-R kinase
(IC50 ) 1.69 µM). On the other hand, compound 15 exhibited more than 100-fold selectivity
against calmodulin kinase 2; casein kinase-1 and -2; CDK1 and -4; mitogen-activated protein
kinase; and protein kinase A, Câ2, and Cγ (IC50 >10 µM). Compound 15 also displayed high
inhibitory potency on VEGF-stimulated human umbilical vein endothelial cell (HUVEC)
proliferation (IC50 ) 0.005 µM) and good selectivity against cell lines such as HUVEC, human
aortic smooth muscle cells, and MRC5 lung fibroblasts. Molecular docking studies were
conducted in an attempt to rationalize the unexpected high VEGFR-2 selectivity of 15.
Introduction
microvascular permeability.14 Therefore, it is expected
that direct inhibition of the kinase activity of VEGFR-2
will result in the reduction of angiogenesis and the
suppression of tumor growth. Furthermore, inhibition
of VEGFR-2 targeting the genetically more stable host
endothelial cells, instead of labile tumor tissues, may
decrease the chance of resistance development.15
A number of VEGF inhibitors are currently undergo-
ing advanced-stage clinical trials for the treatment of
cancer, including a humanized monoclonal antibody to
VEGF (bevacizumab/rhuMab VEGF),16 an anti-VEG-
FR-2 antibody,17 a soluble VEGF receptor,18 and several
small molecule VEGFR-2 inhibitors (PTK787/vata-
lanib,19 SU-11248,20 SU-6668,21 and ZD-6474,22 Scheme
1). Among them, bevacizumab (Avastin) in combination
with chemotherapy,16,23 was accepted by the FDA in
2003 for the treatment of first-line metastatic colorectal
cancer. This first proof-of-concept example will certainly
warrant the development of many more small molecule
VEGF inhibitors24 for anti-angiogenesis therapy. This
paper describes the unexpected discovery of the pyra-
zine-pyridine biheteroaryl scaffold as the basis of a
novel series of potent VEGFR-2 inhibitors.
Angiogenesis, the formation of new capillaries from
preexisting blood vessels,1 is a necessary process for
organ development during embryogenesis and is critical
for the female reproductive cycle, inflammation, and
wound healing in the adult.2 Pathological angiogenesis
is associated with disease states such as cancer,3
diabetic retinopathy,4 rheumatoid arthritis,5 endometri-
osis,6 and psoriasis.7 Solid tumors, in particular, are
dependent on angiogenesis to grow beyond a certain
critical size by inducing new capillaries sprouting from
existing blood vessels to secure their nutrition, oxygen
supply, and waste removal.3,8 In addition, angiogenesis
also promotes metastasis of tumor cells to other sites.9
The new vessel growth and maturation are highly
complex and coordinated processes, requiring the stimu-
lation by a number of growth factors,10 but vascular
endothelial growth factor (VEGF) signaling often rep-
resents a critical rate-limiting step in physiological
angiogenesis and pathological angiogenesis.11 The bio-
logical effects of VEGF are mediated by two receptor
tyrosine kinases (RTKs), VEGFR-1 (Flt-1)12 and VEG-
FR-2 [also known as kinase domain region (KDR) or Flk-
1].13 While the precise function of VEGFR-1 is still
under debate,14 there is much evidence that VEGFR-2
is the major mediator of vascular endothelial cell (EC)
mitogenesis and survival, as well as angiogenesis and
Chemistry
The chloride 3 was synthesized via a palladium-
catalyzed C-N bond formation reaction25 of 4,6-dichloro-
pyrimidine 1 with 3-chloroaniline followed by Boc
protection (Scheme 2). The required organostannane 7
was prepared in three-step sequence: (1) a Curtius
* To whom correspondence should be addressed. Tel: 908-704-4330.
Fax: 908-203-8109. E-mail: gkuo@prdus.jnj.com.
10.1021/jm040099a CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/06/2004

Pyrazine-Pyridine as VEGFR-2 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1887
Scheme 1
Scheme 2a
a (i) 3-chloroaniline, Pd(OAc)2, BINAP, NaOt-Bu, 90 °C; (ii) Boc2O, DMAP; (iii) DPPA, Et3N, t-BuOH, 65-95 °C; (iv) Br(CH2)3OTBS,
Cs2CO3, 70 °C; (v) [(n-Bu)3Sn]2, Pd(OAc)2, P(o-Tol)3, 100 °C; (vi) (a) 3, Pd2(dba)3, AsPh3, 67 °C, (b) TFA.
rearrangement26 of the nicotinic acid 4 with DPPA gave
5, (2) alkylation of 5 with the silyl-protected bromide
gave 6, (3) a palladium-catalyzed transformation of the
organo halide to the organostannane27 provided 7. The
first biheteroaryl target, pyrimidine-pyridine 8, was
synthesized by a palladium-catalyzed Stille coupling
reaction28 of halide 3 with organostannane 7 followed
by deprotection with TFA (Scheme 2).
yield as in chloride 2. The known 3431 was converted to
the organostannane 35, followed by the Stille coupling
reaction with 33 to afford 36. Base hydrolysis, Curtius
rearrangement, alkylation, and deprotection provided
the target molecule 38.
The synthesis of pyrazine-pyridine 42 was shown in
Scheme 5. Starting from the bromide 39, Boc protection,
alkylation and palladium-catalyzed transformation gave
the organostannane 41. Stille coupling reaction of 41
with 33, followed by deprotection afforded target 42.
The synthesis of biheteroaryl 47 was shown in Scheme
6. A Curtius rearrangement was conducted on 4332 to
give 44. Alkylation, organostannane formation, and
Stille coupling reaction, followed by deprotection, gave
the target 47.
The second target, pyrazine-pyridine 15, was pre-
pared by a different reaction sequence (Scheme 3). The
Stille coupling reaction of organostannane 10 with 2,6-
29
dichloropyrazine catalyzed by Pd(PPh3)2Cl2 gave 11.
Palladium-catalyzed C-N bond formation of 11 with
3-chloroaniline in the presence of DPPF30 as the ligand
generated 12 in good yield. Boc-protection and base
hydrolysis, followed by Curtius rearrangement, provided
14. Alkylation of 14 with the silyl-protected bromide,
followed by TFA deprotection, gave the second target
molecule 15. The biheteroaryl targets of 20, 25, and 30
were all prepared in a similar way as 15 (Scheme 3).
The synthesis of pyrazine-pyrazine 38 was shown in
Scheme 4. Under the same reaction conditions used in
the preparation of chloride 2 (Pd(OAc)2, BINAP, NaOt-
Bu), chloride 32 was formed in 90% yield instead of 4%
To our delight, the nucleophilic organostannane 4833
survived the Curtius rearrangement reaction condition
to generate the N-Boc-substituted (trimethylstannanyl)-
pyridine 49 in 57% yield (Scheme 7). A Stille coupling
reaction of 49 with 33, followed by alkylation and
deprotection, generated the pyrazine-pyridine 51.
Transformation of the known triflate 5234 to the
corresponding organostannane, followed by the Stille
coupling reaction, gave 53 (Scheme 8). Base hydrolysis,

1888 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Kuo et al.
Scheme 3a
a (i) [(n-Bu)3Sn]2, Pd(OAc)2, P(o-Tol)3, 100 °C; (ii) ArX1X2, Pd(PPh3)2Cl2, LiCl, 100 °C; (iii) 3-chloroaniline, Pd2(dba)3, DPPF, Cs2CO3,
110 °C; (iv) (a) Boc2O, DMAP, (b) NaOH, 0-20 °C; (v) DPPA, Et3N, t-BuOH, 65-100 °C; (vi) (a) Br(CH2)3OTBS, Cs2CO3, 70 °C, (b) TFA.
Scheme 4a
a (i) 3-chloroaniline, Pd(OAc)2, BINAP, NaOt-Bu, 80 °C; (ii) Boc2O, DMAP; (iii) Pd(PPh3)4, Me6Sn2, LiCl, BHT, 100 °C; (iv) 33,
Pd(PPh3)2Cl2, LiCl, 100 °C; (v) (a) LiOH, (b) DPPA, Et3N, t-BuOH, 60-95 °C; (vi) (a) Br(CH2)3OTBS, Cs2CO3, 70 °C, (b) TFA.
followed by the Curtius rearrangement, alkylation, and
deprotection, afforded the target molecule 55.
gave 60 directly. Iodonation36 followed by the amination
gave the target 62.
The synthesis of the pyrazine-thiazole 59 was started
from the aminothiazole 56 (Scheme 9). Boc-protection
and alkylation gave 58, which was converted to the
corresponding organostannane, followed by the Stille
coupling reaction and deprotection, generating the
target 59.
The target, pyrazine-oxazole 62, was synthesized via
a different method (Scheme 10). A hetero-Heck reac-
tion35 of 33 with oxazole in the presence of Pd(PPh3)4
Results and Discussion
Lead Discovery. We were originally interested in
identifying potent and selective cyclin-dependent ki-
nase-1 (CDK1) inhibitors for the treatment of cancer.37
In a literature review, discovery and lead optimization
efforts have provided many CDK inhibitors over the past
decade;38 two of these agents, flavopiridol and UCN-01,

Pyrazine-Pyridine as VEGFR-2 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1889
Scheme 5a
a (i) (a) Boc2O, DMAP, (b) Br(CH2)3OTBS, Cs2CO3, 70 °C; (ii) Pd(PPh3)4, [(n-Bu)3Sn]2, LiCl, BHT, 100 °C; (iii) (a) 33, Pd(PPh3)2Cl2,
LiCl, 100 °C, (b) TFA.
Scheme 6a
a (i) DPPA, Et3N, t-BuOH, 65-95 °C; (ii) Br(CH2)3OTBS, Cs2CO3, 70 °C; (iii) Me6Sn2,Pd(PPh3)4, LiCl, BHT, 90 °C; (iv) (a) 33, Pd(PPh3)2Cl2,
LiCl, 100 °C, (b) TFA.
Scheme 7a
a (i) DPPA, Et3N, t-BuOH, 65-95 °C; (ii) 33, Pd(PPh3)2Cl2, LiCl, 100 °C; (iii) (a) Br(CH2)3OTBS, Cs2CO3, 70 °C, (b) TFA.
Scheme 8a
a (i) (a) Pd(PPh3)4, Me6Sn2, LiCl, BHT, 100 °C, (b) 33, Pd(PPh3)2Cl2, LiCl, 100 °C; (ii) (a) LiOH, (b) DPPA, Et3N, t-BuOH, 65-95 °C; (iii)
(a) Br(CH2)3OTBS, Cs2CO3, 70 °C, (b) TFA.
Scheme 9a
a (i) Boc2O, DMAP; (ii) Br(CH2)3OTBS, Cs2CO3, 70 °C; (iii) (a) Pd(PPh3)4, [(n-Bu)3Sn]2, LiCl, BHT, 100 °C, (b) 33, Pd(PPh3)2Cl2, LiCl,
100 °C; (c) TFA.
have advanced to clinical evaluation.39,40 However, both
flavopiridol41 and UCN-0142 proved to be potent but
nonselective CDK inhibitors. Recently, a high-affinity
CDK1 inhibitor (CGP-60474, IC50 ) 0.017 µM, Scheme
1) with better selectivity toward other kinases was
reported.43 Therefore, we decided to take advantage of
the recent advances in palladium-catalyzed C-C bond,
C-N bond formation reactions to assemble diverse
biheteroaryl molecules (Scheme 1), which might be
difficult to synthesize otherwise, with the goal to

1890 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Kuo et al.
Scheme 10a
a (i) oxazole, Pd(PPh3)4, AcOK, 80 °C; (ii) LHMDS, -78 °C, diiodoethane; (iii) 3-amino-1-propanol, 100 °C.
Table 1. Binding Affinities at CDK1 and VEGFR-2a
Shifting the (3-chlorophenyl)amino group from the
meta- to the para-position in the upper pyrazine ring
abolished the kinase inhibition of both CDK1 and
VEGFR-2 (20, IC50 >10 µM, IC50 >10 µM). Apparently,
the relative position of the substituted aniline moiety
to the upper ring is also critical to the binding affinities
at the kinases. The poor kinase inhibitory potency of
25 and 30 (IC50 >10 µM at both CDK1 and VEGFR-2)
further suggested the importance of the 1,3-orientation
between the substituted-aniline group and the lower
ring moiety to the kinase activities.
With the discovery of the potent (IC50 ) 0.084 µM)
and selective (>100-fold against CDK1) VEGFR-2 in-
hibitor 15 in hand, we continued to explore the impact
of the lower ring of the biheteroaryl toward the VEG-
FR-2 inhibition. Replacing the pyridine of 15 with
another pyrazine gave 38. The addition of one extra
nitrogen atom to the lower ring reduced by 6-fold the
potency at VEGFR-2 (38, IC50 ) 0.497 µM, Table 2).
Removal of one nitrogen atom from the lower meta-
position of 38 did not restore the potency (42, IC50 > 10
µM for both kinases). By comparing compounds 15 (IC50
) 0.084 µM), 38 (IC50 ) 0.497 µM), and 42 (IC50 >10
µM), the significant contribution of the nitrogen atom
at the meta-position of the lower ring to the VEGFR-2
inhibition was demonstrated; on the other hand, the
nitrogen atom at the ortho-position between the two
substituents on the lower ring was detrimental to the
binding affinity at VEGFR-2.
Replacing the 3-pyridine of 15 with 4-pyridine (as in
CGP-60474) gave 47. Interestingly, compound 47 dis-
played significant inhibition at both VEGFR-2 and
CDK1 (47, IC50 ) 0.5 µM at both kinases). Shifting the
(hydroxypropyl)amino side chain of 47 from the meta-
position to the ortho-position resulted in 51 with re-
duced potency at both kinases (51, IC50 ) 0.761 µM at
VEGFR-2; IC50 ) 2.22 µM at CDK1). By comparison of
a Assay details are described in the Experimental Section.
b SEM: standard error of the mean.
identify a CDK1 inhibitor with optimized potency,
selectivity, and pharmacokinetic profiles.
We first prepared pyrimidine-pyridine 8 (Table 1),
a close analogue of CGP-60474, by moving the bottom
nitrogen atom in the pyridine ring to the meta-position
and moving one of the pyrimidine nitrogen atoms from
the ortho- to the para-position relative to the (3-
chlorophenyl)amino group. A 140-fold decrease in CDK1
inhibitory potency (8, IC50 ) 2.4 µM, Table 1) was
observed together with very poor inhibition against
VEGFR-2 (IC50 >10 µM), one of the kinases in our
selectivity panel.
CGP-60474 (IC50 ) 0.017 µM at CDK1) and 47 (IC50
)
0.5 µM at CDK1) to 15 (IC50 >10 µM at CDK1), the
nitrogen atom at the para-position of the lower ring (4-
pyridine) (such as in CGP-60474 and 47) appears to
contribute to the binding affinity at CDK1 more sig-
nificantly than at VEGFR-2.
If we shifted the (hydroxypropyl)amino side chain of
42 from the meta-position to the para-position to gener-
ate 55, no inhibition was observed at either kinases (55,
IC50 > 10 µM for VEGFR-2 and CDK1). We next
replaced the lower 3-pyridine of 15 with a thiazole to
give 59, and about 8-fold reduced potency at VEGFR-2
We then replaced the upper pyrimidine ring with a
pyrazine ring while the lower pyridine ring remained
unchanged to give 15. To our surprise, the inhibition
at CDK1 was totally abolished (15, IC50 >10 µM) while
a high potency was observed at VEGFR-2 (15, IC50
)
was observed for 59 when compared to 15 (59, IC50 )
0.084 µM). The nitrogen atoms in the upper ring of these
biheteroaryls clearly play a major role in the binding
affinities of the inhibitors for CDK1 and VEGFR-2.
0.708 µM at VEGFR-2). Replacement of the 3-pyridine
of 15 with an oxazole afforded 62 with even lower
potency at VEGFR-2 (62, IC50 ) 2.11 µM). These

Pyrazine-Pyridine as VEGFR-2 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1891
Table 3. IC50 (µM) for Selected Kinase Assays for 15a
Table 2. Binding Affinities at CDK1 and VEGFR-2a
kinase assay
VEGFR-2
IC50
kinase assay
GSK-3
IC50
0.084
>10
>10
>10
>10
>10
1.36
0.21
0.478
1.69
>10
0.36
>10
>10
>10
calmodulin kinase-2
casein kinase-1
casein kinase-2
CDK1
insulin-R kinase
MAPK (ERK2)
PDGF-R
PKA
CDK4
PKCâ2
EGFR
PKCγ
FGFR-2
a Assay details are described in the Experimental Section. IC50
values are reported as the average of at least two separate
determinations.
Table 4. IC50 (µM) for Inhibition of Cell Proliferation by 15a
cell line
IC50 ( SEM cell line IC50 ( SEM
VEGF-stimulated HUVEC 0.005 ( 0.003 HeLa
>10
>10
>10
HUVEC
HASMC
MRC5
1.68 ( 0.146 HCT-116
3.87 ( 0.168 A375
6.6 ( 0.110
a Assay details are described in the Experimental Section.
and is under investigation in the treatment of glioblas-
tomas which exploits its PDGFR inhibitory activity.46
Therefore, although we were targeting the VEGFR
inhibition, the very modest selectivity of compound 15
against FGFR-2 and PDGF-R may not be undesirable.
On the other hand, compound 15 exhibited at least 2
orders of magnitude of selectivity against calmodulin
kinase 2, casein kinase-1 or -2, CDK1, CDK4, mitogen-
activated protein kinase (MAPK), protein kinase A
(PKA), protein kinase Câ2 (PKCâ2), and protein kinase
Cγ (PKCγ) (IC50 >10 µM, Table 3).
Cellular Selectivity. The cellular activity of the
compound was tested for its ability to inhibit VEGF-
stimulated proliferation of human umbilical vein en-
dothelial cells (HUVEC). Compound 15 exhibits excel-
lent cellular potency (IC50 ) 0.005 µM, Table 4) for
inhibiting VEGF-stimulated proliferation.47 On the
other hand, compound 15 only displayed modest effect
on the unstimulated growth of HUVEC (IC50 ) 1.68
µM). The 336-fold difference in potency of compound 15
for inhibition of VEGF-stimulated growth of HUVEC
versus unstimulated growth of HUVEC may suggests
its specificity of action through inhibition of VEGFR
signal transduction. In addition, compound 15 displayed
very minimal inhibition of proliferation of two normal
human cell types, human aortic smooth muscle cells
(HASMC, IC50 ) 3.87 µM) and MRC5 lung fibroblasts
(IC50 ) 6.6 µM), which indicates the high cellular
selectivity of compound 15 for VEGF-mediated pro-
cesses.
The ability of compound 15 to inhibit the proliferation
of cells derived from carcinomas originating from vari-
ous tissues such as HeLa (cervical adenocarcinoma),
HCT-116 (colon carcinoma), and A375 (malignant mela-
noma) was also examined. The lack of inhibition of the
growth of these tumor cell lines (IC50 >10 µM, Table 4)
further confirms that the VEGFR inhibitor 15 does not
exhibit general antiproliferative activity, as would occur
through inhibition of growth regulatory kinases such
as CDK1 inhibitors.
a Assay details are described in the Experimental Section.
observations were consistent with the notion that “the
thiazole is a closer bioisosteres44 of pyridine than
oxazole”.
In summary, while no improvement was made toward
the potency and selectivity at CDK1 with the diverse
biheteroaryls examined, we unexpectedly discovered
that compound 15, having a pyrazine-pyridine scaffold
with 1,3-substitution-orientation at both upper and
lower rings, exhibited high potency at VEGFR-2 and
high selectivity against CDK1.
Kinase Selectivity. We next examined the selectiv-
ity of the most potent VEGFR-2 inhibitor, 15 (IC50
)
0.084 µM), against a representative panel of kinases
(Table 3). Compound 15 showed very modest selectivity
(2-5-fold) against fibroblast growth factor receptor
(FGFR-2, IC50 ) 0.21 µM), platelet-derived growth factor
receptor (PDGF-R, IC50 ) 0.36 µM), and glycogen
synthase kinase-3 (GSK-3, IC50 ) 0.478 µM), while it
exhibited more than 1 order of magnitude of selectivity
against epidermal growth factor receptor (EGFR, IC50
) 1.36 µM) and insulin-R kinase (IC50 ) 1.69 µM).
FGFR-2 and PDGF-R have been implicated indirectly
in inducing VEGF secretion and hence in angiogenesis.45
ST1-571 (Gleevec) is a potent PDGF-R inhibitor and a
v-Abl inhibitor. It has been approved recently for the
treatment of chronic myeloid leukemia and KIT positive
metastatic malignant gastrointestinal stromal tumors
Molecular Docking Studies. In an attempt to
rationalize the unexpected high kinase selectivity of
pyrazine-pyridine biheteroaryl 15 at VEGFR-2 over
CDK1, molecular docking studies of CGP-60474 and 15

1892 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Kuo et al.
VEGFR-2 backbone at the hinge region, i.e., the pyridine
nitrogen and the amide nitrogen of Cys919. Meanwhile,
the anilino nitrogen is moving away from the hinge
region and is hydrogen bonded to the carboxylate group
of Asp1046. Three additional hydrogen bonds may also
be formed, one is between the inner pyrazine nitrogen
with the thiol group of Cys1045, and the other two are
between the hydroxyl group of 15 and the residue
Asn923. The hydrogen bond between the inner pyrazine
nitrogen and the thiol group of Cys1045 may be pivotal,
because it allows the pyrazine ring to get close to the
bulky thiol group of Cys1045, and therefore, 15 may
simultaneously form the above two hydrogen bonds with
Cys919 and Asp1046.
The results of molecular docking studies may provide
us some insights into the SAR observed in Tables 1 and
2 as well. First, the 1,3-disubstitution pattern at both
of the upper and lower rings appears critical, since the
tricyclic scaffold needs to adopt a U-shape conformation
to fit into the ATP-binding site. Any different disubsti-
tution patterns may change the overall shape of the
scaffold and reduce its optimal fit into the binding site
(such as 20, 25, 30, and 55), with the exception of 51.
In the case of 51, the flexibility of the (hydroxylpropyl)-
amino side chain may contribute to the tolerance of its
1,2-substitution pattern. Second, the nitrogen position
in the upper and lower rings seems to contribute
significantly to the binding affinities of different kinases.
A subtle change of the nitrogen position may completely
change the mode of hydrogen bonding and consequently
dramatically alter the selectivity profile of a compound.
For example, comparing 8 and 15 (Table 1), the shift of
a nitrogen atom from the outer ortho-position to the
inner ortho-position, relative to the 3-chloroanilino
group, changes the formation of the hydrogen bond from
8 with Leu83 of CDK1 to 15 with Cys1045 of VEGFR-
2, therefore giving them different selectivity at CDK1
versus VEGFR-2. The addition of the second nitrogen
to the lower ring of 15 (such as 38, Table 2) reduces the
basicity of the first outer nitrogen and weakens the
strength of its hydrogen bond with Cys919 of VEGFR-
2, leading to a decreased potency of 38 at VEGFR-2.
Meanwhile, the removal of the first outer nitrogen of
the lower ring of 38 (such as 42) eliminates its hydrogen
bond with Cys919 and thus abolishes the VEGFR-2
inhibitory potency of 42. Compared to 15, 47 (Table 2)
moves the lower pyridine nitrogen from the meta- to the
para-position, relative to the pyrazine ring. This change
may set the lower nitrogen at a more favorable position
to form the hydrogen bond with Lys33 of CDK1 and a
less favorable position to form the hydrogen bond with
Cys919 of VEGFR-2, therefore restoring some CDK1
inhibitory potency and reducing some VEGFR-2 inhibi-
tory potency at the same time.
Figure 1. Illustration of the binding modes of CGP60474 (a)
and 15 (b) in the ATP-binding sites of CDK1 and VEGFR-2,
respectively, suggested by molecular docking. The ligands and
the key residues that act as their hydrogen-bonding partners
are represented in the tube model, while the proteins are in
the carton model. The hydrogen bonds are indicated by the
yellow dotted lines. (Atom color scheme: hydrogen in white,
oxygen in red, nitrogen in blue, chlorine in dark green, carbon
on ligands in orange, and carbon on proteins in gray.)
on CDK1 and VEGFR-2 were conducted, respectively.
Their favorite docking poses in the ATP-binding sites
are illustrated in Figure 1. Overall, both compounds
show a nice fit in the binding sites and make extensive
favorable van der Waals contacts with proteins through
their tricyclic scaffolds. Interestingly, they adopt two
opposite binding modes, in terms of hydrogen bonding.
CGP-60474 (Figure 1a) adopts a typical bidentate
hydrogen-bonding mode with the CDK1 backbone at the
hinge region, as described by Furet et al.,48 the pyrimi-
dine nitrogen with the amide nitrogen of Leu83, and
the anilino nitrogen with the carbonyl oxygen of Leu83.
Two additional hydrogen bonds may also be formed, one
is between the pyridine nitrogen and the terminal amino
group of Lys33, and the other one is between the
hydroxyl group and the terminal carboxylate group of
Asp86. On the other hand, compound 15 (Figure 1b)
adopts a single hydrogen bond to interact with the
Conclusion
Attempts to optimize a CDK1 inhibitor by using
palladium-catalyzed C-C bond, C-N bond formation
reactions to assemble diverse biheteroaryl molecules led
to the unexpected discovery of a pyrazine-pyridine
biheteroaryl as a novel potent VEGFR-2 inhibitor.
Compound 15, which had IC50 ) 0.084 µM inhibitory
potency at VEGFR-2, showed very modest selectivity
against FGFR-2 (IC50 ) 0.21 µM), PDGF-R (IC50 ) 0.36

Pyrazine-Pyridine as VEGFR-2 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1893
of compound 5 (2.85 g, 10.44 mmol), (3-bromopropoxy)-tert-
butyldimethylsilane (3.96 g, 15.66 mmol), and Cs2CO3 (10.21
g, 31.3 mmol), in anhydrous DMF (55 mL), was stirred at 70
°C for 23 h under nitrogen. The cooled reaction mixture was
diluted with water and extracted with ether (3×), dried (Na2-
SO4), and concentrated. The product was purified by column
chromatography (EtOAc/hexane as solvent) to give 4.2 g (90%)
of compound 6 as yellow oil: 1H NMR (300 MHz, CDCl3) δ
8.45 (brs, 2 H), 7.77 (brs, 1 H), 3.73 (brt, J ) 7.3 Hz, 2 H),
3.62 (t, J ) 5.9 Hz, 2 H), 1.81 (m, 2 H), 1.45 (s, 9 H), 0.84 (s,
9 H), 0.00 (s, 6 H); MS (ES) m/z 445, 447 (M + H+). Anal.
(C19H33N2O3BrSi) C, H, N.
[3-((tert-Butyldimethylsilanyl)oxy)propyl](5-(tributyl-
stannanyl)pyridin-3-yl)carbamic Acid tert-Butyl Ester
7. A mixture of compound 6 (500 mg, 1.12 mmol), bis-
(tributyltin) (780 mg, 1.345 mmol), tri-o-tolylphosphine (88.5
mg, 0.29 mmol), palladium acetate (11.3 mg, 0.05 mmol),
triethylamine (226 mg, 2.24 mmol) in acetonitrile was stirred
at 95-100 °C for 23 h under nitrogen. The cooled reaction
mixture was concentrated, sodium carbonate solution was
added, and the mixture stirred for 10 min before being
extracted with hexane (4×). The combined hexane was filtered
through Celite, dried (Na2SO4), and concentrated. The product
was purified by column chromatography (EtOAc/hexane as
solvent) to give 395 mg (54%) of compound 7 as an oil: 1H
NMR (300 MHz, CDCl3) δ 8.39 (d, J ) 1.2 Hz, 1 H), 8.37 (m,
1 H), 7.55 (m, 1 H), 3.72 (brt, J ) 7.3 Hz, 2 H), 3.63 (t, J ) 6.2
Hz, 2 H), 1.89-1.10 (m, 29 H), 0.89 (t, J ) 7.2 Hz, 9 H), 0.85
(s, 9 H), 0.00 (s, 6 H); MS (ES) m/z 657 (M + H+).
3-{[5-[6-((3-Chlorophenyl)amino)pyrimidin-4-yl]pyri-
din-3-yl]amino}propan-1-ol 8. A mixture of compound 7
(396 mg, 0.6 mmol), compound 3 (293 mg, 0.86 mmol), Pd2-
(dba)3 (16 mg, 0.015 mmol), and triphenylarsine (37 mg, 0.12
mmol) in THF (8 mL) was refluxed for 41 h under nitrogen.
The reaction mixture was concentrated, sodium carbonate
solution was added, and the mixture stirred for 10 min before
being extracted with ether (3×). The combined ether layer was
dried (Na2SO4) and concentrated. The product was purified
by column chromatography (EtOAc/hexane as solvent) to give
63 mg (16%) of the silylated product as an oil: MS (ES) m/z
670 (M + H+).
µM), and GSK-3 (IC50 ) 0.478 µM), while it exhibited
more than 10-fold selectivity against EGFR (IC50 ) 1.36
µM) and insulin-R kinase (IC50 ) 1.69 µM). On the other
hand, compound 15 exhibited more than 100-fold se-
lectivity against calmodulin kinase 2, casein kinase-1
or -2, CDK1, CDK4, MAPK, PKA, PKCâ2, and PKCγ
(IC50 >10 µM). Compound 15 also displayed high
inhibitory potency at VEGF-stimulated HUVEC and
good selectivity against cell lines such as HUVEC,
HASMC, and MRC5. Molecular docking studies were
conducted in an attempt to rationalize the unexpected
high VEGFR-2 selectivity of 15. The high potency, good
kinase selectivity profile, and high cellular potency/
selectivity of compound 15 may render it as valuable
pharmacological tool in elucidating the complex roles
of VEGF signaling pathways and the potential utility
for anti-angiogenesis therapy.
Experimental Section
1
Chemistry. H NMR spectra were measured on a Bruker
AC-300 (300 MHz) spectrometer using tetramethylsilane as
an internal standard. Elemental analyses were obtained by
Quantitative Technologies Inc. (Whitehouse, NJ), and the
results were within 0.4% of the calculated values unless
otherwise mentioned. Melting points were determined in open
capillary tubes with a Thomas-Hoover apparatus and were
uncorrected. Electrospray mass spectra (MS-ES) were recorded
on a Hewlett-Packard 59987A spectrometer. High-resolution
mass spectra (HRMS) were obtained on a Micromass Autospec.
E spectrometer. The term “DMAP” refers to (dimethylamino)-
pyridine, “TFA” refers to trifluoroacetic acid, “NMP” refers to
1-methyl-2-pyrrolidinone, “DPPF” refers to 1,1′-bis(diphen-
ylphosphino)ferrocene, “Pd2(dba)3” refers to tris(dibenzyli-
deneacetone)dipalladium(0)-chloroform adduct, and “DPPA”
refers to diphenylphosphoryl azide.
(3-Chlorophenyl)(6-chloropyrimidin-4-yl)amine 2. A
mixture of 4,6-dichloropyrimidine compound 1 (4 g, 26.9
mmol), 3-chloroaniline (3.44 g, 26.9 mmol), palladium acetate
(121 mg, 38 mmol), BINAP (368 mg, 59 mmol), and NaO-t-Bu
(3.61 g, 217 mmol) in toluene (100 mL) was stirred at 90 °C
for 40 h under nitrogen. The cooled reaction mixture was
concentrated under vacuum. The residue was diluted with
dichloromethane and filtered through Celite, the Celite was
washed with acetone, and the combined filtrates were con-
centrated under vacuum. The product was purified by column
chromatography (100% dichloromethane as solvent) to give 250
mg (4%) of compound 2 as a solid: 1H NMR (300 MHz, DMSO-
d6) δ 8.56 (s, 1 H), 7.94 (s, 1 H), 7.44 (d, J ) 8.4 Hz, 1 H), 7.38
(t, J ) 8.0 Hz, 1 H), 7.12 (d, J ) 8.2 Hz, 1 H), 6.84 (s, 1 H); MS
(ES) m/z 240 (M + H+).
(3-Chlorophenyl)(6-chloropyrimidin-4-yl)carbamic Acid
tert-Butyl Ester 3. A mixture of compound 2 (240 mg, 1
mmol), Boc2O (436 mg, 2 mmol), and DMAP (cat.) in CH2Cl2
was stirred at 20 °C for 3 h and concentrated. The product
was purified by column chromatography (EtOAc/hexane as
solvent) to give 308 mg (91%) of compound 3 as an oil: MS
(ES) m/z 362 (M + Na). Anal. (C15H15N3O2Cl2) C, H, N.
(5-Bromopyridin-3-yl)carbamic Acid tert-Butyl Ester
5. A mixture of 5-bromonicotinic acid compound 4 (7.14 g, 35.4
mmol), t-BuOH (155 mL), triethylamine (6.08 g, 60 mmol), and
DPPA (10.69 g, 38.9 mmol) in toluene (115 mL) was stirred at
65 °C for 30 min then warmed to 95 °C for 15 h under nitrogen.
The cooled reaction mixture was concentrated under vacuum.
The product was purified by column chromatography (SiO2,
ethyl acetate/hexane as solvent) to give 2.9 g (30%) of com-
pound 5 as a white solid: 1H NMR (300 MHz, CDCl3) δ 8.32
(m, 3 H), 6.97 (brs, 1 H), 1.53 (s, 9 H); MS (ES) m/z 273, 275
(M + H+). Anal. (C10H13N2O2Br) C, H, N.
The silylated product was dissolved in TFA (2 mL) and
stirred at room temperature for 5 h before it was concentrated.
An ammonium hydroxide solution was added to the solution
until the pH was about 10-11, water was added, and a yellow
solid was formed. The yellow solid was collected through
filtration, washed with more water, and dried under vacuum.
The yellow solid was purified by column chromatography (dry
loading, methylene chloride/methanol as solvent) to give 26
mg (77%) of compound 8 as yellow solid: 1H NMR (300 MHz,
DMSO-d6) δ 9.94 (s, 1 H), 8.79 (s, 1 H), 8.36 (s, 1 H), 8.08 (brs,
2 H), 7.56 (d, J ) 9.2 Hz, 1 H), 7.47 (s, 1 H), 7.37 (t, J ) 8.1
Hz, 1 H), 7.24 (s, 1 H), 7.07 (d, J ) 7.8 Hz, 1 H), 6.14 (m, 1 H),
4.55 (t, J ) 5 Hz, 1 H), 3.53 (m, 2 H), 3.17 (m, 2 H), 1.75 (m,
2 H); MS (ES) m/z 356 (M + H+
C, H, N.
). Anal. (C18H18N5OCl‚0.2H2O)
5-(Tributylstannanyl)nicotinic Acid Ethyl Ester 10. A
mixture of 5-bromonicotinate 9 (25 g, 108.7 mmol), bis-
(tributyltin) (63.05 g, 108.7 mmol), palladium acetate (1.1 g,
4.89 mmol), tri-o-tolylphosphine (8.6 g, 28.26 mmol), and
triethylamine (21.96 g, 217.4 mmol) in acetonitrile (350 mL)
was stirred at 95-100 °C for 22 h under nitrogen. The cooled
reaction mixture was filtered through Celite. Then the Celite
was washed with more acetonitrile and the combined aceto-
nitrile was concentrated under vacuum. The residue was
diluted with a suitable solvent, such as dichloromethane,
washed with aqueous sodium carbonate, and concentrated. The
residue was further diluted with hexane. The solid was filtered
off and the filtrate was concentrated. The product was purified
by column chromatography (twice, SiO2, dichloromethane/
hexane as solvent) to give 19.09 g (40%) of 10 as a light orange
oil: 1H NMR (300 MHz, CDCl3) δ 9.11 (brs, 1 H), 8.74 (brs, 1
H), 8.35 (brs, 1 H), 4.41 (q, J ) 6.9 Hz, 2 H), 1.72-1.05 (m, 21
H), 0.89 (t, J ) 7.2 Hz, 9 H); MS (ES) m/z 442 (M + H+).
(5-Bromopyridin-3-yl)[3-((tert-butyldimethylsilanyl)-
oxy)propyl]carbamic Acid tert-Butyl Ester 6. A mixture

1894 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Kuo et al.
General Procedure for the Synthesis of 11, 16, 21, and
26. 5-(6-Chloropyrazin-2-yl)nicotinic Acid Ethyl Ester 11.
A mixture of 10 (10 g, 22.7 mmol), 2, 6-dichloropyrazine (6.76
g, 45.4 mmol), dichlorobis(triphenylphosphine)palladium (797
mg, 1.14 mmol), and LiCl (4.77 g, 113.5 mmol) in anhydrous
toluene (85 mL) was stirred at 100 °C for 23 h under nitrogen.
The cooled reaction mixture was concentrated under vacuum.
Aqueous sodium carbonate was added to the residue, and the
mixture stirred for 10 min and was extracted with dichlo-
romethane (3×). The combined dichloromethane solution was
filtered through Celite, dried (Na2SO4), and concentrated. The
product was purified by column chromatography (EtOAc/
hexane as solvent) to give 3.56 g (60%) of 11 as an off-white
solid: 1H NMR (300 MHz, CDCl3) δ 9.43 (brs, 1 H), 9.35 (brs,
1 H), 9.05 (s, 1 H), 8.91 (s, 1 H), 8.64 (s, 1 H), 4.48 (q, J ) 7.3
Hz, 2 H), 1.47 (t, J ) 7.2 Hz, 3 H); MS (ES) m/z 264 (M + H+).
Anal. (C12H10N3O2Cl) C, H, N.
General Procedure for the Synthesis of 12, 17, 22, and
27. 5-[6-((3-Chlorophenyl)amino)-pyrazin-2-yl]nicotinic
Acid Ethyl Ester 12. A mixture of 11 (24.3 g, 92.4 mmol),
3-chloroaniline (16.6 g, 129 mmol), Pd2(dba)3 (2.39 g, 2.31
mmol), DPPF (4.1 g, 7.4 mmol), and Cs2CO3 (60.2 g, 185 mmol)
in anhydrous dioxane (230 mL) was stirred at 110 °C for 46 h
under nitrogen. Dichloromethane (100 mL) was added to the
cooled reaction mixture. The mixture was then filtered through
Celite, the Celite cake was washed with more dichloromethane,
and the combined filtrate was concentrated to give a yellow-
brown solid. Small amounts of dichloromethane were added,
the solid was collected through filtration, and the filtrate was
concentrated. The solid collected was recrystallized from
EtOAc/hexane to give 21.83 g (67%) of compound 12 as a yellow
solid: 1H NMR (300 MHz, CDCl3) δ 9.44 (brs, 1 H), 9.31 (brs,
1 H), 8.90 (s, 1 H), 8.57 (s, 1 H), 8.26 (s, 1 H), 7.72 (s, 1 H),
7.44 (d, J ) 8.1 Hz, 1 H), 7.31 (t, J ) 8.0 Hz, 1 H), 7.08 (d, J
) 8.0 Hz, 1 H), 7.04 (s, 1 H), 4.48 (q, J ) 7.2 Hz, 2 H), 1.46 (t,
J ) 7.0 Hz, 3 H); MS (ES) m/z 355 (M + H+). Anal.
(C18H15N4O2Cl) C, H, N.
hexane as solvent) to give 29.28 g (89%) of 14 as a yellowish
foam: 1H NMR (300 MHz, CDCl3) δ 8.99 (s, 1 H), 8.80 (s, 1
H), 8.67 (d, J ) 1.7 Hz, 1 H), 8.49 (d, J ) 2.4 Hz, 1 H), 8.41
(brs, 1 H), 7.29 (m, 3 H), 7.17 (d, J ) 7.5 Hz, 1 H), 7.08 (s, 1
H), 1.54 (s, 9 H), 1.49 (s, 9 H); MS (ES) m/z 498 (M + H+).
Anal. (C25H28N5O4Cl) C, H, N.
General Procedure for the Synthesis of 15, 20, 25, and
30. 3-[[5-[6-[(3-chlorophenyl)amino]pyrazinyl]-3-pyridin-
yl]amino]-1-propanol 15. A mixture of 14 (3 g, 6 mmol), (3-
bromopropoxy)-tert-butyldimethylsilane (2.28 g, 9 mmol), and
Cs2CO3 (5.9 g, 18 mmol) in DMF (60 mL) was stirred at 70 °C
under nitrogen for 36 h. The reaction mixture was diluted with
water, extracted with ether (3×), dried (Na2SO4), and concen-
trated. The product was purified by column chromatography
(EtOAc/hexane as solvent) to give 3.04 g of the silylated
product as orange oil. The silylated product was dissolved in
TFA (30 mL) and stirred at 20 °C for 1 h before it was
concentrated. An ammonium hydroxide solution was added to
the residue until the pH was about 10-11, water was added,
and a yellow solid was formed. The yellow solid was collected
through filtration, washed with water, and dried under
vacuum. The yellow solid was recrystallized from CH3OH/
EtOAc to give 1.25 g (59%) of 15 as a light yellow solid: 1H
NMR (300 MHz, CD3OD) δ 8.45 (s, 1 H), 8.41 (d, J ) 1.6 Hz,
1 H), 8.12 (brs, 2 H), 8.00 (d, J ) 2.7 Hz, 1 H), 7.66 (brs, 1 H),
7.55 (d, J ) 8.3 Hz, 1 H), 7.30 (t, J ) 8.1 Hz, 1 H), 6.99 (d, J
) 7.9 Hz, 1 H), 3.72 (t, J ) 6.3 Hz, 2 H), 3.33 (t, J ) 6.8 Hz,
2 H), 1.91 (m, 2 H); MS (ES) m/z 356 (M + H+). Anal.
(C18H18N5OCl‚0.5H2O) C, H, N.
5-(5-Chloropyrazin-2-yl)nicotinic Acid Ethyl Ester 16.
Replacing 2,6-dichloropyrazine with 2,5-dichloropyrazine and
following the same procedure as in the preparation of 11 gave
16 as an off-white solid: 54% yield; 1H NMR (300 MHz, CDCl3)
δ 9.33 (d, J ) 2.0 Hz, 1 H), 9.23 (d, J ) 2.2 Hz, 1 H), 8.77 (t,
J ) 2.1 Hz, 1 H), 8.67 (d, J ) 2.4 Hz, 1 H), 8.45 (d, J ) 2.4 Hz,
1 H), 4.46 (q, J ) 7.1 Hz, 2 H), 1.44 (t, J ) 7.1 Hz, 3 H); MS
(ES) m/z 264 (M + H+). Anal. (C12H10N3O2Cl) C, H, N.
5-[5-(3-Chloro-phenylamino)pyrazin-2-yl]nicotinic Acid
Ethyl Ester 17. Replacing 11 with 16 and following the same
procedure as in the preparation of 12 gave 17 as an off-white
General Procedure for the Synthesis of 13, 18, 23, and
28. 5-{6-[tert-Butoxycarbonyl-(3-chlorophenyl)amino]py-
razin-2-yl}nicotinic Acid 13. A mixture of 12 (21.83 g, 61.7
mmol), Boc2O (26.9 g, 123 mmol), and DMAP (∼3 g, cat.) in
dichloromethane (500 mL) was stirred at 20 °C for 4 h. The
reaction mixture was concentrated and the product was
purified by column chromatography (EtOAc/hexane as solvent)
to give 26.7 g (95%) of Boc-12 as yellowish oil: 1H NMR (300
MHz, CDCl3) δ 9.22 (d, J ) 2.0 Hz, 1 H), 9.15 (d, J ) 2.2 Hz,
1 H), 9.10 (s, 1 H), 8.84 (s, 1 H), 8.64 (t, J ) 2.1 Hz, 1 H), 7.35
(m, 3 H), 7.18 (brd, J ) 7.3 Hz, 1 H), 4.44 (q, J ) 7.1 Hz, 2 H),
1.50 (s, 9 H), 1.44 (t, J ) 7.1 Hz, 3 H); MS (ES) m/z 455 (M +
H+).
1
solid: 55% yield; H NMR (300 MHz, CDCl3) δ 9.31 (d, J )
2.0 Hz, 1 H), 9.13 (d, J ) 2.2 Hz, 1 H), 8.65 (t, J ) 2.1 Hz, 1
H), 8.22 (m, 2 H), 7.67 (brs, 1 H), 7.26 (m, 2 H), 7.04 (m, 1 H),
6.63 (s, 1 H), 4.45 (q, J ) 7.1 Hz, 2 H), 1.43 (t, J ) 7.1 Hz, 3
H); MS (ES) m/z 355 (M + H+). Anal. (C18H15N4O2Cl) C, H, N.
5-{5-[(tert-Butoxycarbonyl)(3-chlorophenyl)amino]py-
razin-2-yl}nicotinic acid 18. Replacing 12 with 17 and
following the same procedure as in the preparation of 13 gave
18 as an off-white solid: 55% yield; MS (ES) m/z 425 (M -
H+).
[5-(5-((tert-Butoxycarbonyl)amino)pyridin-3-yl)pyrazin-
2-yl](3-chlorophen yl)carbamic Acid tert-Butyl Ester 19.
Replacing 13 with 18 and following the same procedure as in
the preparation of 14 gave 19 as an white foam: 74% yield;
1H NMR (300 MHz, CDCl3) δ 8.64 (d, J ) 2.1 Hz, 2 H), 8.54
(d, J ) 2.5 Hz, 1 H), 8.50 (d, J ) 2.3 Hz, 1 H), 8.31 (brs, 1 H),
7.26-7.00 (m, 4 H), 6.57 (s, 1 H), 1.52 (s, 9 H), 1.27 (s, 9 H);
MS (ES) m/z 498 (M + H+). Anal. (C25H28N5O4Cl) C, H, N.
3-[[5-[5-[(3-chlorophenyl)amino]pyrazinyl]-3-pyridin-
yl]amino]-1-propanol 20. Replacing 14 with 19 and following
the same procedure as in the preparation of 15 gave 20 as a
light yellow oil: 51% yield; 1H NMR (300 MHz, CDCl3) δ 8.15
(brs, 2 H), 8.08 (d, J ) 2.6 Hz, 1 H), 7.97 (d, J ) 2.6 Hz, 1 H),
7.72 (brs, 1 H), 7.33 (d, J ) 8.2 Hz, 1 H), 7.20 (m, 2 H), 7.09
(brs, 1 H), 6.99 (d, J ) 7.9 Hz, 1 H), 4.57 (m, 1 H), 3.77 (t, J
) 5.6 Hz, 2 H), 3.27 (m, 2 H), 1.86 (m, 2 H); MS (ES) m/z 356
(M + H+). Anal. (C18H18N5OCl‚0.2H2O) C, H, N.
5-(5-Bromopyrimidin-2-yl)nicotinic Acid Ethyl Ester
21. Replacing 2,6-dichloropyrazine with 5-bromo-2-iodopyri-
midine and following the same procedure as in the preparation
of 11 gave 21 as a yellow solid: 18% yield; 1H NMR (300 MHz,
CDCl3) δ 9.76 (d, J ) 2.1 Hz, 1 H), 9.33 (d, J ) 2.0 Hz, 1 H),
9.24 (t, J ) 2.1 Hz, 1 H), 8.91 (s, 2 H), 4.46 (q, J ) 7.1 Hz, 2
H), 1.45 (t, J ) 7.1 Hz, 3 H); MS (ES) m/z 310 (M + H+).
Compound Boc-12 (32.2 g, 71 mmol) was dissolved in
methanol (200 mL) and the mixture was stirred for 10 min
and then cooled to 0 °C. A NaOH(aq) (1 N, 118 mL) solution
was added slowly and the mixture was stirred at 0 °C for 20
min then stirred at 20 °C for another 18 h. Glacial acetic acid
(95 mL) was added to the reaction mixture at 0 °C, slowly
followed by the addition of water (350 mL). A yellow solid was
formed. The yellow solid was collected through filtration,
washed with water (5×), and dried in a vacuum oven overnight
to give 28.3 g (94%) of the carboxylic acid 13 as a yellow solid:
1H NMR (300 MHz, DMSO-d6) δ 9.27 (d, J ) 1.9 Hz, 1 H),
9.22 (s, 1 H), 9.09 (d, J ) 1.6 Hz, 1 H), 9.03 (s, 1 H), 8.65 (brs,
1 H), 7.52-7.31 (m, 4 H), 1.44 (s, 9 H); MS (ES) m/z 425 (M -
H+). Anal. (C21H19N4O4Cl) C, H, N.
General Procedure for the Synthesis of 14, 19, 24, and
29. [6-(5-((tert-Butoxycarbonyl)amino)pyridin-3-yl)pyra-
zin-2-yl](3-chlorophenyl)carbamic Acid tert-Butyl Ester
14. A mixture of the carboxylic acid 13 (28.3 g, 66.5 mmol),
DPPA (21.95 g, 80 mmol), triethylamine (13.43 g, 133 mmol),
and t-BuOH (280 mL) in toluene (200 mL) was stirred under
nitrogen at 70 °C for 30 min and then stirred at 100 °C for 4
h. The reaction mixture was concentrated under vacuum. The
product was purified by column chromatography (EtOAc/

Pyrazine-Pyridine as VEGFR-2 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1895
5-[5-(3-Chlorophenyl)amino)pyrimidin-2-yl]nicotinic
Acid Ethyl Ester 22. Replacing 16 with 21 and following the
same procedure as in the preparation of 17 gave 22 as a light
yellow solid: 27% yield; 1H NMR (300 MHz, CDCl3) δ 9.71 (d,
J ) 2.1 Hz, 1 H), 9.27 (d, J ) 2.0 Hz, 1 H), 9.19 (t, J ) 2.1 Hz,
1 H), 8.66 (s, 2 H), 7.29 (m, 1 H), 7.15 (t, J ) 1.9 Hz, 1 H), 7.04
(m, 2 H), 5.90 (s, 1 H), 4.46 (q, J ) 7.1 Hz, 2 H), 1.45 (t, J )
7.1 Hz, 3 H); MS (ES) m/z 355 (M + H+).
mmol), t-BuONa (3.609 g, 37.6 mmol), 2,6-dichloropyrazine (4
g, 26.85 mmol), and 3-chloroaniline (3.44 g, 26.85 mmol) in
toluene (100 mL) were stirred at 80 °C for 22 h under N2. CH2-
Cl2 was added, the reaction mixture was filtered through
Celite, and the solvent was evaporated. Product was purified
by column chromatography (EtOAc/hexane as solvent) to give
5.8 g (90%) of 32: 1H NMR (300 MHz, CDCl3) δ 8.11 (s, 1 H),
8.03 (s, 1 H), 7.49 (s, 1 H), 7.30 (m, 2 H), 7.12 (m, 1 H), 6.60
(brs, 1 H); MS (ES) m/z 241 (M + H+). Anal. (C10H7N3Cl‚
0.13H2O) C, H, N.
(3-Chlorophenyl)(6-chloropyrazin-2-yl)carbamic Acid
tert-Butyl Ester 33. A mixture of 32 (1.1 g, 4.6 mmol), Boc2O
(3 g, 13.8 mmol), and DMAP (∼200 mg, cat) in dichlo-
romethane (65 mL) was stirred at 20 °C for 16 h. The reaction
mixture was concentrated, and the product was purified by
column chromatography (EtOAc/hexane as solvent) to give 1.6
g (100%) of 33 as a tan wax: 1H NMR (300 MHz, CDCl3) δ
8.87 (s, 1 H), 8.32 (s, 1 H), 7.34-7.27 (m, 2 H), 7.21 (brs, 1 H),
7.10 (dt, J ) 6.9, 2.1 Hz, 1 H), 1.46 (s, 9 H); MS m/z 362 (M +
Na+).
6-(Trimethylstannanyl)pyrazine-2-carboxylicAcidMeth-
yl Ester 35. A mixture of methyl 6-chloro-2-pyrazinecarboxy-
late 34 (4.50 g, 26.1 mmol), bis(trimethyltin) (10.0 g, 30.5
mmol), tetrakis(triphenylphosphine)palladium (1.51 g, 1.31
mmol), LiCl (3.32 g, 78.3 mmol), and 2,6-di-tert-butyl-4-
methylphenol (0.230 g, 1.04 mmol) in anhydrous 1,4-dioxane
(80 mL) was refluxed at 100 °C for 4.5 h under nitrogen. After
being cooled to room temperature, the reaction mixture was
filtered. The solid was washed with dichloromethane and the
filtrate was concentrated and flash chromatographed (ethyl
acetate/hexane as solvent) to give 5.60 g (71%) of organostan-
nane 35 as a yellow oil: 1H NMR (300 MHz, CDCl3) δ 9.09 (s,
1 H), 8.73 (s, 1 H), 4.02 (s, 3 H), 0.44 (s, 9 H).
5-{5-[(tert-Butoxycarbonyl)(3-chlorophenyl)amino]py-
rimidin-2-yl}nicotinic Acid 23. Replacing 17 with 22 and
following the same procedure as in the preparation of 18 gave
23 as a yellow solid: 70% yield; MS (ES) m/z 425 (M - H+).
[2-(5-((tert-Butoxycarbonyl)amino)pyridin-3-yl)pyri-
midin-5-yl](3-chlorophenyl)carbamic Acid tert-Butyl Es-
ter 24. Replacing 18 with 23 and following the same procedure
as in the preparation of 19 gave 24 as a yellow foam: 76%
1
yield; H NMR (300 MHz, CDCl3) δ 9.28 (d, J ) 1.7 Hz, 1 H),
8.90 (s, 1 H), 8.67 (s, 2 H), 8.65 (brs, 1 H), 7.41-7.12 (m, 5 H),
1.55 (s, 9 H), 1.50 (s, 9 H); MS (ES) m/z 498 (M + H+).
3-[[5-[5-[(3-chlorophenyl)amino]-2-pyrimidinyl]-3-py-
ridinyl]amino]-1-propanol 25. Replacing 19 with 24 and
following the same procedure as in the preparation of 20 gave
25 as a yellow foam: 65% yield: 1H NMR (300 MHz, DMSO-
d6) δ 8.91 (s, 1 H), 8.71 (s, 2 H), 8.64 (brs, 1 H), 8.01 (d, J )
2.6 Hz, 1 H), 7.72 (brs, 1 H), 7.31 (t, J ) 8.0 Hz, 1 H), 7.16 (s,
1 H), 7.14 (d, J ) 8.2 Hz, 1 H), 6.97 (d, J ) 7.0 Hz, 1 H), 6.04
(t, J ) 5.3 Hz, 1 H), 4.55 (t, J ) 5.1 Hz, 1 H), 3.53 (q, J ) 5.4
Hz, 2 H), 3.14 (q, J ) 5.9 Hz, 2 H), 1.74 (m, 2 H); MS (ES) m/z
356 (M + H+). Anal. (C18H18N5OCl‚0.5H2O) C, H, N.
5-(5-Bromopyrimidin-4-yl)nicotinic Acid Ethyl Ester
26. Replacing 2,6-dichloropyrazine with 4-chloro-5-bromopy-
rimidine and following the same procedure as in the prepara-
1
tion of 11 gave 26 as an off-white solid: 16% yield; H NMR
(300 MHz, CDCl3) δ 9.35 (d, J ) 2.0 Hz, 1 H), 9.24 (m, 2 H),
9.00 (s, 1 H), 8.78 (t, J ) 2.1 Hz, 1 H), 4.46 (q, J ) 7.1 Hz, 2
H), 1.44 (t, J ) 7.1 Hz, 3 H); MS (ES) m/z 310 (M + H+). Anal.
(C12H10N3O2Br) C, H, N.
6′-[(tert-Butoxycarbonyl)(3-chlorophenyl)amino][2,2′]-
bipyrazinyl-6-carboxylic Acid Methyl Ester 36. A mixture
of 35 (536 mg, 1.78 mmol), compound 33 (666 mg, 1.96 mmol),
dichlorobis(triphenylphosphine)palladium (125 mg, 0.178 mmol),
and LiCl (230 mg, 5.42 mmol) in anhydrous toluene (15 mL)
was stirred at 100 °C for 6.5 h under nitrogen. The cooled
reaction mixture was concentrated under vacuum and purified
by flash chromatography (EtOAc/hexane as solvent) to give
593 mg (75%) of 36 as an off-white solid: 1H NMR (300 MHz,
CDCl3) δ 9.40 (s, 1 H), 9.27 (s, 1 H), 9.20 (s, 1 H), 9.17 (s, 1 H),
7.41-7.32 (m, 2 H), 7.29 (m, 1 H), 7.19-7.16 (m, 1 H), 4.07 (s,
3 H), 1.49 (s, 9 H); MS (ES) m/z 464 (M + Na+). Anal.
(C21H20N5O4Cl) C, H, N.
{6′-[(tert-Butoxycarbonyl)(3-chlorophenyl)amino][2,2′]-
bipyrazinyl-6-yl}carbamic Acid tert-Butyl Ester 37. Com-
pound 36 (1.31 g, 2.97 mmol) was treated with LiOH (0.110 g,
4.58 mmol) in THF (10 mL) and water (2.5 mL) for 2 h at room
temperature. Acetic acid (0.55 mL) was added and the solution
was concentrated to about half of the original volume. After
the addition of water, some solid formed. The solid was
collected through filtration, washed with water, and dried
under vacuum overnight to give 1.22 g (96%) of a carboxylic
acid as a yellow solid: MS (ES) m/z 450 (M + Na+). A mixture
of the carboxylic acid (350 mg, 0.819 mmol), DPPA (340 mg,
1.24 mmol), and triethylamine (0.230 mL, 1.65 mmol) in
t-BuOH (5 mL) and toluene (4 mL) was heated at 60 °C for 30
min and then the temperature was slowly increased to 80 °C
for 2 h and 90 °C for 1 h. After concentration, the reaction
mixture was purified by flash chromatography (EtOAc/hexane
as solvent) to give 278 mg (68%) of 37 as a yellow solid: 1H
NMR (300 MHz, CDCl3) δ 9.31 (s, 1 H), 9.23 (s, 1 H), 9.16 (s,
1 H), 8.70 (s, 1 H), 7.96 (brs, 1 H), 7.39-7.30 (m, 2 H), 7.27
(m, 1 H), 7.19-7.15 (m, 1 H), 1.57 (s, 9 H), 1.48 (s, 9 H); MS
(ES) m/z 521 (M + Na+).
5-[5-(3-Chlorophenyl)amino)pyrimidin-4-yl]nicotinic
Acid Ethyl Ester 27. Replacing 21 with 26 and following the
same procedure as in the preparation of 22 gave 27 as a yellow-
brown foam: 84% yield; 1H NMR (300 MHz, CDCl3) δ 9.14
(brs, 2 H), 8.97 (d, J ) 1.3 Hz, 1 H), 8.79 (s, 1 H), 8.67 (d, J )
1.5 Hz, 1 H), 7.19 (t, J ) 8.4 Hz, 1 H), 6.99 (m, 2 H), 6.86 (brd,
J ) 8.0 Hz, 1 H), 6.21 (brs, 1 H), 4.40 (q, J ) 7.1 Hz, 2 H),
1.40 (t, J ) 7.1 Hz, 3 H); MS (ES) m/z 355 (M + H+).
5-{5-[(tert-Butoxycarbonyl)(3-chlorophenyl)amino]py-
rimidin-4-yl}nicotinic Acid 28. Replacing 22 with 27 and
following the same procedure as in the preparation of 23 gave
28 as a yellow solid: 92% yield; 1H NMR (300 MHz, CDCl3) δ
9.35 (d, J ) 1.7 Hz, 1 H), 9.28 (s, 1 H), 9.18 (d, J ) 2.1 Hz, 1
H), 8.78 (s, 1 H), 8.72 (t, J ) 2.0 Hz, 1 H), 7.11(m, 3 H), 6.90
(brd, J ) 7.4 Hz, 1 H), 1.35 (s, 9 H); MS (ES) m/z 425 (M -
H+).
[4-(5-((tert-Butoxycarbonyl)amino)pyridin-3-yl)pyri-
midin-5-yl](3-chlorophenyl)carbamic Acid tert-Butyl Es-
ter 29. Replacing 23 with 28 and following the same procedure
as in the preparation of 24 gave 29 as a yellowish foam: 81%
yield; 1H NMR (300 MHz, CDCl3) δ 9.22 (s, 1 H), 8.70 (s, 1 H),
8.58 (d, J ) 1.9 Hz, 1 H), 8.52 (d, J ) 2.4 Hz, 1 H), 8.34 (brs,
1 H), 7.16 (m, 3 H), 6.90 (d, J ) 7.9 Hz, 1 H), 6.69 (s, 1 H),
1.52 (s, 9 H), 1.34 (s, 9 H); MS (ES) m/z 498 (M + H+). Anal.
(C25H28N5O4Cl) C, H, N.
3-[[5-[5-[(3-Chlorophenyl)amino]-4-pyrimidinyl]-3-py-
ridinyl]amino]-1-propanol 30. Replacing 24 with 29 and
following the same procedure as in the preparation of 25 gave
1
30 as a yellow solid: 51% yield; H NMR (300 MHz, CD3OD)
δ 8.88 (s, 1 H), 8.75 (s, 1 H), 8.22 (d, J ) 1.6 Hz, 1 H), 7.97 (d,
J ) 2.7 Hz, 1 H), 7.18 (m, 2 H), 7.03 (m, 2 H), 6.90 (dd, J )
7.7, 1.5 Hz, 1 H), 6.24 (s, 1 H), 4.40 (brt, J ) 5.1 Hz, 1 H), 3.79
(t, J ) 5.7 Hz, 2 H), 3.27 (q, J ) 5.7 Hz, 2 H), 1.88 (m, 2 H);
MS (ES) m/z 356 (M + H+). Anal. (C18H18N5OCl‚0.4H2O) C,
H, N.
3-[[6′-((3-Chlorophenyl)amino)[2,2′]bipyrazinyl-6-yl]-
amino]propan-1ol 38. A mixture of compound 37 (60 mg,
0.12 mmol), (3-bromopropoxy)-tert-butyldimethylsilane (40 mg,
0.16 mmol), and Cs2CO3 (80 mg, 0.25 mmol) in dry DMF (1
mL) was heated at 70 °C for 3.5 h. The reaction mixture was
concentrated and purified by column chromatography (EtOAc/
hexane as solvent) to give 71 mg (88%) of the Boc2-38 as an
(3-Chlorophenyl)(6-chloropyrazin-2-yl)amine 32. Pal-
ladium acetate (0.121 g, 0.537 mmol), BINAP (0.368 g 0.591

1896 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Kuo et al.
off-white solid. Anal. Calcd for C33H47N6O5ClSi: C, 59.04; H,
7.06; N, 12.52. Found: C, 59.24; H, 7.00; N, 12.11.
addition of water. The precipitated solid was collected through
filtration, washed with water and Et2O, and dried under
vacuum to provide 28 mg (100%) of 42 as a yellow solid: 1H
NMR (300 MHz, DMSO-d6) δ 9.80 (s, 1 H), 8.85 (s, 1 H), 8.24
(s, 1 H), 8.12 (s, 1 H), 7.64 (d, J ) 8.1 Hz, 1 H), 7.56 (m, 1 H),
7.40-7.35 (m, 2 H), 7.02 (d, J ) 7.7 Hz, 1 H), 6.71 (brs, 1 H),
6.57 (d, J ) 8.3 Hz, 1 H), 4.48 (brs, 1 H), 3.53 (t, J ) 6.3 Hz,
2 H), 3.40 (m, 2 H), 1.76 (m, 2 H); MS (ES) m/z 356 (M + H+).
Anal. (C18H18N5OCl‚0.55H2O) C, H, N.
(4-Iodopyridin-2-yl)carbamic Acid tert-Butyl Ester 44.
A mixture of 4-iodopicolinic acid 43 (1.00 g, 3.30 mmol), DPPA
(1.36 g, 4.95 mmol), triethylamine (1.4 mL, 10 mmol) in
t-BuOH (5.5 mL), and toluene (5 mL) was heated at 65 °C for
1.5 h and then 100 °C for 4 h. After concentration, the reaction
mixture was purified by flash chromatography (EtOAc/hexane
as solvent) to give 515 mg (50%) of 44 as a white solid: 1H
NMR (300 MHz, CDCl3) δ 9.17 (brs, 1 H), 8.48 (s, 1 H), 7.98
(dd, J ) 5.2, 1.5 Hz, 1 H), 7.34 (dd, J ) 5.2, 1.3 Hz, 1 H), 1.56
(s, 9 H). MS (ES) m/z 343 (M + Na+).
[3-(tert-Butyldimethylsiloxy)propyl](4-iodopyridin-2-
yl)carbamic Acid tert-Butyl Ester 45. A mixture of com-
pound 44 (330 mg, 1.03 mmol), (3-bromopropoxy)-tert-bu-
tyldimethylsilane (340 mg, 1.34 mmol), and Cs2CO3 (504 mg,
1.55 mmol) in dry DMF (4 mL) was stirred at 70 °C for 3 h.
The solvent was evaporated under reduced pressure and the
residue was purified by column chromatography (EtOAc/
hexane) to provide 450 mg (89%) of compound 45 as a clear
oil: 1H NMR (400 MHz, CDCl3) δ 8.11 (s, 1 H), 8.00 (d, J )
5.2 Hz, 1 H), 7.33 (dd, J ) 5.2, 1.3 Hz, 1 H), 3.99 (t, J ) 7.3
Hz, 2 H), 3.65 (t, J ) 6.3 Hz, 2 H), 1.84 (m, 2 H), 1.52 (s, 9 H),
0.87 (s, 9 H), 0.02 (s, 6 H); MS (ES) m/z 515 (M + Na+).
[3-(tert-Butyldimethylsiloxy)propyl](4-(trimethylstan-
nanyl)pyridin-2-yl)carbamic Acid tert-Butyl Ester 46. A
mixture of compound 45 (650 mg, 1.32 mmol), bis(trimethyltin)
(870 mg, 2.66 mmol), tetrakis(triphenylphosphine)palladium
(150 mg, 0.130 mmol), LiCl (170 mg, 4.00 mmol), and 2,6-di-
tert-butyl-4-methylphenol (12 mg, 0.054 mmol) in anhydrous
1,4-dioxane (12 mL) was heated at 90 °C for 1.5 h under
nitrogen. The solvent was removed under reduced pressure
and the residue was chromatographed over silica gel (EtOAc/
hexane as solvent) to give 590 mg (84%) of 46 as a clear oil:
1H NMR (300 MHz, CDCl3) δ 8.09 (d, J ) 4.7 Hz, 1 H), 7.56
(s, 1 H), 7.10 (d, J ) 4.7 Hz, 1 H), 3.97 (t, J ) 7.2 Hz, 2 H),
3.64 (t, J ) 6.5 Hz, 2 H), 1.85 (m, 2 H), 1.49 (s, 9 H), 0.86 (s,
9 H), 0.33 (s, 9 H), 0.00 (s, 6 H); MS (ES) m/z 527 (M - H+).
Anal. (C22H42N2O3SiSn) C, H, N.
3-{[4-[6-((3-Chlorophenyl)amino)pyrazin-2-yl]pyridin-
2-yl]amino}propan-1-ol 47. A mixture of compound 46 (550
mg, 1.04 mmol), compound 33 (360 mg, 1.06 mmol), dichloro-
bis(triphenylphosphine)palladium (73 mg, 0.104 mmol), and
LiCl (132 mg, 3.11 mmol) in anhydrous toluene (20 mL) was
stirred at 90 °C for 1 h and then 100 °C for 2 h under nitrogen.
The cooled reaction mixture was concentrated under vacuum
and purified by flash chromatography (EtOAc/hexane as
solvent) to give 380 mg (55%) of Boc2-47 as a yellow oil: 1H
NMR (300 MHz, CDCl3) δ 9.00 (s, 1 H), 8.82 (s, 1 H), 8.40 (d,
J ) 5.2 Hz, 1 H), 8.04 (s, 1 H), 7.37-7.28 (m, 4 H), 7.16 (brd,
J ) 7.3 Hz, 1 H), 4.02 (m, 2 H), 3.65 (t, J ) 6.3 Hz, 2 H), 1.85
(m, 2 H), 1.50 (s, 9 H), 1.49 (s, 9 H), 0.85 (s, 9 H), 0.00 (s, 6 H);
MS (ES) m/z 670 (M + H+).
A solution of Boc2-47 (350 mg, 0.522 mmol) and TFA (4 mL)
was left at room temperature overnight before concentration.
A saturated NH4OH solution was added to the residue until
the mixture was made basic followed by the addition of water.
The precipitated solid was collected through filtration, washed
with water, dried under vacuum, and purified by flash chro-
matography (CH2Cl2/MeOH as solvent) to provide 143 mg
(77%) of 47 as a yellow solid: 1H NMR (300 MHz, DMSO-d6)
δ 9.88 (s, 1 H), 8.54 (s, 1 H), 8.27 (s, 1 H), 8.10 (d, J ) 5.8 Hz,
1 H), 8.00 (s, 1 H), 7.73 (d, J ) 8.2 Hz, 1 H), 7.39 (t, J ) 8.1
Hz, 1 H), 7.10 (s, 2 H), 7.04 (dd, J ) 7.9, 1.0 Hz, 1 H), 6.69
(brt, J ) 5.3 Hz, 1 H), 4.55 (brs, 1 H), 3.51 (t, J ) 6.2 Hz, 2 H),
3.36 (m, 2 H), 1.73 (m, 2 H); MS (ES) m/z 356 (M + H+). Anal.
(C18H18N5OCl‚0.3H2O) C, H, N.
Boc2-38 (250 mg, 0.373 mmol) was dissolved in TFA (4 mL)
and the solution was stirred at room temperature for 3 h before
concentration. A saturated NH4OH solution was added to the
residue until the mixture was made basic followed by the
addition of water. The precipitated solid was collected through
filtration, again washed with water, and dried under vacuum.
The product was purified by flash chromatography on silica
gel (EtOAc/MeOH as solvent) to provide 112 mg (84%) of 38
as a yellow solid: 1H NMR (300 MHz, DMSO-d6) δ 9.89 (s, 1
H), 8.79 (s, 1 H), 8.47 (s, 1 H), 8.28 (s, 1 H), 8.13 (s, 1 H), 8.00
(s, 1 H), 7.62 (d, J ) 8.2 Hz, 1 H), 7.39 (t, J ) 8.0 Hz, 1 H),
7.29 (m, 1 H), 7.04 (d, J ) 8.1 Hz, 1 H), 4.51 (brs, 1 H), 3.53
(m, 2 H), 3.47-3.41 (m, 2 H), 1.82-1.73 (m, 2 H); MS (ES)
m/z 357 (M + H+); FAB-HRMS (M + H+) calcd for C17H18N6-
OCl 357.1230, found 357.1215.
(6-Bromopyridin-2-yl)[3-(tert-butyldimethylsiloxy)pro-
pyl]carbamic Acid tert-Butyl Ester 40. A mixture of
2-amino-6-bromopyridine compound 39 (3.46 g, 20.0 mmol),
di-tert-butyl dicarbonate (4.80 g, 22.0 mmol), and 4-(dimethyl-
amino)pyridine (0.244 g, 2 mmol) in t-BuOH (50 mL) was
stirred at room temperature for more than 24 h. The solution
was concentrated and purified by flash chromatography
(EtOAc/hexane as solvent) to give 1.36 g (25%) of Boc-39 as
clear crystals: 1H NMR (300 MHz, CDCl3) δ 7.88 (d, J ) 8.2
Hz, 1 H), 7.50 (t, J ) 8.0 Hz, 1 H), 7.22 (brs, 1 H), 7.12 (d, J
) 7.7 Hz, 1 H), 1.51 (s, 9 H).
(3-Bromopropoxy)-tert-butyldimethylsilane (1.50 g, 5.93 mmol)
and Cs2CO3 (2.43 g, 7.46 mmol) were added to the solution of
Boc-39 in dry DMF (20 mL). After stirring at 70 °C for 18 h,
the solvent was evaporated under reduced pressure and the
residue was purified by column chromatography (EtOAc/
hexane) to provide 1.93 g (88%) of compound 40 as a clear oil:
1H NMR (300 MHz, CDCl3) δ 7.66 (d, J ) 8.2 Hz, 1 H), 7.44
(m, 1 H), 7.15 (d, J ) 7.6 Hz, 1 H), 3.99 (t, J ) 7.3 Hz, 2 H),
3.67 (t, J ) 6.4 Hz, 2 H), 1.86 (m, 2 H), 1.51 (s, 9 H), 0.88 (s,
9 H), 0.03 (s, 6 H); MS (ES) m/z 467 (M + Na+). Anal.
(C19H33N2O3BrSi) C, H, N.
[3-(tert-Butyldimethylsiloxy)propyl](6-(tributylstan-
nanyl)pyridin-2-yl)carbamic Acid tert-Butyl Ester 41. A
mixture of compound 40 (150 mg, 0.337 mmol), bis(tributyltin)
(0.340 mL, 0.673 mmol), tetrakis(triphenylphosphine)pal-
ladium (40 mg, 0.035 mmol), LiCl (43 mg, 1.0 mmol), and 2,6-
di-tert-butyl-4-methylphenol (3 mg, 0.014 mmol) in anhydrous
1,4-dioxane (3 mL) was refluxed at 100 °C for 4 h under
nitrogen. The solvent was removed under reduced pressure
and the residue was chromatographed over silica gel (ethyl
acetate/hexane as solvent) to give 152 mg (69%) of 41 as a clear
oil: 1H NMR (300 MHz, CDCl3) δ 7.49 (d, J ) 8.5 Hz, 1 H),
7.42 (m, 1 H), 7.08 (d, J ) 7.7 Hz, 1 H), 4.03 (t, J ) 7.3 Hz, 2
H), 3.65 (t, J ) 6.6 Hz, 2 H), 1.88 (m, 2 H), 1.61-1.50 (m, 6
H), 1.52 (s, 9 H), 1.37-1.26 (m, 6 H), 1.10-1.05 (m, 6 H), 0.88
(t, J ) 7.4 Hz, 9 H), 0.87 (s, 9 H), 0.02 (s, 6 H); MS (ES) m/z
656 (M + H+).
3-{[6-[6-((3-Chlorophenyl)amino)pyrazin-2-yl]pyridin-
2-yl]amino}propan-1-ol 42. A mixture of compound 41 (103
mg, 0.157 mmol), N-(tert-butoxycarbonyl)-N-(3-chlorophenyl)-
2-amino-6-chloropyrazine 33 (61 mg, 0.18 mmol), dichlorobis-
(triphenylphosphine)palladium (13 mg, 0.018 mmol), and LiCl
(23 mg, 0.54 mmol) in anhydrous toluene (3 mL) was stirred
at 100 °C for 3 h under nitrogen. The cooled reaction mixture
was concentrated under vacuum and purified by flash chro-
matography (EtOAc/hexane as solvent) to give 63 mg (62%)
of Boc2-42 as a yellow oil: 1H NMR (300 MHz, CDCl3) δ 9.29
(s, 1 H), 8.93 (s, 1 H), 7.73 (dd, J ) 7.8, 1.3 Hz, 1 H), 7.65 (m,
1 H), 7.61-7.58 (m, 1 H), 7.37-7.28 (m, 3 H), 7.19-7.15 (m, 1
H), 4.12 (t, J ) 7.4 Hz, 2 H), 3.71 (t, J ) 6.2 Hz, 2 H), 2.01-
1.92 (m, 2 H), 1.53 (s, 9 H), 1.49 (s, 9 H), 0.85 (s, 9 H), 0.02 (s,
6 H); MS (ES) m/z 692 (M + Na+).
Boc2-42 (53 mg, 0.079 mmol) was dissolved in TFA (1.5 mL)
and the solution was stirred at room temperature for 2 h before
concentration. A saturated NH4OH solution was added to the
residue until the mixture was made basic followed by the

Pyrazine-Pyridine as VEGFR-2 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1897
(4-(Trimethylstannanyl)pyridin-3-yl)carbamic Acid tert-
Butyl Ester 49. A mixture of 4-(trimethylstannyl)nicotinic
acid 48 (300 mg, 1.05 mmol), DPPA (433 mg, 1.57 mmol), and
triethylamine (0.250 mL, 1.79 mmol) in t-BuOH (2 mL) and
toluene (2 mL) was heated at 60 °C for 1.5 h and then 90 °C
for 3.5 h. After concentration, the reaction mixture was
purified by flash chromatography (EtOAc/hexane as solvent)
to give 213 mg (57%) of 49 as a white solid: 1H NMR (300
MHz, CDCl3) δ 8.54 (s, 1 H), 8.31 (d, J ) 4.5 Hz, 1 H), 7.34 (d,
J ) 4.5 Hz, 1 H), 6.30 (brs, 1 H), 1.51 (s, 9 H), 0.37 (s, 9 H).
[6-(3-((tert-Butoxycarbonyl)amino)pyridin-4-yl)pyrazin-
2-yl](3-chlorophenyl)carbamic Acid tert-Butyl Ester 50.
A mixture of 49 (162 mg, 0.454 mmol), 33 (155 mg, 0.454
mmol), dichlorobis(triphenylphosphine)palladium (64 mg, 0.091
mmol), and LiCl (60 mg, 1.42 mmol) in anhydrous toluene (10
mL) was stirred at 100 °C for 4 h under nitrogen. The reaction
mixture was concentrated and purified by flash chromatog-
raphy (EtOAc/hexane as solvent) to give 156 mg (69%) of 50
as a white solid: 1H NMR (300 MHz, CDCl3) δ 9.37 (s, 1 H),
9.23 (s, 1 H), 9.05 (s, 1 H), 8.71 (s, 1 H), 8.37 (d, J ) 5.1 Hz, 1
H), 7.47-7.27 (m, 3 H), 7.20 (dd, J ) 7.0, 2.0 Hz, 1 H), 1.56 (s,
9 H), 1.48 (s, 9 H); MS (ES) m/z 498 (M + H+).
3-{[4-[6-((3-Chlorophenyl)amino)pyrazin-2-yl]pyridin-
3-yl]amino}propan-1-ol 51. A mixture of compound 50 (29
mg, 0.058 mmol), (3-bromopropoxy)-tert-butyldimethylsilane
(22 mg, 0.087 mmol), and Cs2CO3 (38 mg, 0.12 mmol) in dry
DMF (1 mL) was heated at 70 °C for 2 h. The reaction mixture
was concentrated and purified by column chromatography
(EtOAc/hexane as solvent) to give 32 mg (82%) of Boc2-51 as
a yellow oil; MS (ES) m/z 670 (M + H+).
Boc2-51 (60 mg, 0.089 mmol) was dissolved in TFA (2 mL)
and the solution was stirred at room temperature for 1.5 h
before concentration. A saturated NH4OH solution was added
to the residue until the mixture was made basic followed by
the addition of water. The precipitated solid was collected
through filtration, washed with water, and dried under
vacuum. The product was purified by flash chromatography
on silica gel (CH2Cl2/MeOH as solvent) to provide 28 mg (88%)
of 51 as a yellow solid: 1H NMR (300 MHz, DMSO-d6) δ 9.90
(s, 1 H), 8.51 (s, 1 H), 8.28 (s, 1 H), 8.22 (s, 1 H), 7.95 (brd, J
) 4.4 Hz, 1 H), 7.83 (s, 1 H), 7.58 (d, J ) 4.8 Hz, 1 H), 7.51 (d,
J ) 8.5 Hz, 1 H), 7.41 (t, J ) 8.0 Hz, 1 H), 7.11 (m, 2 H), 4.48
(m, 1 H), 3.37 (m, 2 H), 3.29 (m, 2 H), 1.08 (m, 2 H); MS (ES)
m/z 356 (M + H+). Anal. (C18H18N5OCl‚0.6H2O) C, H, N.
6-{6-[(tert-Butoxycarbonyl)(3-chlorophenyl)amino]py-
razin-2-yl}nicotinic Acid Methyl Ester 53. A mixture of
methyl 6-(((trifluoromethyl)sulfonyl)oxy)nicotinate 52 (2.00 g,
7.02 mmol), bis(trimethyltin) (3.50 g, 10.7 mmol), tetrakis-
(triphenylphosphine)palladium (0.810 g, 0.700 mmol), anhy-
drous LiCl (0.900 g, 21.2 mmol), and 2,6-di-tert-butyl-4-
methylphenol (0.062 g, 0.28 mmol) in anhydrous 1,4-dioxane
(60 mL) was refluxed at 100 °C for 2 h under nitrogen. After
being cooled to room temperature, the reaction mixture was
filtered through Celite and washed with dichloromethane. The
filtrate was concentrated under vacuum. Water and Et2O were
added to the residue and the resulting mixture was filtered
again through Celite. The filtrate was separated and the
aqueous layer was extracted with Et2O. The organic layers
were combined and washed with 15% NH4OH (2×), and the
aqueous layer was back-extracted with Et2O. The combined
organic phases were dried over Na2SO4, filtered, and concen-
trated to give 2.31 g of crude methyl 6-(trimethylstannyl)-
nicotinate as a brown oil.
Compound 33 (2.20 g, 6.47 mmol), dichlorobis(triphenylphos-
phine)palladium (0.780 g, 1.11 mmol), LiCl (0.900 g, 21.2
mmol), and anhydrous toluene (80 mL) were added to the
crude organostannane. The reaction mixture was heated at
100 °C for 1 h under nitrogen and then left at room temper-
ature overnight. After filtration through Celite, the filtrate was
concentrated and flash chromatographed (EtOAc/hexane as
solvent) to give 1.19 g (38%, two steps) of 53 as a white solid:
1H NMR (300 MHz, CDCl3) δ 9.43 (s, 1 H), 9.24 (d, J ) 2.1
Hz, 1 H), 9.06 (s, 1 H), 8.31 (dd, J ) 8.3, 2.1 Hz, 1 H), 7.93 (d,
J ) 8.3 Hz, 1 H), 7.39-7.31 (m, 3 H), 7.19-7.15 (m, 1 H), 3.97
(s, 3 H), 1.49 (s, 9 H); MS (ES) m/z 463 (M + Na+). Anal.
(C22H21N4O4Cl) C, H, N.
[6-(5-((tert-Butoxycarbonyl)amino)pyridin-2-yl)pyrazin-
2-yl](3-chlorophenyl)carbamic Acid tert-Butyl Ester 54.
A mixture of 53 (540 mg, 1.22 mmol) and LiOH (44 mg, 1.8
mmol) in THF (16 mL) and water (4 mL) was stirred for 1 h
at room temperature. More LiOH (30 mg, 1.3 mmol) was added
to the cloudy solution. After another 3 h at room temperature,
the solution became clear. Acetic acid (0.30 mL) was added
and the solution was concentrated to about half of the original
volume. After the addition of water, some solid formed. The
solid was collected through filtration, washed with water, and
dried under vacuum overnight to give 522 mg (100%) of a
carboxylic acid as a yellow solid: MS (ES) m/z 425 (M - H+).
A mixture of the carboxylic acid (522 mg, 1.22 mmol), DPPA
(500 mg, 1.82 mmol), and triethylamine (0.340 mL, 2.44 mmol)
in t-BuOH (9 mL) and toluene (9 mL) was heated at 60 °C for
30 min and then the temperature was slowly increased to 80
°C for 2 h and 90 °C for 1 h. After concentration, the reaction
mixture was purified by flash chromatography (EtOAc/hexane
as solvent) to give 393 mg (65%) of 54 as a yellow solid: 1H
NMR (300 MHz, CDCl3) δ 9.31 (s, 1 H), 8.92 (s, 1 H), 8.45 (d,
J ) 2.4 Hz, 1 H), 8.03 (brd, J ) 8.5 Hz, 1 H), 7.84 (d, J ) 8.6
Hz, 1 H), 7.37-7.29 (m, 3 H), 7.19-7.16 (m, 1 H), 6.67 (s, 1
H), 1.54 (s, 9 H), 1.49 (s, 9 H); MS (ES) m/z 520 (M + Na+).
Anal. (C25H28N5O4Cl) C, H, N.
3-{[6-[6-((3-Chlorophenyl)amino)pyrazin-2-yl]pyridin-
3-yl]amino}propan-1-ol 55. A mixture of 54 (43 mg, 0.086
mmol), (3-bromopropoxy)-tert-butyldimethylsilane (30 mg, 0.12
mmol), and Cs2CO3 (45 mg, 0.14 mmol) in dry DMF (1 mL)
was heated at 65 °C for 2 h then 80 °C for 2.5 h. The reaction
mixture was concentrated and purified by column chromatog-
raphy (EtOAc/hexane as solvent) to give 51 mg (88%) of Boc2-
55 as a clear oil.
Boc2-55 (385 mg, 0.575 mmol) was dissolved in TFA (6 mL)
and the solution was stirred at room temperature for 3 h before
concentration. A saturated NH4OH solution was added to the
residue until the mixture was made basic, followed by the
addition of water. The precipitated solid was collected through
filtration, washed with water, and dried under vacuum. The
product was purified by flash chromatography on silica gel
(CH2Cl2/MeOH as solvent) to provide 176 mg (86%) of 55 as a
yellow solid: 1H NMR (300 MHz, DMSO-d6) δ 9.70 (s, 1 H),
8.74 (s, 1 H), 8.11-8.08 (m, 3 H), 7.99 (d, J ) 8.6 Hz, 1 H),
7.63 (d, J ) 8.4 Hz, 1 H), 7.37 (t, J ) 8.1 Hz, 1 H), 7.06 (dd, J
) 8.6, 2.6 Hz, 1 H), 7.01 (d, J ) 7.9 Hz, 1 H), 6.37 (m, 1 H),
4.52 (t, J ) 5.0 Hz, 1 H), 3.53 (m, 2 H), 3.18 (m, 2 H), 1.74 (m,
2 H); MS (ES) m/z 356 (M + H+). Anal. (C18H18N5OCl‚0.24H2O)
C, H, N.
(5-Bromothiazol-2-yl)carbamic Acid tert-Butyl Ester
57. A mixture of (2-amino-5-bromothiazole) 56 (10 g, 38.4
mmol), Boc2O (8.39 g, 38.4 mmol), DMAP (440 mg, 3.6 mmol),
and NaHCO3 (9.6 g, 114 mmol) in t-BuOH (100 mL) was
stirred at 20 °C for 16 h. The reaction mixture was concen-
trated, and the product was purified by column chromatogra-
phy (EtOAc/hexane as solvent) and recrystallized from ethyl
acetate/hexane to give 3.41 g (32%) of 57 as a white solid: 1H
NMR (300 MHz, DMSO-d6) δ 11.70 (brs, 1 H), 7.43 (s, 1 H),
1.48 (s, 9 H). Anal. (C8H11BrN2O2S) C, H, N.
(5-Bromothiazol-2-yl)[3-(tert-butyldimethylsiloxy)pro-
pyl]carbamic Acid tert-Butyl Ester 58. A mixture of
compound 57 (2.2 g, 7.91 mmol), (3-bromopropoxy)-tert-bu-
tyldimethylsilane (3.46 g, 12.7 mmol), and Cs2CO3 (7.7 g, 23.7
mmol) in DMF (24 mL) was stirred at 70 °C under nitrogen
for 18 h. The reaction mixture was diluted with water,
extracted with ether (3×), dried (Na2SO4), and concentrated.
The product was purified by column chromatography (EtOAc/
hexane as solvent) to give 2.6 g (72%) of the silylated product
compound 58 as an oil.1H NMR (300 MHz, CDCl3) δ 7.22 (s, 1
H), 4.08 (t, J ) 7.5 Hz, 2 H), 3.65 (t, J ) 6.3 Hz, 2 H), 1.85 (m,
2 H), 1.53 (s, 9 H), 0.85 (s, 9 H), 0.01 (s, 6 H). Anal. (C17H31-
BrN2O3SSi) C, H, N.
3-{[5-[6-((3-Chlorophenyl)amino)pyrazin-2-yl]thiazol-
2-yl]amino}propan-1-ol 59. A mixture of compound 58 (152

1898 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Kuo et al.
mg, 0.337 mmol), bis(tributyltin) (390 mg, 0.34 mmol), tetrakis-
(triphenylphosphine)palladium (40 mg, 0.035 mmol), LiCl (43
mg, 1.0 mmol), and BHT (3 mg, 0.014 mmol) in dioxane (3
mL) was stirred at 100 °C for 4 h under nitrogen. The cooled
reaction mixture was concentrated under vacuum. The residue
was purified by column chromatography (EtOAc/hexane as
solvent) to give 56 mg (25%) of organostannane as an oil: 1H
NMR (300 MHz, CDCl3) δ 7.27 (s, 1 H), 4.15 (t, J ) 7.4 Hz, 2
H), 3.67 (t, J ) 6.3 Hz, 2 H), 1.87(m, 2 H), 1.54-1.46 (m, 15
H), 1.28 (m, 6 H), 1.05 (t, J ) 8.3 Hz, 6 H), 0.85 (m, 18 H),
0.01 (s, 6 H); MS (ES) m/z 663 (M + 1+).
A mixture of organostannane (140 mg, 0.21 mmol), 33 (72
mg, 0.21 mmol), Pd(PPh3)2Cl2 (15 mg, 0.021 mmol), and LiCl
(27 mg, 0.62 mmol) in toluene (3.5 mL) was stirred at 100 °C
for 3 h under nitrogen. The cooled reaction mixture was
concentrated under vacuum. The residue was purified by
column chromatography (EtOAc/hexane as solvent) to give 96
mg (68%) of the Boc2-59 as an oil.
Boc2-59 was dissolved in dichloromethane (2 mL) and TFA
(2.6 mL) and stirred at 20 °C for 18 h before concentrated.
Ammonium hydroxide solution was added to the residue until
the pH was about 10-11 and a yellow solid was formed. The
yellow solid was collected through filtration, washed with
water, and dried under vacuum to give 59 as a light yellow
solid: 1H NMR (300 MHz, CD3OD) δ 8.23 (s, 1 H), 8.10 (t, J )
2.0 Hz, 1 H), 7.84 (s, 1 H), 7.74 (s, 1 H), 7.45 (dd, J ) 8.3, 1.3
Hz, 1 H), 7.26 (t, J ) 8.1 Hz, 1 H), 6.97 (dd, J ) 7.8, 1.1 Hz,
1 H), 3.68 (t, J ) 6.2 Hz, 2 H), 3.46 (t, J ) 6.9 Hz, 2 H), 1.88
(m, 2 H); MS (ES) m/z 362 (M + 1+). Anal. (C16H16N5OClS) C,
H, N.
(3-Chlorophenyl)(6-oxazol-5-ylpyrazin-2-yl)carbamic
Acid tert-Butyl Ester 60. A mixture of 33 (678 mg, 2.0 mmol),
oxazole (414 mg, 6.0 mmol), potassium acetate (295 mg, 3.0
mmol), and tetrakis(triphenylphosphine)palladium (115 mg,
0.1 mmol) in DMA (5 mL) was heated at 80 °C for 4 days in a
sealed tube. Water was added, the mixture was extracted with
CH2Cl2, and the extracts were dried (Na2SO4) and concen-
trated. The product was purified by column chromatography
(EtOAc/hexane as solvent) to provide 427 mg (57%) of 60 as a
white solid: 1H NMR (300 MHz, CDCl3) δ 8.92 (s, 1 H), 8.66
(s, 1 H), 7.96 (s,1 H), 7.43 (s, 1 H), 7.41-7.26 (m, 3 H), 7.14 (d,
J ) 5.0 Hz, 1 H), 1.48 (s, 9 H). Anal. (C18H17ClN4O3‚0.4H2O)
C, H, N.
(3-Chlorophenyl)[6-(2-iodooxazol-5-yl)pyrazin-2-yl]car-
bamic Acid tert-Butyl Ester 61. LiHMDS (1.0 M in THF,
1.85 mL, 1.85 mmol) was added to a solution of 60 (288 mg,
0.774 mmol) in THF (7.4 mL) at -78 °C, and the mixture was
stirred at -78 °C for 1 h. A solution of 1,2-diiodoethane (492
mg, 1.74 mmol) in THF (4.9 mL) was added at -78 °C, and
the mixture was stirred at -78 °C for 1 h, quenched by adding
10% Na2S2O3, and then extracted with CH2Cl2. The extracts
were dried (Na2SO4) and concentrated. The product was
purified by column chromatography (EtOAc/hexane as solvent)
to provide 252 mg (68%) of 61 as tan solid: 1H NMR (300 MHz,
acetone-d6) δ 9.00 (s, 1 H), 8.72 (s, 1 H), 7.48-7.29 (m, 5 H),
1.47 (s, 9 H); MS (ES) m/z 521 (M + Na+).
(3-Chlorophenyl){6-[2-((3-hydroxypropyl)amino)oxazol-
5-yl]pyrazin-2-yl}carbamic Acid tert-Butyl Ester 62. A
mixture of 61 (198 mg, 0.40 mmol) in 3-amino-1-propanol (4
mL) was heated at 100 °C for 18 h. The reaction mixture was
cooled to room temperature and EtOAc was added. This
solution was washed with water (3×), dried (Na2SO4), and
concentrated. Recrystallization from EtOAc provided 41 mg
(30%) of 62 as a tan solid: 1H NMR (300 MHz, DMSO-d6) δ
9.75 (s, 1 H), 8.07 (s, 1 H), 8.03 (s, 1 H), 8.00 (s, 1 H), 7.71 (t,
J ) 5.7 Hz, 1 H), 7.63 (dd, J ) 8.3, 1.0 Hz, 1 H), 7.44 (s, 1 H),
7.35 (t, J ) 8.0 Hz, 1 H), 7.01 (dd, J ) 7.1, 1.1 Hz, 1 H), 4.50
(t, J ) 5.2 Hz, 1 H), 3.49 (m, 2 H), 3.33 (m, 2 H), 1.72 (m, 2 H).
Anal. (C16H16ClN5O2‚1.1H2O) C, H, N.
test compound and incubated at 30 °C for 1 h in a streptavidin-
coated FlashPlate (Perkin-Elmer, Boston, MA). Biotinylated
peptide substrates were described as follows:49 VEGFR-2 (bio-
tin-KHKKLAEGSAYEEV-amide), calmodulin kinase 2 (biotin-
KKALRRQETVDAL-amide), casein kinase-1 (biotin-KRRRAL-
S(phospho)VASLPGL-amide), casein kinase-2 (biotin-RREEE-
TEEE-amide), CDK1 (biotin-KTPKKAKKPKTPKKAKKL-
amide), CDK4 (GST-retinoblastoma protein construct), EGFR
(biotin-DRVYIHPF-amide), FGFR-2 (biotin-poly(GT) 4:1), GSK-3
(biotin-KRREILSRRP(phospho)SYR-amide), insulin-R kinase
(biotin-TRDIYETDYYRK-amide), MAPK (biotin-APRTPGGRR-
amide), PDGF-R (biotin-KHKKLAEGSAYEEV-amide), PKA
(biotin-GRTGRRNSI-amide), PKCâ2 (biotin-RFARKGSLRQK-
NV-amide), and PKCγ (biotin-RFARKGSLRQKNV-amide).
Reaction conditions and components varied slightly depending
on the protein kinase being assayed. The reaction was termi-
nated by washing with PBS containing 100 mM EDTA, and
plates were counted in a scintillation counter. Inhibition of the
enzymatic activity due to compound was measured by observ-
ing a reduced amount of [33P]-γ-ATP incorporated into the
immobilized peptide relative to untreated controls. Linear
regression analysis of the percent of inhibition by test com-
pound was used to calculate IC50 values (GraphPad Prism 3,
GraphPad Software, SanDiego, CA).
Cell Culture. HUVEC and HASMC cells were obtained
from Cascade Biologicals (Portland, OR) and maintained in
Media 200 containing low serum growth supplement (Cascade
Biologicals). All other cells were obtained from the American
Type Culture Collection (Manassas, VA). HeLa, A375, and
HCT-116 cells were cultured in minimal essential medium
(MEM) with 0.1 mM nonessential amino acids and 1 mM
sodium pyruvate supplemented with 2 mM L-glutamine and
10% FCS (Hyclone, Logan, UT). MRC5 were cultured in Eagles
MEM and A375 in Dulbecco’s modified Eagles medium with
4 mM l-glutamine and 1.5 g/L sodium bicarbonate supple-
mented with 10% FCS. Cells were maintained at 37 °C plus
5% CO2 as exponentially growing monolayers.
VEGF-Stimulated HUVEC Proliferation. To evaluate
inhibition of VEGF-stimulated proliferation, HUVEC cells
were detached with trypsin and seeded into 96-well plates in
F-12K medium (Invitrogen, Carlsbad, CA) containing 0.2%
heat-inactivated fetal bovine serum. Cells were stimulated
with 2 ng/well VEGF (R&D systems) in the presence or
absence of test compounds. BrdU was added 24 h after the
addition of VEGF and incubated with cells for an additional
20-24 h. BrdU incorporation was quantified using cell pro-
liferation ELISA with BrdU reagents (Roche Biochemicals,
Indianapolis, IN).
Cell Proliferation Assay. The ability of test compound to
inhibit the proliferation of cell growth was determined by
measuring [14C]thymidine incorporation into primary cells or
cell lines derived from carcinomas originating from several
tissues. Briefly, cells were trypsinized and seeded into 96-well
CytoStar scintillating microplates (Amersham, Piscataway,
NJ) in complete medium in a volume of 100 µL. Cells were
incubated for 24 h at 37 °C in an atmosphere containing 5%
CO2. Test compound was added, and cells were incubated for
24 more hours. Methyl [14C]thymidine (Perkin-Elmer, Boston,
MA) was diluted in complete medium and 0.2 µCi was added
to each well of the CytoStar plate in a volume of 20 µL. The
plate was incubated for 24 additional hours in the presence of
test compound. Plates were washed twice with 200 µL PBS
and [14C]thymidine incorporation was quantified on a Packard
Top Count. IC50 values reported as >10 indicate that inhibition
did not reach 50% of control at the highest dose tested.
Molecular Docking Procedures. The X-ray structure of
VEGFR-2 has been determined in the unliganded, phospho-
rylated form (PDB code 1VR2).50 This structure was first added
all the missing hydrogen atoms by the modeling program
Maestro,51 followed by the visual inspection to ensure that all
the polar/charged residues form the correct hydrogen bonding
with their neighbors. Then, it was energy minimized in the
aqueous solution using the GB/SA model.52 To prevent the
unrealistic movement that may be caused by using an implicit
Biology. Kinase Activity Assays. A kinase reaction mix-
ture was prepared in 50 mM Tris-Cl (pH 8), 10 mM MgCl2,
0.1 mM Na3VO4, 1 mM DTT, 1% DMSO, 0.25 µM biotinylated
peptide substrate, 0.2-0.8 µCi per well [33P]-γ-ATP [2000-
3000 Ci/mmol], and 5 µM ATP in the presence or absence of

Pyrazine-Pyridine as VEGFR-2 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1899
Yin and yang in angiogenesis. Curr. Top. Microbiol. Immunol.
1999, 237, 173-185. (d) Lindahl, P.; Bostrom, H.; Karlsson, L.;
Hellstrom, M.; Kalen, M.; Betsholtz, C. Role of plate-derived
growth factors in angiogenesis and alveogenesis. Curr. Top.
Pathol. 1999, 93, 27-33.
water model, harmonic position constraints of 100 and 10 kJ/
(Å2‚mol) were applied to the heavy atoms on the protein
backbone and side chain, respectively, while all the hydrogen
atoms were treated without position constraints. The final
structure was used as the target for VEGFR-2 molecular
docking studies.
(11) (a) Cross, M. J.; Claesson-Welsh, L. FGF and VEGF function in
angiogenesis: Signaling pathways, biological responses, and
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Wu, X.-F.; Breltman, M. L.; Schuh, A. C. Failure of blood-island
formation and vasculogenesis in Flk-1-deficient mice. Nature
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receptor.
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and its receptors. Nat. Med. 2003, 9, 669-676.
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truber, D. J.; Topalian, S. L.; Steinberg, S. M.; Chen, H. X.;
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Since no CDK1 X-ray structures are currently available, a
model of CDK1, complexed with its ligand purvalanol B, has
been built on the basis of the X-ray structure of CDK2/
purvalanol B complex (PDB code 1CKP).53 The quality of this
model is promised by the following facts: the amino acid
sequence of human CDK1 (SWISS-PROT code PO6493) shares
high 63% identity and 74% similarity with human CDK2
(SWISS-PROT code P24941); it has only two different residues,
Ser84 and Met85, at the ATP-binding site, as compared to
CDK2; furthermore, the side chains of these two different
residues are extended out of the ATP-binding site and are not
directly involved in ligand binding. The homology-modeling
program Prime54 was used for the model building. The result-
ing CDK1-purvalanol B complex structure was further opti-
mized by full energy minimization in the aqueous solution
using the GB/SA model,52 and the final structure was used as
the target structure for CDK1 molecular docking studies.
CGP60474 and 15 were docked into the ATP-binding sites
of the target structures of CDK1 or VEGFR-2, respectively,
using the docking program Glide.55 The standard precision (SP)
mode of Glide was used to explore the favorable binding poses,
which allowed the ligand conformation be flexibly explored
while holding the protein as a rigid structure during the
docking. The predicted binding modes were determined as the
poses with the best Emodel scores.56 Each predicted complex
structure was then energy-minimized in the aqueous solution
using the GB/SA model.52 Harmonic position constraints of 100
and 10 kJ/(Å2‚mol) were applied to the residues that were more
than 12 Å away or within 6-12 Å from any ligand atom,
respectively, while the ligand and the residues that are less
than 6 Å from it were treated without position constraints.
All the molecular mechanism calculations were done using
the program MacroModel,57 with the OPLS_AA force field.58
Polak-Ribiere conjugate gradient method was used for energy
minimization, and the derivative convergence criterion was
set at 0.05 kJ/(Å‚mol).
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N.; Hugenschmidt, H.; Konerding, M. A.; Allegrini, P. R.; Wood,
J.; Hennig, J.; Unger, C.; Marme´, D. PTK787/ZK222584, a
specific vascular endothelial growth factor-receptor tyrosine
kinase inhibitor, effects the anatomy of the tumor vascular bed
and the functional vascular properties as detected by dynamic
enhanced magnetic resonance imaging. Cancer Res. 2002, 62,
4015-4022. (b) Bold, G.; Altman, K.-H.; Frei, J.; Lang, M.;
Manley, P. W.; Traxler, P.; Wietfeld, B.; Bru¨ggen, J.; Buch-
dunger, E.; Cozens, R.; Ferrari, S.; Furet, P.; Hofmann, F.;
Martiny-Baron, G.; Mestan, J.; Ro¨sel, J.; Sills, M.; Stover, D.;
Acemoglu, F.; Boss, E.; Emmenegger, R.; La¨sser, L.; Masso, E.;
Roth, R.; Schlachter, C.; Vetterli, W.; Wyss, D.; Wood, J. M. New
anilinophthalazines as potent and orally well absorbed inhibitors
of the VEGF receptor tyrosine kinase useful as antagonists of
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J. G.; Li, G.; Schreck, R. E.; Abrams, T. J.; Ngai, T. J.; Lee, L.
B.; Murray, L. J.; Carver, J.; Chan, E.; Moss, K. G.; Haznedar,
J. O.; Sukbuntherng, J.; Blake, R. A.; Sun, L.; Tang, C.; Miller,
T.; Shirazian, S.; McMahon, G.; Cherrington, J. M. In vivo
antitumor activity of SU11248, a novel tyrosine kinase inhibitor
targeting vascular endothelial growth factor and platelet-derived
growth factor receptors: Determination of a pharmacokinetic/
pharmacodynamic relationship. Clin. Cancer Res. 2003, 9(1),
327-337. (b) Sepp-Lorenzino, L.; Thomas, K. A. Expert Opin.
Invest. Drugs 2002, 11, 1-18.
(21) (a) Abdollahi, A.; Lipson, K. E.; Han, X.; Krempien, R.; Trinh,
T.; Weber, K. J.; Hahnfeldt, P.; Hlatky, L.; Debus, J.; Howlett,
A. R.; Huber, P. E. SU5416 and SU6668 attenuate the angiogenic
effects of radiation-induced tumor cell growth factor production
and amplify the direct anti-endothelial action of radiation in
vitro. Cancer Res. 2003, 63(13), 3755-3763. (b) Sun, Li.; 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-dihydroindol-
2-ones as growth factor receptor inhibitors for VEGF-R2 (Flk-
1/KDR), FGF-R1, and PDGF-Râ tyrosine kinases. J. Med. Chem.
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Acknowledgment. We thank Mary Pat Beavers for
calculating the physical properties of some of the
compounds.
Supporting Information Available: Elemental analyses
data for the compounds synthesized. This material is available
free of charge via the Internet at http://pubs.acs.org.
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