New anilinophthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinases useful as antagonists of tumor-driven angiogenesis

Article abstract of DOI:10.1021/jm9909443

The sprouting of new blood vessels, or angiogenesis, is necessary for any solid tumor to grow large enough to cause life-threatening disease. Vascular endothelial growth factor (VEGF) is one of the key promoters of tumor induced angiogenesis. VEGF receptors, the tyrosine kinases Flt-1 and KDR, are expressed on vascular endothelial cells and initiate angiogenesis upon activation by VEGF. 1-Anilino-(4-pyridylmethyl)-phthalazines, such as CGP 79787D (or PTK787/ZK222584), reversibly inhibit Flt-1 and KDR with IC₅₀ values < 0.1 μM. CGP 79787D also blocks the VEGF-induced receptor autophosphorylation in CHO cells ectopically expressing the KDR receptor (ED₅₀ = 34 nM). Modification of the 1-anilino moiety afforded derivatives with higher selectivity for the VEGF receptor tyrosine kinases Flt-1 and KDR compared to the related receptor tyrosine kinases PDGF-R and c-Kit. Since these 1-anilino-(4-pyridylmethyl)phthalazines are orally well absorbed, these compounds qualify for further profiling and as candidates for clinical evaluation.

Full text of DOI:10.1021/jm9909443

2310
J . Med. Chem. 2000, 43, 2310-2323
New An ilin op h th a la zin es a s P oten t a n d Or a lly Well Absor bed In h ibitor s of th e
VEGF Recep tor Tyr osin e Kin a ses Usefu l a s An ta gon ists of Tu m or -Dr iven
An giogen esis
Guido Bold,* Karl-Heinz Altmann, J o¨rg Frei, Marc Lang, Paul W. Manley, Peter Traxler, Bernhard Wietfeld,†
J osef Bru¨ggen, Elisabeth Buchdunger, Robert Cozens, Stefano Ferrari, Pascal Furet, Francesco Hofmann,
Georg Martiny-Baron,‡ J u¨rgen Mestan, J ohannes Ro¨sel, Matthew Sills, David Stover, Figan Acemoglu,
Eugen Boss, Rene´ Emmenegger, Laurent La¨sser, Elvira Masso, Rosemarie Roth, Christian Schlachter,
Werner Vetterli, Dominique Wyss, and J eanette M. Wood
Oncology Research, and Process Research, NOVARTIS Pharma AG, CH-4002 Basel, Switzerland, and
Institute of Molecular Medicine, Tumor Biology Center, D-79106 Freiburg, Germany
Received October 29, 1999
The sprouting of new blood vessels, or angiogenesis, is necessary for any solid tumor to grow
large enough to cause life-threatening disease. Vascular endothelial growth factor (VEGF) is
one of the key promoters of tumor induced angiogenesis. VEGF receptors, the tyrosine kinases
Flt-1 and KDR, are expressed on vascular endothelial cells and initiate angiogenesis upon
activation by VEGF. 1-Anilino-(4-pyridylmethyl)-phthalazines, such as CGP 79787D (or
PTK787 / ZK222584), reversibly inhibit Flt-1 and KDR with IC50 values < 0.1 µM. CGP 79787D
also blocks the VEGF-induced receptor autophosphorylation in CHO cells ectopically expressing
the KDR receptor (ED50 ) 34 nM). Modification of the 1-anilino moiety afforded derivatives
with higher selectivity for the VEGF receptor tyrosine kinases Flt-1 and KDR compared to the
related receptor tyrosine kinases PDGF-R and c-Kit. Since these 1-anilino-(4-pyridylmeth-
yl)phthalazines are orally well absorbed, these compounds qualify for further profiling and as
candidates for clinical evaluation.
In tr od u ction
induced angiogenic signaling selectively targets the
tumor-associated vessels, since proliferation of endot-
helial cells in the normal vasculature is a rare event.
Therefore, antiangiogenic therapy through inhibition of
VEGF-mediated signaling, is expected to be safe and
well-tolerated in cancer patients. Because host tissues
are genetically more stable than tumor cells, targeting
the host endothelial cells should provide a cancer
treatment modality that is less prone to resistance
development.6 Outside the indication cancer, antian-
giogenic drugs also have potential as new therapy for
other neovascularization-related diseases, for example
the treatment of diabetic retinopathy or age-related
maculopathy, the major causes of human blindness7 and
rheumatoid arthritis. In addition, VEGF is a potent
inducer of vascular permeability (and thus also known
as vascular permeability factor, VPF4a) and may also
play a key role in ascites formation and edema associ-
ated with malignant disease.
Recently, 3-substituted indolin-2-ones as inhibitors of
the mouse VEGF-receptor tyrosine kinase Flk-1 (fms-
like kinase) have been disclosed.8 These inhibitors bind
in the ATP binding pocket of the VEGF receptor tyrosine
kinases. One compound from this series, SU 5416 (see
Chart 1), has been selected for clinical trials in cancer
patients.
In this paper we will describe the 1-anilino-(4-py-
ridylmethyl)phthalazines,9 which represent a new class
of kinase inhibitors with high selectivity for the human
VEGF-receptor tyrosine kinases and, in contrast to the
previously described inhibitors, have favorable phar-
macokinetic properties following their oral administra-
tion to animals and men. One of these compounds, CGP
Most solid tumors cannot grow beyond a certain
critical size unless they establish their own blood supply
by inducing formation of new vessels sprouting from
existing host capillaries.1 Growth of blood vessels, or
angiogenesis, also promotes metastasis by providing an
avenue for transmission of the cancer cells to other
sites.2 Specific inhibition of tumor-induced angiogenesis
should prevent growth of many types of solid tumors,
thereby providing a novel approach for the treatment
of cancer.3
Vascular endothelial growth factor (VEGF), an in-
ducer of endothelial cell proliferation, migration, and
survival, is considered to play a key role in angiogenesis3a
and has been shown to be secreted by tumor cells and
macrophages.4 VEGF interacts with cell surface recep-
tors, in humans KDR (kinase domain-containing recep-
tor or VEGFR-2) and Flt-1 (fms-like tyrosine kinase or
VEGFR-1),5 expressed almost exclusively on vascular
endothelial cells. These receptors have intracellular
tyrosine kinase domains, which upon activation by
bound VEGF undergo autophosphorylation, starting the
transmission of the signal to the nucleus. As a conse-
quence, the induction of endothelial cell proliferation
and migration initiates sprouting of new blood vessels
toward the tumor tissue. Tumors have the ability to
upregulate the expression of the ligand as well as the
receptor in endothelial cells of adjacent vessels. In
keeping with this observation, inhibition of VEGF-
* To whom correspondence should be addressed. E-mail:
guido.bold@pharma.novartis.com.
† Process Research, NOVARTIS.
‡ Tumor Biology Center.
10.1021/jm9909443 CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/27/2000

Antagonists of Tumor-Driven Angiogenesis
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2311
Ch a r t 1
Sch em e 2. Synthesis of the Phthalazine Derivatives
41-44a
Sch em e 1. Synthesis of the Phthalazine Derivatives
CGP 79787, 37-40a
a
.
Conditions: (a) melting at 180-210 °C; (b) H2N-NH2 H2O,
vV; or H2N-NH2‚H2O, EtOH, vV; (c) POCl3, CH3CN, HCl, 50 °C; (d)
neat, 110 °C; (e) P2O5, Et3N ‚ HCl, 170 °C.
drazine hydrate. Thus, either base-catalyzed condensa-
tion of phthalides or tetrahydrophthalides with the
corresponding arylcarbaldehydes (Schemes 1, 4, and 5)
or melting of phthalic anhydrides with 2- or 4-meth-
ylnitrogen heterocycles (Schemes 2 and 3) led to inter-
mediates of type A (Scheme 1). This intermediate
rearranged to the required 2-aryl-3-hydroxyinden-1-
ones10 2-9, the 7-hydroxypyrindin-5-ones 10 and 11, or
the tetrahydroinden-1-one 13, respectively. The tetra-
substituted phthalide intermediates, for example, 12,
do not rearrange and can be isolated as such.
Subsequent condensation of 2-7, 12, and 13 with
hydrazine hydrate led to the phthalazones 15-20 and
27 or the tetrahydrophthalazone 28. Conversion of the
unsymmetrical derivatives 8-11 (Schemes 3 and 4)
gave the corresponding regioisomeric products 21-26,
the structures of which were proven by 1H NMR
a
Conditions: (a) MeONa/MeOH, EtCOOEt, 0 °C f vV; (b) H2N-
.
NH2 H2O, vV; (c) POCl3, CH3CN, HCl, 50 °C; or POCl3, CH3CN,
100 °C; (d) P2O5, Et3N ‚ HCl, 4-chloroaniline, 170 °C. (e) N-methyl-
4-chloroaniline, DMPU, 100 °C; or 4-chlorophenol or 4-chloro-
thiophenol, K2CO3, DMSO, 90 °C; or 4-chloroaniline neat, 110 °C.
79787D (also know as P TK787 / ZK222584), is cur-
rently in phase I trials in patients with advanced cancer.
Ch em istr y
Generally, the phthalazines were synthesized from
2-aryl-3-hydroxyinden-1-ones by condensation with hy-

2312 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Bold et al.
Sch em e 3. Synthesis of the Phthalazine Derivatives 45
and 46a
Sch em e 4. Synthesis of the Pyridopyridazine
Derivatives 47-50a
a
Conditions: (a) MeONa/MeOH, EtCOOEt, 0 °C f vV; (b) H2N-
NH2‚H2O, vV; (c) P2O5, Et3N‚HCl, 4-chloroaniline, 170 °C.
tively, the pyridazine-3-ones were activated as the
imidoyl chlorides, 30-36, and then reacted with the
appropriate aniline to afford the 1-anilinophthalazines
37-40, 42, 45, 46, and 51 and the 3-anilinopyridazine
54. The best condition for the chlorination of phthalazin-
1-ones was stirring at 50 °C in a solution of phosphoryl
chloride in slightly acidic acetonitrile. The chloride then
could be mildly substituted by anilines in boiling acidic
ethanol. This method was preferentially used to convert
the chloride 30 into the derivatives 55-66 (Table 3).
a
Conditions: (a) melting at 165 °C; (b) H2N-NH2‚H2O, vV; (c)
(CF3CO)2O; (d) POCl3, CH3CN, HCl, 50 °C; (e) EtOH, HCl, vV; or
n-butanol, 100 °C.
Resu lts a n d Discu ssion
spectroscopy (nuclear Overhauser effects between the
H2C group and the neighboring aromatic HC protons).
During the condensation of the nitro derivative 8 with
hydrazine, the nitro group was reduced to an amino
function as described by Druey and Marxer.11 Whereas
the regioisomeric 6-amino derivative of 21a was not
isolated from the reaction mixture, the methyl deriva-
tive 9 yielded 22, as a ca. 1:1 mixture of regioisomers.
Protection of the amino group as a trifluoracetamide
gave 21b. Condensation of the 7-hydroxypyrindin-5-ones
10 and 11 yielded a chromatographically separable
mixture of the corresponding isomers 23-26. The
monocyclic pyridazine-3-one 29 (Scheme 5) was obtained
in modest yield from hydrazine hydrate and 14, the
addition product of 4-picolyllithium to maleic acid
anhydride.
To identify lead structures for VEGF receptor tyrosine
kinase inhibition, we screened the corporate compound
libraries. Kinase inhibition was evaluated by measuring
the phosphorylation of the artificial substrate poly-Glu-
Tyr by the kinase domain of the Flt-1 VEGF receptor.
Via this approach, we identified the anilinophthalazine
1 (see Chart 1) as a potent inhibitor of Flt-1 (IC50 ) 0.16
µM). Further profiling revealed that 1 is highly selective
for the class III protein tyrosine kinase family:13
1
inhibits the related VEGF receptor tyrosine kinase5b
KDR with an IC50 of 0.05 µM and its mouse homologue
Flk-1 with an IC50 of 0.3 µM. Its succinate salt, CGP
79787D (see Chart 1), blocks the lymphatic endothelial
cell receptor tyrosine kinase Flt-4 with an IC50 of 0.66
µM. Additionally, 1 interacts with the PDGF-â receptor
(platelet derived growth factor-â; IC50 ) 0.7 µM) and
c-Kit (receptor for stem cell factor; IC50 ) 0.7 µM).
However, the IC50 of 1 is higher than 10 µM against a
series of non-class III receptor tyrosine kinases, like the
EGF receptor (epithelial growth factor receptor), v-abl
(virally transduced Abelson oncogene), c-src (protoon-
cogene), or a serine-threonine kinase like PKC-R (pro-
tein kinase C-R ).
Introduction of the 3-anilino group on the pyridazine
core was achieved via two methods. In a one-step
procedure described by Andersen and Pedersen,12
a
mixture of the pyridazine-3-ones, the respective aniline,
phosphorus pentoxide, and triethylamine hydrochloride
was melted together to give the anilino derivatives CGP
79787, 41, 43, 44, 47-50, 52, and 53, directly. Alterna-

Antagonists of Tumor-Driven Angiogenesis
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2313
Sch em e 5. Synthesis of the Derivatives 51-54a
a
Conditions: (a) 90 °C; (b) NaOAc, CH3CONMe2, 180 °C; (c) H2N-NH2‚H2O, EtOH, vV; or H2N-NH2‚H2O, vV; or H2N-NH2‚H2O, BuOH,
120 °C; (d) P2O5, Et3N‚HCl, 170 °C; (e) MeONa/MeOH, EtCOOEt, 0 °C f vV; (f) THF, -78 °C f rt; (g) POCl3, 120 °C; (h) BuOH, 130 °C.
b
Yield for 2 steps
Since a drug must enter the cells in order to inhibit
the tyrosine kinase activity of the receptor, the effect of
1 was tested in cellular, VEGF-induced receptor auto-
phosphorylation assays. CHO cells (chinese hamster
ovarian cells) ectopically expressing the VEGF receptor
KDR were stimulated with VEGF. The degree of recep-
tor autophosphorylation was measured in a sandwich
ELISA assay by trapping KDR with an antibody specific
for KDR and detecting phosphorylation with an anti-
phosphotyrosine antibody. Thus, 1 inhibits the cellular
response with an ED50 of 34 nM. In a similar cell-based
receptor autophosphorylation assay using human um-
bilical vein endothelial cells (HUVEC), which naturally
express the KDR receptor, the compound also inhibits
the VEGF-induced phosphorylation with an ED50 of 17
nM. Additionally, the compound inhibits the autophos-
phorylation of the PDGF receptor induced by PDGF in
BALB/c 3T3 cells (ED50 ) 0.6 µM) as well as the c-Kit
autophosphorylation, induced by stem cell factor (SCF)
in Mo-7e cells (ED50 e 0.1 µM). Again, 1 does not inhibit
the autophosphorylation of other kinases like v-erbB2
(oncogene of avian erythroblastosis virus) or Ins-R
(insulin receptor) in cell based assays, thus indicating
that it inhibits the tyrosine kinase of growth factor
receptors belonging to the same family (class III) with
high selectivity compared to other kinases.
Oral application to mice of 50 mg/kg of 1 as a solution
in water resulted in peak levels of g 20 µM drug at ≈30
min after dosage. Even after 4 h, concentrations were
still above 3 µM. This observation proves that 1 is orally
well absorbed. Additional salt forms of its free base,
CGP 79787 (see Chart 1), were prepared: Among them,
the succinate CGP 79787D has the best physicochem-
ical properties, and when formulated in DMSO/Tween
80, it was also well absorbed orally. In vivo studies with
CGP 79787D which demonstrate that it inhibits an-
giogenesis, tumor growth, and the occurrence of me-
tastasis are described in a separate publication.14
Assuming that CGP 79787 exerts its inhibitory action
by binding to the ATP cleft of the enzyme, a reasonable
hypothesis considering that the available X-ray crystal
structures of protein kinases indicate that no other
binding pockets usually exist in their catalytic domains,
we modeled its binding mode. Thus, CGP 79787 was
docked in a model of the ATP binding site of KDR
constructed using the available X-ray structures15 of the

2314 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Bold et al.
Ta ble 1. Inhibition of VEGF-R Tyrosine Kinases by the Phthalazine Derivatives CGP 79787D, 37-46, 51, and 52a
a
b
The data represent averages of at least three determinations. Mixture of 6- and 7-methyl derivatives.
kinase domain of the fibroblast growth factor receptor
1 (FGFR1), another class III growth factor receptor. The
putative binding mode of CGP 79787 in the ATP
binding cleft of KDR (shown in Chart 2) results from
extensive docking analyses aimed at identifying a model
consistent with the structural complementarity between
the inhibitor and the cleft as well as the available
structure-activity relationships.16 This homology model
for the ATP binding site was recently validated by the
published X-ray of an unligated KDR by Agouron.17
According to our hypothesis, CGP 79787 does not form
direct hydrogen bonds with the peptide backbone of the
hinge region as does ATP and many reported kinase
inhibitors18 but rather occupies the hydrophobic regions
of the binding site. The anilino moiety is located in the
hydrophobic pocket formed by residues Val 914, Val 912,
Val 897, Leu 887, Cys 1043, Phe 1045 and the hydro-
carbon part of the side chain of Lys 866,19 while the
phthalazine bicycle makes hydrophobic contacts with
Leu 1033, Gly 920, and Leu 838. Although no direct
hydrogen bonds with the hinge region are possible in
this binding orientation, the anilino NH group of the
inhibitor is located at distances from the backbone of
Glu 915 and Cys 917, allowing water-mediated hydro-
gen bonds to be formed (Glu 915 and Cys 917 are the
residues of the hinge region that should be involved in
bidentate hydrogen bonding with the adenine ring of
ATP). In addition, the essential pyridyl nitrogen of the
inhibitor is assumed to be engaged in a hydrogen bond
with the side chain of Lys 1060, a residue belonging to
the activation loop of the kinase. Lys 1060 is not
conserved outside the tyrosine kinases of the PDGF

Antagonists of Tumor-Driven Angiogenesis
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2315
Ta ble 2. Enzyme Inhibition of the
1-(4-Chloroanilino)-4-(4-pyridylmethyl)pyridazine Derivatives
47-50, 53, and 54a
by an extra nitrogen atom (51) decreases the activity
significantly, branching the pyridyl-methyl side chain
(52) does not result in a tremendous loss in activity
(
KDRIC50 ) 0.21 µM; for the racemate). A polar substitu-
ent on the phthalazine core (45) leads to a weak
inhibitor of KDR, whereas phthalazines bearing lipo-
philic substitutents (e.g. 46) are weak inhibitors of both
VEGF-receptor kinases.
The effects of substitutions in the condensed benzene
ring of the phthalazine are shown in Table 2: Introduc-
tion of a hydrogen-bond acceptor to possibly pick up
additional interactions with the enzyme by the system-
atic replacements of ring carbons by nitrogens (47-50)
reduced the activity, the only well-tolerated substitution
for KDR-inhibition being the 5-aza one (50). Increasing
the steric demand by hydrogenation of the benzene ring
to the tetrahydrophthalazine 53 also reduced activity.
However, the simple monocyclic analogue (54) retains
some activity against Flt-1.
As previously mentioned, CGP 79787D also inhibits
other class III tyrosine kinases, like the PDGF receptor
kinase and c-Kit (see Table 3). By modification of the
aniline part of the 1-anilino-(4-pyridylmethyl)phthala-
zine we attempted to retain the activity against the
VEGF receptor tyrosine kinases and at the same time
improve the selectivity with respect to the PDGF
receptor and c-Kit: The unsubstituted aniline derivative
55 is a much weaker inhibitor of KDR than the 4-chloro
derivative CGP 79787D. The 4-chlorine can be replaced
by alkyl groups as large as the tert-butyl group in either
the 3- or 4-position of the aniline phenyl ring (56, 57)
or a phenyl (58). Whereas the 3-methoxy derivative 59
is a reasonable inhibitor for both VEGF receptor ki-
nases, the 3-hydroxy derivative 60 potently inhibits only
Flt-1. Disubstituted aniline derivatives retain good
inhibitory activity against KDR tyrosine kinase (61-
66), whereas their activity on the Flt-1 tyrosine kinase
is slightly reduced for 64 and 65. The herein described
disubstituted derivatives tend to be also slightly more
selective toward c-Kit. Most strikingly, compounds 65
and 66 no longer block c-Kit nor the PDGF receptor
tyrosine kinase.
a
The data represent averages of at least three determinations.
family (PDGF-R, c-Kit, KDR, Flt-1, Flk-1) and therefore
may contribute to the selective recognition of CGP
79787 by the members of this family. However, on the
basis of the model one cannot completely rule out an
alternative hydrogen bond with either the proximal Asn
1031 or a residue of the glycine rich loop which may be
prone to conformational rearrangement.15b
In cellular profiling, the potent KDR receptor tyrosine
kinase inhibitors 56, 57, 59, and 61-66 are at least as
potent as CGP 79787D. Among them, 56, 57, and 63
turned out to be clearly superior in inhibiting the
autophosphorylation process. Preliminary pharmacoki-
netic studies (50 mg/kg in mice, formulated in DMSO/
Tween 80) showed that the described phthalazine
derivatives are comparably well-absorbed after oral
administration. The low concentrations of parent com-
pound observed for the hydroxyphenyl derivative 60 are
the result of an apparently good oral absorption followed
by a rapid metabolism. Although the metabolite has not
been identified, by retention time it was more polar than
the parent, and peak heights of the metabolite exceeded
those of the parent by at least 10-fold. The high levels
of the metabolite in the circulation indicate that low
bioavailability of the parent was the result of metabolic
clearance rather than poor absorption.
Tables 1-3 summarize results of structure-activity
investigations on the anilinophthalazine lead, probing
the active site in view of model validation by modifying
the bulk or the donor-acceptor properties first on the
aniline-NH, then on the pyridyl group, the benzene ring,
and finally in the aniline substitution pattern. The
aniline-NH is important for good inhibition of KDR (see
37-39 in Table 1). Even though the ether derivative
38 and the thioether 39 retain the activity against Flt-
1, it appears that for good KDR inhibition a secondary
NH is necessary. The position of the pyridine nitrogen
is crucial for activity, since both the 3- and 2-pyridyl
derivatives (40, 41) are inactive. The 4-pyrimidyl de-
rivative 42, which retains the 4-pyridine nitrogen, still
moderately inhibits the kinases. To hinder interactions
between cytochrome P450 isozymes and the pyridine-
nitrogen, the ortho methyl-derivatives 43 and 44 were
prepared. The one methyl group of 43 is tolerated
In direct comparison with SU 5416,8 the phthalazine
derivatives summarized in Table 3 are equipotent
against KDR on the basis of IC50 values. Some deriva-
tives are more selective VEGF receptor tyrosine kinase
(
KDRIC50 ) 0.24 µM); the dimethyl derivative 44 is
inactive at concentrations up to 10 µM, however.
Whereas the extension of the pyridyl-methyl side chain

2316 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Bold et al.
Ta ble 3. Enzyme Inhibition of the 1-Anilino-(4-pyridylmethyl)phthalazine Derivatives CGP 79787D, 55-66, and SU 5416a
a
b
c
The data represent averages of at least three determinations. PDGF-â receptor. Inhibition of VEGF-driven cellular receptor
d
autophosphorylation in CHO cells transfected with the KDR receptor. The pharmacokinetic studies were performed in mice: Drug
concentrations in blood samples were analyzed by reversed-phase HPLC 30, 60, 90, and 120 min after oral application of 50 mg/kg in a
standardized formulation (DMSO/Tween 80). The value cmax represents the highest observed drug concentration at the indicated time
point (tmax.). Dihydrochloride salt. Low concentration of the parent compound but an apparently high concentration (not determined)
of an unknown metabolite.
e
f
Ch a r t 2. Proposed Binding Mode of CGP 79787 to
KDR
the 1-anilino-(4-pyridylmethyl)phthalazine class of com-
pounds is orally well-absorbed, producing drug concen-
trations in the upper micromolar range upon dosing of
50 mg/kg to mice. This clearly qualifies them for further
profiling as antiangiogenic drugs.
Exp er im en ta l Section
En zym e Assa ys. The kinase domains of Flt-1, KDR, c-Kit,
and PDGF-â were expressed as GST-fusion proteins using the
baculovirus system. The in vitro kinase assays were performed
in 96-well plates using the recombinant GST-fused kinase
domains expressed in baculovirus and purified over gluta-
thione-Sepharose. 33P-ATP (Amersham) was used as the
phosphate donor, and the polyGluTyr (4:1) peptide (Sigma) was
used as the acceptor. For PDGF-â activity, autophosphoryla-
tion was measured.
The buffer conditions were optimized for each kinase: Flt-1
(20 mM Tris-HCl, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 8 µM
ATP, 0.2 µCi 33P-ATP, 3 µg/mL polyGluTyr); KDR (20 mM Tris-
HCl, pH 7.5, 10 mM MgCl2, 1 mM MnCl2, 8 µM ATP, 0.2 µCi
33P-ATP, 8 µg/mL polyGluTyr); c-Kit (20 mM Tris-HCl, pH 7.5,
10 mM MgCl2, 2 mM MnCl2, 1 µM ATP, 0.2 µCi 33P-ATP, 5
µg/mL polyGluTyr); PDGF-â (50 mM Hepes, pH 7.5, 3 mM
MgCl2, 3 mM MnCl2, 3 µM Na3VO4, 1 mM dithiotreitol, 0.1
µM ATP, 0.2 µCi 33P-ATP. The reaction was carried out in a
inhibitors with respect to other class III tyrosine ki-
nases, however. In our experiments, they are also more
potent inhibitors of cellular KDR autophosphorylation
than SU 5416. Additionally and in contrast to SU 5416,

Antagonists of Tumor-Driven Angiogenesis
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2317
volume of 30 µl for 10 min at room temperature in the presence
of either 1% DMSO or the compound at the required concen-
tration in 1% DMSO. The reaction was stopped by the addition
of ethylenediaminetetraacetic acid to a final concentration of
60 mM. The assay mixture was then transferred onto a
Immobilon-PVDF membrane (Millipore), which was subse-
quently washed four times with 0.05% H3PO4 and once with
ethanol. After drying, 10 µl/well of Microscint cocktail (Pack-
ard) was added and scintillation counting was performed
(Hewlett-Packard Top Count).
IC50 values were calculated by linear regression analysis of
the percentage inhibition of each compound in duplicate, at
three concentrations (usually, 0.01, 0.1, and 1 µM or 0.1, 1,
and 10 µM).
RPMI 1640 media supplemented with 10% FCS and 2.5 ng/
mL of GM-CSF. Serum-starved MO7e cells (growth in serum-
free medium for 16 h) were incubated with drug for 90 min
prior to stimulation with SCF (50 ng/mL) for 10 min. Equal
amounts of protein of cell lysates were analyzed by Western
blotting using 4G10 anti-phosphotyrosine antibodies.21 Bound
antibodies were detected using the ECL Western blotting
system from Amersham.
Bioa va ila bility. The compounds were dissolved in DMSO
to a concentration of 100 mg/mL. The stock solution was
diluted 1:20 with an aqueous solution of 1% Tween 80. After
brief, low-power sonication, a milky, homogeneous suspension
was obtained. Then the formulated compounds were given to
female MAG mice that had free access to food and water
throughout the experiments. The mice received an average
dose of 50 mg compound per kilogram by gavage. At allotted
times four mice were sacrificed, heart blood collected into
heparinised tubes, and plasma prepared by centrifugation. The
plasma was either analyzed immediately or stored frozen at
-20 °C. For analysis, the heparinized plasma samples were
deproteinated by the addition of an equal volume of acetoni-
trile. After thorough mixing, the tubes were allowed to stand
for 20-30 min at room temperature, and the precipitated
protein was removed by centrifugation (10000g, 5 min.). The
supernatant was collected and analyzed by reversed phase
HPLC: 100 µL of the supernatant was injected onto a
Nucleosil C18, 5 µm analytical column (125 × 4 mm) with a 8
mm guard column; mobile phase, 5% acetonitrile/0.05% tri-
fluoroacetic acid (TFA) in water/0.05% TFA f 80% acetonitrile/
0.05% TFA during 15 min + 5 min 100% acetonitrile/0.05%
TFA. Compounds were detected by UV absorbance and identi-
fied on the chromatograms by retention time and UV spectrum
compared to control plasma spiked with compound. Quanti-
tation was by the external standard method using peak heights
to quantitate the amounts by reference to a calibration curve.
The calibration curve was constructed by the analysis of
plasma samples containing known amounts of the compound
under evaluation, which had been processed as described
above. The limit of quantitation was 0.1-0.2 µmol/L (com-
pound dependent) under these conditions.
Cellu la r KDR Kin a se In h ibition Assa y. CHO cells,
permanently transfected with the VEGF receptor tyrosine
kinase KDR were seeded in six-well plates and grown to 80%
confluency. Incubation with serial dilutions of compound for
2 h at 37 °C in medium without FCS was followed by addition
of VEGF (final concentration 20 ng/mL). After 5 min incuba-
tion (37 °C) the cells were washed twice with ice-cold PBS and
lysed in 100 µL per well lysis buffer (50 mM Tris/HCl pH 7.4;
150 mM NaCl; 5 mM EDTA; 1 mM EGTA, 1.5 mM MgCl2; 2
mM Na-vanadate; 1% NP40; 10% glycerol; 1mM PMSF; 80
µg/mL aprotinin; 50 µg/mL leupeptin). Nuclei were removed
by full speed centrifugation for 10 min at 4 °C using an
Eppendorf centrifuge. Protein concentrations of the lysates
were determined with the Bio-Rad DC protein assay kit using
BSA as standard. For the second part of the assay a mono-
clonal antibody to the extracytoplasmic domain of the VEGF
receptor was coated to black ELISA plates (OptiPlate HTRF-
96; Packard) as capture antibody. After blocking with 1% BSA
in PBS, the cell lysates (20 µg of protein per well) were added
in triplicates together with PY-20(AP), an alkaline phos-
phatase-labeled anti-phosphotyrosine antibody (Transduction
Laboratories). After overnight incubation at 4 °C the bound
PY-20(AP) was detected with a luminescent alkaline phos-
phatase substrate (CDP-Star Ready to use with Emerald II;
TROPIX). Luminescence was determined using a Packard Top
Count microplate scintillation counter. The difference between
the signals obtained from unstimulated cells (negative control)
and VEGF-stimulated cells (positive control) was considered
as 100% VEGF-induced KDR autophosphorylation. The influ-
ence of a compound on the VEGF-induced KDR autophospho-
rylation was expressed as “% inhibition”; the inhibitory
activities of different compounds were compared by determi-
nation of ED50 (50% effective dose) values from dose-response
curves.
In h ibition of P DGF-Stim u lated P r otein -Tyr osin e P h os-
p h or yla tion . Inhibition of PDGF-stimulated total cellular
tyrosine phosphorylation in Balb/c 3T3 cells was measured
using a microtiter ELISA assay.20 Briefly, Balb/c A31 cells were
grown to conflueny in collagen IV coated 96-well tissue culture
plates. After removal of the medium, cells were treated with
2-fold serial dilutions of compounds in starvation medium
(Dulbecco’s minimal essential medium, 0.1% bovine serum
albumine). Following 2 h incubation at 37 °C, PDGF was added
to a final concentration of 50 ng/mL. After 10 more minutes
at 37 °C, medium was removed, cells were fixed in cold (-20
°C) methanol, washed with phosphate-buffered saline (PBS),
and blocked with PBS containing 0.1% Tween 20 and 3%
bovine serum albumine. Subsequently, the cells were incu-
bated with the monoclonal anti-phosphotyrosine antibody
4G10,21 followed by incubation with an alkaline phosphatase
labeled anti-mouse IgG antibody (SIGMA A3688). Bound
antibody was detected using p-nitrophenyl phosphate (SIGMA)
as substrate. Color development was monitored in an ELISA
reader at 405 nm.
Ch em ist r y. All reactions with air- or moisture-sensitive
reactants and solvents were carried out under nitrogen
atmosphere. In general, reagents and solvents were used as
purchased without further purification. THF was freshly
distilled from sodium/benzophenone. Column flash chroma-
tography was performed on silica gel 60 (230-400 mesh
ASTM, E. Merck) under
a positive nitrogen pressure of
approximately 0.4 atm. Melting points were determined in an
open capillary and are not corrected. 1H NMR spectra were
collected with Bruker DRX-500 (500 MHz), Bruker AM-360
(360 MHz), Varian Gemini-300 (300 MHz), or Varian Gemini-
200 (200 MHz) instruments; chemical shifts of signals are
expressed in parts per million (ppm) and are referenced to the
deuterated solvents used. MS spectra were collected with an
FAB-ZAB, HF (VG Analytical). Elemental analyses were
performed by the Ciba Analytical Department and are within
(0.4% of the calculated values.
Abbr evia tion s: rt, room temperature; DMPU, 1,3-dimeth-
yl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone.
3-Hyd r oxy-2-(p yr id in -4-yl)in d en -1-on e (2). To an ice-
cooled solution of phthalide (201.8 g, 1.50 mol), 4-pyridinecar-
boxaldehyde (141 mL, 1.50 mol), and ethyl propionate (750
mL) in methanol (1.5 L) was portionwise added sodium
methanolate (243 g, 4.5 mol). After 15 min at rt the reaction
mixture was heated to 65 °C for 2.5 h and then cooled back to
rt. Concentration under reduced pressure and dilution with
water (7 L) gave a dark red solution which was extracted with
diethyl ether (5 × 2.5 L). Acidification of the aqueous layer
with acetic acid (350 mL) and stirring produced a yellowish
suspension. Filtration and washing with water (15 L), ethyl
acetate (5 L), and finally diethyl ether (3 L) gave 174 g (52%)
of 2: mp 307-308 °C; 1H NMR (DMSO-d6) δ 8.72 (d, 2H), 8.18
(d, 2H), 7.50 (m, 4H); FAB MS (M + H)+ ) 224. Anal. (C14H9-
NO2‚0.50H2O) C, H, N, H2O.
Deter m in a tion of Liga n d In d u ced c-Kit Au top h osp h o-
r yla tion . MO7e cells are a human promegakaryocytic leuke-
mia cell line that requires either granulocyte macrophage
colony stimulating factor (GM-CSF), interleukin-3 (IL-3), or
SCF for proliferation and were obtained from Grover Bagby,
Oregon Health Sciences University. Cells were cultured in

2318 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Bold et al.
3-Hyd r oxy-2-(p yr id in -3-yl)-in d en -1-on e (3). Preparation
from 3-pyridinecarbaldehyde as described for 2 yielded 61.1 g
(68%) of 3: 1H NMR (DMSO-d6) δ 9.75 (s, 1H), 9.45 (d, 1H),
8.23 (d, 1H), 7.80 (dd, 1H), 7.38 (m, 2H), 7.32 (m, 2H); FAB
MS (M + H)+ ) 224. Anal. (C14H9NO2 ‚ 0.12 H2O) C, H, N.
3-Hyd r oxy-2-(p yr id in -2-yl)-in d en -1-on e (4). A mixture
of phthalic anhydride (98.8 g, 0.667 mol) and 2-methylpyridine
(130 mL, 1.33 mol) was heated to 140 °C for 15 min and then
to 180 °C for 22 h. Recrystallization from ethanol (200 mL)
gave 34.4 g (23%) of 4: mp 288 °C; 1H NMR (DMSO-d6) δ 8.52
(d, 1H), 8.33 (d, 1H), 8.09 (t, 1H), 7.58 (m, 4H), 7.17 (t, 1H);
FAB MS (M + H)+ ) 224. Anal. (C14H9NO2) C, H, N.
2.28 g (12%) of 12 as a mixture of isomers: FAB MS (M+H)+
) 238. Anal. (C15H11NO2) C, H, N.
3-Hyd r oxy-2-(p yr id in -4-yl)-4,5,6,7-tetr a h yd r oin d en -1-
on e (13). To an ice-cooled solution of 4,5,6,7-tetrahydro-3H-
isobenzofuran-1-one25 (1.48 g, 10.7 mmol) and 4-pyridinecarb-
aldehyde (1.01 mL, 10.7 mmol) in methanol (8.7 mL) and ethyl
propionate (5.4 mL) was added sodium methanolate (5.4 M in
methanol; 1.98 mL, 10.7 mmol). After 15 min at rt, the mixture
was heated to 65 °C for 2 h and then cooled to rt again. The
reaction mixture was concentrated under reduced pressure and
the residue stirred in water (5 mL) and filtered, giving 0.62 g
(25%) of 13. The filtrate was extracted twice with diethyl ether,
the aqueous layer neutralized with acetic acid, and the formed
precipitate filtered off, yielding another 1.00 g (totally 66%)
of 13: mp 258-261 °C; 1H NMR (DMSO-d6) δ 8.19 (d, 2H),
3-Hyd r oxy-2-(p yr im id in -4-yl)-in d en -1-on e (5). A mix-
ture of phthalic anhydride (7.87 g, 53.1 mmol) and 4-meth-
ylpyrimidine (22 mL, 239 mmol) was heated to 140 °C for 1 h
and then to 210 °C for 4 h. At rt the partly crystalline material
was stirred in methanol (15 mL). Filtration gave 886 mg of 5;
another 394 mg of product (f1.28 g; 10%) could be isolated
from the filtrate by concentration and crystallization from
7.82 (d, 2H), 2.13 (m, 4H), 1.62 (m, 4H); FAB MS (M + H)+
228. Anal. (C14H13NO2) C, H, N.
)
4-(P yr id in -4-yl)m eth yl-2H-p h th a la zin -1-on e (15). Heat-
ing of a suspension of 2 (174 g, 0.78 mol) in hydrazine
monohydrate (750 mL) to 110 °C gave a dark red solution from
which a new precipitate was formed during 8 h. Cooling to 0
°C, filtration, and washing with ethanol (150 mL) and diethyl
ether (5 × 150 mL) afforded 153 g (82%) of 15: mp 212-213
°C; 1H NMR (DMSO-d6) δ 8.48 (d, 2H), 7.27 (m, 1H), 7.88 (m,
3H), 7.32 (d, 2H), 4.35 (s, 2H); FAB MS (M + H)+ ) 238.
4-(P yr id in -3-yl)m eth yl-2H-p h th a la zin -1-on e (16). Pre-
pared from 3 as described for 15 yielded 50.6 g (79%) of 16:
1
water (30 mL): mp 168-169 °C; H NMR (DMSO-d6) δ 9.22
(s, 1H), 8.88 (d, 1H), 8.32 (d, 1H), 8.04 (m, 2H), 7.94 (t, 1H),
7.77 (t, 1H), 6.95 (s, 1H); FAB MS (M + H)+ ) 225. Anal.
(C13H8N2O2) C, H, N.
3-Hyd r oxy-2-(2-m eth ylp yr id in -4-yl)-in d en -1-on e (6). A
mixture of phthalic anhydride (27.7 g, 187 mmol) and 2,4-
lutidine (21.56 mL, 187 mmol) was heated to 180 °C for 20 h.
The black mass was stirred in boiling ethanol (250 mL) and
filtered. SiO2 was added to the filtrate, and the mixture was
concentrated in vacuo to a powder. Putting this powder on top
of a SiO2 chromatography column and eluation with ethyl
acetate/methanol 49:1 f 19:1 gave 5.7 g (13%) of 6: 1H NMR
(DMSO-d6) δ 8.61 (dd, 1H), 8.56 (s, 1H), 8.07 (d, 1H), 7.47 (m,
1
mp 188-190 °C; H NMR (DMSO-d6) δ 12.6 (s, HN), 8.60 (s,
1H), 8.42 (dd, 1H), 8.28 (d, 1H), 8.00 (m, 1H), 7.88 (m, 2H),
7.69 (d, 1H), 7.32 (dd, 1H), 4.35 (s, 2H). Anal. (C14H11N3O) C,
H, N.
4-(P yr id in -2-yl)m eth yl-2H-p h th a la zin -1-on e (17). Pre-
pared from 4 as described for 15 yielded 22.2 g (61%) of 17:
1H NMR (DMSO-d6) δ 12.6 (s, HN), 8.47 (m, 1H), 8.28 (m, 1H),
8.0-7.7 (m, 4H), 7.38 (d, 1H), 7.24 (dd, 1H), 4.47 (s, 2H); FAB
MS (M + H)+ ) 238.
4H), 2.46 (s, 3H); FAB MS (M + H)+ ) 238. Anal. (C15H11
NO2) C, H, N.
-
3-Hydr oxy-2-(2,6-dim eth ylpyr idin -4-yl)-in den e-1-on e (7).
Preparation from 2,4,6-collidine as described for 6 yielded 5.44
g (11%) of 7: 1H NMR (DMSO-d6) δ 12.9 (sb, 1H), 8.45 (s, 2H),
7.47 (m, 4H), 2.45 (s, 6H); FAB MS (M + H)+ ) 252. Anal.
(C16H13NO2) C, H, N.
3-Hyd r oxy-5-m eth yl-2-(p yr id in -4-yl)-in d en -1-on e (9). A
mixture of 4-methylphthalic anhydride (47.5 g, 293 mmol) and
4-picoline (28.6 mL, 293 mmol) was heated to 165 °C for 18 h.
The resulting material was stirred in boiling ethanol (190 mL),
filtered, and washed with ethanol and diethyl ether, yielding
23.7 g (34%) of 9: 1H NMR (DMSO-d6) δ 8.70 (d, 2H), 8.14 (d,
2H), 7.34 (m, 2H), 7.29 (s, 1H), 2.38 (s, 3H); FAB MS (M +
H)+ ) 238. Anal. (C15H11NO2) C, H, N, O.
4-(P yr im id in -4-yl)m et h yl-2H -p h t h a la zin -1-on e (18).
Heating of a suspension of 5 (1.20 g, 5.35 mmol) in hydrazine
monohydrate (344.8 µL, 6.96 mmol) and ethanol (30 mL) for 5
h to 80 °C, cooling to rt, filtration, and washing with ethanol
yielded 1.12 g (88%) of 18: mp 204-206 °C; 1H NMR (DMSO-
d6) δ 12.60 (s, HN), 9.05 (s, 1H), 8.70 (d, 1H), 8.26 (m, 1H),
7.85 (m, 3H), 7.53 (d, 1H), 4.48 (s, 2H); FAB MS (M + H)+
239.
)
4-(2-Met h ylp yr id in -4-yl)m et h yl-2H -p h t h a la zin -1-on e
(19). Preparation from 6 as described for 15 yielded 3.2 g (55%)
1
of 19: mp 183-184 °C; H NMR (DMSO-d6) δ 12.6 (s, HN),
8.33 (d, 1H), 8.29 (m, 1H), 7.85 (m, 3H), 7.18 (s, 1H), 7.10 (d,
1H), 4.28 (s, 2H), 2.40 (s, 3H); FAB MS (M + H)+ ) 252. Anal.
(C15H13N3O) C, H, N.
7-Hyd r oxy-6-(p yr id in -4-yl)-[1]p yr in d in -5-on e (10). To
an ice-cooled suspension of 7H-furo[3,4-b]pyridin-5-one22 (20.27
g, 150 mmol) and 4-pyridinecarbaldehyde (14.1 mL, 150 mmol)
in methanol (120 mL) and ethyl propionate (75 mL) was added
sodium methanolate (5.4 M in methanol; 27.8 mL, 150 mmol).
After 15 min at rt the reaction mixture was heated to 65 °C
for 2 h and then cooled to rt again. The reaction mixture was
diluted with water (120 mL) and filtered. To the filtrate was
added acetic acid (8.57 mL) and the formed precipitate filtered
off, yielding 9.2 g (27%) of 10: FAB MS (M + H)+ ) 225. Anal.
(C13H8N2O2‚0.3H2O) C, H, N.
7-Hyd r oxy-6-(p yr id in -4-yl)-[2]p yr in d in -5-on e (11). Pre-
pared from a mixture of 1H-furo[3,4-c]pyridin-3-one and 3H-
furo[3,4-c]pyridin-1-one23 and 4-pyridinecarbaldehyde as de-
scribed for 10 yielded 2.8 g (38%) of 11: 1H NMR (DMSO-d6)
δ 8.75 (d, 1H), 8.68 (d, 2H), 8.59 (s, 1H), 8.30 (d, 2H), 7.39 (d,
1H); FAB MS (M + H)+ ) 225.
3-[1-(P yr id in -4-yl)e t h ylid e n e ]-3H -isob e n zofu r a n -1-
on e (12). Phthalic acid anhydride (25.0 g, 169 mmol), 3-pyri-
din-4-ylpropionic acid24 (11.8 g, 78 mmol), sodium acetate (1.06
g, 13 mmol), and dimethylacetamide (40 mL) were stirred for
4 h at 180 °C. The reaction mixture was then poured onto a
mixture of ice and 0.2 N sodium hydroxide solution (250 mL),
stirred, and extracted twice with ethyl acetate. The organic
phases were washed with water and brine, dried (Na2SO4),
and concentrated by evaporation. Chromatography (ethyl
acetate/methanol 19:1) and crystallization from ethanol yielded
4-(2,6-Dim et h ylp yr id in -4-yl)m et h yl-2H -p h t h a la zin -1-
on e (20). Preparation from 7 as described for 15 yielded 3.2 g
1
(57%) of 20: mp 229-230 °C; H NMR (DMSO-d6) δ 12.6 (s,
HN), 8.27 (d, 1H), 7.85 (m, 3H), 6.95 (s, 2H), 4.23 (s, 2H), 2.35
(s, 6H); FAB MS (M + H)+ ) 266. Anal. (C16H15N3O) C, H, N.
4-(P yr id in -4-yl)m et h yl-7-a m in o-2H -p h t h a la zin -1-on e
(21a ).11 Preparation from 5-nitro-3-hydroxy-2-(pyridin-4-yl)-
inden-1-one (8)26 as described for 15 yielded 17.3 g (43%) of
21a : 1H NMR (DMSO-d6) δ 12.1 (s, HN), 8.43 (d, 2H), 7.56 (d,
1H; NOE on dd at 7.00 ppm and s at 4.16 ppm), 7.26 (m, 3H),
7.00 (dd, 1H), 6.17 (s, H2N), 4.16 (s, 2H; NOE on d at 7.56
ppm and d at 7.26 ppm); FAB MS (M + H)+ ) 253. Anal.
(C14H12N4O) C, H, N.
4-(P yr id in -4-yl)m eth yl-7-tr iflu or a ceta m id o-2H-p h th a l-
a zin -1-on e Tr iflu or a ceta te (21b). A suspension of 21a (500
mg, 1.98 mmol) in trifluoracetic anhydride (1.65 mL, 11.9
mmol) was stirred for 48 h at rt. The mixture was diluted with
water, filtrated, and washed with water, yielding 729 mg (80%)
of 21b: 1H NMR (DMSO-d6) δ 12.7 (s, HN), 11.8 (s, HN), 8.72
(d, 2H), 8.67 (d, 1H), 8.15 (dd, 1H), 8.03 (d, 1H), 7.78 (d, 2H),
4.53 (s, 2H); FAB MS (M + H)+ ) 349.
4-(P yr id in -4-yl)m e t h yl-6-m e t h yl-2H -p h t h a la zin -1-
on e a n d 4-(P yr id in -4-yl)m eth yl-7-m eth yl-2H-p h th a la zin -
1-on e (22). Preparation from 9 as described for 15 yielded 13

Antagonists of Tumor-Driven Angiogenesis
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2319
g (60%) of a ca. 1:1 mixture of the regioisomers 22: 1H NMR
(DMSO-d6) δ 12.5 (sb, HN), 8.45 (d, 2H), 8.14 (d, HC6-Me), 8.06
(s, HC7-Me), 7.80 (d, HC7-Me; NOE on signals at 7.69, 7.29, and
4.30 ppm), 7.74 (s, HC6-Me; NOE on signals at 7.31, 4.31, and
2.46 ppm), 7.69 (d, HC7-Me), 7.64 (d, HC6-Me), 7.31 (d, 2HC6-Me),
7.29 (d, 2HC7-Me), 4.31 (s, H2C6-Me), 4.30 (s, H2C7-Me), 2.47 (s,
H3C7-Me), 2.46 (s, H3C6-Me); FAB MS (M + H)+ ) 252. Anal.
(C15H13N3O‚0.16H2O) C, H, N, O, H2O.
5-(P yr id in -4-yl)m eth yl-7H-p yr id o[2,3-d ]p yr id a zin e-8-
on e (23) a n d 8-(P yr id in -4-yl)m eth yl-6H-p yr id o[2,3-d ]p y-
r id a zin e-5-on e (24). Preparation from 10 as described for 15
and chromatography (ethyl acetate/methanol 19:1 f 7:3) gave
1.50 g (16%) of 24 followed by 0.92 g (10%) of 23. 23: 1H NMR
(DMSO-d6) δ 12.83 (s, HN), 9.04 (d, 1H), 8.46 (d, 2H), 8.33
(dd, 1H), 7.86 (dd, 1H), 7.30 (d, 2H), 4.34 (s, 2H; NOE on
signals at 8.33 and 7.30 ppm). Anal. (C13H10N4O) C, H, N. 24:
mp 246-248 °C; 1H NMR (DMSO-d6) δ 12.83 (s, HN), 9.13
(dd, 1H), 8.59 (dd, 1H), 8.43 (d, 2H), 7.85 (dd, 1H), 7.29 (d,
2H), 4.38 (s, 2H; NOE on d at 7.29 ppm). Anal. (C13H10N4O)
C, H, N.
1-(P yr id in -4-yl)m eth yl-3H-p yr id o[3,4-d ]p yr id a zin e-4-
on e (25) a n d 4-(p yr id in -4-yl)m eth yl-2H-p yr id o[3,4-d ]p y-
r id a zin e-1-on e (26). Preparation from 11 as described for 15,
chromatography (toluene/2-propanol 19:1 f toluene/2-pro-
panol/NH3 concentrated 90:10:0.25 f 90:20:0.5), and crystal-
lization from 2-propanol gave 0.71 g (25%) of 26 and 0.34 g
(12%) of 25. 25: mp 204-208 °C; 1H NMR (DMSO-d6) δ 12.93
(s, HN), 9.45 (s, 1H), 9.00 (d, 1H), 8.47 (d, 2H), 7.80 (d, 1H),
7.33 (d, 2H), 4.34 (s, 2H; NOE on d at 7.80 ppm); FAB MS (M
+ H)+ ) 239. 26: mp 236-237 °C; 1H NMR (DMSO-d6) δ 12.90
(s, HN), 9.32 (s, 1H), 8.96 (d, 1H), 8.47 (d, 2H), 8.08 (d, 1H),
7.34 (d, 2H), 4.43 (s, 2H; NOE on s at 9.32 ppm); FAB MS (M
+ H)+ ) 239.
evaporated. The resulting yellow oil was redissolved in dichlo-
romethane and the product was precipitated with diisopropyl
ether to yield 130 mg (6%) of 29 as light-yellow crystals: 1H
NMR (CDCl3) δ 12.3 (sb, 1H), 8.55 (dd, 2H), 7.10 (d, 2H), 3.95
(s, 2H), 2.15 (s, 3H), 2.00 (s, 3H); FAB MS (M + H)+ ) 216.
1-Ch lor o-4-(4-p yr id ylm eth yl)p h th a la zin e (30). To 15
(29 g, 122 mmol) in acetonitrile (450 mL) and HCl/dioxane (4
N; 61 mL, 244 mmol) was added phosphoryl chloride (28 mL,
306 mmol). After stirring for 27 h at 50 °C, a solution of
NaHCO3 (119 g) in water (1.45 L) was added to the ice-cooled
white suspension. Stirring and filtration then afforded 28.7 g
(92%) of 30: 1H NMR (DMSO-d6) δ 8.46 (d, 2H), 8.35 (m, 2H),
8.14 (m, 2H), 7.33 (d, 2H), 4.77 (s, 2H); FAB MS (M + H)+
)
256. Anal. (C14H10N3Cl) C, H, N, Cl.
1-Ch lor o-4-(3-p yr id ylm eth yl)p h th a la zin e (31). To 16
(50 g, 0.21 mol) in ice-cooled acetonitrile (250 mL) was added
phosphoryl chloride (42.4 mL, 0.46 mol). After stirring for 10
h at 100 °C, the reaction mixture was concentrated in vacuo,
and the residue diluted with dichloromethane, ice-water, and
a saturated solution of Na2CO3 (0.4 L). The organic layer was
separated, washed with water and brine, dried (Na2SO4), and
concentrated. Stirring of the residue in ethanol and filtration
then led to 24.4 g (45%) of 31: mp 164-166 °C; 1H NMR
(DMSO-d6) δ 8.65 (s, 1H), 8.43 (m, 2H), 8.32 (m, 1H), 8.15 (m,
2H), 7.70 (d, 1H), 7.30 (dd, 1H), 4.77 (s, 2H). Anal. (C14H10N3-
Cl) C, H, N.
1-Ch lor o-4-(4-pyr im idylm eth yl)ph th alazin e (32): Prepa-
ration from 18 as described for 30 yielded 750 mg (82%) of 32:
FAB MS (M - H)+ ) 255.
1-Ch lor o-4-(4-p yr id ylm e t h yl)-7-t r iflu or a ce t a m id o-
p h th a la zin e (33): Preparation from 21b as described for 30
yielded 404 mg (92%) of 33: FAB MS (M + H)+ ) 367.
1-Ch lor o-4-(4-pyr idylm eth yl)-6-m eth ylph th alazin e an d
1-Ch lor o-4-(4-p yr id ylm eth yl)-7-m eth ylp h th a la zin e (34):
Preparation from 22 as described for 30 yielded 5.0 g (82%) of
34 as a mixture of regioisomers: 1H NMR (DMSO-d6) δ 8.45
(m, 2H), 8.20, 8.09 and 7.95 (m, s, m, 3H), 7.34 and 7.30 (2d,
r a c-4-[1-(4-P yr id yl)eth yl]-2H-p h th a la zin -1-on e (27). In
ethanol (50 mL), 12 (2.20 g, 9.27 mmol) and hydrazine hydrate
(597 µL, 12 mmol) were boiled for 4.5 h under reflux. A white
solid settled out, which was filtered off and discarded. Con-
centration of the filtrate and crystallization from acetonitrile
2H), 4.72 (s, 2H), 2.60 and 2.59 (2s, 3H); FAB MS (M + H)+
270.
)
1
gave 1.75 g (74%) of 27: mp 202-203 °C; H NMR (DMSO-
d6) δ 12.7 (s, HN), 8.45 (d, 2H), 8.24 (m, 1H), 7.83 (m, 3H),
3-C h lo r o -4,5-d im e t h y l-6-(4-p y r id y lm e t h y l)p y r id -
a zin e (36). A solution of 29 (700 mg, 3.26 mmol) in phosphorus
oxychloride (7 mL) was heated for 3 h to 120 °C. The reaction
mixture was then poured onto ice water and the pH adjusted
to alkaline with 2 N NaOH. The aqueous solution was
extracted three times with dichloromethane. The combined
organic extracts were filtered through cotton, and the solvent
was evaporated. The remaining residue was purified by
chromatography (dichloromethane/methanol 19:1) to yield 390
mg (51%) of 36 as brown crystals: 1H NMR (CDCl3) δ 8.50
(dd, 2H), 7.10 (d, 2H), 4.35 (s, 2H), 2.35 (s, 3H), 2.15 (s, 3H);
FAB MS (M + H)+ ) 234.
1-(4-Ch lor oa n ilin o)-4-(4-p yr id ylm et h yl)p h t h a la zin e
(CGP 79787). A mixture of phosphorus pentoxide (142 g, 1.00
mol), triethylamine hydrochloride (137.6 g, 1.00 mol), and
4-chloroaniline (127.6 g, 1.00 mol) was heated to 170 °C for
40 min. To the melt was added 15 (59.3 g, 0.25 mol) during 5
min and stirring continued at 170 °C for additional 2 h. Then
the heating bath was removed. Tetramethylurea (231 mL) was
added during 10 min (135 °C), followed by water (830 mL;
cooling) and a mixture of water (330 mL) and ammonia (30%
in water; 306 mL). At 10 °C, diethyl ether (500 mL) was given
to the yellow suspension. Filtration and washing with water
(3 L) and diethyl ether (1 L) yielded 70.9 g (82%) of CGP
79787: mp 209-212 °C; 1H NMR (DMSO-d6) δ 9.30 (sb, 1 H),
8.60 (d, 1H), 8.46 (d, 2H), 8.0 (m, 5H), 7.40 (d, 2H), 7.33 (d,
2H), 4.59 (s, 2H); FAB MS (M + H)+ ) 347. Anal. (C20H15N4-
Cl) C, H, N.
7.33 (d, 2H), 4.85 (q, 1H), 1.58 (d, 3H); FAB MS (M + H)+
)
.
252. Anal. (C15H13N3O 0.15 H2O) C, H, N.
4-(P yr id in -4-yl)m et h yl-5,6,7,8-t et r a h yd r o-2H -p h t h a l-
a zin -1-on e (28). Preparation from 13 as described for 15
yielded 330 mg (40%) of 28: mp 193-194 °C; 1H NMR (DMSO-
d6) δ 12.6 (s, HN), 8.46 (d, 2H), 7.19 (d, 2H), 3.93 (s, 2H), 2.36
(m, 4H), 1.60 (m, 4H); FAB MS (M + H)+ ) 242. Anal.
(C14H15N3O) C, H, N.
4,5-Dim e t h yl-6-(4-p yr id ylm e t h yl)-2H -p yr id a zin e -3-
on e (29). To a solution of diisopropylamine (26.1 mL, 184
mmol) in THF (200 mL) was added a 1.6 M solution of
butyllithium (124 mL, 198 mmol) in THF at 0 °C. After cooling
to -20 to -30 °C, a solution of 4-picoline (19.3 mL, 198 mmol)
in THF (200 mL) was added dropwise and the reaction mixture
was stirred for 60 min at -30 °C. A solution of maleic acid
anhydride (10 g, 102 mmol) in THF (100 mL) was then added
dropwise at -78 °C and the mixture was stirred for 1 h at
-78 °C and for 2 h at rt. After this time the reaction mixture
was diluted with 2 N HCl (500 mL) and washed twice with
ethyl acetate. This aqueous layer was concentrated by evapo-
ration and the pH adjusted to alkaline with 2 N NaOH. The
solution was washed again with ethyl acetate (2×) and then
reacidified with 2 N HCl and evaporated. The resulting orange
residue was filtered through silica gel (CH2Cl2/MeOH 5:1) and
the material thus obtained [1H NMR (CDCl3): δ 3.20 (s, 2H),
1.25 (s, 3H), 1.20 (s, 3H); FAB MS (M + H)+ ) 220] was
processed without further purification.
A solution of 2 g of the crude product obtained above (14)
and hydrazine hydrate (1.1 mL, 22 mmol) in n-butanol (2 mL)
was heated under nitrogen for 2 h to 120 °C. After cooling to
rt, the resulting emulsion was concentrated by evaporation
and then, after addition of a small amount of water, extracted
three times with dichloromethane. The combined organic
extracts were filtered through cotton, and the solvent was
1-(4-Ch lor oa n ilin o)-4-(4-p yr id ylm et h yl)p h t h a la zin e
Su ccin a te (CGP 79787D). A solution of succinic acid (1.77
g, 15 mmol) in ethanol (35 mL) was added to a hot solution of
CGP 79787 (5.0 g, 14.4 mmol) in ethanol (150 mL). As the
mixture cooled (scraping) to 0 °C, a crystalline precipitate
slowly formed, which was filtered off and washed with ethanol,
yielding 5.06 (75%) of CGP 79787D: mp 195 °C; 1H NMR

2320 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Bold et al.
(DMSO-d6) δ 12.2 (sb, 2 HO2C), 9.26 (s, HN), 8.58 (d, 1H), 8.43
(d, 2H), 8.11 (d, 1H), 7.99 (m, 3H), 7.93 (m, 1H), 7.40 (d, 2H),
7.31 (d, 2H), 4.58 (s, 2H), 2.40 (s, 4H). Anal. (C24H21N4ClO4)
C, H, N.
1-(4-Ch lor oa n ilin o)-4-[2-m et h ylp yr id in -4-ylm et h yl]-
p h th a la zin e (43). Preparation as described for CGP 79787
starting from 19, chromatography (ethyl acetate/methanol 19:
1), and crystallization from acetonitrile gave 193 mg (34%) of
43: mp 158-159 °C; 1H NMR (DMSO-d6) δ 9.27 (s, 1H), 8.59
(d, 1H), 8.30 (d, 1H), 8.10 (m, 1H), 7.97 (m, 4H), 7.40 (d, 2H),
7.18 (s, 1H), 7.10 (d, 1H), 4.53 (s, 2H), 2.39 (s, 3H); FAB MS
(M + H)+ ) 361. Anal. (C21H17N4Cl) C, H, N.
1-(4-Ch lor oa n ilin o)-4-[2,6-d im eth ylp yr id in -4-ylm eth yl]-
p h th a la zin e (44). Preparation as described for CGP 79787
starting from 20, chromatography (ethyl acetate/methanol 19:
1), and crystallization from acetonitrile gave 983 mg (62%) of
44: mp 175-176 °C; 1H NMR (DMSO-d6) δ 9.27 (s, 1H), 8.60
(d, 1H), 8.0 (m, 5H), 7.40 (d, 2H), 6.97 (s, 2H), 4.48 (s, 2H),
2.35 (s, 6H); FAB MS (M + H)+ ) 375. Anal. (C22H19N4Cl) C,
H, N.
1-(4-Ch lor oan ilin o)-4-(4-pyr idylm eth yl)ph th alazin e Di-
h yd r och lor id e (1). A solution of CGP 79787 (115 g, 331
mmol) in methanol (3.25 L) was prepared by heating it up to
50 °C. Then concentrated HCl (37%, 58.05 mL) was added
dropwise at 35 °C. After cooling the solution down to 10 °C,
diethyl ether (3.35 L) was added. Filtration of the precipitate
and washing with diethyl ether (4 L) gave 128 g (84%) of 1:
mp 272-273 °C; 1H NMR (DMSO-d6) δ 11.7 (sb, HN), 9.28
(m, 1H), 8.88 (d, 2H), 8.37 (m, 1H), 8.23 (m, 2H), 8.04 (d, 2H),
7.72 (d, 2H), 7.59 (d, 2H), 5.03 (s, 2H); FAB MS (M + H)+
)
347. Anal. (C20H15N4Cl ‚ 2.00 HCl ‚ 2.17 H2O) C, H, N, Cl, H2O.
(4-Ch lor oph en yl)m eth yl[4-(4-pyr idylm eth yl)ph th alazin -
1-yl]a m in e (37). A solution of 30 (255 mg, 1.0 mmol) and
4-chloro-N-methylaniline (385 µL, 3 mmol) in DMPU (2 mL)
was heated to 100 °C for 15 h. The reaction mixture was
diluted with dichloromethane (20 mL) and aqueous ammonia
(10%; 10 mL), and the organic layer was separated, washed
twice with water (10 mL), dried (Na2SO4), and concentrated.
Chromatography (ethyl acetate/methanol 50:1 f 20:1) and
crystallization by adding hexane gave 75 mg (20%) of 37: mp
128-130 °C; 1H NMR (DMSO-d6) δ 8.48 (d, 2H), 8.18 (d, 1H),
7.86 (t, 1H), 7.74 (t, 1H), 7.55 (d, 1H), 7.36 (d, 2H), 7.29 (d,
2H), 6.97 (d, 2H), 4.68 (s, 2H), 3.54 (s, 3H); HR-FAB MS
(C21H17N4Cl) (M + H)+ calcd 361.1220, obsd 361.1222.
1-(4-C h lo r o a n ilin o )-4-(4-p y r id y lm e t h y l)-7-a m in o -
p h th a la zin e (45). A mixture of 33 (11 g, 30 mmol), 4-chlo-
roaniline (9.2 g, 72 mmol), and n-butanol (90 mL) was heated
to 100 °C for 4 h. The reaction mixture was diluted at rt with
5% Na2CO3 solution (200 mL) and ethyl acetate (250 mL). The
aqueous layer was separated off and extracted with ethyl
acetate (200 mL). The organic phases were washed with water
(4 × 100 mL), dried (Na2SO4), and concentrated by evapora-
tion. Chromatography (dichloromethane/methanol 9:1) gave
2.2 g (20%) of 45: mp 277-279 °C; 1H NMR (DMSO-d6) δ 8.86
(s, HN), 8.42 (d, 2H), 7.88 (d, 2H), 7.80 (d, 1H), 7.33 (d, 2H),
7.28 (d, 2H), 7.26 (d, 1H), 7.15 (dd, 1H), 6.15 (s, H2N), 4.40 (s,
2H); FAB MS (M + H)+ ) 362. Anal. (C20H16N5Cl ‚ 0.2 H2O)
C, H, N, Cl.
1-(4-Ch lor oa n ilin o)-4-(4-p yr id ylm e t h yl)-6-m e t h yl-
ph th alazin e an d 1-(4-Ch lor oan ilin o)-4-(4-pyr idylm eth yl)-
7-m eth ylp h th a la zin e Dih yd r och lor id e (46). A mixture of
34 (500 mg, 1.85 mmol), 4-chloroaniline (248 mg, 1.94 mmol),
ethanol (66 mL), and HCl (4 N in dioxane; 0.5 mL) was heated
for 3 h to 80 °C. Upon standing at rt, product crystallized
slowly from the red solution. Filtration and washing with
ethanol and diethyl ether gave 433 mg (52%) of 46 as a ca. 2:3
mixture of the 6-methyl and the 7-methyl derivative: 1H NMR
(DMSO-d6) δ 11.2 (sb, HN), 8.96 (d, HC6-Me), 8.92 (s, HC7-Me),
8.81 (d, 2HC6-Me), 8.80 (d, 2HC7-Me), 8.26 (d, HC7-Me), 8.20 (s,
HC6-Me), 8.07 (d, HC6-Me), 8.03 (d, HC7-Me), 7.93 (d, 2HC6-Me),
7.90 (d, 2HC7-Me), 7.70 (d, 2HC6-Me), 7.68 (d, 2HC7-Me), 7.57
(d, 2HC6-Me + 2HC7-Me), 4.94 (s, H2C7-Me), 4.91 (s, H2C6-Me),
2.63 (s, H3C7-Me), 2.61 (s, H3C6-Me); FAB MS (M + H)+ ) 361.
Anal. (C21H17N4Cl‚1.94HCl‚0.93H2O) C, H, N, Cl.
1-(4-C h lor op h e n ox y )-4-(4-p yr id ylm e t h yl)p h t h a la -
zin e (38). A mixture of 30 (200 mg, 0.78 mmol), K2CO3 (173
mg, 1.25 mmol), and 4-chlorophenol (120 mg, 0.93 mmol) in
DMSO (2 mL) was heated for 2.5 h to 90 °C. The reaction
mixture was distributed between water (20 mL) and ethyl
acetate (20 mL), and the aqueous phase was separated and
extracted with two portions of ethyl acetate. The organic
phases were washed with water and brine, dried (MgSO4), and
concentrated by evaporation. Chromatography (ethyl acetate/
1
methanol 4:1) gave 96 mg (35%) of 38: mp 207-208 °C; H
NMR (DMSO-d6) δ 8.44 (d, 2H), 8.40 (m, 1H), 8.26 (m, 1H),
8.06 (m, 2H), 7.54 (d, 2H), 7.40 (d, 2H), 7.30 (d, 2H), 4.64 (s,
2H); HR-FAB MS (C20H14N3ClO) (M + H)+ calcd 348.0904,
obsd 348.0904.
1-(4-Ch lor oph en ylsu lfan yl)-4-(4-pyr idylm eth yl)ph th ala-
zin e (39). Preparation as described for 38 starting from
4-chlorothiophenol gave 83 mg (29%) of 39: mp 204-206 °C;
1H NMR (DMSO-d6) δ 8.44 (d, 2H), 8.29 (m, 2H), 8.06 (m, 2H),
7.66 (d, 2H), 7.55 (d, 2H), 7.30 (d, 2H), 4.66 (s, 2H); HR-FAB
MS (C20H14N3ClS) (M + H)+ calcd 364.0675, obsd 364.0679.
1-(4-Ch lor oan ilin o)-4-(3-pyr idylm eth yl)ph th alazin e Hy-
d r och lor id e (40). A mixture of 31 (1.00 g, 3.9 mmol) and
4-chloroaniline (1.5 g, 11.7 mmol) was heated to 110 °C for 4
h. The reaction mixture was diluted with dichloromethane (10
mL) and the crystallized material filtered off. Recrystallization
from methanol yielded 620 mg (41%) of 40: 1H NMR (DMSO-
d6) δ 10.7 (sb, HN), 8.93 (m, 1H), 8.78 (s, 1H), 8.58 (d, 1H),
8.40 (m, 1H), 8.17 (m, 2H), 8.03 (d, 1H), 7.80 (d, 2H), 7.56 (m,
3H), 4.77 (s, 2H); FAB MS (M + H)+ ) 347. Anal. (C20H15N4Cl
‚ HCl) C, H, N.
8-(4-Ch lor oa n ilin o)-5-(4-p yr id ylm eth yl)p yr id o[2,3-d ]-
p yr id a zin e (47). Preparation as described for CGP 79787
starting from 23, chromatography (ethyl acetate/methanol 50:1
f 25:1), and crystallization from acetonitrile gave 268 mg
1
(43%) of 47: mp 196-197 °C; H NMR (DMSO-d6) δ 9.75 (s,
HN), 9.23 (dd, 1H), 8.60 (dd, 1H), 8.46 (d, 2H), 8.25 (d, 2H),
8.00 (dd, 1H), 7.41 (d, 2H), 7.35 (d, 2H), 4.61 (s, 2H); FAB MS
(M + H)+ ) 348. Anal. (C19H14N5Cl) C, H, N.
4-(4-Ch lor oa n ilin o)-1-(4-p yr id ylm eth yl)p yr id o[3,4-d ]-
p yr id a zin e (48). Preparation as described for CGP 79787
starting from 25, chromatography (ethyl acetate/methanol
100:1 f 9:1), and crystallization from acetonitrile and metha-
nol gave 90 mg (19%) of 48: 1H NMR (DMSO-d6) δ 9.96 (s,
1H), 9.63 (s, HN), 9.00 (d, 1H), 8.47 (d, 2H), 8.00 (d, 2H), 7.97
(d, 1H), 7.43 (d, 2H), 7.32 (d, 2H), 4.59 (s, 2H); HR-FAB MS
(C19H14N5Cl) (M + H)+ calcd 348.1016, obsd 348.1017.
1-(4-Ch lor oa n ilin o)-4-(4-p yr id ylm eth yl)p yr id o[3,4-d ]-
p yr id a zin e (49). Preparation as described for CGP 79787
starting from 26, chromatography (ethyl acetate/methanol 50:1
f 19:1), and crystallization from acetonitrile and methanol
1-(4-Ch lor oan ilin o)-4-(2-pyr idylm eth yl)ph th alazin e (41).
Preparation as described for CGP 79787 starting from 17,
chromatography (ethyl acetate), and crystallization from ethyl
1
acetate gave 309 mg (53%) of 41: mp 154-156 °C; H NMR
(DMSO-d6) δ 9.25 (s, 1H), 8.58 (d, 1H), 8.45 (d, 1H), 8.16 (m,
1H), 7.97 (m, 4H), 7.69 (m, 1H), 7.40 (d, 2H), 7.32 (d, 1H), 7.20
(dd, 1H), 4.70 (s, 2H); FAB MS (M + H)+ ) 347. Anal.
(C20H15N4Cl) C, H, N.
1
gave 114 mg (26%) of 49: mp 227-228 °C; H NMR (DMSO-
d6) δ 9.59 (s, 1H), 9.47 (s, HN), 9.08 (d, 1H), 8.47 (m, 3H), 8.02
(d, 2H), 7.43 (d, 2H), 7.35 (d, 2H), 4.69 (s, 2H); HR-FAB MS
(C19H14N5Cl) (M + H)+ calcd 348.1016, obsd 348.1017.
5-(4-Ch lor oa n ilin o)-8-(4-p yr id ylm eth yl)p yr id o[2,3-d ]-
p yr id a zin e (50). Preparation as described for CGP 79787
starting from 24, chromatography (ethyl acetate/methanol 50:1
f 25:1), and crystallization from acetonitrile and methanol
1-(4-Ch lor oa n ilin o)-4-(4-p yr im id ylm e t h yl)p h t h a la -
zin e (42). Preparation as described for 40 starting from 32
and chromatography (ethyl acetate/methanol 19:1) gave 14 mg
(10%) of 42: 1H NMR (DMSO-d6) δ 9.28 (s, 1H), 9.05 (s, 1H),
8.68 (d, 1H), 8.59 (m, 1H), 8.13 (m, 1H), 7.95 (m, 4H), 7.48 (d,
1H), 7.38 (d, 2H), 4.72 (s, 2H); HR-FAB MS (C19H14N5Cl) (M
+ H)+ calcd 348.1016, obsd 348.1015.

Antagonists of Tumor-Driven Angiogenesis
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2321
1
gave 288 mg (39%) of 50: mp 220-222 °C; H NMR (DMSO-
d6) δ 9.46 (s, HN), 9.24 (d, 1H), 9.03 (d, 1H), 8.43 (d, 2H), 8.0
(m, 3H), 7.40 (d, 2H), 7.32 (d, 2H), 4.64 (s, 2H); FAB MS (M +
H)+ ) 348. Anal. (C19H14N5Cl) C, H, N.
1-(4-Bip h en yla m in o)-4-(4-p yr id ylm eth yl)p h th a la zin e
(58). Preparation from 4-aminobiphenyl as described for CGP
79787, chromatography (dichlormethane/methanol 50:1), and
crystallization from acetonitrile gave 396 mg (24%) of 58: mp
189-191 °C; 1H NMR (DMSO-d6) δ 9.29 (s, HN), 8.64 (d, 1H),
8.46 (d, 2H), 8.09 (m, 3H), 7.97 (m, 2H), 7.70 (d, 4H), 7.46 (t,
2H), 7.33 (m, 3H), 4.60 (s, 2H); FAB MS (M + H)+ ) 389. Anal.
(C26H20N4) C, H, N.
1-(4-Ch lor oa n ilin o)-4-(4-p yr id ylm eth yla m in o)p h th a la -
zin e (51). A mixture of 0.500 g (1.54 mmol) of 1-chloro-4-(4-
chloroanilino)phthalazine hydrochloride (35)27 and 2.00 g (18.5
mmol) of 4-aminomethylpyridine was stirred for 36 h at 90
°C. Chromatography (ethyl acetate f ethyl acetate/methanol
19:1) and crystallization from methanol gave 127 mg (23%) of
51: mp 233-236 °C; 1H NMR (DMSO-d6) δ 8.76 (s, HN), 8.44
(d, 2H), 8.38 (m, 2H), 7.97 (m, 2H), 7.80 (d, 2H), 7.75 (m, HN),
1-(3-Me t h oxya n ilin o)-4-(4-p yr id ylm e t h yl)p h t h a la -
zin e (59). Preparation from m-anisidine as described for 56
and crystallization from acetonitrile gave 10.7 g (80%) of 59:
1
mp 158-159 °C; H NMR (DMSO-d6) δ 9.12 (s, HN), 8.61 (d,
7.36 (d, 2H), 7.28 (d, 2H), 4.75 (d, 2H); FAB MS (M + H)+
)
1H), 8.44 (d, 2H), 8.09 (m, 1H), 7.94 (m, 2H), 7.67 (m, 1H),
7.55 (d, 1H), 7.31 (d, 2H), 7.24 (t, 1H), 6.62 (d, 1H), 4.59 (s,
2H), 3.78 (s, 3H); FAB MS (M + H)+ ) 343. Anal. (C21H18N4O)
C, H, N.
362. Anal. (C20H16N5Cl) C, H, N.
r a c-1-(4-Ch lor oa n ilin o)-4-[1-(4-p yr id yl)eth yl]p h th a la -
zin e (52). Preparation as described for CGP 79787 starting
from 27, chromatography (ethyl acetate/methanol 19:1), and
crystallization from methanol gave 145 mg (32%) of 52: mp
132-134 °C; 1H NMR (DMSO-d6) δ 9.28 (s, HN), 8.58 (d, 1H),
8.44 (d, 2H), 8.14 (d, 1H), 8.04 (d, 2H), 7.94 (m, 2H), 7.41 (d,
2H), 7.36 (d, 2H), 5.10 (q, 1H), 1.73 (d, 3H); FAB MS (M +
H)+ ) 361. Anal. (C21H17N4Cl‚0.50MeOH) C, H, N.
1-(3-H yd r oxya n ilin o)-4-(4-p yr id ylm e t h yl)p h t h a la -
zin e (60). Preparation from 3-aminophenol and 30 as de-
scribed for 40, stirring in a mixture of aqueous K2CO3 (20%)
and ethyl acetate, and washing with boiling methanol gave
1
260 mg (51%) of 60: mp 217-219 °C; H NMR (DMSO-d6) δ
9.35 (s, 1H), 9.12 (sb, 1H), 8.61 (d, 1H), 8.45 (d, 2H), 8.10 (d,
1H), 7.93 (m, 2H), 7.56 (s, 1H), 7.33 (d, 2H), 7.26 (d, 1H), 7.12
(t, 1H), 6.45 (dd, 1H), 4.57 (s, 2H); FAB MS (M + H)+ ) 329.
Anal. (C20H16N4O ‚ 0.36 H2O) C, H, N, H2O.
1-(4-Ch lor oan ilin o)-4-(4-pyr idylm eth yl)-5,6,7,8-tetr ah y-
d r op h t h a la zin e (53). Preparation as described for CGP
79787 starting from 28, chromatography (ethyl acetate/
methanol 40:1 f 20:1), and crystallization from acetonitrile
1-(3,4-Dich lor oa n ilin o)-4-(4-p yr id ylm et h yl)p h t h a la -
zin e (61). Preparation from 3,4-dichloroaniline as described
for 56 gave 3.2 g (84%) of 61: mp 254-255 °C; 1H NMR
(DMSO-d6) δ 9.44 (s, HN), 8.60 (d, 1H), 8.45 (m, 3H), 8.15 (d,
1H), 7.97 (m, 3H), 7.59 (d, 1H), 7.32 (d, 2H), 4.60 (s, 2H); FAB
MS (M + H)+ ) 381/383. Anal. (C20H14N4Cl2) C, H, N.
1-(3-Me t h oxy-4-ch lor oa n ilin o)-4-(4-p yr id ylm e t h yl)-
p h t h a la zin e (62). Preparation from 3-methoxy-4-chloro-
aniline as described for 56 and washing in boiling acetonitrile
gave 8.1 g (76%) of 62: mp 243-244 °C; 1H NMR (DMSO-d6)
δ 9.27 (s, HN), 8.61 (d, 1H), 8.44 (d, 2H), 8.12 (m, 1H), 7.97
(m, 2H), 7.86 (m, 1H), 7.72 (dd, 1H), 7.37 (d, 1H), 7.32 (d, 2H),
4.60 (s, 2H), 3.88 (s, 3H); FAB MS (M + H)+ ) 377. Anal.
(C21H17N4ClO) C, H, N.
1
gave 285 mg (68%) of 53: mp 181-183 °C; H NMR (DMSO-
d6) δ 8.46 (d, 2H), 8.00 (s, HN), 7.78 (d, 2H), 7.32 (d, 2H), 7.20
(d, 2H), 4.17 (s, 2H), 2.50 (m, 4H), 1.72 (m, 4H); FAB MS (M
+ H)+ ) 351. Anal. (C20H19N4Cl) C, H, N.
3-(4-Ch lor oa n ilin o)-4,5-d im eth yl-6-(4-p yr id ylm eth yl)-
p yr id a zin e (54). A solution of 36 (70 mg, 0.3 mmol) and
4-chloroaniline (153 mg, 1.2 mmol) in n-butanol (2 mL) was
heated in a sealed tube for 20 h to 130 °C. After cooling to rt,
the residue was diluted with dichloromethane (100 mL) and
the solution washed with saturated aqueous NaHCO3 solution
(100 mL). The organic layer was dried (MgSO4) and the solvent
removed by evaporation. Purification of the residue by chro-
matography (dichloromethane/methanol 19:1) gave 18 mg
(19%) of 54: mp 196-199 °C; 1H NMR (CDCl3) δ 8.45 (sb, 2H),
7.55 (d, 2H), 7.25 (d, 2H), 7.10 (d, 2H), 6.20 (sb, 1H), 4.25 (s,
2H), 2.15 (s, 3H), 2.10 (s, 3H); HR-FAB MS (C18H17N4Cl) (M
+ H)+ calcd 325.1220, obsd 325.1222.
1-(3,5-Dim et h yla n ilin o)-4-(4-p yr id ylm et h yl)p h t h a la -
zin e (63). Preparation from 3,5-dimethylaniline as described
for CGP 79787 and crystallization from acetonitrile gave 820
1
mg (57%) of 63: mp 174-175 °C; H NMR (DMSO-d6) δ 9.02
(s, HN), 8.60 (d, 1H), 8.46 (d, 2H), 8.09 (m, 1H), 7.97 (m, 2H),
7.58 (s, 2H), 7.32 (d, 2H), 6.68 (s, 1H), 4.57 (s, 2H), 2.30 (s,
6H); FAB MS (M + H)+ ) 341. Anal. (C22H20N4) C, H, N.
1-(3-Tr iflu or m eth yl-4-ch lor oa n ilin o)-4-(4-p yr id ylm eth -
yl)p h th a la zin e (64). Preparation from 5-amino-2-chloroben-
zotrifluoride as described for 56 and washing in boiling
1-An ilin o-4-(4-p yr id ylm et h yl)p h t h a la zin e Dih yd r o-
ch lor id e (55): A suspension of 30 (30 g, 117 mmol), aniline
(11.5 g, 123 mmol), ethanol (390 mL) and HCl (4 N in dioxane;
30 mL) was heated for 2 h to 80 °C. During cooling the solution
to rt, the product crystallized. Filtration and washing with
ethanol gave 35 g (71%) of 55: mp 217-220 °C; 1H NMR
(DMSO-d6) δ 11.8 (s, HN), 9.23 (m, 1H), 8.86 (d, 2H), 8.34 (m,
1H), 8.22 (m, 2H), 8.00 (d, 2H), 7.57 (m, 4H), 7.40 (m, 1H),
4.97 (s, 2H); FAB MS (M + H)+ ) 313. Anal. (C20H16N4‚
2HCl‚1.85H2O) C, H, N, H2O.
1-(3-Meth ylan ilin o)-4-(4-pyr idylm eth yl)ph th alazin e (56).
A suspension of 30 (10 g, 39 mmol), m-toluidine (4.45 g, 41.5
mmol), ethanol (80 mL), and HCl (4 N in dioxane; 10.3 mL)
was heated for 3 h to 80 °C. After cooling to rt, the precipitate
was filtered off. The solid was dissolved in water (150 mL).
Precipitation by adding concentrated ammonia (25 mL), filtra-
tion, and recrystallization from acetonitrile/diethyl ether
finally gave 8.25 g (64%) of 56: mp 144-146 °C; 1H NMR
(DMSO-d6) δ 9.08 (s, HN), 8.60 (d, 1H), 8.43 (d, 2H), 8.10 (d,
1H), 7.94 (m, 2H), 7.80 (s, 1H), 7.71 (d, 1H), 7.30 (d, 2H), 7.23
(d, 1H), 6.85 (d, 1H), 4.57 (s, 2H), 2.33 (s, 3H); FAB MS (M +
H)+ ) 327. Anal. (C21H18N4 ‚ 0.2 H2O) C, H, N.
1-(4-t er t -Bu t yla n ilin o)-4-(4-p yr id ylm e t h yl)p h t h a la -
zin e Dih yd r och lor id e (57). Preparation from 4-tert-butyl-
aniline as described for CGP 79787, chromatography (toluene/
acetone 7:3), and crystallization as dihydrochloride salt from
methanol/diethyl ether gave 520 mg (27%) of 57: mp 196-
200 °C; 1H NMR (DMSO-d6) δ 11.8 (s, HN), 9.25 (m, 1H), 8.86
(d, 2H), 8.33 (m, 1H), 8.21 (m, 2H), 7.98 (d, 2H), 7.53 (m, 4H),
4.95 (s, 2H), 1.33 (s, 9H); FAB MS (M + H)+ ) 369. Anal.
(C24H24N4‚2HCl‚1.3H2O) C, H, N, H2O.
1
methanol gave 11.6 g (70%) of 64: mp 218-219 °C; H NMR
(DMSO-d6) δ 9.6 (s, HN), 8.60 (m, 2H), 8.46 (d, 2H), 8.37 (dd,
1H), 8.16 (d, 1H), 8.0 (m, 2H), 7.70 (d, 1H), 7.32 (d, 2H), 4.62
(s, 2H); FAB MS (M + H)+ ) 415. Anal. (C21H14N4ClF3) C, H,
N.
1-(3-Tr iflu or m eth yl-5-br om oa n ilin o)-4-(4-p yr id ylm eth -
yl)p h th a la zin e (65). 3-Amino-5-bromobenzotrifluoride (10.1
g, 42 mmol) and 30 (10.23 g, 40 mmol) in ethanol (160 mL)
and HCl (4 N in dioxane; 10 mL) were heated for 2.5 h to 80
°C. Addition of diethyl ether (100 mL) at rt formed a precipi-
tate which was filtered off at 5 °C and washed with diethyl
ether. Water (100 mL), dichloromethane (350 mL), and con-
centrated ammonia (20 mL) were added to the solid. After 15
min of stirring at 0 °C, the biphasic suspension was filtered,
and the crystals were washed with diethyl ether. Recrystal-
lization from dichloromethane and methanol gave 10.7 g (58%)
of 65: mp 225-226 °C; 1H NMR (DMSO-d6) δ 9.62 (s, HN),
8.70 (s, 1H), 8.62 (d, 1H), 8.45 (m, 3H), 8.17 (d, 1H), 8.0 (m,
2H), 7.55 (s, 1H), 7.33 (d, 2H), 4.63 (s, 2H); FAB MS (M + H)+
) 459/461. Anal. (C21H14N4BrF3) C, H, N.
1-(3-Tr iflu or m eth yl-5-flu or oa n ilin o)-4-(4-p yr id ylm eth -
yl)p h th a la zin e (66). Preparation from 3-amino-5-fluoroben-
zotrifluoride and 30 as described for 40, distributing in a
mixture of aqueous ammonia (10%) and dichloromethane,
chromatography (ethyl acetate f ethyl acetate/methanol 9:1),

2322 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Bold et al.
Endothelial Growth Factor Receptor-2 Tyrosine Kinase (VEG-
FR2 TK), a Key Enzyme in Angiogenesis. Biochemistry 1998,
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and crystallization from acetonitrile yielded 116 mg (29%) of
66: mp 253-255 °C; H NMR (DMSO-d6) δ 9.7 (s, HN), 8.62
(d, 1H), 8.46 (d, 2H), 8.37 (d, 1H), 8.26 (s, 1H), 8.17 (d, 1H),
8.01 (m, 2H), 7.33 (d, 2H), 7.25 (d, 1H), 4.63 (s, 2H); FAB MS
(M + H)+ ) 399. Anal. (C21H14N4F4) C, H, N.
1
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