Anthranilic acid amides: A novel class of antiangiogenic VEGF receptor kinase inhibitors

Article abstract of DOI:10.1021/jm020899q

Two readily synthesized anthranilamide, VEGF receptor tyrosine kinase inhibitors have been prepared and evaluated as angiogenesis inhibitors. 2-[(4-Pyridyl)methyl]amino-N-[3-(trifluoromethyl)phenyl]benzamide (5) and N-3-isoquinolinyl-2-[(4-pyridinylmethyl

Full text of DOI:10.1021/jm020899q

J . Med. Chem. 2002, 45, 5687-5693
5687
An th r a n ilic Acid Am id es: A Novel Cla ss of An tia n giogen ic VEGF Recep tor
Kin a se In h ibitor s
Paul W. Manley,* Pascal Furet, Guido Bold, J osef Bru¨ggen, J u¨rgen Mestan, Thomas Meyer,
Christian R. Schnell, and J eanette Wood
Oncology Research, Novartis Pharma AG, CH-4057 Basel, Switzerland
Martin Haberey, Andreas Huth, Martin Kru¨ger, Andreas Menrad, Eckhard Ottow, Dieter Seidelmann,
Gerhard Siemeister, and Karl-Heinz Thierauch
Schering AG, Research Laboratories, D-13342, Berlin, Germany
Received April 4, 2002
Two readily synthesized anthranilamide, VEGF receptor tyrosine kinase inhibitors have been
prepared and evaluated as angiogenesis inhibitors. 2-[(4-Pyridyl)methyl]amino-N-[3-(trifluo-
romethyl)phenyl]benzamide (5) and N-3-isoquinolinyl-2-[(4-pyridinylmethyl)amino]benzamide
(7) potently and selectively inhibit recombinant VEGFR-2 and VEGFR-3 kinases. As a
consequence of their physicochemical properties, these anthranilamides readily penetrate cells
and are absorbed following once daily oral administration to mice. Both 5 and 7 potently inhibit
VEGF-induced angiogenesis in an implant model, with ED50 values of 7 mg/kg. In a mouse
orthotopic model of melanoma, 5 and 7 potently inhibited both the growth of the primary tumor
as well as the formation of spontaneous peripheral metastases. The anthranilamides 5 and 7
represent a new structural class of VEGFR kinase inhibitors, which possess potent antian-
giogenic and antitumor properties.
In tr od u ction
attractive approach to selectively inhibit tumor growth
and metastasis, without affecting the surrounding
normal tissue.
Angiogenesis is a multistep process, whereby new
capillaries sprout and grow from existing blood vessels.
This process is tightly regulated and only occurs in
normal adult tissues during the female reproductive
cycle and tissue repair. In cancer, angiogenesis is crucial
for supplying growing tumors with a vasculature to
provide nutrients and remove waste products, as well
as providing a conduit for the dispersal of metastases.1,2
A key pro-angiogenic cytokine released by many tumor
types is vascular endothelial growth factor (VEGF). The
angiogenic activity of the VEGF family of proteins is
mediated by two high affinity receptors, VEGFR-1 and
VEGFR-2 located on vascular endothelial cells, with a
third, VEGFR-3 which mediates lymphangiogenesis,
found on lymphatic vessels.3-5 These VEGFR receptors
are members of the receptor tyrosine kinase family and
comprise an extracellular ligand binding domain, a
transmembrane domain, and an intracellular, split
kinase domain. VEGF is a heterodimeric protein, which
binds to two adjacent VEGF receptors, causing them to
dimerize, whereupon the tyrosine kinase domains be-
come activated, bind ATP, and catalyze the transfer of
the γ-phosphate from the ATP-Mg2+ complex to the
hydroxy group of a tyrosine residue. These cytosolic
phosphorylated sites can then serve as binding sites for
other substrates, which in turn may be phosphorylated
as part of a signal transduction cascade. Because of the
tissue localization of the receptors and the tight control
of angiogenesis, VEGFR-kinase inhibition presents an
Because of the difficulties associated with the com-
petitive inhibition of protein-protein interactions by
small molecular weight compounds,6 modulating the
activity of kinases by interfering either with ligand or
substrate binding is difficult and targeting the catalytic
site of kinases with ATP-competitive inhibitors is a more
promising approach for drug intervention.7 To this end,
there are several VEGFR kinase inhibitors (1-3) cur-
rently undergoing clinical evaluation in cancer patients,
belonging, respectively, to either the quinazoline,8,9
phthalazine,10-12 or indolinone13 chemical classes (Chart
1). In this communication, we describe two new com-
pounds, 5 and 7, structurally elaborated from an an-
thranilamide scaffold.
Ch em istr y. The anthranilamides may be conve-
niently prepared in two steps (Scheme 1). Thus, ring
opening of isatoic anhydride with 3-trifluoromethyla-
niline affords the amine 4, which upon reductive alky-
lation with 4-pyridinecarboxaldehye affords the anthra-
nilamide, 5. In an alternative synthesis, reductive
alkylation of methyl anthranilate affords the amine 6,
which is then subjected to Weinreb amidation,14 to give
the anthranilamide 7.
Compounds 5 and 7 are stable crystalline solids.
Although they have relatively low aqueous solubilities
(2 µg/mL) at pH 6.8 in phosphate buffer, as a conse-
quence of the basicity of both compound 5 (pKa 5.2) and
compound 7 (pKa 5.1, 2.7),15 their solubilities markedly
increase with decreasing pH (solubility at pH 1.0 > 200
µg/mL). Furthermore, the topological polar surface areas
of the compounds (TPSA 45.0 and 52.8 Å2 for 5 and 7,
* To whom correspondence should be addressed. Tel: 0041 61
6966878. Fax: 0041 61 6966246. E-mail: paul.manley@pharma.
novartis.com.
10.1021/jm020899q CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/20/2002

5688 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26
Manley et al.
Ch a r t 1
Sch em e 1. Synthesis of Anthranilamidesa
a
Reagents: (i) 3-trifluoromethylaniline, DMF, 100 oC; (ii) 4-pyridinecarboxaldehyde, NaCNBH3, AcOH, MeOH; (iii) 3-isoquinolineamine,
AlMe3, toluene.
Ta ble 1. Comparative Activities of the Anthranilamides 5 and 7, the Phthalazine PTK787 and the Quinazoline ZD6474 against the
VEGF Receptor Kinases and a Selection of Other Tyrosine Kinasesa-c
KINASEb
5
7
ZD6474
PTK787
SU5416
43 ( 11
VEGFR-1
(Flt-1)
VEGFR-2
(human KDR)
VEGFR-3
(Flt-4)
130 ( 81; n ) 5
130 ( 30; n ) 3
90 ( 10; n ) 7
(1600 ( 400)c
17 ( 3; n ) 5
(40 ( 10)c
110 ( 25; n ) 17
23 ( 6; n ) 9
9 ( 1; n ) 4
42 ( 3; n ) 37
220 ( 34
(1300 ( 800)d
50
18 ( 1; n ) 2
52 ( 1; n ) 3
357 ( 130; n ) 3
105 ( 40; n ) 3
99 ( 21; n ) 3
233 ( 52; n ) 6
195 ( 30; n ) 6
490 ( 52; n ) 14
620 ( 56; n ) 21
1240 ( 180; n ) 7
PDGFR-â
640 ( 100; n ) 4
236 ( 60; n ) 4
380 ( 140; n ) 3
477 ( 84; n ) 4
(1100 ( 300)c
343 ( 76; n ) 4
2220 ( 1500
(37 900 ( 8900)d
660 ( 165
(35 ( 5)d
c-Kit
CSF-1R
(c-Fms)
1600 ( 170; n ) 3
84 ( 4
EGFR
(HER-1, ErbB)
FGFR-1
10 400 ( 690; n ) 4
>10 000; n ) 4
>10 000; n ) 3
16 ( 3; n ) 5
(500 ( 100)c
2310 ( 600; n ) 4
(3600 ( 900)c
>10 000
567 ( 27; n ) 6
(2500 ( 1200)c
3370 ( 320; n ) 4
>10 000; n ) 3
186 ( 19; n ) 7
86 ( 21; n ) 5
>10 000; n ) 4
>10 000; n ) 4
>10 000; n ) 4
5100 ( 1660; n ) 4
>10 000; n ) 4
(4200 ( 800)d
>10 000; n ) 3
>10 000; n ) 3
CDK-1
Tie-2
(Tek)
c-Met
IGF-1R
c-Src
>10 000; n ) 3
>10 000; n ) 3
>10 000; n ) 3
6800 ( 2,180; n ) 3
>10 000; n ) 3
>10 000; n ) 4
>10 000; n ) 3
>10 000; n ) 3
>10 000; n ) 3
2820; n ) 1
>10 000; n ) 3
6400 ( 200; n ) 3
>10 000; n ) 2
5330 ( 70; n ) 3
>10 000; n ) 4
>10 000; n ) 3
>10 000; n ) 4
>10 000; n ) 4
>10 000; n ) 3
>10 000; n ) 2
>10 000; n ) 2
>10 000; n ) 1
c-Abl
a
Data represent the mean ( SEM (n determinations) drug concentrations required to inhibit enzyme activity by 50% (IC50 value; nM)
at the following ATP concentrations 1.0 µM (PDGFR-â, c-Kit, CSF-1R, c-Met), 2.0 µM (HER-1), 5.0 µM (c-Abl), 7.5 µM (CDK-1), 8.0 µM
(VEGFR-1, VEGFR-2, FGFR-1, Tie-2), 13.0 µM (VEGFR-3), 20.0 µM (c-Src), 30.0 µM (IGF-1R), and, unless otherwise indicated are data
b
from Novartis Pharma AG. Alternative nomenclature for these kinases is given in parentheses script. c Published data from refs 8 and
d
9. Published data from ref 13.
respectively),16 together with their high permeabilities
(passive diffusion) across Caco-2 cell monolayers (in-
trinsic permeability, Pm ) 45.2 and 21.7 × 10-5 cm/min
for 5 and 7, respectively), are predictive of intestinal
absorption following oral administration.
P h a r m a cology. The in vitro potency and selectivity
profiles of the compounds as kinase inhibitors were
evaluated using recombinant GST-fused kinase domains
expressed in baculovirus. Assays were performed under
conditions as previously described,10 with ATP concen-
trations similar to the Km of the respective enzyme
toward ATP (see legend for Table 1). IC50 values were
calculated by linear regression analysis of the percent-
age inhibition of each compound and are shown in Table
1, expressed as mean ( SEM with n representing the
number of determinations.

Anthranilic Acid Amides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5689
Ta ble 2. Comparative Effects of the Anthranilamides 5 and 7,
the Phthalazine PTK787, the Indolinone SU5416, and the
Quinazoline ZD6474 on VEGF-induced Phosphorylation of
VEGFR-2 in CHO Cellsa
at a standard oral dose of 50 mg/kg, with the exception
of SU5416 which was administered at 50 mg/kg intra-
peritoneally. For the anthranilamides 5 and 7, the dose
dependency of the response was evaluated, with ED50
values being calculated from the dose-response curves
(Figure 1).
The effects of the compounds on tumor growth and
metastasis was evaluated in a mouse orthotopic model
of melanoma.17 Intradermal injection of B16/BL6 mela-
noma cells into the ears of immunocompetent C57BL/6
mice leads to the formation of a primary tumor and
metastases in the regional lymph nodes. The size of the
primary tumor can be quantified microscopically, with
a computerized imaging system. The effect of drug
substances on the formation of metastases is quantified
by the weight of surgically removed cervical lymph
nodes.
inhibition of VEGFR-2
compound
autophosphorylation (IC50 ; nM)
5
7
1.24 ( 0.19 (n ) 9)
1.16 ( 0.12 (n ) 7)
2673 ( 174 (n ) 6)
16.0 ( 1.1 (n ) 127)
884 ( 46 (n ) 12)
1 (ZD6474)
2 (PTK787)
3 (SU5416)
a
Results are given as IC50 values, expressed as mean ( SEM,
n ) number of experiments.
Ta ble 3. Pharmacokinetic Parameters of Compounds 5 and 7,
Following Single Oral Administration to Female MAG Mice (50
mg/kg)a
plasma
C0.5h
plasma
C1.0
plasma
C1.5h
plasma
C2h
compound
(µmol/L)
(µmol/L)
(µmol/L)
(µmol/L)
Resu lts a n d Discu ssion
5
7
9.5 ( 2.1
3.4 ( 0.1
4.7 ( 2.1
1.9 ( 0.4
1.5 ( 0.8
1.9 ( 0.4
1.1 ( 0.3b
1.6 ( 0.2c
The anthranilamides are highly potent and selective
inhibitors of the recombinant VEGFR-2 and VEGFR-3
kinases, with compound 5 possessing IC50 values of 23
and 18 nM and compound 7 of 9 and 52 nM, respec-
tively. At 3- to 5-fold higher concentrations, they also
inhibit VEGFR-1 and, although they possess some
activity against other members of the PDGFR kinase
family at submicromolar concentrations (cKit, CSF-1R,
and PDGFR-â), they do not significantly inhibit any of
the other kinases tested at concentrations <10 µM
(Table 1). The aniline 4 and the ester 6 were devoid of
activity (data not shown). In this respect, compounds 5
and 7 possess a comparable selectivity profile in com-
parison to clinical development compounds such as
PTK787/ZK222584, SU5416, and ZD6474 (Table 1).
Both anthranilamides are capable of penetrating cells
and inhibit the VEGF-stimulated tyrosine autophos-
phorylation of human VEGFR-2 in CHO cells (Table 2).
The potency of compounds 5 and 7 together with that
of PTK787 (2) in this assay parallels that found for the
inhibition of recombinant VEGFR-2 transphosphoryla-
tion. However, a large discrepancy exists for ZD6474
and SU5416 between the two assays. The reason for this
lack of concordance is not clear, particularly since both
compounds have been reported to inhibit the VEGF-
induced proliferation of human umbilical vein endo-
thelial cells.8,9,13
Following oral administration to mice, both 5 and 7
were absorbed (Table 3), with plasma levels after 2 h
being 880-fold and 1100-fold, respectively, greater than
the corresponding IC50 values for the inhibition of
cellular VEGFR-2 autophosphorylation. In addition,
once-daily dosing of 5 (50 mg/kg po) for 20 days had no
effect on the C2h value, indicating that the pharmaco-
kinetic parameters, at least for this compound, remain
stable following chronic dosing. Consequently, oral
administration of 50 mg/kg of either compound would
be expected to inhibit in vivo responses mediated by
VEGFR-2 kinase activity, in a fashion similar to that
demonstrated for other VEGFR-2 kinase inhibitors.8,11,13
Consistent with their in vitro profiles for the inhibition
of tyrosine kinases, coupled with their ability to pen-
etrate cells to inhibit their target enzymes and their oral
bioavailability in mice, in the implant model, at the
standard oral dose of 50 mg/kg, compounds 5 and 7
a
Both 5 and 7 were formulated in 5% DMSO/0.5% TWEEN 80.
At allotted times mice were sacrificed, blood removed, and the
concentration of drug in the plasma was determined by reverse-
phase HPLC. Data are expressed as mean ( SEM, n ) 4.
b
Following once-daily dosing for 20 days, the C2h value for 5 was
1.75 ( 0.23. c At an additional time point after 3 h, the plasma
C3h value for 7 was 1.3 ( 0.4.
Since a VEGFR-kinase inhibitor must enter cells to
bind to the kinase domain of the receptor, the effects
of the compounds were tested in a cellular assay,
using Chinese hamster ovary (CHO) cells transfected
with human VEGFR-2.11 Autophosphorylation of the
VEGFR-2 receptor kinase was induced with VEGF.
After lysis of the cells, the degree of phosphorylation
was measured by capture of the VEGFR-2 with a
specific monoclonal antibody, followed by quantification
of receptor phosphorylation with an anti-phosphoty-
rosine antibody using a chemiluminescent assay. The
influence of the compounds on autophosphorylation was
calculated as percentage inhibition and dose-response
curves were used to calculate IC50 values, which are
presented in Table 2 and expressed as mean ( SEM
with n representing the number of experiments.
Prior to evaluating their effects in vivo, the com-
pounds were first tested to ascertain whether they were
absorbed following oral administration. Plasma concen-
trations of drug substance were determined by HPLC/
UV in normal mice after a single oral dose of 50 mg/kg,
dissolved in a mixture of 5% DMSO and 0.5% Tween
80 in water, and administered by gavage. The pharma-
cokinetic parameters derived from these data are sum-
marized in Table 3.
The capacity of the compounds to modulate angio-
genesis in animals was assessed in a growth factor
implant model.11 In immunocompetent mice, both VEGF
and basic fibroblast growth factor (bFGF) induce the
growth of vascularized tissue around subcutaneous
implants which contain either of these growth factors.
The response is concentration-dependent and can be
quantified by measuring the haemoglobin (blood) con-
tent of the tissue. The response can be specifically
blocked by selective inhibitors of these growth factors
and their signaling pathways. The antiangiogenic activ-
ity of the drug substances in this model were compared

5690 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26
Manley et al.
F igu r e 1. Anti-angiogenic activity of compounds 5 and 7 in a growth factor-stimulated implant model in mice. Results of multiple
independent experiments (n ) 6 animals per group) are expressed as percent inhibition of the growth factor-induced increase in
blood content of the tissue growing around the implanted chamber. ED50 values were estimated from the dose-response curve.
The number of animals per dose is indicated in parentheses alongside each data point.
Ta ble 4. Effects of Compounds on VEGF- and bFGF-induced
Response in a Growth Factor Implant Model and on Primary
Tumor Growth and Formation of Lymph Node Metastasis in a
eral human melanoma cell lines also express VEGF.21,22
B16 Melanoma Model
produce VEGF and bFGF (protein by ELISA and mRNA
by real-time PCR) and it has also been shown that sev-
In this model, both compounds 5 and 7 inhibited
primary tumor growth as well as the formation of
metastases and there were no deaths or overt signs of
mean percent inhibition of response at 50 mg/kg po
B16 melanoma
xenograft model
primary lymph node
tumor growth metastasis
growth factor-induced
angiogenesis
toxicity in any drug treatment group. Oral administra-
tion of 5, from day 7-21 following tumor cell injection,
dose dependently inhibited primary tumor growth (Fig-
ure 2, upper left) with an ED50 value of 50 mg/kg and
the formation of lymph node metastases (Figure 2, lower
left) with an ED50 value less than 25 mg/kg. Effects in
animals receiving once-a-day dosing were similar to
those in which the dose was split and administered
twice-a-day (data not shown). Compound 7 showed
similar efficacy to 5 in these models, although there was
an indication that twice-daily dosing is more efficacious
both against orthotopic tumor growth and the formation
of lymph node metastases (data not shown). The obser-
vation that the development of lymph node metastases
was inhibited to a greater extent than was the growth
of the primary tumor may reflect an additional direct
antimetastatic effect of the compounds. The phthala-
zine, PTK787, and the quinazoline, ZD6474, also showed
efficacy in this melanoma model, with ZD6474 in
comparison with the other compounds, appearing to be
more effective against primary tumor growth (Table 4).
This observation might be a result of ZD6474 having
an advantageous pharmacodynamic profile or possess-
ing an additional mechanism of action affecting primary
tumor growth.
compound
VEGF
bFGF
5
7
84
100
68
77
61
66
87b
47
44
39
85
73
55
n.d.
61
PTK787
SU5416
ZD6474
34
65b
71
n.d.c
60
a
Results are expressed as the mean percent inhibition of g12
b
animals. Data obtained with an intraperitoneal dose of 100 mg/
kg. c n.d. ) not determined.
inhibited the VEGF-induced increase in tissue blood
content by 84% and 100%, respectively (Table 4). In
comparison, PTK787/ZK222584, SU5416 (tested by the
intraperitoneal route),18 and ZD6474 also showed activ-
ity, although with significantly less efficacy. To further
characterize the efficacy of the anthranilamides against
VEGF mediated angiogenesis, the dose-response rela-
tionships of the compounds were investigated: Both
compounds 5 and 7 dose-dependently inhibited angio-
genesis induced by VEGF with an ED50 value of 7 mg/
kg (Figure 1). The efficacy of compound 7 was slightly
greater than that of 5. Surprisingly, despite being devoid
of significant activity against the FGFR-kinase (Table
1), the compounds in this study showed efficacy at
inhibiting bFGF-induced angiogenesis, with 5 for ex-
ample inhibiting bFGF-induced angiogenesis to the
extent of 77% at 50 mg/kg po (ED50 5 mg/kg). We have
reported similar findings with other selective VEGFR-2
kinase inhibitors and taken together these suggest that
in vivo responses to bFGF in this model might be
mediated by the endogenous VEGF/VEGFR system.19,20
The B16/BL6 mouse melanoma is an established
cancer model, in which both a primary tumor and
spontaneous metastases are formed in the cervical
lymph nodes and in the lungs.17 It has been demon-
strated that both the primary tumor and the metastases
Con clu sion
The anthranilamides 5 and 7 are highly potent and
selective inhibitors of recombinant VEGFR-kinase, which
readily penetrate cells to inhibit VEGFR-2 autophos-
phorylation. As a consequence of their physicochemical
properties and their cell permeability, the anthranil-
amides are orally absorbed following administration to
mice. In keeping with these properties, the compounds
potently inhibit angiogenesis with high efficacy in an
implant model in immunocompetent mice. Furthermore,
in an orthotopic melanoma model, the antiangiogenic

Anthranilic Acid Amides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5691
F igu r e 2. Dose-dependent antitumor and antimetastatic effects of compounds 5 (left) and 7 (right) in a B16/BL6 melanoma
orthotopic model in C56BL/6 mice after oral administration. Dose-response effects on primary tumor growth are expressed in
terms of relative mean tumor area quantified on day 14 (A14) and day 21 (A21) after intradermal injection into the ears, compared
to that on day 7 (A7), and are shown in the upper panels; percent inhibition on day 21 (14 days treatment) is given for each curve.
Absolute tumor mean areas at day 7 (A7) were in the range 0.97 ( 0.09 to 1.42 ( 0.13 mm2 and not statistically different. The
influence of the compounds on cervical lymph node metastases, quantified by the weight of the cervical lymph nodes after surgical
*
removal, are shown in the panels below. Values are mean ( SEM., P < 0.5, ANOVA, Dunns, significance compared to vehicle-
treated group.
EtOAc (300 mL) and washed with saturated aqueous am-
monium chloride. The solution was dried (Na2SO4) and filtered
and the solvent was evaporated off under reduced pressure to
yield the crude product which was recrystallized from EtOAcs
hexane to give 4 as colorless needles (6.3 g, 37%): mp 132-
activity of the anthranilamides 5 and 7 translates into
potent inhibition of both primary tumor growth and the
formation of lymph node metastases. Upon chronic oral
dosing in rodents, the compounds are well tolerated with
no overt signs of toxicity. Consequently, these anthra-
nilamides represent a new structural class of VEGFR
kinase inhibitors, possessing potent antiangiogenic and
antitumor properties, with comparable selectivity for
VEGFR-kinases compared to other small molecular
weight compounds currently undergoing clinical evalu-
ation.
o
133 C; NMR (DMSO-d6) δ 6.38 (sbr, 2H), 6.60 (ddd, J ) 8.0,
8.0, 0.7, 1H), 6.77 (dd, J ) 8.2, 0.7 Hz, 1H), 7.22 (ddd, J ) 8.0,
8,0, 1.3 Hz, 1H), 7.43 (dm, J ) 7.7 Hz, 1H), 7.57 (ddd, J ) 8.4,
7.9 Hz, 1H), 7.66 (dd, J ) 8.0, 1.3 Hz, 1H), 7.96 (dm, J ) 8.4
Hz, 1H), 8.21 (s, 1H) and 10.28 (s, 1H). Anal. (C14H11F3N2O)
C, H, N.
2-[(4-P yr id yl)m e t h yl]a m in o-N-[3-(t r iflu or om e t h yl)-
p h en yl]ben za m id e (5). A stirred solution of 4 (28.0 g, 100
mmol) and acetic acid (5 mL) in MeOH (300 mL) was treated
with 4-pyridinecarboxaldehyde (11 mL, 120 mmol) and stirred
at 22 °C for 7 h. Sodium cyanoborohydride (21 g of 95%, 320
mmol) was then added in portions over 1 h and the mixture
was stirred for a further 17 h. The solvent was evaporated off
under reduced pressure and the residue was treated with CH2-
Cl2 (500 mL) and washed with saturated aqueous NaHCO3
(500 mL), followed by saturated aqueous NaCl (100 mL). The
solution was dried (Na2SO4) and filtered and the solvent was
evaporated off under reduced pressure to yield the crude
product which was recrystallized from EtOAcshexane to give
5 as a colorless crystalline solid (23 g, 62%): mp 158-160 °C;
NMR (DMSO-d6) δ 4.51 (d, J ) 6.2 Hz, 2H), 6.55 (d, J ) 8.4
Hz, 1H), 6.67 (ddd, J ) 7.7, 7.3, and 0.6 Hz, 1H), 7.26 (ddd, J
) 8.3, 7.3, and 1.2 Hz, 1H), 7.34 (dm, J ) 6.0 Hz, 2H), 7.45
(dbr, J ) 7.9 Hz, 1H), 7.59 (t, J ) 8.0 Hz, 1H), 7.74 (dd, J )
7.8 and 1.4 Hz, 1H), 7.96 (t, J ) 6.2 Hz, 1H), 7.98 (dbr, J )
Exp er im en ta l Section
Flash chromatography was performed using silica gel (E.
Merck, Grade 60, particle size 0.040-0.063 mm, 230-400
mesh ASTM) with the eluent indicated. Melting points were
determined on a Leitz Kofler hot-stage apparatus and are
uncorrected. Proton NMR spectra were recorded at 300 K with
a Brucker DRX500 instrument (500 MHz) using deuterated
DMSO as internal standard. Chemical analyses, indicated by
the symbols of the elements, were performed by Solvias AG
(Basel) and results obtained were within (0.4% of the theo-
retical values.
2-Am in o N-[3-(tr iflu or om eth yl)p h en yl]ben za m id e (4).
A mixture of 3-(trifluoromethyl)aniline (9.7 g, 60 mmol) and
isatoic anhydride (9.75 g, 60 mmol) in DMF (80 mL) was
stirred at 100 °C for 18 h. The solvent was evaporated off under
reduced pressure to give a residue which was dissolved in

5692 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26
Manley et al.
8.0 Hz, 1H), 8.28 (sbr, 1H), 8.49 (dm, J ) 6.0 Hz, 2H) and
recorded via a low-light color video camera, and quantified
with a computerized imaging system (KS-400 version 3.0,
Zeiss, Germany). After two weeks of daily treatment, the
animals were sacrificed and the cervical lymph nodes collected
and weighed. Effects on primary tumor growth are plotted as
relative tumor mean area (mm2) quantified on day 14 (A14)
and day 21 (A21) compared to the tumor mean area (A7) at the
start of drug treatment (day 7 after tumor cell injection).
Effects on cervical lymph node metastases were quantified by
the relative wet weight (mg) of the collected tissue of animals
treated with drug compared to that of vehicle-treated animals.
10.47 (s, 1H). Anal. (C20H16F3N3O) C, H, N.
2-[(4-P yr id in ylm eth yl)a m in o]ben zoic Acid , Meth yl Es-
ter (6). A mixture of methyl anthranilate (1.51 g, 10 mmol)
and 4-pyridinecarboxaldehyde (1.71 g, 16 mmol) in MeOH (60
mL) was treated with AcOH (0.6 mL) and stirred at 20 °C for
18 h. Sodium cyanoborohydride (1.2 g of 95%, 17 mmol) was
then added in portions over 2 h and the mixture was stirred
for an additional 2 h. The solvent was evaporated off under
reduced pressure and the residue was dissolved in EtOAc. The
solution was washed with saturated aqueous NaHCO3, fol-
lowed by saturated aqueous NaCl, dried (Na2SO4), filtered and
the solvent was evaporated off under reduced pressure. The
crude product was purified by column chromatography (silica
gel, 50% EtOAc in hexane) and recrystallized from 2-propanols
hexane to give 6 as a colorless crystalline solid (1.8 g, 74%):
mp 85-86 °C; NMR (DMSO-d6) δ 3.83 (s, 3H), 4.56 (d, J )
6.1, 2H), 6.58 (ddd, J ) 8.2, 6.9, 1.3 Hz, 1H), 6.60 (dd, J ) 8.2,
6.9, 1.3 Hz, 1H), 7.29 (dd, J ) 6.9, 1.3 Hz, 1H), 7.32 (dm, J )
5.3 Hz, 2H), 7.82 (dd, J ) 8.2, 1.3 Hz, 1H), 8.20 (tbr, J ) 6.1
Hz, 1H) and 8.50 (dm, J ) 5.3 Hz, 2H). Anal. (C14H14N2O2) C,
H, N.
*
Values are mean ( SEM. P < 0.5, ANOVA, Dunns, signifi-
cance compared to vehicle-treated group.
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N-3-Isoq u in olin yl-2-[(4-p yr id in ylm et h yl)a m in o]b en -
za m id e (7). A mixture of 6 (242 mg, 1.0 mmol) and 3-iso-
quinolinamine (202 mg, 1.4 mmol) in toluene (3.5 mL) was
treated with a solution of AlMe3 in toluene (0.75 mL of 2 M,
1.5 mmol) and stirred for 1 h at 20 °C, followed by 1 h at 110
°C. The cooled mixture was treated with saturated aqueous
NaHCO3 and extracted with EtOAc. The combined extracts
were dried (Na2SO4), filtered, and evaporated to yield the crude
product which was recrystallized from EtOAc to give 7 as a
colorless crystalline solid (237 mg, 67%): mp 134-135 °C;
NMR (DMSO-d6) δ 4.55 (d, J ) 6.4 Hz, 2H), 6.60 (d, J ) 7.0
Hz, 1H), 6.66 (dd, J ) 7.0, 7.0 Hz, 1H), 7.28 (ddd, J ) 7.0, 7.0,
1.1 Hz, 1H), 7.39 (dm, J ) 5.3 Hz, 2H), 7.59 (dd, J ) 8.1, 7.0
Hz, 1H), 7.76 (dd, J ) 8.1, 7.0 Hz, 1H), 7.90 (dd, J ) 7.0, 1.1
Hz, 1H), 7.97 (d, J ) 8.1 Hz, 1H), 8.12 (d, J ) 8.1 Hz, 1H),
8.18 (bdr, J ) 6.5 Hz, 1H), 8.53 (dm, J ) 5.3 Hz, 2H), 8.60 (s,
1H), 9.24 (s, 1H) and 10.70 (s, 1H). Anal. (C22H18N4O) C, H,
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