Novel 2,3-dihydro-1,4-benzoxazines as potent and orally bioavailable inhibitors of tumor-driven angiogenesis

Article abstract of DOI:10.1021/jm701129j

Angiogenesis is vital for solid tumor growth, and its prevention is a proven strategy for the treatment of disease states such as cancer. The vascular endothelial growth factor (VEGF) pathway provides several opportunities by which small molecules can act as inhibitors of endothelial proliferation and migration. Critical to these processes is signaling through VEGFR-2 or the kinase insert domain receptor (KDR) upon stimulation by its ligand VEGF. Herein, we report the discovery of 2,3-dihydro-1,4-benzoxazines as inhibitors of intrinsic KDR activity (IC₅₀ < 0.1 μM) and human umbilical vein endothelial cell (HUVEC) proliferation with IC₅₀ < 0.1 μM. More specifically, compound 16 was identified as a potent (KDR: < 1 nM and HUVEC: 4 nM) and selective inhibitor that exhibited efficacy in angiogenic in vivo models. In addition, this series of molecules is typically well-absorbed orally, further demonstrating the 2,3-dihydro-1,4-benzoxazine moiety as a promising platform for generating kinase-based antiangiogenic therapeutic agents.

Full text of DOI:10.1021/jm701129j

J. Med. Chem. 2008, 51, 1695–1705
1695
Novel 2,3-Dihydro-1,4-Benzoxazines as Potent and Orally Bioavailable Inhibitors of
Tumor-Driven Angiogenesis|
Daniel S. La,*,† Julie Belzile,† James V. Bready,‡ Angela Coxon,‡ Thomas DeMelfi,‡ Nicholas Doerr,‡ Juan Estrada,‡
Julie C. Flynn, Shaun R. Flynn,# Russell F. Graceffa,† Shawn P. Harriman, Jay F. Larrow,† Alexander M. Long,§
Matthew W. Martin,† Michael J. Morrison,# Vinod F. Patel,† Philip M. Roveto,† Ling Wang,‡ Matthew M. Weiss,†
Douglas A. Whittington,§ Yohannes Teffera, Zhiyang Zhao, Anthony J. Polverino,‡ and Jean-Christophe Harmange†
Amgen Inc. Chemistry Research and DiscoVery, Cambridge, Massachusetts 02139, and Thousand Oaks, California 91320
ReceiVed September 11, 2007
Angiogenesis is vital for solid tumor growth, and its prevention is a proven strategy for the treatment of
disease states such as cancer. The vascular endothelial growth factor (VEGF) pathway provides several
opportunities by which small molecules can act as inhibitors of endothelial proliferation and migration.
Critical to these processes is signaling through VEGFR-2 or the kinase insert domain receptor (KDR) upon
stimulation by its ligand VEGF. Herein, we report the discovery of 2,3-dihydro-1,4-benzoxazines as inhibitors
of intrinsic KDR activity (IC50 < 0.1 µM) and human umbilical vein endothelial cell (HUVEC) proliferation
with IC50 < 0.1 µM. More specifically, compound 16 was identified as a potent (KDR: < 1 nM and HUVEC:
4 nM) and selective inhibitor that exhibited efficacy in angiogenic in vivo models. In addition, this series
of molecules is typically well-absorbed orally, further demonstrating the 2,3-dihydro-1,4-benzoxazine moiety
as a promising platform for generating kinase-based antiangiogenic therapeutic agents.
Introduction
zyloxy)-5-[3-(4-pyrrolidin-1-ylbutyl)ureido]isothiazole-4-car-
boxylic acid amide (CP547,632)13 in late stage clinical trials
and N-[2-(diethylamino)ethyl]-5-[(Z)-(5-fluoro-1,2-dihydro-2-
oxo-3H-indol-3-ylidine)methyl]-2,4-dimethyl-1H-pyrrole-3-car-
boxamide (SU11248, sunitinib)14–16 and 4-[4-[3-[4-chloro-3-
(trifluoromethyl)phenyl]ureido]phenoxy]-N-methylpyridine-2-
carboxamide (BAY 43-9006, sorafenib),17 which were approved
for the treatment of renal and/or gastrointestinal cancer.
Similarly, our program has been focused on the development
of small-molecule inhibitors of RTKs, namely, KDR. Our initial
efforts resulted in the development of N-(2,3-dihydro-3,3-
dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridi-
necarboxamide (AMG 706, motesanib), a highly selective, oral
agent that is being evaluated in clinical trials for its ability to
inhibit angiogenesis by targeting VEGF receptors 1, 2, and 3,
platelet-derived growth factor receptor (PDGFR), and stem cell
factor receptor (Kit), which may also confer direct antitumor
activity.18,19
Excessive or abnormal angiogenesis has been linked to several
diseases including cancer, psoriasis, arthritis, endometriosis, and
diabetic retinopathy.1 In a cancer setting, a solid tumor that
develops to the size of 2–3 mm in diameter exceeds the limits
of passive diffusion for the purposes of acquiring nutrients and
disposing of metabolic wastes.2 Accordingly, tumor progression
is reliant on the sprouting and maturation of new blood vessels.
Critical to this process is the production of VEGFa in response
to hypoxia or several upstream activators such as oncogenes,
cytokines, or cell growth factors.3,4 VEGF binds to VEGFR-2
(KDR), a receptor tyrosine kinase (RTK), which upon dimer-
ization/activation by VEGF undergoes autophosphorylation and
initiates downstream signaling, ultimately leading to angiogen-
esis, tumor survival, and tumor migration.
The inhibition of the VEGF pathway has received much
attention recently,5 as evidenced by the recent activity in late
stage clinical trials.6 Most notable with respect to validation of
the antiangiogenesis concept is the VEGF monoclonal antibody
bevacizumab,7,8 which has received U.S. Food and Drug
Administration approval. Among the small-molecule inhibitors
are 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine (PTK787/
ZK222584, vatalanib),9,10 N-(4-bromo-2-fluorophenyl)-6-meth-
oxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine
(ZD6474, vandetanib),11,12 and 3-(4-bromo-2,6-difluoro-ben-
We have recently identified a series of naphthylamide
analogues,20 shown in Figure 1A, as effective inhibitors of KDR
at the enzyme and cellular levels. From X-ray cocrystal analysis,
several interactions became apparent and guided our design of
subsequent inhibitors. As depicted in Figure 2, compound 1
(VEGFR-2 kinase IC50 ) 0.03 nM and VEGF-HUVEC IC50
)
8 nM) binds to the ATP active site of the KDR protein in the
inactive, “DFG-out” conformation.21 The amide functional group
participates in hydrogen bond contacts with Glu885 and
Asp1046, allowing the aryl chloride to reside in the extended
hydrophobic pocket. The naphthalene moiety engages the ATP-
binding cleft through van der Waals contacts, and the dimetho-
xyquinoline interacts with the “hinge region” backbone NH of
Cys 919 through a critical hydrogen bond. The high degree of
hydrophobic interactions that ligand 1 makes with the protein
may provide a rationale for the exquisite potency observed.
Consequently, the average calculated log P (cLogP)22 was
greater than 5 for arylamides without solubilizing substituents
(Figure 1) presenting solubility issues. In fact, for compound
|
The PDB codes for cocrystals of compounds 1 and 16 within in the
KDR ATP-binding pocket are 3B8Q and 2RL5, respectively.
* To whom correspondence should be addressed. Phone: 617-444-5187.
Fax: 617-621-3908. E-mail: daniel.la@amgen.com.
†
Department of Chemistry Research and Discovery.
Department of Oncology.
‡
Department of Pharmacokinetics and Drug Metabolism.
Department of HTS and Molecular Pharmacology.
#
§
Department of Molecular Structure.
a Abbreviations: VEGF, vascular endothelial growth factor; KDR, kinase
insert domain receptor; HUVEC, human umbilical vein endothelial cell;
RTK, receptor tyrosine kinase; PDGFR, platelet-derived growth factor
receptor; HTRF, homogenous time-resolved fluorescence.
10.1021/jm701129j CCC: $40.75
2008 American Chemical Society
Published on Web 02/27/2008

1696 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
La et al.
Figure 1. Structural and cLogP analysis of the naphthylamide and 2,3-dihydro-1,4-benzoxazine scaffolds.
Figure 2. Cocrystal of compound 1 within in the ATP-binding pocket of KDR (PDB code 3B8Q). Key hydrogen bond contacts are shown (dotted
lines). In the right diagram, portions of the DFG sequence are omitted for clarity.
1, the solubility was 1.1, <1, and 1.4 µg/mL as measured in
0.01 N HCl, PBS, and SIF, respectively. In addition, the
naphthalene core presented potential bioactivation liabilities that
could lead to an undesired toxicological profile. These charac-
teristics prompted the design of 2,3-dihydro-1,4-benzoxazines
(Figure 1B) that retain the shape and contact elements with the
protein while incorporating polar, heteroatom functional groups
to improve solubility. Within this chemical series, the cLogP
values were approximately 4 calculated log units lower (see
Figure 1) than their naphthylamide counterparts. This paper
describes the effort leading to the discovery of benzoxazine
derivatives as potent, orally available inhibitors of KDR.
the critical linker or hinge region (on the KDR enzyme) binding
moiety. The nucleophilic addition conditions to generate
intermediate 6 employed cesium carbonate at elevated temper-
ature to exclusively provide the O-arylated product (Scheme
2). This protocol was applicable to the formation of various
biaryl ethers, allowing the investigation and optimization of
different linker/hinge region binding groups. The resulting
benzoxazine 6 was acylated with isocyanates or p-nitrophenyl
carbamates to generate the desired urea as KDR inhibitors.
Results and Discussion
Structure–Activity Relationships (SAR). SAR exploration
of the 1,4-benzoxazines series was executed with our KDR
enzyme inhibition and HUVEC proliferation assays. KDR
enzymatic data were obtained utilizing a homogeneous time-
resolved fluorescence (HTRF) assay employing a biotinylated
gastrin substrate. The enzyme and HUVEC proliferation assays
were performed as previously described.18 Simultaneously,
compounds were screened for activity against the cMet, Tie-2,
and Lck enzymes as well as cellular FGF driven HUVEC
proliferation to provide selectivity information (Table 1). We
desired an inhibitor profile free of cMet and Tie-2 activity
because their link to tumor angiogenesis would confound our
mechanism-based analysis.23 A selective KDR inhibitor would
allow for more accurate target coverage and therefore enable
us to reliably account for observed in vivo pharmacology. The
Lck kinase was employed as a representative of the Src kinase
family, which is involved in critical cellular processes such as
cellular growth, differentiation, and signaling.24
Chemistry
Armed with structural information from the naphthylamide
series, the principal design features of the 1,4-benzoxazines were
rooted in the scaffold’s potential to create the appropriate
associations with the target enzyme (KDR) while imparting
favorable physiochemical properties that would increase success
with respect to cell permeability and oral availability. In addition,
urea formation as the penultimate bond disconnection enabled
rapid analoging, which expedited the generation of in vitro
structure–activity relationships (SAR) at the intrinsic, cellular,
pharmacokinetic, and toxicology levels.
The synthesis of the benzoxazine analogues began with the
amidation/cyclization of aminoresorcinol hydrochloride 2 with
chloroacetyl chloride to provide benzoxazinone 3 (Scheme 1).
Initial attempts to reduce 3 with LiAH4 and Red-Al failed
because of decomposition or complications with product isola-
tion. Borane reduction, however, cleanly provided benzoxazine
4. This intermediate was then coupled with chloride 5 containing
Initial scanning of substituents placed at the ortho, meta, and
para positions on the urea phenyl ring revealed that the meta

Potent, Orally AVailable KDR Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1697
Scheme 1. Synthesis of 2,3-Dihydro-1,4-benzoxazinesa
a Reagents and conditions: (a) 2-chloroacetyl chloride, Cs2CO3, MeCN, 20 °C; (b) BH3 ·THF, THF, 20 °C; (c) Cs2CO3, 4-chloro-6,7-dimethoxyquinoline
(5), DMF, 90 °C; (d) RNCO, DCM or R-NHCO2-p-nitrophenyl, NEt3, THF, 50 °C.
Scheme 2. Preparation of Biaryl Ethersa
a Reagents and conditions: Cs2CO3, chlorides 7, 8, or 9, DMF, 90 °C.
and para substituted phenyls were optimal for intrinsic and
cellular potency. However, para substitution maintained selec-
tivity over the cMet, Tie-2, and Lck as well as FGF-driven
HUVEC proliferation (see representative compounds 13 and 16
in Table 1). Substituent at the ortho position resulted in reduced
cell potency as demonstrated by 11 and 14. Compounds bearing
substitution at the meta position were potent in the KDR enzyme
and VEGF driven HUVEC proliferation assays but lacked
selectivity over Tie-2 and Lck (12 and 15).
In an attempt to lower molecular weight, we next turned our
attention toward examining alkylureas, starting with small
substituents and incrementally increasing in size (see representa-
tive compounds 18–25). The smaller compounds were designed
to potentially exploit the active “DFG-in” enzyme conformation.
It was believed and later confirmed by X-ray crystallography
that the arylureas, such as 16, bind in a “DFG-out” manner (see
below) similar to compound 1. Our preliminary study revealed
that an ethylurea was optimal for potency within this series (see
compound 20 in Table 1). The ureas 18, 19 and 21, on the other
hand, were less potent. As exploration expanded to include larger
benzylureas, compound 24 proved to be enzymatically and
cellularly active. Interestingly, its enantiomer, 25, was signifi-
cantly less potent. In general, the alkylureas showed lower
potency compared to the arylureas and were therefore not
investigated further.
With distinct SAR delineated for the urea moiety, the linker
binding portion of the 2,3-dihyro-1,4-benzoxazines was inves-
tigated. Three other substructures were studied as exemplified
by compounds 26-31 (Table 2). The structural design element
of these analogues attempted to exploit a possible two-point
hydrogen bond contact with Cys 919 as shown in Figure 3. It
was thought that the hydrogen bond acceptor of the inhibitor
would engage the amide NH of the cysteine residue. Accord-
ingly, the acidic NH or aryl CH of the compound could enhance
interaction through hydrogen bonding with the cysteine carbo-
nyl.25
The linker binding substituents were evaluated by constructing
the p-chloro- and p-methylarylureas of each structural motif.
Carboxamide analogues 26 and 27 were promising, given their
enzyme and cell potency in addition to the selectivity over the
cMet, Tie-2, Lck and the FGF pathway. The pyrrolopyrimidines

1698 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Table 1. Structure–Activity Relationships of the Urea Moietya
La et al.
IC50 (nM)
compd
R
KDR
HUVEC (VEGF)
HUVEC (FGF)
cMet
Tie-2
Lck
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Ph
1
1
1
0.2
19
1
0.5
3
58
40
1
40
53
26
1
0.6
45
0.6
3
73
0.4
4
340
910
190
280
230
220
1100
300
1100
370
650
410
350
370
290
230
300
1100
>4000
>4000
1400
1200
1600
500
386
1600
1
1000
2200
9
188
27
1
63
2200
4
20
2-Me-Ph
3-Me-Ph
4-Me-Ph
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
4-F-Ph
H
110
2
1500
>4000
>4000
>4000
>4000
>4000
1500
260
90
280
100
23
>4000
2400
1500
1000
500
150
18
Me
Et
780
iPr
52
1100
>1100
1100
300
Bn
237
360
3
4-Me-Bn
(S)-RMe-Bn
(R)-RMe-Bn
460
210
40
250
1100
>4000
78
a All kinases were tested at their apparent Km of ATP with respect to 1 µM peptide substrate. IC50 values are the mean of two or more separate determinations
in duplicate.
Table 2. Structure–Activity Relationships of the Linker Binding Moietya
a All kinases were tested at their apparent Km of ATP with respect to 1 µM peptide substrate. IC50 values are the mean of two or more separate determinations
in duplicate.

Potent, Orally AVailable KDR Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1699
The moderate clearance of the p-methylurea analogues 13 and
27 explains the lower exposure after oral dosing (AUC0-inf
)
1.3 and 0.65 µg h/mL, respectively) in comparison to the
p-chlorourea analogues 16 and 26 (AUC0-inf ) 7.9 and 4.5 µg h/
mL, respectively). However, compounds 16 and 17 exhibited
the most stability in HLM and possessed excellent oral
bioavailability in rats. Half-lives of compounds in Table 4
ranged from 2 to 6 h in rats with low volume of distribution,
which we hypothesized boded well for restricting distribution
to predominantly the blood compartment. The protein binding
of 16 was similar across species (human, dog, rat, and mouse)
and ranged from 99.2% to 99.6%. Because of superior microso-
mal stability, low clearance, and excellent oral bioavailability
(F ) 95%), compound 16 was advanced to selectivity assess-
ment against a broad panel of kinases and to determination of
pharmacodynamic efficacy.
Figure 3. Hydrogen bond interaction between inhibitor and linker/
hinge region.
Table 3. Solubility Comparison between Naphthylamides (A) and
Benzoxazines (B)
Selectivity. Compound 16 was evaluated for activity against
a broader panel of kinases in order to understand its full target
profile. This effort revealed that 16 was selective over a variety
of kinases depicted by the heat map illustrated in Figure 4 (green
most potent, red least potent). Within the protein tyrosine kinase
(PTK) group, 16 was highly selective (>30 fold) over repre-
sentatives from the Src, Abl, Tie/Tck, FGFR, Tec, and hepa-
tocyte growth factor families. Similarly, selectivity was observed
over kinases outside the PTK group (CMGC and AGC), namely,
kinases from the MAP, CDK, and RSK families. Not surpris-
ingly, compound 16 was less selective over kinases within the
PDGFR family presumably because of significant catalytic
domain homology to KDR.
R
0.01 N HCl PBS
SIF
9.4
22
36
28
1.4
33
1.3
41
3.0
130
5.6
14
cLogP cLogD6.5
A
4-Me-Ph
>200
>200
9.5
120
1.1
<1
<1
<1
<1
<1
<1
<1
3.3
1.4
46
6.8
3.0
7.3
3.5
7.3
3.5
6.8
3.0
4.6
1.2
5.4
2.1
6.7
2.9
7.2
3.4
7.2
3.4
6.7
2.9
4.5
1.1
5.3
2.0
B (13) 4-Me-Ph
3-Cl-Ph
B (15) 3-Cl-Ph
4-Cl-Ph
B (16) 4-Cl-Ph
4-F-Ph
B (17) 4-F-Ph
Me
B (19) Me
iPr
B (21) iPr
A
A
28
67
A
Pharmacodynamic Efficacy. Compound 16 was next ex-
amined for in vivo efficacy in a vascular permeability assay
and a rat corneal angiogenesis model. These assays were
performed as previously described. For the vascular permeability
assay27 HEK 293 cells overexpressing murine VEGF were
injected subcutaneously into the abdomen of nude mice.
Approximately 22 h later, a single dose of the inhibitor or
vehicle was orally administered, and after an additional 6 h,
the mice received an iv injection of 0.1 mL 1% Evans blue
dye. The mice were then sacrificed, a 1 cm2 piece of skin
overlaying the cells was harvested, and the extracted Evans blue
dye was measured. Figure 5A illustrates the dose dependent
inhibition of vascular permeability by compound 16. A statisti-
cally significant reduction in extracted Evans blue dye was
detected at doses as low as 3 mg/kg (Table 5). In addition, doses
of 10 and 30 mg/kg lowered vascular permeability to vehicle
control levels.
In vivo efficacy was further demonstrated with a corneal
angiogenesis model employing female Sprague Dawley rats.28
This model entails insertion of either vehicle or VEGF-soaked
disks into a pocket in the corneal stroma of an anesthetized rat.
The vehicle or compound 16 was orally administered for 7 days.
The corneas were then digitally photographed, and the number
of blood vessels intersecting a 2.0 mm perpendicular line at
the midpoint of the disk placement distance was measured.
Figure 5B illustrates the inhibition of angiogenesis in a linear
dose dependent fashion. The ED50 was calculated to be 0.16
mg/kg for compound 16. The associated pharmacokinetic
analysis determined the exposure to be 2.8 µM h and the Cmax
to be 0.18 µM.
>200
>200
>200
>200
>200
A
A
<1
3.0
28 and 29 and N-methylpicolinamides 30 and 31, however, were
less potent and not further pursued.
The solubility of the benzoxazine and their naphthylamide
predecessors was examined in order to test the hypothesis of
introducing heteroatoms to the molecular scaffold to enhance
aqueous solubility (see Figure 1). It was believed that proper
placement of polar substituents would serve to lower log P and
that introduction of the sp3 hybridized ethyl bridge would reduce
the planarity of the central rings and limit the potential for
π-stacking. By use of an equilibrium solubility method previ-
ously described,26 solubility was determined in three media, 0.01
N HCl, PBS (pH 7), and SIF. In all cases where head-to-head
comparisons could be made, the benzoxazines proved to be more
soluble. As shown in Table 3, representative benzoxazines
containing aromatic and aliphatic ureas possessed greater
solubility than their naphthylamide counterparts.
Pharmacokinetic Profiles. On the basis of their desired in
vitro cellular potency (<10 nM) and selectivity, several
compounds were selected for metabolic stability and pharma-
cokinetic analysis. This set of compounds displayed good to
moderate in vitro stability in human and rat liver microsomal
preparations, with the exception of the benzylurea 24 (see Table
4). There was a high correlation between in vitro RLM turnover
and in vivo rat iv Cl. Thus, the poor in vitro stability upon
incubation with both HLM and RLM was consistent with higher
clearance observed after intravenous administration (CL ) 0.98
(L/h)/kg). Similarly, compounds 10, 13, 17, and 27 exhibited
higher clearance than the p-chlorourea analogues 16 and 26.
X-ray Structural Analysis. The cocrystal structure of 16 and
the KDR enzyme (see Figure 6 and Supporting Information
Table S1) highlights the major ligand-target interactions, which
presumably account for the observed potencies of the 1,4-
benzoxazine scaffold. The quinoline moiety engages the linker

1700 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
La et al.
Table 4. Mean Pharmacokinetic Parameters in Male Sprague Dawley Ratsa
a Rats were dosed 0.5 mg/kg iv and 2 mg/kg po.
Figure 4. Selectivity heat map for compound 16.
Figure 5. In vivo analysis of compound 16. (A) Vascular permeability assay. HEK 293 cells transfected with murine VEGF or vector were mixed
with Matrigel and injected sc into CD-1 nu/nu mice. A single dose of compound was given, and vascular permeability in the skin overlying the
Matrigel plug was assessed 6 h later. Data represent the mean ( standard error (n ) 5 animals per group): (/) P e 0.0001. (B) Rat corneal
angiogenesis model. Angiogenesis was induced by implanting a VEGF-soaked or BSA-soaked (control) nylon disk into the corneal stroma. The
number of blood vessels intersecting the midpoint between the disk and the limbus was measured after 7 days of dosing vehicle or drug. Data
represent the mean ( standard error (n ) 8 animals per group): (/) P ) 0.0034.
region of the enzyme in a 2.9 Å hydrogen bond between the
nitrogen and Cys 919. As proposed by the interaction illustrated
in Figure 3, an additional hydrogen bond of 3.2 Å is observed
through the hydrogen at C8. The experimental electron density
mapping shown in Figure 7 illustrates this detailed interaction.25
The ethereal linkage at the 4-position of the quinoline is sp2
hybridized (C-O-C angle ) 119°) and resides such that the
p-orbital lone pair conjugates into the quinoline π-system as
opposed to the benzoxazine ring. Across the molecule, two
hydrogen bond contacts are observed between the urea carbonyl
and Asp 1046 (2.7 Å) and the urea NH and Glu 885 (3.3 Å). In
addition, a water molecule serves as a bridge between Lys 868

Potent, Orally AVailable KDR Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1701
Figure 6. Cocrystal of compound 16 within in the ATP-binding pocket of KDR (PDB code 2RL5). Key hydrogen bond contacts are shown (dotted
lines). In part B, portions of the DFG sequence are omitted for clarity.
optimization of this series to yield our clinical candidate will
be disclosed shortly.
Experimental Section
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification. Dry
organic solvents (CH2Cl2, CH3CN, DMF, etc.) were purchased from
Aldrich packaged under nitrogen in Sure/Seal bottles. Reactions
were monitored using an Agilent 1100 series LC-MS instrument
with UV detection at 254 nm and a low-resonance electrospray
mode (ESI). Medium-pressure liquid chromatography (MPLC) was
performed on a CombiFlash Companion (Teledyne Isco) with
RediSep normal-phase silica gel (35–60 µm) columns and UV
detection at 254 nm. Preparative reversed-phase high-performance
Figure 7. Electron density depicting the interaction of the dimetho-
liquid chromatography (HPLC) was performed on a Gilson (215
liquid handler), YMC-Pack Pro C18, 150–30 mm i.d. column,
eluting with a binary solvent system A and B using a gradient
elution (solvent A, H2O with 0.1% TFA; solvent B, CH3CN with
0.1% TFA) with UV detection at 254 nm. Purity was measured
using Agilent 1100 series HPLC instrument with UV detection at
254 nm (15 min, 1.5 mL/min flow rate; 0-100% 0.1% TFA in
CH3CN/100–0% 0.1% TFA in H2O). 1H NMR spectra were
recorded on a Bruker AV-400 (400 MHz) spectrometer at ambient
temperature or on a Varian 400 MHz or Varian 300 MHz
spectrometer. Chemical shifts are reported in ppm from the solvent
resonance (DMSO-d6, 2.50 ppm). Data are reported as follows:
chemical shift, multiplicity (s ) singlet, d ) doublet, t ) triplet, q
) quartet, br ) broad, m ) multiplet), coupling constants, and
number of protons. Mass spectra were obtained on a high-resolution
electrospray time-of-flight mass spectrometer. Combustion analysis
was performed by Galbraith Laboratories, Inc., Knoxville, TN.
7-Hydroxy-2H-benzo[b][1,4]oxazin-3(4H)-one (3). To a solu-
tion of 4-aminoresorcinol hydrochloride (15.0 g, 92.83 mmol) in
acetonitrile (300 mL) at 0 °C was added cesium carbonate (90.7 g,
278.4 mmol) followed by a dropwise addition of chloroacetyl
chloride (7.76 mL, 97.47 mmol). The reaction mixture was stirred
at room temperature for 18 h and then was diluted with absolute
ethanol (300 mL). The insoluble materials were filtered and rinsed
with absolute ethanol. The filtrate was concentrated under vacuum
to afford the crude product as a dark-purple solid. The solid was
dissolved in methanol (100 mL), silica was added (100 mL), and
the solvent was removed again under vacuum. The solid was loaded
onto a pad of silica (800 mL) and eluted with 1:1 ethyl acetate in
dichloromethane followed by 100% dichloromethane. After removal
of the solvents, the product was obtained as an orange solid (11.71
g, 76%). 1H NMR (DMSO-d6) δ 9.40 (s, 1H), 8.97–8.95 (m, 1H),
8.72 (s, 1H), 7.99–7.94 (m, 1H), 7.93–7.86 (m, 1H), 7.79 (dd, J )
2.5, 6.8 Hz, 1H), 7.74 (d, J ) 9.0 Hz, 1H), 7.64 (s, 1H), 7.50–7.45
(m, 1H), 7.37 (t, J ) 9.1 Hz, 1H), 7.02 (d, J ) 2.8 Hz, 1H), 6.92
(dd, 9.1, 2.7 Hz, 1H), 6.89–6.86 (m, 1H), 4.37–4.33 (m, 2H), 4.08
(s, 3H), 3.94–3.91 (m, 2H). HRMS [M + H] calcd, 166.049 87;
found, 166.050 29.
xyquinoline ring and Cys919 from the hinge region of KDR. A
simulated annealing omit map for the inhibitor (orange, 3σ contour) is
superimposed on the final 2Fo - Fc map (blue, 1σ contour). Dashed
lines indicate hydrogen bonds (distances in angstroms).
Table 5. Pharmacokinetic/Pharmacodynamic Relationship for 16 in the
Vascular Permeability and VEGF-Induced Rat Corneal Angiogenesis
Models
vascular permeability
rat corneal angiogenesis
active
dose
(mg/kg)
exposure at
active dose
(µM)
Cmax at
ED50
(µM)
AUC at
ED50
(µM h)
ED50
(mg/kg)
3
4.6
0.16
0.18
2.8
and Asp 1046 (not shown). Presumably, much of the potency
is acquired through van der Waals contacts between the inhibitor
and the hydrophobic binding pocket generated upon induced
movement of the activation loop by the aryl chloride (compare
16 and 18 of Table 1). Additional van der Waals interactions
are seen with the benzoxazine segment and the surrounding
lipophilic residues (see Figure 6B).
Conclusions
The novel 2,3-dihydro-1,4-benzoxazine inhibitors and com-
pound 16 in particular have been shown to be effective inhibitors
of angiogenesis. This investigation aimed initially to address
physical chemical property liabilities of an alternative naph-
thylamide core, which resulted in the design of the described
benzomorpholine series. Our efforts to optimize potency were
driven by structural analysis and ultimately revealed several
compounds with appropriate levels of activity against KDR and
favorable pharmacokinetic properties. This strategy was further
validated by in vivo efficacy models, which highlighted the
benzoxazines as promising antiangiogenic agents. Further

1702 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
La et al.
3,4-Dihydro-2H-benzo[b][1,4]oxazin-7-ol Hydrochloride
(4). A 500 mL round-bottom flask equipped with a magnetic stir
bar and a pressure equalizing addition funnel was charged with
7-hydroxy-2H-benzo[b][1,4]oxazin-3(4H)-one (9.74 g, 59 mmol)
and anhydrous THF (100 mL) under N2. The orange-pink mixture
was cooled in an ice bath, and borane-THF complex (1 M in THF,
118 mL, 118 mmol, 2 equiv) was added dropwise under a stream
of N2 to remove the H2 generated. Upon complete addition, the
now colorless solution was removed from the ice bath and allowed
to warm to ambient temperature. After 3 h of stirring, an aliquot
was quenched into MeOH and analyzed by LC-MS, which
indicated incomplete consumption of the starting material. After
another 3 h of stirring, the reaction was determined to be complete
by LC-MS, so the mixture was quenched by the slow addition of
MeOH (25 mL) under a stream of N2. The mixture was then stirred
at ambient temperature for 1 h to allow for complete destruction
of the borane reagent. The resulting solution was concentrated by
rotary evaporation, and the pale-yellow residue was dissolved in
MeOH (25 mL). The solution was then cooled in an ice bath under
N2 and a solution of HCl (1 M in Et2O, 100 mL) was added
dropwise, resulting in a white solid suspended in a bright-blue
solution. The suspension was aged for 30 min with cooling and
then was filtered. The pale-blue solid was washed nearly white with
Et2O/MeOH (4:1, 100 mL) and then was dried under vacuum
overnight to yield 3,4-dihydro-2H-benzo[b][1,4]oxazin-7-ol hydro-
2.06 g (83.6%) of product. 1H NMR (CDCl3) δ 9.30 (s, 1H), 8.64
(d, J ) 5.3 Hz, 1H), 8.04 (s, 1H), 7.81 (s, 1H), 7.54 (s, 1H), 6.68
(s, 1H), 6.63–6.66 (m, 1H), 6.61 (d, J ) 2.5 Hz, 1H), 6.59 (d, J )
2.5 Hz, 1H), 6.53 (d, J ) 5.3 Hz, 1H), 4.31 (t, J ) 4.3 Hz, 2H),
4.14 (s, 3H), 3.48 (t, J ) 4.3 Hz, 2H). HRMS [M + H] calcd,
352.129 18; found, 352.129 62.
7-(7H-Pyrrolo[2,3-d]pyrimidin-4-yloxy)-3,4-dihydro-2H-
benzo[b][1,4]oxazine (8). A resealable tube was charged with
4-chloro-7H-pyrrolo[2,3-d]pyrimidine (0.500 g, 3.26 mmol), 3,4-
dihydro-2H-benzo[b][1,4]oxazin-7-ol (0.738 g, 4.88 mmol), cesium
carbonate (3.18 g, 9.77 mmol), and DMF (5.0 mL), and the reaction
mixture was stirred at 80 °C for 17 h. Additional 3,4-dihydro-2H-
benzo[b][1,4]oxazin-7-ol (0.738 g, 4.88 mmol) and cesium carbon-
ate (3.18 g, 9.77 mmol) were added, and the mixture was stirred at
80 °C for 20 h. The reaction mixture was cooled to room
temperature and filtered through a pad of Celite. The filtrate was
concentrated, dissolved in DCM/MeOH (∼1:1), filtered through
Celite, and concentrated to afford a brown solid. This material was
purified twice via column chromatography on silica gel (gradient
elution with 0–5% MeOH/DCM) to afford 7-(7H-pyrrolo[2,3-
d]pyrimidin-4-yloxy)-3,4-dihydro-2H-benzo[b][1,4]oxazine (0.458
g, 1.71 mmol, 52% yield) as an orange solid. 1H NMR (400 MHz,
DMSO-d6) δ ppm 12.12 (s, 1 H), 8.27 (s, 1 H), 7.38–7.41 (m, 1
H), 6.52–6.61 (m, 4 H), 6.29-6.32 (m, 1 H), 4.12–4.17 (m, 2 H),
3.27–3.31 (m, 2 H). HRMS [M + H] calcd, 268.096 03; found,
268.096 03.
1
chloride (10.4 g, 94% yield). H NMR (400 MHz, DMSO-d6) δ
11.22 (br, 1H), 9.85 (br, 1H), 7.07 (d, J ) 8.8, 1H), 6.45 (dd, J )
8.7, 2.6, 1H), 6.37 (d, J ) 2.5, 1H), 4.32 (m, 2H), 3.48 (m, 2H).
HRMS [M + H] calcd, 152.070 60; found, 152.070 60.
4-(3,4-Dihydro-2H-benzo[b][1,4]oxazin-7-yloxy)-N-methylpi-
colinamide (9). Compound 9 was prepared using the same
1
procedure as compound 7. H NMR (DMSO-d6) δ 8.75 (s, 1H),
7-(6,7-Dimethoxyquinolin-4-yloxy)-3,4-dihydro-2H-
benzo[b][1,4]oxazine (6). A 500 mL three-neck flask equipped with
a large magnetic stir bar and a reflux condenser was charged with
cesium carbonate (52.46 g, 161 mmol, 3 equiv), 4-chloro-6,7-
dimethoxyquinoline (12.00 g, 53.7 mmol, 1.0 equiv), 3,4-dihydro-
2H-benzo[b][1,4]oxazin-7-ol hydrochloride (15.1 g, 80.5 mmol, 1.5
equiv), and DMF (100 mL). The mixture was vacuum-purged
(carefully because of CO2 generation) and then switched to N2.
The brown heterogeneous mixture was then heated to 90–100 °C
under N2 with an oil bath. The reaction was periodically monitored
for conversion by LC-MS and showed 75–80% conversion after
3 days. At this point, additional Cs2CO3 (8.75 g, 0.5 equiv) and
benzomorpholine HCl salt (5.0 g, 0.5 equiv) were added and the
mixture was maintained at 100 °C for an additional 26 h until an
aliquot indicated about 95% conversion by LC-MS analysis. At
this point, the mixture was cooled and then quenched by the addition
of water (250 mL). The resulting solid was isolated by vacuum
filtration and washed with water. The gray paste was then slurried
with MeOH/MTBE (2:1) at ambient temperature, filtered, washed
with MeOH, and dried under vacuum to yield 7-(6,7-dimethoxy-
quinolin-4-yloxy)-3,4-dihydro-2H-benzo[b][1,4]oxazine (8.84 g,
49%) containing <5% of the chloroquinoline starting material as
an off-white solid. The filtrate was concentrated to dryness,
reslurried in MeOH, filtered, washed, and dried as before to yield
additional material (6.83 g, 38%) containing slightly more chlo-
roquinoline (<5%) as a tan solid. Analytically pure material could
be obtained by silica gel chromatography (gradient elution with
8.46 (d, J ) 5.6 Hz, 1H), 7.34 (d, J ) 2.7 Hz, 1H), 7.09 (dd, J )
5.6, 2.6 Hz, 1H), 6.59–6.68 (m, 1H), 6.49–6.56 (m, 2H), 5.88 (s,
1H), 4.05–4.24 (m, 2H), 3.26–3.31 (m, 2H), 2.78 (d, J ) 4.8 Hz,
3H). HRMS [M + H] calcd, 286.118 62; found, 286.118 75.
7-((6,7-Bis(methyloxy)-4-quinolinyl)oxy)-N-phenyl-2,3-dihy-
dro-4H-1,4-benzoxazine-4-carboxamide (10). Compound 10 was
prepared using the same procedure as compound 16. 1H NMR
(DMSO-d6) δ 9.13 (s, 1H), 8.50 (d, J ) 5.2 Hz, 1H), 7.64 (d, J )
8.8 Hz, 1H), 7.50 (d, J ) 8.8 Hz, 1H), 7.48 (s, 1H), 7.39 (s, 1H),
7.29 (dd, J ) 7.6, 7.6 Hz, 2H), 7.00 (dd, J ) 8.0, 8.0 Hz, 1H),
6.84 (d, J ) 2.8 Hz, 1H), 6.78 (dd, J ) 8.8, 2.4 Hz, 1H), 6.56 (d,
J ) 5.2 Hz, 1H), 4.31 (t, J ) 4.0 Hz, 2H), 3.94 (s, 3H), 3.93 (s,
3H), 3.90 (t, J ) 3.6 Hz, 2H). HPLC purity: 95%. HRMS [M +
H] calcd, 458.171 05; found, 458.172 22.
7-((6,7-Bis(methyloxy)-4-quinolinyl)oxy)-N-(2-methylphenyl)-
2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide (11). Compound
1
11 was prepared using the same procedure as compound 16. H
NMR (CDCl3) δ 8.53 (d, J ) 5.2 Hz, 1H), 7.84 (d, J ) 8.0 Hz,
1H), 7.53 (s, 1H), 7.51 (d, J ) 8.8 Hz, 1H), 7.45 (s, 1H), 7.27–7.14
(m, 3H), 7.06 (t, J ) 7.2 Hz, 1H), 6.86 (d, J ) 2.4 Hz, 1H), 6.82
(dd, J ) 8.8, 2.4 Hz, 1H), 6.57 (d, J ) 5.2 Hz, 1H), 4.37 (t, J )
4.4 Hz, 2H), 4.06 (s, 6H), 4.01 (t, J ) 4.4 Hz, 2H), 2.20 (s, 3H).
HRMS [M + H] calcd, 472.186 70; found, 472.187 91. Anal.
(C27H25N3O5 ·0.4H2O) C, H.
7-((6,7-Bis(methyloxy)-4-quinolinyl)oxy)-N-(3-methylphenyl)-
2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide (12). Compound
1
1
12 was prepared using the same procedure as compound 16. H
0–5% MeOH in dichloromethane). H NMR (400 MHz, DMSO-
NMR (CDCl3) δ 8.54 (d, J ) 5.6 Hz, 1H), 7.52 (s, 1H), 7.44 (s,
1H), 7.43 (d, J ) 8.8 Hz, 1H), 7.27–7.17 (m, 4H), 6.92 (d, J ) 5.6
Hz, 1H), 6.85 (d, J ) 2.4 Hz, 1H), 6.82 (dd, J ) 8.8, 2.4 Hz, 1H),
6.61 (d, J ) 4.8 Hz, 1H), 4.35 (t, J ) 4.4 Hz, 2H), 4.06 (s, 6H),
4.00 (t, J ) 4.4 Hz, 2H), 2.35 (s, 3H). HRMS [M + H] calcd,
472.186 70; found, 472.187 11. Anal. (C27H25N3O5) C, H.
d6) δ 8.44 (d, J ) 5.2, 1H), 7.47 (s, 1H), 7.36 (s, 1H), 6.67–6.65
(m, 1H), 6.60–6.57 (m, 2H), 6.42 (d, J ) 5.3, 1H), 5.85 (br, 1H),
4.17–4.15 (m, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.32–3.29 (m, 2H).
HRMS [M + H] calcd, 339.133 93; found, 339.135 05.
4-(3,4-Dihydro-2H-benzo[b][1,4]oxazin-7-yloxy)-7-methoxy-
quinoline-6-carboxamide (7). 4-Chloro-7-methoxyquinoline-6-
carboxamide (1.66 g, 7.01 mmol) was added to a sealed round-
bottomed vessel followed by 3,4-dihydro-2H-benzo[b][1,4]oxazin-
7-ol (1.59 g, 10.5 mmol) and cesium carbonate (6.86 g, 21.0 mmol).
DMF (20 mL) was added, and the vessel was sealed and placed in
an oil bath. The resulting mixture was heated to 80 °C and stirred
for 16 h. The reaction mixture was then cooled to room temperature,
and DMF was removed in vacuo. The crude mixture was purified
with silica gel chromotography (5% MeOH in DCM) to provide
7-((6,7-Bis(methyloxy)-4-quinolinyl)oxy)-N-(4-methylphenyl)-
2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide (13). Compound
1
13 was prepared using the same procedure as compound 16. H
NMR (DMSO-d6) δ 9.01 (s, 1H), 8.50 (d, J ) 5.3 Hz, 1 H), 7.64
(d, J ) 8.8 Hz, 1H), 7.48 (s, 1H), 7.39 (s, 2H), 7.37 (s, 1H), 7.10
(d, J ) 8.5 Hz, 2H), 6.82 (d, J ) 2.8 Hz, 1H), 6.77 (dd, J ) 8.9,
2.7 Hz, 1H), 6.56 (d, J ) 5.2 Hz, 1H), 4.24 – 4.34 (m, 2H), 3.94
(s, 3H), 3.93 (s, 3H), 3.85–3.91 (m, 2H), 2.25 (s, 3H). HRMS [M

Potent, Orally AVailable KDR Inhibitors
H] calcd, 472.186 70; found, 472.187 58. Anal.
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1703
+
7-((6,7-Bis(methyloxy)-4-quinolinyl)oxy)-N-ethyl-2,3-dihydro-
4H-1,4-benzoxazine-4-carboxamide (20). Compound 20 was
prepared using the same procedure as compound 16. 1H NMR
(DMSO-d6) δ 8.46 (d, J ) 5.2 Hz, 1H), 7.68 (d, J ) 8.8 Hz, 1H),
7.45 (s, 1H), 7.37 (s, 1H), 6.96 (dd, J ) 5.6, 5.6 Hz, 1H), 6.76 (d,
J ) 2.4 Hz, 1H), 6.73 (dd, J ) 8.4, 2.8 Hz, 1H), 6.50 (d, J ) 5.6
Hz, 1H), 4.19 (t, J ) 4.0 Hz, 2H), 3.92 (s, 3H), 3.90 (s, 3H), 3.72
(t, J ) 4.4 Hz, 2H), 3.12 (p, J ) 6.4 Hz, 2H), 1.06 (t, J ) 7.6 Hz,
3H). HRMS [M + H] calcd, 410.171 05; found, 410.172 38. Anal.
(C22H23N3O5 ·0.2H2O) C, H.
(C27H25N3O5 ·4.6H2O) C, H.
7-((6,7-Bis(methyloxy)-4-quinolinyl)oxy)-N-(2-chlorophenyl)-
2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide (14). Compound
1
14 was prepared using the same procedure as compound 16. H
NMR (CDCl3) δ 8.53 (d, J ) 4.8 Hz, 1H), 8.35 (d, J ) 8.0 Hz,
1H), 7.98 (s, 1H), 7.54 (d, J ) 8.0 Hz, 1H), 7.53 (s, 1H), 7.44 (s,
1H), 7.36–7.27 (m, 2H), 7.02 (t, J ) 7.6 Hz, 1H), 6.86–6.84 (m,
2H), 6.57 (d, J ) 4.8 Hz, 1H), 4.38 (t, J ) 4.0 Hz, 2H), 4.06 (s,
6H), 4.03 (t, J ) 4.0 Hz, 2H). HRMS [M + H] calcd, 492.132 07;
found, 492.132 36. Anal. (C26H22ClN3O5 ·0.1H2O) C, H.
7-((6,7-Bis(methyloxy)-4-quinolinyl)oxy)-N-(1-methylethyl)-
2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide (21). Compound
7-((6,7-Bis(methyloxy)-4-quinolinyl)oxy)-N-(3-chlorophenyl)-
2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide (15). Compound
1
21 was prepared using the same procedure as compound 16. H
1
NMR (DMSO-d6) δ 8.48 (d, J ) 5.3 Hz, 1H), 7.71 (d, J ) 8.9 Hz,
1H), 7.47 (s, 1H), 7.39 (s, 1H), 6.77 (d, J ) 2.9 Hz, 1H), 6.74 (dd,
J ) 8.9, 2.9 Hz, 1H), 6.69 (d, J ) 7.2 Hz, 1H), 6.77 (d, J ) 2.9
Hz, 1H), 6.53 (d, J ) 5.3 Hz, 1H), 4.22 (t, J ) 4.5 Hz, 2H), 3.94
(s, 3H), 3.92 (s, 3H), 3.85–3.83 (m, 1H), 3.73 (t, J ) 4.5 Hz, 2H),
1.14 (d, J ) 6.5 Hz, 6H). HRMS [M + H] calcd, 424.186 70; found,
424.187 87. Anal. (C23H25N3O5 ·0.2H2O) C, H.
15 was prepared using the same procedure as compound 16. H
NMR (DMSO-d6) δ 9.30 (s, 1H), 8.49 (d, J ) 5.2 Hz, 1H), 7.65
(s (br), 1H), 7.61 (d, J ) 9.2 Hz, 1H), 7.46 (s, 1H), 7.42 (d, J )
7.2 Hz, 1H), 7.38 (s, 1H), 7.30 (dd, J ) 7.6, 7.6 Hz, 1H), 7.04 (d
(br), J ) 7.6 Hz, 1H), 6.82 (d, J ) 2.4 Hz, 1H), 6.77 (dd, J ) 8.8,
2.8 Hz, 1H), 6.54 (d, J ) 5.2 Hz, 1H), 4.30 (t, J )4.0 Hz, 2H),
3.92 (s, 3H), 3.91 (s, 3H), 3.88 (t, J ) 4.0 Hz, 1H). HPLC purity:
98%. HRMS [M + H] calcd, 492.132 07; found, 492.134 04.
N-(4-Chlorophenyl)-7-(6,7-dimethoxynaphthalen-1-yloxy)-2,3-
dihydrobenzo[b][1,4]oxazine-4-carboxamide (16). To a hetero-
geneous solution of 1,4-benzoxazine 6 (62.0 mg, 0.183 mmol) in
0.915 mL of anhydrous DCM at room temperature was added
4-chlorophenyl isocyanate (35.2 µL, 0.275 mmol). The resulting
solution was stirred at room temperature for 2 h. The crude reaction
mixture was then loaded directly onto a MPLC silica gel cartridge.
The crude mixture was purified by chromatography (gradient of 100%
DCM to 50% DCM/MeOH/NH4OH (90:10:1)) to yield 48.6 mg of
the final desired product (54% yield). 1H NMR (DMSO-d6) δ 9.16 (s,
1H), 8.40 (d, J ) 5.2 Hz, 1H), 7.53 (d, J ) 8.8 Hz, 1H), 7.43 (d, J )
8.8 Hz, 1H), 7.37 (s, 1H), 7.29 (s, 1H), 7.25 (d, J ) 9.2 Hz, 1H), 6.73
(d, J ) 2.8 Hz, 1H), 6.67 (dd, J ) 8.8 Hz, 1H), 6.45 (d, J ) 5.2 Hz,
1H), 4.21 (t, J ) 4.0 Hz, 2H), 3.84 (s, 3H), 3.83 (s, 3H), 3.79 (t, J )
4.4 Hz, 2H). HRMS [M + H] calcd, 492.132 07; found, 492.133 93.
Anal. (C26H22ClN3O5 ·0.5H2O) C, H.
7-((6,7-Bis(methyloxy)-4-quinolinyl)oxy)-N-(phenylmethyl)-
2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide (22). Compound
1
22 was prepared using the same procedure as compound 16. H
NMR (CDCl3) δ 8.49 (d, J ) 6.2 Hz, 1H), 7.76 (s, 1H), 7.54 (s,
1H), 7.42 (d, J ) 8.43 Hz, 1H), 7.29–7.38 (m, 5H), 6.80 (d, J )
2.6 Hz, 1H), 6.73 (dd, J ) 8.8, 2.6 Hz, 1H), 6.66 (d, J ) 5.5 Hz,
1H), 5.60 (t, J ) 5.1 Hz, 1H), 4.53 (d, J ) 5.5 Hz, 2H), 4.32 (t, J
) 4.13 Hz, 2H), 4.10 (s, 3H), 4.06 (s, 3H), 3.96 (t, J ) 4.1 Hz,
2H). HRMS [M + H] calcd, 472.186 70; found, 472.187 79. Anal.
(C27H25N3O5 ·1.4H2O) C, H.
7-((6,7-Bis(methyloxy)-4-quinolinyl)oxy)-N-((4-methylphenyl-
)methyl)-2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide (23).
Compound 23 was prepared using the same procedure as compound
16. 1H NMR (DMSO-d6) δ 8.48 (d, J ) 5.2 Hz, 1H), 7.72 (d, J )
8.8 Hz, 1H), 7.52 (t, J ) 5.8 Hz, 1H), 7.47 (s, 1H), 7.39 (s, 1H),
7.21 (d, J ) 8.2 Hz, 2H), 7.13 (d, J ) 7.69 Hz, 2H), 6.71–6.83 (m,
1H), 6.52 (d, J ) 5.2 Hz, 1H), 5.76 (s, 1H), 4.29 (d, J ) 5.5 Hz,
2H), 4.24 (t, J ) 4.0 Hz, 2H), 3.94 (s, 3H), 3.92 (s, 3H), 3.80 (t,
J ) 4.1 Hz, 2H), 2.28 (s, 3H). HRMS [M + H] calcd, 486.202 35;
found, 486.203 27. Anal. (C28H27N3O5 ·0.8H2O) C, H.
7-((6,7-Bis(methyloxy)-4-quinolinyl)oxy)-N-(4-fluorophenyl)-
2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide (17). Compound
1
17 was prepared using the same procedure as compound 16. H
7-((6,7-Bis(methyloxy)-4-quinolinyl)oxy)-N-((1S)-1-phenylethyl)-
2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide (24). Compound
NMR (DMSO-d6) δ 9.14 (s, 1H), 8.50 (d, J ) 5.2 Hz, 1H), 7.65
(d, J ) 8.8 Hz, 1H), 7.43–7.55 (m, 3H), 7.39 (s, 1H), 7.14 (t, J )
8.9 Hz, 2H), 6.83 (d, J ) 2.7 Hz, 1H), 6.78 (dd, J ) 8.9, 2.7 Hz,
1H), 6.56 (d, J ) 5.2 Hz, 1H), 4.26–4.35 (m, 2H), 3.94 (s, 3H),
3.93 (s, 3H), 3.86–3.92 (m, 2H). HRMS [M + H] calcd, 476.161 63;
found, 476.162 11. Anal. (C26H22FN3O5 ·0.6H2O) C, H.
1
24 was prepared using the same procedure as compound 16. H
NMR (CDCl3) δ 8.52 (d, J ) 5.5 Hz, 1H), 7.51 (s, 1H), 7.44 (s,
1H), 7.27–7.40 (m, 6H), 6.80 (d, J ) 2.6 Hz, 1H), 6.75 (dd, J )
8.8, 2.6 Hz, 1H), 6.58 (d, J ) 5.5 Hz, 1H), 5.54 (d, J ) 7.3 Hz,
1H), 5.10 (dq, J ) 7.3, 7.0 Hz, 1H), 4.28 (t, J ) 3.7 Hz, 2H), 4.06
(s, 3H), 4.04 (s, 3H), 3.82–4.00 (m, 2H), 1.53 (d, J ) 6.97 Hz,
3H). HRMS [M + H] calcd, 486.202 35; found, 486.203 25. Anal.
(C28H27N3O5) C, H.
7-((6,7-Bis(methyloxy)-4-quinolinyl)oxy)-2,3-dihydro-4H-1,4-
benzoxazine-4-carboxamide (18). To a solution of 7-(6,7-di-
methoxyquinolin-4-yloxy)-3,4-dihydro-2H-benzo[b][1,4]oxazine (100
mg, 0.296 mmol) in toluene (2 mL) was added trichloroacetyl
isocyanate (39.0 µL, 0.325 mmol) at room temperature. The
resulting reaction mixture was stirred for 2 h at 60 °C. The solution
was cooled to room temperature, and 10% HCl/H2O (5 mL) was
added. The solution was concentrated in vacuo and purified through
silica gel chromatography (gradient of 100% CH2Cl2 to 30% 90:10:1
CH2Cl2/MeOH/NH4OH) to provide the desired product (29 mg,
7-((6,7-Bis(methyloxy)-4-quinolinyl)oxy)-N-((1R)-1-phenylethyl)-
2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide (25). Compound
1
25 was prepared using the same procedure as compound 16. H
NMR (CDCl3) δ 8.52 (d, J ) 5.5 Hz, 1H), 7.51 (s, 1H), 7.44 (s,
1H), 7.27–7.39 (m, 6H), 6.79 (d, J ) 2.57 Hz, 1H), 6.75 (dd, J )
8.62, 2.8 Hz, 1H), 6.58 (d, J ) 5.13 Hz, 1H), 5.54 (d, J ) 7.7 Hz,
1H), 5.04–5.15 (m, 1H), 4.24–4.30 (m, 2H), 4.06 (s, 3H), 4.04 (s,
3H), 3.83–3.99 (m, 2H), 1.53 (d, J ) 6.97 Hz, 3H). HRMS [M +
H] calcd, 486.202 35; found, 486.203 19. Anal. (C28H27N3O5 ·
0.1H2O) C, H.
1
25% yield). H NMR (DMSO-d6) δ 8.46 (d, J ) 5.13 Hz, 1H),
7.77 (d, J ) 8.8 Hz, 1H), 7.46 (s, 1H), 7.37 (s, 1H), 6.67–6.81 (m,
2H), 6.53 (s, 2H), 6.50 (d, J ) 5.1 Hz, 1H), 4.21 (t, J ) 3.9 Hz,
2H), 3.92 (s, 3H), 3.91 (s, 3H), 3.73 (t, J ) 4.0 Hz, 2H). HPLC
purity: 97%. HRMS [M + H] calcd, 382.139 75; found, 382.141 11.
7-((6-(Aminocarbonyl)-7-(methyloxy)-4-quinolinyl)oxy)-N-(4-
chlorophenyl)-2,3-dihydro-4H-1,4-benzoxazine-4-carboxam-
ide (26). Compound 26 was prepared using the same procedure as
compound 16. Compound 26 was converted to the HCl salt by
adding boiling ethanol without completely dissolving the urea. Then
1 N HCl in ethanol was added, and the mixture was slowly cooled
to room temperature, with stirring, over 18 h. The solids were
7-((6,7-Bis(methyloxy)-4-quinolinyl)oxy)-N-methyl-2,3-dihy-
dro-4H-1,4-benzoxazine-4-carboxamide (19). Compound 19 was
prepared using the same procedure as compound 16. 1H NMR
(DMSO-d6) δ 8.48 (d, J ) 5.0 Hz, 2H), 7.71 (d, J ) 9.0 Hz, 1H),
7.47 (s, 1H), 7.39 (s, 1H), 6.88 (q, J ) 4.2 Hz, 1H), 6.78 (d, J )
2.8 Hz, 1H), 6.75 (d, J ) 8.7 Hz, 1H), 6.53 (d, J ) 5.0 Hz, 1H),
4.21 (t, J ) 4.3 Hz, 2H), 3.94 (s, 3H), 3.93 (s, 3H), 3.74 (t, J )
4.2 Hz, 2H), 2.68 (d, J ) 4.3 Hz, 3H). HRMS [M + H] calcd,
396.155 40; found, 396.157 25. Anal. (C21H21N3O5 ·0.4H2O) C, H.
1
collected by filtration and rinsed with ethanol. H NMR (DMSO-
d6) δ 9.35 (s, 1H), 8.97 (d, J ) 6.3 Hz, 1H), 8.72 (s, 1H), 7.97 (b,
1H), 7.91 (b, 1H), 7.73 (d, J ) 8.9 Hz, 1H), 7.67 (s, 1H), 7.57–7.53
(m, 2H), 7.37–7.34 (m, 2H), 7.03 (d, J ) 2.7 Hz, 1H), 6.93–6.89

1704 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
La et al.
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(m, 2H), 4.36–4.33 (m, 2H), 4.08 (s, 3H), 3.94–3.91 (m, 2H).
HRMS [M + H] calcd, 505.127 32; found, 505.128 37. Anal.
(C26H21ClN4O5 ·1.9H2O) C, H.
7-((6-(Aminocarbonyl)-7-(methyloxy)-4-quinolinyl)oxy)-N-(4-
methylphenyl)-2,3-dihydro-4H-1,4-benzoxazine-4-carboxam-
ide (27). Compound 27 was prepared using the same procedure as
1
compound 16. H NMR (CDCl3) δ 9.29 (s, 1H), 8.72 (d, J ) 5.1
Hz, 1H), 7.82 (s, 1H), 7.59 (s, 1H), 7.45 (d, J ) 8.6 Hz, 1H), 7.33
(d, J ) 8.2 Hz, 2H), 7.16 (d, J ) 8.7 Hz, 3H), 6.79–6.88 (m, 2H),
6.63 (d, J ) 5.3 Hz, 1H), 5.97 (s, 1H), 4.34–4.40 (m, 2H), 4.16 (s,
3H), 3.98–4.05 (m, 2H), 2.34 (s, 3H). HRMS [M + Na] calcd,
507.163 89; found, 507.164 89. Anal. (C27H24N4O5 ·1.2H2O) C, H.
N-(4-Chlorophenyl)-7-(7H-pyrrolo[2,3-d]pyrimidin-4-yloxy)-
2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide (28). Compound
1
28 was prepared using the same procedure as compound 16. H
NMR (DMSO-d6) δ 12.20 (s, 1H), 9.29 (s, 1H), 8.31 (s, 1H), 7.48
– 7.63 (m, 3H), 7.42–7.50 (m, 1H), 7.34 (d, J ) 8.8 Hz, 2H), 6.82
(d, J ) 2.5 Hz, 1H), 6.76 (dd, J ) 9.0, 2.5 Hz, 1H), 6.49 (d, J )
1.9 Hz, 1H), 4.30 (s, 2H), 3.89 (s, 2H). HRMS [M + H] calcd,
422.101 44; found, 422.103 07. Anal. (C21H16ClN5O3 · 0.8H2O)
C, H.
N-(4-Methylphenyl)-7-(7H-pyrrolo[2,3-d]pyrimidin-4-yloxy)-
2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide (29). Compound
1
29 was prepared using the same procedure as compound 16. H
NMR (DMSO-d6) δ 12.20 (s, 1H), 9.04 (s, 1H), 8.31 (s, 1H), 7.56
(d, J ) 9.0 Hz, 1H), 7.43–7.49 (m, 1H), 7.38 (d, J ) 8.5 Hz, 2H),
7.09 (d, J ) 8.3 Hz, 2H), 6.81 (d, J ) 2.7 Hz, 1H), 6.75 (dd, J )
8.9, 2.7 Hz, 1H), 6.44–6.51 (m, 1H), 4.22–4.33 (m, 2H), 3.78–3.96
(m, 2H), 2.25 (s, 3H). HRMS [M + H] calcd, 402.156 07; found,
402.157 56. Anal. (C22H19N5O3 ·0.9H2O) C, H.
N-(4-Chlorophenyl)-7-((2-((methylamino)carbonyl)-4-pyridi-
nyl)oxy)-2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide (30).
Compound 30 was prepared using the same procedure as compound
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1
16. H NMR (DMSO-d6) δ 9.32 (s, 1H), 8.73–8.83 (m, 1H), 8.52
(d, J ) 5.6 Hz, 1H), 7.62 (d, J ) 9.0 Hz, 1H), 7.53 (d, J ) 8.8 Hz,
2H), 7.43 (d, J ) 2.4 Hz, 1H), 7.34 (d, J ) 8.8 Hz, 2H), 7.19 (dd,
J ) 5.5, 2.6 Hz, 1H), 6.81 (d, J ) 2.7 Hz, 1H), 6.74 (dd, J ) 9.0,
2.7 Hz, 1H), 4.31 (s, 2H), 3.9 (s, 2H), 2.79 (d, J ) 4.8 Hz, 3H).
HRMS [M + H] calcd, 439.116 76; found, 439.117 87. Anal. Calcd
for C22H19ClN4O4: C, 60.21; H, 4.36; N, 12.77. Found: C, 60.55;
H, 4.90; N, 11.76.
7-((2-((Methylamino)carbonyl)-4-pyridinyl)oxy)-N-(4-meth-
ylphenyl)-2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide (31).
Compound 31 was prepared using the same procedure as compound
1
16. H NMR (DMSO-d6) δ 9.08 (s, 1H), 8.73–8.82 (m, 1H), 8.51
(d, J ) 5.6 Hz, 1H), 7.62 (d, J ) 9.0 Hz, 1H), 7.43 (d, J ) 2.5 Hz,
1H), 7.38 (d, J ) 8.5 Hz, 2H), 7.18 (dd, J ) 5.6, 2.5 Hz, 1H), 7.09
(d, J ) 8.3 Hz, 2H), 6.79 (d, J ) 2.8 Hz, 1H), 6.73 (dd, J ) 8.9,
2.8 Hz, 1H), 4.23–4.34 (m, 2H), 3.81–3.95 (m, 2H), 2.79 (d, J )
4.8 Hz, 3H), 2.25 (s, 3H). HRMS [M + H] calcd, 419.171 38;
found, 419.172 44. Anal. (C23H22N4O4) C, H.
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Supporting Information Available: Analytical data for syn-
thesized compounds and data collection and refinement statistics
for X-ray structure of compound 16 bound to KDR. This material
is available free of charge via the Internet at http://pubs.acs.org.
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