Design, synthesis, and evaluation of orally active benzimidazoles and benzoxazoles as vascular endothelial growth factor-2 receptor tyrosine kinase inhibitors

J. Med. Chem. 2007, 50, 4351-4373

4351

Design, Synthesis, and Evaluation of Orally Active Benzimidazoles and Benzoxazoles as

Vascular Endothelial Growth Factor-2 Receptor Tyrosine Kinase Inhibitors

Michele H. Potashman,*^,†James Bready,^‡Angela Coxon,^‡Thomas M. DeMelfi, Jr.,^‡Lucian DiPietro,^†Nicholas Doerr,^‡

Daniel Elbaum,^†Juan Estrada,^‡Paul Gallant,^§Julie Germain,^†Yan Gu,^|Jean-Christophe Harmange,^†Stephen A. Kaufman,

Rick Kendall,^‡Joseph L. Kim,^|Gondi N. Kumar,^#Alexander M. Long,^|Seshadri Neervannan,^OVinod F. Patel,^†

Anthony Polverino,^‡Paul Rose,^|Simon van der Plas,^†Douglas Whittington,^|Roger Zanon,* and Huilin Zhao^|

Department of Medicinal Chemistry, Amgen Inc., One Kendall Square, Building 1000, Cambridge, Massachusetts 02139, and Oncology

Research, Pharmaceutics, and Pharmacokinetic Drug Metabolism, Amgen Inc., One Amgen Center DriVe,

Thousand Oaks, California 91320-1799

ReceiVed January 10, 2007

Inhibition of the VEGF signaling pathway has become a valuable approach in the treatment of cancers.

Guided by X-ray crystallography and molecular modeling, a series of 2-aminobenzimidazoles and

2-aminobenzoxazoles were identified as potent inhibitors of VEGFR-2 (KDR) in both enzymatic and HUVEC

cellular proliferation assays. In this report we describe the synthesis and structure-activity relationship of

a series of 2-aminobenzimidazoles and benzoxazoles, culminating in the identification of benzoxazole 22

as a potent and selective VEGFR-2 inhibitor displaying a good pharmacokinetic profile. Compound 22

demonstrated efficacy in both the murine matrigel model for vascular permeability (79% inhibition observed

at 100 mg/kg) and the rat corneal angiogenesis model (ED₅₀) 16.3 mg/kg).

Introduction

Our independent medicinal chemistry efforts in finding a

selective VEGFR inhibitor led to motesanib diphosphate (AMG

706, 1), currently in phase II clinical trials for the treatment of

various cancers.^10eWe were also interested in identifying a

second generation of KDR inhibitor belonging to a completely

different structural class from the nicotinamide. Urea 2 was

originally described solely as a raf inhibitor, however, we

serendipitously discovered that it inhibited KDR as well, with

a K_iof 2 nM.¹¹As a means to understand the binding mode

and generate a new chemotype for KDR inhibition, we docked

2 into the cocrystal structure of 1 and KDR¹²to identify the

key interactions between this molecule and the protein. One of

the key features of the cocrystal structure of 1 bound KDR is

the reorganization of Phe 1047 (of the Asp 1046, Phe 1047,

Gly 1048 triad, the “DFG” motif) to induce the inactive “DFG-

out” conformation, similar to that observed upon binding of

imatinib to its target kinase Abl.¹³This conformational change

inhibits the ability of the kinase to bind ATP productively and

accommodates the binding of an appropriately substituted

inhibitor into an extended lipophilic pocket. On the basis of

the presumed cis-conformation of the urea moiety and the size

of the terminal aryl group, we reasoned that 2 would also bind

KDR in a DFG-out fashion.

Angiogenesis, the formation of new blood vessels from

existing vasculature, is a normal physiological event that occurs

during embryonic growth, wound healing and the menstrual

cycle. Abnormal regulation of angiogenesis has been implicated

in the pathogenesis of several disorders including diabetic

retinopathy,¹rheumatoid arthritis,²age-related macular degen-

eration,³and cancer.^4,5As angiogenesis is required for tumor

growth and metastasis, the concept of limiting the growth of a

solid tumor by restricting the blood supply has been evaluated

in human cancer and has proven successful.⁶

Vascular endothelial growth factor (VEGF^a) is a known

promoter of angiogenesis, and the increased expression of VEGF

has been implicated in tumor growth and metastasis.^4,5VEGF

signaling through its receptor tyrosine kinase VEGFR-2 (or

KDR, kinase insert domain receptor) promotes several events

required for the formation of new blood vessels, such as

endothelial cell survival, proliferation, migration, and vascular

permeability.

Inhibition of the VEGF signaling pathway has become a

valuable approach in the treatment of cancers. For example,

the use of the neutralizing monoclonal antibody to VEGF,

bevacizumab (Genentech), has demonstrated a prolonged sur-

vival in colorectal cancer patients.⁷The compounds in Figure

1 represent the structural diversity of small molecule, ATP-

competitive, KDR inhibitors under clinical investigation. In

particular, sunitinib (SU-11248)⁸and sorafenib (Bay 43-9006,

2;⁹a dual raf-KDR inhibitor) have recently been approved for

the treatment of cancers.¹⁰

The docking of 2 in the crystal structure of 1 bound to KDR

is depicted in Figure 2. The pyridine ring of 2 is positioned to

overlay with the 4-aminomethylpyridine moiety of the nico-

tinamide to satisfy the critical interaction between the ring

nitrogens and the Cys 919-NH in the kinase hinge region. This

positioning also allows the amide-NH of 2 to engage in a second

hydrogen bond with the backbone amide-CO of Cys 919. The

biaryl-ether linkage projects the substituted phenyl ring into the

lipophilic pocket neighboring the ATP binding site, adjacent to

the gatekeeper residue, Val 916. This portion of 2 overlays well

with the nicotinamide moiety. Further, this model places the

urea carbonyl and its internal NH within hydrogen bond

distances to the backbone of Asp 1046 and side chain of Glu

885, respectively, similar to the amide of 1. The preferred cis-

conformation of the urea places terminal aryl of 2 in a second

* To whom correspondence should be addressed. Phone: (617) 444-

5010. Fax: (617) 577-9822. E-mail: michelep@amgen.com.

^†Department of Medicinal Chemistry.

^‡Department of Cancer Biology.

^§Department of HTS and Molecular Pharmacology.

^|Department of Molecular Structure.

Department of Pathology.

^#Department of Pharmacokinetics and Drug Metabolism.

^ODepartment of Pharmaceutics.

^aAbbreviations: VEGF, vascular endothelial growth factor; KDR, kinase

insert domain receptor; HUVEC, human umbilical vein endothelial cells.

Published on Web 08/15/2007

4352 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18

Potashman et al.

Figure 1. KDR inhibitors under clinical investigation.

Figure 3. Lead compound 3.

3). The cellular activity was assessed by measuring the inhibition

of VEGF-stimulated cellular proliferation of human umbilical

vein endothelial cells (HUVEC). Surprisingly, lead compound

3 exhibited weak cellular activity (IC₅₀g 1140 nM). Guided

by structural information, we varied the three main components

of compound 3 (aniline ring, heterocyclic core, and hinge

binding element) to improve the cellular potency. Herein, we

describe the synthesis, structure-activity relationship, and

pharmacological characterization of this new class of KDR

inhibitors, which culminated in the identification of a potent,

selective, and orally active 2-aminobenzoxazole 22 that dem-

onstrated suppression of capillary formation in a rat-corneal

model of angiogenesis.

Figure 2. Model of urea 2 (yellow) overlaid with cocrystal structure

of nicotinamide 1 (green) and KDR.

lipophilic pocket created by the movement of Phe 1047,

similarly placed as the 3,3-dimethylindoline portion of the

nicotinamide.

Based on the above model, we proposed the cyclization of 2

to the corresponding 2-aminobenzimidazole to enhance con-

formational rigidity while maintaining the key hydrogen-bonding

interactions. Reduced flexibility is expected to improve potency

and oral bioavailablity.¹⁴

Chemistry

The benzo-1,3-azoles described in this paper were prepared

according to the general method represented in Scheme 1.

Benzimidazoles 3-19 were prepared in a two-step, one-pot

protocol by treatment of bis-anilines 39a-i with isothiocyanates

To test this hypothesis, compound 3¹⁵was prepared and found

to be a potent KDR enzyme inhibitor, with a K_iof 9 nM (Figure

Benzimidazoles and Benzoxazoles as VEGFR-2 Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4353

Scheme 1. General Synthesis of 2-Aminobenzimidazoles and

2-Aminobenzoxazoles 3-38^a

Scheme 3. Preparation of N-Methylpyrrolidine-Substituted

Anilines^a

^aReagents: (a) EDC, CH₃CN.

^aReagents and conditions: (a) (S or R)-(-)-1-(tert-butoxycarbonyl)-2-

pyrrolidinemethanol, triphenylphosphine, DIAD, THF; (b) TFA, CH₂Cl₂;

(c) NaBH₃CN, CH₂O, CH₃CN; (d) Pd/C, H₂, MeOH and dioxane/ethyl

acetate or SnCl₂, EtOH.

Scheme 2. Preparation of Substituted Anilines^a

Scheme 4. Synthesis of Thioisocyanates^a

^aReagents: (a) 1,1'-thiocarbonyldiimidazole, CH₃CN.

Scheme 5. Preparation of Phenylene Diamines^a

^aReagents and conditions: (a) neat, 145-170 °C; (b) NEt₃/TFA; (c)

HCl, 145-150 °C; (d) Pd/C, H₂, MeOH and/or ethyl acetate; (e) Zn, AcOH,

THF.

^aReagents and conditions: (a) pyridine‚HCl, 210 °C; (b) HOAc, HBr,

140-180 °C; (c) R-Cl, K₂CO₃, Bu₄N⁺I^-, acetone; (d) R-Cl, K₂CO₃, DMF;

(e) triphenylphosphine, DIAD, R-OH, THF; (f) Pd/C, H₂, ethyl acetate or

MeOH; (g) SnCl₂, EtOH; (h) amine, NaBH(OAc)₃, CH₂Cl₂.

Anilines containing the N-methylpyrrolidine amino group

were prepared using a variation to the chemistry described above

(Scheme 3). Etherification of the phenols 42 with the N-Boc-

pyrrolidinol using Mitsunobu conditions afforded the nitro

intermediates 47 with good yields. Compounds 47 were depro-

tected using TFA and the free amine subjected to reductive

amination conditions to yield compounds 49 in good yields.

Subsequent reduction of the nitro groups to the corresponding

anilines (using either catalytic hydrogenation for the perfluori-

nated compounds or SnCl₂for the halogenated compound)

afforded compounds 50 in excellent yields.

Some arylthioisocyanates were commercially available, how-

ever, the remaining were prepared from condensation of the

corresponding aniline with 1,1′-thiocarbonyldiimidazole (Scheme

4). Intermediates 40 were either purified before use or used crude

in the cyclization sequence (as a 1:2 mixture with imidazole).

The phenylene diamines 39a-h, required for the synthesis

of benzimidazoles 3-18 described in Tables 1-3, were prepared

according to the generic route shown in Scheme 5. The

substitution reactions of 3,4-dinitro phenol with the chlorinated

heterocycles 51 afforded the biarylether 52.¹⁶Subsequent

reduction of the nitro groups using either catalytic hydrogenation

or zinc dust afforded the desired phenylene diamines 39a-h in

good to excellent yields.

40 to afford thiourea intermediates that were directly cyclized

to the desired products employing 1-(3-dimethyl-aminopropyl)-

3-ethylcarbodiimide (EDC). In a sequence similar to that used

to prepare the benzimidazoles, treatment of amino-phenols

39j-t with thioisocyanates 40 and direct cyclization of the urea

intermediates with EDC afforded the benzoxazoles 20-38 in

good yields. The thiourea intermediates were not isolated, but

rather cyclized directly to the desired core structure in situ. This

convergent route allowed for rapid analoging of the 2-aniline

moiety, as each variation could be introduced by simply

employing the corresponding thioisocyanate.

The anilines required for the synthesis of the arylthioisocy-

anates 40 were prepared via one of the methods described in

Schemes 2 and 3. Compounds containing an ether-linked tether

to the amino group were synthesized from the corresponding

nitro anisole 41. Demethylation of 41 using strongly acidic

conditions afforded phenols 42 with excellent yields. Com-

pounds 43 were prepared by either a Mitsunobu etherification

or alkylation of the phenol with generally high yields. Reduction

of the nitro groups using either SnCl₂or catalytic hydrogenation

afforded anilines 44a-d.

Alternatively, anilines 46 containing an all-carbon tether to

the amino group were prepared from 2-chloro-5-nitrobenzal-

dehyde in two steps. Reductive amination of the benzaldehyde

with the appropriate amine afforded intermediate 45. Reduction

of the nitro arenes with SnCl₂-generated anilines 46 in good

yields.

Scheme 6 depicts the synthesis of the N-methylphenylene

diamine 39i, a precursor to N-methylbenzimidazole 19 (Table

4). The substitution reaction of the 4-chloropyridine carboxamide

53 with 4-amino-3-nitro phenol afforded aniline 54. Regiose-

lective monomethylation was achieved in a one-pot procedure

by employing in situ protection of the amine with the trifluo-

roacetate group, followed by methylation with dimethyl sulfate

to afford compound 55 in excellent yield. Reduction of the nitro

4354 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18

Scheme 6. Synthesis of Intermediate 39i^a

Potashman et al.

^aReagents and conditions: (a) KOt-Bu, K₂CO₃, DMSO, rt-110 °C; (b)

(i) TFAA, (ii) TBACl, Me₂SO₄, (iii) NaOH, H₂O; (c) Pd/C, H₂, MeOH/

ethyl acetate.

Scheme 7. Preparation of Aminophenols^a

Figure 4. Crystal structure of 3 bound to KDR. Asp 1046, Phe 1047,

and Gly 1048 (not shown for clarity) constitute the residues of the

“DFG” motif. Potential hydrogen-bond interactions are indicated by

dotted lines.

created by the rearrangement of the protein into the “DFG-out”

conformation. The meta-trifluoromethyl substituent orients

toward a small lipophilic pocket away from the solvent exposed

portion of the protein.

Results and Discussion

All compounds depicted in Tables 1-6 were screened for

inhibition of KDR tyrosine kinase activity with select com-

pounds further assayed in the HUVEC cellular assay. Our initial

work focused on exploring variants to the pyridine carboxamide

moiety of the 2-aminobenzimidazole 3 (Table 1). As shown in

Table 1, removal of the carboxamide group (4 vs 3) led to an

18-fold reduction in enzyme potency, presumably due to the

loss of the hydrogen-bond interaction between the NH of the

carboxamide and the carbonyl of Cys 919. By incorporating

nitrogen-containing heterocycles with a hydrogen-bond donor

and acceptor appropriately spaced to interact with the hinge

greater potency was realized. For example, quinazoline 5 (with

the proton of C8 engaged in a hydrogen bond with the carbonyl

of Cys 919)¹⁷and azaindole-substituted 6 showed improved

enzyme potency by 20-fold and 3-fold, respectively, compared

to pyridine 4. Compound 5 displayed improved potency in the

cellular assay (IC₅₀) 49 nM), however, this was insufficient

for advancement of this compound. Although 3 and 6 exhibited

modest to good enzyme potencies, they lacked the desired

activity in the HUVEC cellular proliferation assay.

In an effort to improve the solubility and potentially improve

cellular potency of the 2-aminobenzimidazoles, analogs contain-

ing a basic amine were investigated (Table 2). Guided by the

structural information, we attached the amine from the 3-position

of the phenyl ring with a 2- to 4-atom tether to direct the group

toward bulk solvent. As exemplified by compound 7, no

significant change was observed at the enzyme level compared

to parent compound 3, however, the enzyme to cellular shift

was 36-fold, resulting in a cellular potency of 146 nM. As shown

by compounds 7-10, varying the amino moiety (e.g., acyclic

vs cyclic), the tether linkage (e.g., O-linked vs C-linked), and

the group at the 4-position (e.g., Cl vs CF₃, vs C₂H₅) resulted

in relatively small differences in the cellular potencies, with

enzyme-to-cell shifts ranging from 10- to 40-fold. Other aniline

substitution patterns (i.e., 3,5-substituted, 11) proved suboptimal

for potency.

^aReagents and conditions: (a) NaH, DMF, rt-85 °C; (b) NEt₃/TFA,

150 °C; (c) Pd/C, H₂, MeOH, ethyl acetate; (d) TFA, heat; (e) HNO₃, AcOH;

(f) Fe, HCl, EtOH; (g) Zn, AcOH, THF; (h) SnCl₂, EtOH.

group of 55, achieved using SnCl₂, then yielded the desired

monomethylated bisaniline 39i.

The amino phenol moieties required for the synthesis of

2-aminobenzoxazoles 20-38 described in Tables 4-6 were

prepared according to the method described in Scheme 7.

Chlorinated nitrogen-containing heterocycles 51 were treated

with the preformed sodium phenolate of a 4-benzyloxyphenol

to yield biarylethers 57 in generally good yields. Debenzylation

of 57 was accomplished using either catalytic hydrogenation

or trifluoroacetic acid (TFA; depending on the substrate) to give

corresponding phenols 58. The benzyl groups of compounds

57c and 57d (R₁) Cl; heterocycle ) quinoline and pyridine

carboxamide, respectively) were removed under the TFA

conditions. Regioselective nitration of phenols 58 using nitric

acid afforded compounds 59 in good yields. Reduction of the

nitro groups yielding amino phenols 39j-t proceeded in

generally excellent yields.

Structural Information on Lead Compound 3. A crystal

structure of 3 bound to KDR (Figure 4) confirmed the proposed

binding mode and allowed us to use structural information to

guide our medicinal chemistry efforts. As shown in Figure 4,

compound 3 binds largely as predicted based on our model of

compound 2 within the AMG 706/KDR structure. Hydrogen

bonds between the hinge-region Cys 919 are observed with both

the nitrogen of the pyridine and the carboxamide-NH. The

benzimidazole core is oriented in the hydrophobic pocket

flanked by Val 917. The endocyclic nitrogen of the benzimi-

dazole hydrogen bonds with the NH of Asp 1046 and either

the exocyclic or the endocyclic NH may interact with the side

chain of Glu 885. Also consistent with the modeling studies,

the aniline aryl ring occupies the second hydrophobic pocket

Benzimidazoles and Benzoxazoles as VEGFR-2 Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4355

Table 3. Further Investigation of Hinge-Binding Moiety^a

Table 1. Investigation of the Hinge-Binding Moiety^a

^aEnzyme binding and cellular IC₅₀data are determined by one single

experiment. See Experimental Section for a description of the assay

conditions. ^bHuman umbilical vein endothelial cells.

Table 2. Investigation of Amino Group to Improve Cellular Potency^a

^aEnzyme binding and cellular IC₅₀data are determined by one single

experiment, except where noted. See Experimental Section for a description

of the assay conditions. ^bHuman umbilical vein endothelial cells. ^cN ) 2

for IC₅₀determination (32.4 nM ( 5.4 nM). ^dN ) 2 for IC₅₀determination

(16.6 nM ( 4.6 nM). ^en.d.) not determined.

Table 4. Investigation of the 1,3-Azole Core^a

K_{i phos}

(nM)

V-HUVEC^b

IC₅₀(nM)

cmpd

X

7

19

20

NH

N-Me

O

4

10

4

146

31

36

^aEnzyme binding and cellular IC₅₀data are determined by one single

experiment. See Experimental Section for a description of the assay

conditions. ^bHuman umbilical vein endothelial cells.

improved potency observed with quinoline derivatives 12-14

reflected a slight decrease in enzyme-to-cell shift. Modifying

either the bicyclic pyrollopyrimidine 15 or the azaindole 16 to

the monocylic 2-aminopyridine 17 resulted in a loss of potency

at the enzyme and cellular level. Although this is consistent

with data in other series (ex: benzoxazole series (vide infra)

and unpublished results), it could not be rationalized by

structural information. As anticipated based on earlier studies,

removal of the hydrogen-bond donor to the hinge region resulted

in a 3-fold decline in enzyme potency (17 vs 18). The

2-aminobenzimidazole 14, containing a 6,7-dimethoxyquinoline

moiety, displayed the best overall potency with a 10-fold

increase in enzyme affinity (K_i) 0.2 nM) compared to the

^aEnzyme binding and cellular IC₅₀data are determined by one single

experiment, except where noted. See Experimental Section for a description

of the assay conditions. ^bHuman umbilical vein endothelial cells. ^cN ) 2

for enzyme binding (3.9 nM ( 3.0 nM). ^dn.d.) not determined.

Encouraged by the improved cellular potency of compounds

7-10 compared to the parent compound 3, we investigated the

effect of substituting the pyridine carboxamide with other

nitrogen-containing heterocycles (Table 3). Consistent with

previous observations (i.e., quinazoline 5, Table 1), compounds

12-14 containing the quinoline moiety were more potent at

the cellular levels than the analogs bearing a pyrrolopyrimidine

(15), an azaindole (16), or a 2-aminopyrimidine (17). The

4356 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18

Potashman et al.

Table 5. Investigation of Pendant Tertiary Amines on the Aniline

Table 6. Benzoxazoles: Investigation of the Hinge-Binding Element^a

Moiety^a

^aEnzyme binding and cellular IC₅₀data are determined by one single

experiment, except where noted. See Experimental Section for a description

of the assay conditions. ^bHuman umbilical vein endothelial cells. ^cN ) 2

for K_idetermination (2.8 nM ( 1.6 nM). ^dN ) 3 for IC₅₀determination

(15.3 nM ( 5.2 nM).

unsubstituted quinoline 13 (K_i) 2 nM). This improved enzyme

potency could result from an increased donor character of the

C8-hydrogen of the 6,7-dimethoxyquinoline moiety binding the

hinge region. In addition, compound 14 displayed an IC₅₀of

17 nM in the HUVEC assay. Although cellular efficacy was

greatly improved by adding a tertiary amino group and varying

the nitrogen-containing heterocycle, the shift between enzyme

potency and cellular potency was difficult to predict and often

greater than 10-fold.

The crystal structure of 3 bound to KDR illustrates the

hydrogen bond between either the exocyclic or the endocyclic

NH of the benzimidazole and the side chain of Glu 885. To

evaluate the possibility of removing the endocyclic hydrogen-

bond donor, we methylated the benzimidazole core. Indeed, the

N-methylbenzimidazole 19 provided a K_iof 10 nM on phos-

phorylated KDR and an IC₅₀of 31 nM in the cellular assay,

exhibiting only a 3-fold enzyme-to-cell shift (compared to the

36-fold shift observed with the parent compound 7; Table 4).

This result indicates that a possible hydrogen bond between the

endocyclic NH and the Glu 885 can be removed due to an

interaction between Glu 885 and the exocyclic NH. Encouraged

by this data, we altered the aminobenzimidazole core to a

2-aminobenzoxazole, as exemplified by compound 20. Com-

pound 20 displayed good potency at the enzyme (K_i) 4 nM)

and cellular (IC₅₀) 36 nM) levels. Based on this finding, we

elected to investigate other structural variants in the 2-ami-

nobenzoxazole series.

^aEnzyme binding and cellular IC₅₀data are determined by one single

experiment, except where noted. See Experimental Section for a description

of the assay conditions. ^bHuman umbilical vein endothelial cells. ^cN ) 2

for K_idetermination (0.5 nM ( 0.1 nM). ^dN ) 2 for K_idetermination (3.6

nM ( 2.3 nM). ^eN ) 2 for K_idetermination (1.6 nM ( 0.5 nM).

potency (Table 5). For example, simply substituted 21 exhibited

a K_iof 12 nM in the enzyme assay, but displayed an IC₅₀of

766 nM in the cellular assay. Appendage of a basic amine (20)

improved the enzyme potency 3-fold (K_i) 4 nM), with a 20-

fold improvement in cellular potency (IC₅₀) 36 nM). A brief

examination of tertiary amine substituents indicated a variety

of groups are tolerated (22-26). In particular, compounds

containing an N-methylpyrrolidine (22), an N,N-diethylamino-

ethyl (24), or an N-methylpiperazine (20) substituent are

optimum for achieving good cellular potency.

To further improve the cellular potency, we also explored

the hinge binding moiety (Table 6). As shown previously, when

combined with the appropriate tertiary amine pendant, com-

pounds containing a pyridine carboxamide were potent KDR

inhibitors (compounds 20, 22, and 24 in Table 5 and compound

27 in Table 6). Similar to the benzimidazole series, compounds

28-30 illustrate that a dimethoxyquinoline and quinazoline

heterocycles were optimal. Although removal of the 6- and

The overall structure-activity relationship of the 2-ami-

nobenzoxazoles followed a similar pattern as that of the

corresponding 2-aminobenzimidazoles (Tables 5 and 6). As

observed in the 2-aminobenzimidazole series, addition of a

pendant basic amine was preferred to achieve good cellular

Benzimidazoles and Benzoxazoles as VEGFR-2 Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4357

Table 7. Investigation of Substitution on Benzoxazole Core^a

Figure 5. Surface diagram of compound 3 bound to KDR. The

7-position is indicated by the arrow.

therefore prepared the 7-fluoro and 7-chloro derivates. Surpris-

ingly, as shown by compounds 36-38 (Table 7), substitution

at the 7-position of the benzoxazole did not yield any significant

improvement in enzyme or cellular potencies compared to the

corresponding unsubstituted analogs.

^aEnzyme binding and cellular IC₅₀data are determined by one single

experiment. See Experimental Section for a description of the assay

conditions. ^bHuman umbilical vein endothelial cells.

7-methoxy groups resulted in an 8-fold drop in enzyme potency,

the cellular activities were comparable (30 vs 31). As observed

in the benzimidazole series, compounds with an amino pyri-

midine hinge binding moiety exemplified exhibited a loss in

cellular activity (32 and 33). Finally, good cellular potency can

also be achieved with an azaindole binding the hinge and an

appropriately substituted aniline (compound 34).

In an attempt to improve the enzyme potency further, we

returned to the crystal structure. Based on the structure of 3

with KDR, we noted a small to medium sized lipophilic pocket

that could be accessed by placing a substituent at the 7-position

of the bicyclic core (Figure 5). We proposed that placement of

a substituent at this position could increase potency. We

Pharmacokinetic Studies

Several 2-aminoazoles with suitable inhibitory activity in the

kinase enzyme and cellular proliferation assays were evaluated

for their in vivo pharmacokinetics in male Sprague-Dawley

rats (Table 8). Compounds were administered by both intrave-

nous (i.v.) and oral (p.o.) dosing routes. Compounds 12 and 34

displayed reasonable clearance values (Cl ) 1.0 and 1.24 L/h/

kg, respectively), but had a low oral bioavailablity (24 and 17

%F, respectively). Dimethoxyquinazoline (28), dimethoxyquin-

oline (30), and pyridine carboxamide (22) had improved

bioavailability (40-67 %F). In general, the 2-aminoazoles tested

exhibited moderate to high Cl and a high volume of distribution

Table 8. PK Studies of Leading Compounds^a

^aIV dosing at 1 mpk (DMSO), with n ) 3 male Sprague-Dawley rats. Oral dosing at 10 mpk (Ora-Plus, pH 2), with n ) 2 rats. ^bHuman umbilical vein

endothelial cells. ^cn ) 2 rats. ^dOral dosing at 3 mpk.

4358 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18

Potashman et al.

Table 9. Cellular Selectivity Profile of Selected Compounds in VEGF-

(16-fold and 6-fold, respectively). Based on its favorable

selectivity profile in both the enzyme and the cellular assays

and pharmacokinetics, compound 22 was further examined in

our animal efficacy models.

and FGF-Induced Cellular Proliferation^a

V-HUVEC^b

IC₅₀(nM)

F-HUVEC^b

IC₅₀(nM)

cmpd

22

28

30

15^c

6

10

420^d

55

101

Animal Efficacy Models. Vascular Permeability. The in

vivo activity of compound 22 on VEGF-induced vascular

permeability was assessed in a modified Miles assay¹⁸(Figure

6). For this assay, HEK 293 cells, transfected with murine VEGF

or vector control were mixed with Matrigel and injected

subcutaneously into CD-1 Nu/Nu mice. Compound 22 was

administered orally 22 h after injection of the cells at 10, 30,

and 100 mg/kg doses. Six hours after administration of

compound, vascular permeability in the skin overlying the

Matrigel plug was measured by the quantization of extravasated

Evan’s blue dye. In mice dosed with 22, linear pharmacokinetics

was observed. Although the responses at 10 and 30 mg/kg were

not statistically significant, a strong inhibition of vascular

permeability at 100 mg/kg (79%) was observed.

Angiogenesis. To evaluate the in vivo VEGF-mediated anti-

angiogenic activity of compound 22, a rat corneal model of

angiogenesis was performed (Figure 7). A disk infused with

rHu-VEGF was placed in the rat cornea to stimulate blood vessel

formation from the limbal vessels of the eye toward the disk.

The rats were then treated with 22 at doses of 1, 3, 10, and 30

mg/kg once a day orally. Treatment began on the day of

implantation and continued for 7 days. Two vascular endpoints

were evaluated: the number of blood vessels at the midpoint

between the limbus and the disk and the mean blood vessel

area. Benzoxazole 22 significantly reduced both vascular

endpoints in a dose-concentration-dependent manner, with an

estimated ED₅₀of 16.3 mg/kg. Terminal PK analysis estimated

the AUC_{(0-24 h)}at the ED₅₀as 17.03 µM‚h.

^aCellular IC₅₀data are determined by one single experiment, except

where noted. See Experimental Section for a description of the assay

conditions. ^bHuman umbilical vein endothelial cells. ^cN ) 3 for IC₅₀

determination (15.3 nM ( 5.2 nM). ^dN ) 3 for IC₅₀determination (420

nM ( 100 nM).

after i.v. dosing. Compounds 22, 28, and 30 all exhibited

acceptable half-lives and oral bioavailability for further in vivo

evaluation.

Selectivity. To evaluate compounds in an in vivo setting, it

was critical to understand the kinase selectivity profile. To

measure the selectivity of this series in a cellular assay,

compounds 22, 28, and 30 were evaluated in a fibroblast growth

factor (b-FGF)-driven HUVEC proliferation assay (Table 9).

While compounds 28 and 30 displayed moderate selectivity (10-

fold) for VEGF-driven cellular proliferation, compound 22

exhibited the best selectivity (28-fold) over FGF-induced

HUVEC proliferation (IC_50(V-HUVEC) ) 15 nM, IC_50(F-HUVEC)

) 420 nM).

Compound 22 was further profiled against an extended panel

of kinase enzymes, including receptor tyrosine kinases and

serine/threonine kinases. As summarized in Table 10, compound

22 exhibited excellent enzymatic selectivity. Within the protein

tyrosine kinase family, 22 showed excellent levels of selectivity

(>100-fold) over Tie-2, FGF, Src, IGFR-1, and EGFR. Similar

selectivity was seen over kinases in the serine/threonine group

(MAP, GSK3-b, CDK). As expected, due to the close homology

of KDR to cFMS and cKit, only modest selectivity was observed

Crystal Structure of 22 Bound to KDR. To confirm the

binding mode of 22, the crystal structure of the benzoxazole

Table 10. Selectivity Profile of 22^a

enzyme inhibition IC₅₀(µM)

PTK group X

PTK group I

Src ) 0.72

LCK ) 1.14

PTK group VI

Zap-70 > 25

CMGC group I

CDK5p25 > 40

CDK1 > 40

EGFR > 25

PTK group XIII

Tie-2 ) 3.18

PTK group XIV

KDR ) 0.005^b

cFMS ) 0.08

cKIT ) 0.03

PTK group XV

FGF ) 4.37

CMGC group II

p38R > 40

p38â > 40

JNK1 > 40

JNK2 > 40

JNK3 > 40

OPK group VII

E2K2 > 40

PTK group XVI

IGFR-1 ) 4.01

PTK group XXI

CMGC group III

cMet ) 13.7

GSK3-b > 40

^aIC₅₀data are determined by one single experiment. See Experimental Section for a description of the assay conditions. ^bN ) 2 for IC₅₀determination

(4.6 nM ( 2.8 nM).

Figure 6. Effect of 22 on VEGF-induced vascular permeability in mice. ([) Indicates drug concentration.

Benzimidazoles and Benzoxazoles as VEGFR-2 Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4359

Figure 7. Inhibition of VEGF-induced angiogenesis in rats by 22. ([) Indicates drug concentration.

upon oral dosing in both the mouse Matrigel and the rat corneal

models. Although compound 22 is less efficacious in the rat

corneal model compared to motesanib,¹⁹it represents one step

toward identification of a novel series of potent KDR inhibitors

with strong antitumor activity in vivo. Reports detailing the

discovery of these compounds will be published hereafter.

Experimental Section

General Considerations. Unless otherwise noted, all materials

were obtained from commercial suppliers and used without further

purification. Anhydrous solvents were obtained from Aldrich and

used directly. All reactions involving air- or moisture-sensitive

reagents were performed under a nitrogen or argon atmosphere.

All final compounds were purified to >95% purity, as determined

by high-performance liquid chromatography (HPLC: A, Zorbax

SB-C8, 4.6 × 150 mm, 15 min; flow rate ) 1.5 mL/min; gradient

) 0 to 100% 0.1% TFA in CH₃CN/100 to 0% 0.1% TFA in water;

B, Zorbax SB-C8, 4.6 × 150 mm, 12 min; flow rate ) 1.5 mL/

min; gradient ) 10 to 90% 0.1% formic acid in CH₃CN/90 to 10%

0.1% formic acid in water; C, Phenomenex Synergi, 2 × 50 mm,

3 min, flow rate ) 1.0 mL/min; gradient ) 5 to 95% 0.1% formic

acid in CH₃CN/95 to 5% 0.1% formic acid in water). Silica gel

chromatography was performed using either glass columns packed

with silica gel (230-400 mesh), prepacked silica gel cartridges (Isco

or Biotage), or preparative thin-layer chromatography plates (An-

altech, 1000 microns). NMR spectra were determined using a Varian

300 or 400 MHz spectrometer. Preparative reverse-phase HPLC

was performed using Gilson 306 gradient elution pump, YMC ODS-

AQ reverse-phase silica gel column and a Gilson variable wave-

length UV/vis-155 detector. High-resolution mass spectrometry

(HRMS) was performed using an Agilent MSD-TOF mass spec-

trometer equipped with an ESI dual channel source. The instrument

was calibrated using Agilent ESI-MS tuning mixture (ES Tuning

Mix PN G2421A). The tuning mixture was continuously infused

on the second ESI channel, and the 622.02895 Da ion was used to

provide a lock-mass correction to the calibration function. All

samples were diluted to an appropriate concentration so that the

number of counts per scan was less that 10e5 to prevent dead-time

distortion of the measurement.

Figure 8. Crystal structure of 22 bound to KDR.

complexed with KDR was obtained. As shown in Figure 8,

compound 22 binds largely as predicted based on our structure

of compound 3 bound to KDR. Hydrogen bonds between the

hinge-region Cys 919 are observed with both the nitrogen of

the pyridine and the carboxamide-NH. The benzoxazole core

is oriented in the hydrophobic pocket flanked by Val 917. The

endocyclic nitrogen of the aminobenzoxazole hydrogen bonds

with the NH of Asp 1046 and the exocyclic NH picks up a key

interaction with side chain of Glu 885. The aniline aryl ring

occupies the second hydrophobic pocket with the N-methyl

pyrrolidine side chain directed to a solvent-exposed portion of

the protein.

Conclusion

Guided by X-ray crystallography and molecular modeling,

we identified a new class of KDR inhibitors: 2-aminobenzimi-

dazoles. While the 2-aminobenzimidazoles were potent enzyme

inhibitors, achieving good cellular potency remained a challenge.

A detailed understanding of the key interactions between the

ligand and the receptor prompted the replacement of the

2-aminobenzimidazole core by a 2-aminobenzoxazole. Optimi-

zation of the hinge binding heterocycle as well as the aryl ring

occupying the second hydrophobic pocket resulted in several

benzoxazoles with good enzyme and cellular potencies. Among

them, compound 22 emerged as the best candidate for in vivo

evaluation. Compound 22 showed on mechanism in vivo activity

4-((2-((4-Chloro-3-(trifluoromethyl)phenyl)amino)-1H-benz-

imidazol-5-yl)oxy)-N-methyl-2-pyridinecarboxamide (3). To a

solution of 4-(3,4-diamino-phenoxy)-pyridine-2-carboxylic acid

methylamide (0.07 g, 0.27 mmol) in CH₃CN (20 mL) was added

dropwise, over 5 min, a solution of 1-chloro-4-isothiocyanato-2-

trifluoromethyl-benzene (0.055 g, 0.27 mmol) in CH₃CN (10 mL).

The reaction was stirred 18 h at rt. The reaction was diluted with

additional CH₃CN (10 mL), followed by addition of EDC (0.078

g, 0.41 mmol). The reaction was heated at 80 °C for 3 h, allowed

to cool to rt, and concentrated in vacuo. The crude mixture was

dissolved in EtOAc and water. The layers were separated, and the

4360 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18

Potashman et al.

organic layer was washed with brine, dried over Na₂SO₄, filtered,

and concentrated in vacuo. The residue was purified by silica gel

column chromatography using a hexane-EtOAc gradient to yield

the title compound as a white solid (74 mg, 59%). ¹H NMR

indicates a mixture of tautomers (400 MHz, DMSO-d₆) δ 11.42

(s, 0.5H), 11.37 (s, 0.5H), 10.13 (s, 0.5H), 10.09 (s, 0.5H), 8.70-

8.80 (m, 1H), 8.45-8.48 (m, 1H), 8.31-8.32 (m, 1H), 8.10-8.14

(m, 1H), 7.62-7.65 (m, 1H), 7.46 (d, J ) 8.4, Hz, 0.5H), 7.30-

7.40 (m, 2.5H), 7.10-7.14 (m, 1H), 6.84-6.88 (m, 1H), 2.75 (d,

J ) 1.6 Hz, 1.5H), 2.74 (d, J ) 2.0 Hz, 1.5H); HRMS calcd for

C₂₁H₁₅ClF₃N₅O₂(M + H)⁺, 462.0939; found, 462.0943; HPLC

purity ) 98% (system A), 98% (system B).

0.5H), 7.05-7.10 (m, 0.5H), 6.85-6.90 (m, 1H), 6.35 (d, J ) 5.8

Hz, 0.5H), 6.30 (d, J ) 5.8 Hz, 0.5H), 6.15-6.20 (m, 1H); Anal.

(C₂₁H₁₃ClF₃N₅O‚0.33 CH₃OH) C, H, N.

4-{2-[4-Chloro-3-(4-methyl-piperazin-1-ylmethyl)-phenylami-

no]-1H-benzimidazol-5-yloxy}-pyridine-2-carboxylic Acid Me-

thylamide (7). To a cooled (0 °C) solution of 1-(2-chloro-5-amino-

benzyl)-4-methyl-piperazine (252 mg, 1.05 mmol) in CH₃CN (10

mL) was added 1,1′-thiocarbonyldiimidazole (225 mg, 1.26 mmol).

The reaction mixture was allowed to warm to rt and stirred for 18

h. The mixture was added dropwise to a solution of 4-(3,4-diamino-

phenoxy)-pyridine-2-carboxylic acid methylamide (271 mg, 1.05

mmol) in CH₃CN (20 mL) at rt and stirred 18 h. The reaction

mixture was further diluted with CH₃CN (15 mL), then EDC (0.20

g, 1.05 mmol) was added, and the vessel was heated at 80 °C for

3 h. The mixture was allowed to cool to rt, concentrated in vacuo,

and the residue was dissolved in EtOAc and water. The layers were

separated, and the organic layer was washed with brine, dried over

Na₂SO₄, filtered, and concentrated in vacuo. The mixture was

purified by a combination of silica gel column chromatography and

silica gel prep plates to obtain the title compound (231 mg, 43%).

¹H NMR indicates a mixture of tautomers (400 MHz, DMSO-d₆)

δ 11.11 (s, 0.5H), 11.05 (s, 0.5H), 9.74 (s, 0.5H), 9.69 (s, 0.5H),

8.70-8.80 (m, 1H), 8.45-8.50 (m, 1H), 7.85-7.90 (m, 1H), 7.70-

7.75 (m, 1H), 7.30-7.45 (m, 3H), 7.10-7.15 (m, 2H), 6.80-6.90

(m, 1H), 3.50-3.54 (m, 2H), 2.75 (s, 1.5H), 2.74 (s, 1.5H), 2.30-

2.50 (m, 8H), 2.17 (s, 1.5H), 2.13 (s, 1.5H); Anal. (C₂₆H₂₈-

ClN₇O₂‚H₂O‚0.5CH₃OH) C, H, N.

4-((2-((3-((2-(Dimethylamino)ethyl)oxy)-4-(trifluoromethyl)-

phenyl)amino)-1H-benzimidazol-5-yl)oxy)-N-methyl-2-pyridin-

ecarboxamide (8). Starting from 4-(3,4-diamino-phenoxy)-pyridine-

2-carboxylic acid methylamide (217 mg, 0.84 mmol) and [2-(5-

isothiocyanato-2-trifluoromethyl-phenoxy)-ethyl]-dimethyl-amine

(prepared following the procedure outlined for 40d from 3-(2-

dimethylamino-ethoxy)-4-trifluoromethylaniline (209 mg, 0.84

mmol) and used crude), 210 mg (48%) of the title compound was

obtained as a tan solid according to the method described for the

synthesis of 3. ¹H NMR indicates a mixture of tautomers (400 MHz,

DMSO-d₆) δ 11.33 (s, 0.5H), 11.27 (s, 0.5H), 10.04 (s, 0.5H), 9.98

(s, 0.5H), 8.75-8.76 (m, 1H), 8.46-8.47 (m, 1H), 7.68-7.70 (m,

1H), 7.32-7.52 (m, 4H), 7.13-7.20 (m, 2H), 6.84-6.87 (m, 1H),

4.18-4.21 (m, 2H), 2.75 (d, J ) 4.8 Hz, 3H), 2.50-2.54 (m, 2H),

2.20-2.30 (m, 6H); Anal. (C₂₅H₂₅F₃N₆O₃‚0.66 H₂O‚CH₃OH) C,

H, N.

(4-Chloro-3-trifluoromethyl-phenyl)-[5-(pyridin-4-yloxy)-1H-

benzimidazol-2-yl]-amine (4). Starting with 4-(3,4-diamino-phe-

noxy)-pyridine (273 mg, 1.36 mmol), 228 mg (42%) of the title

compound was obtained as a white solid according to the method

1

described for the synthesis of 3. H NMR indicates a mixture of

tautomers (400 MHz, DMSO-d₆) δ 11.37 (s, 0.5H), 11.32 (s, 0.5H),

10.10 (s, 0.5H), 10.06 (s, 0.5H), 8.38-8.45 (m, 2H), 8.30-8.35

(m, 1H), 8.11 (d, J ) 8.8 Hz, 1H), 7.64 (dd, J ) 8.8, 3.2 Hz, 1H),

7.44 (d, J ) 8.4 Hz, 0.5H), 7.35 (d, J ) 8.4 Hz, 0.5 H), 7.18 (d,

J ) 2.0 Hz, 0.5H), 7.08 (d, J ) 2.0 Hz, 0.5H), 6.80-6.90 (m,

3H); HRMS calcd for C₁₉H₁₂ClF₃N₄O (M + H)⁺, 405.0725; found,

405.0725; HPLC purity ) 100% (system B), 100% (system C).

(4-Chloro-phenyl)-[5-(6,7-dimethoxy-quinazolin-4-yloxy)-1H-

benzoimidazol-2-yl]-amine (5). A solution of 4-amino-3-nitro-

phenol (0.80 g, 5.34 mmol, 1.2 equiv) in DMSO (3.80 mL) was

treated with KOt-Bu (0.60 g, 5.34 mmol) and the mixture was

stirred at rt for 2 h. To this solution, 4-chloro-6,7-dimethox-

yquinazoline (1.00 g, 4.45 mmol) and K₂CO₃(0.33 g, 2.4 mmol)

were added, and the mixture was heated at 110 °C for 16 h. The

mixture was allowed to cool to rt, diluted with EtOAc, and washed

with NaHCO₃(satd). To remove the emulsion, the mixture was

filtered through Celite, and then the organic layer was washed with

brine, 1 N NaOH, and then brine again. The organic portion was

dried with Na₂SO₄, filtered, and evaporated to give 4-(6,7-

dimethoxyquinazolin-4-yloxy)-2-nitro-phenylamine as an orange

solid (1.0 g, 65%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.54 (s, 1H),

7.86 (d, J ) 3.0 Hz, 1H), 7.50-7.55 (m, 3H), 7.44 (dd, J ) 9.1,

2.5 Hz, 1H), 7.36 (s, 1H), 7.10 (d, J ) 9.1 Hz, 1H), 3.96 (s, 3H),

3.94 (s, 3H).

A flask was charged with 4-(6,7-dimethoxyquinazolin-4-yloxy)-

2-nitro-phenylamine (1.00 g, 2.90 mmol) and the solid was

dissolved in a mixture of EtOH (200 mL) and glacial acetic acid

(10 mL) and placed under nitrogen. Pd/C was added, the reaction

mixture was blanketed with H₂, and the mixture was shaken under

H₂for 18 h at 55 psi. The catalyst was removed by filtration through

Celite and the solution was concentrated in vacuo. The residue was

dissolved in MeOH/water, NH₄OH was added to adjust to pH 10,

and the solvent was evaporated. Partial purification of the residue

by column chromatography using a gradient of 0-100% of a 90:

10:1 CH₂Cl₂/MeOH/NH₄OH eluent afforded 4-(6,7-dimethoxy-

quinazolin-4-yloxy)-benzene-1,2-diamine (impure). A portion of this

(60 mg, est 1.44 mmol) was subjected to the conditions used to

prepare 3 to yield the title compound as a yellow solid (22 mg,

34%). ¹H NMR indicates a mixture of tautomers (400 MHz, DMSO-

d₆) δ 11.05-11.15 (m, 1H), 9.71 (s, 0.5H), 9.66 (s, 0.5H), 8.48-

8.55 (m, 1H), 7.75-7.85 (m, 2H), 7.57 (s, 1H), 7.15-7.40 (m,

5H), 6.80-6.95 (m, 1H), 3.97 (s, 6H); HRMS calcd for C₂₃H₁₈-

ClN₅O₃(M + H)⁺, 448.1170; found, 448.1169; HPLC purity )

100% (system A), 99% (system B).

N-Methyl-4-((2-((3-((((2R)-1-methyl-2-pyrrolidinyl)methyl)-

oxy)-4-(trifluoromethyl)phenyl)amino)-1H-benzimidazol-5-yl)-

oxy)-2-pyridinecarboxamide (9). Starting from 4-(3,4-diamino-

phenoxy)-pyridine-2-carboxylic acid methylamide (83 mg, 0.32

mmol), 109 mg (63%) of the title compound was obtained according

1

to the method described for the synthesis of 3. H NMR indicates

a mixture of tautomers (400 MHz, DMSO-d₆) δ 11.35 (s, 0.5 H),

11.28 (s, 0.5H), 10.05 (s, 0.5H), 9.99 (s, 0.5H), 8.75 (s, 1H), 8.45-

8.47 (m, 1H), 7.69 (s, 1H), 7.30-7.50 (m, 4H), 7.10-7.25 (m,

2H), 6.80-6.90 (m, 1H), 4.05-4.15 (m, 1H), 3.85-3.95 (m, 1H),

2.90-3.00 (m, 1H), 2.70-2.75 (m, 3H), 2.60-2.70 (m, 1H), 2.35-

2.45 (m, 3H), 2.15-2.25 (m, 1H), 1.90-2.00 (m, 1H), 1.55-1.75

(m, 3H); Anal. (C₂₇H₂₇F₃N₆O₃‚1.5 H₂O) C, H, N.

(R)-4-{2-[3-(1-Methyl-pyrrolidin-2-ylmethoxy)-4-pentafluoro-

ethyl-phenylamino]-1H-benzimidazol-5-yloxy}-pyridine-2-car-

boxylic Acid Methylamide (10). To a cooled (0 °C) solution of

(R)-1-methyl-2-(5-amino-2-pentafluoroethyl-phenoxymethyl)-pyr-

rolidine (312 mg, 0.96 mmol) in CH₂Cl₂(3 mL) was added 1,1′-

thiocarbonyldiimidazole (180 mg, 1.00 mmol). The reaction was

allowed to warm to rt and stirred for 18 h. The mixture was

concentrated in vacuo, the residue was dissolved in CH₃CN (7 mL),

and 4-(3,4-diamino-phenoxy)-pyridine-2-carboxylic acid methyla-

mide (312 mg, 0.96 mmol) was added. The reaction mixture stirred

at rt an additional 16 h, followed by the addition of EDC (276 mg,

1.44 mmol) and CH₃CH (20 mL). The mixture was heated at

80 °C for 3 h, allowed to cool to rt, and concentrated in vacuo.

The residue was taken up into CH₂Cl₂, washed with water and brine,

dried with Na₂SO₄, filtered, and evaporated. The crude material

(4-Chloro-3-trifluoromethyl-phenyl)-[6-(1H-pyrrolo[2,3-b]py-

ridin-4-yloxy)-1H-benzimidazol-2-yl]-amine (6). Starting with

4-(1H-pyrrolo[2,3-b]pyridin-4-yloxy)-benzene-1,2-diamine (82 mg,

0.34 mmol), 58 mg (39%) of the title compound was obtained

according to the method described for the synthesis of 3. ¹H NMR

indicates a mixture of tautomers (400 MHz, DMSO-d₆) δ 11.60-

11.65 (m, 1H), 11.30 (s, 0.5H), 11.20 (s, 0.5H), 10.10 (s, 0.5H),

10.00 (s, 0.5H), 8.28-8.35 (m, 1H), 8.10 (d, J ) 7.7 Hz, 1H),

7.95-8.05 (m, 1H), 7.60-7.65 (m, 1H), 7.42 (d, J ) 7.7 Hz, 0.5H),

7.35 (d, J ) 7.7 Hz, 0.5H), 7.28-7.30 (m, 1H), 7.15-7.20 (m,

Benzimidazoles and Benzoxazoles as VEGFR-2 Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4361

was purified by column chromatography using 0-50% of a 90:

10:1 CH₂Cl₂/MeOH/NH₄OH solution as the eluent to yield the title

compound as an off-white solid (170 mg, 29%). ¹H NMR indicates

a mixture of tautomers (400 MHz, DMSO-d₆) δ 11.25-11.40 (m,

1H), 9.95-10.05 (m, 1H), 8.70-8.80 (m, 1H), 8.46 (d, J ) 5.8

Hz, 1H), 7.71 (s, 1H), 7.30-7.50 (m, 4H), 7.10-7.20 (m, 2H),

6.80-6.90 (m, 1H), 3.80-3.90 (m, 1H), 2.90-3.00 (m, 1H), 2.76

(d, J ) 4.8 Hz, 3H), 2.55-2.60 (m, 1H), 2.38 (s, 1.5H), 2.35 (s,

1.5H), 2.15-2.25 (m, 1H), 1.90-2.05 (m, 1H), 1.55-1.70 (m, 3H).

Anal. (C₂₈H₂₇F₅N₆O₃‚CH₃OH‚0.50H₂O) C, H, N.

(m, 8H), 2.35 (s, 3H); HRMS calcd for C₃₀H₃₁ClN₆O₃(M + H)⁺,

559.2219; found, 559.2223; HPLC purity 99% (system A), 96%

(system B).

[4-Chloro-3-(4-methyl-piperazin-1-ylmethyl)-phenyl]-[6-(7H-

pyrrolo[2,3-d]pyrimidin-4-yloxy)-1H-benzimidazol-2-yl]-

amine (15). Starting with 4-(7H-pyrrolo[2,3-d]pyrimidin-4-yloxy)-

benzene-1,2-diamine (100 mg, 0.40 mmol), 15 mg (8%) of the title

compound was obtained according to the method described for the

synthesis of 3. ¹H NMR indicates a mixture of tautomers (400 MHz,

DMSO-d₆) δ 12.08-12.14 (m, 1H), 11.00 (s, 0.5H), 10.94 (s, 0.5H),

9.65 (s, 0.5H), 9.60 (s, 0.5H), 8.23-8.26 (m, 1H), 7.84-7.92 (m,

1H), 7.68-7.74 (m, 1H), 7.24-7.40 (m, 3H), 7.12-7.17 (m, 1H),

6.80-6.88 (m, 1H), 6.24-6.30 (m, 1H), 3.50 (s, 2H), 2.20-2.50

(m, 8H), 2.15 (s, 1.5H), 2.10 (s, 1.5H); HRMS calcd for C₂₅H₂₅-

ClN₈O (M + H)⁺, 489.1912; found, 489.1911; HPLC purity 95%

(system B), 100% (system C).

[4-Chloro-3-(4-methyl-piperazin-1-ylmethyl)-phenyl]-[6-(1H-

pyrrolo[2,3-b]pyridin-4-yloxy)-1H-benzimidazol-2-yl]-amine (16).

Starting with 4-(1H-pyrrolo[2,3-b]pyridin-4-yloxy)-benzene-1,2-

diamine (106 mg, 0.44 mmol), 84 mg (39%) of the title compound

was obtained according to the method described for the synthesis

of 3. ¹H NMR indicates a mixture of tautomers (400 MHz, DMSO-

d₆) δ 11.60-11.70 (m, 1H), 11.05 (s, 0.5H), 10.95 (s, 0.5H), 9.70

(s, 0.5H), 9.65 (s, 0.5H), 7.95-8.05 (m, 1H), 7.85-7.90 (m, 1H),

7.75 (d, J ) 3.2 Hz, 1H), 7.30-7.40 (m, 2H), 7.12 (d, J ) 2.1 Hz,

0.5H), 7.07 (d, J ) 2.1 Hz, 0.5H), 6.80-6.90 (m, 1H), 6.35 (d, J

) 5.5 Hz, 0.5H), 6.30 (d, J ) 5.5 Hz, 0.5H), 6.15-6.20 (m, 1H),

3.52 (s, 1H), 3.50 (s, 1H), 2.20-2.50 (m, 8H), 2.18 (s, 1.5 H),

2.15 (s, 1.5H); Anal. (C₂₆H₂₆ClN₇O‚2H₂0) C, H, N.

N-Methyl-4-((2-((3-((2-(1-pyrrolidinyl)ethyl)oxy)-5-(trifluo-

romethyl)phenyl)amino)-1H-benzimidazol-5-yl)oxy)-2-pyridin-

ecarboxamide (11). A flask was charged with 3-(2-pyrrolin-1-yl-

ethoxy)-5-trifluoromethyl-phenylamine (0.56 m, 2.0 mmol) and

CH₂Cl₂(10 mL) and cooled in an ice bath. To this solution, 1,1-

thiocarbonyldiimidazole (463 mg, 2.6 mmol) was added and the

reaction was allowed to warm to rt. After 4 h, the solvent was

concentrated in vacuo and the residual yellow solid was titrated

with acetone to afford 1-[2-(3-thioisocyanato-5-trifluoromethyl-

phenoxy)-ethyl]-pyrrolidine as a 1/1 mixture with imidazole (242

mg). This mixture was used directly in the cyclization step following

the procedure described for the synthesis of 3 using 4-(3,4-diamino-

phenoxy)-pyridine-2-carboxylic acid methylamide (0.20 g, 0.63

mmol). The title compound was obtained as a yellow solid (0.20

1

g, 58%). H NMR indicates a mixture of tautomers (400 MHz,

DMSO-d₆) δ 11.32 (s, 0.5 H), 11.28 (s, 0.5H), 9.93 (s, 0.5H), 9.88

(s, 0.5H), 8.70-8.80 (m, 1H), 8.46 (d, J ) 5.5 Hz, 1H), 7.77 (s,

1H), 7.65 (s, 1H), 7.45-7.50 (m, 0.5H), 7.30-7.40 (m, 2H), 7.20-

7.25 (m, 0.5H), 7.12 (dd, J ) 5.5, 2.6 Hz, 1H), 6.82-6.90 (m,

1H), 6.78 (s, 1H), 4.10-4.18 (m, 1H), 2.77-2.85 (m, 2H), 2.76

(d, J ) 5.1 Hz, 3H), 2.52-2.58 (m, 4H), 1.60-1.72 (m, 4H); Anal.

(C₂₇H₂₇F₃N₆O₃‚H₂O‚0.50CH₃OH) C, H, N.

N-(3-((((2R)-1-Methyl-2-pyrrolidinyl)methyl)oxy)-4-(trifluo-

romethyl)phenyl)-5-(4-quinolinyloxy)-1H-benzimidazol-2-

amine (12). Starting with 4-(quinolin-4-yloxy)-benzene-1,2-diamine

(200 mg, 0.80 mmol), 121 mg (28%) of the title compound was

obtained according to the method described for the synthesis of 3.

¹H NMR indicates a mixture of tautomers (400 MHz, DMSO-d₆)

δ 11.35 (s, 0.5H), 11.25 (s, 0.5H), 10.05 (s, 0.5H), 9.95 (s, 0.5H),

8.60-8.65 (m, 1H), 8.35 (d, J ) 8.4 Hz, 1H), 8.00 (d, J ) 8.4 Hz,

1H), 7.78-7.85 (m, 1H), 7.70 (s, 1H), 7.60-7.68 (m, 1H), 7.40-

7.55 (m, 3H), 7.30 (s, 0.5H), 7.20 (s, 0.5H), 6.90-6.95 (m, 1H),

6.50-6.60 (m, 1H), 4.10-4.20 (m, 1H), 3.90-4.00 (m, 1H), 2.90-

3.00 (m, 1H), 2.60-2.70 (m, 1H), 2.39-2.42 (m, 3H), 2.20-2.30

(m,3H),1.95-2.05(m,1H),1.50-1.80(m,3H);Anal.(C₂₉H₂₆F₃N₅O₂‚

1.5H₂O) C, H, N.

[4-Chloro-3-(4-methyl-piperazin-1-ylmethyl)-phenyl]-[5-(quin-

olin-4-yloxy)-1H-benzimidazol-2-yl]-amine (13). Starting with

4-(quinolin-4-yloxy)-benzene-1,2-diamine (200 mg, 0.80 mmol),

20 mg (5%) of the title compound was obtained according to the

method described for the synthesis of 7. ¹H NMR indicates a

mixture of tautomers (400 MHz, DMSO-d₆) δ 11.10 (s, 0.5 H),

11.05 (s, 0.5 H), 9.75 (s, 0.5H), 9.65 (s, 0.5H), 8.60-8.70 (m, 1H),

8.34 (d, J ) 7.4 Hz, 1H), 8.00 (d, J ) 7.4 Hz, 1H), 7.85-7.90 (m,

1H), 7.75-7.80 (m, 1H), 7.70-7.80 (m, 1H), 7.60-7.70 (m, 1H),

7.30-7.50 (m ,2H), 7.25 (s, 0.5H), 7.15 (s, 0.5H), 6.80-6.95 (m,

1H), 6.50-6.60 (m, 1H), 3.55 (s, 2H), 2.20-2.60 (m, 8H), 2.18

(s, 1.5H), 2.15 (s, 1.5H); HRMS calcd for C₂₈H₂₇ClN₆O (M + H)⁺,

499.2008; found, 499.2005; HPLC purity 99% (system A), 99%

(system B).

[4-Chloro-3-(4-methylpiperazin-1-ylmethyl)phenyl]-[5-(2-me-

thylamino-pyrimidin-4-yloxy)-1H-benzimidazol-2-yl]-amine (17).

To a solution of 4-(2-methanesulfonyl-pyrimidin-4-yloxy)-benzene-

1,2-diamine (39h; 400 mg, 1.4 mmol) in anhydrous THF (4 mL)

was added CH₃NH₂(2 M in THF, 1 mL, 2.0 mmol). The solution

was heated to 80 °C for 1 h, and the mixture was allowed to cool

to rt and concentrated in vacuo. The material was purified by

column chromatography (0-10% MeOH/CH₂Cl₂with 1% NH₄-

OH) to yield 4-(2-methylamino-pyrimidin-4-yloxy)-benzene-1,2-

diamine (300 mg, 92%), which was used directly in the next step.

To a solution of 4-(2-methylamino-pyrimidin-4-yloxy)-benzene-

1,2-diamine (300 mg, 1.3 mmol) in anhydrous CH₃CN (20 mL)

was added dropwise a solution of 1-(2-chloro-5-isothiocyanato-

benzyl)-4-methyl-piperazine (405 mg [residual imidazole present],

1.3 mmol) in anhydrous CH₃CN (10 mL). The solution was stirred

for 16 h at rt, then EDC (250 mg, 1.3 mmol) was added, and the

reaction mixture was heated at 80 °C for 2 h. The mixture was

allowed to cool to rt and concentrated in vacuo. The residue was

dissolved in a mixture of CH₂Cl₂/MeOH (50 mL) and washed with

H₂O, NaHCO₃(satd), and brine. The aqueous layers were back-

extracted with CH₂Cl₂, and the combined organic layers were dried

over MgSO₄, filtered, and concentrated. The material was purified

by column chromatography (0-10% MeOH/CH₂Cl₂with 1% NH₄-

OH) followed by preparative TLC and reverse-phase HPLC (5-

100% H₂O/CH₃CN with 0.1% TFA) to yield the title compound

1

(57 mg, 9%). H NMR (400 MHz, 325K, DMSO-d₆) δ 10.85-

11.00 (bs, 1H), 9.45-9.55 (bs, 1H), 8.10 (d, J ) 5.3 Hz, 1H), 7.81

(dd, J ) 8.5, 2.3 Hz, 1H), 7.76 (d, J ) 2.6 Hz, 1H), 7.25-7.35

(m, 2H), 7.07 (s, 1H), 6.70-6.90 (m, 2H), 5.95-6.00 (m, 1H),

3.55 (s, 2H), 2.71 (d, J ) 4.6 Hz, 3H), 2.45-2.55 (m, 8H), 2.24

(s, 3H); Anal. (C₂₄H₂₇ClN₈O‚H₂O‚1.66CH₃OH) C, H, N.

[4-Chloro-3-(4-methyl-piperazin-1-ylmethyl)-phenyl]-[5-(2-

methylsulfanyl-pyrimidin-4-yloxy)-1H-benzimidazol-2-yl]-

amine (18). Starting with 4-(2-methylsulfanyl-pyrimidin-4-yloxy)-

benzene-1,2-diamine (400 mg, 1.6 mmol), 13 mg (2%) of the title

compound was obtained according to the method described for the

synthesis of 7. ¹H NMR indicates a mixture of tautomers (400 MHz,

DMSO-d₆) δ 10.95-11.10 (m, 1H), 9.70 (s, 0.5 H), 9.60 (s, 0.5H),

8.40-8.45 (m, 1H), 7.80-7.90 (m, 1H), 7.70-7.78 (m, 1H), 7.25-

7.40 (m, 2H), 7.05-7.18 (m, 1H), 6.75-6.90 (m, 1H), 6.55-6.70

(m, 1H), 3.55 (s, 2H), 2.30-2.50 (m, 1H), 2.20 (s, 3H); HRMS

[4-Chloro-3-(4-methylpiperazin-1-ylmethyl)-phenyl]-[5-(6,7-

dimethoxyquinolin-4-yloxy)-1H-benzimidazol-2-yl]-amine (14).

Starting with 4-(6,7-dimethoxyquinolin-4-yloxy)-benzene-1,2-di-

amine (120 mg, 0.38 mmol), 38 mg (18%) of the title compound

was obtained according to the method described for the synthesis

1

of 7. H NMR (400 MHz, CDCl₃) δ 8.40 (d, J ) 5.0 Hz, 1H),

7.50-7.65 (m, 2H), 7.35-7.50 (m, 2H), 7.30 (d, J ) 8.0 Hz, 1H),

7.20-7.28 (m, 4H), 6.93 (dd, J ) 8.0, 2.0 Hz, 1H), 6.40 (d, J )

5.0 Hz, 1H), 4.05 (s, 3H), 4.03 (s, 3H), 3.70 (s, 2H), 2.60-2.80

4362 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18

Potashman et al.

calcd for C₂₄H₂₆ClN₇OS (M + H)⁺, 496.1681; found, 496.1682;

HPLC purity ) 99% (system B), 99% (system C).

the title compound was obtained as a light yellow solid according

to the method described for the synthesis of 24. H NMR (400

1

MHz, DMSO-d₆) δ 10.91 (s, 1H), 8.76-8.79 (m, 1H), 8.49 (d, J

) 5.2 Hz, 1H), 7.62 (d, J ) 8.4 Hz, 1H), 7.57 (d, J ) 2.0 Hz, 1H),

7.33-7.38 (m, 4H), 7.14-7.16 (m, 1H), 6.98 (dd, J ) 8.4, 2.4 Hz,

1H), 4.12 (t, J ) 6.0 Hz, 2H), 2.75 (d, J ) 4.8 Hz, 3H), 2.67-2.70

(m, 2H), 2.24 (s, 6H); Anal. (C₂₄H₂₄ClN₅O₄‚CH₃OH) C, H, N.

4-{2-[4-Chloro-3-(2-diethylamino-ethoxy)-phenylamino]-ben-

zoxazol-5-yloxy}-pyridine-2-carboxylic Acid Methylamide (24).

To a stirring rt solution of 4-(3-amino-4-hydroxy-phenoxy)-

pyridine-2-carboxylic acid methylamide (1.51 g, 5.824 mmol) in

DMF (8 mL) and CH₃CN (80 mL) was added 1-chloro-2-(2-chloro-

ethoxy)-4-isothiocyanato-benzene (1.39 g, about 5.3 mmol-mixed

with the bromo adduct). The reaction mixture was stirred over 4

days at rt, and EDC (1.02 g, 5.30 mmol) was added. The mixture

was heated at 50 °C for 16 h and allowed to cool to rt. The solvent

was evaporated, and the mixture was diluted with 1 N NaOH and

EtOAc. The organic phase was washed with brine, dried over

Na₂SO₄, filtered, and concentrated in vacuo. Silica gel chromatog-

raphy of the crude residue yielded 4-{2-[4-chloro-3-(2-chloro-

ethoxy)-phenylamino]-benzoxazolo-5-yloxy}-pyridine-2-carboxy-

lic acid methylamide (as a mixture with the bromo). A portion of

this mixture (156 mg, 0.33 mmol) was combined with excess

diethylamine (0.5 mL) and DMSO (1 mL) in a sealed tube, and

the mixture was heated with stirring for 2 days at 85 °C. The

mixture was allowed to cool to rt and treated with 1 N NaOH and

extracted with EtOAc (×3). The combined organic layers were

washed with brine, dried over Na₂SO₄, filtered, and concentrated

in vacuo. Purification of the crude residue by thin layer silica gel

chromatography yielded the title compound as a light yellow solid

4-{2-[4-Chloro-3-(4-methyl-piperazin-1-ylmethyl)-phenylami-

no]-1-methyl-1H-benzimidazol-5-yloxy}-pyridine-2-carboxylic

Acid Methylamide (19). Starting with 4-(3-amino-4-methylamino-

phenoxy)-pyridine-2-carboxylic acid methylamide (25 mg, 0.92

mmol), 5 mg (10%) of the title compound was obtained as an off-

white solid according to the method described for the synthesis of

1

7. H NMR (400 MHz, DMSO-d₆) δ 9.17 (s, 1H), 8.70-8.76 (m,

1H), 8.46 (d, J ) 5.9 Hz, 1H), 7.95 (dd, J ) 9.2, 2.5 Hz, 1H), 7.85

(d, J ) 2.6 Hz, 1 H), 7.40 (d, J ) 8.4 Hz, 1H), 7.30-7.35 (m,

2H), 7.17 (d, J ) 2.2 Hz, 1H), 7.10-7.14 (m, 1H), 6.88 (d, J )

8.5, 2.2 Hz, 1H), 3.74 (s, 3H), 3.51 (s, 2H), 2.75 (d, J ) 4.7 Hz,

3H), 2.30-2.50 (m, 8H), 2.11 (s, 3H); HRMS calcd for C₂₇H₃₀-

ClN₇O₂(M + H)⁺, 519.2149; found, 520.2222; HPLC purity )

100% (system A), 99% (system B).

4-{2-[4-Chloro-3-(4-methyl-piperazin-1-ylmethyl)-phenylami-

no]-benzoxazol-5-yloxy}-pyridine-2-carboxylic Acid Methyl-

amide (20). To a cooled (0 °C) solution of 1-(2-chloro-5-amino-

benzyl)-4-methyl-piperazine (1.87 g, 7.79 mmol) in CH₂Cl₂(30

mL) was added 1,1′-thiocarbonyldiimidazole (2.08 g, 11.69 mmol).

The reaction mixture was allowed to warm to rt and stirred for 2

h. The reaction was concentrated to a small volume and partially

purified by short column silica gel chromatography using 1:3

EtOAc/hexanes as the eluent to obtain 1-(2-chloro-5-isothiocyanato-

benzyl)-4-methyl-piperazine. A portion of this (350 mg, 0.68 mmol)

and 4-(3-amino-4-hydroxy-phenoxy)-pyridine-2-carboxylic acid

methylamide (147 mg, 0.57 mmol) were used to prepare the title

compound according to the method described for the synthesis of

7 (63 mg, 22%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.88 (s, 1H),

8.70-8.80 (m, 1H), 8.45-8.50 (m, 1H), 7.75-7.80 (m, 2H), 7.65-

7.70 (m, 1H), 7.30-7.45 (m, 3H), 7.15-7.20 (m, 1H), 6.95-7.00

(m, 1H), 3.52 (s, 2H), 2.75-2.77 (m, 3H), 2.30-2.50 (m, 8H),

2.14 (s, 3H); HRMS calcd for C₂₆H₂₇ClN₆O₃(M + H)⁺, 507.1906;

found, 507.1906; HPLC purity 100% (system A), 100% (system

B).

1

(105 mg, 63%). H NMR (400 MHz, DMSO-d₆) δ 10.90 (s, 1H),

8.76-8.79 (m, 1H), 8.49 (d, J ) 5.2 Hz, 1H), 7.61 (d, J ) 8.4 Hz,

1H), 7.57 (d, J ) 2.4 Hz, 1H), 7.32-7.40 (m, 4H), 7.14-7.16 (m,

1H), 6.98 (dd, J ) 8.4, 2.8 Hz, 1H), 4.08 (t, J ) 5.6 Hz, 2H),

2.80-2.90 (m, 2H), 2.76 (d, J ) 4.8 Hz, 3H), 2.56 (q, J ) 7.2 Hz,

4H), 0.96 (t, J ) 7.2 Hz, 6H). Anal. (C₂₆H₂₈ClN₅O₄‚H₂O) C, H,

N.

4-((2-((4-Chlorophenyl)-amino)-1,3-benzoxazol-5-yl)oxy)-N-

methyl-2-pyridinecarboxamide (21). Starting from 4-(3-amino-

5-fluoro-4-hydroxyphenoxy)-pyridine-2-carboxylic acid methyl-

amide (100 mg, 0.39 mmol), 39 mg (25%) of the title compound

was obtained according to the method described for the synthesis

4-((2-((4-Chloro-3-(4-morpholinylmethyl)phenyl)amino)-1,3-

benzoxazol-5-yl)oxy)-N-methyl-2-pyridinecarboxamide (25). Start-

ing from 4-(3-amino-4-hydroxy-phenoxy)-pyridine-2-carboxylic

acid methylamide (324 mg, 1.25 mmol), 263 mg (45%) of the title

compound was obtained as an off-white solid according to the

method described for the synthesis of 7. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.89 (s, 1H), 8.77 (q, J ) 4.8 Hz, 1H), 8.49 (d, J )

5.6 Hz, 1H), 7.84 (d, J ) 2.8 Hz, 1H), 7.77 (dd, J ) 8.8, 3.2 Hz,

1H), 7.60 (d, J ) 8.4 Hz, 1H), 7.42 (d, J ) 8.8 Hz, 1H), 7.35 (dd,

J ) 10.4, 2.8 Hz, 1H), 7.14-7.16 (m, 1H), 6.97 (dd, J ) 8.8, 2.0

Hz, 1H), 3.60-3.62 (m, 4H), 3.54 (s, 2H), 2.76 (d, J ) 5.2 Hz,

3H), 2.42-2.47 9 m, 4H); Anal. (C₂₅H₂₄ClN₅O₄‚0.33CH₃OH) C,

H, N.

1

of 7. H NMR (300 MHz, DMSO-d₆) δ 10.95 (s, 1H), 8.70-8.80

(m, 1H), 8.50 (d, J ) 5.6 Hz, 1H), 7.75-7.80 (m, 2H), 7.62 (d, J

) 8.5 Hz, 1H), 7.44 (d, J ) 5.6 Hz, 2H), 7.35-7.38 (m, 2H), 7.15-

7.18 (m, 1H), 7.00 (dd, J ) 8.5, 1.7 Hz, 1H), 2.78 (d, J ) 4.8 Hz,

3H); Anal. (C₂₀H₁₅ClN₄O₃) C, H, N.

4-((2-((4-Chloro-3-((((2S)-1-methyl-2-pyrrolidinyl)methyl)oxy)-

phenyl)amino)-1,3-benzoxazol-5-yl)oxy)-N-methyl-2-pyridinecarboxamide

(22). To a 0 °C solution of (S)-4-chloro-3-(1-methyl-pyrrolidin-2-

ylmethoxy)-phenylamine (3.11 g, 12.92 mmol) in CH₂Cl₂(30 mL)

was added 1,1′-thiocarbonyldiimidazole (2.87 g, 16.15 mmol). The

reaction was allowed to warm to rt and stirred for 2 h. The reaction

was concentrated and purified by short column silica gel chroma-

tography using 1:3 EtOAc/hexanes as the eluent to yield (S)-2-((2-

chloro-5-isothiocyanatophenoxy)methyl)-1-methylpyrrolidine. A por-

tion of this (1.80 g, 6.37 mmol) and 4-(3-amino-4-hydroxy-

phenoxy)-pyridine-2-carboxylic acid methylamide (1.90 g, 7.32

mmol) were used to prepare the title compound according to the

4-{2-[4-Chloro-3-(piperidin-4-ylmethoxy)-phenylamino]-ben-

zoxazol-5-yloxy}-pyridine-2-carboxylic Acid Methylamide (26).

To a cooled (0 °C) solution of 4-(5-amino-2-chloro-phenoxym-

ethyl)-piperidine-1-carboxylic acid tert-butyl ester (54 mg, 0.16

mmol) in CH₂Cl₂(3 mL) was added 1,1′-thiocarbonyldiimidazole

(28 mg, 0.16 mmol). The reaction was allowed to warm to rt and

stirred for 18 h. 4-(3-Amino-4-hydroxy-phenoxy)-pyridine-2-car-

boxylic acid methylamide (41 mg, 0.16 mmol) was added, and the

reaction mixture was stirred an additional 16 h. EDC (45 mg, 0.24

mmol) was added, and the mixture was stirred at rt for 3 h. The

reaction mixture was partially purified by prep HPLC to obtain

4-{2-chloro-5-[5-(2-methylcarbamoyl-pyridin-4-yloxy)-benzoxazol-

2-ylamino]-phenoxymethyl}-piperidine-1-carboxylic acid tert-butyl

ester. This intermediate was stirred with TFA (2 mL) at rt for 3 h

and concentrated in vacuo. The residue was taken up into ethyl

acetate, washed with 1 N NaOH and NaHCO₃(satd), dried with

Na₂SO₄, filtered, and evaporated. Purification by prep HPLC yielded

1

method described for the synthesis of 7 (1.96 g, 60%). H NMR

(400 MHz, DMSO-d₆) δ 10.89 (s, 1H), 8.77 (q, J ) 5.2 Hz, 1H),

8.49 (d, J ) 6.4 Hz, 1H), 7.62 (d, J ) 8.8 Hz, 1H), 7.57-7.58 (m,

1H), 7.32-7.41 (m, 4H), 7.14-7.16 (m, 1H), 6.96-6.99 (m, 1H),

3.91-4.02 (m, 2H), 2.92-2.98 (m, 1H), 2.76 (d, J ) 5.2 Hz, 3H),

2.62-2.68 (m, 1H), 2.40 (s, 3H), 2.16 -2.28 (m, 1H), 1.90-2.02

(m,1H),1.58-1.74(m,3H);Anal.(C₂₆H₂₆ClN₅O₄‚0.50H₂O‚0.50CH₃-

OH) C, H, N.

4-{2-[4-Chloro-3-(2-dimethylamino-ethoxy)-phenylamino]-

benzoxazol-5-yloxy}-pyridine-2-carboxylic Acid Methylamide

(23). Starting from 4-{2-[4-chloro-3-(2-chloro-ethoxy)-phenylamino]-

benzoxazolo-5-yloxy}-pyridine-2-carboxylic acid methylamide (as

a mixture with the bromo) (0.17 g, 0.36 mmol), 89 mg (50%) of

1

the title compound as a white solid (13 mg, 39%). H NMR (400

MHz, DMSO-d₆) δ 10.90 (bs, 1H), 8.76-8.82 (m ,1H), 8.50 (d, J

) 5.9 Hz, 1H), 8.00-8.30 (bs, 1H), 7.58-7.64 (m, 2H), 7.32-

7.42 (m, 4H), 7.15-7.18 (m, 1H), 6.99 (dd, J ) 8.7, 2.4 Hz, 1H),

Benzimidazoles and Benzoxazoles as VEGFR-2 Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4363

3.95 (d, J ) 5.8 Hz, 2H), 3.24-3.30 (m, 2H), 2.54-2.84 (m, 2H),

2.75 (d, J ) 4.9 Hz, 3H), 2.02-2.14 (m, 1H), 1.90-1.98 (m, 2H),

1.42-1.58 (m, 2H); HRMS calcd for C₂₆H₂₆ClN₅O₄(M + H)⁺,

508.1746; found, 508.1747; HPLC purity 99% (system A), 96%

(system B).

reaction mixture was stirred 18 h at rt. The reaction mixture was

diluted with CH₃CN (10 mL), then EDC (0.12 g, 0.62 mmol) was

added, and the mixture was heated at 80 °C for 3 h. The mixture

was allowed to cool to rt and concentrated in vacuo, and the residue

was dissolved in EtOAc and water. The layers were separated, and

the organic layer washed with brine, dried over Na₂SO₄, filtered,

and concentrated in vacuo. The residue was purified by a combina-

tion of silica gel column chromatography and silica gel prep plates

to obtain the title compound (125 mg, 42%). ¹H NMR (400 MHz,

DMSO-d₆) δ 10.80 (s, 1H), 8.12 (bs, 1H), 7.75-7.80 (m, 2H), 7.51

(d, J ) 8.8 Hz, 1H), 7.41 (d, J ) 8.4 Hz, 1H), 7.23 (s, 1H), 6.85-

7.00 (m, 2H), 5.90-6.20 (m, 1H), 3.53 (s, 2H), 2.60-2.70 (m,

4H), 2.30-2.50 (m, 7H), 2.17 (s, 3H); Anal. (C₂₄H₂₆ClN₇O₂‚H₂O)

C, H, N.

4-{2-[4-Chloro-3-((2S)-1-methyl-pyrrolidin-2-ylmethoxy)-phen-

ylamino]-benzooxazol-5-yloxy}-pyridine-2-carboxylic Acid Amide

(27). Starting with 4-(3-amino-4-hydroxy-phenoxy)-pyridine-2-

carboxylic acid amide (0.22 g, 0.88 mmol), 160 g (37%) of the

title compound was obtained as a white solid according to the

1

method described for the synthesis of 22. H NMR (400 MHz,

DMSO-d₆) δ 10.88 (s, 1H), 8.50 (d, J ) 5.2 Hz, 1H), 8.08-8.12

(m, 1H), 7.65-7.70 (m, 1H), 7.61 (d, J ) 8.4 Hz, 1H), 7.57 (d, J

) 2.4 Hz, 1H), 7.30-7.40 (m, 4H), 7.17 (dd, J ) 8.4, 5.2 Hz,

1H), 6.98 (dd, J ) 8.4, 2.4 Hz, 1H), 3.90-4.02 (m, 2H), 2.90-

2.98 (m, 1H), 2.60-2.70 (m, 1H), 2.40 (s, 3H), 2.15-2.25 (m,

1H), 1.90-2.02 (m, 1H), 1.58-1.72 (m, 3H); Anal. (C₂₅H₂₄-

ClN₅O₄‚CH₃OH) C, H, N.

[4-Chloro-3-(4-methyl-piperazin-1-ylmethyl)-phenyl]-[5-(6-

methylamino-pyrimidin-4-yloxy)-benzoxazol-2-yl]-amine (33).

Starting with 2-amino-4-(6-methylamino-pyrimidin-4-yloxy)-phenol

(132 mg, 0.57 mmol), 80 mg (30%) of the title compound was

obtained as a yellow solid according to the method described for

S-[4-Chloro-3-(1-methyl-pyrrolidin-2-ylmethoxy)-phenyl]-[5-

(6,7-dimethoxy-quinazolin-4-yloxy)-benzoxazol-2-yl]-amine (28).

Starting with 2-amino-4-(6,7-dimethoxyquinazolin-4-yloxy)-phenol

(0.22 g, 0.70 mmol), 125 mg (32%) of the title compound was

obtained as a tan solid according to the method described for the

1

the synthesis of 32. H NMR (400 MHz, DMSO-d₆) δ 10.80 (s,

1H), 8.11 (bs, 1H), 7.76-7.80 (m, 2H), 7.50 (d, J ) 8.8 Hz, 1H),

7.41 (d, J ) 8.4 Hz, 1H), 7.25-7.30 (m, 1H), 7.19 (d, J ) 2.4 Hz,

1H), 6.87 (dd, J ) 8.8, 2.4 Hz, 1H), 5.74 (s, 1H), 3.52 (s, 2H),

2.74 (bs, 3H), 2.30-2.50 (m, 8H), 2.15 (s, 3H); Anal. (C₂₄H₂₆-

ClN₇O₂‚H₂O‚0.50CH₃OH) C,H,N.

1

synthesis of 22. H NMR (400 MHz, DMSO-d₆) δ 10.82 (s, 1H),

8.51 (s, 1H), 7.50-7.60 (m, 3H), 7.30-7.40 (m, 4H), 7.00-7.10

(m, 1H), 3.85-4.05 (m, 8H), 2.95-3.00 (m, 1H), 2.60-2.80 (m,

1H), 2.42 (s, 3H), 2.20-2.30 (m, 1H), 1.90-2.05 (m, 1H), 1.60-

1.80 (m, 3H); HRMS calcd for C₂₉H₂₈ClN₅O₅(M + H)⁺, 562.1852;

found, 562.1848; HPLC purity ) 95% (system A), 99% (system

B).

N-(4-Chloro-3-(((2S)-1-methyl-2-pyrrolidinyl)methoxy)phenyl)-

5-(1H-pyrrolo[2,3-b]pyridin-4-yloxy)-1,3-benzoxazol-2-amine (34).

Starting with 4-(1H-indol-4-yloxy)-2-aminophenol (130 mg, 0.50

mmol), 79 mg (32%) of the title compound was obtained according

1

to the method described for the synthesis of 22. H NMR (400

MHz, DMSO-d₆) δ 11.70 (s, 1H), 10.90 (s, 1H), 8.05 (d, J ) 5.8

Hz, 1H), 7.55-7.60 (m, 2H), 7.35-7.45 (m, 3H), 7.30 (d, J ) 2.0

Hz, 1H), 6.95 (dd, J ) 7.7, 2.0 Hz, 1H), 6.38 (d, J ) 5.8 Hz, 1H),

6.20-6.25 (m, 1H), 4.00-4.05 (m, 1H), 3.92-3.97 (m, 1H), 2.92-

2.98 (m, 1H), 2.62-2.70 (m, 1H), 2.40 (s, 3H), 2.16-2.25 (m,

1H), 1.90-2.04 (m, 1H), 1.58-1.78 (m, 3H); HRMS calcd for

C₂₆H₂₄ClN₅O₃(M + H)⁺, 490.1640; found, 490.1642; HPLC purity

) 99% (system B), 100% (system C).

S-[4-Chloro-3-(1-methylpyrrolidin-2-ylmethoxy)-phenyl]-[5-

(6,7-dimethoxyquinolin-4-yloxy)-benzoxazol-2-yl]-amine (29).

Starting with 2-amino-4-(2,3-dimethoxyquinolin-4-yloxy)phenol

(200 mg, 0.64 mmol), 50 mg (14%) of the title compound was

obtained as a tan solid according to the method described for the

1

synthesis of 22. H NMR (300 MHz, DMSO-d₆) δ 10.85 (s, 1H),

8.42 (d, J ) 5.0 Hz, 1H), 7.60 (d, J ) 7.3 Hz, 1H), 7.55 (m, 1H),

7.50 (s, 1H), 7.35-7.40 (m, 4H), 7.00 (dd, J ) 7.3, 2.3 Hz, 1H),

6.40 (d, J ) 5.0 Hz, 1H), 3.90-4.05 (m, 8H), 2.95-3.00 (m, 1H),

2.60-2.70 (m, 1H), 2.40 (s, 3H), 2.15-2.25 (m, 1H), 1.95-2.05

(m, 1H), 1.60-1.80 (m, 3H); HRMS calcd for C₃₀H₂₉ClN₄O₅(M

+ H)⁺, 560.1826; found, 561.18992; HPLC purity ) 99% (system

A), 99% (system B).

N-(4-Chloro-3-((4-methyl-1-piperazinyl)methyl)phenyl)-5-

(1H-pyrrolo[2,3-b]pyridin-4-yloxy)-1,3-benzoxazol-2-amine (35).

Starting with 4-(1H-indol-4-yloxy)-2-aminophenol (0.13 g, 0.50

mmol), 116 mg (48%) of the title compound was obtained according

1

to the method described for the synthesis of 32. H NMR (400

5-((6,7-Bis(methoxy)-4-quinolinyl)oxy)-N-(4-chloro-3-((4-methyl-

1-piperazinyl)methyl)phenyl)-1,3-benzoxazol-2-amine (30). Start-

ing from 2-amino-4-(6,7-dimethoxyquinolin-4-yloxy)phenol (220

mg, 0.70 mmol), 34 mg (9%) of the title compound was obtained

as a tan solid according to the method described for the synthesis

of 20. ¹H NMR (300 MHz, DMSO-d₆) δ 10.90 (s, 1H), 8.42 (d, J

) 3.6 Hz, 1H), 7.80-7.85 (m, 2H), 7.60 (d, J ) 8.9 Hz, 1H), 7.50

(s, 1H), 7.30-7.40 (m, 3H), 7.00-7.05 (m, 1H), 6.45 (d, J ) 3.6

Hz, 1H), 3.90 (s, 6H), 3.50 (s, 2H), 2.20-2.50 (m, 8H), 2.15 (s,

3H); HRMS calcd for C₃₀H₃₀ClN₅O₄(M + H)⁺, 560.2059; found,

560.2058; HPLC purity 99% (system A), 99% (system B).

([4-Chloro-3-(4-methyl-piperazin-1-ylmethyl)-phenyl]-[5-(quin-

olin-4-yloxy)-benzoxazol-2-yl]-amine) (31). Starting from 2-amino-

4-(quinolin-4-yloxy)-phenol (150 mg, 0.59 mmol), 50 mg (17%)

of the title compound was obtained according to the method

MHz, DMSO-d₆) δ 11.70 (s, 1H), 10.85 (s, 1H), 8.05 (d, J ) 5.6

Hz, 1H), 7.80 (s, 1H), 7.78 (d, J ) 7.4 Hz, 1H), 7.55 (d, J ) 7.4

Hz, 1H), 7.40 (d, J ) 7.4 Hz, 1H), 7.30-7.35 (m, 1H), 7.25 (s,

1H), 6.90-7.0 (m, 1H), 6.38 (d, J ) 5.6 Hz, 1H), 6.20-6.25 (m,

1H), 3.55 (s, 2H), 2.30-2.50 (m, 8), 2.15 (s, 3H); Anal. (C₂₆H₂₅-

ClN₆O₂‚0.50CH₃OH) C, H, N.

4-((2-((4-Chloro-3-(((2S)-1-methyl-2-pyrrolidinyl)methoxy)-

phenyl)amino)-7-fluoro-1,3-benzoxazol-5-yl)oxy)-N-methyl-2-py-

ridinecarboxamide (36). Starting from 4-(3-amino-5-fluoro-4-

hydroxyphenoxy)-pyridine-2-carboxylic acid methylamide (256 mg,

0.93 mmol) and 2-(2-chloro-5-isothiocyanato-phenoxymethyl)-1-

methyl-pyrrolidine (249 mg, 0.88 mmol), 244 mg (53%) of the title

compound was obtained according to the method described for the

1

synthesis of 22. H NMR (400 MHz, DMSO-d₆) δ 11.15 (s, 1H),

8.79 (q, J ) 5.2 Hz, 1H), 8.50 (d, J ) 6.0 Hz, 1H), 7.56 (d, J )

2.0 Hz, 1H), 7.39-7.42 (m, 2H), 7.29-7.32 (m, 1H), 7.24 (d, J )

2.0 Hz, 1H), 7.17-7.19 (m, 1H), 7.11-7.14 (m, 1H), 3.90-4.06

(m, 2H), 2.90-3.00 (m, 1H), 2.76 (d, J ) 4.8 Hz, 3H), 2.62-2.70

(m, 1H), 2.40 (s, 3H), 2.18-2.28 (m, 1H), 1.92-2.08 (m, 1H),

1.58-1.76 (m, 3H); Anal. (C₂₆H₂₅ClFN₅O₄‚H₂O) C, H, N.

4-{7-Chloro-2-[4-chloro-3-(4-methyl-piperazin-1-ylmethyl)-

phenylamino]-benzoxazol-5-yloxy}-pyridine-2-carboxylic Acid

Methylamide (37). Starting with 4-(3-amino-5-chloro-4-hydroxy-

phenoxy)-pyridine-2-carboxylic acid methylamide (63 mg, 0.21

mmol), 24 mg (21%) of the title compound was obtained according

1

described for the synthesis of 20. H NMR (300 MHz, CDCl₃) δ

8.65 (d, J ) 4.2 Hz, 1H), 8.38-8.42 (m, 1H), 8.10 (d, J ) 8.3 Hz,

1H), 7.74-7.79 (m, 1H), 7.57-7.67 (m, 4H), 7.32-7.39 (m, 2H),

7.31 (d, J ) 2.1 Hz, 1H), 6.95 (dd, J ) 8.3, 2.8 Hz, 1H), 6.54 (d,

J ) 4.2 Hz, 1H), 3.65 (s, 2H), 2.50-2.70 (m, 8H), 2.30 (s, 3H);

HRMS calcd for C₂₈H₂₆ClN₅O₂(M + H)⁺, 500.1848; found,

500.1847; HPLC purity 98% (system A), 94% (system B).

[4-Chloro-3-(4-methyl-piperazin-1-ylmethyl)-phenyl]-[5-(2-

methylamino-pyrimidin-4-yloxy)-benzoxazol-2-yl]-amine (32).

To a solution of 2-amino-4-(2-methylamino-pyrimidin-4-yloxy)-

phenol (0.14 g, 0.62 mmol) in CH₃CN (30 mL) was added dropwise

over 5 min a solution 1-(2-chloro-5-isothiocyanato-benzyl)-4-

methyl-piperazine (0.17 g, 0.62 mmol) in CH₃CN (10 mL). The

1

to the method described for the synthesis of 20. H NMR (400

MHz, DMSO-d₆) δ 11.21 (s, 1H), 8.75-8.80 (m, 1H), 8.40-8.50

(m, 1H), 7.80-7.90 (m, 1H), 7.70-7.80 (m, 1H), 7.30-7.50 (m,

4364 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18

Potashman et al.

3H), 7.05-7.10 (m, 2H), 3.51 (s, 2H), 2.76 (d, J ) 4.8 Hz, 3H),

2.20-2.50 (m, 8H), 2.15 (s, 3H); Anal. (C₂₆H₂₆Cl₂N₆O₃‚1.50CH₃-

OH) C, H, N.

(d, J ) 3.3 Hz, 1H), 6.29 (d, J ) 5.8 Hz, 1H), 6.16-6.21 (m, 2H),

4.70 (bs, 2H), 4.40 (bs, 2H).

4-(3,4-Diamino-phenoxy)-pyridine (39f). Starting with 4-(3,4-

dinitro-phenoxy)-pyridine (1.36 g, 5.21 mmol), 0.97 g (92%) of

the title compound was obtained as a dark brown solid according

[4-Chloro-3-(4-methylpiperazin-1-ylmethyl)-phenyl]-[7-chloro-

5-(quinolin-4-yloxy)-benzoxazol-2-yl]-amine (38). Starting with

2-chloro-4-(quinolin-4-yloxy)phenol (843 mg, 3.10 mmol), 844 mg

of a yellow solid was obtained (as a 1:1 mixture of starting material

and 2-chloro-6-nitro-4-(quinolin-4-yloxy)phenol) according to the

method described for the synthesis of 59a. This crude mixture was

combined with Fe (3.2 g, excess), 6 N HCl (2 drops), water (5

mL), and EtOH (24 mL) and refluxed for 2.5 h. The hot mixture

was filtered through Celite and concentrated in vacuo. The residue

was partially purified by prep HPLC to give 2-amino-6-chloro-4-

(quinolin-4-yloxy)phenol (31 mg, 0.10 mmol, 9%), which was

dissolved into CH₃CN and subjected to the procedure described

for the synthesis of 20. The title compound was obtained as a white

1

to the method described for the synthesis of 39o. H NMR (400

MHz, DMSO-d₆) δ 8.32-8.38 (m, 2H), 6.75-6.83 (m, 2H), 6.51

(d, J ) 8.0 Hz, 1H), 6.26 (d, J ) 2.4 Hz, 1H), 6.14 (dd, J ) 8.0,

2.4 Hz, 1H), 4.40-4.80 (bm, 4H).

4-(2-Methylsulfanyl-pyrimidin-4-yloxy)-benzene-1,2-di-

amine (39g). A flask was charged with 4-(3,4-dinitro-phenoxy)-

2-methylsulfanyl-pyrimidine (1.00 g, 3.20 mmol) and the solid was

dissolved in MeOH (10 mL). To the argon-degassed solution, a

catalytic amount of Pd/C (10% by wt) was added, and the mixture

was shaken under H₂at 60 psi until the reaction was complete.

The mixture was filtered through a pad of Celite and the material

was purified using column chromatography (0-100% EtOAc in

hexanes) to give the title compound (500 mg, 63%). ¹H NMR (400

MHz, DMSO-d₆) δ 8.41 (d, J ) 6.0 Hz, 1H), 6.62 (d, J ) 7.5 Hz,

1H), 6.33 (d, J ) 6.0 Hz, 1H), 6.37 (d, J ) 3.0 Hz, 1H), 6.24 (dd,

J ) 7.5, 3.0 Hz, 1H), 2.40 (s, 3H).

1

solid (11 mg, 21%). H NMR (400 MHz, DMSO-d₆) δ 11.22 (s,

1H), 8.65-8.70 (m, 1H), 8.32 (d, J ) 8.8 Hz, 1H), 8.00-8.05 (m,

1H), 7.80-7.85 (m, 2H), 7.70-7.78 (m, 1H), 7.60-7.70 (m, 1H),

7.40-7.45 (m, 2H), 7.25-7.30 (m, 1H), 6.65-6.70 (m, 1H), 3.52

(s, 2H), 2.20-2.60 (m, 8H), 2.15 (s, 3H); HRMS calcd for

C₂₈H₂₅C_l2N₅O₂(M + H)⁺, 534.1458; found, 534.1458; HPLC purity

) 95% (system A), 95% (system B).

4-(2-Methylsulfonyl-pyrimidin-4-yloxy)-benzene-1,2-di-

amine (39h). Starting with 4-(3,4-dinitro-phenoxy)-2-methylsul-

fonyl-pyrimidine (1.0 g, 2.9 mmol), 0.80 g (97%) of the title

compound was obtained according to the method described for the

4-(3,4-Diamino-phenoxy)-pyridine-2-carboxylic Acid Methyl-

amide (39a). A flask was charged with 4-(3,4-dinitro-phenoxy)-

pyridine-2-carboxylic acid methylamide (0.71 g, 2.23 mmol),

MeOH (40 mL), and EtOAc (80 mL). To the argon-degassed

solution was added 10% Pd/C (0.20 g). The reaction was vigorously

stirred for 42 h at rt under 1 atm of H₂gas. The reaction was filtered

through Celite and the solvent was removed in vacuo to obtain the

1

synthesis of 39a. H NMR (400 MHz, DMSO-d₆) δ 8.78 (d, J )

5.4 Hz, 1H), 7.15 (d, J ) 5.4 Hz, 1H), 6.55 (d, J ) 8.2 Hz, 1H),

6.35-6.40 (m, 1H), 6.25 (dd, J ) 8.2, 3.0 Hz, 1H), 4.80 (s, 2H),

4.50 (s, 2H), 3.30 (s, 3H).

4-(3-Amino-4-methylamino-phenoxy)-pyridine-2-carboxylic

Acid Methylamide (39i). Starting from 4-(4-methylamino-3-

nitrophenoxy)-pyridine-2-carboxylic acid methylamide (100 mg,

0.33 mmol), the title compound was obtained using the method

described in 39a (28 mg, 31%). ¹H NMR (400 MHz, DMSO-d₆) δ

8.70-8.80 (m, 1H), 8.41 (d, J ) 5.5 Hz, 1H), 7.31 (d, J ) 2.5 Hz,

1H), 7.06 (dd, J ) 5.5, 2.5 Hz, 1H), 6.38 (d, J ) 8.0 Hz, 1H),

6.25-6.35 (m, 2H), 4.80 (s, 2H), 4.60-4.65 (m, 1H), 2.76 (d, J )

4.8 Hz, 3H), 2.72 (d, J ) 5.1 Hz, 3H).

4-(3-Amino-4-hydroxyphenoxy)-pyridine-2-carboxylic Acid

Methylamide (39j). Starting from 4-(4-hydroxy-3-nitrophenoxy)-

pyridine-2-carboxylic acid methylamide (185 mg, 0.69 mmol), 148

mg (89%) of the title compound was obtained as a white foam

according to the method described for the synthesis of 39o. ¹H NMR

(400 MHz, DMSO-d₆) δ 9.18 (s, 1H), 8.70-8.80 (m, 1H), 8.43 (d,

J ) 4.8 Hz, 1H), 7.30-7.35 (m, 1H), 7.06-7.20 (m, 1H), 6.67 (d,

J ) 8.4 Hz, 1H), 6.30-6.40 (m, 1H), 6.10-6.20 (m, 1H), 4.82 (s,

2H), 2.76 (d, J ) 4.8 Hz, 3H).

4-(3-Amino-5-fluoro-4-hydroxyphenoxy)-pyridine-2-carboxy-

lic Acid Methylamide (39k). Starting from 4-(3-fluoro-4-hydroxy-

5-nitrophenoxy)-pyridine-2-carboxylic acid methylamide (0.61 g,

1.98 mmol), 495 mg (90%) of the title compound was obtained

according to the method described for the synthesis of 39o. ¹H NMR

(400 MHz, DMSO-d₆) δ 9.07 (s, 1H), 8.70-8.80 (m, 1H), 8.46 (d,

J ) 5.2 Hz, 1H), 7.36 (d, J ) 2.4 Hz, 1H), 7.09-7.11 (m, 1H),

6.25 (dd, J ) 11.2, 2.8 Hz, 1H), 6.20-6.21 (m, 1H), 5.21 (s, 2H),

2.76 (d, J ) 5.2 Hz, 3H).

1

title compound (71 mg, 95%). H NMR (400 MHz, DMSO-d₆) δ

8.70-8.75 (m, 1H), 8.42 (d, J ) 5.6 Hz, 1H), 7.32 (d, J ) 2.8 Hz,

1H), 7.04-7.06 (m, 1H), 6.53 (d, J ) 8.4 Hz, 1H), 6.27 (d, J )

2.8 Hz, 1H), 6.16 (dd, J ) 8.4, 2.8 Hz, 1H), 4.76 (bs, 1H), 4.51

(bs, 2H), 2.75 (d, J ) 4.4 Hz, 3H).

4-(6,7-Dimethoxyquinolin-4-yloxy)-benzene-1,2-diamine (39b).

Starting with 4-(3,4-dinitrophenoxy)-6,7-dimethoxyquinoline (0.256

g, 0.74 mmol), 122 mg (57%) of the title compounds was obtained

1

according to the method described for the synthesis of 39m. H

NMR (400 MHz, DMSO-d₆) δ 8.40 (d, J ) 5.5 Hz, 1H), 7.45 (s,

1H), 7.30 (s, 1H), 6.55 (d, J ) 8.2 Hz, 1H), 6.40 (d, J ) 5.5 Hz,

1H), 6.35 (d, J ) 2.7 Hz, 1H), 6.25 (dd, J ) 8.2, 2.7 Hz, 1H),

4.40-4.80 (bm, 4H), 3.90 (m 6H).

4-(Quinolin-4-yloxy)-benzene-1,2-diamine (39c). A flask was

charged with 4-(3,4-dinitro-phenoxy)-quinoline (400 mg, 1.2 mmol),

and the compound was dissolved in THF. The mixture was cooled

to 0 °C and AcOH (1.5 mL) and zinc dust (2.5 g, 38 mmol) were

added sequentially. The mixture was allowed to warm to rt, stirred

for 1 h, filtered through a pad of silica gel, and concentrated in

vacuo. The residue was dissolved in CH₂Cl₂and washed with 1 M

NaOH. The organic phases were dried, filtered, and evaporated to

give the title compound as a brown-orange oil (210 mg, 65%). ¹H

NMR (400 MHz, DMSO-d₆) δ 8.61 (d, J ) 5.7 Hz, 1H), 8.24-

8.27 (m, 1H), 7.74-7.79 (m, 1H), 7.56-7.62 (m, 1H), 6.53-6.58

(m, 2H), 6.37 (d, J ) 3.8 Hz, 1H), 6.25 (dd, J ) 7.6, 2.8 Hz, 1H),

5.75 (bs, 2H), 5.50 (bs, 2H).

2-Amino-4-(6,7-dimethoxyquinolin-4-yloxy)phenol (39l). Start-

ing from 4-(6,7-dimethoxyquinolin-4-yloxy)-2-nitrophenol (440 mg,

1.28 mmol), 220 mg (59%) of the title compound was obtained

4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yloxy)benzene-1,2-di-

amine (39d). Starting with 4-(3,4-dinitro-phenoxy)-7,7a-dihydro-

4aH-pyrrolo[2,3-d]pyrimidine (0.16 g, 0.53 mmol), 284 mg (118%)

of the title compound was obtained according to the method

1

according to the method described for the synthesis of 39m. H

NMR (400 MHz, DMSO-d₆) δ 9.30 (bs, 1H), 8.42 (d, J ) 5.2 Hz,

1H), 7.45 (s, 1H), 7.35 (s, 1H), 6.69 (d, J ) 8.8 Hz, 1H), 6.40-

6.45 (m, 2H), 6.24 (dd, J ) 8.8, 3.3 Hz, 1H), 4.80 (bs, 2H), 3.90

(s, 3H).

2-Amino-4-(quinolin-4-yloxy)-phenol (39m). To a cooled

(0 °C) solution of 2-nitro-4-(quinolin-4-yloxy)-phenol (200 mg, 0.79

mmol) in THF (50 mL) and AcOH (0.88 mL), zinc dust (2.3 g,

35.2 mmol) was added. The mixture was stirred at rt for 1.5 h and

filtered through a Celite pad. The solvent was evaporated, and the

residue was dissolved in CH₂Cl₂, washed with 1 M NaOH, dried,

filtered, and concentrated in vacuo to give the title compound as a

1

described for the synthesis of 39o. H NMR (400 MHz, DMSO-

d₆) δ 12.05 (bs, 1H), 8.25 (s, 1H), 7.30-7.35 (m, 1H), 6.50 (d, J

) 7.0 Hz, 1H), 6.35 (s, 1H), 6.20-6.25 (m, 1H), 6.10-6.12 (m,

1H), 4.36-4.70 (bm, 4H).

4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-benzene-1,2-diamine (39e).

Starting with 4-(3,4-dinitro-phenoxy)-1H-pyrrolo[2,3-b]pyridine

(0.284 g, 0.95 mmol), 190 mg (83%) of the title compound was

obtained according to the method described for the synthesis of

1

39o. H NMR (400 MHz, DMSO-d₆) δ 11.55 (bs, 1H), 7.97 (d, J

) 5.8 Hz, 1H), 7.24-7.28 (m, 1H), 6.51 (d, J ) 8.3 Hz, 1H), 6.32

Benzimidazoles and Benzoxazoles as VEGFR-2 Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4365

1

brown solid (150 mg, 86%). H NMR (300 MHz, DMSO-d₆) δ

(R)-2-((5-Isothiocyanato-2-(trifluoromethyl)phenoxy)methyl)-

1-methylpyrrolidine (40b). Starting from (R)-3-(1-methyl-pyrro-

lidin-2-ylmethoxy)-4-trifluoromethyl-phenylamine (225 mg, 0.82

mmol), 94 mg (36%) of the title compound was obtained as an

off-white solid according to the method described for the synthesis

9.20 (s, 1H), 8.65 (d, J ) 4.8 Hz, 1H), 8.30 (d, J ) 9.5 Hz, 1H),

7.95 (d, J ) 9.5 Hz, 1H), 7.70-7.80 (m, 1H), 7.55-7.60 (m, 1H),

6.70 (d, J ) 9.5 Hz, 1H), 6.55 (d, J ) 4.8 Hz, 1H), 6.40-6.45 (m,

1H), 6.20-6.30 (m, 1H), 4.80 (s, 2H).

1

of 40d. H NMR (400 MHz, DMSO-d₆) δ 7.64 (d, J ) 8.1 Hz,

4-(3-Amino-5-chloro-4-hydroxyphenoxy)-pyridine-2-carboxy-

lic Acid Methylamide (39n). Starting with 4-(3-chloro-4-hydroxy-

5-nitrophenoxy)-pyridine-2-carboxylic acid methylamide (394 mg,

approx 1.21 mmol), 66 mg (19%) of the title compound was

obtained as a brown solid according to the method described for

the synthesis of 46a. ¹H NMR (400 MHz, DMSO-d₆) δ 8.80-8.90

(bs, 1H), 8.70-8.80 (m, 1H), 8.47 (d, J ) 5.3 Hz, 1H), 7.35 (d, J

) 2.5 Hz, 1H), 7.10-7.12 (m, 2H), 6.39 (d, J ) 2.9 Hz, 1H), 6.34

(d, J ) 2.9 Hz, 1H), 5.20 (bs, 2H), 2.76 (d, J ) 4.9 Hz, 3H).

2-Amino-4-(2-methylamino-pyrimidin-4-yloxy)-phenol (39o).

A flask was charged with 4-(2-methylamino-pyrimidin-4-yloxy)-

2-nitro-phenol (0.16 g, 0.620 mmol), and the solid was dissolved

in MeOH (7 mL) and EtOAc (15 mL). To the argon-degassed

solution was added 10% by weight Pd/C (0.08 g). The reaction

was stirred vigorously at rt under 1 atm H₂gas for 18 h. The reaction

was filtered through a Celite plug and concentrated in vacuo to

1H), 7.38-7.41 (m, 1H), 7.08-7.12 (m, 1H), 4.08-4.14 (m, 1H),

3.93-4.00 (m, 1H), 2.91-2.98 (m, 1H), 2.52-2.60 (m, 1H), 2.34

(s, 3H), 2.12-2.26 (m, 1H), 1.88-2.00 (m, 1H), 1.50-1.75 (m,

3H).

1-Chloro-2-(2-chloro-ethoxy)-4-isothiocyanato-benzene (40c).

Starting from 4-chloro-3-(2-chloro-ethoxy)-phenylamine (2.54 mg,

11.5 mmol), 2.79 g (98%) of the title compound was obtained (as

a 2:1 mixture with the bromide) as a beige solid according to the

1

method described for the synthesis of 40d. H NMR of the major

product (400 MHz, DMSO-d₆) δ 7.49 (d, J ) 8.4 Hz, 1H), 7.34

(d, J ) 2.4 Hz, 1H), 7.05 (dd, J ) 8.4, 2.4 Hz, 1H), 4.36 (t, J )

5.2 Hz, 2H), 3.95 (t, J ) 5.2 Hz, 1H). ¹H NMR of the minor product

(400 MHz, DMSO-d₆) δ 7.49 (d, J ) 8.4 Hz, 1H), 7.34 (d, J ) 2.4

Hz, 1H), 7.05 (dd, J ) 8.4, 2.4 Hz, 1H), 4.24 (t, J ) 5.2 Hz, 2H),

3.81 (t, J ) 5.2 Hz, 1H).

1

yield the title compound (144 mg, 100%). H NMR (400 MHz,

4-(2-Chloro-5-isothiocyanato-benzyl)-morpholine (40d). To a

0 °C solution of 4-chloro-3-morpholin-4-ylmethyl-phenylamine

(0.33 g, 1.46 mmol) in 10 mL of CH₂Cl₂was added 1,1′-

thiocarbonyldiimidazole (0.39 g, 2.18 mmol). The reaction was

allowed to warm to rt and stirred for 1 h. The mixture was

concentrated down to a small volume and purified by short column

silica gel chromatography using EtOAc as the eluent to obtain the

DMSO-d₆) δ 8.99 (s, 1H), 8.05 (s, 1H), 6.98 (s, 1H), 6.59 (d, J )

8.4 Hz, 1H), 6.31 (s, 1H), 6.10-6.15 (m, 1H), 5.73-6.00 (m, 1H),

4.69 (s, 2H), 2.69 (d, J ) 4.0 Hz, 3H).

2-Amino-4-(6-methylamino-pyrimidin-4-yloxy)-phenol (39p).

Starting with 4-(6-methylamino-pyrimidin-4-yloxy)-2-nitro-phenol

(0.15 g, 0.57 mmol), 133 mg (100%) of the title compound was

obtained as a tan solid according to the method described for the

1

title compound (0.36 mg, 93%). H NMR (400 MHz, DMSO-d₆)

1

synthesis of 39o. H NMR (400 MHz, DMSO-d₆) δ 9.01 (s, 1H),

δ 7.48-7.52 (m, 2H), 7.34-7.38 (m, 1H), 3.56-3.60 (m, 4H),

3.50-3.52 (s, 2H), 2.38-2.42 (m, 4H).

8.09 (s, 1H), 7.17 (q, J ) 4.8 Hz, 1H), 6.60 (d, J ) 8.4 Hz, 1H),

6.30 (d, J ) 2.8 Hz, 1H), 6.10 (dd, J ) 8.4, 2.8 Hz, 1H), 5.56 (s,

1H), 4.70 (s, 2H), 2.70 (s, 3H).

1-Chloro-4-isothiocyanato-2-(2-methoxyethoxy)benzene (40e).

Starting with 1-chloro-2-(2-methoxyethoxy)-4-nitrobenzene (1.30

g, 5.60 mmol), 4-chloro-3-(2-methoxyethoxy)phenylamine was

obtained by the method described for the synthesis of 46a. A portion

of this material (0.20 g, 1.00 mmol) was converted to the title

compound using the procedure described for the synthesis of 40d

4-(1H-Indol-4-yloxy)-2-aminophenol (39q). Starting with 4-(1H-

indol-4-yloxy)phenol (0.80 g, 3.53 mmol), 400 mg (41%) of 4-(1H-

indol-4-yloxy)-2-nitrophenol was obtained according to the method

described for the synthesis of 59e. This compound (400 mg, 1.46

mmol) was subjected to the conditions used in the preparation of

1

(180 mg, 75%). H NMR (400 MHz, DMSO-d₆) δ 7.50 (d, J )

1

39o to yield the title compound (400 mg, 113%). H NMR (400

6.0 Hz, 1H), 7.35 (s, 1H), 7.00-7.05 (m, 2H), 4.20-4.25 (m, 2H),

3.65-3.75 (m, 2H), 3.30 (s, 3H).

MHz, DMSO-d₆) δ 11.60 (s, 1H), 9.08 (s, 1H), 8.00 (d, J ) 5.6

Hz, 1H), 7.30-7.35 (m, 1H), 6.65 (d, J ) 7.4 Hz, 1H), 6.40 (d, J

) 1.8 Hz, 1H), 6.35 (d, J ) 5.6 Hz, 1H), 6.20-6.25 (m, 2H), 4.80

(s, 2H).

5-Nitro-2-trifluoromethylanisole (41b). To a cooled (-40 °C)

solution of pyridine (140 mL), trifluoromethyl iodide (from a gas

cylinder that had been kept in freezer overnight) was bubbled in

over a 20 min period. To this solution was added 2-iodo-5-

nitroanisole (29.76 g, 107 mmol) and copper powder (81.33 g, 128

mol), and the vessel sealed. The reaction was stirred vigorously

for 22 h at 140 °C. The reaction was cooled to -50 °C, and the

vessel was carefully opened and poured onto ice and Et₂O and

allowed to warm slowly to rt. The layers were separated, and the

organic layer was washed with 1 N HCl (×3) and brine, dried over

Na₂SO₄, filtered, and concentrated in vacuo. The crude material

was purified on a short silica gel column (4.5:1 hexanes/CH₂Cl₂)

4-(3-Amino-4-hydroxy-phenoxy)-pyridine-2-carboxylic Acid

Amide (39r). Starting with 4-(4-hydroxy-phenoxy)-pyridine-2-

carboxylic acid amide (6.22 g, 27.0 mmol), 4-(4-hydroxy-3-nitro-

phenoxy)-pyridine-2-carboxylic acid amide was obtained according

to the method described for the synthesis of 59e. A portion of this

material (0.27 g, 0.98 mmol) was subjected to the conditions for

the preparation of 39o to yield the title compound (0.22 g, 90%).

¹H NMR (400 MHz, DMSO-d₆) δ 9.18 (s, 1H), 8.43 (d, J ) 5.6

Hz, 1H), 8.05-8.10 (m, 1H), 7.65-7.68 (m, 1H), 7.33 (d, J ) 2.4

Hz, 1H), 7.08 (dd, J ) 5.6, 2.8 Hz, 1H), 6.70 (d, J ) 8.0 Hz, 1H),

6.34 (d, J ) 2.4 Hz, 1H), 6.16 (dd, J ) 8.4, 3.2 Hz, 1H), 4.82 (s,

2H).

2-Amino-4-(6,7-dimethoxyquinazolin-4-yloxy)-phenol (39s).

Starting with 4-(6,7-dimethoxyquinazolin-4-yloxy)-2-nitrophenol

(0.48 g, 1.40 mmol), 0.22 g (51%) of the title compound was

obtained according to the method described for the synthesis of

39o. ¹H NMR (400 MHz, DMSO-d₆) δ 9.05 (s, 1H), 8.50 (s, 1H),

7.45 (s, 1H), 7.30 (s, 1H), 6.65 (d, J ) 9.0 Hz, 1H), 6.40 (d, J )

3.0, 1H), 6.20 (dd, J ) 9.0, 3.0 Hz, 1H), 4.70 (bs, 2H), 3.95 (s,

3H), 3.90 (s, 3H).

1-(2-Chloro-5-isothiocyanato-benzyl)-4-methyl-piperazine (40a).

Starting from 1-(2-chloro-5-amino-benzyl)-4-methyl-piperazine (1.87

g, 7.79 mmol), 2.03 g (93%) of the title compound was obtained

(as a mixture with imidazole; 3.71 g total) according to the method

described for the synthesis of 40d. The compound was used without

further purification. ¹H NMR of the isothiocyanate (400 MHz,

DMSO-d₆) δ 7.44-7.52 (m, 2H), 7.35 (dd, J ) 8.4, 2.0 Hz, 1H),

3.51 (s, 2H), 2.32-2.46 (m, 8H), 2.17 (s, 3H).

1

to provide the title compound as a yellow oil (10.72 g, 45%). H

NMR (400 MHz, DMSO-d₆) δ 7.85-8.00 (m, 3H), 4.02 (s, 3H).

2-Methoxy-4-nitro-1-pentafluoroethylbenzene (41c). To a

cooled (0 °C) suspension of zinc powder (54.15 g, 828 mmol) in

DMF (200 mL), pentafluoroethyl iodide (excess) was bubbled

through while the mixture was gradually allowed to warm to rt.

The reaction mixture was stirred at rt for 6 h, at which point the

bubbling ceased. The solution was transferred to an addition funnel

and added dropwise to a slurry of CuBr (66.57 g, 464 mmol) in

DMF (200 mL) over 30 min, keeping the temperature below 30

°C. To this mixture, 2-nitro-5-iodoanisole (50.0 g, 179.2 mmol)

was added and the reaction mixture was heated to 70 °C for 6 h,

then allowed to cool to rt and stirred for 8 h. The reaction mixture

was poured into a mixture of ice water (600 mL), 6 N HCl (500

mL), and ethyl acetate (300 mL). The layers were separated and

the organic layer was washed with water then NaHCO₃(satd) and

brine. The solution was dried over Na₂SO₄, filtered, and concen-

trated in vacuo. The residue was purified by column chromatog-

raphy (hexanes/ethyl acetate as the eluent) to give the title

4366 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18

Potashman et al.

1

compound as a yellow oil (41.0 g, 84%). H NMR (400 MHz,

and extracted with EtOAc. The organic layer was dried over Na₂-

SO₄, filtered, and concentrated in vacuo. Purification by silica gel

column chromatography yielded the title compound as a light brown

oil (1.26 g, 27%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.00 (s, 1H),

7.90-7.92 (m, 2H), 4.34 (t, J ) 5.6 Hz, 2H), 2.69 (t, J ) 5.6 Hz,

2H), 2.23 (s, 6H).

1-Chloro-2-(2-methoxyethoxy)-4-nitrobenzene (43d). To a

solution of 5-nitro-2-chlorophenol (1.00 g, 5.80 mmol) and 2-

methyloxychloroethane (2.60 mL, 28.8 mmol) in DMF (20 mL)

was added K₂CO₃(2.40 g, 17.4 mmol). The suspension was heated

at 80 °C for 18 h. After cooling to rt, the mixture was filtered, the

salts were washed with EtOAc, and the solvent was concentrated

in vacuo. The residue was dissolved in EtOAc, washed with 1 N

NaOH and water, dried over MgSO₄, and concentrated in vacuo.

The crude material was purified on silica gel using a hexanes/EtOAc

gradient (100/0 to 50/50) to give the title compound (1.3 g, 96%).

¹H NMR (400 MHz, DMSO-d₆) δ 7.95 (d, J ) 2.6 Hz, 1H), 7.85

(dd, J ) 9.1, 2.6 Hz, 1H), 7.75 (d, J ) 9.1 Hz, 1H), 4.35-4.40

(m, 2H), 3.70-3.75 (m, 2H), 3.35 (s, 3H).

4-Chloro-3-(2-chloro-ethoxy)-phenylamine (44a). To an argon-

degassed solution of 1-chloro-2-(2-chloro-ethoxy)-4-nitro-benzene

(2.9 g, <12.5 mmol due to contamination with the bromide) in

EtOAc (50 mL) was added Pd/C (10% by wt, 1 g). The reaction

was stirred under H₂for 18 h, then filtered through Celite, and

concentrated in vacuo to yield the title compound (as a mixture

with the bromo adduct) (2.54 g, approx 92%). ¹H NMR of the major

product (400 MHz, DMSO-d₆) δ 7.01 (d, J ) 8.4 Hz, 1H), 6.35

(d, J ) 2.8 Hz, 1H), 6.19 (dd, J ) 8.4, 2.8 Hz, 1H), 5.70 (bs, 2H),

4.17 (t, J ) 5.2 Hz, 2H), 3.91 (t, J ) 5.2 Hz, 2H). ¹H NMR of the

minor product (400 MHz, DMSO-d₆) δ 7.01 (d, J ) 8.4 Hz, 1H),

6.35 (d, J ) 2.8 Hz, 1H), 6.19 (dd, J ) 8.4, 2.8 Hz, 1H), 5.70 (bs,

2H) 4.23 (t, J ) 5.2 Hz, 2H), 3.77 (t, J ) 5.2 Hz, 2H).

4-(5-Amino-2-chloro-phenoxymethyl)-piperidine-1-carboxy-

lic Acid tert-Butyl Ester (44b). Starting from 4-(2-chloro-5-nitro-

phenoxymethyl)-piperidine-1-carboxylic acid tert-butyl ester (1.00

g, 2.69 mmol), 54 mg (6%) of the title compound was obtained

according to the method described for the synthesis of 46a. ¹H NMR

(400 MHz, DMSO-d₆) δ 6.93 (d, J ) 8.7 Hz, 1H), 6.28 (d, J ) 2.4

Hz, 1H), 6.10 (dd, J ) 8.8, 2.4 Hz, 1H), 3.95-4.00 (m, 2H), 5.20

(s, 2H), 3.75 (d, J ) 6.3 Hz, 2H), 2.60-2.80 (m, 2H), 1.80-1.95

(m, 1H), 1.70-1.80 (m, 2H), 1.37 (s, 9H), 1.10-1.20 (m, 2H).

3-(2-Dimethylamino-ethoxy)-4-trifluoromethylaniline (44c). A

flask was charged with 1-nitro-3-(2-dimethylamino-ethoxy)-4-

trifluoromethylbenzene (1.26 g, 4.51 mmol) and dissolved in MeOH

(50 mL). To the nitrogen-degassed solution was added 10% Pd/C

(0.10 g). The reaction was stirred vigorously at rt under 1 atm of

H₂gas for 18 h. The reaction was filtered through Celite and

concentrated in vacuo to yield the title compound as a gray solid

(1.03 g, 93%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.14 (d, J ) 7.7

Hz, 1H), 6.27 (s, 1H), 6.13 (d, J ) 7.7 Hz, 1H), 5.78 (s, 2H), 3.99

(t, J ) 6.0 Hz, 2H), 2.60 (t, J ) 6.0 Hz, 2H), 2.19 (s, 3H).

3-(2-Pyrrolin-1-yl-ethoxy)-5-trifluoromethyl-phenylamine (44d).

A flask charged with 1-methoxy-3-nitro-5-trifluoromethyl-benzene

(10 g, 45.2 mmol) and pyridine‚HCl (41.8 g) was heated at 210 °C

open to the air. After 2.5 h, the reaction mixture was allowed to

cool to rt and partitioned between 1 N HCl and EtOAc. The organic

portion was washed with 1 N HCl and brine, dried with Na₂SO₄,

filtered, and concentrated in vacuo to form 3-nitro-5-trifluoromethyl-

phenol (42b) as an off-white solid. A portion of this material (2.00

g, 9.66 mmol) was combined with 1-(2-chloroethyl)pyrrolidine

hydrochloride (3.40 g, 19.3 mmol), K₂CO₃(5.34 g, 38.64 mmol),

and DMF (30 mL) and heated at 90 °C for 3 d. The reaction mixture

was allowed to cool to rt, taken up in EtOAc, and washed with 2

N NaOH and brine. The organic layer was dried with MgSO₄,

filtered, and concentrated in vacuo. The aqueous layer was acidified,

extracted with EtOAc, dried with MgSO₄, filtered, concentrated in

vacuo, and combined with the other portion. The crude 1-[2-(3-

nitro-5-trifluoromethyl-phenoxy)ethyl]-pyrrolidine was reduced to

the amine following the procedure outlined for 58e. The title

compound was obtained as a tan solid (560 mg, 21% over 2 steps).

¹H NMR (400 MHz, DMSO-d₆) δ 6.41 (s, 1H), 6.32 (s, 1H), 6.28

DMSO-d₆) δ 7.70-7.86 (m, 3H), 3.85 (s, 3H).

2-Chloro-5-nitrophenol (42a). To a flask containing HOAc (78

mL) and 48% HBr (97 mL) was added 2-chloro-5-nitroanisole (9.7

g, 49.1 mmol). The mixture was heated for 18 h at 140 °C. The

reaction mixture was allowed to cool to rt, diluted with ice water,

and extracted with EtOAc once the ice had melted. The organic

layer was washed with brine, dried over Na₂SO₄, filtered, concen-

trated, and dried under vacuum to yield the title compound as

yellow-light brown solid (8.3 g, 98%). ¹H NMR (400 MHz, DMSO-

d₆) δ 11.32 (s, 1H), 7.74 (d, J ) 1.6 Hz, 1H), 7.62-7.67 (m, 2H).

5-Nitro-2-trifluoromethylphenol (42b). A round-bottom flask

was charged with 5-nitro-2-trifluoromethylanisole (10.7 g, 48.5

mmol) and pyridine hydrochloride (44.9 g, 388 mmol) and heated

at 210 °C for 2 h. The reaction mixture was allowed to cool to rt

and dissolved into 6 N HCl and EtOAc. The layers were separated,

and the organic layer was washed 4× with 2 N HCl and once with

brine, dried over Na₂SO₄, filtered, and concentrated in vacuo. The

title compound was obtained as a dark red solid (8.98 g, 89%). ¹H

NMR (400 MHz, DMSO-d₆) δ 11.67 (s, 1H), 7.60-7.85 (m, 3H).

5-Nitro-2-pentafluoroethylphenol (42c). A round-bottom flask

was charged with 2-methoxy-4-nitro-1-pentafluoroethylbenzene

(9.35 g) and pyridine hydrochloride and heated at 210 °C for 1 h.

The mixture was allowed to cool to rt and dissolved in EtOAc and

2 N HCl (>500 mL). The organic layer was washed with 2 N HCl

(2×) and concentrated in vacuo. The residue was dissolved in

hexanes and Et₂O and washed with 2 N HCl and then brine. The

organic layer was dried over Na₂SO₄, filtered, concentrated in vacuo,

and dried under high vacuum to provide 5-nitro-2-pentafluorom-

ethylphenol as a dark brown oil (8.77 g, 99%). ¹H NMR (400 MHz,

DMSO-d₆) δ 11.50 (s, 1H), 7.60-7.70 (m, 3H).

1-Chloro-2-(2-chloro-ethoxy)-4-nitro-benzene (43a). A flask

was charged with 2-chloro-5-nitrophenol (4 g, 23 mmol), 1-bromo-

2-chloroethane (9 mL, 115 mmol), K₂CO₃(8 g, 58 mmol), and

DMF (50 mL) and heated at 80 °C for 18 h. The reaction mixture

was allowed to cool to rt, diluted with water, and extracted several

times with EtOAc. The combined organic layers were washed with

1 N NaOH and concentrated in vacuo to yield the title compound

in a 2:1 ratio with the bromo adduct (3.9 g). ¹H NMR of the major

product (400 MHz, DMSO-d₆) δ 7.92-7.94 (m, 1H), 7.84 (d, J )

3.0 Hz, 1H), 7.76 (s, 1H), 4.52 (t, J ) 5.5 Hz, 2H), 4.00 (t, J ) 5.5

1

Hz, 2H); H NMR of the minor product (400 MHz, DMSO-d₆) δ

7.92-7.94 (m, 1H), 7.86 (d, J ) 3 Hz, 1H), 7.72 (s, 1H), 4.58 (t,

J ) 5.5 Hz, 2H), 3.85 (t, J ) 5.5 Hz, 2H).

4-(2-Chloro-5-nitro-phenoxymethyl)-piperidine-1-carboxyl-

ic Acid tert-Butyl Ester (43b). To a cooled solution (-20 °C) of

2-chloro-5-nitro phenol (10 g, 63.497 mmol), N-boc-4-piperidine

methanol (13.67 g, 63.49 mmol), and PPh₃(16.63 g, 63.49 mmol)

in THF (130 mL) was added a solution of DIAD (12.75 mL, 64.76

mmol) in THF (50 mL) dropwise over 1.5 h. The reaction mixture

was allowed to warm to rt and stirred for 18 h. The mixture was

concentrated in vacuo, and the residue was dissolved in Et₂O and

washed with water and then NaHCO₃(satd). The organic layer was

dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue

was treated with a mixture of hexanes and EtOAc (1:1), and the

solid was filtered. The filtrate was evaporated and the residue was

purified by silica gel column chromatography to yield the title

1

compound as a yellow solid (5.59 g, 87%). H NMR (400 MHz,

DMSO-d₆) δ 7.86 (d, J ) 2.5 Hz, 1H), 7.80-7.83 (m, 1H), 7.73

(d, J ) 7.8 Hz, 1H), 4.08 (d, J ) 6.3 Hz, 2H), 3.90-4.00 (m, 2H),

2.60-2.80 (m, 2H), 1.90-2.05 (m, 1H), 1.70-1.80 (m, 2H), 1.37

(s, 9H), 1.10-1.25 (m, 2H).

1-Nitro-3-(2-dimethylamino-ethoxy)-4-trifluoromethylben-

zene (43c). A sealed tube was charged with 5-nitro-2-trifluoro-

methylphenol (3.48 g, 16.8 mmol), 2-(dimethylamino)ethylchloride

hydrochloride (5.32 g, 37.0 mmol), K₂CO₃(9.76 g, 70.7 mmol),

tetrabutylammonium iodide (0.5 g, 1.34 mmol), acetone (114 mL),

and water (33 mL). The mixture was heated at 105 °C for 24 h,

and then the temperature was raised and the mixture was heated at

120 °C for 24 h. The reaction mixture was cooled to rt and partially

concentrated. The solution was diluted with brine and 6 N NaOH

Benzimidazoles and Benzoxazoles as VEGFR-2 Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4367

(s, 1H), 5.55 (s, 2H), 3.98 (t, J ) 5.5 Hz, 2H), 2.72 (t, J ) 5.9 Hz,

2H), 2.50-2.55 (m, 4H), 1.60-1.68 (m, 4H).

(tert-butoxy-carbonyl)-2-pyrrolidinemethanol (5.34 g, 26.55 mmol),

and PPh₃(6.96 g, 26.55 mmol) in THF (46 mL) was added a

solution of DEAD (4.71 g, 27.08 mmol) in THF (23 mL) dropwise

over 1.5 h. The reaction mixture was allowed to warm to rt and

stirred for 18 h. The reaction was concentrated in vacuo and treated

with a small mixture of hexanes and Et₂O. After sonication, the

solids were filtered off, and the filtrate was concentrated in vacuo.

The crude mixture was dissolved in a small amount of EtOAc, then

diluted with hexanes, and washed with 0.1 N HCl, 2 N NaOH, and

brine. The organic layer was dried over Na₂SO₄, filtered, and

concentrated in vacuo. The residue was purified by silica gel column

chromatography using 5% EtOAc in hexanes as the eluent to yield

1-(2-Chloro-5-nitro-benzyl)-4-methyl-piperazine (45a). To a

rt solution of 2-chloro-5-nitrobenzaldehyde (3.28 g, 17.7 mmol)

and 1-methylpiperazine (1.77 g, 17.7 mmol) in CH₂Cl₂(60 mL)

was added NaBH(OAc)₃(5.24 g, 24.7 mmol). The reaction mixture

was stirred overnight at rt. The mixture was allowed to cool to rt,

and 2 N NaOH (150 mL) was added and the mixture was stirred

for 10 min. The reaction mixture was diluted with CH₂Cl₂and

additional 2 N NaOH. The layers were separated, the CH₂Cl₂layer

was washed with 2 N NaOH, and the aqueous layers were back-

extracted with CH₂Cl₂. The combined CH₂Cl₂layers were extracted

with 2 N HCl, which was then basified to pH > 12 by treatment

with solid NaOH. This aqueous layer was extracted with EtOAc,

and the EtOAc layers were combined, washed with a mix of brine

and 2 N NaOH, dried over Na₂SO₄, filtered, and concentrated in

vacuo. The title compound was isolated as an amber oil (4.24 g,

89%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.27 (d, J ) 3.0 Hz, 1H),

8.11 (dd, J ) 8.7, 3.3 Hz, 1H), 7.72 (d, J ) 8.7 Hz, 1H), 3.62 (s,

2H), 2.42-2.50 (m, 4H), 2.30-2.40 (m, 4H), 2.16 (s, 3H).

4-(2-Chloro-5-nitro-benzyl)-morpholine (45b). Starting from

2-chloro-5-nitrobenzaldehyde (6.14 g, 33.0 mmol), 5.96 g (70%)

of the title compound was obtained as a yellow solid according to

the method described for the synthesis of 45a. ¹H NMR (400 MHz,

DMSO-d₆) δ 8.30 (d, J ) 3.2 Hz, 1H), 8.12 (dd, J ) 8.8, 3.2 Hz,

1H), 7.74 (d, J ) 8.8 Hz, 1H), 3.64 (s, 2H), 3.57-3.60 (m, 4H),

2.43-2.46 (m, 4H).

1

the title compound as a light yellow oil (6.36 g, 61%). H NMR

(400 MHz, DMSO-d₆) δ 7.85-8.05 (m, 3H), 4.20-4.35 (m, 2H),

4.00-4.10 (m, 1H), 3.20-3.30 (m, 2H), 1.70-2.05 (m, 4H), 1.37

(s, 9H).

(R)-2-(5-Nitro-2-pentafluoroethyl-phenoxymethyl)-1-(tert-bu-

toxycarbonyl)pyrrolidine (47c). A flask was charged with 5-nitro-

2-pentafluoroethyl-phenol (945.0 mg, 3.7 mmol), PPh₃(965.0 mg,

3.7 mmol), R-(+)-(1-tert-butoxycarbonyl)-2-pyrrolidine-methanol

(740 mg, 3.7 mmol), and THF (9 mL). The mixture was stirred to

dissolve the solids and cooled to -20 °C. DIAD (738 µL, 3.8 mmol)

in THF (4 mL) was added over 2 h, keeping the reaction

temperature between -10 to -20 °C. The reaction was allowed to

warm to rt and stirred for 19 h. The THF was concentrated in vacuo,

and the crude mixture was dissolved in ethyl acetate, washed with

water and brine, dried with MgSO₄, filtered, and evaporated. The

mixture was purified by column chromatography using EtOAc/

hexanes as the eluent. The title compound was obtained as a viscous

liquid (937 mg, 58%). ¹H NMR (rotamers) (400 MHz, DMSO-d₆)

δ 8.00-8.05 (m, 1H), 7.80-7.95 (m, 2H), 4.20-4.30 (m, 2H),

4.00-4.10 (m, 1H), 3.20-3.20 (m, 2H), 1.70-2.00 (m, 4H), 1.36-

1.38 (m, 9H).

(S)-2-(2-Chloro-5-nitro-phenoxymethyl)-pyrrolidine (48a). (S)-

2-(2-Chloro-5-nitro-phenoxymethyl)-pyrrolidine-1-carboxylic acid

tert-butyl ester (6.94 g, 19.45 mmol) was stirred in CH₂Cl₂(30

mL) and TFA (20 mL) for 3 h. The reaction mixture was

concentrated in vacuo, and the residue was dissolved in 0.1 N HCl.

The solution was basified to pH > 12 with solid NaOH and

extracted with EtOAc (×3). The organic layers were combined,

washed with 1 N NaOH and then a mixture of brine and 1 N NaOH,

dried over Na₂SO₄, filtered, and concentrated to dryness. The title

compound was obtained as a yellow solid (4.82 g, 97%). ¹H NMR

(400 MHz, DMSO-d₆) δ 7.87-7.90 (m, 1H), 7.80-7.84 (m, 1H),

7.30 (d, J ) 8.8 Hz, 1H), 4.05 (d, J ) 6.0 Hz, 2H), 3.43-3.49 (m,

1H), 2.79-2.84 (m, 2H), 1.47-1.90 (m, 4H).

1-(2-Chloro-5-amino-benzyl)-4-methyl-piperazine (46a). To a

solution of 1-(2-chloro-5-nitro-benzyl)-4-methyl-piperazine (1.04

g, 3.86 mmol) in EtOH (30 mL) was added SnCl₂(2.2 g, 11.6

mmol), and the reaction mixture was heated at 70 °C for 4.5 h. An

additional amount of SnCl₂(0.7 g, 3.7 mmol) was added, and the

reaction was heated at 80 °C for 1 h. The reaction was allowed to

cool to rt and quenched with 1 N aqueous K₂CO₃. The white slurry

was filtered through a Celite plug, the ethanol was evaporated, and

the resulting aqueous suspension was diluted with EtOAc and 1 N

NaOH. The layers were separated, and the organic layer was washed

with a brine/1 N NaOH mixture. The aqueous layers were back-

extracted with EtOAc. The combined organic layers were dried

over Na₂SO₄, filtered, and concentrated in vacuo to yield the title

1

compound as a light orange solid (793 mg, 86%). H NMR (400

MHz, DMSO-d₆) δ 6.97 (d, J ) 8.4 Hz, 1H), 6.66 (s, 1H), 6.41 (d,

J ) 8.4 Hz, 1H), 5.18 (s, 2H), 3.32 (s, 2H), 2.28-2.42 (m, 8H),

2.13 (s, 3H).

4-Chloro-3-morpholin-4-ylmethyl-phenylamine (46b). Starting

from 4-(2-chloro-5-nitro-benzyl)-morpholine (5.96 g, 23.2 mmol),

2.60 mg (49%) of the title compound was obtained as a light orange

solid according to the method described for the synthesis of 46a.

¹H NMR (400 MHz, DMSO-d₆) δ 6.98 (d, J ) 8.4 Hz, 1H), 6.80

(d, J ) 2.8 Hz, 1H), 6.42 (dd, J ) 8.4, 2.8 Hz, 1H), 5.18 (s, 2H),

3.54-3.58 (m ,4H), 3.37 (s, 2H), 2.35-2.39 (m, 4H).

(R)-2-((5-Nitro-2-(trifluoromethyl)phenoxy)methyl)-

pyrrolidine (48b). To a solution of (R)-tert-butyl 2-((5-nitro-2-

(trifluoromethyl)phenoxy)methyl)pyrrolidine-1-carboxylate (6.36 g,

16.29 mmol) in CH₂Cl₂(20 mL), TFA (20 mL) was added. After

stirring for 1 h at rt, the mixture was concentrated in vacuo and

treated with 6 N NaOH until pH 10. The mixture was taken up

into ethyl acetate and washed with brine, and the organic layer

was dried over Na₂SO₄, filtered, and evaporated. The title compound

(S)-2-(2-Chloro-5-nitro-phenoxymethyl)-pyrrolidine-1-car-

boxylic Acid tert-Butyl Ester (47a). To a cooled solution (-15

°C) of 2-chloro-5-nitrophenol (5.06 g, 29.17 mmol), (S)-(-)-1-(tert-

butoxycarbonyl)-2-pyrrolidinemethanol (5.87 g, 29.17 mmol), and

triphenylphosphine (7.65 g, 29.17 mmol) in THF (50 mL) was

added a solution of DIAD (6.02 g, 29.75 mmol) in THF dropwise

over 75 min. Upon the complete addition, the reaction mixture was

allowed to warm to rt and stirred for 18 h. The mixture was

concentrated in vacuo, and the residue was treated with a mixture

of Et₂O and hexanes. The suspension was sonicated and the solid

was filtered. The filtrate was concentrated in vacuo, and the resulting

residue was purified by silica gel column chromatography using

7% EtOAc in hexanes to yield the title compound as a thick yellow

1

was obtained as a dark yellow oil (4.16 g, 88%). H NMR (400

MHz, DMSO-d₆) δ 7.96 (s, 1H), 7.87-7.92 (m, 2H), 4.05-4.15

(m, 2H), 3.40-3.50 (m, 1H), 3.31 (bs, 1H), 2.47-2.50 (m, 2H),

1.80-1.89 (m, 1H), 1.58-1.77 (m, 2H), 1.46-1.56 (m, 1H).

(R)-2-(5-Nitro-2-pentafluoroethyl-phenoxymethyl)-pyrroli-

dine (48c). To a solution of 2-(5-nitro-2-pentafluoroethyl-phe-

noxymethyl)-1-(tert-butoxycarbonyl)pyrrolidine (727 mg, 1.77

mmol) in CH₂Cl₂(5 mL), TFA (2.5 mL) was added and stirred at

rt for 1 h. The mixture was diluted with CH₂Cl₂(20 mL) and

neutralized with satd NaHCO₃and then 2 N NaOH. The mixture

was transferred to a separatory funnel and the layers were separated.

The aqueous layer was extracted with EtOAc and the combined

organic layers were dried with MgSO₄, filtered, and concentrated

in vacuo to yield the title compound as a yellow solid (603 mg,

1

oil (8.94 g, 97%). H NMR (400 MHz, DMSO-d₆) δ 7.91-7.96

(m, 1H), 7.80-7.86 (m, 1H), 7.72-7.76 (m, 1H), 4.16-4.30 (m,

2H), 4.02-4.10 (m, 1H), 3.24-3.30 (m, 2H), 1.90-2.10 (m, 3H),

1.74-1.84 (m, 1H), 1.36 (s, 9H).

(R)-2-(5-Nitro-2-trifluoromethyl-phenoxymethyl)-1-(tert-bu-

toxycarbonyl)pyrrolidine (47b). To a cooled solution (-20 °C)

of 5-nitro-2-trifluoromethylphenol (5.50 g, 26.55 mmol), (R)-(+)-

1

100%). H NMR (400 MHz, DMSO-d₆) δ 8.74 (bs, 1H), 7.97-

8.04 (m, 1H), 7.89-7.94 (m, 1H), 4.45-4.50 (m, 1H), 4.35-4.40

4368 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18

Potashman et al.

(m, 1H), 3.85-3.95 (m, 1H), 3.10-3.25 (m, 2H), 2.05-2.18 (m,

1H), 1.70-2.00 (m, 3H).

10.89 mmol) in dioxane (80 mL) and methanol (80 mL), Pd/C (10

wt %, 650 mg) was added. The mixture was stirred under H₂for

several days until the starting material was consumed. The reaction

mixture was filtered through Celite and concentrated in vacuo to

yield the title compound as a light yellow solid (2.98 g, 100%). ¹H

NMR (400 MHz, DMSO-d₆) δ 7.13 (d, J ) 8.4 Hz, 1H), 6.25-

6.30 (m, 1H), 6.13 (dd, J ) 8.4, 1.6 Hz, 1H), 5.75 (bs, 2H), 3.88-

3.94 (m, 1H), 3.70-3.88 (m, 1H), 2.90-2.97 (m, 1H), 2.50-2.58

(m, 1H), 2.33 (s, 3H), 2.13-2.22 (m, 1H), 1.88-1.98 (m, 1H),

1.62-1.72 (m, 2H), 1.50-1.58 (m, 1H).

(S)-2-(2-Chloro-5-nitro-phenoxymethyl)-1-methylpyrroli-

dine (49a). To a solution of (S)-2-(2-chloro-5-nitro-phenoxymethyl)-

pyrrolidine (4.82 g, 18.77 mmol) in CH₃CN (167 mL) was added

37% aqueous formaldehyde (7.58 mL) and NaCNBH₃(1.887 g,

30.03 mmol) at rt. The mixture was stirred at rt for 1 h, with the

reaction pH adjusted every 10 min to about 7 by adding small

amounts of HOAc. The reaction mixture was concentrated in vacuo

and dissolved in 2 N NaOH (aq) and Et₂O. The layers were

separated, and the organic layer was washed with 2 N NaOH, and

extracted with 1 N HCl. The combined acidic aqueous layers were

basified to pH > 12 with solid NaOH and extracted with EtOAc

(×3). The EtOAc layers were combined and washed with 2 N

NaOH and a mixture of brine and 2 N NaOH. The organic layer

was dried over Na₂SO₄, filtered, and concentrated in vacuo to yield

the title compound (3.42 g, 67%). ¹H NMR (400 MHz, DMSO-d₆)

δ 7.89-7.91 (m, 1H), 7.80-7.84 (m, 1H), 7.71-7.74 (m, 1H),

4.12-4.14 (m, 2H), 2.93-2.98 (m, 1H), 2.60-2.70 (m, 1H), 2.40

(s, 3H), 2.18-2.40 (m, 1H), 1.90-2.0 (m, 1H), 1.60-1.76 (m, 3H).

(R)-1-Methyl-2-((5-nitro-2-(trifluoromethyl)phenoxy)methyl)-

pyrrolidine (49b). To a solution of (R)-2-((5-nitro-2-(trifluoro-

methyl)phenoxy)methyl)pyrrolidine (4.16 g, 14.33 mmol) in CH₃CN

(128 mL), formaldehyde (5.8 mL, 37% aqueous) was added and

the mixture was stirred. Sodium cyanoborohydride (1.44 g, 22.9

mmol) was added (exotherm observed), and the reaction was stirred

at rt. The pH was monitored every 15 min and adjusted to ∼7 with

AcOH. After 45 min, the mixture was concentrated in vacuo and

the residue was dissolved in Et₂O and washed with 6 N NaOH, 1

N NaOH, and 2 N HCl (3×). The acid washings were combined,

adjusted to ∼pH 10 with solid Na₂CO₃, and extracted with EtOAc

(×2). The EtOAc fractions were combined, dried with Na₂SO₄,

and purified with flash chromatography (SiO₂, 95:5:0.5 CH₂Cl₂/

(R)-1-Methyl-2-(5-amino-2-pentafluoroethyl-phenoxymethyl)-

pyrrolidine (50c). Starting from (R)-1-methyl-2-(5-nitro-2-penta-

fluoroethyl-phenoxymethyl)-pyrrolidine (455 mg, 1.28 mmol), 313

mg (75%) of the title compound was obtained according to the

1

method described for the synthesis of 58e. H NMR (400 MHz,

DMSO-d₆) δ 7.06 (d, J ) 8.5 Hz, 1H), 6.28 (d, J ) 1.5 Hz, 1H),

6.19 (dd, J ) 8.5, 1.8 Hz, 1H), 5.79 (s, 2H), 3.90-3.95 (m, 1H),

3.60-3.70 (m, 1H), 2.90-2.95 (m, 1H), 2.32 (s, 3H), 2.15-2.20

(m, 1H), 1.90-2.00 (M, 1H), 1.60-1.70 (m, 2H), 1.50-1.60 (m,

1H).

4-(3,4-Dinitrophenoxy)pyridine-2-carboxylic Acid Methyl-

amide (52a). A 50 mL flask was charged with 3,4-dinitrophenol

(12.3 g, 66.8 mmol) and 4-chloro-pyridine-2-carboxylic acid

methylamide²⁰(53; 7.79 g, 45.7 mmol) and fitted with a running

water condenser. The mixture was heated at 150 °C for 1 h and at

170 °C for 16 h. The flask was allowed to cool to rt, and the crude

mixture was dissolved in CH₂Cl₂and 2 N NaOH (aq). The layers

were separated, and the organic layer was washed with a mix of

brine and 2 N NaOH. The aqueous layers were back-extracted with

CH₂Cl₂. The combined organic layers were dried over Na₂SO₄,

filtered, and concentrated in vacuo. The crude residue was purified

by silica gel column chromatography using a hexanes to hexanes/

1

EtOAc gradient to yield the title compound (93.5 mg, 15%). H

1

MeOH/NH₄OH) to afford the title compound (3.16 g, 73%). H

NMR (300 MHz, DMSO-d₆) δ 8.80-8.88 (m, 1H), 8.63 (d, J )

5.7 Hz, 1H), 8.34 (d, J ) 9.0 Hz, 1H), 8.12 (d, J ) 2.4 Hz, 1H),

7.70 (dd, J ) 9.0, 2.4 Hz, 1H), 7.66 (d, J ) 2.4 Hz, 1H), 7.40-

7.43 (m, 1H), 2.81 (d, J ) 4.5 Hz, 3H).

NMR (400 MHz, DMSO-d₆) δ 7.80-8.00 (m, 3H), 4.00-4.15 (m,

1H), 4.20-4.30 (m, 1H), 2.90-3.00 (m, 1H), 2.60-2.70 (m, 1H),

2.37 (S, 3H), 2.10-2.20 (m, 1H), 1.80-2.00 (m, 1H), 1.50-1.75

(m, 3H).

4-(3,4-Dinitrophenoxy)-6,7-dimethoxyquinoline (52b). A flask

was charged with 6,7-dimethoxy-4-chloroquinoline (0.35 g, 1.6

mmol) and 3,4-dinitrophenol (0.85 g, 4.6 mmol) and heated at

150 °C for 4 h. The reaction mixture was allowed to cool at rt

suspended in methanol. The suspension was stirred with methanol

for 3 h at which point the precipitate was filtered and washed with

(R)-1-Methyl-2-(5-nitro-2-pentafluoroethyl-phenoxymethyl)-

pyrrolidine (49c). To a solution of 2-(5-nitro-2-pentafluoroethyl-

phenoxymethyl)-pyrrolidine (603.0 mg, 1.8 mmol) and formalde-

hyde (37% in water, 1 mL) in CH₂Cl₂(25 mL), NaBH(OAc)₃(600

mg, 2.8 mmol) was added. The mixture was stirred at rt for 15 h.

The reaction mixture was quenched with water, and the organic

layer was washed with 2 N NaOH, dried with Na₂SO₄, filtered,

and evaporated. The residue was purified by column chromatog-

raphy to give the title compound as a yellow solid (455 mg, 73%).

¹H NMR (400 MHz, DMSO-d₆) δ 8.00 (s, 1H), 7.90-7.94 (m,

1H), 7.84-7.85 (m, 1H), 4.24-4.30 (m, 1H), 3.98-4.04 (m, 1H),

2.90-2.98 (m ,1 H), 2.54-2.62 (m, 1H), 2.34 (s, 3H), 2.16-2.22

(m, 1H), 1.90-2.00 (m, 1H), 1.56-1.72 (m 3H).

1

fresh methanol to afford the title compound (256 mg, 46%). H

NMR (400 MHz, DMSO-d₆) δ 8.93 (d, J ) 6.3 Hz, 1H), 8.65 (d,

J ) 8.4 Hz, 1H), 8.40 (d, J ) 2.1 Hz, 1H), 7.98 (dd, J ) 8.4 Hz,

2.1 Hz, 1H), 7.73 (s, 1H), 7.67 (s, 1H), 7.34 (d, J ) 6.3 Hz, 1H),

4.03 (s, 3H), 3.95 (s, 3H).

4-(3,4-Dinitro-phenoxy)-quinoline (52c). A flask charged with

4-chloroquinoline (4.3 g, 26.3 mmol) and 3,4-dinitrophenol (4.5 g,

24.4 mmol) was heated (neat) at 150 °C for 30 min. The mixture

was allowed to cool to rt, and the residue was dissolved in CH₂Cl₂,

washed with 2 M NaOH, dried with Na₂SO₄, filtered, and

concentrated in vacuo. The residue was dissolved in EtOAc and

filtered through a pad of silica gel, and the solvent was concentrated

in vacuo to give the title compound as a brown solid (2.6 g, 33%).

¹H NMR (400 MHz, DMSO-d₆) δ 8.86 (d, J ) 5.6 Hz, 1H), 8.33

(d, J ) 8.4 Hz, 1H), 8.18-8.21 (m, 1H), 8.15 (d, J ) 8.4 Hz, 1H),

8.10 (d, J ) 8.4 Hz, 1H), 7.83-7.88 (m, 1H), 7.64-7.73 (m, 2H),

7.20 (d, J ) 5.6 Hz, 1H).

4-(3,4-Dinitro-phenoxy)-7,7a-dihydro-4aH-pyrrolo[2,3-d]py-

rimidine (52d). A flask was charged with 4-chloro-7,7a-dihydro-

4aH-pyrrolo[2,3-d]pyrimidine (1.66 g, 10.8 mmol), 3,4-dinitrophe-

nol (2.4 g, 13 mmol), and a TFA/TEA mixture (1.1 mL), and the

reaction mixture was heated at 150 °C for 2 h. The mixture was

allowed to cool to rt and a green solid formed. This material was

purified on silica gel using a hexane/EtOAc gradient (100/0 to 50/

50) to give the title compound (460 mg, 14%). ¹H NMR (400 MHz,

DMSO-d₆) δ 8.36-8.38 (m, 2H), 8.34 (s, 1H), 8.30 (d, J ) 2.9

Hz, 1H), 7.90 (dd, J ) 8.7, 2.9 Hz, 1H), 7.56-7.89 (m, 1H), 6.73-

6.76 (m, 1H).

(S)-4-Chloro-3-(1-methyl-pyrrolidin-2-ylmethoxy)-phenyl-

amine (50a). A flask charged with (S)-2-(2-chloro-5-nitro-phe-

noxymethyl)-pyrrolidine (3.42 g, 12.63 mmol), SnCl₂(7.19 g, 37.9

mmol), and EtOH (45 mL) was heated at 75 °C for 11 h. The

reaction mixture was allowed to cool to rt, treated with 15 mL 1 N

K₂CO₃, and stirred 40 min. The suspension was filtered through

Celite and the ethanol was evaporated. The remaining aqueous

solution was diluted with 1 N NaOH and EtOAc. The layers were

separated, and the organic layer was washed with 1 N NaOH and

a mixture of brine and 1 N NaOH. The aqueous layers were

extracted with EtOAc. The combined organic layers were dried

over Na₂SO₄, filtered, and concentrated in vacuo to yield the title

1

compound as a brown solid (2.77 g, 91%). H NMR (400 MHz,

DMSO-d₆) δ 6.93-6.95 (m, 1H), 6.29-6.30 (m, 1H), 6.08-6.11

(m, 1H), 5.20 (s, 2H), 3.75-3.85 (m, 2H), 2.91-2.94 (m, 1H),

2.53-2.61 (m, 1H), 2.37 (s, 3H), 2.14-2.21 (m, 1H), 1.88-1.98

(m, 1H), 1.63-1.70 (m, 2H), 1.53-1.57 (m, 1H).

(R)-3-((1-Methylpyrrolidin-2-yl)methoxy)-4-(trifluoromethyl)-

benzenamine (50b). To a degassed solution of (R)-1-methyl-2-

((5-nitro-2-(trifluoromethyl)phenoxy)methyl)pyrrolidine (3.16 g,

Benzimidazoles and Benzoxazoles as VEGFR-2 Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4369

4-(3,4-Dinitro-phenoxy)-1H-pyrrolo[2,3-b]pyridine (52e). To

cooled solution of POCl₃(50 mL) was added 1H-pyrrolo[2,3-b]-

pyridine 7-oxide²¹(6.60 g, 55.0 mmol) in portions. Upon complete

addition, the mixture was heated to reflux for 5 h and allowed to

cool to rt. The POCl₃was evaporated under high vacuum with

gentle heating (40-50 °C) to give a black residue. The residue

was diluted with water (50 mL), and the pH was adjusted to 8-9

with Na₂CO₃(first with solid, and then a saturated aqueous

solution). The resulting precipitate was collected by filtration,

washed with cold H₂O, and dried in a vacuum oven (50 °C) to

give 4-chloro-1H-pyrrolo[2,3-b]pyridine as a tan powder (7.7 g,

regioisomeric mixture). A portion of this mixture (0.85 g, 5.50

mmol) was combined with 3,4-dinitrophenol (1.23 g, 6.7 mmol),

and the mixture was heated at 150 °C for 8 h. The reaction mixture

was allowed to cool to rt, and the resulting solid was dissolved in

12 N NaOH and CH₂Cl₂. The aqueous layer was extracted several

times with CH₂Cl₂. The insoluble material was solubilized in

acetone, and the acetone solution was diluted with CH₂Cl₂and

washed with water. The combined CH₂Cl₂layers were dried and

concentrated in vacuo. The crude material was purified on silica

gel using a CH₂Cl₂/EtOH gradient (100/0 to 90/10) to yield the

title compound (350 mg, 21%). ¹H NMR (400 MHz, DMSO-d₆) δ

12.00 (bs, 1H), 8.23-8.29 (m, 2H), 7.99 (d, J ) 2.9 Hz, 1H), 7.46-

7.49 (m, 2H), 6.89 (d, J ) 5.5 Hz, 1H), 6.25-6.35 (m, 1H).

(3,4-Dinitro-phenoxy)-pyridine (52f). A flask charged with

4-chloropyridine (2.45 g, 21.5 mmol) and 3,4-dinitrophenol (4.56

g, 247 mmol) was fitted with a condenser and heated (open to the

air) at 145 °C. After 45 min, 4 N HCl/dioxane (2.1 mL) was added,

and the mixture was heated for an additional 25 min and allowed

to cool to rt. The material was dissolved in a mixture of EtOAc

and 0.5 N HCl/water, and the aqueous layer was basified with 6 N

NaOH. The organic layer was washed with brine, dried with

Na₂SO₄, filtered, and concentrated in vacuo to afford the title

4-(4-Methylamino-3-nitrophenoxy)-pyridine-2-carboxylic Acid

Methylamide (55). To a cooled (0 °C) flask containing CH₂Cl₂

(18 mL) and 4-(4-amino-3-nitro-phenoxy)-pyridine-2-carboxylic

acid methylamide (1.0 g, 3.5 mmol), trifluoroacetic anhydride (0.93

mL, 6.59 mmol) was added dropwise over 15 min and the mixture

was stirred for 45 min. To this cooled solution, tetrabutylammonium

chloride (0.48 g, 1.73 mmol), dimethyl sulfate (0.66 mL, 6.94

mmol), and 50% NaOH/water (by wt, 14 mL) were added. The

mixture was allowed to warm to rt and stirred 16 h. The mixture

was diluted with CH₂Cl₂, washed with water and brine, dried over

Na₂SO₄, filtered, and concentrated in vacuo. The title compound

was obtained as an orange solid after crystallization from 3:1 EtOH/

water (0.87 g, 83% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 8.90-

8.95 (m, 1H), 8.65 (d, J ) 5.7 Hz, 1H), 8.35-8.45 (m, 1H), 8.04

(d, J ) 2.8 Hz, 1H), 7.67 (dd, J ) 9.4, 2.7 Hz, 1H), 7.54 (d, J )

2.6 Hz, 1H), 7.27-7.35 (m, 2H), 3.14 (s, 3H), 2.92 (s, 3H).

4-Benzyloxy-3-chlorophenol (56c).²²To a solution of 4-ben-

zyloxy-3-chlorobenzaldehyde (4 g, 16.2 mmol) in CH₂Cl₂(65 mL),

m-CPBA (3.63 g, 77% max, 21.05 mmol) was added, and the

reaction mixture was stirred at rt for 5 days. The mixture was

washed with a Na₂S₂O₃solution and then NaHCO₃(satd) and

concentrated in vacuo. The residue was suspended in MeOH (150

mL), NaOMe (0.5 M in MeOH, 60 mL) was added, and the mixture

was stirred for 1 h. The mixture was concentrated, and the residue

was dissolved in water and extracted with a mixture of Et₂O/EtOAc.

The aqueous layer was acidified and extracted with EtOAc. The

combined organic portions were dried with Na₂SO₄, filtered, and

evaporated. The mixture was purified by column chromatography

using CH₂Cl₂as the eluent to yield the title compound as an off-

white solid (3.32 g, 87%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.37

(s, 1H), 7.27-7.44 (m, 5H), 7.02 (d, J ) 8.8 Hz, 1H), 6.81 (d, J

) 2.9 Hz, 1H), 6.65 (dd, J ) 8.8, 2.9 Hz, 1H).

4-(4-Benzyloxy-phenoxy)-pyridine-2-carboxylic Acid Methyl-

amide (57a). To a stirred slurry of NaH (2.24 g of 60% oil

dispersion, 55.9 mmol) in DMF (40 mL) was added 4-benzyloxy-

phenol (11.2 g, 55.9 mmol). The mixture was stirred at rt for 10

min, followed by addition of 4-chloro-pyridine-2-carboxylic acid

methylamide (3.18 g, 18.6 mmol). The reaction mixture was stirred

at rt for 5 min, then heated to 75 °C for 2 h, and finally at 85 °C

for 6 h. The reaction was allowed to cool to rt, quenched with

saturated aqueous NaHCO₃, then diluted with Et₂O and 6 N NaOH.

The layers were separated, and the organic layer was washed with

6 N NaOH and brine, dried over Na₂SO₄, filtered, and concentrated

in vacuo to yield the title compound as a light salmon-colored solid

(6.45 g, 103%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.66-8.69 (m,

1H), 8.47 (d, J ) 5.6 Hz, 1H), 7.40-7.47 (m, 2H), 7.29-7.40 (m,

4H), 7.09-7.17 (m, 5H), 5.12 (s, 2H), 2.76 (d, J ) 4.8 Hz, 3H).

4-(4-(Benzyloxy)phenoxy)quinoline (57b). Starting from 4-chlo-

roquinoline (3.0 g, 18.4 mmol), 3.8 g (63%) of the title compound

was obtained as a tan solid according to the method described for

the synthesis of 57a. ¹H NMR (300 MHz, DMSO-d₆) δ 8.64 (d, J

) 5.7 Hz, 1H), 8.27-8.32 (m, 1H), 7.95 (d, J ) 9.5 Hz, 1H), 7.76-

7.82 (m, 1H), 7.58-7.65 (m, 1H), 7.42-7.48 (m, 2H), 7.36-7.42

(m, 2H), 7.30-7.35 (m, 1H), 7.20-7.26 (m, 2H), 7.10-7.16 (m,

2H), 6.50 (d, J ) 5.7 Hz, 1H, 5.12 (s, 2H).

1

compound as a beige solid (1.36 g, 21%). H NMR (400 MHz,

DMSO-d₆) δ 8.58-8.62 (m, 2H), 8.32 (d, J ) 8.8 Hz, 1H), 8.07

(d, J ) 2.4 Hz, 1H), 7.63 (dd, J ) 8.8, 2.4 Hz, 1H), 7.22-7.26

(m, 2H).

4-(3,4-Dinitro-phenoxy)-2-methylsulfanyl-pyrimidine (52g). A

flask charged with 3,4-dinitrophenol (6.10 g, 38.0 mmol) and

2-thiomethyl-4-chloro-1,2-pyrimidine (7.02 g, 38.0 mmol) was

heated at 150 °C for 2 h. The mixture was allowed to cool to rt,

and the resulting solid was finely ground and washed several times

with methyl-t-butylether. The solid was suspended in 2 N NaOH,

recovered by filtration, washed with water and dried under vacuum

1

over P₂O₅to give the title compound (11.26 g, 95%). H NMR

(400 MHz, DMSO-d₆) δ 8.60 (d, J ) 5.2 Hz, 1H), 8.37 (d, J ) 9.0

Hz, 1H), 8.34 (d, J ) 2.6 Hz, 1H), 7.88 (dd, J ) 9.0, 2.6 Hz, 1H),

7.02 (d, J ) 5.2 Hz, 1H), 2.35 (s, 3H).

4-(3,4-Dinitro-phenoxy)-2-methylsulfonyl-pyrimidine (52h).

To a cooled solution (0 °C) of 4-(3,4-dinitro-phenoxy)-2-methyl-

sulfanyl-pyrimidine (0.50 g, 1.60 mmol) in CH₂Cl₂(100 mL) was

added m-CPBA (0.70 g, 4.00 mmol). The solution was allowed to

warm to rt and stirred for 16 h. The reaction mixture was washed

with satd NaHCO₃, dried over MgSO₄, filtered, and concentrated

in vacuo to afford the title compound (0.43 g, 78%). ¹H NMR (300

MHz, DMSO-d₆) δ 9.03 (d, J ) 5.8 Hz, 1H), 8.40-8.42 (m, 2H),

7.98 (dd, J ) 9.2, 2.5 Hz, 1H), 7.65 (d, J ) 5.8 Hz, 1H), 3.30 (s,

3H).

4-(4-Amino-3-nitro-phenoxy)-pyridine-2-carboxylic Acid

Methylamide (54). A solution of 4-amino-3-nitrophenol (97.6 g,

633 mmol) in DMSO (39 g) was treated with KOt-Bu (71.0 g, 633

mmol), and the mixture was stirred at rt for 2 h. The contents of

the flask were treated with 4-chloro-N-methyl-2-pyridine-2-car-

boxylic acid methylamide (90 g, 527.5 mmol) and K₂CO₃(38.6 g,

460 mmol) and heated at 110 °C for 16 h. To the stirred mixture,

water (200 mL) was added slowly. The precipitate was filtered,

washed with water (400 mL), and dried in a vacuum oven overnight

to give the title compound as a purple solid (190 g, 93%). ¹H NMR

(400 MHz, DMSO-d₆) δ 8.80-8.82 (m, 1H), 8.49 (d, J ) 5.7 Hz,

1H), 7.90 (s, 1H), 7.78 (s, 2H), 7.35-7.40 (m, 2H), 7.13-7.16

(m, 2H), 2.80 (s, 3H).

4-(4-(Benzyloxy)-3-chlorophenoxy)quinoline (57c). Starting

with 4-chloroquinoline (725 mg, 5.0 mmol) and 4-benzyloxy-3-

chlorophenol (3 g, 12.78 mmol), 1.37 g (76%) of the title compound

was obtained as a pink solid according to the method described

1

for the synthesis of 57a. H NMR (400 MHz, DMSO-d₆) δ 8.67

(d, J ) 5.7 Hz, 1H), 8.28 (d, J ) 8.4 Hz, 1H), 8.01 (d, J ) 8.4 Hz,

1H), 7.79-7.82 (m, 1H), 7.61-7.64 (m, 1H), 7.46-7.54 (m, 3H),

7.30-7.42 (m, 4H), 7.24-7.30 (m, 1H), 6.58 (d, J ) 5.1 Hz, 1H),

5.23 (s, 2H).

4-(4-(Benzyloxy)-3-chlorophenoxy)-pyridine-2-carboxylic Acid

Methylamide (57d). Starting with 4-chloro-pyridine-2-carboxylic

acid methylamide (367 mg, 2.15 mmol), 609 mg (87%) of the title

compound was obtained as a brown solid according to the method

1

described for the synthesis of 57a. H NMR (400 MHz, DMSO-

d₆) δ 8.75-8.85 (m, 1H), 8.48-8.50 (m, 1H), 7.46-7.50 (m, 3H),

4370 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18

Potashman et al.

7.38-7.44 (m, 2H), 7.32-7.37 (m, 3H), 7.51-7.55 (m, 1H), 7.19-

7.30 (m, 1H), 5.23 (s, 2H), 2.77 (d, J ) 4.8 Hz, 3H).

1H), 8.00 (d, J ) 8.3 Hz, 1H), 7.75-7.85 (m, 1H), 7.60-7.70 (m,

1H), 7.35-7.40 (m ,1H), 7.00-7.15 (m, 2H), 6.57 (d, J ) 5.4 Hz,

1H).

[4-(4-Benzyloxy-phenoxy)-pyrimidin-2-yl]-methyl-amine (57e).

To a stirred rt slurry of NaH (0.5 g of 60% oil dispersion, 12.6

mmol) in DMF (15 mL) was added 4-benzyloxyphenol (2.40 g,

12.0 mmol). The mixture was stirred for 10 min before 2,4-

dichloropyrimidine (1.79 g, 12.0 mmol) was added. A mild

exotherm occurred. The reaction mixture was stirred for 2 h,

quenched with saturated aqueous NaHCO₃, and diluted with EtOAc

and the layers were separated. The organic layer was washed with

2 N NaOH and brine, then dried over Na₂SO₄, filtered, and

concentrated in vacuo to yield crude 4-(4-benzyloxy-phenoxy)-2-

chloro-pyrimidine (4.0 g contaminated with minor 4-Cl impurity).

A portion of this material (2.03 g, approx 6.49 mmol) was dissolved

in 10 mL of DMSO in a sealed tube at 0 °C, and 2 N methylamine

(in THF) was added (4.9 mL, 9.7 mmol) and the tube was sealed.

The mixture was allowed to warm to rt, stirred for 2 h, and then

heated to 70 °C for 2 h. The reaction mixture was allowed to cool

to rt and concentrated in vacuo. The crude solution was diluted

with Et₂O, and washed with 1 N NaOH, water, and then brine.

The organic layer was dried over Na₂SO₄, filtered, and concentrated

in vacuo. The crude material was purified by silica gel column

chromatography using a hexanes/EtOAc gradient to yield the title

4-(3-Chloro-4-hydroxyphenoxy)-pyridine-2-carboxylic Acid

Methylamide (58d). Starting with 4-(4-(benzyloxy)-3-chlorophe-

noxy)-pyridine-2-carboxylic acid methylamide (0.60 g, 1.65 mmol),

578 mg (crude, mixed with salts) of the title compound was obtained

as a tan solid according to the method described for the synthesis

of 58c. H NMR (400 MHz, DMSO-d₆) δ 10.39 (s, 1H), 8.70-

8.85 (m, 1H), 8.47 (d, J ) 5.4 Hz, 1H), 7.30-7.40 (m, 1H), 7.00-

7.15 (m, 4H), 2.76 (d, J ) 4.9 Hz, 3H).

4-(2-Methylamino-pyrimidin-4-yloxy)-phenol (58e). A flask

was charged with [4-(4-benzyloxy-phenoxy)-pyrimidin-2-yl]-meth-

yl-amine (0.75 g, 2.44 mmol) and the solid was dissolved in MeOH

(5 mL) and EtOAc (10 mL). To the argon-degassed solution was

added 10% by weight Pd/C (0.15 g). The reaction mixture was

stirred vigorously at rt under 1 atm H₂gas for 4 days, filtered

through Celite, and concentrated in vacuo to yield the title

compound (517 mg, 98%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.39

(s, 1H), 8.08 (s, 1H), 6.90-7.05 (m, 3H), 6.75 (d, J ) 8.8 Hz,

2H), 5.80-6.00 (m, 1H), 2.66 (s, 3H).

1

4-(6-Methylamino-pyrimidin-4-yloxy)-phenol (58f). Starting

with 6-(4-(benzyloxy)phenoxy)-N-methylpyrimidin-4-amine (1.29

g, 4.20 mmol), 833 mg (97%) of the title compound was obtained

as an off-white solid according to the method described for the

1

compound as a white solid (1.67 g, 84%). H NMR (400 MHz,

DMSO-d₆) δ 8.10 (bs, 1H), 7.30-7.50 (m, 5H), 7.00-7.10 (m,

5H), 5.90-6.10 (m ,1H), 5.08 (s, 2H), 2.60-2.70 (bs, 3H).

6-(4-(Benzyloxy)phenoxy)-N-methylpyrimidin-4-amine (57f).

Starting with 4,6-dichloropyrimidine (4.10 g, 27.6 mmol), 2.89 g

(34%) of the title compound was obtained according to the method

1

synthesis of 58e. H NMR (400 MHz, DMSO-d₆) δ 9.39 (s, 1H),

8.08 (bs, 1H), 7.15-7.25 (m, 1H), 6.88-6.92 (m, 2H), 6.73-6.80

(m, 2H), 5.61 (s, 1H), 2.72 (s, 3H).

4-(4-Hydroxy-phenoxy)-pyridine-2-carboxylic Acid Amide

(58g). Starting with 4-(4-benzyloxy-phenoxy)-pyridine-2-carboxylic

acid amide (8.90 g, 27.78 mmol), 6.22 g (97%) of the title

compound was obtained according to the method described for the

synthesis of 58e. ¹H NMR (400 MHz, DMSO-d₆) δ 9.61 (bs, 1H),

8.46 (d, J ) 5.6 Hz, 1H), 8.08-8.12 (m, 1H), 7.65-7.70 (m, 1H),

7.31 (d, J ) 2.4 Hz, 1H), 7.09 (dd, J ) 5.6, 2.4 Hz, 1H), 6.98-

7.05 (m, 2H), 6.80-6.90 (m, 2H).

1

described for the synthesis of 57a. H NMR (400 MHz, DMSO-

d₆) δ 8.08 (bs, 1H), 7.22-7.48 (m, 6H), 7.00-7.08 (m, 4H), 5.68

(s, 1H), 5.08 (s, 2H), 2.73 (s, 3H).

4-(4-Benzyloxy-phenoxy)-pyridine-2-carboxylic Acid Amide

(57g). Starting with 4-chloro-pyridine-2-carboxylic acid amide²³

(4.27 g, 27.26 mmol), 8.90 g (102%) of the title compound was

obtained according to the method described for the synthesis of

1

57a. H NMR (400 MHz, DMSO-d₆) δ 8.48 (d, J ) 5.6 Hz, 1H),

4-(1H-Indol-4-yloxy)phenol (58i). A mixture of 4-chloro-1H-

pyrrolo[2,3-b]pyridine (3.10 g, 20.0 mmol), 4-benzyloxyphenol

(8.10 g, 40.0 mmol) and Et₃N/TFA 1:1 mixture (3.0 mL) was heated

at 150 °C for 96 h. The crude material was directly purified on

silica gel without workup using a hexanes/EtOAc gradient (100/0

to 0/100) to afford 4-(4-benzyloxy-phenoxy)-1H-pyrrolo[2,3-b]-

pyridine (1.20 g) as a 2:1 mixture with the phenol starting material.

This material was subjected to the conditions for the synthesis of

58e at a pressure of 60 psi to afford the title compound (0.80 g,

8.09-8.12 (m, 1H), 7.68-7.70 (m, 1H), 7.44-7.48 (m, 2H), 7.38-

7.42 (m, 2H), 7.30-7.36 (m, 2H), 7.10-7.18 (m, 5H), 5.15 (s,

2H).

4-(4-Benzyloxyphenoxy)-6,7-dimethoxyquinazoline (57h). Start-

ing with 4-chloro-6,7-dimethoxyquinazoline (2.30 g, 11.1 mmol),

2.0 g (46%) of the title compound was obtained according to the

1

method described for the synthesis of 57a. H NMR (400 MHz,

DMSO-d₆) δ 8.50 (s, 1H), 7.52 (s, 1H), 7.40-7.50 (m, 2H), 7.30-

7.40 (m, 3H), 7.20 (d, J ) 8.7 Hz, 2H), 7.06 (d, J ) 9.0 Hz, 2H),

5.13 (s, 2H), 3.96 (s, 3H), 3.95 (s, 3H).

1

93%). H NMR (400 MHz, DMSO-d₆) δ 11.90 (s, 1H), 9.55 (s,

1H), 8.10 (d, J ) 6.6 Hz, 1H), 7.35-7.40 (m, 1H), 7.05 (d, J )

8.3 Hz, 2H), 6.85 (d, J ) 8.3 Hz, 2H), 6.40 (d, J ) 6.6 Hz, 1H),

6.25-6.30 (m, 1H).

4-(4-Hydroxyphenoxy)-pyridine-2-carboxylic Acid Methyl-

amide (58a). Starting from 4-(4-benzyloxy-phenoxy)-pyridine-2-

carboxylic acid methylamide (0.44 g, 1.32 mmol), 282 mg (88%)

of the title compound was obtained as a pink oil according to the

4-(4-Hydroxy-3-nitrophenoxy)-pyridine-2-carboxylic Acid Me-

thylamide (59a). Starting from 4-(4-hydroxyphenoxy)-pyridine-2-

carboxylic acid methylamide(0.28 g, 1.15 mmol), 187 mg (56%)

of the title compound was obtained as a yellow foam according to

the method described for the synthesis of 59e. ¹H NMR (400 MHz,

DMSO-d₆) δ 11.19 (s, 1H), 8.70-8.80 (m, 1H), 8.50 (d, J ) 5.6

Hz, 1H), 7.80 (d, J ) 2.8 Hz, 1H), 7.44-7.47 (m, 1H), 7.38 (d, J

) 2.4 Hz, 1H), 7.22 (d, J ) 9.2 Hz, 1H), 7.14-7.16 (m, 1H), 2.77

(d, J ) 4.8 Hz, 3H).

1

method described for the synthesis of 58e. H NMR (400 MHz,

DMSO-d₆) δ 9.59 (s, 1H), 8.70-8.80 (m, 1H), 8.45 (d, J ) 5.6

Hz, 1H), 7.31 (d, J ) 2.0 Hz, 1H), 7.06-7.08 (m, 1H), 6.98-7.02

(m, 2H), 6.82-6.86 (m, 2H), 2.76 (d, J ) 4.8 Hz, 3H).

4-(Quinolin-4-yloxy)phenol (58b). Starting from 4-(4-(benzyl-

oxy)phenoxy)quinoline (3.8 g, 11.6 mmol), 800 mg (30%) of the

title compound was obtained according to the method described

1

for the synthesis of 58e. H NMR (300 MHz, DMSO-d₆) δ 9.60

2-Nitro-4-(quinolin-4-yloxy)phenol (59b). Starting from 4-(quin-

olin-4-yloxy)phenol (800 mg, 3.79 mmol), 760 mg (76%) of the

title compound was obtained as an orange solid according to the

(s, 1H), 8.65 (d, J ) 5.8 Hz, 1H), 8.30 (d, J ) 5.8 Hz, 1H), 8.00

(d, J ) 8.7 Hz, 1H), 7.70-7.80 (m, 1H), 7.55-7.65 (m, 1H), 7.10

(d, J ) 8.7 Hz, 2H), 6.85 (d, J ) 8.7 Hz, 2H), 6.50 (d, J ) 3.8 Hz,

1H).

1

method described for the synthesis of 59e. H NMR (300 MHz,

DMSO-d₆) δ 8.68 (d, J ) 7.3 Hz, 1H), 8.25-8.30 (m, 1H), 8.02

(d, J ) 9.7 Hz, 1H), 7.88 (d, J ) 4.8 Hz, 1H), 7.75-7.85 (m, 1H),

7.60-7.70 (m, 1H), 7.55 (d, J ) 4.8 Hz, 1H), 7.50 (d, J ) 4.8 Hz,

1H), 7.25 (d, J ) 12.1 Hz, 1H), 6.65 (d, J ) 5.8 Hz, 1H).

4-(3-Chloro-4-hydroxy-5-nitrophenoxy)-pyridine-2-carboxy-

lic Acid Methylamide (59d). Starting with 4-(3-chloro-4-hydroxy-

phenoxy)-pyridine-2-carboxylic acid methylamide (578 mg, appx

1.83 mmol), 394 mg (crude, with salts) of the title compound was

obtained as an orange solid according to the method described for

2-Chloro-4-(quinolin-4-yloxy)phenol (58c). A solution of 4-(4-

(benzyloxy)-3-chlorophenoxy)quinoline (1.37 g, 3.79 mmol) in TFA

(10 mL) was refluxed for 20 h, and the mixture was allowed to

cool to rt and concentrated in vacuo. The residue was diluted with

water and basified with NH₄OH (concd) and a solid precipitated.

The solid was collected and washed with water and Et₂O, yielding

1

the title compound (983 mg, 95%). H NMR (400 MHz, DMSO-

d₆) δ 10.37 (s, 1H), 8.66 (d, J ) 4.8 Hz, 1H), 8.28 (d, J ) 7.3 Hz,

Benzimidazoles and Benzoxazoles as VEGFR-2 Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4371

1

the synthesis of 59e. H NMR (400 MHz, DMSO-d₆) δ 8.76 (s,

lin-4-yloxy)phenol (200 mg, 0.67 mmol) was converted to 4-(6,7-

dimethoxyquinolin-4-yloxy)-2-nitrophenol (160 mg, 69%) using the

method described in 59e. ¹H NMR (300 MHz, DMSO-d₆) δ 11.40

(bs, 1H), 8.85 (d, J ) 8.0 Hz, 1H), 8.02 (d, J ) 3.3 Hz, 1H), 7.70

(s, 1H), 7.52 (dd, J ) 8.0, 3.3 Hz, 1H), 7.50 (s, 1H), 7.30 (d, J )

10.0 Hz, 1H), 7.00 (d, J ) 8.0 Hz, 1H), 4.04 (s, 3H), 4.01 (s, 3H).

Bioligical Assays. KDR Enzyme Assay: In a 96-well plate, 5

nM of either the phosphorylated or unphosphorylated form of the

KDR kinase domain was incubated with a 10-point titration of

compound ranging from 3 µM to 0.15 nM and 1 µM gastrin

substrate, sequence {N} EEEEA YGWLD F {C}, in 20 mM Tris-

HCl pH 7.5, 10 mM MgCl₂, 100 mM NaCl, 1.5 mM EGTA, 1

mM DTT, 0.2 mM sodium orthovanadate, and 20 µg/mL BSA for

30 min at 25 °C. ATP was added at a final concentration of 11.8

µM and incubated for 60 min at room temperature. From this

reaction mix, 5 µL was transferred to a black 96-well plate

containing 80 µL of 2.75 µg/mL SA-APC (Prozyme, Inc., catalog

#PJ25S) and 0.1125 nM Eu-labeled antiphosphotyrosine pT66

antibody (Perkin-Elmer) in 50 mM Tris-HCl pH 7.5, 100 mM NaCl,

0.1% BSA, 0.05% Tween-20, and incubated for 30 min at 25 °C.

The plate was read on a RubyStar and data was fitted using the

Levenburg-Marquardt algorithm into a four-parameter logistic

equation.

Inhibition of LCK Kinase Domain. In a 96-well black

polypropylene costar plate, 1 µM biotinylated gastrin substrate

(sequence {N} EEEEAYGWLDF {C}) and 0.5 µM ATP (at K_m)

were added to a 10-point, 5-fold titration of compound ranging from

25 µM to 12.8 pM. The reaction was initiated by adding 50 pM of

the GST-tagged form of the LCK kinase domain in 50 mM HEPES

pH 7.5, 20 mM MgCl₂, 5 mM MgCl₂, 50 mM NaCl, 1 mM DTT,

and 0.05% BSA. The reaction was incubated for 90 min at rt. The

reaction was quenched by the addition of 160 µL of detection mix

consisting of 50 mM TRIS pH 7.5, 100 mM NaCl, 0.05% BSA,

0.1% Tween-20, 3 mM EDTA, 40 µg/mL SA-APC (Perkin-Elmer),

25 nM Eu-labeled anti-phosphotyrosine pT66 antibody (Perkin-

Elmer), and incubated at room temperature for 30 min. The plate

was read on a BMG RubyStar. All enzymes were run at the apparent

K_mof ATP, and peptide substrates were dependent upon enzymes:

gastrin (LCK, cMet, Src, EGFR, Zap-70, IGF); biotinyl-GG-

MEDIYFEFMGGKKK (Tie-2, cFMS, cKit); and biotinyl-KEAPED-

LYKDFLTL (FGFR).

Inhibition of p38R, p38â, JNK1, JNK2, JNK3. In a 96-well

black polypropylene costar plate, 100 nM biotinyl-ATF2, and 50

µM ATP(p38R, p38â) or 2 µM ATP(JNK1, JNK2, JNK3) were

added to a 10-point, 3-fold titration of compound. Adding 1 nM

p38R, 5 nM p38â, 0.5 nM JNK1, 1 nM JNK2, or 1.5 nM JNK3 in

50 mM Tris HCl, pH 7.5, 5 mM MgCl₂, 0.5 mM DTT, 0.1 mM

Na₃VO₄, and 0.1 mg/mL BSA initiated the reaction. The 30 µL of

reaction volume was incubated for 60 min at rt. The reaction was

quenched by the addition of 30 µL of detection mix consisting of

50 mM Hepes, pH 7.5, 100 mM NaCl, 0.1% BSA, 0.05% Tween-

20, 10 mM EDTA, 4nM streptavidin allophycocyanin (Prozyme),

and 0.1 nM Eu-labeled antiphosphothreonine proline antibody

(Perkin-Elmer).

Inhibition of CDK1, CDK2, and p25CDK5. In a 96-well black

polypropylene costar plate, 0.5 µM biotinylated histone H1 and 25

µM ATP (<K_m) were added to a 10-point, 3-fold titration of

compound. Adding 5 nM CDK1, 1 nM cyclin E₂-CDK2, or 0.2

nM of p25CDK5 in 50 mM Tris HCl, pH 7.5, 5 mM MgCl₂, 5

mM DTT, 5 mM â-glycerolphosphate, and 0.2 mg/mL BSA

initiated the reaction. The 30 µL of reaction volume was incubated

for 60 min at rt. The reaction was quenched by the addition of 30

µL of detection mix consisting of 100 mM Hepes, pH 7.5, 100

mM NaCl, 0.1% BSA, 0.05% Tween-20, 10 mM EDTA, 1nM

streptavidin allophycocyanin (Prozyme), and 0.05 nM Eu-labeled

antiphosphothreonine proline antibody (Perkin-Elmer) and incubated

at rt for 60 min. The plate was read on a Discovery Plate Reader

(Perkin-Elmer).

1H), 8.40-8.50 (m, 1H), 7.38-7.45 (m, 2H), 7.00-7.25 (m, 3H),

2.75-2.80 (m, 3H).

4-(2-Methylamino-pyrimidin-4-yloxy)-2-nitro-phenol (59e).

A flask was charged with 4-(2-methylamino-pyrimidin-4-yloxy)-

phenol (0.20 g, 0.92 mmol) was the solid dissolved in AcOH (5

mL). To this solution was added fuming HNO₃(64 mg, 0.92 mmol;

diluted with water (∼12 µL) dropwise over 1 min. The reaction

mixture was stirred at rt for 18 h, after which it was poured slowly

to 40 mL saturated aqueous NaHCO₃and extracted with CH₂Cl₂.

The organic layer was washed with NaHCO₃(satd) and brine, dried

over Na₂SO₄, filtered, and concentrated in vacuo. The crude residue

was purified by silica gel column chromatography using hexanes/

1

EtOAc gradient to yield the title compound (163 mg, 68%). H

NMR (400 MHz, DMSO-d₆) δ 10.98 (s, 1H), 8.15 (s, 1H), 7.70-

7.80 (m, 1H), 7.35-7.45 (m, 1H), 7.14 (d, J ) 9.2 Hz, 1H), 6.95-

7.05 (m, 1H), 6.10-6.20 (m, 1H), 2.48 (s, 3H).

4-(6-Methylamino-pyrimidin-4-yloxy)-2-nitro-phenol (59f).

Starting with 4-(6-methylamino-pyrimidin-4-yloxy)-phenol (0.88 g,

4.06 mmol), 740 mg (69%) of the title compound was obtained as

a light yellow solid according to the method described for the

synthesis of 59e. ¹H NMR (400 MHz, DMSO-d₆) δ 10.96 (s, 1H),

8.09 (bs, 1H), 7.65 (d, J ) 2.8 Hz, 1H), 7.33-7.36 (m, 2H), 7.13

(d, J ) 8.4 Hz, 1H), 5.82 (s, 1H), 2.76 (s, 3H).

4-(6,7-Dimethoxyquinazolin-4-yloxy)-2-nitrophenol (59h). Start-

ing with 4-(4-benzyloxy-phenoxy)-6,7-dimethoxyquinazoline (2.0

g, 5.15 mmol), 0.76 g (2.36 mmol, 46%) of 4-(6,7-dimethox-

yquinazolin-4-yloxy)-phenol was obtained according to the method

described for the synthesis of 58e (using a mixture of DMF and

methanol as the solvent). This compound was converted to 4-(6,7-

dimethoxyquinazolin-4-yloxy)-2-nitrophenol using the conditions

described for 59e to obtain the title compound as a yellow solid

1

(0.48 g, 59%). H NMR (400 MHz, DMSO-d₆) δ 11.05 (bs, 1H),

8.60 (s, 1H), 7.85-7.90 (m, 1H), 7.50-7.60 (m, 2H), 7.35-7.40

(m, 1H), 7.15-7.25 (m, 1H), 3.95-4.00 (m, 6H).

4-(3-Fluoro-4-hydroxy-5-nitrophenoxy)-pyridine-2-carboxy-

lic Acid Methylamide (59j). To a stirred rt slurry of NaH (0.10 g

of 60% oil dispersion, 4.51 mmol) in DMF (8 mL) was added

4-(benzyloxy)-3-fluorophenol (0.98 g, 4.50 mmol). The mixture was

stirred at rt for 10 min before adding 4-chloro-pyridine-2-carboxylic

acid methylamide (0.77 g, 4.50 mmol). The reaction mixture was

stirred at rt for 10 min and then heated at 85 °C for 16 h. The

reaction was allowed to cool to rt, quenched with saturated aqueous

NaHCO₃, and then diluted with Et₂O and 6 N NaOH. The layers

were separated, and the organic layer was washed with 6 N NaOH

and then brine, dried over Na₂SO₄, filtered, and concentrated in

vacuo to yield 4-(4-(benzyloxy)-3-fluorophenoxy)-pyridine-2-car-

boxylic acid methylamide as a 1:1 mixture with 4-chloro-pyridine-

2-carboxylic acid methylamide (1.24 g). This crude material was

dissolved in ethanol (20 mL). To the argon-degassed solution was

added Pd/C (10% by wt, 0.40 g), and the reaction was stirred

vigorously at rt under 1 atm H₂gas for 4 days. The mixture was

filtered through Celite and concentrated in vacuo to yield 4-(3-

fluoro-4-hydroxyphenoxy)-pyridine-2-carboxylic acid methylamide

(655 mg) as a 1:1 mixture with pyridine-2-carboxylic acid methy-

lamide (996 mg total). The title compound was further obtained as

a thick oil (611 mg, 80%) according to the method described for

1

the synthesis of 59e. H NMR (300 MHz, DMSO-d₆) δ 11.37 (s,

1H), 8.70-8.80 (m, 1H), 8.51 (d, J ) 5.7 Hz, 1H), 7.55-7.65 (m,

2H), 7.43 (d, J ) 2.4 Hz, 1H), 7.17-7.20 (m, 1H), 2.78 (d, J )

5.1 Hz, 3H).

4-(6,7-Dimethoxyquinolin-4-yloxy)-2-nitrophenol (59k). Start-

ing from 4-chloro-6,7-dimethoxyquinoline²⁴(3 g, 13.4 mmol), 6.43

g of 4-(4-(benzyloxy)phenoxy)-6,7-dimethoxyquinoline (contami-

nated with the phenol) was obtained according to the method

described for the synthesis of 57a. The mixture was treated with

Pd/C according to the procedure described in 39o to give 4-(6,7-

1

dimethoxyquinolin-4-yloxy)phenol. H NMR (300 MHz, DMSO-

d₆) δ 9.60(s, 1H), 8.42 (d, J ) 5.6 Hz, 1H), 7.50 (s, 1H), 7.35 (s,

1H), 7.05 (d, J ) 7.5 Hz, 1H), 6.85 (d, J ) 7.5 Hz, 1H), 6.35 (d,

J ) 5.6 Hz, 1H), 3.95 (s, 6H). A portion of 4-(6,7-dimethoxyquino-

Inhibition of GSK3â. In a 96-well polypropylene costar plate,

0.5 µM biotinylated phosphorylated CREB-T peptide (KKRREIL-

TRRP[pS]YR) and 25 µM ATP (<K_m) were added to a 10-point,

4372 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18

Potashman et al.

3-fold titration of compound. Adding 0.2 nM of GSK3â in 50 mM

Tris HCl, pH 7.5, 5 mM MgCl₂, 5 mM DTT, 10 mM â-glycerol-

phosphate, and 0.2 mg/mL BSA initiated the reaction. The 50 µL

of reaction volume was incubated for 60 min at rt. The reaction

was quenched by a 30-fold dilution into detection mix consisting

of 100 mM Tris HCl, pH 7.5, 100 mM NaCl, 0.1% BSA, 0.05%

Tween-20, 10 mM EDTA, 1 nM ruthenylated antirabbit antibody

(BioVeris), 1 nM antiphosphothreonine antibody (Cell Signaling

Technology), and 7.5 µg streptavidin paramagnetic beads (Dynal)

and incubated with agitation at rt for 60 min. The plate was read

on a M-Series M8 Analyzer (BioVeris). The proportion of substrate

phosphorylated in the kinase reactions in the presence of compound

compared with that phosphorylated in the presence of DMSO

vehicle alone (HI control) was calculated using the formula: %

control (POC) ) (cmpd - average LO)/(average HI - average

LO) × 100. Data (consisting of POC and inhibitor concentration

in µM) was fitted to a four-parameter equation (y ) A + ((B -

A)/(1 + ((x/C)^∧D))), where A is the minimum y (POC) value, B is

the maximum y (POC), C is the x (cmpd concentration) at the point

of inflection, and D is the slope factor) using a Levenburg-

Marquardt nonlinear regression algorithm. The inhibition constant

(K_i) of the inhibitor was estimated from the IC₅₀(cmpd concentra-

tion at the point of inflection; C) using the Cheng-Prussof

equation: K_i) IC₅₀/(1 + S/K_m), where S is the ATP substrate

concentration and K_mis the Michaelis constant for ATP as

determined experimentally.

HUVEC Proliferation Assay. HUVEC proliferation assays were

performed as previously described.^10eBriefly HUVECs were

cultured in endothelial growth medium-2 (Cambrex). One day

before the assay was run, HUVEC were seeded into flat-bottom,

96-well plates (BD Falcon) at 3000 cells per well in DMEM

medium with 10% qualified fetal bovine serum (FBS) and 1×

penicillin, streptomycin, and L-glutamine (1× PSG). After culture

for 22 h, media was removed and HUVEC were preincubated for

2 h with serial dilutions of small molecule diluted 1:400 in DMEM

with 10% FBS plus 1× PSG. HUVEC were then challenged with

50 ng/mL VEGF or 20 ng/mL bFGF and incubated for 72 h at 37

°C, 5% CO₂. Cells were washed 2× with DPBS, and plates were

frozen at -70 °C for 24 h. Plates were thawed, incubated with

CyQuant dye (Molecular Probes), and read on a Victor 1420

Workstation (Perkin-Elmer Corporation). IC₅₀data was calculated

using the Levenburg-Marquardt algorithm into a 4-parameter

logistic equation (ID Business Solutions, Ltd.) and expressed in

µM.

60 °C overnight. The extracted Evan’s blue dye was measured using

a spectrophotometer at OD₆₃₀. Relative Evan’s blue units refer to

the % Evan’s blue, as determined by the standard curve, multiplied

by 10⁴. Data represent mean ( standard error; n ) 5. Statistical

analysis was performed by one-way ANOVA with Bonferroni-

Dunn post hoc test. p < 0.0009 was considered significant.

Rat Corneal Angiogenesis Model. Corneal angiogenesis was

evaluated in female CD rats as described.²⁶Briefly, rats weighing

approximately 250 grams were randomized into one of six treatment

groups. Angiogenesis was induced by surgically implanting a VEGF

(or BSA control)-soaked nylon disk into the corneal stroma (n )

8/group). Rats were treated with 1, 3, 10, or 30 mg/kg orally once

a day with compound or vehicle. Treatment began on the day of

surgery and was continued for 7 days. Two vascular endpoints were

evaluated: the number of blood vessels at the midpoint between

the limbal vessels and the disk and the mean blood vessel area.

After 7 days, the implanted corneas were photographed at 25× using

a Nikon SV-3 Ophthalmic Slit Lamp (Nikon Ophthalmic) equipped

with a Nikon D-1 digital camera back. A reference stage micrometer

was photographed for calibration. The images were transferred to

a desktop PC, reformatted for image analysis, and transferred to a

Metamorph IA system for computerized analysis. Numerical data

were generated from the digital images using the Metamorph image

analysis system (Universal Imaging). Three endpoints were analyzed

on each corneal image: (1) disk placement distance from the limbus,

(2) number of vessels intersecting a perpendicular line at the

midpoint of the disk placement distance, and (3) blood vessel area,

as determined by RGB thresholding and automated pixel counting.

Image analysis was performed in a blinded fashion. Values represent

the group mean ( standard error. Statistical analysis was performed

using one-way ANOVA followed by Fisher’s protected least

significant difference post hoc test.

Acknowledgment. We thank our colleagues Pamela Sproul,

Elizabeth Tominey, Michael Shiue in PKDM; Paul Schnier for

obtaining high-resolution MS analysis; Karen L. Rex for

assistance with the VP assay; Julie Calahan, Gabrielle Palluconi,

Anu Gore, Dennis Lei, and Mark Cappucci from Pharmaceutics;

and Erin DiMauro, Matt Martin, and Mark Duggan for helpful

discussions regarding this manuscript.

Supporting Information Available: Elemental analysis and

X-ray crystallographic data. This material is available free of charge

via the Internet at http://pubs.acs.org.

Pharmacokinetic Studies. Male Sprague-Dawley rats were

dosed via femoral vein (intravenous, DMSO solution, dose 1 mg/

kg) or via oral gavage (suspensions in Ora-Plus, pH adjusted to a

range of 2.0-2.2 using methanesulfonic acid, dose 10 mg/kg).

Concentrations of all formulations were selected as to allow for

dose volumes in accordance with the highest scientific, humane,

and ethical principals as defined by IACUC (Institutional Animal

Care and Use Committees).²⁵Serial blood samples were collected

from jugular vein into heparized tubes over a 12-24 h period.

Plasma was separated by centrifugation, and the sample was

prepared for analysis by protein precipitation with acetonitrile.

Quantitation of the test compounds was accomplished by reversed

liquid chromatography with mass spectral detection in multiple

reaction monitoring mode, with an appropriate internal standard.

Pharmacokinetic parameters such as clearance, volume of distribu-

tion, and terminal half-life were calculated by a noncompartmental

method.

Vascular Permeability Assay. Vascular permeability was

induced using a modified Miles assay.¹⁸HEK 293 cells overex-

pressing murine VEGF were used to induce vascular permeability

in nude mice. Briefly, 2 × 10⁵VEGF-expressing or vector control

HEK 293 cells were mixed with Matrigel and injected subcutane-

ously on the ventral surface of nude mice. Approximately 22 h

later, a single oral dose of compound or vehicle was administered.

After 6 h, the mice received an intravenous injection of 0.1 mL

1% Evan’s blue dye for 10 min prior to sacrifice. A 1 cm²piece of

skin overlying the cells was harvested and placed in formamide at

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JM070034I

Article Doi

Design, synthesis, and evaluation of orally active benzimidazoles and benzoxazoles as vascular endothelial growth factor-2 receptor tyrosine kinase inhibitors

DOI: 10.1021/jm070034i

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Article abstract of DOI:10.1021/jm070034i

Full text of DOI:10.1021/jm070034i

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