Discovery of trans-4-[1-[[2,5-dichloro-4-(1-methyl-3-indolylcarboxamido) phenyl]acetyl]-(4S)-methoxy-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid: An orally active, selective very late antigen-4 antagonist

7974 J. Med. Chem. 2009, 52, 7974–7992

DOI: 10.1021/jm901154c

Discovery of trans-4-[1-[[2,5-Dichloro-4-(1-methyl-3-indolylcarboxamido)phenyl]acetyl]-(4S)-

methoxy-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic Acid: An Orally Active, Selective Very

Late Antigen-4 Antagonist

Fumihito Muro,*^,†Shin Iimura,^†Yuuichi Sugimoto,^†Yoshiyuki Yoneda,^†Jun Chiba,^†Toshiyuki Watanabe,^†

Masaki Setoguchi,^†Yutaka Iigou,^‡Keiko Matsumoto,^†Atsushi Satoh,^†Gensuke Takayama,^†Tomoe Taira,^†

Mika Yokoyama,^†Tohru Takashi,^†Atsushi Nakayama,^†and Nobuo Machinaga^†

^†R&D Division, Daiichi Sankyo Co., Ltd., 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan, and ^‡Research Department,

Daiichi Sankyo RD Associe Co., Ltd., 3-10-2 Kitashinagawa, Shinagawa-ku, Tokyo 140-0001, Japan

Received August 3, 2009

We have focused on optimization of the inadequate pharmacokinetic profile of trans-4-substituted

cyclohexanecarboxylic acid 5, which is commonly observed in many small molecule very late antigen-4

(VLA-4) antagonists. We modified the lipophilic moiety in 5 and found that reducing the polar surface

area of this moiety results in improvement of the PK profile. Consequently, our efforts have led to the

discovery of trans-4-[1-[[2,5-dichloro-4-(1-methyl-3-indolylcarboxamido)phenyl]acetyl]-(4S)-meth-

oxy-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid (14e) with potent activity (IC₅₀= 5.4 nM)

and significantly improved bioavailability in rats, dogs, and monkeys (100%, 91%, 68%), which

demonstrated excellent oral efficacy in murine and guinea pig models of asthma. Based on its overall

profile, compound 14e was progressed into clinical trails. In a single ascending-dose phase I clinical

study, compound 14e exhibited favorable oral exposure as expected and had no serious adverse events.

Introduction

tion of leukocyte infiltration into extravascular tissue and

prevent tissue damage in a wide variety of inflammatory

animal models.¹¹Furthermore, clinical trials in phase III

using a humanized monoclonal anti-R₄antibody, natalizu-

mab, for the treatment of MS and Crohn’s disease provided

excellent results and strongly validated the proof of concept of

this target as a therapeutic agent. Consequently, natalizumab

has been approved by the FDA for the treatment of both MS

and Crohn’s disease.¹²On the other hand, oral small-molecule

VLA-4 antagonists possessing similar efficacy and an appro-

priate half-life in comparison with natalizumab are still

considered to be beneficial from a safety and cost point of

view. Over the past decade or more, intensive efforts to

discover and develop new candidate small molecule VLA-4

antagonists have been seen in the pharmaceutical industry.

However, there are currently only a few candidates in clinical

trials for the treatment of MS and Crohn’s disease, and none

of these compounds has yet reached the marketplace.

The small-molecule VLA-4 antagonists that have so far

been progressed into clinical trials are classified into two

major structures: (1) LDV mimics, whose sequence is respon-

sible for interaction with FN, such as the highly selective

VLA-4 antagonist Bio-1211 (Figure 1)¹³or (2) N-acylpheny-

lalanine-based compounds¹⁴such as valategrast (R411,

Figure 1), many of which exhibit dual inhibitory activity for

integrin R₄β₁, as well as R₄β₇.

The pathogenesis of inflammatory and autoimmune dis-

eases such as asthma,¹rheumatoid arthritis,²type 1 diabetes

mellitus,³psoriasis,⁴multiple sclerosis (MS),⁵inflammatory

bowel disease,⁶and hepatitis C⁷is commonly characterized by

an influx of activated leukocytes to affected tissues. The

sustained accumulation of inflammatory cells keeps chronic

inflammatory conditions in the tissue, resulting in tissue

damage and dysfunction. It is well-known that the integrin

very late antigen-4 (VLA-4, R₄β₁, CD49d/CD29)^ais intrinsi-

cally involved in the development of this pathogenesis. VLA-4

is a heterodimeric glycoprotein receptor consisting of R₄and

β₁chains that is constitutively expressed on the surface of

almost all leukocytes.⁸It binds vascular cell adhesion mole-

cule-1 (VCAM-1, CD106) expressed on cytokine-stimulated

endothelial cells and the alternatively spliced portion of the

type III connecting segment of fibronectin (FN)^9,10and

mediates the process of adhesion, migration, and activation

of inflammatory cells at the site of inflammation. Therefore, a

blockade of the interaction of VLA-4 with the ligands would

alleviate the inappropriate cellular process and would be

useful in the treatment of inflammatory and autoimmune

diseases. To date, it has been reported that anti-R₄antibodies

and small-molecule VLA-4 antagonists demonstrate inhibi-

*To whom correspondence should be addressed: Tel þ81-3-3680-

0151; fax þ81-3-5696-8609; e-mail: muro.fumihito.iy@daiichisankyo.

co.jp Medicinal Chemistry Research Laboratories II.

For several years, our efforts have been directed toward the

development of orally active, potent, and selective VLA-4

antagonists based on LDV-derived compound 3 as the initial

lead (Figure 2),¹⁵which was identified through a hit to lead

research program. The key issue was to address the poor

pharmacokinetic (PK) profile, including the extremely high

a

Abbreviations: VLA-4, very late antigen-4; CD, cluster of differen-

tiation; VCAM-1, vascular cell adhesion molecule-1; PSA, polar surface

area; HBD, hydrogen bond donors; HSA, human serum albumin;

BALF, bronchoalveolar lavage fluid; BHR, bronchial hyper-respon-

siveness; LDV, leucine-aspartate-valine.

r

pubs.acs.org/jmc

Published on Web 11/05/2009

2009 American Chemical Society

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7975

Figure 1

the lipophilic moiety in each compound (36.6 A²for 4; 30.8 A²

for 5), the number of hydrogen bond donors (HBD, 2 for 4; 1

for 5), or both.¹⁹

From these results, we considered that structural modifica-

tion of the lipophilic moiety to reduce PSA and HBD could

lead to further improvement of the PK profile. It might be

presumed that such a modification could enhance the affinity

for certain plasma proteins such as albumin and make it easy

for the modified compounds to employ the plasma proteins as

carriers in the blood. Although the enhancement of plasma

protein binding generally results in a drop of activity in vivo,

with acceptable activity in the presence of plasma and with

sufficient concentrations, we believed that the compounds

could exert in vivo efficacy. Thus, as illustrated in Figure 3,

we focused our efforts on incorporation of the urea in the

(N⁰-phenylureido)phenyl group into various cyclic structures

leading to 2-(arylamino)benzoxazolyl (I), 4-(2-benzoxazoly-

lamino)phenyl (II), and 4-(hetroarylcarboxamido)phenyl

(III) groups while fixing the trans-4-[(4S)-fluoro-(2S)-pyrro-

lidinylmethyloxy]cyclohexanecarboxylic acid scaffold in 5,

which was highly optimized as a novel LDV motif.^18b

We herein report on optimization of the lipophilic moiety in

5, which led to the identification of a series of 1-methyl-3-

indolyl derivatives with a significantly improved PK profile in

rodents, and the subsequent fine-tuning of the substituents in

the 4-(1-methyl-3-indolylcarboxamido)phenyl group and the

pyrrolidine ring as well as the selection of compound 14e as a

clinical candidate.

Chemistry

The target compounds were synthesized according to the

general procedure outlined in Scheme 1. Thus, arylacetic acids

7-9 were condensed with pyrrolidine 10 using EDC and

HOBt. The resulting amideswere subjectedtobasichydrolysis

to afford trans-4-substituted cyclohexanecarboxylic acids

11-14. The syntheses of the intermediates 7-10 in Scheme 1

are fully presented in Schemes 2-5.

The preparation of (6-benzoxazolyl)acetic acids 7a-f is

depicted in Scheme 2. Condensation of methyl (4-amino-3-

hydroxyphenyl)acetate (15)²⁰with o-tolylacetic acid was car-

ried out using triphenylphosphine/hexachloroethane,²¹fol-

lowed by basic hydrolysis to give the [2-(2-methylbenzyl)-6-

benzoxalolyl]acetic acid (7a). After protection of the hydroxyl

group in 2,3-difluoro-6-nitrophenol (16) with a benzyl group,

the benzyl ether was treated with dimethyl malonate in the

presenceofNaH togive17, which was converted tothe methyl

ester 18 by basic hydrolysis and decarboxylation followed by

esterification. Hydrogenation of 18 over Pd/C resulted in

reduction of the nitro group and deprotection of the benzyl

group to give the aminophenol 19. Cyclization of both 15 and

19 to the corresponding benzoxazole derivatives was achieved

Figure 2

plasma clearance (CL = 69.3 (mL/min)/kg) and low oral

bioavailability (F < 1%) in rats, which have commonly been

observed in most VLA-4 antagonists, likely because of their

high biliary excretion¹⁶rate due to a multidrug resistance-

associated protein 2 (MRP2) mediated process,¹⁷while retain-

ing the VLA-4 inhibitory activity. Thus, to establish a perti-

nent structure-PK profile and structure-activity relation-

ships, we have worked on modification of both the LDV motif

and the (N⁰-phenylureido)phenyl group in 3. In the course

of this study, it was revealed that benzoic acid derivative 4

with the chlorine atom at the 3-position of the central benzene

in the (N⁰-phenylureido)phenyl group and cyclohexane-

carboxylic acid derivative 5 with 2-(2-methylphenylami-

no)benzoxazolyl group instead of (N⁰-phenylureido)phenyl

group showed a somewhat improved PK profile in part due to

increased plasma proteinbinding (Figure2).¹⁸In addition, the

difference in the clearance between compounds 4 and 5

seemed to correlate to the polar surface area (PSA) value of

7976 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24

Muro et al.

Figure 3

Scheme 1^a

afford a mixture of the corresponding cis- and trans-4-sub-

stituted cyclohexane derivatives in about a 6:1 ratio, which

without separation were reprotected with a Boc group to

furnish 29a-c. In the case of 29c, the hydroxyl group was

protected with a benzyloxymethyl (BOM) group to give 29d.

Treatment of the cis-rich isomers 29a,b and 29d with sodium

methoxide in refluxing methanol resulted in a mixture of the

cis-and trans-isomersin a ratio of1:1. Followingesterification

with (trimethylsilyl)diazomethane, isolation of the trans-iso-

mers was successfully performed by flash column chromatog-

raphy to afford the trans-isomers 30a-c. Conversion of

the BOMO group in 30c to the substituents of 30d-f was

carried out accoding to the procedure previously reported

by us.²⁵Removal of the Boc function of 30a,b and 30e,f

by TFA treatment produced the methyl trans-4-[(4S)-

substituted-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxy-

lates 10a-d.

^aReagents and conditions: (a) EDC HCl, HOBt, Et₃N, DMF; (b) aq

NaOH, THF.

3

by condensation with commercially avalilable isothiocyanates

or 3-fluoro-2-methylaniline/thiocarbonyl diimidazole and

subsequent treatment with HgO.²⁰The benzoxazoles were

hydrolyzed under a basic condition to provide (2-arylamino-

6-benzoxazolyl)acetic acids 7b-f.

[4-(2-Benzoxazolyl)aminophenyl]acetic acids 8a-e were

prepared as shown in Scheme 3. Nucleophilic displacement

of 2-chlorobenzoxazole with ethyl (4-amino-3-chlorophe-

nyl)acetate (20a)²²and subsequent basic hydrolysis gave

[4-(2-benzoxazolyl)amino-3-chlorophenyl]acetic acid (8a).

Methyl (4-amino-3-chlorophenyl)acetate (20b)²³was treated

with thiophosgene in the presence of CaCO₃to afford iso-

thiocyanate 22,²⁴which was subjected to the cyclization

procedure described in Scheme 2 with aminophenols 23a-d

to afford [4-(2-benzoxazolyl)aminophenyl]acetic acids 8b-e.

[4-(Heteroarylcarboxamido)phenyl]acetic acids 9a-n were

prepared as shown in Scheme 4. Indoline (24) was converted

to 9a,b via urea formation with anilines 20a or 20c using

triphosgene followed by basic hydrolysis. Following treat-

ment of 1-alkylindole-3-carboxylic acid 27c-e with thionyl

chloride, condensation of the resultant acid chlorides with

anilines 20, and basic hydrolysis afforded 9e-i. 1-(4-Methox-

ybenzyl)indazole-3-carboxylic acid (27b) was also converted

to acid chloride, which was condensed with aniline 20c,

followed by deprotection of 4-methoxybenzyl group by

TFA treatment and basic hydrolysis to afford 9d. Alterna-

tively, in the case of 9c and 9j-n, an amide bond forming

reaction of 20 with 1-indole-3-carboxylic acid (27a), benzi-

sothiazole-3-carboxylicacid(27f), benzisoxazole-3-carboxylic

acid (27g), and isoquinoline-1-carboxylic acid (27h) was

performed by using EDC and HOBt.

Results and Discussion

Optimization of Lipophilic Moiety. The trans-4-substituted

cyclohexanecarboxylic acid derivatives 11-14 were initially

screened for their activities to inhibit the binding of CHO

(Chinese hamster ovary) cells expressing VLA-4 to a euro-

pium (Eu)-labeled human VCAM-1/Fc chimera with or

without the addition of 3% human serum albumin (HSA).

In addition, regarding compounds showing potent activity

with IC₅₀’s < 10 nM in the binding assay, we measured the

level of VLA-4 inhibition in the serum collected at 15 min

post-oral dosing at 10 mg/kg in mice. By comparison with a

calibration curve, the compound concentration in serum was

estimated. At this point, because it was found that the T_max

of this series of compounds was within nearly 30 min in the

pharmacokinetic study that we previously reported,^18awe set

the collection time of the blood samples to 15 min.

The evaluation results of 2-(benzyl or phenylami-

no)benzoxazole derivatives are summarized in Table 1. First,

to investigate how the NH at the 2-position in the benzox-

azole unit in 5 affects the activity and oral exposure in mice,

we replaced the NH with CH₂. As a result, it was found that

this modification caused significant loss of potency (11a,

33% inhibition at 2 μM). On the other hand, we reported that

the introduction of a fluorine atom into the 5-position in the

terminal benzene in compound 4 led to somewhat of an

improvement of the pharmacokinetic properties while re-

taining the activity.²²Therefore, we introduced a fluorine

atom to the 5-position in the terminal benzene in 5 as an R1

We next turned our attention to the preparation of methyl

trans-4-substituted cyclohexanecarboxylates 10a-d starting

with the methyl benzoates 28a-c (Scheme 5).²⁵Thus, after

deprotection of the tert-butoxycarbonyl (Boc) group of

28a-c, reduction of the benzene ring in 28a-c was achieved

by hydrogenation (10 atm) using Rh/Al₂O₃as the catalyst to

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7977

Scheme 2^a

^aReagents and conditions: (a) o-tolylacetic acid, hexachloroethane, Ph₃P, Et₃N, MeCN; (b) aq NaOH, THF/MeOH; (c) 5-fluoro-2-methylphenyl

isothiocyanate, MeOH, then HgO, MeOH, reflux; (d) benzyl bromide, K₂CO₃, DMF; (e) dimethyl malonate, NaH, NMP; (f) 2 N NaOH, MeOH, reflux,

then conc. HCl; (g) conc. H₂SO₄, MeOH, reflux; (h) H₂, 5% Pd/C, MeOH; (i) (i) o-tolyl isothiocyanate or 4- or 5-fluoro-2-methylphenyl isothiocyanate,

MeOH, then HgO, MeOH, reflux (for 7b, 7e, and 7f), (ii) 3-fluoro-2-methylaniline, thiocarbonyl diimidazole, THF then HgO, THF (for 7d).

Scheme 3^a

^aReagents and conditions: (a) 2-chlorobenzoxazole, xylene, reflux; (b) aq NaOH, THF; (c) thiophosgene, CaCO₃, H₂O/CHCl₃; (d) HgO, toluene,

reflux.

Scheme 4^a

^aReagents and conditions: (a) anilines 20, triphosgene, pyridine, CH₂Cl₂; (b) aq NaOH, THF; (c) ethyl or isopropyl iodide, NaH, DMF (for 26a,b);

(d) K₂CO₃, PMBCl, DMF (for 26c); (e) (i) (COCl)₂, CH₂Cl₂, then anilines 20, Et₃N, CH₂Cl₂, reflux, (ii) TFA, anisole, reflux (only for 9d), or EDC HCl,

HOBt, Et₃N, DMF, anilines 20.

3

substituent. In addition, we also introduced a fluorine atom to

the 7-position in the benzoxazole ring. Although the introduc-

tions were relatively tolerated in activity without and with 3%

HSA compared with 5, no enhancement of the estimated serum

concentration was observed. Next, we carried out introduction

of a fluorine atom to the 3-, 4-, or 5-position to R1 while fixing

Regarding compounds that had the terminal phenylamino-

carbonyl group in compound 4 transformed into the corres-

ponding benzoxazole ring, we investigated the effect of sub-

stituents (none, 4-Me, 5-F, 6-F, and 7-F) on the benzoxazole

ring on the activity and estimated serum concentration

(Table 2). In this modification, we fixed a chlorine atom at the

3-position on the central benzene ring based on the positive

effect on the pharmacokinetic properties in rodents and dogs

that we previously reported.^18aAmong them, nonsubstituted

benzoxazole 12a and 5-F-benzoxazole 12c retained the activity

(12a, IC₅₀= 5.9 nM; 12c, IC₅₀= 7.3 nM), including in the pre-

sence of 3% HSA (12a, IC₅₀= 138 nM; 12c, IC₅₀= 223 nM).

R2 = F. Only 5-F-benzoxazole 11f retained activity (IC₅₀

=

2.8 nM), showing 10 times less potent activity in the presence of

3% HSA (IC₅₀= 439 nM) compared with 5, which was likely

due to an increase of protein binding. Additionally, it was

found that the estimated serum concentration was 2.4 times

higher than that of 5.

7978 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24

Muro et al.

Scheme 5^a

^aReagents and conditions: (a) TFA, CH₂Cl₂, then 5% Rh/Al₂O₃, H₂(10 atm); (b) Boc₂O, Et₃N, MeCN/H₂O; (c) BOMCl, iPr₂EtN, CH₂Cl₂; (d)

NaOMe, MeOH, reflux; (e) TMSCHN₂, benzene/MeOH then separation by flash column chromatograpy; (f) H₂, 5% Pd/C, MeOH; (g) formic acid,

DIAD, Ph₃P, THF; (h) NaHCO₃, THF, H₂O; (i) MeI, NaH, DMF; (j) TFA, CH₂Cl₂.

Table 1. Inhibitory Activity and Estimated Serum Concentration of 2-(Benzyl or Phenylamino)benzoxazole Derivatives 5 and 11a-f

compd

R1

R2

X

IC₅₀(nM) (-/þ 3% HSA)

2.8/45

estimated serum concentration (ng/mL)

5

H

F

NH

CH₂

NH

373

Not tested

363

11a

11b

11c

11d

11e

11f

H

33% inhibition at 2 μM/7% inhibition at 2 μM

3.0/70

H

5-F

3-F

4-F

5-F

H

F

4.4/131

47/13% inhibition at 1.8 μM

12/1680

2.8/439

448

Not tested

912

F

Table 2. Inhibitory Activity and Estimated Serum Concentration of 4-(2-Benzoxazolylamino)phenyl derivatives 12a-e

compd

R3

IC₅₀(nM) (-/þ 3% HSA)

estimated serum concentration (ng/mL)

12a

12b

12c

12d

12e

H

5.9/138

12/290

7.3/223

20/630

40/1032

508

Not tested

358

4-Me

5-F

6-F

Not tested

7-F

However, 6- and 7-F-benzoxazoles 12d and 12e were clearly

less potent than 5. Next, we evaluated 12a and 12c with the

IC₅₀values of less than 10 nM for estimated serum concentra-

tion but found that those compounds did not demonstrate any

improvement.

We next explored 4-(hetroarylcarboxamido)phenyl deri-

vatives in which the terminal phenylamino group in com-

pound 4 was replaced with the corresponding bicyclic

heteroaryl rings while the central 4-amino-3-chlorophenyl

group was fixed. In addition, introduction of a fluorine atom

at the 2-position (R4) in the central benzene in compound 4

was also investigated. The results are shown in Table 3.

Introduction of a 1-indoline (13a,b), a 3-indole (13c), a

3-indazole (13d), and a 3-benzoisothiazole (13i,j) as the

bicyclic heteroaryl group showed potent activity with

IC₅₀’s of less than 10 nM. However, somewhat of a decrease

of activity in almost all the compounds except for 13c was

observed in the presence of 3% HSA. On the other hand,

introduction of a 1-isoquinoline and a 3-benzoisoxazole

resulted in a loss of potency (13m, IC₅₀= 32 nM; 13k,

IC₅₀= 128 nM). Given the high potency of 13c, we also

explored substituents at the 1-position in the indole ring. As a

result, it was found that the introduction of a methyl group

was well tolerated (13e, IC₅₀= 2.6 nM) but not of an ethyl

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7979

Table 3. Inhibitory Activity and Estimated Serum Concentration of

4-(Heteroarylcarboxamido)phenyl Derivatives 13a-m

Table 4. Pharmacokinetic Properties of Compounds 5, 13c, 13e, and 13f

in Rats

^aCompound 5 was administered intravenously at a dose of 10 mg/kg.

pharmacokinetic properties. In particular, 1-Me-indole de-

rivatives 13e,f displayed extremely high serum concentration

values of 5157 and 11437 ng/mL, respectively. Considering

that the serum concentrations of 13e,f were 56 and 40 times

higher than the IC₅₀(þ3% HSA) values of 92 and 284 nM,

these compounds were expected to show efficacy in vivo.

Pharmacokinetic Properties of Selected 4-(3-Indolylcar-

boxamido)phenyl Derivatives. To investigate what the differ-

ences of the estimated serum concentration in the tested

compounds were based on, we selected 4-(3-indolylcarbox-

amido)phenyl derivatives 13c, 13e,f, and the lead compound

5 on the basis of the serum concentration and conducted a

pharmacokinetic study of these compounds using Spra-

gue-Dawley (SD) rats (1 mg/kg, iv). The results are sum-

marized in Table 4. As we expected, 13e,f were found

to clearly demonstrate low plasma clearance (13e, CL =

5.0 (mL/min)/kg; 13f, CL = 2.6 (mL/min)/kg compared

with 13c and 5 (13c, CL=61.1 (mL/min)/kg; 5, CL=12.2

(mL/min)/kg), consistent with the estimated serum concen-

trations. It was found that the improvement in the PK profile

correlated to decrease of the PSA value of the lipophilic

moiety (39.9 A²for 13c; 28.6 A²for 13e; 27.9 A²for 13f).¹⁹

Considering that compounds 13e,f with improved plasma

clearance were relatively susceptible to HSA in the binding

assay, this result suggests that the improved clearance of

compound 13e,f is at least partially a result of increased

plasma protein binding.

group and an isopropyl group (13g, IC₅₀= 204 nM; 13h,

41% inhibition at 1.7 μM), implying that the size of the

substituent was crucial for the activity. Regarding a sub-

stituent at R4, the fluorine-substituted compounds (13b, 13f,

13j, and 13m) were about 2-4 times less potent than

the nonsubstituted compounds (13a, 13e, 13i, and 13l). We

next carried out evaluation of 13a-f and 13i-j for oral

exposure. Among them, compounds 13a,b and 13e,f with

no proton donor (NH) in the heteroaryl group tended to

show higher oral exposure with estimated serum concentra-

tion values of more than 1000 ng/mL in comparison with

compounds 13c,d with the NH function, indicating that this

modification would lead to a significant improvement in the

Optimization of the (4S)-Substituent in the Pyrrolidine.

With the 4-(1-methyl-3-indolylcarboxamido)phenyl moiety

that showed the improved plasma clearance in hand, we

next turned our attention to optimization of the (4S)-sub-

stituent on the pyrrolidine ring in 13f. Thus, we carried out

replacement of the (4S)-fluorine atom in 13f with other

functional groups (H, OH, OMe), which were selected based

on the SAR in the (N⁰-phenylureido)phenyl derivatives we

7980 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24

Muro et al.

Table 5. Inhibitory Activity and Estimated Serum Concentration of 4-(1-Methyl-3-indolylcarboxamido)phenyl Derivatives 13f and 14a-f

compd

R4

R5

IC₅₀(nM) (-/þ 3% HSA)

estimated serum concentration (ng/mL)

14a

14b

14c

14d

14e

13f

14f

F

H

12/370

6.4/45

7.8/70

2.9/36

5.4/93

5.0/284

5.5/241

Not tested

452

93

F

OH

OMe

F

Cl

F

9405

6153

Cl

F

11437

1878

Cl

F

previously reported.²⁵In addition, we also examined repla-

cement of the fluorine atom with the chlorine atom at the

2-position in the central benzene in 13f. The results are

summarized in Table 5. Almost all the compounds except

for 14a (R5 = H) retained their potency with IC₅₀values of

less than 10 nM. These compounds also showed more potent

activity than 13f in the presence of 3% HSA likely due to a

decrease of lipophylicity. This result was in line with the SAR

of a series of (N⁰-phenylureido)phenyl derivatives. On the

other hand, only 14d,e with R5=MeO showed oral exposure

with estimated serum concentration values of 9405 ng/mL

and 6153 ng/mL, respectively.

In Vivo Evaluation and Safety Assessment. On the basis of

the in vitro activity and estimated serum concentration, we

selected compounds 13e,f and 14d,e and conducted further

biological evaluation. First, compounds 13e,f and 14d,e were

evaluated for their anti-inflammatory effect in an Ascaris-

antigen-induced murine asthma model by measuring the

level of eosinophils in bronchoalveolar lavage fluid

(BALF) at 48 h after an antigen challenge.²²All the com-

pounds were administered orally at three different doses

(1.67, 5, and 15 mg/kg bid (twice a day) for 13e,f and 14e;

5, 15, and 45 mg/kg bid for 14d) to mice. All the com-

pounds (13e, 13f, 14d, and 14e) reduced the eosinophil

accumulation in a dose-dependent manner with 13%, 57%,

30%, and 53% inhibition observed at 5 mg/kg, respec-

tively. In addition, the efficacy of compounds 13f and 14e

(g5 mg/kg bid) was comparable to that of the anti-mouse

R₄antibody²⁷(R1-2, 5 mg/kg, sid (once a day), subcutaneous

(sc), 54-62% inhibition) used as the positive control in

this experiment. At present, the efficacy differences between

these compounds cannot be fully explained from the

activities and PK profiles, and we consider that fur-

ther investigation would be necessary to make the details

explicit.

Table 6. Selectivity of 14e toward Other Integrins

integrin/ligand binding assay^a

IC₅₀

VLA-4/VCAM-1^b

5.4 nM

R_Lβ₂/ICAM-1^c

9% inhibition at 162 μM

41% inhibition at 16 μM

10% inhibition at 16 μM

R₄β₇/MAdCAM-1^b

d

R

_IIbβ₃

^aThe details of the binding assays are described in the Experimental

Section. ^bCell/protein binding assay. ^cProtein/protein binding assay.

^dR_IIbβ₃-dependent platelet aggregation assay.

human ether-a-go-go related gene (hERG), and genotoxi-

city. Based on its good in vivo efficacy, safety, and ADME

profiles, compound 14e was selected as a potential clinical

candidate for further pharmacological and PK evaluation.

Pharmacology of Compound 14e. Integrin Selectivity of

Compound 14e. Compound 14e was tested for its integrin

selectivity in a cell/protein or protein/protein binding assay.

As shown in Table 6, compound 14e was found to show high

selectivity for VLA-4 over other integrins such as R_Lβ₂, R₄β₇,

and R_IIbβ₃.

Asthma Models. To evaluate the efficacy of compound 14e

in animal models, we used actively sensitized guinea pig

and mouse asthma models, assessing its ability to reduce

bronchial hyper-responsiveness (BHR) to acetylcholine

chloride (Ach) at 24 or 48 h after an antigen challenge,

respectively. In the guinea pig model, when dosed orally at

0.8-12.5 mg/kg bid, compound 14e significantly reduced the

bronchial responsiveness in a dose-dependent manner (ID₅₀

=

3.0 mg/kg). Also, compound 14e, administered orally at

12.5 mg/kg bid to mice, reduced the BHR to Ach by 83%

in a murine asthma model.²⁶

Pharmacokinetic Properties. The PK properties of com-

pound 14e were determined in rats, dogs, and monkeys. As

shown in Table 7, compound 14e exhibited favorable plasma

clearance and oral bioavailability in all species.

Next, compounds 13f and 14e showing excellent efficacy

were assessed for their safety profile in mice. According to

the results, it was revealed that compound 13f showed

cholestatic liver injury when dosed intravenously at

50 mg/kg or orally at 25 (mg/kg)/day for 2 days. On the

other hand, when compound 14e was dosed intravenously at

50 mg/kg or orally at 200 (mg/kg)/day for 2 days, no

hepatotoxicity was observed. In addition, compound 14e

demonstrated favorable results in advanced in vitro profiling

assays with regard to CYP inhibition, microsomal stability,

Conclusion

In this study we optimized the PK profile of trans-4-

substituted cyclohexanecarboxylic acid derivative 5 by mod-

ifying its lipophilic moiety. We have found that the replace-

ment of the lipophilic moiety with

a 4-(1-methyl-3-

indolylcarboxamido)phenyl group was effective in improving

the PK profiles in rodents while retaining VLA-4 inhibitory

activity. In addition, it has been found that the improvement

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7981

Table 7. Pharmacokinetic Properties of 14e in Rats, Dogs and Monkeys

po

iv

species

F

^d(%)

AUC^e(ng h/mL)

C_max^f(ng/mL)

T_1/2^g(h)

AUC^e(ng h/mL)

CL^h(mL/min)/kg

V_dssⁱ(L/kg)

T_1/2^g(h)

3

Rat^a

Dog^b

Monkey^c

>100

91

68

975

6091

3480

385

1586

431

1.5

2.0

5.5

865

6712

5141

19.3

1.7

1.222

0.164

0.223

1.0

2.1

6.3

1.8

^aDose: po and iv at 1 mg/kg (n = 4). ^bDose: po and iv at 0.5 mg/kg (n = 3). ^cDose: po and iv at 0.5 mg/kg (n = 3). ^dOral bioavailability.

^ePharmacokinetic area under curve. ^fPharmacokinetic maximum concentration. ^gPlasma half-life. ^hPharmacokinetic clearance. ⁱVolume of distribution.

of the PK profile has correlated to a decrease in the polar

surface area in this moiety. Through subsequent fine-tuning of

substituents in the 4-(1-methyl-3-indolylcarboxamido)phenyl

moiety and the pyrrolidine ring, this study culminated in

the discovery of compound 14e, trans-4-[1-[[2,5-dichloro-

4-(1-methyl-3-indolylcarboxamido)phenyl]acetyl]-(4S)-meth-

oxy-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid.

Compound 14e demonstrated excellent efficacy in animal

models of asthma and PK profiles in rats, dogs, and monkeys.

On the basis of its favorable overall profile, compound 14e

was advanced into clinical trials for the treatment of asthma.

In a single ascending-dose phase I clinical study, compound

14e showed good oral exposure as expected from the results of

PK analysis in preclinical species. In addition, there were no

serious adverse events, and it was well tolerated up to 960 mg/

man in this study. The detailed results of the phase I clinical

study will be reported in due course.

chromatography on silica gel with CHCl₃/MeOH (60:1, v/v) as

an eluent to give methyl trans-4-[(4S)-fluoro-1-[[2-(2-methylben-

zyl)-6-benzoxazolyl]acetyl]-(2S)-pyrrolidinylmethoxy]cyclohexa-

necarboxylate (242 mg, 99%) as a colorless oil. ¹H NMR (CDCl₃)

δ 1.17-1.31 (m, 2H), 1.38-1.52 (m, 2H), 1.96-2.28 (m, 7H), 2.39

(s, 3H), 3.21-3.51 (m, 2H), 3.61-4.02 (m, 8H), 4.24 (s, 2H),

4.19-4.38 (m, 1H), 5.14-5.29 (m, 1H), 7.15-7.19 (m, 4H),

7.28-7.29 (m, 1H), 7.39-7.40 (m, 1H), 7.57-7.61 (m, 1H); MS

(ESI), m/z 523 [M þ H]^þ.

To a stirred solution of methyl trans-4-[(4S)-fluoro-1-[[2-(2-

methylbenzyl)-6-benzoxazolyl]acetyl]-(2S)-pyrrolidinylmeth-

oxy]cyclohexanecarboxylate (242 mg, 0.46 mmol) in THF

(3 mL) was added 0.5 N NaOH (3.0 mL, 1.50 mmol), and the

reaction mixture was stirred at room temperature for 2 h. The

mixture was poured into ice-1 N HCl and extracted with

EtOAc. The extract was washed with brine, dried over Na₂SO₄,

and concentrated in vacuo. The residue was purified by column

chromatography on silica gel with CHCl₃/MeOH (60:1 to 15:1,

v/v) as an eluent to give the title compound (151 mg, 64%) as a

colorless amorphous solid. IR (ATR) 1720, 1610, 1433, 1095,

Experimental Section

1

746 cm^-1; H NMR (CDCl₃) δ 1.21-1.32 (m, 2H), 1.41-1.53

Chemistry. All starting materials and synthesis reagents were

obtained commercially. Column chromatography was per-

formed with a Merck silica gel 60 (particle size 0.060-0.200 or

0.040-0.063). Flash column chromatography was performed

with Biotage FLASH Si or YAMAZEN Hi-Flash packed

columns. Thin-layer chromatography (TLC) was performed

on Merck precoated TLC glass sheets with silica gel 60 F254.

Yields were of purified products and were not optimized.

Optical rotations were measured with a HORIBA SEPA-300

(m, 2H), 2.00-2.34 (m, 7H), 2.39 (s, 3H), 3.23-4.03 (m, 7H),

4.24 (s, 2H), 4.19-4.38 (m, 1H), 5.14-5.31 (m, 1H), 7.16-7.20

(m, 4H), 7.29-7.30 (m, 1H), 7.39-7.40 (m, 1H), 7.58-7.62 (m,

1H); MS (ESI), m/z 509 [M þ H]^þ; HRMS (ESI), m/z calcd for

C₂₉H₃₃FN₂O₅þH, 509.2452; found, 509.2468; anal. calcd for

C₂₉H₃₃FN₂O₅0.5H₂O, C 67.30, H 6.62, N 5.41; found, C 67.13,

3

H 6.53, N 5.23; HPLC t_R= 6.5 min. (98.2%).

Compounds 11b-f, 12a-e, 13a-n, and 14a-f were prepared

according to general procedure A.

1

polarimeter. The H NMR spectra were recorded on a JEOL

trans-4-[1-[[7-Fluoro-2-(2-methylphenyl)amino-6-benzoxazoyl]-

acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarbo-

xylic Acid (11b). Yield 16% (two steps). Colorless solid. IR (ATR)

2937, 2864, 1637, 1579 1454, 1070, 752 cm^-1. ¹H NMR (DMSO-

d₆) δ 1.17-1.40 (m, 6H), 1.85-1.99 (m, 2H), 2.12-2.22 (m, 2H),

2.30 (s, 3H), 3.19 (t, J = 8.3 Hz, 1H), 3.38-4.37 (m, 8H),

5.25-5.47 (m, 1H), 6.99-7.13 (m, 3H), 7.25 (d, J = 6.8 Hz,

2H), 7.80 (d, J = 8.6 Hz, 1H), 9.87 (broad s, 1H), 12.05 (broad s,

1H). MS (ESI), m/z 528 [M þ H]^þ. HRMS (ESI), m/z Calcd for

C₂₈H₃₁F₂N₃O₅þH: 528.2310. Found: 528.2316. Anal. Calcd for

JNM-EX-400 spectrometer, and chemical shifts are given in

ppm (δ) from tetramethylsilane as an internal standard. The

spectral splitting patterns are designated as follows: s, singlet; d,

doublet; dd, doublet of doublets; t, triplet; q, quartet; m, multi-

ple. The IR spectra were recorded on a HORIBA FT-720

spectrometer. The mass spectra were recorded on a SCIEX

API-150EX spectrometer (ESI) or a JEOL JMS-HX110 spec-

trometer (FAB). The high-resolution mass (HRMS) spectra

were recorded on a JEOL JMS-100LP spectrometer. HPLC

analysis was performed on a SHIMADZU 10A series with a

Waters Symmetry C₁₈column (i.d. 4.6 mm ꢀ 250 mm) using

MeCN/0.02 N NaOAc buffer (1:1, v/v) as an eluent. Elemental

analysis was performed using a PerkinElmer CHNS/O 2400II, a

Leco CHNS-932 and a YOKOKAWA analysis IC7000RS.

Elemental data for all tested compounds are within (0.4% of

the theoretical values. Purities of g95% were determined by

elemental analysis (all tested compounds, 11-14) and HPLC

(11a, 11b, 11d, 12b, 12c, 12e).

C₂₈H₃₁F₂N₃O₅0.2H₂O: C, 63.31. H, 5.96; F, 7.15; N, 7.91. Found:

3

C, 63.23; H, 5.97; F, 7.30; N, 7.80. HPLC t_R= 3.6 min. (98.7%).

trans-4-[1-[[2-(5-Fluoro-2-methylphenylamino)-6-benzoxazolyl]-

acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarbo-

xylic Acid (11c). Yield 90% (two steps). Colorless solid. IR

(ATR) 2939, 2864, 1639, 1610, 1576, 1437, 1242, 1097, 804 cm^-1

.

¹H NMR (DMSO-d₆) δ 1.10-1.24 (m, 2H), 1.28-1.40 (m, 2H),

1.82-2.01 (m, 4H), 2.10-2.21 (m, 3H), 2.29 (s, 3H), 3.14-3.95

(m, 7H), 4.14-4.32 (m 1H), 5.24-5.44 (m, 1H), 6.85-6.89 (m,

1H), 7.04-7.09 (m, 1H), 7.24 (t, J = 6.8 Hz, 1H), 7.35 (t, J = 8.3

Hz, 2H), 7.95 (d, J = 11.5 Hz, 1H), 9.77 (broad s, 1H), 12.03

(broad s, 1H). MS (ESI), m/z 528 [M þ H]^þ. HRMS (ESI), m/z

Calcd for C₂₈H₃₁F₂N₃O₅þH: 528.2310. Found: 528.2306. Anal.

General Procedure A: Preparation of trans-4-[(4S)-Fluoro-

1-[[2-(2-methylbenzyl)-6-benzoxazolyl]acetyl]-(2S)-pyrrolidinyl-

methoxy]cyclohexanecarboxylic Acid (11a). A mixture of [2-(2-

methylbenzyl)-6-benzoxazolyl]acetic acid (7a, 131 mg, 0.47

mmol), methyl trans-4-[(4S)-fluoro-(2S)-pyrrolidinylmethoxy]-

Calcd for C₂₈H₃₁F₂N₃O₅0.2H₂O: C, 63.31; H, 5.96; F, 7.15; N,

3

cyclohexanecarboxylate (10b, 120 mg, 0.46 mmol), EDC HCl

7.91. Found: C, 63.13; H, 5.89; F, 7.15; N, 7.71.

3

(134 mg, 0.70 mmol), HOBt (94 mg, 0.70 mmol), and Et₃N (97 μL,

0.70 mmol) in DMF (5 mL) was stirred at room temperature for

3 h. The mixture was diluted with H₂O and extracted with EtOAc.

The extract was washed with brine, dried over Na₂SO₄, and

concentrated in vacuo. The residue was purified by column

trans-4-[1-[[2-(3-Fluoro-2-methylphenylamino)-7-fluoro-6-ben-

zoxazolyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohex-

anecarboxylic Acid (11d). Yield 78% (two steps). Colorless solid.

25

[R]_D-27.8 (c 1.0, THF). IR (ATR) 2939, 1701, 1641, 1581,

1452, 1090, 1063, 779 cm^-1. ¹H NMR (DMSO-d₆) δ 1.15-1.37

7982 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24

Muro et al.

(m, 4H), 1.85-2.03 (m, 5H), 2.08-2.17 (m, 2H), 2.50 (d, J = 2.0

Hz, 3H), 3.19 (t, J = 8.8 Hz, 2H), 3.41-4.06 (m, 4H), 4.12-4.37

(m, 1H), 5.25-5.47 (m, 1H), 7.00 (t, J = 8.8 Hz, 1H), 6.98-7.08

(m, 2H), 7.13-7.15 (m, 1H), 7.28 (dd, J = 14.9, 8.1 Hz, 1H), 7.72

(d, J = 8.1 Hz, 1H). MS (ESI), m/z 546 [M þ H]^þ. HRMS (ESI),

m/z Calcd for C₂₈H₃₀F₃N₃O₅þH: 546.2216. Found: 546.2246.

Anal. Calcd for C₂₈H₃₀F₃N₃O₅: C, 61.64; H, 5.54; F, 10.45; N,

7.70. Found: C, 61.40; H, 5.82; F, 10.46; N, 7.43. HPLC t_R= 6.5

min. (97.4%).

548.1764. Found: 548.1810. Anal. Calcd for C₂₇H₂₈ClF₂N₃O₅:

C, 59.18; H, 5.15; Cl, 6.47; F, 6.93; N, 7.54. Found: C, 59.10; H,

5.14; Cl, 6.18; F, 6.64; N, 7.41. HPLC t_R= 6.5 min. (97.1%).

trans-4-[1-[[3-Chloro-4-(6-fluoro-2-benzoxazolyl)aminophenyl]-

acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]-1-cyclohexane-

carboxylic Acid (12d). Yield 73% (two steps). Colorless solid. IR

(ATR) 1701, 1645, 1595, 1481, 1097, 957 cm^-^{1 1}H NMR

.

(DMSO-d₆) δ 1.10-1.34 (m, 4H), 1.83-1.92 (m, 4H), 2.09-

2.19 (m, 3H), 3.14-3.88 (m, 7H), 4.11-4.31 (m, 1H), 5.22-5.42

(m, 1H), 7.04 (dt, J = 9.2, 2.4 Hz, 1H), 7.21 (dt, J = 8.0, 2.0 Hz,

1H), 7.32-7.37 (m, 2H), 7.44 (d, J = 8.0 Hz, 1H), 7.85-7.88 (m,

1H). HRMS (ESI), m/z Calcd for C₂₇H₂₈ClF₂N₃O₅þH:

548.1764. Found: 548.1774. Anal. Calcd for C₂₇H₂₈ClF₂-

trans-4-[1-[[2-(4-Fluoro-2-methylphenylamino)-7-fluoro-6-benz-

oxazolyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohex-

anecarboxylic Acid (11e). Yield 73% (two steps). Colorless solid.

25

[R]_D-28.9 (c 1.0, THF). IR (ATR) 2941, 1635, 1581, 1496,

1454, 1201, 1097, 1068, 798 cm^-1

.

¹H NMR (DMSO-d₆) δ

N₃O₅1.5H₂O: C, 56.40; H, 5.43; N, 7.31. Found: C, 56.39; H,

5.07; N, 7.02.

3

1.13-1.40 (m, 4H), 1.85-2.01 (m, 4H), 2.08-2.22 (m, 3H),

2.30 (s, 3H), 3.15-3.50 (m, 2H), 3.58-4.03 (m, 5H), 4.13-4.39

(m, 1H), 5.24-5.47 (m, 1H), 6.99-7.16 (m, 4H), 7.76 (dd, J =

8.7, 5.5 Hz, 1H), 9.90 (broad s, 1H), 12.05 (broad s, 1H). MS

(ESI), m/z 546 [M þ H]^þ. HRMS (ESI), m/z Calcd for

C₂₈H₃₀F₃N₃O₅þH: 546.2216. Found: 546.2244. Anal. Calcd

for C₂₈H₃₀F₃N₃O₅: C, 61.64; H, 5.54; N, 7.70. Found: C,

61.51; H, 5.73; N, 7.41.

trans-4-[1-[[3-Chloro-4-[2-(7-fluorobenzoxazolyl)]aminophenyl]-

acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarb-

oxylic Acid (12e). Yield 62% (two steps). Brown solid. IR (ATR)

2937, 1635, 1591, 1444, 1182, 1070, 777 cm^-^{1 1}H NMR

.

(DMSO-d₆) δ 1.15-1.39 (m, 4H), 1.84-2.28 (m, 7H), 3.14-

3.88 (m, 7H), 4.12-4.34 (m, 1H), 5.23-5.44 (m, 1H), 7.00-7.05

(m, 1H), 7.18-7.25 (m, 3H), 7.37-7.39 (m, 1H), 7.87-7.90 (m,

1H). MS (FAB), m/z 548 [Mþ1]^þ. HRMS (ESI), m/z Calcd for

C₂₇H₂₈ClF₂N₃O₅þH: 548.1764. Found: 548.1789. Anal. Calcd

for C₂₇H₂₈ClF₂N₃O₅: C, 59.18; H, 5.15; Cl, 6.47; F, 6.93; N,

7.67. Found: C, 59.15; H, 5.08; Cl, 6.41; F, 6.76; N, 7.64. HPLC

trans-4-[1-[[2-(5-Fluoro-2-methylphenylamino)-7-fluoro-6-benz-

oxazolyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohex-

anecarboxylic Acid (11f). Yield 68% (two steps). Colorless solid.

IR (ATR) 2937, 1639, 1610, 1579, 1452, 1201, 1068, 804 cm^-1

.

¹H NMR (DMSO-d₆) δ 1.12-1.43 (m, 4H), 1.82-2.22 (m, 7H),

2.30 (s, 3H), 3.17-3.22 (m, 1H), 3.40-4.08 (m, 6H), 4.13-4.36

(m, 1H), 5.32-5.40 (m, 1H), 6.89 (dt, J = 8.3, 2.0 Hz, 1H),

7.03-7.10 (m, 1H), 7.21 (dd, J = 8.1, 2.4 Hz, 1H), 7.26 (t, J =

7.8 Hz, 1H), 7.92 (d, J = 11.5 Hz, 1H), 10.04 (broad s, 1H). MS

(ESI), m/z 546 [M þ H]^þ. HRMS (ESI), m/z Calcd for

C₂₈H₃₀F₃N₃O₅þH: 546.2216. Found: 546.2220. Anal. Calcd

t

_R= 7.1 min. (95.5%).

trans-4-[1-[[3-Chloro-4-(1-indolinylcarboxamido)]phenyl]acetyl]-

(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic Acid

(13a). Yield 56% (two steps). Colorless solid. IR (ATR) 2937,

1722, 1681, 1641, 1600, 1581, 1520, 1481, 1092 cm^-1. H NMR

1

(CDCl₃) δ 1.21-1.33 (m, 2H), 1.42-1.54 (m, 2H), 2.00-2.50 (m,

7H), 3.26 (t, J = 8.8 Hz, 2H), 3.24-3.36 (m, 2H), 3.48-4.00 (m,

5H), 4.14 (t, J = 8.8 Hz, 2H), 4.16-4.37 (m, 1H), 5.17-5.32 (m,

1H), 6.97 (t, J = 7.6 Hz, 1H), 7.11-7.24 (m, 4H), 7.33 (s, 1H), 7.93

(d, J = 8.0 Hz, 1H), 8.25 (t, J = 9.2 Hz, 1H). MS (ESI), m/z 558

[M þ H]^þ. HRMS (ESI), m/z Calcd for Calcd for C₂₉H₃₃-

ClFN₃O₅þH: 558.2171. Found: 558.2220. Anal. Calcd for

for C₂₈H₃₀F₃N₃O₅0.4H₂O: C, 60.84; H, 5.62; F, 10.31; N,

3

7.60. Found: C, 60.91; H, 5.49; F, 10.32; N, 7.41.

trans-4-[1-[[4-(2-Benzoxazolyl)amino-3-chlorophenyl]acetyl]-

(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic

Acid (12a). Yield 81% (two steps). Colorless amorphous solid.

25

[R]_D-36.7 (c 1.0, THF). IR (ATR) 1637, 1587, 1570, 1458,

C₂₉H₃₃ClFN₃O₅0.25H₂O: C, 61.92; H, 6.00; N, 7.47. Found: C,

3

1238, 1095, 744 cm^-1. ¹H NMR (CDCl₃) δ 1.11-1.25 (m, 2H),

1.28-1.41 (m, 2H), 1.85-1.95 (m, 4H), 2.08-2.33 (m, 3H),

3.16-3.30 (m, 2H), 3.41-3.74 (m, 3H), 3.81-3.92 (m, 2H),

4.13-4.37 (m, 1H), 5.25-5.46 (m, 1H), 7.09-7.13 (m, 1H),

7.18-7.26 (m, 2H), 7.38-7.40 (m, 2H), 7.46-7.48 (m, 1H),

7.90-7.94 (m, 1H), 9.96 (broad s, 1H), 12.03 (broad s, 1H). MS

(ESI), m/z 530 [M þ H]^þ. HRMS (ESI), m/z Calcd for

C₂₇H₂₉ClFN₃O₅þH: 530.1858. Found: 530.1890. Anal. Calcd

for C₂₇H₂₉ClFN₃O₅: C, 61.19; H, 5.52; Cl, 6.69; F, 3.58; N, 7.93.

Found: C, 61.00; H, 5.40; Cl, 6.76; F, 3.65; N, 7.96.

62.05; H, 6.10; N, 7.17.

trans-4-[1-[[5-Chloro-2-fluoro-4-(1-indolinylcarboxamido)phenyl]-

acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic

Acid (13b). Yield 79% (two steps). Colorless amorphous solid. IR

(ATR) 2941, 1712, 1675, 1642, 1535, 1485, 1113, 871, 745 cm^-1. ¹H

NMR (CDCl₃) δ1.20-1.33(m, 2H),1.41-1.54(m,2H), 2.50-2.55

(m, 7H), 3.27 (t, J = 8.0 Hz, 2H), 3.33-3.97 (m, 7H), 4.14 (t, J =

8.8 Hz, 2H), 4.29-4.38 (m, 1H), 5.19-5.36 (m, 1H), 6.99 (t, J = 7.6

Hz, 1H), 7.19-7.24 (m, 3H), 7.34-7.37 (m, 1H), 7.93 (d, J = 8.4

Hz, 1H), 8.22 (t, J = 11.6 Hz, 1H). MS (ESI), m/z 576 [M þ H]^þ.

HRMS (ESI), m/z Calcd for C₂₉H₃₂ClF₂N₃O₅þH: 576.2077.

Found: 576.2103. Anal. Calcd for C₂₉H₃₂ClF₂N₃O₅: C, 60.47; H,

5.60; Cl, 6.15; F, 6.60; N, 7.29. Found: C, 60.38; H, 5.57; Cl, 6.03; F,

6.38; N, 7.34.

trans-4-[1-[[3-Chloro-4-[2-(4-methylbenzoxazolyl)]aminophenyl]-

acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarbo-

xylic Acid (12b). Yield 29% (two steps). Brown solid. IR (ATR)

2937, 1639, 1591, 1425, 1244, 1095, 746 cm^-^{1 1}H NMR

.

(DMSO-d₆) δ 1.15-1.36 (m, 4H), 1.87-2.20 (m, 7H), 2.39 (s,

3H), 3.15-3.87 (m, 7H), 4.13-4.34 (m, 1H), 5.24-5.44 (m, 1H),

6.97-7.02 (m, 2H), 7.21-7.26 (m, 2H), 7.37-7.38 (m, 1H),

7.89-7.93 (m, 1H). MS (ESI), m/z 544 [M þ H]^þ. HRMS (ESI),

m/z Calcd for C₂₈H₃₁ClFN₃O₅þH: 544.2015. Found: 544.2025.

Anal. Calcd for C₂₈H₃₁ClFN₃O₅: C, 61.82; H, 5.74; Cl, 6.52; F,

3.49; N, 7.72. Found: C, 61.64; H, 5.87; Cl, 6.22; F, 3.37; N, 7.39.

HPLC t_R= 10.7 min. (95.5%).

trans-4-[1-[[3-Chloro-4-[2-(5-fluorobenzoxazolyl)]aminophenyl]-

acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexzanecarbo-

xylic Acid (12c). Yield 68% (two steps). Pale yellow solid. IR

(ATR) 2937, 1637, 1589, 1566, 1442, 1136, 796 cm^-1. ¹H NMR

(DMSO-d₆) δ 1.16-1.38 (m, 4H), 1.88-1.95 (m, 4H), 2.15-2.21

(m, 3H), 3.16-3.88 (m, 7H), 4.13-4.35 (m, 1H), 5.25-5.46 (m,

1H), 6.89-6.93 (m, 1H), 7.21-7.25 (m, 2H), 7.39-7.40 (m, 1H),

7.46-7.49 (m, 1H), 7.83-7.86 (m, 1H). MS (FAB), m/z 548 [M þ

H]^þ. HRMS (ESI), m/z Calcd for C₂₇H₂₈ClF₂N₃O₅þH:

trans-4-[1-[[3-Chloro-4-(3-indolylcarboxamido)phenyl]acetyl]-(4S)-

fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic Acid. (13c).

Yield 74% (two steps). Colorless solid. IR (ATR) 3423, 3210, 2940,

2863, 1697, 1627, 1514, 1433, 1101, 735 cm^-1. ¹H NMR (DMSO-d₆)

δ 1.13-1.41 (m, 4H), 1.86-1.96 (m, 4H), 2.11-2.21 (m, 3H),

3.16-3.28 (m, 2H), 3.42-3.88 (m, 5H), 4.14-4.36 (m, 1H),

5.26-5.46 (m, 1H), 7.13-7.21 (m, 3H), 7.39 (dd, J = 7.3, 1.8 Hz,

1H), 7.48 (d, J = 7.8 Hz, 1H), 7.64 (dd, J = 7.8, 4.6 Hz, 1H), 8.14 (d,

J = 7.3 Hz, 1H), 8.29 (d, J = 2.8 Hz, 1H), 9.33 (s, 1H), 11.76 (s, 1H),

12.06 (broad s, 1H). MS (ESI), m/z 556 [M þ H]^þ. Anal. Calcd for

C₂₉H₃₁ClFN₃O₅1.25H₂O: C, 60.20; H, 5.84; Cl, 6.13; F, 3.28; N,

3

7.26. Found: C, 60.12; H, 5.57; Cl, 6.38; F, 3.37; N, 7.37.

trans-4-[1-[[5-Chloro-2-fluoro-4-(3-indazolylcarboxamido)ph-

enyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexane-

carboxylic Acid (13d). Yield 25% (two steps). Colorless

amorphous solid. IR (ATR) 2937, 1670, 1618, 1589, 1535,

1

1406, 1092, 779 cm^-1. H NMR (DMSO-d₆) δ 1.11-1.41 (m,

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7983

4H), 1.86-2.02 (m, 4H), 2.10-2.27 (m, 3H), 3.17-3.30 (m, 2H),

3.42-3.52 (m, 1H), 3.61-3.95 (m, 4H), 4.14-4.39 (m, 1H),

5.25-5.49 (m, 1H), 7.34 (t, J = 7.6 Hz, 1H), 7.48-7.53 (m, 2H),

7.72 (d, J = 8.3 Hz, 1H), 8.12 (dd, J = 11.5, 4.9 Hz, 1H), 8.22 (d,

J = 8.3 Hz, 1H), 9.79 (s, 1H), 12.04 (broad s, 1H). MS (ESI), m/z

575 [M þ H]^þ. HRMS (ESI), m/z Calcd for C₂₈H₂₉ClF₂-

N₄O₅þH: 575.1873. Found: 575.1908. Anal. Calcd for C₂₈H₂₉-

ClF₂N₄O₅: C, 58.49; H, 5.08; Cl, 6.17; F, 6.61; N, 9.74. Found:

C, 58.29; H, 5.13; Cl, 6.08; F, 6.51; N, 9.55.

trans-4-[1-[[4-(3-Benzo[d]isothiazolylcarboxamido)-5-chloro-

2-fluorophenyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cy-

clohexanecarboxylic Acid (13j). Yield 64% (two steps). Colorless

solid. IR (ATR) 2935, 1691, 1637, 1622, 1525, 1406, 1196, 1093,

741 cm^-^{1 1}H NMR (DMSO-d₆) δ 1.17-1.38 (m, 4H),

.

1.82-2.23 (m, 7H), 3.20-4.02 (m, 7H), 4.13-4.38 (m, 1H),

5.35-5.49 (m, 1H), 7.50-7.55 (m, 1H), 7.64-7.74 (m, 2H),

7.93-7.97 (m, 1H), 8.38 (d, J = 8.0 Hz, 1H), 8.81 (d, J = 8.3 Hz,

1H), 10.30 (s, 1H), 12.05 (broad s, 1H). MS (ESI), m/z 592 [M þ

trans-4-[1-[[3-Chloro-4-(1-methyl-3-indolylcarboxamido)phenyl]-

acetyl-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic

Acid (13e). Yield 72% (two steps). Colorless solid. IR (ATR) 2938,

2863, 1647, 1512, 1465, 1101, 744 cm^-1. ¹H NMR (DMSO-d₆) δ

1.19-1.39 (m, 4H), 1.90-2.11 (m, 4H), 2.14-2.27 (m, 3H),

3.17-3.22 (m, 1H), 3.43-3.87 (m, 6H), 3.89 (s, 3H), 4.16-4.35

(m, 1H), 5.25-5.45 (m, 1H), 7.18-7.28 (m, 3H), 7.37-7.39 (m,

1H), 7.52 (d, J = 8.1 Hz, 1H), 7.69-7.73 (m, 1H), 8.15 (d, J = 7.8

Hz, 1H), 8.27 (d, J = 7.8 Hz, 1H), 9.23 (s, 1H), 11.95 (broad s, 1H).

MS (ESI), m/z 570 [M þ H]^þ. Anal. Calcd for C₃₀H₃₃Cl-

H]^þ. Anal. Calcd for C₂₈H₂₈ClF₂N₃O₅S 0.5H₂O: C, 55.95; H,

3

4.86; N, 6.99. Found: C, 56.01; H, 4.84; N, 6.88.

trans-4-[1-[[4-(3-Benzo[d]isoxazolecarboxamido)-5-chloro-2-

fluorophenyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclo-

hexanecarboxylic Acid (13k). Yield 61% (two steps). Colorless

solid. IR (ATR) 2937, 1736, 1697, 1622, 1533, 1097, 756 cm^-1

.

¹H NMR (DMSO-d₆) δ 1.19-1.40 (m, 4H), 1.88-2.23 (m, 7H),

3.19-4.00 (m, 7H), 4.13-4.38 (m, 1H), 5.36-5.49 (m, 1H),

7.50-7.58 (m, 2H), 7.66-7.68 (m, 1H), 7.77-7.81 (m, 1H),

7.94 (d, J = 8.3 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 10.65

(broad s, 1H), 12.05 (broad s, 1H). MS (ESI), m/z 576 [M þ H]^þ.

Anal. Calcd for C₂₈H₂₈ClF₂N₃O₆: C, 58.39; H, 4.90; N, 7.30.

Found: C, 58.19; H, 4.87; N, 7.18.

FN₃O₅0.75 H₂O: C, 61.75; H, 5.96; N, 7.20. Found: C, 61.85;

3

H, 5.92; N, 6.83.

trans-4-[1-[[5-Chloro-2-fluoro-4-(1-methyl-3-indolylcarboxamido)-

phenyl]acetyl-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecar-

boxylic Acid (13f). Yield 86% (two steps). Colorless solid. IR

(ATR) 2940, 2863, 1652, 1521, 1404, 1227, 1099, 744 cm^-1. ¹H

NMR (DMSO-d₆) δ 1.16-1.41 (m, 4H), 1.86-2.32 (m, 7H),

3.17-3.84 (m, 6H), 3.89 (s, 3H), 4.13-4.38 (m, 1H), 5.25-5.47

(m, 1H), 7.19-7.29 (m, 2H), 7.41-7.46 (m, 1H), 7.54 (d, J = 8.4

Hz, 1H), 7.70-7.72 (m, 1H), 8.15 (d, J = 7.6 Hz, 1H), 8.29-8.30

(m, 1H), 9.28 (s, 1H), 12.01 (broad s, 1H). MS (ESI), m/z 588 [M

þ H]^þ. Anal. Calcd for C₃₀H₃₂ClF₂N₃O₅: C, 61.27; H, 5.48; N,

7.15. Found: C, 61.41; H, 5.48; N, 7.11.

trans-4-[1-[[3-Chloro-4-(1-isoquinolinylcarboxamido)phenyl]-

acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarbo-

xylic Acid (13l). Yield 84% (two steps). Yellow solid. IR (ATR)

2935, 2861, 1716, 1596, 1527, 1442, 1093, 754 cm^-1. ¹H NMR

(DMSO-d₆) δ 1.13-1.41 (m, 4H), 1.85-2.25 (m, 7H), 3.17-3.89

(m, 7H), 4.14-4.36 (m, 1H), 5.25-5.46 (m, 1H), 7.26-7.30 (m,

1H), 7.43-7.45 (m, 1H), 7.81 (t, J = 7.1 Hz, 1H), 7.88 (t, J = 7.1

Hz, 1H), 8.12 (d, J = 8.1 Hz, 1H), 8.16 (d, J = 5.6 Hz, 1H),

8.20-8.24 (m, 1H), 8.67 (d, J = 5.6 Hz, 1H), 9.31 (d, J = 8.5 Hz,

1H), 10.84 (s, 1H), 12.02 (broad s, 1H). MS (ESI), m/z 568 [M þ

H]^þ. Anal. Calcd for C₃₀H₃₁ClFN₃O₅: C, 63.43; H, 5.50; N,

7.40. Found: C, 63.40; H, 5.62; N, 7.13.

trans-4-[1-[[3-Chloro-4-(1-ethyl-3-indolylcarboxamido)phenyl]-

acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarbox-

ylic Acid (13g). Yield 77% (two steps). Colorless amorphous

trans-4-[1-[[5-Chloro-2-fluoro-4-(1-isoquinolinylcarboxamido)-

phenyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclozhexa-

necarboxylic Acid (13m). Yield 87% (two steps). Yellow solid.

IR (ATR) 3282, 2940, 2863, 1722, 1693, 1650, 1619, 1581, 1517

1

solid. IR (ATR) 2937, 1647, 1512, 1209, 1093, 746 cm^-1. H

NMR (DMSO-d₆) δ 1.10-1.40 (m, 4H), 1.45 (t, J = 7.2 Hz, 3H),

1.80-2.04 (m, 4H), 2.04-2.36 (m, 3H), 3.10-3.97 (m, 7H),

4.07-4.39 (m, 1H), 4.29 (q, J = 7.2 Hz, 2H), 5.32-5.39 (m,

1H), 7.10-7.30 (m, 3H), 7.38 (d, J = 6.8Hz, 1H), 7.59(d, J = 8.0

Hz, 1H), 7.65 (m, 1H), 8.16 (d, J = 8.0 Hz, 1H), 8.35 (s, 1H), 9.31

(s, 1H), 12.05 (broad s, 1H). MS (ESI), m/z 584 [M þ H]^þ. Anal.

1

cm^-1. H NMR (DMSO-d₆) δ 1.15-1.41 (m, 4H), 1.86-2.33

(m, 7H), 3.17-4.00 (m, 7H), 4.14-4.37 (m, 1H), 5.26-5.48 (m,

1H), 7.49-7.54 (m, 1H), 7.82 (t, J = 7.3 Hz, 1H), 7.88 (t, J = 7.3

Hz, 1H), 8.12 (d, J = 8.1 Hz, 1H), 8.18-8.23 (m, 2H), 8.67 (d,

J = 5.6 Hz, 1H), 9.38 (d, J = 8.8 Hz, 1H), 10.95-10.96 (m, 1H),

12.02 (broad s, 1H). MS (ESI), m/z 586 [M þ H]^þ. Anal. Calcd

Calcd for C₃₁H₃₅ClFN₃O₅0.25H₂O: C, 63.26; H, 6.08; Cl, 6.02;

3

F, 3.23; N, 7.14. Found: C, 63.20; H, 6.08; Cl, 5.86; F, 3.18;

N, 6.96.

for C₃₀H₃₀ClF₂N₃O₅0.25H₂O: C, 61.02; H, 5.21; N, 7.12.

3

trans-4-[1-[[3-Chloro-4-(1-isopropyl-3-indolylcarboxamido)phe-

nyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecar-

boxylic Acid (13h). Yield 76% (two steps). Colorless amorphous

Found: C, 61.01; H, 5.17; N, 7.00.

trans-4-[1-[[5-Chloro-2-fluoro-4-(1-methyl-3-indolylcarboxamido)-

phenyl]acetyl]-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic Acid

(14a). Yield 74% (two steps). [R]_D²⁵-49.9 (c 1.0, CHCl₃). Colorless

amorphous solid. IR (ATR) 2933, 1724, 1612, 1514, 1404, 1219,

1

solid. IR (ATR) 2933, 1643, 1511, 1203, 1095, 746 cm^-1. H

NMR (DMSO-d₆) δ 1.08-1.44 (m, 4H), 1.53 (d, J = 6.8 Hz,

6H), 1.80-2.05 (m, 4H), 2.05-2.34 (m, 3H), 3.12-3.90 (m,

7H), 4.14-4.40 (m, 1H), 4.83-4.86 (m, 1H), 5.32-5.46 (m,

1H), 7.12-7.28 (m, 3H), 7.39 (d, J = 6.8 Hz, 1H), 7.59-7.63 (m,

2H), 8.17 (d, J = 8.0 Hz, 1H), 8.48 (s, 1H), 9.35 (s, 1H),

12.04 (broad s, 1H). MS (ESI), m/z 598 [M þ H]^þ. Anal. Calcd

1182, 1099, 744 cm^-^{1 1}H NMR (CDCl₃) δ 1.13-1.28 (m,

.

2H), 1.40-1.53 (m, 2H), 1.88-2.06 (m, 8H), 2.23-2.34 (m, 1H),

3.16-3.25 (m, 1H), 3.37-3.80 (m, 6H), 3.88 (s, 3H), 4.13-4.25

(m, 1H), 7.32-7.37 (m, 2H), 7.39-7.44 (m, 2H), 7.81 (s,

1H), 8.11-8.14 (m, 1H), 8.29 (s, 1H), 8.46-8.51 (m, 1H). MS

(FAB), m/z 570 [M þ H]^þ. Anal. Calcd for C₃₀H₃₃ClFN₃-

for C₃₁H₃₇ClFN₃O₅0.25H₂O: C, 63.78; H, 6.27; Cl, 5.88;

3

F, 3.15; N, 6.97. Found: C, 63.62; H, 6.32; Cl, 5.72; F, 3.13;

N, 6.77.

O₅0.25H₂O: C, 62.71; H, 5.88; N, 7.31. Found: C, 62.55; H, 5.99;

3

N, 6.96.

trans-4-[1-[[4-(3-Benzo[d]isothiazolylcarboxamido)-3-chloro-

phenyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohex-

anecarboxylic Acid (13i). Yield 64% (two steps). Colorless solid.

IR (ATR) 2935, 2862, 1691, 1604, 1579, 1520, 1191, 1090, 742

trans-4-[1-[[5-Chloro-2-fluoro-4-(1-methyl-3-indolylcarbox-

amido)phenyl]acetyl]-(4S)-hydroxy-(2S)-pyrrolidinylmethoxy]-

cyclohexanecarboxylic Acid (14b). Yield 75% (two steps). Pale

yellow solid. IR (ATR) 2935, 1633, 1520, 1400, 1227, 1099, 742

1

cm^-1. H NMR (DMSO-d₆) δ 1.11-1.38 (m, 4H), 1.86-1.99

cm^-1. H NMR (DMSO-d₆) δ 1.14-1.37 (m, 4H), 1.75-2.17

(m, 4H), 2.12-2.33 (m, 3H), 3.17-3.89 (m, 7H), 4.14-4.36 (m,

1H), 5.25-5.46 (m, 1H), 7.26 (t, J = 6.4 Hz, 1H), 7.44 (dd, J =

7.7, 1.6 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.69-7.73 (m, 1H),

7.93-7.96 (m, 1H), 8.37 (d, J = 8.3 Hz, 1H), 8.80 (d, J = 8.1 Hz,

1H), 10.27 (s, 1H), 12.05 (broad s, 1H). MS (ESI), m/z 575 [M þ

H]^þ. Anal. Calcd for C₂₈H₂₉ClFN₃O₅S: C, 58.58; H, 5.09; N,

7.32. Found: C, 58.41; H, 5.13; N, 7.02.

(m, 7H), 3.10-3.93 (m, 7H), 3.88 (s, 3H), 4.01-4.31 (m, 2H),

5.04-5.09 (m, 1H), 7.19-7.29 (m, 2H), 7.40-7.45 (m, 1H), 7.55

(d, J = 8.3 Hz, 1H), 7.67-7.71 (m, 1H), 8.14 (d, J = 7.8 Hz, 1H),

8.30 (s, 1H), 9.29 (s, 1H), 12.03 (broad s, 1H). MS (ESI), m/z 586

[M þ H]^þ. Anal. Calcd for C₃₀H₃₃ClFN₃O₆0.25H₂O: C, 61.01;

3

H, 5.72; Cl, 6.00; F, 3.22; N, 7.12. Found: C, 61.04; H, 5.75; Cl,

6.06; F, 3.15; N, 6.95.

7984 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24

Muro et al.

trans-4-[1-[[2,5-Dichloro-4-(1-methyl-3-indolylcarboxamido)phe-

nyl]acetyl]-(4S)-hydroxy-(2S)-pyrrolidinylmethoxy]cyclohexanecar-

boxylic Acid (14c). Yield 56% (two steps). Colorless solid. IR

stirred at room temperature for 2 h. The mixture was poured

into ice-1 N HCl. The resulting precipitate was collected by

suction and dried under vacuum to give the title compound (131

mg, 72%) as a pale yellow solid. H NMR (DMSO-d₆) δ 2.28

(s, 3H), 3.67 (s, 2H), 4.30 (s, 2H), 7.13-7.26 (m, 5H), 7.53-7.58

(m, 2H).

1

(ATR) 2937, 1716, 1630, 1500, 1373, 1219, 1101, 1076, 744 cm^-1

.

¹H NMR (DMSO-d₆) δ 1.13-1.36 (m, 4H), 1.79-2.18 (m, 7H),

3.10-3.95 (m, 7H), 3.88 (s, 3H), 4.00-4.35 (m, 2H), 5.04-5.13 (m,

1H), 7.20 (t, J = 7.3 Hz, 1H), 7.26 (t, J = 7.3 Hz, 1H), 7.47-7.55

(m, 2H), 7.87-7.88 (m, 1H), 8.14 (d, J = 7.6 Hz, 1H), 8.29 (s, 1H),

9.36 (s, 1H). MS (ESI), m/z 602 [M þ H]^þ. Anal. Calcd for

Dimethyl (3-Benzyloxy-2-fluoro-4-nitrophenyl)malonate (17).

To a cooled (0 °C), stirred solution of 2,3-difluoro-6-nitrophe-

nol (16, 314.5 g, 1.79 mol) in DMF (3 L) was added dropwise

benzyl bromide (239.6 mL, 2.02 mol) and K₂CO₃(273.6 g, 1.98

mol). The reaction mixture was stirred at room temperature

for 48 h. After being filtered by suction, the filtrate was poured

into H₂O. The mixture was extracted with EtOAc, washed

with brine, dried over Na₂SO₄, and concentrated in vacuo.

The residue was recrystallized from n-hexane/EtOAc to give

2-benzyloxy-3,4-difluoro-1-nitrobenzene (380.2 g, 80%) as a

pale yellow solid. ¹H NMR (DMSO-d₆) δ 5.30 (s, 2H),

7.36-7.49 (m, 6H), 7.87-7.92 (m, 1H).

To a cooled (-4 °C), stirred solution of dimethyl malonate

(339.1 g, 2.57 mol) in NMP (2 L) was added NaH (60% in oil,

136.8 g, 3.42 mol) for 40 min under nitrogen atmosphere. To the

mixture was added 2-benzyloxy-3,4-difluoro-1-nitrobenzene

(453.7 g, 1.71 mol) in NMP (775 mL) at -4 °C for 1 h, and the

reaction mixture was stirred at 45 °C for 2 h. After being cooled

to room temperature, the mixture was poured into H₂O. After

being made acidic (pH = 3) with 2 N HCl, the mixture was

extracted with EtOAc. The extract was washed with brine, dried

over Na₂SO₄, and concentrated in vacuo. The residue was

purified by column chromatography on silica gel with n-hex-

ane/EtOAc (5:1 to 3:1, v/v) as an eluent to give the title

compound (659.6 g, 100%) as a pale yellow oil. ¹H NMR

(DMSO-d₆) δ 3.74 (s, 6H), 5.21 (s, 2H), 5.40 (s, 1H), 7.35-

7.42 (m, 6H), 7.79 (dd, J = 8.7, 1.8 Hz, 1H). MS (ESI), m/z 378

[M þ H]^þ.

C₃₀H₃₃Cl₂N₃O₆1.25H₂O: C, 57.65; H, 5.72; N, 6.72. Found: C,

3

57.53; H, 5.61; N, 6.44.

trans-4-[1-[[5-Chloro-2-fluoro-4-(1-methyl-3-indolylcarboxamido)-

phenyl]acetyl]-(4S)-methoxy-(2S)-pyrrolidinylmethoxy]cyclohexane-

carboxylic Acid (14d). Yield 96% (two steps). Colorless solid. IR

(ATR) 2937, 1701, 1664, 1626, 1587, 1522, 1402, 1217, 1097, 742

1

cm^-1. H NMR (DMSO-d₆) δ 1.12-1.42 (m, 4H), 1.82-2.19

(m, 7H), 3.14-3.19 (m, 2H), 3.23 and 3.26 (each s, total 3H,

amide isomers), 3.44-3.83 (m, 5H), 3.89 (s, 3H), 3.94-4.25 (m,

2H), 7.22 (t, J = 7.3 Hz, 1H), 7.27 (t, J = 6.8 Hz, 1H), 7.42-7.45

(m, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.68-7.70 (m, 1H), 8.15 (d, J

= 7.8 Hz, 1H), 8.31 (d, J = 2.2 Hz, 1H), 9.31 (s, 1H). MS (ESI),

m/z 600 [M

þ

H]^þ. HRMS (ESI), m/z Calcd for

C₃₁H₃₅ClFN₃O₆þH: 600.2277. Found: 600.2300. Anal. Calcd

for C₃₁H₃₅ClFN₃O₆0.8H₂O: C, 60.59; H, 6.00; N, 6.84. Found:

C, 60.60; H, 5.97; N, 6.74.

3

trans-4-[1-[[2,5-Dichloro-4-(1-methyl-3-indolylcarboxamido)-

phenyl]acetyl]-(4S)-methoxy-(2S)-pyrrolidinylmethoxy]cyclohex-

anecarboxylic Acid (14e). Yield 69% (two steps). Colorless fine

needles. [R]_D²⁵-33.5 (c 1.1, THF). IR (ATR) 2939, 1728, 1600,

1

1498, 1379, 1216, 1174, 1100, 1086, 1076, 743 cm^-1. H NMR

(DMSO-d₆) δ 1.11-1.40 (m, 4H), 1.88-2.20 (m, 7H), 3.14-3.51

(m, 5H), 3.58-3.82 (m, 4H), 3.89 (s, 3H), 3.92-4.26 (m, 3H),

7.21 (t, J = 7.3 Hz, 1H), 7.28 (t, J = 6.9 Hz, 1H), 7.49-7.53 (m,

1H), 7.56(d, J = 8.3Hz, 1H), 7.88-7.90(m, 1H), 8.15(d, J = 7.8

Hz, 1H), 8.31 (s, 1H), 9.39 (d, J = 2.8 Hz, 1H), 12.06 (broad s,

1H). MS (ESI), m/z 616 [M þ H]^þ. HRMS (ESI), m/z Calcd for

C₃₁H₃₅Cl₂N₃O₆þH: 616.1981. Found:616.2003. Anal. Calcdfor

Methyl (3-Benzyloxy-2-fluoro-4-nitrophenyl)acetate (18). To

a stirred solution of 17 (659.6 g, 1.75 mol) in MeOH (2.6 L) was

added 2 N NaOH (2.6 L, 5.20 mol), and the reaction mixture was

heated under reflux for 3 h. After being cooled to room

temperature, the mixture was made acidic (pH = 4) by the

addition of conc. HCl. The resulting solid was collected by

suction, washed with H₂O, and dried under vacuum to give

(3-benzyloxy-2-fluoro-4-nitrophenyl)acetic acid (465.0 g, 89%)

as a colorless solid. ¹H NMR (DMSO-d₆) δ 3.79 (d, J = 1.8 Hz,

2H),5.20(s,2H),7.30-7.44 (m, 6H), 7.73 (dd, J= 8.5, 1.6 Hz, 1H).

To a stirred solution of (3-benzyloxy-2-fluoro-4-nitrophe-

nyl)acetic acid (465.0 g, 1.52 mol) in MeOH (5 L) was added

conc. H₂SO₄(150 mL), and the reaction mixture was heated

under reflux for 4 h. After being cooled to room temperature, the

mixture was concentrated to a small volume. The residue was

diluted with H₂O and extracted with CHCl₃. The extract was

washed with H₂O and brine, dried over Na₂SO₄, and concen-

trated in vacuo to give the title compound (486.3 g, 100%) as a

pale yellow oil. ¹H NMR (CDCl₃) δ 3.74 (s, 2H), 3.75 (s, 3H),

5.24 (s, 2H), 7.10 (dd, J = 8.5, 6.7 Hz, 1H), 7.33-7.41 (m, 3H),

7.46-7.49 (m, 2H), 7.59 (dd, J = 8.7, 1.8 Hz, 1H). MS (ESI), m/z

320 [M þ H]^þ.

C₃₁H₃₅Cl₂N₃O₆0.25H₂O: C, 59.95; H, 5.76; Cl, 11.42; N, 6.77.

3

Found: C, 59.68; H, 5.64; Cl, 11.57; N, 6.80.

trans-4-[1-[[2,5-Dichloro-4-(1-methyl-3-indolylcarboxamido)phe-

nyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarb-

oxylic Acid (14f). Yield 75% (two steps). Colorless solid. IR

(ATR) 2937, 2864, 1726, 1651, 1500, 1379, 1221, 1101, 1076, 741

1

cm^-1. H NMR (DMSO-d₆) δ 1.15-1.38 (m, 4H), 1.85-2.01

(m, 4H), 2.11-2.33 (m, 3H), 3.18-3.23 (m, 1H), 3.46-3.97 (m,

6H), 3.89 (s, 3H), 4.13-4.37 (m, 1H), 5.26-5.49 (m, 1H), 7.21 (t,

J = 7.4 Hz, 1H), 7.28 (t, J = 7.4 Hz, 1H), 7.50 (d, J = 15.7 Hz,

1H), 7.55 (d, J = 8.1 Hz, 1H), 7.88 (d, J = 3.4 Hz, 1H), 8.14 (d,

J = 7.8 Hz, 1H), 8.30 (s, 1H), 9.38 (s, 1H), 12.04 (broad s, 1H).

HRMS (ESI), m/z Calcd for C₃₀H₃₂Cl₂FN₃O₅þH: 604.1781.

Found: 604.1821. Anal. Calcd for C₃₀H₃₂Cl₂FN₃O₅0.25H₂O:

3

C, 59.17; H, 5.38; N, 6.90. Found: C, 59.18; H, 5.38; N, 6.71.

[2-(2-Methylbenzyl)-6-benzoxazolyl]acetic Acid (7a). To a

cooled (0 °C), stirred solution of o-tolylacetic acid (414 mg,

2.76 mmol), methyl (4-amino-3-hydroxyphenyl)acetate (15, 500

mg, 2.76 mmol), Ph₃P (1.81 g, 6.90 mmol), and Et₃N (1.92 mL,

13.8 mmol) in MeCN (20 mL) was added dropwise a solution of

hexachloroethane (1.44 g, 6.08 mmol) in CH₂Cl₂(6 mL) for 5

min. The reaction mixture was stirred at room temperature for

20 h. After being filtered through a Celite pad, the filtrate was

evaporated. The residue was purified by column chromatogra-

phy on silica gel with n-hexane/EtOAc (3:1, v/v) as an eluent to

give methyl [2-(2-methylbenzyl)-6-benzoxazolyl]acetate (190

mg, 23%) as a pale yellow oil. ¹H NMR (CDCl₃) δ 2.38 (s,

3H), 3.68 (s, 3H), 3.71 (s, 2H), 4.24 (s, 2H), 7.18-7.25 (m, 4H),

7.27-7.29 (m, 1H), 7.39-7.40 (m, 1H), 7.59-7.62 (m, 1H). MS

(ESI), m/z 296 [M þ H]^þ.

Methyl (4-Amino-2-fluoro-3-hydroxyphenyl)acetate (19). A

suspension of 18 (486.3 g, 1.52 mmol) and 5% Pd/C (wet)

(48.6 g) in MeOH (5 L) was stirred at room temperature under

hydrogen atmosphere for 14 h. After removal of the catalyst by

filtration, the filtrate was concentrated in vacuo. The residue

was purified by column chromatography on silica gel with

n-hexane/EtOAc (3:1 to 2:1, v/v) as an eluent to give the title

compound (238.2 g, 78%) as a light brown solid. ¹H NMR

(CDCl₃) δ 3.57 (s, 2H), 3.70 (s, 3H), 6.45-6.47 (m, 1H),

6.57-6.61 (m, 1H).

[2-(3-Fluoro-2-methylphenylamino)-7-fluoro-6-benzoxazolyl]-

acetic Acid (7d). To a stirred solution of 3-fluoro-2-methylani-

line (0.57 mL, 5.0 mmol) in THF (20 mL) was added thiocarbo-

nyl diimidazole (990 mg, 5.0 mmol) at room temperature. After

To a stirred solution of methyl [2-(2-methylbenzyl)-6-benzox-

azolyl]acetate (190 mg, 0.64 mmol) in THF (4 mL) was added

0.5 N NaOH (3.9 mL, 1.95 mmol), and the reaction mixture was

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7985

4 h stirring, 19 (996 mg, 5.0 mmol) was added to the mixture, and

the reaction mixture was stirred for 2 days. HgO (1.08 g,

5.0 mmol) was added to the reaction mixture and heated at

70 °C for 4.5 h. After being cooled to room temperature,

the reaction mixture was filtered through a Celite pad, and

the filtered cake was washed with MeOH. The filtrate

was concentrated in vacuo and the residue was purified by

column chromatography on silica gel with n-hexane/EtOAc

(4:1, v/v) as an eluent to give methyl [2-(3-fluoro-2-

methylphenylamino)-7-fluoro-6-benzoxazolyl]acetate (1.21 g,

Ethyl [4-(2-Benzoxazolyl)amino-3-chlorophenyl]acetate (21).

A mixture of 2-chlorobenzoxazole (743 μL, 6.51 mmol) and

ethyl (4-amino-3-chlorophenyl)acetate (20a, 1.30 g, 6.51 mmol)

in xylene (10 mL) was heated under reflux for 2 h. After being

cooled to room temperature, the mixture was diluted with

CHCl₃. The mixture was washed with H₂O and brine, dried

over Na₂SO₄, and evaporated. The residue was purified by

column chromatography on silica gel with n-hexane/EtOAc

(9:1, v/v) as an eluent to give the title compound (1.70 g, 79%)

as a pale yellow solid. ¹H NMR (CDCl₃) δ 1.25-1.28 (m, 3H),

3.58 (s, 2H), 4.14-4.19 (m, 2H), 7.15-7.19 (m, 1H), 7.24-7.30

(m, 3H), 7.36-7.38 (m, 2H), 7.52-7.54 (m, 1H), 8.51-8.53 (m,

1H). MS (ESI), m/z 331 [M þ H]^þ.

1

73%) as a colorless solid. H NMR (CDCl₃) δ 2.27 (s, 3H),

3.72 (s, 3H), 3.76 (s, 2H), 6.87 (t, J = 8.8 Hz, 1H), 7.09 (t, J = 6.6

Hz, 1H), 7.22-7.27 (m, 2H), 7.93 (d, J = 7.8 Hz, 1H). MS (ESI),

m/z 333 [M þ H]^þ.

[4-(2-Benzoxazolyl)amino-3-chlorophenyl]acetic Acid (8a). To

a stirred solution of 21 (1.70 g, 5.14 mmol) in THF (30 mL) was

added 0.5 N NaOH (30 mL, 15.0 mmol), and the reaction

mixture was stirred at room temperature for 20 h. The mixture

was concentrated to a small volume and poured into ice-1 N

HCl. The resulting precipitate was collected by suction and dried

under vacuum to give the title compound (1.24 g, 80%) as a pale

yellow solid. ¹H NMR (DMSO-d₆) δ 3.62 (s, 2H), 7.10-7.19 (m,

1H), 7.21-7.28 (m, 1H), 7.30-7.31 (m, 1H), 7.38-7.40 (m, 1H),

7.45-7.49 (m, 3H), 7.94-7.96 (m, 1H).

To a stirred solution of methyl [2-(3-fluoro-2-methylph-

enylamino)-7-fluoro-6-benzoxazolyl]acetate (1.21 g, 3.64 mmol)

in THF/MeOH (2:1, v/v, 60 mL) was added 1 N NaOH (20 mL).

After stirring at room temperature for 17 h, the mixture was

concentrated in vacuo and acidified with 1 N HCl. The resulting

precipitate was collected, washed with water, and dried under

reduced pressure to give the title compound (1.10 g, 15%) as a

colorless solid. ¹H NMR (DMSO-d₆) δ 2.21 (d, J = 2.0 Hz, 3H),

3.66 (s, 2H), 7.00 (t, J = 8.8 Hz, 1H), 7.10-7.16 (m, 2H),

7.26-7.30 (m, 1H), 7.72 (d, J = 7.1 Hz, 1H), 10.10 (broad s,

1H). MS (ESI), m/z 319 [M þ H]^þ.

Methyl (3-Chloro-4-isothiocyanatophenyl)acetate (22). To a

cooled (0 °C), stirred suspension of CaCO₃(626 mg, 6.25 mmol)

and thiophosgene (191 μL, 2.51 mmol) in CH₂Cl₂-H₂O

(10 mL, 1:1, v/v) was added methyl (4-amino-3-chlorophe-

nyl)acetate (20b, 500 mg, 2.50 mmol) in CH₂Cl₂(5 mL).

The reaction mixture was stirred at 0 °C to room tempera-

ture for 1.5 h. To the mixture was added 1 N HCl, and

the mixture was extracted with CH₂Cl₂. The extract was washed

with brine, dried over Na₂SO₄, and evaporated to give the

title compound (652 mg, 100%) as a yellow oil. MS (ESI) m/z

241 [M]^þ.

General Procedure B: Preparation of [2-(5-Fluoro-2-methyl-

phenylamino)-7-fluoro-6-benzoxazolyl]acetic Acid (7f). To a stirred

solution of 19 (1.0 g, 5.02 mmol) in MeOH (30 mL) was added

5-fluoro-2-methylphenyl isothiocyanate (1.0 g, 5.98 mmol) at

room temperature. After 5 days stirring, HgO (1.14 g, 4.36 mmol)

was added to the reaction mixture, and the mixture was heated at

70 °C for 6 h. After being cooled to room temperature, the reaction

mixture was filtered through a Celite pad, and the filtrate was

concentrated in vacuo. The residue was purified by column

chromatography on silica gel with CHCl₃/EtOAc (30:1, v/v) as

an eluent to give methyl [2-(5-fluoro-2-methylphenylamino)-7-

fluoro-6-benzoxazolyl]acetate (810 mg, 56%) as a pink solid. ¹H

NMR (CDCl₃) δ 2.32 (s, 3H), 3.72 (s, 3H), 3.76 (s, 2H), 6.75 (dt,

J = 8.1, 2.7 Hz, 1H), 6.87 (broad s, 1H), 7.09-7.17 (m, 2H),

7.24-7.25 (m 1H), 8.11 (dd, J = 11.0, 6.7 Hz, 1H). MS (ESI), m/z

333 [M þ H]^þ.

General procedure C: Preparation of [4-[2-(4-Methylbenzox-

azolyl)]amino-3-chlorophenyl]acetic Acid (8b). A mixture of 22

(652 mg, 2.50 mmol) and 2-amino-3-methylphenol (23a, 307 mg,

2.50 mmol) in toluene (15 mL) was heated under reflux for 2 h.

Then HgO (541 mg, 2.50 mmol) was added to the mixture, and

the reaction mixture was heated under reflux for 5 h. After being

cooled to room temperature, the mixture was filtered through a

Celite pad, and the filtrate was evaporated. The residue was

purified by column chromatography on silica gel with n-hexane/

EtOAc (7:1, v/v) as an eluent to give methyl [4-[2-(4-methylben-

zoxazolyl)]amino-3-chlorophenyl]acetate (359 mg, 43%) as a

black oil. ¹H NMR (CDCl₃) δ 2.55 (s, 3H), 3.58 (s, 2H), 3.70 (s,

3H), 7.02-7.06 (m, 2H), 7.11-7.19 (m, 1H), 7.25-7.28 (m, 1H),

7.33-7.34 (m, 1H), 7.50 (broad s, 1H), 8.54-8.56 (m, 1H). MS

(ESI), m/z 331 [M þ H]^þ.

To a stirred solution of methyl [2-(5-fluoro-2-methylphenyl-

amino)-7-fluoro-6-benzoxazolyl]acetate (810 mg, 2.44 mmol) in

THF/MeOH (90 mL, 2:1, v/v) was added 1 N NaOH (30 mL).

After being stirred at room temperature for 12 h, the mixture was

concentrated under reduced pressure and acidified with 1 N HCl.

The precipitate was collected, washed with water, and dried under

reduced pressure to give the title compound (700 mg, 90%) as a

1

pale brown solid. H NMR (DMSO-d₆) δ 2.29 (s, 3H), 3.68 (s,

2H), 6.89 (dt, J = 7.0, 1.7 Hz, 1H), 7.14 (t, J = 6.4 Hz, 1H),

7.20-7.27 (m, 2H), 7.90 (d, J = 11.3 Hz, 1H), 10.06 (broad s, 1H).

MS (ESI), m/z 319 [M þ H]^þ.

To a stirred solution of methyl [4-[2-(4-methylbenzoxazo-

lyl)]amino-3-chlorophenyl]acetate (359 mg, 1.08 mmol) in THF

(6 mL) was added 0.5 N NaOH (6.5 mL, 3.25 mmol), and the

reaction mixture was stirred at room temperature for 4 h. The

mixture was poured into ice-1 N HCl and extracted with

EtOAc. The extract was washed with brine, dried over Na₂SO₄,

and evaporated to give the title compound (281 mg, 82%) as a

Compounds 7b, 7c, and 7e were prepared according to

general procedure B.

[7-Fluoro-2-(2-methylphenylamino)-6-benzoxazolyl]acetic Acid (7b).

Yield 86% (two steps). Light brown solid. ¹H NMR (DMSO-d₆)

δ 2.30 (s, 3H), 3.68 (s, 2H), 7.09-7.18 (m, 3H), 7.25 (d, J = 6.9

Hz, 2H), 7.80 (d, J = 8.1 Hz, 1H), 8.30 (s, 1H), 12.46 (broad s,

1H). MS (ESI), m/z 301 [M þ H]^þ.

1

brown solid. H NMR (CDCl₃) δ 2.55 (s, 3H), 3.57 (s, 2H),

6.72-6.74 (m, 1H), 7.04-7.06 (m, 2H), 7.18-7.20 (m, 2H),

8.46-8.48 (m, 1H). MS (ESI), m/z 317 [M þ H]^þ.

Compounds 8c-e were prepared according to general proce-

dure C.

[2-(5-Fluoro-2-methylphenylamino)-6-benzoxazolyl]acetic Acid (7c).

Yield 45% (two steps). Colorless solid. ¹H NMR (DMSO-d₆) δ 2.49

(s, 3H), 3.64 (s, 2H), 6.87 (dt, J = 8.3, 1.5 Hz, 1H), 7.11 (d, J = 8.1

Hz, 1H), 7.25 (t, J = 7.3 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.41 (s,

1H),7.96(d,J=11.5Hz,1H),9.80(broads,1H).MS(ESI),m/z301

[M þ H]^þ.

[3-Chloro-4-(5-fluoro-2-benzoxazolyl)aminophenyl]acetic Acid (8c).

Yield 7% (two steps). Brown solid. ¹H NMR (DMSO-d₆) δ 3.62 (s,

2H), 6.89-6.94 (m, 1H), 7.21-7.30 (m, 2H), 7.45-7.49 (m, 2H),

7.85-7.87 (m, 1H), 10.16 (broad s, 1H), 12.44 (broad s, 1H). MS

(ESI), m/z 321 [M þ H]^þ.

[2-(4-Fluoro-2-methylphenylamino)-7-fluoro-6-benzoxazolyl]-

acetic Acid (7e). Yield 91% (two steps). Colorless solid. ¹H

NMR (DMSO-d₆) δ 2.26 (s, 3H), 3.68 (s, 2H), 7.06-7.14

(m, 4H), 7.73-7.76 (m, 1H), 9.91 (s, 1H). MS (ESI), m/z 319

[M þ H]^þ.

[3-Chloro-4-(6-fluoro-2-benzoxazolyl)aminophenyl]acetic Acid (8d).

Yield 14% (two steps). Pale yellow solid. ¹H NMR (DMSO-d₆)

δ 3.55 (s, 2H), 7.02-7.07 (m, 1H), 7.26 (dd, J = 8.4, 2.4 Hz, 1H),

7.35 (dd, J = 8.0, 4.4 Hz, 1H), 7.42 (d, J = 1.6 Hz, 1H), 7.47 (dd,

7986 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24

Muro et al.

J = 8.4, 2.4 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 10.0 (broad s,

1H).

7.36-7.40 (m, 1H), 7.95 (s, 1H), 8.16-8.20 (m, 1H). MS

(ESI), m/z 218 [M þ H]^þ.

[3-Chloro-4-(7-fluoro-2-benzoxazolyl)aminophenyl]acetic Acid (8e).

Yield 11% (2 steps). Brown solid. MS (ESI), m/z 321 [M þ H]^þ.

General procedure D: Preparation of [3-Chloro-4-(1-indolinyl-

carboxamido)phenyl]acetic Acid (9a). To a cooled (0 °C), stirred

solution of 20a (2.00 g, 9.36 mmol) in CH₂Cl₂(50 mL) was

added triphosgene (926 mg, 3.12 mmol) and pyridine (5 mL).

After the mixture was stirred at room temperature for 15 h,

indoline (1.05 mL, 9.36 mmol) was added to the mixture,

and the reaction mixture was stirred at room temperature for

15 h. The mixture was diluted with H₂O and extracted with

EtOAc. The extract was washed with brine, dried over MgSO₄,

and evaporated to give ethyl [3-chloro-4-(1-indolinylcarboxa-

mido)phenyl]acetate (3.07 g, 91%) as a light pink crystalline

powder. ¹H NMR (CDCl₃) δ 1.25 (t, J = 7.3 Hz, 3H), 3.20-3.26

(m, 2H), 3.54 (d, J = 2.7 Hz, 2H), 4.08-4.18 (m, 4H), 6.93-6.98

(m, 1H), 7.09-7.31 (m, 5H), 7.94 (d, J = 8.1 Hz, 1H), 8.28

(d, J = 8.1 Hz, 1H). MS (ESI), m/z 359 [M þ H]^þ.

1-Isopropylindole-3-carboxylic Acid (27e). Yield 90%. Light

pink amorphous solid. ¹H NMR (DMSO-d₆) δ 1.50 (d, J = 6.6

Hz, 6H), 4.77-4.82 (m, 1H), 7.17-7.25 (m, 2H), 7.61 (d, J = 8.0

Hz, 1H), 8.04 (d, J = 7.6 Hz, 1H), 8.13 (s, 1H), 11.97 (broad s,

1H).

General Procedure F: Preparation of [5-Chloro-2-fluoro-4-(1-

a

methyl-3-indolylcarboxamido)phenyl]acetic Acid (9f). To

cooled (0 °C), stirred solution of 1-methylindole-3-carboxylic

acid (27c, 1.0 g, 5.71 mmol) in CH₂Cl₂(10 mL) was added

(COCl)₂(735 μL, 8.57 mmol), and the reaction mixture was

stirred at room temperature for 10 min. After removal of the

solvent, the residue was dissolved in CH₂Cl₂(20 mL). To the

mixture was added ethyl (4-amino-5-chloro-2-fluorophe-

nyl)acetate (20c, 1.32 g, 5.71 mmol) and Et₃N (1.60 mL, 11.4

mmol), and the reaction mixture was heated under reflux for

17 h. After being cooled to room temperature, the mixture was

diluted with water and extracted with CHCl₃. The extract was

washed with brine, dried over Na₂SO₄, and evaporated. The

residue was purified by column chromatography on silica gel

with n-hexane/EtOAc (2:1, v/v) as an eluent to give ethyl

[5-chloro-2-fluoro-4-(1-methyl-3-indolylcarboxamido)phenyl]-

acetate (1.55 g, 70%) as a colorless solid. ¹H NMR (CDCl₃) δ

1.27 (t, J = 7.3 Hz, 3H), 3.62 (s, 2H), 3.88 (s, 3H), 4.19 (q, J =

7.3 Hz, 2H), 7.32-7.43 (m, 4H), 7.80 (s, 1H), 8.11-8.16 (m, 1H),

8.29 (broad s, 1H), 8.53 (d, J = 12.0 Hz, 1H). MS (ESI), m/z 389

[M þ H]^þ.

To a stirred solution of ethyl [3-chloro-4-(1-indolinylcarbox-

amido)phenyl]acetate (3.07 g, 8.56 mmol) in THF (70 mL) was

added 0.25 N NaOH (68.4 mL, 17.1 mmol), and the reaction

mixture was heated under reflux for 4 h. After being cooled

to room temperature, the mixture was poured into 1 N HCl

(50 mL). The resulting precipitate was collected by suction and

dried under vacuum to give the title compound (1.71 g, 60%)

1

as a colorless crystalline powder. H NMR (DMSO-d₆) δ 3.16

(t, J = 8.8 Hz, 2H), 3.57 (s, 2H), 4.11 (t, J = 8.8 Hz, 2H), 6.86

(t, J = 7.3 Hz, 1H), 7.07 (t, J = 7.8 Hz, 1H), 7.13-7.20 (m, 2H),

7.38 (s, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 8.3 Hz, 1H),

8.15 (s, 1H), 12.36 (s, 1H). MS (ESI), m/z 331 [M þ H]^þ.

Compound 9b was prepared according to general proce-

dure D.

[5-Chloro-2-fluoro-4-(1-indolinylcarboxamido)phenyl]acetic

Acid (9b). Yield 94% (two steps). Colorless solid. ¹H NMR

(DMSO-d₆) δ 3.19 (t, J = 8.4 Hz, 2H), 3.63 (s, 2H), 4.15 (t, J =

8.8 Hz, 2H), 6.91 (t, J = 7.6 Hz, 1H), 7.12 (t, J = 8.0 Hz, 1H),

7.20 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 7.2 Hz, 1H), 7.59 (d, J =

11.2 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 8.19 (s, 1H), 12.50 (broad

s, 1H).

General procedure E: Preparation of 1-Ethylindole-3-car-

boxylic Acid (27d). To a stirred suspension of NaH (60% in

oil, 274 mg, 6.85 mmol) in DMF (8 mL) was added methyl

indole-3-carboxylate (25a, 400 mg, 2.28 mmol) at 0 °C, and

the mixture was stirred for 40 min. To the mixture was added

ethyl iodide (0.27 mL, 3.42 mmol), and the mixture was stirred

at 0 °C for 2 h. The reaction mixture was quenched by the

addition of H₂O and extracted with EtOAc. The extract was

washed with 1 N HCl, dried over Na₂SO₄, and evaporated to

give methyl 1-ethylindole-3-carboxylate (26a), which was

used in the subsequent reaction without further purification.

¹H NMR (CDCl₃) δ 1.52 (t, J = 7.2 Hz, 3H), 3.91 (s, 3H),

4.20 (q, J = 7.2 Hz, 2H), 7.23-7.28 (m, 2H), 7.33-7.38

(m, 1H), 7.84 (s, 1H), 8.15-8.20 (m, 1H). MS (ESI), m/z 204

[M þ H]^þ.

To a stirred solution of ethyl [5-chloro-2-fluoro-4-(1-methyl-

3-indolylcarboxamido)phenyl]acetate (1.55 g, 3.99 mmol) in

THF (30 mL) was added 0.25 N NaOH (32 mL, 8.0 mmol),

and the reaction mixture was stirred at room temperature for

17 h. After the solution was acidified with 1 N HCl, the resulting

precipitate was collected by suction and dried under vacuum to

1

give the title compound (1.33 g, 92%) as a colorless solid. H

NMR (DMSO-d₆) δ 3.66 (s, 2H), 3.89 (s, 3H), 7.20-7.29 (m,

2H), 7.55 (d, J = 7.8 Hz, 2H), 7.73-7.78 (m, 1H), 8.15 (d, J =

7.8 Hz, 1H), 8.31 (s, 1H), 9.29 (s, 1H), 12.56 (broad s, 1H). MS

(ESI), m/z 361 [M þ H]^þ.

Compounds 9e-i were prepared according to general proce-

dure F.

[3-Chloro-4-(1-methyl-3-indolylcarboxamido)phenyl]acetic Acid

(9e). Yield 28% (two steps). Colorless solid. ¹H NMR (DMSO-d₆)

δ 3.61 (s, 2H), 3.89 (s, 3H), 7.17-7.28 (m, 3H), 7.43 (d, J = 1.7 Hz,

1H), 7.53 (d, J = 8.3 Hz, 1H), 7.69 (d, J = 8.3 Hz, 1H), 8.14 (d,

J = 7.8 Hz, 1H), 8.26 (s, 1H), 9.26 (s, 1H).

[2,5-Dichloro-4-(1-methyl-3-indolecarboxamido)phenyl]acetic

1

Acid (9g). Yield 71%. Colorless solid. H NMR (DMSO-d₆) δ

3.72 (s, 2H), 3.90 (s, 3H), 7.22 (t, J = 8.1 Hz, 1H), 7.28 (t, J = 8.1

Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.64 (s, 1H), 7.92 (s, 1H), 8.15

(d, J = 7.8 Hz, 1H), 8.31 (s, 1H), 9.39 (s, 1H). MS (ESI), m/z 378

[M þ H]^þ.

[3-Chloro-4-(1-ethyl-3-indolylcarboxamido)phenyl]acetic Acid

(9h). Yield 83%. Brown amorphous solid. ¹H NMR (DMSO-d₆)

δ 1.43 (m, 3H), 3.62 (s, 2H), 4.28 (m, 2H), 7.12-7.30 (m, 2H),

7.44 (m, 1H), 7.58 (m, 1H), 7.66 (m, 1H), 7.91-8.10 (m,

1H), 8.15 (m, 1H), 8.35 (m, 1H), 9.31 (s, 1H). MS (ESI), m/z

357 [M þ H]^þ.

To a stirred solution of 26a in THF (9 mL) was added 0.25 N

NaOH (13 mL) at room temperature. The reaction mixture was

stirred at 50 °C for 9 h. After being cooled to room temperature,

the mixture was neutralized with 1 N HCl, and the resulting

precipitate was collected by suction, washed with water, and

dried at 50 °C to give the title compound (388 mg, 90% for two

steps) as a pale pink amorphous solid. ¹H NMR (DMSO-d₆) δ

1.38 (t, J = 7.2 Hz, 3H), 4.26 (q, J = 7.2 Hz, 2H), 7.12-7.28 (m,

2H), 7.55 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 8.07 (s,

1H), 11.93 (broad s, 1H).

Compound 27e was prepared according to general proce-

dure E.

Methyl 1-Isopropylindole-3-carboxylate (26b). Yield 78%.

Light yellow oil. ¹H NMR (CDCl₃) δ 1.56 (d, J = 6.8 Hz,

6H), 3.91 (s, 3H), 4.62-4.72 (m, 1H), 7.24-7.29 (m, 2H),

[3-Chloro-4-(1-isopropyl-3-indolylcarboxamido)phenyl]acetic

Acid (9i). Yield 85%. Brown amorphous solid. ¹H NMR

(DMSO-d₆) δ 1.52 (d, J = 6.4 Hz, 6H), 3.62 (s, 2H), 4.77-

4.89 (m, 1H), 7.13-7.30 (m, 3H), 7.44 (s, 1H), 7.60-7.64 (m,

2H), 8.12-8.20 (m, 1H), 8.48 (s, 1H), 9.36 (s, 1H), 12.32 (broad

s, 1H). MS (ESI), m/z 371 [M þ H]^þ.

General Procedure G: Preparation of [3-Chloro-4-(3-indolyl-

carboxamido)phenyl]acetic Acid (9c). To a stirred solution of

indole-3-carboxylic acid (27a, 1.00 g, 6.21 mmol), 20a (1.33 g,

6.22 mmol) and Et₃N (1.80 mL, 12.9 mmol) in DMF (24 mL)

was added EDC HCl (1.78 g, 9.28 mmol), and the reaction

mixture was stirred at 70 °C for 17 h. After being cooled to room

3

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7987

temperature, the mixture was diluted with H₂O and extracted

with EtOAc. The extract was washed with brine, dried over

Na₂SO₄, and concentrated in vacuo. The residue was purified by

column chromatography on silica gel with n-hexane/EtOAc (4:1

to 1:1, v/v) as an eluent to give ethyl [3-chloro-4-(3-indolylcar-

boxamido)phenyl]acetate (1.25 g, 56%) as a pale yellow solid.

¹H NMR (CDCl₃) δ 1.28 (t, J = 7.1 Hz, 3H), 3.59 (s, 2H), 4.18

(q, J = 7.1 Hz, 2H), 7.23-7.25 (m, 1H), 7.31-7.38 (m, 3H),

7.45-7.49 (m, 1H), 7.92 (d, J = 2.9 Hz, 1H), 8.15-8.18 (m, 1H),

8.32 (broad s, 1H), 8.58 (d, J = 8.5 Hz, 1H), 8.78 (broad s, 1H).

MS (ESI), m/z 357 [M þ H]^þ.

3.76 (s, 3H), 4.05 (s, 3H), 5.64 (s, 2H), 6.82 (d, J = 8.5 Hz, 2H),

7.20 (d, J = 8.5 Hz, 2H), 7.30 (dd, J = 8.1, 4.2 Hz, 1H),

7.36-7.37 (m, 2H), 8.23 (dd, J = 8.1, 1.0 Hz, 1H). MS (ESI), m/z

297 [M þ H]^þ.

1-(4-Methoxybenzyl)-3-indazolylcarboxylic Acid (27b). To a

stirred solution of 26c (830 mg, 2.80 mmol) in THF (22 mL) was

added 0.25 N NaOH (22 mL, 5.60 mmol), and the reaction

mixture was stirred at room temperature for 15 h. The reaction

mixture was poured into 1 N HCl and extracted with CHCl₃.

The extract was dried over MgSO₄and concentrated in vacuo.

The residue was dissolved in CHCl₃, and hexane was added until

a precipitate formed. The resulting precipitate was collected by

suction and dried under vacuum to give the title compound (630

mg, 80%) as a colorless amorphous solid. ¹H NMR (DMSO-d₆)

δ 3.70 (s, 3H), 5.69 (s, 2H), 6.88 (d, J = 8.8 Hz, 2H), 7.26 (d, J =

8.8 Hz, 2H), 7.29-7.32 (m, 1H), 7.43-7.48 (m, 1H), 7.84 (d, J =

8.6 Hz, 1H), 8.08 (d, J = 8.6 Hz, 1H), 13.06 (broad s, 1H). MS

(ESI), m/z 283 [M þ H]^þ.

To a stirred solution of ethyl [3-chloro-4-(3-indolylcarboxa-

mido)phenyl]acetate (1.25 g, 3.50 mmol) in THF (35 mL) was

added 0.25 N NaOH (21 mL, 5.23 mmol), and the reaction

mixture was stirred at room temperature for 4 h. After being

concentrated to a small volume, the residue was made acidic

with 1 N HCl (10 mL). The resulting solid was collected by

suction, washed with H₂O, and dried under vacuum to give the

title compound (1.05 g, 91%) as a colorless solid. ¹H NMR

(DMSO-d₆) δ 3.61 (s, 2H), 7.12-7.20 (m, 2H), 7.24 (d, J = 8.3

Hz, 1H), 7.44 (s, 1H), 7.47 (d, J = 7.3 Hz, 1H), 7.67 (d, J = 8.3

Hz, 1H), 8.14 (d, J = 7.3 Hz, 1H), 8.29 (s, 1H), 9.32 (s, 1H), 11.75

(broad s, 1H).

[5-Chloro-2-fluoro-4-(3-indazolylcarboxamido)phenyl]acetic

Acid (9d). To a stirred suspension of 27b (630 mg, 2.23 mmol)

in CH₂Cl₂(10 mL) were added (COCl)₂(292 μL, 3.53 mmol)

and DMF (1 drop). The mixture was stirred until the suspension

turned into a clear solution. The resulting solution was concen-

trated in vacuo to remove excess (COCl)₂. The solid obtained

was dissolved in CH₂Cl₂(20 mL), and ethyl (4-amino-5-chloro-

2-fluorophenyl)acetate (20c, 517 mg, 2.23 mmol) and Et₃N (621 μL,

4.46 mmol) were added. The mixture was heated under reflux for

15 h. After being cooled to room temperature, the mixture

was quenched by the addition of H₂O and extracted with CHCl₃.

The extract was dried over MgSO₄and concentrated in vacuo.

The residue was purified by column chromatography on silica gel

with n-hexane/EtOAc (4:1, v/v) as an eluent to give ethyl [5-chloro-

2-fluoro-4-[1-(4-methoxybenzyl)-3-indazolylcarboxamido]phenyl]-

acetate (881 mg, 80%) as a pale yellow amorphous solid. ¹H NMR

(CDCl₃) δ 1.27 (t, J = 7.1 Hz, 3H), 3.62 (s, 2H), 3.77 (s, 3H), 4.19

(q, J = 7.1 H, 2H), 5.59 (s, 2H), 6.85 (d, J = 8.8 Hz, 2H), 7.22

(d, J = 8.8 Hz, 2H), 7.24-7.42 (m, 4H), 8.39 (d, J = 8.1 Hz, 1H),

8.54 (d, J = 11.7 Hz, 1H), 9.56 (s, 1H). MS (ESI), m/z 496

[M þ H]^þ.

Compounds 9j-n were prepared according to general proce-

dure G.

[4-(Benzo[d]-3-isothiazolylcarboxamido)-3-chlorophenyl]ace-

1

tic Acid (9j). Yield 92% (two steps). Colorless solid. H NMR

(DMSO-d₆) δ 3.59 (s, 2H), 7.30 (dd, J = 8.2, 1.8 Hz, 1H), 7.49 (d,

J = 1.7 Hz, 1H), 7.61-7.66 (m, 1H), 7.69-7.73 (m, 1H), 7.94 (d,

J = 8.3 Hz, 1H), 8.36 (d, J = 8.1 Hz, 1H), 8.80 (d, J = 8.1 Hz,

1H), 10.26 (s, 1H). MS (ESI), m/z 347 [M þ H]^þ.

[5-Chloro-2-fluoro-4-(benzo[d]-3-isothiazolylcarboxamido)phe-

nyl]acetic Acid (9k). Yield 30% (two steps). Colorless solid. ¹H

NMR (DMSO-d₆) δ 3.68(s, 2H), 7.65(d, J = 7.3 Hz, 1H), 7.66(t,

J = 7.5 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.99 (d, J = 11.2 Hz,

1H), 8.38 (d, J = 8.3 Hz, 1H), 8.81 (d, J = 7.8 Hz, 1H), 10.30 (s,

1H). MS (ESI), m/z 365 [M þ H]^þ.

[4-(Benzo[d]-3-isoxazolylcarboxamido)-5-chloro-2-fluorophe-

1

nyl]acetic Acid (9l). Yield 10% (two steps). Colorless solid. H

NMR (DMSO-d₆) δ 3.66 (s, 2H), 7.54-7.57 (m, 1H), 7.61-7.63

(m, 1H), 7.67-7.70 (m, 1H), 7.78-7.81 (m, 1H), 7.93-7.95

(m, 1H), 8.15-8.17 (m, 1H), 10.62 (s, 1H). MS (ESI), m/z 349

[M þ H]^þ.

To a stirred solution of ethyl [5-chloro-2-fluoro-4-[1-(4-meth-

oxybenzyl)-3-indazolylcarboxamido]phenyl]acetate (880 mg,

1.77 mmol) in TFA (10 mL) was added anisole (288 μL, 2.66 mmol),

and the reaction mixture was heated under reflux for 15 h. After

being cooled to room temperature, the mixture was concen-

trated in vacuo. The residue was diluted with H₂O, and the

resulting precipitate was collected by suction. The solid was

dissolved in CHCl₃-MeOH, and n-hexane was added until a

precipitate formed. The precipitate was collected by suction and

dried under vacuum to give ethyl [5-chloro-2-fluoro-4-(3-inda-

zolylcarboxamido)phenyl]acetate (531 mg, 80%) as a colorless

crystalline powder. ¹H NMR (DMSO-d₆) δ 1.20 (t, J = 7.1 Hz,

3H), 3.75 (s, 2H), 4.12 (q, J = 7.1 Hz, 2H), 7.34 (t, J = 7.8 Hz,

1H), 7.49 (dt, J = 8.1, 1.0 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.71

(d, J = 8.1 Hz, 1H), 8.16 (d, J = 11.5 Hz, 1H), 8.21 (d, J = 8.1

Hz, 1H), 9.79 (s, 1H). MS (ESI), m/z 376 [M þ H]^þ.

[3-Chloro-4-(1-isoquinolinylcarboxamido)phenyl]acetic Acid (9m).

1

Yield 55% (two steps). Brown solid. H NMR (DMSO-d₆) δ

3.63 (s, 2H), 7.33 (dd, J = 8.3, 1.7 Hz, 1H), 7.51 (d, J = 1.7 Hz,

1H), 7.81 (td, J = 8.3, 1.7 Hz, 1H), 7.88 (td, J = 8.3, 1.7 Hz, 1H),

8.12 (d, J = 8.1 Hz, 1H), 8.16 (d, J = 5.6 Hz, 1H), 8.24 (d,

J = 8.3 Hz, 1H), 8.67 (d, J = 5.6 Hz, 1H), 9.34 (d, J = 8.3 Hz,

1H), 10.85 (s, 1H), 12.48 (broad s, 1H). MS (ESI), m/z 341 [M þ

H]^þ.

[5-Chloro-2-fluoro-4-(1-isoquinolinylcarboxamido)phenyl]ace-

tic Acid (9n). Yield 75%. Pale yellow solid. ¹H NMR (DMSO-

d₆) δ 3.66 (s, 2H), 7.62 (d, J = 7.5 Hz, 1H), 7.80-7.90 (m, 2H),

8.12 (d, J = 8.5 Hz, 1H), 8.18 (d, J = 5.6 Hz, 1H), 8.23 (d, J =

11.5 Hz, 1H), 8.67 (d, J = 5.6 Hz, 1H), 9.38 (d, J = 8.5 Hz, 1H),

10.98 (s, 1H), 12.59 (broad s, 1H). MS (ESI), m/z 359 [M þ H]^þ.

Methyl 1-(4-Methoxybenzyl)-3-indazolylcarboxylate (26c).

To a stirred solution of methyl 3-indazolylcarboxylate (25b,

2.0 g, 11.4 mmol) in DMF (20 mL) was added 4-methoxybenzyl

chloride (1.7 mL, 12.5 mmol) and K₂CO₃(2.35 g, 17.0 mmol),

and the reaction mixture was stirred at 80 °C for 15 h. After

being cooled to room temperature, the mixture was poured into

H₂O and extracted with EtOAc. The extract was washed with

brine, dried over MgSO₄, and concentrated in vacuo. The

residue was purified by column chromatography on silica gel

with n-hexane/EtOAc (4:1, v/v) as an eluent to give the title

compound (more polar fractions, 838 mg, 25%) as a pale yellow

oil and 2-PMB isomer (less polar fractions, 500 mg, 15%) as a

To a stirred solution of ethyl [5-chloro-2-fluoro-4-(3-indazo-

lylcarboxamido)phenyl]acetate (531 mg, 1.41 mmol) in MeOH

(50 mL) was added 0.25 N NaOH (12 mL, 3 mmol), and the

reaction mixture was stirred at room temperature for 15 h. The

mixture was poured into 1 N HCl, and the resulting precipitate

was collected by suction and dried under vacuum to give the title

compound (480 mg, 98%) as a colorless crystalline powder. ¹H

NMR (DMSO-d₆) δ 3.63 (s, 2H), 7.34 (t, J = 7.6 Hz, 1H), 7.49

(t, J = 7.6 Hz, 1H), 7.62 (m, 1H), 7.72 (d, J = 8.3 Hz, 1H), 8.13

(d, J = 11.2 Hz, 1H), 8.21 (d, J = 8.1 Hz, 1H), 9.79 (s, 1H), 13.96

(broad s, 1H).

Methyl 4-[N-(tert-Butoxycarbonyl)-(2S)-pyrrolidinylmethox-

y]cyclohexanecarboxylate (29a). To a stirred solution of methyl

4-[N-(tert-butoxycarbonyl)-(2S)-pyrrolidinylmethoxy]benzo-

ate²⁵(28a, 2.47 g, 7.36 mmol) in CH₂Cl₂(60 mL) was added

1

pale yellow oil. For the title compound: H NMR (CDCl₃) δ

7988 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24

Muro et al.

TFA (30 mL), and the reaction mixture was stirred at room

temperature for 3 h. After being concentrated in vacuo, the

residue was dissolved into MeOH/AcOH (55 mL, 10:1, v/v), 5%

Rh on alumina (1.00 g) was added, and the reaction mixture was

hydrogenated at 10 atm at room temperature for 20 h. After

removal of the catalyst by filtration, the filtrate was concen-

trated in vacuo. The residue was dissolved into CHCl₃and

washed with sat. NaHCO₃, dried over MgSO₄, and concen-

trated in vacuo to give methyl 4-[(2S)-pyrrolidinylmethox-

y]cyclohexanecarboxylate (1.71 g, 96% for two steps) as a

colorless oil. ¹H NMR (CDCl₃) δ 1.20-1.53 (m, 4H),

1.62-2.09 (m, 9H), 2.23-2.39 (m, 1H), 2.82-2.88 (m, 1H),

2.95-3.02 (m, 1H), 3.19-3.48 (m, 4H), 3.66 (s, 3H). MS (ESI),

m/z 242 [M þ H]^þ.

1.20-1.85 (m, 6H), 1.99-2.40 (m, 5H), 3.30-3.75 (m, 6H), 3.65

(s, 3H), 4.17 (broad s, 1H), 4.65 (broad s, 1H).

To a stirred solution of methyl 4-[(4R)-hydroxy-(2S)-pyrro-

lidinylmethoxy]cyclohexanecarboxylate (1.41 g, 5.48 mmol) and

Et₃N (1.53 mL, 10.98 mmol) in CH₂Cl₂(30 mL) was added

Boc₂O (1.25 g, 5.73 mmol), and the reaction mixture was stirred

at room temperature for 1 h. After being concentrated in vacuo,

the residue was purified by column chromatography on silica gel

with CHCl₃/MeOH (15:1, v/v) as an eluent to give the title

compound (1.95 g, 100%) as a pale yellow oil. ¹H NMR (CDCl₃)

δ 1.11-1.85 (m, 6H), 1.46 (s, 9H), 1.95-2.05 (m, 3H), 2.12-2.36

(m, 2H), 3.17-3.59 (m, 6H), 3.66 (s, 3H) 4.03 (broad s, 1H),

4.47-4.51 (m, 1H). MS (ESI), m/z 358 [M þ H]^þ.

Methyl 4-[(4R)-Benzyloxymethoxy-N-(tert-butoxycarbonyl)-(2S)-

pyrrolidinylmethoxy]cyclohexanecarboxylate (29d). To a stirred solu-

tion of 29c (1.37 g, 3.83 mmol) and diisopropylethylamine (1.0 mL,

5.75 mmol) in CH₂Cl₂(20 mL) was added benzylchloromethyl ether

(796 μL, 5.75 mmol), and the reaction mixture was stirred at room

temperature for 17 h. The mixture was diluted with H₂O and

extracted with CHCl₃. The extract was washed with brine, dried over

Na₂SO₄, and concentrated in vacuo. The residue was purified by

column chromatography on silica gel with CHCl₃/EtOAc (10:1, v/v)

as an eluent to give the title compound (1.45 g, 79%) as a colorless oil.

¹H NMR (CDCl₃) δ 1.35-1.66 (m, 4H), 1.45 (s, 9H), 1.78-1.86 (m,

4H), 2.05-2.22 (m, 2H), 2.33(broad s, 1H), 3.44-3.73(m, 5H), 3.66

(s, 3H), 3.97-4.04 (m, 1H), 4.40 (broad s, 1H), 4.57-4.64 (m, 2H),

4.78 (s, 2H), 7.28-7.45 (m, 5H).

Methyl trans-4-[(4R)-Benzyloxymethoxy-N-(tert-butoxycarbonyl)-

(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylate (30c). To a stirred

solution of 29d (1.45 g, 3.04 mmol) in MeOH (50 mL) was added

NaOMe (493 mg, 9.12 mmol), and the reaction mixture was heated

under reflux for 2 days under N₂atmosphere. After being cooled to

room temperature, the mixture was quenched by the addition of 1 N

HCl and extracted with CHCl₃/MeOH (5:1, v/v). The extract was

washed with brine, dried over Na₂SO₄, and concentrated in vacuo.

The residue was dissolved in benzene/MeOH (50 mL, 4:1, v/v). To the

solution was added TMSCHN₂(2Minn-hexane, 2.0 mL, 4.0 mmol),

and the reaction mixture was stirred at room temperature for 30 min.

The mixture was quenched by the addition of AcOH and concen-

trated in vacuo. The residue was purified by flash column chroma-

tography (Biotage 40M) with n-hexane/EtOAc (7:1 to 4:1, v/v) as an

eluent to give the title compound (669 mg, 46%) as a colorless oil. ¹H

NMR (CDCl₃) δ 1.18-1.28 (m, 2H), 1.39-1.50 (m, 2H), 1.46 (s,

9H), 1.97-2.28 (m, 7H), 3.16-3.23(m,1H),3.39-3.63 (m, 4H), 3.66

(s, 3H), 3.93-4.04 (m, 1H), 4.34-4.40 (m, 1H), 4.56-4.63 (m, 2H),

4.77 (s, 2H), 7.28-7.37 (m, 5H). MS (ESI), m/z 478 [M þ H]^þ.

Methyl trans-4-[N-(tert-Butoxycarbonyl)-(4R)-hydroxy-(2S)-

pyrrolidinylmethoxy]cyclohexanecarboxylate (30d). A mixture

of 30c (669 mg, 1.40 mmol) and 5% Pd/C (600 mg) in MeOH

(50 mL) was stirred at room temperature under hydrogen

atmosphere for 20 h. After removal of the catalyst by filtration,

the filtrate was concentrated in vacuo to give the title compound

(500 mg, 100%) as a pale yellow oil. ¹H NMR (CDCl₃) δ

1.16-1.30 (m, 2H), 1.41-1.50 (m, 2H), 1.46 (s, 9H),

1.95-2.05 (m, 5H), 2.12-2.18 (m, 1H), 2.23-2.29 (m, 1H),

3.16-3.23 (m, 1H), 3.34-3.62 (m, 5H), 3.66 (s, 3H), 4.02 (broad

s, 1H), 4.44-4.49 (m, 1H).

To a stirred solution of methyl 4-[(2S)-pyrrolidinylmethox-

y]cyclohexanecarboxylate (1.71 g, 7.09 mmol) and Et₃N (1.50

mL, 10.6 mmol) in MeCN/H₂O (40 mL, 1:1, v/v) was added

Boc₂O (1.70 g, 7.80 mmol), and the reaction mixture was stirred

at room temperature for 20 h. The mixture was diluted with

EtOAc, washed with 1 N HCl and brine, dried over MgSO₄, and

concentrated in vacuo. The residue was purified by column

chromatography on silica gel with n-hexane/EtOAc (4:1, v/v) as

an eluent to give the title compound (2.26 g, 93%) as a colorless

oil. ¹H NMR (CDCl₃) δ 1.21-1.53 (m, 4H), 1.46 (s, 9H),

1.61-2.05 (m, 7H), 2.26-2.37 (m, 1H), 3.20-3.74 (m, 6H),

3.66 (s, 3H), 3.82-3.92 (m, 1H).

Methyl trans-4-[N-(tert-Butoxycarbonyl)-(2S)-pyrrolidinyl-

methoxy]cyclohexanecarboxylate (30a). To a stirred solution

of 29a (2.26 g, 6.62 mmol) in MeOH (50 mL) was added Na-

OMe (1.07 g, 19.9 mmol), and the reaction mixture was heated

under reflux for 15 h. After being cooled to room temperature,

the mixture was poured into 1 N HCl (100 mL) and extracted

with CHCl₃. The extract was dried over MgSO₄and concen-

trated in vacuo. The residue was dissolved in benzene/MeOH

(60 mL, 4:1, v/v) and treated with TMSCHN₂(2 M in n-hexane,

1.70 mL, 3.40 mmol) until the carboxylic acid had disappeared

on TLC. The mixture was concentrated in vacuo, and the

residue was purified by flash column chromatography

(Biotage 75M) with n-hexane/EtOAc (7:1 to 4:1, v/v) as

an eluent to give the title compound (780 mg, 35%) as a color-

less oil. ¹H NMR (CDCl₃) δ 1.18-1.30 (m, 2H), 1.41-1.50 (m,

2H), 1.46 (s, 9H), 1.76-2.07 (m, 7H), 2.24-2.29 (m, 1H),

3.18-3.40 (m, 5H), 3.57-3.63 (m, 1H), 3.66 (s, 3H), 3.79-

3.94 (m, 1H).

Methyl trans-4-[(2S)-Pyrrolidinylmethoxy]cyclohexanecarboxylate

(10a). To a stirred solution of 30a (780 mg, 2.28 mmol) in CH₂Cl₂

(5mL) wasaddedTFA(5mL),andthereationmixturewasstirredat

room temperature for 15 h. After being concentrated to a small

volume, the residue was made basic with sat. NaHCO₃and extracted

with CHCl₃. The extract was dried over Na₂SO₄and concentrated in

vacuo to give the title compound (504 mg, 91%) as a pale yellow oil.

¹H NMR (CDCl₃) δ 1.20-1.29 (m, 2H), 1.34-1.51 (m, 3H),

1.66-2.10 (m, 7H), 2.23-2.31 (m, 1H), 2.83-2.89 (m, 1H), 2.95-

3.01 (m, 1H), 3.20-3.28 (m, 2H), 3.32-3.36 (m, 1H), 3.44-3.48 (m,

1H), 3.66 (s, 3H). MS (ESI), m/z 242 [M þ H]^þ.

Methyl 4-[N-(tert-Butoxycarbonyl)-(4R)-hydroxy-(2S)-pyrro-

lidinylmethoxy]cyclohexanecarboxylate (29c). To a stirred solu-

tion of methyl 4-[N-(tert-butoxycarbonyl)-(4R)-hydroxy-(2S)-

pyrrolidinylmethoxy]benzoate²⁵(28c, 4.01 g, 11.4 mmol) in

CH₂Cl₂(60 mL) was added TFA (30 mL), and the reaction

mixture was stirred at room temperature for 3 h. After being

concentrated in vacuo, the residue was dissolved into EtOH

(80 mL). To the mixture was added 5% Rh on alumina (1.01 g),

and the reaction mixture was hydrogenated at 10 atm at room

temperature for 20 h. After removal of the catalyst by filtration,

the filtrate was concentrated in vacuo. The residue was dissolved

into CHCl₃/MeOH (10:1, v.v), washed with sat. NaHCO₃, dried

over Na₂SO₄, and concentrated in vacuo to give methyl 4-[(4R)-

hydroxy-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylate

(2.46 g, 86% for two steps) as a brown oil. ¹H NMR (CDCl₃) δ

Methyl trans-4-[N-(tert-Butoxycarbonyl)-(4S)-hydroxy-(2S)-

pyrrolidinylmethoxy]cyclohexanecarboxylate (30e). To a stirred

soloution of 30d (5.00 g, 14.0 mmol), Ph₃P (4.41 g, 16.8 mmol),

and formic acid (1.58 mL, 42.0 mmol) in THF (50 mL) was

added diisopropyl azodicarboxylate (DIAD, 3.30 mL, 16.8 mmol),

and the reaction mixture was stirred at room temperature for

20 h. The mixture was concentrated in vacuo, and the residue

was purified by column chromatography on silica gel with

n-hexane/EtOAc (4:1, v/v) as an eluent to give methyl trans-

4-[N-(tert-butoxycarbonyl)-(4S)-formyloxy-(2S)-pyrrolidinyl-

methoxy]cyclohexanecarboxylate (5.39 g) as a colorless oil. To

a stirred solution of the product in THF (50 mL) was added sat.

NaHCO₃(50 mL), and the reaction mixture was stirred at room

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7989

temperature for 20 h. After being concentrated to a small

volume, the residue was diluted with H₂O and extracted with

EtOAc. The extract was washed with brine, dried over Na₂SO₄,

and concentrated in vacuo. The residue was purified by column

chromatography on silica gel with n-hexane/EtOAc (1:1, v/v)

as an eluent to give the title compound (4.08 g, 82% for two

2 days. Prior to the assay, the medium was discarded, and each

well was washed twice with 300 μL of chilled wash buffer

(25 mM N-2-hydroxyethylpiperazine-N⁰-2-ethanesulfonic acid

(HEPES), pH 7.5; 150 mM NaCl; 1 mM CaCl₂; 1 mM MgCl₂;

4 mM MnCl₂). Then, 50 μL of compound solution was added to

a well, followed by 50 μL of 2 nM Eu-labeled human VCAM-1/

Fc chimera diluted with the wash buffer (final concentration 1

nM). For assays conducted in the presence of human serum

albumin, 50 μL of compound at various concentrations and an

equal volume of 2 nM Eu-labeled human VCAM-1/Fc chimera

in 6% (w/v) human serum albumin (Sigma Corp.) were dis-

tributed into each well (final concentration 1 nM). The plates

were incubated for 60 min at room temperature, and the wells

were washed 4 times with 300 μL of chilled wash buffer. Finally,

100 μL of the enhancement solution (PerkinElmer Inc.) was

added to each well. The plates were placed on a shaker for 5 min.

Eu fluorescence was then measured using a time-resolved fluo-

rometer (DELFIA Research fluorometer, model 1234-001;

PerkinElmer Inc.). The concentration of compound required

for 50% inhibition in the assay was determined.

r₄β₇/Eu-Human MAdCAM-1 Binding Assay. RPMI8866, a

human B cell line expressing R₄β₇but not β₁integrin, was

maintained in medium, RPMI1640 including 10% FCS (fetal

calf serum), Pn/St. For the RPMI8866/MAdCAM-1 binding

assays, RPMI8866 cells were washed and resuspended in assay

buffer (0.1% BSA, 20 μM diethylene triamine pentaacetic acid

(DTPA), 25 mM HEPES, 150 mM NaCl, 1 mM CaCl₂, 1 mM

MgCl₂, 4 mM MnCl₂) and prepared at 3.0 ꢀ 10⁷cells/mL (final

cell number 1.2 ꢀ 10⁶/well). The compound was diluted from 30

μg/mL to 3 ng/mL in assay buffer (final conc. 10 μg/mL to 1 ng/

mL). Each 40 μL of cells, compound, and Eu-labeled MAd-

CAM-1 chimeric protein at 3 nM (final conc. at 1 nM) was

gently mixed in a round-bottom 96-well plate (Costar no. 3799)

for 1 h at room temperature. The mixture was transferred into a

96-well silent screen plate (Nunc) that had been previously

blocked with 1% BSA in HSM (25 mM HEPES, 150 mM NaCl,

2 mM MgCl₂) for 1 h at room temperature. The free ligands were

removed by centrifugation of the plate at 2000 rpm for 1 min at

4 °C. The plate was washed with wash buffer (25 mM HEPES,

150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 4 mM MnCl₂) three

times. Enhancement solution (100 μL) was added to each well.

After gentle shaking for 5 min, the solution was transferred to an

enzyme immunoassay (EIA) plate (Costar no. 3590), and the

time-resolved fluorescence (TRF) of each well was measured.

mLFA-1/Eu-Human ICAM-1 Binding Assay. Membrane-

bound human LFA-1 (mLFA-1) was immobilized on EIA/

RIA 96-well plates (Corning 3590, Corning) by adding 40 μL

of 1 μg/mL mLFA-1 dissolved in 0.1% OG (N-octyl β-^D-

glucopyranoside)-HSM (25 mM HEPES(pH7.5), 140 mM

NaCl, 2 mM MgCl₂) to each well and incubating overnight at

4 °C. After removal of the nonadherent mLFA-1, each well was

blocked with 200 μL/well of 1% BSA/HSM for 1 h at room

temperature. The plates were washed three times with 200 μL/

well of chilled HSM, and 50 μL/well of 2 nM Eu-labeled human

ICAM-1 D1D5-IgG prepared in dilution buffer (0.01% BSA,

20 μM DTPA-HSM) was added, then allowed to bind to

the immobilized mLFA-1 for 2 h at room temperature. The

inhibitory activity of the compounds was assessed by adding

the compound to the Eu-ICAM-1 preparation with the

DMSO concentration of 5%. The plates were washed three

times with 200 μL/well of chilled HSM, and 150 μL of the

DELFIA enhancement solution (PerkinElmer Inc.) was

added to each well. The Eu-fluorescence was measured using

a time-resolved fluorometer (DELFIA Research fluorometer,

model 1234-001, PerkinElmer Inc.) after gentle shaking for

5 min.

1

steps) as a colorless solid. H NMR (CDCl₃) δ 1.24-1.34 (m,

2H), 1.42-1.52 (m, 2H), 1.46 (s, 9H), 1.86-1.92 (m, 1H),

1.99-2.09 (m, 4H), 2.24-2.36 (m, 2H), 3.30-3.36 (m, 1H),

3.41-3.54 (m, 3H), 3.66 (s, 3H), 3.92-4.21 (m, 3H), 4.83-5.07

(m, 1H). MS (ESI), m/z 358 [M þ H]^þ.

Methyl trans-4-[(4S)-Hydroxy-(2S)-pyrrolidinylmethoxy]cyclohe-

xanecarboxylate (10c). To a stirred solution of 30e (57 mg, 0.16

mmol) in CH₂Cl₂(3 mL) was added TFA (1 mL), and the reation

mixture was stirred at room temperature for 5 h. After being

concentrated to a small volume, the residue was made basic with

sat. NaHCO₃and extracted with CHCl₃. The extract was washed

with brine, dried over Na₂SO₄, and concentrated in vacuo to give the

title compound (38 mg, 92%) as a pale yellow oil. ¹H NMR (CDCl₃)

δ 1.26-1.36 (m, 2H), 1.43-1.54 (m, 2H), 1.79-1.82 (m, 1H),

1.99-2.16 (m, 4H), 2.24-2.34 (m, 2H), 3.04-3.12 (m, 1H),

3.25-3.28 (m, 1H), 3.39-3.48 (m, 1H), 3.61-3.78 (m, 3H), 3.67

(s, 3H), 4.37 (broad s, 1H). MS (ESI), m/z 258 [M þ H]^þ.

Methyl trans-4-[N-(tert-Butoxycarbonyl)-(4S)-methoxy-(2S)-

pyrrolidinylmethoxy]cyclohexanecarboxylate (30f). To a cooled

(0 °C), stirred solution of 30e (3.00 g, 8.39 mmol) and MeI (2.60

mL, 42.0 mmol) in DMF (20 mL) was added NaH (60% in oil)

(336 mg, 8.39 mmol), and the reaction mixture was stirred at

room temperature for 1 h. The reaction mixture was poured into

ice water and extracted with EtOAc. The extract was washed

with brine, dried over Na₂SO₄, and concentrated in vacuo. The

residue was purified by column chromatography on silica gel

with n-hexane/EtOAc (2:1, v/v) as an eluent to give the title

compound (1.82 g, 59%) as a colorless oil. ¹H NMR (CDCl₃) δ

1.18-1.28 (m, 2H), 1.40-1.49 (m, 2H), 1.46 (s, 9H), 1.94-2.08

(m, 5H), 2.15-2.28 (m, 2H), 3.17-3.25 (m, 1H), 3.30 (s, 3H),

3.33-3.56 (m, 3H), 3.66 (s, 3H), 3.69-3.81 (m, 1H), 3.88-3.96

(m, 2H). MS (ESI), m/z 372 [M þ H]^þ.

Methyl trans-4-[(4S)-Methoxy-(2S)-pyrrolidinylmethoxy]cyclohe-

xanecarboxylate (10d). To a stirred solution of 30f (50 mg, 0.135

mmol) in CH₂Cl₂(5 mL) was added TFA (0.5 mL), and the reaction

mixture was stirred at room temperature for 14 h. After being

concentrated to a small volume, the mixture was made basic by the

addition of 1 N NaOH and extracted with CH₂Cl₂. The extract was

washed with brine, dried over Na₂SO₄, and concentrated to give the

title compound (37 mg, 100%) as a yellow oil. ¹H NMR (CDCl₃) δ

1.22-1.35 (m, 2H), 1.41-1.57 (m, 2H), 1.96-2.20 (m, 5H),

2.24-2.35 (m, 2H), 3.33 (s, 3H), 3.34-3.57 (m, 4H), 3.66 (s, 3H),

3.67-3.81 (m, 2H), 3.90-3.96 (m, 1H), 4.11-4.15 (m, 1H). MS

(ESI), m/z 272 [M þ H]^þ.

Biology. All protein labeled with europium (Eu) was prepared

by using Eu-labeling reagent (PerkinElmer Inc.), and purified

using a PD-10 column (Amersham Biosciences KK.). Eu-

labeled protein was stored at -80 °C until use.

VLA-4/Eu-Human VCAM-1 Binding Assay. A human VLA-

4-expressing cell line, 4B4, was established at Pharmacopeia Inc.

by transfecting both the R₄gene and β₁gene of VLA-4 into

CHO-K1 cells. The 4B4 cells were maintained in Ham’s F-12

medium (Sigma Corp.) supplemented with 10% (v/v) fetal calf

serum (REHATUIN fetal bovine serum, Serologicals Corp.),

100 U/mL penicillin (Invitrogen Corp.), 100 μg/mL streptomy-

cin (Invitrogen Corp.), 2 mM ^L-glutamine (Invitrogen Corp.),

and 1 mg/mL G-418 (Geneticin, Invitrogen Corp.). A Eu-

labeling reagent (PerkinElmer Inc.) was used to labeled the

human VCAM-1/Fc chimeria (R&D Systems Inc.). All assays

were performed in duplicate. In preparation for the assay, the

4B4 cells were suspended at 3 ꢀ 10⁵cells/mL in Ham’s F-12

medium. One hundred microliters of the 4B4 cell suspension was

placed into each well of a 96-well-culture plate (Costar Inc.). The

plates were incubated at 37 °C in a 5% CO₂atmosphere for

r

_IIbβ₃-Dependent Platelet Aggregation Assay. Blood was

collected from two in-house volunteers using syringes with

a 1/10 volume of 3.8% sodium citrate (International Reag-

ents Corp.) as an anticoagulant. The blood was centrifuged at

7990 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24

Muro et al.

1500 ꢀ g for 10 min at room temperature, and the platelet-rich

plasma (PRP) supernatant was separated. The residue was

further centrifuged at 2500 ꢀ g for 15 min, and platelet-poor

plasma (PPP) supernatant was obtained. Two-hundred micro-

liters of PRP and 1 μL of compound prepared in DMSO were

added to a cuvette and incubated at 37 °C for 2 min, and platelet

agglutination was induced by adding 2 μL of 10 μg/mL collagen

(Collagenreagent HORM, Nycomed). For 10 min after agglu-

tination induction, the platelet agglutination rate was measured

with a measuring device (HEMA TRACER 313, MC medical).

Estimated Serum Concentration. Female BALB/c mice

(8 weeks old, Charles River Japan, Inc.) were used. Each group

consisted of four animals. The mice were orally administered the

compound dissolved in 0.5% (w/v) methylcellulose (MC) at a

dose of 10 (mg/mL)/kg. After 15 min, blood samples were

collected via the inferior vena cava from the animals under

ether anesthesia. The blood samples were left to stand at room

temperature and centrifuged at 2000 rpm for 10 min at 4 °C. The

serum samples were subsequently stored in a -20 °C freezer

prior to analysis. According to the VLA-4/VCAM-1 binding

assay, instead of the compound solution, 50 μL of serum

samples at various concentrations were added into each well

(final concentration 0.01-10%). As for the calibration curve,

each diluted compound solution was also assayed in the pre-

sence of the same concentration of untreated mouse serum.

Murine Asthma Model. Female BALB/c AnNCrj mice

(8 weeks old, Charles River Japan, Inc.) received an oral

administration of cyclophosphamide dissolved in water at a

dose of 150 mg/kg (day 0). On day 2 and 14, 500 μg of protein of

Ascaris suum extract (LSL Co., Ltd.) in 0.2 mL of saline-

containing 4.5 mg of aluminum hydroxide was injected intra-

peritoneally. On day 22, the mice were challenged intratrache-

ally under anesthesia with 300 μg (30 μL) of protein of Ascaris

suum extract. In the negative control group, sensitized mice were

challenged with saline instead of the antigen.

baseline of lung resistance, ACh, dissolved in saline was cumu-

latively administered (25, 50, 100, and 200 (μg/mL)/kg) via the

tail vein, and the changes in lung resistance were monitered.

Guinea Pig Asthma Model. Using an ultrasonic nebulizer

(NE-U12; OMRON Corp.) and a vinyl chloride box (W 300 ꢀ

H 390 ꢀ D 570 mm³), male guinea pigs (Kud; Hartley (Kudo

Ltd.), n = 8/group) were exposed to an aerosol mist of physio-

logical saline solution containing 1% ovalbumin (OVA) for 10

min/day, for 8 consecutive days. At 1 week after the final

sensitization, each animal was restrained in a Pulmos chamber

(W 115 ꢀ H 140 ꢀ D 410 mm³), and with an ultrasonic nebulizer,

the animals were exposed to an aerosol mist of physiological

saline solution containing 2% OVA for 5 min. At 24 and 1 h

before the OVA challenge, metyrapone (10 (mg/mL)/kg) was

intravenously administered, and at 30 min before the OVA

challenge, pyrilamine (10 (mg/mL)/kg) was intraperitoneally

administered. At about 22-27.5 h after the antigen challenge,

each guinea pig was restrained in the Pulmos chamber (W 115 ꢀ

H 140 ꢀ D 410 mm³), and with the ultrasonic nebulizer,

physiological saline solution and acetylcholine chloride (ACh)

solution at 0.0625, 0.125, 0.25, 0.5, 1, and 2 mg/mL were

stepwise introduced as aerosol mist, allowing each dose to be

inhaled for 1 min. Using an integrated respiratory function

analysis system (Pulmos-I, M.I.P.S. Co.), the measurement of

sRaw was continued until it reached 2-fold the baseline sRaw

value (sRaw following inhalation of physiological saline

solution). From the concentration of ACh and the sRaw con-

centration vs resistance curve, the concentration of ACh neces-

sary to elevate sRaw 100% over the baseline sRaw (PC₁₀₀ACh)

was calculated using CA-Cricket Graph III 1.5.2 software.

Compound 14e was orally administered (0.8, 2, 5, or 12.5 mg/

kg) at 1 h before the antigen challenge, prior to the treatment by

metyrapone, and at 2.5 h after the challenge in each group.

Dexamethasone was orally administered at 16 and 2 h before the

antigen challenge.

Effect on Eosinophil Infiltration. Test compounds dissolved in

0.5% MC containing 0.03% Tween 80 were orally administered

15 min before and 8, 24, and 32 h after the antigen challenge at a

dose of 1.67, 5, or 15 mg/kg (for 13e-f, 14e) and 5, 15, or 45 mg/

kg (for 14d). Forty-eight hours after the antigen challenge, the

mice were sacrificed, and BAL fluid was collected using tracheal

polyethylene cannula with 2 ꢀ 0.5 mL of Hanks’ balanced salt

solution. The cells in the BAL fluid were counted with a particle

analyzer, CDA-500 (Sysmex Corp.). Cytocentrifuged prepara-

tions (Cytospin 2; Shandon) were stained with Wright’s stain

solution (Muto Chemical Co., Ltd.) for differential counts,

based on standard morphologic criteria. The number of eosi-

nophils was calculated by multiplying the total cell number by

the percentage of eosinophils in the cytocentrifuged prepara-

tions.

Effect on Hyper-responsiveness. Compound 14e, which was

dissolved in 0.5% MC containing 0.03% Tween 80, was orally

administered 15 min before and 8, 24, and 32 h after the antigen

challenge at a dose of 2 or 12.5 mg/kg. The bronchial hyper-

responsiveness in each mouse was estimated from the increase in

lung resistance by acetylcholine chloride (ACh; Sigma Corp.)

injection at 48 h after the antigen challenge. Ten minutes before

the start of the measurement, the mice were anesthetized by an

intraperitoneal injection of pentobarbital at a dose of 100 mg/

kg. The trachea was cannulated and connected to a rodent

ventilator (MiniVent type 845; Hugo Sachs Electronik-Harvard

Apparatus) with an in-line pressure transducer (TRD-4510;

Buxco Electronics, Inc.) that was coupled to a pulmonary

mechanics analyzer (Bio-System XA; Buxco Electronics, Inc.).

The flows were determined by measuring the differential pres-

sure (TRD-5100; Buxco Electronics, Inc.) across eight layers of

400-mesh wire cloth covering a 1.3-cm hole in a plethysmograph

box (Plyan-M; Buxco Electorics, Inc.). The mice were placed in

the plethysmogragh box and then ventilated at 140 strokes/min

with a stroke volume of 150 μL. After establishing a stable

Pharmacokinetic Studies on Rats. Male Sprague-Dawley rats

(7 weeks old, SLC Japan) were used. The animals were fasted for

18 h prior to dosing. Each group consisted of four animals. The

rats were orally administered compounds at the dose of 1 mg/kg

dissolved in 0.5% (w/v) MC with 3 equiv of NaOH aqueous

solution. The rats were intravenously administered compounds

at a dose of 1 mg/kg dissolved in saline with 3 equiv of NaOH

solution. Blood samples (0.4 mL) were collected at 0.08 (or 0.25

for po), 0.5, 1, 2, and 6 h after the administration. These

analytical samples were left to stand at room temperature,

followed by centrifugation at 15000 rpm for 10 min at 4 °C.

The plasma fractions were subsequently stored in a -20 °C

freezer until analyzed. The concentrations of the compounds

were determined by an LC/MS/MS method, comprised of an

Alliance 2695 HPLC (Waters), Symmetry Shield RP8, i.d.

2.1 mm ꢀ 50 mm, 3.5 μm column (Waters), and TSQ-700

(Thermo Electron, Waltham, MA). The mobile phase consisted

of 10 mM HCOONH₄in water/methanol; the gradient condi-

tion was 90/10 to 10/90. The plasma concentrations versus time

data were analyzed by noncompartmental approaches using the

WinNonlin software program (version 1.13.1, Pharsight,

Mountain View, CA).

Pharmacokinetic Studies on Dogs. Male beagle dogs (10-12

kg, LSG Corp.) were used. The animals were fasted for 18 h

prior to dosing. Each group consisted of three animals. The test

compounds were dissolved in 0.5% (w/v) MC with 3 equiv of

NaOH for oral cassette dosing or dissolved in saline with 3 equiv

of NaOH for intravenous cassette dosing. The dose in each

experiment was 0.5 mg/kg. Blood samples (1 mL) were collected

after 0.08 (for iv), 0.25 (for po), 0.5, 1, 2, 4, 8, and 24 h. After the

4 h sampling, the animals were provided with food. These

analytical samples were prepared and analyzed according to

the pharmacokinetic studies on rats.

Pharmacokinetic Studies on Monkeys. Female cynomolgus

monkeys (3.5-4 kg, HAMRI Co.) were used. The animals were

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7991

fasted for 18 h prior to dosing. Each group consisted of three

animals. Compound 14e was suspended in 0.5% (w/v) MC for

oral dosing (0.5 mg/kg) or dissolved in saline with 3 equiv

of NaOH for intravenous dosing (0.5 mg/kg). Blood samples

(1 mL) were collected after 0.08 (for iv), 0.25 (for po), 0.5, 1, 2, 4,

8, and 24 h. After the 4 h sampling, the animals were provided

with food. These analytical samples were prepared and analyzed

according to the pharmacokinetic studies on rats.

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Acknowledgment. The authors would like to thank

Dr. Kazuhiko Mori for the safety assessment. We would also

like to thank Dr. Masayuki Takahashi for the pharmaco-

kinetic study.

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Article Doi

Discovery of trans-4-[1-[[2,5-dichloro-4-(1-methyl-3-indolylcarboxamido) phenyl]acetyl]-(4S)-methoxy-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid: An orally active, selective very late antigen-4 antagonist

DOI: 10.1021/jm901154c

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Full text of DOI:10.1021/jm901154c

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