4-(Pyrrolidinyl)methoxybenzoic acid derivatives as a potent, orally active VLA-4 antagonist

Article abstract of DOI:10.1248/cpb.54.1515

A novel series of benzoic acid derivatives as VLA-4 antagonists were synthesized. Optimization, focusing on activity and lipophilicity needed for cell permeability, resulted in the identification of 15b and 15e with good activity (IC₅₀=1.6 nM each) and moderate lipophilicity (Log D=2.0, 1.8). Furthermore, 15e demonstrated efficacy in murine asthma model by an oral dose of 30 mg/kg.

Full text of DOI:10.1248/cpb.54.1515

November 2006
Chem. Pharm. Bull. 54(11) 1515—1529 (2006)
1515
4-(Pyrrolidinyl)methoxybenzoic Acid Derivatives as a Potent, Orally
Active VLA-4 Antagonist
Jun CHIBA,*,a Shin IIMURA,a Yoshiyuki YONEDA,a Yuichi SUGIMOTO,a Takao HORIUCHI,a Fumito MURO,a
Yuichi OCHIAI,a Tomomi OGASAWARA,a Masao TSUBOKAWA,b Yutaka IIGOU,b Gensuke TAKAYAMA,b
Tomoe TAIRA,b Yoshimi TAKATA,b Mika YOKOYAMA,b Tohru TAKASHI,b Atsushi NAKAYAMA,a and
a
Nobuo MACHINAGA
a Medicinal Chemistry Research Laboratory, Daiichi Pharmaceutical, Co., Ltd.; and b New Product Research Laboratories
III, Daiichi Pharmaceutical, Co., Ltd.; 1–16–13 Kitakasai, Edogawa-ku, Tokyo 134–8630, Japan.
Received June 7, 2006; accepted August 10, 2006; published online August 21, 2006
A novel series of benzoic acid derivatives as VLA-4 antagonists were synthesized. Optimization, focusing on
activity and lipophilicity needed for cell permeability, resulted in the identification of 15b and 15e with good ac-
tivity (IC50ꢀ1.6 nM each) and moderate lipophilicity (Log Dꢀ2.0, 1.8). Furthermore, 15e demonstrated efficacy
in murine asthma model by an oral dose of 30 mg/kg.
Key words VLA-4; integrin; human serum albumin; asthma
VLA-4 (very late antigen 4, a4b1 integrin, CD49d/CD29) netic property, structural modifications were made to this
is a key cell receptor expressed on most leukocytes.1) Natural class of compounds. As a result of this study, 3-hydroxypro-
ligands include VCAM-1 (vascular cell adhesion molecule- lyl-1-piperazinylacetic acid 2 (IC50ꢀ1.1 nM) and 3,4-dihy-
1) expressed on cytokine-stimulated endothelial cells, the droxyprolyl-1-piperazinylacetic acid 3 (IC50ꢀ1.4 nM) were
alternatively spliced connecting segment-1 domain of fi- identified; both these compounds have moderate plasma
bronectin on the extracellular matrix,2—4) and junctional ad- clearance (2, 30 ml/min/kg; 3, 21 ml/min/kg) in rats.23) We
hesion molecule 2 on endothelial cells.5) Through interaction also have made it clear that the improvement of plasma clear-
with ligands, VLA-4 plays an important role in the process ance is caused by the change of the main excretion pathway,
of adhesion, migration, and activation of inflammatory from bile for 1 to urine for 2 and 3.12) Unfortunately, neither
leukocytes at sites of inflammation. Anti-VLA-4 antibodies 2 nor 3 showed any oral availability. From result of perme-
or small molecular VLA-4 antagonists6—12) have been re- ability assay using Madin–Darby canine kidney (MDCK)
ported to inhibit leukocyte infiltration into extravascular tis- cell monolayers, it was found that 1, 2, and 3 showed
sue and prevent tissue damage in models of inflammatory low permeability (Papp values, 1: 0.02ꢂ10ꢃ6 cm/s; 2:
such as asthma,13) multiple sclerosis,14,15) rheumatoid arthri- ꢁ0.15ꢂ10ꢃ6 cm/s, 3: ꢁ0.05ꢂ10ꢃ6 cm/s). These results
tis,16) and inflammatory bowel disease.17,18) It has recently stand contrast to value for metoprolol (17.3—20.3ꢂ10ꢃ6
been reported that the humanized monoclonal anti-a4 anti- cm/s), which exhibits good oral availability in human. In ad-
body, Tysabri (Elan Pharmaceuticals Inc. and Biogen Idec dition, it was found that these compounds showed low Log D
Inc.),19—21) demonstrated promising results in treating pa- values (1, ꢃ0.7; 2, 3, ꢃ3), suggesting that their poor perme-
tients with multiple sclerosis, Crohn’s disease, and rheuma- ability is probably caused by their low lipophilicity. Further
toid arthritis in clinical trials. Therefore, the development of attempts to modify 2 and 3 to ethyl-ester, as a pro-drugs,
small molecular VLA-4 antagonists with acceptable oral were conducted as one way to increase the absorption across
pharmacokinetic profiles are viewed as a reasonable ap- the intestinal epithelium. However, the strategy did not lead
proach to a novel class of therapeutic agents.
to a significant improvement of oral availability in rats
We have recently reported the identification of a mor- (Fꢁ3%). Since the extremely low lipophilicity of 2 and 3 is
pholinyl-piperidinylacetic acid 1 as a potent VLA-4 antago- probably responsible at least in part for the improvement in
nist (IC50ꢀ4.4 nM), which showed efficacy in a murine air- their plasma clearance, but, this physicochemical characteris-
way inflammation model by oral administration (Fig. 1).22) tic is at odds with good cell permeability. Thus, further de-
Unfortunately, 1 had a poor pharmacokinetic profiles such as velopment on these compounds were halted.
low oral availability and high plasma clearance (Fꢁ1%,
During the course of the study, we had also identified
69 ml/min/kg) in rats.22) To improve the poor pharmacoki- prolyl-benzoic acid derivative 6 (IC50ꢀ1.6 nM, Fig. 1). This
Fig. 1. VLA-4 Antagonists
∗ To whom correspondence should be addressed. e-mail: chiba2mk@daiichipharm.co.jp
© 2006 Pharmaceutical Society of Japan

1516
Vol. 54, No. 11
compound shows relatively higher lipophilicity (Log Dꢀ ner reaction with ethyl 4-(diethoxyphosphorylmethyl)ben-
0.68) than those of 2 and 3. Additionally, with regard to zoate,26) followed by deprotection of the Boc group to give
plasma clearance, given that compounds with relatively high 8b. On the other hand, 17 was converted to 2-ethynylpyrroli-
lipophilicity tend to bind to serum albumin,24) this character- dine 20 according to the Trybulsk’s protocol.27) Following Pd
istic would improve the maintenance at a given blood con- catalyzed coupling of 20 with ethyl 4-iodobenzoate, depro-
centration. Consequently, we thought that benzoic acid deriv- tection of Boc group afforded 8c.
atives might be useful to achieve a desired compound with
The synthesis of 8e containing a methylthio linker (CH2S)
low plasma clearance and good oral bioavailability. Thus, was achieved in 4 steps as shown in Chart 4. Following tosy-
compound 6 was chosen for further studies. In addition to the lation28) of N-Boc-L-prolinol (22), coupling of 23 with 4-
VLA-4/VCAM-1 binding assay used previous studies, we iodothiophenol29) in the presence of NaH yielded 24. Subse-
performed to evaluate the activities of compounds in the quently, 24 was subjected to Pd catalyzed methoxycarbonyla-
presence of 3% human serum albumin (HSA) to estimate tion reaction with CO and MeOH, followed by deprotection
their effectiveness in vivo.
of the Boc group to afford 8e.
The modification of the amide bond between the pyrroli-
Substituted pyrrolidines 11a—n, which contain a methyl-
dine ring and the benzoic acid is performed firstly, since it oxy linker (CH2O) at 2-position in the pyrrolidine ring, were
has been found that the removal of oxygen atom in the corre- prepared as shown in Chart 5. Substituted proline derivatives
sponding amide bond of prolyl-1-piperazinylacetic acid de- 26a, b, 26d, 26f—j, and 26m, n, which were commercially
rivative 4 (IC50ꢀ1.6 nM, Fig. 1) dose not affect on the activity available or easily prepared from N-Boc-(4R)-hydroxy-L-pro-
(5, IC50ꢀ1.6 nM, Fig. 1).23) Next, appending a variety of sub- line,23,25) were reduced with borane-dimethylsulfide complex,
stituents into pyrrolidine ring and benzene ring of benzoic followed by coupling with methyl 4-hydroxybenzoate em-
acid portion of 6 was focused on.
ploying Mitsunobu reaction to afford 27a, b, 27d, 27f—j,
In this paper, we wish to report the optimization efforts on and 27m, n. Additionally, 27c, 27e, 27k, and 27l were readily
the physicochemical properties of the benzoic acid deriva- prepared from 27b. Thus, the selective hydrolysis of the ace-
tives that retain the activity. We also present the results of toxy group in 27b with K2CO3/MeOH and Mitsunobu reac-
representative compounds in treating a murine model of tion of the resulting (4R)-hydroxypyrrolidine derivative with
asthma and the pharmacokinetic profiles.
acetic acid provided 27c with inversion of configuration at C-
4. The acetoxy group in 27c was also selectively hydrolyzed
under the same condition to give the (4S)-hydroxypyrrolidine
Chemistry
The syntheses of benzoic acid derivatives 9a—g (Syn- derivative, which was subjected to O-methylation using MeI
thetic route A), 12a—p (Synthetic route B), and 15a—k and NaH to afford 27e. The (4S)-hydroxypyrrolidine deriva-
(Synthetic route C) are shown in Chart 1. Pyrrolidine deriva- tive was also converted to 27k via triflation followed by SN2
tives 8a—c, 8e, 11a—n, and 14a—g were condensed with 3- displacement with dimethylamine (Me2NH). In an analogous
methoxy-4-[N-(2-methylphenyl)ureido]phenylacetic acid (7a) manner, 27b was readily converted to 27l. The N-protective
or penetafluorophenyl 3-methoxy-4-[N-(2-methylphenyl)- groups of 27a—n were removed according to standard de-
ureido]phenylacetate (7b) using EDC and HOBt followed by protection protocols to furnish 11a—n.
basic hydrolysis to produce 9a—c, 9e, 12a—n, and 15a—g.
Compounds 14a—g, which have substituents on the ben-
After the hydrolysis, the hydrogenation of CꢀC double bond zene ring of the benzoic acid portion, were synthesized as
in 9b afforded 9d. Oxidation of sulfide 10, which was ob- shown in Chart 6. Compound 22 was coupled with 4-bromo-
tained in the preparation on 9e, by using mCPBA (1 eq or 3-methylphenol, 4-iodo-2-methylphenol, and 3,5-dimethyl-4-
2.2 eq) and basic hydrolysis resulted in 9f and 9g, respec- iodophenol by means of Mitsunobu reaction to give 28a—c,
tively. In the case of 12o and 12p, each of bearing hydroxyl respectively. The methoxycarbonylation was carried out by
group in the pyrrolidine ring, deprotection of the O-protec- treatment of 28a—c with n-BuLi and methyl chloroformate
tive groups was conducted before basic hydrolysis of inter- or Pd(OAc)2 in MeOH under CO atmosphere to afford 29a—
mediates 13m and 13n. Compound 15h was prepared from c, which were followed by deprotection of the Boc group to
15g by SnCl2 reduction of the NO2 group. The subsequent afford 14a—c, respectively. Additionally, N-Boc-L-prolinol
standard N-acylation and N-alkylation protocols provided 15i (22) was also converted to 14d—g using commercially avail-
and 15j, respectively. Compound 15k, with a N-methyl group able 3- or 3,5-substituted benzoic acid derivatives in a man-
at the 3-position of benzoic acid, was synthesized from inter- ner similar to that described for preparation of 11 in Chart 5.
mediate 16, which was obtained in the preparation on 15g,
via mono-N-methylation followed by basic hydrolysis.
Pyrrolidine derivatives 8a—c, 8e, 11a—n, and 14a—g
Results and Discussion
The compounds were evaluated for their VLA-4 inhibitory
were prepared from L-proline derivatives 17, 22, and 2623,25) activities in the receptor binding assay, with or without the
as shown in Chart 2—6.
addition of 3% human serum albumin (HSA). Additionally,
Compound 8a, with a methylamino linker (CH2NH) be- distribution coefficient [Log D, n-Octanol/PBS (pH 7.4)] was
tween the pyrrolidine ring and the benzoic acid moiety, was determined to assess the oral bioavailability. The results are
prepared from N-Boc-L-prolinal (17) with methyl 4- summarized in Tables 1—3.
aminobenzoate by use of NaBH3CN, followed by deprotec-
tion of the Boc group with TFA as shown in Chart 2.
Table 1 shows the results in the modifications of the linker
portion (CONH as in 6) between the pyrrolidine ring and the
Compound 8b with an ethenyl linker (CHꢀCH) and 8c benzoic acid portion. Almost all the linkers maintained the
with an ethynyl linker (CꢀC) were prepared as shown in good activity with IC50 values of less than 10 nM. However,
Chart 3. N-Boc-L-prolinal (17) was subjected to Wittig–Hon- the CH2S linker (9e) and the CH2SO2 linker (9g) slightly de-

November 2006
1517
Reagents: (a) EDC·HCl, HOBt, DMAP or Et3N, DMF or CH2Cl2; (b) aq. NaOH or LiOH, THF/MeOH; (c) H2, Pd/C, MeOH; (d) mCPBA (1 eq for 9f, 2.2 eq for 9g),
CH2Cl2; (e) g. HCl/MeOH; (f) H2, Pd (OH)2/C, THF; (g) H2, Pd/C, MeOH; (h) (CH3CO)2O, DMAP, pyridine; (i), aq. HCHO, NaBH3CN, HOAc, MeOH; (j) SnCl2, EtOH;
(k) (CF3CO)2O, Et3N, CH2Cl2; (l) MeI, K2CO3, DMF.
Chart 1
creased the activity. These modifications suggest that the
amide bond in the liker portion was not necessary to main-
tain an activity, which SAR was similar to that of piperazine
analogue described above. As for the activity in the presence
Reagents: (a) methyl 4-aminobenzoate, toluene then NaBH3CN, MeOH,
HOAc; (b) TFA, CH2Cl2.
of HSA, the order was nearly the same with that of the activ-
ity in the absence of HSA. In addition, considering that the
ratio of IC50 (ꢃHSA) to IC50 (ꢄHSA), it was found that
Chart 2

1518
Vol. 54, No. 11
CH2O linker (12a) would less likely to be affected negative the ratio and the Log D value was not recognized. We con-
by HSA. With regard to the lipophilicity, all compounds sider that potent compound with IC50 value about 1 nM might
measured, except for 9f with the CH2SO linker (Log Dꢀ not be affected in the binding activity in the presence of
ꢃ0.4), showed Log D values of more than 1.0, indicating that HSA. From these results, and the relative ease of synthesis,
modification of CONH linker (6, Log Dꢀ0.68) would in- we considered that compounds contained CH2O linker would
crease of the lipophilicity without lack of the activity. On the be favorable for the further optimization. Thus, next we
other hand, 9a with Log D value of 1.6 showed the ratio value turned our attention to appending substituents on the pyrroli-
of 2.8, whereas 9f with Log D value of ꢃ0.4 showed the ratio dine ring and the benzene ring of benzoic acid portion begin-
value of 6.3, indicating that the apparent correlation between ning with compound 12a.
The result of introduction substitutions onto 3, 4, or both
positions on the pyrrolidine ring is shown in Table 2. As for
the configuration at the 4 position on the pyrrolidine ring, it
was found that the S isomers (12c, WꢀOH; 12e, WꢀOMe;
12h, WꢀF; 12l, WꢀNMe2) bound the receptor about 6 to 7
fold more potent than the corresponding R isomers (12b,
WꢀOH; 12d, WꢀOMe; 12g, WꢀF; 12k, WꢀNMe2). De-
spite the bulkiness (12f, Wꢀ2-O-naphtyl) of the substituents
Reagents: (a) ethyl 4-(diethoxyphosphorylmethyl)benzoate, LiHMDS, THF;
(b) TFA, CH2Cl2; (c) CO, Pd(PPh3)4, CuI, iPr2NH; (d) TFA, CH2Cl2.
at the 4 position, there was almost no effect on activity. As
for piperazine and piperidine acetic acid derivatives,11) the
Chart 3
configuration, bulkiness, and the nature of substituents at the
Reagents: (a) 4-iodothiophenol, NaH, THF; (b) CO, Pd(OAc)2, dppp, Et3N, DMSO/MeOH; (c) TFA, CH2Cl2.
Chart 4
Reagents and conditions: (a) BH3·Me2S, THF; (b) 4-HOPhCO2Me, Ph3P, diisopropyl azodicarboxylate (DIAD), THF; (c) K2CO3, MeOH; (d) HOAc, Ph3P, DIAD, THF;
(e) NaH, MeI, DMF; (f) Tf2O, diisopropylethylamine (DIPEA), THF, then Me2NH; (g) TFA, CH2Cl2; (h) H2, 10% Pd/C, EtOH.
Chart 5
Reagents and conditions: (a) Ph(-X)(-Z)OH, DIAD, PPh3, THF; (b) n-BuLi, TMEDA, methyl chloroformate; (c) Pd(OAc)2, CO, 1,3-bis(diphenylphosphino)propane,
Et3N, DMSO/MeOH; (d) TFA, CH2Cl2; (e) 4-HOPh(-Z)CO2R, DIAD, PPh3, THF.
Chart 6

November 2006
1519
Table 1. Inhibitory Activity and Distribution Coefficient of VLA-4 Antagonists
IC50
(nM)
IC50 (ꢄ3% HSA)
Log Db)
(pH 7.4)
Compounds
R1
X–Y
Ratioa)
(nM)
6
9a
9b
9c
9d
9e
9f
H
–CO–NH–
–CH2–NH–
–CHꢀCH–
–CꢀC–
–CH2–CH2–
–CH2–S–
–CH2–SO–
–CH2–SO2–
–CH2–O–
3.8
1.5
1.7
7.9
3.7
13.1
3.0
10.7
2.4
104
4.2
13
140
62
27.4
2.8
7.6
17.7
16.7
nd.
6.3
54.5
2.5
0.68
1.6
1.3
1.3
1.5
nt.
ꢃ0.4
nt.
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
ꢅ1000
19
9g
12a
583
6.1
1.5
nd., not detected, nt., not tested. a) RatioꢀIC50 (ꢄ3% HSA)/IC50
. b) Log D, n-octanol/PBS (pH 7.4).
Table 2. Inhibitory Activity and Distribution Coefficient of VLA-4 Antagonists
IC50
(nM)
IC50 (ꢄ3% HSA)
Log Db)
(pH 7.4)
Compounds
W
Ratioa)
(nM)
6
—
H
3.8
2.4
3.5
0.62
9.8
1.4
4.2
7.9
1.2
2.0
8.2
5.4
0.83
34
104
6.1
9.1
2.9
14.0
4.6
118
28
3.7
9.1
33
4.0
1.9
373
37
27.4
2.5
2.6
4.7
1.4
3.3
28.1
3.5
3.1
4.6
4.0
0.68
1.5
0.5
0.7
1.0
1.2
4.1
1.1
1.1
1.6
1.8
0.4
0.4
nt.
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
4R-OH
4S-OH
4R-OMe
4S-OMe
4S-O-2-Napthyl
4R-F
4S-F
4,4-diF
4R-Cl
4R-NMe2
4S-NMe2
3R,4S-O-Isopropylidene
3S-OBn
3R,4S-diOH
3S-OH
0.74
2.3
11.0
8.8
3.2
2.3
4.2
1.1
1.4
2.7
0
0.5
3.5
3.2
nd., not detected, nt., not tested. a) RatioꢀIC50 (ꢄ3% HSA)/IC50
. b) Log D, n-octanol/PBS (pH 7.4).
same position on the pyrrolidine ring did not affect the activ- (15e), nitro (15g), amino (15h), methylamino (15k), di-
ity. The fact suggests that the SAR of the benzoic acid deriv- methylamino (15i), and acetylamino (15j), were tolerated at
atives would be subtly different from those of both piper- 3 position on the benzene ring. However, 2-methyl (15a) and
azine and piperidine acetic acid derivatives. The substituents 3,5-dichloro (15f) substituents decreased binding affinity,
[12p, Wꢀ(3R)-OH; 12n, Wꢀ(3R)-OBn] at the 3-position on with IC50 values of 6.0 nM for 15a and 47 nM for 15f. Unex-
the pyrrolidine ring also maintained activity. Although 12o pectedly, the introduction of 2,5-dimethyl substituents (15c)
[Wꢀ(3R,4S)-dihydroxypyrrolidine] showed good activity, caused a loss of activity at 1 mM. Among these compounds,
the shielding of the hydroxyl groups of 12m by isopropyli- 15b and 15e showed higher Log D values (15b, 2.0; 15e, 1.8)
dene ketal was 30-fold loss of activity compared with 12o. than 12a, while the activities in the presence of HSA were
As for lipophilicity, 12f [Wꢀ(4S)-O-2-naphtyl] had a Log D relatively maintained.
of 4.1, 12i (Wꢀ4,4-difluoro) a Log D of 1.6, 12j [Wꢀ(4R)-
On the basis of activity and Log D value, 15b and 15e
chloro] a Log D of 1.8, and 12n [Wꢀ(3S)-OBn] a Log D of were selected for in vivo testing of VLA-4 inhibitory activity.
2.7. However, the most lipophilic compound 12f had signifi- These compounds were evaluated for Ascaris-antigen sensi-
cantly decreased activity in the presence of HSA (IC50ꢀ tized murine airway inflammation model by an oral dose of
118 nM, ratioꢀ28.1).
30 mg/kg/b.i.d. for 2 d (Fig. 2). The result showed that 15e
The results of introduction of several substitutes into the inhibited eosinophil infiltration into bronchial alveolar lavage
benzoic acid portion in 12a are shown in Table 3. The sub- (BAL) fluid by 72% compared with the vehicle alone. Com-
stituents, including methyl (15b), methoxy (15d), chloro pound 15b, however, only weakly inhibited (30%) eosinophil

1520
Vol. 54, No. 11
Table 3. Inhibitory Activity and Distribution Coefficient of VLA-4 Antagonists
IC50
(nM)
IC50 (ꢄ3% HSA)
Log Db)
(pH 7.4)
Compounds
Z
Ratioa)
(nM)
6
—
H
2-Me
3.8
2.4
6.0
1.6
ꢅ1000
1.3
1.6
47
1.4
1.9
104
6.1
118
16
nd.
nt.
37
nt.
nt.
3
27.4
2.5
19.7
10.0
nt.
nt.
23.1
nt.
nt.
1.6
nt.
nt.
6.1
0.68
1.5
1.8
2.0
nt.
1.1
1.8
nt.
0.7
0.5
1.2
0
12a
15a
15b
15c
15d
15e
15f
15g
15h
15i
3-Me
2,5-diMe
3-MeO
3-Cl
3,5-diCl
3-NO2
3-NH2
3-NMe2
3-NHAc
3-NHMe
1.4
1.1
1.8
nt.
nt.
11
15j
15k
1.2
nd., not detected, nt., not tested. a) RatioꢀIC50 (ꢄ3% HSA)/IC50
Table 4. Pharmacokinetic Properties of 15e in Rats
.
b) Log D, n-octanol/PBS (pH 7.4).
i.v.
p.o.
Compounds
c)
c)
AUC(0—inf.)
CL
(ml/min/kg)
MRT
(min)
t1/2l1
(min)
t1/2l2
(min)
AUC(0—inf.)
CL/F
(ml/min/kg)
Fd)
(%)
(ng·h/ml)
(ng·h/ml)
1a)
398
402
69
59
15.5
13.9
6.8
2.9
42.2
85.9
nd.
20
nd.
1670
ꢁ1
5
15eb)
nd., not detected. a) Male Sprague–Dawley rats. Dose: i.v. infusion (2 h) at 2.4 mg/kg; p.o. at 5 mg/kg. b) Male Sprague–Dawley rats. Dose: i.v. infusion (2 h) at
1.5 mg/kg; p.o. at 2 mg/kg. c) AUC were normalized to 1.5 mg/kg. d) F was calculated by dividing CL i.v. with CL/F.
(MDCK) cell monolayers. As a result, 15e demonstrated Paap
values of 0.78ꢂ10ꢃ6 cm/s, indicating that 15e is about 40-
fold more permeable than 1. In comparison, the Papp value of
metoprolol, which is known to show good oral availability
in humans is 17.3—20.3ꢂ10ꢃ6 cm/s. These results suggest
that compound needs to enhance its permeability. As for
the plasma clearance following i.v. administration, there
was no difference between 15e (Clꢀ59 ml/min/kg) and 1
(Clꢀ69 ml/min/kg), disappointingly.
Conclusion
A novel series of benzoic acid derivatives were explored to
obtain VLA-4 antagonists with good pharmacokinetic pro-
files. We focused on increasing the lipophilicity of lead com-
Fig. 2. Effect of VLA-4/VCAM-1 Antagonist 15e on Leukocyte Infiltra-
pounds to enhance the cell permeability while maintaining
tion into BAL Fluid 48 h after Allergen Challenge in Ascaris suum Sensi-
potent VLA-4 inhibitory activity. Thus, modification of the
linker portion between the pyrrolidine ring and benzoic acid,
and introduction of a variety of substituents into the pyrroli-
dine ring and benzene ring of benzoic acid as in 6 were con-
ducted. These modifications yielded several compounds with
potent activity even in the presence of HSA and moderate
lipophilicity. Among them, compound 15e demonstrated effi-
tized Mice
The compound was administered at a dosage at 30 mg/kg/b.i.d. for 2 d. ∗ pꢁ0.05,
∗∗ pꢁ0.01 vs. control (Aspin–Welch test). The total number of cells in BAL fluid iso-
lates were counted separately. Eosinophil numbers are expressed as the mean from each
treatment group (nꢀ7).
infiltration in mice (data not shown).
Next, the pharmacokinetic of 15e in rats was evaluated to cacy in an in vivo model of asthma by an oral dose of
determine whether this compound attained any improvement 30 mg/kg. However, this compound did not show a signifi-
over the initial lead compound 1. Compound 15e showed low cant improvement in oral pharmacokinetic profile compared
bioavailability Fꢀ5%, which indicated a slight improvement with the initial lead 1. Further improvements and structural
compared with 1 (Fꢁ1%) (Table 4). In order to evaluate the modifications of this series of compounds will be presented
causes of the difference in oral absorption, 15e was assessed in forthcoming publications.
for the cell permeability using Madin–Darby canine kidney

November 2006
1521
FAB-MS m/z: 534 (Mꢄꢄ1); Anal. Calcd for C29H31N3O7·0.5H2O: C, 64.20;
H, 5.94; N, 7.74. Found: C, 64.35; H, 5.83; N, 7.68.
Experimental
Melting points were determined on a MP-J3 (YANACO) and were uncor-
rected. Column chromatography was performed with silica gel 60 (Merck,
particle size 0.060—0.200 or 0.040—0.063). Flash column chromatography
was performed using a Biotage Si (Biotage) or a Hi-Flash (YAMAZEN).
Thin-layer chromatography (TLC) was performed on pre-coated TLC glass
4-[(4S)-Hydroxy-1-[4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)-
pyrrolidinyl]methoxybenzoic Acid (12c) Colorless powder (yield, 11%)
crystallized from CHCl3/EtOH/ether; mp 148—152 °C (dec.); IR (KBr)
3356, 2939, 1687, 1604, 1533, 1454, 1255 cmꢃ1 1H-NMR (DMSO-d6) d:
;
1
sheets with silica gel 60 F254 (Merck). H-NMR spectra were recorded on a
1.95—2.09 (2H, m), 2.25 (3H, s), 3.59 (2H, d, Jꢀ5.9 Hz), 3.69—3.73 (1H,
m), 3.81 and 3.85 (total 3H, each s, amide isomers), 4.13—4.47 (4H, m),
5.19 (1H, s), 6.70—7.21 (7H, m), 7.79 (1H, d, Jꢀ7.9 Hz), 7.86 (2H, d,
Jꢀ8.8 Hz), 8.01 (1H, d, Jꢀ8.3 Hz), 8.47 (1H, s), 8.57 (1H, s); ESI-MS m/z:
533 (Mꢄꢄ1); Anal. Calcd for C29H31N3O7·H2O: C, 63.15; H, 6.03; N, 7.62.
Found: C, 63.29; H, 5.76; N, 7.46.
JNM-EX-400 spectrometer (JEOL), and chemical shifts are given in ppm
(d) from tetramethylsilane as the internal standard. IR spectra were recorded
on a 270-30 spectrometer (HITACHI). ESI mass spectra were recorded on
an API-150EX spectrometer (SCIEX). FAB mass spectra were recorded on a
JMS-HX110 spectrometer (JEOL). HR-FAB mass spectra were recorded on
a JMS-700 spectrometer (JEOL).
4-[(4R)-Methoxy-1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenyl-
acetyl]-(2S)-pyrrolidinyl]methoxybenzoic Acid (12d) Colorless crys-
talline powder (yield, 28%) crystallized from CHCl3/MeOH/iPr2O; mp
112—115 °C; IR (ATR) 3348, 2937, 1703, 1682, 1603, 1529, 1485,
4-[1-[3-Methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)-
pyrrolidinyl]methylaminobenzoic Acid (9a) (General Procedure A)
A
mixture of methyl 4-[(2S)-pyrrolidinyl]methylaminobenzoate (6a) (398 mg,
1.69 mmol), 3-methoxy-4-[N-(2-methylphenyl)ureido]phenylacetic acid (7a)
(587 mg, 1.87 mmol), EDC·HCl (486 mg, 2.54 mmol), HOBt (23 mg,
0.17 mmol), and DMAP (21 mg, 0.17 mmol) in DMF (4 ml) was stirred at
room temperature for 12 h. The mixture was poured into water and extracted
with EtOAc. The combined extracts were washed with water, brine, and then
dried over Na2SO4. After the solvent was removed under reduced pressure,
the residue was purified by chromatography on silica gel with CHCl3/
MeOH (50/1, v/v) as an eluent to give methyl 4-[1-[3-methoxy-4-[N-(2S)-
methylphenyl]ureido]phenylacetyl]-(2S)-pyrrolidinyl]methylaminobenzoate
(882 mg, 1.66 mmol) as a brownish amorphous solid.
1
1454 cmꢃ1; H-NMR (DMSO-d6) d: 2.04—2.17 (2H, m), 2.25 (3H, s), 3.21
(3H, s), 3.56—3.75 (4H, m), 3.79 (3H, s), 4.04—4.35 (4H, m), 6.73—7.17
(7H, m), 7.79—7.81 (1H, m), 7.87—7.89 (2H, m), 7.99—8.01 (1H, m), 8.47
(1H, s), 8.55 (1H, s), 12.63 (1H, s); FAB-MS m/z: 548 (Mꢄꢄ1); Anal. Calcd
for C30H33N3O7·0.25H2O: C, 65.26; H, 6.12; N, 7.61. Found: C, 65.36; H,
6.45; N, 7.24.
4-[(4S)-Methoxy-1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenyl-
acetyl]-(2S)-pyrrolidinyl]methoxybenzoic Acid (12e) Colorless amor-
phous solid (yield, 83%); IR (KBr) 3354, 2937, 1709, 1685, 1604, 1533,
1
1454 cmꢃ1; H-NMR (DMSO-d6) d: 2.09—2.23 (2H, m), 2.25 (3H, s), 3.22
To
a stirred solution of methyl 4-[1-[3-methoxy-4-[N-(2S)-methyl-
(3H, s), 3.49—3.78 (4H, m), 3.82 and 3.83 (total 3H, each s, amide iso-
mers), 3.87—4.52 (3H, m), 6.71—7.17 (5H, m), 7.79 (1H, d, Jꢀ8.1 Hz),
7.86—8.03 (3H, m), 8.45—8.57 (2H, m), 12.64 (1H, s); FAB-MS m/z: 548
(Mꢄꢄ1); FAB-MS (HR) m/z: 548.2425 (Calcd for C30H34N3O7: 548.2397).
4-[1-[3-Methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(4S)-(2-
naphthyloxy)-(2S)-pyrrolidinyl]methoxybenzoic Acid (12f) Colorless
amorphous solid (yield, 32%); IR (KBr) 3354, 2937, 1685, 1601, 1533,
phenyl]ureido]phenylacetyl]-(2S)-pyrrolidinyl]methylaminobenzoate (882
mg, 1.66 mmol) in THF/MeOH (18/5 ml) was added 1 M NaOH (5.0 ml, 5.0
mmol), and refluxed for 3 d. After cooling to room temperature, the mixture
was concentrated in vacuo. The residue was diluted with 1 M HCl and ex-
tracted with CH2Cl2. The combined extracts were washed with brine, dried
over Na2SO4, and concentrated. The resulting solid was recrystallized from
n-hexane/diisopropyl ether/CHCl3/MeOH to afford compound 9a (181 mg,
21%) as a pale yellow solid. IR (KBr) 1604, 1535, 1511, 1454, 1255, 1224,
1
1255 cmꢃ1; H-NMR (DMSO-d6) d: 2.24 (3H, s), 2.25—2.43 (2H, m), 3.65
(2H, s), 3.81 (3H, s), 3.81—3.85 (1H, m), 4.05—4.70 (4H, m), 5.21—5.33
(1H, s), 6.76 (1H, d, Jꢀ7.3 Hz), 6.86—7.35 (9H, m), 7.44 (1H, t, Jꢀ7.3 Hz),
7.76—7.89 (6H, m), 8.01 (1H, d, Jꢀ8.3 Hz), 8.48 (1H, s), 8.56 (1H, s);
FAB-MS m/z: 660 (Mꢄꢄ1); FAB-MS (HR) m/z: 660.2715 (Calcd for
C39H38N3O7: 660.2710).
1174 cmꢃ1 1H-NMR (DMSO-d6) d: 1.79—1.99 (4H, m), 2.25 (3H, s),
;
2.90—2.94 (1H, m), 3.35—3.62 (6H, m), 3.87 (3H, s), 4.12—4.15 (1H, s),
6.63—6.78 (4H, m), 6.89—7.17 (3H, m), 7.65 (2H, d, Jꢀ8.3 Hz), 7.80 (1H,
d, Jꢀ8.3 Hz), 8.02 (1H, d, Jꢀ8.3 Hz), 8.47 (1H, s), 8.57 (1H, s), 12.0 (1H,
s); FAB-MS m/z: 517 (Mꢄꢄ1); Anal. Calcd for C29H32N4O5·H2O: C, 65.15;
H, 6.41; N, 10.48. Found: C, 65.45; H, 6.33; N, 10.02.
4-[(4R)-Fluoro-1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenyl-
acetyl]-(2S)-pyrrolidinyl]methoxybenzoic Acid (12g) Pale yellow amor-
phous solid (yield, 70%); IR (KBr) 3354, 3060, 2972, 2937, 2877, 1707,
Compounds 9b, c, 12a—n, and 15g were prepared according to general
procedure A.
1685, 1604, 1533 cmꢃ1 1H-NMR (DMSO-d6) d: 2.24—2.41 (5H, m),
.
4-[2-[1-[3-Methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)-
pyrrolidinyl]ethenyl]benzoic Acid (9b) Colorless crystalline material
(yield, 92%); IR (ATR) 3338, 2976, 2943, 2879, 1701, 1589, 1527,
3.42—4.66 (10H, m), 5.31 and 5.44 (total 1H, each s, amide isomers),
6.71—7.16 (7H, m), 7.79 (1H, d, Jꢀ8.1 Hz), 7.85—7.89 (2H, m), 7.98—
8.00 (1H, m), 8.47 (1H, s), 8.55 (1H, s); FAB-MS m/z: 536 (Mꢄꢄ1); FAB-
MS (HR) m/z: 536.2231 (Calcd for C29H31FN3O6: 536.2197).
1485 cmꢃ1 1H-NMR (DMSO-d6) d: 1.78—2.13 (4H, m), 2.50 (3H, s),
;
3.44—3.68 (4H, m), 3.75 and 3.82 (total 3H, each s, amide isomers), 4.71
(1H, m), 6.26—8.59 (15H, m); FAB-MS m/z: 514 (Mꢄꢄ1); FAB-MS (HR)
m/z: 514.2308 (Calcd for C30H32N3O5: 514.2342)
4-[(4S)-Fluoro-1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenyl-
acetyl]-(2S)-pyrrolidinyl]methoxybenzoic Acid (12h) Pale yellow amor-
phous solid (yield, 30%); IR (KBr) 3338, 3060, 2970, 2937, 2875, 1709,
1685, 1604, 1539, 1485, 1454 cmꢃ1 1H-NMR (DMSO-d6) d: 2.25—2.51
.
4-[2-[1-[3-Methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)-
pyrrolidinyl]ethynyl]phenylacetic Acid (9c) Colorless crystalline powder
(yield, 24%) recrystallized from CHCl3/n-hexane; mp 161—164 °C; IR
(5H, m), 3.33—4.41 (10H, m), 5.30—5.50 (1H, m), 6.75—7.17 (7H, m),
7.79 (1H, d, Jꢀ8.1 Hz), 7.87—8.04 (3H, m), 8.46—8.50 (1H, m), 8.56—
8.60 (1H, m); FAB-MS m/z: 536 (Mꢄꢄ1); FAB-MS (HR) m/z: 536.2174
(Calcd for C29H31FN3O6: 536.2197).
(ATR) 3346, 2979, 2939, 2877, 1691, 1603, 1529, 1485 cmꢃ1 1H-NMR
;
(CDCl3) d: 1.96—2.10 (4H, m), 2.24 (3H, s), 3.32—3.81 (2H, m), 3.62 (2H,
s), 3.81 (3H, s), 4.87—5.10 (1H, m), 6.76—8.58 (13H, m); FAB-MS m/z:
512 (Mꢄꢄ1); FAB-MS (HR) m/z: 512.2181 (Calcd for C30H30N3O5:
512.2185).
4-[4,4-Difluoro-1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenyl-
acetyl]-(2S)-pyrrolidinyl]methoxybenzoate (12i) Colorless crystalline
powder (yield, 61%); mp 135—140 °C; IR (KBr) 3354, 2956, 2935, 1678,
1
1633, 1604, 1531, 1487, 1454 cmꢃ1; H-NMR (DMSO-d6) d: 2.23 (3H, s),
4-[1-[3-Methoxy-4-[N-(2-methylphenyl)ureido]phenylacethyl]-(2S)-
pyrrolidinyl]methoxybenzoic Acid (12a) Colorless amorphous solid
(yield, 40%); IR (KBr) 3348, 2945, 2879, 1680, 1603, 1529, 1485, 1452,
2.49—2.73 (2H, m), 3.36—4.55 (10H, m), 6.73 (1H, d, Jꢀ8.3 Hz), 6.84
(1H, s), 6.93 (1H, t, Jꢀ7.3 Hz), 7.00 (2H, d, Jꢀ8.3 Hz), 7.10—7.16 (2H, m),
7.78 (1H, d, Jꢀ8.3 Hz), 7.86 (2H, d, Jꢀ8.3 Hz), 8.00 (1H, d, Jꢀ8.3 Hz),
8.47 (1H, s), 8.56 (1H, s); FAB-MS m/z: 554 (Mꢄꢄ1); Anal. Calcd for
C29H29F2N3O6·0.75H2O: C, 61.42; H, 5.44; N, 7.06. Found: C, 61.30; H,
5.44; N, 7.06.
1
1415, 1300, 1248 cmꢃ1; H-NMR (DMSO-d6) d: 1.83—2.14 (4H, m), 2.21
(3H, s), 2.46 (2H, s), 3.78 (3H, s), 3.95—4.02 (1H, m), 4.13—4.16 (1H, m),
4.24 (1H, s), 6.51—7.98 (11H, m), 8.43 (1H, s), 8.53 (1H, s), 12.57 (1H, s);
FAB-MS m/z: 518 (Mꢄꢄ1); FAB-MS (HR) m/z: 518.2254 (Calcd for
C29H32N3O6: 518.2291).
4-[(4R)-Chloro-1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenyl-
acetyl]-(2S)-pyrrolidinyl]methoxybenzoic Acid (12j) Colorless amor-
phous solid (yield, 53%); mp 128—132 °C; IR (ATR) 3346, 1684, 1603,
1529, 1454, 1417, 1338 cmꢃ1; 1H-NMR (DMSO-d6) d: 2.25 (3H, s), 2.29—
2.46 (2H, m), 3.57—3.73 (2H, m), 3.78 (3H, s), 3.81—3.99 (2H, m), 4.11—
4.31 (2H, m), 4.43—4.67 (1H, m), 4.83—4.85 (1H, m), 6.71—7.17 (7H, m),
7.78—7.80 (1H, m), 7.87—7.91 (2H, m), 7.99—8.01 (1H, m), 8.47 (1H, s),
8.56 (1H, s), 12.66 (1H, s); FAB-MS m/z: 552 (Mꢄꢄ1); Anal. Calcd for
C29H30ClN3O6·0.75H2O: C, 61.59; H, 5.61; Cl, 6.27; N, 7.43. Found: C,
4-[(4R)-Hydroxy-1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenyl-
acetyl]-(2S)-pyrrolidinyl]methoxybenzoic Acid (12b) Colorless crys-
talline powder (yield, 34%) recrystallized from CHCl3/iPr2O; mp 178—
182 °C; IR (ATR) 3313, 1671, 1651, 1603, 1556, 1452, 1431, 1417 cmꢃ1
;
1H-NMR (DMSO-d6) d: 1.92—2.10 (2H, m), 2.23 (3H, s), 3.40—3.44 (1H,
m), 3.56—3.67 (3H, m), 3.78 (3H, s), 4.05—4.39 (4H, m), 5.04—5.08 (1H,
m), 6.71—7.01 (5H, m), 7.10—7.16 (2H, m), 7.77—7.79 (1H, m), 7.85—
7.89 (2H, m), 7.98—8.00 (1H, m), 8.45 (1H, s), 8.54 (1H, s), 12.59 (1H, s);

1522
Vol. 54, No. 11
61.56; H, 5.51; Cl, 6.68; N, 7.26.
4-[1-[3-Methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)-
4-[(4R)-Dimethylamino-1-[3-methoxy-4-[N-(2-methylphenyl)ureido]- pyrrolidinyl]methoxy-3-methylbenzoic Acid (15b) Colorless crystalline
phenylacetyl]-(2S)-pyrrolidinyl]methoxybenzoic Acid (12k) Colorless powder (yield, 54%) recrystallized from n-hexane/EtOAc/CHCl3/MeOH; IR
1
crystalline solid (yield, 68%); mp 145—150 °C; IR (KBr) 3355, 2948, 1698, (ATR) 3354, 2956, 2875, 1682, 1604, 1529, 1452 cmꢃ1; H-NMR (DMSO-
1604, 1533, 1454, 1417, 1255, 1226, 1166, 1035, 755 cmꢃ1 1H-NMR d6) d: 1.87—2.10 (4H, m), 2.12 (3H, s), 2.25 (3H, s), 3.51—3.71 (4H, m),
;
(DMSO-d6) d: 1.82—1.98 (1H, m), 2.08—2.11 (1H, m), 2.20 (6H, s), 2.25 3.76 (3H, s), 4.08—4.18 (2H, m), 4.32—4.36 (1H, m), 6.74 (1H, dd, Jꢀ1.5,
(3H, s), 3.40—3.60 (3H, m), 3.64 (2H, s), 3.82 (3H, s), 4.01—4.16 (2H, m), 9.8 Hz), 6.84 (1H, d, Jꢀ1.5 Hz), 6.94 (1H, t, Jꢀ6.8 Hz), 7.06 (1H, d,
4.34—4.38 (1H, m), 6.74—7.15 (7H, m), 7.77—8.02 (4H, m), 8.44 (1H, s), Jꢀ8.8 Hz), 7.12 (1H, d, Jꢀ7.8 Hz), 7.16 (1H, d, Jꢀ7.8 Hz), 7.72 (1H, s),
8.54 (1H, s); FAB-MS m/z: 561 (Mꢄꢄ1); Anal. Calcd for C31H36N4O6·
1.2H2O: C, 63.95; H, 6.65; N, 9.62. Found: C, 63.82; H, 6.72; N, 9.44.
7.76 (1H, dd, Jꢀ2.0, 8.3 Hz), 7.79 (1H, d, Jꢀ7.8 Hz), 7.99 (1H, d,
Jꢀ8.3 Hz), 8.46 (1H, s), 8.54 (1H, s); FAB-MS m/z: 532 (Mꢄꢄ1); FAB-MS
4-[(4S)-Dimethylamino-1-[3-methoxy-4-[N-(2-methylphenyl)ureido]- (HR) m/z: 532.2433 (Calcd for C30H34N3O6: 532.2448).
phenylacetyl]-(2S)-pyrrolidinyl]methoxybenzoic Acid (12l) Colorless
2,5-Dimethyl-4-[1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenyl-
crystalline solid (yield, 65%); mp 147—150 °C; IR (KBr) 3353, 2952, 1700, acetyl]-(2S)-pyrrolidinyl]methoxy]benzoic Acid (15c) Pale brown pow-
1604, 1533, 1454, 1415, 1255, 1166, 1035, 755 cmꢃ1; 1H-NMR (DMSO-d6) der (yield, 18%) recrystallized from EtOAc/CHCl3/diisopropylether; IR
d: 1.83—1.84 (1H, m), 2.08—2.10 (1H, m), 2.21 (6H, s), 2.24 (3H, s), (ATR) 3354, 2966, 2877, 1705, 1682, 1599, 1529, 1485 cmꢃ1 1H-NMR
;
2.98—3.02 (2H, m), 3.60 (2H, s), 3.78 (3H, s), 3.85—4.29 (4H, m), 6.71— (DMSO-d6) d: 1.86—2.09 (5H, m), 2.06 (3H, s), 2.25 (3H, s), 3.47—3.67
7.16 (7H, m), 7.77—8.01 (4H, m), 8.46 (1H, s), 8.54 (1H, s); FAB-MS m/z: (6H, m), 3.76 (3H, s), 4.05—4.12 (2H, m), 4.30—4.31 (1H, m), 6.51 (1H,
561 (MꢄH)ꢄ; Anal. Calcd for C31H36N4O6·2H2O: C, 62.40; H, 6.76; N, s), 6.55 (1H, s), 6.73—6.95 (2H, m), 7.11—7.17 (2H, m), 7.64 (1H, s), 7.79
9.39. Found: C, 62.51; H, 6.60; N, 9.36.
(1H, d, Jꢀ7.8 Hz), 7.99 (1H, d, Jꢀ7.8 Hz), 8.47 (1H, s), 8.55 (1H, s); FAB-
4-[(3R,4S)-Isopropylidenedioxy-1-[4-[N-(2-methylphenyl)ureido]-3- MS m/z: 546 (Mꢄꢄ1); FAB-MS (HR) m/z: 546.2567 (Calcd for
methoxyphenylacetyl]-(2R)-pyrrolidinyl]methoxybenzoic Acid (12m) C31H36N3O6: 546.2604).
Colorless amorphous solid (yield, 98%); IR (KBr) 3354, 2983, 2937, 1707,
3-Methoxy-4-[1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenyl-
acetyl]-(2S)-pyrrolidinyl]methoxybenzoic Acid (15d) Yellow amorphous
1
1604, 1533 cmꢃ1; H-NMR (DMSO-d6) d: 1.24 and 1.32 (total 3H, each s,
amide isomers), 1.26 (3H, s), 2.24 (3H, s), 3.40 (1H, dd, Jꢀ14.0, 5.1 Hz), solid (yield, 56%); IR (KBr) 1707 cmꢃ1 1H-NMR (DMSO-d6) d: 1.84—
;
3.58—3.62 (2H, m), 3.69—3.71 (1H, m), 3.76 and 3.82 (total 3H, each s, 2.18 (4H, m), 2.25 (3H, s), 2.49—2.51 (2H, m), 3.29—3.59 (4H, m), 3.80
amide isomers), 3.92—4.96 (5H, m), 6.74 and 6.78 (total 1H, each d, Jꢀ8.3, (3H, s), 3.82 (3H, s), 4.00—4.05 (1H, m), 6.53—8.01 (10H, m), 8.45 (1H,
8.6 Hz respectively, amide isomers), 6.83—7.16 (6H, m), 7.79 (1H, d, s), 8.54 (1H, s), 12.63 (1H, s); FAB-MS m/z: 548 (Mꢄꢄ1); Anal. Calcd for
Jꢀ8.3 Hz), 7.87 (2H, t, Jꢀ9.1 Hz), 7.99—8.03 (1H, m), 8.49 (1H, d, C30H33N3O7·0.75H2O: C, 64.22; H, 6.20; N, 7.59. Found: C, 64.14, H, 6.33,
Jꢀ3.4 Hz), 8.57 (1H, s); FAB-MS m/z: 590 (Mꢄꢄ1); FAB-MS (HR) m/z: N, 6.99.
590.2458 (Calcd for C32H36N3O8: 590.2502).
3-Chloro-4-[1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenyl-
4-[(3R)-Benzyloxy-1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenyl- acetyl]-(2S)-pyrrolidinyl]methoxybenzoic Acid (15e) Colorless crys-
acetyl]-(2S)-pyrrolidinyl]methoxybenzoic Acid (12n) Colorless amor- talline material (yield, 98%) recrystallized from n-hexane/EtOAc/CHCl3; IR
phous solid (yield, 42%); IR (ATR) 3344, 2947, 2875, 1684, 1603, 1529, (ATR) 3346, 2952, 2873, 1685, 1597, 1529, 1500, 1452 cmꢃ1 1H-NMR
;
1454, 1417 cmꢃ1 1H-NMR (CDCl3) d: 2.11—2.25 (1H, m), 2.26—2.30 (DMSO-d6) d: 1.82—2.24 (4H, m), 2.25 (3H, s), 3.48—3.60 (4H, m), 3.78
;
(1H, m), 2.30 (3H, s), 3.58 (3H, s), 3.62 (2H, s), 3.65—3.72 (2H, m), 4.13 (3H, s), 4.16—4.20 (2H, m), 4.29—4.33 (1H, m), 6.74 (1H, dd, Jꢀ1.5,
(1H, dd, Jꢀ8.3, 3.9 Hz), 4.18 (1H, d, Jꢀ3.9 Hz), 4.25 (1H, d, Jꢀ8.3 Hz), 8.3 Hz), 6.84 (1H, d, Jꢀ2.0 Hz), 6.91—6.95 (1H, m), 7.11—7.17 (3H, m),
4.48 (1H, d, Jꢀ12.0 Hz), 4.54—4.57 (2H, m), 6.71 (1H, s), 6.75 (1H, d, 7.79 (2H, dd, Jꢀ2.0, 8.3 Hz), 7.85 (1H, d, Jꢀ2.0 Hz), 7.98 (1H, d,
Jꢀ7.8 Hz), 6.87 (2H, d, Jꢀ8.8 Hz), 7.15—7.33 (10H, m), 7.47 (1H, d, Jꢀ8.3 Hz), 8.53 (1H, s), 8.58 (1H, s); FAB-MS m/z: 552 (Mꢄꢄ1); FAB-MS
Jꢀ7.8 Hz), 7.99 (2H, d, Jꢀ8.8 Hz), 8.02 (1H, d, Jꢀ8.3 Hz); ESI-MS m/z: (HR) m/z: 552.1877 (Calcd for C29H31ClN3O6: 552.1901).
624 (Mꢄꢄ1); Anal. Calcd for C36H38N3O7·0.5H2O: C, 68.34; H, 6.05; N,
6.64. Found: C, 68.38; H, 6.31; N, 6.32.
3,5-Dichloro-4-[1-[3-methoxy-4-(N-(2-methyl)phenylureido)phenyl-
acetyl]-(2S)-pyrrolidinyl]methoxybenzoic Acid (15f) Colorless crys-
4-[1-[3-Methoxy-4-[N-(2-methylphenyl)ureido]phenylacethyl]-(2S)- talline material (yield, 91%) recrystallized from n-hexane/MeOH/CHCl3; IR
pyrrolidinylmethoxy]-3-nitrobenzoic Acid (15g) Yellow crystalline solid (ATR) 3344, 2958, 2879, 1697, 1612, 1589, 1529, 1452 cmꢃ1 1H-NMR
;
(yield, 63%); IR (ATR) 3344, 2974, 2937, 2881, 1697, 1612, 1527, 1485, (DMSO-d6) d: 1.83—2.24 (4H, m), 2.24 (3H, s), 3.50—3.58 (4H, m), 3.84
1
1452 cmꢃ1; H-NMR (CDCl3) d: 1.91—2.09 (4H, m), 2.28 (3H, s), 3.54— (3H, s), 3.98—4.05 (1H, m), 4.15 (1H, dd, Jꢀ2.9, 8.7 Hz), 4.27—4.31 (1H,
3.62 (4H, m), 3.64 (3H, s), 4.15 and 4.59 (total 1H, each d, each Jꢀ7.8 Hz,
amide isomers), 4.41—4.51 (1H, m), 6.66 (1H, s,), 6.72 (1H, d, Jꢀ8.3 Hz),
7.11—7.28 (5H, m), 7.46 (1H, d, Jꢀ7.8 Hz), 7.74 (1H, d, Jꢀ7.8 Hz), 7.85
(1H, s), 8.17 (1H, dd, Jꢀ2.0, 8.8 Hz), 8.48 (1H, d, Jꢀ2.4 Hz); FAB-MS m/z:
563; FAB-MS (HR) Calcd for C29H31N4O8: 563.2142. Found: 563.2150.
Compounds 9e and 15a—f were prepared according to general procedure
A, but with 5b in place of 7a.
m), 6.74 (1H, d, Jꢀ8.3 Hz), 6.87 (1H, s), 6.93 (1H, t, Jꢀ7.3 Hz), 7.11 (1H,
d, Jꢀ7.8 Hz), 7.16 (1H, d, Jꢀ8.3 Hz), 7.79 (1H, d, Jꢀ8.3 Hz), 7.86 (1H, s),
7.87 (1H, d, Jꢀ9.8 Hz), 7.99 (1H, d, Jꢀ8.3 Hz), 8.49 (1H, s), 8.58 (1H, s);
FAB-MS m/z: 586 (Mꢄꢄ1); FAB-MS (HR) m/z: 588.1511 (Calcd for
C29H30Cl2N3O6: 586.1512).
4-[2-[1-[3-Methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)-
pyrrolidinyl]ethyl]benzoic Acid (9d) A suspension of ethyl 4-[2-[1-[3-
4-[1-[3-Methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)- methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinyl]-
pyrrolidinyl]methylthiobenzoic Acid (9e) Colorless crystalline powder ethenyl]benzoic acid (9b) (184 mg, 0.358 mmol) and 5% Pd/C (368 mg) in
(yield, 51%) recrystallized from n-hexane/EtOAc/MeOH; mp 161—164 °C; MeOH (5 ml) was hydrogenated under 1 atom hydrogen atmosphere at room
1
IR (KBr) 3318, 2952, 1596, 1536, 1299, 1155 cmꢃ1; H-NMR (DMSO-d6) temperature for 21 h. After the catalyst was removed by filtration, the filtrate
d: 1.82—2.05 (4H, m), 2.25 (3H, s), 2.91 (1H, dd, Jꢀ9.8, 13.2 Hz), 3.47— was concentrated to dryness. The residue was purified by column chro-
3.52 (3H, m), 3.57 (2H, s), 3.87 (3H, s), 4.14 (1H, m), 6.76 (1H, dd, Jꢀ1.5, matography on silica gel with CHCl3/MeOH (4/1 to 3/1, v/v) as an eluent to
8.3 Hz), 6.89 (1H, d, Jꢀ1.5 Hz), 6.94 (1H, t, Jꢀ7.3 Hz), 7.11—7.19 (2H, give the title compound 9d (123 mg, 66%) as a colorless crystalline solid. IR
m), 7.57 (2H, d, Jꢀ8.3 Hz), 7.80 (1H, d, Jꢀ8.3 Hz), 7.83 (2H, d, Jꢀ8.3 Hz), (ATR) 3345, 2927, 1677, 1589, 1529, 1452, 1414, 1338 cmꢃ1 1H-NMR
;
8.02 (1H, d, Jꢀ8.3 Hz), 8.49 (1H, s), 8.58 (1H, s); FAB-MS m/z: 534 (DMSO-d6) d: 1.55—2.03 (6H, m), 2.24 (3H, s), 2.60 (2H, m), 3.17—3.59
(Mꢄꢄ1); Anal. Calcd for C29H31N3O5S·1.25H2O: C, 62.63; H, 6.07; N, (4H, m), 3.83 (3H, s), 3.95—3.99 (1H, m), 6.61—8.57 (13H, m); FAB-MS
7.36; S, 5.77. Found: C, 62.62; H, 5.74; N, 7.36; S, 5.67.
4-[1-[3-Methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)- 516.2489).
m/z: 516 (Mꢄꢄ1); FAB-MS (HR) m/z: 516.2490 (Calcd for C30H34N3O5:
pyrrolidinyl]methoxy-2-methylbenzoic Acid (15a) Colorless crystalline
Methyl 4-[1-[3-Methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-
powder (yield, 32%) recrystallized from EtOAc/CHCl3/diisopropylether; mp (2S)-pyrrolidinyl]methylthiobenzoate (10) as an Intermediate of 9e
106—109 °C; IR (KBr) 1708, 1602, 1531, 1245 cmꢃ1; 1H-NMR (DMSO-d6) Colorless crystalline powder (yield, 82%). IR (KBr) 1785, 1224, 1216 cmꢃ1
;
d: 1.84—2.03 (4H, m), 2.22 (3H, s), 3.47—3.59 (7H, m), 3.75—3.79 (1H, 1H-NMR (CDCl3) d: 1.88—1.99 (4H, m), 2.30 (3H, s), 2.75 (1H, dd, Jꢀ9.8,
m), 3.80 (3H, s), 3.94—3.98 (1H, m), 4.11—4.26 (1H, m), 6.72 (1H, d, 13.2 Hz), 3.43—3.55 (3H, m), 3.56 (2H, s), 3.64 (1H, dd, Jꢀ1.1, 14.2 Hz),
Jꢀ8.3 Hz), 6.80—6.89 (3H, m), 6.91 (1H, t, Jꢀ7.8 Hz), 7.11 (1H, t,
3.73 (3H, s), 3.88 (3H, s), 4.31—4.35 (1H, m), 6.29 (1H, s), 6.78—6.81
Jꢀ7.8 Hz), 7.14 (1H, d, Jꢀ7.8 Hz), 7.79 (2H, t, Jꢀ8.8 Hz), 7.98 (1H, dd, (2H, m), 7.11—7.26 (5H, m), 7.50 (3H, d, Jꢀ8.3 Hz), 7.93 (2H, d,
Jꢀ2.0, 7.8 Hz), 8.44 (1H, s), 8.54 (1H, s), 12.41 (1H, s); FAB-MS m/z: 514 Jꢀ8.8 Hz), 8.07 (1H, d, Jꢀ7.8 Hz); FAB-MS m/z: 548 (Mꢄꢄ1); Anal. Calcd
(Mꢄꢄ1); Anal. Calcd for C30H33N3O6·0.5H2O: C, 66.65; H, 6.34; N, 7.77. for C30H33N3O5S·0.25H2O: C, 65.26; H, 6.12; N, 7.61. Found: C, 65.48; H,
Found: C, 66.83; H, 6.47; N, 7.42.
6.20; N, 7.47.

November 2006
1523
4-[1-[3-Methoxy-4-[N-(2-Methylphenyl)ureido]phenylacetyl]-(2S)-
pyrrolidinyl]methylsulfinylbenzoic Acid (9f) To a stirred solution of
550.2188 (Calcd for C29H32N3O8: 550.2189).
Methyl 4-[(3R)-Benzyloxy-1-[3-methoxy-4-[N-(2-methylphenyl)urei-
methyl 4-[1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)- do]phenylacetyl]-(2S)-pyrrolidinyl]methoxybenzoate (13n) as an Inter-
1
pyrrolidinyl]methylthiobenzoate (10) (264 mg, 0.482 mmol) in CH2Cl2
(5.2 ml) was added mCPBA (119 mg, 0.482 mmol) at 0 °C, and stirred at
mediate of 12n Yellow amorphous solid (yield, 93%); H-NMR (CDCl3)
d: 2.13—2.17 (1H, m), 2.22—2.26 (1H, m), 2.29 (3H, s), 3.54—3.58 (1H,
room temperature for 1 h. The mixture was diluted with CHCl3, and m), 3.58 (3H, s), 3.62 (2H, s), 3.70—3.74 (1H, m), 3.88 (3H, s), 4.02 (1H,
quenched with sat. Na2S2O3. The separated organic layer was washed with dd, Jꢀ10.0, 7.1 Hz), 4.17 (1H, d, Jꢀ4.2 Hz), 4.28 (1H, dd, Jꢀ10.0, 3.2 Hz),
sat. NaHCO3, brine, and dried over Na2SO4. The solvent was removed 4.46 (1H, d, Jꢀ12.0 Hz), 4.51—4.55 (1H, m), 4.54 (1H, d, Jꢀ12.0 Hz), 6.48
under reduced pressure to afford methyl 4-[1-[3-methoxy-4-[N-(2-methyl- (1H, s), 6.73 (1H, d, Jꢀ1.5 Hz), 6.78 (1H, dd, Jꢀ8.1, 1.5 Hz), 6.87 (1H, d,
phenyl)ureido]phenylacetyl]-(2S)-pyrrolidinyl]methylsulfinylbenzoate as
colorless amorphous solid.
To a stirred solution of methyl 4-[1-[3-methoxy-4-[N-(2-methylphenyl)-
a
Jꢀ8.8 Hz), 7.13—7.16 (2H, m), 7.20—7.36 (7H, m), 7.52—7.56 (1H, m),
7.94 (1H, d, Jꢀ8.8 Hz), 8.05 (1H, d, Jꢀ8.1 Hz); ESI-MS m/z: 638 (Mꢄꢄ1).
4-[(3R)-Hydroxy-1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phen-
ureido]phenylacetyl]-(2S)-pyrrolidinyl]methylsulfinylbenzoate in THF/H2O ylacetyl]-(2S)-pyrrolidinyl]methoxybenzoic Acid (12p) A suspension of
(4/1 ml) was added LiOH (34.6 mg, 1.45 mmol). After 12 h stirring, the mix-
ture was diluted with CHCl3, washed with 1 M HCl, brine, and dried over
methyl 4-[(3R)-benzyloxy-1-[3-methoxy-4-[N-(2-methylphenyl)ureido]-
phenylacetyl]-(2S)-pyrrolidinyl]methoxybenzoate (13n) (1.85 g, 2.90 mmol)
Na2SO4. The solvent was removed under reduced pressure, and the obtained and Pd(OH)2 (1.0 g) in THF (30 ml) was hydrogenated under hydrogen
solid was recrystallized from n-hexane/CHCl3/MeOH to afford compound atmosphere at room temperature for 3 h. After removal of the catalyst by fil-
(9f) (193 mg, 73%) as a colorless crystalline powder. IR (KBr) 3338, 2956,
1708, 1529, 1299, 1207, 1155 cmꢃ1 1H-NMR (DMSO-d6) d: 1.70—2.06 methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinyl]-
(4H, m), 2.24 (3H, s), 2.90 (1H, dd, Jꢀ8.3, 13.2 Hz), 3.02—3.08 (1H, m),
methoxybenzoate (1.56 g, 98%) as a colorless amorphous solid. 1H-NMR
tration, the filtrate was concentrated to afford methyl 4-[(3R)-hydroxy-1-[3-
;
3.16—3.25 (1H, m), 3.41—3.60 (3H, m), 3.84 (3H, s), 4.40 (1H, s), 6.74 (CD3OD) d: 2.07—2.11 (1H, m), 2.29 (3H, s), 2.31—2.35 (1H, m), 3.68
(1H, d, Jꢀ7.8 Hz), 6.87 (1H, s), 6.94 (1H, d, Jꢀ7.3 Hz), 7.11—7.17 (2H, (2H, s), 3.78 (3H, s), 3.72—3.82 (2H, m), 3.84 (3H, s), 4.12 (1H, dd, Jꢀ7.4,
m), 7.75—7.81 (3H, m), 7.98—8.05 (1H, m), 8.10 (2H, d, Jꢀ8.3 Hz), 8.46
4.2 Hz), 4.24—4.30 (2H, m), 4.39—4.43 (1H, m), 6.79 (1H, dd, Jꢀ8.0,
(1H, s), 8.56 (1H, s); FAB-MS m/z: 550 (Mꢄꢄ1), 572 (MꢄꢄNa); Anal. 2.0 Hz), 6.86 (1H, d, Jꢀ1.2 Hz), 6.98 (2H, d, Jꢀ8.8 Hz), 7.02 (1H, td,
Calcd for C29H31N3O6S·1.5H2O: C, 60.40; H, 5.94; N, 7.29. Found: C, Jꢀ7.6, 1.2 Hz), 7.14—7.20 (2H, m), 7.57—7.61 (1H, m), 7.93 (2H, d,
60.15; H, 5.82; N, 6.90.
Jꢀ8.8 Hz), 7.96 (1H, d, Jꢀ8.0 Hz); ESI-MS m/z: 548 (Mꢄꢄ1).
To solution of methyl 4-[(3R)-hydroxy-1-[3-methoxy-4-[N-(2-
4-[1-[3-Methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)-
pyrrolidinyl]methylsulfonylbenzoic Acid (9g) By the procedure de-
scribed for compound 9f using 2.2 eq of mCPBA, compound 9g was
a
methylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinyl]methoxybenzoate
(214 mg, 0.39 mmol) in THF (4 ml) was added 0.25 M NaOH (2.00 ml,
obtained as colorless powder (128 mg, 41%) recrystallized from n- 0.50 mmol) at room temperature. After being stirred for 24 h, the reaction
hexane/EtOAc. IR (KBr) 3388, 2974, 1537, 1298, 1155 cmꢃ1 1H-NMR
;
mixture was poured into 1 M HCl and extracted with CHCl3. The combined
(DMSO-d6) d: 1.80—1.98 (4H, m), 2.24 (3H, s), 2.54 (1H, s), 3.20—3.70 extracts were washed with brine, dried over Na2SO4, and concentrated to af-
(5H, m), 3.82 (3H, s), 4.14—4.18 (1H, m), 6.67 (1H, dd, Jꢀ1.5, 8.3 Hz), ford the title compound 12p (160 mg, 77%) as a colorless amorphous solid.
6.80 (1H, d, Jꢀ1.5 Hz), 6.93 (1H, d, Jꢀ7.3 Hz), 7.10—7.16 (2H, m), 7.78 IR (KBr) 3345, 2954, 1685, 1604, 1536, 1454, 1417 cmꢃ1 1H-NMR
;
(1H, d, Jꢀ7.3 Hz), 7.95 (2H, d, Jꢀ8.3 Hz), 7.98 (1H, d, Jꢀ8.3 Hz), 8.14
(2H, d, Jꢀ8.3 Hz), 8.49 (1H, s), 8.57 (1H, s); FAB-MS m/z: 566 (Mꢄꢄ1); 3.54—3.66 (4H, m), 3.82 (3H, s), 3.90 (1H, dd, Jꢀ9.8, 8.3 Hz), 4.08 (1H,
FAB-MS (HR) m/z: 566.1937 (Calcd for C29H32N3O7S: 566.1961). dd, Jꢀ7.8, 1.9 Hz), 4.16 (1H, dd, Jꢀ10.0, 3.2 Hz), 4.21—4.25 (1H, m), 5.10
(DMSO-d6) d: 1.83—1.87 (1H, m), 2.18—2.22 (1H, m), 2.24 (3H, s),
Methyl 4-[(3R,4S)-Isopropylidenedioxy-1-[4-[N-(2-methylphenyl)ure- (1H, d, Jꢀ3.1 Hz), 6.74 (1H, dd, Jꢀ8.3, 1.7 Hz), 6.87 (1H, d, Jꢀ1.7 Hz),
ido]-3-methoxyphenylacetyl]-(2R)-pyrrolidinyl]methoxybenzoate (13m) 6.92 (1H, t, Jꢀ7.4 Hz), 7.06 (2H, d, Jꢀ8.8 Hz), 7.10—7.16 (2H, m), 7.78
as an Intermediate of 12m Colorless amorphous solid (yield, 100%); IR
(1H, d, Jꢀ7.8 Hz), 7.86 (2H, d, Jꢀ8.8 Hz), 8.00 (1H, d, Jꢀ8.3 Hz), 8.46
(KBr) 3354, 2985, 2939, 1716, 1533, 1254 cmꢃ1; 1H-NMR (CDCl3) d: 1.31
(1H, s), 8.55 (1H, s), 12.60 (1H, s); ESI-MS m/z: 534 (Mꢄꢄ1); Anal. Calcd
(3H, s), 1.42 (3H, s), 2.05 (3H, s), 3.50 (3H, s), 3.55—3.88 (4H, m), 3.89 for C29H31N3O7·1.25H2O: C, 62.64; H, 6.07; N, 7.56. Found: C, 62.66; H,
(3H, s), 4.11—4.15 (1H, m), 4.67 (1H, s), 4.78 (1H, d, Jꢀ6.1 Hz), 4.88 (1H,
t, Jꢀ5.6 Hz), 6.46 (1H, s), 6.62 (1H, d, Jꢀ1.5 Hz), 6.72—6.76 (3H, m), 7.05
5.93; N, 7.17.
3-Amino-4-[1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-
(1H, s), 7.14 (1H, d, Jꢀ7.3 Hz), 7.23 (2H, m), 7.57 (1H, d, Jꢀ7.8 Hz), (2S)-pyrrolidinyl]methoxybenzoic Acid (15h) A suspension of 4-[1-[3-
7.91—8.08 (3H, m); ESI-MS m/z: 604 (Mꢄꢄ1); Anal. Calcd for methoxy-4-[N-(2-methylphenyl)ureido]phenylacethyl]-(2S)-pyrrolidinyl-
C33H37N3O8·0.6H2O: C, 64.50; H, 6.27; N, 6.84. Found: C, 64.38; H, 6.18; methoxy]-3-nitrobenzoic acid (15g) (101 mg, 0.190 mmol) and 5 % Pd/C
N, 6.66.
4-[(3R,4S)-Dihydroxy-1-[4-[N-(2-methylphenyl)ureido]-3-me- sphere at room temperature for 21 h. After the catalyst was removed by fil-
thoxyphenylacetyl]-(2R)-pyrrolidinyl]methoxybenzoic Acid (12o) tration, the filtrate was concentrated to dryness. The residue was purified by
(247 mg) in MeOH (5 ml) was hydrogenated under 1 atom hydrogen atmo-
A
mixture of methyl 4-[(3R,4S)-isopropylidenedioxy-1-[4-[Nꢆ-(2-methyl- column chromatography on silica gel with CHCl3/EtOH (1/1, v/v) as an elu-
phenyl)ureido]-3-methoxyphenylacetyl]-(2R)-pyrrolidinyl]methoxybenzo- ent to give compound 15h (61 mg, 60%) as pale brown crystalline material.
1
ate (13m) (183 mg, 0.303 mmol) and gas.HCl–MeOH (6 ml) was stirred IR (ATR) 3346, 2972, 2941, 2875, 1678, 1589, 1529, 1450 cmꢃ1; H-NMR
at room temperature for 17 h. The mixture was concentrated in vacuo. The (DMSO-d6) d: 1.91—1.99 (4H, m), 2.23 (3H, s), 3.60—4.34 (5H, m), 3.81
residue was purified on TLC [CHCl3/MeOH (10/1, v/v)] to afford meth- (3H, s), 4.88 (2H, m), 6.74 (1H, d, Jꢀ8.3 Hz), 6.86—7.28 (5H, m), 7.78
yl 4-[(3R,4S)-dihydroxy-1-[4-[N-(2-methylphenyl)ureido]-3-methoxy- (1H, d, Jꢀ7.8 Hz), 7.99 (1H, d, Jꢀ8.3 Hz), 8.30 (1H, s), 8.45 and 8.55 (total
phenylacetyl]-(2R)-pyrrolidinyl]methoxybenzoate (162 mg, 95%) as a color- 1H, each s, amide isomers); FAB-MS m/z: 533; FAB-MS (HR) m/z:
less amorphous solid. IR (KBr) 3342, 1716, 1604, 1535, 1255 cmꢃ1
;
1H-
533.3467 (Calcd for C29H33N4O6: 533.3400).
NMR (CDCl3) d: 2.25 (3H, s), 3.33— 3.75 (7H, m), 3.87 (3H, s), 4.10 (1H,
d, Jꢀ8.3 Hz), 4.24 (2H, s), 4.35—4.39 (2H, m), 6.62—7.94 (13H, m); ESI-
MS m/z: 564 (Mꢄꢄ1).
3-Acetylamino-4-[1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenyl-
acetyl]-(2S)-pyrrolidinyl]methoxybenzoic Acid (15i) A solution of 3-
amino-4-[1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)-
pyrrolidinyl]methoxybenzoic acid (15h) (130 mg, 0.244 mmol) and DMAP
To a solution of methyl 4-[(3R,4S)-dihydroxy-1-[4-[N-(2-methylphen-
yl)ureido]-3-methoxyphenylacetyl]-(2R)-pyrrolidinyl]methoxybenzoate (2.9 mg, 0.024 mmol) in pyridine/acetic anhydride (5/5 ml) was stirred at
(63 mg, 0.112 mmol) in THF (0.89 ml) was added 0.25 M NaOH (0.89 ml).
After stirring at room temperature for 3 d, the mixture was acidified with 1 M
room temperature for 2 h. The mixture was poured into water and extracted
with CHCl3. The combined extracts were dried over Na2SO4 and evaporated
HCl and extracted with CHCl3/MeOH (10/1, v/v). The combined extracts in vacuo. The residue was purified by chromatography on silica gel with
were dried over Na2SO4 and concentrated in vacuo, to give compound 12o CHCl3/MeOH (15/1 to 1/1, v/v) as an eluent to afford compound 15i (29 mg,
(54 mg, 88%) as a colorless amorphous solid. IR (KBr) 3356, 2958, 2927, 21%) as white crystalline material. IR (ATR) 3346, 2924, 2852, 1685, 1589,
1685, 1604, 1535, 1255 cmꢃ1
;
1H-NMR (DMSO-d6) d: 2.24 (3H, s),
1529, 1485, 1450, 1415 cmꢃ1; 1H-NMR (DMSO-d6) d: 1.80—2.30 (4H, m),
2.04 (3H, s), 2.26 (3H, s), 3.33 (3H, s), 3.40—4.80 (7H, m), 6.59 (1H, s),
6.74 (1H, d, Jꢀ8.3 Hz), 6.79 (1H, d, Jꢀ8.8 Hz), 7.07—7.57 (6H, m), 7.75
(1H, d, Jꢀ8.8 Hz), 8.07 (1H, d, Jꢀ8.3 Hz), 8.41 and 8.96 (total 1H, each s,
amide isomers); FAB-MS m/z: 575; FAB-MS (HR) m/z: 575.2532 (Calcd for
3.438—3.42 (1H, m), 3.58 (2H, s), 3.66 (1H, dd, Jꢀ9.8, 6.6 Hz), 3.80 (3H,
s), 3.99—4.30 (5H, m), 5.10 (1H, s), 6.72 (1H, d, Jꢀ8.1 Hz), 6.85 (1H, s),
6.93 (1H, t, Jꢀ7.3 Hz), 7.03 (2H, d, Jꢀ8.8 Hz), 7.14 (2H, t, Jꢀ8.8 Hz), 7.79
(2H, d, Jꢀ8.1 Hz), 7.86 (2H, d, Jꢀ8.8 Hz), 7.99 (1H, d, Jꢀ8.3 Hz), 8.46
(1H, s), 8.56 (1H, s); FAB-MS m/z: 550 (Mꢄꢄ1); FAB-MS (HR) m/z: C31H35N4O7: 575.2506).

1524
3-N,N-Dimethylamino-4-[1-[3-methoxy-4-[N-(2-methylphenyl)ure-
Vol. 54, No. 11
1525, 1483 cmꢃ1; 1H-NMR (DMSO-d6) d: 1.90—5.06 (19H, m), 6.71—7.21
(9H, m), 7.78—7.80 (1H, m), 8.00—8.02 (1H, m), 8.46 (1H, s), 8.55 (1H,
s), 12.36 (1H, s); FAB-MS m/z: 547 (Mꢄꢄ1); FAB-MS (HR) m/z: 547.2572
(Calcd for C30H35N4O6: 547.2557).
ido]phenylacetyl]-(2S)-pyrrolidinyl]methoxybenzoic Acid (15j) To a so-
lution of 3-amino-4-[1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenyl-
acetyl]-(2S)-pyrrolidinyl]methoxybenzoic acid (15h) (67 mg, 0.126 mmol) in
MeOH (1.0 ml)/HOAc (0.5 ml), 37% aq. HCHO solution (1.0 ml) and
NaBH3CN (118 mg, 1.88 mmol) was added at room temperature and stirred
4 d. The reaction mixture was diluted with water and extracted with
CHCl3/MeOH (10/1, v/v). The combined extracts were washed with sat.
NaCl, dried over Na2SO4, and evaporated in vacuo. The residue was purified
by TLC with CHCl3/MeOH (5/1, v/v) to afford compound 15j (57 mg, 81%)
as pale yellow amorphous solid. IR (ATR) 3340, 2943, 2873, 2829, 2781,
Methyl 4-[1-(tert-Butoxycarbonyl)-(2S)-pyrrolidinyl]methylaminoben-
zoate (18) A mixture of methyl 4-aminobenzoate (1.52 g, 10.04 mmol) and
N-Boc-L-prolinal (17) (3.00 g, 15.1 mmol) in toluene (30 ml) was refluxed
with stirring for 3 h. The reaction mixture was cooled to room temperature
and dried over Na2SO4. After removing the solvent under reduced pressure,
the resulting precipitate was diluted with MeOH/HOAc (27/3 ml), then
NaBH3CN (1.33 g, 20.1 mmol) was added and the reaction mixture was
stirred at room temperature for 12 h. The reaction mixture was poured into
water and extracted with EtOAc. The combined extracts were washed with
water and brine, dried over Na2SO4. The solvent was removed under reduced
pressure, and the residue was purified by chromatography on silica gel with
n-hexane/EtOAc (3/1, v/v) as an eluent to afford compound 18 (2.17 g, 65%)
as a pale yellow oil. 1H-NMR (CDCl3) d: 1.48 (9H, s), 1.51—2.09 (4H, m),
3.05—3.07 and 3.43—3.48 (total 1H, m, amide isomers), 3.18 (1H, s), 3.36
(2H, s), 3.84 (3H, s), 4.06—4.24 (1H, m), 6.49—6.65 (2H, m), 7.84 (2H, d,
Jꢀ8.3 Hz); FAB-MS m/z: 335 (Mꢄꢄ1).
Methyl 4-[(2S)-Pyrrolidinyl]methylaminobenzoate (8a) (General Pro-
cedure B, Boc Removal Procedure) To a stirred solution of methyl 4-[1-
(tert-butoxycarbonyl)-(2S)-pyrrolidinyl]methylamino- benzoate (18) (2.17 g,
6.49 mmol) in CH2Cl2 (44 ml) was added TFA (9 ml) at 0 °C and stirred at
room temperature for 12 h. After the solvent was removed, the residue was
diluted with 1 M NaOH and extracted with CH2Cl2. The combined extracts
were washed with brine, dried over Na2SO4, and concentrated under reduced
pressure to afford compound 8a (1.34 g, 88%) as a brown oil. 1H-NMR
(CDCl3) d: 1.45—1.50 (1H, m), 1.70—1.94 (3H, m), 2.93 (2H, t, Jꢀ
6.8 Hz), 2.96—3.02 (1H, m), 3.19—3.23 (1H, m), 3.37—3.41 (1H, m), 3.84
(3H, s), 4.73 (1H, s), 6.56 (2H, d, Jꢀ8.8 Hz), 7.84 (2H, d, Jꢀ8.8 Hz); FAB-
MS m/z: 235 (Mꢄꢄ1).
Ethyl 4-[2-[1-(tert-Butoxycarbonyl)-(2S)-pyrrolidinyl]-(E)-ethenyl]-
benzoate (19) To a solution of ethyl 4-(diethoxyphosphorylmethyl)ben-
zoate26) (904 mg, 3.01 mmol) in THF (20 ml) was added LiHMDS (1.0 M in
THF, 3 ml, 3.00 mmol) at ꢃ78 °C. After 1 h stirring, N-Boc-L-prolinal (17)
(500 mg, 2.51 mmol) in THF (10 ml) was added to the reaction mixture, and
allowed to warm up to room temperature over 1 h. After being stirred for 2 h,
the mixture was poured into water and extracted with EtOAc. The combined
extracts were washed with brine, dried over MgSO4, and evaporated. The
residue was purified by column chromatography on silica gel with
n-hexane/EtOAc (8/1, v/v) as an eluent to give compound 19 (713 mg, 82%)
as a colorless crystalline solid. mp 68—70 °C; IR (KBr) 1710, 1697,
1681 cmꢃ1; 1H-NMR (CDCl3) d: 1.39 (12H, m), 1.77—1.93 (3H, m), 2.09—
2.13 (1H, m), 3.45—3.49 (2H, m), 4.34—4.54 (3H, m), 6.20—6.24 (1H, m),
6.43 (1H, d, Jꢀ14.2 Hz), 7.39 (2H, d, Jꢀ8.3 Hz), 7.97 (2H, d, Jꢀ8.3 Hz);
FAB-MS m/z: 346 (Mꢄꢄ1); Anal. Calcd for C20H27NO4: C, 69.54; H, 7.88;
N, 4.05. Found: C, 69.52; H, 8.08; N, 4.07.
1701, 1678, 1589, 1529, 1452 cmꢃ1 1H-NMR (DMSO-d6) d: 1.87—2.14
;
(4H, m), 2.24—2.31 (4H, m), 2.67 (6H, s), 3.45—3.67 (4H, m), 3.72—3.87
(4H, m), 3.98—4.52 (2H, m), 6.66—7.20 (6H, m), 7.42 (1H, d, Jꢀ2.0 Hz),
7.46—7.56 (1H, m), 7.79 (1H, d, Jꢀ7.4 Hz), 7.99 (1H, d, Jꢀ8.1 Hz), 8.46
(1H, s), 8.55 (1H, s); FAB-MS m/z: 561 (Mꢄꢄ1); FAB-MS (HR) m/z:
561.2740 (Calcd for C31H37N4O6: 561.2713).
4-[1-[3-Methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)-
pyrrolidinyl]methoxy-3-methylaminobenzoic Acid (15k) A mixture of
methyl 4-[1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)-
pyrrolidinyl]methoxy-3-nitrobenzoate (16) (5.8 g, 10.1 mmol) and SnCl·
2H2O (8.2 g, 36.3 mmol) in EtOH (80 ml) was refluxed for 1 h. After cooled
to room temperature, the reaction mixture was concentrated. The residue
was poured into 1 M NaOH at 0 °C, and extracted with CHCl3. The com-
bined extracts were washed with sat. NaCl and dried over Na2SO4. After the
solvent was removed, the resulting residue was purified by chromatography
on silica gel with n-hexane/EtOAc (1/1, v/v) as an eluent to afford methyl
3-amino-4-[1-[3-methoxy-4-[N-(2-methylphenyl)ureido]phenylacetyl]-(2S)-
pyrrolidinyl]methoxybenzoate (3.8 g, 69%) as colorless amorphous solid.
IR (KBr) 3458, 3338, 2949, 2877, 2843, 1711, 1620, 1595, 1533, 1485
cmꢃ1 1H-NMR (CDCl3) d: 1.99—2.06 (4H, m), 2.30 (3H, s), 3.46—3.52
;
(3H, m), 3.55 (3H, s), 3.85 (3H, s), 4.14—4.17 (2H, m), 4.50—4.54 (1H,
m), 6.63 (1H, s), 6.76 (1H, s), 6.79 (1H, d, Jꢀ2.0 Hz), 6.80 (1H, d,
Jꢀ8.3 Hz), 7.13 (1H, t, Jꢀ7.8 Hz), 7.18—7.25 (3H, m), 7.33 (1H, d,
Jꢀ2.0 Hz), 7.39 (1H, dd, Jꢀ8.3, 2.0 Hz), 7.56 (1H, d, Jꢀ8.8 Hz), 8.02 (1H,
d, Jꢀ7.8 Hz); FAB-MS m/z: 547 (Mꢄꢄ1).
To a solution of 3-amino-4-[1-[3-methoxy-4-[N-(2-methylphenyl)ureido]-
phenylacetyl]-(2S)-pyrrolidinyl]methoxybenzoate (350 mg, 0.64 mmol) and
Et3N (0.134 ml, 0.96 mmol) in CH2Cl2 (15 ml) was added trifluoroacetic acid
anhydride (0.109 ml, 0.77 ml) at 0 °C. After 1.5 h stirring, the reaction mix-
ture was poured into water and extracted with CHCl3. The combined extracts
were washed with water, dried over MgSO4, and concentrated in vacuo. The
residue was purified by chromatography on silica gel with MeOH/CHCl3
(1/100, v/v) as an eluent to afford methyl 4-[1-[3-methoxy-4-[N-(2-
methylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinyl]methoxy-3-trifluo-
1
roacetylaminobenzoate (302 mg, 74%) as pale orange amorphous solid. H-
NMR (CDCl3) d: 1.97—2.17 (3H, m), 2.30 (3H, s), 3.46—3.60 (8H, m),
3.89 (3H, s), 4.17—4.28 (2H, m), 4.54—4.56 (1H, m), 6.41 (1H, s), 6.73—
6.75 (2H, m), 7.00—7.02 (1H, m), 7.08 (1H, s), 7.12—7.16 (1H, m), 7.22—
7.24 (2H, m), 8.50—8.52 (1H, m), 7.86—7.89 (1H, m), 7.97—7.99 (1H, m),
8.74 (1H, s), 8.80 (1H, s).
Ethyl 4-[2-(2-Pyrrolidinyl)-(E)-ethenyl]benzoate (8b) According to
the general procedure B, 8b was obtained from 19 as a brown oil (yield,
87%). 1H-NMR (CDCl3) d: 1.39 (3H, t, Jꢀ7.3 Hz), 1.52—2.06 (4H, m),
2.93—2.99 (1H, m), 3.07—3.13 (1H, m), 3.74 (1H, q, Jꢀ7.3 Hz), 4.37 (2H,
q, Jꢀ7.3 Hz), 6.34 (1H, dd, Jꢀ15.6, 7.3 Hz), 6.54 (1H, d, Jꢀ15.6 Hz), 7.41
(2H, d, Jꢀ8.3 Hz), 7.97 (2H, d, Jꢀ8.3 Hz).
To
a
solution of methyl 4-[1-[3-methoxy-4-[N-(2-methylphenyl)-
ureido]phenylacetyl]-(2S)-pyrrolidinylmethoxy]-3-trifluoroacetylamino-
benzoate (289 mg, 0.45 mmol) and K2CO3 94 mg (0.68 mmol) in DMF
(10 ml) was added MeI (34 ml, 0.78 mmol) at room temperature. After
12 h stirring, the reaction mixture was poured into 1 M HCl and resulting
solid were collected the resulting solid, which were diluted with CHCl3 and
dried over MgSO4. After the solvent was removed, the residue was purified
by chromatography on silica gel with MeOH/CHCl3 (1/100, v/v) as an
eluent to afford methyl 4-[1-[3-methoxy-4-[N-(2-methylphenyl)ureido]-
phenylacetyl]-(2S)-pyrrolidinylmethoxy]-3-N-trifluoroacetyl-N-methyl-
aminobenzoate (229 mg, 78%) as pale brown amorphous solid. 1H-NMR
(CDCl3) d: 1.92—2.07 (4H, m), 2.29 (3H, s), 3.09—3.71 (10H, m), 3.90
(3H, s), 4.14—4.41 (3H, m), 6.36 (1H, s), 6.77—6.79 (2H, m), 7.06—7.25
(5H, m), 7.48—7.51 (1H, m), 7.84—7.90 (1H, m), 8.04—8.06 (2H, m).
To a stirred solution of methyl 4-[1-(3-methoxy-4-(N-(2-methylphenyl)-
ureido)phenylacetyl)-(2S)-pyrrolidinylmethoxy]-3-N-trifluoroacetyl-N-
methylaminobenzoate (223 mg, 0.34 mmol) in THF (5 ml) was added 0.25 M
NaOH (4 ml). The resulting mixture was stirred at room temperature for
12 h. The mixture was acidified with 1 M HCl and collected the resulting
solid. The resulting solid was diluted with CHCl3 and dried over MgSO4.
After the solvent was removed, the residue was recrystallized with CHCl3/n-
hexane to afford compound 15k (51 mg, 28%) as a colorless crystalline ma-
terial. mp 136—140 °C; IR (ATR) 3346, 2947, 2877, 1701, 1678, 1597,
Ethyl 4-[2-[1-(tert-Butoxycarbonyl)-(2S)-pyrrolidinyl]ethynyl]phenyl-
acetate (21)
A stirred suspension of ethyl 4-iodobenzoate (1.7 ml,
10 mmol), Pd(PPh3)4 (578 mg, 0.5 mmol), and CuI (190 mmol, 1 mmol) in
i-Pr2NH (20 ml) was added a solution of 1-(tert-butoxycarbonyl)-(2S)-
ethynylpyrrolidine (20)27) (1.95 g, 10 mmol) in i-Pr2NH (20 ml) over 10 min
at room temperature under an atmosphere of nitrogen. After 3 h stirring, the
mixture was poured into water and extracted with EtOAc. The combined ex-
tracts were washed with brine, dried over MgSO4, and evaporated. The
residue was purified column chromatography on silica gel with n-
hexane/EtOAc (10/1, v/v) as an eluent to give compound 21 (2.77 g, 81%) as
a colorless oil. 1H-NMR (CDCl3) d: 1.37 (3H, t, Jꢀ6.8 Hz), 1.49 (9H, s),
1.85—2.12 (4H, m), 3.37—3.51 (2H, m), 4.37 (2H, q, Jꢀ6.8 Hz), 4.54—
4.77 (1H, m), 7.44 (2H, d, Jꢀ7.8 Hz), 7.96 (2H, d, Jꢀ7.8 Hz).
Ethyl 4-[2-[(2S)-Pyrrolidinyl]ethynyl]phenylacetate (8c) According
to the general procedure B, 8c was obtained from 21 as a light yellow oil
(yield, 100%). 1H-NMR (CDCl3) d: 1.38 (3H, t, Jꢀ6.8 Hz), 1.82—2.16 (4H,
m), 3.01—3.48 (2H, m), 4.00—4.11 (1H, m), 4.37 (2H, q, Jꢀ6.8 Hz),
4.54—4.77 (1H, m), 7.44—7.46 (2H, m), 7.95—7.97 (2H, m).
1-(tert-Butoxycarbonyl)-(2S)-pyrrolidinylmethyl 4-Iodophenyl Sulfide
(24) To a stirred mixture of p-iodothiophenol29) (1.75 g, 7.43 mmol) and 1-

November 2006
1525
(tert-butoxy)-(2S)-tosyloxymethylpyrrolidine (21)28) (2.39 g, 6.75 mmol) in
pyridine (12.7 ml) was added 8 N KOH (1.27 ml) at room temperature. After
4 h stirring, the reaction mixture was diluted with EtOAc. The solution was
washed with H2O, sat. NH4Cl, brine, and dried over Na2SO4. The organic
layer was concentrated under a reduced pressure. The residue was purified
column chromatography on silica gel with n-hexane/EtOAc (5/1, v/v) as an
Jꢀ9.0 Hz); FAB-MS m/z: 294 (Mꢄꢄ1).
Methyl 4-[1-tert-Butoxycarbonyl-(4S)-(2-naphthyloxy)-(2S)-pyrro-
lidinyl]methoxybenzoate (27f) Colorless oil (yield, 93%); 1H-NMR
(CDCl3) d: 1.49 and 1.51 (total 9H, each s, amide isomers), 2.32—2.36 (1H,
m), 2.53 (1H, d, Jꢀ14.2 Hz), 3.72—3.85 (1H, m), 3.86 and 3.87 (total 3H,
each s, amide isomers), 4.15—4.19 (1H, m), 4.26—4.52 (2H, m), 5.06 (1H,
s), 6.87 (1H, d, Jꢀ8.8 Hz), 6.94 (2H, d, Jꢀ8.8 Hz), 7.04 (2H, s), 7.33 (1H, t,
Jꢀ7.3 Hz), 7.42 (1H, t, Jꢀ7.3 Hz), 7.64—8.02 (5H, m).
1
eluent to afford compound 24 (1.49 g, 53%) as a pale yellow oil. H-NMR
(CDCl3) d: 1.45 (9H, s), 1.78—2.01 (4H, m), 2.69—2.73 (1H, m), 3.32—
3.49 (3H, m), 3.90—4.02 (1H, m), 7.12 (1H, d, Jꢀ7.8 Hz), 7.18 (1H, d,
Jꢀ7.8 Hz), 7.57 (2H, dd, Jꢀ2.0, 8.3 Hz); FAB-MS m/z: 420 (Mꢄꢄ1).
Methyl 4-[(4S)-(2-Naphthyloxy)-(2S)-pyrrolidinyl]methoxybenzoate
1
(11f) Black oil (yield, 100%); H-NMR (CDCl3) d: 1.99 (1H, dd, Jꢀ14.2,
Methyl 4-[(2S)-Pyrrolidinyl]methylthiobenzoate (8e) To
a stirred
5.6 Hz), 2.46—2.50 (1H, m), 3.22 (1H, dd, Jꢀ12.2, 4.6 Hz), 3.43 (1H, d,
Jꢀ12.5 Hz), 3.65—3.69 (1H, m), 3.86 and 3.87 (total 3H, each s, amide iso-
mers), 4.09—4.13 (2H, m), 5.02—5.06 (1H, m), 6.83 (1H, d, Jꢀ8.5 Hz),
6.89 (2H, d, Jꢀ8.8 Hz), 7.07 (1H, d, Jꢀ2.0 Hz), 7.12 (1H, d, Jꢀ9.0 Hz),
7.33 (1H, dt, Jꢀ8.1, 1.2 Hz), 7.44 (1H, dt, Jꢀ6.8, 1.2 Hz), 7.70 (1H, d,
Jꢀ8.1 Hz), 7.75 (2H, dd, Jꢀ9.0, 5.1 Hz), 7.90 (1H, d, Jꢀ8.5 Hz), 7.96 (2H,
dd, Jꢀ6.8, 2.0 Hz); ESI-MS m/z: 377 (Mꢄꢄ1).
mixture of 1-(tert-butoxycarbonyl)-(2S)-pyrrolidinylmethyl 4-iodophenyl
sulfide (24) (1.49 g, 3.56 mmol), Et3N (1.09 ml, 7.84 mmol), Pd(OAc)2
(40 mg, 0.178 mmol), and 1,3-bis(diphenylphosphino)propane (73.4 mg,
0.178 mmol) in DMSO/MeOH (16/13 ml) was induced CO gas for 5 min,
and stirred at 70 °C for 12 h. After cooling to room temperature, the mixture
was concentrated to a small volume. The resulting residue was diluted with
EtOAc, washed with brine, and then dried over Na2SO4. After the solvent
was removed, the residue was purified column chromatography on silica gel
with n-hexane/EtOAc (5/1, v/v) as an eluent to afford methyl 4-[1-(tert-bu-
toxycarbonyl)-(2S)-pyrrolidinyl]methylthiobenzoate (25) (1.16 g, 93%) as a
yellow oil.
To a stirred solution of methyl 4-[1-(tert-butoxycarbonyl)-(2S)-pyrro-
lidinyl]methylthiobenzoate (25) (1.16 g, 3.32 mmol) in CH2Cl2 (20 ml) was
added TFA (4 ml), and the mixture was stirred at room temperature for 1.5 h.
The solvent was removed under a reduced pressure and the residue was
treated with 1 M NaOH. The mixture was extracted with CHCl3. The extract
was washed with brine, dried over KOH, and concentrated under reduced
pressure to afford compound 8e (767 mg, 92%) as a yellow oil. 1H-NMR
(CDCl3) d: 1.69 (1H, dt, Jꢀ3.9, 12.7 Hz), 1.85—2.09 (2H, m) 2.11—2.15
(1H, m), 3.11—3.27 (3H, m), 3.40 (1H, dd, Jꢀ6.8, 13.2 Hz), 3.54 (1H, dd,
Jꢀ7.3, 15.1 Hz), 3.89 (3H, s), 5.07 (1H, s), 7.38 (2H, d, Jꢀ8.3 Hz), 7.91
(2H, d, Jꢀ8.3 Hz); FAB-MS m/z: 252 (Mꢄꢄ1).
Methyl 4-[1-(tert-Butoxycarbonyl)-(4R)-fluoro-(2S)-pyrrolidinyl]me-
thoxybenzoate (27g) Colorless crystalline powder (yield, 65%); mp 122—
1
124 °C; H-NMR (CDCl3) d: 1.49 (9H, s), 2.21—2.44 (2H, m), 3.39—3.69
(1H, m), 3.88 (3H, s), 4.00—4.36 (4H, m), 5.16—5.29 (1H, m), 6.91 (2H, d,
Jꢀ8.3 Hz), 7.97 (2H, d, Jꢀ8.3 Hz); FAB-MS m/z:, 354 (Mꢄꢄ1); Anal.
Calcd for C18H24FNO5: C, 61.18; H, 6.85; N, 3.96. Found: C, 60.90; H, 6.89;
N, 3.98.
Methyl 4-[(4R)-Fluoro-(2S)-pyrrolidinyl]methoxybenzoate (11g) Pale
yellow crystalline powder (yield, 67%); mp 78—80 °C; 1H-NMR (CDCl3) d:
1.71—1.88 (1H, m), 2.24—2.34 (1H, m), 3.06—3.32 (2H, m), 3.81—4.00
(6H, m), 5.20—5.33 (1H, m), 6.91 (2H, d, Jꢀ8.8 Hz), 7.98 (2H, d,
Jꢀ8.8 Hz); FAB-MS m/z:, 254 (Mꢄꢄ1); Anal. Calcd for C13H16FNO3: C,
61.65; H, 6.37; N, 5.53. Found: C, 61.49; H, 6.45; N, 5.65.
Methyl 4-[1-(tert-Butoxycarbonyl)-(4S)-fluoro-(2S)-pyrrolidinyl]me-
thoxybenzoate (27h) Colorless oil (yield, 74%); 1H-NMR (CDCl3) d:
1.49—1.59 (9H, m), 2.05—2.21 (1H, m), 2.45—2.53 (1H, m), 3.56—4.43
(8H, m), 5.19—5.32 (1H, m), 6.95—6.99 (2H, m), 7.98 (2H, d, Jꢀ8.5 Hz),
ESI-MS m/z:, 354 (Mꢄꢄ1).
Methyl 4-[(4S)-Fluoro-(2S)-pyrrolidinyl]methoxybenzoate (11h) Ye l -
low solid (yield, 98%); 1H-NMR (CDCl3) d: 1.89—2.02 (1H, m), 2.16—
2.31 (1H, m), 2.98 (1H, ddd, Jꢀ35.3, 13.0, 3.7 Hz), 3.35 (1H, dd, Jꢀ21.1,
13.0 Hz), 3.46—3.68 (1H, m), 3.86 (3H, s), 4.00—4.08 (2H, m), 5.16 and
5.29 (total 1H, each t, each Jꢀ4.7 Hz), 6.91 (2H, d, Jꢀ8.8 Hz), 7.96 (2H, d,
Jꢀ8.8 Hz); ESI-MS m/z: 254 (Mꢄꢄ1).
Methyl 4-[1-(tert-Butoxycarbonyl)-4,4-difluoro-(2S)-pyrrolidinyl]me-
thoxybenzoate (27i) Colorless oil (yield, 88%); 1H-NMR (CDCl3) d: 1.48
(9H, s), 2.53—2.61 (2H, m), 3.63—4.41 (8H, m), 6.94 (2H, d, Jꢀ8.8 Hz),
7.99 (2H, d, Jꢀ8.8 Hz); ESI-MS m/z: 389 (Mꢄꢄ18).
Preparation of Methyl 4-[1-(tert-Butoxycarbonyl)-(2S)-pyrrolidinyl]-
methoxybenzoate (11a) (General procedure C). N-Boc-L-prolinol (22)
To a stirred solution of N-Boc-L-proline (26a) (20.0 g, 92.9 mmol) in THF
(500 ml) was added BH3·DMS (9.7 ml, 102 mmol) and the mixture was re-
fluxed for 1 h with stirring. After cooling to room temperature, the mixture
was concentrated. The resulting residue was diluted with water and extracted
with CH2Cl2. The combined extracts were washed with sat. NaHCO3 and
brine, which was dried over Na2SO4 and evaporated to afford compound 22
(15.6 g, 83%) as a colorless crystalline powder. N-Boc-L-prolinol (22) is
1
commercially available. H-NMR (CDCl3) d: 1.47 (9H, s), 1.53—1.57 (1H,
m), 1.79—1.83 (2H, m), 1.98—2.02 (1H, m), 3.30—3.34 (1H, m), 3.42—
3.46 (1H, m), 3.51—3.63 (2H, m), 3.94—3.98 (1H, m), 4.72—4.76 (1H, m).
Methyl 4-[1-(tert-Butoxycarbonyl)-(2S)-pyrrolidinyl]methoxybenzoate
Methyl 4-(4,4-Difluoro-(2S)-pyrrolidinylmethoxy)benzoate (11i) Pale
yellow solid (yield, 91%); 1H-NMR (CDCl3) d: 2.17—2.22 (1H, m), 2.41—
2.45 (1H, m), 3.19—3.41 (2H, m), 3.75—3.79 (1H, m), 3.89 (3H, s), 4.00—
4.09 (2H, m), 6.92 (2H, d, Jꢀ9.0 Hz), 7.99 (2H, d, Jꢀ9.0 Hz); FAB-MS
m/z:, 272 (Mꢄꢄ1); Anal. Calcd for C13H15F2NO3: C, 57.56; H, 5.57; N,
5.16. Found: C, 57.65; H, 5.67; N, 5.16.
(27a) To
a stirred solution of methyl 4-hydroxybenzoate (1.96 g,
12.9 mmol), N-Boc-L-prolinol (22) (2.59 g, 12.9 mmol) and Ph3P (4.06 g,
15.5 mmol) in THF (40 ml) was added diisopropyl azodicarboxylate
(3.10 ml, 15.7 mmol) and the reaction mixture was heated under reflux for
14 h. After cooling to room temperature, the mixture was concentrated under
reduced pressure. The resulting residue was purified by column chromatog-
raphy on silica gel with n-hexane/EtOAc (6/1, v/v) as an eluent to afford
1-(tert-Butoxycarbonyl)-(4R)-chloro-(2S)-pyrrolidinylcarboxylic Acid
(26j) To a stirred solution of methyl 1-(tert-butoxycarbonyl)-(4S)-hy-
droxy-(2S)-pyrrolidinylcarboxylate (1.81 g, 7.34 mmol) in CCl4/CH2Cl2
(10/10 ml) was added Ph3P (3.87 mmol, 14.8 mmol) and the reaction mixture
was stirred at room temperature for 2 h. To the mixture was added EtOH
(5 ml) and the reaction mixture was stirred at room temperature overnight.
After removal of the solvent, the residue was purified by column chromatog-
raphy on silica gel with n-hexane/EtOAc (3/1, v/v) as an eluent to afford
methyl 1-(tert-butoxycarbonyl)-(4R)-chloro-(2S)-pyrrolidinylcarboxylate
1
compound (27a) (3.34 g, 77%) as a colorless oil. H-NMR (CDCl3) d: 1.48
(9H, s), 1.67 (1H, d, Jꢀ9.3 Hz), 1.87—2.03 (3H, m), 3.36—3.43 (2H, m),
3.82—4.09 (4H, m), 4.13—4.20 (2H, m), 6.94 (2H, d, Jꢀ8.3 Hz), 7.98 (2H,
d, Jꢀ8.3 Hz); ESI-MS m/z: 336 (Mꢄꢄ1).
Methyl 4-[1-(tert-Butoxycarbonyl)-(2S)-pyrrolidinyl]methoxybenzoate
(11a) According to the general procedure B, 11a was obtained from 27a as
yellow oil (yield, 73%). 1H-NMR (CDCl3) d: 1.54—1.61 (1H, m), 1.77—
1.86 (2H, m), 1.87—1.97 (1H, m), 2.00 (1H, s), 2.93—3.06 (2H, m), 3.52—
3.57 (1H, m), 3.88 (3H, s), 3.90—3.99 (2H, m), 6.92 (2H, d, Jꢀ9.0 Hz),
7.98 (2H, d, Jꢀ9.0 Hz); ESI-MS m/z: 236 (Mꢄꢄ1)
1
(1.36 g, 70%) as a colorless oil. H-NMR (CDCl3) d: 1.42 (9H, s), 2.32—
2.39 (1H, m), 2.49—2.54 (1H, m), 3.66—3.92 (5H, m), 4.44—4.55 (2H, m);
FAB-MS m/z: 264 (Mꢄꢄ1).
The following compound 11b, 11f—j, and 11n were prepared from 26b,
26f—j, and 26n, respectively, according to general procedure C.
To a stirred solution of methyl 1-(tert-butoxycarbonyl)-(4R)-chloro-(2S)-
pyrrolidinylcarboxylate (1.35 g, 5.12 mmol) in THF (10 ml) was added 0.5 M
NaOH (10 ml) and heated under reflux for 1.5 h. After cooling to room tem-
perature, the mixture was poured into 1 M HCl and the mixture was extracted
with CHCl3/MeOH (9/1, v/v). The combined extracts were washed with
brine, dried over Na2SO4 and evaporated to afford compound (26j) (1.28 g,
quant.) as colorless oil. H-NMR (CDCl3) d: 1.44 (9H, s), 2.37—2.54 (2H,
m), 3.68—3.88 (2H, m), 4.42—4.45 (2H, m).
Methyl 4-[1-(tert-Butoxycarbonyl)-(4R)-chloro-(2S)-pyrrolidinyl]me-
thoxybenzoate (27j) Colorless solid (yield, 65%); mp 116—120 °C; 1H-
NMR (CDCl3) d: 1.47 (9H, s), 2.39—2.53 (2H, m), 3.69—4.17 (3H, m),
Methyl 4-(N-tert-Butoxycarbonyl-4-hydroxy-(2S)-pyrrolidinylmethoxy)-
benzoate (27b) Colorless oil (yield, 39%); 1H-NMR (CDCl3) d: 1.46 (9H,
s), 2.06 (3H, s), 2.14—2.41 (3H, m), 3.41—3.75 (2H, m), 3.88 (3H, s),
4.08—4.36 (2H, m), 5.30—5.36 (1H, m), 6.88—6.95 (2H, m), 7.97 (2H, d,
Jꢀ8.6 Hz); ESI–MS m/z: 394 (Mꢄꢄ1).
Methyl 4-[(4R)-Acetoxy-(2S)-pyrrolidinyl]methoxybenzoate (11b)
Brown oil (yield, 95%); 1H-NMR (CDCl3) d: 1.86—1.93 (1H, m), 2.00—
2.12 (5H, m), 3.03—3.29 (1H, m), 3.73—3.80 (1H, m), 3.88 (3H, s), 3.93—
4.01 (2H, m), 5.27—5.30 (1H, m), 6.91 (2H, d, Jꢀ9.0 Hz), 7.98 (2H, d,
1

1526
Vol. 54, No. 11
3.88 (3H, s), 4.30—4.41 (2H, m), 4.50—4.55 (1H, m), 6.90—6.92 (2H, m), thoxybenzoate (27e) To a stirred solution of methyl 4-[(4S)-acetoxy-1-
7.96—7.98 (2H, m); FAB-MS m/z: 370 (Mꢄꢄ1); Anal. Calcd for tert-butoxycarbonyl-(2S)-pyrrolidinyl]methoxybenzoate
(27c) (7.43 g,
C18H24ClNO5: C, 58.46; H, 6.54; Cl; 9.59; N, 3.79. Found: C, 58.35; H, 18.9 mmol) in MeOH (150 ml) was added cat. K2CO3 at room temperature.
6.56; Cl, 9.75; N, 3.77. After 1 d stirring, the mixture was concentrated in vacuo. The resulting
Methyl 4-[(4R)-Chloro-(2S)-pyrrolidinyl]methoxybenzoate (11j) residue was recrystallized with CHCl3/n-hexane to afford methyl 4-[1-tert-
Colorless solid (yield, 95%); mp 61—64 °C; 1H-NMR (CDCl3) d: 1.85 (1H, butoxycarbonyl-(4S)-hydroxy-(2S)-pyrrolidinyl]methoxybenzoate (5.76 g,
s), 2.03—2.10 (1H, m), 2.29—2.35 (1H, m), 3.19—3.31 (2H, m), 3.88 (3H, 87%) as a colorless solid. 1H-NMR (CDCl3) d: 1.46 (9H, s), 2.09—2.13
s), 3.92—4.06 (3H, m), 4.53—4.56 (1H, m), 6.91 (2H, d, Jꢀ8.8 Hz), 7.98
(1H, m), 2.35 (1H, s), 3.27—3.65 (2H, m), 3.89 (3H, s), 4.07—4.54 (4H,
m), 6.96 (2H, d, Jꢀ6.9 Hz), 7.99 (2H, d, Jꢀ6.9 Hz); ESI-MS m/z: 352
(2H, d, Jꢀ8.8 Hz); FAB-MS m/z: 270 (Mꢄꢄ1).
Methyl 4-[3-Benzyloxy-1-tert-butoxycarbonyl-(2S)-pyrrolidinyl]me- (Mꢄꢄ1).
1
thoxybenzoate (27n) Reddish oil (yield, 85%); H-NMR (CDCl3) d: 1.48
To a stirred solution of methyl 4-[1-tert-butoxycarbonyl-(4S)-hydroxy-
(9H, s), 1.95—2.17 (2H, m), 3.36—3.80 (2H, m), 3.89 (3H, s), 4.10—4.19 (2S)-pyrrolidinyl]methoxybenzoate (2.10 g, 5.98 mmol) in THF (60 ml) was
(3H, m), 4.25—4.29 (1H, m), 4.48—4.59 (2H, m), 6.85—6.95 (2H, m), added 60% oil NaH (359 mg, 8.97 mmol) at 0 °C. After 15 min stirring, MeI
7.27—7.44 (5H, m), 7.92—7.80 (2H, m); ESI-MS m/z: 442 (Mꢄꢄ1).
(1.20 ml, 8.97 mmol) was added to the mixture at same temperature, and the
Methyl 4-[3-Benzyloxy-(2S)-pyrrolidinyl]methoxybenzoate (11n) resulting mixture was allowed to room temperature for over 1 h. Then, 60%
Brown oil (yield, 98%); 1H-NMR (CDCl3) d: 1.92—2.05 (2H, m), 2.12 (1H, oil NaH (359 mg, 8.97 mmol) and MeI (1.20 ml, 8.97 mmol) were added to
s), 3.05—3.09 (1H, m), 3.16—3.18 (1H, m), 3.55 (1H, td, Jꢀ5.9, 3.2 Hz),
the reaction mixture at room temperature and stirred for 14 h. The reaction
3.88 (3H, s), 3.92—3.99 (2H, m), 4.00—4.04 (1H, m), 4.52 (1H, d, mixture was poured into ice water and extracted with CHCl3. The combined
Jꢀ11.8 Hz), 4.58 (1H, d, Jꢀ11.8 Hz), 6.89 (1H, d, Jꢀ8.8 Hz), 7.27—7.36 extracts were washed with aq. NaHCO3 and brine. After drying over
(5H, m), 7.97 (2H, d, Jꢀ8.8 Hz); ESI-MS m/z: 342 (Mꢄꢄ1).
Na2SO4, the extracts were concentrated in vacuo. The residue was purified
Methyl 4-[(4S)-Acetoxy-1-tert-butoxycarbonyl-(2S)-pyrrolidinyl]me- by column chromatography on silica gel with n-hexane/EtOAc (4/1, v/v) as
1
thoxybenzoate (27c) To a stirred solution of methyl 4-[(4R)-acetoxy- an eluent to give compound 27e (1.32 g, 60%) as a colorless oil. H-NMR
1-tert-butoxycarbonyl-4-hydroxy-(2S)-pyrrolidinyl]methoxybenzoate (27b) (CDCl3) d: 1.48 (9H, s), 2.03—2.07 (1H, m), 2.29 (1H, d, Jꢀ14.2 Hz), 3.30
(3.31 g, 8.41 mmol) in MeOH (100 ml) was added K2CO3 (1.05 g, 7.57 (3H, s), 3.36—3.68 (2H, m), 3.88 (3H, s), 3.95—4.38 (4H, m), 6.76 (2H, s),
mmol) at room temperature. After 1 h stirring, the mixture was diluted with
H2O and extracted CHCl3. The combined extracts were washed with sat.
NaCl. After dried over Na2SO4, the extracts were concentrated in vacuo. The
7.97 (2H, d, Jꢀ8.8 Hz); ESI-MS m/z: 366 (Mꢄꢄ1).
Methyl 4-[(4S)-methoxy-(2S)-pyrrolidinyl]methoxybenzoate (11e)
According to the general procedure B, 11e was obtained from 27e as a yel-
resulting residue was purified by flush column chromatography with n- low oil (yield, 99%); 1H-NMR (CDCl3) d: 2.16 (1H, t, Jꢀ5.3 Hz), 2.72 (1H,
hexane/EtOAc (10—60%, v/v) as an eluent to afford the methyl 4-[(2S,4R)-
s), 2.95 (1H, d, Jꢀ6.8 Hz), 3.11 (1H, d, Jꢀ11.0 Hz), 3.26 (3H, t, Jꢀ1.9 Hz),
1-tert-butoxycarbonyl-4-hydroxy-(2S)-pyrrolidinyl]methoxybenzoate 3.52 (1H, br), 3.84 (3H, d, Jꢀ1.7 Hz), 3.92 (1H, s), 4.00 (2H, d, Jꢀ4.1 Hz),
(2.84 g, 96%) as a colorless solid. 1H-NMR (CDCl3) d: 1.46 (9H, s), 2.05— 6.86—6.90 (2H, m), 7.92—7.96 (2H, m); ESI-MS m/z: 266 (Mꢄꢄ1).
2.36 (3H, m), 3.51 (1H, dd, Jꢀ11.6, 4.8 Hz), 3.88 (3H, s), 4.07—4.37 (3H,
Methyl 4-[1-tert-Butoxycarbonyl-(4R)-dimethylamino-(2S)-pyrrolidi-
m), 4.51—4.59 (1H, m), 6.92 (2H, d, Jꢀ8.3 Hz), 7.97 (2H, d, Jꢀ8.8 Hz); nyl]methoxybenzoate (27k) To a stirred solution of methyl 4-[1-tert-bu-
ESI-MS m/z: 352 (MꢄꢄNa).
toxycarbonyl-(4S)-hydroxy-(2S)-pyrrolidinyl]methoxybenzoate (300 mg,
To a solution of methyl 4-[(2S,4R)-1-tert-butoxycarbonyl-4-hydroxy-(2S)- 0.85 mmol) in CH2Cl2 (10 ml) was added DIPEA (2.08 ml, 12.0 mmol)
pyrrolidinyl]methoxybenzoate (1.00 g, 2.85 mmol), HOAc (0.48 ml, at room temperature and cooled to ꢃ78 °C. After 30 min stirring, Tf2O
8.54 mmol) and PPh3 (0.90 g, 3.42 mmol) in THF (20 ml) was added diiso- (0.56 ml, 2.42 mmol) was added to the mixture at same temperature and the
propyl azodicarboxylate (0.67 ml, 3.42 mmol) at room temperature. After solution was stirred for 30 min. Two mol/l Me3NH in THF (2.13 ml,
3 d, the reaction mixture was concentrated to dryness. The residue was puri- 4.26 mmol) was added to the reaction mixture at ꢃ78 °C. After 20 min stir-
fied by flush column chromatography with n-hexane/EtOAc (10—80%, v/v) ring at ꢃ78 °C, the reaction mixture was raised to 5 °C and stirred for 18 h.
1
as an eluent to afford compound 27c (1.00 g, 93%) as a colorless solid. H-
The reaction mixture was poured into sat. NaHCO3 solution and extracted
NMR (CDCl3) d: 1.48 (9H, s), 2.03 (3H, s), 2.25—2.29 (2H, m), 3.44—3.48 with CH2Cl2. The combined extracts were washed with brine. After drying
(1H, m), 3.70—3.74 (1H, m), 3.88 (3H, s), 3.98 (1H, t, Jꢀ9.0 Hz), 4.21— over Na2SO4, the extracts were concentrated in vacuo. The residue was puri-
4.47 (2H, m), 5.31 (1H, s), 6.96 (2H, s), 7.98 (2H, d, Jꢀ8.8 Hz); ESI-MS fied by flush column chromatography (Biotage 25S) with MeOH/EtOAc
m/z: 416 (MꢄꢄNa).
(0—40%, v/v) as an eluent, to afford compound 27k (380 mg, over yields)
Methyl 4-[(4S)-Acetoxy-(2S)-pyrrolidinyl]methoxybenzoate (11c) including inseparable materials as a red oily solid. 1H-NMR (CDCl3) d: 1.46
According to the general procedure B, 11c was obtained from 27c as pale (9H, s), 1.80—1.95 (1H, m), 2.20—2.23 (1H, m), 2.24 (6H, s), 2.90—2.95
1
purple solid (1.89 g, 100%); H-NMR (CDCl3) d: 2.10 (3H, s), 2.12—2.16 (1H, m), 3.10—3.30 (1H, m), 3.50—3.65 (1H, m), 3.88 (3H, s), 3.95—4.35
(1H, m), 2.63—2.67 (1H, m), 3.52—3.63 (2H, m), 3.89 (3H, s), 4.16—4.20 (3H, m), 6.93—6.95 (2H, m), 7.96—7.98 (2H, m); ESI-MS m/z: 379
(1H, m), 4.28 (2H, d, Jꢀ5.9 Hz), 5.36—5.40 (1H, m), 6.93 (2H, d, (Mꢄꢄ1).
Jꢀ8.8 Hz), 7.99 (2H, d, Jꢀ8.8 Hz); ESI-MS m/z: 294 (Mꢄꢄ1).
Methyl 4-[(4R)-Dimethylamino-(2S)-pyrrolidinyl]methoxybenzoate
Methyl 4-[(4R)-Methoxy-(2S)-pyrrolidinyl]methoxybenzoate (11d) (11k) According to the general procedure B, 11k was obtained from 27k
1
To a stirred solution of methyl 4-hydroxybenzoate (1.18 g, 7.76 mmol),
as a pale yellow oil (yield 61%). H-NMR (CDCl3) d: 1.77—2.05 (2H, m),
1-(tert-butoxycarbonyl)-(4R)-methoxy-(2S)-pyrrolidinylmethanol (1.79 g, 2.25—2.25 (1H, m), 2.30 (6H, s), 2.72—2.93 (1H, m), 3.26 (1H, dd, Jꢀ9.8,
7.74 mmol) and Ph3P (2.44 g, 9.30 mmol) in THF (30 ml) was added diiso- 6.4 Hz), 3.66—3.75 (1H, m), 3.88 (3H, s), 3.91 (1H, dd, Jꢀ9.1, 6.6Hz), 3.98
propyl azodicarboxylate (1.83 ml, 9.29 mmol) and the reaction mixture was (1H, dd, Jꢀ9.1, 4.9 Hz), 6.91 (2H, d, Jꢀ8.8 Hz), 7.98 (2H, d, Jꢀ8.8 Hz);
heated under reflux for 5 h. After cooling to room temperature, the mixture
was evaporated. The residue was filtered on silica gel with toluene/acetone
(5/1, v/v) as an eluent to afford methyl 4-[1-tert-butoxycarbonyl-(4R)-
ESI-MS m/z: 279 (Mꢄꢄ1).
Methyl 4-[1-tert-Butoxycarbonyl-(4S)-dimethylamino-(2S)-pyrrolidi-
nyl]methoxybenzoate (27l) By the procedure described for 27k, but with
methoxy-(2S)-pyrrolidinyl]methoxybenzoate (27d) including inseparable methyl 4-[1-tert-butoxycarbonyl-(4R)-hydroxy-(2S)-pyrrolidinyl]methoxy-
material.
To a solution of methyl 4-[1-tert-butoxycarbonyl-(4R)-methoxy-(2S)-
pyrrolidinyl]methoxybenzoate (27d) in CH2Cl2 (20 ml) was added TFA (quant.). H-NMR (CDCl3) d: 1.45 (9H, s), 1.70—1.90 (1H, m), 2.26 (6H,
(20 ml) at room temperature. After 12 h stirring, the mixture was concen- s), 2.33 (1H, m), 2.55—2.59 (1H, m), 3.04—3.08 (1H, m), 3.85—4.23 (4H,
trated in vacuo. The residue was made basic by sat. NaHCO3 and extracted m), 3.88 (3H, s), 6.93 (2H, m), 7.95 (2H, m); ESI-MS m/z: 379 (Mꢄꢄ1).
benzoate in place of methyl 4-[1-tert-butoxycarbonyl-(4S)-hydroxy-(2S)-
pyrrolidinyl]methoxybenzoate, compound 27l was obtained as a yellow oil
1
with CHCl3. The combined extracts were washed with brine, dried over
Methyl 4-[1-tert-Butoxycarbonyl-(4S)-dimethylamino-(2S)-pyrrolidi-
K2CO3, and evaporated. The residue was purified by column chromatogra- nyl]methoxybenzoate (11l) According to the general procedure B, 11l
phy on silica gel with CHCl3/MeOH (30/1 to 30/2, v/v) as an eluent to afford was obtained from 27l as a pale yellow oil (yield 79%). 1H-NMR (CDCl3) d:
1
compound 11d (1.67 g, 81% for 2 steps) as a reddish brown oil. H-NMR 1.48—1.58 (1H, m), 2.17—2.29 (1H, m), 2.30 (6H, s), 2.72—2.82 (1H, m),
(CDCl3) d: 1.65—1.72 (1H, m), 1.89 (1H, s), 2.05—2.22 (1H, m), 2.95— 2.87—2.96 (1H, m), 3.17 (1H, dd, Jꢀ10.3, 6.6 Hz), 3.59—3.68 (1H, m),
3.15 (2H, m), 3.31 (3H, s), 3.69—3.76 (1H, m), 3.88 (3H, s), 3.91—4.06 3.88 (3H, s), 3.98 (2H, d, Jꢀ5.9 Hz), 6.91 (2H, d, Jꢀ8.8 Hz), 7.97 (2H, d,
(3H, m), 6.89—6.92 (2H, m), 7.96—7.98 (2H, m); FAB-MS m/z: 266 Jꢀ8.8 Hz); ESI-MS m/z: 279 (Mꢄꢄ1).
(Mꢄꢄ1).
Methyl 4-[1-Benzyloxycarbonyl-(3R,4S)-isopropylidenedioxy-(2R)-
Methyl 4-[1-tert-Butoxycarbonyl-(4S)-methoxy-(2S)-pyrrolidinyl]me- pyrrolidinyl]methoxybenzoate (27m) According to the general procedure

November 2006
1527
C, 27m was obtained from 26m as a colorless oil (yield, 83%); 1H-NMR
(CDCl3) d: 1.01 (7H, s), 1.03 (3H, s), 2.21—2.25 (1H, m), 2.61—2.65 (1H,
m), 3.61 (1H, d, Jꢀ12.5 Hz), 3.80—4.27 (4H, m), 4.84 (1H, s), 5.01 and
5.08 (total 1H, each ABq, each Jꢀ12.2 Hz, amide isomers), 6.75—6.87 (3H,
m), 7.19—7.63 (5H, m).
m), 2.24 (3H, s), 2.97 (1H, dt, Jꢀ6.8, 10.2 Hz), 3.05 (1H, dt, Jꢀ5.9, 6.8 Hz),
3.54—3.58 (1H, m), 3.87 (3H, s), 3.92 (1H, dd, Jꢀ6.3, 9.3 Hz), 3.99 (1H,
dd, Jꢀ4.9, 9.3 Hz), 6.81 (1H, d, Jꢀ8.3 Hz), 7.83 (1H, s), 7.85 (1H, dd,
Jꢀ2.0, 8.3 Hz); FAB-MS m/z: 250 (Mꢄꢄ1).
4-[1-N-(tert-Butoxycarbonyl)-(2S)-pyrrolidinyl]methyloxy-3,5-di-
methyl-1-iodobenzene (28c) The procedure described for 28b, but with
2,5-dimethyl-4-iodophenol in place of 4-iodo-2-methylphenol, compound
Methyl 4-[(3R,4S)-Isopropylidenedioxy-(2R)-pyrrolidinyl]methoxy-
benzoate (11m) A suspension of methyl 4-[benzyloxycarbonyl-(3R,4S)-
isopropylidenedioxy-(2R)-pyrrolidinyl]methoxybenzoate (27m) (2.37 g,
5.76 mmol) and 10% Pd/C (240 mg) in EtOH (170 ml) was stirred at room
temperature under an atmosphere of hydrogen. After 1 d stirring, the catalyst
and solvent were changed for 10% Pd/C (500 mg) and THF (50 ml). The
suspension was stirred at room temperature under an atmosphere of hydro-
gen for 5 d. After removal of the catalyst by filtration, the filtrate was con-
centrated in vacuo. The residue was chromatographed on silica gel [100 g,
CHCl3/Acetone (20/1)], to afford compound (11m) (930 mg, 53%) as a
brown oil. 1H-NMR (CDCl3) d: 1.35 (3H, s), 1.50 (3H, s), 3.02 (1H, dd,
Jꢀ13.7, 4.1 Hz), 3.13 (1H, d, Jꢀ13.7 Hz), 3.58 (1H, t, Jꢀ6.3 Hz), 3.88 (3H,
s), 3.90 (1H, dd, Jꢀ9.3, 6.6 Hz), 4.02 (1H, dd, Jꢀ9.5, 3.9 Hz), 4.74 (1H, d,
Jꢀ5.6 Hz), 4.77—4.81 (1H, m), 6.90 (2H, d, Jꢀ9.0 Hz), 7.98 (2H, d,
Jꢀ9.0 Hz); ESI-MS m/z: 308 (Mꢄꢄ1).
Methyl 2-Methyl-4-[(2S)-pyrrolidinyl]methoxybenzoate (14a) To a
stirred solution of 4-bromo-3-methylphenol (500 mg, 2.67 mmol), N-Boc-L-
prolinol (22) (538 mg, 2.67 mmol), and Ph3P (911 mg, 3.48 mmol) in THF
(5.0 ml) was added diisopropyl azodicarboxylate (690 ml, 3.48 mmol) at
room temperature, and the mixture was stirred for 4 d at 70 °C. After cooling
to room temperature, the reaction mixture was concentrated. The residue
was purified by chromatography on silica gel with n-hexane/EtOAc (7/1,
v/v) as an eluent to afford 1-bromo-4-[1-(tert-butoxycarbonyl)-(2S)-pyrro-
lidinylmethyloxy]-(2S)-methylbenzene (28a) (755 mg, 76%) as a colorless
oil.
1
28c was obtained as a pale red oil (yield, 77%). H-NMR (CDCl3) d: 1.47
(9H, s), 1.83—1.95 (2H, m), 1.97—2.09 (3H, s), 2.13 (3H, s), 2.36 (3H, s),
3.79—3.95 (2H, m), 4.05—4.19 (2H, m), 6.70 (1H, s), 7.51 (1H, s); FAB-
MS m/z: 432 (Mꢄꢄ1).
Methyl 2,5-Dimethyl-4-[(2S)-pyrrolidinyl]methyloxybenzoate (14c)
To a solution of 4-[1-N-(tert-butoxycarbonyl)-(2S)-pyrrolidinyl]methyloxy-
3,5-dimethyl-1-iodobenzene (28c) (400.1 mg, 0.927 mmol) in DMSO (5 ml)
and MeOH (4 ml) was added Et3N (0.283 ml, 2.04 mmol), Pd(OAc)2
(10.4 mg, 0.046 mmol), and 1,3-bis(diphenylphosphino)propane (19.0 mg,
0.046mmol), then CO gas was babbled for 10 min, and the mixture was
stirred at 70 °C for 1 d. The reaction mixture is concentrated in vacuo. The
residue was diluted with EtOAc and washed with brine, dried over Na2SO4.
The solvent was removed under reduced pressure and the residue was chro-
matographed on silica gel eluting with n-hexane/EtOAc (5/1, v/v) to afford
methyl 4-[1-(tert-butoxycarbonyl)-(2S)-pyrrolidinyl]methyloxy-2,5-dimeth-
ylbenzoate 29c (228 mg, 68%) as a yellow oil, which was followed to the
general procedure B to afford 14c (86.9 mg, 53%) as a colorless oil. 1H-
NMR (CDCl3) d: 1.58—1.65 (1H, m), 1.76—1.87 (2H, m), 1.92—1.98 (1H,
m), 2.19 (3H, s), 2.57 (3H, s), 2.93—2.99 (1H, m), 3.02—3.08 (1H, m),
3.54—3.57 (1H, m), 3.85 (3H, s), 3.89—3.99 (2H, m), 6.63 (1H, s), 7.75
(1H, s); FAB-MS m/z: 264 (Mꢄꢄ1).
Ethyl 3-Methoxy-4-[(2S)-pyrrolidinyl]methoxybenzoate (14d) To a
solution of ethyl 4-hydroxy-3-methoxybenzoate (3.00 g, 15.29 mmol), N-
Boc-L-prolinol (22) (3.08 g, 15.3 mmol), Ph3P (4.81 g, 18.3 mmol) in THF
(50 ml) was added diisopropyl azodicarboxylate (3.61 ml, 18.3 mmol) and
the reaction mixture was heated under reflux for 6.5 h. After cooling to room
temperature, the solution was evaporated and the residue was purified by
column chromatography on silica gel with CHCl3/MeOH (50/1, v/v) as elu-
ent to give ethyl 3-methoxy-4-[1-(tert-butoxycarbonyl)-(2S)-pyrrolidinyl]-
methoxybenzoate (29d) containing inseparable compounds.
To a solution of ethyl 3-methoxy-4-[1-(tert-butoxycarbonyl)-(2S)-pyrro-
lidinyl]methoxybenzoate (29d) in CH2Cl2 (50 ml) was added TFA (45 ml) at
room temperature. After stirring for 2 d, the reaction mixture was concen-
trated in vacuo. The residue was made basic with sat. NaHCO3 and extracted
with CH2Cl2. The combined extracts were washed with brine, and dried over
MgSO4. After the solvent was removed, the resulting residue was purified by
column chromatography on silica gel with CHCl3/MeOH (20/1, v/v) as an
eluent to give compound 14d [3.27 g, 77% (2 steps)] as a yellow oil. 1H-
NMR (CDCl3) d: 1.39 (3H, t, Jꢀ7.1 Hz), 1.52—1.59 (1H, m), 1.76—1.88
(2H, m), 1.92—2.01 (1H, m), 2.92—3.06 (2H, m), 3.56—3.63 (1H, m), 3.90
(3H, s), 3.91—4.02 (2H, m), 4.35 (2H, q, Jꢀ7.1 Hz), 6.89 (1H, d,
Jꢀ8.3 Hz), 7.54 (1H, d, Jꢀ2.0 Hz), 7.65 (1H, dd, Jꢀ2.0, 8.3 Hz).
To a cooling stirred solution of 1-bromo-4-[1-(tert-butoxycarbonyl)-(2S)-
pyrrolidinylmethyloxy]-(2S)-methylbenzene (28a) (546 mg, 1.48 mmol) in
THF (6 ml) was added n-BuLi (283 ml, 2.04 mmol), and TMEDA (468 ml,
3.10 mmol) at ꢃ78 °C. The mixture was stirred for 1 h, methyl chlorofor-
mate (429 ml, 3.69 mmol) was added, and the resulting mixture was stirred
for 12 h. The reaction mixture was poured into sat. NH4Cl solution and ex-
tracted with EtOAc. The combined extracts were washed with brine and
dried over Na2SO4. After removal of the solvent, the residue was purified by
chromatography on silica gel with n-hexane/EtOAc (5/1, v/v) as an eluent to
afford methyl 4-[1-(tert-butoxycarbonyl)-(2S)-pyrrolidinylmethyloxy]-(2S)-
methylbenzoate (29a) (108 mg, 21%) as a colorless oil, which was followed
to the general procedure B to afford 14a [75 mg, 97%, (3 steps 15%)] as a
1
pale yellow oil. H-NMR (CDCl3) d: 1.54—1.62 (1H, m), 1.77—2.00 (3H,
m), 2.59 (3H, s), 2.93—3.04 (2H, m), 3.51—3.54 (1H, m), 3.85 (3H, s),
3.87—3.98 (2H, m), 6.74 (1H, dd, Jꢀ3.0, 7.3 Hz), 6.75 (1H, s), 7.91 (1H,
dd, Jꢀ1.5, 7.3 Hz); FAB-MS m/z: 250 (Mꢄꢄ1).
4-[1-N-(tert-Butoxycarbonyl)-(2S)-pyrrolidinylmethyloxy]-1-iodo-3-
methylbenzene (28b) To a stirred solution of 4-iodo-2-methylphenol
(465 mg, 1.99 mmol), N-Boc-L-prolinol (22) (400 mg, 1.99 mmol), and Ph3P
(625 mg, 2.38 mmol) in THF (7 ml) was slowly added diisopropyl azodicar-
boxylate (0.5 ml, 2.40 mmol) at room temperature and the stirring was con-
tinued for 13 h at 70 °C. The reaction mixture was concentrated. The residue
was purified by column chromatography on silica with n-hexane/EtOAc
(9/1, v/v) as an eluent to give compound 28b (645 mg, 78%) as a pale yellow
oil. 1H-NMR (CDCl3) d: 1.47 (9H, s), 1.83—1.89 (1H, m), 1.96—2.04 (3H,
m), 2.16 (3H, s), 3.37—3.43 (2H, m), 3.79—3.96 (1H, m), 4.08—4.18 (2H,
m), 6.62 (1H, s), 7.42 (2H, s); FAB-MS m/z: 418 (Mꢄꢄ1).
The following compound 14e—g were prepared from 22 according to
general procedure C.
Methyl 4-[1-(tert-Butoxycarbonyl)-(2S)-pyrrolidinylmethyloxy]-3-
1
chlorobenzoate (29e) Yellow oil (yield, 97%); H-NMR (CDCl3) d: 1.46
and 1.48 (total 9H, s each, amide isomers), 1.59—1.63 (1H, m), 1.88 (1H,
s), 2.05 (1H, s), 2.05—2.21 (2H, m), 3.34—3.97 (5H, m), 4.21 (2 H, s), 7.05
(1H, d, Jꢀ8.8 Hz), 7.90 (1H, dd, Jꢀ2.0, 8.8 Hz), 8.04 (1H, d, Jꢀ2.0 Hz);
FAB-MS m/z: 370 (Mꢄꢄ1).
Methyl 4-[1-N-(tert-Butoxycarbonyl)-(2S)-pyrrolidinylmethyloxy]-3-
methylbenzoate (29b) To a solution of 4-[1-N-(tert-butoxycarbonyl)-
(2S)-pyrrolidinylmethyloxy]-1-iodo-3-methylbenzene (28b) (645 mg, 1.55
mmol), Et3N (0.47 ml, 3.401 mmol), Pd(OAc)2 (17.4 mg, 0.077 mmol), and
1,3-bis(diphenylphosphino)propane (31.5 mg, 0.077 mmol) in DMSO/
MeOH (7/6 ml) was babbled CO gas for 10 min. After being stirred at 70 °C
for 2 d, the reaction mixture was concentrated. The resulting residue was di-
luted with EtOAc and washed with brine, dried over Na2SO4. After the sol-
vent was removed, the residue was purified by chromatography on silica gel
with n-hexane/EtOAc (5/1, v/v) as an eluent to afford compound 29b
Methyl 3-Chloro-4-[(2S)-pyrrolidinyl]methoxybenzoate (14e) Yellow
oil (yield, 89%); 1H-NMR (CDCl3) d: 1.60—1.67 (1H, m), 1.78—2.02 (3H,
m), 2.93—2.98 (1H, m), 3.03—3.09 (1H, m), 3.59 (1H, dt, Jꢀ2.0, 9.3 Hz),
3.89 (3H, s), 3.98 (1H, dd, Jꢀ6.3, 8.8 Hz), 4.05 (1H, dd, Jꢀ4.9, 9.3 Hz),
6.93 (1H, d, Jꢀ8.8 Hz), 7.90 (1H, dd, Jꢀ2.0, 8.8 Hz), 8.04 (1H, d,
Jꢀ2.0 Hz); FAB-MS m/z: 270 (Mꢄꢄ1).
Methyl 4-[1-(tert-Butoxycarbonyl)-(2S)-pyrrolidinylmethyloxy]-3,5-
dichlorobenzoate (29f) Pale yellow oil (yield, 90%); 1H-NMR (CDCl3) d:
1.44 (9H, s), 1.88—2.15 (3H, m), 2.34 (1H, s), 3.40—3.44 (2H, m), 3.92
(3H, s), 4.14 (1H, m), 4.18 (2H, s), 7.98 (2H, s); FAB-MS m/z: 404 (Mꢄꢄ1).
Methyl 3,5-Dichloro-4-[(2S)-pyrrolidinyl]methoxybenzoate (14f)
Pale yellow oil (yield, 68%); 1H-NMR (CDCl3) d: 1.62—1.69 (1H, m),
1.78—1.86 (2H, m), 1.89—1.99 (1H, m), 2.92—2.98 (1H, m), 3.04—3.09
(1H, m), 3.55—3.60 (1H, m), 3.91 (3H, s), 4.01 (1H, dd, Jꢀ6.8, 8.8 Hz),
4.08 (1H, dd, Jꢀ4.9, 8.8 Hz), 7.97 (2H, s); FAB-MS m/z: 304 (Mꢄꢄ1).
Methyl 4-[1-(tert-Butoxycarbonyl)-(2S)-pyrrolidinyl]methoxybenzoate
1
(302 mg, 56%) as a colorless oil. H-NMR (CDCl3) d: 1.47 (9H, s), 1.86—
2.10 (4H, m), 2.33 (3H, s), 3.32—3.50 (2H, m), 3.88 (3H, s), 3.88—4.04
(1H, m), 4.13—4.20 (2H, m), 6.87—6.89 (1H, m), 7.82 (1H, s), 7.85 (1H,
dd, Jꢀ2.0, 8.8 Hz); FAB-MS m/z: 350 (Mꢄꢄ1).
Methyl 3-Methyl-4-[(2S)-pyrrolidinyl]methyloxybenzoate (14b) Ac-
cording to the general procedure B, 14b was obtained as a colorless oil
(yield, 100%). 1H-NMR (CDCl3) d: 1.58—1.65 (1H, m), 1.78—2.00 (3H,
1
(29g) Yellow oil (yield, 73%); H-NMR (CDCl3) d: 1.58 (9H, s), 1.80—

1528
Vol. 54, No. 11
2.15 (5H, m), 3.93 (3H, s), 4.03—4.22 (2H, m), 4.25—4.39 (2H, m), 7.27— multiplication of the ratio and pooled total cells as the mean from each treat-
7.11 (1H, m), 8.19 (1H, dd, Jꢀ8.8, 2.2 Hz), 8.47—8.52 (1H, m); ESI-MS
m/z: 381 (Mꢄꢄ1).
ment group.
Distribution Coefficient The distribution coefficients (Log D) were de-
Methyl 3-Nitro-4-[(2S)-pyrrolidinyl]methoxybenzoate (14g) Yellow termined by the shake-flask method.30) Milliliters of 400 mM solution of each
oil (yield, 94%); 1H-NMR (CDCl3) d: 1.60—1.96 (4H, m), 2.96—3.05 (2H, compound in a 2 ml n-octanol/2 ml PBS solution was placed on a shaker for
m), 3.53—3.62 (2H, m), 3.93 (3H, s), 4.06 (1H, dd, Jꢀ6.8, 8.8 Hz), 7.12
30 min. After centrifuging each solution separately at for 10 min, an LC/MS
(1H, d, Jꢀ8.8 Hz), 8.19 (1H, dd, Jꢀ2.0, 8.8 Hz), 8.51 (1H, d, Jꢀ2.0 Hz); method was used to assay each layer. The LC/MS system was consisted
ESI-MS m/z: 281 (Mꢄꢄ1).
VLA-4/VCAM-1 Binding Assay A human VLA-4-expressing cell line,
4B4, was established at Pharmacopeia, by transfecting both the a4 gene and
b1 gene of VLA-4 into CHO-K1 cells. The 4B4 cells were maintained in gradient condition 95/5 to 10/90). Analyst software program (version 1.4,
Ham’s F-12 medium (Sigma) supplemented with 10% (v/v) fetal calf serum Applied Bio. Systems) was used to calculated the Log D.
of an 1100 Series LC/MSD (Agilent) and X Terra® MSC18 3.5 mm,
3.0ꢂ30 mm column (Waters). The mobile phase was a 10 mM ammonium
acetate buffer (pH 4.5)/0.05% (v/v) acetic acid mixture in acetonitrile; the
(REHATUIN Fetal Bovine Serum, Serologicals Corporation), 100 U/ml
penicillin (Invitrogen Corporation), 100 mg/ml streptomycin (Invitrogen ity of selected compounds was determined using Madin–Darby canine kid-
Corporation), and 2 mM L-glutamine (Invitrogen Corporation) and 1 mg/ml ney (MDCK, American Type Culture Collection) cells. MDCK cells were
Madin–Darby Canine Kidney Cell Permeability The cell permeabil-
G-418 (Geneticin, Invitrogen Corporation). An Eu-labelling Reagent maintained in Minimum Essential Medium (GIBCO) containing 10% (v/v)
(PerkinElmer Inc.) was used to labeled to human VCAM-1/Fc chimeric anti- fetal bovine serum (Bioproducts Inc.), a penicillin–streptomycin mixture
body (R&D Systems Inc.). The Eu-labelled protein was purified using a PD- (GIBCO), and L-glutamine. For the transport assay, cells were seeded on
10 column (Amersham Biosciences KK.) and stored at ꢃ80 °C until use. All
HTS 24 well transwells (Costar) at 3ꢂ105 cells/ml and grown for 6 d after
assays were performed in duplicate. In preparation for the assay, the 4B4 seeding to allow formation of a cell monolayer. The Transport Buffer was
cells were suspended at 3ꢂ105 cells/ml in Ham’s F-12 medium. One hun- prepared using NaHCO3 (final 0.35 g/l), D-Glucose (final 3.5 g/l), HEPES
dred microliters of the 4B4 cell suspension was into each well of a 96-well-
(Sigma; final 10 mM), CaCl2 (final 0.14 g/l) and MgSO4 (final 0.098 g/l) in
culture plate (Costar). The plates were incubated at 37 °C in a 95% air/5% ꢂ10 Hank’s Balanced Salt Solution (GIBCO) and adjusted to pH 6.0 or 7.4
CO2 atmosphere humidified incubator (Themo Forma, model 3120; Forma
Scientific) for 2 d. Prior to the assay the medium was discarded, and each
well was washed twice with 300 ml of chilled Wash Buffer (25 mM HEPES,
pH 7.5; 150 mM NaCl; 1 mM CaCl2; 1 mM MgCl2; 4 mM MnCl2). Then, 50 ml
of compound solutions was added to a well, followed by 50 ml of 2 nM of Eu-
in 1 M HCl or 1 M NaOH. For each test compound a dosing solution contain-
ing one of the compounds at a concentration of 10 mM in Transport Buffer
(pH 6.0) (100 ml) was added to the apical (A) side of a monolayer. A blank
solution containing 4% (w/v) BSA in Transport Buffer (pH 7.4) (600 ml),
which was re-adjusted to pH 7.4, was added to the basolateral (B) side of the
labelled Human VCAM-1/Fc Chimera diluted with the Wash Buffer (final monolayer. Metoprolol was used as a positive control. After 1 h of incuba-
concentration: 1 nM). For assays conducted in the presence of human serum tion at 37 °C, aliquots of the apical and basal solutions were separately ana-
albumin, 50 ml of compound at various concentrations and an equal volume lyzed on an LC/MS/MS system consisting an Alliance 2790 HPLC (Waters),
of 2 nM Eu-labeled human VCAM-1/Fc Chimera in a 3% (w/v) human Atlantis dC18, 2.1 mm I.D.ꢂ20 mm L, 3 mm particle size column (Waters),
serum albumin (Sigma) were distributed into each well (final concentration: and TSQ7000 mass spectrometer (ThermoQuest). The mobile phase was
1 nM). The plates were incubated for 60 min at room temperature, and the consisted of a 10 mM HCO2NH4–acetonitrile step gradient 100/0 to 80/20 to
wells were washed 4 times with 300 ml of chilled wash Buffer. Finally, 100/0. The concentration of each compound in the apical and the basolateral
100 ml of the enhancement solution (PerkinElmer Inc.) was added to each solution were determined from a peak area versus concentration standard
well. The plates were placed on a shaker for 5 min. Eu fluorescence was then curve. For each compound, Eq. 1 was used to calculate an apparent perme-
measured using a time-resolved fluorometer (DELFIA Research fluorome- ability coefficient (Papp) from the LC/MS/MS-determined concentration in
ter, model 1234-001; PerkinElmer Inc.). The concentration of compound re-
quired for 50% inhibition in the assay was determined.
the basolateral compartment (Cb, mM) and the initial 10 mM concentration in
the donor compartment. In the following equation 3600 s is the total time for
the measurement of compound flux and 0.33 cm2 is the area of transwell fil-
Inhibition of Airway Inflammation in Mice (in Vivo Evaluation) Fe-
male BALB/c AnNCrj mice (18—23 g, 8 weeks old, Charles River Japan, ter.
Inc.) were used. Animals were housed in disposable mouse cage [4 ani-
mals/cage, 220Wꢂ320Dꢂ130H (mm)] with lids in unidirectional air flow
rooms maintained controlled temperature (23ꢇ2 °C) and relative humidity
Papp (10ꢃ6 cm/s)ꢀ(Cbꢂ600 ml)/(10 mMꢂ3600 sꢂ0.33 cm2)
Pharmacokinetic Studies on Rats Male Sprague–Dawley rats (280—
(1)
(55ꢇ20%) on a 12 h light–dark cycle. Animals were provided food and tap 360 g, Hilltop Laboratories and Charles River Laboratories) were used. Ani-
water ad libitum. Norm-Based Group Building ver. 5.0.0 (Visions) was used mals were surgically implanted with catheters in the right and left jugular
to divide the animals into groups. Each group consisted of 7 animals. Mice
veins. The jugular catheters were composed of silastic tubing (i.d. 0.02 inch
received oral administration of cyclophosphamide (Endoxane, Shionogi & and o.d. 0.037 inch). The lock solution for the catheter contained streptoki-
Co. Ltd.) dissolved in 5% (w/v) carboxymethylcellulose (CMC) at a dose of nase (Kabikinase®, 18750 units/ml) and heparin (500 units/ml) in 25% (w/v)
150 mg/10 ml/kg (day 0). A solution containing 500 mg Ascaris suum extract
dextrose. Care was taken to apply only the catheter volume (approx 0.05—
(LSL Co., Ltd.) for every 0.2 ml saline wad prepared. The solution also con- 0.07 ml) of lock solution in order to minimize systemic exposure to this mix-
tained 5 mg aluminum hydroxide as an adjuvant. On day 2, 0.2 ml of the ture. Animals were housed in clear PVC boxes with lids in unidirectional air
antigen solution was injected intraperitoneally into each mouse. On day 14, flow rooms maintained controlled temperature (20ꢇ4 °C) and relative hu-
the same antigen solution was injected intraperitoneally as a booster. Finally, midity (50%ꢇ20%) on a 12 h light–dark cycle. Animals were provided food
on day 22, mice were anesthetized with intraperitoneal injection of and tap water ad libitum, except for the 12 h fasting (food withdrawn) period
70 mg/10 ml/kg of pentobarbital sodium. After 10 min, mice were chal-
prior to test substance administration. For intravenous administration, each
lenged intratracheally with 300 mg protein of Ascaris suum extract. In the compound was dissolved in sterile 0.9% (w/v) saline (pH adjusted to 8.0
negative control group, sensitized mice were challenged with saline instead with sodium bicarbonate) for a target concentration of 1.0 or 0.75 mg of
of antigen. Test compound, which was dissolved in a 5% CMC suspension each compound/ml. Each compound was administered by 2 h intravenous in-
containing 0.03% Tween, was orally administered 1 h before antigen chal- fusion at a targeted dose of 2.4 or 1.5 mg/kg. For oral administration, com-
lenge, 8, 24 and 32 h after antigen challenge at a dose of 30 mg/kg (day 22,
23). Forty-eight hours later, the mice were sacrificed by cutting abdominal cyclodextrin) for a target concentration of 2 mg/ml and was administered by
aorta while the animals were under pentobarbital anesthesia (1.95 mg/0.3 ml oral gavage at a targeted dose of 5 mg/kg. Compound 15e was suspended in
i.p. injection). Lungs were lavaged using tracheal polyethylene cannula (out- 0.5% (w/v) aqueous methylcellulose for a target concentration of 0.2 mg/ml
pound 1 was dissolved in 10% (w/v) aqueous Encapsin® (hydroxypropyl-b-
side diameter 1.33 mm, Hibiki No. 4, Hibiki Co.) with 2ꢂ0.5 ml Hanks’ bal-
anced salt solution supplemented with 0.05 mM potassium EDTA. The BAL
and was administered by oral gavage at a targeted dose of 2 mg/kg. Plasma
samples were analyzed on an LC/MS/MS method consisting HP 1100 Bi-
fluid was immediately centrifuged (5 min, 4 °C, 500ꢂ g). After removing the nary Pump HPLC (Hewlett-Packard), Columbus, C8, 2 mmꢂ100 mm, 5 mm
supernatant, the cells in the BAL fluid were resuspended in 0.2 ml of fetal column (Phenomenex), and Micro Quattro II mass spectrometer (Micro-
calf serum. The cells were counted in a particle analyzer CDA-500 (Sys- mass, Inc) or 2690 Separations Module (Waters), XDB-C18, 2.1ꢂ150 mm,
mex). Cytocentrifuged preparations (Cytospin 2; Shandon) were stained 5 mm column (Zorbax), and TSQ 7000 Triple Stage Quadrupole (Finnigan).
with Wright’s stain solution (Muto Chemicals) for differential counts, based The mobile phase was consisted of 2% (v/v) formic acid in water/acetoni-
on standard morphologic criteria. Taking the ratio for the number of trile; the gradient condition 90/10 to 10/90) or 2% (v/v) acetic acid in
eosinophils in cytocentrifuged preparations, eosinophils were estimated by water/acetonitrile; the gradient condition 76/24 to 55/45). Plasma concentra-

November 2006
1529
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