Scholarly article on ethyl [4-[(4S)-fluoro-(2S)-pyrrolidinylcarbonyl]-1-piperazinyl]acetate 441717-75-5 from Bioorganic and Medicinal Chemistry p. 2725

Bioorganic & Medicinal Chemistry 14 (2006) 2725–2746

Synthesis, biological evaluation, and pharmacokinetic study

of prolyl-1-piperazinylacetic acid and prolyl-4-piperidinylacetic

acid derivatives as VLA-4 antagonists

Jun Chiba,^a,*Gensuke Takayama,^bTohru Takashi,^bMika Yokoyama,^b

Atsushi Nakayama,^aJohn J. Baldwin,^cEdward McDonald,^cKevin J. Moriarty,^c

Christopher R. Sarko,^cKurt W. Saionz,^cRobert Swanson,^cZahid Hussain,^c

Angela Wong^cand Nobuo Machinaga^a

^aMedicinal Chemistry Research Laboratory, Daiichi Pharmaceutical, Co., Ltd, 16-13, Kitakasai 1-chome,

Edogawa-ku, Tokyo 134-8630, Japan

^bNew Product Research Laboratories III, Daiichi Pharmaceutical, Co., Ltd, 16-13, Kitakasai 1-chome,

Edogawa-ku, Tokyo 134-8630, Japan

^cPharmacopeia Drug Discovery, Inc., Princeton, NJ 08543-5350, USA

Received 26 September 2005; revised 25 November 2005; accepted 29 November 2005

Available online 27 December 2005

Abstract—A series of prolyl-1-piperazinylacetic acid and prolyl-4-piperidinylacetic acid derivatives were synthesized and evaluated

for their activity as VLA-4 antagonists. Of 22 compounds synthesized, 19 compounds showed potent activity with low nanomolar

IC₅₀values. In addition, the representative compounds 11o and 11p with a hydroxy group in the pyrrolidine ring showed moderate

plasma clearance in rats (11o, 30 ml/min/kg and 11p, 21 ml/min/kg) and in dogs (11o, 12 ml/min/kg and 11p, 9 ml/min/kg).

1. Introduction

anti-a₄antibody, natalizumab,¹⁰(Elan Pharmaceuticals

Inc. and Biogen Idec Inc.) demonstrated promising re-

sults in patients with multiple sclerosis, Crohnꢀs disease,

and rheumatoid arthritis in clinical trials. Therefore, the

development of small molecular VLA-4 antagonist with

good oral pharmacokinetic proﬁle is viewed as a reason-

able approach to a novel class of therapeutic agents.

VLA-4 (very late antigen 4; a₄b₁integrin; CD49d/CD29)

is a member of the integrin superfamily and is expressed

on most leukocytes.¹It binds a vascular cell adhesion

molecule-1 (VCAM-1) expressed on cytokine-stimulated

endothelial cells, the alternatively spliced connecting

segment-1 domain of ﬁbronectin^2,3and junctional adhe-

sion molecule 2 on endothelial cells.⁴The VLA-4 plays

an important role in the process of inﬂammation includ-

ing extravasation of leukocytes and activation of leuko-

cytes at sites of inﬂammation. Anti-VLA-4 antibodies or

small molecular VLA-4 antagonists⁵have been reported

to inhibit leukocyte inﬁltration to extravascular tissue

and prevent tissue damage in inﬂammatory disease

models of asthma,⁶multiple sclerosis,⁷rheumatoid

arthritis,⁸and inﬂammatory bowel disease.⁹It has

recently been reported that a humanized monoclonal

We have identiﬁed a morpholinyl-4-piperidinylacetic

acid derivative 1 with an IC₅₀value of 4.4 nM as a po-

tent VLA-4 antagonist, which demonstrated eﬃcacy in

the murine airway inﬂammation model by oral dosing

at 30 mg/kg (Fig. 1).¹¹It has been found, however, that

1 showed rapid plasma clearance (69 ml/min/kg) in rats,

resulting in low oral bioavailability (<1%). On the other

hand, we have also identiﬁed a prolyl-1-piperazinylace-

tic acid derivative 2 with an IC₅₀of 1.6 nM (Fig. 1).¹¹

Thus, we next chose 2 as a lead compound to investigate

the structure and physiochemical properties to improve

oral bioavailability while retaining the potent activity.

Several substituents were introduced in the pyrrolidine

ring, and both one and two methyl groups at the a-car-

bon of the acetic acid moiety. The piperidine ring was

Keywords: VLA-4; Integrin.

*

Corresponding author. Tel.: +81 3 3680 0151; fax: +81 3 5696

8609; e-mail: chiba2mk@daiichipharm.co.jp

doi:10.1016/j.bmc.2005.11.058

2726

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

Diphenylurea moiety

O

N

O

N

O

N

H

N

H

N

H

N

H

O

CO₂H

Me

OMe

2 ; IC₅₀= 1.6 nM

1; IC₅₀= 4.4 nM

Figure 1.

also replaced with a piperazine ring to give a piperazi-

nylacetic acid. The diphenylurea moiety, the 4-(2-

(5) using standard amide bond-forming conditions

followed by deprotection of N-protective groups to

give 6a–n, 7b, and 7c. Coupling of 6a–n, 7b, and 7c

with 3-methoxy-4-[N⁰-(2-methylphenyl)ureido]phenyl-

acetic acid (8) and basic hydrolysis aﬀorded 11a–n,

12b, and 12c.

methylphenylureido)-3-methoxyphenylacetyl

group,

was remained ﬁxed, because this moiety has been found

to be useful for maintaining potent activity (Fig. 1).⁶

Herein we report the synthesis, structure–activity rela-

tionships, and physicochemical properties of these com-

pounds. In addition, we examined the pharmacokinetic

proﬁles of some representative compounds and estab-

lished a relationship between the physicochemical prop-

erties and the plasma clearance.

Examples 11o and 12o, bearing a hydroxyl group in the

pyrrolidine ring, were synthesized through O-debenzyla-

tion of 11c and 12c (Scheme 2). In the case of 11p and

11q, the O-deprotection was conducted before the basic

hydrolysis (Scheme 2).

2. Chemistry

The preparation of compounds having a methyl or

dimethyl group at a-position of the acetic acid residue

was performed as shown in Scheme 3. Ethyl 2-bromo-

propionate or ethyl 2-bromo-2-methylpropionate was

coupled by substitution reaction with N-Boc-piperazine

(13) in the presence of K₂CO₃, followed by deprotection

to give 14 and 15. Compounds 14 and 15 were converted

The general synthetic route of prolyl-1-piperazinylace-

tic acids 11a–n and prolyl-4-piperidinylacetic acids

12b and 12c are outlined in Scheme 1. N-Protected pro-

line derivatives 3a–n were condensed with ethyl

1-piperazinylacetate (4) or ethyl 4-piperidinylacetate

O

CO₂H

N

H

N

H

Me

OMe

8

H A

O

c

HN

a, b

P A CO₂H

+

N

Y

CO₂Et

Y

CO₂Et

6a-n, Y = N

7b, c, Y = CH

4, Y = N

5, Y = CH

3a-n

P = Boc, Cbz, Fmoc

d

A

N

Y

A

N

Y

O

N

O

CO₂H

CO₂Et

O

N

H

N

H

N

H

Me

OMe

Me

OMe

9a-n,Y = N

11a-n,Y = N

12b, c,Y = CH

10b, c,Y = CH

X

A =

or

A =

N

a-k

l

m

n

a, X = H

g, X = (3S)-OBn

h, X = (4S)-F

i, X = (4R)-F

b, X = (4R)-OMe

c, X = (4R)-OBn

d, X = (3R,4S)-acetonide j, X = 4,4-diF

e, X = (4S)-OPh

k, X = (4S)-Cl

f, X = (4S)-O-(2-Napthyl)

Scheme 1. Reagents: (a) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl salt (EDCÆHCl), HOBt, Et₃N, THF (CH₂Cl₂or DMF); (b) TFA,

CH₂Cl₂or H₂, Pd/C, EtOH or TBAF, THF; (c) 8, EDCÆHCl, HOBt, Et₃N, THF (CH₂Cl₂or DMF); (d) aq NaOH, THF/MeOH.

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

2727

OH

N

a

O

11c, 12c

N

Y

O

N

H

N

H

O

CO₂H

Me

OMe

11o,Y = N

12o, Y = CH

X

OH

N

O

N

a

b

O

9c, g

9q

N

O

N

O

CO₂Et

N

H

N

H

O

CO₂H

H

Me

OMe

Me

OMe

9o, X = (4R)-OH

9q, X = (3S)-OH

11q

OH

N

b

c

N

9d

O

N

O

N

H

N

H

O

CO₂Et

N

H

N

H

O

CO₂H

Me

OMe

Me

OMe

9p

11p

Scheme 2. Reagents: (a) H₂, Pd/C, EtOH (57% for 9o, 59% for 9q, 58% for 11o, 65% for 12o); (b) aq NaOH, THF or THF/MeOH (90% for 11p, 95%

for 11q); (c) HCl (gas)/MeOH (97%).

X

N

Me

N

Boc

W

O

a, b

c, d

e, f

HN

Me

N

H

W

N

CO₂Et

N

O

NH

N

H

N

H

O

CO₂H

O

CO₂Et

Me W

Me

OMe

13

14, W = H

15, W = Me

16, X = H, W = H

17, X = OBn, W = H

18, X = OBn, W = Me

19, X = H, W = H

20, X = OBn, W = H

21, X = OBn, W = Me

Scheme 3. Reagents: (a) BrCH(CH₃)CO₂Et or BrC(CH₃)₂CO₂Et, K₂CO₃, DMF; (b) TFA, CH₂Cl₂[25% for 14 (2 steps), 98% for 15 (2 steps)];

(c) EDCÆHCl, DMAP, DMF; (d) TFA, CH₂Cl₂[26% for 16 (2 steps), 20% for 17 (2 steps), 26% for 18 (2 steps)]; (e) EDCÆHCl, HOBt, Et₃N, DMF;

(f) aq NaOH, THF/MeOH [12% for 19 (2 steps), 45% for 20 (2 steps), 34% for 21 (2 steps)].

to 19–21, according to the general synthetic route shown

in Scheme 1.

basic hydrolysis to give 3b and 3g, respectively

(Scheme 4). As for the synthesis of the N-Z-3,4-isopro-

pylidenedioxy-^L-proline derivative 3d, ﬁrst, N-Z-trans-

4-hydroxy-^L-proline methyl ester (26) was converted to

N-Z-3,4-dehydro-^L-proline methyl ester (27) in 3 steps

according to a reported procedure¹²(1, tosylation of

the hydroxy group of 26; 2, nucleophilic displacement

of the O-tosyl group with a phenylselenyl group; and

3, oxidative elimination of the phenylselenyl group).

Next, the obtained 27 was dihydroxylated with OsO₄/

NMO to give a mixture of diastereomeric isomers

[(3R,4S)-28:(3S,4R)-28 = ca. 15:1], which were used in

the next step without separation of the isomers. After

both hydroxy groups of the diols 28 were protected as

O-isopropylidene ketal, (3R,4S)-29 was separated by sil-

ica gel column chromatography. Finally, basic hydroly-

sis of the methyl ester group gave 3d (Scheme 5). (4S)-

Aryloxyproline derivatives 3e and 3f were prepared

through introduction of aryloxy groups by the Mitsun-

obu reaction and basic hydrolysis (Scheme 6).

Pyrrolidine intermediates 3b and 3d–k were prepared by

using the commercially available hydroxy-^L-proline

derivatives 22, 24, 26, and 32 as shown in Schemes 4–

7. Thus, N-Boc-trans-4-hydroxy-^L-proline methyl ester

(22) and N-Boc-trans-3-hydroxy-^L-proline methyl ester

(24) were etheriﬁed (MeI or BnBr/NaH) followed by

OR

OH

OR

a

b

CO₂H

CO₂Me

N

Boc

N

Boc

N

Boc

22, (4R)-OH

24, (3S)-OH

3b, (4R)-OMe

3g, (3S)-OBn

23, (4R)-OMe

25, (3S)-OBn

Scheme 4. Reagents: (a) MeI, NaH, DMF (89% for 23) or BnBr, NaH,

tetrabutylammonium iodide, THF (59% for 25); (b) aq NaOH, THF

(quant. for 3b, 59% for 3g).

2728

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

OH

a-c

d

OH

f

e

O

N

Cbz

N

ref. 12

CbzN

CO₂Me

CbzN

Cbz

CO₂Me

26

CbzN

CO₂Me

(3R,4S)-28

(3S,4R)-28

CO₂Na

CO₂Me

27

(3R,4S)-29

3d

Scheme 5. Reagents: (a) 1-(p-toluenesulfonyl)imidazole, TfOMe, N-methylimidazole, THF (97%); (b) PhSeSePh, NaBH₄, MeOH, reﬂux (82%);

(c) H₂O₂, pyridine, CH₂Cl₂(74%); (d) OsO₄, NMO, acetone/H₂O (1:1, v/v) (95%); (e) 2,2-dimethoxypropane, p-TsOHÆH₂O, CH₂Cl₂(cat.) (89%);

(f) aq NaOH, THF (91%).

this position did not aﬀect the activity. As for the pipe-

HO

ArO

ridinylacetic acid derivative, introduction of methoxy

group (12b) and benzyloxy group (12c) also retained

the activity. The piperidine ring was found to have the

same activity as compared with that of the piperazine

ring (12b, 12c, and 12o in comparison with 11b, 11c,

and 11o). Similarly, 3,4- and 4,5-di-substituted pyrroli-

dine derivatives 11d, 11p, 11l, 11m, and 11n showed

good potency. From these results, it appeared that the

3-, 4-, and 5-positions of the pyrrolidine ring do not par-

ticipate in the interaction with VLA-4 protein and there-

fore these positions could be utilized to adjust the

physicochemical properties of the compound. The intro-

duction of methyl groups at a-position of the acetic acid

residue resulted in a signiﬁcant decrease in potency of

19–21 as compared with 2 and 11c.

a

b

CO₂Me

N

CO₂Me

CO₂H

N

Boc

30, Ar = Ph

31, Ar = 2-Napthyl

22

3e, Ar = Ph

3f, Ar = 2-Napthyl

Scheme 6. Reagents: (a) phenol, or 2-naphthol, diisopropyl azodi-

carboxylate, PPh₃, THF (86% for 30, 58% for 31); (b) aq NaOH, THF

(58% for 3e, 100% for 3f).

Y

X

Y

X

c

a or b

CO₂Me

N

Boc

CO₂Me

CO₂H

N

Boc

22, X = OH, Y = H

32, X = H, Y = OH

33, X = H, Y = F

34, X = F, Y = H

35, X = H, Y = Cl

3h, X = H, Y = F

3i, X = F, Y = H

3k, X = H, Y = Cl

Compounds 11o, 11p, 11e, and 11m were next evaluated

for their pharmacokinetic properties in rats. As a result,

11o and 11p bearing a hydroxy group in the pyrrolidine

ring showed improved plasma clearance (Cl = 30 ml/

min/kg for 11o, 21 ml/min/kg for 11p) in comparison

with 1, 11e, and 11m (Cl = 69 ml/min/kg for 1, 77 ml/

min/kg for 11e, 95 ml/min/kg for 11m) as shown in

Table 2. In addition, it was found that both 11o and

11p showed moderate plasma clearance in dogs

(Cl = 12 ml/min/kg for 11o, Cl = 9 ml/min/kg for 11p).

F

O

d

a

c

F

22

CO₂Me

CO₂H

N

Boc

N

Boc

CO₂Me

N

Boc

36

37

3j

Scheme 7. Reagents: (a) DAST, CH₂Cl₂(10% for 33, 40% for 34, 72%

for 37); (b) CCl₄, PPh₃, CH₂Cl₂(76% for 35); (c) aq NaOH, THF (85%

for 3h, quant. for 3i, 92% for 3j, 83% for 3k); (d) PDC, CH₂Cl₂(57%).

To investigate the reason for the improved plasma clear-

ance of 11o, we examined how 1 and 11o were excreted

in rats. As a result, it was found that 44% of the un-

changed 1 was recovered in bile over 6 h, while 36% of

the unchanged 11o was recovered in urine and 4% in bile

over 12 h (Table 3). The results suggested that the

change of the excretion pathway from bile to urine is

responsible for the improvement in plasma clearance,

and signiﬁcant decrease of lipophilicity (logD < 3) ob-

tained by introduction of the hydroxyl group in the pyr-

rolidine ring might explain the change in the excretion

pathway.

4-Fluoroproline derivatives 3h, 3i,¹³and (4S)-chloropro-

line derivative 3k were prepared via ﬂuorination

(DAST) or chlorination (PPh₃/CCl₄) of the hydroxyl

group in 22 and 32, respectively. Similarly, 4,4-diﬂuo-

roproline derivative 3j¹³was synthesized from N-Boc-

4-oxo-^L-proline methyl ester (36) easily prepared by

the treatment of 22 with PDC (Scheme 7).

3. Results and discussion

Compounds obtained were evaluated for their activity in

the VLA-4/VCAM-1 binding assay and the distribution

coeﬃcient [logD, n-octanol/PBS (pH 7.4)]. The results

are summarized in Table 1.

Considering the extremely low lipophilicity of both 11o

and 11p (logD < 3), these compounds would not be

expected to show oral absorption because of poor mem-

brane permeability. Hence, we next attempted a pro-

drug strategy. We selected the ethyl esters 9o of 11o

and 9p of 11p, which were prepared as intermediates,

and examined their oral pharmocokinetic properties in

rats. However, as shown in Table 4, 9o and 9p showed

only 2.7 and 1.5% oral bioavailability, respectively;

and it was found that the prodrugs did not result in sig-

niﬁcant improvement.

Piperazinylacetic acid derivatives bearing methoxy

(11b), benzyloxy (11c), aryoxy (11d–e), hydroxy (11o),

ﬂuoride (11h–j), and chloride groups (11k) at the 4-posi-

tion in the pyrrolidine ring of 2 showed good potency

with IC₅₀values of about 1 nM, suggesting that the

bulkiness, conﬁguration, and nature of substituents at

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

2729

Table 1. Inhibition of VLA-4/VCAM-1 by VLA-4 antagonists

A

N

Y Z

CO₂H

O

N

H

N

H

Me

OMe

Compound

A

Y

Z

IC₅₀(nM)

4.4

logD^a

1

—

ꢀ0.7^b

2

N

–CH₂–

1.6

2.0

ꢀ1.0

N

OMe

OBn

11b

–CH₂–

n.t.

0.4

0.6

N

12b

11c

12c

CH

1.1

N

1.6

CH

0.94

OH

11o

12o

N

–CH₂–

5.1

1.1

<ꢀ3

ꢀ1.2

N

CH

O

11d

11p

11e

N

–CH₂–

1.6

1.4

1.2

n.t.

<ꢀ3

0.1

N

OH

OPh

N

-(2-Naphthyl)

O

11f

N

–CH₂–

1.4

1.3

0.4

N

OBn

11g

N

(continued on next page)

2730

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

Table 1 (continued)

Compound

A

Y

N

Z

IC₅₀(nM)

0.96

logD^a

ꢀ1.4

OH

11q

11h

11i

–CH₂–

N

F

N

–CH₂–

1.0

2.2

ꢀ1.5

ꢀ0.8

N

F

11j

N

–CH₂–

1.4

1.1

1.5

2.6

1.0

ꢀ0.7

ꢀ0.6

0.1

N

Cl

11k

11l

11m

11n

0.2

N

n.t.

N

19

20

N

–CH(CH₃)–

66

n.t.

OBn

231

N

21

N

–C(CH₃)₂–

185

n.t.

n.t., not tested.

^an-Octanol to PBS (pH 7.4) distribution coeﬃcient.

^bn-Octanol to the Japanese Pharmacopoeia Second ﬂuid (pH 6.8) distribution coeﬃcient.

4. Conclusion

molar potency. Among the derived compounds, we

identiﬁed 11o and 11p which had a hydroxyl group in

the pyrrolidine ring, with moderate plasma clearance

in rats and dogs. In addition, we found that these com-

pounds were excreted mainly to urine, whereas 1 with

high plasma clearance was mainly excreted to bile.

Because both compounds had low lipophilicity, we

attempted to apply a pro-drug strategy using the ethyl

In conclusion, we introduced several substituents into

the 3-, 4-, and 5-positions of the pyrrolidine ring and

methyl group into the a-position of acetic acid and re-

placed the piperidine ring with a piperazine ring. Except

for the introduction of the methyl group at a-position of

acetic acid, the other modiﬁcations retained low nano-

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

2731

Table 2. Pharmacokinetic properties of selected VLA-4 antagonists

polymer type synthetic adsorbent). Flash chromato-

graphy was performed with Biotage Si and YAMA-

Compound

Clearance (ml/min/kg)

Dog^b

ZEN

chromatography (TLC) was performed on Merck

pre-coated TLC glass sheets with silica gel 60 F₂₅₄

Hi-Flash

packed

column.

Thin-layer

Rat^a

1

69

30

21

77

95

n.t.

12

.

¹H NMR spectra were recorded on a JEOL JNM-

EX-400 spectrometer, and chemical shifts are given

in ppm (d) from tetramethylsilane as the internal stan-

dard. IR spectra were recorded on a HITACHI 270-

30 spectrometer. ESI mass spectra were recorded on

a SCIEX API-150EX spectrometer. FAB mass spectra

were recorded on a JEOL JMS-HX110 spectrometer.

HR-FAB mass spectra were recorded on a JEOL

JMS-700 spectrometer.

11o

11p

11e

11m

9

n.t.

n.t., not tested.

^aMale Sprague–Dawley rats. Dose: iv infusion (2 h) at 1.2 mg/kg for 1,

1.0 mg/kg for 11o, and 0.75 mg/kg for 11p, 11e, and 11m.

^bMale Beagle dog. Dose: iv infusion (1 h) at 0.75 mg/kg.

esters 9o of 11o and 9p of 11p. However, the strategy did

not result in a signiﬁcant improvement of oral absorp-

tion. Further improvements and structural modiﬁca-

tions of these derivatives toward achieving an orally

active VLA-4 antagonist will be presented in forthcom-

ing publications.

5.2. General procedure A: preparation of 4-[4-methoxy-1-

[3-methoxy-4-[N⁰-(2-methylphenyl)ureido]phenylacetyl]-

(2S)-pyrrolidinylcarbonyl]-1-piperazinylacetic acid (11b)

5.2.1. Ethyl 4-[1-(tert-butoxycarbonyl)-(4R)-methoxy-

(2S)-pyrrolidinylcarbonyl]-1-piperazinylacetate. A mix-

ture of 1-(tert-butoxycarbonyl)-(4R)-methoxypyrroli-

dine-(2S)-carboxylic acid (3b) (910 mg, 3.71 mmol),

ethyl 1-piperazinylacetate (4) (640 mg, 3.72 mmol), ED-

CÆHCl (853 mg, 4.45 mmol), HOBt (602 mg, 4.45 mmol),

and Et₃N (620 ll, 4.45 mmol) in CH₂Cl₂(10 ml) was stir-

red at room temperature for 12 h. The mixture was

poured into water and extracted with CHCl₃. The com-

bined extracts were washed with brine and dried over

Na₂SO₄. After removal of the solvent, the residue was

puriﬁed by column chromatography on silica gel with

5. Experimental

5.1. General

Melting points were determined with a YANACO

MP-J3 and are uncorrected. Column chromatography

was performed with Merck silica gel 60 (particle size

0.060–0.200 or 0.040–0.063) and Daianion HP-20

(MITSUBISHI Chemical Industries, highly porous

Table 3. Excretion of unchanged parent compound in rats

Compound

Cl (iv)^a(ml/min/kg)

Bile

Urine

% Dose

2–4 h

Total

0–6 h

% Dose

3–6 h

Total

0–2 h

4–6 h

0–3 h

6–12 h

0–12 h

1

11o

69

30

31^b

n.t.

13^b

n.t.

nq.

n.t.

44^b

4

n.t.

8^c

n.t.

25^c

n.t.

3^c

n.t.

36^c

nq., compound concentration was below the LLOQ of the assay (5 ng/ml).

n.t., not tested.

^aSee also Table 2.

^bMale Sprague–Dawley rats. Dose: iv infusion (2 h) at 2.4 mg/kg.

^cDose: 1.0 mg/kg iv bolus at 0 and 3 h (total 2 mg/kg).

Table 4. Pharmacokinetic properties of 9o and 9p in rats

OH

N

O

N

O

N

H

N

H

O

CO₂R

N

H

N

H

O

CO₂R

Me

OMe

Me

OMe

11o, R = H

9o, R = Et

11p, R = H

9p, R = Et

Compound

F^a,b(%)

9o

9p

2.7

1.5

^aMale Sprague–Dawley rats. Dose: p.o. at 2 mg/kg; iv see also Table 2.

^bOral plasma concentrations of the parent compounds 9o and 9p were measured.

2732

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

CHCl₃/MeOH (50/1, v/v) as an eluent to give the title

compound (1.50 g, quant.) as a pale yellow oil. ¹H

NMR (CDCl₃) d 1.26–1.30 (3H, m), 1.40 and 1.45 (9H,

s, each, amide isomers), 1.97–2.08 (1H, m), 2.14–2.23

(1H, m), 2.55–2.67 (4H, m), 3.24 (2H, s), 3.32 (3H, s),

3.49–3.75 (6H, m), 3.98–4.07 (1H, m), 4.16–4.22 (2H,

m), 4.64–4.79 (1H, m); MS (ESI), m/z 400 (M⁺+1).

compound 11b (120 mg, 27%) as a pale yellow amor-

phous solid. H NMR (CD₃OD) d 1.91–1.95 (1H, m),

1

2.29 (3H, s), 2.33–2.36 (1H, m), 2.71–2.86 (4H, m),

3.16 (2H, s), 3.23 (3H, s), 3.31 (2H, s), 3.49–3.80

(8H, m), 3.90 (3H, s), 4.05–4.06 (1H, m), 4.92–4.96

(1H, m), 6.80–6.83 (1H, m), 6.93–6.94 (1H, m),

6.99–7.03 (1H, m), 7.13–7.19 (2H, m), 7.56–7.58 (1H,

m), 7.98–8.00 (1H, m); MS (FAB), m/z 568 (M⁺+1);

HR-MS (FAB) calcd for C₂₉H₃₈N₅O₇: 568.2771.

Found: 568.2786; IR (ATR) 3225, 3251, 2935, 2829,

5.2.2. Ethyl 4-[4-methoxy-(2S)-pyrrolidinylcarbonyl]-1-

piperazinylacetate (6b). To a solution of ethyl 4-[1-

(tert-butoxycarbonyl)-(4R)-methoxy-(2S)-pyrrolidinyl-

carbonyl]-1-piperazinylacetate (630 mg, 1.58 mmol) in

CH₂Cl₂(10 ml) was added TFA (10 ml), and the solu-

tion was stirred at room temperature for 12 h. The reac-

tion mixture was concentrated in vacuo and made basic

with NaHCO₃. The mixture was extracted with CHCl₃.

The combined extracts were washed with brine, dried

over Na₂SO₄, and evaporated to give the title compound

6b (245 mg, 52%) as a reddish brown oil. ¹H NMR

(CDCl₃) d 1.28 (3H, t, J = 7.1 Hz), 1.79–1.86 (4H, m),

2.56–2.61 (4H, m), 3.24 (2H, s), 3.31 (3H, s), 3.50–3.73

(4H, m), 3.93–3.97 (1H, m), 4.04–4.08 (2H, m), 4.19

(2H, q, J = 7.1 Hz); MS (ESI), m/z 300 (M⁺+1).

1639, 1620, 1599, 1541 cm^ꢀ1

.

Compounds 11c–n, 12b–c, and 19–22 were prepared

according to procedure A.

5.3. 1-[(4R)-Methoxy-1-[3-methoxy-4-[N⁰-(2-methylphe-

nyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-4-

piperidinylacetic acid (12b)

5.3.1. Ethyl 1-[1-(tert-butoxycarbonyl)-(4R)-methoxy-

(2S)-pyrrolidinylcarbony]-4-piperidinylacetate. Colorless

oil (yield, 44%). ¹H NMR (CDCl₃) d 1.25–1.28 (3H,

m), 1.42 and 1.45 (total 9H, each s, amide isomers),

1.74–1.86 (2H, m), 1.95–2.05 (2H, m), 2.15–2.26 (4H,

m), 2.56–2.64 (1H, m), 3.02–3.16 (1H, m), 3.32 (3H, s),

3.51–3.68 (3H, m), 3.84–4.07 (2H, m), 4.12–4.17 (2H,

m), 4.56–4.82 (2H, m); MS (ESI), m/z 399 (M⁺+1).

5.2.3. Ethyl 4-[4-methoxy-1-[3-methoxy-4-[N⁰-(2-methyl-

phenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-

piperazinylacetate (9b). A mixture of 3-methoxy-4-[N⁰-

(2-methylphenyl)ureido]phenylacetic acid (8) (116 mg,

0.37 mmol), ethyl 4-[(4R)-methoxy-(2S)-pyrrolidinylcar-

bonyl]-1-piperazinylacetate (6b) (110 mg, 0.37 mmol),

EDCÆHCl (85 mg, 0.44 mmol), HOBt (60 mg,

0.44 mmol) and Et₃N (60 ll, 0.43 mmol) in THF

(10 ml) 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

brine and dried over Na₂SO₄. After removal of the sol-

vent, the residue was puriﬁed by TLC with CHCl₃/

MeOH (10/1, v/v) to give the title compound 9b

(183 mg, 83%) as a colorless amorphous solid. ¹H

NMR (CDCl₃) d 1.27 (3H, t, J = 7.1 Hz), 2.01–2.07

(1H, m), 2.13–2.19 (1H, m), 2.29 (3H, s), 2.49–2.65

(4H, m), 3.22 (4H, s), 3.58–3.71 (9H, m), 3.75 (3H, s),

4.08 (1H, m), 4.18 (2H, q, J = 7.1 Hz), 4.93–4.9 (1H,

m), 6.26 (1H, s), 6.82 (2H, s), 7.07 (1H, s), 7.13–7.16

(1H, m), 7.22–7.26 (2H, m), 7.50–7.52 (1H, m), 8.03–

8.05 (1H, m); MS (FAB), m/z 596 (M⁺+1); Anal. Calcd

for C₃₁H₄₁N₅O₇Æ1.25H₂O: C, 60.23; H, 7.09; N, 11.33.

Found: C, 60.28; H, 7.06; N, 11.15.

5.3.2. Ethyl 1-[(4R)-methoxy-(2S)-pyrrolidinylcarbonyl]-

4-piperidinylacetate (7b). Reddish-brown oil (yield,

80%). ¹H NMR (CDCl₃) d 1.24–1.28 (3H, m), 1.74–

1.85 (4H, m), 2.01–2.14 (2H, m), 2.23–2.26 (2H, m),

2.60–2.66 (1H, m), 2.90–3.09 (2H, m), 3.26–3.29 (2H,

m), 3.32 (3H, s), 3.87–3.95 (2H, m), 4.03–4.11 (1H, m),

4.13–4.15 (2H, m), 4.57–4.60 (1H, m); MS (ESI), m/z

299 (M⁺+1).

5.3.3. Ethyl 1-[(4R)-methoxy-1-[3-methoxy-4-[N⁰-(2-

methylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcar-

bonyl]-4-piperidinylacetate (10b). Colorless amorphous

1

solid (yield, 94%). H NMR (CDCl₃) d 1.05–1.29 (5H,

m), 1.68–1.84 (5H, m), 1.99–2.10 (2H, m), 2.13–2.24

(2H, m), 2.26 (3H, s), 2.54–2.62 (1H, m), 3.01–3.18 (1H,

m), 3.21–3.23 (3H, m), 3.69 (2H, s), 3.70 (3H, s), 3.93–

4.17 (4H, m), 4.46–4.59 (1H, m), 4.93–5.00 (1H, m),

6.63–6.65 (1H, m), 6.78–6.80 (2H, m), 7.08–7.12 (1H,

m), 7.20–7.25 (3H, m), 7.55–7.57 (1H, m), 8.03–8.05

(1H, m); MS (FAB), m/z 595 (M⁺+1); Anal. Calcd for

C₃₂H₄₂N₄O₇Æ0.75H₂O: C, 63.19; H, 7.21; N, 9.21. Found:

C, 63.08; H, 7.07; N, 9.01.

5.2.4.

4-[(4R)-Methoxy-1-[3-methoxy-4-[N⁰-(2-methyl-

phenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-

piperazinylacetic acid (11b). To a stirred solution of

ethyl 4-[4-methoxy-1-[3-methoxy-4-[N⁰-(2-methylphe-

nyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-

piperazinylacetate (9b) (460 mg, 0.77 mmol) in THF/

MeOH (10 ml, 1/1, v/v) was added 0.5 N NaOH

(10 ml) and the reaction mixture was heated under re-

ﬂux for 5 h. The mixture was acidiﬁed with 1 N HCl.

After removal of the solvent, the residue was puriﬁed

by ﬁltration through an ion-exchange resin (DIAION,

HP-20) with H₂O to MeOH as an eluent to give a

crude substance. The crude substance was puriﬁed

by TLC with CHCl₃/MeOH (10/1, v/v) to give the title

5.3.4.

1-[(4R)-Methoxy-1-[3-methoxy-4-[N⁰-(2-methyl-

phenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-4-

piperidinylacetic acid (12b). Pale brown crystalline pow-

der (yield, 50%), which was recrystallized from CHCl₃/

Et₂O. Mp 123–127 ꢁC; H NMR (DMSO-d₆) d 0.83–

1

1.32 (2H, m), 1.52–2.70 (11H, m), 2.83–4.93 (15H, m),

6.58–6.92 (2H, m), 7.78 (1H, d, J = 6.8 Hz), 7.14 (2H,

t, J = 7.8 Hz), 8.45 (1H, s), 8.56 (1H, s), 12.12 (1H,

br); MS (FAB), m/z 567 (M⁺+1); Anal. Calcd for

C₃₀H₃₈N₄O₇Æ1.5H₂O: C, 60.69; H, 6.96; N, 9.44. Found:

C, 60.70; H, 6.75; N, 9.36; IR (KBr) 3346, 3059, 2935,

1707, 1624, 1531, 1485, 1454 cm^ꢀ1

.

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

2733

5.4. 4-[(4R)-Benzyloxy-1-[3-methoxy-4-[N⁰-(2-methyl-

phenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-

pipirazinylacetic acid (11c)

5.5.2. Ethyl 1-[(4R)-benzyloxy-(2S)-pyrrolidinylcarbon-

yl]-4-piperidinylacetate (7c). Pale yellow oil (yield, 85%).

¹H NMR (CDCl₃) d 1.11–1.28 (5H, m), 1.75–1.82 (3H,

m), 2.03–2.25 (6H, m), 2.61–2.63 (1H, m), 2.88–3.04

(2H, m), 3.29–3.34 (1H, m), 3.80–3.90 (1H, m),

4.08–4.16 (4H, m), 4.44–4.59 (2H, m), 7.27–7.34 (5H,

m); MS (ESI), m/z 375 (M⁺+1).

5.4.1. Ethyl 1-[(4R)-benzyloxy-1-tert-butoxycarbonyl-

(2S)-pyrrolidinylcarbonyl]-1-piperazinylacetate. Color-

1

less oil (yield, quant.). H NMR (CDCl₃) d 1.28 (3H, t,

J = 6.9 Hz), 1.40 (9H, s), 1.92–2.44 (4H, m), 2.49–2.71

(4H, m), 3.18–3.30 (2H, m), 3.46–3.81 (5H, m), 4.19

(2H, q, J = 7.4 Hz), 4.39–4.86 (3H, m), 7.27–7.39 (5H,

m); MS (ESI), m/z 476 (M⁺+1).

5.5.3. Ethyl 4-[(4R)-Benzyloxy-1-[3-methoxy-4-[N⁰-(2-

methylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcar-

bonyl]-4-piperidinylacetate (10c). Colorless amorphous

1

solid (yield, 81%). H NMR (CDCl₃) d 1.00–1.12 (1H,

5.4.2. Ethyl 1-[(4R)-benzyloxy-(2S)-pyrrolidinylcarbon-

yl]-1-piperazinylacetate (6c). Colorless oil (yield, 94%).

¹H NMR (CDCl₃) d 1.28 (3H, t, J = 7.1 Hz), 1.73–1.90

(3H, m), 2.16 (1H, ddd, J = 13.0, 7.6, 2.7 Hz), 2.53–

2.63 (3H, m), 3.03 (1H, dd, J = 12.3, 3.4 Hz), 3.24

(2H, s), 3.32 (1H, dd, J = 12.0, 5.4 Hz), 3.42–3.77 (4H,

m), 4.08–4.24 (3H, m), 4.41–4.60 (2H, m), 7.27–7.37

(5H, m); MS (ESI) m/z 376 (M⁺+1).

m), 1.22–1.27 (3H, m), 1.45–2.60 (13H, m), 1.97–4.50

(14H, m), 4.98–5.00 (1H, m), 6.44 (1H, s), 6.76–6.79

(2H, m), 7.11–7.33 (9H, m), 7.51–7.53 (1H, m), 8.03–

8.05 (1H, m); MS (FAB), m/z 671 (M⁺+1); HR-MS

(FAB) calcd for C₃₈H₄₇N₄O₇: 671.3445. Found:

671.3474.

5.5.4. 4-[(4R)-Benzyloxy-1-[3-methoxy-4-[N⁰-(2-methyl-

phenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-4-

piperidinylacetic acid (12c). Colorless amorphous solid

5.4.3. Ethyl 4-[(4R)-benzyloxy-1-[3-methoxy-4-[N⁰-(2-

methylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcar-

bonyl]-1-pipirazinylacetate (9c). Colorless amorphous

1

(yield, 86%). H NMR (DMSO-d₆) d 0.98–1.35 (3H,

m), 1.64–2.57 (10H, m), 3.04–4.88 (14H, m), 6.77–

6.79 (1H, m), 6.92–6.95 (2H, m), 7.11–7.17 (2H, m),

7.25–7.35 (5H, m), 7.78–7.80 (1H, m), 8.00–8.03

(1H, m), 8.46 (1H, s), 8.55 (1H, s); MS (FAB), m/z

643 (M⁺+1); Anal. Calcd for C₃₆H₄₂N₄O₇Æ2.5H₂O:

C, 62.87; H, 6.89; N, 8.15. Found: C, 62.26; H,

6.29; N, 7.74; IR (KBr) 3338, 3060, 3006, 2933,

1

solid (yield, 72%). H NMR (CDCl₃) d 1.24–1.30 (4H,

m), 2.00–2.08 (1H, m), 2.17–2.25 (1H, m), 2.29 (3H, s),

2.46–2.71 (4H, m), 3.46–3.85 (12H, m), 4.07–4.56 (5H,

m), 4.97 (1H, dd, J = 6.9, 8.1 Hz), 6.28 and 6.34 (total

1H, each s, amide isomers), 6.69 and 6.79 (total 2H, each

d, J = 7.4 and 8.3 Hz, respectively, amide isomers),

7.02–7.38 (9H, m), 7.49–7.54 (1H, m), 8.05 (1H, d,

J = 7.8 Hz); MS (FAB) m/z 672 (M⁺+1); Anal. Calcd

for C₃₅H₄₅N₅O₇Æ0.75H₂O: C, 64.85; H, 6.84; N, 10.22.

Found: C, 64.93; H, 6.81; N, 10.08; IR (ATR) 3344,

1724, 1624, 1599, 1533 cm^ꢀ1

.

5.6. 4-[1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ureido]phenyl-

acetyl]-(3R,4S)-isopropylidenedioxy-(2S)-pyrrolidinyl-

carbonyl]-1-piperazinylacetic acid (11d)

2979, 2935, 2862, 1736, 1705, 1622, 1589, 1527 cm^ꢀ1

.

5.4.4. 4-[(4R)-Benzyloxy-1-[3-methoxy-4-[N⁰-(2-methyl-

phenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-

1-pipirazinylacetic acid (11c). Colorless amorphous solid

(yield, quant.). ¹H NMR (DMSO-d₆) d 1.68–2.12 (2H,

m), 2.43–2.80 (8H, m), 3.14–3.67 (16H, m), 3.84 and

3.87 (total 3H, each s, amide isomers), 4.19–4.36

(1H, m), 4.85 (1H, t, J = 7.4 Hz), 4.96–5.20 (1H, m),

6.62–6.79 (1H, m), 6.79–6.97 (2H, m), 7.10 (1H, d,

J = 7.8 Hz), 7.15 (1H, d, J = 7.8 Hz), 7.77 (1H, d,

J = 8.1 Hz), 7.98 and 7.99 (total 1H, each d, J = 8.3

and 8.1 Hz, respectively, amide isomers), 8.47 (1H,

s), 8.54 (1H, s); MS (FAB) m/z 644 (M⁺+1); HR-

MS (FAB) calcd for C₃₅H₄₂N₅O₇: 644.3084. Found:

644.3064; Anal. Calcd for C₃₅H₄₁N₅O₇Æ4H₂O: C,

58.73; H, 6.90; N, 9.78. Found: C, 58.39; H, 6.55;

N, 9.65; IR (ATR) 3334, 3028, 2945, 2873, 1620,

5.6.1. Ethyl 4-[N-benzyloxycarbonyl-(3R,4S)-isopropyl-

idenedioxy-(2S)-pyrrolidinylcarbonyl]-1-piperazinylace-

tate. Yellow oil (yield, 79%). H NMR (CDCl₃) d 1.23–

1

1.30 (3H, m), 1.31 (3H, s), 1.43 and 1.46 (total 3H, each

s, amide isomers), 2.24–2.70 (4H, m), 3.12 and 3.23 (total

2H, each s, amide isomers), 3.48–3.84 (5H, m), 3.94 and

3.99 (total 1H, each d, each J = 2.2 Hz, amide isomers),

4.19 (2H, q, J = 7.1 Hz), 4.61 and 4.67 (total each 1H, d,

each J = 5.9 Hz, amide isomers), 4.74 and 4.90 (total

1H, each s, amide isomers), 4.81 (1H, m, 1H), 5.04 and

5.10 (total 1H, each d, each J = 12.5 Hz, amide isomers),

5.16 and 5.19 (total 1H, each d, each J = 12.5 Hz, amide

isomers), 7.25–7.35 (5H, m); MS (FAB) m/z 476 (M⁺+1).

5.6.2. Ethyl 4-[(3R,4S)-isopropylidenedioxy-(2S)-pyrro-

lidinylcarbonyl]-1-piperazinylacetate (6d). A suspension

of ethyl 4-[N-benzyloxycarbonyl-(3R,4S)-isopropyl-

idenedioxy-(2S)-pyrrolidinylcarbonyl]-1-piperazinylac-

etate (1.53 g, 3.22 mmol) and 5% Pd/C (52.2% wet,

1.23 g) in EtOH (30 ml) was hydrogenated under 1

atom hydrogen atmosphere at room temperature for

10 h. After the catalyst was removed by ﬁltration,

the ﬁltrate was concentrated to dryness to aﬀord

the title compound (6d) (0.99 g, 90%) as a yellow

oil. ¹H NMR (CDCl₃) d 1.28 (3H, t, J = 7.3 Hz),

1.33 (3H, s), 1.49 (3H, s), 2.00 (1H, s), 2.53-2.64

(4H, m), 3.00 (1H, dd, J = 13.0, 3.9 Hz), 3.05 (1H,

1529, 1485, 1452 cm^ꢀ1

.

5.5. 4-[(4R)-Benzyloxy-1-[3-methoxy-4-[N⁰-(2-methyl-

phenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-4-

piperidinylacetate (12c)

5.5.1. Ethyl 1-[(4R)-benzyloxy-1-(tert-butoxycarbonyl)-

(2S)-pyrrolidinylcarbonyl]-4-piperidinylacetate. Colorless

1

oil (yield, 58%). H NMR (CDCl₃) d 1.08–2.95 (23H,

m), 3.58–4.81 (10H, m), 7.28–7.36 (5H, m); MS (ESI),

m/z 475 (M⁺+1).

2734

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

dd, J = 13.0, 1.2 Hz), 3.24 (2H, s), 3.60–3.74 (4H, m),

3.96 (1H, s), 4.19 (2H, q, J = 7.3 Hz), 4.77 (1H, m),

4.87 (1H, dd, J = 5.6, 1.2 Hz); MS (FAB) m/z 342

(M⁺+1).

5.7.3. Ethyl 4-[1-[3-methoxy-4-[N⁰-(2-methylphenyl)ure-

ido]phenylacetyl]-(4S)-phenoxy-(2S)-pyrrolidinylcarbonyl]-

1-piperazinylacetate (9e). Colorless amorphous solid

(yield, 86%). ¹H NMR (CDCl₃) d 1.26 (3H, t,

J = 4.4 Hz), 2.15 (1H, m), 2.29 (3H, s), 2.30–2.81 (8H,

m), 2.95–4.30 (12H, m), 4.84–5.05 (2H, m), 6.33 (1H,

m), 6.72–7.52 (6H, m), 7.11–7.52 (6H, m), 8.12 (1H, d,

J = 7.8 Hz); MS (FAB), m/z 658 (M⁺+1); Anal. Calcd

for C₃₆H₄₃N₅O₇Æ1.25H₂O: C, 63.56; H, 6.74; N, 10.29.

Found: C, 63.61; H, 6.64; N, 10.29; IR (ATR) 3340,

5.6.3. Ethyl 4-[1-[3-methoxy-4-[N⁰-(2-methylphenyl)ure-

ido]phenylacetyl]-(3R,4S)-isopropylidenedioxy-(2S)-pyr-

rolidinylcarbonyl]-1-piperazinylacetic

acetate

(9d).

Yellow amorphous solid (yield, 92%). ¹H NMR

(CD₃OD) d 1.27 (3H, t, J = 7.1 Hz), 1.29 (3H, s),

1.38 (3H, s), 2.27 (3H, s), 2.45–2.49 (1H, m), 2.56–

2.70 (3H, m), 3.20 (2H, s), 3.54 (1H, ddd, J = 13.2,

7.6, 3.2 Hz), 3.61–3.86 (10H, m), 4.18 (2H, q,

J = 7.1 Hz), 4.63 (1H, d, J = 6.4 Hz), 4.80–4.84 (1H,

m), 5.11 (1H, s), 6.41 (1H, s), 6.76 (1H, d,

J = 1.7 Hz), 6.80 (1H, dd, J = 8.3, 1.7 Hz), 7.10–7.15

(2H, m), 7.21–7.24 (2H, m), 7.52 (1H, d, J = 7.6

Hz), 8.05 (1H, d, J = 8.3 Hz); MS (FAB) m/z 638

(M⁺+1) ; HR-MS (FAB) calcd for C₃₃H₄₄N₅O₈:

638.3190. Found: 638.3200; IR (KBr) 3351, 2985,

2983, 2937, 1732, 1701, 1626, 1597, 1587 cm^ꢀ1

.

5.7.4. 4-[1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ure-

ido]phenylacetyl]-(4S)-phenoxy-(2S)-pyrrolidinylcarbonyl]-

1-piperazinylacetic acid (11e). Colorless amorphous solid

(yield, quant.). ¹H NMR (CD₃OD) d 2.10–2.83 (7H, m),

2.92 and 3.02 (total 1H, each s, amide isomers), 3.46–

3.88 (10H, m), 3.91 (3H, s), 4.97–5.09 (2H, m), 6.74–

7.04 (9H, m), 7.14–7.28 (3H, m), 7.38 and 7.57 (total

1H, each d, each J = 7.8 Hz, amide isomers), 7.95–8.05

(1H, m); MS (FAB), m/z 630 (M⁺+1); HR-MS (FAB)

calcd for C₃₄H₄₀N₅O₇: 630.2916. Found: 630.2928; IR

2937, 1743, 1708, 1631, 1533, 1455 cm^ꢀ1

.

5.6.4. 4-[1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ureido]pheny-

lacetyl]-(3R,4S)-isopropylidenedioxy-(2S)-pyrrolidinyl-

carbonyl]-1-piperazinylacetic acid (9d). Colorless

amorphous solid (yield, 37%). ¹H NMR (CD₃OD)

d 1.40 (6H, s), 2.13–2.21 (2H, m), 2.89 (3H, s),

2.95–3.20 (4H, m), 3.52 (2H, s), 3.57–3.78 (4H,

m), 3.89 and 3.91 (total 3H, each s, amide isomers),

4.00–4.03 (2H, m), 4.74 (1H, m), 4.92 (1H, m), 5.04

(1H, s), 6.75 (1H, m), 6.81–6.93 (2H, m), 7.02 (1H,

t, J = 7.8 Hz), 7.14–7.20 (2H, m), 7.56 (1H, m),

8.00 and 8.02 (total 1H, each s, amide isomers),

8.32 and 8.35 (total 1H, each s, amide isomers);

MS (FAB) m/z 610 (M⁺+1); HR-MS (FAB) calcd

for C₃₁H₄₀N₅O₈: 610.2877. Found: 610.2841; IR

(ATR) 3329, 2989, 2937, 2860, 1624, 1531,

(ATR) 3328, 3012, 2941, 1626, 1599, 1529, 1487 cm^ꢀ1

.

5.8. 4-[(2S,4S)-1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ure-

ido]phenylacetyl]-4-(2-naphthyloxy)-2-pyrrolidinylcarbon-

yl]-1-piperazinylacetic acid (11f)

5.8.1. Ethyl 4-[1-tert-butoxycarbonyl-(4S)-(2-naphthyl-

oxy)-(2S)-pyrrolidinylcarbonyl]-1-piperazinylacetate.

Yellow oil (yield, 99%). ¹H NMR (CDCl₃) d 1.23

(3H, q, J = 7.1 Hz), 1.43 and 1.48 (total 9H, each s,

amide isomers), 2.21 (1H, m), 2.36 (1H, m), 2.43–

2.80 (4H, m), 3.08 (1H, s), 3.16 (1H, s), 3.42–4.10

(6H, m), 4.18 (2H, t, J = 7.1 Hz), 4.57–5.13 (2H,

m), 7.11 (2H, m), 7.37 (1H, t, J = 7.6 Hz), 7.44

(1H, t, J = 7.1 Hz), 7.73 (3H, m); MS (ESI), m/z

512 (M⁺+1).

1485 cm^ꢀ1

.

5.7. 4-[1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ureido]pheny-

lacetyl]-(4S)-phenoxy-(2S)-pyrrolidinylcarbonyl]-1-piper-

azinylacetic acid (11e)

5.8.2. 4-[(4S)-(2-Naphthyloxy)-(2S)-pyrrolidinylcarbon-

yl]-1-piperazinylacetate (6f). Yellow oil (yield, 94%). H

1

NMR (CDCl₃) d 1.29 (3H, t, J = 7.1 Hz), 2.03 (1H,

m), 2.53 (4H, m), 3.01 (1H, dd, J = 13.0, 4.2 Hz), 3.17

(2H, s), 3.42–3.82 (6H, m), 3.96 (1H, dd, J = 9.3,

6.4 Hz), 4.16 (2H, q, J = 9.3, 6.4 Hz), 4.98 (1H, s), 7.06

(1H, d, J = 2.4 Hz), 7.09 (1H, dd, J = 8.8, 2.4 Hz), 7.33

(1H, dt, J = 8.1, 1.2 Hz), 7.42 (1H, dt, J = 8.1, 1.2 Hz),

7.73 (3H, m); MS (ESI), m/z 412 (M⁺+1).

5.7.1. Ethyl 4-[1-tert-butoxycarbonyl-(4S)-phenoxy-(2S)-

pyrrolidinylcarbonyl]-1-piperazinylacetate. Colorless oil

(yield, 69%). ¹H NMR (CDCl₃) d 1.27 (3H, t,

J = 7.1 Hz), 1.42 and 1.47 (total 9H, each s, amide iso-

mers), 2.13 (1H, dd, J = 11.0, 3.9 Hz), 2.40 (1H, s),

2.56 (4H, m), 3.15 (1H, s), 3.20 (1H, s), 3.49 (1H, s),

3.66 (4H, m), 3.96 (1H, m), 4.17 (2H, ABq,

J = 1.9 Hz), 4.58–4.89 (2H, m), 6.84 (2H, d,

J = 8.1 Hz), 6.96 (1H, t, J = 6.6 Hz), 7.27 (2H, s); MS

(ESI) m/z 462 (M⁺+1).

5.8.3. Ethyl 4-[1-[3-methoxy-4-[N⁰-(2-methylphenyl)ure-

ido]phenylacetyl]-(4S)-(2-naphthyloxy)-(2S)-pyrrolidinyl-

carbonyl]-1-piperazinylacetate HCl salt (9f). Colorless

amorphous solid (yield, 50%). ¹H NMR (CDCl₃) d

1.24 (3H, t, J = 6.9 Hz), 2.19 (1H, s), 2.29 (3H, s),

2.30–2.70 (8H, m), 3.11 (2H, s), 3.32–3.98 (8H, m),

4.14 (2H, q, J = 6.9 Hz), 4.99 (2H, m), 6.33 (1H, m),

6.82 (1H, m), 7.00–7.28 (5H, m), 7.35 (1H, t,

J = 7.1 Hz), 7.44 (1H, t, J = 7.1 Hz), 7.51 (1H, d,

J = 8.1 Hz), 7.67–7.76 (3H, m), 8.06 (1H, d,

J = 8.3 Hz); MS (FAB), m/z 708 (M⁺+1); Anal. Calcd

for C₄₀H₄₅N₅O₇ÆHClÆ2.5H₂O: C, 60.87; H, 6.51; N,

8.87. Found: C, 60.81; H, 6.40; N, 8.79; IR (KBr)

5.7.2. 4-[(4S)-Phenoxy-(2S)-pyrrolidinylcarbonyl]-1-pipe-

razinylacetate (6e). Yellow amorphous solid (yield,

56%). ¹H NMR (CDCl₃) d 1.27 (3H, t, J = 6.8 H),

2.31 (1H, d, J = 13.7 Hz), 2.79 (1H, m), 3.02–3.23

(4H, m), 3.52–3.73 (6H, m), 3.79 (1H, d,

J = 11.9 Hz), 4.18 (3H, m), 5.05 (1H, dd, J = 10.5,

3.4 Hz), 5.09 (1H, s), 6.80 (2H, d, J = 8.0 Hz), 7.04

(1H, t, J = 7.6 Hz), 7.30 (2H, t, J = 7.8 Hz); MS

(ESI) m/z 362 (M⁺+1).

3253, 2978, 1747, 1670, 1628, 1533, 1241 cm^ꢀ1

.

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

2735

5.8.4. 4-[1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ureido]phenyl-

acetyl]-(4S)-(2-naphthyloxy)-(2S)-pyrrolidinylcarbon-

J = 2.7 Hz, amide isomers), 4.54 (1H, d, J = 11.9 Hz),

4.58 (1H, d, J = 11,9 Hz), 4.83 and 5.01 (total 1H, each

s, amide isomers), 6.67 and 6.76 (total 1H, each dd, each

J = 8.1, 1.4 Hz, amide isomers), 6.80 and 6.92 (total 1H,

each d, each J = 1.4 Hz, amide isomers), 6.93 (1H, t,

J = 7.6 Hz), 7.11–7.17 (2H, m), 7.27–7.38 (5H, m),

7.79 (1H, d, J = 7.6 Hz), 8.01 (1H, d, J = 8.1 Hz), 8.46

(1H, s), 8.55 (1H, s); MS (FAB) m/z 644 (M⁺+1); Anal.

Calcd for C₃₅H₄₁N₅O₇Æ0.5H₂O: C, 62.86; H, 6.33; N,

10.47. Found C, 62.69; H, 6.68; N, 10.58; IR (KBr)

yl]-1-piperazinylacetic

acid

(11f).

Colorless

1

amorphous solid (yield, 80%). H NMR (DMSO-d₆)

d 1.74 (1H, s), 1.90 (1H, m), 2.00–3.70 (14H, m),

2.49 (2H, s), 3.85 (3H, s), 3.98–5.20 (2H, m), 6.65–

7.85 (14H, m), 8.46 (1H, s), 8.55 (1H, s); MS

(FAB), m/z 680 (M⁺+1); Anal. Calcd for

C₃₈H₄₁N₅O₇ÆEtOHÆH₂O: C, 64.59; H, 6.64; N, 9.41.

Found: C, 64.34; H, 6.37; N, 9.31; IR (KBr) 3346,

2939, 1628, 1533, 1454, 1257 cm^ꢀ1

.

3353, 2938, 1633, 1533, 1454, 1415 cm^ꢀ1

.

5.9. 4-[(3S)-Benzyloxy-1-[3-methoxy-4-[N⁰-(2-methylphe-

nyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-pip-

erazinylacetic acid (11g)

5.10. 4-[(4S)-Fluoro-1-[3-methoxy-4-[N⁰-(2-methylphenyl)-

ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-piper-

azinylacetic acid (11h)

5.9.1. Ethyl 4-[(3S)-benzyloxy-N-tert-butoxycarbonyl-

(2S)-pyrrolidinylcarbonyl]-1-piperazinylacetate. Yellow

oil (yield, 95%). ¹H NMR (CDCl₃) d 1.28 (3H, t,

J = 7.1 Hz), 1.39 and 1.46 (total 9H, each s, amide iso-

mers), 2.03–2.07 (1H, m), 2.30–3.34 (1H, m), 2.42–2.68

(4H, m), 3.17 (2H, s), 3.41–3.51 (2H, m), 3.53–3.73 (4H,

m), 3.97 (1H, m), 4.19 (2H, q, J = 7.1 Hz), 4.41 and 4.43

(total 1H, each d, each J = 11.9 Hz, amide isomers),

4.61 and 4.63 (total 1H, each d, each J = 11.9 Hz, amide

isomers), 4.53 and 4.70 (total 1H, each s, amide isomers),

7.23–7.37 (5H, m); MS (FAB) m/z 476 (M⁺+1).

5.10.1. Ethyl 4-[1-(tert-butoxycarbonyl)-(4S)-ﬂuoro-(2S)-

pyrrolidinylcarbonyl]-1-piperazinylacetate. Pale yellow oil

(yield, 61%). ¹H NMR (CDCl₃) d 1.28 (3H, t, J = 7.8 Hz),

1.42 and 1.47 (total 9H, each s, amide isomers), 2.11–2.82

(6H, m), 3.22–3.25 (2H, m), 3.42–3.98 (6H, m), 4.19 (2H,

q, J = 7.1 Hz), 4.61 and 4.74 (total 1H, each d, J = 9.3 and

9.6 Hz, respectively, amide isomers), 5.21 (1H, d, J = 53.9

Hz). MS (ESI) m/z, 388 (M⁺+1).

5.10.2. Ethyl 4-[(4S)-ﬂuoro-(2S)-pyrrolidinylcarbonyl]-1-

piperazinylacetate (6h). Yellow oil (yield, 86%). ¹H NMR

(CDCl₃) d 1.28 (3H, t, J = 7.1 Hz), 1.96–2.96 (6H, m), 3.25

(2H, s), 3.46–3.92 (7H, m), 4.19 (2H, q, J = 7.1 Hz), 5.10–

5.25 (1H, m); MS (ESI) m/z, 288 (M⁺+1).

5.9.2. Ethyl 4-[(3S)-benzyloxy-(2S)-pyrrolidinylcarbonyl]-

1-piperazinylacetate (6g). Yellow oil (yield, 100%). H

1

NMR (CDCl₃) d 1.28 (3H, t, J = 7.1 Hz), 1.70–1.74

(1H, m), 1.97–2.01 (1H, m), 2.38 (1H, ddd, J = 14.7,

6.8, 3.4 Hz), 2.46 (1H, ddd, J = 14.7, 6.8, 3.4 Hz),

2.49–2.65 (2H, m), 3.02 (1H, ddd, J = 11.2, 9.3, 6.1

Hz), 3.18 (2H, s), 3.23 (1H, m), 3.52 (1H, ddd,

J = 13.2, 6.8, 3.4 Hz), 3.60 (1H, ddd, J = 13.2, 6.8,

3.4 Hz), 3.67–3.76 (2H, m), 3.91 (1H, d, J = 3.5 Hz),

4.03 (1H, m), 4.19 (2H, q, J = 7.1 Hz), 4.38 (1H, d,

J = 11.5 Hz), 4.60 (1H, d, J = 11.5 Hz), 7.20–7.38 (5H,

m); MS (FAB) m/z 376 (M⁺+1).

5.10.3. Ethyl 4-[(4S)-ﬂuoro-1-[3-methoxy-4-[N⁰-(2-meth-

ylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbon-

yl]-1-piperazinylacetate (9h). Yellow amorphous solid

(yield, 88%). ¹H NMR (CDCl₃) d 1.27 (3H, t,

J = 7.1 Hz), 2.15–2.71 (9H, m), 3.21 and 3.23 (total

3H, each s, amide isomers), 3.40–4.09 (10H, m), 4.15–

4.21 (2H, m), 4.98 (1H, dd, J = 9.6, 2.5 Hz), 5.16–5.29

(1H, m), 6.48–7.27 (7H, m), 7.52–7.54 (1H, m), 8.04-

8.05 (1H, m); MS (FAB) m/z, 584 (M⁺+1); HR-MS

(FAB) calcd for C₃₀H₃₉FN₅O₆: 584.2884. Found:

584.2866; IR (ATR) 3344, 2978, 2937, 2829, 1736,

5.9.3. Ethyl 4-[(3S)-benzyloxy-1-[3-methoxy-4-[N⁰-(2-

methylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbon-

yl]-1-piperazinylacetate (9g). Yellow amorphous solid

1701, 1630, 1589 cm^ꢀ1

.

1

(yield, 81%). H NMR (CDCl₃) d 1.26 (t, J = 7.3 Hz,

5.10.4. 4-[(4S)-Fluoro-1-[3-methoxy-4-[N⁰-(2-methylphe-

nyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-pip-

erazinylacetic acid (11h). Colorless amorphous solid

3H), 1.96 (m, 1H), 2.12 (m, 1H), 2.22 (s, 3H), 2.34–

2.64 (4H, m), 3.10 (2H, s), 3.47–3.57 (4H, m), 3.60

(2H, s), 3.64 (3H, s), 3.69–3.79 (2H, m), 3.95 (1H,

m), 4.17 (2H, q, J = 7.3 Hz), 4.42 (1H, d,

J = 12.0 Hz), 4.56 (1H, d, J = 12.0 Hz), 4.92 (1H, s),

6.71–7.00 (2H, m), 7.02–7.06 (2H, m), 7.11–7.35 (7H,

m), 7.47 (1H, s), 7.61 (1H, d, J = 7.6 Hz), 8.04 (1H,

d, J = 8.3 Hz); MS (FAB) m/z 672 (M⁺+1); HR-MS

(FAB) calcd for C₃₇H₄₆N₅O₇: 672.3397. Found:

672.3386.

1

(yield, 33%). H NMR (DMSO-d₆) d 1.96–3.88 (22H,

m), 4.91 and 5.07 (total 1H, each d, each J = 8.8 Hz,

amide isomers), 5.18–5.38 (1H, m), 6.67–7.17 (5H, m),

7.78 (1H, d, J = 8.1 Hz), 8.00 (1H, d, J = 8.1 Hz), 8.49

(1H, s), 8.57 (1H, s); MS (FAB) m/z, 556 (M⁺+1);

HR-MS (FAB) calcd for C₂₈H₃₅FN₅O₆: 556.2571.

Found: 556.2609; IR (ATR) 3330, 2943, 2873, 2810,

1628, 1587, 1529, 1485, 1448 cm^ꢀ1

.

5.9.4. 4-[(3S)-Benzyloxy-1-[3-methoxy-4-[N⁰-(2-methyl-

phenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-

1-piperazinylacetic acid (11g). Yellow amorphous sol-

5.11. 4-[(4R)-Fluoro-1-[3-methoxy-4-[N⁰-(2-methylphe-

nyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-pip-

erazinylacetic acid (11i)

1

id (yield, 76%). H NMR (DMSO-d₆) d 1.81–2.09 (m,

2H), 2.25 (s, 3H), 2.35–2.51 (m, 4H), 3.13 (s, 2H),

3.17–3.52 (4H, m), 3.63 (2H, s), 3.66–3.99 (2H, m),

3.86 (3H, s), 3.98 and 4.10 (total 1H, each d, each

5.11.1. Ethyl 4-[1-(tert-butoxycarbonyl)-(4R)-ﬂuoro-(2S)-

pyrrolidinylcarbonyl]-1-piperazinylacetate. Colorless oil

(yield, 66%). ¹H NMR (CDCl₃) d 1.28 (3H, t,

2736

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

J = 7.1 Hz), 1.42 and 1.46 (total 9H, each s, amide iso-

mers), 2.02–2.73 (6H, m), 3.25 (2H, d, J = 5.6 Hz),

3.58–3.98 (6H, m), 4.19 (2H, q, J = 7.1 Hz), 4.75 and

4.87 (total 1H, each t, J = 8.2 and 7.8 Hz, respectively,

amide isomers), 5.15–5.31 (1H, m); MS (ESI) m/z 388

(M⁺+1).

6.28 (1H, s), 6.78 (1H, s), 6.79 (1H, d, J = 7.8 Hz),

7.11–7.30 (4H, m), 7.50 (1H, d, J = 7.8 Hz), 8.08 (1H,

d, J = 7.8 Hz). MS (FAB) m/z 602 (M⁺+1); HR-MS

(FAB) calcd for C₃₀H₃₈F₂N₅O₆: 602.2790. Found:

602.2799.

5.12.4. 4-[4,4-Diﬂuoro-1-[3-methoxy-4-[N⁰-(2-methylphe-

nyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-pip-

erazinylacetic acid (11j). Colorless amorphous solid

5.11.2. Ethyl 4-[(4R)-ﬂuoro-(2S)-pyrrolidinylcarbonyl]-1-

piperazinylacetate (6i). Yellow oil (yield, 77%). ¹H NMR

(CDCl₃) d 1.28 (3H, t, J = 7.1 Hz), 1.83–1.99 (1H, m),

2.26–2.37 (1H, m), 2.54–2.64 (4H, m), 3.13–3.77 (8H,

m), 4.13–4.22 (3H, m), 5.27 (1H, dt, J = 54.9, 4.2 Hz);

MS (ESI) m/z 288 (M⁺+1).

1

(yield, 86%). H NMR (DMSO-d₆) d 2.26 (3H, s), 2.30–

4.25 (19H, m), 5.09–5.38 (1H, m), 6.75–7.17 (5H, m),

7.75–7.77 (1H, m), 7.99–8.01 (1H, m), 8.66–8.70 (2H, m);

MS (FAB) m/z, 574 (M⁺+1); HR-MS (FAB) calcd for

C₂₈H₃₄F₂N₅O₆: 574.2477. Found: 574.2488; IR (KBr)

5.11.3. Ethyl 4-[(4R)-ﬂuoro-1-[3-methoxy-4-[N⁰-(2-meth-

ylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-

1-piperazinylacetate (9i). Colorless amorphous solid

(yield, 97%). ¹H NMR (CDCl₃) d 1.27 (3H, t,

J = 7.1 Hz), 2.05–2.22 (1H, m), 2.28 (3H, s), 2.36–2.70

(5H, m), 3.21 (2H, s), 3.53–3.90 (11H, m), 4.18 (2H, q,

J = 7.1 Hz), 5.05 (1H, t, J = 8.1 Hz), 5.20–5.33 (1H,

m), 6.43 (1H, s), 6.79–6.80 (2H, m), 7.11–7.26 (4H, m),

7.52–7.54 (1H, m), 8.06 (1H, d, J = 8.8 Hz); MS

(FAB) m/z 584 (M⁺+1); HR-MS (FAB) calcd for

C₃₀H₃₉FN₅O₆: 584.2884. Found: 584.2870. Anal. Calcd

for C₃₀H₃₈FN₅O₆Æ1.75H₂O: C, 58.57; H, 6.80; N, 11.38.

Found: C, 58.60; H, 6.41; N, 11.26; IR (ATR) 3346,

3400, 3392, 2952, 1641, 1539, 1485, 1454, 1417 cm^ꢀ1

.

5.13. 4-[4-Chloro-1-[3-methoxy-4-[N⁰-(2-methylphenyl)ure-

ido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-piperaz-

inylacetic acid (9k)

5.13.1. Ethyl 4-[1-(tert-butoxycarbonyl)-(4S)-chloro-(2S)-

pyrrolidinylcarbonyl]-1-piperazinylacetate. Colorless oil

(yield, 99%). ¹H NMR (CDCl₃) d 1.28 (3H, t,

J = 7.1 Hz), 1.40 and 1.46 (total 9H, each s, amide iso-

mers), 2.04–2.13 (1H, m), 2.58–2.78 (5H, m), 3.23–3.24

(2H, m), 3.51–3.73 (5H, m), 4.04–4.25 (4H, m), 4.52–

4.68 (1H, m); MS (FAB), m/z 404 (M⁺+1).

2981, 2937, 1739, 1703, 1626, 1589, 1529 cm^ꢀ1

.

5.13.2. Ethyl 4-[(4S)-chloro-(2S)-pyrrolidinylcarbonyl]-1-

piperazinylacetate (6k). Pale yellow oil (yield, 90%). H

1

5.11.4. 4-[(4R)-Fluoro-1-[3-methoxy-4-[N⁰-(2-methylphe-

nyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-pip-

erazinylacetic acid (11i). Colorless amorphous solid

(yield, 39%). ¹H NMR (DMSO-d₆) d 1.91–2.03 (1H,

m), 2.24 (3H, s), 2.40–2.60 (5H, m), 2.98 (2H, s), 3.24–

3.67 (7H, m), 3.85–4.01 (4H, m), 4.91–5.40 (2H, m),

6.65–7.26 (5H, m), 7.77–7.79 (1H, m), 7.98–8.01 (1H,

m), 8.50 (1H, s), 8.58–8.59 (1H, m); MS (FAB) m/z,

556 (M⁺+1); HR-MS (FAB) calcd for C₂₈H₃₄FN₅O₆:

556.2571. Found: 556.2543; IR (KBr) 3338, 2931,

NMR (CDCl₃) d 1.28 (3H, t, J = 7.1 Hz), 1.89 (1H, s),

2.01–2.08 (1H, m), 2.54–2.62 (5H, m), 3.09 (1H, dd,

J = 4.4, 12.7 Hz), 3.25 (2H, s), 3.34 (1H, d,

J = 12.7 Hz), 3.41–3.53 (2H, m), 3.69–3.78 (2H, m),

3.89–3.94 (1H, m), 4.19 (2H, q, J = 7.1 Hz), 4.41–4.43

(1H, m); MS (FAB), m/z 304 (M⁺+1).

5.13.3. Ethyl 4-[(4S)-chloro-1-[3-methoxy-4-[N⁰-(2-methyl-

phenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-

1-piperazinylacetate (9k). Colorless amorphous solid

1635, 1600, 1531, 1495, 1487 cm^ꢀ1

.

1

(yield, 91%). H NMR (CDCl₃) d 1.25–1.29 (3H, m),

5.12. 4-[4,4-Diﬂuoro-1-[3-methoxy-4-[N⁰-(2-methylphe-

nyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-pip-

erazinylacetic acid (11j)

2.04–2.12 (1H, m), 2.25 (3H, s), 2.55–2.75 (5H, m), 3.21

(2H, s), 3.51–3.80 (11H, m), 4.02–4.07 (1H, m), 4.15–

4.21 (2H, m), 4.81–4.85 (1H, m), 6.74–6.83 (3H, m),

7.08–7.34 (4H, m), 7.54–7.56 (1H, m), 8.05–8.07 (1H,

m); MS (FAB), m/z 600 (M⁺+1); HR-MS (FAB) calcd

for C₃₀H₃₉ClN₅O₆: 600.2589. Found: 600.2591; IR

(KBr) 3346, 2979, 2937, 2862, 1743, 1703, 1635,

5.12.1. Ethyl 4-[1-(tert-butoxycarbonyl)-4,4-diﬂuoro-(2S)-

pyrrolidinylcarbonyl]-1-piperazinylacetate. Yellow oil

(yield, 25%). ¹H NMR (CDCl₃) d 1.28 (3H, t,

J = 7.1 Hz), 1.41–1.47 (9H, m), 2.36–2.66 (6H, m), 3.24–

3.25 (2H, m), 3.51–3.95 (6H, m), 4.19 (2H, q, J = 7.1 Hz),

4.71–4.88 (1H, m); MS (ESI) m/z, 406 (M⁺+1).

1599 cm^ꢀ1

.

5.13.4. 4-[(4S)-Chloro-1-[3-methoxy-4-[N⁰-(2-methylphe-

nyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-pip-

erazinylacetic acid (11k). Colorless amorphous solid

(yield, 97%). ¹H NMR (DMSO-d₆) d 2.29 (3H, s),

2.39–2.66 (4H, m), 2.80–3.72 (12H, m), 3.85 and 3.88

(total 3H, each s, amide isomers), 4.09–4.58 (2H, m),

4.78–5.06 (1H, m), 6.66 and 6.76 (total 1H, each d, each

J = 8.0 Hz, amide isomers), 6.90–7.23 (4H, m), 7.77 (1H,

d, J = 9.6 Hz), 8.02 (1H, d, J = 8.0 Hz), 8.45 (1H, s),

8.54 (1H, s); MS (FAB), m/z 572 (M⁺+1); HR-MS

(FAB) calcd for C₂₈H₃₅ClN₅O₆: 572.2276. Found:

572.2281; IR (KBr) 3346, 3022, 2939, 1641, 1600,

5.12.2. Ethyl 4-[4,4-diﬂuoro-(2S)-pyrrolidinylcarbonyl]-1-

piperazinylacetate (11j). Yellow oil (yield, 45%). ¹H NMR

(CDCl₃) d 1.28 (3H, t, J = 7.1 Hz), 2.24–3.77 (15H, m),

4.12–4.23 (3H, m); MS (ESI), m/z 306 (M⁺+1).

5.12.3. Ethyl 4-[4,4-diﬂuoro-1-[3-methoxy-4-[N⁰-(2-meth-

ylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-

1-piperazinylacetate (9j). Yellow oil (yield, quant.). H

1

NMR (CDCl₃) d 1.27 (3H, t, J = 7.1 Hz), 2.30 (3H, s),

2.51–2.68 (4H, m), 3.23 (2H, s), 3.55–3.92 (13H, m),

4.18 (2H, q, J = 7.1 Hz), 5.05 (1H, dd, J = 8.8, 6.6 Hz),

1589, 1531, 1495 cm^ꢀ1

.

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

2737

5.14. ( )-4-[2-[3-Methoxy-4-[N⁰-(2-methylphenyl)ure-

ido]phenylacetyl]-1-isoindolinylcarbonyl]-1-piperazinyl-

acetic acid (11l)

7.41 (8H, m), 7.78—7.80 (1H, m), 8.02–8.04 (1H, m),

8.48 (1H, s), 8.57 (1H, s); MS (FAB), m/z 586

(M⁺+1); Anal. Calcd for C₃₂H₃₅N₅O₆Æ4.5H₂O: C,

57.65; H, 6.65; N, 10.50. Found: C, 57.84; H, 6.44;

N, 10.39; IR (KBr) 3354, 3016, 2931, 1705, 1633,

5.14.1. ( )-Ethyl 4-[2-(9-ﬂuorenylmethoxycarbonyl)-1-

isoindolinylcarbony]-1-piperazinylacetate. A mixture of

( )-2-(9-ﬂuorenylmethoxycarbonyl)-1-isoindolinecarb-

oxylic acid (958 mg, 2.49 mmol), ethyl 1-piperazinylace-

tate (4) (428 mg, 2.49 mmol), EDCÆHCl (572 mg,

2.98 mmol), HOBt (403 mg, 2.98 mmol), and Et₃N

(410 ml, 2.94 mmol) in CH₂Cl₂(10 ml) was stirred at

room temperature for 12 h. The mixture was poured

into water and extracted with CHCl₃. The combined ex-

tracts were washed with brine, dried over Na₂SO₄, and

evaporated. The residue was puriﬁed by column chro-

matography on silica gel with CHCl₃/MeOH (100:1,

v/v) as an eluent to give the title compound (1.07 g,

80%) as a colorless amorphous solid. ¹H NMR (CDCl₃)

d 1.22–1.30 (3H, m), 2.51–2.80 (4H, m), 3.15 and 3.27

(total 2H, each s, amide isomers), 3.59–3.74 (2H, m),

3.88–4.00 (2H, m), 4.11–4.59 (5H, m), 4.83–5.06 (2H,

m), 5.88 and 6.01 (total 1H, each d, each J = 2.2 Hz,

amide isomers), 6.99–7.78 (12H, m); FAB-MS, m/z 540

(M⁺+1).

1589, 1531, 1486 cm^ꢀ1

.

5.15. 4-[1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ure-

ido]phenylacetyl]-2,3-dihydro-1H-(2S)-indolylcarbon-

yl]-1-piperazinylacetic acid (11m)

5.15.1. Ethyl [1-tert-butoxycarbonyl-2,3-dihydro-1H-

(2S)-indolylcarbonyl]-1-piperazinylacetate. Yellow oil.

1

(yield, 54%). H NMR (CDCl₃) d 1.23–1.31 (4H, m),

1.43–1.66 (8H, m), 2.04 (1H, s), 2.53–2.89 (4H, m),

2.91–3.03 (1H, m), 3.26 (2H, s), 3.70–3.76 (5H, m),

4.12 and 4.20 (total 2H, each q, J = 7.4 and 7.1 Hz,

respectively, amide isomers), 5.01–5.29 (1H, m), 6.91

(1H, t, J = 7.5 Hz), 7.08 (1H, d, J = 7.4 Hz), 7.18 (1H,

s); MS (FAB) m/z 417 (M⁺+1).

5.15.2. Ethyl [2,3-Dihydro-1H-(2S)-indolylcarbonyl]-1-

piperazinylacetate (6m). Brown oil (yield, quant.). ¹H

NMR (CDCl₃) d 1.29 (3H, t, J = 7.1 Hz), 2.53–2.72

(4H, m), 3.14 (1H, dd, J = 15.7, 5.4 Hz), 3.27 (2H, s),

3.42–3.73 (5H, m), 4.20 (2H, q, J = 7.1 Hz), 4.54 (1H,

dd, J = 10.5, 5.6 Hz), 4.62 (1H, s), 6.72–6.80 (2H, m),

7.04 (2H, d, J = 7.6 Hz).; MS (FAB) m/z 317 (M⁺+1).

5.14.2. ( )-Ethyl 4-[2-[3-methoxy-4-[N⁰-(2-methylphe-

nyl)ureido]phenylacetyl]-1-isoindolinylcarbonyl]-1-

piperazinylacetate (9l). To a stirred solution of ( )-

ethyl 4-[2-(9-ﬂuorenylmethoxycarbonyl)-1-isoindolinyl-

carbony]-1-piperazinylacetate (410 mg, 0.77 mmol) in

DMF (8 ml) was added TBAF (1 M solution in

THF, 0.8 ml, 0.80 mmol), and the reaction mixture

was stirred at room temperature for 1 h. To the reac-

tion mixture were added 3-methoxy-4-[N⁰-(2-methyl-

phenyl)ureido]phenylacetic acid (239 mg, 0.76 mmol),

EDCÆHCl (175 mg, 0.91 mmol), HOBt (123 mg,

0.91 mmol), and Et₃N (160 ll, 1.14 mmol), and the

reaction mixture was stirred at room temperature for

12 h. The mixture was poured into water and extracted

with EtOAc. The combined extracts were washed with

brine, dried over Na₂SO₄, and evaporated. The residue

was puriﬁed by column chromatography on silica gel with

CHCl₃/MeOH (100:1 to 30:1, v/v) as an eluent to give the

title compound 9l [392 mg, 84% (2 steps)] as a pale yellow

amorphous solid. ¹H NMR (CDCl₃) d 1.28 (3H, t,

J = 7.1 Hz), 2.19 (3H, s), 2.49–2.77 (4H, m), 3.22 (2H,

s), 3.50–3.53 (2H, m), 3.62 (3H, s), 3.73 (2H, s), 3.80–

3.84 (2H, m), 4.01–4.15 (1H, m), 4.18 (2H, q,

J = 7.1 Hz), 4.81–4.84 (1H, m), 4.99–5.02 (1H, m), 6.12

(1H, s), 6.80 (1H, s), 6.82–6.84 (1H, m), 7.05–7.39 (8H,

m), 7.56–7.58 (1H, m), 8.07–8.09 (1H, m); MS (FAB),

m/z 614 (M⁺+1); Anal. Calcd for C₃₄H₃₉N₅O₆Æ1.25H₂O:

C, 64.19; H, 6.57; N, 11.01. Found: C, 64.27; H, 6.44; N,

10.76.

5.15.3. Ethyl 4-[1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ure-

ido]phenylacetyl]-2,3-dihydro-1H-(2S)-indolylcarbonyl]-

1-piperazinylacetate (9m). Colorless amorphous solid

1

(yield, 85%). H NMR (CDCl₃) d 1.24–1.32 (5H, m),

2.30 and 2.31 (total 3H, each s, amide isomers), 2.53–

2.80 (5H, m), 2.88–3.85 (14H, m), 3.97–4.25 (3H, m),

5.12 and 5.54 (total 1H, each d, J = 8.1 and 7.6 Hz, respec-

tively, amide isomers), 6.18 (1H, s), 6.66–7.30 (4H, m),

7.46–7.53 (1H, m), 8.09 (1H, t, J = 9.3 Hz), 8.31 (1H, d,

J = 7.8 Hz); MS (FAB) m/z 614 (M⁺+1); Anal. Calcd

for C₃₄H₃₉N₅O₆Æ0.75H₂O: C, 65.11; H, 6.51; N, 11.17.

Found: C, 65.18; H, 6.61; N, 11.06; IR (ATR) 3351,

2979, 2935, 2823, 1732, 1655, 1589, 1527, 1481 cm^ꢀ1

.

5.15.4. 4-[1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ureido]pheny-

lacetyl]-2,3-dihydro-1H-(2S)-indolylcarbonyl]-1-piperaz-

inylacetic acid (11m). Colorless amorphous solid (yield,

84%). ¹H NMR (DMSO-d₆) d 2.42–2.76 (6H, m),

3.85–2.93 (10H, m), 3.86 (3H, s), 4.61 (1H, q,

J = 5.5 Hz), 5.63 (1H, dd, J = 11.3, 2.9 Hz), 6.54 (1H,

dd, J = 13.6, 7.2 Hz), 6.69–7.27 (5H, m), 7.59 (1H, d,

J = 7.8 Hz), 8.02 and 8.09 (total 1H, each d, each

J = 8.1 Hz, amide isomers), 8.49 (1H, s), 8.59 (1H, s);

MS (FAB) m/z 586 (M⁺+1); HR-MS (FAB) calcd for

C₃₂H₃₅N₅O₆: 586.2666. Found: 86.2652; IR (ATR)

3342, 3016, 1631, 1601, 1529, 1481 cm^ꢀ1

.

5.14.3. ( )-4-[2-[3-Methoxy-4-[N⁰-(2-methylphenyl)ure-

ido]phenylacetyl]-1-isoindolinylcarbonyl]-1-piperazinyl-

acetic acid (11l). Prepared from 9l according to the

basic hydrolysis steps of general procedure A, the title

compound 11l was obtained as a colorless crystalline

powder (48%). Mp 167–171 ꢁC; H NMR (DMSO-d₆)

d 2.42 (3H, s), 2.50–2.75 (4H, m), 3.19 (2H, s), 3.22–

4.10 (10H, m), 4.71–4.99 (2H, m), 6.17 (1H, s), 6.80–

5.16. 4-[1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ureido]pheny-

lacetyl]-(2S)-octahydroindolylcarbonyl]-1-piperazinylace-

tic acid (11n)

1

5.16.1. Ethyl 4-[1-(tert-butoxycarbonyl)-(2S)-octahydro-

indolylcarbonyl]-1-piperazinylacetate. Colorless oil (yield,

1

99%). H NMR (CDCl₃) d 1.12–1.30 (6H, m), 1.38 and

2738

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

1.44 (total 9H, each s, amide isomers), 1.55–1.66 (7H, m),

1.91–1.98 (2H, m), 2.12–2.30 (1H, m), 2.52–2.69 (3H, m),

3.23–3.24 (2H, m), 3.56–3.61 (2H, m), 3.74–3.86 (2H, m),

4.19 (2H, q, J = 7.1 Hz), 4.50–4.62 (1H, m); ESI, m/z 424

(M⁺+1).

tively, amide isomers), 8.47 (1H, s), 8.54 (1H, s). MS

(FAB) m/z 554 (M⁺+1); HR-MS (FAB) calcd for

C₂₈H₃₆N₅O₇: 554.2615. Found: 554.2644; IR (KBr)

3319, 3018, 2945, 2843, 1612, 1529, 1483 cm^ꢀ1

.

5.18. 1-[1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ureido]pheny-

lacetyl]-4-hydroxy-(2S)-pyrrolidinylcarbonyl]-4-piperid-

inyl]acetic acid (12o)

5.16.2. Ethyl 4-[(2S)-octahydroindolylcarbonyl]-1-pipe-

razinylacetate (6n). Pale yellow oil (yield, 86%). ¹H

NMR (CDCl₃) d 1.20-1.30 (5H, m), 1.39–2.02 (9H, m),

2.12–2.19 (1H, m), 2.53–2.63 (4H, m), 3.07–3.11 (1H,

m), 3.24 (2H, s), 3.43–3.54 (2H, m), 3.66–3.77 (2H, m),

3.91–3.94 (1H, m), 4.19 (2H, q, J = 7.1 Hz); ESI, m/z

324 (M⁺+1).

By the procedure described for compound 11o but with

4-[(4R)-benzyloxy-1-[3-methoxy-4-[N⁰-(2-methylphe-

nyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-

pipirazinylacetic acid (12c) in place of 11c, the title com-

pound 12o was obtained as a colorless amorphous solid

(65%). ¹H NMR (DMSO-d₆) d 1.66–2.55 (11H, m),

3.01–4.34 (12H, m), 4.83–5.08 (2H, m), 6.74–6.80 (1H,

m), 6.89–6.93 (2H, m), 7.09–7.15 (2H, m), 7.76–7.78

(1H, m), 7.97–7.99 (1H, m), 8.44 (1H, s), 8.53 (1H, s);

MS (FAB), m/z 553 (M⁺+1); Anal. Calcd for

C₂₉H₃₆N₄O₇Æ2 H₂OÆHCl: C, 55.72; H, 6.61; N, 8.96.

Found: C, 55.70; H, 6.12; N, 8.49; IR (KBr) 3346,

5.16.3. Ethyl 4-[1-[3-methoxy-4-[N⁰-(2-methylphenyl)ure-

ido]phenylacetyl]-(2S)-octahydroindolylcarbonyl]-1-pipe-

razinylacetate (9n). Colorless amorphous solid (yield,

1

52%). H NMR (CDCl₃) d 1.10–1.35 (5H, m), 1.47–

1.50 (1H, m), 1.61–2.02 (7H, m), 2.24 and 2.25 (total

3H, each s, amide isomers), 2.32–3.26 (1H, m), 2.51–

2.64 (4H, m), 3.19 (2H, s), 3.44–3.69 (9H, m), 3.85–

3.91 (1H, m), 4.17 (2H, q, J = 7.1 Hz), 4.78 (1H, t,

J = 9.0 Hz), 6.62–6.83 (3H, m), 7.10–7.11 (1H, m),

7.19–7.24 (3H, m), 7.56–7.58 (1H, m), 8.00–8.04 (1H,

m); MS (FAB), m/z 620 (M⁺+1); HR-MS (FAB) calcd

for C₃₄H₄₆N₅O₆: 620.3448. Found: 620.3433.

3006, 2937, 1709, 1537, 1485 cm^ꢀ1

.

5.19. Ethyl 4-[4-hydroxy-1-[3-methoxy-4-[N⁰-(2-meth-

ylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcar-

bonyl]-1-pipirazinylacetate (9o)

5.16.4. 4-[1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ureido]phenyl-

acetyl]-(2S)-octahydroindolylcarbonyl]-1-piperazinylace-

tic acid (11n). Colorless amorphous solid (yield, 81%).

¹H NMR (DMSO-d₆) d 1.07–1.27 (3H, m), 1.36–1.40

(1H, m), 1.59–1.86 (6H, m), 2.05–2.30 (4H, m), 2.43–

2.63 (4H, m), 3.17 (2H, s), 3.25–3.40 (1H, m), 3.52–

3.73 (4H, m), 3.83–4.03 (5H, m), 4.71–4.91 (1H, m),

6.64–6.75 (1H, m), 6.91–6.94 (2H, m), 7.10–7.26 (2H,

m), 7.78–7.81 (1H, m), 7.97–8.00 (1H, m), 8.46 (1H, s),

8.55 (1H, s); ESI, m/z 592 (M⁺+1); HR-MS (FAB) calcd

for C₃₂H₄₂N₅O₆: 592.3135. Found: 592.3154; IR (ATR)

By the procedure described for compound (11o), but

with ethyl 4-[(4R)-benzyloxy-1-[3-methoxy-4-[N⁰-(2-

methylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcar-

bonyl]-1-pipirazinylacetate in place of 11c, the title com-

pound 9o was obtained as a colorless oil (57%). ¹H

NMR (CDCl₃) d 1.27 (3H, t, J = 7.4 Hz), 1.93–2.18

(2H, m), 2.25 (3H, s), 2.44–2.67 (4H, m), 3.19 (2H, s),

3.44–3.90 (10H, m), 4.17 (2H, q, J = 7.1 Hz), 4.45–4.52

(1H, m), 4.96 (1H, t, J = 7.7 Hz), 6.62–6.83 (3H, m),

7.10 (2H, t, J = 7.6 Hz), 7.20 (2H, t, J = 8.3 Hz), 7.31

(2H, s), 7.95 and 7.99 (total 1H, each d, each

J = 8.1 Hz, amide isomers); MS (ESI) m/z 582 (M⁺+1);

HR-MS (FAB) calcd for C₃₀H₄₀N₅O₇: 582.2928. Found:

582.2936. Anal. Calcd for C₃₀H₃₉N₅O₇ÆH₂O: C, 60.09;

H, 6.89; N, 11.68. Found: C, 59.73; H, 6.92; N, 11.37;

IR (ATR) 3340, 2983, 2831, 1620, 1529, 1485,

3346, 3016, 2927, 2854, 1612, 1529, 1485, 1452 cm^ꢀ1

.

5.17. 4-[4-Hydroxy-1-[3-methoxy-4-[N⁰-(2-methylphenyl)-

ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-pipiraz-

inylacetic acid (11o)

1542 cm^ꢀ1

.

A suspension of 4-[(4R)-benzyloxy-1-[3-methoxy-4-[N⁰-

(2-methylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinyl-

carbonyl]-1-pipirazinylacetic acid (11c) (400 mg,

0.62 mmol) and 20% Pd(OH)₂/C (0.40 g) in EtOH

(20 ml) was hydrogenated under 1 atom hydrogen atmo-

sphere at room temperature for 3 days. After the cata-

lyst was removed by ﬁltration, the ﬁltrate was

concentrated to give the title compound 11o (200 mg,

58%) as a colorless crystalline powder. ¹H NMR

(DMSO-d₆) d 2.25–2.31 (1H, m), 2.40–2.69 (5H, m),

3.05–3.66 (7H, m), 3.71-3.79 (1H, m), 3.81 and 3.84 (to-

tal 3H, each s, amide isomers), 4.07–4.29 (1H, m), 4.35–

4.49 (2H, m), 4.78–5.08 (1H, m), 6.66 and 6.77 (total 1H,

each dd, J = 7.8, 1.7 and 8.1, 1.7 Hz, respectively, amide

isomers), 6.79 and 6.91 (total 1H, each d, J = 1.7 and

1.2 Hz, respectively, amide isomers), 6.92–6.96 (2H,

m), 7.11 (1H, d, J = 10.0 Hz), 7.15 (1H, d, J = 7.8 Hz),

7.24–7.36 (4H, m), 7.78 (1H, dd, J = 8.1, 1.0 Hz), 8.00

and 8.01 (total 1H, each d, J = 8.3 and 8.1 Hz, respec-

5.20. 4-[(3S)-Hydroxy-1-[3-methoxy-4-[N⁰-(2-methylphe-

nyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-pip-

erazinylacetic acid (11q)

5.20.1. Ethyl 4-[(3S)-hydroxy-1-[3-methoxy-4-[N⁰-(2-

methylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcar-

bonyl]-1-piperazinylacetate (9q). By the procedure de-

scribed for compound 11o, but with ethyl 4-[(3S)-

benzyloxy-1-[3-methoxy-4-[N⁰-(2-methylphenyl)ureido]

phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-piperazinyl-

acetate (9g) in place of 11c, the title compound 9q

was obtained as a colorless amorphous solid (59%).

¹H NMR (CD₃OD)

d 1.26 (3H, t, J = 7.1 Hz),

1.97 (1H, m), 2.15 (1H, m), 2.29 (3H, s), 2.54–

2.74 (4H, m), 3.28 (3H, m), 3.47–3.71 (4H, m),

3.73 (2H, s), 3.75–3.81 (2H, m), 3.91 (3H, s),

4.17 (2H, q, J = 7.1 Hz), 4.27–4.33 (total 1H, m),

4.73 and 4.77 (total 1H, each s, amide isomers),

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

2739

6.73 and 6.81 (total 1H, each dd, each J = 8.3, 1.3

Hz, amide isomers), 6.84 and 6.95 (total 1H, each

d, each J = 1.3 Hz, amide isomers), 7.02 (1H, dt,

J = 7.6, 1.3 Hz), 7.13–7.20 (2H, m), 7.57 (1H, dd,

J = 8.3, 1.3 Hz), 7.98 (1H, d, J = 8.3 Hz); MS

(FAB) m/z 582 (M⁺+1).

and 3.87 (total 3H, each s, amide isomers), 3.92 (1H, t,

J = 3.4 Hz), 3.95–4.12 (1H, m), 4.60 (1H, d,

J = 2.7 Hz), 4.80 (1H, s), 6.65 and 6.76 (total 1H, each

d, J = 8.5 and 8.1 Hz, respectively, amide isomers),

6.93 (2H, s), 7.11 (1H, d, J = 7.8 Hz), 7.15 (1H, d,

J = 7.8 Hz), 7.79 (1H, d, J = 7.8 Hz), 8.00 (1H, d,

J = 8.3 Hz), 8.57 (1H, s), 8.62 (1H, s); MS (FAB) m/z

570 (M⁺+1); Anal. Calcd for C₂₈H₃₅N₅O₈Æ2.75H₂O: C,

54.32; H, 6.59; N, 11.37; Found: C, 54.45; H, 6.44; N,

10.96; IR (KBr) 3300, 2937, 2819, 1628, 1601, 1535,

5.20.2. 4-[(3S)-Hydroxy-1-[3-methoxy-4-[N⁰-(2-methyl-

phenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-

piperazinylacetic acid (11q). Prepared from 9q according

to the basic hydrolysis steps of general procedure A, the

title compound 11q was obtained as a yellow amorphous

1485, 1454 cm^ꢀ1

.

1

solid (95%). H NMR (DMSO-d₆) d 1.80 (1H, m), 1.96

5.22. 2-[4-[1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ureido]-

phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-piperazinyl]pro-

pionic acid (19)

(1H, m), 2.25 (3H, s), 2.32–2.64 (4H, m), 2.89 (2H, s),

3.27 (1H, m), 3.48–3.71 (7H, m), 3.85 and 3.87 (total

3H, each s, amide isomers), 4.11 and 4.24 (total 1H, each

d, each J = 2.9 Hz, amide isomers), 4.66 and 4.80 (total

1H, each s, amide isomers), 6.66 and 6.76 (total 1H, each

d, each J = 8.3 Hz, amide isomers), 6.92 (1H, t,

J = 7.3 Hz), 6.81 and 6.94 (1H, s), 7.10–7.16 (2H, m),

7.79 (1H, d, J = 8.3 Hz), 8.00 (1H, d, J = 8.3 Hz), 8.56

(1H, s), 8.61 (1H, s); MS (FAB) m/z 554 (M⁺+1); Anal.

Calcd for C₂₈H₃₅N₅O₇Æ2.5H₂O: C, 56.18; H, 6.73; N,

11.70. Found: C, 56.13; H, 7.05; N, 11.58; IR (KBr)

5.22.1. Ethyl 2-(4-tert-butoxycarbonyl-1-piperazinyl)pro-

pionate. A mixture of tert-butyl-1-piperazinylcarboxylate

(13) (24.22 g, 130 mmol), ethyl 2-bromopropionate

(23.54 g, 130 mmol), and K₂CO₃(27.09 g, 196 mmol)

in DMF (500 ml) was stirred at room temperature for

12 h. The reaction mixture was poured into ice water

and extracted with EtOAc. The combined extracts were

washed with brine and dried over MgSO₄. After removal

of the solvent, the residue was puriﬁed by column chroma-

tography on silica gel with CHCl₃/EtOAc (5:1, v/v) as

an eluent to give the title compound (20.7 g, 56%) as a

colorless oil. ¹H NMR (CDCl₃) d 1.23–1.26 (6H, m), 1.42

(9H, s), 2.52–2.57 (4H, m), 3.23–3.29 (1H, m), 3.39–3.45

(4H, m), 4.10–4.18 (2H, m).

3345, 2927, 1627, 1536, 1454, 1415 cm^ꢀ1

.

5.21. 4-[1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ureido]pheny-

lacetyl]-(3R,4R)-dioxy-(2S)-pyrrolidinylcarbonyl]-1-piperaz-

inylacetic acid (11p)

5.21.1. Ethyl 4-[1-[3-methoxy-4-[N⁰-(2-methylphenyl)ure-

ido]phenylacetyl]-(3R,4R)-dioxy-(2S)-pyrrolidinylcarbon-

yl]-1-piperazinylacetate (9p). A solution of ethyl 4-[1-[3-

methoxy-4-[N⁰-(2-methylphenyl)ureido]phenylacetyl]-(3

R,4R)-isopropylidenedioxy-(2S)-pyrrolidinylcarbonyl]-1

-piperazinylacetic acetate (9d) (168 mg, 0.26 mmol) in

satd HCl (gas)/MeOH (10 ml) was stirred at room tem-

perature for 1 h. The reaction mixture was concentrated

under reduced pressure. The residue was diluted with

sat. NaHCO₃solution and extracted with CHCl₃/

MeOH (5:1, v/v). The extracts were washed with brine,

dried over Na₂SO₄, and concentrated to dryness. The res-

idue was puriﬁed by chromatography on silica gel with

CHCl₃/MeOH (5:1, v/v) as an eluent to give the title com-

pound 9p (151 mg, 97%) as a yellow amorphous solid. ¹H

NMR (CDCl₃) d 1.26 (3H, t, J = 7.1 Hz), 2.22 (3H, s),

2.39–2.66 (4H, m), 3.10–3.76 (13H, m), 4.03–4.28 (5H,

m), 4.58 and 4.72 (total 1H, each d, J = 3.4 and 4.4 Hz,

respectively, amide isomers), 6.59 and 6.69 (total 1H, each

d, J = 8.3 and 7.4 Hz, respectively, amide isomers), 6.74

(1H, s), 7.01–7.24 (4H, m), 7.46–7.61 (2H, m), 7.91 and

7.96 (total 1H, each d, each J = 8.1 Hz, amide isomers);

MS (FAB) m/z 598 (M⁺+1); HR-MS (FAB) calcd for

C₃₀H₄₀N₅O₈: 598.2877. Found: 598.2838; IR (ATR)

5.22.2. Ethyl 2-(1-piperazinyl)propionate (14). To the

solution of ethyl 2-(4-tert-butoxycarbonyl-1-piperazi-

nyl)propionate (20.70 g, 72.3 mmol) in CH₂Cl₂(50 ml),

TFA (50 ml) was added and the solution was stirred

for 1 h. The reaction mixture was concentrated in

vacuo and made basic with NaHCO₃. The mixture

was extracted with CHCl₃/MeOH. The extracts were

washed with brine, dried over Na₂SO₄, and evaporated

to give the title compound 14 (5.90 g, 44%) as a colorless

1

oil. H NMR (CDCl₃) d 1.23–1.27 (6H, m), 2.85–2.94

(8H, m), 3.20–3.24 (1H, m), 4.12–4.17 (2H, m).

5.22.3. Ethyl 2-[4-[1-(tert-butoxycarbonyl)-(2S)-pyrrolid-

inylcarbonyl]-1-piperazinyl]propionate. Pale yellow oil

1

(yield, 58%). H NMR (CDCl₃) d 1.22–1.29 (6H, m),

1.37–1.42 (9H, m), 1.69–2.14 (4H, m), 2.51–2.70 (4H,

m), 3.25–3.69 (7H, m), 4.10–4.16 (2H, m), 4.48–4.64

(1H, m); MS (ESI), m/z 384 (M⁺+1).

5.22.4. Ethyl 2-[4-((2S)-pyrrolidinylcarbonyl)-1-piperazi-

nyl]propionate (16). Pale yellow oil (yield, 45%). ¹H

NMR (CDCl₃) d 1.18–1.25 (6H, m), 1.58–3.60 (15H,

m), 3.93–3.96 (1H, m), 4.07–4.13 (2H, m); MS (ESI),

m/z 284 (M⁺+1).

3340, 2937, 2835, 1736, 1620, 1529, 1485 cm^ꢀ1

.

5.21.2. 4-[1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ureido]pheny-

lacetyl]-(3R,4R)-dioxy-(2S)-pyrrolidinylcarbonyl]-1-pip-

erazinylacetic acid (11p). Prepared from 9p according to

the basic hydrolysis steps of general procedure A, the ti-

tle compound 11p was obtained as a colorless amor-

5.22.5. Ethyl 2-[4-[1-[3-methoxy-4-[N⁰-(2-methylphe-

nyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-pip-

erazinyl]propionate. Pale yellow oil (yield, 27%). ¹H

NMR (CDCl₃) d 1.21–1.31 (6H, m), 1.67–2.71 (13H,

m), 3.23–3.70 (10H, m), 4.10–4.16 (2H, m), 4.84–4.86

(1H, m), 6.43 (1H, s), 6.75–6.83 (2H, m), 7.07–7.24

(4H, m), 7.50–7.52 (1H, m), 7.99–8.01 (1H, m); MS

1

phous solid (yield, 90%). H NMR (DMSO-d₆) d 2.31–

2.68 (5H, m), 2.92 (3H, s), 3.23–3.86 (10H, m), 3.85

2740

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

(FAB), m/z 580 (M⁺+1); HR-MS (FAB) calcd for

C₃₁H₄₂N₅O₆: 580.3135. Found: 580.3112.

with 2-bromo-2-methylpropionate in place of ethyl 2-

bromopropionate, the title compound was obtained as

a yellow oil (yield, 99%). ¹H NMR d 1.27 (3H, t,

J = 7.1 Hz), 1.31 (6H, s), 1.45 (9H, s), 2.52–2.55 (4H,

m), 3.40–3.43 (4H, m), 4.17 (2H, q, J = 7.1 Hz); EI-

MS m/z 300 (M⁺).

5.22.6.

2-[4-[1-[3-Methoxy-4-[N⁰-(2-methylphenyl)ure-

ido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-piperaz-

inyl]propionic acid (19). Pale yellow needles (yield, 44%),

which were crystallized from n-hexane/ether. Mp 165–

168 ꢁC; ¹H NMR (DMSO-d₆) d 1.10–1.14 (3H, m),

1.66–4.81 (24H, m), 6.74–6.79 (1H, m), 6.90–6.94 (2H,

m), 7.09–7.16 (2H, m), 7.77–7.79 (1H, m), 7.96–8.00

(1H, m), 8.50 (1H, s), 8.57–8.58 (1H, m); MS (FAB),

5.24.2. Ethyl 2-(1-piperazinyl)-2-methylpropionate (15).

By the procedure described for compound 14, but with

ethyl 2-[4-(tert-butoxycarbonyl)-1-piperazinyl]-2-meth-

ylpropionate in place of ethyl 2-(4-tert-butoxycarbonyl-

1-piperazinyl)propionate, the title compound was ob-

tained as a yellow oil (yield, 99%). ¹H NMR d 1.28 (3H,

t, J = 7.3 Hz), 1.31 (6H, s), 2.64–2.68 (4H, m), 2.98–3.00

(4H, m), 3.71 (1H, s), 4.17 (2H, q, J = 7.3 Hz); EI-MS

m/z 200 (M⁺).

m/z 552 (M⁺+1)

C₂₉H₃₈N₅O₆: 552.2822. Found: 552.2855; IR (KBr)

3330, 2970, 2945, 2875, 1616, 1589, 1529 cm^ꢀ1

;

HR-MS (FAB) calcd for

.

5.23. 2-[4-[(4R)-Benzyloxy-1-[3-methoxy-4-[N⁰-(2-meth-

ylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-

1-piperazinyl]propionic acid (20)

5.24.3. Ethyl 2-[4-[(4R)-benzyloxy-1-tert-butoxycarbon-

yl-(2S)-pyrrolidinylcarbonyl]-1-piperazinyl]-2-methylpro-

pionate. Colorless oil (yield, 87%). ¹H NMR d 1.27 (3H,

t, J = 7.1 Hz), 1.31 (3H, s), 1.32 (3H, s), 1.40 and 1.45

(total 9H, each s, amide isomers), 1.98–2.08 (1H, m),

2.19–2.29 (1H, m), 2.47–2.74 (4H, m), 3.46–3.78 (6H,

m), 4.16 (2H, q, J = 7.1 Hz), 4.19–4.28 (1H, m), 4.44

and 4.47 (total 1H, each d, each J = 12.0 Hz, amide iso-

mers), 4.52 and 4.56 (total 1H, each d, each J = 12.0 Hz,

amide isomers), 4.73–4.82 (1H, m), 7.28–7.36 (5H, m);

MS (FAB) m/z 504 (M⁺+1).

5.23.1. Ethyl 2-[4-[(4R)-benzyloxy-1-(tert-butoxycarbon-

yl)-(2S)-pyrrolidinylcarbonyl]-1-piperazinyl]propionate.

1

Pale yellow oil (yield, 58%). H NMR (CDCl₃) d 1.22–

1.29 (6H, m), 1.37–1.42 (9H, m), 1.68–2.28 (4H, m),

2.51–2.59 (4H, m), 3.26–3.72 (6H, m), 4.10–4.65 (5H,

m), 7.26–7.34 (5H, m); MS (ESI), m/z 490 (M⁺+1).

5.23.2. Ethyl 2-[(4R)-(4-benzyloxy-(2S)-pyrrolidinylcar-

bonyl)-1-piperazinyl]propionate (17). Pale yellow oil

1

(yield, 45%). H NMR (CDCl₃) d 1.18–1.24 (6H, m),

1.74–3.57 (13H, m), 4.02–4.12 (4H, m), 4.38–4.48 (2H,

5.24.4. Ethyl 2-[4-[(4R)-benzyloxy-2-pyrrolidinyl]-1-pip-

erazinyl]-2-methylpropionate (18). Yellow oil (yield,

m), 7.19–7.29 (5H, m); MS (ESI), m/z 389 (M⁺+1).

1

87%). H NMR d 1.27 (3H, t, J = 7.1 Hz), 1.32 (6H,

5.23.3. Ethyl 2-[4-[(4R)-benzyloxy-1-[3-methoxy-4-[N⁰-

(2-methylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinyl-

carbonyl]-1-piperazinyl]propionate. Pale yellow amor-

phous solid (yield, 81%). ¹H NMR (CDCl₃) d 1.21–

1.29 (6H, m), 1.97–2.68 (9H, m), 3.22–4.40 (17H, m),

4.92–4.96 (1H, m), 6.44–6.48 (1H, m), 6.74–6.76 (2H,

m), 7.08–7.32 (9H, m), 7.49–7.51 (1H, m), 8.01–8.05

(1H, m). MS (FAB) m/z 686 (M⁺+1); HR-MS (FAB)

calcd for C₃₈H₄₈N₅O₇: 686.3554. Found: 686.3586.

s), 1.82 (1H, ddd, J = 13.4, 8.5, 6.3 Hz), 2.15 (1H, ddd,

J = 13.4, 7.6, 2.7 Hz), 2.27 (1H, s), 2.54–2.64 (4H, m),

3.01 (1H, dd, J = 12.2, 3.4 Hz), 3.33 (1H, dd, J = 12.2,

5.1 Hz), 3.39–3.57 (4H, m), 4.09 (1H, t, J = 7.8 Hz),

4.11–4.21 (1H, m), 4.17 (2H, q, J = 7.1 Hz), 4.47 (1H,

d, J = 11.7 Hz), 4.54 (1H, d, J = 11.7 Hz), 7.28–7.38

(5H, m); MS (FAB) m/z 404 (M⁺+1).

5.24.5. Ethyl 2-[4-[(4R)-benzyloxy-1-[3-methoxy-4-[N⁰-

(2-methylphenyl)ureido]phenylacetyl]-2-pyrrolidinylcar-

bonyl]-1-piperazinyl]-2-methylpropionate. Colorless oil

5.23.4.

2-[4-[(4R)-Benzyloxy-1-[3-methoxy-4-[N⁰-(2-

1

methylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcar-

bonyl]-1-piperazinyl]propionic acid (20). Pale yellow crys-

talline powder (yield, 55%), which was crystallized from

n-hexane/ether. Mp 147–150 ꢁC ¹H NMR (DMSO-d₆) d

1.13–1.15 (3H, m), 1.93–4.85 (25H, m), 6.58–6.66 (2H,

m), 6.93–6.97 (1H, m), 7.06–7.09 (2H, m), 7.17–7.29

(5H, m), 7.56–7.58 (2H, m), 7.79 (1H, s), 7.96–7.98

(1H, m); MS (FAB) m/z 658 (M⁺+1); HR-MS (FAB)

calcd for C₃₆H₄₄N₅O₇: 658.3241. Found: 658.3226.

Anal. Calcd for C₃₆H₄₃N₅O₇Æ2H₂O: C, 62.32; H, 6.83;

N, 10.09. Found: C, 61.86; H, 6.62; N, 9.83; ; IR

(yield, 98%). H NMR d 1.25 (3H, t, J = 7.1 Hz), 1.29

(6H, s), 2.03 (1H, ddd, J = 13.2, 7.8, 5.4 Hz), 2.21 (1H,

ddd, J = 13.2, 9.0, 3.9 Hz), 2.27 (3H, s), 2.43–2.54 (2H,

m), 2.63 (1H, m), 2.73 (1H, m), 3.49–3.55 (2H, m),

3.58–3.66 (6H, m), 3.68 (3H, s), 4.15 (2H, q,

J = 7.1 Hz), 4.28 (1H, m), 4.35 (1H, d, J = 12.0 Hz),

4.42 (1H, d, J = 12.0 Hz), 4.97 (1H, dd, J = 8.3,

6.8 Hz), 6.56 (1H, s), 6.76–6.79 (2H, m), 7.11–7.17

(2H, m), 7.21–7.24 (2H, m), 7.27–7.35 (5H, m), 7.53

(1H, d, J = 7.8 Hz), 8.05 (1H, d, J = 8.3 Hz); MS

(FAB) m/z 700 (M⁺+1); HR-MS (FAB) calcd for

C₃₉H₅₀N₅O₇: 700.3710. Found: 700.3676.

(ATR) 3342, 2941, 1620, 1589, 1529, 1485 cm^ꢀ1

.

5.24. 2-[4-[(4R)-Benzyloxy-1-[3-methoxy-4-[N⁰-2-(meth-

ylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-

1-piperazinyl]-2-methylpropionic acid (21)

5.24.6. 2-[4-[(4R)-Benzyloxy-1-[3-methoxy-4-[N⁰-2-(meth-

ylphenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-

piperazinyl]-2-methylpropionic acid (21). Pale yellow

1

amorphous solid (yield, 35%). H NMR d 1.16 (6H, s),

5.24.1. Ethyl 2-[4-(tert-butoxycarbonyl)-1-piperazinyl]-2-

methylpropionate. By the procedure described for ethyl

2-(4-tert-butoxycarbonyl-1-piperazinyl)propionate, but

1.84–1.93 (1H, m), 2.25 (3H, s), 2.24–2.30 (1H, m),

2.41–2.61 (3H, m), 2.62–2.66 (1H, m), 3.23–3.60 (6H,

m), 3.74–3.82 (2H, m), 3.85 (3H, s), 4.19–4.23 (1H, m),

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

2741

4.40 (1H, d, J = 11.7 Hz), 4.46 (1H, d, J = 11.7 Hz), 4.84

(1H, t, J = 7.3 Hz), 6.78 (1H, d, J = 8.3 Hz), 6.91–6.95

(2H, m), 7.11–7.17 (2H, m), 7.26–7.35 (5H, m), 7.78

(1H, d, J = 8.3 Hz), 8.01 (1H, d, J = 8.3 Hz), 8.51 (1H,

s), 8.58 (1H, s); MS (FAB) m/z 672 (M⁺+1); HR-MS

(FAB) calcd for C₃₇H₄₆N₅O₇: 672.3397. Found:

672.3414. Anal. Calcd for C₃₇H₄₅N₅O₇Æ3.5H₂O: C,

60.48; H, 7.13; N, 9.53. Found: C, 60.71; H, 6.46; N,

9.21; IR (KBr) 3345, 2935, 1702, 1631, 1598, 1531,

was puriﬁed by column chromatography on silica gel with

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

1

compound 25 (2.14 g, 59%) as a yellow oil. H NMR

(CDCl₃) d 1.41 and 1.47 (total 9H, each s, amide isomers),

1.62 (1H, m), 2.04 (1H, m), 3.50–3.69 (2H, m), 3.73 and

3.74 (total 3H, each s, amide isomers), 4.12 (1H, m),

4.34–4.71 (3H, m), 7.27–7.39 (5H, m); MS (FAB) m/z

336 (M⁺+1).

1454 cm^ꢀ1

.

5.26.2. (3S)-Benzyloxy-1-(tert-butoxycarbonyl)pyrroli-

dine-(2S)-carboxylic acid (3g). By the procedure de-

scribed for compound 3b, but with methyl 1-(tert-

butoxycarbonyl)-(3S)-hydroxypyrrolidine-2-carboxylate

(25) in place of 23, the title compound 3g was obtained

as a yellow oil (100%). ¹H NMR (CDCl₃) d 1.49 (9H, s),

1.99–2.12 (2H, m), 3.45–3.62 (2H, m), 4.37–4.66 (4H,

m), 7.27–7.39 (5H, m); MS (FAB) m/z 322 (M⁺+1).

5.25. 1-(tert-Butoxycarbonyl)-(4R)-methoxypyrrolidine-

(2S)-carboxylic acid (3b)

5.25.1. Methyl 1-(tert-butoxycarbonyl)-(4R)-methoxy-

pyrrolidine-(2S)-carboxylate (23). To a stirred solution

of N-Boc-trans-4-hydroxy-^L-proline methyl ester (22)

(1.98 g, 8.1 mmol) and MeI (1.22 ml, 19.6 mmol) in

DMF (15 ml) was added 60% oily NaH (0.39 g,

16.2 mmol) at ꢀ10 ꢁC, and the reaction mixture was

stirred at room temperature for 3 h. The mixture was

poured into ice water and extracted with EtOAc. The

combined extracts were washed with brine and dried

over Na₂SO₄. After removal of the solvent, the residue

was puriﬁed by column chromatography on silica gel

with CHCl₃/MeOH (50:1, v/v) as an eluent to give

the title compound 23 (1.87 g, 89%) as a colorless oil.

¹H NMR (CDCl₃) d 1.41 (9H, s), 2.01–2.08 (1H, m),

2.31–2.38 (1H, m), 3.32 (3H, s), 3.48–3.67 (2H, m),

3.73 (s, 3 H), 3.96–3.97 (1H, m), 4.30-4.40 (1H, m);

MS (FAB), m/z 260 (M⁺+1).

5.27. 1-Benzyloxycarbonyl-(3R,4R)-isopropylidenedioxy-

(2S)-pyrrolidinylcarboxylic acid sodium salt (3d)

5.27.1. Methyl 1-benzyloxycarbonyl-(4R)-p-toluenesulfo-

nyloxy-(2S)-pyrrolidinylcarboxylate. To a solution of p-

toluenesulfonylimidazole (10.0 g, 45.0 mmol) in THF

(55 ml) at 0 ꢁC was added methyl triﬂate (5.60 ml,

49.5 mmol). The mixture was stirred for 1h. A solution

of N-Z-trans-(4R)-hydroxy-^L-proline methyl ester (26)

(8.41 g, 30.1 mmol) and N-methylimidazole (3.60 ml,

44.9 mmol) in THF (20 ml) was added to the reaction

mixture, and the resulting mixture was allowed to rise

to room temperature and was stirred for 24 h. The mix-

ture was poured into water and extracted with EtOAc.

The combined extracts were washed with satd. NaHCO₃

solution and brine, dried over Na₂SO₄, and concentrat-

ed to dryness. The residue was puriﬁed by chromatogra-

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

eluent to give the title compound (12.7 g, 97%) as a col-

5.25.2.

1-(tert-Butoxycarbonyl)-(4R)-methoxypyrroli-

dine-(2S)-carboxylic acid (3b). To a stirred solution of

methyl 1-(tert-butoxycarbonyl)-(4R)-methoxypyrroli-

dine-2-carboxylate (23) (1.87 g, 7.2 mmol) in THF

(15 ml) was added 0.25 N NaOH (15 ml) and the reac-

tion mixture was stirred at room temperature for 12 h.

After that, 0.5 N NaOH (3 ml) was added to the reac-

tion mixture and the mixture was heated under reﬂux

for 3 h. The mixture was made acidic with 1 N HCl

and extracted with CHCl₃. The combined extracts

were washed with brine, dried over Na₂SO₄, and evap-

orated to give the title compound 3b (1.80 g, quant.)

as a colorless oil. ¹H NMR (CDCl₃) d 1.49 (9H, s),

2.10–2.43 (2H, m), 3.33 (3H, s), 3.47–3.73 (2H, m),

3.97–3.98 (1H, m), 4.31–4.45 (1H, m); MS (FAB),

m/z 246 (M⁺+1).

1

orless oil. H NMR (CDCl₃) d 2.15–2.29 (1H, m), 2.44

and 2.46 (total 3H, each s, amide isomers), 2.50–2.55

(1H, m), 3.63–3.71 (2H, m), 3.52 and 3.74 (total 3H,

each s, amide isomers), 4.44 and 4.47 (total 1H, each t,

each J = 7.8 Hz, amide isomers), 4.99–5.18 (3H, m),

7.27–7.37 (7H, m), 7.75–7.79 (2H, m).

5.27.2. Methyl 1-benzyloxycarbonyl-(4S)-phenylseleno-

(2S)-pyrrolidinylcarboxylate. To a solution of diph-

enyldiselenide (12.7 g, 29.2 mmol) in MeOH (100 ml)

was added NaBH₄(2.87 g, 75.9 mmol). The reaction

mixture was heated under reﬂux for 24 h and cooled

to room temperature. To the reaction mixture was add-

ed a solution of methyl 1-benzyloxycarbonyl-(4R)-p-tol-

uenesulfonyloxy-(2S)-pyrrolidinylcarboxylate (12.7 g,

29.2 mmol) in MeOH (50 ml), and the mixture was re-

ﬂuxed for 5 h. The mixture was concentrated and diluted

with Et₂O, washed with water and brine, dried over

Na₂SO₄, and concentrated to dryness. The residue was

puriﬁed by chromatography on silica gel with n-hex-

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

5.26. (3S)-Benzyloxy-1-(tert-butoxycarbonyl)pyrrolidine-

(2S)-carboxylic acid (3g)

5.26.1. Methyl (3S)-benzyloxy-1-(tert-butoxycarbon-

yl)pyrrolidine-(2S)-carboxylate (25). To a stirred solution

of methyl 1-(tert-butoxycarbonyl)-(3S)-hydroxypyrroli-

14

dine-2-carboxylate (24)

(2.50 g, 10.2 mmol), benzyl

bromide (1.50 ml, 12.2 mmol), and tetrabutylammonium

iodide (1.13 g, 3.06 mmol) in THF (70 ml) was added 60%

oily NaH (0.45 g, 11.2 mmol) at 0 ꢁC, and the reaction

mixture was stirred at room temperature for 3 h. The mix-

ture was poured into ice water and extracted with EtOAc.

The combined extracts were washed with brine and dried

over Na₂SO₄. After removal of the solvent, the residue

1

pound (10.0 g, 82%) as a yellow oil. H NMR (CDCl₃)

d 2.07–2.11 (1H, m), 2.66–2.70 (1H, m), 3.47–3.64 (2H,

m), 3.57 and 3.75 (total 3H, each s, amide isomers),

4.05–4.09 (1H, m), 4.34–4.38 (1H, m), 5.02 and 5.09 (to-

tal 1H, each d, each J = 12.2 Hz, amide isomers), 5.17

2742

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

(1H, d, J = 12.2 Hz), 7.26–7.36 (8H, m), 7.53–7.55 (2H,

m).

(3H, s), 1.46 (3H, s), 3.61–3.65 (1H, m), 3.64 and 3.77 (to-

tal 3H, each s, amide isomers), 3.91 and 3.96 (total 1H,

each d, each J = 12.7 Hz, amide isomers), 4.54 and 4.63

(total 1H, each s, amide isomers), 4.75–4.77 (2H, m),

5.07 and 5.13 (total 1H, each d, J = 12.5 and 12.4 Hz,

respectively, amide isomers), 5.18 and 5.20 (total 1H, each

d, J = 12.4 and 12.5 Hz, respectively, amide isomers),

7.28–7.37 (5H, m).

5.27.3. Methyl 1-benzyloxycarbonyl-3,4-dehydro-(2S)-

pyrrolidinylcarboxylate (27). To a stirred solution of

methyl

1-benzyloxycarbonyl-(4S)-phenylseleno-(2S)-

pyrrolidinylcarboxylate (10.0 g, 23.9 mmol) and pyri-

dine (3.0 ml, 48.8 mmol) in CH₂Cl₂(120 ml) at 0 ꢁC

was added dropwise H₂O₂(31%, 6.80 g, 59.8 mmol).

The reaction mixture was allowed to warm to room tem-

perature and was stirred for 24 h. The reaction mixture

was diluted with CH₂Cl₂and washed with 0.5 N HCl

solution, satd NaHCO₃solution, 5% Na₂SO₃solution,

and brine. The separated organic layer was dried over

Na₂SO₄and concentrated. The residue was puriﬁed by

chromatography on silica gel with n-hexane:EtOAc

(2:1, v/v) as an eluent to give the title compound 27

5.27.6. 1-Benzyloxycarbonyl-(3R,4S)-isopropylidenedi-

oxy-(2S)-pyrrolidinylcarboxylic acid sodium salt (3d).

To a solution of methyl 1-benzyloxycarbonyl-(3R,4R)-

isopropylidenedioxy-(2S)-pyrrolidinylcarboxylate

[(3R,4S)-29] (1.42 g, 4.23 mmol) in THF/MeOH (4:1, v/

v, 45 ml) was added 0.25 N NaOH (25 ml, 6.35 mmol).

After being stirred at room temperature for 24 h, the

reaction mixture was concentrated to dryness to aﬀord

the title compound 3d (1.32 g, 91%) as a colorless sol-

1

(4.62 g, 74%) as a dark brown oil. H NMR (CDCl₃)

1

d 3.60 and 3.76 (total 3H, each s, amide isomers),

4.24–4.39 (2H, m), 5.07–5.11 (1H, m), 5.14 (1H, d,

J = 12.2 Hz), 5.22 (1H, d, J = 12.2 Hz), 5.73–5.78 (1H,

m), 5.96–6.01 (1H, m), 7.28–7.40 (5H, m); MS (ESI)

m/z 284 (M⁺+Na).

id. H NMR (DMSO-d₆) d 1.22 (3H, s), 1.31 (3H, s),

3.45 (1H, d, J = 2.9 Hz), 3.52 (1H, d, J = 3.9 Hz), 4.00

(1H, d, J = 4.4 Hz), 4.60 (1H, m), 4.65 and 4.70 (total

1H, each d, each J = 6.1 Hz, amide isomers), 4.96 and

5.01 (total 1H, each d, each J = 13.2 Hz, amide isomers),

5.05 and 5.08 (total 1H, each d, each J = 13.2 Hz, amide

isomers), 7.25–7.35 (5H, m); MS (FAB) m/z 344

5.27.4. Methyl 1-benzyloxycarbonyl-3,4-dihydroxy-(2S)-

pyrrolidinylcarboxylate (28). To a solution of methyl 1-

benzyloxycarbonyl-3,4-dehydro-(2S)-pyrrolidinylcarb-

oxylate (27) (4.62 g, 17.7 mmol) and N-methylmorpho-

line-N-oxide (50% in H₂O, 6.51 g, 27.8 mmol) in

acetone (25 ml) was added a catalytic amount of OsO₄

at 0 ꢁC. The reaction mixture was stirred at room tem-

perature for 24 h and quenched by the addition of

10% Na₂SO₃solution. The mixture was stirred for 3 h

and extracted with EtOAc. The extracts were washed

with brine, dried over Na₂SO₄, and concentrated to dry-

ness. The residue was puriﬁed on chromatography by

silica gel with CHCl₃:MeOH (10/1, v/v) as an eluent to

give the title compound 28 (4.98 g, 95%) as a dark

brown oil. ¹H NMR (CDCl₃) d 3.55–3.59 (1H, m),

3.68–3.80 (3H, m), 3.56 and 3.75 (total 3H, each s,

amide isomers), 4.22–4.29 (3H, m), 4.98 and 5.09 (total

1H, each d, J = 12.5 and 12.4 Hz, respectively, amide

isomers), 5.14 and 5.17 (total 1H, each d, J = 12.4 and

12.5 Hz, respectively, amide isomers), 7.28–7.35 (5H,

m); MS (ESI) m/z 296 (M⁺+1).

(M⁺+1); IR (KBr) 1617, 1673 cm^ꢀ1

.

5.28. 1-(tert-Butoxycarbonyl)-(4S)-phenoxy-(2S)-pyrro-

lidinylcarboxylic acid (3e)

5.28.1. Methyl (1-tert-butoxycarbonyl)-(4S)-phenoxy-

(2S)-pyrrolidinylcarboxylate (30). To a stirred mixture

of N-Boc-trans-4-hydroxy-^L-proline methyl ester (22)

(4.69 g, 19.1 mmol), phenol (1.98 g, 21.0 mmol), and

PPh₃(5.51 g, 21.0 mmol) in THF (80 ml) was added

diisopropyl azodicarboxylate (4.13 ml, 21.0 mmol) at

room temperature under a nitrogen atmosphere. The

mixture was stirred for 12 h. After removal of the sol-

vent, the resulting residue was chromatographed on sil-

ica gel with CHCl₃/EtOAc (10:1, v/v) as an eluent to give

the title compound 30 (5.31 g, 86%) as a colorless oil.

¹H NMR (CDCl₃) d 1.43 and 1.48 (total 9H, each s,

amide isomers), 2.48 (1H, m), 3.75 (6H, s), 4.42–4.96

(2H, m), 6.88–7.35 (5H, m); MS (ESI), m/z 322 (M⁺+1).

5.28.2. 1-(tert-Butoxycarbonyl)-(4S)-phenoxy-(2S)-pyr-

rolidinylcarboxylic acid (3e). To a stirred solution of

methyl (1-tert-butoxycarbonyl)-(4S)-phenoxy-(2S)-pyr-

rolidinylcarboxylate (30) (5.31 g, 16.5 mmol) in THF

(132 ml) was added 0.25 N NaOH (132 ml, 33.0 mmol)

at room temperature. The resulting mixture was stirred

for 12 h. After removal of the solvent, the mixture was

acidiﬁed by the addition of 1 N HCl and extracted with

CHCl₃. The combined extracts were washed with brine,

dried over Na₂SO₄, and evaporated. The residue was

recrystallized from n-hexane/CHCl₃, to give the title

5.27.5. Methyl 1-benzyloxycarbonyl-(3R,4R)-isopropyl-

idenedioxy-(2S)-pyrrolidinylcarboxylate [(3R,4S)-29]. To

a solution of methyl 1-benzyloxycarbonyl-3,4-dihydroxy-

(2S)-pyrrolidinylcarboxylate (28)(1.41 g, 4.77 mmol) and

2,2-dimethoxypropane (6.0 ml, 48.8 mmol) in CH₂Cl₂

(20 ml) was added p-TsOHÆH₂O (164 mg, 0.86 mmol).

After being stirred at room temperature for 1 h, the mix-

ture was diluted with CHCl₃and washed with satd NaH-

CO₃solution and brine. The separated organic layer was

dried over Na₂SO₄and concentrated to dryness. The res-

idue was puriﬁed on chromatography by silica gel with

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

compound (3R,4S)-29 (1.42 g, 89%) as a colorless oil.

And further elution gave a stereoisomer of the title com-

pound methyl 1-benzyloxycarbonyl-(3S,4R)-isopropyl-

1

compound (3e) (2.96 g, 58%) as a colorless powder. H

NMR (DMSO-d₆) d 1.36 (9H, s), 2.16 (1H, d,

J = 13.2 Hz), 2.46–2.64 (1H, m), 3.40–3.50 (1H, m),

3.71 (1H, dt, J = 12.0, 5.4 Hz), 4.26 (1H, dt, J = 9.5,

7.1 Hz), 4.95–5.04 (m, 1H), 6.80–6.90 (2H, m), 6.94

(1H, t, J = 7.3 Hz), 7.28 (2H, t, J = 7.3 Hz); MS (ESI),

m/z 308 (M⁺+1).

idenedioxy-(2S)-pyrrolidinylcarboxylate

[(3S,4R)-29]

1

(0.10 g, 6%) as a colorless oil. H NMR (CDCl₃) d 1.31

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

2743

5.29. 1-(tert-Butoxycarbonyl)-(4S)-(2-naphthyloxy)-(2S)-

pyrrolidinylcarboxylic acid (3f)

the title compound 3h (669 mg, 85%) as a colorless crys-

talline powder. Mp 160–162 ꢁC; ¹H NMR (DMSO-d₆) d

1.36 and 1.41 (total 9H, each s, amide isomers), 2.17–

2.58 (2H, m), 3.16–3.66 (2H, m), 4.27 (1H, t,

J = 11.3 Hz), 5.25 (1H, dd, J = 53.6, 3.4 Hz), 12.54

(1H, s); MS (FAB) m/z, 234 (M⁺+1).

5.29.1. Methyl 1-(tert-butoxycarbonyl)-(4S)-(2-naphthyl-

oxy)-(2S)-pyrrolidinylcarboxylate (31). To a stirred mix-

ture of N-Boc-trans-4-hydroxy-^L-proline methyl ester

(22) (4.22 g, 17.2 mmol), 2-naphthol (2.73 g, 18.9 mmol),

and PPh₃(4.96 g, 18.9 mmol) in THF (80 ml) was added

diisopropyl azodicarboxylate (3.72 ml, 18.9 mmol) at

room temperature under an atmosphere of nitrogen.

After stirring for 12 h, the mixture was concentrated in

vacuo. The residue was chromatographed on silica gel

with CHCl₃/EtOAc (10:1, v/v) as an eluent to give the title

compound 31 (5.37 g), which included an inseparable

substrate and was used without further puriﬁcation. MS

(ESI), m/z 372 (M⁺+1).

5.31. 1-(tert-Butoxycarbonyl)-(4R)-ﬂuoropyrrolidine-(2S)-

carboxylic acid (3i)

5.31.1. Methyl 1-(tert-butoxycarbonyl)-(4R)-ﬂuoropyrr-

olidine-(2S)-carboxylate (34). By the procedure de-

scribed for compound 33, but with 32 in place of 22,

the title compound 34 was obtained as a pale yellow

oil (yield, 40%). ¹H NMR (CDCl₃) d 1.42 and 1.47 (total

9H, each s, amide isomers), 2.02–2.19 (1H, m), 2.52–2.64

(1H, m), 3.56–3.96 (5H, m), 4.40–4.47 (1H, m), 5.21 (1H,

dt, J = 52.4, 3.4 Hz); MS (ESI) m/z 270 (M⁺+Na).

5.29.2.

1-(tert-Butoxycarbonyl)-(4S)-(2-naphthyloxy)-

(2S)-pyrrolidinylcarboxylic acid (3f). To a stirred solu-

tion of methyl 1-(tert-butoxycarbonyl)-(4S)-(2-naph-

thyloxy)-(2S)-pyrrolidinylcarboxylate (31) (5.37 g) in

THF (116 ml) was added 0.25 N NaOH (116 ml,

29.0 mmol) at room temperature. The resulting mixture

was stirred for 12 h. After removal of the solvent, the

mixture was acidiﬁed by the addition of 1 N HCl and

extracted with CHCl₃. The combined extracts were

washed with brine, dried over Na₂SO₄, and evaporated.

The residue was recrystallized from n-hexane/CHCl₃, to

give the title compound 3f [4.44 g, 85% (2 steps)] as a

5.31.2. 1-(tert-Butoxycarbonyl)-(4R)-ﬂuoropyrrolidine-

(2S)-carboxylic acid (3i). A mixture of methyl 1-(tert-bu-

toxycarbonyl)-(4R)-ﬂuoropyrrolidine-(2S)-carboxyl-

ate (34) (345 mg, 1.40 mmol), 0.25 N NaOH (11 ml,

2.79 mmol), and THF (10 ml) was stirred for 12 h. The

mixture was acidiﬁed by the addition of 1 N HCl

(3 ml) and extracted with CHCl₃. The combined extracts

were dried over MgSO₄and evaporated to give the title

compound 3i (336 mg, quant) as a colorless oil. ¹H

NMR (CDCl₃) d 1.44–1.58 (9H, m), 2.44–2.56 (2H,

m), 3.42–3.97 (2H, m), 4.43 and 4.45 (total 1H, each t,

each, J = 8.3 Hz, amide isomers), 5.13–5.26 (1H, m);

MS (ESI) m/z 234 (M⁺+1).

1

colorless powder. H NMR (DMSO) d 1.37 and 1.41

(total 9H, each s, amide isomers), 2.26 (1H, d,

J = 13.9 Hz), 2.65 (1H, m), 3.47 (1H, d, J = 11.5 Hz),

3.81 (1H, m), 4.30 (1H, m), 5.14 (1H, m), 7.02–7.86

(7H, m); MS (ESI), m/z 358 (M⁺+1).

5.32. 1-(tert-Butoxycarbonyl)-4,4-diﬂuoropyrrolidine-(2S)-

carboxylic acid (3j)

5.30. 1-(tert-Butoxycarbonyl)-(4S)-ﬂuoropyrrolidine-(2S)-

carboxylic acid (3h)

5.32.1. Methyl 1-(tert-butoxycarbonyl)-4-oxopyrrolidine-

(2S)-carboxylate (36). To a stirred solution of methyl

N-Boc-trans-4-hydroxy-^L-proline methyl ester (22)

5.30.1. Methyl 1-(tert-butoxycarbonyl)-(4S)-ﬂuoropyrr-

olidine-(2S)-carboxylate (33). To a stirred solution of N-

Boc-trans-4-hydroxy-^L-proline methyl ester (22) (2.0 g,

8.15 mmol) in CH₂Cl₂(16 ml) was added DAST

(1.2 ml, 12.2 mmol) at ꢀ78 ꢁC. The mixture was allowed

to warm to room temperature, and the stirring was con-

tinued for 5 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 chromatographed on silica gel with

CHCl₃/EtOAc (20:1, v/v) as an eluent to give the title

compound 33 (830 mg, 41%) as a colorless oil. ¹H

NMR (CDCl₃) d 1.44 and 1.49 (total 9H, s, each, amide

isomers), 2.28–2.53 (2H, m), 3.56–3.90 (5H, m), 4.43 and

4.55 (total 1H, each d, each J = 9.3 Hz, amide isomers),

5.20 (1H, d, J = 9.3 Hz); MS (ESI) m/z, 248 (M⁺+1).

˚

(2.0 g, 8.15 mmol) in CH₂Cl₂were added 3 A molecular

sieves (2 g) and PDC (4.60 g, 12.2 mmol). The mixture

was stirred for 3 days. The mixture was ﬁltered through

a Celite pad, and the ﬁltrate was evaporated. The resi-

due was chromatographed on silica gel with CHCl₃/

MeOH (10:1, v/v) as an eluent to give the title com-

pound 36 (1.13 g, 57%) as a colorless oil. ¹H NMR

(CDCl₃) d 1.46–1.48 (9H, m), 2.56–2.61 (1H, m), 2.88–

3.00 (1H, m), 3.77 (3H, s), 3.82–3.88 (2H, m), 4.71–

4.83 (1H, m); MS (ESI) m/z, 266 (M⁺+Na).

5.32.2. Methyl 1-(tert-butoxycarbonyl)-4,4-diﬂuoropyrr-

olidine-(2S)-carboxylate (37). To a stirred solution of

methyl 1-(tert-butoxycarbonyl)-4-oxopyrrolidine-(2S)-

carboxylate (36) (1.13 g, 4.65 mmol) in CH₂Cl₂(20 ml)

was added DAST (1.1 ml, 11.6 mmol) at ꢀ78 ꢁC. The

mixture was allowed to warm to room temperature.

After 15 h stirring, 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 chromatographed on silica gel with

CHCl₃/EtOAc (20:1, v/v) as an eluent to give the title

5.30.2. 1-(tert-Butoxycarbonyl)-(4S)-ﬂuoropyrrolidine-

(2S)-carboxylic acid (3h). To a stirred solution of methyl

1-tert-butoxycarbonyl-(4S)-ﬂuoropyrrolidine-(2S)-car-

boxylate (33) (830 mg, 3.36 mmol) in THF (25 ml) was

added 0.25 N NaOH (27 ml, 6.71 mmol). The mixture

was stirred for 3 h. The mixture was poured into cold

1 N HCl and extracted with CHCl₃. The combined ex-

tracts were dried over MgSO₄and evaporated to give

1

compound 37 (885 mg, 72%) as a yellow oil. H NMR

(CDCl₃) d 1.42 and 1.47 (total 9H, each s, amide iso-

2744

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

mers), 2.46 (1H, ddd, d = 26.9, 13.7, 5.1 Hz), 2.62–2.78

(1H, m), 3.75–3.95 (5H, m), 4.43–4.57 (1H, m); MS

(ESI) m/z, 288 (M⁺+Na).

Human VCAM-1/Fc Chimera (R&D Systems Inc., Min-

neapolis, USA) was labeled with Eu as follows. The pro-

tein was reconstituted in labeling buﬀer (150 mM NaCl,

50 mM sodium carbonate, pH 8.5). Eu-Labelling Re-

agent (Perkin-Elmer Inc., Wellesley, USA) reconstituted

with the labeling buﬀer was added to the protein solu-

tion. The mixture was then incubated at room tempera-

ture for 4 days. The Eu-labeled protein was puriﬁed with

a PD-10 column (Amersham Biosciences KK, Tokyo,

Japan) and stored at ꢀ80 ꢁC until use.

5.32.3. 1-(tert-Butoxycarbonyl)-4,4-diﬂuoropyrrolidine-(2S)-

carboxylic acid (3j). To a stirred solution of methyl 1-

(tert-butoxycarbonyl)-4,4-diﬂuoropyrrolidine-(2S)-carboxylate

(37) (885 mg, 3.34 mmol) in THF (25 ml) was added 0.25 N

NaOH (26.7 ml, 6.67 mmol), and the stirring was continued

for 1 h. The mixture was poured into 1 N HCl (100 ml)

and extracted with CHCl₃. The combined extracts were

washed with brine, dried over MgSO₄, and evaporated to give

the title compound 3j (775 mg, 92%) as a yellow crystalline

solid. Mp 113–117 ꢁC; ¹H NMR (CDCl₃) d 1.44 and 1.49 (to-

tal 9H, each s, each, amide isomers), 2.53–2.80 (2H, m), 3.71–

3.90 (2H, m), 4.20-4.61 (1H, m); MS (FAB) m/z, 252 (M⁺+1);

Anal. Calcd for C₁₀H₁₅F₂O₄: C, 47.81; H, 6.02; N, 5.58.

Found: C, 48.06; H, 6.05; N, 5.45.

All assays were performed in duplicate. Prior to the as-

say, the 4B4 cells were suspended at 3 · 10⁵cells/ml in F-

12 medium (Hamꢀs F-12) supplemented with 10% fetal

calf serum, 100 U/ml penicillin, 100 lg/ml streptomycin,

and 2 mM ^L-glutamine (Invitrogen Corporation, Carls-

bad, USA). One hundred microliters of the 4B4 cell sus-

pension at 3 · 10⁵cells/ml was distributed into each well

of 96-well culture plates (Costar, 3599, USA). The plates

were incubated in a 5% CO₂humidiﬁed incubator at

37 ꢁC (Themo Forma, model: 3120, Forma Scientiﬁc,

Inc. USA) for 2 days.

5.33. 1-(tert-Butoxycarbonyl)-(4S)-chloropyrrolidine-

(2S)-carboxylic acid (3k)

5.33.1. Methyl 1-(tert-butoxycarbonyl)-(4S)-chloropyrr-

olidine-(2S)-carboxylate (35). To a stirred solution of

methyl N-Boc-trans-4-hydroxy-^L-proline methyl ester

(22) (3.15 g, 12.8 mmol) in CH₂Cl₂/CCl₄(50 ml, 1:1, v/

v) was added Ph₃P (7.11 g, 27.1 mmol), and the reaction

mixture was stirred at room temperature for 2.5 h. To

the mixture was added EtOH (10 ml), and the reaction

mixture was stirred for 12 h. After removal of the sol-

vent, the residue was puriﬁed by column chromatogra-

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

eluent to give the title compound 35 (2.57 g, 76%) as a

The medium was discarded, and each well was washed

twice with 300 ll of chilled wash buﬀer (25 mM Hepes

(pH 7.5), 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂,

and 4 mM MnCl₂). Then 50 ll compound solution was

distributed to the wells, followed by 50 ll of 2 nM of

Eu-labelled Human VCAM-1/Fc Chimera diluted with

the wash buﬀer (ﬁnal concentration: 1 nM). The plates

were incubated for 60 min at room temperature, and

the wells were washed 4 times with 300 ll of chilled wash

buﬀer. Finally, 100 ll of the enhancement solution

(Perkin-Elmer Inc., Wellesley, USA) was added to each

well. The plates were placed on a shaker for 5 min. Eu

ﬂuorescence was then measured in a time-resolved ﬂuo-

rometer (DELFIA Research ﬂuorometer, model: 1234-

001, Perkin-Elmer Inc., Wellesley, USA).

1

colorless oil. H NMR (CDCl₃) d 1.42 (9H, s), 2.33–

2.39 (1H, m), 2.67–2.77 (1H, m), 3.60–3.67 (1H, m),

3.76 (3H, s), 3.80–4.00 (1H, m), 4.32–4.46 (2H, m);

FAB-MS, m/z 264 (M⁺+1).

5.33.2. 1-(tert-Butoxycarbonyl)-(4S)-chloropyrrolidine-

(2S)-carboxylic acid (3k). To a stirred solution of methyl

1-(tert-butoxycarbonyl)-(4S)-chloropyrrolidine-(2S)-carboxyl-

ate (35) (2.57 g, 9.8 mmol) in THF (20 ml) was added

0.5 N NaOH (20 ml), and the reaction mixture was heat-

ed under reﬂux for 2 h. The mixture was poured into 1 N

HCl and extracted with CHCl₃. The combined extracts

were washed with brine, dried over Na₂SO₄, and evapo-

rated to give the title compound 3k (2.02 g, 83%) as a

The concentration of compound required for 50% inhi-

bition in the assay was determined.

5.35. Distribution coeﬃcient

The distribution coeﬃcients (logD) were determined by

the well-known shake-ﬂask method.¹⁵Four hundred

micromolar of compound solution in n-octanol (2 ml)/

PBS (2 ml, pH 7.4) or the Japanese Pharmacopoeia Sec-

ond ﬂuid (2 ml, pH 6.8) was placed on a shaker for

30 min. After centrifuging at 3000 rpm for 10 min, each

layer was assayed using LC–MS methodologies (LC–

Mass spectrometer: 1100 Series LC/MSD, Agilent; Ana-

lytical Column: X Terra^ꢂMSC18 3.5 lm, 3.0 · 30 mm,

Waters; Mobile Phase: 10 mM ammonium acetate buﬀer

(pH 4.5)/0.05% acetic acid in acetonitrile = 95:5 to 10:90

v/v). The values of logD were analyzed using Analyst

software program (version 1.4, Applied Bio. Systems).

1

colorless solid. H NMR (CDCl₃) d 1.44 and 1.48 (total

9H, each s, amide isomers), 2.44–2.76 (2H, m), 3.64 (1H,

m), 3.91–3.97 (1H, m), 4.38–4.46 (2H, m), 8.17 (1H, s);

MS (FAB), m/z 250 (M⁺+1).

5.34. VLA-4/VCAM-1 binding assay

A human VLA-4-expressing cell line, 4B4, was estab-

lished at Pharmacopeia (New Jersey, USA), by trans-

fecting both the a4 gene and b1 gene of VLA-4 into

CHO-K1 cells. The 4B4 cells were maintained in F-12

medium (Hamꢀs F-12) supplemented with 10% fetal calf

serum, 100 U/ml penicillin, 100 lg/ml streptomycin, and

2 mM ^L-glutamine (Invitrogen Corporation, Carlsbad,

USA) and 1 mg/ml G-418 (Geneticin, Invitrogen Corpo-

ration, Carlsbad, USA).

5.36. Pharmacokinetic studies on rats

Male Sprague–Dawley rats (280–360 g, obtained from

Hilltop Laboratories and Charles River Laboratories)

were used for the study. Animals were surgically implant-

J. Chiba et al. / Bioorg. Med. Chem. 14 (2006) 2725–2746

2745

ed with catheters in the right and left jugular veins. The

jugular catheters were composed of silastic tubing (i.d.

0.02 in. and o.d. 0.037 in.). The lock solution for the cath-

eter contained streptokinase (Kabikinase^ꢂ, 18750 U/ml)

and heparin (500 U/ml) in 25% dextrose. Care was taken

to apply only the catheter volume (ꢁ0.05–0.07 ml) of lock

solution in order to minimize systemic exposure to this

mixture. Animals were housed in clear PVC boxes with

lids in unidirectional airﬂow rooms with controlled tem-

perature (20 4 ꢁC) and relative humidity (50 20%)

and a 12 h light/dark cycle. Animals were provided food

and tap water ad libitum, except for the 12 h fasting (food

withdrawn) period prior to dosing.

(72 4 ꢁF) and relative humidity (50 20%) and a

12 h light/dark cycle. Animals were provided food and

tap water ad libitum. During dosing and sampling, the

dogs were restrained in Pavlov slings for ꢁ2 h.

The dosing solution was freshly prepared in a vehicle

comprising 2.9% ethanol, 0.1% DMSO, and 97% sterile

saline (0.9% w/v, pH adjusted to 8.4 with 8% sodium

bicarbonate). The ﬁnal compound concentration in the

dosing mixture was 0.5 mg of each compound/ml. Each

animal received the dosing mixture as a constant 1 h

intravenous infusion via the implanted catheter at a tar-

geted dose rate of 0.75 mg of each compound/kg/h.

Intravenous administration: each compound was dis-

solved in sterile 0.9% saline (pH adjusted to 8.0 with

sodium bicarbonate) for a target concentration of 0.5

or 1.0 mg of each compound per milliliter and adminis-

tered by 2 h intravenous infusion at a targeted dose rate

of 0.75, 1.0, or 1.2 mg/kg, respectively. Oral administra-

tion: each compound was suspended in 0.5% (w/v) aque-

ous methylcellulose for a target concentration of 0.2 mg

of each compound per milliliter and administeredby oral

gavage at a targeted dose rate of 2.0 mg/kg.

Plasma samples were assayed by using LC–MS/MS

methodologies (Mass spectrometer: TSQ 7000 Triple

Stage Quadrupole, Finnigan; HPLC System: 2690 Sepa-

rations Module, Waters; Analytical Column: XDB-C18,

2.1 · 150 mm, 5 lm, Zorbax; Mobile Phase: 0.2% formic

acid in water/acetonitrile = 67:33 v/v). Plasma concen-

trations were analyzed by using the WinNolin software

program (version 1.1, Scientiﬁc Consulting, Inc.).

References and notes

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odologies (Mass spectrometer: Micro Quattro II, Micro-

mass, Inc. or TSQ 7000 Triple Stage Quadrupole,

Finnigan; HPLC System: HP 1100 Binary Pump, Hew-

lett–Packard Company or 2690 Separations Module,

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Article Doi

Synthesis, biological evaluation, and pharmacokinetic study of prolyl-1-piperazinylacetic acid and prolyl-4-piperidinylacetic acid derivatives as VLA-4 antagonists

DOI: 10.1016/j.bmc.2005.11.058

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Article abstract of DOI:10.1016/j.bmc.2005.11.058

Full text of DOI:10.1016/j.bmc.2005.11.058

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