New orally active enkephalinase inhibitors: Their synthesis, biological activity, and analgesic properties

Article abstract of DOI:10.1016/S0968-0896(97)10048-7

A series of (4s)-4-[(2S)-benzyl-3-mercaptopropionylamino]-4-(N- phenylcarbamoyl)-butyric acids has been identified as potent systemically active enkephalinase inhibitors. Structure-activity relationships (SAR) are discussed. Further chemical modification of the inhibitors was carried out in order to identify the inhibitors which are orally active in an animal model. Compounds of particular interest are the prodrug-like analogues, including 5b (ONO-9902). Their analgesic effects after oral administration were evaluated.

Full text of DOI:10.1016/S0968-0896(97)10048-7

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 441±463
New Orally Active Enkephalinase Inhibitors: their Synthesis,
Biological Activity, and Analgesic Properties
Kazuhiko Senokuchi, Hisao Nakai,* Yuuki Nagao, Yasuhiro Sakai,
Nobuo Katsube and Masanori Kawamura
Department of Medicinal Chemistry, Minase Research Institute, Ono Pharmaceutical Co., Ltd.,
Shimamoto, Mishima, Osaka 618, Japan
Received 16 October 1997; accepted 20 November 1997
AbstractÐA series of (4S)-4-[(2S)-benzyl-3-mercaptopropionylamino]-4-(N-phenylcarbamoyl)-butyric acids has been
identi®ed as potent systemically active enkephalinase inhibitors. Structure±activity relationships (SAR) are discussed.
Further chemical modi®cation of the inhibitors was carried out in order to identify the inhibitors which are orally
active in an animal model. Compounds of particular interest are the prodrug-like analogues, including 5b (ONO-9902).
Their analgesic e€ects after oral administration were evaluated. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
physiologically involved in enkephalin degradation.15
Therefore, it is of interest to design compounds which
are able to inhibit the three enkephalin-degrading
enzymes belonging to the group of metallopeptidases.
The discovery of endogenous opioid peptides in the
brain1, enkephalins, which are associated with both a
heterogeneity of binding sites2±4 and a well-de®ned
metabolic pathway might o€er new approaches to the
prevention or treatment of addiction following analgesic
use. In vitro studies performed with rat brain slices
showed that three metalloenzymes are involved in
enkephalin degradation: (1) a neutral metalloendo-
peptidase, EC 3. 4. 24. 11,5 designated enkephalinase,6
cleaving the Gly-Phe (positions 3 and 4) bond, (2) an
aminopeptidase releasing the N-terminal tyrosine, and a
dipeptidylaminopeptidase7 cleaving the Gly-Gly (posi-
tions 2 and 3) amide bond.8 The importance of enke-
phalinase is supported by the analgesic e€ects induced
by the administration of potent enkephalinase inhibitors
such as carboxyalkyl dipeptides9,10, thiorphan 1,11,12
retrothiorphan 2 and kelatorphan 4 (Chart 1)13. The
oral activity of the compounds 3a±c has also been
reported14.
Despite the considerable interest in this ®eld during the
last few decades, therapeutic applications of an enke-
phalinase inhibitor have failed to materialize. This fail-
ure has been due largely to oral activity problems
encountered when the inhibitors are used as a drug.
During the course of a screening of our compound
library, an angiotensin-converting enzyme (ACE) inhi-
bitor 616 was discovered to show moderate inhibitory
activity against enkephalinase (IC50=0.7 mM). The
structural similarity of this compound to thiorphan 1
prompted us to prepare compounds 7a,b and evaluate
their inhibitory activity (Chart 2). Compounds 7a,b
showed potent in vitro inhibitory activity (7a:
IC50=4.25 nM, 7b: IC50=10.5 nM), although they were
not e€ective in systemic administration (Table 1).
According to the reported modes of binding of a sub-
strate and various inhibitors with the active site of
enkephalinase,17 the R residue of general formula I
should be lipophilic, while it was hydrophilic in 7a,b. In
light of this, we were surprised that the glutamic acid
residue was accepted as a surrogate instead of the gly-
cine residue of thiorphan 1. In the present report, we
The analgesic activity induced by the intracerebroven-
tricular (icv) administration of the aminopeptidase inhi-
bitor bestatin suggested that this enzyme could also be
*Corresponding author.
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(97)10048-7

442
K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
describe the successful modi®cation of compound 6 to
obtain new orally active enkephalinase inhibitors. The full
details of the synthesis, structure±activity relationships,
and some pharmacological evaluations are discussed.
of an anilide 32 from 31 was carried out by the acid
anhydride method using pivaloyl chloride followed by
deprotection with tri¯uoroacetic acid to a€ord 33,
which was acylated with 29 to give 34. The aminolysis of
the acetylthio moiety with mercaptoethylamine followed
Table 1. Enkephalinase inhibition and/or the e€ect on the
bradykinin-induced biting-like response of 1, 2, 3a±c, 6 and 7a±b
in male SD rats
Chemistry
The syntheses of 8b and 9c±17c are described in Scheme
1. The preparation of anilide 28 from 27 was carried out
by an acid anhydride method using pivaloyl chloride
followed by acidic deprotection, respectively. The N-
acylation of 28 with an acid chloride 29 followed by
alkaline hydrolysis a€orded 8b. Compounds 9c±17c
were prepared from 27 by the following sequential
reactions: The preparation of acid anhydride with piva-
loyl chloride, the addition of an appropriate amine, and
acidic deprotection a€orded 9a±17a. Compounds 9a±
17a were converted to 9c±17c by the same procedure as
described in the preparation of 8b from 28.
Compound
IC50 (nM)
BK-bitingb (mg/kg ip)
Ne
1
3.0
700
2.5
NEc (30>mg/kg ip)
NTd
7
Ð
6
2
3aa
3b
3c
6
33.3% (5 mg/kg ip)
NTb
33
26
Ð
Ð
4
NTb
NTb
700
4.25
10.5
7a
7b
NEc (30 mg/kg ip)
NEc (10 mg/kg ip)
4
4
aOral activity was reported.14
bBradykinin-induced biting-like response.
cNE: Not e€ective.
dNT: Not tested.
Scheme 2 demonstrates the syntheses of 18a and 18b.
Compound 18a was prepared from 31. The preparation
H
O
HS
N
COOH
HS
COOH
N
O
H
1 (Thiorphan)
2 (Retrothiorphan)
O
H
HS
NHR
N
COOH
HOHN
O
Me
O
4 (Kelatorphan)
3a (Ru 42827) : R =
3b : R =
N • HCl
3c : R =
OMe
O
H
S
N
N
H
O
O (CH2)n
COOH
O
5a : n = 1
5b : n = 2 (ONO-9902)
Chart 1. Enkephalinase inhibitors.

K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
443
by alkaline hydrolysis provided 18a. An anilide 37 was
prepared from 36 by the same procedure as described in
the preparation of 32 from 31. Compound 37 was con-
verted to 18b by these sequential reactions: hydrogena-
tion; acylation with 29; acidic deprotection; aminolysis
of thioacetate; hydrolysis with sodium hydroxide.
The treatment of 44(S) and 44(R) with oxalyl chloride
followed by the addition of 45a provided 46a(S) and
46a(R), respectively, which were converted to 8a(S) and
8a(R) by methanolysis followed by deprotection with
tri¯uoroacetic acid (TFA), respectively. The N-acylation
of 45b,c with 44(S) and 44(R) provided 46b(S), 46c(S)
and 46b(R), 46c(R), respectively, which were converted
to 8b(S), 11(S) and 8b(R) and 11(R) by alkaline hydro-
lysis, respectively.
The preparation of 19a±c was carried out as shown in
Scheme 3. The alkaline hydrolysis of 41a±c followed by
the Michael addition of thiobenzoic acid or thioacetic
acid provided 42a±c, respectively. The conversion of
42a±c to the corresponding acid chloride with oxalyl
chloride followed by the addition of 28 a€orded 43a±c,
respectively. The alkaline hydrolysis of 43a±c gave 19a±c,
respectively.
Compounds 20±24 were prepared as shown in Scheme 5.
The condensation of 47 with aniline provided 48. The
hydrogenation of 48 followed by the addition of 1
equivalent of hydrogen chloride a€orded 49, which was
acylated with an acid chloride prepared from 44(S),
a€ording 50a. The methanolysis of 50a provided 50b,
which was acylated with an appropriate acyl chloride
and then deprotected to give 20 and 24, respectively.
The following sequential reactions: The protection of
The preparation of optically active diastereomers 8a(S),
8a(R), 8b(S), 8b(R), 11(S) and 11(R) are described in
Scheme 4.
• CF CO H
3
2
O
O
a), b), c)
d)
O
BocHN
H
OH
H N
2
AcS
N
N
N
(CH )
2 2
H
H
(CH )
2 2
COOBn
O (CH )
2 2
COOBn
COOBn
27
28
30
O
e)
H
HS
N
N
H
O (CH )
2 2
COOH
8b
• CF CO H
3
2
O
O
a), f), c)
d)
H
H N
2
27
AcS
N
NHR
NHR
(CH )
2 2
O (CH )
2 2
COOBn
COOBn
9b - 17b
9a - 17a
9 : R = 2-Naphthyl 10 : R = 4-Pyridyl 11 : R = 2-Benzthiazolyl
12 : R = 4-F-Phenyl 13 : R = 4-Cl-Phenyl 14 : R = 4-I-Phenyl
15 : R = 4-MeO-Phenyl 16 : R = 4-Ac-Phenyl
O
e)
H
N
HS
NHR
17 : R = 4-SO NH -Phenyl
2
2
O (CH )
2 2
COOH
9c - 17c
Reagents: a) t-BuCOCl, Et N, CH Cl , 5 °C, 0.5 h; b) PhNH , 5 °C, 1 h; c) CF CO H, CH Cl ,
3
2
2
2
3
2
2
2
25 °C, 2.5 h; d) 2-benzyl-3-acetylthiopropionyl chloride (29), Py, CH Cl , 5 °C then 25 °C, 15 min;
2
2
e) LiOH, DME - THF - H O, 25 °C, 0.5 h; f) RNH , 5 °C, 1 h.
2
2
Scheme 1. Synthesis of 8b and 9c±17c.

444
K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
Scheme 2. Synthesis of 18a and 18b.

K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
445
the carboxylic acid moiety of 8b(S) with t-butyl-
chlorodiphenylsilane; acylation with an appropriate acyl
chloride or with an appropriate carboxylic acid in the
presence of DPPA; deprotection with n-butylammoniun
¯uoride, a€orded 21±23, respectively.
21 nM of 8b or 3 nM of thiorphan 1), this orally active
compound suppressed all of the above experimental pain
responses in a dose-dependent manner. The potentiation
of the SIA-induced antinociceptive action of rodents in
the hot-plate test was also observed. An evaluation of 5b
using the BK-induced nociceptive biting-like and
AcOH-induced writhing responses indicated a 30 mg/kg
ED50 and 10 mg/kg as the minimum e€ective dose (MED)
values in oral administration. Though this enkephalin-
ase inhibitor was e€ective in the above tests, it did not
elicit signi®cant suppressive e€ects on naive rodents
examined with the hot-plate test, a property intrinsic to
the opioid agonist±antagonist pentazocine, as well.
Compounds 5a,b were prepared from 46a and 8b(S) by
S-alkylation with 3-chlorophthalide followed by depro-
tection with TFA and simple S-alkylation with 3-chlor-
ophthalide, respectively. The preparation of 7b, 25 and
26 is illustrated in Scheme 6. After protection of the
carboxylic acid function of 11c(S) as a silyl ester, the S-
alkylation with 3-chlorophthalide followed by acidic
deprotection a€orded 25. The alkaline hydrolysis of 51
a€orded 7b(S). The methanolysis of 44(S) provided 26.
Enkephalinase is a zinc metallopeptidase homologous to
ACE, and previous work provided some precedents for
the design of enkephalinase inhibitors.10 The design of
enkephalinase inhibitors of the general formula II was
based on the subsite preferences. According to the
reported modes of binding of a substrate and various
inhibitors with the active site of enkephalinase, enke-
phalinase inhibitors such as SCH-3261520, which was
reported during our work, were estimated to possess a
strong interaction with the two subsites S10 and S20 of
enkephalinase using the two aromatic moieties, besides
the regular interaction using zinc ligand and acidic
moiety, as illustrated in Chart 3(a) and (b). Based on
this information, the conversion of the a-carboxylic acid
moiety of 7b to an anilide provided 8b, whose modes of
interaction with the enzyme were expected to be similar
to those of SCH-32615 as shown in Chart 3(c), with a
Results and discussion
The enkephalinase inhibitory activity of the synthetic
inhibitors was studied using an in vitro approach.18
Their antinociceptive e€ect was evaluated with the
bradykinin (BK)-induced biting-like response,19 the
formalin-induced licking method, the acetic acid
(AcOH)-induced writhing method, and potentiation
e€ects on stress-induced analgesia (SIA) in a hot-plate
test.
Although the selected compound 5b showed low in vitro
enkephalinase inhibition (IC50=0.3 mM as compared to
R1
O
R1
H
R1
a), b)
c), d)
R2
S
N
R2
S
N
COOH
H
COOEt
41a-c
O
O (CH2)2
O
COOBn
43a - c
42a - c
R1
O
H
19a : R1 = 4-Methoxyphenyl
19b : R1 = 4-Methylphenyl
19c : R1 = Cyclohexyl
HS
N
e)
N
H
O (CH2)2
COOH
Reagents: a) KOH, EtOH, reflux, 1.5 h; b) PhSH, CH2Cl2, reflux or AcSH, Ph-H, reflux, 2 h;
c) (COCl)2, CH2Cl2, 5 - 25 °C, 0.5 h; d) L-GluNHPh(OBn) • CF3CO2H (28), Py, CH2Cl2, 5 °C, 1 h;
e) LiOH, DME - THF, 25 °C, 1 h.
Scheme 3. Synthesis of 19a±19c.

446
K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
Scheme 4. Synthesis of 8a(S), 8a(R), 8b(S), 8b(R), 11c(S) and 11c(R).

K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
447
Scheme 5. Synthesis of 20±24 and 5a and 5b.

448
K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
a), b), c)
11c(S)
a)
H
PhCOS
N
COOH
O
O (CH2)2
H
N
S
COOMe
S
N
51
N
H
O
O (CH2)2
COOH
O
25
Reagents: a) t-BuPh2SiCl, imidazole, DMF, 5 °C;
b) 3-chlorophthalide, K2CO3, acetone - DMF,
25 °C, 2h; c) AcOH - THF - H2O, 25 °C, 15 h.
H
HS
N
COOH
O (CH2)2
COOH
7b(S)
a)
Reagents: a) 2N NaOH, MeOH, 25 °C, 1.5 h.
44(S)
HS
COOH
26
Reagents: a) K2CO3, MeOH, 25 °C.
Scheme 6.
slightly less in vitro potency. However, compound 8b
showed systemic activity after intraperitoneal (ip)
administration (Table 3), while 7b at 10 mg/kg ip did not
a€ect the BK-induced biting-like response of rats
(Table 1). The compounds shown in Table 2 were made
in order to explore the subsite generalization and to test
our hypothesis described in Chart 3(c). The naphthyl-
amide moiety of 9c was estimated to be the one which is
dicult to ®t into the S20 pocket of enkephalinase,
based on its lesser in vitro activity compared to the
other compounds listed in Table 2. Modi®cations of the
anilide moiety provided 12c±17c with retained in vitro
activity (Table 3). The compounds 8±17 demonstrated
antinociceptive e€ects on the BK-induced biting-like
response after ip administration regardless of their in
vitro potency.
H
HS
N
COOH
1 (Thiorphan)
O (CH2)2
COOH
6
Regioisomers 18a,b (Table 4) exhibited less anti-
nociceptive e€ects in the BK-induced biting-like model
described above regardless of their in vitro potency. As
a result, the conversion of one carboxylic acid group of
7a,b to an anilide was found to improve the pharmaco-
dynamics of the inhibitors. We had to revise our
hypothesis at this stage.
H
HS
N
COOH
O (CH2)n
COOH
7a : n = 1
7b : n = 2
In order to obtain more e€ective compounds for sys-
temic administration, a further chemical modi®cation of
the anilide moiety was made. As shown in Table 3, a
Chart 2. Hybridization of 1 and 6.

K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
449
signi®cant increase in the in vitro activity was not
obtained with this modi®cation.
S1'
S2'
(a)
Arg+
Zn++
SMe
Additionally, the in vivo e€ects of 8±17 did not show a
close relationship to their in vitro potency. These results
strongly suggested that the in vivo ecacy of these
compounds is largely dependent upon their metabolic
pathway and/or rate (Scheme 7). In addition, the per-
centage of the parent compound and the active meta-
bolites, and their pharmacodynamic parameters such as
the permeability through the blood±brain barrier (BBB)
were estimated to give a reasonable explanation of the
in vivo ecacy of compounds 8±17.
O
CH2
O
Tyr-Gly-Gly) C N C C N C C O
H
H
H
H
O
[Met5] enkephalin
(Km = 2 µM)
S1'
S2'
(b)
Arg+
Zn++
O
CH2 CH2
C C N C C NH
O C
Among compounds 8±17, 8b exhibited the most potent
antinociceptive e€ects on the BK-induced biting-like
response by ip administration.
O
O
CH2
CH2
H H
H
O
SCH 32615
(IC50 = 21.0 nM)
In order to further optimize the benzyl moiety, a mod-
i®cation of R of the general formula 4 was carried out.
As shown in Table 5, the aromatic moiety such as p-
methoxyphenyl of 19a or p-methylphenyl group of 19b
was more preferable to the aliphatic moiety such as the
cyclohexyl group of 19c. A phenyl group was found to
be the best among the synthesized compounds.
S1'
S2'
Arg+
(c)
Zn++
O
O
HN
CH2
C O C
S
CH2 C C N CCH2 CH2
H
H
H
O
As illustrated in Table 6, a series of the synthetic inhi-
bitors possessing an S con®guration of the benzyl moi-
ety showed more potent inhibitory activity than their
corresponding R-isomers. The BK-induced biting-like
response was also more potently suppressed by the S-
isomer 8b(S) than its R-isomer 8b(R), based on the
comparison of the e€ects of 8b(S) and 8b(R). None of
the synthesized inhibitors were orally active, although
they showed an in vivo e€ect by ip administration.
8b
(IC50 = 26 nM)
Chart 3. Model of active site of enkephalinase with proposed
models of binding of [Met5]-enkephalin, and the synthetic
inhibitors.
O
H
H
HS
N
COOH
HS
N
N
H
O (CH2)2
O (CH2)2
COOH
COOH
8b(S)
7b
O
H
S
N
HS
N
COOH
H
O
O (CH2)2
COOH
O
26
5b (ONO-9902)
Scheme 7. Plausible metabolic pathway and metabolites of 5b (ONO-9902) in rats and dogs after oral administration.

450
K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
The purpose of this investigation was to identify an
orally active enkephalinase inhibitor as a drug candi-
date. The metabolic instability of the naked thiol group
was considered to be one of the most plausible reasons
of the oral inactivity encountered. The protection of the
thiol group as a prodrug was regarded as one reasonable
way to solve this problem.
possesses metabolically unstable functions such as one
ester and two amides in its molecule. The metabolic
pathway of 5b in rat was presumed as described in
Scheme 7 based on its metabolites. The metabolism of
5b was estimated to proceed di€erently in the rate and/
or the pathway dependent upon the species, judging
from the di€erent percentage of its metabolites as shown
in Table 9.
As described in Tables 7 and 8, several kinds of pre-
sumed prodrugs, 5a,b and 20±25, were prepared as the
candidates and evaluated in the BK-induced biting test
and formalin- induced licking test following oral
administration. Compounds 5a,b, 20, 21, 23 and 25
exhibited analgesic e€ects on at least the BK-induced
biting-like response. Among the synthesized com-
pounds, 5b a€orded the best result in all the test pain
models and was least toxic in the acute toxicity test. A
more detailed comparison was then performed to dif-
ferentiate 5a and 5b, because of their structural similarity.
A main metabolite of 5b in rat plasma and dog plasma
was 26. The parent compound was not detected in rat
plasma, while it was detected in dog plasma as a minor
metabolite. Compound 7b was detected as one of the
metabolites in rat plasma. It was also detected in dog
Table 3. Enkephalinase inhibition and the e€ect on the
bsdykinin-induced biting-like response of 12c±17c in male SD
rats
As shown in Table 8, a simultaneous comparison of the
synthetic inhibitors 5a and 5b by an in vivo evaluation
was carried out. In this evaluation, 5b gave better results
than 5a, especially in acute toxicity. As a result, 5b
(ONO-9902) was selected for further evaluation. It was
of interest to investigate the metabolism of 5b because it
Compound
X
IC50 (nm)
BK-bitinga
(mg/kg ip) (EN/N)b
Table 2. Enkephalinase inhibition and the e€ect on the
bradykinin-induced biting-like response of 8a(S),b and 9c±11c
in male SD rats
12c
13c
14c
15c
16c
17c
F
CI
94
49
10(2/4)
10 (3/4)
10 (5/8)
10 (2/4)
10 (3/4)
10 (1/4)
I
48
OMe
COMe
SO2NH2
31.9
20.8
23.3
O
H
S
R1
N
NHR2
aBradykinin-induced biting-like response.
bEN/N: Number of a€ected rats/number of tested rats.
O (CH2)2
I
COOH
Compound
n
R
IC50 (nm) BK-bitinga
(mg/kg ip)
(EN/N)b
Table 4. Enkephalinase inhibition and the e€ect on the
bradykinin-induced biting-like response of 18a and 18b in male
SD rats
8a(S)
1
2
30
21
10 (4/8)
8b
3 (4/8)
10 (7/8)
9c
2
349
38
10 (2/4)
Compound n IC50 (nM) BK-bitinga (mg/kg ip) (EN/N)b
10c
11c
2
2
10 (1/4)
10 (3/4)
18a
18b
1
2
40
68
10 (1/4)
10 (3/6)
43.8
aBradykinin-induced biting-like response.
bEN/N: Number of a€ected rats/number of tested rats.
aBradykinin-induced biting-like response.
bEN/N: Number of a€ected rats/number of tested rats.

K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
451
Table 5. Enkephalinase inhibition and the e€ect on the
bradykinin-induced biting-like response of 19a±c in male SD
rats
Table 6. Enkephalinase inhibition and the e€ect on the
bradykinin-induced biting-like response of 8a(S), 8a(R), 8b(S),
8b(R), 11(S) and 11(R) in male SD rats
Compound n R1 R2
R3
IC50 (nM)
BK-bitinga
(mg/kg ip) (nM)
(EN/N)b
Compound
19a
R
IC50 (nM)
BK-bitinga
(mg/kg ip) (EN/N)b
51
71
10 (2/4)
10 (2/4)
NTCc
8a(S)
8a(R)
8b(S)
8b(R)
11(S)
11(R)
1 H Bn
1Bn H
2 H Bn
2Bn H
2 H Bn
2Bn H
30
47
10 (4/8)
NTc
19b
19c
400
23
10(7/8)
10(0/8)
10 (5/8)
NTc
aBradykinin-induced biting-like response.
bEN/N: Number of a€ected rats/number of tested rats.
cNT: Not tested.
175
16.8
240
plasma. The bioavailability of 5b after oral administra-
tion was 40% in rats with a half-life of about 7 h.
In summary, structurally new enkephalinase inhibitors
were discovered based on the chemical modi®cation of
an ACE inhibitor, compound 6. Systemically e€ective
enkephalinase inhibitors were designed based on the
reported modes of interaction between the enzyme and
the inhibitor, although they showed less in vitro activity
than the lead compound 7b. Thus, these newly found
synthetic inhibitors possessing a naked thiol group were
not orally active because of their presumed instability
(mainly against oxidative metabolism) and/or an oral
absorption problem, although they exhibited systemic
e€ects by ip administration. The protection of the thiol
group with the known prodrug-like moiety illustrated in
Table 7 provided 5b, 20, 21 and 23, which were orally
active. Some of these orally active enkephalinase inhi-
bitors elicited potent analgesic e€ects when tested with
experimental nociceptive methods, and could be of sig-
ni®cant bene®ts in clinical application.
aBradykinin-induced biting-like response.
bEN/N: Number of a€ected rats/number of tested rats.
cNT: Not tested.
Germany). Thin layer chromatography was performed
on silica gel (Merck Art. no. 5715). All solvents were
distilled before use. Thiorphan (1), retrothiorphan (2),
3a±c and kelatorphan (4) were prepared according to
the reported methods.12±14,21
Preparation of 2-substituted-3-acylthiopropionic acids
(42a-c): 2-(4-methoxybenzyl)-3-benzoylthiopropionic acid
(42a). To a stirred solution of 41a (6.49 g, 29.5 mmol),
which was prepared from diethyl malonate (10 mL,
0.065 mol), p-anisaldehyde (9.8 mL, 0.081 mol) and par-
aformaldehyde22 was added potassium hydroxide
(3.89 g, 59.0 mmol), and the mixture was re¯uxed for
1.5 h. After evaporation, the reaction mixture was dis-
solved in water (30 mL), acidi®ed with concentrated
hydrogen chloride (6 mL) and extracted with dichloro-
methane. The organic layer was washed with distilled
water and dried (Na2SO4). The resulting product was
puri®ed by chromatography on silica-gel (n-hexane:
ethyl acetate=5:2) to yield a corresponding carboxylic
acid (3.38 g, 59.6%) as a white powder: Rf=0.42 (n-
hexane:ethyl acetate=2:1); 1H-NMR (CDC13) d 3.67
(2H, s), 3.80 (3H, s, -OMe), 5.45±5.56 (1H, m, vinylic),
6.36 (1H, brd, J=1.0 Hz), 6.85 (2H, brd, J=8.5 Hz),
7.14 (2H, brd, J=8.5 Hz).
Experimental
Chemistry
1
General directions. All H-NMR spectra were taken on
a JEOL FX- 90Q, Varian VXR-200s, or 500s spectro-
meter. MS spectra were obtained on a JEOL JMS-DX-
303HF. IR spectra were measured on a Perkin Elmer
FT-IR 1760X. Melting points were uncorrected. Col-
umn chromatography was carried out on silica gel
(particle size 0.063±0.02 mm; E. Merck, Darmstadt,

452
K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
Table 7. Enkephalinase inhibition, analgesic properties and acute toxicity of 5b and 20±25
Compound
R1
R2
IC50 (nM)
Analgesic test (po)
Acute toxicityf
ip N=5, 500 mg/kg
BK testb N=8, % mg/kg Lickingc N=6, 300 mg/kg
5b
20
Ph
Ph
340
50 (30)
75 (100)
++
NEe
( )
(1600)a
37.5 (30)
50 (100)
(+)
(+)
NTd
21
22
Ph
Ph
1900
160
37.5 (30)
81.5 (100)
+
0 (100)
NTd
23
24
Ph
Ph
135
62.5 (100)
0 (100)
NEe
NTd
( )
3300
NTd
25
310
0 (30)
62.5 (100)
NEe
( )
aThese compounds were studied as diastereomeric mixtures of R, S and S, S isomers.
bBK test: Bradykinin-induced biting-like response. Values are inhibition %.
cLicking: Formalin-induced licking test. Signi®cant di€erence from the control group: ++, p< 0.01 at 0±60 min; +, p<0.01 at 0±
15 min.
dNT: Not tested.
eNE: Not e€ective.
fAcute toxicity: ( ), no e€ect; (+), slight loss of body weight.
To a stirred solution of the resulting carboxylic.acid
(3.37 g, 17.6 mmol) in dichloromethane (30 mL) was
added thiobenzoic acid (2.18 mL, 17.6 mmol), and the
mixture was re¯uxed for 15 h. After evaporation, the
residue was puri®ed by chromatography on silica-gel (n-
hexane:ethyl acetate=4:1 followed by 2:1) to yield 42a
(3.29 g, 56.6%): Rf=0.25 (n-hexane:ethyl acetate=2:1);
1H-NMR (CDC13) d 2.92±3.40 (5H, m), 3.79 (3H, s, -
OMe), 6.85 (2H, brd, J=8.5 Hz), 7.17 (2H, brd,
J=8.5 Hz), 7.32±7.62 (3H, m), 7.96 (2H, brd,
J=8.5 Hz).
Compound 42b was prepared according to the same
method as described above (43.0% in 2 steps).
2-(4-Methylbenzyl)-3-benzoylthiopropionic acid (42b).
Rf=0.28 (n-hexane:ethyl acetate=2:1); 1H-NMR
(CDC13) d 2.33 (3H, s, Me), 2.80±3.40 (SH, m), 7.12
(4H, brs), 7.30±7.61 (3H, m), 7.97 (2H, brd, J=8.5 Hz).
2-Cyclohexylmethyl-3-acetylthiopropionic acid (42c).
To a stirred solution of carboxylic acid (3.38 g,
20.1 mmol) which was prepared from diethyl malonate,

K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
453
Table 8. Enkephalinase inhibition, analgesic properties and acute toxicity of 5a,b and 26
Compound
n
R
IC50 (nM)
Analgesic test (po)
Lickingb Acetic acidc
Acute toxicitye
(ip) N=5
500 mg/kg
BK testa
N=8, % (mg/kg) N=6, 300 mg/kg N=10, % (mg/kg)
SIAd N=5
(mg/kg)
5a
5b
1
2
135
340
50 (30)
75 (100)
++
20.1(10)
37.6 (30)
+++ (30)
+++ (100)
(‹)
(-)
50 (30)
75 (100)
+++
40.7 (10)
49.6 (30)
++ (30)
+++ (100)
aBK test: Bradykinin-induced biting-like response. Values are inhibition %.
bLicking: Formalin-induced licking test. Signi®cant di€erence from the control group: +++, p<0.01 at 0±60 min; ++, p>0.01 at
0±15 min.
cAcetic acid: Acetic acid-induced writhing test. Values are inhibition %.
dSIA: Stress-induced analgesia in the hot-plate test. Signi®cant di€erence from the control group: +++, p<0.001; ++, p<0.01.
eAcute toxicity: ( ), no e€ect; (‹), slight loss of body weight.
cyclohexylaldehyde and paraformaldehyde according to
the method described above, was added thioacetic acid
(2.5 mL, 35.0 mmol), and the mixture was re¯uxed for
2 h. After evaporation, the residue was puri®ed by
chromatography on silica-gel (n-hexane:ethyl acetate) to
yield 42c (3.76 g, 76.0%): 1H-NMR (CDC13) d 0.64±
1.20 (6H, m), 1.30±1.65 (6H, m), 2.08 (lH, brd,
J=7.5 Hz), 2.32 (3H, s, -SCOMe), 2.50±2.71 (1H, m),
3.02 (2H, brt, J=7.0 Hz); Mass m/e 244 (M+), 201 (M+
±Ac), 168 (M+±AcSH).
acetonitrile three times to a€ord optically active salt
(1080 g, 27.6%) as a white solid.
A suspension of the salt (1080 g) in ethyl acetate (25 L)
was treated with 0.5 N hydrochloric acid (24 L). The
organic layer was washed with water and dried
(MgSO4), followed by evaporation of the solvent to give
a residue which was crystallized from dichloromethane±
n-hexane to yield 44(S) (505 g, 79.9%) as a white pow-
der: Rf=0.30 (n-hexane:ethyl acetate=1:1); 1H-NMR
(CDCl3) d 2.90-3.20 (3H, m), 3.20±3.40 (2H, m), 7.15±
7.38 (5H, m), 7.38±7.52 (2H, m), 7.52±7.62 (1H, m),
7.90±8.00 (2H, m); Mass m/e 300 (M+), 282, 254, 196
(M+ -AcSH); IR (KBr, cm 1) 3030, 1696, 1597, 1581,
1496, 1448, 1423; [a]d 40.8ꢀ (c=1.035, chloroform).
Preparation of optically active carboxylic acid (44):
(2S)-Benzyl-3- benzoylthiopropionic acid [44(S)]. To a
stirred solution of dl-44 (2292 g, 7.64 mol) in acetonitrile
(27 L) was added (1R, 2S)±(±)±2-amino-1,2-diphenyl-
ethanol (814 g, 3.82 mol), and the mixture was stirred at
25 ꢀC for 2 h. The resulting salt was recrystallized from
(2R)-Benzyl-3-benzoylthiopropionic acid [44(R)]. 44(R)
was prepared according to the method described in ref
23: [a]d+42.48ꢀ (c=1.025, chloroform).
Table 9. Percentage of the identi®ed metabolites of 5b in rats
and dogs after its oral administration
Compounda
Rat (%)
Dog (%)
Preparation of (4S)-4-(2-substituted-3-mercaptopropionyl-
amino)-4-(N-arylcarbamoyl)-butyric acid and (3S)-3-(2-
substituted-3-mercapto-propionylamino)-3-(N-arylcarb-
amoyl)-propionic acid derivatives
5b
NDb
2.79
0.53
11.38
15.91
72.17
8b(S)
7b
26
24.00
73.21
(1) General procedure for (4S)-4-(2-substituted-3-mer-
captopropionyl-amino)-4-(N-arylcarbamoyl)-butyric acid
derivatives: (4S)-4-(2-benzyl-3-mercaptopropionylamino)-
4-(N-phenylcarbamoyl)-butyric acid (8b). Step 1: To a
stirred solution of t-butoxycarbonyl-l-glutamic acid
aThe identi®ed metabolites were 8b(S), 7b and 26. The percen-
tage of the each metabolite and 5b was obtained based on the
total AUC of these four compounds as 100%.
bND: Not detected.

454
K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
g-benzyl ester (3.37 g, 10 mmol) in dichloromethane
(30 mL) were added pivaloyl chloride (1.35 mL,
11 mmol) and triethylamine (1.4 mL, 10 mmol) at 5 ꢀC,
followed by stirring at 25 ꢀC for 30 min. The reaction
mixture was treated with aniline (1 mL, 11 mmol) at
5 ꢀC, then stirred at 25 ꢀC for 1 h. After dilution with
ethyl acetate, the reaction mixture was washed with 1N
hydrochloric acid, 1N sodium hydroxide, brine and
dried (MgSO4). After evaporation, the residue was
crystallized from n-hexane to a€ord t-butoxycarbonyl-
l-glutamylanilide g-benzyl ester (2.9 g, 70.4%) as a
white powder: Rf=0.74 (n-hexane:ethyl acetate=1:1);
1H-NMR (CDCl3) d 1.42 (9H, s), 1.70±2.40 (2H, m),
2.40±2.80 (2H, m), 4.10±4.40 (1H, m), 5.10(2H, s), 5.35
(1H, brd, J=7.5 Hz, -NH-), 6.90±7.55 (10H, m),
8.40 (1H, brs, -NHPh); Mass m/e 412 (M+), 356, 339,
292.
treated with 1N hydrochloric acid at 5 ꢀC and extracted
with ethyl acetate. The organic layer was washed with
brine and dried (MgSO4), and evaporated. The residue
was puri®ed by chromatography on silica-gel (chlor-
oform:acetic acid=99:1) to yield 8b (387 mg, 69.1%):
mp.
158.5±160.0ꢀC; Rf=0.48 (chloroform:acetic
1
acid=95:5); H-NMR (CDCl3+DMSO-d6) d 1.54 (1H,
t, J=7.5 Hz), 1.68±2.40 (4H, m), 2.40±3.02 (6H, m),
4.46±4.72 (1H, m), 6.92±7.36 (9H, m), 7.40±7.56 (2H,
m), 8.86±9.04 (1H, m); Mass m/e 400 (M+), 382, 366,
353, 335; IR (KBr, cm 1) 3275, 1700, 1640, 1600, 1540,
1440, 1300, 1250, 750, 695.
Compounds 9c±17c were prepared from 27b according
to the same method as described above for the prepara-
tion of 8b from 27. Compounds 19a±c were prepared by
the acylation of 28 with 42a±c, respectively. Compounds
8b(S) and 8b(R) were prepared by the acylation of 45b
with 44(S) and 44(R), respectively. Compounds 11c(S)
and 11c(R) were prepared by the acylation of 45c with
44(S) and 44(R), respectively.
Step 2: To a stirred solution of t-butoxycarbonyl-l-glu-
tamylanilide g-benzyl ester (2.9 g, 7.04 mmol) in
dichloromethane (2 mL) was added tri¯uoroacetic acid
(5.4 mL, 70 mmol) at 5 ꢀC, followed by stirring at 25 ꢀC
for 2.5 h. The solvent was evaporated to yield 31 (3.0 g,
quantitative yield): Rf=0.23 (ethyl acetate); 1H-NMR
(CDCl3) d 1.90±2.50 (2H, m), 2.50±2.70 (2H, m), 4.30±
4.60 (1H, m), 5.00 (2H, s), 6.80±7.60 (10H, m), 9.50 (1H,
brs, -NHPh); Mass m/e 312 (M+), 204, 193, 192.
(4S)-4-(2-Benzyl-3-mercaptopropionylamino)-4-[N-(2-
naphthyl)-carbamoyl]-butyric acid (9c). Rf=0.25 (5%
1
acetic acid in chloroform); H-NMR (CDCl3+DMSO-
d6) d 1.50±3.60 (10H, m), 4.50±4.88 (1H, m), 6.80±8.30
(13H, m), 9.35, 9.57 (1H, s); Mass m/e 450 (M+), 432,
416, 389, 385; IR (KBr, cm 1) 3260, 3025, 1695, 1630,
1525, 1500, 1430, 1395, 1265.
Step 3: 2-benzyl-3-acetylthiopropionic acid (833 mg,
3.5 mmol) was treated with excess oxalyl chloride in
dichloromethane (1 mL) at 25 ꢀC for 30 min. After eva-
poration, the resulting 29 was used for the next reaction
without puri®cation. To a stirred mixture of 28 (1.64 g,
3.83 mmol) and pyridine (2.83 mL, 35 mmol) in dichloro-
methane (16 mL) was added a solution of 29 in dichloro-
methane (2 mL) at 5 ꢀC, followed by stirring at 25 ꢀC for
15 min. The reaction mixture was poured into water and
extracted with ethyl acetate. The organic layer was
washed with 1N hydrochloric acid, 1N sodium hydro-
xide, brine and dried (MgSO4), and then evaporated.
The residue was puri®ed by chromatography on silica-
gel (eluted with n-hexane:dichloromethane=1:2 fol-
lowed by methanol:dichloromethane=1:20) followed by
recrystallization from ethyl acetate and n-hexane to
yield 30 (743 mg, 40.0%): Rf=0.48 (n-hexane:ethyl
acetate=1:1); 1H-NMR (CDCl3) d 1.82±2.20 (2H, m),
2.22, 2.28 (3H, s), 2.34±2.76 (3H, m), 2.82±3.00, 3.00±
3.24 (4H, m), 4.30±4.60 (1H, m), 5.10, 5.12 (2H, s),
6.24±6.44 (1H, m), 7.00±7.34 (15H, m), 8.38, 8.52 (1H,
brs, -NHPh); Mass m/e 532 (M+), 489, 440, 370.
(4S)-4-(2-Benzyl-3-mercaptopropionylamino)-4-[N-(4-
pyridyl)-carbamoyl]-bubric acid (10c). Rf=0.18 (chloro-
form:methanol:acetic acid=30:5:1) H-NMR (CDCl3+
CD3OD) d 1.64±2.22 (3H, m), 2.35±2.45 (1H, m), 2.50±
3.00 (5H, m), 4.35±4.50 (1H, m), 7.00±7.35 (5H, m), 7.55
1
(2H, brt, J=6.0 Hz), 8.38 (2H, brt, J=6.0 Hz); Mass m/
1
e 401 (M+), 383, 368, 349, 336, 310; IR (KBr, cm
3250, 1720, 1700, 1630, 1590, 1500.
)
(4S)-4-(2-Benzyl-3-mercaptopropionylamino)-4-[N-(2-
benzothiazoly)-carbamoyl]-butyric acid (11c). Rf=0.52
(chloroform:methanol:acetic acid=30:3:1); 1H-NMR
(CDCl3+DMSO-d6) d 1.52±3.20 (10H, m), 4.56±4.85
(1H, m), 6.95±7.50 (8H, m), 7.67±8.06 (3H, m); Mass m/
e 439 (M+), 405, 392, 367; IR (KBr, cm 1) 3275, 1700,
1640, 1530, 1440, 1305, 1285.
(4S)-4-(2-Benzyl-3-mercaptopropionylamino)-4-[N-(4-
¯uorophenyl)-carbamoyl]-bubric acid (12c). Rf=0.19
(8% acetic acid in chloroform); 1H-NMR (CDCl3+
DMSO-d6) d 1.56, 1.60 (1H, t, J=8.0 Hz), 1.70±2.30
(3H, m), 2.30±3.04 (6H, m), 4.40±4.64 (1H, m), 6.86±
7.02 (2H, m), 7.04±7.32 (5H, m), 7.40±7.64 (3H, m),
9.18, 9.20 (1H, brs); Mass m/e 418 (M+), 400, 368, 353,
308; IR (KBr, cm 1) 3275, 1705, 1640, 1540, 1505, 1440,
1405.
Step 4: To a stirred solution of 29 (743 mg, 1.4 mmol) in
tetrahydrofuran (9 mL)-1,2-dimethoxyethane (1.4 mL)
was added lithium hydroxide monohydrate (283 mg,
6.74 mmol) in water (5 mL) under argon at 25 ꢀC, fol-
lowed by stirring for 30 min. The reaction mixture was

K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
455
(4S)-4-(2-Benzyl-3-mercaptopropionylamino)-4-[N-(4-
chlorophenyl)-carbamoyl]-butyric acid (13c). Rf=0.28
(4S)-4-[2-(4-Methylbenzyl)-3-mercaptopropionylamino]-
4-(N-phenyl-carbamoyl]-bubric acid (19b). Rf=0.47 (10%
acetic acid in chloroform); 1H-NMR (CDCl3) d 1.42±
2.98 (13H, m), 4.75±5.01 (1H, m), 6.62±7.56 (10H, m),
9.12, 9.16 (1H, s); Mass m/e 414 (M+), 467, 349, 321,
294; IR (KBr, cm 1) 3270, 3040, 2925, 1705, 1640, 1600.
(8%
acetic
acid
in
chloroform);
1H-NMR
(CDCl3+DMSO-d6) d 1.77, 1.82 (1H, t, J=8.0 Hz),
1.70±2.30 (3H, m), 2.30±3.06 (6H, m), 4.40±4.66 (1H,
m), 7.00±7.34 (7H, m), 7.35±7.70 (3H, m), 9.34 (1H,
1
brs); Mass m/e 434 (M+), 416, 368, 308; IR (KBr, cm
3275, 1705, 1640, 1600, 1530, 1495.
)
(4S)-4-(2-Cyclohexylmethyl-3-mercaptopropionylamino)-
4-(N-phenyl-carbamoyl]-butyric acid (19c). Rf=0.33
(10% acetic acid in chloroform); 1H-NMR (CDCl3) d
0.70±1.10 (3H, m), 1.10±1.40 (6H, m), 1.40±1.90 (5H, m),
2.00±2.20 (1H, m), 2.20±2.40 (1H, m), 2.40±2.82 (5H,
m), 4.80±5.10 (1H, m), 6.90±7.30 (2H, m), 7.30±7.60
(3H, m), 7.55 (2H, brd, J=8.0 Hz), 9.27 (1H, brs); Mass
m/e 406 (M+), 388, 373, 355, 326, 277, 259, 184; IR
(KBr, cm 1) 3600±2400, 1710, 1640, 1540, 1440, 1245.
(4S)-4-(2-Benzyl-3-mercaptopropionylamino)-4-[N-(4-
iodophenyl)-carbamoyl]-bubric acid (14c). Rf=0.34 (8%
1
acetic acid in chloroform); H-NMR (CDCl3+DMSO-
d6) d 1.55, 1.60 (1H, t, J=8.0 Hz), 1.80±2.25 (2H, m),
2.30±3.10 (7H, m), 4.40±4.65 (1H, m), 7.00±7.38 (6H,
m), 7.50±7.70 (3H, m), 9.30, 9.32 (1H, s); Mass m/e 526
(M+), 508, 474, 461, 417, 331; IR (KBr, cm 1) 3300,
1700, 1650, 1601, 1580, 1520, 1480, 1390.
(4S)-4-[(2S)-Benzyl-3-mercaptopropionylamino]-4-(N-
phenylcarbamoyl)-butyric acid [8b(S)]. mp 153±155 ꢀC;
Rf=0.39 (chloroform:tetrahydrofuran:acetic acid=
15:4:1); 1H-NMR (CDCl3+DMSO-d6) d 1.60 (1H, t,
J=8.0 Hz), 1.82±2.27 (2H, m), 2.42 (2H, t, J=8.0 Hz),
2.50±3.54 (6H, m), 4.58 (1H, ddd, J=4.0, 8.0 Hz), 6.99±
7.20 (6H, m), 7.25 (2H, t, J=8.0 Hz), 7.50 (2H, d,
J=8.0 Hz), 7.76 (1H, d, J=8.0 Hz), 9.30 (1H, s); Mass
m/e 400 (M+), 382, 353, 335, 307, 291, 280, 274,262;IR
(KBr,cm 1) 3275,3050, 1700, 1640, 1530, 1500, 1440;
(4S)-4-(2-Benzyl-3-mercaptopropionylamino)-4-[N-(4-
methoxyphenyl)-carbamoyl]-butyric acid (15c). Rf=0.25
(20% acetic acid in ethyl acetate); 1H-NMR
(CDCl3+DMSO-d6) d 1.55, 1.63 (1H, t, J=8.0 Hz),
1.70±2.30 (3H, m), 2.40±3.10 (6H, m), 3.78, 3.81 (3H, s),
4.40±4.70 (1H, m), 6.80, 6.83 (2H, d, J=8.0 Hz), 7.00±
7.30 (4H, m), 7.30±7.60 (3H, m), 8.95, 9.05 (1H, brs);
Mass m/e 430 (M+), 412,398, 368, 334, 321, 307; IR
(KBr, cm 1) 3275, 1700, 1640, 1510, 1440, 1420, 1300,
1240.
[a]d
C21H24N2O4S: C, 62.98; N, 6.04; S, 8.01. Found: C,
62.93; N, 7.02; S, 8.10.
16.11ꢀ (c 1.00, methanol); Anal. Calcd.
(4S)-4-(2-Benzyl-3-mercaptopropionylamino)-4-[N-(4-
acetylphenyl)-carbamoyl]-butyric acid (16c). Rf=0.34
(10% acetic acid in chloroform); 1H-NMR
(CDCl3+DMSO-d6) d 1.52±3.40 (13H, m), 4.42±4.66
(1H, m), 6.98±7.36 (5H, m), 7.59±7.76 (2H, m), 7.80±
8.00 (3H, m), 9.76, 9.82 (1H s); Mass m/e 442 (M+),
424, 390, 377, 333; IR (KBr, cm 1) 3350, 1710,
1640, 1595, 1525, 1405, 1360, 1320, 1270, 1180, 840,
700.
(4S)-4-[(2R)-Benzyl-3-mercaptopropionylamino]-4-(N-
phenylcarbamoyl)-bubric acid [8b(R)]. mp 164±168 ꢀC;
Rf=0.30 (5% acetic acid in chloroform); 1H-NMR
(CDCl3+DMSO-d6) d 1.58 (1H, t, J=8.0 Hz), 1.70±
1.90 (1H, m), 1.90±2.34 (2H, m), 2.54±3.04 (5H, m),
4.42±4.60 (1H, m), 7.04 (1H, brt, J=8.0 Hz), 7.10±7.34
(8H, m), 7.40 (1H, t, J=8.0 Hz), 7.50 (2H, dd, J=1.0,
8.0 Hz), 9.09 (1H, s); Mass m/e 400 (M+), 382, 353, 335,
307; IR (KBr, cm 1) 3275, 1700, 1640, 1600, 1530; [a]d
91.72ꢀ (c 1.01, ethanol); Anal. Calcd. C21H24N204S: C,
62.98; N, 6.04; S, 8.01. Found: C, 63.12; N, 7.06; S, 8.17.
(4S)-4-(2-Benzyl-3-mercaptopropionylamino)-4-[N-(4-
sulfamoylphenyl)-carbamoyl]-butyric acid (17c). Rf=0.52
(5% acetic acid in ethyl acetate); 1H-NMR
(CDCl3+CD3OD) d 1.11, 1.20 (1H, t, J=8.0 Hz), 1.60±
2.30 (3H, m), 2.30±2.52 (1H, m), 2.54±3.10 (5H, m),
4.40±4.60 (1H, m), 6.96±7.36 (5H, m), 7.58±7.75 (2H,
m), 7.75±7.90 (2H, m); Mass m/e 427 (M+ -H2O - H2S),
414, 383, 368, 320, 284; IR (KBr, cm 1) 3300, 1700,
1645, 1595, 1520, 1400, 1330, 1255, 1160.
(4S)-4-[(2S)-Benzyl-3-mercaptopropionylamino]-4-[N-(2-
benzothiazolyl)-carbamoyl]-bubric acid [11(S)]. Rf=0.26
(5% acetic acid in chloroform); 1H-NMR (CDCl3) d 1.56
(1H, t, J=8.0 Hz), 1.85±3.00 (9H, m), 4.99 (1H, ddd,
J=4.0, 8.0 Hz), 6.64 (1H, d, J=8.0 Hz), 6.83±7.20 (6H,
m), 7.29±7.55 (2H, m), 7.70±7.85 (2H, m); Mass m/e 457
(M+), 439, 392; IR (KBr, cm 1) 3280, 2910, 2525, 1640,
1600, 1530, 1440, 1300, 1270; [a]d 5.27ꢀ (c 1.12,
chloroform).
(4S)-4-[2-(4-Methoxybenzyl)-3-mercaptopropionylamino]-
4-(N-phenyl-carbamoyl]-bubric acid (19a). Rf=0.40
(10% acetic acid in chloroform); 1H-NMR (CDCl3) d
1.40±2.96 (10H, m), 3.50, 3.77 (3H, s), 4.76±5.00 (1H,
m), 6.54±7.15 (10H, m), 9.13, 9.16 (1H, s); Mass m/e 430
(M+), 412, 337, 310; IR (KBr, cm 1) 3270, 3040, 2920,
1630, 1505, 1440, 1295, 1240, 1 175.
(4S)-4-[(2R)-Benzyl-3-mercaptopropionylamino]-4-[N-(2-
benzothiazolyl)-carbamoyl]-bubric acid [11(R)]. Rf=
0.29 (chloroform:tetrahydrofuran:acetic acid=30:8:1);

456
K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
1H-NMR (CDCl3+CD3OD) d 1.60±1.90 (1H, m), 2.00±
2.30 (4H, m), 2.55±3.00 (5H, m), 4.60±4.70 (1H, m),
7.05±7.50 (7H, m), 7.60±795 (2H, m); Mass m/e 457
(M+), 439, 392, 368, 278; IR (KBr, cm 1) 3250, 1710±
1666, 1640, 1600, 1530, 1440; [a]d 35.6ꢀ (c 1.00,
chloroform)
hydrofuran:acetic acid=15:4:1); 1H-NMR (CDCl3+
DMSO-d6) d 1.56 (1H, t, J=7.5 Hz), 2.50±3.00 (7H, m),
3.00±4.00 (1H, m), 4.91 (1H, q, J=7.5 Hz), 7.00±7.20
(6H, m), 7.29 (2H, t, J=7.5 Hz), 7.43 (2H, d, J=7.5 Hz),
7.58 (1H, d, J=7.5 Hz), 8.69 (1H, s); Mass m/e 386 (M+),
368, 335, 321; IR (KBr, cm 1) 3250, 1675, 1640, 1601,
1520, 1435; [a]d 28.6ꢀ (c 1.00, methanol).
General procedure for (3S)-3-(2-substituted-3-mercapto-
acid
propionyl-amino)-3-(N-arylcarbamoyl)-propionic
derivatives
Compound 8a(R) was prepared from 44(R) according to
the same method as described above (19.4% in three
steps).
(3S)-3-[(2S)-Benzyl-3-mercaptopropionylamino]-3-(N-
phenylcarbamoyl)-propionic acid[8a(S)]. Step 1: To a
stirred mixture of 45a (832 mg, 3.2 mmol) and pyridine
(0.5 mL, 6.2 mmol) in dichloromethane (10 mL) was
added a solution of an acyl chloride which was prepared
from 44(S) by the usual method. After being stirred for
30 min, the reaction mixture was poured into water and
extracted with ethyl acetate. The organic layer was
washed with 1N hydrochloric acid, 1N sodium hydrox-
ide, brine and dried (MgSO4). Removal of the solvent
by evaporation a€orded a residue which was puri®ed by
chromatography on silica-gel (ethyl acetate:dichloro-
methane=1:4) to yield 46a(S) (1.45 g, 88.4%) as a white
powder: Rf=0.40 (n-hexane:ethyl acetate=2:1); 1H-
NMR (CDCl3) d 1.40 (9H, s), 2.35±3.20 (4H, m), 3.35
(2H, brd, J=8.0 Hz), 4.70±5.00 (1H, m), 6.83 (1H, brd,
J=8.0 Hz), 7.00±7.70 (13H, m), 8.00 (2H, brdd, J=2.0,
8.0 Hz), 8.25 (1H, brs); Mass m/e 546 (M+), 473
(M+ -t-BuO), 454, 398.
(3S)-4-[(2R)-Benzyl-3-mercaptopropionylamino]-3-(N-
phenylcarbamoyl)-propionic acid [8a(R)]. Rf=0.46 (5%
acetic acid in chloroformÂ2); 1H-NMR (CDCl3+
DMSO-d6) d 1.75 (1H, brt, J=7.5 Hz), 2.40±3.00 (7H,
m), 4.80±5.00 (1H, m), 7.05 (1H, t, J=7.5 Hz), 7.09±7.38
(6H, m), 7.53 (2H, d J=7.5 Hz), 7.86 (1H, d, J=7.5 Hz),
9.05 (1H, s); Mass m/e 386 (M+), 368, 335, 321; IR
(KBr, cm 1) 3300, 1700, 1680, 1670, 1645, 1600, 1530,
1440; [a]d 86.0ꢀ (c 0.82, methanol).
Preparation of (2S)-2-(2-benzyl-3-mercapto-propionyl-
amino)-2-(N-phenylcarbamoylmethyl)-acetic acid and
(2S) - 2 - (2 - benzyl - 3 - mercapto-propionylamino) - 2 - (N-
phenylcarbamoylethyl)-acetic acid derivatives
(1)(2S)-2-(2-Benzyl-3-mercapto-propionylamino)-2-(N-
phenylcarbamoyl-methyl)-acetic acid (18a). Step 1: To a
stirred solution of 31(4.00 g, 12.4 mmol) and triethyl-
amine (1.90 mL, 13.6 mmol) in dichloromethane (40 mL)
was added pivaloyl chloride (1.68 mL, 13.6 mmol) at
5 ꢀC and stirred at 25 ꢀC for 1 h. The resulting solution
was treated with aniline (1.23 mL, 13.6 mmol) and tri-
ethylamine (1.90 mL, 13.6 mmol) at 5 ꢀC and then stirred
at 25 ꢀC for 1 h. The reaction mixture was diluted with
ethyl acetate and washed with 1N hydrochloric acid,
brine, dried (Na2SO4) and evaporated to give a residue.
The residue was triturated with ethyl acetate:n-hexane
to a€ord 32 (3.80 g, 77.0%) as a white powder: Rf=0.43
(n-hexane:ethyl acetate=1:1); 1H-NMR (CDC13) d 1.63
(9H, s), 2.64±3.20 (2H, m), 4.40±4.70 (1H, m), 5.16 (2H,
s), 5.68 (1H, brd, J=8.0 Hz), 6.90±7.60 (11H, m); Mass
m/e 398 (M+), 342, 250; IR (KBr, cm 1) 3290, 1725,
1670, 1595, 1535, 1295.
Step 2: 46a(S) (1.41 g, 2.58 mmol) was dissolved in
methanol (20 mL). To this solution was added potas-
sium carbonate (714 mg, 5.16 mmol), followed by stir-
ring under argon at 25 ꢀC for 30 min. The reaction
mixture was acidi®ed with 1N hydrochloric acid and
extracted with diethyl ether. The organic layer was washed
with brine, dried (MgSO4), and then evaporated to give a
residue which was puri®ed by chromatography on silica-
gel (ethyl acetate:n-hexane=1:2) to a€ord thiol (1.04 g,
91.2%) as a white amorphous powder: Rf=0.50 (n-hex-
ane:ethyl acetate=1:1) 1H-NMR (CDCl3) d 1.43 (9H, s),
1.50 (1H, t, J=8.0 Hz), 2.50±3.04 (7H, m), 4.78±4.91 (1H,
m), 6.86 (1H, brd, J=8.0 Hz), 7.02±7.22 (5H, m), 7.25±
7.50 (5H, m), 8.27 (1H, brs); Mass m/e 442 (M+), 386, 369.
Step 3: The resulting thiol (913 mg, 2.07 mmol) was
treated with tri¯uoroacetic acid (3.2 mL, 41.54 mmol) in
dichloromethane (5 mL) at 25 ꢀC for 2 h. The reaction
mixture was poured into water and extracted with ethyl
acetate. The organic layer was washed with distilled
water, brine, dried (Na2SO4) and evaporated to give a
residue which was puri®ed by chromatography on silica-
gel (eluted with dichloromethane followed by ethyl
acetate:dichloromethane=1:4) to yield 8a(S) (284 mg,
35.6%) as a white powder: Rf=0.43 (chloroform:tetra-
Step 2: The solution of 32 (2.5 g, 6.27 mmol) in
dichloromethane (5 mL) was treated with tri¯uoroacetic
acid (4.83 mL, 62.7 mmol) at 25 ꢀC for 15 h. The reac-
tion mixture was poured into water and extracted with
ethyl acetate. The organic layer was washed with 1N
sodium hydroxide, brine, dried (MgSO4) and evapo-
rated to give a residue. The residue was puri®ed by
chromatography on silica-gel (ethyl acetate followed
by 10% methanol in ethyl acetate) to yield 33 (1.74 g,
93.0%): Rf=0.46 (10% methanol in ethyl acetate);

K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
457
1H-NMR (CDC13) d 2.48±2.95 (2H, m), 3.00 (2H, s),
3.88 (1H, dd, J=7.5 Hz), 5.02 (2H, s), 6.90±7.60 (10H,
m), 9.36 (1H, brs).
(2)(2S)-2-(2-benzyl-3-mercapto-propionylamino)-2-(N-
phenylcarbamoyl-ethyl)-acetic acid (18b). Step 1: To a
stirred solution of 36 (2.00 g, 6.0 mmol) and triethyl-
amine (0.92 mL, 6.6 mmol) in dichloromethane (30 mL)
was added pivaloyl chloride (0.74 mL, 6.0 mmol) at 5 ꢀC,
followed by stirring at 25 ꢀC for 10 min. The resulting
solution was treated with aniline (0.66 mL, 7.2 mmol)
and triethylamine (0.92 mL, 6.6 mmol) at 5 ꢀC, followed
by stirring at 25 ꢀC for 15 h. The reaction mixture was
diluted with ethyl acetate. The organic layer was washed
with 1N hydrochloric acid, 1N sodium hydroxide, brine,
dried (MgSO4) and evaporated. The residue was tritu-
rated with ethyl acetate:n-hexane (1:9) to yield 37
(2.07 g, 83.9%) as a white powder: Rf=0.48 (n-hex-
Step 3: To a stirred solution of 33 (1.3 g, 4.35 mmol) and
pyridine (0.53 mL, 6.53 mmol) in dichloromethane
(20 mL) was added a solution of 29 which was prepared
from the corresponding acid (1.04 g, 4.35 mmol) and
excess oxalyl chloride in dichloromethane (10 mL) at
5 ꢀC, and then stirred at 25 ꢀC for 1 h. The reaction
mixture was poured into water and extracted with ethyl
acetate. The organic layer was washed with 1N hydro-
chloric acid, brine, dried (MgSO4). Removal of the sol-
vent by evaporation a€orded a residue which was
puri®ed by chromatography on silica-gel (dichloro-
methane:ethyl acetate=10:1 to 6:1) to yield 34 (1.37 g,
60.7%). Rf=0.29 (n-hexane:ethyl acetate=1:1); 1H-
NMR (CDC13) d 2.05, 2.23 (3H, s), 2.28±3.20 (7H, m),
4.62±4.84 (1H, m), 5.12, 5.13 (2H, s), 6.55±6.70 (1H, m),
6.95±7.78 (16H, m); Mass m/e 518 (M+), 429, 410; IR
(KBr, cm 1) 3280, 1720, 1680, 1650, 1535, 690.
1
ane:ethyl acetate=1:1); H-NMR (CDC13) d 1.42 (9H,
s), 1.70±2.60 (4H, m), 4.05±4.40 (1H, m), 5.08 (2H, s),
5.40±5.70 (1H, m), 6.80±7.60 (10H, m), 8.00±8.30 (1H,
m); Mass m/e 356 (M+-56), 311 (M+-COOt-Bu).
Step 2: To a solution of 37 (2.14 g, 5.20 mmol) in
methanol (20 mL) was added 10% Pd on carbon
(214 mg). The mixture was stirred under hydrogen at
25 ꢀC for 50 min. The removal of the catalyst by ®ltra-
tion followed by evaporation a€orded 38 (1.37 g,
94.2%) as a colorless oil: Rf=0.42 (10% methanol in
chloroform); 1H-NMR (CDC13) d 1.43 (9H, s), 2.40±
1.80 (2H, m), 2.40±2.60 (2H, m), 3.40 (1H, dd, J=4.0,
8.0 Hz), 6.90±7.60 (5H, m), 8.30±8.62 (1H, m).
Step 4: 34 (1.31 g, 2.52 mmol) was treated with 2-amino-
ethanethiol (292 mg, 3.8 mmol) in acetonitrile (20 mL)±
dichloromethane (4 mL) at 45 ꢀC for 2 h under argon.
The reaction mixture was poured into 1N hydrochloric
acid and extracted with ethyl acetate. The organic layer
was washed with 1N hydrochloric aid, brine, dried
(MgSO4) and evaporated. The residue was puri®ed by
chromatography on silica-gel (dichloromethane:ethyl
acetate=6:1) to yield 35 (860 mg, 72.0%): mp. 148±
Step 3: To a cold solution of 38 (285 mg, 1.03 mmol) and
pyridine (0.5 mL, 6.18 mmol) in dichloromethane (5 mL)
was added a solution of 29, which was prepared from
the corresponding acid (268.4 mg, 1.13 mmol) and excess
oxalyl chloride, at 5 ꢀC. The mixture was stirred at 25 ꢀC
for 2 h, and then poured into water and extracted with
ethyl acetate. The organic layer was washed with 1N
hydrochloric acid, 1N sodium hydroxide, brine, dried
(Na2SO4) and evaporated. The residue was puri®ed by
chromatography on silica-gel (ethyl acetate:n-hex-
ane=1:2 followed by 1:1) to yield 39 (302 mg, 59.1%):
Rf=0.42 (n-hexane:ethyl acetate=1:1); 1H-NMR
(CDC13) d 1.39, 1.42 (9H, s), 1.60±2.48 (4H, m), 2.32,
2.36 (3H, s), 2.58±2.84 (1H, m), 2.84±3.00 (2H, m), 3.04±
3.28 (2H, m), 4.22±2.50 (1H, m), 6.12 (1H, brt,
J=8.0 Hz), 6.98±7.40 (8H, m), 7.48±7.80 (2H, m), 8.56,
9.08 (1H, brs); Mass m/e 498 (M+), 422, 425, 409, 397.
154.5 ꢀC; Rf=0.24 (ethyl acetate); H-NMR (CDCl3) d
1
1.36±1.60 (1H, m), 2.37±3.34 (7H, m), 4.74±4.92 (1H, m),
5.10±5.20 (2H, m), 6.65±6.90 (1H, m), 6.96±7.60 (16H,
m); Mass m/e 476 (M+), 429, 368, 335, 321; IR (KBr,
cm 1) 3275, 2950, 1760, 1650, 1595, 1540, 1440, 690.
Step 5: To a stirred solution of 35 (667 mg, 1.40 mmol)
in methanol (10 mL)±tetrahydrofuran (3 mL) was added
a solution of potassium hydroxide (184 mg, 2.80 mmol)
in distilled water (2 mL) at 25 ꢀC. The mixture was stir-
red for 4 h, and then diluted with ethyl acetate. The
organic layer was washed with 1N hydrochloric acid,
1N sodium hydroxide, brine, dried (MgSO4) and eva-
porated. The residue was puri®ed by chromatography
on silica-gel (dichloromethane:tetrahydrofuran:meth-
anol=60:2:1 followed by 40:2:1) to a€ord thiol (330 mg,
0.90 mmol). The resulting thiol (330 mg, 0.90 mmol) was
treated with 1N sodium hydroxide (0.9 mL) in methanol
(2 mL) at 25 ꢀC followed by evaporation to a€ord 18a
(367 mg, 64.1% in 2 steps): Rf=0.35 (chloroform:tetra-
hydrofuran:acetic acid=15:4:1); 1H-NMR (CD3OD+
CDCl3) d 2.25±3.30 (7H, m), 4.34±4.58 (1H, m), 6.85±
7.56 (10H, m).; Mass m/e 386 (M+), 368, 335, 321, 293.
(as a carboxylic acid); IR (KBr, cm 1) 3640±2400, 1650,
1630, 1595, 1530, 1380, 1300.
Step 4: A solution of 39 (301 mg, 0.60 mmol) in dichloro-
methane (2 mL) was treated with tri¯uoroacetic acid
(1.15 mL, 15 mmol) at 25 ꢀC for 2 h. The reaction mix-
ture was poured into water and extracted with ethyl
acetate. The organic layer was washed with distilled
water, brine, dried (Na2SO4) and evaporated. The resi-
due was triturated with ethyl acetate:n-hexane (1:2) to
yield 40 (150 mg, 56.6%) as a white powder: Rf=0.10
(ethyl acetate); Mass m/e 442 (M+), 424.

458
K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
Step 5: Compound 40 (442 mg, 1.0 mmol) was treated
with 2-aminoethanethiol (154 mg, 2.0 mmol) in acetoni-
trile (12 mL) at 50 ꢀC for 30 min under argon. The reac-
tion mixture was poured into 1N hydrochloric acid and
extracted with ethyl acetate. The organic layer was
washed with 1N hydrochloric acid, brine, dried
(Na2SO4) and evaporated. The residue was puri®ed by
chromatography on silica-gel (ethyl acetate:n-hex-
ane=1:1) to a€ord thiol (242 mg, 0.60 mmol) which was
treated with 1N sodium hydroxide (0.6 mL) in methanol
(2 mL) at 25 ꢀC followed by evaporation to give 18b
(235 mg, 55.6% in 2 steps): mp 131±135 ꢀC; Rf=0.23
(chloroform:tetrahydrofuran:acetic acid=16:3:1); 1H-
NMR (CD3OD) d 1.09±1.42 (1H, m), 1.68±2.09 (2H,
m), 2.10±2.57 (2H, m), 2.58±3.12 (5H, m), 4.12±4.36
(1H, m), 6.96±7.36 (8H, m), 7.52 (2H, brd, J=8.0 Hz);
Mass m/e 309 (M+-NHPh), 237, 213, 160, 145, 117;
IR(KBr, cm 1) 3300, 1660, 1640, 1600, 1540, 1500,
1440.
added a solution of an acid chloride, which was pre-
pared from 44(S) by the usual method, in dichloro-
methane (8 mL) at 5 ꢀC followed by stirring for 1 h. The
reaction mixture was poured into water and extracted
with ethyl acetate. The organic layer was washed with
1N hydrochloric acid, 1N sodium hydroxide, brine,
dried (MgSO4) and evaporated. The residue was pur-
i®ed by chromatography on silica-gel (ethyl acetate:di-
chloromethane=1:10 followed by 1:6) to yield 50
(8.46 g, 73.2% in 2 steps): Rf=0.80 (n-hexane:ethyl
acetate=1:1) 1H-NMR (CDCl3) d 1.48 (9H, s), 1.73±
3.20 (6H, m), 3.35 (2H, d, J=7.0 Hz), 4.28±4.64 (1H,
m), 6.36±6.60 (2H, m), 6.85±8.07 (15H, m), 8.53 (1H,
brs); Mass m/e 560 (M+), 504, 487, 469, 432, 412, 399,
384; IR (KBr, cm 1) 3300, 1730, 1640, 1600, 1540, 1450.
(4S)-4-[(2S)-Benzyl-3-mercaptopropionylamino]-4-(N-
phenylcarbamoyl)-bubric acid t-butyl ester (50b). 50a
(8.46 g, 15.1 mmole) was dissolved in methanol (180 mL)
degassed with argon. To the stirred solution was added
potassium carbonate (4.38 g, 31.7 mmole) under argon
at 25 ꢀC for 1 h. The reaction mixture was acidi®ed with
1N hydrochloric acid and extracted with diethyl ether.
The organic layer was washed with brine, dried
(MgSO4) and evaporated. The residue was puri®ed by
chromatography on silica-gel (ethyl acetate:dichloro-
methane=1:9) to yield 50b (5.6 g, 81.2%) as a white
amorphous powder: Rf=0.43 (ethyl acetate:dichloro-
Preparation of 5a, b and 20±25
(a) General Procedure for (4S)-4-amino-4-(N-arylcarb-
amoyl)-butyric acid derivatives: Preparation of (4S)-4-
[(2S)-benzyl-3-nicotinoylthio-propionylamino]-4-(N-phe-
nylcarbamoyl)-butyric acid (20)
(4S)-4-(Benzyloxycarbamoyl)-4-(N-phenylcarbamoyl)-
butyric acid t-butyl ester (48). To a stirred solution of
47 (8.00 g, 23.7 mmol) and aniline (2.27 mL, 24.9 mmol)
in dichloromethane (60 mL) was added N, N-dicyclo-
hexylcarbodiimide (DCC, 5.13 g, 24.9 mmol) at 5 ꢀC.
After being stirred for 1 h, the resulting urea was
removed by ®ltration and the ®ltrate was evaporated.
The residue was triturated with ethyl acetate:n-hex-
ane(1:9) to yield 48 (8.5 g, 86.9%) as a white powder:
Rf=0.80 (ethyl acetate:n-hexane=1:1); 1H-NMR
(CDCl3) d 1.50 (9H, s), 1.70±2.70 (4H, m), 4.37 (1H, dt,
J=7.5 Hz), 5.16 (2H, s), 5.84 (1H, d, J=7.5 Hz), 7.04±
7.64 (10H, m), 8.53 (1H, brs).; Mass m/e 412 (M+), 336,
295.; IR (KBr, cm 1) 3300, 1740, 1700, 1660, 1600,
1520, 1450.
1
methane=1:6); H-NMR (CDC13) d 1.49 (9H, s), 1.78±
3.17 (9H, m), 4.51 (1H, dt, J=7.0 Hz), 6.79 (1H, d,
J=7.0 Hz), 6.89±7.69 (10H, m), 8.62 (1H, brs); Mass m/
e 456 (M+), 400, 383, 364.
(4S)-4-[(2S)-Benzyl-3-nicotinoylthiopropionylamino]-4-
(N-phenyl-carbamoyl)-butyric acid (20). To a stirred
solution of 50b (2.20 g, 4.82 mmol) and triethylamine
(2.69 mL, 19.3 mmol) in dichloromethane (20 mL) was
added nicotinoyl chloride hydrochloride (0.994 g,
5.30 mmol) at 5 ꢀC followed by stirring at 25 ꢀC for 3 h.
The reaction mixture was diluted with ethyl acetate and
washed with 1N sodium hydroxide, and then brine. The
organic layer was dried (MgSO4) and evaporated. The
residue was puri®ed by chromatography on silica-gel
(ethyl acetate:dichloromethane=1:6 followed by 1:4)
to a€ord 2.20 g (3.92 mmol, 81.3%) of (4S)-4-[(2S)-
benzyl-3-nicotinoylthiopropionylamino]-4-(N-phenyl-c-
arbamoyl)-butyric acid t-butyl ester as a white powder:
Rf=0.24 (ethyl acetate:n-hexane=1:1); 1H-NMR
(CDC13) d 1.49 (9H, s), 1.80±3.10 (7H, m), 3.26±3.42
(2H, m), 4.46 (1H, dt, J=7.5 Hz), 6.80 (1H, d,
J=7.5 Hz), 7.00±7.20 (5H, m), 7.11±7.50 (5H, m), 8.19
(1H, ddd, J=1.5, 5.0, 8.0 Hz), 8.61 (1H, brs), 8.77 (1H,
dd, J=2.0, 5.0 Hz), 9.15 (1H, d, J=2.0 Hz).
(4S)-4-Amino-4-(N-phenylcarbamoy)-butyric acid t-butyl
ester (49). To a solution of 48 (8.50 g, 20.6 mmol) in
ethyl acetate±acetic acid (160 mL±0.1 mL) was added
10% Pd on carbon (850 mg) followed by stirring under a
hydrogen atmosphere for 1 h. Removal of the catalyst by
®ltration followed by treatment with 1 equivalent of
hydrogen chloride in ethyl acetate a€orded 49 as a hydro-
chloride salt, which was used without further puri®cation
in the next step: Rf=0.20 (ethyl acetate:n-hexane=1:1)
(4S)-4-[(2S)-Benzyl-3-benzoylthiopropionylamino]-4-(N-
phenyl-carbamoyl)-bubric acid t-butyl ester (50). To a
stirred solution of 49 (6.48 g, 20.6 mmol) and pyridine
(12 mL, 0.15 mol) in dichloromethane (110 mL) was
The resulting solution of t-butyl ester (2.20 g, 3.92 mmol)
in ethyl acetate±dichloromethane (20 mL±10 mL) was

K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
459
treated with 4N hydrogen chloride in ethyl acetate
(20 mL) at 25 ꢀC and stirred for 2 h. The resulting pre-
cipitate was collected by ®ltration and washed with ethyl
acetate to yield 20 (1.96 g, 75.0% in 2 steps) as a white
(1.61 g, 23.6 mmol) in DMF (30 mL) was treated
with t-butylchlorodiphenylsilane (3.13 mL, 11.8 mmol)
at 5 ꢀC followed by stirring at 25 ꢀC for 4 h. The
reaction mixture was diluted with ethyl acetate, washed
with 1N sodium hydroxide and then brine. The organic
layer was dried (MgSO4) and evaporated. To a stirred
solution of the residue in dichloromethane (30 mL)
was added triethylamine (3.9 mL, 28 mmol) and then
diphenylacetyl chloride, which was prepared from the
corresponding acid (2.97 g, 14.0 mmol) and an excess
amount of oxalyl chloride (10 mL). The reaction
mixture was diluted with diethyl ether and washed with
water and then brine. The organic layer was dried
(MgSO4) and evaporated. The resulting silyl ester was
treated with acetic acid:tetrahydrofuran: water (3:1:1)
at 25 ꢀC for 15 h and evaporated. The residue was
triturated with n-hexane-diethyl ether to yield 21
(3.20 g, 50.3%) as a white powder: Rf=0.40 (10%
1
powder: Rf=0.35 (10% acetic acid in chloroform); H-
NMR (CDCl3+DMSO-d6) d 1.82±3.45 (9H, m), 4.58
(1H, dt, J=7.5 Hz), 7.00±7.36 (8H, m), 7.53 (2H, d,
J=7.0 Hz), 7.98 (1H, dd, J=5.0, 8.0 Hz), 8.19 (1H, d,
J=9.0 Hz), 8.65 (1H, d, J=8.0 Hz), 8.97 (1H, dd,
J=2.0, 5.0 Hz), 9.19 (1H, d, J=2.0 Hz), 9.50 (1H, s);
Mass m/e 505 (M+), 487; IR (KBr, cm 1) 3270, 3040,
1720, 1665, 1640, 1595, 1520, 1440, 1210, 930, 810, 750,
695, 680; [a]d 50.6ꢀ (c=1.00, methanol).
Compounds 24 was prepared from 50b according to the
same method as described above.
(4S)-4-[(2S)-Benzyl-3-benzoylthiopropionylamino]-4-(N-
phenyl-carbamoyl)-butyric acid (24). Rf=0.29 (5% acetic
acid in chloroform); 1H-NMR (CDCl3+DMSO-d6) d
1.80 (4H, m), 2.79±3.38 (5H, m), 4.47±4.63 (1H, m),
6.99±7.33 (8H, m), 7.40±7.65 (5H, m), 7.85±8.00 (3H,
m), 9.28 (1H, s); Mass m/e 504 (M+), 486, 412, 395, 384;
IR (KBr, cm 1) 3270, 3050, 1705, 1655, 1640, 1600,
1530, 1490, 1445, 1205, 910, 750, 685; [a]d 77.9ꢀ
(c=0.96, methanol).
1
methanol in chloroform); H-NMR (CDCl3+CD3OD)
d 1.60±2.35 (4H, m), 2.65±3.25 (5H, m) 4.45±4.70 (1H,
m), 5.19 (1H, s), 6.92±7.51 (20H, m); Mass m/e 594 (M+),
557, 548, 502; IR (KBr, cm 1) 3270, 1690, 1640, 1600,
1535, 1490, 1440; [a]d 52.9ꢀ (c=1.035, chloroform).
(4S)-4-[(2S)-Benzyl-3-(2,2-dimethyl- 1,3-dioxolane-4S-
carbonyl)thio-propionylamino]-4-(N-phenyl-carbamoyl)-
butyric acid (23). To a solution of diphenylphosphoryl
azide (DPPA, 1.80 mL, 8.00 mmol) and silyl ester of
8b(S) (5.117 g, 8.00 mmol) ,which was prepared accord-
ing to the method described above, in DMF (8 mL) was
added triethylamine (1.12 mL, 8.00 mmol) at 5 ꢀC fol-
lowed by stirring at 25 ꢀC for 24 h. The reaction mixture
was diluted with ethyl acetate±diethyl ether (2:1),
washed with distilled water and then brine. The organic
layer was dried (MgSO4) and evaporated. The residue
was dissolved in tetrahydrofuran (10 mL) and treated
with tetra-n-butylammonium ¯uoride (1 M in tetra-
hydrofuran, 2.32 mL, 2.32 mmol) at 5 ꢀC for 5 min. The
reaction mixture was diluted with ethyl acetate, washed
with water and then brine. The organic layer was dried
(MgSO4) and evaporated. The residue was puri®ed by
chromatography on silica-gel (eluted with ethyl acetate
followed by 10% methanol in ethyl acetate) to yield 23
(1.50 g, 35.0%) as a white powder: Rf=0.25 (ethylace-
Preparation of 21±23 from 8b(S)
Direct preparation of (4S)-4-[(2S)-benzyl-3-[(1RS)-1,3-
dihydro-3-isobenzofuran-1-ylthio]-propionylamino]-4-(N-
phenylcarbamoyl)-butyric acid (5b) from 8b(S). To a
stirred solution of 8b(S) (221.4 g, 0.55 mol) and 3-
chlorophthalide (140 g, 0.83 mol) in acetone (1.2 L)-N,
N-dimethylformamide (DMF, 600 mL) was added
potassium carbonate (152.8 g, 1.11 mol) at 5 ꢀC followed
by stirring at 25 ꢀC for 1.5 h. The reaction mixture was
poured into ice-water and extracted with ethyl acetate.
The organic layer was washed with diluted water, brine,
dried (MgSO4) and evaporated. The residue was recrys-
tallized from acetone-n-hexane (1:4) to yield 5b (234 g,
79.3%) as a white amorphous powder: mp 180±183 ꢀC;
Rf=0.45 (5% methanol in ethyl acetate); 1H-NMR
(CDCl3+DMSO-d6) 1.85±2.30 (2H, m), 2.35±2.58 (2H,
m), 2.65±3.11 (5H, m), 4.50±4.70 (1H, m), 6.68, 6.78
(1H, s, 1:1.6), 7.00±7.38 (8H, m), 7.48±7.65 (4H, m),
7.65±7.80 (2H, m), 9.22±9.37 (1H, m); Mass m/e 532
(M+), 514, 496, 440, 412.; IR (KBr, cm 1) 3250, 1750,
1640, 1600, 1525, 1440; Anal. Calcd. C29H28N2O6S:
C, 65.40; N, 5.26; S, 6.02. Found: C, 64.92; N, 5.32; S,
6.07.
1
tate); H-NMR (CDCl3+DMSO-d6) 1.35 (3H, s), 1.50
(3H, s), 1.80±3.50 (9H, m), 4.01 (1H, dd, J=5.0, 9.0 Hz),
4.18 (1H, dd, J=7.0, 9.0 Hz), 4.50±4.70 (2H, m), 6.95±
7.30 (8H, m), 7.58 (2H, d, J=7.0 Hz), 8.18 (1H, d,
J=8.0 Hz), 9.78 (1H, brs); Mass m/e 510 (M+ H2O),
495, 424; IR (KBr, cm 1) 3280, 2990, 1640, 1590, 1535,
1440, 1380, 1310, 1250, 1220; [a]d 58.8ꢀ (c=1.085,
chloroform).
General procedure for the preparation of 21±23 via silyl
ester of 8b(S)(4S)-4-[(2S)-benzyl-3-diphenylacetylthio-
propionylamino]-4-(N-phenyl-carbamoyl)-butyric acid (21).
A solution of 8b(S) (4.30 g, 10.7 mmol) and imidazole
Compound 22 was prepared from 8b(S) according to
the same procedure as described in the preparation of 21
or 23.

460
K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
(4S)-4-[(2S)-Benzyl-3-isonicotinoylthio-propionylamino]-
4-(N-phenyl-carbamoyl)-bubric acid (22). Rf=0.36
(chloroform:tetrahydrofuran:acetic acid=15:4:1); 1H-
NMR (CDCl3+CD3OD) 1.75±2.00, 2.00±2.25 (2H, m),
2.38 (2H, brt, J=7.5 Hz), 2.75±3.13 (3H, m), 3.20±3.50
(2H, m), 4.43±4.60 (1H, m), 7.00±7.21 (6H, m), 7.31
(2H, brt, J=8.0 Hz), 7.40±7.50 (2H, m), 7.80 (2H. dd,
J=2.5, 5.0 Hz), 8.71 (2H, dd, J=2.5, 5.0 Hz); Mass m/e
505 (M+), 487, 413, 385; IR (KBr, cm 1) 3275, 1700,
1670, 1640, 1600, 1530, 1500, 1440, 1410; [a]d 77.5ꢀ
(c=1.00, methanol).
7.44 (11H, m), 7.51±7.78 (3H, m), 7.88, 7.90 (1H, d,
J=8.0 Hz), 8.30, 8.35 (1H, s); Mass m/e 574 (M+), 518,
501, 482.
A solution of S-alkylated product (6.35 g, 11 mmol) in
dichloromethane (20 mL) was treated with tri-
¯uoroacetic acid (25 mL, 0.32 mol) at 25 ꢀC for 1 h. The
reaction mixture was poured into water and extracted
with ethyl acetate. The organic layer was washed with
1N sodium hydroxide, brine, dried (Na2SO4) and eva-
porated. The residue was triturated with diethyl ether:n-
hexane to yield 5a (4.89 g, 58.8% in 3 steps) as a white
powder: Rf=0.51 (chloroform:tetrahydrofuran:acetic
acid=15:4:1); 1H-NMR (CDCl3) d 2.50±3.10 (7H, m),
4.85±5.06 (1H, m), 6.50, 6.53 (1H, s), 6.95±7.88 (15H,
m), 9.46, 9.60 (1H, s); Mass m/e 500 (M+ H2O), 482,
407, 367; IR (KBr, cm 1) 3280, 3030, 1760, 1640, 1600,
1525, 1440, 1285, 1170, 940, 760, 725, 695.
(4S)-4-[(2S)-Benzyl-3-[(1RS)-1,3-dihydro-3-isobenzofuran-
1-ylthio]-propionylamino]-4-[N-(2-benzothiazoly)carbam-
oyl] -butyric acid (25). Compound 26 was prepared
from 11(S) according to the same procedure as descri-
bed in the preparation of 21 or 23. Rf=0.25 (2% acetic
acid in chloroform); 1H-NMR (CDCl3+CD3OD) d
1.90±2.30 (2H, m), 2.30±2.60 (2H, m), 2.65±3.05 (5H,
m), 4.60±4.72 (1H, m), 6.75 (1H, s), 6.95±7.18 (5H, m),
7.27±7.68 (4H. m), 7.75±7.90 (4H, m); Mass m/e 571
(M+ H2O), 454, 438, 421, 405; IR (KBr, cm 1) 3300,
1760, 1640, 1600, 1540, 1440.
Preparation of metabolites 7b(S) and 26(4S)-4-[(2S)-
benzyl-3-mercaptopropionyl]-4-carboxybutyric acid (7b).
To a stirred solution of cyclohexylamine salt of 5124
(5.42 g, 10 mmol) in methanol (25 mL) was added 2N
sodium hydroxide (16.5 mL, 33 mmol). After being stir-
red at 25 ꢀC for 1.5 h under argon, the reaction mixture
was poured into cold 0.5N hydrochloric acid (100 mL,
50 mmol) and extracted with ethyl acetate. The organic
layer was washed with water, brine, dried (MgSO4) and
evaporated. The residue was recrystallized from ethyl
acetate±n-hexane to yield 7b(S) (2.6 g, 80.0%) as a white
powder: Rf=0.50 (chloroform:tetrahydrofuran:acetic
acid=20:2:1); 1H-NMR (CDCl3+DMSO-d6) d 1.60±
1.75 (1H, m), 1.78±2.23 (2H, m), 2.25±2.45 (4H, m),
2.63±2.85 (2H, m), 2.80±4.00(2H, m, OH), 2.90±3.15
(1H, m), 4.37±4.50 (1H, m), 7.12±7.32 (5H, m), 7.88
(1H, d, J=8.5 Hz); Mass m/e 325 (M+), 292, 278, 260,
234; [a]d 31.5ꢀ (c=0.93, methanol).
(b) General procedure for (3S)-3-amino-3-(N-arylcar-
bamoyl)-propionic acid derivative: Preparation of (3S)-3-
[(2S)-benzyl-3-[(1RS)-1,3-dihydro-3-isobenzofuran-1-yl-
thio]propionylamino]-3 -(N-phenylcarbamoyl)-propionic
acid (5a)
(3S)-3-[(2S)-Benzyl-3-[(1RS)-1,3-dihydro-3-isobenzofuran-
1-ylthio]-propionylamino]-3-(N-phenylcarbamoyl)-prop-
ionic acid (5a). 46a(S) (8.95 g, 16.8 mmol), which was
prepared according to the general procedure for the
preparation of (3S)-3-(2-substituted-3-mercaptopropio-
nyl-amino)-3-(N-arylcarbamoyl)-propionic acid deriva-
tives, was dissolved in methanol (80 mL)±dichloro-
methane (20 mL). To the solution was added potassium
carbonate (4.52 g, 32.7 mmol) under argon at 25 ꢀC for
1.5 h. The reaction mixture was acidi®ed with 1N hydro-
chloric acid and extracted with ethyl acetate. The organic
layer was washed with brine, dried (MgSO4) and evapo-
rated. The residue was puri®ed by chromatography on
silica-gel (ethyl acetate:dichloromethane=1:15) to a€ord
thiol (8.95 g, 12.0 mmol). To a solution of the resulting
thiol (5.20 g, 11.8 mmol) and 3-chlorophthalide (3.28 g,
17.6 mmol) in acetone (40 mL) was added potassium
carbonate (2.65 g, 19.4 mmol) at 25 ꢀC followed by stir-
ring for 5 h. The reaction mixture was diluted with ethyl
acetate±diethyl ether (1:1) and washed with water, then
brine. The organic layer was dried (MgSO4) and evapo-
rated. The residue was puri®ed by chromatography on
silica-gel (ethyl acetate:n-hexane=1:2 followed by 2:3)
to a€ord 6.36 g (11.1 mmol) of S-alkylated product as a
white amorphous powder: Rf=0.17 (ethyl acetate:n-
(2S)-Benzyl-3-mercaptopropionic acid (26). To a stirred
solution of 44(S) (6.0 g 20 mmol) in methanol (50 mL)
was added potassium carbonate (5.52 g, 40 mmol). After
being stirred under argon at 25 ꢀC for 2 h, the reaction
mixture was acidi®ed with 1N hydrochloric acid and
extracted with ethyl acetate. The organic layer was
washed with brine, dried (MgSO4) and evaporated. The
residue was puri®ed by chromatography on silica-gel (n-
hexane:dichloromethane=1:1 followed by dichloro-
methane) to yield 26 (3.6 g, 92.0%): Rf=0.68 (chloro-
form:tetrahydrofuran:acetic acid=20:2:1); 1H-NMR
(CDCl3) d 1.55 (1H, t, J=10 Hz), 2.58±3.23 (5H, m),
7.03±7.21 (5H, m); Mass m/e 196 (M+), 162, 117.
Biological evaluation. Assay for enkephalinase inhibitory
activity. Mouse striatal membranes were used as the
enzyme source. Male ddY mice (20±25 g) were sacri®ced
by decapitation; striata were rapidly dissected out on ice
1
hexane=1:2); H-NMR (CDCl3) d 1.40, 1 .45 (9H, s),
2.55±3.15 (7H, m), 4.77±4.91 (1H, m), 6.55 (1H, s), 6.92±

K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
461
and homogenized by sonication in 10 mL of ice-cold
50 mM Tris-HCl bu€er, pH 7.4. The homogenate was
centrifuged immediately for 1 min at 200,000 g. The
resulting pellet was sonicated in the same volume of cold
bu€er and used as an enzyme source. Aliquots of the
suspension (50 mL) were preincubated for 5 min at
37 ꢀC with 30 mL of 100 mM bestatin, 100 mM puromycin
and 1 mM captopril with or without the inhibitor. Incu-
bation for 180 min at 37 ꢀC was started by adding
rat treated with formalin. No restraint was applied
to the rat during the behavioral observations.
3. Acetic acid-induced writhing test: Male ddY mice
weighing 20±23 g were used after overnight star-
vation. Compounds and vehicle were adminis-
tered po at a 20 mL/kg dose 30 min prior to the ip
injection of 0.6% acetic acid (AcOH) at 10 mL/kg
to conscious animals. Mice were then placed into
perspex observation cages, and the number of
writhings was counted from 5 min to 20 min after
the ip injection of AcOH.
4. Stress-induced analgesia (SIA) in the hot-plate test:
Male ICR mice weighing 20±23 g were used after
overnight starvation. As inescapable stress (a
2 mA electric footshock) was delivered in
60Â16 msec pulses once every 2 s for 3 min to
mice. After 1, 5, and 10 min of the stress exposure,
jump latencies on a 55 ꢀC hot-plate test were
measured. The vehicle or compound was admi-
nistered 60 min prior to the stress exposure. The
potentiation e€ects of the compound/vehicle on
SIA in the mice were judged by determining the
extension seen in the jump latencies before and
10 min after the stress. Student's t-test was used
for statistical analysis.
3
300 mM Leu5-enkephalin and 10 nM [H]Leu5-enkepha-
lin (1472.6 Gbq/mmol) and terminated by the addition
of 0.2 M HCl. Aliquots of the incubation medium were
spotted on TLC silica gel sheets, and the metabolites
Tyr (aminopeptidase metabolite), Tyr-Gly (dipeptidyl
aminopeptidase metabolite), and Tyr-Gly-Gly (enke-
phalinase metabolite) were separated according to their
Rf values.18 Their radioactivity was estimated by a
liquid scintillation counter.
Pharmacological evaluation. Antinociceptive test
1. Bradykinin (BK)-induced biting-like response: Male
Sprague±Dawley rats weighing 200±250 g were
used after overnight starvation. Implantation of
the bradykinin-fed cannula onto the tooth pulp
and the ®xation of it on the lower incisor surfaces
were carried out under diethyl ether anesthesia
according to the previous report19. At least 2 h
were allowed for recovery from anesthesia.
Microapplications of BK (0.5±1 mL, 10 7 M) onto
the tooth pulp of the right mandibular incisor of
the animals produced biting-like responses, and
these responses were taken as the measure for
assaying the analgesic potencies of compounds.
Only rats that showed two serial biting-like
responses with a latency less than 1 min and a
duration of more than 20 min were used for the
analgesic assays. BK was applied at 30 min inter-
vals. When the biting duration was shortened to
4 min or less after drug administration, the e€ect
of the compound was considered analgesic.
Pharmacokinetic study
The bioavailability of 5b in rats was obtained from the
area under the plasma concentration±time curves
(AUC0±1), which were determined by high-performance
liquid chromatography (HPLC), after oral (10 mg/kg)
and intravenous (iv) (1 mg/kg) administrations of 5b.
The half-life of 5b in rats was obtained from the plasma
concentration-time curve after the oral (10 mg/kg)
administration of 5b.
The percentages of metabolites of 5b were obtained
from their AUC0±48 values after the oral (100 mg/kg)
administration of 5b to fasted male rats and beagle
dogs.
2. Formalin-induced licking test: Male Sprague±
Dawley rats weighing 200±300 g were used after
overnight starvation. A rat was placed in a metal
cage and left there for 10 min or more. A group of
three animals was used for each treatment. For
noxious stimulation, the rat was injected with 5 mL
of diluted formalin (5% formaldehyde solution)
into the plantar region of the left hindpaw, and
the duration of the licking response was measured
by stopwatch from 10 min to 60 min after the
injection. Compounds were administered per os
(po) 30 min prior to the injection of formalin.
Saline (1 mL/kg) was given as a vehicle. A mirror
was placed on the opposite side of the enclosure
for the unhindered observation of the paw of the
The metabolites of 5b were detected as their derivatives
by reduction with tri-n-butylphosphine followed by
treatment
(ABD-F).
with
4-¯uoro-7-sulfamoylbenzofurazan
(1) Evaluation of the metabolism of 5b in rats. Four
male Sprague±Dawley rats weighing 210±260 g (8 weeks
old, Charles River Japan, Atsugi, Japan) were fasted for
20 h prior to and for 12 h after the drug administration,
but were allowed free access to water. 5b suspended in
0.5% carboxymethyl cellulose (CMC) in water was
administered orally at a dose of 100 mg/5 mL/kg. Blood
samples were taken from the jugular vein with hepar-
inized syringes. Then, 0.1 M potassium borate bu€er

462
K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
containing 2M EDTA (0.5 mL, pH8.2), 11c(S) as an
internal standard (1 mg/mL, 0.05 mL) and 2% tri-n-
butylphosphine in DMF (10 mL) were added to the
plasma (0.2 mL) prepared from the above-mentioned
blood. After incubation at 37 ꢀC for 10 min, 0.1 M citric
acid (0.1 mL) was added to the samples in an ice bath.
The resulting samples were charged to Bond Elut C18
and eluted with CH3CN (1 mL). After treatment with
0.1 M potassium borate bu€er containing 2M EDTA
(0.5 mL, pH8.2) and ABD-F (0.1 mL, 1 mg/mL in 0.1 M
pH8.2 potassium borate bu€er) at 37 ꢀC for 45 min,
0.1 M citric acid (0.5 mL) was added in the ice bath.
Samples were extracted with ethyl acetate (2 mL) twice
and evaporated. The residue was dissolved in the mobile
phase (0.24 mL), and 80 mL of this solution was loaded
on an HPLC column.
5b:
Eluent A: 0.02 M KH2PO4 (adjusted to pH2.1 with
H3PO4)/CH3CN=70/30 (v/v)
Eluent B: 0.02 M KH2PO4 (adjusted to pH2.1 with
H3PO4)/CH3CN=30/60 (v/v)
Linear gradient (Eluent A/Eluent B=100/0 at 0 min
followed by Eluent A/Eluent B=34/66 at 30 min)
Flow rate: 0.8 mL/min. Detection: 236 nm.
References
1. Hugues, J.; Smith, T. W.; Kosterlitz, H. W.; Fothergill, H. W.;
Morgan, L. A.; Morris, H. R. Nature (London) 1975, 258, 577.
2. Lord, J. A.; Water®eld, A. A.; Hugues, J.; Kosterlitz, H. W.
Nature (London) 1977, 267, 495.
3. Chang, K. J.; Cuatrecasas, P. J. Biol. Chem. 1979, 254, 2160.
(2) Evaluation of the metabolism of 5b in beagle dogs.
Three male beagle dogs weighing 13 to 15 kg were fas-
ted for 20 h prior to and for 12 h after the drug admin-
istration, but were allowed free access to water. 5b
charged in 1/2 oz capsule was administered orally at a
dose of 100 mg/kg. Blood samples were taken from the
cephalic vein with heparinized syringes. Potassium
borate bu€er (0.1 M) containing 2M EDTA (1 mL,
pH8.2), 11c(S) as an internal standard (1 mg/mL,
0.5 mL) and 2% tri-n-butylphosphine in DMF (10 mL)
were added to the plasma (0.2 mL) prepared from the
aforementioned blood. After incubation at 37 ꢀC for
10 min, 0.1 M citric acid (0.1 mL) was added to the
samples cooling with an ice bath. The resulting samples
were charged to Bond Elut C18 and eluted with CH3CN
(1 mL). After treatment with 0.1 M potassium borate
bu€er containing 2M EDTA (0.5 mL, pH8.2) and ABD-
F (0.1 mL, 1 mg/mL in 0.1 M pH8.2 potassium borate
bu€er) at 37 ꢀC for 45 min, 0.1 M citric acid (0.5 mL) was
added cooling with the ice bath. Samples were extracted
with ethyl acetate (2 mL) twice and evaporated. The
residue was dissolved in the mobile phase (0.24 mL), and
80 mL of this solution was loaded on an HPLC column.
4. Goodman, R. R.; Snyder, S. H.; Kuhar, H. J.; Young, III
W. S. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 6239.
5. Fulcher, I. S.; Matsas, R.; Turner, A. J.; Kenny, A. J. Bio-
chem. J. 1982, 203, 519.
6. Malfroy, B.; Swerts, J. P.; Guyon, A.; Roques, B. P.;
Schwartz, J. C. Nature (London) 1978, 276, 523.
7. Hambrook, J. M.; Morgan, B. A.; Rance, M. J.; Smith, C.
F. Nature (London) 1976, 262, 782.
8. Gorenstein, C.; Snyder, S. H. Life Sci. 1979, 25, 2065.
9. Fournie-Zaluski, M. C.; Soroca-Lucas, E.; Waksman, G.;
Llorens, C.; Schwartz, J. C.; Roques, B. P. Life Sci. 1982, 31,
2947.
10. Fournie-Zaluski, M. C.; Chaillet, P.; Soroca-Lucas, E.;
Marcais-Collado, H.; Costentin, J.; Roques, B. P. J. Med.
Chem. 1983, 26, 60.
11. Roques, B. P.; Fournie-Zaluski, C.; Soroca, E.; Lecomte, J.
M.; Malfroy, B.; Llorens, C.; Schwartz, J. C. Nature (London)
1980, 288, 286.
12. Fournie-Zaluski, M. C.; Lucas, E.; Waksman, G.; Roques,
B. P. Eur, J, Biochem. 1984, 139, 267.
13. Roques, B. P.; Soroca-Lucas, E.; Chaillet, P.; Costentin, J.;
Fournie-Zaluski, M. C. Proc. Natl. Acad. Sci. U.S.A. 1983, 80,
3178.
14. Vevert, J. P.; Delevallee, F. F.; Deraedt, R. F. Japan Kokai
Tokkyo Koho 1984, 59±148759.
The HPLC was carried out using a YMC A-302 ODS
(150Â4.6 mm i.d.) column, and the HPLC analysis of
metabolites 8b(S), 7b, 26 and 5b was carried out under
the following conditions:
15. Carenzi, A.; Frigeni, V.; Della-Bella, D. Advances in Endo-
geneous and Exogenous Opioids; Takagi, H., Simon, E. J., Eds.;
Elsevier/North-Holland Biochemical Press: Amsterdam, 1981;
p 267.
16. Compound 6 was prepared as follows:
Metabolites 8b(S), 7b and 26:
Eluent A: 0.02 M KH2PO4 (adjusted to pH2.1 with
H3PO4)/CH3CN=70/30 (v/v)
Eluent B: 0.02 M KH2PO4 (adjusted to pH2.1 with
H3PO4)/CH3CN=30/60 (v/v)
Linear gradient (Eluent A/Eluent B=100/0 at 0 min
followed by Eluent A/Eluent B=34/66 at 30 min)
Flow rate: 0.8 mL/min. Detection: Ex=380 nm,
Em=510 nm.
6:Rf=0.13 (chloroform:tetrahydrofuran:acetic acid=10:2:1);
1H-NMR (CDCl3+acetone-d6) d 1.21 (3H, d, J=7.5 Hz), 1.63
(1H, t, J=7.5 Hz), 1.80±3.00 (7H, m), 4.48±4.80 (1H, m), 7.10±

K. Senokuchi et al./Bioorg. Med. Chem. 6 (1998) 441±463
463
1
7.70 (3H, m); Mass m/e 249 (M+), 216, 205; IR (KBr, cm
)
3600±3200, 3200±2300, 1700, 1640, 1520, 1240; [a]d 39.2ꢀ (c 1.01,
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