Cysteine derivatives as inhibitors for carboxypeptidase A: Synthesis and structure-activity relationships

Article abstract of DOI:10.1021/jm010272s

A series of cysteine (Cys) derivatives having an alkyl or arylalkyl moiety on the α-amino group of the amino acid have been synthesized as a novel type of inhibitor for carboxypeptidase A. These compounds are readily prepared starting with Cys in an optic

Full text of DOI:10.1021/jm010272s

J . Med. Chem. 2002, 45, 911-918
911
Cystein e Der iva tives a s In h ibitor s for Ca r boxyp ep tid a se A: Syn th esis a n d
Str u ctu r e-Activity Rela tion sh ip s
J ung Dae Park and Dong H. Kim*
Department of Chemistry, Division of Molecular and Life Science, Pohang University of Science and Technology, San 31,
Hyojadong, Namku, Pohang 790-784, Korea
Received J une 18, 2001
A series of cysteine (Cys) derivatives having an alkyl or arylalkyl moiety on the R-amino group
of the amino acid have been synthesized as a novel type of inhibitor for carboxypeptidase A.
These compounds are readily prepared starting with Cys in an optically active form. The
structure-activity relationship study revealed that the inhibitors prepared from D-Cys are
much more potent than the corresponding inhibitors obtained from L-Cys, and the most potent
inhibitor in the series, (S)-1j with a Ki value of 55 ( 4 nM, is obtained by introducing a phenethyl
moiety on the amino group of D-Cys. In comparison, the most active inhibitor in the series of
2-substituted 3-mercaptopropanoic acid is found to be 20, in which the phenyl ring is linked to
the mercaptocarboxylic acid at the R-position with a methylene unit. A proposal that accounts
for the different structural requirement for the maximum activity between the two series of
inhibitors is provided.
In tr od u ction
Resu lts a n d Discu ssion
Design Ra tion a le. The topology of the CPA active
site and roles of the catalytic residues for the enzymic
reaction have been well-characterized. The catalytic zinc
ion is bound tightly to the enzyme by coordination of
His-69, Glu-72, and His-196. A molecule of water that
is coordinated to the metal ion as the fourth ligand
functions as the nucleophile that attacks the scissile
peptide bond of the enzyme-bound substrate. Glu-270
and Arg-145 have been identified as residues that play
important roles in catalysis and substrate recognition,
respectively. The former functions as a general base,
enhancing the nucleophilicity of the zinc-bound water
molecule, and the latter forms hydrogen bonds with the
C-terminal carboxylate of substrate.2,10 In addition,
there exists a hydrophobic pocket at the active site, the
primary function of which is to recognize the substrate
by accommodating the aromatic side chain in the P1′
residue of the substrate.10 On the basis of the foregoing
structural and functional feature of the active site of
CPA, numerous inhibitors for the enzyme have been
designed mostly by incorporating a zinc coordinating
functionality into the substrate-like structural frame
that can be recognized by the enzyme.11 Among several
zinc-coordinating groups known, the sulfhydryl group
has been used most frequently by the virtue of its strong
affinity for zinc ion.11
Carboxypeptidase A (CPA) is a zinc-containing pro-
teolytic enzyme that catalyzes selectively the removal
of the carboxy-terminal amino acid residue having a
hydrophobic side chain.1,2 It is one of the most exten-
sively studied zinc proteases and represents a large
family of pathologically important zinc-containing pro-
teolytic enzymes such as matrix metalloproteases.3 The
X-ray crystal structure of CPA has been refined to 1.54
Å.4 Because CPA is a well-characterized prototypical
zinc enzyme, it has served as a model for inhibitor
design protocols that can be applied to other zinc
proteases. For example, captopril,5 an inhibitor of an-
giotensin converting enzyme (ACE), was obtained by the
application of the design strategy that originated from
the synthesis of 2-benzysuccinic acid as a potent inhibi-
tor of CPA.6 ACE, also a zinc-containing protease,
catalyzes the conversion of angiotensin I to angiotensin
II, a potent vasoconstrictor. The inhibitors of ACE have
found widespread uses not only in the treatment of
hypertension but also for the clinical management of
congestive heart failure.7,8 Accordingly, the inhibitor
design strategies developed using CPA as a model
enzyme bear considerable importance to medicinal
chemistry.
We have been involved in the development of design
strategies for CPA inhibitors that can be applied to other
zinc proteases.9 We describe in this paper the design
and synthesis of a series of cysteine (Cys) derivatives
and their structure-activity relationships (SAR) as
inhibitors of CPA. Unlike most of the other CPA
inhibitors, these compounds are readily synthesized in
an optically active form starting with enantiomerically
pure Cys.
We have envisioned that Cys, which carries a sulf-
hydryl group at the â-position, may serve as a viable
structural frame that is useful for the preparation of a
novel type of CPA inhibitor with high potency. In the
binding of Cys to CPA, the carboxylate and sulfhydryl
groups in Cys are expected to interact with the guani-
dinium moiety of Arg-145 and the active site zinc ion,
respectively. It was thought that the CPA inhibitory
potency of Cys would be improved by the incorporation
of a hydrophobic group that can be accommodated in
* To whom correspondence should be addressed. Tel: +82-54-279-
2101. Fax: +82-54-279-5877. E-mail: dhkim@postech.ac.kr.
10.1021/jm010272s CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/17/2002

912 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Park and Kim
Sch em e 3a
a
Reagents, conditions, and yield: (a) CH2dC(Br)CO2H, toluene,
90 °C, 1 h, 92%; (b) 48% HBr, reflux, 2 h, 82%.
The synthesis of N-benzyl-N-methylcysteine 8 was
started with the methyl ester of serine, which upon
treatment with benzyl bromide in the presence of
potassium carbonate, gave exclusively the N-monoben-
zylated product, 5. The latter was allowed to react with
methyl iodide to form 6, which was then converted into
thioester 7 by reacting with thioacetic acid under
Mitsunobu conditions. The hydrolysis of 7 in diluted
hydrochloric acid afforded 8 (Scheme 4).
F igu r e 1. Rationale used for preparing N-substituted Cys
derivatives as potential inhibitors for CPA.
Sch em e 1a
To probe the effect of the amino group on the binding
of the N-substituted Cys inhibitors to CPA, we have also
synthesized 13, 14, and 19, in which the amino groups
are replaced with a methylene unit. Scheme 5 depicts
the synthesis of 2-(mercaptomethyl)-4-methylpentanoic
acid (13). The reaction of 3-hydroxypropionitrile in the
presence of 2 equiv of LDA with isobutyl bromide in
hexamethylphosphoramide (HMPA) afforded 9. The
latter was then treated with aqueous potassium hy-
droxide solution under reflux conditions, whereby the
nitrile group was converted into carboxylate, which was
then transformed into methoxycarbonyl to give 11. The
hydroxyl group in 11 was converted into an acetylmer-
capto group by allowing it to react with thioacetic acid
under Mitsunobu conditions. The hydrolysis of 12 in
48% HBr solution under reflux conditions afforded 13
(Scheme 5). In an analogous fashion, 14 was prepared.
2-(Mercaptomethyl)-6-phenylhexanoic acid (19) was
prepared according to the synthetic route reported by
Ondetti et al.15 (Scheme 6). Diethyl malonate was
allowed to react with 1-bromo-4-phenylbutane to obtain
15, which was subsequently subjected to saponification
to give 16. The latter was converted under Mannich-
type conditions into R,â-unsaturated carboxylic acid 17
and then into 18 by treatment with thioacetic acid. The
acetyl group in 18 was removed by treatment with
methoxyethylamine to afford 19 (Scheme 6).
SAR. The compounds thus synthesized were evalu-
ated as competitive inhibitors for CPA by a standard
method using hippuryl-L-phenylalanine (Hipp-L-Phe) as
a substrate at pH 7.5,15 and the inhibitory constants
(Ki values) were estimated from the respective Dixon
plot.16 Table 1 lists Ki values of N-alkylcysteine for the
inhibition of CPA. It can be seen from Table 1 that the
introduction of an alkyl group at the amino nitrogen of
rac-Cys does not affect the Ki value much, suggesting
that small alkyl groups such as ethyl or propyl may be
accommodated in the S1′ pocket, but their effects on the
binding of the inhibitors to CPA are marginal probably
due to limited van der Waals contacts of the small alkyl
groups with the surface of the pocket. Bulky alkyl
groups such as cyclohexyl and isobutyl affect adversely,
which may possibly be due to the difficulty of the large
moiety getting in the S1′ pocket. It has been reported
that the opening of the substrate recognition pocket is
relatively narrower than the pocket, and thus, large
groups experience difficulty in entering the pocket.17
a
Reagents, conditions, and yield: (a) Aldehydes or ketones,
NaBH3CN, MeOH, room temperature, 18-36 h, 30-50%.
Sch em e 2a
a
Reagents, conditions, and yield: (a) Acetone, reflux, 8 h, 96%;
(b) MeI, K2CO3, DMF, room temperature, 4 h, 45%; (c) 3 N HCl,
reflux, 8 h, 88%.
the S1′ pocket of CPA. Figure 1 depicts schematically
the probable binding mode of the Cys-based inhibitors
that we have designed. While our study was in progress,
Chong and Auld12 reported that D-Cys is a potent
inhibitor for CPA with a Ki value of 2.3 µM and
subsequently the X-ray crystal structure of the CPA
complex formed with D-Cys was determined to establish
that the sulfhydryl group in the amino acid is coordi-
nated to the active site zinc ion.13
Syn th esis. Derivatives of Cys, 1b-g a n d 1i-n ,
which carry alkyl or arylalkyl moieties at the R-amino
group, were prepared from unprotected Cys simply by
the reductive amination with the appropriate aldehyde
or ketone using sodium cyanoborohydride in methanol
in moderate yields (Scheme 1). The individual stereoi-
somers of 1d , 1f, 1i, and 1j were prepared starting with
optically active Cys.
An attempt at N-methylation to obtain N-methylcys-
teine by reductive amination with formaldehyde gave
a mixture of mono- and dimethylated Cys, which could
not be purified. Accordingly, an alternative synthetic
path was devised as follows: The condensation of the
methyl ester of Cys with acetone afforded thiazolidine
2, which was then treated with methyl iodide to give 3.
The hydrolysis of 3 under acidic conditions provided the
desired compound 1a in 38% overall yield (Scheme 2).
N-Phenylcysteine (1h ) was synthesized by a literature
method14 that involves the formation of 4 from N-
phenylthioacetamide and R-bromoacrylic acid and sub-
sequent hydrolysis under acidic conditions (Scheme 3).

Cysteine Derivatives for Carboxypeptidase A
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 913
Sch em e 4a
a
Reagents, conditions, and yield: (a) Benzyl bromide, K2CO3, DMF, room temperature, 8 h, 51%; (b) MeI, K2CO3, DMF, room temperature,
2 h, 84%; (c) PPh3, DEAD, CH3COSH, THF, 0 °C, 4 h, 36%; (d) 6 N HCl, reflux, 2 h, 90%.
Sch em e 5a
Ta ble 1. Inhibitory Potencies (Ki Values) of N-Alkylcysteines
for CPA Inhibition
compd no.
R
Ki (µM)a
L-Cys
D-Cys
1a
1b
1c
hydrogen
hydrogen
methyl
ethyl
propyl
isopropyl
isopropyl
isopropyl
isobutyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexylmethyl
350 ( 5b
2.3 ( 1.6b
2.4 ( 0.17
1.4 ( 0.19
5.6 ( 0.5
1d
1.1 ( 0.16
2.9 ( 0.23
0.56 ( 0.02
7.5 ( 0.42
9.6 ( 0.87
7.9 ( 0.63
2.9 ( 1.6
(R)-1d
(S)-1d
1e
1f
(R)-1f
(S)-1f
1g
a
Reagents, conditions, and yield: (a) LDA, isopropyl bromide,
HMPA-THF, 0 °C, 6 h, 36%; (b) aqueous KOH (4 equiv), reflux,
8 h, quantitative; (c) SOCl2, MeOH, reflux, 4 h, quantitative; (d)
PPh3, DEAD, CH3COSH, THF, 0 °C, 2 h, 70%; (e) 48% HBr, reflux,
2 h, 82%.
9.4 ( 0.2
a
b
Ki values are expressed as mean ( SEM (n ) 3). Ref 12.
the CPA inhibitory activity. Second, to explore the
stereochemistry associated with the inhibitions, repre-
sentative inhibitors (1d , if, 1i, and 1j) were prepared
in an optically active form and it was found that the
inhibitory activities of these compounds reside mostly
on compounds having the (S) configuration, that is,
inhibitors derived from D-Cys (Tables 1 and 2). Appar-
ently, inhibitors belonging to the D-series bear molecular
configurations more complementary to the active site
geometry of CPA. Third, it is noteworthy that the Ki
value of compound 1m with a 1-naphthylmethyl group
at the amino nitrogen is over 620-fold more potent as
As expected, the introduction of an aromatic ring at
the amino group of Cys augmented the binding affinity
of the inhibitors to CPA (Table 2). Several features are
apparent from the analysis of the data in Table 2. First,
the binding affinity increases as the number of meth-
ylene units of the linker that bridges between the phenyl
ring and the amino group in the inhibitors increases,
maximizing with an ethylene chain. The highest binding
affinity was observed with 1j that carries a phenethyl
group at the amino nitrogen of D-Cys. Figure 2 shows
the relationships between the length of the linker and
Sch em e 6a
a
Reagents, conditions, and yield: (a) 1-Bromo-4-phenylbutane, NaOEt, EtOH, room temperature, 8 h, 82%; (b) 4 N KOH, reflux, 4 h,
88%; (c) Et2NH‚HCl, HCHO, reflux, 12 h, 62 %; (d) CH3COSH, reflux, 4 h, 88%; (e) MeOCH2CH2NH2, room temperature, 1 h, 90%.

914 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Park and Kim
Ta ble 2. Inhibitory Potencies (Ki Values) of
N-Arylalkylcysteines for CPA Inhibition
Ta ble 3. Inhibitory Constants (Ki Values) for CPA Inhibition
compd no.
R
Ki (µM)a
compd no.
R
R1
Ki (µM)a
13
14
20
21
22
19
isopropyl
cyclohexyl
phenyl
benzyl
phenethyl
phenylpropyl
0.16 ( 0.01
2.35b
1h
1i
(R)-1i
(S)-1i
8
phenyl
benzyl
benzyl
benzyl
benzyl
H
H
H
H
Me
H
H
H
H
H
H
H
5.5 ( 0.71
0.27 ( 0.01
33.0 ( 1.0
0.010c
2.1b
1.35b
0.19 ( 0.01
12.0 ( 0.86
0.065 ( 0.0014
0.61 ( 0.058
0.055 ( 0.004
1.3 ( 0.12
1.3 ( 0.12
1j
phenethyl
phenethyl
phenethyl
phenylpropyl
(p-methoxy)benzyl
1-naphthylmethyl
2-naphthylmethyl
Ki values are expressed as mean ( SEM (n ) 3). b Ref 19. c Ref
a
(R)-1j
(S)-1j
1k
1l
1m
15.
molecule may rest in such a way that its positively
charged ammonium ion has maximum interactions with
the Glu-270 carboxylate anion. Such interactions would
be oriented closer to Glu-270 than the nonCys-based
sulfhydryl-type CPA inhibitors. The 20-fold increase in
the Ki value by the replacement of the amino group in
1j with a methylene (to obtain 19) may reflect that the
amino group may indeed participate in electrostatic
interactions with the carboxylate of Glu-270. As a result,
the benzene ring in 1h may not be fully accommodated
in the S1′ pocket. In order for the aromatic ring in the
Cys-based inhibitor to fit in the pocket fully and to have
maximum hydrophobic interactions, the inhibitor would
require a longer linker than an amino unit. Apparently,
a chain consisting of three atoms, including the amine
nitrogen, appears to be optimal, as shown by 1j. If the
chain is longer than the optimal length, the benzene
ring, upon inserting into the S1′ pocket, may experience
repulsive interactions with the surface of the pocket,
causing the inhibitor molecule to be displaced somewhat
from the position of maximum interactions with the
enzyme. The reduction of the binding affinity of 1k by
20-fold, as compared with that of 1j, may thus be
rationalized.
0.85 ( 0.074
0.81 ( 0.14
1n
>500
a
Ki values are expressed as mean ( SEM (n ) 3).
F igu r e 2. Relationships between the length of the linker that
bridges a phenyl ring and 3-mercaptopropanoic acid and the
pKi (-log Ki) in the inhibition of CPA.
compared with its isomer in which the naphthyl moiety
is linked at the 2-position. This result may be rational-
ized in terms of the narrow mouth of the S1′ pocket: the
naphthyl ring in 1n experiences a difficulty in entering
the pocket.17 Last, the substitution of the amino hydro-
gen in 1i with a methyl group decreased the binding
affinity by 44-fold, suggesting that the methyl group
may experience repulsive steric interactions with neigh-
boring amino acid residues at the active site of the
enzyme.
It is instructive to compare the CPA binding affinity
of the Cys-based inhibitors with those of inhibitors in
which the amino group of Cys is replaced with a
methylene unit. While the most potent compound in the
series of Cys-based inhibitors is 1j as described above,
in the series of 2-substituted 3-mercaptopropanoic acid,
the compound in which the benzene ring is linked to
the mercaptopropanoic acid by a methylene unit, i.e.,
20,15 is most active (Table 3 and Figure 2). The seem-
ingly perplexing SAR may be reconciled as follows: The
X-ray crystal structure of the CPA complex formed with
D-Cys revealed that in addition to the strong interaction
of the sulfhydryl group of the inhibitor with the zinc
ion at the active site, the positively charged ammonium
ion of the zwitterionic form of Cys interacts with the
negatively charged carboxylate of Glu-270.13 It has also
been shown that upon binding of D-Cys, the side chains
of Glu-270, Arg-145, and Arg-127 move toward the
ammonium ion of the bound D-Cys.13 It is not unreason-
able to assume that in the CPA‚D-Cys complex the Cys
Con clu sion
In summary, a series of Cys derivatives (15 racemic
and 8 optically active compounds) with an alkyl or
arylalkyl moiety on the R-amino group of Cys have been
synthesized as novel types of inhibitors for CPA. The
SAR study revealed that in cases of optically active
inhibitors, those prepared from D-Cys are much more
potent than the corresponding inhibitors obtained from
L-Cys, and the most potent inhibitor in this study is (S)-
1j, obtained by introducing a phenethyl moiety on the
amino group of D-Cys. The Ki value of 55 ( 4 nM
exhibited by (S)-1j corresponds to a 42-fold decrease as
compared with that of D-Cys, and this increase in
binding affinity (2.2 kcal mol-1) may be attributed to
the hydrophobic interactions between the phenyl ring
of the inhibitor and the S1′ pocket of CPA and the ionic
interactions between the amino group in (S)-1j and the
carboxylate of Glu-270. It appears therefore that in
addition to the three most important binding groups
(carboxylate, sulfhydryl, and aromatic) that participate
in binding interactions with the enzyme, the amino
group in these inhibitors also engages in binding in the
form of electrostatic interactions with the Glu-270
carboxylate. The CPA inhibitor design approach that
uses Cys as the starting point may be applied to other

Cysteine Derivatives for Carboxypeptidase A
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 915
(S)-N-Cycloh exylcystein e ((S)-1f). Compound (S)-1f was
prepared from (S)-Cys and cyclohexanone. [R]25D +26.2° (c 0.5,
H2O). mp and spectral data are identical with those of rac-1f.
(R)-N-Cycloh exylcystein e ((R)-1f). Compound (R)-1f was
prepared from (R)-Cys and cyclohexanone. [R]25D -28.4° (c 0.25,
H2O). mp and spectral data are identical with those of rac-1f.
r a c-N-(Cycloh exylm eth yl)cystein e (1g). Compound 1g
was prepared from rac-Cys and cyclohexylcarboxaldehyde.
zinc-containing proteases of medicinal interest such as
ACE and matrix metalloproteases. We are currently
exploring these applications.
Exp er im en ta l Section
Melting points were determined on a Thomas-Hoover capil-
lary melting point apparatus and were uncorrected. 1H nuclear
magnetic resonance (NMR) and 13C NMR spectra were re-
corded with a Bruker AM 300 (300 MHz) instrument using
tetramethylsilane as the internal standard. IR spectra were
recorded on a Bruker Equinox 55 Fourier transform infrared
spectrometer. Mass spectra were obtained by Korea Basic
Science Insititute. Silica gel 60 (230-400 mesh) was used for
flash chromatography, and thin-layer chromatography was
carried out on silica-coated glass sheets (Merck silica gel 60
F-254). Elemental analyses were performed at the Center for
Biofunctional Molecules, Pohang University of Science and
Technology, Pohang, Korea, and results were within (0.4%
of the theoretical value.
All solutions for kinetic study were prepared using doubly
distilled and deionized water. Stock assay solutions were
filtered (GHP Acrodic syringe filter, pore size 0.2 µm) before
use. CPA was purchased from Sigma Chemical Co. (Allan form,
twice crystallized from bovine pancreas, aqueous suspension
in toluene) and used without further purification. CPA stock
solutions were prepared by dissolving the enzyme in 0.05 M
Tris/0.5 M NaCl, pH 7.5, buffer solution. Hipp-L-Phe purchased
from Sigma Chemical Co. was used as substrate for CPA, and
the decrease in the absorbance at 254 nm was followed at 25
°C. An HP 8453 UV/vis spectrometer was used in enzyme
inhibition studies.
Yield, 26%; mp 216-218 °C (dec). IR (KBr): 1580, 2562 cm-1
.
1H NMR (D2O, 300 MHz): δ 0.79 (m, 5H), 1.33 (m, 6H), 2.59
(d, 2H), 2.80 (t, 2H), 3.94 (t, 1H). 13C NMR (D2O, 300 MHz):
δ 22.6, 25.0, 25.5, 30.0, 34.5, 52.6, 61.1, 168.2. Anal. (C10H19
NO2S‚1/4H2O) C, H, N.
-
r a c-N-Ben zylcystein e (1i). Compound 1i was prepared
from rac-Cys and benzaldehyde. Yield, 30%; mp 212-214 °C
(dec). IR (KBr): 1595, 2562 cm-1 1H NMR (D2O, 300 MHz):
.
δ 3.08 (ddd, 2H), 4.21 (t, 1H), 4.23 (s, 2H), 7.37 (s, 5H). 13C
NMR (D2O, 300 MHz): δ 23.2, 50.4, 60.4, 129.6, 130.1, 130.3,
130.5, 169.5. Anal. (C10H13NO2S‚HCl) C, H, N.
(S)-N-Ben zylcystein e ((S)-1i). Compound (S)-1i was pre-
pared from (S)-Cys and benzaldehyde. [R]25 +20.4° (c 0.5, 3
D
N HCl). mp and spectral data are identical with those of rac-
1i.
(R)-N-Ben zylcystein e ((R)-1i). Compound (R)-1i was pre-
pared from (R)-Cys and benzaldehyde. [R]25 -18.0° (c 0.4, 3
D
N HCl). mp and spectral data are identical with those of rac-
1i.
r a c-N-P h en eth ylcystein e (1j). Compound 1j was pre-
pared from rac-Cys and phenylacetaldehyde. Yield, 30%; mp
1
220-221 °C (dec). IR (KBr): 1600, 2567 cm-1. H NMR (D2O,
300 MHz): δ 2.96 (t, 2H), 3.03 (m, 2H), 3.27 (t, 2H), 4.22 (t,
1H), 7.24 (m, 5H). 13C NMR (D2O, 300 MHz): δ 22.9, 31.8,
47.8, 61.0, 127.7, 129.1, 129.4, 136.5, 169.4. Anal. (C11H15NO2S‚
1/4H2O) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of N-Alk ylcys-
tein e. To a suspension of Cys and NaBH3CN (1 equiv) in
methanol (10 mL) was added the appropriate aldehyde (1.5
equiv) or ketone (1.1 equiv) at room temperature and stirred
for 18-36 h. The white precipitate formed was collected and
washed with MeOH to give the product.
(S)-N-P h en eth ylcystein e ((S)-1j). Compound (S)-1j was
prepared from (S)-Cys and phenylacetaldehyde. [R]25 +23.2°
D
(c 0.25, 3 N HCl). mp and spectral data are identical with those
of rac-1j.
r a c-N-Eth ylcystein e (1b). Compound 1b was prepared
from rac-Cys and acetaldehyde. Yield, 32%; mp 216-217 °C
(R)-N-P h en eth ylcystein e ((R)-1j). Compound (R)-1j was
prepared from (R)-Cys and phenylacetaldehyde. [R]25D -22.7°
(c 0.25, 3 N HCl). mp and spectral data are identical with those
of rac-1j.
(dec). IR (KBr): 1596, 2587 cm-1 1H NMR (D2O, 300 MHz):
.
δ 1.39 (t, 3H) 3.15 (m, 2H) 3.18 (d, 2H) 3.96 (t, 1H). 13C NMR
(D2O, 300 MHz): δ 10.9, 23.9, 42.5, 62.7, 172.0. Anal. (C5H11
NO2S) C, H, N.
-
r a c-N-P h en ylp r op ylcystein e (1k ). Compound 1k was
prepared from rac-Cys and 3-phenylpropionaldehyde. Yield,
r a c-N-P r op ylcystein e (1c). Compound 1c was prepared
from rac-Cys and propionaldehyde. Yield, 37%; mp 229-230
43%; mp 205-206 °C (dec). IR (KBr): 1582, 2561 cm-1 1H
.
°C (dec). IR (KBr): 1553, 2596 cm-1 1H NMR (D2O, 300
.
NMR (D2O, 300 MHz): δ 1.69 (m, 2H), 2.35 (t, 2H), 2.76 (t,
2H), 2.78 (ddd, 2H), 3.93 (t, 1H), 6.92 (m, 5H). 13C NMR (D2O,
300 MHz): δ 22.7, 27.1, 31.9, 46.2, 60.7, 126.6, 128.6, 128.9,
140.7, 169.3. Anal. (C12H17NO2S‚1/4H2O) C, H, N.
MHz): δ 0.99 (t, 3H) 1.75 (m, 2H) 3.06 (m, 2H) 3.10 (ddd, 2H)
3.90 (t, 1H). 13C NMR (D2O, 300 MHz): δ 5.5, 10.6, 19.5, 23.9,
48.8, 63.0, 172.0. Anal. (C6H13NO2S‚2/3H2O) C, H, N.
r a c-N-(p-Meth oxy)ben zylcystein e (1l). Compound 1l was
prepared from rac-Cys and p-anisaldehyde. Yield, 47%; mp
r a c-N-Isop r op ylcystein e (1d ). Compound 1d was pre-
pared from rac-Cys and acetone. Yield, 51%; mp 228-230 °C
1
209-211 °C (dec). IR (KBr): 1596, 2568 cm-1. H NMR (D2O,
(dec). IR (KBr): 1585, 2576 cm-1 1H NMR (D2O, 300 MHz):
.
δ 1.34 (q, 6H), 3.16 (ddd, 2H), 3.54 (m, 2H), 4.35 (t, 1H). 13C
NMR (D2O, 300 MHz): δ 18.5, 18.6, 23.6, 51.0, 59.1, 170.3.
Anal. (C6H13NO2S‚HCl‚1/4H2O) C, H, N.
300 MHz): δ 2.87 (ddd, 2H), 3.51 (s, 3H), 4.00 (s, 2H), 4.01 (t,
1H), 6.71 (d, 2H), 7.13 (d, 2H). 13C NMR (D2O, 300 MHz): δ
23.1, 49.9, 55.6, 59.9, 114.8, 122.4, 132.1, 160.1, 169.3. Anal.
(C11H15NO2S‚1/4H2O) C, H, N.
(S)-N-Isop r op ylcystein e ((S)-1d ). Compound (S)-1d was
prepared from (S)-Cys and acetone. [R]25D -32.6° (c 0.5, H2O).
mp and spectral data are identical with those of rac-1d .
(R)-N-Isop r op ylcystein e ((R)-1d ). Compound (R)-1d was
prepared from (R)-Cys and acetone. [R]25D +35.4° (c 0.25, H2O).
mp and spectral data are identical with those of rac-1d .
r a c-N-Isobu tylcystein e (1e). Compound 1e was prepared
from rac-Cys and isobutyraldehyde. Yield, 52%; mp 208-209
r a c-N-(1-Na p h th ylm eth yl)cystein e (1m ). Compound 1m
was prepared from rac-Cys and 1-naphthaldehyde. Yield, 40%;
mp 204-205 °C (dec). IR (KBr): 1598, 2568 cm-1 1H NMR
.
(D2O, 300 MHz): δ 2.77 (d, 2H), 3.98 (t, 1H), 4.27 (s, 2H), 7.18
(m, 4H), 7.58 (m, 3H). 13C NMR (D2O, 300 MHz): δ 23.3, 47.6,
60.9, 122.7, 125.7, 125.9, 126.8, 127.7, 129.2, 130.5, 131.0,
131.2, 133.7, 169.3. Anal. (C14H15NO2S‚1/4H2O) C, H, N.
r a c-N-(2-Na p h th ylm eth yl)cystein e (1n ). Compound 1n
was prepared from rac-Cys and 2-naphthaldehyde. Yield, 45%;
°C (dec). IR (KBr): 1595, 2582 cm-1 1H NMR (D2O, 300
.
MHz): δ 1.03 (d, 6H), 2.07 (m, 1H), 2.95 (m, 2H), 3.10 (m, 2H),
3.89 (t, 1H). 13C NMR (D2O, 300 MHz): δ 19.4, 19.5, 23.8, 25.9,
54.3, 63.5, 171.8. Anal. (C7H15NO2S) C, H, N.
mp 177-179 °C (dec). IR (KBr): 1622, 2578 cm-1 1H NMR
.
(DMSO-d6, 300 MHz): δ 2.78 (m, 2H), 3.33 (t, 1H), 4.00 (q,
2H), 7.50 (m, 3H), 7.87 (m, 4H). 13C NMR (DMSO-d6, 300
MHz): δ 27.0, 51.4, 62.6, 126.3, 127.0, 127.7, 128.4, 128.5,
128.7, 133.2, 137.7, 173.2. Anal. (C14H15NO2S‚1/4H2O) C, H,
N.
r a c-N-Cycloh exylcystein e (1f). Compound 1f was pre-
pared from rac-Cys and cyclohexanone. Yield, 38%; mp 233-
1
235 °C (dec). IR (KBr): 1596, 2585 cm-1. H NMR (D2O, 300
MHz): δ 1.21 (m, 5H), 1.48 (d, 1H), 1.65 (d, 2H), 1.92 (t, 2H),
3.01 (m, 3H), 4.31 (t, 1H). 13C NMR (D2O, 300 MHz): δ 23.4,
24.1, 24.3, 24.6, 29.1, 54.6, 57.5, 58.3, 169.9. Anal. (C9H17NO2S‚
1/4H2O) C, H, N.
r a c-N-2,2-Dim eth yl-4-th ia zolid in eca r boxyla te Meth yl
Ester (2). A mixture of rac-Cys methyl ester hydrochloride
(3.4 g, 20 mmol) and acetone (50 mL) was refluxed for 8 h and

916 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Park and Kim
then evaporated under reduced pressure. The residue was
diluted with ethyl acetate, washed successively with water,
5% aqueous sodium bicarbonate solution (50 mL × 3), and
brine (50 mL × 3), and then dried over anhydrous MgSO4. The
solution was concentrated, and the crude product was purified
by column chromatography (EtOAc/n-hexane ) 1/8) to give 2
product as a pale yellow oil (2.5 g, 84%). IR (CHCl3): 1732,
3446 cm-1 1H NMR (CDCl3, 300 MHz): δ 2.34 (s, 3H), 3.55
.
(q, 1H), 3.76 (m, 2H), 3.78 (s, 3H), 3.82 (dd, 2H), 7.31 (m, 5H).
13C NMR (CDCl3, 300 MHz): δ 37.8, 51.8, 59.3, 59.6, 66.2,
127.8, 128.9, 129.3, 138.9, 171.6.
r a c-N-Ben zyl-N-m eth yl-S-a cetylcystein e Meth yl Ester
(7). To a solution of triphenylphosphine (1.0 g, 4 mmol) in
tetrahydrofuran (THF, 15 mL) was slowly added diethyl
azodicarboxylate (0.63 mL, 4 mmol) at 0 °C under a nitrogen
atmosphere, and the resulting mixture was stirred for 30 min
at 0 °C. To the reaction mixture was added slowly a mixture
of 6 (0.44 g, 2 mmol) and thioacetic acid (0.3 mL, 4 mmol) by
a cannular and stirred for 4 h at room temperature. The
reaction mixture was diluted with ethyl acetate and washed
with a saturated NaHCO3 solution (10 mL × 3). After the
organic layer was collected and acidified with 3 N HCl, the
aqueous layer was washed with ethyl acetate (10 mL × 3),
then basified with 2 N NaOH, and extracted with ethyl acetate
(10 mL × 3). The combined extracts were dried over anhydrous
MgSO4 and concentrated in vacuo, and the crude product was
purified by column chromatography (EtOAc/n-hexane ) 1/4)
as a pale yellow oil (3.4 g, 96%). IR (CHCl3): 1744 cm-1 1H
.
NMR (CDCl3, 300 MHz): δ 1.52 (d, 3H), 1.70 (d, 3H), 2.51 (s,
1H), 3.24 (m, 2H), 3.79 (s, 3H), 4.08 (s, 1H). 13C NMR (CDCl3,
300 MHz): δ 30.7, 30.9, 33.3, 53.4, 64.4, 65.1, 172.4.
r a c-N -Me t h yl-2,2-d im e t h yl-4-t h ia zolid in e ca r b oxyl-
a te Meth yl Ester (3). A mixture of 2 (1.2 g, 6.9 mmol),
potassium carbonate (0.95 g, 6.9 mmol), and methyl iodide (0.5
mL, 8.3 mmol) in dimethylformamide (DMF, 30 mL) was
stirred at room temperature for 4 h. The residue was diluted
with ethyl acetate, washed successively with water, 5% sodium
thiosulfate (50 mL × 3), and brine (50 mL × 3), and dried over
anhydrous MgSO4. The solution was evaporated in vacuo, and
the crude product was purified by column chromatography
(EtOAc/n-hexane ) 1/4) to give 3 as a pale yellow oil (0.58 g,
1
45%). IR (CHCl3): 1747 cm-1. H NMR (CDCl3, 300 MHz): δ
to give 7 as an oil (0.2 g, 36%). IR (CHCl3): 2244, 3446 cm-1
.
1.37 (s, 3H), 1.46 (s, 3H), 2.24 (s, 3H), 3.10 (m, 2H), 3.70 (s,
3H), 3.72 (t, 3H). 13C NMR (CDCl3, 300 MHz): δ 27.3, 29.4,
32.3, 34.6, 52.8, 70.0, 75.1, 172.5.
1H NMR (CDCl3, 300 MHz): δ 0.92 (d, 6H), 1.45 (m, 2H), 1.79
(m, 1H), 2.78 (m, 1H), 3.69 (d, 2H). 13C NMR (CDCl3, 300
MHz): δ 21.9, 23.3, 26.5, 33.7, 37.5, 63.3, 121.6.
r a c-N-Meth ylcystein e Hyd r och lor id e (1a ). A mixture of
3 (0.50 g, 2.6 mmol) and 6 N HCl (10 mL) was refluxed for 8
h and then evaporated under reduced pressure. The residue
was recrystallized from ether and ethanol to give a white
powder (0.40 g, 88%): mp 127-129 °C (literature 127 °C).18
1H NMR (D2O, 300 MHz): δ 2.59 (s, 3H), 3.07 (m, 2H), 4.15 (t,
1H). 13C NMR (D2O, 300 MHz): δ 22.8, 31.7, 62.3, 170.0.
4-Car boxy-2-m eth yl-3-ph en yl-∆2-th iazolin iu m Br om ide
(4). A mixture of thioacetamide (1.0 g, 6.6 mmol) and R-bro-
moacrylic acid (1.1 g, 7.3 mmol) in dry toluene (20 mL) was
heated at 90 °C for 1 h. The reaction mixture was cooled to
room temperature, and then, the precipitate was collected on
a filter and washed with acetone (30 mL × 3), and the filter
cake was recrystallized from MeOH/EtOAc/hexane (1:1:2) to
give a white solid (1.8 g, 90%): mp 216-218 °C (literature14
215-216 °C). IR (KBr): 1735 cm-1 (literature14 1722 cm-1).
1H NMR (TFA, 300 MHz): δ 2.45 (s, 3H), 4.21 (ddd, 2H), 5.75
(dd, 1H), 7.36 (s, 5H).
r a c-N-Ben zyl-N-m eth ylcystein e (8). A mixture of 7 (0.07
g, 0.25 mmol) and concentrated HCl (5 mL) was refluxed for
2 h and then evaporated under reduced pressure. The residue
was recrystallized from ether and ethanol to give a white
powder (0.66 g, 90%): mp 68-70 °C. IR (KBr): 1739, 2585
1
cm-1. H NMR (D2O, 300 MHz): δ 2.71 (s, 3H), 3.05 (m, 2H),
4.06 (t, 1H), 4.67 (s, 2H), 7.33 (m, 5H). 13C NMR (D2O, 300
MHz): δ 21.3, 37.8, 59.5, 67.5, 128.7, 129.7, 130.8, 131.7, 169.5.
Anal. (C11H15NO2S‚1/2H2O) C, H, N.
r a c-2-(Hyd r oxym eth yl)-4-m eth ylva ler on itr ile (9). To a
solution of diisopropylamine (5.6 mL, 40 mmol) in THF (100
mL) under a nitrogen atmosphere at 0 °C was added a n-BuLi
(2.5 M in hexane, 16 mL, 40 mmol) by a microsyringe. The
mixture was stirred for 30 min at that temperature and cooled
to -78 °C. To the chilled and stirring solution of LDA was
added slowly a solution of 3-hydroxypropanitrile (1.4 mL, 20
mmol) in 30 mL of THF, and the stirring was continued for
an additional 1 h at -78 °C. Isobutyl bromide (2.2 mL, 20
mmol) was added to the reaction mixture at -78 °C, and the
resulting mixture was warmed to 0 °C slowly and stirred at
the temperature for 12 h. The reaction was quenched by the
addition of 6 N HCl solution (30 mL). The organic layer was
separated, and the aqueous layer was extracted with ethyl
acetate (10 mL × 3). The combined organic layers were washed
with 5% Na2S2O3 (10 mL × 3) and brine (10 mL × 3), dried
under anhydrous MgSO4, and then evaporated in vacuo to
dryness. The crude product was purified by column chroma-
tography (EtOAc/n-hexane ) 1/1) to give 9 as an oil (0.91 g,
r a c-N-P h en ylcystein e Hyd r obr om id e (1h ). A mixture
of 4 (1.0 g, 3.3 mmol) and 1.5 N HBr solution (20 mL) was
refluxed for 3 h and then evaporated under reduced pressure.
The residue was dissolved in 48% HBr solution (10 mL) and
evaporated in vacuo to give a brown solid. The solid was
recrystallized from n-PrOH and benzene to give the product
as a white solid (0.83 g, 90%): mp 173-175 °C (literature14
1
173-174 °C). IR (KBr): 1739, 2543 cm-1. H NMR (TFA, 300
MHz): δ 3.22 (dd, 2H), 6.87 (t, 1H), 7.51 (m, 5H).
r a c-N-Ben zylser in e Meth yl Ester (5). A mixture of rac-
serine methylester (4.7 g, 30 mmol), potassium carbonate (8.3
g, 60 mmol), and benzyl bromide (4.0 mL, 33 mmol) in DMF
(100 mL) was stirred at room temperature for 8 h. The residue
was diluted with ethyl acetate, washed successively with
water, 5% aqueous sodium thiosulfate solution (50 mL × 3),
and brine (50 mL × 3), and then dried over anhydrous MgSO4.
The solution was concentrated, and the crude product was
purified by column chromatography (EtOAc/n-hexane ) 1/2)
to give the product as a pale yellow oil (3.2 g, 51%). IR
(CHCl3): 1736, 3321 cm-1. 1H NMR (CDCl3, 300 MHz): δ 3.43
(t, 1H), 3.69 (m, 2H), 3.75 (dd, 2H), 7.33 (m, 5H). 13C NMR
(CDCl3, 300 MHz): δ 52.5, 52.6, 62.3, 62.9, 127.7, 128.7, 128.9,
139.6, 173.9.
r a c-N-Ben zyl-N-m eth ylser in e Meth yl Ester (6). A mix-
ture of 5 (3.2 g, 15.3 mmol), potassium carbonate (4.2 g, 30.6
mmol), and methyl iodide (1.9 mL, 30.6 mmol) in DMF (100
mL) was stirred at room temperature for 2 h. The residue was
diluted with ethyl acetate, washed successively with water,
5% aqueous sodium thiosulfate solution (50 mL × 3), and brine
(50 mL × 3), and then dried over anhydrous MgSO4. The
solution was concentrated, and the crude product was purified
by column chromatography (EtOAc/n-hexane ) 1/8) to give the
36%). IR (CHCl3): 2244, 3446 cm-1 1H NMR (CDCl3, 300
.
MHz): δ 0.92 (d, 6H), 1.45 (m, 2H), 1.79 (m, 1H), 2.78 (m, 1H),
3.69 (d, 2H). 13C NMR (CDCl3, 300 MHz): δ 21.9, 23.3, 26.5,
33.7, 37.5, 63.3, 121.6.
r a c-2-(Hyd r oxym eth yl)-4-m eth ylp en ta n oic Acid (10).
A mixture of 9 (1.3 g, 10 mmol) and 4 N KOH (10 mL, 40 mmol)
was refluxed for 8 h. The reaction mixture was cooled to room
temperature and was acidified with 3 N HCl. The product was
extracted with ethyl acetate (10 mL × 3). The combined
extracts were dried over anhydrous MgSO4 and evaporated
on the reduced pressure to give 10 as an oil (1.39 g, 95%). IR
(CHCl3): 1714, 3346 cm-1. 1H NMR (CDCl3, 300 MHz): δ 0.90
(m, 6H), 1.31 (m, 1H), 1.62 (m, 2H), 2.69 (m, 1H), 3.74 (d, 2H).
13C NMR (CDCl3, 300 MHz): δ 22.7, 22.9, 26.2, 37.6, 46.0, 63.7,
181.2.
r a c-2-(Hyd r oxym eth yl)-4-m eth ylp en ta n oic Acid Meth -
yl Ester (11). To a solution of 10 (0.90 g, 6.2 mmol) in MeOH
(20 mL) was added slowly thionyl chloride (2.3 mL, 31.0 mmol)
at 0 °C, and the resulting mixture was refluxed for 4 h. The
reaction mixture was evaporated in vacuo, diluted with ethyl
acetate, washed with 5% aqueous NaHCO3 solution (10 mL ×

Cysteine Derivatives for Carboxypeptidase A
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 917
cm-1 1H NMR (CDCl3, 300 MHz): δ 1.59 (m, 2H), 1.68 (m,
2H), 2.36 (m, 2H), 2.68 (m, 2H), 5.65 (s, 1H), 6.31 (s, 1H), 7.27
(m, 5H). 13C NMR (CDCl3, 300 MHz): δ 28.4, 31.4, 31.7, 36.1,
126.1, 127.5, 128.7, 128.8, 140.5, 142.9, 173.3.
3) and brine (10 mL×3), and then dried over anhydrous
MgSO4. The solution was concentrated, and the crude product
was purified by column chromatography (EtOAc/n-hexane )
1/10) to give the product as an oil (0.99 g, quantitative). IR
(CHCl3): 1696, 1738 cm-1. 1H NMR (CDCl3, 300 MHz): δ 0.92
(d, 6H), 1.36 (m, 1H), 1.61 (m, 2H), 2.34 (s, 3H), 2.68 (m, 1H),
3.05 (m, 2H), 3.70 (s, 3H). 13C NMR (CDCl3, 300 MHz): δ 22.6,
23.0, 26.4, 31.0, 31.2, 41.7, 44.3, 52.2, 175.5, 195.7.
.
r a c-r-(Acetylth iom eth yl)-6-p h en ylh exa n oic Acid (18).
A mixture of 17 (0.38 g, 1.9 mmol) and thioacetic acid (0.2 mL,
5.7 mmol) in benzene (10 mL) was refluxed for 12 h. The
solution was concentrated under reduced pressure, diluted
with ethyl acetate, and washed with water (50 mL × 3). The
organic phase was dried over anhydrous MgSO4 and concen-
trated under reduced pressure to give 18 as a pale yellow oil
r a c-2-(Acetylth iom eth yl)-4-m eth ylp eta n oic Acid Meth -
yl Ester (12). To a solution of triphenylphosphine (1.0 g, 4
mmol) in THF (15 mL) was added slowly diethyl azodicar-
boxylate (0.63 mL, 4 mmol) at 0 °C under a nitrogen atmo-
sphere and stirred for 30 min at 0 °C. To the resulting mixture
was added slowly a mixture of 11 (0.44 g, 2 mmol) and
thioacetic acid (0.3 mL, 4 mmol) by a cannular and stirred for
4 h at room temperature. The reaction mixture was diluted
with ethyl acetate and washed with saturated NaHCO3
solution (10 mL × 3), then dried over anhydrous MgSO4, and
concentrated in vacuo, and the crude product was purified by
column chromatography (EtOAc/n-hexane ) 1/4) to give 12 as
(0.47 g, 88%). IR (CHCl3): 1698 cm-1 1H NMR (CDCl3, 300
.
MHz): δ 1.43 (m, 2H), 1.66 (m, 2H), 1.71 (m, 2H), 2.32 (s, 3H),
2.61 (m, 2H), 2.62 (m, 1H), 3.07 (m, 2H), 7.21 (m. 5H). 13C NMR
(CDCl3, 300 MHz): δ 27.0, 30.5, 31.0, 31.6, 32.0, 36.0, 46.0,
126.1, 128.7, 128.8, 142.7, 180.7, 195.9.
r a c-2-(Mer ca p tom eth yl)-6-p h en ylh exa n oic Acid (19).
A mixture of 18 (0.10 g, 0.4 mmol) and 2-methoxyethylamine
(5 mL) was stirred at room temperature for 1 h. The resulting
mixture was acidified with 3 N HCl (10 mL) and extracted
with ethyl acetate (10 mL × 3). The organic layer was dried
over MgSO4 and evaporated in vacuo to give 18 as an oil (0.09
an oil (0.2 g, 36%). IR (CHCl3): 2244, 3446 cm-1 1H NMR
.
(CDCl3, 300 MHz): δ 0.92 (d, 6H), 1.45 (m, 2H), 1.79 (m, 1H),
2.78 (m, 1H), 3.69 (d, 2H). 13C NMR (CDCl3, 300 MHz): δ 21.9,
23.3, 26.5, 33.7, 37.5, 63.3, 121.6.
1
g, 90%). IR (CHCl3): 1705, 2565 cm-1. H NMR (CDCl3, 300
MHz): δ 1.41 (m, 2H), 1.61 (m, 4H), 2.60 (t, 2H), 2.82 (m, 2H),
2.98 (m, 1H). 13C NMR (CDCl3, 300 MHz): δ 27.0, 31.6, 31.7,
36.0, 40.5, 45.6, 126.2, 128.7, 128.8, 142.7, 181.2. HRMS
(FAB+) (M + H)+: calcd for C11H16NO2S, 239.1106; found,
239.1108.
r a c-2-(Mer ca p tom eth yl)-4-m eth ylp en ta n oic Acid (13).
A mixture of 12 (0.11 g, 0.5 mmol) and 48% HBr (5 mL) was
refluxed for 2 h. To the reaction mixture was added deoxy-
genated water (10 mL) and was extracted with ethyl acetate
(10 mL × 3). The organic layer was dried over MgSO4 and
evaporated in vacuo to give 13 as an oil (0.08 g, 92%). IR
(CHCl3): 1708, 2574 cm-1. 1H NMR (CDCl3, 300 MHz): δ 0.93
(d, 6H), 1.44 (m, 2H), 1.63 (m, 2H), 2.11 (m, 3H), 2.71 (m, 2H),
2.95 (m, 1H). 13C NMR (CDCl3, 300 MHz): δ 22.6, 23.1, 26.3,
26.5, 40.9, 47.8, 181.5. High-resolution mass spectroscopy
(HRMS) (fast atom bombardment (FAB+)) (M + H)+: calcd
for C11H15NO2S, 162.0715; found, 162.0714.
Deter m in a tion of Ki Va lu e. Typically, the enzyme stock
solution was added to a solution containing Hipp-L-Phe (final
concentrations, 150 and 300 µM) and inhibitor (five different
final concentrations in the range of 0.023-1.0 µM) in 0.05 M
Tris/0.5 M NaCl, pH 7.5, buffer (1 mL cuvette), and the change
in absorbance at 254 nm was measured immediately. The final
concentration of CPA was 68.5 nM. Initial velocities were then
calculated from the linear initial slopes of the change in
absorbance where the amount of substrate consumed was less
than 10%. The Ki values were then estimated from the
semireciprocal plot of the initial velocity vs the concentration
of the inhibitors according to the method of Dixon.16 The
correlation coefficients for the Dixon plots were above 0.992.
r a c-2-(Mer ca p tom eth yl)-3-cycloh exylp r op a n oic Acid
(14). Compound 14 was prepared as described in the litera-
ture.19
r a c-2-(P h en ylbu tyl)p r op a n ed ioic Acid Dieth yl Ester
(15). To absolute EtOH (100 mL) at 0 °C was added sodium
metal (1.03 g, 44.8 mmol). To the solution were added diethyl
malonate (6.8 mL, 44.8 mmol) and 1-bromo-4-phenylbutane
(6.4 g, 30 mmol). After the mixture was stirred at room
temperature for 8 h, the solution was concentrated, acidified
with 3 N HCl, and extracted with ether. The combined extracts
were dried over anhydrous MgSO4 and evaporated to give a
crude product, which was purified by column chromatography
(EtOAc/n-hexane ) 1/4) to give 15 as a pale yellow oil (7.2 g,
Ack n ow led gm en t. This work was supported by the
Brain Korea 21 Project.
Refer en ces
(1) Lipscomb, N. W.; Stra¨ter, N. Recent Advances in Zinc Enzymol-
ogy. Chem. Rev. 1996, 96, 2375-2433.
(2) Christianson, D. W.; Lipscomb, N. W. Carboxypeptidase A. Acc.
Chem. Res. 1989, 22, 62-69.
1
82%). IR (CHCl3): 1733 cm-1. H NMR (CDCl3, 300 MHz): δ
(3) Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A. J . H. Design
and Therapeutic Application of Matrix Metalloproteinase Inhibi-
tors. Chem. Rev. 1999, 99, 2735-2776.
1.24 (t, 6H), 1.36 (m, 2H), 1.64 (m, 2H), 1.88 (m, 2H), 2.60 (t,
2H), 3.30 (t, 1H), 4.17 (q, 4H), 7.16 (m, 5H). 13C NMR (CDCl3,
300 MHz): δ 14.5, 27.3, 29.0, 31.4, 36.0, 52.4, 61.7, 126.1,
128.7, 128.8, 142.7, 169.9.
(4) Rees, D. C.; Lewis, M.; Lipscomb, W. N. Refined Crystal
Structure of Carboxypeptidase A at 1.54 Å Resolution. J . Mol.
Biol. 1983, 168, 367-387.
r a c-(2-P h en ylbu tyl)p r op a n ed ioic Acid (16). A mixture
of 15 (2.9 g, 10.0 mmol) and 4 N KOH solution (8.7 mL) was
refluxed for 2 h. The solution was diluted with water, washed
with ether (50 mL × 3), acidified with 3 N HCl, and extracted
with ether (50 mL × 3). The organic phase was dried over
anhydrous MgSO4 and concentrated under the reduced pres-
sure to give 16 as a white solid (2.08 g, 88%): mp 93-94 °C.
(5) (a) Ondetti, M. A.; Rubin, B.; Cushman, D. W. Design of Specific
Inhibitors of Angiotensin-Converting Enzyme: New Class of
Orally Active Antihypertensive Agents. Science 1977, 196, 441-
444. (b) Cushman, D. W.; Cheung, H. S.; Sabo, E. F.; Ondetti,
M. A. Design of Potent Competitive Inhibitors of Angiotensin-
Converting Enzyme. Carboxyalkanoyl and Mercaptoalkanoyl
Amino Acids. Biochemistry 1977, 16, 5484-5491.
(6) Byers, L. D.; Wolfenden, R. Binding of the By-Product Analogue
Benzylsuccinic Acid by Carboxypeptidase A. Biochemistry 1973,
12, 2070-2078.
IR (CHCl3): 1716 cm-1 1H NMR (CDCl3, 300 MHz): δ 1.44
.
(m, 2H), 1.65 (m, 2H), 1.98 (m, 2H), 2.62 (m, 2H), 3.43 (t, 1H),
7.23 (m, 5H). 13C NMR (CDCl3, 300 MHz): δ 27.2, 28.9, 31.3,
35.9, 52.0, 126.2, 128.7, 128.8, 142.5, 175.5.
(7) Horovitz, Z. P., Ed. Angiotensin Converting Enzyme Inhibitors.
Mechanisms of Action and Clinical Implications; Urbans &
Schwarzenberg: Baltimore-Munich, 1981.
r a c-r-Meth ylen e-6-p h en ylh exa n oic Acid (17). To a mix-
ture of 16 (1.8 g, 7.6 mmol) and diethylamine hydrochloride
(1.0 g, 9.1 mmol) was added 37% aqueous formaldehyde
solution (1.1 mL, 15.1 mmol) at room temperature, and the
resulting mixture was refluxed for 12 h. The solution was
washed with ether (50 mL × 3), acidified with 3 N HCl, and
extracted with ether (50 mL × 3). The organic phase was dried
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