2-Benzyl-2-methylsuccinic acid as inhibitor for carboxypeptidase A. synthesis and evaluation

Article abstract of DOI:10.1016/S0968-0896(99)00110-8

Recently, Asante-Appiah et al. (Asante-Appiah, E.; Seetharaman, J.; Sicheri, F.; Yang, D. S.-C.; Chan, W. W.-C. Biochemistry 1997, 36, 8710-8715) reported that 2-ethyl-2-methylsuccinic acid is a highly potent inhibitor for carboxypeptidase A (CPA), a prototypic zinc protease. The X-ray crystal structure of the complex of the enzyme formed with 2-ethyl-2-methylsuccinic acid revealed that at the active site of CPA there is present a small cavity which accommodates the methyl group of the inhibitor. These investigators postulated that incorporation of a methyl group at the α-position to the carboxylate of existing inhibitors of CPA would improve the inhibitory potency. We have synthesized racemic and optically active 2-benzyl-2-methylsuccinic acids and evaluated their inhibitory activities for CPA to find the K(i) values to be 0.28, 0.15, and 17μM for racemic form, (R)-, and (S)-enantiomer, respectively. Contrary to the expectation, the effect on the binding affinity by the incorporation of the methyl group is minimal. The validity of the proposition that the small cavity may be utilized for the improvement of the inhibitory potency appears questionable. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00110-8

Bioorganic & Medicinal Chemistry 7 (1999) 1755±1760
2-Benzyl-2-methylsuccinic Acid as Inhibitor for
Carboxypeptidase A. Synthesis and Evaluation
Mijoon Lee, Yonghao Jin and Dong H. Kim*
Center for Biofunctional Molecules and Department of Chemistry, Pohang University of Science and Technology, San 31 Hyoja-dong,
Pohang 790-784, South Korea
Received 22 February 1999; accepted 19 April 1999
AbstractÐRecently, Asante-Appiah et al. (Asante-Appiah, E.; Seetharaman, J.; Sicheri, F.; Yang, D. S.-C.; Chan, W. W.-C. Biochem-
istry 1997, 36, 8710±8715) reported that 2-ethyl-2-methylsuccinic acid is a highly potent inhibitor for carboxypeptidase A (CPA), a
prototypic zinc protease. The X-ray crystal structure of the complex of the enzyme formed with 2-ethyl-2-methylsuccinic acid
revealed that at the active site of CPA there is present a small cavity which accommodates the methyl group of the inhibitor. These
investigators postulated that incorporation of a methyl group at the a-position to the carboxylate of existing inhibitors of CPA
would improve the inhibitory potency. We have synthesized racemic and optically active 2-benzyl-2-methylsuccinic acids and eval-
uated their inhibitory activities for CPA to ®nd the Ki values to be 0.28, 0.15, and 17 mM for racemic form, (R)-, and (S)-enantio-
mer, respectively. Contrary to the expectation, the e€ect on the binding anity by the incorporation of the methyl group is
minimal. The validity of the proposition that the small cavity may be utilized for the improvement of the inhibitory potency appears
questionable. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
2-ethylsuccinic acid, and their evaluation as inhibitors
for CPA in an e€ort to probe the proposition that the
methyl hole may be utilized to obtain inhibitors of
enhanced potency in designing inhibitors for CPA.
Carboxypeptidase A (CPA), a well studied zinc pro-
tease, selectively cleaves o€ the C-terminal amino acid
residue having a hydrophobic side chain from protein
substrate.1 The enzyme has been served as a model in
devising design strategies for inhibitors that are e€ective
for zinc containing proteases (for example, refs 2±5).
Recently, Asante-Appiah et al. reported that 2-ethyl-2-
methylsuccinic acid (1) is a potent inhibitor for carboxy-
peptidase A (CPA).6 The X-ray crystal structure of the
complex of CPA formed with (R)-1 revealed that the
methyl group at the 2-position of the inhibitor occupies
a small cavity of hydrophobic nature. They proposed to
call the cavity a `methyl hole' and attributed the high
inhibitory potency of 1 to the additional interactions
that result from the a-methyl group in 1 occupying the
cavity.6 These investigators invited evaluation of 2-ben-
zyl-2-methylsuccinic acid (2) that resembles more closely
optimal substrates by having a benzyl side chain at the
a-position, expecting it would be an inhibitor of con-
siderably improved potency over 1. This paper reports
syntheses of racemic and optically active 2 as well as
Results and Discussion
Racemic 2 was prepared in three steps starting with
commercially available 2-benzylpropanoic acid. The
latter was converted into the corresponding tert-butyl
ester. The enolate that was generated by the treatment
of the tert-butyl ester with LDA was allowed to react
with tert-butyl bromoacetate to a€ord 2-benzyl-2-
methylsuccinic acid di-tert-butyl ester which upon
treatment with tri¯uoroacetic acid gave 2 in an overall
yield of 47% (Scheme 1).
Key words: Zinc proteases; carboxypeptidase A; enzyme inhibitors;
inhibitor design.
* Corresponding author. Tel.: +82-562-279-2101; fax: +82-562-279-
5877; e-mail: dhkim@vision.postech.ac.kr
(R)-Benzylmethylpropanedioic acid monomethyl ester
(6) that was obtained by enzymatic hydrolysis of di-
methyl benzylmethylmalonate using a-chymotrypsin
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00110-8

1756
M. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1755±1760
Scheme 1. (a) Isobutylene, H2SO4, CH2Cl2, rt, 3 d, 90%; (b) LDA (1.2
equiv), THF, 0ꢀC; butyl bromoactate (2.0 equiv), 0ꢀC to rt, 4 h, 65%;
(c) CF3CO2H, rt, 1 h, 80%.
according to the literature method7 was regioselectively
reduced to give 7 following the method of Martinez et
al.8 The hydroxyl group in 7 was converted into iodo by
the method of Garegg and Samuelsson9 with a minor
modi®cation to give 8 which was then transformed to 9
by treatment with KCN. Hydrolysis of 9 under alkaline
conditions a€orded (R)-2 in an overall yield of 47%
(Scheme 2).
Scheme 3. (a) Isobutyl chloroformate (1 equiv), NMM (1 equiv),
DME, 15ꢀC, 15 min; NH4OH (5 equiv), 15ꢀC to rt, 2 h, 64%; (b)
LiOH, THF/MeOH/H2O, rt, 12 h, 95%; (c) isobutyl chloroformate (1
equiv), NMM (1 equiv), DME, 15ꢀC; NaBH4 (3 equiv), 15ꢀC to rt,
1 h, 70%; (d) MeOH, HCl, re¯ux, 24 h, 80%; (e) PPh3 (1.2 equiv), I2
(1.2 equiv), imidazole (1.2 equiv), toluene, 80ꢀC, 18 h, 89%; (f) KCN,
18-crown-6, DMSO, 80ꢀC, 10h, 90%; (g) 20% NaOH, 90ꢀC, 24h, 90%.
The synthesis of (S)-2 is outlined in Scheme 3. The
carboxylate of 6 was converted into carboxamide to
give 10 which upon treatment with lithium hydroxide in
a solution of THF±MeOH±water a€orded 11. The car-
boxylate in 11 was reduced to hydroxymethyl and the
carboxamide was converted into methoxycarbonyl fol-
lowing the method reported in the literature10 to give
13. The hydroxyl in 13 was converted into carboxylate
to give (S)-2 in three steps in an analogous manner that
was used for the synthesis of (R)-2. The overall yield for
this synthesis amounted to 25%.
than its enantiomer. This stereochemistry which is
consistent with the stereospeci®city of the enzyme
shown for peptide substrates is in agreement with the
observation of Asante-Appiah et al. who found that the
stereochemistry of the CPA-bound 1 bears the R-con-
®guration.6 It was expected that 2 which resembles more
closely the substrate than 1 of Asante-Appiah et al. by
having a benzyl group instead of the ethyl at the 2-
position would be much more potent inhibitor for CPA
than 1.6 Contrary to the expectation, however, 2 exhib-
ited the Ki value of 0.28 mM,13 which corresponds to the
potency improvement of 2.0-fold.14 This may re¯ect
that the binding force involved in the interaction of the
2-Ethylsuccinic acid (18) was synthesized as outlined
in Scheme 4, starting with 2-(triphenylphosphoranyl-
idene)succinic acid 1-methyl ester (16) which was pre-
pared as described in the literature.11 Treatment of 16
with acetaldehyde followed by alkaline hydrolysis
a€orded 17 which upon hydrogenation in the presence
of palladium-charcoal gave the target compound (18).
0
aromatic ring of 2 with S1 pocket (primary substrate
recognition pocket) is minimal, if at all. The not so sig-
ni®cant binding interaction between the aromatic ring
and the pocket is inconsistent with the view that the
0
The synthesized compounds were assayed for their
inhibitory activities for CPA. As expected, they are
found to be competitive inhibitors for CPA as shown by
the Dixon plot (Fig. 1).12 The inhibitory constants of
these compounds were estimated from the respective
Dixon plot12 and are listed in Table 1. The (R)-form of
2, which corresponds to the stereochemistry of the l-
series of a-amino acids is bound 113-fold more tightly
primary role of S1 pocket is simply to provide an
0
anchoring space for the aromatic side chain of P1 resi-
due of substrate, and the anchored ring is held by the
side chain of Tyr-248.1,15 It has been well established
that Tyr-248 moves downward to the mouth of the pri-
mary substrate recognition pocket upon substrates and
inhibitors bind the enzyme, and holds physically the
ligands in place at the active site.1,14
2-Benzylsuccinic acid (BSA) is a prototypic CPA inhi-
bitor designed by Byers and Wolfenden.16 The X-ray
crystal structure of the CPA complex formed with BSA
has been reported.17 It is instructive to compare the Ki
Scheme 2. (a) Isobutyl chloroformate (1 equiv), NMM (1 equiv),
DME, 15ꢀC, 15 min; NaBH4 (3 equiv), 15ꢀC, 10 min, 65%; (b)
PPh3 (1.2 equiv), I2 (1.2 equiv), imidazole (1.2 equiv), toluene, 80ꢀC,
18 h, 90%; (c) KCN, 18-crown-6, DMSO, 80ꢀC, 10 h, 90%; (d) 20%
NaOH, 90ꢀC, 24 h, 90%.
Scheme 4. (a) PPh3, acetone, rt, 83%; (b) MeOH, rt, 24 h, 75%; (c) (i)
CH3CHO, benzene, rt, 24 h; (ii) 1 N NaOH, rt, 24 h, 80%; (d) Pd/C,
MeOH, rt, 3 h, 95%.

M. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1755±1760
1757
basis of the distinctive binding mode of 1 to CPA as
revealed by the X-ray structural analysis.6 The X-ray
crystal structure of the enzyme complex formed with
inhibitor 1 is noticeably di€erent from the crystal struc-
ture of complex formed with BSA: in the case of CPA-1
complex the aromatic side chain of Tyr-248 is appeared
as if it is pushed outward by the ethyl group. Further-
more, it is noteworthy that whereas the carboxylate at
the 1-position of BSA forms a hydrogen bond with the
phenolic hydrogen of Tyr-248, the carboxylate of 1
hydrogen bonds to Arg-127 instead, in addition to the
hydrogen bondings that both carboxylates form with
Arg-145 and Asn-144. This di€erence in the binding
mode between the two molecules, i.e. 1 is being pushed
outward compared with BSA in the complex formation
appears to be arisen as a result of the less than satisfac-
tory ®tting of the methyl group in the small cavity. That
is the methyl hole is most probably not suciently large
or deep enough to accommodate a methyl group snug-
gly. It is expected that the binding of 2 which carries a
bulkier benzyl group than the ethyl in 1 would push the
Tyr-248 side chain further outward. This would result in
diminished binding interactions, and as a consequence
no signi®cant improvement is e€ected in the binding of
2 to CPA compared with that for binding of 1. An
alternative interpretation may be o€ered:8 The methyl
hole is suciently large so that the methyl group may be
readily accommodated, but by 0doing so the ring would
not be ®tted in snugly in the S1 pocket. As a result, the
optimum contribution of the phenyl ring to the overall
binding anity in the formation of the enzyme complex
would not be achieved.
Figure 1. The Dixon plot for the inhibition of CPA with racemic 2-
benzyl-2-methylsuccinic acid (substrate, Hipp-l-Phe; Tris bu€er of pH
7.5; temperature, 25ꢀC).
Table 1. Inhibitory constants determined for CPA
Inhibitor
Ki (mM)
(R,S)-2-Ethylsuccinic acid (18)
(R,S)-2-Ethyl-2-methylsuccinic acid (1)
(R,S)-2-Benzylsuccinic acid
(R)-2-Benzylsuccinic acid
(S)-2-Benzylsuccinin acid
(R,S)-2-Benzyl-2-methylsuccinic acid (2)
(R)-2-Benzyl-2-methylsuccinic acid
(S)-2-Benzyl-2-methylsuccinic acid
25
0.11a
0.55b (0.2c, 1.1d)
0.27b (0.45d)
3d
0.28
0.15
17
a
ref 6.
These are values obtained under the same kinetic conditions as those
b
used for compound 2 and ethylsuccinic acid (18).
c
ref 22.
ref 14.
d
The role of the small cavity in the enzymic machinery is
not apparent presently, but it is highly unlikely that the
small cavity is designed to accommodate any speci®c
group in binding of substrate. However, it is con-
ceivable, as Asante-Appiah et al. postulated,6 that the
cavity serves as a space for the protonated carboxylate
of0Glu-270 to traverse to the leaving amino group of the
P1 residue transferring its acidic proton to the amino
value of 2 for the CPA inhibition with that of 0.55 mM
obtained for BSA because the di€erence in the inhibi-
tory constants re¯ects the e€ect that brings about on the
binding anity as a result of replacing the proton at the
2-position of BSA with a methyl group. The introduc-
tion of a methyl group, however, brought an improve-
ment of the binding anity only by 2.0-fold. In the case
of (R)-2-benzylsuccinic acid the improvement by the
introduction of a methyl group is 1.8-fold. On the basis
of the binding modes of BSA and 1 to CPA established
by the X-ray crystal structure analyses,6,17 it is expected
that the methyl group of 2 would be positioned at the
locus where the a-hydrogen of BSA lies, namely the
methyl hole, although it may not ®t in perfectly.
0
group of the departing P1 residue in the catalytic pro-
cess. In a proposed reaction path of the enzymic cata-
lytic reaction of CPA,1 it has been postulated that a
transient intermediate is generated by the nucleophilic
attack of an activated water molecule on the carbonyl
carbon of the scissile peptide bond of substrates. The
subsequent collapse of the transition state to the pro-
ducts is thought to be facilitated by the protonation of
the amino group by the protonated carboxylate of Glu-
270. The proton delivery from the carboxylate to the
departing amino group of the tetrahedral transition
state in the catalytic process may be achieved in the
small cavity.
In order to examine the e€ect of the methyl group in 1
on the binding anity, we have synthesized 2-ethylsuc-
cinic acid (18) and evaluated its inhibitory activity for
CPA. The Ki value of 25 mM which we obtained with 18
re¯ects that the introduction of a methyl group at the 2-
position of the ethylsuccinic acid improves the binding
anity by 227-fold.
Conclusion
Considering the minor increase of the binding anity
resulted by the introduction of a methyl group on the 2-
benzylsuccinic acid as described above, the 227-fold
improvement is remarkable. This dramatic increase in
the binding anity may, however, be explained on the
Asante-Appiah et al. invited people to investigate zinc
protease inhibitors which bear a gem-dialkyl structure,
postulating that an introduction of a small alkyl group
preferably of a methyl onto known inhibitors at an
appropriate position (the a-position next to the terminal

1758
M. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1755±1760
carboxylate in the case of inhibitors for CPA) would
result in an improvement of the potency.6 The postula-
tion of Asante-Appiah et al. was made on the basis of
their unexpected observation that 1 shows high binding
anity for CPA and the X-ray crystal structure of CPA-
1 complex which reveals the presence of a small cavity
that accommodates the methyl group at the active site
of CPA. However, the present study suggests that the
high binding anity exhibited by 1 is an exceptional
occurrence unique to the compound rather than a
general phenomenon. Accordingly, the validity of the
proposition of Asante-Appiah et al. that the small
cavity at the active site of CPA may be utilized for
designing CPA inhibitors of improved potency appears
questionable.
layer was separated and the aqueous layer was washed
with ethyl acetate (3Â10 mL). The combined organic
layers were washed with brine, dried under anhydrous
MgSO4 and evaporated in vacuo to dryness. The crude
product thus obtained was puri®ed by chromatography
(silica gel) to give the title compound (5) as an oil
1
(1.48 g, 65%). IR (neat) 1725 cm 1; EI-MS m/z 334; H
NMR 300 MHz (CDCl3) d 1.18 (s, 3H), 1.41±1.45 (m,
18H), 2.26±2.56 (dd, 2H), 2.87±2.97 (m, 2H), 7.12±7.2
(m, 5H); 13C NMR 300 MHz (CDCl3) d 22.69, 28.33,
28.42, 43.02, 44.42, 45.59, 126.91, 128.36, 131.01,
137.64, 171.12, 175.54.
2-Benzyl-2-methylsuccinic acid (2). Compound 5 (1.40 g,
4.2 mmol) dissolved in tri¯uoroacetic acid (3.2 mL,
42 mmol) was stirred at room temperature for 1 h. Eva-
poration of the excess tri¯uoroacetic acid yielded 2
(0.74 g, 80%) which was recrystallized from diethyl
ether and hexane. Mp=144±146ꢀC (lit.19 117±119ꢀC);
Experimental
1
Melting points were taken on a Thomas±Hoover capil-
1
IR (neat) 3300, 1652 cm 1; EI-MS m/z 222; H NMR
lary melting point apparatus and were uncorrected. H
300 MHz (CDCl3) d 1.24 (s. 3H), 2.37±2.69 (dd, 2H),
2.98 (s, 2H), 7.16±7.28 (m, 5H); 13C NMR 300 MHz
(CDCl3) d 27.09, 46.64, 49.12, 49.76, 131.35, 132.80,
135.32, 142.26, 178.60, 183.48; Anal. calcd for
C12H14O4: C, 64.85; H, 6.35. Found: C, 64.47; H, 6.47.
NMR and 13C NMR spectra were recorded on a Bruker
AM 300 (300 MHz) instrument using tetramethylsilane
as the internal standard. IR spectra were recorded on a
Bruker Equinox 55 FT-IR spectrometer. Mass spectra
were obtained with a Micro Mass Platform II 8410E
spectrometer. Silica gel 60 (230±400 mesh) was used for
¯ash chromatography and thin layer chromatography
(TLC) was carried out on silica coated glass sheets
(Merck silica gel 60 F±254). Elemental analyses were
performed at the Center of Biofunctional Molecules,
Pohang University of Science and Technology, Pohang,
Korea.
(R)-Methyl 2-benzyl-2-methyl-3-hydroxypropanate (7).
To a cold ( 15ꢀC) solution of (R)-2-benzyl-2-methyl-
malonic acid monomethyl ester (6)7 (2.22 g, 10 mmol)
dissolved in 1,2-dimethoxyethane (DME, 10 mL), were
added successively N-methyl morpholine (1.1 mL,
10 mmol) and isobutyl chloroformate (1.3 mL,
10 mmol). The resulting mixture was stirred in an ice-
salt bath for 15 min. To the chilled stirring mixture was
added at once a solution of sodium borohydride
(1140 mg, 30 mmol) dissolved in water (10 mL), whereby
a strong evolution of gas was observed. The mixture
was stirred for 1 h. The reaction was quenched with 0.5
N HCl (100 mL), then was extracted with diethyl ether
(3Â30 mL). The combined extracts were washed with
brine and dried under anhydrous MgSO4. After eva-
poration of the solvent, the residue was puri®ed by silica
gel column chromatography to give a colorless oil
(1.67 g, 86%). [a]d=+16.3ꢀ (c 2.1, MeOH); IR (neat)
tert-Butyl (‹)-ꢀ-methylhydrocinnamate (4). To a solu-
tion of (‹)-a-methylhydrocinnamate (3) (1.64 g,
10 mmol) in dichloromethane (20 mL), concentrated
sulfuric acid (0.5 mL) was added and the mixture was
saturated with isobutylene at 78ꢀC. This resulted in an
increase of the volume to about 30 mL. The ¯ask was
stoppered and kept at room temperature for three days.
After the addition of a solution of sodium carbonate
(1 g) in water (15 mL), the two layers were separated and
the organic phase was washed with water (3Â10 mL),
brine and dried under anhydrous MgSO4. Evaporation
of the solvent under reduced pressure yielded the
desired product (4) as an oil (1.98 g, 90%). IR (neat)
1
3418, 1725 cm 1; EI-MS m/z 190 (M±H2O); H NMR
300 MHz (CDCl3) d 1.15 (s, 3H), 2.89±3.01 (dd, 2H),
3.58±3.59 (d, 2H), 3.73 (s, 3H), 7.15±7.33 (m, 5H); 13C
NMR 300 MHz (CDCl3) d 19.66, 41.41, 49.31, 52.26,
67.48, 127.01, 128.52, 130.64, 137.08, 177.53.
1
1720 cm 1; H NMR 300 MHz (CDCl3) d 1.10±1.12 (d,
3H), 1.36 (s, 9H), 2.59±2.66 (m, 2H), 2.92±3.01 (m, 1H),
7.16±7.30 (m, 5H); 13C NMR 300 MHz (CDCl3) d
17.38, 28.39, 40.27, 42.69, 80.43, 126.30, 126.56, 128.62,
129.46, 175.89.
(S)-Methyl 2-benzyl-2-methyl-3-iodopropanate (8). Tri-
phenylphosphine (2.27 g, 8.6 mmol) was added to
iodine (2.19 g, 8.6 mmol) solution dissolved in toluene
(1.50 mL) and the mixture was heated at 60ꢀC for 0.5 h.
To the resulting solution was added 7 (1.50 g, 7.2 mmol)
dissolved in toluene (20 mL) followed by the addition of
imidazole (0.59 g, 8.6 mmol). The mixture was then stir-
red at 60ꢀC for 18 h. After completion of the reaction,
the mixture was diluted with ethyl acetate, washed with
water, sodium thiosulfate (10%), and brine, and dried
over anhydrous MgSO4. After evaporation of the
solvent, the residue was puri®ed by silica gel column
chromatography to give a colorless oil (2.06 g, 90%).
Di-tert-butyl 2-benzyl-2-methylsuccinate (5). Compound
4 (1.50 g, 6.8 mmol) dissolved in THF (10 mL) was
added dropwise to a solution of lithium diisopropyl-
amide (8.2 mmol) at 0ꢀC. The mixture was stirred for
2 h, then warmed slowly to room temperature. tert-
Butyl bromoacetate (2.0 mL, 13.7 mmol) was added
dropwise to the reaction mixture at 0ꢀC and the resul-
tant mixture was stirred at room temperature for 4 h.
The reaction was quenched by the addition of saturated
solution of ammonium chloride (25 mL). The organic

M. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1755±1760
1759
[a]d= 0.76ꢀ (c 0.8, MeOH); IR (neat) 1734 cm 1; EI-
MS m/z 318; 1H NMR 300 MHz (CDCl3) d 1.29 (s, 3H),
2.92±3.06 (dd, 2H), 3.22±3.46 (dd, 2H), 3.71 (s, 3H),
7.13±7.30 (m, 5H); 13C NMR 300 MHz (CDCl3) d
15.43, 23.52, 44.03, 48.51, 52.58, 127.46, 128.69, 130.34,
136.86, 174.35.
under reduced pressure, the residue was acidi®ed with
2 N HCl (10 mL) and extracted with ethyl acetate
(3Â5 mL). The combined extracts were washed with
brine and dried over anhydrous MgSO4. Evaporation of
the solvent gave 11 (1.23 g, 95%) which was recrys-
tallized from diethyl ether and hexane. Mp=120±
121ꢀC; [a]d= 4.4ꢀ (c 0.5, MeOH); IR (KBr) 3412,
1
(R)-Methyl 2-benzyl-2-methyl-3-cyanopropanate (9). The
mixture of 8 (2.00 g, 6.3 mmol), potassium cyanide
(0.49 g, 7.5 mmol), and catalytic amount of 18-crown-6
in DMSO (5 mL) was stirred at 80ꢀC for 10 h. After
completion of the reaction, the mixture was diluted with
ethyl acetate (50 mL), washed with water (5Â10 mL)
and brine and dried over anhydrous MgSO4. Evapora-
1715, 1677 cm 1; EI-MS m/z 207; H NMR 300 MHz
(CDCl3) d 1.36 (s, 3H), 3.06±3.27 (dd, 2H), 6.22 (br s,
1H), 7.14±7.50 (m, 6H); 13C NMR 300 MHz (CDCl3) d
21.70, 43.98, 54.85, 127.20, 128.49, 130.34, 137.10,
175.49, 176.46.
(S)-2-Benzyl-2-methyl-3-hydroxypropamide (12). The
synthesis was carried out as described for 7. Compound
12 (white solid, 0.70 g) was obtained in 70% yield from
11 (1.00 g, 4.9 mmol). Mp=88±89ꢀC; [a]d= 21.0ꢀ (c
0.5, MeOH); IR (KBr) 3358, 1655 cm 1; EI-MS m/z
tion of the solvent yielded 9 as an oil (1.30 g, 95%).
1
[a]d=+3.30ꢀ (c 0.5, MeOH); IR (neat) 2247, 1734 cm
;
1
EI-MS m/z 217; H NMR 300 MHz (CDCl3) d 1.45 (s,
3H), 2.46±2.62 (dd, 2H), 2.91±3.17 (dd, 2H), 3.75 (s,
3H), 7.12±7.34 (m, 5H); 13C NMR 300 MHz (CDCl3) d
23.75, 25.56, 44.13, 46.37, 52.85, 118.19, 127.78, 129.02,
130.10, 136.03, 174.78.
1
193; H NMR 300 MHz (CDCl3) d 1.08 (s, 3H), 2.86±
3.01 (dd, 2H), 3.07 (br s, 1H), 3.54±3.63 (dd, 2H), 5.46
(br s, 1H), 5.93 (r s, 1H), 7.19±7.31 (m, 5H); 13C NMR
300 MHz (CDCl3) d 19.80, 42.03, 48.20, 68.44, 126.30,
127.10, 128.60, 130.78, 137.12.
(R)-2-Benzyl-2-methylsuccinic acid ((R)-2). Compound 9
(1.00 g, 4.6 mmol) dissolved in NaOH solution (20%,
5 mL) was stirred at 90ꢀC for 24 h. After acidi®ng with
2 N HCl, the mixture was extracted with ethyl acetate
(3Â10 mL). The combined extracts were washed with
brine and dried over anhydrous MgSO4. Evaporation of
the solvent yielded (R)-2 (0.86 g, 95%), which was
recrystallized from diethyl ether and hexane. Mp=159±
160ꢀC; [a]d=+0.7ꢀ (c 5.0, dioxane); IR (neat) 3300,
Methyl (S)-2-benzyl-2-methyl 3-hydroxypropanate (13).
To dry methanol (10 mL) saturated with gaseous HCl,
12 (0.70 g, 3.4 mmol) was added and re¯uxed for 24 h.
The solution is poured into ice water (20 mL). A satu-
rated aqueous solution of sodium bicarbonate is added
until the mixture is faintly alkaline. The mixture was
extracted with diethyl ether (3Â20 mL), washed with
brine and dried over anhydrous MgSO4. After eva-
poration of the solvent, the residue was puri®ed by silica
gel column chromatography to give an oily product
(0.56 g, 80%). [a]d= 16.3ꢀ (c 0.5, MeOH); IR (neat)
1
1652 cm 1; H NMR 300 MHz (CDCl3) d 1.22 (s. 3H),
2.34±2.66 (dd, 2H), 2.96 (s, 2H), 7.15±7.28 (m, 5H); 13C
NMR 300 MHz (CDCl3) d 27.15, 46.73, 49.17, 49.75,
131.65, 133.11, 135.59, 142.39, 178.28, 183.05.
1
3418, 1725 cm 1; H NMR 300 MHz (CDCl3) d 1.15 (s,
(R)-Methyl 2-benzyl-2-methylmalonamate (10). To a
cold ( 15ꢀC) solution of 66 (2.22 g, 10 mmol) dissolved
in 1,2-dimethoxyethane (DME, 10 mL), were added
successively N-methyl morpholine (1.1 mL, 10 mmol)
and isobutyl chloroformate (1.3 mL, 10 mmol). The
resulting mixture was stirred in an ice-salt bath for
15 min. To the chilled stirring mixture was added at
once NH4OH solution (30%, 3.5 mL, 30 mmol). The
mixture was stirred for 2 h. The reaction mixture was
diluted with diethyl ether (30 mL), the organic layer was
collected and was washed with brine and dried over
anhydrous MgSO4. After evaporation of the solvent,
the residue was recrystallized from diethyl ether to a
give white solid product (1.50 g, 64%). Mp=104±
106ꢀC; [a]d=+9.8ꢀ (c 0.5, CHCl3); IR (neat) 3358,
3H), 2.89±3.01 (dd, 2H), 3.58±3.59 (d, 2H), 3.73 (s, 3H),
7.15±7.33 (m, 5H); 13C NMR 300 MHz (CDCl3) d
19.66, 41.41, 49.31, 52.26, 67.48, 127.01, 128.52, 130.64,
137.08, 177.53.
Methyl (R)-2-benzyl-2-methyl-3-iodopropanate (14). The
synthesis was carried out as described for 8, using 13
(0.50 g, 2.4 mmol) to give 14 (oil, 0.68 g, 89%).
[a]d=+0.76ꢀ (c 0.5, MeOH); IR (neat) 1734 cm 1; H
1
NMR 300 MHz (CDCl3) d 1.29 (s, 3H), 2.92±3.06 (dd,
2H), 3.22±3.46 (dd, 2H), 3.71 (s, 3H), 7.13±7.30 (m, 5H);
13C NMR 300 MHz (CDCl3) d 15.43, 23.52, 44.03,
48.51, 52.58, 127.46, 128.69, 130.34, 136.86, 174.35.
Methyl (S)-2-benzyl-2-methyl-3-cyanopropanate (15).
The synthesis was carried out as described for 9, using
14 (0.60 g, 1.9 mmol) to give 15 (oil, 0.39 g, 95%).
1
1732, 1673 cm 1; EI-MS m/z 221; H NMR 300 MHz
(CDCl3) d 1.43 (s, 3H), 3.08±3.37 (dd, 2H), 3.73 (s, 3H),
5.59 (br s, 1H). 6.87 (br s, 1H), 7.11±7.29 (m, 5H); 13C
NMR 300 MHz (CDCl3) d 21.19, 44.02, 53.00, 55.48,
127.43, 128.67, 130.27, 136.78, 173.89, 175.05.
[a]d= 3.30ꢀ (c 0.5, MeOH); IR (neat) 2247, 1734 cm
;
1
1H NMR 300 MHz (CDCl3) d 1.41 (s, 3H), 2.42±2.59
(dd, 2H), 2.87±3.12 (dd, 2H), 3.71 (s, 3H), 7.09±7.31 (m,
5H); 13C NMR 300 MHz (CDCl3) d 23.69, 25.57, 44.15,
46.35, 52.82, 118.18, 127.76, 129.00, 130.10, 136.04,
174.74.
(R)-2-Benzyl-2-methylmalonic acid monoamide (11). An
.
aqueous lithium hydroxide solution (0.324 g of LiOH H2O
in 3 mL of water, 7.7 mmol) was added to a solution of
10 (1.50 g, 6.4 mmol) dissolved in THF:MeOH (3:1,
12 mL). The reaction mixture was stirred at room tem-
perature for 12 h. After evaporation of the solvent
(S)-2-Benzyl-2-methylsuccinic acid ((S)-2). The synth-
esis was carried out as described for (R)-2 from 15
(0.30 g, 1.4 mmol) in 90% yield (white solid, 0.28 g).

1760
M. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1755±1760
Mp=152±154ꢀC; [a]d= 0.7ꢀ (c 5.0, dioxane); IR (KBr)
inhibitors according to the method of Dixon.12 Two
concentrations of the substrate of Hipp-l-Phe were
used. Typically, enzyme stock solution was added to
various concentrations of inhibitors in 0.05 M Tris/0.5
M NaCl, pH 7.5 bu€er (1 mL cuvette), and the initial
rates were measured immediately.
1
cm 1; H NMR 300 MHz (CDCl3) d 1.27 (s, 3H), 2.39±
2.71 (dd. 2H), 3.02 (s, 2H), 7.20±7.32 (m, 5H); 13C
NMR 300 MHz (CDCl3) d 27.19, 46.79, 49.21, 49.77,
131.65, 133.37, 135.60, 142.41, 180.90, 184.86.
2-Ethylidene succinic acid (17). Acetaldehyde (7.0 mL,
125 mmol) was added to a benzene solution (10 mL)
containing 2-(triphenylphosphoranylidene)succinic acid
1-methyl ester (16)11 (4.92 g, 12.5 mmol). After stirring
for 24 h, the mixture was washed with water, and the
ethylidene compound was extracted with a 1 N NaOH
solution (30 mL). The alkaline solution was stirred at
room temperature for 24 h, washed with diethyl ether,
acidi®ed with 4 N HCl solution, and extracted with
ethyl acetate (3Â30 mL). The collected organic layers
were washed with water, brine and dried over anhy-
drous MgSO4. The solvent was concentrated in vacuo.
The residue was recrystallized from ethyl acetate and
hexane to give a white powder (1.44 g, 80%). Mp=166±
168ꢀC (lit.20 mp 165±167ꢀC); 1H NMR (300 MHz) d
1.82±1.84 (d. 3H), 3.31 (s, 2H), 7.03±7.06 (q, 1H); 13C
NMR (300 MHz) d 19.72, 36.97, 132,42, 145.16, 145.61,
174.18, 178.10.
Acknowledgements
The authors gratefully acknowledge the Korea Science
and Engineering Foundation for the generous support
of this work.
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1
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General remarks for kinetic experiments
All solutions were prepared by dissolving in doubly
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®ltered before use. Carboxypeptidse A was purchased
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from bovine pancrease, aqueous suspension in toluene)
and used without further puri®cation. CPA stock solu-
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phenylalanine (Hipp-l-Phe) purchased from Sigma
chemical Co. was used as substrate for CPA and the
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25ꢀC. A Perkin±Elmer HP 8453 UV±vis spectrometer
was used in enzyme inhibition studies.
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Determination of Ki
Initial velocities were 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 versus the concentration of the