Synthesis and evaluation of α,α -disubstituted-3-mercaptopropanoic acids as inhibitors for carboxypeptidase A and implications with respect to enzyme inhibitor design

Article abstract of DOI:10.1016/j.bmc.2003.08.017

2-Ethyl-2-methyl-3-mercaptopropanoic acid (6) and 2-benzyl-2-methyl-3-mercaptopropanoic acid (7) were synthesized and evaluated as inhibitors for carboxypeptidase A (CPA), a prototypical zinc protease with the expectation that the binding affinities of these inhibitors would be augmented over those of 2-ethyl-3-methylsuccinic acid (2) and 2-benzyl-3-methylsuccinic acid (3), respectively, in light of the fact that the sulfhydryl group is a better zinc coordinating moiety than the carboxylate group. Contrary to the expectation, however, the inhibitory potency of 6 was not improved and that of 7 was rather attenuated by the replacement. A probable explanation for the unexpected results is offered.

Full text of DOI:10.1016/j.bmc.2003.08.017

Bioorganic & Medicinal Chemistry 11 (2003) 4685–4691
Synthesis and Evaluation of ꢀ,ꢀ-Disubstituted-3-
mercaptopropanoic Acids as Inhibitors for Carboxypeptidase A
and Implications with Respect to Enzyme Inhibitor Design
Hyun Soo Lee and Dong H. Kim*
Center for Integrated Molecular Systems, and Division of Molecular and Life Sciences,
Pohang University of Science and Technology, San 31 Hyojadong, Pohang 790-784, South Korea
Received 2 May 2003; accepted 15 August 2003
Abstract—2-Ethyl-2-methyl-3-mercaptopropanoic acid (6) and 2-benzyl-2-methyl-3-mercaptopropanoic acid (7) were synthesized
and evaluated as inhibitors for carboxypeptidase A (CPA), a prototypical zinc protease with the expectation that the binding affi-
nities of these inhibitors would be augmented over those of 2-ethyl-3-methylsuccinic acid (2) and 2-benzyl-3-methylsuccinic acid (3),
respectively, in light of the fact that the sulfhydryl group is a better zinc coordinating moiety than the carboxylate group. Contrary
to the expectation, however, the inhibitory potency of 6 was not improved and that of 7 was rather attenuated by the replacement.
A probable explanation for the unexpected results is offered.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
CPA.8 The X-ray crystal structure of the CPA complex
formed with l-2-benzylsuccinic acid revealed that the
inhibitor binds CPA with its carboxylate at the b-posi-
tion coordinating the zinc ion at the enzyme active site
in an asymmetric bidentate fashion.9 The other carbox-
ylate forms a salt link with the guanidinium moiety of
Arg-145 and also engages in hydrogen bonding with the
phenolic hydroxyl of Tyr-248 and the amide group of
Asn-144. The side chain aromatic ring of 1 is accom-
modated in the S10 pocket of CPA. Recently, Asante-
Appiah et al.10 reported that 2-ethyl-2-methylsuccinic
acid (2) is a highly potent inhibitor for CPA. The X-ray
Zinc proteases that are characterized by having a cata-
lytically essential zinc ion at their active site constitute a
major family of proteolytic enzymes.1 Physiologically
and etiologically important enzymes such as angiotensin
converting enzyme2 and matrix metalloproteases3
belong to this family, and thus zinc proteases have
received much attention as target enzymes for drug
development. Captopril4 and enalapril5 that are widely
prescribed for the treatment of hypertension, congestive
heart failure and myocardial infarction are inhibitors of
angiotensin converting enzyme.2 Of numerous zinc pro-
teases, carboxypeptidase A (CPA)6 is most extensively
studied and has served as a model target enzyme for the
development of inhibitor design strategies7 that can be
applied to zinc proteases of medicinal interest. The enzyme
catalyzes the hydrolysis of the C-terminal amide bond of
peptide substrate and shows specificity for oligopeptide
having the C-terminal residue with a hydrophobic side
chain such as Phe.6
It has been known that 2-benzylsuccinic acid (1) and
closely related compounds are effective inhibitors for
*Corresponding author. Tel.: +82-54-279-2101; fax: +82-54-279-
5877; e-mail:
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.08.017

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H. S. Lee, D. H. Kim / Bioorg. Med. Chem. 11 (2003) 4685–4691
Results and Discussion
crystal structure of the complex of CPA formed with
(R)-2 showed that the methyl group of the inhibitor
rests in a small hydrophobic cavity present next to the
S10 pocket. They have proposed to call the cavity a
‘methyl hole’ and attributed the unexpectedly high
inhibitory potency of 2 to the additional binding inter-
actions resulted from its 2-methyl group occupying the
cavity.10 We have evaluated 2-benzyl-2-methylsuccinic
acid (3) as a CPA inhibitor, expecting it to be more
potent than 2, but found that the compound is slightly
less potent than 2.11
Compound 5 was prepared according to the synthetic
route described in the literature12 starting with diethyl-
malonate as outlined in Scheme 1.
Scheme 2 represents the synthetic route for the prep-
aration of 6. Treatment of 12 with sodium hydride fol-
lowed by addition of ethyl iodide afforded 13 in an
excellent yield. The latter was then reduced with lithium
aluminumhydride to obtain 14. When 14 was allowed to
react with trimethyl orthoacetate in the presence of p-
toluenesulfonic acid yielded 15 which was oxidized with
the Jones reagent to give 16. The latter was converted
into 17 by treatment with hydrobromic acid under
reflux. The treatment of 17 with potassium thioacetate
afforded 18, and subsequent treatment of 18 with 6 N
hydrochloric acid yielded 6. By an analogous route,
compound 7 was synthesized.
Sulfhydryl group has been extensively utilized as a zinc
ligating functionality in designing inhibitors for zinc
proteases including CPA. 2-Benzyl-3-mercaptopropa-
noic acid (4) is a CPA inhibitor whose inhibitory
potency exceeds that of 1 by at least 50-fold.12 The
enhanced CPA inhibitory activity of 4 compared with 1
is attributable to the thiol group that forms a stronger
coordinative bond to the active site zinc ion than car-
boxylate does.13 It was, therefore, thought to be of
interest to evaluate 2-ethyl-2-methyl-3-mercaptopropa-
noic acid (6) and 2-benzyl-2-methyl-3-mercaptopropa-
noic acid (7) as inhibitors for CPA. It was expected that
the replacement of the carboxylate at the 3-positon of 2
and 3 with a thiol group to give 6 and 7, respectively,
would improve the inhibitory potency due to the
increased ligating affinity of the sulfhydryl to the active
site zinc ion. This paper reports syntheses of 2-ethyl-3-
mercaptopropanoic acid (5) and 6 as well as racemic
and optically active 7, and their evaluation as inhibitors
for CPA.
Synthesis of optically active 7 is illustrated in Scheme 3.
Compound 19, an intermediate in the synthesis of 7 was
treated with 2 N sodium hydroxide solution in methanol
to give 20 which was then allowed to react with (S)-
phenylethylamine in the presence of 1-ethyl-3-(3-dime-
thylaminopropyl)-carbodiimide hydrochloride (EDCI)
to obtain 21 and its diastereoisomer as an 1:1 diaster-
eoisomeric mixture. The mixture was readily separated
by flash column chromatography to give optically pure
21, the absolute stereochemistry of which was estab-
lished by the X-ray crystallographic method.14 The dia-
stereoisomer 21 thus separated was treated with
concentrated hydrobromic acid under reflux to give (S)-
22, which was then converted into (S)-7 by the same
route as that was used for the conversion of 17 into 7.
(R)-7 was similarly prepared from the diastereoisomer
of 21.
The synthesized compounds were assayed as competi-
tive inhibitors for CPA by a standard method using
hippuryl-l-phenylalanine (Hipp-l-Phe) as substrate at
pH 7.5. Inhibitory constants (Ki values) were estimated
from the respective Dixon plot11,15 and are collected in
Table 1. It can be seen from Table 1 that the substitu-
tion of 3-carboxylate in 2 with a sulfhydryl group failed
Scheme 1. (a) 4 N KOH, reflux, 4 h, 94%; (b) HCHO, Et2NH HCl, H2O, reflux, 4 h, 64%; (c) CH3COSH, benzene, reflux, 12 h, 82%; (d) HCl,
reflux, 4 h, 97%.

H. S. Lee, D. H. Kim / Bioorg. Med. Chem. 11 (2003) 4685–4691
4687
Scheme 2. (a) NaH, EtI, DMF, reflux, 2 h, 98%; (b) LAH, diethyl ether, 4 h, 96%; (c) CH3C(OCH3)3, p-TsOH, CH2Cl2, 1 h, 91%; (d) Jones
reagent, acetone, 2 h, 71%; (e) HBr, reflux, 1 day, 85%; (f) KSAc, Et3N, THF, 3 h, 90%; (g) 6 N HCl, reflux, 4 h, 95%.
Scheme 3. (a) 2 N NaOH, MeOH, reflux, 4 h, 97%; (b) (S)-phenylethylamine, EDCI, HOBT, Et3N, CH2Cl2, 1 h, 97%, then chromatographic
separation; (c) concd HBr, reflux, 1 day, 78%; (d) KSAc, Et3N, THF, 2 h, 90%; (e) 6 N HCl, 4 h, 95%.
to improve the binding affinity. The replacement of 3-
carboxylate in 3 with a sulfhydryl group to obtain 7 did
not enhance the binding affinity but rather attenuated
the affinity slightly. However, when there is present no
methyl group at the 2-positon, surprisingly the replace-
ment of the zinc liganting carboxylate with a sulfhydryl
moiety improved the binding affinity drastically: the
binding affinity of 5 corresponds to 250-fold that of 2-
ethylsuccinic acid.11 These observations indicate that
the methyl group in 6 and 7 effects adversely in binding
of the inhibitors to CPA, which may be reconciled with
the assumption that due to the tight binding of the
sulfhydryl group to the active site zinc ion these inhi-
bitor molecules reside closer towards the zinc ion than
molecules of 2 and 3 in forming complexes with CPA,
and as a consequence the 2-methyl group in these inhi-
bitors cannot fit in the methyl hole, bringing about
lowering of the binding affinity of these inhibitors. In
this conjunction, the lack of stereochemical preference
shown by the enantiomeric pair of 7 in inhibiting CPA
is noteworthy and may be accounted for by invoking a
proposition that the methyl group in both the enantio-
mers find a room for its resting by pointing towards
bulk water. On the other hand, in cases of 2 and 3, due
to the relatively weak ligating propensity of the carbox-
ylate to the zinc ion, these molecules may have con-
siderable freedom of movement in forming complexes
with the enzyme, and as a result, their methyl group
would fit in the small methyl hole, reinforcing the
binding interactions of the inhibitors to CPA. Espe-
cially, in the binding of 2 to CPA, since the S10 pocket
is occupied by a methyl group of the 2-ethyl chain,10
there remains in the pocket a sufficient room for the
inhibitor molecule to maneuver to form an enzy-
Table 1. Ki values for CPA inhibition
Inhibitor
Ki (mM)
(RS)-1
(RS)-2
(RS)-3
(RS)-4
(RS)-5
(RS)-6
(RS)-7
(R)-7
0.5511 (0.2)24
0.1110
0.2810
0.01112
0.10ꢀ0.019
0.11ꢀ0.017
0.38ꢀ0.030
0.43ꢀ0.073
0.37ꢀ0.059
(R)-7

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H. S. Lee, D. H. Kim / Bioorg. Med. Chem. 11 (2003) 4685–4691
!
me [currency]inhibitor complex with its 2-methyl group
being accommodated in the methyl hole.
2-Ethylacrylic acid (10). To a mixture of 9 (1.00 g, 7.6
mmol) and diethylamine hydrochloride (1.24 g, 11.4
mmol) in water was added 37% aqueous formaldehyde
solution (1.2 mL, 15.2 mmol) and the resulting mixture
was refluxed for 12 h. The solution was extracted with
ether, and the organic phase was dried over MgSO4 and
concentrated under reduced pressure to give 1018 as an
oil (0.48 g, 64%). 1H NMR (CDCl3, 300 MHz) d 1.11 (t,
3H), 2.34 (q, 2H), 5.66 (d, 1H), 6.29 (d, 1H); 13C NMR
(CDCl3, 300 MHz) d 13.03, 24.83, 126.34, 141.95,
172.85.
We have previously reported that an introduction of a
methyl group at the 2-position of 2-benzylsuccinic acid
improves the binding affinity by 2-fold.11 In this study,
we have observed that such an introduction of a methyl
group into 4 to obtain 7 deminished the binding affinity
by 35-fold. This attenuation of the binding affinity by
the methyl introduction into 4 may as well be accounted
for by the proposition discussed above: in the case of 7
that bears a methyl group at the 2-position, the 2-
methyl group deters the binding of the molecule to the
active site as discussed above, but in the binding of 4,
since there is no methyl group at the 2-position, no such
adverse effect is in operation, and 4 can form a complex
with CPA much more tightly than 7.
2-Acetylsufanylmethylbutyric acid (11). A mixture of 10
(0.54 g, 4.5 mmol) and thioacetic acid (1.2 mL, 17.0
mmol) in benzene was refluxed for 12 h. The solution
was concentrated under reduced pressure, diluted with
ethyl acetate, and washed with 1 N HCl. The organic
phase was dried over MgSO4, and concentrated under
reduced pressure to give 1118 as a pale yellow oil (0.65 g,
1
82%). H NMR (CDCl3, 300 MHz) d 1.00 (t, 3H), 1.71
Conclusion
(m, 2H), 2.35 (s, 3H), 2.58 (m, 1H), 3.03 (dd, 1H), 3.14
(dd, 1H); 13C NMR (CDCl3, 300 MHz) d 11.72, 25.36,
29.98, 31.00, 47.36, 180.87, 195.89.
The replacement of the zinc coordinating carboxylate in
CPA inhibitors such as 2 and 3 with a sulfhydryl group,
a stronger zinc ligating moiety than carboxylate, failed
to improve the binding affinity. The present investiga-
tion together with our previous studies16,17 tends to
suggest that the expectation that one may design CPA
inhibitors of improved potency by taking advantage of
the methyl hole at the active site is hardly achievable
especially when the zinc ligating moiety has a high
binding affinity towards the active site zinc ion.
2-Mercaptomethylbutyric acid (5). Compound 11 (400
mg, 2.27 mmol) was suspended in 6 N HCl and refluxed
for 4 h. The reaction mixture was cooled to room tem-
perature and extracted with EtOAc. The organic layer
was dried over MgSO4 and concentrated under reduced
pressure to give a crude product which was purified by
column chromatography (hexane/EtOAc=3/1) to yield
1
5 as a colorless oil (295 mg, 97%). H NMR (CDCl3,
300 MHz) d 0.98 (t, 3H), 1.55 (t, 1H), 1.72 (m, 2H), 2.55
(m, 1H), 2.63–2.83 (m, 2H); 13C NMR (CDCl3,
300 MHz) d 11.79, 24.69, 25.58, 51.00, 181.30. FAB
HRMS calcd for C5H11O2S (MH+) 135.0480, found
135.0479.
Experimental
Melting points were taken on a Thomas-Hoover capil-
lary melting point apparatus and were uncorrected. IR
spectra were recorded on a Bruker Equinox 55 FT-IR
spectrometer. 1H NMR and 13C NMR spectra were
obtained with a Bruker AM 300 (300 MHz) NMR
spectrometer using tetramethylsilane as the internal
standard. Mass spectra were obtained with a Micro
Mass Platform II 8410E spectrometer and high resolu-
tion mass spectra were obtained at Korea Basic Science
Institute, Daejeon, Korea. Silica gel 60 (230–400 mesh)
was used for flash chromatography and thin-layer
chromatography (TLC) was carried out on silica coated
glass sheets (Merck silica gel 60 F-254). Elemental ana-
lyses were performed at Pohang University of Science
and Technology, Pohang, Korea.
Diethyl 2-ethyl-2-methylmalonate (13). Diethyl methyl-
malonate (10.0 mL, 58 mmol) was added to the suspen-
sion of NaH (60% dispersion in mineral oil, 2.55 g, 64
mmol) in DMF at 0 ꢁC. When hydrogen gas evolution
was ceased, iodoethane (5.62 mL, 70 mmol) was added
to the solution and the reaction mixture was stirred for
2 h at 100 ꢁC. The mixture was cooled to room tem-
perature and diluted with ethyl acetate. The solution
was washed with 5% aqueous Na2S2O3 solution to
remove DMF. The organic layer was dried over MgSO4
and concentrated under reduced pressure. The crude
product was purified by flash column chromatography
(hexane/EtOAc=10/1) to give 1319 (11.4 g, 97%) as a
colorless oil. 1H NMR 300 MHz (CDCl3) d 0.87 (t, 3H),
1.25 (t, 6H), 1.39 (s, 3H), 1.91 (q, 2H), 4.18 (q, 4H); 13C
NMR 300 MHz (CDCl3) d 9.30, 14.69, 19.97, 29.22,
54.78, 61.65, 127.27, 173.06.
2-Ethylmalonic acid (9). A mixture of diethyl ethylma-
lonate (10 mL, 53.2 mmol) and 4 N KOH solution (50
mL) was refluxed for 2 h. The solution was diluted with
water, washed with ether, acidified with 6 N HCl, and
extracted with ether. The organic phase was dried over
MgSO4, and concentrated under reduced pressure to
give 918 as a white solid (6.61 g, 94%). Mp 108–110 ꢁC;
1H NMR (MeOH-d4, 300 MHz) d 0.95 (t, 3H), 1.85 (m,
2H), 3.20 (t, 1H); 13C NMR (CDCl3, 300 MHz) d 11.20,
22.42, 53.57, 172.33.
Diethyl 2-benzyl-2-methylmalonate.20 This was similarly
prepared from diethyl methylmalonate and benzyl bro-
mide in 98% yield as a colorless oil. 1H NMR 300 MHz
(CDCl3) d 1.25 (t, 6H), 1.34 (s, 3H), 3.23 (s, 2H), 4.19
(q, 4H), 7.10–7.26 (m, 5H); 13C NMR 300 MHz
(CDCl3) d 14.42, 20.12, 41.50, 55.22, 61.70, 127.27,
128.56, 130.60, 136.64, 172.35.

H. S. Lee, D. H. Kim / Bioorg. Med. Chem. 11 (2003) 4685–4691
4689
2-Ethyl-2-methyl-1,3-propanediol (14). An ice-chilled
solution of LiAlH4 (6.07 g, 160 mmol) in diethyl ether
was treated dropwise with a solution of 13 (8.08 g, 40
mmol) in diethyl ether. After stirring for 4 h at room
temperature, the reaction mixture was treated succes-
sively by the careful dropwise addition of 5.8 mL of
H2O, 11.7 mL of 15% aqueous NaOH solution, and
17.5 mL of H2O. The mixture was filtered and the fil-
trate was concentrated under reduced pressure. The
residue was purified by flash column chromatography
(hexane/EtOAc=3/1) to give 14 (4.44 g, 94%) as a color-
less oil. 1H NMR 300 MHz (CDCl3) d 0.81 (s, 3H), 0.87 (t,
3H), 1.37 (q, 2H), 2.72 (br, 2H), 3.53 (dd, 4H); 13C NMR
300MHz (CDCl3) d 7.97, 18.24, 26.60, 39.23, 70.59.
3H), 4.09–4.23 (dd, 2H); 13C NMR 300 MHz (CDCl3) d
8.87, 19.29, 21.20, 28.94, 46.71, 68.83, 171.35, 181.93;
MS (EI) m/z 175 (M+). HRMS (FAB+) (M+H)+:
calcd for C8H14O4, 175.0970; found 175.0966.
2-Acetoxymethyl-2-methyl-3-phenylpropanoic acid. This
was similarly prepared in 71% yield as a white. Mp 98–
100 ꢁC (solid diethyl ether and hexane); IR (KBr) 2978,
1747, 1699 cmꢂ1; 1H NMR 300 MHz (CDCl3) d 1.22 (s,
3H), 2.10 (s, 3H), 2.97 (dd, 2H), 4.13 (dd, 2H), 7.13-7.29
(m, 5H); 13C NMR 300 MHz (CDCl3) d 19.93, 21.12,
41.60, 47.44, 68.22, 127.34, 128.69, 130.55, 136.36,
170.93, 180.03. Anal. calcd for C13H16O4: C, 66.09; H,
6.83. Found: C, 66.18; H, 6.82.
2-Benzyl-2-methyl-1,3-propanediol.21 This was similarly
prepared in 96% yield as a white solid. Mp 53–55 ꢁC; 1H
NMR 300 MHz (CDCl3) d 0.76 (s, 3H), 2.58 (br, 2H),
2.70 (s, 2H), 3.55 (s, 4H), 7.20–7.29 (m, 5H); 13C NMR
300 MHz (CDCl3) d 18.97, 40.23, 40.51, 70.39, 126.55,
128.41, 130.98, 138.20.
2-Bromomethyl-2-methylbutyric acid (17). Compound
16 (1.70 g, 9.8 mmol) was suspended in concentrated
hydrobromic acid and refluxed for 24 h. The reaction
mixture was cooled to room temperature, diluted with
water, and extracted with EtOAc. The organic layer was
dried over MgSO4, and concentrated under reduced
pressure to give a crude oil which was purified by col-
umn chromatography (hexane/EtOAc=5/1) to yield
1722 (1.58 g, 83%) as a white solid which was recrys-
2-Acetoxymethyl-2-methylbutanol (15). A mixture of 14
(3.00 g, 25.4 mmol), trimethylorthoacetate (3.56 mL,
27.9 mmol), and a catalytic amount of p-toluenesulfonic
acid monohydrate (0.48 g, 2.5 mmol) in CH2Cl2 was
stirred for 1 h at room temperature. The reaction mix-
ture was concentrated under reduced pressure and the
residue was purified by flash column chromatography
(EtOAc/hexane=1/5) to give 15 (3.70 g, 91%) as a col-
tallized from hexane. Mp 48–50 ꢁC; H NMR 300 MHz
1
(CDCl3) d 0.93 (t, 3H), 1.31 (s, 3H), 1.66–1.83 (m, 2H),
3.44–3.64 (dd, 2H); 13C NMR 300 MHz (CDCl3) d 9.21,
14.55, 21.14, 30.71, 39.45, 48.11, 65.25, 181.47.
2-Bromomethyl-2-methyl-3-phenylpropanoic acid. This
was similarly prepared in 85% yield as a white solid
which was recrytallized from hexane. Mp 115–116 ꢁC;
IR (KBr) 2939, 1696 cmꢂ1; 1H NMR 300 MHz (CDCl3)
d 1.33 (s, 3H), 3.04 (s, 2H), 3.43 (d, 1H), 3.57 (d, 1H),
7.21–7.32 (m, 5H); 13C NMR 300 MHz (CDCl3) d
21.94, 39.07, 42.53, 48.98, 127.55, 128.82, 130.53,
136.30, 181.08. Anal. calcd for C19H21NO: C, 51.38; H,
5.10. Found: C, 51.28; H, 5.01.
1
orless oil. IR (neat) 3450, 2967, 1740 cmꢂ1; H NMR
300 MHz (CDCl3) d 0.85 (m, 6H), 1.31 (m, 2H), 2.09 (s,
3H), 3.33 (dd, 2H), 3.96 (dd, 2H); 13C NMR 300 MHz
(CDCl3) d 7.79, 18.32, 21.16, 26.80, 39.20, 44.30, 66.96,
68.40, 172.02; MS (EI) m/z 161 (M+). HRMS (FAB+)
(M+H)+: calcd for C8H16O3, 161.1178; found
161.1174.
2-Acetoxymethyl-2-methyl-3-phenylbutanol. This was
similarly prepared in 90% yield as a colorless oil. IR
2-Acetylsulfanylmethyl-2-methylbutanoic
acid
(18).
(neat) 3470, 2927, 1737 cmꢂ1
;
1H NMR 300 MHz
Potassium thioacetate (322 mg, 2.82 mmol) was added
to a stirred solution of 17 (500 mg, 2.56 mmol) and
triethylamine (393 mL, 2.82 mmol) in THF, and the
reaction mixture was stirred for 3 h at room tempera-
ture. The mixture was diluted with EtOAc and washed
with 1 N HCl. The organic layer was dried over MgSO4
and concentrated under reduced pressure to give a crude
oil which was purified by column chromatography
(hexane/EtOAc=4/1) to yield 1823 (439 mg, 90%) as a
colorless oil. 1H NMR 300 MHz (CDCl3) d 0.91 (t, 3H),
1.18 (s, 3H), 1.61–1.77 (m, 2H), 2.35 (s, 3H), 3.19 (dd,
2H); 13C NMR 300 MHz (CDCl3) d 9.31, 20.89, 30.95,
31.81, 36.03, 47.29, 182.51, 195.63.
(CDCl3) d 0.83 (s, 3H), 2.13 (s, 3H), 2.34 (br, 1H), 2.60
(dd, 2H), 3.32 (dd, 2H), 3.95 (dd, 2H), 7.16–7.32 (m,
5H); 13C NMR 300 MHz (CDCl3) d 18.93, 21.35, 40.33,
40.55, 66.57, 68.08, 126.73, 128.48, 130.99, 137.40,
172.32; MS (EI) m/z 223 (M+). HRMS (FAB+)
(M+H)+: calcd for C13H19O3, 223.1334; found
223.1333.
2-Acetoxymethyl-2-methylbutyric acid (16). To an ice-
cooled solution of 15 (3.20 g, 20.0 mmol) in acetone was
added slowly the Jones reagent until brownish color of
the solution remains over 20 min, then 2-propanol was
added until the solution became clear. The precipitate
was filtered and the filtrate was concentrated under
reduced pressure. The residue was diluted with ethyl
acetate and extracted with saturated aqueous NaHCO3
solution. The aqueous layer was acidified with 6 N HCl
and extracted with CH2Cl2. The organic layer was dried
over MgSO4 and concentrated under reduced pressure
to give 16 (2.47 g, 71%) as a colorless oil. IR (neat)
2-Acetylsulfanylmethyl-2-methyl-3-phenylpropanoic acid.
This was similarly prepared in 90% yield as a colorless
;
oil. IR (neat) 3029, 1699 cmꢂ1 1H NMR 300 MHz
(CDCl3) d 1.21 (s, 3H), 2.36 (s, 3H), 2.97 (dd, 2H), 3.19
(dd, 2H), 7.13–7.30 (m, 5H); 13C NMR 300 MHz
(CDCl3) d 20.37, 30.08, 35.32, 44.14, 47.52, 126.55,
127.86, 129.61, 135.72, 181.02, 194.48; MS (EI) m/z 253
(M+). HRMS (EI): calcd for C13H17O3S, 252.0820;
found 252.0819.
1
2975, 1746, 1705 cmꢂ1; H NMR 300 MHz (CDCl3) d
0.92 (t, 3H), 1.22 (s, 3H), 1.55–1.76 (m, 2H), 2.07 (s,

4690
H. S. Lee, D. H. Kim / Bioorg. Med. Chem. 11 (2003) 4685–4691
2-Mercaptomethyl-2-methylbutanoic acid (6). Com-
pound 18 (250 mg, 1.31 mmol) was suspended in 6 N
HCl and refluxed for 4 h. The reaction mixture was
cooled to room temperature and extracted with EtOAc.
The organic layer was dried over MgSO4 and con-
centrated under reduced pressure to give a crude oil
which was purified by column chromatography (hex-
ane/EtOAc=3/1) to yield 6 (185 mg, 95%) as a col-
(2R,10S)-21. Mp 129–131 ꢁC; [a]D ꢂ50.8ꢁ (c 1.89,
MeOH); IR (KBr) 3250, 2915, 1635, 1545 cmꢂ1 1H
;
NMR 300 MHz (CDCl3) d 1.05 (s, 3H), 1.34 (d, 3H),
2.80 (d, 1H), 3.03 (d, 1H), 3.53–3.61 (m, 3H), 5.06 (m, 1H),
6.20 (br, 1H), 7.14–7.31 (m, 10H); 13C NMR 300 MHz
(CDCl3) d 19.72, 22.13, 42.26, 47.70, 49.03, 68.94, 126.35,
127.03, 127.68, 128.56, 129.08, 130.80, 137.44, 143.66,
176.40. Anal. calcd for C19H23NO2: C, 76.73; H, 7.80; N,
4.71. Found: C, 76.82; H, 7.88; N, 4.73.
1
orless oil. H NMR 300 MHz (CDCl3) d 0.90 (t, 3H),
1.24, (s, 3H), 1.43 (t, 1H), 1.60–1.79 (m, 2H), 2.58
(dd, 1H), 2.86 (dd, 1H); 13C NMR 300 MHz (CDCl3)
d 9.28, 20.46, 31.19, 32.58, 48.65, 182.85. FAB
HRSM calcd for C6H13O2S (MH+) 149.0636, found
149.0643.
(2S,10S)-21. Mp 95–97 ꢁC; [a]D ꢂ81.5ꢁ (c 1.34,
MeOH); IR (KBr) 3292, 2929, 1631, 1545 cmꢂ1 1H
;
NMR 300 MHz (CDCl3) d 1.04 (s, 3H), 1.45 (d, 3H),
2.80 (d, 1H), 3.03 (d, 1H), 3.30 (dd, 1H), 3.59 (m, 2H),
5.10 (m, 1H), 6.09 (br, 1H), 7.07–7.32 (m, 10H); 13C
NMR 300 MHz (CDCl3) d 19.66, 22.06, 42.20, 47.71,
49.07, 69.08, 126.57, 126.89, 127.69, 128.52, 129.01,
130.71, 137.29, 143.29, 176.27. Anal. calcd for
C19H23NO2: C, 76.73; H, 7.80; N, 4.71. Found: C,
76.73; H, 7.82; N, 4.73.
2-Mercaptomethyl-2-methyl-3-phenylpropanoic acid (7).
This was similarly prepared in 95% yield as a crystal-
line solid. Mp 62–64 ꢁC; IR (KBr) 3244, 1731, 1696
1
cmꢂ1; H NMR 300 MHz (CDCl3) d 1.25 (s, 3H), 1.50
(t, 1H), 2.58 (dd, 1H), 2.84 (dd, 1H), 2.99 (dd, 2H),
7.15–7.31 (m, 5H); 13C NMR 300 MHz (CDCl3) d
21.11, 32.40, 43.71, 49.67, 127.32, 128.69, 130.58,
136.82, 181.77. Anal. calcd for C19H21NO: C, 62.83; H,
6.71. Found: C, 63.20; H, 6.65.
(R)-2-Bromomethyl-2-methyl-3-phenyl-propanoic
acid
[(R)-22]. Compound (2R,10S)-21 (800 mg, 2.7 mmol)
was suspended in concentrated hydrobromic acid and
refluxed for 24 h. The reaction mixture was cooled to
room temperature, diluted with water, and extracted
with EtOAc. The organic layer was dried over MgSO4
and concentrated under reduced pressure to give a col-
orless oil which was purified by column chromato-
graphy (hexane/EtOAc=5/1) to yield (R)-22 (630 mg,
78%) as a white solid which was recrystallized from
hexane. Mp 115–116 ꢁC; [a]D +3.37ꢁ (c 1.33, MeOH).
Anal. calcd for C11H13BrO2: C, 51.38; H, 5.10. Found:
C, 51.29; H, 4.75.
2-Hydroxymethyl-2-methyl-3-phenylpropanoic acid (20).
Compound 19 (4.00 g, 16.9 mmol) was dissolved in
MeOH containing 2 N NaOH (17 mL) and the solution
was refluxed for 4 h. The reaction mixture was cooled to
room temperature and concentrated under reduced
pressure. The residue was acidified with 6 N HCl and
extracted with CH2Cl2. The organic layer was dried
over MgSO4 and concentrated under reduced pressure
to give 20 (3.19 g, 97%) as a white solid which was
recrystallized from the mixed solvent of diethyl ether
and hexane. Mp 103–105 ꢁC; 1H NMR 300 MHz
(CDCl3) d 1.14 (s, 3H), 2.95 (dd, 2H), 3.60 (dd, 2H),
7.18–7.30 (m, 5H); 13C NMR 300 MHz (CDCl3) d
19.60, 40.99, 48.97, 67.03, 127.21, 128.64, 130.81,
136.60, 182.75. Anal. calcd for C11H14O3: C, 68.02; H,
7.27. Found: C, 68.10; H, 7.30.
(S)-2-Bromomethyl-2-methyl-3-phenyl-propanoic
acid
[(S)-22]. This was similarly prepared from (2S,10S)-21
in 77% yield as a crystalline solid. Mp 115-116 ꢁC; [a]D
ꢂ3.32ꢁ (c 1.17, MeOH). Anal. calcd for C11H13BrO2: C,
51.38; H, 5.10. Found: C, 51.59; H, 5.21.
(R)-2-Acetylsulfanylmethyl-2-methyl-3-phenylpropanoic
acid [(R)-23]. Potassium thioacetate (151 mg, 1.32
mmol) was added to a stirred solution of (R)-22 (225
mg, 0.88 mmol) and triethylamine (0.134 mL,
0.96mmol) in THF and stirred for 3 h at room tem-
perature. The reaction mixture was diluted with EtOAc
and washed with 1 N HCl. The organic layer was dried
over MgSO4 and concentrated under reduced pressure
to give an yellowish oil which was purified by column
chromatography (hexane/EtOAc=4/1) to yield (R)-23
(199 mg, 90%) as an oil. [a]D +9.7ꢁ (c 1.00, CHCl3).
HRMS (EI): calcd for C13H16O3S, 252.0820; found
252.0823.
(2R,10S)- and (2S,10S)-2-hydroxymethyl-2-methyl-3-
phenyl-N-(10-phenylethyl)-propionamide [(2R,10S)- and
(2S,10S)-21]. 1-Ethyl-3-(3-dimethylaminopropyl)-carbo-
diimide hydrochloride (EDCI, 2.87 g, 14.9 mmol), 1-
hydroxybenzotriazole hydrate (2.01 g, 14.9 mmol), and
triethylamine (2.29 mL, 16.4 mmol) were added to the
stirred solution of 20 (2.90 g, 14.9 mmol) in CH2Cl2 at
0 ꢁC, and the solution was stirred for 10 min. (S)-Phe-
nylethylamine (2.11 mL, 16.4 mmol) was added to the
reaction mixture at 0 ꢁC, and the mixture was stirred for
1 h at room temperature. The solution was washed with
10% aqueous solution of citric acid, saturated aqueous
NaHCO3 solution, and brine, and the organic layer was
dried over MgSO4. The dried solution was concentrated
under reduced pressure to give the product (4.31 g,
97%) as a diastereomeric mixture which was separated
by flash column chromatography (hexane/EtOAc=4/1
to 2/1) to give the optically pure product, (2R,10S)-21
(1.95 g) and (2S,10S)-21 (2.08 g). The analytical sample
was prepared by recrystallization from the mixed
solvent of diethyl ether and hexane.
(S)-2-Acetylsulfanylmethyl-2-methyl-3-phenylpropanoic
acid [(S)-23]. This was similarly prepared from (S)-22 in
90% yield as an oil. [a]D ꢂ10.3ꢁ (c 1.43, CHCl3). HRMS
(EI): calcd for C13H16O3S, 252.0820; found 252.0824.
(R)- and (S)-2-Mercaptomethyl-2-methyl-3-phenylpropa-
noic acid [(R)- and (S)-7]. These were prepared from (R)-
and (S)-23, respectively, in 95% yield in an analogous

H. S. Lee, D. H. Kim / Bioorg. Med. Chem. 11 (2003) 4685–4691
4691
fashion to that used for preparation of (R,S)-7 from
(R,S)-23.
2. Ehlers, M. R. W.; Riordan, J. F. Biochemistry 1989, 28, 5312.
3. Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A. J. H.
Chem. Rev. 1999, 99, 2735.
(R)-7. Mp 62–64 ꢁC; [a]D ꢂ5.5ꢁ (c 0.87, CHCl3). Anal.
calcd for C19H21NO: C, 62.83; H, 6.71. Found: C,
63.05; H, 6.62.
4. Ondetti, M. A.; Rubin, B.; Cushman, D. W. Science 1977,
196, 441.
5. Patchett, A. A.; Harris, E.; Tristram, E. W.; Wyvratt, M. J.;
Wu, M. T.; Taub, D.; Perterson, E. R.; Ikeler, T. J.; ten
Broeke, J.; Payne, L. G.; Ondeyka, D. L.; Thorsett, E. D.;
Greenlee, W. J.; Lohr, N. S.; Hoffsommer, R. D.; Joshua, H.;
Ruyle, W. V.; Rothrock, J. W.; Aster, S. D.; Maycock, A. L.;
Robinson, F. M.; Hirschmann, R.; Sweet, C. S.; Ulm, E. H.;
Gross, D. H.; Vassil, T. C.; Stone, C. A. Nature (London)
1980, 288, 280.
(S)-7. Mp 62–64 ꢁC; [a]D +5.5ꢁ (c 0.70, CHCl3). Anal.
calcd for C19H21NO: C, 62.83; H, 6.71. Found: C,
63.15; H, 6.72.
General remarks for enzyme kinetic experiments
6. Christianson, D. W.; Lipscomb, N. W. Acc. Chem. Res.
1989, 22, 62.
CPA was purchased from Sigma Chemical Co. (Allan
form, twice crystallized from bovine pancrease, aqueous
suspension in toluene) and used without further pur-
ification. Hippuryl-l-phenylalanine (Hipp-l-Phe) pur-
chased from Sigma Chemical Co. was used as substrate.
All solutions were prepared by dissolving in doubly
distilled and deionized water. CPA stock solutions were
prepared by dissolving CPA in 0.05 M Tris/0.5 M NaCl,
pH 7.5 buffer solution and their concentrations were
estimated from the absorbance at 278 nm
(e278=64,200). The stock assay solutions were filtered
(GHP Acrodic syringe filter, pore size 0.2 mm) before
use. A Perkin-Elmer HP 8453 UV–vis spectrometer was
used for UV absorbance measurements.
7. (a) Kim, D. H.; Kim, K. B. J. Am. Chem. Soc. 1991, 113,
3200. (b) Kim, D. H.; Chung, S. J. Bioorg. Med. Chem. Lett.
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14. Crystallographic data (excluding structure factors) for the
structure have been deposited with the Cambridge Crystal-
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CCDC 206138. Copies of the data can be obtained, free of
charge, on application to CCDC, 12 Union Road, Cambridge,
CB2 1EZ, UK (fax: +44-1223-336033 or e-mail: deposit@
ccdc.cam.ac.uk).
Determination of Ki values
Typically, an aliquot of the enzyme stock solution
was added to the solution of the inhibitor and the
substrate in 0.05 M Tris/0.5 M NaCl, pH 7.5 buffer
solution containing 0.1 mM glutathione (1 mL cuv-
ette), and the absorption increase at 254 nm was
recorded immediately. The initial velocities were
obtained from the linear plot of the substrate hydro-
lysis monitored by the increase of absorbance at 254
nm. The Ki values were then estimated from the semi-
reciprocal plot of the initial velocity versus the con-
centrations of the inhibitors according to the method of
Dixon. Two concentrations of substrate of Hipp-l-Phe
were used.
15. Dixon, M. Biochem. J. 1953, 55, 170.
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Acknowledgements
The authors express their thanks to the Korean Science
and Engineering Foundation for the financial support
of this work and the Ministry of Education and
Human resources for the BK21 fellowship to HSL.
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References and Notes
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1. Lipscomb, N. W.; Strater, N. Chem. Rev. 1996, 96, 2375.