Inhibition of α-chymotrypsin with thiol-bearing substrate analogues in the presence of zinc ion

Article abstract of DOI:10.1016/j.bmcl.2003.11.058

We have demonstrated that thiol-bearing analogues of α-chymotrysin (α-CT) substrates such as (S)-(1-benzyl-2-thiolethyl)-carbamic acid, benzyl ester (3) inhibits α-CT, a prototypical serine protease, in the presence of Zn(II) ion. They constitute a novel class of small molecule inhibitors for α-CT believed to inhibit the enzyme by forming a ternary complex consisting of α-CT, Zn(II) ion, and the inhibitor.

Full text of DOI:10.1016/j.bmcl.2003.11.058

Bioorganic & Medicinal Chemistry Letters 14 (2004) 701–705
Inhibition of ꢀ-chymotrypsin with thiol-bearing substrate
analogues in the presence of zinc ion
Min Su Han, Dong Ju Oh and Dong H. Kim*
Center for Integrated Molecular System and Department of Chemistry, Division of Molecular and Life Sciences,
Pohang University of Science and Technology, San 31Hyojadong, Pohang 790-784, South Korea
Received 29 September 2003;accepted 14 November 2003
Abstract—We have demonstrated that thiol-bearing analogues of a-chymotrysin (a-CT) substrates such as (S)-(1-benzyl-2-thiol-
ethyl)-carbamic acid, benzyl ester (3) inhibits a-CT, a prototypical serine protease, in the presence of Zn(II) ion. They constitute a
novel class of small molecule inhibitors for a-CT believed to inhibit the enzyme by forming a ternary complex consisting of a-CT,
Zn(II) ion, and the inhibitor.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
analogues that bear a methylmercapto moiety in place
of hydrolysable peptide bond in the substrate of a-CT.
Serine proteases have received increasing attention in
recent years because of their roles in the pathology of
numerous diseases such as rheumatoid arthritis, throm-
bosis, and pulmonary emphysema.1 These enzymes are
characterized by having a catalytic triad consisting of
Ser, His, and Asp as the essential catalytic machinery.
The serine hydroxyl group in the catalytic triad in colla-
boration with the His and Asp residues attacks the scissile
peptide or ester bond of the enzyme bound substrates,
generating an acyl-enzyme intermediate which, in turn, is
hydrolyzed by the active site water molecule to yield the
second product with regeneration of the free enzyme.
a-Chymorypsin (a-CT) is a much studied serine protease
whose structure and catalytic mechanism have been well
established.2 There is present at the active site of a-CT a
large hydrophobic pocket (S1 subsite), the primary func-
tion of which involves accommodation of the P1 residue
having a bulky hydrophobic side chain such as Phe. As
a prototypical enzyme for a large family of proteases,
a-CT has served as a model target enzyme for the
development of design strategies that can be utilized to
inhibit serine proteases of medicinal interest. As a con-
sequence, numerous design protocols for a-CT inhibi-
tors are known and a vast number of a-CT inhibitors
have been reported.3 We wish to report herein a new
class of inhibitors that bind strongly to a-CT in the
presence of Zn(II) ion. These inhibitors are substrate
Recently, Katz et al. have developed a novel strategy for
designing inhibitors that are highly effective against
trypsin and other serine proteases.4 The design proto-
col takes advantage of the high propensity of Zn(II)
ion to form a tetrahedrally coordinated complex. The
binding affinity of bis-(5-amidino-2-benzimidazolyl)-
methane (BABIM) toward trypsin was enhanced by as
much as 1.7Â104-fold in the presence of Zn(II) ion.
The X-ray crystallographic structural analysis of the
complex of trypsin formed with BABIM revealed that
the Zn(II) ion is coordinated by one nitrogen from each
of the two benzimidazoles in BABIM, the hydroxyl of
Ser-195, and the imidazole of His-57 of the catalytic
* Corresponding author: Tel.: +82-54-279-2101;fax: +82-54-279-
5877;e-mail: dhkim@postech.ac.kr
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.058

702
M. S. Han et al. / Bioorg. Med. Chem. Lett. 14 (2004) 701–705
triad of the enzyme, thus to bridge the inhibitor and
trypsin.
synthesized.11 In the synthesis of 3, the hydroxyl group in
5 that was obtained by the reduction of 8 was converted
into the corresponding bromide using tetra-
bromomethane and triphenylphophine,12 then the pro-
duct was subjected to thioacetylation using potassium
thioacetate to give 10. Hydrolysis of the latter compound
afforded 313 (Scheme 2). Inhibitor 4 was synthesized
starting with 2-(1-benzyl-2-hydroxy-ethylcarbamoyl)-
pyrrolidine-1-carboxylic acid, benzyl ester,14 whose
hydroxyl group was converted directly into the thioace-
tate by the modified Mitsunobu reaction,15 and the
product thus obtained was hydrolyzed to yield 4.16
It has been well established that a mercapto group has a
high tendency to form a coordinative bond to Zn(II)
ion.5 In fact, this property of mercapto group has been
extensively utilized in the design of inhibitors for zinc-
containing metalloenzymes.6 We have envisioned that
appropriately modified mercapto-bearing analogues of
a-CT substrate would bind strongly to a-CT in the pre-
sence of Zn(II) ion, forming a ternary complex, as
depicted in Figure 1. The phenyl ring in such inhibitors
is expected to anchor in the primary substrate recogni-
tion pocket (S1 pocket) and the thiol group would ligate
the zinc ion that is held by the side chains of Ser-195
and His-57 present at the active site of a-CT. A water
molecule is thought to bind the metal ion as the fourth
ligand. It is known that when sulfur is bound, the Zn(II)
ion prefers to have the coordination number of 4.7,8 The
water molecule may be coordinated to the Zn(II) in a
deprotonated form. It has been known that the pKa
value of the water molecule coordinated to Zn(II) can
be lowered as much as 9 units.9
The a-CT inhibition potencies are expressed by the IC50
value which is estimated from the plot of the percent
enzymic activity remaining in the presence of an inhibitor
and Zn(II) ion versus concentration of the inhibitor, as
exemplified by Figure 2. The assay was performed using
Suc-Ala-Ala-Pro-Phe-p-NO2-anilide17 as substrate in a
Tris buffer solution of pH 7.5. The IC50 values18 thus
obtained are listed in Table 1. None of the inhibitors
exhibited inhibitory activity against a-CT up to the
2. Results and discussion
2.1. Synthesis
Compound 6 that was prepared by the literature10
method was treated with hydrochloric acid, then the
product was allowed to react with acetic anhydride to
give 7 which was treated with potassium carbonate in
an aqueous methanol solution to obtain inhibitor 111
(Scheme 1). In a similar fashion, inhibitor 2 was
Scheme 2.
Figure 1. Schematic representation of inhibitor design rationale: mer-
capto-bearing substrate analogues inhibit a-chymotrypsin in the pre-
sence of Zn(II) ion by forming ternary complexes.
Figure 2. Plot of the percent enzymic activity remaining in the presence
of an inhibitor and Zn(II) ion versus concentration of the inhibitor (S)-3.
Scheme 1.

M. S. Han et al. / Bioorg. Med. Chem. Lett. 14 (2004) 701–705
703
revealed that (S)-3 that carries a Cbz moiety on the
amino group is most potent, suggesting that the phenyl
ring in the Cbz may play a role in binding of the inhi-
bitor to the enzyme, possibly the phenyl group being
accommodated in the S2 pocket. It has been known that
the S2 pocket of a-CT prefers a large residue.21 Segel
and others showed that substrates of a-CT having a
proline at the P2 position bind the enzyme with high
affinity by virtue of its hydrophobic interactions possi-
bly with the side chain of Ile-99 as well as a smaller
entropy decrease upon binding to the enzyme due to its
restricted rotational freedom, in addition to hydrogen
bondings between the substrate and active site back-
bone residues of the enzyme.22 Accordingly, we have
synthesized 4 and evaluated it as an a-CT inhibitor in
the presence of Zn(II) ion. Contrary to the expectation,
4 was found to be less effective than 3. The replacement
of the thiol group in (S)-3 with a hydroxyl essentially
abolishes the a-CT inhibitory activity in accordance
with the proposition that the enzyme inhibition is effec-
ted via the formation of the metal-centered ternary
complex, and indicates that the thiol group coordinates
the metal ion much more strongly than a hydroxyl.
Recently, Vahrenkamp and associates have demon-
strated that a thiol has a stronger coordination power
toward Zn(II) than a hydroxyl in forming the tetra-
hedral Zn(II) complex.23
Table 1. Kinetic parameters for the inhibition of a-CT in the pre-
sence of Zn(II) (500 mM)
Inhibitor
IC50 (mM)
1
2
>800
23
(S)-3
(R)-3
4
3.8
6.0
8.0
(S)-5
(R)-5
>3000
>3000
concentration of 2 mM when tested in the absence of
Zn(II) ion, suggesting that the metal ion plays a critical
role in the inhibition, most likely by bridging the inhi-
bitor to the enzyme with the formation of a ternary
complex as depicted in Figure 1. Since some proteases
such as carboxypeptidase A are known to be inactivated
by Zn(II) ion,19 we have investigated the effect of Zn(II)
ion alone on a-CT to find that the enzymic activity is
essentially unchanged by Zn(II) ion up to the concen-
tration of 2 mM. These results suggest strongly that in
the inhibitions of a-CT both the thiol and Zn(II) ion are
involved, which is in line with the proposed design
rationale depicted schematically in Figure 1.
As can be seen in Figure 3, the extent of Zn(II) medi-
ated inhibition of a-CT by (S)-3 is dependent on the
concentration of Zn(II) ion. The enzymic activity is
essentially abolished when the concentration of Zn(II)
ion reaches 150 mM. We were interested in knowing the
effect of other metal ions than Zn(II) on the a-CT inhi-
bitory property of the thiol-bearing inhibitors, and eval-
uated four different metal ions including Zn(II) ion to
find that Cu(II) ion is as effective as Zn(II) ion in med-
iating (S)-3 to inhibit the a-CT. Although the extent of
the inhibition caused by the two metal ions is compar-
able, Cu(II) ion is much more efficient than Zn(II) ion.
That is, the maximum inhibition is achieved by a much
smaller amount of Cu(II) ion than Zn(II) ion (Fig. 3).20
Zn(II) ion has a filled d-shell and thus is redox-inert
which is a desirable feature for the metal ion being used
as a cofactor of proteolytic enzymes. In fact, Zn(II) ion
is the most abundantly and widely distributed transition
metal ion next to Fe(II) and Fe(III) ions in the biologi-
cal systems: The concentrations of Zn(II) ion in human
tissue and blood amount to 60–120 mM.24 Comparative
trace metal analyses of cancerous and noncancerous
human tissue have revealed that the concentration of
Zn(II) ion in cancerous cell such as breast carcinoma is
much higher by 700% compared with that in normal
breast cells.25 Hence, the design protocol reported in this
communication may be applied for developing inhibitors
that selectively inhibit serine proteases in cells of high
Zn(II) ion concentrations. In employing the present pro-
tocol in designing inhibitors that are effective in the living
system, there is a strong possibility of the inhibitors may
undergo dimerization with loss of the inhibitory activity.
Use of the inhibitors in a form of prodrug such as
S-acylated derivatives may circumvent the problem.
The limited structure–activity relationships study on the
Zn(II) ion mediated inhibition of a-CT by thiol derivatives
3. Conclusion
We have demonstrated that thiol-bearing analogues of
a-CT substrate functions as inhibitors for a-CT in the
presence of Zn(II) ion. In the inhibitions, the Zn(II) plays
a mediating role by bridging the inhibitors to the enzyme,
forming the ternary complexes: The Zn(II) ion is coordi-
nated with the thiol of the inhibitors, the hydroxyl of Ser-
195 and the imidazole of His-57 in a-CT, and a water
molecule. In addition to the high selectivity towards ser-
ine proteases in cells of high Zn(II) ion concentrations,
this type of inhibitors are thought to manifest high
inhibition power in light of the fact that coordinative
Figure 3. Inhibition of a-CT by (S)-3 (5.0 mM) in the presence of dif-
ferent metal ion (0–500 mM) in Tris buffer 7.5 (substrate, Suc-Ala-Ala-
Pro-Phe-p-NO2-anilide (300 mM);[E]=0.4 mg/mL).

704
M. S. Han et al. / Bioorg. Med. Chem. Lett. 14 (2004) 701–705
bonds to a metal ion are in general much stronger than
hydrogen bonds or hydrophobic interactions that are
involved in the binding of inhibitors to enzymes.
Furthermore, these compounds are structurally simple
having an amino acid structural frame, and hence there
is an ample room for structural modifications for the
activity improvements.
to afford the product (0.58 g, 82%). Mp 121–122 ꢀC;[ a]20
À18.7 (c 1, CHCl3);IR (KBr) 3299, 1731, 1678, 1605
cmÀ1 1H NMR (CDCl3) d 7.27 (m, 5H), 5.90 (b, 1H),
;
4.28 (m, 1H), 2.99 (m, 3H), 2.81 (m, 1H), 2.34 (s, 3H),
1.89 (s, 3H); 13C NMR (CDCl3) d 196.99, 170.29, 137.60,
129.70, 128.97, 127.12, 51.35, 40.48, 32.78, 30.98, 23.68.
Anal. calcd for C13H17NO2S: C, 62.12;H, 6.82;N, 5.57.
Found: C, 62.41;H, 6.95;N, 5.60. Thioacetic acid, (S)-(2-
benzoylamino-3-phenyl-propyl) ester (11) was prepared
similarly starting with thioacetic acid, (S)-(2-tert-butoxy-
carbonylamino-3-phenyl-propyl) ester. Benzoyl chloride
was used in lieu of acetic anhydride. Yield 71%. Mp 137–
138 ꢀC;[ a]20 +1.3 (c 1, CHCl3);IR (KBr) 3350, 1703,
Acknowledgements
The work was supported by Korea Research Founda-
tion Grant (KRF-2000-015-ODS0025) and Korea
Science and Engineering Foundation.
1
1654, 1505 cmÀ1; H NMR (CDCl3) d 7.71 (d, 2H), 7.41
(m, 3H), 7.26 (m, 5H), 6.65 (b, 1H), 4.45 (m, 1H), 3.12 (m,
3H), 2.85 (m, 1H), 2.30 (s, 3H); 13C NMR (CDCl3) d
197.97, 167.40, 137.64, 134.59, 131.89, 129.84, 129.06,
128.97, 127.31, 127.19, 52.48, 40.70, 32.63, 31.00. Anal.
calcd for C18H19NO2S: C, 68.98;H, 6.11;N, 4.47. Found:
References and notes
C, 68.84;H, 6.14;N, 4.58.
(S)-N-(1-Mercaptomethyl-2-
1. Wharton C. W. In Comprehensive Biological Catalysis, A
Mechanistic Reference;Sinnott, M., Ed.;Academic: New
York, 1997;Vol. 1, p 345–379.
2. Barrett, A. J. In Handbook of Proteolytic Enzymes;
Rawlings, N. D., Woessner, J. F. Eds.;Academic: New
York, 1998;p 30.
3. (a) Powers, J. C.;Harper, J. W. In Protease Inhibitors;
Barrett, A. J., Salvesen, G. Eds.;Elsevier: Amsterdam,
1986;p 55–142 (b) Demuth, H.-U. J. Enzyme Inhibition
1990, 3, 249.
4. Katz, B. A.;Clark, J. M.;Finer-Moore, J. S.;Jenkins,
T. E.;Johnson, C. R.;Ross, M. J.;Luong, C.;Moore,
W. R.;Stroud, R. M. Nature 1998, 391, 608.
5. Thorp, H. H. Chem. Biol. 1998, 5, R125.
6. (a) Ondetti, M. A.;Rubin, B.;Cushman, D. W. Science
1977, 196, 441. (b) Chong, C. R.;Auld, D. S. Biochemistry
2000, 39, 7580. (b) Ondetti, M. A.;Condon, M. E.;Reid,
J.;Sato, E. F.;Cheung, H. S.;Cushman, D. W. Biochem-
istry 1979, 18, 1427. (c) Park, J. D.;Kim, D. H. J. Med.
Chem. 2002, 45, 911.
7. In general, Zn(II) ion complexes may have coordination
numbers of 4, 5, and 6, although in the biological systems,
Zn(II) complexes having coordination number 4 are most
common (Dudev, T.;Lim, C . J. Am. Chem. Soc. 2000,
122, 11146).
phenyl-ethyl)-acetamide (1). Compound 7 (0.25 g, 1.0
mmol) was added to a solution of potassium carbonate
(0.027 g, 0.2 mmol) in MeOH/H2O (10 mL, 95/5). The
reaction mixture was stirred for 3 h at room temperature.
The reaction mixture was diluted with methylene chloride
and washed with 0.1 N hydrochloric acid solution, dried
over magnesium sulfate, and concentrated under reduced
pressure. The crude product was purified by column
chromatography (EtOAc–hexane=1/1) to afford the pro-
duct (0.18 g, 85%) as a white solid. Mp 70–71 ꢀC;[ a]20
1
À18.8 (c 1, CHCl3);IR (KBr) cm À1; H NMR (CDCl3) d
7.28 (m, 5H), 5.74 (d, 1H), 4.38 (m, 1H), 2.94 (m, 2H),
2.64 (m, 2H), 1.95 (s, 3H), 1.32 (t, 1H); 13C NMR
(CDCl3) d 170.32, 137.95, 129.95, 129.34, 127.43, 51.65,
38.96, 28.46, 24.05. HRMS-EI m/z: calcd for C11H15NOS,
209.0874, found: 209.0872. (S)-N-(1-Mercaptomethyl-2-
phenyl-ethyl)-benzamide (2) was prepared in a similar
fashion from 11. Yield 59%. Mp 135–136 ꢀC;[ a]20 À19.7
1
(c 1, CHCl3);IR (KBr) 3299, 1632, 1530 cm À1; H NMR
(CDCl3) d 7.73–7.24 (m, 10H), 6.34 (d, 1H), 4.56 (m, 1H),
3.03 (m, 2H), 2.76 (m, 2H), 1.38 (t, 1H); 13C NMR
(CDCl3) d 167.66, 137.86, 135.11, 132.30, 129.44, 129.31,
127.55, 52.00, 39.02, 28.55. HRMS-EI m/z: calcd for
C16H17NOS, 271.1031, found: 271.1029.
12. Kocienski, P. J.;Cernigliaro, G.;Feldstein, G. J. Org.
Chem. 1977, 42, 353.
8. Bock, C. W.;Tatz, A. K.;Glusker, J. P. J. Am. Chem.
Soc. 1995, 177, 3754.
13. (S)-2-(Benzyloxycarbonylamino)-3-phenyl-1-propanol [(S)-
5]. To an ice-chilled stirred solution of 8 (13.6 g, 46 mmol)
and N-hydroxysuccinimide (5.52 g, 48 mmol) in ethylene
glycol dimethyl ether (80 mL) was added 1,3-
dicyclohexylcarbodiimide (9.88 g, 48 mmol). The mixture
was stirred overnight and then filtered through a Celite
pad, the filtrate was evaporated under reduced pressure,
and the residue was dissolved in THF (80 mL). To the ice-
chilled stirred solution of THF was added sodium boro-
hydride (4.53 g, 120 mmol), and stirred for 18 h, then
poured into vigorously stirred ice-water. The product was
extracted with ethyl acetate, and the extract was washed
with water and brine, dried over magnesium sulfate, and
concentrated to give (S)-5 (7.3 g, 92%) mp 89–91 ꢀC. lit
(Loeffler, L. J.;Sajadi, Z.;Hall, I. H. J. Med. Chem. 1977,
20, 1578) mp 89–90 ꢀC. (S)-(1-Benzyl-2-bromoethyl)-car-
bamic-acid, benzyl ester [(S)-9]. To an ice-chilled stirred
solution of (S)-5 (13.6 g, 47.7 mmol) and triphenylphos-
pine (13.75 g, 52.4 mmol) in methylene chloride (80 mL)
was added tetrabromomethane (17.4 g, 52.4 mmol). The
resulting mixture was stirred overnight and concentrated
under reduced pressure. The crude product was purified
by column chromatography (EtOAc–hexane=1/4) to
9. (a) Kimura, E.;Shiota, T.;Koike, T.;Shiro, M.;Kodama,
M. J. Am. Chem. Soc. 1990, 112, 5805. (b) Groves, J. T.;
Olson, I. R. Inorg. Chem. 1985, 24, 2715.
10. Fournie-Zaluski, M. C.;Coric, P.;Turcaud, S.;Bruets-
chy, L.;Lucas, E.;Noble, F.;Roques, B. P.
Chem. 1992, 35, 1259.
J. Med.
11. Thioactic acid, (S)-(2-acethylamino-3-phenyl-propyl) ester
(7). Thioacetic acid, (S)-(2-tert-butoxycarbonylamino-3-
phenylpropyl) ester (Fournib-Zaluski, M.-C.;Coric, P.;
Turcaud, S.;Bruetschy, L.;Lucas, E.;Noble, F.;Roques,
B. P. J. Med. Chem. 1992, 35, 1259) (4.0 g, 12.93 mmol)
was added to a solution of saturated hydrochloric acid in
ethyl acetate (80 mL). The reaction mixture was stirred
overnight, evaporated under reduced pressure, and the
residue was dissolved in methylene chloride (50 mL). To
the solution was added triethylamine (0.64 g, 6.23 mmol)
and acetic anhydride (0.35 g, 3.39 mmol) at 0 ꢀC, then
stirred for 1 h at room temperature. The reaction mixture
was washed with 0.1 N hydrochloric acid solution, 1 N
sodium bicarbonate solution, dried over magnesium sul-
fate, and concentrated under reduced pressure. The crude
product was purified by recrystallization (toluene/hexane)

M. S. Han et al. / Bioorg. Med. Chem. Lett. 14 (2004) 701–705
705
afford 9 (11.95 g, 72%). Mp 69–70 ꢀC;[ a]20 À5.3 (c 1,
3.10 (m, 1H), 2.87 (m, 1H), 2.79 (m, 2H), 2.28 (m, 3H),
2.03 (m, 1H), 1.76 (m, 3H); 13C NMR (DMSO, 100 ꢀC) d
193.87, 170.76, 153.62, 137.51, 137.51, 136.51, 128.33,
127.51, 127.39, 126.85, 126.59, 125.42, 65.38, 59.54, 49.15,
46.17, 32.30, 29.59, 22.54;HRMS-EI m/z: calcd for
C24H28O4N2S, 440.1770, found: 440.1765. (2R,3S)-2-(1-
Benzyl-2-mercapto-ethylcarbamoyl)-pyrrolidine-1-carboxylic
acid, benzyl ester (4). Compound 12 (1.2 g, 2.72 mmol)
was added to a solution of potassium carbonate (0.07 g,
0.5 mmol) in MeOH/H2O (50 mL, 95/5). The reaction
mixture was stirred for 3 h at room temperature, then
diluted with methylene chloride, washed with 0.1 N
hydrochloric acid solution, dried over magnesium sulfate,
and concentrated under reduced pressure. The crude pro-
duct was purified by column chromatography (EtOAc–
hexane=1/1) to afford 4 (0.68 g, 63%). Mp 87–89 ꢀC;
1
CHCl3);IR (KBr) 3321, 1698 cm À1; H NMR (CDCl3) d
7.30 (m, 10H), 5.09 (s, 2H), 5.05 (s, 1H), 4.12 (m, 1H),
3.40 (dd, 2H), 2.91 (m, 2H); 13C NMR (CDCl3) d 155.89,
137.19, 136.65, 129.66, 129.18, 128.99, 128.65, 128.52,
127.37, 67.34, 52.43, 39.17, 37.43. Anal. calcd for
C17H18BrNO2: C, 58.63;H, 5.21;N, 4.02. Found: C,
58.51;H, 5.20;N, 4.08. (S)-2-[(benzyloxycarbonyl)amino]-
3-phenylpropyl ethanethioate [(S)-10]. Potassium thioace-
tate (3.07 g, 27 mmol) was added to a solution of (S)-9
(7.8 g, 22 mmol) in DMF and the reaction mixture was
stirred for 1 h at room temperature. The reaction mixture
was diluted with ethyl acetate and washed with 5% aqu-
eous solution of sodium thiosulfate to remove DMF. The
organic layer was washed with 0.1 N hydrochloric acid
solution, dried over magnesium sulfate, and concentrated
under reduced pressure to afford the product (7.5 g, 98%).
Mp 89–90 ꢀC, Lit. (Higashiura, K.;Ienga, K. J. Org.
Chem. 1992, 57, 764) mp 89–90 ꢀC;[ a]20 À6.2 (c 1,
1
[a]20 À88 (c 0.8, CHCl3);IR (KBr) 2551, 1691 cm À1; H
NMR (DMSO, 100 ꢀC) d 7.28 (m, 10H), 5.06 (m, 2H),
4.20 (m, 1H), 3.41 (t, 1H), 2.49 (m, 2H), 2.18 (m, 2H),
2.12 (m, 1H), 1.93 (m, 1H), 1.78 (m, 3H); 13C NMR
(CDCl3) d 170.88, 153.69, 137.83, 136.49, 128.34, 127.54,
127.41, 126.86, 126.62 125.37, 65.43, 59.62, 51.88, 46.22,
42.64, 37.97, 27.12, 22.60;HRMS-EI m/z: calcd for
C22H26N2O3S, 398.1664, Found: 398.1664.
1
CHCl3);IR (KBr) 3333, 1692 cm À1; H NMR (CDCl3) d
7.29 (m, 10H), 5.07 (s, 2H), 4.88 (m, 1H), 4.05 (m, 1H),
2.94 (m, 4H), 2.32 (s, 3H). 13C NMR (CDCl3) d 196.31,
156.27, 137.55, 136.99, 129.77, 129.02, 128.92, 128.48,
128.41, 127.16, 67.01, 52.94, 40.82, 33.15, 30.99. (S)-(1-
Benzyl-2-thiolethyl)-carbamic acid, benzyl ester [(S)-3].
Compound (S)-10 (0.4 g, 1.14 mmol) was added to a
solution of potassium carbonate (0.03 g, 0.23 mmol) in
MeOH/H2O (50 mL, 95/5). The reaction mixture was
stirred for 3 h at room temperature, then diluted with
methylene chloride, and washed with 0.1 N hydrochloric
acid solution, dried over magnesium sulfate, and con-
centrated under reduced pressure. The crude product was
purified by column chromatography (EtOAc–hexane=1/
4) to afford (S)-3 (0.23 g, 68%) Mp 56–57 ꢀC;[ a]20 À13.0
17. DelMar, E. G.;Largman, C.;Brodrick, J. W.;Giokas,
M. C. Anal. Biochem. 1979, 99, 316.
18. Determination of IC50 values: Assay mixtures were pre-
pared by dissolving Suc-AAPF-pNA (300 mM), zinc
chloride (500 mM), and various concentrations of the
inhibitor in pH 7.5 buffer (50 mM CaCl2, 50 mM
TRIZMA). Enzyme stock solution was added to the assay
mixture to afford a final concentration of 0.4 mg/mL.
Residual enzyme activities were determined by using
initial rates monitored at 405 nm at 25 ꢀC. IC50 of inhibi-
tors were determined by plotting the percentage of resi-
dual enzyme activity versus the concentration of
inhibitors.
À1
;
1H
(c 0.67, CHCl3);IR (KBr) 3323, 2550, 1697 cm
NMR (CDCl3) d 7.27 (m, 10H), 5.08 (s, 2H), 4.95 (d, 1H),
4.07 (m, 1H), 2.87 (m, 2H), 2.65 (m, 2H), 1.33 (t, 1H); 13
C
NMR (CDCl3) d 156.07, 137.64, 136.90, 129.61, 129.04,
128.89, 128.49, 128.39, 127.13, 67.17, 53.75, 39.24, 28.56;
HRMS-EI m/z: calcd for C17H19NO2S, 301.1137, Found:
301.1135.
19. (a) Mock, W. L.;Wang, L. Chem. Biophys. Res. Commun.
1999, 257, 239. (b) Han, M. S.;Kim, D. H. Bioorg. Med.
Chem. Lett. 2001, 11, 1425.
20. a-CT is inhibited significantly by Cu(II) ion alone: in the
presence of Cu(II) ion at 2 mM, the activity of a-CT is
reduced by 30%.
14. Pfeiffer, F. R.;Chambers, P. A.;Hilbert, E. E.;Woodward,
P. W.;Ackerman, D. M. J. Med. Chem. 1984, 27, 325.
15. Higashiura, K.;Ienaga, K. J. Org. Chem. 1992, 57, 764.
16. (2R,3S)-2-(2-Acetylsulfanyl-1-benzyl-ethylcarbamoyl)-pyr-
rolidine-1-carboxylic acid benzyl ester (12). To the mixture
of 2-(1-benzyl-2-hydroxy-ethylcarbamoyl)-pyrrolidine-1-
carboxylic acid, benzyl ester (Pfeiffer, F. R.;Chambers, P.
A.;Hilbert, E. E.;Woodward, P. W.;Ackerman, D. M. J.
Med. Chem. 1984, 27, 325) (0.3 g, 0.73 mmol) and triphen-
ylphospine (0.23 g, 0.86 mmol) in THF (10 mL) were
added dropwise dimethyl azodicarboxylate (0.32 mL of
40% solution in toluene, 0.86 mmol) and thioacetic acid
(0.06 mL, 0.86 mmol), and stirred for 1 h. The reaction
mixture was evaporated under reduced pressure to give a
reddish oil which was purified by column chromato-
graphy (EtOAc–hexane=1/2) to yield 12 (0.26 g, 76%).
Mp 75–77 ꢀC;[ a]20 À77 (c 0.8, CHCl3);IR (KBr) 3309,
21. Schellenberger, V.;Braune, K.;Hofmann, H.-J.;Jakubke,
H.-D. Eur. J. Biochem. 1991, 199, 623.
22. (a) Segel, D. M. Biochemistry 1972, 11, 349. (b) Segel,
D. M.;Powers, J. C.;Cohen, G. H.;Davies, D. R.;Wil-
cox, P. E. Biochemistry 1971, 10, 3728. (c) Baumann,
W. K.;Bizzoreo, S. A.;Dutler, H. Eur. J. Biochem. 1973,
39, 381.
23. Vahrenkamp, H. Acc. Chem. Res. 1999, 32, 589.
24. (a) Lenter, C., Ed. Geigy Scientific Tables, Vol. 3. Physical
Chemistry Composition of Blood Hematology Somato-
metric Data;Ciba-Geigy: Basle, Switzerland, 1984;p 87.
(b) Hambridge, K. M.;Casey, C. E.;Krebs, N. E. In
Trace Elements in Human and Animal Nutrition, 5th ed.;
Mertz, W., Ed.;Academic: Orlando, FL, 1996;Vol. 2, p
15.
ꢀ
1
1691 cmÀ1; H NMR (DMSO, 100 C) d?7.28 (m, 10H),
25. Mulay, I. L.;Roy, R.;Knox, B. E.;Suhr, N. H.;Delaney,
W. E. J. Natl. Cancer Inst. 1971, 47, 1.
5.07 (m, 2H), 4.17 (m, 1H), 4.15 (m, 1H), 3.41 (m, 2H),