Sulfamide-based inhibitors for carboxypeptidase A. Novel type transition state analogue inhibitors for zinc proteases

Article abstract of DOI:10.1021/jm020258v

N-Sulfamoylphenylalanine and its derivatives having varied alkyl groups on the terminal amino group were designed rationally as transition state analogue inhibitors for carboxypeptidase A (CPA) and synthesized. In CPA inhibitory assays the parent compound having the (S)-configuration, i.e., (S)-1a, showed potent inhibitory activity with the K_i value of 0.64 μM. Its enantiomer was shown to be much less potent (K_i = 470 μM). Introduction of an alkyl group such as methyl or isopropyl group on the terminal amino group of (S)-1a lowered the inhibitory potency drastically. Introduction of a methyl group on the internal amino group of (S)-1a also caused a drastic reduction of the inhibitory activity. The structure of the CPA-(S)-1a complex determined by single-crystal X-ray diffraction reveals that the sulfamoyl moiety interacts with the zinc ion and functional groups at the active site of CPA, which is reminiscent of the postulated stabilization mode of a tetrahedral transition state in the CPA-catalyzed hydrolysis of a peptide substrate. On the basis of the design rationale and the binding mode of (S)-1a to CPA shown by X-ray crystallographic analysis, the present inhibitors are inferred to be a novel type of transition state analogue inhibitor for CPA.

Full text of DOI:10.1021/jm020258v

J . Med. Chem. 2002, 45, 5295-5302
5295
Su lfa m id e-Ba sed In h ibitor s for Ca r boxyp ep tid a se A. Novel Typ e Tr a n sition
Sta te An a logu e In h ibitor s for Zin c P r otea ses
J ung Dae Park,† Dong H. Kim,†,* Seung-J un Kim,‡ J oo-Rang Woo,‡ and Seong Eon Ryu‡,*
Center for Integrated Molecular Systems, Division of Molecular and Life Sciences, Pohang University of Science and
Technology, San 31 Hyoja-dong, Namku, Pohang 790-784 Korea, and Center for Cellular Switch Protein Structure,
Korea Research Institute of Bioscience and Biotechnology, 52 Euh-eun-dong, Yusong-gu, Deajeon 305-806 Korea
Received J une 19, 2002
N-Sulfamoylphenylalanine and its derivatives having varied alkyl groups on the terminal amino
group were designed rationally as transition state analogue inhibitors for carboxypeptidase A
(CPA) and synthesized. In CPA inhibitory assays the parent compound having the (S)-
configuration, i.e., (S)-1a , showed potent inhibitory activity with the Ki value of 0.64 µM. Its
enantiomer was shown to be much less potent (Ki ) 470 µM). Introduction of an alkyl group
such as methyl or isopropyl group on the terminal amino group of (S)-1a lowered the inhibitory
potency drastically. Introduction of a methyl group on the internal amino group of (S)-1a also
caused a drastic reduction of the inhibitory activity. The structure of the CPA‚(S)-1a complex
determined by single-crystal X-ray diffraction reveals that the sulfamoyl moiety interacts with
the zinc ion and functional groups at the active site of CPA, which is reminiscent of the
postulated stabilization mode of a tetrahedral transition state in the CPA-catalyzed hydrolysis
of a peptide substrate. On the basis of the design rationale and the binding mode of (S)-1a to
CPA shown by X-ray crystallographic analysis, the present inhibitors are inferred to be a novel
type of transition state analogue inhibitor for CPA.
Zinc proteases are a family of enzymes having a
catalytically essential zinc ion at their active sites.
These enzymes play key roles in a wide variety of
physiological and pathological processes.1 Among the
enzymes, carboxypeptidase A (CPA) has been most
extensively investigated and serves as a leading repre-
sentative.2 Furthermore, CPA has been much used as
a model target enzyme3 for developing inhibitor design
strategies that can be applied to zinc proteases of
pathological importance such as angiotensin converting
enzyme4 and matrix metalloproteases.5
CPA cleaves preferentially the peptide bond of the
C-terminal amino acid having an aromatic side chain.
The X-ray crystal structure of the enzyme has been
elucidated to the resolution of 1.54 Å,6 and its catalytic
mechanism has been the subject of numerous investiga-
tions.2 The catalytically essential zinc ion is coordinated
to side chain functional groups of His-69, Glu-72, and
His-196. A water molecule is loosely bound to the zinc
ion as the fourth ligand. The most important residues
present at the active site are Glu-270 and Arg-145. The
former is intimately involved in the hydrolysis of
substrate, and the latter forms hydrogen bonds with the
C-terminal carboxylate of substrate. In addition, there
is present a hydrophobic pocket, the primary function
of which is to accommodate the aromatic ring in the side
chain of the P1′ residue of the substrate. It is generally
believed that the CPA-catalyzed hydrolysis of peptide
substrates is initiated by the attack of the zinc-bound
water molecule on the scissile peptide bond to generate
a tetrahedral transition state which is known to be
stabilized by the zinc ion and the guanidinium moiety
of Arg-127.2,7
Mechanistic theories for enzymic reactions conspire
that chemically stable analogues of a transition state
generated along the reaction path of an enzymic reaction
would bind tightly to the enzyme, hampering the
catalytic activity,8 and this proposition has been suc-
cessfully exploited.9 Thus, for example, in the case of
CPA, the substrates in which the scissile peptide bond
is replaced with a tetrahedral phosphorus ester or amide
moiety were shown to be potent inhibitors for the
enzyme. The X-ray crystal structures of the CPA
complexes formed with these inhibitors revealed that
the phosphorus moieties bear close structural resem-
blances to the transition state postulated for the enzy-
mic hydrolytic reaction.10 In addition to the phosphorus
ester and amides, sulfoximine,11 sulfodiimine,11 and
sulfonimidamide12 moieties have also been incorporated
into substrate analogues of CPA to obtain potent inhibi-
tors for CPA. We wish to report herein a novel type of
transition state analogue inhibitors for CPA, in which
a readily obtainable sulfamide moiety is incorporated
into the basic structural frame of CPA substrate.
Resu lts a n d Discu ssion
Although sulfamide was first reported in 1892 by
Traube who prepared it from sulfuryl chloride and
gaseous ammonia,13 it received only scant attention.
Recently, however, the utility of sulfamide as an isostere
for a peptide bond has been recognized.14 Sulfamide is
a water soluble and chemically stable compound, and
its X-ray crystal structure showed that the compound
has the shape of tetrahedron with its sulfur atom
occupying the center.15 The nitrogen atoms in sulfamide
are conceivably sufficiently basic to allow metal ion
* To whom correspondence should be addressed (E-mail: dhkim@
postech.ac.kr or ryuse@mail.krib.re.kr).
† Pohang University of Science and Technology.
‡ Korea Research Institute of Bioscience and Biotechnology.
10.1021/jm020258v CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/23/2002

5296 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24
Park et al.
Sch em e 1a
Sch em e 2a
a
Reagents, conditions, and yield: (a) (i) BnOH, CH2Cl2, 0 °C,
a
Reagents, conditions, and yield: (a) TMSCl (4 equiv), MeOH,
rt, 4 h, 98%; (b) CbzNH2SO2Cl prepared from OCNSO2Cl and
BnOH (CH2Cl2, 0 °C, 30 min), Et3N, CH2Cl2, rt, 6 h, 88%; (c) (i)
H2, Pd-C, MeOH, rt, 2 h; (ii) 1 N LiOH, MeOH, rt, 2 h, 91%.
30 min; (ii) phenylalanine benzyl ester, Et3N, CH2Cl2, rt, 1 h, 96%;
(b) H2, Pd-C, MeOH, rt, 1 h, quantitative; (c) ROH (1.1 equiv),
PPh3 (1.5 equiv), DIAD (1.5 equiv), CH2Cl2, rt, 1-12 h, 69-93%.
coordination. From the physicochemical properties, mo-
lecular geometry, and electronic feature, it was inferred
that sulfamide would serve as a viable mimic for the
tetrahedral transition state postulated for the proteoly-
sis catalyzed by CPA, and its incorporation into sub-
strate molecule would result in potent inhibitors for the
enzyme. Compounds 1a -f have been synthesized and
evaluated as inhibitors of CPA. We have also prepared
and tested N-sulfamoylated derivatives of â-phenylala-
nine, 2, as inhibitors for CPA.
F igu r e 1. The Dixon plot for the inhibition of CPA-catalyzed
hydrolysis of ClCPL by (S)-1a at pH 7.5. The inset represents
the replot of slopes in the Dixon plot against the corresponding
1/[S].
2 was prepared as its methyl ester according to the
method reported by J in and Kim:18 (3R)-N-Benzyloxy-
3-benzyl-2-azetidine (5) that was obtained from 3-phen-
ylpropanoic acid in eight steps was treated with metha-
nol in the presence of TMSCl to give 6. The latter was
then allowed to react with N-(benzyloxycarbonyl)ami-
nosulfonyl chloride that was obtained by the reaction
of chlorosulfonyl isocyanate with benzyl alcohol to give
7. The subsequent hydrogenolysis followed by alkaline
hydrolysis using lithium hydroxide afforded (R)-2
(Scheme 2). In an analogous fashion (S)-2 was synthe-
sized.
In h ibition of CP A a n d Str u ctu r e-Activity Re-
la tion sh ip s. The compounds thus synthesized were
evaluated as inhibitors for CPA using O-(trans-p-chlo-
rocinnamoyl)-L-phenyllactic aicd (ClCPL)19 as substrate
at pH 7.5, and the inhibitory constants (Ki values) were
estimated from the respective Dixon plot20 (Figure 1)
and collected in Table 1. As can be seen in the inset of
Figure 1, (S)-1a is a competitive inhibitor for CPA.21 The
unsubstituted N-sulfamoylPhe having the (S)-configu-
ration, i.e., (S)-1a , binds CPA most tightly with the Ki
value of 0.64 µM. The binding affinity of its enantiomer
is reduced dramatically to the Ki value of 470 µM. Since
Ch em istr y. Chlorosulfonyl isocyanate was used for
the N-sulfamoylation of phenylalanine benzyl ester:
Addition of benzyl alcohol to a chlorosulfonyl isocyanate
solution in dry methylene chloride generated benzyl
N-chlorosulfonylcarbamate which was then allowed to
react with (S)-phenylalanine benzyl ester in the pres-
ence of an equivalent amount of triethylamine to obtain
2 in 96% yield. Hydrogenolysis of 2 using 10% Pd-C
provided (S)-1a in quantitative yield (Scheme 1). In a
similar fashion, (R)-1a was prepared. N-(N-Alkylsulfa-
moyl)phenylalanines, (S)-1b and (S)-1d -f were pre-
pared in 69-93% yield by allowing 2 to react with the
appropriate alcohol under the Mitsunobu conditions16
followed by hydrogenolysis. Likewise, (S)-1c was syn-
thesized using N-methylphenylalanine methyl ester17
in place of phenylalanine benzyl ester in the above
reactions. In this case, the Cbz group was removed
under the hydrogenolysis conditions and the methyl
ester moiety was hydrolyzed with 1 N lithium hydroxide
solution. â-Phenylalanine required for the synthesis of

Sulfamide-Based Inhibitors for Carboxypeptidase A
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24 5297
Ta ble 2. Crystallographic Data of Inhibitor A
Ta ble 1. Inhibitory Potencies (Ki values) of
N-Sulfamoylphenylalanines for CPA Inhibition
space group
P21
compd no.
Ki (µM)a
unit cell
a, b, c (Å)
51.69, 60.47, 47.26
(S)-1a
(R)-1a
(S)-1b
(S)-1c
(S)-1d
(S)-1e
(S)-1f
(S)-2
0.65 ( 0.05b
470 ( 110
180 ( 50
3500 ( 900
610 ( 130
35 ( 21
180 ( 60
2900 ( 930
1400 ( 190
R, â, γ (°)
90, 82.55, 90
99-2.0
19491
99.2
resolution range (Å)
number of unique reflections
overall completeness (%)
Rmerge (%)a
10.8
number of atoms
protein atoms
nonprotein atoms
Rcryst (%)b
2437
165
(R)-2
17.5
20.8
0.005
1.2
a
b
Rfree (%)c
Ki values are expressed as mean ( SEM (n ) 3). Ki value of
1.6 ( 0.3 µM was obtained when Hipp-Phe was used as the
substrate.
rms deviations
bonds (Å)
angles (deg)
dihedrals (deg)
impropers (deg)
23.2
0.71
most of the inhibitory activity of 1 was found to be
associated with the enantiomer 1a that belongs to the
L-series, structural modifications for the improvement
of the inhibitory activity were performed with N-
sulfamoylPhe having the (S)-configuration. The S1 site
of CPA is known to consist of a cavity that can
accommodate a hydrophobic side chain of amino acids
such as Phe and Leu.10c Accordingly, we have introduced
varied alkyl groups on the terminal amino nitrogen of
(S)-1. However, contrary to the expectation, the substi-
tution attenuated the inhibitory activity by 2 orders of
magnitude except in the case of the benzyl. The drastic
reduction of the binding affinity caused by the alkyl
substitution may be due to unfavorable steric interac-
tions of the alkyl group possibly with the zinc ion at
the active site. The substitution of the methyl group on
the nitrogen with a benzyl group to give 1e, i.e., an
introduction of a benzene ring on the methyl group of
1b, improved the binding affinity by 5-fold, which may
be rationalized on the ground that the benzene ring may
undergo favorable interactions with the side chains of
the amino acid residues that constitute the S1 pocket.
Introduction of a methyl group at the internal nitrogen
to yield (S)-1c reduced the binding affinity by 3 orders
of magnitude. The drastic reduction of the binding
affinity shown by (S)-1c may be explained on the basis
that the methyl group would experience a severe steric
hindrance possibly by the phenolic hydroxyl of the down
positioned Tyr-248 and the carboxylate of Glu-270 upon
its binding to the enzyme. Some of â-phenyalanine
derivatives are known to be potent inhibitors for CPA.22
It was thus thought to be of interest to evaluate
N-sulfamoylated â-phenylalanine (2) as a CPA inhibitor.
Both enantiomers of 2 were, however, found to be only
marginally active as inhibitors for CPA. As with N-
sulfamoylated â-Phe, (R)-2, that belongs to the L-series
is 2-fold more potent than its enantiomer. The limited
study for the SAR tends to suggest that the improve-
ment for the inhibitory activity of the parent compound,
(S)-1a , may only be realized by incorporating substit-
uents that can interact with cavities at the S1 subsite
and beyond.
a
Rmerge ) Σi|Ii
-
I
/Σ I |, where I is the intensity for the ith
measurement of an equivalent reflection with the indices h,k,l.
b
Rcryst ) Σ|Fo - Fc|/Σ Fo, where Fo and Fc are the observed and
calculated structure factor amplitudes, respectively. c The Rfree
value was calculated from 5% of all data that were not used in
the refinement.
1a and the mode of binding of the inhibitor to CPA.
Distances of important interactions between CPA and
the inhibitor in the complex are estimated from Figure
2 and listed in Table 3.
The carboxylate of (S)-1a forms a salt bridge with the
guanidinium moiety of Arg-145 with bond distances of
2.93 and 2.73 Å. The amide group of the side chain of
Asn-144 forms a hydrogen bond (2.87 Å) to O2 of the
carboxylate. The aromatic side chain of Tyr-248 is found
in the “so-called” down position, and its phenolic oxygen
is separated from one of the carboxylate oxygen atoms
of the inhibitor by 2.60 Å to form a hydrogen bond. This
type of hydrogen bonding is commonly observed in X-ray
crystal structures of CPA‚inhibitor complexes. For
example, the phenolic oxygen atom of the down posi-
tioned Tyr-248 in the complex of CPA‚Gly-L-Tyr, a
slowly hydrolyzed substrate of CPA, was shown to be
separated by 2.8 Å from one of the terminal carboxylate
oxygens of the dipeptide.24 The benzene ring of (S)-1a
is anchored in the S1′ hydrophobic pocket, the primary
substrate recognition site for CPA. One (O4) of the two
oxygen atoms of the sulfamide moiety in (S)-1a is
engaged in a hydrogen bonding with a nitrogen atom
of the guanidinium group in Arg-127 with the bond
distance of 2.76 Å. The binding mode of sulfamide
moiety in (S)-1a is very reminiscent of those seen in
CPA complexes formed with phosphonate-bearing tran-
sition state analogues. For example, in the CPA com-
plexes formed with Cbz-Ala-GylP(O)Phe, Cbz-Ala-AlaP-
(O)Phe, or Cbz-Phe-ValP(O)Phe, the guanidinium
nitrogen of Arg-127 was shown to interact with one of
the phosphinyl oxygens of the phosphonate.10c The other
oxygen atom in the sulfamide moiety is shown to rest
in an open space exposed to bulk water, being separated
from the oxygen atoms of Glu-270 carboxylate by 4.13
and 4.28 Å. The Glu-270 carboxylate forms a hydrogen
bond with N1 of the sulfamide: one of the Glu-270
carboxylate oxygens is separated from the N1 of the
sulfamide moiety by 2.87 Å. It is also noteworthy that
not an oxygen but the terminal nitrogen atom (N2) of
sulfamide coordinates to the zinc ion at the active site
of CPA with the bond distance of 1.92 Å. The binding
mode of (S)-1a to CPA is schematically depicted in
X-r a y Str u ctu r a l An a lysis of CP A‚(S)-1 Com -
p lex.23 Crystal structure of the CPA‚(S)-1a complex was
determined at 2.0 Å resolution by using the native CPA
structure (pdb code: 5CPA) as the starting model (Table
2). The final model including all residues of CPA and
the inhibitor is very well defined with the Rcryst and Rfree
values of 17.5% and 20.8%, respectively. Figure 2 depicts
the stereoview of the difference electron density in the
region of the active site of CPA that is occupied by (S)-

5298 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24
Park et al.
F igu r e 2. Difference electron density map for CPA‚(S)-1a complex generated with Fourier coefficient |Fo| - |Fc| phases calculated
from the final model omitting the bound inhibitor.
Ta ble 3. Selected CPA‚(S)-1a Interactions
Ta ble 4. Bond Angles and Bond Lengths between Atoms in
the Sulfamide Moiety of (S)-1
atom in (S)-1a
enzyme residue
separation (Å)
bond angle (deg)
bond length (Å)
O1
O2
O1
O2
O2
O3
O3
O4
O4
O4
N1
N1
N2
Arg-145 guanidinium N1
Arg-145 guanidinium N2
Asn-144 side chain amide N
Try-248 phenolic O
Arg-127 guanidinium N1
Glu-270 carboxylate O1
Glu-270 carboxylate O2
Arg-127 guanidinium N1
Arg-127 guanidinium N2
Zn2+
2.73
2.73
2.87
2.60
3.54
4.13
4.28
3.22
2.76
3.19
2.87
3.16
1.92
N1-S-O3
109.9
107.5
102.3
97.5
117.7
122.1
N1-S
1.75
1.70
1.43
1.43
N1-S-O3
N2-S-O3
N2-S-O4
N1-S-N2
O3-S-O4
N2-S
O3-S
O4-S
The sulfamoyl moiety in (S)-1a bears an ill-shaped
tetrahedral configuration. Bond distances and angles in
the moiety are collected in Table 4. On the basis of the
structural feature and the binding mode of the sulfa-
moyl moiety in (S)-1a to CPA, it may be concluded that
(S)-1a is a transition state analogue inhibitor for CPA
as it has been so designed. The observed stereochemical
preference in the binding of 1a to CPA, i.e., the (S)-
isomer of 1a that belongs to the L-series binds CPA more
tightly than the (R)-diastereoisomer by 2 orders of
magnitude is in agreement with the stipulation that
transition state analogue inhibitors exhibit the same
stereochemical preference as that of substrate in inhib-
iting the target enzyme.25 The inhibitor-bound CPA
shows no significant changes in its conformation from
that of the native enzyme except that the rotation along
the CR-Câ bond of Tyr-248 to cause its aromatic ring
to move down from the surface of the enzyme molecule
toward the opening of the S1′ pocket and the movement
of Arg-127 side chain toward the sulfamide moiety to
form hydrogen bonds with one of sulfamide oxygens of
the inhibitor.
Glu-270 carboxylate O1
Try-248 phenolic O
Zn2+
F igu r e 3. Schematic representation of the binding mode of
(S)-1a to CPA.
Figure 3. The binding mode of (S)-1a to CPA is signifi-
cantly different from those of phosphorus transition
state analogue inhibitors: In the binding of phospho-
ramidate transition state analogue such as 810a the two
oxygen atoms of its tetrahedral phosphinyl group coor-
dinate to the zinc ion and one of these oxygens interacts
with Arg-127. The phosphonate transition state ana-
logue inhibitor such as 910b binds CPA with the both
oxygen atoms of its phosphinyl group coordinating to
the zinc ion. In this case, one of the phosphinyl oxygens
is also engaged in hydrogen bonding with the carboxy-
late of Glu-270.
Con clu sion
Transition state analogue inhibitors constitute an
important class of inhibitors whose binding affinities
usually surpass those of the other classes of reversible
inhibitors.26 This study demonstrated that the sulfamide
moiety would serve as a viable transition state mimic
when incorporated judiciously into substrate molecules
to afford potent inhibitors for zinc proteases. The
prototypical sulfamide-bearing transition state analogue
inhibitor, (S)-1a is 3-fold more potent in the inhibition
of CPA than the corresponding phosphonamidate type
transition state analogue inhibitor,9b suggesting that the
sulfamide moiety may possibly better mimic the tetra-
hedral transition state postulated for the proteolytic

Sulfamide-Based Inhibitors for Carboxypeptidase A
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24 5299
temperature and concentrated in vacuo. The crude product was
purified by column chromatography (EtOAc/n-hexane ) 1/8)
and recrystallized from ethyl acetate and n-hexane to afford
a white crystalline solid.
reaction path than the phosphonamidate. Sulfamide
bearing inhibitors are easily prepared and stable under
physiological conditions, which are important require-
ments with respect to developing enzyme inhibitors of
therapeutic uses. Investigation for the application of the
present design protocol to pathologically important zinc
proteases with the aim of obtaining therapeutically
useful agents is in progress.
(S)-N-(N-(Ben zyloxyca r b on yl)-N-m et h ylsu lfa m oyl)-
p h en yla la n in e Ben zyl Ester ((S)-4b). Compound (S)-4b was
prepared from (S)-3 and methanol. Yield, 69%; [R]20 +18.3°
D
(c 0.65, CHCl3); IR (neat) 1379, 1738 cm-1; H NMR (CDCl3,
1
300 MHz) δ 3.05 (d, 2H), 3.13 (s, 3H), 4.40 (m, 1H), 5.02 (m,
2H), 5.14 (m, 2H), 5.78 (d, 1H), 7.02-7.38 (m, 15H); 13C NMR
(CDCl3, 300 MHz) δ 34.8, 39.5, 58.2, 68.0, 69.4, 127.8, 128.7,
129.1, 129.8, 135.0, 135.2, 170.8. HRMS (FAB+) (M + H)+:
calcd for C25H26N2O6S, 483.1590; found 483.1590.
Exp er im en ta l Section
Melting points were determined on a Thomas-Hoover capil-
1
lary melting point apparatus and were uncorrected. H NMR
(S)-N-(N-(Ben zyloxyca r bon yl)-N-isop r op ylsu lfa m oyl)-
p h en yla la n in e Ben zyl Ester ((S)-4d ). Compound (S)-4d was
prepared from (S)-3 and isopropyl alcohol. Yield, 80%; mp 94-
and 13C NMR spectra were recorded with 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. 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). Mass spectra were obtained at Korea Basic
Science Institute, Daejeon, Korea. Elemental analyses were
performed at Center for Biofunctional Molecules, Pohang
University of Science and Technology, Pohang, Korea and
results were within ( 0.4% of the theoretical values.
95 °C; [R]20D +20.7° (c 1.03, CHCl3); IR (neat) 1366, 1740 cm-1
;
1H NMR (CDCl3, 300 MHz) δ 1.31 (t, 6H), 3.03-3.06 (m, 2H),
4.03-4.35 (m, 1H), 4.52-4.57 (m, 1H), 5.02 (s, 2H), 5.13 (q,
2H), 5.87 (d, 1H), 7.00-7.36 (m, 15H); 13C NMR (CDCl3, 300
MHz) δ 21.4, 21.5, 39.7, 52.7, 58.0, 68.0, 69.3, 127.7, 128.7,
129.0, 129.1, 129.2, 129.8, 135.0, 135.1, 135.2, 153.2, 170.7.
Anal. (C27H30N2O6S) C, H, N.
(S )-N -(N -Be n zyl-N -(b e n zyloxyca r b on yl)su lfa m oyl)-
p h en yla la n in e Ben zyl Ester ((S)-4e). Compound (S)-4e was
prepared from (S)-3 and benzyl alcohol. Yield, 93%; mp 82-
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. Carboxypeptidase A 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.
O-(trans-p-Chlorocinnamoyl)-L-phenyllactic acid (ClCPL) was
synthesized by the literature method19 and hippuryl L-phenyl-
alanine (Hipp-L-Phe) was purchased from Sigma Chemical Co.
An HP 8453 UV/VIS spectrometer was used in enzyme
inhibition kinetic studies.
83 °C; [R]20 -4.7° (c 0.98, CHCl3); IR (neat) 1368, 1735 cm-1
;
D
1H NMR (CDCl3, 300 MHz) δ 2.93 (m, 2H), 4.09 (m, 1H), 4.64-
4.85 (m, 2H), 4.99 (s, 2H), 5.07-5.19 (m, 2H), 5.76 (d, 2H),
6.93-7.32 (m, 20H); 13C NMR (CDCl3, 300 MHz) δ 39.4, 46.1,
51.3, 57.7, 67.9, 69.6, 127.7, 128.3, 128.8, 129.0, 129.8, 135.1,
137.3, 153.2, 170.6. Anal. (C31H30N2O6S) C, H, N.
(S)-N-(N-(Ben zyloxyca r bon yl)-N-p h en eth ylsu lfa m oyl)-
p h en yla la n in e Ben zyl Ester ((S)-4f). Compound (S)-4f was
prepared from (S)-3 and phenethyl alcohol. Yield, 90%; mp
82.5-83 °C; [R]20D +13.9° (c 1.03, CHCl3); IR (neat) 1368, 1729
1
cm-1; H NMR (CDCl3, 300 MHz) δ 2.84 (t, 2H), 3.03 (d, 2H),
(S)-N-(N-(Ben zyloxyca r b on yl)su lfa m oyl)p h en yla la -
n in e Ben zyl Ester ((S)-3). Benzyl alcohol (2.4 mL, 23 mmol)
was added slowly at 0 °C to the stirring chlorosulfonyl
isocyanate (2 mL, 23 mmol) solution in anhydrous dichlo-
romethane (20 mL), and the stirring was continued for 30 min
at 0 °C. Triethylamine (10 mL) in anhydrous dichloromethane
was added to the solution. The resulting mixture was then
added dropwise to an ice-chilled suspension of (S)-phenylala-
nine benzyl ester p-toluenesulfonate (10.3 g, 23 mmol) and
triethylamine (6 mL) in anhydrous dichloromethane (50 mL).
The solution thus obtained was stirred for 2 h and evaporated
under reduced pressure. The residue was dissoloved in ethyl
acetate (50 mL), washed successively with 1 N HCl solution
(50 mL × 3) and brine (50 mL × 3), and dried over anhydrous
MgSO4. The ethyl acetate solution was concentrated in vacuo
to afford a white solid, which was recrystallized from dichlo-
romethane to give (S)-3 as a white crystal (10.7 g, 96%): mp
109-110 °C; [R]25D +5.2° (c 1.06, CHCl3); IR (neat) 1360, 1741
cm-1; 1H NMR (CDCl3, 300 MHz) δ 3.07 (d, 2H), 4.55 (q, 1H),
5.03 (s, 2H), 5.06 (d, 2H), 5.29 (s, 1H), 5.77 (d, 1H), 7.02-7.63
(m, 15H); 13C NMR (D2O, 300 MHz) δ 39.4, 58.1, 68.1, 68.9,
127.7, 128.8, 129.1, 129.9, 135.0, 135.1, 151.3, 171.0. Anal.
(C24H24N2O6S) C, H, N.
(R)-N-(N-(Ben zyloxyca r b on yl)su lfa m oyl)p h en yla la -
n in e Ben zyl Ester ((R)-3). Compound (R)-3 was prepared
from (R)-phenylalanine benzyl ester p-toluenesulfonate in a
manner analogous to that used for the preparation of (S)-3.
[R]25D -5.8° (c 1.06, CHCl3). Mp and spectral data are identical
with those of (S)-3. Anal. (C24H24N2O6S) C, H, N.
Gen er a l P r oced u r e for Alk yla tion of (S)-N-(N-(Ben z-
yloxyca r bon yl)su lfa m oyl)p h en yla la n in e Ben zyl Ester
To P r ep a r e (S)-4b a n d (S)-4d -4f. To an ice-chilled solution
of triphenylphosphine (1.5 equiv) in anhydrous dichloromethane
(20 mL) was added slowly diisopropyl azodicarboxylate (1.5
equiv). To the resulting solution was added the mixture of (S)-3
and the appropriate alcohol (1.1 equiv) in anhydrous dichlo-
romethane (10 mL). The solution was stirred for 1 h at room
3.75 (t, 2H), 3.36 (q, 1H), 5.00 (m, 2H), 5.09 (q, 2H), 5.77 (d,
1H), 7.00-7.35 (m, 20H); 13C NMR (CDCl3, 300 MHz) δ 36.5,
39.5, 49.9, 58.2, 68.0, 69.4, 121.4, 126.3, 127.0, 127.7, 128.2,
128.7, 129.1, 129.4, 129.8, 135.1, 135.2, 138.2, 153.0, 170.7.
Anal. (C32H32N2O6S) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of N-(N-Alk yl)-
su lfa m oylp h en yla la n in es, ((S)-1a , (R)-1a , (S)-1b, a n d (S)-
1d -f). A solution of (S)-3, (R)-3, (S)-4b, or (S)-4d -f in MeOH
(20 mL) was stirred for 1 h under hydrogen atmosphere in
the presence of 10% Pd-C. The resulting mixture was filtered
and the filtrate was evaporated under reduced pressure to give
(S)-1a , (R)-1a , (S)-1b, and (S)-1d -f, respectively.
(S)-N-Su lfa m oylp h en yla la n in e Ben zyl Ester ((S)-1a ).
Compound (S)-1a was prepared from (S)-3 (quantitative). mp
113.5-114 °C; [R]20 +16.4° (c 0.22, MeOH); IR (neat) 1358,
D
1634 cm-1; 1H NMR (D2O, 300 MHz) δ 2.85-3.07 (m, 2H), 4.13
(q, 1H), 7.18-7.28 (m, 5H); 13C NMR (D2O, 300 MHz) δ 38.2,
57.8, 127.6, 129.0, 129.8, 136.6, 175.8. Anal. (C9H12N2O4S) C,
H, N.
(R)-N-Su lfa m oylp h en yla la n in e ((R)-1a ). Compound (R)-
1a was prepared from (R)-3 (quantitative). [R]20 -16.0° (c
D
0.53, MeOH). Mp and spectral data are identical with those
of (S)-1a . Anal. (C9H12N2O4S) C, H, N.
(S)-N-(N-Meth ylsu lfam oyl)ph en ylalan in e ((S)-1b). Com-
pound (S)-1b was prepared from (S)-4b. mp 93-94 °C; [R]20
D
-22.7° (c 0.3, CHCl3); IR (neat) 1331, 1746 cm-1
;
1H NMR
(CDCl3, 300 MHz) δ 2.35 (s, 3H), 2.97 (m, 1H), 3.21 (dd, 1H),
4.26 (q, 1H), 5.08 (d, 1H), 7.24-7.35 (m, 5H); 13C NMR (CDCl3,
300 MHz) δ 29.0, 38.9, 57.7, 127.6, 129.1, 130.0, 136.4, 176.6.
Anal. (C10H14N2O4S‚1/4H2O) C, H, N.
(S)-N-(N-Isop r op ylsu lfa m oyl)p h en yla la n in e ((S)-1d ).
Compound (S)-1d was prepared from (S)-4d (quantitative) and
isolated as a dicyclohexylamine salt. mp 198-200 °C; [R]20
D
+1.3° (c 0.54, CHCl3); IR (neat) 1371, 1730 cm-1
;
1H NMR
(CDCl3, 300 MHz) δ 0.95-1.09 (dd, 6H), 2.95-3.20 (m, 2H),
3.25 (m, 1H), 4.22 (q, 1H), 5.13 (d, 1H), 5.29 (d, 1H), 7.22-

5300 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24
Park et al.
7.34 (m, 5H); 13C NMR (CDCl3, 300 MHz) δ 23.8, 23.9, 39.1,
46.7, 57.5, 127.7, 129.1, 130.0, 136.0, 176.6. Anal. (C24H41N3O4S)
C, H, N.
(R)-6. [R]20 -25.7° (c 0.8, EtOH). Mp and spectral data are
D
identical with those of (R)-6. Anal. (C18H22ClNO3) C, H, N.
(R)-2-Ben zyl-3-(N-b en zyloxy-N-(N-b en zyloxyca r b on -
yl)su lfa m oyl)a m in op r op a n oic Acid Meth yl Ester ((R)-7).
Benzyl alcohol (0.13 mL, 1.2 mmol) was added slowly to an
ice-chilled chlorosulfonyl isocyanate (0.11 mL, 1.2 mmol)
solution in anhydrous dichloromethane (5 mL). After being
stirred for 30 min, the solution and triethylamine (0.5 mL) in
anhydrous dichloromethane were concurrently added dropwise
to an ice-chilled solution of (R)-6 (0.4 g, 1.2 mmol) in dichlo-
romethane (10 mL). The resulting solution was stirred for 6 h
and then evaporated under reduced pressure. The residue was
dissoloved in ethyl acetate (20 mL), washed successively with
1 N HCl solution (30 mL × 3) and brine (30 mL × 3), and
dried over anhydrous MgSO4. The solution was concentrated
(S)-N-(N-Ben zylsu lfa m oyl)ph en yla la n in e ((S)-1e). Com-
pound (S)-1e was prepared from (S)-4e (quantitative) and
isolated as a dicyclohexylamine salt. mp 201-202 °C; [R]20
D
-5.2° (c 0.52, CHCl3); IR (neat) 1324, 1729 cm-1
;
1H NMR
(CDCl3, 300 MHz) δ 2.88-3.15 (m, 2H), 3.70-3.96 (dd, 2H),
4.25 (q, 1H), 5.13 (d, 1H), 5.29 (d, H), 7.13-7.32 (m, 10H); 13
C
NMR (CDCl3, 300 MHz) δ 38.9, 47.2, 57.4, 127.8, 128.3, 129.1,
129.2, 130.0, 135.8, 136.8, 176.5. Anal. (C28H41N3O4S) C, H,
N.
(S)-N-(N-P h en eth ylsu lfa m oyl)p h en yla la n in e ((S)-1f).
Compound (S)-1f was prepared from (S)-4f. mp 116.5-117.5
°C; [R]20 -3.4° (c 1.04, CHCl3); IR (neat) 1317, 1731 cm-1; 1H
D
in vacuo to give the product as an oil (0.53 g, 88%): [R]25
D
NMR (CDCl3, 300 MHz) δ 2.64 (m, 2H), 2.89-3.17 (m, 4H),
4.19 (m, 1H), 5.00 (d, 1H), 5.29 (d, 1H), 7.08-7.32 (m, 10H);
13C NMR (CDCl3, 300 MHz) δ 35.9, 39.0, 44.3, 57.3, 127.2,
127.8, 129.1, 129.9, 135.7, 138.2, 176.6. Anal. (C17H20N2O4S)
C, H, N.
-18.9° (c 1.1, CHCl3); IR (neat) 1386, 1738 cm-1
;
1H NMR
(CDCl3, 300 MHz) δ 2.80 (m, 1H), 2.91 (m, 1H), 3.04 (m, 1H),
3.44 (d, 1H), 3.81 (q, 1H), 5.00 (d, 2H), 5.15 (s, 2H), 7.10-7.39
(m, 15H); 13C NMR (CDCl3, 300 MHz) δ 36.5, 46.3, 52.3, 55.5,
69.4, 80.0, 127.2, 129.0, 129.1, 129.2, 129.4, 129.5, 130.0, 134.8,
134.9, 136.8, 139.4, 153.2, 174.5.
(S)-N-Meth yl-N-su lfa m oylp h en yla la n in e ((S)-1c). Benz-
yl alcohol (0.4 mL, 3.9 mmol) was added slowly to an ice-chilled
chlorosulfonyl isocyanate (0.36 mL, 3.9 mmol) solution in
anhydrous dichloromethane (10 mL). After being stirred for
30 min, the solution and triethylamine (1.8 mL) in anhydrous
dichloromethane were concurrently added dropwise to an ice-
chilled solution of N-methylphenylalanine benzyl ester (0.75
g, 3.9 mmol) in dichloromethane (10 mL). The resulting
solution was stirred for 2 h and then evaporated under reduced
pressure. The residue was dissoloved in ethyl acetate (30 mL),
washed successively with 1 N HCl solution (50 mL × 3) and
brine (50 mL × 3), and dried over anhydrous MgSO4. The
solution was concentrated in vacuo to give (S)-N-(N-(benzyl-
oxycarbonyl)sulfamoyl)-N-methylphenylalanine methyl ester
(S)-2-Ben zyl-3-(N-b en zyloxy-N-(N-b en zyloxyca r b on -
yl)su lfa m oyl)a m in op r op a n oic Acid Meth yl Ester ((S)-7).
Compound (S)-7 was prepared from (S)-6 in a manner analo-
gous to that used for the preparation of (R)-7. [R]20 -19.6° (c
D
0.94, CHCl3). Mp and spectral data are identical with those of
(R)-7.
(R)-2-Ben zyl-3-(N-su lfam oyl)am in opr opan oic Acid ((R)-
2). A solution of (R)-7 (0.53 g, 1.03 mmol) in anhydrous MeOH
(10 mL) was stirred under hydrogen atmosphere in the
presence of 10% Pd-C (30 mg) for 2 h. The resulting mixture
was filtered, and the filtrate was evaporated under reduced
pressure to give (R)-2-benzyl-3-(N-sulfamoyl)aminopropanoic
acid methyl ester as an oil. The ester was dissolved in a
mixture of anhydrous MeOH (5 mL) and 1 N lithium hydroxide
solution (1.1 mL), and the resulting mixture was stirred for 2
h, then acidified with 1 N HCl solution and extracted with
ethyl acetate. The organic layer was evaporated under reduced
pressure to afford a white solid, which was recrystallized from
ethyl acetate to give a white solid (0.24 g, 91%). mp 111-113
as an oil (1.3 g, 82%): [R]25 -18.5° (c 0.41, CHCl3); IR (neat)
D
1359, 1746 cm-1; H NMR (CDCl3, 300 MHz) δ 2.93 (q, 1H),
1
3.00 (s, 3H), 3.28 (q, 1H), 3.63 (s, 3H), 4.88 (q, 1H), 5.09 (s,
2H), 7.19-7.38 (m, 10H); 13C NMR (D2O, 300 MHz) δ 32.2,
36.0, 52.9, 61.8, 68.7, 127.6, 128.8, 129.1, 129.4, 136.3, 151.2,
171.1.
A solution of (S)-N-(N-(benzyloxycarbonyl)sulfamoyl)-N-
methylphenylalanine methyl ester (1.3 g, 3.2 mmol) in anhy-
drous MeOH (20 mL) was stirred under hydrogen atmosphere
in the presence of 10% Pd-C (100 mg) for 2 h. The resulting
mixture was filtered, and the filtrate was evaporated under
reduced pressure to give N-methyl-N-sulfamoylphenylalanine
methyl ester as an oil. The ester was dissolved in a mixture of
anhydrous MeOH (20 mL) and 1 N lithium hydroxide solution
(3.2 mL), and the resulting mixture was stirred for 1 h, then
acidified with 1 N HCl solution and extracted with ethyl
acetate. The organic layer was evaporated under reduced
pressure to afford a white solid, which was recrystallized from
ethyl acetate to give a white crystal (0.82 g, quantitative). mp
°C; [R]20 -60.4° (c 0.65, CHCl3); IR (neat) 1341, 1693 cm-1
;
D
1H NMR (DMSO-d6, 300 MHz) δ 2.70 (m, 2H), 2.88 (m, 1H),
3.08 (m, 2H), 6.56 (s, 2H), 7.14-7.29 (m, 5H), 7.88 (t, 1H); 13
C
NMR (DMSO-d6, 300 MHz) δ 35.0, 42.5, 42.6, 127.6, 129.3,
129.5, 137.4, 173.2. Anal. (C10H14N2O4S) C, H, N.
(S)-2-Ben zyl-3-(N-su lfam oyl)am in opr opan oic Acid ((S)-
2). Compound (S)-2 was prepared from (S)-7 in a manner
analogous to that used for the preparation of (R)-2. [R]20
D
-59.1° (c 1.1, CHCl3). Mp and spectral data are identical with
those of (R)-2. Anal. (C10H14N2O4S) C, H, N.
Deter m in a tion of Ki Va lu e. Typically, the enzyme stock
solution was added to a solution containing ClCPL (final
concentrations: 50 µM and 100 µM) and inhibitor (five
different final concentrations in the range of 0.5Ki to 2Ki µM)
in 0.05 M Tris/0.5 M NaCl, pH 7.5 buffer (1 mL cuvette), and
the change in absorbance at 320 nm was measured im-
mediately. The final concentration of CPA was 12 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
versus the concentration of the inhibitor according to the
method of Dixon.20 The correlation coefficients for the Dixon
plots were above 0.990.
X-r a y Cr ysta llogr a p h y. The complex of CPA and (S)-1a
was prepared by incubating the protein and the inhibitor in a
1:10 molar ratio overnight at 4 °C. Crystals were grown by
the microdialysis method by equilibrating the protein-inhibi-
tor complex in a solution of 1.2 M LiCl and 20 mM Tris-HCl
buffer (pH 7.5) against a reservoir containing 0.2 M LiCl and
20 mM Tris-HCl buffer (pH 7.5) as described previously.6
Single crystals grew in the P21 space group with one molecule
in the asymmetric unit. Crystallographic data were collected
152-153 °C; [R]20 -108.8° (c 1, CHCl3); IR (neat) 1310, 1731
D
cm-1; 1H NMR (CDCl3, 300 MHz) δ 2.61 (s, 3H), 3.08 (m, 1H),
3.2 (m, 1H), 4.06 (q, 1H), 7.22-7.35 (m, 5H); 13C NMR (D2O,
300 MHz) δ 33.2, 37.1, 69.2, 127.9, 129.2, 130.0, 135.4, 168.2.
Anal. (C10H14N2O4S‚1/3H2O) C, H, N.
(R)-2-Ben zyl-3-(N-b en zyloxy)a m in op r op a n oic Acid
Meth yl Ester ((R)-6). To the solution of (R)-517 (2.2 g, 8.2
mmol) in anhydrous MeOH (20 mL) was added slowly TMSCl
(4.18 mL, 32.8 mmol) under nitrogen atmosphere. The result-
ing solution was stirred for 2 h and evaporated under reduced
pressure to afford a white solid which was recrystallized from
methanol and ether to give a white crystal (2.7 g, 98%). mp
152.5-153 °C; [R]20D +25.4° (c 1.1, EtOH); IR (KBr) 1726 cm-1
;
1H NMR (DMSO-d6, 300 MHz) δ 2.87 (m, 2H), 3.16 (m, 1H),
3.23 (m, 1H), 3.38 (s, 3H), 3.42 (m, 1H), 5.01 (s, 2H), 7.12-
7.37 (m, 10H); 13C NMR (DMSO-d6, 300 MHz) δ 36.6, 43.7,
50.4, 52.5, 75.6, 127.5, 129.3, 129.4, 129.7, 129.9, 138.5, 173.8.
Anal. (C18H22ClNO3) C, H, N.
(S)-2-Ben zyl-3-(N-b en zyloxy)a m in op r op a n oic
Acid
Meth yl Ester ((S)-6) Compound (S)-6 was prepared from (S)-
517 in a manner analogous to that used for the preparation of

Sulfamide-Based Inhibitors for Carboxypeptidase A
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MOSFLM27 and the CCP4 program suite.28
Model refinement was carried out with the programs O29
and CNS.30 The structure of native CPA (pdb code: 5CPA) was
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body refinement was followed by cycles of simulated annealing
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calculation. Water molecules were gradually added to the
model with the WATERPICK routine in the program CNS.30
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refinement. The final model includes all residues of CPA
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