Aziridinyl peptides as inhibitors of cysteine proteases: Effect of a free carboxylic acid function on inhibition

Article abstract of DOI:10.1016/S0968-0896(00)00058-4

Peptides containing aziridine-2,3-dicarboxylate (Azi) as electrophilic building block are evaluated as inhibitors of the cysteine proteases papain, cathepsin B, cathepsin L and clostripain. The influence of a free carboxylic acid as functional group at di

Full text of DOI:10.1016/S0968-0896(00)00058-4

Bioorganic & Medicinal Chemistry 8 (2000) 1281±1291
Aziridinyl Peptides as Inhibitors of Cysteine Proteases: E€ect of a
Free Carboxylic Acid Function on Inhibition
T. Schirmeister* and M. Peric
Department of Pharmaceutical Chemistry, Pharmaceutical Institute, Albert-Ludwigs-University of Freiburg,
Hermann-Herder-Str. 9, 79104 Freiburg i. Br., Germany
Received 23 November 1999; accepted 20 January 2000
AbstractÐPeptides containing aziridine-2,3-dicarboxylate (Azi) as electrophilic building block are evaluated as inhibitors of the
cysteine proteases papain, cathepsin B, cathepsin L and clostripain. The in¯uence of a free carboxylic acid as functional group at
di€erent positions of the inhibitor molecule on inhibition is analyzed. Structure±activity relationships and binding mode hypotheses
are discussed. In contrast to the bacterial enzyme clostripain, the papain like mammalian proteases (cathepsins) are irreversibly
inactivated by aziridinyl peptides. N-Unsubstituted aziridines are much more potent inhibitors of papain and cathepsins if they
contain the free carboxylic acid attached to the aziridine ring (HOAzi-Leu-ProOBzl). Two free carboxylic acid functions at the
aziridine ring are necessary for good inhibition of these enzymes by N-acylated aziridinyl peptides (BOC-Phe-Azi(OH)2). Chimeric
bispeptidyl derivatives are selective CB inhibitors if the free acid is located at the C-terminus of the peptide (BOC-Phe-(EtO)Azi-
Leu-ProOH). Clostripain is only inhibited by aziridinyl peptide esters. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
residue. This leads to alkylation and therefore irrever-
sible inhibition of the proteases. Besides this common
mechanism of enzyme inactivation epoxysuccinyl and
aziridinyl peptides show remarkable di€erences in their
respective behaviour as cysteine protease inhibitors.
They concern inhibition activity, stereospeci®city, pH-
dependency of inhibition, and selectivity between the
di€erent cathepsins.18 First results have indicated that
there are also di€erences in the in¯uence exerted on
inhibition properties by a free carboxylic acid function
within the inhibitor structure.18a,c This is very well
investigated and elucidated in the case of the epoxide
derived inhibitors with the results shown in Table 1.
Epoxides containing only Leu as amino acid (I±V) are
much more potent with a free carboxylic acid function
at the epoxide ring and, simultaneously, an ester or
amide as C-terminal functional group (II±IV versus I,
V). These inhibitors do not discriminate between CB
and CL and are known to interact in an anti-substrate
orientation with the S-subsite of the enzymes.4,5,27 The
reverse holds true for epoxides containing the dipeptide
Leu(Ile)-Pro (VI±X). These compounds are selective and
highly potent inhibitors of CB if they contain an ester or
amide function at the epoxide ring and, simultaneously,
a free carboxylic acid function at the C-terminus (IX,
X). X-ray structure analysis showed these inhibitors to
interact with the S0-subsite of CB in a substrate-like
direction.28 In contrast, this in¯uence of a free car-
boxylic acid function on inhibition of cysteine proteases
Until today more than 10 lysosomal papain-like
cysteine proteases have been characterized, including
the well known cathepsins B,1 C,2 H,3 K,4 L,5 and S6 as
well as the recently discovered cathepsins F,7 O,8 V,9
W,10 X,11 and Z.12 They are supposed to be key
enzymes responsible for disregulated protein turnover in
a variety of diseases. Examples are muscular dystro-
phy,13 chronic in¯ammatory diseases,14 tumour progres-
sion,15 and osteoporosis.16 Inhibitors of these enzymes
could therefore be promising therapeutic agents. Epoxy-
succinyl peptides17 (Table 1) and aziridinyl peptides
(peptide derivatives of trans-aziridine-2,3-dicarboxylic
acid)18 have previously been shown to be irreversible
and selective inhibitors of cysteine proteases. Inactiva-
tion of enzymes by both inhibitor classes proceeds from
nucleophilic ring opening of the epoxide and aziridine
ring, respectively, by the enzyme's active site cysteine
*Corresponding author. Tel.: +49-761-203-6347; fax: +49-761-203-
6351; e-mail: schirmei@uni-freiburg.de
Abbreviations: Amino acids are written in the three letter code and are
l
con®gured. Azi aziridine-2,3-dicarboxylate, AMC aminomethyl
coumarine, CB cathepsin B, CL cathepsin L, CL(Pt) CL from Para-
mecium tetraurelia, CL(h) CL from human liver, DCC dicyclohex-
ylcarbodiimide, DMAP 4-(dimethylamino)pyridine, DPPA diphenyl
phosphorazidate, DPSI diphenylsul®mine, DTT dithiothreitol, EEDQ
1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, TEA triethylamine,
TFA tri¯uoro acetic acid, iAm isoamyl.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00058-4

1282
T. Schirmeister, M. Peric / Bioorg. Med. Chem. 8 (2000) 1281±1291
Table 1. Epoxysuccinyl peptides as inhibitors of cysteine proteases
No.
R1
Aa
R2
Ring
con®guration
Papain k2nd
1
CB k2nd
1
CL k2nd
1
Ref
1
1
1
(M
s
)
(M
s
)
(M
s )
I
II
Et
H
H
H
H
Et
H
H
Et
Et
Ð
Ð
Ð
Ð
NHiAm
NHiAm
NHiAm
OBzl
OH
OBzl
OBzl
OH
S,S
S,S
R,R
S,S
S,S
S,S
S,S
S,S
S,S
R,R
750
357,000
38,000
19
23
23
24
23
25
25
25
25
26
298,000
6900
206,000
791,000
III
IV
V
VI
VII
VIII
IX
X
Ð
207
110
176,000
30,000
760
388
30
8700
2200
13,800
567,000
Pro
Pro
Pro
Pro
Pro
OH
OH
100
26
aA: amino acid Pro or none.
has not been studied systematically with aziridinyl pep-
tides, especially with N-acylated ones, since up to now
only few aziridine derivatives containing a free car-
boxylic acid function have been synthesized and tested.
The present paper provides a systematic study concern-
ing the in¯uence of a free carboxylic acid as functional
group at di€erent positions of the inhibitor molecule on
inhibition of cysteine proteases. Syntheses and inhibi-
tion pro®les are described and structure±activity rela-
tionships are discussed comparing the inhibition
constants obtained with the acids to those obtained with
the recently published corresponding esters.18
Table 2. Inhibition of papain by aziridine building blocks
1
1
No.
R1
R2
R3
k2nd (M min
)
Ref
1a
1b
2a+b
Et
Et
Et
Et
Et
Bzl
H
H
H
H
11‹0.5a
12‹0.5a
141a,b
18b
18b
3a+b Bzl Bzl
273a
5b
27b
28b
Et
Et
Et
H
Et
Et
H
4692‹1000a
8‹2.5a
98
18c
CH2CO2Bzl
CH2CO2H
1
ki 0.033‹0.0012 (min
Ki 0.34‹0.058 (mM)
)
Results
Syntheses
aMeasurements were limited to the linear range, with [I] ꢀKi, therefore
only the second-order rate constant could be obtained.
bCB: k2nd=204, ki=0.037 min 1, Ki=0.18 mM.
Starting materials for the syntheses of aziridinyl pep-
tides are the aziridine building blocks 1±4 (Table 2,
Scheme 1). Diethyl aziridine-2,3-dicarboxylates 1a and
1b were synthesized as pure (R,R) (=a-series) and (S,S)
(=b-series) trans enantiomers by the literature known
®ve step synthesis starting from (S,S) and (R,R) diethyl
tartrate, respectively.18c The building blocks 2a+b,18c
3a+b, and 4a+b were obtained as racemic mixtures of
trans (R,R) and (S,S) enantiomers by Michael type
addition of diphenylsul®mine to the corresponding
fumarates (Scheme 1). Selective alkaline hydrolysis of
one ester group of diethyl esters 1a and 1b, respectively,
lead to the monoesters 5a and 5b (Table 2).18c The
dipeptides 6a, 6b and the tripeptides 10a, 10b (Table 3)
were synthesized by DPPA mediated peptide coupling
of the monoesters with LeuOBzl and Leu-ProOBzl,
respectively, as published recently.18c Compound 7a
(Table 3) with a free C-terminal carboxylic acid function
was obtained by hydrogenolytic cleavage of the benzyl
ester of 6a.18c Compounds 8a,b, 9a,b and 11a,b (Table
3) have been synthesized and tested by Martichonok et
al.18a Since these inhibitors complete the list of acid/
ester pairs of N-unsubstituted aziridinyl peptides their
inhibition constants are included for discussion. Com-
pounds 14a+b with the free carboxylic acid function
attached to the aziridine ring were synthesized as a dia-
stereomeric mixture starting from building blocks
4a+b. The crude monoesters 12a+b, obtained by
alkaline hydrolysis, were coupled to Leu-ProOBzl via
the DPPA procedure leading to tripeptides 13a+b.
Subsequent acid mediated cleavage of the tertbutyl
esters lead to 14a+b (Scheme 2).
The syntheses of the N-acylated aziridinyl dipeptides
15±17 (Table 4) (Scheme 3) with BOC-Phe as N-term-
inal amino acid were performed by DMAP catalyzed
acylation18b,c of the building blocks 1±3 with BOC-Phe
anhydride. The N-acylated tripeptides 20, 21 and 23 were
synthesized by EEDQ mediated peptide coupling18c of
BOC-Phe-Ala or BOC-Leu-Gly to the aziridine building
blocks 1 and 2, respectively.

T. Schirmeister, M. Peric / Bioorg. Med. Chem. 8 (2000) 1281±1291
1283
Scheme 1.
Table 3. Inhibition of papain and cathepsins by N-unsubstituted aziridinyl peptides
No.
R1
Aa
Ð
R2
Papain k2nd
1
CB k2nd
1
CL(Pt) k2nd
1
Ref
18c
1
1
1
(M min
)
(M min
)
(M min
)
6a
EtO
OBzl
1533‹41b
1607‹395b
41‹8b
16,261
ki 0.44‹0.0004 (min
1
)
Ki 0.027‹0.0001 (mM)
6b
EtO
Ð
OBzl
214
3130
ki 0.055‹0.009 (min
18c
1
1
ki 0.036‹0.0015 (min
Ki 0.17‹0.02 (mM)
63‹18b
)
)
Ki 0.018‹0.0021 (mM)
7a
8a
8b
9a
9b
10a
EtO
EtO
EtO
HO
HO
EtO
Ð
Ð
Ð
Ð
Ð
Pro
OH
nd
nd
nd
nd
nd
188
nd
nd
nd
nd
nd
18c
18a
18a
18a
18a
18c
NHiAm
NHiAm
NHiAm
NHiAm
OBzl
3300
180
864,000‹42,000
108,000‹6000
466‹37b
1728
1
1
ki 0.025‹0.0001 (min
Ki 0.13‹0.0011 (mM)
405
ki 0.039‹0.02 (min
Ki 0.096‹0.01 (mM)
)
ki 0.043‹0.0033 (min
Ki 0.025‹0.057 (mM)
2142
ki 0.047‹0.005 (min
Ki 0.022‹0.0085 (mM)
3000‹60
)
10b
EtO
Pro
OBzl
227
ki 0.021‹0.0002 (min
Ki 0.094‹0.0034 (mM)
18c
1
1
1
)
)
)
11a
11b
14a+b
iBuNH
iBuNH
HO
Pro
Pro
Pro
OH
OH
OBzl
ni
108‹6
780‹180
ni
3870
18a
18a
402‹6
16,930c
ki 0.51‹0.13 (min
7660
ki 0.24‹0.025 (min
1
1
1
)
Ki 0.031‹0.0009 (mM)
ki 0.066‹0.047 (min
Ki 0.017‹0.004 (mM)
)
)
Ki 0.03‹0.011 (mM)
aA: amino acid Pro or none; nd: not determined; ni: no time-dependent inhibition.
bMeasurements were limited to the linear range, with [I]ꢀKi, therefore only the second-order rate constant could be obtained.
cCL(h).
Again, the peptides 15a, 20a and 15b, 20b derived from
the building blocks 1a and 1b, respectively, were syn-
thesized as pure isomers, whereas peptides 16a+b,
17a+b, 21a+b and 23a+b are derivatives of the
racemic building blocks 2a+b or 3a+b and are there-
fore diastereomeric mixtures.
The corresponding monoesters 18a+b, 22a+b and
24a+b as well as the diacids 19a+b were obtained by
hydrogenolysis of the benzyl esters (Scheme 3). This
synthetic pathway, which leads to the N-acylated azir-
idines with one or two carboxylic acid functions
attached to the aziridine ring, had to be chosen because

1284
T. Schirmeister, M. Peric / Bioorg. Med. Chem. 8 (2000) 1281±1291
Scheme 2.
Table 4. Inhibition of papain and cathepsins by N-acylated aziridinyl peptides
No.
R1
Aa
Bb
R2
Papain k2nd
1
CB k2nd
1
CL(Pt) k2nd
1
Ref
1
1
1
(M min
)
(M min
)
(M min
)
15a
15b
16a+b
18a+b
Et
Et
Bzl
H
BOC-Phe
BOC-Phe
BOC-Phe
BOC-Phe
Ð
Ð
Ð
Ð
OEt
OEt
OEt
OEt
42‹9d
408‹16d
76‹9d
505
18‹6d
125‹19d
ndc
16‹6d
65‹12d
nd
18b
18b
18c
18c
1230
ki 0.83‹0.019 (min )
Ki 0.68‹0.03 (mM)
807‹81d
188‹6d
1
1
ki 0.21‹0.013 (min
Ki 0.41‹0.07 (mM)
3544
ki 0.2‹0.014 (min
)
19a+b
H
BOC-Phe
Ð
OH
38,081e
ki 1.02‹0.08 (min )
1
1
)
Ki0.057‹0.015 (mM)
91‹17d
1370
Ki 0.027‹0.005 (mM)
nd
469
20a
20b
Et
Et
BOC-Phe-Ala
BOC-Phe-Ala
Ð
Ð
OEt
OEt
nd
455‹15d
18c
18c
1
1
ki 0.028‹0.0026 (min
Ki 0.02‹0.009 (mM)
1232
)
ki 0.051‹0.019 (min
Ki 0.11‹0.08 (mM)
281‹2d
)
21a+b
22a+b
23a+b
24a+b
Bzl
H
BOC-Phe-Ala
BOC-Phe-Ala
BOC-Leu-Gly
BOC-Leu-Gly
Ð
Ð
Ð
Ð
OEt
OEt
OEt
OEt
433‹71d
676‹82d
240‹61d
1
ki 0.020‹0.001 (min
Ki 0.016‹0.001 (mM)
)
1456
843‹42d,e
1
ki 0.67‹0.059 (min
Ki0.46‹0.046 (mM)
149
ki 0.01‹0.001 (min )
Ki 0.069‹0.0012 (mM)
)
Bzl
H
3237
ki 0.028‹0.00015 (min )
Ki 0.0088‹0.0015 (mM)
18c
1
1
575
766
ki 0.062‹0.0032 (min
Ki 0.081‹0.021 (mM)
1938‹440d
450
ki 0.037‹0.0022 (min
Ki 0.082‹0.013 (mM)
6538
ki 0.85‹0.064 (min
Ki 0.13‹0.018 (mM)
13,030f
ki 0.030‹0.002 (min
1
1
1
ki 0.021‹0.0003 (min
Ki 0.037‹0.0046 (mM)
768‹125d
)
)
)
)
Ki 0.0022‹0.001 (mM)
5869‹409d
25a
25b
Et
Et
BOC-Phe
BOC-Phe
Leu-Pro
Leu-Pro
OBzl
OBzl
18c
18c
321
1210‹260d
1
1
ki 0.016‹0.0019 (min
Ki 0.05‹0.029 (mM)
176‹29d
)
26a
26b
Et
Et
BOC-Phe
BOC-Phe
Leu-Pro
Leu-Pro
OH
OH
655‹76d,e
1
)
704
ki 0.052‹0.0054 (min
Ki0.073‹0.03 (mM)
6859
ki 0.78‹0.053 (min
Ki 0.11‹0.026 (mM)
212
ki 0.027‹0.0031 (min
Ki 0.12‹0.04 (mM)
1
1
1
)
)
)
aA: amino acid BOC-Phe or dipeptides BOC-Phe-Ala and BOC-Leu-Gly, respectively.
bB: dipeptide Leu-Pro or none.
cnd: not determined.
dMeasurements were limited to the linear range, with [I]ꢀKi, therefore only the second-order rate constant could be obtained.
eCL(h).
1
fCL(h): k2nd=10,950 (M min 1), ki=0.0220‹0.0025 (min 1), Ki=0.002‹0.0007 (mM).

T. Schirmeister, M. Peric / Bioorg. Med. Chem. 8 (2000) 1281±1291
1285
of the lability of the aziridide bond towards alkaline
hydrolysis.18c
family. As a cysteine protease non-related to the papain
clan the bacterial enzyme clostripain21 isolated from
Clostridium histolyticum was included in the test series.
This protease which belongs to the C11 cysteine pro-
tease family is one of few known bacterial cysteine
proteases and does not share any sequence homology
with the papain-like enzymes. Inhibitors of clostripain
may be of therapeutic interest since this protease is
supposed to act as a virulence factor in infections.
The N-acylated tetrapeptide esters 25a, 25b18c and acids
26a, 26b result from N-acylation of 10a and 10b,
respectively, via the symmetric anhydride procedure and
subsequent hydrogenolytic benzyl ester cleavage
(Scheme 4).
N-Alkylated aziridine building block esters 27a, 27b20 and
acids 28a, 28b were obtained by alkylation with benzyl
bromoacetate followed by hydrogenolysis (Scheme 5).
1
The second-order rate constants of inhibition k2nd (M
min 1) (k2nd=ki/Ki) for irreversible inactivation of
papain and cathepsins B and L were determined by
continuous22a or dilution22b assays as described pre-
viously.18c This irreversibility is evident from the time-
dependency of inhibition and was con®rmed by dialysis
experiments.18c In most cases both individual inhibition
constants, the dissociation constant Ki (mM) and the ®rst-
Inhibition of cysteine proteases
Aziridine building blocks and aziridinyl peptides were
tested against papain and the papain-like cathepsins B,
L(Pt) and L(h), all belonging to the C1 cysteine protease
Scheme 3.
Scheme 4.

1286
T. Schirmeister, M. Peric / Bioorg. Med. Chem. 8 (2000) 1281±1291
Scheme 5.
Table 5. Inhibition of clostripain by aziridinyl peptides
In the case of the N-acylated inhibitors (Table 4) only a
slight inhibition improvement with an acid/ester ratio of
2±10 is caused by one free carboxylic acid at the aziridine
ring (18, 22, 24 versus 15, 16, 20, 21, 23). No di€erence
is found between CL(Pt) and CL(h) (compound 24). A
second acidic function, however, leads to the desired
high increase of inhibition constants with acid/ester
ratios up to 2000, especially for CL (19 versus 15, 16,
18). The main reason for this good inhibition of CL by
the diacid 19 is the high ki value of 1 min 1. This is in
contrast to compound 24 for which the high inhibition
constant is mainly caused by a low Ki value (2 mM).
No.
Compound
Ki (mM)
5a
6a
18a+b
23a+b
26b
EtOAziOH
EtOAzi-LeuOBzl
BOC-Phe-(EtO)AziOH
BOC-Leu-Gly-(EtO)AziOBzl
BOC-Phe-(EtO)Azi-Leu-ProOH
5.05‹0.38
0.056‹0.0043
1.07‹0.09
0.068‹0.018
0.066‹0.0019
order rate constant ki (min 1), could be determined. Dis-
sociation constants Ki (mM) for the reversible inhibition
of clostripain were obtained by Dixon plots as described
in previous experiments.18c The results of the assays are
listed in Tables 2±5.
An inhibition improvement is also reached by N-acylation
of the Leu-Pro derivatives 10a, 10b (Table 3) with BOC-
Phe to yield the bispeptidyl derivatives 25a, 25b (Table 4).
For these compounds the free acid at the C-terminal Pro
residue (26a, 26b) (Table 4) leads to higher inhibition
constants for CB but not for CL or papain. That means,
the increase in selectivity, which one could have expected
for the Leu-Pro derivatives 11a, 11b without N-sub-
stituent, is achieved by introduction of a third amino acid
at the N-terminus of the peptide.
Discussion
In earlier publications,18b,c we have reported on the
structure±activity relationships concerning activity, ste-
reoselectivity, pH-dependency of inhibition, and selec-
tivity between di€erent cathepsins. Herein we will focus
the discussion on inhibition of cysteine proteases by the
new aziridinyl peptides containing a free carboxylic acid
function.
Opposite acid/ester ratios of 0.01±0.05 are found for
inhibition of clostripain (Table 5). Dissociation con-
stants in the micromolar range are only reached with
aziridine esters. In contrast to cysteine proteases of the
papain family the inhibition of clostripain by aziridinyl
peptides is reversible. This is comparable to results
obtained with epoxysuccinyl peptides.30
N-Unsubstituted aziridinyl peptides (Table 3) contain-
ing a Leu derivative as the only amino acid exhibit very
high inhibition constants if they contain a free car-
boxylic acid function at the aziridine ring (9a, 9b).
Esteri®cation at this position (6, 8) or a free acid at
the C-terminus (7a) lead to a dramatic decrease in
inhibition potency. These results are similar to those
obtained with epoxysuccinyl peptides (Table 1). The
results obtained with the Leu-Pro derivatives, how-
ever, are contrary to those of the epoxides. A free
carboxylic acid at the C-terminus of these inhibitors
neither leads to higher inhibition constants nor to
higher selectivity between CB and CL (11 versus 10).
For these derivatives, however, an inhibition improve-
ment (but not an increase in selectivity) can be reached
with an additional free acid function at the aziridine
ring (14). These ®ndings apply also to the aziridine
building blocks (5 versus 1±3) (Table 2). An acid/ester
ratio of 10 is found for the N-alkylated inhibitors 28/27
with the free acid attached to a methylene spacer at the
aziridine nitrogen.29
These results, in combination with the proved binding
modes of epoxysuccinyl peptides to cysteine proteases of
the papain family, allow ®rst hypotheses on the binding
of aziridinyl peptides to these enzymes. One reason for
the good inhibition of cysteine proteases by epoxy-
succinyl peptides containing a Leu amide or ester on
the one hand and a free carboxylic acid at the epoxide
ring on the other is an ionic interaction between the
histidinium cation of the active site and the carboxylate
anion of the inhibitor.27 This interaction could also be
possible with the carboxylate of N-unsubstituted azir-
idinyl peptides and aziridine building blocks (5, 9, 14)
and would explain the similar behaviour of these inhi-
bitors compared to the epoxides. Obviously this ionic
bond is not possible with the one carboxylate of N-acy-
lated aziridines (18, 22, 24). Previous molecular model-
ing results18c indicated that the aziridide structure leads

T. Schirmeister, M. Peric / Bioorg. Med. Chem. 8 (2000) 1281±1291
1287
to a less favourable inhibitor conformation in which the
free carboxylate is possibly turned away from the histi-
dinium ion. Therefore, we reasoned that this ionic
interaction which is the main factor for good inhibition
will only become possible if the aziridine bears a second
carboxylate at the three-membered ring (19).
prepared by well established literature procedures.32
Reagent grade chemicals were purchased from the fol-
lowing companies: EEDQ from Novabiochem, DPSI,
LiOH from Merck, DCC, DMAP from Fluka, DPPA
from Aldrich, Pd±C 10% type E 10 N/D from Degussa.
Preparative ¯ash column chromatography was per-
formed using silica gel 60, 40±63 mm, from Merck.
Melting points are uncorrected and were obtained on
Mel-Temp II capillary melting point apparatus. Optical
rotations were measured on a Perkin±Elmer 241 polari-
meter. IR spectra were determined on a Perkin±Elmer
841 IR spectrometer. Mass spectra were measured on a
Finnigan MAT 312 by the Chemisches Laboratorium of
the University of Freiburg. Elemental analyses were
determined on a Perkin±Elmer Elemental Analyzer 240
by the Chemisches Laboratorium of the University of
Freiburg. NMR spectra were recorded on Varian Unity
In contrast, the inhibition improvement by a free acid
attached to the methylene spacer of N-alkyated aziridines
(28) should have another explanation. As shown ear-
lier,18a,c the aziridine-N exhibits a very low basicity leading
to increased inhibition constants at pH 4. A similarly low
basicity could be expected for the N-alkylated aziridine-N.
We could prove this by determination of pH-dependency
of inhibition of papain by compound 27b.29 A free car-
boxylic acid function at the methylene spacer (28b) should
now lead to enhancement of basicity compared to the
ester (compare pKa values of GlyOH/GlyOMe: 9.8/7.8)
and as a consequence to a higher degree of protonation
and activation towards nucleophilic ring opening under
the assay conditions (pH 6.5).
1
300 (300 and 75.43 MHz). H NMR chemical shifts are
reported in ppm relative to the CHCl3 peak at d=7.26.
13C NMR chemical shifts are reported in ppm relative
1
to the CHCl3 peak at d=77.00. All H NMR assign-
ments were supported by homonuclear decoupling
experiments or by 2-D COSY experiments and all 13C
NMR assignments by 2-D HETCOR experiments.
The reasons for the increase in selectivity between CB
and CL by epoxides containing Leu-ProOH are ionic
interactions with the C-terminal carboxylate of the
inhibitors and two protonated histidinium residues
located at the occluding loop speci®c for CB.28 A cor-
responding interaction between the C-terminal Pro car-
boxylate of the N-unsubstituted aziridinyl peptides 11
containing Leu-ProOH and these His residues in CB
seems, based on the obtained inhibition constants, very
unlikely. It may, however, be possible for the N-acy-
lated derivatives 26 since these inhibitors exhibit a
selective improvement in inhibition of CB. The N-term-
inal BOC-Phe residue of these peptides may additionally
bind to the hydrophobic S2-subsite of CB as shown by
Schaschke et al.26 for chimeric bispeptidyl derivatives of
the epoxysuccinic acid which are highly selective and
potent CB inhibitors.
Syntheses
The syntheses of the following compounds have been
published previously: (1a, 1b, 15a, 15b),18b (2a+b, 5a,
5b, 6a, 6b, 7a, 10a, 10b, 16a+b, 18a+b, 20b, 21a+b,
23a+b, 25a, 25b, 29a(I,II)).18c
General procedures
Synthesis of aziridine building blocks and enamino esters
starting from fumarates. DPSI (5.2 g, 24 mmol) and
20 mmol of the corresponding fumarate were dissolved
in 60 mL toluene and heated at 80 ꢁC for 24 h. The
mixture was evaporated in vacuo. Column chromato-
graphy (silica gel, cyclohexane:ethyl acetate, 4:1) yielded
the aziridines (30±35%) 3a+b and 4a+b, respectively,
and the enamino esters (40±50%) 29b and 29c(I,II),
respectively.
Unlike other cysteine protease inhibitors (e.g., peptidyl
sulfonium salts)31 aziridines and epoxides are only rever-
sible clostripain inhibitors. In contrast to the papain-like
cysteine proteases, however, no X-ray data are available
for clostripain. Therefore, at the moment no discussion is
possible on binding modes or inhibition mechanism.
Catalytic hydrogenolysis of benzyl esters. An evacuated
solution of benzyl ester in MeOH or EtOH and Pd±C
(10%) was vigorously stirred at rt and atmospheric
pressure for 0.5±3 h (TLC control) under a slow stream
of hydrogen. The catalyst was removed by ®ltration
over Celite and washed with MeOH or EtOH. The ®l-
trate was evaporated in vacuo and the residue was
recrystallized.
To ®nally prove the binding mode hypotheses presented
herein, X-ray analyses of enzyme inhibitor complexes are
underway.
Experimental
(2R,3R)+(2S,3S) Dibenzyl aziridine-2,3-dicarboxylate
(3a+b). The general procedure A with 5.9 g dibenzyl
fumarate yielded 1.8 g (30%) 3a+b as a yellowish solid.
General methods
Enzymes were purchased from the following companies
and used without further puri®cation: papain from
Fluka, CB (bovine spleen) from Sigma, CL(Pt) and
CL(h) from Calbiochem, clostripain from Sigma. The
substrate Z-Phe-Arg-AMC, the dipeptide BOC-Leu-
GlyOH and all amino acids were purchased from
Bachem. Leu-ProOBzl TFA and BOC-Phe-AlaOH were
Rf 0.27; mp 60±61 ꢁC. IR (ethyl acetate): n=3276, 3064,
1
2956, 1733, 1497, 1455, 1384, 1340 cm
.
1H NMR
(CDCl3): d=1.90 (bs, 1H, NH), 3.00 (s, 2H, ring-H), 5.20
(2Âd, J=12.0 Hz, 2Â2 H), 7.40 (m, 10H). 13C NMR
(CDCl3): d=35.68, 67.48, 127.27, 128.31, 128.48,
134.28, 170.0. Anal. (C18H17NO4): calcd: C 69.44, H
5.50, N 4.50; found: C 69.26, H 5.47, N 4.42.

1288
T. Schirmeister, M. Peric / Bioorg. Med. Chem. 8 (2000) 1281±1291
(2R,3R)+(2S,3S) 2-tertButyl-3-ethyl aziridine-2,3-dicar-
boxylate (4a+b). The general procedure A with 4.0 g
tertbutyl ethyl fumarate yielded 1.5 g (35%) 4a+b as a
yellowish liquid. Rf 0.18. IR (ethyl acetate): n=3279,
2982, 2939, 1725, 1477, 1460, 1444, 1394, 1371, 1344
solvent was removed in vacuo, 5 mL CH2Cl2 were added
and the solvent was evaporated again. This was repeated
two times. The residue was dried in vacuo. Yield: 556 mg
(100%), colourless viscous liquid. [a]d20= 42.5 (c 1.13,
EtOH). IR (ethyl acetate): n=3987, 3784, 2956, 1732,
1
1
cm 1. H NMR (CDCl3): d=1.27 (t, 3H, J=7.0 Hz),
1634, 1444, 1180 cm 1. H NMR (CDCl3): d=0.8±1.0
1.35 (s, 9H), 1.67 (bs, 1H), 2.60 and 2.62 (2Âd,
J=2.2 Hz, 2H), 4.06 (q, J=7.0 Hz, 2H). 13C NMR
(CDCl3): d=13.60, 27.43, 35.03, 35.97, 61.19, 82.07,
168.85, 169.76. MS (CI, C4H10): m/z (%)=215 (28)
[M+], 158 (100). Anal. (C10H17NO4): calcd: C 55.80, H
7.96, N 6.51; found: C 55.18, H 7.71, N 6.75.
(m, 6H, Leu), 1.25±1.60 (m, 2H, bH Leu), 1.60±1.80 (m,
1H, gH Leu), 1.90±2.10 (m, 3H, bH Pro, 2ÂgH Pro),
2.15±2.25 (m, 1H, bH Pro), 2.95 (d, J=1.9 Hz, 1H, ring-
H), 3.05 (d, J=1.9 Hz, 1H, ring-H), 3.50±3.70 (m, 1H,
dH Pro), 3.70±3.90 (m, 1H, dH Pro), 4.42±4.60 (m, 1H,
aH Pro), 4.65±4.75 (m, 1H aH Leu), 5.05±5.20 (m, 2H,
benzyl), 7.00±7.45 (m, 6H, arom., NH Leu), 8.80 (bs, 3H,
NH+2 , CO2H). 13C NMR (CDCl3): d=21.21, 22.89 (dC
Leu), 23.69 (gC Leu), 24.60 (gC Pro), 28.73, 29.61 (ûC
Pro), 38.68, 40.0 (ring-C), 40.05 (bC Leu), 47.26 (dC
Pro), 49.50 (aC Leu), 59.48 (aC Pro), 67.24 (benzyl),
115.06 (Q, J (C,F)=286 Hz, CF3), 126.27, 126.29, 128.17,
128.54, 129.47, 130.05, 130.06, 135.13, 159.90 (Q, 2J
(C,F)=40 Hz, CˆO, TFA), 171.13, 171.85. MS (ESI):
m/z (%)=478 (100) [M++2Na], 432 (2.5) [M++1].
(2R,3R)+(2S,3S) 3-tertButoxycarbonyl aziridine-2-car-
boxylate, Li salt (12a+b). 4a+b (280 mg, 1.3 mmol)
were dissolved in 20 mL EtOH. LiOH monohydrate
(54 mg, 1.3 mmol) was added. After 2 h at 0 ꢁC the mix-
ture was stirred at rt for 2 days. The residue remaining
after evaporation of the solvent was used without fur-
ther puri®cation: mp 178 ꢁC. 1H NMR (pyridine-d5):
d=1.36 (s, 9H), 3.0±3.12 (m, 2H, ring-H), 4.9 (bs, 1H).
MS (EI, 70 eV): m/z (%)=186 (32.85) [M+], 85 (100).
.
Anal. (C22H29N3O6 C2F3HO2): calcd: C 52.84, H 5.54,
N 7.70; found: C 53.87, H 5.68, N 7.57.
(2R,3R)+(2S,3S) N-{[3-tertButoxycarbonyl aziridine-2-
yl]carbonyl} - (S) - leucyl - (S) - proline benzyl ester (R,R/
S,S=1:1) (13a+b). Crude 12a+b (193mg, 1.0 mmol)
and 432 mg (1.0mmol) Leu-ProOBzl TFA were dissolved
in 10 mL DMF abs. under N2 atmosphere. At 0 ꢁC 240 mL
(1.1 mmol) DPPA and 170 mL (1.2 mmol) TEA were
added. The solution was stirred for 10h at 0 ꢁC and for
further 12h at rt. Ethyl acetate (100mL) was added. The
solution was washed with water (3Â25mL), 5% NaHCO3
(1Â25mL) and brine (2Â25mL). The organic layer was
dried with MgSO4 and evaporated in vacuo. Flash col-
umn chromatography (silica gel, cyclohexane:ethyl ace-
tate, 1:1) gave 210 mg (43%) 13a+b as a colourless
viscous liquid. Rf 0.43. [a]2d0= 59.6 (c 0.51, EtOH). IR
(2R,3R)+(2S,3S) Dibenzyl-1-[N-(tertbutoxycarbonyl)-(S)-
phenylalanyl]aziridine-2,3-dicarboxylate (R,R/S,S=1:1)
(17a+b). BOC-Phe (3.2 g, 12 mmol) and 1.24 g
(6 mmol) DCC were dissolved in 15 mL CH2Cl2 at 0 ꢁC
and stirred for 45 min at rt. Insoluble dicyclohexyl urea
was ®ltered o€ and 930 mg (3 mmol) 3a+b were added
together with a few crystals of DMAP (10 mol%). The
solvent was removed after 1 d at rt and the residue was
puri®ed by ¯ash column chromatography (silica gel,
cyclohexane:ethyl acetate, 10:1). Yield: 810 mg (48.4%),
yellowish viscous liquid. Rf 0.1. [a]d18= 5.8 (c 0.86,
EtOH). IR (ethyl acetate): n=3371, 2928, 1739, 1604,
1
1497, 1447, 1369 cm 1. H NMR (CDCl3): d=1.40 (s,
(ethyl acetate): n=3268, 2957, 1739, 1636, 1549, 1447
9H, BOC), 3.06 (mc) and 3.23 (mc) (together 2H, bH
Phe), 3.41 (s) and 3.55 (s) (together 2H, ring-H), 4.51 (m)
and 4.63 (m) (together 1H, aH Phe), 5.00 (d) and 5.13 (d)
(J=8.2Hz, together 1H, NH Phe), 5.20 (m, 4H, OCH2),
7.13±7.43 (m, 15H). 13C NMR (CDCl3): d=28.02, 28.07
(BOC), 38.01, 39.00, 39.76, 40.08 (ring-C, bC Phe), 55.92,
56.38 (aC Phe), 67.97, 67.99 (OCH2), 79.53, 79.71 (BOC),
126.58, 126.62, 128.23, 128.41, 128.44, 128.47, 128.52,
128.59, 129.43, 134.92, 134.33, 135.92, 136.06, 154.59,
154.75 (BOC), 165.59, 165.63, 178.73, 178.86. MS (EI,
70eV): m/z (%)=558 (0.3) [M+], 164 (100). HREIMS:
calcd: 558.2366; found: 558.2374. Anal. (C32H34N2O7):
calcd: C 68.80, H 6.13, N 5.01; found: C 69.36, H 5.56,
N 4.41.
1
cm
.
1H NMR (CDCl3): d=0.80±1.0 (m, 6H, Leu),
1.32±1.60 (m, 11H, butyl, bH Leu), 1.60±1.80 (m, 2H,
NH azi, gH Leu), 1.85±2.05 (m, 3H, bH Pro, 2ÂgH
Pro), 2.15±2.25 (m, 1H, bH Pro), 2.58, 2.65, 2.75, 2.85
(d each, J=1.9 Hz, together 2H, ring-H), 3.45±3.60 (m,
1H, dH Pro), 3.62±3.80 (m, 1H, dH Pro), 4.55 (m, 1H,
aH Pro), 4.70 (m, 1H, aH Leu), 5.12 (m, 2H, benzyl),
6.80 and 6.92 (2Âbs, together 1H, NH Leu), 7.10±7.40
(m, 5H). 13C NMR (CDCl3): d=21.7, 23.27 (dC Leu),
24.62 (gC Leu), 24.84 (gC Pro), 27.91 (tbutyl), 28.91
and 29.63 (bC Pro), 35.78, 36.30 (double peak), 37.09
(ring-C), 41.25 and 41.57 (bC Leu), 46.81 (dC Pro),
48.27 (aC Leu), 58.85 and 58.89 (aC Pro), 66.88
(OCH2 benzyl), 83.0 (tbutyl), 126.88, 128.09, 128.11,
128.26, 128.50, 135.51, 168.07, 170.77, 170.86, 171.62.
MS (EI, 70eV): m/z (%)=487.1 (2.2) [M+], 69.9 (100).
HREIMS: calcd: 487.2682; found: 487.2667. Anal.
(C26H37N3O6): calcd: C 64.05, H 7.65, N 8.62; found:
C 63.96, H 7.67, N 8.81.
(2R,3R)+(2S,3S) 1-[N-(tertButoxycarbonyl)-(S)-pheny-
lalanyl]aziridine - 2,3 - dicarboxylic acid (R,R/S,S=1/1)
(19a+b). The general procedure
B with 160 mg
(0.28 mmol) 17a+b, 40 mL EtOH and 100 mg Pd-C
gave after 1.5 h 106 mg (100%) 19a+b as colourless
crystals: mp 77 ꢁC. [a]1d8= 15.6 (c 0.50, EtOH). IR
(ethyl acetate): n=3432, 2076, 1740, 1700, 1643, 1370
(2R,3R)+(2S,3S) N-{[3-Carboxy aziridine-2-yl]carbo-
nyl}-(S)-leucyl-(S)-proline benzyl ester, tri¯uoro acetate
(R,R/S,S=1:1) (14a+b). 13a+b (500 mg, 1.02 mmol)
was dissolved in 5 mL CH2Cl2 abs. at 0 ꢁC. TFA (5 mL)
was added and the solution was stirred at rt for 1 h. The
1
cm 1. H NMR (CDCl3): d=1.10±1.42 (m, 9H, BOC),
3.60±3.76 (m, 4H, bH Phe, ring-H), 4.40 and 4.62 (2Âm,
together 1H, aH Phe), 5.32 (bs, 1H, NH Phe, disappears
with D2O), 7.0±7.4 (m, 7H, 2ÂCO2H, disappears with

T. Schirmeister, M. Peric / Bioorg. Med. Chem. 8 (2000) 1281±1291
1289
D2O, 5H, arom.). 13C NMR (CDCl3): d=28.21 (BOC),
38.42, 38.74, 40.34 (bC Phe, ring-C), 56.46 (aC Phe),
80.46 (BOC), 126.83, 127.02, 128.38, 128.58, 129.34,
129.62, 136.08, 155.71 (BOC), 166.11, 169.17, 174.74.
MS (EI, 70 eV): m/z (%)=378.2 (0.8) [M+], 57 (100).
HREIMS: calcd: 378.1427; found: 378.1437. Anal.
colourless crystals: mp 79 ꢁC. [a]2d0= 20.0 (c 0.66,
EtOH). IR (ethyl acetate): n=3984, 3784, 3312, 2959,
1739, 1522, 1443, 1369 cm
1
.
1H NMR (CDCl3):
d=0.85±0.95 (m, 6H, dH Leu), 1.27 (t, J=7.15 Hz, 3H,
ester), 1.37±1.55 (m, 11H, BOC, bH Leu), 2.60±2.76 (m,
1H, gH Leu), 3.47 (m, 2H, ring-H), 3.95±4.08 (m, 1H,
Gly), 4.10±4.28 (m, 4H, ester, aH Gly, aH Leu), 4.85
(bs, 1H, NH Leu), 6.70 (bs, 1H, NH Gly). 13C NMR
(CDCl3): d=13.97, 21.77, 22.99 (dC Leu), 24.74 (gC
Leu), 28.28 (BOC), 39.52, 39.62, 39.85 (ring-C), 41.32
(bC Leu), 43.80 (Gly), 53.26, 53.29 (aC Leu), 62.23,
62.79 (OCH2), 80.33 (BOC), 155.64 (BOC), 165.94,
.
(C18H22N2O7 C2H5OH): calcd: C 55.80, H 7.80, N 5.42;
found: C 55.60, H 7.61, N 5.56.
(2R,3R) Diethyl-1-[N-(tertbutoxycarbonyl)-(S)-phenyl-
alanyl-(S)-alanyl]aziridine-2,3-dicarboxylate (20a). 1a
(101 mg, 0.54 mmol) and 180 mg (0.54 mmol) BOC-Phe-
Ala were dissolved in 5 mL DMF abs. EEDQ (142 mg,
0.57 mmol) was added and the mixture was stirred at
50 ꢁC. After 2 days 50 mL ethyl acetate were added and
the solution was washed with 5% NaHCO3 (1Â50 mL)
and water (1Â50 mL). The organic layer was dried with
MgSO4 and removed in vacuo. The residue was puri®ed
by ¯ash column chromatography (silica gel, cyclohex-
ane:ethyl acetate, 3:1). Yield: 100 mg (40%), colourless
166.41, 166.47, 172.68, 176.49, 176.53. MS (ESI): m/z
+
.
(%)=428 (66) [M ], 287 (100). Anal. (C19H31N3O8 1/2
MeOH): calcd: C 52.60, H 7.46, N 9.44; found: C 52.49,
H 7.48, N 9.79.
(2R,3R) N-{1-[N-(tertButoxycarbonyl)-(S)-phenylalanyl]-
3-ethoxycarbonyl aziridine-2-yl]carbonyl}-(S)-leucyl-(S)-
proline (26a). The general procedure B with 40 mg
(0.057 mmol) 25a, 20 mL MeOH and 20 mg Pd±C gave
after 0.5 h 35 mg (100%) 26a as colourless crystals: mp
109 ꢁC. [a]2d0= 32.6 (c 1.0, EtOH). IR (ethyl acetate):
viscous liquid. Rf 0.13. [a]d20= 12.6 (c 0.46, EtOH). IR
1
(ethyl acetate): n=3785, 2924, 1741, 1663, 1595 cm
.
1H NMR (CDCl3): d=1.15±1.21 (m, 18H, CH3 ester,
bH Ala, BOC), 3.05 (m, 2H, bH Phe), 3.42 (s, 2H, ring-
H), 4.15±4.4 (m, 5H, ester, aH Phe), 4.50 (m, 1H, aH
Ala), 4.91 (bd, J=8.5 Hz, 1H, NH Phe), 6.42 (bd,
J=8.6 Hz, 1H, NH Ala), 7.2 (m, 5H). 13C NMR
(CDCl3): d=13.98 (CH3 ester), 18.31 (bC Ala), 28.19
(BOC), 38.31 (bC Phe), 40.10 (ring-C), 48.08, 49.49 (aC
Phe, Ala), 62.63 (OCH2 ester), 80.05 (BOC), 128.64,
129.33, 129.36, 136.37, 155.27 (BOC), 165.88, 170.72,
179.31. MS (CI, C4H10): m/z (%)=506 (7) [M++1],
319 (100). Anal. (C25H35N3O8): calcd: C 59.39, H 6.98,
N 8.31; found: C59.22, H 7.08, N 8.85.
1
n=3278, 2960, 1740, 1701, 1630, 1530, 1448 cm 1. H
NMR (CDCl3): d=0.95 (m, 6H, Leu), 1.30±1.40 (m,
12H, BOC, CH3), 1.40±2.10 (m, 7H, bH, gH Leu, Pro),
3.10±3.30 (m, 4H, bH Phe, ring-H), 3.40±3.60 (m, 1H,
bH Pro), 3.60±3.80 (m, 1H, dH Pro), 4.20 (m, 2H,
OCH2), 4.40 (m, 1H, H Pro), 4.60 (m, 1H, aH Phe), 4.75
(m, 1H, aH Leu), 5.30 (bs, 1H, NH Phe), 6.90 (bs, 1H,
NH Leu), 7.09±7.20 (m, 5H, arom.). 13C NMR
(CDCl3): d=13.90 (CH3), 21.37, 23.41 (dC Leu), 24.74
(gC Leu), 24.86 (gC Pro), 28.14 (BOC), 28.90 (bC Pro),
36.37 (bC Phe), 38.39 (ring-C), 40.42 (bC Leu), 41.02
(ring-C), 47.23 (dC Pro), 49.11 (aC Leu), 56.06 (aC
Phe), 58.10 (aC Pro), 62.26 (OCH2), 79.53 (BOC),
126.94, 128.20, 129.09, 136.38, 154.89 (BOC), 162.46,
(2R,3R)+(2S,3S) 1-[N-(tertButoxycarbonyl)-(S)-phenyl-
alanyl - (S) - alanyl] - 3 - ethoxycarbonyl aziridine - 2 - car-
boxylic acid (R,R/S,S=1:1.3) (22a+b). The general
procedure B with 160 mg (0.28 mmol) 21a+b, 20 mL
EtOH and 50 mg Pd±C gave after 1 h 130 mg (100%)
22a+b as colourless crystals: mp 73 ꢁC. [a]1d8= 3.4 (c
0.5, EtOH). IR (ethyl acetate): n=3296, 2976, 1735, 1524,
164.70, 171.10. MS (EI, 70 eV): m/z (%)=616 (0.2)
+
.
[M ], 90.9 (100). Anal. (C31H44N4O9 MeOH): calcd: C
59.24, H 7.46, N 8.64; found: C 59.09, H 7.21, N 8.84.
(2S,3S) N-{1-[N-(tertButoxycarbonyl)-(S)-phenylalanyl]-
3-ethoxycarbonyl aziridine-2-yl]carbonyl}-(S)-leucyl-(S)-
proline (26b). The general procedure B with 400 mg
(0.57 mmol) 25b, 50 mL MeOH and 100 mg Pd±C gave
after 1 h 350 mg (100%) 26b as colourless crystals: mp
119 ꢁC. [a]2d3= 33.4 (c 1.05, EtOH). IR (ethyl acetate):
n=3287, 2980, 1740, 1712, 1637, 1543, 1529, 1498, 1454
1
1449 cm 1. H NMR (CDCl3): d=1.03±1.50 (m, 15H,
BOC, CH3 ester, bH Ala), 2.85±3.10 (m, 2H, bH Phe),
3.40±3.70 (m, 2H, ring-H), 4.12±4.28 (m, 2H, OCH2),
4.40 (m, 1H, aH Phe), 4.68 (m, 1H, aH Ala), 5.30 and
5.62 (2Âbs, together 1H, NH Phe), 6.80 (bs, 2H, NH Ala,
CO2H), 7.03±7.35 (m, 5H). 13C NMR (CDCl3): d=13.77,
13.90 (CH3 ester), 17.89, 17.97 (bC Ala), 28.08 (BOC),
37.72, 38.63, 39.50, 39.64, 41.67 (bC Phe, ring-C), 49.58,
49.77, 52.30, 55.57 (aC Phe, aC Ala), 62.16, 62.22
(OCH2), 80.24 (BOC), 126.74, 128.46, 128.61, 129.20,
129.28, 136.53, 155.60, 155.74 (BOC), 166.49, 166.61,
169.49, 171.49, 179.98. MS (EI, 70eV): m/z (%)=477.3
(0.9) [M+], 120 (100). HREIMS: calcd: 477.2111; found:
1
cm 1. H NMR (CDCl3): d=0.92 (d, J=6.6 Hz, 6H),
1.29 (t, J=7.0 Hz, 3H, CH3), 1.38 (s, 9H, BOC), 1.46±
1.52 (m, 3H, bH, gH Leu), 2.0±2.20 (m, 3H, bH, 2ÂgH
Pro), 2.20±2.32 (m, 1H, bH Pro), 3.0±3.40 (m, 4H, bH
Phe, ring-H), 3.50±3.64 (m, 1H, dH Pro), 3.68±3.80 (m,
1H, dH Pro), 4.20 (m, 2H, OCH2), 4.40 (mc, 1H, aH
Pro), 4.60 (mc, aH Phe), 4.75 (mc, aH Leu), 5.31 (bs,
1H, NH Phe), 6.81 (bs, 1H, NH Leu), 7.12±7.40 (m,
5H). 13C NMR (CDCl3): d=13.85 (CH3), 21.59, 23.21
(dC Leu), 24.52 (gC Leu), 24.80 (gC Pro), 28.15 (BOC),
28.70 (bC Pro), 39.15 (bC Phe), 39.74 (ring-C), 40.95
(bC Leu), 41.61 (ring-C), 47.07 (dC Pro), 49.23 (aC
Leu), 56.75 (aC Phe), 59.51 (aC Pro), 62.34 (OCH2),
79.38 (BOC), 126.56, 128.23, 129.61, 136.61, 154.73
(BOC), 162.82, 164.65, 166.45, 171.17, 179.62. MS
.
477.2121. Anal. (C23H31N3O8 C2H5OH): calcd: C 57.35,
H 7.12, N 8.03; found: C 57.42, H 7.08, N 7.37.
(2R,3R)+(2S,3S) 1-[N-(tertButoxycarbonyl)-(S)-leucyl-
glycyl] - 3 - ethoxycarbonyl aziridine - 2 - carboxylic acid
(R,R/S,S=1:1.5) (24a+b). The general procedure B
with 340 mg (0.65 mmol) 23a+b, 50 mL MeOH and
100 mg Pd±C gave after 3 h 280 mg (100%) 24a+b as

1290
T. Schirmeister, M. Peric / Bioorg. Med. Chem. 8 (2000) 1281±1291
(ESI): m/z (%)=655 (66) [M++K], 639 (100)
[M +Na], 617 (3) [M ]. Anal. (C31H44N4O9 MeOH):
calcd: C 59.24, H 7.46, N 8.64; found: C 59.99, H 7.05,
N 8.60.
CO2H). 13C NMR (CDCl3): d=13.32, 36.04, 51.22,
61.20, 169.19, 172.33. MS (CI, C4H10): m/z (%)=258
+
+
.
(39) [M+ CO2+C4H9], 244 (22) [M+]. Anal.
1
.
(C10H15NO6 H2O): calcd: C 47.31, H 6.34, N 5.52;
2
found: C 47.04, H 5.94, N 5.28.
(2R,3R) Diethyl -1-(benzyloxycarbonylmethyl)aziridine-
2,3 - dicarboxylate (27a). 1a (1.0 g, 5.3 mmol), 555 mg
(5.3 mmol) Na2CO3, a few crystals of KI and 1.7 mL
(10.5 mmol) benzyl bromoacetate were stirred at 60 ꢁC
in 20 mL DMF abs. for 2 days. Ice water (100 mL) was
added and the mixture was extracted with ethyl acetate
(4Â100 mL). The organic layer was washed with water
(1Â100 mL) and brine (1Â100 mL), dried with MgSO4
and removed in vacuo. The residue was puri®ed by ¯ash
column chromatography (silica gel, cyclohexane:ethyl
acetate, 5:1). Yield: 890 mg (52.3%), yellowish solid. Rf
0.2; mp 45±47 ꢁC. [a]2d0=+20.1 (c 0.76, EtOH). IR (ethyl
acetate): n=2984, 1750, 1732, 1498, 1455 cm 1. 1H NMR
(CDCl3): d=1.30 (t, J=7.1Hz, 6H), 2.84 (bs) and 3.15
(bs) (together 2H, ring-H), 3.80 (d) and 3.95 (d,
J=16.8 Hz, together 2H, NCH2), 4.19 (q, J=7.1 Hz, 2H),
5.20 (s, 2H, OCH2), 7.21±7.41 (m, 5H). 13C NMR
(CDCl3): d=13.95, 39.89 and 43.65 (ring-C), 51.56
(NCH2), 61.69 (OCH2), 66.59 (OCH2), 128.21, 128.23,
128.41, 135.31, 167.5 and 168.5 (CO2Et), 169.18 (CO2Bzl).
MS (EI, 70eV): m/z (%)=334.9 (0.83) [M+], 261.9 (100).
MS (ESI): m/z (%)=374 (70) [M++K], 358 (100)
[M++Na], 336 (7.4) [M++1]. Anal. (C17H21NO6): calcd:
C 60.89, H 6.31, N 4.18; found: C 60.98, H 6.31, N 3.98.
(2S,3S) 2-[2,3-(Bis(ethoxycarbonyl)aziridine-1-yl]acetic
acid (28b). The general procedure B with 900 mg
(2.7 mmol) 27b, 100 mL EtOH and 300 mg Pd±C gave
after 1 h of hydrogenolysis and recrystallization with
water/EtOH 590 mg (89%) 28b as colourless crystals; mp
158±160 ꢁC. [a]2d4=+4.8 (c 1.33, EtOH). IR (ethyl ace-
1
tate): n=3459, 3000, 2956, 2850, 1738, 1676, 1440 cm
.
1H NMR (CDCl3): d=1.20 (t, J=7.1Hz, 6H), 2.85 (s, 2H,
ring-H), 3.51±3.90 (m, 2H), 4.18 (q, J=7.1 Hz, 4H), 9.0
(bs, 1H, CO2H). 13C NMR (CDCl3): d=13.32, 36.04,
51.22, 61.20, 169.19, 172.33. MS (CI, C4H10): m/z
(%)=258 (100) [M+ CO2+C4H9], 244 (23) [M+]. Anal.
.
(C10H15NO6 H2O): calcd: C 45.63, H 6.51, N 5.32; found:
C 45.30, H 6.02, N 5.82.
Dibenzyl-2-amino fumarate (29b). The general proce-
dure A with 5.9 g dibenzyl fumarate yielded 2.5 g (40%)
29b as a yellowish liquid. Rf 0.60. IR (ethyl acetate):
n=3483, 3352, 3091, 3066, 3035, 2956, 2892, 1728, 1679,
1
1620, 1554, 1498, 1455, 1380 cm 1. H NMR (CDCl3):
d=5.21 (s, 2H), 5.30 (s, 2H), 5.66 (s, 1H), 6.51 (bs, 2H,
NH2), 7.25±7.50 (m, 10H). 13C NMR (CDCl3): d=65.0,
67.5, 88.0, 127.5±128.4 (6 peaks), 134.1, 136.1, 146.1,
162.5, 168.5. Anal. (C18H17NO4): calcd: C 69.44, H 5.50,
N 4.50; found: C 69.70, H 5.56, N 4.41. GC-MS:
Rt=15.0 min, m/z (%)=311 (5) [M+], 91 (100).
(2S,3S) Diethyl - 1 - (benzyloxycarbonylmethyl)aziridine -
2,3 - dicarboxylate (27b). 1b (1.6 g, 8.5 mmol), 880 mg
(8.5 mmol) Na2CO3, a few crystals of KI and 2.6 mL
(16 mmol) benzyl bromoacetate were stirred at 60 ꢁC in
40 mL DMF abs. for 2 days. Ice water (120 mL) was
added and the mixture was extracted with ethyl acetate
(4Â120 mL). The organic layer was washed with water
(1Â120 mL) and brine (1Â120 mL), dried with MgSO4
and removed in vacuo. The residue was puri®ed by ¯ash
column chromatography (silica gel, cyclohexane:ethyl
acetate, 5:1). Yield: 1.5 g (52.6%), yellowish solid. Rf
0.2; mp 49±50 ꢁC. [a]2d0= 20.7 (c 0.92, EtOH). IR (ethyl
acetate): n=2984, 1750, 1732, 1498, 1455 cm 1. 1H NMR
(CDCl3): d=1.30 (t, J=7.1 Hz, 6H), 2.84 (bs) and 3.15
(bs) (together 2H, ring-H), 3.80 (d) and 3.95 (d, J=16.8
Hz, together 2H, NCH2), 4.19 (q, J=7.1 Hz, 2H), 5.20
(s, 2H, OCH2), 7.21±7.41 (m, 5H). 13C NMR (CDCl3):
d=13.95, 39.89 and 43.65 (ring-C), 51.56 (NCH2), 61.69
(OCH2), 66.59 (OCH2), 128.21, 128.23, 128.41, 135.31,
167.5 and 168.5 (CO2Et), 169.18 (CO2Bzl). MS (CI,
C4H10): m/z (%)=336 (100) [M++1]. HREIMS: calcd:
335.1368; found: 335.1380. Anal. (C17H21NO6): calcd: C
60.89, H 6.31, N 4.18; found: C 60.82, H 6.39, N 3.69.
1-tertButyl-4-ethyl-2-amino fumarate (29c(I))/1-ethyl-4-
tertbutyl-2-amino fumarate (29c(II)), I/II=(1:1.6). The
general procedure A with 4.0 g tertbutyl ethyl fumarate
yielded 2.1 g (49%) 29c(I)+29c(II) as a yellowish liquid.
Rf 0.63. IR (ethyl acetate): n=3492, 3349, 2983, 1724,
1677, 1619, 1581, 1477, 1459, 1394 cm
1
.
1H NMR
(CDCl3): d=1.20 and 1.22 (2Ât, J=7.1 Hz, together
3H), 1.42 and 1.44 (2Âs, together 9H), 4.09 and 4.20
(2Âq, J=7.1 Hz, together 2H), 5.35 and 6.70 (2Âs,
together 1H). 13C NMR (CDCl3): d=13.95, 14.27,
27.66, 27.79, 59.20, 60.97, 81.61, 83.01, 87.94 (2 peaks),
147.62 (2 peaks), 162.40, 163.96, 164.98, 169.85. MS
(EI, 70 eV): m/z (%)=215 [M+], 87 (100). Anal.
(C10H17NO4): calcd: C 55.80, H 7.96, N 6.51; found: C
56.16, H 7.59, N 6.75. Assignment of NMR signals was
carried out by INEPT long range NMR spectroscopy.
Enzyme assays
Kinetic measurements were carried out for papain, CB
and CL(Pt) as described previously.18c The assay bu€er
for clostripain (3.0±5.9 mg/mL) was 50 mM phosphate
bu€er, pH 7.6, 200 mM NaCl, 2.5 mM DTT, and for
CL(h) (0.005 mg/mL) 50 mM citrate bu€er, pH 6.0,
5 mM EDTA, 2.5 mM DTT, 0.005% Brij 35, 200 mM
NaCl. Z-Phe-Arg-AMC (0.011±0.094 mM) was used as
substrate for both enzymes. The following Km values
were used: CL(h) 3.8 mM, clostripain 0.023 mM. The
kinetic constants were obtained by non-linear or linear
regression analysis using the program GraFit.33
(2R,3R) 2-[2,3-(Bis(ethoxycarbonyl)aziridine-1-yl]acetic
acid (28a). The general procedure B with 810 mg
(2.4 mmol) 27a, 100 mL EtOH and 150 mg Pd±C gave
after 1 h of hydrogenolysis and recrystallization with
water/EtOH 488 mg (83%) 28a as colourless crystals;
mp 151 ꢁC. [a]1d8 9.4 (c 0.51, EtOH). IR (ethyl ace-
=
1
tate): n=2955, 1740, 1676, 1440 cm
.
1H NMR
(CDCl3): d=1.20 (t, J=7.1Hz, 6H), 2.90 (s, 2H, ring-H),
3.51±3.90 (m, 2H), 4.18 (q, J=7.1 Hz, 4H), 9.0 (bs, 1H,

T. Schirmeister, M. Peric / Bioorg. Med. Chem. 8 (2000) 1281±1291
1291
Acknowledgements
16. Drake, F. H.; Dodds, R. A.; James, I. E.; Connor, J. R.;
Debouck, C.; Richardson, S.; Lee-Rykaczewski, E.; Coleman,
L.; Rieman, D.; Barthlow, R.; Hastings, G.; Gowen, M. J.
Biol. Chem. 1996, 271, 12511.
17. Hanada, K.; Tamai, M.; Yamagishi, M.; Ohmura, S.;
Sawada, J.; Tanaka, I. Agric. Biol. Chem. 1978, 42, 523.
18. (a) Martichonok, V.; Plou€e, C.; Storer, A.; Menard, R.;
Jones, J.B. J. Med. Chem. 1995, 38, 3078. (b) Schirmeister, T.
Arch. Pharm. Pharm. Med. Chem. 1997, 329, 239. (c) Schir-
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Financial support of this work by the Deutschen For-
schungsgemeinschaft DFG and the Faculty of Chem-
istry and Pharmacy, University of Freiburg, is gratefully
acknowledged. We would like to thank Professor Dr. A.
W. Frahm, University of Freiburg, Germany, for his
kind support during the last years.
19. Meara, J. P.; Rich, D. H. J. Med. Chem. 1996, 39, 3357.
20. The two peaks at 2.84 and 3.15 ppm for the ring protons in
1H NMR spectra as well as the two signals at 39.9, 43.7 ppm
and 167.5, 168.5 ppm for the ring and ethyl ester carboxyl car-
bons, respectively, in 13C NMR spectra are caused by restricted
pyramidal inversion of the aziridine-N. The inversion barrier
for compound 27b has been calculated as 16.7 (kcal mol 1) at a
coalescence temperature of 55 ꢁC in DMSO-d6.
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