3-Acylamino-azetidin-2-one as a novel class of cysteine proteases inhibitors

Article abstract of DOI:10.1016/S0960-894X(02)00831-4

A new class of inhibitors for cysteine proteases cathepsin B, L, K and S is described. These inhibitors are based on the β-lactam ring designed to interact with the nucleophilic thiol of the cysteine in the active site of cysteine proteases. Some 3-acylamino-azetidin-2-one derivatives showed very potent inhibition activities for cathepsins L, K and S at the nanomolar or subnanomolar IC₅₀ values.

Full text of DOI:10.1016/S0960-894X(02)00831-4

Bioorganic & Medicinal Chemistry Letters 13 (2003) 139–141
3-Acylamino-azetidin-2-one as a Novel Class of Cysteine
Proteases Inhibitors
Nian E. Zhou,a Deqi Guo,a George Thomas,a Andhe V. N. Reddy,a Jadwiga Kaleta,a
Enrico Purisima,b Robert Menard,b Ronald G. Miceticha and Rajeshwar Singha,*
aSynPhar Laboratories, currently NAEJA Pharmaceutical Inc., 4290-91A Street, Edmonton, Alberta, Canada T6E 5V2
bNational Research Council Canada, BRI, 6100 Royalmount Ave, Montreal, Quebec, Canada H4P 2R2
Received 5 July 2002; accepted 14 September 2002
Abstract—A new class of inhibitors for cysteine proteases cathepsin B, L, K and S is described. These inhibitors are based on the b-
lactam ring designed to interact with the nucleophilic thiol of the cysteine in the active site of cysteine proteases. Some 3-acylamino-
azetidin-2-one derivatives showed very potent inhibition activities for cathepsins L, K and S at the nanomolar or subnanomolar
IC50 values.
# 2002 Elsevier Science Ltd. All rights reserved.
The cysteine proteases cathepsin B, L, K and S are
involved in diseases1 such as osteoporosis, cancer meta-
stasis, rheumatoid arthritis, and infectious diseases.
These proteases may be important targets for the
development of inhibitors as therapeutic agents.2 Sev-
eral types of chemical functionalities served as the cen-
tral pharmacophore for cysteine proteases inhibitors,3
such as aldehydes,4 nitriles,5 a-ketocarbonyl compounds,6
ketones7 (halomethyl, acyloxymethyl, mercaptomethyl,
cyclic), epoxide,8 vinyl sulfones,9 and aryaminoethyl
amides.10 To explore new classes of pharmacophores for
cysteine proteases inhibitors, a series of 4-substituted-3-
Cbz-Phe-b-lactam compounds are identified as a novel
class of cysteine proteases inhibitors. The design,
synthesis and activities of these inhibitors are reported
in this communication.
fragment was selected as the substituent at C3 of the b-
lactam ring to explore novel pharmacophore.
The synthesis of the b-lactam compounds reported in
this communication is shown in Scheme 1. The inter-
mediate 1 was prepared according to a known method14
from D-Ser. Using a similar method; its 3S-isomer 3
was also prepared. Compounds 2, 4, and 6 were pre-
pared by coupling Cbz-Phe-OH with (3R)-3-amino-
monobactam 1, (3S)-3-amino-monobactam 3 or (3S)-3-
amino-4-acetoxy-monobactam15 5, respectively, via the
unsymmetrical anhydride method or via an active ester.
The compounds 7 and 8 were obtained by reacting 6
with the carboxylic acids in presence of NaOH. Only
trans lactam products 9–12 were obtained by refluxing
of the acetate 6 with the corresponding alcohols in the
presence of zinc acetate dihydrate in benzene–toluene
with the azeotropic removal of water.16 Substitution of
the acetate 6 with phenol or thiophenol in presence of
NaOH gave both trans b-lactam compound 13 and 16
and its cis-isomer 14 and 15, respectively. The trans- and
cis-isomers were separated by silica gel column chro-
matography. The trans-isomer 13 and 16 could also be
prepared by reacting 6 with phenol or thiophenol in the
presence of zinc acetate dihydrate in benzene–toluene in
low yield. The sulfone compound 17 was obtained by
oxidation of 16 with KMnO4 in acetic acid.
The b-lactam skeleton is a well known pharmacophore
for antibiotics11 and for inhibitors of serine proteases.12
The chemical reactivity of the b-lactam ring can be
adjusted by substitution at the C3 and C4 positions. In
addition, substituents at C3 and C4 of b-lactam skele-
ton may extend interactions into both the S and S0 sub-
sites which may increase potency and specificity. Known
substrate selectivity for the papain type of cysteine pro-
teases provided us with the template for exploration of a
new pharmacophore. Since papain has a high specificity
for phenylalanine at the P2 position,13 the Cbz-Phe
The inhibitory activities of these compounds against
cathepsins B, L, K, and S were determined according to
the procedure described in the literature17 by using Cbz-
*Corresponding author. Tel.: +1-780-462-4044; fax: +1-780-461-
0196; e-mail: rsingh@naeja.com
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00831-4

140
N. E. Zhou et al. / Bioorg. Med. Chem. Lett. 13 (2003) 139–141
Scheme 1. Reagents and conditions: (a) TFA, DCM, anisole; (b) Cbz-Phe-OH, Cl–COOEt, TEA, 2: 70%, 4: 90%; (c) H2, Pd/C, EtOAc; (d) Cbz-
Phe-OH, DCC, HOBT, THF, 6: 90%; (e) RCOOH, NaOH, H2O, acetone, 7: 20%, 8: 24%; (f) ROH, Zn(OAc)2–2H2O, benzene/toluene, reflux 4 h,
9: 14%, 10: 7%, 11: 14%, 12: 26%; (g) Ph–OH, NaOH, H2O, THF, 13: 47%, 14: 25%; (h) Ph–SH, NaOH, H2O, THF, 15: 15%, 16: 33%;
(i) KMnO4, AcOH, H2O, 17: 70%.
Phe-Arg-AMC as substrate for cathepsin B, L and K
and Cbz-Val-Val-Arg-AMC for cathepsin S. The inhib-
itory activities of the compounds are shown in Table 1.
All of these b-lactam compounds can selectively inhibit
the papain type of cysteine proteases (cathepsins) with
no inhibition or non-significant inhibition of serine pro-
tease, human leukocyte elastase. They are more potent
towards cathepsin L and K, closely related enzymes,
than for cathepsin B. The more active compound 4 in
comparison to 2 against cathepsin B suggested the
importance of the 3S stereo configuration at C-3, which
is equal to the configuration of natural l-amino acids,
on the interactions with the enzymes. The substitution
at C-4 showed a significant effect on inhibitory potency.
The substitutions at C-4 such as –OR, –OCOR, –OPh,
–SPh and –S(O)2Ph appear to be necessary for a good
inhibitory activity. For the alkoxy substitution, the linear
group as in 9 showed better activities against cathepsins
K and S than branched groups 10–11. In terms of ether
group, phenoxy (13 and 14) is much better than alkoxy
substitutions (9–12). However, the substituent at C-4
may not function just as a leaving group, it is also
involved in interactions with the S0 subsite of the
enzyme. The stereo configuration requirement of C-4
depends upon the nature of the substituents. For the
phenoxy substituent, the cis-isomer 14 is more active
than trans-isomer 13. However, the opposite effect was
observed for the phenylthio substituent, the trans-iso-
mer 16 being more potent than the cis-isomer 15. The
Table 1. In vitro inhibitory activity of azetidin-2-one derivatives with
cysteine and serine proteases
Compd
IC50 (mM)
Cath B
Cath L
Cath K
Cath S
HLE
2
4
6
7
8
9
10
11
12
13
14
15
16
17
13.6
2.70
0.47
27.5
9.71
7.01
6.46
11.4
32.57
10.9
0.43
39.7
10.5
7.39
37.4
nd
>5
nd
>5
nd
>50
>50
>10
>50
>50
>50
>50
>50
>50
>30
>30
>50
>50
>50
0.042
0.166
0.047
0.163
0.091
0.78
0.100
0.100
0.038
0.100
2.30
0.207
0.020
0.041
0.355
>2.5
>2.5
>2.5
0.33
0.0
2.5
2.5
3.96
0.017
0.0026
0.015
0.0001
0.0001
0.100
0.016
0.072
0.0084
0.0065
2.5
0.33
0.048
Cath L, K and S are recombinant human enzymes; cath B is recom-
binant rat enzyme; HLE is human leukocyte elastase as an example of
a serine protease; nd, not determined; IC50 values were usually derived
from single experiments using a range of inhibitor concentration.

N. E. Zhou et al. / Bioorg. Med. Chem. Lett. 13 (2003) 139–141
141
most potent compounds have the phenylthiol (16) or
phenylsulfonyl (17) group at C-4 with IC50 values in the
subnanomolar range for cathepsin L.
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