Design, synthesis and stability of N-acyloxymethyl- and N-aminocarbonyloxymethyl-2-azetidinones as human leukocyte elastase inhibitors

Article abstract of DOI:10.1016/S0960-894X(01)00131-7

A series of N-acyloxymethyl- and N-aminocarbonyloxymethyl derivatives of 2-azetidinones, 3, with different substituent patterns at the β-lactam C-3 and C-4 positions, were designed as potential mechanism-based inhibitors for human leukocyte elastase and f

Full text of DOI:10.1016/S0960-894X(01)00131-7

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1065–1068
Design, Synthesis and Stability of N-Acyloxymethyl- and
N-Aminocarbonyloxymethyl-2-azetidinones as Human
Leukocyte Elastase Inhibitors
A. Clemente,a A. Domingos,a A. P. Grancho,a J. Iley,b R. Moreira,c,* J. Neres,c
N. Palma,d,e A. B. Santana c and E. Valente c
aINETI, Dep. Biotecnologia, Estrada das Palmeiras, 1649 Lisboa, Portugal
bChemistry Department, The Open University, Milton Keynes MK7 6AA, UK
c
´
CECF, Faculdade de Farmacia, Universidade de Lisboa, Av. Forc¸as Armadas, 1600 Lisboa, Portugal
dCentro de Quimica Fina e Biotecnologia, Universidade Nova de Lisboa, 2825 Monte de Caparica, Portugal
eInstituto Superior de Cieˆncias da Sau´de-Sul, 2829 Monte de Caparica, Portugal
Received 27 November 2000; revised 13 February 2001; accepted 23 February 2001
Abstract—A series of N-acyloxymethyl- and N-aminocarbonyloxymethyl derivatives of 2-azetidinones, 3, with different substituent
patterns at the b-lactam C-3 and C-4 positions, were designed as potential mechanism-based inhibitors for human leukocyte elas-
tase and found to exhibit inhibitory potency and selectivity for the enzyme. # 2001 Elsevier Science Ltd. All rights reserved.
Human leukocyte elastase (HLE, EC 3.4.21.37) is a
serine protease that has received great attention because
it efficiently degrades various tissue matrix proteins such
as elastin.1 The imbalance between HLEand its endo-
genous inhibitors and the subsequent excessive elastin
proteolysis has been implicated in acute and chronic
inflammatory diseases of the lungs.2,3 Thus, specific
inhibitors of HLEcapable of restoring the protease/
antiprotease imbalance might be beneficial in such
pathologies.4,5
leaving group with pKa>4 would be expected to
increase substantially the stability of 3 in aqueous buf-
fers and thus increase their ability to reach their target.
We now report the synthesis and biochemical evaluation
of C-3 and C-4 substituted N-acyloxymethyl and N-
aminocarbonyloxymethyl-2-azetidinones.
Compounds 3 were prepared (Scheme 2) by known
methods,11 using the appropriate 4-acetoxy-2-azeti-
dinones 411b as starting materials.
Cephalosporin sulfones, e.g., 1, are potent, mechanism-
6ꢀ8
Compounds 3 were found to be time-dependent irrever-
sible inhibitors of HLE, the inhibitory activity (Table 1)
based irreversible HLEinhibitors.
A structural
requirement of 1 is the presence of a good leaving group
L (e.g., acetate) at the 30-position, which is thought to
provide an electrophilic center for the nucleophilic
attack of histidine-57 following initial serine-195 attack
at the b-lactam carbonyl atom (Scheme 1). We reasoned
that simple b-lactams 3, in which L is a good leaving
group such as a carboxylic or carbamic acid, would also
be capable of inhibiting HLEvia a similar ‘double-hit’
mechanism. From our previous experience with the
chemistry of N-acyloxymethylamides,9,10 appending a
*Corresponding author. Tel.: +351-21-794-6416; fax: +351-21-794-
6470; e-mail: rmoreira@ff.ul.pt
Scheme 1.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00131-7

1066
A. Clemente et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1065–1068
Substitution at C-3
Substitution at this position generally leads to more
potent inhibitors. Incorporation of two ethyl groups at
C-3 increases inhibition up to ca. 4-fold (compounds 3c
and 3g). This finding is in agreement with the known S1
subsite specificity of HLEtoward small hydrophobic
substituents with three or four carbon atoms15 and is
identical to the reported preference of HLEtoward C-3
substituted N-acyl-2-azetidinones.11,16
Substitution at C-4
Scheme 2. Reagents and conditions: (i) R3SH/NaOH/acetone; (ii)
MCPBA; (iii) HCHO/aq K2CO3; (iv) RCOCl/Et3N; RNCO/Et3N.
The phenyl sulfones 3c and 3g are more potent inhibi-
tors of HLEthan the corresponding sulfides, 3d and 3h,
respectively. This result compares to the superior HLE
inhibitory activity displayed by cephalosporin sulfones 1
relative to their sufide counterparts.6,7 However, there is
not an absolute requirement for a sulfone at C-4 to
achieve HLEinhibition, as shown by the inhibition dis-
played by the sulfides 3e and 3f as well as by the 2-fold
greater inhibitory potency of 3a over its sulfone-con-
taining analogue 3c. The superior activity displayed by
the 4-phenylsulfone and 4-(20-thiobenzoxazolyl) deriva-
tives can be ascribed to the electronegativity of these
substituents, which is likely to increase the reactivity of
the b-lactam carbonyl group. Alternatively, both sul-
fone and 2-mercaptobenzoxazole may function as leav-
ing groups after the b-lactam ring opening (Scheme 3,
path B). A similar mechanism, in which a sulfone serves as
a leaving group, has been suggested for the b-lactamase
inhibition by sulbactam.17 Indeed, it was recently repor-
ted that the use of 2-mercaptobenzoxazole and related
sulfides as leaving groups in the 1,2,5-thiadiazolidin-3-
being expressed by the bimolecular rate constant kobs/[I]
determined by Kitz and Wilson’s pre-incubation
method.12,13 The irreversible nature of the inactivation
was shown when no significant reactivation of enzyme
activity was detected upon dialyzing HLEagainst pH
7.2 HEPES buffer for 24 h. Independently, the native
enzyme itself was found to retain activity after dialysis.
Inhibition toward pancreatic porcine elastase (PPE) was
also evaluated,14 but none of the compounds studied
inactivated this enzyme at [I]/[E]=200. The compounds
3 displayed variable inhibitory potencies towards HLE,
the unsubstituted prototype 3a being a fair inhibitor
(Table 1). Unfortunately, compound 3b could not be
evaluated under the assay conditions due to solubility
problems. For comparison, data for two cephalosporins
is also presented in Table 1. Inspection of the data in
Table 1, allows the following conclusions to be drawn.
Table 1. Second-order rate constants, kobs/[I], for the inhibition of HLEand PPEby N-acyloxymethyl- and N-aminocarbonloxymethyl-2-azetidi-
nones 3 at 25 ꢁC, and half-lives in pH 7.4 phosphate buffer at 37 ꢁC
kobs/[I], Mꢀ1 sꢀ1
Compound
R1
R2
R3
L
HLEPPSEtability
t1/2/h
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
8b
9
1a7a
1b7b
H
H
H
H
H
Et
E
E
E
E
E
E
E
H
MeO
MeO
H
H
H
H
H
Et
H
H
OCOC6H5
B
35.5
ND
18.7
NI
43.2
52.8
67.5
2.2
41.9
60.9
100.3
ND
NIa
NDb
NI
ND
NI
NI
NI
ND
NI
NI
NI
ND
ND
ND
—
1.3
ND
978
ND
ND
316
2995
ND
ND
ND
SO2C6H5
SC6H5
A
OCOC6H5
OCOC6H5
OCOC6H5
OCOC6H5
OCSOOC6H5
OCOSC6H5
OCOSNOHC6H5
SOC
A
2C6H5
6H5
2C6H5
2C6H5
2C6H5
2C6H5
2C6H5
H
t
E
t
t
t
t
t
t
t
t
t
t
t
t
t
E
E
E
E
E
E
SOD
>5000
SOE32.3
SOOH
OH
OAc
F
NI
NI
19000
0.11 mMc
ND
ND
–
H
H
H
—
—
—
19
aNo inhibition.
bNot determined.
cIC50
.

A. Clemente et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1065–1068
1067
one 1,1-dioxide scaffold resulted in highly potent HLE
inhibitors.18
substituent (Table 1). Indeed, while the unsubstituted
compound 3a is rapidly hydrolyzed with t1/2 of ca. 80
min, its 4-phenylsulfone analogue 3c presents a 750-fold
increase in stability. Introducing the C3 substituents
leads to further increase in stability (3c vs 3g). Carba-
mate leaving groups lead to improved stability, when
compared to a carboxylate leaving group (3g vs 3k).
Nature of the Leaving Group L
Both carboxylate and carbamic acid leaving groups lead
to active compounds, the most active compound being
the 4-methoxyphenylcarbamic acid derivative 3k. Use of
a poor leaving group, L=OH, (8b and 9) leads to the
loss of inhibitory activity. Modification of the leaving
group by using an ionizable functionality to increase
solubility reduced inhibitory potency (compare com-
pounds 3j and 3l, the latter of which has pKa ca. 319).
Molecular Modeling
The molecular interactions between the inhibitors 3 and
HLE(PDB code 1hne) were studied with the program
GOLD.20ꢀ22 Docking results show that the presence of
ethyl groups at C3 consistently restricts the degrees of
freedom of ligand binding as compared to the non-sub-
stituted analogues (compounds 3d/3h, 3c/3g and 3e/3f),
and helps anchor the ligand in the S1 subsite forcing the
carbonyl C2 into close vicinity of the Ser195 hydroxyl
oxygen (d<3A).
Stability
The stability study carried out in pH 7.4 phosphate buf-
fer reveals that reactivity is mainly dependent on the C-4
Substitution at C4 greatly enhances the van der Waals
contacts with Leu99 and His57. Moreover, the presence
of a sulfone (compounds 3c and 3g compared to 3d and
3h, respectively) enables a further stabilization of the
complex by hydrogen bonding to the NH of Val216 (2.5
A) (Fig. 1). The larger benzoxazolyl moiety of 3f seems
to force the b-lactam ring deeper inside the active site,
reducing the distance between C2 and Ser195 hydroxyl
oxygen. Comparative docking experiments with the C4
substituted inhibitors (e.g., 3k) indicate that only R-
enantiomers consistently interact with the b-lactam car-
bonyl carbon atom accessible to the serine hydroxyl
oxygen. This finding suggests that a stereospecific
synthesis of the R-enantiomers alone should improve
inhibitor potency.
Interestingly, our docking results with the intermediate
11 do not support the hypothetical mechanism in which
the substituents at C4 would function as the leaving
group (Scheme 3, path B). In fact, all docked config-
urations of intermediate 11 show that the electron defi-
cient C4 atom becomes sterically inaccessible to the
Scheme 3.
Figure 1. Molecular docking of inhibitors into the active site of HLE(see text for details of docking procedure). A: Inhibitor 3k interacting with
active site by van der Waals contacts and hydrogen bonding (green dashed lines). B: Best scoring docked configuration of intermediate 10, covalently
bound to Ser195. Orange arrows show close contacts between enzyme nucleophiles and electron deficient atoms of the ligand.

1068
A. Clemente et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1065–1068
His57 nitrogen atom. In contrast, the alternative
mechanism involving the intermediate 10 (Scheme 3,
path A) appears to be compatible with our docking
experiments. Figure 1B shows this intermediate bound
to the active site Ser195 and with the methylene group
facing the NE2 atom of His57 (d=2.7 A).
9. Iley, J.; Moreira, R.; Rosa, E. J. Chem. Soc., Perkin Trans.
2 1991, 563.
10. Iley, J.; Moreira, R.; Calheiros, T.; Mendes, E. Pharm.
Res. 1997, 14, 1634.
11. (a) Gu, H.; Fedor, L. R. J. Org. Chem. 1990, 55, 5655.
(b) Shah, S. K.; Dorn, C. P.; Finke, P. E.; Hale, J. J.; Hag-
mann, W. K.; Brause, K. A.; Chandler, G. O.; Kissinger,
A. L.; Ashe, B. M.; Weston, H.; Knight, W. B.; Maycock,
A. L.; Dellea, P. S.; Fletcher, D. S.; Hand, K. M.; Mumford,
R. A.; Underwood, D. J.; Doherty, J. B. J. Med. Chem. 1992,
35, 3745.
12. Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245.
13. HLE(Calbiochem, Germany) was assayed spectro-
photometrically at 25 ꢁC by monitoring the release of 4-
nitroaniline from MeO-Suc-Ala-Pro-Val-4-nitroanilide at
410 nm. Typically, the inhibitor in DMSO was mixed with the
enzyme in pH 7.2 HEPES buffer and placed in a constant-
temperature bath. Aliquots were withdrawn at different time
intervals and transferred to a cuvette containing the substrate
in HEPES buffer. After incubating for 30 s, the absorbance
change was followed for 60 s.
In conclusion, N-acyloxymethyl- and N-aminocarbonyl-
oxymethyl-2-azetidinones 3 are time-dependent irrever-
sible inhibitors of HLE. Derivatives 3 containing either
a phenyl sulfone or a 20-mercaptobenzoxazolyl sub-
stituent at C-4 and a gem-diethyl at C-3 were the most
potent inhibitors. The results are consistent with,
though do not prove, the mechanism A depicted in
Scheme 3, with substituents at C-4 enhancing the reac-
tivity of the b-lactam carbonyl. Ongoing studies aimed
at optimizing inhibitory potency and elucidating the
mechanism of action of these compounds are currently
in progress.
14. Inhibition toward PPEwas assayed as for HLE, using
Suc-Ala-Ala-Ala-4-nitroanilide as substrate.
15. Bode, W.; Meyer, E.; Powers, J. C. Biochemistry 1989, 28,
1451.
Acknowledgements
16. Firestone, R. A.; Barker, P. L.; Pisano, J. M.; Ashe, B. M.;
Dahlgren, M. E. Tetrahedron 1990, 46, 2255.
17. Mossova, I.; Mobashery, S. Acc. Chem. Res. 1997, 30,
162.
This project was supported by the contract PRAXIS/
QUI/10039/98.
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