Synthesis, hydrolysis, biochemical and theoretical evaluation of 1,4-bis(alkoxycarbonyl)azetidin-2-ones as potential elastase inhibitors

Article abstract of DOI:10.1016/S0040-4020(02)00112-6

A series of 1,4-bis(alkoxycarbonyl)azetidin-2-ones, designed as potential suicide-inhibitors of serine proteases, has been synthesized and evaluated against porcine pancreatic elastase (PPE). The most active compound (K_i～10 μM; reversible inhibitor) was equipped with phenethyloxycarbonyl and benzyloxycarbonyl side-chains at positions N1 and C4, respectively, with the (S)-configuration. ¹H NMR spectroscopic analysis of the reaction mixtures showed that the ester function is preferentially hydrolyzed, in both chemical and enzyme-catalyzed reactions, with regard to the azetidinone and urethane functions. Considering the three potentially sensitive carbonyl functions and the two stereoisomers, ab initio calculations were performed to determine the energetic barriers required to reach the transition state structures of hydrolysis in a model of the enzyme pocket.

Full text of DOI:10.1016/S0040-4020(02)00112-6

TETRAHEDRON
Pergamon
Tetrahedron 58 (2002) 2423±2433
Synthesis, hydrolysis, biochemical and theoretical evaluation
of 1,4-bis(alkoxycarbonyl)azetidin-2-ones as potential
elastase inhibitors
a
b
a
c
Â
Â
Stephane Gerard, Georges Dive, Brigitte Clamot, Roland Touillaux
and Jacqueline Marchand-Brynaerta,p
a
   Ã
Unite de Chimie Organique et Medicinale, Universite Catholique de Louvain, Batiment Lavoisier, Place Louis Pasteur 1,
B-1348Louvain-la-Neuve, Belgium
Á Ã Â
Centre d'Ingenierie des Proteines, Universite de Liege, Batiment B6, allee de la chimie 17, B-4000 Sart-Tilman, Liege, Belgium
b
Â
Â
Â
c
  Ã
Unite d'Analyse Structurale, Universite Catholique de Louvain, Batiment Lavoisier, Place Louis Pasteur 1,
B-1348Louvain-la-Neuve, Belgium
Received 11 September 2001; revised 20 December 2001; accepted 24 January 2002
AbstractÐA series of 1,4-bis(alkoxycarbonyl)azetidin-2-ones, designed as potential suicide-inhibitors of serine proteases, has been
synthesized and evaluated against porcine pancreatic elastase (PPE). The most active compound (Ki,10 mM; reversible inhibitor) was
equipped with phenethyloxycarbonyl and benzyloxycarbonyl side-chains at positions N1 and C4, respectively, with the (S)-con®guration. 1H
NMR spectroscopic analysis of the reaction mixtures showed that the ester function is preferentially hydrolyzed, in both chemical and
enzyme-catalyzed reactions, with regard to the azetidinone and urethane functions. Considering the three potentially sensitive carbonyl
functions and the two stereoisomers, ab initio calculations were performed to determine the energetic barriers required to reach the transition
state structures of hydrolysis in a model of the enzyme pocket. q 2002 Elsevier Science Ltd. All rights reserved.
1.Introduction
promote irreversible inhibition via
mechanism.17±22
a
suicide-type
The search for mechanism-based inhibitors1 of serine
proteases is still an attractive approach for the discovery
of new active compounds. Important target-enzymes are
b-lactamases2 and DD-transpeptidases3 for antibacterial
drugs, and elastases4 for anti-in¯ammatory drugs. Various
reactive chemical structures have been considered to inter-
act with the essential serine nucleophile of the enzymic
cavity, as for instance, a-¯uoroketones,5 halo-enol-
lactones,6 benzisothiazolones,7 phthalimides,8 thiadiazo-
lidinones,9 and coumarinic derivatives.10,11 However, the
b-lactam heterocycle (azetidin-2-one) remains one of the
most exploited lead-structures,12±14 due to the remarkable
activity of the naturally occurring antibiotics (penicillins
and cephalosporins) which are bicyclic azetidinones.15
Monocyclic azetidinones also behave as serine-protease
inhibitors, provided that they are adequately functionalized
with substituents raising speci®c enzyme recognition and
chemical activation towards nucleophilic attack.16 More-
over, the presence of a potential leaving group could
Recently, we became interested in the development of new
b-lactamic inhibitors bearing both the activating substituent
(EWG: electron-withdrawing group) and the leaving group
(LG) in position N1.23 A ®rst combination of EWG and LG
substituents resulted in the synthesis of N-[a-(hetero-
functionalized)-carboxymethylated]-azetidinones
which
could unmask a reactive Schiff base under processing by
the target-enzymes. However, the compounds tested were
found to be inactive against b-lactamases of classes A, B, C,
and D, and to behave as weak reversible inhibitors of
porcine pancreatic elastase (PPE).23 Another series of
azetidinones (general structure A) was equipped with
N-(alkoxycarbonyl) substituents designed to unmask an iso-
cyanate function24 into the enzymic cavity, by ring opening
under nucleophilic attack of Ser-195 followed by expulsion
of the alkoxyl group; suicide-inhibition was expected by
quenching of the isocyanate intermediate with a nucleo-
philic residue (His-57), as illustrated in Scheme 1. Only a
few studies of activated (N-acyl or N-sulfonyl) carbamate
hydrolysis have been described in the literature;25 however,
formation of electrophilic intermediates (isocyanates) over
a wide pH range has been unambiguously proved by kinetics
and trapping experiments. In this chemical process, the
quality of the leaving group appears important; it could be
Keywords: b-lactam; serine-protease inhibition; elastase; ab initio
calculation.
p
Corresponding author. Tel.: 132-10-47-27-40; fax: 132-10-47-41-68;
e-mail: marchand@chim.ucl.ac.be
0040±4020/02/$ -see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)00112-6

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S. Gerard et al. / Tetrahedron 58(2002) 2423±2433
2424
Scheme 1. Expected mechanism of suicide-inhibition of serine proteases.
of less signi®cance in an enzymic process where proton
transfer and speci®c interaction with the protein occur.
Experimentally, we found that 3-bromo-derivatives A
(R3ˆ(R)-Br; R2ˆH; R1ˆCH2Ph, CH2CCl3, CH2CF3) were
transient inhibitors of PPE (low kcat and Km values); the
enzyme catalyzes ring opening, but without expulsion of
the leaving group.26
and H30 protons at 4.45
d
(dd, Jcisˆ6.6 Hz and
Jtransˆ3.3 Hz), 3.30 d (dd, JABˆ15.9 and 6.6 Hz) and 3.00
d (dd, JABˆ15.9 and 3.3 Hz), respectively. The three
carbonylic carbons appeared at 168.6 d (ester), 161.7 d
(b-lactam) and 147.9 d (carbamate). The b-lactam carbonyl
stretching at 1812 cm21 for compounds 3,4 shifted to
1825 cm21 for compounds 5±9 bearing the ester substituent
(electron-withdrawing substituent at C4).
With a view to improving the biological activity of the
N-(alkoxycarbonyl)azetidinones towards elastase, we
considered new derivatives bearing a side chain in position
C4, combining enzymic recognition and activation factors,
namely an alkyloxycarbonyl group. Such an electron-
withdrawing substituent has been previously reported to ®t
well into the active site of elastases (human leukocyte
elastase (HLE) and PPE) in the case of azetidinones
equipped with a sulfonyl substituent,27 or a benzoyl
group28 in position N1; enzymic hydrolysis induced the
cleavage of the activated b-lactam bond. In this paper, we
describe the synthesis and the hydrolysis (chemical and
enzymic) of azetidinones A bearing alkoxycarbonyl sub-
stituents in both positions N1 and C4. Unexpected results
stimulated a theoretical study of reactivity.
Enantiomerically pure azetidinone 6a was obtained starting
from 2a [(S)-enantiomer], while 6b [(R)-enantiomer] was
isolated by preparative HPLC of racemic 6 over a chiral
stationary phase.
Compounds 10a (methyl ester) and 11a (i-propyl ester)
(Scheme 3), prepared by transesteri®cation32 of 2a, were
N-functionalized with the phenethyloxycarbonyl side-
chain as above; the NMR spectroscopic features of the
2.Results and discussion
2.1. Synthesis
Scheme 2. Synthesis of compounds.
Table 1. Yields of compounds
N-Acyl-azetidinones could be prepared from N-unsubsti-
tuted precursors by deprotonation with lithium hexamethyl-
disilazide at low temperature and quenching of the resulting
anion with chloroformates.26 This procedure has been
applied to the synthesis of compounds 3±9 (Scheme 2,
Table 1), starting from the commercially available azetidi-
none 1 and azetidinone 2 obtained in four steps from
aspartic acid [(S)-enantiomer or racemic mixture].29,30 The
chloroformates (except benzylchloroformate) were
prepared by reacting the corresponding alcohols, namely
phenethyl alcohol, 3-phenylpropyl alcohol, 2-indanol and
2-cyclohexylethanol, with triphosgene and pyridine in
toluene at 08C.31 The N-substituted derivatives 3±9 were
puri®ed by chromatography on silica gel, and were charac-
terized by NMR spectroscopy. The 4-substituted azetidi-
nones 5±9 showed a typical ABX pattern for the H4, H3
Entry
Compound
R2
R1
Yielda (%)
1
2
3
4
5
3
4
H
H
CH2Ph
CH2CH2Ph
75
98
68
50
75
5b
6b
7b
CO2CH2Ph CH2Ph
CO2CH2Ph CH2CH2Ph
CO2CH2Ph CH2CH2CH2Ph
6
8b
9b
CO2CH2Ph
CO2CH2Ph
56
69
7
a
Isolated by column-chromatography.
Racemic mixture.
b

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S. Gerard et al. / Tetrahedron 58(2002) 2423±2433
2425
Scheme 3. Synthesis of compounds.
resulting azetidinone 12a [(S)-enantiomer] and 13a [(S)-
enantiomer] were similar to those of 6. Lastly, the free
acid 14 (Scheme 3) resulted from hydrogenolysis of the
benzyl ester 6.
where the slope corresponds to 1/Ki. The measured
inhibition constants (Ki) are collected in Table 2.
Table 2. PPE inhibition
Compound R1
R2
R3 Ki (mM)
2.2. Elastase inhibition
2
3
4
5
±
CH2Ph
CH2CH2Ph
CH2Ph
CH2CH2Ph
CH2CH2Ph
CH2CH2Ph
CH2CH2CH2Ph
CO2CH2Ph
H
H
CO2CH2Ph
CO2CH2Ph
CO2CH2Ph
CO2CH2Ph
CO2CH2Ph
H
H
H
H
H
H
H
H
No inhibition
No inhibition
Compounds 2±14 were evaluated for their potential inhibi-
tory effect on PPE. This readily available enzyme could be
considered as a good model of HLE; crystallographic
studies have demonstrated33 the structural similarity
between the active sites of both enzymes.
Low (.103)
110
12
10
330
50
6
6a (S)
6b (R)
7
The rates of elastase-catalyzed hydrolysis of N-succinyl±
Ala±Ala±Ala±p-nitroanilide (substrate) were measured
([E]ˆ2£1027 M; [S]ˆ3£1024 M, pH 7.5) in the presence
5£1025 and 1025 M). The variation of absorbance at 410 nm
was recorded as a function of time (Fig. 1A); transient
inhibitions were observed, then complete enzymic activity
was restored after about 180 min. The control of the
reversibility was assessed as usual by the incubation\
dilution method (see Section 4).
8
9
CO2CH2Ph
CO2CH2Ph
H
H
No inhibition
No inhibition
of various concentrations of potential inhibitors ([I]ˆ1024
,
12a (S)
13a (S)
14
CH2CH2Ph
CH2CH2Ph
CH2CH2Ph
CH2Ph
CO2CH3
CO2CH(CH3)2
CO2H
H
H
H
68
87
$700
15
H
Br 1.5
See Scheme 1 for R1/R2/R3 description.
Plots of V/VI, corresponding to the ratios of initial rates of
hydrolysis in the absence and in the presence of inhibitor
versus the concentration of inhibitor gave a straight line
The presence of the N1-alkoxycarbonyl substituent is
required for activity, since 2 was inactive. However, a
second alkoxycarbonyl substituent at C4 dramatically
Figure 1. Inhibition of PPE by compound 6a (A) (0, [I]ˆ0; a, [I]ˆ1024 M; b, [I]ˆ5£1025 M and c, [I]ˆ1025 M) and compound 15 (B) (0, [I]ˆ0; a,
[I]ˆ1024 M; b, [I]ˆ5£1025 M and c, [I]ˆ1025 M).

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S. Gerard et al. / Tetrahedron 58(2002) 2423±2433
2426
1
improved the inhibitory effect, as shown by comparison of 3
and 4 to, respectively, 5 and 6. Evaluation of each enantio-
mer of 6 revealed that the activity is mainly due to the
(S)-enantiomer 6a giving a Ki value of 10 mM. This enantio-
selectivity is opposite to that observed with related
C4-substituted inhibitors (such as Merck's L-694,458),34
and could suggest a different positioning in the active site.
Also, the best enzymic ®t required the presence of a phenyl
residue on the R1 side-chain (compare 6 with 9) with a two-
methylene spacer (compare 6 with 5 and 7), and some ¯exi-
bility (compare 6 with 8). Replacing the benzyl ester of the
R2 side-chain with methyl or isopropyl esters did not greatly
reduce the inhibitory activity (compare 6 with 12 and 13),
while the corresponding free carboxylate 14 was found to be
practically inactive. Thus, by varying the N1 and C4 side-
chains, we could not ®nd a better inhibitor than 6a.
However, this compound remained about 10 times less
active than the 3-bromo-azetidinone 15 (Table 2) described
previously.26 Moreover, the inhibition curves of 6 (Fig. 1A),
and related C4 substituted compounds (5, 7, 12, 13)
displayed a totally different shape compared to the reference
15 (Fig. 1B), suggesting a different mode of action. It has
been demonstrated by NMR spectroscopic experiments that
the b-lactam ring of compound 15 was easily opened by
chemical or enzymic hydrolysis, but without expulsion of
R1OH. The reactions of 4 and 6 were similarly monitored by
1H NMR spectroscopy.
analyzed by H NMR spectroscopy at 500 MHz as a func-
tion of time. No detectable reaction occurred within 24 h.
Addition of PPE (6£1025 M) led to the slow transformation
of 4 into b-aminoacid derivative 16 (Scheme 4); the
b-lactam ring opening was ascertained by the signi®cant
deshielding of the former's C3 and C4 protons. As in
the case of compound 15, no cleavage of the urethane
function was observed. This is consistent with the kinetic
measurements, and the curve shapes (recorded by spectro-
photometry) of 4 which look like to those of 15.
The inhibitor 6 behaved totally different. In the absence of
enzyme, chemical hydrolysis slowly occurred (1023 M
solution of 6 in deuteriated phosphate buffer, pD 7.5,
50 mM15% DMSO-d6), leading to the acid 14 (Scheme 5)
as a result of ester cleavage without b-lactam ring opening;
benzyl alcohol was formed (singlet at 4.44 d), but not
phenethyl alcohol (two triplets at 2.68 d and 3.65 d). Acid
14 was unambiguously identi®ed by comparison with a
reference sample prepared by hydrogenolysis of 6.
Nucleophilic attack on activated b-lactams (N1-EWG
derivatives) is a known methodology for the construction
of functionalized b-aminoacid derivatives.35 However,
examples involving
a possible competition between
azetidinone ring opening and attack on an ester substituent
are not described very often. Carbon based nucleophiles
(sulfone stabilized carbanions, for instance) showed low
selectivity for the b-lactam carbonyl (N1-Boc or N1-Cbz
derivatives) over benzyl ester, but complete selectivity
towards ring opening occurred in the presence of a t-butyl
ester.36 Glycosyl anions,37 cyanide and azide 38 exclusively
2.3. NMR spectroscopic study
A solution of b-lactam 4 (1023 M) in deuteriated phosphate
buffer (pD 7.5; 50 mM) containing 5% of DMSO-d6 was
Scheme 4. Enzymic hydrolysis of 4.
Scheme 5. Chemical and enzymic hydrolysis of 6a.

Â
S. Gerard et al. / Tetrahedron 58(2002) 2423±2433
2427
reacted on the azetidinone, leaving benzyl or methyl ester
attack of Ser-195 was considered from the a face of the
azetidinone. At the transition state, the amide N±H gener-
ates a pseudo seven-membered ring including the oxygen
atom of the carbonyl (b-lactam carbonyl as shown in Fig. 2,
or ester/urethane carbonyl) and the g-oxygen atom of the
nucleophile. An analogous model has been studied to high-
light the catalytic mechanism of class-A b-lactamases.40 In
the trypsin family, the activation barriers thus estimated
have been correlated with the free energy variation observed
between native and mutated enzymes.11,41
2
substituents untouched. Accordingly, CN2 and N3 were
used as catalysts for the chemoselective aminolysis or
alcoholysis of N-alkoxycarbonyl azetidinones bearing a
C4 ester substituent.38 As far as basic hydrolysis is
concerned, one publication reported that N-arylsulfonylaze-
tidinones equipped with an alkoxycarbonyl substituent at
C4 are hydrolyzed in phosphate buffer,28b giving the
cleavage of the b-lactam ring without ester saponi®cation.
We did not identify hydrolysis studies related to our
compounds 5±13 (Schemes 2 and 3). Thus, our results
show that the N1 alkoxycarbonyl substituent of 6 is not
suf®ciently electron-withdrawing to promote nucleophilic
attack of hydroxide anion on the b-lactam carbonyl over
the ester carbonyl, as it was the case with a N1 arylsulfonyl
substituent.
With more than 80 atoms, the molecular system could only
be handled at the ab initio level with a minimal basis set.
The accuracy of the results derived from the Huzinaga basis
has been demonstrated in previous studies.40,41 The DG
values have been computed by reference to the isolated
partners. The high values obtained give an indication of
the complexation effort that has to be performed by the
enzyme to generate the Michaelis complex as the starting
point of the reaction path toward the transition state. On the
other hand, the optimization of the complexes connected by
the transition state could generate nonrepresentative struc-
tures as the active site geometric constraints are not taken
into account in the ab initio calculations.
Although another hydrolysis pro®le could be expected
under enzyme processing, in the presence of PPE
(6£1025 M), the catalyzed hydrolysis of 6a rapidly
proceeded to the same products, i.e. acid 14a and benzyl
alcohol. Products resulting from b-lactam ring opening
could not be identi®ed, nor phenethyl alcohol, as expected
in the case of a suicide-type mechanism of serine-enzyme
inhibition (Scheme 5). Amongst the three carbonyl groups
(azetidinone, urethane and ester) of 6a being potentially
sensitive to Ser-195 nucleophilic attack, only the ester was
hydrolyzed by the enzyme. Thus, 6a (and related
compounds 5, 7, 12 and 13) behaved differently from 15,
and most probably were transient inhibitors via a different
mode of action and ®t in the enzymic cavity.
For compound 6, six combinations have been studied
depending on the broken C±N/C±O bond for the (S) and
(R) enantiomers. The activation barrier is lower for 6b, the
molecule having the (R) con®guration. The C±N transition
state (p in Table 3) could not be quenched as a stationary
point; the highest ®rst derivative component remains higher
then the usual threshold in spite of very careful optimization
runs. The given value can be seen as a lower bound of the
energy involved in the C±N cleavage.
Enzyme-catalyzed hydrolysis of C4 ester groups has never
been mentioned for previously reported elastase inhibitors
bearing N1 arylsulfonyl or benzoyl substituents.27,28 There-
fore, we turned to theoretical chemistry as a possible predic-
tive tool in this subtle competition between the different
carbonyl groups of A (Scheme 1, R2ˆCO2CH2Ph) towards
nucleophilic attack.
Table 3. Calculated energies (kcal/mol) at the transition states
Compound
Cleaved bonda
DE
DH
DG
3
15
(S)-5a
C±N
C±N
C±N
C±O1
C±N
C±O1
C±O2
C±N (p)
C±O1
C±O2
17.46
14.26
17.95
19.54
19.41
20.78
17.97
13.45
16.20
16.00
17.94
14.73
18.44
19.78
19.90
21.12
18.45
13.40
16.43
16.48
40.07
36.87
40.27
41.15
44.05
42.79
41.50
38.38
40.00
39.13
2.4. Theoretical evaluation
(S)-6a
(R)-6b
The reactivity of b-lactams 3, 5, 6 and 15, has been
evaluated by theoretical methods. The mechanism of
cleavage of the N1±C2 azetidinone bond has been exten-
sively studied in the context of chemical stability of anti-
biotics and their hydrolysis by b-lactamases.39,40 In our case,
we have also considered the cleavage of the (O)C±O ester
bond (5 and 6) and (O)C±O urethane bond (3, 15, 5 and 6).
a
C±N azetidinone bond, C±O1 ester bond, C±O2 urethane bond.
The alkoxycarbonyl side-chain at C4 signi®cantly increased
the barrier height for compound 6a with the (S) con®gura-
tion, comparatively to its (R) enantiomer 6b, and
compounds 3 and 15 devoid of such a side-chain. In this
last case, the electronic effect of the bromine was expressed
by the decrease of the energy barrier. The lower reactivity of
the azetidinone function in 6a could result from a lack of
conjugation with the N1 carbonyl substituent due to an elec-
tronic repulsion effect with the C4 carbonyl group at the
transition state, a phenomenon which does not occur in 6b
(Fig. 3 shows the respective diastereoisomeric transition
states).
The catalytic environment was mimicked by an imidazole
as a model of His-57, a water molecule as a transient vehicle
of the proton and the side-chain of Ser-195 including the
amide bond of the backbone which is involved in the
stabilization of the oxyanion hole (Fig. 2). Nucleophilic
Considering the C±O1 and the C±O2 cleavages, the activa-
Figure 2. Model of elastase active site.

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S. Gerard et al. / Tetrahedron 58(2002) 2423±2433
2428
Figure 3. Stereoscopic view of the transition state of C±N cleavage in: (a) 6a (S) and (b) 6b (R). Atom coloring: red, oxygen; blue, nitrogen; green, carbon;
black, hydrogen.
Figure 4. Stereoscopic view of the transition state of C±O1 cleavage in 6a (S). Atom coloring: red, oxygen; blue, nitrogen; green, carbon; black, hydrogen.

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S. Gerard et al. / Tetrahedron 58(2002) 2423±2433
2429
tion barriers are also lower for 6b than for 6a. Moreover, in
the active compound 6a, the differences between C±N and
C±O breaking processes are not very high. The three
possible hydrolysis routes are energetically accessible, and
therefore would depend on the best ®t of the molecule
into the enzymic cavity. Fig. 4 shows the transition state
of the C±O1 cleavage in 6a, the process experimentally
observed.
dried with sodium/benzophenone, then distilled. Column
chromatographies were carried out with silicagel 60 (70±
230 mesh ASTM) supplied by Merck. The IR spectra were
recorded with a Perkin±Elmer 1710 instrument, only the
most signi®cant absorption bands being reported. The
mass spectra were obtained with a Finnigan MAT TSQ-70
instrument. The microanalyses were performed at the
Christopher Ingold Laboratories of the University College,
London (Dr A. Stones). The melting points were determined
with an Electrothermal microscope and are uncorrected.
1
The H and 13C NMR spectra were recorded on Varian
3.Conclusions
Gemini 300 (at 300 MHz for proton and 75 MHz for carbon)
or Bruker AM-500 spectrometers (at 500 MHz for proton
and 125 MHz for carbon); the chemical shifts are reported in
ppm (d) down®eld from tetramethylsilane (internal
standard) or dimethylsulfoxide.
A series of monocyclic b-lactams sharing the general
structure A (Scheme 1) has been prepared. They are
characterized by various lipophilic R1 substituents and an
alkoxycarbonyl side-chain at position C4. The compounds
were designed to promote the suicide-inhibition of serine-
enzymes such as elastases. When tested against PPE,
b-lactams 5±7 and 12±13 were found to be reversible
inhibitors of the enzyme (Ki of about 10±100 mM), while
b-lactams 8, 9 and 14 were inactive.
4.2. Synthesis, general procedure for the preparation of
chloroformates
To a cold solution (ice-bath) of triphosgene (0.4 equiv.) in
toluene (5 mL/mmol) was added dropwise (over 2 h)
pyridine (1.25 equiv.). After 1 h stirring at 08C, alcohol
(1 equiv.) was added dropwise (over 30 min). The mixture
was stirred for 17 h at 208C, then ®ltered. The precipitate
was rinsed twice with toluene. Concentration of the
®ltrate under vacuum gave crude chloroformate (about
85% yield).
NMR analysis of the crude mixtures resulting from chemi-
cal or enzymic hydrolysis of 6a (chosen as model for all the
series of compounds A), revealed an unexpected reaction:
the C4 ester group was cleaved instead of the azetidinone
amide bond. A similar observation has been reported
occasionally in the case of one ester of 6-chloromethyl-2-
oxo-2H-1-benzopyran-3-carboxylic acid tested against
HLE; the enzyme reacted with the exocyclic ester.42
4.2.1. Phenethyl chloroformate. It was obtained from
594 mg (2 mmol) of triphosgene, 10 mL of toluene,
505 mL (6.25 mmol) of pyridine and 597 mL (5 mmol) of
Geometry optimization of compound 6a showed a particular
conformation of the N1 side-chain due to the repulsion
between the C5 and C6 carbonyls which occurs when C2
suffers nucleophilic attack from the a face. Considering the
three potentially sensitive carbonyl functions (C2, C5 and
C6), ab initio calculations were performed to determine the
energetic barriers required to reach the transition state struc-
tures of hydrolysis in a model of enzymic cavity. The most
reactive compound was the (R)-enantiomer of 6; but experi-
mentally, we have demonstrated that this isomer 6b is not
well recognized by PPE. On the other hand, the active
isomer 6a appeared somewhat less reactive than 6b, con-
sidering the three hydrolysis processes. Also, discrimination
between the three hydrolysis processes of 6a could not be
made from our theoretical approach. Thus, the best ®t into
the enzymic pocket is de®nitively the crucial factor
determining the chemoselectivity of the substrate/inactiva-
tor hydrolysis pathway. This has been con®rmed by docking
experiments.43 The possibility of increasing the chemical/
enzymic reactivity of the b-lactam carbonyl C2 by
suppressing the C5/C6 carbonyl repulsion is currently
under investigation.
1
phenethyl alcohol. H NMR (200 MHz, CDCl3): d 7.15±
7.39 (m, 5H), 4.50 (t, 2H, Jˆ7 Hz), 3.02 (t, 2H, Jˆ7 Hz).
13C NMR (50 MHz, CDCl3): d 150.7, 136.4, 129.0, 128.9,
127.2, 72.3, 34.9.
4.2.2. Phenylpropyl chloroformate. It was obtained from
594 mg (2 mmol) of triphosgene, 10 mL of toluene, 505 mL
(6.25 mmol) of pyridine and 676 mL (5 mmol) of phenyl-
1
propyl alcohol. H NMR (200 MHz, CDCl3): d 7.10±7.35
(m, 5H), 4.27 (t, 2H, Jˆ6.6 Hz), 2.69 (t, 2H, Jˆ6.6 Hz),
2.01 (quint, 2H, Jˆ6.6 Hz). 13C NMR (50 MHz, CDCl3):
d 150.2, 140.3, 128.5, 128.3, 126.2, 71.3, 31.5, 29.6.
4.2.3. 2-Indanyl chloroformate. It was obtained from
476 mg (1.6 mmol) of triphosgene, 9 mL of toluene,
401 mL (5 mmol) of pyridine and 537 mL (4 mmol) of
2-indanol in 3 mL of toluene. 1H NMR (200 MHz,
CDCl3): d 7.25 (m, 5H), 5.62 (m, 1H), 3.38 (dd, 2H,
Jˆ17.4 and 6.2 Hz), 3.18 (dd, 2H, Jˆ17.4 and 2.8 Hz).
13C NMR (50 MHz, CDCl3): d 150.2, 139.1, 127.0, 124.5,
83.7, 39.1.
4.2.4. Cyclohexylethyl chloroformate. It was obtained
from 594 mg (2 mmol) of triphosgene, 10 mL of toluene,
505 mL (6.25 mmol) of pyridine and 696 mL (5 mmol) of
2-cyclohexylethanol in 3 mL of toluene. 1H NMR
(200 MHz, CDCl3): d 4.42 (t, 2H, Jˆ6.8 Hz), 1.76 (m,
2H), 1.71 (t, 2H, Jˆ6.8 Hz), 1.48 (m, 1H), 1.28 (m, 4H),
1.02 (m, 4H). 13C NMR (50 MHz, CDCl3): d 150.8, 70.8,
37.8, 34.3, 33.2, 33.1, 26.5, 26.3, 26.2.
4.Experimental
4.1. General
Reagents and solvents were purchased from Acros chimica,
Aldrich or Fluka. Porcine pancreatic elastase (type 1) and
N-succinyl±l-alanyl±l-alanyl±l-alanyl±p-nitroanilide were
obtained from Sigma Chemical Co. Tetrahydrofuran was

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S. Gerard et al. / Tetrahedron 58(2002) 2423±2433
2430
4.3. General procedure for 1-alkoxycarbonylazetidin-2-
ones
to yield 6 (177 mg, 50%) as a white solid. Mp 100±1018C.
Found: C, 67.79; H, 5.39; N, 3.84%. C20H19O5N requires C,
67.98; H, 5.42; N, 3.96%. IR (KBr): 2965, 1823, 1732 cm21
.
1H NMR (300 MHz, CDCl3): d 7.18±7.40 (m, 10H), 5.20
(AB, 2H, Jˆ12.2 Hz), 4.43 (dd, 1H, Jˆ6.6, 3.3 Hz), 4.37 (t,
1H, Jˆ7.2 Hz), 4.36 (t, 1H, Jˆ7.2 Hz), 3.29 (dd, 1H,
Jˆ15.9, 6.6 Hz), 3.01 (dd, 1H, Jˆ15.9, 3.3 Hz), 2.93 (t,
1H, Jˆ7.2 Hz), 2.92 (t, 1H, Jˆ7.2 Hz). 13C NMR
(75 MHz, CDCl3): d 168.6 (CO ester), 161.7 (CO azetidi-
none), 147.9 (CO carbamate), 136.8, 134.6, 128.6, 128.4,
128.3, 68.2, 67.5, 67.3, 49.2 (C-4), 41.6 (C-3), 34.7. MS (CI)
m/z 354 (M11), 262, 204, 135, 122, 105, 91.(S)-Enantiomer
A solution of LiHMDS (1 equiv.) in dry THF (5 mL/mmol)
was dropwise added to a solution of azetidin-2-one
(1 equiv.) in dry THF (5 mL/mmol), cooled at 2788C
under argon atmosphere. The mixture was stirred for
30 min at 2788C, then chloroformate (1 equiv.) was
added with a syringe through a rubber septum. Stirring
was continued for 1 h at this temperature and the mixture
was allowed to reach room temperature and stirred for
45 min at 208C. After addition of water and extraction
with CH2Cl2, the organic layer was washed with brine,
dried over MgSO4, ®ltered and concentrated under vacuum.
The resulting b-lactam was puri®ed by column chromato-
graphy on silica gel (CH2Cl2±EtOAc, 90/10 to 70/30).
6a was similarly prepared from 2a:29,30 [a]D ˆ255.9
25
(cˆ0.086, CHCl3).
(R)-Enantiomer 6b was recovered by preparative HPLC of
the racemic mixture with a chiralpak AD@ column and
elution with hexane±isopropanol (90:10) at 1 mL/min;
retention times were 21.1 and 23.8 min for the (R) and the
4.3.1. 1-Benzyloxycarbonylazetidin-2-one (3). This
compound was obtained from 1 (106 mg, 1.5 mmol) by
treatment with 362 mg of LiHMDS (1.5 mmol) and
214 mL of benzyl chloroformate (1.5 mmol) to yield 3
(224 mg, 75%) as a white solid. Mp 72±738C. Found: C,
64.54; H, 5.45; N, 6.63%. C11H11O3N requires C, 64.38; H,
5.40; N, 6.82%. IR (KBr): 3054, 1812, 1726 cm21. 1H NMR
(300 MHz, CDCl3): d 7.34±7.41 (m, 5H), 5.27 (s, 2H), 3.62
(t, 2H, Jˆ5.3 Hz), 3.04 (t, 2H, Jˆ5.3 Hz). 13C NMR
(75 MHz, CDCl3): d 164.1 (CO azetidinone), 148.9 (CO
carbamate), 135.1, 128.6, 128.5, 128.3, 67.9, 37.8, 36.6.
MS (CI) m/z 206 (M11), 107, 91.
25
(S) enantiomers, respectively; [a]D ˆ138.5 (cˆ0.500,
CHCl3).
4.3.5. 1-Phenylpropyloxycarbonyl-(4-benzyloxycarbonyl)-
azetidin-2-one (7). This compound was obtained from 2
(205 mg, 1 mmol) by treatment with 241 mg of LiHMDS
(1 mmol) and 198 mg of phenylpropyl chloroformate
(1 mmol) to yield 7 (275 mg, 75%) as a yellow oil.
Found: C, 68.79; H, 5.84; N, 3.81%. C21H21O5N requires
C, 68.65; H, 5.76; N, 3.81%. IR (®lm): 2987, 1824,
1
1736 cm21. H NMR (300 MHz, CDCl3): d 7.09±7.39 (m,
10H), 5.23 (AB, 2H, Jˆ12.3 Hz), 4.45 (dd, 1H, Jˆ6.6,
3.3 Hz), 4.21 (t, 2H, Jˆ6.6 Hz), 3.31 (dd, 1H, Jˆ15.9,
6.6 Hz), 3.02 (dd, 1H, Jˆ15.9 Hz, 3.3 Hz), 2.67 (t, 2H,
Jˆ7.2 Hz), 1.97 (q, 2H, Jˆ7.2 Hz). 13C NMR (75 MHz,
CDCl3): d 168.6 (CO ester), 161.7 (CO azetidinone),
147.9 (CO carbamate), 140.6, 134.6, 128.5, 128.2, 125.9,
67.4, 65.9, 49.3 (C-4), 41.5 (C-3), 31.5, 29.8. MS (EI) m/z
368 (M11), 149, 118, 91.
4.3.2. 1-Phenethyloxycarbonylazetidin-2-one (4). This
compound was obtained from 1 (106 mg, 1.5 mmol) by
treatment with 362 mg of LiHMDS (1.5 mmol) and
280 mg of phenethyl chloroformate (1.5 mmol) to yield 4
(315 mg, 98%) as a white solid. Mp 64±658C. Found: C,
66.06; H, 6.24; N, 5.94%. C12H13O3N requires C, 65.74; H,
5.98; N, 6.38%. IR (KBr): 2958, 1813, 1729 cm21. 1H NMR
(300 MHz, CDCl3): d 7.34±7.41 (m, 5H), 4.41 (t, 2H,
Jˆ7.2 Hz), 3.58 (t, 2H, Jˆ5.2 Hz), 3.04 (t, 2H,
Jˆ5.2 Hz), 3.02 (t, 2H, Jˆ7.2 Hz). 13C NMR (75 MHz,
CDCl3): d 163.8 (CO azetidinone), 148.5 (CO carbamate),
136.8, 128.6, 128.1, 126.2, 66.4, 37.4, 36.1, 34.7. MS (CI)
m/z 220 (M11), 114, 105, 70.
4.3.6.
1-Indanyloxycarbonyl-(4-benzyloxycarbonyl)-
azetidin-2-one (8). This compound was obtained from 2
(205 mg; 1 mmol) by treatment with 241 mg of LiHMDS
(1 mmol) and 196 mg of indanyl chloroformate (1 mmol) to
yield 8 (204 mg, 56%) as a yellow solid. Mp 100±1018C.
Found: C, 69.26; H, 5.37; N, 3.61%. C21H19O5N requires C,
4.3.3. 1-Benzyloxycarbonyl-(4-benzyloxycarbonyl)-aze-
tidin-2-one (5). This compound was obtained from 2
(205 mg, 1 mmol) by treatment with 241 mg of LiHMDS
(1 mmol) and 143 mL of benzyl chloroformate (1 mmol) to
yield 5 (218 mg, 68%) as a white solid. Mp 73±748C.
Found: C, 67.29; H, 4.96; N, 4.08%. C19H17O5N requires
C, 67.25; H, 5.05; N, 4.13%. IR (KBr): 2958, 1826,
69.03; H, 5.24; N, 3.83%. IR (KBr): 2959, 1823, 1732 cm21
.
1H NMR (300 MHz, CDCl3): d 7.15±7.41 (m, 9H), 5.53 (m,
1H, Jˆ3.3 Hz), 5.08 (AB, 2H, Jˆ12.0 Hz), 4.42 (dd, 1H,
Jˆ6.6, 3.3 Hz), 3.34 (dd, 1H, Jˆ15.0, 6.6 Hz), 3.28 (dd, 2H,
Jˆ17.0, 6.6 Hz), 3.03 (t, 1H, Jˆ17.0 Hz), 3.02 (t, 1H,
Jˆ17.0 Hz), 2.98 (dd, 1H, Jˆ15.0 Hz, 3.3 Hz). 13C NMR
(75 MHz, CDCl3): d 168.7 (CO ester), 161.7 (CO azetidi-
none), 147.8 (CO carbamate), 139.7, 134.6, 128.5, 128.2,
128.1, 126.8, 124.6, 78.2, 67.6, 49.6 (C-4), 41.7 (C-3), 39.3.
MS (CI) m/z 366 (M11), 274, 133, 117, 91.
1
1737 cm21. H NMR (300 MHz, CDCl3): d 7.23±7.39 (m,
10H), 5.24 (s, 2H), 5.20 (s, 2H), 4.48 (dd, 1H, Jˆ6.6,
3.3 Hz), 3.31 (dd, 1H, Jˆ15.9, 6.6 Hz), 3.03 (dd, 1H,
Jˆ15.9, 3.3 Hz). 13C NMR (75 MHz, CDCl3): d 168.7
(CO ester), 161.7 (CO azetidinone), 147.9 (CO carbamate),
134.6, 128.5, 128.2, 128.1, 68.2, 67.5, 49.2 (C-4), 41.6
(C-3). MS (CI) m/z 340 (M11), 206, 181, 107, 91.
4.3.7. 1-Cyclohexylethyloxycarbonyl-(4-benzyloxycar-
bonyl)-azetidin-2-one (9). This compound was obtained
from 2 (205 mg; 1 mmol) by treatment with 241 mg of
LiHMDS (1 mmol) and 190 mg of cyclohexyl chloro-
formate (1 mmol) to yield 9 (246 mg, 69%) as an oil.
Found: C, 67.29; H, 7.05; N, 3.70%. C20H25O5N requires
C, 66.83; H, 7.01; N, 3.89%. IR (®lm): 2947, 1826,
4.3.4. 1-Phenethyloxycarbonyl-(4-benzyloxycarbonyl)-
azetidin-2-one (6). This compound was obtained from 2
(205 mg, 1 mmol) by treatment with 241 mg of LiHMDS
(1 mmol) and 190 mg of phenethyl chloroformate (1 mmol)

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S. Gerard et al. / Tetrahedron 58(2002) 2423±2433
2431
1
1733 cm21. H NMR (300 MHz, CDCl3): d 7.27±7.49 (m,
5H), 5.24 (AB, 2H, Jˆ12.0 Hz), 4.48 (dd, 1H, Jˆ6.6,
3.3 Hz), 4.24 (t, 1H, Jˆ7.2 Hz), 4.23 (t, 1H, Jˆ6.9 Hz),
3.31 (dd, 1H, Jˆ15.9, 6.6 Hz), 3.02 (dd, 1H, Jˆ15.9,
3.3 Hz), 1.44±1.76 (m, 7H), 1.33 (m, 2H), 1.23 (m, 2H),
0.91 (m, 2H). 13C NMR (75 MHz, CDCl3): d 168.6 (CO
ester), 161.5 (CO azetidinone), 147.9 (CO carbamate),
134.5, 128.3, 128.2, 128.1, 68.2, 67.2, 65.1, 49.1 (C-4),
41.3 (C-3), 35.4, 34.0, 32.7, 26.1, 25.8. MS (CI) m/z 360
(M11), 268, 111, 91.
4.4. NMR spectroscopic study
Chemical hydrolysis. 2.1022 M solutions of 4 and 6 in
DMSO-d6 were added to phosphate buffer in D2O
1
(pDˆ7.5; ®nal concentration 1023 M). H NMR spectra
(500 MHz) were recorded every 60 min; 4 was not hydro-
lyzed, while about 50% of 6 was hydrolyzed within 36 h.
Enzymic hydrolysis. 2.1022 M solutions of 4 and 6a in
DMSO-d6 were added to a solution of PPE (6£1025 M) in
deuteriated phosphate buffer (50 mM, pD 7.5; ®nal concen-
tration 5.1024 M). 1H NMR spectra (500 MHz) were
recorded every 60 min during 48 h; about 50% of 4 was
hydrolyzed within 15 h (b-lactam ring opening); 6a was
totally hydrolyzed after 8 h (ester cleavage).
4.3.8. 1-Phenethyloxycarbonyl-(4-methyloxycarbonyl)-
azetidin-2-one (12a). This compound was obtained from
10a32 (205 mg, 1 mmol) by treatment with 241 mg of
LiHMDS (1 mmol) and 190 mg of phenethylchloroformate
(1 mmol) to yield 12a (177 mg, 50%) as a white solid. Mp
100±1018C. Found: C, 60.64; H, 5.45; N, 5.05%.
C14H15O5N requires C, 61.68; H, 5.75; N, 4.78%.
4.5. Assay of porcine pancreatic elastase44
25
[a]D ˆ245.5 (cˆ1.22, CHCl3). IR (KBr): 2965, 1823,
To 2 mL of the solution of substrate (solution of N-succi-
nyl±l-alanyl±l-alanyl±l-alanyl±p-nitroanilide (2.7 mg in
200 mL of N-methyl pyrrolidone (NMP) diluted with Tris
buffer (20 mL at 100 mM, pH 7.5 solution)) were added
1
1732 cm21. H NMR (300 MHz, CDCl3): d 7.15±7.38 (m,
5H), 4.43 (dd, 1H, Jˆ6.6, 3.3 Hz), 4.33 (t, 1H, Jˆ7.2 Hz),
4.36 (t, 1H, Jˆ7.2 Hz), 3.78 (s, 3H), 3.31 (dd, 1H, Jˆ15.9,
6.5 Hz), 3.05 (dd, 1H, Jˆ15.9, 3.3 Hz), 2.93 (t, 1H,
Jˆ7.2 Hz), 2.92 (t, 1H, Jˆ7.2 Hz). 13C NMR (75 MHz,
CDCl3): d 168.6 (CO ester), 161.7 (CO azetidinone),
147.9 (CO carbamate), 136.8, 134.6, 128.6, 128.4, 128.3,
68.2, 67.5, 67.3, 49.2 (C-4), 41.6 (C-3), 34.7. MS (CI) m/z
354 (M11), 262, 204, 135, 122, 105, 91.
20 mL of the solution of the tested compound (1022
±
1024 M in NMP) and 67 mL of the solution of elastase
(6£1026 M in acetate buffer (50 mM, pH 5)). The appear-
ance of the substrate hydrolysis product (p-nitroaniline) was
measured (with a Cary 210) at 410 nm as a function of time.
Plots of V/VI versus [I] (ratios of initial rates of hydrolysis in
the absence and in the presence of inhibitors) gave the
inhibition constants indicated in the Table 2. All experi-
ments were performed two or three times.
4.3.9. 1-Phenethyloxycarbonyl-(4-isopropyloxycarbonyl)-
azetidin-2-one (13a). This compound was obtained from
11a32 (113 mg, 0.72 mmol) by treatment with 174 mg of
LiHMDS (0.72 mmol) and 135 mg of phenethyl chloro-
formate (0.72 mmol) to yield 13a (167 mg, 76%) as an
oil. Found: C, 62.94; H, 6.27; N, 4.58%. C16H19O5N
The reversibility of the inhibition was controlled by the
incubation/dilution method. The incubation solution
consisted of 5 mL of inhibitor solution (1023 M in
DMSO), 34 mL of elastase solution (2.5£1024 M in acetate
buffer, 50 mM, pH 5) and 161 mL of Tris buffer (100 mM,
pH 7.5). At various times (0, 5, 10, 15, 20, 30 min), 10 mL of
the incubation solution were dissolved in 2 mL of the
substrate solution (1024 M in Tris buffer), and the enzyme
activity was measured as before. No decrease of activity was
recorded as a function of incubation time.
25
requires C, 62.87; H, 6.42; N, 4.42%. [a]D ˆ246.4
1
(cˆ0.78, CHCl3). IR (®lm): 2976, 1825, 1732 cm21. H
NMR (300 MHz, CDCl3): d 7.18±7.40 (m, 5H), 5.11
(sext, 1H, Jˆ6.0 Hz), 4.37 (dd, 1H, Jˆ6.7, 3.4 Hz), 4.42
(t, 2H, Jˆ7.1 Hz), 4.36 (t, 1H, Jˆ7.2 Hz), 3.31 (dd, 1H,
Jˆ15.6, 6.7 Hz), 3.03 (dd, 1H, Jˆ15.6, 3.4 Hz), 3.01 (t,
2H, Jˆ7.1 Hz), 1.36 (d, 3H, Jˆ6.0 Hz), 1.32 (d, 3H,
Jˆ6.0 Hz). 13C NMR (75 MHz, CDCl3): d 168.4 (CO
ester), 161.7 (CO azetidinone), 148.5 (CO carbamate),
138.2, 129.0, 128.9, 128.6, 69.9, 67.4, 49.6 (C-4),
41.6 (C-3), 34.9, 21.5. MS (CI) m/z 306 (M11), 105,
91, 86.
4.6. Theoretical calculations
All the calculations have been performed at the ab initio
RHF level using the minimal basis set MINI-10.45,46 They
were performed with Gaussian 9447,48 on two computers, a
Dec Alpha 8400 8-processor and a Dec Alpha 4100
4-processor running Digital Unix. The starting geometries
were sketched drawn by standard fragments and then
completely optimized following all the 3N-6 degrees of
freedom either for the minima or the transition state struc-
tures. For each equilibrium structure, the thermochemistry
data are derived from the analytical frequency calculation at
298.15 K and 1 atm.
4.3.10. 1-Phenethyloxycarbonyl-(4-hydroxycarbonyl)-
azetidin-2-one (14). This compound was obtained by
hydrogenation in Parr apparatus (12 h under 30 psi, 208C)
of 6 (90 mg, 0.25 mmol), in solution in AcOEt (10 mL) with
12 mg of Pd/C (50 mg/mmol), to yield 14 (65 mg, 97%) as a
white solid. Mp 104±1058C. Found: C, 58.95; H, 5.29; N,
4.99%. C13H13O5N requires C, 59.31; H, 4.98; N, 5.32%. IR
.
(®lm): 3550, 1815, 1734 cm21 1H NMR (300 MHz,
CDCl3): d 10.25 (sl, 1H), 7.18±7.40 (m, 5H), 4.38 (m,
3H), 3.31 (dd, 1H, Jˆ15.6, 6.7 Hz), 3.06 (dd, 1H, Jˆ15.6,
3.4 Hz), 2.97 (t, 2H, Jˆ7.1 Hz). 13C NMR (75 MHz,
CDCl3): d 173.3 (CO acid), 162.2 (CO azetidinone), 148.5
(CO carbamate), 136.9, 129.0, 128.9, 128.7, 67.8, 49.6
(C-4), 41.9 (C-3), 35.1. MS (CI) m/z 262 (M21), 172,
105, 91.
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