4-Oxo-β-lactams (azetidine-2,4-diones) are potent and selective inhibitors of human leukocyte elastase

Article abstract of DOI:10.1021/jm901082k

Human leukocyte elastase (HLE) is a serine protease stored in and secreted from neutrophils that plays a determinant role in the pathogenesis of several lung diseases. 4-Oxo-β-lactams, previously reported as acylating agents of porcine pancreatic elastase, were found to be selective and potent inhibitors of HLE. Structure - activity relationship analysis showed that inhibitory activity is very sensitive to the nature of C-3 substituents, with small alkyl substituents such as a gem-diethyl group improving the inhibitory potency when compared to gem-methyl benzyl or ethyl benzyl counterparts. 4-Oxo-β-lactams containing a heteroarylthiomethyl group on the para position of an N ¹-aryl moiety afforded highly potent and selective inhibition of HLE, even at a very low inhibitor to enzyme ratio, as shown by the k_on value of 3.24 x 10⁶ M^-1 s^-1 for 6f. The corresponding ortho isomers were 40- to 90-fold less potent.

Full text of DOI:10.1021/jm901082k

J. Med. Chem. 2010, 53, 241–253 241
DOI: 10.1021/jm901082k
4-Oxo-β-lactams (Azetidine-2,4-diones) Are Potent and Selective Inhibitors of Human Leukocyte
Elastase
Jalmira Mulchande,† Rudi Oliveira,† Marta Carrasco,† Luıs Gouveia,† Rita C. Guedes,† Jim Iley,‡ and Rui Moreira*,†
´
†iMed.UL, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal and ‡Department of Chemistry and
Analytical Sciences, The Open University, Milton Keynes, MK7 6AA, U.K.
Received July 21, 2009
Human leukocyte elastase (HLE) is a serine protease stored in and secreted from neutrophils that plays a
determinant role in the pathogenesis of several lung diseases. 4-Oxo-β-lactams, previously reported as
acylating agents of porcine pancreatic elastase, were found to be selective and potent inhibitors of HLE.
Structure-activity relationship analysis showed that inhibitory activity is very sensitive to the nature of
C-3 substituents, with small alkyl substituents such as a gem-diethyl group improving the inhibitory
potency when compared to gem-methyl benzyl or ethyl benzyl counterparts. 4-Oxo-β-lactams contain-
ing a heteroarylthiomethyl group on the para position of an N1-aryl moiety afforded highly potent and
selective inhibition of HLE, even at a very low inhibitor to enzyme ratio, as shown by the kon value of
3.24 ꢀ 106 M-1 s-1 for 6f. The corresponding ortho isomers were 40- to 90-fold less potent.
Introduction
which is a result of an imbalance between protease and its
endogenous inhibitors (R1-antitrypsin and secretory leukopro-
tease inhibitor), leads to an excessive tissue destruction.11 This
protease/antiprotease imbalance appears to be a key patho-
genic determinant in COPD as well in other inflammatory
disorders such as acute respiratory distress syndrome (ARDS)
and cystic fibrosis.14-17 Thus, HLE is an attractive target for
therapeutic intervention, since selective inhibitors of this en-
zyme can reestablish the protease/antiprotease balance, which
makes this a promising strategy for the treatment of COPD and
other inflammatory disorders.18-20
β-Lactams are well-known acylating agents of serine en-
zymes, including the bacterial transpeptidases and class A and
class C β-lactamases.21 The first irreversible inhibitors of HLE
based on the β-lactam scaffold were the cephalosporin sul-
fones derivatives, 1 (Chart 1).22 Molecular simplification of
the β-lactam template led to the development of azetidin-
2-ones, e.g., 2 (L-680,833), as potent, orally available, and
hydrolytically more stable inhibitor of HLE.23-26 Increasing
the intrinsic chemical reactivity of the β-lactam motif has been
a strategy to increase the rate of acylation of the catalytic
serine residue and improve inhibitory properties.27-31 We
recently described the design rationale and some preliminary
biochemical evaluation findings of a novel class of acylating
agents, the 4-oxo-β-lactams (azetidine-2,4-diones), 3.32 These
compounds were found to be time dependent inhibitors of
porcine pancreatic elastase, PPE, which shares ∼40% homo-
logy and the catalytic triad with HLE.33 The most potent PPE
inhibitor also emerged as very potent inhibitor of the HLE.32
This promising result has prompted us to investigate the
structure-activity relationships (SAR) of 4-oxo-β-lactams,
3, toward the therapeutic target HLE. In this context, new
compounds have been synthesized to explore the structural
motifs at C-3 and N-1 of the 4-oxo-β-lactam scaffold required
for optimal molecular recognition by HLE. These studies
enable us to describe the mechanism of HLE inhibition and
Chronic obstructive pulmonary disease (COPDa) is a
progressive and ultimately fatal chronic illness1 that is one
of the fastest growing causes of death worldwide.2-4 Active
exposure to cigarette smoke is the cause of the vast majority of
COPD cases in Europe and the U.S.1,5,6 Aside from smoking
cessation and domiciliary oxygen therapy, all current treat-
ment strategies for COPD are largely aimed at improving
symptoms and reducing exacerbations. Thus, there is an
urgent need to develop novel therapeutic strategies to slow
the progression of COPD and to improve the management of
COPD patients beyond that achieved by current therapies.7
The inflammatory basis of COPD is complex, and it is
known to involve the recruitment of macrophages, neutrophils,
and CD8þ T cells into the lungs upon an inflammatory sti-
mulus.1,6 Release of proteases from neutrophils is one of the
several mechanisms involved in the pathogenesis of COPD.
Human leukocyte elastase (HLE), along with proteinase 3 and
cathepsin G, is a neutrophil serine protease member of the
chymotrypsin superfamily, being expressed primarily in neu-
trophils and stored in their azurophil granules.8 These neutro-
phil serine proteases are involved in pathogen killing within
phagolysomes, while extracellularly they degrade extracellular
matrix proteins, such as elastin, collagen, laminin, fibronectin,
and proteoglycans, and play an important role in the modula-
tion of the inflammatory response.8,9 In particular, HLE is the
most efficient neutrophil serine protease in the turnover of
matrix proteins and, in addition, activates other proteases (e.g.,
metalloproteinases) and up-regulates inflammation.10-13 Un-
regulated activity of HLE during pulmonary inflammation,
*To whom correspondence should be addressed. Phone: þ351
217946477. Fax: þ351 217946470. E-mail: rmoreira@ff.ul.pt.
a Abbreviations: HLE, human leukocyte elastase; COPD, chronic
obstructive pulmonary disease; ARDS, acute respiratory distress syn-
drome; PPE, porcine pancreatic elastase.
r
2009 American Chemical Society
Published on Web 11/11/2009
pubs.acs.org/jmc

242 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Mulchande et al.
Chart 1. Chemical Structures of Cephalosporin Sulfone, 1,
Azetidin-2-one, 2, and 4-Oxo-β-lactams, 3
absorbance at time t = 0, vi and vs are the initial and final
rates of product formation, and kobs is the pseudo-first-order
rate constant for the approach to the steady state.42 Such
kinetic behavior was reported for other acylating agents of
serine proteases, including β-lactams.19 For the more potent
inhibitors, 6e-j, extremely rapid enzyme inhibition was
observed at very low inhibitor concentrations. In these
experimental conditions, where the inhibitor concentration
was not much higher than that of HLE (i.e., <10[E]), the
progress curves had the same shape as those described by eq 1
but were fitted to the more general eq 2.43
the target protein selectivity, while a molecular modeling
study based on the reported crystal structure of the enzyme
helps to rationalize the binding modes of these compounds in
the active site.
A ¼ vst þ ½ðvi - vsÞð1 - γÞ=ðkobsγÞꢁ
lnf½1 - γexpð - kobstÞꢁ=ð1 - γÞg þ A0
(2Þ
(3Þ
where
Design and Chemistry
2
γ ¼ ð½Eꢁ=½IꢁÞ½1 - ðvs=viÞꢁ
4-Oxo-β-lactams 3a-r (see Table 1 for a full listing of R1,
R2, and R3 substituents) were readily prepared from the
appropriate 2,2-disubstituted malonic acid dichlorides, 4,
and amines, as described previously (Scheme 1).32 Compound
3m was synthesized by reacting ethylbutylketene, generated in
situ, with phenyl isocyanate.34 Substituents at C-3 of the 4-
oxo-β-lactam scaffold were varied in order to assess the effect
of size and lipophilicity on the inhibitor specificity for HLE
versus cathepsin G and proteinase-3. Cathepsin G has both
chymotrypsin- and trypsin-like specificities, and thus, the
specificity of cathepsin G is directed toward substrates with
a Phe or a Lys residue at the P1 position (accommodated atthe
primary specificity enzyme pocket, S1),8,35-37 while protei-
nase-3 is more restrictive for the P1 position than HLE.8,38
Previous studies reporting that substituents at the N-1
position of β-lactam inhibitors interact preferentially with
Typical progress curves for compounds 3h and 6f determined
at different concentrations are shown in Figure 1.
For most inhibitors (3a, 3c, 3f-q, 6d-e, 6h-j), the pseudo-
first-order rate constant for the approach to the steady-state,
kobs, presented a linear dependence with inhibitor concentra-
tion, consistent with the simple time-dependent inhibition via
the slow association mechanism A depicted in Scheme 4,42
where kobs has the relationship
kobs ¼ kon½Iꢁ=ð1 þ ½Sꢁ=KmÞ þ koff
(4Þ
and where kon is the second-order rate constant for the
formation of the enzyme-inhibitor complex, EI, and koff is
the first-order rate for the decomposition of EI. The steady-
state dissociation constant of the enzyme-inhibitor complex,
Ki, was calculated using the steady-state velocity, vs, together
with vi, and fitting them by nonlinear regression to eq 5,44 and
koff was calculated from eq 6:
the S1 subsite of HLE23,39 suggested that the N-aryl moiety
0
in 4-oxo-β-lactams could be explored as an anchor to probe
the interaction with Sn subsites. The fact that aryl and
0
v0
heterocyclic sulfides had been successfully used as a highly
flexible design element in the design of serine protease inhibi-
tors40,41 prompted us to design N-aryl-4-oxo-β-lactams with a
para- or ortho-thioether function, 6 (Scheme 2), as potential
mechanism-based inhibitors featuring a latent electrophilic
quinone methide imine function upon ring-opening, which
upon further reaction with an active site nucleophilic residue
(e.g., His-57) would ultimately lead to irreversible inactivation
of the enzyme (Scheme 3).41 Thioether derivatives 6a-j were
synthesized by first converting 4-oxo-β-lactams 3b, 3f, and 3h
into their bromomethyl derivatives 5a-c using NBS and then
reacting these with the appropriate thiol in the presence of
triethylamine (Scheme 2).
vs ¼
(5Þ
½Iꢁ=fKið1 þ ½Sꢁ=KmÞg þ 1
Ki ¼ koff =kon
(6Þ
where v0 is the rate for product formation in the absence of
inhibitor. As an example, the analysis of inhibition kinetics by
4-oxo-β-lactam 3h is presented in Figures 2 and 3. For the
remaining inhibitors (3b, 3d-e, 6a-c, 6f,g), a hyperbolic plot
of kobs versus [I]/(1 þ [S]/Km) was observed, consistent with a
mechanism involving rapid equilibration between the enzyme
and inhibitor to form the preassociation complex EI, which
then isomerizes relativelyslowly to the tight or stable complex,
EI* (mechanism B, Scheme 4). Accordingly, kobs is given by eq
7
Results and Discussion
k5½Iꢁ
Inhibition Kinetics. All 4-oxo-β-lactams 3 and 6 displayed
time-dependent inhibition of HLE, and their inhibitory
activity was determined using the progress curve method.
kobs
¼
þ k6
(7Þ
½Kdð1 þ ½Sꢁ=KmÞꢁ þ ½Iꢁ
where Kd is the dissociation constant for EI. As an example,
the analysis of inhibition kinetics by compound 6f is presented
in Figure 4. The second-order rate constant for the formation
of EI*, kon, and the dissociation constant for EI*, Ki*, were
calculated through eqs 8 and 9, respectively:
23,27
The progress curves presented an initial exponential
phase followed by a linear steady-state turnover of the
substrate and were analyzed by the formalism of slow
binding kinetics:
A ¼ vst þ ðvi - vsÞ½1 - expð - kobstÞꢁ=kobs þ A0 (1Þ
kon ¼ k5=Kd
(8Þ
(9Þ
where A is the absorbance at 410 nm (related to the con-
centration of 4-nitroaniline formed upon hydrolysis of the
MeOSuc-Ala-Ala-Pro-Val-p-nitroanilide substrate through
an extinction coefficient of 8250 M-1 cm-1), A0 is the
ꢀ
ꢁ
k6
ꢂ
Ki ¼ Kd
k5 þ k6

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 243
Table 1. 4-Oxo-β-lactams Prepared and the Corresponding Kinetic Data for HLE Inhibitionc

244 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Table 1. Continued
Mulchande et al.
a Value for Kd (see eq 7). b kobs/[I] determined at [3r] = 0.5 μM. c See text for definition of each kinetic parameter.
The relevant kinetic data for the whole set of 4-oxo-β-
lactams are outlined in Table 1. The kon values, ranging from
2 ꢀ 102 to 3 ꢀ 106 M-1 s-1, were used as an index of
inhibitory potency. Remarkably, these association rate con-
stants compare favorably with those of other very potent
HLE inhibitors. For example, the kon value for the 4-oxo-β-
lactam 6f is 1 order of magnitude higher than the second-
order rate constant for HLE inactivation by β-lactam 2, (L-
680,833, kon = 6.22 ꢀ 105 M-1 s-1).26 Modeling studies (see
next section) suggest that the tight complex inferred from
progress curves resulted from acylation of Ser-195 due to
attack on one of the carbonyl carbon atoms of the 4-oxo-β-
lactam. In this case, intermediates EI and EI* in mechanisms
A and B depicted in Scheme 4, respectively, refer to the acyl-
enzyme, and thus, k6 and koff values reflect the deacylation
step. An acylation-deacylation mechanism has been put
forward for the reaction of porcine pancreatic elastase with
oxo-β-lactams,32 and a similar mechanism has also been
reported for other acylating agents of serine proteases.44-46
The magnitude of koff and k6 is very small when compared to
kobs and is close to the deacylation rate constants reported
for other HLE inhibitors; the value of k6 for compounds 3b,
Scheme 1. Synthesis of 4-Oxo-β-lactams 3a-l and 3n-r
Scheme 2. Synthesis of 4-Oxo-β-lactams 6a-ja
a Reagents: (i) NBS, CCl4; (ii) ArSH, Et3N, THF.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 245
3b, and 6c could not be distinguished from zero, and thus, the
corresponding Ki* were not determined.
SAR and Modeling Studies. Inspection of the data in
Table 1 indicates that inhibitory activity of compounds 3 is
strongly dependent on the nature of substituents at C-3 of the
4-oxo-β-lactam ring. For example, replacing one ethyl sub-
stituent in 3c by a larger benzyl moiety, i.e., 3n, decreased the
kon value by 120-fold and increased Ki about 100-fold, while
changing the gem-diethyl substitution pattern of 3c to a
methyl benzyl one, i.e., 3o, decreased the kon value by 900-
fold and dramatically increased Ki nearly 3700-fold. In
addition, the ethyl isobutyl derivative, 3l, exhibited only an
8-fold decrease in the kon value when compared to its gem-
diethyl counterpart, 3c. In contrast, the most potent inhibi-
tor in the N-phenyl series was the ethyl n-butyl derivative,
3m, with kon = 2.9 ꢀ 105 M-1 s-1 and Ki = 0.85 nM. Overall,
these results are consistent with the known preference of
HLE for small hydrophobic substituents at C-3 of mono-
cyclic β-lactams for optimal interaction with the S1 binding
pocket of the enzyme.23,27
To study the efficiency of inactivation, titration of enzy-
matic activity studies was performed in which HLE was
incubated during 30 min with different concentrations of 3e
at pH 7.2. The fractional remaining activity was found to be
proportional to the molar ratio of inhibitor to enzyme, as
illustrated in Figure 5. The extrapolation of the line to v/vi =
0 gives the partition ratio; approximately 1.5 molecules of 3e
are required to completely inactivate 1 molecule of enzyme.
This result suggests that 3e is a very efficient irreversible
inhibitor of HLE and is consistent with the formation of a
tight complex as revealed by the hyperbolic dependence of
kobs versus [I]/(1 þ [S]/Km). Similar low partition ratios were
also reported for monocyclic β-lactam inhibitors of elas-
tase.27 For example, 1.3 molecules of β-lactam 2 (L-680,833)
are required to inactivate HLE to approximately 99%.47
Derivatives with different N-aryl and N-alkyl substituents
on N-1 were assayed in order to evaluate the effect of the
ability of the amide leaving group by C-N bond fission on
the inhibitory potency of 3,3-diethyl-4-oxo-β-lactams. Re-
placing the N-benzyl by an N-phenyl derivative significantly
increases in the rate of enzyme inhibition, as the kon value for
3c is nearly 137-fold higher than that of 3j. This effect can be
ascribed to the different leaving group abilities of the amide
formed from the decomposition of the tetrahedral intermedi-
ate 7 generated from 3c and 3j. However, the amide leaving
group ability is not the only factor affecting inhibitory
potency. For example, the N-1-naphthyl derivative, 3i, is
55 times less reactive toward HLE than its N-phenyl counter-
part, 3c, suggesting that rigid substituents at this position
may be responsible for some nonproductive binding inter-
actions.
The inhibitory potency of 4-oxo-β-lactams 3a-e toward
HLE does not correlate with the Hammett σp value of
substituents at the aromatic moiety. Analysis of the data
for the N-aryl, 3a-e, and N-(30-pyridyl), 3g,h, series indicates
that potency is not significantly affected by the electronic
Scheme 4. Slow-Binding Mechanisms for the Reaction of 4-
Oxo-β-lactams with HLE in the Presence of Substrate
Figure 1. Progress curves for the slow-binding inhibition of HLE
by 3h and 6f. Reaction conditions are as follows: [HLE] = 20 nM,
[MeOSuc-Ala-Ala-Pro-Val-p-NA] = 1 mM, 0.1 M HEPES buffer,
pH 7.2, 25 °C. Part A shows results for [3h]/nM: (a) 0; (b) 75; (c) 100;
(d) 250; (e) 375; (f) 500; (g) 750. Part B shows results for [6f]/nM: (a)
0; (b) 6.25; (c) 12.5; (d) 25; (e) 50; (f) 75.
Scheme 3. Postulated “Double-Hit” Mechanism for HLE Inactivation by 4-Oxo-β-lactams 6, Containing a Potential Thiol Leaving
Group

246 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Mulchande et al.
Figure 2. Effect of inhibitor concentration on the onset of inhibition of HLE by 3h. The values of kobs were obtained as an average of at least
duplicate assays from fits to eq 1 of the data shown in Figure 1A.
Figure 3. Plot of the steady-state rates versus [3h] for the inhibition of HLE. The values for the steady-state rate were obtained from fits to the
data shown in Figure 1A. The inset is a Dixon plot to show the linearity.
properties of substituents in the aromatic moiety. For ex-
ample, the 4-methylphenyl and 4-chlorophenyl derivatives
(3b and 3d, respectively) have roughly the same kon values
(about 6.5 ꢀ 105 M-1 s-1), which were about 3- and 6-fold
higher than those of the unsubstituted and 30-pyridyl coun-
terparts, 3c and 3g, respectively. This can be explained by a
significant degree of molecular recognition of these deriva-
tives by HLE. In contrast, previous work showed a good
Hammett correlation of the same series of compounds
toward PPE,32 indicating that PPE is much more susceptible
to intrinsic chemical reactivity effects rather than favorable
binding interactions with 3. Ortho substitution has a detri-
mental effect on potency, with compound 3b being 583-fold
more potent than 3f, the ortho positional isomer. This may
reflect the less stereochemically hindered carbonyl carbon of
3b toward nucleophilic attack by Ser-195.
low Ki values, ranging from 0.50 to 9.5 nM. These results
suggest that the heterocyclic and aryl moieties play an
important role in the molecular recognition of 6e-j by
HLE. In contrast, the ortho-substituted derivatives 6a-d
demonstrated inferior levels of inhibitory potency (kon) and
much higher Ki values than the corresponding para-analo-
gues, in line with results observed for their unfunctionalized
analogues, 3b and 3f. Two inhibitors from the para-substi-
tuted set, 6f and 6g, exhibited a hyperbolic dependence of
kobs versus [I]/(1 þ [S]/Km). Since both are derivatives of the
thiol 2-mercaptobenzoxazole, we suspect that this moiety
allows specific interaction within the active site of the
enzyme, thereby facilitating formation of a specific preasso-
ciation enzyme-inhibitor complex. Replacing the oxygen
atom of the benzoxazole moiety of 6f by a sulfur atom, giving
a benzothiazole moiety (as in 6e), decreases the inhibitory
potency by ∼3-fold. This reinforces the idea that the benzox-
azole substituent effects a gain in molecular recognition, with
its oxygen atom involved in hydrogen bonding within the
active site. Interestingly, the incorporation of a meta nitro-
gen atom into the aromatic ring, as in 6i, improves inhibitory
N-Functionalized derivatives that contain a thioether
function in the position para to the azetidine ring nitrogen,
6e-j, present improved inhibitory potency when compared
to the unfunctionalized counterparts 3b and 3h, with kon
values ranging from 1.2 ꢀ 106 to 3.2 ꢀ 106 M-1 s-1, and very

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 247
Figure 4. Effect of inhibitor concentration on the onset of inhibition of HLE by 6f. The values for the steady-state rate were obtained from fits
to the data shown in Figure 1B. The solid line was obtained using nonlinear regression analysis to eq 7.
observed. The OγSer195-CO distances for 6e are 2.6 and
2.8 A, suggesting that nucleophilic attack by Ser-195 at the
4-oxo-β-lactam is likely to be a facile process. Moreover, the
carbonyl of 6e closest to Ser-195 is involved in the H-bond
network of the so-called oxyanion hole defined by the backbone
NHs of Gly-193 and Ser-195, suggesting that the oxyanion hole
is used to stabilize the tetrahedral intermediate derived from 6e.
When 6f and 6i were docked to HLE, two binding poses
were identified: the normal pose (12 and 14 conformations
out of the top 15 solutions, respectively), in which the ethyl
groups at C-3 of the 4-oxo-β-lactam moiety sit in the S1
pocket and the carbonyl carbon atoms lie close to the Ser-195
hydroxyl oxygen atom (OγSer195-CO = 2.5/2.7 and 2.5/
2.8 A, respectively, Figure 6B and Figure 6C); and the
inverted pose, in which the benzothiazole (6i) and benzox-
azole (6f) moieties are within the S1 site, while the four-
membered ring is sitting outside the active site. Although the
inverted binding mode does not favor HLE acylation (the
Figure 5. Inactivation of HLE as
a function of the ratio
average shortest OγSer195-CO distance is 5.3 A), its low
frequency of occurrence is consistent with the observed
inhibitory efficiency, as shown by the ∼3-fold higher kon
values for 6f and 6i compared with that for 6e. The presence
of such inverted binding modes in top ranking conforma-
tions has been reported for other potent irreversible serine
protease inhibitors.49 In contrast, all top ranking docking
solutions of 3o correspond to the inverted pose, with the
N-phenyl group inside the S1 pocket and an average shortest
OγSer195-CO distance of 5.5 A (Figure 6D), a result entirely
consistent with the weaker HLE inhibition profile of 3o.
Selectivity Studies. Representative compounds were tested
against human neutrophil serine proteases PR3 and cathe-
psin G and the cysteine protease papain (Table 2). With the
exception of 3e and 3o, all 4-oxo-β-lactams assayed were
found to be time-dependent inhibitors of PR3, although with
significantly lower activity in comparison with HLE inhibi-
tion. In contrast, most of 4-oxo-β-lactams assayed were
inactive or only weakly active against cathepsin G. For
example, compound 6f was ∼105 times more potent an
inhibitor of HLE than PR3 and was inactive against cathe-
psin G. A similar selectivity pattern was also observed for 6g
and 6i. Dual inhibition of HLE and PR3 is not surprising, as
it reflects the close similarity of the primary specificity sites of
[3e]/[enzyme] at a fixed [HLE] of 10 μM. Various amounts of
inhibitor 3e (25-800 nM) in 0.1 M pH 7.2 HEPES buffer were
incubated at 25 °C for 30 min. Then aliquots were withdrawn and
measured for the remaining enzyme activity.
potency when compared to its carba counterpart, 6e. This
finding contrasts with the lower potency of 3g and 3h,
compared with the corresponding N-phenyl analogues.
To obtain a clearer insight into the binding modes between
4-oxo-β-lactams and HLE, we studied the molecular inter-
actions between compounds 3o (the least potent compound),
6e, 6f, and 6i (the most potent compounds) and the enzyme
(PDB code 1HNE) using the program GOLD.48 The top 15
conformations (i.e., those with the highest GoldScore fitness
score) were visually analyzed for both the hydrophobic
interactions between the ligand and the residues defining
the S1 pocket, and the distances between the Oγ oxygen atom
of Ser-195 and the two 4-oxo-β-lactam carbonyl carbon
atoms (OγSer195-CO). Modeling the interaction of 6e with
HLE revealed that both ethyl substituents at C-3 of the
4-oxo-β-lactam moiety are accommodated in the S1 pocket
where they interact with Val-190, Phe-192, and Val-216
(Figure 6A). Enhanced van der Waals contacts between the
benzothiazole moiety with Leu-99 and His-57 were also

248 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Mulchande et al.
Figure 6. Docking poses of 6e (A), 6f (B), 6i (C), and 3o (D) in the active site of HLE, obtained from PDB 1HNE. Pictures were prepared using
Pymol.66
The general lack of cathepsin G inhibition shown by 4-
oxo-β-lactams is consistent with the known substrate speci-
ficity, since this enzyme has a trypsin and a chymotrypsin-
like preference for P1, accepting aromatic and positively
charged side chains.8 Surprisingly, neither the methyl benzyl,
3n, or the ethyl benzyl, 3o, derivatives inhibit cathepsin G at
60 μM, despite the presence of an aromatic side chain at P1.
This result may be ascribed to the restricted conformational
mobility imparted by the geminal ethyl and methyl groups.
While the N-aryl-4-oxo-β-lactams 3a-e were previously
studied against porcine pancreatic elastase (PPE),32 these
compounds are much more potent inhibitors of HLE. In
fact, the second-order rate constant for HLE inactivation for
Table 2. Selectivity of Selected 4-Oxo-β-lactams toward HLE, Cathe-
psin G, Proteinase 3, and Papaina
kobs/[I], M-1 s-1
kon, M-1 -1
HLE
s
compd
proteinase 3
cathepsin G
papain
3e
3i
5.08 ꢀ 105
4.35 ꢀ 103
2.86 ꢀ 105
2.00 ꢀ 103
2.67 ꢀ 102
3.49 ꢀ 104
3.24 ꢀ 106
1.77 ꢀ 106
3.18 ꢀ 106
1.17 ꢀ 106
16%b
ND
ND
20.2
22%c
361
36.5
35.6
47.3
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
3m
3n
3o
6b
6f
NI
NI
29.6
689
NI
NI
6g
6i
these compounds ranges from 2.4 ꢀ 105 to 6.6 ꢀ 105 M-1 s-1
,
4080
122
NI
ND
6j
some 3 orders of magnitude larger than those reported for
PPE inhibition. This result accords with the S1 binding
pocket of HLE being larger than that of PPE,33 thereby
allowing optimal interaction with the diethyl group at the C-
3 of the 4-oxo-β-lactam moiety. Furthermore, the tight
preassociation inhibitor-HLE complex observed for com-
pounds 3b, 3d, and 3e (Table 1) contrasts with the simple
association mechanism reported for PPE inhibition.32 Final-
ly, all 4-oxo-β-lactams tested were inactive against the
cysteine protease, papain, thus suggesting a high degree of
a NI: no inhibition at 60 μM. ND: not determined. b At 10 μM (highest
concentration used). c At 60 μM (highest concentration used).
both enzymes8 and is consistent with similar observations
previously described for other scaffolds.40 As both of these
neutrophil serine proteases are involved in inflammation and
extracellular matrix destruction observed in pulmonary dis-
eases, a potent inhibition of HLE combined with a weaker
inhibition of PR3 may be an advantage for therapeutics.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 249
Table 3. Aqueous and Plasma Stability at 37 °C for Compounds 6a, 6e,
and 6i
potency. In addition, derivatives containing a benzoxazole- or
benzothiazolethiomethyl group in the N-aryl moiety embody
a general motif that allows an extremely potent and time-
dependent irreversible inhibition of HLE, with some com-
pounds displaying kon values higher than 106 M-1 s-1. Dock-
ing studies reveal that ethyl substituents at C-3 of the 4-oxo-β-
lactam moiety are well accommodated in the hydrophobic S1
pocket, allowing proper orientation between the Oγ oxygen
atom of Ser-195 and at least one of the 4-oxo-β-lactam
carbonyl carbon atoms. These inhibitors were relatively spe-
cific for HLE when compared to proteinase 3 and cathepsin
G but displayed absolute specificity when compared to pa-
pain. Some of the most potent and selective inhibitors are
relatively stable in pH 7.4 buffer but reactive in 80% human
plasma, suggesting that they might be susceptible to esterases.
To the best of our knowledge, this is the first comprehensive
SAR and selectivity study of 4-oxo-β-lactam HLE inhibitors,
which reveals the key structural determinants for inhibitory
potency and suggests that the 4-oxo-β-lactam scaffold repre-
sents a novel lead for further optimization.
t1/2, h
compd
buffer, pH 7.4
80% human plasma
6a
6e
6i
37
10
13
1.6
0.4
0.2
Scheme 5. Products for Hydrolysis of 6a, 6e, and 6i in pH 7.4
Buffer and in Human Plasma
selectivity of the azetidine-2,4-dione scaffold toward serine
proteases and, in particular, HLE.
Hydrolytic Stability. Stability toward hydrolysis is a pre-
requisite for maintaining the therapeutically useful concen-
trations in biological fluids, such as pulmonary epithelial
lining fluid and plasma. Thus, the stability of the three
compounds containing a benzothiazolylthiomethyl moiety,
6a, 6e, and 6i, was evaluated in pH 7.4 phosphate buffer
saline at 37 °C. These were observed to have reasonable
stability under physiological conditions, compound 6a being
the most stable with a half-life of 37 h (Table 3). This value is
well within the range of hydrolytic half-lives (pH 7.4 and
37 °C) reported for cephalosporin sulfones,22,50 N-acyl- and
N-sulfonylazetidin-4-ones,51 and other β-lactams52 but low-
er than those reported for N-carbamoylazetidin-4-ones.53
The product of hydrolysis in all cases was the corresponding
N-aryl-2,2-diethylpropanedioic acid monoamide 8 (Scheme
5), which implies that departure of the benzothiazolylthiol
leaving group is not an efficient process under physiological
conditions. This result also suggests that inhibition of HLE
by compounds 6 does not involve departure of the leaving
group and subsequent formation of an electrophilic quinone
methide imine.
In human plasma, compounds 6a, 6e, and 6i were rapidly
hydrolyzed, with half-lives ranging from 0.2 to ∼2 h. 4-Oxo-
β-lactams are thus susceptible to plasma enzymes, in line
with the pronounced susceptibility of neutral β-lactam deri-
vatives (e.g., penicillin esters) to undergo plasma-catalyzed
hydrolysis of the β-lactam ring.52 Overall, these results
indicate that although the oral availability of 4-oxo-β-lac-
tams might be limited by compound stability, these inhibi-
tors might be suitable for aerosolization, a route of
administration commonly employed for other agents used
for lung diseases.54 Aerosolization circumvents the problems
of absorption and metabolism associated with systemic
administration and should also lessen any possible side
effects.55
Experimental Section
General. Melting points were determined using a Kofler
camera Bock monoscope M and are uncorrected. The infrared
spectra were collected on a Nicolet Impact 400 FTIR infrared
spectrophotometer and the NMR spectra on a Bruker 400
Ultra-Shield (400 MHz) in CDCl3; chemical shifts, δ, are
expressed in ppm, and coupling constants, J, are expressed in
Hz. Low-resolution mass spectra were recorded using an
HP5988A spectrometer, by RIAIDT, University of Santiago
de Compostela, Spain. Compounds were determined to be
>95% pure by elemental analysis (for C, H, and N) performed
at Medac Ltd., Brunel Science Centre, Englefield Green,
ꢀ
Egham, TW20 0JZ, U.K., or by ITN, Chemistry Unit, Sacavem,
Portugal. UV spectra and spectrophotometric assays were
recorded using either a Shimadzu UV-1603 or UV-2100 PC
spectrophotometer. Thin layer chromatography was performed
using Merck silica gel 60 F254 aluminum plates and visualized by
UV light and/or iodine. Preparative column chromatography
was performed using Merck silica gel 60 (70-230 mesh ASTM).
Dioxane, tetrahydrofuran, and triethylamine were dried before
use. Solvents and buffer materials for enzyme assays were of
analytical reagent grade and were purchased from Merck or
Sigma. Cathepsin G (EC 3.4.21.20), MeOSuc-Ala-A-Ala-Pro-
Val-p-NA, and Suc-Ala-Ala-Pro-Phe-p-NA were purchased
from Sigma, and HLE (EC 3.4.21.37) and proteinase 3
(EC.3.4.21.76) were purchased from Calbiochem. Compounds
3a-e, 3j-k, 3m, 3p-r were synthesized as described in the
literature.32,34
General Procedure for the Synthesis of 4-Oxo-β-lactam Deri-
vatives 3. The necessary primary amine (0.015 mol) in dioxane
(15 mL) was added under a nitrogen atmosphere to a solution of
the appropriate malonyl dichloride (0.015 mol) in dioxane (15
mL). A solution of triethylamine (0.036 mol) in dioxane (15 mL)
was then added dropwise during 1.5 h and the resulting mixture
subsequently refluxed for 6 h, reaction progress being moni-
tored by TLC. After the mixture was cooled, triethylamine
hydrochloride was filtered off and the solvent removed under
reduced pressure, yielding a solid (the corresponding malon-
diamide). The mother liquor was concentrated under reduced
pressure and the residue purified by chromatography on silica
gel, affording the desired azetidine-2,4-dione.
Conclusion
4-Oxo-β-lactam HLE inhibitors have been designed to
incorporate the structural requirements for optimal interac-
tion at the S1 primary selectivity pocket of the enzyme. The
SAR data indicate that substituents at C-3 of the four-
membered ring are a major determinant of the inhibitory
3,3-Diethyl-1-o-tolylazetidine-2,4-dione (3f). 3f was prepared
using diethylmalonyl dichloride (0.015 mol) and o-toluidine
(0.015 mol). The product was purified by chromatography on
silica gel using hexane/ethyl acetate (8:2) and subsequently by
preparative TLC, yielding the product as a colorless oil (6%). δ

250 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Mulchande et al.
1H NMR 1.15 (6H, t, J = 7.6); 1.90 (4H, q, J = 7.6); 2.38 (3H, s);
7.26-7.27 (2H, m); 7.30-7.32 (2H, m); δ 13C NMR 9.52, 18.83,
24.11, 70.80, 125.59, 126.68, 129.03, 129.83, 131.43, 133.83,
172.84.
3,3-Diethyl-1-(pyridin-3-yl)azetidine-2,4-dione (3g). 3g was
prepared using diethylmalonyl dichloride (0.015 mol) and 3-
aminopyridine (0.015 mol). The product was purified by column
chromatography on silica gel using dichloromethane/ethyl acet-
ate (9.5:0.5) to give a light-yellow solid, mp 38 °C (47%). δ 1H
NMR 1.10 (6H, t, J = 7.6); 1.90 (4H, q, J = 7.6); 7.38 (1H, ddd,
J = 8.4, 4.8, 0.4); 8.35 (1H, ddd, J = 8.4, 2.8, 1.6); 8.55 (1H, dd,
J = 4.8, 1.6); 9.16 (1H, d, J = 2.8); δ 13C NMR 9.22, 23.93,
72.40, 123.84, 126.21, 130.83, 140.77,147.71, 171.73.
corresponding bromomethylarylazetidine-2,4-dione, 5. Deriva-
tives 6 were prepared by adding triethylamine (2.2 mmol) to a
solution of the appropriate bromomethyl derivatives 5a-c (2
mmol) and thiol (2.2 mmol) in dry THF (5 mL). The mixture was
stirred at room temperature for 1-8 h, being monitored by
TLC. Upon completion, triethylamine hydrochloride was re-
moved by filtration, the solvent removed under reduced pres-
sure, and the residue purified first by chromatography on silica
gel and subsequently by preparative TLC.
1-(2-((Benzo[d]thiazol-2-ylthio)methyl)phenyl)-3,3-diethylaze-
tidine-2,4-dione, 6a. 6a was prepared as described above using 5a
(0.193 mmol) and 2-mercaptobenzothiazole (0.212 mmol). Elu-
tion with dichloromethane-hexane, 7:3, yielded a white solid,
mp 73-75 °C (68%). δ 1H NMR 1.15 (6H, t, J = 7.6); 1.93 (4H,
q, J = 7.6); 4.72 (2H, s); 7-28-7.36 (4H, m); 7.44 (1H, t, J =
8.0); 7.67-7.68 (1H, ddd, J = 8.0, 1.2, 0.4); 7.75 (1H, d, J = 8.0);
7.91 (1H, d, J = 8.0); δ 13C NMR 9.45, 23.98, 34.02, 70.80,
121.02, 121.69, 124.36, 125.98, 126.09, 128.84, 129.25, 129.89,
131.42, 132.06, 135.39, 153.05, 165.42, 172.87.
1-(2-((Benzo[d]oxazol-2-ylthio)methyl)phenyl)-3,3-diethylaze-
tidine-2,4-dione, 6b. 6b was prepared as described above using 5a
(0.284 mmol) and 2-mercaptobenzoxazole (0.314 mmol) to give
a white solid (95%), mp 63-66 °C. δ 1H NMR 1.17 (6H, t, J =
7.6); 1.95 (4H, q, J = 7.6); 4.66 (2H, s); 7.28 (1H, dt, J = 7.6,
1.2); 7.30 (1H, dt, J = 7.6, 1.2); 7.33-7.38 (3H, m); 7.43 (1H,
ddd, J = 7.6, 1.2, 0.4); 7.62 (1H, ddd, J = 7.6, 1.2, 0.4);
7.68-7.71 (1H, m); δ 13C NMR 9.42, 23.94, 32.92, 70.79,
109.92, 118.62, 123.98, 124.34, 125.88, 128.98, 129.24, 129.88,
131.46, 131.83, 141.83, 151.98, 163.74, 172.85.
3,3-Diethyl-1-(6-methylpyridin-3-yl)azetidine-2,4-dione (3h).
3h was prepared as described for 3a, using diethylmalonyl
dichloride (0.0075 mol) and 5-amino-2-methylpyridine (0.0075
mol). The product was purified by column chromatography on
silica gel using dichloromethane/ethyl acetate (9.5:0.5) and
recrystallized from dichloromethane-hexane to give white
1
crystals (39%), mp 44-45 °C. δ H NMR 1.09 (6H, t, J =
7.6); 1.89 (4H, q, J = 7.6); 2.59 (3H, s); 7.22 (1H, d, J = 8.4);
8.04 (1H, dd, J = 8.4, 2.4); 9.01 (1H, d, J = 2.4); δ 13C NMR
9.22, 23.91, 24.25, 72.25, 123.34, 126.74, 129.24, 140.13, 156.83,
171.80.
3,3-Diethyl-1-(naphthalen-1-yl)azetidine-2,4-dione (3i). 3i was
prepared using diethylmalonyl dichloride (0.015 mol) and
naphthalen-1-amine (0.015 mol). The product was purified by
column chromatography on silica gel using hexane/ethyl acetate
(8:2), recrystallized from hexane, to yield a white solid (5%), mp
49-50 °C. δ 1H NMR 1.26 (6H, t, J = 7.6); 2.00 (4H, q, J = 7.6);
7.50-7.64 (4H, m); 7.89-7.93 (3H, m); δ 13C NMR 9.63, 24.25,
71.32, 123.06, 123.32, 125.21, 126.77, 127.17, 127.20, 127.90,
128.47, 129.43, 134.36, 173.26.
3-Ethyl-3-isobutyl-1-phenylazetidine-2,4-dione (3l). 3l was
prepared using 2-ethyl-2-isobutylmalonyl dichloride (0.014
mol) and aniline (0.014 mol). The product was purified by
chromatography on silica gel using hexane/ethyl acetate (9:1),
yielding the desired product as a colorless oil (12%). δ 1H NMR
1.00 (6H, d, J = 6.4); 1.08 (3H, t, J = 7.6); 1.75 (2H, d, J = 6.4);
1.81-1.91 (3H, m); 7.29 (1H, dt, J = 7.6, 1.2); 7.44 (2H, dt, J =
7.6, 1.2); 7.86 (2H, dd, J = 7.6, 1.2); δ 13C NMR 9.09, 23.66,
25.04, 25.26, 39.80, 70.89, 119.20, 126.77, 129.29, 133.93, 172.46.
3-Benzyl-3-ethyl-1-phenylazetidine-2,4-dione (3n). 3n was pre-
pared using 2-benzyl-2-ethylmalonyl dichloride (0.0081 mol)
and aniline (0.0081 mol). The product was purified by chroma-
tography on silica gel using hexane/ethyl acetate (8:2) and
recrystallized from hexane, yielding white crystals (22%), mp
76-77 °C. δ 1H NMR 1.11 (3H, t, J = 7.6); 1.95 (2H, q, J = 7.6);
3.10 (2H, s); 7.20-7.24 (2H, m); 7.27-7.28 (4H, m); 7.33 (2H, dt,
J = 7.6, 1.2); 7.52 (2H, dd, J = 7.6, 1.2); δ 13C NMR 9.33, 24.42,
37.38, 73.37, 119.61, 126.90, 127.38, 128.57, 129.11, 129.63,
132.99, 134.86, 171.36.
1-(2-((5-Phenyl-1,3,4-oxadiazol-2-ylthio)methyl)phenyl)-3,3-
diethylazetidine-2,4-dione, 6c. 6c was prepared as described
above, using 5a (0.177 mmol) and 5-phenyl-1,3,4-oxadiazole-
2-thiol (0.194 mmol), and purified using dichloromethane-
hexane, 8:2, as eluant to yield white crystals (87%), mp
1
110-112 °C. δ H NMR 1.18 (6H, t, J = 7.6); 1.95 (4H, q,
J =7.6); 4.67 (2H, s);7.35-7.40 (3H, m);7.48-7.56(3H, m); 7.71
(1H, dd, J = 6.0, 2.0); 7.99 (2H, dd, J = 8.0, 1.6); δ 13C NMR
9.47, 23.99, 33.57, 70.83, 123.50, 125.96, 126.69, 129.05, 129.24,
129.27, 129.96, 131.18, 131.73, 131.83, 163.35, 166.03, 172.74.
1-(2-((1-Methyl-1H-imidazol-2-ylthio)methyl)phenyl)-3,3-di-
ethylazetidine-2,4-dione, 6d. 6d was prepared as described above,
using 5a (0.169 mmol) and 1-methyl-1H-imidazole-2-thiol
(0.186 mmol), and purified using ethyl acetate-acetone, 9:1,
as eluant to yield a pale-yellow solid (70%), mp 61-63 °C. δ 1H
NMR 1.17 (6H, t, J = 7.6); 1.94 (4H, q, J = 7.6); 3.11 (3H, s);
4.22 (2H, s); 6.80 (1H, s); 6.93 (1H, d, J = 7.6); 7.10 (1H, s); 7.15
(1H, dt, J = 7.6, 1.6); 7.25-7.32 (2H, m)); δ 13C NMR 9.50,
23.95, 33.19, 37.31, 70.68, 122.77, 126.32, 128.60, 128.96, 129.32,
129.42, 130.90, 133.82, 139.92, 172.89.
1-(4-((Benzo[d]thiazol-2-ylthio)methyl)phenyl)-3,3-diethylaze-
tidine-2,4-dione, 6e. 6e was prepared as described above, using
5b (0.097 mmol) and 2-mercaptobenzothiazole (0.106 mmol),
and purified using dichloromethane-hexane, 7:3, as eluant to
yield a white solid (73%), mp 102-104 °C. δ 1H NMR 1.07 (6H,
t, J = 7.6); 1.86 (4H, q, J = 7.6); 4.60 (2H, s); 7.32 (1H, dt, J =
7.6, 1.2); 7.45 (1H, dt, J = 7.6, 1.2); 7.53 (2H, d, J = 8.4); 7.76
(1H, dd, J = 7.6, 0.4); 7.83 (2H, d, J = 8.4); 7.92 (1H, dd, J =
7.6, 0.4); δ 13C NMR 9.25, 23.96, 37.07, 72.25, 119.37, 121.06,
121.60, 124.41, 126.13, 130.06, 133.22, 135.08, 135.36, 153.06,
165.77, 172.10.
1-(4-((Benzo[d]oxazol-2-ylthio)methyl)phenyl)-3,3-diethylaze-
tidine-2,4-dione, 6f. 6f was prepared as described above, using 5b
(0.145 mmol) and 2-mercaptobenzoxazole (0.160 mmol), and
purified using dichloromethane-hexane, 7:3, as eluant to yield
white crystals (59%), mp 96-98 °C. δ 1H NMR 1.07 (6H, t, J =
7.6); 1.86 (4H, q, J = 7.6); 4.57 (2H, s); 7.28 (1H, dt, J = 7.6,
1.2); 7.32 (1H, dt, J = 7.6, 1.2); 7.47 (1H, d, J = 7.6); 7.55 (2H, d,
J = 8.4); 7.65 (1H, d, J = 7.6); 7.84 (2H, d, J = 8.4); δ 13C NMR
9.25, 23.97, 36.02, 72.30, 109.98, 118.50, 119.42, 124.12, 124.44,
130.03, 133.40, 134.63, 141.83, 151.93, 164.05, 172.09.
3-Benzyl-3-methyl-1-phenylazetidine-2,4-dione (3o). 3o was
prepared using 2-benzyl-2-methylmalonyl dichloride (0.016
mol) and aniline (0.016 mol). The product was purified by
column chromatography on silica gel using hexane/ethyl acetate
(8:2), subsequently by preparative TLC, and recrystallized from
dichloromethane-hexane to yield white crystals (5%), mp
1
54-55 °C. δ H NMR 1.57 (3H, s); 3.10 (2H, s); 7.20-7.24
(2H, m); 7.27-7.29 (4H, m); 7.33 (2H, dt, J = 7.6, 1.2); 7.56 (2H,
dd, J = 7.6, 1.2); δ 13C NMR 16.27, 38.65, 73.03, 119.48, 126.92,
127.56, 128.60, 129.12, 129.56, 134.86, 136.16, 171.51.
General Procedure for the Synthesis of 4-Oxo-β-lactam Deri-
vatives 6. To a solution of the appropriate ortho or para-methyl
substituted 4-oxo-β-lactam (3b, 3f, or 3h, 0.282 mmol) in tetra-
chloromethane, N-bromosuccinimide (NBS, 0.310 mmol) and
benzoyl peroxide (0.0282 mmol) were added, and the reaction
mixture was heated under reflux for 12 h with monitoring by
TLC. The benzoic acid byproduct was removed by filtration and
the solvent was removed under reduced pressure to yield the

Article
2-(4-(3,3-Diethyl-2,4-dioxoazetidin-1-yl)benzylthio)benzo[d ]-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 251
the inhibitor was omitted, ran linearly. Data were treated as
described above.
oxazole-6-carboxylic acid, 6g. 6g was prepared as described
above, using 5b (0.225 mmol) and 2-mercaptobenzoxazole-5-
carboxylic acid, 9 (0.247 mmol), and triethylamine (0.472
mmol). The reaction mixture was acidified with HCl until pH
2 was obtained and extracted with ethyl acetate. After the
mixture was dried and the solvent evaporated off, the residue
was purified by column chromatography on silica gel, and
subsequently by preparative TLC (elution with ethyl acetate),
to yield a yellow solid (24%), mp 166-171 °C. δ 1H NMR 1.07
(6H, t, J = 7.6); 1.87 (4H, q, J = 7.6); 4.65 (2H, s); 7.52 (1H, d,
J = 8.8); 7.56 (2H, d, J = 8.4); 7.86 (2H, d, J = 8.4); 8.11 (1H, d,
J = 8.8); 8.41 (1H, s); δ 13C NMR 9.25, 23.97, 36.12, 72.32,
109.88, 119.47, 121.05; 126.78, 130.09, 133.48, 134.35, 135.76,
142.02, 151.52, 166.02, 169.87, 172.09.
1-(4-((5-Phenyl-1,3,4-oxadiazol-2-ylthio)methyl)phenyl)-3,3-
diethylazetidine-2,4-dione, 6h. 6h was prepared as described
above, using 5b (0.290 mmol) and 5-phenyl-1,3,4-oxadiazole-
2-thiol (0.319 mmol), and purified using dichloromethane-
hexane, 9:1, to yield white crystals (71%), mp 120-122 °C; δ
1H NMR 1.07 (6H, t, J = 7.6); 1.87 (4H, q, J = 7.6); 4.53 (2H, s);
7.49-7.54 (3H, m); 7.55 (2H, d, J = 8.4); 7.85 (2H, d,
J = 8.4); 8.01 (2H, dd, J = 8.4, 1.6); δ 13C NMR 9.22, 23.97,
36.27, 72.29, 119.49, 123.53, 126.68, 129.06, 130.10, 131.74,
133.34, 134.30, 147.38, 163.50, 172.07.
1-(6-((Benzo[d]thiazol-2-ylthio)methyl)pyridin-3-yl)-3,3-diethyl-
azetidine-2,4-dione, 6i. 6i was prepared as described above by
reaction of 5c with 2-mercaptobenzothiazole (0.166 mmol) and
purified using dichloromethane as eluant to yield a white solid
(61%); mp 129-131 °C. δ 1H NMR 1.08 (6H, t, J = 7.6); 1.88
(4H, q, J = 7.6); 4.76 (2H, s); 7.33 (1H, dt, J = 8.4, 1.6); 7.44
(1H, dt, J = 8.4, 1.6); 7.66 (1H, d, J = 8.4); 7.77 (1H, dd, J =
8.4, 0.4); 7.91 (1H, d, J = 8.4); 8.12 (1H, dd, J = 8.4, 2.4); 9.11
(1H, d, J = 2.4); δ 13C NMR 9.23, 23.94, 38.60, 72.46, 121.09,
121.56, 123.85, 124.42, 126.12, 126.87, 129.72, 135.4, 140.36,
152.99, 154.85, 165.69, 171.61.
3,3-Diethyl-1-(4-((phenylthio)methyl)phenyl)azetidine-2,4-di-
one, 6j. 6j was prepared as described above by reaction of 5b
(0.229 mmol) with thiophenol (0.252 mmol) and purified using
dichloromethane-hexane, 7:3, to yield a colorless oil (79%). δ
1H NMR 1.08 (6H, t, J = 7.6); 1.86 (4H, q, J = 7.6); 4.12 (2H, s);
7.20-7.31 (5H, m); 7.34 (2H, d, J = 8.4); 7.77 (2H, d, J = 8.4); δ
13C NMR 9.26, 23.95, 38.71, 72.15, 119.24, 126.65, 128.95,
129.61, 130.13, 132.76, 135.72, 136.28, 172.17.
Enzyme Kinetics: Progress Curve Method. Inactivation of
HLE was studied at 25 °C by mixing 10 μL of HLE stock
solution (2 μM in 0.05 M acetate buffer, pH 5.5) to a solution
containing 10 μL of inhibitor in DMSO, 20 μL of the MeOSuc-
Ala-Ala-Pro-Val-p-nitroanilide substrate (50 mM in DMSO),
and 960 μL of 0.1 M pH 7.2 HEPES buffer, and the absorbance
was continuously monitored at 410 nm for 20 min. Control
assays, in which the inhibitor was omitted, ran linearly. Progress
curves were fitted to eqs 1 or 2, depending on the inhibitor
concentration relative to that of the enzyme.26 The fitting
residuals were typically less than (0.001 absorbance units,
and typical residual standard deviation for both vs and kobs
was in the range 2-8%. The kobs values calculated from the
progress plot were fitted to the slow association or the isomer-
ization mechanisms (Scheme 4) using eqs 3 and 7, respectively.
The error associated with Ki was usually ∼5% and did not show
any relationship to the inhibitory potency. The Km value of 0.16
mM for the hydrolysis of MeO-Suc-Ala-Ala-Pro-Val-p-NA by
HLE was determined as described previously.39
Inactivation of proteinase 3 was studied at 25 °C by mixing
70 μL of enzyme stock solution (325 nM in 0.05 M acetate
buffer, pH 6, 150 mM NaCl) with a solution containing 10 μL of
inhibitor in DMSO, 75 μL of MeOSuc-Ala-Ala-Pro-Val-p-
nitroanilide substrate (50 mM in DMSO), and 835 μL of 0.1
M pH 7.2 HEPES buffer, and the absorbance was continuously
monitored at 410 nm for 20 000 s. Control assays, in which the
inhibitor was omitted, ran linearly. Data were treated as de-
scribed above.
Enzyme Kinetics: Incubation Method. Papain (1 mg/mL) was
activated as described,56 and the activated enzyme (300 μL) was
incubated at 25 °C with 50 μL of the inhibitor in 650 μL of 50
mM pH 7.0 K2HPO4/KH2PO4 containing 2.5 mM EDTA. At
different times, aliquots (100 μL) were withdrawn and trans-
ferred to a cuvette thermostated at 25 °C, containing 100 μL of
(S)-N-benzoylarginine-4-nitroanilide (BAPA) substrate (10
mM in DMSO) and 800 μL of 50 mM K2HPO4/KH2PO4, pH
7.0, containing 2.5 mM EDTA. The absorbance was monitored
during 4 min at 410 nm. Control assays, without the inhibitor,
were carried out as described above.56
Titration of HLE. An amount of 10 μL of HLE stock solution
(20 μM in 0.05 M acetate buffer, pH 5.5) was incubated at 25 °C
with different concentrations of 3e (25-800 nM) in DMSO (10
μL) and 980 μL of 0.1 M HEPES buffer, pH 7.2. After incuba-
tion of the reaction mixture for 30 min, a 100 μL aliquot was
withdrawn and assayed for remaining enzyme activity, as
described previously. The partition ratio was determined from
the intercept on the x-axis of the linear plot of the remaining
activity ([E]r/[E]0), versus the initial ratio of inhibitor to enzyme
([I]0/[E]0).
HPLC Analysis. The analytical high-performance liquid
chromatography (HPLC) system comprised a Merck Hitachi
L-7100 pump with an Shimadzu SPD-6AV UV detector, a
manual sample injection module equipped with a 20 μL loop,
and a Merck LlchroCART 250-4 RP8 (5 μm) column equipped
with a Merck Lichrocart precolumn (Merck, Germany). An
isocratic solvent system of acetonitrile/water (65:35) was used
for compounds 6a, 6e, and 6i, The column effluent was moni-
tored at 225 nm for 6a and 220 nm for compounds 6e and 6i.
Hydrolysis in Buffer Solution. The kinetics of hydrolysis of the
compounds was studied using HPLC. Usually, a 15 μL aliquot
of a 10-2 M stock solution of the compound in DMSO was
added to 2.5 mL of thermostated phosphate buffer solution (37
°C). At regular intervals, samples of the reaction mixture were
analyzed by HPLC using a mobile phase of acetonitrile/water
(the composition of which depended on the compound) and a
flow rate of 1.0 mL/min.
Hydrolysis in Human Plasma. Human plasma was obtained
from the pooled, heparinized blood of healthy donors and was
frozen and stored at -20 °C prior to use. For the hydrolysis
experiments, the compounds were incubated at 37 °C in human
plasma that had been diluted to 80% (v/v) with pH 7.4 isotonic
phosphate buffer. At appropriate intervals, aliquots of 100 μL
were added to 200 μL of DMSO to both quench the reaction and
precipitate plasma proteins. These samples were centrifuged,
and the supernatant was analyzed by HPLC for the presence of
initial compound and hydrolysis products.
Molecular Modeling. Geometries of compounds 3o, 6a, 6e,
and 6i were energy-minimized using density functional theory.57
These calculations were performed with the B3LYP58,59 hybrid
functional and the 6-31þG(d,p) basis set implemented in Gauss-
ian 03 software package.60 After geometry optimizations, par-
tial charges were included using Amber’s antechamber module61
(included with Chimera software).62 The HLE structure (PDB
code 1HNE63) was obtained by deletion from the active site of
the ligand (MeOSuc-Ala-Ala-Pro-Ala chloromethyl ketone)
present in the crystal structure. Hydrogen atoms were added,
and the correct protonation state of histidine residues was
Inactivation of cathepsin G was studied at 25 °C by
mixing 100 μL of enzyme stock solution (680 nM in 0.05 M
acetate buffer, pH 5.5) with a solution containing 10 μL of
inhibitor in DMSO, 20 μL of Suc-Ala-Ala-Pro-Phe-p-nitro-
anilide substrate (42.5 mM in DMSO), and 870 μL of 0.1 M
pH 7.5 HEPES buffer, and the absorbance was continuously
monitored at 410 nm for 20 min. Control assays, in which

252 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Mulchande et al.
assigned according to their surrounding environment. The PPE
structure was energy minimized, and charges were added using
the Amber united atom force field64 implemented in Chimera
software.62 Molecular docking studies of 4-oxo-β-lactams into
the active site of HLE enzyme were performed with the flexible
GOLD48 docking program using the GoldScore scoring func-
tion.65 Each ligand was initially energy-minimized and then
subjected to 10 000 docking runs (with a population size of
100, 100 000 genetic algorithm operations, 5 islands). The top 15
solutions (i.e., those with the highest fitness score) were visually
analyzed for (i) the hydrophobic and hydrophilic interactions
between the ligand and enzyme surfaces and (ii) the distance
between the Ser-195 hydroxyl oxygen atom and the carbonyl
carbon atoms of each 4-oxo-β-lactam.
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~
Acknowledgment. This work was funded by Fundac-ao
para a Ciencia e Tecnologia (FCT, Portugal) through Project
^
PTDC/QUI/64056/2006. J.M. acknowledges FCT for the Ph.
D. Grant SFRH/BD/17534/2004.
Supporting Information Available: Analytical data for com-
pounds 3 and 6; infrared and mass spectra for compounds 3 and
5; synthesis of compounds 5 and 9; complete listing of ref 60.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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