N-Acyl and N-sulfonyloxazolidine-2,4-diones are pseudo-irreversible inhibitors of serine proteases

Article abstract of DOI:10.1016/j.bmcl.2012.04.093

The synthesis, inhibitory activity and mode of action of oxazolidine-2,4-diones against porcine pancreatic elastase, here used as a model for human neutrophil elastase, are reported. The nature of N-substitution at the oxazolidine-2,4-dione scaffold has large effect on the inhibitory potency against elastase. N-Acyl and N-sulfonyloxazolidine-2,4-diones emerged as potent pseudo-irreversible inhibitors, displaying high second-order rate constants for PPE inactivation. The title compounds were also shown to be potent inhibitors of human neutrophil elastase (HNE) and proteinase-3, and weak inhibitors of human cathepsin G. The results herein presented show that the oxazolidine-2,4-diones represent a new promising class of serine protease inhibitors.

Full text of DOI:10.1016/j.bmcl.2012.04.093

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3993–3997
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Acyl and N-sulfonyloxazolidine-2,4-diones are pseudo-irreversible
inhibitors of serine proteases
Ana Bela Santana a, Susana D. Lucas a, Lídia M. Gonçalves a, Henrique F. Correia a, Teresa A.F. Cardote a,
Rita C. Guedes a, Jim Iley b, Rui Moreira a,
⇑
a Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal
b Department of Chemistry and Analytical Sciences, The Open University, Milton Keynes MK7 6AA, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, inhibitory activity and mode of action of oxazolidine-2,4-diones against porcine pancreatic
elastase, here used as a model for human neutrophil elastase, are reported. The nature of N-substitution
at the oxazolidine-2,4-dione scaffold has large effect on the inhibitory potency against elastase. N-Acyl
and N-sulfonyloxazolidine-2,4-diones emerged as potent pseudo-irreversible inhibitors, displaying high
second-order rate constants for PPE inactivation. The title compounds were also shown to be potent
inhibitors of human neutrophil elastase (HNE) and proteinase-3, and weak inhibitors of human cathepsin
G. The results herein presented show that the oxazolidine-2,4-diones represent a new promising class of
serine protease inhibitors.
Received 23 September 2011
Revised 19 April 2012
Accepted 20 April 2012
Available online 28 April 2012
Keywords:
Oxazolidine-2,4-diones
Porcine pancreatic elastase
Human neutrophil elastase
Pseudo-irreversible inhibitors
Serine proteases
Ó 2012 Elsevier Ltd. All rights reserved.
Human neutrophil elastase (HNE), a serine protease of the
chymotrypsin superfamily stored in the primary azurophilic gran-
ules of polymorphonuclear neutrophils, is a major etiologic factor
in chronic inflammatory diseases such as acute respiratory distress
syndrome (ARDS), chronic obstructive pulmonary disease (COPD)
and cystic fibrosis (CF).1–4 During these inflammatory processes,
neutrophils release an excess of HNE to the extracellular medium
where, in absence of appropriated levels of endogenous inhibitors,
can hydrolyze a variety of matrix proteins such as elastin and
collagen.5,6 Hence, HNE inhibition has been recognized as a valid
therapeutic approach to protect the lungs from HNE-mediated
tissue damage but also to control over exuberant inflammatory
responses.7
a leaving group (a sulfonate for 2 and a carboxylate for 3) then
can trigger a suicide-type inactivation mechanism in the active site
of HNE.9,13,14
The rate of serine acylation is controlled by the molecular
recognition by the target enzyme and intrinsic reactivity of the
inhibitor. Improving the rate of serine acylation by increasing the
OPh
O
O
N
O
N
OTs
N
O
Bn
O
O
In our previous work, the unexpected synthesis of azetidin-2-
ones containing a oxazolidin-2,4-dione moiety, for example 1,
drew our attention to the potential of oxazolidine-2,4-diones as
elastase inhibitors.8 Structurally, oxazolidine-2,4-diones are isos-
teric to succinimide, 2, and 1,2,5-thiadiazolin-3-one 1,1-dioxide,
3, derivatives, which are two well-known classes of HNE inhibi-
tors.9–12 The first step in the mechanism of HNE inhibition by 2
and 3 involves nucleophilic attack of the active site serine residue
(Ser-195) to the carbonyl carbon atom at the five-membered ring
to generate a tetrahedral intermediate that collapses via ring-
opening to give a relatively stable acyl enzyme. The departure of
1
2
R2
O
O
O
O
R1
O
Cl
N
R3
N
N
S
Cl
O
O
O
3
4
R1, R2 = H, Me, Ph
R3 = Ar, COAr, SO2Ar
intrinsic chemical reactivity of acylating agents such as b- and
-lactams has been a successful strategy to design more potent
elastase inhibitors.15–17 Usually, this has been performed by mod-
c
⇑
Corresponding author. Tel.: +351 21 7946477; fax: +351 21 7946470.
E-mail address: rmoreira@ff.ul.pt (R. Moreira).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.093

3994
A. B. Santana et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3993–3997
R2
R1
OH
R1
O
(i)
R2
O
O
RO2C
N
R3
5a, R = Et, R1 = Me, R2 = H
5b, R = Me, R1 = R2 = Me
4a, R1 = Me, R2 = H, R3 = CH2Ph
, R1 = Me, R2 = H, R3 = C6H4-4-OMe
, R1 = Me, R2 = H, R3 = C6H4-4-NO2
4b
4c
4d, R1 = R2 = Me, R3 = CH2Ph
4e, R1 = R2 = Me, R3 = C6H4-4-OMe
4f, R1 = R2 = Me, R3 = C6H4-4-NO2
R2
R1
OH
O
(ii)
R2
Figure 1. Time-dependent loss of enzymatic activity. Porcine pancreatic elastase
(5 M) was incubated with and various amounts of 4g (50–500 M) in 0.1 M HEPES
buffer, pH 7.2. Aliquots were removed periodically and assayed for enzymatic
O
O
MeO2C
N
R1
l
l
R3
activity using N-Suc-(L-Ala)3-p-nitroanilide.
6a, R1 = Me, R2 = H
6b, R1 = Ph, R2 = H
7a, R1 = Me, R2 = R3 = H
7b, R1 = Ph, R2 = R3 = H
7c, R1 = R2 = Me, R3 = H
Table 1
(iii) or (iv)
Half-lives in pH 7.4 phosphate buffer at 37 °C, second-order rate constant for
inactivation, kobs/[I], second-order rate constant for the alkaline hydrolysis kOHꢀ, and
enzyme-rate-enhancement factor for oxazolidine-2,4-diones 4a–j
4g, R1 = R2 = Me, R3 = COC6H4-4-OMe
4h, R1 = R2 = Me, R3 = SO2C6H4-4-Me
4i, R1 = Me, R2 = H, R3 = COC6H4-4-OMe
a
, R1 = Ph, R2 = H, R3 = COC6H4-4-OMe
Compound
t
(min)
kobs/[I]/Mꢀ1sꢀ1
kb ꢀ/Mꢀ1sꢀ1
EREFc
½
OH
4j
4a
4b
4c
4d
4e
4f
4g
4h
4i
NDd
ND
38.7
ND
ND
ND
16.7
6.86
ND
NIe
NI
NI
3.02
NI
NI
467
255
133
1320
ND
2.14
ND
ND
ND
—
—
—
—
—
—
53
3
Scheme 1. Synthesis of oxazolidine-2,4-diones 4a–j. Reagents and conditions: (i)
R3NCO, DCM or THF, TEA, reflux; (ii) urea, MeONa/MeOH; (iii) 4g, 4i and 4j: R3COCl,
THF, TEA, reflux; (iv) 4h: R3SO2Cl, Na, THF, reflux.
ND
8.75
92.2
ND
ulating the electrophilic nature of the acyl center and the leaving
group ability of the group that results from the collapse of the tet-
rahedral intermediate and lactam ring-opening.
—
—
4j
ND
ND
To assess the potential of oxazolidine-2,4-diones as elastase
inhibitors, we synthesized derivatives, 4, bearing N-aryl-, N-benzyl,
N-acyl- and N-sulfonyl moieties, which were selected to convey
intrinsic chemical reactivity required for a fast acylation of the
Ser-195. In this study, compounds 4 were initially tested in vitro
towards porcine pancreatic elastase (PPE), a model enzyme that
shares 40% of homology and the catalytic triad with HNE.4 The
enzymatic recognition by S1 subsite may be achieved by append-
ing methyl groups to C-5 of the oxazolidine-2,4-dione scaffold,
as PPE and HNE prefer small lipophilic groups for molecular
recognition.18,19
a
b
c
pH 7.4 phosphate buffer, T = 37 °C.
Borate buffer containing 20% (v/v) of acetonitrile, T = 25 °C.
Enzyme-rate-enhancement factor, EREF = kobs/kOHꢀ[I].
ND, not determined.
d
e
NI, no inhibition.
Oxazolidine-2,4-diones 4a–j were synthesized as illustrated in
Scheme 1. The reflux of 2-hydroxypropanoates 5 with the appro-
priate isocyanate in anhydrous DCM or THF and in the presence
of triethylamine afforded directly the N-benzyl- and N-aryl-oxazol-
idine-2,4-diones 4a–f. N-Acyl- and N-sulfonyloxazolidine-diones
4g–j were prepared starting from 2-hydroxy esters 6, which were
converted to the oxazolidine-2,4-diones 7 by reaction with urea
and sodium methoxide. Reaction of 7 with 4-methoxybenzoyl
chloride or 4-methylbenzene-sulfonyl chloride in anhydrous
THF containing triethylamine or sodium gave the N-acyl- and
N-sulfonyloxazolidinediones 4g–j.
The inhibitory activity of compounds 4a–j towards PPE was
determined using Kitz and Wilson’s pre-incubation method.20 No
inhibition of the enzymatic activity was observed in the incubation
of 600-fold molar excess of 4a or 4b, and 300-fold molar excess of
4c, 4e and 4f with PPE. In contrast, 4d and 4g–j inhibited PPE in a
time- and concentration-dependent manner. For example, expo-
sure of PPE to 100-fold molar excess of compounds 4g or 4h re-
sulted in a very rapid loss of enzymatic activity which reached
ca. 96% and 92% of inhibition, respectively. However, the enzymatic
activity was almost completely recovered within 72 min when the
Figure 2. Plots of first-order rate constants, kobs, for the inactivation of PPE versus
the concentration of inhibitors 4g (d) and 4h (s).
inhibition was carried out by 4h, suggesting that an instable acyl–
enzyme complex was formed, whereas no recovery of activity was
observed after 61 min of PPE inhibition by 4g (Fig. 1). The inhibi-
tory potency of compounds 4d and 4g–j against PPE was expressed
as kobs/[I] (second-order rate constant for inactivation) and is listed
in Table 1. These values were determined from the plots of first-or-

A. B. Santana et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3993–3997
3995
der inactivation rate constant, kobs versus [I] (Fig. 2). Since the inac-
tivation of PPE by 4g–j was extremely rapid with less than 20% of
the residual enzymatic activity being observed at the first time
points at higher [I]/[E] (Fig. 1), the calculated inactivation rate con-
stants should be considered to be lower limits.21,22 Alternate kinet-
ics methods such as the progress curve method could not be
applied due to the instability of the acyl–enzyme complex. Inspec-
tion of the data present in Table 1 shows that:
scaffold would be expected to have a large effect on rate-limiting
C–N fission (either at the C4–N or C2–N bond) but a much smaller
one if nucleophilic attack on the carbonyl carbon and formation of
the tetrahedral intermediate is the rate-limiting step. Inspection of
Table 1 indicates that the N-sulfonyl derivative 4h is ca. 40 times
more reactive that its N-aryl counterpart 4b. Taking in account
the large difference of ca. 10 pKa units between anilide and N-acyl-
sulfonamide leaving groups, the relative rate between 4b and 4h
can be considered small and suggests that the rate-limiting step
is the hydroxide-ion attack and formation of the tetrahedral
intermediate.
The half-lives for the hydrolysis of 4g and 4h in pH 7.4 phos-
phate buffer are 17 and 7 min, respectively (Table 1). The higher
reactivity of the N-sulfonyl derivative 4h compared to its N-acyl
counterpart 4g is in line with the reactivity pattern observed for
the alkaline hydrolysis of these inhibitors, and indicates that the
N-acyloxazolidinedione scaffold presents adequate chemical sta-
bility for further development.
N-Acyloxazolidinedione 4g can undergo nucleophilic attack by
OHꢀ at endocyclic and exocyclic carbonyl carbon atoms. In order
to determine the preferred pathway, we investigated the reaction
of 4g in the presence of sodium methoxide (generated in situ) in
methanol at room temperature. TLC analysis of the reaction mix-
ture showed the formation of two major products, which were iso-
lated and identified as methyl 4-methoxybenzoate and 824
(Scheme 2). Monitoring the reaction by 1H NMR in a CD3ONa/
CD3OD solution, revealed that consumption of 4g was complete
in 15 min, with formation of a 1:1 mixture of 8 and 9 (Supplemen-
tary data). In an independent experiment, compound 8 was shown
to be stable in the time-scale of the methanolysis, thus indicating
that benzoate 9 arises exclusively from the reaction of methoxide
at the exocyclic carbonyl group of 4g. Overall, these results indicate
that reaction of 4g with nucleophiles such as hydroxide and meth-
oxide occurs approximately at the same rate at the lactam (C-4)
and exocyclic carbonyl carbon atoms.
It has been suggested that the effectiveness of an enzyme in
using its catalytic apparatus in an acylation reaction can be indi-
cated by the rate enhancement factor, EREF, which is evaluated
by dividing the second-order rate constant for the enzyme cata-
lyzed reaction, kobs/[I], by that for hydrolysis of the same substrate
catalyzed by hydroxide ion, kOHꢀ.16,23 Inhibitor 4g exhibits an EREF
value ca. 20 higher than that of its N-sulfonyl counterpart 4h (Table
1), suggesting that 4g may establish better interactions with PPE
binding pockets. These interactions probably position one of the
carbonyl carbon atoms within the oxyanion hole, facilitating suc-
cessful nucleophilic attack by Ser-195.
1. N-Acyloxazolidinedione 4j, containing a phenyl group at C-5,
was nearly 2- and 10 times more potent than its 5,5-dimethyl
and 5-methyl counterparts, 4g and 4i, respectively.
2. N-Acyloxazolidinedione 4g, was ca. 2- and 155 times more
effective than its N-sulfonyl and N-benzyl counterparts, 4h and
4d, respectively, in the inactivation of PPE.
3. Both 5-methyl- and 5,5-dimethyl-N-aryloxazolidinediones (i.e.,
4b,c and 4e,f) were inactive against PPE; in contrast, 3,3-dimethyl
N-benzyloxazolidinedione 4d inhibited PPE, while its 5-methyl
counterpart, 4a, was inactive.
These results indicate that lipophilic substituents are preferred
at C-5 of the oxazolidine-2,4-dione moiety, while N-acyl substitu-
tion leads to the highest rates of PPE inactivation. Further evidence
of the efficiency of N-acyloxazolidinediones as inactivators of PPE
comes from the titration of enzyme activity with 4g. The number
of equivalents required to inactivate PPE was calculated by plotting
the fraction of remaining enzyme activity, vt/v0, after a 15 min
incubation period versus the initial ratio of inhibitor to enzyme,
that is [I]/[E]0. For levels of inhibition up to 70% of the original en-
zyme activity, the extent of inactivation was found to depend lin-
early on the inhibitor to enzyme molar ratio (Fig. 3), and
extrapolation of the line to vt/v0 = 0 shows that it required approx-
imately 9.3 equiv of 4g to completely inactivate PPE. This inhibi-
tory efficiency is of the same order of magnitude to that reported
for PPE inactivation by 3,3-diethyl monobactams.23
The second-order rate constant, kOHꢀ, for the alkaline hydroly-
sis of the oxazolidinediones 4 were also determined for compari-
son with those for PPE inhibition (Table 1). It has been suggested
that the magnitude of the second-order rate-constant, kOHꢀ, for
the alkaline hydrolysis of potential inhibitors of enzymes contain-
ing a catalytic serine is a crude indicator for their ability to be
effective and therapeutically useful acylating agents, as it gives
an insight to their acylating power as well as to the hydrolytic
and metabolic stability.15 N-Substitution at the oxazolidinedione
In order to further evaluate the potential of oxazolidine-2,4-
dione scaffold as serine protease inhibitors, we screened
compounds 4a–j against the three neutral serine proteases of poly-
morphonuclear neutrophils: HNE, cathepsin G, and proteinase-3
(PR3).4 The resulting IC50 values are presented in Table 2. Inspec-
tion of the activity data shows that most of the compounds that
inhibited PPE (4d, 4g–h and 4j; Table 1) were also inhibitors of
HNE, with the N-sulfonyloxazolidinedione 4h being the most po-
tent with an IC50 value of 2.6 lM. The exception was 5-methyl-
oxazolidine-2,4-dione, 4i, which was inactive against HNE. This
result is consistent with HNE’s preference for larger or branched
amino acids at position P1 of substrates, when compared with
PPE.18,19 Another important observation is that compounds 4d,
4g–h and 4j also inhibit proteinase-3, and to a lesser extent,
cathepsin G. The most selective inhibitor in this series was com-
pound 4h, with IC50CatG/IC50HNE and IC50PR3/IC50HNE ratios of
140 and 6, respectively. Interestingly, its N-acyl counterpart, 4g,
was the most potent inhibitor against PR3, with an IC50 value of
Figure 3. Inactivation of PPE as a function of the molar ratio of inhibitor 4g
to enzyme. PPE (5 lM) and various amounts of 4g (2.5–500 lM) in pH 7.25
HEPES buffer, were incubated for 15 min, aliquots were withdrawn at the end of
the incubation period and assayed for remaining enzyme activity (vt/v0) using
1.4
lM. Dual inhibition of HLE and PR3 is not surprising as it re-
N-Suc-(L-Ala)3-p-nitroanilide.
flects the close similarity of the primary specificity sites of both en-

3996
A. B. Santana et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3993–3997
O
CO2Me
O
MeO2C
OMe
N
H
N
O
MeONa
MeOH
O
O
O
O
OMe
OMe
4g
8
9
Scheme 2. Products isolated from the methanolysis of 4g.
PPE.26 Each compound was constructed and its energy was mini-
mized using Molecular Operating Environment (MOE 2009.10)
software and MMFF94x forcefield.27 The coordinates of enzyme
structures were obtained from Protein Data Bank selecting the
structures with accession codes 1HNE (X-ray coordinates at
1.84 Å resolution) and 1BTU (X-ray coordinates at 1.6 Å resolution)
for HNE and PPE, respectively. Compounds were docked within the
active site and the conformations were evaluated with Goldscore
fitness function. The top 20 solutions, that is, those showing the
highest score, were visually inspected for the hydrophobic interac-
tions between the ligands and the residues defining the S1 pocket
Table 2
IC50 values for the inhibition of human neutrophile elastase (HNE), cathepsin G (Cat
G) and proteinase-3 (PR3) by oxazolidine-2,4-diones 4a–j
Compound
IC50 (lM)
HNE
Cat G
PR3
4a
4b
4c
4d
4e
4f
4g
4h
4i
>500
>500
>500
15.6 0.9
>500
NDa
ND
ND
2.1 1.0
ND
ND
ND
ND
12.8 1.6
ND
>500
ND
14.2 1.1
ND
10.2 2.1
2.6 0.2
>500
1.4 0.8
14.5 1.6
ND
and the distance between the O
c oxygen atom Ser-195 and oxa-
369
ND
1
zolidine-2-4-dione carbonyl carbon atoms.
4j
15.6 0.4
100
2
5.0 1.1
Modeling the interaction of 4g and 4h with HNE revealed that
all top ranking conformations for both inhibitors present the two
methyl substituents at C-5 sitting in the S1 primary recognition site
of the enzyme (Fig. 4A and B). However, the interaction of N-sul-
fonyloxazolidinedione 4h with HNE presents the lactam (C-4) car-
bonyl carbon atom as slightly more accessible to the Ser-195
a
ND, not determined.
zymes.4 As both of these neutrophil serine proteases are involved
in inflammation and extracellular matrix destruction observed in
pulmonary diseases, a dual inhibition of HNE and PR3, may be an
advantage for therapeutics.
hydroxyl O
c oxygen when compared with its N-acyl counterpart,
In silico ligand docking studies using GOLD 5.0 software25 were
performed to get some insight into the binding mode of oxazoli-
dine-2-4-diones 4g, 4h and 4j in the active sites of HNE and
4g (2.4 vs 3.0 Å). This result is consistent with the observation that
4h inhibits HNE nearly 4 times more efficiently than 4g (Table 2).
Interestingly, 4g interact with HNE in a manner that seems to force
Figure 4. Docking poses of 4g (A,C) and 4h (B,D) in the active site of HNE and PPE, respectively. Pictures were prepared using MOE 2009.10.

A. B. Santana et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3993–3997
3997
the oxazolidinedione ring deep inside the active site, orienting the
lactam (C-4) carbonyl carbon atom closer to Ser-195 hydroxyl oxy-
gen, when compared to the exocyclic carbonyl (3.0 vs 4.2 Å). This is
gratefully acknowledges financial support from FCT with a post-
doctoral fellowship (SFRH/BPD/64265/2009).
due, in part, to enhanced p–p stacking between the aromatic moi-
Supplementary data
ety of 4g and His-57 (Fig. 4A). Finally, docking 4j with HNE re-
vealed that the C-5 phenyl group sits inside the S1 primary
recognition site stabilized by enhanced van der Waals contacts
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.04.
093.
with Val-190, Phe-192 and Ala-213, and
the 4-methoxybenzoyl moiety and His-57. The distance between
the carbonyl carbon atom C-4 and the O atom of Ser-195 is
p–p stacking between
c
References and notes
3.7 Å, consistent with the lower potency of 4j when compared to
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4g and 4i.
When 4h was docked close to the PPE active site, an inverted
binding mode was observed, with the ligand sitting along the S1
0
4. Korkmaz, B.; Moreau, T.; Gauthier, F. Biochimie 2008, 90, 227.
5. Chua, F.; Laurent, G. J. Proc. Am. Thorac. Soc. 2006, 3, 424.
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Am. J. Physiol. 1998, 275, H385.
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pocket pocket, that is, with the oxazolidine-2,4-dione carbonyl
carbon atoms lying at a much longer distance (4.1 and 5.3 Å for
C-2 and C-4, respectively) from the catalytic serine. Hence, in an
attempt to simulate a complex where the ligand was closer to the
active site of PPE, a constraint was applied so that distance from
carbonyl carbon atom C-4 to O
c atom of Ser-195 would be lower
than 4.0 Å. The resulting best pose obtained is depicted in Figure
4D and reveals an inverted binding mode, in which the aromatic
moiety lies on the S1 pocket, while the two methyl groups are close
0
to the S1 pocket, and with a distance between the carbonyl carbon
12. Li, Y.; Yang, Q.; Dou, D.; Alliston, K. R.; Groutas, W. C. Bioorg. Med. Chem. 2008,
16, 692.
atom C-4 and the Oc atom of Ser-195 of 3.3 Å. Examination of the
PPE–4g complex revealed that the methyl substituents at the C-5
position are well accommodated in the hydrophobic S1 pocket, ori-
enting the oxazolidine-2,4-dione lactam carbon atom close (2.5 Å)
13. Groutas, W. C.; Stanga, M. A.; Brubaker, M. J. J. Am. Chem. Soc. 1989, 111, 1931.
14. Huang, W.; Yamamoto, Y.; Li, Y.; Dou, D.; Alliston, K. R.; Hanzlik, R. P.;
Williams, T. D.; Groutas, W. C. J. Med. Chem. 2008, 51, 2003.
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16. Mulchande, J.; Guedes, R. C.; Tsang, W. Y.; Page, M. I.; Moreira, R.; Iley, J. J. Med.
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17. Mulchande, J.; Oliveira, R.; Carrasco, M.; Gouveia, L.; Guedes, R. C.; Iley, J.;
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to O
vealed a similar binding pose to that of the 4j–HNE complex, with
a distance between the carbonyl carbon atom C-4 and the O atom
c oxygen atom of Ser-195 (Fig. 4C). Docking 4j with PPE re-
c
of Ser-195 of 3.2 Å. Overall, these results are consistent with a
poorer molecular recognition of 4h by PPE, when compared to 4g
and 4j, which might be ascribed by the lower flexibility imparted
to the sulfonyl moiety.
20. Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245.
21. Harper, J. W.; Powers, J. C. Biochemistry 1985, 24, 7200.
22. Kerrigan, J. E.; Oleksyszyn, J.; Kam, C.; Selzler, J.; Powers, J. C. J. Med. Chem.
1995, 38, 544.
In summary, we report the synthesis and inhibitory activity of
oxazolidine-2,4-diones against porcine pancreatic elastase (PPE),
human neutrophil elastase (HNE), cathepsin G and proteinase-3.
N-Substitution at the oxazolidine-2,4-dione scaffold has large ef-
fect on the inhibitory potency against serine proteases, with the
N-acyl and N-sulfonyloxazolidine-2,4-dione derivatives inhibiting
HNE in the micromolar range, while their N-aryl counterparts
being inactive. Both N-acyl, 4g, and N-sulfonyloxazolidine-2,4-
dione, 4h, derivatives behave as pseudo-irreversible inhibitors of
PPE, displaying high second-order rate constants for enzyme inac-
tivation and recovery of enzyme activity. The results herein pre-
sented reveal that the oxazolidine-2,4-dione scaffold can be used
to develop novel serine protease inhibitors.
23. Mulchande, J.; Martins, L.; Moreira, R.; Archer, M.; Oliveira, T. F.; Iley, J. Org.
Biomol. Chem. 2007, 5, 2617.
24. Compound 8 was obtained as described in the Supplementary data. Oil; 1H
NMR d 1.67 (6H, s, (CH3)2CO), 3.79 (3H, s, CH3OCO), 3.89 (3H, s, CH3OAr), 6.97
(2H, d, J = 8.8, ArH), 7.83 (2H, d, J = 8.8, ArH), 8.11 (1H, brs, NH); MS-EI m/z (%)
295 (35.7, M+), 177 (36.1), 135 (100), 107 (11.4).
25. CCDC Software Ltd; Cambridge, UK. (www.ccdc.cam.ac.uk/products/
gold_suite).
26. The 3D structure coordinates of HNE and PPE were obtained from the Protein
Data Bank, and the elected structures were 1HNE with a 1.84 Å resolution and
1BTU with a 1.6 Å resolution, respectively. To prepare the enzymes for the
docking studies, the co-crystallized inhibitors as well as crystallographic
waters, included in the PDB structures, were removed. Hydrogen atoms were
added and the protonation states were correctly assigned using the Protonate-
3D tool within the Molecular Operating Environment (MOE) 2009.10 software
package, energy was minimized using MMFF94x forcefield. Molecular docking
studies were then performed using the GoldScore scoring function from GOLD
software package and each ligand was subjected to 1000 docking runs. For
Acknowledgements
docking compound 4h at PPE active site
a
constraint distance between
carbonyl carbon atom C- 4 and the O
added.
27. MOE, Chemical Computing Group Inc.: Montreal, 2010 (www.chemcomp.com).
c atom of Ser-195 of maximum 4.0 Å was
This work was supported by Fundação para a Ciência e Tecnolo-
gia (FCT, Portugal) through project PTDC/QUI/64056/2006. S.D.L.