In vitro evaluation of the antielastase activity of polycyclic β-lactams

Article abstract of DOI:10.1016/j.bioorg.2012.08.001

A series of bi- and tricyclic β-lactam compounds was synthesized and evaluated as inhibitors of cleavage of synthetic substrates in vitro by the serine proteases Human Leukocyte Elastase (HLE), Human Leukocyte Proteinase 3 (HLPR3) and Porcine Pancreatic Elastase (PPE). The obtained results have permitted us to describe a homobenzocarbacephem compound as HLE and HLPR3 inhibitor, to observe the positive effect that the styryl group exerts on the HLE inhibitory activity in polycyclic β-lactam compounds and to conclude that the hydroxyl function decreases the HLE inhibitory activity or rules it out completely.

Full text of DOI:10.1016/j.bioorg.2012.08.001

Bioorganic Chemistry 45 (2012) 29–35
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
In vitro evaluation of the antielastase activity of polycyclic b-lactams
a
Laura M. Monleón a, Fernando Díez-García b, Héctor Zamora b, Josefa Anaya a, , Manuel Grande ,
⇑
Juana G. de Diego b, F. David Rodríguez b
a Departamento de Química Orgánica, Facultad de Ciencias Químicas, Universidad de Salamanca, Plaza de los Caídos s/n, E-37008 Salamanca, Spain
b Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Salamanca, C/Donantes de sangre s/n, E-37007 Salamanca, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 May 2012
Available online 23 August 2012
A series of bi- and tricyclic b-lactam compounds was synthesized and evaluated as inhibitors of cleavage
of synthetic substrates in vitro by the serine proteases Human Leukocyte Elastase (HLE), Human Leuko-
cyte Proteinase 3 (HLPR3) and Porcine Pancreatic Elastase (PPE). The obtained results have permitted us
to describe a homobenzocarbacephem compound as HLE and HLPR3 inhibitor, to observe the positive
effect that the styryl group exerts on the HLE inhibitory activity in polycyclic b-lactam compounds and
to conclude that the hydroxyl function decreases the HLE inhibitory activity or rules it out completely.
Ó 2012 Elsevier Inc. All rights reserved.
Keywords:
Epoxybenzonitrile
Polycyclic b-lactam
Homobenzocarbacephem
Protease
Inflammation
Elastase inhibitory activity
1. Introduction
natural molecules showing antiprotease activity. However, the action
of theseinhibitors isalsocontrolled andcounteracted bymechanisms
The HLE (EC 3.4.21.37) [1], also called neutrophil elastase
(HNE), polymorphonuclear leukocyte elastase or granulocyte elas-
tase, is a 29 kDa glycoprotein with serine peptidase activity [2]. The
HLE, together with other neutrophil serine peptidases, including
HLPR3, hydrolyses the contents of phagolysosomes, degrades
extracellular matrix proteins and regulates inflammation [3]. As a
consequence of deregulation in enzyme synthesis or alteration of
its physiological inhibition, the protease/antiprotease equilibrium
is disrupted and the enzyme may exert its proteolytic activity on
non-physiological targets.
The HLE hydrolytic activity is very potent and it is capable of
degrading several substrates, including elastin, collagen or fibrin.
The consequences of its exacerbated activity can be devastating
and are associated to serious pathologies [4], such as, pulmonary
emphysema [5], rheumatoid arthritis [6], psoriasis, acute respira-
tory distress syndrome, cystic fibrosis, and tumor progression [7].
In an analogous way, pancreatitis [8] can be provoked by pancre-
atic tissue digestion when the inactive form of HPE, Human Pan-
creatic Elastase (zymogen), becomes pathologically activated
inside the pancreas instead of in the intestine.
related to the formation of the enzyme-substrate complex and to the
oxidation state of the environment [10]. A number of synthetic inhib-
itors of HLE has been produced over the last years and applied, for in-
stance, in specific lung pathologies [11]. Various compounds have
been used as the starting point for the synthesis of specific proteases
inhibitors, such as acyl compounds, pyrrolpyrrolones, coumarins,
azapeptides, chloromethylketones, trifluoromethylketones, and sul-
phonil-fluorides. b-Lactam compounds, traditionally used as anti-
bacterial agents, have also been tested as inhibitors of mammalian
serine proteases, such as HLE [12], thrombin [13], prostate specific
antigen (PSA) [14], human cytomegalovirus (HCMV) protease [15],
and human chymase [16].
The abilities of mono- and polycyclic b-lactams to inhibit
serin-enzymes lie mainly in the b-lactam ring. Its action consists
of kidnapping the Ser residue at the active site of the enzyme by
a nucleophilic attack that opens the b-lactam ring (Fig. 1) and
produces a stable acyl-enzyme covalent complex that turns the
enzyme inactive [17].
b-Lactams could be classified as mechanism-based inhibitors of
HLE which could promote irreversible inhibition via a suicide-type
mechanism by the presence of a potential leaving group in the
inhibitor structure. Thus, they could represent a good model base
for designing powerful drugs able to inhibit HLE and restore the al-
tered protease/antiprotease ratio at the inflammatory sites. There
are a lot of examples in the literature of elastase inhibitors with
a b-lactam skeleton [18], some of which have been proofed to be
orally active [19].
Thus, HLE has been the object of extensive research to develop
potent inhibitors [9] that target its destructive and pro-inflammatory
action by restoring the protease/antiprotease imbalance. Alpha-1-
antitrypsin and the secretory leukocyte protease inhibitor are some
⇑
Corresponding author. Fax: +34 923 294 574.
E-mail address: janay@usal.es (J. Anaya).
0045-2068/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2012.08.001

30
L.M. Monleón et al. / Bioorganic Chemistry 45 (2012) 29–35
OH
H H
OH
H H
MeO
Ph
MeO
Ph
5
5
N
HLE
6
4
3
6
4
7
7
H
8
8
N
3
2
O
H
2
N
N
O
O
O
1
1
O
O
Ser
H Sg
H Sg
CO2Me
CO2Me
Ser
1
3
5
7
(4β,5α,6α,7α)
(4α,5β,6α,7α)
(4β,5α,6β,7β)
(4α,5β,6β,7β)
2
4
6
8
(4β,5α,6α,7α)
(4α,5β,6α,7α)
(4β,5α,6β,7β)
(4α,5β,6β,7β)
22a
Fig. 1. Proposed mechanism for the inhibition of HLE by b-lactam compounds.
22a
HO
H H
Our research group, with a vast experience in the synthesis of b-
lactam compounds [20], has evaluated a series of monolactams
against the enzymes Human Leukocyte Elastase (HLE), Porcine
Pancreatic Elastase (PPE) and Rat Leukocyte Elastase (RLE) [21].
The obtained results revealed that only the monolactams A
(Fig. 2) bearing the styryl and moreover the methylstyryl groups
at the C4 position of the b-lactam ring and a D-glucosamine derived
moiety (Sg) at the N-substituent, showed specific HLE inhibitory
activity.
In this work, we analyse as lead inhibitors of HLE the polycyclic
b-lactams 1–18 (Fig. 3) which have been obtained by cyclisation
reactions of benzyl [22] and homobenzyl [23] radicals generated
from epoxyolefin-, and epoxynitrile-2-azetidinones with titano-
cene monochloride (Cp2TiCl) [24].
Ph
MeO
4
5
6
3
7
2
N
1
CO2Me
H H
O
Sg
H
9
H H
N
MeO
MeO
Ph
O
MeO
O
O
N
O
O
1022b
1123
H H
H H
O
MeO
N
N
O
O
2. Results and discussion
1223
1323
2.1. Chemistry
OH
OH
Ph
Ph
Ph
H H
H H
H H
As it has been above reported, the synthesis of the target mole-
cules 1–16 is based in a radical cyclisation induced by Cp2TiCl in
the epoxy-monolactams 19–25 (Scheme 1). These compounds
were prepared by standard Staudinger reactions between
methoxyacetyl chloride in the presence of TEA and the imines
obtained by condensation of the amines I [21b], 2-amino-2,2-
dimethyletanol and o-cyanoaniline with cinnamaldehyde or
methyl-cinnamaldehyde. Several chemical transformations on the
formed 2-azetidinones yielded the unsaturated epoxy-esters 19,
the epoxy-nitriles 20 and 21 and the cis/trans epoxy-benzonitriles
22–25. Finally, the treatment of the epoxides 19–25 with Cp2TiCl
afforded the bi- and tricyclic b-lactams 1–16.
The reported studies on the radical cyclisation reactions in-
duced by Cp2TiCl in epoxy-esters 19 [22b] were carried out by in-
verse addition of the reagents; that is, by addition of a solution of
the epoxide in THF to a green suspension of Cp2TiCl in THF. Under
these conditions, only the isomers 19ab and 19ba afforded the car-
bacephams 3 and 6, respectively. In this work, we report the phys-
ical properties of the carbacephams 1–8 and the carbapenam 9,
which were obtained by treatment of the four optically pure iso-
mers 19 with Cp2TiCl by direct addition of the reagents (see exper-
imental section). The stereochemistry for these new bicyclic
compounds (Fig. 3) was rigorously established by IR and NMR
spectroscopy.
MeO
MeO
MeO
O
6
6
5
5
7
7
8 7
8
8
O
O
N
N
N
O
O
O
22b
22b
22b
22b
1422b
17 (7,8-cis)
18 (7,8-trans)
15 (5β,6α)
16 (5α,6β)
Fig. 3. Polycyclic b-lactams analyzed as candidate inhibitors of HLE.
ported for the monolactams A [21], also the trilactams 17 and 18
were obtained by dehydratation of the epoxy-benzonitriles 14–
16 with p-Me-C6H4SO2Cl.
2.2. Inhibition of Elastases
In vitro analysis of the antielastase activity [25] of the com-
pounds 1–18 was carried out by a spectrophotometric microassay
based on the use of multi-well plates (see experimental section for
details). With this procedure several compounds can simulta-
neously be screened by recording absorbances over defined peri-
ods of time with a Multiscan spectrophotometer connected to a
computer that records all data. Another advantage is the use of
small reaction volumes, with significant saving of reactants. Kinetic
and dose-response experiments were done.
In order to evaluate if the role the styryl group plays on the
antielastase activity of the polyciclic b-lactams is similar to that re-
In a preliminary screening, all compounds shown in Fig. 3, ex-
cept 7, 15 and 16, were evaluated at a 50 lM concentration. Se-
R2
H H
lected compounds showing more than 25% inhibition of
enzymatic activity were further assayed and the IC50 values (half
maximal inhibitory concentration) determined (Table 1).
R1
O
Ph
S
3
4
N
Asshown in Table 1, only the trilactams17 and 18 exhibitedhigh-
er than 25% HLE-inhibition activities. All the studied bilactams, ex-
cept 10 and 11, bear a hydroxyl group in the second ring of their
structures. Since none of them has a significant HLE inhibitory activ-
ity, we guess that the hydroxyl function decreases the anti-HLE
activity. This conclusion was supported by the inhibition displayed
by the tricyclic b-lactam 14, the hydroxylated precursor of 17.
H
S
O
O
O
O
= Sg
A
Fig. 2. Monocyclic b-lactams with specific HLE inhibitory activity [21].

L.M. Monleón et al. / Bioorganic Chemistry 45 (2012) 29–35
31
CH(SEt)2
H
O
H H
O
H2N
MeO
Ph
5
1 + 2 / 3 + 4 (+ 9)
5 + 6 / 7 + 8
g
a, b, c, d
3
4
N
CO2Me
O
O
O
O
H
Sg
= Sg
19aa/19ab (3α,4α,5α-epoxy/3α,4α,5β-epoxy)
19ba/19bb (3β,4β,5α-epoxy/3β,4β,5β-epoxy)
I
Me
H H
O
H H
O
MeO
Ph MeO
CN
Ph
CN
5
5
g
a, e, b, f, d
H2N
3
4
3 4
N
10
11
OH
CN
N
O
O
20a
21a,b
na = 5α-epoxy
nb = 5β-epoxy
Me
H H
O
H H
O
NH2
MeO
Ph
CN
MeO
Ph
CN
5
5
a, b, d
14
15,16
12
13
3
4
N
g
3
4
N
O
O
h
22b (3,4-cis
)
24a (3,4-cis
)
17
18
23a (3,4-trans
)
25a,b (3,4-trans
)
Scheme 1. Reagents and conditions: (a) cinnamaldehyde, for 19, 20, 24 and 25 or trans-a-methyl-cinnamaldehyde, for 21–23; CH2Cl2 (toluene for 19), rt. (b)
methoxyacetylchloride, TEA, CH2Cl2, rt. (c) 1: PhI(CF3CO2)2/NaHCO3, 9:1 CH3CN/H2O, rt. 2: Ph3P@CHACO2Me, THF, rt. (d) m-CPBA, NaHCO3, CH2Cl2, rt. (e) tBu(Me)2SiCl, pyr/
DMAP, rt. (f) 1: HCl (0.1 M), MeOH, rt. 2: (COCl)2, DMSO, CH2Cl2, ꢀ78 °C, then TEA. 3: Me2N-NH2, MeOH, rt; then MMPP, 0 °C. (g) 1: Cp2TiCl, THF, rt; 2: KH2PO4 aq. (h) p-Me-
C6H4SO2Cl, TEA, DMAP, CH2Cl2, rt.
Table 1
Percentage inhibition of HLE for compounds 1–18 at 50
lM. IC50
(l
M) measurements and Ki (
l
M) values for 17 and 18.a
Comp. no.
% Inhibition at 50
lM
Comp. no.
% Inhibition at 50
lM
IC50 M) Ki ( M)
(
l
l
1
2
3
4
5
6
8
9
13
10
8
9
1
5
1
12
10
11
12
13
14
17
18
0
14
16
9
8
97
98
21.0 1.2
22.1 2.6
12.6 0.7
13.4 1.6
a
IC50 and Ki are expressed as mean SEM (standard error of the mean).
The trilactam 14, with a hydroxyl group in its structure, shows
only an 8% inhibition of the enzymatic activity. However, the enon-
es 17 and 18, with a styryl moiety in their structures, resulted to be
Compounds 17 and 18, which showed higher than 25% HLE-
inhibition values, were tested at lower concentration (1, 5, 10
and 20
lM) in order to determine the IC50 parameters shown in
the most active compounds at a concentration of 50
l
M, showing
Table 1 (21.0 1.2
l
M, for 17; 22.1 2.6 M, for 18). According
l
97% and 98% inhibition of the enzymatic activity, respectively.
On the contrary, their benzocarbacephem homologues, compounds
12 and 13, gave lower values of inhibition, probably due to the ab-
sence of a double bond in the second ring [26].
Although the styryl group seems to be responsible for the great-
er inhibitory activity shown by the tricyclic systems 17 and 18,
bilactam 10 showed no inhibition at all, being even less active than
the carbacephams 1–8, which are influenced by the negative effect
of the hydroxyl groups present in their structures. Perhaps the
gem-dimethyl moiety that the carbacepham 10 bears at the C2 po-
sition has a bad influence on the anti-elastase activity in this kind
of bilactams, overruling the positive effect of the styryl group. It
was also evident that the positive effect of the D-glucosamine de-
rived moiety, Sg, is apparently stronger than the negative influence
of the hydroxyl group.
The positive effect exerted by the styryl group could be due to
the fact that the phenyl ring may stabilize the enzyme-inhibitor
complex previous to the b-lactam ring opening because this phenyl
group could form hydrophobic bonds with the residues at the
oxyanion hole of the enzyme, which is responsible for the charges
stabilization during the enzymatic catalysis process.
to these results, the relative cis/trans stereochemistry of the
b-lactam ring seems to have no effect on the HLE inhibitory activity
of these tricyclic compounds.
The inhibitory effect of compounds 17 and 18 against HLPR3
was tested under the same experimental conditions used for HLE
in the presence of the same substrate concentration (100 lM
methoxysuccinyl–Ala–Ala–Pro–Val–p-nitroanilide). Both com-
pounds significantly inhibited HLPR3, rendering the following
IC50 values: 16.2 3.9
trilactam 18. Ki values were 18.3 5.2
27.9 11.2 M for compound 18. The measurements (expressed
l
M for trilactam 17 and 24.6 4.9
lM for
l
M for compound 17 and
l
as mean SEM) are similar to the ones obtained for HLE (Table 1).
Therefore, compounds 17 and 18 are mutual inhibitors of the
tested neutrophil serine proteases.
Both trilactams 17 and 18 were used to undertake the specific-
ity study with the enzymes HLE and Porcine Pancreatic Elastase
(PPE). The assays with PPE were carried out under the same exper-
imental conditions as the previous HLE–inhibition activity assays,
except the buffer pH value, that in this case was pH = 8.0. The
obtained results were 35% and 70% of enzyme inhibition at
50 lM for 17 and 18, respectively.

32
L.M. Monleón et al. / Bioorganic Chemistry 45 (2012) 29–35
Fig. 4. Lower energy conformers of the tricyclic b-lactams 17 and 18.
These latter values, as compared with those obtained against
HLE (Table 1), indicate that compound 18 at 50 M shows high
inhibition values for both elastases, whereas compound 17 pre-
sents lower inhibition of PPE at the same concentration. These re-
sults seem to point out that the cis-trilactam 17 can be a selective
inhibitor of the HLE (compared to PPE). Furthermore, the IC50 value
silica gel plates (Merck F-254). Mass spectra (MS) were recorded
l
on an Applied Biosystems QSTAR XL (HRMS, 5 kV). IR spectra were
recorded as neat film on a Nicolet IR-100 instrument. 1H and 13C
NMR spectra were obtained on Bruker WP200SY and Bruker
Avance 400DRX instruments (200 and 400 MHz respectively) in
CDCl3 solutions with tetramethylsilane as internal standard. Sol-
vents and reagents were purified according to standard techniques.
of compound 17 in the presence of PPE was 170
lM (range: 140–
201 M), almost nine times higher than the IC50 calculated for HLE.
l
The low toxicity of b-lactam compounds makes homobenzocar-
bacephem 17 an attractive HLE inhibitor despite the obtained IC50
for compound 17 against HLE is much higher than those exhibited
by the natural inhibitors studied under the same experimental
conditions [10].
4.1.2. Preparation of epoxy-monolactams 19–25
Epoxy-monolactams 19–25 were prepared according to the
protocols reported in references 22 (esters 19, 22a; nitriles 20, 24
and 25, 22b) and 23 (nitriles 21–23).
The observed specific effect of compound 17 against the HLE
(compared to PPE) is probably due to minor differences in the res-
idues of both the catalytic active site environment in the enzymes
HLE and PPE and the oxyanion hole [27], which therein helps mol-
ecule adaptation and charge stabilization. These slight differences
give rise to different interactions with those substituents of the se-
lected compound that are mainly involved in the enzyme-inhibitor
complex stability.
4.1.3. Preparation of bicyclic b-lactams 1–9
In situ Generation of Titanocene Monochloride (Cp2TiCl). To
548 mg (2.2 mmol) of titanocene dichloride in anhydrous and
strictly deoxygenated THF (12.5 mL, c 0.176 M), 262 mg (4.0 mmol)
of activated zinc granules were added. The resulting red mixture
was then vigorously stirred under argon atmosphere with rigorous
exclusion of oxygen until a green color was observed (about
20 min.).
On the other hand, as the HLE-inhibitory effect observed for
compound 17 (compared to PPE) appears to be related to the cis
disposition of the substituents of the 2-azetidinone ring, it seems
clear that the configuration of this ring affects the interactions
occurring at the enzyme catalytic site and its environment.
The minimized structures [28] of both isomeric compounds 17
and 18 show a very unlike spatial disposition in each case (Fig. 4).
This different spatial disposition may be responsible for the specific
HLE-inhibitory activity (compared to PPE) of compound 17, which
should fit better in the catalytic site environment of the HLE enzyme.
Radical Reactions with Cp2TiCl. A THF green suspension of Cp2-
TiCl, generated in situ from Cp2TiCl2 and Zn0 as described above,
was added drop-wise through cannula to a solution of the corre-
sponding epoxide 19 (1.0 mmol) in THF (17.0 mL, c 0.058 M). The
resulting reaction mixture was stirred until a color change from
green to orange occurred or until the disappearance of the starting
material was observed by TLC. Then the reaction was quenched
with 10% v/v aqueous KH2PO4 (30.0 mL) and the resulting aqueous
phase was extracted with ethyl acetate. The organic combined ex-
tracts were filtered through CeliteÒ, dried (anhydrous Na2SO4) and
concentrated in vacuo. The crude product obtained was purified by
column chromatography using hexanes/EtOAc mixtures as the
eluent.
3. Conclusions
Carbacephams 1 and 2: From the epoxide 19aa (262 mg,
0.51 mmol), 69 mg of 1 (26%) and 85 mg of 2 (32%) were isolated
in 3 h.
From the results reported here, we conclude that the homo-
benzocarbacephem 17 may be an interesting HLE inhibitor and
that the styryl group also exerts a positive effect on the HLE inhib-
itory activity in polycyclic b-lactam compounds as the formerly ob-
served for monolactams A (Fig. 2) [21].
1: Rf (4:6 hexanes/EtOAc) 0.26; ½a D25
ꢁ
+56 (c 1.0,CHCl3); IR (film)
t
3509, 1761, 1738, 1215, 1070, 700 cmꢀ1 1H NMR (400 MHz,
;
CDCl3) d 1.26 (3H,s), 1.34 (3H,s), 1.35 (3H,s), 1.54 (3H,s), 2.31
(1H, dd, J = 4.6 and 16.8 Hz), 2.58 (1H,dd,J = 9.0 and 16.8 Hz),
3.25–3.32 (2H,m), 3.58 (3H,s), 3.65 (3H,s), 3.90 (1H, d,
J = 8.0 Hz), 3.95–4.00 (3H,m), 4.11 (1H,t,J = 4.7 Hz), 4.14
(1H,dd,J = 5.2 and 9.2 Hz), 4.37 (1H,d,J = 7.2 Hz), 4.50
(1H,dd,J = 4.7 and 9.4 Hz), 4.76 (1H,d,J = 4.7 Hz), 7.20–7.40 (5H,
m) ppm; 13C NMR (100 MHz, CDCl3) d 25.4, 26.3, 26.8 (2C), 32.9,
34.5, 49.7, 50.6, 51.6, 53.1, 59.7, 67.8, 68.6, 77.3, 78.9, 79.1, 85.3,
109.4, 110.1, 126.8, 128.2 (2C), 129.6 (2C), 138.6, 168.6,
4. Experimental
4.1. Chemistry
4.1.1. General methods
Flash chromatographies were run on silica gel (Merck 60 230–
400 mesh) and thin layer chromatographies (TLC), on commercial

L.M. Monleón et al. / Bioorganic Chemistry 45 (2012) 29–35
33
172.6 ppm; HRMS (Q-TOF) calcd for C27H38NO9 (M+ + 1) 520.2541,
found 520.2573.
d 1.34 (3H,s), 1.36 (3H,s), 1.42 (3H,s), 1.59 (3H,s), 2.17 (1H, dd,
J = 8.1 and 15.7 Hz), 2.44 (1H, dd, J = 5.3 and 15.7 Hz), 3.35–3.45
(1H,m), 3.50 (1H, dd, J = 3.2 and 4.8 Hz), 3.57 (3H,s), 3.60 (3H,s),
3.85 (1H, dd, J = 1.8 and 4.8 Hz), 4.00–4.12 (4H,m), 4.15 (1H, dd,
J = 3.5 and 6.0 Hz), 4.19 (1H, t, J = 8.3 Hz), 4.30 (1H, dd, J = 1.9 and
8.3 Hz), 4.51 (1H, d, J = 4.8 Hz), 7.20–7.40 (5H,m) ppm; 13C NMR
(100 MHz, CDCl3) d 25.2, 25.3, 26.7, 26.8, 32.1, 34.4, 49.9, 51.5,
51.8, 56.6, 59.4, 68.0, 69.1, 76.5, 77.3, 77.7, 84.0, 109.3, 109.7,
127.2, 128.7 (2C), 130.1 (2C), 138.4, 163.7, 172.1 ppm; HRMS (Q-
TOF) calcd for C27H38NO9 (M+ + 1) 520.2541, found 520.5980.
2: Rf (4:6 hexanes/EtOAc) 0.30; ½a D25
ꢁ
+64 (c 1.0,CHCl3); IR (film)
t
3511, 1761, 1738, 1165, 1020 cmꢀ1
;
1H NMR (400 MHz, CDCl3) d
1.32 (3H,s), 1.39 (3H,s), 1.43 (3H,s), 1.47 (3H,s), 2.20–2.35
(2H,m), 2.55–2.65 (1H,m), 2.82 (1H,t,J = 11.2 Hz), 3.47
(1H,d,J = 10.8 Hz), 3.54 (3H,s), 3.68 (3H,s), 3.70 (1H,d, J = 8.4 Hz),
3.96 (1H,dd,J = 4.2 and 8.6 Hz), 4.01–4.06 (2H,m), 4.08
(1H,d,J = 6.4 Hz), 4.13 (1H,dd,J = 6.4 and 8.6 Hz), 4.24
(1H,dd,J = 4.4 and 7.2 Hz), 4.41 (1H,ddd,J = 7.2, 10.8 and 11.2 Hz),
4.81 (1H,d,J = 4.4 Hz), 7.20–7.35 (5H,m) ppm; 13C NMR
(100 MHz,CDCl3) d 25.2, 26.5, 26.7, 27.0, 38.3, 40.6, 50.7, 51.5,
53.8, 54.3, 59.7, 67.7, 72.9, 77.3, 77.7, 81.7, 86.6, 109.6, 109.8,
127.2, 128.6 (2C), 128.7 (2C), 139.4, 171.0, 172.1 ppm; HRMS (Q-
TOF) calcd for C27H38NO9 (M+ + 1) 520.2541, found 520.2539.
Carbacephams 3 and 4; carbapenam 9: From the epoxide 19ab
(220 mg, 0.43 mmol), 45 mg of 3 (20%), 16 mg of 4 (7%) and 11 mg
of 9 (5%) were isolated in 4 h.
8: Rf (4:6 hexanes/EtOAc) 0.25; ½a D25
ꢁ
ꢀ6 (c 1.0,CHCl3); IR (film)
t
3480, 1761, 1738, 1215, 1070, 700 cmꢀ1; 1H NMR (400 MHz,CDCl3)
d 1.26 (3H,s), 1.30 (3H,s), 1.33 (3H,s), 1.37 (3H,s), 2.75–2.85 (2H,m),
3.00–3.10 (1H,m), 3.35 (1H,brs), 3.63 (3H,s), 3.66 (3H,s), 3.74 (1H,
d, J = 6.4 Hz), 3.90 (1H, dd, J = 2.8 and 4.8 Hz), 3.93 (1H, dd, J = 6.4
and 8.8 Hz), 4.00–4.05 (2H,m), 4.15 (1H, dd, J = 6.0 and 8.7 Hz),
4.23 (1H,d,J = 3.2 Hz), 4.45 (1H, dd, J = 2.8 and 3.2 Hz), 4.55 (1H, t,
J = 6.4 Hz), 4.76 (1H, dd, J = 0.9 and 4.8 Hz), 7.20–7.35 (5H, m)
ppm; 13C NMR (100 MHz,CDCl3) d 25.3, 26.3, 27.3, 27.4, 29.6, 37.4,
51.4, 53.7, 55.2, 59.4, 59.5, 68.0, 69.5, 77.2, 77.9, 79.4, 84.5, 109.9,
110.1, 126.7, 127.7, 128.7 (3C), 141.3, 165.5, 173.3 ppm; HRMS (Q-
TOF) calcd for C27H38NO9 (M+ + 1) 520.2541, found 520.2568.
3: The properties of this compound were in agreement with
those reported in Ref. [22a].
4: Rf (4:6 hexanes/EtOAc) 0.28; ½a D25
ꢁ
+26 (c 1.0,CHCl3); IR (film) t
3480, 1761, 1738, 1212, 1065, 700 cmꢀ1; 1H NMR (400 MHz,CDCl3)
d 1.31 (3H,s), 1.39 (3H,s), 1.46 (3H,s), 1.50 (3H,s), 2.10 (1H,dd,J = 4.8
and 12.8 Hz), 2.20 (1H,dd,J = 10.8 and 12.8 Hz), 2.33 (1H,br s), 2.65–
2.75 (1H,m), 3.05 (1H,t, J = 10.6 Hz), 3.55 (3H,s), 3.59 (3H,s), 3.70
(1H,t,J = 7.9 Hz), 3.85 (1H,dd,J = 4.1 and 8.6 Hz), 3.95–4.00
(3H,m), 4.12–4.16 (2H,m), 4.27 (1H,d,J = 6.4 Hz), 4.68
4.1.4. Preparation of polycyclic b-lactams 10–18
Compounds 10–18 were prepared according to the protocols re-
ported in references 22b (bilactam 10 and trilactams 14–18) and
23 (compounds 11–13).
(1H,d,J = 4.1 Hz),
7.20–7.40
(5H,m)
ppm;
13C
NMR
(100 MHz,CDCl3) d 25.3, 26.3, 26.8, 27.0, 35.5, 40.2, 48.2, 50.6,
51.7, 58.8, 59.0, 67.7, 70.4, 77.3, 78.4, 78.8, 85.0, 110.0, 110.3,
127.8 (2C), 128.7, 129.0, 129.2, 138.5, 167.5, 171.7 ppm; HRMS (Q-
TOF) calcd for C27H38NO9 (M+ + 1) 520.2541, found 520.2527.
4.2. Biological evaluation
4.2.1. Materials
Human Leukocyte Elastase (HLE) and Human Leukocyte Pro-
teinase 3 (HLPR3) were obtained form Calbiochem–Novabiochem;
Porcine Pancreatic Elastase (PPE) was from Elastin Products Co.
Inc.; substrate methoxysuccinyl–Ala–Ala–Pro–Val–p-nitroanilide,
reference inhibitor 3,4–dichloroisocoumarin (DCI) and HEPES
[4-(2-hydroxyethyl)-1-piperazinethanesulphonic acid] were from
Sigma–Aldrich. All other reagents were from Panreac Química S.A.
Biological assays [29] were carried out on flat bottom 96-well
microtiter plates (BIOTECH, S.L.) in a final reaction volume of
9: Rf (4:6 hexanes/EtOAc) 0.32; ½a D25
ꢁ
+14 (c 1.0,CHCl3); IR (film)
t
3488, 1761, 1738, 1215, 1070, 700 cmꢀ1 1H NMR
;
(400 MHz,CDCl3) d 1.32 (3H,s), 1.45 (3H,s), 1.47 (3H,s), 1.49
(3H,s), 2.25–2.35 (2H,m), 2.62 (1H, dt, J = 5.7 and 11.3 Hz), 2.85–
2.95 (1H,m), 3.65 (6H,s), 3.77 (1H,t,J = 8.2 Hz), 3.98
(1H,dd,J = 4.3 and 8.5 Hz), 4.00 (1H,dd,J = 4.9 and 11.3 Hz), 4.05–
4.10 (1H,m), 4.10 (1H,d,J = 11.3 Hz), 4.14 (1H,dd,J = 1.5 and
5.5 Hz), 4.15 (1H,t,J = 8.5 Hz), 4.39 (1H,dd,J = 1.5 and 8.2 Hz),
4.70 (1H,d,J = 4.9 Hz), 7.20–7.35 (5H,m) ppm; 13C NMR
(100 MHz,CDCl3) d 25.2, 26.6, 26.8, 27.0, 32.5, 39.6, 48.8, 51.6,
54.3, 59.5, 60.1, 67.6, 68.6, 76.9, 77.5, 83.3, 85.0, 109.2, 109.9,
127.1, 128.5 (3C), 129.2, 139.6, 172.0, 172.6 ppm; HRMS (Q-TOF)
calcd for C27H38NO9 (M+ + 1) 520.2541, found 520.2560.
300 lL. Reactions were monitorized at 405 nm using an Ascent
Multiskan Spectrophotometer (Thermo Electron Corporation) con-
nected to a computer (Main Program of Ascent Software, 2.6 Version,
Thermo Labsystems).
Carbacephams
5
and 6: From the epoxide 19ba
4.2.2. Enzyme assays for the inhibition of HLE and HLPR3
[30] Enzymatic activities of HLE and HLPR3 were assayed with
the chromogenic substrate, methoxysuccinyl–Ala–Ala–Pro–Val–p-
nitroanilide, in 50 mM HEPES buffer at pH = 7.8, containing 10%
DMSO (dimethyl sulphoxide), by continuous reading of the release
of p-nitroaniline from the peptide at 405 nm [30]. Enzyme concen-
trations were 20.4 nM (0.0038 IU per well) for HLE and 70 nM
(0.0038 IU per well) for HLPR3.
The reaction evolution was recorded every 30 s for 10 min and
the inhibitory activity was calculated at a time of 5 min, when
the reaction is still linear and assuming that a 100% of anti–enzyme
activity, or 0% of inhibition, corresponds to the absorbance value
registered for the control well containing only enzyme + substrate.
All experiments were conducted at 37 °C, in triplicate, and re-
peated at least three times.
(400 mg,0.77 mmol), 88 mg of 5 (22%) and 152 mg of 6 (38%) were
isolated in 16 h.
5: Rf (4:6 hexanes/EtOAc) 0.30; ½a D25
ꢁ
ꢀ20 (c 1.0,CHCl3); IR (film)
t
3466, 1761, 1750, 1215, 1065, 700 cmꢀ1 1H NMR
;
(400 MHz,CDCl3) d 1.31 (3H,s), 1.32 (3H,s), 1.35 (3H,s), 1.59
(3H,s), 2.08 (1H, dd, J = 3.2 and 16.2 Hz), 2.37 (1H, dd, J = 8.0 and
16.2 Hz), 2.45–2.55 (1H,m), 3.00 (1H,t,J = 11.2 Hz), 3.35
(1H,dd,J = 4.3 and 7.5 Hz), 3.52 (3H,s), 3.55–3.65 (1H,m), 3.58
(3H,s), 3.95–4.05 (3H,m), 4.05–4.15 (2H,m), 4.20 (1H, dd, J = 3.4
and 8.1 Hz), 4.50 (1H,d,J = 4.3 Hz), 7.20–7.40 (5H, m) ppm; 13C
NMR (100 MHz, CDCl3) d 25.4, 26.5, 26.6, 26.8, 32.7, 40.2, 51.6,
53.3, 58.4, 58.5, 59.0, 68.0, 70.2, 76.7, 77.3, 77.7, 83.3, 109.4,
109.7, 127.8 (2C), 129.1 (3C), 138.6, 163.2, 171.7 ppm; HRMS (Q-
TOF) calcd for C27H38NO9 (M+ + 1) 520.2541, found 520.2568.
6: The properties of this compound were in agreement with
those reported in Ref. [22a].
3,4-Dichloroisocoumarin (DCI), at a concentration of 10
was throughout used as a reference inhibitor.
lM,
Carbacephams 7 and 8: From the epoxide 19bb (200 mg,
0.39 mmol), 30 mg of 7 (15%) and 16 mg of 8 (8%) were isolated in 5 h.
4.2.3. Specificity assays
Only the most effective HLE inhibitors among the target mole-
cules were selected for the specificity assays. These assays were
7: Rf (4:6 hexanes/EtOAc) 0.29; ½a D25
ꢁ
ꢀ23 (c 1.0,CHCl3); IR (film)
t
3480, 1759, 1738, 1215, 1070, 700 cmꢀ1; 1H NMR (400 MHz,CDCl3)

34
L.M. Monleón et al. / Bioorganic Chemistry 45 (2012) 29–35
Fig. 5. Graphic plots of absorbance versus time for trilactam 17 (E+S: enzyme + sustrate; RI: reference inhibitor 3,4-dichloroisocoumarin).
carried out following the same experimental procedure described
for the HLE and HLPR3 inhibition assays but at pH = 8.0, instead
of 7.8, following the recommendations of the commercial supplier
of the enzyme PPE (HLE activity was not significantly affected by
the change in the pH value). HLE and PPE concentration was
20.4 nM.
References
[1] International Union of Biochemistry and Molecular Biology, Recommendations
on Biochemical and Organic Nomenclature, Symbols and Terminology. <http://
www.chem.qmw.ac.uk/iubmb/> (accessed 10.01.2012).
[2] (a) A.J. Barret, Mehtods Enzymol. 80 (1981) 581–588;
(b) J.W. Harper, R.R. Cook, C.J. Roberts, B.J. McLaughlin, J.C. Powers,
Biochemistry 23 (1984) 2995–3002.
[3] (a) B. Korkmaz, T. Moreau, F. Gauthier, Biochimie 90 (2008) 227–242;
(b) C.T.N. Pham, Int. J. Biochem. Cell. Biol. 40 (2008) 1317–1333.
[4] (a) R.A. Briggaman, N.M. Schechter, J. Fraki, G.S. Lazarus, J. Exp. Med. 160
(1984) 1027–1042;
4.2.4. Data analysis
All the obtained data were treated and analyzed with MicrosoftÒ
Office ExcelÒ 2007 Software (12.0.6504.5011 Version, Microsoft Cor-
porationÒ). The backgrounds were subtracted from the crude data
and the data corresponding to a medium time among the linear re-
gion (reaction time of 5 min) were chosen to calculate inhibition
percentages. The observed enzymatic activity in the absence of
inhibitor (control well) was considered 100% of activity (0% inhibi-
tion) and the inhibition percentages were calculated in respect to
that value. Progress graphic plots of absorbance versus time were
obtained for each assay. A representative one is shown in Fig. 5.
IC50 values were calculated from dose-response studies per-
formed in triplicate and repeated at least three times. A calculus
sheet designed for that purpose with MicrosoftÒ Office ExcelÒ
2007 Software (12.0.6504.5011 Version, Microsoft CorporationÒ)
was used for these calculations. Both, forecasting analysis based
on linear regressions and calculations from the equations of trend
curves in each case, produced the same results. Ki values were cal-
culated from IC50 measurements according to the expression for a
competitive inhibition in the presence of tightly bound inhibitors
[31].
(b) G.L. Snider, Drug Devel. Res. 10 (1987) 235–253;
(c) P.J.N. Barnes, Engl. J. Med. 343 (2000) 269–280;
(d) N. Hilbert, J. Schiller, J. Arnhold, K. Arnold, Bioorg. Chem. 30 (2002) 119–
132;
(e) K. Kawabata, T. Hagio, S. Matsuoka, Eur. J. Pharmacol. 451 (2002) 1–10;
(f) S. Tamakuma, M. Ogawa, N. Aikawa, T. Kubota, H. Hirasawa, A. Ishizaka, N.
Taenaka, C. Hamada, S. Matsuoka, T. Abiru, Pulm. Pharm. Ther. 17 (2004) 271–
279.
[5] (a) J.C. Powers, Am Rev. Respir. Dis. 127 (1983) S54–S58;
(b) A. Janoff, Am. Rev. Respir. Dis. 132 (1985) 417–433;
(c) J.C. Powers, Z.H. Bengali, Am. Rev. Respir. Dis. 134 (1986) 1097–1100;
(d) S. Eriksson, Eur. Respir. J. 4 (1991) 1041–1043;
(e) H. Parfrey, R. Mahadeva, D.A. Lomas, Int. J. Biochem. Cell. Biol. 35 (2003)
1009–1014.
[6] (a) J. Saklatvala, A.J. Barrett, Biochim. Biophys. Acta 615 (1980) 167–177;
(b) K. Kawabata, A.R. Moore, D.A. Willoughby, Ann. Rheum. Dis. 55 (1996) 248–
252.
[7] (a) Z. Sun, P. Yang, Lancet Oncol. 5 (2004) 182–190;
(b) D. Kunh, C. Coates, K. Daniel, D. Chen, M. Bhuiyan, A. Kazi, E. Turos, Q.P.
Dou, Front. Biosci. 9 (2004) 2605–2617.
[8] G. Lungarella, C. Gardi, M.M. De Santi, P. Luzi, Exp. Mol. Path. 42 (1985) 44–
59.
[9] (a) For reviews, see: R.L. Stein, D.A. Trainor, R.A. Wildonger, Annual Reports in
Medicinal Chemistry, vol. 20, Academic Press, London, 1985, pp. 237ꢀ246.;
(b) P.D. Edwards, P.R. Bernstein, Med. Res. Rev. 14 (1994) 127–194;
(c) J.C. Powers, J.L. Asgian, O.D. Ekici, K.E. James, Chem. Rev. 102 (2002) 4639–
4750.
[10] (a) M. Padrines, J.G. Bieth, Am. J. Respir. Cell Mol. Biol. 4 (1991) 187–193;
(b) W.L. Lee, G.P. Downey, Am. J. Respir. Crit. Care Med. 164 (2001) 896–
904;
Kið MÞ ¼ ðIC50 ꢀ ½Eꢁ=2Þ=ð½Sꢁ=Km þ 1Þ
l
(c) B. Siedle, A. Hrenn, I. Merfort, Planta Med. 73 (2007) 401–420.
[11] For example, see: (a) M. Takayama, M. Ishibashi, H. Ishii, T. Kuraki, T. Nishida,
M. Yoshida, J Appl. Physiol. 91 (2001) 1800–1807;
The concentrations of the reagents in our microassays were as fol-
lows: [HLE] = 0.02 M, [HLPR3] = 0.07 M, [S] = 100 M. Km (S0.5
determined for HLE was 152 M and Km (S0.5) determined for HLPR3
was 112 M.
l
l
l
)
(b) Y. Nakayama, Y. Odagaki, S. Fujita, S. Matsuoka, N. Hamanaka, H. Nakai, M.
Toda, Bioorg. Med. Chem. Lett. 12 (2002) 2349–2353;
(c) Y. Kadoi, H. Hinohara, F. Kunimoto, S. Saito, F. Goto, T. Kosaka, K. Ieta,
Anesth. Analg. 99 (2004) 872–877;
l
l
(d) C. Guay, M. Laviolette, G.M. Tremblay, Curr. Top. Med. Chem. 6 (2006) 393–
402.
[12] (a) J.B. Doherty, B.M. Ashe, L.W. Argenbright, P.L. Barker, R.J. Bonney, G.O.
Chandler, M.E. Dahlgren, C.P. Dorn, P.E. Finke, R.A. Firestone, D. Fletcher, W.K.
Hagmann, R. Mumford, L. O’Grady, A.L. Maycok, J.M. Pisano, S.K. Shah, K.R.
Thompson, M. Zimmerman, Nature 322 (1986) 192–194;
(b) O.A. Mascaretti, C.E. Boschetti, G.O. Danelon, E.G. Mata, O.A. Roveri, Curr.
Acknowledgment
Financial support for this work from the Junta de Castilla y León
(SA046A08) is gratefully acknowledged.

L.M. Monleón et al. / Bioorganic Chemistry 45 (2012) 29–35
35
Med. Chem. 1 (1995) 441–470;
(c) G. Veinberg, M. Vorona, I. Shestakova, I. Kanepe, E. Lukevics, Curr. Med.
Chem. 10 (2003) 1741–1757.
[20] (a) D.H.R. Barton, A. Gateau-Olesker, J. Anaya-Mateos, J. Cléophax, S.D. Gero, A.
Chiaroni, C. Riche, J. Chem. Soc., Perkin Trans. 1 (1990) 3211–3212;
(b) D.H.R. Barton, J. Anaya, A. Gateau-Olesker, S.D. Géro, Tetrahedron Lett. 33
(1992) 6641–6642;
[13] For example, see: (a) W.T. Han, A.K. Trehan, J.J.K. Wright, M.E. Federici, S.M.
Seiler, N.A. Meanwell, Bioorg. Med. Chem. 3 (1995) 1123–1143;
(b) J.S. Plummer, K.A. Berryman, C. Cai, W.L. Cody, J. DiMaio, A.M. Doherty, S.
Eaton, J.J. Edmunds, D.R. Holland, D. Lafleur, S. Levesque, L.S. Narasimhan, J.R.
Rubin, S.T. Rapundalo, M.A. Siddiqui, A. Susser, Y. St-Denis, P. Winocour,
Bioorg. Med. Chem. Lett. 9 (1999) 835–840;
(c) J. Anaya, D.H.R. Barton, S.D. Gero, M. Grande, N. Martín, C. Tachdjian,
Angew. Chem., Int. Ed. 32 (1993) 867–869;
(d) J. Anaya, D.H.R. Barton, S.D. Gero, M. Grande, J.I.M. Hernando, N.M. Laso,
Tetrahedron: Asymm. 6 (1995) 609–624;
(e) J.I.M. Hernando, N.M. Laso, J. Anaya, S.D. Gero, M. Grande, Synlett (1997)
281–282;
(f) G. Ruano, J. Anaya, M. Grande, Synlett (1999) 1441–1443;
(g) G. Ruano, M. Grande, J. Anaya, J. Org. Chem. 67 (2002) 8243–8246;
(h) J. Anaya, A. Fernández-Mateos, M. Grande, J. Martiáñez, G. Ruano, M.R.
Rubio-González, Tetrahedron 59 (2003) 241–248.
(c) S. Lévesque, Y. St-Denis, B. Bachand, P. Préville, L. Leblond, P.D. Winocour,
J.J. Edmunds, J.R. Rubin, M.A. Siddiqui, Bioorg. Med. Chem. Lett. 11 (2001)
3161–3164.
[14] (a) R.M. Adlington, J.E. Baldwin, G.W. Becker, B. Chen, L. Cheng, S.L. Cooper, R.B.
Hermann, T.J. Howe, W. McCoull, A.M. McNulty, B.L. Neubauer, G.J. Pritchard, J.
Med. Chem. 44 (2001) 1491–1508;
[21] (a) M. Adonias, J. Anaya, J. Cámara, E. Canet, A. Gateau-Olesker, S.D. Géro, M.
Grande, J.I.M. Hernando, Bioorg. Med. Chem. Lett. 3 (1993) 2547–2552;
(b) J. Anaya, S.D. Gero, M. Grande, J.I.M. Hernando, N.M. Laso, Bioorg. Med.
Chem. 7 (1999) 837–850.
[22] (a) G. Ruano, J. Martiáñez, M. Grande, J. Anaya, J. Org. Chem. 68 (2003) 2024–
2027;
(b) R. Annunziata, M. Benaglia, M. Cinquini, F. Cozzi, A. Puglisi, Bioorg. Med.
Chem. 10 (10) (2002) 1813–1818;
(c) P. Singh, S.A. Williams, M.H. Shah, T. Lectka, G.J. Pritchard, J.T. Isaacs, S.R.
Denmeade, Proteins 70 (2008) 1416–1428.
[15] (a) A.D. Borthwick, G. Weingarten, T.M. Haley, M. Tomaszewski, W. Wang, Z.
Hu, J. Bedard, H. Jin, L. Yuen, T.S. Mansour, Bioorg. Med. Chem. Lett. 8 (1998)
365–370;
(b) L.M. Monleón, M. Grande, J. Anaya, Tetrahedron 63 (2007) 3017–
3025.
(b) W.W. Ogilvie, C. Yoakim, F.DôB. Haché, L. Lagacé, J. Naud, J.A. O’Meara, R.
Déziel, Bioorg. Med. Chem. 7 (1999) 1521–1531;
(c) G. Gerona-Navarro, M.J. Pérez de Vega, M.T. García-López, G. Andrei, R.
Snoeck, E. De Clercq, J. Balzarini, R. González-Muñiz, J Med. Chem. 48 (2005)
2612–2621.
[23] (a) L.M. Monleón, M. Grande, J. Anaya Synlett (2007) 1243–1282;
(b) L.M. Monleón, M. Grande, J. Anaya Tetrahedron, in press (doi:10.016/
j.tet.2011.11.015).
[24] For reviews, see: (a) A.F. Barrero, J.F. Quílez del Moral, E.M. Sánchez, J.F.
Arteaga, Eur. J. Org. Chem. (2006) 1627–1641;
[16] Y. Aoyama, M. Uenaka, M. Kii, M. Tanaka, T. Konoike, Y. Hayasaki-Kajiwara, N.
Naya, M. Nakakima, Bioorg. Med. Chem. 9 (2001) 3065–3075. and references
therein.
[17] (a) R.C. Wilmouth, S. Kassamally, N.J. Westwood, R.J. Sheppard, T.D.W.
Claridge, R.T. Aplin, P.A. Wright, G.J. Pritchard, C.J. Schofield, Biochemistry 38
(1999) 7989–7998;
(b) J.M. Cuerva, J. Justicia, J.L. Oller-López, B. Bazdi, J.E. Oltra, Mini-Rev. Org.
Chem. 3 (2006) 23–35;
(c) J.M. Cuerva, J. Justicia, J.L. Oller-López, J.E. Oltra, Top. Curr. Chem. 264
(2006) 63–91;
(d) A. Gansäuer, J. Justicia, C.-A. Fan, D. Worgull, F. Piestert, Top. Curr. Chem.
279 (2007) 25–52.
(b) B. Walker, J.F. Lynas, Cell. Mol. Life Sci. 58 (2001) 596–624;
(c) E. Hedstrom, Chem. Rev. 102 (2002) 4501–4524.
[25] B.M. Ashe, R.L. Clark, H. Jones, M. Zimmerman, J. Biol. Chem. 256 (1981)
11603–11606.
[18] For recent examples, see: (a) M.I. Konaklieva, Med. Chem. Rev. 1 (2004) 133–
140;
[26] (a) A.V. Narender Reddy, C.Y. Fiakpui, D.P. Czajkowski, J. Kaleta, R.G. Micetich,
S.N. Maiti, Chem. Het. Comp. 34 (1998) 1282–1288;
(b) G. Cainelli, P. Galletti, S. Garbisa, D. Giacomini, L. Sartor, A. Quintavalla,
Bioorg. Med. Chem. 11 (2003) 5391–5399.
(b) S. Gérard, M. Galleni, G. Dive, J. Marchand-Brynaert, Bioorg. Med. Chem. 12
(2004) 129–138;
(c) R. Moreira, A.B. Santana, J. Iley, J. Neres, K.T. Douglas, P.N. Horton, M.B.
Hursthouse, J. Med. Chem. 48 (2005) 4861–4870;
(d) G. Cainelli, P. Galletti, S. Garbisa, D. Giacomini, L. Sartor, A. Quintavalla,
Bioorg. Med. Chem. 13 (2005) 6120–6132.
[27] (a) W. Bode, E. Meyer Jr., J.C. Powers, Biochemistry 28 (1989) 1951–1963;
(b) M. Geller, A. Laczkowski, S.M. Swanson, E.F. Meyer Jr, Computers Chem. 15
(1991) 261–272.
[28] Conformational minima derived from analysis with CS Chem3D Ultra 11.0
software, CambridgeSoft, Cambridge, MA, 2008 using MM2 calculations.
[29] K. Nakajima, J.C. Powers, B.M. Ashe, M. Zimmerman, J. Biol. Chem. 254 (1979)
4027–4032.
[30] B.F. Erlanger, N. Kokowsky, W. Cohen, Arch. Biochem. Biophys. 95 (1961) 271–
278.
[31] R.Z. Cer, U. Mudunuri, R. Stephens, F.J. Lebeda, Nucl. Acids Res. 37 (2009)
W441–W445.
[19] For examples, see: (a) P.E. Finke, S.K. Shah, D.S. Fletcher, B.M. Ashe, K.A. Brause,
G.O. Chandler, P.S. Dellea, K.M. Hand, A.L. Maycock, D.G. Osinga, D.J.
Underwood, H. Weston, P. Davies, J.B. Doherty, J. Med. Chem. 38 (1995)
2449–2462;
(b) B. Macchia, D. Gentili, M. Macchia, F. Mamone, A. Martinelli, E. Orlandini, A.
Rossello, G. Cercignani, R. Pierotti, M. Allegretti, C. Asti, G. Caselli, Eur. J. Med.
Chem. 35 (2000) 53–67. and references here contained.