Design, synthesis, and biological evaluation of 4-alkyliden-beta lactams: New products with promising antibiotic activity against resistant bacteria

Article abstract of DOI:10.1021/jm0580510

The design, synthesis, and antibacterial activity of 4-alkyliden-azetidin- 2-ones as new antimicrobial agents against multidrug-resistant pathogens is reported. 4-Alkyliden-azetidin-2-ones were easily obtained using an original protocol starting from 4-ac

Full text of DOI:10.1021/jm0580510

2804
J. Med. Chem. 2006, 49, 2804-2811
Design, Synthesis, and Biological Evaluation of 4-Alkyliden-beta Lactams: New Products with
Promising Antibiotic Activity Against Resistant Bacteria
Francesco Broccolo,§ Gianfranco Cainelli,*,† Gianluigi Caltabiano,‡ Clementina E. A. Cocuzza,§ Cosimo G. Fortuna,‡
Paola Galletti,† Daria Giacomini,† Giuseppe Musumarra,*,‡ Rosario Musumeci,§ and Arianna Quintavalla†
Department of Chemistry “G. Ciamician”, UniVersity of Bologna, Via Selmi 2, I-40126 Bologna, Italy, Department of Chemical Sciences,
UniVersity of Catania, Viale A. Doria 6, I-95125 Catania, Italy, and Department of Clinical Medicine, PreVention and Biotechnology,
UniVersity of Milano-Bicocca, Via Cadore 48, I-20052 Monza, Italy
ReceiVed NoVember 17, 2005
The design, synthesis, and antibacterial activity of 4-alkyliden-azetidin-2-ones as new antimicrobial agents
against multidrug-resistant pathogens is reported. 4-Alkyliden-azetidin-2-ones were easily obtained using
an original protocol starting from 4-acetoxy-azetidinones and diazoesters. Parent compounds were further
elaborated to obtain a small library of 4-alkylidene derivatives. A molecular modeling approach using GRID
descriptors based on the concept of VRS identified attractive drug candidates and contributed to the
rationalization of functional group effects in QSARs. The in vitro antibacterial activity of the new agents
was evaluated against 43 recent clinical isolates of antibiotic-susceptible and -resistant Gram-positive and
Gram-negative pathogens by determining their minimum inhibitory concentrations (MICs). The most active
compound showed MIC values ranging from 0.25 to 32 mg/L against some of the bacterial species tested.
Interestingly, some compounds demonstrated similar activity against methicillin-susceptible and -resistant
strains of Staphylococcus aureus suggesting possible alternative mechanisms of action of these agents,
supported by citotoxicity and preliminary scanning electron microscopy studies.
Introduction
resistance forces the continuous modification of side chains and
structures of known active compounds and the development of
new ones.
Since their introduction, â-lactam antibiotics proved to be
chemotherapeutics of incomparable effectiveness, conjugating
a broad spectrum of activity with low toxicity.1 â-Lactams are
a large class of antibiotics characterized by the presence of the
azetidin-2-one ring, which is the core of the biological activity,
and differentiated by side chains, unsaturations, heteroatoms,
and, in many cases, by the presence of another five- or six-
membered ring.
The great vivacity of the research in this field led to the
development of classical â-lactam substrates such as penicillins
and cephalosporins together with nonclassical ones such as
carbapenems and monobactams obtained via semi or total
syntheses. To complete and increase the importance of â-lac-
tams, nonclassical bicyclic and monocyclic â-lactam substrates
with different biological activities have appeared in recent
literature: serine-dependent enzyme inhibitors,2 matrix-metallo
protease inhibitors,3 and even apoptosis inductors.4
The problem of microbial resistance, related to the use of
these agents over the last 50 years, is nowadays widely
recognized, and treatment options in clinical practice are limited
by multidrug-resistant bacteria.5 Despite the need for new
antibiotics, large pharmaceutical companies are devoting fewer
resources to their development because these agents do not
provide as great a return on investment relative to that of
compounds for some other therapies.6 However, in the United
States, antimicrobial drugs have had the first or second shortest
mean and median clinical development times in every four-
year period since 1982.7 Therefore, the phenomenon of bacterial
Herein we report the synthesis and antibacterial activity of a
new class of monocyclic beta-lactams substituted in C-4 with
an alkyliden carboxy side chain named 4-alkyliden-azetidin-2-
one with the aim to contribute to the development of new
antimicrobial agents against multidrug-resistant pathogens.
Preliminary results of the antibacterial activity of some of
these 4-alkyliden-beta lactams disclosed the opportunity for the
application of molecular modeling to relate chemical structures
to antibiotic activity and to point out structural modifications
that might increase antibiotic potency.
Despite significant advances in the elucidation of the struc-
tures of penicillin-binding proteins (PBPs), the overall structural
basis for multidrug bacterial resistance has not been clarified.
PBPs are a heterogeneous family of enzymes with transpeptidase
and transcarboxylase activities involved in the synthesis and
cross-linking of the peptidoglycan component of bacterial cell
walls, which is fundamental for the manteinance of bacterial
cell morphology and integrity. The above considerations suggest
that molecular descriptors based on the concept of virtual
receptor site (VRS) should be adopted.8 We selected a molecular
interaction field (MIF) approach using grid independent descrip-
tors (GRIND),8 calculated by using the program ALMOND,9
which is based on the assumption that the process of ligand-
receptor interaction can be represented with the help of the MIF.
Accordingly, when a drug binds a receptor of unknown structure,
some of the regions defined in its VRS are expected to overlap
groups of the real receptor site; therefore, a subset of the VRS
regions should be relevant for representation of the binding
properties of the ligand.
* To whom correspondence should be addressed. Tel: +39-051-20995-
10. Fax: +39-051-20994-56. E-mail: gianfranco.cainelli@unibo.it (G.C.).
Tel: +39-095-7385012. Fax: +39-095-334175. E-mail: gmusumarra@unict.it
(G.M.).
Results and Discussion
† University of Bologna.
Very few examples of alkyliden-azetidin-2-ones have been
reported in the literature. In the 1980s, some C-4 alkyliden
‡ University of Catania.
§ University of Milano-Bicocca.
10.1021/jm0580510 CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/08/2006

4-Alkyliden-beta Lactam Antibiotic ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2805
Scheme 3
Scheme 1
Scheme 2
4-alkyliden beta lactams 1-5 and 18 are reported. As reference
compounds, we tested two drugs in clinical use, such as
amoxicillin 27, ceftriaxone 28, and a saturated analogue 26,
which is important for the evaluation of the influence of
unsaturation on C-4 in alkyliden-beta-lactams (Chart 1). Some
of these 4-alkyliden derivatives demonstrated antibacterial
activity.
derivatives were synthesized and tested for antimicrobial activity
with poor results.10 Recently, we developed a process to obtain
4-alkyliden-azetidin-2-ones11 (Scheme 1), which could be further
modified to obtain a small library of 4-alkyliden compounds.
Synthesis of 4-Alkyliden-beta-lactams. 4-Alkyliden-azeti-
din-2-ones can be easily obtained using an original and well-
established protocol starting from 4-acetoxy-azetidinones and
diazoesters in the presence of Lewis acids.11 Compounds 1-6
were obtained starting from 3-chloro- or 3-(2-tert-butyldimeth-
ylsilyloxyethyl)-4-acetoxy-azetidin-2-ones and ethyl- or benzyl-
diazoacetate, with TiCl4 or AlCl3 (Scheme 1). The products are
obtained in good yields with diastereomeric Z/E ratios depending
on the Lewis acid and reactants: with ethyldiazoacetate and
TiCl4, we obtained (1/2) Z/E ) 80:20 and with AlCl3 Z/E )
53:47, with benzyldiazoacetate and TiCl4 (3/4) Z/E ) 90:10,
and with AlCl3 Z/E ) 60:40. Starting from 3-chloro 4-acetoxy
azetidinone, we obtained only Z isomers 5 and 6 with ethyl
and benzyl diazoacetate, respectively. In all cases, Z and E
diastereoisomers could be easily separated by flash chromatog-
raphy.
Parent compounds 1-6 offer two points for a further
elaboration: the C-3 hydroxy side chain and the C-4 ester
function. Exploiting modifications on the C-4 side chain, benzyl
esters 3 and 6 were quantitatively cleaved using H2 (1 atm)
and Pd on C in THF/MeOH, 1/1 to afford the corresponding
acids 7 and 8 as outlined in Scheme 2. Acid 7 can, in turn, be
easily transformed in esters or amides 9-12 by DCC coupling
using benzyl alcohols, phenols, or an amino-ester (Scheme 2).
The C-3 hydroxy-ethyl chain can be further derivatized to
several aromatic esters by preliminary deprotection of the silyl
group followed by acylation in the presence of SnCl4 (5%) in
refluxing benzene (Scheme 3) giving products 16-22.
Compounds 23 and 24 were obtained from corresponding
benzyl esters 21 and 22 via hydrogenolysis in THF/MeOH, 1/1,
and Pd 10% on C. With the same hydrogenolysis procedure,
compound 15 from 14 and 25 from 19 were obtained (Scheme
3).
Table 1 shows that compounds 4 and 5 exhibited some
antibacterial activity against the bacterial strains tested. Com-
pound 4 appeared to have some activity (MIC50 of 32-64 mg/
L) against Gram-positive pathogens, such as S. pyogenes, S.
pneumoniae, and Enterococcus spp. Against the latter bacterial
strains, compound 4 appeared a possible alternative to inactive-
marketed compound 28 (ceftriaxone). Compound 5 showed
some antibacterial activity against the Gram-positive strains of
Staphylococcus aureus and Staphylococcus epidermidis as well
as against the Gram-negative isolates of H. influenzae. In
particular, its antibacterial activity against Staphylococcus
epidermidis was comparable to that of reference compound 28
(ceftriaxone). The other alkyliden-azetidinones shown in Table
1 demonstrated relatively poor antibacterial activity.
Considering that these 4-alkyliden-beta lactams apart from
the monocyclic azetidinone framework have a very different
structure with respect to monobactam antibiotics (e.g., Aztre-
onam) their antibacterial activity results are significant. To
follow up and assess these microbiological results, we ap-
proached a rational design of these new compounds. Difficulties
in finding a single receptor target for beta-lactam action against
a wide panel of microorganisms prevents the use of specific
docking procedures. Therefore, we adopted a VRS approach,
where ligands interact with a complex receptor of unknown
structure, and QSARs are aimed at identifying pieces of the
structure that could be valuable for improving antibacterial
activity.
The structures in Chart 1 were selected to derive the first
model by a novel methodology adopting GRIND8 descriptors.
The inclusion of totally different structures, such as those of
currently marketed drugs amoxicillin 27 and ceftriaxone 28, is
possible because of an important peculiarity in these novel
descriptors insensitive to the position and orientation of the
molecular structures in the space. Therefore, because the GRIND
program does not need the alignment of compounds, 3D-QSAR
analysis requires less time compared to that of other 3D-QSAR
approaches where the need for the alignment of compounds is
Microbiological Evaluation. In Table 1 the preliminary
results of antibacterial activity toward sensible and antibiotic-
resistant bacteria (both Gram-positive and Gram-negative) of

2806 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Broccolo et al.
Table 1. MICs (mg/L) for Compounds 1-5, 18, 26, and Reference Compounds 27 and 28a
S. pyogenes (6)
S. pneumoniae (9)
MIC
range
Enterococcus spp (2)
MIC
MRSA (3)
MIC
MIC
range
compd
range
MIC50
MIC90
MIC50
MIC90
range
MIC50
MIC90
MIC50
MIC90
1
2
3
4
128 ->128
128
128 ->128
32-64
64-128
128 ->128
>128
<0.06
<0.06
128
128
128
64
128
128
>128
<0.06
<0.06
128
128
>128
64
128
128->128
128
16-32
128
128
>128
<0.06-8
<0.06-2
128
128
128
32
128
128
>128
0.12
0.12
128
128
128
32
128
128
>128
8
>128
>128
>128
64
>128
>128
>128
2
>128
>128
>128
64
>128
>128
>128
2
>128
>128
>128
64
>128
>128
>128
2
>128
>128
>128
>128
>128
128
>128
>128
64
>128
>128
>128
>128
128
>128
>128
128
>128
>128
128->128
64-128
64->128
>128
5
128
18
26
27
28
>128
>128
<0.06
<0.06
64-128
>128
2
>128
>128
>128
>128
>128
MSSA (3)
S. epidermidis (6)
H. influenzae (4)
other Gram - (10)
MIC
range
MIC
range
MIC
range
MIC
range
compd
MIC50
MIC90
MIC50
MIC90
MIC50
MIC90
MIC50
MIC90
1
2
3
4
8->128
32->128
>128
>128
>128
>128
128
>128
>128
>128
>128
64
>128
>128
8
128 >128
128 >128
128 >128
128 >128
16-32
>128
128
>128
128
16
128
>128
1
>128
>128
>128
>128
32
>128
>128
4
128
128
128->128
128
128
128
128
128
32
128
128
1
128
128
>128
128
128->128
128->128
128->128
128->128
64-128
>128
>128
>128
>128
128
>128
>128
128
>128
>128
>128
>128
128
>128
>128
>128
>128
128->128
32-64
5
32
16-32
32
18
26
27
28
16->128
16->128
0.5-8
>128
>128
1
32->128
>128
128->128
128->128
0.5-64
>128
>128
64
128->128
128->128
1->128
0.25-4
1-32
2
2
2
8
32
<0.06 0.12
<0.06
0.12
<0.06 >128
<0.06
a The number of clinical isolates for each species is indicated in parentheses.
Chart 1. Structures of New 4-Alkyliden Beta Lactams 1-5, 18,
and Reference Compounds 26-28 Tested for the Antibacterial
Activity Reported in Table 1.
Figure 1. Three-component PCA scores plot for compounds 1-5, 18,
and 26-28.
an extremely difficult and time-consuming step. Moreover, the
use of these descriptors is not limited to 3D-QSAR and allows
the extention of their application in 3D searching, pharmaco-
phore identification, and structure-metabolism relations.
The 3D structures of the compounds in Chart 1were imported
in Mol-file and coded as ALMOND descriptors following the
procedure described in the Computational Methods section.
To obtain relevant VRSs, it is necessary to adopt probes
resembling potentially important groups of the binding site. For
compounds interacting with proteins, it seems reasonable to use
the DRY probe to represent hydrophobic interactions, the O
probe (carbonyl oxygen) to represent hydrogen bond acceptor
groups, and the N1 probe (amide nitrogen) to represent hydrogen
bond donor groups.
Principal components analysis (PCA) of ALMOND descrip-
tors relative to compounds tested afforded a five-principal-
components (PC) model explaining 93.4% of variance, 68.3%
1st PC, 5.2% 2nd PC, 6.2% 3rd PC, 6.0% 4th PC, and 7.7%
5th PC. Figure 1, the scores plot for the above model, depicting
the data structure elucidated by 3 PC (already explaining more
than 79% of variance), appears to be a simplified graphical
representation for an immediate evaluation of 3D structural MIF
results for the compounds when interacting with the three above-
mentioned probes.
Figure 1 shows that commercially available drugs, amoxicillin
27 and ceftriaxone 28, exhibit a high value in the x axis, the
main direction representative of interactions with the VRS
expected to be related to overall antibacterial activities. Ac-
cordingly, all 4-alkyliden-azetidin-2-ones, exhibiting low activ-
ity, lie in the left part of the plot with negative x values. It is
worth mentioning that 4 and 5, moderate antibacterials with
negative x values in Table 1, are characterized by the lowest
and the highest y values, respectively in Figure 1. The latter
features suggest that their activity could be ascribed to peculiar
interactions with the selected probes evidenced by the 2nd PC.
Relevant information on the changes in molecular structures
in relation to their interactions with the selected probes (Dry,
O, and N1) can be obtained by an inspection of the correlograms
(Computational methods). In the correlograms for the com-
pounds in Chart 1with the hydrophobic probe, the O probe, and
the N1 probe (from left to right) shown in Figure 2, striking
differences can be envisaged in their interactions with the O

4-Alkyliden-beta Lactam Antibiotic ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2807
Figure 4. Correlograms for compounds 1-30 with the O probe. The
four curves indicate profiles for 27-30.
Figure 2. Correlograms for compounds 1-5, 18, and 26-28 with the
hydrophobic probe, the O probe, and the N1 probe (from left to right).
The two curves indicate profiles for 27 and 28.
the O probe, which are comparable with the correlogram profiles
of marketed drugs 27-30.
Table 2 shows that compound 6 was the most active of the
new antibacterial compounds tested (MIC50 ranging from 1 to
32 mg/L) with the broadest spectrum of antibacterial activity
(S. pyogenes, Enterococcus spp., MRSA, MSSA, S. epidermidis,
and H. influenzae). Compound 8 was less active but with a
similar spectrum of activity as that of compound 6. The
antibacterial activity of chloro derivatives 5, 6, and 8, exhibiting
negative x values in Figure 3, could be explained by complex
processes involving nonspecific toxic effects12 rather than
specific interactions with PBPs, as evidenced by the citotoxicity
studies described below.
Compounds 21 and 22 were shown to have a relatively good
antibacterial activity, specifically, against methicillin-susceptible
staphylococci (MIC50 ranging from 2 to 16 mg/L). In particular,
the lowest MIC values were shown by compound 22 against
Staphylococcus epidermidis (0.25 mg/L), value comparable to
clinically used antibiotics.
Figure 3. Three-component PCA scores plot for compounds 1-30.
probe (center). Taking into account that every peak in the
correlogram indicates the VRS at a distance corresponding to
the abscissa of the peak it is evident that apart from 18 showing
some interactions in the range 6-10 Å alkyliden-azetidinones
do not interact at all with the O probe at distances in the range
2-21 Å. The insertion of hydroxyl groups at appropriate
distances in alkyliden-azetidinones is expected to provide better
interactions that might result in increased antibacterial activities.
Thus, further structural modifications on the set of 4-alkyliden
beta lactams were aimed at verifying the effects of the carboxylic
function in relation to the corresponding ethyl or benzyl esters
and the mono or polyphenolic substituents at appropriate
positions. A principal components analysis (PCA) of ALMOND
descriptors relative to compounds 1-30 afforded a second five-
principal-components (PC) model explaining 65.3% of variance,
32.5% 1st PC, 15.8% 2nd PC, 7.3% 3rd PC, 5.5% 4th PC, and
4.2% 5th PC. Figure 3, the scores plot depicting the data
structure in the above model elucidated by 3 PC (explaining
55.6% of variance), shows clearly that compounds 1-30 are
equally distributed in the eight octants of the 3D plot. The four
commercially available active compounds (27-30) exhibit
highly positive x values. Consequently, in the present model,
compounds with positive x values, whose structures are
characterized by phenolic (9, 10, 12, 17, 20, 22, and 25) and
carboxylic functions (23 and 24), are shown to interact better
with the selected probes of the VRS and are expected to exhibit
antimicrobial activity. Interestingly, Figure 4 points out that
several designed compounds exhibit significant interactions with
Compounds 9 and 10 were found to have moderate antibacte-
rial activity against some Gram-positive bacteria, such as S.
pyogenes, S. pneumoniae, and Staphylococci spp. More interest-
ing, however, was the finding that compounds 6, 9, 10, and 22
showed very similar antibacterial activity against both MRSA
and MSSA. Methicillin resistance in strains of Staphylococcus
spp. is brought about by the acquisition of a new PBP, PBP 2A
with low affinity for all beta-lactam agents. Therefore, the
methicillin-resistant phenotype usually brings about cross
resistance of the isolate to all other beta-lactam agents. The
antibacterial activity of compounds 6, 9, 10, and 22 may,
therefore, be indicative of an alternative target of action.
To investigate the mechanisms of action of compounds 6 and
22, which were found to be most active against MRSA, further
studies were carried out to determine the possibility of non-
specific toxicity of these agents as well as their effect on
bacterial cell wall formation.
Cytotoxicity studies using a human cell lines were carried
out evaluating the effect of the new compounds on cell viability
at concentrations above and below those shown to be antimi-
crobial.
The highest cytotoxic effect was observed with compound
6. The cells showed a decreased viability to 48% at 128 µM
following a 3 h exposure. After 21 h, a viability of 81% was
observed at a concentration of 8 µM, with a progressive decrease
at higher concentrations, reaching 23% at 128 µM. Compound
22 showed little or no cytotoxicity on the cell line after 3 h.

2808 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Broccolo et al.
Chart 2. Structures of New 4-Alkyliden Beta Lactams 6-12, 15-17, and 19-25 and Reference Compounds 29-30 Tested for the
Antibacterial Activity Reported in Table 2.
Upon exposure of the cell culture for 21 hours, a slight
progressive cytotoxic effect was seen starting at a concentration
of 32 µM with a viability of 81%, reaching 64% at 128 µM,
compared to that of the control.
the cooperative functioning of PBP2, PBP4, and PBP2A in the
antibiotic susceptibility of Staphylococcus aureus provided.15
However, the role of other PBPs remains to be explored, and
specific drug interactions with different bacterial strains have
not been fully elucidated. In this context, the modeling VRS
approach demonstrates its utility in suggesting structural
modifications for the design of more active compounds.
However, it is worth mentioning that an increased in silico
drug-receptor interaction does not necessarily imply an in-
creased in vitro antimicrobial activity because it is well known
that the penetration of cell membranes is related to the lipid
solubility of the drug, an important property governing the
passage through membrane barriers. Therefore, the drug partition
coefficient plays a fundamental role in its absorption and
consequently, in its activity.
Table 2 shows clearly that the increased activity of com-
pounds 9, 10, 17, 20, 21, 22, and 25, paralleled by positive x
values in Figure 3, due to the presence of phenolic OH and/or
OMe groups,16 results in higher affinities of the designed
structures for the VRS. Partition coefficients (clogP), calculated
by HINT!17 for the above drugs (3.66 for 9, 2.98 for 10, 1.99
for 17, 5.76 for 20, 2.34 for 21, 3.67 for 22, and 1.33 for 25)
point out hydrophobicity values consistent with an acceptable
permeability and with the observed in vitro activities.
Preliminary in vitro cell morphology studies by scanning
electron microscopy (SEM) based on the exposure of a MSSA
and a MRSA strain to a 8 × MIC concentration of compound
6, 22, and penicillin at several time-points pointed out that these
new 4-alkyliden-beta lactams bring about similar cellular
morphological changes with both MSSA and MRSA. This
finding is in contrast to the behavior of penicillin, which, as
expected, produced cell lysis in the MSSA strain but not with
the MRSA isolate. Compound 6 showed a massive reduction
of the bacterial cellular pellet during bacterial preparation for
microscopy, which was also associated with large amounts of
cell debris on microscopic visualization. This finding together
with the results of cytotoxic studies would point to a nonspecific
cellular toxicity of 6, particularly, at high concentrations.
Exposure to compound 22 brought about the formation of large
abnormally shaped cells. This dysfunction in bacterial cell wall
formation may arise from the interaction of this compound with
another PBP involved in the synthesis of peptidoglycan and
present in both MRSA and MSSA.
The role of PBP2A in the resistance for methicillin-resistant
Staphylococcus aureus (MRSA)13 or PBP2X for Streptococcus
pneumoniae14 has been pointed out and elegant evidences for
However, the presence of carboxylic functionalities results
in the lack of activity for compounds 7, 8, 15, 23, and 24. The

4-Alkyliden-beta Lactam Antibiotic ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2809
Table 2. MIC (mg/L) for Compounds 6-12, 15-17, 19-25 and Reference Compounds 29-30a
S. pyogenes (6)
S. pneumoniae (9)
MIC
Enterococcus spp (2)
MIC
range
MRSA (3)
MIC50
MIC
range
MIC
range
compd
MIC50
32
MIC90
range
MIC50
MIC90
MIC50
MIC90
MIC90
6
16-128
128
>128
128
16
32
64-128
128
128
128
128
128
128
16
16
16
16
16-32
>128
16
>128
128
32
64
>128
>128
>128
>128
>128
>128
>128
128
32
7
8
128->128
4-128
128
16
16
128
128
128
>128
128
64-128
8-16
128
16
32
128->128
128
64
>128
64
16-128
32-64
64-128
>128
9
8-16
64
64
10
11
12
15
16
17
19
20
21
22
23
24
25
29
30
32
32
32
32
128
128
128
128
128->128
128->128
128
128
>128
128
>128
>128
128
>128
>128
>128
>128
>128
128
>128
>128
>128
<0.06
<0.06
128->128
64->128
128
128
>128
128
>128
>128
128
>128
128
>128
>128
>128
128
128
128
>128
1
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
128
>128
>128
128
>128
>128
128
>128
>128
>128
128
>128
>128
128->128
>128
32->128
128->128
128
64-128
128->128
16->128
>128
>128
8->128
>128
>128
>128
>128
128->128
128->128
>128
2-128
1-64
128
128
128
32
64
128->128
128->128
128->128
<0.06
>128
>128
>128
<0.06
<0.06
128
128
>128
>128
>128
0.5
>128
>128
>128
>128
128
64
32
>128
>128
128
128
32
128
128
>128
>128
0.5->128
2->128
128->128
<0.06-1
<0.06-2
128
0.12
<0.06
8-128
32-128
32
<0.06
2
2
MSSA(3)
MIC50
S. epidermidis (6)
MIC
range
H. influenzae (4)
MIC
range
other Gram - (10)
MIC
MIC
range
compd
MIC90
MIC50
MIC90
MIC50
MIC90
range
MIC50
MIC90
6
7
8
9
8-16
8
16
>128
64
32
64
>128
>128
>128
>128
>128
>128
>128
16
8-32
8
32
>128
64
1-2
1
2
128->128
128->128
16-128
128
>128
32
>128
>128
128
>128
32-64
32
>128
32
32
64
>128
>128
>128
>128
>128
>128
>128
8
>128
>128
64
32
>128
8-64
>128
64
>128
64
32-64
16-64
64
128->128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
<0.06
<0.06
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
0.12
10
11
12
15
16
17
19
20
21
22
23
24
25
29
30
64
64->128
>128
128
>128
>128
>128
>128
8
>128
>128
4
>128
>128
>128
>128
>128
>128
>128
>128
16
>128
>128
>128
>128
16->128
>128
>128
8-16
8-128
>128
>128
64-128
<0.06
<0.06
>128
>128
>128
>128
-
>128
>128
128->128
32-128
128
128
128
<0.06-0.12
<0.06
>128
>128
>128
>128
-
>128
>128
>128
64
128
128
128
<0.06
<0.06
>128
>128
>128
>128
-
>128
>128
>128
128
128
128
128
0.12
>128
>128
>128
>128
>128
64->128
8->128
>128
>128
>128
128->128
128->128
128->128
128->128
128->128
128->128
128->128
<0.06-0.12
<0.06
>128
2-16
0.25-64
64->128
64->128
8-64
16
128
2
64
>128
>128
128
<0.06
<0.06
>128
>128
128
<0.06
<0.06
>128
>128
16
<0.06
0.12
>128
>128
64
1
64
<0.06-1
<0.06-64
<0.06
<0.06
a The number of clinical isolates for each species is indicated in parentheses.
above structure-activity relationships are more evident when
comparing active ester precursors 21 (clogP 2.34) and 22 (clogP
3.67) with the corresponding inactive carboxylic acids 23 (clogP
-1.48) and 24 (clogP -0.16). The lack of activity of the latter
acids (exhibiting positive x values in Figure 3 and, therefore,
significant interactions with the VRS) as compared to that of
the corresponding esters could be related to the permeability
difficulties indicated by their higher hydrophilicities.
action as compared to that of typical beta-lactams. A molecular
modeling approach using GRID descriptors based on the concept
of the VRS allowed the identification of interactions through
oxygenated functions such as phenolic OH, which are valuable
for the antibacterial activity and contributed to the rationalization
of functional group effects in QSARs. Although the compounds
reported here do not exhibit outstanding antibacterial activities,
they could represent lead structures for the development of new
4-alkyliden-beta-lactams specifically designed against resistant
bacteria.
Conclusions
In conclusion, the reported 4-alkyliden-beta-lactams constitute
a family of new compounds with interesting antibacterial
properties, possessing, apart from the monocyclic azetidinone
framework, a very different structure with respect to monobac-
tam antibiotics.
The antibacterial activity of the 4-alkyliden-beta-lactams
tested was, overall, not outstanding; however, some compounds
were found to have an inhibitory effect, generally, more marked
against Gram-positive pathogens, although the spectrum of
activity varied. Interestingly, the generally undifferentiated
antibacterial activity of some of the studied compounds against
both methicillin-susceptible and -resistant strains of Staphylo-
coccus aureus was suggestive of an alternative mechanism of
Experimental Section
General Procedures. Commercial reagents were used as re-
ceived without additional purification. Anhydrous solvents were
1
obtained commercially and used without further drying. H- and
13C NMR values were recorded on a VARIAN Mercury 400,
INOVA 300, or GEMINI 200 instrument using a 5 mm probe. All
chemical shifts have been quoted relative to deuterated solvent
signals, δ in ppm and J in Hz. Data and spectra are reported in the
Supporting Information. FT-IR: Nicolet 205 measured as films
between NaCl plates and reported in cm-1. TLC: Merck 60 F254
.
Column chromatography: Merck silica gel 200-300 mesh. HPLC-
MS, HPLC: Agilent Technologies HP1100, column ZOBRAX-
Eclipse XDB-C8 Agilent Technologies. The compounds were eluted

2810 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Broccolo et al.
with CH3CN/H2O, gradient from 30 to 80% of CH3CN. Acid
compounds were eluted with a CH3CN/H2O mixture containing
0.1% of formic acid. MS: Agilent Technologies MSD1100 single-
quadrupole mass spectrometer, full-scan mode from m/z 50 to 2600,
scan time 0.1 s in positive ion mode, ESI spray voltage 4500 V,
nitrogen gas 35 psgi, drying gas flow 11.5 mL/min, fragmentor
studied by determining their minimum inhibitory concentrations
(MICs) by means of the broth microdilution method according to
the CLSI (Clinical and Laboratory Standards Institute, formerly the
NCCLS) guidelines. Briefly, serial 2-fold dilutions of each antibiotic
were made using the Mueller-Hinton broth in microtiter plates with
96 wells. An equal volume of the bacterial inoculum (5 × 105 CFU/
mL) was added to each well on the microtiter plate containing 0.05
mL of the serial antibiotic dilutions. The microtiter plate was then
incubated at 35 °C for 16-20 h after which each well was analyzed
for the presence of bacterial growth. The MIC was defined as the
lowest concentration of antimicrobial agent able to cause inhibition
of bacterial growth as shown by the lack of turbidity of the culture
medium. The results have been reported as MIC ranges, MIC50
and MIC90 (the minimum concentrations able to inhibit 50 and 90%
of the tested isolates, respectively), for the different bacterial species,
despite the small number of strains tested to show the different
spectrum of activity of the new compounds.
The in vitro antibacterial activity of 23 new 4-alkyliden-azetidin-
2-ones was tested and compared to that of reference beta-lactam
agents in clinical use: amoxicillin (Sigma-Aldrich), ceftriaxone
(Sigma-Aldrich), imipenem (Merk Sharp Dohme), and Meropenem
(AstraZeneca).
Cell Viability Assay. Cells from a human cell line (TPC1) were
seeded into 96-well plates (Nunc, Roskilde, Denmark) at 40 000
cells/well in 200 µL of supplemented DMEM medium and cultured
overnight at 37 °C and 5% CO2. On the following day, the medium
was changed to a fresh medium and compounds 6 and 22 at
concentrations of 0, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 µM
in 200 µL of supplemented medium was added to the wells. Each
concentration and controls was tested using six parallel wells. The
cells were cultured at 37 °C and 5% CO2 for 3, 6, and 21 h, and
the viability of the cells was determined using an MTT-assay (van
de Loosdrecht AA).
voltage 20 V. The [R]25 values were determined with a Perkin-
D
Elmer 343 polarimeter. Melting points were determined using a
Bu¨chi apparatus and are uncorrected. The purities of the target
compounds were assessed as being >95% using HPLC with two
diverse systems (see Supporting Information for details).
Compounds 1, 2, 5, 7, and 26 have been prepared according to
ref 11. Compounds 3, 4, and 6 have been prepared according to
the same reported procedure11 using benzyldiazoacetate18 as the
diazocarbonyl compound.
Compounds 8, 15, 23, and 24 were obtained by hydrogenolysis
of the corresponding benzylesters. A representative procedure is
the following: Compound 21 (0.444 g, 0.976 mmol) was dissolved
in an anhydrous mixture THF/MeOH (4 mL/4 mL) and Pd. A 10
wt % of activated carbon, (0.045 g) was added. Finally, the reaction
mixture was treated with H2 (1 atm), and it was stirred at room
temperature for 4 h. Then it was filtered and concentrated to give
compound 23.
Compounds 9, 10, 11, and 12 were obtained starting from 7 via
DCC coupling with the corresponding amine, amino acid, or alcohol
(Scheme 3). A representative procedure is as follows: Compound
7 (0.065 g, 0.228 mmol) was dissolved in anhydrous CH2Cl2 (3
mL), and the solution was brought to 0 °C. Then DCC (0.052 g,
0.251 mmol), 1,3,5-trihydroxybenzene (0.045 g, 0.274 mmol), and
catalytic DMAP (5.6 mg, 0.0456 mmol) were added at 0 °C. The
reaction mixture was stirred at room temperature for 4 h, then
concentrated EtOAc was added, and the mixture was filtered. The
liquid residue was concentrated and purified by flash chromatog-
raphy (cyclohexanes-ethyl acetate 60:40) to give compound 9. Note
that in the preparation of 10, 11, and 12 catalytic DMAP was not
necessary.
Compounds 16, 17, 18, 19, 20, 21, and 22 were obtained by
SnCl4 catalyzed O-acylation of the 3-hydroxyethyl chain. Com-
pounds 1 and 3 were deprotected on the 3-hydroxyethyl chain by
means of HCl 1N in CH3CN. A representative procedure for
compound 13 is as follows: Compound 1 (0.313 g, 1 mmol) was
dissolved in CH3CN (6 mL), and HCl 1N (1 mL) was added. The
reaction was monitored by TLC, and other 1 mL portions of HCl
1N were added till total conversion. After that, saturated NaCl
solution was added, and the mixture was extracted with CH2Cl2 (5
× 10 mL), dried on Na2SO4, concentrated, and the residue purified,
when necessary, by flash chromatography (cyclohexane/ethyl
acetate 4:6) or by trituration in pentane/Et2O. Yield 87%.
The obtained desilylated compound 13 (0.045 g, 0.226 mmol)
was dissolved in anhydrous benzene (4 mL), and then 3,4,5-
trimethoxybenzoyl chloride (0.104 g, 0.452 mmol) and, finally,
SnCl4 (0.023 mmol, 23 µL of a solution of 1M in CH2Cl2) were
added. The solution was heated to reflux and was stirred for 4 h.
Then the reaction mixture was quenched in a solution of NaHCO3
(5%), extracted with ethyl acetate (4 × 10 mL), dried on Na2SO4,
and concentrated. The residue was purified by flash chromatography
(cyclohexane/ethyl acetate 8/2) to give compound 18. Yield 50%.
Compound 25 was obtained in quantitative yield by hydro-
genolysis (THF/MeOH 1/1, Pd 10% on C, H2 1 atm) of the
corresponding tribenzyloxyderivative 19.
The MTT-solution (5 mg/mL) was prepared by dissolving 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and thiazolyl
blue (MTT) (Sigma, St. Louis, MO) in sterile D-PBS (Gibco or
BioWhittaker, Verviers, Belgium) and mixing with the medium at
a ratio of 1:15. The medium was removed, and the cells were
washed twice with the medium. After the addition of 100 µL of
MTT-solution to each well, the plates were incubated at 37 °C and
5% CO2 for 3 h. After removing the MTT-solution, the cells were
washed with D-PBS, and 100 µL of acidified 2-propanol solvent
was added to dissolve the intracellular crystals that may have
formed. The plates were then incubated at 37 °C for 15 min with
occasional shaking, and the optical density of each well was
measured at 600 nm using a micro-ELISA reader.
Computational Methods. MIFs were obtained using the pro-
gram GRID, version 20.19 GRIND were generated, analyzed, and
interpreted using the program ALMOND version 3.0.0 (www.mol-
discovery.com). Computations and graphical displays were per-
formed on SGI O2 workstations (MIPS R12000 processor). The
process of ligand-receptor interactions, represented with the help
of the MIF, is described using molecular descriptors based on the
concept of VRS. Basically, GRIND are a small set of variables
representing the geometrical relationships between relevant regions
of the VRS and, as such, are independent of the coordinate frame
of the space where the MIF is computed. The procedure for
obtaining GRIND involves three steps: (i) computing a set of MIFs,
(ii) filtering the MIFs to extract the most relevant regions that define
the VRS, and (iii) encoding the VRSs into the GRIND variables.
(i) Computing the MIF. To obtain relevant VRSs, it seems
reasonable to use the DRY probe to represent the hydrophobic
interactions, the O probe (carbonyl oxygen) to represent hydrogen
bond acceptor groups, and the N1 probe (amide nitrogen) to
represent hydrogen bond donor groups. In default mode, a grid-
spacing of 0.5 Å is used with the grid extending 5 Å beyond a
molecule.
Microbiological Assays. (i) Bacterial Strains. A total of 43
clinical isolates were used for the determination of the in vitro
antibacterial activity of the studied compounds. The bacterial strains
included the following Gram-positive (29) and Gram-negative (14)
species: S. pyogenes (6), S. pneumoniae (9), Enterococcus spp.
(2), methicillin-resistant S. aureus (MRSA) (3), methicillin-
susceptible S. aureus (MSSA) (3), S. epidermidis (6), H. influenzae
(4), M. catarrhalis (1), E. coli (3), P. mirabilis (2), S. marcescens
(2), and K. pneumoniae (2).
(ii) Filtering the MIF. A previously decribed procedure,8
defining the most interesting regions as those characterized by
intense favorable (negative) energies of interaction by means of
an algorithm to select from each MIF a fixed number of nodes
(ii) Determination of Minimum Inhibitory Concentrations
(MICs). The in vitro antibacterial activity of the new agents was

4-Alkyliden-beta Lactam Antibiotic ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2811
(3) Cainelli, G.; Galletti, P.; Garbisa, S.; Giacomini, D.; Sartor, L.;
Quintavalla A. 4-Alkylidene-azetidin-2-ones: Novel inhibitors of
leukocyte elastase and gelatinase. Bioorg. Med. Chem. 2003, 11,
5391-5399.
(4) Kazi, A.; Hill, R.; Long, T. E.; Kuhn, D. J.; Turos, E.; Dou, Q. P.
Novel N-thiolated-beta-lactam antibiotics selectively induce apoptosis
in human tumor and transformed, but not normal or nontransformed,
cells. Biochem. Pharmacol. 2004, 67, 365-374.
(5) World Health Organization. OVercoming antimicrobial resistance;
WHO Report on Infectious Diseases, 2000. www.who.int/infectious-
disease-report/2000/index.html.
(6) Power, J. H. Development of drugs for antimicrobial-resistant
pathogens. Curr. Opin. Infect. Dis. 2003, 16, 547-551.
(7) Reichert, J. M. Trends in drug development and approval times for
new therapeutics in the United States. Nat. ReV. Drug DiscoVery 2003,
2, 695-702.
(8) Pastor, M.; Cruciani, G.; McLay, I.; Pickett, S.; Clementi, S. Grid-
independent descriptors (GRIND): A novel class of alignment-
independent three-dimensional molecular descriptors. J. Med. Chem.
2000, 43, 3233-3243.
optimizing a scoring function, was adopted. This function, including
the intensity of the field at a node and the mutual distances between
the chosen nodes as optimality criteria, extracts from each field a
number of nodes (in the order of 150-200) that represent
independent, favorable, probe-ligand interaction regions.
(iii) Encoding the VRS into GRIND. GRIND encodes the
geometrical relationships between the VRS regions so that they
are independent of their positions in the 3D space by using an auto-
and cross-correlation transform.8 For each probe, the procedure
computes the product of the interaction energy for each pair of
filtered nodes, storing only the highest product, a peculiarity
responsible for the reversibility properties of GRIND. This method
is called maximum auto- and cross-correlation (MACC) or, more
specifically, MACC-2.9 The values obtained from the analysis can
be represented directly in correlogram plots, where the products of
the node-node energies are reported versus the distance separating
the nodes.
(9) ALMOND Molecular Discovery, Ltd. www.moldiscovery.com.
(10) Bachi, M. D.; Goldberg, O.; Grass, A.; Vaya, J. Properties and
reactions of 4-thioxo-2-azetidinones. J. Org. Chem. 1980, 45, 1481-
1484. (b) Prasad, K.; Kneussel, P.; Schulz, G.; Stutz, P. A new
method of carbon extension at C-4 of azetidinones. Tetrahedron Lett.
1982, 23, 1247-1250; (c) Greengrass, C. W.; Hoople, D. W. Reaction
of 4-acetoxy-2-azetidinone with tertiary carbanions: Preparation of
4-alkyl- and 4-alkylidene-2-azetidinones. Tetrahedron Lett. 1981, 22,
1161-1164. (d) Battaglia, A.; Cainelli, G.; Giacomini, D.; Martelli,
G.; Panunzio, M. Cycloaddition reaction of heterocumulenes and ester
enolates: A novel synthesis of 4-alkylidene-azetidin-2-ones. Tetra-
hedron Lett. 1987, 28, 4347-4350.
(11) Cainelli, G.; Giacomini, D.; Galletti, P.; Quintavalla, A. Synthesis
of novel 4-(2-oxoethylidene)azetidin-2-ones by a Lewis acid mediated
reaction of acyldiazo compounds. Eur. J. Org. Chem. 2003, 9, 1765-
1774
(12) Bogdanov, P. M.; Dabbene, V. G.; Albesa, I.; de Bertorello, M. M.;
Brino`n, M. C. Mechanism of antibacterial and degradation behavior
of a chlorinated isoxazolynaphthoquinone. Biochem. Biophys. Res.
Commun. 1999, 263, 301-307
(13) Lim, D.; Strynadka, C. J. Structural basis for the â-lactam resistance
of PBP2a from methicillin-resistance Staphylococcus aureus. Nat.
Struct. Biol. 2002, 9, 870-876.
(14) Dessen, A.; Mouz, N.; Gordon, E.; Hopkins, J.; Dideberg, O. Crystal
structure of PBP2x from a highly penicillin-resistant Streptococcus
pneumoniae clinical isolate: a mosaic framwork containing 83
mutations. J. Biol. Chem. 2001, 276, 45106-45112.
(15) Leski, T. A.; Tomasz, A. Role of Penicillin-Binding Protein 2 (PBP2)
in the antibiotic susceptibility and cell wall cross-linking of Staphy-
lococcus aureus: Evidence for cooperative functioning of PBP2,
PBP4, and PBP2A. J. Bacteriol. 2005, 187, 1815-1824.
(16) Jones, R. N.; Erwin, M. E. In vitro evaluation of Ro 09-1227, a novel
catechol-substituted cephalosporin. Antimicrob. Agents Chemother.
1992, 36, 233-238.
(17) Kellogg, G. E.; Semus, S. F.; Abraham, D. J. HINT - A new method
of empirical hydrophobic field calculation for CoMFA. J. Comput.-
Aided Mol. Des. 1991, 5, 545-552.
Molecular Description: GRID Force Field. The GRID pro-
gram20 was used to describe the molecular structures. GRID is a
computational procedure calculating, at each point, the interaction
energy between the probe and the target molecule as the sum of
Lennard-Jones (ELJ), hydrogen bond (EHB) electrostatic interactions
(EEL) and, for specific probes, the entropic contribution (EENT
)
N
N
N
N
Ex,y,z
)
ELJ
+
EHB
+
EEL
+
EENT
∑
∑
∑
∑
i ) 1
i ) 1
i ) 1
i ) 1
GRID contains parameters describing each type of atom in each
ligand molecule. These parameters define the strength of the
Lennard-Jones, hydrogen bond, and electrostatic interactions used
to evaluate the energy functions. GRID give precise spatial
information, and this specificity and sensitivity are an advantage
because they may be representative of the important chemical
groups present in the active site.
Calculation of clogP. The partition coefficients calculations
(clogP) were performed by HINT! on the basis of the hydrophobic
fragment constant approach of Hansch and Leo21 in which fragment
constants (and some atom constants) for a variety of organic species
of biological importance are tabulated.
Acknowledgment. We thank Dr. L. Giurato (Department
of Pharmaceutical Sciences, University of Catania) for the
calculation of clogP, Mr. Andrea Garelli for HPLC/MS analysis,
Mrs. Diana Rambaldi for experimental assistance, and Dr.
Antonello Villa (Consorzio MIA, University of Milano-Bicocca)
for the expertise on experiments using SEM. This work was
supported by MURST (ex-60% and COFIN 2004).
(18) Benzyldiazoacetate is a known compound: Angle, S. R.; Belanger,
D. S. Stereoselective synthesis of 3-hydroxyproline benzyl esters from
N-protected-beta-aminoaldehydes and benzyl diazoacetate. J. Org.
Chem. 2004, 69, 4361-4368, and references citated herein. We
synthesized this compound using the procedure as described for
similar compounds in: Danheiser, R. L.; Miller, R. F.; Brisbois, R.
G.; Park, S. Z. An improved method for the synthesis of alpha-diazo
ketones. J. Org. Chem. 1990, 55, 1959-1964.
Supporting Information Available: 1HNMR and 13CNMR data
and spectra for all new compounds and HPLC for key target
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
(19) GRID, version 20; Molecular Discovery, Ltd. (www.
moldiscovery.com).
(20) Goodford, P. J. A computational procedure for determining energeti-
cally favourable binding sites on biologically important macro-
molecules. J. Med. Chem. 1985, 28, 849-857. (b) Boobbyer, D. N.
A.; Goodford, P. J.; Mcwhinnie, P. M.; Wade, R. C. New hydrogen-
bond potentials for use in determining energetically favourable
binding sites on molecules of known structure. J. Med. Chem. 1989,
32, 1083-1094. (c) Wade, R.; Clerk, K. J.; Goodford, P. J. Further
development of hydrogen bond function for use in determining
energetically favourable binding sites on molecules of known
structure. Ligand probe groups with the ability to form two hydrogen
bonds. J. Med. Chem. 1993, 36, 140-147.
References
(1) Wright A. J. The penicillins. Mayo Clin. Proc. 1999, 74, 290-307.
(2) Konaklieva, M. I. Beta-lactams as inhibitors of serine enzymes. Curr.
Med. Chem.:-Anti-Infect. Agents 2002, 1, 215-238. As recent
examples of beta-lactam inhibitors of HLE see: (b) Singh, R.;
Micetich, R. G. Monobactams as enzyme inhibitors. IDrugs 2000,
3, 512-517. (c) Clemente, A.; Domingos, A.; Grancho, A. P.; Iley,
J.; Moreira, R.; Neres, J.; Palma, N.; Santana, A. B.; Valente, E.
Design, synthesis and stability of N-acyloxymethyl- and N-amino-
carbonyloxymethyl-2-azetidinones as human leukocyte elastase in-
hibitors. Bioorg. Med. Chem. Lett. 2001, 11, 1065-1068. (d) Koteva,
K. P.; Cantin, A. M.; Neugebauer, W. A.; Escher, E. Synthesis and
evaluation of novel â-lactam inhibitors of human leukocyte elastase.
Can. J. Chem. 2001, 79, 377-38.
(21) Hansch, C.; Leo, A. Substituent Constant for Correlation Analysis
in Chemistry and Biology; Wiley and Sons: NewYork, 1979.
JM0580510