Antibacterial Agents and Cystic Fibrosis: Synthesis and Antimicrobial Evaluation of a Series of N-Thiomethylazetidinones

Article abstract of DOI:10.1002/cmdc.201100282

The increasing emergence of multidrug-resistant microorganisms is one of the greatest challenges in the clinical management of infectious disease. New antimicrobial agents are therefore urgently required, particularly in the treatment of chronic and recurrent infections often associated with antibiotic-resistant pathogens, as in the case of cystic fibrosis (CF) patients. This study reports the antibacterial activity of a series of monocyclic β-lactams with an alkylidenecarboxyl chain or electron-withdrawing groups such as 4-OAc, 4-SAc, and 4-SO₂Ph at the C4 position of the ring. N-Unsubstituted and N-thiomethyl derivatives were compared. A total of 33 azetidinones were tested for their activity against Gram-positive and Gram-negative bacterial clinical isolates. The combination of an N-thiomethyl group and a benzyl ester on the 4-alkylidene side chain were found to increase the potency against Gram-positive bacteria. The N-thiomethyl group clearly elevated the activity of 4-acetoxyazetidinones relative to the corresponding NH derivatives. The most active compounds showed minimum inhibitory concentration (MIC) values of 4 and 8mgL^-1 against methicillin-resistant Staphylococcus aureus isolated from pediatric patients with CF.

Full text of DOI:10.1002/cmdc.201100282

DOI: 10.1002/cmdc.201100282
Antibacterial Agents and Cystic Fibrosis: Synthesis and
Antimicrobial Evaluation of a Series of
N-Thiomethylazetidinones
Paola Galletti,[a] Clementina E. A. Cocuzza,*[b] Matteo Pori,[a] Arianna Quintavalla,[a]
Rosario Musumeci,[b] and Daria Giacomini*[a]
The increasing emergence of multidrug-resistant microorgan-
isms is one of the greatest challenges in the clinical manage-
ment of infectious disease. New antimicrobial agents are there-
fore urgently required, particularly in the treatment of chronic
and recurrent infections often associated with antibiotic-resist-
ant pathogens, as in the case of cystic fibrosis (CF) patients.
This study reports the antibacterial activity of a series of mono-
cyclic b-lactams with an alkylidenecarboxyl chain or electron-
withdrawing groups such as 4-OAc, 4-SAc, and 4-SO2Ph at the
C4 position of the ring. N-Unsubstituted and N-thiomethyl de-
rivatives were compared. A total of 33 azetidinones were
tested for their activity against Gram-positive and Gram-nega-
tive bacterial clinical isolates. The combination of an N-thio-
methyl group and a benzyl ester on the 4-alkylidene side chain
were found to increase the potency against Gram-positive bac-
teria. The N-thiomethyl group clearly elevated the activity of 4-
acetoxyazetidinones relative to the corresponding NH deriva-
tives. The most active compounds showed minimum inhibitory
concentration (MIC) values of 4 and 8 mgLꢀ1 against methicil-
lin-resistant Staphylococcus aureus isolated from pediatric pa-
tients with CF.
Introduction
The widespread emergence of resistance to antibiotics in
pathogenic bacteria over the past 30 years is now a serious
threat in the treatment of infections.[1] Whereas antibiotic re-
sistance was once largely confined to hospitals and long-term
care facilities, it has now emerged in community settings and
represents one of the most pressing global public health con-
cerns. In extreme cases, multidrug-resistant bacteria are no
longer susceptible to any of the licensed antibacterial agents.
Some patients are especially vulnerable to acquiring multi-
drug-resistant bacterial infections as a consequence of either
underlying illnesses, such as cystic fibrosis (CF) and cancer, or
medical procedures such as organ transplantation and hemo-
dialysis. Resistance is an inevitable side effect of antibiotic use
and requires the search for new bacterial targets and novel
therapeutic agents to treat drug-resistant microorganisms.
For approximately four decades (from the early 1940s to the
late 1970s) the pharmaceutical industry provided a steady flow
of new antibiotics, including several agents with new mecha-
nisms of action. Since then only two new classes of antibiotics,
oxazolidinones and lipopeptides, which include agents such as
linezolid and daptomycin, have been marketed in Europe to
treat infections caused by multidrug-resistant Gram-positive
bacteria. Meanwhile, multidrug resistance among Gram-nega-
tive bacteria has been increasing relentlessly, with only few
new active antibacterial agents reaching the market in the last
decade.[1c]
bacterial cell wall by covalently binding to nucleophilic active-
site serine residues in d,d-transpeptidases (called penicillin-
binding proteins, PBPs), which are involved in the extracellular
assembly of bacterial peptidoglycan, the essential component
of the cell wall.[3]
However, just a few years after the introduction of penicillin
in clinical practice, penicillin-resistant strains of Staphylococcus
aureus were isolated, a Gram-positive pathogen causing severe
illnesses such as surgical wound infections, pneumonia, osteo-
myelitis, endocarditis, and sepsis. Resistant S. aureus destroys
penicillin by expressing and secreting the enzyme b-lactamase.
Although little is known about the genotypes of the original
resistant strains, it is believed that the b-lactamase gene was
spread from strain to strain primarily on plasmids, probably via
S. aureus bacteriophages. Today most staphylococcal strains
are known to be b-lactamase producers. Shortly after the intro-
duction of b-lactamase-resistant semi-synthetic penicillins such
as methicillin, a new resistance mechanism to these drugs was
reported with the emergence of methicillin-resistant S. aureus
(MRSA).[4]
[a] Dr. P. Galletti, M. Pori, Dr. A. Quintavalla, Prof. Dr. D. Giacomini
Department of Chemistry “G. Ciamician”
University of Bologna, Via Selmi 2, 40126 Bologna (Italy)
Fax: (+39)051 209 9456
E-mail: daria.giacomini@unibo.it
[b] Prof. C. E. A. Cocuzza, Dr. R. Musumeci
Department of Clinical Medicine and Prevention
University of Milano-Bicocca, Via Cadore 48, 20052 Monza (Italy)
Fax: (+39)02 6448 8363
b-Lactam antibiotics are still the main class of agents used
to treat bacterial infections.[2] Their activity is strictly related to
the presence of the four-membered azetidinone ring. They
share a common mode of action, inhibiting biosynthesis of the
E-mail: clementina.cocuzza@unimib.it
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C. E. A. Cocuzza, D. Giacomini, et al.
MED
against resistant bacteria,[11] and
as effective enzyme inhibitors.[12]
In these derivatives the carbon–
carbon double bond directly
linked to the azetidinone ring
confers an increased aptitude
toward ring-opening reactions.
In this perspective, the C=C acts
as
an
electron-withdrawing
group following the model for
inhibitory activity of monocyclic
b-lactams.[13] We focused the
design of the new 4-alkylidene-
b-lactams on monocyclic N-un-
substituted azetidinones bearing
a polyphenolic side chain at the
C3 or C4 position of the b-
lactam ring. Concerning antibac-
Figure 1. Overview of all monocyclic azetidinones tested.
terial activity, the 4-alkylidenea-
zetidinones show significant in-
hibitory activity against Gram-
Methicillin resistance—the major staphylococcal resistance
challenge today—is mediated by the acquisition of a novel
penicillin-binding protein 2a (PBP2a), encoded by the mecA
gene located in a mobile genetic element called the staphylo-
coccal cassette chromosome (SCC), which often carries resist-
ance genes to other classes of antibacterial agents. PBP2a
takes over the functions of other PBPs and evades inhibition
by methicillin and all other b-lactamase-insensitive b-lactams.[5]
The major spread of antibiotic resistance started in the early
1980s and continues today with the worldwide diffusion of vir-
ulent clones of MRSA in both healthcare-associated (HA-MRSA)
and community settings (CA-MRSA). A rise in S. aureus infec-
tions has also been reported as cause of respiratory tract infec-
tions in CF patients, with an increase in the prevalence of
highly virulent, multidrug-resistant MRSA.[6,7] Persistent MRSA
infections represent a new threat to the CF community, as
they are not only difficult to treat but are also associated with
biofilm formation, Panton–Valentine leukocidin (PVL) produc-
tion, as well as decreased lung function and patient survival.[8]
As respiratory failure continues to be the most common cause
of death in CF, appropriate antibiotic treatment constitutes a
major challenge for the future development of new active
agents against multidrug-resistant MRSA strains.
positive pathogens, generally undifferentiated against both
MRSA and methicillin-susceptible S. aureus (MSSA).[11]
Herein we report the synthesis and antibacterial evaluation
of a series of new monocyclic b-lactam derivatives with an al-
kylidenecarboxyl chain or electron-withdrawing groups at the
C4 position of the ring (Figure 1); among those, N-unsubstitut-
ed and N-thiomethyl derivatives were compared. Some of the
new N-thiomethylated b-lactams were found to have promis-
ing antibacterial activity.
Turos and co-workers reported the first synthesis and biolog-
ical evaluation of monocyclic b-lactams with an alkylthio group
on the lactam nitrogen atom.[14] These compounds were found
to be highly selective toward Staphylococcus and Bacillus spp.
and to possess completely unprecedented structure–activity
profiles for a b-lactam antibiotic. Unlike penicillin, which inhib-
its cell wall cross-linking enzymes, thus conferring broad-spec-
trum bactericidal activity, N-thiolated lactams are bacteriostatic
in their behavior and act through a different mechanistic
model.[15]
Results and Discussion
Beside bicyclic b-lactam substrates such as penicillins, cepha-
losporins, and carbapenems, monocyclic compounds have
emerged for their interesting and variegated biological activi-
ties. The first classes of monocyclic b-lactam antibacterial
agents were isolated from natural sources in the late 1970s
and early 1980s. The discovery of nocardicines and monobac-
tams was the initial demonstration that a conformationally
constrained bicyclic structure is not necessary for antibacterial
activity.[9] Research in this field is quite active, and reports of
new derivatives have appeared recently.[10]
Chemistry
A series of new azetidinones were evaluated in this study and
classified accordingly to core structures shown in Figure 1. Our
initial objective was to extend our previous study on 4-alkyli-
deneazetidinones by further modification of the b-lactam nitro-
gen atom by the addition of the thiomethyl group. The syn-
thesis of parent 4-alkylidene-b-lactams was conducted accord-
ing to a well-established protocol by starting from commercial-
ly available (3R,4R)-4-acetoxy-3-[(1R)-1-(tert-butyldimethylsily-
loxy)ethyl]azetidin-2-one and 4-acetoxyazetidin-2-one with
ethyl or benzyl diazoacetate, thus obtaining compounds 1, 3,
5, 7, 8, and 9 (Scheme 1).[16]
We have actively contributed to this field with the design
and synthesis of monocyclic b-lactams, having discovered 4-al-
kylideneazetidinones as interesting scaffolds for antibiotics
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Antibacterial Agents and CF
Scheme 1. Reagents and conditions: a) LDA or LiHMDSA, THF, ꢀ788C, then
MeSSO2Me (33–60%).
E and Z diastereomers were easily separated by flash chro-
matography. E isomers can be successfully thiomethylated on
the nitrogen atom after treatment with lithium diisopropyla-
mide (LDA) or lithium hexamethyldisilylamide (LiHMDSA) and
subsequent addition of S-methyl methanethiosulfonate to give
compounds 2, 4, and 6. Unfortunately, under the same reac-
tion conditions, Z isomers did not react, thus confirming a pe-
culiar reactivity of 4-(Z)-alkylideneazetidinones, as we previous-
ly observed in N-acylation reactions.[17] The C3 hydroxyethyl
chain can be further derivatized to phenolic aromatic esters by
preliminary deprotection of the tert-butyldimethylsilyl (TBS)
group with BF3·OEt2 (giving compound 13) followed by subse-
quent treatment in the presence of an aryl acid chloride and
tin(IV) chloride (5%) to give products 14 and 15 (Scheme 2).
Compounds 11 and 12, which lack the C=C double bond, were
synthesized as models for comparison with 4-alkylidene deriva-
tives (Scheme 2).[18]
Scheme 3. Reagents and conditions: a) MeCOSK/ZnBr2/dioxane or PhSO2Na/
H2O (quant); b) LDA or LiHMDSA, THF, ꢀ788C, then MeSSO2Me (31–88%);
c) BF3·OEt2, CH3CN, 08C (88–99%).
commercially available 4-acetoxyazetidinones 16 and 28 via
substitution reactions (Scheme 3).[19,20] Further elaborations
such as N-thiomethylation (compounds 17, 29, 21, 25, 31, and
33) and TBS removal (19, 23, and 27) were conducted with
procedures as described above.
Microbiological evaluation
Antibacterial activity screening was carried out for all new
compounds against a total of 116 recent clinical isolates. Gram-
positive and Gram-negative bacterial pathogens tested in-
clude: Streptococcus pyogenes, Streptococcus pneumoniae, En-
terococcus faecalis, Staphylococcus aureus, Staphylococcus epi-
dermidis, Haemophilus influenzae, Escherichia coli, Klebsiella
pneumoniae, Proteus mirabilis, and Serratia marcescens. Antimi-
crobial activities of the most active compounds are listed in
Table 1, with potency being expressed as the range of mini-
mum inhibitory concentrations (MICs, in mgLꢀ1) for the total
number of tested isolates belonging to each bacterial species
(MIC range), as well as the concentrations required to inhibit
the growth of 50 and 90% of the tested microorganisms
(MIC50 and MIC90, respectively). Only the results for compounds
showing MIC values of ꢁ32 mgLꢀ1 for at least one of the bac-
terial species tested are listed. Data are not shown for com-
pounds with no demonstrated antibacterial activity and for
bacterial species not found to be susceptible to any of the
tested molecules.
We then selected a second series of azetidinones in which
the 4-alkylidene side chain is substituted by electron-withdraw-
ing groups such as 4-acetoxy, 4-thioacetate, or 4-phenylsulfon-
yl derivatives. 4-Thioacetates 20 and 30, and 4-phenylsulfonyl
derivatives 24 and 32, were easily obtained by starting from
Promising potency against some of the bacterial strains
tested, particularly among Gram-positive pathogens, was ob-
served for some monocyclic b-lactams NH or N-thiomethyl.
Concerning 4-alkylidene derivatives, N-thiomethyl compounds
Scheme 2. Reagents and conditions: a) BF3·OEt2, CH3CN, 08C (80–100%);
b) ArylCOCl, SnCl4, benzene, reflux; c) LDA or LiHMDSA, THF, ꢀ788C, then
MeSSO2Me (33–74%).
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C. E. A. Cocuzza, D. Giacomini, et al.
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2 and 4 showed interesting activity against both susceptible
(MSSA) and resistant (MRSA) S. aureus strains. For compound 4
the best activity was observed against S. pyogenes (MIC range:
4–8 mgLꢀ1). Results for the corresponding NH 4-alkylideneaze-
tidinones previously reported[11] showed compound 1 to be in-
active, whereas 3 appeared to have some activity against
Gram-positive pathogens (MIC range: 16–32 mgLꢀ1 against
S. pneumoniae). Unprotected derivative 13 and aryl esters 14
and 15 were inactive (data not shown). Regarding C3-unsubsti-
tuted 4-alkylideneazetidinones, NH derivatives 7 and 9 showed
only modest activity against E. coli strains, whereas N-thio-
methyl compound 6 is inactive; C4 saturated analogues 10, 11,
and 12, tested for comparison, were also inactive (data not
shown).
In the 4-acetoxy series the N-thiomethyl compound 17
showed activity against MSSA, MRSA, and S. epidermidis. The
C3-unsubstituted analogue 29 showed decreased activity
against MSSA, MRSA, E. faecalis, and E. coli. 4-Sulfonylphenyla-
zetidinones showed very low activity, with some potency ob-
served for N-thiomethyl compounds 25, 27, and 33; these
showed a MIC range of 64–128 mgLꢀ1 against MSSA, MRSA,
S. pyogenes, and E. coli (data not shown). The 4-thioacetylazeti-
dinone series showed interesting antibacterial activities exerted
by compounds 22, 23, 30, and 31, particularly against Gram-
positive pathogens, as shown in Table 1.
Compounds demonstrating the most promising activities
against S. aureus were subsequently tested against some addi-
tional clinical strains of both MSSA (10) and MRSA (10) isolated
from pediatric patients with CF (Table 2). Whereas compounds
30 and 31 confirmed the same potency as previously shown
(Table 1), compounds 4 and 17 interestingly showed greater
antibacterial activity against S. aureus strains isolated from CF
patients (Table 2) than toward previously tested isolates. More-
over, compound 17 was found to have similar, or in some
cases greater, activity against MRSA than against MSSA isolates.
Generally, similar antibacterial activities against MSSA and
MRSA were observed with the new azetidinones. Minimum
bactericidal concentration (MBC) determinations demonstrated
MBC/MIC ratios >4 for compounds 4, 17, 30, and 31, thus
confirming the bacteriostatic nature of their mechanism of
action against both MSSA and MRSA strains.
Results listed in Tables 1 and 2 prompted us to undertake a
preliminary structure–activity relationship (SAR) study to char-
acterize the structural determinants of tested compounds re-
sponsible for antibacterial activity. Comparison of 4-alkylide-
neazetidinones 1–4 shows that the combination of an N-thio-
methyl group and a benzyl ester on the 4-alkylidene side chain
increases the potency against Gram-positive bacteria. The ab-
sence of any substituent at C3 conferred a specific activity
toward S. pyogenes and induced potency, although to modest
levels, against E. coli for compounds 7 and 9; the presence of
an N-thiomethyl group resulted in complete inactivation. In
our earlier findings,[11] the presence of a polyphenolic ester at
the C3 hydroxyethyl side chain increased antibacterial potency.
Unfortunately, combining a phenolic ester with an N-thiometh-
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Antibacterial Agents and CF
Table 2. Antimicrobial activity of the four most active monocyclic b-lactams against MSSA and MRSA strains isolated from CF patients.[a]
MSSA
Compd
S.a.CF1
S.a.CF2
S.a.CF3
S.a.CF4
S.a.CF5
S.a.CF6
S.a.CF7
S.a.CF8
S.a.CF9
S.a.CF10
MIC
MIC50
MIC90
range
4
17
30
31
CTX[b]
4
8
16
32
2
8
4
8
16
1
8
4
8
8
2
8
8
8
8
16
32
1
8
8
4
8
16
32
2
16
8
32
32
2
4–16
4–32
8–32
8–64
1–2
8
8
16
32
1
8
16
32
64
1
16
16
32
2
16
32
32
1
32
32
64
2
16
32
64
2
MRSA
Compd
S.a.215
S.a.216
S.a.217
S.a.218
S.a.219
S.a.220
S.a.221
S.a.222
S.a.223
S.a.224
MIC
MIC50
MIC90
range
4
17
30
31
CTX[b]
4
4
16
32
8
8
4
8
16
2
32
8
32
64
4
16
4
16
32
4
8
8
16
32
4
8
8
4
16
64
8
8
8
16
32
2
64
8
32
32
8
8
4
16
64
8
4–64
4–16
8–32
16–64
2–8
8
4
16
32
4
32
8
32
64
8
16
32
64
2
[a] Data expressed as MIC values [mgLꢀ1]. [b] CTX=ceftriaxone.
yl group resulted in inactivation (compare compounds 14 and
15), as is the case for the OH derivative 13.
reported herein, and the activity of some N-unsubstituted 4-al-
kylidene-b-lactams previously reported,[11] attests that the pres-
ence of an N-thiomethyl group and its contribution to the in-
crease in lipophilicity relative to NH-azetidinones is not essen-
tial for conferring potency. Finally, the similar activities against
both MRSA and MSSA provide evidence that PBP2a does not
represent the biological target of these new azetidinones.
Concerning 4-acetoxyazetidinones 17, 19, and 29, the N-thi-
omethyl group clearly increased potency over the correspond-
ing NH derivatives. In comparing the 4-OAc, 4-SAc, and 4-
SO2Ph variations, the thioacetate was shown to be the most ef-
ficient group, whilst the N-thiomethylation did not improve
potency.
Turos and colleagues investigated and discussed a tentative
mechanism of action for N-thiolated azetidinones.[15] Their hy-
pothesis is that these derivatives primarily block type II fatty
acid biosynthesis in S. aureus through an initial transfer of the
N-alkylthio moiety from the ring nitrogen atom onto a cellular
target. In particular, they should react rapidly with coenzyme A
(CoA) within the bacterial cell through “in vivo” transfer of the
N-thio group to produce an alkyl-CoA mixed disulfide species,
which then interferes with fatty acid biosynthesis. In this per-
spective the b-lactams of Turos et al. appeared to act as pro-
drugs without any special structural features needed for the
activity, other than being relatively lipophilic and able to serve
as a leaving group during nucleophilic attack on sulfur. Gram-
positive bacterial species are known to have lower cytoplasmic
levels of glutathione than Gram-negative pathogens; this sup-
ports the thesis of Turos et al., who suggested that glutathione
helps protect the organism against the inhibitory effects of N-
thiolated lactams, thus lowering the activity of these com-
pounds against Gram-negative bacterial strains. Another sug-
gested mechanism could be that N-thiolated lactams show a
delayed bacteriostatic effect by inhibiting protein biosynthesis
in bacteria.
Conclusions
This study reports the synthesis of a series of new monocyclic
azetidinones modified at the ring C4 position with an alkylide-
necarboxyl chain or electron-withdrawing groups such as OAc,
SAc, and SO2Ph. N-Unsubstituted and N-thiomethyl derivatives
were synthesized; in all, a total of 33 azetidinones were synthe-
sized and evaluated for their antibacterial activity against 116
Gram-positive and Gram-negative clinical isolates. The combi-
nation of an N-thiomethyl group and a benzyl ester on the 4-
alkylidene side chain were found to strengthen potency
against Gram-positive bacteria.
Regarding 4-acetoxyazetidinones, the N-thiomethyl group
clearly increased potency with respect to the corresponding
NH derivatives. The best results were obtained for compounds
4 and 17, which were found to have the best activity against
MRSA isolated from pediatric CF patients. These compounds
showed similar activity against both MSSA and MRSA; there-
fore, as methicillin resistance in S. aureus is brought about by
the acquisition of the PBP2a protein, a different pharmacologi-
cal target could be suggested. In conclusion, the azetidinones
studied in this work represent potential lead structures for the
development of new antibacterials particularly useful against
highly virulent and multidrug-resistant MRSA strains such as
those isolated from CF patients, providing further insight into
the mechanism of action of the new b-lactams.
Staphylococcal bacterial strains are known to have high
levels of CoA. Therefore, the bacteriostatic activity of our N-thi-
omethylazetidinones 4, 17, and 31 could suggest a mechanism
of action such as that reported by Turos and colleagues as thi-
omethylating agents. However, the relevant antibacterial activi-
ty of the N-unsubstituted azetidinone 30 against Gram-positive
strains, its considerable activity toward Gram-negative strains
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C. E. A. Cocuzza, D. Giacomini, et al.
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EtOAc 9:1); [a]D25 = +93.3 (c=0.84 in CHCl3); 1H NMR (200 MHz,
CDCl3): d=0.09 (s, 3H), 0.11 (s, 3H), 0.90 (s, 9H), 1.18 (d, J=6.6 Hz,
3H), 1.30 (t, J=7.4 Hz, 3H), 2.48 (s, 3H), 4.20 (q, J=7.4 Hz, 2H),
4.64 (dq, J=6.6, 4.0 Hz, 1H), 4.22 (m, 1H), 5.62 ppm (d, J=1.0 Hz,
1H); 13C NMR (50 MHz, CDCl3): d=ꢀ4.8, ꢀ4.6, 14.4, 18.0, 19.8, 21.6,
25.7, 60.1, 65.1, 65.9, 92.6, 157.0, 165.6, 169.3 ppm; HPLC–MS (ESI),
tR =24.35, m/z (%): 360 (100) [M+H]+, 382 (17) [M+Na]+; GC–MS
(EI, 70 eV): tR =23.29, m/z (%): 73 (85), 103 (30), 115 (32) 159 (28),
302 (100), 315 (39); IR (film): n˜ =2952, 2926, 2853, 1825, 1713,
1660, 1255, 1162, 1063, 831 cmꢀ1; Anal. calcd for C16H29NO4SSi: C
53.45, H 8.13 N 3.90, S 8.92, found: C 53.54, H 8.16, N 3.83, S 8.78.
Experimental Section
Commercially available reagents were used as received without ad-
ditional purification. 1H and 13C NMR values were recorded on
Inova 400, 300, or Gemini 200 instruments with a 5 mm probe. All
chemical shifts (d) are reported in ppm relative to deuterated sol-
vent signals, and coupling constants (J) are reported in Hz. FTIR:
Thermo Nicolet 380 measured as films between NaCl plates, with
wavenumbers reported in cmꢀ1. TLC: Merck 60 F254. Column chro-
matography: Merck silica gel 200–300 mesh. GC–MS: Agilent Tech-
nologies MSD1100 single-quadrupole mass spectrometer, EI volt-
age: 70 eV, gradient from 50!2808C over 30 min, column HP5 5%
Ph-Me silicon. HPLC–MS: Agilent Technologies HP1100, column
Zobrax-Eclipse XDB-C8 Agilent Technologies, mobile phase: H2O/
CH3CN, gradient from 30!80% CH3CN over 8 min, 80% CH3CN
until 25 min, flow rate: 0.4 mLminꢀ1 coupled with Agilent Technol-
ogies MSD1100 single-quadrupole mass spectrometer, full-scan
mode from m/z 50 to m/z 2600, scan time 0.1 s in positive ion
mode, ESI spray voltage: 4500 V, N2 gas at 240 kPa, drying gas flow
rate: 11.5 mLminꢀ1, fragmentor voltage: 20 V. Elemental analysis:
Thermo Flash 2000 CHNS/O analyzer.
(E)-Benzyl 2-{(S)-3-[(1R)-1-(tert-butyldimethylsilanyloxy)ethyl]-1-
(methylthio)-4-oxoazetidin-2-ylidene}acetate (4): Obtained with
LDA. Transparent oil (181 mg, 43%: yield): Rf =0.9 (cyclohexane/
EtOAc 9:1); [a]D25 = +69.1 (c=0.73 in CHCl3); 1H NMR (200 MHz,
CDCl3): d=0.07 (s, 3H), 0.08 (s, 3H), 0.89 (s, 9H), 1.17 (d, J=6.2 Hz,
3H), 2.48 (s, 3H), 4.21 (dd, J=4.0, J=1.0 Hz, 1H), 4.62 (dq, J=6.2,
J=4.0 Hz, 1H), 5.19 (s, 2H), 5.68 (d, J=1.0 Hz, 1H), 7.35–7.39 ppm
(m, 5H); 13C NMR (50 MHz, CDCl3): d=ꢀ5.1, ꢀ4.9, 17.8, 19.7, 21.4,
25.6, 64.9, 65.8, 65.9, 92.1, 128.0, 128.1, 128.5, 135.9, 157.7, 165.4,
169.1 ppm; IR (film): n˜ =2955, 2929, 2895, 2856, 1825, 1714, 1661,
1256, 1157, 1057, 831 cmꢀ1; HPLC–MS (ESI), tR =12.87 min, m/z (%):
422 [M+H]+, 440 [M+Na]+, 460 [M+K]+; Anal. calcd for
C21H31NO4SSi: C 59.82, H 7.41, N 3.32, S 7.61; found: C 59.69, H
7.38, N 3.24, 7.49.
4-Alkylideneazetidinones 1,[11,16] 3,[11,16] 5,[11] and 8[11] are known and
were prepared by following published procedures. Azetidinones
16 and 28 are commercially available. Azetidinones 10,[18] 20,[19]
24,[21] 29,[22] 30,[23] 32,[20] 18,[24] 22,[25] 26,[26] and 34,[11] are known.
Benzyl 2-(4-oxoazetidin-2-ylidene)acetate (E)-7 and (Z)-9: Com-
pounds 7 and 9 were obtained with the same procedure as for
1[11,16] by starting from the commercially available 4-acetoxyazetidi-
none in 43% yield and in a E/Z ratio of 41:59. The two isomers
were separated by flash chromatography.
(E)-Ethyl 2-[1-(methylthio)-4-oxoazetidin-2-ylidene]acetate (6):
Obtained with LiHMDSA. Transparent oil (66 mg, 33% yield): Rf =
0.7 (cyclohexane/EtOAc 5:5); H NMR (200 MHz, CDCl3): d=1.30 (t,
J=7.2 Hz, 3H), 2.51 (s, 3H), 3.89 (d, J=1.2 Hz, 2H), 4.20 (q, J=
7.2 Hz, 2H), 5.65 ppm (dd, J=1.2, J=1.4 Hz, 1H); 13C NMR (CDCl3,
50 MHz): d=14.3, 21.6, 46.8, 60.2, 92.5, 155.0, 166.0, 168.4 ppm; IR
(neat): n˜ =2958, 2923, 1810, 1702, 1660, 1057, 835 cmꢀ1; HPLC–MS
(ESI): tR =6.38 min, m/z (%): 156 (82) [MꢀOEt]+, 174 (46), 202 (100)
[M+H]+; Anal. calcd for C8H11NO3S: C 47.75, H 5.51, N 6.96, S
15.93, found: C 47.67, H 5.48, N 6.84, S 15.78.
1
(E)-7: white solid: Rf =0.5 (cyclohexane/EtOAc 5:5); mp: 98–1038C;
1H NMR (300 MHz, CDCl3): d=3.80 (d, J=1.2 Hz, 2H), 5.17 (s, 2H),
5.40 (d, J=1.2 Hz, 1H), 7.22 (brs, 1H), 7.30–7.38 ppm (m, 5H);
13C NMR (50 MHz, CDCl3) d=45.9, 65.9, 91.8, 128.0, 128.1, 128.5,
135.9, 151.6, 166.3, 166.5 ppm; IR (KBr): n˜ =3193, 1827, 1698, 1639,
1284, 1046 cmꢀ1; HPLC–MS, (ESI): tR =6.40 min, m/z (%): 200 (90),
218 (38) [M+H]+, 240 (100) [M+Na]+; Anal. calcd for C12H11NO3: C
66.35, H 5.10, N 6.45, found: C 66.28, H 5.01, N 6.32.
Ethyl 2-[1-(methylthio)-4-oxoazetidin-2-yl]acetate (11): Obtained
with LiHMDSA. Transparent oil (67 mg, 33% yield): Rf =0.3 (CH2Cl2/
acetone 98:2); 1H NMR (300 MHz, CDCl3): d=1.30 (t, J=7.2 Hz,
3H), 2.44 (s, 3H), 2.57 (dd, J=15.9, 7.8 Hz, 1H), 2.85 (dd, J=15.0,
J=2.7 Hz, 1H), 2.90 (dd, J=15.9, J=5.4 Hz, 1H), 3.27 (dd, J=15.0,
J=5.4 Hz, 1H), 4.07 (m, 1H), 4.20 ppm (q, J=7.2 Hz, 2H); 13C NMR
(75 MHz, CDCl3): d=14.1, 22.4, 38.0, 44.3, 51.8, 61.0, 169.8,
170.1 ppm; IR (neat): n˜ =2922, 2852, 1763, 1735, 1420, 1319,
(Z)-9: white solid: Rf =0.8 (cyclohexane/EtOAc 5:5); mp: 167–
1
1658C; H NMR (300 MHz, CDCl3): d=3.55 (d, J=0.9 Hz, 1H), 3.56
(d, J=0.9 Hz, 1H), 5.19 (s, 2H), 5.24 (dd, J=0.9 Hz, J=0.6 Hz, 1H),
7.30–7.39 (m, 5H), 8.59 ppm (brs, 1H); 13C NMR (CDCl3, 50 MHz):
d=44.9, 66.0, 90.3, 128.1, 128.3, 128.6, 135.9, 150.3, 164.5,
166.8 ppm; IR (KBr): n˜ =231, 1825, 1687, 1642, 1251, 1141 cmꢀ1
;
1260 cmꢀ1
; HPLC–MS (ESI): tR =3.73 min, m/z (%): 162 (100)
HPLC–MS, (ESI): tR =6.87 min, m/z (%): 218 (12) [M+H]+, 236 (15)
[M+H2O+H]+, 262 (100), 278 (42); Anal. calcd for C12H11NO3: C
66.35, H 5.10, N 6.45, found: C 66.31, H 4.99, N 6.34.
[MꢀCH2CO+H]+, 204 (32) [M+H]+, 226 (35) [M+Na]+; GC–MS (EI,
70 eV): tR =16.18 min, m/z (%): 61 (27), 72 (30), 86 (77), 88 (75), 89
(74), 114 (22), 116 (23), 141 (100), 161 (71) [MꢀCOCH2]+, 203 (11)
[M]+; Anal. calcd for C8H13NO3S: C 47.27, H 6.45, N 6.89, S 15.78,
found: C 47.16, H 6.36, N 6.70, S 15.69.
General procedure for N-methylthiolation: A freshly prepared 1m
solution of LDA (or LiHMDSA) in THF (110 mL, 1.1 mmol) was added
to a solution of b-lactam (N1-unsubstituted, 1 mmol) in THF
(10 mL) at ꢀ788C under inert an atmosphere of N2, and then S-
methyl methanethiosulfonate (236 mL, 2.5 mmol) was added. The
solution was allowed to warm at room temperature and was moni-
tored by TLC. After 4 h the reaction was quenched with aqueous
NH4Cl (10 mL) and extracted with EtOAc (3ꢁ10 mL). The organic
extracts were dried over Na2SO4, filtered and concentrated in va-
cuo. Flash chromatography of the extracts gave the N-thiomethyl-
b-lactams.
Ethyl
{(3S,2R)-3-[(1R)-1-(tert-butyldimethylsilanyloxy)ethyl]-1-
(methylthio)-4-oxoazetidin-2-yl}acetate (12): Obtained starting
from 34 with LDA. Transparent oil (267 mg, 74% yield): Rf =0.8 (cy-
clohexane/EtOAc 8:2); [a]D25 = +71.3 (c=1.3 in CHCl3); 1H NMR
(300 MHz, CDCl3): d=0.03 (s, 3H), 0.04 (s, 3H), 0.83 (s, 9H), 1.13 (d,
J=6.3 Hz, 3H), 1.26 (t, J=7.2 Hz, 3H), 2.38 (s, 3H), 2.57 (dd, J=
15.0, J=6.6 Hz, 1H), 2.77 (dd, J=15.0, J=6.0 Hz, 1H), 2.99 (dd, J=
3.3, J=3.0 Hz, 1H), 4.13–4.20 (m, 1H), 4.15 (q, J=7.2 Hz, 2H),
4.20 ppm (dq, J=6.3, J=3.3 Hz, 1H); 13C NMR (75 MHz, CDCl3): d=
ꢀ5.0, ꢀ4.6, 14.0, 17.8, 22.6, 22.5, 25.6, 37.9, 53.7, 60.8, 64.3, 64.8,
170.2, 170.8 ppm; IR (neat): n˜ =3019, 2956, 2930, 2857, 1763, 1734,
1265, 1216, 838 cmꢀ1; GC–MS (EI, 70 eV): tR =23.90 min, m/z (%): 73
(E)-Ethyl
2-{(S)-3-[(1R)-1-(tert-butyldimethylsilanyloxy)ethyl]-1-
(methylthio)-4-oxoazetidin-2-ylidene}acetate (2): Obtained with
LDA. Transparent oil (215 mg, 60% yield): Rf =0.8 (cyclohexane/
1924
www.chemmedchem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1919 – 1927

Antibacterial Agents and CF
(90), 75 (100), 103 (35), 143 (35), 304 (100); Anal. calcd for
C16H31NO4SSi: C 53.15, H 8.64, N 3.87, S 8.87; found: C 53.12, H
8.76, N 3.89, S 8.79.
116 (67) [MꢀSO2Ph]+, 125 (100) [PhSO]+, 258 (72) [M+H]+, 280
(70) [M+Na]+; Anal. calcd for C10H11NO3S2: C 46.67, H 4.31, N 5.44,
S 24.92, found: C 46.54, H 4.24, N 5.38, S 24.78.
(2R,3R)-3-[(1R)-1-(tert-Butyldimethylsilanyloxy)ethyl]-1-(methyl-
thio)-4-oxoazetidin-2-yl acetate (17): Obtained with LDA. Trans-
parent oil (100 mg, 30% yield): Rf =0.9 (CH2Cl2/acetone 98:2);
Deprotection of the tert-butyldimethylsilanyloxy group: BF3·OEt2
(226 mL, 1.8 mmol) was added to a solution of b-lactam (1.5 mmol)
in CH3CN (15 mL) at 08C under inert atmosphere, and the reaction
was allowed to warm to room temperature and was monitored by
TLC. The reaction was quenched with phosphate buffer (0.1m,
pH 6, 15 mL) and extracted with CH2Cl2 (3ꢁ15 mL). The organic ex-
tracts were dried (Na2SO4), filtered and concentrated in vacuo.
Flash chromatography of the extracts gave the deprotected b-
lactam.
1
[a]D25 = +7.0 (c=1.4 in CHCl3); H NMR (200 MHz, CDCl3): d=0.03
(s, 3H), 0.06 (s, 3H), 0.84 (s, 9H), 1.23 (d, J=6.2 Hz, 3H), 2.14 (s,
3H), 2.45 (s, 3H), 3.18 (dd, J=3.0, J=1.6 Hz, 1H), 4.22 (dq, J=6.2,
J=3.0 Hz, 1H), 6.22 ppm (d, J=1.6 Hz, 1H); 13C NMR (50 MHz,
CDCl3): d=ꢀ5.1, ꢀ4.5, 17.9, 21.0, 22.2, 22.8, 25.7, 64.0, 66.3, 80.2,
169.3, 169.8 ppm; IR (film): n˜ =2929, 2857, 1793, 1752, 1252, 1066,
838 cmꢀ1
;
HPLC–MS (ESI): tR =17.69, m/z (%): 274 (100)
[MꢀCH3COO]+, 290 (26) [MꢀCH3CO]+, 356 (9) [M+Na]+; GC–MS
(EI, 70 eV): tR =17.69, m/z (%): 234 (75), 190 (100), 117 (46), 75 (45);
Anal. calcd for C14H27NO4SSi: C 50.42, H 8.16, N 4.20, S 9.61, found:
C 50.29, H 8.09, N 4.18, S 9.47.
(E)-Benzyl 2-[(S)-3-((R)-1-hydroxyethyl)-1-(methylthio)-4-oxoazeti-
din-2-ylidene)acetate (13): Transparent oil (419 mg, 91% yield):
Rf =0.4 (cyclohexane/EtOAc 9:1); [a]D25 = +36.3 (c=0.78 in CHCl3);
1H NMR (300 MHz, CDCl3): d=1.39 (d, J=6.0 Hz, 3H), 2.50 (s, 3H),
4.13 (d, J=7.8 Hz, 1H), 4.25 (m, 1H), 5.13 (brs, 1H), 5.21 (s, 2H),
5.79 (s, 1H), 7.37–7.41 ppm (m, 5H); 13C NMR (75 MHz, CDCl3): d=
20.9, 21.6, 66.2, 66.6, 66.9, 92.7, 128.5, 128.7, 135.4, 159.3, 167.4,
168.5, 834 ppm; IR (film): n˜ =3400, 3010, 1820, 1699, 1652, 1274,
1162, 1067 cmꢀ1; HPLC–MS (ESI): tR =11.24, m/z (%): 308 (100) [M+
H]+; Anal. calcd for C15H17NO4S: C 58.61, H 5.57, N 4.56, S 10.43,
found: C 58.74, H 5.66, N 5.48, S 10.38.
S-(2R,3S)-3-[(1R)-1-(tert-Butyldimethylsilanyloxy)ethyl]-1-(methyl-
thio)-4-oxoazetidin-2-yl ethanethioate (21): Obtained with
LiHMDSA. Transparent oil (230 mg, 66% yield): Rf =0.8 (cyclohex-
ane/EtOAc 7:3); [a]D25 = +45.2 (c=1.7 in CHCl3); 1H NMR
(200 MHz, CDCl3): d=0.05 (s, 3H), 0.07 (s, 3H), 0.86 (s, 9H), 1.17 (d,
J=6.2 Hz, 3H), 2.43 (s, 3H), 2.44 (s, 3H), 3.29 (dd, J=3.4, J=2.6 Hz,
1H), 4.26 (dq, J=6.2, J=3.4 Hz, 1H), 5.62 ppm (d, J=2.6 Hz, 1H);
13C NMR (75 MHz, CDCl3): d=ꢀ5.1, ꢀ4.6, 17.8, 22.0, 22.2, 25.6,
30.9, 59.0, 64.4, 65.3, 169.4, 193.5 ppm; IR (film): n˜ =2929, 2986,
2956, 1782, 1707, 1252, 1127, 836 cmꢀ1; HPLC–MS (ESI): tR =
10.70 min, m/z (%): 372 (100) [M+Na]+, 721 (50) [2M+Na]+; Anal.
calcd for C14H27NO3S2Si: C 48.10, H 7.78, N 4.01, S 18.35, found: C
48.18, H 7.84, N 3.89, S 18.31.
(2R,3R)-3-((1R)-1-hydroxyethyl)-1-(methylthio)-4-oxoazetidin-2-yl
acetate (19): Transparent oil (263 mg, 80% yield): Rf =0.3 (cyclo-
hexane/EtOAc, 5:5); [a]D25 =ꢀ33.3 (c=1.8 in CHCl3); 1H NMR
(200 MHz, CDCl3): d=1.33 (d, J=6.2 Hz, 3H), 2.18 (s, 3H), 2.22
(brs, 1H, OH), 2.51 (s, 3H), 3.21 (dd, J=1.6 Hz, J=6.4 Hz, 1H), 4.18
(quint, J=6.4 Hz, 1H), 6.01 ppm (d, J=6 Hz, 1H); 13C NMR
(50 MHz, CDCl3): d=20.9, 21.0, 22.8, 64.2, 66.7, 82.1, 168.5,
170.7 ppm; IR (film): n˜ =3458, 2974, 2924, 1781, 1752, 1229,
1040 cmꢀ1; HPLC–MS (ESI): tR =min 2.45, m/z (%): 116 (100), 242
(45) [M+Na]+, 461 (10) [2M+Na]+; Anal. calcd for C8H13NO4S: C
43.82, H 5.98, N 6.39, S 14.92, found: C 43.69, H 5.88, N 6.37, S
14.81.
(3S,4R)-3-[(1R)-1-(tert-Butyldimethylsilanyloxy)ethyl]-1-(methyl-
thio)-4-(phenylsulfonyl)azetidin-2-one (25): Obtained with
LHMDSA. Pale-yellow oil (365 mg, 88% yield): Rf =0.7 (cyclohex-
1
ane/EtOAc 7:3); [a]D25 =ꢀ75.0 (c=1.2 in CHCl3); H NMR (400 MHz,
CDCl3): d=0.01 (s, 3H), 0.03 (s, 3H), 0.84 (s, 9H), 0.90 (d, J=6.0 Hz,
3H), 2.52 (s, 3H), 3.47 (dd, J=4.0, J=2.4 Hz, 1H), 4.24 (dq, J=6.0,
J=4.0 Hz, 1H), 4.83 (d, J=2.4 Hz, 1H), 7.62–7.66 (m, 2H), 7.72–7.76
(m, 2H), 7.97–8.00 ppm (m, 1H); 13C NMR (50 MHz, CDCl3): d=
ꢀ5.1, ꢀ4.8, 17.7, 21.9, 22.9, 25.6, 63.0, 63.7, 73.0, 129.1, 129.5,
134.7, 136.3, 169.1 ppm; IR (neat): n˜ =3066, 2955, 2929, 2857, 2481,
1792, 1326, 1153, 835 cmꢀ1; HPLC–MS (ESI): tR =12.24, m/z (%): 116
(69) [MꢀSO2Ph]+, 309 (30), 372 (69), 433 (100) [M+H2O]+, 854 (10)
[2M+Na]+; Anal. calcd for C18H29NO4S2Si: C 52.01, H 7.03, N 3.37, S
15.43, found: C 52.19, H 7.08, N 3.39, S 15.23.
S-(2R,3S)-3-((1R)-1-hydroxyethyl)-1-(methylthio)-4-oxoazetidin-2-
yl ethanethioate (23): Transparent oil (349 mg, 99% yield): Rf =0.3
(cyclohexane/EtOAc 6:4); mp: 100–1038C; [a]D25 = +19.8 (c=2.4 in
1
CHCl3); H NMR (300 MHz, CDCl3): d=1.30 (d, J=6.6 Hz, 3H), 2.45
(s, 3H), 2.49 (s, 3H), 3.36 (dd, J=5.4 Hz, J=2.7 Hz, 1H), 4.27 (dq,
J=6.6 Hz, 5.4 Hz, 1H), 5.52 ppm (d, J=2.7 Hz, 1H); 13C NMR
(100 MHz, CDCl3): d=20.9, 22.3 31.0, 60.1, 64.4, 66.0, 169.5,
194.3 ppm; IR (film): n˜ =3402, 1753, 1695, 1130 cmꢀ1; HPLC–MS
(ESI): tR =2.19 min, m/z (%): 116 (100), 192 (20) [MꢀCOCH3]+, 236
(15) [M+H]+, 258 (28) [M+Na]+, 493 (9) [2M+Na]+; Anal. calcd
for C8H13NO3S2: C 40.83, H 5.57, N 5.95, S 27.25, found: C 40.89, H
5.68, N 5.91, S 27.18.
S-1-(Methylthio)-4-oxoazetidin-2-yl ethanethioate (31): Obtained
with LiHMDSA. Transparent oil (59 mg, 31% yield): Rf =0.8 (cyclo-
1
hexane/EtOAc 6:4); H NMR (200 MHz, CDCl3): d=2.45 (s, 3H), 2.49
(s, 3H), 3.12 (dd, J=15.4, J=3.0 Hz, 1H), 3.58 (dd, J=15.4, J=
5.6 Hz, 1H), 5.50 ppm (dd, J=5.6, J=3.0 Hz, 1H); 13C NMR
(75 MHz, CDCl3): d=22.4, 31.0, 45.5, 57.9, 167.2, 193.8 ppm; IR
(neat): n˜ =2918, 1773 cmꢀ1; HPLC–MS (ESI): tR =4.03 min, m/z (%):
116 (100) [MꢀSAc]+, 134 (61), 192 (26) [M+H]+, 214 (35) [M+Na]+
; Anal. calcd for C6H9NO2S2: C 37.68, H 4.74, N 7.32, S 33.53, found:
C 37.74, H 4.81, N 7.26, S 33.21.
(3S,4R)-3-((1R)-1-hydroxyethyl)-1-(methylthio)-4-(phenylsulfon-
yl)azetidin-2-one (27): White solid (406 mg, 90% yield): Rf =0.4
(cyclohexane/EtOAc 5:5); mp: 167–1698C; [a]D25 =ꢀ107.4, (c=1.7
in CHCl3); 1H NMR (400 MHz, CDCl3): d=1.14 (d, J=6.8 Hz, 3H),
1.85 (d, J=4.4 Hz, 1H), 2.48 (s, 3H), 3.59 (dd, J=3.2 Hz, 2.8 Hz, 1H),
4.30 (ddq, J=6.8 Hz, J=4.4 Hz, J=3.2 Hz, 1H), 4.86 (d, J=2.8 Hz,
1H), 7.62–7.66 (m, 2H), 7.73–7.77 (m, 1H), 7.97–8.00 ppm (m, 1H);
13C NMR (50 MHz, CDCl3): d=21.4, 22.4, 62.2, 63.4, 72.7, 129.2,
129.6, 134.8, 136.2, 169.7 ppm; IR (KBr): n˜ =3433, 2979, 2925, 1763,
1647, 1310, 1147 cmꢀ1; HPLC–MS (ESI): tR =4.90 min, m/z (%): 116
(15) [MꢀSO2Ph]+, 319 (100) [M+H2O]+, 625 (72) [2M+Na]+; Anal.
calcd for C12H15NO4S2: C 47.82, H 5.02, N 4.65, S 21.28; found: C
47.79, H 4.95, N 4.54, S 20.88.
1-(Methylthio)-4-(phenylsulfonyl)azetidin-2-one (33): Obtained
with LiHMDSA. Pale-yellow oil (203 mg, 79% yield): Rf =0.6 (CH2Cl2/
1
acetone 98:2); H NMR (200 MHz, CDCl3): d=2.52 (s, 3H), 3.30 (d,
J=4.0 Hz, 2H), 4.74 (t, J=4.0 Hz, 1H), 7.58–7.79 (m, 3H), 7.92–
7.98 ppm (m, 2H); 13C NMR (50 MHz, CDCl3): d=22.8, 42.0, 71.0,
129.1, 129.6, 134.9, 135.8, 167.3; IR (neat): n˜ =3063, 2964, 1781,
1584, 1309, 1149, 722 cmꢀ1; HPLC–MS (ESI): tR =4.27 min, m/z (%):
ChemMedChem 2011, 6, 1919 – 1927
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1925

C. E. A. Cocuzza, D. Giacomini, et al.
MED
(R)-1-{(S,E)-2-[2-(benzyloxy)-2-oxoethylidene]-1-(methylthio)-4-
oxoazetidin-3-yl)ethyl 4-hydroxybenzoate (14): Oxalyl chloride
(66 mL, 0.78 mmol) was added to a solution of para-hydroxybenzoic
acid (54 mg, 0.39 mmol) in CH2Cl2 (6 mL) under an inert N2 atmos-
phere. The solution was held at reflux for 2 h; after removal of
CH2Cl2, compound 13 (58 mg, 0.19 mmol) was introduced, along
with benzene (4 mL) and SnCl4 (1m in CH2Cl2, 9.7 mL, 0.0097 mmol),
and the mixture was held at reflux for 2.5 h followed by TLC. After
completion, the reaction was washed with 5% NaHCO3 (10 mL), ex-
tracted with EtOAc (2ꢁ10 mL) and CH2Cl2 (2ꢁ10 mL), dried on
Na2SO4 and concentrated in vacuo. Flash chromatography (cyclo-
hexane/EtOAc 8:2) of the extracts gave 14 (44 mg, 54% yield). Rf =
0.7 (cyclohexane/EtOAc 8:2); [a]D25 = +93.0 (c=0.84 in CHCl3);
1H NMR (200 MHz, CDCl3): d=1.35 (d, J=6.6 Hz, 3H), 2.52 (s, 3H),
4.55 (dd, J=4.8 Hz, J=1.5 Hz, 1H), 5.23 (s, 2H), 5.75 (d, J=1.5 Hz,
1H), 5.87 (dq, J=6.6 Hz, J=4.8 Hz, 1H), 6.34 (brs, 1H, OH), 6.86–
6.90 (m, 2H, Ar), 7.27–7.43 (m, 5H, Ar), 7.96–8.00 ppm (m, 2H, Ar);
13C NMR (50 MHz, CDCl3): d=15.5, 21.7, 62.7, 66.4, 66.6, 93.3, 115.2,
122.2, 128.5, 128.6, 132.2, 135.7, 156.1, 160.2, 165.2, 168.8 ppm; IR
(film): n˜ =3392, 1817, 1702, 1825, 1713, 1668, 1061 cmꢀ1; HPLC–MS
(ESI), tR =12.94, m/z (%): 290 (100) [MꢀCOOPh(OH)]+, 428 (20)
[M+H]+, 450 (20) [M+Na]+, 877 (50) [2M+Na]+; Anal. calcd for
C22H21NO6S: C 61.81, H 4.95, N 3.28, S 7.50, found: C 61.70, H 4.88,
N 3.25, S. 7.41.
Determination of minimum inhibitory concentrations (MICs)
The in vitro antibacterial activity of the new compounds was stud-
ied by determining the minimum inhibitory concentrations (MICs)
by means of the broth microdilution method, according to the
Clinical and Laboratory Standards Institute.[27] Briefly, serial twofold
dilutions of each antibiotic stock solution were obtained using Mu-
eller–Hinton broth in 96-well microtiter plates. An equal volume of
bacterial inoculum (5ꢁ105 CFUmLꢀ1) was added to each well of
the microtiter plate containing serial antibiotic concentrations in
0.05 mL volumes. The microtiter plate was then incubated at 358C
for 16–20 h; subsequently each well was analyzed for the presence
of visible bacterial growth. MIC was defined as the lowest concen-
tration of the tested compound able to inhibit visible growth of
the microorganism after overnight incubation. MIC50 and MIC90 are
defined as the concentrations required to inhibit the growth of 50
and 90% of the tested microorganisms, respectively. The in vitro
antibacterial activity of 33 new monocyclic azetidin-2-ones was
tested and compared against that of reference b-lactam agents in
clinical use: amoxicillin (Sigma–Aldrich) and ceftriaxone (Sigma–Al-
drich). MSSA S. aureus ATCC 29213, MSSA S. aureus ATCC 25923,
MRSA S. aureus ATCC 700699 (MU50), S. pyogenes ATCC 19615, and
E. coli ATCC 25922 were used as controls for MIC determinations.
Determination of minimum bactericidal concentrations
(MBCs)
(R)-1-{(S,E)-2-[2-(benzyloxy)-2-oxoethylidene]-1-(methylthio)-4-
oxoazetidin-3-yl}ethyl 4-hydroxy-3,5-dimethoxybenzoate (15):
Compound 15 was obtained with the same procedure as 14 using
syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid): Transparent
oil (6.5 mg, 7% yield): Rf =0.8 (cyclohexane/EtOAc 8:2); [a]D25 = +
The minimum bactericidal concentration (MBC) of the test solu-
tions against MSSA ATCC 25923 and MRSA 215 strains was deter-
mined by inoculating Mueller–Hinton agar plates with the solution
mixture in wells representing the MIC and the next two higher-
concentration wells (2ꢁMIC and 4ꢁMIC). Pure cell cultures were
used as the positive controls plated on separate Mueller–Hinton
agar plates. The contents of wells representing the MIC and the
next two higher-concentration wells were streaked onto Mueller–
Hinton agar plates. This process was performed for compounds 4,
17, 30, and 31. The inoculated agar plates were placed in an aero-
bic incubator at 37.58C for the previously specified amount of time
for each test bacteria. At the end of the incubation period, the
plate’s division with the lowest concentration of test solution that
exhibited no growth was determined to be the MBC (in mgLꢀ1).
1
63.3 (c=1.01 in CHCl3); H NMR (200 MHz, CDCl3): d=1.36 (d, J=
6.6 Hz, 3H), 2.52 (s, 3H), 3.97 (s, 6H), 4.55 (dd, J=5.2, 1.4 Hz, 1H),
5.19 (d, JAB =12.4 Hz, 1H), 5.26 (d, JAB =12.4 Hz, 1H), 5.75 (d, J=
1.4 Hz, 1H), 5.89 (dd, J=6.6 Hz, 5.2 Hz, 1H), 5.93 (brs, 1H, OH),
7.32–7.42 ppm (m, 7H); 13C NMR (75 MHz, CDCl3): d=15.5, 21.7,
56.4, 62.7, 66.3, 66.9, 93.3, 106.9, 120.9, 128.3, 128.5, 128.6, 135.8,
139.3, 146.6, 156.0, 165.0, 165.1, 168.4 ppm; IR (film): n˜ =3403,
2923, 1819, 1713, 1653, 1335, 1222, 1063 cmꢀ1 HPLC–MS (ESI): tR =
13.05, m/z (%): 290 (100) [MꢀCOOPh(OCH3)2(OH)]+, 488 (10) [M+
1]+, 510 (15) [M+Na]+, 998 [2M+Na]+; Anal. calcd for C24H25NO8S:
C 59.13, H 5.17, N 2.87, S 6.58, found: C 58.95, H 5.08, N 2.81, S
6.44.
Acknowledgements
Financial support was from Ministero Istruzione Universitꢀ e Ri-
cerca (MIUR), the University of Bologna, and the Fondazione del
Monte di Bologna e Ravenna. The authors thank Mr. Andrea Van-
nini, Mr. Michel Chiarucci, Mr. Nicola Castellucci, and Ms. Fran-
cesca Casoni for technical assistance, as well as Dr. Laura Garla-
schi, Dr. Cristina Garlaschi, and Dr. Lisa Cariani for providing the
bacterial starins isolated at the Department of Pediatrics, CF
Center, University of Milan (Italy).
Microbiological assays
Bacterial strains
A total of 116 clinical isolates were used for the in vitro antibacteri-
al activity determination of the studied compounds. The following
Gram-positive and Gram-negative bacterial species were included:
Streptococcus pyogenes (10), Streptococcus pneumoniae (10), Entero-
coccus faecalis (10), methicillin-resistant Staphylococcus aureus
(MRSA) (13), methicillin-susceptible S. aureus (MSSA) (13), Staphylo-
coccus epidermidis (10), Haemophilus influenzae (10), Escherichia coli
(10), Proteus mirabilis (10), Serratia marcescens (10), and Klebsiella
pneumoniae (10). In addition, a total of 20 MSSA and MRSA strains
had been recently isolated from pediatric patients with cystic fibro-
sis at the Department of Pediatrics, CF Center, Foundation IRCCS
CA’ GRANDA Ospedale Maggiore Policlinico, University of Milan
(Italy); these were further tested for their susceptibility toward the
most active compounds.
Keywords: antibiotics · azetidinones · cystic fibrosis · drug-
resistant bacteria · lactams
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Received: June 8, 2011
Revised: July 7, 2011
Published online on August 10, 2011
ChemMedChem 2011, 6, 1919 – 1927
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www.chemmedchem.org
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