Novel penicillin-type analogues bearing a variable substituted 2-azetidinone ring at position 6: Synthesis and biological evaluation

Article abstract of DOI:10.3390/molecules201219828

The synthesis and the biological activity of novel semi-synthetic β-Lactam compounds containing an azetidinone moiety joined to the amino-nitrogen of the (+)-6-aminopenicillanic acid (6-APA) as new antibacterial agents is reported. The synthesized compounds were screened for their in vitro antimicrobial activity against a panel of Gram positive and Gram negative pathogens and environmental bacteria. Tested compounds displayed good antimicrobial activity against all tested Gram positive bacteria and for Staphylococcus aureus and Staphylococcus epidermidis antimicrobial activity resulted higher than that of the reference antibiotic. Additionally, in vitro cytotoxic screening was also carried out indicating that the compounds do not cause a cell vitality reduction effective at concentration next to and above those shown to be antimicrobial.

Full text of DOI:10.3390/molecules201219828

Article
Novel Penicillin-Type Analogues Bearing a Variable
Substituted 2-Azetidinone Ring at Position 6:
Synthesis and Biological Evaluation
Margherita De Rosa 1,*, Giovanni Vigliotta 1,*, Giuseppe Palma 2, Carmela Saturnino 3
and Annunziata Soriente 1
Received: 28 September 2015 ; Accepted: 2 December 2015 ; Published: 10 December 2015
Academic Editor: Peter J. Rutledge
1
Dipartimento di Chimica e Biologia, Università degli Studi di Salerno, via Giovanni Paolo II, 132,
Fisciano 84084, Italy; titti@unisa.it
S.S.D. “Sperimentazione Animale”, National Cancer Institute, I.R.C.C.S. “Fondazione Pascale”,
2
Naples 80131, Italy; palma.giuseppe@icloud.com
Dipartimento di Farmacia, Università degli Studi di Salerno, via Giovanni Paolo II, 132, Fisciano 84084,
3
Italy; saturnino@unisa.it
*
Correspondence: maderosa@unisa.it (M.D.R.); gvigliotta@unisa.it (G.V.); Tel.: +39-089-969-553 (M.D.R.);
+39-089-969-294 (G.V.); Fax: +39-89-969-663 (M.D.R. & G.V.)
Abstract: The synthesis and the biological activity of novel semi-synthetic β-lactam compounds
containing an azetidinone moiety joined to the amino-nitrogen of the (+)-6-aminopenicillanic acid
(6-APA) as new antibacterial agents is reported. The synthesized compounds were screened for their
in vitro antimicrobial activity against a panel of Gram positive and Gram negative pathogens and
environmental bacteria. Tested compounds displayed good antimicrobial activity against all tested
Gram positive bacteria and for Staphylococcus aureus and Staphylococcus epidermidis antimicrobial
activity resulted higher than that of the reference antibiotic. Additionally, in vitro cytotoxic screening
was also carried out indicating that the compounds do not cause a cell vitality reduction effective at
concentration next to and above those shown to be antimicrobial.
Keywords: 6-aminopenicillanic acid (6-APA); 2-azetidinone; β-lactam antibiotics; antibacterial;
Staudinger reaction
1. Introduction
Since the discovery of penicillin, and shortly there after cephalosporin, the key role of β-lactam
framework was recognized thus catching the attention of medicinal researchers and organic chemists.
The β-lactam nucleus is the structural feature and the core of the biological activity of one of the most
successful classes of therapeutic agents to date characterized by a broad spectrum of activity and low
toxicity (Figure 1) [1–9]. Unfortunately, long-term use related to the overuse and misuse of β-lactam
antibiotics have resulted in the proliferation of resistant organisms among a variety of clinically
significant species of bacteria becoming an important worldwide problem. Their effectiveness has
been seriously compromised by the bacterial ability to develop different competitive mechanisms in
order to survive [10–14]. In particular, resistance to β-lactams in many bacteria is usually due to the
production of β-lactamases [15–19], enzymes that inactivate the drug by hydrolysis of the β-lactam
ring preventing the action against its original cellular targets, or the modification of penicillin-binding
proteins (PBPs) or cellular permeability. In response to this challenge, substantial research is devoted
to the design and synthesis of new classes of antimicrobial agents with greater potency, stability and
efficacy than existing drugs. In fact, attention in this field has not yet been exhausted because the
basic difficulty is the rapid development of drug resistant pathogenic bacteria.
Molecules 2015, 20, 22044–22057; doi:10.3390/molecules201219828
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Molecules 2015, 20, 22044–22057
H
N
H
N
R2
H
N
H
N
H
N
R1
S
R1
O
O
R
S
OH
R3
O
R2
N
O
O
O
O
OH
COOH
OH
OH
O
O
O
Penicillin
Cephalosporin
Clavulanic acid
Carbapenem
OH
O
O
H
N
H
H
N
R2
R1
S
R
R1
R3
S
R
N
R4
OH
O
N
O
O
R2
OH
O
O
O
Monobactam
Penam sulfone
Penem
Figure 1. Representative β-lactam structures.
In line with this rationale and as a part of our ongoing study on the synthesis of antimicrobial
active molecules [20], we have planned a synthetic strategy to synthesize a set of new penicillanic acid
(6-APA) derivatives containing an additional appropriately functionalized β-lactam nucleus joined
to the amino-nitrogen of 6-APA with the hypothesis that a synergistic effect should be the result
of the presence of the additional 2-azetidinone ring on the 6-APA scaffold thus enhancing the drug
stability and biological activity. The synthesis and characterization of a number of new penicillanic
acid derivatives is described and their biological activities are screened.
2. Results and Discussion
2.1. Chemistry
Scheme 1 shows the retrosynthetic analysis of the penicillanic acid derivatives 12a–d. The
construction of 12 is implemented with a simple strategy based on the coupling of the 6-APA nucleus
with a functionalized 2-azetidinone ring (6a–d) built through [2+2] cycloaddition reaction of a ketene
with an imine (Staudinger reaction) [21–24]. The presence of an acid functionality in C-3 position of
the β-lactam derivatives 6a–d directly engages the β-lactam unit with 6-APA nucleus by standard
amide coupling methodology and provides an amide functionality in the connecting linker directly
attached at position 6 of 6-APA, a structural feature common to various bio- and semi-synthetic
penicillins [25].
R3
R3
R1
H
H
R1
R2
H2N
O
S
H
N
H
H
N
+
R2
S
OH
N
H
N
O
N
H
OH
O
O
O
O
O
O
Na
12a-d
O
6-APA
(+/-)-trans-6a-d
Scheme 1. General retrosynthetic strategy.
It is reported that the presence of a phenyl group at the C-4 position of the azetidinone ring
benefits a hydrophobic interaction with the active site of beta-lactamases, thus increasing the ability
of the molecules to inhibit them [1]. Structure-activity studies show the significant influence on the
biological activity exerted also by the presence of a phenyl group at the N-1 position and of the
different substituents on the N-1 aromatic ring [3,4,26]. Therefore, keeping these studies in view,
we decided to combine 1,4-diaryl-2-azetidinones with 6-APA in search of a cooperative effect on the
biological activity, possibly resulting in an improvement of antibacterial activity, and our hypothesis
is confirmed by the biological data.
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Molecules 2015, 20, 22044–22057
The target compounds 12a–d were synthesized as depicted in Scheme 2. The preparation
of 2-azetidinones 5a–d (Scheme 2A) is performed by the Staudinger [2+2] cycloaddition
reaction between properly substituted imines 3 and a ketene prepared in situ from methyl
3-(chloroformyl)propionate 4 [24,27,28]. The preparation of the imine precursor 3a–d was achieved
in good yields (85%–96%) by condensation of the properly substituted aldehydes and anilines
in ethanol [29–32]. The Staudinger reaction is one of the most widely employed methods in
the preparation of β-lactam nucleus because it provides direct access to variously functionalized
2-azetidinones and allows to modulate the stereochemical outcome of the reaction. Under our
experimental conditions, we obtained trans-5-cycloadduct only in moderate yield (45%–60%). The
stereoselectivity of this reaction was unambiguously assigned by 1H-NMR coupling constant analysis
regarding the protons at C3 and C4 in accordance with literature data for trans-β-lactam [21]. With the
aim of increasing the reaction efficiency, different protocols were used, changing solvent, temperature
and order of addition of the reagents, but we did not obtain any improvement.
A
O
H2N
N
H
i
+
R2
R
R2
R
R2
R
R1
R1
R1
iv
3a-d
2 a: R1=R2= H
2b: R1=R2=CH3
1a: R= H
1b: R= NO2
N
O
2c: R1=H, R2=OCH3
O
OMe
R
(+/-) 5a-d
v
R2
R1
O
ii
O
iii
O
O
O
Cl
OMe
OH
O
O
OMe
4
N
O
O
OH
(+/-) 6a-d
a: R=R1=R2=H
b: R=H, R1=R2=CH3
c: R=R1=H, R2=OCH3
d: R=NO2, R1=H, R2= OCH3
Reagents and conditions: (i)EtOH, reflux; (ii) CH3OH, reflux; (iii)
SOCl2, reflux; (iv) n-Bu3N, toluene, reflux; (v) LiOH, THF/H2O, rt
B
H
H
H
H
H
Br
H
O
S
S
S
HN
HN
H2N
,ii
MeO
O
N
MeO
O
N
N
i
O- Et3NH+
O
OH
O
O
8
O
O
O
7
6-APA
R2
R
iii, iv
R1
H
H
S
N
O
v
H2N
H
H
S
N
O
N
O
6
H
O
N
O
10
O
11a-d
O
O
vi
R2
R
R1
a: R=R1=R2=H
b: R=H, R1=R2=CH3
c: R=R1=H, R2=OCH3
N
O
H
H
O
S
O
N
d: R=NO2, R1=H, R2= OCH3
H
12a-d
N
O-Na+
O
Reagents and conditions: (i) MeCOCH2CO2Me, Et3N, DCM, rt;(ii)DMF, rt; (iii) TsOH.H2O, acetone,
rt; (iv) DCM,Et3N and NaOH; (v) DCC, DCM, rt; (vi) H2, 10% Pd/C, NaHCO3/H2O/THF, rt
Scheme 2. (A) General synthetic route to compounds 5a–d; (B) General synthetic route to
compounds 12a–d.
Next, the hydrolysis of 5 with LiOH solution afforded corresponding acid 6 in almost
quantitative yield [24]. This intermediate was then directly used for the coupling reaction with
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Molecules 2015, 20, 22044–22057
6-amino penicillanic nucleus. At first, we tried to perform the direct coupling of 6 with the
commercially available and inexpensive (+)-6-APA, but the formation of a complex mixture of
products was observed. Therefore, we considered a different strategy based on the coupling of
azetidinone key intermediate 6 to the benzyloxy ester of (+)-6-APA [33], followed by hydrogenolysis
in a mixture of THF/aqueous NaHCO3 to deprotect the benzyl ester and directly furnish the target
compounds as sodium salt (Scheme 2B) [34]. (+)-6-APA was N-protected using ethyl acetoacetate,
then esterified with benzyl bromide and next the amine function of (+)-6-APA ester was liberated
by treatment with p-toluensulfonic acid affording 10 [33]. The free amine 10 was so added to a
preformed mixture of acid 6 and DCC in order to afford compound 11 as mixture of diastereomers,
with the 2-azetidinone ring directly coniugated to 6-APA residue [33]. Thus, derivatization of 6-APA
led to higher coupling reaction yields and easier isolation of target compounds. Finally, to ensure
water-solubility of the final compounds hydrogenation of 11 over Pd/C (10%) in THF/aqueous
NaHCO3 at room temperature afforded the target molecules 12 in high yield [34]. All the reported
compounds were characterized by analytical spectroscopic methods (1H- and 13C-NMR, MS) and
elemental analysis.
2.2. Biological Work
2.2.1. Antimicrobial Activity of Compounds 12a–d
The antibacterial activity of new compounds was evaluated by determining the minimum
inhibitory concentration (MIC), according to the CLSI (Clinical and Laboratory Standards Institute,
formerly the NCCLS) guidelines and minimum lethal dose (MLD) [35]. All the synthesized
compounds 12a–d were tested against pathogenic and non-pathogenic strains, representative of
Gram positive (Staphylococcus aureus, Staphylococcus epidermidis, Bacillus sp.) and Gram negative
bacteria (Escherichia coli, Salmonella entericaserovar Typhimurium, Pseudomonas fluorescens and
Pseudomonas aeruginosa). Ampicillin was used as the reference compound, a derivative of the
6-aminopenicillanic acid (6-APA), clinically used for the treatment of infections caused by
Gram positive (Streptococcus spp., Staphyloccus spp.) and few Gram negative bacteria (Neisseria,
some Enterobacteriaceae).
Table 1 shows that four compounds, on the whole, did not exhibit antimicrobial activity towards
Gram negative considered, although the analysis of MIC50 evidenced that 12a showed some growth
inhibition activity against Salmonella (MIC50 > 64 µg¨ mL´1). In contrast, they were very effective
against all examined Gram positive (Tables 1 and 2). In order to better interpret the results, MIC and
MLD were expressed also in terms of molarity (values in parentheses in Tables 1 and 2). The MIC100
and/or MLD analysis indicated that all four modifications of the 6-APA improved the antimicrobial
activity against S. aureus, compared to ampicillin. The compound most efficient was 12a, with
both the MIC100 and MLD two times as massic concentration and almost three times lower molar
concentration than that of ampicillin (Table 2). Similar results were obtained against S. epidermidis,
but in this case improvements were more evident by comparing the molar concentrations. For this
bacterial strain, the derivative 12a turned out the most active in terms of MIC, two times lower than
MIC of ampicillin as molar concentration, while the 12c presented the highest biocidal action (Tables 1
and 2). All new compounds were very effective also against the Bacillus strain, although by MIC and
MLD analysis there were no obvious improvements compared to the activity of ampicillin. Among
the four new compounds, the highest antimicrobial activity was observed for 12a and 12b, the first
with the lowest values of MIC and MLD (Tables 1 and 2).
Thus, all synthesized compounds were characterized by a strong antimicrobial action towards
Gram positive, and as reported in Table 2 (column MICa´d/MICa) 12a was the one with greater
activity. In addition, 12a was the only compound with a slight activity also against Gram negative,
confirming its best antibacterial ability.
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Molecules 2015, 20, 22044–22057
Table 1. Minimal inhibitory concentration (MIC) and Minimal lethal dose (MLD) as µg¨ mL´1. The numbers in parentheses indicate the molar concentration (µM).
Salmonella
Pseudomonas spp.
Escherichia coli
Staphylococcus aureus
Staphylococcus epidermidis
Bacillus sp.
MIC100
Compd.
typhimurium
(fluorescens, aeruginosa)
MIC100 MLD
MIC100 MLD
MIC100
MLD
MIC100
MLD
MIC100
MLD
MLD
Amp
12a
12b
12c
12d
48
ď256
>256
>256
>256
>256
8.0
ď256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
2.0 (5.8)
1.0 (2.1)
8.0 (23.2)
4.0 (8,4)
32.0 (63.4)
<4.0 (<7.9)
32.0 (57.6)
0.35 (1.0)
0.25 (0.52)
0.35 (0.69)
<0.35 (<0.69)
0.75 (1.35)
16.0 (46.0)
16.0 (33.6)
0.25 (0.72)
0.5 (1.05)
8.0 (23.2)
32.0 (67.2)
48.0 (86.4)
128.0 (252.2)
64.0 (115.2)
>256
>256
>256
>256
>256
>256
>256
>256
<1.5(3.9)
ě16.0 (ě31.7)
>1.0; <1.5 (>1.98; <2.97)
2.0(3.94)
>2.0; <3.0 (>3.9; <5.9)
1.75 (3.45)
8.0(15.8)
ě16.0 (ě28.8)
2.0 (3.6)
MIC100 = Minimal compound concentration that completely inhibits bacterial growth; Amp = ampicillin. Each value represents the mean of three independent determinations.
Table 2. Antimicrobial efficiency of compounds evaluated by MICs and MLDs ratios. In parentheses are indicate the ratios of the MIC expressed as molarity (µM).
Each value was obtained from the ratio of MIC100 indicated in Table 1.
S. aureus
MLDCmp/MLDAmp
0.5 (0.36)
S. epidermidis
MLDCmp/MLDAmp
1.0 (0.73)
Bacillus sp.
MLDCmp/MLDAmp
4.0 (2.89)
Compd.
MICCmp/MICAmp
MICa´d/MICa
MICCmp/MICAmp
MICa´d{MICa
MICCmp/MICAmp
MICa´d{MICa
12a
12b
0.5 (0.36)
1
0.71 (0.52)
1
2.0 (1.46)
1
>4.0, <6.0
(>2.75, <4.12)
>2.0, <3.0
(>1.88, <2.83)
<0.75 (<0.67)
4.0 (2.73)
<1.5 (<1.86)
1.0 (0.69)
ě1.0 (ě0.69)
1.4 (1.33)
6.0 (3.72)
>1.0, <1.5
>2.0, <3.0
12c
<0.5 (<0.34)
4.0 (2.48)
<1.0 (<0.69)
2.14 (1.35)
0.5 (0.34)
<1.4 (<1.33)
2.14 (2.6)
8.0 (5.47)
16.0 (10.87)
8.0 (4.96)
4.0 (3.75)
(>0,67, <1.01)
(>1.87, <2.81)
12d
0.87 (0.6)
1.75 (1.64)
ě1.0 (ě0.63)
8.0 (5.0)
4.0 (3.43)
MICCmp = MIC of indicated compounds (a–d); MICAmp = MIC ampicillin; MICa´d = MIC of compounds 12a, b, c, d; MICa = MIC of compound 12a; MLDCmp = MLD of indicated
compounds (a–d); MLDAmp = MLD of ampicillin.
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Molecules 2015, 20, 22044–22057
2.2.2. Cytotoxicity Assays
In vitro cytotoxicity of the new compounds was carried out evaluating viability of NIH-3T3 cells
after 72 h of incubation in the presence of increasing concentrations of antibiotics (2–50 µM) (Figure 2).
Cell viability was assessed by using the MTT assay as described in the Experimental Section. As
shown in Figure 2, the presence of 12a, 12b and 12d induced a mild reduction of vitality respect to the
control (0.5% DMSO alone), however for 12d higher values (10–50 µM) a dose-dependent reduction
of cell viability was observed indicating a potential toxicity. Compound 12c showed slight or no
cytotoxic effect on the cell line at concentration between 2 and 50 µM. Thus, these results suggest that
tested compounds do not show in vitro relevant effects on the cell viability at concentration tens of
times higher than MICs found in examined Gram-positive bacteria (see Table 1).
120
12a
12b
12c
12d
100
80
60
40
20
0
2
5
10
20
50
Compound concentration (μM)
Figure 2. Effect of synthesized compounds on cell viability. NIH-3T3 cells were exposed 72 h at
indicated concentration of 12a, b, c, d. Inhibition activity of compounds was expressed as percentages
of control (0.5% DMSO). Each point represents the mean ˘ SD of the six experiments.
3. Experimental Section
3.1. Chemistry
3.1.1. Material and Methods
All reagents and anhydrous solvents were obtained from commercial sources and used
without further purification. All reactions requiring anhydrous conditions were performed under
N2 atmosphere and all glassware were flame dried. Elemental analyses were performed on the
FlashEA 1112 Series with Thermal Conductivity Detector (Thermo Scientific Corporation, Waltham,
MA, USA). 1H- and 13C-NMR spectra were recorded on Bruker (Rheinstetten, Germany) DRX
600, 400, 300, 250 spectrometers (600 MHz, 400 MHz , 300 MHz, 250 MHz for 1H; 151 MHz,
100 MHz, 75 MHz, 62.89 MHz for 13C. J values are given in Hz. The 1H chemical shifts were
referenced to the solvent peak: CDCl3 (7.26 ppm), and the 13C chemical shifts were referenced to
the solvent peak: CDCl3 (77.0 ppm). For D2O, 13C-NMR spectra were referenced to the signal for
the carbonyl group of acetone (one drop, added as an internal standard), which was set to 215.94
ppm. ESI(+)-MS measurements were performed on a Waters 4 micro quadrupole mass spectrometer
equipped with electrospray ion source (Waters, Milford, MA, USA). IR spectra were recorded on an
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Molecules 2015, 20, 22044–22057
FT-IR instrument Bruker Vector 22 (Bruker, Ettlingen, Germany). Melting points were performed
on DSC 2920 TA INSTRUMENTS (TA Instruments-Division of Waters, Milano, Italy). Thin-layer
chromatography was performed on Macherey-Nagel (Macherey-Nagel GmbH & Company, Düren,
Germany) pre-coated aluminum sheets (0.20 mm, silica gel 60 with fluorescent indicator UV254
)
in appropriate solvent. Column chromatography was carried out using silica gel 60 (70–230 mesh
ASTM, Merck, Darmastdt, Germany).
Compounds 3a–dwere prepared according to the previously described procedure and their
spectral features matched with those reported in literature [29–32].
3.1.2. Synthesis of Compounds 3a–d. General Method
The aldehyde 1 (11 mmol) was added to a solution of anilines 2 (11 mmol) in absolute ethanol
(40 mL). The resulting mixture was heated at reflux for 4 h and left standing overnight at room
temperature. After the reaction was complete, the solution was cooled and the precipitate formed was
collected by filtration. The product was washed with cold water and purified by recrystallization.
˝
N-Benzylidenebenzenamine (3a). Recrystallized from ethanol as pale colorless needles; m.p. 50–51 C.
1H-NMR (300 MHz, CDCl3): δ 7.25–7.28 (m, 3H), 7.44 (t, J = 8.3, 2H), 7.50–7.54 (m, 3H), 7.94–7.97 (m,
2H), 8.49 (s, 1H); 13C-NMR (75 MHz, CDCl3): δ 120.8, 125.9, 128.7, 128.8, 129.1, 131.3, 136.2, 152.0,
160.3 [29,30].
N-Benzylidene-3,4-dimethylbenzenamine (3b). Recrystallized from ethanol as yellow solid [31]; m.p.
˝
1
42–43 C. H-NMR (400 MHz, CDCl3): δ = 2.28 (s, 3H), 2.30 (s, 3H), 6.99–7.00 (m, 1H), 7.05 (s, 1H),
7.15 (d, J = 7.9, 1H), 7.46–7.48 (m, 3H), 7.88–7.91 (m, 2H), 8.47 (s, 1H); 13C-NMR (100 MHz, CDCl3):
δ = 19.2, 19.7, 117.9, 122.2, 128.6 (ˆ2), 130.1, 131.0, 134.4, 136.2, 137.2, 149.7, 159.3.
N-Benzylidene-(4-methoxybenzenamine) (3c). The reaction was performed at room temperature
˝
affording the title compound after 3 h [30]. Recrystallized from ethanol as gray plates; m.p. 71–72 C.
1H-NMR (300 MHz, CDCl3): δ = 3.83 (s, 3H), 6.93 (d, J = 8.4, 2H), 7.23 (d, J = 8.4, 2H), 7.43–7.47
(m, 2H), 7.86–7.92 (m, 2H), 8.48 (s, 1H); 13C-NMR (100 MHz, CDCl3): δ = 55.4, 114.2, 122.0, 128.4,
128.5, 130.8, 136.3, 144.7, 158.1, 158.2.
N-(4-Nitrobenzylidene)-4-methoxybenzenamine (3d). Recrystallized from ethanol as yellow solid [32];
˝
1
m.p. 126–128 C. H-NMR (400 MHz, CDCl3): δ = 3.85 (s, 3H), 6.96 (d, J = 8.8, 2H), 7.31 (d, J = 8.8,
2H), 8.06 (d, J = 8.4, 2H), 8.31 (d, J = 8.4, 2H), 8.58 (s, 1H); 13C-NMR (100 MHz, CDCl3): δ = 55.4,
114.20, 122.1, 128.6, 130.9, 136.4, 142.4, 144.6, 150.6, 158.2.
3.1.3. Methyl 4-chloro-4-oxobutanoate (4)
The product was prepared as a pale yellow oil from succinic anhydride according to the literature
procedure in 98% yield and was used without further purification [27,28].
1H-NMR (300 MHz, CDCl3): δ = 2.68 (t, J = 6.4, 2H), 3.21 (t, J = 3.4, 2H), 3.71 (s, 3H); 13C-NMR
(75 MHz, CDCl3): δ = 173.0, 171.2, 52.1, 41.6, 29.0.
3.1.4. General procedure for the synthesis of azetidin-2-ones 5a–d
A flame-dried round-bottom flask was charged with a solution of imine 3 (2 mmol) and
n-tributylamine (6 mmol, 3 equiv.) in dry toluene (8 mL). The solution was heated to reflux and the
acyl chloride 4 (4 mmol, 2 equiv.) was then added dropwise through a syringe. The resulting solution
was kept at reflux temperature for 22 h. After cooling, 1M HCl was added and the mixture was
stirred for an additional 15 min. The mixture was transferred to a separatory funnel and extracted
with AcOEt (2 ˆ 10 mL). The combined organic layers were washed first with saturated NaHCO3
aqueous solution (20 mL), brine (20 mL), and finally dried over Na2SO4, and evaporated. The oily
crude mixture was purified by column chromatography (silica gel, 7:3 hexane/AcOEt) affording
trans-2-azetidinone compound 5 [24].
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Molecules 2015, 20, 22044–22057
˝
Methyl 2-(2-oxo-1,4-diphenylazetidin-3-yl)acetate (5a). Brown solid (45% yield, m.p. 144–146 C). IR (KBr
1
pellet, cm´1): ν 2958, 1746, 1597, 1498, 1389; H-NMR (300 MHz, CDCl3): δ 2.83 (dd, J = 10.2, 16.7,
1H), 2.99 (dd, J = 4.7, 16.1, 1H), 3.36–3.42 (m, 1H), 3.70 (s, 3H), 4.85 (d, J = 2.3, 1H), 6.91–7.06 (m, 2H),
7.14–7.38 (m, 8 H); 13C-NMR (75 MHz, CDCl3): δ 172.2, 166.0, 140.1, 139.9, 130.4 (ˆ2), 128.0, 127.1,
126.3 (ˆ2), 125.8 (ˆ2), 125.2 (ˆ2), 61.8, 55.1, 52.1, 32.5. MS: m/z 295 (M+). Anal. calcd. for C18H17NO3:
C, 73.20; H, 5.80; N, 4.74%; found: C, 73.16; H, 5.83, N, 4.64 %.
Methyl 2-(1-(3,4-dimethylphenyl)-2-oxo-4-phenylazetidin-3-yl)acetate (5b). Colorless oil (60% yield). IR
(KBr pellet, cm´1): ν 2950, 1736, 1612, 1513, 1315; 1H-NMR (400 MHz, CDCl3): δ 2.16 (s, 3H), 2.17 (s,
3H), 2.81 (dd, J = 10.2, 16.7, 1H), 2.97 (dd, J = 4.5, 16.7 , 1H), 3.32–3.37 (m, 1H), 3.69 (s, 3H), 4.81 (d,
J = 2.3, 1H), 6.84–6.86 (m, 1H), 6.95–6.97 (m, 2H), 7.22–7.29 (m, 5H); 13C-NMR (75 MHz, CDCl3): δ
171.0, 165.7, 137.6, 135.3, 132.3, 129.9, 129.0 (ˆ2), 128.4, 126.0 (ˆ2), 118.6, 114.3, 61.0, 55.7, 52.1, 32.9,
29.7, 19.9, 19.3. MS: m/z 323 (M+). Anal. calcd. for C20H21NO3: C, 74.28; H, 6.55; N, 4.33%; found: C,
74.31; H, 6.53, N, 4.32%.
Methyl-2-(1-(4-methoxyphenyl)-2-oxo-4-phenylazetidin-3-yl)acetate (5c). Yellow solid (55% yield, m.p.
˝
111–113 C). IR (KBr pellet, cm´1): ν 2954, 1741, 1647, 1592, 1503; 1H-NMR (300 MHz, CDCl3):
δ 2.82 (dd, J = 10.3, 16.7, 1H), 2.98 (dd, J = 4.6, 16.7, 1H), 3.31–3.38 (m, 1H), 3.69 (s, 3H), 3.73 (s, 3H),
4.81 (d, J = 2.1, 1H), 6.78 (d, J = 9.0, 2H), 7.22 (d, J = 9.0, 2H), 7.26–7.36 (m, 5H); 13C-NMR (75 MHz,
CDCl3): δ 171.3, 156.1, 137.4, 131.1, 129.0 (ˆ2), 128.5, 127.6, 126.1 (ˆ2), 118.4 (ˆ2), 114.3 (ˆ2), 61.1,
55.7, 55.4, 52.0, 32.9. MS: m/z 325 (M+). Anal. calcd. for C19H19NO4: C, 70.14; H, 5.89; N, 4.31%;
found: C, 70.16; H, 5.86; N, 4.30%.
Methyl 2-(1-(4-methoxyphenyl)-2-(4-nitrophenyl)-4-oxoazetidin-3-yl)acetate (5d). Brown solid (52% yield.
˝
m.p. 124–126 C). IR (KBr pellet, cm´1): ν 2944, 1741, 1607, 1513, 1354; 1H-NMR (400 MHz, CDCl3):
δ, 2.86 (dd, J = 11.2, 17.2, 1H), 3.04 (dd, J = 4.4, 17.2, 1H), 3.33–3.38 (m, 1H), 3.71 (s, 3H), 3.74 (s, 3H),
4.93 (d, J = 2.0, 1H), 6.79 (d, J = 9.2, 2H), 7.16 (d, J = 9.2, 2H), 7.57 (d, J = 8.8, 2H), 8.24 (d, J = 8.8,
2H); 13C-NMR (100 MHz, CDCl3): δ 171.2, 164.3, 156.5, 148.0, 144.9, 130.5, 127.2 (ˆ2), 124.3 (ˆ2), 118.3
(ˆ2), 114.5 (ˆ2), 60.3, 55.9, 55.4, 52.2, 32.8. MS: m/z 370 (M+). Anal. calcd. for C19H18N2O6: C, 61.62;
H, 4.90; N, 7.56%; found: C, 61.65; H, 4.87, N, 7.58%.
3.1.5. General Procedure for the Synthesis of 6a–d
A 0.98M aqueous solution of LiOH (0.10 mL, 0.096 mmol) was added to a solution of trans-5
(0.08 mmol) at room temperature in THF. The reaction mixture was stirred at room temperature for
5h and quenched with HCl 1 M (0.2 mL). The reaction mixture was diluted with ethyl acetate (1 mL)
and washed with HCl 1M (1 mL), water (1 mL) and brine (1 mL). The combined organic layers were
dried over magnesium sulfate and concentrated in vacuo to give crude acid 6 of at purity such as to
be used without further purification [24].
2-(2-Oxo-1.4-diphenylazetidin-3-yl)acetic acid (6a). Brown solid (>99% yield). 1H-NMR (250 MHz,
CDCl3): δ 2.89 (dd, J = 9.5, 17.0, 1H), 3.03 (dd, J = 5.2, 17.0, 1H), 3.38–3.49 (m, 1H), 4.84 (d, J = 2.5, 1H),
7.00–7.12 (m, 2H), 7.16–7.38 (m, 8H); 13C-NMR (75 MHz, CDCl3): δ 176.0, 165.6, 140.3, 139.9, 129.4
(ˆ2), 129.1 (ˆ2), 128.5 (ˆ2), 126.3, 124.7, 122.0 (ˆ2), 61.0, 55.2, 32.3.
2-(1-(3.4-Dimethylphenyl)-2-oxo-4-phenylazetidin-3-yl)acetic acid (6b). White solid (>99% yield).
1H-NMR (300 MHz, CDCl3): δ 2.16 (s, 3H), 2.17 (s, 3H), 2.86 (dd, J = 9.8, 17.0 Hz, 1H), 3.00 (dd,
J = 5.0, 17.0 Hz, 1H), 3.34–3.40 (m, 1H), 3.69 (s, 3H), 4.81 (d, J = 2.2 Hz, 1H), 6.84–6.87 (m, 1H),
6.95–7.00 (m, 2H), 7.22–7.34 (m, 5H); 13C-NMR (75 MHz, CDCl3): δ 175.1, 165.8, 138.0, 137.4, 134.8,
132.5, 130.0, 128.9 (ˆ2), 128.4, 126.1, 118.6 (ˆ2), 114.5, 60.8, 55.2, 32.5, 19.3, 19.1.
2-(1-(4-Methoxyphenyl)-2-oxo-4-phenylazetidin-3-yl)acetic acid (6c). Brown oil (>99% yield). 1H-NMR
(250 MHz, CDCl3): δ 2.02 (s, 3H), 2.84 (dd, J = 9.8, 17.0, 1H), 2.99 (dd, J = 5.0, 17.0, 1H), 3.29–3.39 (m,
1H), 3.70 (s, 3H), 4.80 (d, J = 2.25, 1H), 6.75 (d, J = 9.0, 2H), 7.19 (d, J = 9.0, 2H), 10.4 (bs, 1H); 13C-NMR
22051

Molecules 2015, 20, 22044–22057
(75 MHz, CDCl3): δ 175.5, 165.5, 156.2, 141.9, 137.1, 130.9, 129.1, 128.6, 126.1, 122.3, 118.5, 114.3, 61.1,
55.4, 55.3, 32.8.
2-(1-(4-Methoxyphenyl)-2-(4-nitrophenyl)-4-oxoazetidin-3-yl)acetic acid (6d). Brown oil (>99% yield).
1H-NMR (400 MHz, CDCl3): δ 2.92 (dd, J = 10.4, 17.6, 1H), 3.07 (d, J = 4.4, 17.6, 1H), 3.36–3.39 (m, 1H),
3.74 (s, 3H), 4.92 (d, J = 2.0, 1H), 6.79 (d, J = 9.2, 2H), 7.16 (d, J = 9.2, 2H), 7.54 (d, J = 8.8, 2H), 8.22 (d,
J = 8.8, 2H); 13C-NMR (75 MHz, CDCl3): δ 175.0, 164.5, 156.5, 148.0, 144.6, 130.2, 127.1 (ˆ2), 124.3
(ˆ2), 118.3 (ˆ2), 114. 5 (ˆ2), 60.2, 55.4 (ˆ2), 32.5.
3.1.6. Synthesis of (2S,5R,6R)-Benzyl 6-amino-3,3-dimethyl-7-oxo-4-thia-1-aza-bicyclo[3.2.0]
heptane-2-carboxylate 10
A mixture of 6-APA (1g, 4.6 mmol) and triethylamine (1.30 mL, 9.2 mmol) in anhydrous
dichloromethane (10 mL) was stirred at rt until dissolution before adding methyl acetoacetate
(4.6 mmol, 0.502 mL). The stirring was continued for 3h and the solvent was then removed in vacuo
affording 7. To the crude 7 dissolved in DMF (10 mL) benzyl bromide (4.6 mmol, 0.54 mL) was
added and the reaction mixture was stirred overnight. The solution was diluted with ethyl acetate
and washed with brine (3 ˆ 15 mL). The combined organic layers were dried over magnesium sulfate,
filtered and concentrated under reduced pressure to give crude material 8 as a solid. A solution of 8 in
acetone (10 mL) was treated with p-toluenesulfonic acid monohydrate (967 mg). The salt 9 precipitate
after 10 min. was filtered and washed with ethyl ether (5 mL). Finally, crude 9 was solubilized with
triethylamine (1.30 mL) in dichloromethane (50 mL) and stirred for 2 h. NaOH 1 M (10 mL) was
added and the organic phase removed. The aqueous phase was extracted with dichloromethane and
the resulting organic phase were combined, washed with brine (10 mL), dried and concentrated under
vacuum to give 10 as yellow thick oil [33].
1H-NMR (300 MHz, CDCl3): δ 1.40 (s, 3H), 1.60 (s, 3H), 4.41 (s, 1H), 4.54 (d, J = 4.3, 1H), 5.17
(s, 2H), 5.49 (d, J = 4.3, 1H), 7.33–7.44 (m, 5H); 13C-NMR (75 MHz, CDCl3): δ 177.7, 168.0, 134.8, 128.7,
69.9, 67.4, 64.0, 62.8, 31.7, 27.0.
3.1.7. General Procedure for the Synthesis of 11a–d
DCC (0.33 mmol) was added to a solution of the acid 6 (0.30 mmol) in anhydrous
dichloromethane (3.8 mL) at room temperature and stirred for 30 min. After filtration on Celite,
10 was added to the solution and the reaction mixture was stirred overnight at room temperature.
The solvent was then evaporated to dryness under vacuum [33]. The residue obtained was purified
by chromatography on silica gel using dichloromethane/AcOEt (9/1 to 7/3) as eluent to give 11 as
inseparable mixture of diastereomers.
(2S,5R,6R)-Benzyl 6-(2-(2-oxo-1,4-diphenylazetidin-3-yl)acetamido)-3,3-dimethyl-7-oxo-4-thia-1-aza-bicyclo
heptane-2-carboxylate (11a). Colorless oil isolated as inseparable mixture of diastereomers (55% yield).
IR (neat, cm´1): ν 3320, 3047, 1746, 1676, 1501, 1461, 1389; 1H-NMR (250 MHz, CDCl3): δ 1.39 (s, 3H),
1.61 (s, 3H), 2.81 (dd, J = 8.0, 15.8, 1H), 2.92 (dd, J = 7.0, 15.8, 1H), 3.34–3.40 (m, 1H), 4.47 (s, 1H), 4.84
(d, J = 2.2, 1H ), 4.87 (d, J = 2.2, 1H), 5.18 (s, 2H), 5.51 ( d, J = 4.2, 1H), 5.54 (d, J = 4.2, 1H), 5.61–5.69
(m, 1H), 7.01–7.37 (m, 15H); 13C-NMR (62.89 MHz, CDCl3): δ173.4, 169.3, 167.5, 166.6, 137.2, 136.9,
136.8, 134.7, 129.1, 128.7, 128.6, 126.0, 124.2, 117.2, 70.3, 68.0, 67.5, 64.9, 60.6, 60.4, 59.1, 58.9, 55.5, 32.0,
26.8, 21.0, 14.2 MS: m/z 570 (M+). Anal. calcd. for C32H31N3O5S: C, 67.47; H, 5.48; N, 7.38%; found: C,
67.44; H, 5.50, N, 7.36%.
(2S,5R,6R)-Benzyl 6-(2-(1-(3,4-dimethylphenyl)-2-oxo-4-phenylazetidin-3-yl)acetamido)-3,3-dimethyl-7-oxo-4
-thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylate (11b). Yellow oil isolated as inseparable mixture of
diastereomers (69% yield). IR (neat, cm´1): ν 3463, 2958, 1746, 1671, 1508, 1463, 1394; 1H-NMR
(300 MHz, CDCl3): δ 1.40 (s, 3H), 1.41 (s, 3H), 1.63 (s, 3H), 1.64 (s, 3H), 2.17 (s, 12H), 2.80 (dd, J = 7.1,
15.7, 2H), 2.91 (dd, J = 7.1, 15.7, 2H), 3.30–3.37 (m, 2H), 4.47 (s, 2H), 4.79 (d, J = 2.3, 1H ), 4.82 (d,
J = 2.3, 1H), 5.19 (s, 4H), 5.52 (d, J = 4.2, 1H), 5.54 (d, J = 4.2, 1H), 5.63 (dd, J = 4.2, 8.4, 1H), 5.66 (dd,
22052

Molecules 2015, 20, 22044–22057
J = 4.2, 8.4, 1H), 6.85–6.98 (m, 1H), 7.15 (m, 4 H), 7.37 (m, 12H); 13C-NMR (75 MHz, CDCl3): δ 173.4,
169.2, 167.5, 166.2, 166.1, 137.5, 137.1, 135.1, 134.6, 132.7, 129.9, 129.1, 128.7, 125.9, 118.6, 114.4, 70.4,
68.0, 67.4, 64.9, 60.8, 60.4, 59.1, 58.9, 55.3, 32.0, 31.9, 26.8, 19.8, 19.2. MS: m/z 620 [M+Na]+. Anal. calcd.
for C34H35N3O5S: C, 68.32; H, 5.90; N, 7.03%;found: C, 68.36; H, 5.84, N, 7.05%.
(2S,5R,6R)-Benzyl 6-(2-(1-(4-methoxylphenyl)-2-oxo-4-phenylazetidin-3-yl)acetamido)-3,3-dimethyl-7-oxo-4-
thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylate (11c). Yellowish oil isolated as inseparable mixture of
diastereomers (54% yield). IR (neat, cm´1): ν 3379, 2949, 1746, 1681, 1518, 1246; 1H-NMR (300 MHz,
CDCl3): δ 1.40 (s, 6H), 1.63 (s, 6H), 2.80 (dd, J = 7.1, 15.7, 2H), 2.91 (dd, J = 7.1, 15.7, 2H), 3.30-3.37 (m,
2H), 3.73 (s, 6H), 4.47 (s, 2H), 4.79 (d, J = 2.3, 1H ), 4.82 (d, J = 2.3, 1H), 5.19 (s, 4H), 5.52 (d, J = 4.2,
1H), 5.54 (d, J = 4.2, 1H), 5.62–5.69 (m, 2H), 6.77 (d, J = 9.0, 4H), 7.21 (d, J = 9.0, 4 H), 7.27–7.37 (m,
20H); 13C-NMR (100.03 MHz, CDCl3): δ 173.6, 169.2, 167.5, 156.2, 141.2, 139.9, 134.6, 130.6, 129.1(ˆ2),
128.7, 126.1 (ˆ3), 118.5 (ˆ2), 114.4 (ˆ2), 70.4, 68.0, 67.5, 65.0, 61.2, 60.7, 59.1, 55.5, 35.4, 34.9, 32.2, 31.5,
26.8 . MS: m/z 620 [M+Na]+. Anal. calcd. for C33H33N3O6S: C: 66.09, H: 5.55, N: 7.01;found: C: 66.06,
H: 5.57, N: 7.04.
(2S,5R,6R)-Benzyl 6-(2-(1-(4-methoxylphenyl)-2-(4-nitrophenyl)-4-oxoazetidin-3-yl)acetamido)-3,3-dimethyl-
7-oxo-4-thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylate (11d). Yellowish oil isolated as inseparable
mixture of diastereomers (45% yield). IR (neat, cm´1): ν 2929, 1751, 1666, 1513, 1349, 1246; 1H-NMR
(400 MHz, CDCl3): δ 1.40 (s, 6H), 1.63 (s, 6H), 2.82 (dd, J = 8.0, 16.0, 2H), 2.95 (dd, J = 5.2, 16.0, 2H),
3.32–3.36 (m, 2H), 3.74 (s, 6H), 4.47 (s, 2H), 4.95 (d, J = 2.0, 1H), 4.98 (d, J = 2.0, 1H), 5.18 (s, 4H),
5.50 (d, J = 4.4, 1H), 5.55 (d, J = 4.4, 1H), 5.60–5.68 (m, 2H), 6.79 (d, J = 9.2, 4H), 7.16 (d, J = 9.2, 4 H),
7.36–7.39 (m, 10H), 7.55-7.59 (m, 4 H), 8.22 (d, J = 8.4, 4H); 13C-NMR (100.03 MHz, CDCl3): δ 173.2,
169.0, 167.3, 164.8, 156.5, 148.0, 144.6, 138.1, 134.6, 130.3, 128.7, 127.2, 127.0, 124.3, 118.4, 114.5, 70.4,
67.8, 65.1, 60.3, 58.7, 55.4, 32.0, 29.7, 26.9 . MS: m/z 644 [M]+. Anal. calcd. for C33H32N4O8S: C: 61.48,
H: 5.00, N: 8.69; found: C: 61.51, H: 4.98, N: 8.72.
3.1.8. General Procedure for the Synthesis of 12a–d
10 mol % Pd/C (46 mg) was added to a solution of 11 (0.039 mmol) in a mixture of
tetrahydrofuran (3.9 mL), water (3.9 mL) and 0.006 g (0.08 mmol) of NaHCO3. The reaction mixture
was left at rt overnight connected to a double layer balloon of hydrogen. The catalyst was filtered
and washed with ethanol (4 mL) and water (4 mL). The combined filtrate was extracted with diethyl
ether (2 ˆ 8 mL) and the aqueous phase was freeze-dried yielding in high purity the compound 12 as
a sodium salt [34].
Sodium (2S,5R,6R)-6-(2-(2-oxo-1,4-diphenylazetidin-3-yl)acetamido-3,3-dimethyl-7-oxo-4-thia-1-aza-bicyclo
[3.2.0]heptane-2-carboxylate (12a). Light gry solid isolated as inseparable mixture of diastereomers (76%
˝
1
yield m.p. 240–243 C). IR (KBr pellet, cm´1): ν 3400, 2924, 1751, 1662, 1592, 1389, 1503; H-NMR
(300 MHz, D2O): δ 1.39 (s, 3H), 1.40 (s, 3H), 1.54 (s, 3H), 1.55 (s, 3H), 2.81–3.00 (m, 4H), 3.49–3.57 (m,
2H), 4.18 (s, 2H), 5.03 (d, J = 2.5, 1H), 5.07 (d, J = 2.5, 1H), 5.34 ( d, J = 4.2, 1H), 5.38 (d, J = 4.2, 1H), 5.45
(d, J = 4.2, 1H), 5.51 (d, J = 4.2, 1H), 7.12–7.45 (m, 20H); 13C-NMR (100.03 MHz, D2O): δ 175.7, 175.3,
173.3, 170.1, 169.5, 137.6, 137.4, 137.1, 130.2, 130.0, 129.6, 127.4, 125.9, 118.8, 74.0, 72.4, 65.4, 65.2, 61.3,
59.0, 31.5, 28.5, 27.2. MS: m/z 501 [M+Na]+. Anal. calcd. for C25H24N3O5SNa: C: 59.87, H: 4.82, N:
8.38; found: C: 59.85, H: 4.80, N: 8.36.
Sodium (2S,5R,6R)-6-(2-(1-(3,4-Dimethylphenyl)-2-oxo-4-phenylazetidin-3-yl)acetamido)-3,3-dimethyl-7-oxo
-4-thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylate (12b). Pearly white solid as inseparable mixture of
˝
diastereomers (67% yield. m.p. 235–238 C). IR (KBr pellet, cm´1): ν 3439, 2963, 1751, 1676, 1617,
1508, 1399; 1H-NMR (250 MHz, CDCl3): δ 1.40 (s, 3H), 1.43 (s, 3H), 1.55 (s, 6H), 2.13(s, 12H), 2.87–2.94
(m, 4H), 3.43–3.52 (s, 2H), 4.19 (s, 2H), 5.03 (d, J = 2.8, 1H), 5.07 (d, J = 2.8, 1H), 5.34 (d, J = 4.0, 1H),
5.37 (d, J = 4.0, 1H), 5.46 (d, J = 4.0, 1H), 5.51 (d, J = 4.0, 1H), 6.98–7.10 (m, 6 H), 7.35–7.43 (m, 10H);
13C-NMR (100.03 MHz, D2O): δ 174.9, 172.3, 172.1,168.1, 137.7, 137.6, 135.4, 132.9, 130.4, 129.4, 129.0,
22053

Molecules 2015, 20, 22044–22057
126.7, 119.5, 119.1, 115.9, 115.3, 74.1, 67.2, 64.9, 60.7, 58.6, 56.5, 55.6, 34.1, 31.0, 30.7, 27.5, 27.3, 19.8,
19.1. MS: m/z 529 [M + Na]+. Anal. Calcd. for C27H28N3 O5SNa: C: 61.23, H: 5.33, N: 7.93; found: C:
61.26, H: 5.34, N: 7.91.
Sodium (2S,5R,6R)-6-(2-(1-(4-methoxyphenyl)-2-oxo-4-phenylazetidin-3-yl)acetamido)-3,3-dimethyl-7-oxo-4-
thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylate (12c). Yellow-white solid as inseparable mixture of
˝
diastereomers (80% yield, m.p. 245–248 C). IR (KBr pellet, cm´1): ν 3439, 2934, 1741, 1657, 1602,
1518, 1399, 1251; 1H-NMR (600 MHz, D2O): δ 1.44 (s, 3H), 1.48 (s, 3H), 1.58 (s, 3H), 1.59 (s, 3H), 2.89
(dd, J = 9.6, 15.0, 2H), 2.96–3.00 (m, 2H), 3.50–3.58 (m, 2H), 3.77 (s, 6H), 4.23 (s, 2H), 5.08 (d, J = 2.4,
1H), 5.11 (d, J = 2.4, 1H), 5.38 (d, J = 3.6, 1H), 5.41 (d, J = 4.3, 1H), 5.50 (d, J = 3.6, 1H), 5.55 (d, J = 4.3,
1H), 6.94 (d, J = 7.4, 4H), 7.27 (d, J = 7.4, 4H), 7.42–7.48 (m, 10H) ); 13C-NMR (151 MHz, D2O): δ 175.4,
175.1, 174.7, 173.4, 172.9, 168.7, 155.9, 144.5, 137.2, 130.7, 129.9, 129.0, 128.8, 126.4, 119.6, 119.4, 114.5,
79.2, 76.7, 73.8, 73.0, 67.3, 67.1, 66.9, 65.1, 64.4, 61.5, 61.3, 60.7, 58.9, 58.0, 56.3, 55.4, 30.4, 28.6, 28.3, 27.1,
27.0. MS: m/z 531 [M + Na]+. Anal. Calcd. for C26H26N3 O6SNa: C: 58.75, H: 4.93, N: 7.91; found: C:
58.73, H: 4.90, N: 7.92.
Sodium (2S,5R,6R)-6-(2-(1-(4-Methoxyphenyl)-2-(4-nitrophenyl)-4-oxo-azetidin-3-yl)acetamido)-3,3-dimethyl-7-
oxo-4-thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylate (12d). Dark brown solid as inseparable mixture of
˝
diastereomers (68% yield, m.p. 123–125 C). IR (KBr pellet, cm´1): ν 3443, 2964, 2919, 1736, 1657,
1
1612, 1513, 1399, 1384, 1251; H-NMR (400 MHz, D2O): δ 1.44 (s, 3H), 1.49 (s, 3H), 1.56 (s, 3H), 1.58
(s, 3H), 2.85–3.00 (m, 4H), 3.37–3.51 (m, 2H), 3.64 (s, 3H), 3.65 (s, 3H), 4.22 (s, 2H), 4.95–5.01 (m, 2H),
5.35 (d, J = 2.4, 1H), 5.40 (d, J = 2.4, 1H), 5.47 (d, J = 3.6, 1H), 5.54 (d, J = 3.6, 1H), 6.72–7.21 (m, 16H);
13C-NMR (100 MHz, D2O):δ 174.8, 172.8,168.6, 157.1, 156.19, 143.3, 145.1, 130.3, 128.3, 137.3, 124.9,
124.4, 120.1, 19.1, 116.9, 114.9, 114.6, 77.5, 66.9, 66.8, 64.7, 60.9, 58.5, 55.8, 46.4, 37.6, 30.9, 26.7; MS: m/z
576 [M + Na]+. Anal. calcd. for C26H25N4 O8SNa: C: 54.16, H: 4.37, N: 9.72; found: C: 54.13, H: 4.34,
N: 9.76.
3.2. Microbiological Assays and Bacterial Strains
The minimum inhibitory concentrations (MICs) of the different antibiotics was estimated
by means of the broth microdilution method, using the densities (5 ˆ 105 CFU¨ mL´1) and
protocolsrecommendedby the Clinical and Laboratory Standards Institute (CLSI), formerly the
National Committee for Clinical Laboratory Standards (NCCLS) [35]. Slight modifications were the
˝
incubation at 37 C and shaking at 200 rpm of the bacterial cultures. The effects of the different
antibiotics concentrations on the microbial growth was evaluated by turbidity, by measuring optical
density at 600 nm (OD600). MIC100 was defined as the minimum antibiotic concentration that does not
change the sample turbidity compared to time 0, while the MIC50 as the minimal concentration that
reduced OD600 of 50% compared to that ofinoculated sample without antibiotic. Minimum lethal dose
´1
(MLD) was estimated by CFU¨ mL determination. Briefly, for each examined antibiotic, different
dilutions of each bacterial inoculum growth for 24 h in the presence of antibiotic concentrations
ě MIC100 were spread on plate count agar (PCA) and CFU were counted after incubation 24 h
˝
´1
at 37 C. The MLD was the minimum antibiotic concentrationat which the number of CFU¨ mL
resulted equal to 0.
The in vitro antibacterial activity of the new compound was compared to that of the ampicillin
(Sigma-Aldrich), a reference beta-lactam agent in clinical use for the treatment of infections caused
from Gram positive (Streptococcus spp., Staphyloccus spp.) and few Gram negative bacteria (Neisseria,
some Enterobacteriaceae).
Bacterial strain used for in vitro antibiotic activity determination included patogenic
and not patogenic clinical and environmental isolated, both Gram negative Escherichia coli,
Salmonella, Pseudomonas fluorescens, Pseudomonas aeruginosa and Gram positive Staphylococcus aureus,
Staphylococcus epidermidis, Bacillus sp. Escherichia coli (strain JM109) was purchased from Promega [36].
Salmonella enterica subsp. enterica serovar thyphimurium, strain LT2 ATCC 700720 was purchased
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Molecules 2015, 20, 22044–22057
from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture [37]. The
remaining strains were derived from the collection deposited in the microbiology laboratory directed
by G. Vigliotta.
3.3. In vitro NIH-3T3 Cells Line Cytotoxicity Testing
3.3.1. Cell Cultures
NIH-3T3 cells (murine fibroblast) were obtained from ATCC (American Type Culture Collection,
Rockville, MD, USA). Cells were grown in DMEM medium with heat-inactivated 10% FCS,
´1
´1
100 mg¨ mL penicillin, 100 mg¨ mL streptomycin, by incubating in a humidified atmosphere of
˝
95% air/5% CO2 at 37 C.
3.3.2. Cell Viability Assay
The cytotoxicity of the synthesized compounds was detectedby the mitochondrial cytotoxic test,
using thiazolyl blue tetrazolium bromide (MTT) as an indicator of metabolically active cells. The
assay was performed in 96-well microplates. NIH-3T3 cells were seeded at a density of 1.0 ˆ 105 cells
˝
per well and growth 24 h at 37 C before the addition of the compounds. Antibiotics were dissolved
in 50% DMSO (v/v, in water) and subsequently added to the cells to the final concentration of 2 µM
˝
to 50 µM (final concentration of DMSO 0.5%). Microplates were incubated for 72 h at 37 C. After
´1
incubation thiazolyl blue tetrazolium bromide (3.33 mg¨ mL phosphate buffered saline, pH = 7.4)
was added to each well and left to incubate for further 4 h to allow for intracellular reduction of the
soluble yellow MTT to the insoluble purple formazan dye. Then the medium with MTT solution was
removed. Formazan crystals in viable cells were dissolved in the lysis solution (4 M MHCl and 0.1%
Nonidet P40 in ethanol) and absorbance was measured at 540 nm. As a control, the same cells were
exposed to 0.5% DMSO alone and inhibition activity of compounds was expressed as percentages of
control. Each value is the mean of 6 wells with standard deviation.
4. Conclusions
In summary, the present work describes a new and easy protocol for the synthesis of some
novel semi-synthetic penicillin analogues by the conjunction of substituted β-lactam nucleus with
a penicillanic acid framework. The involvement in the synthetic route of a Staudinger reaction
constitutes a convenient approach for the functionalization of the intermediate β-lactam units
and consequently for the introduction of different substituents on the target scaffold. All target
compounds were screened for their antibacterial activities, and from the biological evaluation it
appeared that the introduction of a β-lactam moiety at the amino end of 6-amino penicillanic
acid positively affected the antibacterial ability. In general, all target compounds exhibited potent
antibacterial activity against Gram positive bacteria in most of the cases higher than that of reference
antibiotic ampicillin. Further, some title compounds were also assessed for their cytotoxic activity
against NIH-3T3 cell lines using MTT assay method. The cytotoxic screening displayed that these
compounds did not cause any cytotoxic effect at the concentrations higher than that required to exert
antibiotic activity. The promising antimicrobial activity and low-toxicity profile of targets make them
promising molecules for further studies. The development of new derivatives and the investigations
devoted to the mode of antibacterial action of these new compounds are now underway.
Acknowledgments: We thank the Ministero dell’Istruzione, dell’Università e dellaRicerca (MIUR) for the
financial support and the Centro di Tecnologie Integrate per la Salute (Project PONa3_00138), Università di
Salerno, for the 600 MHz NMR instrumental time. Thanks are due to Ivano Immediata, Patrizia Iannece and
Patrizia Oliva for IR, ESI-MS and NMR measurements, respectively.
Author Contributions: M.D.R., A.S. and C.S. conceived and designed the study. M.D.R., G.V. and G.P. performed
the experiments and interpreted the results. M.D.R., A.S. and G.V. wrote the paper. All authors read, revised and
approved the manuscript.
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Molecules 2015, 20, 22044–22057
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Troisi, L.; Granito, C.; Pindinelli, P. Novel and recent synthesis and applications of β-lactams. In Heterocyclic
Scaffolds I-β-Lactams; Banik, B.K., Ed.; Springer-Verlag: Berlin, Germany, 2010; Volume 22, pp. 101–209.
Fernandes, R.; Amador, P.; Prudêncio, C. β-lactams: Chemical structure, mode of action and mechanisms
of resistance. Rev. Med. Microbiol. 2013, 24, 7–17. [CrossRef]
Galletti, P.; Giacomini, D. Monocyclic β-lactams: New Structures for New Biological Activities. Curr. Med.
Chem. 2011, 18, 4265–4283. [CrossRef] [PubMed]
Mehta, P.D.; Sengar, N.P.S.; Pathak, A.K. 2-Azetidinone—A new profileof various pharmacological
activities. Eur. J. Med. Chem. 2010, 45, 5541–5560. [CrossRef] [PubMed]
Singh, G.S. β-Lactams in the New Millenium. Part-I: Monobactams and Carbapenems. Mini Rev.
Med. Chem. 2004, 4, 69–92. [CrossRef] [PubMed]
Singh, G.S. β-Lactams in the New Millenium. Part-II: Cephems, Oxacephems, Penams and Sulbactam.
Mini Rev. Med. Chem. 2004, 4, 93–109. [CrossRef] [PubMed]
Veinberg, G.; Vorona, M.; Shestakova, I.; Kanepe, I.; Lukevics, E. Design of β-Lactams with Mechanism
Based Nonantibacterial Activities. Curr. Med. Chem. 2003, 10, 1741–1757. [CrossRef] [PubMed]
Page, M.I. Structure-activity relationships: Chemical. In The Chemistry of β-Lactams; Page, M.I., Ed.;
Springer-Science: Dordrecht, The Netherlands, 1992; pp. 79–99.
Holden, K.G. Chemistry and Biology of β-Lactam Antibiotics; Morin, R.B., Gorman, M., Eds.; Academic Press:
London, UK, 1982–1983; Volume 1–3, pp. 101–158.
10. Fisher, J.F.; Meroueh, S.O.; Mobashery, S. Bacterial Resistance to β-Lactams Antibiotics: Compelling
Opportunism, Compelling Opportunity. Chem. Rev. 2005, 105, 395–424. [CrossRef] [PubMed]
11. Llarrull, L.I.; Testero, S.A.; Fisher, J.F.; Mobashery, S. The future of the β-lactams. Curr. Opin. Microbiol.
2010, 13, 551–557. [CrossRef] [PubMed]
12. Kong, K.F.; Schneper, L.; Mathee, K. Beta-lactam antibiotics: From antibiosis to resistance and bacteriology.
APMIS 2010, 118, 1–36. [CrossRef] [PubMed]
13. Wilke, M.S.; Lovering, A.L.; Strynadka, N.C.J. β-Lactam antibiotic resistance: A current structural
perspective. Curr. Opin. Microbiol. 2005, 8, 525–533. [CrossRef] [PubMed]
14. Rice, L.B. Mechanisms of Resistance and Clinical Relevance of Resistance to β-Lactams, Glycopeptides, and
Fluoroquinolones. Mayo Clin. Proc. 2012, 87, 198–208. [CrossRef] [PubMed]
15. Powers, J.C.; Asgian, J.L.; Ekici, O.D.; James, K.E. Irreversible Inhibitors of Serine, Cysteine, and Threonine
Proteases. Chem. Rev. 2002, 102, 4639–4750. [CrossRef] [PubMed]
16. Hubschwerlen, C. β-Lactam Antibiotics. In Comprehensive Medicinal Chemistry, 2nd ed.; Taylor, J.B.,
Triggle, D.J., Eds.; Elsevier: Oxford, UK, 2007; pp. 479–517.
17. Lakshmi, R.; Nusrin, K.S.; Ann, G.S.; Sreelakshmi, K.S. Role of Beta Lactamases in Antibiotic Resistance:
A Review. Int. Res. J. Pharm. 2014, 5, 37–40. [CrossRef]
18. Drawz, S.M.; Bonomo, R.A. Three Decades of β-Lactamase Inhibitors. Clin. Microbiol. Rev. 2010, 23,
160–199. [CrossRef] [PubMed]
19. Lewis, K. Platforms for antibiotic discovery. Nat. Rev. Drug Dis. 2013, 12, 371–386. [CrossRef] [PubMed]
20. De Rosa, M.; Zanfardino, A.; Notomista, E.; Wichelhaus, T.A.; Saturnino, C.; Varcamonti, M.;
Soriente, A. Novel promising linezolid analogues: Rational design, synthesis and biological evaluation.
Eur. J. Med. Chem. 2013, 69, 779–785. [CrossRef] [PubMed]
21. Wang, Y.; Liang, Y.; Du, D.-M.; Xu, J. Do Reaction Conditions Affect the Stereoselectivity in the Staudinger
Reaction? J. Org. Chem. 2006, 71, 6983–6990. [CrossRef] [PubMed]
22. Staudinger, H. Ketenes. 1. Diphenylketene. Justus Liebigs Ann. Chem. 1907, 356, 51–123. [CrossRef]
23. Jiao, L.; Liang, Y.; Xu, J. Origin of the Relative Stereoselectivity of the β-Lactam Formation in the Staudinger
Reaction. J. Am. Chem. Soc. 2006, 128, 6060–6069. [CrossRef] [PubMed]
24. Wang, Y.; Qian, H.; Zhou, J.; Zhang, H.; Ji, J.; Huang, W. Synthesis and Bioactivities of 2-Azetidinone
Derivatives as Cholesterol Absorption Inhibitors. Med. Chem. 2011, 7, 534–542. [CrossRef] [PubMed]
25. Page, M.G.P. β-Lactam Antibiotics. In Antibiotic Discovery and Development; Dougherthy, T.J., Pucci, M.J.,
Eds.; Springer: US, 2012; Volume XVII, pp. 79–117.
22056

Molecules 2015, 20, 22044–22057
26. Arya, N.; Jagdale, A.Y.; Patil, T.A.; Yeramwar, S.S.; Holikatti, S.S.; Dwivedi, J.; Shishoo, C.J.; Jain, K.S.
The chemistry and biological potential of azetidin-2-ones. Eur. J. Med. Chem. 2014, 74, 619–656. [CrossRef]
[PubMed]
27. Burchacka, E.; Walczak, M.; Sienczyk, M.; Dubin, G.; Zdzalik, M.; Potempa, J.; Oleksyszyn, J.
The development of first Staphylococcus aureus SpIB protease inhibitors: Phosphonic analogues of
glutamine. Bioorg. Med. Chem. 2012, 22, 5574–5578.
28. Regourd, J.; Al-Sheikh Ali, A.; Thompson, A. Synthesis and Anti-Cancer Activity of C-Ring-Functionalized
Prodigiosin Analogues. J. Med. Chem. 2007, 50, 1528–1536. [CrossRef] [PubMed]
29. Ibrahim, M.N.; Hamad, K.J.; Al-Joroshi, S.H. Synthesis and characterization of some Schiff bases.
Asian J. Chem. 2006, 18, 2404–2406.
30. Denmark, S.E.; Nakajima, N.; Stiff, C.M.; Nicaise, O.J.-C.; Kranz, M. Studies on the Bisoxazoline and
(–)-Sparteine Mediated Enantioselective Addition of Organolithium Reagents to Imines. Adv. Synth. Catal.
2008, 350, 1023–1045. [CrossRef] [PubMed]
31. Hwu, J.R.; Tseng, W.N.; Patel, H.V.; Wong, F.F.; Horng, D.-N.; Liaw, B.R.; Lin, L.C. Mono-deoxygenation
of Nitroalkanes, Nitrones, and Heterocyclic N-Oxides by Hexamethyldisilane through 1,2-Elimination:
Concept of “Counterattack Reagent”. J. Org. Chem. 1999, 64, 2211–2118. [CrossRef]
32. Zarei, M.; Mohamadzadeh, M. 3-Thiolaterd 2-azetidinones: synthesis and in vitro antibacterial and
antifungal activities. Tetrahedron 2011, 67, 5832–5840. [CrossRef]
33. Favre, A.; Grugier, J.; Brans, A.; Joris, B.; Marchand-Brynaert, J. 6-Aminopenicillanic acid (6-APA)
derivatives equipped with anchoring arms. Tetrahedron 2012, 68, 10818–10826. [CrossRef]
34. Josephine, H.R.; Charlier, P.; Davies, C.; Nicholas, R.A.; Pratt, R.F. Reactivity of Penicillin-Binding Proteins
with Peptidoglycan-Mimetic β-Lactams: What’s Wrong with These Enzymes? Biochemistry 2006, 45,
15873–15883. [CrossRef] [PubMed]
35. National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility
Tests for Bacteria that Grow Aerobically, 5th ed.; Approved Standard. NCCLS Document M7-A5; National
Committee for Clinical Laboratory Standards: Wayne, PA, USA, 2001.
36. Promega. http://www.promega.com/products (accessed on 4 December 2015).
37. DSMZ. Available online: http://www.dsmz.de/ (accessed on 4 December 2015).
Sample Availability: Samples of the compounds are available upon request.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open
access article distributed under the terms and conditions of the Creative Commons by
Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
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