4-Alkyliden-azetidinones modified with plant derived polyphenols: Antibacterial and antioxidant properties

Article abstract of DOI:10.1016/j.ejmech.2017.09.048

Antimicrobial resistance is one of the major and growing concerns in hospital- and community acquired infections, and new antimicrobial agents are therefore urgently required. It was reported that oxidative stress could contribute to the selection of resistant bacterial strains, since reactive oxygen species (ROS) revealed to be an essential driving force. In the present work 4-alkylidene-azetidinones, a new class of antibacterial agents, were functionalized with phytochemical polyphenolic acids such as protocatechuic, piperonyl, caffeic, ferulic, or sinapic acids and investigated as dual target antibacterial-antioxidant compounds. The best candidates showed good activities against multidrug resistant clinical isolates of MRSA (MICs 2–8 μg/mL). Among the new compounds, two revealed the best antioxidant capacity with TEAC-DPPH and TEAC-ABTS being significantly more active than Trolox.

Full text of DOI:10.1016/j.ejmech.2017.09.048

Accepted Manuscript
4-Alkyliden-azetidinones modified with plant derived polyphenols: Antibacterial and
antioxidant properties
Daria Giacomini, Rosario Musumeci, Paola Galletti, Giulia Martelli, Lorenzo
Assennato, Gianni Sacchetti, Alessandra Guerrini, Enrico Calaresu, Marianna
Martinelli, Clementina Cocuzza
PII:
S0223-5234(17)30763-8
10.1016/j.ejmech.2017.09.048
EJMECH 9763
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 July 2017
Revised Date: 21 September 2017
Accepted Date: 21 September 2017
Please cite this article as: D. Giacomini, R. Musumeci, P. Galletti, G. Martelli, L. Assennato, G.
Sacchetti, A. Guerrini, E. Calaresu, M. Martinelli, C. Cocuzza, 4-Alkyliden-azetidinones modified with
plant derived polyphenols: Antibacterial and antioxidant properties, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.09.048.
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ACCEPTED MANUSCRIPT
4-Alkyliden-azetidinones modified with plant derived polyphenols: antibacterial
and antioxidant properties.
Daria Giacomini,a* Rosario Musumeci,b* Paola Galletti,a Giulia Martelli,a Lorenzo Assennato,a
Gianni Sacchetti,c Alessandra Guerrini,c Enrico Calaresu,b Marianna Martinelli,b and Clementina
Cocuzzab
Addresses
a
Department of Chemistry "G. Ciamician", University of Bologna, Via Selmi 2, 40126 Bologna,
Italy.
b
Department of Medicine and Surgery, University of Milano-Bicocca, Via Cadore 48, 20900,
Monza, Italy.
c Department of Life Sciences and Biotechnology – Pharmaceutical biology lab., UR7 Tecnopolo
Terra&Acqua Tech, University of Ferrara, P.le Chiappini 3, 44123 Ferrara, Italy

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Abstract
Antimicrobial resistance is one of the major and growing concerns in hospital- and community
acquired infections, and new antimicrobial agents are therefore urgently required. It was
reported that oxidative stress could contribute to the selection of resistant bacterial strains,
since reactive oxygen species (ROS) revealed to be an essential driving force. In the present
work 4-alkylidene-azetidinones, a new class of antibacterial agents, were functionalized with
phytochemical polyphenolic acids such as protocatechuic, piperonyl, caffeic, ferulic, or sinapic
acids and investigated as dual target antibacterial-antioxidant compounds. The best candidates
showed good activities against multidrug resistant clinical isolates of MRSA (MICs 2-8 µg/mL).
Among the new compounds, two revealed the best antioxidant capacity with TEAC-DPPH and
TEAC-ABTS being significantly more active than Trolox®.
Keywords:
Azetidinones; Gram-positive; Antibacterials; Lactams; Resistant bacteria; Antioxidant capacity;
ROS
Highlights:
•
•
•
•
Design and synthesis of new β-lactams targeting resistant bacteria.
Synthesis of new 4-alkyliden-azetidinones decorated with phenolic esters.
Antibacterial activity was tested on multidrug-resistant bacterial strains.
Antioxidant potency of the new compounds was evaluated.

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Graphical Abstract

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1. Introduction
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in
regulation of cell life [1, 2]. In general, moderate levels of ROS/RNS may function as signals to
promote cell proliferation and survival, whereas severe increase of ROS/RNS can also induce
cell death in vitro [3].
Oxidative stress is generated by an imbalance increase of ROS/RNS which can damage all
classes of cellular macromolecules as proteins, lipids and nucleic acids with strong effects on
human metabolic pathways and causing the onset of several disorders and diseases. In fact,
oxidative stress is generally considered the starting point for the onset of several diseases (i.e.
tissues chronic inflammation, cancers) and undoubtedly one of the major pathophysiological
hallmarks of severe obstructive lung diseases, including chronic obstructive pulmonary disease
and cystic fibrosis (CF) [4, 5]. In CF, a severe genetic disease, the presence of oxidative stress is
due to an increased production of ROS and to an impaired antioxidant status [6, 7] that
particularly arise during chronic pulmonary infections.
In CF patients, a persistent colonization by pathogenic bacteria occurs and the repeated use of
antibacterial agents selects for specific resistant strains such as multidrug resistant
Pseudomonas aeruginosa (incidence 43%) and Staphylococcus aureus (incidence 73.1%) strains
[8]. A rise in S. aureus infections has been reported in CF patients, with an increase in the
prevalence of highly virulent, methicillin-resistant S. aureus strains (MRSA) [9-11]. Persistent
MRSA infections represent an underestimated new threat to the CF community, as they are not
only difficult to treat but also associated with factors complicating patient outcome as well as
biofilm formation, Panton–Valentine leukocidin (PVL) production and decreased lung function,
with an impairment of patient survival [12].
The involvement of oxidative stress in the mechanism of antibiotic-mediated cell death is
unclear and subject to debate [13]. It has been shown that under aerobic conditions
bactericidal antibiotics with specific targets in bacterial cell stimulate the production of harmful
reactive oxygen species (ROS), which contribute to killing by these drugs [14]. On the contrary,
several research groups provided convincing arguments against ROS-mediated killing of
bacteria by antibiotics [15, 16].

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Moreover, it was recently reported that oxidative stress could contribute to the phenomenon
of selection of pro-biofilm variants and H2O2-resistance, since ROS revealed to be an essential
driving force for the selection of variants of Pseudomonas aeruginosa strains [17]. This evidence
was also recently confirmed by clinical evaluations in patients affected by diabetes and/or
related cardiovascular diseases, becoming more susceptible to bacterial infections (nosocomial
infections and infections related to specific diseases) and less reactive to pharmacological
treatments [18].
The described phenomena point to the need of new therapeutic strategies in targeting anti-
oxidant pathways together with new antibacterial agents able to fight chronic infections caused
by multidrug resistant bacterial strains. In this article, we report our recent efforts on this topic,
as part of an ongoing project on the synthesis of dual-target monocyclic β-lactams, specifically
designed to target resistant bacterial strains [19, 20, 21], and to have antioxidant activities [22,
23, 24].
We focused our attention on the synthesis of 4-alkyliden-azetidinones armed with polyphenolic
residues as possible antibacterial-antioxidant dual-active compounds. In these derivatives a
double C=C bond on the C-4 position of the β-lactam ring acts as a strong electron withdrawing
group [25], thus conferring an increased aptitude toward ring-opening reactions by suitable
enzymes. Indeed some 4-alkyliden-azetidinones were strong inhibitors of serine-dependent
enzymes such as human leucocyte elastase [26], for the treatment of pulmonary emphysema,
or matrix-metallo proteases (MMPs) involved in cancer invasion and metastasis [27, 28].
Concerning the antibacterial activity, some structural features resulted particularly important
for valuable potency: the alkylidene function on the C4 position with a (Z) stereochemistry
showed to favor the biological activity if compared to (E) stereoisomers; the presence of a
benzyl ester strongly improved the potency in respect to the ethyl ester or the carboxylic acid
[19]. Furthermore, it was observed that coupling a phenolic ester with N-methylthio-4-
alkylidene-β-lactams resulted in inactivation [23], hence showing the need to include an
unsubstituted-NH atom in these compounds.
Our aim was then to broad the scope of our previous studies by functionalizing the
antibacterial-core structure of 4-alkylidenazetidin-2-ones with different polyphenolic moieties
from some plant derived benzoic acids such as protocatechuic- or piperonyl acids at the C3

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position as to efficiently combine in one structure both antioxidant and antibacterial activities.
In a previous work it was found out that polyphenol-substituted β-lactams presented a
decreased antioxidant activity if compared with their corresponding polyhydroxybenzoates,
pointing out a steric hindrance phenomenon [24]. Hence, we considered that an extension of
the side chain linking the phenolic aromatic ring to the rest of the structure might provide a
greater exposure of the phenolic OH residues to affect the antioxidant activity without
disturbing the antibacterial activity. Relying on the commonly occurring structures in natural
phytochemical polyphenols, we identified also the C6-C3 leitmotiv of hydroxy-cinnamic acids
such as coumaric, caffeic, ferulic, or sinapic acids as promising groups (Chart 1) [29, 30, 31].
Hydroxycinnamic acids are a group of polyphenols highly abundant in food that may account
for about one-third of the phenolic compounds in our diet. Moreover, they have gained an
increasing interest in health because they are known to be potent antioxidants [32]. These
compounds have been described as chain-breaking antioxidants acting through radical
scavenging activity that is related to their hydrogen or electron donating capacity and to the
ability to delocalize/stabilize the resulting phenoxyl radical within their structure.
Moreover, in the study of the phenol-protection strategy, we considered the methylenedioxy
moiety, present in many natural product, as an interesting substructure to be held in the target
compounds for evaluating its antibacterial activity. As a result, a number of novel compounds
with good antibacterial activity against staphylococci, particularly multidrug resistant S. aureus
(MRSA), and with antioxidant potency have been discovered and qualified for further in vivo
evaluations.
Chart 1. Novel β-lactam derivatives synthesized and evaluated in this study

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2. Results and Discussion
2.1 Synthesis of azetidinones
All target compounds were prepared with a convergent synthetic strategy that comprised the
preparation of the 3-hydroxyethyl-4-alkylidene β-lactam A, obtained in turn from the
commercially available (3R,4R)-4-acetoxy-3-[(1R)-1-(tert-butyldimethylsilyloxy) ethyl]azetidin-2-
one and benzyl diazoacetate [25], and its esterification with polyphenolic acids duly protected
on the phenolic OH groups (Scheme 1).
Scheme 1. Convergent synthesis of new 4-alkylidene β-lactams with polyphenolic residues.
The O-protected aryl acids were prepared in a two-step procedure starting from their
corresponding methyl esters exploiting
a
phenolic oxygen protection with
chloromethylethylether (EOM) followed by alkaline hydrolysis (Scheme 2), according to what
previously optimized for the O-protected caffeic acid [22].
Scheme 2. Synthesis of O-protected phenolic acids; a) NaH, EOMCl, THF, 0°C to rt; b) NaOH 5 M,
THF/MeOH 1:1, 40°C. Yields of isolated products after flash chromatography reported in brackets.
Letters in compound numbering referred to the substituents as in Chart 1 for target products.

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The EOM ether turned out to be an effective protective group that can be easily inserted, and
selectively removed at the late stage of the synthesis with a mild procedure without damaging
the β-lactam ring in final compounds. The dioxymethylene-group was inserted on the suitable
methyl esters (i.e. 2,3-dihydroxy-methyl-benzoate, 3,4-dihydroxy-methyl-benzoate), with
diiodomethane and potassium carbonate as reported by Alam et al. (Scheme 3) [33].
Scheme 3. Synthesis of methylenedioxy-protected acids; a) CH2I2, K2CO3, DMF, 110 °C; b) NaOH
5 M, THF/MeOH 1:1, 40°C. Yields of isolated products after flash chromatography in brackets.
Letters in compound numbering referred to the substituents as in Chart 1 for target products.
The 3-hydroxyethyl side chain of β-lactam A was then derivatized as phenolic or polyphenolic
aromatic ester with the appropriate O-protected acid 3a-j by treatment with N,N’-
dicyclohexylcarbodiimide (DCC) and a catalytic amount of 4-dimethylaminopyridine (DMAP) in
dichloromethane (Scheme 4) according to the Steglich procedure [34]. Yields were slightly
affected by this step probably due to a low reactivity of the secondary alcohol of the
intermediate A due to a steric hindrance that could obstruct the nucleophilic attack to the acid-
DCC adduct. Esters 4c, 4d, and 4f-j were finally treated with trifluoroacetic acid (TFA) to
eliminate the EOM protection [22], affording the target β-lactams 1c, 1d, and 1f-j in
quantitative yields.

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Scheme 4. Synthesis of target compounds a) DCC, DMAP, DCM, 0 °C to rt; b) CF3COOH, DCM, 0 °C to
rt. Yields of isolated compounds are reported in brackets.
2.2 Antibacterial activity
Antibacterial activity screening of compounds 1a-j was carried out against recent, well
characterized clinical isolates. A selection of Gram-positive and Gram-negative bacterial
pathogens used for the in vitro antimicrobial susceptibility testing included: 11 Gram-positive (8
staphylococci and 3 enterococci) and 5 Gram-negative strains.
Most of the bacterial strains were specifically selected to exhibit a multidrug-resistant
phenotype against penicillins/cephalosporins, linezolid, or vancomycin. Antimicrobial activities
of the compounds are listed in Table 2, with potency being showed as minimum inhibitory
concentration values (MICs) expressed in µg/mL, and only MIC values equal to or less than 128
µg/mL against corresponding bacterial species were reported. Commercially available
vancomycin and cefuroxime were used as reference compounds in antibacterial susceptibility
testing.
The in vitro antimicrobial susceptibility analysis indicated antibacterial activity only against
Gram-positive bacteria, while none of the tested compounds exhibited an appreciable
antibacterial effect against studied Gram-negative bacteria (results not included in Table 2 as
for them MIC values of tested compounds were > 128 µg/mL). Recently, lauryl ester of ferulic
acid showed antibacterial activity rather toward E. coli than S. aureus in agar diffusion assays,
but here the presence of ferulic moiety (compound 1i) did not contribute to antibacterial
activity against Gram-negative bacteria [35].

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Table 1 MIC values (µg/mL) for compounds 1a-j, vancomycin (VA) and cefuroxime (FUR) as reference compounds.
Organism ID
Phenotype
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
VA
FUR
2
2
4
8
4
4
4
8
16
2
2
2
16
16
8
>128
>128
>128
>128
>128
8
4
4
8
4
4
8
8
2
1
>128
>128
4
4
4
8
8
4
4
8
>128 0.5
2
>128
8
S. aureus ATCC 29213 CLSI control
S. aureus SAU-1
8
8
1
4
LZD-R MRSA
S. aureus 44674
>128 >128
>128 >128
>128 >128
>128 >128
>128 >128
>128 >128
MRSA
S. aureus 69856
4
>128 0.5
1
MSSA
S. aureus 39249
4
8
2
1
2
1
2
2
MSSA
8
16
>128 128
2
>128
4
8
S. hominis α26
LZD-R MRSH
>128 >128
>128 >128
16
16
>128
>128
>128 >128
>128 >128
>128
>128
32
S. epidermidis G1027 LZD-R MRSE
S. epidermidis 3226
0.5
>128a
MSSE
>128 >128 >128 128 >128 >128 >128 >128 >128 >128
E. faecalis ATCC 29212 CLSI control
>128 0.25 >128a
E. faecalis 4150
>128 >128 >128 128 >128
64
>128 >128
8
all-S
>128 >128 >128 128 >128 >128 >128 >128 >128 >128 >128 >128a
E. faecium VRE 2
LZD-R VRE
a Enterococci are inherently resistant to cephalosporins such as cefuroxime.
However, the lack of activity against Gram-negative of the tested compounds confirmed a
general trend of this class of monocyclic β-lactam derivatives as previously observed [19, 20,
23]. This inefficiency could be due to a reduced uptake through the outer membrane of Gram-
negative bacteria, and unfortunately, the presence of one cathecol moiety, as in compounds
reported here, was not sufficient to allow bacterial cell penetration via the siderophore
receptors of Gram-negative bacteria [36, 37].
On the contrary, compounds 1a, 1f, and 1i produced significant antibacterial activity against the
tested Gram-positive bacteria grouped in staphylococci with MIC values between 2 and 8
µg/mL (Table 1). Other compounds, such as 1b, 1c, 1g, and 1j showed a selective activity
towards some S. aureus and the S. hominis resistant strains (MICs 1-16 µg/mL). Compound 1g
showed the most prominent activity against strains of S. aureus ATCC 29213, S. aureus SAU-1,
and S. hominis α26, the latter two strains characterized by an extensive condition of multidrug
resistance (MDR) and high resistance to linezolid (MIC values 2, 1, and 2 µg/mL, respectively). It

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is noteworthy that against S. aureus SAU-1 1g was as active as vancomycin, whereas
cefuroxime, a second-generation cephalosporin, were completely inactive.
Among the three methylenedioxy-derivatives, 1a was the most active, 1e completely inactive,
whereas 1b with an oxygen atom at an ortho position, resulted in a selective activity against S.
aureus ATCC 29213 and S. aureus SAU-1. The association of ortho substitution vs selectivity was
observed also for azetidinones 1f vs 1g.
The cathecol- and caffeoyl-derivatives 1d and 1h possessing two vicinal OH groups, are less or
no active, whereas the monomethylether 1i recovers an appreciable activity, as well as the
coumaroyl derivative 1f (against staphylococci MICs 4-8 µg/mL, respectively). This result is
consistent with some data recently reported for some xanthone derivatives where a loss of
antibacterial activity of OH phenolics compared to the corresponding isoprenyl ethers was
reported [38]. Relying on the cinnamyl series, the most and the less lipophilic compounds 1e
and 1h were both inactive, whereas compounds with an intermediate lipophilicity 1f, 1g, 1i,
and 1j showed good activities.
Except for compounds 1f and 1i, active also against E. faecalis 4150 strain (MIC values = 64 and
8 µg/mL, respectively), all other compounds exhibited an exclusively anti-staphylococcal
activity.
2.3 Antioxidant activity: TEAC-DPPH and TEAC-ABTS evaluations.
Antioxidant activity determination based on different approaches was carried out, considering the
importance of a multidimensional evaluation of the antioxidant activity [39]. With the objective to
ensure a better comparison of the results and covering a wider range of possible applications of
novel molecules, the use of more than a single method – employing radicals such as DPPH (2,2-
diphenyl-1-picrylhydrazyl) and ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) - is
highly advisable [40]. Therefore, the β-lactams were then subjected to TEAC (Trolox® equivalent
antioxidant capacity) assays, based on the ability of the compounds to scavenge the DPPH (2,2-
diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid))
radical cations, compared to the same scavenging ability of Trolox®, a water-soluble derivative of
vitamin E with potent antioxidant properties [41].

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Compounds 1b and 1e were not tested because the absence of OH groups precludes from
antioxidant activity, as obtained for 1a (Table 2). The presence of one OH as in compounds 1c,
1f, and 1g was not enough to give radical-scavenging ability. The best performances were
instead recorded with compounds with a cathecol group 1d and 1h in both the assays (Table 2).
In particular, 1d and 1h showed antioxidant capacity with DPPH assay 2.6 and 1.7 times
respectively more effective than that expressed by 1 mmol of Trolox® as reference compound.
Analogously, 1d and 1h evidenced the same outcomes with ABTS test, even if with lower values
than those of TEAC-DPPH. The quantitative difference is due to the different type of radical
employed, but it should be however pointed out that for both the assays the 1,2 dihydroxy
groups provided in those azetidinones excellent values of antioxidant capacity. Moreover, the
1d and 1h data are comparable with those expressed by natural compounds known for their
important applications thanks to their valuable antioxidant properties, i.e. ascorbic acid and α-
tocopherol [42].
Table 2 Antioxidant activity of eight β-lactam derivatives based on DPPH and
ABTS assays. The results are expressed as mmol of Trolox Equivalent
Antioxidant Capacity (TEAC) ± Standard Deviation (SD).
Comp.
TEAC-DPPH
TEAC-ABTS
(mmol eq. Trolox ± SD) (mmol eq. Trolox ± SD)
1a
1c
1d
1f
0.012 ± 0.001
0.007 ± 0.001
2.590 ± 0.220
0.003 ± 0.001
0.002 ± 0.001
1.670 ± 0.178
0.142 ± 0.015
0.191 ± 0.020
nd
0.010 ± 0.001
1.300 ± 0.138
0.053 ± 0.006
0.143 ± 0.015
1.030 ± 0.110
0.284 ± 0.030
0.232 ± 0.025
1g
1h
1i
1j
nd = not detectable
Values obtained for the other compounds - ranging from 0.284 ± 0.030 to 0.002 ± 0.001 are very
low and not significant for antioxidant applications, even though ferulic and sinapic esters
performed a little bit better than the others, in line with results regarding some polyphenolic-β-
lactams, as previously reported [22, 24].

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It would be interesting now to compare the most active compounds reported here, with some
monocyclic β-lactams with polyphenolic esters on the 3-hydroxylethyl side-chain previously
studied with reference to their antibacterial and antioxidant activities (Chart 2). Compounds A is a
4-alkylideneazetidinone syringoyl ester which showed a good antibacterial activity against MRSA
(MICs range 1-64 µg/mL) strains [19], but the syringic moiety get scarce antioxidant potency as
observed for compound D (TEAC 0.037 mmol equiv. TROLOX) [22]. Compounds B and C are good
antioxidants (TEAC 1.23 and 0.98 mmol equiv. TROLOX, respectively) but have a lower
antibacterial activity against MRSA (MICs 32-128 and 16-128 µg/mL, respectively) [22]. It is
important to highlight that in this class of monocyclic β-lactam derivatives the antibacterial
potency is mainly activated by the presence of a good electron withdrawing group on the C4
position of the β-lactam ring such as 4-alkylidene- or 4-acetoxy groups. Whenever such
substituents were absent, as in compounds E and F, the antibacterial potency was completely lost
[43], notwithstanding the presence of cinnamoyl esters which in some other derivatives showed a
certain potency [30]. Finally compounds 1a, 1d, and 1i of the present work are good anti-MRSA
agents with a narrower MIC range than compound A, and 1d showed also a very good antioxidant
capacity (TEAC 2.59 mmol equiv. TROLOX®).
Chart 2. Best antibacterial and antioxidant activities of the compounds reported in this work in
comparison with data obtained for monocyclic β-lactam derivatives previously studied [19, 22]. In
bracket MIC ranges in µg/mL toward MRSA strains, and TEAC-DDPH expressed as mmol eq. Trolox.

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3. Conclusions
The synthesis of new 4-alkylidene-β-lactams functionalized with some plant phenolic
compounds was accomplished through a convergent synthetic strategy. The new derivatives
were tested in vitro for antibacterial and antioxidant activities. Compounds with a good
antibacterial potency against multidrug resistant staphylococcal strains were found. Moreover,
compounds with a catechol moiety on the side-chain showed anti-radical capacity against DPPH
and ABTS, with TEAC values 2.5 times higher than those known for compounds currently used
as antioxidants (for e.g. Vitamin E, citric acid). The present study reinforces the idea that
phytochemicals moieties can be used for the development of new 4-alkylidene-β-lactams that
combine in one molecule the antibacterial and the antioxidant activities thus realizing dual-
active compounds with a synergistic action for the potential treatment of those infections
where the oxidative stress could increase specific resistant variants [44].
4. Experimental Section
4.1. General
Commercial reagents (reagent grade, >99%) were used as received without additional
purification. Anhydrous solvents (CH2Cl2, THF, MeOH, DMF) were obtained commercially. All
1
13
reactions were performed under an inert atmosphere (N2) unless unspecified. H and C NMR
spectra were recorded with an INOVA 400 instrument with a 5 mm probe. All chemical shifts
are quoted relative to deuterated solvent signals (δ in ppm and J in Hz). Polarimetric Analyses
were conducted on Unipol L 1000 “Schmidt-Haensch” Polarimeter at 598 nm. FTIR spectra:
Brucker instrument, measured as films between NaCl plates or CH2Cl2 solutions for solid
compounds; wave numbers are reported in cm-1. The purities of the target compounds were
assessed as being >95% using HPLC-MS. Elemental analysis were performed on a Thermo Flash
2000 CHNS/O Analyzer. HPLC-MS: Agilent Technologies HP1100 instrument, equipped with a
ZOBRAX-Eclipse XDB-C8 Agilent Technologies column; mobile phase: H2O/CH3CN, 0.4 mL/min,
gradient from 30 to 80% of CH3CN in 8 min, 80% of CH3CN until 25 min, coupled with an Agilent
Technologies MSD1100 single-quadrupole mass spectrometer, full scan mode from m/z = 50 to
2600, in positive ion mode. Elemental analysis were performed on a Thermo Flash 2000
CHNS/O analyzer.

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4.2 Synthesis
Starting polyhydroxy benzoic and polyhydroxy cinnamic methyl esters are all known and were prepared
according to reported procedures with methanol and sulfuric acid as catalyst [45]. EOM-protected esters
2a, 2b, 2e are known and were prepared by following a published procedure [33]; acids 3a, 3b, and 3e
are known; alcohol A, ester 2h and acid 3h were prepared as previously reported [22].
General procedure for EOM-protection (GP1). A solution of the desired methyl ester (1 equiv) in THF (1.5
mL/mmol) was added dropwise to a suspension of NaH (60% in mineral oil) in THF (5 mL/mmol) at 0°C
under inert atmosphere. After 10 min a solution of chloromethylethylether (EOMCl) in THF (2.2
mL/mmol) was added dropwise and the reaction mixture was allowed to warm to room temperature
and monitored by TLC. After 2 h the reaction was quenched with a saturated NaHCO3 solution until pH
10 at 0°C and extracted with EtOAc (3 x 15 mL). The organic extracts were dried over Na2SO4 and
concentrated to obtain the desired EOM-protected compound (2c, 2d, 2f, 2g, 2i or 2j). The crude was
purified by column chromatography only when specified.
General procedure for hydrolysis of methyl esters (GP2). A stirred solution of the desired methyl ester
(2c, 2d, 2f, 2g, 2i or 2j) in a mixture of THF/MeOH 4:1 (4 mL/mmol) was treated with 5 M NaOH (1
mL/mmol). The reaction mixture was heated at 40°C until consumption of the starting material. At
completion, EtOAc (10 mL) was added and the organic phase was separated and discarded. The aqueous
phase was then cooled to 0 °C, adjusted to pH 5 with HCl 1M and then extracted with EtOAc (3 x 15 mL).
The collected organic layers were dried on Na2SO4 and concentrated to afford the corresponding
carboxylic acid (3c, 3d, 3f, 3g, 3i or 3j).
General procedure for alcohol-acid coupling (GP3). To a solution of alcohol A (1 equiv), in CH2Cl2 (10
mL/mmol) under inert atmosphere, the desired acid (3a-j) (1.58 equiv) and DMAP (0.2 equiv) were
added. The mixture was then cooled to 0°C and DCC (1.58 equiv) was added; the system was allowed to
reach room temperature in 15 min and left under stirring overnight. After 16h (TLC monitoring) the
reaction mixture was quenched with water at 0°C and extracted with CH2Cl2 (3 x 15 mL). The collected
organic phases were dried on Na2SO4 and evaporated. The residue was treated with EtOAc (2 x 20 mL)
and filtered to separate dicyclohexylurea precipitate. The solution was then evaporated and purified by
flash-chromatography to afford the desired β-lactams functionalized with O-protected polyphenolic
residues (1a, 1b, 1e, 4c, 4d, or 4f-j).
General procedure for EOM-deprotection (GP4). A stirred solution of the EOM-protected β-lactam (4c,
4d, 4f, 4g, 4i, 4h, or 4j) in CH2Cl2 under inert atmosphere was treated with aliquots of trifluoroacetic acid

ACCEPTED MANUSCRIPT
at 0°C every 60 min. After reaction completion (TLC monitoring), the solvent and the trifluoroacetic acid
were evaporated to obtain the phenolic compounds (1c, 1d, 1f, 1g, 1i, 1h or 1j).
4.2.1. Methyl 4-(ethoxymethoxy)benzoate (2c)
Following GP1, p-hydroxybenzoic acid methyl ester (395 mg, 2.60 mmol), NaH (208 mg, 5.20 mmol) and
1
EOMCl (482 µL, 5.20 mmol) yielded compound 2c (502 mg, 91% yield) as a colorless oil. H NMR (400
MHz, CDCl3) δ (ppm) 1.21 (t, J = 7.1 Hz, 3H), 3.72 (q, J = 7.1 Hz, 2H), 3.87 (s, 3H), 5.26 (s, 2H), 7.05 (d, J =
8.9 Hz, 2H), 7.97 (d, J = 8.9 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ (ppm) 15.0, 51.8, 64.5, 92.8, 115.6,
123.4, 131.4, 161.1, 166.7; HPLC-MS (ESI+): Rt = 8.83 min; m/z = 211 [M+H]+; IR (film, cm-1): 2978, 1720,
1607, 1510, 1435, 1279.
4.2.2. Methyl 3,4-bis(ethoxymethoxy)benzoate (2d)
Following GP1, 3,4-dihydroxy-benzoic acid methyl ester (193 mg, 1.15 mmol), NaH (184 mg, 4.60 mmol)
and EOMCl (427 µL, 4.60 mmol) yielded compound 2d (163 mg, 50% yield) as a colorless oil after flash
chromatography purification (cyclohexane/EtOAc 3:1). 1H NMR (400 MHz, CDCl3) δ (ppm) 1.18 – 1.25 (m,
6H), 3.72 – 3.81 (m, 4H), 3.88 (s, 3H), 5.32 (d, J = 9.6 Hz, 4H), 7.20 (d, J = 8.6 Hz, 1H), 7.66 – 7.71 (m, 1H),
7.80 – 7.83 (m, 1H); 13C NMR (100 MHz, CDCl3) δ (ppm) 15.0, 52.0, 64.5, 64.6, 93.6, 94.1, 115.1, 117.7,
123.9, 124.7, 146.6, 151.5, 166.6; HPLC-MS (ESI+): Rt = 9.51 min; m/z = 285 [M+H]+; IR (film, cm-1): 2978,
1719, 1602, 1510, 1440, 1290.
4.2.3. (E)-methyl 3-(4-(ethoxymethoxy)phenyl)acrylate (2f)
Following GP1, methyl 3-(4-hydroxyphenyl)acrylate (523 mg, 2.93 mmol), NaH (235 mg, 5.88 mmol) and
1
EOMCl (546 µL, 5.88 mmol) yielded compound 2f (701 mg, 98% yield) as a colorless oil. H NMR (400
MHz, CDCl3) δ (ppm) 1.20 (t, J = 7.0 Hz, 3H), 3.70 (q, J = 7.0 Hz, 2H), 3.77 (s, 3H), 5.22 (s, 2H), 6.31 (d, J =
16.0 Hz, 1H), 7.02 (d, J = 8.7 Hz, 2H), 7.44 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 16.0 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ (ppm) 15.0, 51.4, 64.4, 92.8, 115.6, 116.4, 127.9, 129.5, 144.3, 159.1, 167.5; HPLC-MS (ESI+): Rt
= 10.79 min; m/z = 237 [M+H]+; IR (film, cm-1): 2950, 1714, 1634, 1604, 1511, 1434, 1230.
4.2.4. (E)-methyl 3-(2-(ethoxymethoxy)phenyl)acrylate (2g)
Following GP1, methyl (2E)-3-(2-hydroxyphenyl)-2-propenoate (539 mg, 3.03 mmol), NaH (242 mg, 6.06
mmol) and EOMCl (562 µL, 6.06 mmol) yielded compound 2g (584 mg, 82% yield) as a colorless oil after
1
flash chromatography (cyclohexane/EtOAc 7:3). H NMR (400 MHz, CDCl3) δ (ppm) 1.22 (t, J = 7.1 Hz,
3H), 3.75 (q, J = 7.1 Hz, 2H), 3.81 (s, 3H), 5.30 (s, 2H), 6.50 (d, J = 16.2 Hz, 1H), 6.99 – 7.03 (m, 1H), 7.19
(dd, J = 8.4, 0.8 Hz, 1H), 7.31 – 7.35 (m, 1H), 7.53 (dd, J = 7.7, 1.6 Hz, 1H), 8.03 (d, J = 16.2 Hz, 1H); 13C
NMR (100 MHz, CDCl3) δ (ppm) 15.1, 51.6, 64.6, 93.2, 114.8, 118.3, 121.7, 124.0, 128.4, 131.5, 140.1,
156.2, 167.8; HPLC-MS (ESI+): Rt = 9.41 min; m/z = 237 [M+H]+; IR (film, cm-1): 2961, 1716, 1636, 1601,
1511, 1448, 1251.

ACCEPTED MANUSCRIPT
4.2.5. (E)-methyl 3-(4-(ethoxymethoxy)-3-methoxyphenyl)acrylate (2i)
Following GP1, methyl 3-(4-hydroxy-3-methoxyphenyl)acrylate (139 mg, 0.67 mmol), NaH (67 mg, 1.68
1
mmol) and EOMCl (124 µL, 1.34 mmol) yielded compound 2i (159 mg, 89% yield) as a colorless oil. H
NMR (400 MHz, CDCl3) δ (ppm) 1.17 (t, J = 7.1 Hz, 3H), 3.70 (q, J = 7.1 Hz, 2H), 3.74 (s, 3H), 3.85 (s, 3H),
5.25 (s, 2H), 6.28 (d, J = 15.9 Hz, 1H), 7.01 – 7.13 (m, 3H), 7.58 (d, J = 15.9 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ (ppm) 14.9, 51.4, 55.7, 64.3, 93.7, 110.2, 115.7, 115.8, 122.0, 128.4, 144.5, 148.6, 149.6, 167.3;
HPLC-MS (ESI+): Rt = 8.67 min; m/z = 267 [M+H]+; IR (film, cm-1): 2923, 1702, 1637, 1582, 1510, 1461,
1256.
4.2.6. (E)-methyl 3-(4-(ethoxymethoxy)-3,5-dimethoxyphenyl)acrylate (2j)
Following GP1, methyl 3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate (470 mg, 1.97 mmol), NaH (197 mg,
4.93 mmol) and EOMCl (366 µL, 3.94 mmol) yielded compound 2j (582 mg, 99% yield) as a colorless oil.
1H NMR (400 MHz, CDCl3) δ (ppm) 1.13 (t, J = 7.1 Hz, 3H), 3.72 (s, 3H), 3.79 (s, 6H), 3.81 (q, J = 7.1 Hz,
2H), 5.11 (s, 2H), 6.28 (d, J = 15.9 Hz, 1H), 6.68 (s, 2H), 7.53 (d, J = 15.9 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ (ppm) 14.8, 51.4, 55.8, 64.7, 96.4, 105.0, 116.9, 130.1, 136.5, 144.7, 153.4, 167.1; HPLC-MS
(ESI+): Rt = 8.72 min; m/z (%) = 297 [M+H]+; IR (film, cm-1): 2926, 1714, 1636, 1587, 1505, 1454, 1277.
4.2.7. 4-(ethoxymethoxy)benzoic acid (3c)
1
Following GP2, ester 2c (210 mg, 1.00 mmol) yielded acid 3c (121 mg, 62% yield) as a white solid. H
NMR (400 MHz, CD3OD) δ (ppm) 1.19 (t, J = 7.1 Hz, 3H), 3.72 (q, J = 7.1 Hz, 2H), 5.30 (s, 2H), 7.08 (d, J =
8.8 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H); 13C NMR (100 MHz, CD3OD) δ (ppm) 15.5, 65.5, 93.9, 116.7, 125.0,
132.7, 162.7, 169.7; HPLC-MS (ESI-): Rt = 5.85 min; m/z = 195 [M-H]+; IR (film, cm-1): 3418, 2983, 1688,
1603, 1427, 1291; m.p. = 118- 119 °C.
4.2.8. 3,4-bis(ethoxymethoxy)benzoic acid (3d)
3d. Following GP2, ester 2d (163 mg, 0.57 mmol) yielded acid 3d (138 mg, 90% yield) as a waxy white
1
solid. H NMR (400 MHz, CDCl3) δ (ppm) 1.18 – 1.24 (m, 6H), 3.72 – 3.80 (m, 4H), 5.31 (s, 2H), 5.34 (s,
2H), 7.22 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 8.6 Hz, 1H), 7.88 (s, 1H); 13C NMR (100 MHz, CDCl3) δ (ppm) 15.0,
64.6, 64.7, 93.6, 94.1, 115.0, 118.2, 123.0, 125.5, 146.6, 152.2, 171.6; HPLC-MS (ESI+): Rt = 10.02 min;
m/z = 288 [M+H2O]+; IR (film, cm-1): 3404, 2976, 1687, 1599, 1512, 1443, 1296.
4.2.9. (E)-3-(4-(ethoxymethoxy)phenyl)acrylic acid (3f)
1
Following GP2, ester 2f (236 mg, 1 mmol) yielded acid 3f (212 mg, 95% yield) as a white solid. H NMR
(400 MHz, CDCl3) δ (ppm) 1.23 (t, J = 7.1 Hz, 3H), 3.73 (q, J = 7.1 Hz, 2H), 5.26 (s, 2H), 6.33 (d, J = 15.9 Hz,

ACCEPTED MANUSCRIPT
1H), 7.06 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 8.6 Hz, 2H), 7.74 (d, J = 15.9 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ
(ppm) 15.1, 64.5, 92.9, 115.1, 116.5, 127.7, 130.0, 146.6, 159.5, 172.5; HPLC-MS (ESI+): Rt = 6.76 min;
m/z = 223 [M+H]+; IR (film, cm-1): 3424, 2973, 1670, 1627, 1603, 1511, 1429, 1264; m.p. = 152 - 154°C.
4.2.10. (E)-3-(2-(ethoxymethoxy)phenyl)acrylic acid (3g)
1
Following GP2, ester 2g (236 mg, 1 mmol) yielded acid 3g (164 mg, 74% yield) as a waxy white solid. H
NMR (400 MHz, CD3OD) δ (ppm) 1.16 (t, J = 7.1 Hz, 3H), 3.70 (q, J = 7.1 Hz, 2H), 5.27 (s, 2H), 6.49 (d, J =
16.2 Hz, 1H), 6.98 (t, J = 7.5 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.55 (d, J = 7.7 Hz,
1H), 8.01 (d, J = 16.2 Hz, 1H); 13C NMR (100 MHz, CD3OD) δ (ppm) 15.4, 65.6, 94.4, 116.0, 119.5, 122.9,
125.1, 129.3, 132.6, 141.4, 157.3, 170.7; HPLC-MS (ESI-): Rt = 6.96 min; m/z = 221 [M-H]+; IR (film, cm-1):
3419, 2974, 1677, 1634, 1601, 1510, 1435, 1271.
4.2.11. (E)-3-(4-(ethoxymethoxy)-3-methoxyphenyl)acrylic acid (3i)
1
Following GP2, ester 2i (267 mg, 1 mmol) yielded acid 3i (217 mg, 86% yield) as a white solid. H NMR
(400 MHz, CDCl3) δ (ppm) 1.22 (t, J = 7.1 Hz, 3H), 3.77 (q, J = 7.1 Hz, 2H), 3.91 (s, 3H), 5.32 (s, 2H), 6.33
(d, J = 15.9 Hz, 1H), 7.08 – 7.21 (m, 3H), 7.73 (d, J = 15.9 Hz, 1H); 13C NMR (100MHz, (CD3)2CO) δ (ppm)
15.4, 56.2, 64.9, 94.6, 111.9, 117.3, 117.4, 123.0, 129.8, 145.5, 149.7, 151.4, 167.9; HPLC-MS (ESI+): Rt =
5.90 min; m/z = 253 [M+H]+; IR (film, cm-1): 3423, 2904, 1671, 1625, 1583, 1509, 1424, 1254; m.p. = 131 -
133°C.
4.2.12. (E)-3-(4-(ethoxymethoxy)-3,5-dimethoxyphenyl)acrylic acid (3j)
Following GP2, ester 2j (237 mg, 0.8 mmol) yielded acid 3j (187 mg, 83% yield) as a white solid. 1H NMR
(400 MHz, (CD3)2CO) δ (ppm) 1.14 (t, J = 7.1 Hz, 3H), 3.85 (q, J = 7.1 Hz, 2H), 3.90 (s, 6H), 5.10 (s, 2H),
6.50 (d, J = 15.9 Hz, 1H), 7.03 (s, 2H), 7.61 (d, J = 15.9 Hz, 1H); 13C NMR (100 MHz, (CD3)2CO) δ (ppm)
15.4, 56.2, 64.9, 94.6, 111.9, 117.2, 117.3, 123.0, 129.7, 145.5, 149.7, 151.4, 168.2; HPLC-MS (ESI+): Rt =
6.44 min; m/z = 283 [M+H]+; IR (film, cm-1): 3423, 2921, 1675, 1627, 1449, 1250; m.p. = 110 - 112°C.
4.2.13.
(R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethylbenzo[d][1,3]dioxole-5-
carboxylate (1a)
Following GP3, alcohol A (55 mg, 0.21 mmol) and acid 3a (55 mg, 0.33 mmol) yielded compound 1a (28
1
mg, 33% yield) as a waxy solid after flash chromatography purification (CH2Cl2/acetone 99:1). H NMR
(400 MHz, CDCl3) δ (ppm) 1.50 (d, J = 6.4 Hz, 3H), 4.00 (d, J = 6.8 Hz, 1H), 5.14 (d, JAB = 12.3 Hz, 1H), 5.19
(d, JAB = 12.3 Hz, 1H), 5.25 (s, 1H), 5.45 (quintet, J = 6.4 Hz, 1H), 6.04 (s, 2H), 6.83 (d, J = 8.2 Hz, 1H), 7.30
– 7.39 (m, 5H), 7.42 (d, J = 1.5 Hz, 1H), 7.62 (dd, J = 1.5, 8.2 Hz, 1H), 8.65 (bs, 1H); 13C NMR (100 MHz,
CDCl3) δ (ppm) 18.0, 61.6, 66.2, 67.3, 90.6, 101.8, 108.1, 109.5, 123.5, 125.6, 128.2, 128.3, 128.6, 135.7,

ACCEPTED MANUSCRIPT
147.8, 151.9, 152.1, 164.5, 164.6, 166.6; HPLC- MS (ESI+) Rt = 10.08 min; m/z = 427 [M+H2O]+; IR (film,
cm-1): 3286, 2980, 1820, 1701, 1657, 1489, 1259; [α]20D = - 0.24 (c = 0.8, CH2Cl2); found C, 64.72; H, 4.65;
N, 3.40%; C22H19NO7 requires C, 64.54; H, 4.68; N, 3.42%.
4.2.14. (R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl benzo[d][1,3]dioxole-4-
carboxylate (1b)
Following GP3, alcohol A (35 mg, 0.13 mmol) and acid 3b (34 mg, 0.21 mmol) yielded compound 1b (24
1
mg, 44% yield) as a waxy solid after flash chromatography purification (CH2Cl2/acetone 99:1). H NMR
(400 MHz, CDCl3) δ (ppm) 1.53 (d, J = 6.4 Hz, 3H), 3.99 (d, J = 6.7 Hz, 1H), 5.17 (s, 2H), 5.29 (s, 1H), 5.46
(quintet, J = 6.4 Hz, 1H), 5.97 – 6.01 (m, 2H), 6.85 (t, J = 8.0 Hz, 1H), 6.96 (dd, J = 1.1, 7.7 Hz, 1H), 7.30 –
7.38 (m, 6H), 8.67 (bs, 1H); 13C NMR (100 MHz, CDCl3) δ (ppm) 18.3, 61.5, 66.1, 67.7, 90.7, 101.8, 112.4,
112.5, 121.3, 122.5, 128.2, 128.3, 128.6, 135.8, 148.7, 148.8, 152.2, 163.2, 164.6, 166.7; HPLC-MS (ESI+):
Rt =10.00 min; m/z = 427 [M+H2O]+; IR (film, cm-1): 3287, 2924, 1820, 1700, 1656, 1457, 1288; [α]20D = -
0.17 (c=1.2, CH2Cl2); found C, 64.69 ; H, 4.74; N, 3.37%; C22H19NO7 requires C, 64.54; H, 4.68; N, 3.42%.
4.2.15. (R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl 4-
(ethoxymethoxy)benzoate (4c)
Following GP3, alcohol A (39 mg, 0.15 mmol) and acid 3c (47 mg, 0.24 mmol) yielded compound 4c (27
1
mg, 41% yield) as a waxy solid after flash chromatography purification (CH2Cl2/acetone 99:1). H NMR
(400 MHz, CDCl3) δ (ppm) 1.22 (t, J = 7.0 Hz, 3H), 1.51 (d, J = 6.3 Hz, 3H), 3.73 (q, J = 7.0 Hz, 2H), 4.01 (d, J
= 6.7 Hz, 1H), 5.15 (d, JAB = 12.3 Hz, 1H), 5.19 (d, JAB = 12.3 Hz, 1H), 5.26 (s, 1H), 5.27 (s, 2H), 5.48
(quintet, J = 6.3 Hz, 1H), 7.06 (d, J = 8.7 Hz, 2H), 7.32 – 7.40 (m, 5H), 7.96 (d, J = 8.7 Hz, 2H), 8.67 (bs, 1H);
13C NMR (100 MHz, CDCl3) δ (ppm) 15.0, 18.1, 61.6, 64.6, 66.2, 67.1, 90.6, 92.8, 115.8, 122.9, 128.2,
128.3, 128.6, 131.6, 135.8, 152.2, 161.4, 164.6, 164.9, 166.7; HPLC-MS (ESI+): Rt = 10.86 min; m/z = 457
[M+H2O]+; IR (film, cm-1): 3300, 2926, 1821, 1702, 1656, 1606, 1509, 1270; [α]20 = - 0.10 (c=1.05,
D
CH2Cl2).
4.2.16.
(R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl-3,4-bis(ethoxymethoxy)
benzoate (4d)
Following GP3, alcohol A (60 mg, 0.23 mmol) and acid 3d (98 mg, 0.36 mmol) yielded compound 4d (44
1
mg, 37% yield) as a yellow oil after flash chromatography purification (CH2Cl2/diethyl ether 95:5). H
NMR (400 MHz, CDCl3) δ (ppm) 1.20 (t, J = 7.0 Hz, 3H), 1.21 (t, J = 7.0 Hz, 3H), 1.51 (d, J = 6.4 Hz, 3H),
3.72 – 3.81 (m, 4H), 4.00 (d, J = 6.7 Hz, 1H), 5.13 (d, JAB = 12.3 Hz, 1H), 5.18 (d, JAB = 12.3 Hz, 1H), 5.26 –
5.33 (m, 5H), 5.43 (quintet, J = 6.4 Hz, 1H), 7.20 (d, J = 8.6 Hz, 1H), 7.30 – 7.35 (m, 5H), 7.65 (dd, J = 1.8,
8.6 Hz, 1H), 7.81 (d, J = 1.8 Hz, 1H), 8.76 (bs, 1H); 13C NMR (100 MHz, CDCl3) δ (ppm) 14.99, 15.01, 18.2,

ACCEPTED MANUSCRIPT
61.6, 64.5, 64.6, 66.1, 67.3, 90.6, 93.5, 94.0, 115.1, 117.7, 123.3, 124.7, 128.2, 128.3, 128.6, 135.7,
146.7, 151.7, 152.2, 164.7, 164.8, 166.6; HPLC-MS (ESI+): Rt = 11.52 min; m/z = 531 [M+H2O]+; IR (film,
cm-1): 3287, 2976, 1822, 1704, 1658, 1603, 1509, 1263; [α]20D = - 0.12 (c= 0.6, CH2Cl2).
4.2.17. (E)-(R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl 3-(benzo[d][1,3]dioxol-
5-yl)acrylate (1e)
Following GP3, alcohol A (67 mg, 0.25 mmol) and acid 3e (77 mg, 0.40 mmol) yielded compound 1e (38
1
mg, 35% yield) as a waxy solid after flash chromatography purification (CH2Cl2/acetone 98:2). H NMR
(400 MHz, CDCl3) δ (ppm) 1.46 (d, J = 6.4 Hz, 3H), 3.95 (d, J = 6.8 Hz, 1H), 5.15 (d, JAB = 12.3 Hz, 1H), 5.18
(d, JAB = 12.3 Hz, 1H), 5.27 (d, J = 0.6 Hz, 1H), 5.36 (quintet, J = 6.4 Hz, 1H), 6.01 (s, 2H), 6.23 (d, J = 15.9
Hz, 1H), 6.81 (d, J = 7.9 Hz, 1H), 6.98 – 7.03 (m, 2H), 7.32 – 7.37 (m, 5H), 7.59 (d, J = 15.9 Hz, 1H), 8.51
(bs, 1H); 13C NMR (100 MHz, CDCl3) δ (ppm) 18.0, 61.5, 66.2, 66.7, 90.6, 101.6, 106.5, 108.5, 115.2,
124.7, 128.2, 128.3, 128.5, 128.6, 135.7, 145.5, 148.3, 149.8, 152.1, 164.7, 165.9, 166.7; HPLC-MS (ESI+):
Rt = 10.44 min; m/z = 392 [M-43]+; IR (film, cm-1): 3286, 2924, 1821, 1704, 1657, 1510, 1256; [α]20 = +
D
0.09 (c=1.0, CH2Cl2); found C, 66.47; H, 4.89; N, 3.28%; C24H21NO7 requires C, 66.20; H, 4.86; N, 3.22%.
4.2.18. (E)-(R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl-3-(4-ethoxymethoxy)
phenyl)acrylate (4f)
Following GP3, alcohol A (67 mg, 0.25 mmol) and acid 3f (89 mg, 0.40 mmol) yielded compound 4f (67
mg, 58% yield) as a waxy solid after flash chromatography purification (cyclohexane/EtOAc 3:1). 1H NMR
(400 MHz, CDCl3) δ (ppm) 1.22 (t, J = 7.1 Hz, 3H), 1.47 (d, J = 6.4 Hz, 3H), 3.73 (q, J = 7.1 Hz, 2H), 3.96 (d, J
= 6.9 Hz, 1H), 5.14 (d, JAB = 12.3 Hz, 1H), 5.18 (d, JAB = 12.3 Hz, 1H), 5.25 (s, 2H), 5.27 (s, 1H), 5.37
(quintet, J = 6.4 Hz, 1H), 6.29 (d, J = 15.9 Hz, 1H), 7.04 (d, J = 8.7 Hz, 2H), 7.33 – 7.38 (m, 5H), 7.47 (d, J =
8.7 Hz, 2H), 7.65 (d, J = 15.9 Hz, 1H), 8.44 (bs, 1H); HPLC-MS (ESI+): Rt = 11.57 min; m/z = 422 [M-43]+; IR
(film, cm-1): 3286, 2924, 1821, 1704, 1658, 1454, 1256.
4.2.19. (E)-(R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl 3-(2-(ethoxymethoxy)
phenyl)acrylate (4g)
Following GP3, alcohol A (40 mg, 0.15 mmol) and acid 3g (40 mg, 0.18 mmol) yielded compound 4g (45
mg, 65% yield) as a waxy solid after flash chromatography purification (cyclohexane/EtOAc 3:1). 1H NMR
(400 MHz, CDCl3) δ (ppm) 1.21 (t, J = 7.1 Hz, 3H), 1.48 (d, J = 6.4 Hz, 3H), 3.74 (q, J = 7.1 Hz, 2H), 3.97 (d, J
= 7.0 Hz, 1H), 5.16 (d, JAB = 12.3 Hz, 1H), 5.20 (d, JAB = 12.3 Hz, 1H), 5.29 (s, 3H), 5.38 (quintet, J = 6.4 Hz,
1H), 6.48 (d, J = 16.2 Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 7.32– 7.36 (m, 6H), 7.53 (d, J
= 7.7 Hz, 1H), 8.06 (d, J = 16.2 Hz, 1H), 8.78 (bs, 1H); 13C NMR (100 MHz, CDCl3) δ (ppm) 15.0, 18.1, 61.5,
64.5, 66.1, 66.8, 90.5, 93.2, 114.9, 117.7, 121.7, 123.7, 128.2, 128.3, 128.4, 128.6, 131.7, 135.8, 141.0,

ACCEPTED MANUSCRIPT
152.3, 156.2, 164.7, 166.1, 166.7; HPLC-MS (ESI+): Rt = 11.74 min; m/z = 483 [M+H2O]+; IR (film, cm-1):
3284, 2933, 1821, 1702, 1657, 1631, 1542, 1457, 1264; [α]20D = - 0.16 (c=1.0, CH2Cl2).
4.2.20. (E)-(R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl3-(3,4-bis(ethoxy
methoxy) phenyl) acrylate (4h)
Following GP3, alcohol A (65 mg, 0.25 mmol) and acid 3h (116 mg, 0.39 mmol) yielded compound 4h (51
1
mg, 38% yield) as a yellow oil after flash chromatography purification (CH2Cl2/diethyl ether 95:5). H
NMR (400 MHz, CDCl3) δ (ppm) 1.20 – 1.25 (m, 6H), 1.46 (d, J = 6.3 Hz, 3H), 3.73 – 3.80 (m, 4H), 3.96 (d, J
= 7.0 Hz, 1H), 5.14 (d, JAB = 12.4 Hz, 1H), 5.19 (d, JAB = 12.4 Hz, 1H), 5.27 (s, 1H), 5.30 (s, 2H), 5.31 (s, 2H),
5.37 (quintet, J = 6.3 Hz, 1H), 6.29 (d, J = 15.9 Hz, 1H), 7.12 – 7.19 (m, 2H), 7.32 – 7.37 (m, 6H), 7.61 (d, J
= 15.9 Hz, 1H), 8.66 (bs, 1H); 13C NMR (100 MHz, CDCl3) δ (ppm) 15.0, 15.1, 18.0, 61.5, 64.5, 64.6, 66.2,
66.8, 90.6, 93.7, 94.0, 115.5, 115.6, 115.9, 123.1, 123.5, 128.2, 128.3, 128.6, 135.7, 145.5, 147.4, 149.5,
152.1, 164.7, 165.9, 166.7; HPLC-MS (ESI+): Rt = 12.10 min; m/z 557 [M+H2O]+; IR (film, cm-1): 3266,
2973, 1820, 1701, 1655, 1638, 1510, 1251; [α]20D = + 0.06 (c= 1.0, CH2Cl2).
4.2.21. (E)-(R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl 3-(4-(ethoxymethoxy)-
3-methoxyphenyl)acrylate (4i)
Following GP3, alcohol A (40 mg, 0.15 mmol) and acid 3i (61 mg, 0.24 mmol) yielded compound 4i (27
1
mg, 36% yield) as a waxy solid after flash chromatography purification (CH2Cl2/acetone 98:2). H NMR
(400 MHz, CDCl3) δ (ppm) 1.22 (t, J = 7.1 Hz, 3H), 1.47 (d, J = 6.4 Hz, 3H), 3.77 (q, J = 7.1 Hz, 2H), 3.92 (s,
3H), 3.97 (d, J = 6.8 Hz, 1H), 5.14 (d, JAB = 12.3 Hz, 1H), 5.18 (d, JAB = 12.3 Hz, 1H), 5.28 (s, 1H), 5.32 (s,
2H), 5.38 (quintet, J = 6.4 Hz, 1H), 6.29 (d, J = 15.9 Hz, 1H), 7.06 (s, 1H), 7.07 (d, J = 8.1 Hz, 1H), 7.18 (d, J
= 8.1 Hz, 1H), 7.33 – 7.37 (m, 5H), 7.64 (d, J = 15.9 Hz, 1H), 8.47 (bs, 1H); 13C NMR (100MHz, (CD3)2CO) δ
(ppm) 15.4, 18.4, 56.2, 62.3, 65.0, 66.0, 67.5, 90.4, 94.6, 111.8, 116.5, 117.2, 123.5, 128.8, 129.0, 129.3,
129.5, 137.8, 146.2, 151.4, 153.5, 154.2, 166.2, 166.4, 166.7; HPLC-MS (ESI+): Rt = 10.85 min; m/z = 452
[M-43]+; IR (film, cm-1): 3287, 2924, 1821, 1704, 1657, 1510, 1256.
4.2.22. (E)-(R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl 3-(4-(ethoxymethoxy)-
3,5-dimethoxyphenyl)acrylate (4j)
Following GP3, alcohol A (40 mg, 0.15 mmol) and acid 3j (68 mg, 0.24 mmol) yielded compound 4j (30
1
mg, 38% yield) as a waxy solid after flash chromatography purification (CH2Cl2/acetone 98:2). H NMR
(400 MHz, CDCl3) δ (ppm) 1.21 (t, J = 7.1 Hz, 3H), 1.47 (d, J = 6.4 Hz, 3H), 3.87 (s, 6H), 3.88 (q, J = 7.1 Hz,
2H), 3.97 (d, J = 6.7 Hz, 1H), 5.15 (d, JAB = 12.3 Hz, 1H), 5.18 (d, JAB = 12.3 Hz, 1H), 5.19 (s, 2H), 5.28 (s,
1H), 5.38 (quintet, J = 6.4 Hz, 1H), 6.32 (d, J = 15.9 Hz, 1H), 6.75 (s, 2H), 7.33 – 7.36 (m, 5H), 7.61 (d, J =
15.9 Hz, 1H), 8.56 (bs, 1H); 13C NMR (100 MHz, CDCl3) δ (ppm) 15.0, 17.9, 56.1, 61.5, 62.1, 65.0, 66.2,

ACCEPTED MANUSCRIPT
66.8, 90.7, 96.6, 105.3, 116.5, 128.2, 128.4, 128.7, 130.0, 135.7, 136.8, 145.9, 152.0, 153.6, 164.5, 165.7,
166.7; HPLC-MS (ESI+): Rt = 11.46 min; m/z = 543 [M+H2O]+; IR (film, cm-1): 3289, 2925, 1736, 1649, 1584,
1504, 1454.
4.2.23. (R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl 4-hydroxybenzoate (1c)
Following GP4, β-lactam 4c (27 mg, 61 µmol) treated with TFA (126 µL, 1.68 mmol) yielded compound 1c
(23 mg, 99% yield) as an oil. 1H NMR (400 MHz, CDCl3) δ (ppm) 1.51 (d, J = 6.3 Hz, 3H), 4.02 (d, J = 6.5 Hz,
1H), 5.14 (d, JAB = 12.0 Hz, 1H), 5.19 (d, JAB = 12.0 Hz, 1H), 5.30 (d, J = 1.7 Hz, 1H), 5.47 (quintet, J = 6.3 Hz,
1H), 6.83 (d, J = 7.2 Hz, 2H), 7.31 – 7.40 (m, 5H), 7.89 (d, J = 7.2 Hz, 2H), 8.67 (bs, 1H); 13C NMR (100 MHz,
CDCl3) δ (ppm) 18.0, 61.6, 66.5, 67.1, 91.0, 115.4, 121.5, 128.2, 128.4, 128.7, 132.1, 135.4, 152.1, 160.6,
165.6, 165.7, 167.0; HPLC-MS (ESI+): Rt = 8.64 min; m/z = 399 [M+H2O]+; IR (film, cm-1): 3304, 2982,
1818, 1697, 1662, 1608, 1592, 1269; [α]20D = - 0.05 (c=1.00, CH2Cl2); found C, 66.20; H, 4.90; N, 3.58%;
C21H19NO6 requires C, 66.13; H, 5.02; N, 3.67%.
4.2.24. (R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl 3,4-dihydroxybenzoate
(1d)
Following GP4, β-lactam 4d (36 mg, 70 µmol) treated with TFA (210 µL, 2.85mmol) yielded compound 1d
(27 mg, 99% yield) as an oil. 1H NMR (400 MHz, (CD3)2CO) δ (ppm) 1.48 (d, J = 6.4 Hz, 3H), 4.22 (d, J = 6.3
Hz, 1H), 5.14 (d, JAB = 12.4 Hz, 1H), 5.20 (d, JAB = 12.4 Hz, 1H), 5.25 (s, 1H), 5.42 (quintet, J = 6.3 Hz, 1H),
6.89 (d, J = 8.3 Hz, 1H), 7.29 – 7.50 (m, 7H), 9.89 (bs, 1H); 13C NMR (100 MHz, (CD3)2CO) δ (ppm) 18.4,
62.4, 65.9, 67.8, 90.4, 115.8, 117.2, 122.5, 123.4, 128.8, 128.9, 129.2, 137.8, 145.7, 151.1, 154.2, 165.5,
166.3, 166.6; HPLC-MS (ESI+): Rt = 8.82 min; m/z = 415 [M+H2O]+; IR (film, cm-1): 3356, 2925, 1815, 1694,
1657, 1604, 1524, 1293; [α]20D = - 1.27 (c= 0.9, MeOH); found C, ; H, ; N, %; C21H19NO7 requires C, 63.47;
H, 4.82; N, 3.52%.
4.2.25. (E)-(R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl 3-(4-hydroxyphenyl)
acrylate (1f)
Following GP4, β-lactam 4f (50 mg, 107 µmol) treated with TFA (85 µL, 1.13 mmol) yielded compound 1f
(43 mg, 99% yield) as an oil. 1H NMR (400 MHz, CDCl3) δ (ppm) 1.46 (d, J = 6.4 Hz, 3H), 3.75 (bs, 1H), 3.97
(d, J = 6.6 Hz, 1H), 5.15 (d, JAB = 12.4 Hz, 1H), 5.19 (d, JAB = 12.4 Hz, 1H), 5.28 (s, 1H), 5.37 (quintet, J = 6.4
Hz, 1H), 6.25 (d, J = 15.9 Hz, 1H), 6.84 (d, J = 8.5 Hz, 2H), 7.33 – 7.38 (m, 5H), 7.40 (d, J = 8.5 Hz, 2H), 7.62
(d, J = 15.9 Hz, 1H), 8.53 (bs, 1H); 13C NMR (101 MHz, CDCl3) δ (ppm) 18.0, 61.5, 66.3, 66.6, 90.7, 114.4,
115.9, 126.6, 128.2, 128.4, 128.6, 130.1, 135.7, 145.7, 152.1, 158.4, 164.9, 166.2, 166.7; HPLC-MS (ESI+):
Rt = 8.93 min; m/z = 364 [M-43]+; IR (film, cm-1): 3405, 3286, 2929, 1819, 1650, 1515, 1453, 1257, 1159,

ACCEPTED MANUSCRIPT
1038, 832, 698; [α] 20 = + 0.06 (c=1.0, CH2Cl2); found C, 67.71; H, 5.13; N, 3.38%; C23H21NO6 requires C,
D
67.80; H, 5.20; N, 3.44%.
4.2.26. (E)-(R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl 3-(2-
hydroxyphenyl)acrylate (1g)
Following GP4, β-lactam 4g (38 mg, 82 µmol) treated with TFA (120 µL, 1.60 mmol) yielded compound
1g (33 mg, 99% yield) as an oil. 1H NMR (400 MHz, (CD3)2CO) δ 1.45 (d, J = 6.3 Hz, 3H), 4.16 (d, J = 6.6 Hz,
1H), 5.15 (d, JAB = 12.5 Hz, 1H), 5.19 (d, JAB = 12.5 Hz, 1H), 5.28 (s, 1H), 5.37 (quintet, J = 6.3 Hz, 1H), 6.64
(d, J = 16.1 Hz, 1H), 6.89 (t, J = 7.4 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H), 7.26 (t, J = 7.4 Hz, 1H), 7.30 – 7.40 (m,
5H), 7.59 (d, J = 7.6 Hz, 1H), 8.02 (d, J = 16.1 Hz, 1H), 9.84 (bs, 1H); 13C NMR (100 MHz, CDCl3) δ (ppm)
18.0, 61.5, 66.4, 66.8, 90.9, 116.3, 117.0, 120.6, 121.2, 128.2, 128.4, 128.6, 129.1, 131.8, 141.6, 152.1,
155.5, 165.7, 166.9, 167.1; HPLC-MS (ESI-): Rt = 11.74 min; m/z = 406 [M-H]+; IR (film, cm-1): 3386, 3282,
2934, 1822, 1702, 1662, 1629, 1604, 1458, 1249; [α]20 = - 0.14 (c=1.0, CH2Cl2); found C, 67.99; H, 5.12;
D
N, 3.43%; C23H21NO6 requires C, 67.80; H, 5.20; N, 3.44%.
4.2.27. (E)-(R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl 3-(3,4-dihydroxyphenyl)
acrylate (1h)
Following GP4, β-lactam 4h (44 mg, 82 µmol) treated with TFA (490 µL, 6.56 mmol) yielded compound
1h (34 mg, 99% yield) as an oil. 1H NMR (400 MHz, (CD3)2CO) δ (ppm) 1.43 (d, J = 6.4 Hz, 3H), 4.14 (d, J =
6.5 Hz, 1H), 5.15 (d, JAB = 12.5 Hz, 1H), 5.19 (d, JAB = 12.5 Hz, 1H), 5.26 (s, 1H), 5.33 (quintet, J = 6.4 Hz,
1H), 6.28 (d, J = 15.9 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 7.03 (dd, J = 1.6, 8.2 Hz, 1H), 7.16 (d, J = 1.6 Hz, 1H),
7.30 – 7.40 (m, 5H), 7.56 (d, J = 15.9 Hz, 1H),9.85 (bs, 1H); 13C NMR (100 MHz, (CD3)2CO) δ (ppm) 18.5,
62.3, 66.0, 67.5, 90.4, 115.1, 115.3, 116.4, 122.7, 127.4, 128.8, 129.0, 129.3, 137.8, 146.3, 146.5, 149.0,
154.2, 166.2, 166.4, 166.7; HPLC-MS (ESI+): Rt = 8.90 min; m/z = 441 [M+H2O]+; IR (film, cm-1): 3331,
2925, 1816, 1698, 1657, 1604, 1515, 1260; [α]D20= + 0.11 (c=0.6, MeOH); found C, 65.29; H, 5.04; N,
3.23%; C23H21NO7 requires C, 65.24; H, 5.00; N, 3.31%.
4.2.28. (E)-(R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl 3-(4-hydroxy-3-
methoxyphenyl)acrylate (1i)
Following GP4, β-lactam 4i (20 mg, 40 µmol) treated with TFA (150 µL, 2.02 mmol) yielded compound 1i
(17 mg, 99% yield) as an oil. 1H NMR (400 MHz, CDCl3) δ (ppm) 1.46 (d, J = 6.3 Hz, 3H), 3.93 (s, 3H), 3.96
(d, J = 6.8 Hz, 1H), 5.14 (d, JAB = 12.4 Hz, 1H), 5.18 (d, JAB = 12.4 Hz, 1H), 5.28 (s, 1H), 5.37 (quintet, J = 6.3
Hz, 1H), 6.26 (d, J = 15.9 Hz, 1H), 6.92 (d, J = 8.1Hz, 1H), 7.03 (s, 1H), 7.06 (d, J = 8.1Hz, 1H), 7.31 – 7.39
(m, 5H), 7.62 (d, J = 15.9 Hz, 1H), 8.53 (bs, 1H); 13C NMR (100 MHz, CDCl3) δ (ppm) 18.0, 56.0, 61.5, 66.2,
66.7, 90.7, 109.3, 114.6, 114.7, 123.5, 126.7, 128.2, 128.4, 128.7, 146.0, 146.7, 148.2, 152.1, 164.7,

ACCEPTED MANUSCRIPT
166.1, 166.4, 166.7; HPLC-MS (ESI+): Rt = 9.16 min; m/z = 394 [M-43]+; IR (film, cm-1): 3397, 3293, 2930,
1819, 1702, 1659, 1514, 1454, 1267; [α]20 = + 0.13 (c=1.0, CH2Cl2); found C, 66.92; H, 5.35; N, 3.15%;
D
C24H23NO7 requires C, 65.90; H, 5.30; N, 3.20%.
4.2.29. (E)-(R)-1-((S,Z)-2-(2-(benzyloxy)-2-oxoethylidene)-4-oxoazetidin-3-yl)ethyl 3-(4-hydroxy-3,5-
dimethoxyphenyl)acrylate (1j)
Following GP4, β-lactam 4j (30 mg, 57 µmol) treated with TFA (210 µL, 2.85 mmol) yielded compound 1j
(26 mg, 99% yield) as an oil. 1H NMR (400 MHz, CDCl3) δ (ppm) 1.46 (d, J = 6.4 Hz, 3H), 3.92 (s, 6H), 3.97
(d, J = 6.8 Hz, 1H), 5.14 (d, JAB = 12.4 Hz, 1H), 5.18 (d, JAB = 12.4 Hz, 1H), 5.28 (s, 1H), 5.38 (quintet, J = 6.4
Hz, 1H), 6.27 (d, J = 15.8 Hz, 1H), 6.77 (s, 2H), 7.33 – 7.36 (m, 5H), 7.60 (d, J = 15.8 Hz, 1H), 8.54 (bs, 1H);
13C NMR (100 MHz, CDCl3) δ (ppm) 18.0, 56.4, 61.6, 62.1, 66.2, 66.6, 90.7, 105.2, 114.9, 125.6, 128.3,
128.4, 128.7, 135.7, 137.3, 146.1, 147.2, 152.1, 160.0, 164.6, 165.9, 166.7; HPLC-MS (ESI+): Rt = 9.04 min;
m/z = 424 [M-43]+; IR (film, cm-1): 3398, 3286, 2923, 1819, 1702, 1657, 1514, 1457, 1255; [α]20D = + 0.11
(c=1.0, CH2Cl2); found C, 64.31; H, 5.41; N, 2.94%; C25H25NO8 requires C, 64.23; H, 5.39; N, 3.00%.
4.3. Bacterial strains and antimicrobial susceptibility testing procedures
Representative Gram-positive and Gram-negative bacterial pathogens, hosted in the microbial
Biobank (MicroMiB Biobank) of the Laboratory of Clinical Microbiology and Virology of the
University of Milano-Bicocca, Monza, were used for the in vitro antimicrobial susceptibility
testing; these included Staphylococcus aureus, Staphylococcus hominis, Staphylococcus
epidermidis, Enterococcus faecalis and Enterococcus faecium as Gram-positive species and
Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli as Gram-negative species,
respectively. In particular, laboratory stocks of methicillin-susceptible S. aureus (MSSA) strain
No. 69856, S. aureus (MSSA) strain No. 39249, methicillin-resistant S. aureus (MRSA) strain No.
44674, linezolid and methicillin-resistant S. aureus (LIN-R MRSA) strain No. SAU-1, methicillin-
susceptible Staphylococcus epidermidis (MSSE) strain No. 3226, linezolid and methicillin-resistant
S. epidermidis (LIN-R MRSE) strain No. G1027, linezolid and methicillin-resistant Staphylococcus
hominis (LIN-R MRSH) strain No. α26, E. faecalis strain No. 4150 and linezolid and vancomycin-
resistant Enterococcus faecium (LIN-R VRE) strain No. VRE-2 were used as test strains.
Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were purchased from
the American Type Culture Collection (Manassas, VA, USA) and used as additional test strains.
The in vitro antibacterial activity of the new synthetized compounds 1a-j was studied by
determining their minimum inhibitory concentrations (MICs) by means of the broth
microdilution method according to the Clinical and Laboratory Standards Institute (CLSI)

ACCEPTED MANUSCRIPT
guidelines [46]. All chemicals used in this study were of analytical grade. Vancomycin and
cefuroxime (Sigma Aldrich, Italy) were used as reference antibiotic compounds for MIC
determinations.
Briefly, serial 2-fold dilutions of each compound were made using the Mueller-Hinton broth in
96 wells microtitre plates. Dimethyl sulfoxide (DMSO) was used as solvent for all the synthetized
compounds. An equal volume of the bacterial inoculum (1x106 CFU/mL) was added to each well
on the microtitre plate containing 0.05 mL of the serial antibiotic dilutions. The microtitre plate
was then incubated at 37 °C for 18-24 h after which each well was analyzed for the presence of
bacterial growth.
The MIC was defined as the lowest concentration of antimicrobial agent able to cause inhibition
of bacterial growth as shown by the lack of visible turbidity of the culture medium. Standard
strains S. aureus ATCC 29213 and E. faecalis ATCC 29212 were used as control and to MIC testing
validation.
4.4. Trolox equivalent antioxidant capacity assay (TEAC)
Antioxidant capacity was checked through TEAC-DPPH and TEAC-ABTS assays, performed following the
methods described by Cheng et al. [47] and Marecek et al. [48] and properly modified.
4.4.1. TEAC-DPPH
The DPPH (2,2-diphenyl-1-picrylhydrazyl; Sigma-Aldrich) working sample was prepared in the dark at
room temperature dissolving the radical (0.208 mM) in a 50% acetone solution. The stock solution of
Trolox® (0.160 mM) and the solutions of sample compounds 1a-j (7.00 mM) were prepared following
the same procedure. Trolox® and 1a-j stock solutions were then serially diluted to obtain solutions
progressively less concentrated (dilution ratio 1: 2) to be tested. To 100 µL of each of these solutions
were added 100 µL of DPPH solution, and then each 200 µL sample was poured into a 96-microwells
plate, incubated in agitation (100 rpm) for 40 min at room temperature in the dark. The DPPH acetone
solution (200 µL) was used as negative control, while 200 µL of acetone solution (50%) was considered
as blank sample. After incubation, the 96-microwells plate was checked with microplate reader
(Microplate Reader 680 XR, Biorad) at 515 nm. The content of antioxidants in the sample is determined
as inhibition percentage of DPPH radical related to the negative control sample (see the below reported
formula). The total antioxidant capacity of 1a-j compounds was then expressed as mmol equivalent of
Trolox® (TEAC).
DPPH radical inhibition (%) = ꢀꢁꢂ.ꢃꢄꢂꢅꢆꢄꢇꢈꢁꢉꢊꢋꢌꢇꢍꢎ x100
ꢁꢂ.ꢃꢄꢂꢅꢆꢄꢇ
Asample: absorbance value of 1a-j sample compounds

ACCEPTED MANUSCRIPT
An.control: absorbance value of negative control
4.4.2. TEAC-ABTS
An aqueous solution of the radical cation ABTS (ABTS•+; 2,2'-azino-bis(3-ethylbenzothiazoline-6-
sulphonic acid); Sigma-Aldrich) was prepared by mixing 10 mL of a 7.0 mM aqueous solution of ABTS
with 0.5 mL of a 51.4 mM aqueous solution of K2S2O4 (pH=5.0), and then incubated overnight in the dark
with the object to achieve an absorbance of 0.700 ± 0.02 at 734 nm. Both the acetone (50%) stock
solution of Trolox® (0.160 mM) and of those of sample compounds 1a-j (7.0 mM) were prepared in the
dark at room temperature. Trolox®; 1a-j stock solutions were then serially diluted to obtain solutions
progressively less concentrated (dilution ratio 1: 2) to be tested. To 10 µL of each of these solutions, 290
µL of radical cation ABTS aqueous solution (ABTS•+) were added and poured into a 96-microwells plate,
incubated for 6 min at room temperature in the dark and then assayed with Microplate reader
(Microplate Reader 680 XR, Biorad). The TEAC-ABTS antioxidant capacity was measured as reported for
TEAC-DPPH assay.
Acknowledgements
This research was supported by University of Bologna (RFO 2016) and by University of Ferrara
(FAR 2016). D.G. would like to thank S. Forni, S. Cerofolini and M. Cirillo for the fruitful technical
assistance.
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