New linezolid-like 1,2,4-oxadiazolesactive against Gram-positive multiresistant pathogens

Article abstract of DOI:10.1016/j.ejmech.2013.03.069

The synthesis and the in vitro antibacterial activity of novel linezolid-like oxadiazoles are reported. Replacement of the linezolid morpholine C-ring with 1,2,4-oxadiazole results in an antibacterial activity against Staphylococcus aureus both methicillin-susceptible and methicillin-resistant comparable or even superior to that of linezolid. While acetamidomethyl or thioacetoamidomethyl moieties in the C(5) sidechain are required, fluorination of the phenyl B ring exhibits a slight effect on an antibacterial activity but its presence seems to reduce the compounds cytotoxicity. Molecular modeling performed using two different approaches - FLAP and Amber software - shows that in the binding pose of the newly synthesized compounds as compared with the crystallographic pose of linezolid, the 1,2,4-oxadiazole moiety seems to perfectly mimic the function of the morpholinic ring, since the H-bond interaction with U2585 is retained.

Full text of DOI:10.1016/j.ejmech.2013.03.069

European Journal of Medicinal Chemistry 65 (2013) 533e545
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New linezolid-like 1,2,4-oxadiazoles active against Gram-positive
multiresistant pathogens
,
a
b
c
Cosimo G. Fortuna a , Carmela Bonaccorso , Alessandra Bulbarelli , Gianluigi Caltabiano ,
*
Laura Rizzi b, Laura Goracci a, Giuseppe Musumarra a, Andrea Pace d
,
,e,
**
Antonio Palumbo Piccionello d e, Annalisa Guarcello d e, Paola Pierro d
***
,
,
,
,e
Clementina E.A. Cocuzza f, Rosario Musumeci f
,
a Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Viale Andrea Doria 6, I-95125 Catania, Italy
b Dipartimento di Scienze della Salute, Università di Milano-Bicocca, Via Cadore 48, I-20900 Monza, MB, Italy
c Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
d Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari (STEMBIO), Sez. Chimica Organica “E. Paternò”, Università degli Studi di Palermo,
Viale delle Scienze Ed. 17 e Parco D’Orleans II, I-90128 Palermo, Italy
e Istituto EuroMediterraneo di Scienza e Tecnologia (IEMEST), Via Emerico Amari 123, I-90139 Palermo, Italy
f Dipartimento di Chirurgia e Medicina Interdisciplinare, Università di Milano-Bicocca, Via Cadore 48, I-20900 Monza, MB, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and the in vitro antibacterial activity of novel linezolid-like oxadiazoles are reported.
Replacement of the linezolid morpholine C-ring with 1,2,4-oxadiazole results in an antibacterial activity
against Staphylococcus aureus both methicillin-susceptible and methicillin-resistant comparable or even
superior to that of linezolid. While acetamidomethyl or thioacetoamidomethyl moieties in the C(5) side-
chain are required, fluorination of the phenyl B ring exhibits a slight effect on an antibacterial activity but
its presence seems to reduce the compounds cytotoxicity. Molecular modeling performed using two
different approaches e FLAP and Amber software e shows that in the binding pose of the newly syn-
thesized compounds as compared with the crystallographic pose of linezolid, the 1,2,4-oxadiazole
moiety seems to perfectly mimic the function of the morpholinic ring, since the H-bond interaction
with U2585 is retained.
Received 26 August 2012
Received in revised form
25 February 2013
Accepted 31 March 2013
Available online 14 May 2013
Keywords:
Oxazolidinones
Linezolid
Drug design
Ó 2013 Elsevier Masson SAS. All rights reserved.
Antimicrobial activity
Cell viability
Staphylococcus aureus
1. Introduction
pneumoniae (PNSSP), have emerged as major public health concern,
both in hospital and community settings worldwide. The need for
Use and misuse of antibacterial agents have resulted in the
development of bacterial resistance to all antibiotics in clinical use
irrespective of the chemical class or molecular target of the drug.
Infections caused by multiresistant Gram-positive cocci, such as
methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-
resistant enterococci (VRE) and penicillin-resistant Streptococcus
newantibiotics urged the Infectious Disease Societyof America (IDSA)
to issue the challenge to develop ten new antibiotics by 2020 [1].
Oxazolidinones are a class of antibacterial agents which dis-
played activity against a variety of Gram-positive pathogens and are
highly potent against multidrug-resistant bacteria. In particular,
oxazolidinones are used to treat skin and respiratory tract in-
fections caused by S. aureus and streptococci strains, as well as
being active against vancomycin-resistant Enterococcus faecium
[2,3]. Linezolid (Fig. 1), the first oxazolidinone antibiotic approved
for clinical use, has been shown to inhibit translation at the initi-
ation phase of protein synthesis in bacteria by binding to the 50S
ribosomal subunit [4]. Since 2001, however, linezolid resistance
began to appear in S. aureus and E. faecium clinical isolates and the
rate of resistance raised especially among enterococci and Staphy-
lococcus epidermidis strains with its usage [5e8].
* Corresponding author. Tel.: þ39 0957385010; fax: þ39 095580138.
** Corresponding author. Dipartimento di Scienze e Tecnologie Molecolari e Bio-
molecolari (STEMBIO), Sez. Chimica Organica “E. Paternò”, Università degli Studi di
Palermo, Viale delle Scienze Ed. 17 e Parco D’Orleans II, I-90128 Palermo, Italy.
Tel.: þ39 09123897543; fax: þ39 091596825.
*** Corresponding author. Tel.: þ39 (0)264488103; fax: þ39 (0)264488363.
E-mail addresses: cg.fortuna@unict.it (C.G. Fortuna), andrea.pace@unipa.it
(A. Pace), rosario.musumeci@unimib.it (R. Musumeci).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.069

534
C.G. Fortuna et al. / European Journal of Medicinal Chemistry 65 (2013) 533e545
Compound R1
R2
I
O
O
O
H3C
1a
H
N
O
O
N
N
H
N
O
N
1b
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
F
H
F
H
F
H
F
H
F
H
F
H
F
I
O
N
N3
N3
R1
1-7a,b
F
R2
NHC(=O)CH3
NHC(=O)CH3
NHC(=S)CH3
NHC(=S)CH3
pyrazol-1-yl
pyrazol-1-yl
imidazol-1-yl
imidazol-1-yl
1,2,4-triazol-1-yl
1,2,4-triazol-1-yl
C-ring
B-ring
A-ring
C5 side-chain
Linezolid
Fig. 1. Structure and portions nomenclature of linezolid.
A number of solutions to the problem of bacterial resistance are
possible. Successful strategies include combination of existing
antibacterial agents with other drugs as well as the development of
improved diagnostic procedures that may lead to rapid identifica-
tion of the causative pathogen and permit the use of antibacterial
agents with a narrow spectrum of activity. Another strategy is the
discovery of novel classes of antibacterial agents acting through
new mechanisms of action. However, the most common approach,
and still the most promising one, is the modification of existing
classes of antibacterial agents to provide new analogs with
improved activities.
Chart 1.
2. Results and discussion
2.1. Chemistry
In this context many researchers tried to modify the structure of
linezolid to improve the antibacterial activity [9e14]. In order to
rationalize the site of modifications, the structure of linezolid can
formally be divided into four portions according to oxazolidinone
antibacterials nomenclature [9]: i) the A-ring, consisting of the
oxazolidinone central heterocycle; ii) the B-ring, consisting of a N-
aryl moiety linked to the oxazolidinone nitrogen; iii) the C-ring,
consisting of either a carbo- or heterocyclic-functional group, not
necessarily aromatic; iv) the side-chain, consisting of any functional
group linked to the oxazolidinone C(5) or in an isosteric position
with respect to an A-ring of general type (Fig. 1).
Different types of modifications are reported in literature; the
most common one regards the C-ring, while only few modifications
were reported for the A-ring, and in some cases good activity was
retained [15,16].
Our group previously reported that the replacement of the
oxazolidinone (A-ring) with an isosteric 1,2,4-oxadiazole hetero-
aromatic ring resulted in a lack of activity [17]. Therefore, these
compounds have been chosen as references for inactive linezolid-
like compounds in a virtual screening approach.
Due to the lack of pharmacological targets, in previous studies
[18] some of us already applied a Virtual Receptor Site (VRS)
approach adopting a Molecular Interaction Field (MIF) approach
using Grid Independent Descriptors (GRIND) calculated using the
program Pentacle, which is based on the assumption that the
process of ligandereceptor interaction can be represented with the
help of the MIF.
The same approach, when applied to synthetically accessible
linezolid-like derivatives, indicated that the introduction of a 3-
methyl-1,2,4-oxadiazol-5-yl moiety as a C-ring could be a prom-
ising modification of the oxazolidinone antibiotics’ scaffold. In the
present paper we report the synthesis and the in vitro antibacterial
activity of novel linezolid-like oxadiazoles 1e7a,b (Chart 1) to
investigate the change in biological activity due to: the replacement
of the morpholine ring by the 1,2,4-oxadiazole in the C-ring, the
presence of a fluorine substituent in the B-ring, and the variety of
substituents on the C(5) side-chain of the oxazolidinone [17].
Additionally, the recently developed algorithm called Finger-
prints for Ligands and Proteins (FLAP), that can be used to describe
proteins and ligands based on a common reference framework [19],
has been used to compare the position of the synthesized active
compounds with that of linezolid into the binding site.
In order to prepare the desired scaffold, the synthetic route to-
ward compounds 1e7a,b has been planned starting from the oxa-
diazole end of the molecule by following the classic amidoxime
route (Scheme 1) [20]. Thus, acetamidoxime 9 was reacted with
either mono- or di-fluorobenzoyl chlorides 8a,b producing 5-
fluoroarylated 1,2,4-oxadiazoles 10a,b. In the latter compounds,
according to previously reported reactivity [21e24], the para po-
sition of the 5-aryl substituent is activated to undergo a SNAr with
allylamine, yielding compounds 11a,b. Finally, reaction with di-(t-
butyl)-dicarbonate and subsequent iodocyclocarbamation [25] of
the resulting derivatives 12a,b, yielded oxazolidinones1a,b as ideal
precursors for further side-chain modifications.
For the subsequent functionalization, the considered side-
chains included the acetamidomethyl moiety (see 3a,b), typical of
linezolid, as well as the corresponding thioamide derivatives (4a,b).
Their azide precursors 2a,b were obtained by reaction of com-
pounds 1a,b with sodium azide in dimethylformamide. Subsequent
reduction with triphenylphosphine [26] yielded the corresponding
amino derivatives 13a,b. After isolation and characterization, the
amino derivatives 13 were readily reacted with acetyl chloride, thus
avoiding their decomposition, presumably due to oxidative pro-
cesses occurring upon air exposure. The resulting acetamidomethyl
derivatives 3a,b, were finally reacted with the Lawesson’s Reagent
yielding thioamide derivatives 4a,b (Scheme 2).
Additionally, since the introduction of five-membered hetero-
aromatic moieties into the C(5) side-chain was shown to maintain
or enhance the antibacterial activity [27], the synthesis of com-
pounds 5e7a,b, containing various azole rings in the side-chain,
was planned.
All compounds 5e7a,b were obtained by reaction under
solvent-free conditions of the corresponding iodomethylene de-
rivatives 1a,b with pyrazole, imidazole, or 1,2,4-triazole, respec-
tively (Scheme 3). Reaction conditions were carefully chosen in
order to avoid the formation of by-products due to
observed in alkaline solution conditions.
b-elimination, as
2.2. Minimum inhibitory concentration testing
The fourteen new compounds were tested to evaluate their
antibacterial activity against both standard references and clinical
strains of methicillin-susceptible (MSSA) and methicillin-resistant

C.G. Fortuna et al. / European Journal of Medicinal Chemistry 65 (2013) 533e545
535
O
R
Cl
CH3
CH3
F
N
N
NH2
K2CO3
i) Acetone / K2CO3
8a,b
N
N
ii) Δ/solvent-free
O
O
HN
F
R
R
11a,b
H2N
CH3
NOH
10a,b
t
( BuOCO)2O
DMAP
a
: R = H
ACN
9
b
: R = F
CH3
CH3
N
N
O
N
N
N
O
O
I2
N
O
CH2Cl2
R
R
1a,b
12a,b
O
O
I
Scheme 1.
S. aureus (MRSA). Antimicrobial activities, summarized in Table 1,
were determined by the “gold standard” broth microdilution
method using the Clinical and Laboratory Standards Institute (CLSI)
recommendations (See Experimental). Minimal Inhibitory Con-
centrations (MIC) values were expressed in mg/mL and cell viability
tests were performed to evaluate the selective antibacterial toxicity
of the most active compounds.
performed a first level assay in different types of eukaryotic cell
lines to screen the new compounds for their general cytotoxic ac-
tivity. Data obtained from the first level assay reported below will
allow us to select the most promising compounds that in a next-
step analysis will be evaluate for the known oxazolidinone mech-
anisms of toxicity, such as the inhibition of mitochondrial protein
synthesis. All tested cell lines were treated with increasing con-
Four out of the fourteen tested compounds (see Table 1) showed
MIC values against both MSSA and MRSA strains with potency
comparable or superior to that of linezolid. Moreover, better ac-
tivity against MSSA and MRSA as compared to linezolid was shown
by the sulfur containing derivatives 4a and 4b, whereas compounds
3a,b were less active than linezolid, except for MRSA 433 strain.
Comparison with linezolid must take into account that the tested
compounds were used as racemic mixture, therefore the antibac-
terial activity for 3a, 3b, 4a and 4b is possibly underestimated as
compared to the potency of the pure active enantiomer.
centrations (5e400 mg/mL) of 4a, 4b, and linezolid as reference
compound. Another control was DMSO used as solvent. In PK15
(porcine kidney epithelial) cell line, all the tested concentrations of
compound 4a induced a significant reduction (P < 0.01) of cells
viability. These effects were concentration-dependent (Fig. 2) and
the reduction of cell viability reached about 70% at the 100
concentration of 4a and more than 90% cell death at 200 and
400 g/mL.
mg/mL
m
Compound 4a negatively affected also the viability of HaCaT
(human keratinocytes) cell line. In fact, it significantly reduced cell
viability already at 5 mg/mL (P < 0.05) and it induced more than 60%
cell death at all concentrations higher or equal to 25
mg/mL
2.3. Cell viability assay
(P < 0.01) (Fig. 3).
Similar results were obtained using the HepG2 (human hepa-
tocellular carcinoma) cell line (Fig. 4).
To assess if the effect shown against bacterial cells could be
related to a selected toxicity or to a more general toxic effect, we
CH3
CH3
CH3
N
N
N
O
O
O
N
N
N
NaN3
DMF
PPh3
THF
O
O
O
N
N
N
O
O
O
R
R
R
1a,b
2a,b
13a,b
I
NH2
N3
O
CH2Cl2
N
/ Py
Cl
H3C
O
CH3
CH3
N
Lawesson's
O
N
N
N
reagent
O
O
N
O
O
THF
R
R
O
S
HN
HN
4a,b
3a,b
CH3
CH3
Scheme 2.

536
C.G. Fortuna et al. / European Journal of Medicinal Chemistry 65 (2013) 533e545
CH3
CH3
CH3
N
N
N
N
N
O
N
O
I
O
N
N
H
Δ
N
H
N
N
N
O
O
O
N
N
N
N
N
Δ
O
O
O
R
R
R
5a,b
1a,b
6a,b
a: R = H
b: R = F
N
N
Δ
N
H
CH3
N
O
N
O
N
O
R
N
7a,b
N
N
Scheme 3.
Compound 4b showed a moderate viability reduction (less than
10%) on PK15 cells, which reached statistically significance at the 25
(P < 0.01), 50 (P < 0.05) and 200 g/mL (P < 0.05) concentrations,
respectively (Fig. 2). This trend was comparable to that exhibited by
linezolid at the same concentrations.
2.4. Structureeactivity relationships
m
Structureeactivity relationship (SAR) for oxazolidinones has
been extensively reported. In the case of the 5-acetoamidomethyl
moiety, it was previously believed that this group could play a
key role on the antimicrobial activity; however, good alternatives
such as 1,2,3-triazol-2yl-methyl [28], the pyrid-2-yl-oxymethyl and
the isoxazol-3-yl-oxymethyl groups [29] were able to maintain or
enhance the antibacterial activity [27]. In this study the substitu-
tion of the 5-acetamidomethyl group with other five-membered
heterocyclic rings such as 1,2,4-triazole and 1,3-diazole led to a
complete loss of the activity against both MSSA and MRSA (see
Table 1). A loss of antibacterial activity was also observed for
the synthetic precursors where R ¼ I or N3. On the contrary, the
most active compounds reported in the present study (see Chart 2)
retain the 5-acetamidomethyl (3a and 3b) or possess the 5-
thioacetamidomethyl (4a and 4b) groups.
The reduction of cell viability caused by 4b was more pro-
nounced in the HaCaT, reaching more than 50% cell death for the
200 and 400
HepG2 cells showed a significant reduction (P < 0.01) of viability
beginning from the 50 g/mL of 4b, with a maximum of toxicity at
g/mL concentration, with a cellular decrease of about 40%
mg/mL concentrations (P < 0.01; Fig. 3).
m
the 400
(Fig. 4).
m
Linezolid demonstrated no viability reduction on PK15 cells, and
its 400 g/mL concentration paradoxically stimulated cell replica-
m
tion (P < 0.01; Fig. 2). The cell viability of HaCaT was decreased by
incubation with linezolid, with the higher effects at the 200 and
400 mg/mL concentrations (P < 0.01; Fig. 3). Interestingly, HepG2
well tolerated the incubation with linezolid, and no negative effects
on cell viability were measured (Fig. 4).
In particular, the replacement of the acetoamidomethyl group
with a thioacetoamidomethyl group led to a 8-fold decrease of the
MIC value. This finding confirms results reported by L.M. Thomasco
et al. [30] who demonstrated that the 2-aminomethyl thiadiazole
oxazolidinones with a thioamide side-chain are extraordinarily
potent. In particular, they showed that the 2-aminomethyl-1,3,4-
thiadiazolyl phenyl oxazolidinone thioamide (PNU-182347) has
Table 1
Antimicrobial activities, expressed in MIC values (mg/mL) of compounds 1e7 against
MSSA and MRSA strains; linezolid was used as reference antibiotic agent.
MIC values of <0.5 mg/mL against S. aureus UC 9213, S. pneumoniae
Compd
MIC (mg/mL)
UC 9912 and Haemophilus influenzae UC 30063. More recently, a
series of thiocarbonyl groups such as thioamide, dithiocarbamate,
thiourea, and thiocarbamate were tested as possible modifications of
the C(5) side-chain of linezolid [31] and those compounds resulted to
be in vivo more active than linezolid tested as the internal reference.
In addition to the acetoamide group, the 3-(30-fluorophenyl)-
oxazolidinone ring was also considered to play a key role for anti-
microbial activity. In particular, in all SAR studies the tendency is to
maintain the fluorine atom at the 3 position of the phenyl ring,
because for linezolid a fluorine atom at the 30 position was reported
to practically double both in vitro and in vivo activity [12,32]. In vivo
studies for linezolid also showed that the addition of a fluorine
substituent on the phenyl 3-position in the B-ring eliminated the
toxic bone marrow effects, decreased adverse reactions, and
enhanced activity [32]. Finally, the size of the 3-substituents is very
ATCC 29213 MSSA M923 MRSA MU50 MRSA 433 MRSA F511
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
>50
>50
>50
>50
12.5
12.5
3.13
1.6
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
6.25
6.25
1.6
50
50
25
50
>50
>50
1.6
1.6
1.6
0.8
>50
>50
>50
>50
>50
>50
50
>50
>50
>50
12.5
12.5
1.6
>50
>50
6.25
6.25
ꢀ0.4
ꢀ0.4
>50
>50
>50
>50
>50
>50
1.6
1.6
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
Linezolid ꢀ0.4
3.13
0.8
1.6
3.13

C.G. Fortuna et al. / European Journal of Medicinal Chemistry 65 (2013) 533e545
537
Fig. 2. Cell viability of 4a, 4b, and linezolid in PK15 cells. *P < 0.05; **P < 0.01.
important; the larger the substituent, the lower the antimicrobial
activity [33,34].
many other antibiotics [3]. A model of 23S subunit for S. aureus (see
Methods) has been made based over the 23S subunit of the D.
radiodurans 50S ribosomal subunit, in complex with linezolid [3].
Sequence alignment of D. radiodurans and S. aureus shows that the
bases directly involved in the binding site of linezolid are 100%
conserved between the two bacteria, allowing the hypothesis that
the designed oxadiazole compounds dock at the same spot of
linezolid.
In this study, a comparison of the antimicrobial activity for 3a
and 3b, and for 4a and 4b, respectively, shows that the fluorine
atom in 3 position of the phenyl ring does not play any role when
the morpholinic ring of linezolid is replaced with the 1,2,4-
oxadiazole ring. Thus, the modification of the C-ring seems to
drastically change the SAR behavior of 3a, 3b, 4a, and 4b with
respect to linezolid, although the antimicrobial activity is retained.
Both R and S stereoisomers of 3a, 3b, 4a and 4b dock super-
posing the best conformation of each compound over linezolid, as
shown in Fig. 5AeD.
3. Modeling studies
Fig. 5A shows the S-enantiomer of 4b, the most active com-
pound, in complex with S. aureus, superposed over linezolid (LZD)
in complex with D. radiodurans. After minimization, 4b perfectly
overlaps linezolid and small adjustment over S. aureus binding site
are observed. Designed drugs have an aromatic C-ring moiety, and
this difference versus linezolid C-ring confers a more rigid system
with delocalized aromaticity between C-ring and B-ring. This re-
sults, see Fig. 5B, in a more organized packing of the bases around
An in silico study of the binding mode for the tested compounds
based on the eubacterial Deinococcus radiodurans 50S ribosomal
subunit resolved at 3.5 A in complex with linezolid (pdb code:
3DLL) was performed. Considering the low resolution of the crystal,
two independent and complementary approaches were used: a
ligand-based method, driven by the X-ray linezolid conformation,
and a structure-based approach, only driven by the 50S subunit
structure.
ꢀ
C
and B-rings, A2451, C2452 and U2504 (Escherichia coli
numbering), that force a small rotation over drug vertical axis with
respect to linezolid. B-ring of the tested compounds is tightly
sandwiched between A2451 and C2452 and this forces the co-
planar C-ring moiety to interact, through hydrogen bonding
involving the N(2) of the 1,2,4 oxadiazole moiety with U2585,
whose importance in linezolid binding has been documented [3].
The stacking of the oxazolidinone ring moiety (A-ring) with U2504
observed in the crystal structure is also observed in our set of
compounds.
3.1. Ligand-based binding mode
To discuss the mechanism of action of the most active com-
pounds, docking studies have been carried out on 3a, 3b, 4a, and 4b
(Chart 2), also comparing their binding position with that of the
reference drug, linezolid.
Linezolid binds to Peptidyl Transferase Center (PTC), specifically
to the A-site domain, partially overlapping the aminoacyl moiety as
Fig. 3. Cell viability of 4a, 4b, and linezolid in HaCaT cells. *P < 0.05; **P < 0.01.

538
C.G. Fortuna et al. / European Journal of Medicinal Chemistry 65 (2013) 533e545
Fig. 4. Cell viability of 4a, 4b, and linezolid in HepG2 cells. *P < 0.05; **P < 0.01.
Designed compounds, thus, share a mostly common binding
clinical MSSA and MRSA strains used to test our drugs, thus the
models do not yet take in consideration the mutated bases of our
strains, which actually make the difference. The docking study also
suggests a higher affinity of S stereoisomers for the binding site,
which might lead to higher activity of the S compounds in the
racemic mixture. Indeed, the C(5) side-chain of R enantiomers are
predicted to clash with U2504 thus forcing a rotation of U2504 base
and altering the fundamental stacking interaction of this base with
the A-ring. A similar effect has been reported for 23S rRNA crystal
structure of Haloarcula marismortui in complex with anisomycin
[39], which shows a rotated U2504 base, although anisomycin does
not occupy the same binding site, and U2504 does not form
stacking interactions with any ring of this drug.
mode with linezolid. The active role of Mg2þ ion is still uncertain,
since Wilson et al. discussed about a putative Mg ion [3]. However,
no clear interaction with magnesium ions could apparently be
forecasted in the present case. Nevertheless, by using this ligand-
based approach, a possible interaction between 4b-S and Mg2þ
,
due to a coordination involving the thionyl moiety and the F atom,
is observed (Fig. 5B).
Resistance to linezolid in S. aureus has been associated to mu-
tations in both 23S rRNA and L3 ribosomal protein. In 23S rRNA
documented mutations are G2447T, T2500A, T2504A and G2576T
[5,35e41], which, unlike U2504, are not in direct contact with the
oxazolidinone compounds, but interact with bases involved in drug
binding. In fact, T2500 interacts with U2504, which is stacked with
the A-ring moiety; G2447 interacts with both C2452 and A2451
which, in our model of S. aureus, tightly pack the B-ring moiety;
G2576 forms stacking interaction with G2505, that interacts with
the C(5) side-chain of the oxazolidinone. In L3 ribosomal protein
A157R mutation has been reported [38]. A157 of L3, as seen for
most of the 23S rRNA mutants, does not directly interact with
linezolid but, again, interacts with both G2505 and U2506, both
involved in the binding site. Reported mutations, with the excep-
tion of U2504, seem to act as modulators for shape of the binding
site, indirectly allowing or hindering linezolid binding. Fig. 5C
shows all designed S-enantiomers of the tested compounds docked
in PTC A-site binding pocket. As shown, no important differences
are observed among these drugs, and no relevant changes in
binding are observed between the drugs here reported and line-
zolid. This is due to the absence, to date, of a characterization of the
3.2. Structure-based binding mode
FLAP structure-based approach was used to investigate the
possible binding modes of the four active compounds 3a, 3b, 4a,
and 4b, reported in Chart 2.
FLAP (Fingerprints for Ligands and Proteins) [19,42,43] is a
Virtual-Screening and Model-Development method based on 3D
molecular similarity, measured through common Molecular Inter-
action Field (MIF) volumes [44e46]. FLAP is an extremely flexible
method that enables a wide variety of applications, from ligand-
based Virtual-Screening [47,48] and 3D pharmacophore hypothe-
sis generation [49], to structure based virtual screening [50] and
high-throughput proteins pockets clusterization [51].
In this study, the 50S ribosomal subunit from D. radiodurans co-
crystallized with linezolid (PDB code: 3DLL) [3] was used as a
O
O
H3C
N
H3C
N
O
N
O
N
N
N
N
H
N
H
N
O
O
O
O
S
H
H
F
F
H3C
H3C
3a
3b
O
O
H3C
H3C
N
O
N
O
O
O
N
N
N
H
N
H
N
S
H3C
H3C
4a
4b
Chart 2.

C.G. Fortuna et al. / European Journal of Medicinal Chemistry 65 (2013) 533e545
539
Fig. 5. A) Superimposition of crystal structure of D. radiodurans (yellow) 23S subunit binding site in complex with linezolid (ZLD, in yellow sticks) and S. aureus model (light blue) in
complex with 4b-S (red, in sticks). A small rotation over 4b-S axis is observed with respect to ZLD. B) S. aureus in complex with 4b-S, the small rotation observed over ZLD favors a
“sandwich-like” stacking of B-ring with A2451 and C2452. As a result, N-(2) of the 1,2,4-oxadiazole directly hydrogen bonds U2585, a key interaction for linezolid activity. C)
Superimposition of S-stereoisomers of 3a, 3b, 4a and 4b, all sharing a similar binding mode. D) Difference among S (red, in transparency) and R (gray) stereoisomers of 4b. R
compound might clash with the key base U2504, involved in stacking with the oxazolidinone moiety of linezolid-like compounds. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
target. A few X-ray structures of the linezolid-binding site in the
50S subunit have been published so far, and Wilson et al. [3]
recently demonstrated that no significant conformational change
in the RNA forming binding pocket can be observed upon binding
of linezolid, with an exception for U2585. Thus, our target was
used as a rigid conformation, having the U2585 in the interactive
orientation.
A scheme of the computational procedure is reported in
Fig. 6AeC. The X-ray structure of linezolid was removed from the
3DLL structure, and the GRID [46] molecular interaction fields were
computed in the cavity.
As expected, the ribosomal cavity is highly polar (blue and red
fields), due to several RNA bases able to donate and/or accept
Hydrogen-bonds. In the FLAP docking approach, the possible
Fig. 6. FLAP structure-based approach. From the crystallographic structure (A), the structure of the co-crystallized linezolid is removed (B). Afterward, the GRID molecular
interaction fields were computed in the cavity (C). (Green fields: hydrophobic interaction; blue fields: H-bond donor interaction; red fields: H-bond acceptor interaction). (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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C.G. Fortuna et al. / European Journal of Medicinal Chemistry 65 (2013) 533e545
binding modes of the compounds in the cavity are automatically
identified without using the coordinates of the co-crystallized
ligand. In fact, starting from a 2D structure of linezolid, FLAP was
able to generate a pose possessing high similarity with the cavity
interaction fields, and being well superposed to the X-ray structure
of linezolid (see Fig. 7).
The same procedure used for linezolid was then repeated for
each compound 3a, 3b, 4a, and 4b with the intent to find their
docked conformation and 3D-protein interactions.
Since the measured antimicrobial activity (Table 1) was referred
to a racemic mixture, both enantiomers of each structure were
included in the dataset. In the structure-based approach, the FLAP
software generates the GRID molecular interaction fields (MIFs)
both for the ribosomal cavity and for each compound in the dataset,
and then it evaluates the similarity between the MIFs of the 50S
subunit and those of the compounds. Thus, FLAP automatically
selects the conformation and the enantiomeric form for each
compound that best fits the cavity. Details for the FLAP procedure
are reported in the Experimental section.
A2451 is less effective (green region facing this residue). The overall
hydrophobic interaction computed by GRID is really weak, and the
greatest effect is estimated at only ꢁ1.30 kcal/mol, involving the A
ring. About the side-chain, the thionyl group is oriented toward a
hydroxyl group of the ribose in A2503, and the sulfurehydroxy
hydrogen bond interaction energy computed by GRID is close
to ꢁ2.0 kcal/mol. The A2503 is not generally ascribed among the
critical residues for linezolid binding. Nevertheless, the modification
made in the C-ring could favor a possible interaction with A2503.
Compounds 3a, 4a, and 3b displayed an analog orientation into the
binding site, having the methyl-1,2,4-oxadiazole pointing toward
U2585, indicating that neither the lack of the fluorine atom nor the
use of an amide groups modify orientation in the cavity (data not
shown).
In order to verify whether the activity of the tested compounds
was related to the linezolid-like binding mode, the procedure was
repeated for the inactive compound 5b, in which the side-chain of
linezolid was replaced by an pyrazol-1-yl moiety (Fig. 8C and D). The
orientation of 5b is similar to the linezolid X-ray pose and to the
computed poses for the tested active compounds, having the 1,2,4
oxadiazole interacting with U2585. This is not surprising consid-
ering that all the synthesized compounds mimic the linezolid shape.
Nevertheless, the putative interaction with A2503 proposed for the
four active compounds is not yet possible not only for 5b (Fig. 8C and
D), but also for all the other inactive compounds in our series. The
possible relevance of A2503 in the binding of our linezolid-like
1,2,4-oxadiazole compounds is currently under investigation.
In this study, for all the compounds FLAP automatically selected
the S-enantiomer as the most likely one interacting with the target,
according to the ligand-based approach.
Fig. 8A and B shows the best pose for the most active compound
4b against all the MSSA and MSRA strains.
The pose of compound 4b suggested by FLAP is very similar to that
of the crystallographic linezolid (Fig. 8A). The morpholinic ring is
replaced by the N(2) of the 1,2,4-oxadiazole in its H-bond interaction
with the nitrogen of U2585, in agreement with the ligand-based re-
sults and the crystallographic evidences [3]. Moreover, the methyl
group in the 1,2,4-oxadiazole is perfectly oriented toward a hydro-
phobic region (green color). In this pose the A-ring is differently
oriented, and the putative interaction between the Fluorine substit-
uent and the Mg2þ ion proposed by Wilson [3] seems not to play a
role in this binding mode. The electrostatic interaction of the sulfur
atom with the magnesium ion is weak and estimated at
about ꢁ0.5 kcal using GRID [46]. Looking at the GRID fields (Fig. 8B),
due to the different orientation of the A-ring, the stacking with the
4. Conclusions
Replacement of the linezolid morpholine C-ring with 1,2,4-
oxadiazole and the substitution of the carbonyl group with a thi-
onyl one in the side-chain results in the maintenance of the anti-
bacterial activity against S. aureus resistant clinical isolates, with a
better activity demonstrated against MRSA strains for compound 4b
and a similar cell viability as compared to linezolid. A comparison of
the possible binding modes for the tested compounds obtained by
two complementary (ligand-based and structure-based) ap-
proaches suggests that the binding mode of the tested compounds is
very similar to that reported for the ribosomeelinezolid complex
crystal structure. In particular, the 1,2,4-oxadiazole moiety seems to
perfectly mimic the function of the morpholinic ring, since the H-
bond interaction with U2585 is retained. This interaction was
observed in silico for all the tested compounds, despite their activity.
However, the performed activity experiments have suggested that
the modifications made at the side-chain level could be responsible
for the activity. An in silico study of the possible interactions of the
side chain in the target cavity identified a possible weak interaction
with A2503 for the active compounds 3a, 3b, 4a, and 4b. For all the
tested inactive compounds such interaction cannot occur. Thus the
possible role of A2503 is currently under investigation.
Fluorination of the phenyl B-ring, reported to improve the anti-
bacterial activity of oxazolidinones with morpholine or piperazine C-
ring [11], exhibits a slighter effect in the presence of 1,2,4-oxadiazole
as C-ring. Indeed, an effect can be noticed comparing only 4a and 4b,
thus with a thioacetoamidomethyl moietyas the C(5) side-chain, and
only for some strains. Moreover, acetamidomethyl or thio-
acetoamidomethyl moieties in the C(5) side-chain are essential for
activity, likely due to their interactions with the H-bond acceptor
region of the ribosomal pocket. On the basis of these experimental
evidences and of the modeling information, we hypothesize that the
interaction between the F atom and the Mg2þ ions for the tested
compounds is less important for activity, while a possible interaction
of the thionyl group is proposed. However, cell viability studies
performed with 4a and 4b on three different cell lines of excretory
Fig. 7. Comparison of the crystallographic linezolid (in brown) and the FLAP pose (in
green). (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)

C.G. Fortuna et al. / European Journal of Medicinal Chemistry 65 (2013) 533e545
541
Fig. 8. FLAP poses for 4b and 5b. A) Comparison of the X-ray pose for linezolid (brown) and the FLAP pose for 4b (gray); B) FLAP pose for 4b according to the GRID fields (green field:
hydrophobic interaction; blue fields: H-bond donor interaction; red fields: H-bond acceptor interaction); C) comparison of the X-ray pose for linezolid (brown) and the FLAP pose
for 4b (light blue); D) FLAP pose for 5b according to the GRID fields. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)
organs demonstrated that the presence of fluorine atom in the
phenyl B ring (in 4b), compared to 4a in which it is absent, decreases
the cytotoxicity to levels similar to those of linezolid. Other modifi-
cations seem not to influence the cytotoxicity. Finally, compounds 4a
and 4b displayed a greater activity with respect to the analogs 3a and
3b. It is well-known that thioamide substitutions can improve sta-
bility in proteolitic degradation and can also improve ADME prop-
erties with respect to the corresponding amide compounds [52].
Thus, whereas thioamides have been considered as an isosteric
replacement of amides, the structural differences (i.e., the larger
sulfur atom, the elongated C]S bond), the different propensity for
hydrogen bond formation and the fact that thioamides are more
difficult to hydrolyze than the corresponding amides appear to in-
crease the stability of thioamides in biological systems. The inactive
compounds displayed in silico a similar binding mode; however, only
the interaction with U2585 seems to occur.
determined with a Shimadzu FTIR-8300 instrument; 1H NMR
spectra were recorded on a Bruker 300 Avance spectrometer using
TMS as an internal standard. GCeMS determinations were carried
out on a Shimadzu GCMS-QP2010 system. Flash chromatography
was performed by using silica gel (0.040e0.063 mm) and mixtures
of ethyl acetate and petroleum ether (fraction boiling in the range
of 40e60 ꢂC) in various ratios. The purity of compounds, in all cases
higher than 95%, has been checked by both NMR and HPLC ana-
lyses. Elemental analysis (C, H, N) was performed through a vario EL
III Element Analyzer, by Elementar Analysensysteme gmbh Hanau,
Germany.
5.1.2. General procedure for the preparation of compounds 10a,b
A solution of hydroxylamine hydrochloride (1.00 g, 14.4 mmol)
and NaOH (0.57 g, 14.4 mmol) in water (5 mL) was added (in about
15 min) to 15 mL of CH3CN. The reaction mixture was stirred at room
temperature for 24 h. The solvent was removed under reduced
pressure and the residue treated with ethanol; the resulting sus-
pension was filtered and the solvent was removed under reduced
pressure producing 1.659 g of acetamidoxime 9 (77%). Then, either
4-fluorobenzoyl (8a) chloride or 2,4-difluorobenzoyl chloride (8b)
(14.8 mmol) were added to a solution of 9 (1.00 g; 13.5 mmol) in
Acetone (35 mL) containing also K2CO3 (2.05 g, 14.8 mmol). The
mixture was stirred at room temperature for about 90 min after
5. Experimental section
5.1. Synthesis
5.1.1. Materials and methods
Melting points were determined on a Reichert-Thermovar hot-
stage apparatus and are uncorrected. IR spectra (Nujol) were

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C.G. Fortuna et al. / European Journal of Medicinal Chemistry 65 (2013) 533e545
which the solvent was removed under reduced pressure. The res-
idue was treated with water and the solid precipitate was collected
by filtration. The obtained O-acylamidoxime was heated, without
any further purification, at about 130 ꢂC for 90 min in a sealed tube.
The obtained residue was chromatographed yielding the corre-
sponding 1,2,4-oxadiazoles 10a and 10b.
2H, J ¼ 9.0 Hz, Ar); 7.84 (d, 2H, J ¼ 9.0 Hz, Ar). Anal. Found (calc) for
17H21N3O3 (%): C, 64.70 (64.74); H, 6.80 (6.71); N, 13.35 (13.32).
C
5.1.5.2. tert-Butyl-N-allyl-(3-fluoro-4-(30-methyl-1,2,4-oxadiazol-50-
yl)-phenyl)-carbamate (12b). oil; Yield (72%); IR (Nujol) 1713
(NCO2), 1615 (C]N) cmꢁ1; 1H NMR (300 MHz; CDCl3)
d 1.53 (s, 9H,
t-Bu); 2.53 (s, 3H, Me); 4.36 (d, 2H, J ¼ 5.1 Hz, CH2); 5.21e5.28 (m,
2H, eCH]CH2); 5.91e6.02 (m, 1H, eCH]CH2); 7.28e7.36 (m, 2H,
Ar); 8.02e8.08 (m, 1H, Ar). Anal. Found (calc) for C17H20FN3O3 (%):
C, 61.25 (61.25); H, 6.10 (6.05); N, 12.65 (12.61).
5.1.2.1. 3-Methyl-5-(40-fluorophenyl)-1,2,4-oxadiazole
(72%); mp 80.0e81.0 ꢂC; 1H NMR (300 MHz; CDCl3)
7.16e7.23 (m, 2H, Ar); 8.08e8.14 (m, 2H, Ar). Anal. Found (calc) for
(10a). Yield
2.45 (s, 3H, Me);
d
C9H7FN2O (%): C, 60.65 (60.67); H, 3.90 (3.96); N, 15.70 (15.72).
5.1.6. General procedure for the preparation of compounds 1a,b
To a solution of 1.70 mmol of either compound 12a or 12b in
CH2Cl2 (10 mL) was added I2 sublimate (1.29 g; 5.10 mmol). The
solution was stirred for 24 h, after which the reaction was treated
with a solution of Na2SO3; the organic layer was dried over anhy-
drous Na2SO4, filtered and the solvent removed. The residue was
chromatographed yielding the corresponding compounds 1a
and 1b.
5.1.2.2. 3-Methyl-5-(20,40-difluorophenyl)-1,2,4-oxadiazole
Yield (72%); mp 57.0e60.0 ꢂC; 1H NMR (300 MHz; CDCl3)
3H, Me); 6.95e7.07 (m, 2H, Ar); 8.04e8.14 (m, 1H, Ar). Anal. Found
(calc) for C9H6F2N2O (%): C, 55.15 (55.11); H, 3.10 (3.08); N, 14.25
(14.28).
(10b).
2.46 (s,
d
5.1.3. Preparation of N-allyl-4-(30-methyl-1,2,4-oxadiazol-50-yl)-
aniline (11a)
Compound 10a (0.61 g; 3.43 mmol) was heated, with allylamine
(3.0 mL; 2.28 g; 40.0 mmol) and K2CO3 (2.00 g; 14.5 mmol), at about
60 ꢂC for 8 days. The reaction mixture was treated with water and
extracted with EtOAc. The organic layers were collected, dried over
anhydrous Na2SO4, filtered and the solvent removed. The residue
was chromatographed yielding compound 11a: Yield (54%); mp
5.1.6.1. 3-(40-(300-Methyl-1,2,4-oxadiazol-500-yl)-phenyl)-5-(iodo-
methyl)-oxazolidin-2-one (1a). Yield (89%); mp 145.0e147.0 ꢂC; IR
(Nujol) 1763 (NCO2), 1618 (C]N) cmꢁ1; 1H NMR (300 MHz; DMSO-
d6)
d 2.47 (s, 3H, Me); 3.62e3.73 (m, 2H, CH2eI); 3.80 (dd, 1H,
J1 ¼ 9.3 Hz, J2 ¼ 6.0 Hz, C4eH); 4.34 (dd, 1H, J1 ¼ 9.3 Hz, J2 ¼ 9.0 Hz,
C4eH); 4.81e4.90 (m, 1H, C5eH); 7.88 (d, 2H, J ¼ 9.0 Hz, Ar); 8.17 (d,
2H, J ¼ 9.0 Hz, Ar). Anal. Found (calc) for C13H12IN3O3 (%): C, 40.55
(40.54); H, 3.15 (3.14); N, 10.85 (10.91).
63.9e65.5 ꢂC; IR (Nujol) 3335 (NH), 1607 (C]N) cmꢁ1
;
1H NMR
(300 MHz; DMSO-d6) 2.31 (s, 3H, Me); 3.76e3.79 (m, 2H, CH2);
d
5.12 (dd, 1H, J1 ¼ 10.5 Hz, J2 ¼ 1.8 Hz, eCH]CH2); 5.22 (dd, 1H,
J1 ¼17.1 Hz, J2 ¼ 1.8 Hz, eCH]CH2); 5.82e5.93 (m, 1H, eCH]CH2);
6.68 (d, 2H, J ¼ 9.0 Hz, Ar); 6.87 (t, 1H, J ¼ 5.7 Hz, NH, exch. with
D2O); 7.76 (d, 2H, J ¼ 9.0 Hz, Ar). Anal. Found (calc) for C12H13N3O
(%): C, 66.95 (66.96); H, 6.10 (6.09); N, 19.45 (19.52).
5.1.6.2. 3-(30-Fluoro-40-(300-methyl-1,2,4-oxadiazol-500-yl)-phenyl)-5-
(iodomethyl)-oxazolidin-2-one (1b). Yield (76%); mp 148.0e
149.0 ꢂC; IR (Nujol) 1743 (NCO2), 1637 (C]N) cmꢁ1 1H NMR
;
(300 MHz; DMSO-d6) d 2.48 (s, 3H, Me); 3.61e3.72 (m, 2H, CH2eI);
3.81 (dd, 1H, J1 ¼ 9.6 Hz, J2 ¼ 6.0 Hz, C4eH); 4.33 (dd, 1H, J1 ¼ 9.6 Hz,
J2 ¼ 9.0 Hz, C4eH); 4.83e4.93 (m, 1H, C5eH); 7.68 (dd, 1H,
J1 ¼8.7 Hz, J2 ¼ 2.1 Hz, Ar); 7.80 (dd,1H, J1 ¼13.8 Hz, J2 ¼ 2.1 Hz, Ar);
8.16 (dd, 1H, J1 ¼ 8.7 Hz, J2 ¼ 8.5 Hz, Ar). Anal. Found (calc) for
5.1.4. Preparation of N-allyl-3-fluoro-4-(30-methyl-1,2,4-oxadiazol-
50-yl)-aniline (11b)
To a solution of 10b (0.86 g; 4.38 mmol) in DMF (2.0 mL) was
added allylamine (1.64 mL; 1.25 g; 22.0 mmol). The reaction mixture
was stirred for 2 days, after which the solution was treated with
water and extracted with EtOAc. The organic layers were collected,
dried over anhydrous Na2SO4, filtered and the solvent removed. The
residue was chromatographed yielding compound 11b: Yield (49%);
mp 57.9e59.9 ꢂC; IR (Nujol) 3335 (NH), 1626 (C]N) cmꢁ1; 1H NMR
C
13H11FIN3O3 (%): C, 38.75 (38.73); H, 2.55 (2.75); N, 10.35 (10.42).
5.1.7. General procedure for the preparation of compounds 2a,b
To a solution of 0.75 mmol of compound 1a or 1b in DMF (6 mL)
was added NaN3 (0.39 g; 6.00 mmol). The solution was stirred for
24 h, after which the reaction was treated with water and extracted
with EtOAc; the organic layers were dried over anhydrous Na2SO4,
filtered and the solvent removed. The residue was chromato-
graphed yielding the corresponding compounds 2a and 2b.
(300 MHz; DMSO-d6)
d 2.34 (s, 3H, Me); 3.77e3.81 (m, 2H, CH2);
5.13 (dd, 1H, J1 ¼ 13.2 Hz, J2 ¼ 1.2 Hz, eCH]CH2); 5.23 (dd, 1H,
J1 ¼17.4 Hz, J2 ¼ 1.2 Hz, eCH]CH2); 5.81e5.93 (m, 1H, eCH]CH2);
6.46 (dd, 1H, J1 ¼ 14.4 Hz, J2 ¼ 1.8 Hz, Ar); 6.56 (dd, 1H, J1 ¼ 8.7 Hz,
J2 ¼1.8 Hz, Ar); 7.17e7.21 (bs,1H, NH, exch. with D2O); 7.72e7.77 (m,
1H, Ar). Anal. Found (calc) for C12H12FN3O (%): C, 61.80 (61.79); H,
5.10 (5.19); N, 18.15 (18.02).
5.1.7.1. 3-(40-(300-Methyl-1,2,4-oxadiazol-5-yl)-phenyl)-5-(azidome-
til)-oxazolidin-2-one (2a). Yield (94%); mp 133.9e135.0 ꢂC; IR
(Nujol) 2095 (N3), 1765 (NCO2), 1727 (NCO2), 1618 (C]N) cmꢁ1; 1H
NMR (300 MHz; DMSO-d6)
d 2.46 (s, 3H, Me); 3.75e3.88 (m, 2H,
5.1.5. General procedure for the preparation of compounds 12a,b
Either compound 11a or 11b (2.15 mmol) were dissolved in
CH3CN (25 mL); di-(t-butyl)-dicarbonate (0.51 g; 2.36 mmol) and 4-
dimethylaminopyridine (0.29 g; 2.36 mmol) were added and the
mixture was stirred for 2 days or 2.5 h, respectively. The solvent was
removed under reduced pressure and the obtained residue was
chromatographed yielding the corresponding compounds 12a
and 12b.
CH2eN3); 3.92 (dd, 1H, J1 ¼ 9.3 Hz, J2 ¼ 6.0 Hz, C4eH); 4.28 (t, 1H,
J ¼ 9.3 Hz, C4eH); 4.96e5.03 (m, 1H, C5eH); 7.86 (d, 2H, J ¼ 9.0 Hz,
Ar); 8.16 (d, 2H, J ¼ 9.0 Hz, Ar). Anal. Found (calc) for C13H12N6O3
(%): C, 52.05 (52.00); H, 4.10 (4.03); N, 27.85 (27.99).
5.1.7.2. 3-(30-Fluoro-40-(300-methyl-1,2,4-oxadiazol-5-yl)-phenyl)-5-
(azidometil)-oxazolidin-2-one (2b). Yield (99%); mp 126.2e127.7 ꢂC;
IR (Nujol) 2107 (N3), 1758 (NCO2), 1743 (NCO2), 1630 (C]N) cmꢁ1
;
1H NMR (300 MHz; DMSO-d6)
d 2.41 (s, 3H, Me); 3.69e3.82 (m, 2H,
5.1.5.1. tert-Butyl-N-allyl-(4-(30-methyl-1,2,4-oxadiazol-50-yl)-
CH2eN3); 3.86 (dd, 1H, J1 ¼ 9.3 Hz, J2 ¼ 6.0 Hz, C4eH); 4.21 (t, 1H,
J ¼ 9.3 Hz, C4eH); 4.91e4.99 (m, 1H, C5eH); 7.60 (dd, 1H, J1 ¼ 9.0 Hz,
J2 ¼ 1.8 Hz, Ar); 7.72 (dd, 1H, J1 ¼ 13.5 Hz, J2 ¼ 1.8 Hz, Ar); 8.08e8.14
(m, 1H, Ar). Anal. Found (calc) for C13H11FN6O3 (%): C, 49.10 (49.06);
H, 3.50 (3.48); N, 26.45 (26.41).
phenyl)-carbamate (12a). Oil; Yield (73%); IR (Nujol) 1711 (NCO2),
1614 (C]N) cmꢁ1; 1H NMR (300 MHz; CDCl3)
d 1.27 (s, 9H, t-Bu); 2.25
(s, 3H, Me); 4.10 (d, 2H, J ¼ 5.1 Hz, CH2);4.95e4.97 (m,1H, eCH]CH2);
4.99e5.01 (m,1H, eCH]CH2); 5.67e5.78 (m,1H, eCH]CH2); 7.23 (d,

C.G. Fortuna et al. / European Journal of Medicinal Chemistry 65 (2013) 533e545
543
5.1.8. General procedure for the preparation of compounds 13a,b
To a solution of 0.45 mmol of compound 2a or 2b in THF (15 mL)
was added PPh3 (0.16 g; 0.60 mmol). The solution was stirred for
(d, JCeF ¼ 14 Hz), 114.1, 131.4, 144.3 (d, JCeF ¼ 14 Hz), 153.9, 160.4 (d,
JCeF 305 Hz), 167.5, 170.2, 171.6. Anal. Found (calc) for
15H15FN4O4 (%): C, 53.90 (53.89); H, 4.65 (4.52); N, 16.65 (16.76).
¼
C
about 90 min, after which 100 ml of distilled water was added and
the resulting mixture was refluxed for 4 h. The THF was removed
under reduced pressure, the resulting residue was neutralized with
hydrochloric acid and extracted with EtOAc. A solution of NaOH (pH
w9) was added to the aqueous phase, which was extracted with
EtOAc; the organic layers were dried over anhydrous Na2SO4,
filtered and the solvent removed, yielding the corresponding
compounds 13a and 13b.
5.1.10. General procedure for the preparation of compounds 4a,b
The Lawesson’s reagent (0.2 g; 0.49 mmol) was added to a so-
lution of either 3a or 3b (0.49 mmol) in THF (14 mL). The reaction
mixture was refluxed for 2 h, after which the solvent was removed
under reduced pressure. The residue was chromatographed
yielding the corresponding compounds 4a and 4b.
5.1.10.1. 3-(40-(300-Methyl-1,2,4-oxadiazol-5-yl)-phenyl)-5-(N-thio-
5.1.8.1. 3-(40-(300-Methyl-1,2,4-oxadiazol-5-yl)-phenyl)-5-(amino-
acetylaminomethyl)-oxazolidin-2-one (4a). Yield (77%); mp 199.ꢁ41e
methyl)-oxazolidin-2-one (13a). Yield (66%); mp 139.3e141.3 ꢂC; IR
201.0 ꢂC; IR (Nujol) 3217 (NH), 1721 (NCO2), 1618 (thioamide) cm
;
(Nujol) 3390 (NH), 3361 (NH), 1748 (NCO2), 1616 (C]N) cmꢁ1
;
1H
1H NMR (300 MHz; DMSO-d6)
d 2.47 (s, 3H, Me); 2.51 (s, 3H, CSMe);
NMR (300 MHz; DMSO-d6)
d
2.22 (bs, 2H, NH2, exch. with D2O);
3.95e4.03 (m, 3H, overlapped signals); 4.28e4.34 (m, 1H, C4eH);
5.01e5.11 (m, 1H, C5eH); 7.85 (d, 2H, J ¼ 9.0 Hz, Ar); 8.18 (d, 2H,
J ¼ 9.0 Hz, Ar); 10.45 (bs,1H, NH, exch. with D2O). Anal. Found (calc)
for C15H16N4O3S (%): C, 54.15 (54.20); H, 4.85 (4.85); N, 16.90
(16.86).
2.39 (s, 3H, Me); 2.77e2.91 (m, 2H, CH2eNH2); 3.94 (dd, 1H,
J1 ¼ 9.0 Hz, J2 ¼ 6.3 Hz, C4eH); 4.13 (t, 1H, J ¼ 9.0 Hz, C4eH); 4.61e
4.70 (m, 1H, C5eH); 7.80 (d, 2H, J ¼ 9.0 Hz, Ar); 8.09 (d, 2H,
J ¼ 9.0 Hz, Ar). Anal. Found (calc) for C13H14N4O3 (%): C, 56.90
(56.93); H, 5.15 (5.14); N, 20.45 (20.43).
5.1.10.2. 3-(30-Fluoro-40-(300-methyl-1,2,4-oxadiazol-5-yl)-phenyl)-5-
(N-thioacetylaminomethyl)-oxazolidin-2-one (4b). Yield (93%); mp
166.5e167.7 ꢂC; IR (Nujol) 3262 (NH), 1746 (NCO2), 1633 (thio-
5.1.8.2. 3-(30-Fluoro-40-(300-methyl-1,2,4-oxadiazol-5-yl)-phenyl)-5-
(aminomethyl)-oxazolidin-2-one (13b). Yield (88%); mp 137.0e
140.0 ꢂC; IR (Nujol) 3372 (NH), 1743 (NCO2), 1630 (C]N) cmꢁ1; 1H
amide) cmꢁ1; 1H NMR (300 MHz; DMSO-d6)
d 2.48 (s, 3H, Me); 2.51
NMR (300 MHz; DMSO-d6)
d
2.21 (bs, 2H, NH2, exch. with D2O);
(s, 3H, CSMe); 3.94e4.00 (m, 3H, overlapped signals); 4.28e4.34
(m, 1H, C4eH); 5.04e5.12 (m, 1H, C5eH); 7.65 (dd, 1H, J1 ¼ 9 Hz,
J2 ¼ 1.8 Hz, Ar); 7.78 (dd, 1H, J1 ¼13.5 Hz, J2 ¼ 1.8 Hz, Ar); 8.16e8.22
(m, 1H, Ar); 10.45 (bs, 1H, NH exch. with D2O). Anal. Found (calc) for
2.41 (s, 3H, Me); 2.77e2.91 (m, 2H, CH2eNH2); 3.93 (dd, 1H,
J1 ¼ 9.3 Hz, J2 ¼ 6.3 Hz, C4eH); 4.13 (t, 1H, J ¼ 9.0 Hz, C4eH); 4.63e
4.71 (m, 1H, C5eH); 7.60 (dd, 1H, J1 ¼ 9.0 Hz, J2 ¼ 2.1 Hz, Ar); 7.73
(dd, 1H, J1 ¼ 10.8 Hz, J2 ¼ 2.1 Hz, Ar); 8.08e8.14 (m, 1H, Ar). Anal.
Found (calc) for C13H13FN4O3 (%): C, 53.40 (53.42); H, 4.45 (4.48); N,
19.25 (19.17).
C15H14FN4O3S (%): C, 51.35 (51.42); H, 4.30 (4.32); N, 16.05 (15.99).
5.1.11. General procedure for the preparation of compounds 5a,b
and 6a,b
5.1.9. General procedure for the preparation of compounds 3a,b
Either 1a or 1b (0.26 mmol) was heated in presence of pyrazole
or imidazole (0.18 g; 2.6 mmol) for 30 min. The mixture was
recovered with EtOAc and the solvent removed under reduced
pressure. The residue was chromatographed yielding the corre-
sponding compounds 5a,b and 6a,b.
Acetyl chloride (40 ml; 44 mg; 0.56 mmol) was added to a so-
lution of either compound 13a or 13b (0.28 mmol) in CH2Cl2 (3 mL)
containing also pyridine (1 mL; 0.97 g; 12.3 mmol). The solution
was stirred for 30 min after which the solvent was removed and the
residue treated with HCl 1 M (20 mL) and extracted with EtOAc; the
organic layers were dried over anhydrous Na2SO4, filtered and the
solvent removed. The residue was chromatographed yielding the
corresponding compounds 3a and 3b.
5.1.11.1. 3-(40-(300-Methyl-1,2,4-oxadiazol-5-yl)-phenyl)-5-((pyrazol-
1-yl)-methyl)-oxazolidin-2-one (5a). Yield (56%); mp 192.2e
194.5 ꢂC; IR (Nujol) 1745 (NCO2), 1618 (C]N) cmꢁ1 1H NMR
;
(300 MHz; CDCl3)
d 2.53 (s, 3H, Me); 4.16e4.30 (m, 2H, C4eH);
5.1.9.1. 3-(40-(300-Methyl-1,2,4-oxadiazol-5-yl)-phenyl)-5-(N-acetyla-
4.60e4.62 (m, 2H, CH2epyrazole); 5.08e5.16 (m, 1H, C5eH); 6.36e
6.38 (m, 1H, Pyrazole C3eH); 7.55 (d, 1H, J ¼ 1.5 Hz, Pyrazole C4eH);
7.64 (d, 1H, J ¼ 1.5 Hz, Pyrazole C5eH); 7.70 (d, 2H, J ¼ 9.6 Hz, Ar),
8.15 (d, 2H, J ¼ 9.6 Hz, Ar). Anal. Found (calc) for C16H15N5O3 (%): C,
59.10 (59.07); H, 4.70 (4.65); N, 21.45 (21.53).
minomethyl)-oxazolidin-2-one (3a). Yield (58%); mp 214.0e216.0 ꢂC;
IR (Nujol) 3257 (NH), 1751 (NCO2), 1646 (amide), 1616 (C]N) cmꢁ1
;
1H NMR (300 MHz; DMSO-d6)
d 1.89 (s, 3H, COMe); 2.46 (s, 3H, Me);
3.50 (t, 2H, J ¼ 5.7 Hz, CH2eNHCOMe); 3.88 (dd, 1H, J1 ¼ 9.0 Hz,
J2 ¼ 6.6 Hz, C4eH); 4.25 (t, 1H, J ¼ 9.0 Hz, C4eH); 4.79e4.87 (m, 1H,
C5eH); 7.84 (d, 2H, J ¼ 8.7 Hz, Ar); 8.16 (d, 2H, J ¼ 8.7 Hz, Ar); 8.32 (t,
1H, J ¼ 5.7 Hz, NH, exch. with D2O); 13C NMR (75 MHz; DMSO-d6)
5.1.11.2. 3-(30-Fluoro-40-(300-methyl-1,2,4-oxadiazol-5-yl)-phenyl)-5-
((pyrazol-1-yl)-methyl)-oxazolidin-2-one (5b). Yield (40%); mp
d
11.4, 22.6, 41.5, 47.2, 72.0, 118.1 (overlapped signals), 128.9, 142.6,
179.2e181.0 ꢂC; IR (Nujol) 1745 (NCO2) cmꢁ1 1H NMR (300 MHz;
;
154.1,167.7,170.2,174.5. Anal. Found (calc) for C15H16N4O4 (%): C, 56.95
(56.96); H, 5.05 (5.10); N, 17.85 (17.71).
CDCl3) d 2.55 (s, 3H, Me); 4.19e4.22 (m, 2H, C4eH); 4.60e4.62 (m,
2H, CH2epyrazole); 5.09e5.17 (m, 1H, C5eH); 6.36e6.37 (m, 1H,
Pyrazole C3eH); 7.35e7.38 (m, 1H, Ar); 7.54 (d, 1H, J ¼ 1.8 Hz,
Pyrazole C4eH); 7.59e7.64 (m, 2H, overlapped signals), 8.08e8.14
(m, 1H, Ar). Anal. Found (calc) for C16H14FN5O3 (%): C, 56.00 (55.98);
H, 4.10 (4.11); N, 20.35 (20.40).
5.1.9.2. 3-(30-Fluoro-40-(300-methyl-1,2,4-oxadiazol-5-yl)-phenyl)-5-
(N-acetylaminomethyl)-oxazolidin-2-one (3b). Yield (62%); mp
184.0e186.0 ꢂC; IR (Nujol) 3343 (NH), 1751 (NCO2), 1666 (amide),
1628 (C]N) cmꢁ1 1H NMR (300 MHz; DMSO-d6)
; d 1.89 (s, 3H,
COMe); 2.48 (s, 3H, Me); 3.50 (t, 2H, J ¼ 5.4 Hz, CH2eNHCOMe);
3.88 (dd, 1H, J1 ¼ 9.3 Hz, J2 ¼ 6.3 Hz, C4eH); 4.25 (t, 1H, J ¼ 9.0 Hz,
C4eH); 4.81e4.88 (m, 1H, C5eH); 7.64 (dd, 1H, J1 ¼ 9.0 Hz,
J2 ¼ 1.8 Hz, Ar); 7.77 (dd, 1H, J1 ¼13.8 Hz, J2 ¼ 1.8 Hz, Ar); 8.15e8.21
(m, 1H, Ar), 8.31 (m, 1H, NH, exch. with D2O); 13C NMR (75 MHz;
5.1.11.3. 3-(40-(300-Methyl-1,2,4-oxadiazol-5-yl)-phenyl)-5-((imida-
zol-1-yl)-methyl)-oxazolidin-2-one (6a). Yield (72%); mp 206.0e
209.0 ꢂC; IR (Nujol) 1747 (NCO2) cmꢁ1; 1H NMR (300 MHz; DMSO-
d6)
d
2.46 (s, 3H); 3.95 (dd, 1H, J1 ¼ 9.3 Hz, J2 ¼ 6.0 Hz, C4eH); 4.28e
4.34 (t, 1H, J ¼ 9.3 Hz, C4eH); 4.47e4.49 (m, 2H, CH2eimidazole);
DMSO-d6)
d
11.32, 22.6, 41.5, 47.3, 72.2, 105.7 (d, JCeF ¼ 32 Hz), 106.2
5.06e5.14 (m, 1H, C5eH); 6.98 (s, 1H, Imidazole C5eH); 7.30 (s, 1H,

544
C.G. Fortuna et al. / European Journal of Medicinal Chemistry 65 (2013) 533e545
Imidazole C4eH); 7.76 (s, 1H, Imidazole C2eH); 7.81 (d, 2H
J ¼ 8.7 Hz, Ar); 8.15 (d, 2H, J ¼ 8.7 Hz, Ar). Anal. Found (calc) for
plate was then incubated at 37 ꢂC for 18e24 h after which each well
was analyzed for the presence of bacterial growth. The MIC was
defined as the lowest concentration of antimicrobial agent able to
cause inhibition of bacterial growth as shown by the lack of
turbidity of the culture medium. The in vitro antibacterial activities
of 14 new linezolid-like 1,2,4-oxadiazoles were tested and
compared to that of reference oxazolidinone in clinical use: line-
zolid (Zyvox(R), Pfizer in Sigma-Aldrich, Italy). Final DMSO con-
centrations were also taken into account in all the biological assays.
C
16H15N5O3 (%): C, 59.10 (59.07); H, 4.65 (4.65); N, 21.55 (21.53).
5.1.11.4. 3-(30-Fluoro-40-(300-methyl-1,2,4-oxadiazol-5-yl)-phenyl)-5-
((imidazol-1-yl)-methyl)-oxazolidin-2-one (ꢁ61b). Yield (55%); mp
185.6e188.9 ꢂC; IR (Nujol) 1748 (NCO2) cm
;
1H NMR (300 MHz;
DMSO-d6)
d
2.48 (s, 3H, Me); 3.96 (dd, 1H, J1 ¼ 9.3 Hz, J2 ¼ 6.0 Hz,
C4eH); 4.28e4.34 (m, 1H, J ¼ 9.3 Hz, C4eH); 4.47e4.48 (m, 2H,
CH2eimidazole); 5.07e5.16 (m, 1H, C5eH); 6.97 (s, 1H, Imidazole
C5eH); 7.30 (s, 1H, Imidazole C4eH); 7.62e7.77 (m, 3H, overlapped
signals); 8.16e8.22 (m, 1H, Ar). Anal. Found (calc) for C16H14FN5O3
(%): C, 55.95 (55.98); H, 4.10 (4.11); N, 20.40 (20.40).
5.2.3. Cell viability assay
The effects of 4a, 4b, and linezolid on cells viability were studied
in vitro on PK15 (porcine kidney epithelial), HaCaT (human kera-
tinocytes), and HepG2 (human hepatocellular carcinoma) cell lines
[54e56]. HepG2 and HaCat cells were grown in Dulbecco’s modi-
fied eagles medium (DMEM) whereas PK15 in DMEM/M199 (1:1).
All media were supplemented with 10% heat inactivated fetal
5.1.12. General procedure for the preparation of compounds 7a,b
Either 1a or 1b (0.45 mmol) was heated in the presence of 1,2,4-
triazole (0.12 g; 1.8 mmol) for 1 h. The mixture was recovered with
EtOAc and the solvent removed under reduced pressure. The res-
idue was chromatographed yielding the corresponding compounds
7a and 7b.
bovine serum (FBS), 2 mM
L-glutamine, 100 units/mL penicillin and
100
m
g/mL streptomycin. Cells were maintained at 37 ꢂC in a 5% CO2
atmosphere. All reagents for cell culture were from Euroclone
(Pero, Italy).
5.1.12.1. 3-(40-(300-Methyl-1,2,4-oxadiazol-5-yl)-phenyl)-5-((1,2,4-
Cell viability was measured by the MTT assay [57]. Briefly, MTT
triazol-1-yl)-methyl)-oxazolidin-2-onꢁe1(7a). Yield (73%); mp 208.0e
[3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyltetrazolium
bromide]
210.0 ꢂC; IR (Nujol) 1751 (NCO2) cm
;
1H NMR (300 MHz; CDCl3)
stock solution (5 mg/mL) was added to each well to a final con-
centration of 1.2 mM, and cells were incubated for 1 h and 30 min at
37 ꢂC. After removing MTT solution, the reaction was stopped by
adding 90% ethanol. Resuspended cells were centrifuged 10 min at
800ꢃ g. The absorbance was measured with the multilabel Victor3
spectrophotometer (Perkin Elmer, Turku, Finland) at wavelength of
570 nm. Data are means ꢄ S.E. of 3 separate experiments performed
in triplicate.
d
2.48 (s, 3H, Me); 4.13 (dd, 1H, J1 ¼ 9.0 Hz, J2 ¼ 6.1 Hz, C4eH); 4.25
(t, 1H, J ¼ 9.0 Hz, C4eH); 4.59e4.61 (m, 2H, CH2etriazole); 5.06e
5.12 (m, 1H, C5eH); 7.64 (d, 2H, J ¼ 9.0 Hz, Ar); 7.96 (s, 1H, Triazole
C3eH); 8.12 (d, 2H, J ¼ 9.0 Hz, Ar); 8.26 (s, 1H, Triazole C5eH). Anal.
Found (calc) for C15H14N6O3 (%): C, 55.20 (55.21); H, 4.30 (4.32); N,
25.70 (25.75).
5.1.12.2. 3-(30-Fluoro-40-(300-methyl-1,2,4-oxadiazol-5-yl)-phenyl)-5-
((1,2,4-triazol-1-yl)-methyl)-oxazolidin-2-onꢁe1(7b). Yield (64%); mp
5.2.4. Statistical analysis
176.2e177.8 ꢂC; IR (Nujol) 1751 (NCO2) cm
;
1H NMR (300 MHz;
Statistical significance was obtained with Student’s t test in
comparison with controls *P < 0.05, **P < 0.001. Data are
means ꢄ S.E. of 3 separate experiments performed in triplicate.
CDCl3)
d
2.48 (s, 3H, Me); 4.07 (dd, 1H, J1 ¼ 9.5 Hz, J2 ¼ 5.7 Hz, C4e
H); 4.31e4.39 (m, 1H, J ¼ 9.5 Hz, C4eH); 4.61e4.79 (m, 2H, CH2e
triazole); 5.15e5.27 (m, 1H, C5eH); 7.60 (dd, 1H, J1 ¼ 9.0 Hz,
J2 ¼ 2.2 Hz, Ar); 7.73 (dd,1H, J1 ¼13.5 Hz, J2 ¼ 2.0 Hz, Ar); 8.07 (s,1H,
Triazole C3eH); 8.14e8.21 (m, 1H, Ar) 8.64 (s, 1H, Triazole C5eH).
Anal. Found (calc) for C15H13FN6O3 (%): C, 52.35 (52.33); H, 3.80
(3.81); N, 20.45 (24.41).
6. Computational methods
6.1. Methods
6.1.1. Ligand-based approach
5.2. Microbiological assays
The model of wild type S. aureus 23S ribosomal rRNA (genebank
id: X68425) has been made with mode RNA program [58] using as a
template the 23S subunits of the 50S rRNA subunit of D. radiodurans
(pdb ID: 3DLL).
5.2.1. Bacterial strains
A total of 5 (2 MSSA and 3 MRSA) well characterized for their
antibiotic-susceptibility phenotype S. aureus isolates were used for
the determination of the in vitro antibacterial activity of the studied
compounds. In particular, S. aureus ATCC 29213 reference standard
strain and S. aureus M923 (collection strain) were used as MSSA
strains. Among MRSA, S. aureus MU50 (ATCC 700699) reference
standard strain and two collection strains (433 and F511) were used
for susceptibility assays.
3a, 3b, 4a and 4b molecules, as the most representative com-
pounds of this study, have been drawn, in both R and S stereoiso-
mers, with MOE [59]. Minimization and a conformational analysis
of the compounds have been performed with the same program.
The complexes of S. aureus 23S ribosomal rRNA with drawn drugs
3a,b and 4a,b have been made taking in consideration the similarity
between designed compounds and linezolid, thus docking have
been made manually, superposing the best conformations over
linezolid, using PyMOL [60]. The general Amber force field [61] and
HF/6-31G*-derived RESP atomic charges were used for the ligands.
Minimizations of complexes have been performed with the Sander
module of AMBER10 [62,63]. Restrictions over atoms farther than
5.2.2. Determination of minimum inhibitory concentrations (MICs)
The in vitro antibacterial activity of the new agents 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) guidelines [53]. Briefly,
serial 2-fold dilutions of each compound were made using the
MuellereHinton broth in microtitre plates with 96 wells. Dimethyl
sulfoxide (DMSO) was used as solvent for all the synthetized
compounds. An equal volume of the bacterial inoculum
(1 ꢃ 106 CFU/mL) was added to each well on the microtitre plate
containing 0.05 mL of the serial antibiotic dilutions. The microtitre
ꢀ
20 A from binding site have been applied.
6.1.2. Structure-based approach
The computational tools employed in this work are mainly part
of FLAP package [19]. A number of applications of FLAP for virtual-
screening was already reported, and its use as Model-Development
program was recently published [64]. In this paper, FLAP was used

C.G. Fortuna et al. / European Journal of Medicinal Chemistry 65 (2013) 533e545
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Financial supports from the Italian MIUR within the “FIRB-
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