Synthesis and preliminary antibacterial evaluation of Linezolid-like 1,2,4-oxadiazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.02.002

In the present study the synthesis of new Linezolid-like molecules has been achieved by substitution of the oxazolidinone central heterocyclic moiety with a 1,2,4-oxadiazole ring. Two series of 1,2,4-oxadiazoles, bearing different side-chains and containing a varying number of fluorine atoms, were synthesized and preliminarily tested for biological activity against some Gram-positive and Gram-negative bacteria using Linezolid and Ceftriaxone as reference drugs.

Full text of DOI:10.1016/j.ejmech.2012.02.002

European Journal of Medicinal Chemistry 50 (2012) 441e448
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis and preliminary antibacterial evaluation of Linezolid-like
1,2,4-oxadiazole derivatives
,
b
c
Antonio Palumbo Piccionello a, Rosario Musumeci b , Clementina Cocuzza , Cosimo Gianluca Fortuna ,
**
Annalisa Guarcello a d, Paola Pierro a d, Andrea Pace a
,
,
,d,
*
a Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari (STEMBIO), Sez. Chimica Organica “E. Paternò”, Università degli Studi di Palermo, Viale delle Scienze, Ed. 17,
Parco D’Orleans II, I-90128, Palermo, Italy
b Dipartimento di Medicina Clinica e Prevenzione, Ed. U8 - Università degli Studi di Milano-Bicocca, Via Cadore 48, I-20900, Monza (MB), Italy
c Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Viale Andrea Doria 6, I-95125, Catania, Italy
d Istituto EuroMediterraneo di Scienza e Tecnologia (IEMEST), Via Emerico Amari 123, I-90139, Palermo, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study the synthesis of new Linezolid-like molecules has been achieved by substitution
Received 13 September 2011
Received in revised form
4 January 2012
Accepted 1 February 2012
Available online 9 February 2012
of the oxazolidinone central heterocyclic moiety with
a 1,2,4-oxadiazole ring. Two series of
1,2,4-oxadiazoles, bearing different side-chains and containing a varying number of fluorine atoms, were
synthesized and preliminarily tested for biological activity against some Gram-positive and Gram-
negative bacteria using Linezolid and Ceftriaxone as reference drugs.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Oxazolidinone
1,2,4-Oxadiazole
Linezolid
Antimicrobial activity
Fluorinated heterocycles
1. Introduction
by MRSA and VRE [10e12]. It is the only marketed oxazolidinone,
although others are in development. It binds the 50S ribosomal
The alarming rate of increase of the multidrug resistance
phenomenon among bacterial pathogens in hospitals represents
one of the major challenges for researchers [1e7]. For approxi-
mately four decades, from the 40’s until the mid 70’s, the phar-
maceutical industry provided a steady flow of new antibiotics,
including several with new mechanisms of action that circum-
vented the problems caused by bacterial resistance to earlier agents.
Since then only three antibiotics, Quinupristin/Dalfopristin,
Linezolid, and Daptomycin, have been marketed in Europe to treat
infections caused by multidrug-resistant Gram-positive bacteria [4].
Inparticular, Linezolid (Fig.1) is the lead compoundof oxazolidinones
[8,9] and was approved in 2000 by the FDA for the treatment of
community-acquired and nosocomial pneumonia, complicated and
uncomplicated skin and soft-tissue infections, and infections caused
subunit preventing the formation of a functional initiation complex
70S for the protein synthesis [13,14]. This unique mechanism, among
protein synthesis inhibitors, explains why Linezolid retained anti-
bacterial activity against Gram-positive organisms which were
resistant to others antibacterials as well as why cross-resistance does
not occur with other classes of antibiotics [8,15]. Nevertheless,
resistance towards Linezolid has been recently triggered by muta-
tions within regions of 50S large-subunit ribosomal proteins, which
closely interact with the oxazolidinone binding site in the peptidyl
transferase center, as well as by enhanced efflux pumps or inactiva-
tion of the endogenous ribosomal methyltransferase [16,17].
In order to rationalize the type of modifications, the structure of
Linezolid can formally be divided into four portions according to
oxazolidinone antibacterials nomenclature [8]: i) the A-Ring, con-
sisting 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 func-
tional 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).
* Corresponding author. Dipartimento di Scienze
e Tecnologie Molecolari
e Biomolecolari (STEMBIO), Sez. Chimica Organica “E. Paternò”, Università degli
Studi di Palermo, Viale delle Scienze, Ed. 17, Parco D’Orleans II, I-90128, Palermo,
Italy. Tel.: þ39 091 23897543; fax: þ39 091 596825.
** Corresponding author.
E-mail address: andrea.pace@unipa.it (A. Pace).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.002

442
A. Palumbo Piccionello et al. / European Journal of Medicinal Chemistry 50 (2012) 441e448
O
O
O
N
N
H
N
O
F
C-ring
B-ring A-ring
Linezolid
C5 side-chain
Fig. 1. Structure and nomenclature of Linezolid.
Chart 2.
To date, numerous structural modifications of Linezolid have
been reported, many of them involving the introduction of hetero-
cyclic moieties replacing the A-, B- or C-ring [8,10,11,15,18e21]. For
instance, 1,2,4-oxadiazoles have been introduced in compound 1
(Chart 1) as terminal heterocyclic rings, far from the oxazolidinonic
core [21]. Additionally, it has been shown that the antibacterial
activity of Linezolid analogues could be maintained by replacing the
oxazolidinone ring with five-membered heteroaromatic rings, such
as isoxazole in compound 2 (Chart 1). Contrary to initial claims [15],
this result reveals that the presence of the oxazolidinone C5 asym-
metric center is not necessary for antibacterial activity [18,22].
Surprisingly, despite the well-known biological activity of
1,2,4-oxadiazoles [23e25] and the non-fluorinated series of
3-morpholinophenyl-1,2,4-oxadiazoles [26], there are no reports
of Linezolid analogues containing a 1,2,4-oxadiazole as the A-ring
portion.
We, therefore, decided to exploit the synthesis of a series of
Linezolid-like derivatives bearing a 1,2,4-oxadiazole moiety in place
of the oxazolidinone ring. As for the choice of the side-chain linked to
the C(3) of the 1,2,4-oxadiazole ring, in addition to the typical
methyleneeacetamide moiety of Linezolid, we decided to introduce
a carboxamide moiety. In fact, it has been reported that oxazolidi-
nones 3(Chart1), endowedwitha reversedamideside-chain, showed
a reduction of toxic effects and an extension of the spectrum of
activity associated with monoamine oxidase (MAO) inhibition [27].
Additionally, since the content of fluorine atoms on the B-ring
can affect the biological activity [28,29], we planned the synthesis
of derivatives with a varying number of fluorine atoms on the
phenyl ring (B-ring). The general structure of our target molecules
is illustrated in Chart 2.
obtained by initially building the oxadiazole heterocycle by
following the amidoxime synthetic route (Scheme 1) [23]. When
taking advantage of this approach, one should consider that the
amidoxime should have been previously functionalized either
with the final side-chain or with one of its precursors. Therefore,
2-aminoacetonitrile 6 was previously acetylated to obtain the
N-(cyanomethyl)acetamide 7 which was subsequently treated with
hydroxylamine to produce amidoxime 8.
The latter was acylated by reaction with benzoyl chloride and
subsequent thermal cyclization of the obtained crude O-aroylami-
doxime under solvent-free conditions produced the corresponding
oxadiazole 9. The same approach was also applied to the synthesis
of fluorinated oxadiazoles 11aee by using the corresponding fluo-
rinated benzoyl chlorides 10aee. In the last step, compounds 4aee
were obtained by reaction of the corresponding oxadiazoles 11aee
with morpholine through a SNAr which involved the displacement
of the fluorine atom occupying the para position with respect to the
oxadiazole ring [30e34].
Similarly, 1,2,4-oxadiazole-3-carboxamides 5aee, bearing
a reversed amide moiety with respect to the side-chain of Linezolid,
were obtained by initially constructing the oxadiazole ring through
acylation of amidoxime 12 with aroyl chlorides 10aee and subse-
quent solvent-free thermal cyclization of the corresponding
O-acylamidoxime into 1,2,4-oxadiazoles 13aee (Scheme 2).
3-Carboxyethyl-5-fluoroaryl-1,2,4-oxadiazoles 13aed were
then converted into the corresponding 3-carboxamides 14aed by
treatment with methanolic ammonia solution at room tempera-
ture. In the final step, compounds 14aed were functionalized by
a SNAr introducing morpholine on the 40 position of the aryl moiety
and producing target compounds 5aed (Scheme 2).
2. Results and discussion
This approach could not be applied to the more reactive
pentafluorophenyl-oxadiazole 13e, which upon treatment with the
ammonia solution underwent a displacement of the 40-fluorine
atom by the methanolic solvent. In turn, the pronounced reactivity
of the pentafluorophenyl moiety of compound 13e allowed the
introduction of the morpholine moiety and this gave rise to
2.1. Chemistry
5-Morpholinoaryl-substituted 1,2,4-oxadiazoles 4aee, contain-
ing a different number of fluorine atoms in the phenyl ring, were
N
S
O
O
CH3
O
HN
HN
O
NH2
O
N
N
O
O
N
R
F
N
N
N
N
O
2
3
1
N
CH3
Chart 1.

A. Palumbo Piccionello et al. / European Journal of Medicinal Chemistry 50 (2012) 441e448
443
Scheme 1.
Scheme 2.
Table 1
Antimicrobial activities, expressed in MIC values (mg/L) of synthetized compounds against Gram-positive and Gram-negative pathogens.
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
Ceftriaxone
Linezolid
S. pyogenes ATCC 19615
S. pyogenes EryR 6
S. pyogenes EryR 24
S. pyogenes EryR 47
S. pyogenes EryS 3
S. pyogenes EryS 9
S. pyogenes EryS 12
S aureus ATCC 29213
S aureus ATCC 25923
S aureus ATCC 43300
S aureus GISA MU 50
MRSA C530
128
128
64
64
128
128
128
>256
>256
>256
>256
>256
128
>256
128
128
128
128
128
128
128
>256
128
128
128
>256
128
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
128
128
128
>256
>256
128
128
128
128
>256
128
128
128
>256
128
128
128
>256
128
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
128
<0.12
<0.12
<0.12
<0.12
<0.12
<0.12
<0.12
4
1
0,12
0,12
1
1
1
0,25
4
1
2
2
2
1
4
>256
>256
>256
128
>256
>256
>256
>256
>256
>256
128
>256
>256
>256
>256
>256
>256
>256
>256
>256
128
>256
>256
>256
>256
>256
>256
>256
>256
>256
128
>256
>256
>256
>256
>256
>256
>256
>256
>256
128
>256
>256
>256
>256
>256
>256
>256
>256
>256
128
128
128
>256
128
>256
>256
>256
>256
>256
128
>256
128
128
>256
>256
>256
>256
>256
>256
128
1
8
4
>256
0.12
0.12
4
S. pneumoniae PEN I
E. coli ATCC 25922
E. coli 3350
128
>256
128
>256
128
>256
>256
>256
128
128
128
>256
128
>256
>256
>256
>256
128
S. marcescens Mel 522
<0.12

444
A. Palumbo Piccionello et al. / European Journal of Medicinal Chemistry 50 (2012) 441e448
compound 15. Finally, compound 15 was converted into the target
carboxamide 5e by reaction with ammonia.
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 ꢀC) in various ratios. Compound 12 was prepared
according to reported procedures [36]. Biological assays were
performed by using a broth microdilution method as described by
CLSI [37].
It is worth noting that the synthetic strategy used to obtain
compounds 5aee, the reverse approach (i.e. an initial reaction of
13aee with morpholine followed by reaction in methanolic
ammonia), could not be applied to all compounds 13aee since
reaction of morpholine with the ester moiety was observed for the
less fluorinated substrates.
2.2 Biological activity
4.2. N-(Cyanomethyl)-acetamide (7)
In this preliminary study, a screening of the antibacterial activity
was carried out on compounds 4aee and 5aee against a series of
clinical isolates. Tested Gram-positive and Gram-negative bacterial
pathogens included: Streptococcus pyogenes, Streptococcus pneu-
moniae, Staphylococcus aureus, Escherichia coli, and Serratia mar-
cescens. Antimicrobial activities are summarized in Table 1 and
were determined by the microbroth dilution method using the
Clinical Laboratory Standards Institute (CLSI) recommendations
(See Experimental). MIC (Minimal Inhibition Concentrations)
values were expressed in mg/L.
The synthesized compounds showed weak or no antibacterial
activity against tested strains. Considering that the 1,2,4-oxadiazole
ring is isosteric with the oxazolidinone ring and possesses very
similar hydrogen bond acceptors sites, one may ascribe the
observed weak activity to the introduction of an heteroaromatic
core which could affect the cellular uptake. Although weak, the
activity was observed more frequently for the non-fluorinated
or less fluorinated compounds. In particular, only compound 4a
demonstrated a better activity against S. pyogenes (64 mg/L).
Interestingly, this activity was shown for two clinical isolates
(EryR 24 and EryR 47) both expressing ribosomal constitutive
Acetic anhydride (2.03 g, 19.9 mmol) was added at 0 ꢀC to
a solution of 2-aminoacetonitrile sulfate (2.04 g, 13.2 mmol) in
Pyridine. The reaction mixture was stirred at room temperature for
24 h. The solvent was removed under reduced pressure and the
residue treated with water and rendered alkaline (pH 8) by adding
NaOH(aq) 1 M. The aqueous phase was extracted with EtOAc
(3 ꢁ 75 mL). The organic layers were collected, dried over anhy-
drous Na2SO4, filtered and the solvent removed under reduced
pressure to give the required product 7 as a white solid (0.62 g,
48%); mp 73e74 ꢀC (lit. 77 ꢀC [38]); IR (Nujol) 3301, 3060, 2258,
1634 cmꢂ1; GCeMS m/z: 98 (Mþ, 100%); 1H NMR (300 MHz-CDCl3)
d
7.80 (bs, 1H, exch. with D2O), 4.10 (d, 2H, J ¼ 5.7 Hz), 2.03 (s, 3H);
Anal. Found (calc) for C4H6N2O (%): C, 48.97 (48.90); H, 6.16 (6.10);
N, 28.56 (28.50).
4.3. 2-(N-Acetylamino)-acetamidoxime (8)
A solution of hydroxylamine hydrochloride (0.65 g, 9.5 mmol)
and NaOH (0.3 g, 9.5 mmol) in water (8 mL) was added at 0 ꢀC to
a solution of 7 (0.62 g, 6.3 mmol) in MeOH (10 mL). The reaction
mixture was stirred at room temperature for 24 h. The solvent was
removed under reduced pressure and the residue treated with
water (50 mL) and extracted with EtOAc (3 ꢁ 75 mL). The organic
layers were collected, dried over anhydrous Na2SO4, filtered and the
solvent removed under reduced pressure to give the required
product 8 as a white solid (0.74 g, 90%); mp 123e125 ꢀC; IR (Nujol)
3366, 3228, 1670, 1625 cmꢂ1; GCeMS m/z: 131 (Mþ, 100%); 1H NMR
resistance
for
macrolide
class
compounds
(MIC
of
Erythromycin ¼ >256 mg/L).
3. Conclusion
Two series of 1,2,4-oxadiazoles analogues of Linezolid were
synthesized by using the amidoxime route approach [23]. The two
series differ for the type of side-chain, while, within a given series,
the number of fluorine atoms on the phenyl moiety varies. The
sequence of the synthetic steps was crucial for the synthesis of
compounds 5aee since the fluorine content was strongly affecting
the competition between the ester and the fluoroaryl moiety for
the nucleophilic reagent. Compounds 4aee and 5aee were tested
to evaluate their antibacterial activity against a series of standard
and clinical isolates. Tested Gram-positive and Gram-negative
bacterial pathogens included: S. pyogenes, S. pneumoniae,
S. aureus, E. coli and S. marcescens. The antibacterial assay against
other Gram-positive strains is in progress, because none of the
presented 1,2,4-oxadiazoles was effective against the tested
microorganisms in comparison with used drugs. In order to
optimize the pharmacological properties of this class of
compounds, the obtained biological activity data will be used as
inactive compounds references in the construction of a QSAR
model based on a chemoinformatic approach recently used by
some of us [35].
(300 MHz-DMSO-d6)
d 9.07 (bs, 1H, exch. with D2O), 8.16 (bs, 1H,
exch. with D2O), 5.35 (bs, 2H, exch. with D2O), 3.66 (d, 2H,
J ¼ 5.7 Hz), 1.93 (s, 3H); Anal. Found (calc) for C4H9N3O2 (%): C,
36.64 (36.60); H, 6.92 (6.90); N, 32.04 (32.00).
4.4. General procedure for the preparation of compounds 9 and
11aee
Either benzoyl chloride or fluorinated aroyl chlorides 10aee
(2.2 mmol) were added to a solution of 8 (0.26 g; 2 mmol) in
Acetone (20 mL) containing also K2CO3 (0.30 g, 2.2 mmol). The
mixture was stirred at room temperature for 1 h after which the
solvent was removed under reduced pressure. The residue was
treated with water and the solid precipitate was collected by
filtration. The O-acylamidoxime so formed was heated, without any
further purification, at 170 ꢀC for 5 min in a sealed tube. The crude
material obtained was purified by column chromatography to give
the desired products 1,2,4-oxadiazoles 9 and 11aee.
4 Experimental
4.4.1. N-((5-Phenyl-1,2,4-oxadiazol-3-yl)-methyl)-acetamide (9)
White solid (0.39 g, 90%); mp 128e130 ꢀC; IR (Nujol) 3261,
1646 cmꢂ1; GCeMS m/z: 217 (Mþ, 100%); 1H NMR (300 MHz;
4.1. Materials and methods
DMSO-d6)
d
8.67 (t, 1H, J ¼ 5.4 Hz; exch. with D2O), 8.17e8.14
Melting points were determined on a Reichart-Thermovar hot-
stage apparatus and are uncorrected. IR spectra (Nujol) were
determined with a Shimadzu FTIR-8300 instrument; 1H NMR
(m, 2H), 7.80e7.67 (m, 3H), 4.50 (d, 2H, J ¼ 5.4 Hz), 1.94 (s, 3H).
Anal. Found (calc) for C11H11N3O2 (%): C, 60.82 (60.80); H, 5.10
(5.15); N, 19.34 (19.30).

A. Palumbo Piccionello et al. / European Journal of Medicinal Chemistry 50 (2012) 441e448
445
4.4.2. N-((5-(40-Fluorophenyl)-1,2,4-oxadiazol-3-yl)-methyl)-
acetamide (11a)
by filtration as a white solid (0.23 g, 72%); mp 202e204 ꢀC; IR
(Nujol) 3281, 1659 cmꢂ1; GCeMS m/z: 320 (Mþ, 100%); 1H NMR
White solid (0.30 g, 64%); mp 146e148 ꢀC; IR (Nujol) 3300,
(300 MHz; DMSO-d6)
d
8.63 (t, 1H, J ¼ 6.0 Hz; exch. with D2O), 7.91
1651 cmꢂ1; GCeMS m/z: 235 (Mþ, 100%); 1H NMR (300 MHz-
(d, 1H, J ¼ 9.0 Hz), 7.84 (d, 2H, J ¼ 14.7 Hz), 7.27 (t, 1H, J ¼ 9.0 Hz),
DMSO-d6)
d
8.64 (t, 1H, J ¼ 5.6 Hz; exch. with D2O), 8.26e8.19
4.46 (d, 2H, J ¼ 5.7 Hz), 3.81 (t, 4H, J ¼ 4.2 Hz), 3.24 (t, 4H,
(m, 2H), 7.58e7.51 (m, 2H), 4.49 (d, 2H, J ¼ 5.6 Hz), 1.94 (s, 3H).
Anal. Found (calc) for C11H10FN3O2 (%): C, 56.17 (56.10); H, 4.29
(4.20); N, 17.86 (17.80).
J ¼ 4.2 Hz), 1.94 (s, 3H). 13C NMR (62.5 MHz; DMSO-d6)
d 174.4,
169.7, 169.1, 154.0 (d, JCeF ¼ 244.6 Hz), 143.8, 125.3, 119.5, 115.9,
115.5 (d, JCeF ¼ 23.7 Hz), 66.1, 49.9, 34.6, 22.6. Anal. Found (calc) for
C15H17FN4O3 (%): C, 56.24 (56.20); H, 5.35 (5.30); N, 17.49 (17.40).
4.4.3. N-((5-(30,40-Difluorophenyl)-1,2,4-oxadiazol-3-yl)-methyl)-
acetamide (11b)
4.5.3. N-((5-(30,50-Difluoro-40-(morpholin-N0-yl)-phenyl)-1,2,4-
oxadiazol-3-yl)-methyl)-acetamide (4c)
White solid (0.30 g, 59%); mp 145e146 ꢀC; IR (Nujol) 3302,
1622 cmꢂ1; GCeMS m/z: 253 (Mþ, 100%); 1H NMR (300 MHz;
Morpholine (0.18 g; 2 mmol) was added to a solution of oxa-
diazole 11c (0.27 g; 1 mmol) in DMF (3 mL). The mixture was
refluxed for 1 h, concentrated under reduced pressure and treated
with water. The formed precipitate of compound 4c was collected
by filtration as a white solid (0.23 g, 68%); mp 196e198 ꢀC; IR
(Nujol) 3292, 1659 cmꢂ1; GCeMS m/z: 338 (Mþ, 100%); 1H NMR
DMSO-d6)
d
8.65 (t, 1H, J ¼ 5.8 Hz; exch. with D2O), 8.25e8.19
(m, 1H), 8.06e8.03 (m, 1H), 7.83e7.74 (m, 1H), 4.50 (d, 2H,
J ¼ 5.8 Hz), 1.94 (s, 3H). Anal. Found (calc) for C11H9F2N3O2 (%): C,
52.18 (52.10); H, 3.58 (3.50); N, 16.60 (16.65).
4.4.4. N-((5-(30,40,50-Trifluorophenyl)-1,2,4-oxadiazol-3-yl)-
methyl)-acetamide (11c)
(300 MHz; DMSO-d6)
d
8.62 (t, 1H, J ¼ 5.7 Hz; exch. with D2O), 7.78
(d, 2H, J ¼ 9.0 Hz), 4.48 (d, 2H, J ¼ 5.7 Hz), 3.77 (t, 4H, J ¼ 4.5 Hz),
3.33 (t, 4H, J ¼ 4.5 Hz), 1.94 (s, 3H). Anal. Found (calc) for
C15H16F2N4O3 (%): C, 53.25 (53.20); H, 4.77 (4.70); N, 16.56 (16.50).
White solid (0.35 g, 65%); mp 161e163 ꢀC; IR (Nujol) 3292, 1659,
cmꢂ1; GCeMS m/z: 271 (Mþ, 100%); 1H NMR (300 MHz; DMSO-d6)
d
8.64 (t, 1H, J ¼ 6.0 Hz; exch. with D2O), 8.14 (t, 2H, J ¼ 7.2 Hz), 4.50
(d, 2H, J ¼ 6.0 Hz), 1.94 (s, 3H). Anal. Found (calc) for C11H8F3N3O2
4.5.4. N-((5-(20,30,50,-Trifluoro-40-(morpholin-N0-yl)-phenyl)-1,2,4-
oxadiazol-3-yl)-methyl)-acetamide (4d)
(%): C, 48.72 (48.70); H, 2.97 (2.90); N, 15.49 (15.40).
Morpholine (0.18 g; 2 mmol) was added to a solution of oxa-
diazole 11d (0.29 g; 1 mmol) in DMF (3 mL). The mixture was
stirred for 24h at r.t, concentrated under reduced pressure and
treated with water. The formed precipitate of compound 4d was
4.4.5. N-((5-(20,30,40,50-Tetrafluorophenyl)-1,2,4-oxadiazol-3-yl)-
methyl)-acetamide (11d)
White solid (0.50 g, 87%); mp 117e119 ꢀC; IR (Nujol) 3300,
1651 cmꢂ1; GCeMS m/z: 289 (Mþ,100%); 1H NMR (300 MHz; DMSO-
collected by filtration as
199e201 ꢀC; IR (Nujol) 3292, 1659 cmꢂ1; GCeMS m/z: 356
(Mþ, 100%); 1H NMR (300 MHz; DMSO-d6)
a white solid (0.30 g, 84%); mp
d6)
d
8.67 (t, 1H, J ¼ 5.7 Hz; exch. with D2O), 8.22e8.13 (m, 1H), 4.50
(d, 2H, J ¼ 5.7 Hz), 1.94 (s, 3H). Anal. Found (calc) for C11H7F4N3O2
d
8.64 (t, 1H, J ¼ 5.7 Hz;
(%): C, 45.69 (45.70); H, 2.44 (2.40); N, 14.53 (14.50).
exch. with D2O), 7.72 (ddd, 1H, J1 ¼ 8.4 Hz, J2 ¼ 6.3 Hz, J3 ¼ 2.1 Hz),
4.50 (d, 2H, J ¼ 5.7 Hz), 3.77 (t, 4H, J ¼ 4.5 Hz), 3.41 (t, 4H, J ¼ 4.5 Hz),
1.94 (s, 3H). Anal. Found (calc) for C15H15F3N4O3 (%): C, 50.56
(50.55); H, 4.24 (4.20); N, 15.72 (15.70).
4.4.6. N-((5-Pentafluorophenyl-1,2,4-oxadiazol-3-yl)-methyl)-
acetamide (11e)
White solid (0.46 g, 75%); mp 130e132 ꢀC; IR (Nujol) 3296,
1653 cmꢂ1; GCeMS m/z: 307 (Mþ, 100%); 1H NMR (300 MHz;
4.5.5. N-((5-(20,30,50,60-Tetrafluoro-40-(morpholin-N0-yl)-phenyl)-
1,2,4-oxadiazol-3-yl)-methyl)-acetamide (4e)
DMSO-d6)
d
8.70 (t, 1H, J ¼ 6.0 Hz; exch. with D2O), 4.56 (d, 2H,
J ¼ 6.0 Hz), 1.94 (s, 3H). Anal. Found (calc) for C11H6F5N3O2 (%): C,
Morpholine (0.18 g; 2 mmol) was added to a solution of oxa-
diazole 11e (0.31 g; 1 mmol) in DMF (3 mL). The mixture was stirred
for 24 h at r.t, concentrated under reduced pressure and treated
with water. The formed precipitate of compound 4e was collected
by filtration as a white solid (0.30 g, 80%); mp 189e191 ꢀC; IR
(Nujol) 3288, 1653 cmꢂ1; GCeMS m/z: 374 (Mþ, 100%); 1H NMR
43.01 (43.00); H, 1.97 (1.90); N, 13.68 (13.60).
4.5. Synthesis of compounds 4aee
4.5.1. N-((5-(40-(Morpholin-N0-yl)-phenyl)-1,2,4-oxadiazol-3-yl)-
methyl)-acetamide (4a)
(300 MHz; DMSO-d6)
d
8.69 (t, 1H, J ¼ 6.0 Hz, exch. with D2O), 4.52
Compound 11a (0.23 g; 1 mmol) was dissolved in morpholine
(3 mL) and the mixture refluxed for 5 h. The reaction mixture was
concentrated under reduced pressure and treated with water
(25 mL). The formed precipitate of compound 4a was collected by
filtration as a white solid (0.21 g, 70%); mp 197e199 ꢀC; IR (Nujol)
3283, 1653 cmꢂ1; GCeMS m/z: 302 (Mþ, 100%); 1H NMR (300 MHz;
(d, 2H, J ¼ 6.0 Hz), 3.78 (t, 4H, J ¼ 4.5 Hz), 3.45 (t, 4H, J ¼ 4.5 Hz),1.94
(s, 3H). Anal. Found (calc) for C15H14F4N4O3 (%): C, 48.13 (48.10); H,
3.77 (3.70); N, 14.97 (14.90).
4.6. General procedure for the preparation of compounds 13aee
DMSO-d6)
d
8.59 (t, 1H, J ¼ 5.7 Hz; exch. with D2O), 7.96 (d, 2H,
A 10% excess of the appropriate fluorinated aroyl chlorides
10aee (4.4 mmol) was added to a solution of compound 12 (0.52 g,
4.0 mmol) in acetone (20 mL) together with K2CO3 (0.60 g,
4.4 mmol). The mixture was stirred at room temperature for 1 h
after which the solvent was removed under reduced pressure. The
residue was treated with water and the solid collected by filtration
and heated, without any previous purification, at 180 ꢀC for 30 min
in a sealed tube. Chromatography of the residue yielded the cor-
responding oxadiazole compounds 13aee.
J ¼ 9.0 Hz), 7.16 (d, 2H, J ¼ 9.0 Hz), 4.44 (d, 2H, J ¼ 5.7 Hz), 3.80
(t, 4H, J ¼ 4.8 Hz), 3.37 (t, 4H, J ¼ 4.8 Hz), 1.94 (s, 3H); 13C NMR
(62.5 MHz; DMSO-d6) d 175.5, 169.7, 168.8, 154.1, 129.3, 114.1, 112. 5,
66.0, 46.9, 34.6, 22.6. Anal. Found (calc) for C15H18N4O3 (%): C, 59.59
(59.50); H, 6.00 (6.05); N, 18.53 (18.50).
4.5.2. N-(5-(30-Fluoro-(40-(morpholin-N0-yl)-phenyl)-1,2,4-
oxadiazol-3-yl)-methyl)-acetamide (4b)
Morpholine (0.18 g; 2 mmol) was added to a solution of oxa-
diazole 11b (0.25 g; 1 mmol) in DMF (3 mL). The mixture was
refluxed for 1 h, concentrated under reduced pressure and treated
with water. The formed precipitate of compound 4b was collected
4.6.1. 3-Carboxyethyl-5-(40-fluorophenyl)-1,2,4,oxadiazole (13a)
White solid (0.81 g, 86%); mp 75e76 ꢀC (lit. 65 ꢀC [39]); IR
(Nujol) 1743, 1608 cmꢂ1; GCeMS m/z: 236 (Mþ, 100%); 1H NMR

446
A. Palumbo Piccionello et al. / European Journal of Medicinal Chemistry 50 (2012) 441e448
(300 MHz; CDCl3)
d
8.26e8.22 (m, 2H), 7.30e7.22 (m, 2H), 4.56
4.7.4. 5-(20,30,40,50-Tetrafluorophenyl)-1,2,4-oxadiazole-3-
(q, 2H, J ¼ 7.2 Hz), 1.48 (t, 3H, J ¼ 7.2 Hz). Anal. Found (calc) for
carboxamide (14d)
C11H9FN2O3 (%): C, 55.93 (55.90); H, 3.84 (3.80); N, 11.86 (11.80).
White solid (0.64 g, 98%); mp 173e176 ꢀC; IR (Nujol) 3481, 3331,
1686 cmꢂ1; GCeMS m/z: 261 (Mþ, 100%); 1H NMR (300 MHz-
DMSO-d6) d 8.49 (bs, 1H, exch. with D2O), 8.29 (bs, 1H exch. with
D2O), 8.28e8.19 (m, 1H). Anal. Found (calc) for C9H3F4N3O2 (%): C,
41.40 (41.40); H, 1.16 (1.10); N, 16.09 (16.00).
4.6.2. 3-Carboxyethyl-5-(30,40-difluorophenyl)-1,2,4,oxadiazole
(13b)
White solid (0.75 g, 74%); mp 86e87 ꢀC; IR (Nujol) 1741 cmꢂ1
;
GCeMS m/z: 254 (Mþ, 100%); 1H NMR (300 MHz-CDCl3)
d
8.07e8.00 (m, 2H), 7.42e7.33 (m, 1H), 4.55 (q, 2H, J ¼ 6.9 Hz), 1.48
4.8. 3-Carboxyethyl-5-(20,30,50,60-tetrafluoro-40-(morpholin-N-yl)-
phenyl)-1,2,4-oxadiazole 15
(t, 3H, J ¼ 6.9 Hz). Anal. Found (calc) for C11H8F2N2O3 (%): C, 51.98
(51.90); H, 3.17 (3.10); N, 11.02 (11.0).
A solution of13e (0.31 g, 1 mmol) in DMF (3 mL) containing
morpholine (0.09 g, 1 mmol) was stirred at room temperature for 3
days. The reaction mixture was concentrated under reduced pres-
sure, treated with water (50 mL) and extracted with EtOAc
(3 ꢁ 75 mL). The organic layers were collected, dried over anhy-
drous Na2SO4, filtered and the solvent removed. The residue was
chromatographed to give the required product 15 as a white solid
(0.30 g, 80%); mp 160e162 ꢀC; IR (Nujol) 1748 cmꢂ1; GCeMS m/z:
4.6.3. 3-Carboxyethyl-5-(30,40,50-trifluorophenyl)-1,2,4,oxadiazole
(13c)
White solid (0.80 g, 74%); mp 103e105 ꢀC; IR (Nujol) 1747 cmꢂ1
;
GCeMS m/z: 272 (Mþ, 100%); 1H NMR (300 MHz; CDCl3)
d 7.91
(dd, 2H, J1 ¼ 7.2 Hz, J2 ¼ 6.3 Hz), 4.57 (q, 2H, J ¼ 7.2 Hz), 1.48 (t, 3H,
J ¼ 7.2 Hz). Anal. Found (calc) for C11H7F3N2O3 (%): C, 48.54 (48.50);
H, 2.59 (2.50); N, 10.29 (10.20).
375 (Mþ, 100%); 1H NMR (300 MHz-CDCl3)
d
4.56 (q, 2H, J ¼ 7.2 Hz),
4.6.4. 3-Carboxyethyl-5-(20,30,40,50-tetrafluorophenyl)-
1,2,4,oxadiazole (13d)
3.84 (t, 4H, J ¼ 4.5 Hz), 3.46 (t, 4H, J ¼ 4.5 Hz), 1.48 (t, 3H, J ¼ 7.2 Hz).
Anal. Found (calc) for C15H13F4N3O4 (%): C, 48.01 (48.00); H, 3.49
(3.50); N, 11.20 (11.25).
White solid (0.86 g, 74%); mp 77e78 ꢀC; IR (Nujol) 1749 cmꢂ1
;
GCeMS m/z: 290 (Mþ, 100%); 1H NMR (300 MHz; CDCl3)
d
7.92e7.88 (m, 1H), 4.56 (q, 2H, J ¼ 7.2 Hz), 1.48 (t, 3H, J ¼ 7.2 Hz).
4.9. General procedure for the preparation of compounds 5aed
Anal. Found (calc) for C11H6F4N2O3 (%): C, 45.53 (45.50); H, 2.08
(2.00); N, 9.65 (9.60).
Morpholine (1.74 g; 20 mmol) was added to a solution of 14aed
(2.5 mmol) in DMF (5 mL). The resulting solution was refluxed for
1 h after which the reaction mixture was concentrated under
reduced pressure, treated with water (50 mL) and extracted with
EtOAc (3 ꢁ 75 mL). The organic layers were collected and dried over
anhydrous Na2SO4, filtered and the solvent evaporated under
reduced pressure. The residue was chromatographed yielding the
corresponding compounds 5aed.
4.6.5. 3-Carboxyethyl-5-(pentafluorophenyl)-1,2,4,oxadiazole (13e)
White solid (0.92 g, 75%); mp 69e71 ꢀC; IR (Nujol) cmꢂ1 1749;
GCeMS m/z: 308 (Mþ, 100%); 1H NMR (300 MHz-CDCl3)
d 4.56
(q, 2H, J ¼ 7.0 Hz), 1.48 (t, 3H, J ¼ 7.0 Hz); Anal. Found (calc) for
C11H5F5N2O3 (%): C, 48.87 (48.80); H, 1.64 (1.60); N, 9.09 (9.0).
4.7. General procedure for the preparation of carboxamides 14aed
4.9.1. 5-(40-(Morpholin-N-yl)-phenyl)-1,2,4-oxadiazole-3-
carboxamide (5a)
An ammonia saturated methanolic solution (2 mL) was added to
a solution of the appropriate esters 13aed (2.5 mmol) in MeOH
(10 mL). The mixture was stirred for 30 min at room temperature,
after which the solvent was removed under reduced pressure and
the residue re-crystallized from MeOH.
White solid (0.51 g, 74%); mp 247e249 ꢀC; IR (Nujol) 3487, 3240,
1686 cmꢂ1; GCeMS m/z: 274 (Mþ, 100%); 1H NMR (300 MHz-
DMSO-d6) d 8.37 (s, 1H, exch. with D2O), 8.16 (s, 1H, exch. with D2O),
8.01 (d, 2H, J ¼ 9.0 Hz), 7.18 (d, 2H, J ¼ 9.0 Hz), 3.80 (t, 4H, J ¼ 4.5 Hz),
3.40 (t, 4H, J ¼ 4.5 Hz); 13C NMR (62.5 MHz; DMSO-d6)
d 176.2,
164.4, 158.3, 154.3, 129.6, 114.1, 111.9, 66.0, 46.8. Anal. Found (calc)
for C13H14N4O3 (%): C, 56.93 (56.90); H, 5.14 (5.10); N, 20.43 (20.40).
4.7.1. 5-(40-Fluorophenyl)-1,2,4-oxadiazole-3-carboxamide (14a)
White solid (0.50 g, 97%); mp 215e216 ꢀC; IR (Nujol) 3477, 3217,
1689 cmꢂ1; GCeMS m/z: 207 (Mþ, 100%); 1H NMR (300 MHz-
4.9.2. 5-(30-Fluoro-40-(morpholin-N-yl)-phenyl)-1,2,4-oxadiazole-
3-carboxamide (5b)
DMSO-d6)
d 8.46 (bs, 1H, exch. with D2O), 8.30e8.24 (m, 3H,
overlapped signals), 7.60e7.54 (m, 2H). Anal. Found (calc) for
C9H6FN3O2 (%): C, 52.18 (52.10); H, 2.92 (2.90); N, 20.28 (20.25).
White solid (0.61 g, 84%); mp 217e218 ꢀC; IR (Nujol) 3469, 1721,
1687 cmꢂ1; GCeMS m/z: 292 (Mþ, 100%); 1H NMR (300 MHz-
DMSO-d6) d 8.41 (s,1H, exch. with D2O), 8.20 (s,1H, exch. with D2O),
4.7.2. 5-(30,40-Difluorophenyl)-1,2,4-oxadiazole-3-carboxamide
(14b)
7.94 (dd, 1H, J1 ¼ 13.8 Hz, J2 ¼ 2.1 Hz), 7.90 (dd, 1H, J1 ¼ 19.2 Hz,
J2 ¼ 2.1 Hz), 7.33e7.27 (m, 1H), 3.82 (t, 4H, J ¼ 4.5 Hz), 3.27 (t, 4H,
White solid (0.52 g, 92%); mp 201e203 ꢀC; IR (Nujol) 3419, 3283,
J ¼ 4.5 Hz); 13C NMR (62.5 MHz; DMSO-d6)
d 175.2, 164.5, 158.0,
1686 cmꢂ1; GCeMS m/z: 225 (Mþ, 100%); 1H NMR (300 MHz-
153.9 (d, JCeF ¼ 244.2 Hz), 144.0 (d, JCeF ¼ 6.7 Hz), 125.6, 119.5, 115.8
(d, JCeF ¼ 24.2 Hz), 115.5, 66.1, 49.8. Anal. Found (calc) for
C13H13FN4O3 (%): C, 53.42 (53.40); H, 4.48 (4.40); N, 19.17 (19.10).
DMSO-d6)
d 8.48 (bs, 1H, exch. with D2O), 8.30e8.24 (m, 2H), 8.08
(bs, 1H, exch. with D2O), 7.86e7.77 (m, 1H). Anal. Found (calc) for
C9H5F2N3O2 (%): C, 48.01 (48.00); H, 2.24 (2.20); N, 18.66 (18.60).
4.9.3. 5-(30,50-Difluoro-40-(morpholin-N-yl)-phenyl)-1,2,4-
oxadiazole-3-carboxamide (5c)
4.7.3. 5-(30,40,50-Trifluorophenyl)-1,2,4-oxadiazole-3-carboxamide
(14c)
White solid (0.70 g, 90%); mp 208e210 ꢀC; IR (Nujol) 3371, 3193,
White solid (0.59 g, 97%); mp 200e202 ꢀC; IR (Nujol) 3367, 3180,
1684 cmꢂ1; GCeMS m/z: 310 (Mþ, 100%); 1H NMR (300 MHz-
1689, cmꢂ1; GCeMS m/z: 243 (Mþ, 100%); 1H NMR (300 MHz-
DMSO-d6)
d 8.44 (s, 1H, exch. with D2O), 8.23 (s, 1H, exch. with
DMSO-d6)
d
8.48 (bs, 1H exch. with D2O), 8.27 (bs, 1H exch. with
D2O), 7.86e7.80 (m, 2H), 3.77 (t, 4H, J ¼ 4.5 Hz), 3.35 (t, 4H,
J ¼ 4.5 Hz). Anal. Found (calc) for C13H12F2N4O3 (%): C, 50.33
(50.30); H, 3.90 (3.95); N, 18.06 (18.00).
D2O), 8.19 (dd, 2H, J1 ¼ 7.8 Hz, J2 ¼ 6.6 Hz). Anal. Found (calc) for
C9H4F3N3O2 (%): C, 44.46 (44.40); H, 1.66 (1.60); N, 17.28 (17.20).

A. Palumbo Piccionello et al. / European Journal of Medicinal Chemistry 50 (2012) 441e448
447
4.9.4. 5-(20,30,50,-Trifluoro-40-(morpholin-N-yl)-phenyl)-1,2,4-
oxadiazole-3-carboxamide (5d)
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White solid (0.70 g, 85%); mp 204e206 ꢀC; IR (Nujol) 3441, 3256,
1697 cmꢂ1; GCeMS m/z: 328 (Mþ, 100%); 1H NMR (300 MHz-
DMSO-d6)
d 8.44 (s, 1H, exch. with D2O), 8.25 (s, 1H, exch. with
D2O), 7.82 (ddd, 1H, J1 ¼12.6 Hz, J2 ¼ 6.3 Hz, J3 ¼ 2.1 Hz), 3.78 (t, 4H,
J ¼ 4.5 Hz), 3.42 (t, 4H, J ¼ 4.5 Hz). Anal. Found (calc) for
C13H11F3N4O3 (%): C, 47.57 (47.50); H, 3.38 (3.40); N, 17.07 (17.00).
4.10. 5-(2,3,5,6-Tetrafluoro-4-morpholinphenyl)-1,2,4-oxadiazole-
3-carboxamide (5e)
An ammonia saturated methanolic solution (2 mL) was added to
a solution of compound 15 (0.94 g, 2.5 mmol) in MeOH (20 mL). The
mixture was stirred for 30 min at room temperature, after which
the solvent was removed under reduced pressure and the residue
re-crystallized from MeOH to give oxadiazole 5e as a white solid
(0.67 g, 77%); mp 252e253 ꢀC; IR (Nujol) 3367, 3201, 1683 cmꢂ1
;
GCeMS m/z: 346 (Mþ, 100%); 1H NMR (300 MHz-DMSO-d6)
d 8.47
(s, 1H, exch. with D2O), 8.29 (s, 1H, exch. with D2O), 3.78 (t, 4H,
J ¼ 4.5 Hz), 3.47 (t, 4H, J ¼ 4.5 Hz). Anal. Found (calc) for
C13H10F4N4O3 (%): C, 45.10 (45.15); H, 2.91 (2.90); N, 16.18 (16.10).
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4.11. Determination of minimum inhibitory concentrations (MICs)
The in vitro antibacterial activity of compounds 4aee and 5aee
was studied by determining the Minimum Inhibitory Concentra-
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according to the protocols approved by the Clinical and Laboratory
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Serial two-fold dilutions of each antibiotic stock solution were
obtained using MuellereHinton broth in 96 wells microtitre plates.
Compound stocks were prepared in 100% DMSO at 1000 g/L. Serial
dilutions were made for compound concentrations of
0.12e>256 mg/L. An equal volume of 1 ꢁ 106 CFU/mL bacterial
inoculum Colony Forming Unit/mL was added to each well of the
microtitre plate containing 0.05 mL of serial antibiotic concentra-
tions. The microtitre plate was then incubated at 35 ꢀC for 16e20 h;
subsequently each well was analyzed for the presence of visible
bacterial growth. MIC was defined as the lowest concentration of
the tested compound able to inhibit visible growth of the micro-
organism after overnight incubation. Controls with DMSO and
uninoculated media were run parallel to the tested compounds
under the same conditions. The in vitro antibacterial activity of
compounds 4aee and 5aee was tested and compared to that of
Linezolid used as oxazolidinones reference. S. pyogenes ATCC 19615,
S. aureus ATCC 29213(MSSA), S. aureus ATCC 25923(MSSA), S. aureus
ATCC 700699, also known as MU50 (MRSA) and E. coli 25922 were
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Acknowledgements
a new lead compound, J. Med. Chem. 50 (2007)
5886e5889.
Financial support from the Italian MIUR within the “FIRB-Futuro
in Ricerca 2008” Program - Project RBFR08A9V1 is gratefully
acknowledged.
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