Easy access to evans' oxazolidinones. Stereoselective synthesis and antibacterial activity of a new 2-oxazolidinone derivative

Article abstract of DOI:10.3390/molecules19067429

An interesting new approach was developed for the synthesis of Evans' chiral auxiliaries with excellent yields. In turn, another new stereoselective and efficient strategy has also allowed for the preparation of a 2-oxazolidinone derivative in 34% overall yield from the Morita-Baylis-Hillman adduct. The antibacterial activity of this oxazolidinone was tested against Staphylococcus aureus strains isolated from animals with mastitis infections.

Full text of DOI:10.3390/molecules19067429

Molecules 2014, 19, 7429-7439; doi:10.3390/molecules19067429
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Easy Access to Evans’ Oxazolidinones. Stereoselective Synthesis
and Antibacterial Activity of a New 2-Oxazolidinone Derivative
Gaspar Diaz 1,*, Michelle A. A. de Freitas 1, Maria E. Ricci-Silva 2 and Marisa A. N. Diaz 2
1
Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais,
Belo Horizonte–MG 31270-901, Brazil; E-Mail: adrianeamantea@gmail.com
2
Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa,
Viçosa–MG 36570-000, Brazil; E-Mails: esthericci@hotmail.com (M.E.R-S.);
marisanogueira@ufv.br (M.A.N.D.)
* Author to whom correspondence should be addressed; E-Mail: gaspardm@qui.ufmg.br or
dmgbras@hotmail.com; Tel.: +55-31-3409-5728; Fax: +55-31-3409-5700.
Received: 26 March 2014; in revised form: 28 May 2014 / Accepted: 28 May 2014 /
Published: 6 June 2014
Abstract: An interesting new approach was developed for the synthesis of Evans’ chiral
auxiliaries with excellent yields. In turn, another new stereoselective and efficient strategy
has also allowed for the preparation of a 2-oxazolidinone derivative in 34% overall yield
from the Morita-Baylis-Hillman adduct. The antibacterial activity of this oxazolidinone was
tested against Staphylococcus aureus strains isolated from animals with mastitis infections.
Keywords: Evans’ oxazolidinones; stereoselective synthesis; Morita-Baylis-Hillman
adduct; Staphylococcus aureus; mastitis bovina
1. Introduction
1.1. Evans’ Oxazolidinones
The use of enantiomerically pure oxazolidin-2-one derivatives as chiral auxiliaries in asymmetric aldol
condensations was first reported by Evans et al. [1], in 1981 (Figure 1), and the enormous utility of these
and related oxazolidinones has been amply demonstrated [2–5].

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Figure 1. Structures of the Evans’ oxazolidinones 1 and 2.
O
HN
R
O
1 R=Bn
2 R=i-Pr
Many sources for oxazolidin-2-one structures have been documented; however, preparative access
to chiral auxiliaries has proven to be only moderately efficient due to the β-aminoalcohol cyclization
step using phosgene, urea, or diethyl carbonate, which gives moderate yields [6]. Of the procedures
used for amino-alcohols cyclization, those involving phosgene proved to be most convenient and
produced the best yields. However, phosgene has proven to be a dangerous gas for humans, therefore,
other alternative methods are needed to properly prepare these oxazolidinones.
Therefore, this study describes a novel, short, and direct alternative strategy, for the preparation of the
oxazolidinones 1 and 2, in nearly quantitative yields and without the purification of intermediates.
1.2. Oxazolidinone Derivatives
Several compounds containing 4-substituted and 4,5-disubstituted oxazolidin-2-one rings have
shown promising pharmacological activities. For instance, in the 1980s, DuPont researchers found that
certain compounds containing 2-oxazolidinone rings showed potent antibacterial activities through a
unique mechanism of action [7]. Linezolid (Figure 2) was the first 2-oxazolidinone derivative with
antibiotic action to be introduced into clinical trials by Pharmacia [8] and was approved in 2000 by the
Food and Drug Administration for use in the treatment of infections caused by Gram-positive bacteria.
Figure 2. Structures of the linezolid and N-thiolated 2-oxazolidinones.
O
O
MeS N
R
O
O
N
N
O
CO2Me
F
NHAc
N-thiolated
linezolid
2-oxazolidinones
Turos et al. [9] recently reported the discovery of compounds with N-thiolated 2-oxazolidinone
rings (Figure 2) as a new family of antibacterial agents, since many of these N-thiolated derivatives
showed a potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) [10].
In conjunction with the first portion of this work, and as part in another ongoing research work
aimed at the synthesis of natural products with potential antibacterial activities, the present study also
proposes a novel approach for the stereoselective synthesis of a new 2-oxazolidinone derivative 5.
This was possible following the same strategy of N,O-heterocyclization of the oxazolidinones 1 and 2,
respectively, from diol 12. Thus, as a supplement to the work previously published by Coelho et al. [11],
the synthesis of this 2-oxazolidinone provided the opportunity to explore the conversion of a
Morita-Baylis-Hillman (MBH) adduct as a key intermediate into 4,5-disubstituted 2-oxazolidinone 5.

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To the best of our knowledge, this approach is the first that examines the major MBH stereoisomer,
which in turn produced the acetonide derivative 14 as an advanced intermediate used to synthesize
2-oxazolidinone products.
2. Results and Discussion
2.1. Preparation of Evans’ Oxazolidinones 1 and 2
The commercially available L-phenylalanine ethyl ester hydrochloride 3 was converted to the
corresponding N-Boc ethyl ester in the presence of di-tert-butyl dicarbonate ((Boc)2O), followed by
the reduction of the ester function using LiBH4. The cyclization was performed by treating the
resulting alcohol with NaH, producing the corresponding oxazolidin-2-one derivative 1 with a yield of
98% (for three steps), [α]D25 −62.5 (c 1.0, CHCl3) (Scheme 1).
Scheme 1. Preparation of Evans’ oxazolidinones 1 and 2.
O
O
(i) Boc2O, 0.5 M aq. sln. of Na2CO3
dioxane/H2O (2:1), rt, 5 h
(ii) LiCl, NaBH4, EtOH, 0 oC to rt
18 h
2
R
HN
R
O
OEt
4 5
NH2.HCl
3, R = Bn
4, R = iPr
(iii) NaH, DMF, 0 oC to rt, 3 h
1, R = Bn
2, R = iPr
98% (three steps)
In a similar fashion to 1, the commercially available L-valine ethyl ester hydrochloride 4 was
25
transformed into oxazolidinone 2 (98%, three steps), [α]D −15.3 (c 7.0, CHCl3) (Scheme 1);
the characterization data of both 1 and 2 are in good agreement with reported values [1].
2.2. Preparation of 2-Oxazolidinone Derivative 5
Initially, the stereoselective synthesis of 2-oxazolidinone derivative 5 was proposed according to
the retrosynthetic analysis shown in Scheme 2. The Morita-Baylis-Hillman adduct 6 was obtained
according to the procedure described by Coelho et al. [11]. N-Boc protection of L-phenylalanine ethyl
ester hydrochloride 3 using di-tert-butyl dicarbonate and reduction of the ester function of crude
product by treating with DIBAL-H at −78 °C produced aldehyde 7 (note: the crude aldehyde was used
immediately after preparation to avoid or minimize racemization). The L-phenylalaninal 7 was treated
with DABCO and methyl acrylate and was kept in an ultrasonic bath for 36 h to produce the
diastereomeric mixture (3:7; anti:syn) according to chiral gas chromatography (see Supplementary
Material for details). The crude mixture was purified by column chromatography to produce the major
Morita-Baylis-Hillman adduct 6 (56%, 3 steps) in 99% ee as determined by chiral GC.
At first glance, the N,O-heterocyclization from the MBH adduct 6 appeared to be quite simple.
However, the preliminary results contradicted this prediction. Any attempt, including inter alia direct
N,O-heterocyclization from this adduct with the intact ester function and free hydroxyl led to
a complex mixture of products, as witnessed when 6 was treated with NaH or phosgene,
for example. Due to the unsatisfactory results obtained from the initial proposal to attempt the direct

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N,O-heterocyclization from MBH adduct 6, a new route was proposed, this time in four steps from the
adduct 6, as shown in Scheme 3.
Scheme 2. Retrosynthetic analysis of 2-oxazolidinone derivative 5.
O
OH
OH
O
R
R
MBH reaction
R
H
OCH3
+
OCH3
NHBoc
HN
O
HN
O
Boc
O
5, R = Bn
6, R = Bn (MBH adduct)
7, R = Bn
8
Scheme 3. Synthesis of 2-oxazolidinone derivative 5 from 1,2-syn-MBH adduct 6.
8
OCH3
OH
O
7
(i) DIBAL-H, CH2Cl2
−30 oC, 1 h
(ii) PTSA, rt, 48 h
R
DMP, PTSA
rt, 48 h
R
6
OCH3
O
4 5
2
BocN
O
HN
88%
Boc
21
22
1,2-syn-MBH adduct 6, R = Bn
acetonide 14, R = Bn
J = 5.2 Hz
H H
OH
(iii) NaH, THF, rt, 2 h
OH
8
6
9
4 5
2
7
HN
OH
HN
O
(38%, three steps)
Boc
O
12
oxazolidinone derivative 5
The step performed to protect the Morita-Baylis-Hillman adduct 6 had to be radically modified for
the N,O-protected acetonide derivative (Scheme 3). For this purpose, this adduct was treated with
2,2-dimethoxypropane and catalytic amounts of p-toluenesulfonic acid, producing the acetonide
derivative 14 in 88% yield. The one pot sequence, starting from the reduction of the ester function of
acetonide 14 using DIBAL at −30 °C, a chemoselective deprotection using p-toluenesulfonic acid, and
cyclization of the resultant diol 12 in the presence of NaH produced the corresponding oxazolidinone
derivative 5 with a yield of 38% (three steps). The relative stereochemistry of trans-oxazolidinone
derivative 5 can readily be assigned by comparing it to the ring-proton coupling constant (JH5-H4) [12–14].
The observed coupling constant of JH5-H4 = 5.2 Hz indicates that the protons are on opposite faces of
the heterocyclic ring; therefore, the trans-oxazolidinone 5 is derived from the 1,2-syn MBH adduct 6
(Scheme 3).
2.3. Antibacterial Activity
Compounds 5 and 14 were assayed against Staphylococcus aureus strains. Ciclopirox olamine was
used as positive control since this compound possesses a broad-spectrum of antibacterial and
anti-inflammatory activities [15,16] and it is one of the most used drugs to treat bovine mastitis in
some farms in Brazil. According to Table 1, oxazolidinone derivative 5 has shown good antimicrobial

Molecules 2014, 19
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activity against the five S. aureus strains isolated from animals with bovine mastitis infection.
However, the results of bioassays performed on acetonide 14 showed little or no activity. These results
are in agreement with findings from DuPont researchers [7,8] and Turos et al. [9] who affirm that
some compounds containing oxazolidinone rings present a potent antimicrobial activity against
methicillin-resistant S. aureus (MRSA) and other Gram-positive bacteria. It is believed that,
in addition to the substituents on carbons 4 and 5, respectively, this activity is likely related to the
presence of a more rigid and almost planar ring. This ring rigidity is more probable in the presence of
a carbonyl group at 2-oxazolidinone 5, when compared with the ring of the acetonide derivative 14
(Scheme 3) since this most likely provides a more flexible conformation bias.
Table 1. Antibacterial activity of compounds 5 and 14 (inhibition zones in mm ± SD).
S. aureus
680
Compound 5
19.0 ± 0.58
18.0 ± 0.60
21.0 ± 0.33
15.0 ± 0.67
16.0 ± 0.58
Compound 14 Ciclopirox Olamine DMSO
2.0 ± 0.48
NA
17.0 ± 0.58
18.0 ± 0.60
17.0 ± 0.58
17.0 ± 0.58
17.0 ± 0.50
0.00
0.00
0.00
0.00
0.00
2221
4006
2.0 ± 0.58
NA
4075
4127
2.0 ± 0.50
NA = not active.
Table 2 shows the results of the minimum inhibitory concentration (MIC) for compound 5 against
S. aureus (4006 strain) which presented the best value in the antibacterial assay. The MIC value was
lower than the standard antibiotic value, demonstrating that this compound, containing a 2-oxazolidinone
ring, belongs to a new family of compounds with potential antimicrobial activity.
Table 2. MIC value obtained from compound 5 against Staphylococcus aureus (4006 strain).
MIC (μg mL−1)
Compound 5
0.03
0.05
Ciclopirox olamine
3. Experimental
3.1. General Procedures
All reactions were carried out under N2 in flame-dried glassware with magnetic stirring. CH2Cl2,
Et3N, and DMF were distilled from CaH2. THF was distilled from sodium/benzophenone ketyl.
Chromatographic purification was carried out on silica gel (mesh particle size 70–230) and thin layer
chromatography (TLC) was performed on silica GF254 Merck. TLC visualization was accomplished
with UV light and by spraying with 7% ethanolic phosphomolybdic acid and heating. 1H- and 13C-NMR
(nuclear magnetic resonance) spectra were recorded on a Varian Gemini BB at 300 MHz and 75.4 MHz,
respectively. FTIR spectra were determined using KBr disks on a FTIR Perkin Elmer Spectrum
200 spectrometer. Uncorrected melting points were determined using METTLER equipment, model
FP82. GC analyses were recorded on HP6890 equipment with a flame ionization detector, using an
HP-5 capillary (cross-linked 5% phenylmethylsiloxane, 30 m, 0.25 mm × 25 µm) column (Agilent
Technology, Santa Clara, CA, USA). Mass spectra were recorded on a Prominence HPLC (Shimadzu,

Molecules 2014, 19
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Kyoto, Japan) coupled to the MicroQTOF II mass spectrometer (Bruker Daltonics, Fremont, CA,
USA). Optical rotation was measured through Perkin-Elmer equipment using a Na lamp at 585 nm.
Diastereoselectivity of MBH adduct 6 was determined from GC analysis. The Morita-Baylis-Hillman
reaction was sonicated in a UNIQUE model GA 10.25 (1200 W, 25 kHz) ultrasonic bath.
3.2. Synthesis
3.2.1. General Procedure for the Synthesis of Evans’ Oxazolidinones 1 and 2
To the respective L-amino ethyl ester hydrochloride (1 mmol) dissolved in dioxane-distilled water
(2:1, 3.9 mL), 0.5 mol L−1 of an aqueous solution of Na2CO3 (4 mL) was added at room temperature.
The resulting solution was cooled to 0 °C, at which time the di-tert-butyldicarbonate (192 mg, 1.1 mmol)
was added and the ice-bath was removed. This mixture was stirred for 3 h at room temperature and
extracted with EtOAc (3 × 10 mL). The combined organic layers were dried with MgSO4, filtered, and
concentrated to give a slightly yellow solid that was dissolved in EtOH (4 mL). NaBH4 (113.5 mg,
3.0 mmol) and LiCl (127.2 mg, 3.0 mmol) were added to this flask at 0 °C, at which time the ice-bath
was removed. This mixture was stirred for 18 h at room temperature and quenched by the addition of
a saturated aqueous solution of citric acid (5 mL) followed by Et2O (10 mL). The layers were
separated, and the aqueous layer was additionally extracted with Et2O (2 × 10 mL). The combined
organic layers were treated with a saturated aqueous solution of NaHCO3 (5 mL), washed with brine
(5 mL), dried with MgSO4. The solvent was concentrated to produce a slightly yellow solid, which was
dissolved in DMF (3 mL), stirred, and cooled at 0 °C. Next, NaH (72 mg, 3.0 mmol) was added, and
the resulting mixture was warmed gradually to room temperature, stirred for 3 h, and subsequently
quenched by adding a 5% aqueous solution of hydrochloric acid (5 mL) followed by Et2O (10 mL).
The layers were separated, and the aqueous layer was extracted using Et2O (2 × 10 mL). The combined
organic layers were treated with a saturated aqueous solution of NaHCO3 (5 mL), washed with brine
(5 mL), dried with MgSO4, and concentrated. The residues were separately purified by column
chromatography on silica gel using 60% EtOAc/hexanes as the eluent to produce compounds 1 and 2,
respectively, with an average yield of 98% after the three steps.
25
(S)-(−)-4-Benzyl-2-oxazolidinone (1). [α]D −62.5 (c 1.0, CHCl3); mp 89–90 °C; IR (KBr) νmax/cm−1
3280, 3023, 2853, 1960, 1752, 1548, 1475, 1454, 1403, 1021, 943; 1H-NMR (CDCl3) δ 7.24–7,37 (m,
3H, H9/H9'/H10), 7.17–7,19 (m, 2H, H8/H8'), 5.78 (brs, 1H, NH), 4.44 (dd, J = 12.0, 8.1 Hz, 1H, H5),
4.04–4.14 (m, 2H, H4/H5), 2.87 (d, J = 6.3 Hz, 2H, H6); 13C-NMR (CDCl3) δ 159.6 (C2), 136.2 (C7),
129.2 (C8/C8'/C9/C9'), 127.5 (C10), 69.8 (C5), 54.0 (C4), 41.6 (C6).
25
(S)-(−)-4-Isopropyl-2-oxazolidinone (2). [α]D −15.3 (c 7.0, CHCl3); mp 72–73 °C; IR (neat)
1
ν
max/cm−1 3259, 2960, 2914, 1720, 1471, 1404, 1327, 1242, 1089, 1007, 933, 706; H-NMR (CDCl3)
−7.39 (brs, 1H, NH), 4.44 (dd, J = 8.6, 6.4 Hz, 1H, H5), 4.10 (dd, J = 8.6, 6.4 Hz, 1H, H5), 3.57–3.68
(m, 1H, H4), 1.64–1.81 (m, 1H, H6), 0.96 (d, J = 6.6 Hz, 3H, H7'), 0.90 (d, J = 6.6 Hz, 3H, H7);
13C-NMR (CDCl3) δ 160.6 (C2), 68.4 (C5), 58.2 (C4), 32.5 (C6), 17.7 (C7'), 17.4 (C7).

Molecules 2014, 19
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3.2.2. (−)-(3S,4S)-4-Tert-butoxycarbonylamino-3-hydroxy-2-methylene-5-phenylpentanoic Acid Methyl
Ester (6) [11]
To a solution of L-phenylalanine ethyl ester hydrochloride (3, 400 mg, 1.74 mmol) in MeOH
(8.8 mL) at room temperature, di-tert-butyl dicarbonate (456 mg, 2.08 mmol), and NaHCO3 (432 mg,
5.2 mmol) were added. The resultant mixture was kept in an ultrasonic bath for 4 h at room
temperature. The solvent was concentrated under reduce pressure. The residue was redissolved in Et2O
(40 mL) and treated with a saturated aqueous solution of NaHCO3 (20 mL). The layers were then
separated, and the aqueous layer was extracted with Et2O (3 × 20 mL). The combined organic layers
were washed with brine (20 mL) and dried over MgSO4. After removal of the solvent, the crude N-Boc
amino ester was obtained in a quantitative yield. To the crude carbamate dissolved in anhydrous
toluene (5 mL), kept under an argon atmosphere, and cooled at −78 °C, DIBAL-H (1.3 mL, 1.9 mmol,
1.5 mol L−1 in toluene) was slowly added. The reaction mixture was additional stirred over 30 min at
−78 °C and quenched by adding a methanol (0.2 mL) and Rochelle solution (2 mL) at same
temperature. The heterogeneous mixture was then stirred vigorously for 3 h at room temperature.
The reaction mixture was filtered over Celite, washed with EtOAc (3 × 20 mL), dried over MgSO4,
and concentrated under reduced pressure to afford the crude L-phenylalaninal (90% yield; the crude
aldehyde was used immediately after preparation to avoid or minimize racemization). The L-phenylalaninal
was treated with DABCO (100 mg, 0.89 mmol) and methyl acrylate (2 mL, 23 mmol) and was kept in
an ultrasonic bath for 36 h at room temperature. After that, the reaction mixture was diluted in
dichloromethane (20 mL). The mixture was then washed with a saturated solution of NaHCO3,
a solution of 10% HCl, brine, and finally dried over anhydrous sodium sulfate. After solvent
evaporation under reduced pressure, the diastereomeric mixture (3:7; anti:syn) according chiral gas
chromatography, was purified by column chromatography on silica gel using 20% EtOAc/hexanes as
an elutant to produce 326 mg (56%, 3 steps) of the major Morita-Baylis-Hillman adduct (6) as a slight
yellow oil in 99% ee as determined by chiral GC. [α]D −1.80 (c 1.0, CHCl3); IV (neat) νmax/cm−1
25
1
3404, 2979, 2930, 1714, 1694, 1509, 1440, 1172; H-NMR (CDCl3) δ 7.31–7.16 (m, 5H, Ph-H), 6.31
(brs, 1H, CH2), 5.92 (brs, 1H, CH2), 4.87 (sh, 1H, NH), 4.51 (brs, 1H, OCH), 4.03 (brq, 1H, J = 7.0 Hz,
NCH), 3.71 (s, 3H, OCH3), 3.08 (sh, 1H, OH), 3.03–2.90 (m, 2H, Bn-H), 1.36 (brs, 9H, C(CH3)3);
13C-NMR (CDCl3) δ 166.3 (C0), 156.1 (C0), 140.3 (C0), 138.2 (C0), 129.1 (CH), 128.2 (CH),
126.2 (CH), 125.7 (CH2), 79.4 (C0), 70.7 (CH), 54.8 (CH), 51.9 (CH3), 38.1 (CH2), 28.3 (CH3);
HRMS (ESI+) m/z, calcd. for C18H25NO5 [M+1]+: 336.1733, found: 336.1793.
3.2.3. (+)-(4S,5S)-4-Benzyl-5-(1-Methoxycarbonylvinyl)-2,2-dimethyloxazolidine-3-carboxylic Acid
Tert-Butyl Ester (14)
To a solution of the Morita-Baylis-Hillman adduct 6 (335.4 mg, 1 mmol) in 2,2-dimethoxypropane
(3.0 mL, 24.4 mmol), camphorsulfonic acid (46.5 mg, 0.20 mmol) was added and the mixture stirred
for 48 h at room temperature. The reaction was quenched by adding distilled water (10 mL) followed
by Et2O (10 mL). Subsequently, both layers were separated. The organic layer was washed with
a saturated aqueous solution of NaHCO3 (5 mL) and brine (5 mL). The organic layer was dried with
MgSO4, filtered, and concentrated. Purification by flash chromatography on silica gel using 3%

Molecules 2014, 19
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25
EtOAc/hexanes as the eluent produced compound 14 in 80% yield. [α]D +0.03 (c 10.0, CHCl3);
IR (neat) νmax/cm−1 3063, 2985, 2951, 2936, 1696, 1633, 1452, 1437, 1376, 1363, 1279, 1252, 1165,
1129, 1067, 851, 700; 1H-NMR (CDCl3) δ 7.10–7.28 (m, 5H, H11-15), 6.25 (brs, 1H, H6a'), 5.89 (brs,
1H, H6b'), 4.84 (brs, 1H, H5), 4.16 (brs, 1H, H4), 3,63 (brs, 3H, H8), 3.14 (brs, 2H, H9), 1.52 (s, 9H,
H18-20), 1.50 (s, 3H, H21), 1.45 (s, 3H, H22); 13C-NMR (CDCl3) δ 166.1 (C7), 151.5 (C16),
138.0 (C6), 135.5 (C10), 130.0 (C12), 127.9 (C11), 126.6 (C6'), 125.7 (C13), 92.7 (C2), 80.1 (C17),
74.5 (C5), 60.2 (C4), 51.7 (C8), 36.9 (C9), 28.4 (C18-20), 27.2 (C21), 23.8 (C22); HRMS (ESI+) m/z
calcd for C21H29NO5 [M+1]+ 376.2046, found 376.2120.
3.2.4. (−)-(3S,4S)-4-Benzyl-5-(1-Hydroxymethylvinyl)-2-oxazolidinone (5)
To a stirred solution under argon of the corresponding acetonide derivative 14 (120 mg, 0.32 mmol)
in 2 mL of dichloromethane at −30 °C, 1.2 mol L−1 in toluene of DIBAL-H (1 mL, 0.83 mmol) was
slowly added. After 90 min of stirring, the solution was warmed to 0 °C, stirred for 30 min, and
quenched by adding a methanol (0.15 mL) and Rochelle solution (1 mL). The heterogeneous mixture
was then stirred vigorously for 3 h. Following the addition of water (5 mL) and Et2O (5 mL), the layers
were separated. The aqueous layer was extracted with Et2O (3 × 5 mL). The combined organic extracts
were dried over MgSO4 and evaporated under reduced pressure to give an oily residue. This residue
was dissolved in methanol (3 mL). Next, p-toluenesulfonic acid (17.2 mg, 0.1 mmol) was added and
stirred for 14 h at room temperature. The reaction mixture was dissolved in EtOAc (5 mL) and treated
with a saturated aqueous solution of NaHCO3 (3 mL). The layers were then separated, and the aqueous
layer was extracted with EtOAc (3 × 5 mL). The combined organic layers were washed with brine
(5 mL) and dried with anhydrous Na2SO4. The solvent was concentrated to give a slightly yellow
residual product that was dissolved in anhydrous THF (2 mL) and, stirred at 0 °C under argon.
A suspension of NaH (24 mg, 1.0 mmol) in anhydrous THF (2 mL) was added in a dropwise manner
to this mixture and stirred at 0 °C for 2 h. The reaction was quenched with H2O (3 mL), and the layers
were separated. The aqueous layer was extracted with EtOAc (3 × 5 mL). The combined organic layers
were washed with brine, dried with anhydrous Na2SO4, and concentrated. The residue was purified by
column chromatography using EtOAc/hexanes 50% as an eluent to produce 28.4 mg (38% yield,
3 steps) of oxazolidinone derivative 5 as a slightly yellow oil in 99% ee as determined by chiral GC.
[α]D25 −0.04 (c 9.0, CHCl3); IR (neat) νmax/cm−1 3283, 3029, 2922, 2855, 1734, 1390, 1231, 1019, 918,
733; 1H-NMR (CDCl3) δ 7.18–7.34 (m, 5H, H10-14), 5.65 (s, 1H, NH), 5.24 (s, 1H, H6a'), 5.13 (s, 1H,
H6b'), 4.82 (d, J = 5.2 Hz, 1H, H5), 4.14 (s, 2H, H7), 3.97 (brd, J = 6.4 Hz, 1H, H4), 2.96 (dd, J = 13.6,
4.8 Hz, 1H, H8a), 2.84 (dd, J = 13.6, 4.8 Hz, 1H, H8b), 2.13 (brs, 1H, OH); 13C-NMR (CDCl3) δ 158.9 (C2),
144.6 (C6), 136.0 (C9), 129.3 (C10), 128.9 (C11), 127.2 (C12), 114.3 (C6'), 82.2 (C5), 62.2 (C7),
58.7 (C4), 41.5 (C8); HRMS (ESI+) m/z calcd. for C13H15NO3 [M+H]+ 234.1052, found 234.1069.
3.3. Antibacterial Assays
3.3.1. Bacterial Strains and Culture Media
The bacterial strains used in this study, which were isolated from animals with mastitis infections,
were kindly provided by Embrapa Dairy Cattle/Laboratory of Milk Microbiology (Juiz de Fora, Minas

Molecules 2014, 19
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Gerais, Brazil). Five S. aureus strains (680, 2221, 4006, 4075, and 4127) were used to determine
the antimicrobial activity of compounds 5 and 14. Bacteria were routinely cultured on brain heart
infusion (BHI) agar at 37 °C for 16 h prior to the experiments. The cell concentration was adjusted to
106 CFU mL−1 with an optical density set at 600 nm. Stock cultures were maintained in BHI agar
containing 25% glycerol at −80 °C.
3.3.2. Antibacterial Screening Assay
Hole-plate diffusion assay was initially performed to test the antibacterial activity of compounds.
To accomplish this, the bacteria were cultivated overnight, and a suspension containing 106 CFU mL−1
was spread on plates containing Müeller-Hinton agar (Himedia®, Mumbai, India). Holes of approximately
5 × 3 mm were made in the agar and filled with 30 μL of the stock with 10 µg mL−1 for compounds 5
and 14. After incubation at 37 °C for 24 h, inhibition zones were measured in millimeters and
compared to the controls. The antibiotic ciclopirox olamine (Uci-Farma®, São Bernardo do Campo, SP,
Brazil) was used as the positive control due to its antibacterial properties [15,16]. Dimethylsulfoxide
(DMSO) was used as a negative control. Tests were performed twice in triplicate. The minimum
inhibitory concentration (MIC) of compound 5 was determined by applying a broth microdilution
method followed by incubation at 37 °C for 24 h and by observing media turbidity [17]. Tests were
performed twice in triplicate.
4. Conclusions
The Evans’ chiral auxiliaries 1 and 2 were prepared in high yields (98%, three steps) through
a simple and straightforward alternative strategy. It is worth mentioning that either of the chiral
auxiliaries can be obtained in gram scale. By contrast, a novel stereoselective synthetic approach allowed
for the preparation of oxazolidin-2-one derivative 5 with an overall yield of 34% in four steps, starting from
the Morita-Baylis-Hillman adduct 6.
The bioassay of oxazolidinone 5 against Staphylococcus aureus isolated from animals with bovine
mastitis infections showed a potent antibacterial activity, with an MIC value of 0.03 μg mL−1. Further
studies on the synthesis of other oxazolidinone derivatives beginning with different amino acids are
currently in progress.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/6/7429/s1.
Acknowledgments
The authors thank FAPEMIG, Grant CEX-APQ-00614-08, for financial support.
Author Contributions
Gaspar Diaz conceived the concept, design of study, and wrote the article. Michelle A. A. de Freitas.
synthesized and characterized compounds. Maria E. Ricci-Silva and Marisa A. N. Diaz tested the
biological activity of the compounds and interpreted the results.

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Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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