Discovery of a novel nitroimidazolyl-oxazolidinone hybrid with potent anti Gram-positive activity: Synthesis and antibacterial evaluation

Article abstract of DOI:10.1016/j.ejmech.2010.10.015

A number of linezolid analogues containing a nitroaryl-1,3,4-thiadiazole moiety, were prepared and evaluated as antibacterial agents against a panel of Gram-positive and Gram-negative bacteria. Among synthesized compounds, nitrofuran analogue 1b exhibited more potent inhibitory activity, with respect to other synthesized compounds and reference drug linezolid. The target compounds were also assessed for their cytotoxic activity against normal mouse fibroblast (NIH/3T3) cells using MTT assay. The results indicated that compound 1c exhibit potent antibacterial activity against Gram-positive bacteria at non-cytotoxic concentrations.

Full text of DOI:10.1016/j.ejmech.2010.10.015

European Journal of Medicinal Chemistry 46 (2011) 65e70
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of a novel nitroimidazolyleoxazolidinone hybrid with potent anti
Gram-positive activity: Synthesis and antibacterial evaluation
Ali Khalaj a, Maryam Nakhjiri b, Amir Soheil Negahbani b, Marjaneh Samadizadeh c,
Loghman Firoozpour b, Saeed Rajabalian d, Nasrin Samadi e, Mohammad Ali Faramarzi f,
,d,
Neda Adibpour g, Abbas Shafiee a, Alireza Foroumadi a
*
a Department of Medicinal Chemistry, Faculty of Pharmacy & Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran
b Drug Design & Development Research Center, Tehran University of Medical Sciences, Tehran, Iran
c Department of Chemistry, Science and Research Campus, Islamic Azad University, Tehran, Iran
d Kerman Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran
e Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
f Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14174, Iran
g Department of Medicinal Chemistry, School of Pharmacy, Ahwaz Jundishapur University of Medical Sciences, Ahwaz, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of linezolid analogues containing a nitroaryl-1,3,4-thiadiazole moiety, were prepared and
evaluated as antibacterial agents against a panel of Gram-positive and Gram-negative bacteria. Among
synthesized compounds, nitrofuran analogue 1b exhibited more potent inhibitory activity, with respect
to other synthesized compounds and reference drug linezolid. The target compounds were also assessed
for their cytotoxic activity against normal mouse fibroblast (NIH/3T3) cells using MTT assay. The results
indicated that compound 1c exhibit potent antibacterial activity against Gram-positive bacteria at non-
cytotoxic concentrations.
Received 1 May 2010
Received in revised form
13 October 2010
Accepted 13 October 2010
Available online 10 November 2010
Keywords:
Oxazolidinone
Ó 2010 Elsevier Masson SAS. All rights reserved.
Nitroimidazole
In vitro antibacterial activity
Cytotoxicity
1. Introduction
However, DuP 721 was discontinued following Phase I clinical trials
because it was shown that DuP 721 exhibited lethal toxicity in rats in
Antibiotic resistance is a major problem in hospitals as well as in
community settings. Gram-positive pathogens such as Staphylo-
coccus aureus, Enterococcus faecalis, Enterococcus faecium and
Streptococcus pneumoniae are becoming resistant to most of the
existing antibiotics [1]. Oxazolidinones are a new class of totally
synthetic antibiotics with activity against Gram-positive organisms
such as methicillin-resistant Staphylococcus aureus (MRSA) and
vancomycin-resistant enterococci (VRE) [2e5]. E. I. du Pont de
Nemours & Company scientists were the first to discover, in the late
1970s, the antimicrobial properties of oxazolidinones during their
research on plant antimicrobials [6]. The oxazolidinone, exemplified
by DuP 721 (Fig. 1), is a relatively new class of orally active, totally
synthetic antibacterial agent discovered by scientists at Du Pont [7].
drug safety studies conducted at the Upjohn company. Subsequent
studies at Pharmacia and Upjohn Co. have resulted in two potent
oxazolidinone antibacterial agents, eperezolid and linezolid (Fig. 1)
[8]. These oxazolidinones inhibit protein synthesis by acting against
the formation of the 70S initiation complex, and they are generally
considered to be bacteriostatic [9,10]. The approval of linezolid
(LZD), expanded therapeutic options to include other enterococcal
species and permitted use of oral therapy [11]. Linezolid is currently
approved to treat skin and respiratory infections caused by Gram-
positive pathogens, including multidrug-resistant staphylococci,
enterococci, and streptococci [2]. Although this is a rather expensive
drug, treatments are considered more cost effective than using
competing drugs [12]. Linezolid is considered a relatively safe drug
and the main toxicity issue reported to date refers to the possible
development of thrombocytopenia upon prolonged treatment
[13e15]. Thus, the synthesis and antibacterial evaluation of this
group of compounds in order to modify and eliminate their
unwanted side effects, are in progress [16]. Ranbezolid (Fig. 1), an
* Corresponding author. Department of Medicinal Chemistry, Faculty of Phar-
macy & Pharmaceutical Sciences Research Center, Tehran University of Medical
Sciences, Tehran, Iran. Tel.: þ98 21 66954708; fax: þ98 21 66461178.
E-mail address: aforoumadi@yahoo.com (A. Foroumadi).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.10.015

66
A. Khalaj et al. / European Journal of Medicinal Chemistry 46 (2011) 65e70
O
O
O
O
O
O
N
N
N
H
N
H
N
F
Dup-721
O
O
Linezolid
O
O
N
O
O
O
N
N
F
N
O2N
H
N
N
N
F
H
N
O
HO
O
O
O
Eperezolid
Ranbezolid
a:
O2N
S
N
N
S
N
O
N
N
H
N
Ar =
b:
O2N
Ar
O
F
O
N
1a-c
c:
O2N
N
CH3
Fig. 1. Structures of four recognized oxazolidinone drugs and novel oxazolidinone molecules (1aec).
investigational oxazolidinone [17], with an additional 5-nitrofuran
moiety in oxazolidinone class of antibacterial drugs, showed excel-
lent in vitro activity against Gram-positive pathogens [18]. Ranbe-
zolid has some advantages over linezolid in the oxazolidinone series
and its mode of action against Staphylococci and structural
modeling studies of its interaction with ribosomes have been
recently reported [19].
In recent years, we have reported the synthesis and antibacterial
activity of several nitroaryl-1,3,4-thiadiazole-quinolone hybrids
[20e23], which some of them showed potent antibacterial activity
against Gram-positive bacteria. In continuation our research
program to find new antibacterial agents for the treatment of
infectious diseases, herein we would like to report the synthesis
and in vitro antibacterial profile of a series of nitroaryl-1,3,4-thia-
diazole-oxazolidinone hybrids 1aec (Fig. 1) which led to the
discovery of N-((3-(3-fluoro-4-(4-(5-(1-methyl-5-nitro-1H-imida-
zol-2-yl)-1,3,4-thiadiazol-2-yl)piperazin-1-yl)phenyl)-2-oxoox-
azolidin-5-yl)methyl)acetamide 1c as a potent antibacterial agent
against Gram-positive bacteria.
required 1,3,4-thiadiazoles 6aec were obtained according to the
method previously reported by us [20,24]. The key intermediate
oxazolidinone 16 was prepared according to the method reported
in the literature [25] with some modifications. Thus, the reaction of
commercially available 3,4-difluoronitrobenzene 7 with excess
piperazine, selectively gave the p-substituted nitrobenzene 8.
Catalytic reduction of 8 and subsequent reaction of the amine with
benzylchloroformate afforded carbamate 10. Reaction of 10 with n-
BuLi (THF, ꢀ78 ꢁC) followed by addition of (R)-glycidyl butyrate (11)
provided (5R)-(hydroxymethyl)-2-oxazolidinone 12. Activation of
hydroxyl group as mesylate 13 and subsequent displacement of
mesyl group with phthalimide gave compound 14. Compound 14
was deprotected with methylamine to afford the intermediate 5-
(aminomethyl)oxazolidinone which was reacted with Ac2O to
provide compound 15. Catalytic deprotection of 15 provided the
key intermediate piperazine 16 [25]. Finally, reaction of compounds
6aec with piperazine 16 afforded target compounds 1aec.
3. Pharmacology
2. Chemistry
Compounds 1aec were evaluated for their antibacterial activity
against Gram-positive (Staphylococcus aureus ATCC 6538p, Staphy-
lococcus epidermidis ATCC 12228, Staphylococcus warneri, Staphylo-
coccus lentus, Staphylococcus xylosus, Staphylococcus saprophyticus,
Our synthetic pathways to nitroaryl-1,3,4-thiadiazoles 6aec and
target compounds 1aec are presented in Schemes 1 and 2. The
Ar-CH(OCOCH3)2
i
H
N
N
H
N
N
N
2a,b
ii
NH2
iii
Ar
NH2
Ar
N
Ar
Cl
S
S
S
N
i
6a-c
5a-c
4a-c
O2N
CHO
N
N
CH3
3
c:
a:
O2N
b: O2N
O2N
S
O
N
CH3
Scheme 1. Synthetic procedure for the synthesis of the intermediate compounds 6aec. Reagents and conditions: (i) thiosemicarbazide, EtOH, HCl, reflux; (ii) (NH4) Fe(SO4)2, H2O,
reflux; (iii) NaNO2, HCl, Cu.

A. Khalaj et al. / European Journal of Medicinal Chemistry 46 (2011) 65e70
67
i
iii
ii
F
NO2
HN
N
NO2
HN
N
NH2
F
F
F
7
8
9
O
O
Cbz
Cbz
N
N
N
H
O
iv, v
O
vi
Cbz
Cbz
N
N
N
OH
O
F
F
10
12
O
O
O
O
vii
O
N
N
N
N
N
N
N
O
SO2Me
F
F
O
14
13
O
O
H
N
viii
ix
O
x
Cbz
N
N
N
N
HN
N
N
H
N
F
F
O
16
15
O
O
N
1a:
O2N
S
N
O
xi
N
N
H
N
1b: O2N
1c: O2N
Ar =
O
S
Ar
F
N
O
1a-c
N
CH3
Scheme 2. Synthetic procedure for the synthesis of the target compounds 1aec. Reagent and condition: (i) Piperazine, CH3CN, reflux; (ii) H2, 5% Pd/C, THF; (iii) Benzylox-
ycarbonyleCl (CbzeCl), NaHCO3 (or Na2CO3), acetoneeH2O; (iv) n-BuLi, THF ꢀ78 ꢁC; (v) (R)-glycidyl butyrate (11); (vi) MsCl, Et3N, CH2Cl2; (vii) potassium phthalimide, CH3CN, H2O,
reflux; (viii) aqueous MeNH2, EtOH, reflux; (ix) Ac2O, pyr; (x) Pd/C, H2, MeOHeCH2Cl2; (xi) (6aec), Et3N, EtOH, reflux.
Micrococcus luteus, Corynebacterium glutamicum, Bacillus subtilis
PTCC 1023, MRSA 3, MRSA 5 and MRSA 17) and Gram-negative
(Escherichia coli ATCC 8739, Klebsiella pneumonia ATCC 10031, and
Pseudomonas aeruginosa ATCC 9027) bacteria using conventional
agar-dilution method [26]. The MIC (minimum inhibitory concen-
tration) values were determined by comparison to linezolid and
ciprofloxacin as reference drugs.
Antibacterial evaluation of compounds 1aec and 16 against
Micrococcus luteus reveals that compounds 1c (MICs 0.195 g/mL)
followed by 1b (MICs 0.391 g/mL) possessed a significant activity.
In addition compounds 1b and 1c were the most potent against
C. glutamicum, S. sarophyticus and MRSA 5 with MIC values of
m
m
0.024e0.195
mg/mL. Moreover the latter compounds were more
potent than reference drugs. Furthermore, compound 1b was the
most active compound against S. aureus, S. epidermidis and MRSA
3. Generally, most of the compounds have shown poor or no
4. Results
activity (MIC ꢂ 6.25
the exception of compound 1c against K. pneumonia which
showed potency (MIC ¼ 0.781 g/mL), although not superior to
those of ciprofloxacin and linezolid (MICs ¼ 0.195 and 0.391 g/
mg/mL) against Gram-negative bacteria with
The individual minimum inhibitory concentrations (MICs, mg/
mL) obtained for compounds 1aec against Gram-positive and
Gram-negative are presented in Table 1. The IC50 values obtained
for in vitro cytotoxic activity of the test compounds 1aec and 16
against normal mouse fibroblast cell line (NIH/3T3) are shown in
Table 2.
m
m
mL, respectively). Eventually it can be inferred from Table 1 that
compound 16 exhibited weak activity against all of the Gram-
positive and Gram-negative bacteria; moreover, among
compounds tested, the 5-nitrofuran counterpart 1b, showed the
highest activity against nearly all of the tested Gram-positive
bacteria, superior to the reference drugs linezolid and
ciprofloxacin.
5. Discussion
MIC values of the tested derivatives indicate that compounds
1aec and 16 showed a higher antibacterial activity against Gram-
positive rather than Gram-negative bacteria. Table 1 reveals that
For further pharmacological study, in vitro cytotoxic activity of
the test compounds 1aec and 16 against normal mouse fibroblast
cell line (NIH/3T3) was investigated using MTT colorimetric assay
[27]. The IC50 values obtained for these compounds are shown in
Table 2. While nitrothiophene and nitrofuran derivatives (1a and
1b) showed moderate toxicity against mouse fibroblast cell line,
nitroimidazole analogue (1c) showed no toxicity at concentration
compounds 1b and 1c (MICs 0.006 mg/mL) followed by 1a were
superior in inhibiting the growth of Staphylococcus warneri in
comparison to linezolid and ciprofloxacin as reference drugs.
Compound 1b was the most active compound against S. lentus, its
activity was found to be 65e130 times better than reference drugs.

68
A. Khalaj et al. / European Journal of Medicinal Chemistry 46 (2011) 65e70
Table 1
In vitro antibacterial activities of compounds 1aec, 16 and reference drugs against selected strains (MICs in
mg/mL).
Microorganismsa
1a
1b
1c
16
Linezolid
Ciprofloxacin
B. s
C. g
E. c
K. p
0.781
6.25
>100
100
6.25
0.391
0.195
>100
50
0.391
0.098
0.195
>100
0.781
12.5
1.563
50
25
1.563
25
0.391
0.781
>100
6.25
0.195
0.391
0.012
0.003
3.125
0.391
0.391
0.391
0.0391
0.195
0.391
0.781
0.098
0.195
0.391
M. l
0.195
0.391
0.195
0.024
0.781
0.781
0.781
0.781
>100
0.781
0.781
1.563
0.781
0.781
0.781
MRSA 3
MRSA 5
MRSA 17
P. a
S. a
S. e
S. l
S. s
S. w
S. x
0.195
6.25
0.098
0.049
0.195
0.012
12.5
25
>100
>100
>100
>100
12.5
6.25
12.5
3.125
18
3.125
0.098
0.024
0.098
0.195
0.049
0.098
0.012
0.006
0.012
0.024
0.006
0.024
0.098
0.024
0.098
0.024
0.006
0.049
a
B. s: Bacillus subtilis PTCC 1023; C. g: Corynebacterium glutamicum ATCC 13032; E. c: Escherichia coli ATCC 8739; K. p: Klebsiella pneumonia ATCC 10031; M. l: Micrococcus
luteus ATCC 9341; MRSA 3; MRSA 5; MRSA 17; P. a: Pseudomonas aeruginosa ATCC 9027; S. a: Staphylococcus aureus ATCC 6538p; S. e: Staphylococcus epidermidis ATCC 12228; S.
l: Staphylococcus lentus ATCC 29070; S. s: Staphylococcus saprophyticus ATCC 15305; S. w: Staphylococcus warneri ATCC 27836; S. x: Staphylococcus xylosus ATCC 29971.
of 200
m
g/mL (IC50 > 200
m
g/mL). The same result was obtained for
solvents and reagents were purchased from Merck, Aldrich and
compound 16 (Table 2).
Fluka and used as such without purification.
Among target compounds, the linezolid analogue 1c, showed
the highest inhibition against nearly all of the tested Gram-positive
bacteria, superior to the reference drugs, and displayed antibacte-
rial activity at non-cytotoxic concentrations.
7.1. 1-(2-Fluoro-4-nitrophenyl)piperazine (8)
A solution of 12.0 g (75.42 mmol) of 3,4-difluoronitrobenzene in
150 mL of acetonitrile was treated with 16.24 g (188.6 mmol) of
piperazine followed by warming at reflux for 3 h. The solution was
cooled to room temperature and concentrated in vacuo. The
resulting residue was diluted with 200 mL of water and extracted
with ethyl acetate (3 ꢃ 250 mL). The combined organic layers were
extracted with water (200 mL) and saturated sodium chloride
solution (200 mL) followed by drying on sodium sulfate. The
solution was concentrated in vacuo to afford an orange solid which
was purified by gradient chromatography eluting initially with
dichloromethane until the least polar fractions had eluted, and then
elution was continued with 2% (v/v) ethanoledichloromethane and
then with 10% (v/v) ethanoledichloromethane. These procedures
afforded compound 8, Yield 85%; mp 68.5e71 ꢁC; IR: ymax (KBr)
3022 (NH), 1518 and 1336 (NO2) cmꢀ1; 1H NMR (CDCl3): 7.94 (ddd,
1H, J ¼ 9, 2.5 and 1 Hz, H5 nitrobenzene), 7.85 (dd, 1H, J ¼ 13 and
2.5 Hz, H3 nitrobenzene), 6.88 (t, 1H, J ¼ 9 Hz, H6 nitrobenzene),
3.25e3.23 (m, 4H, piperazine), 3.04e3.01 (m, 4H, piperazine); MS:
m/z 225 (37, Mþ), 183 (100), 137 (37), 58 (43); Anal. Calcd for
C10H12FN3O2: C, 53.33; H, 5.37; F, 8.44; N, 18.66. Found: C, 52.97; H,
5.21; F, 8.65; N, 18.52.
6. Conclusion
In light of this study, it can be inferred that the antibacterial and
cytotoxic properties of target compounds are mainly determined
by heteroaryl substituent and this scaffold can be served as
a promising substituent for oxazolidinone class of antibacterial
drugs.
In conclusion, we report the discovery of N-((3-(3-fluoro-4-
(4-(5-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thiadiazol-2-yl)
piperazine-1-yl)phenyl)-2-oxooxazolidine-5-yl)methyl)acetamide
1c as a potent antibacterial agent against Gram-positive bacteria.
7. Experimental protocols
Melting points were taken on a Kofler hot stage apparatus and
are uncorrected. The FT-IR spectra were obtained using a Nicolet
550 spectrometer (Potassium bromide disks). The 1H NMR and 13
C
NMR spectra were recorded on Bruker FT-500 and Bruker FT-80.
Chemical shifts ( ) are in parts per million relative to internal tet-
d
7.2. N-Carbobenzoxy-3-fluoro-4-(N-carbobenzoxypiperazinyl)
aniline (10)
ramethylsilane. The mass spectra were run on a Finnigan mat TSQ-
70 spectrometer at 70 eV. Thin layer chromatography (TLC) was
performed on plates of silica gel 60 F254 plates. Silica gel 60,
0.040e0.063 mm (230e400 mesh) was used for flash column
chromatography. All solvents used were reagent grade. Most of the
A mixture of 12.5 g (0.056 mol) of 8 and 1.4 g of 5% palladium on
carbon in 115 mL of THF was stirred under 40 psi of H2 for 1.5 h,
while maintaining the reaction at room temperature. The reaction
mixture was filtered through diatomaceous earth and the pad
washed with 2 ꢃ 50 mL of THF. The filtrate was concentrated, and
the solid 9 was azeotroped with 43 mL of acetone. The crude amine
was immediately dissolved in 69 mL of acetone and added to
a 500 mL three-neck flask equipped with a mechanical stirrer,
containing 138 mL of 10% aqueous sodium carbonate. The mixture
was cooled to 0 ꢁC, and 18 mL (0.127 mol) of benzylchloroformate
was added dropwise over 20 min while maintaining the tempera-
ture between 0 and 5 ꢁC. The mixture was then stirred for 1 h at 5 ꢁC
and then allowed to stir overnight at room temperature. The
mixture was decanted, and the product was extracted with
Table 2
Cytotoxic activity of compounds 1aec and 16, against
mouse fibroblast (NIH/3T3) cell line.
Compound
IC50 (m
g/mL)a
1a
1b
1c
16
184 ꢄ 4.2
21.8 ꢄ 3
>200
>200
a
IC50 is the concentration required to inhibit 50% of
cell growth. The values represent mean ꢄ SD.

A. Khalaj et al. / European Journal of Medicinal Chemistry 46 (2011) 65e70
69
chloroform (3 ꢃ 50 mL) and dried over sodium sulfate then dried in
vacuo, and then was decolorized by activated charcoal in hot
methanol and filtrated to give off-white solid compound 10. Yield
52%; mp 159e161 ꢁC; IR: ymax (KBr) 3283 (NH), 1716 and 1690
was triturated with 100 mL of ethyl acetateehexane (1:1) followed
by the addition of 50 mL of ethyl acetate. Upon concentration of the
mixture under reduced pressure to 25 mL, a white precipitate
formed; this was cooled to 4 ꢁC, and the solids were collected to
provide compound 14. The filtrate was passed over a silica gel
column, eluting with methylene chloride. The appropriate fractions
were combined to provide 14 as a light yellow solid. Yield 66%; mp
(C]O) cmꢀ1 1H NMR (CDCl3): 7.39e7.32 (m, 10H, phenyl), 6.98
;
(d, 1H, J ¼ 9 Hz, H2 nitrobenzene), 6.85 (t, 1H, J ¼ 9 Hz, H6 nitro-
benzene), 6.70 (s (brs),1H, H5 nitrobenzene), 5.19 (s, 2H, CH2O), 5.17
(s, 2H, CH2O), 3.67 (t, 4H, J ¼ 5 Hz, piperazine), 2.99 (s (brs), 4H,
piperazine); MS: m/z 463 (54, Mþ), 328 (40), 91 (100), 88 (66); Anal.
Calcd for C26H26FN3O4: C, 67.37; H, 5.65; F, 4.10; N, 9.07. Found: C,
67.58; H, 5.26; F, 4.32; N, 8.84.
127e129 ꢁC; IR: ymax (KBr) 1746 and 1710 (C]O) cmꢀ1 1H NMR
;
(CDCl3): 8.00e7.52 (m, 4H, H phtalimide), 7.56e7.30 (m, 7H, H
Phenyl, H2 and H6 flurobenzene), 5.14 (s, 2H, CH2 benzylic),
5.00e4.58 (m, 1H, H5 oxazolidinone), 4.40e4.20 (m, 2H, CH2N),
4.15e3.90 (m, 2H, H4 oxazolidinone), 3.69 (t, 4H, J ¼ 5 Hz, pipera-
zine), 3.00 (t, 4H, J ¼ 5 Hz, piperazine); Anal. Calcd for C30H27FN4O6:
C, 64.51; H, 4.87; F, 3.40; N, 10.03. Found: C, 64.32; H, 5.21; F, 3.31;
N, 10.23.
7.3. (R)-[N-3-[3-fluoro-4-[N-1-(4-carbobenzoxy)piperazinyl]-
phenyl]-2-oxo-5-oxazolidinyl] methanol (12)
To a mixture of 4.33 g (10.06 mmol) of 10 in 50 mL of dry THF at
ꢀ78 ꢁC under argon was added 6.8 mL (10.88 mmol) of 1.6 M
n-butyllithiumehexane dropwise over 5 min. After 1.5 h, 1.55 mL of
(R)-glycidyl butyrate 11 was added and the mixture allowed stirring
at ꢀ78 ꢁC for 1 h and then at room temperature for 3.5 h (the
mixture became thick with solid precipitation). Saturated aqueous
ammonium chloride (25 mL) was added followed by 25 mL of ethyl
acetate and 5 mL of water. The layers were separated, and the
aqueous layer was extracted with ethyl acetate (3 ꢃ 25 mL). The
combined organic layers were dried (Na2SO4) and concentrated
under reduced pressure to provide 12 as a white solid. The solid was
triturated with ethyl acetateehexane (1: 1) in a warm water bath
for 30 min and then refrigerated; the solids were collected by
vacuum filtration to provide 12 as white solids. Yield 82%; mp
7.5. (S)-N-[[3-[3-Fluoro-4-[N-1-(4-carbobenzoxy)piperazinyl]-
phenyl]-2-oxo-5-oxazolidinyl] methyl]acetamide (15)
A mixture of 1.51 g (2.7 mmol) of 14 and 3 mL of 40% methyl-
amine in water (34.85 mmol) and 30 mL of ethanol was heated to
reflux for 5.5 h; then additional methylamine solution (0.5 mL) was
added. The mixture was heated to reflux for 1 h and then
concentrated under reduced pressure. The residue was dissolved in
15 mL of pyridine, the mixture cooled to 0 ꢁC, and acetic anhydride
(5 mL) added. The mixture was allowed to stir at room temperature
overnight and then concentrated under reduced pressure to
provide crude 15 as a white solid. The crude was placed upon
a silica gel column and eluted with an ethanolemethylene chloride
gradient (0e7% EtOH); the appropriate fractions were combined to
provide compound 15 as a white solid. Yield 33%; mp 174e176 ꢁC;
IR: ymax (KBr) 3313 (NH), 1751, 1685 and 1644 (C]O) cmꢀ1; 1H NMR
(CDCl3): 7.45 (dd, 1H, J ¼ 14.5 and 2.5 Hz, H2 fluorobenzene),
7.39e7.37 (m, 4H, H2, H3, H5 and H6 phenyl), 7.36e7.32 (m, 1H, H4
phenyl), 7.09e7.06 (m, 1H, H6 fluorobenzene), 6.92 (t, 1H, J ¼ 9 Hz,
H5 fluorobenzene), 5.97 (t, 1H, J ¼ 6 Hz, CONH), 4.80e4.74 (m, 1H,
H5 oxazolidinone), 4.50e4.00 (t, 1H, J ¼ 9 Hz, CH2NH), 3.76e3.71
(m, 2H, CH2NH and H4 oxazolidinone), 3.68 (t, 4H, J ¼ 5 Hz,
piperazine), 3.64e3.58 (m, 1H, H4 oxazolidinone), 3.08e2.98 (m,
4H, J ¼ 5 Hz, piperazine), 2.03 (s, 3H, CH3CO); MS: m/z 470 (12, Mþ),
426 (12), 335 (5), 177 (5), 105 (5), 91 (100), 56 (38); Anal. Calcd for
C25H29FN4O5: C, 61.97; H, 6.03; F, 3.92; N, 11.56. Found: C, 61.74; H,
5.69; F, 3.99; N, 11.27.
149e150 ꢁC; IR: ymax (KBr) 3426 (OH), 1741 and 1664 (C]O) cmꢀ1
;
1H NMR (CDCl3): 7.46 (dd, 1H, J ¼ 14.5 and 2.5 Hz, H2 fluo-
robenzene), 7.39e7.37 (m, 4H, H2, H3, H5 and H6 phenyl), 7.36e7.32
(m, 1H, H4 phenyl), 7.13 (dd,1H, J ¼ 9 and 2.5 Hz, H6 fluorobenzene),
6.92 (t, 1H, J ¼ 9 Hz, H5 fluorobenzene), 5.17 (s, 2H, benzylic CH2),
4.77e4.72 (m, 1H, H5 oxazolidinone), 4.03e3.98 (m, 2H, CH2OH),
3.97e3.93 (m, 1H, H4 oxazolidinone), 3.8e3.74 (m, 1H, H4 oxazoli-
dinone), 3.68 (t, 4H, J ¼ 5 Hz, piperazine), 3.02 (t, 4H, J ¼ 5 Hz,
piperazine); MS: m/z 429 (66, Mþ), 338 (25), 294 (33), 265 (40), 239
(25), 91 (100); Anal. Calcd for C22H24FN3O5: C, 61.53; H, 5.63; F,
4.42; N, 9.78. Found: C, 61.32; H, 5.54; F, 4.67; N, 10.02.
7.4. (R)-N-[[3-[3-Fluoro-4-[N-1-(4-carbobenzoxy)piperazinyl]-
phenyl]-2-oxo-5-oxazolidinyl]methyl]phthalimide (14)
To a mixture of 3.5 g (8.15 mmol) of 12 and 2.5 mL (17.97 mmol)
of triethylamine in 88 mL of methylene chloride at 0 ꢁC was added
0.75 mL (9.69 mmol) of methanesulfonyl chloride dropwise over
2 min. The mixture was stirred at 0 ꢁC for 1.5 h and at room
temperature for 3 h, the mixture was washed with 74 mL water, and
the aqueous layer was extracted with methylene chloride (13 mL).
Ethyl acetate (63 mL) was added to the combined organic layers;
these were dried (Na2SO4) and concentrated under reduced pres-
sure to give 13 as a yellow foamy solid.
7.6. (S)-N-[[3-[3-Fluoro-4-(N-1-piperazinyl)phenyl]-2-oxo-5-
oxazolidinyl]methyl]acetamide, hydrochloride (16)
A mixture of 0.4 g (0.85 mmol) of 15 and 0.06 g of 10% palladium
on carbon in 19 mL of methanol and 6 mL of methylene chloride
was stirred under hydrogen for 48 h. The mixture was then filtered
through diatomaceous earth. The filter cake was washed with
50 mL of methylene chloride followed by 25 mL of ethyl acetate,
and the filtrate was concentrated to give off-white solid. The solid
was triturated with 50 mL of 10% methanoleethyl acetate in
a warm water bath for 30 min, cooled to 0 ꢁC and then was collected
to provide 16 as a white solid. Yield 91%; mp 198e200 ꢁC; IR: ymax
(KBr) 3436 and 3329 (NH), 1741 (C]O) and 1644 (CONH) cmꢀ1; 1H
NMR (DMSO): 9.03 (s (brs), 2H, NHþ2 ), 8.26 (t, 1H, J ¼ 6 Hz, CONH),
7.50 (dd, 1H, J ¼ 15 and 2.5 Hz, H2 fluorobenzene), 7.19 (dd, 1H, J ¼ 9
and 2.5 Hz, H6 fluorobenzene), 7.12 (t, 1H, J ¼ 9 Hz, H5 fluo-
robenzene), 4.71e4.68 (m, 1H, H5 oxazolidinone), 4.07 (t, 1H,
J ¼ 9 Hz, CH2NH), 3.69 (dd, 1H, J ¼ 9 and 6 Hz, CH2NH), 3.39 (t, 2H,
J ¼ 6 Hz, H4 oxazolidinone), 3.22e3.21 (m, 4H, piperazine),
3.17e3.16 (m, 4H, piperazine), 1.81 (s, 3H, CH3CO); MS: m/z 337 (45,
Mþ), 295 (100), 250 (53), 191 (32), 57 (32); Anal. Calcd for
IR: ymax (KBr) 1751 and 1695 (C]O) cmꢀ1 1H NMR (CDCl3):
;
7.56e7.32 (m, 6H, H Phenyl, H2 fluorobenzene), 7.1e6.53 (m, 2H, H5
and H6 fluorobenzene), 5.16 (s, 2H, CH2 benzylic), 5.05e4.53 (m, 1H,
H5 oxazolidinone), 4.54e4.40 (m, 2H, CH2O), 4.30e3.53 (m, 2H, H4
oxazolidinone), 3.52e3.56 (m, 4H, piperazine), 3.15e2.95 (m, 7H,
CH3 and piperazine); Anal. Calcd for C23H26FN3O7S: C, 54.43; H,
5.16; F, 3.74; N, 8.28; S, 6.32. Found: C, 54.79; H, 4.88; F, 3.51; N,
8.39; S, 6.04.
This was taken up into 126 mL of acetonitrile and 0.63 mL of
water and 4.57 g (24.55 mmol) of potassium phthalimide was
added. The mixture was heated to reflux for 48 h and then
concentrated under reduced pressure to a yellow gum. The gum

70
A. Khalaj et al. / European Journal of Medicinal Chemistry 46 (2011) 65e70
C17H24ClFN4O3: C, 52.78; H, 6.25; Cl, 9.16; F, 4.91; N, 14.48. Found: C,
52.94; H, 6.10; Cl, 9.24; F, 4.73; N, 14.60.
J ¼ 9 Hz, H5 fluorobenzene), 5.94 (s (brs), 1H, CONH), 4.78e4.75 (m,
1H, H5 oxazolidinone), 4.50 (s, 3H, NMe), 4.03 (t, 1H, J ¼ 9 Hz,
CH2NH), 3.82 (t, 4H, J ¼ 5 Hz, piperazine), 3.75 (dd, 1H, J ¼ 9 and
7 Hz, CH2NH), 3.71e3.70 (m, 1H, H4 oxazolidinone), 3.69e3.59
(m, 1H, H4 oxazolidinone), 3.22 (t, 4H, J ¼ 5 Hz, piperazine), 2.02
(s, 3H, CH3CO); 13C NMR (DMSO, 125 MHz): 172.2, 169.9, 155.6,
153.9, 153.6, 149.3, 141.0, 140.4, 133.8, 133.1, 119.9, 114.1, 106.7, 106.5,
71.5, 49.5, 47.3, 41.3, 39.6, 35.0, 22.4; MS: m/z 545 (13, Mþ), 502
(43), 307 (57), 263 (100), 235 (57), 57 (37); Anal. Calcd for
C23H26FN9O5S: C, 49.37; H, 4.68; F, 3.40; N, 22.53; S, 5.73. Found: C,
49.62; H, 4.49; F, 3.72; N, 22.82.
7.7. General procedure for the synthesis of (S)-N-[[3-[3-fluoro-4-
[4-(5-aryl-1,3,4-thiadiazol-2-yl)-1-piperazinyl] phenyl]-2-oxo-5-
oxazolidinyl]methyl]acetamides (1aec)
To a mixture of 0.06 g (0.16 mmol) of 16 and 0.16 mmol of
appropriate chloro-1,3,4-thiadiazole 6aec in 20 mL of ethanol,
0.2 mL triethylamine was added. The mixture was heated under
reflux for 6 h (3 h for imidazole derivative) and then concentrated
under reduced pressure. The crude product was placed on a silica
gel column and eluted with ethanolemethylene chloride 5% to
provide compounds 1aec.
Acknowledgments
This work was supported by grants from Tehran University of
Medical Sciences and Iran National Science Foundation (INSF).
7.7.1. (S)-N-[[3-[3-Fluoro-4-[4-[5-(5-nitrothiophene-2-yl)-1,3,4-
thiadiazol-2-yl]-1-piperazinyl] phenyl]-2-oxo-5-oxazolidinyl]
methyl]acetamide (1a)
Appendix. Supplementary data
Yield 41%; mp 271e273 ꢁC (dec); IR: ymax (KBr) 3339 (NH), 1746
(C]O), 1659 (CONH), 1521 and 1347 (NO2) cmꢀ1; 1H NMR (CDCl3):
7.87 (d, 1H, J ¼ 4 Hz, H4 thiophene), 7.56e7.46 (m, 1H, H2 fluo-
robenzene), 7.40 (d, 1H, J ¼ 4 Hz, H3 thiophene), 7.10 (dd, 1H, J ¼ 9
and 2.5 Hz, H6 fluorobenzene), 6.95 (t, 1H, J ¼ 9 Hz, H5 fluo-
robenzene), 5.92 (t, 1H, J ¼ 6 Hz, CONH), 4.78e4.75 (m, 1H, H5
oxazolidinone), 4.03 (t, 1H, J ¼ 9 Hz, CH2NH), 3.80 (t, 4H, J ¼ 5 Hz,
piperazine), 3.78e3.74 (m, 1H, CH2NH), 3.72e3.69 (m, 1H, H4 oxa-
zolidinone), 3.64e3.58 (m, 1H, H4 oxazolidinone), 3.21 (t, 4H,
J ¼ 5 Hz, piperazine), 2.02 (s, 3H, CH3CO); 13C NMR (DMSO,
125 MHz): 172.7, 169.9, 155.6, 153.9, 153.6, 150.2, 149.6, 139.6, 133.8,
130.7, 119.9, 114.0, 106.7, 106.5, 71.5, 49.5, 47.3, 41.3, 39.4, 22.4; MS:
m/z 547 (5, Mþ), 528 (2), 504 (12), 320 (19), 306 (100), 263 (25), 234
(19), 15 (19), 57 (19); Anal. Calcd for C23H24FN7O5S2: C, 49.19; H,
4.31; F, 3.38; N, 17.46. Found: C, 49.39; H, 4.08; F, 3.11; N, 17.69.
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2010.10.015.
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7.52e7.47 (m, 1H, H2 fluorobenzene), 7.44 (d, 1H, J ¼ 4 Hz, H4 furan),
7.18 (d, 1H, J ¼ 4 Hz, H3 furan), 7.10 (dd, 1H, J ¼ 9 and 2 Hz, H6
fluorobenzene), 6.96 (t, 1H, J ¼ 9 Hz, H5 fluorobenzene), 5.92 (t, 1H,
J ¼ 6 Hz, CONH), 4.78e4.75 (m, 1H, H5 oxazolidinone), 4.03 (t, 1H,
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1H, CH2NH), 3.72e3.69 (m, 1H, H4 oxazolidinone), 3.64e3.58 (m,
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CH3CO); 13C NMR (DMSO, 125 MHz): 172.4, 169.9, 155.6, 153.9,
153.6, 151.5, 147.2, 145.5, 133.9, 131.6, 119.9, 114.0, 106.6, 106.5, 71.5,
49.5, 47.3, 41.3, 39.5, 22.3; MS: m/z 531 (31, Mþ), 512 (38), 488 (52),
319 (52), 306 (100), 263 (100), 235 (72), 177 (58), 152 (64), 57 (50);
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5.88. Found: C, 50.92; H, 4.68; F, 3.21; N, 18.09.
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7.7.3. (S)-N-[[3-[3-Fluoro-4-[4-[5-(1-methyl-5-nitro-1H-
imidazole-2-yl)-1,3,4-thiadiazol-2-yl]-1-piperazinyl] phenyl]-2-
oxo-5-oxazolidinyl]methyl]acetamide (1c)
Yield 80%; mp 261e262 ꢁC; IR: ymax (KBr) 3431 (NH), 1741 (C]
O), 1654 (CONH), 1516 and 1362 (NO2) cmꢀ1; 1H NMR (DMSO): 8.05
(s,1H, imidazole), 7.49 (dd,1H, J ¼ 14 and 2.5 Hz, H2 fluorobenzene),
7.10 (dd, 1H, J ¼ 9 and 2.5 Hz, H6 fluorobenzene), 6.96 (t, 1H,
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