Synthesis and in vitro activity of new methylenepiperidinyl and methylenepyrrolidinyl oxazolidinone antibacterial agents

Article abstract of DOI:10.1016/S0960-894X(03)00249-X

We have prepared and evaluated the antibacterial activities of a series of substituted methylenepiperidinyl and methylenepyrrolidinyl oxazolidinones against several gram-positive strains including the resistant strains of Staphyloccus and Enterococcus, such as MRSA, CRSA, MSSA and VRE. Some of them showed comparable or superior in vitro activities (MIC) to vancomycin.

Full text of DOI:10.1016/S0960-894X(03)00249-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2227–2230
Synthesis and In Vitro Activity of New Methylenepiperidinyl and
Methylenepyrrolidinyl Oxazolidinone Antibacterial Agents
Hye Yeon Kim, Jae Seok Lee, Joo Hwan Cha, Ae Nim Pae,
Yong Seo Cho, Moon Ho Chang and Hun Yeong Koh*
Biochemicals Research Center, Korea Institute of Science and Technology, Cheongryang, Seoul 130-650, South Korea
Received 4 January 2003; revised 12 March 2003; accepted 13 March 2003
Abstract—We have prepared and evaluated the antibacterial activities of a series of substituted methylenepiperidinyl and methyl-
enepyrrolidinyl oxazolidinones against several gram-positive strains including the resistant strains of Staphyloccus and Enterococcus,
such as MRSA, CRSA, MSSA and VRE. Some of them showed comparable or superior in vitro activities (MIC) to vancomycin.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
The oxazolidinones are an exciting new development in
the effective treatment of Gram-positive bacterial infec-
tions, including those infections caused by strains resis-
tant to other antibiotics. The rising prevalence of
multidrug resistant Gram-positive bacteria requires the
discovery of novel active agents against these patho-
gens. New classes of antibacterial agents with novel
mechanisms of action are urgently needed to combat the
increase in multidrug resistant infections. The oxazoli-
dinones, a new class of totally synthetic antibacterial
agents, are active against a variety of clinically
important susceptible and resistant Gram-positive
organisms such as methicillin-resistant Staphylococcus
aureus (MRSA), vancomycin-resistant Enterococcus
faecium (VRE), and penicillin-resistant Streptococcus
pneumoniae (PRSP). Scientists at DuPont originally
discovered this class of agents in the late 1980’s.1
However, development of DuP-721, the drug candi-
date that emerged from these initial studies, was dis-
Recently, our laboratory revealed that two series of
oxazolidinones having an isoxazole as a rigid bioisostere
of hydroxymethyl group of eperezolid were synthesized
and the effect of introducing the isoxazole moiety on the
activity was investigated.3 To improve biological activ-
ity, in the present work we describe the synthesis and
antibacterial activity of a related family of compounds 5
and 6 in which the substituted methylenepiperidinyl and
methylenepyrrolidinyl ring were introduced. Several
potent compounds were studied in vivo activities and
pharmakokinetic profile.
continued following Phase
I
clinical trials.
Subsequently, researchers at Pharmacia and Upjohn
identified two clinical candidates, eperezolid (1) and
linezolid (2).2 Linezolid is currently marketed for the
treatment of multidrug resistant Gram-positive infec-
tions such as nosocomial and community-acquired
pneumonia and skin infections.
Chemistry
The synthesis of the oxazolidinone derivatives is out-
lined in Scheme 1. To prepare the oxazolidinone deri-
vatives shown in Table 1, we could use the Knoevenagel
condensation reaction as a key step.4 The ketone inter-
mediates 3 and 4, which were prepared by known
*Corresponding author. Tel.: +822-958-5146; fax: +822-958-5189;
e-mail: hykoh@kist.re.kr
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00249-X

2228
H. Y. Kim et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2227–2230
Scheme 2.
reflux was required to confirm the reactions completed
in 15–98%.9
Scheme 1.
As reported by Upjohn,10 thioacetamide of 5-position
of oxazolidinone showed 2-fold good activity compared
to the acetamide form. Thus, the most potent com-
pound 6c synthesized was treated with Lawesson’s
reagent to transform into thioacetamide 6u (Scheme 2)
and it was also evaluated in vitro activity.
Table 1. Synthesized oxazolidinones of 5 and 6
Compd
Methoda
R1
R2
Yield (%)b
5a
5b
5c
5d
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
A
A
B
B
A
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
CN
CN
H
CH3
CN
CN
H
H
H
CH3
H
Cl
CN
H
H
H
H
H
H
H
H
CN
CO2Et
CN
CN
CN
CO2Et
CN
CO2Et
COCH3
CO2Et
COONa
CO2Et
CH3
CHO
51
50
98
98
69
46
64
64
91
54
13
35
32
8
71
57
43
74
77
57
70
69
In Vitro and In Vivo Activity
In vitro antibacterial activities of all the compounds
prepared and references were determined by the Muel-
ler–Hinton agar dilution method.11 The activities of
compounds synthesized were compared with linezolid,
and vancomycin as references. Data for selected Gram-
positive organisms are reported as a minimum inhibi-
tory concentration (MIC) expressed in umg/mL (Tables
2and 3 ). Most of the compounds synthesized exhibited
good antibacterial activities against Gram-positive
strains and the resistant strains including MRSA
(methicillin-resistant Staphylococcus aureus), CRSA
(ciprofloxacin-resistant Staphylococcus aureus), MSSA
(methicillin-susceptible Staphylococcus aureus) and
VRE (vancomycin-resistant Enterococcus faecium).
CH(NOH)
CH(NOCH3)
C(NOH)CH3
C(NOCH3)CH3
CH(OH)CH3
CH(OAc)CH3
C(OCOCH2Cl)2CH3
C(OCOCHCl2)CH3
6m
6n
6o
6p
6q
6r
H
6s
6t
B
B
H
H
20
15
As results of screening of compounds 6a–t, compound
6c and 6u showed the most potent activities in this series
relative to linezolid and vancomycin against Gram-
positive organisms (Table 2). Especially, 6u showed 8-
fold and 4-fold good activity than linezolid against Sta-
phylococcus pyogenes C6003 (S.p.2) and VRE, respec-
tively. The cyano group might be playing crucial role
for the activity as a substituent.
aMethod A; Knoevenagel condensation, Method B; Wadsworth–
Horner–Emmons reaction.
bIsolated yield.
method,5,6 with the malononitrile and cyanoacetic ester
using aluminum oxide gave the product 5 and 6 in 46–
69% yield after chromatographic purification on silica
gel (5% methanol-ethyl acetate).7
In our previous study,3 5-isoxazolyl moiety was effective
to the activity as a rigid bioisostere of the hydroxy-
acetamide functionality of eperezolid (1). According to
this result, heterocyclyl methylene 6s–t were synthesized
and evaluated. The replacement of cyanomethylene with
heterocyclyl methylene led to less potent compounds.
By investigation of the effect of substituents R1 and R2,
the introduction of polar, simple functional group to the
oxzolidinone derivatives were effective for the activity.
The replacement by thioacetamide achieved 2-fold
increase of activities against most of strains tested and
especially 8-fold enhancement against VRE. For the
In another way, the new oxazolidinone derivatives also
can be prepared via Wadsworth–Horner–Emmons
reaction.8 As shown in Scheme 1, a variety of phospho-
nate anions have been prepared via reaction of phospho-
nates with sodium hydride or n-butyllithium in freshly
distilled tetrahydrofuran for 1 h. Then compounds 3 or
4 were added to the preformed phosphonate anions,
and the reaction mixture was stirred for a few hours, for
some time, relatively rigorous reaction condition such as

H. Y. Kim et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2227–2230
2229
Table 2. In vitro antibacterial activity of oxazolidinone derivatives
against 11 bacterial strains (MIC, mg/mL)a
Table 4. Single dose pharmacokinetic parameters for oxazolidinone
antibiotics in male rates
Compd
Microorganismb
Compd
AUC
(mg.h/mL)
Tmax (h)
Cmax (mg/mL)
T1/2(h)
S.a. MRSA CRSA MSSA S.e. S.e. E.f. E.f. S.p. S.p. VRE
2
1
1
2
1
2
6c
Linezolid
6.807
4.239
0.5
0.333
1.321
1.389
8.139
3.093
6a
6b
6c
6d
6e
2
4
21
16
8
16
2
4
4
1
4
2
1
4
21
4
4
4
4
8
4
2
2 4 0.5 1 0.522 2 4
21
4
2
Each compound was dissolved in 50% PEG solution and orally
administered to rats at a dose of 15 mg/Kg.
8
16
8
16
16
16
8
4
1
4
1
8
8
8
1
8
8 28
16 8 0.25 16
8
16
8
8
8
Table 5. In vivo activity of selected compounds
6f
8
8
8 4
4 2
4
16
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
LZc
VCMd
8
3216
22 2 02.50.522 2
8
8
8
8
16
8
16
16
16
3216
16
4
8
8
8
4
a
Microorganism Challenge dose Route Compd
(cfu/mouse)
MIC
PD50
16
8
1
24
4 228
(ugmg/mL) (mg/kg)
4
4
4
4
8
4
8
8
8
4
8
4
8
16
8
16
16
16
8
8
8
8
16
16
16 28
16 28
16 28
1
1
1
1
8
2
2
8
4
4 2
4 2
4 1
4 1
8 28
4 2
8
8
4
S. aureus Smith
1.8Â107
po
6c
6i
24.60
2
4
2
4
8
4
4
2
>8.0
>8.0
6k
Linezolid
8
24.07
4
8
4
4
4
8
aEach test compound was suspended in 0.5% carboxymethyl cellulose
and administered orally 1 h following intraperitoneally bacterial
inoculation in mice.
4
8
8
4
8 50.28
16 8 2 8
8 2 8
16 16 216
8 2 8
4
4
4
8
16
16
4
8
16 24
16
8
8
8
4
active than 6c. The pharmacokinetic profile of some
active compounds were studied, only 6c showed mean-
ingful result by comparing with Linezolid (Table 4). In
vivo activity of 6c (PD50=4.60 mg/kg) was comparable
to that of linezolid (PD50=4.07 mg/kg) against S. aur-
eus Smith (Table 5). Further optimization of the struc-
ture of oxazolidinone analogues for in vitro and in vivo
activities are in progress.
22 2 02.5 0.5
4
1
1
1 0.5 5 0.20.5
224 0.50.25 222
21 1 1 2240.12
1
>32
aAgar dilution method, Mueller–Hinton agar, 104 CFU/spot.
bS.a.=Staphylococcus aureus ATCC 29213, MRSA=methicillin-
resistant Staphylococcus aureus C6068, CRSA=ciprofloxacin-resistant
Staphylococcus aureus C6043, MSSA=methicillin-susceptible Staphy-
lococcus aureus C2214, S.e. 1=Staphylococcus epidermis ATCC 1228,
S.e. 2 =Staphylococcus epidermis C2235, E.f. 1=Enterococcus faecalis
C6291, E.f. 2 =Enterococcus faecalis C6301, S.p. 1=Staphylococcus
pyogenes ATTCC8668, S.p.
2 =Staphylococcus pyogenes C6003,
Acknowledgements
VRE=vancomycin-resistant Enterococcus faecium, C6487.
cLZ=linezolid.
dVCM=vancomycin.
This work was supported by the Korea Ministry of
Science and Technology.
Table 3. In vitro antibacterial activity of oxazolidinone derivatives
against 11 bacterial strains (MIC, mg/mL)a
References and Notes
Compd
Microorganismb
S.a. S.a. MRSA MRSA S.e. S.e. S.e. E.f. E.f. VRE VRE
1.
1. Gregory, W.; Brittelli, D.; Wang, C.-L.; Manninen, P.; Slee,
A.; Forbes, M. J. Med. Chem. 1989, 32, 1673.
2
1
2
1
2
3
1
2
1
2
2. Bricker, S.; Hutchinson, D.; Barbachyn, M.; Manninen, P.;
Ulanowicz, D.; Garmon, S.; Grega, K.; Hendges, S.; Toops,
D.; Ford, C.; Zurenko, G. J. Med. Chem. 1996, 39, 673.
3. Pae, A. N.; Kim, H. Y.; Joo, H. J.; Kim, B. H.; Cho, Y. S.;
Choi, K. I.; Choi, J. H.; Koh, H. Y. Bioorg. Med. Chem. Lett.
1999, 9, 2679.
4. Francoise, T. B.; Foucaud, A. Tetrahedron Lett. 1982,
4927.
5. Hutchinson, D. K. WO 9613502.
6. Yamada, H.; Munesada, K.;Taniguchi, M. WO 9525106.
7. Representative procedure: 6a To a solution of N-[3-{3-
fluoro-4-(4-oxo-piperidin-1-yl)-phenyl}-2-oxo-oxazolidin-
5-ylmethyl]-acetamide (20 mg, 0.06 mmol), aluminium oxide
(Basic, I, Aldrich, 17.2mg) and malononitrile (3.8 mg, 0.06
mmol) in dichloromethane (0.5 mL) was stirred at 40 ꢀC for 15
h. The reaction mixture was poured into a mixture of di-
chloromethane and water. The organic layer was separated
and the aqueous layer was extracted with dichloromethane (2
mLÂ3). The collected organic layer was washed with brine,
dried over anhydrous MgSO4, evaporated and purified by
column chromatography (5% methanol–ethyl acetate) to give
the product 6a (14.8 mg, 69%). 1H NMR (300 MHz, CDCl3) d
7.47 (dd, J=1.2, 1.2 Hz, 1H), 7.09 (dd, J=1.1, 6.6 Hz, 1H),
5a
5b
5c
5d
6c
LZc
VCMd
8 16
16 3216
8
16
4
8
4
8
16 16 16
16
16
16
8
16 332126
0.5 0.5 0.5 0.5
0.5
1
1
2
0.5
1
1
1
1
1
1
1
1
1
1
4
4
2
2
0.5 0.5
0.5
1
2
0.5 0.25 0.5 0.5
0.5 0.5 0.5
1
21
1
1
0.5
1
1
1
1
1
1
64
1
1
1
1
4
64 64
aAgar dilution method, Mueller–Hinton agar, 104 CFU/spot.
bS.a. 1=Staphylococcus aureus 77, S.a. 2 =Staphylococcus aureus
SA011, MRSA 1=methicillin-resistant Staphylococcus aureus 241,
MRSA 2=methicillin-resistant Staphylococcus aureus K283, S.e.
1=Staphylococcus epidermiidis Q004, S.e. 1=Staphylococcus epi-
dermiidis Q033, S. e. 1=Staphylococcus epidermiidis R005, E. f.
1=Enterococcus faecalis 29212A, E. f. 2 =Enterococcus faecalis 2009,
VRE 1=vancomycin-resistant Enterococcus faecium 2006, VRE
1=vancomycin-resistant Enterococcus faecium 2153.
cLZ: linezolid.
dVCM=vancomycin.
investigation of the effectiveness of ring size, a series of
the five membered pyrrolidinyl derivatives 5 were syn-
thesized and evaluated (Table 3). Cyanomethylenyl
analogue 5c showed also the best activity but 2-fold less

2230
H. Y. Kim et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2227–2230
6.92(t, J=9.1 Hz, 1H), 6.31 (m, 1H), 4.76 (m, 1H), 4.02(t,
J=8.8 Hz, 1H), 3.76 (t, J=8.7 Hz, 1H), 3.62 (m, 2H), 3.26 (m,
4H), 2.92(m, 4H), 1.92(s, 3H), IR (KBr, cm À1) 2232, 1750,
1660, 1518, 1418, 1382, 1216, 1012, 752, 13C NMR(CDCl3,
300 MHz) CDCl3, 300 MHz), d 180.21, 171.83, 157.38, 154.68,
135.65, 134.25, 134.36, 134.22, 120.44, 114.25, 111.53, 108.46,
107.79, 84.52, 73.36, 51.39, 47.28, 42.31, 34.72, 23.51, HRMS
(FAB, M+H) Calcd. for C20H21N5O3 398.1628, found
398.1579.
d 7.40 (dd, 1H, J=2.5, 2.5 Hz), 6.70 (m, 2H), 5.38 (d, 1H,
J=2.4 Hz), 4.77 (m, 1H), 4.13 (s, 1H), 4.01 (t, 1H, J=8.9 Hz),
3.76 (t, 1H, J=7.9 Hz), 3.66 (d, 2H, J=2.7 Hz), 3.48 (m, 2H),
2.92 (m, 2H), 2.02 (s, 3H); 13C NMR (CDCl3, 75 MHz) d
171.69, 166.61, 154.95, 154.84, 151.40, 133.31, 133.20, 133.17,
131.38, 131.25, 117.10, 117.03, 116.37, 116.40, 114.80, 114.76,
108.41, 108.06, 91.91, 91.58, 77.89, 77.46, 77.05, 72.40, 56.02,
55.93, 50.04, 49.98, 49.66, 49.60, 48.13, 42.31, 32.96, 32.12,
23.40. HRMS (FAB, M+H) Calcd. for C18H20FN4O3
359.1519, found 359.1471.
8. (a) Wadsworth, W. S.; Emmons, W. D. J. Org. Chem. 1961,
1733. (b) Corey, E. J.; Cane, D. E. J. Org. Chem. 1969, 3053.
9. Representative procedure: 5c; To a solution of tert-but-
oxide (0.84 g, 1.49 mmol) in THF (30 mL) was added drop-
wise diethyl cyanomethyl phosphonate (1.35 g, 7.6 mmol) in
THF (30 mL). After stirring for 1 h, N-[3-{3-fluoro-4-(3-oxo-
pyrrolidin-1-yl)-phenyl}-2-oxo-oxazolidin-5-ylmethyl]-acetamide
(0.50 g, 0.24 mmol) in THF (90 mL) was dropwise for 1 h at
À78 ꢀC. After stirring for 7 h at the same temperature, The
reaction mixture was poured into a mixture of dichloro-
methane and water. The organic layer was separated and the
aqueous layer was extracted with dichloromethane (20
mLÂ3). The collected organic layer was washed with brine,
dried over anhydrous MgSO4, evaporated and purified by
column chromatography (5% methanol–ethyl acetate) to give
1
6c; H NMR (300 MHz, CDCl3) d 7.46 (dd, J=2.70, 2.40
Hz, 1H), 7.08 (dd, J=2.49, 2.46 Hz, 1H), 6.93 (t, J=9.00
Hz, 1H), 6.05 (m, 1H), 5.20 (s, 1H) 4.78 (m, 1H), 4.03 (t,
J=9.3, 1H), 3.78–3.59 (m, 3H), 3.16 (m, 4H), 2.78 (t,
J=4.80 Hz, 2H), 2.55 (t, J=5.10 Hz, 2H), 2.03 (s, 3H).
13C NMR (75 MHz, CDCl3)
d 171.92, 164.45, 157.01,
155.10, 136.80, 133.98, 120.56, 114.65, 108.33, 108.15, 94.57,
72.65, 52.49, 52.05, 48.34, 42.62, 35.98, 33.50, 23.70. HRMS
(FAB, M+H) cacld. for C19H22FN4O3 373.1676, found
373.1676.
10. Thomasco, L.; Yang, M.; Lucy, X. W.O. patent 9,902,525,
1999. Chem. Abstr. 1999, 123, 2870.
11. Leitner, F.; Misiek, M.; Pursiano, T. A.; Buck, R. E.;
Chisholm, D. R.; Deregis, R. G.; Tsai, Y. H.; Price, K. E.
Antimicrob. Agents Chemother. 1976, 10, 426.
1
the product 5c (525.7 mg, 98%). H NMR (CDCl3, 300 MHz)