Synthesis and in vitro activity of novel isoxazolyl tetrahydropyridinyl oxazolidinone antibacterial agents

Article abstract of DOI:10.1016/j.bmcl.2003.08.021

A series of isoxazolyl tetrahydropyridinyl oxazolidinones with various substituents at the 3-position of the isoxazole ring have been synthesized and their in vitro antibacterial activities (MIC) were evaluated against several Gram-positive strains including the resistant strains of Staphyloccus and Enterococcus, such as MRSA and VRE. One of the most potent compounds synthesized, 4f, showed comparable or better activity against selected bacterial strains than those of linezolid and vancomycin.

Full text of DOI:10.1016/j.bmcl.2003.08.021

Bioorganic & Medicinal ChemistryLetters 13 (2003) 4117–4120
Synthesis and In Vitro Activity of Novel Isoxazolyl
Tetrahydropyridinyl Oxazolidinone Antibacterial Agents
Jae Seok Lee,a Yong Seo Cho,a Moon Ho Chang,a
Hun Yeong Koh,a Bong Young Chungb and Ae Nim Paea,*
aBiochemicals Research Center, Korea Institute of Science and Technology, Cheongryang, Seoul 130-650, South Korea
bDepartment of Chemistry, Korea University, 1-Anamdong, Seoul 136-701, South Korea
Received 9 July2003; revised 8 August 2003; accepted 8 August 2003
Abstract—A series of isoxazolyl tetrahydropyridinyl oxazolidinones with various substituents at the 3-position of the isoxazole ring
have been synthesized and their in vitro antibacterial activities (MIC) were evaluated against several Gram-positive strains includ-
ing the resistant strains of Staphyloccus and Enterococcus, such as MRSA and VRE. One of the most potent compounds synthe-
sized, 4f, showed comparable or better activityagainst selected bacterial strains than those of linezolid and vancomycin.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
Subsequent studies at Pharmacia and Upjohn Co. have
resulted in two potent oxazolidinone antibacterial
agents, eperezolid (2) and linezolid (3).3
The oxazolidinones represent a new class of anti-
bacterial agents which showed activityagainst a wide
spectrum of Gram-positive bacterial infections, including
those infections caused bystrains resistant to other anti-
biotics. The oxazolidinones are active against a varietyof
clinicallyimportant susceptible and resistant Gram-posi-
tive organisms such as methicillin-resistant Staphylo-
Linezolid (Zyvox1) is the first member of this class
recentlylicensed in the USA and Europe for the treat-
ment of multidrug resistant Gram-positive infections
such as nosocomial and community-acquired pneumo-
nia and skin infections.
coccus
aureus
(MRSA),
vancomycin
resistant
Enterococcus faecium (VRE) and penicillin-resistant
Streptococcus pneumoniae (PRSP).
The mechanism of action of the oxazolidinones has
been revealed that theyinhibit bacterial protein synth-
esis at an earlyevent in the initiation step of protein
synthesis.1
The oxazolidinone, exemplified byDuP 721 ( 1), is a
relativelynew class of orallyactive, totallysynthetic-
2
antibacterial agent discovered byscientists at DuPont.
However, DuP 721 was discontinued following Phase I
clinical trials because it was shown that DuP 721
exhibited lethal toxicityin rats in drug safetystudies
conducted at the Upjohn Co.
In our previous study, we have described the synthesis
of two series of oxazolidinones having an isoxazole as a
rigid bioisostere of hydroxymethyl group of eperezolid
and the investigation of the effect of introducing the
4
isoxazole moietyon the activity.
Recently, we have reported on another oxazolidinone
analogues having methylenepiperidinyl and methylene-
*Corresponding author. Tel.:+82-295-85185; fax:+82-295-85189;
e-mail: anpae@kist.re.kr
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.021

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J. S. Lee et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4117–4120
Scheme 1. (a) LDA, PhNTf2, HMPA/THF, À50 ꢀC; (b) Pd2(dba)3 CHCl3, PPh3, 9 (or 10), ZnCl2, NMP, THF, À78 ꢀC to rt.
pyrrolidinyl groups and their evaluation of their anti-
bacterial activities.5 From the continuing studyon anti-
infectives, we have explored the possibilityof synthesis
of novel antibacterials based on the conformationally
constrained analogues of the oxazolidinones. Herein, we
describe the synthesis and antibacterial activity of a
related familyof compound 4 and 5 in which the sub-
stituted isoxazolyl tetrahydropyridinyl groups were
introduced.
Table 1. Prepared tributylstannanyl isoxazoles of 9 and 10
Substrates
Methoda
R
X
Products (yield, %)b
9
10
8a
8b
8c
8d
8e8
A
B
B
B
B
CH3
CN
Cl
CF3
CO2Et
H
Cl
Cl
Cl
Cl
47
46
51
50
82
—
48
27
—
—
aMethod A: NCS, tributylethynyltin, Et3N in THF at 65 ꢀC; method
B: K2CO3, tributylethynyltin in CH2Cl2 at room temperature.
bIsolated yield.
As shown in Table 1, most of oximes gave 3-substituted
5-tributylstannanyl isoxazoles 9. In the cases of cycli-
zation reaction of 8b and 8c, however, two regioisomers,
9 and 10, were obtained.
To prepare 3-methyl-5-tributylstannanyl isoxazole (9a),
we used different method because chlorination of
methyl oxime with NCS was needed for the isoxazole
ring cyclization. Chlorinated acetaldoxime with
N-chlorosuccinimide in THF at 65 ꢀC was formed in
situ. Subsequent addition of tributyletynyltin and tri-
ethylamine gave the product successfully (47%).
Chemistry
The synthesis of the novel oxazolidinone derivatives
having isoxazolyl tetrahydropyridinyl groups is shown
in Scheme 1. The keyintermediate 6 was prepared
according to the known method.6 To synthesize the
oxazolidinone derivatives shown in Table 2, we sur-
veyed reaction conditions suitable for Pd0-catalysed
Stille coupling reaction with triflate 7 and various tri-
butylstannanyl isoxazoles.
The Stille-coupling reaction (method C) of triflate 7 and
various
tributylstannanyl
isoxazoles
with
.
[Pd2(dba)2 CH3Cl], PPh3 and ZnCl2 in THF/NMP at
room temperature for 3 days afforded the correspond-
ing products.
Lithium enolate of 6 was prepared with LDA in THF/
HMP at À50 ꢀC, and then PhNTf2 was added and the
mixture was kept at À50 ꢀC for 1.5 h. This sequence
provided the triflate 7 which is an intermediate of the
coupling reactions.7
Table 2. Synthesized oxazolidinones of 4 and 5
Compd
Methoda
R
Yield (%)b
As shown in Scheme 2, a variety of tributylstannanyl
isoxazoles has been prepared via [2+3]cycloaddition
with various substituted chloro oximes in the presence
of tributylethynyltin and potassium carbonate in
CH2Cl2.
4a
4b
4c
4d
4e9
4f10
4g
5a
5b
C
C
C
C
C
D
D
C
C
CH3
CN
Cl
CF3
86
58
52
52
61
80
63
93
91
CO2Et
CONH2
CON(CH3)2
C l
CN
aMethod C: Stille-coupling reaction; method D: amidation reaction of
4e.
Scheme 2.
bIsolated yield.

J. S. Lee et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4117–4120
4119
Table 3. In vitro antibacterial activityof oxazolidinone derivatives against 11 bacterial strains (MIC, mg/mL)a
Compd
Microorganismb
S. a.1
S. a.2
S. a.3
MRSA
S. e. 1
S. e. MR
E. f.1
E. f.2
VRE.1
VRE.2
VRE.3
4a
4b
4c
4d
4e
4f
4g
5a
5b
LZc
VCMd
2
2
8
1
1
8
4
4
32
>64
4
2
2
4
1
1
8
2
4
16
>64
16
1
4
4
64
>64
16
1
4
4
64
2
4
32
>64
16
1
8
8
2
2
64
2
2
4
32
>64
8
4
32
>64
8
>64
8
>64
4
>64
4
>64
4
>64
8
1
8
8
2
2
4
0.5
4
2
1
0.5
0.5
0.5
8
2
1
0.5
1
0.5
4
8
2
2
0.5
8
2
1
0.5
2
0.5
2
2
0.5
0.5
1
1
8
8
2
1
8
8
2
8
8
4
1
8
8
2
2
2
>64
2
>64
1
2
4
aAgar dilution method, Mueller-Hinton agar, 104 CFU/spot.
bS. a.1, Staphylococcus aureus 6538p; S. a.2, Satphylococcus aureus 77; S. a.3, Staphylococcus aureus 29213A; MRSA, methicillin-resistant Staphy-
lococcus aureus 241; S. e. 1, Staphylococcus epidermidis Q004; S. e. MR, Staphylococcus epidermidisMR 887E; E. f. 1, Enterococcus faecalis 29212A; E.
f. 2, Enterococcus faecalis EFS008; VRE.1, vancomycin-resistant Enterococcus faecium or2; VRE.2, vancomycin-resistant Enterococcus faecium
2006; VRE.3, vancomycin-resistant Enterococcus faecium2164.
cLZ, linezolid.
dVCM, vancomycin.
MRSA and VRE (Table 3). The amide group of iso-
xazole moietyof 4f might be playing crucial role for the
activityas a substituent. Although 4b, 4c and 4d have
strong electronegative group such as –CN, –Cl and
–CF3 at the same position as amide group of 4f, they
showed lower activitythan 4f. Therefore we suggest that
the hydrophilic group of 3-position of isoxazole in 4f
might have important function for antibacterial activity.
Acknowledgements
.
Scheme 3. (a) aq NH4OH, NH4Cl, MeOH, rt; (b) (CH3)2NH HCl,
InBr3, K2CO3, MeOH, rt.
This work was supported bythe Korea Ministryof
Science and Technology.
In another way, the new oxazolidinone derivatives,
amides 4f and 4g, could be synthesized via amidation
reaction (method D) of isoxazolyl ethylester 4e with
NH4OH or dimethyl amine in methanol at room temp-
erature as shown in Scheme 3. In the case of 4g, how-
ever, InBr3 was required as a Lewis acid to activate the
ester group of 4e.
References and Notes
1. Eustice, D. C.; Feldman, P. A.; Zajac, I.; Slee, A. M. Anti-
microb. Agents Chemother. 1988, 32, 1218.
2. Gregory, W.; Brittelli, D.; Manninen, P.; Slee, A.; Forbes,
M. J. Med. Chem. 1989, 32, 1673.
3. 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.
4. 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.
5. Kim, H. Y.; Lee, J. S.; Cha, J. H.; Pae, A. N.; Cho, Y. S.;
Chang, M. H.; Koh, H. Y. Bioorg. Med. Chem. Lett. 2003, 13,
2227.
6. Yamada, H.; Munesada, K.;Taniguchi, M. WO 9525106.
7. Experimental Procedure: 7; To a solution of diisopropyl-
amine (92 mL, 0.66 mmol) in THF (2 mL) was added dropwise
n-BuLi (1.45 M in hexane, 452 mL, 0.66 mmol) at 0 ꢀC under
N2. After stirring for 30 min, 6 (100 mg, 0.30 mmol) in THF (7
mL)/HMPA (2.7 mL) was added dropwise for 20 min at
À50 ꢀC. After stirring for 3 h, N-phenyltrifluoromethane-
sulfonimide (117 mg, 0.33 mmol) in THF (2 mL) was added
dropwise. After stirring for 1.5 h at À50 ꢀC, The reaction
mixture was poured into a mixture of ethyl acetate and water.
The organic layer was separated and the aqueous layer was
extracted with ethyl acetate (20 mLÂ3). The collected organic
layer was washed with brine, dried over anhydrous MgSO4,
In vitro activity
In vitro antibacterial activities of all the compounds
prepared and references were determined bythe Muel-
ler-Hinton agar dilution method11 against Gram-posi-
tive strains and the resistant strains including MRSA
(methicillin-resistant Staphylococcus aureus) and VRE
(vancomycin-resistant Enterococcus faecium). The
activities of the compounds synthesized were compared
with linezolid and vancomycin as references. Data for
selected Gram-positive organisms are reported as a
minimum inhibitoryconcentration (MIC) expressed in
mg/mL. The results are presented in Table 3. Most of the
compounds synthesized exhibited good antibacterial
activities.
The carbamoyl isoxazole 4f demonstrated MIC values
comparable or 2–4-fold better than those of linezolid
and vacomycin against all of tested organisms including

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J. S. Lee et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4117–4120
evaporated and purified bycolumn chromatographywith
neutralized SiO2 byEt N (5% methanol-ehtyl acetate) to give
the product 7 (54 mg, 37%).
M+H) calcd for C23H26FN4O6 473.1836, found 473.1836.
4c: 1H NMR (DMSO, 300 MHz) d 8.26 (m, 1H), 7.53 (dd,
J=1.80, 2.40, 1H), 7.17 (m, 2H), 6.95 (s, 1H), 6.75 (s, 1H),
4.73 (m, 1H), 4.11 (t, J=9.3, 1H), 3.75 (m, 3H), 3.34–3.27 (m,
2H), 1.85 (s, 3H). IR (KBr, cmÀ1) 1740, 1654, 1567, 1520,
1424, 1226, 1192. 13C NMR (DMSO, 75 MHz) d 172.01,
170.76, 156.93, 154.80, 153.67, 135.57, 135.45, 134.22, 134.09,
129.61, 123.69, 120.64, 114.83, 107.56, 107.21, 101.13, 72.28,
49.86, 49.21, 48.03, 47.55, 42.13, 25.76, 23.15, 17.93. HRMS
(FAB, M+H) calcd for C20H21ClFN4O4 435.1235, found
435.1235.
10. Representative procedure: 4f: To a solution of 4e (18 mg,
0.04 mmol) in methanol (3 mL) was added NH4OH (28% in
water, 500 mL, excess) at room temperature, followed by
NH4Cl (50 mg, excess) and stirred for 21 h. After removing
methanol, a mixture of CH2Cl2 and water was poured into the
reaction pot. The organic layer was separated and the aqueous
layer was extracted with CH2Cl2 (20 mLÂ3). The collected
organic layer was washed with brine, dried over anhydrous
MgSO4, evaporated and purified bycolumn chromatography
(2% methanol–dichloromethane) to give the product 4f (13
mg, 80%).
1H NMR (DMSO, 300 MHz) d 8.09 (s, 1H), 7.80 (s, 1H), 7.49
(dd, J=1.5, 1.8, 1H), 7.16 (m, 2H), 6.82 (s, 1H), 6.71 (s, 1H),
4.69 (m, 1H), 4.07 (t, J=8.70, 1H), 3.76 (s, 2H), 3.69 (m, 1H),
2.55 (s, 2H), 2.05 (s, 3H). IR (KBr, cmÀ1) 11738, 1684, 1659,
1520, 1232. 13C NMR (DMSO, 75 MHz) d 171.14, 170.87,
161.05, 160.10, 154.92, 135.77, 134.16, 128.64, 123.98, 120.69,
114.92, 107.69, 107.34, 100.01, 72.40, 49.98, 48.14, 47.77,
42.26, 26.19, 23.30. HRMS (FAB, M+H) calcd for
C21H23FN5O5 444.1683, found 444.1683.
4g: 1H NMR (CDCl3, 300 MHz) d 7.46 (dd, J=2.43, 2.37,
1H), 7.10 (d, J=8.79, 1H), 6.96 (t, J=9.03, 1H), 6.68 (s, 1H),
6.47 (s, 1H), 5.99 (m, 1H), 4.77 (m, 1H), 4.03 (t, J=8.91, 1H),
3.84 (s, 1H), 3.76 (m, 2H), 3.36 (t, J=5.64, 2H), 3.31 (s, 3H),
2.63 (s, 2H), 2.03 (s, 3H). IR (KBr, cmÀ1) 1732, 1672, 1628,
1518, 1230. 13C NMR (DMSO, 75 MHz) d 171.14, 170.87,
161.05, 160.10, 154.92, 135.77, 134.16, 128.64, 123.98, 120.69,
114.92, 107.69, 107.34, 100.01, 72.40, 49.98, 48.14, 47.77,
42.26, 26.19, 23.30. HRMS (FAB, M+H) calcd for
C23H27FN5O5 472.1996, found 472.1996.
3
8. Representative procedure: 8e: To a solution of ethyl chloro
oximidoacetate (100 mg, 0.66 mmol) in CH2Cl2 (2 mL) was
added tributyl ethynyl tin (191 mL, 0.66 mmol) followed by
K2CO3 (100 mg, 0.73 mmol). After stirring 5 h at room tem-
perature, the reaction mixture was poured into a mixture of
CH2Cl2 and water. The organic layer was separated and the
aqueous layer was extracted with CH2Cl2 (20 mLÂ3). The
collected organic layer was washed with brine, dried over
anhydrous MgSO4, evaporated and purified bycolumn chro-
matography(hexane–ethyl acetate=40:1) to give the product
8e (232 mg, 82%).
1H NMR (CDCl3, 300 MHz) d 6.81 (s, 1H), 4.45 (q, J=13.8,
2H), 1.57 (m, 6H), 1.44 (t, J=6.6, 3H), 1.33 (m, 6H), 1.23 (t,
J=8.29, 6H), 0.92 (t, J=7.05, 9H).
9. Representative procedure: 4e; To a solution of 7 (53 mg,
.
0.11 mmol) in THF (1 mL) was added [Pd2(dba)3] CHCl3
(2.3 mg, 8 mol%) at À78 ꢀC, followed byPPh (2.3 mg, 2
3
mol%) and ethyl 5-tributylstannanyl-3-isoxazole carboxylate
(8e) (48 mg, 0.11 mmol) in THF (1 mL). Finally, a solu-
tion of ZnCl2 in hexane (1 M, 110 mL, 0.11 mmol) was
added. The cooling bath was removed and the reaction
temperature was rapidlyraised to ambient temperature using a
water bath. An intense red color developed on warming. After
stirring 3 days at ambient temperature, the reaction mixture
was poured into a mixture of ethyl acetate and water. The
organic layer was separated and the aqueous layer was
extracted with ethyl acetate (20 mLÂ3). The collected
organic layer was washed with brine, dried over anhydrous
MgSO4, evaporated and purified bycolumn chromato-
graphy(2% methanol–ethyl acetate) to give the product 4e
(34 mg, 61%).
1H NMR (CDCl3, 300MHz) d 7.48 (dd, J=2.40, 2.40, 1H),
7.11 (d, J=9.60, 1H), 6.98 (t, J=8.70, 1H), 6.73 (s, 1H), 6.59
(s, 1H), 6.20 (m, 1H), 4.80 (m, 1H), 4.45 (q, J=7.20 1H), 4.06
(t, J=8.70, 1H), 3.86 (s, 1H), 3.73 (m, 2H), 3.38 (m, 2H), 2.66
(s, 2H), 2.05 (s, 3H), 1.45 (t, J=7.2, 3H). IR (KBr, cmÀ1
)
1736, 1652, 1522, 1422, 1226.
13C NMR (CDCl3, 75 MHz) d 171.33, 160.31, 157.44, 156.88,
154.56, 154.18, 136.08, 133.33, 133.19, 128.03, 123.86, 119.80,
114.20, 108.02, 107.67, 100.22, 99.82, 72.15, 62.48, 49.74,
47.91, 47.60, 42.23, 29.96, 26.18, 23.40, 14.42. HRMS (FAB,
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.