Synthesis of novel optically pure α-amino acid functionalised-7- trifluoromethyl substituted quinolone derivatives and their antibacterial activity

Article abstract of DOI:10.1007/s00044-012-0020-3

7-Trifluoromethyl-4-hydroxy quinoline-3-carboxylic acid ethyl ester was prepared from 3-amino benzotrifluoride and EMME. The hydroxyquinoline was reacted with amino acids to furnish amino acid funtionalised quinolines (5, 6, 7). The compounds were alkylated by ethyl iodide followed by hydrolysis afforded the title compounds in good yields. They were screened for their antibacterial activity for in vitro against Gram +ve and Gram -ve bacterial strains and found 11e and 11b as promising compounds.

Full text of DOI:10.1007/s00044-012-0020-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:186–194
DOI 10.1007/s00044-012-0020-3
ORIGINAL RESEARCH
Synthesis of novel optically pure a-amino acid functionalised-7-
trifluoromethyl substituted quinolone derivatives and their
antibacterial activity
•
B. P. Venkat Lingaiah T. Yakaiah A. Ravi Kumar G. Sathaiah
•
•
•
•
•
•
•
A. Chandra Shekhar K. Raju P. Shanthan Rao B. Narsaiah
U. S. N. Murthy
Received: 22 August 2011 / Accepted: 7 March 2012 / Published online: 21 March 2012
Ó Springer Science+Business Media, LLC 2012
Abstract 7-Trifluoromethyl-4-hydroxy quinoline-3-car-
boxylic acid ethyl ester was prepared from 3-amino ben-
zotrifluoride and EMME. The hydroxyquinoline was
reacted with amino acids to furnish amino acid funtiona-
lised quinolines (5, 6, 7). The compounds were alkylated
by ethyl iodide followed by hydrolysis afforded the title
compounds in good yields. They were screened for their
antibacterial activity for in vitro against Gram ?ve and
Gram -ve bacterial strains and found 11e and 11b as
promising compounds.
well documented. Several fluoro (Hooper and Wolfson,
1993; Violetta et al., 2000; Radl and Bouzard, 1992) and
trifluoromethyl substituted quinolones (Lopez et al., 2003;
Uray and Niederreiter, 2003; Bridges and Domagala, 1988 )
are known in prior art and they found to possess broad
spectrum of antibacterial activity against a wide range of
gram ?ve and gram -ve aerobes and few being active
against aerobic organisms as well. Several quinolones are
already being marketed as antibacterial drugs, e.g. Nor-
floxacin (Koga et al., 1980), Enoxacin (Matsumoto et al.,
1984), Ciprofloxacin (Wise et al., 1983), Tosufloxacin (Chu
et al., 1985) etc. Quinolones having trifluoromethyl func-
tionality at N-1 or C-8 (Sanchz et al., 1992) positions
showed promising activity, however, the known quinolone
drugs are showing resistance to certain bacterial strains over
a period of time. To overcome this problem, intense
research is under progress worldwide to identify new lead
molecules. In continuation of our ongoing programme on
the synthesis of fluoroquinolones (Venkat Reddy et al.,
2009), we wish to report here the synthesis of 7-trifluoro-
methyl substituted quinolones coupled with optically pure
a-amino acid functionality at C-3 carbonyl moiety to see the
effect on their bioefficacy.
Keywords 3-Aminobenzotrifluoride Á Proline Á
D/L-amino acids Á EMME Á Antibacterial activity
Introduction
Fluoro or trifluoromethyl substituted organic compounds
are more lipophilic in nature compared to non fluorinated
molecules due to the unique properties of fluorine. Tri-
fluoromethyl substituted benzodiazepines (Filler et al.,
1993) as calcium channel blockers, and dihydro naphthyl
derivatives as antiprotozoal (Filler et al., 1993) activity are
Chemistry
B. P. Venkat Lingaiah Á T. Yakaiah Á A. Ravi Kumar Á
G. Sathaiah Á A. Chandra Shekhar Á K. Raju Á
P. Shanthan Rao (&) Á B. Narsaiah
The 3-amino benzotrifluoride was reacted with diethyl
2-(ethoxymethylene) malonate to furnish initial uncyclised
product 2 which on heating in diphenylether at 260 °C
gave exclusively 7-trifluoromethyl quinoline 3. The pres-
ence of trifluoromethyl group in benzene ring promotes
nucleophilic substitution in para position rather than in
ortho position which facilitated cyclisation of 2 results an
exclusive formation of product 3. The proton NMR of 3
Fluoroorganics Division, Indian Institute of Chemical
Technology, Tarnaka, Hyderabad 500 607, India
e-mail: shanthanpp1954@gmail.com
U. S. N. Murthy
Biology Division (Bioinformatics), Indian Institute of Chemical
Technology, Tarnaka, Hyderabad 500 607, India
123

Med Chem Res (2013) 22:186–194
187
showed two sets of doublets and two singlets in aromatic
region indicating the structure of 3 as 7-trifluoromethyl-
4-hydroxy quinoline-3-carboxylic acid ethyl ester. Thus,
the formation of 4 was eliminated. The sequence of reac-
tions are outlined in Scheme 1.
Conclusion
In conclusion we have successfully synthesized a series of
amino acid functionalized 7-trifluoromethylated quinolone
derivatives and evaluated for antibacterial activity. Com-
pounds 11e and 11b showed promising activity and 12a, 13
showed moderate activities.
The trifluoromethyl quinoline 3 was further indepen-
dently reacted with optically pure amino acids such as
L-amino acid, D-amino acid and L-proline in DMSO at
reflux temperature for 8 h furnished amino acid function-
alised derivatives 5, 6 and 7, respectively. Ethylation of
compounds 5, 6 and 7 using ethyl iodide in the presence of
a base afforded N-ethylated esters 8, 9 and 10. The ester
derivatives 8, 9 and 10 on hydrolysis using LiOH resulted
the desired compounds 11, 12 and 13, respectively. The
sequence of reactions outlined in Scheme 2, and products
are tabulated in Table 1.
Experimental section
All required chemicals were purchased from Sigma-Aldrich
Company. Melting points were recorded on Casia-siamia
(VMP-AM) melting point apparatus and are uncorrected. IR
spectra were recorded on a Perkin-Elmer FT-IR 240-C
spectrophotometer using KBr optics. 1H NMR spectra were
recorded on Bruker AV 300 MHz instrument in DMSO-d6 or
CDCl3 or TFA using TMS as an internal standard. Electron
impact (EI) and chemical ionization mass spectra were
recorded on a VG 7070 H instrument at 70 eV. All reactions
were monitored by thin layer chromatography (TLC) on
precoated silica gel 60 F254 (mesh); spots were visualized
with UV light. Merck silica gel (60–120 mesh) was used for
column chromatography. CHN analyses were recorded on a
Vario EL analyzer.
Results and discussion
Antibacterial activity
A series 7-trifluoromethyl-1-ethyl-4-oxo-3-quinolyl car-
bonyl amino acids 11a–e, 12a, b and 13 and their penul-
timate diesters 8a–e, 9a, b and 10 are tested against gram
?ve and gram -ve bacterial strains in vitro by using micro
titration techniques and their minimum inhibitory concen-
trations (MIC in lg/ml) were compared with standard
sample ciprofloxacin. The antibacterial activity of 11e is
promising towards Staphylococcus epidermis (gram ?ve)
and Pseudomonas aeruginosa (gram -ve) species and
of 11b is towards Klebsiella aerogens (gram -ve). The
presence of phenyl group on amino acid in 11e may be
responsible for some activity. The presence of trifluoro-
methyl group at 7th position of quinolone ring did not have
any enhanced activity than the standard molecule. The
results of activity data is tabulated in Table 2.
Preparation of diethyl 2-(3-trifluoromethyl aniline)
malonate, 2
Procedure
A mixture of 3-trifluoromethyl aniline (50 g, 344 mmol)
and diethyl ethoxy methylene malonate (75 g, 350 mmol)
was heated at 120–130 °C for 2 h without any solvent and
the resulting byproduct ethanol was distilled. The crude
malonate was recrystallised from n-hexane.
Scheme 1 Synthesis of ethyl
4-hydroxy-7-
(trifluoromethyl)quinoline-
3-carboxylate
EtOOC
EtOOC
COOEt
OEt
COOEt
F3C
NH2
H
F3C
N
H
1
2
Diphenylether
260°C
CF3 OH
COOEt
N
OH
4
COOEt
F3C
N
3
123

188
Med Chem Res (2013) 22:186–194
Scheme 2 Synthesis of amino
acid functionalized
trifluoromethyl quinolones
OH
N
O
OEt
F3C
3
L-Amino acid
D-Amino acid
L-Proline
OH
N
O
OH
N
O
R
COOH
OH
N
O
R
N
N
COOH
F3C
N
COOH
F3C
H
H
F3C
7
5
6
Et-I
Et-I
Et-I
O
N
O
COOEt
O
N
O
R
O
N
O
R
N
N
COOEt
F3C
H
N
COOEt
F3C
H
F3C
9
10
8
LiOH
LiOH
O
LiOH
O
O
COOH
O
N
R
O
O
R
N
N
H
COOH
F3C
N
H
COOH
F3C
N
F3C
N
13
12
11
1
IR (KBr, cm-1): 1680 (CO–NH), 1695 (Ester CO). H
NMR CDCl3, 300 MHz): d 1.2–1.4 (m, 6H, CH3), 4.1–4.4
(m, 4H, –CH2–), 7.71 (d, 1H, Ar–H, J = 8.6), 7.97 (s, 1H,
Ar–H), 8.64 (d, 1H, Ar–H, J = 8.6), 8.96 (s, 1H, Ar–H),
10.25 (d, 1H, –NH, J = 8). MS (EI, 70 eV): m/z 331 (M?).
IR (KBr, cm-1): 1680 (Ester CO). 1H NMR (TFA,
300 MHz): d 1.56 (t, 3H, –CH3, J = 7 Hz), 4.74 (q, 2H,
–CH2, J = 7 Hz), 7.70 (d, 1H, Ar–H, J = 8.2), 7.78
(s, 1H, Ar–H), 8.59 (d, 1H, Ar–H, J = 8.2), 8.90 (s, 1H,
Ar–H). MS (EI, eV): m/z 285 (M?).
Preparation of 7-trifluoromethyl-4-hydroxy
quinoline-3-carboxylic acid ethyl ester, 3
Preparation of trifluoromethyl substituted a-amino acid
functionalised quinoline compounds, 5, 6, 7
Procedure
General procedure
The compound 2 (80 g, 253 mmol) charged in diphenyl
ether (200 ml) and refluxed for 1 h at 260 °C. The solution
was cooled, and the resulting precipitate was filtered off,
washed with hexane and dried. The solid was recrystallised
from dimethylformamide to give 3.
To a mixture of 7-trifluoromethyl-4-hydroxy quinoline-3-
carboxylic acid ethyl ester 3 (6 mmol), amino acid
(12 mmol), potassium carbonate (18 mmol) and dimethyl
sulphoxide (10 ml) was added and refluxed for 8 h. The
123

Med Chem Res (2013) 22:186–194
189
(2S)-2-({[4-hydroxy-7-(trifluoromethyl)-3-
quinolyl]carbonyl}amino)-3-methyl butanoic acid (5b)
Table 1 Physical properties of compounds 2, 3, 5–13
S. no. Compound
R
Yield MP
1
2
–
78.2 Gummymaterial
Specific rotation [a]: -86.2232 (c = 0.0049 g/ml, EtOH). IR
(KBr, cm-1) 1719 (Acid CO), 1645 (CO–N). 1H NMR
(DMSO-d6, 300 MHz): d 0.99 (d, 6H, –CH–(CH3)2, J =
7 Hz), 2.30–2.40 (m, 1H, CH–(CH3)2, J = 7 Hz), 4.52–4.62
(m, 1H, –NH–CH–CO2H), 7.60 (d, 1H, Ar–H, J = 8.2),
8.00(s, 1H, Ar–H), 8.46 (d, 1H, Ar–H, J = 8.2), 8.80 (s, 1H
Ar–H,,), 10.35 (d, 1H, –NH, J = 7 Hz). MS (EI, eV): m/z 356
(M?). Anal. Calcd. for C16H15N2F3O4: C, 53.94; H, 4.24; N,
7.86 %. Found: C, 53.89; H, 4.28; N, 7.90 %.
2
3
–
82.3 [280
3
5a
5b
5c
–CH3
78
76
[290
[290
[290
[290
[290
[290
[290
[290
250.5
213.5
249.2
208.7
236.4
250.5
236.4
248.9
184.6
202.6
222.6
193.9
147.2
184.6
147.2
239.5
4
–CH((CH3)2)
5
–CH(CH3)–CH2–CH3 79
6
5d
5e
–CH2–CH(CH3)2
–CH2–Ph
–CH3
73
76
74
77
72
88
84
7
8
6a
6b
7
9
–CH2–Ph
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
(2S,3R)-2-({[4-hydroxy-7-(trifluoromethyl)-3-quinolyl]
carbonyl}amino)-3-methyl pentanoic acid, 5c
8a
8b
8c
–CH3
–CH((CH3)2)
–CH(CH3)–CH2–CH3 87
Specific rotation [a]: 55.6535 (c = 0.0054 g/ml, EtOH); IR
8d
8e
–CH2–CH(CH3)2
–CH2–Ph
–CH3
82
81
88
81
83
46
89
1
(KBr, cm-1): 1732 (Acid CO), 1645 (CO–N). H NMR
(DMSO-d6, 300 MHz): d 0.92–1.20 (m, 6H, –CH–CH3,
–CH–CH2–CH3, J = 7 Hz), 1.40–1.45 (m, 2H, –CH–CH2–
CH3), 2.18–2.24 (m, 1H, CH–CH2–CH3), 4.62 (m, 1H,
–NH–CH–CO2H), 7.56 (d, 1H, Ar–H, J = 8.0), 8.00 (s,
1H, Ar–H), 8.46 (d, 1H, Ar–H, J = 8.0), 8.85 (s, 1H Ar–
H), 10.40 (d, 1H, –NH, J = 8 Hz). MS (EI, eV): m/z 370
(M?). Anal. Calcd. for C17H17N2F3O4: C, 55.14; H, 4.63;
N, 7.56 %. Found: C, 55.19; H, 4.59; N, 7.51 %.
9a
9b
10
11a
11b
11c
11d
11e
12a
12b
13
–CH2–Ph
–
–CH3
–CH((CH3)2)
–CH(CH3)–CH2–CH3 92
–CH2–CH(CH3)2
–CH2–Ph
–CH3
93
90
92
90
89
(2S)-2-({[4-hydroxy-7-(trifluoromethyl)-3-quinolyl]
carbonyl}amino)-4-methyl pentanoicacid, 5d
–CH2–Ph
–
Specific rotation [a]: -89.5296 (c = 0.0016 g/ml, EtOH).
1
IR (KBr, cm-1): 1732 (Acid CO), 1645 (CO–N). H NMR
reaction mixture was cooled and diluted with water. The
aqueous material was neutralized with 1N HCl until
the pH is reached to 4. The precipitated solid was filtered,
dried and purified by column chromatography using
MeOH:CH2Cl2 (1:1) as eluent to furnish pure compounds
5, 6, 7.
(DMSO-d6 300 MHz): d 1.07 (d, 6H, –CH (CH3)2,
J = 7.2 Hz), 1.96–2.08 (m, 1H, –CH (CH3)2), 2.22–2.30
(m, 1H, –CH–CH (CH3)2), 4.56–4.64 (m, 1H, –NH–CH–
CO2H), 7.57 (d, 1H, Ar–H, J = 8.2), 8.10 (s, 1H Ar–H),
8.44 (d, 1H, Ar–H, J = 8.0), 8.80 (s, 1H, Ar–H), 10.30 (d,
1H, –NH, J = 11 Hz). MS (EI, eV): m/z 370 (M?). Anal.
Calcd. for C17H17N2F3O4: C, 55.14; H, 4.63; N, 7.56 %.
Found: C, 55.10; H, 4.69; N, 7.52 %.
(2S)-2-({[4-hydroxy-7-(trifluoromethyl)-3-
quinolyl]carbonyl}amino) propanoic acid (5a)
(2S)-2-({[4-hydroxy-7-(trifluoromethyl)-3-quinolyl]
carbonyl}amino)-3-phenyl propanoicacid, 5e
Specific rotation [a]: -14.2865 (c = 0.0043 g/ml, EtOH).
1
IR (KBr, cm-1): 1711 (Acid CO), 1655 (CO–N). H NMR
Specific rotation [a]: -66.9389 (c = 0.0048 g/ml, EtOH).
1
(TFA, 300 MHz): d 1.58 (d, 3H, –NH–CH–CH3, J =
18 Hz), 4.50–4.70 (m, 1H, –NH–CH–CH3, J = 18 Hz),
7.80 (d, 1H, Ar–H, J = 8.2), 7.95(s, 1H, Ar–H), 8.52 (d,
1H, Ar–H, J = 8.2), 8.80 (s, 1H, Ar–H), 9.35 (d, 1H,
–NH, J = 8.6), 12.60–12.80 (s, br, 1H, –CO2H). MS (EI,
eV): m/z 328 (M?). Anal. Calcd. for C14H11N2F3O4: C,
51.23; H, 3.38; N, 8.53 %. Found: C, 51.28; H, 3.32; N,
8.50 %.
IR (KBr, cm-1): 1745 (Acid CO), 1655 (CO–N). H NMR
(DMSO-d6, 300 MHz):
d 2.55 (d, 2H, –CH2–Ph,
J = 9 Hz), 4.60–4.80 (m, 1H, –NH–CH–CO2H), 7.00–7.38
(m, 5H, Ar–H), 7.50 (d, 1H, Ar–H, J = 8.6), 7.80 (s, 1H),
8.23 (d, 1H, Ar–H, J = 8.6), 8.80 (s, 1H, Ar–H), 10.30 (d,
1H, –NH, J = 9 Hz). MS (EI, eV): m/z 404 (M?). Anal.
Calcd. for C20H15N2F3O4: C, 59.41; H, 3.74; N, 6.93 %.
Found: C, 59.38; H, 3.69; N, 6.99 %.
123

190
Med Chem Res (2013) 22:186–194
Table 2 Minimum inhibitory
concentration (MIC in lg/ml) of
compounds for various bacterial
microorganisms
Compound
B. subtilis
Microorganisms
Gram positive
S. epidermis
Gram negative
S. aureus
E. coli
K. aerogens
P. aureginosa
5a
50
50
50
50
50
50
50
50
25
25
50
50
50
25
25
50
50
50
25
25
50
50
25
25
0.048
25
25
25
50
25
5b
25
25
25
25
25
5c
25
25
25
25
25
5d
25
25
25
25
25
5e
25
12.5
25
25
25
25
6a
12.5
25
25
25
6.125
6.125
25
6b
25
25
12.5
25
7
50
25
25
8a
6.25
25
50
25
12.5
12.5
12.5
12.5
6.25
12.5
25
25
8b
25
25
25
8c
25
25
25
25
8d
25
25
25
6.25
6.25
6.125
25
8e
6.25
25
25
12.5
25
9a
25
9b
25
25
25
10
25
25
25
12.5
12.5
3.125
12.5
12.5
6.25
25
6.25
25
11a
6.25
6.25
6.25
6.25
3.125
25
12.5
12.5
25
12.5
12.5
12.5
12.5
12.5
12.5
25
11b
6.25
6.25
6.25
3.125
12.5
25
11c
11d
25
11e
12.5
25
12a
12b
25
25
12.5
6.25
0.012
Std.: Ciprofloxacin-positive
control (conc. 50 lg/ml);
DMSO-negative control showed
no activity
13
6.25
0.012
25
12.5
0.024
6.25
0.012
Ciprofloxacin
0.048
(2R)-2-({[4-hydroxy-7-(trifluoromethyl)-3-
quinolyl]carbonyl}amino) propanoic acid, 6a
4.60–4.80 (m, 1H, –NH–CH–CO2H), 7.00–7.38 (m, 5H, Ar–
H), 7.50 (d, 1H, Ar–H, J = 8.6), 7.80 (s, 1H), 8.23 (d, 1H,
Ar–H, J = 8.6), 8.80 (s, 1H, Ar–H), 10.30 (d, 1H, –NH,
J = 9 Hz). MS (EI, eV): m/z 404 (M?). Anal. Calcd. for
C20H15N2F3O4: C, 59.41; H, 3.74; N, 6.93 %. Found: C,
59.38; H, 3.69; N, 6.99 %.
Specific rotation [a]: ?18.8563 (c = 0.0037 g/ml, EtOH);
IR (KBr, cm-1): 1711 (Acid CO), 1655 (CO–N). H NMR
1
(DMSO-d6, 300 MHz): d 1.52–1.58 (d, 3H, –NH–CH–CH3,
J = 18 Hz), 4.50–4.70 (m, 1H, –NH–CH–CH3, J = 18 Hz),
7.80 (d, 1H, Ar–H, J = 8.2), 7.95 (s, 1H, Ar–H), 8.52 (d,
1H, Ar–H, J = 8.2), 8.80 (s, 1H, Ar–H), 9.35 (d, 1H,
–NH, J = 8.6), 12.60–12.80 (s, br, 1H,–CO2H). MS (EI,
eV): m/z 328 (M?). Anal. Calcd. for C14H11N2F3O4: C,
51.23; H, 3.38; N, 8.53 %. Found: C, 51.28; H, 3.32; N,
8.50 %.
(2S)-1-{[4-hydroxy-7-(trifluoromethyl)-3-
quinolyl]carbonyl}tetrahydro-1H-2-pyrrole carboxylic
acid, 7
Specific rotation [a]: -81.1231 (c = 0.0065 g/ml, EtOH);
1
IR (KBr, cm-1): 1755 (Acid CO), 1659 (CO–N). H NMR
(DMSO-d6, 300 MHz): d 2.00–2.24 (m, 4H, –CH2–CH2–
CH2–, and –CH2–CH2–CH–CO2H), 4.05–4.18 (m, 2H,
–N–CH2–CH2–), 4.50 (s, 1H, –CH–CO2H) 7.41 (d, 1H,
Ar–H J = 8.2), 7.87 (s, 1H, Ar–H), 8.28 (d, 1H, Ar–H,
J = 8.6), 8.40 (s, 1H Ar–H). MS (EI, eV): m/z 354 (M?).
Anal. Calcd. for C16H13N2F3O4: C, 54.24; H, 3.70; N,
7.91 %. Found: C, 54.29; H, 3.64; N, 7.87 %.
(2R)-2-({[4-hydroxy-7-(trifluoromethyl)-3-quinolyl]
carbonyl}amino)-3-phenyl propanoicacid, 6b
Specific rotation [a]: ?58.2176 (c = 0.0037 g/ml, EtOH);
1
IR (KBr, cm-1): 1745 (Acid CO), 1655 (CO–N). H NMR
(DMSO-d6, 300 MHz): d 2.55 (d, 2H, –CH2–Ph, J = 9 Hz),
123

Med Chem Res (2013) 22:186–194
191
Preparation of a-amino acid functionalised di ethylated
quinoline compounds 8, 9, 10
(4-keto), 1026 (C–O–C). 1H NMR (DMSO-d6, 300 MHz): d
0.95–1.18 (m, 6H, –CH–CH3, –CH–CH2–CH3,), 1.38 (t, 3H,
–CO2–CH2–CH3, J = 8.0), 1.48–1.52 (m, 2H, –CH–CH2–
CH3), 1.65 (t, 3H, –N–CH2–CH3, J = 6.0), 2.28–2.34 (m,
1H, –CH–CH2–CH3), 4.32 (q, 2H, –CO2–CH2–CH3,
J = 8.0), 4.58 (q, 2H,–N–CH2–CH3, J = 6.0), 4.68–4.72
(m, 1H, –NH–CH–CO2H), 7.57 (d, 1H, Ar–H J = 8.2), 8.05
(s, H, Ar–H), 8.50 (d, 1H, Ar–H J = 8.2), 8.90 (s, 1H, Ar–H),
10.35 (d, 1H, –NH, J = 8 Hz). MS (EI, eV): m/z 426 (M?).
Anal. Calcd. for C21H25N2F3O4: C, 59.15; H, 5.91; N,
6.57 %. Found: C, 59.10; H, 5.98; N, 6.54 %.
General procedure
Quinoline 5, 6, 7 (3 mmol), potassium carbonate (9 mmol)
and ethyl iodide (15 mmol) were charged in a flask and
added DMF (15 ml). The mixture was heated at 80–90 °C
with stirring for 15 h. The solvent was evaporated to dry-
ness by distilling DMF from reaction mixture and extracted
with chloroform. The CHCl3 layer was washed with water,
dried and concentrated to dryness. The dried crude com-
pound was purified by passing through column chroma-
tography and isolated the title compounds.
Ethyl (2S)-2-({[1-ethyl-4-oxo-7-(trifluoromethyl)-
1,4-dihydro-3-quinolinyl] carbonyl} amino)-4-methyl
pentanoate, 8d
Ethyl (2S)-2-({[1-ethyl-4-oxo-7-(trifluoromethyl)-1,4-
dihydro-3-quinolinyl] carbonyl} amino) propanoate, 8a
Specific rotation [a]: -17.5329 (c = 0.0039 g/ml, CHCl3);
IR (KBr, cm-1): 1764 (Ester CO), 1670 (CO–NH), 1649
1
(4-keto), 1027 (C–O–C). H NMR (DMSO-d6, 300 MHz):
Specific rotation [a]: -42.8570 (c = 0.0028 g/ml, CHCl3).
IR (KBr, cm-1): 1734 (Ester CO), 1674 (CO–NH), 1612
(4-keto), 1022 (C–O–C). 1H NMR (DMSO-d6, 300 MHz): d
1.30 (t, 3H, –CO2–CH2–CH3, J = 8.0), 1.50–1.70 (m, 6H, N–
CH2–CH3, –CH–CH3, J = 8 and 6.0), 4.24 (q, 2H, –CO2–
CH2–CH3, J = 8.0), 4.47 (q, 2H, N–CH2–CH3, J = 6.0),
4.62–4.75 (m, 1H, –NH–CH–CO2H), 7.71 (d, 1H, Ar–H,
J = 8.0), 7.84 (s, 1H, Ar–H), 8.65 (d, 1H, ArH, J = 8.0), 8.88
(s, 1H, Ar–H), 10.24 (d, 1H, –NH, J = 8.0 Hz). MS (EI, eV):
m/z 384 (M?). Anal. Calcd. for C18H19N2F3O4: C, 56.25; H,
4.98; N, 7.29 %. Found: C, 56.28; H, 4.92; N, 7.34 %.
d 0.96 (d, 6H, –CH–(CH3)2), 1.40 (t, 3H, CO2–CH2–CH3,
J = 8.2), 1.72–1.84 (t, 3H, –N–CH2–CH3, J = 6.0),
1.89–1.95 (m, 1H, –CH–(CH3)2), 2.26–2.38 (m, 1H, CH–
CH–(CH3)2), 4.37 (q, 2H, CO2–CH2–CH3, J = 8.2), 4.65
(q, 2H, –N–CH2–CH3, J = 6.0), 4.82–4.88(m, 1H, –NH–
CH–CO2H), 7.58 (d, 1H, Ar–H J = 9.0), 8.08 (s, 1H, Ar–
H),8.49 (d, 1H, Ar–H, J = 8.2), 8.82 (s, 1H, Ar–H), 10.36
(d, 1H, –NH, J = 8.0 Hz). MS (EI, eV): m/z 426 (M?).
Anal. Calcd. for C21H25N2F3O4: C, 59.15; H, 5.91; N,
6.57 %. Found: C, 59.12; H, 5.96; N, 6.62 %.
Ethyl (2S)-2-({[1-ethyl-4-oxo-7-(trifluoromethyl)-1,4-
dihydro-3-quinolinyl] carbonyl} amino)-3-methyl
butanoate, 8b
Ethyl (2S)-2-({[1-ethyl-4-oxo-7-(trifluoromethyl)-1,4-
dihydro-3-quinolinyl] carbonyl} amino)-3-phenyl
propanoate, 8e
Specific rotation [a]: -18.4485 (c = 0.0017 g/ml, CHCl3);
IR (KBr, cm-1): 1754 (Ester CO), 1670 (CO–NH), 1618
(4-keto), 1029 (C–O–C). 1H NMR (DMSO-d6, 300 MHz): d
1.16 (d, 6H, –CH–(CH3)2, J = 9), 1.36 (t, 3H, –CO2–CH2–
CH3, J = 8.0),1.60 (t, 3H, –N–CH2–CH3, J = 6.0), 2.20–
2.40 (m, 1H, –CH–(CH3)2), 4.26 (q, 2H, –CO2–CH2–CH3,
J = 8.0), 4.42 (q, 2H, –N–CH2–CH3, J = 6.0), 4.60–4.70 (m,
1H, –NH–CH–CO2H), 7.65 (d, 1H, Ar–H, J = 8.2), 7.74 (s,
1H, Ar–H), 8.70 (d, 1H, Ar–H, J = 8.2), 8.88 (s, 1H, Ar–H),
10.30 (d, 1H, –NH, J = 9 Hz). MS (EI, eV): m/z 412 (M?).
Anal. Calcd. for C20H23N2F3O4: C, 58.25; H, 5.62; N, 6.79 %.
Found: C, 58.29; H, 5.69; N, 6.72 %.
Specific rotation [a]: -38.4386 (c = 0.0048 g/ml, CHCl3);
IR (KBr, cm-1): 1769 (Ester CO), 1650 (CO–NH), 1638
1
(4-keto), 1037 (C–O–C). H NMR (DMSO-d6, 300 MHz):
d 1.25 (t, 3H, –CO2–CH2–CH3, J = 8.0), 1.62 (t, 3H, –N–
CH2–CH3,J = 6.0), 3.20 (d, 2H, CH2–Ph, J = 12), 4.20 (q,
2H, –CO2–CH2–CH3, J = 8.0), 4.36 (q, 2H, –N–CH2–
CH3, J = 6.0), 4.88–5.00 (m, 1H, –NH–CH–CO2C2H5),
7.20–7.40 (m, 5H, Ar–H), 7.65 (d, 1H, Ar–H, J = 8.2),
7.76 (s, 1H, Ar–H), 8.72 (d, 1H, Ar–H J = 6), 8.82 (s, 1H,
Ar–H), 10.30 (d, 1H, –NH, J = 9 Hz). MS (EI, eV):
m/z 460 (M?). Anal. Calcd. for C24H23N2F3O4: C, 62.60;
H, 5.03; N, 6.08 %. Found: C, 62.56; H, 5.10; N, 6.03 %.
Ethyl (2S, 3R)-2-({[1-ethyl-4-oxo-7-(trifluoromethyl)-
1,4-dihydro-3-quinolinyl] carbonyl} amino)-3-methyl
pentanoate, 8c
Ethyl(2S)-2-({[1-ethyl-4-oxo-7-(trifluoromethyl)-1,4-
dihydro-3-quinolinyl] carbonyl} amino) propanoate, 9a
Specific rotation [a]: ?37.5535 (c = 0.0043 g/ml, CHCl3);
Specific rotation [a]: -22.5356 (c = 0.0039 g/ml, CHCl3);
IR (KBr, cm-1): 1734 (Ester CO), 1674 (CO–NH), 1612
IR (KBr, cm-1): 1744 (Ester CO), 1676 (CO–NH), 1638
(4-keto), 1022 (C–O–C). H NMR (DMSO-d6, 300 MHz):
1
123

192
Med Chem Res (2013) 22:186–194
d 1.30 (t, 3H, –CO2–CH2–CH3, J = 8.0), 1.50–1.70 (m,
6H, N–CH2–CH3, –CH–CH3, J = 8 and 6.0), 4.24 (q, 2H,
–CO2–CH2–CH3, J = 8.0), 4.47 (q, 2H, N–CH2–CH3,
J = 6.0), 4.62–4.75 (m, 1H, –NH–CH–CO2H), 7.71 (d,
1H, Ar–H, J = 8.0), 7.84 (s, 1H, Ar–H), 8.65 (d, 1H, Ar H,
J = 8.0), 8.88 (s, 1H, Ar–H), 10.24 (d, 1H, –NH,
J = 8.0 Hz). MS (EI, eV): m/z 384 (M?). Anal. Calcd. for
C18H19N2F3O4: C, 56.25; H, 4.98; N, 7.29 %. Found: C,
56.28; H, 4.92; N, 7.34 %.
temperature for 3 h. The reaction mixture was neutralized
with 1N HCl, and solvent was removed under vacuum. The
resulted material was extracted with ethyl acetate. The
organic layer was washed with water, dried and concen-
trated to dryness. The dried residue was purified by passing
through column chromatography and isolated the acid
derivatives.
(2S)-2-({[1-ethyl-4-oxo-7-(trifluoromethyl)-1,4-dihydro-
3-quinolinyl] carbonyl} amino) propanoic acid, 11a
Ethyl (2S)-2-({[1-ethyl-4-oxo-7-(trifluoromethyl)-1,4-
dihydro-3-quinolinyl] carbonyl} amino)-3-
phenylpropanoate, 9b
Specific rotation [a]: -39.5388 (c = 0.0025 g/ml, EtOH).
IR (KBr, cm-1): 1759 (Acid CO), 1635 (CO–N), 1612
(4-keto). 1H NMR (DMSO-d6, 300 MHz): d 1.40–1.60 (m,
6H, N–CH2–CH3 and –NH–CH–CH3, J = 8.2 and 6 Hz),
4.42–4.70 (m, 3H, –CH–CH3 and N–CH2–CH3, J = 8.2
and 6.0), 7.71 (d, 1H, Ar–H, J = 8.6), 7.97 (s, 1H, Ar–H),
8.7 (d, 1H, Ar–H, J = 8.6), 8.96 (s, 1H, Ar–H), 10.25 (d,
1H, –NH, J = 9 Hz). MS (EI, eV): m/z 356 (M?). Anal.
Calcd. for C16H15N2F3O4: C, 53.94; H, 4.24; N, 7.86 %.
Found: C, 53.98; H, 4.23; N, 7.81 %.
Specific rotation [a]: ?33.1847 (c = 0.0032 g/ml, CHCl3);
IR (KBr, cm-1): 1769 (Ester CO), 1650 (CO–NH), 1630
1
(4-keto), 1037 (C–O–C). H NMR (DMSO-d6, 300 MHz):
d 1.25 (t, 3H, –CO2–CH2–CH3, J = 8.0), 1.62 (t, 3H, N–
CH2–CH3, J = 6.0), 3.20 (d, 2H, CH2–Ph, J = 12), 4.20
(q, 2H, –CO2–CH2–CH3, J = 8.0), 4.36 (q, 2H, –N–CH2–
CH3, J = 6.0), 4.88–5.00 (m, 1H, –NH–CH–CO2C2H5),
7.20–7.40 (m, 5H, Ar–H), 7.65 (d, 1H, Ar–H, J = 8.2),
7.76 (s, 1H, Ar-H), 8.72 (d, 1H, Ar–H, J = 6), 8.82 (s, 1H,
Ar–H), 10.30 (d, 1H, –NH, J = 9 Hz). MS (EI, eV):
m/z 460 (M?). Anal. Calcd. for C24H23N2F3O4: C, 62.60;
H, 5.03; N, 6.08 %. Found: C, 62.56; H, 5.10; N, 6.03 %.
(2S)-2-({[1-ethyl-4-oxo-7-(trifluoromethyl)-1,4-dihydro-
3-quinolinyl] carbonyl} amino)-3-methyl butanoic acid, 11b
Specific rotation [a]: -49.5372 (c = 0.0035 g/ml, EtOH);
Ethyl (2S)-1-{[1-ethyl-4-oxo-7-(trifluoromethyl)-1,4-
dihydro-3-quinolinyl] carbonyl} tetrahydro-1H-2-pyrrole
carboxylate, 10
IR (KBr, cm-1): 1749 (Acid CO), 1645 (CO–N), 1630
1
(4-keto). H NMR (DMSO-d6, 300 MHz): d 0.97 (d, 6H,
–CH–(CH3)2, J = 6 Hz,), 1.46 (t, 3H, N–CH2–CH3, J =
6.0), 2.24–2.34 (m, 1–CH–(CH3)2), 4.53 (q, 2H, –N–CH2–
CH3, J = 6.0), 4.58–4.67 (m, 1H, –NH–CH–CO2H,), 7.70
(d, 1H, Ar–H J = 8.2), 7.78 (s, 1H, Ar–H), 8.59 (d, 1H,
Ar–H J = 8.2), 8.90 (s, 1H, Ar–H), 10.30 (d, 1H, NH,
J = 8 Hz). MS (EI, eV): m/z 384 (M?). Anal. Calcd. for
C18H19N2F3O4: C, 56.25; H, 4.98; N, 7.29 %. Found: C,
56.28; H, 4.90; N, 7.30 %.
Specific rotation [a]: -18.1114 (c = 0.0023 g/ml, CHCl3);
IR (KBr, cm-1): 1777 (ESTER CO), 1648 (CO–N), 1630
1
(4-keto), 1039 (C–O–C). H NMR (DMSO-d6, 300 MHz):
d 1.22–1.38 (m, 6H, –CO2–CH2–CH3, –N–CH2–CH3,
J = 6.0 and 8.2), 2.10–2.28 (m, 4H, –N–CH2–CH2–CH2–),
3.78–4.00 (m, 2H, –N–CH2–CH2), 4.31 (q, 2H, –CO2–
CH2–CH3, J = 8.2), 4.78 (q, 2H, –N–CH2–CH3, J = 6.0),
4.89 (m, 1H, –CH–CO2CH2CH3), 7.40 (d, 1H, Ar–H
J = 8.0), 7.78 (s, 1H, Ar–H), 8.30 (d, 1H, Ar–H, J = 8.0),
8.38 (s, 1H, Ar–H). MS (EI, eV): m/z 410 (M?). Anal.
Calcd. for C20H21N2F3O4: C, 58.53; H, 5.16; N, 6.83 %.
Found: C, 58.59; H, 5.10; N, 6.86 %.
(2S)-2-({[1-ethyl-4-oxo-7-(trifluoromethyl)-1,4-dihydro-3-
quinolinyl] carbonyl} amino)-3-methyl pentanoic acid, 11c
Specific rotation [a]: -77.8268 (c = 0.0033 g/ml, EtOH);
IR (KBr, cm-1): 1789 (Acid CO), 1655 (CO–N), 1622 (4-
keto). 1H NMR (DMSO-d6, 300 MHz): d 0.93–1.08 (m, 6H,
–CH–CH3, –CH–CH2–CH3, J = 11 and 9 Hz),1.42–1.48
(m, 2H, CH–CH2–CH3), 1.69 (t, 3H, –N–CH2–CH3, J =
6.0), 2.39–2.45 (m, 1H, –CH–CH2–CH3), 4.70 (q, 2H, N–
CH2–CH3, J = 6.0), 4.76–4.84 (m, 1H, –NH–CH–CO2H),
7.69 (d, 1H, Ar–H J = 8.6), 8.09 (s, 1H, Ar–H), 8.61 (d, 1H,
Ar–H J = 8.6), 8.93 (s, 1H, Ar–H), 10.32 (d, 1H, –NH,
J = 8 Hz). MS (EI, eV): m/z 398 (M?). Anal. Calcd. for
C19H21N2F3O4: C, 57.28; H, 5.31; N, 7.03 %. Found: C,
57.22; H, 5.26; N, 7. 09 %.
Preparation of a-amino acid functionalised
1-N-ethylated quinolone compounds, 11, 12, 13
General procedure
Quinolone 8, 9, 10 (1.5 mmol) and lithium hydroxide
(3.0 mmol) were added to the solvent mixture of THF:H2O
(3:1) (5 ml) and the contents were stirring at room
123

Med Chem Res (2013) 22:186–194
193
(2S)-2-({[1-ethyl-4-oxo-7-(trifluoromethyl)-1,4-dihydro-3-
quinolinyl] carbonyl} amino)-4-methyl pentanoic acid, 11d
CH–CO2H), 7.10–7.30 (m, 5H, Ar–H), 7.90 (d, 1H, Ar–H,
J = 8.6), 7.96 (s, 1H, Ar–H), 8.64 (d, 1H, Ar–H, J = 8.6),
8.92 (s, 1H, Ar–H), 10.20 (d, 1H, –NH, J = 8 Hz). MS (EI,
eV): m/z 432 (M?). Anal. Calcd. for C22H19N2F3O4: C,
61.11; H, 4.43; N, 6.48 %. Found: C, 61.17; H, 4.40; N,
6.47 %.
Specific rotation [a]: -74.7827 (c = 0.0046 g/ml, EtOH);
IR (KBr, cm-1): 1799 (Acid CO), 1659 (CO–N), 1618
1
(4-keto). H NMR (DMSO-d6, 300 MHz): d 0.95 (d, 6H,
–CH–(CH3)2, J = 8 Hz), 1.72 (t, 3H, N–CH2–CH3,
J = 6.7),1.83–1.92 (m, 1H, CH–(CH3)2), 2.30–2.42 (m,
1H, –CH–CH–(CH3)2), 4.65 (q, 2H, N–CH2–CH3
J = 6.7), 4.78–4.85 (m, 1H, –NH–CH–CO2H,), 7.58 (d,
1H, Ar–H J = 8.8), 8.10 (s, 1H, Ar–H), 8.46 (d, 1H, Ar–H,
J = 8.8), 8.81 (s, 1H, Ar–H), 10.30 (d, 1H, –NH,
J = 11 Hz). MS (EI, eV): m/z 398 (M?). Anal. Calcd. for
C19H21N2F3O4: C, 57.28; H, 5.31; N, 7.03 %. Found: C,
57.32; H, 5.26; N, 7. 05 %.
(2S)-1-{[1-ethyl-4-oxo-7-(trifluoromethyl)-1,4-dihydro-
3-quinolinyl] carbonyl} tetra hydro-1H-2-pyrrole
carboxylic acid, 13
Specific rotation [a]: -78.5585 (c = 0.004 g/ml, EtOH); IR
(KBr, cm-1): 1799 (Acid CO), 1689 (CO–N), 1620 (4-keto).
1H NMR (DMSO-d6, 300 MHz): d 1.31 (t, 3H, –N–CH2–
CH3, J = 6.0), 2.24–2.32 (m, 4H, –N–CH2–CH2–CH2–),
3.92–4.04 (m, 2H, –N–CH2–CH2 J = 8 Hz), 4.85 (q, 2H,
–N–CH2–CH3, J = 6.0), 4.88–4.92 (m, 1H, –CH–CO2H),
7.32 (d, 1H, Ar–H J = 8.2), 7.80 (s, 1H, Ar–H), 8.38 (d, 1H,
Ar–H J = 8.2), 8.95 (s, 1H, Ar–H). MS (EI, eV): m/z 382
(M?). Anal. Calcd. for C18H17N2F3O4: C, 56.55; H, 4.48; N,
7.33 %. Found: C, 56.54; H, 4.52; N, 7.26 %.
(2S)-2-({[1-ethyl-4-oxo-7-(trifluoromethyl)-1,4-dihydro-3-
quinolinyl] carbonyl} amino)-3-phenyl propanoic acid, 11e
Specific rotation [a]: -64.2423 (c = 0.0033 g/ml, EtOH);
IR (KBr, cm-1): 1780 (Acid CO), 1680 (CO–NH), 1620
1
(4-keto). H NMR (DMSO-d6, 300 MHz): d 1.54 (t, 3H,
Acknowledgments The authors are thankful to Dr. J.S.Yadav,
Director, IICT, Hyderabad for his constant encouragement and
support.
–N–CH2–CH3, J = 6.7), 3.20 (d, 2H, CH2–Ph, J =
12 Hz), 4.51 (q, 2H, N–CH2–CH3, J = 6.7), 4.60–4.76 (m,
1H, –NH–CH–CO2H), 7.10–7.30 (m, 5H, Ar–H), 7.90 (d,
1H, Ar–H J = 8.6), 7.96 (s, 1H, Ar–H), 8.64 (d, 1H, Ar–H
J = 8.6), 8.92 (s, 1H, Ar–H), 10.20 (d, 1H, –NH,
J = 8 Hz). MS (EI, eV): m/z 432 (M?). Anal. Calcd. for
C22H19N2F3O4: C, 61.11; H, 4.43; N, 6.48 %. Found: C,
61.17; H, 4.40; N, 6.47 %.
References
Bridges AJ, Domagala JM (1988) 8-trifluoromethyl quinolones as
antibacterial agents. US Patent 4,780,468, 25 Oct 1988
Chu DTW, Prabhavathi BF, Akiyo KC, Pihuleae E, Nordeen CW,
Maleczka RE, Pernet AG (1985) Synthesis and structure–activity
relationships of novel arylfluoroquinolone antibacterial agents.
J Med Chem 28:1558–1564
(2S)-2-({[1-ethyl-4-o-7-(trifluoromethyl)-1,4-dihydro-3-
quinolinyl]carbonyl}amino)propanoic acid, 12a
Filler R et al (eds) (1993) Organofluorine compounds in medicinal
chemistry and biomedical applications. Elsevier, Amsterdam,
pp 165
Hooper DC, Wolfson JS (1993) Quinolone antimicrobial agents, 2nd
edn. American Society for Microbiology, Washington, DC,
pp 3–51
Koga H, Itoh A, Murayama S, Suzue S, Irikura T (1980) Structure–
activity relationships of antibacterial 6, 7- and 7, 8-disubstituted
1-alkyl-1,4-dihydro-4-oxoquinoline-3-carboxylic acids. J Med
Chem 23:1358–1363
Lopez SE, Rebollo O, Salazar J, Charris JE, Yanez C (2003)
Synthesis of 2-trifluoromethyl-1 (substituted aryl)-4(1H)-quino-
lones using trifluoroacetamidoyl chlorides. J Fluor Chem 120:
71–75
Specific rotation [a]: ?45.5629 (c = 0.0025 g/ml, EtOH);
IR (KBr, cm-1): 1728 (Acid CO), 1656 (CO–N), 1622
(4-keto). 1H NMR (DMSO-d6, 300 MHz): d 2.48–2.62 (m,
6H, N–CH2–CH3 and –CH–CH3, J = 8.2 and 6 Hz),
4.42–4.70 (m, 3H, –NH–CH–CO2H, N–CH2–CH3, J = 8.2
and 6.0), 7.71 (d, 1H, Ar–H, J = 8.6), 7.97 (s, 1H, Ar–H),
8.64 (d, 1H, Ar–H, J = 8.6), 8.96 (s, 1H, Ar–H), 10.25 (d,
1H, –NH, J = 9 Hz). MS (EI, eV): m/z 356 (M?). Anal.
Calcd. for C16H15N2F3O4: C, 53.94; H, 4.24; N, 7.86 %.
Found: C, 53.98; H, 4.23; N, 7.81 %.
Matsumoto J, Miyamoto T, Minamida A, Nishimura Y, Egawa H,
Nishimura H (1984) 1,4-dihydro-4-oxopyridine carboxylic acids
as antibacterial agents. Synthesis and structure activity relation-
ships of 1,6,7-trisubstituted 1,4-dihydro-4-oxo-1,8-naphthyri-
dine-3-carboxylic acids including enoxacin, a new antibacterial
agent. J Med Chem 27:292–301
Radl S, Bouzard D (1992) Recent advances in the synthesis of
antibacterial quinolones. Heterocycles 34:2143
Sanchez JP, Bridges AJ, Bucsh R, Domagala JM, Gogliotti RD,
Hagen SE, Heifetz CL, Joannides ET, Sesnie JC (1992) New
8-(trifluoromethyl)-substituted quinolones. The benefits of the
(2S)-2-({[1-ethyl-4-oxo-7-(trifluoromethyl)-1,4-dihydro-3-
quinolinyl] carbonyl} amino)-3-phenyl propanoic acid, 12b
Specific rotation [a]: ?59.8396 (c = 0.0036 g/ml, EtOH);
IR (KBr, cm-1): 1780 (Ester CO), 1680 (CO–N), 1612 (4-
1
keto). H NMR (DMSO-d6, 300 MHz): d 1.54 (t, 3H, –N–
CH2–CH3, J = 6.7), 3.20 (d, 2H, CH2–Ph, J = 12), 4.51
(q, 2H, N–CH2–CH3, J = 6.7), 4.60–4.76 (m, 1H, –NH–
123

194
Med Chem Res (2013) 22:186–194
8-fluoro group with reduced phototoxic risk. J Med Chem
35:361–367
fused quinolones carboxamides. Eur J Med Chem 44:1570–
1578
Uray G, Niederreiter K (2003) Luminescent 4-trifluoromethyl 1-2-
quinolones with longwave UV absorption and use thereof. US
Patent 6,635,759, 21 Oct 2003
Venkat Reddy G, Ravikanth S, Maitraie D, Narsaiah B, Shanthan
Rao P, Harakishore K, Murthy USN, Ravi B, Ashok kumar B,
Parthasarathy T (2009) Design, synthesis, structure–activity
relationship and antibacterial activity series of novel imidazo
Violetta C, Cristina P, Stefano M, Teresa P, Enrica F, Arianna C,
Oriana T, Barbara G, Manlio P, Arnaldo F, Giorgio P (2000)
6-Aminoquinolones as new potential anti-HIV agents. J Med
Chem 43:3799–3802
Wise R, Andrews JM, Edwards LJ (1983) In vitro activity of Bay
09867, a quinoline derivative, compared with these of other
antimicrobial agents. Antimicrob Agents Chemother 23:559–564
123