Synthesis and antimycobacterial evaluation on arylsulfonyl and arylcarbonyl derivatives of ofloxacin

Article abstract of DOI:10.1002/jccs.200900054

Four ofloxacin derivatives 3, 5, 6, and 11 were found to exhibit > 90 % inhibition on the growth of M. tuberculosis at a concentration of 6.25μg/mL. Compounds 3, 5 and 11 have also exhibited a broad spectrum of antibacterial activities while 8-fluoro-3-me

Full text of DOI:10.1002/jccs.200900054

374
Journal of the Chinese Chemical Society, 2009, 56, 374-380
Synthesis and Antimycobacterial Evaluation on Arylsulfonyl and
Arylcarbonyl Derivatives of Ofloxacin
Yeh-Long Chena (
Chai-Lin Kaoa (
Chen-Wen Yaob (
), Yu-Wen Chena (
), Yu-Shuan Liub (
) and Cherng-Chyi Tzenga,* (
), We-Fen Loa (
),
),
)
aFaculty of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University,
Kaohsiung City 807, Taiwan, R.O.C.
bDepartment of Pathology, Tri-Service General Hospital, Taipei 114, Taiwan, R.O.C.
Four ofloxacin derivatives 3, 5, 6, and 11 were found to exhibit > 90 % inhibition on the growth of M.
tuberculosis at a concentration of 6.25 mg/mL. Compounds 3, 5 and 11 have also exhibited a broad spec-
trum of antibacterial activities while 8-fluoro-3-methyl-9-[4-(4-nitrophenylsulfonyl)piperazin-1-yl)-6-
oxo-2,3-dihydro-6H-1-oxo-3a-azaphenalene-5-carboxylic acid (6), which exhibited potent activity
against the growth of TB with the MIC of 2.23 mg/mL and a selectivity index (SI) of > 14.80, was inactive
against the growth of G(+)- and G(-)-bacteria. Selective anti-TB activity was achieved by the introduction
of an arylsulfonyl group at C-7 piperazin-4-yl of N-demethyl ofloxacin. Compound 6 is species-specific,
exhibiting no significant activity against the growth of bacterial species other than M. tuberculosis, which
implied the possibility of developing new specific anti-TB drug candidates without inducing cross resis-
tance with other currently used antibacterial drugs. Structural optimization of 6 is on-going.
Keywords: Fluoroquinolone; Antimycobacterial activity; Ofloxacin derivatives.
INTRODUCTION
The drugs currently used for the treatment of tubercu-
their antibacterial and anticancer activities.16-22 Since the
antibacterial mechanism of these fluoroquinolones is
unique and absolutely different from the currently used
anti-TB drugs, it will become a very important anti-TB
drug candidate if proved to be active against the growth of
M. tuberculosis. Therefore, we have synthesized and evalu-
ated certain hybrid compounds for anti-TB evaluation.21 To
continue our search for more potent anti-TB agents, we re-
port herein synthesis and evaluation of ofloxacin deriva-
tives with an arylsulfonyl group substituented at C-7 piper-
azine moiety on the ground that a number of sulfonylated
derivatives such as 123 and 224 exhibited significant anti-TB
losis (TB) infection are streptomycin, isoniazid, etham-
butol, pyrazinamide, and rifampicin.1 However, the current
TB treatment regimens, although highly effective, are far
from idea. Recently, the emergence of multi-drug resistant
tuberculosis (MDRTB)2,3 has caused an urgent need in
search of alternative chemotherapeutics for Mycobacte-
rium tuberculosis infection. Several fluoroquinolone anti-
bacterial drugs were examined as potential chemothera-
peutics for M. tuberculosis infection.4-10 A number of
newer fluoroquinolone derivatives have been synthesized
and evaluated for their anti-TB activity.11-15 However, the
optimization of the fluoroquinolones against TB infection
has not been thoroughly explored especially the optimum
substituent at the C-7 position which has a great impact on
potency, antibacterial spectrum, solubility, and pharmaco-
kinetics, has not been precisely defined.
Over the past few years, we were particularly inter-
ested in the synthesis and evaluation of fluoroquinolons for
* Corresponding author. Tel: +886-7-3121101 ext 2123; Fax: +886-7-3125339; E-mail: tzengch@kmu.edu.tw (Cherng-Chyi Tzeng);
Tel: +886-2-87923311 ext 16756; Fax: +886-2-87929095; E-mail: yaocw329@gmail.com (Chen-Wen Yao)

Anti-TB Evaluation of Ofloxacin Derivatives
J. Chin. Chem. Soc., Vol. 56, No. 2, 2009 375
activities. The bio-isosteric arylcarbonyl counterparts of
arylsulfonyl ofloxacin derivatives were also synthesized
for evaluation.
MTT into a formazan product using the Promega Cell Titer
96 Non-radioactive Cell Proliferation Assay and the results
are summarized in Table 1. 8-Fluoro-3-methyl-9-[4-(4-ni-
trophenylsulfonyl)piperazin-1-yl)-6-oxo-2,3-dihydro-6H-
1-oxo-3a-azaphenalene-5-carboxylic acid (6),26 with 4-ni-
tro group substituted at benzenesulfonyl moiety, exhibited
96% inhibition on the growth of M. tuberculosis at a con-
centration of 6.25 mg/mL, which was comparable to the
positive N-demethyl ofloxacin 3 (99%) and its 4-fluoro
counterpart 5 (92%) but was more active than its 4-meth-
oxy counterpart 7 (62%). These results implied that the
electron-withdrawing groups, NO2 and F, enhanced while
the electron-donating group, MeO, decreased anti-TB ac-
tivity. However, the above structure-activity relationships
can not be applied to their bio-isosteric benzoyl derivatives
in which 9 (4-F, 64%) and 10 (4-NO2, 73%), bearing elec-
tron-withdrawing groups, exhibited only marginal activity
while 11 (4-OMe, 94%) was much more potent. The cyto-
toxicity (IC50) against mammalian VERO cells as well as
the selectivity index (SI), defined as IC50/MIC, for com-
pounds 3, 5, 6, and 11 were also determined. Compounds
with an SI of > 10 are of interest for further development as
potential anti-TB agents. Results indicated that compounds
3, 5, and 6 exhibited significant activities against the
growth of M. tuberculosis with MIC values of 1.58, 1.83,
and 2.23 mg/mL respectively. Compounds 3 and 6 have also
exhibited SI values of greater than 20.89 and 14.80 respec-
RESULTS AND DISCUSSION
Synthesis
Preparation of ofloxacin derivatives with arylsulfo-
nyl and arylcarbonyl substituents at C-7 piperazine moiety
is depicted in the Scheme I. Treatment of N-demethyl
ofloxacin (3) with substituted benzenesulfonyl chloride or
benzoyl chloride in the presence of K2CO3 afforded ben-
zenesulfonyl derivatives 4-7 and their bio-isosteric benzo-
yl isomers 8-11, respectively.
Biological activities
Primary screening is conducted against M. tuberculo-
sis H37Rv (ATCC 27294) in BACTEC 12B medium using a
broth microdilution assay to determine the actual minimum
inhibitory concentration (MIC) using the Microplate Alamar
Blue Assay (MABA).25 The MIC is defined as the lowest
concentration effecting a reduction in fluorescence of 90%
relative to controls. Concurrent with the determination of
MICs, compounds are screened by serial dilutions to assess
cytotoxicity (IC50) to a VERO cell. After 72 h exposure, vi-
ability is assessed on the basis of cellular conversion of
Scheme I

376 J. Chin. Chem. Soc., Vol. 56, No. 2, 2009
Chen et al.
Table 1. Anti-Mycobacterium tuberculosis H37Rv (ATCC 27294) activity of ofloxacin
derivatives
O
F
COOH
N
N
N
O
Me
R
% Inhibition at
R
MIC (mg/mL) IC50 (mg/mL) SI (IC50/MIC)
6.25 mg/mL
3
H
99
66
92
96
62
86
64
73
94
1.58
nda
> 33
nd
> 20.89
nd
4
SO2Ph
5
SO2Ph-4-F
SO2Ph-4-NO2
SO2Ph-4-OMe
CO-Ph
1.81
2.23
nd
16.60
> 33
nd
9.16
> 14.80
nd
6
7
8
nd
nd
nd
9
COPh-4-F
COPh-4-NO2
COPh-4-OMe
nd
nd
nd
10
11
nd
nd
nd
> 6.25
> 10
nd
a Not determined.
tively.
(3) exhibited a broad spectrum of antibacterial activity.
However, its arylsulfonyl derivatives such as compounds 4
and 7 were much less active while compound 6 was inac-
tive. With exception of 9, the antibacterial activity of all the
arylcarbonyl derivatives 8, 10, and 11 were very active and
The zone of inhibition in diameter at a concentration
100 mg/mL of each tested agent was determined by the disk
diffusion (Kirby-Bauer) methods27,28 and the results are
summarized in Table 2. The positive N-demethyl ofloxacin
Table 2. Antimicrobial activity of ofloxacin derivatives
Eca
Pa
Ef
Sp
Spn
Sa
Sa-a Sa-b Sa-c Ml-a Ml-b Ms
3
26b
Nc
22
N
20
N
17
N
24
N
24
+/-
20
N
20
16
25
9
23
15
24
+/-
13
28
16
30
26
24
14
30
11
13
27
17
27
23
22
15
22
7.5
12
33
12
32
29
24
9
28
N
40
N
4
5
N
16.5
N
20
11
12
22
14
21
21
22
N
22
N
35
N
6
N
7
14
22
15
24
24
N
9
15
25
8
16.5
22
14
24
22
8
8
+/-
52
+/-
47
50
8
15
N
16
7.5
15
15
29
9
27
8
9
10
11
12
14
25
24
25
22
30
23
a Ec, Escherichia coli; Pa, Pseudomonas aeruginosa; Ef, Enterococcus faecalis; Sp,
Streptococcus pyogenes; Spn, Streptococcus pneumoniae; Sa, Staphylococcus aureus; Sa-a,
Staphylococcus aureus (ATCC12692) resistant to novobiocin; Sa-b, Staphylococcus aureus
(ATCC12715) resistant to tetracycline; Sa-c, Staphylococcus aureus (ATCC14154) resistant
to tetracycline, penicillin, streptomycin, and erythromycin, sensitive to carbomycin,
neomycin, chloramphenicol, and novobiocin; Ef-s, Enterococcus faecalis (vancomycin
resistant strain, clinical isolated); Ml-a, Micrococcus luteus (ATCC10240A) resistant to
dihydrostreptomycin sulfate and streptomycin; Ml-b.
b Diameter of the zone of inhibition (in mm), each compound was tested at concentration of
100 mg/mL.
c No inhibition.

Anti-TB Evaluation of Ofloxacin Derivatives
J. Chin. Chem. Soc., Vol. 56, No. 2, 2009 377
comparable to that of 3.
e): 230 (4.20), 297 (4.50) in MeOH. IR nmax cm-1: 1171,
1458, 1525, 1620, 1713 in KBr. 1H-NMR (DMSO-d6): 1.41
(d, 3H, J = 6.8 Hz, 2-CH3), 3.03, 3.34 (m, 8H, 9-piper-
azinyl-H), 4.31, 4.53 (AB, 2H, J = 11.6 Hz, 2H-C(1)), 4.90
(q, 1H, J = 6.8 Hz, 1H-C(2)), 7.56 (d, 1H, J = 12.0 Hz,
1H-C(7)), 7.69 (m, 2H, Ar-H), 7.79 (m, 3H, Ar-H), 8.96 (s,
1H, 1H-C(4)), 15.12 (br s, COOH). ESI MS m/z: 488 (M +
H)+.
EXPERIMENTAL PART
1. General
All reactions were monitored by TLC on silica gel 60
F-254 plates purchased from EM Laboratories, Inc. and
were detected by UV light at 254 nm. All flash column
chromatographic separations were performed using silica
gel (Merck 60 230-400 mesh). Melting points were deter-
mined on an Electrothermal IA9100 melting point appara-
tus and are uncorrected. UV Spectra (lmax (log e) in nm)
were recorded on a Shimadzu UV-Visible UV-160A spec-
trophotometer. IR Spectra (cm-1): Perkin-Elmer System-
2000 infrared spectrophotometer. Nuclear magnetic reso-
nance (1H and 13C) spectra were recorded on a Varian Gem-
ini 200 spectrometer or Varian-Unity-400 spectrometer.
Chemical shifts were expressed in parts per million (d) with
tetramethylsilane (TMS) as an internal standard. Elemental
analyses were carried out on a Heraeus CHN-O-Rapid ele-
mental analyzer, and results were within ± 0.4% of calcu-
lated values. Electrospray ionization mass spectra (ESI MS
or HRMS (ESI)) were recorded on Bruker APEX II FT-
MS.
8-Fluoro-9-[4-(4-fluorophenylsulfonyl)piperazin-1-yl)-
3-methyl-6-oxo-2,3-dihydro-6H-1-oxo-3a-azaphenalene-
5-carboxylic acid (5)
Compound 5 was obtained by recrystallization from
EtOH to give 0.38 g (76% yield) as a white powder. Mp
245-246 °C. UV lmax nm (log e): 230 (4.32), 294 (4.62) in
MeOH. IR nmax cm-1: 1158, 1472, 1522, 1617, 1718 in KBr.
1H-NMR (DMSO-d6): 1.41 (d, 3H, J = 6.4 Hz, 2-CH3),
3.04, 3.35 (m, 8H, 9-piperazinyl-H), 4.32, 4.53 (AB, 2H, J
= 10.4 Hz, 2H-C(1)), 4.91 (m, 1H, 1H-C(2)), 7.56 (m, 3H,
Ar-H and 1H-C(7)), 7.87 (m, 2H, Ar-H), 8.97 (s, 1H, 1H-
C(4)), 15.12 (br s, COOH). Anal. Calc. for C23H21F2N3O6S:
C 54.65, H 4.19, N 8.31; found: C 54.48, H 4.36, N 7.32.
ESI MS m/z: 506 (M + H)+.
8-Fluoro-3-methyl-9-[4-(4-nitrophenylsulfonyl)piper-
azin-1-yl)-6-oxo-2,3-dihydro-6H-1-oxo-3a-azaphenalene-
5-carboxylic acid (6)26
2. Syntheses
General Procedure for the Synthesis of N-Benzoyl or
N-Phenylsulfonyl Ofloxacins
Compound 6 was obtained by FC (MeOH/DCM =
1:50) and recrystallized from EtOH to give 0.38 g (71%
yield) as a pale yellow powder. Mp 286-287 °C. UV lmax
nm (log e): 296 (4.54) in MeOH. IR nmax cm-1: 1167, 1351,
1469, 1529, 1620, 1712 in KBr. 1H-NMR (DMSO-d6): 1.41
(d, 3H, J = 6.4 Hz, 2-CH3), 3.12, 3.37 (m, 8H, 9-piper-
azinyl-H), 4.31, 4.52 (AB, 2H, J = 12.0 Hz, 2H-C(1)), 4.89
(q, 1H, J = 6.4 Hz, 1H-C(2)), 7.55 (d, 1H, J = 12.0 Hz,
1H-C(7)), 8.07 (m, 2H, Ar-H), 8.47 (m, 2H, Ar-H), 8.97 (s,
1H, 1H-C(4)), 15.11 (br s, COOH). Anal. Calc. for
C23H21FN4O8S·1.0 H2O: C 50.18, H 4.21, N 10.18; found:
C 50.47, H 4.03, N 9.80. ESI MS m/z: 533 (M + H)+.
8-Fluoro-9-[4-(4-methoxyphenylsulfonyl)piperazin-1-yl]-
3-methyl-6-oxo-2,3-dihydro-6H-1-oxo-3a-azaphenalene-
5-carboxylic acid (7)
A mixture of N-demethyl ofloxacin (1 mmol), K2CO3
(0.28 g, 2 mmol), KI (70 mg), and corresponding phenyl-
sulfonyl chlorides or benzoyl chlorides (4 mmol) in dry
DMF (8 mL) was heated at 80 °C (monitored until the reac-
tant disappeared by TLC). Evaporation of the solvent gave
a residue, which was participated into biphasic mixture of
saturated NaHCO3 (50 mL) and CH2Cl2 (50 mL). The lay-
ers were separated, and the aqueous layer was extracted
with CH2Cl2 (50 mL). The organic extracts were washed
with H2O (50 mL ´ 2) and brine (50 mL ´ 1), dried over
Na2SO4, filtered, and concentrated in a vacuum. The crude
product was purified by flash column chromatography
and/or crystallization.
8-Fluoro-3-methyl-6-oxo-9-[4-(phenylsulfonyl)piper-
azin-1-yl]-2,3-dihydro-6H-1-oxo-3a-aza-phenalene-5-
carboxylic acid (4)26
Compound 7 was obtained by recrystallization from
EtOH, to give 0.42 g (82% yield) as a white powder. Mp
245-246 °C. UV lmax nm (log e): 238 (4.44), 296 (4.58) in
MeOH. IR nmax cm-1: 1160, 1463, 1525, 1620, 1716 in KBr.
1H-NMR (DMSO-d6): 1.40 (d, 3H, J = 6.8 Hz, 2-CH3),
Compound 4 was obtained by FC (MeOH/DCM =
1:100) and recrystallized from EtOH to give 0.39 g (79%
yield) as a white powder. Mp 279-280 °C. UV lmax nm (log

378 J. Chin. Chem. Soc., Vol. 56, No. 2, 2009
Chen et al.
2.99, 3.34 (m, 8H, 9-piperazinyl-H), 3.88 (s, 3H, OCH3),
4.32, 4.53 (AB, 2H, J = 11.2 Hz, 2H-C(1)), 4.89 (q, 1H, J =
6.8 Hz, 1H-C(2)), 7.20 (m, 2H, Ar-H), 7.55 (d, 1H, J = 12.4
Hz, 1H-C(7)), 7.72 (m, 2H, Ar-H), 8.96 (s, 1H, 1H-C(4)),
15.12 (br s, COOH). Anal. Calc. for C24H24FN3O7S·0.4
H2O: C 54.94, H 4.76, N 8.01; found: C 54.91, H 4.75, N
7.99. ESI MS m/z: 518 (M + H)+. HRMS (ESI): Calcd for
C24H25FN3O7S: 518.1397; found: 518.1398.
258 °C (dec). UV lmax nm (log e): 213 (4.33), 296 (4.57) in
MeOH. IR nmax cm-1: 1467, 1619, 1727 in KBr. 1H-NMR
(DMSO-d6): 1.54 (d, 3H, J = 6.8 Hz, 2-CH3), 3.32, 3.61 (m,
8H, 9-piperazinyl-H), 4.48, 4.69 (AB, 2H, J = 11.6 Hz,
2H-C(1)), 5.02 (q, 1H, J = 6.8 Hz, 1H-C(2)), 7.70 (d, 1H, J
= 12.4 Hz, 1H-C(7)), 8.22 (m, 2H, Ar-H), 8.42 (m, 2Ar-H),
9.07 (s, 1H, 1H-C(4)), 15.16 (br s, COOH). Anal. Calc. for
C24H21FN4O7·0.3 H2O: C 57.44, H 4.34, N 11.16; found: C
57.13, H 4.52, N 11.09. ESI MS m/z: 497 (M + H)+.
8-Fluoro-3-methyl-9-[4-(4-methoxybenzoyl)piperazin-
1-yl]-6-oxo-2,3-dihydro-6H-1-oxo-3a-azaphenalene-5-
carboxylic acid (11)
9-(4-Benzoylpiperazin-1-yl)-8-fluoro-3-methyl-6-oxo-
2,3-dihydro-6H-1-oxo-3a-azaphenalene-5-carboxylic
acid (8)
Compound 8 was obtained by FC (MeOH/DCM = 1:
100) and recrystallized from EtOH, to give 0.38 g (85%
yield) as a white powder. Mp 223-225 °C. UV lmax nm (log
e): 297 (4.44) in MeOH. IR nmax cm-1: 1475, 1620, 1706 in
Compound 11 was obtained by recrystallization from
EtOH, to give 0.39 g (82% yield) as a yellow powder. Mp
256-258 °C. UV lmax nm (log e): 221 (3.96), 297 (4.82) in
MeOH. IR nmax cm-1: 1458, 1619, 1721 in KBr. 1H-NMR
(CDCl3): 1.61 (d, 3H, J = 6.4 Hz, 2-CH3), 3.38, 3.78 (m,
8H, 9-piperazinyl-H), 3.85 (s, 3H, OCH3), 4.39-4.56 (M,
3H, 2H-C(1) and 1H-C(2)), 6.95 (m, 2H, Ar-H), 7.43 (m,
2H, Ar-H), 7.74 (d, 1H, J = 12.0 Hz, 1H-C(7)), 8.64 (s, 1H,
1H-C(4)), 14.91 (br s, COOH). Anal. Calc. for C25H24FN3O6·
0.2 H2O: C 61.90, H 5.07, N 8.66; found: C 61.83, H 5.29,
N 8.57. ESI MS m/z: 482 (M + H)+. HRMS (ESI): Calcd for
C25H25FN3O6: 482.1727; found: 482.1725.
1
KBr. H-NMR (DMSO-d6): 1.45 (d, 3H, J = 6.0 Hz, 2-
CH3), 3.36-3.53 (m, 8H, 9-piperazinyl-H), 4.39, 4.60 (AB,
2H, J = 11.2 Hz, 2H-C(1)), 4.93 (q, 1H, J = 6.0 Hz, 1H-
C(2)), 7.50-7.67 (m, 3Ar-H and 1H-C(7)), 7.92-7.97 (m,
2Ar-H), 8.98 (s, 1H, 1H-C(4)), 15.11 (br s, COOH). Anal.
Calc. for C24H22FN3O5·0.3 H2O: C 63.10, H 4.99, N 9.20;
found: C 63.14, H 5.16, N 9.18. ESI MS m/z: 452 (M + H)+.
HRMS (ESI): Calcd for C24H23FN3O5: 452.1622; found:
452.1619.
8-Fluoro-9-[4-(4-fluorobenzoyl)piperazin-1-yl]-3-meth-
yl-6-oxo-2,3-dihydro-6H-1-oxo-3a-azaphenalene-5-car-
boxylic acid (9)
3. Anti-mycobacterium activity
Primary screening is conducted against Mycobacte-
rium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B
medium using a broth microdilution assay to determine the
actual minimum inhibitory concentration (MIC) using the
Microplate Alamar Blue Assay (MABA).25 The MIC is de-
fined as the lowest concentration effecting a reduction in
fluorescence of 90% relative to controls. Concurrent with
the determination of MICs, compounds are screened by se-
rial dilutions to assess cytotoxicity (IC50) to a VERO cells.
After 72 h exposure, viability is assessed on the basis of
cellular conversion of MTT into a formazan product using
the Promega Cell Titer 96 Non-radioactive Cell Prolifera-
tion Assay.
Compound 9 was obtained by FC (MeOH/DCM =
1:50) and recrystallized from EtOH, to give 0.34 g (72%
yield) as a white powder. Mp 270-272 °C. UV lmax nm (log
e): 232 (4.28), 297 (4.56) in MeOH. IR nmax cm-1: 1467,
1619, 1727 in KBr. 1H-NMR (DMSO-d6): 1.45 (d, 3H, J =
6.8 Hz, 2-CH3), 3.49, 3.74 (m, 8H, 9-piperazinyl-H), 4.38,
4.60 (AB, 2H, J = 10.4 Hz, 2H-C(1)), 4.93 (q, 1H, J = 6.8
Hz, 1H-C(2)), 7.30 (m, 2H, Ar-H), 7.53 (m, 2Ar-H), 7.60
(d, 1H, J = 12.4 Hz, 1H-C(7)), 8.99 (s, 1H, 1H-C(4)), 15.18
(br s, COOH). Anal. Calc. for C24H21F2N3O5: C 61.41, H
4.51, N 8.95; found: C 61.16, H 4.70, N 8.72. ESI MS m/z:
470 (M + H)+. HRMS (ESI): Calcd for C24H22F2N3O5:
470.1527; found: 470.1525.
4. Antimicrobial assays
Disk diffusion (Kirby-Bauer) is one of the most com-
monly used antimicrobial susceptibility testing (AST)
methods among diagnostic laboratories. This method is a
well-established procedure for which there are accepted
standards including those endorsed by the National Com-
mittee for Clinical Laboratory Standards (NCCLS).27,28
8-Fluoro-3-methyl-9-[4-(4-nitrobenzoyl)piperazin-1-yl]-
6-oxo-2,3-dihydro-6H-1-oxo-3a-azaphenalene-5-carbox-
ylic acid (10)
Compound 10 was obtained by recrystallization from
EtOH to give 0.34 g (68% yield) as a yellow powder. Mp

Anti-TB Evaluation of Ofloxacin Derivatives
J. Chin. Chem. Soc., Vol. 56, No. 2, 2009 379
Growth inhibitory activity of the target compounds
was tested against five strains (Staphylococcus aureus,
Pseudomonas aeruginosa, Entercocus species, Streptococ-
cus pyrogenes, and Streptoccus pneumoniae). These micro-
organisms were provided from the Microbiology Labora-
tory Culture Collection, Department of pathology, Tri-ser-
vice General Hospital, Taipei, Taiwan. The bacteria were
first incubated at 37 ± 0.1 °C for 24 h in nutrient broth
(Difco). The standard NCCLS disk diffusion test was per-
formed on each bacteria strain using unsupplemented
Mueller-Hinton agar (Becton Dickinson Microbiology
System, Cocleysville, Md) and standard 10-mg gentamicin
dis, 10-mg penicillin disk and 30-mg vancomycin (Becton
Dickinson). With the Bauer-Kirby method, an activity
growing broth culture is diluted until the turbidity matches
that of a MacFarland 0.5 BaSO4 standard (ca. 108 colony-
forming units [CPU]/mL), 15 mL of mueller hinton agar
(MHA, Oxoid) and sabouraud dextrose agar (SDA) (steril-
ized in a flask and cooled to 40-50 °C) were homogenously
distributed onto the sterilized petri dishes. Sterilized blank
paper discs 6 mm in diameter were saturated with 80-mg of
tested compounds per disc, then placed onto the agar plates
which had previously been inoculated with the above or-
ganisms. Standard paper discs treated with gentamycin,
penicillin and vancomycin-saturated antibiotics were used
as postive controls. Following incubation for 16 to 18 h at
37 ± 0.1 °C, inhibition zones appearing around the discs
were measured and recorded in mm.
knowledged. We also thank National Center for High-Per-
formance Computing for providing computer resources
and chemical database services. Antimycobacterial data
were provided by Tuberculosis Antimicrobial Acquisition
& Coordinating Facility (TAACF) through a research and
development contract with the US National Institute of
Allergy and Infectious Diseases.
Received August 28, 2008.
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In summary, the positive N-demethyl ofloxacin (3)
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