Synthesis, and antimycobacterial and cytotoxic evaluation of certain fluoroquinolone derivatives

Article abstract of DOI:10.1002/hlca.200390201

Certain 1-ethyl- and 1-aryl-6-fluoro-1,4-dihydroquinol-4-one derivatives were synthesized and evaluated for antimycobacterial and cytotoxic activities. Preliminary results indicated that, for 1-aryl-6-fluoroquinolones, both 7-(piperazin-1-yl)- and 7-(4-me

Full text of DOI:10.1002/hlca.200390201

Helvetica Chimica Acta Vol. 86 (2003)
2481
Synthesis, and Antimycobacterial and Cytotoxic Evaluation of Certain
Fluoroquinolone Derivatives
by Jia-Yuh Sheu, Yeh-LongChen , and Cherng-Chyi Tzeng*
School of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University, Kaohsiung
City, Taiwan (phone:(886)7-3121101 ext 6985; fax. (886)7-3125339; e-mail:tzengch@cc.kmu.edu.tw)
and Shu-Lin Hsu, Kuo-ChangFang , and Tai-Chi Wang
Department of Environmental Engineering & Health, Tajen Institute of Technology, Pingtong, Taiwan
Certain 1-ethyl- and 1-aryl-6-fluoro-1,4-dihydroquinol-4-one derivatives were synthesized and evaluated
for antimycobacterial and cytotoxic activities. Preliminary results indicated that, for 1-aryl-6-fluoroquinolones,
both 7-(piperazin-1-yl)- and 7-(4-methylpiperazin-1-yl) derivatives, 9b and 11a, are able to completely inhibit
the growth of M. tuberculosis at a concentration of 6.25 mg/ml, while the 7-[4-(2-oxo-2-phenylethyl)piperazin-1-
yl] derivative 13 exhibits only 31% growth inhibition at the same concentration. For 1-ethyl-6-fluoroquinolones,
both 7-[4-(2-oxopropyl)piperazin-1-yl]- and 7-[4-(2-oxo-2-phenylethyl)piperazin-1-yl]-derivatives, 2a and 2b,
respectively, show complete inhibition, while their 2-iminoethyl and substituted phenyl counterparts 3a and 2c
are less active. In addition, the 6,8-difluoro derivative was a more-favorable inhibitor than its 6-fluoro
counterpart (2b vs. 2d). These results deserve full attention especially because 2a, 2b, 9b, and 11a are non-
cytotoxic at a concentration of 100 mm. Furthermore, compound 9b proved to be a potent anti-TB agent with
selective index (SI) > 40 and an EC90 value of 5.75 mg/ml.
Introduction. The drugs currently used for the treatment of tuberculosis (TB)
infection are streptomycin, isoniazid, ethambutol, pyrazinamide, and rifampicin [1].
However, the current TB treatment regimens, although highly effective, are far from
ideal. With the optimal combination of available drugs, the duration of treatment
required for curing patients cannot be reduced to less than six months. As a result,
noncompliance, i.e., patients stopping therapy as soon as an improvement in the
symptoms is felt, allows the remaining, more-resistant bacteria to survive and spread.
Recently, the incidence of TB infection has been further complicated by an increase in
cases that are resistant to conventional drug therapy, especially the emergence of multi-
drug resistant tuberculosis (MDRTB) [2][3]. Furthermore, the association of TB and
HIV infection is so dramatic that, in some cases, nearly two-thirds of the patients
diagnosed with TB are also HIV-1 seropositive [4]. Numerous studies showing that TB
may be a cofactor in the progression of HIV has caused an urgent need to search for
alternative chemotherapeutics for Mycobacterium tuberculosis infection [5 8]. During
the past decade, several of the fluoroquinolone antibacterial drugs have been examined
as potential chemotherapeutics for M. tuberculosis infection because of their favorable
pharmacokinetic profiles, such as easy absorbtion after oral administration and ready
penetration into mammalian cells [9 14]. These properties are important for the
treatment of intracellular pathogens, such as TB [15]. Synthesis of new fluoroquino-
lones and the evaluation of these agents for antimycobacterial activity has continued,

2482
Helvetica Chimica Acta Vol. 86 (2003)
and a series of SAR studies has been performed with M. avium, M. tuberculosis, and
other mycobacteria [16 20]. However, the structural modification of the fluoroqui-
nolones with respect to their optimum anti-TB activity has not been thoroughly
explored.
Over the past few years, we were particularly interested in the synthesis and
evaluation of fluoroquinolones for their antibacterial and anticancer activities [21 24].
Since the antibacterial mechanism of these fluoroquinolones is unique and absolutely
different from that of the currently used anti-TB drugs, they will become very
important drug candidates if proved to be active against the growth of M. tuberculosis.
Results and Discussion. The preparation of (E)-1-ethyl-6,8-difluoro-1,4-dihydro-
7-{4-[2-(hydroxyimino)propyl]piperazin-1-yl}-4-oxoquinoline-3-carboxylic acid (3a) is
outlined in Scheme 1. 8-Fluoronorfloxacin (1a) was treated with NaHCO3 and
chloroacetone to give its N-(2-oxopropyl) intermediate 2a, which, upon treatment
with NH2OH in EtOH, afforded 3a in 79% overall yield. Synthesis and antibacterial
activities of 3b 3e have been reported previously [22][23].
Scheme 1
i) NaHCO3, R2COCH2Cl in DMF. ii) NH2OH in EtOH.
The synthetic pathways for preparation of 1-aryl-6-fluoro-1,4-dihydro-7-(piperazin-
1-yl)-4-oxoquinoline-3-carboxylic acids 6 12 are described in Scheme 2. Reaction of
ethyl 6,7-difluoro-1-(2-fluoro-4-nitrophenyl)-1,4-dihydro-4-oxoquinoline-3-carboxy-
late (4a) with piperazine or 1-methylpiperazine in MeCN afforded ethyl 6-fluoro-1-
(2-fluoro-4-nitrophenyl)-1,4-dihydro-4-oxo-7-(piperazin-1-yl)quinoline-3-carboxylate
(6a) and its 7-(4-methylpiperazin-1-yl) derivative 6b, respectively. Accordingly, ethyl 6-
fluoro-1,4-dihydro-7-(4-methylpiperazin-1-yl)-1-(4-nitrophenyl)-4-oxoquinoline-3-
carboxylate (6c) was obtained by the treatment of 4b with 1-methylpiperazine.
Compounds 6a 6c were reduced by catalytic hydrogenation on Pd/C to their
corresponding 1-(4-aminophenyl) counterparts 7a 7c. Reaction of 6a with 28%
NH4OH in a sealed bomb afforded 6-fluoro-1-(2-fluoro-4-nitrophenyl)-1,4-dihydro-4-

Helvetica Chimica Acta Vol. 86 (2003)
2483
oxo-7-(piperazin-1-yl)quinoline-3-carboxamide (8a), which was reduced by catalytic
hydrogenation to give 1-(4-amino-2-fluorophenyl)-6-fluoro-1,4-dihydro-4-oxo-7-
(piperazin-1-yl)quinoline-3-carboxamide (9a) in 59% overall yield. Compounds 8b
and 9b were synthesized according to our previous report [23].
Scheme 2
i) Piperazine or 1-methylpiperazine in MeCN. ii) H2, Pd/C in CH2Cl2. iii) NH4OH in a sealed bomb (for 7a from
5a); HCl/AcOH 1:4 (for 7b from 5a). iv) Ac2O, pyridine.
1-(4-Amino-2-fluorophenyl)-6-fluoro-1,4-dihydro-7-(4-methylpiperazin-1-yl)-4-oxo-
quinoline-3-carboxylic acid (11a) was obtained by catalytic hydrogenation of its nitro
precursor 10a, which, in turn, was prepared by the reaction of 6,7-difluoro-1-(2-fluoro-
4-nitrophenyl)-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (5a) and 1-methylpiper-
azine. Acetylation of 11a with Ac2O afforded 1-[4-(acetylamino)-2-fluorophenyl]-6-
fluoro-1,4-dihydro-7-(4-methylpiperazin-1-yl)-4-oxoquinoline-3-carboxylic acid (12a)

2484
Helvetica Chimica Acta Vol. 86 (2003)
in 93% yield. Accordingly, compound 12b was synthesized from 5b. Compound 13 was
prepared from 5a according to our previous report [23].
The anti-TB activity of fluoroquinolones is summarized in Table 1. Compound 2a
was found to be a very potent inhibitor, being able to inhibit 99% growth of M.
tuberculosis at a concentration of 6.25 mg/ml. Of the 6-fluoro derivatives, compound 2d
exhibited 53% inhibition of the growth of M. tuberculosis and was more active than its
4-Cl and the oxime congeners (2d vs. 2e and 2d vs. 3d). The same order has been
observed for 6,8-difluoro derivatives, in which 2a is a more-potent anti-TB agent than
its oxime counterpart 3a, and 2b was more potent than its 4-MeO derivative 2c.
Compound 2b was able to completely inhibit the growth of M. tuberculosis, while its 6-F
counterpart 2d showed only marginal inhibitory activity, indicating that the 6,8-difluoro
derivative was a more-favorable inhibitor than its 6-F counterpart. The enhancement of
anti-TB activity by 8-F group could be attributed to the higher lipophilicity, which
facilitated its penetration into mammalian cells.
Table 1. Inhibitory Activity of Fluoroquinolones against M. tuberculosis at a Concentration of 6.25 mg/ml
Compound
R1
R2
R3
X
Inhibition [%]
2a
2b
2c
2d
2e
3a
3d
7a
7b
7c
8a
8b
9a
9b
11a
11b
12a
12b
13
MeCOCH2
PhCOCH2
4-MeOC6H4COCH2
PhCOCH2
MeCH2
MeCH2
MeCH2
MeCH2
MeCH2
MeCH2
MeCH2
4-NH2,2F-C6H3
4-NH2,2F-C6H3
4-NH2C6H4
2-F,4-NO2-C6H3
2-F,4-NO2-C6H3
4-NH2,2-F-C6H3
4-NH2,2-F-C6H3
4-NH2,2-F-C6H3
4-NH2C6H4
OH
OH
OH
OH
OH
OH
OH
EtO
EtO
EtO
NH2
OH
NH2
OH
OH
OH
OH
OH
OH
F
F
F
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
99
100
29
53
24
34
28
13
0
0
0
37
1
100
100
84
18
15
31
4-ClC6H4CO
MeC(ˆNOH)CH2
PhC(ˆNOH)CH2
H
Me
Me
H
H
H
H
Me
Me
Me
Me
4-(NHAC),2-F-C6H3
4-(NHAC)-C6H4
4-NH2,2-F-C6H3
4-MeOC6H4COCH2
The esters 7a 7c and amides 8a and 9a were devoid of anti-TB activity. Compound
11b exhibited good inhibitory activity (84% inhibition), while its 4-amino-2-fluoro-
phenyl derivative 11a further improved the potency. Both 9b and 11a were able to
completely inhibit the growth of M. tuberculosis, indicating that the 7-(piperazin-1-yl)
can be substituted with a Me group. However, a [(4-methoxyphenyl)carbonyl]methyl

Helvetica Chimica Acta Vol. 86 (2003)
2485
substituent (instead of Me; i.e., 13) drastically decreased potency. Acetylation of amino
group led to decreased anti-TB activity (11a vs. 12a and 11b vs. 12b).
Compounds 2a, 2b, and 9b, which demonstrated at least 90% inhibition in the
primary screen, were retested at lower concentrations against M. tuberculosis H37Rv to
determine the actual minimum inhibitory concentration (MIC) and the cytotoxicity
(IC50) [25] (Table 2). Among them, 9b, which possessed the selective index (SI, IC50/
MIC) > 40, was then tested for killing of M. tuberculosis Erdman (ATCC 35801) in
monolayers of mouse bone marrow macrophages [26] at four-fold concentrations
equivalent to 0.25, 1, 4, and 16 Â the MIC. The results indicated 9b to be a potent anti-
TB fluoroquinolone with an EC90 value of 5.75 mg/ml (EC90 > 16 Â MIC are considered
inactive).
Table 2. Anti-TB Activity of 2a, 2b, and 9b
Compound
MIC [mg/ml]a)
IC50 [mg/ml]b)
SIc)
EC90 [mg/ml)d)
2a
2b
9b
3.13
3.13
1.56
> 10
> 10
> 62.5
> 3.19
> 3.19
> 40.06
n.d.e)
n.d.
5.75
a) Minimium inhibitory concentration (MIC) against M. tuberculosis H37Rv. b) Cytotoxicity (IC50) in VERO
cells at concentrations of 62.5 mg/ml or 10 Â the MIC for M. tuberculosis H37Rv. c) Selective index (SI, IC50/
MIC). d) Lowest concentration effecting a 90% reduction in colony forming units at 7 days compared to drug-
free controls. e) Not determined.
All compounds were evaluated in vitro against a three-cell-line panel consisting of
MCF 7 (Breast), NCI-H460 (Lung), and SF-268 (CNS). In this protocol, each cell line
is inoculated and preincubated on a microtiter plate. Test agents are then added at a
single concentration (100 mm), and the culture was incubated for 48 h. End-point
determinations are made with sulforhodamine B, a protein-binding dye. Results for
each test agent are reported as the percent of growth of the treated cells when
compared to the untreated control cells. Compounds, which reduced the growth of any
one of the cell lines to 32% or less (negative numbers indicate cell kill), are passed on
for evaluation in the full panel of 60 cell lines over a 5-log dose range [27]. Results from
Table 3 indicated that all of them, with the exception of compounds 2c 2e, 3d, and 13,
whose mean GI50 value ranged from 22 to 83 mm, are inactive.
Conclusions. A number of fluoroquinolone derivatives were synthesized, and
evaluated for antimycobacterial and cytotoxic activities. Preliminary results are 1) for
1-ethyl 6,8-difluoroquinolones, 7-[4-(2-oxopropyl)piperazin-1-yl] and 7-[4-(2-oxo-2-
phenylethyl)piperazin-1-yl] derivatives, 2a and 2b, respectively, are active anti-TB
agents, while their 2-iminoethyl and substituted phenyl counterparts are inactive. 2) For
1-aryl-6-fluoroquinolones, both 7-(piperazin-1-yl) and 7-(4-methylpiperazin-1-yl) de-
rivatives, 9b and 11a, respectively, are active against the growth of M. tuberculosis,
while the 7-[4-(2-oxo-2-phenylethyl)piperazin-1-yl] counterpart, 13, becomes inactive.
These results deserve full attention, especially because 2a, 2b, 9b, and 11a are non-
cytotoxic. Furthermore, compound 9b proved to be a potent anti-TB agent with the
selective index (SI) > 40 and an EC90 value of 5.75 mg/ml.

2486
Helvetica Chimica Acta Vol. 86 (2003)
Table 3. In Vitro Anticancer Assay of Fluoroquinolone Derivatives
Compound
Growth Percentages
Mean GI50 [mm]a)
NCI-H460 (Lung)
MCF 7 (Breast)
SF-268 (CNS)
2a
2b
81
50
66
37
72
23
Inactive
> 100
2c
2d
84
93
69
91
À 2
83.0
69.9
6
2e
94
89
22
72.0
3d
9
25
3
29.4
6a
6b
6c
7a
7b
9a
9b
11a
11b
12a
12b
13
92
74
76
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
22.1
101
104
96
111
96
116
95
100
85
114
116
64
94
105
89
96
109
90
108
11
120
115
109
78
89
86
106
118
84
121
29
96
4
a) Mean values over all 60 cancer cell lines tested [27].
Experimental Part
General. M.p.: Electrothermal IA9100 digital melting-point apparatus; uncorrected. TLC:silica gel 6 0 F-
254 plates from EM Laboratories, Inc.; detection by UV light (254 nm). NMR (1H and 13C) spectra; Varian
Unity-400 spectrometer or Varian Gemini-200 spectrometer, chemical shifts d in ppm with Me4Si as an internal
standard (ˆ0 ppm), coupling constants J in Hz. Elemental analyses were carried out on a Heraeus CHN-O-
Rapid elemental analyzer, and results were within Æ 0.4% of calc. values.
1-Ethyl-6,8-difluoro-1,4-dihydro-4-oxo-7-[4-(2-oxopropyl)piperazin-1-yl]quinoline-3-carboxylic acid (2a).
A mixture of 8-fluoronorfloxacin (0.5 g, 1.48 mmol), KHCO3 (0.15 g, 1.48 mmol), KI (0.08 g, 0.5 mmol), and
chloroacetone (0.19 g, 2 mmol) in DMF (40 ml) was stirred at r.t. for 24 h, poured into ice-water (50 ml), and
filtered to give a solid, which was washed with H2O, crystallized from EtOH/CH2Cl2 10 :1 to give 2a (0.49 g,
85%). M.p. 1988 (dec.). 1H-NMR (400 MHz, DMSO):1.44 ( t, J ˆ 6.0, Me); 2.11 (s, MeCO); 2.58, 3.28 (2 br. s,
8 H-(piperazine)); 3.15 (s, NCH2CO); 4.59 (m, N(1)CH2); 7.90 (d, J ˆ 11.6, HÀC(5)); 8.92 (s, HÀC(2)); 14.90
(br. s, COOH). Anal. calc. for C19H21F2N3O4 ¥ 0.2 H2O:C 57.34, H 5.42, N 10.56; found:C 57.48, H 5.37, N 10.52.
1-Ethyl-6,8-difluoro-1,4-dihydro-7-{(E)-4-[2-(hydroxyimino)propyl]piperazin-1yl}-4-oxoquinoline-3-carb-
oxylic Acid (3a). To a suspension of 2a (0.39 g, 1 mmol) in MeOH (20 ml) was added a soln. of NH2OH ¥ HCl
(0.14 g, 2 mmol) and NaHCO3 (0.17 g, 2 mmol) in H2O (2 ml). The mixture was stirred for at r.t. 4 h, H2O
(20 ml) and CH2Cl2 (50 ml) were then added. The org. phase was washed successively with H2O and brine, dried
(Na2SO4), and evaporated to give a residual solid, which was purified by flash column chromatography (FC)
(silica gel; CH2Cl2/MeOH 10 :1) and crystallized from CH2Cl2/MeOH 10 :1 to give 3a (0.38 g, 93%). M.p. 2318
(dec.). 1H-NMR (400 MHz, DMSO):1.44 ( t, J ˆ 6.6, Me); 1.81 (s, MeCˆN); 2.52, 3.36 (2 br. s, 8 H
(piperazine)); 3.02 (s, NCH2CˆN); 4.58 (m, N(1)CH2); 7.84 (d, J ˆ 13.2, HÀC(5)); 8.91 (s, HÀC(2)); 10.61 (s,
NOH); 15.30 (br. s, COOH). Anal. calc. for C19H22F2N4O4:C 55.88, H 5.43, N 13.72; found:C 55.69, H 5.23,
N 13.57.
Ethyl 6-Fluoro-1-(2-fluoro-4-nitrophenyl)-1,4-dihydro-4-oxo-7-(piperazin-1-yl)quinoline-3-carboxylate
(6a). A mixture of 4a (2.68 g, 6.83 mmol) and piperazine (1.72 g, 20 mmol) in MeCN (100 ml) was refluxed
under N2 for 5 h (TLC monitoring). The solvent was evaporated to give a residual solid, which was purified by
1
FC (CH2Cl2/MeOH 10 :1) and crystallized from EtOH to afford 6a (2.66 g, 85%). M.p. 2888 (dec.). H-NMR
(400 MHz, DMSO):1.24 ( t, J ˆ 7.2, Me); 2.77, 2.91 (2 br. s, 8 H (piperazine)); 4.19 (q, J ˆ 7.2, CH2O); 6.24 (d,
J ˆ 6.0, HÀC(8)); 7.80 (d, J ˆ 13.2, HÀC(5)); 8.18 (dd, J ˆ 8.8, 8.0, HÀC(6')); 8.35 (ddd, J ˆ 8.8, 2.8, 2.0,

Helvetica Chimica Acta Vol. 86 (2003)
2487
HÀC(5')); 8.51 (dd, J ˆ 9.6, 2.8, HÀC(3')); 8.54 (s, HÀC(2)). Anal. calc. for C22H20F2N4O5 ¥ 0.2 H2O:C 57.02,
H 4.45, N 12.13; found:C 57.09, H 4.46, N 11.96.
Ethyl 6-Fluoro-1-(2-fluoro-4-nitrophenyl)-1,4-dihydro-7-(4-methylpiperazin-1-yl)-4-oxoquinoline-3-car-
boxylate (6b). Compound 6b was obtained from 4a and 1-methylpiperazine as described for 6a. FC (CH2Cl2/
MeOH 20 :1): 63% yield. M.p. 184 185 8. 1H-NMR (200 MHz, DMSO):1.26 ( t, J ˆ 7.2, Me); 2.21 (s, MeN);
2.44, 3.03 (2 br. s, 8 H (piperazine)); 4.21 (q, J ˆ 7.2, CH2O); 6.28 (d, J ˆ 7.0, HÀC(8)); 7.83 (d, J ˆ 13.4,
HÀC(5)); 8.18 (dd, J ˆ 8.4, 8.0, HÀC(6')); 8.36 (dd, J ˆ 8.8, 2.0, HÀC(5')); 8.53 (m, HÀC(3', HÀC(2)). Anal.
calc. for C23H22F2N4O5 ¥ 4.0 H2O:C 50.73, H 5.55, N 10.29; found:C 50.63, H 5.29, N 10.54.
Ethyl 6-Fluoro-1,4-dihydro-7-(4-methylpiperazin-1-yl)-1-(4-nitrophenyl)-4-oxoquinoline-3-carboxylate
(6c). Compound 6c was obtained from 4b and 1-methylpiperazine as described for 6a. FC (CH2Cl2/MeOH
20 :1):90% yield. M.p. 231 233 8. 1H-NMR (200 MHz, DMSO):1.24 ( t, J ˆ 7.2, Me); 2.17 (s, MeN); 2.38, 2.96 (2
br. s, 8 H (piperazine)); 4.20 (q, J ˆ 7.2, CH2O); 6.33 (d, J ˆ 7.2, HÀC(8)); 7.82 (d, J ˆ 13.6, HÀC(5)); 7.97 (m,
HÀC(2'), HÀC(6')); 8.48 (m, HÀC(3'), HÀC(5'), HÀC(2)). Anal. calc. for C23H23FN4O5 ¥ 0.5 H2O:C 59.61,
H 5.22, N 12.09; found:C 59.25, H 5.23, N 11.91.
Ethyl 1-(4-Amino-2-fluorophenyl)-6-fluoro-1,4-dihydro-4-oxo-7-(piperazin-1-yl)quinoline-3-carboxylate
(7a). A mixture of 6a (3.40 g, 7.42 mmol) and 5% Pd/C (0.50 g) in CH2Cl2/EtOH 3 :1 (100 ml) was stirred
under H2 for 24 h (TLC monitoring). The resulting mixture was filtered, and the soln. was evaporated under
reduced pressure. The residual solid was purified by FC (CH2Cl2/MeOH 30 :1) and crystallized from EtOH to
give 7a (2.45 g, 77%). M.p. 260 2618. 1H-NMR (400 MHz, DMSO):1.26 ( t, J ˆ 7.2, Me); 3.40 (br. s, 8 H
(piperazine)); 4.20 (q, J ˆ 7.2, CH2O); 6.04 (br. s, NH2); 6.36 (d, J ˆ 7.2, HÀC(8)); 6.59 (m, HÀC(3'),
HÀC(5')); 7.34 (dd, J ˆ 9.2, 8.4, HÀC(6')); 7.85 (d, J ˆ 13.2, HÀC(5)); 8.37 (s, HÀC(2)). Anal. calc. for
C22H22F2N4O3 ¥ 2.5 H2O:C 55.80, H 5.75, N 11.83; found:C 55.57, H 5.74, N 11.60.
Ethyl 1-(4-Amino-2-fluorophenyl)-6-fluoro-1,4-dihydro-7-(4-methylpiperazin-1-yl)-4-oxoquinoline-3-car-
boxylate (7b). Compound 7b was obtained from 6b as described for 7a. FC (CH2Cl2/MeOH 20 :1): 47% yield.
M.p. 132 1338. 1H-NMR (200 MHz, DMSO):1.25 ( t, J ˆ 7.2, Me); 2.18 (s, MeN); 2.42, 2.99 (2 br. s, 8 H
(piperazine)); 4.18 (q, J ˆ 7.2, CH2O); 5.99 (br. s, NH2); 6.33 (d, J ˆ 7.2, HÀC(8)); 6.57 (m, HÀC(3'),
HÀC(5')); 7.32 (dd, J ˆ 9.2, 8.4, HÀC(6')); 7.79 (d, J ˆ 13.6, HÀC(5)); 8.32 (s, HÀC(2)). Anal. calc. for
C23H24F2N4O3 ¥ 0.8 H2O:C 60.46, H 5.65, N 12.26; found:C 60.68, H 5.68, N 11.98.
Ethyl 1-(4-Aminophenyl)-6-fluoro-1,4-dihydro-7-(4-methylpiperazin-1-yl)-4-oxoquinoline-3-carboxylate
(7c). Compound 7c was obtained from 6c as described for 7a. FC (CH2Cl2/MeOH 30 :1): 60% yield. M.p.
227 2298. 1H-NMR (400 MHz, DMSO):1.25 ( t, J ˆ 7.2, Me); 2.38 (s, MeN); 2.71, 3.06 (2 br. s, 8 H
(piperazine)); 4.19 (q, J ˆ 7.2, CH2O); 5.65 (br. s, NH2); 6.43 (d, J ˆ 7.6, HÀC(8)); 6.74 (m, 2 HÀC(2', 6')); 7.23
(m, 2 HÀC(3', 5'); 7.79 (d, J ˆ 13.6, HÀC(5)); 8.30 (s, HÀC(2)). Anal. calc. for C23H25FN4O3 ¥ 2.0 H2O:C 59.99,
H 6.35, N 12.17; found:C 60.23, H 6.05, N 12.03.
6-Fluoro-1-(2-fluoro-4-nitrophenyl)-1,4-dihydro-4-oxo-7-(piperazin-1-yl)quinoline-3-carboxamide (8a). A
mixture of the 6a (2.18 g, 4.75 mmol) and 28% NH4OH (40 ml) in MeOH (10 ml) was heated in a steel bomb at
1258 for 5 h. The mixture was evaporated to give a residual solid, which was purified by FC (CH2Cl2/MeOH
20 :1) and crystallized from EtOH to afford 8a (2.66 g, 76%). M.p. 259 2608 (dec.). 1H-NMR (200 MHz,
DMSO):2.78, 2.94 (2 br. s, 8 H (piperazine)); 6.31 (dd, J ˆ 7.0, 1.4, HÀC(8)); 7.64 (d, J ˆ 4.4, CONH); 7.92 (d,
J ˆ 13.6, HÀC(5)); 8.17 (dd, J ˆ 8.8, 7.6, HÀC(6')); 8.37 (ddd, J ˆ 8.8, 2.4, 1.0, HÀC(5')); 8.55 (dd, J ˆ 9.8, 2.4,
HÀC(3')); 8.65 (s, HÀC(2)); 9.13 (d, J ˆ 4.4, CONH). Anal. calc. for C20H17F2N5O4 ¥ 0.8 H2O:C 54.13, H 4.22,
N 15.78; found:C 54.44, H 4.08, N 15.41.
1-(4-Amino-2-fluorophenyl)-6-fluoro-1,4-dihydro-4-oxo-7-(piperazin-1-yl)quinoline-3-carboxamide (9a).
Compound 9a was obtained from 8a as described for 7a:78% yield. M.p. 276 278 8. 1H-NMR (400 MHz,
DMSO):2.80, 2.92 (2 br. s, 8 H (piperazine)); 6.01 (br. s, NH2); 6.38 (d, J ˆ 6.8, HÀC(8)); 6.59 (m, HÀC(3'),
HÀC(5')); 7.34 (dd, J ˆ 9.2, 8.4, HÀC(6')); 7.57 (d, J ˆ 4.4, CONH); 7.88 (d, J ˆ 13.2, HÀC(5)); 8.44 (s,
HÀC(2)); 9.20 (d, J ˆ 4.4, CONH). Anal. calc. for C21H21F2N5O2 ¥ 1.0 H2O:C 58.46, H 5.37, N 16.23; found:
C 58.38, H 5.11, N 16.05.
6-Fluoro-1-(2-fluoro-4-nitrophenyl)-1,4-dihydro-7-(4-methylpiperazin-1-yl)-4-oxoquinoline-3-carboxylic
Acid (10a). Compound 10a was obtained from 5a and 1-methylpiperazine as described for 6a. FC (CH2Cl2/
1
MeOH 3 :1): 91% yield. M.p. 288 289 8. H-NMR (400 MHz, DMSO):2.18 ( s, MeN); 2.40, 3.09 (2 br. s, 8 H
(piperazine)); 6.37 (d, J ˆ 7.2, HÀC(8)); 8.00 (d, J ˆ 13.2, HÀC(5)); 8.18 (dd, J ˆ 8.8, 8.0, HÀC(6')); 8.39 (d,
J ˆ 8.0, HÀC(5')); 8.57 (dd, J ˆ 8.8, 2.4, HÀC(3')); 8.92 (s, HÀC(2)); 14.80 (br. s, COOH). Anal. calc. for
C21H18F2N4O5:C 56.76, H 4.08, N 12.61; found:C 56.74, H 4.08, N 12.61.
6-Fluoro-1,4-dihydro-7-(4-methylpiperazin-1-yl)-1-(4-nitrophenyl)-4-oxoquinoline-3-carboxylic Acid
(10b). Compound 10b was obtained from 5b and 1-methylpiperazine as described for 6a. FC (CH2Cl2/MeOH

2488
Helvetica Chimica Acta Vol. 86 (2003)
2 :1):72% yield. M.p. 297 298 8. 1H-NMR (400 MHz, DMSO/CF3COOH):2.88 ( s, MeN); 3.14, 3.59 (2 br. s, 8 H
(piperazine)); 6.53 (d, J ˆ 7.2, HÀC(8)); 8.06 (m, HÀC(5), HÀC(2'), HÀC(6')); 8.54 (m, HÀC(3'), HÀ(5'));
8.77 (s, HÀC(2)). Anal. calc. for C23H22F2N4O4 ¥ 0.3 H2O:C 58.41, H 4.57, N 12.98; found:C 58.46, H 4.55,
N 13.12.
1-(4-Amino-2-fluorophenyl)-6-fluoro-1,4-dihydro-7-(4-methylpiperazin-1-yl)-4-oxoquinoline-3-carboxylic
Acid (11a). Compound 11a was obtained from 10a as described for 7a. FC (CH2Cl2/MeOH 10 :1): 77% yield.
1
M.p. 278 2808. H-NMR (400 MHz, DMSO):2.18 ( s, MeN); 2.42, 3.07 (2 br. s, 8 H (piperazine)); 6.03 (br. s,
NH2); 6.43 (d, J ˆ 7.2, HÀC(8)); 6.58 (m, HÀC(3'), HÀC(5')); 7.34 (dd, J ˆ 8.8, 8.4, HÀC(6')); 7.94 (d, J ˆ 13.2,
HÀC(5)); 8.59 (s, HÀC(2)). Anal. calc. for C21H20F2N4O3:C 60.86, H 4.86, N 13.52; found:C 60.83, H 5.07,
N 13.27.
1-(4-Aminophenyl)-6-fluoro-1,4-dihydro-7-(4-methylpiperazin-1-yl)-4-oxoquinoline-3-carboxylic Acid
(11b). Compound 11b was obtained from 10b as described for 7a. FC (CH2Cl2/MeOH 20 :1): 51% yield. M.p.
268 2698. 1H-NMR (200 MHz, DMSO):2.19 ( s, MeN); 2.44, 3.06 (2 br. s, 8 H (piperazine)); 5.69 (br. s, NH2);
6.55 (d, J ˆ 7.0, HÀC(8)); 6.76 (m, HÀC(3'), HÀC(5')), 7.27 (m, HÀC(2'), HÀC(6')); 8.50 (s, HÀC(2)). Anal.
calc. for C21H21FN4O3:C 63.63, H 5.34, N 14.13; found:C 63.41, H 5.40, N 13.94.
1-[4-(Acetylamino)-2-fluorophenyl]-6-fluoro-1,4-dihydro-7-(4-methylpiperazin-1-yl)-4-oxoquinoline-3-car-
boxylic Acid (12a). A mixture of 11a (0.23 g, 0.55 mmol), Ac2O (30 ml), and pyridine (30 ml) was stirred at r.t.
for 24 h. The resulting mixture was concentrated under reduced pressure, and then CH2Cl2 (100 ml) was added,
and the mixture was washed with H2O (3 Â 80 ml), dried (Na2SO4), and evaporated. The residual solid was
purified by FC (CH2Cl2/MeOH 50 :1) to give 12a (0.23 g, 93%). M.p. 246 2478. 1H-NMR (200 MHz, DMSO):
2.14 (s, MeCO); 2.18 (s, MeN); 2.42, 3.08 (2 br. s, 8 H (piperazine)); 6.38 (d, J ˆ 7.0, HÀC(8)); 7.53 (dd, J ˆ 8.8,
1.4, HÀC(5')); 7.74 (dd, J ˆ 8.8, 8.4, HÀC(6')); 7.95 (m, HÀC(3'), HÀC(5)); 8.75 (s, HÀC(2)); 10.54 (br. s,
CONH). Anal. calc. for C23H22F2N4O4 ¥ 0.5 H2O:C 57.14, H 5.18, N 10.66; found:C 56.82, H 5.33, N 10.85.
1-[4-(Acetylamino)phenyl]-6-fluoro-1,4-dihydro-7-(4-methylpiperazin-1-yl)-4-oxoquinoline-3-carboxylic
Acid (12b). Compound 12b was obtained from 11b as described for 12a. FC (CH2Cl2/MeOH 50 :1):71% yield.
M.p. 287 2888. 1H-NMR (200 MHz, DMSO):2.12 ( s, MeCO); 2.19 (s, MeN); 2.43, 3.06 (2 br. s, 8 H
(piperazine)); 6.46 (d, J ˆ 7.4, HÀC(8)); 7.61 (m, HÀC(2'), HÀC(6')); 7.86 (m, HÀC(3'), HÀC(5')); 7.96 (d,
J ˆ 13.4, HÀC(5)); 8.58 (s, HÀC(2)); 10.35 (br. s, CONH). Anal. calc. for C23H22FN4O4 ¥ AcOH ¥ 0.2 H2O:
C 59.81, H 5.51, N 11.16; found:C 59.73, H 5.59, N 11.09.
Antimycobacterium Activity. Primary screening is conducted at 6.25 mg/ml against Mycobacterium
tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium with a broth microdilution assay, the Microplate
Alamar Blue Assay (MABA) [25]. Compounds exhibiting fluorescence are tested in the BACTEC 460
radiometric system [25]. Compounds demonstrating at least 90% inhibition in the primary screen are retested at
lower concentrations against M. tuberculosis H37Rv to determine the actual minimum inhibitory concentration
(MIC) with MABA. 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 tested for cytotoxicity
(IC50) in VERO cells at concentrations of 62.5 mg/ml or 10 Â the MIC for M. tuberculosis H37Rv. After 72-h
exposure, viability is assessed on the basis of cellular conversion of MTT to a formazan product with the
Promega Cell Titer 96Nonradioactive Cell Proliferation Assay . Compounds for which the selective index (SI,
IC50/MIC) > 10 will have in vitro activity confirmed in the BACTEC 460 at 6.25 mg/ml. Compounds are then
tested for killing of M. tuberculosis Erdman (ATCC 35801) in monolayers of mouse bone marrow macrophages
[26] (EC90; lowest concentration effecting a 90% reduction in colony forming units at seven days compared to
drug-free controls) at four-fold concentrations equivalent to 0.25, 1, 4, and 16 Â the MIC. Compounds with EC90
>16 Â MIC are considered inactive in the model.
Financial support of this work by the National Science Council of the Republic of China is gratefully
acknowledged. Antimycobacterial data were provided by the Tuberculosis Antimicrobial Acquisition and
Coordinating Facility (TAACF) through a research and development contract with the U.S. National Institute of
Allergy and Infectious Diseases. We also thank U.S. National Cancer Institute (NCI) for the anticancer screenings
and the National Center for High-Performance Computing of the Republic of China for providing computer
resources and chemical database services.

Helvetica Chimica Acta Vol. 86 (2003)
2489
REFERENCES
[1] S. Houston, A. Fanning, Drugs 1994, 48, 689.
[2] R. F. Jacobs, Clin. Infec. Dis. 1994, 19, 1.
[3] A. C. Weltman, D. N. Rose, Arch. Intern. Med. 1994, 154, 2161.
[4] D. Alland, G. E. Kalkut, A. R. Moss, R. A. McAdam, J. A. Hahn, W. Bosworth, E. Drucker, B. R. Bloom,
New Engl. J. Med. 1994, 330, 1710.
[5] R. S. Wallis, M. Vjecha, M. Amir-Tahmasseb, A. Okwera, F. Byekwaso, S. Nyole, S. Kabengera, R. D.
Mugerwa, J. J. Eliner, J. Infec. Dis. 1993, 167, 43.
[6] A. K. Bakkestuen, L. L. Gundersen, G. Langli, F. Liu, J. M. J. Nolsoe, Bioorg. Med. Chem. Lett. 2000, 10,
1207.
[7] Y. M. Lin, M. T. Flavin, C. S. Cassidy, A. Mar, F. C. Chen, Bioorg. Med. Chem. Lett. 2001, 11, 2101.
[8] Y. M. Lin, Y. Zhou, M. T. Flavin, L. M. Zhou, W. Nie, F. C. Chen, Bioorg. Med. Chem. 2002, 10, 2795.
[9] D. C. Leysen, A. Haemers, S. R. Pattyn, Antimicrob. Agents Chemother. 1989, 33, 1.
[10] A. Haemers, D. C. Leysen, W. Bollaert, M. Zhang, S. R. Pattyn, Antimicrob. Agents Chemother. 1990, 34,
496.
[11] J. A. Garcia-Rodriguez, A. C. Gomez Garcia, J. Antimicrob. Chemother. 1993, 32, 797.
[12] T. E. Renau, J. P. Sanchez, M. A. Shapiro, J. A. Dever, S. J. Gracheck, J. M. Domagala, J. Med. Chem. 1995,
38, 2974.
[13] T. E. Renau, J. P. Sanchez, J. W. Gage, J. A. Dever, M. A. Shapiro, S. J. Gracheck, J. M. Domagala, J. Med.
Chem. 1996, 39, 729.
[14] S. E. Berning, Drugs 2001, 61, 9.
[15] M. Neuman, Int. J. Clin. Pharmcol. Res. 1987, 7, 173.
[16] B. Y. Zhao, R. Pine, J. Domagala, K. Drlica, Antimicrob. Agents Chemother. 1999, 43, 661.
[17] T. E. Renau, J. W. Gage, J. A. Dever, G. E. Roland, E. T. Joannides, M. A. Shapiro, J. P. Sanchez, S. J.
Gracheck, J. M. Domagala, M. R. Jacobs, R. C. Reynolds, Antimicrob. Agents Chemother. 1996, 40, 2363.
[18] G. Klopman, D. Fercu, T. E. Renau, M. R. Jacobs, Antimicrob. Agents Chemother. 1996, 40, 2637.
[19] M. R. Jacobs, Activity of Quinolones Against Mycobacteria, Drugs, 1999, 58 (Suppl. 2), 19.
[20] C. C. Tzeng, Y. L. Chen, Chin. Pharm. J. 2002, 54, 229.
[21] J. Y. Sheu, Y. L. Chen, K. C. Fang, T. C. Wang, C. F. Peng, C. C. Tzeng, J. Heterocyclic Chem. 1998, 35, 955.
[22] K. C. Fang, Y. L. Chen, J. Y. Sheu, T. C. Wang, C. C. Tzeng, J. Med. Chem. 2000, 43, 3809.
[23] Y. L. Chen, K. C. Fang, J. Y. Sheu, S. L. Hsu, C. C. Tzeng, J. Med. Chem. 2001, 44, 2374.
[24] S. L. Hsu, Y. L. Chen, C. C. Tzeng, K. C. Fang, J. Y. Sheu, Helv. Chim. Acta 2001, 84, 874.
[25] L. Collins, S. G. Franzblau, Antimicrob. Agents Chemother. 1997, 41, 1004.
[26] P. S. Skinner, S. K. Furney, M. R. Jacobs, G. Klopman, J. J. Ellner, I. M. Orme, Antimicrob. Agents
Chemother. 1994, 38, 2557.
[27] A. Monks, D. Scuderio, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose, J. Langlay, P. Cronise, A.
Vaigro-Wolff, M. Gray-Goodrich, H. Campbell, J. Mayo, M. Boyd, J. Natl. Cancer Inst. 1991, 83, 757.
Received March 24, 2003