Synthesis, antibacterial, and cytotoxic evaluation of certain 7-substituted norfloxacin derivatives

Article abstract of DOI:10.1021/jm000153x

We report herein the synthesis and biological evaluation of two series of 7-substituted norfloxacin derivatives. Most compounds tested in this study demonstrated better activity against methicillin-resistant Staphylococcus aureus than norfloxacin. Prelimi

Full text of DOI:10.1021/jm000153x

J . Med. Chem. 2000, 43, 3809-3812
3809
Syn th esis, An tiba cter ia l, a n d Cytotoxic Eva lu a tion of Cer ta in 7-Su bstitu ted
Nor floxa cin Der iva tives
Kuo-Chang Fang,† Yeh-Long Chen,† J ia-Yuh Sheu,† Tai-Chi Wang,‡ and Cherng-Chyi Tzeng*,†
School of Chemistry, Kaohsiung Medical University, Kaohsiung City 807, Taiwan, Republic of China, and Department of
Pharmacy, Tajen Institute of Technology, Pingtong 907, Taiwan, Republic of China
Received April 3, 2000
We report herein the synthesis and biological evaluation of two series of 7-substituted
norfloxacin derivatives. Most compounds tested in this study demonstrated better activity
against methicillin-resistant Staphylococcus aureus than norfloxacin. Preliminary in vitro
evaluation indicated that the 7-[4-(2-hydroxyiminoethyl)piperazin-1-yl] derivatives 3b-e possess
distinct cytotoxicity profiles as compared with their R-methylene-γ-butyrolactone counterparts,
4b,e: i.e., excellent activities against the renal cancer subpanel. Among them, 1-ethyl-6-fluoro-
7-{4-[2-(4-chlorophenyl)-2-hydroxyiminoethyl]-1-piperazinyl}-4-oxo-1,4-dihydro-3-quinolinecar-
boxylic acid (3d ) demonstrated the most significant activities against renal cancer cell lines,
with log GI50 values of -6.40 against CAK-1, -6.14 against RXF 393, and -7.54 against UO-
31, compared with a mean log GI50 value of -5.03.
In tr od u ction
Herein we report on the synthesis of certain norfloxa-
cin derivatives with an additional functional moiety,
such as a 4-hydroxyaminoalkyl on the C-7 piperazin-1-
yl group, with the aim of providing extra hydrogen-
bonding capacities with the target DNA gyrase and,
therefore, to increase potency and broaden the antibac-
terial spectrum. A number of fluoroquinolones with an
oxime or a substituted oxime attached to the pyrrolidine
or piperazine ring at C-7 position were also synthesized
and evaluated for antibacterial activities.14-17 Indeed,
although fluoroquinolones are generally classified as
broad-spectrum antibacterial agents, their activities
against the majority of methicillin-resistant staphylo-
cocci (especially Staphylococcus aureus) are limited. We
have also prepared R-methylene-γ-butyrolactone-bear-
ing analogues in hope that the R-methylene-γ-butyro-
lactone moiety will provide an extra covalent bonding
capacity with DNA gyrase.18-20 Due to structural and
functional similarities between bacterial DNA gyrase
and mammalian topoisomerase II, the cytotoxicities of
these norfloxacin analogues were also evaluated.
Since the discovery of nalidixic acid by Lesher in
1962,1 a number of analogues have been synthesized,
and some of them, in particular the fluoroquinolone
derivatives such as norfloxacin,2 pefloxacin,3 enoxacin,4
ofloxacin,5 and ciprofloxacin,6 exhibited broader anti-
bacterial spectrum and enhanced potencies. These
agents were shown to be specific inhibitors of the
bacterial DNA gyrase,7-9 an enzyme which is respon-
sible for negatively supercoiling covalently closed cir-
cular DNA, and also in catenation and decatenation
reactions.10
The antibacterial activity of fluoroquinolones depends
not only on the bicyclic heteroaromatic pharmacophore
but also on the nature of the peripheral substituents
and their spatial relationship. These substituents exert
their influence on antibacterial activity by providing
additional affinity for the bacterial enzymes, enhancing
the cell penetration, or altering the pharmacokinet-
ics.11-13 The structure-activity relationships (SAR) of
antibacterial quinolones have been the subject of ex-
tensive review. In general, the upper portion of the
molecule which includes the C-3 carboxy and C-4 keto
moieties is required for hydrogen-bonding interactions
with DNA bases in the single-stranded regions of duplex
DNA created by the action of the enzyme, and therefore
it is essential. The lower portion of the molecule (i.e.,
N-1 and C-8) should be relatively small and lipophilic
to enhance self-association.11-13 Groups at C-5 and C-6
have also been optimized in which an amino and fluoro
substituent, respectively, at C-5 and C-6 appear to be
the best. The features for substitution at C-7 position
were more difficult to formulate; therefore, the struc-
tural requirements were not precisely defined. Exten-
sively investigated substituents are piperazin-1-yl and
its 4-substituted derivatives.
Ch em istr y
Preparations of 6-fluoro-1,4-dihydro-7-[4-(2-hydroxy-
iminoethyl)piperazin-1-yl]-4-oxoquinoline-3-carboxylic ac-
ids 3a -e and their R-methylene-γ-butyrolactone ana-
logues 4 are described in Scheme 1. Norfloxacin was
treated with K2CO3 and bromoacetone to provide 6-fluoro-
1,4-dihydro-4-oxo-7-[4-(2-oxopropyl)piperazin-1-yl]-4-
oxoquinoline-3-carboxylic acid (2a )21 which was then
reacted with hydroxylamine to give exclusively (E)-6-
fluoro-1,4-dihydro-7-[4-(2-hydroxyiminopropyl)piperazin-
1-yl]-4-oxoquinoline-3-carboxylic acid (3a ). The config-
uration of the oxime moiety was determined by through-
space nuclear Overhauser effect spectroscopy (NOESY)
which revealed coupling connectivity to CH3 protons.
Accordingly, 3b-e were obtained by hydroxylamination
of the respective 2b-e which were synthesized via
alkylation of norfloxacin with bromoacetophenone or its
4-substituted derivatives. However, compounds 3b-e
were found to be a mixture of E- and Z-isomers. The
* To whom correspondence should be addressed. Tel: (886) 7-3121101,
ext 2198. Fax: (886) 7-3125339. E-mail: tzengch@cc.kmu.edu.tw.
† Kaohsiung Medical University.
‡ Tajen Institute of Technology.
10.1021/jm000153x CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/16/2000

3810 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Ta ble 1. In Vitro Antimicrobial Activity of Norfloxacin Derivatives
Brief Articles
MIC (µM)
3e
1.62
104
25.9
organisma
3a
3b
0.86
55.3
13.8
0.22
3.45
0.44
3c
6.38
>200
>200
3d
6.16
>200
>200
4b
2e
0.83
>200
13.4
0.053
0.83
6.70
>200
>200
Nfb
E. coli
0.51
98.9
0.63
9.80
1.22
0.041
0.16
4.89
19.6
9.80
M. ranae
32.0
16.0
0.13
2.00
0.51
>200
>200
12.4
>200
P. aeruginosa
K. pneumoniae
P. vulgaris
S. aueus M-R
S. pneumoniae E&A-R
E. faecalis V-R
0.21
6.38
2.13
0.21
6.16
0.027
3.23
0.41
>200
>200
>200
1.54
30.0
30.0
>200
>200
>200
>200
13.0
3.23
ndc
nd
a
Organisms selected are as follows: Escherichia coli, Mycobacterium ranae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus
vulgaris, Staphylococcus aureus methicillin-resistant, Staphylococcus pneumoniae erythromycin- and ampicillin-resistant, Enterococcus
b
faecalis vancomycin-resistant. Nf, norfloxacin. c nd, not determined.
Ta ble 2. Inhibitory Activity of Norfloxacin Derivatives in a
Panel of Human Renal Cancer Cell Lines
Sch em e 1
log GI50 (M)a
compd
CAKI-1
RXF 393
UO-31
meanc
3a
3b
3c
3d
3e
4b
4e
>-4.0
-4.49
-5.72
-6.40
-5.16
-5.73
-5.29
>-4.0
-4.44
-6.77
-6.14
ndb
-4.40
-6.51
-6.88
-7.54
-6.26
-5.80
-5.56
>-4.0
-4.53
-4.67
-5.03
-4.50
-5.47
-5.19
nd
nd
a
Data obtained from NCI’s in vitro disease-oriented tumor cells
screen.23 nd, not determined. c Mean values over all cell lines
b
tested.
vancomycin-resistant E. faecalis. However, its ketone
precursor 2e is relatively inactive against the growth
of resistant strains. The R-methylene-γ-butyrolactone
counterpart 4b was inactive against most of the bacteria
tested.
These norfloxacin derivatives 2-4 were also evalu-
ated in the NCI human cancer cell line panel,23 and the
in vitro inhibitory activity for human renal cancer cell
lines are presented in Table 2 as log GI50 values (the
concentration of drug resulting in inhibition of cell
growth to 50% of controls), together with the mean value
(the average log GI50 value for the compound over all
cell lines). Compound 3a is inactive with the exception
of its weak activity against renal UO-31. A more potent
cytotoxicity was observed for its phenyl counterpart 3b
(log GI50 ) -4.53), and the potency was further en-
hanced by the introduction of an electron-withdrawing
substituent at the phenyl (3c, log GI50 ) -4.67; 3d , log
GI50 ) -5.03). Compounds 4b,e which bear an alkylat-
ing R-methylene-γ-butyrolactone, are more cytotoxic
than their respective hydroxyimino counterparts 3b,e.
However, it was especially surprising that the screening
of 3b-e revealed one of the most striking examples of
subpanel specificity: excellent inhibitory activities against
the renal cancer subpanel. Among them, 3d demon-
strated the most significant activities against renal
cancer cell lines, with log GI50 values of -6.40 against
CAK-1, -6.14 against RXF 393, and -7.54 against UO-
31, compared with a mean log GI50 value of -5.03.
Besides, the renal UO-31 cancer cell was found to be
very sensitive to 3b-e with log GI50 values of -6.51,
-6.88, -7.54, and -6.26, respectively. However, these
compounds are relatively inactive against leukemia
cancer cells.
carbon of 1′-CH2 which is anti to the oxime moiety
shifted downfield (δ 61.61 ppm for E-3a and 61.30 ppm
for E-3b), while that of the syn isomer shifted upfield
(δ 52.53 ppm for Z-3b).22 The Z-form of 3b was sepa-
rated by silica gel column chromatography. Preliminary
antibacterial assay indicated the same inhibitory activi-
ties between the mixture and the pure Z-isomer of 3b,
and separation of the respective 3c-e was not at-
tempted. Reformatsky-type condensation of 2b21 and 2e
respectively afforded 1-ethyl-6-fluoro-7-{4-[(2,3,4,5-tet-
rahydro-4-methylene-2-phenyl-5-oxofuran-2-yl)methyl]-
piperazinyl}-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(4b) and its 4-methoxy derivative 4e.
Resu lts a n d Discu ssion
Two series of norfloxacin derivatives prepared for this
study were tested in vitro against five susceptible
strains and three resistant strains. The minimum
inhibitory concentrations (MICs, µM) are presented in
Table 1. The data for norfloxacin is included for com-
parison. The first information obtained in this study is
that 6-fluoro-1,4-dihydro-7-[4-(2-hydroxyiminoethyl)pip-
erazin-1-yl]-4-oxoquinoline-3-carboxylic acids 3a -c are
less active than norfloxacin except for the inhibitory
activity against methicillin-resistant S. aureus. A meth-
yl substituent (3a , R1 ) Me) at the C-7 piperazinyl side
chain is more favorable than a phenyl (3b, R1 ) Ph).
Any substitution at the phenyl group of 3b decreased
the antibacterial activities with an exception of 3e which
exhibited the most significant activities against K.
pneumoniae, methicillin-resistant S. aureus, erythro-
mycin- and ampicillin-resistant S. pneumoniae, and
Con clu sion
Two series of 7-substituted norfloxacin derivatives
were synthesized and evaluated for antibacterial and

Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3811
dried over Na2SO4, filtered and concentrated in vacuo to give
a solid which was purified by flash column chromatography
(silica gel, with CH2Cl2-MeOH (10:1) as the eluent) and
crystallization from CH2Cl2-MeOH (5:1) to give 3a as an off-
white amorphous solid (0.28 g, 72%): mp 218 °C dec; 1H NMR
(200 MHz, DMSO-d6) δ 1.42 (t, 3H, J ) 7.2 Hz), 1.81 (s, 3H),
2.50 (m, 4H), 3.03 (s, 2H), 3.33 (m, 4H), 4.59 (q, 2H, J ) 7.2
Hz), 7.18 (d, 1H, J ) 7.3 Hz), 7.91 (d, 1H, J ) 13.5 Hz), 8.95
(s, 1H), 10.58 (s, 1H), 15.32 (br s, 1H); 13C NMR (100 MHz,
DMSO-d6) δ 12.19, 14.33, 49.04, 49.47, 49.51, 52.29, 61.61,
105.91, 107.06, 111.12 (J CF ) 22.8 Hz), 119.25 (J CF ) 7.5 Hz),
137.20, 145.50 (J CF ) 10.7 Hz), 148.48, 152.91 (J CF ) 247.3
Hz), 153.29, 166.11, 176.16 (J CF ) 3.0 Hz). Anal. (C19H23FN4O4‚
0.5H2O) C, H, N.
cytotoxic activities. Preliminary results indicated that
most compounds tested in this study demonstrated
better activity against methicillin-resistant S. aureus
than norfloxacin. Among them, 3e exhibited the most
significant activities against K. pneumoniae, methicillin-
resistant S. aureus, erythromycin- and ampicillin-
resistant S. pneumoniae, and vancomycin-resistant E.
faecalis. Although the alkylating R-methylene-γ-butyro-
lactone-bearing derivatives 4b,e are more cytotoxic than
their respective hydroxyimino counterparts 3b,e, their
cytotoxicity profiles are quite different. Compounds
3b-e possess a distinct renal cancer subpanel specific-
ity.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of 1-E t h yl-
6-flu or o-4-oxo-7-{4-[2-h ydr oxyim in o-2-(4-su bstitu ted-ph en -
yl)eth yl]-1-p ip er a zin yl}-1,4-d ih yd r oqu in olin e-3-ca r box-
ylic Acid s 3b-e. To a solution of 1-ethyl-6-fluoro-1,4-dihydro-
4-oxo-7-(4-phenacyl-1-piperazinyl)quinoline-3-carboxylic acid21
(2b; 0.44 g, 1 mmol) in absolute methanol (20 mL) was added
a solution of hydroxylamine hydrochloride (0.14 g, 2 mmol)
and sodium bicarbonate (0.17 g, 2 mmol) in water (2 mL). The
mixture was heated at reflux for 36 h and allowed to cool to
room temperature. CH2Cl2 (50 mL) was added, and the layers
were separated. The organic phase was washed successively
with water and brine, dried over Na2SO4, filtered and concen-
trated in vacuo to give a solid which was purified by flash
column chromatography (silica gel, with CH2Cl2-MeOH (10:
1) as the eluent) and crystallization from CH2Cl2-MeOH (5:
1) to give 1-ethyl-6-fluoro-4-oxo-7-[4-(2-hydroxyimino-2-phen-
ylethyl)-1-piperazinyl]-1,4-dihydroquinoline-3-carboxylic acid
(3b; E/Z ) 1:2.2) as an off-white amorphous solid (0.31 g,
Exp er im en ta l Section
Melting points were determined on a Fargo MP-ID melting
point apparatus and are uncorrected. Nuclear magnetic reso-
nance (1H and 13C) spectra were recorded on a Varian Gemini
200 spectrometer or Varian Unity 400 spectrometer. Chemical
shifts were expressed in parts per million (δ) with tetrameth-
ylsilane (TMS) as an internal standard. Thin-layer chroma-
tography was performed on silica gel 60 F-254 plates pur-
chased from E. Merck and Co. The elemental analyses were
performed at the Instrument Center of National Science
Council at National Cheng-Kung University and National
Chung-Hsing University using a Heraeus CHN-O Rapid EA,
and all values are within (0.4% of the theoretical composi-
tions.
Gen er a l P r oced u r e for th e P r ep a r a tion of 1-Eth yl-6-
flu or o-7-{4-[2-(4-su bstitu ted -p h en yl)-2-oxoeth yl]-1-p ip er -
a zin yl}-4-oxo-1,4-d ih yd r oq u in olin e-3-ca r b oxylic Acid s
2c-e. A mixture of norfloxacin (0.5 g, 1.56 mmol), sodium
bicarbonate (0.13 g, 1.56 mmol), potassium iodide (0.08 g, 0.5
mmol), and 2-bromo-4′-fluoroacetophenone (0.40 g, 1.86 mmol)
in DMF (40 mL) was stirred at room temperature for 3 h, then
poured into ice-water (50 mL), and extracted with CH2Cl2 (50
mL × 3). The extract was washed with water, dried over Na2-
SO4, and evaporated. Recrystallization from EtOH-CHCl3 (5:
1) yielded 1-ethyl-6-fluoro-7-{4-[2-(4-fluorophenyl)-2-oxoethyl]-
1-piperazinyl}-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2c)
(61% yield): mp 208 °C dec; 1H NMR (200 MHz, DMSO-d6) δ
1.41 (t, 3H, J ) 7.0 Hz), 2.73 and 3.3 (two m, 8H), 3.94 (s,
2H), 4.58 (q, 2 H, J ) 7.0 Hz), 7.18 (d, 1H, J ) 7.0 Hz), 7.36
(m, 2H), 7.90 (d, 1H, J ) 13.2 Hz), 8.11 (m, 2H), 8.94 (s, 1H),
15.30 (br s, 1H). Anal. (C24H23F2N3O4) C, H, N.
1
68%): mp 203 °C dec; H NMR (DMSO-d6) δ 1.39 (t, 3H, J )
7.2 Hz), 2.64 and 3.25 (two m, 8H), 3.43 and 3.74 (two s, 2 H),
4.56 (q, 2H, J ) 7.2 Hz), 7.15 (d, 1H, J ) 7.2 Hz), 7.36-7.81
(m, 5H), 7.89 (d, 1H, J ) 13.2 Hz), 8.93 (s, 1H), 10.99 (s, E-form
OH), 11.47 (s, Z-form OH), 15.07 (br s, 1H); 13C NMR (50 MHz,
DMSO-d6) δ 14.28, 48.98, 49.43, 49.51, 49.95, 52.16, 52.48,
61.30, 105.88, 105.94, 107.02,0.107.07, 110.76, 111.22, 119.13,
119.28, 126.23, 127.68, 127.95, 128.03, 128.27, 128.36, 138.45,
133.40, 136.03, 137.08, 145.35, 145.56, 148.36, 152.04, 152.93,
155.33, 166.04, 176.04, 176.09. Anal. (C24H25FN4O4‚1.5 H2O)
C, H, N.
The Z-form of 3b was separated by silica gel column
chromatography with CH2Cl2-MeOH (30:1) as the eluent: 1H
NMR (200 MHz, DMSO-d6) δ 1.39 (t, 3H, J ) 7.0 Hz), 2.64
and 3.24 (two m, 8H), 3.74 (s, 2 H), 4.5 (q, 2H, J ) 7.0 Hz),
7.15 (d, 1H, J ) 7.0 Hz), 7.36 (m, 3H), 7.79 (m, 2H), 7.90 (d,
1H, J ) 13.3 Hz), 8.93 (s, 1H), 11.47 (s, 1H), 15.28 (br s, 1H);
13C NMR (100 MHz, DMSO-d6) δ 14.31, 49.06, 49.50, 49.54,
49.98, 52.53, 106.08, 107.06, 111.09 (J CF ) 22.8 Hz), 119.30
(J CF ) 7.6 Hz), 126.28, 128.09, 128.52, 136.07, 137.18, 145.56
(J CF ) 10.6 Hz), 148.48, 152.95 (J CF ) 247.3 Hz), 153.02,
166.13, 176.19.
1-Eth yl-6-flu or o-7-{4-[2-(4-ch lor op h en yl)-2-oxoeth yl]-
1-p ip er a zin yl}-4-oxo-1,4-d ih yd r oq u in olin e-3-ca r b oxyl-
ic Acid (2d ). The general procedure using 2-bromo-4′-
1
chloroacetophenone gave 2d (67% yield): mp 207 °C dec; H
NMR (200 MHz, DMSO-d6) δ 1.41 (t, 3H, J ) 7.2 Hz), 2.73
and 3.29 (two m, 8H), 3.95 (s, 2H), 4.58 (q, 2H, J ) 7.2 Hz),
7.19 (d, 1H, J ) 7.2 Hz), 7.59 and 8.02 (m, 4H), 7.92 (d, 1H,
J ) 13.2 Hz), 8.95 (s, 1H), 15.41 (br s, 1H). Anal. (C24H23
ClFN3O4‚0.25H2O) C, H, N.
-
1-Eth yl-6-flu or o-7-{4-[2-(4-flu or op h en yl)-2-h yd r oxyim -
in oet h yl]-1-p ip er a zin yl}-4-oxo-1,4-d ih yd r oq u in olin e-3-
ca r boxylic a cid (3c, E/Z ) 1:1.3): 66% yield (starting with
1-Eth yl-6-flu or o-7-{4-[2-(4-m eth oxyph en yl)-2-oxoeth yl]-
1-p ip er a zin yl}-4-oxo-1,4-d ih yd r oq u in olin e-3-ca r b oxyl-
ic Acid (2e). The general procedure using 2-bromo-4′-
methoxyacetophenone gave 2e (75% yield): mp 202 °C dec;
1H NMR (200 MHz, DMSO-d6) δ 1.41 (t, 3H, J ) 7.0 Mz), 2.73
and 3.3 (two m, 8H), 3.85 (s, 3H), 3.88 (s, 2H), 4.58 (q, 2H,
J ) 7.0 Mz), 7.02-7.2 (m, 3H), 7.87 (d, 1H, J ) 13.8 Mz), 8.01
(m, 2H), 8.94 (s, 1H), 15.33 (br s, 1H). Anal. (C25H26FN3O5) C,
H, N.
(E)-1-Eth yl-6-flu or o-7-[4-(2-h ydr oxyim in opr opyl)-1-pip-
er a zin yl]-4-oxo-1,4-d ih yd r oqu in olin e-3-ca r boxylic Acid
(3a ). To a suspension of the 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-
7-[4-(2-oxopropyl)-1-piperazinyl]quinoline-3-carboxylic acid21
(2a ; 0.38 g, 1 mmol) in absolute methanol (20 mL) was added
a solution of hydroxylamine hydrochloride (0.14 g, 2 mmol)
and sodium bicarbonate (0.17 g, 2 mmol) in water (2 mL). The
mixture was stirred for 20 h at room temperature, then CH2-
Cl2 (50 mL) was added, and the layers were separated. The
organic phase was washed successively with water and brine,
1
0.5 mmol of 2c); mp 227 °C dec; H NMR (200 MHz, DMSO-
d6) δ 1.39 (t, 3H, J ) 7.2 Hz), 2.62 and 3.25 (two m, 8H), 3.44
and 3.73 (two s, 2H), 4.57 (q, 2H, J ) 7.2 Hz), 7.15 (d, 1H,
J ) 7.2 Hz), 7.42-7.83 (m, 4H),7.89 (d, 1H, J ) 13.3 Hz), 8.93
(s, 1H), 11.19 (s, E-form OH), 11.62 (s, Z-form OH). Anal.
(C24H24F2N4O4‚0.5H2O) C, H, N.
1-E t h yl-6-flu or o-7-{4-[2-(4-ch lor op h en yl)-2-h yd r oxy-
im in oeth yl]-1-p ip er a zin yl}-4-oxo-1,4-d ih yd r oqu in olin e-
3-ca r boxylic a cid (3d , E/Z ) 1:1.2): 73% yield (starting with
1
0.5 mmol of 2d ); mp 222 °C dec; H NMR(200 MHz, DMSO-
d6) δ 1.39 (t, 3H, J ) 7.0), 2.61 and 3.24 (two m, 8H), 3.44 and
3.73 (two s, 2H), 4.56 (q, 2H, J ) 7.0 Hz), 7.24 and 7.78 (two
m, 5H), 7.89 (d, 1H, J ) 13.4 Hz), 8.93 (s, 1H), 11.10 (s, E-form
OH), 11.49 (s, Z-form OH), 15.35 (br s, 1H). Anal. (C24H24
FClN4O4‚0.25H2O) C, H, N.
-
1-E t h yl-6-flu or o-7-{4-[2-h yd r oxyim in o-2-(4-m et h oxy-
ph en yl)eth yl]-1-piper azin yl}-4-oxo-1,4-dih ydr oqu in olin e-

3812 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
3-ca r boxylic a cid (3e, E/Z ) 1:1.3): 74% yield (starting with
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1
0.5 mmol of 2e); mp 221 °C dec; H NMR (200 MHz, DMSO-
d6) δ 1.39 (t, 3H, J ) 7.2 Hz), 2.62 and 3.26 (two m, 8H), 3.42
and 3.71 (two s, 2H), 3.78 (s, 3H), 4.57 (q, 2H, J ) 7.2 Hz),
6.92 (m, 2H),7.16 (d, 1H, J ) 7.6 Hz),7.72 (m, 2H), 7.89 (d,
1H, J ) 13.6 Hz), 8.93 (s, 1H), 10.97 (s, E-form OH), 11.28 (s,
Z-form OH), 15.29 (br s, 1H). Anal. (C25H27FN4O5‚0.25H2O)
C, H, N.
1-Eth yl-6-flu or o-7-{4-[(2,3,4,5-tetr a h yd r o-4-m eth ylen e-
2-p h en yl-5-oxofu r a n -2-yl)m eth yl]p ip er a zin yl}-1,4-d ih y-
d r o-4-oxo-3-qu in olin eca r boxylic Acid (4b). To a suspen-
sion of 2b21 (0.44 g, 1 mmol) in dry THF (20 mL) were added
activated Zn powder (0.13 g, 2 mmol), hydroquinone (6 mg),
and ethyl 2-(bromomethyl)acrylate (0.26 g, 1.3 mmol). The
mixture was refluxed under nitrogen atmosphere for 15 h (TLC
monitoring). After cooling, it was poured into an ice-cold 5%
HCl solution (100 mL), neutralized with 1.0 N NaHCO3, and
extracted with CH2Cl2 (60 mL × 3). The CH2Cl2 extracts were
combined and washed with H2O, dried (Na2SO4), and then
evaporated to give a residual solid which was purified by
column chromatography on silica gel using CH2Cl2-MeOH (10:
1). The proper fractions were combined and evaporated to
furnish a residual solid which was crystallized from CH2Cl2-
EtOH (5:1) to afford 4b (0.42 g, 83% yield): mp 199-200 °C
dec; 1H NMR (200 MHz, CF3COOD) δ 1.71 (t, 3H, J ) 7.1 Hz),
3.28-3.75 (m, 7H), 3.98-4.42 (m, 5H), 4.82 (q, 2H, J ) 7.1
Hz), 6.02 (br s, 1H,), 6.59 (br s, 1H), 7.39 (d, 1H, 6.6 Hz), 7.53
(m, 5H), 8.27 (d, 1H, J ) 12.5 Hz), 9.28 (s, 1H, 2-H). Anal.
(C28H28FN3O5‚0.25H2O) C, H, N.
(9) Chu, D. T. W. Recent Development in Antibacterial Research.
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Agents. J . Med. Chem. 1986, 29, 1531-1534.
(13) Domagala, J . M.; Heifetz, C. L.; Hutt, M. P.; Mich, T. F.; Nichols,
J . B.; Solomon, M.; Worth, D. F. 1-Substituted 7-[3-[(Ethylami-
no)methyl]-1-pyrrolidinyl]-6,8-difluoro-1,4-dihydro-4-oxo-3-quino-
linecarboxylic Acids. New Quantitative Structure-Activity Re-
lationships at N1 for the Quinolone Antibacterials. J . Med. Chem.
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R. N.; Plattner, J . J . Preparation and in Vitro and in Vivo
Evaluation of Quinolones with Selective Activity against Gram-
Positive Organisms. J . Med. Chem. 1992, 35, 1392-1398.
(15) Foroumadi, A.; Emami, S.; Davood, A.; Moshafi, M. H.; Sharifian,
A.; Tabatabaie, M.; Tarhimi Farimani, H.; Sepehri, G.; Shafiee,
A. Synthesis and in vitro antibacterial activities of N-substituted
piperazinyl quuinolones. Pharm. Sci. 1997, 3, 559-563.
(16) Hong, C. Y.; Kim, Y. K.; Lee, Y. H.; Kwak, J . H. Methyloxime-
substituted Aminopyrrolidine: A New Surrogate for 7-Basic
Group of Quinolone. Bioorg. Med. Chem. Lett. 1998, 8, 221-
226.
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Synthesis and In-vitro Antibacterial Activity of New N-Substi-
tuted Piperazinyl Quinolones. Pharm. Pharmacol. Commun.
1999, 5, 591-594.
(18) Wang, T.-C.; Lee, K.-H.; Chen, Y.-L.; Liou, S.-S.; Tzeng, C.-C.
Synthesis and Anticancer Evaluation of Certain γ-Aryloxy-
methyl-R-Methylene-γ-Phenyl-γ-Butyrolactones. Bioorg. Med.
Chem. Lett. 1998, 8, 2773-2776.
(19) Tzeng, C.-C.; Chen, Y.-L.; Wang, C.-J .; Wang, T.-C.; Chang, Y.-
L.; Teng, C.-M. Synthesis and Evaluation of 2-{[(2-Oxo-1H-
quinolin-8-yl)oxy]methyl}-Substituted R-Methylidene-γ-butyro-
lactones. Helv. Chim. Acta 1997, 80, 1161-1168.
1-Eth yl-6-flu or o-7-{4-{[2,3,4,5-tetr a h yd r o-2-(4-m eth oxy-
p h en yl)-4-m eth ylen e-2-p h en yl-5-oxofu r a n -2-yl]m eth yl}-
piper azin yl}-1,4-dih ydr o-4-oxoqu in olin e-3-car boxylic Acid
1
(4e). From 2e as described for 4b: 88% yield; mp 191 °C; H
NMR (200 MHz, CF3COOD) δ 1.76 (t, 3H, J ) 7.0 Hz,), 3.28-
3.79 (m, 7H), 4.02-4.42 (m, 8H), 4.84 (q, 2H, J ) 7.0 Hz), 6.06
(br s, 1H), 6.62 (br s, 1H), 7.19-7.55 (m, 5H), 8.31 (d, 1H, J )
12.5 Hz), 9.32 (s, 1H) Anal. (C29H30FN3O6) C, H, N.
In Vitr o An tiba cter ia l Assa y. Deter m in a tion of MIC:
2 mg of each test compound was dissolved in an appropriate
solvent (100% DMSO) and serially diluted with DMSO into
the desired testing concentration ranges. Each series of testing
solution (0.01 mL) was added into the 48-well plate with 0.99
mL of media broth containing 1-5 × 105 CFU/mL testing
microorganism. Thus the final maximal concentration of
DMSO was 1% and the initial concentration of testing solution
was 300 µM. Media used were as follows: nutrient broth (NB,
DIFCO) for Escherichia coli, Pseudomonas aeruginosa and
Klebsiella pneumoniae; Mueller-Hinton broth (DIFCO) for
Staphylococcus aureus, methicillin-resistant (MRSA) and Pro-
teus vulgaris; brain heart infusion broth (BHI, DIFCO) for
Mycobacterium ranae; tryptic soy broth (DIFCO) containing
of 7% calf serum for Streptococcus pneumoniae (EM & AM
Research Clinical Isolates) and Enterococcus faecalis (VRE,
Clinical Isolates). The plates were incubated for 20-72 h at
37 °C, then the MIC was determined by visual turbidity
readout or by microscope observation of microorganism growth.
Vehicle and reference agents were used in every test as the
negative and positive controls, and the assays were performed
in duplicate.
(20) Chen, Y.-L.; Wang, T.-C.; Chang, N.-C.; Chang, Y.-L.; Teng, C.-
M. Tzeng, C.-C. R-Methylidene-γ-butyrolactones: Synthesis and
Vasorelaxing Activity Assay of Coumarin, Naphthalene, and
Quinoline Derivatives. Chem. Pharm. Bull. 1998, 46, 962-965.
(21) Kondo, H.; Sakamoto, F.; Kodera, Y.; Tsukamoto, G. Studies on
Prodrugs. 5. Synthesis and Antibacterial Activity of N-(Oxo-
alkyl)norfloxacin Derivatives. J . Med. Chem. 1986, 29, 2020-
2024.
Ack n ow led gm en t. Financial support of this work
by the National Science Council of the Republic of China
is gratefully acknowledged. We also thank the National
Cancer Institute (NCI) for the anticancer screenings.
(22) Silverstein, R. M.; Webster, F. X. 13C NMR Spectrometry. In
Spectrometric Identification of Organic Compounds, 6th ed.;
Rose, N., Ed.; J ohn Wiley and Sons: New York, 1998; pp 217-
249.
(23) Monks, A.; Scudiero, D.; Skehaan, P.; Shoemaker, R.; Paull, K.;
Vistica, D.; Hose, C.; Langley, J .; Cronise, P.; Vaigro-Wolff, A.;
Gray-Goodrich, M.; Campbell, H.; Mayo, J .; Boyd, M. Feasibility
of a High-flux Anticancer Drug Screen Using a Diverse Panel
of Cultured Human Tumor Cell Lines. J . Natl. Cancer Inst. 1991,
83, 757-766.
Su p p or tin g In for m a tion Ava ila ble: Table containing
data for inhibition of cancer cell lines for 3a -e and 4b,e. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Refer en ces
(1) Lesher, G. Y.; Froelich, E. J .; Gruett, M. D.; Bailey, J . H.;
Brundage, R. P. 1,8-Naphthyridine Derivatives. A New Class
of Chemotherapeutic Agents. J . Med. Chem. 1962, 5, 1063-1065.
J M000153X