Synthesis and in vitro antibacterial activity of 7-(3-Alkoxyimino-4-amino- 4-methylpiperidin-1-yl) fluoroquinolone derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.03.026

A series of novel 7-(3-alkoxyimino-4-amino-4-methylpiperidin-1-yl) fluoroquinolone derivatives were designed, synthesized and evaluated for their in vitro antibacterial activity and cytotoxicity. All of the target compounds have potent antibacterial activ

Full text of DOI:10.1016/j.ejmech.2011.03.026

European Journal of Medicinal Chemistry 46 (2011) 2421e2426
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro antibacterial activity of 7-(3-Alkoxyimino-4-amino-
4-methylpiperidin-1-yl) fluoroquinolone derivatives
1
1
*
Ju-Xian Wang , Yi-Bin Zhang , Ming-Liang Liu , Bo Wang, Yun Chai, Su-Jie Li, Hui-Yuan Guo
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 7-(3-alkoxyimino-4-amino-4-methylpiperidin-1-yl)fluoroquinolone derivatives were
designed, synthesized and evaluated for their in vitro antibacterial activity and cytotoxicity. All of the
target compounds have potent antibacterial activity against the tested Gram-positive and Gram-negative
strains, and exhibit good potency in inhibiting the growth of Staphylococcus aureus including MRSA,
Received 31 July 2010
Received in revised form
5 March 2011
Accepted 14 March 2011
Available online 23 March 2011
Staphylococcus epidermidis including MRSE and Streptococcus pneumoniae (MICs: 0.125e4 mg/mL).
Compound 22, with the best activity against Gram-positive strains, is 4e16 fold more potent than
gemifloxacin, gatifloxacin and levofloxacin against Enterococcus faecalis, and 16- and 4-fold more potent
than levofloxacin against S. epidermidis 09-6 and S. pneumoniae 08-4, respectively.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Fluoroquinolone
Synthesis
Antibacterial activity
1. Introduction
the three, piperidinyl analogs are the least studied [4]. As part of an
ongoing program to find potent new quinolones displaying strong
Quinolone antibacterial agents are among the most attractive
drugs in the anti-infective chemotherapy field. These antibiotics act
against bacteria by selectively inhibiting two type II topoisomerase
enzymes, DNA gyrase and topoisomerase IV, which both play
a critical role in bacterial cell growth and division [1].
Gram-positive antibacterial activity, we have focused our attention
on introducing new functional groups to the piperidine ring [5e8].
Previous work on pyrrolidine analogs suggests that introduction of
a methyl group in the 3-position of the pyrrolidine ring can increase
Gram-positive antibacterial activity. For example, an analog of
gemifloxacin (GMFX), DW 286 (Fig. 1), possessing an additional
methyl group at 3-position of pyrrolidine ring, displays far more
potent antibacterial activity than GMFX against important Gram-
positive organisms, MRSA and ofloxacin resistant organisms, while
maintaining an excellent pharmacokinetic profile [9]. We applied
this structural modification to DZH (Fig. 1), which shows good in
vitro activity against Gram-positive and Gram-negative organisms,
including MRSA and Pseudomonas aeruginosa [4]. A series of fluo-
roquinolone compounds containing piperidinyl substitution at the
C-7 position were designed and synthesized. These derivatives are
structurally novel, having both an amino and a methyl group at the
4-position and an alkoxyimino group at 3-position of the piperidine
ring. Our primary objective was to optimize the potency of these
compounds against Gram-positive and Gram-negative organisms.
Because of increasing prevalence of bacterial infections, partic-
ularly those such as the clinically important pathogenic bacteria,
Staphylococci (including methicillin-resistant Staphylococcus
aureus, MRSA and methicillin-resistant Staphylococcus epidermidis,
MRSE), Streptococci and Enterococci, drug-discovery efforts have
been intensified in the past years to search for more effective
antibacterial agents with a broad spectrum of activity, and activity
against Gram-positive pathogens, particularly resistant pathogens
[2,3]. The ideal strategy to such challenges is to find novel agents
that inhibit new targets in bacteria. However, despite advances in
drug development, finding new antibacterial agents with novel
mechanisms of action remains extremely difficult. A more practical
approach is to modify the structures of existing antibacterial agents
to increase potency, and to overcome resistance.
From the chemical structural point of view, one of the most
important features of the fluoroquinolone antibacterials is the
presence of a 5- or 6-membered nitrogenheterocycle as side chain at
the C-7 position, including piperazine, pyrrolidine and piperidine. Of
2. Chemistry
The new piperidine derivatives 10a,b and novel fluoroquino-
lones 12e25 described herein were synthesized as shown in
Schemes 1 and 2, respectively. Catalytic hydrogenation of ethyl
N-benzyl-3-oxopiperidine-4- carboxylate hydrochloride 1 gave
* Corresponding author. Tel.: þ86 10 63036965; fax: þ86 10 63047865.
E-mail address: lmllyx@yahoo.com.cn (M.-L. Liu).
1
These authors contributed equally to the work.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.026

2422
J.-X. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 2421e2426
Fig. 1. Chemical structures of quinolone compounds.
secondary amine 2. The amine 2 was subsequently treated with
di-tert-butyl dicarbonate (Boc2O) in ethanol to form Boc-protected
compound 3, which was methylated with methyl iodide at the
presence of anhydrous potassium carbonate to produce methyl
ketone 4. The oximes 5a,b were obtained from the reaction of 4
with alkoxylamines at 60 ꢀC and converted to acids 6a,b by
saponification and acidification. The acids 6a,b were subsequently
reacted with isobutyl chloroformate to give activated esters 7a,b,
which upon ammonolysis afforded amides 8a,b by pumping
ammonia gas in methylene chloride. Hoffmann degradation of the
amides 8a,b used freshly prepared sodium hypobromite to give
amines 9a,b. Deprotection of Boc protective group on the amines
9a,b was carried out by pumping dry hydrogen chloride gas in
methylene chloride to afford new piperidine derivative dihydro-
chlorides 10a,b in a good yield.
Finally, the target compounds 12e25 were obtained by coupling
the new piperidine derivatives 10a,b with various compounds
containing quinolone or naphthyridine core according to the well-
established literature procedures [10]. In the case of quinolones
12e21, condensation of 10a,b with 11a-f was carried out in the
presence of triethylamine. However for 22e25, boric chelates 11g,h
were used to increase reactivity. Table 1 shows structures and
cytotoxicity of the novel fluoroquinolones 12e25.
3. Results and discussion
3.1. Antibacterial activity
The novel fluoroquinolones 12e25 were evaluated for their in
vitro antibacterial activity against ten Gram-positive and six Gram-
negative strains using conventional agar-dilution method [12].
Minimum inhibitory concentration (MIC) is defined as the
concentration of the compound required to give complete inhibi-
tion of bacterial growth and MICs of the synthesized compounds
along with the reference drugs GMFX, moxifloxacin (MXFX), gati-
floxacin (GTFX) and levofloxacin (LVFX) for comparison are repor-
ted in Table 2.
All of the target compounds 12e25 have generally potent anti-
bacterial activity against the sixteen tested strains. They exhibit
good potency in inhibiting the growth of S. aureus including MRSA,
S. epidermidis including MRSE and Streptococcus pneumoniae, with
MICs in the range of 0.125e4 mg/mL, which were comparable to
that of the reference drugs. Compound 22, with the best activity
against Gram-positive strains, is found to be 4e16 fold more potent
than GMFX, GTFX and LVFX against Enterococcus faecalis, and 16-
and 4-fold more potent than LVFX against S. epidermidis 09-6 and
S. pneumoniae 08-4, respectively.
Since the oxime group can exist in the E or Z configuration, it was
necessary to determine the geometries of all the oxime target
compounds 12e25. It was a pity that we were not successful in
preparing X-ray quality single crystals of compounds 12e25. In NOE
spectrums of 12e25, there was no correlation between the oxime
group and its neighbor group (amino group or methyl group). So it is
quite probable that the geometry of the oxime group is the E
configuration. Finally, we were able to obtain X-ray data for the
compound 8a [11]. As expected, the geometry of the methyloxime
group on the piperidine ring which adopts a boat conformation is
the E configuration (Fig. 2).
As for Gram-negative strains, the novel fluoroquinolones 12e25
have poor or no activity against Escherichia coli, but show moderate
activity against P. aeruginosa. For example, compounds 12, 14e16,
Scheme 1. Synthesis of new piperidine derivatives 10a,b. Reagents and conditions: (a)
Pd/C 5%, H2 0.5 MPa, EtOH, rt, 4 h; (b) NaHCO3, Boc2O, EtOH 50%, rt, 1 h; (c) K2CO3, MeI,
Me2CO, 50 ꢀC, 1.5 h; (d) R1ONH2$HCl, Et3N, EtOH 80%, 60 ꢀC, 2 h; (e) aq NaOH, EtOH, rt,
5 h; (f) HOAc, H2O, rt, 0.5 h; (g) Et3N, ClCOOBu-i, CH2Cl2, ꢁ15 ꢀC, 2 h; (h) NH3, CH2Cl2,
Scheme 2. Synthesis of novel fluoroquinolones 12e25. Reagents and conditions: (k)
10a,b, Et3N, MeCN, rt, 3e10 h; (l) aq NaOH, rt, 1e4 h; (m) aq HCl, rt.
0
ꢀC, 2 h; (i) aq NaBrO, MeCN, 5 ꢀC, 10 h; (j) HCl gas, CH2Cl2, rt, 1 h.

J.-X. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 2421e2426
2423
Table 1
1506
(CC50 > 1000
m
M. Among them, cytotoxicity of compounds 20, 24 and 25
The structures and cytotoxicity of compounds 12e25.
mM) is much less than the three reference drugs. The
cytotoxicity of the substitution pattern at C-7 position depends on
the quinolone nuclei. For example, fluoroquinolones fearturing
ethyloxime-incorporated piperidino-substitution are less cytotoxic
than the analogs containing methyloxime for naphthyridine nuclei,
which is contrary to the cytotoxicity profiles for the quinolone
nuclei of substitution linkage to the C-8 position though an oxygen
atom.
O
F
COOH
N
X
N
R2
Me
H2N
NOR1
4. Conclusion
Compd.
R1
X
R2
CC50 (mM)
12
13
Me
Et
N
N
77.4
300
In summary, a series of novel 7-(3-alkoxyimino-4-amino-
4-methylpiperidin-1-yl)fluoroquinolone derivatives weredesigned,
synthesized and evaluated for their in vitro antibacterial activity and
cytotoxicity. Generally, all of the target compounds 12e25 demon-
strate potent antibacterial activity against the sixteen tested Gram-
positive and Gram-negative strains. In particular, they exhibit
good potency in inhibiting the growth of S. aureus including MRSA,
S. epidermidis including MRSE and S. pneumoniae (MICs: 0.125e4 mg/
mL), and some of them are much less cytotoxic than GMFX, MXFX
and GTFX.
14
15
16
17
18
Me
Et
Me
Et
N
N
CF
CF
CF
2,4-F2-C6H3
2,4-F2-C6H3
Et
93.0
128
433
592
36.6
Et
Me
2-F-C2H4
19
20
21
22
23
24
Et
CF
288
Me
Et
COCHF2
COCHF2
COMe
COMe
1068
518
Me
Et
729
280
5. Experimental protocols
Me
1506
O
O
5.1. Chemistry
25
Et
1157
321
161
396
GMFX
MXFX
GTFX
All chemical reagents and solvents used in this study were
purchased from Beihua Fine Chemicals Company (Beijing, China). The
starting material 1 was purchased from Bayoupharm Company
(Chengdu, China). The compounds 8a,b were prepared from 1
according to the literature [5]. Melting points were determined in
open glass capillaries and are uncorrected. 1H-NMR spectra were
recorded on a Varian Mercury-400 or an INOVA-500 spectrometer
(both: Varian Inc., Palo Alto, CA, USA) using tetramethylsilane as
internal standard. Electron spray ionization (ESI) mass spectra and
high-resolution mass spectra (HRMS) were recorded on a MDSSCIEX
Q-Tap mass spectrometer (Applied Biosystems, USA). Merck silica gel
ART555460F254 plates (Merck, Germany) were used foranalytical TLC.
18, 20, 21 and 23 are more active than or comparable to LVFX
against these strains. In particular, compound 14 is 64-fold more
potent than GTFX and LVFX, and comparable to GMFX and MXFX
against P. aeruginosa 09-32. However, Compound 12, possessing the
same quinolone nucleus as DZH, is not superior to GMFX against
the tested Gram-positive and Gram-negative strains, except Kleb-
siella pneumoniae 09-23. These results suggest that introduction of
another methyl group into 4-position of piperidine ring does not
cause increased antibacterial activity, which is not consistent with
the activity profiles of the pyrrolidine-containing fluoroquinolones.
5.1.1. N-tert-Butoxycarbonyl-4-amino-3-methoxyimino-4-
methylpiperidine (9a)
To a stirring solution of 8a (2.8 g, 10.0 mmol) in acetonitrile
(150 mL) was added dropwise a freshly prepared sodium hypo-
bromite solution (25 mL) at 5 ꢀC. The reaction mixture was stirred
for 10 h at the same temperature. After removal of the acetonitrile
under reduced pressure, the reaction mixture was diluted with
water (30 mL), adjusted to pH 2.5e3 with 2 N hydrochloric acid and
washed with methylene chloride (3 ꢂ 30 mL). Then the water layer
was adjusted to pH 12 with 6 N sodium hydroxide solution and
extracted with methylene chloride (3 ꢂ 50 mL). The combined
extracts were washed with saturated brine, dried over anhydrous
sodium sulfate and concentrated under reduced pressure to afford
compound 9a (2.1 g). Yield: 83%, colorless liquid.1H-NMR (400 MHz,
3.2. Cytotoxicity
Cytotoxicity of compounds 12e25 were also assessed in
comparison with the reference drugs GMFX, MXFX and GTFX using
CPE assay against a mammalian MDCK cell line. As listed in Table 1,
14 target compounds 12e25 show CC50 values ranging from 36.6 to
CDCl3) d: 1.33 (3H, s, CH3), 1.46 (9H, s, Boc-9H), 1.68e1.80 (4H, m),
3.32e3.38 (1H, m), 3.67e3.72 (1H, m), 3.85 (3H, s, OCH3), 4.02 (1H, d,
J ¼ 17.2 Hz), 4.61 (1H, d, J ¼ 17.2 Hz). ESI-MS m/z: 258 (M þ H)þ.
5.1.2. N-tert-Butoxycarbonyl-4-amino-3-ethoxyimino-4-
methylpiperidine (9b)
The title compound was obtained in a similar manner as for the
preparation of 9a. Yield: 58%, colorless liquid. 1H-NMR (400 MHz,
CDCl3)
d
: 1.22 (3H, t, OCH2CH3, J ¼ 7.2 Hz),1.31 (3H, s, CH3),1.45 (9H, s,
Boc-9H), 1.64e1.78 (4H, m), 3.31e3.38 (1H, m), 3.62e3.66 (1H, m),
Fig. 2. X-ray structure of compound 8a.
4.01e4.09(3H, m), 4.61 (1H, d, J ¼ 17.2 Hz). ESI-MS m/z: 272 (M þ H)þ.

2424
J.-X. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 2421e2426
Table 2
In vitro antibacterial activity of compounds 12e25.
MIC (
m
g/mL)
13
Compd.
12
14
15
16
17
18
19
20
21
22
23
24
25
GMFX
MXFX
GTFX
LVFX
S.a.1a
S.a.2
S.a.3
S.e.1
S.e.2
S.p.
E.fa.1
E.fa.2
E.fm.1
E.fm.2
E.c.1
E.c.2
K.p.1
K.p.2
P.a.1
P.a.2
0.25
0.25
0.25
0.25
0.25
4
16
16
64
16
8
16
16
2
4
16
0.125
0.25
0.25
0.25
0.25
0.25
>128
>128
>128
>128
128
0.25
0.25
0.25
0.25
0.25
1
0.25
0.25
2
0.25
0.25
0.25
8
32
64
>128
16
32
0.5
0.25
0.25
0.25
0.25
0.25
0.25
4
>128
>128
>128
>128
>128
>128
>128
4
0.125
0.25
0.25
0.25
0.25
2
128
32
32
128
32
32
16
32
0.5
0.5
0.5
0.5
0.5
4
0.25
0.25
0.25
0.25
0.25
0.25
8
>128
>128
>128
128
>128
2
0.125
0.25
0.25
0.25
0.25
0.25
8
0.125
0.25
0.25
0.25
0.25
0.5
0.25
0.25
0.25
0.25
0.25
0.25
1
128
64
128
128
>128
1
0.25
0.25
0.25
0.25
0.25
0.25
16
>128
>128
>128
128
>128
2
0.25
0.25
0.25
0.25
0.25
2
32
64
32
>128
32
0.125
0.06
0.25
0.25
0.125
0.25
16
16
2
16
4
0.125
0.125
0.25
0.125
0.25
0.125
1
2
2
2
2
2
1
1
2
0.25
0.06
0.06
0.25
0.25
0.25
0.25
8
4
2
4
4
8
0.25
2
1
16
0.25
0.125
0.125
4
0.25
2
8
8
4
16
2
0.25
0.25
0.25
0.25
0.25
32
64
64
4
1
1
64
>128
>128
>128
128
>128
4
>128
8
4
32
>128
>128
16
>128
64
64
4
0.25
>128
>128
128
>128
32
64
4
4
128
128
16
128
16
8
4
16
>128
>128
>128
>128
>128
>128
16
>128
>128
>128
8
64
64
2
4
16
8
8
2
16
16
16
4
32
32
4
0.5
>128
4
2
>128
4
0.5
>128
8
128
2
0.5
2
64
8
64
0.25
16
a
Abbreviations: S.a.1, Staphylococcus aureus ATCC25923; S.a.2, methicillin-sensitive Staphylococcus aureus 08-1; S.a.3, methicillin-resistant Staphylococcus aureus 08-1;
S.e.1, methicillin-sensitive Staphylococcus epidermidis 09-6; S.e.2, methicillin-resistant Staphylococcus epidermidis 09-2; S.p., Streptococcus pneumoniae 08-4; E.fa.1, Entero-
coccus faecalis 08-10; E.fa.2, Enterococcus faecalis 08-12; E.fm.1, Enterococcus faecium 08-2; E.fm.2, Enterococcus faecium 06-7; E.c.1, Escherichia coli ATCC25922; E.c.2,
Escherichia coli 08-21; K.p.1, Klebsiella pneumoniae 09-22; K.p.2, Klebsiella pneumoniae 09-23; P.a.1, Pseudomonas aeruginosa ATCC27853; P.a.2, Pseudomonas aeruginosa 09-32.
5.1.3. 4-Amino-3-methoxyimino-4-methylpiperidine
dihydrochloride (10a)
(1C, s, C]N),165.66(1C, s, COOH),176.40 (1C, s, C]O). HRMS-ESI m/z:
Calcd for C19H23FN5O4: 404.17341. Found: 404.17228 (M þ H)þ.
To a stirring solution of 9a (2.6 g, 10.0 mmol) dissolved in
methylene chloride (50 mL) was pumped dry hydrogen chloride gas
at room temperature for 0.5 h. The reaction mixture was allowed to
stir for another 0.5 h at the same temperature. The resulting solid
was collected by suction, and dried in vacuo to give the title
compound 10a (2.3 g). Yield: 98%, white solid, mp: 178e180 ꢀC.
5.1.5.2. 1-Cyclopropyl-6-fluoro-7-(4-amino-3-ethoxyimino-4-meth-
ylpiperidin-1-yl)-1,4-dihydro-4-oxo-1,8- naphthyridine-3-carboxylic
acid (13). Yield: 80%, off-white solid, mp: 209e211 ꢀC. 1H-NMR
(400 MHz, CDCl3) d: 1.05e1.10 (2H, m, cyclopropyl CH2), 1.23e1.32
(5H, m, cyclopropyl CH2, OCH2CH3),1.44 (3H, s, CH3),1.88e2.03 (2H,
m), 3.61e3.66 (1H, m), 3.86e3.92 (1H, m), 4.09e4.22 (3H, m), 4.63
(1H, d, J ¼ 17.2 Hz), 5.07 (1H, d, J ¼ 17.2 Hz), 8.08 (1H, d, C5-H,
1H-NMR (400 MHz, DMSO-d6)
d: 1.54 (3H, s, CH3), 2.18e2.21 (2H, m),
3.23e3.25 (1H, m), 3.39e3.42 (1H, m), 3.87e3.96 (4H, m), 4.34e4.38
(1H, m), 8.80 (3H, s, NH3þ), 9.70 (2H, s, NH2þ). ESI-MS m/z: 158
(M þ H)þ.
J ¼ 13.2 Hz), 8.74 (1H, s, C2-H). 13C-NMR (400 MHz, DMSO-d6)
d:
6.83 (2C, s, cyclopropyl CH2), 14.34 (1C, s, NOCH2CH3), 27.73 (1C, s),
34.92 (1C, s), 38.43 (1C, s), 40.40 (1C, s), 42.77 (1C, s), 51.25 (1C, s),
68.71 (1C, s, NOCH2), 107.60 (1C, s), 112.16 (1C, s), 119.32 (1C, d,
J ¼ 88.4 Hz), 146.57 (1C, s), 146.74 (1C, d, J ¼ 1030.8 Hz, C-6), 147.21
(1C, s), 149.37 (1C, d, J ¼ 34.4 Hz), 158.15 (1C, s, C]N), 165.69 (1C, s,
COOH), 176.40 (1C, s, C]O). HRMS-ESI m/z: Calcd for C20H25FN5O4:
418.18906. Found: 418.19072 (M þ H)þ.
5.1.4. 4-Amino-3-ethoxyimino-4-methylpiperidine dihydrochloride
(10b)
The title compound was obtained in a similar manner as for
the preparation of 10a. Yield: 95%, white solid, mp: 152e154 ꢀC.
1H-NMR (400 MHz, DMSO-d6)
1.54 (3H, s, CH3), 2.14e2.24 (2H, m), 3.21e3.23 (1H, m), 3.29e3.42
d
: 1.24 (3H, t, OCH2CH3, J ¼ 7.2 Hz),
(1H, m), 3.89 (1H, d, J ¼ 17.2 Hz), 4.15 (2H, q, OCH2CH3, J ¼ 7.2 Hz),
5.1.5.3. 1-(2,4-Difluorophenyl)-6-fluoro-7-(4-amino-3-methoxyimino-
4-methylpiperidin-1-yl)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-
carboxylic acid (14). Yield: 60%, light yellow solid, mp: 168e171 ꢀC.
þ
4.36 (1H, d, J ¼ 17.2 Hz), 8.80 (3H, s, NH3þ), 9.68 (2H, d, NH2
J ¼ 46.0 Hz). ESI-MS m/z: 172 (M þ H)þ.
,
1H-NMR (400 MHz, CDCl3)
d: 1.35 (3H, s, CH3), 1.43e1.74 (2H, m),
5.1.5. General procedure for the synthesis of 12e21
3.64e3.64 (1H, m), 3.82 (3H, s, OCH3), 3.82e3.93 (1H, m), 4.16e4.26
(1H, m), 4.59e4.69 (1H, m), 7.07e7.09 (2H, m, ph-2H), 7.36e7.39 (1H,
m, ph-H), 8.12 (1H, d, C5-H, J ¼ 13.2 Hz), 8.68 (1H, s, C2-H). HRMS-ESI
m/z: Calcd for C22H21F3N5O4: 476.15456. Found: 476.15426 (M þ H)þ.
A mixture of 11a-f (1.0 mmol),10a,b (1.2 mmol), dry triethylamine
(8.0 mmol) and dry acetonitrile (20 mL) was stirred at room temper-
ature under an atmosphere of nitrogen for 3e8 h. The resulting solid
was collected by suction, and dried in vacuo to give 12e21.
5.1.5.4. 1-(2,4-Difluorophenyl)-6-fluoro-7-(4-amino-3-ethoxyimino-4-
methylpiperidin-1-yl)-1,4-dihydro-4-oxo-1,8- naphthyridine-3-carboxy-
lic acid (15). Yield: 60%, light yellow solid, mp: 169e171 ꢀC. 1H-NMR
5.1.5.1. 1-Cyclopropyl-6-fluoro-7-(4-amino-3-methoxyimino-4-meth-
ylpiperidin-1-yl)-1,4-dihydro-4-oxo-1,8- naphthyridine-3-carboxylic
acid (12). Yield: 67%, off-white solid, mp: 168e170 ꢀC. 1H-NMR
(400 MHz, CDCl3) d: 1.20e1.27 (3H, m, OCH2CH3), 1.41 (3H, s, CH3),
(400 MHz, CDCl3)
d
: 1.05e1.10 (2H, m, cyclopropyl CH2), 1.26e1.31
1.90 (2H, brs), 3.61e3.63 (1H, m), 3.90e3.97 (1H, m), 4.05e4.10 (2H,
m), 4.15e4.23 (1H, m), 4.65e4.72 (1H, m), 7.05e7.39 (3H, m, ph-3H),
8.13 (1H, d, C5-H, J ¼ 12.8 Hz), 8.68 (1H, s, C2-H). HRMS-ESI m/z: Calcd
for C23H23F3N5O4: 490.17021. Found: 490.16883 (M þ H)þ.
(2H, m, cyclopropyl CH2), 1.50 (3H, s, CH3), 2.04e2.05 (2H, m),
3.62e3.65 (1H, m), 3.84e3.92 (4H, m), 4.19e4.23 (1H, m), 4.57 (1H, d,
J ¼ 16.8 Hz), 5.12 (1H, d, J ¼ 16.8 Hz), 8.08 (1H, d, C5-H, J ¼ 13.2 Hz),
8.72 (1H, s, C2-H). 13C-NMR (400 MHz, DMSO-d6)
d: 6.85 (2C, s,
cyclopropyl CH2), 26.94 (1C, s), 34.94 (1C, s), 37.90 (1C, s), 40.32 (1C, s),
42.74 (1C, s), 51.67 (1C, s), 61.41 (1C, s, NOCH3), 107.62 (1C, s), 112.30
(1C, s), 119.44 (1C, d, J ¼ 88.0 Hz), 146.53 (1C, s), 146.72 (1C, d,
J ¼ 1032.4 Hz, C-6), 147.22 (1C, s), 149.33 (1C, d, J ¼ 34.4 Hz), 157.70
5.1.5.5. 1-Ethyl-6,8-fluoro-7-(4-amino-3-methoxyimino-4-methylpiper-
idin-1-yl)-1,4-dihydro-4-oxo-quinoline-3- carboxylic acid (16). Yield:
65%, off-white solid, mp: 199e201 ꢀC.1H-NMR (500 MHz, DMSO-d6)
d:
1.29e1.59 (8H, m), 2.01e2.12 (2H, m), 3.54e3.85 (2H, m), 3.87e4.11

J.-X. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 2421e2426
2425
(5H, m), 7.90(1H, d, C5-H, J ¼ 11.5 Hz), 8.94 (1H, s, C2-H). HRMS-ESI m/z:
Calcd for C19H23F2N4O4: 409.16874. Found: 409.17062 (M þ H)þ.
CH2), 1.61 (3H, s, CH3), 2.10e2.21 (2H, m), 3.54 (2H, brs), 3.75 (3H, s,
OCH3), 3.87 (3H, s, NOCH3), 3.94 (1H, d, J ¼ 15.2 Hz), 4.15e4.19 (1H,
m), 4.65 (1H, d, J ¼ 15.2 Hz), 7.76 (1H, d, C5-H, J ¼ 12.0 Hz), 8.69 (1H,
s, C2-H), 14.85 (1H, brs, COOH). HRMS-ESI m/z: Calcd for
C21H26FN4O5: 433.18872. Found: 433.18958 (M þ H)þ.
5.1.5.6. 1-Ethyl-6,8-difluoro-7-(4-amino-3-ethoxyimino-4-methylpiper-
idin-1-yl)-1,4-dihydro-4-oxo-quinoline-3- carboxylic acid (17). Yield:
1
55%, off-white solid, mp: 182e184 ꢀC. H-NMR (400 MHz, CDCl3)
d:
1.25 (3H, t, OCH2CH3, J ¼ 6.8 Hz),1.43 (3H, s, CH3),1.59 (3H, t, NCH2CH3,
J ¼ 7.2 Hz), 1.91e1.99 (2H, m), 3.46e3.56 (2H, m), 4.08e4.14 (3H, m),
4.47e4.51 (3H, m), 7.99 (1H, d, C5-H, J ¼ 10.8 Hz), 8.62 (1H, s, C2-H).
HRMS-ESI m/z: Calcd for C20H25F2N4O4: 423.18439. Found: 423.18460
(M þ H)þ.
5.1.6.2. 1-Cyclopropyl-6-fluoro-7-(4-amino-3-ethoxyimino-4-methyl-
piperidin-1-yl)-8-methoxyl-1,4-dihydro-4-oxo- quinoline-3-carboxylic
acid (23). Yield: 51%, light yellow solid, mp: 79e81 ꢀC. 1H-NMR
(400 MHz, CDCl3)
d:
1.01e1.25 (7H, m, 2ꢂ cyclopropyl CH2,
OCH2CH3), 1.44 (3H, s, CH3), 1.88e1.97 (2H, m), 3.48e3.59 (2H, m),
3.76 (3H, s, OCH3), 4.02e4.13 (4H, m), 4.53 (1H, d, J ¼ 15.2 Hz), 7.89
(1H, d, C5eH, J ¼ 12.0 Hz), 8.82 (1H, s, C2-H). HRMS-ESI m/z: Calcd for
C22H28FN4O5: 447.20437. Found: 447.20451 (M þ H)þ.
5.1.5.7. 1-(2-Fluoroethyl)-6,8-difluoro-7-(4-amino-3-methoxyimino-
4-methylpiperidin-1-yl)-1,4-dihydro-4-oxo-quinoline-3-carboxylic
acid (18). Yield: 64%, light yellow solid, mp: 205e207 ꢀC. 1H-NMR
(500 MHz, DMSO-d6)
d
: 1.10e1.41 (7H, m), 1.79e1.90 (2H, m),
5.1.6.3. 9-Fluoro-3(S)-methyl-10-(4-amino-3-methoxyimino-4-meth-
ylpiperidin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzox-
azine-6-carboxylic acid (24). Yield: 47%, light yellow solid, mp:
0
3.55e3.81 (5H, m), 4.14e4.35 (2H, m), 7.89 (1H, d, C5-H, J ¼ 10.5 Hz),
8.87 (1H, s, C2-H). HRMS-ESI m/z: Calcd for C19H22F3N4O4: 427.15931.
Found: 427.15679 (M þ H)þ.
166e168 ꢀC. 1H-NMR (400 MHz, CDCl3)
d: 1.45 (3H, s, C4 -CH3), 1.62
(3H, d, C3-CH3, J ¼ 6.8 Hz), 1.93e1.96 (2H, m), 3.40e3.46 (2H, m), 3.85
(3H, s, OCH3), 4.01e4.08 (1H, m), 4.38e4.51 (4H, m), 7.73 (1H, d, C8-H,
J ¼ 12.0 Hz), 8.63 (1H, s, C5-H). HRMS-ESI m/z: Calcd for C20H24FN4O5:
419.17307. Found: 419.17502 (M þ H)þ.
5.1.5.8. 1-Cyclopropyl-6,8-difluoro-7-(4-amino-3-ethoxyimino-4-met-
hylpiperidin-1-yl)-1,4-dihydro-4-oxo-quinoline-3-carboxylic acid (19).
Yield: 76%, off-white solid, mp: 164e166 ꢀC. 1H-NMR (400 MHz,
CDCl3) d: 1.18e1.20 (2H, m, cyclopropyl CH2), 1.25 (3H, t, OCH2CH3,
J ¼ 6.8 Hz), 1.28e1.33 (2H, m, cyclopropyl CH2), 1.48 (3H, s, CH3), 1.99
(2H, brs), 3.47e3.58 (2H, m), 3.99e4.01 (1H, m), 4.07e4.14 (3H, m),
4.53 (1H, d, J ¼ 15.6 Hz), 7.94 (1H, d, C5-H, J ¼ 11.2 Hz), 8.80 (1H, s,
C2-H). HRMS-ESI m/z: Calcd for C21H25F2N4O4: 435.18439. Found:
435.18382 (M þ H)þ.
5.1.6.4. 9-Fluoro-3(S)-methyl-10-(4-amino-3-ethoxyimino-4-meth-
ylpiperidin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzox-
azine-6-carboxylic acid (25). Yield: 46%, light yellow solid, mp:
122e124 ꢀC. 1H-NMR (400 MHz, CDCl3)
d: 1.24 (3H, t, OCH2CH3),
0
1.41 (3H, s, C4 -CH3), 1.62 (3H, d, C3-CH3, J ¼ 4.4 Hz), 1.90 (2H, t,
J ¼ 5.6 Hz), 3.42e3.48 (2H, m), 4.03e4.12 (3H, m), 4.38e4.49 (4H,
m), 7.74 (1H, d, C8-H, J ¼ 11.6 Hz), 8.62 (1H, s, C5-H). HRMS-ESI m/z:
Calcd for C21H26FN4O5: 433.18872. Found: 433.18662 (M þ H)þ.
5.1.5.9. 1-Cyclopropyl-6-fluoro-7-(4-amino-3-methoxyimino-4-
methylpiperidin-1-yl)-8-difluoromethoxyl-1,4- dihydro-4-oxo-quino-
line-3-carboxylic acid (20). Yield: 54%, off-white solid, mp:
164e166 ꢀC. 1H-NMR (400 MHz, CDCl3)
d
: 0.98e1.28 (4H, m, 2ꢂ
cyclopropyl CH2), 1.84 (3H, s, CH3), 2.42e2.57 (2H, m), 3.54e3.64
(3H, m), 3.94 (3H, s, OCH3), 4.10 (1H, s), 4.71 (1H, d, J ¼ 15.2 Hz),
6.56 (1H, OCHF2, t, J ¼ 68.4 Hz), 8.05 (1H, d, C5eH, J ¼ 11.6 Hz), 8.86
(1H, s, C2eH), 14.27 (1H, brs, COOH). HRMS-ESI m/z: Calcd for
C21H24F3N4O5: 469.16988. Found: 469.17033 (M þ H)þ.
5.2. Antibacterial activity
Compounds 12e25 were evaluated for their in vitro antibacterial
activity using standard techniques in comparison to the reference
drugs GMFX, MXFX, GTFX and LVFX. Drugs (10.0 mg) were dissolved
in 0.1 N sodium hydroxide solution and water (10 mL). Further
progressive twofold serial dilutionwith melted Mueller-Hinton agar
was performed to obtain the required concentrations of 128, 64, 32,
5.1.5.10. 1-Cyclopropyl-6-fluoro-7-(4-amino-3-ethoxyimino-4-meth-
ylpiperidin-1-yl)-8-difluoromethoxyl-1,4-dihydro-4-oxo-quinoline-3-
carboxylic acid (21). Yield: 64%, off-white solid, mp: 235e238 ꢀC.
16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, and 0.03 mg/mL. Petri dishes were
1H-NMR (400 MHz, CDCl3)
d
: 0.99e1.28 (7H, m, 2ꢂ cyclopropyl
incubated with 104 colony-forming units (cfu) and incubated at
35 ꢀC for 18 h. MIC was the lowest concentration of the test
compound, which resulted in no visible growth on the plate.
CH2, OCH2CH3), 1.69 (3H, s, CH3), 2.27 (2H, brs), 3.53e3.64 (2H, m),
3.96 (1H, d, J ¼ 15.2 Hz), 4.11e4.19 (3H, m), 4.67 (1H, d, J ¼ 15.2 Hz),
6.52 (1H, t, OCHF2, J ¼ 74.8 Hz), 8.01 (1H, d, C5-H, J ¼ 11.6 Hz), 8.84
(1H, s, C2-H). HRMS-ESI m/z: Calcd for C22H26F3N4O5: 483.18553.
Found: 483.18588 (M þ H)þ.
5.3. Cytotoxicity
5.1.6. General procedure for the synthesis of 22e25
Compounds 12e25 were examined for their toxicity (CC50) in
A mixture of 11g,h (1.0 mmol), 10a,b (1.2 mmol), dry triethyl-
amine (8.0 mmol) and dry acetonitrile (20 mL) was stirred for
6e10 h at room temperature under an atmosphere of nitrogen.
After completion of the condensation, the reaction mixture was
concentrated under reduced pressure. The residue was dissolved in
2% sodium hydroxide solution (20 mL) and stirred for 1e4 h at
room temperature. The reaction mixture was adjusted to pH 7 with
6 N hydrochloric acid. The resulting solid was collected by suction,
and dried in vacuo to give 22e25.
a mammalian MDCK cell line till 3.9 mg/mL concentrations. The
MDCK cells were maintained in culture medium (Minimum Essen-
tial Medium with Earle’s salt, supplemented with 10% fetal bovine
serum) at 37 ꢀC under 5% CO2. Cells were seeded in 96-well plate at
the plating density of 2.5ꢂ104 cells per well and allowed to recover
for 24 h. Culture medium was replaced by assay medium containing
the compound to be tested or drug-free. After 48 h of exposure, cells
wereharvested and cell viability was assessed byCPE assay. The CC50
values were calculated by ReedeMuench analyses.
5.1.6.1. 1-Cyclopropyl-6-fluoro-7-(4-amino-3-methoxyimino-4-
methylpiperidin-1-yl)-8-methoxyl-1,4-dihydro-4-oxo-quinoline-3-
carboxylic acid (22). Yield: 69%, light yellow solid, mp: 229e231 ꢀC.
Acknowledgements
1H-NMR (400 MHz, DMSO-d6)
d
: 1.02e1.13 (4H, m, 2ꢂ cyclopropyl
This work was supported by the IMB Research Foundation.

2426
J.-X. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 2421e2426
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