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

Article abstract of DOI:10.1016/j.ejmech.2009.04.041

A series of novel 7-(4-alkoxyimino-3-amino-3-methylpiperidin-1-yl)fluoroquinolone derivatives were designed, synthesized and characterized by ¹H NMR, MS and HRMS. These fluoroquinolones were evaluated for in vitro antibacterial activity against

Full text of DOI:10.1016/j.ejmech.2009.04.041

European Journal of Medicinal Chemistry 44 (2009) 4063–4069
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro antibacterial activity of 7-(4-alkoxyimino-3-amino-3-
methylpiperidin-1-yl)fluoroquinolone derivatives
1
1
*
Yun Chai , Zhi-Long Wan , Bo Wang, Hui-Yuan Guo, Ming-Liang Liu
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, 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-(4-alkoxyimino-3-amino-3-methylpiperidin-1-yl)fluoroquinolone derivatives were
designed, synthesized and characterized by 1H NMR, MS and HRMS. These fluoroquinolones were
evaluated for in vitro antibacterial activity against representative Gram-positive and Gram-negative
strains. All of the title compounds have considerable activity against the twelve strains, and exhibit
exceptional potency in inhibiting the growth of Staphylococcus aureus, Staphylococcus epidermidis and
Received 17 December 2008
Received in revised form
16 April 2009
Accepted 29 April 2009
Available online 8 May 2009
Klebsiella pneumoniae (minimum inhibitory concentration (MIC): 0.06–8 mg/mL). The most active
compound 17 is 4-fold more potent than levofloxacin against S. aureus and S. epidermidis, 32-fold more
potent than levofloxacin against Streptococcus pneumoniae, and 16-fold more potent than IMB against
K. pneumoniae.
Keywords:
Fluoroquinolone
Synthesis
Ó 2009 Elsevier Masson SAS. All rights reserved.
Antibacterial activity
1. Introduction
position is the most adaptable site for chemical change and is an
area that greatly influences potency, spectrum and safety [7–9]. In
Quinolone antibacterial agents are among the most attractive
drugs in the anti-infective chemotherapy field. These antibiotics
exert their antimicrobial activity by binding to two type II bacterial
topoisomerase enzymes, DNA gyrase (subunits encoded by gyrA
and gyrB) and topoisomerase IV (subunits encoded by grlA and grlB
for Staphylococcus aureus). This binding induces permanent double-
stranded DNA breaks, and results in cell death [1,2].
Although most of the quinolones currently on the market or
under development are generally characterized by a broad anti-
microbial spectrum, their activity against clinically important
Gram-positive pathogens (including S. aureus, Streptococcus pyo-
genes, Streptococcus pneumoniae and Enterococcus) is relatively
moderate, which has not only limited their use in infections caused
by these organisms, but has also contributed to rapidly developing
quinolone resistance. Thus, recent efforts have been directed
toward the synthesis of new quinolones that can provide improved
Gram-positive antibacterial activity, while retaining the good
Gram-negative activity of early fluoroquinolones, such as cipro-
floxacin and ofloxacin [3–6].
general, 5- and 6-membered nitrogen heterocycles including
piperazinyl, pyrrolidinyl and piperidinyl type side chains have
proven to be optimal substituents. Of the three, piperidinyl analogs
are the least studied [9,10].
As part of an ongoing program to find potent new quinolones
displaying strong Gram-positive activity, we have focused our
attention on introducing new functional groups to the piperidine
ring. Previous work on pyrrolidine analogs suggests that intro-
duction of a methyl group in the 3-position of the pyrrolidine ring
can increase Gram-positive activity [11]. For example, an analog of
gemifloxacin (GMFX), DW 286 (Fig. 1) possessing an additional
methyl group at the 3-position of the pyrrolidine ring displays far
more potent antibacterial activity than GMFX against important
Gram-positive organisms, methicillin-resistant S. aureus (MRSA)
and ofloxacin resistant organisms, while maintaining an excellent
pharmacokinetic profile [12]. We applied this structural modifica-
tion to IMB (Fig. 1), which shows good in vitro and in vivo anti-
bacterial activity against Gram-negative and Gram-positive
organisms, including MRSA and methicillin-resistant S.taphylo-
Structure–activity relationship (SAR) studies of quinolone anti-
bacterial agents have demonstrated that the basic group at the C-7
coccus epidermidis (MRSE) [13]. A series of fluoroquinolone
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 3-position
and an alkoxyimino group at 4-position of the piperidine ring. Our
primary objective was to optimize the potency of these compounds
against Gram-positive and Gram-negative organisms.
* Corresponding author. Tel.: þ86 010 63036965; fax: þ86 010 63047865.
E-mail address: lmllyx@yahoo.com.cn (M.-L. Liu).
1
These authors contributed equally to the work.
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.041

4064
Y. Chai et al. / European Journal of Medicinal Chemistry 44 (2009) 4063–4069
O
N
O
N
O
O
N
COOH
COOH
F
F
F
COOH
F
COOH
N
N
N
N
H3CON
H3CON
H3C
H2N
N
N
OCH3
N
H3CN
O
H3CON
NH2
H2N
IMB
Gemifloxacin
DW 286
Levofloxacin
Fig. 1.
2. Results and discussion
9a,b with 10a–f was performed in the presence of triethylamine.
However for 24–27, boric chelates 11g–h were required to increase
reactivity. Table 1 shows the novel fluoroquinolone analogs, the
yield of the final coupling step, and melting points of the purified
compounds.
2.1. Chemistry
Detailed synthetic pathways to new piperidine derivatives 9a,b
and novel fluoroquinolones 12–27 are depicted in Schemes 1 and 2,
respectively. According to published procedures [14–17], N-tert-
butoxycarbonyl-4-piperidone 1 reacted with dimethyl carbonate in
the presence of sodium hydride in dry toluene to give the keto ester
2, which was methylated with methyl iodide to provide methyl
ketone 3. Ketone 3 was smoothly converted to the oximes 4a,b by
reaction with O-alkylhydroxyamines in ethanol at 55–60 ꢀC.
However, direct ammonolysis of the ester moiety in oximes 4a,b
was unsuccessful.
Instead, the esters 4a,b were first converted to acids 5a,b by
saponification and acidification. The acids 5a,b were subsequently
reacted with isobutyl chloroformate to give activated esters 6a,b,
which upon ammonolysis afforded amides 7a,b, in an overall yield
of 84% for the three steps.
2.2. Pharmacology
Fluoroquinolones 12–27 were evaluated for their in vitro anti-
bacterial activity against representative Gram-positive and Gram-
negative strains using standard techniques [19]. Minimum inhibi-
tory concentration (MIC) is defined as the concentration of the
compound required to give complete inhibition of bacterial growth
and MICs of the synthesized compounds along with the standard
drugs – IMB, levofloxacin (LVFX) and GMFX for comparison are
reported in Table 2.
The novel fluoroquinolones 12–27 have generally potent anti-
bacterial activity against the twelve strains. They exhibit good
potency in inhibiting the growth of S. aureus, S. epidermidis and
Hoffmann degradation of amides 7a,b used freshly prepared
sodium hypobromite instead of commercial sodium hypochlorite.
The N-tert-butoxycarbonyl protecting group on amines 8a,b was
removed with hydrogen chloride gas in methylene chloride to
afford the new piperidine derivative dihydrochlorides 9a,b in good
yield.
The coupling of the piperidine derivatives 9a,b with various
compounds containing quinolone and naphthyridine cores was
carried out according to well-established literature procedures
(Scheme 2) [18]. In the case of quinolones 12–23, condensation of
Klebsiella pneumoniae (MIC: 0.06–8 mg/mL). The most active
compound 17 is 4-fold more potent than LVFX against S. aureus and
S. epidermidis, 32-fold more potent than LVFX against S. pneumo-
niae, and 16-fold more potent than IMB against K. pneumoniae. The
compounds 16 and 26 are 4-fold more potent than LVFX against
Escherichia coli, 32-fold more potent than IMB and 4-fold more
potent than LVFX against K. pneumoniae.
Some compounds were further examined for toxicity (CC50
)
in a mammalian Vero cell line till 31.25 g/mL concentration.
m
After 72 h of exposure, viability was assessed on the basis of
O
O
O
COOCH3
R1ONH2.HCl
CH3
COOCH3
(CH3O)2CO
NaH
CH3I
Et3N
K2CO3
N
Boc
N
Boc
1
N
Boc
2
3
NOR1
NOR1
NOR1
1)NaOH
COOH
CH3
COOCH3
CH3
COOCOOBu-i
CH3
ClCOOBu-i
2)HOAc
N
Boc
N
Boc
4a,b
N
Boc
6a,b
5a,b
NOR1
NOR1
NOR1
NH2
CH3
CONH2
CH3
NH2
CH3
NH3
NaBrO
HCl
a: R1=CH3
N
Boc
b: R1=CH2CH3
N
Boc
N
H
.2HCl
7a,b
8a,b
9a,b
Scheme 1. Synthesis of piperidine derivatives 9a,b.

Y. Chai et al. / European Journal of Medicinal Chemistry 44 (2009) 4063–4069
4065
O
O
F
COOH
F
COOH
9a,b
CH3
H2N
(Cl)F
X
N
N
X
N
R2
R2
R1ON
10a-10f
12-23
10a: X:N, R2=cyclopropyl
10b: X:N, R2=2,4-difluorophenyl
10c: X:CF, R2=cyclopropyl
10d: X:difluoromethoxyl, R2=cyclopropyl
10e: X:CF, R2=ethyl
10f: X:CF, R2=2-fluoroethyl
H3COCO
O
OCOCH3
O
B
O
1) 9a,b
F
COOH
F
F
O
CH3
2) OH-
H2N
N
X
N
X
N
3) H3O+
R2
R1ON
R2
24-27
11g-11h
11g: X:methoxyl, R2=cyclopropyl
11h: X, R2=
Ο
Scheme 2. Synthesis of novel fluoroquinolones 12–27.
cellular conversion of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) into a formazan product and the
results are reported in Table 1. Fifteen compounds when tested
4. Experimental protocols
4.1. Chemistry
showed CC50 values ranging from 31.4 to 791.2 mM. A compar-
ison of the substitution pattern at C-7 position demonstrated
that ethyloxime-incorporated piperidino-substitutions were
generally more cytotoxic than the analogs containing
methyloxime.
Melting points were determined in open capillaries and are
uncorrected. 1H NMR spectra were determined on a Varian
Mercury-400 spectrometer in DMSO-d6 or CDCl3 using tetrame-
thylsilane as an internal standard. Electrospray ionization (ESI)
mass spectra and high resolution mass spectra (HRMS) were
obtained on a MDSSCIEX Q-Tap mass spectrometer. Fast Atom
Bombardment (FAB) mass spectra and high resolution mass spectra
(HRMS) were obtained on a MICROMASS AutoSpec Ultima-TOF
mass spectrometer. The reagents were all of analytical grade or
chemically pure. TLC was performed on silica gel plates (Merck,
ART5554 60F254).
3. Conclusion and discussion
In summary, a series of novel 7-(4-alkoxyimino-3-amino-3-
methylpiperidin-1-yl) fluoroquinolone derivatives were designed,
synthesized and evaluated for their in vitro antibacterial activity
against representative Gram-positive and Gram-negative strains.
Generally, all of the novel fluoroquinolones demonstrated potent
antibacterial activity. In particular, they exhibited good potency in
inhibiting the growth of S. aureus, S. epidermidis and K. pneumoniae.
However, overall the piperidinyl analogs were not superior to
pyrrolidinyl analogs such as IMB and GMFX.
Novel fluoroquinolones 12–27 exhibited less activity than IMB
against the tested Gram-positive, including resistant strains and
Gram-negative strains, except K. pneumoniae. These results showed
that introduction of another methyl group into 3-position of
piperidine ring caused reduced antibacterial activity, which was
contrary to the activity profiles of pyrrolidine-containing
fluoroquinolones.
The activities of the quinolone nuclei in this study are in the
order 1-cyclopropyl-8-fluoroquinolone > levofloxacin nuclei > 1-
cyclopropyl-8-methoxylquinolone > 1-cyclopropyl-8-difluoromethoxyl
quinolone > 1-ethyl-8-fluoroquinolone z 1-(2-fluoroethyl)-8-flu-
oroquinolone z naphthyridine. In addition, fluoroquinolones
featuring methyloxime-incorporated piperidino-substitution at
C-7 position are comparable to analogs containing ethyloxime.
4.2. Synthesis
4.2.1. Methyl N-tert-butoxycarbonyl-4-oxopiperidine-3-
carboxylate 2
To a suspension of 70% sodium hydride (30.20 g, 0.88 mol) in dry
toluene (500 mL) was added dropwise dimethyl carbonate (43.20 g,
0.48 mol) over 0.5 h at room temperature under an atmosphere of
nitrogen. After addition of a few drops of methanol, a solution of
N-tert-butoxycarbonyl-4-piperidone
1 (48.00 g, 0.24 mol) dis-
solved in dry toluene (200 mL) was added dropwise to the reaction
mixture while stirring at 80 ꢀC over 35 min. The reaction mixture
was stirred for 3 h at the same temperature and then cooled to 0 ꢀC
(ice bath), adjusted to pH 6–6.5 with acetic acid. The resulting
mixture was diluted with cold water (100 mL) and adjusted to pH 8
with 5% sodium hydroxide solution. The toluene layer was sepa-
rated and the aqueous layer was extracted with toluene
(3 ꢁ 20 mL). The combined toluenes were dried over anhydrous
sodium sulfate, and concentrated under reduced pressure. The solid
obtained was dried in vacuo to give the title compound 2 (54.50 g,

4066
Y. Chai et al. / European Journal of Medicinal Chemistry 44 (2009) 4063–4069
88.0%) as a off-white solid, mp: 32–34 ꢀC. 1H NMR (CDCl3,
400 MHz) dH 1.47 (9H, s, Boc-9H), 2.35–2.38 (2H, t, J 6.0,C5–2H),
3.54–3.57 (2H, t, J 6.0, C6–2H), 3.76 (3H, s, OCH3), 4.05 (2H, s, C2–
2H), 11.97 (1H, s, C3-H). ESI-MS: m/z 258 (M þ H)þ.
Table 1
The structures, physical data and cytotoxicity of novel fluoroquinolones 12–27.
Compd.
R1
X
N
R2
Yield (%)
68
mp (ꢀC)
CC50 (mM)
12
CH3
231–232
169.7
92.8
4.2.2. Methyl N-tert-butoxycarbonyl-3-methyl-4-oxopiperidine-3-
carboxylate 3
13
CH2CH3
N
70
187–188
To a suspension of methyl N-tert-butoxycarbonyl-4-oxopiper-
idine-3-carboxylate 2 (53.68 g, 0.21 mol) and anhydrous potassium
carbonate (100.93 g, 0.73 mol) in dry acetone (500 mL) was added
a solution of methyl iodide (23.38 mL, 0.38 mol) dissolved in dry
acetone (250 mL) at room temperature under an atmosphere of
nitrogen. The reaction mixture was heated to 40 ꢀC and stirred for
6 h, cooled to room temperature and filtered. The filtrate was
concentrated under reduced pressure. The residue was diluted with
methylene chloride (300 mL) and washed with water then satu-
rated saline, dried over anhydrous sodium sulfate, filtered and
concentrated. The yellow oily residue was treated with petroleum
ether (300 mL) and filtered. The solid obtained was washed twice
with petroleum ether and dried in vacuo to give the title compound
3 (45.36 g, 80.1%) as a white solid, mp: 42–43 ꢀC. 1H NMR (CDCl3,
400 MHz) dH 1.31 (3H, s, CH3), 1.49 (9H, s, Boc-9H), 2.45–2.51 (1H,
m), 2.76 (1H, s), 3.06–3.10 (1H, m), 3.30–3.37 (1H, m), 3.72 (3H, s,
OCH3), 4.12 (1H, s), 4.48–4.51 (1H, m). ESI-MS: m/z 272 (M þ H)þ.
14
15
CH3
CH2CH3
N
N
2,4-F2-C6H3
2,4-F2-C6H3
66
64
215–216
210–211
258.7
31.4
16
17
18
19
CH3
CF
67
65
59
59
212–213
194–195
216–217
166–169
575.3
146.9
791.2
721.9
CH2CH3
CH3
CF
COCHF2
COCHF2
CH2CH3
20
21
22
23
CH3
CH2CH3
CH3
CF
CF
CF
CF
CH2CH3
CH2CH3
CH2CH2F
CH2CH2F
62
65
55
50
214–215
236–239
216–217
192–194
337.9
219.2
626.1
243.2
CH2CH3
24
25
CH3
COCH3
COCH3
67
70
204–205
155–157
219.0
342.6
4.2.3. Methyl N-tert-butoxycarbonyl-4-methoxyimino-3-
methylpiperidine-3-carboxylate 4a
CH2CH3
To
a stirring solution of methyl N-tert-butoxycarbonyl-3-
methyl-4-oxopiperidine-3-carboxylate 3 (15.00 g, 55.3 mmol) dis-
solved in ethanol (50 mL) was added dropwise a solution of
methoxylamine hydrochloride (5.05 g, 60.5 mmol) and triethyl-
amine (8.37 mL, 60.5 mmol) in 80% ethanol (25 mL) at 55–60 ꢀC
over 15 min. The reaction mixture was stirred at the same
temperature for 1.5 h and concentrated under reduced pressure.
The residue was diluted with distilled water (30 mL) and extracted
with ethyl acetate (3 ꢁ 50 mL). The combined extracts were washed
with 1 N HCl then saturated brine, dried over anhydrous sodium
sulfate and concentrated under reduced pressure. The crude
product was recrystallized from petroleum ether to give the title
compound 4a (14.71 g, 88.6%) as a white solid, mp: 75–76 ꢀC. 1H
O
O
26
27
CH3
55
53
228–230
185–188
NT
CH2CH3
732.2
348.5
IMB
LVFX
–
–
–
–
–
–
–
–
>1000
CC50: The 50% cytotoxic concentration; NT: not tested.
Table 2
In vitro antibacterial activity of compounds 12–27 against selected strains.
Compd.
Strains MIC (
m
g/mL)
S. a.
MRSA
S. e.
MRSE
S. p.
S. py.
E. f.
E. co.
K. p.
P. a.
S. s.
E. cl.
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
4
2
1
1
128
128
>128
128
16
16
64
64
>128
>128
>128
>128
16
8
16
16
1
4
2
2
1
32
64
>128
>128
16
16
64
64
>128
>128
>128
>128
16
8
16
16
1
32
16
8
16
2
1
8
8
16
16
16
16
8
4
2
8
2
64
128
>128
128
2
32
16
8
4
0.5
0.5
2
4
8
16
8
16
2
0.5
1
2
2
0.06
1
4
2
8
8
0.25
0.5
2
4
2
8
4
8
1
0.5
0.25
0.5
0.5
0.06
1
4
4
8
8
0.25
0.5
2
4
2
8
4
4
2
32
32
32
64
4
16
8
8
16
0.25
0.5
4
8
16
16
8
16
2
1
0.5
1
0.125
0.06
1
4
4
16
16
0.5
1
8
16
4
16
8
16
4
2
1
0.125
0.06
0.25
0.5
1
2
1
2
0.25
0.125
0.25
0.5
<0.03
<0.03
0.25
0.125
0.125
1
2
1
2
1
2
0.5
0.25
0.25
0.5
<0.03
<0.03
0.5
4
8
8
32
64
32
64
32
128
16
8
>128
32
32
16
32
8
16
8
32
4
1
0.25
0.5
8
0.5
1
4
8
2
2
IMB
GMFX
LVFX
0.25
0.06
0.125
4
32
2
32
2
32
2
4
0.125
0.5
S. a., Staphylococcus aureus ATCC 29213; MRSA, Methicillin-resistant Staphylococcus aureus 05-3; S. e., Staphylococcus epidermidis ATCC 12228; MRSE, Methicillin-resistant
Staphylococcus epidermidis 05-1; S. p., Streptococcus pneumoniae 97100; S. py., Streptococcus pyogenes 9619; E. f., Enterococcus faecalis ATCC 29212; E. co., Escherichia coli 26; K. p.,
Klebsiella pneumoniae 7; P. a., Pseudomonas aeruginosa 17; S. s., Shigella sonnei 51592; E. cl., Enterobacter cloacae 45301.

Y. Chai et al. / European Journal of Medicinal Chemistry 44 (2009) 4063–4069
4067
NMR (CDCl3, 400 MHz) dH 1.36 (3H, s, CH3), 1.46 (9H, s, Boc-9H),
2.39–3.16 (4H, m), 3.70, 3.86 (6H, s, s, 2ꢁ OCH3), 3.77–3.82 (1H, m),
4.32–4.36 (1H, m). ESI-MS: m/z 301 (M þ H)þ.
4.2.5.1. N-tert-Butoxycarbonyl-3-amino-4-ethoxyimino-3-methyl-
piperidine 8b. The title compound was obtained in a similar
manner as for the preparation of 8a (66.0%). 1H NMR (CDCl3,
400 MHz) dH 1.22–1.26 (3H, t, J 7.2, OCH2CH3), 1.28 (3H, s, CH3), 1.46
(9H, s, Boc-9H),1.79 (2H, br, NH2), 2.28–3.93 (6H, m), 4.05–4.10 (2H,
q, J 7.2, OCH2CH3). ESI-MS: m/z 272 (M þ H)þ.
4.2.3.1. Methyl
N-tert-butoxycarbonyl-4-ethoxyimino-3-methyl-
piperidine-3-carboxylate 4b. The title compound was obtained in
a similar manner as for the preparation of 4a (90.3%). mp: 51–53 ꢀC.
1H NMR (CDCl3, 400 MHz) dH 1.22–1.26 (3H, t, J 7.2, OCH2CH3), 1.36
(3H, s, CH3), 1.46 (9H, s, Boc-9H), 2.42–3.19 (4H, m), 3.70 (3H, s,
OCH3), 4.07–4.12 (2H, q, J 7.2, OCH2CH3), 3.79 (1H, s), 4.30–4.33 (1H,
m). ESI-MS: m/z: 315 (M þ H)þ.
4.2.6. 3-Amino-4-methoxyimino-3-methylpiperidine
dihydrochloride 9a
To a stirring solution of N-tert-butoxycarbonyl-3-amino-4-
methoxyimino-3-methylpiperidine 8a (2.58 g, 10 mmol) dissolved
in methylene chloride (50 mL) was pumped dried hydrochloride
gas at 0–5 ꢀC for 0.5 h. The reaction mixture was allowed to stir
for another 0.5 h at room temperature, the resulting solid was
collected by suction, and the solid was dried in vacuo to give the
title compound 9a (2.27 g, 98.7%) as a white solid, mp: 121–
122 ꢀC. 1H NMR (DMSO-d6 þ D2O, 400 MHz) dH 1.65 (3H, s, CH3),
2.54–2.62 (1H, m), 2.90–2.98 (1H, m), 3.11–3.17 (1H, m), 3.26–3.32
(2H, m), 3.57–3.60 (1H, m), 3.88 (3H, s, OCH3). ESI-MS: m/z 158
(M þ H)þ.
4.2.4. N-tert-Butoxycarbonyl-3-carbamyl-4-methoxyimino-3-
methylpiperidine 7a
To
a stirring solution of methyl N-tert-butoxycarbonyl-4-
methoxyimino-3-methylpiperidine-3-carboxylate 4a (17.00 g,
56.6 mmol) in methanol (100 mL) was added dropwise a solution of
sodium hydroxide (4.53 g, 113.2 mmol) dissolved in distilled water
(20 mL) at room temperature. The reaction mixture was heated to
50 ꢀC and stirred for 2 h at the same temperature. After removal of
the methanol under reduced pressure, the reaction mixture was
diluted with distilled water (30 mL), adjusted to pH 6–6.5 with
acetic acid and filtered to give the acid 5a as a white solid. The solid
5a was dissolved in methylene chloride (150 mL), and to this
solution was added triethylamine (8.8 mL, 63.6 mmol). The reac-
tion mixture was cooled to ꢂ12 to ꢂ14 ꢀC using an ice-salt bath,
isobutyl chloroformate (9.0 mL, 69.2 mmol) was added and stirred
for 0.5 h at the same temperature to give the ester 6a. To the
reaction mixture containing the ester 6a was pumped ammonia gas
cautiously at 0–5 ꢀC for 0.5 h. The mixture was then washed with
1 N HCl and saturated brine, dried over anhydrous sodium sulfate
and concentrated under reduced pressure. The resulting yellow
residue was recrystallized from ethyl acetate to give the title
compound 7a (13.50 g, 83.6%) as a white solid, mp: 126–127 ꢀC. 1H
NMR (CDCl3, 400 MHz) dH 1.37 (3H, s, CH3), 1.47 (9H, s, Boc-9H),
2.14–3.86 (5H, m), 3.89 (3H, s, OCH3), 4.35–4.37 (1H, m), 5.37 (1H,
br, CONH), 6.19, 6.78 (1H, 2s, CONH). ESI-MS: m/z 286 (M þ H)þ.
4.2.6.1. 4-Amino-3-ethoxyimino-4-methylpiperidine dihydrochloride
9b. The title compound was obtained in a similar manner as for the
preparation of 9a (98.6%). mp: 135–137 ꢀC. 1H NMR (DMSO-
d6 þ D2O, 400 MHz) dH 1.24 (3H, t, J 7.2, OCH2CH3), 1.62 (3H, s, CH3),
2.45–2.53 (1H, m), 2.89–3.16 (1H, m), 3.17–3.53 (3H, m), 3.57–3.60
(1H, m), 4.12 (2H, q, J 7.2, OCH2CH3). ESI-MS: m/z 172 (M þ H)þ.
4.2.7. General procedure for the synthesis of 7-(4-alkoxyimino-3-
amino-3- methylpiperidin-1-yl) fluoroquinolone derivatives 12–23
A mixture of 10a–f (1 mmol), 9a,b (1.3 mmol), triethylamine
(5 mmol) and dry acetonitrile (10 mL) was stirred at 30–60 ꢀC for
1–6 h. The resulting solid was collected by suction, and dried in
vacuo to give the title compounds 12–23.
4.2.7.1. 1-Cyclopropyl-6-fluoro-7-(3-amino-4-methoxyimino-3-methyl-
piperidin-1-yl)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylic acid
12. The title compound was obtained as off-white solid. 1H
NMR (CDCl3, 400 MHz) dH 1.09–1.10 (2H, m, cyclopropyl CH2),
1.30–1.32 (2H, m, cyclopropyl CH2), 1.41 (3H, s, CH3), 2.59–2.66
(1H,m), 3.09–3.12 (1H, m), 3.68–3.73 (3H, m), 3.89 (3H, s,
OCH3), 4.22–4.25 (2H, m), 8.11 (1H, d, J 13.2, C5–H), 8.73(1H, s,
C2–H). ESI-MS: m/z 404 (M þ H)þ. HRMS-ESI: m/z Calcd. for
C19H23FN5O4 (M þ H)þ: 404.17341; Found 404.17398.
4.2.4.1. N-tert-Butoxycarbonyl-3-carbamyl-4-ethoxyimino-3-methyl-
piperidine 7b. The title compound was obtained in a similar
manner as for the preparation of 7a (78.58%). mp: 108–109 ꢀC. 1H
NMR (CDCl3, 400 MHz) dH 1.24–1.27 (3H, t, J 7.2, OCH2CH3), 1.38
(3H, s, CH3), 1.52 (9H, s, Boc-9H), 2.20–3.78 (5H, m), 4.11–4.16 (2H,
q, J 7.2, OCH2CH3), 4.33–4.35(1H, m), 5.46 (1H, br, CONH), 6.19, 6.66
(1H, 2s, CONH). ESI-MS: m/z 300 (M þ H)þ.
4.2.7.2. 1-Cyclopropyl-6-fluoro-7-(3-amino-4-ethoxyimino-3-meth-
ylpiperidin-1-yl)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylic
acid 13. The title compound was obtained as off-white solid. 1H
NMR (CDCl3, 400 MHz) dH 1.08–1.12 (2H, m, cyclopropyl CH2), 1.26–
1.30 (5H, m, cyclopropyl CH2, OCH2CH3), 1.35 (3H, s, CH3), 2.62–2.70
(1H,m), 3.08–3.14 (1H, m), 3.59–3.77 (3H, m), 4.09–4.24 (4H, m),
8.10 (1H, d, J 13.2, C5–H), 8.74 (1H, s, C2-H). FAB-MS: m/z 418
(M þ H)þ. HRMS-FAB: m/z Calcd. for C20H25FN5O4 (M þ H)þ:
418.1891; Found 418.1874.
4.2.5. N-tert-Butoxycarbonyl-3-amino-4-methoxyimino-3-
methylpiperidine 8a
To a solution of N-tert-butoxycarbonyl-3-carbamyl-4-methox-
yimino-3-methylpiperidine 7a (2.00 g, 7.0 mmol) in acetonitrile
(80 mL) was added dropwise a freshly prepared sodium hypo-
bromite solution (14 mL) at 5 ꢀC for 0.5 h. The reaction mixture was
stirred at the same temperature for 3 h, and then concentrated
under reduced pressure. The residue was dissolved in distilled water
(30 mL), then the solution was adjusted to pH 2.5 with 2 N HCl,
washed with ethyl acetate (3 ꢁ 30 mL). The aqueous layer was
adjusted to pH 12 with 5% sodium hydroxide solution, and then
extracted with ethyl acetate (3 ꢁ 50 mL). The combined organic
extracts were washed with saturated brine, dried over anhydrous
sodium sulfate, and then concentrated under reduced pressure to
give the title compound 8a (0.91 g, 50.6%) as a colorless liquid. 1H
NMR (CDCl3, 400 MHz) dH 1.27 (3H, s, CH3),1.43 (9H, s, Boc-9H),1.75
(2H, br, NH2), 2.30–3.33 (1H, m), 2.90–3.00 (3H, m), 3.67–3.70 (1H,
m), 3.83 (3H, s, OCH3), 3.84–3.87 (1H, m). ESI-MS: m/z 258 (M þ H)þ.
4.2.7.3. 1-(2,4-Difluorophenyl)-6-fluoro-7-(3-amino-4-methoxyimino-
3-methylpiperidin-1-yl)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-car-
boxylic acid 14. The title compound was obtained as off-white
solid. 1H NMR (CDCl3, 400 MHz) dH 1.20 (3H, s, CH3), 2.36–2.48 (1H,
m), 2.78–2.88 (1H, m), 3.33–3.51 (2H, m), 3.67–3.82 (2H, m), 3.85
(3H, s, OCH3), 7.08–7.14 (2H, m, ph-2H), 7.38–7.44 (1H, m, ph-H),
8.14 (1H, d, J 13.2, C5–H), 8.68 (1H, s, C2–H). ESI-MS: m/z 476
(M þ H)þ. HRMS-ESI: m/z Calcd. for C22H21F3N5O4 (M þ H)þ:
476.15456; Found 476.15598.

4068
Y. Chai et al. / European Journal of Medicinal Chemistry 44 (2009) 4063–4069
4.2.7.4. 1-(2,4-Difluorophenyl)-6-fluoro-7-(3-amino-4-ethoxyimino-
3-methylpiperidin-1-yl)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carb-
oxylic acid 15. The title compound was obtained as off-white solid.
1H NMR (CDCl3, 400 MHz) dH 1.23–1.27 (6H, m, OCH2CH3, CH3),
2.37–2.49 (1H, m), 2.82–2.91 (1H, m), 3.34–3.52 (2H, m), 3.70–3.89
(2H, m), 4.07–4.12 (2H, m, OCH2CH3), 7.09–7.14 (2H, m, ph-2H),
7.39–7.45 (1H, m, ph-H), 8.14 (1H, d, J 13.2, C5–H), 8.68 (1H, s, C2–H).
FAB-MS: m/z 490 (M þ H)þ. HRMS-FAB: m/z Calcd. for
C23H23F3N5O4 (M þ H)þ: 490.1702; Found 490.1713.
(1H, m), 3.46–3.58 (3H, m), 4.12–4.17 (2H, m, OCH2CH3), 4.59–4.60
(2H, m, NCH2CH3), 7.95 (1H, d, J 11.6, C5–H), 8.97 (1H, s, C2–H). FAB-
MS: m/z 423 (M þ H)þ. HRMS-FAB: m/z Calcd. for C20H25F2N4O4
(M þ H)þ: 423.1844; Found 423.1809.
4.2.7.11. 1-(2-Fluoroethyl)-6,8-difluoro-7-(3-amino-4-methoxyimino-
3-methylpiperidin-1-yl)-1,4-dihydro-4-oxo-quinoline-3-carboxylic acid
22. The title compound was obtained as off-white solid. 1H NMR
(CDCl3, 400 MHz) dH 1.40 (3H, s, CH3), 2.73–2.78 (1H, m), 2.95–2.99
(1H, m), 3.22–3.41 (4H, m), 3.88 (3H, s, OCH3), 4.70–4.89 (4H, m,
NCH2CH2F), 8.02 (1H, d, J 11.2, C5–H), 8.61 (1H, s, C2–H). ESI-MS: m/z
427 (M þ H)þ. HRMS-ESI: m/z Calcd. for C19H22F3N4O4 (M þ H)þ:
427.15931; Found 427.15950.
4.2.7.5. 1-Cyclopropyl-6,8-difluoro-7-(3-amino-4-methoxyimino-3-
methylpiperidin-1-yl)-1,4-dihydro-4-oxo-quinoline-3-carboxylic acid
16. The title compound was obtained as off-white solid. 1H NMR
(DMSO-d6, 400 MHz) dH 1.15–1.22 (4H, m, 2ꢁ cyclopropyl CH2), 1.58
(3H, s, CH3), 2.53–2.59 (1H, m), 3.08–3.14 (1H, m), 3.22–3.28 (1H,
m), 3.49–3.55 (2H, m), 3.60–3.63 (1H, m), 3.90 (3H, s, OCH3), 4.11–
4.13 (1H, m), 7.90 (1H, d, J 10.8, C5–H), 8.69 (1H, s, C2–H). ESI-MS: m/
z 421 (M þ H)þ. HRMS-ESI: m/z Calcd. for C20H23F2N4O4 (M þ H)þ:
421.16874; Found 421.17060.
4.2.7.12. 1-(2-Fluoroethyl)-6,8-difluoro-7-(3-amino-4-ethoxyimino-
3-methylpiperidin-1-yl)-1,4-dihydro-4-oxo-quinoline-3-carboxylic
acid 23. The title compound was obtained as off-white solid. 1H
NMR (CDCl3, 400 MHz) dH 1.27 (3H, t, OCH2CH3), 1.39 (3H, s, CH3),
2.74–2.79 (1H, m), 2.95–3.02 (1H, m), 3.21–3.44 (4H, m), 4.09–4.15
(2H, m, OCH2CH3), 4.69–4.88 (4H, m, NCH2CH2F), 8.01 (1H, d, J 11.2,
C5–H), 8.60 (1H, s, C2–H). FAB-MS: m/z 441 (M þ H)þ. HRMS-FAB:
m/z Calcd. for C20H24F3N4O4 (M þ H)þ: 441.1750; Found 441.1752.
4.2.7.6. 1-Cyclopropyl-6,8-difluoro-7-(3-amino-4-ethoxyimino-3-
methylpiperidin-1-yl)-1,4-dihydro-4-oxo-quinoline-3-carboxylic acid
17. The title compound was obtained as off-white solid. 1H NMR
(CDCl3, 400 MHz) dH 1.18 (2H, m, cyclopropyl CH2), 1.24–1.34 (5H,
m, OCH2CH3, cyclopropyl CH2), 1.40 (3H, s, CH3), 2.73–2.79 (1H, m),
2.99–3.05 (1H, m), 3.25–3.46 (4H, m), 3.99–4.01 (1H, m), 4.10–4.15
(2H, m, OCH2CH3), 7.95 (1H, d, J 11.2, C5–H), 8.80 (1H, s, C2–H). FAB-
MS: m/z 435 (M þ H)þ. HRMS-FAB: m/z Calcd. for C21H25F2N4O4
(M þ H)þ: 435.1844; Found 435.1843.
4.2.8. General procedure for the synthesis of 7-(4-alkoxyimino-3-
amino-3- methylpiperidin-1-yl) fluoroquinolone derivatives 24–27
A mixture of 11g–h (1 mmol), 9a,b (1.3 mmol), triethylamine
(5 mmol) and dry acetonitrile (10 mL) was stirred at 30–60 ꢀC for
1–6 h. After completion of the condensation, the reaction mixture
was concentrated under reduced pressure. The residue was dis-
solved in 5% sodium hydroxide solution (6.0 mL), heated to 40–
55 ꢀC and stirred for 2–5 h at the same temperature. The reaction
mixture was cooled to room temperature and adjusted to pH 7–7.5
with 2 N HCl. The solid product was collected by suction, and dried
in vacuo to give the title compounds 24–27.
4.2.7.7. 1-Cyclopropyl-6-fluoro-7-(3-amino-4-methoxyimino-3-meth-
ylpiperidin-1-yl)-8-difluoromethoxyl-1,4-dihydro-4-oxo-quinoline-
3-carboxylic acid 18. The title compound was obtained as off-white
solid. 1H NMR (DMSO-d6, 400 MHz) dH 0.967 (2H, m, cyclopropyl
CH2), 1.13–1.15 (2H, m, cyclopropyl CH2), 1.26 (3H, s, CH3), 2.63–2.68
(1H, m), 2.84–2.88 (1H, m), 3.20–3.32 (4H, m), 3.78 (3H, s, OCH3),
4.03 (1H, m), 6.98 (1H, t, J 73.2, OCHF2), 7.91 (1H, d, J 11.6, C5–H),
8.75 (1H, s, C2–H), ESI-MS: m/z 469 (M þ H)þ. HRMS-ESI: m/z Calcd.
for C21H24F3N4O5 (M þ H)þ: 469.16988; Found 469.16722.
4.2.8.1. 1-Cyclopropyl-6-fluoro-7-(3-amino-4-methoxyimino-3-meth-
ylpiperidin-1-yl)-8-methoxyl-1,4-dihydro-4-oxo-quinoline-3-carbox-
ylic acid 24. The title compound was obtained as pale yellow
solid. 1H NMR (DMSO-d6, 400 MHz) dH 0.93–1.18 (4H, m, 2ꢁ
cyclopropyl CH2), 1.53 (3H, s, CH3), 2.49–2.57 (1H, m), 3.09–3.13
(1H, m), 3.17–3.23 (1H, m), 3.41–3.55 (3H, m), 3.71 (3H, s, OCH3),
3.89 (3H, s, OCH3), 4.13–4.15 (1H, m), 7.81 (1H, d, J 11.6, C5–H),
8.73 (1H, s, C2–H), ESI-MS: m/z 433 (M þ H)þ. HRMS-ESI: m/z
Calcd. for C21H26FN4O5 (M þ H)þ: 433.18872; Found 433.18866.
4.2.7.8. 1-Cyclopropyl-6-fluoro-7-(3-amino-4-ethoxyimino-3-meth-
ylpiperidin-1-yl)-8-difluoromethoxyl-1,4-dihydro-4-oxo-quinoline-3-
carboxylic acid 19. The title compound was obtained as off-white
solid. 1H NMR (DMSO-d6, 400 MHz) dH 0.98 (2H, m, cyclopropyl
CH2), 1.14–1.15 (2H, m, cyclopropyl CH2), 1.19–1.23 (3H, t, OCH2CH3),
1.37 (3H, s, CH3), 2.58–2.63 (1H, m), 2.95–2.98 (1H, m), 3.23–3.43
(4H, m), 4.04–4.11 (3H, m), 7.01 (1H, t, J 73.2, OCHF2), 7.94 (1H, d, J
11.6, C5–H), 8.77 (1H, s, C2–H), FAB-MS: m/z 483 (M þ H)þ. HRMS-
FAB: m/z Calcd. for C22H26F3N4O5 (M þ H)þ: 483.1855; Found
483.1864.
4.2.8.2. 1-Cyclopropyl-6-fluoro-7-(3-amino-4-ethoxyimino-3-meth-
ylpiperidin-1-yl)-8-methoxyl-1,4-dihydro-4-oxo-quinoline-3-carbox-
ylic acid 25. The title compound was obtained as pale yellow solid.
1H NMR (CDCl3, 400 MHz) dH 0.93–1.04 (2H, m, cyclopropyl CH2),
1.18–1.31 (5H, m, cyclopropyl CH2, OCH2CH3), 1.59 (3H, s, CH3),
2.68–2.70 (1H, m), 3.12–3.16 (1H, m), 3.34–3.60 (4H, m), 3.71 (3H, s,
OCH3), 3.99–4.00 (1H, m), 4.10–4.16 (2H, m, OCH2CH3), 7.87 (1H, d, J
11.6, C5–H), 8.83 (1H, s, C2–H), FAB-MS: m/z 447 (M þ H)þ. HRMS-
FAB: m/z Calcd. for C22H28FN4O5 (M þ H)þ: 447.2044; Found
447.2026.
4.2.7.9. 1-Ethyl-6,8-fluoro-7-(3-amino-4-methoxyimino-3-methylpipe-
ridin-1-yl)-1,4-dihydro-4-oxo-quinoline-3-carboxylic acid 20. The title
compound was obtained as off-white solid. 1H NMR (DMSO-d6,
400 MHz) dH 1.40–1.44 (3H, t, NCH2CH3,), 1.54 (3H, s, CH3), 2.53–2.58
(1H, m), 3.04–3.08 (1H, m), 3.20–3.25 (1H, m), 3.42–3.53 (3H, m), 3.88
(3H, s, OCH3), 4.53–4.56 (2H, m, NCH2), 7.91 (1H, d, J 10.8, C5–H), 8.87
(1H, s, C2–H), ESI-MS: m/z 409 (M þ H)þ. HRMS-ESI: m/z Calcd. for
C19H23F2N4O4 (M þ H)þ: 409.16874; Found 409.16956.
4.2.8.3. 9-Fluoro-3(S)-methyl-10-(3-amino-4-methoxyimino-3-meth-
ylpiperidin-1-yl)-7-oxo-2,3-dihydro-7H-pyrrido[1,2,3-de][1,4]benzox-
azine-6-carboxylic acid 26. The title compound was obtained as pale
yellow solid. 1H NMR (CDCl3, 400 MHz) dH 1.46 (3H, s, CH3),1.62 (3H,
d, J 6.8, CHCH3), 2.75–2.82 (1H, m), 2.88–2.95 (1H, m), 3.25–3.44
(4H, m), 3.88 (3H, s, OCH3), 4.36–4.52 (3H, m), 7.75 (1H, d, J 11.6, C8–
H), 8.63 (1H, s, C5–H). ESI-MS: m/z 419 (M þ H)þ. HRMS-ESI: m/z
Calcd. for C20H24FN4O5 (M þ H)þ: 419.17307; Found 419.17072.
4.2.7.10. 1-Ethyl-6,8-difluoro-7-(3-amino-4-ethoxyimino-3-methyl-
piperidin-1-yl)-1,4-dihydro-4-oxo-quinoline-3-carboxylic acid 21. The
title compound was obtained as off-white solid. 1H NMR (DMSO-d6,
400 MHz) dH 1.25 (3H, t, J 6.8, OCH2CH3), 1.44 (3H, t, J 7.2, NCH2CH3),
1.55 (3H, s, CCH3), 2.53–2.61 (1H, m), 3.07–3.11 (1H, m), 3.22–3.28

Y. Chai et al. / European Journal of Medicinal Chemistry 44 (2009) 4063–4069
4069
4.2.8.4. 9-Fluoro-3(S)-methyl-10-(3-amino-4-ethoxyimino-3-methyl-
Acknowledgements
piperidin-1-yl)-7-oxo-2,3-dihydro-7H-pyrrido[1,2,3-de][1,4]benzox-
azine-6-carboxylic acid 27. The title compound was obtained as
pale yellow solid. 1H NMR (CDCl3, 400 MHz) dH 1.22 (3H, t,
OCH2CH3), 1.37 (3H, s, CH3), 1.45 (3H, d, J 6.4,CHCH3), 2.63–2.66 (1H,
m), 2.90–2.91 (1H, m), 3.27–3.33 (4H, m), 4.05–4.07 (2H, m,
OCH2CH3), 4.37–4.41 (1H, m), 4.59–4.63 (1H, m), 4.92–4.94 (1H, m),
7.62 (1H, d, J 11.6, C8–H), 8.99 (1H, s, C5–H). FAB-MS: m/z 433
(M þ H)þ. HRMS-FAB: m/z Calcd. for C21H26FN4O5 (M þ H)þ:
433.1887; Found 433.1871.
This work was supported by the IMB Research Foundation.
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