Synthesis and antibacterial activity of naphthyridone derivatives containing mono/difluoro-methyloxime pyrrolidine scaffolds

Article abstract of DOI:10.1016/j.ejmech.2011.10.048

A series of novel naphthyridone derivatives containing mono/difluoro- methyloxime pyrrolidine scaffolds were designed and synthesized. These derivatives were initially evaluated for their in vitro antibacterial activity and compounds 13a1, b1 were chosen

Full text of DOI:10.1016/j.ejmech.2011.10.048

European Journal of Medicinal Chemistry 47 (2012) 619e625
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and antibacterial activity of naphthyridone derivatives containing
mono/difluoro-methyloxime pyrrolidine scaffolds
1
,1
*
Kai Lv , Ming-Liang Liu , Lian-Shun Feng, Lan-Ying Sun, Ye-Xin Sun, Zeng-Quan Wei, Hui-Quan Guo
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 naphthyridone derivatives containing mono/difluoro-methyloxime pyrrolidine scaffolds
were designed and synthesized. These derivatives were initially evaluated for their in vitro antibacterial
activity and compounds 13a1, b1 were chosen for further evaluation their in vivo activity against
systemic infections in mice. The results indicate that all of the target compounds have considerable
in vitro antibacterial activity. In the in vivo experiments, 13b1 was found to be more effective than the
parent drug gemifloxacin against the tested five strains, and especially its activity (ED50:21.27 mg/kg) is
5.2e6.1 times more potent than gemifloxacin and ciprofloxacin against clinically important Gram-
negative pathogen Pseudomonas aeruginosa.
Received 3 August 2011
Received in revised form
26 October 2011
Accepted 26 October 2011
Available online 4 November 2011
Keywords:
Antibacterial activity
Naphthyridones
Ó 2011 Elsevier Masson SAS. All rights reserved.
Fluoro-methyloxime pyrrolidine
1. Introduction
not only limited their use in infections caused by these organisms,
but has also contributed to rapidly developing quinolone resistance
Quinolones have become a major class of antibacterial agents
mainly used to fight both community-acquired and serious
hospital-acquired infections. These antibiotics act by binding two
type II bacterial topoisomerase enzymes, DNA gyrase and topo-
isomerase IV [1,2].
[6]. Thus, recent efforts have been directed toward the synthesis of
new quinolones that can provide improved Gram-positive anti-
bacterial activity, while retaining the good Gram-negative activity
of early fluoroquinolones, such as CPFX and OFLX [7,8].
Previous work on pyrrolidinyl quinolones suggested the
importance of the methyloxime functional group with respect to
biological activity. For example, GMFX displays much more anti-
bacterial activity than its desmethyloximino analog [5]. DW286
(Fig.1), a methylation analog of GMFX, shows improved in vitro and
in vivo activity than GMFX against important Gram-positive
bacteria including methicillin-resistant Staphylococcus aureus
(MRSA) and quinolone-resistant S. aureus, while maintaining an
excellent pharmacokinetic profile [9,10]. It was reported that some
cephalosporins possessing a monofluoro-methyloxime moiety,
such as CP6679 [11] and cefluprenam (E-1077) [12] have superior
antibacterial activity to the corresponding methyloxime analogs
against MRSA and Pseudomonas aeruginosa.
Inspired by these research results, we considered replacement
of the methyloxime moiety in GMFX and DW286 with the bio-
isosteric fluoro-methyloximes which can be readily obtained from
the corresponding oximes in a single operation. A series of novel
naphthyridone compounds containing fluoro-methyloxime func-
tionalized pyrrolidine side chains at the C-7 position were
designed and synthesized. These derivatives are structurally
unique, having an aminomethyl (and a methyl) and a mono/
difluoro-methoxyimino group at the 3- and 4-positions of the
Since the discovery of norfloxacin by Koga et al. in 1980 [3], most
of the research concerning the quinolone antibacterials has been
focused on the basic group at the C-7 position which greatly
influences their potency, spectrum and safety [4]. As a result,
ciprofloxacin (CPFX), lomefloxacin, fleroxacin, ofloxacin (OFLX),
sparfloxacin and so on containing a piperazinyl group at the C-7
position were successfully introduced into the market. These early
fluoroquinolones are currently used for the treatment of infections
mainly caused by most Gram-negative organisms. Recently, the
introduction of the noted pyrrolidine derivatives to the quinolones
resulted in a dramatic improvement of Gram-positive activity
compared to piperazinyl analogs [5]. The pyrrolidinyl-based fluo-
roquinolones, such as trovafloxacin, tosufloxacin, sitafloxacin,
moxifloxacin (MXFX) and gemifloxacin (GMFX, Fig. 1), are generally
characterized by a broad antimicrobial spectrum, but their activity
against clinically important Gram-positive cocci including Staphy-
lococci, Streptococci and Enterococci is relatively moderate. This has
* Corresponding author. Tel.: þ86 010 63036965; fax: þ86 010 63046965.
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.2011.10.048

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K. Lv et al. / European Journal of Medicinal Chemistry 47 (2012) 619e625
product in a similar manner as for preparation of 8a, b even though
variousattemptsweremade.
Although quinolone agents are usually synthesized by direct
condensation of quinolone nuclei and the side chain compounds
[20e22], preparation of naphthyridones 13aec met with no
success through this strategy. In this case, the reaction was complex
and it was very difficult to obtain the desired compounds with
enough purity even though column chromatography separation
technique was used. This was due partly to the fact that both the
primary and second amino groups of pyrrolidine derivatives 8aec
could attack 6-des/fluoronaphthyridone cores 9e11 [15e17]
simultaneously. Therefore, the primary amino group of 8aec was
first protected by iminization with benzaldehyde, and subsequently
condensation with 9e11 in the presence of triethylamine gave
compounds 12aec. Finally, the resulting condensates 12aec were
treated with methanesulfonic acid or through silica gel column
with weak acidity to give the target naphthyridones 13aec
(Scheme 2).
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 13aec. It was a pity that we were not successful in
preparing X-ray quality single crystals of any oxime intermediate or
product. We have done some NOE experiments with final
compounds 13a1, b1, but we did not observe any positive correla-
tion between the crucial protons. Anyway, the oxime geometry
would be expected to have the Z configuration, like GMFX [9]. It is
also obvious that the target compounds 13aec and intermediates
8aec are all racemes.
Fig. 1. Structures of Gemifloxacin and DW286.
pyrrolidine ring, respectively. Our primary objective was to opti-
mize the potency of these naphthyridones against Gram-positive
and Gram-negative organisms.
2. Results and discussion
2.1. Chemistry
Detailed synthetic pathways to new pyrrolidine derivatives
8aec and novel naphthyridones 13aec are depicted in Schemes 1
and 2, respectively. Catalytic hydrogenation of readily available
tert-butyl 3-cyano-4-oxopyrrolidine-1-carboxylate (1) [13,14] yiel-
ded the primary amine with in situ N-tert-butoxycarbonyl (Boc)
protection in a poor yield (43%). The resulting ketone 2 was reacted
with hydroxyamine hydrochloric acid in the presence of pyridine to
give oxime 3 (R1 ¼ H).
Nucleophilic substitution of
1 with iodomethane in the
presence of anhydrous K2CO3 gave cyano ketone 4. Selective
hydrogenation of the cyano group of 4 by 5% Pd/C to the primary
amine with in situ Boc protection yielded bis-Boc protected
compound 5 in a good yield (78%) by conventional treatments.
The methylated oxime 6 (R1 ¼ CH3) was then obtained by
introduction of an oxime group to 5 in a similar manner as for
the preparation of 3.
2.2. Antibacterial activity
2.2.1. In vitro activity
The oximes 3, 6 were condensed with bromofluoromethane
or chlorodifluoromethane in the presence of tetra-n-butyl-
ammonium bromide (TBAB) as the phase transfer catalyst to give
fluoro-methyloximes7aec,whichuponremovalofbis-Bocprotected
groups afforded the desired pyrrolidine derivatives 8aec by meth-
anesulfonic acid in ethanol or hydrogen chloride gas in methanol.
Surprisingly,condensationof3withtrifluoromethyliodideyieldedthe
bis-Boc protected compound containing a trifluoro-methoxyimino
groupsuccessfully[19],butthelatterwasnotconvertedtothedesired
The target compounds 13aec were screened for their in vitro
antibacterial activity against representative Gram-positive and
Gram-negative organisms including standard strains and clinical
isolates by means of standard two fold serial dilution method using
agar media [18]. Minimum inhibitory concentration (MIC) is
defined as the concentration of the compound required to give
complete inhibition of bacterial growth and MICs of the synthe-
sized compounds along with the reference drugs CPFX, GMFX and
MXFX for comparison are reported in Table 1.
Scheme 1. Synthesis of pyrrolidine derivatives 8aec.

K. Lv et al. / European Journal of Medicinal Chemistry 47 (2012) 619e625
621
Scheme 2. Synthesis of naphthyridone derivatives 13aec.
Table 1
In vitro antibacterial activity of naphthyridone derivatives 13aec against selected strains.
Strains
MIC (mg/mL)
13a1
13a2
0.25
64
16
64
64
13a3
0.25
>128
64
>128
>128
13b1
13b2
0.008
16
4
16
16
13c1
0.008
13c2
0.03
128
8
128
128
CPFX
0.25
>128
16
>128
>128
GMFX
MXFX
S.a. ATCC25923
MRSA 10-1
MRSA 10-2
MRSA 10-3
MRSA 10-4
MSSA 10-1
MSSA 10-2
MSSA 10-3
MSSA 10-4
MSSA 10-5
MSSA 10-6
MRSE 10-1
MRSE 10-2
MSSE 10-1
MSSE 10-2
S.p.10e1
E.fm. 09-1
E.fm. 09-2
E.fm. 09-3
E.fm. 09-4
E.fm. 09-5
E.fm. 09-6
E.fs. 09-1
0.015
32
2
32
64
0.015
16
1
16
32
0.008
16
0.5
16
0.008
8
8
8
8
4
0.25
4
4
16
0.06
8
0.06
4
0.03
128
0.25
0.25
0.125
0.125
0.015
4
0.03
1
0.06
1
0.5
32
0.015
0.015
0.03
0.03
4
0.015
64
0.06
0.06
0.03
0.125
16
8
8
0.008
2
0.008
4
0.03
0.25
0.125
0.5
2
0.125
0.125
0.125
0.125
4
0.06
0.5
0.008
0.06
4
0.25
64
32
0.03
32
32
16
0.125
16
0.06
4
8
0.008
16
32
16
16
0.5
8
32
0.015
0.008
0.015
0.015
2
0.25
0.06
0.03
0.125
1
0.5
8
64
0.5
16
16
8
0.06
16
0.06
0.06
0.015
0.5
0.008
0.25
0.5
2
16
0.015
16
16
4
0.03
16
0.03
2
0.008
0.008
0.03
0.008
0.5
1
32
1
8
64
16
32
1
8
4
32
64
0.06
32
32
16
0.06
32
0.03
8
32
0.008
32
32
32
32
0.5
16
32
32
32
0.125
64
64
32
0.5
64
0.5
32
32
32
64
0.06
32
32
16
0.125
32
0.125
8
16
0.008
32
>128
64
>128
4
2
64
0.015
32
32
2
0.5
32
0.5
32
0.015
0.015
>128
>128
>128
>128
1
64
0.03
16
0.008
16
16
2
0.008
16
0.125
8
16
0.008
16
32
16
16
0.5
4
0.25
64
64
64
2
16
1
64
64
0.06
>128
>128
>128
>128
4
>128
>128
128
0.5
64
0.5
64
64
0.008
32
32
64
32
0.03
4
4
1
E.fs. 09-2
4
E.co. ATCC25922
E.co. 09-1
E.co. 09-2
E.co. 09-3
E.co. 10-1
E.co. 10-2
E.co. 10-3
K.p. 09-2
0.125
0.008
64
>128
32
64
0.125
32
128
0.008
16
32
16
16
0.25
4
16
128
>128
>128
128
4
32
>128
>128
8
64
128
>128
>128
32
16
K.p. 10-1.
0.03
0.5
4
16
0.5
4
4
0.015
0.5
0.5
0.5
32
0.25
2
4
1
128
8
0.015
0.25
2
1
0.5
64
8
0.06
0.015
0.5
0.5
0.25
16
0.03
0.5
1
0.5
16
K.p. 10e3
P.a. ATCC27853
P.a. 10-1
P.a. 10-2
P.a. 10-3
0.5
0.5
0.5
32
4
0.125
0.125
0.008
16
4
0.25
0.25
0.06
4
2
>128
32
>128
32
>128
0.5
4
2
4
CPFX: ciprofloxacin; GMFX: gemifloxacin; MXFX: moxifloxacin. S.a., Staphylococcus aureus; MRSA, Methicillin-resistant Staphylococcus aureus; MSSA, Methicillin-sensitive
Staphylococcus aureus; MRSE, Methicillin-resistant Staphylococcus epidermidis; MSSE, Methicillin-sensitive Staphylococcus epidermidis; S.p., Streptococcus pneumonia; E.fm.,
Enterococcus faecium; E.fs., Enterococcus faecalis; E.co., Escherichia coli; K.p., Klebsiella pneumoniae; P.a., Pseudomonas aeruginosa.

622
K. Lv et al. / European Journal of Medicinal Chemistry 47 (2012) 619e625
Table 2
In vivo efficacy of compounds 13a1, b1 against systemic infections in mice.
b
Infected bacteria [challenge dose (cfu/mL)]
Compda
MIC (
m
g/mL)
ED50 (mg/kg)
95% confidence limit (mg/kg)
MRSA10-2 (5.2 ꢁ 105)
13a1
13b1
GMFX
CPFX
13a1
13b1
GMFX
CPFX
13a1
13b1
GMFX
CPFX
13a1
13b1
GMFX
CPFX
13a1
13b1
GMFX
CPFX
2
1
1
16
2
2
16.91
14.30
14.86
15.92
8.00
16.91
24.00
24.54
8.79
28.53e11.30
21.46e9.91
26.46e9.49
28.55e10.27
11.95e5.15
28.53e11.30
55.92e15.30
46.91e16.64
16.92e5.62
8.78e3.94
MRSE10-4 (5.0 ꢁ 105)
4
8
E. coli10-2 (3.0 ꢁ 105)
0.5
0.5
0.25
0.06
0.5
5.83
6.86
4.00
11.59e4.43
5.97e2.58
K. pneumoniae10-3 (3.0 ꢁ 105)
P.aeruginosa10-1 (5.2 ꢁ 105)
>160
100.26
108.46
87.45
39.28
21.27
129.40
110.89
ec
0.5
119.79e84.66
130.43e91.42
101.30e74.08
93.64e23.96
30.30e14.97
188.85e105.99
139.41e92.62
0.25
0.25
1
1
0.25
0.25
CPFX: ciprofloxacin; GMFX: gemifloxacin.
MRSA, Methicillin-resistant Staphylococcus aureus; MRSE, Methicillin-resistant Staphylococcus epidermidis.
a
Antimicrobial agents were orally administrated twice at 1 and 4 h after infection.
ED50:50% effective dose.
e, confidence limits could not be calculated.
b
c
The data shows that all of the target compounds 13aec have
generally potent in vitro antibacterial activity against the tested
strains. The activity of compounds 13a1, b1, b2, c1 against Gram-
positive organisms is better than CPFX, and comparable to GMFX
13b1 is marginally more effective than GMFX against E. coli10-2
and K. pneumoniae10-3.
3. Conclusion
and MXFX. For example, compound 13c1 (MICs: 0.25e1 mg/mL) is
2- to 64-fold more potent than the three reference drugs against
MRSA 10-2 and Enterococcus faecalis 09-2. For Gram-negative
In conclusion, a series of novel naphthyridone derivatives con-
taining mono/difluoro-methyloxime pyrrolidine scaffolds were
designed, synthesized and characterized by 1H NMR, MS, HRMS
and 13C NMR. These derivatives were initially evaluated for their
in vitro activity against representative Gram-positive strains
including MRSA and MRSE, and Gram-negative strains including
P. aeruginosa. Compounds 13a1, b1 were chosen for further evalu-
ation using their in vivo activity against systemic infections in mice.
Our results demonstrate that all of the synthesized compounds
have considerable in vitro antibacterial activity. For example, the
activity of compounds 13a1, b1, b2, c1 is better than CPFX, and
comparable to GMFX and MXFX against Gram-positive organisms.
Compounds 13a1, b1 are comparable to the three reference drugs
against Gram-negative organisms. It is important to note that 13b1
is more effective than the parent drug GMFX against the tested five
strains, and especially its activity (ED50:21.27 mg/kg) is 5.2e6.1
times more potent than the references GMFX and CPFX against
clinically important Gram-negative pathogen P. aeruginosa in the
in vivo experiments.
organisms, compounds 13a1, b1 (MICs: 0.015e32
mg/mL) are
comparable to the reference drugs.
Generally, the activity of the naphthyridone nuclei in this study
is in the order: 1-cyclopropyl-6-fluoro-1,8-naphthyridone > 1-(2,4-
difluorophenyl) -6-fluoro ꢀ1,8- naphthyridone > 1-cyclopropyl-
1,8-naphthyridone. In addition, naphthyridones featuring difluoro-
methyloxime-incorporated pyrrolidino substitution at C-7 position
are more potent than the analogs containing a monofluoro-
methyloxime.
2.2.2. In vivo activity
Mice protection tests were used to evaluate further in vivo
efficacy of compounds 13a1, b1 having better in vitro activity,
and CPFX and GMFX were used as control drugs (Table 2). The
efficacy of these compounds was tested against five clinical
isolate strains: MRSA 10-2 and methicillin-resistant Staphylo-
coccus epidermidis (MRSE) 10-4 were selected for Gram-positive
bacteria, and Escherichia coli10-2, Klebsiella pneumoniae10-3
and P. aeruginosa 10-1 were chosen for Gram-negative bacteria.
The data suggests that monofluoro-methyloxime derivative
13a1 exhibits lower in vivo efficacy than the corresponding
difluoro-methyloxime analog 13b1 against the tested strains
except for MRSE10-4, which is approximately consistent with
the in vitro activity results. The ED50s of 13a1, b1 are comparable
to those of GMFX and CPFX against the Gram-positive strain
MRSA 10-2, but both of them (ED50s of 8 and 16.91 mg/kg,
respectively) are 1.4e3.1 times stronger than the two reference
drugs (ED50s of 24 and 24.54 mg/kg, respectively) against
MRSE10-4. For Gram-negative strain P. aeruginosa 10-1,
compounds 13a1, b1 (ED50s of 39.28 and 21.27 mg/kg, respec-
tively) are both more effective than GMFX and CPFX (ED50s of
129.4 and 110.89 mg/kg, respectively). In addition, compound
4. Experimental protocol
4.1. Chemistry
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 ob-
tained on an 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

K. Lv et al. / European Journal of Medicinal Chemistry 47 (2012) 619e625
623
chemically pure. TLC was performed on silica gel plates (Merck,
ART5554 60F254).
6.94 (1H, brs), 3.96 (2H, s), 3.50e2.98 (4H, m), 1.40 (9H, s), 1.36
(9H, s), 1.06 (3H, s). ESI-MS (m/z): 344 (M þ H)þ.
4.2. Synthesis
4.2.6. tert-Butyl 3- (tert-butoxycarbonyl)aminomethyl-4-
(fluoromethoxyimino) pyrrolidine-1-carboxylate 7a
4.2.1. tert-Butyl 3-(tert-butoxycarbonyl)aminomethyl-4-
oxopyrrolidine-1-carboxylate 2
To a stirring suspension of 3 (6.59 g, 20 mmol) in toluene
(400 mL) was added 12N NaOH solution (10 mL, 120 mmol) and
bromofluoromethane (3.93 g, 30 mmol) at 0 ꢂC. The reaction
mixture was then stirred for 10 min at room temperature, and
washed successively with water and saturated saline solution,
dried over anhydrous MgSO4, and filtered. The filtrate was
concentrated under reduced pressure to afford the title compound
7a (6.86 g, 95.0%) as a pale yellow oil. 1H NMR (400 MHz, DMSO-d6)
A
mixture of
tert-butyl 3-cyano-4-oxopyrrolidine-1-
carboxylate 1 (21.02 g, 100 mmol), (Boc)2O (26.19 g, 120 mmol)
and 5% Pd/C (6.00 g) in methanol (250 mL) was pressurized at
70 psi of hydrogen at room temperature for 12 h, and then filtered.
The filtrate was concentrated under reduced pressure. The residue
was purified by column chromatography (silica gel) eluted with
petroleum ether and ethyl acetate (v: v ¼ 5: 1) to give the title
compound 2 (13.53 g, 43.1%) as a colorless oil. 1H NMR (400 MHz,
d
(ppm): 7.07 (1H, brs), 5.68 (2H, d, J ¼ 56 Hz), 4.05 (2H, brs), 3.57
(1H, brs), 3.31e3.27 (1H, m), 3.19 (1H, brs), 3.10e3.03 (2H, m), 1.45
(9H, s), 1.40 (9H, s). MS-ESI (m/z): 362 (M þ H)þ.
DMSO-d6)
d (ppm): 6.97 (1H, brs), 3.79e3.63 (3H, m), 3.51e3.36
(1H, m), 3.27e3.18 (1H, m), 3.10e3.01 (1H, m), 2.85e2.73 (1H, m),
1.41 (9H, s), 1.39 (9H, s). MS-ESI (m/z): 315 (M þ H)þ.
4.2.7. tert-Butyl 3- (tert-butoxycarbonyl)aminomethyl-4-
(difluoromethoxyimino) pyrrolidine-1-carboxylate 7b
4.2.2. tert-Butyl 3-(tert-butoxycarbonyl)aminomethyl-4-
(hydroxyimino)pyrrolidine-1-carboxylate 3
To a stirring suspension of 3 (3.29 g, 10 mmol) and TBAB (0.97 g,
3 mmol) in toluene (300 mL) was added 12N NaOH solution (5 mL,
60 mmol) at 0e5 ꢂC, and pumped chlorodifluoromethane gas for 1 h
at the same temperature. The reaction mixture was then stirred for
48 h at 70 ꢂC, and washed successively with water and saturated
saline solution, dried over anhydrous MgSO4, and filtered. The
filtrate was concentrated under reduced pressure. The residue was
purified by column chromatography (silica gel) eluted with petro-
leum etherand ethyl acetate (v: v ¼ 6: 1) to affordthe title compound
7b (1.07 g, 28.4%) as a colorless oil. 1H NMR (400 MHz, DMSO-d6)
To a solution of 2 (9.43 g, 30 mmol) in methanol (200 mL)
was added hydroxylamine hydrochloride (2.50 g, 36 mmol) and
pyridine (2.85 g, 36 mmol). The reaction mixture was stirred for
5 h at room temperature, and concentrated under reduced
pressure. The residue was treated with ethyl acetate (50 mL),
washed successively with water and saturated saline solution,
dried over anhydrous MgSO4, and filtered. The filtrate was
concentrated under reduced pressure, and the crude product
was recrystallized from petroleum ether (400 ml) to afford the
d
(ppm): 7.07 (1H, brs), 7.06 (1H, t, J ¼ 72 Hz), 4.09 (2H, brs), 3.58 (1H,
title compound
3
(7.91 g, 80.1%) as
a
white solid, mp:
brs), 3.32e3.28 (1H, m), 3.20 (1H, brs), 3.11e3.07 (2H, m),1.41 (9H, s),
1.37 (9H, s). MS-ESI (m/z): 380 (M þ H)þ.
138e140 ꢂC 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 10.91 (1H, s),
6.94 (1H, s), 3.92e3.85 (2H, m), 3.54e3.48 (1H, m), 3.32e3.12
(2H, m), 3.00e3.90 (2H, m), 1.44 (18H, s). MS-ESI (m/z): 330
(M þ H)þ.
4.2.8. tert-Butyl 3- (tert-butoxycarbonyl)aminomethyl-3-methyl-4-
(fluoromethoxyimino)pyrrolidine-1-carboxylate 7c
The title compound 7c was obtained from 6 in a similar manner
4.2.3. tert-Butyl 3-cyano-3-methyl-4-oxopyrrolidine-1-carboxylate 4
To a mixture of 1 (21.02 g, 100 mmol) and anhydrous K2CO3
(41.46 g, 300 mmol) in dry acetone (800 mL) was added dropwise
iodomethane (17.03 g, 120 mmol) over a period of 30 min at room
temperature. The reaction mixture was stirred for 3 h at the same
temperature, and then filtered. The filtrate was concentrated under
reduced pressure. The residue was treated with petroleum ether
(300 mL) and filtered, dried to afford the title compound 4 (18.83 g,
84.0%) as a white solid, mp: 71e73 ꢂC 1H NMR (400 MHz, CDCl3)
as for the preparation of 7a (96.1%) as a colorless oil. 1H NMR
(400 MHz, CDCl3)
d
(ppm): 7.08 (1H, brs), 5.66 (2H, t, J ¼ 56 Hz),
4.09 (2H, s), 3.53e2.97 (4H, m), 1.40 (9H, s), 1.36 (9H, s), 1.09 (3H, s).
ESI-MS (m/z): 376 (M þ H)þ.
4.2.9. 3-Aminomethyl-4-(fluoromethoxyimino)pyrrolidine
dimethanesulfonate 8a
To a solution of 7a (3.61 g, 10 mmol) in ethanol (40 mL) was
added methylsulfonic acid (2.88 g, 30 mmol). The reaction
mixture was stirred for 16 h at room temperature, and then
filtered. The precipitate was washed with ethanol, and dried in
vacuo to yield the title compound 8a (2.57 g, 72.6%) as a white
d
(ppm): 4.15e3.69 (4H, m), 1.58 (3H, s), 1.49 (9H, s). MS-ESI (m/z):
225 (M þ H)þ.
4.2.4. tert-Butyl 3-(tert-butoxycarbonyl)aminomethyl-3-methyl-4-
(hydroxyimino) pyrrolidine-1-carboxylate 5
solid, mp: 137e139 ꢂC 1H NMR (400 MHz, DMSO-d6)
d (ppm):
9.32 (2H, brs), 7.99 (3H, brs), 5.74 (2H, d, J ¼ 56 Hz), 4.16e3.99
(2H, m), 3.75e3.71 (1H, m), 3.36e3.28 (2H, m), 3.19e3.07 (2H,
m), 2.38 (6H, s). MS-ESI (m/z): 162 (M þ H)þ.
A mixture of 4 (11.20 g, 50 mmol), (Boc)2O (13.09 g, 60 mmol)
and 5% Pd/C (3.0 g) in methanol (200 mL) was pressurized at 75 psi
of hydrogen at room temperature for 8 h and then filtered. The
filtrate was concentrated under reduced pressure. The residue was
treated with petroleum ether (200 mL), filtered and dried to afford
the title compound 5 (12.80 g, 78.0%) as a white solid, mp:
4.2.10. 3-Aminomethyl-4-(difluoromethoxyimino)pyrrolidine
dimethanesulfonate 8b
The title compound 8b was obtained from 7b in a similar manner
106e108 ꢂC 1H NMR (400 MHz, CDCl3)
d (ppm): 4.77 (1H, brs), 3.83
asforthepreparationof8a(76.4%)asawhitesolid, mp:168e170 ꢂC1H
(2H, s), 3.64e3.26 (4H, m), 1.50 (9H, s), 1.43 (9H, s), 1.15 (3H, s). MS-
NMR (400 MHz, DMSO-d6) d (ppm): 9.35 (2H, brs), 7.97 (3H, brs), 7.16
ESI (m/z): 329 (M þ H)þ.
(1H, t, J ¼ 72 Hz), 4.22e4.05 (2H, m), 3.77e3.73 (2H, m), 3.45e3.40
(1H, m), 3.16e3.12 (2H, m), 2.37 (6H, s). MS-ESI (m/z): 180 (M þ H)þ.
4.2.5. tert-Butyl 3-(tert-butoxycarbonyl)aminomethyl-3-methyl-4-
(hydroxyimino) pyrrolidine-1-carboxylate 6
4.2.11. 3-Aminomethyl-3-methyl-4-(fluoromethoxyimino)
pyrrolidine dihydrochloride 8c
The title compound 6 was obtained from 5 in a similar
manner as for the preparation of 3 (93.1%) as a white solid, mp:
To a stirring solution of 7c (7.50 g, 20 mmol) dissolved in
149e150 ꢂC 1H NMR (400 MHz, CDCl3)
d (ppm): 10.86 (1H, brs),
methanol (50 mL) was pumped dried hydrochloride gas at 0e5 ꢂC

624
K. Lv et al. / European Journal of Medicinal Chemistry 47 (2012) 619e625
for 30 min. The reaction mixture was allowed to stir for another
30 min at room temperature, and the precipitate was collected by
suction, and dried in vacuo to give the title compound 8c (4.47 g,
90.0%) as a off-white solid easily absorbing moisture.
4.2.13.2. 7-(3-Aminomethyl-4-fluoromethoxyimino-1-pyrrolidinyl)-
1- (2,4-difluorophenyl)-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthy-
ridine-3-carboxylic acid methanesulfonate 13a2. The title
compound 13a2 was obtained from 12a2 as a white solid (69.4%),
mp: 168e170 ꢂC 1H NMR (400 MHz, DMSO-d6)
d (ppm): 8.84 (1H,
4.2.12. General procedure for the synthesis of condensates 12a1, a2,
b1
s), 8.17 (1H, d, J ¼ 13 Hz), 7.86e7.78 (4H, m), 7.59e7.56 (1H, m),
7.36e7.32 (1H, m), 5.72 (2H, d, J ¼ 56 Hz), 4.46e3.90 (3H, m),
3.72e3.30 (2H, m), 3.20e3.00 (2H, m), 2.32 (3H, s). MS-FAB (m/z):
480 (M þ H)þ. HRMS-FAB (m/z): Calcd. for C21H18N5O4F4 (M þ H)þ:
480.1295; Found 480.1279.
To a suspension of 8a, b (1.1 mmol) in dry acetonitrile (15 mL)
was added benzaldehyde (1.2 mmol) and triethylamine (3 mmol),
and then stirred for 2 h at room temperature. To the reaction
mixture was added 9, 10 (1 mmol), and stirred for another 12 h at
the same temperature. The precipitate was filtered, washed with
acetonitrile, and dried in vacuo to give the title compounds 12a1,
a2, b1.
4.2.13.3. 7-(3-Aminomethyl-4-difluoromethoxyimino-1-pyrrolidinyl)-
1-cyclopropyl
-6-fluoro-4-oxo-1,4-dihydro-
1,8-naphthyridine-3-
carboxylic acid methanesulfonate 13b1. The title compound 13b1
was obtained from 12b1 as a white solid (65.4%), mp: 235e237 ꢂC 1H
4.2.12.1. 7-(3-Benzylideneaminomethyl-4-fluoromethoxyimino-1-
pyrrolidinyl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-
NMR (400 MHz, DMSO-d6) d (ppm): 15.19 (1H, brs), 8.59 (1H, s), 8.08
(1H, d, J ¼ 13 Hz), 8.00 (3H, brs), 7.18 (1H, t, J ¼ 72 Hz), 4.77e4.66 (2H,
m), 4.46e4.41 (1H, m), 3.91e3.87 (1H, m), 3.74e3.68 (1H, m),
3.61e3.53 (1H, m), 3.45e3.41 (1H, m), 3.22e3.20 (1H, m), 2.32 (3H, s),
naphthyridine-3-carboxylic acid 12a1. The title compound 12a1
was obtained from 8a and 9 as an off-white solid (85.4%), mp:
176e178 ꢂC 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 15.22 (1H, s),
1.24e1.02 (4H, m). 13C NMR (400 MHz, DMSO-d6)
d (ppm): 180.87,
8.53 (1H, s), 8.38 (1H, s), 7.91 (1H, d, J ¼ 13 Hz), 7.58 (2H, d, J ¼ 8 Hz),
7.36e7.27 (3H, m), 5.77 (2H, d, J ¼ 56 Hz), 4.71e4.62 (2H, m),
4.13e4.09 (2H, m), 3.93e3.85 (2H, m), 3.67e3.62 (1H, m),
3.52e3.49 (1H, m), 1.13e0.99 (4H, m). MS-FAB (m/z): 496 (M þ H)þ.
170.20, 151.89 (d, J ¼ 12 Hz), 151.53, 151.36, 150.60 (d, J ¼ 257 Hz),
122.81 (t, J ¼ 247 Hz), 122.56 (d, J ¼ 12 Hz), 116.10,112.07, 55.19, 53.55,
53.50, 44.38, 43.54, 11.45, 11.28. MS-FAB (m/z): 426 (M þ H)þ. HRMS-
ESI (m/z): Calcd. for C18H19N5O4F3 (M þ H)þ: 426.1404; Found
426.1415.
4.2.12.2. 7-(3-Benzylideneaminomethyl-4-fluoromethoxyimino-1-
pyrrolidinyl)-1- (2,4-difluorophenyl)-6-fluoro- 4-oxo-1,4-dihydro-
1,8-naphthyridine-3-carboxylic acid 12a2. The title compound 12a2
was obtained from 8a and 10 as a white solid (69.5%), mp:
4.2.13.4. 7-(3-Aminomethyl-4-fluoromethoxyimino-1-pyrrolidinyl)-
1-cyclopropyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic
acid methanesulfonate 13a3. To
a suspension of 8a (0.39 g,
248e250 ꢂC 1H NMR (400 MHz, CDCl3)
d
(ppm): 14.74 (1H, s), 8.64
1.1 mmol) in dry acetonitrile (10 mL) was added benzaldehyde
(0.13 g, 1.2 mmol) and triethylamine (0.3 g, 3 mmol) at room
temperature and stirred for 30 min at the same temperature. To the
reaction mixture was added 11 (0.26 g, 1.0 mmol) and stirred for
another 12 h at room temperature, and then concentrated under
reduced pressure. The residue was treated with ethanol (10 mL)
and filtered. To the filtrate was added methylsulfonic acid (0.77 g,
8 mmol) and few drops of water. The reaction mixture was stirred
for 10 h at room temperature. The precipitate was filtered and dried
in vacuo to yield the title compound 13a3 [0.20 g, 42.1% (from 11)]
as a white solid, mp: >240 ꢂC 1H NMR (400 MHz, DMSO-d6)
(1H, s), 8.27 (1H, s), 8.08 (1H, d, J ¼ 13 Hz), 7.66e7.52 (2H, m),
7.43e7.32 (4H, m), 7.03e6.99 (2H, m), 5.61 (2H, d, J ¼ 56 Hz),
4.46e3.36 (7H, m). MS-ESI (m/z): 568 (M þ H)þ.
4.2.12.3. 7-(3-Benzylideneaminomethyl-4-difluoromethoxyimino-1-
pyrrolidinyl)-1- cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthy-
ridine-3-carboxylic acid 12b1. The title compound 12b1 was ob-
tained from 8b and 9 as an white solid (69.3%), mp: 205e207 ꢂC 1H
NMR (400 MHz, DMSO-d6) d (ppm): 15.19 (1H, brs), 8.55 (1H, s), 8.39
(1H, s), 7.94 (1H, d, J ¼ 13 Hz), 7.58 (2H, d, J ¼ 8 Hz), 7.36e7.27 (3H,
m), 7.16 (1H, t, J ¼ 72 Hz), 4.77e4.70 (2H, m), 4.20e4.13 (2H, m),
3.91e3.89 (2H, m), 3.68e3.62 (1H, m), 3.59e3.56 (1H, m), 1.14e1.00
(4H, m). MS-FAB (m/z): 514 (M þ H)þ.
d
(ppm): 15.43 (1H, s), 8.59 (1H, s), 8.34 (1H, brs), 7.95 (3H, brs), 6.93
(1H, brs), 5.80 (2H, d, J ¼ 56 Hz), 4.61e4.40 (2H, m), 4.29e4.00 (1H,
m), 3.80e3.08 (5H, m), 2.31 (3H, s), 1.22e1.06 (4H, m). 13C NMR
(400 MHz, DMSO-d6) d (ppm): 176.79, 165.98,163.48,157.25, 151.24,
4.2.13. General procedure for the synthesis of naphthyridine
derivatives 13a1, a2, b1
147.22, 135.26, 110.93, 108.70, 107.61, 104.19 (d, J ¼ 217 Hz), 49.13,
48.61, 47.54, 39.08, 34.63, 6.98, 6.83. MS-ESI (m/z): 390 (M þ H)þ.
HRMS-ESI (m/z): Calcd. for C18H21N5O4F1 (M þ H)þ: 390.1577;
Found 390.1586.
To a suspension of 12a1, a2, b1 (1 mmol) in ethanol (20 mL) was
added methylsulfonic acid (3 mmol) and water (0.2 mL), and the
reaction mixture was then stirred for 16 h at room temperature. The
precipitate was filtered, washed with ethanol, and dried in vacuo to
yield the title compounds 13a1, a2, b1.
4.2.13.5. 7-(3-Aminomethyl-4-difluoromethoxyimino-1-pyrrolidinyl)-
1- (2,4-difluorophenyl)-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-
3-carboxylic acid methanesulfonate 13b2. To a suspension of 8b
(0.39 g, 1.1 mmol) in DMSO (10 mL) was added benzaldehyde (0.13 g,
1.2 mmol) and triethylamine (0.30 g, 3.0 mmol) and stirred for 30 min
at room temperature. 10 (0.35 g, 1.0 mmol) was added to the reaction
mixture and stirred for another 12 h at room temperature. The
reaction mixture was treated with distilled water (50 mL) and
filtered. The obtained solid was diluted with chloroform (10 mL), and
then methylsulfonic acid (0.38 g, 4 mmol) and few drops of water
were added. The reaction mixture was stirred for 16 h at room
temperature, and then treated with ether (40mL). The precipitate was
filtered and dried in vacuo to yield the title compounds 13b2 [0.36 g,
60.1% (from 10)] as a white solid, mp: 145e147 ꢂC 1H NMR (400 MHz,
4.2.13.1. 7-(3-Aminomethyl-4-fluoromethoxyimino-1-pyrrolidinyl)-1-
cyclopropyl- 6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxy-
lic acid methanesulfonate 13a1. The title compound 13a1 was ob-
tained from 12a1 as a white solid (68.9%), mp: 220e222 ꢂC 1H NMR
(400 MHz, DMSO-d6) d (ppm): 15.22 (1H, brs), 8.60 (1H, s), 8.08 (1H,
d, J ¼ 13 Hz), 7.95 (3H, brs), 5.79 (2H, d, J ¼ 56 Hz), 4.71e4.64 (2H, m),
4.44e4.39 (1H, m), 3.88e3.83 (1H, m), 3.74e3.68 (1H, m), 3.52e3.49
(1H, m), 3.20e3.14 (2H, m), 2.31 (3H, s), 1.24e1.02 (4H, m). 13C NMR
(400 MHz, DMSO-d6)
d (ppm): 176.38, 165.67, 162.81, 148.33 (d,
J ¼ 15 Hz), 146.96, 146.90, 146.13 (d, J ¼ 257 Hz), 118.15 (d, J ¼ 21 Hz),
111.55, 107.60, 104.12 (d, J ¼ 219 Hz), 50.57, 48.81, 48.54, 40.13, 34.95,
6.92, 6.76. MS-FAB (m/z): 408 (M þ H)þ. HRMS-FAB (m/z): Calcd. for
C18H20N5O4F2 (M þ H)þ: 408.1483; Found 408.1495.
DMSO-d6)
d
(ppm): 14.96 (1H, s), 8.88 (1H, s), 8.19 (1H, d, J ¼ 13 Hz),
7.92e7.78 (4H, m), 7.58e7.51 (1H, m), 7.36e7.32 (1H, m), 7.10 (1H, t,

K. Lv et al. / European Journal of Medicinal Chemistry 47 (2012) 619e625
625
J ¼ 72 Hz), 4.46e4.00 (3H, m), 3.92e3.60 (1H, m), 3.53e3.40 (1H, m),
3.18e3.03 (2H, m), 2.29 (3H, s). 13C NMR (400 MHz, DMSO-d6):
177.68, 165.78, 161.89, 159.09, 156.44, 148.83, 148.80, 146.71 (d,
J ¼ 255 Hz), 146.37, 131.36, 124.50, 118.95 (d, J ¼ 21 Hz), 118.63 (t,
J ¼ 243 Hz), 112.85,111.84, 109.55, 105.51, 50.64, 49.42, 49.40. MS-FAB
(m/z): 498 (M þ H)þ. HRMS-FAB (m/z): Calcd. for C21H17N5O4F5
(M þ H)þ: 498.1201; Found 498.1180.
organisms for infection were cultured on MH(Mueller-Hinton) Agar
Medium (Difco) at 35 ꢂC for 18 h and were suspended in gastric
mucin (OXOID). Male KM mice (weight 19e21 g) in four groups of
10 mice each were infected intraperitoneally with 0.5 mL of
a bacterial suspension corresponding to an inoculum range of 5e10
times the MLD (minimal lethal dose) of bacteria. Four dose levels
were used for each antibiotic, depending on the in vitro antimi-
crobial activity of the compounds. Mice were orally administered
twice at 1 and 4 h post infection with various dose regimens of
antibiotics. Mortality was recorded for 7 days, and the median
effective dose needed to protect 50% of mice (ED50) was calculated
by the method of Bliss. All untreated mice died within 2 days after
infection.
4.2.14. 7-(3-Aminomethyl-3-methyl-4-fluoromethoxyimino-1-
pyrrolidinyl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxylic acid 13c1
To a suspension of 8c (2.5g, 10 mmol) in dry acetonitrile (20 mL)
was added benzaldehyde (1.27 g, 12 mmol) and triethylamine
(3.03 g, 30 mmol) at room temperature under an atmosphere of
nitrogen and then stirred for 2 h at the same temperature. To the
reaction mixture was added 9 (2.5g, 9mmol) and stirred for another
12 h at room temperature, and then concentrated under reduced
pressure. The residue was purified by column chromatography
(silica gel) eluted with methanol and methylene chloride (v: v ¼ 1:
5) to afford the title compound 13c1 [0.95 g, 25.0% (from 9)] as an
off-white solid. mp: 195e196 ꢂC 1H NMR (400 MHz, DMSO-d6)
Acknowledgments
The work was supported by the National S&T Major Special
Project on Major New Drug Innovation (No. 2009ZX09301-003).
References
d
(ppm): 8.58 (1H, s), 8.03 (1H, d, J ¼ 13 Hz), 5.76 (2H, d, J ¼ 64 Hz),
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4.71 (2H, s), 4.28 (1H, d, J ¼ 11 Hz), 3.75e3.71 (2H, m), 3.31 (2H, s),
2.73 (2H, q, J ¼ 12 Hz), 1.25 (3H, s), 1.22e1.17 (2H, m), 1.12e1.09 (2H,
m). ESI-MS (m/z): 422 (M þ H)þ. HRMS-ESI (m/z): Calcd for
C19H22F2N5O4 (M þ H)þ: 422.1640; Found 422.1656.
4.2.15. 7-(3-Aminomethyl-3-methyl-4-fluoromethoxyimino-1-
pyrrolidinyl)-1- cyclopropyl-4-oxo-1,4-dihydro-1,8-naphthyridine-
3-carboxylic acid 13c2
The title compound was obtained from 8c and 11 in a similar
manner as for the preparation of 13c2 (23.5%, from 11) as a white
solid, mp: 184e186 ꢂC 1H NMR (400 MHz, DMSO-d6)
d (ppm): 8.57
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H.J. Kwon, Y.H. Jung, M.J. Shim, E.C. Choi, Antimicrob. Agents Chemother. 46
(2002) 3071e3074.
(1H, s), 8.29 (1H, s), 6.94 (1H, s), 5.76 (2H, d, J ¼ 65 Hz), 4.53 (2H, s),
4.14e4.02 (1H, m), 3.77e3.71 (1H, m), 3.58e3.31 (1H, m), 2.70 (2H,
q), 1.25 (3H, s), 1.20e1.12 (2H, m), 1.09e1.03 (2H, m). ESI-MS (m/z):
404 (M þ H)þ. HRMS-ESI (m/z): Calcd. for C19H23FN5O4 (M þ H)þ:
404.1734; Found 404.1739.
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4.3. MIC determination
Compounds 13aec were evaluated for their in vitro antibacterial
activity using standard techniques in comparison to the reference
drugs CPFX, GMFX and MXFX. Drugs (10.0 mg) were dissolved in
0.1 N NaOH solution and water (10 mL). Further progressive two
fold serial dilution with melted Mueller-Hinton agar was per-
formed to obtain the required concentrations of 128, 64, 32, 16, 8, 4,
2, 1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015 and 0.008 mg/mL. Petri dishes
[18] Performance standards for antimicrobial susceptibility testing: 17th infor-
mational supplement. Clinical and Laboratory Standards Institute, Wayne, PA,
2007, M100eS17.
were incubated with 104 colony-forming units (cfu) and incubated
at 35 ꢂC for 18 h. MIC was the lowest concentration of the test
compound, which resulted in no visible growth on the plate.
[19] 1H NMR (400 MHz, DMSO-d6)
d (ppm): 4.92 (1H, brs), 4.19 (1H, m), 3.53e3.42
(2H, m), 3.09e3.06 (1H, m), 3.00e2.97(1H, m), 1.45 (9H, s), 1.44 (9H, s).
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4.4. ED50 determination
[21] M.L. Liu, Y.G. Wei, L.Y. Sun, H.Y. Guo, Chin. J. Pharmaceuticals 35 (2004)
129e131.
The in vivo activity of compounds 13a1, b1, CPFX and GMFX
[22] M.L. Liu, B.Q. Liu, L.Y. Sun, H.Y. Guo, Chin. J. Pharmaceuticals 35 (2004)
were determined against systemic infection model in mice. Test
385e387.