Synthesis, antimycobacterial and antibacterial activity of fluoroquinolone derivatives containing an 3-alkoxyimino-4-(cyclopropylanimo)methylpyrrolidine moiety

Article abstract of DOI:10.1016/j.ejmech.2015.09.030

A series of novel fluoroquinolone derivatives containing an 3-alkoxyimino-4-(cyclopropylanimo)methylpyrrolidine moiety were designed, synthesized and evaluated for their biological activity. Our results revealed that 19b2 shows good activity against MTB H

Full text of DOI:10.1016/j.ejmech.2015.09.030

European Journal of Medicinal Chemistry 104 (2015) 73e85
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis, antimycobacterial and antibacterial activity of
fluoroquinolone derivatives containing an 3-alkoxyimino-4-
(cyclopropylanimo)methylpyrrolidine moiety
,
b
,
,
,
Tingting Zhang a 1, Weiyi Shen b 1, Mingliang Liu a *, Rui Zhang a, Minghua Wang a,
Linhu Li a, Bin Wang c, Huiyuan Guo a, Yu Lu c
,
**
a Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
b Zhejiang Starry Pharmaceutical Co. Ltd., Xianju 317300, China
c Beijing Key Laboratory of Drug Resistance Tuberculosis Research, Department of Pharmacology, Beijing Tuberculosis and
Thoracic Tumor Research Institute, Beijing Chest Hospital, Capital Medical University, Beijing 101149, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 July 2015
Received in revised form
15 September 2015
Accepted 24 September 2015
Available online 30 September 2015
A series of novel fluoroquinolone derivatives containing an 3-alkoxyimino-4-(cyclopropylanimo)meth-
ylpyrrolidine moiety were designed, synthesized and evaluated for their biological activity. Our results
revealed that 19b2 shows good activity against MTB H37Rv ATCC 27294 (MIC: <0.25
MTB 6133 clinical isolate (MIC: 0.11 g/mL). Most of them have potent potency against Gram-positive
strains, although they are generally poor active against Gram-negative strains. Especially, compounds
22b1 and 23a3 (MICs: <0.008e8 g/mL) were found to 2e128 times more potent than ciprofloxacin and
mg/mL) and MDR-
m
m
levofloxacin against all of the tested Gram-positive strains including quinolone-resistant MRSA, MRSE,
Enterococcus faecium and Enterococcus faecalis.
Keywords:
Fluoroquinolone derivatives
Synthesis
© 2015 Published by Elsevier Masson SAS.
Antimycobacterial activity
Antibacterial activity
1. Introduction
and Gram-positive species as well as MTB, due mainly to the high
level of use and to some degree of abuse [7,8]. The ideal strategy to
Fluoroquinolones (FQs) represent an extremely successful
family of antibiotics that have a broad spectrum of antibacterial
activity and relatively few side effects [1]. Targeting two type II
bacterial topoisomerase enzymes, DNA gyrase and topoisomerase
IV, FQs are used mainly to fight community-acquired and serious
hospital-acquired infections [2]. Among of them, ciprofloxacin
(CPFX), ofloxacin and levofloxacin (LVFX) are frequently used for
the treatment of tuberculosis (TB) including multi-drug resistant TB
(MDR-TB) as components of second-line regiments [3]. Two C-8
methoxy FQs gatifloxacin (GTFX) and moxifloxacin (MXFX),
currently in Phase III clinical trials [4], show particularly strong
in vitro and in vivo activity against Mycobacterium tuberculosis
(MTB) and MDR-MTB [5,6].
such challenges is to find novel agents that inhibit new targets in
pathogens, but it now remains extremely difficult. A more practical
approach is to modify the structures of existing antibacterial agents
to increase potency and to overcome resistance [9].
From the chemical structural point of view, FQs consist of a 4-
quinolone/naphthyridone-3-carboxylic acid core and a secondary
amino group attached to the C-7 position of the heterocyclic core
(Fig. 1). CPFX, GTFX, LVFX and gemifloxacin (GMFX) represent the
most common cores of important FQs on the market. Some new
FQs, such as sitafloxacin (STFX), delafloxacin (DLFX) and AM-1954
have novel cores which are different from the traditional ones.
On the other hand, the basic substituent at C-7 position, playing an
important role in the antibacterial potency, spectrum and safety of
FQs [10], is recognized as the most adaptable site for chemical
change, and the presence of five- or six-membered nitrogen het-
erocycle including pyrrolidine, piperazine and piperidine at this
position is particularly structural feature of FQs [11].
However, FQ resistance increases in almost all Gram-negative
* Corresponding author.
Recently, methyloxime-functionalized pyrrolidines as novel C-7
substituents have attracted great attention and led to the discovery
** Corresponding author.
E-mail addresses: lmllyx@126.com (M. Liu), luyu4876@hotmail.com (Y. Lu).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2015.09.030
0223-5234/© 2015 Published by Elsevier Masson SAS.

74
T. Zhang et al. / European Journal of Medicinal Chemistry 104 (2015) 73e85
Fig. 1. Structures of some fluoroquinolones.
of new FQs (GMFX, zabofloxacin and DW286) [12e14]. In our
previous works, some FQs containing oxime-functionalized azeti-
dines, pyrrolidines or piperidines were found to have considerable
biological activity [1,15e18]. These studies suggest the importance
of the oxime functional group with respect to biological activity and
pharmacokinetic profiles of FQs. Therefore, we intended to apply
this modification strategy to the C-7 substituent of AM-1954 (Fig. 1)
which has good in vitro activity against MDR-Gram-positive or-
ganisms [19]. More specifically, introduction of an oxime group of
GMFX (and an alkyl group) instead of the fluorine atom on the
pyrrolidine ring of AM-1954 develops 3-alkoxyimino-4-(cyclo-
of the esters 3a,b with LiAlH4 in tetrahydrofuran (THF) and then
oxidation of the resulting alcohols (4a,b) with Dess-Martin Peri-
odinane (DMP). Condensation of 5a,b and cyclopropanamine pro-
duced secondary amines (6a,b), which were protected by treatment
with di-tert-butyl dicarbonate (Boc2O) and then deprotection of the
hydroxyl groups gave the desired bis-Boc-protected amino alcohols
(8a,b). Oxidation of compounds 8a,b by DMP afforded the corre-
sponding ketones (9a,b), on which the oxime function groups were
introduced via condensation with alkoxylamines to yield amines
(10a,b). The bis-Boc-protecting groups of 10a,b were removed by
pumping hydrogen chloride gas in methylene chloride to afford the
pyrrolidine derivative dihydrochlorides (11a,b) (Scheme 1).
Finally, the target compounds 19e25 were obtained by coupling
the new pyrrolidine derivatives 11a,b with various compounds
containing quinolone and naphthyridone cores according to well-
established literature procedures (Scheme 2) [22]. In the case of
naphthyridones 19e21, direct condensation of 11a,b with 12e14
was performed in the presence of triethylamine. However for
quinolones 22e25, boric chelates 15e18 were required to increase
reactivity. All of the synthetic compounds were well characterized
through the spectral characteristics.
propylanimo)methylpyrrolidine
and
3-alkoxyimino-4-(cyclo-
propylanimo)methylpyrrolidine (Fig. 2) as new side chains at the 7-
position of FQs. A series of novel FQ derivatives were designed and
synthesized by condensation of the side chains with the traditional
and new FQ cores. Our primary objective was to optimize the po-
tency of these compounds against clinically important pathogens
(especially Gram-positive ones) and MTB including MDR-MTB. A
preliminary structure-activity relationship (SAR) study was also
explored to facilitate the further development of FQs.
2. Results and discussion
2.2. Anti-MTB activity
2.1. Chemistry
Fifteen of the target compounds were selected initially for
evaluation their in vitro activity against MTB H37Rv ATCC 27294
(MTB-1) using the Microplate Alamar Blue Assay (MABA) [23,24].
The minimum inhibitory concentration (MIC) is defined as the
lowest concentration effecting a reduction in fluorescence of ꢀ90%
relative to the mean of replicate bacterium-only controls and MICs
of these compounds along with isoniazid (INH) and rifampicin
(RFP) for comparison are presented in Table 1. The data reveal that
Detailed synthetic pathways to pyrrolidine derivatives 11a,b and
target compounds 19e25 are depicted in Schemes 1 and 2,
respectively. Reduction of readily available pyrrolidones (1a,b)
[20,21] with NaBH4 in methanol gave alcohols (2a,b), which upon
hydroxyl protection by treatment with 3,4-dihydro-2H-pyran
(DHP) in the presence of 4-methylbenzenesulfonic acid (p-TsOH)
yielded esters (3a,b). Aldehydes (5a,b) were prepared via reduction
Fig. 2. Design of the new pyrrolidyl side chains.

T. Zhang et al. / European Journal of Medicinal Chemistry 104 (2015) 73e85
75
Scheme 1. Synthesis of pyrrolidine derivatives 11a,b.
Scheme 2. Synthesis of target compounds 19e25.

76
T. Zhang et al. / European Journal of Medicinal Chemistry 104 (2015) 73e85
six compounds (19a1, 19b2, 20a2, 20b1, 20b2, 22b1) have consid-
erable activity against MTB-1 (MICs: <0.25e0.72 g/mL). The most
active 19b2 was chosen for further evaluation its activity against
MDR-MTB 6133 (MTB-2) clinical isolate which is resistant to both of
INH and RFP, and it was found to have good activity against MTB-2
mL) against all of the tested Gram-positive strains including both of
CPFX- and LVFX-resistant MRSA, MRSE, E. faecium and E. faecalis.
However, our results suggested that the lipophilicity seems not to
be an important parameter affecting both the anti-MTB and anti-
bacterial activity.
m
(MIC: 0.11
mg/mL). Although it is generally believed that simply
increasing the lipophilicity could improve the anti-MTB and anti-
bacterial activity of FQs [25], our results suggest that the lip-
ophilicity of these compounds which is expressed in the term of
their Clog P values (Table 1) seems not to be an important
parameter affecting the anti-MTB activity (20a1 vs 20a2 vs 20a3,
21b1 vs 21b2 vs 21b3).
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, D2O or CDCl3 using tetra-
methylsilane 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. The reagents
were all of analytical grade or chemically pure. TLC was performed
on silica gel plates (Merck, ART5554 60F254).
2.3. Antibacterial activity
The target compounds 19e25 were evaluated for their in vitro
antibacterial activity against representative strains using standard
techniques [26]. Minimum inhibitory concentration (MIC) is
defined as the concentration of the compound required to give
complete inhibition of bacterial growth, and MIC values of 19e25
against Gram-negative and Gram-positive strains along with CPFX
and LVFX for comparison, are listed in Tables 2 and 3, respectively.
These data suggest that most of the target compounds 19e25 have
considerable potency against Gram-positive strains, although they
are generally less active than CPFX and LVFX against the Gram-
negative strains. For example, these target compounds except for
19a, 21a,b, 22a1 and 24a1, show good activity (MICs:
4.2. Synthesis
4.2.1. tert-Butyl 3-ethyl 3-methyl-4-oxopyrrolidine-1,3-
dicarboxylate 1b
To a stirring solution of 1a (43.53 g, 169.2 mmol) dissolved in
anhydrous acetone (846 mL) was added potassium carbonate
(70.14 g, 507.6 mmol). Iodomethane (21.08 mL, 338.4 mmol) was
added dropwise at 0 ꢁC for about 28 min. After the addition was
complete, the reaction mixture was stirred at the same temperature
for 0.5 h and then allowed to stir for another 3 h at room temper-
ature, and then filtered. The filtrate was concentrated under reduce
pressure at below 35 ꢁC to give a yellow oil. The residue was dis-
solved with dichloromethane (600 mL), and washed with water
(100 mL ꢂ 3), brine (80 mL ꢂ 2), dried over anhydrous sodium
sulfate, and evaporated under vacuo. The residue was purified by
column chromatography (silica gel) eluted with petroleum ether
and ethyl acetate (v: v ¼ 5: 1) to afford the title compound 1b
(26.67 g, 58.1%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3)
<0.008e32
mg/mL), against all of the tested seventeen Gram-
positive strains including methicillin-resistant Staphylococcus
aureus (MRSA, two strains) and methicillin-resistant Staphylococcus
epidermidis (MRSE, four strains). Among of them, compounds 22b1
and 23a3 (MICs: <0.008e8
more potent than the two reference drugs CPFX and LVFX (MIC:
0.125 / 128 g/mL) against all of the tested Gram-positive strains
mg/mL) were found to 2e128 times
m
including both of CPFX- and LVFX-resistant MRSA, MRSE, Entero-
coccus faecium and Enterococcus faecalis.
In the case of Gram-positive strains, the activity of some groups
at the N-1 position of naphthyridone derivatives (19e21) is in the
order (1R, 2S)-2-fluorocyclopropyl ꢀ cyclopropyl >> 6-amino-3,5-
difluoro-2-pyridinyl against S.a. and MSSA1-3. In the series of FQ
derivatives (22e25) with same group at the C-7 position, the
contribution of the FQ nucleus to the activity is as follows: AM-1594
nucleus ꢀ LVFX nucleus (22a3 vs 23a3) ꢀ GTFX nucleus (22a1 vs
24a1), but LVFX nucleus ꢀ AM-1594 nucleus (22b1 vs 23b1) when
R2 ¼ Bn. On the other hand, introduction of a methyl group at the 3-
position of the pyrrolidine ring can increase Gram-positive anti-
bacterial activity (19a vs 19b, 20a vs 20b, 21a vs 21b, 22a1 vs 22b1),
which is consistent with the SAR in Yun's study [27]. In addition,
d
(ppm): 4.28 (d, J ¼ 11.8 Hz, 1H), 4.19 (qd, J ¼ 7.1, 1.5 Hz, 2H), 4.05 (t,
J ¼ 19.7 Hz, 1H), 3.81 (d, J ¼ 19.4 Hz, 1H), 3.47 (d, J ¼ 11.8 Hz, 1H),
1.49 (s, 9H), 1.41 (s, 3H), 1.25 (t, J ¼ 7.1 Hz, 3H). MS-ESI (m/z): 289.13
(M þ NH4)þ.
4.2.2. tert-Butyl 3-ethyl 4-hydroxypyrrolidine-1,3-dicarboxylate 2a
To a suspension of NaBH4 (2.74 g, 72.3 mmol) in anhydrous
methanol (290 mL) was added 1a (37.19 g, 144.6 mmol) at below
10 ꢁC. The reaction mixture was stirred at the same temperature for
50 min, and then 20% aqueous acetic acid was added dropwise to
adjust pH 7.0 at 0 ꢁC, and then concentrated under reduced pres-
sure. The residue was dissolved with ethyl acetate (400 mL), and
washed with water (50 mL ꢂ 3), brine (40 mL ꢂ 2), dried over
anhydrous sodium sulfate, and evaporated under vacuo to afford
the title compound 2a (37.11 g, 98.9%) as a pale yellow oil. 1H NMR
naphthyridone derivatives (19e21) featuring
a methyloxime-
incorporated pyrrolidino-substitution at C-7 position are compa-
rable to corresponding analogs containing an ethyloxime or a
benzyloxime with a few exceptions.
(400 MHz, CDCl3)
d (ppm): 4.53e4.42 (m, 1H), 4.16e4.08 (m, 2H),
3. Conclusion
3.36e3.60 (m, 2H), 3.43 (brs, 1H), 3.28e3.25 (brs, 2H), 3.01e2.97
(brs, 1H), 1.42 (s, 9H), 1.28e1.21 (m, 3H). MS-ESI (m/z): 282.75
(M þ Na)þ.
In summary, a series of novel FQ derivatives containing an 3-
alkoxyimino-4-(cyclopropylanimo)methylpyrrolidine moiety were
designed, synthesized and evaluated for their biological activity.
Our results revealed that compound 19b2 shows good activity
4.2.3. tert-Butyl 3-ethyl 4-hydroxy-3-methylpyrrolidine-1,3-
dicarboxylate 2b
against MTB H37Rv ATCC 27294 (MIC: <0.25
6133 (MIC: 0.11 g/mL). On the other hand, most of the target
compounds 19e25 have potent potency against Gram-positive
strains (MICs: <0.008e32 g/mL) with a few exceptions, although
they are generally poor active against Gram-negative strains.
Notedly, 22b1 and 23a3 shows useful activity (MICs: <0.008e8 g/
m
g/mL) and MDR-MTB
The title compound 2b (60.2%) was obtained from 1b in a similar
manner as for the preparation of 2a as a white solid, mp:
m
68.8e70.6 ꢁC. 1H NMR (500 MHz, CDCl3)
d
(ppm): 4.50 (t, J ¼ 6.3 Hz,
m
1H), 4.22e4.16 (m, 3H), 3.74e3.64 (m, 1H), 3.59 (s, 1H), 3.40e3.22
(m, 2H), 1.45 (d, J ¼ 3.8 Hz, 9H), 1.31e1.28 (m, 3H), 1.25 (d, J ¼ 7.3 Hz,
3H). MS-ESI (m/z): 296.16 (M þ Na)þ.
m

T. Zhang et al. / European Journal of Medicinal Chemistry 104 (2015) 73e85
77
Table 1
Structures, physical data and antimycobacterial activity of compounds 19e25.
Compd.
19a1
19a2
19a3
19b1
19b2
19b3
20a1
20a2
20a3
20b1
20b2
20b3
21a1
R1
R2
R3
Y
Mp
½aꢃ2D0
Clog Pc
ꢄ0.38
0.15
MIC (
mg/mL)
[ꢁC]a
(c, CH3OH)b
MTB-1
MTB-2
H
CH3
C2H5
Bn
N
N
N
N
N
N
N
N
N
N
N
N
N
190e192
156e157
160e161
193e195
182e184
127e128
136e139
151e153
112e114
150-152
165e166
>300
0.44
H
H
1.25
CH3
CH3
CH3
H
CH3
C2H5
Bn
0.14
0.67
<0.25
0.11
1.77
CH3
C2H5
Bn
NT
ꢄ0.15
0.38
1.25
0.72
2.10
0.45
0.60
H
41.176ꢁ
(0.034)
H
NT
1.48
CH3
CH3
CH3
H
CH3
C2H5
Bn
40.698ꢁ
(0.086)
0.37
NT
NT
0.90
1.99
CH3
132e133
ꢄ0.45
24.26
22.00
21a2
21a3
21b1
21b2
21b3
22a1
H
C2H5
Bn
N
N
N
N
N
141e142
161e162
155e157
120e121
125e126
287e288
0.07
1.17
0.06
0.59
1.69
0.20
H
>32
CH3
CH3
CH3
H
CH3
C2H5
Bn
15.63
27.74
15.98
CH3
NT
(continued on next page)

78
T. Zhang et al. / European Journal of Medicinal Chemistry 104 (2015) 73e85
Table 1 (continued )
Compd.
R1
R2
R3
Y
Mp
½aꢃ2D0
Clog Pc
1.83
MIC (
mg/mL)
[ꢁC]a
(c, CH3OH)b
MTB-1
MTB-2
22a3
22b1
22b2
H
Bn
149e150
185e186
186-187
NT
NT
CH3
CH3
CH3
C2H5
0.72
0.64
2.76
ꢄ66.667ꢁ
1.25
(0.054)
23a3
23b1
H
Bn
142e143
189e190
NT
NT
1.70
0.59
CH3
CH3
24a1
25a2
H
H
CH3
CeOCH3
CeOCH3
116e118
168e170
0.38
1.13
>32
C2H5
NT
INH
RFP
0.05
0.05
4
>40
a
Melting points are uncorrected.
b
c
NT: not tested due to their limited solubilities or/and poor optical activities.
The Clog P is calculated by Chemoffice 2010 software; INH: Isoniazid; RFP: Rifampicin; MTB-1: MTB H37Rv ATCC 27294; MTB-2: MDR-MTB 6133 resistant to INH and RFP.
4.2.4. tert-Butyl 3-ethyl 4-((tetrahydro-2H-pyran-2-yl)oxy)
pyrrolidine-1,3-dicarboxylate 3a
To a solution of 2a (37.11 g, 143.1 mmol) in dichloromethane
(150 mL) was added p-toluenesulfonic acid monohydrate (1.36 g,
7.1 mmol) at 0 ꢁC. 3,4-2H-dihydropyran (26.1 mL, 286.1 mmol) was
added dropwise at the same temperature for about 40 min. The
reaction mixture was stirred at room temperature for 2 h. The re-
action mixture was diluted with 10% aqueous sodium carbonate
solution (30 mL). The organic phase was washed with water
(50 mL ꢂ 3) and brine (40 mL ꢂ 2), dried over anhydrous sodium
Table 2
In vitro antibacterial activity of compounds 19e25 against Gram-negative strains.
Compd
Strains MIC (mg/mL)
E. coli
E.co.1
E.co.2
E.co.3
E.co.4
K.p.1
K.p.2
K.p.3
K.p.4
K.p.5
K.p.6
K.p.7
P.a.
P.a.1
P.a.2
32
32
64
P.a.3
P.a.4
32
16
64
64
32
128
32
32
64
32
64
64
64
128
128
>128
>128
>128
64
32
32
64
32
64
128
32
2
1
19a1
19a2
19a3
19b1
19b2
19b3
20a1
20a2
20a3
20b1
20b2
20b3
21a1
21a2
21a3
21b1
21b2
21b3
22a1
22a3
22b1
22b2
23a3
23b1
24a1
25a2
LVFX
CPFX
0.5
2
4
0.5
1
1
4
0.03
0.25
1
2
4
4
4
8
16
32
64
0.5
1
2
2
1
2
32
2
0.125
<0.008
16
8
>128
128
>128
128
>128
128
16
8
128
64
64
128
32
32
64
64
64
16
8
128
64
64
128
32
32
64
64
128
64
32
8
128
64
64
128
64
64
128
32
64
>64
64
128
128
>128
>128
>128
8
64
32
32
32
128
32
64
64
64
64
64
32
128
32
128
32
64
128
64
64
64
32
64
64
128
128
128
>128
128
>128
>128
128
128
128
128
64
>128
128
>128
>128
>128
128
>64
64
128
>128
64
128
>128
>32
32
>128
128
16
8
8
8
32
32
128
64
64
128
64
32
64
64
64
32
32
32
32
16
16
16
16
32
32
16
32
64
128
64
>128
>64
2
>128
>128
>128
>128
>128
128
>128
>128
>128
>64
>128
>128
>128
>128
>128
>128
>64
>128
>128
>128
>128
>128
128
>128
>128
>128
>64
>128
>128
>128
>128
>128
>128
>64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>64
>128
>128
>128
128
128
>128
>64
>128
>128
>128
>128
>128
128
>128
>128
>128
>64
>128
>128
>128
>128
>128
>128
>64
64
32
32
64
8
64
16
32
64
8
64
128
128
32
32
64
64
128
64
128
128
128
>128
128
128
>64
64
32
128
64
128
128
32
16
32
16
32
64
16
32
128
128
128
128
64
32
16
64
32
32
128
16
1
8
8
16
32
32
32
32
128
128
64
128
64
16
8
64
32
32
128
32
64
64
32
64
128
64
32
64
64
64
64
128
128
128
128
128
128
>64
64
32
64
64
128
128
32
2
1
128
128
>128
>128
>128
4
32
16
32
64
128
>128
>128
>128
>128
4
32
16
32
32
64
64
128
>128
>128
>128
32
64
16
64
32
>128
>128
>32
8
>128
>128
>128
32
32
16
64
64
>128
>128
>32
16
>64
>64
64
>64
64
64
4
8
4
8
128
4
4
>128
>128
>64
>128
>128
>32
>128
>128
>64
128
>128
>32
>128
>64
>128
>128
>32
>128
>64
>128
>128
>32
32
>128
32
32
>128
32
>128
>128
>32
64
8
32
8
8
8
8
0.5
0.5
0.5
0.5
16
32
0.5
0.5
2
1
8
64
32
16
>128
0.5
E. coli: E. coli ATCC 25922. E.co.1: Extended-spectrum b
-lactamase-producing (ESBLþ) E. coli 14-1. E.co.2: ESBLþ E. coli 14-2. E.co.3: E. coli 14-1. E.co.4: E. coli 14-2. K.p.1: ESBLþ
K. pneumoniae 14e17. K.p.2: ESBLþ K. pneumoniae 14e18. K.p.3: ESBLþ K. pneumoniae 14e19. K.p.4: K. pneumoniae 14-1. K.p.5: K. pneumoniae 14-2. K.p.6: K. pneumoniae 14-3.
K.p.7: K. pneumoniae 14-4. P.a.: P. aeruginosa ATCC 27853. P.a.1: P. aeruginosa 14-9. P.a.2: P. aeruginosa 14-14. P.a.3: P. aeruginosa 14e15. P.a.4: P. aeruginosa 14e16. LVFX:
Levofloxacin. CPFX: Ciprofloxacin.

T. Zhang et al. / European Journal of Medicinal Chemistry 104 (2015) 73e85
79
Table 3
In vitro antibacterial activity of compounds 19e25 against Gram-positive strains.
Compd Strains MIC ( g/mL)
m
S.a.
MSSA1
MSSA2
MSSA3
MRSA1 MRSA2 MSSE1 MRSE1 MRSE MRSE3 MRSE4 S.p.
2
E.fm. 1 E.fm. 2 E.fm. 3 E. fs.1 E. fs.2
19a1
19a2
19a3
19b1
19b2
19b3
20a1
20a2
20a3
20b1
20b2
20b3
21a1
21a2
21a3
21b1
21b2
21b3
22a1
22a3
22b1
22b2
23a3
23b1
24a1
25a2
LVFX
CPFX
<0.008
<0.008
<0.008
<0.008
<0.008
<0.008
<0.008
<0.008
<0.008
<0.008
0.015
0.125
0.25
8
8
8
0.5
0.25
2
8
4
4
4
8
4
8
8
8
0.5
0.25
0.5
16
8
16
2
1
8
16
32
32
8
>128
128
>128
4
8
8
16
4
4
2
8
8
8
2
>128
128
64
16
8
16
>128
64
32
16
16
16
16
4
4
8
32
16
16
4
16
16
16
2
4
4
16
8
8
4
4
16
32
32
128
32
128
128
>64
4
2
8
2
4
32
32
16
4
2
2
64
16
16
2
16
16
32
2
4
2
32
32
64
4
4
2
8
0.25
0.25
0.5
4
1
1
32
32
16
4
4
4
16
4
8
4
4
16
32
64
64
64
128
32
>64
4
16
16
32
4
8
2
32
16
16
4
32
8
8
2
4
64
64
64
16
16
64
128
32
64
16
16
64
16
128
64
128
64
32 16
>64 16
16
8
32
1
0.5
0.125
1
0.5
0.125
0.25
0.5
0.125
1
1
1
2
4
4
8
8
8
0.25
0.5
0.5
8
4
4
1
2
2
16
32
32
16
16
8
16
0.125
0.015
0.03
0.015
0.03
4
2
4
>128
16
32
4
64
32
32
2
1
1
8
0.5
0.25
1
4
2
16
64
4
2
4
2
4
16
64
64
64
32
64
16
>64
4
16
16
8
16
64
128
64
128
64
32
>64
8
16
64
64
128
64
128
64
64
2
2
4
2
16
32
16
64
64
128
64
128
>64
4
2
8
2
2
32
16
32
64
128
128
>128
64
8
32
32
128
128
64
64
128
>128
16
8
16
16
16
16
32
16
64
2
2
4
1
4
128
>128
128
128
128
64
>64
8
8
32
8
16
>128
16
32
0.125
0.015
0.25
0.5
1
16
16
4
16
8
8
32
64
32
64
4
4
16
4
4
4
4
16
0.06
0.015
0.06
0.03
0.03
8
<0.008
<0.008
<0.008
<0.008
<0.008
8
0.06
0.25
0.5
0.125
0.5
0.25
0.125
0.25
0.5
0.5
1
0.25
0.5
4
64
2
4
8
2
4
16
1
4
4
8
4
16
8
8
4
16
>128
8
64
64
8
16
>128
16
64
>128
16
>128
8
8
32
>128
>128
>128
8
64
>128
>128
16
8
>128 >128
8
32
>128
4
64
128
64
>128
8
32
32
>128
4
64
128
>128 64
0.03
0.125
0.125
0.06
0.5
0.5
8
64
128
0.25
0.5
2
32
128
64
2
1
1
>128
>128
16
128
S.a.: S. aureus CMCC 26003. MSSA1: Methicillin-sensitive S. aureus 14-1. MSSA2: Methicillin-sensitive S. aureus 14-3. MSSA3: Methicillin-sensitive S. aureus 14-4. MRSA1:
Methicillin-resistant S. aureus 14-4. MRSA2: Methicillin-resistant S. aureus 14-5. MSSE1: Methicillin-sensitive S. epidermidis 14-2. MRSE1: Methicillin-resistant S. epidermidis
14-21. MRSE2: Methicillin-resistant S. epidermidis 14-22. MRSE3: Methicillin-resistant S. epidermidis 14-37. MRSE4: Methicillin-resistant S. epidermidis 14-39. S.p.:
S. pneumoniae ATCC 19615. E. fm. 1: E. faecium 14-2. E. fm. 2: E. faecium 14-5. E. fm. 3: E. faecium 14-6. E. fs. 1: E. faecalis 14-1. E. fs. 2: E. faecalis 14-2. LVFX: Levofloxacin. CPFX:
Ciprofloxacin.
sulfate, and filtered. The filtrate was concentrated under reduce
pressure. The residue was purified by column chromatography
(silica gel) eluted with petroleum ether and ethyl acetate (v: v ¼ 6:
1) to give the crude product which was recrystallized from petro-
leum ether (about 180 mL) to give the title compound 3a (26.85 g,
54.6%) as a white solid, mp: 89.4e90.4 ꢁC. The mother liquor was
then concentrated to afford an additional 21.20 g (43.2%) of the
the mixture became too thick to stir at 0 ꢁC. The mixture was
filtered and the filter cake was dissolved ethyl acetate (200 mL),
washed successively with water (50 mL ꢂ 2), brine (30 mL). The
organic layer was dried over anhydrous sodium sulfate, and filtered
and concentrated under reduced pressure. The residue was purified
by column chromatography (silica gel) eluted with petroleum ether
and ethyl acetate (v: v ¼ 2: 1) to give the title compound 4a as a
material. 1H NMR (400 MHz, CDCl3)
d
(ppm): 4.73e4.47 (m, 1H),
colorless oil (16.66 g, 72.1%). 1H NMR (400 MHz, CDCl3)
d (ppm):
4.24e4.15 (m, 2H), 3.85e3.52 (m, 4H), 3.15e2.99 (m, 1H), 1.66e1.50
(m, 9H), 1.47 (s, 9H), 1.31e1.25 (m, 3H). MS-ESI (m/z): 366.24
(M þ Na)þ.
4.69e4.50 (m, 1H), 4.23e4.09 (m, 1H), 4.00e3.75 (m, 2H),
3.68e3.61 (m, 2H), 3.53e3.42 (m, 3H), 3.16e3.09 (m, 1H),
2.45e2.40 (brs, 1H), 1.81e1.69 (m, 2H), 1.62e1.54 (m, 4H), 1.45 (s,
9H). MS-ESI (m/z): 324.26 (M þ Na)þ.
4.2.5. tert-Butyl 3-ethyl 3-methyl-4-((tetrahydro-2H-pyran-2-yl)
oxy)pyrrolidine-1,3-dicarboxylate 3b
4.2.7. tert-Butyl 3-(hydroxymethyl)-3-methyl-4-((tetrahydro-2H-
pyran-2-yl)oxy)pyrrolidine-1-carboxylate 4b
The title compound 3b (60.4%) was obtained from 2b in a similar
manner as for the preparation of 3a as a yellow oil. 1H NMR
The title compound 4b (80.6%) was obtained from 3b in a similar
(500 MHz, CDCl3)
d
(ppm): 4.60 (d, J ¼ 18.3 Hz, 1H), 4.44 (dd,
manner as for the preparation of 4a as a colorless oil. 1H NMR
J ¼ 48.5, 4.9 Hz,1H), 4.07 (q, J ¼ 6.9 Hz, 2H), 3.73 (dd, J ¼ 18.7, 9.2 Hz,
2H), 3.64e3.54 (m, 1H), 3.49e3.41 (m, 1H), 3.32e3.13 (m, 2H), 1.71
(d, J ¼ 5 Hz, 1H), 1.62 (d, J ¼ 10 Hz, 1H), 1.49e1.44 (m, 4H), 1.36 (s,
9H), 1.27e1.17 (m, 6H). MS-ESI (m/z): 380.24 (M þ Na)þ.
(400 MHz, CDCl3)
d
(ppm): 4.47 (d, J ¼ 8 Hz, 1H), 3.96 (t, J ¼ 10.4 Hz,
2H), 3.86e3.80 (m, 1H), 3.59e3.27 (m, 6H), 3.14e3.03 (m, 1H),
1.78e1.73 (m, 2H), 1.51 (d, J ¼ 4.6 Hz, 4H), 1.44 (s, 9H), 1.07 (s, 3H).
MS-ESI (m/z): 316.39 (M þ H)þ.
4.2.6. tert-Butyl 3-(hydroxymethyl)-4-((tetrahydro-2H-pyran-2-yl)
oxy)pyrrolidine-1-carboxylate 4a
4.2.8. tert-Butyl 3-formyl-4-((tetrahydro-2H-pyran-2-yl)oxy)
pyrrolidine-1-carboxylate 5a
To a solution of tetrahydrofuran (500 mL) was added Lithium
aluminum hydride (3.49 g, 92.0 mmol) in portions at 0 ꢁC, and 3a
(26.34 g, 76.7 mmol) in tetrahydrofuran (100 mL) was added
dropwise within 20 min. The reaction mixture was stirred at room
temperature for 2 h. Water (3.5 mL), 7% aqueous sodium hydroxide
solution (3.5 mL), and water (10.5 mL) was added successively until
To a solution of 4a (16.66 g, 55.3 mmol) and in dichloromethane
(150 mL) was added Dess-Martin periodinane (30.47 g, 71.8 mmol)
in portions at room temperature, and then added slowly
dichloromethane (1300 mL) containing water (1.3 mL). The clear
solution grew cloudy toward the end of wet dichloromethane
addition, which required 30 min. The reaction mixture was stirred

80
T. Zhang et al. / European Journal of Medicinal Chemistry 104 (2015) 73e85
at room temperature for 3.5 h and then filtered. The filtrate was
washed with 10% Na2S2O3: saturated NaHCO3 (300 mL, v: v ¼ 1: l),
water (200 mL ꢂ 2) and brine (100 mL). The organic layer was dried
over anhydrous sodium sulfate, filtered and concentrated under
reduced pressure to give the title compound 5a as a colorless oil
oil (10.57 g, 52.0%). 1H NMR (400 MHz, CDCl3)
d (ppm): 4.68 (m,1H),
4.10e3.97 (m,1H), 3.87e3.77 (m,1H), 3.65e3.38 (m, 2H), 3.34e3.27
(m, 2H), 3.28e3.03 (m, 2H), 2.64 (brs, 1H), 2.58 (brs, 1H), 1.80e1.67
(m, 2H), 1.57e1.53 (m, 5H), 1.45 (s, 18H), 0.87e0.85 (m, 2H),
0.59e0.55 (m, 2H). MS-ESI (m/z): 463.28 (M þ Na)þ.
(15.08 g, 91.1%). 1H NMR (400 MHz, CDCl3)
d (ppm): 9.86 (s, 1H),
4.84e4.64 (m, 3H), 3.82e3.75 (m, 3H), 3.62e3.51 (m, 2H),1.77e1.62
4.2.13. tert-Butyl 3-(((tert-butoxycarbonyl) (cyclopropyl)amino)
methyl)-3-methyl-4-((tetrahydro-2H-pyran-2-yl)oxy)pyrrolidine-
1-carboxylate 7b
(m, 7H), 1.53 (s, 9H). MS-ESI (m/z): 322.26 (M þ Na)þ.
4.2.9. tert-Butyl 3-formyl-3-methyl-4-((tetrahydro-2H-pyran-2-yl)
oxy)pyrrolidine-1-carboxylate 5b
The title compound 7b (62.1%) was obtained from 6b in a similar
manner as for the preparation of 7a as a light yellow oil. 1H NMR
The title compound 5b (74.9%) was obtained from 4b in a similar
(500 MHz, CDCl3)
d
(ppm): 4.63 (d, J ¼ 13.9 Hz, 1H), 4.03e3.96 (m,
manner as for the preparation of 5a as a colorless oil. 1H NMR
1H), 3.87e3.77 (m, 2H), 3.59e3.38 (m, 4H), 3.35e3.06 (m, 2H),
2.55e2.43 (m, 1H), 1.79 (d, J ¼ 14.8 Hz, 1H), 1.75e1.68 (m, 1H),
1.58e1.53 (m, 4H),1.47 (d, J ¼ 5.0 Hz, 9H),1.46 (s, 9H), 1.04e0.96 (m,
3H), 0.89e0.79 (m, 1H), 0.74 (s, 1H), 0.62e0.54 (m, 2H). MS-ESI (m/
z): 477.34 (M þ Na)þ.
(500 MHz, CDCl3)
d
(ppm): 9.77 (t, J ¼ 17.0 Hz, 1H), 4.65 (d,
J ¼ 5.0 Hz, 1H), 4.30 (d, J ¼ 17.8 Hz, 1H), 3.93e3.86 (m, 1H), 3.72 (t,
J ¼ 9.9 Hz, 1H), 3.59e3.40 (m, 3H), 3.27e3.17 (m, 1H), 1.69 (d,
J ¼ 11.4 Hz, 2H), 1.54 (d, J ¼ 18.4 Hz, 4H), 1.47 (s, 9H), 1.22 (s, 3H).
MS-ESI (m/z): 314.00 (M þ H)þ.
4.2.14. tert-Butyl 3-(((tert-butoxycarbonyl) (cyclopropyl)amino)
methyl)-4-hydroxypyrrolidine-1-carboxylate 8a
4.2.10. tert-Butyl 3-((cyclopropylamino)methyl)-4-((tetrahydro-
2H-pyran-2-yl)oxy)pyrrolidine-1-carboxylate 6a
To a solution of 7a (1.14 g, 2.6 mmol) in the anhydrous methanol
was added methanesulfonic acid (0.19 mL, 2.9 mmol) at room
temperature. The reaction mixture was stirred at the same tem-
perature for 25 min and then saturated aqueous sodium bicar-
bonate solution (about 2 mL) was added to adjust pH to 7.0e8.0.
The solvent was evaporated, and the residue was dissolved in 50 mL
of ethyl acetate, washed with water (15 mL ꢂ 2), brine (20 mL),
dried over anhydrous sodium sulfate, filtered and evaporated. The
residue was purified by column chromatography (silica gel) eluted
with petroleum ether and ethyl acetate (v: v ¼ 5 / 3: 1) to give the
title compound 8a as a yellow oil (0.70 g, 75.9%). 1H NMR (400 MHz,
To
a solution of 5a (15.08 g, 50.4 mmol) and in 1,2-
dichloroethane (150 mL) was added cyclopropanamine (3.8 mL,
54.8 mmol) at room temperature. The reaction mixture was stirred
at the same temperature for 1e5 h, and then added sodium tri-
acetoxyborohydride solid (12.80 g, 60.4 mmol) in portions at the
same temperature. The reaction mixture was stirred at the same
temperature for 4 h and then added saturated aqueous sodium
bicarbonate solution (160 mL) cautiously. The organic phase was
separated and the aqueous phase was extracted with dichloro-
methane (50 mL ꢂ 2). The combined organic phases were washed
with brine (40 mL ꢂ 3), dried over sodium sulfate, filtered and
evaporated, the residue was purified by column chromatography
(silica gel) eluted with methanol and dichloromethane (v: v ¼ 1: 5)
to give the title compound 6a as a light yellow oil (15.71 g, 91.6%).
CDCl3)
d (ppm): 4.06 (s, 1H), 3.81e3.75 (m, 1H), 3.51e3.44 (m, 3H),
3.07 (t, J ¼ 8 Hz, 1H), 2.92e2.88 (m, 1H), 2.52e2.50 (m, 1H),
2.28e2.26 (brs, 1H), 1.56 (s, 1H), 1.48 (s, 9H), 1.45 (s, 9H), 0.86e0.82
(m, 1H), 0.79e0.73 (m, 2H), 0.70e0.54 (m, 1H). MS-ESI (m/z):
379.25 (M þ Na)þ.
1H NMR (400 MHz, CDCl3)
d (ppm): 4.70 (s, 1H), 4.20 (s, 1H),
3.88e3.85 (brs, 1H), 3.53e3.48 (m, 4H), 3.16e3.12 (m, 1H),
2.91e2.89 (m, 1H), 2.75e2.72 (m, 1H), 2.33 (brs, 1H), 2.13e2.10 (m,
1H), 1.83e1.70 (m, 3H), 1.71e1.58 (m, 4H), 1.53 (s, 9H), 0.45e0.33
(m, 4H). MS-ESI (m/z): 341.22 (M þ H)þ.
4.2.15. tert-Butyl 3-(((tert-butoxycarbonyl) (cyclopropyl)amino)
methyl)-3-methyl-4-hydroxypyrrolidine-1-carboxylate 8b
The title compound 8b (72.6%) was obtained from 7b in a similar
manner as for the preparation of 8a as a white solid, mp:
4.2.11. tert-Butyl 3-((cyclopropylamino)methyl)-3-methyl-4-
((tetrahydro-2H-pyran-2-yl)oxy)pyrrolidine-1-carboxylate 6b
The title compound 6b (82.2%) was obtained from 5b in a similar
manner as for the preparation of 6a as a light yellow oil. 1H NMR
108.3e109.2 ꢁC. 1H NMR (500 MHz, CDCl3)
d (ppm): 5.36 (s, 1H),
3.80 (t, J ¼ 15.6 Hz, 1H), 3.71 (s, 1H), 3.57 (ddd, J ¼ 28.6, 12.0, 4.1 Hz,
1H), 3.48e3.36 (m, 1H), 3.21e3.13 (m, 1H), 3.05 (d, J ¼ 10 Hz, 1H),
2.76 (dd, J ¼ 31.2, 14.7 Hz, 1H), 2.63e2.58 (m, 1H), 1.48 (s, 9H), 1.45
(s, 9H), 1.04 (d, J ¼ 5.4 Hz, 3H), 0.91e0.85 (m, 1H), 0.79e0.70 (m,
1H), 0.66e0.57 (m, 2H). MS-ESI (m/z): 393.42 (M þ Na)þ.
(500 MHz, CDCl3)
d (ppm): 4.66e4.52 (m, 1H), 3.90e3.75 (m, 2H),
3.66e3.55 (m, 1H), 3.51e3.46 (m, 2H), 3.30e3.25 (m, 1H), 3.10e3.02
(m, 1H), 2.85e2.82 (m, 1H), 2.66e2.59 (m, 1H), 2.12e2.07 (m, 1H),
1.78 (d, J ¼ 8.9 Hz, 1H), 1.71e1.68 (m, 2H), 1.52 (d, J ¼ 4.4 Hz, 3H),
1.44 (s, 9H), 1.24 (s, 1H), 1.03 (d, J ¼ 3.6 Hz, 1H), 0.99 (d, J ¼ 2.5 Hz,
2H), 0.39 (d, J ¼ 5.9 Hz, 2H), 0.26 (d, J ¼ 2.3 Hz, 2H). MS-ESI (m/z):
355.25 (M þ H)þ.
4.2.16. tert-Butyl 3-(((tert-butoxycarbonyl) (cyclopropyl)amino)
methyl)-4-oxopyrrolidine-1-carboxylate 9a
The title compound 9a was obtained from 8a and Dess-Martin
periodinane in a similar manner as for the preparation of 5a from
4a, and then purified by column chromatography (silica gel) eluted
with petroleum ether and ethyl acetate (v: v ¼ 4: 1) to give the title
compound 9a as a yellow oil (72.8%). 1H NMR (400 MHz, CDCl3)
4.2.12. tert-Butyl 3-(((tert-butoxycarbonyl) (cyclopropyl)amino)
methyl)-4-((tetrahydro-2H-pyran-2-yl)oxy)pyrrolidine-1-
carboxylate 7a
d
(ppm): 3.99 (brs, 1H), 3.79e3.74 (m, 2H), 3.63 (brs, 1H), 3.47e3.42
To a solution of 6a (15.71 g, 46.1 mmol) in methanol (150 mL)
was added di-tert butyl dicarbonate (10.58 g, 48.5 mmol) at room
temperature. The reaction mixture was stirred at the same tem-
perature for 3 h and then evaporated. The remaining yellow oil was
dissolved in 100 mL of ethyl acetate, washed with water
(30 mL ꢂ 2), brine (30 mL), dried over anhydrous sodium sulfate,
filtered and evaporated. The residue was purified by column
chromatography (silica gel) eluted with petroleum ether and ethyl
acetate (v: v ¼ 7: 1) to give the title compound 7a as a light yellow
(m, 2H), 2.93e2.90 (m, 1H), 2.47 (s, 1H), 1.47 (s, 18H), 0.76 (brs, 2H),
0.61 (brs, 2H). MS-ESI (m/z): 377.36 (M þ Na)þ.
4.2.17. tert-Butyl 3-(((tert-butoxycarbonyl) (cyclopropyl)amino)
methyl)-3-methyl-4-oxopyrrolidine-1-carboxylate 9b
The title compound 9b (91.4%) was obtained from 8b in a similar
manner as for the preparation of 9a as a yellow oil. 1H NMR
(500 MHz, CDCl3)
1H), 3.27 (dd, J ¼ 39.5, 14.1 Hz, 1H), 2.43 (d, J ¼ 30.5 Hz, 1H), 1.48 (s,
d
(ppm): 3.96e3.59 (m, 4H), 3.44 (d, J ¼ 11.5 Hz,

T. Zhang et al. / European Journal of Medicinal Chemistry 104 (2015) 73e85
81
9H), 1.46 (s, 9H), 1.18 (s, 3H), 0.89e0.81 (m, 1H), 0.74e0.66 (m, 1H),
4.2.23. tert-Butyl 3-((benzyloxy)imino)-3-methyl-4-(((tert-
butoxycarbonyl) (cyclopropyl)amino)methyl)pyrrolidine-1-
carboxylate 10b3
The title compound 10b3 (59.8%) was obtained from 9b and O-
benzylhydroxylamine hydrochloride in a similar manner as for the
preparation of 10a1 as a light yellow oil. 1H NMR (500 MHz, CDCl3)
0.64e0.59 (m, 1H), 0.55e0.53 (m, 1H). MS-ESI (m/z): 391.41
(M þ Na)þ.
4.2.18. tert-Butyl 3-(((tert-butoxycarbonyl) (cyclopropyl)amino)
methyl)-4-(methoxyimino)pyrrolidine-1-carboxylate 10a1
d
(ppm): 7.34 (s, 2H), 7.31 (s, 3H), 5.13e5.05 (m, 2H), 4.18e4.08 (m,
To a solution of methoxyamine hydrochloride (0.30 g, 3.6 mmol)
in the methanol (20 mL) was added sodium hydroxide (0.15 g,
3.7 mmol) at room temperature. The reaction mixture was stirred
at the same temperature for 30 min. After the addition of 9a (1.17 g,
3.3 mmol) was complete, the mixture was heated at reflux for 8 h.
The solvent was evaporated, and the residue was dissolved in 20 mL
of water. The mixture was extracted with ethyl acetate (40 mL ꢂ 3).
The combined organic layers were washed with brine (20 mL),
dried over anhydrous sodium sulfate, filtered and evaporated. The
residue was purified by column chromatography (silica gel) eluted
with petroleum ether and ethyl acetate (v: v ¼ 6: 1) to give the title
compound 10a1 as a light yellow oil (0.68 g, 53.7%). 1H NMR
2H), 3.70e3.57 (m, 2H), 3.30e3.11 (m, 2H), 2.49e2.43 (m, 1H), 1.46
(s, 18H), 1.26 (d, J ¼ 4.5 Hz, 3H), 0.89e0.77 (m, 2H), 0.63e0.48 (m,
2H). MS-ESI (m/z): 474.14 (M þ H)þ.
4.2.24. 4-((Cyclopropylamino)methyl)pyrrolidin-3-one O-methyl
oxime dihydrochloride 11a1
To a stirring solution of 10a1 (3.06 g, 8.0 mmol) dissolved in
anhydrous dichloromethane (200 mL) was pumped dried hydro-
chloride gas at room temperature for 2 h. The reaction mixture was
stirred for 6 h at the same temperature, and then filtered. The
precipitate was washed with anhydrous ethyl acetate (20 mL), and
dried in vacuo at 45 ꢁC for 10 h to yield the title compound 11a1
(1.70 g, 83.1%) as an off-white solid, mp: 157.4e158.2 ꢁC. 1H NMR
(400 MHz, CDCl3) d (ppm): 4.12 (2H, s), 3.88 (3H, s), 3.65e3.53 (1H,
m), 3.41 (1H, brs), 3.30e3.23 (1H, m), 2.60 (1H, s), 1.66e1.62 (2H,
m), 1.51 (18H, s), 0.82e0.79 (2H, m), 0.71e0.62 (2H, m). MS-ESI (m/
z): 383.88 (M þ H)þ.
(400 MHz, DMSO-d6 þ D2O exchanged)
d (ppm): 4.05e3.91 (m, 2H),
3.89 (s, 3H), 3.74e3.70 (m, 1H), 3.38e3.35 (m, 2H), 3.32e3.24 (m,
2H), 2.78e2.71 (m, 1H), 0.86e0.82 (m, 4H). MS-ESI (m/z): 184.11
(M þ H)þ.
4.2.19. tert-Butyl 3-(((tert-butoxycarbonyl) (cyclopropyl)amino)
methyl)-3-methyl-4-(methoxyimino)pyrrolidine-1-carboxylate
10b1
The title compound 10b1 (81.5%) was obtained from 9b and
methoxyamine hydrochloride in a similar manner as for the prep-
aration of 10a1 as a light yellow oil. 1H NMR (500 MHz, CDCl3)
4.2.25. 4-((Cyclopropylamino)methyl)-4-methylpyrrolidin-3-one
O-methyl oxime dihydrochloride 11b1
The title compound 11b1 (92.8%) was obtained from 10b1 in a
similar manner as for the preparation of 11a1 as an off-white solid,
d
(ppm): 4.16e4.05 (m, 2H), 3.86 (s, 3H), 3.71 (s, 1H), 3.60 (t,
mp: 117.6e118.9 ꢁC. 1H NMR (500 MHz, DMSO-d6)
d (ppm):
J ¼ 16.1 Hz, 1H), 3.25e3.19 (m, 1H), 3.10 (d, J ¼ 13.9 Hz, 1H), 2.57 (s,
1H), 1.46 (s, 9H), 1.46 (s, 9H), 1.29 (s, 3H), 0.85 (s, 1H), 0.74e0.69 (m,
1H), 0.63e0.58 (m,1H), 0.55 (d, J ¼ 4.3 Hz,1H). MS-ESI (m/z): 398.11
(M þ H)þ.
4.05e3.98 (m, 2H), 3.87 (s, 3H), 3.70e3.57 (m, 3H), 3.40e3.32 (m,
1H), 2.76 (s, 1H), 1.37 (s, 3H), 1.05e1.01 (m, 2H), 0.75 (d, J ¼ 6.9 Hz,
2H). MS-ESI (m/z): 198.10 (M þ H)þ.
4.2.26. 4-((Cyclopropylamino)methyl)pyrrolidin-3-one O-ethyl
oxime dihydrochloride 11a2
4.2.20. tert-Butyl 3-(((tert-butoxycarbonyl) (cyclopropyl)amino)
methyl)-4-(ethoxyimino)pyrrolidine-1-carboxylate 10a2
The title compound 10a2 (88.7%) was obtained from 9a and
ethoxyamine hydrochloride in a similar manner as for the prepa-
ration of 10a1 as an off-white solid, mp: 158.7e160.1 ꢁC. 1H NMR
The title compound 11a2 (83.1%) was obtained from 10a2 in a
similar manner as for the preparation of 11a1 as an off-white solid,
mp: 180.7e182.1 ꢁC. 1H NMR (400 MHz, CDCl3)
d (ppm): 4.13 (q,
J ¼ 7.0 Hz, 2H), 3.92 (dd, J ¼ 37.7, 17.1 Hz, 2H), 3.74 (dd, J ¼ 10.7,
7.4 Hz, 1H), 3.42 (d, J ¼ 8 Hz, 1H), 3.33e3.23 (m, 3H), 2.75 (s, 1H),
1.22 (t, J ¼ 7.0 Hz, 3H), 0.99e0.68 (m, 4H). MS-ESI (m/z): 198.05
(M þ H)þ.
(400 MHz, CDCl3)
d (ppm): 4.17e4.06 (m, 4H), 3.66e3.19 (m, 4H),
2.55 (s, 1H), 1.69 (s, 1H), 1.46 (s, 9H), 1.45 (s, 9H), 1.23 (t, J ¼ 6.7 Hz,
3H), 0.87e0.58 (m, 4H). MS-ESI (m/z): 398.05 (M þ H)þ.
4.2.21. tert-Butyl 3-(((tert-butoxycarbonyl) (cyclopropyl)amino)
methyl)-3-methyl-4-(ethoxyimino)pyrrolidine-1-carboxylate 10b2
The title compound 10b2 (81.5%) was obtained from 9b and
ethoxyamine hydrochloride in a similar manner as for the prepa-
ration of 10a1 as a light yellow oil. 1H NMR (500 MHz, CDCl3)
4.2.27. 4-((Cyclopropylamino)methyl)-4-methylpyrrolidin-3-one
O-ethyl oxime dihydrochloride 11b2
The title compound 11b2 (68.2%) was obtained from 10b2 in a
similar manner as for the preparation of 11a1 as a yellow solid, mp:
146.5e147.3 ꢁC. 1H NMR (500 MHz, DMSO-d6)
d (ppm): 4.11 (t,
d
(ppm): 4.07 (q, J ¼ 17.7 Hz, 4H), 3.70 (s, 1H), 3.57 (t, J ¼ 15.3 Hz,
J ¼ 7.0 Hz, 2H), 4.00 (s, 2H), 3.69 (d, J ¼ 11.9 Hz,1H), 3.41 (s, 1H), 3.33
(d, J ¼ 12.0 Hz, 1H), 3.26 (s, 1H), 2.75 (s, 1H), 1.37 (s, 3H), 1.21 (t,
J ¼ 7.0 Hz, 3H),1.04 (dd, J ¼ 27.6, 8.0 Hz, 2H), 0.78e0.72 (m, 2H). MS-
ESI (m/z): 212.14 (M þ H)þ.
1H), 3.20 (dd, J ¼ 23.2, 12.3 Hz, 1H), 3.08 (d, J ¼ 14.3 Hz, 1H), 2.53 (s,
1H), 1.44 (s, 9H), 1.43 (s, 9H), 1.26 (s, 3H), 1.23e1.20 (m, 3H),
0.84e0.76 (m, 1H), 0.70e0.66 (m, 1H), 0.59e0.52 (m, 2H). MS-ESI
(m/z): 412.05 (M þ H)þ.
4.2.22. tert-Butyl 3-((benzyloxy)imino)-4-(((tert-butoxycarbonyl)
(cyclopropyl)amino)methyl)pyrrolidine-1-carboxylate 10a3
4.2.28. 4-((Cyclopropylamino)methyl)pyrrolidin-3-one O-benzyl
oxime dihydrochloride 11a3
The title compound 10a3 (80.6%) was obtained from 9a and O-
benzylhydroxylamine hydrochloride in a similar manner as for the
preparation of 10a1 as a light yellow oil. 1H NMR (400 MHz, CDCl3)
The title compound 11a3 (89.3%) was obtained from 10a3 in a
similar manner as for the preparation of 11a1 as an off-white solid,
mp: 179.4e180.6 ꢁC. 1H NMR (500 MHz, DMSO-d6)
d (ppm):
d
(ppm): 7.37e7.32 (m, 5H), 5.08 (s, 2H), 4.14e4.11 (m, 2H),
7.40e7.12 (m, 5H), 5.15 (s, 2H), 3.98 (dd, J ¼ 42.5, 16.9 Hz, 2H), 3.75
(dd, J ¼ 10.9, 7.8 Hz, 1H), 3.41 (dd, J ¼ 32.3, 21.5 Hz, 4H), 2.72 (s, 1H),
0.94 (dd, J ¼ 34.9, 11.2 Hz, 2H), 0.77e0.68 (m, 2H). MS-ESI (m/z):
260.70 (M þ H)þ.
3.68e3.61 (m, 1H), 3.50e3.21 (m, 3H), 2.51 (s, 1H), 1.70e1.58 (m,
1H), 1.46 (s, 18H), 0.86e0.57 (m, 4H). MS-ESI (m/z): 460.16
(M þ H)þ.

82
T. Zhang et al. / European Journal of Medicinal Chemistry 104 (2015) 73e85
4.2.29. 4-((Cyclopropylamino)methyl)-4-methylpyrrolidin-3-one
O-benzyl oxime dihydrochloride 11b3
112.74, 108.93, 70.47, 51.40, 50.37, 48.85, 34.93, 30.64, 29.85, 14.82,
7.62, 7.55, 5.85, 5.72. MS-ESI (m/z): 444.20 (M þ H)þ. HRMS-ESI (m/
z): Calcd. for C22H27O4N5F (M þ H)þ: 444.20416; Found: 444.20380.
The title compound 11b3 (36.3%) was obtained from 10b3 in a
similar manner as for the preparation of 11a1 as a yellow solid
easily absorbing moisture. 1H NMR (500 MHz, DMSO-d6)
d
(ppm):
4.2.30.4. 1-Cyclopropyl-7-(3-((cyclopropylamino)methyl)-4-(ethox-
yimino)-3-methylpyrrolidin-1-yl)-6-fluoro-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxylic acid 19b2. The title compound 19b2
was obtained from 11b2 and 12 as a yellow solid (31.0%). 1H NMR
7.36e7.16 (m, 5H), 5.14e5.14 (m, 2H), 3.91e3.80 (m, 2H), 3.68 (dd,
J ¼ 11.1, 8.8 Hz, 1H), 3.23e3.18 (m, 1H), 3.11e2.96 (m, 2H), 2.72 (s,
1H), 1.48e1.25 (m, 3H), 1.08e0.95 (m, 2H), 0.76e0.67 (m, 2H). MS-
ESI (m/z): 274.18 (M þ H)þ.
(500 MHz, CDCl3)
d
(ppm): 8.68 (s, 1H), 8.00 (d, J ¼ 12.5 Hz, 1H),
4.64e4.55 (m, 2H), 4.18e4.12 (m, 3H), 3.75 (d, J ¼ 11.0 Hz, 1H), 3.65
(td, J ¼ 7.3, 3.7 Hz, 1H), 2.98 (d, J ¼ 12.0 Hz, 1H), 2.83 (d, J ¼ 12.0 Hz,
1H), 2.20e2.16 (m, 1H), 1.32 (s, 3H), 1.28 (dd, J ¼ 7.1, 2.4 Hz, 5H),
1.08e1.06 (m, 2H), 0.46e0.41 (m, 2H), 0.31 (s, 2H). 13C NMR
4.2.30. General procedure for the synthesis of target compounds
19e21
To a suspension of 11a,b (1.6 mmol) in dry acetonitrile (20 mL)
was added triethylamine (7.9 mmol), and then stirred for 0.5 h at
room temperature. To the reaction mixture was added 12e14
(0.8 mmol), and then stirred at 50 ꢁC for another 3 h under an at-
mosphere of nitrogen and then concentrated under reduced pres-
sure. The residue was dissolved with 5% aqueous acetic acid
solution (6.5 mL), and then stirred for 0.5 h at 70 ꢁC. After cooling to
room temperature, ammonia water (12%) was added dropwise to
adjust pH 6.5e7.0 and then filtered. The precipitate was washed
with water (20 mL), and dried in vacuo at 60 ꢁC for 10 h to yield the
title compounds 19e21.
(125 MHz, CDCl3)
d
(ppm): 177.20, 167.04, 149.46 (d, J ¼ 12.5 Hz),
147.81, 147.43, 146.37, 145.75, 118.80 (d, J ¼ 21.3 Hz), 112.55, 108.94,
70.13, 58.13, 55.90, 49.05, 49.00, 34.86, 31.15, 23.06,14.82, 7.59, 7.52,
6.76 ꢂ 2. MS-ESI (m/z): 458.37 (M þ H)þ. HRMS-ESI (m/z): Calcd. for
C
23H29O4N5F (M þ H)þ: 458.21981; Found: 458.22025.
4.2.30.5. 7-(3-((Benzyloxy)imino)-4-((cyclopropylamino)methyl)
pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxylic acid 19a3. The title compound 19a3
was obtained from 11a3 and 12 as a yellow solid (11.2%). 1H NMR
(600 MHz, CDCl3)
d
(ppm): 8.68 (s, 1H), 8.02 (d, J ¼ 12.3 Hz, 1H),
4.2.30.1. 1-Cyclopropyl-7-(3-((cyclopropylamino)methyl)-4-
(methoxyimino)pyrrolidin-1-yl)-6-fluoro-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxylic acid 19a1. The title compound 19a1
was obtained from 11a1 and 12 as an off-white solid (35.6%). 1H
7.39e7.36 (m, 4H), 7.35e7.26 (m, 1H), 5.16 (s, 2H), 4.63 (s, 2H), 4.24
(s, 1H), 3.86 (s, 1H), 3.66e3.62 (m, 1H), 3.27 (s, 1H), 3.00e2.94 (m,
2H), 2.14 (dt, J ¼ 9.8, 3.2 Hz, 1H), 1.27 (q, J ¼ 6.5 Hz, 2H), 1.06 (q,
J ¼ 6.1 Hz, 2H), 0.46e0.45 (m, 2H), 0.34 (s, 2H). 13C NMR (151 MHz,
NMR (400 MHz, DMSO-d6)
d
(ppm): 8.60 (s, 1H), 8.04 (d,
CDCl3)
d
(ppm): 177.04, 166.80, 149.09 (d, J ¼ 12.2 Hz), 147.43,
J ¼ 12.7 Hz, 1H), 4.57 (s, 2H), 4.17 (s, 1H), 3.92 (s, 1H), 3.84 (s, 3H),
3.73 (d, J ¼ 3.9 Hz, 1H), 3.19 (s, 1H), 2.92 (dd, J ¼ 12.0, 5.2 Hz, 1H),
2.73 (dd, J ¼ 11.9, 8.8 Hz, 1H), 2.12e2.09 (m, 1H), 1.19 (d, J ¼ 6.8 Hz,
2H), 1.10 (d, J ¼ 3.8 Hz, 2H), 0.39e0.37 (m, 2H), 0.22 (dd, J ¼ 9.9,
147.20, 146.31, 145.71, 137.55, 128.47, 128.19 ꢂ 2, 128.08 ꢂ 2, 118.78
(d, J ¼ 20.7 Hz), 112.55, 108.83, 76.53, 51.35, 50.62, 49.05, 49.01,
34.73, 30.18, 7.44,7.41, 6.41, 6.3. MS-ESI (m/z): 506.19 (M þ H)þ.
HRMS-ESI (m/z): Calcd. for C27H29O4N5F (M þ H)þ: 506.21981;
Found: 506.21951.
3.2 Hz, 2H). 13C NMR (101 MHz, DMSO-d6)
d (ppm): 177.04, 166.24,
149.12 (d, J ¼ 12.2 Hz), 147.54, 147.29, 146.47, 145.82, 118.96 (d,
J ¼ 20.1 Hz), 112.74, 108.93, 62.24, 51.34, 50.37, 48.85, 35.40, 30.44,
29.85, 7.34 ꢂ 2, 6.92, 6.48. MS-ESI (m/z): 430.29 (M þ H)þ. HRMS-
ESI (m/z): Calcd. for C21H25O4N5F (M þ H)þ: 430.18851; Found:
430.18799.
4.2.30.6. 7-(4-((Benzyloxy)imino)-3-((cyclopropylamino)methyl)-3-
methylpyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-
1,8-naphthyridine-3-carboxylic acid 19b3. The title compound 19b3
was obtained from 11b3 and 12 as a yellow solid (15.1%). 1H NMR
(400 MHz, CDCl3)
d
(ppm): 8.62 (s, 1H), 8.03 (d, J ¼ 12.3 Hz, 1H),
4.2.30.2. 1-Cyclopropyl-7-(3-((cyclopropylamino)methyl)-4-
(methoxyimino)-3-methylpyrrolidin-1-yl)-6-fluoro-4-oxo-1,4-
dihydro-1,8-naphthyridine-3-carboxylic acid 19b1. The title com-
pound 19b1 was obtained from 11b1 and 12 as an off-white solid
7.41e7.25 (m, 5H), 5.17 (s, 2H), 4.67 (s, 2H), 4.21 (s, 1H), 3.87 (s, 1H),
3.67e3.63 (m, 1H), 3.01e2.92 (m, 2H), 2.18e2.15 (m, 1H), 1.32 (s,
3H), 1.27 (q, J ¼ 6.5 Hz, 2H), 1.06 (q, J ¼ 6.1 Hz, 2H), 0.46e0.45 (m,
2H), 0.34 (s, 2H). MS-ESI (m/z): 520.40 (M þ H)þ. HRMS-ESI (m/z):
Calcd. for C28H31O4N5F (M þ H)þ: 520.23546; Found: 520.23592.
(23.8%). 1H NMR (600 MHz, CDCl3)
d (ppm): 8.70 (s, 1H), 8.03 (d,
J ¼ 12.4 Hz, 1H), 4.64e4.55 (m, 2H), 4.14 (d, J ¼ 10.6 Hz, 1H), 3.93 (s,
3H), 3.75 (d, J ¼ 8.2 Hz, 1H), 3.66 (td, J ¼ 7.4, 3.7 Hz, 1H), 2.98 (d,
J ¼ 12.0 Hz, 1H), 2.83 (d, J ¼ 12.0 Hz, 1H), 2.19e2.16 (m, 1H), 1.32 (s,
3H), 1.29 (td, J ¼ 6.9, 3.6 Hz, 2H), 1.09e1.07 (m, 2H), 0.47e0.41 (m,
4.2.30.7. 7-(3-((Cyclopropylamino)methyl)-4-(methoxyimino)pyrro-
lidin-1-yl)-6- fluoro-1-((1R,2S)-2-fluorocyclopropyl)-4-oxo-1,4-
dihydro-1,8-naphthyridine-3-carboxylic acid 20a1. The title com-
pound 20a1 was obtained from 11a1 and 13 as a yellow solid
2H), 0.30 (s, 2H). 13C NMR (151 MHz, CDCl3)
d (ppm): 177.21, 167.04,
149.40 (d, J ¼ 11.7 Hz), 147.61, 147.41, 146.38, 145.89, 118.88 (d,
J ¼ 20.7 Hz), 112.60, 108.96, 62.42, 58.08, 55.91, 49.00, 48.96, 34.86,
31.13, 23.03, 7.61, 7.53, 6.83 ꢂ 2. MS-ESI (m/z): 444.50 (M þ H)þ.
HRMS-ESI (m/z): Calcd. for C22H27O4N5F (M þ H)þ: 444.20416;
Found: 444.20435.
(33.8%). 1H NMR (600 MHz, DMSO-d6)
d (ppm): 8.70 (s, 1H), 8.04 (d,
J ¼ 12.6 Hz, 1H), 5.15 (d, J ¼ 64.9 Hz, 1H), 4.58 (s, 2H), 4.17 (s, 1H),
3.86 (s, 3H), 3.84 (d, J ¼ 1.1 Hz, 1H), 3.75 (s, 1H), 3.19 (s, 1H),
2.94e2.91 (m, 1H), 2.75 (dd, J ¼ 12.0, 8.7 Hz, 1H), 2.11 (dt, J ¼ 9.8,
3.3 Hz, 1H), 1.91e1.84 (m, 1H), 1.64 (qd, J ¼ 15.0, 9.0 Hz, 1H),
0.42e0.35 (m, 2H), 0.25e0.19 (m, 2H). 13C NMR (151 MHz, CDCl3)
4.2.30.3. 1-cyclopropyl-7-(3-((cyclopropylamino)methyl)-4-(ethox-
yimino)pyrrolidin-1-yl)-6-fluoro-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxylic acid 19a2. The title compound 19a2
was obtained from 11a2 and 12 as a yellow solid (11.5%). 1H NMR
d
(ppm): 176.45, 165.48, 148.87 (d, J ¼ 12.3 Hz), 147.75, 147.15,
146.96, 145.44, 117.77 (d, J ¼ 20.6 Hz), 110.97, 108.09, 70.59, 69.13,
61.76, 51.35, 50.81, 48.60, 36.09 (d, J ¼ 8.5 Hz), 29.93, 11.91 (d,
J ¼ 10.6 Hz), 6.39, 5.97. MS-ESI (m/z): 448.40 (M þ H)þ. HRMS-ESI
(m/z): Calcd. for C21H24O4N5F2 (M þ H)þ: 448.17909; Found:
448.17903.
(600 MHz, CDCl3)
d
(ppm): 8.67 (s, 1H), 7.99 (d, J ¼ 11.7 Hz, 1H), 4.59
(s, 2H), 4.35 (s, 1H), 4.18 (q, J ¼ 7.0 Hz, 2H), 3.84e3.12 (m, 5H), 2.36
(s, 1H), 1.29 (dd, J ¼ 21.1, 14.1 Hz, 7H), 1.05 (s, 2H), 0.62 (s, 2H). 13
C
NMR (151 MHz, CDCl3)
d
(ppm): 177.14, 166.97, 149.12 (d,
4.2.30.8. 7-(3-((Cyclopropylamino)methyl)-4-(methoxyimino)-3-
J ¼ 12.2 Hz), 147.54, 147.29, 146.47, 145.82, 118.96 (d, J ¼ 20.1 Hz),
methylpyrrolidin-1-yl)-6-fluoro-1-((1R,2S)-2-fluorocyclopropyl)-4-

T. Zhang et al. / European Journal of Medicinal Chemistry 104 (2015) 73e85
83
oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid 20b1. The title
compound 20b1 was obtained from 11b1 and 13 as a yellow solid
4.2.30.12. 7-(4-((Benzyloxy)imino)-3-((cyclopropylamino)methyl)-3-
methylpyrrolidin-1-yl)-6-fluoro-1-((1R,2S)-2-fluorocyclopropyl)-4-
oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid 20b3. The title
compound 20b3 was obtained from 11b3 and 13 as a yellow solid
(58.3%). 1H NMR (600 MHz, CDCl3)
d (ppm): 8.72 (s, 1H), 8.02 (d,
J ¼ 12.6 Hz, 1H), 4.97 (dd, J ¼ 63.2, 2.5 Hz, 1H), 4.59e4.57 (m, 2H),
4.14 (s, 1H), 3.92 (s, 3H), 3.74 (s, 1H), 3.61 (dd, J ¼ 12.8, 6.3 Hz, 1H),
2.96 (dd, J ¼ 12.2, 7.1 Hz, 1H), 2.82 (d, J ¼ 12.0 Hz, 1H), 2.16e2.15 (m,
1H), 1.71e1.63 (m,2H), 1.31 (d, J ¼ 2.4 Hz, 3H), 0.44e0.40 (m, 2H),
(18.9%). 1H NMR (400 MHz, DMSO-d6)
d (ppm): 8.73 (s, 1H), 8.02 (d,
J ¼ 12.5 Hz, 1H), 7.39e7.33 (m, 5H), 5.18e5.07 (m, 3H), 4.65 (s, 2H),
4.17 (s, 1H), 3.74 (s, 1H), 3.61(dd, J ¼ 12.7, 6.0 Hz, 1H), 2.91e2.89 (m,
1H), 2.82 (t, J ¼ 12.0 Hz, 1H), 2.13 (d, J ¼ 3.3 Hz, 1H), 1.71e1.63 (m,
2H), 1.31 (s, 3H), 0.44e0.40 (m, 2H), 0.23e0.16 (m, 2H). MS-ESI (m/
z): 538.31 (M þ H)þ. HRMS-ESI (m/z): Calcd. for C28H30O4N5F2
(M þ H)þ: 538.22604; Found: 538.22656.
0.28 (s, 2H). 13C NMR (151 MHz, CDCl3)
d (ppm): 176.39, 165.82,
148.56 (d, J ¼ 12.1 Hz), 146.71, 146.31, 146.08, 144.99, 117.87 (d,
J ¼ 21.1 Hz), 111.38, 108.56, 68.87(d, J ¼ 6.0 Hz), 67.38(d, J ¼ 4.5 Hz),
61.42, 57.09, 55.01, 47.98, 35.27 (dd, J ¼ 9.2, 4.0 Hz), 30.13, 30.12,
21.97 (d, J ¼ 12.1 Hz), 11.48 (d, J ¼ 10.6 Hz), 5.87, 5.82. MS-ESI (m/z):
462.35 (M þ H)þ. HRMS-ESI (m/z): Calcd. for C22H26O4N5F2
(M þ H)þ: 462.19474; Found: 462.19476.
4.2.30.13. 1-(6-Amino-3,5-difluoropyridin-2-yl)-7-(3-((cyclo-
propylamino)methyl)-4-(methoxyimino)pyrrolidin-1-yl)-6-fluoro-4-
oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid 21a1. The title
compound 21a1 was obtained from 11a1 and 14 as an off-white
4.2.30.9. 7-(3-((Cyclopropylamino)methyl)-4-(ethoxyimino)pyrroli-
din-1-yl)-6-fluoro-1-((1R,2S)-2-fluorocyclopropyl)-4-oxo-1,4-
dihydro-1,8-naphthyridine-3-carboxylic acid 20a2. The title com-
pound 20a2 was obtained from 11a2 and 13 as a yellow solid
solid (20.5%). 1H NMR (400 MHz, CDCl3)
d (ppm): 8.78 (s, 1H),
8.08 (d, J ¼ 12.3 Hz, 1H), 7.35 (t, J ¼ 8.2 Hz, 1H), 4.77 (s, 2H), 4.38 (s,
1H), 3.91 (s, 3H), 3.90 (s, 1H), 3.59 (s, 1H), 3.15 (s, 1H), 2.99 (dd,
J ¼ 12.2, 6.7 Hz, 1H), 2.86 (dd, J ¼ 12.1, 7.2 Hz, 1H), 2.18 (d, J ¼ 3.3 Hz,
1H), 1.28e1.24 (m, 1H), 0.49e0.29 (m, 4H). 13C NMR (126 MHz,
(33.3%). 1H NMR (600 MHz, DMSO-d6)
d (ppm): 8.71 (s, 1H), 8.03 (d,
J ¼ 12.5 Hz, 1H), 5.14 (d, J ¼ 65.4 Hz, 1H), 4.58 (s, 2H), 4.17 (s, 1H),
4.11 (q, J ¼ 7.0 Hz, 2H), 3.89 (s, 1H), 3.74 (s, 1H), 3.18 (s, 1H), 2.92
(ddd, J ¼ 11.9, 5.1, 3.1 Hz, 1H), 2.77e2.73 (m, 1H), 2.12 (dt, J ¼ 9.4,
3.0 Hz, 1H), 1.91e1.84 (m, 1H), 1.64 (td, J ¼ 15.2, 8.9 Hz, 1H), 1.23 (t,
J ¼ 7.0 Hz, 3H), 0.42e0.35 (m, 2H), 0.27e0.18 (m, 2H). 13C NMR
CDCl3)
d
(ppm): 177.80, 166.36, 149.24 (d, J ¼ 13.1 Hz), 147.64, 147.11,
146.29 ꢂ 2, 145.57, 145.51, 144.52, 144.41, 118.94 (d, J ¼ 21.3 Hz),
113.07 (t, J ¼ 21.4 Hz), 111.81, 110.11, 62.36, 51.17, 51.13, 50.79, 48.58,
30.18, 6.52, 6.30. MS-ESI (m/z): 518.29 (M þ H)þ. HRMS-ESI (m/z):
Calcd. for C23H23O4N7F3 (M þ H)þ: 518.17581; Found: 518.17606.
(151 MHz, DMSO-d6)
d (ppm): 176.46, 165.49, 148.85 (d,
J ¼ 12.2 Hz), 147.79, 147.16, 146.96, 145.44, 117.76 (d, J ¼ 20.9 Hz),
110.97, 108.09, 70.60, 69.10, 51.35, 50.91, 50.85, 48.59, 36.11, 29.95,
14.62, 11.93 (d, J ¼ 10.1 Hz), 6.42 (d, J ¼ 3.4 Hz), 6.01 (d, J ¼ 5.8 Hz).
MS-ESI (m/z): 462.41 (M þ H)þ. HRMS-ESI (m/z): Calcd. for
4.2.30.14. 1-(6-Amino-3,5-difluoropyridin-2-yl)-7-(3-((cyclo-
propylamino)methyl)-4-(methoxyimino)-3-methylpyrrolidin-1-yl)-6-
fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid 21b1.
The title compound 21b1 was obtained from 11b1 and 14 as a
C
22H26O4N5F2 (M þ H)þ: 462.19474; Found: 462.19468.
yellow solid (29.8%). 1H NMR (600 MHz, CDCl3)
d (ppm): 8.77 (s,
4.2.30.10. 7-(3-((Cyclopropylamino)methyl)-4-(ethoxyimino)-3-
methylpyrrolidin-1-yl)-6-fluoro-1-((1R,2S)-2-fluorocyclopropyl)-4-
oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid 20b2. The title
compound 20b2 was obtained from 11b2 and 13 as a yellow solid
1H), 8.05 (d, J ¼ 12.3 Hz, 1H), 7.33 (s, 1H), 4.81 (s, 2H), 4.34 (s, 2H),
3.88 (s, 4H), 2.87 (d, J ¼ 12.1 Hz, 1H), 2.74 (d, J ¼ 12.2 Hz, 1H), 2.11 (s,
1H), 1.22 (s, 4H), 0.40 (s, 2H), 0.22 (d, J ¼ 14.3 Hz, 2H). 13C NMR
(151 MHz, CDCl3)
d
(ppm): 177.92, 166.55, 149.49 (d, J ¼ 12.1 Hz),
(8.9%). 1H NMR (500 MHz, CDCl3)
d
(ppm): 8.73 (s, 1H), 8.02 (d,
147.63, 147.05, 146.35, 145.91, 145.65, 145.31, 144.56 (d, J ¼ 13.6 Hz),
131.84 (d, J ¼ 10.6 Hz), 118.95 (d, J ¼ 19.6 Hz), 113.17 (t,
J ¼ 21.1 Hz),111.81, 110.19, 62.39, 57.85, 55.82, 48.78, 48.74, 31.01,
22.78, 6.87, 6.76. MS-ESI (m/z): 532.39 (M þ H)þ. HRMS-ESI (m/z):
Calcd. for C24H25O4N7F3 (M þ H)þ: 532.19146; Found: 532.19207.
J ¼ 12.5 Hz, 1H), 5.03e4.89 (m, 1H), 4.60 (s, 2H), 4.19e4.14 (m, 3H),
3.74 (d, J ¼ 9.5 Hz, 1H), 3.61 (dd, J ¼ 12.7, 5.8 Hz, 1H), 2.98 (t,
J ¼ 12.0 Hz, 1H), 2.83 (d, J ¼ 12.0 Hz, 1H), 2.17e2.16 (m, 1H),
1.73e1.61 (m, 2H), 1.32e1.27 (m, 6H), 0.44e0.40 (m, 2H), 0.30 (s,
2H). 13C NMR (125 MHz, CDCl3)
d (ppm): 177.43, 167.84, 149.70 (d,
J ¼ 12.5 Hz), 147.91, 147.33, 147.13, 145.85, 118.88 (d, J ¼ 20.0 Hz),
112.41, 109.62, 70.10 (dd, J ¼ 14.1, 4.2 Hz), 68.26 (d, J ¼ 6.3 Hz), 58.11,
56.01, 55.86, 49.01, 36.30 (d, J ¼ 10.0 Hz), 31.16 (d, J ¼ 5.0 Hz), 23.06
(d, J ¼ 23.8 Hz), 14.85, 12.51 (d, J ¼ 7.7 Hz), 6.80, 6.73. MS-ESI (m/z):
476.34 (M þ H)þ. HRMS-ESI (m/z): Calcd. for C23H28O4N5F2
(M þ H)þ: 476.21039; Found: 476.21055.
4.2.30.15. 1-(6-Amino-3,5-difluoropyridin-2-yl)-7-(3-((cyclo-
propylamino)methyl)-4-(ethoxyimino)pyrrolidin-1-yl)-6-fluoro-4-
oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid 21a2. The title
compound 21a2 was obtained from 11a2 and 14 as a yellow solid
(43.3%). 1H NMR (600 MHz, CDCl3)
d (ppm): 8.79 (s, 1H), 8.08 (d,
J ¼ 12.0 Hz, 1H), 7.34 (s, 1H), 4.77 (s, 2H), 4.33 (s, 1H), 4.15 (d,
J ¼ 5.4 Hz, 2H), 3.94 (s, 1H), 3.51 (s, 1H), 3.15e2.86 (m, 3H), 2.16 (s,
1H), 1.27 (s, 4H), 0.47e0.30 (m, 4H). 13C NMR (151 MHz, CDCl3)
4.2.30.11. 7-(3-((Benzyloxy)imino)-4-((cyclopropylamino)methyl)
pyrrolidin-1-yl)-6-fluoro-1-((1R,2S)-2-fluorocyclopropyl)-4-oxo-1,4-
dihydro-1,8-naphthyridine-3-carboxylic acid 20a3. The title com-
pound 20a3 was obtained from 11a3 and 13 as a yellow solid
d
(ppm): 177.98, 166.49, 149.12 (d, J ¼ 12.2 Hz), 147.54, 147.29,
146.47, 146.36, 145.95 145.63, 144.85, 144.45, 119.11 (d, J ¼ 19.4 Hz),
113.21, 111.95, 110.29, 70.20, 51.25, 50.99, 50.67, 48.83, 30.32, 14.65,
6.57, 6.41. MS-ESI (m/z): 532.23 (M þ H)þ. HRMS-ESI (m/z): Calcd.
for C24H25O4N7F3 (M þ H)þ: 532.19146; Found: 532.19238.
(27.0%). 1H NMR (600 MHz, DMSO-d6)
d (ppm): 8.71 (s, 1H), 8.03 (d,
J ¼ 12.5 Hz, 1H), 7.39e7.36 (m, 4H), 7.32 (td, J ¼ 5.7, 2.7 Hz, 1H),
5.18e5.07 (m, 3H), 4.65 (s, 2H), 4.17 (s, 1H), 3.88 (s, 1H), 3.74 (s, 1H),
3.19 (s, 1H), 2.89e2.87 (m, 1H), 2.74 (t, J ¼ 8.9 Hz, 1H), 2.08 (d,
J ¼ 3.3 Hz, 1H), 1.91e1.84 (m, 1H), 1.64e1.62 (m, 1H), 0.36e0.32 (m,
4.2.30.16. 1-(6-Amino-3,5-difluoropyridin-2-yl)-7-(3-((cyclo-
propylamino)methyl)-4-(ethoxyimino)-3-methylpyrrolidin-1-yl)-6-
fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid 21b2.
The title compound 21b2 was obtained from 11b2 and 14 as a grey
2H), 0.21e0.17 (m, 2H). 13C NMR (151 MHz, DMSO-d6)
d (ppm):
176.46, 165.48, 148.86 (d, J ¼ 12.2 Hz), 147.78, 147.16, 146.95, 145.44,
137.90, 128.29 ꢂ 2, 127.77, 127.69 ꢂ 2, 117.75 (d, J ¼ 20.7 Hz), 110.99,
108.10, 75.32, 70.58, 69.12, 51.30, 50.74 (d, J ¼ 8.7 Hz), 48.78, 36.11,
29.91, 11.92 (d, J ¼ 10.3 Hz), 6.36 (d, J ¼ 4.1 Hz), 6.01 (d, J ¼ 5.5 Hz).
MS-ESI (m/z): 524.11 (M þ H)þ. HRMS-ESI (m/z): Calcd. for
solid (27.9%). 1H NMR (500 MHz, CDCl3)
d (ppm): 8.77 (s, 1H), 8.04
(d, J ¼ 12.3 Hz, 1H), 7.32 (t, J ¼ 8.3 Hz, 1H), 4.82 (s, 2H), 4.12 (q,
J ¼ 7.0 Hz, 1H), 3.71 (q, J ¼ 7.0 Hz, 2H), 2.87 (d, J ¼ 12.0 Hz, 1H), 2.74
(d, J ¼ 12.2 Hz, 1H), 2.11 (s, 1H), 1.27e1.21 (m, 7H), 0.40 (s, 2H), 0.24
C
27H28O4N5F2 (M þ H)þ: 524.21039; Found: 524.21069.
(s, 2H). 13C NMR (125 MHz, CDCl3)
d (ppm): 177.96, 166.55, 149.56

84
T. Zhang et al. / European Journal of Medicinal Chemistry 104 (2015) 73e85
(d, J ¼ 12.1 Hz), 147.83, 147.22, 146.29, 145.76, 145.67, 145.16 (d,
J ¼ 2.5 Hz), 144.52 (t, J ¼ 12.5 Hz), 131.89 (d, J ¼ 18.8 Hz), 118.91 (d,
J ¼ 20.0 Hz), 113.17 (t, J ¼ 21.1 Hz), 111.79, 110.22, 69.99, 57.90, 55.74,
48.97, 48.92, 31.02, 22.84, 14.58, 6.83 ꢂ 2. MS-ESI (m/z): 546.30
(M þ H)þ. HRMS-ESI (m/z): Calcd. for C25H27O4N7F3 (M þ H)þ:
546.20711; Found: 546.20826.
compound 22b1 was obtained from 11b1 and 15 as a yellow solid
(15.9%). 1H NMR (500 MHz, CDCl3)
(ppm): 8.60 (s, 1H), 7.68 (d,
d
J ¼ 13.3 Hz, 1H), 4.52e4.47 (m, 2H), 4.42 (t, J ¼ 14.3 Hz, 2H),
4.36e4.34 (m, 1H), 3.89e3.82 (m, 4H), 3.53 (ddd, J ¼ 25.6, 10.0,
2.1 Hz, 1H), 2.93 (dd, J ¼ 12.0, 5.0 Hz, 1H), 2.83 (d, J ¼ 12.1 Hz, 1H),
2.15 (s, 1H), 1.61 (d, J ¼ 6.5 Hz, 3H), 1.29 (s, 3H), 0.42 (d, J ¼ 6.4 Hz,
2H), 0.31 (t, J ¼ 9.7 Hz, 2H). 13C NMR (125 MHz, CDCl3)
d (ppm):
4.2.30.17. 1-(6-Amino-3,5-difluoropyridin-2-yl)-7-(3-((benzyloxy)
imino)-4-((cyclopropylamino)methyl)pyrrolidin-1-yl)-6-fluoro-4-
oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid 21a3. The title
compound 21a3 was obtained from 11a3 and 14 as a yellow solid
177.13, 167.32, 163.79, 155.68, 144.83, 137.42 (d, J ¼ 7.5 Hz), 130.55
(d, J ¼ 7.5 Hz), 124.97, 119.04 (d, J ¼ 10.0 Hz), 107.87, 105.61 (d,
J ¼ 23.8 Hz), 68.27, 62.14, 61.22(t, J ¼ 7.5 Hz), 55.66, 55.58, 50.96,
45.58, 31.20, 21.98 (d, J ¼ 18.8 Hz), 18.47 (d, J ¼ 2.5 Hz), 6.74. MS-ESI
(m/z): 459.72 (M þ H)þ. HRMS-ESI (m/z): Calcd. for C23H28O5N4F
(M þ H)þ: 459.20382; Found: 459.20369.
(18.8%). 1H NMR (600 MHz, CDCl3)
d (ppm): 8.77 (s, 1H), 8.07 (d,
J ¼ 12.2 Hz, 1H), 7.40 (d, J ¼ 7.0 Hz, 2H), 7.35 (d, J ¼ 7.3 Hz, 3H), 7.13
(s, 1H), 5.12 (s, 2H), 4.69 (s, 2H), 4.35 (s, 1H), 3.14 (s, 1H), 2.91 (s, 1H),
2.84 (s, 1H), 2.10 (s, 1H), 1.25 (s, 1H), 0.43 (s, 2H), 0.27 (s, 2H). 13
C
4.2.31.3. (3S)-10-(3-((Cyclopropylamino)methyl)-4-(ethoxyimino)-
3-methylpyrrolidin-1-yl)-9-fluoro-3-methyl-7-oxo-3,7-dihydro-2H-
[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid 22b2. The title
compound 22b2 was obtained from 11b2 and 15 as a yellow solid
NMR (151 MHz, CDCl3)
d (ppm): 177.98, 166.45, 149.34 (d,
J ¼ 12.2 Hz), 147.57, 147.47, 146.97, 146.39, 145.85, 145.57, 144.48,
144.39, 137.37, 128.66 ꢂ 2, 128.50, 128.34 ꢂ 2, 119.12 (d, J ¼ 20.7 Hz),
113.15 (t, J ¼ 21.4 Hz),111.95,110.32, 76.79, 51.27, 50.98, 50.67, 49.04,
30.24, 6.70, 6.56. MS-ESI (m/z): 594.47 (M þ H)þ. HRMS-ESI (m/z):
Calcd. for C29H27O4N7F3 (M þ H)þ: 594.20711; Found: 594.20740.
(19.6%). 1H NMR (500 MHz, CDCl3)
d (ppm): 8.61 (s, 1H), 7.68 (d,
J ¼ 13.3 Hz, 1H), 4.55e4.35 (m, 5H), 4.13 (q, J ¼ 7.0 Hz, 2H), 3.86
(ddd, J ¼ 22.3, 10.0, 2.3 Hz, 1H), 3.54 (ddd, J ¼ 24.5, 10.0, 2.3 Hz, 1H),
2.93 (dd, J ¼ 12.0, 4.8 Hz, 1H), 2.84 (dd, J ¼ 12.0, 1.7 Hz, 1H),
2.16e2.15 (m, 1H), 1.62 (d, J ¼ 6.8 Hz, 3H), 1.27 (dd, J ¼ 13.6, 6.5 Hz,
6H), 0.41 (t, J ¼ 7.0 Hz, 2H), 0.30 (dd, J ¼ 12.7, 6.1 Hz, 2H). 13C NMR
4.2.30.18. (6-Amino-3,5-difluoropyridin-2-yl)-7-(4-((benzyloxy)
imino)-3-((cyclopropylamino)methyl)-3-methylpyrrolidin-1-yl)-6-
fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid 21b3.
The title compound 21b3 was obtained from 11b3 and 14 as a
(125 MHz, CDCl3)
d (ppm): 177.13, 167.33, 163.36, 155.72, 144.82,
137.45 (d, J ¼ 8.3 Hz), 130.63 (d, J ¼ 13.8 Hz), 124.98, 119.01 (d,
J ¼ 10.0 Hz), 107.86, 105.58 (d, J ¼ 25.0 Hz), 69.83, 68.27, 61.26(t,
J ¼ 6.3 Hz), 55.65, 55.59, 51.00 (d, J ¼ 5.0 Hz), 45.58, 31.21, 22.02 (d,
J ¼ 16.3 Hz), 18.47 (d, J ¼ 2.5 Hz), 14.87, 6.71. MS-ESI (m/z): 473.35
(M þ H)þ. HRMS-ESI (m/z): Calcd. for C24H30O5N4F (M þ H)þ:
473.21947; Found: 473.21965.
yellow solid (16.9%). 1H NMR (400 MHz, CDCl3)
d (ppm): 8.77 (s,
1H), 8.04 (d, J ¼ 12.3 Hz, 1H), 7.38 (d, J ¼ 7.0 Hz, 2H), 7.33 (d,
J ¼ 7.0 Hz, 3H), 7.11 (s, 1H), 5.11 (s, 2H), 4.73 (s, 2H), 4.31 (s, 1H), 2.91
(s, 1H), 2.85 (s, 1H), 2.74 (d, J ¼ 12.2 Hz, 1H), 2.05 (s, 1H), 1.22 (s, 4H),
0.38 (s, 2H), 0.22 (s, 2H). MS-ESI (m/z): 608.64 (M þ H)þ. HRMS-ESI
(m/z): Calcd. for C30H29O4N7F3 (M þ H)þ: 608.22277; Found:
608.22328.
4.2.31.4. (3S)-10-(3-((Benzyloxy)imino)-4-((cyclopropylamino)
methyl)pyrrolidin-1-yl)-9-fluoro-3-methyl-7-oxo-3,7-dihydro-2H-
[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid 22a3. The title
compound 22a3 was obtained from 11a3 and 15 as a yellow solid
4.2.31. General procedure for the synthesis of target compounds
22e25
To a suspension of 11a,b (0.5 mmol) in dry acetonitrile (7.0 mL)
was added triethylamine (2.2 mmol), and then stirred for 0.5 h at
room temperature. To the reaction mixture was added 15e18
(0.5 mmol), and then stirred at 35 ꢁC for another 40 h under an
atmosphere of nitrogen and then concentrated under reduced
pressure. The residue was purified by column chromatography
(silica gel) eluting with dichloromethane and acetone (v: v ¼ 2: 1)
to get the chelates. The chelates (0.3 mmol) was dissolved with 5%
aqueous acetic acid (4.5 mL), and then stirred for 2 h at 80 ꢁC. After
cooling to room temperature, the mixture was extracted with ethyl
acetate (3 mL ꢂ 2), and then 12% ammonia water was added
dropwise to adjust pH 6.5e7.0 and then filtered. The precipitate
was washed with water (3 mL), and dried in vacuo at 60 ꢁC for 10 h
to yield the title compounds 22e25.
(4.5%). 1H NMR (500 MHz, CDCl3)
d (ppm): 8.63 (s, 1H), 7.73 (dd,
J ¼ 13.1, 2.2 Hz, 1H), 7.40 (d, J ¼ 4.3 Hz, 4H), 7.36 (td, J ¼ 8.4, 4.5 Hz,
1H), 5.16 (s, 2H), 4.50 (dt, J ¼ 22.6, 14.5 Hz, 3H), 4.38 (t, J ¼ 12.6 Hz,
2H), 4.00 (dt, J ¼ 17.4, 7.8 Hz, 1H), 3.69e3.63 (m, 1H), 3.15 (d,
J ¼ 6.0 Hz,1H), 3.03e2.99 (m,1H), 2.95 (dd, J ¼ 11.9, 6.9 Hz,1H), 2.19
(s, 1H), 1.64 (d, J ¼ 6.4 Hz, 3H), 0.49 (d, J ¼ 4.8 Hz, 2H), 0.38 (s, 2H).
MS-ESI (m/z): 521.42 (M þ H)þ. HRMS-ESI (m/z): Calcd. for
C
28H30O5N4F (M þ H)þ: 521.21947; Found: 521.21956.
4.2.31.5. (3R)-10-(3-((Cyclopropylamino)methyl)-4-(methox-
yimino)-3-methylpyrrolidin-1-yl)-9-fluoro-3-(fluoromethyl)-7-oxo-
3,7-dihydro-2H- [1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid
23b1. The title compound 23b1 was obtained from 11b1 and 16 as
a yellow solid (9.8%). 1H NMR (500 MHz, CDCl3)
d (ppm): 8.63 (s,
1H), 7.67 (d, J ¼ 13.3 Hz, 1H), 4.86e4.60 (m, 4H), 4.42 (dd, J ¼ 28.3,
17.7 Hz, 3H), 3.89e3.83 (m, 4H), 3.57e3.48 (m, 1H), 2.93 (dd,
J ¼ 12.1, 4.8 Hz, 1H), 2.84e2.82 (m, 1H), 2.15 (d, J ¼ 3.2 Hz, 1H), 1.28
(s, 3H), 0.42 (d, J ¼ 6.5 Hz, 2H), 0.31 (t, J ¼ 9.6 Hz, 2H). 13C NMR
4.2.31.1. (3S)-10-(3-((Cyclopropylamino)methyl)-4-(methoxyimino)
pyrrolidin-1-yl)-9-fluoro-3-methyl-7-oxo-3,7-dihydro-2H- [1,4]oxa-
zino[2,3,4-ij]quinoline-6-carboxylic acid 22a1. The title compound
22a1 was obtained from 11a1 and 15 as a yellow solid (6.5%). 1H
(125 MHz, CDCl3)
d (ppm): 177.38, 167.14, 163.61, 155.59, 146.78,
NMR (500 MHz, CDCl3)
d
(ppm): 8.72 (s, 1H), 7.90 (s, 1H), 4.56e4.47
136.74 (d, J ¼ 7.5 Hz), 131.12 (d, J ¼ 12.5 Hz), 124.89, 118.79 (d,
J ¼ 8.8 Hz), 108.09, 106.17 (d, J ¼ 2.5 Hz), 81.62, 80.20, 63.91, 62.26,
61.32(d, J ¼ 7.5 Hz), 58.79(d, J ¼ 21.3 Hz), 55.65 (d, J ¼ 10.0 Hz), 51.09
(d, J ¼ 6.3 Hz), 45.63, 31.31, 22.10 (d, J ¼ 20.0 Hz), 6.84. MS-ESI (m/z):
(m, 4H), 3.91 (s, 1H), 3.80e3.73 (m, 1H), 3.60 (s, 1H), 3.51e3.34 (m,
1H), 2.96 (s, 1H), 2.88 (s, 1H), 2.56 (s, 1H), 2.48 (s, 1H), 1.66 (s, 7H),
1.26 (s, 1H), 0.87 (s, 1H). MS-ESI (m/z): 445.34 (M þ H)þ. HRMS-ESI
(m/z): Calcd. for C22H26O5N4F (M þ H)þ: 445.18817; Found:
445.18790.
477.48 (M
þ
H)þ. HRMS-ESI (m/z): Calcd. for C23H27O5N4F2
(M þ H)þ: 477.19440; Found: 477.19406.
4.2.31.2. (3S)-10-(3-((Cyclopropylamino)methyl)-4-(methoxyimino)-
3-methylpyrrolidin-1-yl)-9-fluoro-3-methyl-7-oxo-3,7-dihydro-2H-
[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid 22b1. The title
4.2.31.6. (3R)-10-(3-((Benzyloxy)imino)-4-((cyclopropylamino)
methyl)pyrrolidin-1-yl)-9-fluoro-3-(fluoromethyl)-7-oxo-3,7-
dihydro-2H- [1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid 23a3.

T. Zhang et al. / European Journal of Medicinal Chemistry 104 (2015) 73e85
85
The title compound 23a3 was obtained from 11a3 and 16 as a
yellow solid (4.7%). 1H NMR (500 MHz, CDCl3)
(ppm): 8.63 (s, 1H),
Appendix A. Supplementary data
d
7.69 (d, J ¼ 13.2 Hz, 1H), 4.86e4.60 (m, 4H), 4.49e4.28 (m, 3H),
3.99e3.95 (m, 1H), 3.90 (s, 3H), 3.65e3.63 (m, 1H), 3.12e3.07 (m,
1H), 3.04e3.00 (m, 1H), 2.92e2.88 (m, 1H), 2.22e2.16 (m, 1H),
0.49e0.43 (m, 2H), 0.38e0.33 (m, 2H). 13C NMR (126 MHz, CDCl3)
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2015.09.
030. These data include MOL files and InChiKeys of the most
important compounds described in this article.
d
(ppm): 177.30, 166.96, 161.05, 155.60, 153.61, 146.70, 130.78 (d,
J ¼ 13.7 Hz), 124.73, 119.06 (d, J ¼ 9.4 Hz), 108.09, 106.11 (d,
J ¼ 24.9 Hz), 81.50, 80.08, 63.87, 62.25, 58.67 (d, J ¼ 21.5 Hz), 54.80,
50.48, 41.59, 30.36, 6.63, 6.44. MS-ESI (m/z): 463.18 (M þ H)þ.
HRMS-ESI (m/z): Calcd. for C22H25O5N4F2 (M þ H)þ: 463.17875;
Found: 463.17849.
References
[1] Y.B. Zhang, G.Q. Li, M.L. Liu, X.F. You, L.S. Feng, K. Lv, J. Cao, H.Y. Guo, Bioorg.
Med. Chem. Lett. 21 (2011) 928e931.
[2] Y. Chai, J. Wang, M.L. Liu, H. Yi, L.Y. Sun, X.F. You, H.Y. Guo, Bioorg. Med. Chem.
Lett. 21 (2011) 3377e3380.
[3] A. Zumla, P. Nahid, S.T. Cole, Nat. Rev. Drug Discov. 12 (2013) 388e404.
[4] Z. Ma, C. Lienhardt, H. McIlleron, A.J. Nunn, X.X. Wang, Lancet 375 (2010)
2100e2109.
[5] B.J. Bradbury, M.J. Pucci, Curr. Opin. Pharmacol. 8 (2008) 574e581.
[6] A.S. Ginsburg, J.H. Grosset, W.R. Bishai, Lancet. Infect. Dis. 3 (2003) 432e442.
[7] H.M. Liu, J. Huang, J.Y. Wang, M.H. Wang, M.L. Liu, B. Wang, H.Y. Guo, Y. Lu,
Eur. J. Med. Chem. 57 (2014) 628e638.
[8] A. Dalhoff, Infection 40 (2012) 239e262.
[9] J.X. Wang, Y.B. Zhang, M.L. Liu, B. Wang, Y. Chai, S.J. Li, H.Y. Guo, Eur. J. Med.
Chem. 46 (2011) 2421e2426.
[10] L.S. Feng, M.L. Liu, S. Wang, K. Lv, J. Cao, S.J. Li, H.Y. Guo, Tetrahedron 67 (2011)
8264e8270.
[11] Y. Chai, M.L. Liu, K. Lv, L.S. Feng, S.J. Li, L.Y. Sun, S. Wang, H.Y. Guo, Eur. J. Med.
Chem. 46 (2011) 4267e4273.
[12] C.Y. Hong, Y.K. Kim, J.H. Chang, S.H. Kim, H. Choi, D.H. Nam, Y.Z. Kim,
J.H. Kwak, J. Med. Chem. 40 (1997) 3584e3593.
4.2.31.7. 1-Cyclopropyl-7-(3-((cyclopropylamino)methyl)-4-
(methoxyimino)pyrrolidin-1-yl)-6-fluoro-8-methoxy-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid 24a1. The title compound 24a1
was obtained from 11a1 and 17 as a yellow solid (9.2%). 1H NMR
(500 MHz, CDCl3)
d
(ppm): 8.81 (s, 1H), 7.87 (d, J ¼ 13.1 Hz, 1H), 4.33
(dd, J ¼ 39.2, 17.8 Hz, 2H), 4.00e3.97 (m, 2H), 3.93 (s, 3H), 3.64 (s,
3H), 3.62e3.61 (m, 1H), 3.15 (d, J ¼ 6.8 Hz, 1H), 3.06 (d, J ¼ 12.0 Hz,
1H), 2.94 (dd, J ¼ 11.7, 7.4 Hz, 1H), 2.21 (s, 1H), 1.22 (d, J ¼ 6.8 Hz,
2H), 1.00 (s, 2H), 0.48 (d, J ¼ 6.3 Hz, 2H), 0.39 (s, 2H). MS-ESI (m/z):
459.37 (M
þ
H)þ. HRMS-ESI (m/z): Calcd. for C23H28O5N4F
(M þ H)þ: 459.20382; Found: 459.20360.
4.2.31.8. 7-(3-((Cyclopropylamino)methyl)-4-(ethoxyimino)pyrroli-
din-1-yl)-6-fluoro-1-((1R,2S)-2-fluorocyclopropyl)-8-methoxy-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid 25a2. The title com-
pound 25a2 was obtained from 11a2 and 18 as a yellow solid (3.0%).
[13] D.R. Choi, J.H. Shin, J. Yang, S.H. Yoon, Y.H. Jung, Bioorg. Med.Chem. Lett. 14
(2004) 1273e1277.
[14] A.R. Kwon, Y.H. Min, J.M. Ryu, D.R. Choi, M.J. Shim, E.C. Choi, J. Antimicrob.
Chemother. 58 (2006) 684e688.
[15] K. Lv, Y.X. Sun, L.Y. Sun, H.Y. Guo, J.W. Wu, M.L. Liu, Chem. Med. Chem. 7
(2012) 1230e1236.
[16] X.Y. Wang, Q. Guo, Y.C. Wang, B.Q. Liu, M.L. Liu, L.Y. Sun, H.Y. Guo, Acta.
Pharm. Sin. 43 (2008) 819e827.
[17] L.S. Feng, K. Lv, M.L. Liu, S. Wang, J. Zhao, X.F. You, S.J. Li, J. Cao, H.Y. Guo, Eur. J.
Med. Chem. 55 (2012) 125e136.
[18] K. Lv, M.L. Liu, L.S. Feng, L.Y. Sun, Y.X. Sun, H.Y. Guo, Eur. J. Med. Chem. 47
(2012) 619e625.
[19] Y. Asahina, M. Takei, T. Kimura, Y. Fukuda, J. Med. Chem. 51 (2008)
3238e3249.
[20] L.S. Feng, M.L. Liu, K. Lv, S. Wang, H.Y. Guo, Asian. J. Chem. 25 (2013)
2327e2328.
[21] Z.L. Wan, Y. Chai, M.L. Liu, H.Y. Guo, Chin. J. Med. Chem. 19 (2009) 109e111.
[22] Y. Chai, Z.L. Wan, B. Wang, H.Y. Guo, M.L. Liu, Eur. J. Med. Chem. 44 (2009)
4063e4069.
[23] L. Collins, S.G. Franzblau, Antimicrob. Agents Chemother. 41 (1997)
1004e1009.
[24] Y. Lu, M.Q. Zheng, B. Wang, L. Fu, W.J. Zhao, P. Li, J. Xu, H. Zhu, H.X. Jin, D.L. Yin,
H.H. Huang, A.M. Upton, Z.K. Ma, Antimicrob. Agents Chemother. 55 (2011)
5185e5193.
[25] D. Sriram, A. Aubry, P. Yogeeswaria, L.M. Fisher, Bioorg. Med. Chem. Lett. 16
(2006) 2982e2985.
[26] MIC values were determined as described by the NCCLS (see: National
Committee for Clinical Laboratory Standards, Performance Standards for
Antimicrobial Susceptibility Testing: 11th Informational Supplement, vol. 21;
NCCLS: Wayne, PA, USA, 2001, M100eS11). MIC was Defined as the Lowest
Concentration of Each Compound that Inhibits Visible Growth of Bacteria after
Incubation at 35ꢁC for 18e24 h.
[27] H.J. Yun, Y.H. Min, J.A. Lim, J.W. Kang, S.Y. Kim, M.J. Kim, J.H. Jeong, Y.J. Choi,
H.J. Kwon, Y.H. Jung, M.J. Shim, E.C. Choi, Antimicrob. Agents. Chemother. 46
(2002) 3071e3074.
1H NMR (500 MHz, CDCl3)
d
(ppm): 8.75 (s, 1H), 7.86 (d, J ¼ 11.7 Hz,
1H), 4.92e4.60 (m, 1H), 4.46e4.22 (m, 1H), 4.16 (q, J ¼ 7.0 Hz, 2H),
3.91 (s, 2H), 3.67 (s, 3H), 3.57 (s, 1H), 3.25e2.98 (m, 2H), 2.23 (dd,
J ¼ 24.5, 17.0 Hz, 1H), 2.02e1.99 (m, 1H), 1.66e1.61 (m, 2H), 1.29 (t,
J ¼ 7.0 Hz, 3H),1.26 (d, J ¼ 5.6 Hz, 2H), 0.88 (t, J ¼ 6.9 Hz,1H), 0.54 (s,
2H). MS-ESI (m/z): 491.57 (M þ H)þ. HRMS-ESI (m/z): Calcd. for
C
24H29O5N4F2 (M þ H)þ: 491.21005; Found: 491.20977.
4.3. MIC determination
All compounds were screened for their in vitro antibacterial
activity against representative Gram-positive and Gram-negative
strains, by means of standard twofold serial dilution method us-
ing agar media [26]. Minimum inhibitory concentration (MIC) is
defined as the minimum concentration of the compound required
to give complete inhibition of bacterial growth after incubation at
35 ꢁC for 18e24 h.
Acknowledgments
This work was supported by the National S&T Major Special
Project on Major New Drug Innovations (2012ZX09301002-001-
017/023, 2014ZX09507009-003) and NSFC 81373267-003.