Synthesis and antibacterial activity of amino acid and dipeptide prodrugs of IMB-070593, a fluoroquinolone candidate

Article abstract of DOI:10.3390/molecules19056822

A series of amino acid and dipeptide prodrugs of IMB-070593, a fluoroquinolone candidate discovered in our lab, were synthesized and evaluated for their water solubility and then antibacterial activity. Our results reveal that four amino acid prodrugs 4a,b,e,f and two dipeptide prodrugs 4k,l have much greater solubility (>85 mg/mL) than IMB-070593 mesylate (22.5 mg/mL). Compounds 4a and 4k show good in vivo efficacy against MSSA 12-1 (p.o./i.v., 5.32-7.68 mg/kg) and S. pneumoniae12-10 (p.o., 18.39-23.13 mg/kg) which is 1.19-1.50 fold more active than the parent drug.

Full text of DOI:10.3390/molecules19056822

Molecules 2014, 19, 6822-6837; doi:10.3390/molecules19056822
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antibacterial Activity of Amino Acid and Dipeptide
Prodrugs of IMB-070593, a Fluoroquinolone Candidate
1
,2,†
2,†
2
3
4
1
Tingting Zhang
, Jinwei Wu , Shihong Chen , Kaixiang Liu , Yabin Lin , Huiyuan Guo
1
,
and Mingliang Liu *
1
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, China
2
Zhejiang Starry Pharmaceutical Co. Ltd., Xianju 317300, China
3
China Resources Double-Crane Pharmaceutical Co. Ltd., Beijing 100102, China
4
Harbin Stomatological Hospital, Harbin 150010, China
†
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed; E-Mail: lmllyx@126.com;
Tel./Fax: +86-10-6303-6965.
Received: 14 April 2014; in revised form: 20 May 2014 / Accepted: 21 May 2014 /
Published: 23 May 2014
Abstract: A series of amino acid and dipeptide prodrugs of IMB-070593, a fluoroquinolone
candidate discovered in our lab, were synthesized and evaluated for their water solubility and
then antibacterial activity. Our results reveal that four amino acid prodrugs 4a,b,e,f and two
dipeptide prodrugs 4k,l have much greater solubility (>85 mg/mL) than IMB-070593
mesylate (22.5 mg/mL). Compounds 4a and 4k show good in vivo efficacy against MSSA
1
2-1 (p.o./i.v., 5.32–7.68 mg/kg) and S. pneumoniae12-10 (p.o., 18.39–23.13 mg/kg)
which is 1.19–1.50 fold more active than the parent drug.
Keywords: IMB-070593; prodrugs; synthesis; water solubility; antibacterial activity
1
. Introduction
Fluoroquinolone (FQ) antibacterial agents which target two type II bacterial topoisomerase
enzymes, DNA gyrase and/or topoisomerase IV, are among the most attractive drugs in the anti-
infective chemotherapy field [1].

Molecules 2014, 19
6823
The structure activity relationship (SAR) studies of FQs show that the basic substituent at the C-7
position, the most amenable site for chemical changes, greatly influences their antibacterial potency,
spectrum and safety [2]. The presence of five- or six- membered nitrogen heterocycle including
pyrrolidine, piperazine and piperidine at this position is a particularly favorable structural feature of
important FQs on the market [3]. Moreover, methyloxime-functionalized pyrrolidines as novel C-7
substituents have also been proved to be of importance with respect to biological activity and led to the
discovery of some new FQ agents, such as gemifloxacin, zabofloxacin and DW286 [4–6].
Recently, as a part of an ongoing program to optimize FQs against bacterial pathogens and
mycobacterium tuberculosis (MTB), we have focused our attention on exploring the effect of
introducing an oxime group into azetidine, pyrrolidine or piperidine side chains at the C-7 position of
FQs, and some of them were found to have considerable biological activity [7–10]. For example, IMB-
070593 (Figure 1), a piperidinyl-based FQ candidate discovered in our lab and in late pre-clinical stage of
development currently, possesses potent antibacterial and anti-MTB activity [9,11] as well as extremely
low phototoxicity, hepatotoxicity and cardiac toxicity (unpublished data). However, IMB-070593 has low
water solubility (0.4 mg/mL) and its mesylate salt still exhibits poor solubility (22.5 mg/mL). Since water
solubility at physiological pH is important for preclinical testing, in vivo efficacy, and parenteral
formulation [12], we decided to improve the solubility of IMB-070593 by employing a prodrug strategy.
Figure 1. Structure of IMB-070593.
One of the best strategies for increasing solubility of a drug containing a hydroxyl, carboxyl or an
amino moiety is, in pharmaceutical field, to transform it into an amino acid prodrug, as exemplified by
valaciclovir (a valine prodrug of acyclovir) [13]. We had previously reported that some of the amino
acid prodrugs of tosufloxacin and moxifloxacin (MXFX) have better solubility and in vivo activity
against clinically pathogens than the parent drugs [14,15].
Inspired by the above research results, it was decided to introduce common amino acids onto the
nitrogen of the 3-amino-4-(methoxyimino)piperidine side chain of IMB-070593 by covalent binding.
Thus, a series of amino acid and dipeptide prodrugs of IMB-070593 were designed, synthesized and
evaluated for their water solubility and then antibacterial activity in this study. Our primary objective
was to improve the solubility of IMB-070593, followed by optimizing its in vivo potency so as to
finally develop the parenteral formulation.

Molecules 2014, 19
6824
2
. Results and Discussion
2
.1. Chemistry
Synthetic pathways to the prodrugs 4a–l of IMB-070593 are depicted in Scheme 1. According to
published procedures [14,15], treatment of various amino acids 1a–j with di-tert-butyl dicarbonate
Boc O) in methanol gave N-Boc-protected acids 2a–j, which were coupled with IMB-070593 in the
(
2
presence of dicyclohexylcarbodiimide (DCC) to yield the condensation products 3a–j. The N-Boc
protecting group on 3a–j was removed with trifluoroacetic acid (TFA) to provide the amino acid
prodrugs 4a–j. Similarly, Gly-Gly-amino prodrug 4k or L-Ala-L-Ala-amino prodrug 4l could be
conveniently obtained from the corresponding mono-Gly-amino prodrug 4a and N-Boc-protected
glycine 2a or mono-L-Ala-amino prodrug 4b and N-Boc-protected L-alanine 2b according to the same
procedures used for preparation of 4a–j.
Scheme 1. Synthesis of the prodrugs 4a–l of IMB-070593.
Reagents and Conditions: (i) (1) (Boc)
2 2 5 3 3
O, (C H ) N, CH OH, (2) 2 N HCl; (ii) IMB-070593, DCC,
CH Cl ; (iii)/(v) (1) TFA, (2) NH OH; (iv) 2a or 2b, DCC, CH Cl .
2
2
4
2
2
Because the methyloxime group of all the prodrugs 4a–l may be present in the E- or Z-
configuration, it was necessary to determine their geometries. Although we were unable to prepare
X-ray quality single crystals of any methyloxime intermediate or product in this study, we had
previously obtained the ingle crystals of 4-(methoxyimino)-3-methylaminopiperidine dihydrochloride,
a N-methylated analogue of the side chain at C-7 position of IMB-070593, in which the piperidine ring
adopts a chair conformation and the methyloxime geometry exists in an E-configuration [9].
Accordingly, we can speculate that the methyloxime group of the prodrugs in this study should have
the same E-configuration.

Molecules 2014, 19
.2. Water Solubility
The prodrugs 4a–l were initially evaluated for their water solubility which was determined by
6825
2
HPLC measurement of the concentration of a micromembrane filtered saturated solution [16]. The
solubility of 4a–l along with the parent IMB-070593 and IMB-070593 mesylate for comparison is
presented in Table 1. The data reveal that all of 4a–l have greater solubility (0.6–795.1 mg/mL) than
that of IMB-070593 (0.4 mg/mL), and two dipeptide prodrugs 4k and 4l (235.0 and 281.5 mg/mL,
respectively) are more soluble than the corresponding amino acid prodrugs 4a and 4b (97.0 and
1
12.0 mg/mL, respectively). It is encouraging that prodrugs 4a,b,e,f,k,l have also much greater
solubility (>85 mg/mL) than the parent IMB-070593 mesylate (22.5 mg/mL) which suggest that the
six prodrugs could display better in vivo efficacy than IMB-070593 mesylate.
Table 1. Structures, physical data and solubility of compounds 4a–l.
Water
Solubility
m.p.
1
2
20
Compd.
R
R
[α] (c, CH
3
OH)
a
D
[°C]
(
mg/mL)
112.0
97.0
15.6
5.3
4
a
H
H
H
H
H
136–137
133–134
154–156
109–110
157–158
0° (0.50)
4
b
CH
3
−2.80°(0.50)
−24.30°(0.50)
−20.37°(0.11)
−19.17°(0.24)
−26.68°(0.40)
−13.32°(0.77)
−9.01° (0.44)
−14.14°(0.50)
−15.25°(0.12)
0° (0.13)
4
c
(CH ) CH
3 2
4
d
(CH ) CHCH
3 2 2
4
e
(CH2)3
795.1
87.4
1.0
4
f
H
H
H
H
H
H
H
(S)-CH CH CH(CH ) 136–138
3 2 3
4
g
C H CH
2
123–125
114–116
138–140
111–113
121–123
120–121
6
5
4
h
CH OH
50.2
0.6
2
4
i
4-(OH)C H CH
2
6
4
4
j
(R)-CH CH(OH)
72.3
235.0
281.5
0.4
3
4
k
H
4
l
CH3
−27.19°(0.23)
IMB
IMB mesylate
22.5
a
Melting points are uncorrected; IMB: IMB-070593.
2
.3. Antibacterial Activity
2
.3.1. In Vitro Activity
The prodrugs 4a–l were evaluated for their in vitro antibacterial activity against representative
bacterial strains using standard techniques [17]. Minimum inhibitory concentration (MIC) is defined as
the concentration of the compound required to give complete inhibition of bacterial growth, and MIC
values of 4a–l against Gram-positive and Gram-negative strains along with IMB-070593 mesylate,
MXFX and levofloxacin (LVFX) for comparison, are listed in Table 2.

Molecules 2014, 19
6826
Table 2. In vitro activity of compounds 4a–l against Gram-positive and Gram-negative strains.
Compd.
Strains MIC (μg/mL)
S.a.
0.25
MSSA
0.5
MRSA
32
MSSE
MRSE
32
S.p.
0. 5
E.coli
E.co.1
16
E.co.2
128
E.co.3
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
K.p.1
8
K.p.2
32
P.a.1 P.a.2 P.a.3
4
a
1
1
4
4
32
64
16
16
32
64
128
32
64
32
32
32
64
64
4b
0.125
0.25
0.25
0.25
0.5
0.25
0.25
0.25
2
64
32
1
16
128
8
64
4
c
64
2
64
1
4
16
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
16
32
32
64
4d
64
4
64
2
4
16
16
128
64
128
64
64
4
e
64
4
64
2
4
32
16
128
64
4
f
0.5
64
2
64
1
8
32
16
128
>128
>128
>128
64
128
128
128
128
64
4
g
0.125
1
0.25
1
64
1
64
1
1
16
64
64
128
128
128
64
4h
64
2
64
16
128
128
64
32
4
i
2
2
>128
128
>128
>128
2
8
64
2
16
32
4
j
2
1
2
64
2
16
32
4k
4
8
16
32
0.06
0.125
0.5
64
8
16
128
>128
0.5
0.5
0.25
64
>128
>128
0.5
>128 >128
128
128
16
4
l
4
4
64
8
32
64
128
4
>128
2
IMB mesylate
MXFX
<0.008
<0.008
0.06
<0.008
<0.008
0.06
0.125
0.25
0.03
0.03
0.06
0.125
0.06
<0.008
<0.008
0.06
0.03
0.03
8
16
16
0.5
2
16
8
LVFX
64
4
16
0.5
0.5
32
8
S.a.: S. aureus ATCC 25923. MSSA: Methicillin-sensitive S. aureus 12-4. MRSA: Methicillin-resistant S. aureus 12-1. MSSE: Methicillin-sensitive S.epidermidis 12-3.
MRSE: Methicillin-resistant S. epidermidis 12-1. S.p.: S. pneumoniae ATCC 49619. E.coli: E. coli ATCC 25922. E.co.1: E. coli 12-6. E.co.2: E. coli 12-11.
+
+
E.co.3: Extended-spectrum β-lactamase-producing (ESBL ) E. coli 12-14. K.p.1: K. pneumoniae 12-4. K.p.2: ESBL K. pneumoniae 12-7. P.a.1: P. aeruginosa 12-12.
P.a.2: P. aeruginosa 12-14. P.a.3: P. aeruginosa 12-20. IMB mesylate: IMB-070593 mesylate. MXFX: Moxifloxacin. LVFX: Levofloxacin.

Molecules 2014, 19
6827
The prodrugs 4a–l show significantly less activity than the parent IMB-070593, MXFX and LVFX
which is consistent with the biological character of common prodrugs. Moreover, 4a–l are generally
more active against Gram-positive strains except methicillin-resistant S. aureus (MRSA) and
methicillin-resistant S. epidermidis (MRSE) than Gram-negative ones, and they have virtually no
activity against all of the tested Gram-negative strains except for few exceptions.
2
.3.2. In Vivo Activity
Mice protection tests were used to evaluate in vivo efficacy of the prodrugs 4a,b,e,f,k,l with greater
solubility than IMB-070593 mesylate, given the fact that there is no obvious difference among of 4a–l
with regard to their in vitro activity. The efficacy of them was initially tested against two clinical
isolate strains (methicillin-sensitive S. aureus / MSSA 12-1, E. Coli 12-1), and then 4a of the amino
acid prodrugs and 4k of the dipeptide ones with better efficacy were chosen for further evaluation their
protective effects in vivo (p.o.) against other four clinical isolates and that (i.v.) against the above two
strains, IMB-070593 mesylate was used as the control drug (Table 3).
The results illustrate that all of the six prodrugs have better oral activity against MSSA 12-1 (6.44–
9.39 mg/kg) than IMB-070593 mesylate (9.40 mg/kg), but weaker against E. coli 12-1. Furthermore,
4a and 4k show stronger efficacy than the parent drug against S. pneumonia 12-10 (p.o., 18.39 and
23.13 mg/kg, respectively) and MSSA 12-1 (i.v., 5.71 and 5.32 mg/kg, respectively), but weaker oral
activity against MRSE 12-1, MRSA 12-5 and K. pneumonia 12-1. In a word, the prodrugs seem to be
more active against drug-sensitive Gram-positive strains (p.o. or i.v., such as MSSA12-1 and S.
pneumonia 12-10) than IMB-070593 mesylate. Conversely, they are much weaker against drug-
resistant Gram-positive strains (p.o., such as MRSE 12-1and MRSA 12-5) and Gram-negative strains
(
p.o., such as E. Coli 12-1 and K. pneumonia 12-1), compared with the parent drug.
Table 3. In vivo efficacy of selected compounds against systemic infections in mice.
a
Infected strain
cfu/mL)
MIC
ED50 (mg/kg)
Compd
(
(μg/mL)
[95% confidence limit (mg/kg)]
b
c
MSSA 12-1
4a
4b
1
0.5
2
7.16 (10.13–4.97)
5.71 (7.97–4.11)
4
d
(
3.0 × 10 )
9.30 (13.43–6.41)
8.74 (12.17–6.27)
6.44 (9.75–3.83)
NT
4
e
NT
NT
4
f
1
4
k
8
7.68 (10.76–5.42)
9.39 (13.85–6.31)
9.40 (13.36–6.60)
17.53 (25.25–12.04)
25.17 (37.89–17.12)
21.44 (31.18–14.83)
17.53 (25.25–12.04)
18.80 (26.72–13.20)
23.43 (36.23–15.57)
8.76 (12.63–6.02)
5.32 (7.58–3.74)
4
l
8
NT
IMB mesylate
<0.008
16
7.15 (10.73–4.95)
E. coli 12-1
4a
4b
12.51 (19.41–8.37)
5
(
6.0 × 10 )
16
NT
4
e
32
NT
NT
4
f
32
4
k
128
>128
0.5
14.30 (21.46–9.91)
NT
4
l
IMB mesylate
7.67 (11.83–5.27)

Molecules 2014, 19
6828
Table 3. Cont.
a
Infected strain
Compd
MIC
ED50 (mg/kg)
(
cfu/mL)
(μg/mL)
[95% confidence limit (mg/kg)]
MRSE 12-1
4a
32
64
2
50.55 (39.57–66.50)
54.03 (40.89–77.35)
NT
6
(
4.5 × 10 )
4k
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
IMB mesylate
15.33 (11.47–20.06)
72.80 (55.03–123.15)
86.06 (67.10–152.78)
25.36 (20.10–32.68)
18.39 (14.00–23.60)
23.13 (17.30–31.81)
27.57 (21.85–36.07)
33.91 (26.82–44.56)
33.30(25.16–47.78)
MRSA 12-5
4a
32
>128
2
6
(
4.5 × 10 )
4k
IMB mesylate
S.pneumonia12-10
4a
1
8
(
5.2 × 10 )
4k
IMB mesylate
4a
2
0.06
4
K.pneumoniae 12-1
6
(
4.5 × 10 )
4k
16
0.06
IMB mesylate
18.56(14.55–23.97)
a
b
ED50: 50% effective dose [95% confidence limit (mg/kg)]; Antimicrobial agents were orally administrated
c
twice at 0 h and 6 h after infection.; Antimicrobial agents were intravenous injected once at 0 h after infection;
d
NT: not tested. MRSE: MRSE: Methicillin-resistant S. epidermidis. MRSA: Methicillin-resistant S. aureus.
3. Experimental
3.1. General Information
1
Melting points were determined in open capillaries and are uncorrected. H-NMR spectra were
determined on a Varian Mercury-400/500/600 spectrometer in DMSO-d or CDCl using
6
3
tetramethylsilane (TMS) as an internal standard. Electrospray ionization (ESI) mass spectra and high
resolution mass spectra (HRMS) were obtained on a MDSSCIEX Q-Tap mass spectrometer and
AccuTOF CS JMS-T100CS (JEOL) mass spectrometer. Unless otherwise noted, the reagents were
obtained from commercial supplier and used without further purification. TLC was performed on silica
gel plates (Merck, ART5554 60F254).
3.2. Synthesis
3
.2.1. General Procedure for the Synthesis of 3a–j
To a solution of amino acids 1a–j (20 mmol) in methanol (90 mL) was added di-tert-butyl
carbonate (8.73 g, 40 mmol) and triethylamine (11.1 mL, 80 mmol). The reaction mixture was heated
to refluxing and stirred for 3 h at the same temperature, and concentrated under reduced pressure. The
residue was diluted with water (40 mL), adjusted to pH 2.0–3.0 with 2 N HCl at 0–5 °C, and then extracted
with ethyl acetate (50 mL × 3). The combined extracts were washed with saturated brine (30 mL),
2 4
dried over anhydrous Na SO , and then concentrated under reduced pressure to provide 2a–j as white
solids [18–23] (64.8%–91.4%). A mixture of IMB-070593 (2.08 g, 4.97 mmol), 2a–j (5.71 mmol),
dicyclohexylcarbodiimide (1.18 g, 5.71 mmol) and dry dichloromethane (42 mL) was stirred at room
temperature for 1 h and filtered. The filtrate was concentrated under reduced pressure, and the residue
was treated with diethyl ether (20 mL), and then filtered. The solid was purified by column

Molecules 2014, 19
6829
chromatography (silica gel) eluted with dichloromethane and methanol (v:v = 55:1) to afford the title
compounds 3a–j (36%–79%, from 1a–j) as white or yellow solids.
7
-(3-(2-(tert-Butoxycarbonylamino)acetamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-
fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3a). Obtained from 2a and IMB-
1
0
70593 as a white solid (86.1%), m.p.: 139–140 °C. H-NMR (400 MHz, CDCl ) δ (ppm) 14.68 (s, 1H,
3
COOH), 8.83 (s, 1H, C
2
5
-H), 7.90 (d, J = 11.7 Hz, 1H, C -H), 7.12 (d, J = 5.4 Hz, 1H), 5.09 (s, 1H),
4
1
1
1
.70–4.59 (m, 1H), 4.18–4.14 (m, 1H), 4.04–4.01 (m, 1H), 3.92 (s, 3H, CH O-N), 3.85 (d, J = 5.4 Hz,
3
3
H), 3.79 (s, 3H, CH O-C), 3.72 (q, J = 6.9 Hz, 1H), 3.62 (d, J = 9.0 Hz, 1H), 3.34 (d, J = 14.2 Hz,
H), 3.26 (t, J = 11.9 Hz, 1H), 3.04 (t, J = 10.3 Hz, 1H), 2.34–2.32 (m, 1H), 1.47 (s, 9H, Boc), 1.30–
+
.19 (m, 2H, cyclopropyl CH ), 1.12–0.91 (m, 2H, cyclopropyl CH ). MS-ESI (m/z): 576.31(M+H) .
2
2
7
6
-(3-((S)-2-(tert-Butoxycarbonylamino)propanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-
-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3b). Obtained from 2b as a off-
1
white solid (84.6%), m.p.: 150–152 °C. H-NMR (400 MHz, CDCl ) δ (ppm) 14.68 (s, 1H, COOH),
3
8
.83 (s, 1H, C
2
-H), 7.91 (d, J = 11.6 Hz, 1H, C
5
-H), 7.27–7.07 (m, 1H), 4.98 (s, 1H), 4.63 (s, 1H), 4.21
O-N), 3.81 (s, 3H, CH O-C),
.61 (d, J = 7.7 Hz, 1H), 3.36–3.24 (m, 2H), 3.03 (t, J = 10.8 Hz, 1H), 2.32 (s, 1H), 1.46 (d, J = 6.8 Hz,
(
s, 1H), 4.22–4.10 (m, 1H), 4.03 (s, 1H), 3.92 (d, J = 2.6 Hz, 3H, CH
3
3
3
9
H, Boc), 1.38–1.26 (m, 3H, CH
3
), 1.25–1.13 (m, 2H, cyclopropyl CH
2
), 1.01 (s, 2H, cyclopropyl
+
CH
2
). MS-ESI (m/z): 590.32(M+H) .
7
-(3-((S)-2-(tert-Butoxycarbonylamino)-3-methylbutanamido)-4-(methoxyimino)piperidin-1-yl)-1-
cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3c). Obtained from 2c
1
as a white solid (78.6%), m.p.: 130–131 °C. H-NMR (400 MHz, CDCl
3
) δ (ppm) 14.69 (s, 1H,
-H), 7.01–6.89 (m, 1H), 5.06 (s, 1H),
.73–4.58 (m, 1H), 4.19–4.08 (m, 1H), 4.07–4.01 (m, 1H), 3.92 (d, J = 8.3 Hz, 3H, CH O-N), 3.80 (s,
H, CH O-C), 3.61 (s, 1H), 3.48 (q, J = 7.0 Hz, 1H), 3.39–3.20 (m, 2H), 3.06–2.99 (m, 1H), 2.31 (s,
2 5
COOH), 8.83 (s, 1H, C -H), 7.91 (d, J = 11.6 Hz, 1H, C
4
3
1
1
3
3
H), 2.21–2.08 (m, 1H, CH(CH
3
)
2
), 1.45 (d, J = 2.3 Hz, 9H, Boc), 1.23–1.21(m, 2H, cyclopropyl CH
2
),
+
.09–0.88 (m, 8H, cyclopropyl CH
2
, CH(CH
3
)
2
). MS-ESI (m/z): 618.65 (M+H) .
7
-(3-((S)-2-(tert-Butoxycarbonylamino)-4-methylpentanamido)-4-(methoxyimino)piperidin-1-yl)-1-
cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3d). Obtained from 2d
1
as a yellow solid (58.2%), m.p.: 142–143 °C. H-NMR (500 MHz, CDCl ) δ (ppm) 14.67 (s, 1H,
3
COOH), 8.83 (s, 1H, C
2
-H), 7.91 (d, J = 11.6 Hz, 1H, C
H), 4.64 (s, 1H), 4.14 (d, J = 17.1 Hz, 1H), 4.06–4.00 (m, 1H), 3.92 (d, J = 9.4 Hz, 3H, CH
.80 (d, J = 2.6 Hz, 3H, CH O-C), 3.61 (s, 1H), 3.46 (d, J = 10.3 Hz, 1H), 3.39–3.21 (m, 2H), 3.03 (t,
5
-H), 7.26–7.02 (m, 1H), 4.88 (d, J = 19.8 Hz,
1
3
3
O-N),
3
J = 10.5 Hz, 1H), 2.38–2.24 (m, 1H), 1.94 (d, J = 9.8 Hz, 1H), 1.70 (s, 2H), 1.45 (d, J = 6.4 Hz, 9H,
Boc), 1.38–1.30 (m, 1H), 1.27 (d, J = 10.0 Hz, 1H), 1.21–1.13 (m, 2H, cyclopropyl CH
2
), 1.04–1.00
+
(
m, 1H), 0.98–0.95 (m, 5H). MS-ESI (m/z): 632.63 (M+H) .
7
-(3-((S)-1-(tert-Butoxycarbonyl)pyrrolidine-2-carboxamido)-4-(methoxyimino)piperidin-1-yl)-1-
cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3e). The title
1
compound 3e was obtained from 2e as a off-white solid (83.6%), m.p.: 186–187 °C. H-NMR (400 MHz,

Molecules 2014, 19
6830
CDCl ) δ (ppm) 14.67 (s, 1H, COOH), 8.83 (s, 1H, C -H), 7.91 (d, J = 11.5 Hz, 1H, C -H), 7.00 (s, 1H),
3
2
5
4.63 (s, 1H), 4.22 (s, 1H), 4.10 (d, J = 15.4 Hz, 1H), 4.03 (d, J = 3.9 Hz, 1H), 3.91 (d, J = 2.6 Hz, 3H,
CH O-N), 3.81 (d, J = 4.8 Hz, 3H, CH O-C), 3.70–3.17 (m, 6H), 3.03 (s, 1H), 2.47–2.10 (m, 2H),
3
3
2
.00–1.84 (m, 2H), 1.46 (d, J = 10.9 Hz, 9H, Boc), 1.29–1.20 (m, 2H, cyclopropyl CH ), 1.00 (d,
2
+
J = 4.4 Hz, 2H, cyclopropyl CH
2
). MS-ESI (m/z): 616.67 (M+H) .
7
1
2
-(3-((2S,3S)-2-(tert-Butoxycarbonylamino)-3-methylpentanamido)-4-(methoxyimino)piperidin-1-yl)-
-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3f). Obtained from
1
f as a white solid (96.5%), m.p. : 126–127 °C. H-NMR (400 MHz, CDCl ) δ (ppm) 14.68 (s, 1H,
3
COOH), 8.83 (s, 1H, C
2
5
-H), 7.91 (d, J = 11.6 Hz, 1H, C -H), 6.99 (d, J = 5.9 Hz, 1H), 5.08 (s, 1H),
4
3
.67 (d, J = 4.1 Hz, 1H), 4.21–3.98 (m, 3H), 3.92 (d, J = 8.5 Hz, 3H, CH O-N), 3.80 (s, 3H, CH O-C),
3
3
.61 (s, 1H), 3.35 (d, J = 14.1 Hz, 1H), 3.26 (s, 1H), 3.03 (d, J = 9.6 Hz, 1H), 2.30 (s, 1H), 1.90–1.82
(
(
m, 2H), 1.46 (s, 9H, Boc), 1.29–1.16 (m, 3H), 1.01 (s, 2H), 0.95–0.91 (m, 6H). MS-ESI (m/z): 632.35
+
M+H) .
7
-(3-((S)-2-(tert-Butoxycarbonylamino)-3-phenylpropanamido)-4-(methoxyimino)piperidin-1-yl)-1-
cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3g). Obtained from 2g
1
as a white solid (79.1%), m.p.: 149–150 °C. H-NMR (400 MHz, CDCl
3
) δ (ppm) 14.68 (s, 1H,
-H), 7.40–7.22 (m, 5H, ph-H), 6.90–6.77 (m,
H), 5.07–5.01 (m, 1H), 4.56 (s, 1H), 4.40 (s, 1H), 4.16–3.97 (m, 2H), 3.85 (d, J = 10.0 Hz, 3H,
2 5
COOH), 8.84 (s, 1H, C -H), 7.92 (d, J = 11.6 Hz, 1H, C
1
3 3
CH O-N), 3.79 (s, 3H, CH O-C), 3.56 (s, 1H), 3.31–2.75 (m, 5H), 2.28 (t, J = 12.4 Hz, 1H), 1.43 (s,
9
H, Boc), 1.28–1.20 (m, 2H, cyclopropyl CH
2
), 1.03 (d, J = 11.3 Hz, 2H, cyclopropyl CH
2
). MS-ESI
+
(
m/z): 666.31 (M+H) .
7
-(3-((S)-2-(tert-Butoxycarbonylamino)-3-hydroxypropanamido)-4-(methoxyimino)piperidin-1-yl)-1-
cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3h). Obtained from 2h
1
as a off-white solid (83.3%), m.p. : 133–135 °C. H-NMR (400 MHz, CDCl
3
) δ (ppm) 14.64 (s, 1H,
-H), 7.15 (s, 1H), 5.48 (s, 1H), 4.84–4.61 (m,
H), 4.23 (s, 1H), 4.17–3.97 (m, 3H), 3.93 (d, J = 11.7 Hz, 3H, CH O-N), 3.80 (d, J = 2.6 Hz, 3H,
CH O-C), 3.70–3.68 (m, 1H), 3.64–3.43 (m, 1H), 3.35–3.25 (m, 2H), 3.12 (t, J = 10.3 Hz, 1H), 2.37–2.31
2 5
COOH), 8.83 (s, 1H, C -H), 7.92 (d, J = 11.6 Hz, 1H, C
1
3
3
(
2
m, 1H), 1.91 (brs, 1H, OH), 1.47 (d, J = 10.2 Hz, 9H, Boc), 1.29–1.19 (m, 2H, cyclopropyl CH ), 1.03 (t,
+
J = 7.7 Hz, 2H, cyclopropyl CH
2
). MS-ESI (m/z): 606.24 (M+H) .
7-(3-((S)-2-(tert-Butoxycarbonylamino)-3-(4-hydroxyphenyl)propanamido)-4-(methoxyimino)piperidin-1-
yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3i). Obtained
1
from 2i as a off-white solid (43.9%), m.p.: 151–153 °C. H-NMR (400 MHz, CDCl
3
) δ (ppm) 14.73 (s,
1
6
3
H, COOH), 8.82 (s, 1H, C
.72 (d, J = 8.3 Hz, 2H), 5.14 (d, J = 35.9 Hz, 1H), 4.56 (s, 1H), 4.37 (s, 1H), 4.06 (d, J = 12.0 Hz, 2H),
.87 (d, J = 10.9 Hz, 3H, CH O-N), 3.80 (d, J = 4.7 Hz, 3H, CH O-C), 3.76–3.46 (m, 2H), 3.22 (d,
2 5
-H), 7.89–7.84 (m, 1H, C -H), 7.02 (d, J = 8.1 Hz, 2H), 6.98–6.76 (m, 1H),
3
3
J = 11.9 Hz, 2H), 3.03–2.89 (m, 2H), 2.38–2.23 (m, 1H), 1.44 (s, 9H, Boc), 1.26–1.19 (m, 2H,
+
cyclopropyl CH ), 1.12–0.94 (m, 2H, cyclopropyl CH ). MS-ESI (m/z): 682.22 (M+H) .
2
2

Molecules 2014, 19
6831
7
1
2
-(3-((2S,3R)-2-(tert-Butoxycarbonylamino)-3-hydroxybutanamido)-4-(methoxyimino)piperidin-1-yl)-
-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3j). Obtained from
1
j as a white solid (47.7%), m.p.: 134–136 °C. H-NMR (400 MHz, CDCl
3
) δ (ppm) 14.64 (s, 1H,
COOH), 8.83 (s, 1H, C -H), 7.92 (d, J = 11.6 Hz, 1H, C -H), 7.33 (s, 1H), 5.42 (s, 1H), 4.74–4.65 (m,
2
5
1
3 3
H), 4.42–4.33 (m, 1H), 4.17–4.03 (m, 3H), 3.92 (t, J = 4.3 Hz, 3H, CH O-N), 3.80 (s, 3H, CH O-C),
3
.61 (s, 1H), 3.29 (dd, J = 22.8, 13.8 Hz, 2H), 3.11 (t, J = 11.4 Hz, 1H), 2.43–2.26 (m, 1H), 1.80 (brs,
H, OH), 1.46 (d, J = 11.6 Hz, 9H, Boc), 1.26–1.20 (m, 5H, cyclopropyl CH , CH CH), 1.01 (s, 2H,
1
2
3
+
2
cyclopropyl CH ). MS-ESI (m/z): 620.21 (M+H) .
3
.2.2. General Procedure for the Synthesis of 4a–j
To a stirring solution of trifluoroacetic acid (8.0 mL) was added 3a–j (1.80 mmol) in portions over
a period of 0.5 h at −5–0 °C and stirred for 3 h at the same temperature, and then concentrated under
reduced pressure. The residue was treated with diethyl ether (10 mL) and then filtered. The solid was
dissolved in methanol (2 mL) and adjusted to pH 7.0 with the ammonia water, and then extracted with
dichloromethane (30 mL × 3). The combined extracts were washed with saturated brine (10 mL) and
dried over anhydrous Na SO , and then concentrated under reduced pressure to provide the title
2
4
compounds 4a–j.
7
1
-(3-(2-Aminoacetamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-
1
,4-dihydroquinoline-3-carboxylic acid (4a). Obtained from 3a as a yellow solid (71.8%). H-NMR
(400 MHz, CDCl
3
2 5
) δ (ppm) 8.83 (s, 1H, C -H), 8.14 (s, 1H), 7.90 (d, J = 8.9 Hz, 1H, C -H), 4.71 (s,
1
H), 4.03–3.81 (m, 6H), 3.62 (s, 1H), 3.49–3.12 (m, 6H), 2.41 (s, 2H), 1.25–1.21 (m, 2H, cyclopropyl
13
2 2 6
CH ), 1.02 (d, J = 17.5 Hz, 2H, cyclopropyl CH ). C-NMR (150 MHz, DMSO-d ) δ 176.33, 172.48,
1
6
65.59, 156.28, 154.37, 150.61, 146.23, 138.59(d, J = 12.2 Hz), 134.06, 121.49, 106.69, 106.46, 63.24,
+
1.33, 55.74, 49.15, 44.50, 40.73, 24.85, 18.55, 9.03, 8.86. MS-ESI (m/z): 476.23(M+H) . HRMS-ESI
+
+
(
m/z): C H O N F, Calcd: 476.193899(M+H) ; Found: 476.19389(M+H) .
22 27 6 5
7
-(3-((S)-2-Aminopropanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-
4
-oxo-1,4-dihydroquinoline-3-carboxylic acid (4b). Obtained from 3b as a yellow solid (88.3%).
1
H-NMR (400 MHz, CDCl
-H), 4.67 (s, 1H), 4.06 (s, 2H), 3.93 (s, 3H, CH
H), 3.31 (d, J = 11.1 Hz, 2H), 3.11 (s, 1H), 2.55–2.30 (m, 1H), 1.38–1.33 (m, 2H), 1.26–1.19 (m, 3H),
3
) δ (ppm) 8.81 (s, 1H, C
2
-H), 8.38–8.00 (m, 1H), 7.87 (d, J = 11.7 Hz, 1H,
C
5
3
O-N), 3.82 (s, 3H, CH
3
O-C), 3.57 (d, J = 13.5 Hz,
2
1
1
2
4
1
3
3
.03 (s, 2H). C-NMR (100 MHz, CDCl ) δ 177.15, 175.65, 166.78, 157.70, 153.47, 150.12, 146.20,
39.02 (d, J = 12 Hz), 133.91, 122.77, 108.40, 107.96, 62.84, 62.06, 56.65, 51.00, 50.64, 50.24, 40.70,
+
5.67, 21.77, 9.86, 9.53. MS-ESI (m/z): 490.25 (M+H) .HRMS-ESI (m/z): C23
H
29
O
6
N
5
F, Calcd:
+
+
90.20964 (M+H) ; Found: 490.20968 (M+H) .
7
-(3-((S)-2-Amino-3-methylbutanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-
methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4c). Obtained from 3c as a light yellow solid
1
(
70.0%). H-NMR (400 MHz, DMSO-d
6
) δ (ppm) 8.70 (s, 1H, C
2
-H), 8.29–8.24 (m, 1H), 7.77 (d,
J = 10.6 Hz, 1H, C -H), 4.59–4.55 (m, 1H), 4.16 (s, 1H), 3.83 (s, 3H, CH O-N), 3.76 (d, J = 5.2 Hz,
5
3
3
3
H, CH O-N), 3.73–3.58 (m, 1H), 3.56–3.41 (m, 1H), 3.28–3.16 (m, 1H), 3.03 (d, J = 3.5 Hz, 2H),

Molecules 2014, 19
6832
2
0
.60–2.50 (m, 1H), 1.98–1.94 (m, 1H), 1.91–1.72 (m, 1H), 1.21–0.91 (m, 4H, 2 × cyclopropyl CH ),
2
1
3
6
.87 (d, J = 6.8 Hz, 1H), 0.80 (d, J = 6.8 Hz, 2H), 0.74–0.70 (m, 3H). C-NMR (100 MHz, DMSO-d )
δ 176.30, 174.16, 165.61, 156.74, 154.45, 150.62, 146.08(d, J = 18 Hz), 138.60, 134.04, 121.33,
06.65, 106.42, 63.11, 61.27, 59.66, 55.47, 49.25, 40.71, 30.30, 24.76, 19.29, 16.65, 16.53, 8.98, 8.88.
1
+
+
MS-ESI (m/z): 518.35 (M+H) .HRMS-ESI (m/z): C H O N F, Calcd: 518.24094 (M+H) ; Found:
2
5
33
6
5
+
5
18.24080 (M+H) .
7
-(3-((S)-2-Amino-4-methylpentanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-
methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4d). Obtained from 3d as a yellow solid
1
(
71.5%). H-NMR (400 MHz, DMSO-d ) δ (ppm) 8.71 (s, 1H, C -H), 8.46–8.23 (m, 1H), 7.77 (d,
6
2
J = 10.6 Hz, 1H, C -H), 4.55–4.49 (m, 1H), 4.17 (s, 1H), 3.83 (s, 3H, CH O-N), 3.76 (d, J = 8.3 Hz,
5
3
3
3H, CH O-C), 3.69 (s, 1H), 3.48–3.21 (m, 3H), 3.03 (t, J = 16.1 Hz, 1H), 2.64 (d, J = 6.4 Hz, 1H),
1
.72 (s, 1H), 1.56 (s, 1H), 1.41 (s, 1H), 1.35–0.96 (m, 5H), 0.86–0.81 (m, 3H), 0.77–0.73 (m, 3H).
1
3
6
C-NMR (100 MHz, DMSO-d ) δ 176.31, 175.04, 165.60, 156.72, 154.44, 150.60, 146.02 (d, J = 33 Hz),
1
2
38.66, 134.03, 121.43, 106.65, 106.42, 63.13, 61.27, 55.51, 52.99, 49.16, 44.07, 40.70, 24.87, 23.97,
+
3.12, 23.03, 21.85, 8.97, 8.93. MS-ESI (m/z): 532.40 (M+H) . HRMS-ESI (m/z): C26
H
35
O
6
N
5
F,
+
+
Calcd: 532.25659 (M+H) ; Found: 532.25671 (M+H) .
7
-(4-(methoxyimino)-3-((S)-pyrrolidine-2-carboxamido)piperidin-1-yl)-1-Cyclopropyl-6-fluoro-8-
methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4e). Obtained from 3e as a yellow solid
1
(
66.8%). H-NMR (400 MHz, CDCl
3
) δ (ppm) 8.81 (s, 1H, C
2
-H), 8.45–8.39 (m, 1H), 7.87 (d, J = 11.7 Hz,
O-N), 3.81 (s, 4H), 3.57 (d,
J = 12.5 Hz, 1H), 3.31–3.27 (m, 2H), 3.15 (t, J = 10.7 Hz, 1H), 3.05–2.93 (m, 2H), 2.51–2.31 (m, 1H),
.15–2.10 (m, 1H), 2.00–1.95 (m, 1H), 1.88–1.56 (m, 2H), 1.24–1.19 (m, 2H, cyclopropyl CH ), 1.02
) δ 177.12, 175.17, 166.76, 157.61, 153.61, 150.11, 146.10,
38.97 (d, J = 12 Hz), 133.91, 122.64, 108.41, 107.97, 62.81, 61.96, 60.82, 56.59, 50.52, 49.88, 47.37,
1
5 3
H, C -H), 4.67 (s, 1H), 4.04–3.98 (m, 2H), 3.92 (d, J = 7.8 Hz, 3H, CH
2
2
1
3
(
s, 2H). C-NMR (100 MHz, CDCl
3
1
4
+
31 6 5
0.68, 30.92, 26.21, 25.61, 9.86, 9.53. MS-ESI (m/z): 516.36 (M+H) .HRMS-ESI (m/z): C25H O N F,
+
+
Calcd: 516.22529 (M+H) ; Found: 516.22545 (M+H) .
7
-(3-((2S,3S)-2-Amino-3-methylpentanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-
fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4f). Obtained from 3f as a yellow
1
solid (88.3%). H-NMR (400 MHz, CDCl ) δ (ppm) 8.73 (d, J = 3.5 Hz, 1H, C -H), 7.91–7.57 (m, 1H,
3
2
C
5
-H), 7.46 (s, 1H), 4.71 (s, 1H), 4.13–4.04 (m, 3H), 3.90 (d, J = 6.9 Hz, 3H, CH
3
O-N), 3.79 (s, 3H,
CH O-C), 3.57–3.47 (m, 1H), 3.34–3.19 (m, 2H), 2.41 (s, 1H), 2.13 (s, 1H), 1.63–1.58 (m, 1H), 1.22
3
1
3
(
3
d, J = 6.4 Hz, 4H), 1.06–0.97 (m, 6H), 0.92 (s, 2H). C-NMR (100 MHz, CDCl ) δ 176.74, 175.34,
1
6
66.81, 154.83, 153.05, 150.25, 145.93(d, J = 12 Hz), 138.91, 134.05, 122.08, 107.94, 107.61, 63.12,
2.15, 58.19, 55.96, 50.94, 50.48, 40.87, 37.01, 25.38, 14.86, 11.75, 9.76. MS-ESI (m/z): 532.33
+
+
+
(
M+H) .HRMS-ESI (m/z): C26
35 6 5
H O N F, Calcd: 532.25659 (M+H) ; Found: 532.25653 (M+H) .
7
-(3-((S)-2-Amino-3-phenylpropanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-
methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4g). Obtained from 3g as a yellow solid
1
(
82.4%). H-NMR (400 MHz, DMSO-d
6
2
) δ (ppm) 8.72 (s, 1H, C -H), 8.28 (d, J = 4.6 Hz, 1H),
7
.90–7.70 (m, 1H, C
5
-H), 7.40–6.90 (m, 5H, ph-H), 4.54 (s, 1H), 4.17 (s, 1H), 3.83 (d, J = 8.0 Hz, 3H,

Molecules 2014, 19
6833
CH O-N), 3.76–3.66 (m, 4H), 3.47 (d, J = 5.7 Hz, 2H), 3.31 (s, 2H), 3.23–3.07 (m, 1H), 2.96–2.91 (m,
3
1
3
2
1
1
2 6
H), 2.74–2.51 (m, 1H), 1.26–0.83 (m, 4H, 2 × cyclopropyl CH ). C-NMR (100 MHz, DMSO-d ) δ
76.29, 173.76, 165.66, 156.71, 154.33, 150.64, 146.24, 138.35(d, J = 29 Hz), 134.00, 129.24, 128.02,
26.01, 121.57, 106.69, 106.46, 63.14, 61.30, 56.00, 55.68, 49.25, 40.68, 40.45, 24.70, 16.37, 8.95.
+
+
MS-ESI (m/z): 566.40 (M+H) . HRMS-ESI (m/z): C H O N F, Calcd: 566.24094 (M+H) ; Found:
2
9
33
6
5
+
5
66.24108 (M+H) .
7
8
-(3-((S)-2-Amino-3-hydroxypropanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-
-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4h). Obtained from 3h as a yellow solid
1
(
66.2%). H-NMR (400 MHz, DMSO-d ) δ (ppm) 8.71 (s, 1H, C -H), 8.36 (d, J = 5.1 Hz, 1H), 7.78 (d,
6
2
J = 8.0 Hz, 1H, C -H), 4.76 (s, 1H), 4.56–4.52 (m, 1H), 4.17 (s, 1H), 3.84 (d, J = 0.9 Hz, 3H, CH O-N),
5
3
3
1
1
6
.81–3.70 (m, 4H), 3.60–3.35 (m, 4H), 3.26–3.25 (m, 2H), 3.11–2.96 (m, 1H), 2.56–2.51 (m, 1H),
1
3
2 6
.13–0.96 (m, 4H, 2 × cyclopropyl CH ). C-NMR (100 MHz, DMSO-d ) δ 176.36, 172.77, 165.60,
56.82, 154.37, 150.61, 146.29 (d, J = 11 Hz), 138.67, 134.05, 121.49, 106.69, 106.42, 64.90, 63.18,
+
1.34, 56.76, 55.65, 49.40, 40.75, 24.85, 15.16, 8.92. MS-ESI (m/z): 506.28 (M+H) .HRMS-ESI (m/z):
+
+
C H O N F, Calcd: 506.20455 (M+H) ; Found: 506.20465 (M+H) .
2
3
29
7
5
7
6
-(3-((S)-2-Amino-3-(4-hydroxyphenyl)propanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-
-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4i). Obtained from 3i as a light
1
yellow solid (83.4%). H-NMR (400 MHz, DMSO-d
6
) δ (ppm) 9.11 (d, J = 15.9 Hz, 1H), 8.71 (d,
-H), 6.95 (t, J = 7.6 Hz,
H), 6.59 (dd, J = 14.6, 8.3 Hz, 2H), 4.53 (s, 1H), 4.18 (dd, J = 6.8, 3.8 Hz, 1H), 3.83 (d, J = 7.8 Hz,
H, CH O-N), 3.74 (d, J = 10.8 Hz, 3H, CH O-C), 3.67 (d, J = 7.8 Hz, 1H), 3.54–3.44 (m, 1H),
.42–3.34 (m, 2H), 3.26–3.16 (m, 1H), 3.15–2.89 (m, 2H), 2.79 (d, J = 11.0 Hz, 1H), 2.65–2.53 (m,
2 5
J = 4.5 Hz, 1H, C -H), 8.23 (t, J = 8.0 Hz, 1H), 7.79 (dd, J = 11.8, 6.1 Hz, 1H, C
2
3
3
1
1
1
9
3
3
1
3
2 6
H), 1.16–1.01 (m, 4H, 2 × cyclopropyl CH ). C-NMR (100 MHz, DMSO-d ) δ 176.40, 173.94,
65.64, 156.74, 155.78, 154.30, 150.63, 146.26, 138.72(d, J = 38 Hz), 134.07, 130.27, 130.09, 128.33,
27.95, 121.53, 114.88, 106.69, 106.48, 63.15, 61.31, 56.18, 55.80, 49.71, 49.24, 40.75, 24.87, 15.15,
+
+
.06, 8.96. MS-ESI (m/z): 582.30 (M+H) .HRMS-ESI (m/z): C29
H
33
O N
7 5
F, Calcd: 582.23585 (M+H) ;
+
Found: 582.23593 (M+H) .
7
-(3-((2S,3R)-2-Amino-3-hydroxybutanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-
fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4j). Obtained from 3j as a yellow
1
solid (73.3%). H-NMR (400 MHz, DMSO-d
6
2
) δ (ppm) 8.71 (s, 1H, C -H), 8.38 (d, J = 7.0 Hz, 1H),
7
3
2
.78 (dd, J = 11.8, 3.1 Hz, 1H, C
5
-H), 4.63–4.50 (m, 1H), 4.22–4.10 (m, 1H), 3.91–3.80 (m, 4H),
.79–3.59 (m, 4H), 3.51–3.47 (m, 1H), 3.31–3.12 (m, 2H), 3.10–3.02 (m, 1H), 2.99 (d, J = 4.1 Hz, 1H),
.60–2.51 (m, 1H), 1.29–1.23 (m, 1H), 1.13–1.11 (m, 2H), 1.04–0.97 (m, 3H), 0.91–0.73 (m, 1H).
C-NMR (100 MHz, DMSO-d6) δ 176.34, 173.04, 165.60, 156.84, 154.39, 150.61, 146.10, 138.56 (d,
1
3
J = 38 Hz), 134.06, 122.24, 106.66, 106.42, 67.14, 63.17, 61.32, 60.19, 55.60, 49.47, 39.94, 24.85,
+
2
0.08, 19.94, 8.94. MS-ESI (m/z): 520.28 (M+H) . HRMS-ESI (m/z): C24
H
31
O
7
N
5
F, Calcd: 520.22020
+
+
(
M+H) ; Found: 520.22083 (M+H) .

Molecules 2014, 19
.2.3. General Procedure for the Synthesis of 3k–l
A mixture of 4a–b (0.82 mmol), 2a–b (0.82 mmol), dicyclohexylcarbodiimide (0.17 g, 0.82 mmol)
6834
3
and dry dichloromethane (20 mL) was stirred at room temperature for 4 h, filtered and concentrated
under reduced pressure. The solid was treated with diethyl ether (3 mL) and filtered. The solid
was purified by column chromatography (silica gel) eluted with dichloromethane and methanol
(
v:v = 60:1) to afford the title compounds 3k–l.
7
-(3-(2-(2-(tert-Butoxycarbonylamino)acetamido)acetamido)-4-(methoxyimino)piperidin-1-yl)-1-
cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3k). Obtained from 4a
1
and 2a as a light yellow solid (30.8%), m.p.: 122–124 °C. H-NMR (400 MHz, CDCl ) δ (ppm) 14.71
3
(
s, 1H, COOH), 8.83 (s, 1H, C -H), 7.90 (d, J = 11.6 Hz, 1H, C -H), 6.97 (d, J = 5.6 Hz, 1H), 6.75 (s,
2
5
1
3
H), 5.07 (s, 1H), 4.72–4.56 (m, 1H), 4.11 (d, J = 7.0 Hz, 1H), 4.06–3.95 (m, 3H), 3.93 (s, 3H, CH O-
N), 3.86 (d, J = 3.7 Hz, 2H), 3.78 (s, 3H, CH O-C), 3.61 (d, J = 10.0 Hz, 1H), 3.34–3.24 (m, 2H),
3
3
1
.07 (t, J = 10.5 Hz, 1H), 2.42–2.26 (m, 1H), 1.45 (s, 9H, Boc), 1.26–1.21 (m, 2H, cyclopropyl CH ),
2
+
.07–0.98 (m, 2H, cyclopropyl CH
2
). MS-ESI (m/z): 633.17 (M+H) .
7-(3-((S)-2-((S)-2-(tert-Butoxycarbonylamino)propanamido)propanamido)-4-(methoxyimino)piperidin-1-
yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3l). Obtained
1
from 4b and 2b as a yellow solid (71.5%), m.p.: 126–128 °C. H-NMR (400 MHz, CDCl
3
) δ (ppm)
-H), 7.18–6.92 (m, 1H), 6.71 (d,
J = 20.8 Hz, 1H), 4.93 (s, 1H), 4.70–4.58 (m, 1H), 4.53–4.48 (m, 1H), 4.16 (s, 1H), 4.16–4.03 (m, 2H),
1
2 5
4.68 (s, 1H, COOH), 8.83 (s, 1H, C -H), 7.90 (d, J = 11.6 Hz, 1H, C
3 3
3.92 (d, J = 4.0 Hz, 3H, CH O-N), 3.79 (d, J = 1.5 Hz, 3H, CH O-C), 3.59 (s, 1H), 3.40–3.18 (m, 2H),
3
.14–2.97 (m, 1H), 2.34–2.32 (m, 1H), 1.63–1.29 (m, 15H, Boc, 2 × CH
3
+
), 1.28–1.16 (m, 2H, cyclopropyl
CH
.2.4. General Procedure for the Synthesis of 4k–l
To a stirring solution of trifluoroacetic acid (3.0 mL) was added 3k–l (0.47 mmol) in portions over
2 2
), 1.09–0.87 (m, 2H, cyclopropyl CH ). MS-ESI (m/z): 661.33 (M+H) .
3
a period of 0.5 h at −5–0 °C, stirred for 2 h at the same temperature and then concentrated under
reduced pressure. The residue was treated with diethyl ether (7 mL) and filtered. The solid was
dissolved in methanol (1 mL) and adjusted to pH 7.0 with the ammonia water, and then extracted with
dichloromethane (50 mL × 3). The combined extracts were washed with saturated brine (10 mL), dried
over anhydrous Na
2 4
SO , and concentrated under reduced pressure to provide the title compounds 4k–l.
7
-(3-(2-(2-Aminoacetamido)acetamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-
methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4k). Obtained from 3k as a yellow solid
1
(
73.3%). H-NMR (400 MHz, CDCl
3
) δ (ppm) 8.81 (s, 1H, C
2
-H), 7.98–7.80 (m, 2H), 7.11 (d, J = 5.9 Hz,
1
H), 4.75–4.56 (m, 1H), 4.14–4.10 (m, 1H), 4.07–4.00 (m, 2H), 3.97 (d, J = 5.6 Hz, 1H), 3.92 (s, 3H,
CH O-N), 3.79 (s, 3H, CH O-C), 3.60 (d, J = 8.0 Hz, 1H), 3.52–3.44 (m, 1H), 3.44 (s, 1H), 3.35–3.19
3
3
(
(
m, 2H), 3.09 (t, J = 10.7 Hz, 1H), 2.46–2.22 (m, 1H), 1.27–1.18 (m, 2H, cyclopropyl CH
2
), 1.13–0.95
) δ 177.07, 173.52, 168.75, 166.74, 157.52,
52.99, 150.12, 146.05, 138.85(d, J = 12 Hz), 133.94, 122.74, 108.33, 107.96, 62.95, 62.11, 56.43,
1
3
2 3
m, 2H, cyclopropyl CH ). C-NMR (100 MHz, CDCl
1

Molecules 2014, 19
6835
+
5
0.57, 50.15, 44.79, 42.87, 40.70, 25.66, 9.86, 9.53. MS-ESI (m/z): 533.32 (M+H) .HRMS-ESI (m/z):
+
+
C
24 30 7 6
H O N F, Calcd: 533.21545 (M+H) ; Found: 533.21660 (M+H) .
7
6
-(3-((S)-2-((S)-2-Aminopropanamido)propanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-
-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4l). Obtained from 3l as a yellow
1
solid (34.7%). H-NMR (400 MHz, CDCl ) δ (ppm) 8.81 (s, 1H, C -H), 7.86 (dd, J = 11.6, 6.2 Hz, 1H,
3
2
C
5
-H), 7.75 (t, J = 7.8 Hz, 1H), 7.28–7.10 (m, 1H), 4.66–4.62 (m, 1H), 4.56–4.47 (m, 1H), 4.13–4.00
(
m, 2H), 3.92 (d, J = 4.6 Hz, 3H, CH O-N), 3.80 (s, 3H, CH O-C), 3.68–3.44 (m, 2H), 3.32–3.25 (m,
3
3
2
2
1
4
H), 3.08 (t, J = 10.4 Hz, 1H), 2.34 (t, J = 12.7 Hz, 1H), 1.44–1.33 (m, 5H), 1.26–1.21 (m, 3H), 1.03 (s,
1
3
3
H). C-NMR (100 MHz, CDCl ) δ 177.11, 175.83, 172.12, 166.76, 159.46, 153.12, 150.11,
46.14(d, J = 12 Hz), 138.80, 133.90, 122.82, 108.39, 107.98, 62.90, 62.07, 56.37, 50.78, 50.60, 48.68,
+
8.47, 40.69, 25.69, 21.66, 18.13, 9.80, 9.58. MS-ESI (m/z): 561.44 (M+H) .HRMS-ESI (m/z):
+
+
C H O N F, Calcd: 561.24675 (M+H) ; Found: 561.24691 (M+H) .
2
6
34
7
6
3.3. 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 using agar
media [17]. 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 18–24 h.
4
. Conclusions
In summary, a series of amino acid and dipeptide prodrugs of IMB-070593 were designed,
synthesized and evaluated for their water solubility and antibacterial activity in this study. Our results
reveal that the solubility (>85 mg/mL) of four amino acid prodrugs 4a,b,e,f and two dipeptide
prodrugs 4k,l was much greater than that of IMB-070593 mesylate (22.5 mg/mL). Compounds 4a and
4
k were found to have lower ED50 values when administered by intravenous injection, compared with
orally. Moreover, both of them show stronger efficacy against drug-sensitive Gram-positive strains
than the parent drug, as opposed to Gram-negative and drug-resistant Gram-positive strains.
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.
Author Contributions
Conceived and designed the experiments: Mingliang Liu, Huiyuan Guo. Performed the
experiments: Tingting Zhang, Jinwei Wu and Kaixiang Liu. Analyzed the data: Tingting Zhang, Jinwei
Wu, Shihong Chen, Kaixiang Liu and Yabin Lin. Wrote the paper: Tingting Zhang, Mingliang Liu,
Huiyuan Guo. All authors read and approved the final manuscript.

Molecules 2014, 19
6836
Conflicts of Interest
The authors declare no conflict of interest.
References and Notes
1
.
Feng, L.S.; Liu, M.L.; Wang, S.; Chai, Y.; Lv, K.; Shan, G.Z.; Cao, J.; Li, S.J.; Guo, H.Y.
Synthesis of naphthyridone derivatives containing 8-alkoxyimino-1,6-dizaspiro[3.4]octane
scaffolds. Tetrahedron 2011, 67, 8264–8270.
2
.
Liu, M.L.; Guo, H.Y. Evolution of quinolone antibacterial drugs. World. Notes. Antibiot. (Chin.)
2
006, 27, 69–75.
3
.
Dang, Z.; Yang, Y.S.; Ji, R.Y.; Zhang, S.H. Synthesis and antibacterial activity of novel
fluoroquinolones containing substituted piperidines. Bioorg. Med. Chem. Lett. 2007, 17, 4523–4526.
Hong, C.Y.; Kim, Y.K.; Chang, J.H.; Kim, S.H.; Choi, H.; Nam, D.H.; Kim, Y.Z.; Kwak, J.H.
Novel fluoroquinolone antibacterial agents containing oxime-substituted (aminomethyl)pyrrolidines:
Synthesis and antibacterial activity of 7-(4-(aminomethyl)-3-(methoxyimino)pyrrolidin-1-yl)-1-
cyclopropyl-6-fluoro-4-oxo-1,4-dihydro[1,8]naphthyridine-3-carboxylic acid (LB20304). J. Med.
Chem. 1997, 40, 3584–3593.
4
.
5
6
7
8
9
1
1
1
.
.
.
.
.
Choi, D.R.; Shin, J.H.; Yang, J.; Yoon, S.H.; Jung, Y.H. Syntheses and biological evaluation of
new fluoroquinolone antibacterials containing chiral oxiimino pyrrolidine. Bioorg. Med. Chem. Lett.
2
004, 14, 1273–1277.
Kwon, A.R.; Min, Y.H.; Ryu, J.M.; Choi, D.R.; Shim, M.J.; Choi, E.C. In vitro and in vivo
activities of DW-224a, a novel fluoroquinolone antibiotic agent. Chin. J. Antimicrob. Chemother.
2
006, 58, 684–688.
Lv, K.; Sun, Y.X.; Sun, L.Y.; Wei, Z.Q.; Guo, H.Y.; Wu, J.W.; Liu, M.L. Design, synthesis, and
in vitro antibacterial activity of fluoroquinolone derivatives containing a chiral 3-(alkoxyimino)-2-
(
aminomethyl)azetidine moiety. Chem. Med. Chem. 2012, 7, 1230–1236.
Lv, K.; Liu, M.L.; Feng, L.S.; Sun, L.Y.; Sun, Y.X.; Wei, Z.Q.; Guo, H.Y. Synthesis and
antibacterial activity of naphthyridone derivatives containing mono/difluoro-methyloxime
pyrrolidine scaffolds. Eur. J. Med. Chem. 2012, 47, 619–625.
Wang, X.Y.; Guo, Q.; Wang, Y.C.; Liu, B.Q.; Liu, M.L.; Sun, L.Y.; Guo, H.Y. Synthesis and
antibacterial activity of 7-(3-amino-4-alkoxyimino-1-piperidyl)quinolones. Acta Pharm. Sin.
2
008, 43, 819–827.
0. Zhang, Y.B.; Li, G.Q.; Liu, M.L.; You, X.F.; Feng, L.S.; Lv, K.; Cao, J.; Guo, H.Y. Synthesis and
in vitro antibacterial activity of 7-(3-alkoxyimino-5-amino/methylaminopiperidin-1-yl)fluoroquinolone
derivatives. Bioorg. Med. Chem. Lett. 2011, 21, 928–931.
1. Guo, H.Y.; Liu, M.L.; Liu, B.Q.; Hu, J.S.; Wu, J.W.; Wang, Z. Application of 7-(4-alkoxyimino-
3
2
-aminopiperidin-1-yl)fluoroquinolones and their combinations. CN Patent 101863876,
0 October 2010.
2. Sanchez, J.P.; Domagala, J.M.; Heifetz, C.L.; Priebe, S.R.; Sesnie, J.A.; Trehan, A.K. Quinolone
antibacterial agents. Synthesis and structure-activity relationships of a series of amino acid

Molecules 2014, 19
6837
prodrugs of racemic and chiral 7-(3-amino-1-pyrrolidinyl)quinolones. Highly soluble quinolone
prodrugs with in vivo pseudomonas activity. J. Med. Chem. 1992, 35, 1764–1773.
1
1
1
1
3. De Clercq, E. Dancing with chemical formulae of antivirals: A personal account. Biochem. Pharm.
2
013, 86, 711–725.
4. Liu, M.L.; Guo, H.Y. Synthesis and in vivo antibacterial activity of tosufloxacin prodrugs.
Chin. J. Antibiot. (Chin.) 2004, 29, 590–591.
5. Liu, M.L.; Wei, Y.G.; Sun, L.Y.; Zhang, S.H.; Guo, H.Y. Synthesis and the in vivo and in vitro
antibacterial activity of moxifloxacin prodrugs. Chin. J. Antibiot. (Chin.) 2003, 28, 647–653.
6. Furfine, E.S.; Baker, C.T.; Hale, M.R.; Reynolds, D.J.; Salisbury, J.A.; Searle, A.D.;
Studenberg, S.D.; Todd, D.; Tung, R.D.; Spaltenstein, A. Preclinical pharmacology and
pharmacokinetics of GW433908, a water-soluble prodrug of the human immunodeficiency virus
protease inhibitor amprenavir. Antimicrob. Agents. Chemother. 2004, 48, 791–798.
7. 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, Volume 21; NCCLS: Wayne, PA, USA, 2001, M100–S11). MIC was
defined as the lowest concentration of each compound that inhibits visible growth of bacteria after
incubation at 35 °C for 18–24 h.
1
1
8. Jahani, F.; Tajbakhsh, M.; Golchoubian, H.; Khaksar, S. Guanidine hydrochloride as an
organocatalyst for N-Boc protection of amino groups. Tetrahedron. Lett. 2011, 52, 1260–1264.
9. Ling, Y.; Ye, X.L.; Ji, H.; Zhang, Y.H.; Lai, Y.S.; Peng, S.X.; Tian, J.D. Synthesis and
evaluation of nitric oxide-releasing derivatives of farnesylthiosalicylic acid as anti-tumor agents.
Bioorg. Med. Chem. 2010, 18, 3448–3456.
1
2
0. Sutherland, A.; Willis, C.L. Chemoenzymatic synthesis of 4-amino-2-hydroxy acids: A comparison of
mutant and wild-type oxidoreductases. J. Org. Chem. 1998, 63, 7764–7769.
2
1. Gowda, D.C.; Abiraj, K.; Augustine, P. New hydrogenation catalyst: An advantageous method for
the removal of hydrogenolysable protecting groups in peptide synthesis. Lett. Pept. Sci. 2002, 9,
4
3–47.
2
2
2. Otsuka, H.; Inouye, K.; Kanayama, M.; Shinozaki, F. The synthesis and biological activities of a
hexadecapeptide corresponding to a modified sequence in the corticotropin structure. Bull. Chem.
Soc. Jpn. 1965, 38, 679–680.
3. Barcelo, G.; Senet, J.P.; Sennyey, G.; Bensoam, J.; Loffet, A. Alkyl 1-chloroalkyl carbonates:
Reagents for the synthesis of carbamates and protection of amino groups. Synthesis (Stuttg.).
1
986, 627–623.
Sample Availability: Samples of the compounds 3b–j, 3l and 4a–i and 4k are available from
the authors.
©
2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(