Synthesis and in vitro antibacterial activity of novel fluoroquinolone derivatives containing substituted piperidines

Article abstract of DOI:10.1016/j.bmcl.2010.07.006

We report herein the synthesis of novel 7-(4-alkoxyimino-3-aminomethyl-3- methylpiperidin-1-yl) fluoroquinolone derivatives. The antibacterial activity of the newly synthesized compounds was evaluated and correlated with their physicochemical properties.

Full text of DOI:10.1016/j.bmcl.2010.07.006

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5195–5198
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro antibacterial activity of novel fluoroquinolone
derivatives containing substituted piperidines
*
Yun Chai, Mingliang Liu , Bo Wang, Xuefu You, Lianshun Feng, Yibin Zhang, Jue Cao, Huiyuan Guo
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein the synthesis of novel 7-(4-alkoxyimino-3-aminomethyl-3-methylpiperidin-1-yl) fluo-
roquinolone derivatives. The antibacterial activity of the newly synthesized compounds was evaluated
and correlated with their physicochemical properties. Results reveal that all of the target compounds
have good potency in inhibiting the growth of Staphylococcus aureus and Staphylococcus epidermidis
Received 15 March 2010
Revised 1 July 2010
Accepted 2 July 2010
Available online 8 July 2010
including MRSE (MIC: 0.125–4 lg/mL). Compounds 12, 13 are more potent than or comparable to levo-
floxacin against MRSA, Streptococcus pyogenes, Escherichia coli, Klebsiella pneumoniae, and Shigella sonnei.
Compound 17 is more active than or comparable to levofloxacin against S. aureus including MRSA, S. epi-
dermidis and S. pyogenes.
Keywords:
Fluoroquinolone
Synthesis
Ó 2010 Elsevier Ltd. All rights reserved.
Antibacterial activity
Quinolone antibacterial agents represent a fast growing group
of antibiotics. These antibiotics exert their antimicrobial activity
by binding to two type II bacterial topoisomerase enzymes, DNA
gyrase (subunits encoded by gyrA and gyrB) and topoisomerase
IV (subunits encoded by grlA and grlB for Staphylococcus aureus).
This binding induces permanent double-stranded DNA breaks,
and results in cell death.1
Although most of the quinolones currently on the market or
under development are generally characterized by a broad antimi-
crobial spectrum, their activity against clinically important Gram-
positive cocci including Staphylococci, Streptococci, and Enterococci
is relatively moderate, which has not only limited their use in
infections caused by these organisms, but has also contributed to
rapidly developing quinolone resistance. Thus, recent efforts have
been directed toward the synthesis of new quinolones that can
provide improved Gram-positive antibacterial activity, while
retaining the good Gram-negative activity of early fluoroquino-
lones, such as ciprofloxacin and ofloxacin.2
The structure–activity relationship (SAR) study of fluoroquino-
lone antibacterial agents showed that the basic group at the C-7
position is the most adaptable site for chemical change and an area
that greatly influences their potency, spectrum and safety.3,4 In
general, five- and six-membered nitrogen heterocycles including
piperazinyl, pyrrolidinyl, and piperidinyl type side chains have
proven to be the optimal substituents. However, piperidinyl ana-
logs of the three are the least studied.5,6
positive activity, we also have focused on introducing new func-
tional groups to the piperidine ring.7–10 It was reported that
DW286 (Fig. 1), possessing an additional methyl group at the 3-po-
sition of the pyrrolidine ring displays more antibacterial activity
than Gemifloxacin against important Gram-positive organisms,
methicillin-resistant Staphylococcus aureus (MRSA) and ofloxacin
resistant organisms, while maintaining an excellent pharmacoki-
netic profile,11,12 and IMB (Fig. 1), a 8-methoxyl fluoroquinolone
with a methoxyimino group attached to the piperidine ring at C-7
position, shows good in vitro and in vivo antibacterial activity.7 In-
spired by those research results, a series of new fluoroquinolone
derivatives were designed and synthesized. These derivatives are
structurally novel, having both an aminomethyl and a methyl group
at the 3-position and an alkoxyimino group at 4-position of the
piperidine ring. Our primary objective was to optimize the potency
of these compounds against Gram-positive and Gram-negative
organisms.
The synthetic routes of new piperidine derivatives 9a,b and no-
vel fluoroquinolones 12–26 are shown in Schemes 1 and 2, respec-
tively. Addition reaction of ethyl 3-aminopropionate hydrochloride
1 with acrylonitrile in the presence of sodium hydroxide gave the
secondary amine 2, which was subsequently treated with di-tert-
butoxycarbonyl dicarbonate (Boc2O) to produce Boc-protected
cyano ester 3. The compound 3 was cyclized to the ketone 4 by
sodium hydride in refluxing toluene, with an overall yield of 83%
for the three steps. The ketone 4 was methylated with methyl
iodide to provide methyl ketone 5. However, selective reduction
of the cyano group to the primary amine, in the presence of the
ketone moiety of cyano ketone 5 was unsuccessful.
Recently, as part of an ongoing program to find potent and
broad-spectrum antibacterial agents that display strong Gram-
Therefore, catalytic hydrogenation of both functional groups of
the cyano ketone 5 and subsequent protection of the resulting
* Corresponding author. Tel.: +86 10 63036965; fax: +86 10 63047865.
E-mail address: lmllyx@yahoo.com.cn (M. Liu).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.006

5196
Y. Chai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5195–5198
O
O
N
O
N
COOH
COOH
F
F
F
COOH
N
N
N
N
N
MeON
Me
H2N
MeON
N
OMe
MeON
NH2
H2N
DW 286
Gemifloxacin
IMB
Figure 1. Structures of Gemifloxacin, DW286 and IMB.
O
O
CO2Et
CO2Et
CN
CN
Me
(c)
(d)
(b)
CO2Et
(e)
CN
CN
(a)
.
HCl
N
H
N
Boc
H2N
N
Boc
4
N
Boc
5
1
2
3
NOR1
NOR1
OH
O
(g)
NHBoc
NHBoc
(h)
NHBoc
(f)
NH2
2HCl
a: R1=Me
b: R1=Et
Me
Me
Me
Me
.
N
Boc
6
N
Boc
7
N
Boc
8a,b
N
H
9a,b
Scheme 1. Reagents and conditions: (a) acrylonitrile, NaOH, EtOH, 50 °C, 5 h; (b) Boc2O, MeOH, 50 °C, 3 h; (c) 60% NaH, toluene, reflux, 2 h, 83% (three steps); (d) MeI, K2CO3,
acetone, 40 °C, 6 h, 82%; (e) H2(g), 5% Pd/C, Boc2O, MeOH, rt, 8 h, 65%; (f) Jone’s reagent, acetone, 0 °C, 1 h, 86%; (g) R1ONH2ÁHCl, Et3N, MeOH, 55–60 °C, 2 h, 88–91%; (h) HCl(g),
CH2Cl2, rt, 1 h, 98%.
AcO OAc
O
B
O
O
O
F
COOH
F
COOH
9a,b
H2N
Me
F
F
O
or
(Cl)F
X
N
R2
i or j
N
X
N
R2
X
N
R2
11g,11h
R1ON
10a-10f
12-26
10a: X=N, R2=cyclopropyl
11g: X:C-methoxyl, R2=cyclopropyl
10b: X=N, R2=2,4-difluorophenyl
10c: X=CF, R2=cyclopropyl
11h: X, R2=
Ο
10d: X=C-difluoromethoxyl, R2=cyclopropyl
10e: X=CF, R2=ethyl
10f: X=CF, R2=2-fluoroethyl
Scheme 2. Reagents and conditions: (i) Et3N, MeCN, 25–50 °C, 2–10 h, 50–70%; (j) a—Et3N, MeCN, 25–50 °C, 2–24 h; b—5% NaOH/H2O, 40 °C, 0.5–2 h; c—2 N HCl, rt, 50–60%.
amine by Boc group yielded the bis-Boc-protected amino alcohol 6.
Oxidation of compound 6 by Jone’s reagent afforded the corre-
sponding ketone 7, on which the oxime function group was intro-
duced via condensation with methoxylamine/ethoxylamine to
yield amines 8a,b. The bis-Boc-protecting groups of amines 8a,b
were removed by pumping hydrogen chloride gas in methylene
chloride to afford the new piperidine derivative dihydrochlorides
9a,b in good yield.
Finally, the target compounds 12–26 were obtained by coupling
the new piperidine derivatives 9a,b with various compounds
containing quinolone and naphthyridone cores according to
well-established literature procedures (Scheme 2).13 In the case
of quinolones 12–23, condensation of 9a,b with 10a–f was per-
formed in the presence of triethylamine. However for 24–26, boric
chelates 11g–h were required to increase reactivity. All of the syn-
thetic compounds were well characterized through the spectral
characteristics.14
successful in preparing X-ray quality single crystals of compounds
12–26, we were able to obtain X-ray data for the intermediate 9a.
As expected, the six-membered piperidine ring adopts a chair con-
formation and the methyloxime geometry of 9a was confirmed to
have the E-configuration (Fig. 2).15
Fluoroquinolones 12–26 were evaluated for their in vitro
antibacterial activity against representative Gram-positive and
Gram-negative strains using standard techniques. The minimum
inhibitory concentration values (MICs) were compared with those
of levofloxacin (LVFX) (Table 1).
The novel fluoroquinolones 12–26 have potent antibacterial
activity against the tested strains. They exhibit good potency in
inhibiting the growth of S. aureus and Staphylococcus epidermidis
including MRSE (MIC: 0.125–4 lg/mL). Compounds 12, 13 are
more potent than or comparable to LVFX against MRSA, Strepto-
coccus pyogenes, Escherichia coli, Klebsiella pneumoniae, and Shigella
sonnei. Compound 17 is more active than or comparable to LVFX
against S. aureus including MRSA, S. epidermidis and S. pyogenes.
However, most of the target compounds exhibit less activity than
LVFX against the tested Gram-positive and Gram-negative strains.
These results show that introduction of a methyl group into 3-po-
It is obvious that the target compounds 12–26 and intermedi-
ates 9a,b are all racemes. Since the oxime group can exist in the
E- or Z-configuration, it was necessary to determine the geometries
of all the oxime target compounds 12–26. Although we were not

Y. Chai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5195–5198
5197
Table 1
In vitro antibacterial activity and the cytotoxicity of the target compounds 12–26
Strains
MIC (lg/mL)
CC50 (lg/mL)
Compound
R1
X
R2
S.a.
MRSA
8
S.e.
MRSE
0.25
S.p.
S.py.
E.f.
32
E.co.
K.p.
0.5
P.a.
S.s.
E.cl.
12
Me
N
0.5
1
32
8
0.5
8
1
4
176.78
13
14
15
Et
Me
Et
N
N
N
0.25
1
2
8
128
128
0.5
2
4
0.25
2
2
4
64
128
16
64
64
4
>128
>128
1
8
8
1
128
64
16
32
128
1
8
16
8
32
32
44.19
88.39
78.75
2,4-F2–C6H3
2,4-F2–C6H3
16
17
18
Me
Et
CF
0. 25
0.125
0.25
16
8
0.25
0.25
1
0.25
0.25
1
32
8
32
16
64
64
2
0. 5
2
64
2
16
16
32
1
2
4
4
16
8
88.39
62.5
CF
Me
COCHF2
32
128
128
4
4
88.39
19
20
21
22
23
Et
Me
Et
Me
Et
COCHF2
CF
CF
CF
CF
1
1
2
2
2
16
32
16
64
64
1
2
4
4
4
2
2
2
2
4
16
128
64
128
128
128
64
64
128
128
2
128
128
>128
128
8
8
16
8
8
64
16
64
64
64
64
128
64
16
8
32
16
64
16
32
128
64
44.19
314.98
78.75
314.98
157.49
CH2CH3
CH2CH3
CH2CH2F
CH2CH2F
16
128
64
24
25
Me
Et
COCH3
COCH3
0.5
2
16
4
4
1
2
64
16
64
32
8
8
8
64
16
8
2
32
157.49
19.69
128
128
16
32
128
26
Me
1
64
16
2
1
128
2
64
16
128
4
1
128
1
32
8
1
16
176.78
O
LVFX
0.125
0.25
0.125
0.5
0.25
0.125
>1000
S.a., Staphylococcus aureus ATCC 29213; MRSA, methicillin-resistant Staphylococcus aureus 08-52; S.e., Staphylococcus epidermidis ATCC 12228; MRSE, methicillin-resistant
Staphylococcus epidermidis 08-18; S.p., Streptococcus pneumoniae ATCC 49619; S.py., Streptococcus pyogenes 06-1; E.f., Enterococcus faecalis ATCC 29212; E.co., Escherichia coli
26; K.p., Klebsiella pneumoniae 7; P.a., Pseudomonas aeruginosa 17; S.s., Shigella sonnei 51592; E.cl., Enterobacter cloacae 45301.
sition of piperidine ring possessing an aminomethyl group in place
of the amino group of IMB reduces antibacterial activity, which is
contrary to our expected.
oxime-incorporated piperidino-substitutions were generally more
cytotoxic than the analogs containing methyloxime.
In summary, we report herein the synthesis of some novel 7-(4-
alkoxyimino-3-aminomethyl-3-methylpiperidin-1-yl) fluoroquin-
olone derivatives. The antibacterial activities of the newly synthe-
sized compounds were evaluated and correlated with their
physicochemical properties. Results reveal that all of the target
compounds have good activity against S. aureus and S. epidermidis
including MRSE, but most of them exhibit less activity than LVFX
against the remaining Gram-positive and Gram-negative strains.
Generally, the activity of the quinolone nuclei in this study are in
the order 1-cyclopropyl-1,8-naphthyridone ꢀ 1-cyclopropyl-8-fluo-
roquinolone > 1-cyclopropyl-8-methoxylquinolone ꢀ 1-cyclopropyl-
8-difluoromethoxylquinolone > levofloxacin nuclei ꢀ 1-(2,4-difluo-
rophenyl)-1,8-naphthyridone ꢀ 1-ethyl-8-fluoroquinolone > 1-(2-
fluoroethyl)-8-fluoroquinolone. In addition, fluoroquinolones
featuring methyloxime- incorporated piperidino-substitution at C-
7 position are comparable to analogs containing ethyloxime.
Some compounds were further examined for toxicity (CC50) in a
Acknowledgment
mammalian Vero cell line from 1000 to 7.81 lg/mL concentrations.
After 48 h of exposure, viability was assessed and the results are
reported in Table 1. Fifteen compounds when tested showed CC50
values ranging from 314.98 to 19.69 lg/mL. A comparison of the
The work was supported by the National S&T Major Special Pro-
ject on Major New Drug Innovation. Item No. 2009ZX09301-003.
substitution pattern at C-7 position demonstrated that ethyl-
References and notes
1. Hoshino, K.; Kitamura, A.; Morrissey, I.; Sato, K.; Kato, J.; Ikeda, H. Antimicrob.
Agents Chemother. 1994, 38, 2623.
2. Srivastava, B. K.; Solanki, M.; Mishra, B.; Soni, R.; Jayadev, S.; Valani, D.; Jain, M.;
Patel, P. R. Bioorg. Med. Chem. Lett. 2007, 17, 1924.
3. Bryskier, A.; Chantot, J. F. Drugs 1995, 49, 16.
4. Forroumadi, A.; Emami, S.; Mehni, M.; Moshafi, M. H.; Shafiee, A. Bioorg. Med.
Chem. Lett. 2005, 15, 4536.
5. Domagala, J. M. J. Antimicrob. Chemother. 1994, 33, 685.
6. Dang, Z.; Yang, Y. S.; Ji, R. Y.; Zhang, S. H. Bioorg. Med. Chem. Lett. 2007, 17, 4523.
7. Wang, X. Y.; Guo, Q.; Wang, Y. C.; Liu, B. Q.; Liu, M. L.; Sun, L. Y.; Guo, H. Y. Acta
Pharm Sin 2008, 43, 819.
8. Wang, J. X.; Guo, Q.; Chai, Y.; Feng, L. S.; Guo, H. Y.; Liu, M. L. Chin. Chem. Lett.
2010, 21, 55.
9. Chai, Y.; Wan, Z. L.; Wang, B.; Liu, M. L.; Guo, H. Y. Eur. J. Med. Chem. 2009, 44,
4063.
10. Zhang, Y. B.; Feng, L. S.; You, X. F.; Guo, Q.; Guo, H. Y.; Liu, M. L. Arch. Pharm.
Chem. Life Sci. 2010, 343, 143.
11. Suto, M. J.; Domagala, J. M.; Roland, G. E.; Mailloux, G. B.; Cohen, M. A. J. Med.
Chem. 1992, 35, 4745.
12. Yun, H. J.; Min, Y. H.; Lim, J. A.; Kang, J. W.; Kim, S. Y.; Kim, M. J.; Jeong, J. H.;
Choi, Y. J.; Kwon, H. J.; Jung, Y. H.; Shim, M. J.; Choi, E. C. Antimicrob. Agents
Chemother. 2002, 46, 3071.
13. MICs were determined by the agar dilution method as recommended by the
National Committee for Clinical Laboratory Standards. The MIC was defined as
the lowest concentration of each compound resulting in inhibition of visible
growth of bacteria after incubation at 35 °C for 18–24 h. The cytotoxicity was
tested in a mammalian Vero cell line and was assessed by CPE assay.
Figure 2. X-ray structure of 9a.

5198
Y. Chai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5195–5198
14. A mixture of 10a (1 mmol), 9a (1.3 mmol), triethylamine (5 mmol), and dry
acetonitrile (10 mL) was stirred at 30 °C for 3 h. After the reaction was
completed, the reaction mixture was filtered. The resulting solid was purified
via silica gel column chromatography (chloroform/methanol, 10:1, v/v) to give
the title compound 12 as white solid. 1H NMR (CDCl3, 400 MHz) dH 1.09–1.10
(2H, m, cyclopropylCH2), 1.17 (3H, s, CH3), 1.27–1.29 (2H, m, cyclopropylCH2),
2.78–2.94 (4H, m), 3.62–3.64 (1H, m), 3.86–3.87 (2H, m), 3.89 (3H, s, OCH3),
3.99–4.10 (2H, m), 8.10 (1H, d, J = 13.2, C5-H), 8.74 (1H, s, C2–H). ESI-MS: m/z
418 (M+H)+. HRMS-ESI: m/z Calcd for C20H25FN5O4 (M+H)+: 418.18906; found
418.18798. A mixture of 11g (1 mmol), 9a (1.3 mmol), triethylamine (5 mmol)
and dry acetonitrile (10 mL) was stirred at 60 °C for 6 h. After the reaction was
completed, the reaction mixture was concentrated under reduced pressure.
The residue was dissolved in 5% sodium hydroxide solution (6.0 mL), heated to
45 °C and stirred for 2 h at the same temperature. The reaction mixture was
cooled to room temperature and adjusted to pH 7–7.5 with 2 N HCl. The solid
product was collected by suction, purified via silica gel column
chromatography (chloroform/methanol, 10:1, v/v) to give the title compound
24 as pale yellow solid. 1H NMR (CDCl3, 400 MHz) dH 0.99–1.04 (2H, m,
cyclopropylCH2), 1.19–1.22 (2H, m, cyclopropylCH2), 1.27 (3H, s, CH3), 2.48–
2.54 (1H, m), 2.71–2.78 (1H, m), 2.83–2.91 (2H, m), 2.95–3.06 (2H, m), 3.61–
3.66 (1H, m), 3.72 (3H, s, OCH3), 3.76–3.81 (1H, m), 3.91 (3H, s, OCH3), 3.95–
3.98 (1H, m), 7.85 (1H, d, J = 12.4, C5–H), 8.75 (1H, s, C2–H), ESI-MS: m/z 447
(M+H)+. HRMS-ESI: m/z Calcd for C22H28FN4O5 (M+H)+: 447.19858; found
447.20148.
15. The crystallographic data of 9a has been deposited at the Cambridge
Crystallographic Data Center (CCDC No. 767681).