Synthesis and in vitro antimycobacterial activity of balofloxacin ethylene isatin derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.04.027

A series of novel balofloxacin ethylene isatin derivatives with remarkable improvement in lipophilicity,as compared to the parent compound balofloxacin,were designed,synthesized and characterized by ¹H NMR,MS and HRMS. These derivatives were in

Full text of DOI:10.1016/j.ejmech.2010.04.027

European Journal of Medicinal Chemistry 45 (2010) 3407e3412
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro antimycobacterial activity of balofloxacin ethylene
isatin derivatives
1
,1
*
Lian-Shun Feng , Ming-Liang Liu , Bo Wang, Yun Chai, Xue-Qin Hao, Shuai Meng, Hui-Yuan Guo
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel balofloxacin ethylene isatin derivatives with remarkable improvement in lipophilicity,
as compared to the parent compound balofloxacin, were designed, synthesized and characterized by 1H
NMR, MS and HRMS. These derivatives were initially evaluated for their in vitro antimycobacterial
activity against M. phlei CMCC 93201 and M. smegmatis CMCC 93202. Compounds 3b, 3d, 3gej and 3l
were chosen for further evaluation their in vitro activity against MTB 09710 clinical isolate, and then
compounds 3h and 3g against MTB H37Rv ATCC 27294. All of the synthesized compounds were less
active than balofloxacin against M. phlei CMCC 93201 and M. smegmatis CMCC 93202, but compounds
Received 6 July 2009
Received in revised form
24 March 2010
Accepted 25 April 2010
Available online 20 May 2010
Keywords:
Balofloxacin derivatives
Synthesis
3gej (MIC: <0.5e8
m
g/mL) were more potent than balofloxacin (MIC: 16
m
g/mL) against MTB 09710. In
particular, compound 3h (MIC: 0.25e < 0.5
m
g/mL) was found to be comparable to moxifloxacin, and ꢀ32
fold more potent than balofloxacin against MTB 09710 and MTB H37Rv ATCC 27294. The results
demonstrated that the lipophilicity of the tested compounds was not the sole parameter affecting
antimycobacterial activity, as well as the potential and importance of developing new fluoroquinolone
derivatives against mycobacterial infections.
Antimycobacterial activity
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
enzymes, DNA gyrase (the principal target in gram-negative
bacteria) and topoisomerase IV (the main target in gram-positive
Tuberculosis (TB), caused predominantly by M. tuberculosis (MTB),
is one of the most common infectious diseases known to man. The
WHO has estimated that 9.2 million new incident cases in 2006, with
approximately 1.7 million individual deaths in the same year [1], and
one-third of the world population (around 1.7 billion) is latently
infected with MTB [2]. In India alone, one person dies of TB every
minute [3]. The major concerns for current TB treatment are its
latency, co-infection with HIV, poor patient compliance, and drug
resistance issues caused by the emergence of multidrug resistant
tuberculosis (MDR-TB) and the recent advent of extensively drug
resistant tuberculosis (XDR-TB) [4,5]. It is estimated that between
2002 and 2020, approximately 1 billionpeoplewill be newly infected,
morethan150millionpeoplewillgetsick, and36millionwilldieofTB
if new disease prevention and treatment measures are not developed
[6]. Hence, thereisanurgentneedtodevelopnovel, higheffective, and
fast acting anti-TB drugs with low toxicity profiles and performing
activity against both actively growing and latent infections [7].
Fluoroquinolones (FQs) exert their bactericidal activity by
interfering with the function of two type II bacterial topoisomerase
bacteria) [8]. The incidence of mycobacterial resistance to FQs is
relatively low at the present time, and there are no reports of cross-
resistance or antagonism with other classes of anti-TB agents [9].
Several FQs, including ciprofloxacin, ofloxacin and sparfloxacin are
currently recommended as second-line agents by WHO for the
treatment of TB primarily in cases involving resistance or intoler-
ance to first-line anti-TB therapy [10]. Interestingly, C-8 methoxy
FQ derivatives with N1-cyclopropyl substitution are much more
active against resistant MTB than C-8 hydrogen compounds [11].
For example, moxifloxacin (MXFX) and gatifloxacin (Fig. 1) were
observed to have a particularly strong in vitro and in vivo activity
(similar to isoniazid) against MTB and may be able to reduce the
length of therapy owing to activity against both replicating and
non-replicating bacilli [12,13].
Structure-activity relationship (SAR)studies of FQs indicated that
the substituents at C-7 position greatly influence their potency,
antibacterial spectrum as well as safety [14], and substitution of
bulky functional group is permitted at the C-7 position [15]. More-
over, the lipophilicity of the FQs plays an important role in the
penetration of these compounds into bacterial cells, and simply
increasing the lipophilic characterat C-7 position could also increase
the anti-TB activity [16]. Therefore, reasonable modification at C-7
position is likely to provide more effective anti-TB agents.
* Corresponding author.
E-mail address: lmllyx@yahoo.com.cn (M.-L. Liu).
These authors contributed equally to the work.
1
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.04.027

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L.-S. Feng et al. / European Journal of Medicinal Chemistry 45 (2010) 3407e3412
Fig. 1. Chemical structures of Moxifloxacin, Gatifloxacin and Balofloxacin.
Balofloxacin (Fig. 1, BLFX), one of C-8 methoxy FQs with abroad
2. Results and discussion
spectrum against gram-positive and gram-negative bacteria, is
comparable to that of ofloxacin or levofloxacin against MTB,
M. kansasii and M. fortuitum [17]. However, to date few studies have
been undertaken to optimize BLFX against mycobacteria.
2.1. Chemistry
Detailed synthetic pathways to BLFX ethylene isatin derivatives
3aen are depicted in Scheme 1. Isatins 1a,b were alkylated with 1,2-
dibromoethane in the presence of anhydrous potassium carbonate
to give N-(2-bromoethyl)isatins 2a,b in a yield of 53e68% according
to the reported procedures [20]. Nucleophilic substitution reactions
of compounds 2a,b with BLFX [1-cyclopropyl-6-fluoro-8-methoxy-
7-(3-methylamino- piperidin-1-yl)-1,4-dihydro-4-oxo-quinoline-3-
carboxylic acid] were performed in the presence of anhydrous
potassium carbonate in N,N-dimethylformamide at 40 ꢁC to afford
BLFX derivatives 3a,b (57e59%). Subsequent condensations of
compounds 3a,b with requisite substituted amine hydrochlorides in
the presence of sodium bicarbonate formed other derivatives
(Schiff’s bases) 3cen (63e85%).
Recently, asa partof an ongoingprogramto optimize C-8 methoxy
FQs against mycobacteria, we also have focused our attention on
exploring the effect of increasing the lipophilic character at C-7
position of FQs by forming methylene- or ethylene-linked isatin-FQ
conjugates. Isatin(1H-indole-2,3-dione)isanendogenouscompound
identified in many organisms [18], and its derivatives are reported to
show variety of biological activity like antibacterial [19], antifungal
[20], anti-HIV [21] and anti-TB activity [22], so great interests have
been placed on these bioactive substances. In the present report,
a series of novel BLFX ethylene isatin derivatives were designed and
synthesized. Our primary objective was to optimize the potency of
these compounds against mycobacteria.
Scheme 1. Synthesis of Balofloxacin derivatives 3a-n.

L.-S. Feng et al. / European Journal of Medicinal Chemistry 45 (2010) 3407e3412
3409
Table 1
Structures, lipophilicity, antimycobacterial activity and cytotoxicity of compounds 3aen.
O
N
F
CO2H
CH3
N
R1
N
N
OCH3
O
R2
3a-n
c
Compound
R1
R2
Clog P (log P)
MIC (
m
g/mL)
CC50 (mM)
M.p.a
M sb
MTB 09710
MTB H37Rv TCC 27294
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
H
F
H
F
H
F
H
F
H
F
H
F
H
F
O
O
NOH
NOH
NOCH3
NOCH3
NOCH2CH3
NOCH2CH3
NNHCSNH2
NNHCSNH2
NNHCONH2
NNHCONH2
NNHC6H5
NNHC6H5
1.10 (2.44)
1.43 (2.60)
0.79 (2.83)
0.93 (2.99)
1.38 (3.10)
1.52 (3.25)
1.91 (3.43)d
2.05 (3.59)d
0.79 (2.36)d
0.94 (2.52)d
0.13 (1.80)
0.27 (1.96)
2.24 (4.08)
2.38 (4.24)
ꢂ0.27(1.74)d
ꢂ0.08(1.68)
25
25
12.5
6.25
3.12
3.12
3.12
3.12
3.12
3.12
25
e
e
136.9
96.2
148.2
305.6
15.0
12.8
60.5
53.1
12.5
12.5
12.5
6.25
6.25
6.25
6.25
6.25
6.25
12.5
6.25
6.25
6.25
0.39
0.10
64
e
e
e
16
e
e
e
e
e
8
<0.5
8
4
e
e
0.25
e
293.8
312.9
56.1
8.0
e
12.5
3l
6.25
3.12
3.12
0.78
0.20
32
e
e
57.0
3m
3n
BLFX
MXFX
e
<6.1
<6.0
317.0
160.8
e
e
16
<0.5
8.0
0.25
a
M.p.: M. phlei CMCC 93201.
M.s.: M. smegmatis CMCC 93202.
CC50: The 50% cytotoxic concentration.
The experimental log P values by HPLC of compounds 3gej and BLFX are 0.96, 1.29, 1.40, 1.80 and ꢂ1.11, respectively.
b
c
d
2.2. Lipophilicity
presented in Table 1. These data suggested that the target
compounds 3aen showed considerable potency in inhibiting the
growth of M. phlei CMCC 93201 and M. smegmatis CMCC 93202
Lipophilicity of the target compounds 3aen and the parent
compound BLFX and MXFX, is expressed in the term of their Clog P
or log P values. As shown in Table 1, a remarkable improvement in
the lipophilicity of the target compounds 3aen, as seen from the
Clog P or log P values (0.13e2.38 or 1.80e4.24, statistically signifi-
cant at p < 0.001 using t test), which were much more than that of
BLFX (ꢂ0.27 or 1.74).
(MIC: 3.12e25
mg/mL), although less active than that of the parent
BLFX (MIC: 0.39e0.78
mg/mL).
Compounds 3b, 3d, 3gej and 3l, characterized by their better
activity, were chosen for further evaluation their in vitro activity
against MTB 09710 clinical isolate, and then compounds 3h and 3g
against MTB H37Rv ATCC 27294 using rapid direct susceptibility
test technique [24,25]. The MICs of the seven derivatives, along
with BLFX and MXFX for comparison, are presented in Table 1.
Compounds 3gej were chosen for further evaluation their
experimentallogPvalues byHPLC, and comparedwithBLFX(Table 1).
These results indicated that the lipophilicity of derivatives 3gej
(experimental log P values arrange from 0.96 to 1.80) was much more
than BLFX (ꢂ1.11), which was consistent with lipophilicity profiles
based on Clog P or log P values calculated by softwares. This may be
rendering them more capable of penetrating various biomembrane,
consequently improving their permeation properties toward myco-
bacterial cell membrane. In other words, the improvement of the
lipophilic character of compounds 3aen probably enhances their
antimycobacterial activity.
Among them, compounds 3gej (MIC: <0.5e8
active than the parent BLFX (MIC: 16 g/mL) against MTB 09710. In
particular, compound 3h (MIC: 0.25e < 0.5 g/mL) was found to be
mg/mL) were more
m
m
comparable to MXFX, and ꢀ32 fold more potent than BLFX against
MTB 09710 and MTB H37Rv ATCC 27294.
The target compounds 3aen were subsequently examined for
toxicity (CC50) in a mammalian Vero cell line [23]. After 72 h of
exposure, viability was assessed on the basis of cellular conversion
of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) into a formazan product and the results are reported in
Table 1. Herein, the cytotoxicity of compounds 3e, 3f, 3m and 3n
2.3. Pharmacology
(CC50: <6.0e15.0
(CC50: 53.1e312.9
m
M) is much more than the other derivatives
mM).
The target compounds 3aen were initially evaluated for their in
vitro antimycobacterial activity against M. phlei CMCC 93201 and
M. smegmatis CMCC 93202 using serial double dilution technique in
duplicate [16]. The minimum inhibitory concentration (MIC) is
defined as the concentration of the compound required to give
complete inhibition of mycobacterial growth, and MICs of the
target compounds along with BLFX and MXFX for comparison are
2.4. Conclusion
In summary, a series of novel BLFX ethylene isatin derivatives
3aen were designed, synthesized and characterized by 1H NMR,
MS and HRMS. These derivatives were initially evaluated for their in

3410
L.-S. Feng et al. / European Journal of Medicinal Chemistry 45 (2010) 3407e3412
vitro antimycobacterial activity against M. phlei CMCC 93201 and
M. smegmatis CMCC 93202. Compounds 3b, 3d, 3gej and 3l were
further evaluated their activity against MTB 09710 clinical isolate,
and then compounds 3h and 3g against MTB H37Rv ATCC 27294.
The preliminary evaluation results showed that all of the synthe-
sized compounds with much more lipophilic than that of the
parent BLFX, were less active than BLFX against M. phlei CMCC
93201 and M. smegmatis CMCC 93202. Compounds 3gej were more
active than BLFX against MTB 09710, while the other three
compounds (3b, 3d and 3l) were not. It was noted that the activity
of compound 3h was comparable to MXFX, and ꢀ32 fold more
potent than BLFX against MTB 09710 and MTB H37Rv ATCC 27294.
The above results demonstrated that the lipophilicity of the tested
compounds was not the sole parameter affecting antimycobacterial
activity. However, improvement in the lipophilicity of the
compounds containing a 5-fluoroisatin moiety did increase the
antimycobacterial activity, when compared with corresponding
analogs containing an isatin moiety against MTB 09710, as seen
NMR (CDCl3, 400 MHz) dH 3.62 (2H, t, J ¼ 6.8, CH2CH2Br), 4.15 (2H, t,
J ¼ 6.8, CH2CH2Br), 7.00 (1H, d, J ¼ 7.6), 7.15 (1H, t, J ¼ 7.6), 7.60e7.65
(2H, m). ESI-MS: m/z 254 (M þ H)þ, 256 (M þ 2 þ H)þ.
3.2.1.1. N-(2-Bromoethyl)-5-fluoroisatin (2b). The title compound
was obtained in a similar manner as for the preparation of 2a (53%) as
anacaratsolid, mp:109e110 ꢁC.1H NMR (CDCl3, 400 MHz)dH 3.62(2H,
t, J ¼ 6.4, CH2CH2Br), 4.11 (2H, t, J ¼ 6.4, CH2CH2Br), 6.98e7.00 (1H, m),
7.26e7.35 (2H, m). ESI-MS: m/z 272 (M þ H)þ, 274 (Mþ2 þ H)þ.
3.2.2. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{3-[N-methyl-N-
(2-isatinylethyl)] aminopiperidin-1-yl}-1,4-dihydro-4-oxoquinoline-
3-carboxylic acid (3a)
A suspension of 2a (5.08 g, 20 mmol), BLFX (7.78 g, 20 mmol),
anhydrous potassium carbonate (8.34 g, 60 mmol) in N,N-dime-
thylformamide (200 mL) was stirred at 40 ꢁC for 27.5 h and
concentrated under reduced pressure. To the residue was added
water (300 mL), stirred at room temperature for 0.5 h and then
filtered. The solid obtained was dissolved in 4 N HCl (150 mL), and
washed with methylene chloride (3 ꢃ 100 mL). The water layer was
adjusted pH 7.0 with 10% NaOH solution and filtered. The crude
product was purified by column chromatography (silica gel) eluted
with methylene chloride and methanol (v: v ¼ 20: 1) to give the
title compound 3a (6.65 g, 59%) as anacaratsolid, mp: 151e154 ꢁC.
1H NMR (DMSO-d6, 400 MHz) dH 0.84e0.91 (2H, m, cyclopropyl-H),
0.97e1.02 (2H, m, cyclopropyl-H), 1.54e1.56 (1H, m), 1.64e1.67 (1H,
m), 1.83e1.86 (1H, m), 2.16e2.20 (1H, m), 2.55 (3H, s, NCH3), 3.03
(1H, t, J ¼ 11.6), 3.16e3.18 (2H, m), 3.30 (1H, d, J ¼ 11.6), 3.65 (1H, d,
J ¼ 11.6), 3.74 (3H, s, OCH3), 3.92e3.95 (1H, m, cyclopropyl-H), 4.05
(2H, t, J ¼ 5.2, CH2 of linker), 4.36 (2H, t, J ¼ 5.2, CH2 of linker),
7.11e8.34 (6H, m, Ar-H), 8.98 (brs, 1H, D2O exchangeable, COOH).
ESI-MS: m/z 563 (M þ H)þ. HRMS-ESI: m/z Calcd. for C30H32FN4O6
(M þ H)þ: 563.23059; Found 563.22917.
from the MICs of compounds 3h (<0.5
or 3j (4 g/mL) versus 3i (8 g/mL).
mg/mL) versus 3g (8 mg/mL),
m
m
Antimycobacterial activity of Schiff’s bases (3cen) of derivatives
3a,b generally improved. The relative contribution of imines of the
Schiff’s bases to activity against MTB 09710 is as follows:
ethyloxime > thiosemicarbazone > oxime > semicarbazone. The
relative contribution of imines to cytotoxicity as follows:
phenylhydrazone > methyloxime > semicarbazone z ethyloxime
> oxime > thiosemicarbazone.
3. Experimental section
3.1. Chemistry
Melting points were determined in open capillaries and
uncorrected. Clog P was calculated by CLOGP module in sybyl 7.3
software. Log P values were calculated with Chem office 2009
software. HPLC was preformed using a Shimadzu LC-10Avp with
SPD-10Avp UV detector (Shimadzu) and a Class VP 6.x workstation.
3.2.2.1. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{3-[N-methyl-N-2-(5-
fluoroisatinyl)ethyl] aminopiperidin-1-yl}-1,4-dihydro-4-oxoquino-
line-3-carboxylic acid (3b). The title compound was obtained in
a similar manner as for the preparation of 3a (57%) as anacar-
atsolid, mp: 155e158 ꢁC. 1H NMR (DMSO-d6, 400 MHz) dH
0.84e0.90 (2H, m, cyclopropyl-H), 0.97e1.01 (2H, m, cyclopropyl-
H), 1.41e1.44 (1H, m), 1.63e1.66 (1H, m), 1.80e1.83 (1H, m), 2.50
(3H, s, NCH3), 3.00e3.06 (3H, m), 3.30 (1H, d, J ¼ 11.2), 3.60 (1H, d,
J ¼ 11.6), 3.74 (3H, s, OCH3), 3.92e3.95 (1H, m, cyclopropyl-H), 4.05
(2H, t, J ¼ 5.2, CH2 of linker), 4.35 (2H, t, J ¼ 5.2, CH2 of linker),
7.39e8.35 (5H, m, Ar-H). ESI-MS: m/z 581 (M þ H)þ. HRMS-ESI: m/
z Calcd. for C30H31F2N4O6 (M þ H)þ: 581.22117; Found 581.22235.
The column used was a Diamonsil C18 5-
m
m 250 ꢃ 4.6 mm column
(Dikma Technologies). 1H NMR and 13C NMR spectra were deter-
mined on a Varian Mercury-400 spectrometer in DMSO-d6 or CDCl3
using 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, respec-
tively. Fast Atom Bombardment (FAB) mass spectra and high reso-
lution mass spectra (HRMS) were obtained on a MICROMASS
AutoSpec Ultima-TOF mass spectrometer. Unless otherwise noted,
the reagents were obtained from commercial supplier and were
used without further purification. TLC was performed on silica gel
plates (Merck, ART5554 60F254).
3.2.3. General procedure for the preparation of compounds 3cen
The general procedure for preparing compounds 3cen was
described as follows. To a solution of substituted amine hydro-
chlorides (3 mmol) and sodium bicarbonate (0.25 g, 3 mmol) dis-
solved in water (10 mL) was added dropwise a solution of 3a,
b (1 mmol) in methanol (10 mL) at room temperature over 5 min.
The reaction mixture was stirred at the same tempareture for
12e24 h (monitored until the substrate disappeared by TLC, vme-
thylene chloride: vmethanol ¼ 5: 1). After removal of the methanol under
reduced pressure, the reaction mixture was diluted with water
(20 mL) and stirred for 10 min, and then filtered. The solid crude
product was purified by column chromatography (silica gel) eluted
with methylene chloride and methanol (v: v ¼ 20: 1) to give the
title compounds 3cen (63e85%).
3.2. Synthesis
3.2.1. N-(2-Bromoethyl)isatin (2a)
A suspension of isatin (1a, 4.41 g, 30 mmol), anhydrous potas-
sium carbonate (12.51 g, 90 mmol) and 1,2-dibromoethane (16.83 g,
90 mmol) in N,N-dimethylformamide (100 mL) was stirred at room
temperature for 24 h and filtered. The filtrate was concentrated
under reduced pressure. The residue was poured into water (50 mL)
and extracted with ethyl acetate (3 ꢃ 50 mL). The combined extracts
were washed with saturated brine, dried over anhydrous sodium
sulfate, and concentrated under reduced pressure. The crude
product was purified by column chromatography (silica gel) eluted
with petroleum ether and ethyl acetate (v: v ¼ 5: 1) to give the title
compound 2a (5.19 g, 68%) as anacaratsolid, mp: 126e128 ꢁC. 1H
3.2.3.1. 1-Cyclopropyl-6-fluoro-7-{3-[N-methyl-N-2-(isatinyl-b-hydrox-
imino)ethyl] aminopiperidin-1-yl}-1,4-dihydro-8-methoxy–4-oxoquino-
line-3-carboxylic acid (3c). Yield: 85%. mp: 188e192 ꢁC. 1H NMR

L.-S. Feng et al. / European Journal of Medicinal Chemistry 45 (2010) 3407e3412
3411
(DMSO-d6, 400 MHz) dH 0.83e0.91 (2H, m, cyclopropyl-H), 0.98e1.02
(2H, m, cyclopropyl-H), 1.48e1.51 (1H, m), 1.64e1.67 (1H, m),
1.82e1.85 (1H, m), 2.13e2.16 (1H, m), 2.54 (3H, s, NCH3), 3.04 (1H, t,
J ¼ 10.8), 3.11e3.13 (2H, m), 3.28e3.31 (1H, m), 3.62 (1H, d, J ¼ 8.0),
3.74 (3H, s, OCH3), 3.91e3.96 (1H, m, cyclopropyl-H), 4.08 (2H, t,
J ¼ 4.8, CH2 of linker), 4.37 (2H, t, J ¼ 4.8, CH2 of linker), 7.05e8.35 (6H,
m, Ar-H). ESI-MS: m/z 578 (M þ H)þ. HRMS-ESI: m/z Calcd. for
C30H33FN5O6 (M þ H)þ: 578.24149; Found 578.23997.
(1H, m),1.82e1.85 (1H, m), 2.08 (1H, t, J ¼ 12.0), 2.53 (3H, s, NCH3), 2.80
(1H, s), 2.98 (1H, t, J¼ 11.2), 3.13 (1H, t, J¼ 11.2), 3.37 (1H, d, J¼ 12.0), 3.62
(1H, d, J ¼ 12.0), 3.72 (3H, s, OCH3), 3.86e3.90 (1H, m, cyclopropyl-H),
4.13 (2H, t, J ¼ 5.2, CH2 of linker), 4.48e4.58 (4H, m, CH2 of linker,
NOCH2CH3), 7.05e8.44 (5H, m, Ar-H). 13C NMR (CDCl3, 400 MHz) dC
:
9.16, 9.34, 14.61, 24.63, 29.82, 32.99, 38.87, 39.60, 51.30, 55.86, 56.14,
61.10, 62.41, 72.91, 108.39, 108.59, 109.16, 115.73, 122.88, 127.69, 132.42,
132.75, 138.59, 143.25, 143.41, 145.47, 150.62, 154.73, 157.20, 163.75,
163.99, 172.93. ESI-MS: m/z 624 (M þ H)þ. HRMS-ESI: m/z Calcd. for
C32H36F2N5O6 (M þ H)þ: 624.26336; Found 624.26651.
3.2.3.2. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{3-[N-methyl-N-2-(5-
fluorinisatinyl-
b
-
hydroximino)ethyl]aminopiperidin-1-yl}-1,4-
dihydro-4-oxoquinoline-3-carboxylic acid (3d). Yield: 82%. mp:
193e194 ꢁC. 1H NMR (DMSO-d6, 400 MHz) dH 0.84e0.87 (2H, m,
cyclopropyl-H), 1.00e1.01 (2H, m, cyclopropyl-H), 1.41e1.44 (1H,
m), 1.62e1.65 (1H, m), 1.79e1.82 (1H, m), 2.09e2.12 (1H, m), 2.48
(3H, s, NCH3), 3.00e3.03 (3H, m), 3.20 (1H, d, J ¼ 10.8), 3.58 (1H, d,
J ¼ 10.8), 3.72 (3H, s, OCH3), 3.92e3.95 (1H, m, cyclopropyl-H), 4.07
(2H, t, J ¼ 4.8, CH2 of linker), 4.35 (2H, t, J ¼ 4.8, CH2 of linker),
7.27e8.33 (5H, m, Ar-H). ESI-MS: m/z 596 (M þ H)þ. HRMS-ESI: m/
z Calcd. for C30H32F2N5O6 (M þ H)þ: 596.23206; Found 596.22956.
3.2.3.7. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{3-[N-methyl-N-2-(b-
thiosemicarbazo isatinyl)ethyl]aminopiperidin-1-yl}-1,4-dihydro-4-
oxoquinoline-3-carboxylic acid (3i). Yield: 80%. mp: 174e176 ꢁC.
1H NMR (DMSO-d6, 400 MHz) dH 0.85e0.89 (2H, m, cyclopropyl-
H), 1.05e1.07 (2H, m, cyclopropyl-H), 1.22e1.25 (1H, m), 1.60e1.63
(1H, m), 1.74e1.77 (1H, m), 1.89e2.00 (1H, m), 2.35 (3H, s, NCH3),
2.67e2.72 (1H, m), 2.80e2.85 (1H, m), 3.00e3.06 (1H, m), 3.29
(1H, d, J ¼ 10.0), 3.52 (1H, d, J ¼ 10.0), 3.72 (3H, s, OCH3), 3.97e4.00
(1H, m, cyclopropyl-H), 4.13 (2H, t, J ¼ 4.8, CH2 of linker), 4.37 (2H,
t, J ¼ 4.8, CH2 of linker), 7.13e8.40 (6H, m, Ar-H), 8.73 (brs, 1H, D2O
exchangeable, NNHCSNH2), 9.06 (brs, 1H, D2O exchangeable,
COOH). HRMS-FAB: m/z Calcd. for C31H35FN7O5S (M þ H)þ:
636.2404; Found 636.2355.
3.2.3.3. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{3-[N-methyl-N-2-(b-
methoxyiminoisatinyl) ethyl]aminopiperidin-1-yl}-1,4-dihydro-4-
oxoquinoline-3-carboxylic acid (3e). Yield: 85%. mp: 156e158 ꢁC.
1H NMR (DMSO-d6, 400 MHz) dH 0.84e0.91 (2H, m, cyclopropyl-
H), 0.99e1.04 (2H, m, cyclopropyl-H), 1.54e1.56 (1H, m), 1.65e1.68
(1H, m), 1.83e1.87 (1H, m), 2.17e2.19 (1H, m), 2.58 (3H, s, NCH3),
3.04 (1H, t, J ¼ 10.4), 3.17 (2H, m), 3.28e3.32 (1H, m), 3.65 (1H, d,
J ¼ 10.4), 3.74 (3H, s, OCH3), 3.92e3.96 (1H, m, cyclopropyl-H), 4.06
(2H, t, J ¼ 5.2, CH2 of linker), 4.20 (3H, s, NOCH3), 4.36 (2H, t,
J ¼ 5.2, CH2 of linker), 7.06e8.34 (6H, m, Ar-H), 8.99 (brs, 1H, D2O
exchangeable, COOH). ESI-MS: m/z 592 (M þ H)þ. HRMS-ESI: m/z
Calcd. for C31H35FN5O6 (M þ H)þ: 592.25714; Found 592.26033.
3.2.3.8. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{3-[N-methyl-N-2-(5-
fluorinisatinyl-b- thiosemicarbazo)ethyl]aminopiperidin-1-yl}-1,4-
dihydro-4-oxoquinoline-3-carboxylic acid (3j). Yield: 70%. mp:
191e193 ꢁC. 1H NMR (DMSO-d6, 400 MHz) dH 0.86e0.90 (2H, m,
cyclopropyl-H), 0.98e1.09 (2H, m, cyclopropyl-H), 1.25e1.34 (1H,
m), 1.61e1.64 (1H, m), 1.75e1.78 (1H, m), 2.02e2.05 (1H, m), 2.40
(3H, s, NCH3), 2.75e2.79 (1H, m), 2.90 (1H, t, J ¼ 11.2), 3.02 (1H, t,
J ¼ 11.2), 3.29 (1H, d, J ¼ 12.0), 3.54 (1H, d, J ¼ 9.6), 3.72 (3H, s,
OCH3), 3.95e4.00 (1H, m, cyclopropyl-H), 4.12 (2H, t, J ¼ 4.8, CH2 of
linker), 4.36 (2H, t, J ¼ 4.8, CH2 of linker), 7.24e8.38 (5H, m, Ar-H),
8.81 (brs, 1H, D2O exchangeable, NNHCSNH2), 9.14 (brs, 1H, D2O
exchangeable, COOH). ESI-MS: m/z 654 (M þ H)þ. HRMS-ESI: m/z
Calcd. for C31H34F2N7O5S (M þ H)þ: 654.23102; Found 654.23211.
3.2.3.4. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{3-[N-methyl-N-2-(5-
fluorinisatinyl-
b
-
methoxyimino)ethyl]aminopiperidin-1-yl}-1,4-
dihydro-4-oxoquinoline-3-carboxylic acid (3f). Yield: 79%. mp:
169e171 ꢁC. 1H NMR (DMSO-d6, 400 MHz) dH 0.85e0.91 (2H, m,
cyclopropyl-H), 0.99e1.03 (2H, m, cyclopropyl-H), 1.54e1.67 (2H,
m), 1.82e1.86 (1H, m), 2.17e2.20 (1H, m), 2.55 (3H, s, NCH3), 3.03
(1H, t, J ¼ 10.4), 3.16e3.19 (2H, m), 3.28e3.31 (1H, m), 3.64e3.66
(1H, m), 3.75 (3H, s, OCH3), 3.93e3.97 (1H, m, cyclopropyl-H), 4.07
(2H, t, J ¼ 4.8, CH2 of linker), 4.21 (3H, s, NOCH3), 4.36 (2H, t,
J ¼ 4.8, CH2 of linker), 7.28e8.32 (5H, m, Ar-H), 9.04 (brs, 1H, D2O
exchangeable, COOH). ESI-MS: m/z 610 (M þ H)þ. HRMS-ESI: m/z
Calcd. for C31H34F2N5O6 (M þ H)þ: 610.24771; Found 610.24786.
3.2.3.9. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{3-[N-methyl-N-2-(
b-
semicarbazoisatinyl) ethyl]aminopiperidin-1-yl}-1,4-dihydro-4-
oxoquinoline-3-carboxylic acid (3k). Yield: 73%. mp: 199e201 ꢁC.
1H NMR (DMSO-d6, 400 MHz) dH 0.84e0.90 (2H, m, cyclopropyl-
H), 0.99e1.06 (2H, m, cyclopropyl-H), 1.53e1.55 (1H, m), 1.65e1.68
(1H, m), 1.83e1.86 (1H, m), 2.16e2.18 (1H, m), 2.72 (3H, s, NCH3),
3.04 (1H, t, J ¼ 10.4), 3.13e3.23 (2H, m), 3.28e3.31 (1H, m), 3.64
(1H, d, J ¼ 10.0), 3.74 (3H, s, OCH3), 3.92e3.96 (1H, m, cyclopropyl-
H), 4.10 (2H, t, J ¼ 5.0, CH2 of linker), 4.37 (2H, t, J ¼ 5.0, CH2 of
linker), 6.09 (brs, 2H, D2O exchangeable, NNHCONH2), 7.07e8.36
(6H, m, Ar-H), 8.89 (brs, 1H, D2O exchangeable, NNHCONH2), 10.33
(brs, 1H, D2O exchangeable, COOH). ESI-MS: m/z 620 (M þ H)þ.
HRMS-FAB: m/z Calcd. for C31H35FN7O6 (M þ H)þ: 620.2633;
Found 620.2667.
3.2.3.5. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{3-[N-methyl-N-2-(b-
ethoxyiminoisatinyl) ethyl]aminopiperidin-1-yl]-1,4-dihydro-4-oxo-
quinoline-3-carboxylic acid (3g). Yield: 79%. mp: 101e103 ꢁC. 1H
NMR (CDCl3, 400 MHz) dH 0.82e0.88 (2H, m, cyclopropyl-H),
1.07e1.11 (2H, m, cyclopropyl-H), 1.41e1.45 (4H, m, NOCH2CH3,
piperidinyl-H), 1.71e1.74 (1H, m), 1.82e1.84 (1H, m), 2.06e2.10 (1H,
m), 2.52 (3H, s, NCH3), 2.79e2.83 (1H, m), 2.99 (1H, t, J ¼ 10.8), 3.12
(1H, t, J ¼ 10.8), 3.36 (1H, d, J ¼ 12.4), 3.62 (1H, d, J ¼ 12.4), 3.73 (3H,
s, OCH3), 3.84e3.90 (1H, m, cyclopropyl-H), 4.13 (2H, t, J ¼ 5.6, CH2
of linker), 4.48e4.56 (4H, m, CH2 of linker, NOCH2CH3), 7.00e8.43
(6H, m, Ar-H). ESI-MS: m/z 606 (M þ H)þ. HRMS-ESI: m/z Calcd. for
C32H37FN5O6 (M þ H)þ: 606.27279; Found 606.27614.
3.2.3.10. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{3-[N-methyl-N-2-(5-
fluorinisatinyl-b- semicarbazo)ethyl]aminopiperidin-1-yl}-1,4-dihy-
dro-4-oxoquinoline-3-carboxylic acid (3l). Yield: 68%. mp:
197e199 ꢁC. 1H NMR (DMSO-d6, 400 MHz) dH 0.83e0.89 (2H, m,
cyclopropyl-H), 0.99e1.05 (2H, m, cyclopropyl-H), 1.45e1.47 (1H, m),
1.63e1.66 (1H, m), 1.80e1.83 (1H, m), 2.11e2.14 (1H, m), 2.49 (3H, s,
NCH3), 3.01e3.16 (3H, m), 3.27 (1H, d, J ¼ 12.0), 3.60 (1H, d, J ¼ 8.4),
3.74 (3H, s, OCH3), 3.92e3.96 (1H, m, cyclopropyl-H), 4.08e4.13 (2H,
m, CH2 of linker), 4.35e4.38 (2H, m, CH2 of linker), 6.94 (brs, 2H,
D2O exchangeable, NNHCONH2), 7.21e8.34 (5H, m, Ar-H), 8.80 (brs,
1H, D2O exchangeable, NNHCONH2). ESI-MS: m/z 638 (M þ H)þ.
3.2.3.6. 1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-{3-[N-methyl-
N-2-(5- fluorinisatinyl-b-ethoxyimino)ethyl]aminopiperidin-1-yl}-4-oxo-
quinoline-3-carboxylic acid (3h). Yield: 71%. mp: 107e110 ꢁC. 1H NMR
(CDCl3, 400 MHz) dH 0.87e0.91 (2H, m, cyclopropyl-H),1.11e1.14 (2H, m,
cyclopropyl-H), 1.41e1.46 (4H, m, NOCH2CH3, piperidinyl-H), 1.72e1.76

3412
L.-S. Feng et al. / European Journal of Medicinal Chemistry 45 (2010) 3407e3412
HRMS-ESI: m/z Calcd. for C31H34F2N7O6 (M þ H)þ: 638.25386;
96-well plates at the plating density of 1 ꢃ 104 cells per well and
allowed to recover for 24 h. Culture medium was replaced by assay
medium containing the compound to be tested or drug-free. After
72 h of exposure, cells were harvested and cell viability was assessed
by MTT assay. The CC50 values were calculated by Bliss analyses.
Found 638.24956.
3.2.3.11. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{3-[N-methyl-N-2-(
b-
phenylhydrazo isatinyl)ethyl]aminopiperidin-1-yl}-1,4-dihydro-4-
oxoquinoline-3-carboxylic acid (3m). Yield: 78%. mp: 151e153 ꢁC.
1H NMR (DMSO-d6, 400 MHz) dH 0.80e0.84 (2H, m, cyclopropyl-H),
0.93e1.00 (2H, m, cyclopropyl-H), 1.16e1.22 (1H, m), 1.34e1.37 (1H,
m), 1.61e1.63 (1H, m), 1.77e1.80 (1H, m), 2.05e2.08 (1H, m), 2.49
(3H, s, NCH3), 2.89e3.03 (3H, m), 3.54 (1H, d, J ¼ 10.0), 3.69 (3H, s,
OCH3), 3.86e3.89 (1H, m, cyclopropyl-H), 4.17 (2H, t, J ¼ 5.0, CH2 of
linker), 4.44 (2H, t, J ¼ 5.0, CH2 of linker), 7.03e8.66 (11H, m, Ar-H),
12.69 (brs, 1H, D2O exchangeable, COOH). ESI-MS: m/z 653
(M þ H)þ. HRMS-ESI: m/z Calcd. for C36H38FN6O5 (M þ H)þ:
653.28877; Found 653.28580.
Acknowledgments
This work was supported by the key project of Major infectious
disease (No. 2008ZX10003-006), National S&T Major Special
Project on Major New Drug Innovation (No. 2009Zꢃ09301-003)
and IMB Research Foundation (No. IMBF 2009013).
References
3.2.3.12. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{3-[N-methyl-N-2-(5-
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