Synthesis and in vitro antimycobacterial activity of 8-OCH3 ciprofloxacin methylene and ethylene isatin derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.11.023

A series of novel 8-OCH₃ ciprofloxacin methylene and ethylene isatin derivatives with remarkable improvement in lipophilicity were synthesized in this study. These derivatives were evaluated for their in vitro activity against some mycobacteria. All of the synthesized compounds were less active than the parent 8-OCH₃ ciprofloxacin against Mycobacteriumsmegmatis CMCC 93202, but most of the methylene isatin derivatives were more active than 8-OCH₃ ciprofloxacin, ciprofloxacin, isoniazid and rifampin against MTB H37Rv ATCC 27294. It was noted that compound 3b (MIC: 0.074 μM) was 2-13 fold more potent than the reference compounds against MTB H37Rv ATCC 27294, and compounds 3f and 3i-k (MIC: 6.72-7.05 μM) were around 1.6 fold more potent than the parent 8-OCH₃ ciprofloxacin, 3.5 fold more potent than ciprofloxacin against MDR-MTB 09710.

Full text of DOI:10.1016/j.ejmech.2010.11.023

European Journal of Medicinal Chemistry 46 (2011) 341e348
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 8-OCH3 ciprofloxacin
methylene and ethylene isatin derivatives
,
b
a
a
a
a
Lian-Shun Feng a, Ming-Liang Liu a , Shu Zhang , Yun Chai , Bo Wang , Yi-Bin Zhang , Kai Lv ,
*
Yan Guan a, Hui-Yuan Guo a, Chun-Ling Xiao a
,
**
a Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China
b Department of Chemistry and Chemical Engineering, College of Environmental and Energy Engineering, Beijing University of Technology, Beijing 100124, PR 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 8-OCH3 ciprofloxacin methylene and ethylene isatin derivatives with remarkable
improvement in lipophilicity were synthesized in this study. These derivatives were evaluated for their in
vitro activity against some mycobacteria. All of the synthesized compounds were less active than the
parent 8-OCH3 ciprofloxacin against Mycobacteriumsmegmatis CMCC 93202, but most of the methylene
isatin derivatives were more active than 8-OCH3 ciprofloxacin, ciprofloxacin, isoniazid and rifampin
Received 20 July 2010
Received in revised form
13 October 2010
Accepted 14 November 2010
Available online 24 November 2010
against MTB H37Rv ATCC 27294. It was noted that compound 3b (MIC: 0.074
potent than the reference compounds against MTB H37Rv ATCC 27294, and compounds 3f and 3iek
(MIC: 6.72e7.05 M) were around 1.6 fold more potent than the parent 8-OCH3 ciprofloxacin, 3.5 fold
more potent than ciprofloxacin against MDR-MTB 09710.
Ó 2010 Elsevier Masson SAS. All rights reserved.
mM) was 2e13 fold more
Keywords:
8-OCH3 ciprofloxacin derivatives
Synthesis
m
Antimycobacterial activity
1. Introduction
bacterial infections in both community and hospital settings [7].
These antibacterial agents act by inhibiting bacterial type II DNA
topoisomerases, DNA gyrase (the principal target in gram-negative
bacteria) and topoisomerase IV (the primary target in gram-posi-
tive bacteria) [8]. Surprisingly, there is no evidence of the top-
oisomerase IV parC and parE gene homologs in the genome of MTB,
so it seems that DNA gyrase is the sole topoisomerase drug target of
FQs in MTB [9]. Resistance to FQs remains relatively low in clinical
isolates of MTB currently, and there are no reports of cross-resis-
tance or antagonism with other classes of antimycobacterial agents
[10,11]. Some early FQs, including ciprofloxacin (CPFX, Fig. 1),
ofloxacin and sparfloxacin were recommended as second-line
agents for the treatment of TB mainly in cases involving resistance
or intolerance to first-line anti-TB therapy by WHO in 1996 [12].
Interestingly, 8-OCH3 FQ derivatives with N1-cyclopropyl substi-
tution are much more potent against resistant MTB than 8-H
analogs [13]. The newer 8-OCH3 FQs moxifloxacin (MXFX) and
gatifloxacin (GTFX) having a particularly strong in vitro and in vivo
activity against MTB, could be promising agents for the treatment
of TB [14]. It was noted that 8-OCH3 ciprofloxacin (8-OCH3 CPFX,
Fig. 1) exhibits excellent in vitro activity against gram-positive,
gram-negative and anaerobic bacteria [15]. However, to our
knowledge, no studies aiming at optimizing 8-OCH3 CPFX against
mycobacteria have been reported in the literature.
Tuberculosis (TB) is a common, communicable and even fatal
disease caused by mycobacteria, mainly by Mycobacterium tuber-
culosis (MTB) [1]. The WHO has estimated that approximately one-
third of the world population (more than 2 billion) is infected with
MTB, and 9.4 million new incident cases occurred in 2008, with
around 1.3 million individual deaths in the same year [2,3]. The
increasing emergence of drug-resistant TB (DR-TB), especially
multidrug-resistant TB (MDR-TB) resistant to at least two major
first-line anti-TB drugs (isoniazid/INH and rifampin/RIF) is causing
particularly concern [3,4]. MDR-TB has already caused several fatal
outbreaks, and posed a significant threat to the treatment and
control of the disease in some parts of the world [5]. For example, of
the new TB cases in Azerbaijan in 2007, the proportion of MDR-TB
reached as high as 22.3% [2]. In addition, TB is also a frequent HIV co-
infection, which is having a devastating impact in some regions,
such as African where 38% of new TB cases were attributable to HIV
co-infection in 2008 [2,6]. Therefore, it’s imperative to develop
novel, high effective and fast acting anti-TB drugs.
Fluoroquinolones (FQs) have emerged as one of the dominant
classes of chemotherapeutic drugs for the treatment of various
* Corresponding author. Tel.: þ86 10 63036965; fax: þ86 10 63047865.
** Corresponding author. Tel./fax: þ86 10 63020226.
Recently, the research concerning FQs has been focused on the
basic group at the C-7 position which is the most adaptable site for
E-mail addresses: lmllyx@yahoo.com.cn (M.-L. Liu), xiaocl@163.com (C.-L. Xiao).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.023

342
L.-S. Feng et al. / European Journal of Medicinal Chemistry 46 (2011) 341e348
Isatins 1a, b were alkylated with 1,2-dibromoethane to give N-(2-
bromoethyl)isatins 4a, b (53e68%) according to the reported
procedures [20,21]. Nucleophilic substitution reactions of
compounds 4a, b with 8-OCH3 CPFX were performed in DMF at
40 ꢀC to afford 8-OCH3 CPFX ethylene derivatives 5a, b (34e45%).
Subsequent condensations of compounds 5a, b with requisite
substituted amine hydrochlorides in the presence of NaHCO3
formed other derivatives (Schiff’s bases) 5cel (48e77%) (Scheme 2).
The structures of the synthesized 8-OCH3 CPFX methylene and
ethylene isatin derivatives 3aek and 5ael were established by MS,
HRMS, 1H NMR and 13C NMR spectra. For example, the 1H NMR
chemical shifts of some respective protons in the title compounds
Fig. 1. Chemical structures of CPFX and 8-OCH3 CPFX.
chemical change and an area that greatly influences their potency,
spectrum and safety [16e18]. On the other hand, it was suggested
that the lipophilicity of the FQs plays an important role in the
penetration of these compounds into bacterial cells, and simply
increasing the lipophilic character at C-7 position could also increase
the anti-TB activity [11,19]. Therefore, reasonable modification at C-7
position is likely to produce more effective anti-TB agents.
were as follows: two sets of singlet at
zine-4H, two sets of multiplet at 0.72e1.22 and 3.88e4.15 ppm for
cyclopropyl-5H, a singlet at 3.72e3.82 for 8-OCH3 group, a singlet at
4.48e4.65 ppm corresponding to eNCH2Ne group for methylene
derivatives (around 62 ppm in 13C NMR), two sets of triplet at
4.07e4.15 and 4.35e4.53 ppm corresponding to eNCH2CH2Ne
d 2.66e3.76 ppm for pipera-
d
d
d
group for ethylene derivatives.
Sriram et al. reported GTFX and CPFX methylene isatin deriva-
tives, and a few compounds were more potent than the parent
drugs against MTB [11,19]. In our previous work, a series of balo-
floxacin (BLFX) ethylene isatin derivatives were synthesized and
evaluated for their in vitro activity against some mycobacteria. The
2.2. Lipophilicity
Lipophilicity of the synthesized 8-OCH3 CPFX derivatives 3aek
and 5ael, the parent compound 8-OCH3 CPFX and CPFX is
expressed in the term of their log P values which were calculated
with Chem office 2009 software. As shown in Table 1, a remarkable
improvement in the lipophilicity of the derivatives 3aek and 5ael
as evidenced by log P values (1.26e3.33) which were much more
than that of the parent 8-OCH3 CPFX (1.20) (statistically significant
at p < 0.001 using t test).
Compounds 3b, 3d, 3e, 3j and 3k were chosen for further
evaluation their experimental log P values by HPLC, and compared
with 8-OCH3 CPFX (Table 1). These results indicated that the lip-
ophilicity of derivatives 3b, 3d, 3e, 3j and 3k (experimental log P
values arrange from 1.08 to 1.67) was much more than 8-OCH3
CPFX (0.79), which was consistent with lipophilicity profiles based
on log P values calculated by softwares. This may be rendering them
more capable of penetrating various biomembranes, consequently
improving their penetrability towards mycobacterial cell
membrane. In other words, the improvement of the lipophilic
character of the target compounds 3aek and 5ael probably
enhances their antimycobacterial activity.
derivative containing 3-[N-methyl-N-2-(5-fluorinisatinyl-b-ethox-
yimino)ethyl]aminopiperidin-1-yl group at the C-7 position of
BLFX was found to be far more potent than the parent BLFX, and
comparable to MXFX [20].
Inspired by the above research results, a series of 8-OCH3 CPFX
methylene and ethylene isatin derivatives containing oxime,
methyloxime, ethyloxime, semicarbazone and thiosemicarbazone
were designed and synthesized in this study. Our primary objective
was to optimize the potency of these compounds against
mycobacteria.
2. Results and discussion
2.1. Chemistry
Synthetic pathways to 8-OCH3 CPFX methylene and ethylene
isatin derivatives 3aek and 5ael are depicted in Schemes 1 and 2,
respectively. Condensations of compounds 1a, b with requisite
substituted amine hydrochlorides in the presence of NaHCO3
formed isatin derivatives (Schiff’s bases) 2aei (62e88%). Mannich
reactions of the compounds 1a, b or 2aei with paraformaldehyde
[(CH2O)n] and 8-OCH3 CPFX were performed in refluxing EtOH
under an atmosphere of nitrogen to provide 8-OCH3 CPFX methy-
lene isatin derivatives 3aek (40e85%) (Scheme 1) [11,19].
In addition, 8-OCH3 CPFX methylene isatin derivatives (log P:
1.54e3.33) were more lipophilic than that of the corresponding
ethylene isatin analogs (log P: 1.26e3.05), and compounds con-
taining a 5-fluoroisatin moiety (log P: 1.42e3.33) were more than
the corresponding isatin analogs (log P: 1.26e3.17) (statistically
significant at p < 0.001 using t test).
Scheme. 1. Synthesis of 8-OCH3 CPFX methylene isatin derivatives 3aek.

L.-S. Feng et al. / European Journal of Medicinal Chemistry 46 (2011) 341e348
343
Scheme. 2. Synthetic route of 8-OCH3 CPFX ethylene isatin derivatives 5ael.
2.3. Pharmacology
93202 using serial double dilution technique in duplicate [20].
Considering the fact that the activity against M. smegmatis is often
correlated poorly with that against MTB, most of the compounds
with relatively good activity, such as 3bek, 5e, 5f, 5k and 5l, and
compound 3a with least activity against M. smegmatis, were chosen
The target compounds 3aek, 5ael along with 8-OCH3 CPFX,
CPFX, RIF and INH were initially evaluated for their in vitro anti-
mycobacterial activity against Mycobacterium smegmatis CMCC
Table 1
Structures, lipophilicity, antimycobacterial activity and cytotoxicity of compounds 3aek and 5ael.
O
N
F
COOH
R1
R
N
O
N
OCH3
N
n
Log Pa
MIC (
m
M)
CC50
(
m
M)
SIf
e
Compd.
n
R
R1
M. s.b
MTBc
MDR-MTBd
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
H
F
H
F
H
F
H
F
H
F
H
H
F
H
F
H
F
O
O
NOH
NOH
NOCH3
NOCH3
NOC2H5
NOC2H5
NNHCONH2
NNHCONH2
NNHCSNH2
O
2.18
2.34g
2.57
2.73g
2.83g
2.99
3.17
3.33
1.54
1.70g
2.10g
1.91
2.06
2.29
2.45
2.56
2.71
2.89
3.05
1.26
1.42
1.82
1.98
1.20g
1.32
24.04
2.90
2.92
1.41
5.68
2.75
2.77
2.68
1.35
2.62
2.63
5.84
2.83
11.38
22.05
2.77
2.68
5.41
1.31
5.28
5.12
2.57
2.50
0.54
2.36
3.79
5.69
0.15
0.074
0.15
0.14
0.15
0.14
0.14
0.55
0.14
0.13
0.13
30.77
14.87
29.91
14.47
14.57
7.05
70.8
70.6
74.4
73.8
76.5
46.8
91.5
85.8
472
954
496
527
510
334
654
156
28.42
6.93
6.72
6.75
266.3
168.4
153.6
1902
1295
1182
O
NOH
NOH
NOCH3
NOCH3
NOC2H5
NOC2H5
NNHCONH2
NNHCONH2
NNHCSNH2
NNHCSNH2
1.14
1.10
305.6
12.8
268
12
H
F
H
F
H
F
1.05
1.02
0.22
0.97
0.19
0.29
56.1
57.0
242.1
600.8
180
53
56
1100
619
947
21665
8-OCH3 CPFX
CPFX
RIF
11.08
24.17
>311
467
INH
6282.8
a
The log P is calculated by Chem office 2009 software.
M.s.: M. smegmatis CMCC 93202.
MTB: MTB H37Rv ATCC 27294.
MDR-MTB: MDR-MTB 09710 was resistant to RIF, INH, EMB and SM.
CC50: The 50% cytotoxic concentration.
SI: Selectivity index for MTB H37Rv ATCC 27294, CC50/MICMTB
b
c
d
e
f
.
g
The experimental log P values by HPLC of compounds 3b, 3d, 3e, 3j, 3k and 8-OCH3 CPFX are 1.08, 1.57, 1.67, 1.44, 1.34 and 0.79, respectively.

344
L.-S. Feng et al. / European Journal of Medicinal Chemistry 46 (2011) 341e348
for evaluation of their in vitro activity against MTB H37Rv ATCC
27294. Finally, compounds 3aeg and 3iek with better activity than
the parent against MTB H37Rv ATCC 27294, were further evaluated
their in vitro activity against MDR-MTB 09710 clinical isolate using
rapid direct susceptibility test technique [20].
than the corresponding ethylene isatin analogs against MTB H37Rv
ATCC 27294.
4. Experimental section
The minimum inhibitory concentration (MIC) is defined as the
concentration of the compound required to give complete inhibi-
tion of mycobacterial growth and MICs of the synthesized
compounds along with 8-OCH3 CPFX, CPFX, RIF and INH for
comparison are presented in Table 1. These data indicated that all of
the target compounds showed considerable potency in inhibiting
4.1. General
Melting points were determined in open capillaries and
uncorrected. Log P was 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. The column
the growth of M. smegmatis CMCC 93202 (MIC: 1.31e24.04
although less active than the parent 8-OCH3 CPFX (MIC: 0.54
m
m
M),
M).
used was a Diamonsil C18 5
mm 250 ꢁ 4.6 mm column (Dikma
Technologies). 1H NMR and 13C NMR spectra were determined 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 used
without further purification. TLC was performed on silica gel plates
(Merck, ART5554 60F254).
As for MTB H37Rv ATCC 27294, all of the methylene isatin
derivatives except compound 3h (MIC: 0.074e0.15 M) were more
active than 8-OCH3 CPFX, CPFX, RIF and INH (MIC: 0.19e0.97 M).
In particular, the most active compound 3b (MIC: 0.074 M) was
m
m
m
found to be 2e13 fold more potent than the reference compounds.
On the other hand, the activity of four ethylene isatin analogs 5e, 5f,
5k and 5l was less than the references and the corresponding
methylene isatin analogs.
Compounds 3aeg and 3iek with better activity than the refer-
ence compounds against MTB H37Rv ATCC 27294, were also found
to have considerable activity (MIC: 6.72e30.77 mM) against MDR-
MTB 09710 resistant to INH, RIF, ethambutol/EMB and strepto-
mycin/SM. Among them, compounds 3f and 3iek (MIC:
6.72e7.05
mM) were around 1.6 fold more potent than the parent 8-
4.2. Synthesis
OCH3 CPFX, and 3.5 fold more potent than CPFX against this strain.
Finally, compounds 3aek, 5e, 5f, 5k and 5l were examined for
toxicity (CC50) in a mammalian Vero cell line [22]. 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. The fifteen derivatives when tested showed CC50 values
4.2.1. General procedure for the preparation of compounds 2aei
To a solution of substituted amine hydrochloride (1.5 mmol) and
NaHCO3 (0.12 g, 1.4 mmol) dissolved in water (10 mL) was added
dropwise a solution of 1a, b (1 mmol) in MeOH (10 mL) at room
temperature over 5 min. The reaction mixture was stirred at the
same temperature for 1 h and filtered. The solid obtained was
washed with water and EtOH to give the title compounds 2aei
(62e88%).
ranging from 12.8 to 266.3 mM. A comparison demonstrated that
the derivatives containing a 5-fluoroisatin moiety were generally
more cytotoxic than that of the corresponding isatin analogs.
Herein, compounds 3i and 5e showed similar toxicity as the
parent 8-OCH3 CPFX, and the selectivity index (SI: 1182e1902) of
compounds 3iek was more than 8-OCH3 CPFX (1100) for MTB
H37Rv ATCC 27294.
4.2.1.1.
b
-Hydroximinoisatin (2a). Yield: 81%. mp: 215e218 ꢀC. 1H
NMR (DMSO-d6, 400 MHz) dppm: 6.85e7.93 (4H, m, Ar-H), 10.66
(1H, s, D2O exchangeable, NH), 13.25 (1H, s, D2O exchangeable,
NOH). FAB-MS: m/z 163 (M þ H)þ.
3. Conclusion
4.2.1.2. 5-Fluoro-
b
-hydroximinoisatin
(2b). Yield:
88%.
mp:
262e264 ꢀC. 1H NMR (DMSO-d6, 400 MHz) dppm: 6.84e7.70 (3H, m,
Ar-H), 10.70 (1H, s, D2O exchangeable, NH), 13.53 (1H, s, D2O
exchangeable, NOH). FAB-MS: m/z 181 (M þ H)þ.
In summary, a series of novel 8-OCH3 CPFX methylene and
ethylene isatin derivatives were designed, synthesized and charac-
terized by 1H NMR, 13C NMR, MS and HRMS. These derivatives were
initially evaluated for their in vitro antimycobacterial activity against
M. smegmatis CMCC 93202, and then some of them against MTB
H37Rv ATCC 27294 and MDR-MTB 09710, respectively. The results
showed that all of the target compounds with improved lipophilicity
were less active than the parent 8-OCH3 CPFX against M. smegmatis
CMCC 93202, but most of the methylene isatin derivatives were
more active than 8-OCH3 CPFX, which suggested the activity of these
compounds against M. smegmatis was correlated poorly with that
4.2.1.3.
b
-Methoxyiminoisatin (2c). Yield: 80%. mp: 172e174 ꢀC. 1H
NMR (DMSO-d6, 400 MHz) dppm: 4.17 (3H, s, NOCH3), 6.86e7.83
(4H, m, Ar-H), 10.75 (1H, s, D2O exchangeable, NH). FAB-MS: m/z
177 (M þ H)þ.
4.2.1.4. 5-Fluoro-b-methoxyiminoisatin (2d). Yield: 82%. mp:
228e231 ꢀC. 1H NMR (DMSO-d6, 400 MHz) dppm: 4.19 (3H, s,
NOCH3), 6.86e7.63 (3H, m, Ar-H), 10.79 (1H, s, D2O exchangeable,
NH). FAB-MS: m/z 195 (M þ H)þ.
against MTB. It was noted that compound 3b (MIC: 0.074
2e13 fold more potent than the reference compounds, and the
activity of compounds 3f and 3iek (MIC: 6.72e7.05
mM) was
m
M) was better
4.2.1.5. b
-Ethoxyiminoisatin (2e). Yield: 72%. mp: 136e139 ꢀC. 1H
than 8-OCH3 CPFX against MDR-MTB 09710.
NMR (DMSO-d6, 400 MHz) dppm: 1.36 (3H, t, J ¼ 7.2 Hz, NOCH2CH3),
4.42 (2H, q, J ¼ 7.2 Hz, NOCH2CH3), 6.86e7.85 (4H, m, Ar-H), 10.74
(1H, s, D2O exchangeable, NH). FAB-MS: m/z 191 (M þ H)þ.
The relative contribution of isatin moieties of 8-OCH3 CPFX
methylene isatin derivatives to activity against MDR-MTB 09710 is
as follows: thiosemicarbazone z semicarbazone > methyloxime >
ketone z oxime z ethyloxime, when R ¼ H; semicarbazone z
methyloxime > ketone z oxime, when R ¼ F; 5-fluoroisatin >
isatin. In addition, methylene isatin derivatives were more potent
4.2.1.6. 5-Fluoro-
b-ethoxyiminoisatin
(2f). Yield:
62%.
mp:
170e174 ꢀC. 1H NMR (DMSO-d6, 400 MHz) dppm: 1.36 (3H, t,
J ¼ 6.8 Hz, NOCH2CH3), 4.45 (2H, q, J ¼ 6.8 Hz, NOCH2CH3),

L.-S. Feng et al. / European Journal of Medicinal Chemistry 46 (2011) 341e348
345
6.86e7.62 (3H, m, Ar-H), 10.78 (1H, s, D2O exchangeable, NH). FAB-
4.2.2.4. 1-Cyclopropyl-6-fluoro-8-methoxy-7-[4-(5-fluorinisatinyl-b-
MS: m/z 209 (M þ H)þ.
hydroximino) methylpiperazin-1-yl]-1,4-dihydro-4-oxoquinoline-3-
carboxylic acid (3d). Yield: 74%. mp: 172e176 ꢀC.1H NMR (DMSO-d6,
400 MHz) dppm: 1.02 (2H, m, cyclopropyl-H), 1.10 (2H, m, cyclo-
propyl-H), 2.76 (4H, s, piperazine-4H), 3.30 (4H, s, piperazine-4H),
3.74 (3H, s, OCH3), 4.14 (1H, m, cyclopropyl-H), 4.48 (2H, s, CH2 of
linker), 7.33e8.68 (5H, m, Ar-H), 14.92 (1H, brs, D2O exchangeable,
COOH). FAB-MS: m/z 554 (M þ H)þ. HRMS-FAB: m/z Calcd. for
C27H26F2N4O6 (M þ H)þ: 554.1851; Found 554.1863.
4.2.1.7.
b
-Semicarbazoisatin (2g). Yield: 85%. mp: 260e265 ꢀC. 1H
NMR (DMSO-d6, 400 MHz) dppm: 6.87 (2H, brs, D2O exchangeable,
NNHCONH2), 7.00e8.06 (4H, m, Ar-H), 10.66 (1H, s, D2O
exchangeable, NH), 13.25 (1H, brs, D2O exchangeable, NNHCONH2).
FAB-MS: m/z 205 (M þ H)þ.
4.2.1.8. 5-Fluoro-
b
-semicarbazoisatin
(2h). Yield:
72%.
mp:
273e276 ꢀC. 1H NMR (DMSO-d6, 400 MHz) dppm: 6.86 (2H, brs, D2O
exchangeable, NNHCONH2), 6.84e8.08 (3H, m, Ar-H), 10.34 (1H, s,
D2O exchangeable, NH), 10.69 (1H, brs, D2O exchangeable,
NNHCONH2). FAB-MS: m/z 223 (M þ H)þ.
4.2.2.5. 1-Cyclopropyl-6-fluoro-8-methoxy-7-[4-(b-methox-
yiminoisatinyl)methylpiperazin-1-yl]-1,4-dihydro-4-oxoquinoline-3-
carboxylic acid (3e). Yield: 72%. mp: 132e135 ꢀC. 1H NMR (CDCl3,
400 MHz) dppm: 0.99 (2H, m, cyclopropyl-H), 1.19e1.22 (2H, m,
cyclopropyl-H), 2.81 (4H, s, piperazine-4H), 3.42 (4H, s, piperazine-
4H), 3.75 (3H, s, OCH3), 4.01 (1H, m, cyclopropyl-H), 4.31 (3H, s,
NOCH3), 4.58 (2H, s, CH2 of linker), 7.06e8.80 (6H, m, Ar-H), 14.75
(1H, brs, D2O exchangeable, COOH). FAB-MS: m/z 550 (M þ H)þ.
HRMS-FAB: m/z Calcd. for C28H29FN5O6 (M þ H)þ: 550.2102; Found
550.2076.
4.2.1.9.
b
-Thiosemicarbazoisatin (2i). Yield: 77%. mp: 251e253 ꢀC.
1H NMR (DMSO-d6, 400 MHz) dppm: 6.90e7.65 (4H, m, Ar-H), 8.66,
9.02 (2H, brs, D2O exchangeable, NNHCSNH2), 11.18 (1H, s, D2O
exchangeable, NH), 12.46 (1H, brs, D2O exchangeable, NNHCSNH2).
FAB-MS: m/z 221 (M þ H)þ.
4.2.2. General procedure for the preparation of compounds 3aek
A solution of 1-cyclopropyl-6-fluoro-8-methoxy-7-(piperazin-
1-yl)-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (8-OCH3 CPFX,
2.89g, 8 mmol) and (CH2O)n (0.36 g, 12 mmol) in EtOH (100 mL)
was heated to refluxing for 1.5 h under an atmosphere of nitrogen.
To the reaction mixture was added isatin 1a, b or Schiff’s base 2aei
(5 mmol), stirred at refluxing for 12 h, and cooled to room
temperature. The precipitate was filtered and recrystallized from
DMF and water to give the title compounds 3aek (40e85%).
4.2.2.6. 1-Cyclopropyl-6-fluoro-8-methoxy-7-[4-(5-fluorinisatinyl-b-
methoxyimino)methylpiperazin-1-yl]-1,4-dihydro-4-oxoquinoline-3-
carboxylic acid (3f). Yield: 75%. mp: 134e138 ꢀC.1H NMR (DMSO-d6,
400 MHz) dppm: 1.01 (2H, m, cyclopropyl-H), 1.09e1.12 (2H, m,
cyclopropyl-H), 2.72 (4H, s, piperazine-4H), 3.31 (4H, s, piperazine-
4H), 3.75 (3H, s, OCH3), 4.14 (1H, m, cyclopropyl-H), 4.23 (3H, s,
NOCH3), 4.53 (2H, s, CH2 of linker), 7.29e8.69 (5H, m, Ar-H), 14.92
(1H, brs, D2O exchangeable, COOH). 13C NMR (DMSO-d6, 400 MHz)
dC: 8.93, 40.77, 50.08, 50.68, 61.44, 62.68, 64.72, 106.47, 111.89,
114.04, 115.47, 119.14, 120.86, 134.00, 138.99, 140.65, 142.89, 145.88,
150.48, 154.29, 156.80, 159.17, 162.99, 165.59, 176.26. FAB-MS: m/z
568 (M þ H)þ. HRMS-FAB: m/z Calcd. for C28H28F2N5O6 (M þ H)þ:
568.2008; Found 568.2008.
4.2.2.1. 1-Cyclopropyl-6-fluoro-8-methoxy-7-[4-(isatinylmethyl)
piperazin-1-yl]-1,4-
dihydro-4-oxoquinoline-3-carboxylic
acid
(3a). Yield: 85%. mp: 218e222 ꢀC. 1H NMR (CDCl3, 400 MHz) dppm
:
1.00 (2H, m, cyclopropyl-H),1.21 (2H, m, cyclopropyl-H), 2.93 (4H, s,
piperazine-4H), 3.53 (4H, s, piperazine-4H), 3.78 (3H, s, OCH3), 4.01
(1H, m, cyclopropyl-H), 4.64 (2H, s, CH2 of linker), 7.19e8.82 (6H, m,
Ar-H), 14.67 (1H, brs, D2O exchangeable, COOH). FAB-MS: m/z 521
(M þ H)þ. HRMS-FAB: m/z Calcd. for C27H26FN4O6 (M þ H)þ:
521.1836; Found 521.1801.
4.2.2.7. 1-Cyclopropyl-6-fluoro-8-methoxy-7-[4-(b-ethox-
yiminoisatinyl)methylpiperazin-1-yl]-1,4-dihydro-4-oxoquinoline-3-
carboxylic acid (3g). Yield: 65%. mp: 117e120 ꢀC. 1H NMR (DMSO-
d6, 400 MHz) dppm: 1.01 (2H, m, cyclopropyl-H), 1.09 (2H, m,
cyclopropyl-H), 1.38 (3H, t, J ¼ 7.2 Hz, NOCH2CH3) 2.73 (4H, s,
piperazine-4H), 3.31 (4H, s, piperazine-4H), 3.72 (3H, s, OCH3), 4.13
(1H, m, cyclopropyl-H), 4.36 (2H, q, J ¼ 7.2 Hz, NOCH2CH3), 4.54
(2H, s, CH2 of linker), 7.10e8.68 (6H, m, Ar-H), 14.92 (1H, brs, D2O
exchangeable, COOH). FAB-MS: m/z 564 (M þ H)þ. HRMS-FAB: m/z
Calcd. for C29H31FN5O6 (M þ H)þ: 564.2258; Found 564.2256.
4.2.2.2. 1-Cyclopropyl-6-fluoro-8-methoxy-7-[4-(5-fluo-
roisatinylmethyl)piperazin-1-yl]-1,4-dihydro-4-oxoquinoline-3-
carboxylic acid (3b). Yield: 78%. mp: 124e127 ꢀC. 1H NMR (CDCl3,
400 MHz) dppm: 1.01 (2H, m, cyclopropyl-H), 1.18e1.22 (2H, m,
cyclopropyl-H), 2.88 (4H, s, piperazine-4H), 3.50 (4H, s, pipera-
zine-4H), 3.79 (3H, s, OCH3), 4.02 (1H, m, cyclopropyl-H), 4.59 (2H,
s, CH2 of linker), 7.26e8.81 (5H, m, Ar-H), 14.68 (1H, brs, D2O
exchangeable, COOH). 13C NMR (DMSO-d6, 400 MHz) dC: 8.93,
40.79, 50.10, 50.50, 61.85, 62.74, 106.48, 111.03, 113.45, 118.53,
120.88, 123.79, 124.03, 134.03, 139.03, 145.93, 147.59, 150.53,
154.34, 157.05, 159.34, 165.62, 176.30, 182.55. FAB-MS: m/z 539
(M þ H)þ. HRMS-FAB: m/z Calcd. for C27H25F2N4O6 (M þ H)þ:
539.1742; Found 539.1729.
4.2.2.8. 1-Cyclopropyl-6-fluoro-8-methoxy-7-[4-(5-fluorinisatinyl-b-
ethoxyimino) methylpiperazin-1-yl]-1,4-dihydro-4-oxoquinoline-3-
carboxylic acid (3h). Yield: 57%. mp:125e128 ꢀC.1H NMR (DMSO-d6,
400 MHz) dppm: 0.98e1.02 (2H, m, cyclopropyl-H),1.04e1.09 (2H, m,
cyclopropyl-H), 1.39 (3H, t, J ¼ 7.6 Hz, NOCH2CH3) 2.66 (4H, s,
piperazine-4H), 3.35 (4H, s, piperazine-4H), 3.75 (3H, s, OCH3), 4.15
(1H, m, cyclopropyl-H), 4.49 (2H, q, J ¼ 7.6 Hz, NOCH2CH3), 4.53 (2H,
s, CH2 of linker), 7.29e8.69 (5H, m, Ar-H), 14.92 (1H, brs, D2O
exchangeable, COOH). FAB-MS: m/z 582 (M þ H)þ. HRMS-FAB: m/z
Calcd. for C29H30F2N5O6 (M þ H)þ: 582.2164; Found 582.2159.
4.2.2.3. 1-Cyclopropyl-6-fluoro-8-methoxy-7-[4-(b-hydrox-
iminoisatinyl) methylpiperazin-1-yl]-1,4-dihydro-4-oxoquinoline-3-
carboxylic acid (3c). Yield: 79%. mp: 165e170 ꢀC. 1H NMR (DMSO-
d6, 400 MHz) dppm: 1.00e1.05 (2H, m, cyclopropyl-H), 1.09e1.12
(2H, m, cyclopropyl-H), 2.73 (4H, s, piperazine-4H), 3.30 (4H, s,
piperazine-4H), 3.72 (3H, s, OCH3), 4.13 (1H, m, cyclopropyl-H), 4.55
(2H, s, CH2 of linker), 7.09e8.69 (6H, m, Ar-H), 13.46 (1H, s, D2O
exchangeable, NOH), 14.92 (1H, brs, D2O exchangeable, COOH).
FAB-MS: m/z 536 (M þ H)þ. HRMS-FAB: m/z Calcd. for C27H26FN5O6
(M þ H)þ: 536.1945; Found 536.1936.
4.2.2.9. 1-Cyclopropyl-6-fluoro-8-methoxy-7-[4-(b-semi-
carbazoisatinyl)methylpiperazin-1-yl]-1,4-dihydro-4-oxoquinoline-
3-carboxylic acid (3i). Yield: 62%. mp: 174e177 ꢀC. 1H NMR (DMSO-
d6, 400 MHz) dppm: 0.98e1.02 (2H, m, cyclopropyl-H), 1.04e1.09
(2H, m, cyclopropyl-H), 2.66 (4H, s, piperazine-4H), 3.35 (4H, s,
piperazine-4H), 3.75 (3H, s, OCH3), 4.14 (1H, m, cyclopropyl-H), 4.61
(2H, s, CH2 of linker), 7.14 (2H, brs, D2O exchangeable, NNHCONH2),
7.16e8.69 (6H, m, Ar-H), 11.66 (1H, brs, D2O exchangeable,

346
L.-S. Feng et al. / European Journal of Medicinal Chemistry 46 (2011) 341e348
NNHCONH2), 14.92 (1H, brs, D2O exchangeable, COOH). FAB-MS: m/
z 578 (M þ H)þ. HRMS-FAB: m/z Calcd. for C28H29FN7O6 (M þ H)þ:
578.2163; Found 578.2162.
cyclopropyl-H), 3.40 (4H, s, piperazine-4H), 3.70 (4H, s, piperazine-
4H), 3.82 (3H, s, OCH3), 3.90 (1H, m, cyclopropyl-H), 4.15 (2H, t,
J ¼ 5.2 Hz, CH2 of linker), 4.56 (2H, t, J ¼ 5.2 Hz, CH2 of linker),
7.09e8.53 (6H, m, Ar-H). ESI-MS: m/z 535 (M þ H)þ. HRMS-ESI: m/z
Calcd. for C28H28FN4O6 (M þ H)þ: 535.1992; Found 535.2028.
4.2.2.10. 1-Cyclopropyl-6-fluoro-8-methoxy-7-[4-(5-fluorinisatinyl-
b-semicarbazo) methylpiperazin-1-yl]-1,4-dihydro-4-oxoquinoline-
3-carboxylic acid (3j). Yield: 54%. mp: 182e186 ꢀC. 1H NMR
(DMSO-d6, 400 MHz) dppm: 1.00e1.03 (2H, m, cyclopropyl-H),
1.06e1.10 (2H, m, cyclopropyl-H), 2.74 (4H, s, piperazine-4H), 3.32
(4H, s, piperazine-4H), 3.73 (3H, s, OCH3), 4.14 (1H, m, cyclopropyl-
H), 4.59 (2H, s, CH2 of linker), 7.23 (2H, brs, D2O exchangeable,
NNHCONH2), 7.25e8.67 (5H, m, Ar-H), 11.58 (1H, brs, D2O
exchangeable, NNHCONH2), 14.91 (1H, brs, D2O exchangeable,
COOH). FAB-MS: m/z 596 (M þ H)þ. HRMS-FAB: m/z Calcd. for
C28H38F2N7O6 (M þ H)þ: 596.2069; Found 596.2064.
4.2.4. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{4-[2-(5-fluoroisatinyl)
ethyl]piperazin- 1-yl}-1,4-dihydro-4-oxoquinoline-3-carboxylic acid
(5b). The title compound was obtained in a similar manner as for the
preparation of 5a (34%) as anacaratsolid, mp: 225e227 ꢀC. 1H NMR
(DMSO-d6, 400 MHz) dppm: 0.87 (2H, m, cyclopropyl-H), 0.98e1.01
(2H, m, cyclopropyl-H), 2.84 (4H, s, piperazine-4H), 3.19 (4H, s,
piperazine-4H), 3.76 (3H, s, OCH3), 3.93 (1H, m, cyclopropyl-H), 4.07
(2H, t, J ¼ 5.2 Hz, CH2 of linker), 4.36 (2H, t, J ¼ 5.2 Hz, CH2 of linker),
7.28e8.32 (5H, m, Ar-H). ESI-MS: m/z 553 (M þ H)þ. HRMS-ESI: m/z
Calcd. for C28H27F2N4O6 (M þ H)þ: 553.1898; Found 553.1924.
4.2.2.11. 1-Cyclopropyl-6-fluoro-8-methoxy-7-[4-(b-thio-
semicarbazoisatinyl) methylpiperazin-1-yl]-1,4-dihydro-4-oxoquino-
line-3-carboxylic acid (3k). Yield: 40%. mp: 180e183 ꢀC. 1H NMR
(DMSO-d6, 400 MHz) dppm: 0.97e1.00 (2H, m, cyclopropyl-H),
1.09e1.12 (2H, m, cyclopropyl-H), 2.77 (4H, s, piperazine-4H), 3.33
(4H, s, piperazine-4H), 3.73 (3H, s, OCH3), 4.13 (1H, m, cyclopropyl-
H), 4.65 (2H, s, CH2 of linker), 7.15e8.67 (6H, m, Ar-H), 8.73, 9.08
(2H, brs, D2O exchangeable, NNHCSNH2), 12.39 (1H, brs, D2O
exchangeable, NNHCSNH2), 14.92 (1H, brs, D2O exchangeable,
COOH). FAB-MS: m/z 594 (M þ H)þ. HRMS-FAB: m/z Calcd. for
C28H29FN7O5S (M þ H)þ: 594.1935; Found 594.1934.
4.2.5. General procedure for the preparation of compounds 5cel
To a solution of substituted amine hydrochloride (3 mmol) and
NaHCO3 (0.25 g, 3 mmol) dissolved in water (10 mL) was added
dropwise a solution of 5a, b (1 mmol) in MeOH (10 mL) at room
temperature over 5 min. The reaction mixturewas stirred at the same
temperature for 24 h. After removal of the MeOH under reduced
pressure, the reaction mixture was diluted with water (20 mL) and
stirred for 10 min, and then filtered. The crude product was purified
bycolumn chromatography (silica gel) eluted with CH2Cl2 and MeOH
(v: v ¼ 20: 1) to give the title compounds 5cel (48e77%).
4.2.3. N-(2-Bromoethyl)isatin (4a)
4.2.5.11-Cyclopropyl-6-fluoro-8-methoxy-7-{4-[2-(b-hydrox-
A suspension of isatin (1a, 4.41 g, 30 mmol), anhydrous K2CO3
(12.51 g, 90 mmol) and 1,2-dibromoethane (16.83 g, 90 mmol) in
DMF (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 ꢁ 50 mL). The combined extracts were washed with
saturated brine, dried over anhydrous Na2SO4, 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 4a (5.19 g, 68%) as
iminoisatinyl) ethyl]piperazin-1-yl}-1,4-dihydro-4-oxoquinoline-3-
carboxylic acid (5c). Yield: 66%. mp: 220e225 ꢀC. 1H NMR (DMSO-
d6, 400 MHz) dppm: 0.89 (2H, m, cyclopropyl-H), 1.01e1.06 (2H, m,
cyclopropyl-H), 3.23 (4H, s, piperazine-4H), 3.46 (4H, s, piperazine-
4H), 3.78 (3H, s, OCH3), 3.94 (1H, m, cyclopropyl-H), 4.07 (2H, t,
J ¼ 5.2 Hz, CH2 of linker), 4.37 (2H, t, J ¼ 5.2 Hz, CH2 of linker),
7.06e8.36 (6H, m, Ar-H), 9.14 (1H, brs, D2O exchangeable, NOH),
13.46 (1H, brs, D2O exchangeable, COOH). ESI-MS: m/z 550
(M þ H)þ. HRMS-ESI: m/z Calcd. for C28H29FN5O6 (M þ H)þ:
550.2101; Found 550.2133.
anacaratsolid, mp: 126e128 ꢀC. 1H NMR (CDCl3, 400 MHz) dppm
:
3.62 (2H, t, J ¼ 6.8 Hz, CH2CH2Br), 4.15 (2H, t, J ¼ 6.8 Hz, CH2CH2Br),
7.00 (1H, d, J ¼ 7.6 Hz, Ar-H), 7.15 (1H, t, J ¼ 7.6 Hz, Ar-H), 7.60e7.65
(2H, m, Ar-H). ESI-MS: m/z 254 (M þ H)þ, 256 (Mþ2 þ H)þ.
4.2.5.2. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{4-[2-(5-fluo-
rinisatinyl-b-hydroximino) ethyl]piperazin-1-yl}-1,4-dihydro-4-oxo-
quinoline-3-carboxylic acid (5d). Yield: 60%. mp: 189e192 ꢀC. 1H
NMR (DMSO-d6, 400 MHz) dppm: 0.85 (2H, m, cyclopropyl-H), 1.00
(2H, m, cyclopropyl-H), 2.49 (4H, s, piperazine-4H), 3.19 (4H, s,
piperazine-4H), 3.75 (3H, s, OCH3), 3.93 (1H, m, cyclopropyl-H), 4.07
(2H, t, J ¼ 4.8 Hz, CH2 of linker), 4.36 (2H, t, J ¼ 4.8 Hz, CH2 of linker),
7.27e8.32 (5H, m, Ar-H). ESI-MS: m/z 568 (M þ H)þ. HRMS-ESI: m/z
Calcd. for C28H28F2N5O6 (M þ H)þ: 568.2007; Found 568.2030.
4.2.3. N-(2-Bromoethyl)-5-fluoroisatin (4b). The title compound
was obtained in a similar manner as for the preparation of 4a (53%)
as anacaratsolid, mp: 109e110 ꢀC. 1H NMR (CDCl3, 400 MHz) dppm
:
3.62 (2H, t, J ¼ 6.4 Hz, CH2CH2Br), 4.11 (2H, t, J ¼ 6.4 Hz, CH2CH2Br),
6.99 (1H, m, Ar-H), 7.26e7.35 (2H, m, Ar-H). ESI-MS: m/z 272
(M þ H)þ, 274 (Mþ2 þ H)þ.
4.2.5.3. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{4-[2-(b-methox-
4.2.4. 1-Cyclopropyl-6-fluoro-8-methoxy-7-[4-(2-isatinylethyl)
piperazin-1-yl]-1,4- dihydro-4-oxoquinoline-3-carboxylic acid (5a)
A suspension of 4a (5.08 g, 20 mmol), 8-OCH3 CPFX (7.22 g,
20 mmol), anhydrous K2CO3 (8.34 g, 60 mmol) in DMF (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
yiminoisatinyl) ethyl]piperazin-1-yl}-1,4-dihydro-4-oxoquinoline-3-
carboxylic acid (5e). Yield: 71%. mp: 156e159 ꢀC. 1H NMR (CDCl3,
400 MHz) dppm: 0.88 (2H, m, cyclopropyl-H),1.13 (2H, m, cyclopropyl-
H), 3.36 (4H, s, piperazine-4H), 3.67 (4H, s, piperazine-4H), 3.81 (3H,
s, OCH3), 3.88 (1H, m, cyclopropyl-H), 4.15 (2H, t, J ¼ 5.2 Hz, CH2 of
linker), 4.29 (3H, s, NOCH3), 4.53 (2H, t, J ¼ 5.2 Hz, CH2 of linker),
7.03e8.51 (6H, m, Ar-H). ESI-MS: m/z 564 (M þ H)þ. HRMS-ESI: m/z
Calcd. for C29H31FN5O6 (M þ H)þ: 564.2258; Found 564.2239.
dissolved in
4 N HCl (150 mL), and washed with CH2Cl2
(3 ꢁ 100 mL). The pH of the aqueous layer was adjusted to 7.0 with
10% NaOH solution and filtered. The solid was washed with water
and purified by column chromatography (silica gel) eluted with
CH2Cl2 and MeOH (v: v ¼ 20: 1) to give the title compound 5a
(4.81 g, 45%) as anacaratsolid, mp: 165e167 ꢀC. 1H NMR (CDCl3,
4.2.5.4. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{4-[2-(5-fluo-
rinisatinyl-
b
-
methoxyimino)ethyl]piperazin-1-yl}-1,4-dihydro-4-
oxoquinoline-3-carboxylic acid (5f). Yield: 65%. mp: 131e132 ꢀC. 1H
NMR (CDCl3, 400 MHz) dppm: 0.87 (2H, m, cyclopropyl-H), 1.01 (2H,
m, cyclopropyl-H), 2.94 (4H, s, piperazine-4H), 3.27 (4H, s,
400 MHz) dppm: 0.91 (2H, m, cyclopropyl-H), 1.15 (2H, m,

L.-S. Feng et al. / European Journal of Medicinal Chemistry 46 (2011) 341e348
347
piperazine-4H), 3.76 (3H, s, OCH3), 3.94 (1H, m, cyclopropyl-H),
4.06 (2H, t, J ¼ 4.8 Hz, CH2 of linker), 4.21 (3H, s, NOCH3), 4.35 (2H, t,
J ¼ 4.8 Hz, CH2 of linker), 7.32e8.32 (5H, m, Ar-H). ESI-MS: m/z 582
(M þ H)þ. HRMS-ESI: m/z Calcd. for C29H30F2N5O6 (M þ H)þ:
582.2164; Found 582.2148.
NMR (DMSO-d6, 400 MHz) dppm: 0.88 (2H, m, cyclopropyl-H), 1.06
(2H, m, cyclopropyl-H), 3.02 (4H, s, piperazine-4H), 3.31 (4H, s,
piperazine-4H), 3.78 (3H, s, OCH3), 3.99 (1H, m, cyclopropyl-H),
4.12 (2H, t, J ¼ 4.8 Hz, CH2 of linker), 4.36 (2H, t, J ¼ 4.8 Hz, CH2 of
linker), 7.26e8.38 (5H, m, Ar-H), 8.81, 9.14 (2H, 2s, D2O exchange-
able, CSNH2). FAB-MS: m/z 626 (M þ H)þ. HRMS-FAB: m/z Calcd. for
C29H30F2N7O5S (M þ H)þ: 626.1997; Found 626.2057.
4.2.5.5. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{4-[2-(b-ethox-
yiminoisatinyl) ethyl]piperazin-1-yl}-1,4-dihydro-4-oxoquinoline-3-
carboxylic acid (5g). Yield: 75%. mp: 161e165 ꢀC. 1H NMR (DMSO-
d6, 400 MHz) dppm: 0.87 (2H, m, cyclopropyl-H), 0.99e1.02 (2H, m,
cyclopropyl-H), 1.36 (3H, t, J ¼ 7.2 Hz, NOCH2CH3), 3.07 (4H, s,
piperazine-4H), 3.36 (4H, s, piperazine-4H), 3.78 (3H, s, OCH3), 3.94
(1H, m, cyclopropyl-H), 4.07 (2H, t, J ¼ 4.8 Hz, CH2 of linker), 4.36
(2H, t, J ¼ 4.8 Hz, CH2 of linker), 4.43 (2H, q, J ¼ 7.2 Hz, NOCH2CH3),
7.06e8.33 (6H, m, Ar-H). ESI-MS: m/z 578 (M þ H)þ. HRMS-ESI: m/z
Calcd. for C30H33FN5O6 (M þ H)þ: 578.2414; Found 578.2391.
4.3. MIC determination
All of the target compounds 3aek, 5ael along with 8-OCH3
CPFX, CPFX, RIF and INH were initially screened for their in vitro
activity against M. smegmatis CMCC 93202 using serial double
dilution technique in duplicate [20]. Bacteria were incubated in
20 mL L-broth at 37 ꢀC for 48 h. The culture was diluted to 150
mL,
5 ꢁ 105 colony-forming units per well in the 96-well microplates.
The tested compounds were dissolved in DMSO and two-fold
4.2.5.6. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{4-[2-(5-fluo-
diluted at the final concentrations of 200, 100, 50, //, 0.1 mg/mL,
rinisatinyl-
b-ethoxyimino) ethyl]piperazin-1-yl}-1,4-dihydro-4-oxo-
and then mixed with the culture. The plates were incubated at 37 ꢀC
for 72 h. The MIC was defined as the minimum concentration at
which the culture in the well was clear.
quinoline-3-carboxylic acid (5h). Yield: 61%. mp: 162e164 ꢀC. 1H
NMR (CDCl3, 400 MHz) dppm: 0.90 (2H, m, cyclopropyl-H), 1.13e1.17
(2H, m, cyclopropyl-H), 1.45 (3H, t, J ¼ 7.2 Hz, NOCH2CH3), 3.45 (4H,
s, piperazine-4H), 3.73 (4H, s, piperazine-4H), 3.82 (3H, s, OCH3),
3.89 (1H, m, cyclopropyl-H), 4.15 (2H, t, J ¼ 4.8 Hz, CH2 of linker),
4.53e4.58 (4H, m, CH2 of linker, NOCH2CH3), 7.07e8.52 (5H, m, Ar-
Compounds 3aek, 5e, 5f, 5k, 5l along with 8-OCH3 CPFX, CPFX,
RIF and INH were chosen for further evaluation of their in vitro
activity against MTB H37Rv ATCC 27294, and then compounds 3aeg
and 3iek against MDR-MTB 09710 using rapid direct susceptibility
test technique [20]. All the tested compounds were two-fold diluted
with the two-fold concentration medium (2 ꢁ medium, Advanced
Middlebrook 7H9 broth) at the final concentrations of 1.28, 0.64,
H). ESI-MS: m/z 596 (M
þ
H)þ. HRMS-ESI: m/z Calcd. for
C30H32F2N5O6 (M þ H)þ: 596.2320; Found 596.2309.
4.2.5.7. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{4-[2-(
b
-semi-
0.32, //, 0.005
64, //, 1 g/ml (for MDR-MTB 09710). The wells of a sterile 48-
well plate were filled with 100 two-fold diluted tested
compounds and 100 L MTB H37Rv ATCC 27294 or MDR-MTB 09710
mg/ml (for MTB H37Rv ATCC 27294) or 256, 128,
carbazoisatinyl) ethyl]piperazin-1-yl}-1,4-dihydro-4-oxoquinoline-3-
carboxylic acid (5i). Yield: 59%. mp: 220e225 ꢀC.1H NMR (DMSO-d6,
400 MHz) dppm: 0.86 (2H, m, cyclopropyl-H),1.01 (2H, m, cyclopropyl-
H), 3.20 (4H, s, piperazine-4H), 3.45 (4H, s, piperazine-4H), 3.78 (3H,
s, OCH3), 3.94 (1H, m, cyclopropyl-H), 4.09 (2H, s, CH2 of linker), 4.37
(2H, s, CH2 of linker), 6.98 (2H, brs, D2O exchangeable, NNHCONH2),
7.06e8.33 (6H, m, Ar-H), 9.58 (2H, brs, D2O exchangeable,
NNHCONH2, COOH). ESI-MS: m/z592 (Mþ H)þ. HRMS-ESI:m/zCalcd.
for C29H31FN7O6 (M þ H)þ: 592.2319; Found 592.2344.
m
mL
m
suspension containing 4 ꢁ 10ꢂ3 mg cells. Pure medium replaced the
diluted compounds in two wells as the positive control of growth,
and deionized water instead of the culture in other two wells as the
negative control of growth in the plates. The plates were covered
and sealed, then incubated at 37 ꢀC in a wet box. The positive and
negative control wells should show obvious difference after 3 days.
The MIC was determined by observing the quantity and state of the
cells in each test well by a continuous visual high magnification
system (Nanjing Shengguang Photoelectricity Medical Device Co.,
Ltd), and redetermined 7 days later. The MIC is defined as the
concentration of the compound required to give complete inhibi-
tion of bacterial growth. The two strains were obtained from Jiangsu
Province Hospital, Nanjing, China.
4.2.5.8. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{4-[2-(5-fluo-
rinisatinyl-b-semicarbazo) ethyl]piperazin-1-yl}-1,4-dihydro-4-oxo-
quinoline-3-carboxylic acid (5j). Yield: 53%. mp: 189e190 ꢀC. 1H
NMR (DMSO-d6, 400 MHz) dppm: 0.79e0.82 (2H, m, cyclopropyl-H),
1.00 (2H, m, cyclopropyl-H), 2.88 (4H, s, piperazine-4H), 3.33 (4H, s,
piperazine-4H), 3.74 (3H, s, OCH3), 3.93 (1H, m, cyclopropyl-H), 4.10
(2H, t, J ¼ 4.8 Hz, CH2 of linker), 4.36 (2H, t, J ¼ 4.8 Hz, CH2 of linker),
5.78 (2H, brs, D2O exchangeable, NNHCONH2), 6.88 (1H, brs, D2O
exchangeable, NNHCONH2), 7.17e8.31 (5H, m, Ar-H), FAB-MS: m/z
610 (M þ H)þ. HRMS-FAB: m/z Calcd. for C29H30F2N7O6 (M þ H)þ:
610.2226; Found 610.2246.
4.4. Cytotoxicity
Compounds 3aek, 5e, 5f, 5k, 5l were further examined for
toxicity (CC50) in a mammalian Vero cell line at concentrations from
1000 to 4
mg/mL. The Vero cells were maintained in culture
4.2.5.9. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{4-[2-(
b
-thio-
medium (Minimum Essential Medium with Earle’s salt, supple-
mented with 10% fetal bovine serum) at 37 ꢀC under 5% CO2. Cells
were seeded in 96-well plates at the plating density of 1 ꢁ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 calcu-
lated by Bliss analyses.
semicarbazoisatinyl) ethyl]piperazin-1-yl}-1,4-dihydro-4-oxoquino-
line-3-carboxylic acid (5k). Yield: 48%. mp: 160e165 ꢀC. 1H NMR
(DMSO-d6, 400 MHz) dppm: 0.85e0.89 (2H, m, cyclopropyl-H),
1.03e1.08 (2H, m, cyclopropyl-H), 2.88 (4H, s, piperazine-4H), 3.21
(4H, s, piperazine-4H), 3.75 (3H, s, OCH3), 3.99 (1H, m, cyclopropyl-
H), 4.13 (2H, t, J ¼ 4.8 Hz, CH2 of linker), 4.38 (2H, t, J ¼ 4.8 Hz, CH2 of
linker), 7.13e8.39 (6H, m, Ar-H), 8.73, 9.06 (2H, 2s, D2O exchange-
able, CSNH2). ESI-MS: m/z 608 (M þ H)þ. HRMS-ESI: m/z Calcd. for
C29H31FN7O5S (M þ H)þ: 608.2091; Found 608.2078.
Acknowledgment
4.2.5.10. 1-Cyclopropyl-6-fluoro-8-methoxy-7-{4-[2-(5-fluo-
This work was supported by the key project of Major infectious
disease (No. 2008ZX10003-006) and National S&T Major Special
Project on Major New Drug Innovation (No. 2009ZX09301-003).
rinisatinyl-
b-
thiosemicarbazo)ethyl]piperazin-1-yl}-1,4-dihydro-4-
oxoquinoline-3-carboxylic acid (5l). Yield: 64%. mp: 218e220 ꢀC. 1H

348
L.-S. Feng et al. / European Journal of Medicinal Chemistry 46 (2011) 341e348
[11] D. Sriram, P. Yogeeswari, J.S. Basha, D.R. Radha, V.J. Nagaraja, Bioorg. Med.
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