Ciprofloxacin–Isatin Hybrids and Their Antimycobacterial Activities

Article abstract of DOI:10.1002/jhet.3382

A series of diethylene glycol tethered ciprofloxacin–isatin hybrids 5a–j were designed, synthesized, and evaluated for their in vitro antimycobacterial activity against both drug-sensitive and multidrug-resistant (MDR) Mycobacterium tuberculosis strains i

Full text of DOI:10.1002/jhet.3382

Month 2018
Ciprofloxacin–Isatin Hybrids and Their Antimycobacterial Activities
a
Zhi Xu,
Shi-Jia Zhao,b Jia-Lun Deng,c Qin Wang,d and Zao-Sheng Lvb
*
aHuanghuai University Industry Innovation & Research and Development Institute of Zhumadian, Zhumadian 463000,
People’s Republic of China
bKey Laboratory of Hubei Province for Coal Conversion and New Carbon Materials, Wuhan University of Science and
Technology, Wuhan 430081, People’s Republic of China
cHaiso Technology Co., Ltd., Wuhan 430080, People’s Republic of China
dWuhan Changqing No. 1 High School, Wuhan 430024, People’s Republic of China
*E-mail: chemorgchem@126.com
Received July 5, 2018
DOI 10.1002/jhet.3382
Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com).
A series of diethylene glycol tethered ciprofloxacin–isatin hybrids 5a–j were designed, synthesized, and
evaluated for their in vitro antimycobacterial activity against both drug-sensitive and multidrug-resistant
(MDR) Mycobacterium tuberculosis strains in this paper. The preliminary results revealed that all hybrids
with greater lipophilicity than the parent ciprofloxacin displayed considerable activity against the tested
strains with minimum inhibitory concentration (MIC) in a range of 1.56–64 μg/mL. In particular, hybrid
5f (MIC: 1.56 and 2 μg/mL) with low cytotoxicity in VERO cell line was comparable with the parent cip-
rofloxacin (MIC: 0.78 and 2 μg/mL) against M. tuberculosis H37Rv and MDR tuberculosis strains and
≥16-fold more potent than isoniazid and rifampicin (MIC: >128 and 32 μg/mL, respectively) against
MDR tuberculosis, suggesting that it may serve as a new and promising candidate for further study.
J. Heterocyclic Chem., 00, 00 (2018).
INTRODUCTION
Furthermore, MTB isolates expressing resistance to both
INH and RIF are sensitive to fluoroquinlones generally
[5,6]. Consequently, great efforts have been made by
medicinal chemists to discover new fluoroquinolones as
potential anti-TB agents.
Some early fluoroquinlones such as ciprofloxacin
(CPFX, Fig. 1) have been recommended as the second-
line anti-TB agents by the World Health Organization for
the treatment of TB mainly in cases involving resistance
or intolerance to first-line anti-TB therapy [2]. CPFX
Tuberculosis (TB), which is caused mainly by
Mycobacterium tuberculosis (MTB), is still one of the
most common, communicable, and leading deadliest
diseases and kills around 5000 people every day [1]. The
first-line anti-TB agents such as isoniazid (INH),
rifampicin (RIF), pyrazinamide, and ethambutol are still
crucial therapeutics for the treatment of drug-susceptible
MTB-infected patients [2]. However, the evolution of
MTB new virulent forms such as drug-resistant TB,
multidrug-resistant TB (MDR-TB), and extremely drug-
resistant TB as well as MTB co-infection with HIV has
created a global epidemic problem [3,4]. Thus, there is an
urgent need to develop novel, fast-acting, and more
effective anti-TB drugs.
exhibits
notable
anti-TB
activity,
excellent
pharmacokinetic properties, and fewer side effects, so
CPFX derivatives have kept drawing continuous
attentions from the health-care community over these
years [7]. The previous research indicated that the
lipophilicity of fluoroquinlones plays an important role in
the penetration of these compounds into bacterial cells,
and simply incensement of the lipophilicity may increase
the anti-TB activity accordingly [8,9]. Isatin is a flexible
lead molecule, and its derivatives were reported to
Fluoroquinolones are emerged as a family of synthetic
broad spectrum antibiotics with extensive indications for
infections, and some fuloroquinolones demonstrated
excellent in vitro and in vivo anti-TB activity [3,4].
© 2018 Wiley Periodicals, Inc.

Z. Xu, S.-J. Zhao, J.-L. Deng, Q. Wang, and Z.-S. Lv
Vol 000
Figure 1. Chemical structures of isoniazid, rifampicin, ciprofloxacin, and isatin.
possess a variety of biological properties such as
antibacterial, antimalarial, anticancer, and in vitro and
in vivo anti-TB activities, and several isatin-based
compounds that are exemplified by sunitinib, nintedanib,
and semaxanib have already been used in clinics [10].
Introduction of isatin in to C-7 position of
fluoroquinolones could improve the lipophilic character
and consequently boost up the anti-TB activity [10], and
some CPFX–isatin hybrids exhibited excellent anti-TB
activities [11]. The structure–activity relationship
suggested that the linker between the fluoroquinolones
and isatin played a pivotal role in the exertion of the anti-
TB activity and the linker that could exert noncovalent
bind interactions benefited the activity [12].
As a part of an ongoing program to discover new anti-
TB candidates, we recently developed various
fluoroquinlone–isatin hybrids with different linkers
including methylene, ethylene, acetyl, and 1,2,3-triazole,
and some of them displayed promising inhibitory activity
against MTB H37Rv and MDR-TB [13–18]. Inspired by
the aforementioned research results, a series of diethylene
glycol tethered CPFX–isatin hybrids were designed,
synthesized, and evaluated for their in vitro
antimycobacterial activity against MTB H37Rv and
MDR-TB because diethylene glycol fragment has the
potential to exert various noncovalent interactions, which
may facilitate binding with active site. Our primary
objective was to identify optimized linker between CPFX
and isatin to facilitate the further development. The
illustration of the design strategy is depicted in Figure 2.
Figure 2. The design strategy of diethylene glycol tethered ciprofloxa-
cin–isatin hybrids. TB, tuberculosis. [Color figure can be viewed at
wileyonlinelibrary.com]
Compared with the parent CPFX (Log P: 1.32), all
diethylene glycol tethered CPFX–isatin hybrids 5a–j
(Log P: 2.03–3.01) showed higher lipophilicity, and this
character may improve their permeation properties toward
mycobacterial cell membrane. All hybrids 5a–j together
with the references CPFX, INH, and RIF were examined
for their in vitro antimycobacterial activities against MTB
H37Rv and MDR-TB (resistant to INH, RIF, and
ethambutol) strains, and the results were presented in
Table 1. The minimum inhibitory concentration (MIC) is
defined as the lowest concentration that inhibits the
visible bacterial growth.
As shown in Table 1, all diethylene glycol tethered
CPFX–isatin hybrids 5a–j displayed considerable activity
against both drug-sensitive and MDR-MTB strains with
MIC in a range of 1.56–64 μg/mL but were less active
than the references CPFX, INH, and RIF (MIC: 0.78,
0.05, and 0.39 μg/mL, respectively) against drug-
sensitive MTB H37Rv. Compared with the parent CPFX,
the antimycobacterial activity of the CPFX–isatin hybrids
5a–j did not increase with the incensement of the
lipophilicity, suggesting that the lipophilicity was not the
sole factor affected the antimycobacterial activity [8,9].
The structure–activity relationship revealed that the
antimycobacterial activity of the synthesized hybrids was
influenced greatly by substituents on C-3 and C-5
positions of isatin moiety, which was in accordance with
RESULTS AND DISCUSSION
Detailed synthetic route for diethylene glycol tethered
CPFX–isatin hybrids 5a–j was described in Scheme 1.
Treatment of diethylene glycol 1 with tosyl chloride in
presence of triethylamine yielded intermediate 2, which
was then alkylated with C-5 substituted isatins 3 provided
the key intermediates 4a–c. Introduction of 4a–c into
CPFX gave the desired CPFX–isatin hybrids 5a–c,
which was consequently condensation with the
corresponding hydroxylamine hydrochlorides gave the
other hybrids 5d–j.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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Ciprofloxacin–Isatin Hybrids and Their Antimycobacterial Activities
Scheme 1. Synthetic route for diethylene glycol tethered CPFX–isatin hybrids 5a–j.
Table 1
Structures, lipophilicity, and in vitro antimycobacterial activity of diethylene glycol tethered CPFX–isatin hybrids 5a-j.
MIC (μg/mL)
b
CC50
Compd.
R1
R2
Log Pa
MTB H37Rv
MDR-TB
(μg/mL)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
CPFX
INH
RIF
O
O
O
NOH
NOH
H
Me
F
H
Me
F
H
Me
F
1.88
2.36
2.03
2.26
2.75
2.42
2.53
3.01
2.69
2.87
1.32
À0.67
3.71
12.5
25
16
16
8
8
16
2
32
64
8
64
2
512
216
64
128
512
128
256
256
128
64
12.5
6.25
12.5
1.56
50
50
25
50
NOH
NOMe
NOMe
NOMe
NOEt
H
0.78
0.05
0.39
128
128
512
>128
32
CPFX, ciprofloxacin; INH, isoniazid; MDR-TB, multidrug-resistant tuberculosis; MIC, minimum inhibitory concentration; MTB, Mycobacterium tu-
berculosis; RIF, rifampicin.
aThe Log P is calculated with ChemOffice 2012 software.
bCC50: The 50% cytotoxic concentration in a mammalian VERO cell line.
our previous studies [13–18]. For C-3 position, the relative
contribution of imines of the Schiff’s bases to the activity
was –NOH > –O > –NOMe > –NOEt, indicating that
hydrogen bond donor favored the activity; introduction of
electron-donating –Me could not improve the activity,
while electron-withdrawing –F was favorable to the
activity generally. In particular, hybrid 5f (MIC: 1.56 and
2 μg/mL), which was found to be most active against
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Z. Xu, S.-J. Zhao, J.-L. Deng, Q. Wang, and Z.-S. Lv
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MTB H37Rv and MDR-TB, was comparable with the parent
CPFX (MIC: 0.78 and 2 μg/mL) against the tested two
strains and ≥16-fold more potent than INH and RIF
(MIC: >128 and 32 μg/mL, respectively) against MDR-TB.
The diethylene glycol tethered CPFX–isatin hybrids 5a–
j were subsequently examined for toxicity (CC50) in a
mammalian VERO cell line by MTT assay [9], and the
results were reported in Table 1. All CPFX–isatin hybrids
(CC50: 64–512 μg/mL) showed excellent cytotoxic
profiles, and the majority of them were less toxic than the
parent CPFX (CC50: 128 μg/mL). The most active
hybrid 5f (CC50: 128 μg/mL) also displayed acceptable
cytotoxic profile, could act as a lead for further
optimization.
1-Cyclopropyl-7-(4-(2-(2-(2,3-dioxoindolin-1-yl)ethoxy)ethyl)
piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
1
carboxylic acid (5a). Yellow solid, yield: 26%. H NMR
(400 MHz, DMSO-d6)
δ
1.10–1.24 (4H, m,
2 × cyclopropyl-CH2), 2.70 (2H, t, –NCH2), 2.92 (4H, s,
piperazine-4H), 3.18 (4H, s, piperazine-4H), 3.64–3.76
(3H, m, cyclopropyl-CH and –OCH2–), 3.86 (2H, t,
–OCH2), 4.20 (2H, t, –OCH2), 7.06 (1H, t, Ar–H), 7.32
(1H, d, Ar–H), 7.43–7.48 (2H, m, Ar–H), 7.63 (1H, t,
Ar–H), 7.76 (1H, d, Ar–H), 8.47 (1H, s, C2-H). ESI-MS
m/z: 549 [M + H]+. Elemental Anal. Calcd (%) for
C29H29FN4O6: C, 63.50; H, 5.33; N, 10.21; found: C,
63.31; H, 5.14; N, 10.07.
1-Cyclopropyl-6-fluoro-7-(4-(2-(2-(5-methyl-2,3-
dioxoindolin-1-yl)ethoxy)ethyl)piperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (5b).
Light yellow
solid, yield: 27%. 1H NMR (400 MHz, DMSO-d6) δ
1.12–1.26 (4H, m, 2 × cyclopropyl-CH2), 2.22 (3H, s,
–CH3), 2.68 (2H, t, –NCH2), 2.98 (4H, s, piperazine-4H),
3.20 (4H, s, piperazine-4H), 3.66–3.75 (3H, m,
cyclopropyl-CH and –OCH2–), 3.86 (2H, t, –OCH2),
4.19 (2H, t, –OCH2), 7.21 (1H, d, Ar–H), 7.29 (1H, s,
Ar–H), 7.41 (2H, t, Ar–H), 7.75 (1H, d, Ar–H), 8.42
(1H, s, C2-H). ESI-MS m/z: 563 [M + H]+. Elemental
Anal. Calcd (%) for C30H31FN4O6: C, 64.05; H, 5.55; N,
CONCLUSIONS
In conclusion, a series of diethylene glycol tethered
CPFX–isatin hybrids were designed and synthesized as
new anti-TB candidates. All the synthesized hybrids
exhibited promising activity against both drug-sensitive
and MDR-MTB strains as well as low cytotoxicity in
VERO cell line. In particular, hybrid 5f not only showed
great potency against the tested two strains (MIC: 1.56
and 2 μg/mL) but also displayed low cytotoxicity (CC50:
128 μg/mL), worth to be further optimization.
9.96; found: C, 63.87; H, 5.29; N, 9.83.
1-Cyclopropyl-6-fluoro-7-(4-(2-(2-(5-fluoro-2,3-
dioxoindolin-1-yl)ethoxy)ethyl)piperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (5c).
Light yellow
solid, yield: 21%. 1H NMR (400 MHz, DMSO-d6) δ
1.13–1.26 (4H, m, 2 × cyclopropyl-CH2), 2.70 (2H, t,
–NCH2), 2.96 (4H, s, piperazine-4H), 3.16 (4H, s,
piperazine-4H), 3.66–3.76 (3H, m, cyclopropyl-CH and
–OCH2–), 3.87 (2H, t, –OCH2), 4.20 (2H, t, –OCH2),
7.16 (1H, t, Ar–H), 7.29 (1H, t, Ar–H), 7.39 (1H, d,
Ar–H), 7.61 (1H, d, Ar–H), 7.73 (1H, d, Ar–H), 8.48
(1H, s, C2-H). ESI-MS m/z: 567 [M + H]+. Elemental
Anal. Calcd (%) for C29H28F2N4O6: C, 61.48; H, 4.98;
N, 9.89; found: C, 61.31; H, 4.79; N, 9.73.
The general procedure for preparing targets 5d–j. To a
solution of substituted amine hydrochlorides (6 mmol)
and sodium bicarbonate (6 mmol) dissolved in water
(10 mL) and methanol (10 mL) was added CPFX–isatin
hybrids 5a–c (5 mmol). The reaction mixture was stirred
at 50°C for 12 h. After removal of the solvent, the
residue 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 DCM to vDCM:vMeOH = 5:1 to give the title hybrids
EXPERIMENTAL SECTION
Synthesis. The general procedure for preparing hybrids
5a–c. To a mixture of diethylene glycol 1 (100 mmol)
and triethylamine (500 mmol) in DCM (1 L), tosyl
chloride (300 mmol) was added. The mixture was stirred
at room temperature overnight. The mixture was
concentrated under reduced pressure, and the residue was
purified by silica gel chromatography eluted with PE:
EA = 1:2 to give the desired product 2 (yield: 72%). The
mixture of intermediate
2 (1.5 mmol), potassium
carbonate (5 mmol), and isatins 3 (1 mmol) in DMF
(30 mL) was stirred at room temperature overnight. After
filtration, the filtrate was concentrated under reduced
pressure. The residue was purified by silica gel
chromatography eluted with PE:EA = 1:2 to give the
intermediates 4a–c (yields: 47%, 76%, and 63%,
respectively).
A
mixture of CPFX (1 mmol),
intermediates 4a–c (1 mmol), and potassium carbonate
(10 mmol) in DMF (10 mL) was stirred at room
temperature for 2 days. After filtration, the filtrate was
concentrated under reduced pressure. The residue was
purified by silica gel chromatography eluted with DCM:
MeOH = 5:1 to give the desired diethylene glycol
tethered CPFX–isatin hybrids 5a–c (yield: 21–27%).
5d–j (yield: 26–49%).
1-Cyclopropyl-6-fluoro-7-(4-(2-(2-(3-(hydroxyimino)-2-
oxoindolin-1-yl)ethoxy)ethyl)piperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (5d). Light yellow solid,
1
yield: 45%. H NMR (400 MHz, DMSO-d6) δ 1.12–1.24
(4H, m, 2 × cyclopropyl-CH2), 2.64 (2H, t, –NCH2), 2.94
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Ciprofloxacin–Isatin Hybrids and Their Antimycobacterial Activities
(4H, s, piperazine-4H), 3.18 (4H, s, piperazine-4H),
3.65–3.76 (3H, m, cyclopropyl-CH and –OCH2–), 3.88
(2H, t, –OCH2), 4.20 (2H, t, –OCH2), 7.09 (1H, t, Ar–H),
7.29 (1H, d, Ar–H), 7.41–7.48 (2H, m, Ar–H), 7.61 (1H,
t, Ar–H), 7.78 (1H, d, Ar–H), 8.44 (1H, s, C2-H), 13.46
(1H, brs, NOH). ESI-MS m/z: 564 [M + H]+. Elemental
Anal. Calcd (%) for C29H30FN5O6: C, 61.80; H, 5.37; N,
3.18 (4H, s, piperazine-4H), 3.70–3.78 (3H, m,
cyclopropyl-CH and –OCH2–), 3.86 (2H, t, –OCH2), 4.13
(3H, s, NOCH3), 4.19 (2H, t, –OCH2), 7.17 (1H, d,
Ar–H), 7.25 (1H, s, Ar–H), 7.38 (2H, t, Ar–H), 7.73 (1H,
d, Ar–H), 8.44 (1H, s, C2-H). ESI-MS m/z: 592 [M + H]+.
Elemental Anal. Calcd (%) for C31H34FN5O6: C, 62.93;
H, 5.79; N, 11.84; found: C, 62.76; H, 5.55; N, 11.67.
1-Cyclopropyl-6-fluoro-7-(4-(2-(2-(5-fluoro-3-
(methoxyimino)-2-oxoindolin-1-yl)ethoxy)ethyl)piperazin-1-yl)-
12.43; found: C, 61.69; H, 5.14; N, 13.21.
1-Cyclopropyl-6-fluoro-7-(4-(2-(2-(3-(hydroxyimino)-5-
methyl-2-oxoindolin-1-yl)ethoxy)ethyl)piperazin-1-yl)-4-oxo-
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5i).
Light
yellow solid, yield: 49%. 1H NMR (400 MHz,
DMSO-d6) δ 1.14–1.26 (4H, m, 2 × cyclopropyl-CH2),
2.71 (2H, t, –NCH2), 2.98 (4H, s, piperazine-4H), 3.26
(4H, s, piperazine-4H), 3.68–3.76 (3H, m, cyclopropyl-
CH and –OCH2–), 3.88 (2H, t, –OCH2), 4.20 (2H, t,
–OCH2), 7.19 (1H, t, Ar–H), 7.31 (1H, t, Ar–H), 7.39
(1H, d, Ar–H), 7.61 (1H, d, Ar–H), 7.72 (1H, d, Ar–H),
8.46 (1H, s, C2-H). ESI-MS m/z: 596 [M + H]+.
Elemental Anal. Calcd (%) for C30H31F2N5O6: C, 60.50;
1,4-dihydroquinoline-3-carboxylic acid (5e).
Light yellow
solid, yield: 39%. 1H NMR (400 MHz, DMSO-d6) δ
1.13–1.26 (4H, m, 2 × cyclopropyl-CH2), 2.22 (3H, s,
–CH3), 2.66 (2H, t, –NCH2), 2.98 (4H, s, piperazine-4H),
3.20 (4H, s, piperazine-4H), 3.65–3.74 (3H, m,
cyclopropyl-CH and –OCH2–), 3.84 (2H, t, –OCH2), 4.20
(2H, t, –OCH2), 7.24 (1H, d, Ar–H), 7.29 (1H, s, Ar–H),
7.40 (2H, t, Ar–H), 7.71 (1H, d, Ar–H), 8.43 (1H, s, C2-
H), 13.48 (1H, brs, NOH). ESI-MS m/z: 578 [M + H]+.
Elemental Anal. Calcd (%) for C30H32FN5O6: C, 62.38;
H, 5.25; N, 11.76; found: C, 60.35; H, 5.03; N, 11.59.
1-Cyclopropyl-7-(4-(2-(2-(3-(ethoxyimino)-2-oxoindolin-1-yl)
ethoxy)ethyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-
H, 5.58; N, 12.12; found: C, 62.17; H, 5.33; N, 11.95.
1-Cyclopropyl-6-fluoro-7-(4-(2-(2-(5-fluoro-3-
(hydroxyimino)-2-oxoindolin-1-yl)ethoxy)ethyl)piperazin-1-yl)-
dihydroquinoline-3-carboxylic acid (5j). Light yellow solid,
1
yield: 41%. H NMR (400 MHz, DMSO-d6) δ 1.12–1.24
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5f).
Light
yellow solid, yield: 31%. 1H NMR (400 MHz, DMSO-d6)
δ 1.13–1.25 (4H, m, 2 × cyclopropyl-CH2), 2.72 (2H, t,
–NCH2), 2.97 (4H, s, piperazine-4H), 3.22 (4H, s,
piperazine-4H), 3.68–3.78 (3H, m, cyclopropyl-CH and
–OCH2–), 3.89 (2H, t, –OCH2), 4.20 (2H, t, –OCH2),
7.18 (1H, t, Ar–H), 7.29 (1H, t, Ar–H), 7.36 (1H, d,
Ar–H), 7.66 (1H, d, Ar–H), 7.75 (1H, d, Ar–H), 8.46 (1H,
s, C2-H), 13.44 (1H, brs, NOH). ESI-MS m/z: 626
[M + H]+. Elemental Anal. Calcd (%) for C29H29F2N5O6:
C, 59.89; H, 5.03; N, 12.04; found: C, 59.67; H, 4.89;
(4H, m, 2 × cyclopropyl-CH2), 1.32–1.36 (3H, m,
NOCH2CH3), 2.66 (2H, t, –NCH2), 2.98 (4H, s,
piperazine-4H), 3.20 (4H, s, piperazine-4H), 3.68–3.78
(3H, m, cyclopropyl-CH and –OCH2–), 3.87 (2H, t,
–OCH2), 4.20 (2H, t, –OCH2), 4.40–4.44 (2H, m,
NOCH2CH3), 7.03 (1H, t, Ar–H), 7.22 (1H, d, Ar–H),
7.40–7.46 (2H, m, Ar–H), 7.58 (1H, t, Ar–H), 7.72 (1H,
d, Ar–H), 8.46 (1H, s, C2-H). ESI-MS m/z: 592 [M + H]+.
Elemental Anal. Calcd (%) for C31H34FN5O6: C, 62.93;
H, 5.79; N, 11.84; found: C, 62.79; H, 5.56; N, 11.67.
N, 11.77.
Minimum inhibitory concentration determination. The
CPFX–isatin hybrids 5a–j along with the references
CPFX, RIF, and INH were screened for their in vitro
antimycobacterial activities against MTB H37Rv and
MDR-MTB by rapid direct susceptibility test technique
[17,18]. The wells of a sterile 48-well plate were filled
with 100-mL twofold diluted tested compounds and
100-mL MTB H37Rv or MDR-MTB suspension
containing 4 × 10À3 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 and 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 and redetermined 7 days later. The
MIC is defined as the concentration of the compound
required to give 90% inhibition of bacterial growth.
1-Cyclopropyl-6-fluoro-7-(4-(2-(2-(3-(methoxyimino)-2-
oxoindolin-1-yl)ethoxy)ethyl)piperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (5g).
Light yellow
solid, yield: 26%. 1H NMR (400 MHz, DMSO-d6) δ
1.08–1.20 (4H, m, 2 × cyclopropyl-CH2), 2.64 (2H, t,
–NCH2), 2.94 (4H, s, piperazine-4H), 3.18 (4H, s,
piperazine-4H), 3.70–3.78 (3H, m, cyclopropyl-CH and
–OCH2–), 3.88 (2H, t, –OCH2), 4.12 (3H, s, NOCH3),
4.20 (2H, t, –OCH2), 7.04 (1H, t, Ar–H), 7.28 (1H, d,
Ar–H), 7.40–7.45 (2H, m, Ar–H), 7.60 (1H, t, Ar–H),
7.76 (1H, d, Ar–H), 8.45 (1H, s, C2-H). ESI-MS m/z:
578 [M
+
H]+. Elemental Anal. Calcd (%) for
C30H32FN5O6: C, 62.38; H, 5.58; N, 12.12; found: C,
62.16; H, 5.41; N, 11.99.
1-Cyclopropyl-6-fluoro-7-(4-(2-(2-(3-(methoxyimino)-5-
methyl-2-oxoindolin-1-yl)ethoxy)ethyl)piperazin-1-yl)-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (5h). Light yellow
solid, yield: 41%. 1H NMR (400 MHz, DMSO-d6) δ
1.11–1.26 (4H, m, 2 × cyclopropyl-CH2), 2.22 (3H, s,
–CH3), 2.70 (2H, t, –NCH2), 2.98 (4H, s, piperazine-4H),
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Z. Xu, S.-J. Zhao, J.-L. Deng, Q. Wang, and Z.-S. Lv
Vol 000
[5] Zhang, G. F.; Zhang, S.; Pan, B. F.; Liu, X. F.; Feng, L. S. Eur
J Med Chem 2018, 143, 710.
[6] Hu, Y. Q.; Zhang, S.; Xu, Z.; Lv, Z. S.; Liu, M. L.; Feng, L. S.
Eur J Med Chem 2017, 141, 335.
[7] Zhang, G. F.; Liu, X. F.; Zhang, S.; Pan, B. F.; Liu, M. L. Eur J
Med Chem 2018, 146, 599.
[8] Sriram, D.; Aubry, A.; Yogeeswari, P.; Fisher, L. M. Bioorg
Med Chem Lett 2006, 16, 2982.
[9] Xu, Z.; Song, X. F.; Hu, Y. Q.; Qiang, M.; Lv, Z. S. Eur J Med
Chem 2017, 138, 66.
[10] Xu, Z.; Zhang, S.; Gao, C.; Zhao, F.; Lv, Z. S.; Feng, L. S.
Chin Chem Lett 2017, 28, 159.
[11] Sriram, D.; Yogeeswari, P.; Basha, J. S.; Radha, D. R.;
Nagaraja, V. Bioorg Med Chem 2005, 13, 5774.
[12] Jiang, D.; Wang, G. Q.; Liu, X. F.; Zhang, Z. B.; Feng, L. S.;
Liu, M. L. J Heterocyclic Chem 2018, 55, 1263.
[13] Feng, L. S.; Liu, M. L.; Wang, B.; Chai, Y.; Guo, H. Y. Eur J
Med Chem 2010, 45, 3407.
Cytotoxicity. The CPFX–isatin hybrids 5a–j together
with the references CPFX, RIF, and INH were further
assessed for their toxicity in VERO cell line dissolved in
DMSO at concentrations from 1024 to 1 μg/mL. The
VERO cells were maintained in culture medium 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 media were replaced by assay
medium containing the tested hybrid or drug free. After
72 h of exposure, cells were harvested, and cell viability
was assessed by MTT assay. The CC50 was calculated by
Bliss analyses.
[14] Feng, L. S.; Liu, M. L.; Zhang, S.; Chai, Y.; Guo, H. Y.; Xiao,
C. L. Eur J Med Chem 2011, 46, 341.
REFERENCES AND NOTES
[15] Xu, Z.; Lv, Z. S.; Song, X. F.; Qiang, M. J Heterocyclic Chem
2018, 55, 97.
[1] Zhang, S.; Xu, Z.; Gao, C.; Ren, Q. C.; Chang, L.; Lv, Z. S.;
Feng, L. S. Eur J Med Chem 2017, 138, 501.
[16] Xu, Z.; Song, X. F.; Fan, J.; Lv, Z. S. J Heterocyclic Chem
2018, 55, 77.
[17] Hu, Y. Q.; Xu, Z.; Qiang, M.; Lv, Z. S. J Heterocyclic Chem
2018, 55, 187.
[2] Guidelines for the Management of Drug-Resistant Tuberculo-
sis WHO/TB/96-210 (Rev.1); World Health Organization: Geneva, 1997.
[3] Fan, Y. L.; Wu, J. B.; Cheng, X. W.; Zhang, F. Z.; Feng, L. S.
Eur J Med Chem 2018, 146, 554.
[18] Xu, Z.; Song, X. F.; Qiang, M.; Lv, Z. S. J Heterocyclic Chem
2017, 54, 3735.
[4] Liu, B.; Li, F.; Zhou, T.; Tang, X. Q.; Hu, G. W. J Heterocy-
clic Chem 2018, 55, 1863.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet