Ciprofloxacin-isatin-1H-1,2,3-triazole Hybrids: Design, Synthesis, and in vitro Anti-tubercular Activity against M.?tuberculosis

Article abstract of DOI:10.1002/jhet.3010

A new set of ciprofloxacin (CPFX)-isatin-1H-1,2,3-triazole hybrids 6a–l with greater lipophilicity compared with the parent CPFX was designed, synthesized, and assessed for their in vitro anti-mycobacterial activity against Mycobacterium tuberculosis (MTB) H₃₇Rv as well as cytotoxicity in VERO cell line. The preliminary results showed that all hybrids (MIC: 0.39–50?μg/mL) exhibited promising activities against MTB H₃₇Rv, and six of them (MIC: 0.39–1.56?μg/mL) were more active than the parent CPFX (MIC: 3.12?μg/mL). In particular, the most active conjugate 6h (MIC: 0.39?μg/mL) was comparable with RIF (MIC: 0.39?μg/mL), and eight times more potent than CPFX. All conjugates (CC₅₀: 4–64?μg/mL) were more toxic than the parent (CC₅₀: 128?μg/mL) in VERO cell lines, and the most active hybrids, which also displayed the highest cytotoxicity, should be further optimized.

Full text of DOI:10.1002/jhet.3010

Month 2017
Ciprofloxacin-isatin-1H-1,2,3-triazole Hybrids: Design, Synthesis, and
in vitro Anti-tubercular Activity against M. tuberculosis
a
b
a
Zhi Xu,a
*
Zao-Sheng Lv, Xu-Feng Song, and Min Qiang
aKey Laboratory of Hubei Province for Coal Conversion and New Carbon Materials, Wuhan University of Science and
Technology, Hubei, People’s Republic of China
bBeijing University of Technology, Beijing 100124, People’s Republic of China
*
E-mail: chemmedchem@163.com
Received June 14, 2017
DOI 10.1002/jhet.3010
Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com).
A new set of ciprofloxacin (CPFX)-isatin-1H-1,2,3-triazole hybrids 6a–l with greater lipophilicity
compared with the parent CPFX was designed, synthesized, and assessed for their in vitro
anti-mycobacterial activity against Mycobacterium tuberculosis (MTB) H37Rv as well as cytotoxicity in
VERO cell line. The preliminary results showed that all hybrids (MIC: 0.39–50 μg/mL) exhibited
promising activities against MTB H37Rv, and six of them (MIC: 0.39–1.56 μg/mL) were more active
than the parent CPFX (MIC: 3.12 μg/mL). In particular, the most active conjugate 6h (MIC: 0.39 μg/
mL) was comparable with RIF (MIC: 0.39 μg/mL), and eight times more potent than CPFX. All
conjugates (CC50: 4–64 μg/mL) were more toxic than the parent (CC50: 128 μg/mL) in VERO cell
lines, and the most active hybrids, which also displayed the highest cytotoxicity, should be further
optimized.
J. Heterocyclic Chem., 00, 00 (2017).
INTRODUCTION
recommended as the second-line agents for the treatment
of TB by WHO [2] (Fig. 1).
Tuberculosis (TB) threats human being for thousands of
years and remains one of the leading causes of deaths,
which results in more than 1 million deaths annually
according to the World Health Organization (WHO)
report [1,2]. The spread of drug-resistant TB including
multidrug-resistant TB, extensively drug-resistant TB,
and totally drug-resistant throughout the world as well as
TB co-infected with HIV made the TB prevention and
control situation even worse. Therefore, it is imperative
to develop new anti-TB agents.
The isatin derivatives exhibited diverse biological
profiles such as anti-tumor, anti-bacterial, anti-malarial,
and anti-TB activities [3]. Meanwhile, fluoroquinolones
(FQs) demonstrated potential anti-TB activity, and some
of them such as ciprofloxacin (CPFX) are currently
On the other hand, the extreme complication and
hydrophobic cell envelop of mycobacteria is the main
reason that prevents many drugs from penetrating into the
bacteria, thus simply increasing the lipophilic character of
FQs may also increase the anti-TB activity [3–5]. To
verify the aforementioned hypothesis, plenty of FQs
derivatives with remarkable improvement in lipophilicity
have been screened for their anti-mycobacterial activities
[4–16]. All of them displayed considerable activity, and
CPFX-isatin hybrid 1 was found very effective both
in vitro and in vivo, which warrant further investigation
[5]. Moreover, I-A09, which contains a 1,2,3-triazole
motif, is in clinical evaluations currently and may be
used to treat TB infection in the near future [17].
Therefore, incorporation of the 1,2,3-triazole and isatin
© 2017 Wiley Periodicals, Inc.

Z. Xu, Z.-S. Lv, X.-F. Song, and M. Qiang
Vol 000
Figure 1. Chemical structures of CPFX and CPFX-isatin conjugate 1.
moieties into the FQs may give more active candidates
(Fig. 2).
formed CPFX-isatin-1H-1,2,3-triazole hybrids 6a–l
(18–41%) [11].
Inspired by the aforementioned research results and as a
part of an ongoing program to optimize FQs against
mycobacteria, a new class of CPFX-isatin-1H-1,2,3-
triazole hybrids 6a–l with greater lipophilicity compared
with the parent CPFX was designed, synthesized,
and evaluated for their in vitro anti-mycobacterial
activity against Mycobacterium tuberculosis (MTB)
H37Rv as well as cytotoxicity in VERO cell line in this
study.
All CPFX-isatin-1H-1,2,3-triazole hybrids 6a–l (log P:
1.36–3.01) with greater lipophilicity compared with the
parent CPFX (log P: 1.32), so their capacity of
penetration of various biomembrane may be prompted,
consequently
increasing
their
anti-mycobacterial
activities. The hybrids 6a–h were screened for their
in vitro anti-mycobacterial activities against MTB H37Rv
strains [14]. The minimum inhibitory concentration
(MIC) is defined as the minimum concentration of
compound required to give 90% inhibition of bacterial
growth, and MICs of the compounds are reported in
Table 1. All hybrids showed promising in vitro activities
against MTB H37Rv strains with MIC in a range of 0.39
to 50 μg/mL, but less active than INH (MIC: 0.05 μg/
mL). Seven of them (MIC: 0.39–3.12 μg/mL) were
comparable with or more active than the parent CPFX
(MIC: 3.12 μg/mL), and two conjugates 6h and 6l (MIC:
0.39 and 0.78 μg/mL) were eight and four times more
potent than CPFX, respectively. In particular, the most
active 6h was as potent as RIF (MIC: 0.39 μg/mL). The
SAR revealed that the relative contribution of imines of
the Schiff’s bases to anti-mycobacterial activity is as
RESULTS AND DISCUSSION
The synthetic route for the desired CPFX-isatin-1H-
1,2,3-triazole hybrids 6a–l is depicted in Scheme 1. N-(2-
Bromoethyl)isatins 2a–d (yield: 51–67%) and propargyl
CPFX 4 (yield: 42%) via literature methods [18,19] by
alkylation of the corresponding isatins and CPFX with
1,2-dibromoethane and propargyl bromide in the presence
of potassium carbonate. Treatment of N-(2-bromoethyl)
isatins 2a–d with sodium azide at 60°C provided the
desired azido precursors 3a–d [20], which was used
along with 4 to synthesize 5a–d (yield: 31–46%) via
Cu-promoted azide-alkyne cycloaddition reaction in the
presence of Cu(OAc)2 in DMF [14]. Finally,
condensations of targets 5a–d with various amine
hydrochloride in the presence of sodium bicarbonate
follows: thiosemicarbazone/semicarbazone
>
oxime;
hybrids with the electron-withdrawing –F at C-5 position
of isatin motif boost up the activity, while
electron-donating group –OMe decreased the activity
generally, indicating further modification should focus on
incorporation of various electron-withdrawing groups
like –NO2.
The hybrids 6a–l were subsequently examined for
toxicity (CC50) in a mammalian VERO cell line [11].
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 also reported in Table 1. All
hybrids (CC50: 4–64 μg/mL) were much more toxic than
the parent CPFX (CC50: 128 μg/mL). Unfortunately, the
cytotoxicity of the most potency 6h (CC50: 4 μg/mL) is
much more than the other hybrids. General speaking, the
relative contribution of imines of the Schiff’s bases to the
cytotoxicity
is
as
follows:
thiosemicarbazone/
Figure 2. Illustration of the design strategy for the hybrids. [Color figure
can be viewed in the online issue, which is available at wileyonlinelibrary.
com.]
semicarbazone > oxime, which was in accordance with
the activity; 5-F > 5-H ≈ 5-OCH3 > 5-CH3.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Ciprofloxacin-isatin-1H-1,2,3-triazole Hybrids: Design, Synthesis, and in vitro
Anti-tubercular Activity against M. tuberculosis
Scheme 1. Synthetic route for hybrids 6a–l.
Table 1
Structures, lipophilicity, anti-mycobacterial activity, and cytotoxicity of hybrids 6a–l.
a
Compound
R1
R2
Log P
MIC (μg/mL)
CC50 b(μg/mL)
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
Me
OMe
F
H
Me
OMe
F
NOH
NOH
NOH
NOH
NNHCONH2
NNHCONH2
NNHCONH2
NNHCONH2
NNHCSNH2
NNHCSNH2
NNHCSNH2
NNHCSNH2
2.52
3.01
2.39
2.68
1.49
1.97
1.36
1.65
2.05
2.53
1.92
2.21
50
12.5
50
64
128
64
16
32
16
32
4
16
32
16
4
12.5
1.56
1.56
6.25
0.39
3.12
1.56
1.56
0.78
H
6j
6k
6l
Me
OMe
F
(Continues)
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Z. Xu, Z.-S. Lv, X.-F. Song, and M. Qiang
Vol 000
Table 1
(Continued)
CPFX
INH
RIF
1.32
À0.67
3.71
3.12
0.05
0.39
128
1024
512
aThe Log P is calculated with CHEMOFFICE 2014 software.
bCC50: The 50% cytotoxic concentration in a mammalian VERO cell line.
CONCLUSIONS
then filtered. The solid crude product was purified by
column chromatography (silica gel) eluted with DCM to
v(DCM):v(MeOH) = 10:1 to give the title compounds
In summary, 12 novel CPFX-isatin-1H-1,2,3-triazole
hybrids were synthesized and evaluated for their in vitro
anti-mycobacterial activity against MTB H37Rv as well
as cytotoxicity in VERO cell line. The preliminary results
showed that all hybrids exhibited promising activity
against MTB H37Rv, and half of them were more active
than the parent CPFX. Among them, the most active
conjugate 6h (MIC: 0.39 μg/mL) was comparable with
RIF, and eight times more potent than CPFX. All
conjugates were more toxic than the parent (CC50:
128 μg/mL) in VERO cell lines, and the most active
hybrids, which also displayed the highest cytotoxicity,
should be further optimized. The SAR was enriched, and
the results warrant further development of the anti-TB
properties of CPFX-isatin-1H-1,2,3-triazole hybrids.
6a–l (18–41%).
1-Cyclopropyl-6-fluoro-7-(4-((1-(2-(3-(hydroxyimino)-2-
oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-
yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6a).
1
Yield: 36%. H NMR (400 MHz, DMSO-d6) δ 1.02–1.21
(4H, m, 2 × cyclopropyl-CH2), 2.43–2.58 (4H, m,
piperazine-4H), 2.92–3.21 (4H, m, piperazine-4H),
3.61–3.83 (2H, m, ÀCH2 chain), 4.01–4.14 (3H, m,
cyclopropyl-CH, and –CH2 of linker), 4.66 (2H, s, ÀCH2
of linker), 6.93–8.69 (8H, m, Ar–H), 13.42 (1H, brs,
NOH), 14.89 (1H, brs, COOH). ESI-MS m/z: 601
[M + H]+. HRMS (ESI), m/z calcd. for C30H30FN8O5
[M + H]+: 601.2323; found 601.2327. Elemental Anal.
Calcd (%) for C30H29FN8O5: C, 59.99; H, 4.87; N,
18.66; found: C, 59.87; H, 4.84; N, 18.63.
1-Cyclopropyl-6-fluoro-7-(4-((1-(2-(3-(hydroxyimino)-5-
methyl-2-oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)
piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
EXPERIMENTAL
1
(6b).
Yield: 41%. H NMR (400 MHz, DMSO-d6) δ
1.02–1.16 (4H, m, 2 × cyclopropyl-CH2), 2.21 (3H, s,
ÀCH3), 2.46–2.55 (4H, m, piperazine-4H), 3.17–3.26
(4H, m, piperazine-4H), 3.72 (2H, q, ÀCH2 chain),
4.13–4.17 (3H, m, cyclopropyl-CH, and –CH2 of linker),
4.61–4.66 (2H, m, ÀCH2 of linker), 6.67–8.69 (7H, m,
Ar–H), 13.70 (1H, brs, NOH), 14.74 (1H, brs, COOH).
ESI-MS m/z: 615 [M + H]+. HRMS (ESI), m/z calcd. for
C31H32FN8O5 [M + H]+: 615.2480; found 615.2477.
Elemental Anal. Calcd (%) for C31H31FN8O5: C, 60.58;
Synthesis.
5a–d.
General procedure for the preparation of
N-(2-Azidoethyl)isatins 3a–d and propargyl
CPFX 4 (yield: 42%) were prepared via literature
methods [11–13]. To a mixture of N-(2-azidoethyl)isatins
3a–d (1.0 mmol) and propargyl CPFX 4 (1.0 mmol) in
DMF (20 mL), Cu(OAc)2 (20 mg) was added under N2
atmosphere. The mixture was allowed to stir at room
temperature overnight. After removal of the solvent, the
residue was purified by silica gel column chromatography
eluted with DCM to v(DCM):v(MeOH) = 10:1 to give
the desired 5a–d (yield: 31–46%).
H, 5.08; N, 18.23; found: C, 60.47; H, 5.03; N, 18.16.
1-Cyclopropyl-6-fluoro-7-(4-((1-(2-(3-(hydroxyimino)-5-
methoxy-2-oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)
The general procedure for preparing targets 6a–l. To a
solution of the corresponding amine hydrochloride
(6 mmol) and sodium bicarbonate (6 mmol) dissolved in
water (10 mL) and methanol (10 mL) was added 5a–d.
The reaction mixture was stirred at room temperature for
24 h. After removal of the solvent, the residue was
diluted with water (20 mL) and stirred for 10 min, and
piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
1
(6c).
Yield: 27%. H NMR (400 MHz, DMSO-d6) δ
1.02–1.18 (4H, m, 2 × cyclopropyl-CH2), 2.51–2.57 (4H,
m, piperazine-4H), 3.17–3.26 (4H, m, piperazine-4H),
3.70–3.82 (5H, m, ÀCH2 chain, and OCH3), 4.12–4.17
(3H, m, cyclopropyl-CH, and –CH2 of linker), 4.63–4.67
(2H, m, ÀCH2 of linker), 6.69–8.68 (7H, m, Ar–H),
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Ciprofloxacin-isatin-1H-1,2,3-triazole Hybrids: Design, Synthesis, and in vitro
Anti-tubercular Activity against M. tuberculosis
13.68 (1H, brs, NOH), 14.92 (1H, brs, COOH). ESI-MS m/
z: 631 [M + H]+. HRMS (ESI), m/z calcd. for
C31H32FN8O6 [M + H]+: 631.2429; found 631.2428.
Elemental Anal. Calcd (%) for C31H31FN8O6: C, 59.04;
H, 4.95; N, 17.77; found: C, 58.99; H, 4.94; N, 17.75.
1-Cyclopropyl-6-fluoro-7-(4-((1-(2-(5-fluoro-3-
(hydroxyimino)-2-oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)
methyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (6d). Yield: 30%. 1H NMR (400 MHz, DMSO-d6) δ
1.02–1.16 (4H, m, 2 × cyclopropyl-CH2), 2.21 (3H, s,
ÀCH3), 2.46–2.55 (4H, m, piperazine-4H), 3.17–3.26
(4H, m, piperazine-4H), 3.72 (2H, q, ÀCH2 chain),
4.13–4.17 (3H, m, cyclopropyl-CH, and –CH2 of linker),
4.61–4.66 (2H, m, ÀCH2 of linker), 6.67–8.69 (7H, m,
Ar–H), 13.70 (1H, brs, NOH), 14.74 (1H, brs, COOH).
ESI-MS m/z: 619 [M + H]+. HRMS (ESI), m/z calcd. for
C30H29F2N8O5 [M + H]+: 619.2229; found 619.2227.
Elemental Anal. Calcd (%) for C30H28F2N8O5: C, 58.25;
linker), 4.61–4.67 (2H, m, ÀCH2 of linker), 6.67–9.01
(9H, m, Ar–H, and NNHCONH2), 12.13 (1H, brs,
NNHCONH2), 14.84 (1H, brs, COOH). ESI-MS m/z: 673
[M + H]+. HRMS (ESI), m/z calcd. for C32H34FN10O6
[M + H]+: 673.2647; found 673.2646. Elemental Anal.
Calcd (%) for C32H33FN10O6: C, 57.14; H, 4.94; N,
20.82; found: C, 57.09; H, 4.94; N, 20.76.
7-(4-((1-(2-(3-(2-Carbamoylhydrazono)-5-fluoro-2-
oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-
yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
1
carboxylic acid (6h). Yield: 23%. H NMR (400 MHz,
DMSO-d6) δ 1.05–1.21 (4H, m, 2 × cyclopropyl-CH2),
2.56–2.61 (4H, m, piperazine-4H), 3.21–3.28 (4H, m,
piperazine-4H), 3.71–3.83 (2H, m, ÀCH2 chain),
4.11–4.20 (3H, m, cyclopropyl-CH, and –CH2 of linker),
4.65–4.69 (2H, m, ÀCH2 of linker), 6.71–9.00 (9H, m,
Ar–H,
and
NNHCONH2),
12.16
(1H,
brs,
NNHCONH2), 14.92 (1H, brs, COOH). ESI-MS m/z:
661 [M
+
H]+. HRMS (ESI), m/z calcd. for
H, 4.56; N, 18.11; found: C, 58.16; H, 4.51; N, 18.03.
7-(4-((1-(2-(3-(2-Carbamoylhydrazono)-2-oxoindolin-1-yl)
ethyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-1-
C31H31F2N10O5 [M + H]+: 661.2447; found 661.2443.
Elemental Anal. Calcd (%) for C31H30F2N10O5: C,
56.36; H, 4.58; N, 21.20; found: C, 56.27; H, 4.51; N,
21.19.
cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (6e). Yield: 26%. 1H NMR (400 MHz, DMSO-d6) δ
1.05–1.17 (4H, m, 2 × cyclopropyl-CH2), 2.46–2.59 (4H,
m, piperazine-4H), 2.92–3.22 (4H, m, piperazine-4H),
3.63–3.83 (2H, m, ÀCH2 chain), 4.01–4.12 (3H, m,
cyclopropyl-CH, and –CH2 of linker), 4.67 (2H, s, ÀCH2
of linker), 6.74–9.00 (10H, m, Ar–H, and NNHCONH2),
12.10 (1H, brs, NNHCONH2), 14.89 (1H, brs, COOH).
ESI-MS m/z: 643 [M + H]+. HRMS (ESI), m/z calcd. for
C31H32FN10O5 [M + H]+: 643.2541; found 643.2541.
Elemental Anal. Calcd (%) for C31H31FN10O5: C, 57.94;
7-(4-((1-(2-(3-(2-Carbamothioylhydrazono)-2-oxoindolin-1-
yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-1-
cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
1
acid (6i). Yield: 21%. H NMR (400 MHz, DMSO-d6) δ
1.08–1.19 (4H, m, 2 × cyclopropyl-CH2), 2.43–2.58 (4H,
m, piperazine-4H), 2.95–3.21 (4H, m, piperazine-4H),
3.65–3.83 (2H, m, ÀCH2 chain), 4.02–4.11 (3H, m,
cyclopropyl-CH, and –CH2 of linker), 4.66 (2H,
s, ÀCH2 of linker), 6.69–8.69 (7H, m, Ar–H), 8.78 (1H,
s, NNHCSNH2), 9.11 (1H, s, NNHCSNH2), 12.08 (1H,
s, NNHCSNH2), 14.90 (1H, brs, COOH). ESI-MS m/z:
H, 4.86; N, 21.80; found: C, 57.81; H, 4.77; N, 21.68.
7-(4-((1-(2-(3-(2-Carbamoylhydrazono)-5-methyl-2-
oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-
yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
659 [M
+
H]+. HRMS (ESI), m/z calcd. for
C31H32FN10O4S [M + H]+: 659.2313; found 659.2311.
Elemental Anal. Calcd (%) for C31H31FN10O4S: C,
56.52; H, 4.74; N, 21.26; found: C, 56.41; H, 4.66; N,
21.09.
1
carboxylic acid (6f). Yield: 23%. H NMR (400 MHz,
DMSO-d6) 1.06–1.18 (4H, m, 2 × cyclopropyl-CH2),
2.22 (3H, s, ÀCH3), 2.46–2.57 (4H, m, piperazine-4H),
3.15–3.28 (4H, m, piperazine-4H), 3.71 (2H, q, ÀCH2
chain), 4.12–4.16 (3H, m, cyclopropyl-CH and –CH2 of
linker), 4.62–4.66 (2H, m, ÀCH2 of linker), 6.69–9.00
(9H, m, Ar–H, and NNHCONH2), 12.12 (1H, brs,
NNHCONH2), 14.96 (1H, brs, COOH). ESI-MS m/z: 657
[M + H]+. HRMS (ESI), m/z calcd. for C32H34FN10O5
[M + H]+: 657.2698; found 657.2695. Elemental Anal.
Calcd (%) for C32H33FN10O5: C, 58.53; H, 5.07; N,
7-(4-((1-(2-(3-(2-Carbamothioylhydrazono)-5-methyl-2-
oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-
yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
1
carboxylic acid (6j).
Yield: 23%. H NMR (400 MHz,
DMSO-d6) 1.05–1.18 (4H, m, 2 × cyclopropyl-CH2),
2.23 (3H, s, ÀCH3), 2.45–2.57 (4H, m, piperazine-4H),
3.13–3.29 (4H, m, piperazine-4H), 3.73 (2H, q, ÀCH2
chain), 4.10–4.18 (3H, m, cyclopropyl-CH, and –CH2 of
linker), 4.61–4.66 (2H, m, ÀCH2 of linker), 6.70–8.68
(7H, m, Ar–H), 8.80 (1H, s, NNHCSNH2), 9.13 (1H, s,
NNHCSNH2), 12.10 (1H, s, NNHCSNH2), 14.92 (1H,
brs, COOH). ESI-MS m/z: 673 [M + H]+. HRMS (ESI),
m/z calcd. for C32H34FN10O4S [M + H]+: 673.2469;
found 673.2468. Elemental Anal. Calcd (%) for
C32H33FN10O4S: C, 57.13; H, 4.94; N, 20.82; found: C,
57.10; H, 4.88; N, 20.71.
21.33; found: C, 58.51; H, 5.01; N, 21.26.
7-(4-((1-(2-(3-(2-Carbamoylhydrazono)-5-methoxy-2-
oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-
yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
1
carboxylic acid (6g). Yield: 19%. H NMR (400 MHz,
DMSO-d6) δ 1.03–1.17 (4H, m, 2 × cyclopropyl-CH2),
2.53–2.59 (4H, m, piperazine-4H), 3.17–3.27 (4H, m,
piperazine-4H), 3.72–3.81 (5H, m, ÀCH2 chain and
OCH3), 4.12–4.18 (3H, m, cyclopropyl-CH, and –CH2 of
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Z. Xu, Z.-S. Lv, X.-F. Song, and M. Qiang
Vol 000
7-(4-((1-(2-(3-(2-Carbamothioylhydrazono)-5-methoxy-2-
oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-
yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
concentration of the compound required to give complete
inhibition of bacterial growth.
Cytotoxicity.
The synthesized conjugates 6a–l were
1
carboxylic acid (6k). Yield: 18%. H NMR (400 MHz,
further examined for toxicity (CC50) in a mammalian
VERO cell line at concentrations from 1024 to 1 μg/mL
[11]. The VERO cells were maintained in culture
medium (minimum essential medium with Earle’s salt,
supplemented 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 calculated by Bliss analyses.
DMSO-d6) δ 1.06–1.18 (4H, m, 2 × cyclopropyl-CH2),
2.51–2.61 (4H, m, piperazine-4H), 3.14–3.26 (4H, m,
piperazine-4H), 3.73–3.84 (5H, m, ÀCH2 chain, and
OCH3), 4.13–4.18 (3H, m, cyclopropyl-CH, and –CH2 of
linker), 4.64–4.68 (2H, m, ÀCH2 of linker), 6.68–8.68
(7H, m, Ar–H), 8.81 (1H, s, NNHCSNH2), 9.12 (1H, s,
NNHCSNH2), 12.11 (1H, s, NNHCSNH2), 14.88 (1H,
brs, COOH). ESI-MS m/z: 689 [M + H]+. HRMS (ESI),
m/z calcd. for C32H34FN10O5S [M + H]+: 689.2418;
found 689.2414. Elemental Anal. Calcd (%) for
C32H33FN10O5S: C, 55.80; H, 4.83; N, 20.34; found: C,
55.76; H, 4.75; N, 20.31.
7-(4-((1-(2-(3-(2-Carbamothioylhydrazono)-5-fluoro-2-
oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-
REFERENCES AND NOTES
yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
1
carboxylic acid (6l).
Yield: 20%. H NMR (400 MHz,
[1] World Health Organization, Global Tuberculosis Report 2016
(WHO/HTM/TB/2016.10). World Health Organization, Geneva (2016).
[2] Crofton, J.; Choculet, P.; Maher, D. Guidelines for the manage-
ment of drug-resistant tuberculosis WHO/TB/96-210 (Rev.1). World
Health Organization, Geneva (1997).
[3] Xu, Z.; Zhang, S.; Gao, C.; Zhao, F.; Lv, Z. S.; Feng, L. S.
Chin Chem Lett 2017, 2, 159.
[4] Sriram, D.; Aubry, A.; Yogeeswaria, P.; Fisher, L. M. Bioorg
Med Chem Lett 2006, 16, 2982.
[5] Sriram, D.; Yogeeswaria, P.; Basha, J. S.; Radhaet, D. R.;
Nagaraja, V. Bioorg Med Chem 2005, 13, 5774.
[6] Pandeya, S. N.; Sriram, D.; Yogeeswaria, P.; Ananthan, S.
Chemother 2001, 47, 266.
[7] Zhao, S. H.; Pine, R.; Domagala, J.; Drlica, K. Antimicrob
Agents Chemother 1999, 43, 661.
[8] Feng, L. S.; Liu, M. L.; Zhang, S. Eur J Med Chem 2011, 46,
341.
DMSO-d6) δ 1.08–1.20 (4H, m, 2 × cyclopropyl-CH2),
2.58–2.64 (4H, m, piperazine-4H), 3.24–3.30 (4H, m,
piperazine-4H), 3.74–3.83 (2H, m, ÀCH2 chain),
4.14–4.20 (3H, m, cyclopropyl-CH, and –CH2 of linker),
4.64–4.69 (2H, m, ÀCH2 of linker), 6.69–8.71 (7H, m,
Ar–H), 8.83 (1H, s, NNHCSNH2), 9.10 (1H,
s, NNHCSNH2), 12.08 (1H, s, NNHCSNH2), 14.96 (1H,
brs, COOH). ESI-MS m/z: 677 [M + H]+. HRMS (ESI),
m/z calcd. for C31H31F2N10O4S [M + H]+: 677.2219;
found 677.2218. Elemental Anal. Calcd (%) for
C31H30F2N10O4S: C, 55.02; H, 4.47; N, 20.70; found: C,
54.98; H, 4.45; N, 20.57.
MIC determination. Hybrids 6a–l along with CPFX,
RIF, and INH were evaluated in vitro activities against
MTB H37Rv via rapid direct susceptibility test technique
[14]. All the tested compounds were twofold diluted
with the twofold concentration medium at the
concentrations from 0.025 to 200 μg/mL. The wells of a
sterile 48-well plate were filled with 100-mL twofold
diluted tested compounds and 100-mL Mycobacterium
smegmatis or MTB H37Rv 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, 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 re-
determined 7 days later. The MIC is defined as the
[9] Feng, L. S.; Liu, M. L.; Zhang, Y. B. Chem Res Chin Univ
2012, 28, 61.
[10] Wan, Z. L.; Liu, M. L.; Feng, L. S.; Wang, B.; Zheng, X. D.;
Guo, H. Y. Chin J Antibiot 2011, 36, 37.
[11] Xu, Z.; Song, X. F.; Hu, Y. Q.; Qiang, M.; Lv, Z. S. Eur J Med
Chem 2017, 138, 66.
[12] Zhang, S.; Xu, Z.; Gao, C.; Ren, Q. C.; Le, C.; Lv, Z. S.; Feng,
L. S. Eur J Med Chem 2017, 138, 501.
[13] Xu, Z.; Qiang, M.; Lv, Z. S. Asian J Chem 2017, 29, 1039.
[14] Xu, Z.; Zhang, S.; Song, X. F.; Qiang, M.; Qiang, M.; Lv, Z. S.
Bioorg Med Chem Lett . https://doi.org/10.1016/j.bmcl.2017.07.023.
[15] Xu, Z.; Zhang, S.; Feng, L. S.; Huang, G. C.; Chai, Y.; Lv, Z.
S.; Guo, H. Y.; Liu, M. L. Molecules. https://doi.org/10.3390/
molecules22071171.
[16] Xu, Z.; Song, X. F.; Qiang, M.; Lv, Z. S. J Heterocyclic Chem .
https://doi.org/10.1002/jhet.2933.
[17] Zhou, B.; He, Y.; Zhang, X.; Xu, J.; Luo, Y.; Wang, Y.;
Franzblau, S. G.; Yang, Z.; Chan, R. J.; Liu, Y.; Zheng, J.; Zhang, Z. Y.
Proc Natl Acad Sci U S A 2010, 107, 4573.
[18] Lin, H. N.; Walsh, C. T. J Am Chem Soc 2004, 126, 13998.
[19] McPherson, J. C.; Royce, R.; Thomas, B. B.; Hartmann, J. F.;
Farcasiu, D.; Bereczki, I.; Rȍth, E.; Tollas, S.; Ostorházi, E.; Rozgonyi, F.;
Herczegh, P. Eur J Med Chem 2012, 47, 615.
[20] Kumar, K.; Pradines, B.; Madamet, M.; Amalvict, R.; Benoit,
N.; Kumar, V. Eur J Med Chem 2014, 87, 801.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet