Design, Synthesis, and in vitro Anti-mycobacterial Evaluation of Propylene-1H-1,2,3-triazole-4-methylene-tethered (Thio)semicarbazone-isatin-moxifloxacin Hybrids

Article abstract of DOI:10.1002/jhet.3004

A new class of propylene-1H-1,2,3-triazole-4-methylene-tethered (thio)semicarbazone-isatin-moxifloxacin hybrids 6a–h was designed, synthesized, and screened for their in vitro anti-mycobacterial activities against Mycobacterium tuberculosis (MTB) H₃₇Rv and MDR-TB as well as cytotoxicity in VERO cell line. All the synthesized hybrids (MIC: 0.05–2.0?μg/mL) exhibited excellent activities against M.?tuberculosis H₃₇Rv and MDR-TB; in particular, conjugate 6c (MIC: 0.05 and 0.12?μg/mL) was no inferior to the three references MXFX (MIC: 0.10 and 0.12?μg/mL), RIF (MIC: 0.39 and 32?μg/mL), and INH (MIC: 0.05 and >128?μg/mL) against the tested two strains. All hybrids (CC₅₀: 2–8?μg/mL) were much more cytotoxic than the parent MXFX (CC₅₀: 128?μg/mL) should be further optimized.

Full text of DOI:10.1002/jhet.3004

Month 2017
Design, Synthesis, and in vitro Anti-mycobacterial Evaluation of
Propylene-1H-1,2,3-triazole-4-methylene-tethered
(Thio)semicarbazone-isatin-moxifloxacin Hybrids
b
c
a
Zhi Xu,a
*
Xu-Feng Song, Jing Fan, and Zao-Sheng Lv
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
cHengshui University, Hebei, People’s Republic of China
*
E-mail: chemorgchem@126.com
Received July 29, 2017
DOI 10.1002/jhet.3004
Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com).
A
new class of propylene-1H-1,2,3-triazole-4-methylene-tethered (thio)semicarbazone-isatin-
moxifloxacin hybrids 6a–h was designed, synthesized, and screened for their in vitro anti-mycobacterial ac-
tivities against Mycobacterium tuberculosis (MTB) H37Rv and MDR-TB as well as cytotoxicity in VERO
cell line. All the synthesized hybrids (MIC: 0.05–2.0 μg/mL) exhibited excellent activities against M. tuber-
culosis H37Rv and MDR-TB; in particular, conjugate 6c (MIC: 0.05 and 0.12 μg/mL) was no inferior to the
three references MXFX (MIC: 0.10 and 0.12 μg/mL), RIF (MIC: 0.39 and 32 μg/mL), and INH (MIC: 0.05
and >128 μg/mL) against the tested two strains. All hybrids (CC50: 2–8 μg/mL) were much more cytotoxic
than the parent MXFX (CC50: 128 μg/mL) should be further optimized.
J. Heterocyclic Chem., 00, 00 (2017).
INTRODUCTION
around 10 million newly clinical cases and 1 million
deaths annually according to the World Health
Organization (WHO) report [1]. In spite of the first-line
drugs, isoniazid (INH), rifampicin (RIF), pyrazinamide
Tuberculosis (TB), caused predominately by the
pathogen Mycobacterium tuberculosis (MTB), resulted in
© 2017 Wiley Periodicals, Inc.

Z. Xu, X.-F. Song, J. Fan, and Z.-S. Lv
Vol 000
(PZA), and ethambutol (EMB) are crucial therapeutics for
the treatment of TB; these drugs are becoming less and less
effective because of the increasing prevalence of
drug-resistant TB, multidrug-resistant TB (MDR-TB),
extremely drug-resistant TB, and totally drug-resistant TB
[2–5]. Therefore, it is imperative to develop new agents
for efficient treatment.
Numerous of fluoroquinolone derivatives were
synthesized for searching more potent anti-TB agents,
among them, fluoroquinolone-isatin hybrids caused great
interests attribute to their promising in vitro and in vivo
activities [9–26]. The previous work demonstrated that
the linkers between fluoroquinolones and isatin have
great influence on the anti-TB activity of these
hybrids, that is, methylene-linked gatifloxacin-isatin
hybrid 1 (Fig. 1) exhibited higher in vitro and in vivo
potency than the parent gatifloxacin [9]. Our work
showed that hybrids with 1,2,3-triazole linker could
boost up the activity against MTB H37Rv and MDR-
TB strains, and as the most emblematic example, the
1,2,3-triazole-tethered gatifloxacin-isatin hybrid 2 was
4≥512 times more potent in vitro than the three
references gatifloxacin, RIF, and INH against the
tested two strains [25–27].
Fluoroquinolones
exhibit
considerable
anti-TB
activities, although some of them such as ciprofloxacin,
ofloxacin, and levofloacin are presently recommended as
second-line anti-TB agents by WHO [6]; these drugs
such as moxifloxacin (MXFX) are potential first-line
agents and are under study for this indication [7]. In
general, MTB isolates expressing resistance to both INH
and RIF are susceptible to fluoroquinolones, while
MXFX retains activity against MTB strains with various
levels of fluoroquinolones resistance [8].
Figure 1. Illustration of the design strategy for propylene-1H-1,2,3-triazole-4-methylene-tethered (thio)semicarbazone-isatin-MXFX hybrids. [Color
figure can be viewed at wileyonlinelibrary.com]
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(thio)semicarbazone-isatin-moxifloxacin Hybrids
Currently, MXFX is under phase III clinical trial for the
treatment of TB, and compared with the standard regimen,
MXFX exhibited equivalent or even slightly better efficacy
in the clinical trial [28]. Thus, incorporation of isatin into
MXFX with propylene-1H-1,2,3-triazole-4-methylene as
linker may provide more effective candidates.
Based on the aforementioned considerations, series of
propylene-1H-1,2,3-triazole-4-methylene-tethered (thio)
semicarbazone-isatin-MXFX hybrids were designed,
synthesized, and screened for their in vitro anti-
mycobacterial activities against MTB H37Rv and MDR-
TB as well as cytotoxicity in VERO cell line in this study.
Illustration of the design strategy is depicted in Figure 1.
33–59%) and propargyl MXFX 4 (yield: 69%) via
literature methods [25,26]. Introduction of azido by
treatment of C-5 substituted N-(3-bromoproyl)isatins 2a–d
with sodium azide at 60°C to provide N-(3-azidoproyl)
isatins 3a–d, which was utilized together with propargyl
MXFX 4 for the synthesis of the precursors 5a–d (yield:
41–63%) via Cu-promoted azide-alkyne cycloaddition
reaction in the presence of Cu(OAc)2 in DMF [25].
Finally, condensations of conjugates 5a–d with
semicarbazide or thiosemicarbazide hydrochlorides in the
presence of sodium bicarbonate formed other hybrids
(16–33%) [26].
All the synthesized propylene-1H-1,2,3-triazole-4-
methylene-tethered
(thio)semicarbazone-isatin-MXFX
hybrids 6a–h were screened for their in vitro anti-
mycobacterial activities against MTB H37Rv and MDR-
TB strains by rapid direct susceptibility test technique
[25]. The MDR-TB strain was resistant to INH, RIF,
and ethambutol (EMB). 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 targets
were reported in Table 1.
RESULTS AND DISCUSSION
Detailed pathways for synthesis of propylene-1H-1,2,3-
triazole-4-methylene-tethered (thio)semicarbazone-isatin-
MXFX hybrids 6a–h are depicted in Scheme 1.
Alkylation of C-5 substituted isatins and MXFX with
1,3-dibromopropane and propargyl bromide respectively
yielded the desired N-(3-bromoproyl)isatins 2a–d (yield:
Scheme 1. Synthesis of propylene-1H-1,2,3-triazole-4-methylene-tethered (thio)semicarbazone-isatin-MXFX hybrids 6a–h.
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Z. Xu, X.-F. Song, J. Fan, and Z.-S. Lv
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Table 1
Structures, anti-mycobacterial activity, and cytotoxicity of compounds 6a–h.
MIC (μg/mL)
Compound
R1
R2
MTB H37Rv
MDR-TBa
CC50 b(μg/mL)
6a
6b
6c
6d
6e
6f
6g
6h
NNHCONH2
NNHCONH2
NNHCONH2
NNHCONH2
NNHCSNH2
NNHCSNH2
NNHCSNH2
NNHCSNH2
H
Me
F
Cl
H
Me
F
0.78
0.78
0.05
0.39
1.56
0.78
0.39
0.78
0.10
0.05
0.39
0.5
1.0
0.12
0.25
1.0
8
8
2
4
8
4
2
4
2.0
0.5
Cl
0.50
0.12
>128
32
MXFX
INH
RIF
128
128
512
aMDR-TB: resistant to INH, RIF and EMB.
bCC50: The 50% cytotoxic concentration in a mammalian VERO cell line.
All the synthesized hybrids (MIC: 0.05–2.0 μg/mL)
exhibited considerable activities against both MTB H37Rv
and MDR-TB strains, but the majority of them were less
active than the parent MXFX (MIC: 0.10 and 0.12 μg/mL).
The SAR indicated that introduction of halogen atoms –
Cl and –F at C-5 position of isatin moiety favored the
anti-TB activity, and hybrids with semicarbazone moiety
at C-5 position of isatin motif were more active than the
corresponding thiosemicarbazone analogs. In particular,
conjugate 6c (MIC: 0.05 and 0.12 μg/mL) was
comparable with the parent MXFX, and 256≥1024 folds
more potent than RIF (MIC: 32 μg/mL) and INH (MIC:
>128 μg/mL) against MDR-TB, and was comparable
with INH (MIC: 0.05 μg/mL) and twofold to eightfold
more potent than MXFX and INH (MIC: 0.39 μg/mL)
against MTB H37Rv. Moreover, the resistance index (RI:
MICMDR-TB: MICMTB H37Rv) of the most targets was
around 1, suggesting this kind of hybrids could reduce
the cross-resistant to some extent.
CONCLUSION
In conclusion, a set of novel propylene-1H-1,2,3-
triazole-4-methylene-tethered (thio)semicarbazone-isatin-
MXFX hybrids was designed, synthesized, and evaluated
for their in vitro anti-mycobacterial activities against
MTB H37Rv and MDR-TB as well as cytotoxicity in
VERO cell line. All hybrids exhibited excellent activities
against the tested MTB H37Rv and MDR-TB, and the
most active 6c, which was no inferior to the three
references MXFX, RIF and INH against the tested two
strains, warrant further investigations. The SAR of 1H-
1,2,3-triazole-tethered isatin-FQs hybrids was enriched,
and the results warrant further development of the anti-
TB properties of this kind of conjugates.
EXPERIMENTAL
The conjugates 6a–h were subsequently examined for
toxicity (CC50) in a mammalian VERO cell line [26].
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. All
hybrids (CC50: 2–8 μg/mL) were much more cytotoxic
than the parent MXFX (CC50: 128 μg/mL), and hybrids
with halogen atoms at C-5 position of isatin motif showed
highest cytotoxicity. Thus, reduce the cytotoxicity is the
main direction for further modification.
Synthesis. General Procedure for the Preparation of 6a–h.
The key intermediates 5a–d were prepared via the methods
we previously reported [25–27]. To a solution of
semicarbazide or thiosemicarbazide hydrochlorides
(6 mmol) and sodium bicarbonate (6 mmol) dissolved in
water (10 mL) and methanol (10 mL) was added 5a–d
(3 mmol). 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 then filtered. The solid crude product was
purified by column chromatography (silica gel) eluted
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Month 2017
(thio)semicarbazone-isatin-moxifloxacin Hybrids
with DCM to v(DCM):v(MeOH) = 10:1 to give targets 6a–
h (16–33%).
m), 2.75–2.77 (1H, m), 2.99–3.01 (1H, m), 3.61–3.80 (11H,
m), 4.11–4.13 (1H, m), 4.36–4.38 (2H, m), 7.06–8.64 (6H,
m), 8.70 (1H, s), 9.12 (1H, s), 12.10 (1H, brs), 15.20 (1H,
brs). ESI-MS m/z: 743 [M + H]+. Elemental Anal. Calcd
(%) for C36H39FN10O5S: C, 58.21; H, 5.29; N, 18.86;
7-((4aR,7aR)-1-((1-(3-(3-(2-carbamoylhydrazono)-2-
oxoindolin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)
hexahydro-1H-pyrrolo[3,4-b]pyridin-6(2H)-yl)-1-cyclopropyl-6-
fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(6a). Yellow solid, yield: 31%; mp: 191–193°C. 1H NMR
(400 MHz, DMSO-d6) δ 1.12–1.62 (8H, m), 2.12–2.33 (4H,
m), 2.75–2.77 (1H, m), 2.99–3.00 (1H, m), 3.63–3.78 (11H,
m), 4.11–4.13 (1H, m), 4.36–4.38 (2H, m), 6.76–7.28 (6H,
m), 7.58 (1H, d), 8.06 (1H, s), 8.64 (1H, s), 11.50 (1H,
brs), 15.20 (1H, brs). ESI-MS m/z: 727 [M + H]+.
Elemental Anal. Calcd (%) for C36H39FN10O6: C, 59.50;
H, 5.41; N, 19.27; found: C, 59.29; H, 5.33; N, 19.18.
7-((4aR,7aR)-1-((1-(3-(3-(2-carbamoylhydrazono)-5-methyl-
2-oxoindolin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)
hexahydro-1H-pyrrolo[3,4-b]pyridin-6(2H)-yl)-1-cyclopropyl-6-
fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(6b). Light yellow solid, yield: 16%; mp: 173–174°C. 1H
NMR (400 MHz, DMSO-d6) δ 1.01–1.59 (m, 8H), 2.12–
2.35 (m, 7H), 2.73–2.75 (m, 1H), 3.00–3.01 (m, 1H),
3.54–3.72 (m, 11H), 3.94–3.96 (m, 1H), 4.39–4.48 (m,
2H), 7.04–8.60 (7H, m), 8.68 (1H, s), 9.02 (1H, s), 11.52
(1H, brs), 15.16 (1H, brs). ESI-MS m/z: 741 [M + H]+.
Elemental Anal. Calcd (%) for C37H41FN10O6: C, 59.99;
H, 5.58; N, 18.91; found: C, 59.78; H, 5.43; N, 18.82.
7-((4aR,7aR)-1-((1-(3-(3-(2-carbamoylhydrazono)-5-fluoro-
2-oxoindolin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)
hexahydro-1H-pyrrolo[3,4-b]pyridin-6(2H)-yl)-1-cyclopropyl-6-
fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(6c). Light yellow solid, yield: 21%; mp: 201–203°C. 1H
found: C, 58.03; H, 5.11; N, 18.63.
7-((4aR,7aR)-1-((1-(3-(3-(2-carbamothioylhydrazono)-5-
methyl-2-oxoindolin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)
hexahydro-1H-pyrrolo[3,4-b]pyridin-6(2H)-yl)-1-cyclopropyl-6-
fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(6f). Light yellow solid, yield: 33%; mp: 159–161°C. 1H
NMR (400 MHz, DMSO-d6) δ 1.08–1.61 (m, 8H), 2.11–
2.34 (m, 7H), 2.71–2.73 (m, 1H), 3.00–3.01 (m, 1H),
3.60–3.78 (m, 11H), 4.01–4.02 (m, 1H), 4.38–4.42 (m,
2H), 7.00–8.62 (6H, m), 8.72 (1H, s), 9.10 (1H, s), 12.18
(1H, brs), 15.12 (1H, brs). ESI-MS m/z: 757 [M + H]+.
Elemental Anal. Calcd (%) for C37H41FN10O5S: C, 58.72;
H, 5.46; N, 18.51; found: C, 58.59; H, 5.33; N, 18.37.
7-((4aR,7aR)-1-((1-(3-(3-(2-carbamothioylhydrazono)-5-
fluoro-2-oxoindolin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)
hexahydro-1H-pyrrolo[3,4-b]pyridin-6(2H)-yl)-1-cyclopropyl-6-
fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(6g). Light yellow solid, yield: 27%; mp: 182–184°C. 1H
NMR (400 MHz, DMSO-d6) δ 1.02–1.71 (8H, m), 2.13–
2.38 (4H, m), 2.75–2.77 (1H, m), 3.00–3.01 (1H, m),
3.63–3.80 (11H, m), 4.11–4.13 (1H, m), 4.34–4.37 (2H,
m), 7.12–7.64 (6H, m), 8.76 (1H, s), 9.10 (1H, s), 12.12
(1H, brs), 15.16 (1H, brs). ESI-MS m/z: 761 [M + H]+.
Elemental Anal. Calcd (%) for C36H38F2N10O5S: C,
56.83; H, 5.03; N, 18.41; found: C, 56.75; H, 4.92; N, 18.33.
7-((4aR,7aR)-1-((1-(3-(3-(2-carbamothioylhydrazono)-5-
NMR (400 MHz, DMSO-d6) δ 1.05–1.71 (8H, m), 2.11–
2.37 (4H, m), 2.75–2.77 (1H, m), 2.99–3.01 (1H, m),
3.61–3.80 (11H, m), 4.11–4.12 (1H, m), 4.36–4.38 (2H,
m), 7.00–8.62 (6H, m), 8.66 (1H, s), 9.01 (1H, s), 11.52
(1H, brs), 15.16 (1H, brs). ESI-MS m/z: 745 [M + H]+.
Elemental Anal. Calcd (%) for C36H38F2N10O6: C, 58.06;
H, 5.14; N, 18.81; found: C, 57.95; H, 5.06; N, 18.72.
7-((4aR,7aR)-1-((1-(3-(3-(2-carbamoylhydrazono)-5-chloro-
2-oxoindolin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)
chloro-2-oxoindolin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)
hexahydro-1H-pyrrolo[3,4-b]pyridin-6(2H)-yl)-1-cyclopropyl-6-
fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(6h). Light yellow solid, yield: 16%; mp: 163–165°C. 1H
NMR (400 MHz, DMSO-d6) δ 0.95–1.64 (m, 8H), 2.10–
2.36 (m, 4H), 2.76–2.77 (m, 1H), 3.01–3.02 (m, 1H),
3.62–3.80 (m, 11H), 4.12 (s, 1H), 4.37–4.40 (m, 2H),
7.06–8.64 (6H, m), 8.78 (1H, s), 9.12 (1H, s), 12.10 (1H,
brs), 15.12 (1H, brs). ESI-MS m/z: 777 [M + H]+, 779
[M + 2 + H]+. Elemental Anal. Calcd (%) for
C36H38FClN10O5S: C, 55.63; H, 4.93; N, 18.02; found:
C, 55.39; H, 4.77; N, 17.91.
hexahydro-1H-pyrrolo[3,4-b]pyridin-6(2H)-yl)-1-cyclopropyl-6-
fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(6d). Light yellow solid, yield: 18%; mp: 192–194°C. 1H
NMR (400 MHz, DMSO-d6) δ 1.08–1.66 (8H, m), 2.12–
2.36 (4H, m), 2.76–2.77 (1H, m), 2.99–3.00 (1H, m),
3.58–3.77 (11H, m), 4.10–4.11 (1H, m), 4.36–4.38 (2H,
m), 7.12–8.64 (6H, m), 8.70 (1H, s), 9.04 (1H, s), 11.64
(1H, brs), 15.18 (1H, brs). ESI-MS m/z: 761 [M + H]+,
763 [M + 2 + H]+. Elemental Anal. Calcd (%) for
C36H38FClN10O6: C, 56.80; H, 5.03; N, 18.40; found: C,
MIC determination. Hybrids 6a–h along with MXFX,
RIF, and INH were evaluated in vitro activity against
MTB H37Rv and MDR-TB via rapid direct susceptibility
test technique [23]. The compounds together with the
references MXFX, RIF, and INH were dissolved in
dimethyl sulfoxide (DMSO) and twofold diluted at
concentrations from 0.0125 to 200 μg/mL (for MTB
H37Rv) or 0.062 to 128 μg/mL (for MDR-MTB). 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
56.57; H, 4.81; N, 18.19.
7-((4aR,7aR)-1-((1-(3-(3-(2-carbamothioylhydrazono)-2-
oxoindolin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)
hexahydro-1H-pyrrolo[3,4-b]pyridin-6(2H)-yl)-1-cyclopropyl-6-
fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(6e). Yellow solid, yield: 26%; mp: 183–185°C. 1H NMR
(400 MHz, DMSO-d6) δ 1.06–1.58 (8H, m), 2.12–2.32 (4H,
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Cytotoxicity. The synthesized hybrids 6a–h along with
the references MXFX, RIF, and INH were further
examined for toxicity (CC50) in a mammalian VERO cell
line dissolved in DMSO at concentrations from 1024 to
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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
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