Synthesis and biological evaluation of moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids as potential anti-tubercular agents against both drug-susceptible and drug-resistant Mycobacterium tuberculosis strains

Article abstract of DOI:10.1016/j.ejmech.2019.07.057

Herein, synthesis and biological evaluation of fourteen moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids as potential anti-tubercular agents against both drug-susceptible (MTB H₃₇Rv), rifampicin-resistant and multidrug-resistant Mycobacterium tuberculosis strains were reported, and cytotoxicity towards VERO cells as well as inhibitory activity against MTB DNA gyrase were also discussed in this paper. The structure-activity relationship and structure-cytotoxicity relationship demonstrated that substituents on the C-3 and C-5/C-7 positions of isatin framework were closely related with the anti-mycobacterial activity and cytotoxicity. The most active hybrids 8h and 8l (MIC: 0.12–0.5 μg/mL) showed excellent activity which was no inferior to the parent moxifloxacin against the tested drug-susceptible, rifampicin-resistant and multidrug-resistant Mycobacterium tuberculosis strains, demonstrating their potential application as novel anti-tubercular candidates.

Full text of DOI:10.1016/j.ejmech.2019.07.057

Accepted Manuscript
Synthesis and biological evaluation of moxifloxacin-acetyl-1,2,3-1H-triazole-
methylene-isatin hybrids as potential anti-tubercular agents against both drug-
susceptible and drug-resistant Mycobacterium tuberculosis strains
Feng Gao, Zijian Chen, Long Ma, Yilei Fan, Linjun Chen, Guangming Lu
PII:
S0223-5234(19)30678-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.057
EJMECH 11554
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 June 2019
Revised Date: 17 July 2019
Accepted Date: 20 July 2019
Please cite this article as: F. Gao, Z. Chen, L. Ma, Y. Fan, L. Chen, G. Lu, Synthesis and biological
evaluation of moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids as potential anti-tubercular
agents against both drug-susceptible and drug-resistant Mycobacterium tuberculosis strains, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.07.057.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis and biological evaluation of
moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids
as potential anti-tubercular agents against both drug-susceptible
and drug-resistant Mycobacterium tuberculosis strains
Feng Gaoa, Zijian Chena, Long Maa, Yilei Fanb, Linjun Chenb*, Guangming Lua*
a Department of Medical Imaging, Jinling Hospital, Medical School of Nanjing University,
Nanjing, PR China
b SUMHS-SHUANG JIA Institute of Emergency Medical Rescue Technology, Shanghai
University of Medicine and Health Sciences, Shanghai, P. R. China

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of
moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids
as potential anti-tubercular agents against both drug-susceptible
and drug-resistant Mycobacterium tuberculosis strains
Feng Gaoa, Zijian Chena, Long Maa, Yilei Fanb, Linjun Chenb*, Guangming Lua*
a Department of Medical Imaging, Jinling Hospital, Medical School of Nanjing University, Nanjing,
PR China
b SUMHS-SHUANG JIA Institute of Emergency Medical Rescue Technology, Shanghai University
of Medicine and Health Sciences, Shanghai, P. R. China
Abstract:
Herein,
synthesis
and
biological
evaluation
of
fourteen
moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids as potential anti-tubercular agents
against both drug-susceptible (MTB H37Rv), rifampicin-resistant and multidrug-resistant
Mycobacterium tuberculosis strains were reported, and cytotoxicity towards VERO cells as well as
inhibitory activity against MTB DNA gyrase were also discussed in this paper. The
structure-activity relationship and structure-cytotoxicity relationship demonstrated that
substituents on the C-3 and C-5/C-7 positions of isatin framework were closely related with the
anti-mycobacterial activity and cytotoxicity. The most active hybrids 8h and 8l (MIC: 0.12-0.5
µg/mL) showed excellent activity which was no inferior to the parent moxifloxacin against the
tested drug-susceptible, rifampicin-resistant and multidrug-resistant Mycobacterium tuberculosis
strains, demonstrating their potential application as novel anti-tubercular candidates.
Keywords: moxifloxacin; 1,2,3-triazole; isatin; anti-tubercular; drug-resistant Mycobacterium
tuberculosis; structure-activity relationship; structure-cytotoxicity relationship
1. Introduction
The World Health Organization (WHO) End TB Strategy aimed to halt the global
tuberculosis (TB) epidemic by 2035, with the target to decline TB deaths by 95% and
to reduce new cases by 90% [1,2]. An estimated 54 million lives were saved between
2000 and 2017 with the technology development of disease detection and the
*Corresponding author: chenlj@sumhs.edu.cn(L. Chen); cjr.luguangming@vip.163.com (G. Lu).
1

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improvement of treatment success rates [3,4]. However, TB continues to ravage the
world, leading to tremendous morbidity and mortality as evidenced by that over 10
million new TB cases (1 million children) and 1.6 million (230,000 children) deaths
occurred in 2017 [5]. The main obstacle to elimination of TB is the widely spread of
drug-resistant TB (DR-TB, resistance to at least one first-line anti-TB drugs)
especially multi-drug resistant TB (MDR-TB, resistance to at least both isoniazid and
rifampicin) [6], and 3.5% of new cases and 18% of previous treated cases were
MDR-TB or rifampicin-resistant (RR) TB patients with 580,000 incident cases and
230,000 deaths in 2017 [3]. The second-line anti-TB agents (such as levofloxacin) and
newly developed anti-TB agents (such as bedaquiline and delamanid) occupy an
important position in the treatment of DR-TB and MDR-TB patients, but these agents
are generally less effective (treatment success remains low, at 55% globally in 2017)
and more toxic [7,8]. Moreover, there are only 20 drugs in Phase I, II or III trials for
the treatment of drug-susceptible TB, MDR-TB or latent TB infection, and they are
still not sufficient [3]. All the above facts made an urgent demand to develop new
anti-TB agents with unique mechanisms and favorable profiles such as high efficacy,
low toxicity and short therapy duration.
Moxifloxacin (7-[(4aS,7aS)-octahydro-1H-pyrrolo[3,4-b]pyridin-6-yl]-1-cyclopropyl-
6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid), as the fourth
generation of fluoroquinolone antibiotic, could act on both DNA gyrase and
topoisomerase IV, possess broad-spectrum antimicrobial activity [9,10]. Moxifloxacin
exhibits excellent in vitro and in vivo potency against drug-susceptible and
drug-resistant Mycobacterium tuberculosis (MTB) strains including various levels of
fluoroquinolones resistance strains [11-13], and the clinical trial demonstrated that the
efficacy of moxifloxacin was no inferior to the standard regimen in early phase of
treatment TB patients [14,15]. Currently, moxifloxacin is under phase III clinical trial,
and it may be used in the near future for the treatment of TB, even MDR-TB.
However, the moxifloxacin-resistant MTB strains have already emerged, and serious
adverse effects like irreversible peripheral neuropathy [16], hepatitis [17], and
phototoxicity reactions [18] may occur as a result of moxifloxacin therapy. To
overcome the resistance and reduce the adverse effects, modification of moxifloxacin
is needed.
2

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Isatin and 1,2,3-triazole derivatives exhibit a variety of pharmacological properties
such as antibacterial [19,20], anticancer [21,22], and anti-TB [23,24] activities which
may be attributed to that these heterocycles can serve as a useful tool to manipulate
lipophilicity, polarity, and hydrogen bonding capacity of molecules, and consequently
improve pharmacological, pharmacokinetic, toxicological, and physicochemical
properties of drug candidates and the ultimate drugs. Moreover, several isatin- or
1,2,3-triazole-based compounds are under clinical evaluations for the treatment of
various diseases including TB [23,24], demonstrating isatin and 1,2,3-triazole
moieties are useful pharmacophores in the development of new drugs.
R1
O
O
F
COOH
N
R2
N
N
n
H
N
N
N
N
OMe
H
1
Isatin moiety with potential anti-TB activity
Moxifloxacin with excellent anti-TB activity
acetyl linker
O
N
N
N
N
R1
Electronic effect
F
COOH
N
O
N
O
H
R2
N
OMe
H
Control the lipophilicity
1,2,3-triazole motif with diverse non-covalent interactions
Figure 1 Design strategy for moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin
hybrids
Moxifloxacin-1,2,3-triazole-isatin 1 (Figure 1) tethered via various alkyl linkers
displayed promising anti-TB activity against both drug-susceptible and drug-resistant
(DR) MTB strains [25,26], and the structure-activity relationship (SAR) as well as
structure-cytotoxicity relationship demonstrated that the linker between the
3

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fluoroquinolone and 1,2,3-triazole frameworks as well as substituents on the isatin
fragment influenced the anti-TB activity and cytotoxicity significantly [27-30]. Thus,
further optimization of the linker between the moxifloxacin and 1,2,3-triazole moieties
and substituents on the isatin motif may provide more effective anti-TB candidates
which are highly active against both drug-susceptible and drug-resistant MTB strains.
Based
on
the
above
facts,
a
series
of
novel
moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids were designed,
synthesized and evaluated for their in vitro antimycobacterial activity against
drug-susceptible (MTB H37Rv) and DR including MDR MTB strains. The design
strategy was depicted in Figure 1.
2. Results and discussion
The
synthetic
pathway
for
the
desired
moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids 8a-n was depicted in
Scheme 1. 2-Bromoacetic acid 1 was converted to 2-azidoacetic acid 2 by treatment
with sodium azide in H2O, and then 2-azidoacetyl chloride 3 was obtained by
treatment of 2-azidoacetic acid 2 with oxalyl chloride in presence of catalytic amount
of DMF in DCM. Acylation reaction between moxifloxacin and 2-azidoacetyl
chloride 3 with pyridine as base in DCM provided the 2-azidoacetyl moxifloxacin 4.
Isatin/5-methylisatin/5-fluoroisatin/7-fluoroisatin 5a-d reacted with propargyl
bromide with K2CO3 as base in DMF, giving N-propargyl isatin intermediates 6a-d.
Condensation of N-propargyl isatins 6a-d with the requested amine hydrochlorides in
presence of NaHCO3 in a mixture of THF and H2O yielded isatin intermediates 7a-j.
Finally, cyclization of 2-azidoacetyl moxifloxacin 4 with N-propargyl isatin
intermediates 6a-d or 7a-j with copper acetate (Cu(OAc)2) as catalyst generated the
desired moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids 8a-n.
4

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Scheme 1 Synthesis of moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin
hybrids 8a-n
The
chemical
structures
and
yields
of
moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids 8a-n were presented
in Table 1.
Table
1.
Chemical
structures
and
yields
of
moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids 8a-n
5

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O
N
N
N
R1
F
COOH
N
O
N
O
H
R2
N
N
OMe
H
8a-n
Compd
8a
R1
H
R2
O
O
O
O
Yield (%)
41%
43%
36%
24%
28%
37%
19%
35%
39%
46%
27%
19%
15%
11%
5-F
5-Me
7-F
H
8b
8c
8d
8e
NOH
NOH
5-F
7-F
H
8f
NOH
8g
NOMe
NOMe
NOMe
NOMe
8h
8i
5-F
5-Me
7-F
H
8j
8k
8l
NNHCSNH2
NNHCSNH2
NNHCSNH2
5-F
7-F
8m
8n
Moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids 8a-n together with
references gatifloxacin, moxifloxacin, isoniazid and rifampicin were initially
evaluated for their in vitro anti-mycobacterial activity against MTB H37Rv and
cytotoxicity towards mammalian VERO cells, and MIC as well as CC50 values were
presented in Table 2. The selectivity index (SI) expressed in terms of CC50/MIC, and
the results were also listed in Table 2.
6

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Table 2. In vitro anti-mycobacterial activity against MTB H37Rv, cytotoxicity and
selectivity index of moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids
8a-n
MICMTB H37Rv (µg/mL)
CC50 (µg/mL)
SI
128
16
Compd.
0.25
1
32
16
8a
8b
2
64
32
8c
8
32
4
8d
1
32
32
8e
2
16
8
8f
16
0.5
1
16
1
8g
128
32
256
32
8h
8i
4
64
16
8j
8k
16
0.12
0.25
8
16
1
16
128
32
8l
8
8m
8
1
8n
0.5
0.12
0.12
0.06
64
128
1,024
1,024
>512
Gatifloxacin
Moxifloxacin
Isoniazid
Rifampicin
128
128
>128
7

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From
Table
2,
it
can
be
seen
that
all
moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids 8a-n (MIC: 0.12-16
µg/mL) showed considerable activity against MTB H37Rv, and the anti-mycobacterial
activities of hybrids 8a, 8h, 8l and 8m (MIC: 0.06-0.5 µg/mL) were in the same level
with those of the references gatifloxacin, moxifloxacin, isoniazid and rifampicin (MIC:
0.12-0.5 µg/mL). The SAR indicated that introduction of thiosemicarbazone at C-3
position of isatin skeleton was favorable to the activity, while oxime and methyloxime
was harmful to the activity when compared with the ketone, and the relative
contribution order of the substituents was thiosemicarbazone > ketone >
methyloxime > oxime. Hybrids with either electron-donating methyl or
electron-withdrawing fluoro at C-5 position of isatin moiety exhibited reduced
activity when compared with unsubstituted analogues, and movement of fluoro to C-7
position led to great loss of activity, suggesting this position may not suitable for
modification.
All hybrids 8a-n (CC50: 8-64 µg/mL) except 8h (CC50: 128 µg/mL) were more toxic
than the parent moxifloxacin (CC50: 128 µg/mL), and the structure-cytotoxicity
relationship revealed that installation of oxime and thiosemicarbazone at C-3 position
or fluoro at either C-5 or C-7 position of isatin fragment could increase the
cytotoxicity, while methyloxime at C-3 position and methyl at C-5 position of isatin
motif could decrease cytotoxicity to some extent.
The SI of the synthesized hybrids were in a range of 1 to 256, and three of them 8a,
8h and 8l were comparable to or higher than gatifloxacin (SI: 128), but all of them
were lower than moxifloxacin, isoniazid and rifampicin (SI: >512). The most active
four hybrids 8a, 8h, 8l and 8m (SI: 32-256) which also showed relatively high SI were
selected for further evaluation of their inhibitory activity against MTB DNA gyrase
and anti-mycobacterial activity against one rifampicin-resistant (RR-MTB) and two
MDR (resistant to isoniazid and rifampicin) MTB strains, and the results were
presented in Table 3 and 4, respectively.
Table 3. In vitro inhibitory activity of hybrids 8a, 8h, 8l and 8m against MTB DNA
gyrase
8

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Comp.
IC50 (µg/mL)
4.0
6.0
5.0
8.0
5.0
5.5
8a
8h
8l
8m
Gatifloxacin
Moxifloxacin
DNA gyrase is one of the targets for the anti-TB agents, and isatin structural scaffolds
as well as moxifloxacin could bind DNA gyrase [31,32]. Thus, hybridization of isatin
with moxifloxacin may enhance of the inhibitory activity against DNA gyrase, and
consequently improve the anti-TB activity. From Table 3, all the selected four
hybrids with IC50 values of 4.0-8.0 µg/mL were in the same level with the references
gatifloxacin (IC50: 5.0 µg/mL) and moxifloxacin (IC50: 5.5 µg/mL) against MTB DNA
gyrase, and the most active hybrid 8a (IC50: 4.0 µg/mL) was slightly more active than
the two references.
Table 4. In vitro anti-mycobacterial activity of hybrids 8a, 8h, 8l and 8m against RR
and MDR MTB strains
MIC (µg/mL)
Compd.
RR-MTB
MDR-MTB1
MDR-MTB2
0.5
1
0.5
0.5
0.25
1
8a
8h
8l
0.25
0.25
0.5
9

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1
1
1
2
8m
0.25
0.25
64
0.5
Gatifloxacin
Moxifloxacin
Isoniazid
0.5
0.5
>128
0.25
>128
>128
>128
Rifampicin
From Table 4, all of the four selected hybrids showed promising activity against the
tested RR-MTB and two MDR-MTB strains with MIC values ranging from 0.25 to 2
µg/mL, and they were far more potent than the first-line anti-TB agents isoniazid and
rifampicin (MIC: ≥64 µg/mL) against two MDR-MTB strains. Two of them 8h and 8l
with MIC of 0.25-0.5 µg/mL were no inferior to the fluoroquinolone references
gatifloxacin (MIC: 0.25-1 µg/mL) and moxifloxacin (MIC: 0.25-0.5 µg/mL) against
the tested three strains, demonstrating their potential to fight against DR-TB even
MDR-TB.
The two hybrids 8h and 8l with promising activity against drug-susceptible,
rifampicin-resistant and MDR MTB strains, together with low cytotoxicity towards
CHO cells were selected for further investigation on their in vivo pharmacokinetics in
mice (100 mg/kg, subcutaneous injection/s.c. administration), and the results were
presented in Table 5.
Table 5 The in vivo pharmacokinetic values of hybrids 8h and 8l and moxifloxacin in
mice
Pharmacokinetics (s.c.)
Compd.
Cmax
(ng/mL)
t1/2 (h)
Tmax (min)
AUC0-inf (ng•h/mL)
3,247
3.1
4.3
2.6
39
52
27
6,435
3,269
8h
8l
1,398
10,783
19,243
moxifloxacin
10

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Cmax: the peak concentration;
t1/2: half-life;
Tmax: the time to reach peak concentration;
AUC: the area under the curve.
From Table 5, the in vivo pharmacokinetic profiles of the selected hybrids 8h and 8l
were worse than the parent moxifloxacin in terms of the peak concentration (Cmax
:
3,247 and 1,398 ng/mL vs 10,783 ng/mL), the time to reach peak concentration (Tmax
:
39 and 52 min vs 27 min) and the area under the curve (AUC0-inf: 6,435 and 3,269
ng•h/mL vs 19,243 ng•h/mL), and this may be attributed to that the existence of
1,2,3-triazole and amide functional groups led to poor solubility and bioavailability.
However, the half-life (t1/2: 3.1 and 4.3 h vs 2.6 h) was longer than moxifloxacin.
3. Conclusions
In summary, fourteen moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids
were designed, synthesized and assessed for their in vitro anti-mycobacterial activity
against drug-susceptible (MTB H37Rv), RR and MDR MTB strains. Cytotoxicity
towards VERO cells as well as inhibitory activity against MTB DNA gyrase of the
hybrids were also investigated in this study. Among them, the most active hybrids 8h
and 8l (MIC: 0.12-0.5 µg/mL, CC50: 128 and 16 µg/mL) showed excellent activity
which was no inferior to the parent moxifloxacin against all tested drug-susceptible
and drug-resistant MTB strains, and acceptable cytotoxicity towards VERO cells,
suggesting these two hybrids could act as a platform for further investigation.
Figure 2 The SAR and structure-cytotoxicity relationship of
moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids
11

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The SAR and structure-cytotoxicity relationship (Figure 2) revealed that the
substituents on the C-3, C-5 and C-7 positions of isatin motif influenced the activity and
cytotoxicity remarkably, and the enriched SAR and structure-cytotoxicity relationship
may help global efforts for identification of new chemical entities as potent anti-TB
agents.
4. Experimental section
4.1 Chemistry
All chemicals for this study were chemically pure or analytical grade. TLC were
1
performed on silica plates (Merk, ART5554 60F254). H NMR spectra were
determined on a Varian Mercury-400 instrument in CDCl3 or DMSO-d6. HRMS-ESI
were obtained on an MDSSCIEX Q-Tap mass spectrometer.
4.2 Synthesis
2-Bromoacetic acid 1 (20 mmol) and sodium azide (30 mmol) in H2O (100 mL) was
stirred at room temperature for 48 h, and then aq. HCl (12 mol/L, 10 mL) was added
dropwise. After stirring for additional 30 min, the mixture was extracted with methyl
tert-butyl ether (100 mL×3). The combined organic layers were washed with H2O
(100 mL), brine (100 mL×2), dried over anhydrous Na2SO4, filtered and concentrated
under reduced pressure to give 2-azidoacetic acid 2 (yield: 94%) as yellow oil.
To a solution of 2-azidoacetic acid 2 (18 mmol) and oxalyl chloride (50 mL) in DCM
(200 mL), 5 drops of DMF was added. The mixture was stirred at room temperature
overnight, and then evaporated under reduced pressure to give crude 2-azidoacetyl
chloride 3 as brown oil which was used directly in the next step.
To a solution of moxifloxacin (10 mmol) in a mixture of pyridine (50 mL) and DCM
(200 mL), a solution of 2-azidoacetyl chloride 3 (18 mmol) in DCM (50 mL) was
added dropwise. The mixture was stirred at room temperature overnight, and then
evaporated under reduced pressure. The residue was purified by silica gel
chromatography eluted with DCM:MeOH=10:1 to give the key intermediate
2-azidoacetyl moxifloxacin 4 (yield: 44%) as yellow solid.
12

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The suspension of isatin/5-methylisatin/5-fluoroisatin/7-fluoroisatin 5a-d (10 mmol),
propargyl bromide (20 mmol) and K2CO3 (30 mmol) in DMF (50 mL) was stirred at
room temperature overnight, and then the mixture was poured into ice-water (200 g).
The solid was filtered, washed with H2O (100 mL) and petroleum ether (20 mL) to
give crude N-propargyl isatin intermediates 6a-d as brown solid which was used
directly in the next step.
The mixture of N-propargyl isatins 6a-d (5 mmol), the requested amine hydrochlorides
(6 mmol) and NaHCO3 (10 mmol) in a mixture of THF (30 mmol) and H2O (30 mmol)
o
was stirred at 50 C for 12 h, and then cooled to room temperature. The mixture was
extracted with ethyl acetate (50 mL×2), and the combined organic layers were washed
with brine (50 mL×3), and then concentrated under reduced pressure to yield isatin
intermediates 7a-j as yellow solid which was used directly in the next step.
Finally, cyclization of N-propargyl isatin precursors 6a-d or 7a-j (1.1 mmol) with
2-azidoacetyl moxifloxacin 4 (1 mmol) by “click chemistry” in presence of copper
o
acetate (Cu(OAc)2, 0.1 mmol) in DMF (10 mL) at 40 C for 12 h. The mixture was
filtered, and the filtrated was concentrated under reduced pressure. The residue was
purified
by
Prep-HPLC
to
generate
the
desired
moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin hybrids 8a-n (yield: 12-46%).
4.2.1
1-cyclopropyl-7-((4aR,7aR)-1-(2-(4-((2,3-dioxoindolin-1-yl)methyl)-1H-1,2,3-triazol-
1-yl)acetyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-6-fluoro-8-methoxy-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (8a)
1
Yellow solid, yield: 41%. H NMR (400 MHz, DMSO-d6) δ 0.85-1.52 (m, 7H),
1.77-1.80 (m, 2H), 2.24-2.27 (m, 2H), 3.27-3.31 (m, 1H), 3.60 (d, 3H), 3.80-4.42 (m,
4H), 4.57 (s, 1H), 4.96 (s, 2H, -CH2- linker), 5.41-5.67 (m, 2H, -CH2- linker), 7.14 (t,
1H, J = 8.0 Hz, Ar-H), 7.18 (d, 1H, J = 8.0 Hz, Ar-H), 7.57 (d, 1H, J = 8.0 Hz, Ar-H),
7.62-7.66 (m, 2H, Ar-H), 8.09 (s, 1H, Ar-H), 8.66 (s, 1H, Ar-H), 15.14 (brs, 1H,
13
COOH). C NMR (101 MHz, DMSO-d6) δ 183.56, 181.55, 176.46, 175.27, 166.35,
158.82, 158.51, 158.28, 150.78, 150.66, 141.54, 140.54, 138.60, 134.95, 128.15,
125.85, 124.94, 123.88, 118.01, 111.72, 106.81, 81.90, 61.94, 61.51, 51.72, 50.74,
13

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41.14, 24.18, 24.06, 23.92, 10.40, 8.52. HRMS-ESI: m/z Calcd. for C34H33FN7O7
[M+H]+: 670.2420; Found: 670.2411.
4.2.2
1-cyclopropyl-6-fluoro-7-((4aR,7aR)-1-(2-(4-((5-fluoro-2,3-dioxoindolin-1-yl)methyl
)-1H-1,2,3-triazol-1-yl)acetyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-8-methoxy-4
-oxo-1,4-dihydroquinoline-3-carboxylic acid (8b)
1
Yellow solid, yield: 43%. H NMR (400 MHz, DMSO-d6) δ 0.86-1.53 (m, 6H),
1.77-1.80 (m, 3H), 2.32-2.44 (m, 1H), 2.74-2.78 (m, 1H), 3.60 (d, 3H), 3.80-4.04 (m,
4H), 4.14-4.16 (m, 1H), 4.86-4.88 (m, 1H), 5.00 (s, 2H, -CH2- linker), 5.41-5.71 (m,
2H, -CH2- linker), 7.20 (d, 1H, J = 4.0 Hz, Ar-H), 7.48-7.53 (m, 2H, Ar-H), 7.64-7.68
(m, 1H, Ar-H), 8.09 (s, 1H, Ar-H), 8.66 (s, 1H, Ar-H), 15.15 (brs, 1H, COOH).
HRMS-ESI: m/z Calcd. for C34H32F2N7O7 [M+H]+: 688.2326; Found: 688.2319.
4.2.3
1-cyclopropyl-6-fluoro-8-methoxy-7-((4aR,7aR)-1-(2-(4-((5-methyl-2,3-dioxoindolin
-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8c)
Yellow solid, yield: 36%. 1H NMR (400 MHz, CDCl3) δ 0.84-1.44 (m, 6H), 1.91-1.95
(m, 3H), 2.19 (s, 3H, -Me), 3.30-3.37 (m, 2H), 3.53-3.63 (m, 4H), 3.82-4.06 (m, 4H),
4.58-4.60 (m, 1H), 5.04 (s, 2H, -CH2- linker), 5.16-5.35 (m, 2H, -CH2- linker),
7.11-7.22 (m, 1H, Ar-H), 7.40 (s, 1H, Ar-H), 7.60 (d, 1H, J = 8.0 Hz, Ar-H), 7.82 (d,
2H, J = 8.0 Hz, Ar-H), 8.80 (s, 1H, Ar-H), 14.96 (brs, 1H, COOH). HRMS-ESI: m/z
Calcd. for C35H35FN7O7 [M+H]+: 684.2577; Found: 684.2568.
4.2.4
1-cyclopropyl-6-fluoro-7-((4aR,7aR)-1-(2-(4-((7-fluoro-2,3-dioxoindolin-1-yl)methyl
)-1H-1,2,3-triazol-1-yl)acetyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-8-methoxy-4
-oxo-1,4-dihydroquinoline-3-carboxylic acid (8d)
14

ACCEPTED MANUSCRIPT
Yellow solid, yield: 24%. 1H NMR (400 MHz, CDCl3) δ 0.83-1.60 (m, 8H), 2.42-2.48
(m, 1H), 3.30-3.49 (m, 3H), 3.59 (d, 3H), 3.82-4.02 (m, 4H), 4.52-4.60 (m, 1H),
5.13-5.38 (m, 4H, 2×-CH2- linker), 7.07-7.09 (m, 1H, Ar-H), 7.32-7.34 (m, 1H, Ar-H),
7.40 (d, 1H, J = 8.0 Hz, Ar-H), 7.72-7.86 (m, 2H, Ar-H), 8.09 (s, 1H, Ar-H), 8.76 (s,
1H, Ar-H), 15.00 (brs, 1H, COOH). HRMS-ESI: m/z Calcd. for C34H32F2N7O7
[M+H]+: 688.2326; Found: 688.2319.
4.2.5
1-cyclopropyl-6-fluoro-7-((4aR,7aR)-1-(2-(4-((3-(hydroxyimino)-2-oxoindolin-1-yl)
methyl)-1H-1,2,3-triazol-1-yl)acetyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-8-met
hoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8e)
1
Yellow solid, yield: 28%. H NMR (400 MHz, DMSO-d6) δ 0.87-1.53 (m, 7H),
1.76-1.80 (m, 2H), 2.25-2.28 (m, 2H), 3.29-3.33 (m, 1H), 3.62 (d, 3H), 3.82-4.14 (m,
4H), 4.61 (s, 1H), 4.98 (s, 2H, -CH2- linker), 5.42-5.61 (m, 2H, -CH2- linker), 7.12 (t,
1H, J = 8.0 Hz, Ar-H), 7.16 (d, 1H, J = 8.0 Hz, Ar-H), 7.54 (d, 1H, J = 8.0 Hz, Ar-H),
7.64-7.68 (m, 2H, Ar-H), 8.12 (s, 1H, Ar-H), 8.68 (s, 1H, Ar-H), 12.42 (brs, 1H,
NOH), 15.12 (brs, 1H, COOH). HRMS-ESI: m/z Calcd. for C34H34FN8O7 [M+H]+:
685.2529; Found: 685.2524.
4.2.6
1-cyclopropyl-6-fluoro-7-((4aR,7aR)-1-(2-(4-((5-fluoro-3-(hydroxyimino)-2-oxoindol
in-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl
)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8f)
1
Yellow solid, yield: 37%. H NMR (400 MHz, DMSO-d6) δ 0.88-1.52 (m, 6H),
1.78-1.80 (m, 3H), 2.35-2.45 (m, 1H), 2.76-2.79 (m, 1H), 3.61 (d, 3H), 3.80-4.08 (m,
4H), 4.14-4.16 (m, 1H), 4.85-4.88 (m, 1H), 5.01 (s, 2H, -CH2- linker), 5.44-5.65 (m,
2H, -CH2- linker), 7.18 (d, 1H, J = 4.0 Hz, Ar-H), 7.39-7.46 (m, 2H, Ar-H), 7.62-7.65
(m, 1H, Ar-H), 8.08 (s, 1H, Ar-H), 8.65 (s, 1H, Ar-H), 12.38 (brs, 1H, NOH), 15.18
(brs, 1H, COOH). HRMS-ESI: m/z Calcd. for C34H33F2N8O7 [M+H]+: 703.2435;
Found: 703.2427.
15

ACCEPTED MANUSCRIPT
4.2.7
1-cyclopropyl-6-fluoro-7-((4aR,7aR)-1-(2-(4-((-7-fluoro-3-(hydroxyimino)-2-oxoindo
lin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-y
l)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8g)
1
Yellow solid, yield: 19%. H NMR (400 MHz, DMSO-d6) δ 0.87-1.79 (m, 8H),
2.25-2.42 (m, 1H), 3.18-4.12 (m, 9H), 5.06-5.09 (m, 3H), 5.38-5.61 (m, 2H, -CH2-
linker), 7.09-7.11 (m, 1H, Ar-H), 7.29-7.34 (m, 1H, Ar-H), 7.62-7.66 (m, 1H, Ar-H),
7.88 (d, 1H, J = 4.0 Hz, Ar-H), 7.97 (s, 1H, Ar-H), 8.66 (s, 1H, Ar-H), 15.14 (brs, 1H,
COOH). HRMS-ESI: m/z Calcd. for C34H33F2N8O7 [M+H]+: 703.2435; Found:
703.2427.
4.2.8
1-cyclopropyl-6-fluoro-8-methoxy-7-((4aR,7aR)-1-(2-(4-((3-(methoxyimino)-2-oxoin
dolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6
-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8h)
1
Yellow solid, yield: 35%. H NMR (400 MHz, DMSO-d6) δ 0.81-1.42 (m, 6H),
2.25-2.39 (m, 3H), 3.29-3.50 (m, 3H), 3.60 (d, 3H), 3.79-4.09 (m, 3H), 4.28 (d, 3H,
NOMe), 4.53-4.56 (m, 1H), 5.04-5.37 (m, 4H, 2×-CH2- linker), 7.05 (t, 1H, J = 8.0
Hz, Ar-H), 7.15-7.19 (m, 1H, Ar-H), 7.35-7.39 (m, 1H, Ar-H), 7.53-7.78 (m, 2H,
Ar-H), 7.62-7.66 (m, 2H, Ar-H), 7.92 (d, 1H, J = 8.0 Hz, Ar-H), 8.81 (s, 1H, Ar-H),
15.02 (brs, 1H, COOH). HRMS-ESI: m/z Calcd. for C35H36FN8O7 [M+H]+: 699.2686;
Found: 699.2678.
4.2.9
1-cyclopropyl-6-fluoro-7-((4aR,7aR)-1-(2-(4-((5-fluoro-3-(methoxyimino)-2-oxoindo
lin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-y
l)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8i)
Yellow solid, yield: 39%. 1H NMR (400 MHz, CDCl3) δ 0.84-1.52 (m, 8H), 2.32-2.39
(m, 1H), 3.30-3.36 (m, 2H), 3.51-3.62 (m, 4H), 3.82-4.06 (m, 4H), 4.31 (d, 3H,
NOMe), 4.54-4.58 (m, 1H), 5.07 (s, 2H, -CH2- linker), 5.27-5.38 (m, 2H, -CH2-
linker), 7.08-7.18 (m, 2H, Ar-H), 7.66 (d, 1H, J = 8.0 Hz, Ar-H), 7.72-7.79 (m, 2H,
16

ACCEPTED MANUSCRIPT
13
Ar-H), 8.78 (s, 1H, Ar-H). C NMR (101 MHz, CDCl3) δ 176.72, 173.46, 167.17,
164.69, 163.14, 161.47, 149.83, 142.52, 139.07, 134.35, 133.05, 124.96, 119.04,
116.15, 115.45, 115.19, 110.57, 108.14, 107.90, 107.59, 105.63, 65.10, 61.24, 56.31,
51.49, 51.07, 49.27, 48.20, 41.12, 40.50, 35.46, 24.93, 18.73, 10.51, 8.59. HRMS-ESI:
m/z Calcd. for C35H35F2N8O7 [M+H]+: 717.2591; Found: 717.2582.
4.2.10
1-cyclopropyl-6-fluoro-8-methoxy-7-((4aR,7aR)-1-(2-(4-((3-(methoxyimino)-5-meth
yl-2-oxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetyl)octahydro-6H-pyrrolo[3,4-
b]pyridin-6-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8j)
1
Yellow solid, yield: 46%. H NMR (400 MHz, CDCl3) δ 0.84-1.60 (m, 8H), 2.32 (s,
4H), 3.30-3.34 (m, 2H), 3.53-3.89 (m, 5H), 4.00-4.09 (m, 2H), 4.30 (d, 3H, NOMe),
4.54-4.58 (m, 1H), 5.07 (s, 2H, -CH2- linker), 5.15-5.32 (m, 3H), 7.06 (d, 1H, J = 8.0
Hz, Ar-H), 7.20 (d, 1H, J = 8.0 Hz, Ar-H), 7.77-7.83 (m, 3H), 8.80 (s, 1H, Ar-H),
14.98 (brs, 1H, COOH). HRMS-ESI: m/z Calcd. for C36H38FN8O7 [M+H]+: 713.2842;
Found: 713.2836.
4.2.11
1-cyclopropyl-6-fluoro-7-((4aR,7aR)-1-(2-(4-((7-fluoro-3-(methoxyimino)-2-oxoindo
lin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-y
l)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8k)
Yellow solid, yield: 27%. 1H NMR (400 MHz, CDCl3) δ 0.83-1.92 (m, 8H), 2.30-2.32
(m, 1H), 3.29-3.32 (m, 2H), 3.50-3.61 (m, 4H), 3.83-4.00 (m, 4H), 4.30 (d, 3H,
NOMe), 4.52-4.58 (m, 1H), 5.14-5.34 (m, 5H), 6.98-7.02 (m, 1H, Ar-H), 7.11-7.16
(m, 1H, Ar-H), 7.75-7.78 (m, 3H, Ar-H), 8.77 (s, 1H, Ar-H), 14.99 (brs, 1H, COOH).
HRMS-ESI: m/z Calcd. for C35H35F2N8O7 [M+H]+: 717.2591; Found: 717.2587.
4.2.12
7-((4aR,7aR)-1-(2-(4-((3-(2-carbamothioylhydrazono)-2-oxoindolin-1-yl)methyl)-1H-
1,2,3-triazol-1-yl)acetyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-1-cyclopropyl-6-fl
uoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8l)
17

ACCEPTED MANUSCRIPT
Yellow solid, yield: 19%. 1H NMR (400 MHz, CDCl3) δ 0.83-1.93 (m, 9H), 3.30-3.35
(m, 2H), 3.52-3.61 (m, 4H), 3.82-4.12 (m, 4H), 5.08 (s, 2H, -CH2- linker), 5.26-5.39
(m, 2H, -CH2- linker), 6.71 (s, 1H, NH), 7.09-7.12 (m, 1H, Ar-H), 7.25-7.27 (m, 1H,
Ar-H), 7.39-7.41 (m, 1H, Ar-H), 7.52-7.55 (m, 2H, Ar-H and NH), 7.79-7.86 (m, 2H,
Ar-H), 8.79 (s, 1H, Ar-H), 12.78 (s, 1H, NNHCS), 14.98 (brs, 1H, COOH).
HRMS-ESI: m/z Calcd. for C35H36FN10O6S [M+H]+: 743.2513; Found: 743.2519.
4.2.13
7-((4aR,7aR)-1-(2-(4-((3-(2-carbamothioylhydrazono)-5-fluoro-2-oxoindolin-1-yl)me
thyl)-1H-1,2,3-triazol-1-yl)acetyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-1-cyclopr
opyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8m)
Yellow solid, yield: 19%. 1H NMR (400 MHz, CDCl3) δ 0.83-1.64 (m, 8H), 2.23-2.24
(m, 1H), 3.30-3.35 (m, 2H), 3.52-3.62 (m, 4H), 3.74-4.05 (m, 4H), 4.58-4.59 (m, 1H),
5.07 (s, 2H, -CH2- linker), 5.26-5.37 (m, 2H, -CH2- linker), 6.71 (s, 1H, NH),
7.09-7.11 (m, 1H, Ar-H), 7.22-7.27 (m, 2H, Ar-H), 7.54-7.56 (m, 1H, Ar-H),
7.79-7.87 (m, 2H, Ar-H and NH), 8.80 (s, 1H, Ar-H), 12.78 (s, 1H, NNHCS).
HRMS-ESI: m/z Calcd. for C35H35F2N10O6S [M+H]+: 761.2424; Found: 761.2421.
4.2.14
7-((4aR,7aR)-1-(2-(4-((3-(2-carbamothioylhydrazono)-7-fluoro-2-oxoindolin-1-yl)me
thyl)-1H-1,2,3-triazol-1-yl)acetyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-1-cyclopr
opyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8n)
Yellow solid, yield: 11%. 1H NMR (400 MHz, CDCl3) δ 0.83-1.96 (m, 8H), 2.33-2.34
(m, 1H), 3.30-3.35 (m, 2H), 3.52-3.62 (m, 4H), 3.82-4.10 (m, 4H), 4.70-4.72 (m, 1H),
5.24-5.38 (m, 4H, 2×-CH2- linker), 6.68 (s, 1H, NH), 7.02-7.14 (m, 2H, Ar-H),
7.27-7.29 (m, 1H, Ar-H), 7.58 (s, 1H, NH), 7.80-7.89 (m, 2H, Ar-H), 8.80 (s, 1H,
Ar-H), 12.76 (s, 1H, NNHCS), 14.97 (brs, 1H, COOH). HRMS-ESI: m/z Calcd. for
C35H35F2N10O6S [M+H]+: 761.2424; Found: 761.2422.
4.3. MIC determination
18

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The in vitro activities of moxifloxacin-acetyl-1,2,3-1H-triazole-methylene-isatin
hybrids 8a-n, along with the references gatifloxacin, moxifloxacin, isoniazid and
rifampicin against MTB H37Rv, RR-MTB and MDR-MTB were evaluated via rapid
direct susceptibility test technique [25,26]. The wells of a sterile 48-well plate were
filled with 100 mL two-fold diluted tested compounds and 100 mL MTB H37Rv (or
MDR-TB) suspension containing 4×10-3 mg cells. The plates were incubated at 37 °C
in a wet box. 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.
4.4. Cytotoxicity
The hybrids 8a-n were dissolved in DMSO and examined for toxicity (CC50) in a
mammalian CHO cell line at concentrations from 128 to 4 µg/mL [33]. 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.
4.5 Pharmacokinetic Profiles determination
SPF female ICR mice (20-25 g) were used in the pharmacokinetic study [34,35]. Total
area under the concentration time curve (AUC), the elimination half-time (t1/2), the
peak concentration (Cmax) and the time to reach peak concentration (Tmax) of samples
were etermined directly from the experimental data using WinNonlin V6.2.1.
Acknowledgements
This study was supported by grants from the Natural Science Foundation of Jiangsu
Province (Grant No. BK20170626 to G. F.) and China Postdoctoral Science
Foundation (Grant No. 2016M603061 to G.F.).
19

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ACCEPTED MANUSCRIPT
1. Hybrids 8h and 8l (MIC: 0.12-0.5 µg/mL) were no inferior to the parent
moxifloxacin against all tested strains
2. The structure-activity relationship and structure-cytotoxicity relationship were
enriched