Azide-alkyne cycloaddition towards 1H-1,2,3-triazole-tethered gatifloxacin and isatin conjugates: Design, synthesis and in?vitro anti-mycobacterial evaluation

Article abstract of DOI:10.1016/j.ejmech.2017.05.057

Twelve novel 1H-1,2,3-triazole-tethered gatifloxacin (GTFX) isatin conjugates 5a-l with greater lipophilicity compared with GTFX were designed, synthesized and evaluated for their in vitro anti-mycobacterial activities against M. tuberculosis (MTB) H₃₇Rv and MDR-TB as well as cytotoxicity. The preliminary results showed that all the targets (MIC: 0.10–8 μg/mL) exhibited excellent inhibitory activity against MTB H₃₇Rv and MDR-TB, but eight of them (CC₅₀: 7.8–62.5 μg/mL) were much more toxic than the parent GTFX (CC₅₀: 125 μg/mL). Among them, 5g (MIC: 0.10 μg/mL) was 4–8 times more potent in vitro than the references GTFX (MIC: 0.78 μg/mL) and RIF (MIC: 0.39 μg/mL) against MTB H₃₇Rv, but less active than INH (MIC: 0.05 μg/mL). The most potent 5g and 5h (MIC: 0.25 μg/mL) were 4->512 times more active than the three references (MIC: 1.0->128 μg/mL) against MDR-TB. Unfortunately, both of the two hybrids (CC₅₀: 7.8 μg/mL) were much more cytotoxic than the other derivatives, need to be further optimized.

Full text of DOI:10.1016/j.ejmech.2017.05.057

Accepted Manuscript
Azide-alkyne cycloaddition towards 1H-1,2,3-triazole-tethered gatifloxacin and isatin
conjugates: Design, synthesis and in vitro anti-mycobacterial evaluation
Xianghua Xia, Qianhua Zhang, Lichun Zhao, Yuanqiang Hu
PII:
S0223-5234(17)30428-2
10.1016/j.ejmech.2017.05.057
EJMECH 9487
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 February 2017
Revised Date: 26 May 2017
Accepted Date: 27 May 2017
Please cite this article as: X. Xia, Q. Zhang, L. Zhao, Y. Hu, Azide-alkyne cycloaddition towards
1H-1,2,3-triazole-tethered gatifloxacin and isatin conjugates: Design, synthesis and in vitro
anti-mycobacterial evaluation, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.05.057.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
Azide-alkyne cycloaddition towards 1H-1,2,3-triazole-tethered
gatifloxacin and isatin conjugates: design, synthesis and in
vitro anti-mycobacterial evaluation
Xianghua Xiaa, Qianhua Zhangb, Lichun Zhao b, , Yuanqiang Huc,
aInstitute of Guangxi Medicinal Plant, National Engineering Laboratory of Southwest Endangered Medicinal
Resources Development, Nanning, P R China;
bCollege of Pharmacy, Guangxi University of Chinese Medicine, Nanning, P R China;
cSchool of Chemistry and Materials Science, Hubei Engineering University, Hubei, P R China.
A new class of 1H-1,2,3-triazole-tethered gatifloxacin isatin conjugates (MIC: 0.10-8
µg/mL) with greater lipophilicity compared with gatifloxacin exhibited excellent
inhibitory activity against MTB H37Rv and MDR-TB.

ACCEPTED MANUSCRIPT
Azide-alkyne cycloaddition towards 1H-1,2,3-triazole-tethered
gatifloxacin and isatin conjugates: design, synthesis and in
vitro anti-mycobacterial evaluation
Zhi Xua, Xu-Feng Songb, Yuan-Qiang Huc, Min Qianga, Zao-Sheng Lva,
aWuhan University of Science and Technology, 430081, Hubei, P R China;
bBeijing University of Technology, 100124, Beijing, P R China;
cSchool of Chemistry and Materials Science, Hubei Engineering University, Hubei, P R China.
Abstract: Twelve novel 1H-1,2,3-triazole-tethered gatifloxacin (GTFX) isatin conjugates 5a-l
with greater lipophilicity compared with GTFX were designed, synthesized and evaluated for
their in vitro anti-mycobacterial activities against M. tuberculosis (MTB) H37Rv and MDR-TB as
well as cytotoxicity. The preliminary results showed that all the targets (MIC: 0.10-8 µg/mL)
exhibited excellent inhibitory activity against MTB H37Rv and MDR-TB, but eight of them (CC50:
7.8-62.5 µg/mL) were much more toxic than the parent GTFX (CC50: 125 µg/mL). Among them,
5g (MIC: 0.10 µg/mL) was 4-8 times more potent in vitro than the references GTFX (MIC: 0.78
µg/mL) and RIF (MIC: 0.39 µg/mL) against MTB H37Rv, but less active than INH (MIC: 0.05
µg/mL). The most potent 5g and 5h (MIC: 0.25 µg/mL) were 4->512 times more active than the
three references (MIC: 1.0->128 µg/mL) against MDR-TB. Unfortunately, both of the two hybrids
(CC50: 7.8 µg/mL) were much more cytotoxic than the other derivatives, need to be further
optimized.
Keywords: Gatifloxacin; Isatin; Triazole; Conjugates; Anti-tuberculosis activity;
Anti-mycobacterial activity; Structure-activity relationship
1. Introduction
Tuberculosis (TB) remains one of the most widespread and leading deadliest diseases
result in 1.4 million deaths and 10.4 million clinical cases, and is in continual increase,
*Corresponding author email address: chemorgchem@126.com.
1

ACCEPTED MANUSCRIPT
especially in developing countries according to the World Health Organization (WHO)
2016 report [1]. The emergence of drug-resistant TB (DR-TB), multidrug-resistant TB
(MDR-TB, only 40-80% cure rate, results in 0.48 million deaths in 2015), extensively
drug-resistant TB (XDR-TB) and the recently cases of totally drug resistant (TDR) as
well as co-infection with HIV (a major cause of death among people living with
HIV/AIDS, leads to 0.4 million deaths in 2015) alarming the serious problem in TB
control and demanding the need for new drugs more potent than earlier with safe
ADME profile. However, no new chemical entity has been emerged in last 50 years
after the discovery of rifampicin (RIF), creating an urgent need to develop new drugs
and strategies for efficient treatment. Several strategies are being pursued in order to
identify new leads, although only a few leads are being optimized to generate drug
candidates. Most of the efforts have been directed towards making new analogues or
modifying old drugs or existing compounds with an established activity for shortening
and improving TB treatment, since it’s the most promising strategy to develop new
anti-TB agents in the short time.
The isatin (1H-indole-2,3-dione, Fig. 1) moiety is a privileged scaffold for chemical
modification and is responsible for a broad spectrum of biological properties such as
anti-TB, anti-bacterial, anti-fungal, anti-virus, anti-tumor, anti-HIV, and well
tolerance in humans [2-4]. The traditional antibiotics fluoroquinolones (FQs, Fig. 1)
exhibited excellent anti-bacterial activity, and some of them are currently
recommended as the second-line agents by the WHO for the treatment of TB [5].
Moreover, the lipophilicity of the FQs plays an important role in the penetration of
these compounds into bacterial cells, which suggests that increasing the lipophilic
character at C-7 position could also increase the anti-TB activity [6]. Therefore,
several series of FQs-isatin derivatives with remarkable improvement in lipophilicity
have been synthesized and their anti-TB activity has been explored [7-12]. Among
these derivatives, gatifloxacin (GTFX, Fig. 1, has been withdrawn from the therapy
because of its side effects (dysglycaemia), but still worth to be investigated attribute
2

ACCEPTED MANUSCRIPT
to its excellent in vitro and in vivo anti-TB activities) methylene isatin hybrid 1
exhibited higher in vitro (16 and 64 folds against MTB H37Rv and MDR-TB) and in
vivo potency than the parent GTFX [6]. Further investigation indicated that the
anti-TB activity of these derivatives was greatly influenced by the linkers between
FQs and isatin [7-12]. Chemical structures of isatin, FQs, GTFX, GTFX-isatin
derivative 1 and I-A09 are shown in Fig. 1.
< Fig 1. Chemical structures of isatin, FQs, GTFX, GTFX-isatin derivative 1 and I-A09>
Azoles, are one of the most important classes of nitrogen containing heterocycles,
exhibited various biological activities. In particular, 1,2,3-triazole and its derivatives,
which are easily to be obtained by ‘Click chemistry’, have attracted continuous
interest. Indeed this particular application of ‘Click chemistry’ has become one of the
mainstays of currently favored approaches for the introduction of structural diversity
in medicinal chemistry. Moreover, some drugs currently in use are based on
1,2,3-triazole moiety such as anti-HIV agent TSAO, antibiotic Cefatrizine,
anti-bacterial agent Tazobactum as well as anti-cancer agent CAI [13]. In addition,
I-A09 is in clinical evaluations currently, and may be used to treat TB infection in the
3

ACCEPTED MANUSCRIPT
near future [14]. Obviously, the favorable properties of 1,2,3-triazole ring like
moderate dipole character, hydrogen bonding capability, rigidity and stability under in
vivo conditions are responsible for their enhanced biological activities [15].
< Fig 2. Illustration of the design strategy for triazole-tethered GTFX-isatin derivatives>
Inspired by the above research results, we designed and synthesized a series of
triazole-tethered GTFX-isatin derivatives in this study. Illustration of the design
strategy for triazole-tethered GTFX-isatin derivatives is depicted in Fig 2. These
derivatives were initially evaluated for their in vitro anti-mycobacterial activity
against MTB H37Rv and MDR-TB strains, with Isoniazid (INH), rifampicin (RIF)
and GTFX as references.
2. Results and discussion
Detailed synthetic pathways to triazole-tethered GTFX-isatin derivatives 5a-l are
depicted in Scheme 1. C-5 substituted isatins and GTFX were alkylated with
1,2-dibromoethane and propargyl bromide, respectively, in the presence of anhydrous
potassium carbonate to give the corresponding N-(2-bromoethyl)isatins 2a-d (yield:
51-67%) and propargyl GTFX 4 (yield: 39%) via literature methods [10, 16, 17]. The
subsequent treatment of C-5 substituted N-(2-bromoethyl)isatins 2a-d with sodium
azide at 60 °C resulted in the formation of the desired azido precursors 3a-d [18]. The
4

ACCEPTED MANUSCRIPT
precursors 3a-d and 4 were utilized for the synthesis of desired 1,2,3-triazole-tethered
conjugates via Cu-promoted azide-alkyne cycloaddition reaction in the presence of
CuI in DMF gave targets 5a-d (yield: 28-39%) [17]. Subsequent condensations of
targets 5a-d with requisite substituted amine hydrochlorides in the presence of sodium
bicarbonate formed other derivatives 5e-l (43-67%) [10].
< Scheme 1 Synthesis of 1H-1,2,3-triazole-tethered GTFX isatin conjugates 5a-l>
All hybrids 5a-l (Log P: 2.32-3.53) with greater lipophilicity compared with GTFX
(Log P: 1.51), and this profile may be rendering them more capable of penetrating
various biomembrane, consequently improving their permeation properties toward
mycobacterial cell membrane. The conjugates 5a-l were initially evaluated for their in
vitro anti-mycobacterial activity against MTB H37Rv and MDR-TB by rapid direct
susceptibility test technique [10]. The MDR-TB strain was resistant to INH, RIF and
ethambutol (EMB). The minimum inhibitory concentration (MIC) is defined as the
5

ACCEPTED MANUSCRIPT
minimum concentration of compound required to give 90% inhibition of bacterial
growth and MICs of the compounds are reported in Table 1. All triazole-tethered
GTFX-isatin conjugates exhibited considerable in vitro activity against MTB H37Rv
and MDR-TB with MIC ranging from 0.10 to 8 µg/mL. Against MTB H37Rv, three of
them (5c, 5g and 5h) were more active than that of the parent GTFX (MIC: 0.78
µg/mL), particularly, 5g (MIC: 0.10 µg/mL) was 4-8 times more potent in vitro than
the references GTFX (MIC: 0.78 µg/mL) and RIF (MIC: 0.39 µg/mL) against MTB
H37Rv, but less active than INH (MIC: 0.05 µg/mL). Against MDR-TB, five
conjugates (5c, 5d, 5f, 5g and 5h) with MIC of 0.25-0.5 µg/mL were more active than
the references GTFX (MIC: 1.0 µg/mL), INH (MIC: 32 µg/mL) and RIF (MIC: >128
µg/mL), and the most potent derivatives 5g and 5h (MIC: 0.25 µg/mL) were 4->512
times more active than the three references.
The lipophilicity of the synthesized targets (Clog P: 2.32-3.46) was much greater than
the parent GTFX (Clog P: 1.51), to some extent, increasing the lipophilicity of targets
could increase the anti-TB activity as evidenced by the fact that the most lipophilic
targets (5c, 5g and 5k) in each series also exhibited the highest activity. However, in
general, the anti-TB activity did not boost up with the incensement of the lipophilicity,
indicating simply increasing lipophilicity of the tested compounds does not improve
the anti-mycobacterial activity accordingly which could also be associated with other
steric factors [10,11].
The resistance index (RI: MICMDR-TB : MICMTB H Rv) of the targets was in a range of
37
0.32 to 2.56, and eight of them showed RI around 1. In particular, the RI of 5b, 5d
and 5f was less than 1, indicated this kind of hybrids could reduce the cross-resistant
to some extent.
Table 1
Structures, lipophilicity, anti-mycobacterial activity and cytotoxicity of compounds 5a-l
6

ACCEPTED MANUSCRIPT
MIC (µg/mL)
Compd.
R1
R2
Clog P a
CC50 c(µg/mL)
MTB H37Rv
1.56±0.52
1.56±0.52
0.39±0.13
0.78±0.26
0.78±0.26
1.56±0.52
0.10±0.03
0.20±0.06
3.12±1.04
6.25±2.08
0.78±0.26
1.56±0.52
0.78±0.26
bMDR-TB
4.0±1.3
O
O
H
Me
Cl
F
2.32
2.81
2.88
2.48
2.97
3.46
3.53
3.13
2.71
3.20
3.27
2.85
1.51
125±41.7
250±83.3
62.5±20.8
125±41.7
62.5±20.8
31.2±10.4
7.8±2.6
5a
5b
5c
1.0±0.33
0.5±0.17
0.5±0.17
1.0±0.33
0.5±0.17
0.25±0.08
0.25±0.08
8.0±2.7
O
O
5d
5e
NOCH3
NOCH3
NOCH3
NOCH3
NOH
NOH
NOH
NOH
H
Me
Cl
F
5f
5g
7.8±2.6
5h
5i
H
250±83.3
62.5±20.8
31.2±10.4
7.8±2.6
Me
Cl
F
8.0±2.7
5j
2.0±0.67
1.0±0.33
1.0±0.33
5k
5l
125±41.7
GTFX
7

ACCEPTED MANUSCRIPT
-0.67
3.71
0.05±0.16
0.39±0.13
>128
125±41.7
500±167
INH
RIF
32±10.7
aThe Clog P is calculated with ChemOffice 2004 software.
bMDR-TB: resistant to INH, RIF and EMB.
cCC50: The 50% cytotoxic concentration in a mammalian VERO cell line.
Against MTB H37Rv, the relatively order of substituents at C-5 position on isatin
affecting activity was -Cl > -F > -H > -Me, while against MDR-TB, the order was -F >
-Cl > -Me > -H. The relative contribution of imines/carbonyl of Schiff’s base to
activity against both strains is as follows: methyloxime > carbonyl > oxime. The SAR
revealed that conjugates with electron-withdrawing groups (-Cl and -F) at C-5
position on isatin boost up the activity against both MTB H37Rv and MDR-TB strains,
suggesting further modification should focus on introduction of various
electron-withdrawing groups like -NO2.
The conjugates 5a-l were subsequently examined for toxicity (CC50) in a mammalian
VERO cell line [10]. 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. Eight of
them (CC50: 7.8-62.5 µg/mL) were much more toxic than the parent GTFX (CC50:
125 µg/mL), and four hybrids (CC50: 125-250 µg/mL) were equal to or less toxic than
the parent. Unfortunately, the cytotoxicity of the most potency 5g and 5h (CC50:
7.8µg/mL) is much more than the other derivatives. In general, the carbonyl hybrids
were less toxic than methyloxime and oxime derivatives, indicating the introduction
of imines increased the cytotoxicity. In addition, halogen atoms at C-5 position on
isatin also increased the cytotoxicity.
In summary, a series of novel triazole-tethered GTFX isatin conjugates with greater
lipophilicity compared with GTFX were synthesized and evaluated for their in vitro
anti-mycobacterial activity against MTB H37Rv and MDR-TB. All the targets
8

ACCEPTED MANUSCRIPT
exhibited excellent inhibitory activity against the tested MTB H37Rv and MDR-TB,
and the most active derivatives were more potent than the parent GTFX, INH and
RIF against both strains. The SAR of this kind of hybrids was enriched, and the
results warrant further development of the anti-TB properties of
1,2,3-triazole-tethered FQs isatin conjugates.
3. Experimental section
3.1.Synthesis
3.1.1. General Procedure for the Preparation of 5a-d
N-(2-azidoethyl)isatins 3a-d and propargyl GTFX 4 (yield: 39%) were prepared via
literature methods [10, 16, 17]. To a mixture of N-(2-azidoethyl)isatins 3a-d (1.0
mmol) and propargyl GTFX 4 (1.0 mmol) in DMF (50 mL), CuI (30 mg) was added
under N2 atmosphere. The mixture was allowed to react for 4 h at room temperature.
After removal of the solvent, the residue was purified by silica gel column
chromatography eluted with DCM to v(DCM):v(MeOH)=10:1.
3.1.1.1. 1-cyclopropyl-7-(4-((1-(2-(2,3-dioxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)
methyl)-3-methylpiperazin-1-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-ca
rboxylic acid (5a)
1
Yellow solid, yield: 36%. Mp: 167-169 °C. H NMR (400 MHz, DMSO-d6) δ
1.02-1.22 (7H, m, 2×cyclopropyl-CH2 and -CH3), 2.31-2.37 (2H, m, piperazine-2H),
2.62-2.64 (1H, m, piperazine-1H), 2.94-3.26 (4H, m, piperazine-4H), 3.61-3.83 (5H,
m, -CH2 chain and -OCH3), 4.01-4.14 (3H, m, cyclopropyl-CH and -CH2 of linker),
4.66 (2H, s, -CH2 of linker), 6.91 (1H, d, Ar-H), 7.04 (1H, t, Ar-H), 7.49-7.57 (2H, m,
Ar-H), 7.70 (1H, s, Ar-H), 8.13 (1H, s, triazole-H), 8.69 (1H, s, C2-H), 14.91 (1H, brs,
13
COOH). C NMR (100 MHz, DMSO-d6) δ 9.38, 16.10, 41.19, 47.33, 48.13, 50.98,
51.25, 54.32, 57.46, 63.43, 106.92, 107.12, 110.71, 117.74, 121.21, 123.65, 124.93,
125.17, 125.34, 134.56, 138.59, 139.36, 139.49, 143.27, 146.16, 150.76, 150.97,
158.54, 166.21, 176.67, 183.39. ESI-MS m/z: 630 [M+H]+.
3.1.1.2. 1-cyclopropyl-6-fluoro-8-methoxy-7-(3-methyl-4-((1-(2-(5-methyl-2,3-dioxoi
ndolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-4-oxo-1,4-dihydroquin
9

ACCEPTED MANUSCRIPT
oline-3-carboxylic acid (5b)
1
Light yellow solid, yield: 28%. Mp: 154-155 °C. H NMR (400 MHz, DMSO-d6) δ
1.03-1.13 (7H, m, 2×cyclopropyl-CH2 and -CH3), 2.20 (3H, s, -CH3), 2.33-2.36 (2H,
m, piperazine-2H), 2.62 (1H, d, piperazine-1H), 2.96 (1H, t, piperazine-1H),
3.23-3.31 (3H, m, piperazine-3H), 3.70 (3H, s, -OCH3), 3.73 (2H, q, -CH2 chain),
4.10-4.16 (3H, m, cyclopropyl-CH and -CH2 of linker), 4.63-4.66 (2H, m, -CH2 of
linker), 6.76 (1H, d, Ar-H), 7.32 (1H, s, Ar-H), 7.36 (1H, d, Ar-H), 7.73 (1H, d, Ar-H),
13
8.10 (1H, s, triazole-H), 8.77 (1H, s, C2-H), 14.95 (1H, brs, COOH). C NMR (100
MHz, DMSO-d6) δ 8.92, 15.58, 19.92, 40.79, 46.91, 47.67, 50.48, 50.50, 50.79, 53.83,
56.99, 62.97, 106.50, 110.01, 117.21, 120.63, 120.72, 124.66, 132.49, 134.13, 138.34,
138.97, 139.09, 142.81, 145.75, 148.17, 150.48, 154.17, 156.65, 158.10, 165.64,
176.27, 183.11. ESI-MS m/z: 664 [M+H]+.
3.1.1.3. 7-(4-((1-(2-(5-chloro-2,3-dioxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)meth
yl)-3-methylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquin
oline-3-carboxylic acid (5c)
1
Light yellow solid, yield: 39%. Mp: 182-183°C. H NMR (400 MHz, DMSO-d6) δ
1.02-1.14 (7H, m, 2×cyclopropyl-CH2 and -CH3), 2.31-2.35 (2H, m, piperazine-2H),
2.64 (1H, d, piperazine-1H), 2.96 (1H, t, piperazine-1H), 3.21-3.30 (3H, m,
piperazine-3H), 3.64-3.83 (5H, m, -CH2 chain and -OCH3), 4.15-4.17 (3H, m,
cyclopropyl-CH and -CH2 of linker), 4.65 (2H, s, -CH2 of linker), 6.89 (1H, d, Ar-H),
7.53-7.58 (2H, m, Ar-H), 7.71-7.74 (1H, m, Ar-H), 8.15 (1H, s, triazole-H), 8.81 (1H,
13
s, C2-H), 14.95 (1H, brs, COOH). C NMR (100 MHz, DMSO-d6) δ 9.45, 16.07,
41.27, 47.41, 48.12, 48.52, 50.97, 51.31, 54.24, 57.44, 63.51, 107.00, 112.38, 113.27,
119.08, 121.21, 124.40, 125.30, 127.82, 134.60, 137.39, 139.46, 143.25, 146.26,
149.31, 150.96, 154.67, 157.15, 158.33, 166.14, 176.74, 182.29. ESI-MS m/z: 664
[M+H]+.
3.1.1.4. 1-cyclopropyl-6-fluoro-7-(4-((1-(2-(5-fluoro-2,3-dioxoindolin-1-yl)ethyl)-1H
-1,2,3-triazol-4-yl)methyl)-3-methylpiperazin-1-yl)-8-methoxy-4-oxo-1,4-dihydroquin
oline-3-carboxylic acid (5d)
1
Light yellow solid, yield: 33%. Mp: 171-174 °C. H NMR (400 MHz, DMSO-d6) δ
0.92-1.08 (7H, m, 2×cyclopropyl-CH2 and -CH3), 2.31-2.37 (2H, m, piperazine-2H),
2.62 (1H, d, piperazine-1H), 2.90-2.93 (1H, m, piperazine-1H), 3.19-3.33 (3H, m,
piperazine-3H), 3.64-3.83 (5H, m, -CH2 chain and -OCH3), 4.01-4.14 (3H, m,
10

ACCEPTED MANUSCRIPT
cyclopropyl-CH and -CH2 of linker), 4.65 (2H, s, -CH2 of linker), 6.90 (1H, s, Ar-H),
7.42-7.72 (3H, m, Ar-H), 8.13 (1H, s, triazole-H), 8.69 (1H, s, C2-H). ESI-MS m/z:
648 [M+H]+.
3.1.2. The general procedure for preparing targets 5e-l
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 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 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 5e-l
(43-67%).
3.1.2.1. 1-cyclopropyl-6-fluoro-8-methoxy-7-(4-((1-(2-(3-(methoxyimino)-2-oxoindoli
n-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-3-methylpiperazin-1-yl)-4-oxo-1,4-dihydro
quinoline-3-carboxylic acid (5e)
1
Light yellow solid, yield: 67%. Mp: 136-137 °C. H NMR (400 MHz, DMSO-d6) δ
1.03-1.14 (7H, m, 2×cyclopropyl-CH2 and -CH3), 2.31-2.37 (2H, m, piperazine-2H),
2.63 (1H, d, piperazine-1H), 2.96 (1H, t, piperazine-1H), 3.21-3.31 (3H, m,
piperazine-3H), 3.70 (3H, s, -OCH3), 3.72 (2H, q, -CH2 chain), 4.09-4.17 (6H, m,
cyclopropyl-CH, -CH2 of linker and NOCH3), 4.66 (2H, s, -CH2 of linker), 6.88 (1H,
d, Ar-H), 6.99 (1H, t, Ar-H), 7.33 (1H, t, Ar-H), 7.73 (1H, d, Ar-H), 7.81 (1H, d,
Ar-H), 8.02 (1H, s, triazole-H), 8.70 (1H, s, C2-H), 14.96 (1H, brs, COOH). ESI-MS
m/z: 659 [M+H]+.
3.1.2.2. 1-cyclopropyl-6-fluoro-8-methoxy-7-(4-((1-(2-(3-(methoxyimino)-5-methyl-2-
oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-3-methylpiperazin-1-yl)-4-oxo-1,
4-dihydroquinoline-3-carboxylic acid (5f)
1
Light yellow solid, yield: 58%. Mp: 126-129 °C. H NMR (400 MHz, DMSO-d6) δ
1.03-1.14 (7H, m, 2×cyclopropyl-CH2 and -CH3), 2.20-2.30 (5H, m, -CH3 and
piperazine-2H), 2.63 (1H, d, piperazine-1H), 2.98 (1H, t, piperazine-1H), 3.17-3.26
(3H, m, piperazine-3H), 3.59-3.81 (5H, m, -CH2 chain and -OCH3), 4.12-4.20 (6H, m,
cyclopropyl-CH, -CH2 of linker and NOCH3), 4.68 (2H, s, -CH2 of linker), 6.70-6.86
(1H, m, Ar-H), 7.11-7.31 (1H, m, Ar-H), 7.69-7.77 (2H, m, Ar-H), 8.04 (1H, s,
triazole-H), 8.71 (1H, s, C2-H), 14.91 (1H, brs, COOH). ESI-MS m/z: 644 [M+H]+.
3.1.2.3. 7-(4-((1-(2-(5-chloro-3-(methoxyimino)-2-oxoindolin-1-yl)ethyl)-1H-1,2,3-tri
11

ACCEPTED MANUSCRIPT
azol-4-yl)methyl)-3-methylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,
4-dihydroquinoline-3-carboxylic acid (5g)
1
Light yellow solid, yield: 61%. Mp: 156-158 °C. H NMR (400 MHz, DMSO-d6) δ
1.03-1.14 (7H, m, 2×cyclopropyl-CH2 and -CH3), 2.31-2.34 (2H, m, piperazine-2H),
2.61 (1H, d, piperazine-1H), 2.96 (1H, t, piperazine-1H), 3.22-3.31 (3H, m,
piperazine-3H), 3.66-3.79 (5H, m, -CH2 chain and -OCH3), 4.13-4.20 (6H, m,
cyclopropyl-CH, -CH2 of linker and NOCH3), 4.65 (2H, d, -CH2 of linker), 6.88 (1H,
d, Ar-H), 7.40 (1H, d, Ar-H), 7.71-7.77 (2H, m, Ar-H), 8.04 (1H, s, triazole-H), 8.69
(1H, s, C2-H), 14.95 (1H, brs, COOH). 13C NMR (100 MHz, DMSO-d6) δ 9.45, 16.05,
41.37, 47.36, 48.09, 50.99, 51.26, 54.21, 57.44, 63.51, 65.25, 107.00, 110.98, 116.34,
121.20, 125.01, 126.90, 126.97, 132.61, 134.61, 139.48, 139.57, 142.56, 142.64,
143.17, 146.20, 150.97, 154.65, 157.14, 162.31, 166.13, 176.78. ESI-MS m/z: 693
[M+H]+.
3.1.2.4. 1-cyclopropyl-6-fluoro-7-(4-((1-(2-(5-fluoro-3-(methoxyimino)-2-oxoindolin-
1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-3-methylpiperazin-1-yl)-8-methoxy-4-oxo-1,
4-dihydroquinoline-3-carboxylic acid (5h)
1
Light yellow solid, yield: 63%. Mp: 139-141 °C. H NMR (400 MHz, DMSO-d6) δ
1.02-1.13 (7H, m, 2×cyclopropyl-CH2 and -CH3), 2.33-2.35 (2H, m, piperazine-2H),
2.61 (1H, d, piperazine-1H), 2.94 (1H, t, piperazine-1H), 3.25-3.31 (3H, m,
piperazine-3H), 3.70 (3H, s, -OCH3), 3.72 (2H, q, -CH2 chain), 4.12-4.19 (6H, m,
cyclopropyl-CH, -CH2 of linker and NOCH3), 4.66 (2H, d, -CH2 of linker), 6.90 (1H,
d, Ar-H), 7.24 (1H, t, Ar-H), 7.58 (1H, d, Ar-H), 7.73 (1H, d, Ar-H), 8.03 (1H, s,
triazole-H), 8.70 (1H, s, C2-H), 14.96 (1H, brs, COOH). 13C NMR (100 MHz,
DMSO-d6) δ 9.44, 16.07, 41.25, 47.31, 48.11, 50.96, 51.25, 54.28, 57.46, 63.47, 65.16,
107.01, 110.57, 114.59, 114.85, 115.67, 119.33, 119.56, 121.16, 124.97, 134.64,
139.46, 140.13, 143.13, 143.20, 146.17, 150.98, 154.64, 157.10, 159.46, 162.50,
166.14, 176.79. ESI-MS m/z: 677 [M+H]+.
3.1.2.5. 1-cyclopropyl-6-fluoro-7-(4-((1-(2-(3-(hydroxyimino)-2-oxoindolin-1-yl)ethyl
)-1H-1,2,3-triazol-4-yl)methyl)-3-methylpiperazin-1-yl)-8-methoxy-4-oxo-1,4-dihydro
quinoline-3-carboxylic acid (5i)
1
Light yellow solid, yield: 54%. Mp: 188-190 °C. H NMR (400 MHz, DMSO-d6) δ
1.03-1.14 (7H, m, 2×cyclopropyl-CH2 and -CH3), 2.30-2.37 (2H, m, piperazine-2H),
2.60 (1H, d, piperazine-1H), 2.95 (1H, t, piperazine-1H), 3.20-3.21 (3H, m,
12

ACCEPTED MANUSCRIPT
piperazine-3H), 3.69 (3H, s, -OCH3), 3.72 (2H, q, -CH2 chain), 4.16-4.17 (3H, m,
cyclopropyl-CH and -CH2 of linker), 4.67-4.68 (2H, m, -CH2 of linker), 6.84 (1H, d,
Ar-H), 6.97 (1H, t, Ar-H), 7.29 (1H, t, Ar-H), 7.73 (1H, d, Ar-H), 7.91 (1H, d, Ar-H),
8.04 (1H, s, triazole-H), 8.70 (1H, s, C2-H), 13.42 (1H, brs, NOH), 14.94 (1H, brs,
COOH). 13C NMR (100 MHz, DMSO-d6) δ 9.40, 9.46, 16.03, 41.28, 47.46, 48.16,
51.03, 51.19, 54.33, 57.42, 63.48, 107.00, 109.05, 115.58, 121.06, 121.16, 122.98,
124.90, 127.27, 132.25, 134.64, 139.60, 143.12, 143.27, 143.66, 146.18, 150.98,
154.67, 157.15, 163.49, 166.16, 176.79. ESI-MS m/z: 645 [M+H]+.
3.1.2.6. 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)-3-methylpiperazin-1-yl)-8-methoxy-4-oxo-1,
4-dihydroquinoline-3-carboxylic acid (5j)
1
Light yellow solid, yield: 47%. Mp: 175-177 °C. H NMR (400 MHz, DMSO-d6) δ
1.02-1.14 (7H, m, 2×cyclopropyl-CH2 and -CH3), 2.19 (3H, s, -CH3), 2.28-2.32 (2H,
m, piperazine-2H), 2.59 (1H, d, piperazine-1H), 2.96 (1H, t, piperazine-1H),
3.17-3.26 (3H, m, piperazine-3H), 3.69 (s, 3H, -OCH3), 3.72 (2H, q, -CH2 chain),
4.13-4.16 (3H, m, cyclopropyl-CH and -CH2 of linker), 4.64-4.67 (2H, m, -CH2 of
linker), 6.69 (1H, d, Ar-H), 7.10 (1H, d, Ar-H), 7.70-7.76 (2H, m, Ar-H), 8.00 (1H, s,
13
triazole-H), 8.69 (1H, s, C2-H), 13.70 (1H, brs, NOH), 14.74 (1H, brs, COOH). C
NMR (100 MHz, DMSO-d6) δ 9.42, 16.00, 20.89, 41.25, 47.54, 48.19, 51.01, 51.20,
54.34, 57.43, 63.48, 107.05, 108.73, 115.63, 121.21, 124.87, 127.78, 131.86, 132.35,
134.60, 139.47, 140.89, 143.29, 143.77, 146.22, 150.95, 154.66, 157.14, 163.55,
166.14, 176.76. ESI-MS m/z: 659 [M+H]+.
3.1.2.7. 7-(4-((1-(2-(5-chloro-3-(hydroxyimino)-2-oxoindolin-1-yl)ethyl)-1H-1,2,3-tri
azol-4-yl)methyl)-3-methylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,
4-dihydroquinoline-3-carboxylic acid (5k)
1
Light yellow solid, yield: 48%. Mp: 194-196 °C. H NMR (400 MHz, DMSO-d6) δ
1.00-1.14 (7H, m, 2×cyclopropyl-CH2 and -CH3), 2.31-2.33 (2H, m, piperazine-2H),
2.60 (1H, d, piperazine-1H), 2.96 (1H, t, piperazine-1H), 3.27-3.31 (3H, m,
piperazine-3H), 3.65-3.80 (5H, m, -CH2 chain and -OCH3), 4.16-4.19 (3H, m,
cyclopropyl-CH and -CH2 of linker), 4.65-4.67 (2H, m, -CH2 of linker), 6.83 (1H, d,
Ar-H), 7.37 (1H, d, Ar-H), 7.73 (1H, d, Ar-H), 7.87 (1H, t, Ar-H), 8.01 (1H, s,
13
triazole-H), 8.70 (1H, s, C2-H), 13.77 (1H, brs, NOH), 14.98 (1H, brs, COOH). C
NMR (100 MHz, DMSO-d6) δ 9.45, 15.99, 41.25, 47.45, 48.13, 50.98, 51.23, 51.77,
13

ACCEPTED MANUSCRIPT
54.23, 57.42, 63.53, 107.01, 110.63, 116.65, 121.21, 124.99, 126.50, 126.70, 131.63,
134.63, 139.61, 141.91, 142.91, 143.23, 146.19, 150.99, 154.68, 157.16, 163.15,
165.25, 166.15, 176.80. ESI-MS m/z: 679 [M+H]+.
3.1.2.8. 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)-3-methylpiperazin-1-yl)-8-methoxy-4-oxo-1,
4-dihydroquinoline-3-carboxylic acid (5l)
1
Light yellow solid, yield: 43%. Mp: 191-192 °C. H NMR (400 MHz, DMSO-d6) δ
1.02-1.14 (7H, m, 2×cyclopropyl-CH2 and -CH3), 2.31-2.35 (2H, m, piperazine-2H),
2.60 (1H, d, piperazine-1H), 2.96 (1H, t, piperazine-1H), 3.27-3.30 (3H, m,
piperazine-3H), 3.69 (3H, s, -OCH3), 3.72 (2H, q, -CH2 chain), 4.16-4.19 (3H, m,
cyclopropyl-CH and -CH2 of linker), 4.65-4.68 (2H, m, -CH2 of linker), 6.84-6.87 (1H,
m, Ar-H), 7.18 (1H, t, Ar-H), 7.29 (1H, t, Ar-H), 7.65 (1H, d, Ar-H), 7.72 (1H, d,
Ar-H), 8.01 (1H, s, triazole-H), 8.70 (1H, s, C2-H), 13.70 (1H, brs, NOH), 14.97 (1H,
13
brs, COOH). C NMR (100 MHz, DMSO-d6) δ 9.39, 16.00, 41.25, 47.40, 48.14,
50.96, 51.21, 54.31, 57.40, 63.48, 107.00, 110.11, 114.09, 115.99, 116.08, 118.31,
118.55, 121.07, 124.96, 134.62, 139.46, 143.24, 143.36, 146.18, 150.96, 154.65,
157.08, 159.44, 163.34, 166.14, 176.77. ESI-MS m/z: 663 [M+H]+.
3.2. MIC determination
Conjugates 5a-l along with GTFX, RIF and INH were evaluated in vitro activity
against MTB H37Rv and MDR-TB via rapid direct susceptibility test technique [9].
The compounds along with the references GTFX, RIF and INH were dissolved in
dimethyl sulfoxide (DMSO) and two-fold 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 two-fold diluted tested compounds and
100 mL MTB H37Rv or MDR-MTB suspension containing 4×10-3 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
14

ACCEPTED MANUSCRIPT
re-determined 7 days later. The MIC is defined as the concentration of the compound
required to give complete inhibition of bacterial growth.
3.3. Cytotoxicity
The synthesized conjugates 5a-l along with the references GTFX, RIF and INH were
further examined for toxicity (CC50) in a mammalian VERO cell line dissolved in
DMSO at concentrations from 1000 to 4 µg/mL [10]. 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.
References
[1] World Health Organization, Global Tuberculosis Report 2016 (WHO/HTM/TB/2016.10).
World Health Organization, Geneva (2016).
[2] Z. Xu, S. Zhang, C. Gao, F. Zhao, Z. S. Lv, L. S. Feng, Chin. Chem. Lett. 2 (2017) 159-167
[3] Y. Q. Hu, S. Zhang, F. Zhao, C. Gao, L. S. Feng, Z. S. Lv, Z. Xu, X. Wu, Eur. J. Med. Chem.
133 (2017) 255-267
[4] Y. Q. Hu, Z. Xu, S. Zhang, X. Wu, J. W. Ding, Z. S. Lv, L. S. Feng, Eur. J. Med. Chem. 136
(2017) 122-130
[5] J. Crofton, P. Choculet, D. Maher, Guidelines for the Management of Drug-Resistant
Tuberculosis WHO/TB/96-210 (Rev.1). World Health Organization, Geneva (1997).
[6] D. Sriram, A. Aubry, P. Yogeeswaria, L. M. Fisher, Bioorg. Med. Chem. Lett. 16 (2006)
2982-2985
[7] D. Sriram, P. Yogeeswaria, J. S. Basha, D. R. Radhaet, V. Nagaraja, Bioorg. Med. Chem. 13
(2005) 5774-5778.
[8] S. N. Pandeya, D. Srirama, P. Yogeeswari, S. Ananthan, Chemother. 47 (2001) 266-269
[9] S. H. Zhao, R. Pine, J. Domagala, K. Drlica, Antimicrob. Agents Chemother. 43 (1999)
661-666.
[10] L. S. Feng, M. L. Liu, S. Zhang, Eur. J. Med. Chem. 46 (2011) 341-348
[11] L. S. Feng, M. L. Liu, Y. B. Zhang, Chem. Res. Chin. Univ. 28 (2012) 61-66
15

ACCEPTED MANUSCRIPT
[12] Z. L. Wan, M. L. Liu, L. S. Feng, B. Wang, X. D. Zheng, H. Y. Guo, Chin. J. Antibiot. 36
(2011) 37-43.
[13] H. N. Nagesh, K. M. Naidu, D. H. Rao, J. P. Sridevi, D. Sriram, P. Yogeeswari, K. V. G. C.
Sekhar, Bioorg. Med. Chem. Lett. 23 (2013) 6805-6810.
[14] B. Zhou, Y. He, X. Zhang, J. Xu, Y. Luo, Y. Wang, S. G. Franzblau, Z. Yang, R. J. Chan, Y.
Liu, J. Zheng, Z. Y. Zhang, Proc. Natl. Acad. Sci. U.S.A. 107 (2010) 4573-4578.
[15] K. D. Thomas, A. V. Adhikari, I. H. Chowdhury, E. Sumesh, N. K. Pal, Eur. J. Med. Chem.
46 (2011) 2503-2512
[16] H. N. Lin, C. T. Walsh, J. Am. Chem. Soc. 126 (2004) 13998 -14003
[17] J. C. McPherson, R. Royce, B. B. Thomas, J. F. Hartmann, D. Farcasiu, I. Bereczki, E. Rȍth,
S. Tollas, E. Ostorházi, F. Rozgonyi, P. Herczegh, Eur. J. Med. Chem. 47 (2012) 615-618
[18] K. Kumar, B. Pradines, M. Madamet, R. Amalvict, N. Benoit, V. Kumar, Eur. J. Med. Chem.
87 (2014) 801-804
16

ACCEPTED MANUSCRIPT
5g was 4-8 times more potent in vitro than the references GTFX and RIF against MTB H37Rv.
The most potent 5g and 5h were 4->512 times more active than the three references (MIC:
1.0->128 µg/mL) against MDR-TB, and the results warrant further development of the anti-TB
properties of triazole-tethered FQs isatin conjugates.