Synthesis and in vitro evaluation of novel substituted isatin-propylene-1H-1,2,3-triazole-4-methylene-moxifloxacin hybrids for their anti-mycobacterial activities

Article abstract of DOI:10.1016/j.ejmech.2017.11.090

Twelve novel substituted isatin-propylene-1H-1,2,3-triazole-4-methylene-moxifloxacin hybrids 5a-l were designed, synthesized and screened for their in vitro anti-mycobacterial activities against drug-sensitive and multidrug-resistant Mycobacterium tubercu

Full text of DOI:10.1016/j.ejmech.2017.11.090

Accepted Manuscript
Synthesis and in vitro evaluation of novel substituted isatin-propylene-1H-1,2,3-
triazole-4-methylene-moxifloxacin hybrids for their anti-mycobacterial activities
Xinjia Yan, Zaosheng Lv, Jing Wen, Shijia Zhao, Zhi Xu
PII:
S0223-5234(17)30993-5
10.1016/j.ejmech.2017.11.090
EJMECH 9965
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 July 2017
Revised Date: 22 November 2017
Accepted Date: 28 November 2017
Please cite this article as: X. Yan, Z. Lv, J. Wen, S. Zhao, Z. Xu, Synthesis and in vitro evaluation
of novel substituted isatin-propylene-1H-1,2,3-triazole-4-methylene-moxifloxacin hybrids for their
anti-mycobacterial activities, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2017.11.090.
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
Synthesis and in vitro evaluation of novel substituted
isatin-propylene-1H-1,2,3-triazole-4-methylene-moxifloxacin
hybrids for their anti-mycobacterial activities
Xinjia Yan1,*, Zaosheng Lv2, Jing Wen1, Shijia Zhao3, Zhi Xu2,*
1College of Pharmacy, Harbin University of Commerce, Harbin, Heilongjiang Province, P. R.
China 150076
2Key Laboratory of Hubei Province for Coal Conversion and New Carbon Materials, Wuhan
University of Science and Technology, Hubei, P R China
3DXC PharmaTech (Hubei) Co. Ltd., Hubei, P R China
.
The isatin-propylene-1H-1,2,3-triazole-4-methylene-moxifloxacin hybrids (MIC:
0.05-2.0 µg/mL) exhibited excellent in vitro activities against drug-sensitive and
multidrug-resistant Mycobacterium tuberculosis, but all hybrids (CC50: 2-32 µg/mL)
were much more cytotoxic than the parent moxifloxacin (CC50: 128 µg/mL) against
VERO cell line.

ACCEPTED MANUSCRIPT
Synthesis and in vitro evaluation of novel substituted
isatin-propylene-1H-1,2,3-triazole-4-methylene-moxifloxacin
hybrids for their anti-mycobacterial activities
Xinjia Yan1,*, Zaosheng Lv2, Jing Wen1, Shijia Zhao3, Zhi Xu2,*
1College of Pharmacy, Harbin University of Commerce, Harbin, Heilongjiang Province, P. R.
China 150076
2Key Laboratory of Hubei Province for Coal Conversion and New Carbon Materials, Wuhan
University of Science and Technology, Hubei, P R China
3DXC PharmaTech (Hubei) Co. Ltd., Hubei, P R China
Abstract: Twelve novel substituted isatin-propylene-1H-1,2,3-triazole-4-methylene-moxifloxacin
hybrids 5a-l were designed, synthesized and screened for their in vitro anti-mycobacterial
activities against drug-sensitive and multidrug-resistant Mycobacterium tuberculosis as well as
cytotoxicity in VERO cell line. All hybrids exhibited excellent activities against two
Mycobacterium tuberculosis strains with minimum inhibitory concentration in the range from 0.05
to 2.0 µg/mL. The most active hybrid 5i was 2-8 times more potent than the reference agents
(moxifloxacin and rifampicin) in vitro against Mycobacterium tuberculosis H37Rv, while 2->2,048
times more potent than the reference agents (moxifloxacin, rifampicin and isoniazid) in vitro
against multidrug-resistant Mycobacterium tuberculosis. However, all hybrids (the 50% cytotoxic
concentration/CC50: 2-32 µg/mL) were much more cytotoxic than the parent moxifloxacin (CC50:
128 µg/mL) against VERO cell line. Therefore, our further optimization will focus on their
cytotoxicity reducing as well as activity enhancing. The structure-activity relationship of
1H-1,2,3-triazole-tethered isatin-fluoroquinolone hybrids was investigated, and the results could
promote further development of the anti-tuberculosis properties of this kind of hybrids.
Keywords: Moxifloxacin; Isatin; Triazole; Anti-mycobacterial activity; Structure-activity
relationship
1. Introduction
Tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis (MTB), has
*Corresponding authors email address: yanxinjia@yeah.net (X. J. Yan); chemorgchem@126.com
(Z. Xu).
1

ACCEPTED MANUSCRIPT
threatened human health for thousands of years [1,2]. With the discovery of the
first-line anti-TB agents such as isoniazid (INH), rifampicin (RIF), pyrazinamide
(PZA) and ethambutol (EMB), the epidemic of TB has been under control. In spite of
the first-line anti-TB agents are still highly effective for the treatment of
drug-susceptible MTB infected patients, the reemergence and wide spread of new
virulent forms of MTB such as drug-resistant TB (DR-TB, 15 million),
multidrug-resistant TB (MDR-TB, 480,000), extremely drug-resistant TB (XDR-TB,
around 10% of MDR-TB) and totally drug resistant TB (TDR-TB) making them less
and less effective [3,4]. Therefore, there is an urgent need to develop new effective
agents to treat these new virulent forms of MTB.
In the past 50 years, only a handful of compounds have entered human trials since the
discovery of RIF, reflecting the inherent difficulties of developing new anti-TB
agents. Several promising derivatives with potential new action mechanism such as
target MTB nucleoid-associated protein HU and perturb its function [5-7], have been
developed in recent years. However, the number is still insufficient to control this
disease.
Moxifloxacin (MXF), the fourth generation fluoroquinolone, not only exhibits
excellent in vitro and in vivo potency against MTB including MDR-TB [8], but also
retains activity against MTB strains with various levels of FQs resistance [9].
Moreover, compared with the standard regimen, MXF exhibits equivalent or even
slightly better efficacy in clinical trials [10]. Currently, MXF is in phase III clinical
trial for the treatment of TB, and is a potential first-line agent.
< Fig 1. Chemical structures of MXF and triazole-tethered MXF-isatin hybrid 1 >
Isatin derivatives also demonstrate promising anti-TB activity [3]. Various
fluoroquinolone-isatin hybrids with different linkers including methylene, ethylene,
2

ACCEPTED MANUSCRIPT
acetyl, carbohydrazide and 1,2,3-triazole have been screened for their anti-TB
activities [11-27]. The structure-activity relationship (SAR) indicates that the anti-TB
activity of these hybrids is greatly influenced by the linkers between fluoroquinolone
and isatin. In general, the linkers’ affect the anti-TB activity in the order of
1,2,3-triazole > methylene > ethylene > acetyl. In our previous studies, several
ethylene-1H-1,2,3-triazole-4-methylene linked gatifloxacin and MXF hybrids were
evaluated for their anti-mycobacterial activities against MTB H37Rv and MDR-TB
strains, and some of them displayed excellent activity [26-28]. Among them, the most
active MXF hybrid 1 was 2->2,048 times more potent than the parent MXF, and two
reference agents (RIF and INH) in vitro against two MTB strains [28].
< Fig 2. Illustration of the design strategy for substituted
isatin-propylene-1H-1,2,3-triazole-4-methylene-moxifloxacin hybrids >
Based on the above research results and to continue our effort to develop new anti-TB
agents, we designed, synthesized a series of propylene-1H-1,2,3-triazole-4-methylene
linked isatin-MXF hybrids and evaluated their anti-mycobacterial activities as well as
cytotoxicity in this study.
2. Results and discussion
The
synthetic
route
for
substituted
isatin-propylene-1H-1,2,3-triazole-4-methylene-moxifloxacin hybrids 5a-l are
depicted in Scheme 1. The N-(3-bromoproyl)isatins 2a-c (yield: 37-59%) and
propargyl MXF 4 (yield: 69%) were obtained according to literature methods [26-28]
by alkylation of C-5 substituted isatins and MXF with 1,3-dibromopropane and
propargyl bromide, respectively. Introduction of azido by treating C-5 substituted
N-(3-bromoproyl)isatins 2a-c with sodium azide at 60 °C to provide precursors 3a-c,
which were utilized along with propargyl MXF 4 for the synthesis of desired
3

ACCEPTED MANUSCRIPT
substituted isatin-propylene-1H-1,2,3-triazole-4-methylene-moxifloxacin hybrids 5a-c
(yield: 46-63%) via Cu-promoted azide-alkyne cycloaddition reaction in the presence
of Cu(OAc)2 in DMF [26]. In the last step, condensations of conjugates 5a-c with
requisite substituted amine hydrochlorides in the presence of sodium bicarbonate
formed other hybrids (55-71%) [27].
< Scheme 1 Synthesis of 1H-1,2,3-triazole-tethered isatin-MXF hybrids 5a-l>
All
the
synthesized
substituted
isatin-propylene-1H-1,2,3-triazole-4-methylene-moxifloxacin hybrids 5a-l were
screened for their in vitro anti-mycobacterial activities against MTB H37Rv and
MDR-TB strains by rapid direct susceptibility test technique [26-28]. 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.
All the synthetic hybrids (minimum inhibitory concentration/MIC: 0.05-2.0 µg/mL)
exhibited promising activities against MTB H37Rv and MDR-TB strains, but most of
them were less active than the parent MXF (MIC: 0.10 and 0.12 µg/mL). The SAR
revealed that the relative importance of various moieties of the Schiff’s bases to
anti-mycobacterial activity is as follows: methyloxime > ethyloxime > ketone >
oxime. In general, hybrids with the electron-withdrawing groups such as -F at C-5
position of isatin motif boost the activity, while electron-donating group -Me
decreased the activity. The most active hybrid 5i (MIC: 0.05 and 0.06 µg/mL) was
comparable to the reference INH (MIC: 0.05 µg/mL), 2-8 times more potent than
MXF and RIF (MIC: 0.39 µg/mL) against MTB H37Rv, and 2->2,048 times more
potent than MXF, RIF and INH (MIC: 0.12, 32 and >128 µg/mL, respectively)
against MDR-TB.
The resistance index (RI: MICMDR-TB : MICMTB H Rv) of the majority targets was
37
around 1, and six of them showed RI less than 1, indicating that this kind of hybrids
could reduce the cross-resistant to some extent.
4

ACCEPTED MANUSCRIPT
<Table 1 Structures, anti-mycobacterial activity and cytotoxicity of hybrids 5a-l>
The conjugates 5a-l were subsequently examined for toxicity (the 50% cytotoxic
concentration/CC50, causing visible morphological changes in 50% of the cells with
respect
to
cell
control,
determined
with
the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assay) in a
mammalian VERO cell line [27]. 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 were reported in Table 1. All hybrids (CC50: 2-32 µg/mL) were
much more cytotoxic than the parent MXF (CC50: 128 µg/mL). In general, hybrids
containing imine moieties were more cytotoxic than those containing ketone moieties,
and -F containing hybrids showed the highest cytotoxicity in each series. The most
active hybrid 5i (CC50: 32 µg/mL) displayed high cytotoxicity, would be further
optimized. The selectivity index (SI: CC50/MICMTB H37Rv) for all hybrids were in a
range of 2.6 to 160, which were less than the references MXF, RIF and INH (SI:
1280, 2560 and 1312, respectively).
<Table 2 Metabolic stability and in vivo pharmacokinetic values of hybrid 5i in mice>
The metabolic stability and in vivo PK profiles of the most active hybrid 5i were
evaluated in mice after a single oral administration of 50 mg/kg. As shown in Table 2,
hybrid 5i displayed much lower microsomal stability than the parent MXF, and was
also inferior to MXF in PK profiles in terms of t1/2, Cmax, Tmax and AUC0 inf
.
‑
In
summary,
a
new
class
of
substituted
isatin-propylene-1H-1,2,3-triazole-4-methylene-moxifloxacin hybrids 5a-l 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 MTB H37Rv and MDR-TB, and the most
5

ACCEPTED MANUSCRIPT
active 5i was 2-8 times more potent than the reference agents MXF and RIF in vitro
against MTB H37Rv, and 2->2,048 times more potent than MXF, RIF and INH in
vitro against MDR-TB. The SAR of 1H-1,2,3-triazole-tethered isatin-FQs hybrids was
investigated, and the results could promote further development of this kind of
hybrids for better anti-TB properties.
3. Experimental section
3.1.Synthesis
3.1.1. General Procedure for the Preparation of 5a-c
N-(2-azidoethyl)isatins 3a-c and propargyl MXF 4 were prepared via literature
methods [26,27]. To a mixture of N-(2-azidoethyl)isatins 3-c (1.0 mmol) and
propargyl MXF 4 (1.0 mmol) in DMF (50 mL), Cu(OAc)2 (100 mg) was added under
N2 atmosphere. The mixture was allowed to react for 48 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. The analytical data were in the
supplementary material.
3.1.1.1. 1-cyclopropyl-7-((4aR,7aR)-1-((1-(3-(2,3-dioxoindolin-1-yl)propyl)-1H-1,2,3
-triazol-4-yl)methyl)hexahydro-1H-pyrrolo[3,4-b]pyridin-6(2H)-yl)-6-fluoro-8-metho
xy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5a)
1
Yellow solid, yield: 56%. Mp: 166-168 °C. H NMR (400 MHz, DMSO-d6) δ
0.98-1.60 (m, 8H), 2.12-2.46 (m, 4H), 2.73-2.77 (m, 1H), 2.98-3.00 (m, 1H),
3.16-3.18 (m, 1H), 3.56-3.80 (m, 11H), 4.11-4.13 (m, 1H), 4.40-4.44 (m, 2H),
7.10-7.14 (m, 2H, Ar-H), 7.53 (d, 1H, Ar-H), 7.60-7.65 (m, 2H, Ar-H), 7.95 (1H, s,
triazole-H), 8.66 (1H, s, C2-H), 15.19 (1H, brs, COOH). ESI-MS m/z: 670 [M+H]+.
3.1.1.2. 7-((4aR,7aR)-1-((1-(2-(5-chloro-2,3-dioxoindolin-1-yl)ethyl)-1H-1,2,3-triazo
l-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 (5b)
1
Light yellow solid, yield: 63%. Mp: 143-145 °C. H NMR (400 MHz, DMSO-d6) δ
0.84-1.59 (m, 8H), 2.12-2.35 (m, 7H), 2.73-2.75 (m, 1H), 2.99-3.01 (m, 1H),
3.54-3.73 (m, 11H), 3.95-3.97 (m, 1H), 4.39-4.51 (m, 2H), 7.00-7.05 (m, 1H, Ar-H),
6

ACCEPTED MANUSCRIPT
7.31 (s, 1H, Ar-H), 7.35-7.46 (m, 1H, Ar-H), 7.97 (s, 1H, Ar-H), 8.15 (1H, s,
triazole-H), 8.44 (1H, s, C2-H). ESI-MS m/z: 684 [M+H]+.
3.1.1.3. 1-cyclopropyl-6-fluoro-7-((4aR,7aR)-1-((1-(3-(5-fluoro-2,3-dioxoindolin-1-yl
)propyl)-1H-1,2,3-triazol-4-yl)methyl)hexahydro-1H-pyrrolo[3,4-b]pyridin-6(2H)-yl)
-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5c)
1
Light yellow solid, yield: 46%. Mp: 175-177 °C. H NMR (400 MHz, DMSO-d6) δ
1.06-1.61 (m, 8H), 2.11-2.38 (m, 4H), 2.73-2.75 (m, 1H), 2.99-3.00 (m, 1H),
3.55-3.79 (m, 11H), 4.11-4.13 (m, 1H), 4.40-4.43 (m, 2H), 7.19-7.20 (m, 1H, Ar-H),
7.42-7.51 (m, 2H, Ar-H), 7.61 (d, 1H, Ar-H), 7.92 (1H, s, triazole-H), 8.79 (1H, s,
C2-H). 15.16 (1H, brs, COOH). ESI-MS m/z: 684 [M+H]+.
3.1.2. The general procedure for preparing other targets
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-c. 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 other targets (55-71%). The
analytical data were in the supplementary material.
3.1.2.1. 1-cyclopropyl-6-fluoro-7-((4aR,7aR)-1-((1-(3-(3-(hydroxyimino)-2-oxoindoli
n-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)hexahydro-1H-pyrrolo[3,4-b]pyridin-6(2
H)-yl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5d)
1
Light yellow solid, yield: 71%. Mp: 191-193 °C. H NMR (400 MHz, DMSO-d6) δ
0.96-1.72 (m, 8H), 2.10-2.37 (m, 4H), 2.75-2.77 (m, 1H), 2.99-3.00 (m, 1H),
3.69-3.83 (m, 11H), 4.11-4.13 (m, 1H), 4.36-4.40 (m, 2H), 7.08-7.11 (m, 1H, Ar-H),
7.26-7.29 (m, 1H, Ar-H), 7.63 (d, 1H, Ar-H), 7.73-7.75 (m, 1H, Ar-H), 7.95 (1H, s,
triazole-H), 8.64 (1H, s, C2-H), 15.16 (1H, brs, COOH). ESI-MS m/z: 685 [M+H]+.
3.1.2.2. 1-cyclopropyl-6-fluoro-7-((4aR,7aR)-1-((1-(3-(3-(hydroxyimino)-5-methyl-2-
oxoindolin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)hexahydro-1H-pyrrolo[3,4-b]py
ridin-6(2H)-yl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5e)
1
Light yellow solid, yield: 68%. Mp: 184-185 °C. H NMR (400 MHz, DMSO-d6) δ
0.92-1.62 (m, 8H), 2.11-2.35 (m, 7H), 2.74-2.75 (m, 1H), 3.00-3.01 (m, 1H),
7

ACCEPTED MANUSCRIPT
3.51-3.76 (m, 11H), 3.97-3.99 (m, 1H), 4.41-4.51 (m, 2H), 7.02-7.05 (m, 1H, Ar-H),
7.34 (s, 1H, Ar-H), 7.36-7.47 (m, 1H, Ar-H), 7.99 (s, 1H, Ar-H), 8.16 (1H, s,
triazole-H), 8.44 (1H, s, C2-H). ESI-MS m/z: 699 [M+H]+.
3.1.2.3. 1-cyclopropyl-6-fluoro-7-((4aR,7aR)-1-((1-(3-(5-fluoro-3-(hydroxyimino)-2-
oxoindolin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)hexahydro-1H-pyrrolo[3,4-b]py
ridin-6(2H)-yl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5f)
1
Light yellow solid, yield: 67%. Mp: 197-199 °C. H NMR (400 MHz, DMSO-d6) δ
0.96-1.72 (m, 8H), 2.10-2.37 (m, 4H), 2.76-2.77 (m, 1H), 3.00-3.01 (m, 1H),
3.64-3.83 (m, 11H), 4.11-4.13 (m, 1H), 4.38-4.40 (m, 2H), 7.08-7.11 (m, 1H, Ar-H),
7.26-7.29 (m, 1H, Ar-H), 7.63 (d, 1H, Ar-H), 7.72-7.75 (m, 1H, Ar-H), 7.95 (1H, s,
triazole-H), 8.64 (1H, s, C2-H). ESI-MS m/z: 703 [M+H]+.
3.1.2.4. 1-cyclopropyl-6-fluoro-8-methoxy-7-((4aR,7aR)-1-((1-(3-(3-(methoxyimino)-
2-oxoindolin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)hexahydro-1H-pyrrolo[3,4-b]
pyridin-6(2H)-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5g)
1
Light yellow solid, yield: 55%. Mp: 155-157 °C. H NMR (400 MHz, DMSO-d6) δ
0.95-1.61 (m, 8H), 2.10-2.37 (m, 4H), 2.76-2.77 (m, 1H), 3.01-3.02 (m, 1H),
3.60-3.76 (m, 11H), 4.12 (s, 1H), 4.20 (s, 3H), 4.37-4.40 (m, 2H), 7.06-7.10 (m, 1H,
Ar-H), 7.42-7.47 (m, 1H, Ar-H), 7.63 (d, 1H, Ar-H), 7.87 (d, 1H, Ar-H), 7.94 (1H, s,
triazole-H), 8.64 (1H, s, C2-H), 15.19 (1H, brs, COOH). ESI-MS m/z: 699 [M+H]+.
3.1.2.5. 1-cyclopropyl-6-fluoro-8-methoxy-7-((4aR,7aR)-1-((1-(3-(3-(methoxyimino)-
5-methyl-2-oxoindolin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)hexahydro-1H-pyrro
lo[3,4-b]pyridin-6(2H)-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5h)
1
Light yellow solid, yield: 67%. Mp: 146-147 °C. H NMR (400 MHz, DMSO-d6) δ
0.94-1.71 (m, 8H), 2.09-2.37 (m, 7H), 2.76-2.77 (m, 1H), 2.99-3.01 (m, 1H),
3.66-3.72 (m, 11H), 4.11 (s, 1H), 4.20 (s, 3H), 4.37-4.40 (m, 2H), 6.95-6.97 (m, 1H,
Ar-H), 7.25-7.27 (m, 1H, Ar-H), 7.61-7.64 (m, 1H, Ar-H), 7.71 (s, 1H, Ar-H), 7.95
(1H, s, triazole-H), 8.64 (1H, s, C2-H), 15.05 (1H, brs, COOH). ESI-MS m/z: 713
[M+H]+.
3.1.2.6. 1-cyclopropyl-6-fluoro-7-((4aR,7aR)-1-((1-(3-(5-fluoro-3-(methoxyimino)-2-
oxoindolin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)hexahydro-1H-pyrrolo[3,4-b]py
ridin-6(2H)-yl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5i)
1
Light yellow solid, yield: 71%. Mp: 165-166 °C. H NMR (400 MHz, DMSO-d6) δ
0.96-1.61 (m, 8H), 2.09-2.38 (m, 4H), 2.76-2.77 (m, 1H), 2.99-3.01 (m, 1H),
8

ACCEPTED MANUSCRIPT
3.57-3.62 (m, 11H), 4.14 (s, 1H), 4.21 (s, 3H), 4.36-4.39 (m, 2H), 7.11-7.13 (m, 1H,
Ar-H), 7.34-7.36 (m, 1H, Ar-H), 7.60-7.67 (m, 2H, Ar-H), 7.93 (1H, s, triazole-H),
8.64 (1H, s, C2-H), 15.18 (1H, brs, COOH). ESI-MS m/z: 717 [M+H]+.
3.1.2.7. 1-cyclopropyl-7-((4aR,7aR)-1-((1-(3-(3-(ethoxyimino)-2-oxoindolin-1-yl)pro
pyl)-1H-1,2,3-triazol-4-yl)methyl)hexahydro-1H-pyrrolo[3,4-b]pyridin-6(2H)-yl)-6-fl
uoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5j)
1
Light yellow solid, yield: 63%. Mp: 123-124 °C. H NMR (400 MHz, DMSO-d6) δ
1.06-1.61 (m, 11H), 2.10-2.38 (m, 4H), 2.76-2.77 (m, 1H), 2.99-3.01 (m, 1H),
3.67-3.77 (m, 11H), 4.14 (s, 1H), 4.38-4.46 (m, 4H), 7.06-7.11 (m, 2H, Ar-H),
7.42-7.48 (m, 1H, Ar-H), 7.62-7.65 (m, 1H, Ar-H), 7.88-7.90 (m, 1H, Ar-H), 7.93 (1H,
s, triazole-H), 8.64 (1H, s, C2-H), 15.18 (1H, brs, COOH). ESI-MS m/z: 713 [M+H]+.
3.1.2.8. 1-cyclopropyl-7-((4aR,7aR)-1-((1-(3-(3-(ethoxyimino)-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)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5k)
1
Light yellow solid, yield: 58%. Mp: 120-121 °C. H NMR (400 MHz, DMSO-d6) δ
1.06-1.61 (m, 11H), 2.10-2.38 (m, 7H), 2.76-2.77 (m, 1H), 2.99-3.01 (m, 1H),
3.56-3.83 (m, 11H), 4.14 (s, 1H), 4.36-4.46 (m, 4H), 6.91-6.97 (m, 1H, Ar-H),
7.24-7.27 (m, 1H, Ar-H), 7.61-7.65 (m, 1H, Ar-H), 7.72 (s, 1H, Ar-H), 7.95 (1H, s,
triazole-H), 8.64 (1H, s, C2-H), 15.20 (1H, brs, COOH). ESI-MS m/z: 727 [M+H]+.
3.1.2.9. 1-cyclopropyl-7-((4aR,7aR)-1-((1-(3-(3-(ethoxyimino)-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)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5l)
1
Light yellow solid, yield: 66%. Mp: 130-131 °C. H NMR (400 MHz, DMSO-d6) δ
1.06-1.71 (m, 11H), 2.10-2.38 (m, 7H), 2.76-2.77 (m, 1H), 2.99-3.01 (m, 1H),
3.61-3.83 (m, 4H), 4.14 (s, 1H), 4.38-4.46 (m, 4H), 7.10-7.12 (m, 1H, Ar-H),
7.32-7.35 (m, 1H, Ar-H), 7.58-7.62 (m, 2H, Ar-H), 7.94 (1H, s, triazole-H), 8.64 (1H,
s, C2-H), 15.16 (1H, brs, COOH). ESI-MS m/z: 731 [M+H]+.
3.2. MIC determination
Hybrids 5a-l along with MXF, RIF and INH were evaluated in vitro activity against
MTB H37Rv and MDR-TB via rapid direct susceptibility test technique [26]. The
compounds together with the references MXF, RIF and INH were dissolved in
9

ACCEPTED MANUSCRIPT
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 with the cell density of the inoculum
4×10-5 CFU/mL. 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
concentration of the compound required to give complete inhibition of bacterial
growth.
3.3. Cytotoxicity
The synthesized hybrids 5a-l along with the references MXF, RIF and INH were
further
examined
for
toxicity
(CC50)
determined
with
the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assay in a
mammalian VERO cell line dissolved in DMSO at concentrations from 1024 to 1
µg/mL [27]. 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.
3.4 Pharmacokinetic Profiles determination
SPF female ICR mice weighing 20–25 g were used in the pharmacokinetic study. The
rats were fasted overnight before dosing. Every treatment group contained 3 mice.
10

ACCEPTED MANUSCRIPT
Mice were dosed with the tested compounds suspension at 50 mg/kg (p.o.).
Compounds were suspended in 0.5% CMC for oral administration. Blood was
collected from the jugular vein of each animal at the following times after
administration of drugs: 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h after a single oral dosing. All
blood samples were centrifuged at 3000 r/min for 10 min to obtain serum which was
then stored at -20 °C. 150 µL of the serum was added to 500 µL of acetonitrile and the
mixture was centrifuged at 13000 r/min for 10 min to remove protein. The supernatant
was dried and dissolved in 100 µL of acetonitrile, the solution was centrifuged at
13000 r/min for 10 min. The supernatant was moved to a sample bottle for HPLC
analysis. 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 determined directly from the experimental data using
WinNonlin V6.2.1.
Acknowledgements
We gratefully acknowledge the financial support by University Nursing Program for
Young Scholars with Creative Talents in Heilongjiang Province (UNPYSCT-2017).
References
[1] 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
[2] S. Zhang, Z. Xu, C. Gao, Q. C. Ren, C. Le, Z. S. Lv, L. S. Feng, Eur. J. Med. Chem. 138 (2017)
501-513
[3] Z. Xu, S. Zhang, C. Gao, F. Zhao, Z. S. Lv, L. S. Feng, Chin. Chem. Lett. 2 (2017) 159-167
[4] 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
[5] T. Bhowmick, S. Ghosh, K. Dixit, V. Ganesan, U. A. Ramagopal, D. Dey, S. P. Sarma, S.
Ramakumar, V. Nagaraja. Nat Commun. 11 (2014) 4124-4135
[6] M. A. Suarez, J. Valencia, C. C. Cadena, R. Maiti, C. Datta, G. Puerto, J. H. Isaza, H. S. Juan,
V, Nagaraja, J. D. Guzman. Molecules 22 (2017) e1245
[7] A. Sipos, J. Pató, R. Székely, R. C. Hartkoorn, L Kékesi,L. Õrfi, C. Szántai-Kis, K. Mikušová,
Z. vetlíková,J.Korduláková, V. Nagaraja, A. A. Godbole, N. Bush,F. Collin, A. Maxwell, S. T.
Cole, G. Kéri. Lead selection and characterization of antitubercular compounds using the
Nested Chemical Library. Tuberculosis 95 (2015) S200-S206.
11

ACCEPTED MANUSCRIPT
[8] L. S. Feng, M. L. Liu, Y. B. Zhang, Chem. Res. Chin. Univ. 28 (2012) 61-66
[9] Y. Q. Hu, S. Zhang, Z. Xu, Z. S. Lv, M. L. Liu, L. S. Feng, Eur. J. Med. Chem. 141 (2017)
335-345.
[10] Q. L. Ruan, Q. H. Liu, F. Sun, L. Y. Shao, J. L. Jin, S. L. Yu, J. W. Ai, B. Y. Zhang, W. H.
Zhang, Emerg. Microbes Infect. 5 (2016) e12
[11] D. Sriram, A. Aubry, P. Yogeeswaria, L. M. Fisher, Bioorg. Med. Chem. Lett. 16 (2006)
2982-2985.
[12] D. Sriram, P. Yogeeswaria, J. S. Basha, D. R. Radha, V. Nagaraja, Bioorg. Med. Chem. 13
(2005), 5774-5778.
[13] S. N. Pandeya, D. Sriram, P. Yogeeswari, S. Ananthan, Chemother. 47 (2001) 266-269.
[14] S. Talath, B. A. Bhongade, Am. J. Pharm. Tech. Res. 3 (2013) 570-581.
[15] T. Aboul-Fadl, F. A. S. Bin-Jubair, O. Aboul-Wafa, Eur. J. Med. Chem. 45 (2010) 4578-4586.
[16] T. R. Bal, B. Anand, P. Yogeeswari, D, Sriram, Bioorg. Med. Chem. Lett. 15 (2005)
4451-4455.
[17] D. Sriram, T. R. Bal, P. Yogeeswari, Med. Chem. Res. 14 (2005) 211-228
[18] D. Sriram, T. R. Bal, P. Yogeeswari, J. Pharm. Pharmaceut. Sci. 8 (2005) 565-577.
[19] D. Banerjee, P. Yogeeswari, P. Bhat, A. Thomas, M. Srividya, D. Sriram, Eur. J. Med. Chem.
46 (2011) 106-121.
[20] D. Sriram, P. Yogeeswari, G. Gopal, Eur. J. Med. Chem. 40 (2005) 1373-1376.
[21] D. Sriram, P. Yogeeswari, K. Meena, Pharmazie 61 (2006) 274-277
[22] L. S. Feng, M. L. Liu, S. Zhang, Y. Chai, B. Wang, Y. B. Zhang, K. Lv, Y. Guan, H. Y. Guo, C.
L. Xiao, Eur. J. Med. Chem. 46 (2011) 341-348.
[23] L .S. Feng, M. L. Liu, B. Wang, Y. Chai, X. Q. Hao, S. Meng, H. Y. Guo, Eur. J. Med. Chem.
45 (2010) 3407-3412.
[24] L. S. Feng, M. L. Liu, Y. B. Zhang, Chem. Res. Chin. Univ. 28 (2012) 61-66
[25] Z. Xu, M. Qiang, Z. S. Lv, Asian J. Chem. 29 (2017) 1039-1041
[26] Z. Xu, S. Zhang, X. F. Song, M. Qiang, Z. S. Lv, Bioorg. Med. Chem. Lett. 27 (2017)
3643-3646
[27] Z. Xu, X. F. Song, Y. Q. Hu, M. Qiang, Z. S. Lv, Eur. J. Med. Chem. 138 (2017) 66-71.
12

ACCEPTED MANUSCRIPT
Table 2 Metabolic stability and in vivo pharmacokinetic values of hybrid 5i in mice
pharmacokinetics (p.o.)
microsomal
Compd.
Cmax (ng·mL−1)
243
tl/2 (h)
3.87
Tmax (h) AUC0-inf (ng·h·mL−1)
stability (t1/2, min)
5i
13.7
>60
1.33
1.08
1236
7032
MXF
2731
11.35

ACCEPTED MANUSCRIPT
Table 1
Structures, anti-mycobacterial activity and cytotoxicity of compounds 5a-l
MIC (µg/mL)
Compd.
R1
R2
CC50b(µg/mL)
SIc
MTB H37Rv
1.56
MDR-TBa
1.0
O
H
Me
F
16
32
16
8
10.2
82.0
20.5
10.2
5.1
5a
5b
5c
5d
5e
5f
O
0.39
1.0
O
0.78
0.25
1.0
NOH
NOH
NOH
NOMe
NOMe
NOMe
NOEt
H
0.78
Me
F
1.56
2.0
8
0.78
0.25
0.25
0.50
0.06
0.25
2
2.6
H
0.10
16
8
160.0
20.5
80.0
41.0
5g
5h
5i
Me
F
0.39
0.05
4
H
0.39
16
5j

ACCEPTED MANUSCRIPT
NOEt
NOEt
Me
F
0.39
0.20
0.10
0.05
0.39
0.25
0.12
0.12
>128
32
4
10.2
20.0
5k
5l
4
128
128
512
1,280
2,560
1,312
MXF
INH
RIF
aMDR-TB: resistant to INH, RIF and EMB.
bCC50: The 50% cytotoxic concentration in a mammalian VERO cell line.
cSI: CC50/MICMTB H37Rv
.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
1. All the synthesized hybrids exhibited excellent activities against drug-sensitive
and multidrug-resistant Mycobacterium tuberculosis
2. conjugate 5i was 2->2,048 times more potent in vitro than the references
moxifloxacin, rifampicin and/or isoniazid against drug-sensitive and
multidrug-resistant Mycobacterium tuberculosis
3. all hybrids were much more cytotoxic than moxifloxacin, warrant further
optimization.