Ciprofloxacin-1,2,3-triazole-isatin hybrids tethered via amide: Design, synthesis, and in vitro anti-mycobacterial activity evaluation

Article abstract of DOI:10.1016/j.bmcl.2019.07.041

The purpose of this study was to prepare various novel amide tethered ciprofloxacin-1,2,3-triazole-isatin hybrids 7a-l, and evaluate their in vitro anti-mycobacterial activity as well as cytotoxicity in VERO cells. The synthesized hybrids showed considerable in vitro activity against both MTB H₃₇Rv and MDR-MTB with MIC of 0.12 to 32 μg/mL, and acceptable cytotoxicity in VERO cells (CC₅₀: 8.0–>128.0 μg/mL). In particular, the most active hybrid 7a (MIC_{MTB H37Rv}: 0.5 μg/mL and MIC_MDR-MTB: 0.12 μg/mL) had the activity in the same level with the first-line anti-tubercular agents isoniazid (MIC: 0.12 μg/mL) and rifampicin (MIC: 0.25 μg/mL), and it was 2-fold more active than the parent ciprofloxacin (MIC: 1.0 μg/mL) against MTB H₃₇Rv, and ≥16 folds more potent than ciprofloxacin (MIC: 2.0 μg/mL), isoniazid (MIC: >64 μg/mL) and rifampicin (MIC: >64 μg/mL) against MDR-MTB. Moreover, hybrid 7a (CC₅₀: 16.0 μg/mL) also displayed considerable cytotoxicity towards VERO cells. Thus, hybrid 7a could act as a platform for further investigations.

Full text of DOI:10.1016/j.bmcl.2019.07.041

Accepted Manuscript
Ciprofloxacin-1,2,3-triazole-isatin hybrids tethered via amide: Design, synthe-
sis, and in vitro anti-mycobacterial activity evaluation
Rongxing Chen, Hao Zhang, Tianwei Ma, Huarui Xue, Zhong Miao, Liyan
Chen, Xiangkui Shi
PII:
S0960-894X(19)30497-4
https://doi.org/10.1016/j.bmcl.2019.07.041
BMCL 26582
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 May 2019
21 July 2019
23 July 2019
Please cite this article as: Chen, R., Zhang, H., Ma, T., Xue, H., Miao, Z., Chen, L., Shi, X., Ciprofloxacin-1,2,3-
triazole-isatin hybrids tethered via amide: Design, synthesis, and in vitro anti-mycobacterial activity evaluation,
Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.07.041
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Ciprofloxacin-1,2,3-triazole-isatin hybrids tethered via amide:
Design, synthesis, and in vitro anti-mycobacterial activity
evaluation
Rongxing Chena,1, Hao Zhangb,1, Tianwei Maa, Huarui Xuea, Zhong Miaoa, Liyan
Chena, Xiangkui Shia,*
aXuzhou Women&Children’s Healthcare Hospital, Xuzhou 221009, Jiangsu province,
PR China;
bXuzhou Central Hospital, Department of Neurosurgery, Second Division, Xuzhou
221009, Jiangsu province, PR China;
1These authors contributed equally
Abstract: The purpose of this study was to prepare various novel amide tethered
ciprofloxacin-1,2,3-triazole-isatin hybrids 7a-l, and evaluate their in vitro anti-
mycobacterial activity as well as cytotoxicity in VERO cells. The synthesized hybrids
showed considerable in vitro activity against both MTB H37Rv and MDR-MTB with
MIC of 0.12 to 32 μg/mL, and acceptable cytotoxicity in VERO cells (CC50: 8.0-
>128.0 μg/mL). In particular, the most active hybrid 7a (MICMTB H37Rv: 0.5 μg/mL and
MICMDR-MTB: 0.12 μg/mL) had the activity in the same level with the first-line anti-
tubercular agents isoniazid (MIC: 0.12 μg/mL) and rifampicin (MIC: 0.25 μg/mL),
and it was 2-fold more active than the parent ciprofloxacin (MIC: 1.0 μg/mL) against
MTB H37Rv, and ≥16 folds more potent than ciprofloxacin (MIC: 2.0 μg/mL),
isoniazid (MIC: >64 μg/mL) and rifampicin (MIC: >64 μg/mL) against MDR-MTB.
Moreover, hybrid 7a (CC50: 16.0 μg/mL) also displayed considerable cytotoxicity
towards VERO cells. Thus, hybrid 7a could act as a platform for further
investigations.
Keywords: ciprofloxacin; isatin; 1,2,3-triazole; anti-mycobacterial; anti-tubercular;
drug-resistant
*Corresponding author: ysk83907278@163.com
1

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), mainly occurs in
the lungs[1,2]. TB is widespread (one-third of the world population infected with MTB,
and 5-10% of them will develop TB disease during their lifetime)[3], infectious (one
contagious patient can infect 10-20 contacts)[4], and of high mortality (over 1 million
death annually)[3], which has serious influence on the global healthcare system. The
widespread of drug-resistant TB (DR-TB, especially multi-drug resistant TB/MDR-
TB) and HIV/TB co-infection (TB is the leading cause of death among HIV/TB co-
infection patients) are main reasons for the incremental prevalence of TB[5,6]. It has
been estimated by The World Health Organization (WHO) that 558,000 new cases of
rifampicin-resistant TB (RR-TB) occurred in 2017, of which 82% were MDR-TB
patients [3]. Around 3.5% of new TB cases and 18% of previously treated cases had
MDR-TB or RR-TB. However, only a handful of drug candidates are under clinical
trials currently, and a few new clinically approved anti-TB drugs have emerged
during the past 30 years[7]. Therefore, it is urgent to develop novel anti-TB drugs with
great potency against both drug-sensitive TB and DR-TB especially MDR-TB.
Ciprofloxacin, the third-generation quinolone antibiotic, has already used as the
second-line anti-TB agent to treat the MDR-TB[8]. However, the emergency of
extensively drug-resistant TB (XDR-TB) and totally drug-resistant TB (TDR-TB)
make ciprofloxacin less and less effective[9,10]. Isatin structural scaffolds and
ciprofloxacin could act on DNA gyrase, so hybridization of isatin with ciprofloxacin
has the potential to enhance the inhibitory activity against DNA gyrase, and
consequently improve the anti-TB activity[11,12]. Some ciprofloxacin-1,2,3-triazole-
isatin hybrids showed excellent potency against both drug-sensitive MTB and MDR-
MTB[13,14], and the structure-activity relationship (SAR) revealed that the linker
between ciprofloxacin and 1,2,3-triazole has great influence on the activity, and the
most common linker was alkyl with different length[15-18]. Moreover, N-acylated
ciprofloxacin derivatives showed enhanced biological activity when compared with
the parent ciprofloxacin, so amide may be preeminent linker[19-23]
.
2

Ciprofloxacin with excellent anti-TB activity
Isatin moiety with potential anti-TB activity
Control the lipophilicity
O
N
F
COOH
R2
O
N
Electronic effect
R1
N
N
N
N
O
N
Amide linker
1,2,3-triazole motif with considerable anti-TB activity
Figure 1 Design strategy for amide tethered ciprofloxacin-1,2,3-triazole-isatin
hybrids
Based on the above facts, a class of novel amide tethered ciprofloxacin-1,2,3-triazole-
isatin hybrids were designed, synthesized, and the in vitro anti-mycobacterial activity
against both drug-susceptible MTB H37Rv and MDR-MTB strains as well as
cytotoxicity in VERO cells were evaluated to search novel anti-TB candidates in this
paper. The design strategy was depicted in Figure 1.
The novel amide tethered ciprofloxacin-1,2,3-triazole-isatin hybrids 7a-l could be
achieved by the synthetic route depicted in Scheme 1. The 2-azidoacetic acid 2 was
obtained by the treatment of 2-bromoacetic acid 1 with sodium azide in H2O, and then
2-azidoacetic acid 2 reacted with oxalyl chloride with DMF as catalyst to give 2-
azidoacetyl chloride 3[24]. Condensation of ciprofloxacin with 2-azidoacetyl chloride 3
with pyridine as base yielded the amide 4. Isatin/5-methylisatin/5-fluoroisatin/7-
fluoroisatin 5a-d were alkylated with propargyl bromide and generated isatin
intermediates 6a-d[25,26]. Cyclization of amide 4 and isatin intermediates 6a-d with
Cu(OAc)2 as catalyst in DMF provided the desired amide tethered ciprofloxacin-
1,2,3-triazole-isatin hybrids 7a-d[27]. Finally, condensations of 7a-d with
methoxylamine hydrochloride or ethoxylamine hydrochloride in the presence of
sodium bicarbonate afforded the rest ciprofloxacin-1,2,3-triazole-isatin hybrids 7e-l.
3

O
N
F
COOH
O
O
(COCl)2
ciprofloxacin
pyridine
NaN3
O
N
N3
Br
N3
Cl
HO
HO
N
N3
O
1
2
3
4
Cu(OAc)2
O
O
5
6
O
Br
K2CO3, DMF
R1
N
O
R1
N
7
H
8
6a-d
5a-d
R1 = H, 5-F, 5-Me, 7-F.
O
N
O
N
F
COOH
F
COOH
R2
N
O
RONH2
O
O
N
N
R1
R1
N
N
N
N
N
N
N
O
O
N
N
7e-l
7a-d
7b
7e
7f
: R1 = H, R2 = NOMe;
: R1 = 5-F, R2 = NOMe;
7a
7c
: R1 = H; : R1 = 5-F;
7g
7h
: R1 = 5-Me, R2 = NOMe;
: R1 = 7-F, R2 = NOMe;
: R1 = H, R2 = NOEt;
7d
: R1 = 5-Me; : R1 = 7-F.
7i
7j
: R1 = 5-F, R2 = NOEt;
7k
7l
: R1 = 5-Me, R2 = NOEt;
: R1 = 7-F, R2 = NOEt.
Scheme 1 Synthesis of amide tethered ciprofloxacin-1,2,3-triazole-isatin hybrids 7a-l
The anti-mycobacterial activity of the desired amide tethered ciprofloxacin-1,2,3-
triazole-isatin hybrids 7a-l against MTB H37Rv and MDR-MTB (resistant to isoniazid
and rifampicin) strains and cytotoxicity towards VREO cells were investigated, and
the results were presented in Table 1.
Table 1 In vitro anti-mycobacterial activity and cytotoxicity of amide tethered
ciprofloxacin-1,2,3-triazole-isatin hybrids 7a-l
MIC (μg/mL)
Compounds
CC50 (μg/mL)
SI
MTB H37Rv
MDR-MTB
4

7a
0.5
1.0
0.12
1.0
16.0
32.0
16.0
8.0
32.0
32.0
16.0
2.0
7b
7c
1.0
2.0
7d
4.0
2.0
7e
8.0
8.0
128.0
64.0
8.0
16.0
8.0
7f
8.0
16.0
8.0
7g
32.0
32.0
32.0
16.0
32.0
32.0
1.0
0.25
1.0
7h
32.0
16.0
16.0
32.0
32.0
2.0
32.0
64.0
8.0
7i
2.0
7j
7k
0.5
32.0
16.0
128.0
64.0
128.0
1.0
7l
0.5
Ciprofloxacin
Isoniazid
Rifampicin
128.0
512.0
512.0
0.12
0.25
>64
>64
aSI: selectivity index, CC50/MICMTB H37Rv
From Table 1, it can be concluded that all the synthesized hybrids 7a-l were active
against both drug-sensitive MTB H37Rv and MDR-MTB with MIC values in a range
of 0.12 to 32 μg/mL. The SAR indicated that introduction of alkyloxime onto C-3
position of isatin moiety was detrimental to the activity. Compared with unsubstituted
5

analogs, hybrids with either electron-withdrawing (fluoro and chloro) or electron-
donating group (methyl) could reduce the activity. The potency of the synthesized
hybrids against MDR-MTB were higher than that against drug-sensitive MTB H37Rv,
demonstrating that this kind of hybrids might have novel mechanism on MDR-MTB.
In particular, conjugate 7a (MICMTB H37Rv: 0.5 μg/mL and MICMDR-TB: 0.12 μg/mL)
which was found to be the most active compound against MTB H37Rv and MDR-
MTB strains, has the same level activity with the first-line anti-TB agents isoniazid
(MIC: 0.12 μg/mL) and rifampicin (MIC: 0.25 μg/mL), and it was 2-fold more active
than the parent ciprofloxacin (MIC: 1.0 μg/mL) against MTB H37Rv. Moreover,
hybrid 7a was ≥16 folds more potent than ciprofloxacin (MIC: 2.0 μg/mL), isoniazid
(MIC: >64 μg/mL) and rifampicin (MIC: >64 μg/mL) against MDR-MTB. The above
results demonstrated that hybrid 7a has the potential to treat both drug-sensitive MTB
and MDR-MTB infections.
All hybrids also displayed acceptable toxicological properties with CC50 ranging from
8.0 to 128.0 μg/mL, but the cytotoxicity of all the hybrids except 7e was higher than
the parent ciprofloxacin (CC50: 128.0 μg/mL). The structure-cytotoxicity relationship
study indicated that hybrids with alkyloxime at C-3 position of isatin moiety had
lower cytotoxicity compared to the corresponding ketones, and hybrids with
substituents at C-5 position of isatin motif were much more toxic than the
unsubstituted analogs. In particular, the most active hybrid 7a (CC50: 16.0 μg/mL)
displayed moderate cytotoxicity against VERO cells, and the selective index (SI) was
32.0, worth to be further investigated.
In summary, a series of amide tethered ciprofloxacin-1,2,3-triazole-isatin hybrids 7a-l,
were designed, synthesized, and the in vitro anti-mycobacterial activity as well as
cytotoxicity in VERO cells were evaluated in this study. All synthesized hybrids
showed promising in vitro activity against both drug-sensitive and MDR MTB strains,
and acceptable cytotoxicity in VERO cells. Among them, the most active hybrid 7a
was no inferior to the first-line anti-TB agents isoniazid, rifampicin, and the parent
ciprofloxacin against the tested strains in vitro. The enriched SAR may help to
identify new chemical entities as potent anti-TB agents.
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1. The novel hybrids 7a-l showed considerable antimycobacterial
activity;
2. Hybrid 7a was no inferior to the first-line anti-TB;
3. The SAR and structure-cytotoxicity relationship were discussed.
Graphical Abstract
Ciprofloxacin-1,2,3-triazole-isatin hybrids tethered via amide:
Design, synthesis, and in vitro anti-mycobacterial activity
evaluation
Rongxing Chena,1, Hao Zhangb,1, Tianwei Maa, Huarui Xuea, Zhong Miaoa, Liyan
Chena, Xiangkui Shia,*
aXuzhou Women&Children’s Healthcare Hospital, Xuzhou 221009, Jiangsu province,
PR China;
8

bXuzhou Central Hospital, Department of Neurosurgery, Second Division, Xuzhou
221009, Jiangsu province, PR China;
1These authors contributed equally
Ciprofloxacin with excellent anti-TB activity
Isatin moiety with potential anti-TB activity
Control the lipophilicity
O
F
COOH
R2
O
N
N
Electronic effect
R1
N
N
N
N
O
N
Amide linker
1,2,3-triazole motif with considerable anti-TB activity
The purpose of this study was to prepare various novel amide tethered ciprofloxacin-
1,2,3-triazole-isatin hybrids 7a-l, and evaluate their in vitro and in vivo anti-
mycobacterial activity as well as cytotoxicity in VERO cells. Among them, the most
active hybrid 7a was no inferior to the first-line anti-TB agents isoniazid, rifampicin,
and the parent ciprofloxacin against the tested strains in vitro. The enriched structure-
activity relationship may help global efforts for identification of new chemical entities
as potent anti-TB agents.
9