Benzofuran-isatin-hydroxylimine/thiosemicarbazide hybrids: Design, synthesis and in vitro anti-mycobacterial activity evaluation

Article abstract of DOI:10.1016/j.cclet.2018.11.032

A series of novel benzofuran-isatin-hydroxylimine/thiosemicarbazide hybrids were designed, synthesized and evaluated for their in vitro anti-TB activities against drug-sensitive MTB H₃₇Rv and MDR-TB isolates as well as cytotoxicity. All benzofu

Full text of DOI:10.1016/j.cclet.2018.11.032

Accepted Manuscript
Title: Benzofuran-isatin-hydroxylimine/thiosemicarbazide
hybrids: Design, synthesis and in vitro anti-mycobacterial
activity evaluation
Authors: Yaohuan Zhang, Ruo Wang, Tesen Zhang, Weitao
Yan, Yihong Chen, Yanping Zhang, Muyang Zhou
PII:
DOI:
Reference:
S1001-8417(18)30459-5
https://doi.org/10.1016/j.cclet.2018.11.032
CCLET 4736
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Chinese Chemical Letters
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Accepted date:
9 November 2018
29 November 2018
30 November 2018
Please cite this article as: Zhang Y, Wang R, Zhang T, Yan W, Chen Y, Zhang Y, Zhou
M, Benzofuran-isatin-hydroxylimine/thiosemicarbazide hybrids: Design, synthesis and
in vitro anti-mycobacterial activity evaluation, Chinese Chemical Letters (2018),
https://doi.org/10.1016/j.cclet.2018.11.032
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Communication
Benzofuran-isatin-hydroxylimine/thiosemicarbazide hybrids: Design, synthesis and in
vitro anti-mycobacterial activity evaluation
Yaohuan Zhang 1, Ruo Wang1,* , Tesen Zhang, Weitao Yan, Yihong Chen, Yanping Zhang, Muyang Zhou
College of Chemistry, Fuzhou University, Fuzhou 350116, China
* Corresponding author.
E-mail address: wangruo1201@gmail.com (R. Wang).
1 These authors contributed equally to this work.
Graphical abstract
A series of novel benzofuran-isatin-hydroxylimine/-thiosemicarbazide hybrids were designed, synthesized and evaluated for their in vitro anti-
TB activities against drug-sensitive MTB H37Rv and MDR-TB isolates as well as cytotoxicity. All benzofuran-isatin-hydroxylimine/-
thiosemicarbazide hybrids exhibited considerable in vitro anti-mycobacterial activities against the tested three MTB strains, and all of them also
showed acceptable cytotoxicity. The most active hybrid 7f was >4.8 and >51 folds more potent than the first line anti-TB agents RIF and INH
against both drug-sensitive MTB H37Rv and MDR-TB isolates, respectively. The results demonstrated the potential utility of benzofuran-isatin-
hydroxylimine/-thiosemicarbazide hybrids as anti-TB agents.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of novel benzofuran-isatin-hydroxylimine/-thiosemicarbazide hybrids 7a−l tethered
through propylene, butylene and pentylene were designed, synthesized and evaluated for their in
vitro anti-TB activities against drug-sensitive MTB H37Rv and MDR-TB isolates as well as
cytotoxicity. All benzofuran-isatin-hydroxylimine/-thiosemicarbazide hybrids exhibited
considerable in vitro anti-mycobacterial activities against the tested three MTB strains, and all
hybrids (MIC: <0.06−0.062 μg/mL) except 7i were more potent than the first-line anti-TB agents
INH and RIF (MIC: 0.078 μg/mL), and nine of them 7a, 7b, 7e−h and 7j−l (MIC: <0.06 μg/mL)
were comparable to the parent compound TAM16 (MIC: <0.06 μg/mL). The most active hybrid
7f (MIC: <0.06, 0.22 and 0.86 μg/mL, respectively) was >4.8 folds more potent than RIF and
INH against both drug-sensitive MTB H37Rv and MDR-TB isolates. The results demonstrated
the potential utility of benzofuran-isatin-hydroxylimine/-thiosemicarbazide hybrids as anti-TB
agents.
Available online
Keywords:
Benzofuran
Isatin
Hydroxylamine
Thiosemicarbazide
Hybrid compounds
Anti-mycobacterial
Anti-tubercular
Multi-drug resistant
Structure-activity relationship
Tuberculosis (TB), mainly caused by the bacterium Mycobacterium tuberculosis (MTB), is one of the top 10 causes of death and
the leading cause from a single infectious agent (above HIV/AIDS) [1]. Approximately 5%−10% of the estimated 1.7 billion people
infected with MTB will develop TB disease during their lifetime, and about 90% of cases occur among adults [2]. The latest World
Health Organization (WHO) global TB report has estimated that around 10 million people including 5.8 million men, 3.2 million women
and 1.0 million children developed TB disease in 2017, and 1.3 million deaths occurred in the same year [1].
There are several reasons for the incremental prevalence of TB, and the widely spread of HIV/TB co-infection and drug-resistant
TB (DR-TB, especially multi-drug resistant TB (MDR-TB)) are the two major reasons [3,4]. TB is one of the most common opportunistic
infection among HIV-positive people, and is the leading cause of death among HIV/TB co-infection patients, which resulted in 300,000
deaths in 2017 [1]. Globally in 2017, there were an estimated 558,000 new cases of rifampicin (RIF)-resistant TB (RR-TB), and 82% of

them were MDR-TB patients. The latest treatment outcome data show treatment success rates of 82% for TB, 77% for HIV-associated
TB, 55% for MDR/RR-TB and 34% for extensively drug-resistant TB (XDR-TB) [1]. Specific targets set in the End TB Strategy include
a 95% reduction in TB deaths and a 90% reduction in TB incidence by 2035, compared with levels in 2015. The most immediate
milestones, set for 2020, are a 35% reduction in TB deaths and a 20% reduction in the TB incidence rate, compared with levels in 2015
[5,6]. Obviously, novel anti-TB agents effective against both drug-susceptible and DR-TB especially MDR-TB are needed to achieve
this goal.
Fig. 1. Chemical structures of TAM16, isatin and 1,2,3-triazole tethered benzofuran-isatin-imine hybrid 1.
Benzofuran and isatin derivatives possess diverse biological properties [7-10], and some of them which are exemplified by TAM16
(Fig. 1) endow with promising in vitro and in vivo anti-TB potential [11,12]. TAM16 not only showed broad-spectrum in vitro anti-TB
activities, but also exhibited excellent in vivo physiochemical, toxicological and pharmacological properties [12]. 1,2,3-Triazole tethered
benzofuran-isatin-imine hybrids displayed excellent in vitro anti-TB activities against both drug-susceptible and MDR-TB strains, and
the most active hybrid 1 (MIC: 0.25 and 0.5ꢀμg/mL) was highly active against MTB H37Rv and MDR-TB strains [13], suggesting
benzofuran-isatin-imine hybrids are potential anti-TB candidates. The structure-activity relationship (SAR) for isatin-containing hybrids
indicated that the linker between the isatin and the other pharmacophores played a pivotal role in exertion of the anti-TB activities [14-
19], and introduction of hydrogen bond donors such as hydroxylimine and thiosemicarbazide were favorable to the activity [12,13].
Fig. 2. Design strategy for benzofuran-isatin-hydroxylimine/-thiosemicarbazide hybrids tethered via different linkers.
Based on the above facts and as a continuous program to develop novel anti-TB candidates, a series of novel benzofuran-isatin-
hydroxylimine/-thiosemicarbazide hybrids 7a−l tethered via propylene, butylene and pentylene were designed, synthesized and evaluated
for their anti-mycobacterial activity in vitro against both drug-susceptible and MDR-TB strains as well as cytotoxicity in VERO cells.
The design strategy was depicted in Fig. 2.
All of the desired benzofuran-isatin-hydroxylamine/-thiosemicarbazide hybrids 7a−l can be obtained by the synthetic route depicted
in Scheme 1. 5-Methoxyisatin/5-fluoroisatin/isatin 1a−c were alkylated with 1,3-dibromopropane, 1,4-dibromobutane, or 1,5-
dibromopentane 2a−c generated N-(3-bromopropyl/4-bromobutyl/5-bromopentyl)isatin derivatives 3a−f (yield: 63%−81%) [15,16].
Cyclization of ethyl 3-(4-substituted phenyl)-3-oxopropanoate 4a−c and benzoquinone with copper(II) triflate (Cu(OTf)2) as catalyst
provided benzofuran intermediates 5a−c [13]. Treatment of isatin derivatives 3a−f and benzofuran intermediate 5a−c with potassium
carbonate as base yielded the key intermediates 6a−h (51%−77%). Finally, condensations of 6a−h with hydroxylamine hydrochloride
or thiosemicarbazide hydrochloride in the presence of sodium bicarbonate provided benzofuran-isatin-hydroxylamine/-
thiosemicarbazide hybrids 7a−l (19%−54%).

Scheme 1. Synthesis of benzofuran-isatin-hydroxylimine/-thiosemicarbazide hybrids 7a−l.
The anti-mycobacterial activity of benzofuran-isatin-hydroxylimine/-thiosemicarbazide hybrids 7a−l, precursor 6a along with the
first-line anti-TB agents isoniazid (INH) and rifampicin (RIF) against MTB H37Rv strains and cytotoxicity towards VREO cells were
investigated (Table 1).
All benzofuran-isatin-hydroxylimine/-thiosemicarbazide hybrids 7a−l and precursor 6a with MIC values ranging from <0.016
μg/mL to 0.156 μg/mL exhibited great anti-mycobacterial activities against MTB H37Rv, and all hybrids except 7i were more potent than
the first-line anti-TB agents INH and RIF (MIC: 0.078 μg/mL), and nine of them 7a, 7b, 7e−h and 7j−l and precursor 6a (MIC: <0.06
μg/mL) were comparable to the parent compound TAM16 (MIC: <0.06 μg/mL). The SAR revealed that for benzofuran-isatin-
hydroxylimine hybrids, shorter linker between isatin and benzofuran was more favorable to the activity. In general, hybrids with
thiosemicarbazide fragment were more potent than the corresponding hydroxylamine analoges, and introduction of -F at para-position
of phenyl position (R3) was detrimental to the activity.
All hybrids also displayed acceptable toxicological profiles with CC50 ranging from 8 μg/mL to 128 μg/mL. The structure-
cytotoxicity relationship study indicated that compared with ketone, introduction of either hydroxylimine or thiosemicarbazide at C-3
position of isatin motif could increase the cytotoxicity as evidenced by ketone 6a (CC50: 512 μg/mL) was less toxic than hybrids 7a (CC50:
128 μg/mL) and 7e (CC50: 128 μg/mL), and the relative contribution order was ketone > hydroxylimine > thiosemicarbazide. Introduction
of -F at C-5 position of isatin and para-position of phenyl position resulted in the increment of cytotoxicity. In general, hybrids with
longer linkers also showed higher cytotoxicity.
Table 1
In vitro anti-mycobacterial activity and cytotoxicity of benzofuran-isatin-hydroxylimine/-thiosemicarbazide hybrids 7a−l.
Compounds
6a
7a
7b
MIC (μg/mL)
<0.016
<0.016
<0.016
0.062
CC50 (μg/mL)
SI
512
128
32
>32000
>8000
>2000
1032
7c
64
7d
7e
7f
7g
7h
7i
0.031
128
128
8
16
16
4129
>8000
>500
>1000
>1000
205
<0.016
<0.016
<0.016
<0.016
0.156
32
7j
7k
7l
TAM16
INH
RIF
<0.016
<0.016
<0.016
<0.016
0.078
128
64
64
>1024
256
512
>8000
>4000
>4000
>64000
3282
0.078
6564
SI: selectivity index, CC50/MICMTB H37Rv
To evaluate the selectivity profiles of these hybrids, the selectivity index (SI) values from the ratio of CC50/MICMTB H37Rv were
calculated (Table 1). All hybrids possessed high SI values (SI: ≥205), suggesting that this kind of hybrids hold excellent selectivity
profiles.
Encouraged by their strong potency against the drug sensitive MTB H37Rv strain, the most active nine benzofuran-isatin-
hydroxylimine/-thiosemicarbazide hybrids 7a, 7b, 7e−h and 7j−l and precursor 6a were selected for further evaluation for in vitro activity
against two clinical MDR-TB isolates (resistant to INH and RIF). The in vitro activity of selected compounds against MDR-TB isolates
was listed in Table 2.
Table 2
In vitro activity of selected hybrids against MDR-TB isolates.

Compounds
MIC (μg/mL)
MDR-TB-1
2.47
MDR-TB-2
5.92
6a
7a
1.59
1.83
7b
1.94
2.22
7e
0.90
0.94
7f
0.22
0.86
7g
1.41
1.83
7h
2.84
4.26
7j
2.26
3.89
7k
3.24
2.92
7l
12.96
<0.016
>40
14.18
<0.016
>40
TAM16
INH
RIF
>40
>40
All of the selected nine benzofuran-isatin-hydroxylimine/-thiosemicarbazide hybrids 7a, 7b, 7e−h and 7j−l and precursor 6a
demonstrated considerable activity against the tested two clinical MDR-TB isolates with MIC values ranging from 0.22 μg/mL to 14.18
μg/mL. All hybrids were more potent than RIF and INH (MIC: >40 μg/mL), but were less active than TAM16 (MIC: <0.016 μg/mL)
against the two clinical MDR-TB isolates. The SAR indicated that benzofuran-isatin-thiosemicarbazide hybrids were more active than
the corresponding hydroxylimine analogs. Hybrid 7f (MIC: 0.22 and 0.86 μg/mL, respectively) was found to be most active against the
tested two clinical MDR-TB isolates, and it was >51-fold more potent than RIF and INH (MIC: >40 μg/mL).
In conclusion, a series of novel benzofuran-isatin-hydroxylimine/-thiosemicarbazide hybrids were designed, synthesized and
evaluated for their in vitro anti-TB activities against drug-sensitive MTB H37Rv and MDR-TB isolates as well as cytotoxicity. All
benzofuran-isatin-hydroxylimine/-thiosemicarbazide hybrids exhibited considerable in vitro anti-mycobacterial activities against the
tested three MTB strains, and all of them also showed acceptable cytotoxicity. The most active hybrid 7f was >4.8 and >51 folds more
potent than the first line anti-TB agents RIF and INH against both drug-sensitive MTB H37Rv and MDR-TB isolates, respectively. The
results demonstrated the potential utility of benzofuran-isatin-hydroxylimine/-thiosemicarbazide hybrids as anti-TB agents.
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