Benzofuran-isatin-imine hybrids tethered via different length alkyl linkers: Design, synthesis and in vitro evaluation of anti-tubercular and anti-bacterial activities as well as cytotoxicity

Article abstract of DOI:10.1016/j.ejmech.2019.01.042

Herein we report the design and synthesis of twenty-two novel benzofuran-isatin-imine hybrids 7a-v tethered through propylene, butylene, pentylene and hexylene, and for the evaluation of their in vitro anti-tubercular and anti-bacterial activities as well

Full text of DOI:10.1016/j.ejmech.2019.01.042

Accepted Manuscript
Benzofuran-isatin-imine hybrids tethered via different length alkyl linkers: Design,
synthesis and in vitro evaluation of anti-tubercular and anti-bacterial activities as well
as cytotoxicity
Feng Gao, Tengfei Wang, Meixiang Gao, Xia Zhang, Zhuqing Liu, Shi-Jia Zhao, Zao-
Sheng Lv, Jiaqi Xiao
PII:
S0223-5234(19)30052-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.042
EJMECH 11049
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 October 2018
Revised Date: 16 January 2019
Accepted Date: 17 January 2019
Please cite this article as: F. Gao, T. Wang, M. Gao, X. Zhang, Z. Liu, S.-J. Zhao, Z.-S. Lv, J. Xiao,
Benzofuran-isatin-imine hybrids tethered via different length alkyl linkers: Design, synthesis and in vitro
evaluation of anti-tubercular and anti-bacterial activities as well as cytotoxicity, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.01.042.
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
Benzofuran-isatin-imine hybrids tethered via different length
alkyl linkers: Design, synthesis and in vitro evaluation of
anti-tubercular and anti-bacterial activities as well as
cytotoxicity
Feng Gao1,2,3*, Tengfei Wang4, Meixiang Gao1, Xia Zhang1, Zhuqing Liu1, Shi-Jia Zhao5,
Zao-Sheng Lv5, Jiaqi Xiao1,2*
1 Academy of Advanced Interdisciplinary Studies, Qilu University of Technology (Shandong Academy of
Sciences), Jinan, PR China;
2 School of Chemistry and Pharmaceutical Engineering, Qilu University of Technology (Shandong Academy of
Sciences), Jinan, PR China;
3 Department of Medical Imaging, Jinling Hospital, Medical School of Nanjing University, Nanjing, PR China;
4 State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology
(Shandong Academy of Sciences), Jinan, PR China;
5 Key Laboratory of Hubei Province for Coal Conversion and New Carbon Materials, Wuhan University of
Science and Technology, Wuhan, P R China.
All benzofuran-isatin-imine hybrids 7a-v exhibited considerable in vitro anti-TB and anti-bacterial

ACCEPTED MANUSCRIPT
activities, as well as acceptable cytotoxicity towards VERO. The most active hybrid 7j (MIC:
<0.016, 0.062 and 0.16 µg/mL, respectively) was >4.8 and ≥48 folds more potent than the first
line anti-TB agents RIF and INH against both drug-sensitive MTB H37Rv and MDR-TB isolates,
respectively. Moreover, hybrid 7j also demonstrated promising anti-bacterial activities with MIC
values of ≤1 µg/mL against the majority of the tested Gram-negative and Gram-positive pathogens,
which was comparable to vancomycin (MIC: 0.5-4 µg/mL) and CPFX (MIC: 0.125-8 µg/mL)
against Gram-positive bacteria, but slightly less potent than CPFX (MIC: ≤0.03-0.5 µg/mL)
against Gram-negative bacteria. The results indicated that benzofuran-isatin-imine hybrids could
act as candidates for the development of anti-TB and anti-bacterial agents.

ACCEPTED MANUSCRIPT
Benzofuran-isatin-imine hybrids tethered via different length
alkyl linkers: Design, synthesis and in vitro evaluation of
anti-tubercular and anti-bacterial activities as well as
cytotoxicity
Feng Gao1,2,3*, Tengfei Wang4, Meixiang Gao1, Xia Zhang1, Zhuqing Liu1, Shi-Jia Zhao5,
Zao-Sheng Lv5, Jiaqi Xiao1,2*
1 Academy of Advanced Interdisciplinary Studies, Qilu University of Technology (Shandong Academy of
Sciences), Jinan, PR China;
2 School of Chemistry and Pharmaceutical Engineering, Qilu University of Technology (Shandong Academy of
Sciences), Jinan, PR China;
3 Department of Medical Imaging, Jinling Hospital, Medical School of Nanjing University, Nanjing, PR China;
4 State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology
(Shandong Academy of Sciences), Jinan, PR China;
5 Key Laboratory of Hubei Province for Coal Conversion and New Carbon Materials, Wuhan University of Science
and Technology, Wuhan, P R China.
Abstract: Herein we report the design and synthesis of twenty-two novel
benzofuran-isatin-imine hybrids 7a-v tethered through propylene, butylene, pentylene
and hexylene, and for the evaluation of their in vitro anti-tubercular and anti-bacterial
activities as well as cytotoxicity. All benzofuran-isatin-imine hybrids exhibited
considerable in vitro anti-TB (MIC: <0.016-0.218 µg/mL and 0.062-14.15 µg/mL
against drug-sensitive and MDR MTB, respectively) and anti-bacterial (MIC: 0.25-64
µg/mL and 0.06-16 µg/mL against Gram-positive and Gram-negative strains,
respectively) activities. All of them also showed acceptable cytotoxicity towards
VERO (CC50: 8-128 µg/mL). The most active hybrid 7j (MIC: <0.016, 0.062 and 0.16
µg/mL, respectively) was >4.8 and ≥48 folds more potent than the first line anti-TB
agents RIF and INH against both drug-sensitive MTB H37Rv and MDR-TB isolates,
respectively. Moreover, hybrid 7j also demonstrated promising anti-bacterial activities
with MIC values of ≤1 µg/mL against the majority of the tested Gram-negative and
Gram-positive pathogens, which was comparable to vancomycin (MIC: 0.5-4 µg/mL)
and CPFX (MIC: 0.125-8 µg/mL) against Gram-positive bacteria, but slightly less
potent than CPFX (MIC: ≤0.03-0.5 µg/mL) against Gram-negative bacteria. The
*Corresponding author: rggaofeng@hotmail.com (Feng Gao); jiaqixiao@qlu.edu.cn (Jiaqi
Xiao)
1

ACCEPTED MANUSCRIPT
results indicated that benzofuran-isatin-imine hybrids could act as candidates for the
development of anti-TB and anti-bacterial agents.
Keywords: benzofuran; isatin; imine; hybrid compounds; anti-tubercular;
anti-bacterial; multi-drug resistant; structure-activity relationship
1. Introduction
Infections, caused by Gram-positive and Gram-negative bacteria, have already put a
heavy burden on the global health system [1,2]. Antibiotics are the most important
weapons for human to fight against bacterial infection, but the overuse and misuse of
antibiotics have already triggered increased drug resistance [3]. Approximately
700,000 drug-resistant pathogens caused deaths annually throughout the world and
the number may increase to 10 million in the year 2050 if current trends continue.
Tuberculosis (TB), mainly caused by the bacterium Mycobacterium tuberculosis
(MTB), is the leading cause from a single infectious agent (above HIV/AIDS) [4]. The
World Health Organization (WHO) report estimated that that around 10 million
people developed TB disease and around 1.3 million deaths happened in 2017 [4].
Drug-resistant TB (DR-TB), especially multidrug-resistant TB (DMR-TB), is one of
the major reasons for the incremental prevalence of TB [5]. Obviously, novel anti-TB
agents which are effective against both drug-susceptible TB and DR-TB especially
MDR-TB are needed to control and eradication of this plague.
There are several anti-TB and anti-bacterial candidates which are under different
stages of clinical trials. The efficiency of the candidates rely on the structure.
Heterocyclic compounds play a dominate role in the development of new drugs as
evidenced by the fact that 80% of top-selling drugs contain heterocycles [6].
Heterocycles can serve as useful tools to manipulate lipophilicity, polarity, and
hydrogen bonding capacity of molecules, which may lead to improved
pharmacological, pharmacokinetic, toxicological, and physicochemical properties of
drug candidates and ultimately drugs [7].
2

ACCEPTED MANUSCRIPT
Figure 1 Chemical structures of benzofuran, isatin and TAM16
Benzofuran (constituted by fused benzene and furan rings) and isatin
(1H-indole-2,3-dione, Figure 1), are fundamental structure units in a variety of
biologically active natural products [8,9]. Benzofuran and isatin have the ability to
exert several noncovalent interactions such as Van der Waals force and hydrogen
bonds [10,11], and their derivatives possess various biological properties such as
anti-cancer [12,13], anti-viral [14,15], anti-bacterial [16,17] and anti-tubercular [18,19]
activities. Many benzofuran- or isatin-based derivatives have already been used for
the treatment various diseases, so benzofuran and isatin derivatives occupy an
important position in the development of new drugs.
Molecular hybridization, a method to form the novel compounds which has the
potential to overcome cross resistance and improve efficacy compared to the parent
drugs, represents a promising strategy in the development of new drugs [20,21].
Benzofuran derivative TAM16 (Figure 1) not only showed broad-spectrum in vitro
anti-bacterial activities, but also exhibited excellent in vivo physiochemical,
toxicological and pharmacological properties [22,23]. Our previous results indicated
that benzofuran-isatin hybrids 1 exhibited promising in vitro anti-TB activities against
both drug-susceptible and MDR-TB strains, demonstrating that benzofuran-isatin
hybrids could act as potential anti-TB candidates [24-31]. The structure-activity
relationship (SAR) for isatin-containing hybrids indicated that the linker between the
isatin and the other pharmacophores and imines at C-3 position of isatin moiety played
a pivotal role in exertion of the anti-TB and anti-bacterial activities [32-36].
3

ACCEPTED MANUSCRIPT
Figure 2 Design strategies for benzofuran-isatin-imine hybrids tethered via different
length alkyl linkers
Based on the facts stated above and as a continuous program to develop novel anti-TB
and anti-bacterial candidates, a series of novel benzofuran-isatin-imine hybrids 7a-v
tethered via different length alkyl linkers were designed and synthesized. In vitro
anti-mycobacterial and anti-bacterial activities, together with cytotoxicity of the
hybrids in VERO cells were also investigated. The design strategy was depicted in
Figure 2.
2
Results and discussion
Benzofuran-isatin-imine hybrids 7a-k were prepared following the synthetic route
described in Scheme 1. Alkylation of 5-methoxyisatin/5-fluoroisatin/isatin 1a-c with
1,3-dibromopropane,
1,6-dibromohextane
1,4-dibromobutane,
2a-d
1,5-dibromopentane
and
provided
N-(3-bromopropyl/4-bromobutyl/5-bromopentyl/6-bromohexyl)isatin derivatives 3a-i
with yields 62-76% [15,16]. The reaction of ethyl 3-(4-substituted
4

ACCEPTED MANUSCRIPT
phenyl)-3-oxopropanoates 4a-c with benzoquinone in the presence of copper (II)
triflate (Cu(OTf)2) as catalyst yielded benzofuran intermediates 5a-c [13]. Treatment
of isatin derivatives 3a-i and benzofuran intermediate 5a-c with potassium carbonate
as base afforded the key intermediates 6a-m (51-68%). Finally, condensations of
6a-m with the corresponding amine hydrochlorides in the presence of sodium
bicarbonate gave benzofuran-isatin-imine hybrids 7a-v (18-73%). The chemical
structures and yields of target compounds 7a-v were listed in Table 1.
Scheme 1 Synthesis of benzofuran-isatin-imine hybrids 7a-v
The anti-mycobacterial activity of benzofuran-isatin-imine hybrids 7a-v, their
precursors 6h,i and 6l,m, together with the references TAM16, ciprofloxacin (CPFX),
5

ACCEPTED MANUSCRIPT
rifampicin (RIF) and isoniazid (INH) against MTB H37Rv strains and the cytotoxicity
towards VREO cells were studied and the results were presented in Table 2.
Table 1. Chemical structures and yields of benzofuran-isatin-imine hybrids 7a-v
R2
O
O
OEt
N
O
n
R3
R1
O
7
Compounds
n
R1
F
R2
R3
Yield (%)
59
1
1
1
1
1
1
1
1
1
2
2
3
NOMe
NOMe
NOEt
H
7a
7b
7c
7d
7e
7f
F
OMe
OMe
H
79
F
49
OMe
OMe
OMe
H
NOMe
NOMe
NOEt
83
OMe
OMe
H
58
46
NOMe
NOMe
NOEt
45
7g
7h
7i
H
OMe
OMe
OMe
OMe
OMe
68
H
51
H
NOMe
NOEt
69
7j
H
53
7k
7l
F
NOMe
67
6

ACCEPTED MANUSCRIPT
3
4
4
4
3
3
3
4
4
4
F
F
NOEt
NOMe
OMe
OMe
OMe
F
43
42
18
49
36
73
39
71
62
27
7m
7n
7o
7p
7q
7r
7s
F
NNHCSNH2
NOMe
F
OMe
H
NOMe
OMe
OMe
OMe
OMe
OMe
OMe
NOH
H
NOMe
H
NOMe
7t
H
NOEt
7u
H
NNHCSNH2
7v
All benzofuran-isatin-imine hybrids 7a-v and their precursors 6h,i and 6l,m exhibited
excellent in vitro anti-mycobacterial activities against MTB H37Rv, and MIC values
ranging from < 0.016 to 0.218 µg/mL were found. All hybrids were more active than
the reference CPFX (MIC: 0.291 µg/mL), and the majority of them were also more
potent than the first-line anti-TB agents INH and RIF (MIC: 0.078 µg/mL). In
particular, among all the hybrids, sixteen compounds 6i, 6m, 7d-l, 7n, 7o and 7t-v
(MIC: < 0.016 µg/mL), held comparable activities to TAM16 (MIC: < 0.016 µg/mL)
against MTB H37Rv.
Table 2 In vitro anti-mycobacterial activity and cytotoxicity of
benzofuran-isatin-imine hybrids 7a-v and their precursors 6h,i and 6l,m
Compounds
MIC (µg/mL)
CC50 (µg/mL)
SI
0.064
512
8,000
6h
7

ACCEPTED MANUSCRIPT
<0.016
0.062
1,024
256
512
64
>64,000
4,129
>32,000
516
6i
6l
<0.016
0.062
6m
7a
7b
7c
7d
7e
7f
0.031
32
1,032
410
0.156
32
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
<0.016
0.031
>4,000
>8,000
>4,000
>2,000
>1,000
>8,000
>2,000
>4,000
>2,000
1,032
>1,000
>500
64
128
32
7g
7h
7i
64
128
16
7j
32
7k
7l
64
32
32
16
8
7m
7n
7o
7p
7q
<0.016
<0.016
0.218
16
16
73
0.156
102
8

ACCEPTED MANUSCRIPT
0.062
0.049
64
32
1,032
1,032
7r
7s
7t
<0.016
<0.016
<0.016
<0.016
0.291
32
>2,000
>1,000
>500
16
7u
7v
8
>1,024
512
>64,000
1,759
TAM16
CPFX
INH
0.078
3,282
256
512
0.078
6,564
RIF
aSI: selectivity index, CC50/MICMTB H37Rv
All hybrids 7a-v also displayed acceptable toxicological profiles with CC50 values
ranging from 8 to 128 µg/mL. The structure-cytotoxicity relationship study indicated
that introduction of imines at C-3 position of isatin motif increased the cytotoxicity
greatly, and the relative contribution order was -O > -NOH > -NOMe ≈ -NOEt >
NNHCSNH2. Introduction of -F at para-position of phenyl position resulted in the
increment of cytotoxicity. In general, hybrids with longer linkers displayed higher
cytotoxicity.
In order to test the selectivity of these hybrids, the selectivity index (SI) values from the
ratio of CC50/MICMTB H37Rv were calculated (Table 2). All hybrids possessed high SI
values (SI: ≥73), and some of them were much greater than references CPFX, INH
and RIF (SI: 1,759, 3,282 and 6,564, respectively), suggesting that this kind of
hybrids hold excellent selectivity properties.
Encouraged by their strong potency against the drug sensitive MTB H37Rv strain, the
most active hybrids 6i, 6m, 7d-l, 7n, 7o and 7t-v were selected for further evaluation
about in vitro activity against two clinical MDR-TB isolates (resistant to INH and
9

ACCEPTED MANUSCRIPT
RIF). The in vitro activity of the selected hybrids against MDR-TB isolates was listed
in Table 3.
Table 3. In vitro activity of selected hybrids against MDR-TB isolates
MIC (µg/mL)
Compounds
MDR-TB-1
14.15
5.89
MDR-TB-2
12.94
2.98
6i
6m
7d
7e
7f
0.21
0.27
0.49
0.29
2.24
2.12
7g
7h
7i
0.94
0.91
0.16
0.21
4.25
5.88
7j
0.062
3.23
0.16
7k
7l
3.42
3.46
4.24
7n
7o
7t
2.85
3.56
0.95
0.90
1.73
1.87
7u
7v
2.69
3.86
0.47
0.48
10

ACCEPTED MANUSCRIPT
TAM16
CPFX
INH
<0.016
0.39
>40
<0.016
0.27
>40
RIF
>40
>40
All of the selected hybrids 6i, 6m, 7d-l, 7n, 7o and 7t-v showed promising activity
against the tested two clinical MDR-TB isolates with MIC values in the range of
0.062 to 14.15 µg/mL. All of these hybrids were more potent than RIF and INH
(MIC: >40 µg/mL), and three of them 7d (MIC: 0.21 and 0.27 µg/mL), 7h (MIC: 0.16
and 0.21 µg/mL) and 7j (MIC: 0.062 and 0.16 µg/mL) were more potent than CPFX
(MIC: 0.39 and 0.27 µg/mL). However, all of these hybrids were less active than
TAM16 (MIC: <0.016 µg/mL) against the two clinical MDR-TB isolates.
The SAR indicated that hybrids with shorter propylene and butylene linkers were more
potent than the corresponding analogs with longer hexylene and pentylene linkers
against both drug-sensitive and MDR MTB strains generally. Introduction of imines
could enhance the anti-mycobacterial activity, and the relative contribution was
-NNHCSNH2 > -NOMe > -NOEt > -O > -NOH. Introduction of -F at the para-position
of phenyl position (R3) was detrimental to the activity generally. Hybrid 7j (MIC:
0.062 and 0.16 µg/mL) was found to be the most active compound against the two
clinical MDR-TB isolates used in this study. The result displayed that hybrid 7j was
≥48 folds more potent than first-line anti-TB agents RIF (MIC: >40 and >40 µg/mL)
and INH (MIC: 2.98 and >40 µg/mL), and also superior to CPFX (MIC: 0.39 and 0.27
µg/mL).
In vitro antibacterial activities of all benzofuran-isatin-imine hybrids 7a-v and their
precursors 6h,i and 6l,m against a panel of Gram-positive and Gram-negative
pathogens were evaluated [37]. The minimum inhibitory concentration (MIC) is
defined as the minimum concentration of compound required to give 90% inhibition of
bacterial growth. The results were listed in Table 4 and 5, respectively.
11

ACCEPTED MANUSCRIPT
Table 4 In vitro antibacterial activity of benzofuran-isatin-imine hybrids 7a-v and their
precursors 6h,i and 6l,m against Gram-positive strains
MIC (µg/mL)
Compd.
MSSE
1
MRSE
MSSA
MRSA
E.fa.
4
E.fm.
64
16
8
8
8
2
2
1
1
1
1
8
1
4
4
8
4
8
1
1
4
8
4
1
2
1
2
2
1
2
2
4
4
4
2
8
2
4
4
1
1
2
4
4
1
1
1
6h
6i
1
2
0.25
1
4
2
6l
4
2
16
8
6m
7a
7b
7c
7d
7e
7f
0.25
0.50
1
1
2
2
2
16
32
16
8
16
0.5
2
4
0.5
0.25
1
1
1
8
8
64
8
0.125
0.5
1
0.5
1
1
7g
7h
7i
4
32
16
8
8
4
0.125
0.5
0.5
1
0.5
2
1
7j
4
64
16
32
16
32
8
7k
7l
4
1
4
4
7m
7n
7o
7p
7q
0.5
0.5
0.25
1
16
2
2
1
8
1
4
1
16
12

ACCEPTED MANUSCRIPT
0.25
0.5
0.5
1
4
4
8
8
4
4
1
2
2
2
4
2
4
8
7r
7s
32
16
32
64
8
2
2
4
7t
1
1
2
7u
1
1
0.5
0.25
1
7v
0.125
1
0.25
0.5
0.5
4
CPFX
VAN
1
1
Abbreviations:
MSSE,
methicillin-sensitive
Staphylococcus
epidermidis;
MRSE,
methicillin-resistant Staphylococcus epidermidis; MSSA, methicillin-sensitive Staphylococcus
aureus; MRSA, methicillin-resistant Staphylococcus aureus; E.fa., Enterococcus faecalis ATCC;
E.fm., Enterococcus faecium; CPFX, ciprofloxacin; VAN, vancomycin.
Table 5 In vitro antibacterial activity of benzofuran-isatin-imine hybrids 7a-v and their
precursors 6h,i and 6l,m against Gram-positive strains against Gram-negative strains
MIC (µg/mL)
Compd.
E.co.
K.p.
P.a.
A.c.
E.c.
E.a.
P.m.
C.f.
0.25
2
8
8
1
1
0.25
1
6h
6i
0.125
0.06
0.25
0.125
0.25
0.5
1
0.5
1
4
4
4
2
4
1
4
8
2
0.5
0.125
0.5
0.25
0.5
0.125
0.125
0.06
0.25
0.25
0.25
2
1
0.25
0.06
0.25
0.25
0.5
6l
2
0.125
0.125
0.5
6m
7a
7b
7c
7d
0.25
2
0.5
8
1
0.5
2
8
0.25
0.5
0.5
0.125
0.5
1
2
0.5
13

ACCEPTED MANUSCRIPT
0.25
0.5
1
2
1
4
4
4
0.25
1
0.125
2
0.25
4
0.5
2
7e
7f
0.25
0.25
0.5
0.5
1
1
1
2
0.25
1
0.5
0.5
1
7g
2
1
0.5
4
0.5
7h
7i
1
4
2
1
2
2
0.5
0.25
2
4
1
0.25
1
0.06
2
0.125
2
0.25
2
7j
1
2
0.5
1
7k
7l
0.125
0.125
0.25
0.125
0.5
0.5
4
1
2
0.125
0.5
0.5
2
0.06
0.25
0.125
1
0.125
0.125
0.5
0.25
0.125
0.125
0.5
1
4
8
0.25
0.5
0.5
0.25
0.06
1
7m
7n
7o
2
8
4
1
4
16
4
2
2
0.25
0.125
0.5
1
0.125
0.06
0.25
0.5
7p
7q
7r
0.125
0.25
0.25
0.125
0.5
2
2
2
1
4
2
2
1
1
0.25
0.125
0.5
1
7s
4
2
4
0.25
0.25
2
0.125
0.25
1
0.125
1
7t
1
1
2
7u
7v
1
8
4
4
2
0.06
0.5
0.25
0.5
0.06
0.06
≤0.03
0.03
CPFX
Abbreviations: E.co., Escherichia coli; K.p., Klebsiellapneumoniae; P.a., Pseudomonas aeruginosa
ATCC 27853; A.c., Acinetobactercal coacetious; E.c., Enterobacter cloacae; E.a., Enterobacter
aerogenes; P.m., Proteus mirabilis; C.f., Citrobacter freundii; CPFX, ciprofloxacin.
14

ACCEPTED MANUSCRIPT
As shown in Table 4 and 5, all benzofuran-isatin-imine hybrids 7a-v and their
precursors 6h,i and 6l,m exhibited considerable antibacterial activities against all the
tested pathogens including drug-resistant MRSA and MRSE with MIC ranging from
0.06 to 64 µg/mL. In general, the hybrids were more potent against Gram-negative
pathogens than Gram-positive strains. Substitution of the ketone at C-3 position of
isatin motif could improve the anti-bacterial activity against both Gram-positive and
Gram-negative organisms. In particular, the most active anti-TB hybrid 7j
demonstrated promising anti-bacterial activities with MIC values of ≤1 µg/mL against
the majority of the tested Gram-negative and Gram-positive pathogens, which further
suggested that in vitro activity of hybrid 7j was comparable to vancomycin (MIC: 0.5-4
µg/mL) and CPFX (MIC: 0.125-8 µg/mL) against Gram-positive bacteria, and slightly
less potent than CPFX (MIC: ≤0.03-0.5 µg/mL) against Gram-negative bacteria.
Table 6 The metabolic stability and in vivo pharmacokinetic values of hybrid 7j,
TAM16 and CPFX in mice
Pharmacokinetics (p.o.)
Compd.
metabolic stability
(t1/2, min)
AUC0-inf
(ng•h/mL)
Cmax (ng/mL)
t1/2 (h)
Tmax (h)
19.2
>60
>60
927
3.33
9.88
5.2
1.65
3.46
1.8
3,024
39,329
17,835
7j
TAM16[24]
CPFX[17]
t1/2: half-life;
5,064
3,129
Cmax: the peak concentration;
Tmax: the time to reach peak concentration;
AUC: the area under the curve.
With the promising anti-TB and anti-bacterial activities, hybrid 7j was selected for the
further investigation of metabolic stability and in vivo pharmacokinetics in mice (25
mg/kg, single oral administration) [24]. The results were listed in Table 6. Comparing
to TAM16 (t1/2: >60 min) and CPFX (t1/2: >60 min), hybrid 7j showed much lower
15

ACCEPTED MANUSCRIPT
metabolic stability (t1/2: 19.2 min). Pharmacokinetic study showed that hybrid 7j was
also inferior to TAM16 and CPFX in terms of Cmax (927 ng/mL vs 5,064 and 3,129
ng/mL), t1/2 (3.33 h vs 9.88 and 5.2 h), Tmax (1.65 h vs 3.46 and 1.8 h) and AUC0-inf
(3,024 ng•h/mL vs 39,329 and 17,835 ng•h/mL).
3 Conclusions
In conclusion, a series of novel benzofuran-isatin-imine hybrids tethered by different
length alkyl linkers propylene, butylenes, pentylene and hexylene were designed and
synthesized. The evaluation of all the hybrids about the in vitro anti-TB activities
against both drug-sensitive MTB H37Rv and MDR-TB isolates, anti-bacterial
activities against a panel of Gram-positive and Gram-negative pathogens as well as
cytotoxicity towards VERO were performed. All benzofuran-isatin-imine hybrids not
only exhibited considerable in vitro anti-TB and anti-bacterial activities, but also
showed acceptable cytotoxicity. The most active hybrid 7j was >4.8 and ≥48 folds
more potent than the first line anti-TB agents RIF and INH against both
drug-sensitive MTB H37Rv and MDR-TB isolates, respectively. Moreover, hybrid 7j
also demonstrated promising anti-bacterial activities which was comparable to
vancomycin and CPFX against Gram-positive bacteria, but slightly less potent than
CPFX against Gram-negative bacteria. The results suggested the potential utility of
benzofuran-isatin-imine hybrids as anti-TB and anti-bacterial agents.
4. Synthesis methods
N-(3-bromopropyl/4-bromobutyl/5-bromopentyl/6-bromohexyl)isatin
3a-i (yield: 62-76%) and ethyl
derivatives
2-(4-substituted
phenyl)-5-(prop-2-yn-1-yloxy)benzofuran-3-carboxylate 5-c were synthesized via
literature methods [13,15].
A mixture of N-(3-bromopropyl/4-bromobutyl/5-bromopentyl/6-bromohexyl)isatin
derivatives
3a-i
(1
mmol),
ethyl
2-(4-substituted
phenyl)-5-(prop-2-yn-1-yloxy)benzofuran-3-carboxylate 5a-c (1 mmol) and K2CO3 (3
mmol) in DMF (20 mL) was stirred at room temperature for 48 h. Then the mixture
was filtered and the filtrate was concentrated under reduced pressure. The residue was
16

ACCEPTED MANUSCRIPT
purified by silica gel column chromatography with PE:EA=1:1 to give the precursors
6a-m (51-68%).
Precursors 6a-m (1 mmol), sodium bicarbonate (2 mmol), and the corresponding
amine hydrochlorides (1.5 mmol) were dissolved in water (10 mL) and tetrahydrofuran
o
(THF, 30 mL), and the mixturewas stirred at 60 C for 12 h. After cooling to room
temperature, the mixture was extracted with EA (20 mL×3). The combined organic
phases were washed with water (50 mL×2) and brine (50 mL), and then dried with
Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give a
residue, which was further purified by silica gel chromatography with PE: EA=1:8 as
eluant to give the desired benzofuran-isatin-imine hybrids 7a-v (18-79%).
4.1.
ethyl
5-((5-(5-fluoro-2,3-dioxoindolin-1-yl)pentyl)oxy)-2-(4-methoxyphenyl)benzofuran-3-
carboxylate (6h)
1
Yellow solid, yield: 65%. H NMR (400 MHz, DMSO-d6) δ 1.32 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 1.51-1.54 (2H, m, -CH2-), 1.66-1.70 (2H, m, -CH2-), 1.78-1.82 (2H, m,
-CH2-), 3.68 (2H, t, J = 8.0 Hz, -CH2-), 3.85 (3H, s, OCH3), 4.01 (2H, t, J = 8.0 Hz,
-CH2-), 4.32 (2H, q, J = 4.0 Hz, CO2CH2CH3), 6.96 (1H, dd, J = 4.0, 8.0 Hz, Ar-H),
7.09 (2H, d, J = 8.0 Hz, Ar-H), 7.41-7.45 (2H, m, Ar-H), 7.49-7.57 (2H, m, Ar-H),
7.57 (1H, d, J = 8.0 Hz, Ar-H), 7.95 (2H, d, J = 8.0 Hz, Ar-H). 13C NMR (101Hz,
DMSO-d6) δ 183.40, 163.64, 161.43, 161.22, 158.61, 157.69, 156.25, 148.25, 147.42,
131.41, 127.97, 124.35, 121.73, 118.89, 118.82, 114.45, 114.16, 112.62, 112.55,
112.29, 112.03, 111.79, 107.69, 105.67, 68.35, 60.81, 55.87, 28.83, 26.89, 23.31,
14.47. ESI-MS m/z: 568 [M+Na]+.
4.2.
ethyl
5-((6-(5-fluoro-2,3-dioxoindolin-1-yl)hexyl)oxy)-2-(4-methoxyphenyl)benzofuran-3-
carboxylate (6i)
1
Yellow solid, yield: 67%. H NMR (400 MHz, DMSO-d6) δ 1.32 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 1.35-1.49 (4H, m, 2×-CH2-), 1.59-1.66 (2H, m, -CH2-), 1.70-1.77 (2H,
m, -CH2-), 3.66 (2H, t, J = 8.0 Hz, -CH2-), 3.85 (3H, s, OCH3), 4.00 (2H, t, J = 8.0 Hz,
-CH2-), 4.32 (2H, q, J = 8.0 Hz, CO2CH2CH3), 6.94 (1H, dd, J = 4.0, 8.0 Hz, Ar-H),
17

ACCEPTED MANUSCRIPT
7.07 (2H, d, J = 8.0 Hz, Ar-H), 7.19-7.22 (1H, m, Ar-H), 7.40-7.43 (2H, m, Ar-H),
7.49-7.54 (2H, m, Ar-H), 7.96 (2H, d, J = 8.0 Hz, Ar-H). 13C NMR (101Hz, DMSO-d6)
δ 183.38, 163.62, 161.40, 161.17, 160.07, 158.53, 157.67, 156.23, 148.22, 147.42,
131.37, 127.97, 124.56, 124.32, 121.73, 118.85, 114.43, 114.11, 112.54, 112.47,
112.21, 111.99, 107.66, 105.64, 68.40, 60.78, 55.83, 49.07, 29.08, 27.13, 26.45, 25.73,
14.44. ESI-MS m/z: 582 [M+Na]+.
4.3.
ethyl
5-((6-(2,3-dioxoindolin-1-yl)hexyl)oxy)-2-(4-methoxyphenyl)benzofuran-3-carboxyla
te (6l)
1
Yellow solid, yield: 76%. H NMR (400 MHz, CDCl3) δ 1.43 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 1.48-1.56 (4H, m, 2×-CH2-), 1.76-1.87 (4H, m, 2×-CH2-), 3.76 (2H, t, J
= 8.0 Hz, -CH2-), 3.88 (3H, s, OCH3), 4.05 (2H, t, J = 8.0 Hz, -CH2-), 4.43 (2H, q, J =
8.0 Hz, CO2CH2CH3), 6.91-6.94 (2H, m, Ar-H), 7.02 (2H, d, J = 8.0 Hz, Ar-H), 7.12
(1H, t, J = 4.0 Hz, Ar-H), 7.40 (1H, d, J = 8.0 Hz, Ar-H), 7.53 (1H, d, J = 4.0 Hz,
Ar-H), 7.57-7.63 (2H, m, Ar-H), 8.00 (2H, d, J = 8.0 Hz, Ar-H). ESI-MS m/z: 565
[M+Na]+.
4.4.
ethyl
ethyl
5-((6-(5-fluoro-2,3-dioxoindolin-1-yl)hexyl)oxy)-2-(4-fluorophenyl)benzofuran-3-car
boxylate (6m)
1
Yellow solid, yield: 62%. H NMR (400 MHz, DMSO-d6) δ 1.31 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 1.39-1.51 (4H, m, 2×-CH2-), 1.62-1.65 (2H, m, -CH2-), 1.73-1.77 (2H,
m, -CH2-), 3.68 (2H, t, J = 4.0 Hz, -CH2-), 4.03 (2H, t, J = 4.0 Hz, -CH2-), 4.32 (2H, q,
J = 8.0 Hz, CO2CH2CH3), 7.00 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 7.21-7.24 (2H, m,
Ar-H), 7.36-7.54 (5H, m, Ar-H), 7.58 (1H, d, J = 8.0 Hz, Ar-H), 8.00 (2H, d, J = 8.0
Hz, Ar-H). 13C NMR (101Hz, DMSO-d6) δ 183.39, 163.39, 162.41, 160.00, 158.57,
157.68, 156.36, 148.51, 147.45, 13.36, 13.27, 127.65, 126.04, 124.58, 124.34, 115.90,
115.68, 115.06, 112.58, 112.51, 108.94, 105.63, 68.45, 60.96, 29.05, 27.13, 26.44,
25.73, 14.29. ESI-MS m/z: 570 [M+Na]+.
4.5
ethyl
5-(4-(5-fluoro-3-(methoxyimino)-2-oxoindolin-1-yl)butoxy)-2-phenylbenzofuran-3-ca
rboxylate (7a)
18

ACCEPTED MANUSCRIPT
1
Yellow solid, yield: 59%. H NMR (400 MHz, DMSO-d6) δ 1.32 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 2.10 (2H, t, J = 4.0 Hz, -CH2-), 3.92 (2H, t, J = 4.0 Hz, -CH2-), 4.08 (2H,
t, J = 4.0 Hz, -CH2-), 4.21 (3H, s, NOCH3), 4.32 (2H, q, J = 8.0 Hz, CO2CH2CH3),
6.94 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 7.18-7.21 (1H, m, Ar-H), 7.30-7.32 (1H, m,
Ar-H), 7.38 (1H, d, J = 4.0 Hz, Ar-H), 7.53-7.61 (4H, m, Ar-H), 7.68 (1H, d, J = 4.0
Hz, Ar-H), 7.93-7.96 (2H, m, Ar-H). 13C NMR (101Hz, DMSO-d6) δ 163.41, 162.70,
160.96, 159.54, 157.17, 156.07, 148.64, 143.50, 140.62, 130.95, 129.70, 129.45,
128.67, 127.68, 119.63, 119.42, 116.02, 114.96, 114.71, 112.49, 110.97, 108.98,
105.70, 66.35, 65.12, 60.93, 37.40, 27.21, 14.40. ESI-MS m/z: 553 [M+Na]+.
4.6.
ethyl
5-(4-(5-fluoro-3-(methoxyimino)-2-oxoindolin-1-yl)butoxy)-2-(4-methoxyphenyl)ben
zofuran-3-carboxylate (7b)
1
Yellow solid, yield: 79%. H NMR (400 MHz, DMSO-d6) δ 1.32 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 2.10 (2H, t, J = 4.0 Hz, -CH2-), 3.86 (3H, s, OCH3), 3.92 (2H, t, J = 4.0
Hz, -CH2-), 4.08 (2H, t, J = 4.0 Hz, -CH2-), 4.22 (3H, s, NOCH3), 4.34 (2H, q, J = 8.0
Hz, CO2CH2CH3), 6.91 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 7.10 (2H, d, J = 4.0 Hz, Ar-H),
7.20-7.23 (1H, m, Ar-H), 7.32-7.37 (2H, m, Ar-H), 7.58 (1H, d, J = 8.0 Hz, Ar-H),
7.70 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 7.95-7.97 (2H, m, Ar-H). ESI-MS m/z: 583
[M+Na]+.
4.7.
ethyl
5-(4-(5-fluoro-3-(ethoxyimino)-2-oxoindolin-1-yl)butoxy)-2-(4-methoxyphenyl)benz
ofuran-3-carboxylate (7c)
1
Yellow solid, yield: 49%. H NMR (400 MHz, DMSO-d6) δ 1.29-1.39 (6H, m,
NOCH2CH3 and CO2CH2CH3), 2.10 (2H, t, J = 4.0 Hz, -CH2-), 3.85 (3H, s, OCH3),
3.92 (2H, t, J = 4.0 Hz, -CH2-), 4.08 (2H, t, J = 4.0 Hz, -CH2-), 4.32 (2H, q, J = 8.0 Hz,
CO2CH2CH3), 4.48 (2H, q, J = 8.0 Hz, NOCH2CH3), 6.91 (1H, dd, J = 4.0, 8.0 Hz,
Ar-H), 7.10 (2H, d, J = 4.0 Hz, Ar-H), 7.20-7.23 (1H, m, Ar-H), 7.32-7.37 (2H, m,
Ar-H), 7.58 (1H, d, J = 8.0 Hz, Ar-H), 7.70 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 7.95-7.97
(2H, m, Ar-H). ESI-MS m/z: 597 [M+Na]+.
4.8.
ethyl
5-(3-(5-methoxy-3-(methoxyimino)-2-oxoindolin-1-yl)propoxy)-2-phenylbenzofuran-
3-carboxylate (7d)
19

ACCEPTED MANUSCRIPT
1
Yellow solid, yield: 83%. H NMR (400 MHz, DMSO-d6) δ 1.30 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 2.10 (2H, dd, J = 4.0, 8.0 Hz, -CH2-), 3.74 (3H, s, OCH3), 3.90 (2H, t, J
= 8.0 Hz, -CH2-), 4.08 (2H, t, J = 4.0 Hz, -CH2-), 4.20 (3H, s, NOCH3), 4.34 (2H, q, J
= 8.0 Hz, CO2CH2CH3), 6.98 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 6.99-7.13 (2H, m, Ar-H),
7.39 (1H, d, J = 4.0 Hz, Ar-H), 7.48 (1H, d, J = 4.0 Hz, Ar-H), 7.54-7.58 (3H, m,
Ar-H), 7.62 (1H, d, J = 8.0 Hz, Ar-H), 7.94-7.97 (2H, m, Ar-H). ESI-MS m/z: 551
[M+Na]+.
4.9.
ethyl
5-(3-(5-methoxy-3-(methoxyimino)-2-oxoindolin-1-yl)propoxy)-2-(4-methoxyphenyl
)benzofuran-3-carboxylate (7e)
1
Yellow solid, yield: 59%. H NMR (400 MHz, DMSO-d6) δ 1.32 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 2.09 (2H, dd, J = 4.0, 8.0 Hz, -CH2-), 3.75 (3H, s, OCH3), 3.86-3.91
(5H, m, -CH2- and OCH3), 4.05-4.20 (5H, m, -CH2- and NOCH3), 4.34 (2H, q, J = 8.0
Hz, CO2CH2CH3), 6.94 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 7.01-7.11 (4H, m, Ar-H), 7.48
(1H, d, J = 4.0 Hz, Ar-H), 7.56 (1H, d, J = 4.0 Hz, Ar-H), 7.62 (1H, d, J = 8.0 Hz,
Ar-H), 7.95-7.97 (2H, m, Ar-H). ESI-MS m/z: 581 [M+Na]+.
4.10.
ethyl
5-(3-(5-methoxy-3-(ethoxyimino)-2-oxoindolin-1-yl)propoxy)-2-(4-methoxyphenyl)b
enzofuran-3-carboxylate (7f)
1
Yellow solid, yield: 45%. H NMR (400 MHz, DMSO-d6) δ 1.30 (3H, J = 8.0 Hz,
CO2CH2CH3), 2.11 (2H, t, J = 4.0 Hz, -CH2-), 3.93 (2H, t, J = 4.0 Hz, -CH2-), 4.03 (2H,
t, J = 4.0 Hz, -CH2-), 4.34 (2H, q, J = 8.0 Hz, CO2CH2CH3), 4.45 (2H, q, J = 8.0 Hz,
NOCH2CH3), 6.90-7.09 (5H, m, Ar-H), 7.36-7.56 (3H, m, Ar-H), 7.93-7.96 (2H, m,
Ar-H). 13C NMR (101Hz, DMSO-d6) δ 163.61, 162.74, 161.22, 156.01, 155.54,
148.31, 143.96, 137.73, 131.38, 127.91, 121.71, 118.52, 117.73, 116.12, 114.48,
112.23, 110.33, 107.69, 105.78, 72.78, 66.30, 60.80, 56.11, 55.85, 37.21, 27.36, 14.90,
14.38. ESI-MS m/z: 595 [M+Na]+.
4.11.
ethyl
5-(3-(3-(methoxyimino)-2-oxoindolin-1-yl)propoxy)-2-phenylbenzofuran-3-carboxyla
te (7g)
20

ACCEPTED MANUSCRIPT
1
Yellow solid, yield: 45%. H NMR (400 MHz, DMSO-d6) δ 1.29-1.38 (6H, m,
NOCH2CH3 and CO2CH2CH3), 2.08 (2H, dd, J = 4.0, 8.0 Hz, -CH2-), 3.75 (3H, s,
OCH3), 3.83-3.89 (5H, m, -CH2- and OCH3), 4.06 (2H, t, J = 4.0 Hz, -CH2-), 4.20 (3H,
s, NOCH3), 4.32 (2H, q, J = 8.0 Hz, CO2CH2CH3), 4.45 (2H, q, J = 8.0 Hz,
NOCH2CH3), 6.98-7.20 (3H, m, Ar-H), 7.40-7.62 (4H, m, Ar-H), 7.89-7.97 (3H, m,
Ar-H). ESI-MS m/z: 521 [M+Na]+.
4.12.
ethyl
5-(3-(3-(methoxyimino)-2-oxoindolin-1-yl)propoxy)-2-(4-methoxyphenyl)benzofuran
-3-carboxylate (7h)
1
Yellow solid, yield: 68%. H NMR (400 MHz, CDCl3) δ 1.43 (3H, J = 8.0 Hz,
CO2CH2CH3), 2.25 (2H, t, J = 4.0 Hz, -CH2-), 3.90 (3H, s, OCH3), 4.02 (2H, t, J = 4.0
Hz, -CH2-), 4.12 (2H, t, J = 4.0 Hz, -CH2-), 4.31 (3H, s, NOCH3), 4.43 (2H, q, J = 8.0
Hz, CO2CH2CH3), 6.92-6.97 (2H, m, Ar-H), 7.01-7.07 (3H, m, Ar-H), 7.28-7.33 (1H,
m, Ar-H), 7.42 (1H, d, J = 8.0 Hz, Ar-H), 7.52 (1H, d, J = 4.0 Hz, Ar-H), 7.96 (1H, d,
13
J = 8.0 Hz, Ar-H), 8.02 (2H, d, J = 8.0 Hz, Ar-H). C NMR (101Hz, CD3OD) δ
164.20, 163.66, 161.75, 161.19, 155.67, 148.69, 143.99, 143.69, 132.55, 131.14,
128.15, 127.97, 122.89, 122.17, 115.81, 113.97, 113.52, 111.50, 108.70, 107.83,
106.05, 65.69, 64.77, 60.53, 55.40, 37.13, 27.70, 14.36. ESI-MS m/z: 551 [M+Na]+.
4.13.
ethyl
5-(3-(3-(ethoxyimino)-2-oxoindolin-1-yl)propoxy)-2-(4-methoxyphenyl)benzofuran-3
-carboxylate (7i)
Yellow solid, yield: 51%. 1H NMR (400 MHz, CDCl3) δ 1.31-1.39 (6H, m, NOCH2CH3
and CO2CH2CH3), 2.11 (2H, t, J = 8.0 Hz, -CH2-), 3.86 (3H, s, OCH3), 3.93 (2H, t, J =
8.0 Hz, -CH2-), 4.08 (2H, t, J = 4.0 Hz, -CH2-), 4.32 (2H, q, J = 8.0 Hz, CO2CH2CH3),
4.46 (2H, q, J = 8.0 Hz, NOCH2CH3), 6.92 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 7.07-7.11
(3H, m, Ar-H), 7.18 (1H, d, J = 8.0 Hz, Ar-H), 7.38-7.43 (2H, m, Ar-H), 7.56 (1H, d, J
= 8.0 Hz, Ar-H), 7.92 (1H, d, J = 8.0 Hz, Ar-H), 7.97 (2H, d, J = 8.0 Hz, Ar-H).
ESI-MS m/z: 565 [M+Na]+.
4.14.
ethyl
5-(4-(3-(methoxyimino)-2-oxoindolin-1-yl)butoxy)-2-(4-methoxyphenyl)benzofuran-
3-carboxylate (7j)
21

ACCEPTED MANUSCRIPT
1
Yellow solid, yield: 69%. H NMR (400 MHz, CDCl3) δ 1.32 (3H, J = 8.0 Hz,
CO2CH2CH3), 1.74-1.78 (4H, m, 2×-CH2-), 3.78 (2H, t, J = 4.0 Hz, -CH2-), 3.85 (3H,
s, OCH3), 4.04 (2H, t, J = 4.0 Hz, -CH2-), 4.19 (3H, s, NOCH3), 4.32 (2H, q, J = 8.0
Hz, CO2CH2CH3), 6.93 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 7.06-7.09 (3H, m, Ar-H), 7.16
(1H, d, J = 8.0 Hz, Ar-H), 7.37-7.42 (2H, m, Ar-H), 7.52 (1H, d, J = 12.0 Hz, Ar-H),
7.86 (1H, d, J = 8.0 Hz, Ar-H), 7.96 (2H, d, J = 8.0 Hz, Ar-H). 13C NMR (101Hz,
DMSO-d6) δ 163.62, 162.74, 161.41, 161.20, 156.08, 148.26, 144.13, 143.75, 133.38,
131.38, 127.94, 127.83, 123.12, 122.85, 121.73, 115.40, 114.47, 112.24, 109.91,
107.69, 105.79, 67.96, 64.89, 60.75, 55.85, 26.48, 24.12, 14.45. ESI-MS m/z: 565
[M+Na]+.
4.15.
ethyl
5-(4-(3-(ethoxyimino)-2-oxoindolin-1-yl)butoxy)-2-(4-methoxyphenyl)benzofuran-3-
carboxylate (7k)
Yellow solid, yield: 53%. 1H NMR (400 MHz, CDCl3) δ 1.30-1.39 (6H, m, NOCH2CH3
and CO2CH2CH3), 1.78-1.81 (4H, m, 2×-CH2-), 3.79 (2H, t, J = 8.0 Hz, -CH2-), 3.85
(3H, s, OCH3), 4.06 (2H, t, J = 4.0 Hz, -CH2-), 4.33 (2H, q, J = 8.0 Hz, CO2CH2CH3),
4.46 (2H, q, J = 8.0 Hz, NOCH2CH3), 6.95 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 7.08-7.11
(3H, m, Ar-H), 7.18 (1H, d, J = 8.0 Hz, Ar-H), 7.38-7.41 (2H, m, Ar-H), 7.56 (1H, d, J
= 8.0 Hz, Ar-H), 7.90 (1H, d, J = 8.0 Hz, Ar-H), 7.97 (2H, d, J = 8.0 Hz, Ar-H). 13C
NMR (101Hz, DMSO-d6) δ 163.63, 162.86, 161.43, 161.22, 156.11, 148.27, 144.06,
143.66, 133.29, 131.39, 127.94, 127.74, 123.12, 121.73, 115.47, 114.16, 112.27,
109.90, 107.70, 105.80, 72.78, 67.98, 60.80, 55.86, 26.49, 24.14, 14.94, 14.47.
ESI-MS m/z: 579 [M+Na]+.
4.16.
ethyl
5-((5-(5-fluoro-3-(methoxyimino)-2-oxoindolin-1-yl)pentyl)oxy)-2-(4-methoxyphenyl
)benzofuran-3-carboxylate (7l)
1
Yellow solid, yield: 67%. H NMR (400 MHz, DMSO-d6) δ 1.32 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 1.46-1.49 (2H, m, -CH2-), 1.66-1.69 (2H, m, -CH2-), 1.77-1.80 (2H, m,
-CH2-), 3.72 (2H, t, J = 8.0 Hz, -CH2-), 3.86 (3H, s, OCH3), 4.00 (2H, t, J = 8.0 Hz,
-CH2-), 4.22 (3H, s, NOCH3), 4.32 (2H, q, J = 8.0 Hz, CO2CH2CH3), 6.96 (1H, dd, J =
4.0, 8.0 Hz, Ar-H), 7.09 (2H, d, J = 8.0 Hz, Ar-H), 7.17-7.21 (1H, m, Ar-H), 7.31-7.37
(1H, m, Ar-H), 7.40 (1H, d, J = 4.0 Hz, Ar-H), 7.56 (1H, d, J = 8.0 Hz, Ar-H), 7.68
22

ACCEPTED MANUSCRIPT
(1H, dd, J = 4.0, 8.0 Hz, Ar-H), 7.96 (2H, d, J = 8.0 Hz, Ar-H). ESI-MS m/z: 597
[M+Na]+.
4.17.
ethyl
5-((5-(3-(ethoxyimino)-5-fluoro-2-oxoindolin-1-yl)pentyl)oxy)-2-(4-methoxyphenyl)
benzofuran-3-carboxylate (7m)
1
Yellow solid, yield: 43%. H NMR (400 MHz, CDCl3) δ 1.26 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 1.44 (3H, t, J = 8.0 Hz, NOCH2CH3), 1.59-1.65 (2H, m, -CH2-),
1.76-1.82 (2H, m, -CH2-), 3.72 (2H, t, J = 8.0 Hz, -CH2-), 3.82 (3H, s, OCH3), 4.05
(2H, t, J = 8.0 Hz, -CH2-), 4.43 (2H, q, J = 8.0 Hz, CO2CH2CH3), 4.58 (2H, q, J = 4.0
Hz, NOCH2CH3), 6.80 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 6.92 (1H, dd, J = 4.0, 8.0 Hz,
Ar-H), 7.02 (2H, d, J = 8.0 Hz, Ar-H), 7.11 (1H, t, J = 4.0 Hz, Ar-H), 7.38 (1H, d, J =
12.0 Hz, Ar-H), 7.52 (1H, d, J = 4.0 Hz, Ar-H), 7.74 (1H, dd, J = 4.0, 8.0 Hz, Ar-H),
8.02 (2H, d, J = 8.0 Hz, Ar-H). 13C NMR (101Hz, CD3OD) δ 164.27, 163.41, 161.65,
161.15, 156.08, 148.55, 143.08, 139.82, 131.14, 128.10, 122.24, 118.56, 118.32,
116.50, 115.58, 115.33, 114.09, 113.50, 111.43, 109.12, 109.05, 107.83, 105.77,
73.37, 68.29, 60.48, 55.40, 39.96, 29.00, 27.25, 23.54, 14.71, 14.36. ESI-MS m/z: 611
[M+Na]+.
4.18.
ethyl
5-((6-(5-fluoro-3-(methoxyimino)-2-oxoindolin-1-yl)hexyl)oxy)-2-(4-methoxyphenyl
)benzofuran-3-carboxylate (7n)
Yellow solid, yield: 52%. 1H NMR (400 MHz, DMSO-d6) δ 1.30-1.38 (5H, m, -CH2-
and CO2CH2CH3), 1.46-1.49 (2H, m, -CH2-), 1.62-1.64 (2H, m, -CH2-), 1.72-1.75 (2H,
m, -CH2-), 3.71 (2H, t, J = 8.0 Hz, -CH2-), 3.85 (3H, s, OCH3), 3.99 (2H, t, J = 8.0 Hz,
-CH2-), 4.22 (3H, s, NOCH3), 4.33 (2H, q, J = 8.0 Hz, CO2CH2CH3), 6.95 (1H, dd, J =
4.0, 8.0 Hz, Ar-H), 7.08 (2H, d, J = 8.0 Hz, Ar-H), 7.15-7.18 (1H, m, Ar-H), 7.32-7.34
(1H, m, Ar-H), 7.40 (1H, d, J = 4.0 Hz, Ar-H), 7.56 (1H, d, J = 8.0 Hz, Ar-H), 7.66
(1H, dd, J = 4.0, 8.0 Hz, Ar-H), 7.96 (2H, d, J = 8.0 Hz, Ar-H). ESI-MS m/z: 611
[M+Na]+.
4.19.
ethyl
5-((6-(3-(2-carbamothioylhydrazono)-5-fluoro-2-oxoindolin-1-yl)hexyl)oxy)-2-(4-met
hoxyphenyl)benzofuran-3-carboxylate (7o)
23

ACCEPTED MANUSCRIPT
1
Yellow solid, yield: 18%. H NMR (400 MHz, CDCl3) δ 1.32 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 1.41-1.59 (4H, m, 2×-CH2-), 1.65-1.85 (4H, m, 2×-CH2-), 3.78 (2H, t, J
= 8.0 Hz, -CH2-), 3.90 (3H, s, OCH3), 4.05 (2H, t, J = 8.0 Hz, -CH2-), 4.43 (2H, q, J =
8.0 Hz, CO2CH2CH3), 6.86 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 6.93 (1H, dd, J = 4.0, 8.0
Hz, Ar-H), 7.00 (2H, d, J = 8.0 Hz, Ar-H), 7.11 (1H, t, J = 8.0 Hz, Ar-H), 7.30 (1H, dd,
J = 4.0, 8.0 Hz, Ar-H), 7.40 (1H, d, J = 4.0 Hz, Ar-H), 6.68, 7.53 (3H, s, Ar-H and
NNHCSNH2), 8.02 (2H, d, J = 8.0 Hz, Ar-H), 12.91 (1H, s, NNHCSNH2). ESI-MS
m/z: 655 [M+Na]+.
4.20.
ethyl
5-((6-(5-fluoro-3-(methoxyimino)-2-oxoindolin-1-yl)hexyl)oxy)-2-(4-fluorophenyl)be
nzofuran-3-carboxylate (7p)
Yellow solid, yield: 49%. 1H NMR (400 MHz, DMSO-d6) δ 1.30 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 1.37-1.39 (2H, m, -CH2-), 1.46-1.48 (2H, m, -CH2-), 1.61-1.64 (2H, m,
-CH2-), 1.72-1.76 (2H, m, -CH2-), 3.71 (2H, t, J = 8.0 Hz, -CH2-), 4.00 (2H, t, J = 8.0
Hz, -CH2-), 4.22 (3H, s, NOCH3), 4.33 (2H, q, J = 8.0 Hz, CO2CH2CH3), 6.99 (1H, dd,
J = 4.0, 8.0 Hz, Ar-H), 7.16-7.20 (1H, m, Ar-H), 7.32-7.44 (4H, m, Ar-H), 7.60 (1H, d,
J = 8.0 Hz, Ar-H), 7.68 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 8.04 (2H, d, J = 8.0 Hz, Ar-H).
ESI-MS m/z: 599 [M+Na]+.
4.21.
ethyl
5-((5-(5-methoxy-3-(methoxyimino)-2-oxoindolin-1-yl)pentyl)oxy)-2-(4-methoxyphe
nyl)benzofuran-3-carboxylate (7q)
1
Yellow solid, yield: 36%. H NMR (400 MHz, DMSO-d6) δ 1.32 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 1.72-1.81 (6H, m, 3×-CH2-), 3.67-3.74 (5H, m, -CH2- and OCH3), 3.86
(3H, s, OCH3), 4.05 (2H, t, J = 8.0 Hz, -CH2-), 4.21 (3H, s, NOCH3), 4.33 (2H, q, J =
8.0 Hz, CO2CH2CH3), 6.94-7.10 (6H, m, Ar-H), 7.40-7.45 (2H, m, Ar-H), 7.54 (1H, d,
J = 8.0 Hz, Ar-H), 7.96 (2H, d, J = 8.0 Hz, Ar-H). ESI-MS m/z: 609 [M+Na]+.
4.22.
ethyl
5-((5-(3-(hydroxyimino)-2-oxoindolin-1-yl)pentyl)oxy)-2-(4-methoxyphenyl)benzofu
ran-3-carboxylate (7r)
1
Yellow solid, yield: 73%. H NMR (400 MHz, DMSO-d6) δ 1.31 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 1.46-1.52 (2H, m, -CH2-), 1.65-1.71 (2H, m, -CH2-), 1.78-1.81 (2H, m,
24

ACCEPTED MANUSCRIPT
-CH2-), 3.74 (2H, t, J = 8.0 Hz, -CH2-), 3.85 (3H, s, OCH3), 4.00 (2H, t, J = 8.0 Hz,
-CH2-), 4.31 (2H, q, J = 8.0 Hz, CO2CH2CH3), 6.96 (1H, dd, J = 4.0, 8.0 Hz, Ar-H),
7.07-7.14 (4H, m, Ar-H), 7.28-7.32 (2H, m, Ar-H), 7.54 (1H, d, J = 8.0 Hz, Ar-H),
7.95 (2H, d, J = 8.0 Hz, Ar-H), 8.00 (1H, d, J = 8.0 Hz, Ar-H), 13.42 (1H, brs, NOH).
13C NMR (101Hz, DMSO-d6) δ 163.64, 163.57, 161.42, 161.20, 156.24, 148.25,
143.98, 143.46, 132.37, 131.40, 127.96, 127.28, 122.94, 121.74, 115.75, 114.47,
114.15, 112.27, 109.60, 107.71, 105.70, 68.37, 60.80, 55.86, 28.82, 27.29, 23.40,
14.46. ESI-MS m/z: 565 [M+Na]+.
4.23.
ethyl
5-((5-(3-(methoxyimino)-2-oxoindolin-1-yl)pentyl)oxy)-2-(4-methoxyphenyl)benzofu
ran-3-carboxylate (7s)
1
Yellow solid, yield: 39%. H NMR (400 MHz, CDCl3) δ 1.42 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 1.59-1.65 (2H, m, -CH2-), 1.77-1.93 (4H, m, 2×-CH2-), 3.80 (2H, t, J =
8.0 Hz, -CH2-), 3.89 (3H, s, OCH3), 4.05 (2H, t, J = 8.0 Hz, -CH2-), 4.27 (3H, s,
NOCH3), 4.43 (2H, q, J = 8.0 Hz, CO2CH2CH3), 6.86 (1H, d, J = 8.0 Hz, Ar-H), 6.92
(1H, dd, J = 4.0, 8.0 Hz, Ar-H), 7.02 (2H, d, J = 8.0 Hz, Ar-H), 7.08 (1H, t, J = 8.0 Hz,
Ar-H), 7.38 (1H, d, J = 8.0 Hz, Ar-H), 7.52 (1H, d, J = 4.0 Hz, Ar-H), 7.96 (1H, dd, J =
13
4.0, 8.0 Hz, Ar-H), 8.02 (2H, d, J = 8.0 Hz, Ar-H). C NMR (101Hz, CD3OD) δ
164.28, 163.52, 16.64, 161.14, 156.09, 148.5, 14.89, 14.65, 132.45,131.13, 128.07,
128.02, 122.84, 122.24, 115.86, 114.11, 113.50, 111.43, 108.66, 107.83, 105.80,
68.33, 64.75, 60.50, 55.39, 39.83, 29.02, 27.32, 23.55, 14.36. ESI-MS m/z: 579
[M+Na]+.
4.24.
ethyl
5-((6-(3-(methoxyimino)-2-oxoindolin-1-yl)hexyl)oxy)-2-(4-methoxyphenyl)benzofur
an-3-carboxylate (7t)
1
Yellow solid, yield: 71%. H NMR (400 MHz, CDCl3) δ 1.42 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 1.45-1.60 (4H, m, 2×-CH2-), 1.74-1.78 (2H, m, -CH2-), 1.82-1.86 (2H,
m, -CH2-), 3.79 (2H, t, J = 8.0 Hz, -CH2-), 3.91 (3H, s, OCH3), 4.05 (2H, t, J = 8.0 Hz,
-CH2-), 4.31 (3H, s, NOCH3), 4.43 (2H, q, J = 8.0 Hz, CO2CH2CH3), 6.87 (1H, d, J =
8.0 Hz, Ar-H), 6.92 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 7.02 (2H, d, J = 4.0 Hz, Ar-H),
7.08 (1H, t, J = 8.0 Hz, Ar-H), 7.38-7.41 (2H, m, Ar-H), 7.52 (1H, d, J = 4.0 Hz, Ar-H),
25

ACCEPTED MANUSCRIPT
7.98 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 8.02 (2H, d, J = 8.0 Hz, Ar-H). ESI-MS m/z: 593
[M+Na]+.
4.25.
ethyl
5-((6-(3-(ethoxyimino)-2-oxoindolin-1-yl)hexyl)oxy)-2-(4-methoxyphenyl)benzofura
n-3-carboxylate (7u)
Yellow solid, yield: 62%. 1H NMR (400 MHz, CDCl3) δ 1.41-1.49 (6H, m, NOCH2CH3
and CO2CH2CH3), 1.51-1.58 (4H, m, 2×-CH2-), 1.74-1.78 (2H, m, -CH2-), 1.82-1.86
(2H, m, -CH2-), 3.79 (2H, t, J = 8.0 Hz, -CH2-), 3.90 (3H, s, OCH3), 4.05 (2H, t, J =
8.0 Hz, -CH2-), 4.45 (2H, q, J = 8.0 Hz, CO2CH2CH3), 4.56 (2H, q, J = 8.0 Hz,
NOCH2CH3), 6.88 (1H, d, J = 8.0 Hz, Ar-H), 6.92 (1H, dd, J = 4.0, 8.0 Hz, Ar-H),
7.02 (2H, d, J = 4.0 Hz, Ar-H), 7.08 (1H, t, J = 4.0 Hz, Ar-H), 7.37-7.41 (2H, m, Ar-H),
7.53 (1H, d, J = 4.0 Hz, Ar-H), 8.00 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 8.02 (2H, d, J =
8.0 Hz, Ar-H). ESI-MS m/z: 607 [M+Na]+.
4.26.
ethyl
5-((6-(3-(2-carbamothioylhydrazono)-2-oxoindolin-1-yl)hexyl)oxy)-2-(4-methoxyphe
nyl)benzofuran-3-carboxylate (7v)
1
Yellow solid, yield: 27%. H NMR (400 MHz, DMSO-d6) δ 1.32 (3H, t, J = 8.0 Hz,
CO2CH2CH3), 1.40-1.50 (4H, m, 2×-CH2-), 1.66-1.77 (4H, m, 2×-CH2-), 3.77 (2H, t, J
= 8.0 Hz, -CH2-), 3.86 (3H, s, OCH3), 4.01 (2H, t, J = 8.0 Hz, -CH2-), 4.45 (2H, q, J =
8.0 Hz, CO2CH2CH3), 4.32 (2H, q, J = 8.0 Hz, NOCH2CH3), 6.96 (1H, dd, J = 4.0, 8.0
Hz, Ar-H), 7.10 (2H, d, J = 4.0 Hz, Ar-H), 7.16 (1H, t, J = 4.0 Hz, Ar-H), 7.22 (1H, d,
J = 8.0 Hz, Ar-H), 7.27-7.41 (2H, m, Ar-H), 7.57 (1H, d, J = 8.0 Hz, Ar-H), 7.72 (1H,
d, J = 8.0 Hz, Ar-H), 7.96 (2H, dd, J = 4.0, 8.0 Hz, Ar-H), 8.73, 9.08 (2H, s,
NNHCSNH2), 12.44 (1H, s, NNHCSNH2). ESI-MS m/z: 637 [M+Na]+.
References
[1] M. Garland, S. Loscher, M. Bogyo. Chem. Rev. 117 (2017) 4422-4461
[2] Y. Q. Hu, S. Zhang, Z. Xu, Z. S. Lv, M. L. Liu, L. S. Feng. Eur. J. Med. Chem. 141 (2017)
335-345
[3] Z. W. Chen, Z. Z. Wang, J. S. Ren, X. G. Qu. Acc. Chem. Res. 51 (2018) 789-799
[4] World Health Organization. Global Tuberculosis Report 2018.
[5] Y. L. Fan, J. B. Wu, X. W. Cheng, F. Z. Zhang, L. S. Feng. Eur. J. Med. Chem. 146 (2018)
554-563
26

ACCEPTED MANUSCRIPT
[6] 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
[7] A. Gomtsyan. Chem. Heterocycl. Comp. 48 (2012) 7-10
[8] A. Radadiya, A. Shah. Eur. J. Med. Chem. 97 (2015) 356-376.
[9] Z. Xu, X. F. Song, Y. Q. Hu, M. Qiang, Z. S. Lv. Eur. J. Med. Chem. 138 (2017) 66-71.
[10] R. Naik, D. S. Harmalkar, X. Z. Xu, K. Jang, K. Lee. Eur. J. Med. Chem. 97 (2015) 379-397.
[11] Z. Xu, S. Zhang, C. Gao, F. Zhao, Z. S. Lv, L. S. Feng. Chin. Chem. Lett. 28 (2017) 159-167
[12] R. J. Nevagi, S. N. Dighe, S. N. Dighe. Eur. J. Med. Chem. 97 (2015) 561-581.
[13] H. Singh, J. V. Singh, M. K. Gupta, A. K. Saxena, S. Sharma, K. Nepali, P. M. S. Bedi.
Bioorg. Med. Chem. Lett. 27 (2017) 3974-3979
[14] S. A. Galal, A. S. A. El-All, M. M. Abdallah, H. I. El-Diwani. Bioorg. Med. Chem. Lett. 19
(2009) 2420-2428
[15] D. Sriram, T. R. Bal, P. Yogeeswari. J. Pharm. Pharmaceut. Sci. 8 (2005) 565-577
[16] X. Z. Jiang, W. L. Liu, W. Zhang, F. Q. Jiang, Z. Gao, H. Zhuang, L. Fu. Eur. J. Med. Chem.
46 (2011) 3526-3530
[17] R. Wang, X. Y. Yin, Y. H. Zhang, W. T. Yan. Eur. J. Med. Chem. 156 (2018) 580-586
[18] X. Y. Lu, J. Tang, Z. Y. Liu, M. Li, T. Y. Zhang, X. T. Zhang, K. Ding. Bioorg. Med. Chem.
Lett. 26 (2016) 5916-5919
[19] Z. Xu, S. Zhang, X. F. Song, M. Qiang, Z. S. Lv. Bioorg. Med. Chem. Lett. 27 (2017)
3643-3646
[20] M. Matias, S. Silvestre, A. Falcao, G. Alves. Mini. Rev. Med. Chem. 17 (2017) 486-494.
[21] C. Viegas-Junior, A. Danuello, V. S. Bolzani, E. J. Barreiro, C. A. M. Fraga. Curr. Med. Chem.
14 (2007) 1829-1836.
[22] W. Zhang, S. C. Lun, S. H. Wang, X. W. Jiang, F. Yang, J. Tang, A. L. Manson, A. M. Earl, H.
Gunosewoyo, W. R. Bishai, L. F. Yu. J. Med. Chem. 61 (2018) 791-803
[23] A. Aggarwal, M. K. Parai, N. Shetty, D. Wallis, L. Woolhiser, C. Hastings, N. K. Dutta, S.
Galaviz, R. C. Dhakal, R. Shrestha, S. Wakabayashi, C. Walpole, D. Matthews, D. Floyd, P.
Scullion, J. Riley, O. Epemolu, S. Norval, T. Snavely, G. T. Robertson, E. J. Rubin, T. R.
Loerger, F. A. Sirgel, R. vam der Merwe, P. D. Helden, P. Keller, E. C. Böttger, P. C.
Karakousis, A. J. Lenaerts, J. C. Sacchettini. Cell 170 (2017) 249-259
[24] F. Gao, H. Yang, T. Y. Lu, Z. J. Chen, L. Ma, Z. Xu, G. M. Lu. Eur. J. Med. Chem. 159 (2018)
277-281
[25] C. J. Carroux, G. M. Rankin, J. Moeker, L. F. Bornaghi, K. Katneni, J. Morizzi, S. A. Charman,
D. Vullo, C. T. Superan, S. A. Poulsen. J. Med. Chem. 56 (2013) 9623-9634.
[26] G. Wu, W. A. Zalloum, M. E. Meuser, L. Jiang, D. Kang, C. H. Chen, Y. Tian, F. F. Zhang, S.
Cocklin, K. H. Lee, X. Liu, P. Zhan. Eur. J. Med. Chem. 158 (2018) 478-492.
[27] G. Wu, Y. Gao, D. Kang, B. Huang, Z. Huo, H. Liu, V. Poongavanam, P. Zhan, X. Liu.
MedChemComm 19 (2018)149-159.
[28] P. Gao, L. Zhang, L. Sun, T. Huang, J. Tan, J. Zhang, Z. Zhou, T. Zhao, L. Menendez-Arias, C.
Pannecouque, E. D. Clercq, P. Zhang, X. Liu. Bioorg. Med. Chem. 25(2017) 5779-5789.
[29] D. Kang, H. Zhang, Z. Zhou, B. Huang, L. Naesens, P. Zhang, X. Liu. Bioorg. Med. Chem. Lett.
26 (2016) 5182-5186.
[30] Z. J. Fang, D. Kang, L. Zhang, B. Huang, H. Liu, C. Pannecouque, E. D. Clercq, P. Zhan, X.
Liu. Chem. Biol. Drug Des. 86 (2015) 614-618.
27