Discovery of hybrids of indolin-2-one and nitroimidazole as potent inhibitors against drug-resistant bacteria

Article abstract of DOI:10.1038/s41429-018-0076-5

With antibiotics resistance developing rapidly, new antibacterial agents are needed to be discovered. We readily synthesized 11 indolin-2-one compounds and found a hybrid of indolin-2-one and nitroimidazole 3-((1-methyl-5-nitro-1H-imidazol-2-yl)methylene)

Full text of DOI:10.1038/s41429-018-0076-5

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The Journal of Antibiotics
https://doi.org/10.1038/s41429-018-0076-5
ARTICLE
Discovery of hybrids of indolin-2-one and nitroimidazole as potent
inhibitors against drug-resistant bacteria
Yuanzheng Zhou1 Yuan Ju1 Yang Yang1 Zitai Sang1 Zhenling Wang1 Gu He1 Tao Yang1 Youfu Luo1
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Received: 3 April 2018 / Revised: 20 May 2018 / Accepted: 4 June 2018
© The Author(s) under exclusive licence to the Japan Antibiotics Research Association 2018
Abstract
With antibiotics resistance developing rapidly, new antibacterial agents are needed to be discovered. We readily synthesized
11 indolin-2-one compounds and found a hybrid of indolin-2-one and nitroimidazole 3-((1-methyl-5-nitro-1H-imidazol-2-yl)
methylene)indolin-2-one to be effective on Staphylococcus aureus strains. Six derivatives of this compound were further
designed and synthesized in order to enhance its efficacy. After a second turn of structural refinement, a novel hybrid of
indolin-2-one and nitroimidazole 3-((1-methyl-5-nitro-1H-imidazol-2-yl)methylene)-5-nitroindolin-2-one with a nitro group
on C-5 position of indolin-2-one was shown to exhibit remarkable antibacterial activities with a low MIC value against
MRSA ATCC 33591. Besides, this molecule demonstrated its potency on Gram-negative bacteria and VRE strain. The time-
killing curve experiment showed its good bactericidal activity. Low hemolytic rate suggested its promising safety profile.
Introduction
the greatest threats to human health” in 2017 for their ability
to resist the “last resort” antibiotics carbapenem [2]. Since the
therapeutic options for these pathogens are increasingly lim-
ited due to multidrug resistance development, novel anti-
bacterial agents with new antimicrobial profiles are
desperately needed to be discovered.
The growing crisis of antibiotics has placed enormous threat
upon mankind. Such crisis stems mainly from the resistance
of pathogenic bacteria toward antibiotics. Among all those
drug-resistant microbes, the notorious “ESKAPE pathogens,”
a group of bacteria comprising Enterococcus faecium, Sta-
phylococcus aureus, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa, and Enterobacter
species, was believed to cause various hospital infections, and
to be able to effectively “escape” the effects of multiple
antibacterial drugs [1]. Furthermore, A. baumannii, P. aeru-
ginosa, and Enterobacter species occupy the top three posi-
tions of WHO’s list of “the drug-resistant bacteria that pose
Indolin-2-one is a chemical scaffold ubiquitous in nature,
ranging from tissues and fluids of mammals to products
produced by plants, bacteria, and invertebrates [3]. The
indolin-2-one derivatives have been known to possess a
broad spectrum of biological and pharmacological activ-
ities, including kinase inhibitory activity [4, 5], anticancer
[6, 7], anti-Alzheimer’s [8, 9], antinociceptive, anti-
inflammatory [10, 11], antioxidant [12, 13], neuroprotec-
tive [14, 15], and antibacterial activity [16, 17]. Derivatives
with 3-(substitutedmethylene)indolin-2-one scaffold have
been reported to exhibit inhibitory activities against various
bacteria. Compound A (violacein, Fig. 1) was implicated to
interfere S. aureus planktonic cells and biofilms [18].
Compound B (Fig. 1) has effect both on two Gram-negative
bacteria, Escherichia coli, P. aeruginosa, and on two Gram-
positive bacteria, Streptococcus pyogenes and S. aureus
[19]. Compound C [20], compound D [21], and compound
E [22] are able to inhibit growth of Agrobacterium tume-
faciens, Erythrobacter litoralis, and Candida albicans,
respectively (Fig. 1). These instances imply 3-(sub-
stitutedmethylene)indolin-2-one as a promising antibacterial
scaffold.
These authors contributed equally: Yuanzheng Zhou, Yuan Ju.
Electronic supplementary material The online version of this article
(https://doi.org/10.1038/s41429-018-0076-5) contains supplementary
material, which is available to authorized users.
* Tao Yang
michael_2012@126.com
* Youfu Luo
luo_youfu@scu.edu.cn
1
Cancer Center, West China Hospital, Sichuan University and
Collaborative Innovation Center for Biotherapy,
Chengdu, Sichuan, China

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Y. Zhou et al.
of 0.0625–0.125 μg/mL against MRSA strain ATCC 33591;
we also found this molecule to be effective on two
Gram-negative bacteria strains E. coli ATCC 25922,
P. aeruginosa ATCC 27853 and one Gram-positive bacteria
strain vancomycin-resistant Enterococcus (VRE) strain
B148, respectively. The time-kill curve experiment was
performed to test the bactericidal activity of compound 5c
on MSSA ATCC 6538, MRSA ATCC 33591, and E. coli
ATCC 25922; a total eradication of bacteria cells at a
concentration of 16* MIC of 5c was observed among all
three strains. Next, the hemolytic rates of 5c and 3g were
determined and low hemolytic rates were observed, sug-
gesting a promising safety profile of this chemical class.
Synthesis and biological evaluation
At the beginning of the study, a one-step simple synthetic
strategy (Scheme 1) was used to quickly obtain several 3-
(arylmethylene)indolin-2-ones. The synthesis was carried
out by aldol condensation using 1,3-dihydro-2H-indole-2-
one with 11 aromatic aldehydes in the presence of
piperidine in methyl alcohol to afford 11 corresponding
3-substituted indolin-2-ones 3a–3k (Table 1). Various
aldehydes with different aromatic rings were chosen for
SAR investigation, including benzpyrole, anthracene,
phenanthrene, quinoline, pyridine, pyrrole, furan, nitro-
benzene, nitroimidazole, and nitrothiophene. The Z/E
configuration of each compound was determined by their
2D ROESY NMR spectrum (Supporting Information),
investigating whether the olefinic hydrogen has a co-
signal with hydrogen at C-4 position of indolin-2-one, or
the C-4 position hydrogen has a co-signal with hydrogens
on aromatic ring of aldehyde moiety (Fig. 2). The repor-
ted compound 3g, of which the configuration was not
disclosed in the literature [23], along with the compounds
3a–3b, 3d–3f, 3j, and5a–5f adopted an E configuration,
compounds 3h, 3i, and 3k a Z one, while 3c was a Z/E
mixture. With eleven 3-(argiomethylene)indolin-2-ones in
hand, we tested their antibacterial activities against
both MRSA and MSSA strains. The MICs of these
compounds (Table 1) are determined by broth micro-
dilution method (see Experimental Procedures for
detail). Compound 3g, a hybrid of indolin-2-one and
nitroimidazole, exhibited a low MIC of 2 μg/mL and
0.5–1 μg/mL against MSSA ATCC 25923 and MRSA
ATCC 33591, respectively. Compound 3d showed a MIC
of 16 μg/mL on MSSA ATCC 25923, while its MIC on
MRSA ATCC 33591 was higher than 64 μg/mL. Other
compounds exerted MIC values higher than 64 μg/mL on
both stains.
Fig. 1 The chemical structures of the reported compounds with anti-
microbial activities (A–E)
Although a large number of molecules with 3-(sub-
stitutedmethylen)indolin-2-one skeleton has been depicted
to exhibit antibacterial activities, but to the best of our
knowledge these reported compounds exhibit minimum
inhibitory concentration (MIC) values mainly ranging from
10 to 64 μg/mL against reported bacteria, none of these
molecules are able to enter the drug development pipeline.
Herein, with the intention of discovering new indolin-2-one
antibiotics and disclosing the antibacterial structure-activity
relationship (SAR) of this scaffold, we investigated the
antibacterial properties of a class of 3-(substitutedm-
ethylene)indolin-2-one compounds, and presented one
indolin-2-one compound possessing a nitroimidazole moi-
ety with a remarkable MIC of 0.0625–0.125 μg/mL
against methicillin-resistant S. aureus (MRSA) strain ATCC
33591.
Results and discussion
Aiming at seeking effective antibacterial indolin-2-one
agents, we firstly synthesized 11 different 3-(substituted-
methylene)indolin-2-ones, and screened their antibacterial
activities on S. aureus strains. A hybrid of indolin-2-one
and nitroimidazole 3-((1-methyl-5-nitro-1H-imidazol-2-yl)
methylene)indolin-2-one (3g) was found to exhibit a MIC
of 2 μg/mL and 1 μg/mL on methicillin-sensitive S. aureus
(MSSA) ATCC 25923 and MRSA ATCC 33591, respec-
tively. Then we investigated the initial SAR of this chemical
class, and found one compound, 3-((1-methyl-5-nitro-1H-
imidazol-2-yl)methylene)-5-nitroindolin-2-one (5c), to
possess a high potency of antibacterial activity with a MIC
The SAR study above indicated that nitroimidazole
moiety plays an essential role in enhancing the antibacterial
activities of indolin-2-ones. This pharmacophore is widely

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Discovery of hybrids of indolin-2-one and nitroimidazole as potent inhibitors against drug-resistant. . .
Scheme 1 The synthesis of
compounds 3a–3k
known for its ability to combat a broad spectrum of Gram-
positive and Gram-negative bacteria, such as Clostridium
difficile and Bacteroides fragilis [24]. We consequently
wondered whether compound 3g has an effect on Gram-
negative bacteria and other drug-resistant bacteria or not.
Two clinical related Gram-negative strains, E. coli ATCC
25922 and P. aeruginosa ATCC 27853, and one Gram-
positive drug-resistant bacteria strain, VRE B148, were
chosen and treated with 3g, but no MICs below 64 μg/mL
were observed (Table 1).
To disclose the SAR of 3g as well as to discover more
effective antibacterial agents, six hybrids (5a–5f) of indolin-
2-one and nitroimidazole with various substituents on the
benzene ring of indolin-2-one were prepared. Compounds
5a–5f were obtained from aldol condensation of compounds
4a–4f with 1-methyl-5-nitro-1H-imidazole-2-carbaldehyde
(2g) (Scheme 2). Compounds 4a–4c and 4e were com-
mercially available, while compounds 4d and 4f were
synthetic intermediates. Compound 4d was obtained by
condensing 3-(piperidin-1-yl)propanoic acid with inter-
mediate 6, which was obtained by reduction of compound
4c (Scheme 2). Compound 4f was synthesized starting from
compound 1, after substitution with chlorosulfonic acid at
C-5 position to afford intermediate 7 and condensing with
morpholine; the intermediate 4f thus formed was finally
reacted with 1-methyl-5-nitro-1H-imidazole-2-carbaldehyde
(2g) to give compound 5f (Scheme 2). All of these six
hybrids of indolin-2-one and nitroimidazole adopted an E
configuration (Supporting Information). The MIC values
against ATCC 33591 and ATCC 25923 of these six
molecules were screened (Table 1).
significantly increased the antibacterial activities of this
class; a 16-fold and 8-fold increase in efficacy of 5c
(compared with 3g) against ATCC 25923 and ATCC 33591
were found, while 5e exhibited a fourfold and twofold
increased efficacy (compared with 3g) against ATCC 25923
and ATCC 33591, respectively. A derivative with a 3-
(piperidin-1-yl)propanamide substituent (5d) at C-5 posi-
tion resulted in a fourfold drop in potency on both stains.
However, introduction of a trifluoromethyl moiety, a bro-
mine atom, and a morpholinosulfonyl moiety almost com-
pletely abolished inhibitory activities. Interestingly,
compound 5c also showed potential activity against two
Gram-negative bacteria strains and VRE strain mentioned
above (Table 1). These initial SAR studies showed great
antibacterial potential of hybrids of indolin-2-one and
nitroimidazole; especially, introduction of a nitro group on
indolin-2-one remarkably enhanced the antimicrobial
activities as well as broadened the antibacterial spectrum.
we also found this molecule to be effective on two Gram-
negative bacteria strains E. coli ATCC 25922, P. aeruginosa
ATCC 27853 and one Gram-positive bacteria strain
vancomycin-resistant Enterococcus (VRE) strain B148,
respectivelyThe most effective compound 5c was experi-
mented for its time-kill ability. The time-kill curve experi-
ments (see Experimental Procedures for detail) were
performed at concentrations of 1/4* MIC, 1* MIC, 4* MIC,
and 16* MIC of compound 5c against two S. aureus strains,
MSSA ATCC 6538, MRSA ATCC 33591, and one E. coli
strain, ATCC 25922. When treated with 16* MIC com-
pound concentration of 5c, bacterial growth was absent
among all three strains, while 1/4* MIC of 5c almost did not
kill any microbes. A slight bacterial regrowth phenomenon
was observed after 4–6 h of incubation with bacteria with
Introduction of a nitro group at either C-5 position (5c) or
C-6 position (5e) of the phenyl ring of indolin-2-one

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Y. Zhou et al.
Table 1 Structure of
Compounds MICs (μg/mL)
synthesized compounds and
their MICs against MSSA
ATCC 25923, MRSA ATCC
33591, E. coli ATCC 25922,
P. aeruginosa ATCC 27853,
and VRE B148
MSSA ATCC
25923
MRSA ATCC
33591
E. coli ATCC P. aeruginosa ATCC VRE
25922
27853
B148a
3a
3b
3c
3d
3e
3f
>64
>64
N.D
N.D
N.D
N.D
N.D
N.D
>64
N.D
N.D
N.D
N.D
N.D
N.D
0.25
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
>64
N.D
N.D
N.D
N.D
N.D
N.D
>64
N.D
N.D
N.D
N.D
N.D
N.D
4
>64
>64
>64
>64
16
>64
>64
>64
>64
>64
3g
3h
3i
2
0.5–1
>64
>64
N.D
N.D
N.D
N.D
N.D
N.D
0.25–0.5
N.D
N.D
N.D
64
64
3j
>64
>64
3k
5a
5b
5c
5d
5e
5f
>64
>64
>64
>64
>64
>64
0.0625–0.125
0.0625–0.125
2–4
8
N.D
N.D
N.D
0.5
>64
0.25
>64
aThe bacteria strain VRE B148 was a clinical isolate
1* MIC or 4* MIC of 5c within three strains, especially in
E. coli (Fig. 3). Furthermore, compound 3g and 5c were
tested for their hemolytic activities (see Experimental Pro-
cedures for detail), both 3g and 5c showed significant low
hemolytic rates even at 500 μM (Fig. 4), indicating a pro-
mising safety profile.
Conclusion
In conclusion, we have discovered a chemical class of
hybrids of indolin-2-one and nitroimidazole that pos-
sessed a high potency of killing several bacteria strains,
including drug-resistant bacteria. The initial SAR study
showed great potential in development of this class, and
the low hemolytic rates suggested a promising safety
profile for this scaffold. The introduction of the nitro
group resulted in significant enhancement of potency
and broad antibacterial spectrum of this class. This result
suggested the presumption that compound 5c may have an
effect on the nitroreductase of bacteria, and our
further work should concentrate on proof of this hypoth-
esis. This study presented a preliminary insight into this
chemical structure; we consider that our further research
on this class of compounds should consist in exploration
of the mechanism of action and investigation of the
pharmacodynamics and pharmacokinetics of the hit
compound.
Fig. 2 The 2D ROSEY NMR spectrum of compound 3b (a) and
compound 3h (b); Z/E configuration was judged from the existence of
co-signal of olefinic hydrogen with hydrogen at C-4 position, and C-4
position hydrogen with hydrogens on the aromatic ring of aldehyde
moiety