Discovery of novel nitroimidazole enols as Pseudomonas aeruginosa DNA cleavage agents

Article abstract of DOI:10.1016/j.bmc.2017.10.028

A series of nitroimidazole enols as new bacterial DNA-targeting agents were for the first time designed, synthesized and characterized by NMR, IR and HRMS spectra. The antimicrobial screening revealed that 2-methoxyphenyl nitroimidazole enol 3i possessed stronger anti-P. aeruginosa efficacy (MIC = 0.10 μmol/mL) than reference drugs Norfloxacin and Metronidazole. Time-kill kinetic assay manifested that the active molecule 3i could rapidly kill the tested strains. Molecular docking indicated that the interactions between compound 3i and topoisomerase II were driven by hydrogen bonds. Quantum chemical study was also performed on 3i to understand the structural features essential for activity. Further research found that compound 3i was not able to effectively intercalate into bacterial DNA but could cleave DNA isolated from the standard P. aeruginosa strain, which might block DNA replication to exert the efficient bioactivities, and this active molecule was also able to be stored and carried by human serum albumin via hydrophobic interactions and hydrogen bonds.

Full text of DOI:10.1016/j.bmc.2017.10.028

Accepted Manuscript
Discovery of novel nitroimidazole enols as Pseudomonas aeruginosa DNA
cleavage agents
Zhen-Zhen Li, Lavanya Gopala, Vijai Kumar Reddy Tangadanchu, Wei-Wei
Gao, Cheng-He Zhou
PII:
S0968-0896(17)31682-6
https://doi.org/10.1016/j.bmc.2017.10.028
BMC 14033
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
23 August 2017
18 October 2017
20 October 2017
Please cite this article as: Li, Z-Z., Gopala, L., Tangadanchu, V.K.R., Gao, W-W., Zhou, C-H., Discovery of novel
nitroimidazole enols as Pseudomonas aeruginosa DNA cleavage agents, Bioorganic & Medicinal Chemistry (2017),
doi: https://doi.org/10.1016/j.bmc.2017.10.028
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Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Discovery of novel nitroimidazole enols as Pseudomonas aeruginosa DNA cleavage
agents
Zhen-Zhen Li, Lavanya Gopala†, Vijai Kumar Reddy Tangadanchu‡, Wei-Wei Gao, Cheng-He Zhou*
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing Municipality, School of Chemistry and Chemical
Engineering, Southwest University, Chongqing 400715, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of nitroimidazole enols as new bacterial DNA-targeting agents were for the first time
designed, synthesized and characterized by NMR, IR and HRMS spectra. The antimicrobial
screening revealed that 2-methoxyphenyl nitroimidazole enol 3i possessed stronger anti-P.
aeruginosa efficacy (MIC
= 0.10 µmol/mL) than reference drugs Norfloxacin and
Available online
Metronidazole. Time-kill kinetic assay manifested that the active molecule 3i could rapidly kill
the tested strains. Molecular docking indicated that the interactions between compound 3i and
topoisomerase II were driven by hydrogen bonds. Quantum chemical study was also performed
on 3i to understand the structural features essential for activity. Further research found that
compound 3i was not able to effectively intercalate into bacterial DNA but could cleave DNA
isolated from the standard P. aeruginosa strain, which might block DNA replication to exert
the efficient bioactivities, and this active molecule was also able to be stored and carried by
human serum albumin via hydrophobic interactions and hydrogen bonds.
Keywords:
Nitroimidazole
Antibacterial
Antifungal
Pseudomonas aeruginosa
DNA cleavage
HSA study
2017 Elsevier Ltd. All rights reserved.
structural characteristics, nitroimidazole-based compounds
exhibit a variety of biological activities such as antiparasitic, anti-
1. Introduction
mycobacterial, antibacterial13 and antitumor14 efficiencies etc. So
far many nitroimidazoles as clinical drugs like Metronidazole,
Ornidazole, Secnidazole and Nimorazole have been witnessed
extensive uses in treating several diseases.15–17 Increasing efforts
Microbial infection has been a major global threat for public
health. Pseudomonas aeruginosa represents a primary cause of
infections including nosocomial pneumonia, urinary tract
diseases and so on.1 These emerging and evolving threats have
been leading to an increase in the morbidity and mortality rates.2
Owing to its large genome and regulatory capacity, P.
aeruginosa possesses numerous virulence factors and about 26%
of its isolates lead to an intrinsic resistance to many antimicrobial
agents and disinfectants.3 Nevertheless, the efficiency and
specificity of antibiotics towards P. aeruginosa infection are
barely satisfactory.4 All these make the fight against P.
aeruginosa bacterium a challenging global issue and underscore
the urgent need in developing the most appropriate
antimicrobials.5
have been directing towards the development of simple
19
nitroimidazole-based drugs.18,
However, to the best of our
knowledge, the combination of enol moiety with nitroimidazole
nucleus has been seldom reported. Therefore, it is of great
interest to develop novel nitroimidazole enols as new bacterial
DNA-targeting agents.
It is well known that azolyl alcohol structure such as
imidazole and triazole ones presents in many biologically active
compounds in which this structural fragment plays a key role in
exerting biological effect including enhancing the water
solubility and overcoming the drug resistance to a certain extent
by forming hydrogen bond at resistance mutation region of
bacteria.20–22 Especially, imidazole ethanol in many
nitroimidazole drugs has been revealed to be an important
molecular structure in treating infective diseases23 When the
unsaturated double bond is coupled to nitroimidazole nucleus, the
resulting nitroimidazole enol is able to extend the molecular π-
conjugated system, and this might be beneficial for forming π-π
stacking and electronic interactions with biological targets like
DNA in organism.24
Nitroimidazole is a significant class of nitrogen-containing
heterocycle with aromatic high electron density6 and can readily
form various non-covalent interactions with biomolecules like
DNA.7,8 The nitro group can be reduced to reactive radical
species9, 10 as soon as the molecule enters into the cells through
passive diffusion, and then the radicals react with DNA or
proteins and cause DNA-strand breaks.11, 12 Due to these special
———
∗Corresponding author. Tel.: +86-23-68254967; fax: +86-23-68254967; e-
mail: zhouch@swu.edu.cn (Cheng-He Zhou).
†Postdoctoral Researcher from Hyderabad Central University, India.
‡Postdoctoral Researcher from Indian Institute of Chemical Technology, India.
1

Fig. 1. Design of novel nitroimidazole enols.
In view of the above observations, as an extension of our
NMR and HRMS spectra and screened against Gram-positive
bacteria, Gram-negative bacteria and fungi. To further assess the
antimicrobial ability of the most active compound, bactericidal
kinetic assay, quantum chemical and molecular docking studies
were investigated. Moreover, the standard P. aeruginosa DNA
was isolated and the targeting DNA interaction with the most
active nitroimidazole enol was performed by UV-vis absorption
spectroscopy and agarose gel electrophoresis. Also, the binding
behavior of the highly active molecule to human serum albumin
(HSA) was carried out to preliminarily study its absorption,
distribution and metabolism.30
research,25 a series of nitroimidazole enols as new bacterial
DNA-targeting antimicrobial agents were designed, synthesized,
characterized, evaluated for their antimicrobial activity and
preliminarily explored for the antibacterial action mode.26
Diverse conjugated aromatic rings including phenyl, indolyl and
carbazolyl groups were introduced into target compounds to
investigate their effects on bioactivities (Fig. 1).27 Meanwhile, the
substituents on aromatic ring were helpful to regulate the
physicochemical properties and thus their incorporation into
target molecules was beneficial to understand structure-activity
relationships.28,29 All the new compounds were confirmed by IR,
Scheme 1 Synthetic route of nitroimidazole enols 2–10. Reagents and conditions: (i) 1-chloropropan-2-one, acetonitrile, potassium carbonate, 80 oC; (ii) aryl
o
aldehyde, piperidine, glacial acetic acid, toluene, refluxed, 4–6 h; (iii) sodium borohydride, 0 C–r.t.; (iv) dimethylformamide, phosphorus oxychloride, 0 oC; (v)
ethyl bromide, sodium hydride, dry DMF, r.t.; (vi) phosphorus oxychloride, dimethylformamide, 1,2-dichloroethane, 100 oC.

indole aldehyde 8, which was prepared by the formylation of
indole with dimethylformamide and phosphorus oxychloride at 0
oC, and the reduction of compound 4 with sodium borohydride in
methanol produced nitroimidazole enol 5 in good yield of 68.8%.
Intermediate 9 was prepared in yield of 70.1% by the N-
alkylation of carbazole with ethyl bromide and sodium hydride,
and then the formylation reaction of 9 with dimethylformamide
2. Results and discussion
2.1. Chemistry
The desired nitroimidazole enols were synthesized according
to the synthetic routes outlined in Scheme 1. Commercially
availiable 2-methyl-5-nitro-1H-imidazole was reacted with 1-
chloropropan-2-one in the presence of potassium carbonate to
produce compound 1 in yield of 76.6%, and then further
condensation with a series of aromatic aldehydes in toluene using
piperidine and glacial acetic acid as catalysts at 120 oC afforded
nitroimidazole ketones 2a–i with yields ranging from 21.4% to
39.4%.31 The reduction of intermediates 2a–i with sodium
borohydride gave the target nitroimidazole enols 3a–i in yields of
o
and phosphorus oxychloride in 1,2-dichloroethane at 100 C
produced carbazole aldehyde 10 in 65.4% yield. The later was
coupled with compound
1
to afford carbazole-derived
nitroimidazole enone 6 in 23.4% yield, and then further reduction
by sodium borohydride in methanol gave nitroimidazole enol 7 in
yield of 65.1%. All the new compounds were confirmed by NMR,
IR and HRMS spectra. The HPLC analysis showed that all the
target compounds were pure, and the related HPLC spectra were
provided in the Supporting Information.
74.9–86.3%. Nitroimidazole-derived indole ketone
obtained in 47.2% yield by the condensation of compound 1 with
Table 1 Antibacterial data as MIC (µmol/mL) for compounds 2–7.a,b
4 was
Gram-positive bacteria
Gram-negative bacteria
Compds
S. aureus
S. aureus
E. coli
ATCC 25922
1.65
P. aeruginosa
ATCC 27853
0.83
MRSA S. aureus
E. coli
P. aeruginosa
ATCC 25923 ATCC 29213
2a
2b
2c
2d
2e
2f
2g
2h
2i
1.65
1.64
1.64
1.79
1.72
1.76
1.62
1.88
1.60
1.50
1.67
1.67
1.78
1.71
1.76
1.62
1.87
1.60
0.83
0.81
1.64
1.31
1.60
2.97
1.65
0.21
1.64
0.22
1.72
1.76
1.62
0.94
1.60
1.50
0.83
1.67
1.78
1.71
1.71
1.62
1.87
1.60
1.65
1.63
0.82
1.31
0.20
2.97
0.83
1.64
0.82
0.90
0.86
0.88
1.62
0.47
0.40
1.50
1.67
1.67
0.89
1.71
0.88
0.81
1.87
0.80
0.83
0.41
0.82
1.31
0.10
2.97
1.65
1.64
0.82
0.45
0.86
1.76
0.41
0.94
1.60
1.50
0.83
1.67
1.78
1.71
1.71
1.62
1.87
0.80
0.83
0.81
1.64
0.66
0.025
2.97
1.65
1.64
1.64
0.90
1.72
1.76
0.81
0.94
0.80
1.50
1.67
1.67
1.78
1.71
1.71
1.62
1.87
1.60
1.65
0.81
1.64
1.31
1.60
2.97
1.65
1.64
0.83
1.79
0.43
0.88
1.62
0.47
0.80
1.50
1.67
1.67
0.89
1.71
0.88
1.62
0.94
0.40
0.42
0.81
1.64
1.31
1.60
2.97
0.41
0.82
0.41
0.83
0.056
0.86
0.45
0.86
0.11
0.44
0.81
0.41
0.23
0.47
0.10
0.40
3a
3b
3c
3d
3e
3f
3g
3h
3i
1.50
0.75
1.67
0.41
0.21
0.83
0.44
0.44
0.86
0.86
0.44
0.11
0.81
0.40
1.87
0.23
0.40
0.10
4
0.42
0.42
5
0.41
0.41
6
1.64
1.64
7
1.31
1.31
A
0.05
1.60
B
2.97
2.97
a Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates.
b MRSA, Methicillin-Resistant Staphylococcus aureus N315; S. aureus, drug-resistant Staphylococcus aureus, Staphylococcus aureus ATCC25923; S. aureus,
Staphylococcus aureus ATCC29213; E. Coli, drug-resistant Escherichia coli, Escherichia coli ATCC25922; P. aeruginosa, drug-resistant Pseudomonas
aeruginosa, Pseudomonas aeruginosa ATCC27853; C. albicans, drug-resistant Candida albicans, Candida albicans ATCC90023; C. tropicals, Candida
tropicals.
c A = Norfloxacin; B = Metronidazole.
bacteria and fungi by two folds serial dilution technique
according to Clinical and Laboratory Standards Institute (CLSI)
with the positive control of clinical drugs Norfloxacin,
Metronidazole and Fluconazole.32 Minimum inhibitory
2.2. Antimicrobial activities
The prepared nitroimidazole enones and enols 2–7 were
evaluated for their antimicrobial activities in vitro against several
3

concentration (MIC, µmol/mL) is defined as the lowest
concentration of new compounds that completely inhibit the
growth of fungi or bacteria.33 The tested strains including clinical
drug-resistant strains isolated from infected patients are four
Gram-positive bacteria (Methicillin-Resistant Staphylococcus
aureus N315, Staphylococcus aureus ATCC 25923 and
The antifungal activities of the synthesized compounds were
showed in Fig. 2 and Fig. 3. It was observed that most of the
target compounds showed comparable or higher anti-C. tropicals
activity to reference drug Fluconazole, except for compounds 2a,
3c, 3e and 4. Noticeably, the inhibitory concentrations towards C.
tropicals of nitroimidazole enols 3f–i (MIC = 0.40–0.47
µmol/mL) were lower than those of nitroimidazole enones 2f–i
(MIC = 0.47–0.88 µmol/mL), which was consistent with their
antibacterial tendency. Derivative 3i could efficiently inhibit the
growth of standard C. tropicals with MIC value of 0.40 µmol/mL,
being 4-fold more active than the reference drug Fluconazole
(MIC = 0.84 µmol/mL). It was noteworthy that 4-chlorophenyl
derived enol 3b and phenyl one 3h exhibited equivalent
inhibitions against C. albicans with MIC value of 0.05 µmol/mL
to Fluconazole.
Staphylococcus
aureus
ATCC
29213,
drug-resistant
Staphylococcus aureus),
four Gram-negative bacteria
(Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC
25922, drug-resistant Escherichia coli and Pseudomonas
aeruginosa) and three fungi (Candida albicans ATCC 90023,
drug-resistant Candida albicans and Candida tropicals).
Preliminary active screening showed that all the prepared
compounds displayed superior inhibitory activities to
metronidazole, and some target compounds gave moderate to
good inhibitory potencies towards Gram-negative bacteria but
weak activities against Gram-positive bacteria. As shown in
Table 1, standard P. aeruginosa was more sensitive to 2d, 2f–i,
3d, 3f–i, 4 and 5 with MIC values ranging from 0.10 to 0.47
µmol/mL, which were 4- to 16-fold more potent than clinical
3.0
C. albicans
C. albicans ATCC 90023
2.5
2.0
1.5
1.0
0.5
0.0
C. tropicals
Norfloxacin (MIC
=
1.60 µmol/mL). Especially 3-
methoxyphenyl nitroimidazole enol 3i showed the strongest anti-
P. aeruginosa efficiency (MIC = 0.10 µmol/mL), and 2-
fluorophenyl compound 3f also gave a low MIC value of 0.11
µmol/mL towards standard P. aeruginosa. Meanwhile, phenyl
derivative 3h (MIC = 0.23 µmol/mL) could restrain the growth of
the above strain. The replacement of 3-methoxyphenol moiety in
compound 3i by 4-methylphenyl and 4-dimethylaminophenyl
groups yielded nitroimidazole enols 3d (MIC = 0.44 µmol/mL)
and 3g (MIC = 0.40 µmol/mL) with 4 times higher activity than
Norfloxacin (MIC = 1.60 µmol/mL) and 7 times more active than
Metronidazole (MIC = 2.97 µmol/mL) against standard P.
aeruginosa. However, derivatives 3a–c and 3e with electron-
withdrawing moieties on benzene rings showed weaker activity
than molecule 3i. The similar antibacterial tendency could also be
concluded from 2d and 2f–i. When it came to drug-resistant P.
aeruginosa, only derivatives 2e, 2h and 3i possessed better
inhibitory concentrations than Norfloxacin with MIC values of
0.43, 0.47 and 0.40 µmol/mL, respectively. As for standard E.
coli strain, 4-methylphenyl nitroimidazole ketone 2d gave almost
equivalent activity (MIC = 0.056 µmol/mL) to Norfloxacin (MIC
= 0.050 µmol/mL). Compounds 2g and 2i exhibited relatively
low concentrations of 0.81 and 0.80 µmol/mL towards drug-
resistant E. coli than Norfloxacin (MIC = 1.60 µmol/mL). All the
results could be explained that the incorporation of electron-
donating groups into phenyl rings resulted in positive MIC values,
and the hydroxyl group played a positive role in antibacterial
efficiency by forming hydrogen bond at resistance mutation
region of the tested strains
2a 2b 2c 2d 2e 2f 2g 2h 2i
Compound
4
6
C
Fig. 2. Antifungal activity as MIC (µmol/mL) for nitroimidazole enones 2, 4
and 6. C = Fluconazole.
3.0
C. albicans
2.5
2.0
1.5
1.0
0.5
0.0
C. albicans ATCC 90023
C. tropicals
3a 3b 3c 3d 3e 3f 3g 3h 3i
Compound
5
7
C
Fig. 3. Antifungal activity as MIC (µmol/mL) for nitroimidazole enols 3, 5
and 7. C = Fluconazole.
Compound 3i
10
Control
8
Continual studies were concentrated on investigating the
influence of different conjugated structures on antimicrobial
activities. It was observed that indolyl-derived nitroimidazole
enone 4 (MIC = 0.42 µmol/mL) and enol 5 (MIC = 0.41
µmol/mL) displayed poorer anti-P. aeruginosa efficiency than
phenyl series 3 but superior activity to carbazole-derived one 6
(MIC = 1.64 µmol/mL) and 7 (MIC = 1.31 µmol/mL).
6
4
2
120
150
180
210
240
270
Time (min)
Analogously, indolyl-derived compounds
4
and
5
also
demonstrated better efficiency than carbazole-derived
compounds 6 and 7 against MRSA, drug-resistant P. aeruginosa
and standard E. coli strains. These showed that the larger
conjugated skeletons linked to nitroimidazole might be
disadvantageous for antibacterial efficiency, for that the steric
hindrance of compounds made it difficult to access the active site
of validated target. More needs to be done to elucidate them on
antibacterial activity.
Fig. 4. Time-kill kinetics of compound 3i (4 × MIC) against sensitive P.
aeruginosa.
2.3. Bactericidal kinetic assay
The antibacterial ability of an agent to eradicate sensitive P.
aeruginosa will rapidly decline as soon as this bacterium shows
resistance towards that agent.34 Time-kill assay was performed by
4

checking the viability of exponentially growing sensitive P.
aeruginosa against compound 3i with the purpose of having a
good command of antibacterial potency to the prepared
compound. Fig. 4 revealed that there was more than 3.5 log (CFU
mL-1) reduction of 3i in the number of viable bacteria within two
hours at a concentration of 4 × MIC, which manifested that the
molecule 3i had rapid killing effect against sensitive P.
aeruginosa.
increase with the increasing concentration of molecule 3i.
Meanwhile, the absorption values of simply sum of free DNA
and free 3i were a little greater than the measured values of 3i-
DNA complex, which meant that a weak hypochromic effect
existed between DNA and molecule 3i.
Based on variations in the absorption spectra of DNA upon
binding to compound 3i, equation (1) could be utilized to
calculate the binding constant (K).39
2.4. Interactions of compound 3i with sensitive P. aeruginosa
DNA
A0
A − A0 ξD−C − ξC ξD−C − ξC K[Q]
ξC
ξC
1
=
+
×
(1)
It is well known that DNA is regarded as one of the most
important targets for the study of biologically important small
molecules such as antimicrobial drugs. Much attention has been
paid to investigate the interaction of small molecules with DNA
and construction of new and efficient drug targeting DNA.35, 36
To explore the possible antimicrobial action mechanism, the
binding behavior of the active molecule 3i with sensitive P.
aeruginosa DNA was studied on molecular level in vitro using
neutral red (NR) dye as a spectral probe by UV-vis spectroscopic
methods. The tested DNA was isolated from sensitive P.
aeruginosa bacteria to the active molecule 3i (in Supporting
information).
A0 and A are the absorbance of DNA in the absence and
presence of compound 3i at 260 nm, ξC and ξD-C represent the
absorption coefficients of compound 3i and 3i-DNA complex
respectively. The plot of A0 / (A − A0) versus 1/ [compound 3i]
was constructed by using the absorption titration data and linear
fitting (Fig. 6), yielding the binding constant, K = 1.83 × 104
L/mol, R = 0.999, SD = 0.091 (R is the correlation coefficient.
SD is standard deviation).
g
0.3
1.0
0.2
a
DNA+NR
0.8
DNA+3i
400
425
450
475
500
DNA-3i complex
Wavelength (nm)
0.5
1.0
0.6
0.4
h
DNA+NR
300
0.4
0.6
0.8
-5
1.0
1.2
0.0
0.5
0.0
10 [Q]
250
350
400
450
500
550
Wavelength (nm)
Fig. 7. UV absorption spectra of NR in the presence of DNA at pH 7.4 and
room temperature. c (NR) = 2 × 10-5 mol/L, c (DNA) = 0–5.71 × 10-5 mol/L
for curves a-g respectively at increment 0.48 × 10-5.
a
DNA
250
275
300
325
350
Wavelength (nm)
2.4.2. Absorption spectra of NR interactions with DNA
Fig. 5. UV absorption spectra of DNA with different concentrations of
compound 3i (pH = 7.4, T = 290 K). Inset: comparison of absorption at 260
nm between the 3i-DNA complex and the sum values of free DNA and free
compound 3i. c (DNA) = 5.71 × 10-5 mol/L, and c (compound 3i) = 3.81 × 10-
6 mol/L for curves a-h respectively at increment 0.25 × 10-5.
Neutral Red (NR) is a planar phenazine dye with the features
of low toxicity, high stability and convenient application. It is
commonly considered that the binding of NR with DNA is an
intercalation binding confirmed by spectrophotometric and
electrochemical techniques. In the present study, the interaction
of compound 3i with sensitive P. aeruginosa DNA was
investigated in vitro using NR dye as a spectral probe by the
application of UV-vis absorption. The absorption spectra of the
NR dye upon the addition of DNA was showed in Fig. 7. The
absorption peak of the NR at around 460 nm did not appear
gradually increase with the increase of DNA concentration,
turning out that there might be weak or no formation of the new
DNA-NR complex and DNA intercalation was not the main
reason to exert antimicrobial activity.
7
6
5
4
3
2
1
1
2
3
4
5
6
-5
-1
10 [Q] ( L/mol )
Fig. 6. The plot of A0 / (A - A0) versus 1/[compound 3i].
2.4.1. Absorption spectra of DNA in the presence of
compound 3i
Absorption spectroscopy plays an important role in DNA-
binding studies. For that hyperchromism and hypochromism are
certain spectral features which can distinguish the change of
DNA double-helical structure, and the observed large
hypochromism powerfully illustrated that the aromatic
chromophore is in close proximity to the DNA bases.37, 38 The
UV-vis absorption spectrum was recorded with the increasing
amount of compound 3i and the constant concentration of DNA.
As shown in Fig. 5, UV-vis spectra displayed that the maximum
absorption peak of DNA at 260 nm exhibited proportional
Fig. 8. Cleavage of P. aeruginosa DNA by compound 3i. Lane 1: DNA
control; Lane 2: DNA + 75 µM compound 3i; Lane 3: DNA + 15 U/µL EcoR
I enzyme.
5

2.5. DNA cleavage study of compound 3i towards sensitive P.
aeruginosa
was probably oriented towards the outer part of the molecules
and therefore was accessible to interact with their surroundings.
A comparison of the MEP surfaces of target compounds 3g and
3i indicated that the electronegative zones mainly located on O-H
and the O-CH3 groups in compound 3i, while there was only N-
(CH3)2 in the 3g which led to a considerable decrease of the
electron density, and the changes mentioned above would be in
favor of the possible formation of hydrogen bonds. Therefore, it
was supposed that orientating drug candidate 3i possessed better
biological activity among all the compounds.
The DNA cleavage property of compound 3i in variety
concentrations was confirmed in Fig. 8. The performed
experiment was proceeded under physiological conditions (37 oC
and pH = 7.4)40 and the obtained result suggested that the P.
aeruginosa DNA could not be cleaved in the absence of
compound 3i (lane 1). Nevertheless, when the concentration of
compound 3i was about 75 µM (lane 2), almost all of the Form I
was completely converted into the Form II and the result was not
consistent with the effect of EcoR I enzyme cleavage (lane 3). It
indicated that the active molecule 3i acted as a DNA cleavage
agent whose cleavage efficacy was better than EcoR I enzyme.
The experimental result implied that nitroimidazole derivative 3i
might inhibit the growth of sensitive P. aeruginosa and further
destroy DNA by the action mode of cleaving DNA, which might
be an encouraging start in the discovery of novel antimicrobial
agents with the ability to overcome drug resistance.
Table 2 Comparison of the MEP surfaces of some target nitroimidazoles.
2.6. Molecular docking
To understand the possible binding behaviors and justify the
observed antibacterial activity, topoisomerase II receptor (PDB
code: 1ZXN) docking investigation was undertaken41 and the
docking mode with the lowest binding free energy (-9.6 kJ mol-1)
was shown in Fig. 9. The nitro group could interact with Arg162,
Asn150 and Ser148 residues of Top II through hydrogen bonds
with the distance of 1.8 Å, 2.3 Å and 2.5 Å, respectively. The
hydroxyl group could form hydrogen bond with Ser148 residue
in nearly 2.1 Å distance. Furthermore, the nitrogen atom of
imidazole ring was in close vicinity to the residue Ala167 of
topoisomerase II through a hydrogen bond with a distance of 2.0
Å. All of these bindings might be favorable to stabilize the
compound 3i-topoisomerase II supramolecular complex, which
might be responsible for the good inhibitory efficacy of
compound 3i against the tested strains.
2.8. Interactions of compound 3i with HSA
HSA is the main extracellular protein of the circulatory system
and all the distribution, metabolism and absorption of drug can
be changed by their affinity to HSA. So far many promising new
drugs have been rendered ineffectiveness because of their
unusually high affinity to this protein.44 So the investigations of
interactions between drugs or bioactive small molecules and
HSA are not only beneficial to thoroughly understand the
pharmacokinetic behavior of drug, but also significant to design,
modify and screen drug molecules.
2.8.1. Fluorescence quenching mechanism
Fluorescence quenching is usually classified as dynamic
quenching and static quenching, which depends on temperature
and viscosity. The larger diffusion coefficients often result from
the higher temperature, and the quenching constants can increase
along with a gradually increasing temperature in dynamic
quenching. However, the increasing of temperature is likely to
result in a smaller static quenching constant due to the
dissociation of weakly bound complexes.45 The fluorescence
quenching data can be analyzed by the well-known Stern-Volmer
equation46:
Fig. 9. Three-dimensional conformation of 3i-topoisomerase II
supramolecular complex.
2.7. Quantum chemical studies
The molecular electrostatic potential (MEP) surface is able to
indicate that the charged surface area might be responsible for the
hydrophilicity of compounds and can explain the orientation of
drug candidates for their activities. Accordingly, MEP map was
given to investigate the key structural features of the target
compounds such as steric, electrostatic interactions, hydrogen
donor/acceptor properties and lipophilicity.42,43 Comparing the
MEP surfaces of the most active antimicrobial series 3 with the
less active series 2 (Table 2), it was easy to find out that both of
them had almost the same electropositive regions (in blue) but
different electronegative regions (in red). The most obvious
difference came from whether the substituent was carbonyl or
hydroxyl group, the electronegative area had decreased once the
carbonyl group of series 2 was turned into hydroxyl group, which
F0
= 1+ KSV [Q] = K τ [Q]
(2)
q
0
F
F0 and F represent fluorescence intensity in the absence and
presence of compound 3i, respectively. KSV (L/mol) is the Stern-
Volmer quenching constant, Kq is the bimolecular quenching rate
constant (L/mol/s), τ0 is the fluorescence lifetime of the
fluorophore in the absence of quencher, assumed to be 6.4 × 10-9
s for HSA, and [Q] is the concentration of compound 3i. The
6

results (Fig. 10) manifested that the binding constants were
moderate.
F0
1
fa Ka
1
1
=
+
(3)
∆F
[
Q
]
fa
The values of KSV and Kq for the interaction of compound 3i
with HSA at different temperatures were shown in (Table 3). The
Ksv values were inversely correlated with the temperature,
indicating that the fluorescence quenching of HSA was probably
initiated by the formation of 3i-HSA complex rather than
dynamic collision. The Kq values obtained at different
temperatures were in 1013 L mol-1 s-1, which had already far
exceeded the diffusion controlled rate constants of various
quenchers with a biopolymer (2.0 × 1010 L mol-1 s-1), and
manifested that the quenching was not initiated by the dynamic
diffusion process but occurred in the static formation of 3i-HSA
complex.
The fluorescence intensity ∆F is different in the absence and
presence of compound 3i at the concentration [Q], fa is the
fraction of accessible fluorescence, and Ka is the effective
quenching constant for the accessible fluorophore, which is
analogous to associative binding constants for the quencher-
acceptor system. The dependence of F0/∆F on the reciprocal
value of quencher concentration [Q]-1 is linear with the slope
equal to the value of (fa Ka)-1. The value fa-1 is fixed on the
ordinate. The constant Ka is a quotient of the ordinate fa-1 and the
slope (fa Ka)-1. The modified Stern-Volmer plots were shown in
Fig. 11 and the calculated results were depicted in Table 4.
The Scatchard equation48 can be used to estimate the
equilibrium binding constant (Kb) and the number of binding sites
(n), it is described as:
1.0
T = 290K
T = 300K
0.8
0.6
0.4
0.2
T = 313K
log[(F / F)−1] =logKb +nlog[Q]
(4)
0
The interaction between HSA and compound 3i at various
temperatures could be concluded from Fig. 12. Kb and n were
obtained from the Scatchard plots. The decreasing trend of Ka
and Kb with the increase in temperature was conformed to the
temperature dependence of the KSV values. The value of the
binding site approximates to 1, indicating that there was only one
high affinity binding site in the interaction of compound 3i with
HSA. It also could be concluded that the binding constants were
moderate and the effects of temperatures were not obvious, thus
compound 3i might be stored and carried by this protein.
0.2
0.4
0.6
5
0.8
1.0
1.2
10 [Q] (mol/L)
Fig. 10. Sterne-Volmer plots of 3i-HSA system at different temperatures.
Table 3 Sterne-Volmer quenching constants for the interaction of compound
3i with HSA at various temperatures.
pH T (K) KSV (L/mol) Kq (L mol-1 s-1)
ra
S.D.b
290
300
313
7.54 × 104
6.12 × 104
4.99 × 104
1.18 × 1013
9.56 × 1013
7.79 × 1013
0.997 0.019
0.999 0.006
0.997 0.014
T = 290 K
T = 300 K
-0.25
7.4
T = 313 K
-0.50
-0.75
-1.00
ra is the correlation coefficient.
S. D. b is standard deviation
T = 290 K
12
10
8
T = 300 K
T = 313 K
-5.8
-5.6
-5.4
-5.2
-5.0
-5
log [Q] (mol/L)
Fig. 12. Scatchard plots of the 3i-HSA system at different temperatures.
6
2.8.3. Binding mode and thermodynamic parameters
4
Generally, there are four types of non-covalent interactions
between the binding of small molecules and proteins including
hydrogen bonds, van der Waals forces, electrostatic interactions
and hydrophobic bonds.49 The thermodynamic parameters
enthalpy (∆H) and entropy (∆S) change of binding reaction are
the main evidence for confirming the interactions between small
molecules and protein. If the ∆H does not vary significantly over
the studied temperatures range, then its value and ∆S can be
evaluated from the van’t Hoff equation50:
2
1
2
3
4
5
6
10 [Q]-1 (L/mol)
-5
Fig. 11. Modified Sterne-Volmer plots of 3i-HSA system at different
temperatures.
Table 4 Binding constants and sites of 3i-HSA system at pH = 7.4
Modified Sterne−Volmer
Scatchard method
method
T
(K)
10-4 Ka
Kb × 104
(L/mol)
r
S.D.
r
S.D.
n
∆H ∆S
(L/mol)
ln K = −
+
(5)
RT
R
290
300
313
8.22
6.18
4.74
0.999 0.067
0.999 0.081
0.998 0.022
4.77
4.77
4.83
0.999 0.015 0.980
0.999 0.009 0.997
Where K is analogous to the associative binding constants at
the corresponding temperature and R is the gas constant. To
elucidate the binding model between compound 3i and HSA, the
thermodynamic parameters were calculated from the van't Hoff
plots. The enthalpy change (∆H) was estimated from the slope of
the van't Hoff relationship (Fig. 13). The free energy change (∆G)
was then calculated from the following equation51:
0.998 0.019
1.03
2.8.2. Binding constant and site
For a static quenching process, the data can be described by
the Modified Sterne-Volmer equation47:
7

(6)
∆G = ∆H −T∆S
All the target compounds were monitored by analytical thin-
layer chromatography (TLC) using pre-coated silica gel plates.
1H NMR and 13C NMR spectra were recorded on a Bruker AV
300 spectrometer and AVANCE III 600 spectrometer in CDCl3
or DMSO-d6. The chemical shifts were reported in parts per
million (ppm), the coupling constants (J) were expressed in hertz
(Hz) and signals were described as singlet (s), doublet (d), triplet
(t) as well as multiplet (m). The following abbreviations were
used to designate aryl groups: Im = imidazole, Ph = phenyl.
Melting points were recorded on X–6 melting point apparatus
and were uncorrected. Infrared absorption spectrum was carried
out on Bruker RFS100/S spectrophotometer (Bio-Rad,
Cambridge, MA, USA) using KBr pellets in the 400–4000 cm-1
range. The high resolution mass spectra (HRMS) were recorded
on an IonSpec FTICR mass spectrometer. UV spectra were
recorded at room temperature on a TU-2450 spectrophotometer
(Puxi Analytic Instrument Ltd. of Beijing, China) equipped with
1.0 cm quartz cells. NR was obtained from Sigma-Aldrich (St.
Louis, MO, USA). Tris, NaCl and HCl were analytical purity,
and the HPLC spectra were recorded on LC-20AT
chromatography by Chiralpak IA column, hexane / i-PrOH = 85 /
15; flow rate 1.0 mL/min; λ = 254 nm. Sample masses were
weighed on a microbalance with a resolution of 0.1 mg. All
chemicals and solvents were commercially available and were
used without further purification.
Table 5 summarized the values of ∆H, ∆G and ∆S. The
binding process of the interaction between compound 3i and
HSA was spontaneous because that the free energy ∆G was
negative, and the negative value of ∆H indicated that the binding
was mainly enthalpy-driven and involved an exothermic reaction,
the ∆S was unfavorable for it. A positive ∆S value was frequently
taken as a typical evidence for hydrophobic interaction, which
was consistent with the above discussion. The negative ∆H value
(-33.530 kJ/mol) observed could not be mainly attributed to
electrostatic interactions since the ∆H value of electrostatic
interactions was very small, almost zero. Therefore, ∆H < 0 and
∆S > 0 demonstrated that the hydrophobic interaction and
hydrogen bond played an important role in the binding of
compound 3i to HSA, and electrostatic interaction might also be
involved in the binding process.52
11.3
ln K = 2.11 + 4033/T
a
R = 0.997, S.D. = 0.029
11.2
11.1
11.0
10.9
10.8
4.1.1. Synthesis of 1-(2-methyl-5-nitro-1H-imidazol-1-yl)
propan-2-one (1)
3.20
3.25
3.30
3.35
-1
3.40
3.45
1000/T (K
)
Target compound 1 (11 g) was prepared according to the
literature procedure, starting from 2-methyl-5-nitro-1H-imidazole
(10 g, 79 mmol), potassium carbonate (21.75 g, 157 mmol) and
acetonitrile (30 mL), then the 1-chloropropan-2-one (6.9 mL)
Fig. 13. Van’t Hoff plots of the 3i-HSA system.
Table Thermodynamic parameters of 3i-HSA system at different
temperatures.
T (K) ∆H (kJ/mol) ∆G (kJ/mol) ∆S (J/mol K)
5
o
was added to the mixture at 80 C for 5 h. Yield: 76.6%; mp:
116–118 oC.53
290
-38.617
-38.793
-39.021
300
313
4.1.2. Synthesis of 4-(2,4-dichlorophenyl)-3-(2-methyl-5-nitro-
1H-imidazol-1-yl)but-3-en-2-one (2a)
-33.530
17.542
A mixture of compound 1 (200 mg, 1.08 mmol), piperidine
(0.011 mL) and glacial acetic acid (0.040 mL) was stirred in
toluene (30 mL) at 45 oC for 0.5 h, then 2,4-
dichlorobenzaldehyde (94.50 mg, 0.54 mmol) was added, then
3. Conclusion
In conclusion, a series of nitroimidazole enols were for the
first time designed and synthesized in an efficient, easy and
economic synthetic way. All the target compounds were
characterized by IR, NMR and HRMS spectra. The in vitro
antimicrobial evaluation revealed that some target compounds
could effectively inhibit the growth of the tested strains in
comparison with the reference drugs Norfloxacin, Metronidazole
and Fluconazole. Especially 2-methoxyphenyl nitroimidazole
enol 3i with electron-donating group gave the most potent anti-P.
aeruginosa efficiency with MIC value of 0.10 µmol/mL. The
preliminary research displayed that active molecule 3i could
rapidly kill sensitive P. aeruginosa and had the ability to directly
cleave P. aeruginosa DNA, but there was no significant
intercalation into P. aeruginosa DNA. Molecular docking
suggested that compound 3i could bind with topoisomerase II
through hydrogen bonds. The transportation behavior of
derivative 3i by HSA demonstrated that the binding process was
spontaneous and the hydrophobic interactions and hydrogen
bonds played important roles in the association of compound 3i
with HSA. Therefore, it is of great potential for hybrid 3i to be a
new DNA cleavage agent with broad antimicrobial spectrum to
treat bacterial infections.
o
the resulting system was stirred at 120 C. Upon completion of
the reaction, the mixture was cooled to room temperature and
evaporated under reduced pressure, the residue was extracted
with chloroform (3 × 20 mL), and the combined organic phase
was dried over anhydrous sodium sulfate and further purified by
silica gel column chromatography (eluent, chlorlform/acetone,
8/1, V/V) to give compound 2a (135.23 mg) as yellow solid.
o
Yield: 36.7%; mp: 178−179 C; IR (KBr, cm-1) ν: 3097 (Ar-H,
C=C-H), 2922 (CH3), 1689 (C=O), 1642 (C=C), 1544, 1426,
1379; 1H NMR (600 MHz, CDCl3, ppm) δ: 8.14 (s, 1H), 7.62 (s,
1H), 7.29 (s, 1H), 7.16 (dd, J = 8.5 Hz, 1H), 6.51 (d, J = 8.5 Hz,
1H), 2.60 (s, 3H), 2.10 (s, 3H); 13C NMR (151 MHz, CDCl3,
ppm) δ: 191.2 (C=O), 146.7, 144.1, 137.5, 136.5, 135.3, 134.7,
132.6, 129.6, 127.5, 126.5, 119.3, 24.7, 12.0; HRMS calcd. for
C14H11Cl2N3O3 [M+H]+, 340.0256; found, 340.0257; [M+Na]+,
362.0075; found, 362.0080; [M+K]+, 377.9815; found, 377.9812.
4.1.3. Synthesis of 4-(4-chlorophenyl)-3-(2-methyl-5-nitro-1H-
imidazol-1-yl)but-3-en-2-one (2b)
Compound 2b was prepared according to the procedure
described for compound 2a, starting from compound 1 (200 mg,
1.08 mmol) and 4-chlorobenzaldehyde (75.91 mg, 0.54 mmol).
The desired compound 2b (70.95 mg) was obtained as yellow
4. Experimental
o
solid. Yield: 21.4%; mp: 145−146 C; IR (KBr, cm-1) ν: 3066
4.1. General methods
(Ar-H, C=C-H), 2923 (CH3), 1675 (C=O), 1630 (C=C), 1539,
1433, 1378; H NMR (600 MHz, CDCl3, ppm) δ: 7.79 (s, 1H),
1
8

7.61 (s, 1H), 7.36 (d, J = 8.4 Hz, 2H), 6.89 (d, J = 8.4 Hz, 2H),
2.52 (s, 3H), 2.14 (s, 3H); 13C NMR (151 MHz, CDCl3, ppm) δ:
192.3 (C=O), 147.9, 145.0, 139.2, 138.8, 131.7, 131.4, 130.1,
128.7, 119.7, 25.6, 12.9; HRMS calcd. for C14H12ClN3O3
[M+H]+, 306.0645; found, 306.0646.
1380; 1H NMR (600 MHz, CDCl3, ppm) δ: 7.70 (s, 1H), 7.63 (s,
1H), 6.77 (d, J = 9.0 Hz, 2H), 6.60 (d, J = 8.7 Hz, 2H), 3.06 (s,
6H), 2.45 (s, 3H), 2.18 (s, 3H);13C NMR (151 MHz, DMSO-d6,
ppm) δ: 187.5 (C=O), 148.1, 142.8, 140.8, 136.6, 128.2, 121.5,
115.8, 112.6, 107.4, 35.0, 20.5, 8.0; HRMS calcd. for
C16H18N4O3 [M+Na]+, 337.1277; found, 337.1276; [M+K]+,
353.1016; found, 353.0991.
4.1.4. Synthesis of 4-(2-chlorophenyl)-3-(2-methyl-5-nitro-1H-
imidazol-1-yl)but-3-en-2-one (2c)
Compound 2c (77.91 mg) was obtained as yellow solid by
compound 1 (200 mg, 1.08 mmol) and 2-chlorobenzaldehyde
(37.95 mg, 0.27 mmol) according to the procedure described for
compound 2a. Yield: 23.5%; mp: 197−198 oC; IR (KBr, cm-1) ν:
3065 (Ar-H, C=C-H), 2927 (CH3), 1679 (C=O), 1631 (C=C),
4.1.9. Synthesis of 3-(2-methyl-5-nitro-1H-imidazol-1-yl)-4-
phenylbut-3-en-2-one (2h)
Compound 2h (92.49 mg) was obtained as yellow solid.
o
Yield: 31.4%; mp: 221−222 C; IR (KBr, cm-1) ν: 3060 (Ar-H,
C=C-H), 2928 (CH3), 1680 (C=O), 1637 (C=C), 1542, 1446,
1383; 1H NMR (600 MHz, DMSO-d6, ppm) δ: 8.36 (s, 1H), 8.24
(s, 1H), 7.45 (m, 3H), 7.07 (d, J = 7.2 Hz, 2H), 2.57 (s, 3H), 1.99
(s, 3H); 13C NMR (151 MHz, DMSO-d6, ppm) δ: 194.1 (C=O),
147.2, 145.2, 141.3, 132.1, 131.8, 130.5, 129.8, 123.1, 26.1, 12.7;
HRMS calcd. for C14H13N3O3 [M+Na]+, 294.0857; found,
294.0855.
1
1541, 1429, 1379; H NMR (600 MHz, CDCl3, ppm) δ: 8.21 (s,
1H), 7.60 (s, 1H), 7.54 (d, J = 4.7 Hz, 1H), 7.38 (d, J = 9.1 Hz,
1H), 7.16 (t, J = 7.0 Hz, 1H), 6.58 (t, J = 7.8 Hz, 1H), 2.60 (s,
3H), 2.09 (s, 3H); 13C NMR (151 MHz, CDCl3, ppm) δ: 192.2
(C=O), 145.1, 136.9, 135.5, 133.5, 132.7, 130.6, 128.9, 127.9,
120.3, 25.7, 13.0; HRMS calcd. for C14H12ClN3O3 [M+Na]+,
328.0465; found, 328.0467; [M+K]+, 344.0204; found, 344.0209.
4.1.10. Synthesis of 4-(4-hydroxy-3-methoxyphenyl)-3-(2-
methyl-5-nitro-1H-imidazol-1-yl)but-3-en-2-one (2i)
4.1.5. Synthesis of 3-(2-methyl-5-nitro-1H-imidazol-1-yl)-4-(p-
tolyl)but-3-en-2-one (2d)
Compound 2i (90.97 mg) was prepared as yellow solid. Yield:
26.4%; mp: 179−180 oC; IR (KBr, cm-1) ν: 3469 (OH), 3073 (Ar-
H, C=C-H), 2944 (CH3), 1670 (C=O), 1626 (C=C), 1543, 1424,
Compound 2d (105.95 mg) was prepared as yellow solid by
compound 1 (200 mg, 1.08 mmol) and 4-methylbenzaldehyde
(65 mg, 0.54 mmol) according to the procedure described for
compound 2a. Yield: 34.2%; mp: 184−185 oC; IR (KBr, cm-1) ν:
3031 (Ar-H, C=C-H), 2923 (CH3), 1651 (C=O), 1628 (C=C),
1
1380; H NMR (600 MHz, DMSO-d6, ppm) δ: 10.09 (s, 1H),
8.35 (s, 1H), 8.09 (s, 1H), 6.82 (d, J = 8.3 Hz, 1H), 6.64 (d, J =
8.3 Hz, 1H), 6.41 (d, J = 1.1 Hz, 1H), 3.56 (s, 3H), 2.51 (s, 3H),
2.03 (s, 3H); 13C NMR (151 MHz, DMSO-d6, ppm) δ: 193.7
(C=O), 151.3, 148.3, 147.2, 145.6, 141.6, 128.9, 126.3, 123.1,
122.8, 116.7, 113.7, 55.6, 25.9, 12.7; HRMS calcd. for
C15H15N3O5 [M+H]+, 317.1012; found, 317.0983.
1
1538, 1438, 1381; H NMR (600 MHz, CDCl3, ppm) δ: 7.81 (s,
1H), 7.62 (s, 1H), 7.17 (d, J = 7.6 Hz, 2H), 6.84 (d, J = 8.0 Hz,
2H), 2.53 (s, 3H), 2.36 (s, 3H), 2.13 (s, 3H);13C NMR (151 MHz,
CDCl3, ppm) δ: 192.6 (C=O), 147.6, 145.2, 143.7, 141.0, 130.5,
127.6, 120.2, 25.5, 21.6, 12.8; HRMS calcd. for C15H15N3O3
[M+H]+, 286.1192; found, 286.1204; [M+Na]+, 308.1011; found,
308.1012.
4.1.11. Synthesis of 4-(2, 4-dichlorophenyl)-3-(2-methyl-5-
nitro-1H-imidazol-1-yl)but-3-en-2-ol (3a)
Compound 2a (100 mg, 0.29 mmol) was dissolved in
methanol, and sodium borohydride (22.70 mg, 0.60 mmol) was
4.1.6. Synthesis of 4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)-3-
oxobut-1-en-1-yl) benzonitrile (2e)
o
slowly added to the mixture at 0 C. Then the resulting system
Compound 2e (99.56 mg) was obtained prepared as yellow
was kept stir for 1 h at room temperature, the solvent was
evaporated under reduced pressure and the residue was extracted
with chloroform (3 × 20 mL), the combined organic phase was
dried over anhydrous sodium and the crude product was purified
o
solid. Yield: 29.7%; mp: 131−132 C; IR (KBr, cm-1) ν: 3045
(Ar-H, C=C-H), 2926 (CH3), 2227 (C≡N), 1687 (C=O), 1610
1
(C=C), 1542, 1431, 1380; H NMR (600 MHz, DMSO-d6, ppm)
δ: 8.35 (s, 2H), 7.90 (d, J = 8.2 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H),
2.59 (s, 3H), 1.98 (s, 3H);13C NMR (151 MHz, DMSO-d6, ppm)
δ: 194.1 (C=O), 147.3, 145.2, 139.5, 136.4, 133.6, 130.9, 123.2,
118.6, 113.8, 26.3, 12.9; HRMS calcd. for C15H12N4O3 [M+H]+,
297.0988; found, 297.0985; [M+Na]+, 319.0807; found,
319.0753.
via
silica
gel
column
chromatography
(eluent,
chlorlform/acetone, 10/1, V/V) to give compound 3a (86.81 mg)
as yellow oily liquid. Yield: 86.3%; mp: 52−53 oC; IR (KBr, cm-
1) ν: 3364 (OH), 2975, 2930 (CH3, CH2), 1664 (C=C), 1546,
1471, 1381; 1H NMR (600 MHz, DMSO-d6, ppm) δ 8.59 (s, 1H),
7.72 (s, 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.09 (s, 1H), 6.73 (d, J =
8.0 Hz, 1H), 5.69 (s, 1H), 4.66 (s, 1H), 1.93 (s, 3H), 1.22 (s, 3H);
13C NMR (151 MHz, DMSO-d6, ppm) δ 145.1, 133.2, 131.9,
130.6, 130.1, 129.5, 128.0, 123.0, 68.4, 20.9, 12.8; HRMS calcd.
for C15H16N3O3 [M+H]+, 342.0412; found, 342.0411; [M+Na]+,
364.0232; found, 364.0231.
4.1.7. Synthesis of 4-(4-fluorophenyl)-3-(2-methyl-5-nitro-1H-
imidazol-1-yl)but-3-en-2-one (2f)
Compound 2f (87.64 mg) was synthesised as yellow solid.
o
Yield: 27.9%; mp: 176−177 C; IR (KBr, cm-1) ν: 3063 (Ar-H,
C=C-H), 2928 (CH3), 1675 (C=O), 1636 (C=C), 1541, 1435,
1379; 1H NMR (600 MHz, CDCl3, ppm) δ: 7.82 (s, 1H), 7.63 (s,
1H), 7.08 (dd, J = 13.2, 5.2 Hz, 2H), 6.98 (dd, J = 7.1, 4.6 Hz,
2H), 2.54 (s, 3H), 2.16 (s, 3H); 13C NMR (151 MHz, CDCl3,
ppm) δ: 192.4 (C=O), 165.6, 147.8, 145.1, 139.4, 132.7, 131.0,
126.6, 119.9, 117.2, 25.5, 12.9; HRMS calcd. for C14H12FN3O3
[M+H]+, 290.0941; found, 290.0940; [M+Na]+, 312.0760; found,
312.0775.
4.1.12. Synthesis of 4-(4-chlorophenyl)-3-(2-methyl-5-nitro-
1H-imidazol-1-yl)but-3-en-2-ol (3b)
Compound 3b (83.44 mg) was synthesised as yellow oily
liquid. Yield: 82.9%; mp: 53−54 oC; IR (KBr, cm-1) ν: 3394
(OH), 2976, 2930 (CH3, CH2), 1662 (C=C), 1544, 1496, 1381; 1H
NMR (600 MHz, DMSO-d6, ppm) δ 8.38 (s, 1H), 7.38 (d, J = 8.1
Hz, 2H), 7.00 (s, 1H), 6.85 (d, J = 7.2 Hz, 2H), 5.54 (s, 1H), 4.55
(s, 1H), 2.00 (s, 3H), 1.21 (s, 3H);13C NMR (151 MHz, DMSO-
d6, ppm) δ 146.0, 138.9, 133.7, 132.4, 131.2, 130.2, 129.5, 128.7,
122.5, 68.8, 18.9, 13.1; HRMS calcd. for C15H16N3O3 [M+H]+,
308.0802; found, 308.0811; [M+Na]+, 330.0621; found,
330.0620.
4.1.8. Synthesis of 4-(4-(dimethylamino) phenyl)-3-(2-methyl-
5-nitro-1H-imidazol-1-yl)but-3-en-2-one (2g)
Compound 2g (134.47 mg) was prepared as yellow solid.
o
Yield: 39.4%; mp: 242−243 C; IR (KBr, cm-1) ν: 3061 (Ar-H,
C=C-H), 2907 (CH3), 1650 (C=O), 1627 (C=C), 1533, 1432,
9

4.1.13. Synthesis of 4-(2-chlorophenyl)-3-(2-methyl-5-nitro-
1H-imidazol-1-yl)but-3-en-2-ol (3c)
phenylbut-3-en-2-ol (3h)
Compound 3h (76.96 mg) was prepared as yellow solid. Yield:
76.4%; mp: 49−51 oC; IR (KBr, cm-1) ν: 3404 (OH), 2971, 2929
(CH3, CH2), 1630 (C=C), 1542, 1449, 1379; 1H NMR (600 MHz,
DMSO-d6, ppm) δ 8.38 (s, 1H), 7.33 (m, 3H), 7.00 (s, 1H), 6.84
(d, J = 5.4 Hz, 2H), 5.54 (s, 1H), 4.58 (s, 1H), 1.98 (s, 3H), 1.22
(s, 3H); 13C NMR (151 MHz, DMSO-d6, ppm) δ 146.9, 145.1,
138.2, 133.5, 129.4, 129.1, 128.5, 122.6, 68.9, 21.5, 13.1; HRMS
calcd. for C14H15N3O3 [M+H]+, 274.1192; found, 274.1194.
Compound 3c (66.25 mg) was prepared as yellow oily liquid.
Yield: 79.4%; mp: 43−44 oC; IR (KBr, cm-1) ν: 3376 (OH), 2976,
2928 (CH3, CH2), 1667 (C=C), 1543, 1471, 1381;1H NMR (600
MHz, DMSO-d6, ppm) δ 8.50 (s, 1H), 7.53 (d, J = 8.0 Hz, 1H),
7.31 (t, J = 7.6 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 7.11 (d, J = 15.8
Hz, 1H), 6.72 (d, J = 7.6 Hz, 1H), 5.66 (s, 1H), 4.66 (s, 1H), 1.91
(s, 3H), 1.22 (s, 3H); 13C NMR (151 MHz, DMSO-d6, ppm) δ
162.7, 145.0, 134.2, 133.1, 132.9, 130.7, 129.7, 129.3, 128.4,
127.9, 120.4, 68.3, 19.0, 12.8; HRMS calcd. for C15H16N3O3
[M+H]+, 308.0802; found, 308.0805; [M+Na]+, 330.0621; found,
330.0620.
4.1.19. Synthesis of 4-(3-hydroxy-2-(2-methyl-5-nitro-1H-
imidazol-1-yl)but-1-en-1-yl)-2-methoxyphenol (3i)
Compound 3i (78.69 mg) was obtained as yellow solid. Yield:
78.2%; mp: 48−49 oC; IR (KBr, cm-1) ν: 3396 (OH), 2982, 2925
(CH3, CH2), 1658 (C=C), 1545, 1467, 1380; 1H NMR (600 MHz,
DMSO-d6, ppm) δ 9.39 (s, 1H), 8.34 (s, 1H), 6.84 (s, 1H), 6.69 (d,
J = 8.2 Hz, 1H), 6.34 (d, J = 7.9 Hz, 1H), 6.15 (d, J = 18.5 Hz,
1H), 5.44 (s, 1H), 4.54 (s, 1H), 3.52 (s, 3H), 2.05 (s, 3H), 1.21 (s,
3H); 13C NMR (151 MHz, DMSO-d6, ppm) δ 147.9, 146.9, 145.8,
145.5, 134.6, 127.5, 124.4, 122.6, 116.3, 111.8, 68.9, 55.5, 21.7,
13.1; HRMS calcd. for C15H17N3O5 [M+H]+, 320.1246; found,
320.1245.
4.1.14. Synthesis of 3-(2-methyl-5-nitro-1H-imidazol-1-yl)-4-
(p-tolyl)but-3-en-2-ol (3d)
Compound 3d (76.84 mg) was prepared as yellow solid. Yield:
76.3%; mp: 46−47 oC; IR (KBr, cm-1) ν: 3385 (OH), 2955, 2924
(CH3, CH2), 1661 (C=C), 1539, 1455, 1378; 1H NMR (600 MHz,
DMSO-d6, ppm) δ 8.35 (s, 1H), 7.11 (d, J = 7.7 Hz, 2H), 6.94 (s,
1H), 6.70 (s, 2H), 5.50 (s, 1H), 4.56 (s, 1H), 2.24 (s, 3H), 1.98 (s,
3H), 1.20 (s, 3H); 13C NMR (151 MHz, DMSO-d6, ppm) δ 147.0,
145.1, 138.9, 137.1, 130.6, 130.1, 128.4, 127.4, 126.9, 122.6,
68.9, 21.2, 13.1; HRMS calcd. for C15H16N3O3 [M+H]+, 288.1348;
found, 288.1347; [M+Na]+, 310.1168; found, 310.1180.
4.1.20. Synthesis of 4-(3H-indol-3-yl)-3-(2-methyl-5-nitro-1H-
imidazol-1-yl)but-3-en-2-one (4)
Compound 4 was prepared following the procedure described
for compound 2a, starting from compound 1 (200 mg, 1.08
mmol), 3H-indole-3-carbaldehyde (156.6 mg, 1.08 mmol). The
target compound 4 (159.04 mg) was obtained as yellow solid.
4.1.15. Synthesis of 4-(3-hydroxy-2-(2-methyl-5-nitro-1H-
imidazol-1-yl)but-1-en-1-yl) benzonitrile (3e)
Compound 3e (75.41 mg) was obtained as yellow solid. Yield:
74.9%; mp: 44−46 oC; IR (KBr, cm-1) ν: 3397 (OH), 2976, 2930
Yield: 47.2%; mp: 164−166 C; IR (KBr, cm ) ν: 3165 (NH),
-1
o
1
(CH3, CH2), 2227 (C≡N), 1663 (C=C), 1544, 1417, 1380; H
3027 (Ar-H, C=C-H), 2929 (CH3), 1664 (C=O), 1664 (C=C),
1
NMR (600 MHz, DMSO-d6, ppm) δ 8.41 (s, 1H), 7.78 (d, J = 7.8
Hz, 2H), 7.11 (s, 1H), 7.03 (d, J = 7.8 Hz, 2H), 5.63 (s, 1H), 4.57
(s, 1H), 1.97 (s, 3H), 1.22 (s, 3H); 13C NMR (151 MHz, DMSO-
d6, ppm) δ 162.7, 147.3, 144.8, 141.1, 138.3, 133.3, 129.3, 122.5,
118.8, 111.4, 68.8, 26.8, 13.0; HRMS calcd. for C15H16N3O3
[M+Na]+, 321.0964; found, 321.0966.
1541, 1458, 1380; H NMR (600 MHz, DMSO-d6, ppm) δ11.94
(s, 1H), 8.44 (s, 1H), 8.34 (s, 1H), 7.96 (d, J = 7.7 Hz, 1H), 7.49
(d, J = 7.6 Hz, 1H), 7.26 (d, J = 6.9 Hz, 1H), 7.22 (d, J = 7.1 Hz,
1H), 6.34 (s, 1H), 2.58 (s, 3H ), 2.06 (s, 3H); 13C NMR (151
MHz, DMSO-d6, ppm) δ 192.7 C=O, 147.2, 145.3, 136.4, 134.4,
129.6, 123.7, 122.9, 121.8, 25.7, 12.5; HRMS calcd. for
C16H14N4O3 [M+Na]+, 333.0964; found, 333.0964.
4.1.16. Synthesis of 4-(4-fluorophenyl)-3-(2-methyl-5-nitro-
1H-imidazol-1-yl)but-3-en-2-ol (3f)
4.1.21. Synthesis of 4-(3a,7a-dihydro-3H-indol-3-yl)-3-(2-
methyl-5-nitro-1H-imidazol-1-yl)but-3-en-2-ol (5)
Compound 3f (81.86 mg) was synthesised as yellow solid
according to the procedure described for compound 3a. Yield:
81.3%; mp: 45−47 oC; IR (KBr, cm-1) ν: 3437 (OH), 2986, 2927
(CH3, CH2), 1636 (C=C), 1543, 1416, 1381; 1H NMR (600 MHz,
DMSO-d6, ppm) δ 8.45 (s, 1H), 7.16 (d, J = 8.7 Hz, 2H), 7.01 (s,
1H), 6.89 (s, 2H), 5.56 (s, 1H), 4.56 (s, 1H), 2.00 (s, 3H), 1.22 (s,
3H); 13C NMR (151 MHz, DMSO-d6, ppm) δ 163.1, 147.0, 145.0,
138.0, 130.8, 129.9, 126.4, 122.5, 116.4, 68.9, 21.5, 13.0; HRMS
calcd. for C14H13FN3O3 [M+H]+, 292.1097; found, 292.1094;
[M+Na]+, 314.0915; found, 314.0917.
Compound 5 was prepared by compound 4 (100 mg, 0.32
mmol) according to the procedure described for compound 3a.
The target compound 5 (69.24 mg) was obtained as yellow solid.
Yield: 68.8%; mp: 54−55 oC; IR (KBr, cm-1) ν: 3537 (OH), 3157
(NH), 2954, 2924 (CH3, CH2), 1658 (C=C), 1538, 1459, 1379; 1H
NMR (600 MHz, DMSO-d6, ppm) δ 11.26 (s, 1H), 8.32 (s, 1H),
7.73 (d, 1H) 7.39 (d, J = 8.0 Hz, 1H), 7.22 (s, 1H), 7.15 (d, J =
7.4 Hz, 1H), 7.10 (d, J = 7.4 Hz, 1H ), 5.95 (s, 1H), 5.36 (s, 1H),
4.64 (s, 1H), 2.50 (s, 3H), 2.10 (s, 3H); 13C NMR (151 MHz,
DMSO-d6, ppm) δ 206.8, 146.9, 145.3, 135.8, 123.7, 122.6,
108.2, 68.9, 31.1, 21.8, 13.2; HRMS calcd. for C16H16N4O3
[M+Na]+, 335.1120; found, 335.1121.
4.1.17. Synthesis of 4-(4-(dimethylamino)phenyl)-3-(2-methyl-
5-nitro-1H-imidazol-1-yl)but-3-en-2-ol (3g)
Compound 3g was synthesised according to the procedure
described for compound 3a, starting from compound 2g (100 mg,
0.32 mmol). The target compound 3g (83.13 mg) was obtained as
4.1.22. Synthesis of 4-(9-ethyl-9H-carbazol-3-yl)-3-(2-methyl-
5-nitro-1H-imidazol-1-yl)but-3-en-2-one (6)
Compound 6 was synthesised by compound 1 (200 mg, 1.08
mmol) and 9-ethyl-9H-carbazole-3-carbaldehyde (120.57 mg,
0.54 mmol) according to the procedure described for compound
2a. The target compound 6 (98.69 mg) was obtained as yellow
-1
o
yellow solid. Yield: 82.6%; mp: 176−178 C; IR (KBr, cm ) ν:
3285 (OH), 2972, 2926 (CH3, CH2), 1638 (C=C), 1529, 1442,
1381; 1H NMR (600 MHz, DMSO-d6, ppm) δ 8.35 (s, 1H), 6.77
(s, 1H), 6.58 (s, 3H), 6.55 (d, J = 8.4 Hz, 1H), 5.35 (s, 1H), 4.51
(s, 1H), 2.88 (s, 6H), 2.05 (s, 3H), 1.16 (s, 3H); 13C NMR (151
MHz, DMSO-d6, ppm) δ 150.7, 146.9, 133.1, 129.7, 127.3, 122.6,
120.3, 112.5, 69.3, 49.1, 18.9, 13.1; HRMS calcd. for
C16H20N4O3 [M+H]+, 317.1614; found, 317.1617; [M+Na]+,
339.1433; found, 339.1435.
-1
o
solid. Yield: 23.4%; mp: 106−107 C; IR (KBr, cm ) ν: 3052
(Ar-H, C=C-H), 2929 (CH3), 1670 (C=O), 1617 (C=C), 1537,
1
1429, 1379; H NMR (600 MHz, DMSO-d6, ppm) δ: 8.46 (s,
1H), 8.40 (s, 1H), 8.01 (s, 1H), 7.97 (d, J = 7.7 Hz, 1H), 7.68 (d,
J = 3.6 Hz, 1H), 7.67 (d, J = 3.8Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H),
7.30 (t, J = 7.4 Hz, 1H), 6.91 (d, J = 8.7 Hz, 1H), 4.44 (d, J = 6.9
Hz, 2H), 2.61 (s, 3H), 2.05 (s, 3H), 1.30 (t, J = 7.0 Hz, 3H);13C
4.1.18. Synthesis of 3-(2-methyl-5-nitro-1H-imidazol-1-yl)-4-
10

NMR (151 MHz, DMSO-d6, ppm) δ: 193.7 (C=O), 147.2, 145.7,
141.7, 140.6, 129.0, 127.5, 125.0, 123.3, 123.1, 122.4, 122.2,
120.6, 110.9, 110.4, 47.1, 24.5, 14.2, 12.7; HRMS calcd. for
C22H20N4O3 [M+Na]+, 333.0964; found, 333.0964; [M+K]+,
427.1172; found, 427.1177.
This work was partially supported by National Natural
Science Foundations of China [(No. 21372186, 21672173), the
Research Fund for International Young Scientists from
International (Regional) Cooperation and Exchange Program
(No. 81650110529)] and Chongqing Special Foundations for
Postdoctoral Research Proposal (No. Xm2016039, Xm2017185),
Program for Overseas Young Talents from State Administration
of Foreign Experts Affairs, China (No. WQ2017XNDX047).
4.1.23. Synthesis of 4-(9-ethyl-9H-carbazol-2-yl)-3-(2-methyl-
5-nitro-1H-imidazol-1-yl)but-3-en-2-ol (7)
Compound 7 (65.43 mg) was obtained as yellow solid. Yield:
65.1%; mp: 54−55 oC; IR (KBr, cm-1) ν: 3421 (OH), 2974, 2929
(CH3, CH2), 1659 (C=C), 1541, 1472, 1380; 1H NMR (600 MHz,
DMSO-d6, ppm) δ 8.45 (s, 1H), 7.93 (d, J = 6.2 Hz, 1H), 7.67 (s,
1H), 7.60 (d, J = 8.2 Hz, 1H), 7.53 (d, J = 8.6 Hz, 1H), 7.47 (t, J
= 7.6 Hz, 1H), 7.21 (t, J = 7.4 Hz, 1H), 7.13 (s, 1H), 6.75 (d, J =
8.0 Hz, 1H), 5.51 (s, 1H), 4.62 (s, 1H), 4.39 (q, J = 6.9 Hz, 2H),
1.99 (s, 3H), 1.30 (m, 6H); 13C NMR (151 MHz, DMSO-d6, ppm)
δ 147.0, 145.4, 140.4, 139.9, 135.3, 134.8, 128.3, 126.7, 125.7,
123.9, 122.8, 122.4, 120.4, 110.2, 109.9, 69.1, 49.1, 37.5, 14.1,
13.1; HRMS calcd. for C22H22N4O3 [M+Na]+, 413.1590; found,
413.1592; [M+K]+, 429.1329; found, 429.1339.
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12

Graphical Abstract
Discovery of novel nitroimidazole enols as
Leave this area blank for abstract info.
Pseudomonas aeruginosa DNA cleavage agents
Zhen-Zhen Li, Lavanya Gopala†, Vijai Kumar Reddy Tangadanchu‡, Wei-Wei Gao, Cheng-He Zhou*
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing
Municipality, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715,
China.
H3C
N
OH
R3
R2
H3C
N
R1
NO2
3i R1 = H, R2 = OCH3, R3 = OH
MIC = 0.10 umol/mL (
)
P. aeruginosa
bacterium
Rapidly kill
P. aeruginosa
Efficiently cleave DNA
Form hydrogen bonds and be
stored and carried by HSA
13