Design, synthesis and biological evaluation of amino organophosphorus imidazoles as a new type of potential antimicrobial agents

Article abstract of DOI:10.1007/s11426-016-9009-6

A series of amino organophosphorus imidazoles were designed and synthesized as a novel structural type of antimicrobial agents. Bioactive evaluation in vitro showed that compound 3f exhibited equipotent or superior anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) and anti-S. cerevisiae efficiencies (minimal inhibitory concentration (MIC)=2 μg/mL) to clinical drugs, and the combinations with antibacterial or antifungal drugs enhanced the antimicrobial efficiency. Highly active molecule 3f showed low propensity for bacteria to develop resistance, and the preliminary action mechanism studies demonstrated that 3f was membrane-active, but had no significant intercalation towards MRSA DNA. The computational study on 3f reasonably explained its high antimicrobial activity. Experimental data revealed that ground-state 3f-HSA complexes were formed mainly through hydrophobic interactions and hydrogen bonds with a spontaneous process, and the non-radioactive energy transfer from HSA to 3f occurred beyond F?rster resonance energy transfer theory. The participation of metal ions in 3f-HSA supramolucular system could increase the concentration of free compound 3f, and shorten its storage time and half-life in the blood to improve the maximum antimicrobial efficacy.

Full text of DOI:10.1007/s11426-016-9009-6

SCIENCE CHINA
Chemistry
doi: 10.1007/s11426-016-9009-6
•
ARTICLES •
Design, synthesis and biological evaluation of amino
organophosphorus imidazoles as a new type of potential
antimicrobial agents
1
1
1
1
1
Wei-Wei Gao , Syed Rasheed , VijaiKumarReddy Tangadanchu , Yi Sun , Xin-Mei Peng ,
1
1
2*
1*
Yu Cheng , Feng-Xiu Zhang , Jian-Mei Lin & Cheng-He Zhou
1
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing Municipality,
School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
2
School of Medicine University of Electronic Science and Technology of China, Chengdu 610072, China
Received December 1, 2016; accepted January 23, 2017; published online May 9, 2017
A series of amino organophosphorus imidazoles were designed and synthesized as a novel structural type of antimicrobial agents.
Bioactive evaluation in vitro showed that compound 3f exhibited equipotent or superior anti-methicillin-resistant Staphylococcus
aureus (anti-MRSA) and anti-S. cerevisiae efficiencies (minimal inhibitory concentration (MIC)=2 μg/mL) to clinical drugs,
and the combinations with antibacterial or antifungal drugs enhanced the antimicrobial efficiency. Highly active molecule 3f
showed low propensity for bacteria to develop resistance, and the preliminary action mechanism studies demonstrated that 3f
was membrane-active, but had no significant intercalation towards MRSA DNA. The computational study on 3f reasonably
explained its high antimicrobial activity. Experimental data revealed that ground-state 3f-HSA complexes were formed mainly
through hydrophobic interactions and hydrogen bonds with a spontaneous process, and the non-radioactive energy transfer from
HSA to 3f occurred beyond Förster resonance energy transfer theory. The participation of metal ions in 3f-HSA supramolucular
system could increase the concentration of free compound 3f, and shorten its storage time and half-life in the blood to improve
the maximum antimicrobial efficacy.
organophosphorus, imidazole, antibacterial, antifungal, HSA
Citation:
Gao WW, Rasheed S, Tangadanchu VKR, Sun Y, Peng XM, Cheng Y, Zhang FX, Lin JM, Zhou CH. Design, synthesis and biological evaluation of
amino organophosphorus imidazoles as a new type of potential antimicrobial agents. Sci China Chem, 2017, 60: doi: 10.1007/s11426-016-9009-6
1
Introduction
challenged to cure infections caused by resistant pathogens.
In addition, both Pseudomonas aeruginosa and Escherichia
coli also have shown rapid development of resistance against
several classes of antibiotics that have been used for a long
time [1]. As a result, many traditional antibiotics decrease
or totally lose the effects and activities, and the use of an-
timicrobial drugs is being more and more limited. Thus, it is
an increasingly active topic to develop antimicrobial agents
with structural novelty and functional peculiarity for these
Gram-positive and Gram-negative pathogens.
Multidrug-resistant pathogens like methicillin-resistant
Staphylococcus aureus (MRSA), methicillin-resistant
Staphylococcus epidermidis (MRSE) and vancomycin-re-
sistant Enterococcus faecium (VRE), have become a major
public health concern. Despite of a half century of efforts
to find effective treatments, healthcare practitioners are still
*Corresponding authors (email: linjianmei@med.uestc.edu.cn; zhouch@swu.edu.cn)
It is well known that heterocyclic imidazole plays an ex-
©
Science China Press and Springer-Verlag Berlin Heidelberg 2017
chem.scichina.com link.springer.com

2
Gao et al. Sci China Chem
tremely important role in medicinal field, numerous imida-
zole derivatives as drugs have been used in clinic, especially
in antimicrobial drugs such as the antibacterial Metronida-
zole, Ornidazole and Secnidazole as well as the antifungal
Eniconazole, Miconazole and Sertaconazole (Figure 1) [2].
The imidazole nucleus has also been identified as an effec-
tive isostere of thiazole, oxazole, triazole, pyrazole, tetra-
zole, etc. in drug design, which strongly supports the large
developmental value and spacious potentialities, and the ex-
ploitation of all possible application of imidazole-based com-
pounds as medicinal drugs has been being one of the attract-
ing highlighted topics. Numerous outstanding achievements
have been revealed that imidazole nucleus in combination
with antimicrobial moieties like quinolone, berberine, naph-
thalimide and sulfanilamide has wide potentiality in antimi-
crobial use [3]. However, the combination of the imidazole
nucleus with organophosphorus fragments has been seldom
observed. Therefore, it is importantly meaningful to com-
bine the organophosphorus fragment with the imidazole core
in order to investigate the contribution to antimicrobial activ-
ities.
Phosphorus element is ubiquitous in nature and plays
positive roles in living organisms, and organophosphorus
compounds have received much attention owing to their
various bioactivities. Some of organophosphorus antimi-
crobial drugs have been widely used in clinic, such as the
phosphonic acid derivatives Fosfomycin, Fosfomochlorin
and Fosmidomycin behaving a broad spectrum of activi-
ties against a variety of Gram-positive and Gram-negative
pathogens. Phosphonates are extensively used as prodrugs
to improve biological activities through regulating solubility
like prodrug Fosfluconazole, exhibiting better antifungal
activity than Fluconazole (Figure 2) [4]. In spite of enormous
potentiality of organophosphorus compounds against micro-
Figure 2 Structures of some antimicrobial organophosphorus compounds
color online).
(
bes, probably owning to the difficulty of synthetic method-
ology to access antimicrobial organophosphorus derivatives,
the related work is relatively seldom. Thus, we have an over-
whelming interest to develop organophosphorus imidazoles
as novel antimicrobial agents with structural novelty and
functional peculiarity.
In view of the above observations, as an extension of our
research on bioactive heterocyclic compounds [5], herein a
series of structurally novel compounds with a combination
of amine, organophosphorus and imidazole nucleus as poten-
tially antimicrobial agents were developed (Figure 3). The
designed target compounds were based on the following con-
siderations.
(1) The unique imidazole nucleus is a two-nitrogen
five-numbered aromatic heterocycle, this type of structural
fragment can readily accept or donate proton and exert
various non-covalent interactions with the enzymes and
receptors in organisms via coordination bonds, hydrogen
bonds, ion-dipole, cation-π, π-π stacking, hydrophobic effect
and van der Waals force etc., which helpfully modulates the
pharmacokinetic properties. More importantly, imidazole
nucleus can coordinate with a variety of inorganic metal ions
to produce supramolecular complexes, which may not only
exert the bioactivities of imidazoles themselves, but also
exhibit the advantages of supramolecular complexes, possi-
Figure 1 Structures of some antimicrobial imidazole compounds (color on-
line).
Figure 3 Design of novel organophosphorus imidazoles (color online).

Gao et al. Sci China Chem
3
bly exerting double action mechanisms to overcome the drug
resistance [6]. Rationally, imidazole moiety is chosen as an
important fragment of the target molecule.
nine bacteria and five fungi. The pharmacokinetics properties
including drug combination study, bactericidal kinetic assay
and resistance investigation were done to further evaluate the
biological activity of the most active molecule. Also, com-
putational methods were used to predict these pharmacoki-
netic properties of the drug candidate and to rationalize its
biological activity. The preliminary interactions between the
highly active compound and calf thymus DNA or bacterial
membrane were investigated to provide the possible binding
mechanism and to make further help for the design, modifica-
tion and screening of new drug molecules. Moreover, the ef-
fects of metal ions on their transportation by human serum al-
bumin (HSA) were also investigated in order to preliminarily
evaluate their transportation, distribution, and metabolism by
fluorescence and UV-Vis absorption spectroscopy on molec-
ular level.
(2) The organophosphorus group can provide unique bind-
ing interactions with biological targets because of their di-
or trivalent chelating properties and possibly dual function
as a hydrogen acceptor and donor at physiological pH [7].
Moreover, the introduction of organophosphorus moiety
would play important roles in regulating the physicochem-
ical properties of target molecules through changing the
intermolecular forces and molecular polarity, then possi-
bly improving the lipoid/water solubility. Therefore, the
organophosphorous fragment was incorporated into the tar-
get molecules to discover new molecular scaffolds with high
efficiency and lower resistance.
(3) Amino group is one of the most important moieties with
prominent functionality in organic molecules, and 84% of
small-molecule pharmaceuticals contain at least one [8]. It
can target biomacromolecules in organisms to exhibit biolog-
ical activity. Thus, it may be necessary to introduce amino
group into the antimicrobial nucleus.
2
Experimental
2
.1 Synthesis
The procedures of synthesizing compounds 2–7 and 9 are pro-
(4) The coordination properties of phosphorus and oxy-
vided in the Supporting Information online.
gen atoms in organophosphorus moiety or nitrogen atoms
both in amino group and imidazole nucleus might make them
form supramolecular complexes which could interact with
DNA, HSA, enzymes, and other biomacromolecules to be-
have multi-targeting property.
2
.2 Biological assays
The in vitro minimal inhibitory concentrations (MICs) (in
µg/mL) of the target compounds were determined using the
two-fold serial dilution technique in 96-well microtest plates,
according to the National Committee for Clinical Laboratory
Standards (NCCLS) [11]. The tested microorganism strains
were provided by the School of Pharmaceutical Sciences,
Southwest University and the College of Pharmacy, Third
Military Medical University, China.
(5) Aliphatic chain could significantly influence bioactiv-
ities through regulating the flexibility of molecules. Thus,
alkyl organophosphorous imidazole 2 was prepared to ex-
plore the effect of aliphatic substituents on the antimicrobial
activity.
(6) It is well known that the substituted aromatic ring can
regulate the rigidity of target molecule, and substituents on
benzene rings would improve the binding affinity to targets.
In view of this, electron withdrawing and donating groups
substituted phenyl organophosphorous imidazoles 3, 4 and
2
.2.1 Antibacterial assays
The prepared compounds 2a–2b, 3a–3i, 4a–4f, 5a–5c, 6 and
7 were evaluated for their antibacterial activities against four
Gram-positive bacteria (Staphylococcus aureus ATCC25923,
MRSA, Micrococcus luteus ATCC 4698 and Bacillus subtilis
ATCC 21216), and five Gram-negative bacteria (Escherichia
coli JM109, Pseudomonas aeruginosa ATCC 27853, Bacillus
proteus ATCC 13315, Eberthella typhosa ATCC 14028 and
Shigella dysenteriae ATCC 49550). The bacterial suspension
5
were prepared with the aim to explore the effect of sub-
stituents on biological activity.
7) Pyrimidine, an important aromatic hetertocycle with
(
two nitrogen atoms, has been prevalently employed in drug
design molecules for treating a variety of diseases [9].
Herein, the pyrimidine ring as a nitrogen containing moiety
was introduced into the target molecule 6 to evaluate the
antimicrobial activity.
5
was adjusted with sterile saline to a concentration of 1×10
CFU. The tested compounds were dissolved in dimethyl sul-
foxide (DMSO) to prepare the stock solutions. The tested
compounds and reference drugs were prepared in Mueller-
Hinton broth (Guangdong Huaikai Microbial Sci.& Tech Co.,
Ltd., Guangzhou, China) by two fold serial dilution to obtain
the required concentrations of 512, 256, 128, 64, 32, 16, 8,
4, 2, 1, 0.5 µg/mL. These dilutions were inoculated and in-
cubated at 37 °C for 24 h. To ensure that the solvent had no
effect on bacterial growth, a control test was performed with
(8) Coumarin has a rigid fused structure of benzene ring
and α-pyrone, its derivatives exhibit extensively medicinal
application and multi-targeting properties [10]. Herein, the
coumarin moiety as a larger fragment was introduced into the
target molecule 7 to investigate its effect on the antimicrobial
activity.
All the newly synthesized compounds were evaluated for
their antibacterial and antifungal activities in vitro against

4
Gao et al. Sci China Chem
test medium supplemented with DMSO at the same dilutions
as used in the experiment. The MICs for 2a–2b, 3a–3i, 4a–4f,
tested compounds and reference antifungal drug Flucona-
zole, dilutions in sterile RPMI 1640 medium (Neuronbc
Laboraton Technology CO., Ltd., Beijing, China) were made,
resulting in eleven desired concentrations (0.5 to 512 μg/mL)
of each tested compound. These dilutions were inoculated
and incubated at 36 °C for 24 h.
5a–5c, 6 and 7 were summarized in Table1.
2.2.2 Antifungal assays
The newly synthesized compounds 2a–2b, 3a–3i, 4a–4f,
a–5c, 6 and 7 were evaluated for their antifungal activities
5
2
.2.3 Drug combination assays
against Candida albicans ATCC 76615, Candida utilis
ATCC 9950, Candida mycoderma ATCC 9888, Saccha-
romyces cerevisiae ATCC 9763 and Aspergillus flavus ATCC
The drug combination studies between the most active
compound 3f and standard drugs Chloromycin, Norfloxacin,
Ciprofloxacin and Fluconazole were investigated by 2-fold
dilution checkerboard assay method with concentration
values from 1/32 to 4 times the MIC value of each molecule.
The drug combination effect is usually expressed using frac-
204304. A spore suspension in sterile distilled water was
prepared from a culture of the fungi growing on Sabouraud
agar (SA) media, aged 1 d. The final spore concentration
3
3
was 1×10 –5×10 spore/mL. From the stock solutions of the
a)
Table 1 In vitro antibacterial data as MIC (μg/mL) for compounds 2–7
b)
b)
Gram-positive bacteria
Gram-negative bacteria
Com-
pounds
S.
B.
proteus
S. aureus
MRSA
B. subtilis
M. luteus
E. coli
E. typhosa
P. aeruginosa
dysenteriae
2
a
16±2.4
16±2.4
16±2.4
32±6.4
128±25.6
128±0.0
32±6.4
64±9.6
128±25.6
16±2.4
256±38.4
256±38.4
16±2.4
256±38.4
128±25.6
64±0.0
256±0.0
128±25.6
32±6.4
16±2.4
64±9.6
4±1.2
64±9.6
128±25.6
16±2.4
2b
3a
32±6.4
32±2.4
3
b
64±9.6
8±1.6
64±3.6
64±9.6
128±25.6
128±25.6
256±0.0
16±0.0
128±9.6
64±9.6
32±6.4
4±0.2
32±6.4
3
c
32±0.0
16±2.4
128±25.6
128±25.6
128±25.6
32±6.4
16±2.4
128±25.6
32±6.4
64±9.6
4±1.2
64±9.6
3
d
128±25.6
128±9.6
8±0.0
64±9.6
64±0.0
256±38.4
64±6.4
128±25.6
128±0.0
16±2.4
3
e
128±25.6
2±0.8
64±3.6
256±25.6
16±2.4
16±2.4
2±0.0
3
f
32±0.0
16±2.4
32±0.0
3
g
128±25.6
32±6.4
32±6.4
128±25.6
256±0.0
256±38.4
256±0.0
128±25.6
256±0.0
128±25.6
64±9.6
64±9.6
32±6.4
256±9.6
256±38.4
512±0.0
256±38.4
128±25.6
64±9.6
256±0.0
256±38.4
256±38.4
128±25.6
256±38.4
128±25.6
256±38.4
64±6.4
16±2.4
32±2.4
128±25.6
64±9.6
128±9.6
64±9.6
128±25.6
128±25.6
128±25.6
4±1.2
32±6.4
3
h
128±25.6
128±9.6
128±25.6
256±0.0
128±25.6
128±25.6
128±25.6
64±9.6
64±6.4
512±38.4
256±38.4
256±0.0
256±38.4
128±25.6
128±25.6
256±38.4
128±25.6
128±0.0
128±25.6
128±0.0
128±25.6
256±38.4
32±6.4
128±25.6
256±38.4
256±38.4
128±25.6
128±25.6
64±9.6
3
i
128±25.6
64±9.6
256±25.6
128±25.6
128±25.6
128±9.6
256±38.4
256±0.0
128±25.6
256±38.4
64±0.0
4
a
4
b
128±25.6
128±25.6
64±3.6
4
c
4
d
128±25.6
128±25.6
256±0.0
512±0.0
256±38.4
256±0.0
256±0.0
256±38.4
32±6.4
4
e
128±25.6
128±9.6
128±25.6
64±9.6
128±25.6
64±6.4
4
f
128±25.6
256±0.0
128±25.6
256±38.4
128±25.6
256±38.4
32±6.4
128±25.6
128±25.6
64±9.6
5
a
128±25.6
128±25.6
128±25.6
32±6.4
128±25.6
64±9.6
5
b
4±1.2
5
c
64±6.4
128±25.6
128±25.6
128±25.6
8±0.0
32±6.4
8±1.6
128±25.6
64±9.6
6
32±6.4
256±38.4
256±38.4
32±0.0
16±0.0
8±1.6
7
32±6.4
64±0.0
128±0.0
32±6.4
c)
c)
A
16±2.4
16±2.4
32±6.4
16±2.4
0.5±0.0
B
C
0.5±0.0
4±1.2
8±1.6
1±0.0
2±0.2
16±2.4
4±0.0
4±1.2
8±1.6
c)
2±0.0
2±0.8
4±0.0
1±0.2
2±0.0
0.5±0.0
2±0.0
a) Minimal inhibitory concentrations were determined by micro broth dilution method for microdilution plates, and the data are average of multiple replicates.
b) S. aureus, Staphylococcus aureus (ATCC 25923); MRSA, methicillin-resistant Staphylococcus aureus (N315); B. subtilis, Bacillus subtilis (ATCC 21216);
M. luteus, Micrococcus luteus (ATCC 4698); E. coli, Escherichia coli (JM109); E. typhosa, Eberthella typhosa (ATCC 14028); S. dysenteriae, Shigella dysente-
riae (ATCC 49550); P. aeruginosa, Pseudomonas aeruginosa (ATCC 27853); B. proteus, Bacillus proteus (ATCC 13315). c) A=Chloromycin; B=Norfloxacin;
C=Ciprofloxacin.

Gao et al. Sci China Chem
5
tional inhibitory concentration (FIC) index. The FIC value
can be calculated as FIC=MIC of compound A in mix-
ture/MIC of compound A alone+MIC of compound B in
mixture/MIC of compound B alone. Using this method,
FIC≤0.5 represents synergism; FIC>0.5 and ≤1.0 represents
additivism; FIC>1 and ≤2 represents an indifferent effect;
and FIC>2 represents antagonism.
different time intervals (0, 60, 90, 120, 150, 180, 210, 240,
300 and 360 min), 20 µL of aliquots from the solution were
taken out and serially diluted (10-fold serial dilution) in 0.9%
saline. Then 20 µL of the dilutions was plated on respective
agar plates and incubated at 37 °C for 24 h. The bacterial
colonies were counted, and results represented in logarithmic
scale: log10 (CFU/mL) vs. time (in min).
2.2.4 Resistance assays
2.2.6 Bacterial membrane permeabilization assays
Both strains of S. aureus and MRSA were exposed to
sub-MICs of compound 3f for sustained passages, and then
the MIC of compound 3f was determined against each pas-
sage of the strains. The freshly diluted S. aureus and MRSA
The 6 h grown culture (mid log phase) of MRSA and P. aerug-
inosa was harvested (3500 r/min, 5 min), washed, and resus-
pended in 5 mM glucose and 5 mM HEPES buffer (pH 7.2)
in 1:1 ratio. Then an amount of 10 μL of tested compound 3f
(12×MIC) was added to a cuvette containing 2 mL of bacterial
suspension and 10 μM propidium iodide (PI). Fluorescence
was monitored at excitation wavelength of 535 nm (slit width
of 10 nm) and emission wavelength of 617 nm (slit width of 5
nm). As a measure of inner membrane permeabilization, the
uptake of PI was monitored by the increase in fluorescence
for 2 h.
5
strains (1.0×10 CFU) in the broth medium were cultured in
1.3 μg/mL (2/3 MIC) of compound 3f at 37 °C for 12 h on
a shaker bed at 90 r/min, and the sensitivity of each strain
passage to compound 3f was tested. To make comparative
analysis, parallel cultures were exposed to 2-fold dilutions
with the reference drugs Chloromycin, Norfloxacin and
Ciprofloxacin as positive controls.
2
.2.5 Bactericidal kinetic assays
3
Results and discussion
The rate of which the compound killed bacteria was evaluated
by performing time-kill kinetics. MRSA was cultured in suit-
able medium at 37 °C for 6 h and diluted in respective media.
Compound 3f was added to the bacterial solution (MRSA of
approximately 1.8×10 CFU/mL) at concentration of 4×MIC
in a 96-well plate. The plate was then incubated at 37 °C. At
3
.1 Chemistry
A series of amino organophosphorus imidazoles were conve-
niently synthesized according to the synthetic route outlined
in Scheme 1. Alkyl imidazoles 2a and 2b were prepared in
5
Scheme 1 The synthetic route of amino organophosphorus imidazoles. Reagents and conditions are: i) diethyl phosphonate, aliphatic amines, toluene, reflux;
ii) diethyl phosphonate, phenyl amines, toluene, reflux; iii) diethyl phosphonate, 3-amino-2H-chromen-2-one, toluene, reflux; iv) conc. hydrochloric acid,
8
9
0 °C; v) conc. hydrochloric acid, 50 °C; vi) diethyl phosphonate, pyrimidin-2-amine, toluene, reflux; vii) methyl glycinate hydrochloride, Et
0 °C, pH 9–10.
3
N, 30 min,

6
Gao et al. Sci China Chem
yields of 50% and 49% from commercially available
-butyl-4-chloro-1H-imidazole-5-carbaldehyde, diethyl
of P–OCH
nals for P–OCH, P–OCH and P–OCH
2
were different, and there were three or four sig-
in δ 3.87–4.12 ppm,
2
2
phosphonate and aliphatic amines in toluene. Phenyl type
of imidazole compound 3 was obtained in 60%−80% yields
under the same reaction condition starting from a series
of anilines, and then further hydrolyzed in concentrated
hydrochloric acid to afford imidazole phosphonic acids 4 and
3.75–4.02 ppm and 4.16–4.37 ppm, respectively, due to the
adjacency of chiral carbon. The chemical shifts for the pro-
tons of chiral carbon of all the targets as double peaks in δ
4.16–5.06 ppm. The signals of the aromatic protons appeared
ranging from 6.36 ppm to 7.59 ppm. The aromatic protons
showed various multiplicities such as doublets due to several
5
with yields ranging from 40% to 60%. Pyrimidine-based
1
molecule 6 was obtained under the same reaction condi-
tion starting from 2-aminopyrimidine in the yield of 50%.
Coumarin derivative 7 was efficiently synthesized in good
yield by the Mannish reaction of 3-aminocoumarin which
was easily prepared from the one-pot cyclization of 2-hy-
droxyl benzaldehyde and methyl glycinate hydrochloride.
The HPLC analysis showed that almost all of the prepared
target compounds were pure, and the related HPLC spectra
were provided in the Supporting Information online.
couplings and the influence of F atom. The H resonances of
aliphatic groups such as CH
2
and CH were observed in the
3
region between 2.83 and 0.78 ppm mainly influenced by the
imidazole ring, because of the effect of electron-withdrawing
imidazole ring, the protons of CH
2
linked to 2-position gave
large downfield chemical shifts around δ 2.41–2.83 ppm.
1
3
3
.2.3 C NMR spectra
1
3
The C NMR spectra of all the target imidazole phospho-
nates and imidazole phosphonic acids were consistent with
1
3
the assigned structures. No large difference was found in C
chemical shifts for the chiral carbon and 2-, 4-, 5-position of
imidazole ring. The impalpable distinctions of some chemi-
cal shifts were mainly attributed to the substituent at differ-
ent positions of aromatic carbons and to the various amines.
Additionally, all the aliphatic carbons appeared at the appro-
priate chemical shift regions.
3
.2 Spectral analysis
All the newly synthesized target imidazol phosphonates and
imidazol phosphonic acids were characterized by infrared
spectroscopy (IR), nuclear magnetic resonance of H ( H
NMR), C NMR, P NMR and high resolution mass spec-
trometry (HRMS). The spectral analyses were in accordance
with the assigned structures, and all the spectral data were
listed in the experimental protocols. The HRMS for each new
1
1
13
31
3
1
3
.2.4 P NMR spectra
3
1
+
+
There was only one signal peak in each P NMR spectrum of
all the target compounds. For imidazole phosphonates 2, 3, 6
and 7, the chemical shifts were almost in δ 18.95–21.60 ppm,
but for imidazole phosphonic acids 4, δ 12.99–20.13 ppm,
and for compound 5, δ 16.23–18.17 ppm due to the adjacency
of hydroxyl group.
compounds gave a major fragment of [M+H] or [M+Na]
according to their molecular formula.
3
.2.1 IR spectra
Almost all the target compounds gave similar IR spectra and
there was no large difference for the characteristic absorption
peaks. Characteristic NH stretching bands were observed
between 3361 and 3302 cm . In addition, three sharp
and strong peaks at 2970–2957 cm , 2935–2931 cm and
−
1
3.3 Biological activity
−
1
−1
−
1
3.3.1 Antibacterial activity
2
875–2866 cm were ascribed to the vibration of aliphatic
C−H bonds. The stretching of P=O groups showed frequen-
The antibacterial assay showed that some target compounds
exhibited moderate to good efficacy against some tested
bacterial strains (Table 1). Noticeably, compound 3f with
3-chloro-4-fluorophenyl group was the most active molecule,
which deserved to be further investigated as potentially
antimicrobial agent.
−
1
cies around 1250–1218 cm . The characteristic absorption
peaks of aromatic frame for imidazole phosphonates 3, 6
and 7, imidazole phosphonic acids 4 and 5 appeared in the
−
1
region of 1650–1450 cm , and the broad absorption around
−
1
3
445–3394 cm was attributed to the P–OH group in com-
pounds 4 and 5. All the other absorption bands were also
observed at expected regions.
Table 1 showed that the phenyl series of compound 3
exhibited moderate to good activities against Gram-negative
bacteria P. aeruginosa with MIC values of 4–32 μg/mL,
except for 4-fluorophenyl derivative 3c (MIC=64 μg/mL)
and 3-trifluoromethylphenyl compound 3i (MIC=128
μg/mL). Compound 3a with 2-fluorophenyl moiety gave a
MIC value of 16 μg/mL against E. coli, which was supe-
rior to Chloromycin (MIC=32 μg/mL) and equipotent to
Norfloxacin (MIC=16 μg/mL). 3,4-Difluorophenyl deriva-
tive 3b showed good activity against MRSA with MIC
1
3
.2.2 H NMR spectra
1
In H NMR spectra, it was noticed that the protons of sec-
ondary amine appeared sometimes, Im-NH and Ph-NH ex-
isted as single peak in δ 11.07–11.47 ppm and δ 5.06–5.95
ppm, respectively. Most of the protons of hydroxyl group
in imidazole phosphonic acids 4 and 5 did not appear at all.
For imidazole phosphonates 2, 3, 6 and 7, the chemical shifts

Gao et al. Sci China Chem
7
value of 8 μg/mL, which was better than Chloromycin
tested bacterial strains, which might result from its decreased
liposolubility after hydrolysis. Alkyl-derived molecules
2a and 2b with ethyl and hydroxyethyl groups also exhib-
ited equipotent anti-S. aureus efficacy (MIC=16 μg/mL)
to Chloromycin, while pyrimidine derivative 6 with hete-
rocycle or coumarin one 7 with larger skeleton in contrast
with phenyl derivative 3 only displayed moderate activity
against P. aeruginosa with MIC value of 16 and 8 μg/mL,
respectively. More work is necessary to elucidate them on
antibacterial activity.
(MIC=16 μg/mL) and almost equipotent to Norfloxacin
(MIC=8 μg/mL). Compounds 3g and 3h with non-halo
substituted benzene rings only possessed acceptable anti-P.
aeruginosa activities with MIC values of 16 and 32 μg/mL,
respectively. Moreover, 3-chloro-4-fluorophenyl derivative
3f exhibited superior activity against Gram-negative strains
E. coli (MIC=16 μg/mL), S. dysenteriae (MIC=16 μg/mL),
P. aeruginosa (MIC=2 μg/mL) and B. proteus (MIC=16
μg/mL) to Chloromycin (MIC=32 μg/mL). It also pos-
sessed superior anti-MRSA (MIC=2 μg/mL) efficiency to
Chloromycin (MIC=16 μg/mL) and Norfloxacin (MIC=8
μg/mL), and almost equipotent to Ciprofloxacin (MIC=2
μg/mL). These implied that compound 3f exhibited moderate
to good efficacy with MIC values between 2 and 32 μg/mL,
and behaved large potentiality with a broad antibacterial
spectrum. However, the hydrolyzed imidazole phosphoric
acids 5a–5c exhibited decreased activities in comparison to
the corresponding imidazole phosphonates against the tested
bacterial strains, and they only showed moderate anti-P.
aeruginosa efficacy with MIC values of 4−8 μg/mL. The
corresponding phosphoric acid monoesters 4a–4f almost
possessed moderate antibacterial efficacies against all the
3
.3.2 Antifungal activity
The antifungal data in Table 2 revealed that all target com-
pounds displayed moderate inhibition activities against five
tested fungi. The antifungal abilities for the synthesized
compounds were similar to their antibacterial efficiencies.
It was obviously found that all target compounds possessed
anti-A. flavus activities with MIC values of 32–256 μg/mL,
which were equipotent or superior to Fluconazole (MIC=256
μg/mL). Compounds 3a and 3b exhibited equipotent efficien-
cies against C. utilis to Fluconazole (MIC=8 μg/mL), and 3b
gave 4-fold better anti-S. cerevisiae activity than Fluconazole
(MIC=16 μg/mL). Moreover, 3-chloro-4-fluorophenyl deriv-
a)
Table 2 In vitro antifungal data as MIC (μg/mL) for compounds 2–7
Compounds
C. albicans
256±38.4
128±25.6
64±9.6
C. mycoderma
64±6.4
C. utilis
16±2.4
A. flavus
128±25.6
128±25.6
32±6.4
S. cerevisiae
256±0.0
256±38.4
32±6.4
2
a
2
b
64±9.6
64±9.6
3
a
128±0.0
128±25.6
64±9.6
8±1.6
3
b
128±25.6
64±9.6
8±1.6
128±25.6
128±9.6
128±25.6
64±0.0
4±1.2
3
c
16±0.0
128±25.6
128±0.0
256±38.4
2±0.8
3
d
128±25.6
512±76.8
128±25.6
512±76.8
256±38.4
512±0.0
128±25.6
512±76.8
256±38.4
128±25.6
128±0.0
64±9.6
32±6.4
128±25.6
256±38.4
8±1.6
3
e
64±9.6
3
f
32±6.4
32±6.4
3
g
64±9.6
128±0.0
32±6.4
64±9.6
128±25.6
256±0.0
32±6.4
3
h
128±25.6
256±0.0
256±0.0
256±38.4
128±25.6
64±9.6
256±38.4
256±0.0
256±38.4
128±0.0
64±9.6
3
i
64±9.6
4
a
128±25.6
64±9.6
64±9.6
4
b
128±25.6
128±0.0
256±38.4
64±6.4
4
c
128±25.6
128±25.6
256±0.0
128±25.6
16±2.4
4
d
128±25.6
128±25.6
64±0.0
4
e
128±25.6
128±25.6
256±0.0
128±25.6
32±6.4
4
f
64±9.6
5
a
256±38.4
128±25.6
256±0.0
128±25.6
256±38.4
1±0.0
256±38.4
64±9.6
128±25.6
256±0.0
32±6.4
5
b
32±6.4
5
c
128±25.6
64±6.4
256±38.4
128±25.6
256±0.0
256±38.4
6
256±38.4
128±25.6
4±0.0
128±25.6
256±38.4
16±2.4
7
16±2.4
b)
D
8±1.6
a) C. albicans, Candida albicans (ATCC 76615); C. mycoderma, Candida mycoderma (ATCC 96918); C. utilis, Candida utilis; A. flavus, Aspergillus flavus;
S. cerevisiae, Saccharomyces cerevisiae (ATCC 9763); the data are average of multiple replicates. b) D=Fluconazole.

8
Gao et al. Sci China Chem
ative 3f exhibited significant inhibition activity against S.
cerevisiae with MIC value of 2 μg/mL, which was 8-fold
more potent than Fluconazole.
manifested that the combinations of compound 3f with
antibacterial drugs could enhance antibacterial activity, over-
come drug resistance and broaden antibacterial spectrum.
The combination of Fluconazole with compound 3f dis-
played good activities against fungi C. utilis, A. flavus and S.
cerevisia with the FIC index being no more than 1.0 (Table 4).
But the combination use of 3f with Fluconazole against C.
albicans and C. mycoderma produced the indifferent effect.
Moreover, these combinations gave high activities with less
dosage against Fluconazole-insensitive A. flavus. It clearly
showed that the combination of Fluconazole with 3f broad-
ened antifungal spectrum.
The above discussion implied that the antimicrobial effi-
cacies should be closely related to substituents on amines
to some extent. Phenyl derivatives seemed to be more ac-
tive than those of aliphatic chain, pyrimidine and coumarin
rings, halo-substituted phenyl groups resulted in more potent
against the tested strains than those of non-halo substituted
ones, and both the extremely strong electron withdrawing
groups such as nitro and trifluoromethyl ones and the elec-
tron donating groups like methoxyl one were unfavourable
to some extent. Phosphonates seemed to be better than their
hydrolyzed ones, it might be due to a relative decrease in li-
posolubility after hydrolysis.
3
.3.4 Resistance study
Resistance to antibiotics has been increasing in recent
years and becoming a serious and global challenge to the
chemotherapy or drug discovery. The mutagenic properties
of bacteria raise the possibility of the resistance even in
smaller populations of bacteria exposed to low concentra-
tion of antibacterial drugs, which may promote the ease of
selection of low-level resistance mutations [13]. Therefore,
it is significantly vital to study the resistance of the potent
compound against bacterial strains. To investigate the drug
resistance of compound 3f, the susceptible Gram-positive
pathogens S. aureus and MRSA, Gram-negative strain P.
aeruginosa were employed, and Chloromycin, Norfloxacin,
Ciprofloxacin as positive control. The experimental results
3.3.3 Drug combination study
Much research has shown that the combination therapy in
clinic with two or more agents might improve the efficiency
and bioavailability, reduce or even eliminate side effects and
allergic reactions and overcome multi-drug resistances as
well as treat mixed diseases which cannot be cured by single
drug [12]. The drug combination use between the most
active molecule 3f and clinical Chloromycin, Norfloxacin,
Ciprofloxacin and Fluconazole were investigated. Most of
the combinations of 3f with Chloromycin, Norfloxacin, and
Ciprofloxacin showed excellent antibacterial efficacy with
less dosage and broad antimicrobial spectrum (Table 3).
The FIC index was no more than 1.0, which meant that the
combinations had excellent synergistic or additive effects.
Except for the combination use of 3f with both Chloromycin
and Norfloxacin against B. subtilis, the FIC index was more
than 1.0, which represented an indifferent effect. It was
worthy to note that the combination of 3f with clinical drugs
could effectively inhibit the growth of MRSA. These results
(
Figure 4) showed that the low propensity of both Gram-posi-
tive and Gram-negative bacteria to develop resistance against
-chloro-4-fluorophenyl derivative 3f as there was almost no
or little change in the MIC values even after 12 passages.
3
3
.3.5 Bactericidal kinetic study
The bactericidal efficiency for the highly active compound 3f
was also evaluated against MRSA by time-kill kinetics exper-
a)
Table 3 Drug combinations of compound 3f with antibacterial drugs Chloromycin, Norfloxacin and Ciprofloxacin
Chloromycin
Norfloxacin
Ciprofloxacin
Effect
Compound
3f MIC
Bacteria
Compound 3f
MIC (μg/mL)
Compound 3f
MIC (μg/mL)
FIC
index
Effect
FIC index
Effect
FIC index
(μg/mL)
S. aureus
MRSA
0.25±0.04
0.12±0.02
2±0.8
additivism
additivism
indifference
synergism
synergism
synergism
synergism
synergism
additivism
0.531
0.562
1.062
0.187
0.187
0.500
0.250
0.125
0.625
0.25±0.04
0.062±0.01
1±0.3
additivism
synergism
indifference
synergism
synergism
additivism
additivism
synergism
synergism
0.531
0.281
1.031
0.187
0.156
0.625
0.625
0.125
0.156
0.25±0.04
synergism
0.094
0.039
0.156
0.094
0.312
0.047
0.281
0.249
0.094
0.062±0.01 synergism
B. subtilis
M. luteus
1±0.3
1±0.3
synergism
synergism
synergism
synergism
synergism
synergism
synergism
2±0.8
2±0.8
E. coli
1±0.3
0.5±0.2
4±1.2
1±0.3
E. typhosa
S. dysenteriae
P. aeruginosa
B. proteus
8±1.6
1±0.3
2±0.8
2±0.8
0.5±0.2
0.25±0.04
0.5±0.2
0.12±0.02
2±0.8
0.12±0.02
0.5±0.2
a) Average of multiple replicates.

Gao et al. Sci China Chem
9
a)
Table 4 Drug combinations of compound 3f with antifungal drug Fluconazole
Fungi
C. albicans
MIC (μg/mL)
Effect
FIC index
1.031
4±1.2
4±1.2
indifference
indifference
synergism
additivism
synergism
C. mycoderma
C. utilis
1.125
1±0.3
0.375
A. flavus
1±0.3
0.531
S. cerevisiae
0.25±0.04
0.375
a) Average of multiple replicates.
Figure 4 Evaluation of resistant development against compound 3f in bacterial strains S. aureus (ATCC 25923) (a), MRSA (N 315) (b) and P. aeruginosa
ATCC 27853) (c) (color online).
(
3
iment. Figure 5 revealed nearly 10 CFU/mL reduction in the
number of viable bacteria within 60 min at a concentration of
4-times MIC. The result manifested that 3f had rapidly killing
effect against MRSA [14].
3.3.6 Bacterial membrane permeabilization study
Bacterial membrane has been considered as an important and
intriguing antibacterial target, and membrane-active nature
induces low propensity for bacteria to develop resistance.
Therefore, it is reasonable to speculate that antibacterial
agents would have satisfactory efficacy and low drug-resis-
tance if they target on or interact with cell membrane. Herein,
the most active antimicrobial compound 3f was chosen to
investigate its membrane permeabilization against MRSA
and P. aeruginosa bacteria by fluorescence spectra using
propidium iodide (PI), a common dye that only can pass
through the membrane of compromised cells and fluoresces
Figure 5 Bactericidal kinetics of compound 3f at 4× MIC against MRSA
color online).
(

10
Gao et al. Sci China Chem
upon binding to the DNA [15]. For the increase of fluores-
cence intensity within 90 min in Figure 6, which might be
due to the formed PI-DNA complexes as the gradual dam-
age of bacterial membrane in the presence of 3f at 12-times
MIC. Also, the number of viable bacteria was nearly de-
creased by 6 log CFU/mL within 150 min at a concentration
of 12-times MIC (Figure 7). These results manifested that
compound 3f could effectively interact with the membranes
of both Gram-positive (MRSA) and Gram-negative (P. aerug-
inosa) bacteria.
These meant that there was no remarkable intercalation to-
wards calf thymus DNA or very weak interaction between 3f
and calf thymus DNA. The further investigation like its test
toward MRSA DNA cleavage is necessary to explore the pos-
sible antimicrobial action mechanism.
3
.5 Partition coefficient and computational analysis
The partition coefficient is used to define the lipophilic char-
acter of a drug, and the suitable values would make contri-
bution to ideal pharmacokinetic and pharmacodynamic prop-
erties of drugs. In general, most of the target compounds
3.4 Interactions with calf thymus DNA
(Figure 8) were lipophilic (clogP=1.33–5.17). The clogP
In order to explore the possible antimicrobial action mecha-
nism, the binding behavior of compound 3f with calf thymus
DNA was studied on molecular level in vitro using NR dye
as a spectral probe by UV-Vis spectroscopic methods (Figure
S1 and Figure S2, Supporting Information online). However,
the maximum absorption peak of DNA at 260 nm exhibited
no noticeable increase with the increasing concentration of
compound 3f. Besides, the absorption spectra of competitive
interactions of 3f and NR with DNA showed that the maxi-
mum absorption around 530 nm of the DNA-NR complexes
did not decrease with the addition of 3f, and no intensity in-
crease was observed in the developing band around 460 nm.
values of phenyl series of compounds 3a–3i were related to
types of substituents on the benzene rings. The chloro group
containing derivatives generally showed greater lipophilicity
than fluoro, nitro or methoxyl group containing ones. More-
over, alkyl imidazoles 2, imidazole phosphonic acids 4 and
5, and pyrimidine-based molecule 6 possessed lower clogP
values than phenyl imidazoles 3, except for 4b and 4f. Com-
pound 2b with a hydroxyethyl group had the lowest clogP
value. Molecules 3b, 3d and 3f with relatively higher clogP
values showed better antibacterial and antifungal activities in
comparison with other target compounds. These might be re-
sponsible for the possibility that higher lipophilic compounds
Figure 6 Bacterial membrane permeabilization of compound 3f at concentrations of 12× MIC. (a) Against MRSA; (b) against P. aeruginosa (color online).
Figure 7 The growth curves of MRSA (a) and P. aeruginosa (b) in the presence of compound 3f at 12-times MIC (color online).

Gao et al. Sci China Chem
11
the physicochemical properties. In addition, the LUMOs
of 3f and 3b were mainly located in the halo-substituted
benzene ring which did good nucleophilic attack.
The molecular electrostatic potential (MEP) surface gives
an indication of the charged surface area, an idea for explain-
ing the hydrophilicity of compounds and the orientation of
drug candidates for their activities [16]. Thus, MEP maps
were given to investigate the similarities and differences in
electrostatic binding characteristic of the surfaces of the most
active antimicrobial 3f and the less active one 3b (Table 5).
Comparing the MEP surfaces of 3f and 3b revealed that both
of them had almost the same electropositive regions (in blue)
but different electronegative regions (in red). The electroneg-
ative region mainly located on one P–O and the P=O groups in
compound 3b, while the electronegative region of 3f moved
towards the imidazole ring, which might be influenced by the
chlorine atom on the benzene ring. This moving would be
in favor of orientating drug candidate 3f towards better bio-
logical activity, which indicated that the possible formation
of hydrogen bonds with a share of N1-position of imidazole
ring and the oxygen atom of P–O or P=O group. Therefore, it
reasonably explained that compound 3f possessed better bio-
logical activity than its analog 3b.
Figure 8 clogP values of compounds 2a–2b, 3a–3i, 4a–4f, 5a–5c, 6, and 7
color online).
(
were easy to be delivered to the binding sites.
Computational methods are usually used to predict im-
portant pharmacokinetic properties of drug candidates and
to rationalize biological activity. The frontier molecular
orbitals (FMO) theory, that is the highest occupied molecular
orbital (HOMO) and the lowest unoccupied molecular orbital
(LUMO) can always dominate the intermolecular interac-
3
.6 Interactions of compound 3f with HSA
tions. As shown in Table 5, the plots of HOMOs and LUMOs
of the most active compound 3f and its structurally similar
compound 3b were obtained to analyze the main atomic
contributions for these orbitals. The HOMOs of 3f and 3b
were mainly focused on imidazole ring, halo-substituted
benzene ring, chiral carbon, phosphoryl (P=O) and P–O
groups, which indicated that those rings and groups might
be active sites and the intermolecular interactions could take
place between these sites and positively charged molecules.
It was also found that the n-Bu on imidazole ring and ethyl
groups of phosphonates did not contribute directly to the
HOMO, manifesting that these groups might mainly adjust
HSA is the principal extracellular protein in the circulatory
system, and the distribution, metabolism, and efficacy of
drugs can be changed by their affinity to HSA. Many promis-
ing new drugs have been rendered to be ineffective because
of their unusually high affinity to this protein. So the in-
vestigation of interactions between drugs or bioactive small
molecules and HSA is not only significant to design, modify
and screen drug molecules but also helpful to understand the
pharmacokinetic properties of drugs. Herein, highly active
molecule 3f-albumin binding was investigated by UV-Vis
absorption and fluorescence spectra on the molecular level to
Table 5 Plots of the HOMOs, LUMOs and MEP maps of compounds 3f and 3b (color online)
Compounds Simulation structures
HOMO
LUMO
MEPs
3
f
3
b

12
Gao et al. Sci China Chem
preliminarily study the absorption, distribution, and metabo-
lism.
3.6.1 Absorption spectra of HSA in the presence of com-
pound 3f
The UV-Vis absorption peak at 278 nm should be at-
tributed to the aromatic rings in Tryptophan (Trp-214),
Tyrosine (Tyr-411) and Phenylalanine (Phe) residues in
HSA (Figure 9). With the addition of compound 3f, the
peak intensity increased, indicating that 3f could interact
with HSA and the peptide strands of HSA were extended.
However, the maximum absorption wavelength remained
unchanged, implying that the noncovalent interactions were
formed between 3f and HSA via the π-π stacking between
aromatic rings of 3f and Trp-214, Tyr-411 and Phe residues
in the binding cavity of HSA [17].
Figure 10 Emission spectra of HSA in the presence of various concen-
trations of compound 3f. c(HSA)=1.0×10 mol/L; c(compound 3f) (×10
mol/L): (a–j) from 0.0 to 2.25 at increments of 0.25. Red line shows the
emission spectrum of compound 3f only. T=298 K, λex=295 nm (color on-
line).
−
5
−5
3.6.2 Fluorescence quenching mechanism with HSA
Fluorescence competition is another approach to explore the
binding behaviors of small molecules with HSA. Trp 214
in HSA is a dominant fluorophore capable of fluorescence
quenching that absorbs near 280 nm and emits near 340 nm.
The emission may be blue-shifted if the group is buried within
a native protein, and its emission may be red-shifted when
the protein is unfolded. Hence, changes in the fluorescence
intensity can reflect the interaction of small molecules with
Trp-214 in HSA.
to the single Trp-214 residue, exhibiting a regular decrease as
the concentration of compound increased, but the maximum
emission wavelength of HSA remained unchanged. This sug-
gested that the complexes between 3f and HSA were formed,
and compound 3f was likely to interact with HSA via the hy-
drophobic region located in HSA [18]. According to the fol-
lowing well-known Stern-Volmer Eq. (1), the fluorescence
quenching data of HSA could be analyzed [20]:
The effect of compound 3f on the fluorescence intensity to
HSA (T=298 K, λex=295 nm) was shown in Figure 10. The red
solid line was the only emission spectrum of the active small
molecule 3f, which indicated that its fluorescence intensity
was very weak and could be negligible in comparison with
the fluorescence of HSA at the excitation wavelength. The
maximum emission peak of HSA appeared at 348 nm owing
F0
=
1 + K [Q] = K 0[Q]
(1)
SV
q
F
where F and F represent fluorescence intensity in the absence
and presence of compound 3f, respectively. KSV (L/mol) is the
Stern-Volmer quenching constant, [Q] is the concentration of
compound 3f, K
L/(mol s)), and τ
0
q
is the bimolecular quenching rate constant
is the fluorescence lifetime of the fluo-
(
0
−
9
rophore in the absence of quencher, assumed to be 6.4×10 s
for HSA. Hence, the Stern-Volmer plots of HSA in the pres-
ence of compound 3f at different concentrations and temper-
atures could be calculated and were showed in Figure 11.
Fluorescence quenching occurs by different mechanisms,
commonly classified as dynamic quenching and static
quenching depending on temperature and viscosity. Higher
temperatures usually result in larger diffusion coefficients,
and the quenching constants are expected to increase with
a gradually increasing temperature in dynamic quenching.
However, due to the dissociation of weakly bound com-
plexes, the increasing temperature is likely to result in a
smaller static quenching constant.
The values of KSV and K
f with HSA at different temperatures (298, 303, and 310
K) were showed in Table 6. The results indicated that the
ground-state complexes were formed between compound 3f
and HSA, and the static quenching governed the quenching
q
for the interaction of compound
Figure
c(HSA)=1.0×10 mol/L; c(compound 3f) (×10 mol/L): (a–k) from 0.0 to
.33 at the increment of 0.33 (T=298 K, pH 7.4). The inset corresponds
to the absorbance at 278 nm with different concentrations of compound
f (color online).
9 Effect of compound 3f on HSA UV-Vis absorption.
3
−
5
−5
3
3

Gao et al. Sci China Chem
13
Figure 11 Stern-Volmer plots of 3f-HSA system at different temperatures
color online).
Figure 12 Modified Stern-Volmer plots of 3f-HSA system at different tem-
peratures (color online).
(
Table 6 Stern-Volmer quenching constants for the interaction of compound
f with HSA at various temperatures
The equilibrium binding constant (K ) and the number of
b
3
binding sites (n): can be calculated according to the Scatchard
Eq. (3):
a)
b)
pH
T (K)
98
303
10
K
SV (L/mol)
K
q
(L/(mol s))
R
S.D.
4
12
2
4.16×10
3.84×10
3.57×10
6.50×10
6.00×10
5.58×10
0.9995
0.9992
0.9993
0.010
0.115
0.010
4
4
12
12
7
.4
r
Df
=
nKb
rKb
(3)
3
a) R is the correlation coefficient; b) S.D. is the standard deviation.
where D
f
and r are the molar concentration of free small
molecules and the moles of small molecules bound per mole
of protein, respectively, n is binding sites multiplicity per
mechanism, because the K values at different temperatures
q
12
were in the range of 10 L/(mol s), which far exceeded the
diffusion controlled rate constants of various quenchers with
class of binding sites, and K
stant. Figure 13 showed the Scatchard plots, and the binding
constants K and site n values of the compound 3f-HSA sys-
b
is the equilibrium binding con-
10
a biopolymer (2.0×10 L/(mol s)) [19].
b
3.6.3 Binding sites and constants
tem were listed in Table 7.
For a static quenching process, the equilibrium binding
For a static quenching process, the data could be analyzed by
the modified Stern-Volmer Eq. (2):
constant (K
b
) and the number of binding site (n) were also
and K with
calculated (Table 7). The decreased trend of K
a
b
F0
F
1
1
1
=
+
(2)
increased temperatures was in accordance with the depen-
dence of KSV on temperatures. The value of the binding site
n was approximate 1, which showed that one binding site
was present in the interaction of 3f with HSA. The results
also showed that the binding constants were suitable and the
effects of temperatures were not significant, thus compound
3f could be stored and transported by HSA.
f K [Q] fa
a
a
where ΔF is the fluorescence difference of HSA in the
presence and absence of compound 3f, and f and K are
a
a
the fraction of accessible fluorescence and the effective
quenching constant for the accessible fluorophore, respec-
tively. The dependence of F
0
/ΔF on the reciprocal value
−
1
of the concentration [Q] , which is linear with the slope
−
1
3.6.4 Binding mode and thermodynamic parameters
equaling to the (f
a
K
a
)
value. The modified Stern-Volmer
plots were showed in Figure 12 and the calculated results
were depicted in Table 7.
It is well-known that there are four types of non-covalent in-
teractions such as hydrogen bonds, electrostatic interactions,
Table 7 Binding constants and sites of 3f-HSA system at pH 7.4
Modified Stern-Volmer method
Scatchard method
T (K)
−
4
−4
1
0
K
a
(L/mol)
R
S.D.
0.091
0.257
0.132
10
K
b
(L/mol)
R
S.D.
0.005
0.020
0.031
n
2
3
3
98
03
10
3.25
2.26
1.91
0.9996
0.9976
0.9995
3.31
2.27
2.01
0.9965
0.9976
0.9954
1.28
1.39
1.41

14
Gao et al. Sci China Chem
Figure 13 Scatchard plots of 3f-HSA system at different temperatures
color online).
Figure 14 van’t Hoff plots of the 3f-HSA system (color online).
(
van der Waals forces and hydrophobic bonds, which play im-
portant roles in small molecules binding to proteins. The
main evidence for confirming the interactions between small
molecules and protein is the thermodynamic parameters en-
thalpy (ΔH) and entropy (ΔS) change of binding reaction. The
ΔH and ΔS values can be evaluated from the van’t Hoff equa-
tion:
Table 8 Thermodynamic parameters of 3f-HSA system at different temper-
atures
T (K)
ΔH (kJ/mol)
ΔG (kJ/mol)
–24.98
ΔS (J/(mol K))
2
3
3
98
03
10
–14.69
–25.15
34.53
–25.39
H
RT
S
R
lnK =
+
(4)
tensity decreased regularly without no significant change in
wavelength with the addition of compound 3f, indicating that
the interactions between 3f and HSA did not affect the con-
formation of Tyr-411 micro-region. While there was a blue
shift of 4 nm in the maximum emission wavelength (from
where K and R are analogous to the associative binding
constants at the corresponding temperature and gas constant,
respectively. The thermodynamic parameters were calcu-
lated from the van’t Hoff plots in order to explain the binding
model between compound 3f and HSA. The ΔH was esti-
mated according to the slope of the van’t Hoff relationship in
Figure 14. Then, the free energy change (ΔG) was calculated
from the following equation:
2
85 nm to 281 nm) with gradual decrease in synchronous flu-
orescence intensity of Trp-214, this indicated that the confor-
mation of HSA was changed and the polarity in the vicinity
of Trp-214 residue decreased, leading to the increase of hy-
drophobicity [20]. Furthermore, as the gradually increased
concentration of 3f, there was a notable decrease in fluores-
cence intensity at Δλ=60 nm while no appreciable decrease
was observed at Δλ=15 nm (Figure 15(C)), which also ex-
plained reasonably that compound 3f had probability to cause
conformational changes close to Trp-214 residue rather than
Tyr-411.
G = H T S
(5)
The main evidence to confirm the interactions between
small molecules and protein is the thermodynamic param-
eters enthalpy (ΔH) and entropy (ΔS) change of binding
reaction [19]. Herein, ΔH<0 and ΔS>0 obtained in this case
indicated that the hydrophobic interactions and hydrogen
bonds played important roles in the binding process of
compound 3f and HSA (Table 8).
3
.6.6 Energy transfer between HSA and compound 3f
Förster resonance energy transfer (FRET) theory is typically
used to describe energy transfer between donor and acceptor
chromophores. According to FRET theory, an excited donor
fluorophore can transfer the excited state energy to a nearby
acceptor [21]. The non-radioactive energy transfer between
drugs and HSA can be explained and determined according to
FRET theory. The efficiency of energy transfer is dependent
on the extent of overlap between the fluorescence emission
spectrum of a donor and the absorption spectrum of an accep-
tor, the relative angular orientation and the distance between
3
.6.5 Synchronous fluorescence spectra
Synchronous fluorescence spectroscopy, a simple and effec-
tive technique, was used to explore the molecular microenvi-
ronment in the vicinity of the fluorophore in HSA (Figure 15).
When the wavelength interval (Δλ) between excitation and
emission wavelength was fixed at Δλ=15 nm, the synchronous
fluorescence would only exhibit the characteristic spectrum
of Tyr-411 residue. If at Δλ=60 nm, it would yield the spectral
information of Trp-214 residue. The Tyr-411 fluorescence in-

Gao et al. Sci China Chem
15
−
5
Figure 15 Synchronous fluorescence spectra of HSA in the presence of increasing concentration of 3f. (A) Δλ=15 nm. (B) Δλ=60 nm.c(HSA)=1.0×10
mol/L; c(compound 3f) (10 mol/L): (a–k) from 0.0 to 2.25 at increments of 0.25. (C) Variation (quenching) curves of relative (F/F
intensity of HSA as a function of increasing concentration of 3f (color online).
−
5
0
) synchronous fluorescence
donor and acceptor. From the above results, it was estab-
lished that compound 3f had probability to cause conforma-
tional changes close to Trp-214 residue, which confirmed that
namely that the distance between the moieties should be
much larger than the size of the individual donor or acceptor
dipoles. The efficiency of energy transfer depends on the
distance between donor and acceptor, their spectral charac-
teristics, and their relative orientation. In order for transfer to
be efficient, the donor and acceptor absorption must be well
separated, while the donor emission spectrum must overlap
with the absorption of the acceptor. The efficiency of energy
E can be evaluated according to Eq. (6):
3f mainly interacted with Trp-214 residue of HSA by non-
radioactive energy transfer since there was a suitable spec-
tral overlap between HSA emission and 3f absorption spectra
(Figure 16).
FRET theory is based on a point dipole approximation,
6
0
F
F0
R
E = 1
=
(6)
6
6
0
R + r
0
where R
efficiency of energy transfer is 50%, and r
between the donor (HSA) and the acceptor (compound 3f).
F and F are the fluorescence intensity of HSA in the pres-
ence and absence of compound 3f, respectively. R can be
0
is the Förster distance (critical distance) when the
0
is the distance
0
0
calculated from HSA emission and compound 3f absorption
spectra using Eq. (7):
6
0
25
2
4
R = 8.8 × 10 K n
J
(7)
2
where K is the orientation factor related to the geometry of
the HSA-3f dipole, n is the refractive index of the medium, φ
is the fluorescence quantum yield of HSA, and J is the spectral
overlap integral between the HSA emission spectrum and the
Figure 16 The overlap of fluorescence spectrum of HSA (a) and absorp-
tion spectrum of compound 3f (b). c(HSA)=c(compound 3f)=1.0×10
mol/L;T=298 K, pH 7.4 (color online).
−
5

16
Gao et al. Sci China Chem
Table 9 Effect of metal ions on the binding constants of 3f-HSA complexes at 298 K
−
4
0
a)
b)
c)
Systems
f-HSA
10
K
a
(L/mol)
K
a
/K
a
R
S.D.
3
3.25
6.33
6.44
5.77
5.62
7.02
5.63
7.12
7.46
1.00
1.95
1.98
1.77
1.73
2.16
1.73
2.19
2.30
0.9996
0.9999
0.9999
0.9999
0.9999
0.9998
0.9999
0.9998
0.9998
0.091
0.013
0.031
0.037
0.032
0.047
0.033
0.038
0.038
+
3
f-HSA-K
+
3
f-HSA-Na
2
+
3
f-HSA-Mg
2
+
3
f-HSA-Ni
2
+
3
f-HSA-Zn
f-HSA-Ca
2
+
3
2
+
3
f-HSA-Cu
+
3
f-HSA-Ag
0
a a
a) K is the binding constant of 3f-HSA complexes in the absence of metal ions, K is the binding constants of 3f-HSA complexes with metal ions; b) R is
the correlation coefficient; c) S.D. is standard deviation.
compound 3f absorbance spectrum, which was calculated ac-
cording to Eq. (8). For HSA, K , φ, and n were taken as 2/3,
of free compound 3f and shorten its storage time and half-life
in the blood, thus improve the maximum efficacy [24], or
possibly produce 3f-ion supramolecular complexes to exhibit
biological activities.
2
0.118, and 1.33, respectively.
4
F( ) ( )
0
J =
(8)
F( )
4 Conclusions
0
where F(λ) is the fluorescence intensity of HSA at wavelength
λ, ε(λ) is the molar absorption coefficient of compound 3f at
wavelength λ.
In summary, a series of amino organophosphorus imidazoles
as a novel type of antimicrobial agents have been success-
fully developed for the first time. The in vitro antimicrobial
activities revealed that 3-chloro-4-fluorophenyl derivative 3f
showed superior activities against MRSA and S. cerevisiae
(MIC=2 μg/mL) to clinical drugs, and its combinations with
antibacterial or antifungal drugs enhanced the antimicrobial
efficiency. Compound 3f exhibited low resistant develop-
ment for bacteria and was membrane-active, but had no sig-
nificant intercalation towards MRSA DNA. Computational
study of 3f supported its good antimicrobial activity. Ex-
perimental data revealed that HSA could form ground-state
complexes with 3f through hydrophobic interactions and hy-
drogen bonds with the spontaneous binding process, and the
non-radioactive energy could transfer from HSA to 3f be-
yond FRET theory. Competitive interactions suggested that
According to the above equations, it could be calculated
13
3
the spectral overlap integral J=1.01×10 cm L/mol, the effi-
ciency of energy E=0.27, the Förster distance R =2.62 nm and
the distance between HSA and 3f r =2.96 nm, suggesting that
the non-radioactive energy transfer from HSA to 3f occurred
with high possibility based on the prerequisite of 2<r <8 nm
22]. Furthermore, the acquisition of 0.5R <r <1.5R indi-
0
0
0
[
0
0
0
cated that the existence of static quenching due to complex
formation between HSA (Trp-214) and compound 3f [23].
3.6.7 Effect of common metal ions
HSA plays a key role in the transport of metal ions in blood
plasma. It is reported that metal ions can form complexes
with HSA and then influence drug binding to HSA. There-
+
+
2+
2+
2+
2+
2+
the participation of K , Na , Mg , Cu , Ca , Zn , Ni and
+
+
2+
2+
fore, the effect of the eight metal ions Na , K , Mg , Cu ,
+
Ag ions in 3f-HSA system could increase the concentration
2+
2+
2+
+
Ca , Zn , Ni and Ag ions on the binding of compound
f to HSA was studied at 298 K (Figure S5–Figure S12). It
of free compound 3f, shorten its storage time and half-life in
the blood to improve the maximum antimicrobial efficacy, or
possibly produce 3f-ion supramolecular complexes to exhibit
biological activities.
3
was worthy to note that the competition between the metal
ions and 3f led to the binding constants of HSA-3f complexes
ranging from 172.9% to 229.5% of the value of binding con-
stant in absence of metal ions (Table 9).
The altered binding constants might be explained by the
conformational changes of HSA when bound to metal ions, or
the formation of supramolecular interactions between metal
ions and 3f, which in turn affected HSA binding to the agent.
Clearly, the presence of the above metal ions increased the
binding constants of 3f-HSA-ions complexes, suggesting the
presence of these metal ions could increase the concentration
Acknowledgments This work was partially supported by the National
Natural Science Foundation of China (21672173, 21372186), Research Fund
for International Young Scientists from International (Regional) Coopera-
tion and Exchange Program (81350110523), Chongqing Special Foundation
for Postdoctoral Research Proposal (Xm2014127, Xm2016039), and Fun-
damental Research Funds for the Central Universities (XDJK2016E059).
Conflict of interest
The authors declare that they have no conflict of
interest.

Gao et al. Sci China Chem
17
Supporting information The supporting information is available online
at http://chem.scichina.com and http://link.springer.com/journal/11426.
The supporting materials are published as submitted, without typesetting
or editing. The responsibility for scientific accuracy and content remains
entirely with the authors.
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