Synthesis and biological evaluation of a new class of quinazolinone azoles as potential antimicrobial agents and their interactions with calf thymus DNA and human serum albumin

Article abstract of DOI:10.1039/c4md00281d

A series of novel quinazolinone azoles were synthesized and characterized by NMR, IR, MS and HRMS spectra. Bioactive assays showed that some target compounds exhibited significant antimicrobial potency. Especially, nitroimidazole derivative 3a displayed comparable or even superior antibacterial efficacies (MIC = 0.03-0.05 μmol mL^-1) in contrast with norfloxacin (MIC = 0.01-0.05 μmol mL^-1) and chloromycin (MIC = 0.02-0.10 μmol mL^-1). The preliminary interactive investigations of compound 3a with calf thymus DNA by UV-vis spectroscopy revealed that compound 3a could bind to DNA to form compound 3a-DNA complex by an intercalative mode and further block DNA replication to exert powerful antibacterial and antifungal activities. Hydrogen bonds and van der Waals forces played important roles in the association of compound 3a-HSA.

Full text of DOI:10.1039/c4md00281d

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Medicinal Chemistry Communications
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ARTICLE TYPE
Synthesis and biological evaluation of a new class of quinazolinone
azoles as potential antimicrobial agents and their interactions with calf
thymus DNA and human serum albumin
†
‡
Li-Ping Peng, Sangaraiah Nagarajan , Syed Rasheed , Cheng-He Zhou*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
A series of novel quinazolinone azoles were synthesized and characterized by NMR, IR, MS and HRMS
spectra. Bioactive assay showed that some target compounds exhibited significant antimicrobial potency.
Especially, nitroimidazole derivative 3a displayed comparable or even better antibacterial efficacies (MIC
= 0.03–0.05 µmol/mL) in contrast with norfloxacin (MIC = 0.01–0.05 µmol/mL) and chloromycin (MIC
1
0
=
0.02–0.10 µmol/mL). The preliminary interactive investigations of compound 3a with calf thymus
DNA by UVꢀvis spectroscopic method revealed that compound 3a could bind to DNA to form compound
aꢀDNA complex by an intercalative mode and further block DNA replication to exert their powerful
3
antibacterial and antifungal activities. The hydrogen bonds and van der Waals forces played important
1
5
roles in the association of compound 3aꢀHSA.
5
1
. Introduction
the related research has become increasingly active.
Quinazolinones have been found to possess extensively
Quinolones are a well known class of synthetic antimicrobial
drugs with excellent safety profile, favorable pharmacokinetic
characteristics and broad antibacterial spectrum. It is generally
considered that quinolones can bind with the enzymeꢀDNA
binary complex to form ternary complexes, thereby blocking
DNA replication and leading to bacterial cells’ death. Since the
first generation of quinolone drugs was found to be active in the
early 1960s, four generations of quinolones have been clinically
used in succession, such as nalidixic acid, pipemidic acid,
lomefloxacin, moxifloxacin and so on.
prevalently clinical use of this class of antiꢀinfective drugs has led
to increasingly worrisome resistance at a disturbing rate.
Furthermore, their shortcomings including phototoxicity, lipid
peroxidation, photohemolysis and bad waterꢀsolubility also limit
the administrable mode. This situation stimulates the urgent
need to design and develop powerful antimicrobial quinolone
agents, especially for the exploration of new quinoloneꢀlike
compounds. Quinazolinones with benzopyrimidone skeleton are a
type of important compounds structurally similar to clinical
benzopyridone quinolone drugs. Reasonably, a great deal of work
has recently been directed towards quinazolinone compounds and
5
6
7
4
4
5
5
6
6
0
5
0
5
0
5
biological activities such as antibacterial, antifungal, antiviral,
8
9
10
11
antiinflammatory,
antimalarial,
anticancer properties
antioxidant,
12
13
antimycobacterial,
and so on.
2
2
3
3
0
5
0
5
Numerous efforts have been devoted to the research and
development of quinazolinones as potential medicinal drugs by
the separation and purification of naturally occurring alkaloids
from a variety of plants as well as artificial synthesis of
quinazolinone compounds with novel structures and properties.
So far some quinazolinones, for example, methaqualone as a
hypnotic and anticonvulsant agent and quinethazone as a diuretic
1
Nevertheless, the
5a
drug have been used in clinic. More importantly, an increasing
number of quinazolinone compounds have displayed great
2
5,14
potency in the treatment of microbial infections.
15
Azole compounds such as monoꢀnitrogen thiazoles, oxazoles
bisꢀnitrogen imidazoles
benzimidazoles, triꢀnitrogen triazoles and benzotriazoles,
3
16
17
18
and carbazoles,
and
19
20
21
4
22
and tetraꢀnitrogen tetrazoles are an important class of nitrogen
heterocycles with multiple heteroatoms, aromaticity and electron
rich property. Their special structure endows azoleꢀbased
derivatives easily bind with the enzymes and receptors in
organisms through noncovalent interactions such as coordination
and hydrogen bonds, thereby possessing various applications in
23
medicinal chemistry, especially their protrudent effects such as
4
a,18,24
4b,20,25
imidazoles
and triazoles
against microbial strains. In
Institute of Bioorganic & Medicinal Chemistry, School of Chemistry and
Chemical Engineering, Southwest University, Chongqing 400715, China
*
recent years, some work has manifested that the introduction of
some nitrogenꢀcontaining heterocyclic moieties such as pyrazole
and thiazole into quinazolinone backbone can significantly
increase the antimicrobial efficiency and broaden their
However, to the best of our
knowledge, other quinazolinone azoles have been seldom
Corresponding authors; Eꢀmail: zhouch@swu.edu.cn (C. H. Zhou);
Tel.: +86ꢀ23ꢀ68254967; Fax: +86ꢀ23ꢀ68254967
†
Postdoctoral fellow from School of Chemistry, Madurai Kamaraj
University, India
5,26
antimicrobial spectrum.
‡
Postdoctoral fellow from Department of Chemistry, Hyderabad
University, India
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1

Medicinal Chemistry Communications
Page 2 of 7
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DOI: 10.1039/C4MD00281D
reported. In view of the above observations and as an extension
The target quinazolinone azoles were synthesized according to
of our studies on the development of azole compounds, herein we 35 the synthetic route outlined in Scheme 1. Commercially available
incorporated different azole fragments into the Nꢀ3 position of
quinazolinone to generate a novel class of quinazolinone azoles.
Research provides evidence that alkyl linkers can modulate the
physicochemical properties and thus improve biological potency.
With the aim of better understanding of structureꢀactivity 40 were further treated with a series of alkyl dibromides in acetone
relationship and increasing flexibilities, different lengths of alkyl
chains were introduced into the target compounds to investigate
the influences of linkers on bioactive profiles. The prepared
compounds might be expected to exhibit potential activities
fluoro or chloro substituted 2ꢀaminobenzoic acid was reacted
with formamidine acetate in the presence of 2ꢀmethoxyethanol to
produce 7ꢀfluoroquinazolinꢀ4(3H)ꢀone or 7ꢀchloroquinazolinꢀ
5
0
5
0
5
0
4(3H)ꢀone 1a-b in high yield of 80%, and then compounds 1a-b
2
7
using potassium carbonate as base to afford bromides 2a-j with
the yields of 50–76% according to the previously reported
31
1
1
2
2
3
method. The target quinazolinone azoles 3a-j, 4a-j and 5a-d
were conveniently and efficiently obtained in 50–67% yields by
against bacterial and fungal strains including drugꢀresistant 45 the reaction of bromides 2a-j respectively with 2ꢀmethylꢀ5ꢀ
microorganisms. Therefore, their antibacterial and antifungal
potency for all newly synthesized compounds were evaluated in
vitro against four Gramꢀpositive bacteria including Methicillinꢀ
Resistant Staphylococcus aureus N315 (MRSA), four Gramꢀ
nitroimidazole, 4ꢀnitroimidazole, imidazole or triazole in
acetonitrile at 50 ºC in the presence of potassium carbonate as
base. Unfortunately, under the same condition above, imidazoles
5a-b were obtained in relatively low yields, which demonstrated
negative bacteria and five fungi. The preliminary antimicrobial 50 that potassium carbonate was unfavorable for this reaction,
mechanism was investigated by evaluating the interactions of the
probably its basicity was too weak to form imidazole carbanion.
Therefore, sodium hydride was selected as base in the reaction of
imidazole with bromides, which easily afforded the imidazole
derivatives 5a-b with good yields ranging from 58% to 69% at 50
28
prepared highly active compound with calf thymus DNA. It is
well accepted that the overall distribution, metabolism, and
efficacies of drugs can be changed by their affinity to human
serum albumin (HSA), and many promising new drugs have been 55 ºC in anhydrous tetrahydrofuran under a stream of nitrogen. All
1
13
rendered ineffective because of their unusually high affinity to
the new compounds were confirmed by H NMR, C NMR, IR,
MS and HRMS spectra (Supporting Information 1).
2
9
this protein. So the full investigations of interactions between
drugs or bioactive small molecules and HSA not only provide a
proper understanding of the absorption, transportation,
distribution, metabolism and excretion properties of drugs, but
3. Results and discussion
3
.1. Antimicrobial activities
30
also are helpful to design, modify and screen drug molecules.
Thus, further binding behaviors between the synthesized active 60 The obtained results (Supporting Information 2) as depicted in
compound and HSA were investigated to evaluate their
transportation and pharmacokinetic properties by fluorescence
and UVꢀvis absorption spectroscopy.
Table 1 revealed that quinazolinone nitroimidazoles 3a-j could
effectively inhibit the growth of all the tested bacterial strains
except for P. aeruginosa, while triazole compounds 5c-d showed
potent activity against M. luteus and P. aeruginosa.
2
. Chemistry
O
CH3
n
2-4: a, R = F, n = 2;
b, R = F, n = 4;
c, R= F, n = 5;
d, R = F, n = 6;
e, R= F, n = 10;
f, R = Cl, n = 2;
g, R = Cl, n = 4;
h, R = Cl, n = 5;
i, R = Cl, n = 6;
j, R = Cl, n = 10
N
O
N
N
COOH
R
N
iii
O2N
3a-j
R
NH2
O
N
iv
N
n N
N
i
N
n Br
R
N
R
4a-j
O
N
NO2
ii
2a-j
O
N
NH
v
5: a, R = F, Z = C;
b, R = Cl, Z = C;
c, R = F, Z = N;
d, R = Cl, Z = N
N
N
Z
R
1a-b
5
a-d
R
N
1
: a, R = F; b, R = Cl
65
Scheme 1 Synthetic routes of quinazolinone azoles. Reagents and conditions: (i) formamidine acetate, 2ꢀmethoxyethanol, reflux, 18 h; (ii) alkyl
dibromides, K CO , acetone, 50 ºC, 5 h; (iii) 2ꢀmethylꢀ5ꢀnitroimidazole, K CO , acetonitrile, 50 ºC, 5 h; (iv) 4ꢀnitroimidazole, K CO , acetonitrile, 50 ºC,
h; (v) imidazole or triazole, K CO , acetonitrile, 50 ºC, 5 h.
2
3
2
3
2
3
5
2
3
Especially the hybrid of quinazolinone and 2ꢀmethylꢀ5ꢀ
nitroimidazole 3a gave comparable or even superior antibacterial 75 (MIC
efficacies (MIC = 0.03–0.05 µmol/mL) to norfloxacin (MIC =
.01–0.05 µmol/mL) and chloromycin (MIC 0.02–0.10
µmol/mL). 4ꢀNitroimidazoles 4a-j showed stronger antiꢀB.
subtilis activity (MIC = 0.02–0.05 µmol/mL) than chloromycin
70
=
0.10 µmol/mL). The results also showed that
intermediates 2a-j and imidazoles 5a-b displayed weak or no
obvious activities in inhibiting the growth of microorganisms.
The fluoro or chloro substituent of quinazolinone at Cꢀ7 position
0
=
2
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

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Medicinal Chemistry Communications
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DOI: 10.1039/C4MD00281D
did not exert significantly different influence on antimicrobial
potency.
It was known that MRSA was one of the most virulent
organisms that showed severe multiꢀdrug resistance to numerous
currently available agents. Excitingly, 2ꢀmethylꢀ5ꢀnitroimidazoles
explained by the high hydrophobicity of long alkyl chain, making
it easy to penetrate the cytomembrane and therefore improve the
antibacterial activity.
The antifungal evaluation in vitro revealed that most of
nitroimidazole compounds showed antifungal efficiency to some
extent, which were similar to the antibacterial results. Noticeably,
2ꢀmethylꢀ5ꢀnitroimidazoles 3a-j showed good inhibitory
efficiency against all the tested fungal strains, which further
20
5
3
a-j could effectively inhibit the growth of MRSA at the
concentrations of 0.02–0.05 µmol/mL, which were comparable or
even more active in comparison with clinical chloromycin (MIC
=
0.05 µmol/mL). The results in Table 2 also demonstrated the 25 implied the fact that nitroimidazole nucleus was in favor of
1
0
great effects of alkyl chain lengths on biological activities. Longꢀ
chain alkyl 4ꢀnitroimidazoles 4c-e and 4h-j possessed better
antimicrobial activities than shortꢀchain derivatives 4a-b and 4f-g
against most of the tested strains, respectively. Particularly for B.
antimicrobial potency. Especially, compound 3d with (CH2)6
linker could efficiently inhibit the growth of A. flavus strain in
vitro with MIC value of 0.01 µmol/mL, which was 84ꢀfold more
potent than fluconazole (MIC = 0.84 µmol/mL). The results
subtilis, compounds 4c-e with (CH ) , (CH ) and (CH ) linkers 30 suggested that this compound may be further investigated as antiꢀ
2
5
2 6
2 10
1
5
respectively were fiveꢀfold more potent (MIC = 0.02 µmol/mL)
A. flavus agent.
than chloromycin (MIC = 0.10 µmol/mL). This might be
a, b, c
Table 1. In vitro antimicrobial data as MIC (µmol/mL)
for compounds 2–5
Fungi
Gramꢀpositive bacteria
Gram-negative bacteria
Compds
C.
A.
B.
C.
C.
M.
luteus
S.
aureus
B.
P.
E.
coli
B.
E.
MRSA
utilis flavus yeast albicans mycoderma
subtilis aeruginosa
proteus typhosa
2
2
2
a
b
c
0.94
0.43
0.41
0.39
0.33
0.89
0.41
0.39
0.74
0.32
0.03
0.05
0.02
0.02
0.02
0.05
0.02
0.02
0.02
0.02
0.42
0.39
0.37
0.36
0.15
0.40
0.37
0.35
0.34
0.15
0.99
0.47
0.99
0.46
1.89
1.71
0.82
1.56
1.34
>1.78 0.89
>1.62 0.81
>1.55 0.78
>1.49 0.74
>1.28 0.64
0.03
0.02
0.02
0.01
0.04
0.02
0.04
0.02
0.04
0.02
0.94
0.86
0.82
0.78
0.67
0.94
0.86
0.82
0.78
0.67
>1.78
>1.62
>1.55
>1.49
>1.28
0.03
0.02
0.04
0.02
0.04
0.05
0.02
0.02
0.02
0.02
0.05
0.10
0.09
0.04
0.04
0.10
0.09
0.09
0.04
0.04
0.99
0.93
0.99
0.93
>1.89
>1.71
>1.63
>1.56
>1.34
>1.78
>1.62
>1.55
>1.49
>1.28
0.03
0.05
0.04
0.02
0.02
0.05
0.04
0.02
0.02
0.04
0.84
0.77
0.19
0.04
0.04
0.80
0.74
0.18
0.04
0.04
>1.98
>1.86
0.49
0.47
0.94
0.43
0.82
0.39
0.33
0.89
0.81
0.78
0.74
0.64
0.03
0.02
0.02
0.04
0.02
0.05
0.02
0.02
0.02
0.04
0.21
0.19
0.19
0.18
0.15
0.20
0.18
0.18
0.17
0.15
0.99
0.93
0.49
0.46
0.05
0.03
>1.67
>1.89
>1.71
>1.63
>1.56
>1.34
>1.78
>1.62
>1.55
>1.49
>1.28
0.03
0.05
0.04
0.04
0.02
0.10
0.04
0.04
0.02
0.02
0.84
0.39
0.19
0.36
0.31
0.80
0.37
0.18
0.34
0.30
>1.98
>1.86
0.99
0.93
0.05
1.89
1.71
1.63
1.56
1.34
1.78
1.62
1.55
1.49
1.28
0.03
0.05
0.02
0.02
0.02
0.05
0.04
0.02
0.04
0.02
0.05
0.05
0.02
0.02
0.02
0.05
0.05
0.04
0.04
0.04
>1.98
>1.86
0.49
0.46
0.10
0.01
>1.67
>1.89
>1.71
>1.63
>1.56
>1.34
>1.78
>1.62
>1.55
>1.49
>1.28
>1.61
>1.48
>1.42
>1.37
>1.19
>1.53
>1.42
>1.36
>1.31
>1.15
>1.69
0.77
0.37
0.36
0.08
>1.60
0.74
0.35
0.34
0.07
0.99
1.89
1.71
1.63
0.78
1.34
1.78
0.81
1.55
1.49
0.64
0.03
0.05
0.04
0.04
0.02
0.10
0.04
0.02
0.04
0.02
0.84
0.77
0.37
0.18
0.15
0.80
0.74
0.35
0.17
0.15
1.98
1.86
0.25
0.23
0.10
0.05
>1.67
0.47
0.86
0.41
0.39
0.33
0.89
0.41
0.39
0.74
0.32
0.05
0.02
0.02
0.04
0.02
0.05
0.02
0.02
0.02
0.02
0.42
0.39
0.37
0.09
0.08
0.40
0.37
0.35
0.09
0.07
1.98
1.86
0.99
0.93
0.10
0.01
>1.67
>1.89
>1.71
>1.63
>1.56
>1.34
>1.78
>1.62
>1.55
>1.49
>1.28
0.03
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.42
0.39
0.37
0.36
0.31
0.40
0.37
0.35
0.34
0.30
>1.98
>1.86
0.99
0.93
0.10
0.86
0.41
0.39
0.67
0.45
0.81
0.39
0.37
0.32
0.05
0.05
0.02
0.04
0.04
0.10
0.04
0.04
0.04
0.02
0.05
0.05
0.05
0.04
0.04
0.05
0.05
0.04
0.04
0.04
0.50
0.47
0.03
0.03
0.02
0.01
>1.67
2
d
2
2
e
f
g
2
2
h
2
2
i
j
a
3
3
0.03
0.05
0.02
0.04
0.02
0.05
0.04
0.04
0.02
0.04
b
3
c
3
d
3
3
e
f
g
3
3
h
3
3
i
j
a
4
4
>1.69 0.21
b
0.77
0.74
0.18
0.15
0.10
0.09
0.09
0.08
4
c
4
d
4
4
e
f
g
>1.60 0.20
4
4
0.74
0.71
0.17
0.15
0.50
0.93
0.99
0.93
0.09
0.09
0.09
0.07
0.50
0.93
0.49
0.46
h
4
4
i
j
a
5
5
b
0.93
0.03
0.03
0.10
0.01
>1.67
5
c
5
d
0.46
A
B
C
>1.58 >1.58 >1.58 >1.58
>1.60 >1.60 >1.60 >1.60
0.03
>1.58
>1.60
0.01
0.01
>1.67
0.01
>1.67
0.84
0.05
<0.01
a
Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates.
b
C. utilis, Candida utilis ATCC9950; A. flavus, Aspergillus flavus ATCC204304; B. yeast, Beer yeast ATCC9763; C. albicans, Candida albicans
3
5
ATCC10231; C. mycoderma, Candida mycoderma ATCC9888; M. luteus, Micrococcus luteus ATCC4698; MRSA, MethicillinꢀResistant Staphylococcus
aureus N315; S. aureus, Staphylococcus aureus ATCC25923; B. subtilis, Bacillus subtilis ATCC6633; P. aeruginosa, Pseudomonas aeruginosa
ATCC27853; E. Coli, Escherichia coli DH52; B. proteus, Bacillus proteus ATCC13315; E. typhosa, Eberthella typhosa ATCC14028.
c
A = chloromycin; B = norfloxacin; C = fluconazole.
40
Deoxyribonucleic acid (DNA) is the informational molecule
encoding the genetic instructions used in the development and
3
.2 Interactions with calf thymus DNA
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Journal Name, [year], [vol], 00–00 | 3

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function of almost all known living organisms. It is one of the
targets for the studies of biologically important small molecules
such as antimicrobial drugs. The binding studies of small
molecules with DNA are important and helpful for developing
presence of compound 3a at 260 nm, ξC and ξDꢀC are the
absorption coefficients of compound 3a and 3aꢀDNA complex
respectively. The plot of A /(AꢀA ) versus 1/[compound 3a] is
0
0
constructed by using the absorption titration data and linear
4
5
novel and more efficient drugs, which have been attracting 50 fitting (Figure 2), yielding the binding constant, K = 1.41 × 10
considerable attention in biomedical science. To explore the
possible antimicrobial action mechanism, the binding behavior of
active compound 3a with calf thymus DNA (a DNA model with
medical importance, low cost and ready availability properties)
was studied on molecular level in vitro using neutral red (NR)
L/mol, R = 0.998, SD = 0.34 (R is the correlation coefficient, and
SD is standard deviation).
20
10
16
32
dye as a spectral probe by UVꢀvis spectroscopic methods.
12
3
.2.1 Absorption spectra of DNA in the presence of
8
4
compound 3a
The use of absorption spectroscopy is quite useful technique in
DNAꢀbinding studies. In absorption spectroscopy, hypochromism
and hyperchromism are very important spectral features to
distinguish the change of DNA doubleꢀhelical structure. Due to
the strong interactions between the electronic states of
intercalating chromophore and that of the DNA base, the
15
20
25
30
0.5
1.0
1.5
2.0
2.5
-1
3.0
3.5
4.0
4.5
-5
1
0
[Q] (L/mol)
33
0
0
Figure 2 The plot of A /(AꢀA ) versus 1/[compound 3a]
55
3.2.2 Absorption spectra of NR interactions with DNA
observed large hyperchromism strongly suggests
a
close
Neutral Red (NR) is a planar phenazine dye and structurally
similar to other planar dyes such as acridines, thiazines and
xanthenes, which shows lower toxicity, higher stability and
convenient application. Furthermore, it has been sufficiently
proximity of the aromatic chromophore to the DNA bases. With a
fixed concentration of DNA, UVꢀvis absorption spectra were
recorded with the increasing amount of compound 3a. As shown
in Figure 1, UVꢀvis spectra displayed that the maximum 60 demonstrated by spectrophotometric and electrochemical
absorption peak of DNA at 260 nm exhibited proportional
increase with the increasing concentration of compound 3a.
Meanwhile the absorption value of 3aꢀDNA complex was a little
greater than the measured value of simply sum of free DNA and
free compound 3a. These meant a weak hyperchromism effect
existed between DNA and compound 3a. Furthermore, the
intercalation of the aromatic chromophore of compound 3a into
the helix and the strong overlap of πꢀπ* states in the large πꢀ
conjugated system with the electronic states of DNA bases were
consistent with the observed spectral changes.
techniques that the binding of NR with DNA is an intercalation
28b
binding.
Therefore, NR was employed as a spectral probe to
investigate the binding mode of 3a with DNA in the present
work. The absorption spectra of the NR dye upon the addition of
65 DNA were shown in Figure 3. It was apparent that the absorption
peak of the NR at around 460 nm displayed gradual decrease
with the increasing concentration of DNA, and a new band at
around 530 nm developed. This was attributed to the formation of
the new DNAꢀNR complex. An isosbestic point at about 504 nm
70 provided evidence of DNAꢀNR complex formation.
0.4
1.0
0.8
0.6
0.4
0.2
0.0
DNA-3acomplex
DNA+3a
a
0
.75
i
0.70
0.3
i
0.65
i
0.60
0.2
0.1
0.0
a
0.5
1.0
1.5
2.0
a
10
-5 -1
[Q] (L/mol)
DNA
3
50
400
450
500
550
600
Wavelength (nm)
240
270
300
330
Wavelength (nm)
Figure 3 UV absorption spectra of NR in the presence of DNA at pH 7.4
3
5
0
ꢀ5
Figure 1 UV absorption spectra of DNA with different concentrations of
compound 3a (pH = 7.4, T = 290 K). Inset: comparison of absorption at
and room temperature. c(NR) = 2.0 × 10 mol/L, and c(DNA) = 0–5.25 ×
ꢀ5 ꢀ5
10 mol/L for curves aꢀi respectively at increment 0.66 × 10 .
2
60 nm between the 3aꢀDNA complex and the sum values of free DNA
ꢀ5
75 3.2.3 Absorption spectra of competitive interactions of
compound 3a and NR with DNA
and free compound 3a. c(DNA) = 8.62 × 10 mol/L, and c(compound 3a)
= 0–2.0 × 10 mol/L for curves aꢀi respectively at increment 0.25 × 10 .
ꢀ5
ꢀ5
4
On the basis of the variations in the absorption spectra of DNA
upon binding to 3a, equation 1 can be utilized to calculate the
Figure 4 displayed the absorption spectra of a competitive
binding between NR and 3a with DNA. As shown, with the
increasing concentration of 3a, the maximum absorption around
460 nm of the DNAꢀNR complex increased. Compared with the
absorption band at around 460 nm of the free NR in the presence
of the increasing concentrations of DNA (Figure 3), the spectra in
Figure 4 (inset) exhibited the reverse process. The results
intrinsic binding constant (K).
0
80
A
ξC
ξC
1
(1)
=
+
×
0
A− A
ξD−C −ξC ξD−C − ξC K[Q]
0
45
A and A represent the absorbance of DNA in the absence and
| Journal Name, [year], [vol], 00–00
4
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Medicinal Chemistry Communications
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DOI: 10.1039/C4MD00281D
40
absorbance at 278 nm with different concentrations of compound 3a.
.3.2 Fluorescence quenching mechanism
suggested that compound 3a intercalated into the double helix of
DNA by substituting for NR in the DNAꢀNR complex.
3
0.4
i
1.5
1.0
0.5
0.0
i
a
Fluorescence spectroscopy is also an effective method to study
the interactions of small molecules with HSA. The fluorescence
intensity of Trpꢀ214 may change when HSA interacts with other
small molecules, which could be reflected in the fluorescence
spectra of HSA in the UV region. The effect of compound 3a on
the fluorescence intensity to HSA at 298 K was shown in Figure
0
.3
0.2
a
0.1
DNA+NR
45
0.0
4
00
500
600
Wavelength (nm)
6
(Supporting Information 3). It was obvious that HSA had a
strong fluorescence emission with a peak at 358 nm owing to the
single Tryꢀ214 residue. The intensity of this characteristic broad
emission band regularly decreased with the increased
concentrations of compound 3a, but the maximum emission
wavelength of HSA remained unchanged. This suggested that
DNA+NR
00
5
0
3
400
500
600
Wavelength (nm)
Figure 4 UV Absorption spectra of the competitive reaction between 3a
ꢀ5
5
and neutral red with DNA. c(DNA) = 4.31 × 10 mol/L, c(NR) = 2.0 ×
ꢀ5 ꢀ5
29b
Trpꢀ214 did not undergo any change in polarity.
The fluorescence quenching data can be analyzed by the wellꢀ
1
0 mol/L, and c(compound 3a) = 0–2.0 × 10 mol/L for curves aꢀi
ꢀ5
respectively at increment 0.25 × 10 . (Inset) Absorption spectra of the
55
35
system with the increasing concentration of 3a in the wavelength range of
50–600 nm absorption spectra of competitive reaction between
known SternꢀVolmer equation:
3
F0
1
0
compound 3a and NR with DNA.
=1+ K [Q] =1+ Kqτ0[Q]
(2)
SV
F
3
.3 Interactions of compound 3a with HSA
Where F and F represent fluorescence intensity in the absence
o
and presence of compound 3a, respectively. KSV (L/mol) is the
SternꢀVolmer quenching constant, Kq is the bimolecular
HSA is the most abundant serum proteins in the systemic
circulation comprising about 60% in plasma and provides about
6
0
ꢀ
1
ꢀ1
quenching rate constant (L mol s ), τ is the fluorescence
8
0% of the colloid osmotic pressure of blood. Full investigations
0
lifetime of the fluorophore in the absence of quencher, assumed
1
5
of interactions of drugs with HSA are not only beneficial to
thoroughly understand the pharmacokinetic behavior of drugs,
ꢀ
9
to be 6.4×10 s for HSA, and [Q] is the concentration of
compound 3a. Hence, the SternꢀVolmer plots of HSA in the
presence of compound 3a at different concentrations and
temperatures could be calculated and were shown in Figure 7.
30
but also to design, modify and screen drug molecules.
65
3
.3.1 UV-vis absorption spectral study
2
.0
UVꢀvis absorption measurement is a very easy operational
method applicable to explore the structural change of protein and
to identify the complex formation. In our binding experiment,
UVꢀvis absorption spectroscopic method was adopted to evaluate
the binding behaviors between compound 3a and HSA. As
shown in Figure 5, the absorption peak observed at 278 nm was
attributed to the aromatic rings in Tryptophan (Trpꢀ214), Tyrosine
T = 298 K
T = 303 K
T = 310 K
2
2
3
3
0
5
0
5
1.5
1.0
0.5
(
Tyrꢀ411) and Phenylalanine (Phe) residues in HSA. With the
0.0
addition of compound 3a, the peak intensity increased, indicating
that compound 3a could interact with HSA and the peptide
strands of HSA were extended. However, the maximum
absorption wavelength remained unchanged, implying that the
interactions of compound 3a and HSA were a noncovalent
interaction. It occurred via the πꢀπ stacking between aromatic
rings of compound 3a and Trp, Tyr and Phe residues, which
possessed conjugated πꢀelectrons and located in the binding
0.0
0.5
1.0
1.5
2.0
5
10 [Q] (mol/L)
Figure 7 SternꢀVolmer plots of 3aꢀHSA system at different temperatures
Fluorescence quenching occurs by different mechanisms,
usually classified as dynamic quenching and static quenching
depending on temperature and viscosity. Because higher
temperatures result in larger diffusion coefficients, the quenching
constants are expected to increase with a gradually increasing
temperature in dynamic quenching. However, the increase of
70
34
cavity of HSA.
1.0
0
0
0
0
0
.65
.60
.55
.50
.45
λ
R
max
0.9999
= 278 nm
75 temperature is likely to result in a smaller static quenching
constant due to the dissociation of weakly bound complexes.
=
i
0.40
.35
The values of KSV and K for the interactions of compound 3a
0
q
0
.0
0.5
1.0
1.5
2.0
0.5
a
Concentration( 10-5 mol/L)
with HSA at different temperatures were shown in Table 2
(Supporting Information 3). The KSV values were inversely
8
0
correlated with the temperature, which indicated that the
fluorescence quenching of HSA was probably initiated by the
formation of 3aꢀHSA complex rather than dynamic collisions.
The Kq values obtained at different temperatures were in the
range of 10 –10 L/mol s , which far exceeded the diffusion
controlled rate constants of various quenchers with a biopolymer
HSA
Compound 3a only
0.0
250
300
350
Wavelength (nm)
Figure 5 Effect of compound 3a to HSA UVꢀvis absorption, c(HSA) =
.0 × 10 mol/L; c(compound 3a)/(10 mol/L): 0, 0.25, 0.5, 0.75, 1, 1.25,
.5, 1.75, 2 (T = 298 K, pH = 7.40). The inset corresponds to the
12
13
ꢀ1
ꢀ5
ꢀ5
1
1
85
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Journal Name, [year], [vol], 00–00 | 5

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DOI: 10.1039/C4MD00281D
1
0
ꢀ1
(
2.0 × 10 L/mol s ), and indicated that the quenching was not
the corresponding temperature and R is the gas constant. In order
to explain the binding model between compound 3a and HSA,
55 the thermodynamic parameters were calculated from the van’t
Hoff plots. The ꢁH was estimated from the slope of the van’t
Hoff relationship (Figure 10, Supporting Information 3). The free
energy change (ꢁG) was then calculated from the following
equation:
initiated by the dynamic diffusion process but occurred in the
statical formation of 3aꢀHSA complex.
3
.3.3 Binding constant and site
5
For a static quenching process, the data could be described by the
Modified SternꢀVolmer equation:
F0
3
6
ꢀG = ꢀH −TꢀS
1
1
1
60
(6)
(3)
=
+
ꢀF
f K [Q] fa
a a
Table 4 (Supporting Information 3) summarized the values of
ꢁH, ꢁG and ꢁS. The negative values of free energy ꢁG of the
Where ꢁF is the difference in fluorescence intensity in the
interactions between compound 3a and HSA suggested that the
binding process was spontaneous. The negative entropy ꢁS and
enthalpy ꢁH values of the interactions of compound 3a with
HSA indicated that the binding was mainly enthalpyꢀdriven and
the entropy was unfavorable for it, moreover, hydrogen bonds
and van der Waals forces played a major role in the reaction.
absence and presence of compound 3a at concentration [Q], f is
a
10
15
20
the fraction of accessible fluorescence, and K is the effective
a
65
quenching constant for the accessible fluorophores, which are
analogous to associative binding constants for the quencherꢀ
acceptor system. The dependence of F /ꢁF on the reciprocal
o
ꢀ
1
value of quencher concentration [Q] is linear with the slope
ꢀ
1
ꢀ1
equaling to the value of (f K ) . The value f is fixed on the
a
a
a
ꢀ
1
4. Conclusion
ordinate. The constant K is a quotient of the ordinate f and the
slope (f K ) . The Modified SternꢀVolmer plots were shown in
a
a
ꢀ
1
a
a
70 In conclusion, a novel class of quinazolinone azoles were
successfully synthesized in good yields via an easy, convenient
and efficient synthetic route. All the newly synthesized
Figure 8 and the calculated results were depicted in Table 3
(Supporting Information 3).
When small molecules bind to a set of equivalent sites on a
1
13
compounds were characterized by H NMR, C NMR, MS, IR
and HRMS spectra. The biological results revealed that some
target compounds exhibited significant antibacterial and
antifungal activities against most of the tested strains. Especially
nitroimidazole derivative 3a gave comparable or even better
antibacterial efficacies (MIC = 0.03–0.05 µmol/mL) in contrast
with norfloxacin (MIC = 0.01–0.05 µmol/mL) and chloromycin
(MIC = 0.02–0.10 µmol/mL). The specific interactions of
compound 3a with DNA were studied by UVꢀvis absorption
spectroscopy. Experimental results displayed that compound 3a
could intercalate DNA to form compound 3aꢀDNA complex
which might further block DNA replication to exert their
powerful antibacterial and antifungal activities. The calculated
parameters indicated that the binding process was spontaneous,
and hydrogen bonds and van der Waals forces played important
roles in the strong association of compound 3aꢀHSA. Further
research, including the in vivo bioactive evaluation along with
toxicity investigation and the effect factors on antimicrobial
macromolecule, the equilibrium binding constants and the
numbers of binding sites can also be calculated according to the
Scatchard equation:
7
8
8
9
9
5
0
5
0
5
3
7
r
=
nK − rKb
b
(4)
Df
25
30
35
40
Where D is the molar concentration of free small molecules, r
f
is the moles of small molecules bound per mole of protein, n is
binding sites multiplicity per class of binding sites, and K is the
b
equilibrium binding constant. The Scatchard plots were shown in
Figure 9 and the K and n were listed in Table 3 (Supporting
b
Information 3).
Figure 9 showed the Modified SternꢀVolmer and Scatchard
plots for the 3aꢀHSA system at different temperatures. The
decreased trend of K and K with increased temperatures was in
a
b
accordance with K ’s depended on temperatures. The value of
SV
the binding site n was approximately 1, which showed one high
affinity binding site was present in the interactions of compound
23a,39
3
a with HSA. The results also showed that the binding constants
activities such as other heterocyclic rings
(benzotriazole,
were moderate and the effects of temperatures were not
significant, thus compound 3a might be stored and carried by this
protein.
benzimidazole and their derivatives etc.) as well as spacers with
different types of linkers (aralkyl, aryl and heterocyclic types and
their lengths of chains) are active in progress. All these will be
discussed in a future paper.
3
.3.4 Binding mode and thermodynamic parameters
Generally, there are four types of nonꢀcovalent interactions
including hydrogen bonds, van der Waals forces, electrostatic
interactions and hydrophobic bonds, which play important roles
Acknowledgments
This work was partially supported by National Natural Science
Foundation of China (No. 21172181, 21372186), the Research
Fellowship for International Young Scientists from International
3
8
45
in small molecules binding to proteins.
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 equation:
1
00 (Regional) Cooperation and Exchange Program (No.
8
1350110523, 81450110095), the key program from Natural
Science Foundation of Chongqing (CSTC2012jjB10026) and the
Specialized Research Fund for the Doctoral Program of Higher
Education of China (SRFDP 20110182110007) and Chongqing
05 Special Fund for Postdoctoral Research Proposal (Xm2014127).
50
ꢀ
RT
H
ꢀS
R
ln K = −
+
(5)
1
Where K is analogous to the associative binding constants at
6
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Medicinal Chemistry Communications
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DOI: 10.1039/C4MD00281D
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