Synthesis of tetrazole compounds as a novel type of potential antimicrobial agents and their synergistic effects with clinical drugs and interactions with calf thymus DNA

Article abstract of DOI:10.1039/c4md00266k

A series of tetrazole derivatives were synthesized and characterized by NMR, IR, MS and HRMS spectroscopy. The bioactive assay manifested that most of the target compounds exhibited good antifungal activity, especially compound 6g displayed comparable or even stronger antifungal efficiency in comparison with the reference drug Fluconazole. The combination of tetrazole derivative 6g with antibacterials Chloromycin and Norfloxacin, or antifungal Fluconazole respectively was more sensitive to methicillin-resistant MRSA and Fluconazole-insensitive Aspergillus flavus. Further research revealed that compound 6g could effectively intercalate into Calf Thymus DNA to form a 6g-DNA complex which might block DNA replication to exert its good antimicrobial activities. This journal is

Full text of DOI:10.1039/c4md00266k

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Synthesis of tetrazole compounds as a novel type
of potential antimicrobial agents and their
synergistic effects with clinical drugs and
interactions with calf thymus DNA†
Cite this: DOI: 10.1039/c4md00266k
Ling-Ling Dai, Hui-Zhen Zhang, Sangaraiah Nagarajan,‡ Syed Rasheed§
and Cheng-He Zhou*
A series of tetrazole derivatives were synthesized and characterized by NMR, IR, MS and HRMS
spectroscopy. The bioactive assay manifested that most of the target compounds exhibited good
antifungal activity, especially compound 6g displayed comparable or even stronger antifungal efficiency
in comparison with the reference drug Fluconazole. The combination of tetrazole derivative 6g with
antibacterials Chloromycin and Norfloxacin, or antifungal Fluconazole respectively was more sensitive to
methicillin-resistant MRSA and Fluconazole-insensitive Aspergillus flavus. Further research revealed that
compound 6g could effectively intercalate into Calf Thymus DNA to form a 6g–DNA complex which
might block DNA replication to exert its good antimicrobial activities.
Received 21st June 2014
Accepted 22nd September 2014
DOI: 10.1039/c4md00266k
www.rsc.org/medchemcomm
Tetrazole is an important ve-membered aromatic hetero-
cyclic compound with poly-nitrogen electron-rich planar struc-
1
. Introduction
Microbial infections have become alarming recently due to the tural features. This unique structure allows tetrazole derivatives
increasing emergence of multidrug-resistant strains, intractable to readily bind with various enzymes or receptors in organisms
1
–4
pathogenic microorganisms and newly arising pathogens.
via weak interactions such as coordination bonds, hydrogen
Various synthetic and semi-synthetic antimicrobial agents have bonds, cation–p, p–p stacking, hydrophobic effect, van der
been discovered and extensively used in the clinic to treat Waals force and so on, thus displaying a broad spectrum of
various community and hospital acquired microbial infections. biological activities and a considerable role in the pharmaceu-
12,13
Especially, the FDA approved drug auranon exerts an incred- tical eld.
The tetrazole ring can be used as an attractive
ible effect on Gram-positive and multidrug-resistant bacteria linker to combine or stabilize different pharmacophore frag-
5
,6
14,15
and has attracted much attention recently. However, there are ments to generate special functionalized molecules.
The
16,17
18
still some unresolved problems such as narrow antimicrobial tetrazole ring is also an isostere of carboxyl,
spectrum, adverse effects, high toxicity and so on for clinical some heterocycles (triazole,
amide and
19
20
21
benzotriazole,
imidazole,
22
23
drugs. The combination therapy with two or more agents, which benzimidazole, carbazole, etc.) in designing various new
could usually overcome multi-drug resistance, is one of the types of drug molecules. So far many tetrazole-based derivatives
important strategies to improve the efficiency and bioavail- have been successfully developed and prevalently used as
7,8
24
ability as well as treat mixed diseases in the clinic. However, clinical drugs such as antihypertensives Lorsartan and
25
26
27
the discovery and development of structurally novel antimi- Valsartan, antibiotics Flomoxef and Cefonicid, and anti-
crobial agents with good pharmacological proles and excellent nociceptive Alfentanil to treat various diseases. Some litera-
28
9
–11
activity towards resistant strains are highly desirable.
ture has revealed that tetrazole derivatives could effectively
29,30
prevent the biosynthesis of microbial proteins
to inhibit the
growth of various microorganisms in recent years, which
suggests the large potentiality of tetrazole compounds as a new
type of antimicrobial agents.
Fluconazole is one of the most important triazole-based
compounds recommended as the rst-line antifungal drug by
the World Health Organization (WHO). It has been preva-
Institute of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical
Engineering, Southwest University, Chongqing 400715, China. E-mail: zhouch@swu.
edu.cn; Fax: +86-23-68254967; Tel: +86-23-68254967
†
1
‡
Electronic supplementary information (ESI) available. See DOI:
0.1039/c4md00266k
Postdoctoral fellow from School of Chemistry, Madurai Kamaraj University, lently employed to treat fungal infection by Candida albicans,
India.
Cryptococcus neoformans, Dermatitis blastomycosis, etc. due to
its potent activity, excellent safety prole, and favorable
§
Postdoctoral fellow from Department of Chemistry, University of Hyderabad,
India.
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3
1,32
pharmacokinetic characteristics.
The triazole ring of
2
. Chemistry
Fluconazole could efficiently coordinate with the iron(II) ion
of heme to inhibit the biosynthesis of ergosterol and thus The target tetrazole compounds 4a–d and 6a–l were prepared via
inhibits the growth of fungi. However, the emergence of multi-step reactions and the synthetic process was outlined in

uconazole-resistant Candida albicans isolates, increasingly Scheme 1. Commercially available substituted anilines were
serious drug resistance, narrow antifungal spectrum, and low treated by chloroacetonitrile in acetonitrile to give N-phenyl gly-
activity against invasive mycoses have attracted great efforts cinonitriles 2a–c in 50–65% yields. The N-alkylation of
towards modifying the side chain of Fluconazole or exploit- compounds 2a–c with a series of alkyl bromides in reuxing
3
3
34–36
ing its new analogues. In our previous work,
it has been ethanol using potassium carbonate as the base respectively
demonstrated that the tertiary amine type of Fluconazole afforded the intermediates 3a–d in yields of 50–55%, which
analogues displayed large potentiality as a new type of showed that the length of the aliphatic chain in alkyl bromides
antimicrobial agents in which the tertiary alcohol moiety in exerted a slight effect on the formation of tertiary amines. Hal-
Fluconazole was replaced by a tertiary amine fragment obenzyl halides and intermediates 2a–c underwent N-alkylation
that may exert the same function with the active site residue to produce compounds 5a–l in yields of 65–70%. Compounds 4a–
H310 as the tertiary alcohol moiety in Fluconazole. However, d and 6a–l were easily synthesized in high yields ranging from
to the best of our knowledge, so far the combination of a 75% to 80% by the cycloaddition of compounds 3a–d and 5a–l
tetrazole ring with a tertiary amine fragment has not been respectively with sodium azide using ammonium chloride as the
ꢀ
reported.
catalyst in DMF at 120 C. The total yields of target compounds
In view of the above considerations and as an extension of were ranging from 18% to 36%. Moreover, in the procedure for
our continuous work, we have great interest in investigating the preparation of N-phenyl glycinonitriles, it was found that no
tetrazole tertiary amines as a novel type of potential antimi- anticipated products were observed when the aniline ring was
crobial agents. Herein, we would like to report the synthesis substituted by electron-withdrawing groups such as uoro,
of tetrazole compounds 4a–d and 6a–l with different lengths chloro, nitro and so on. This phenomenon might be attributed to
of alkyl chains or halogen-substituted aralkyl moieties. All the electron-withdrawing properties of these groups which made
the new compounds were screened for their antibacterial and the electron density of the nitrogen atom in the aniline ring low
antifungal activities in vitro, and the synergistic effects of the and made it difficult to perform the nucleophilic substitution. All
1
13
most active tetrazole compound with clinical drugs were the new compounds were conrmed by IR, H NMR, C NMR,
also evaluated in vitro. The ionization constants (pK ) and MS and HRMS spectroscopy (See the ESI 1†).
a
lg P of title compounds were determined by the UV-vis
absorption spectroscopic method to evaluate the antimicro-
bial activity. In this work, with the consideration to explore
3
. Results and discussion
the preliminary mechanism of action, the interaction of the 3.1 Antimicrobial activities
most active compound with calf thymus DNA was also
investigated.
The results of antibacterial activity (ESI 2†) in Table 1 showed
that intermediates 3a–d and 5a–l exhibited weak or no
ꢀ
Scheme 1 Reagents and conditions: (i) chloroacetonitrile, potassium carbonate, CH
3
CN, 80 C; (ii) substituted halobenzyl halide, potassium
ꢀ
carbonate, CH CH
3 2
OH, reflux; (iii) sodium azide, ammonium chloride, DMF, 120 C; (iv) alkyl bromide, potassium carbonate, CH
3 2
CH OH, reflux.
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antibacterial efficacy against all the tested bacterial strains. In to their antibacterial activities. The halobenzyl substituted
addition, some of the halobenzyl tertiary amines among 5a–l target compounds 6a–l and alkyl substituted target compounds
exerted relatively better activities in inhibiting the growth of 4a–d exhibited moderate to excellent antifungal activities
tested strains in comparison with alkyl tertiary amines 3a–d. against the tested strains. Especially the target 2-chlorobenzyl
Moreover, the bioactivity of target compounds 4a–d and 6a–l compound 6g displayed comparable or even a stronger anti-
ꢁ1
were improved by the introduction of a tetrazole ring into fungal efficiency (MIC ¼ 4–8 mg mL ) against the tested fungi
substituted benzyl tertiary amines, which indicated that the in comparison with the reference drug Fluconazole (MIC ¼ 1–
ꢁ
1
tetrazole ring was helpful for enhancing antibacterial activity. 256 mg mL ). The length of the aliphatic chain also exhibited
The types of substituents on the aniline ring exhibited no obvious effects on antifungal activity. The suitable length of the
signicant effects on biological activity. Moreover, substituents alkyl chain to exert the best antifungal efficacy was observed to
on benzyl moieties displayed notable effects on antibacterial be (CH ) , and decyl tetrazole 4c gave generally better activity in
2
9
ꢁ
1
activity, especially 6g (MIC ¼ 16–32 mg mL ) with the chlorine comparison with other alkyl tetrazole compounds with shorter
atom on the 2-position of the benzyl ring gave better bioactivity or longer chain length.
against all the tested bacterial strains than others.
In addition, the combinations of tetrazole compound 6g
The antifungal evaluation in vitro revealed that the activities with clinical antibacterials Chloromycin and Noroxacin, or
of most of the compounds were relatively better in comparison antifungal Fluconazole respectively against the tested bacteria
ꢁ
1 a,b,c
Table 1 Antimicrobial activities in vitro for the prepared compounds expressed as MIC (mg mL
)
Gram-positive bacteria Gram-negative bacteria
Fungi
C. A
Compds
S. A
MRSA
B. S
M. L
B. P
E. C
P. A
S. D
C. M
C. U
A. F
3
3
3
3
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
a
b
c
d
a
b
c
d
e
f
g
h
i
j
k
l
a
b
c
d
e
f
g
h
i
256
256
128
256
128
128
64
128
256
256
128
64
128
256
128
128
128
128
256
128
32
128
64
64
64
64
32
64
32
64
512
256
128
512
256
128
32
128
256
256
128
128
256
128
128
256
128
256
256
128
64
128
64
64
32
64
32
128
16
64
64
256
256
64
256
128
128
32
256
256
128
128
128
64
256
256
128
512
128
128
64
256
256
256
512
128
256
256
128
128
512
128
256
128
32
128
64
64
64
128
16
32
32
64
64
128
128
64
256
128
64
512
512
256
512
128
256
128
256
256
256
128
256
128
256
128
64
256
256
128
256
32
64
64
64
32
512
512
512
512
128
128
64
256
256
128
512
128
256
128
128
128
256
128
256
128
64
64
128
32
64
64
32
64
32
64
256
256
128
256
64
256
256
256
256
64
128
16
64
128
256
128
128
512
128
64
128
64
128
128
128
8
256
64
128
256
64
32
32
64
64
128
128
128
256
128
64
128
512
128
256
64
8
16
16
32
16
16
4
16
8
16
16
8
128
256
128
128
64
128
32
32
256
256
128
256
256
128
128
256
128
64
128
128
16
32
64
32
32
32
8
64
32
32
32
64
128
256
256
256
256
256
512
256
128
512
128
128
256
64
128
64
64
64
256
32
128
128
256
128
256
64
128
256
128
256
128
256
256
16
64
64
32
64
64
16
32
16
128
128
64
128
64
128
64
128
128
64
64
128
64
16
64
32
32
64
32
8
16
16
32
128
128
256
128
256
128
256
128
512
256
128
256
32
64
32
32
64
64
16
32
64
32
32
16
16
16
4
32
16
64
16
64
32
32
64
32
64
32
64
64
32
16
32
32
j
k
l
64
64
32
64
64
32
32
16
8
64
32
64
32
32
16
64
32
32
16
A
B
C
8
4
16
1
—
32
2
—
8
1
32
4
—
16
4
—
16
1
—
32
2
—
—
—
—
—
—
—
—
—
256
—
—
1
4
8
a
b
Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates. MRSA, Methicillinresistant
Staphylococcus aureus N315; S. A, Staphylococcus aureus ATCC25923; B. S, Bacillus subtilis ATCC6633; M. L, Micrococcus luteus ATCC 4698; E. C,
Escherichia coli DH52; S. D, Shigella dysenteriae ATCC51252; P. A, Pseudomonas aeruginosa ATCC27853; B. P, Bacillus proteus ATCC13315; C. A,
c
Candida albicans ATCC90029; C. M, Candida mycoderma; C. U, Candida utilis ATCC9950; A. F, Aspergillus avus. A ¼ Chloromycin; B ¼
Noroxacin; C ¼ Fluconazole.
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a,b,c
Table 2 Synergistic effects of compound 6g with antibacterial Chloromycin and Norfloxacin
Bacteria
S. A
Compds
MIC
FIC index
0.750
0.500
0.375
0.500
0.750
0.750
0.500
0.500
Effect
Compds
MIC
FIC index
0.500
0.750
0.625
0.750
0.500
0.625
0.750
0.625
Effect
A
4
16
4
8
4
16
2
8
8
32
8
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
B
6g
B
6g
B
6g
B
6g
B
6g
B
6g
B
6g
B
2
16
0.5
8
1
8
0.5
8
1
16
0.5
8
0.5
16
0.5
4
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
6g
MRSA
B. S
A
6g
A
6g
M. L
B. P
A
6g
A
6g
E. C
A
6g
16
4
16
8
P. A
A
6g
S. D
A
6g
8
6g
a
b
Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates. MRSA, Methicillinresistant
Staphylococcus aureus N315; S. A, Staphylococcus aureus ATCC25923; B. S, Bacillus subtilis ATCC6633; M. L, Micrococcus luteus ATCC 4698; E. C,
Escherichia coli DH52; S. D, Shigella dysenteriae ATCC51252; P. A, Pseudomonas aeruginosa ATCC27853; B. P, Bacillus proteus ATCC13315; C. A,
c
Candida albicans ATCC90029; C. M, Candida mycoderma; C. U, Candida utilis ATCC9950; A. F, Aspergillus avus. A ¼ Chloromycin; B ¼
Noroxacin; C ¼ Fluconazole.
and fungi were investigated. To our excitement, the tested as described in Table 3. Especially, this combination showed
results showed excellent antimicrobial efficacies with less excellent activity against Fluconazole-insensitive Aspergillus a-
ꢁ
1
dosage and a broad antimicrobial spectrum as described in vus in comparison with Fluconazole alone (MIC ¼ 256 mg mL ).
Tables 2 and 3. The FIC index was less than 0.8 which suggested These results manifested that the combinations of tetrazoles
that the combinations of compound 6g with clinical drugs have with clinical drugs could efficiently enhance antimicrobial
good synergistic effects. As shown in Table 2, the combinations activity, overcome drug resistance and broaden the antimicro-
ꢁ1
of compound 6g with Chloromycin (4 mg mL ) or Noroxacin bial spectrum. The advantages of combinations might be
ꢁ
1
(
0.5 mg mL ) were four- or two-fold more potent than them- attributed to the different binding sites of these compounds
selves alone against MRSA. Interestingly, the combination of towards the tested microorganism.
compound 6g with Fluconazole also displayed good activity
ꢁ1
against all the tested fungi with low MIC values of 2–4 mg mL
3
.2 Values of ionization constant (pK ) and lg P
a
a
The ionization constant (pK ) of a molecule is an important
physicochemical parameter which has signicant inuence on
its lipophilicity, solubility, protein binding as well as perme-
Table
Fluconazole
3 Synergistic effects of compound 6g with antifungal
a,b,c
a
ability. Therefore the pK values are usually used to evaluate the
ꢁ
1
Fungi
C. A
Compds
MIC (mg mL
)
FIC index
0.750
Effect
extent of ionization of bioactive molecules at different pH
values, which is of fundamental importance in the consider-
ation of their pharmacokinetic behavior such as absorption,
C
0.5
2
1
2
2
4
64
4
Synergistic
6g
37,38
C. M
C. U
A. F
C
0.500
Synergistic distribution, metabolism, and excretion in organisms.
A
6g
a
compound with a pK < 4 or > 10 will be charged at physiological
C
0.500
Synergistic
Synergistic
pH and display slower diffusion rate across biological
membranes such as the blood–brain barrier. UV spectroscopy is
an efficient method for the determination of ionization
constants which employs readily available equipment with a
fast determination. Structural transposition of title tetrazole
6g
C
0.500
6g
a
Minimum inhibitory concentrations were determined by micro broth
dilution method for microdilution plates. MRSA, Methicillinresistant
b
Staphylococcus aureus N315; S. A, Staphylococcus aureus ATCC25923; B. compounds would be generated in buffer solutions at different
S, Bacillus subtilis ATCC6633; M. L, Micrococcus luteus ATCC 4698; E.
C, Escherichia coli DH52; S. D, Shigella dysenteriae ATCC51252; P. A,
pH values, thus resulting in the changes in UV absorbance. The
changes observed in the UV spectrum of compound 6g in
Pseudomonas aeruginosa ATCC27853; B. P, Bacillus proteus ATCC13315;
C. A, Candida albicans ATCC90029; C. M, Candida mycoderma; C. U, different buffer solutions are clearly displayed in Fig. 1. With
c
Candida utilis ATCC9950; A. F, Aspergillus avus. A ¼ Chloromycin; B
the pH values ranging from 2.0 to 8.5, the structural formula of
compound 6g was changed from I to II (Scheme 2). As shown in
Fig. 2, the spectral differences between 283 and 309 nm were
¼
Noroxacin; C ¼ Fluconazole.
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a
chosen to analyze the pK of target tetrazole compounds. Data
analysis included normalization of the raw scans (Abs330 nm ¼
0), and then the spectral difference between the acid spectra
and the spectra obtained at every other pH was calculated. The
wavelengths of maximum positive and negative deviations were
determined graphically, and the absolute values of the absor-
bance difference at the chosen wavelengths were summed. The
total absorbance difference was then plotted versus pH, and the
data were t to eqn (1)
Â
Ã
ðpH ꢁ pKaÞ
3
HA ꢁ 3Aꢁ ꢂ 10
Absorbance total ¼
ꢂ ½S
t
ꢃ
(1)
ðpH ꢁ pKaÞ
1
þ 10
Fig. 2 Plot of the spectral difference between different solutions of
where 3HA and 3Aꢁ are the extinction coefficients of the acid and compound 6g. The maximum positive deviation occurred at 309 nm,
while the maximum negative deviation occurred at 283 nm.
t
base forms of the compound, respectively, and [S ] is the total
compound concentration. When using absorbance differences,
39
3HA and 3Aꢁ are simply the minima and maxima of the curve.
As shown in Table 4, the pK
a
values of compounds 4a–d and 6a–l decreased in that of compounds 4c–d, these might be explained
displayed an appropriate range 4.13–4.83, which suggested by the possibility that higher lipophilic compounds were
their good pharmacokinetic behavior as bioactive agents.
unfavourable for being delivered to the binding sites. This
Lipophilicity/hydrophilicity plays a signicant role in deter- phenomenon also indicted that suitable lipophilicity is neces-
mining where drugs are distributed and how rapidly they are sary for good activities in drug design (Fig. 3).
metabolized and excreted in the body, for example, hydro-
phobic drugs are preferentially distributed to hydrophobic
compartments such as lipid bilayers of cells while hydrophilic 3.3 Interaction with calf thymus DNA
drugs are preferentially found in hydrophilic compartments
3.3.1 UV-vis absorption spectral study. The application of
40
like blood serum. Therefore, the lipophilicity/hydrophilicity
expressed as lg P was calculated theoretically using ChemDraw
Ultra 10.0 soware and experimentally by a traditional satura-
tion shake ask approach combined with a UV-vis spectropho-
tometric method. The obtained results are given in Table 4, the
c lg P of compounds 4a–d increased with the increase in the
length of the alkyl chain, and an enhancement of the antimi-
crobial activities was observed in compounds 4a–c, but
UV-vis absorption measurement is one of the most important
techniques in DNA-binding studies. Generally, hypochromism
and hyperchromism are important spectral features to distin-
guish the change of DNA double-helical structure. The hyper-
chromism originates from the breakage of the DNA duplex
secondary structure; while the hypochromism generates from
the stabilization of the DNA duplex by either the intercalation
41
binding mode or the electrostatic effect of small molecules.
The hypochromism demonstrated a close proximity of the
aromatic chromophore to the DNA bases, which might be
attributed to the strong interaction between the electronic
a
a
Table 4 pK , lg P and c lg P of target compounds 4a–d and 6a–l
Compds
pK
a
lg P
c lg P
4
4
4
4
6
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
a
b
c
d
e
f
g
h
i
j
k
l
4.78
4.83
4.87
4.92
4.54
4.49
4.22
4.19
4.13
4.20
4.34
4.38
4.26
4.23
4.31
4.29
2.18 ꢄ 0.16
2.24 ꢄ 0.24
4.92 ꢄ 0.18
NE
4.11
5.17
6.23
7.28
4.56
4.44
3.28
3.28
3.42
3.28
3.85
3.85
5.06
3.92
3.45
4.59
Fig. 1 UV spectra (l ¼ 260–330 nm) of compound 6g in different
aqueous buffer solutions ranging from pH 2.0 to 8.5. The absorbances
are normalized to zero for l ¼ 330 nm.
1.67 ꢄ 0.19
1.63 ꢄ 0.14
1.28 ꢄ 0.11
1.29 ꢄ 0.13
1.36 ꢄ 0.14
1.26 ꢄ 0.16
1.09 ꢄ 0.11
1.39 ꢄ 0.21
1.99 ꢄ 0.12
1.42 ꢄ 0.13
1.37 ꢄ 0.11
1.58 ꢄ 0.10
a
Scheme 2 The structural formula of compound 6g was changed from
I to II in buffer solutions with the increase of pH values.
NE ¼ No experimental data. It is difficult to obtain the lg P data of
compounds 4d due to their quite low water solubility.
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1/[compound 6g] is constructed by using the absorption titra-
tion data and linear tting (Fig. 5), yielding the binding
4
ꢁ1
constant, K ¼ 4.16 ꢂ 10 L mol , R ¼ 0.999, and SD ¼ 0.17 (R is
the correlation coefficient. SD is standard deviation).
3.3.2 Absorption spectra of NR interaction with DNA.
Neutral Red (NR) is a planar phenazine dye and is structurally
similar to other planar dyes like acridine, thiazine and xanthene
kind. Recently, it has been demonstrated that the binding of NR
44
with DNA is an intercalation binding. Therefore, NR was
employed as a spectral probe to investigate the binding mode of
Fig. 3 Plot of the total absorbance difference vs. pH to determine the
pK of compound 6g. The total absorbance difference is the sum of the
a
absolute absorbance difference values at the chosen wavelengths
6g with DNA in the present work.
from 283 to 309 nm.
The absorption spectra of the NR dye upon the addition of
DNA are shown in Fig. 6. It is apparent that the absorption peak
of the NR at around 460 nm showed a gradual decrease with the
states of the intercalating chromophore and that of the DNA increasing concentration of DNA, and a new band at around 530
base. With a xed concentration of DNA, UV-vis absorption nm developed. This was attributed to the formation of the DNA–
spectra were recorded with the increasing amount of compound NR complex. An isosbestic point at 504 nm provided evidence of
6
g. As shown in Fig. 4, UV-vis spectra showed that the maximum DNA–NR complex formation.
absorption of DNA at 260 nm displayed a proportional increase
3.3.3 Absorption spectra of competitive interaction of
with the increasing concentration of compound 6g. Simulta- compound 6g and NR with DNA. As shown in Fig. 7, the
neously, the absorption value of the sum of free DNA and free competitive binding between NR and 6g with DNA was
compound 6g was a little greater than the measured value of the observed in the absorption spectra. With the increasing
6
g–DNA complex, which was observed in the inset of Fig. 4. concentration of compound 6g, an apparent intensity increase
These indicated that a weak hypochromic effect existed between was observed around 276 nm. In comparison with the
DNA and compound 6g. Moreover, the intercalation of the absorption around 276 nm of the NR–DNA complex, the
aromatic chromophore of compound 6g into the helix and the absorbance at the same wavelength exhibited the reverse
strong overlap of p–p* states in the large p-conjugated system process (inset of Fig. 5). These various spectral changes were
with the electronic states of DNA bases were consistent with the
42,43
observed spectral changes.
On the basis of the variations in the absorption spectra of
DNA upon binding to tetrazole derivative 6g, eqn (2) can be
utilized to calculate the binding constant (K).
0
A
xC
ꢁ xC
xC
1
¼
þ x
ꢂ
(2)
0
A ꢁ A
x
ꢁ xC K½Qꢃ
DꢁC
DꢁC
0
A and A represent the absorbance of DNA in the absence
and presence of compound 6g at 260 nm, xC and x are
DꢁC
the absorption coefficients of compound 6g and 6g–DNA
0
ꢁ1
0
complex respectively. The plot of
A
(A ꢁA ) versus
0
ꢁ1
0
Fig. 5 The plot of A (A ꢁA ) versus 1/[compound 6g].
Fig. 4 UV absorption spectra of DNA with different concentrations of
compound 6g (pH ¼ 7.4, T ¼ 303 K). Inset: comparison of absorption
at 260 nm between the 6g–DNA complex and the sum values of free Fig. 6 UV absorption spectra of NR in the presence of DNA at
ꢁ
5
ꢁ1
ꢁ5
ꢁ1
DNA and free compound 6g. c(DNA) ¼ 4.52 ꢂ 10 mol L , and pH 7.4 under room temperature. c(NR) ¼ 2 ꢂ 10 mol L , and
ꢁ5
ꢁ1
ꢁ5
ꢁ1
c(compound 6g) ¼ 0–1.6 ꢂ 10 mol L for curves a–i respectively at c(DNA) ¼ 0–3.61 ꢂ 10
mol L for curves a–i respectively at
ꢁ5
ꢁ5
increment 0.2 ꢂ 10
.
increment 0.45 ꢂ 10
.
Med. Chem. Commun.
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