Synthesis of novel calix[4]arene: P -benzazole derivatives and investigation of their DNA binding and cleavage activities with molecular docking and experimental studies

Article abstract of DOI:10.1039/d0ra07486a

In this study, novel p-benzimidazole-derived calix[4]arene compounds with different structures, and a benzothiazole-derived calix[4]arene compound, were synthesized by a microwave-assisted method and their structures were determined by FTIR, 1H NMR, 13C NMR, MALDI-TOF mass spectroscopy, and elemental analysis. The effects of functional calixarenes against bacterial (pBR322 plasmid DNA) and eukaryotic DNA (calf thymus DNA = CT-DNA) were investigated. The studies with plasmid DNA have shown that compounds 6 and 10 containing methyl and benzyl groups, respectively, have DNA cleavage activity at the highest concentrations (10 000 μM). Interactions withplasmid DNA using some restriction enzymes (BamHI and HindIII) were also investigated. The binding ability of p-substituted calix[4]arene compounds towards CT-DNA was examined using UV-vis and fluorescence spectroscopy and it was determined that some compounds showed efficiency. In particular, itwas observed that the functional compounds (10 and 5) containing benzyl and chloro-groups had higher activity (Kb binding constants were found to be 7.1 × 103 M-1 and 9.3 × 102 M-1 respectively) on DNA than other compounds. Competitive binding experiments using ethidium bromide also gave an idea about the binding properties. Docking studies of the synthesized compounds with DNA were performed to predict the binding modes, affinities and noncovalent interactions stabilizing the DNA-compound complexes at the molecular level. Docking results were in good agreement with the experimental findings on the DNA binding activities of compounds. Based on these results, this preliminary study could shed light on future experimental antibacterial and/or anticancer research.

Full text of DOI:10.1039/d0ra07486a

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PAPER
Synthesis of novel calix[4]arene p-benzazole
derivatives and investigation of their DNA binding
and cleavage activities with molecular docking and
experimental studies†
Cite this: RSC Adv., 2020, 10, 38695
Seyda Cigdem Ozkan, Fatma Aksakal and Aydan Yilmaz *b
ab
c
In this study, novel p-benzimidazole-derived calix[4]arene compounds with different structures, and
a benzothiazole-derived calix[4]arene compound, were synthesized by a microwave-assisted method
1
13
and their structures were determined by FTIR, H NMR, C NMR, MALDI-TOF mass spectroscopy, and
elemental analysis. The effects of functional calixarenes against bacterial (pBR322 plasmid DNA) and
eukaryotic DNA (calf thymus DNA ¼ CT-DNA) were investigated. The studies with plasmid DNA have
shown that compounds 6 and 10 containing methyl and benzyl groups, respectively, have DNA cleavage
activity at the highest concentrations (10 000 mM). Interactions with plasmid DNA using some restriction
enzymes (BamHI and HindIII) were also investigated. The binding ability of p-substituted calix[4]arene
compounds towards CT-DNA was examined using UV-vis and fluorescence spectroscopy and it was
determined that some compounds showed efficiency. In particular, it was observed that the functional
compounds (10 and 5) containing benzyl and chloro-groups had higher activity (K
b
binding constants
3
ꢁ1
2
ꢁ1
were found to be 7.1 ꢀ 10
M
and 9.3 ꢀ 10
M
respectively) on DNA than other compounds.
Competitive binding experiments using ethidium bromide also gave an idea about the binding properties.
Docking studies of the synthesized compounds with DNA were performed to predict the binding modes,
affinities and noncovalent interactions stabilizing the DNA–compound complexes at the molecular level.
Docking results were in good agreement with the experimental findings on the DNA binding activities of
compounds. Based on these results, this preliminary study could shed light on future experimental
antibacterial and/or anticancer research.
Received 31st August 2020
Accepted 14th September 2020
DOI: 10.1039/d0ra07486a
rsc.li/rsc-advances
completely cured and pose daily threats to human life. Various
studies on biological applications of calixarenes, such as anti-
Introduction
7
8
9
10
One of the developments in the eld of chemistry in recent years bacterial, antifungal, antiviral, anti-HIV, anti-inamma-
11
12
13
has been the birth and progress of supramolecular chemistry. tory, anticancer, and biotechnological studies, have been
The discovery and many different features of macrostructures carried out in recent years. Many studies on the interaction of
1
2
3
14
such as cyclodextrins, crown ethers and calixarenes have calixarenes with DNA have been previously described; DNA
contributed to the advancement in this area.
cleavage studies were performed using plasmid DNA, and
7b,15
Calixarenes are excellent products of phenol-formaldehyde highly effective results were obtained.
However, there are
4
chemistry, which naturally act as sensors for many cations,
also studies using eukaryotic DNA, and similar results have
5
6
16
anions and neutral molecules due to their inherent structural been obtained in these studies.
properties. However, the idea that calixarenes can be used in
Preliminary anticancer studies on interaction of candidate
areas such as medicine and biology has led scientists to work on molecules with DNA are mostly examined. Most of the calixar-
this subject due to the increase in the occurrence of diseases ene molecules that have been studied and those that bind to or
12a,b,16b
(
such as cancer, Alzheimer's, and Parkinson's) that cannot be interact with DNA are cationic structures.
However, as an
alternative to these molecules, it is necessary to develop mole-
cules that are uncharged but contain functional groups that can
bind to DNA. For this purpose, the effects of calixarene
compounds containing uncharged and functional groups on
a
Department of Chemical and Chemical Processing Technologies, Acigol Vocational
School of Technical Sciences, Nevsehir Haci Bektas Veli University, Nevsehir, Turkey.
E-mail: aydan@selcuk.edu.tr; Fax: +90 332 2412499; Tel: +90 332 2233866
b
Department of Chemistry, Faculty of Science, Selcuk University, 42075, Konya, Turkey cancer cells were investigated and good results were obtai-
c
12c–e
Department of Chemistry, Faculty of Science, Hacettepe University, Ankara, Turkey
ned.
In our study, we focus on synthesizing molecules that
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/d0ra07486a
are uncharged but contain functional group(s) that can bind to
1
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DNA. We chose the benzimidazole groups and benzothiazole
group, which have aromatic structures containing hetero
groups as the functional groups.
The benzimidazole compounds are among the bioactive
heterocyclic compounds, which exhibit a wide range of biological
17
activities due to their imidazole nuclei. In particular, functional
benzimidazole derivatives have been synthesized as anticancer
drugs in medicinal chemistry and their efficacy has been
18
demonstrated by patents. Benzimidazole compounds are fusion
molecules, composed of the imidazole ring with a positive charge
and a benzene ring with hydrophobic properties and a partial
negative charge. Therefore, they are capable of interacting with
both positive and negative regions of biological molecules. As is
known, the DNA molecules have a partial negative charge due to
the phosphate groups they contain, so the positive portions of the
benzimidazole compounds tend to bind to DNA. There are a lot
of studies on this topic and as a result, effective and remarkable
17
data have been obtained and reported.
Scheme 1 Schematic route for the synthesis of p-benzimidazole
In the literature, DNA binding modes include electrostatic derivatives of calix[4]arenes.
interactions, intercalation interactions, binding to major and
small grooves, and single-stranded DNA (ssDNA) base binding.
Electrostatic interaction is the deterioration of the DNA charge
To obtain benzimidazole compounds that have different
balance due to interactions between the negatively charged structures, the dialdehyde-derived calix[4]arene compound 3
phosphate backbone of DNA and the positively charged ends of was reacted with o-phenylenediamine and its 4-substituted
18
the molecules. Intercalation occurs when an aromatic planar derivatives separately in the presence of KI in DMF (Scheme 1).
substituent is inserted between the DNA base pairs causing the Before the mixtures were boiled in a domestic microwave oven
1
9
ꢂ
unwinding and lengthening of the DNA helix. Groove binding for a few minutes, they were heated at 80 C under a reux
is caused by groove binders connected to the major or minor condenser. The reactions were monitored by thin-layer chro-
grooves of the DNA. This involves hydrogen bonding or van der matography (TLC). Aer purication, the FTIR spectrum
ꢁ
1
Waals interactions of the molecule with the nucleic acid showed that the peak at 1680 cm of the aldehyde carbonyl was
without substantially disrupting the duplex structure, or bases, completely gone and the peaks belonging to the benzimidazole
as opposed to intercalation. ssDNA base binding results from group (approximately 1660 cm and 1620 cm ) were formed.
20
ꢁ1
ꢁ1
21
binding to the single-stranded DNA of some proteins.
Also, –NH protons belonging to the benzimidazole group were
1
In this study, we report the synthesis and characterization of determined by H NMR at 12.54 ppm for compound 4,
new p-tert-butylcalix[4]arenes with benzimidazole derivatives 12.76 ppm for compound 5, 12.80 ppm for compound 6, and
and a benzothiazole derivative. The DNA cleavage effects of the 13.30 ppm for compound 7; NH protons were in resonance. The
synthesized compounds were investigated using pBR322 conrmations of the cone conformations of the compounds
1
plasmid DNA by the agarose gel electrophoresis method. The obtained were carried out by H NMR to give the following
2
determination of the site of DNA cleavage with BamHI and results for ArCH Ar protons: compound 4 had a doublet pair at
HindIII restriction enzymes was carried out for all compounds. 3.57 ppm and 4.24 ppm J ¼ 13.1 Hz; compound 5 had a doublet
In another study, the effects of synthesized calixarene pair at 3.61 ppm and 4.27 ppm, J ¼ 12.8 Hz; compound 6 had
compounds against CT-DNA were investigated using uores- a doublet pair at 3.60 ppm and 4.28 ppm, J ¼ 12.8 Hz;
cence and UV-vis. spectrometry. Binding constants (K ) and compound 7 had a doublet pair at 3.60 ppm and 4.26 ppm, J ¼
b
quenching constants (K ) were calculated for some compounds 13.1 Hz. The characterizations of the synthesized compounds
sv
1
3
that were effective in uorescence spectrometry. Finally, were supported by C NMR and MALDI TOF MS analyses
competitive binding experiments with ethidium bromide were (Fig. S1 and S2†).
performed and quenching constants (Ksv) were determined. To
In the second part of the synthesis, to obtain N-substituted
support the experimental results with theoretical studies, benzimidazole derivatives, o-phenylenediamine was reacted
molecular docking studies of the synthesized compounds with with benzyl bromide and p-nitrobenzyl bromide and converted
the DNA were also performed.
into N-substituted o-phenylenediamine derivatives (8 and 9)
25
(
Scheme 2) as in the literature, and the structural analyses
1
were performed using FTIR and H NMR. The obtained
compounds were reacted with dialdehyde-derived calixarene (3)
and compounds 10 and 11 were synthesized. In the FTIR anal-
Results and discussion
Synthesis of p-benzazole derivatives of calix[4]arenes
The starting compound, p-tert-butylcalix[4]arene (1) and deriv- ysis of these compounds, the peak of the aldehyde group dis-
1
ative compounds 2 and 3 were synthesized according to the appeared and the benzimidazole imine peak was formed; H
22–24
literature procedures (Scheme 1).
NMR spectra were also determined. The conrmations of the
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slower-moving open circular Form II, and the linear Form III
26
will all be generated, with migration in between.
Firstly, in order to determine whether dimethylformamide
DMF) as a solvent in the various concentrations of the
(
compound samples affected pBR322 plasmid DNA, incubation
with plasmid DNA for the purpose of control in the same
proportions was performed using the same conditions.
According to the obtained results, the solvent had little effect on
the plasmid DNA (Fig. 1a). According to the electrophoresis
image seen in Fig. 1a, compound 4 is not effective on the
plasmid DNA at all the studied concentrations. In the embodi-
ment of Fig. 1b, compound 5 destroyed the Form II (open
circular form) structure that was present in the plasmid DNA at
the rst three concentrations. However, the Form I (supercoiled
form) structure remained. In the same image, for compound 6
on the right side of the gel, all bands of plasmid DNA (Form I
and Form II) at the maximum concentration of 10 000 mM dis-
appeared, while the Form I structure was formed in more dilute
concentrations, and the Form II structure was observed as very
slim at the low concentration. We can say that this compound
showed the cleavage effect on plasmid DNA at this
7b
concentration.
Scheme 2 Schematic route for the synthesis of functional p-benz-
imidazole derivatives and a benzothiazole derivative of calix[4]arenes.
The image on the le (column 2–5) of Fig. 2a shows the
interaction of the pBR322 plasmid DNA with compound 7.
Accordingly, at all concentrations of this compound, a higher
band was observed showing that the molecular weight of the
Form I structure (supercoiled) was increased. A similar situation
was observed in the 100% DMF sample used for the control, but
a band closer to Form I was observed below. From this, we can
say that compound 7 decreases the mobility and density of the
Form I structure. Similarly, results were obtained in the litera-
ture where functional calixarene molecules reduced the
cone conformations of the compounds were again provided by
1
H NMR analysis. For the synthesis of the benzothiazole deriv-
ative of calixarene (12), the synthetic pathway is given in Scheme
2. Compound 3 and 2-aminothiophenol were heated in the
presence of KI in DMF by using the synthesis-type microwave
oven for 3 hours and compound 12 was obtained. Structure
1
analysis was performed by FTIR and H NMR. In the FTIR
7b,15
mobility of the Form I structure of plasmid DNA.
Calixarene
spectrum of this compound, the carbonyl band of the aldehyde
group disappeared while the imine band appeared at
molecules, which are large in structure, can bind to the super-
coiled structure of DNA and limit its mobility. The same image
ꢁ
1
1
1
2
658 cm . In the H NMR spectrum, ArCH Ar protons were
(Fig. 2a) on the right side (column 6–9) shows the interaction of
exposed as 3.57 ppm and 4.36 ppm as a doublet pair (J ¼ 13.3
1
3
compound 11 with pBR322 plasmid DNA. According to this, at
the highest concentration of this compound (11), the super-
coiled DNA fragment (Form I) and the open circular form (Form
II) are broken and are, therefore, converted into smaller DNA
fragments. At the lower concentrations, the Form I band was
Hz). C NMR and MALDI-TOF MS analyses were performed for
all compounds and their structures were illuminated (Fig. S3
and S4†).
DNA cleavage studies with pBR322 plasmid DNA
Plasmid DNAs are fragments of protective DNA that are con-
tained within the bacterial cell that can copy itself, apart from
the chromosomal DNA of the bacterium. When bacteria are
exposed to any external agent (various chemicals etc.), they use
their DNA to protect themselves. Therefore, molecules that can
damage or break down the plasmid DNA cause the bacteria to
die by preventing their growth. For all these reasons, we inves-
tigated the activity of the synthesized molecules on this DNA
using the pBR322 plasmid DNA. These studies were carried out
with the aim of pioneering DNA studies, presenting the rst
ideas about the interaction of compounds with DNA.
Fig. 1 Electrophoresis images of pBR322 plasmid DNA interacting
with compounds. P: only plasmid DNA, M: DNA marker. (a) Compound
The interactions of synthesized calixarene compounds with
supercoiled pBR322 DNA were investigated using agarose gel
electrophoresis. When circular plasmid DNA is subjected to
4
(
5
, 1/4: 10 000 mM, 5000 mM, 2500 mM, 1250 mM; 5/8: only solvent
DMF) 100%, 50%, 25% and 12.5%. (b) Compound 5, 1/4: 10 000 mM,
000 mM, 2500 mM, 1250 mM; compound 6, 5/8: 10 000 mM, 5000
electrophoresis, the fastest migrating supercoiled Form I, the mM, 2500 mM, 1250 mM. (c) Standard DNA marker (1 Kb DNA ladder).
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Fig. 3 Electrophoresis images of compound-pBR322 plasmid DNA
Fig. 2 Electrophoresis images of pBR322 plasmid DNA interacting mixture after interactions with the enzymes BamHI (a) and HindIII (b).
with compounds. P: only plasmid DNA. (a) 1: DMF control (100%), P: plasmid DNA, CP: cut plasmid DNA, 1/4: only solvent (DMF) 100%,
compound 7, 2/5: 10 000 mM, 5000 mM, 2500 mM, 1250 mM; 50%, 25%, 12.5%; compound 4, 5/8: 10 000 mM, 5000 mM, 2500 mM,
compound 11, 6/9: 10 000 mM, 5000 mM, 2500 mM, 1250 mM. (b) 1250 mM.
Compound 10, 1: DMF control (100%), 2/5: 10 000 mM, 5000 mM,
2
2
500 mM, 1250 mM. (c) Compound 12, 1/4: 10 000 mM, 5000 mM,
500 mM, 1250 mM.
observed, albeit higher. In the electrophoresis image shown in
Fig. 2b (column 2–5) is the interaction of compound 10 with
plasmid DNA. Accordingly, all bands of the plasmid DNA (Form
I and Form II) at the highest concentration (10 000 mM) dis-
7b
appeared. A cleavage effect was observed at this concentration.
Form I structure was observed at lower concentrations. On the
Fig. 4 Electrophoresis images of compound-pBR322 plasmid DNA
mixture after interactions with the enzymes BamHI (a) and HindIII (b).
other hand, the Form II structure was not observed. Fig. 2c P: plasmid DNA, CP: cut plasmid DNA, compound 5, 1/4: 10 000 mM,
5
000 mM, 2500 mM, 1250 mM; compound 6, 5/8: 10 000 mM, 5000
shows the interaction of plasmid DNA with compound 12 on the
le side (column 1–4) of the electrophoresis image. At the
highest concentration of this compound, both the density and
the mobility of the supercoiled Form I structure of the plasmid
DNA decreased. On the other hand, the density of the open
circular structure, Form II, increased. At lower concentrations,
the bands appeared as in the control plasmid.
mM, 2500 mM, 1250 mM.
Accordingly, the enzyme made the cuts in the highest DMF
ratios. In Fig. 3b, a gel image of the HindIII segment is shown.
On the right side of this gure, the enzyme cuts the plasmid
DNA at all concentrations of compound 4. On the le side of the
same gure, the enzyme again cuts in the samples containing
only the solvent.
At the highest concentration of compound 5 on the le side
of Fig. 4a, it was observed that plasmid DNA was almost absent
and the linear Form III structure had very low density and
BamHI enzyme was cut at lower concentrations of this
compound. On the right side of the same gure, plasmid DNA
was not seen at the rst high concentration of compound 5 and
the BamHI enzyme was cut at lower concentrations. In Fig. 4b,
the gel image of the HindIII segment is given. From the gure, it
is seen that at the highest concentrations of compounds 5 and 6
do not have visible plasmid DNA bands (columns 1 and 5),
whereas, at lower concentrations of these compounds, the
enzyme cut the plasmid DNA.
According to the obtained results, the two most effective
compounds were compounds 6 and 10, which have methyl and
benzyl groups, respectively. The benzyl group has a rigid and
planar structure due to the benzene ring it contains. Therefore,
it was expected that compound 10 would approach the aromatic
core bases of DNA. Unlike other benzimidazole compounds, the
methyl group in compound 6 may bind hydrophobically to DNA
due to its hydrophobic properties, as reported studies in the
37,38
literature.
In the second part of the study, to determine the ability of the
synthesized heterocyclic p-tert-butylcalix[4]arene derivatives to
cleave DNA molecules at the positions at which particular short
sequences of bases are present, we used BamHI and HindIII
enzymes. Restriction endonuclease analyses of compound-
pBR322 plasmid DNA inserts were performed using these
enzymes. BamHI and HindIII enzymes cut DNA at specic
0
0
0
0
sequences as follows: 5 -G/GATCC-3 and 5 -A/AGCTT-3 . As
a result of the cutting process, Form I (supercoiled form) DNA
and Form II (nicked form) DNA constructs were converted into
Form III DNA in linear form.
Fig. 3a shows the results of the experiment with the BamHI
enzyme. On the right side of the gure, it was found that the
BamHI enzyme cut the plasmid DNA at all concentrations of
compound 4. The electropherograms on the le side of the Fig. 5 Electrophoresis images of compound-pBR322 plasmid DNA
mixture after interactions with the enzymes BamHI (a) and HindIII (b).
P: plasmid DNA, CP: cut plasmid DNA, compound 7, 1/4: 10 000 mM,
same gure were formed by the control samples made as
a control.
5000 mM, 2500 mM, 1250 mM; compound 11, 5/8: 10 000 mM, 5000
mM, 2500 mM, 1250 mM.
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Fig. 6 Electrophoresis images of the compound-pBR322 plasmid
DNA mixture after interaction with the enzymes BamHI (a and c) and
HindIII (b and d), (a and b). P: plasmid DNA, CP: cut plasmid DNA,
compound 10, 1/4: 10 000–1250 mM; (c and d) compound 12, 1/4:
1
0 000–1250 mM.
In Fig. 5a, it was found that plasmid DNA did not appear in
both compounds 7 and 11 at the rst high concentrations,
whereas in lower concentrations, the BamHI enzyme cut the
plasmid DNA. The HindIII enzyme cut image in Fig. 5b shows
that there are no plasmid DNA bands at the highest concen-
trations of both compounds 7 and 11, while the enzyme cut the
plasmid DNA partially at the lower concentrations of these
compounds.
Fig. 6a and c show the results of the experiments with
compound 10 and compound 12 of the BamHI enzyme,
respectively. At the rst high concentration of both compounds,
all the bands of plasmid DNA were not found whereas, at lower
concentrations of these compounds, the BamHI enzyme cut the
plasmid DNA. Similar results were observed in the experimental
results with the HindIII enzyme (Fig. 6b and d).
Fig. 7 The obtained UV-vis absorption spectra of the compounds on
adding CT-DNA in increasing concentrations (from bottom to top (0–
5
0 mM)) in increments of 2.5 mM of CT-DNA, additionally 75 mM and
100 mM CT-DNA. (a) Compound 4, (b) compound 5, (c) compound 6,
d) Compound 10, (e) compound 11, (f) compound 12. The arrows
(
The obtained results showed that the compounds had no
interest in the specic binding regions of the enzymes.
show the changes upon increasing the amounts of CT-DNA.
As shown in Fig. 7, there was a regular increase in the
DNA interaction studies with calf thymus DNA
absorbance of compound 4 at 318 and 333 nm, compound 5 at
3
22 and 338 nm, and compound 6 at 317 and 320 nm on
DNA-binding studies by UV-vis absorption spectroscopy. UV-
vis spectroscopy is one of the basic and useful tools that give an
idea about whether the compound or substance interacts with
calf thymus DNA (CT-DNA) or its ability to bind. CT-DNA gives
a band at 258–260 nm, resulting from the p–p* transitions
between the base pairs of DNA. When the concentration of the
compound or the substance is kept constant and the concen-
tration of DNA is regularly increased, the increase or decrease in
the absorption of DNA is one of the proofs that the interaction
has occurred. These interactions may be the distortion of the
hydrogen bond between the base pairs, covalent bonding or
intercalation. On the other hand, following the absorption of
the compound/compounds is another method used for inves-
tigating compound–DNA interactions.
increasing the CT-DNA concentration. Likewise, a steady
increase in CT-DNA concentration was observed with the
absorbances of compound 10 at 316 and 330 nm, compound 11
at 304 nm and compound 12 at 330 nm. When the all spectra
were examined, it was observed that there was no shi in
wavelengths and only the intensity of absorption increased.
Although there were no appreciable changes in the absorp-
tion spectra of the compounds, the positions of the intra-
compound bands were observed, and a strong chromic effect
was observed with increased CT-DNA concentration. This
observed change shows that the compounds most likely inter-
acted with DNA in the groove binding mode.
hyperchromism may have occurred as a result of external
contact, such as surface bonding with the DNA double-
strand. This surface contact may have occurred as a result of
hydrogen bonding, electrostatic interactions, or p-interactions
between aromatic groups. The compounds that contain imid-
27a–c
This observed
In this study, we also followed the absorbances of the
compounds (4–7 and 10–12). Upon the addition of increasing
amounts of CT-DNA, a signicant hyperchromism was observed
27a
26
for all compounds except compound 7 (Fig. 7). In the
absorption spectrum of compound 7, a partial increase in
electronic absorption (at 396 nm) was observed with the
increase in the DNA concentration as in other compounds;
however, the graph was not included here because the increase
remained low as compared to other compounds.
azole groups, other functional groups (such as Cl, NO , or
2
methyl) and aromatic rings can cause these conditions.
Based on these results, we can say or suggest that the
compounds most likely bind to CT-DNA. However, extra studies
were needed to obtain accurate and reliable results and to
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determine the mode of binding of the compounds with CT-
DNA; hence, uorimetry and competitive binding studies with
EtBr, known as an intercalator, were performed to clarify the
DNA-binding mode.
DNA-binding studies by uorescence spectroscopy. All the
compounds showed uorescence properties and exhibited good

uorescence, except compound 7. Since the uorescence
intensity of compound 7 was very low, experimental evaluation
could not be performed. Interestingly, the uorescence inten-
Fig.
9
Fluorescence spectra obtained by adding CT-DNA in
increasing concentrations (from bottom to top (0–30 mM) with
sity of compound 6 did not change on adding CT-DNA; as such, increments of 5 mM of CT-DNA, additionally 75 mM and 100 mM CT-
constant
the spectra are not shown herein. The monitoring of uores- DNA) of compound 5 (a); the linear graph from which the K
b
was calculated (b). The arrow shows the changes upon increasing the
amounts of CT-DNA.
cence intensities by the addition of CT-DNA was performed only
for the compounds that gave emission spectra. The concentra-
tions of the compounds were kept constant at 25 mM; changes in
the emission spectra of the compounds were recorded on add-
ing CT-DNA up to 50 mM, with increments of 2.5 or 5 mM, and
also at concentrations of 75 mM and 100 mM. Due to the high
emission intensities, compounds 4 and 5 were studied at 10 mM.
While working with these compounds (4 and 5), CT-DNA was
increased to a maximum of 30 mM in increments of 5 mM. It was
concluded that the compounds interacted with DNA or bound
to DNA in the event of a decrease or increase in the emission
spectra of the compounds. As a result of the computational
processes, taking the effects into account, DNA binding
constant, n is the number of binding sites of the compound to
CT-DNA, and [DNA] is the concentration of DNA in base pairs.
An increase in the emission intensity of compound 4 at
401 nm was observed with increasing DNA concentration
according to the emission spectrum obtained using 330 nm
excitation (Fig. 8a). The binding constant K was determined
b
from the plot of log(F ꢁ F)/F vs. log[DNA] and was found to be
0
2
ꢁ1
8
.6 ꢀ 10 M (Fig. 8b). A regular increase in the emission
intensity of compound 5 at 431 nm was observed with
increasing the DNA concentration; the emission maximum
wavelength of this compound shied slightly (1 nm) (Fig. 9a).
28
constants or extinguishing constants were calculated.
The quantitative estimation of extinguishing experiments in
terms of the binding constant was conducted using the
b
The K constant calculated from the linear graph obtained was
2
ꢁ1
28
9.3 ꢀ 10 M (Fig. 9b).
following Stern–Volmer equation:
The uorescence spectra obtained using the excitation
wavelength at 300 nm for compound 10 are shown in Fig. 10a.
When we look at the uorescence spectrum of compound 10, it
F
0
/F ¼ 1 + Ksv[Q]
F0 and F are the uorescence intensities of the compounds in can be said that a different state occurred. A slight decrease in
the absence and presence of CT-DNA, respectively. Ksv is the uorescence the intensity was observed with the addition of
3
ꢁ1
linear Stern–Volmer quenching constant and [Q] is the DNA. The quenching constant Ksv 9.8 ꢀ 10 M was calculated
concentration of CT-DNA.
from the uorescence quenching measurement at 381 nm
Another modied Stern–Volmer equation was used to
calculate the DNA binding constants of the compounds from
28
both the increase and decrease in emission intensities:
log(F ꢁ F)/F ¼ log K + n log[DNA]
0
b
F
0
and F are the uorescence intensities of the compounds in
the absence and presence of DNA, respectively, K is the binding
b
Fig. 10 Fluorescence spectra obtained by adding CT-DNA in
increasing concentrations [from bottom to top (0–50 mM) with
Fig.
8 Fluorescence spectra obtained by adding CT-DNA in increments of 2.5 mM of CT-DNA and additionally, 75 mM and 100 mM
increasing concentrations (from bottom to top (0–30 mM)) with CT-DNA] of compound 10 (a); the linear graph from which the K
increments of 5 mM of CT-DNA of compound 4 (a); the linear graph constant was calculated (b); the linear graph from which the K
b
b
from which the K
changes upon increasing the amounts of CT-DNA.
b
constant was calculated (b). The arrow shows the constant was calculated (c). The arrows show the changes upon
increasing the amounts of CT-DNA.
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b
Table 1 The calculated quenching (Ksv) and binding constants (K )
ꢁ
1
(
M ), the number of binding sites of the compound to CT-DNA (n),
2
and correlation coefficient values (R ) of the compounds that interact
with DNA
a
b
2
A
B
K
b
n
K
sv
R
2
2
3
2
2
4
5
—
Chloro
Benzyl
p-Nitro benzyl
Sulfur (S)
8.6 ꢀ 10
9.3 ꢀ 10
7.1 ꢀ 10
7.2 ꢀ 10
1.4 ꢀ 10
0.37
0.45
0.78
0.39
0.52
—
—
9.8 ꢀ 10
7.8 ꢀ 10
3.3 ꢀ 10
0.991
0.996
0.991/0.993
0.99/0.993
3
3
3
c
c
10
11
12
c
0.993/0.989
a
b
c
2
Compound. Functional Group.
R
values are given for K
b
and Ksv
constants, respectively.
Fig. 11 Fluorescence spectra obtained by adding CT-DNA in
increasing concentrations (from bottom to top (0–50 mM)) with
3
ꢁ1
K
sv, 3.3 ꢀ 10
M , was calculated using the Stern–Volmer
increments of 5 mM of CT-DNA of compound 11 (a); the linear graph equation (Fig. 12b).
from which the Ksv constant was calculated (b); the linear graph from
which the K constant was calculated (c). The arrow shows the
changes upon increasing the amounts of CT-DNA.
According to the obtained results, the uorescence intensi-
ties of some of the compounds increased with the addition of
CT-DNA while others decreased. The compounds that had
decreased uorescence intensities were compounds 10, 11 and
b
1
2. The compounds that had increased emission intensities
(
b
Fig. 10b). The calculated K constant from the linear graph
were compounds 4 and 5. The calculated mathematical data are
given in Table 1. According to these results, the compound that
had the highest binding constant was 10. DNA binding strength
ranking was 5 > 4 > 11 > 12. The quenching constants were also
calculated from here, as the emission intensity of the
compounds decreased with increasing DNA concentration for
compounds 4, 5 and 12. The found Ksv constants are compatible
3
ꢁ1
obtained was 7.1 ꢀ 10 M (Fig. 10c).
Compound 11 also showed a result similar to compound 10;
the uorescence spectra obtained using the excitation wave-
length at 300 nm are shown in Fig. 11a. A slight decrease in

uorescence intensity was observed with the addition of DNA.
3
ꢁ1
The quenching constant Ksv 7.8 ꢀ 10 M was calculated from
the uorescence quenching measurement at 386 nm (Fig. 11b).
Fig. 12a shows the uorescence spectra obtained at the
excitation wavelength of 300 nm for 12. With increasing DNA
concentration, the emission band of the compound at 384 nm
showed a steady decrease (Fig. 12a). The quenching constant
b
with the results in the K binding constants. The numbers n
found for all compounds are approximately equal to one, indi-
cating that the compounds bind 1 : 1 with DNA. Both results
showed that the functional calixarene compounds interacted by
binding to CT-DNA.
Competitive binding experiments in the presence of ethidium
bromide
One of the useful experimental methods for understanding
whether the compounds interact with CT-DNA is to monitor the

uorescence intensity of the EtBr–DNA complex using ethidium
bromide (EtBr). EtBr is one of the most sensitive uorescent
probes due to its planar structure and is connected to the DNA
29
by the intercalative mode. When the uorescence spectrum in
EtBr alone is taken up, an emission peak at about 600 nm is
29
observed and its intensity is very low due to the solvent action.
However, when CT-DNA is added to the environment, the
emission intensity of EtBr increases due to the formed EtBr +
29
DNA complex. If there is a substance that binds to DNA in the
environment, the uorescence intensity of the EtBr–DNA
29
complex decreases because the substance inhibits EtBr inter-
Fig. 12 Fluorescence spectra obtained by adding CT-DNA in action with DNA. From the resulting decrease, Stern–Volmer
increasing concentrations (from bottom to top (0–50 mM) with
increments of 2.5 mM of CT-DNA and additionally, 75 mM and 100 mM
CT-DNA) of compound 12 (a); the linear graph from which the Ksv
28
quenching constants can be calculated and the binding
strength of molecules or substances to DNA can be compared.
For these experiments, the CT-DNA + EtBr complex was
formed by adding 2 mM EtBr to 20 mM CT-DNA. The prepared
compound solutions were added to CT-DNA/EtBr solutions in
constant was calculated (b); the linear graph from which the K
b
constant was calculated (c). The arrow shows the changes upon
increasing the amounts of CT-DNA.
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concentrations from 2.5 mM to 35 mM increasing by 2.5 mM and
incubated for 3 hours. Emission spectra ranking between 500–
750 nm were recorded in each experiment using 480 nm as the
excitation wavelength.
In the obtained emission spectra, the intensities at approx-
imately 600 nm where maximum emission occurred were taken
into consideration and the necessary calculations were made
28
using the Stern–Volmer equation below:
F /F ¼ 1 + K [Q]
0
sv
0
F and F are the uorescence intensities of the CT-DNA + EtBr
complex measured in the absence and presence of the
compounds, respectively, Ksv is a linear Stern–Volmer quench-
Fig. 14 Fluorescence emission spectra of EtBr bound with CT-DNA
ꢁ5
ꢁ6
ing constant, and [Q] is the concentration of the compound(s). ([DNA] ¼ 2.0 ꢀ 10 M, [EtBr] ¼ 2.0 ꢀ 10 M) in the absence (black)
According to the results of the experiments with EtBr and and presence (other colours) of compounds 10 (a) and 12 (c) in Tris–
HCl/NaCl buffer upon the addition of CT-DNA and EtBr [from top to
compounds 4 and 5, the emission of the EtBr–DNA complex
bottom (0–35 mM)] with increments of 2.5 mM of the compounds.
Arrows represent the changes in fluorescence intensity upon
increasing the compound concentration. For compounds 10 (b) and
decreased with the increasing concentration of the compound
(Fig. 13a and c). Here, we can say that the compounds bound to
CT-DNA, preventing EtBr from binding. The Ksv constants 12 (d) linear fitting graphs were used to calculate the quenching
calculated using the Stern–Volmer equation were found to be constant Ksv based on the Stern–Volmer equation.
3
ꢁ1
3
ꢁ1
4
.3 ꢀ 10 M and 5.9 ꢀ 10 M , respectively (Fig. 13b and d).
Both compounds led to a gradual decrease in the emission
3
ꢁ1
spectrum and very close Ksv values appeared.
constant was found to be 2.4 ꢀ 10
M
(Fig. 13f). As in
Fig. 13e shows that the emission intensity of the EtBr–DNA compounds 4 and 5, it can be interpreted that this compound
complex at about 600 nm decreased with the addition of performed intercalative binding with DNA.
compound 6 at increasing concentrations. The calculated Ksv
Compounds 10 and 12 had the same effect as the other
compounds competitively substituted with EtBr, causing
a slight reduction in the emission of the EtBr–DNA complex
(
Fig. 14a and c). Stern–Volmer quenching constants were also
3
ꢁ1
2
ꢁ1
found to be 5.8 ꢀ 10 M and 1.1 ꢀ 10 M , respectively
(
Fig. 14b and d). Unlike other compounds, compound 11
increased the EtBr emission intensity. The obtained emission
spectrum is not included here because a different effect
occurred. Some studies in the literature have shown that some
molecules can bind to the cavity of the DNA by the groove
binding mode through hydrophobic interactions, and in this
case, the EtBr–DNA complex can cause an increase in the
36
emission intensity. The same effect may apply for compound
1.
1
The calculated quenching constants of the compounds using
the reductions in the emission intensity of the CT-DNA + EtBr
complex are given in Table 2. According to the calculated Ksv
values, compounds 5 and 10 achieved the strongest binding
with CT-DNA, while the compounds following them were 4 > 6 >
ꢁ1
Table 2 The calculated quenching constants (Ksv) (M ) and correla-
2
tion coefficient values (R ) of the compounds that interact with CT-
DNA
Fig. 13 Fluorescence emission spectra of EtBr bound with CT-DNA
(
and presence (other colours) of compounds 4 (a), 5 (c), and 6 (e) in
Tris–HCl/NaCl buffer upon addition of CT-DNA and EtBr [from top to
bottom (0–35 mM)] with increments of 2.5 mM of the compounds.
Arrows represent the changes in the fluorescence intensity upon
[DNA] ¼ 2.0 ꢀ 10ꢁ5 M, [EtBr] ¼ 2.0 ꢀ 10 M) in the absence (black) Compound
ꢁ6
ꢁ1
2
Functional group
Ksv (M
)
R
3
3
3
3
2
4
5
6
—
4.3 ꢀ 10
5.9 ꢀ 10
2.4 ꢀ 10
5.8 ꢀ 10
1.1 ꢀ 10
0.989
0.989
0.987
0.997
0.988
Chloro
Methyl
Benzyl
Sulfur(S)
increasing the compound concentration. For compounds 4 (b), 5 (d) 10
and 6 (f), linear fitting graphs were used to calculate the quenching 12
constant Ksv based on the Stern–Volmer equation.
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Table
3 The binding free energy scores for the synthesized
compounds with DNA
ꢁ1
Compound
Binding free energy (kcal mol )
4
5
6
7
1
1
1
ꢁ10.9
ꢁ15.2
ꢁ11.4
ꢁ10.9
ꢁ20.1
ꢁ13.5
ꢁ10.0
0
1
2
12, respectively. Compound 10 had the highest binding
constant in uorescence studies and it gave the same value as
compound 5 in competitive displacement studies with EtBr.
Molecular docking studies
To predict the noncovalent binding conformations and affini-
ties of the newly synthesized compounds toward DNA, molec-
ular docking was carried out. The predicted free energy values of
binding are given in Table 3. Active compounds 10, 5, and
inactive compound 12, were selected for comparison according
to the experimental results of their binding strength to DNA.
The most energetically protable poses of 10, 5 and 12 in the
DNA binding site are given in Fig. 15 with both the three-
dimensional (3D) structures and the molecular surface repre-
sentations (Fig. 15a–c) and two-dimensional (2D) interaction
diagrams (Fig. 15d–f). Docking gures for the other compounds
4, 6, 7 and 11 are presented as ESI (Fig. S5†). As seen in Fig. 15,
the interaction of the compounds with the DNA was dominated
by noncovalent C–H/p, hydrogen bonding and water-
mediated contacts. For the docking of the compounds 10, 5
and 12 to DNA, binding free energy values of ꢁ20.1, ꢁ15.2, and
ꢁ
1
ꢁ
10.0 kcal mol were predicted, respectively. Lower binding
energy values indicate more preferable bonding interactions
between compound–DNA complexes. It was observed that
compound 12 with the highest binding energy, interacts less
Fig. 15 Three-dimensional (3D) docked structures with molecular
with DNA than other compounds. Five- and six-membered rings surface representations (left column) and two-dimensional (2D)
of benzothiazole groups of 12 interacted with the DNA residues interaction plots (right column) obtained for the most energetically
profitable poses of the compound–DNA complexes: (a) and (d)
A17 and A18 via C–H/p interactions. In the case of compound
compound 10; (b) and (e) compound 5; (c) and (f) compound 12 (PDB
ID: 1BNA).
10, it was predicted that there were many more C–H/p inter-
action networks with DNA residues as compared to 12: two
between the toluene ring bound to benzimidazole group and
DNA residues C11 and A17, ve- and six-membered rings of
other benzimidazole groups and G12, the ring of the tert-butyl
methoxybenzene group of 10 and the G16 residue. For the
docking of compound 5, hydrogen bonding and O–H/p
interactions as well as C–H/p interactions with G12, T19, A18
residues, and water molecules were observed.
purication. DNA (supercoiled pBR322) and restriction
enzymes (BamHI and HindIII) were purchased from Thermo-
Fischer Scientic. Calf thymus DNA was supplied by Sigma.
1
13
Measurements of H NMR and C NMR spectra were recorded
in CDCl and DMSO-d on a Varian MR 400 MHz spectrometer
3
6
using TMS as an internal standard. A Perkin Elmer 100 FTIR
spectrophotometer was used to record the infrared spectra of all
ꢁ1
compounds (4000–400 cm ). Mass spectra were obtained using
Bruker Microex LT MALDI-TOF mass spectrometer.
Elemental analyses (C, H, and N) were performed using a Leco
Experimental
Reagents and techniques
a
Chemicals and solvents were obtained from commercial sour-
ces (Merck and Sigma) and were used without further
932 CHNS analyzer. The UV-vis. measurements were performed
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t
on a Shimadzu UV 1800 UV-vis spectrometer. A Perkin Elmer LS NMR (DMSO-d
6
): d ppm 1.07 (s, 18H, Bu ), 3.61 (d, J ¼ 12.8 Hz,
5
5 Fluorimeter was used for uorescence spectra. Microwave 4H, ArCH Ar), 3.97 (s, 6H, OCH ), 4.27 (d, J ¼ 12.8 Hz, 4H,
2
3
irradiated reactions were performed using a CEM MDS-2000, ArCH Ar), 7.07–7.29 (overlapped, 6H, Calix. ArH), 7.43–7.72 (m,
2
and a household microwave oven. The determination of the 4H, Calix. ArH, Benz. ArH), 8.05 (s, 4H, Benzyl. ArH), 8.81 (s, 2H,
1
3
melting points was performed using a B u¨ chi B-540 instrument. OH), 12.76 (s, 2H, Benzyl. NH). C NMR (DMSO-d
Analytical TLC was performed on precoated silica gel plates 155.3, 147.7, 145.4, 142.7, 135.1, 134.8, 132.3, 132.1, 128.8, 128,
SiO , Merck PF254). All aqueous solutions were prepared with 127.7, 126.5, 125.3, 124.7, 118.1, 64.0, 34.5, 31.6, 31.5. MALDI-
): d ppm
6
(
2
+
puried water or ultrapure water puried by a Millipore Milli-Q TOF MS: (CHClNO ¼ 864.32 m/z) 864.57 [M ] (Fig. S1†). Anal.
Plus water purication device.
2 4 4
calc. for C52H50Cl N O : C, 72.1; H, 5.82; N, 6.47%. Found: C,
7
1.6; H, 6.2; N, 5.9%.
Compound 6. Compound 6 was obtained in 45% yield. Mp
Synthetic procedures
ꢂ
ꢁ1
1
2
59–261 C. FTIR: 1664, 1628 cm (C]N). H NMR (DMSO-d ):
6
The p-tert-butylcalix[4]arene 1 and its derivatives 2 and 3, and 8
t
d ppm 1.08 (s, 18H, Bu ), 2.46 (s, 6H, ArCH
H, ArCH Ar), 3.97 (s, 6H, OCH
), 4.28 (d, J ¼ 12.8 Hz, 4H,
ArCH
3
), 3.60 (d, J ¼ 12.8 Hz,
and
procedures.
9
were synthesized according to the literature
4
2
3
22–25
2
Ar), 6.99 (d, J ¼ 8.2 Hz, 2H, Benzyl. ArH), 7.15 (s, 4H,
Synthesis of compound 4. Here, 0.62 g (1 mmol) of
compound 3 and 1.08 g (10 mmol) of o-phenylenediamine were
weighed and 0.83 g (5 mmol) of potassium chloride (KI) was
added. Then, 50 mL of dimethylformamide (DMF) was added
and reuxed in a laboratory-type microwave device for about 3
hours, or 2 minutes in the domestic microwave oven, and then
Calix. ArH), 7.35 (s, 2H, Benz. ArH), 7.44 (d, J ¼ 8.2 Hz, 2H,
Benzyl. ArH), 7.77–8.19 (m, 5H, Calix. ArH, OH), 8.77 (s, 1H,
1
3
OH), 12.8 (bs, 2H, Benz) NH. C NMR (DMSO-d ): d ppm 154.9,
6
1
1
51.8, 151.7, 147.7, 132.4, 132.1, 131.3, 130.8, 129.3, 128.8,
27.4, 126.5, 126.4, 123.5, 121.5, 64, 34.5, 31.5, 31.4, 21.8.
+
MALDI-TOF MS: (CHNO ¼ 824.43 m/z) 824.07 [M ] (Fig. S2†).
ꢂ
heated to 80 C for 48 hours. To the cooled mixture was added
56 4 4
Anal. Cal. for C54H N O : C, 78.6; H, 6.84; N, 6.79%. Found: C,
50 mL of ethyl acetate and water. The mixture was taken into the
7
7.6; H, 7.2; N, 6.5%.
Compound 7. Compound 7 was obtained in 25% yield. Mp
separating funnel, shaken and allowed to separate. The nal
product formed on the interface was obtained and puried from
ethanol. Compound 4 was obtained in 35% yield. Mp 284–
ꢂ
ꢁ1
ꢁ1
1
2
2
75–277 C. FTIR: 1624 cm (C]N), 1521 cm (NO ). H NMR
t
(
DMSO-d ): d ppm 1.09 (bs, 18H, Bu ), 3.60 (d, J ¼ 13.1 Hz, 4H,
ꢂ
ꢁ1
1
6
2
1
86 C. FTIR: 1620 cm (C]N). H NMR (DMSO-d
6
): d ppm
Ar), 3.94 (s, 6H,
Ar), 7.12 (bs, 8H, Calix.
ArH, Benzim. ArH), 7.54 (dd, J ¼ 7.04 Hz, 4H, Benzim. ArH), 8.05
s, 4H, Calix. ArH), 8.74 (s, 2H, OH), 12.54 (s, 2H, Benzim. NH).
ArCH Ar), 3.96 (bs, 6H, OCH ), 4.26 (d, J ¼ 13.1 Hz, 4H,
t
2
3
.05 (s, 18H, Bu ), 3.57 (d, J ¼ 13.1 Hz, 4H, ArCH
), 4.24 (d, J ¼ 13.1 Hz, 4H, ArCH
2
ArCH Ar), 6.64–6.81 (m, 4H, Calix. ArH), 7.07–7.37 (m, 4H,
2
OCH
3
2
Calix. ArH), 7.85–7.93 (m, 4H, Benzyl. ArH), 8.04–8.21 (bs, 2H,
Benz. ArH), 8.59 (s, 1H, OH), 9.04 (s, 1H, OH), 13.32 (s, 2H,
Benzyl. NH). C NMR (DMSO-d
(
1
3
6
): d ppm 159.6, 156.6, 151.6,
1
3
C NMR (DMSO-d ): d ppm 155, 153.2, 152.2, 151.7, 150.5,
6
1
1
8
7
51.3, 147.9, 147.7, 136.5, 135.0, 132.8, 130.5, 129.2, 128.3,
1
1
47.7, 132.4, 130.7, 128.8, 127.5, 126.4, 123.7, 121.9, 121.6,
26.5, 124.2, 112.8, 64, 34, 31.5, 31.3. MALDI-TOF MS: (CHNO ¼
17.7, 64.0, 34.5, 31.6, 31.5. MALDI-TOF MS: (CHNO ¼ 796.4 m/
+
86.37 m/z) 886.60 [M ] (Fig. S2†). Anal. cal. for C H N O : C,
+
52 50 4 4
z) 796.12 [M ] (Fig. S1†). Anal. cal. for C52
H
52
N
4
O
4
: C, 78.36; H,
0.4; H, 5.68; N, 9.47%. Found: C, 70.6; H, 6.2; N, 9.5%.
6.58; N, 7.03%. Found: C, 78.0; H, 6.8; N, 7.1%.
Compound 10. Compound 10 was obtained in 35% yield. Mp
ꢂ
ꢁ1
1
1
90–192 C. FTIR: 1624 cm
(C]N). H NMR (DMSO-d
Ar), 3.95
Ar), 5.31 (s, 4H, NCH ),
.691 mmol of o-phenylenediamine derivatives [4-methyl-o- 6.73–7.37 (m, 12H, Calix. ArH, ArH), 7.37–7.78 (m, 6H, Calix.
6
):
General procedure for synthesis of compounds 5–7 and 10, 11
t
d ppm 1.08 (s, 18H, Bu ), 3.61 (d, J ¼ 13.5 Hz, 4H, ArCH
2
Here, 0.5 g (0.805 mmol) of the dialdehyde compound (3), (bs, 6H, OCH
1
3
), 4.10–4.45 (m, 4H, ArCH
2
2
phenylenediamine (0.21 g), 4-nitro-o-phenylenediamine (0.26 ArH, ArH), 7.79–8.17 (m, 5H, ArH) 8.18–8.45 (m, 2H, ArH), 8.55
g), and 4-chloro-o-phenylenediamine (0.24 g), N-benzyl-o-phe- (s, 1H, ArH), 8.82 (s, 2H, ArOH). MALDI-TOF MS: (CHNO ¼
+
nylenediamine (0.34 g), and N-p-nitrobenzyl-o-phenylenedi- 976.49 m/z) 976.04 [M ] (Fig. S3†). Anal. cal. for C66
H
N
64
O
: C,
4
4
amine (0.41 g), respectively] were weighed. Then, 0.28 g (1.691 81.1; H, 6.60; N, 5.73%. Found: C, 80.6; H, 6.8; N, 5.6%.
mmol) of KI was added, followed by 50 mL of DMF, and kept in
Compound 11. Compound 11 was obtained in 45% yield. Mp
the household microwave device for approximately 1–2 min. 259–262 C. FTIR: 1624 cm (C]N), 1519 cm (NO
ꢂ
ꢁ1
ꢁ1
1
2
). H NMR
): d ppm 0.96 (s, 18H, Bu ), 3.51 (d, J ¼ 13.0 Hz, 4H,
Ar), 3.91 (bs, 6H, OCH
), 4.18 (d, J ¼ 13.0 Hz, 4H,
Ar), 5.71 (s, 4H, NCH ), 6.89 (m, 2H, Calix. ArH), 7.03–7.50
t
Aer being removed from the device and cooled slightly, it was (DMSO-d
6
ꢂ
heated to 80 C by connecting it back to the condenser for 24–96 ArCH
hours (96 hours for 5, 10 and 11, 72 hours for 6 and 24–48 hours ArCH
2
2
3
2
for 7). It was cooled when the reaction was determined to be (m, 11H, Calix. ArH, ArH), 7.50–7.89 (m, 6H, ArH), 7.97–8.06 (m,
over by TLC and FTIR. To the mixture was added 50 mL of ethyl 1H, ArH), 8.16 (d, J ¼ 8.61 Hz, 4H, ArH), 8.30–8.47 (m, 1H,
1
3
acetate and water. The mixture was taken into the separating ArOH), 8.55–8.77 (m, 1H, ArOH). C NMR (DMSO-d ): d ppm
6
funnel, shaken and allowed to separate. The ethyl acetate phase 159.2, 154.9, 154.1, 151.6, 147.3, 145.3, 143.2, 136, 132.2, 131.3,
was obtained, dried with CaCl , then the solvent was removed 130.8, 129.9, 128.9, 127.8, 126.4, 124.4, 122.7, 119.5, 111.2, 63.9,
2
and washed with ethanol.
47.7, 34.3, 34.1, 31.3. MALDI-TOF MS: (CHNO ¼ 1066.46 m/z)
+
Compound 5. Compound 5 was obtained in 40% yield. Mp 1066.05 [M ] (Fig. S3†). Anal. cal. for C66
62 6 8
H N O : C, 74.3; H,
ꢂ
ꢁ1
1
>
300 C (decomposition). FTIR: 1664, 1621 cm (C]N). H 5.86; N, 7.87%. Found: C, 74.6; H, 6.0; N, 7.7%.
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Synthesis of compound 12
A
260/A280 ratio between 1.8–1.9, showing that the DNA was
30
sufficiently transformed into the free form. Taking into
account the absorbance at 260 nm, the 3 (molar absorption
coefficient) value of 6600 M
Here, 0.62 g (1 mmol) of compound 3 was weighed and
1.068 mL (10 mmol) of 2-aminothiophenol and 0.83 g (5 mmol)
ꢁ
1
ꢁ1
cm
was determined. The
of KI were added. Next, 50 mL of DMF was added and reuxed
for 2–3 hours in a laboratory microwave. To the cooled mixture
was added 50 mL of ethylacetate and water. The mixture was
taken into the separating funnel, shaken and allowed to sepa-
rate. The ethyl acetate phase was separated off, dried with
compounds were prepared by dissolving in 0.125 mM stock
solutions in DMF. The proportion of DMF in the measured
solution was kept at 2%. This ratio was used because smaller
amounts of the materials precipitated. The volumes were
completed by adding a buffer solution (10 mM Tris–HCl +
50 mM NaCl pH 7.2) to a nal volume of 3 mL. The prepared
samples were incubated at room temperature and in the dark
for 24 hours. Fluorescence spectrophotometer measurements
were performed in a 3 mL cuvette. UV-vis spectrophotometer
measurements were made using the same samples as in uo-
rescence spectroscopy. In EtBr studies, EtBr was dissolved with
the same buffer solution and prepared freshly for each
measurement. Samples prepared with EtBr were incubated for 3
hours at room temperature and in the dark.
MgSO , and the solvent removed and puried from hot ethanol.
4
ꢂ
Compound 12 was obtained in 55% yield. Mp 258–262 C FTIR:
ꢁ
1
ꢁ1
1
1
0
658 cm (C]N), 1373 cm (C–S). H NMR (CDCl
3
): d ppm
Ar), 4.03 (s, 6H,
Ar), 6.60 (bs, 1H, Calix.
ArH), 6.72 (bs, 1H, Calix. ArH), 6.84 (s, 1H, Calix. ArH), 6.94 (s,
t
.99 (s, 18H, Bu ), 3.57 (d, J ¼ 13.3 Hz, 4H, ArCH
), 4.36 (d, J ¼ 13.3 Hz, 4H, ArCH
2
OCH
3
2
2
6
1
H, Calix. ArH), 7.18 (d, J ¼ 6.5 Hz, 2H, Thia. ArH), 7.35 (t, J ¼
.5 Hz, 2H, Thia. ArH), 7.48 (t, J ¼ 6.5 Hz, 2H, Thia. ArH), 7.69 (s,
H, Calix. ArH), 7.82–7.90 (m, 4H, Calix. ArH, ArOH), 8.04–8.08
1
3
(m, 2H, Thia. ArH). C NMR (CDCl ): d ppm 156.2, 154.3, 151.3,
3
1
1
8
48.6, 148, 137, 136.8, 134.8, 131.6, 129.1, 127.8, 125.9, 122.5,
21.4, 118.3, 63.6, 34.1, 31.2, 31.1. MALDI-TOF MS: (CHNOS ¼ Molecular docking computations
+
50 2 4 2
30.32 m/z) 830.70 [M ] (Fig. S4†). Anal. cal. for C52H N O S :
Before the molecular docking, the geometries of the initial
C, 75.15; H, 6.06; N, 3.37; S, 7.72%. Found: C, 74.8; H, 6.2; N, 3.4;
S, 7.80%.
structures of the ligands considered were optimized by using
33
the Gaussian 09 soware with the semi-empirical parametric
34
method 6 (PM6). The crystal structure of the DNA dodecamer
was retrieved from the RCSB Protein Data Bank (http://
DNA studies
2
8b
www.rcsb.org/pdb/), under the accession code 1BNA. Molec-
ular Operating Environment (MOE) soware was used for
molecular docking studies. The co-crystallized water molecules
pBR322 plasmid DNA studies. For these studies, each
material and prepared buffer solution was autoclaved and
sterilized before use. pBR322 plasmid DNA was prepared as
3
5
ꢁ
1
farther than 4.5 A˚ from ligand or DNA were deleted from the
a 0.5 mg mL plasmid DNA solution with buffer prepared as
0 mM Tris–HCl and 1 mM EDTA at pH 7.4. The compounds
crystal structure. DNA–ligand complexes were energy-
minimized to a gradient of 0.01 kcal (mol
1
ꢁ
1
ꢁ1
˚
A ), and proton-
were prepared as stock in DMF at 10 000 MM. The buffer
solution prepared for both DMF and DNA was used in half-
dilution processes (5000, 2500, 1250, 625 mM). The prepared
stock DNA solution contained 0.5 mg mL plasmid DNA for
each substance (30 mL samples) and was incubated in the dark
in a water bath at 37 C for 24 hours. At the end of this period, 10
mL samples were taken from each sample and mixed with 3 mL
of dye and loaded onto 1% agarose gel [prepared with 1XTAE
ated by the force eld AMBER99. Charges on the DNA and
ligands were assigned using the force elds AMBER99 and
MMF94X, correspondingly. Possible ligand-binding sites were
identied by the “Site Finder” module of the MOE. The Triangle
Matcher Algorithm and scoring functions of London dG and
GBVI/WSA dG were used to produce 30 poses of each ligand. All
poses generated with docking were analyzed and the best-
scored pose with the lowest binding energy and root mean
square value for each compound was selected for further
investigation of interactions with the DNA.
ꢁ1
ꢂ
(Tris + acetic acid + EDTA)] and electrophoresed in 1XTAE buffer
at 60 V for 2 or 3 hours. At the end of this time, the gel was
ꢁ1
removed and 10 mL of 0.5 mg mL (100 mM) EtBr solution was
shaken for 30 minutes, aer which the images of the gels were
recorded under a UV lamp. In experiments with restriction
enzymes, 8 mL samples were taken from samples incubated
Conclusions
overnight for both BamHI and HindIII. Then, 1 unit of enzyme In this study, the synthesis and characterization of six different
(
0.1 mL) and buffer (1 mL) was added to these samples and p-benzimidazole-derived and a benzothiazole-derived p-tert-
ꢂ
incubated for one hour at 37 C in a hot water bath. At the end of butylcalix[4]arene compounds were performed and the inter-
this period, the samples were removed from the hot water bath actions of the compounds obtained with pBR322 plasmid DNA
and immediately transferred to an ice bath. The same opera- and CT-DNA were evaluated by various methods. Compounds
tions described above were performed and the images were showing cleavage effects on pBR322 plasmid DNA were 6 and 10
recorded.
(at the highest concentration, 10 000 mM), while other
CT-DNA studies. CT-DNA was dissolved in 10 mM Tris–HCl + compounds 5, 7, 11 and 12 caused different changes in the
ꢂ
5
0 mM NaCl pH 7.2 buffer solution. It was kept at 4 C for 1–2 structure of the plasmid DNA. CT-DNA interaction experiments
days at equilibrium. At the end of this period, the DNA was were evaluated using UV-vis and molecular uorescence spec-
taken and its absorbances at 260 nm and 280 nm were tral studies. The results obtained from UV-vis studies showed
measured and a stock solution was prepared according to the that there was a hyperchromic effect, proving that all of the
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compounds, except compound 7, interact with DNA. The stoi- scary day by day. For this reason, the design and synthesis of
chiometries of the binding modes of the compounds with DNA new compounds with antibacterial properties have been among
and the evaluation of the interaction strengths of the complexes the subjects that scientists have worked hard on. For this
formed were performed by uorescence spectroscopy. The rst purpose, it was seen in this study that two of the synthesized
of these studies showed that the emission intensities of compounds could break down the plasmid DNA, which is the
compounds 4 and 5 were increased by the addition of DNA, protective DNA of bacteria.
whereas those of compounds 10–12 decreased. DNA binding
constants (K ) and the number of molecules that bind to DNA completely cure cancer, one of the biggest health problems of
were calculated for the compounds giving both conditions. today, requires the synthesis of new and effective molecules. For
On the other hand, the absence of the drug(s) that
b
2
Compounds 7 and 10 had the highest K constants (9.3 ꢀ 10
this purpose, the studies carried out with CT-DNA are prelimi-
b
ꢁ1
3
ꢁ1
M
and 7.1 ꢀ 10
M
, respectively). In the displacement nary studies and the active compounds can be used in future
experiments performed with EtBr, results in line with previous experimental in vitro anticancer studies.
studies were obtained. Compounds 5 and 10 had the highest Ksv
M , respectively).
Consequently, when the obtained results were combined, it was
12,16,30,32,37–39
3
ꢁ1
3
ꢁ1
constants (5.9 ꢀ 10
M
and 5.8 ꢀ 10
Conflicts of interest
evaluated that the compounds showed binding strengths There are no conicts to declare.
toward CT-DNA of the order of 10 > 5 > 4 > 11 > 6 > 12.
Docking of the synthesized compounds into the DNA
binding site was carried out. Docking results complemented
Acknowledgements
well the experimental results on binding strength of the We would like to thank the Research Foundation of Selcuk
compounds to DNA. Lower binding free energies and much University, Konya Turkey [grant number BAP 2017/17201115]
more interaction networks were observed for compounds that for its nancial support of this work produced from a part of
¨
more strongly interacted with the DNA than weaker ones. For ¸S . Ç. Ozkan's PhD thesis. In addition, the author would like to
the docking of compounds 10, 5 and 12, the DNA residues C11, thank the Department of Chemistry of Gebze Technical
G12, G16, A17, A18 and T19 played a major role in the binding University for its laboratory and device facilities.
site.
Experimental results have shown that the benzyl ring of
compound 10 is more potent between the nucleotides of DNA
Notes and references
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36
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