Synthesis of 6,12-Methanobenzo[d]pyrano[3,4-g][1,3]dioxocin-1(12H)-ones and Study of Their Interaction with DNA and β-Lactoglobulin

Article abstract of DOI:10.1002/ejoc.201701224

An efficient synthesis of 6,12-methanobenzo[d]pyrano[3,4-g][1,3]dioxocin-1(12H)-ones, a new class of 2,8-dioxabicyclo[3.3.1]nonanes, starting from 2-hydroxychalcones or their analogues and 4-hydroxy-6-methyl-2H-pyran-2-one has been achieved by use of amberlyst-15, a sulfonated polystyrene resin, as a recyclable heterogeneous catalyst. The methodology involves a domino sequence of Michael addition and two-stage heterocyclization. Among the synthesized products, two show significant ct-DNA-binding properties and all show strong binding with the carrier protein β-lactoglobulin (β-lg). A study of antibacterial properties conducted on two bacterial species by using four compounds showed that two of them are moderately active.

Full text of DOI:10.1002/ejoc.201701224

A Journal of
Accepted Article
Title: Synthesis of 6,12-methanobenzo[d]pyrano[3,4-g]
[1,3]dioxocin-1(12H)-ones and study of their interaction with DNA
and β-lactoglobulin
Authors: Asok Kumar Mallik, Nayim Sepay, Chayan Guha, and Sanhita
Maity
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201701224
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10.1002/ejoc.201701224
European Journal of Organic Chemistry
FULL PAPER
Synthesis of 6,12-Methanobenzo[d]pyrano[3,4-g][1,3]dioxocin-
1(12H)-ones and Study of Their Interaction with DNA and β-
Lactoglobulin
Nayim Sepay,[a] Chayan Guha,[a] Sanhita Maity,[a] Asok K. Mallik*[a]
Abstract: An efficient synthesis of 6,12-methanobenzo[d]pyrano-
[3,4-g][1,3]dioxocin-1(12H)-ones, a new class of 2,8-dioxabicyclo-
OH
OH
HO
[3.3.1]nonanes, starting from 2-hydroxychalcones or their analogues
OH
and 4-hydroxy-6-methyl-2H-pyran-2-one has been achieved by use
O
O
of amberlyst-15, a sulfonated polystyrene resin, as a recyclable
heterogeneous catalyst. The methodology involves domino
O
O
O
O
O
O
HO
OH
O
R1O
a
OMe
HO
HO
OH
H
H
H
sequence of Michael addition and two-stage heterocyclisation.
Among the synthesized products, two show significant ct-DNA-
binding property and all show strong binding with the carrier protein
β-lactoglobulin. Study of antibacterial property done on two bacterial
species using four compounds showed that two of them are
moderately active.
O
OH
OH
Me
OR
R1
R2
HO
OH
HO
R2
HO
Diinsininol:R1= b-glucose, R2 = OH
Diinsinin:R1= b-glucose, R2 = H
Procyanidin A1 : R1=H, R2 =OH Dracoflavan C : R=Me
Procyanidin A2 : R1=OH, R2 =H
Dracoflavan D : R=H
Figure 1. Several bioactive 2,8-dioxabicyclo[3.3.1]nonanes of natural origin.
Introduction
OMe
OMe
Compounds possessing a bicyclo[3.3.1]nonane framework with
one or more heteroatom are found to be very important due to
their cleft shaped structure.[1] A good number of natural products
O
O
O
OMe
H
OMe
O
HO
O
O
O
having
a 2,8-dioxabicyclo[3.3.1]nonane framework in their
OH
O
MeO
HO
4'-Hydroxy-5,6-
dehydrokawain
structural motif have been reported to show interesting biological
activities like antioxidant, anti-inflammatory, COX-2 enzyme
inhibitory, antiviral, immunomodulatory and antitumor activities.[2]
Several such compounds[2b,3] are shown in Figure 1. Again,
compounds containing a 2-pyrone moiety are known to occur in
different types of fungi[4] and they can be easily obtained by
conventional organic synthesis[4a,b,5] as well as in vitro
synthesis.[6] They are known to show antifungal, antibiotic,
anticancer, cell toxin, neurotoxin and plant toxin activities.[4,6,7] 4-
Hydroxy-6-methyl-2H-pyran-2-one (triacetic acid lactone) is the
simplest natural product of the 2-pyrone family[4a,e] and available
OH
Terreulactone D
Neurymenolide A
Figure 2. Several bioactive compounds containing a 2-pyrone moiety.
2-hydroxychalcones (1) and cyclopentane-1,3-dione (2) under
amberlyst-15 catalyzed condition and observed that when the
reaction was conducted in an aerial atmosphere it gave fused
2,8-dioxabicyclo[3.3.1]nonanes (3) as major product in a short
reaction time (3-4 h) but 2-(2-phenyl-4H-chromen-4-ylidene)-
cyclopentane-1,3-diones (4) in a longer reaction time (10-12 h).
In an inert atmosphere, however, 3 was found to be the only
product (Scheme 1).[9]
commercially. Other complex molecules containing either
a
fused or attached 2-pyrone moiety are also known as natural
products.[5,7] Several bioactive 2-pyrone derivatives[7] are shown
in Figure 2.
Being interested from the reported biological activities of
2,8-Dioxabicyclo[3.3.1]nonanes
having
V-shaped
rigid
geometry are structurally analogous to Tröger’s base.[8b]
Considering the importance of 2,8-dioxabicyclo[3.3.1]nonanes
and 2-pyrones for their biological activities briefly mentioned
above, ability of some Tröger’s base derived bicyclo[3.3.1]-
nonanes having cleft-like structure to bind with DNA[10] and
natural
2,8-dioxabicyclo[3.3.1]nonanes,
recently
several
research groups have developed synthetic routes to different
compounds containing this framework.[8] On the other hand,
development of synthetic routes to simple, attached and fused 2-
pyrones is an interesting research area in organic chemistry
even up to the present time.[4a,5,6] The common features of the
synthetic routes to 2,8-dioxabicyclo[3.3.1]nonanes are Michael
addition followed by two stage heterocyclisation. However, some
of these methods suffer from drawbacks like use of expensive
catalysts[8a] and application of harsh reaction condition like
heating in a sealed tube.[8b] Recently, we studied the reaction of
OH
R2
R1
R2
R1
R2
O
O
R1
O
1
O
H
O
O
O
O
O
3
4
2
[a]
Department of Chemistry, Jadavpur University, Kolkata 700 032,
India
Scheme 1. Formation of 2,8-dioxabicyclo[3.3.1]nonanes in our previous study.
E-mail: mallikak52@yahoo.co.in
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10.1002/ejoc.201701224
European Journal of Organic Chemistry
FULL PAPER
12
CH3CN
Amb.-15
40
110
4
47
reported Michael donor property of 4-hydroxy-6-methyl-2H-
pyran-2-one (5), [11] we set our objective to study the reaction of
2-hydroxychalcones (1) and their analogues (6) with 5 in boiling
toluene under amberlyst-15 catalyzed condition with a view to
synthesizing molecules incorporating both 2,8-dioxabicyclo-
[3.3.1]nonane and 2-pyrone moieties in their structures. As an
outcome of our endeavor, we could develop a simple and
efficient methodology for synthesis of 6,12-methanobenzo[d]-
pyrano[3,4-g][1,3]dioxocin-1(12H)ones (7 and 8). Getting these
new 2,8-dioxabicyclo[3.3.1]nonane derivatives in hand, we first
undertook a study of their interaction with DNA. Out of a total of
sixteen compounds of the series 7 and 8, two showed significant
DNA-binding property. -Lactogobulin (-lg) is a well-known
carrier protein.[12] The current literature shows that there are a
number of reports of its use for transport of several types of
biologically active molecules.[12] Since the compounds of the
series 7 and 8 are almost insoluble in water and some of them
show significant DNA binding property, it was then our interest to
investigate their interaction with -lg. This investigation also
produced some interesting results. Lastly, study of antibacterial
property done on two bacterial species using four compounds
showed that two of them are moderately active. Our findings on
the above-said synthetic, binding and antibacterial studies are
presented in this paper. It may be mentioned here that none of
the recent papers dealing with synthesis of 2,8-dioxabicyclo-
[3.3.1]nonane derivatives[8] reports any biophysical or
antibacterial study of the synthesized compounds. Moreover,
any independent study in the said areas with those compounds
did not appear in the literature subsequently.
[a] For 1mmol of each of 1a and 5; [b] Isolated yield; [c] Reflux temp.; [d]
Anhydrous; [e] Fused; Amb.-15 = Amberlyst-15
(5) under amberlyst-15 catalyzed reaction condition. The initial
success of the reaction to produce the 2,8-dioxabicyclo[3.3.1]-
nonane derivative 7a encouraged us to optimize the process by
variation of the solvent, catalyst, amount of catalyst and reaction
time under reflux condition. The results of the optimization study
are given in Table 1. Under the optimized reaction conditions,
eleven more compounds of the series 7 could be synthesized in
good to very good yield by using different 2-hydroxychalcones
(1); use of 6 instead of 1 gave four more structurally analogous
compounds (8) (Table 2). All the synthesized compounds were
new and they were characterized from their analytical and
spectral data. In case of compound 7d confirmation of the
structure was done from single crystal X-ray crystallography
Table 2. Reaction of 2-hydroxychalcones and their analogues (1) with 4-
hydroxy-6-methyl-2H-pyran-2-one (5)
Results and Discussion
Synthesis of the New Compounds 7a-l and 8a-d
The present study started with the reaction of 2-
hydroxychalcone (1a) and 4-hydroxy-6-methyl-2H-pyran-2-one
Table 1. Optimization of the reaction condition for synthesis of 7a
Entry
Solvent
Catalyst
Amt. of
Temp.
(oC)
Time
(h)
Yield[b]
(%)
Catal.[a]
1
2
Ethanol
Toluene
-
-
-
-
78[c]
24
24
110[c]
12
5
[d]
3
4
Toluene
Toluene
FeCl3
5
5
110
110
10
10
[e]
ZnCl2
10
5
6
7
8
Toluene
Toluene
Toluene
Toluene
PTSA
10
10
20
30
110
110
110
110
10
10
4
28
58
70
79
Amb.-15
Amb.-15
Amb.-15
4
9
10
11
Toluene
Ethanol
DMF
Amb.-15
Amb.-15
Amb.-15
40
50
40
110
110
110
4
4
4
75
32
55
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10.1002/ejoc.201701224
European Journal of Organic Chemistry
FULL PAPER
(CCDC No. CCDC 1548651) also. Cleft-like geometry of the
molecule is clearly evident from the ORTEP diagram (Table 2).
The mechanism of this transformation appears very simple.
The first step is an acid catalyzed Michael addition involving 1 /
6 and 5. The intermediates (9 / 10) thus formed undergoes two
consecutive heterocyclisation processes in two possible routes
leading to compounds 7 / 8 (Scheme 2).
Study of Absorption Spectra
Small molecule-DNA interaction can be easily studied and
illustrated using UV-vis spectroscopy, simple and most
a
commonly used technique.[10,15d,e] The study can be carried out
by monitoring changes in absorption properties of either DNA or
the small molecule. In aqueous 10 mM phosphate buffer solution,
i.e., at the physiological pH condition (pH = 7.4), each of the
synthesized compound of the series
7
and
8
exhibited
absorption peak near 300 nm along with
a
high energy
OH
O
Ar/R
OH
O
Ar/R
absorption. There was considerable change in the absorbance
of 7b and 7c near 230 nm and 340 nm upon binding to ct-DNA
under the above mentioned condition as shown in Figures 3 (a)
and (b). For both the compounds 7b and 7c, it was observed
that on gradual addition of ct-DNA (0 to 70 M) the absorption
intensity decreased in the 200-220 nm region with formation of a
hump near 220 nm and the same increased slightly around 340
nm, these were accompanied with ca. 8 nm bathochromic shift
of the 300 nm peak. The results thus suggest a significant
binding of these two compounds with ct-DNA.
R1
R1
a
OH
H
R1
-H2O
a
b
Ar/R
OH
O
O
OH
O
O
b
O
O
7
8
/
OH
Me
OH
Me
OH
Me
OH
Me
9 / 10
R1
R1
Ar/R
Ar/R
H
-H2O
O
O
O
O
O
O
Me
Scheme 2. Proposed mechanism for the formation of 7 / 8.
The heterogeneous catalyst was found to be recoverable
after the reaction and can be recycled effectively up to the fifth
cycle (Table 3).
Table 3. Recycling of the catalyst in the synthesis of 7a
Entry
Condition of
the catalyst
Fresh
Time (h)
Yield (%)
1
2
4
4
79
75
Run 1
3
4
Run 2
Run 3
4
4
72
69
5
Run 4
4
65
Study of Interaction with DNA
DNA is an important genetic bio-macromolecule and it
regulates the structure and function of the cell. The mechanisms
of action of drugs are related to a binding process, which
generally involves an interaction of small drug molecules with
bio-macromolecules, such as DNA, RNA or protein.[13]
Furthermore, such binding can interfere with DNA replication
and RNA transcription, providing valuable insight into how the
drugs control the gene expression.[13,14] Understanding of the
interaction mechanism between small molecules and DNA is a
matter of paramount importance as this gives very useful
information for designing new and more efficient therapeutic
agents.[15] Since the synthesized compounds 7a-l and 8a-d
possess a molecular geometry similar to Tröger’s base derived
bicyclo[3.3.1]nonanes which are known to show interesting
DNA-binding property,[10] we got interested in undertaking a
study of their binding with ct-DNA with the help of UV-vis and
fluorescence spectroscopy. In this study, it was observed that
two compounds (7b and 7c) of the series showed significant
binding with ct-DNA.
Figure 3. UV-visible spectral changes of (a) 7b (15 µM) and (b) 7c (15 µM)
and fluorescence spectral changes of (c) 7b (10 µM) and (d) 7c (10 µM) with
the gradual addition of ct-DNA at pH 7.4. The insets of fluorescence spectra
shows the plot of (Fα – F0)/(F – F0) vs 1/[DNA].
Study of Fluorescence Spectra
In order to elucidate the interaction of the synthesized
compounds 7b and 7c with ct-DNA, we studied their steady
state fluorescence. We investigated the fluorescence spectra of
both these compounds for the study as the endogenous
fluorescence property of ct-DNA is very insignificant.[15d,e] The
emission spectra of 7b (upon excitation at 295 nm) in 10 mM
phosphate buffer (pH 7.4) showed two maxima around 323 and
363 nm (Figure 3 (c)). A hyperchromism of fluorescence with a 2
nm red shift of the 363 nm peak was observed on addition of ct-
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10.1002/ejoc.201701224
European Journal of Organic Chemistry
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DNA, which collectively indicated a binding interaction of 7b with
ct-DNA. For having an insight of this interaction it was our
endeavor to get Benesi-Hildebrand plot[16] which is a plot of (Fα –
F0)/ (F – F0) vs inverse of DNA concentration, where Fα and F0
are the fluorescence intensities in presence of large excess and
Competitive Displacement Assay
Extensive applications of DNA binding dyes, whose binding
mode to DNA is well established, are found for studying the
mode of small molecule-DNA interaction. Competitive
displacement of a bound dye from DNA helix by a small
molecule indicates that the latter binds to the DNA in a similar
fashion as the former.[19] Ethidium bromide (EB), a fluorescent
DNA-intercalating dye, is often used to explore the binding mode
of a molecule with DNA. To confirm the binding mode of each of
7b and 7c with DNA, we monitored the change of fluorescence
absence of DNA, respectively, and
F is the fluorescence
intensities in presence of DNA at different concentrations. The
linearity of this plot indicates a1:1 binding of the compound with
DNA. Again, the plot of (Fα – F0)/(F – F0) vs 1/[DNA]2 shows a
non-linear plot (provided in Supporting Information (SI)), which
confirms
a
1:1 binding interaction.[16] Similar results were
obtained with 7c also. Binding constants for the 7b- and 7c-DNA
systems have been calculated from the slope of the linear plots
and their values are 7.57×104 M-1 and 3.031×105 M-1,
respectively. These values suggest that the binding between
each of 7b and 7c with ct-DNA is moderately strong.
peak at 618 nm of a preformed DNA-EB complex with
successive addition of the compound. In this experiment it was
observed that there was almost no change in intensity of the
emission spectra (Figures 4(c) and 4(d)). This indicates that
none of the compounds 7b and 7c is able to replace EB from the
DNA-EB complex, thereby confirming that their binding is of non-
intercalative type.
Iodide Quenching Experiment
Negatively charged iodide ions, efficient fluorescence
quencher for small fluorescent molecules in their aqueous
solution, provide great help in understanding the mode of
interaction between DNA and small molecules. As DNA
containing a negatively charged phosphate framework can repel
iodide ions, any small molecule bound to the DNA helix by
intercalation is well protected to approach of anionic quenchers.
The situation is different for molecules bound to DNA by
electrostatic or groove binding as they are easily approachable
by the said quenchers.[17] The binding mode of 7b and 7c to ct-
DNA was investigated using fluorescence quenching of these
compounds by KI as anionic quencher in the presence and
absence of ct-DNA. The data obtained from the experiment were
analyzed by calculating Stern-Volmer quenching constant Ksv in
the Stern-Volmer equation.[18]
F0/F = 1 + Ksv[Q]
(1)
where F0 and
F
denote the highest steady-state
fluorescence intensity of the compounds in the absence
and presence of the quencher (KI). The slope obtained from
[F0/F] vs [Q] plot is the quenching constant Ksv. The binding
mode of the small molecule can be understood from the
values of Ksv obtained in absence and presence of DNA.
Any unchanged Ksv in presence of DNA signifies
electrostatic or groove binding interaction while a decrease
of this constant under the same condition indicates
intercalation.[15d,e] It is apparent from Figure 4 (a) that KI
could effectively quench the fluorescence of 7b both in
absence and presence of ct-DNA in a buffer solution, and a
Ksv value of 8.61 M-1 and 8.28 M-1, respectively, was
obtained. A similar experiment for 7c gave analogous
results (Figure 4 (b)) (without DNA Ksv = 16.7 M-1 and in
presence of DNA Ksv = 15.1 M-1). The slight difference of the
Ksv values with and without DNA for both the compounds
indicates groove binding for them.
Figure 4. Plots of F0/F versus [KI] at λex 295 nm at pH 7.4 for (a) 7b and (b) 7c.
Fluorescence spectral changes of DNA-EB (2 µM) at pH 7.4 with the gradual
addition of (c) 7b and (d) 7c.
Molecular Docking Study
Molecular docking, a very effective theoretical tool, helps to
predict the mode and site of binding at the target specific region
of proteins, DNA etc.[12h,i] These macromolecules are generally
amphiphilic in nature and interact with an amphiphilic organic
small molecule through hydrogen bonding and weak forces like
van der Waals forces, electrostatic effects and stacking
interactions. Here, the binding of the compounds 7b and 7c with
ct-DNA (PDB code 2BNA) was realized by utilizing molecular
docking. The results of this study reveal that both the
compounds bind at a groove of DNA through weak electrostatic
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10.1002/ejoc.201701224
European Journal of Organic Chemistry
FULL PAPER
12d]
12f]
12f]
12f]
and/or π-staking interactions (Figure 5) as the molecules have
large hydrophobic parts and aromatic rings (Figures 5(b) and
5(d) ). The compound 7b interacts with G4, A5, A6, T7 and G22
residues of DNA present at the groove. On the other hand, 7c
binds at the groove of DNA where D4, A5, T7 and C23 residues
are in its vicinity. The first compound has few stacking
interactions of which the interaction of the pyrone methyl group
with A5 and A6 bases and that of the p-methylphenyl group with
sugar hydrogens of DNA backbone are noteworthy. In case of
7c, the p-methoxyphenyl group interacts through π-stacking with
DNA bases G4 and A5. Its pyrone methyl and methoxy groups
are also involved in other type of stacking interactions with DNA.
genistein,[
quercetin,[
quercitrin[
and rutin[
and
12b,g]
steroids and their derivatives[
etc. We observed that two
compounds of the series 7 show significant binding with ct-DNA.
As in the biological systems small organic molecules are carried
to the cells by carrier proteins,[20] it was our interest to study the
binding of the compounds of the series 7 and 8 with the carrier
protein β-lg. The presence or absence of interaction was
investigated using fluorescence in 5% aqueous ethanolic
phosphate buffer (pH
fluorescence study that all the compounds of the series 7 and 8
showed strong binding interaction, 7k giving the best result.
= 7.4). It was evident from the
Study of Fluorescence Spectra
The phenylalanine (Phe), tyrosine (Tyr) and two tryptophan
(Trp-19 and Trp-61) amino acid residues of the protein β-lg are
responsible for its intrinsic emission property. It shows emission
spectrum with a maximum at 337 nm on excitation at 295 nm.
The tryptophan residue Trp-19 located inside the hydrophobic
environment of β-barrel, a core structure of
β-lg consisting of
eight anti-parallel beta-strands, contributes about 80% of the
total fluorescence when the protein is excited at 295 nm. The
second tryptophan residue (Trp-61) located at the hole of the
barrel near the loop is “fluorescence silent” due to fluorescence
quenching by a disulfide bond in its vicinity.[22] The fluorescence
property of the protein often changes owing to protein–small
molecule interactions. This is why the emission spectral
techniques are widely used to investigate the binding ability of
small molecules to the protein. Here, we investigated the effect
of the compounds 7a-l and 8a-d on the intrinsic fluorescence
property of
β-lg through monitoring the change of emission
spectra of the protein. It was observed that the emission
intensity at 337 nm of β-lg decreases in the presence of
increasing concentrations of 7a-l and 8a-d at pH 7.4 as shown in
Figure 6. The observed decrease in fluorescence intensity may
be ascribed to the binding of 7a-l and 8a-d with β-lg, because
the intensity depends only on how much the Trp-19 residues of
the protein is exposed by the change of solvent polarity or the
presence of a specific quencher in its proximity. Therefore, it
may be concluded that the compounds of the series 7 and 8
probably bind at a site near to the Trp-19.[23]
Figure 5. Docking poses (a) and (c) showing the groove binding of the
compounds 7b and 7c, respectively, inside the ct-DNA. A close view of the
binding pocket of DNA demonstrating the mode of interactions of 7b and 7c
with some DNA bases, (b) and (d).
Study of Interaction with β-lg, a Carrier Protein
Carrier proteins play a key role in the transport of
molecules across the cell boundary through active transport
as well as facilitated diffusion, a type of passive transport.[20]
These macromolecules have suitable pockets where they
can bind small molecules which are transported by
them.[16a,18] β-Lactoglobulin (β-lg) is one of the main
lipocalin proteins of whey, the binding properties of which
with varieties of organic molecules have been studied
exhaustively.[12] Several important features such as its
abundance from a cheap protein source, easy purification,
possession of a number of natural nutrient binding sites,
high water solubility and relative insensitivity to proteases at
the acidic pH, make this globular protein a potential carrier
for various small organic molecules.[12a,b,21] Thus, the
current literature shows that this protein acts as carrier for a
The fluorescence intensity of the protein was found to depend
on the concentration of the small molecules 7a-l and 8a-d, which
was analyzed using the Stern-Volmer plots. The plots of F0/F vs
concentration for each of 7a-l and 8a-d gave a straight line with
an intercept as shown in the insets of the plots given in the SI
file. Only four such plots are shown in Figures 6(a)-(d). The
Stern-Volmer quenching constant Ksv at pH 7.4 for each of the
compounds (Table 1, SI) indicated strong binding between β-lg
and the compound. In this respect, the most strong binding was
observed with 7k (Ksv = 5.3×107 M-1). The Ksv values for 7b and
7c, both able to bind with ct-DNA, were 3.9×107M-1 and 2.7×107
M-1, respectively.
12c]
variety of bioactive small molecules such as folic acid,[
12d,18]
12d]
resveratrol,[
curcumin,[
and ciprofloxacin,[
fluorinated antibiotics like
For this quenching, the binding sites (n) and the binding
constant (KS) can be calculated using the following equation[24]
12b]
12e]
norfloxacin[
polyphenol flavanoids like
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10.1002/ejoc.201701224
European Journal of Organic Chemistry
FULL PAPER
log [(F0-F)/F] = log Ks + n log[Q]
(2)
Molecular Docking Study
The protein -lg having 162 amino acid residues forms eight
where the parameters F0, F and [Q] bear the same significance
as in the Stern-Volmer equation. The linearity of the plot of log
[(F0-F)/F] vs log [compound 7 / 8] at pH 7.4 as shown in SI was
found for all the compounds when the protein is excited at 295
nm. The number of binding sites (n) and Ks (binding constant)
can be obtained from the slope and intercept, respectively, of
the linear plot, which have been shown in Table 2 in SI. The
values of n at pH 7.4 are very close to 1 for all the cases.
β
long anti-parallel β-strands to build a β-barrel, called calyx, and
one α-helix at the outer surface of the barrel. This calyx is a
hydrophobic pocket capable to provide a suitable place to small
hydrophobic molecules inside it. There are also two more
binding pockets at its outer surface, a channel between α-helix
and the barrel and the region near Trp19–Arg124, which prefer
binding with amphiphilic aromatic molecules through
electrostatic and hydrogen bonding interactions.[21a]
It may be mentioned here that the excitation wavelength of
the protein and the compounds of the series 7 is same, i.e., 295
nm, and their emission maxima are 337 and 364 nm,
respectively (Figures 3(a)-(d) show the values for 7b and 7c). In
all the cases, except for 7c, 7i and 7j, only one emission peak
was observed at 337 nm instead of the expected pair of peaks,
which definitely indicates a strong binding interaction between
the partners. It is interesting to note that with increasing
concentration of the compounds 7c and 7i the intensity of the
protein fluorescence at 337 nm decreases with the formation of
a new peak at 393 nm (Figure 6), 7j also gives analogous result
(shown in SI). In these cases the new peak is far away from the
corresponding peak of the compounds (at ca. 364 nm) and the
peak saturates at their higher concentration. This interesting
observation suggests the formation of an exciplex between -lg
and the compound at the excited state.[25] The stabilization
associated with the formation of the exciplex may be assessed
from the difference between the protein peak and the new peak
which was found to be ca. 56 nm red shifted. This is indicative of
the presence of hydrogen bonding interaction between 7c / 7i /
7j and the protein.[25]
Here, all the molecules of the series 7 / 8 are amphiphilic and
bear aromatic ring in their structures. We performed docking
study of the compounds 7k and 7c with β-lg (PDB code 1bsy) to
find the binding pocket and the mode of interaction. As
mentioned above, the compound 7k shows highest binding
affinity towards β-lg while 7c forms exciplex with the protein. The
molecule 7k binds at a pocket 7.7 Å away from the Trp19
residue on the surface of the protein through various
electrostatic interactions. Possibly, this binding changes the
protein conformation in such a way that Trp19 moves inside the
hydrophobic cavity. This may be the reason why the
fluorescence quenching was observed on addition of 7k and its
analogues into the protein solution. The amino acid residues
Lys101, Val123, Arg124, Glu127, Asp129 and Asp130 are inside
the pocket (Figure 7 (a)). On the other hand, 7c binds inside the
calyx. In this case the distance between the molecule and Trp19
is about 15.5 Å. The molecule gets the amino acid residues
Leu39, Val41, Lys60, Ile71, Ile84, Ala86 and Met107 around
itself for electrostatic and π-stacking interactions and Lys69 for
H-bonding interaction (Figure 7 (b)). These interactions may be
involved in exciplex formation.
Figure 7. Docking poses showing the interaction sites of (a) the compound 7k
at the surface of β-lg and (b) the compound 7c at the calyx of this protein.
Study of Antibacterial Properties
From the photophysical studies done on sixteen
synthesized compounds it is evident that 7b and 7c show
significant binding with ct-DNA and 7k shows highest binding
Figure 6. The changes of emission spectrum of β-lg (10 µM) with increasing
concentration of the compounds (a) 7k, (b) 7b, (c) 7c and (d) 7i. The inset of
each shows the plot of F0/F versus [Concn.] at λex 295 nm at pH 7.4.
affinity towards
β-lg. In the last part of the present work we
undertook a study of antibacterial properties of these three
compounds of the series 7 and of 8a of the other series on one
gram-positive (Staphylococcus aureus) and one gram-negative
(Escherichia coli) bacteria. It was observed that among these
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10.1002/ejoc.201701224
European Journal of Organic Chemistry
FULL PAPER
four compounds 7c and 8a showed moderate antibacterial
properties towards both these bacteria (Figure 8).
Waters Xevo G2QT mass spectrometers. Elemental
analyses were done using two Perkin-Elmer 2400 Series II
C, H, N analyzers. UV-vis and Fluorescence spectra were
recorded on
Technologies
a
SIMADZU UV1700 and an Agilent
Cary Eclipse Fluorescence
spectrophotometer, respectively.
Calf thymus DNA (ct-DNA) was purchased from Sigma
Aldrich Corporation (MO, USA) and used without further
purification. The homogeneous stock solutions of ct-DNA
were prepared in 10 mM phosphate buffer (pH∼7.4) by
magnetically stirring for 24 h at 40C. The concentration of
ct-DNA was estimated spectrophotometrically using the
molar absorption coefficient, ε DNA = 6600 M−1 cm−1 at 260
nm. β-Lactoglobulin (β-lg) was isolated and purified from
cow milk.[26] The final product was lyophilized and stored at
4
0C. Using the known extinction coefficient of β-lg (0.96
Figure 8 Results of antibacterial study done for 7b, 7c, 7k and 8a using well
mg-1 mL-1 cm-1 at 280 nm) different concentrations of this
protein were prepared by dissolving β-lg samples in milli-Q-
water and then measuring the O.D. at 280 nm.
diffusion method in Nutrient Agar (NA) medium
2-Hydroxychalcones and their analogues (1) required for this
study were synthesized by base catalyzed condensation of
salicylaldehydes and methyl ketones in ethanolic KOH and all of
them were known compounds.[9,12h,27]
Conclusions
Thus, we report here
a
facile synthesis of 6,12-
methanobenzo[d]pyrano[3,4-g][1,3]dioxocin-1(12H)-ones, a new
class of 2,8-dioxabicyclo[3.3.1]nonanes, using readily available
3.2 General Procedure for the Synthesis of 6,12-
Methanobenzo-[d]pyrano[3,4-g][1,3]dioxocin-1(12H)-
ones (7 / 8)
starting materials by use of amberlyst-15,
a sulfonated
polystyrene resin, as a recyclable heterogeneous catalyst. The
methodology involves a domino sequence of Michael addition
and two-stage heterocyclisation. Among the sixteen synthesized
products, two show significant ct-DNA-binding property and all
show strong binding with the carrier protein β-lactoglobiulin.
These interesting biophysical properties are indicative of the
compounds having potential biological activities. Antibacterial
property studied on two bacterial species (one gram-positive and
one gram-negative) using four new compounds showed that two
of them are moderately active.
To a solution of a 2-hydroxychalcone or its analogue (
1 /
6
) (1 mmol) and 4-hydroxy-6-methyl-2H-pyran-2-one ( ) (1
5
mmol) in anhydrous toluene (10 mL) was added amberlyst-
15 (40 mg) at room temperature. The resulting mixture was
refluxed with stirring for 4 h. After completion of the
reaction, toluene was removed by distillation and
dichloromethane was added to dissolve the crude product
and then the catalyst was filtered off. The filtrate was
concentrated and the resulting material was subjected to
column chromatography over silica gel. The pure product
thus obtained was finally crystallized from DCM-petroleum
ether.
Experimental Section
General
3-Methyl-6-phenyl-6,12-methanobenzo[d]pyrano[3,4-
g][1,3]-dioxocin-1(12H)-one (7a): Colorless crystals (262
mg, 79% yield). M.p. 115-116 ○C. IR (KBr): ν = 1710, 1652,
1591, 1481, 1457, 1441, 1400, 1339, 1319, 1242, 1141,
1116, 1019, 995, 865, 849, 832, 763, 698 cm-1. 1H NMR
(300 MHz, CDCl3): 7.69-7.65 (m, 2H, Ar-H), 7.50-7.45
(m, 4H, Ar-H), 7.22 (dt, J = 7.2 and 1.5 Hz, 1H, H-9), 7.04
(d, J = 8.1 Hz, 1H, H-8), 6.97 (t, J = 7.2 Hz, 1H, H-10), 5.93
(s, 1H, H-4), 4.23 (t, J = 2.7 Hz, 1H, H-12), 2.37 (dd, J =
14.4 and 3.0 Hz, 1H, H-13A), 2.29 (dd, J = 14.4 and 3.3 Hz,
1H, H-13B), 2.19 (s, 3H, 3-CH3) ppm. 13C NMR (75 MHz,
CDCl3): 163.3, 162.9, 161.0, 151.4, 139.7, 129.3, 128.5,
128.2, 128.0, 125.6, 122.0, 116.3, 103.3, 100.0, 99.9, 32.8,
Standard literature procedures were followed to dry the
solvents used in the experiments. Oven-dried round-
bottomed flasks were used to perform the reactions. Thin-
layer chromatography plates (Silica gel G) were visualized
by exposure to ultraviolet light and/or iodine vapour. IR
spectra were recorded on
a
Perkin Elmer FT-IR
Spectrophotometer (Spectrum BX II) as KBr pellets.1H NMR
and 13C NMR spectra were recorded in CDCl3 on a Bruker
300 MHz NMR spectrometer. 1H chemical shifts (δ) were
reported in parts per million (ppm) taking CHCl3 peak at δ
7.26 and 13C chemical shifts taking the value for CDCl3 at
δ 77.0. Coupling constants (J) are quoted in hertz (Hz).
HRMS were recorded on Waters Xevo G2-S QTOF and
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10.1002/ejoc.201701224
European Journal of Organic Chemistry
FULL PAPER
26.7, 19.9 ppm. HRMS (TOF): calcd. for C21H16NaO4
(M+Na)+ 355.0946; found 355.0933. Anal. calcd for
C21H16O4: C, 75.89; H, 4.85. Found: C, 75.72; H, 4.96.
Anal. calcd for C21H15ClO4: C, 68.77; H, 4.12. Found: C,
68.48; H, 4.09.
10-Chloro-3-methyl-6-phenyl-6,12-methanobenzo[d]-
pyrano[3,4-g][1,3]dioxocin-1(12H)-one (7e): Colorless
crystals (264 mg, 72 % yield). M.p. 123-124 ○C. 1H NMR
3-Methyl-6-(p-tolyl)-6,12-methanobenzo[d]pyrano-[3,4-
g][1,3]dioxocin-1(12H)-one
(
7b
):
Colorless crystals (242
= 1709, 1651,
mg, 70% yield). M.p. 118-119 ○C. IR (KBr):
ν
(300 MHz, CDCl3):  = 7.65-7.62 (m, 2H, Ar-H), 7.47-7.45
1591, 1486, 1457, 1400, 1344, 1227, 1178, 1142, 1121,
1105, 1020, 1004, 992, 935, 891, 838, 814, 762 cm-1. 1H
NMR (300 MHz, CDCl3): = 7.54 (d, J = 8.1 Hz, 2H, H-
2′,6′), 7.48 (dd, J = 7.5 and 1.5 Hz, 1H, H-11), 7.27 (d, J =
8.1 Hz, 2H, H-3′,5′), 7.18 (dt, J = 7.5 and 1.6 Hz, 1H, H-9),
7.03 (br. d, J = 7.5 Hz, 1H, H-8), 6.96 (dt, J = 7.4 and 1.2
Hz, 1H, H-10), 5.92 (s, 1H, H-4), 4.21 (t, J = 2.7 Hz, 1H, H-
12), 2.40 (s, 3H, 4′-CH3), 2.40-2.28 (m, 2H, CH2), 2.18 (s,
3H, 3-CH3) ppm. 13C NMR (75 MHz, CDCl3): = 163.3,
162.9, 160.9, 151.4, 139.3, 136.8, 129.2, 128.1, 128.0,
125.6, 125.5, 121.9, 116.3, 103.3, 100.1, 99.9, 32.8, 26.7,
21.2, 19.9 ppm. HRMS (TOF): calcd. for C22H18NaO4
(M+Na)+ 369.1103; found 369.1072.
(m, 4H, Ar-H), 7.14 (dd, J = 8.7 and 2.5 Hz, 1H, H-9), 6.96
(d, J = 8.7 Hz, 1H, H-8), 5.94 (s, 1H, H-4), 4.19 (t, J = 2.7
Hz, 1H, H-12), 2.39-2.27 (m, 2H, CH2), 2.20 (s, 3H, 3-CH3)
ppm. 13C NMR (75 MHz, CDCl3):
 = 163.1, 162.6, 161.1,
151.2, 138.3, 135.4, 128.7, 128.3, 128.1, 127.1, 125.4,
122.2, 116.2, 103.3, 99.7, 99.6, 32.8, 26.6, 19.9 ppm.
HRMS (TOF): calcd. for C21H16ClO4 (M+H)+ 367.0737;
found 367.0747.
10-Chloro-3-methyl-6-(p-tolyl)-6,12-methano-benzo[d]-
pyrano[3,4-g][1,3]dioxocin-1(12H)-one
crystals (304 mg, 80 % yield). M.p. 126-127 ○C. 1H NMR
(300 MHz, CDCl3): = 7.52 (d, J = 8.2 Hz, 2H, H-2′,6′), 7.46
(7f
):
Colorless

(d, J = 2.4 Hz, 1H, H-11), 7.27 (d, J = 8.1 Hz, 2H, H-3′, 5′),
7.13 (dd, J = 8.7 and 2.5 Hz, 1H, H-9), 6.95 (d, J = 8.6 Hz,
1H, H-8), 5.92 (s, 1H, H-4), 4.17 (br. s, 1H, H-12), 2.41 (s,
3H, 4′-CH3), 2.37-2.25 (m, CH2 2H), 2.19 (s, 3H, 3-CH3)
ppm. 13C NMR (75 MHz, CDCl3): 163.1, 163.0, 161.2,
150.1, 139.4, 136.4, 129.2, 128.7, 128.6, 128.1, 127.6,
127.1, 126.8, 125.4, 117.5, 102.7, 100.1, 99.8, 32.4, 26.6,
21.2, 19.9 ppm. HRMS (TOF): calcd. for C22H17ClNaO4
(M+Na)+ 403.0713; found 403.0679.
6-(4-Methoxyphenyl)-3-methyl-6,12-methanobenzo[d]-
pyrano[3,4-g][1,3]dioxocin-1(12H)-one (7c): Colorless
crystals (253 mg, 70 % yield). M.p. 127-128 ○C. IR (KBr): ν
= 1715, 1652, 1617, 1589, 1515, 1483, 1456, 1444, 1400,
1334, 1259, 1243, 1173, 1110, 1027, 999, 937, 882, 834,
814, 768 cm-1. 1H NMR (300 MHz, CDCl3): 7.58 (d, J =
8.7 Hz, 2H, H-2′,6′), 7.48 (dd, J = 7.5 and 1.5 Hz, 1H, H-11),
7.18 (dt, J = 7.5 and 1.5 Hz, 1H, H-9), 7.02 (br. d, J = 7.5
Hz, 1H, H-8), 6.98-6.93 (m, 3H, Ar-H), 5.91 (s, 1H, H-4),
4.21 (t, J = 3.0 Hz, 1H, H-12), 3.86 (s, 3H, OCH3), 2.39-2.18
(m, 2H, CH2), 2.18 (s, 3H, 3-CH3) ppm. 13C NMR (75 MHz,
CDCl3 = 163.3, 162.9, 160.9, 160.3, 151.4, 131.9, 128.1,
128.0, 126.9, 125.6, 121.9, 116.2, 113.8, 103.3, 100.1,
99.9, 55.4, 32.9, 26.7, 19.9 ppm. HRMS (TOF): calcd. for
C22H18NaO5 (M+Na)+ 385.1052; found 385.1039.
10-Chloro-6-(4-methoxyphenyl)-3-methyl-6,12-methano-
benzo[d]pyrano[3,4-g][1,3]dioxocin-1(12H)-one
Colorless crystals (329 mg, 83 % yield). M.p. 152-153 ○C.
1H NMR (300 MHz, CDCl3):
= 7.55 (d, J = 8.7 Hz, 2H, H-2′
(7g):

and H-6′), 7.46 (d, J = 2.4 Hz, 1H, H-11), 7.12 (dd, J = 8.4
and 2.4 Hz, 1H, C-9), 6.97 (d, J = 8.7 Hz, H-3′ and H-5′),
6.94 (1H, d, J = 8.7 Hz, H-8), 5.91 (1H, s, 2H, H-4), 4.17
(br. s, 1H, H-12), 3.85 (s, 3H, OMe), 2.35-2.26 (m, 2H,
CH2), 2.91 (s, 3H, 3-CH3) ppm. 13C NMR (75 MHz, CDCl3):
163.1, 163.0, 161.2, 160.4, 150.1, 131.5, 128.1, 127.6,
127.1, 126.9, 126.8, 117.5, 113.8, 102.7, 100.1, 99.8, 55.4,
32.5, 26.7, 19.9 ppm. HRMS (TOF): calcd. for
C22H17ClNaO5 (M+Na)+ 419.0662; found 419.0647.
6-(4-Chlorophenyl)-3-methyl-6,12-methanobenzo[d]-
pyrano[3,4-g][1,3]dioxocin-1(12H)-one (7d): Colorless
crystals (293 mg, 80 % yield). M.p. 131-132 ○C. IR (KBr): ν
= 1718, 1654, 1590, 1487, 1447, 1399, 1339, 1244, 1176,
1136, 1112, 1088, 1016, 1000, 972, 937, 893, 793, 757 cm-
1
.
1H NMR (300 MHz, CDCl3): = 7.60 (d, J = 6.9 Hz, 2H,
H-3′,5′), 7.48 (dd, J = 7.8 and 1.5 Hz, 1H, H-9), 7.43 (d, J =
8.4 Hz, 2H, H-2′,6′), 7.19 (dt, J = 8.9 and 1.5 Hz, 1H, H-9),
7.02 (d, J = 8.2 Hz, 1H, H-8), 6.98 (dt, J = 7.3 Hz, 1H, H-
10), 5.92 (s, 1H, H-4), 4.21 (t, J = 2.8 Hz, 1H, H-12), 2.28
(dd, J = 13.5 and 3.0 Hz, 1H, H-13A), 2.34 (dd, J = 13.5
and 3.0 Hz, 1H, H-13B), 2.16 (s, 3H, 3-CH3) ppm. 13C NMR
3,10-Dimethyl-6-(p-tolyl)-6,12-methanobenzo[d]-pyrano
[3,4-g][1,3]dioxocin-1(12H)-one (7h): Colorless crystals
1
(284 mg, 79 % yield). M.p. 121-122 ○C. H NMR (300 MHz,
CDCl3): 7.54 (d, J = 8.4 Hz, 2H,H-2′,6′),7.36-7.24 (m,
2H, Ar-H), 6.97 (dt, J = 8.1 and 1.8 Hz, 1H, H-9), 6.93 (d,
1H, J = 8.2 Hz, H-8), 5.91 (s, 1H, H-4), 4.17 (t, J = 2.7 Hz,
1H, H-12), 2.40 (s, 3H, 4′-CH3), 2.35-2.23 (m, 2H, CH2),
2.30 (s, 3H, 10-CH3), 2.17 (3H, s, 3-CH3) ppm. 13C NMR (75
(75 MHz, CDCl3):
 = 163.2, 162.6, 161.1, 151.2, 138.3,
135.4, 128.7, 128.3, 128.1, 127.1, 125.4, 122.2, 116.2,
103.3, 99.7, 99.6, 32.8, 26.6, 19.9 ppm. HRMS (TOF):
calcd. for C21H16ClO4 (M+H)+ 367.0737; found 367.0747.
MHz, CDCl3):
 = 163.4, 163.0, 160.8, 149.2, 139.2, 137.0,
131.4, 129.1, 128.7, 128.3, 125.5, 125.3, 116.0, 103.3,
100.1, 100.0, 32.9, 26.7, 21.2, 20.5, 19.9 ppm. HRMS
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10.1002/ejoc.201701224
European Journal of Organic Chemistry
FULL PAPER
(ESI): calcd. for C23H20O4 (M+Na)+ 383.1259; found
383.0748; Anal. calcd for C23H20O4: C, 76.65; H, 5.59.
Found: C, 76.70; H, 5.54.
1H,H-10), 5.90 (s, 1H, H-4), 4.24 (br. s, 1H, H-12), 2.52 (dd,
J = 13.5 and 2.7 Hz, 1H, H-13A), 2.45 (dd, J = 13.5 and 3
Hz, 1H, H-13B), 2.17 (s, 3H, 3-CH3) ppm. 13C NMR (75
MHz, CDCl3): 163.2, 162.5, 161.0, 151.0, 142.9, 128.2,
128.0, 127.0, 126.5, 125.4, 125.3, 122.2, 116.3, 103.3,
99.8, 98.9, 33.0, 26.7, 19.9 ppm. Anal. calcd for C19H14O4S:
C, 67.44; H, 4.17. Found: C, 67.27; H, 4.26.
6-(4-Methoxyphenyl)-3,10-dimethyl-6,12-methanobenzo-
[d]pyrano[3,4-g][1,3]dioxocin-1(12H)-one (7i): Colorless
crystals (305 mg, 81 % yield) M.p. 135-136 ○C. 1H NMR
(300 MHz, CDCl3): 7.58 (d, J = 8.4 Hz, 2H, H-2′,6′)7.28
(s, 1H, H-11), 6.97-6.92 (m, 3H, Ar-H), 6.91 (d, J = 8.1 Hz,
1H, H-8), 5.90 (s, 1H, H-4), 4.16 (br. s, 1H, H-12), 3.84 (s,
3H, 4′-OCH3), 2.28 (s, 3H, 10-CH3), 2.34-2.22 (m, 2H, CH2),
2.17 (s, 3H, 3-CH3). 13C NMR (75 MHz, CDCl3):  163.4,
163.0, 160.3, 149.2, 132.1, 131.4, 128.7, 128.3, 126.9,
125.3, 116.0, 113.8, 103.3, 100.1, 99.9, 55.4, 33.0, 26.8,
20.5, 19.9. HRMS (ESI): calcd. for C23H21O5 (M+Na)+
399.1208; found 399.1067; Anal. calcd for C23H20O5: C,
73.39; H, 5.36. Found: C, 73.42; H, 5.08.
3,6-Dimethyl-6,12-methanobenzo[d]pyrano[3,4-g][1,3]-
dioxocin-1(12H)-one (8a): Colorless crystals (197 mg, 73
% yield). M.p. 112-113 ○C. 1H NMR (300 MHz, CDCl3):
7.41 (br. d, J = 7.4 Hz, 1H, H-11), 7.12 (br. t, J = 7.8 Hz,
1H, H-9), 6.90 (d, J = 7.4 Hz, 1H, H-8), 6.85 (t, J = 8.1 Hz,
1H, H-10), 5.79 (s, 1H, H-4), 4.12 (br. s, 1H, H-12), 2.19
(dd, J = 13.5 and 2.7 Hz, 1H, H-13A), 2.14 (s, 3H, 3-CH3),
2.10 (dd, J = 13.5 and 2.7 Hz, 1H, H-13B), 1.85 (s, 3H, 6-
CH3) ppm. 13C NMR (75 MHz, CDCl3):  163.3, 162.7,
160.7, 151.1, 128.0, 125.4, 121.6, 116.0, 103.2, 99.8, 99.4,
30.8, 26.7, 26.2, 19.8 ppm. HRMS (ESI): calcd. for
C16H15O4 (M+Na)+ 293.0790; found 293.0454. Anal. calcd
for C16H14O4: C, 71.10; H, 5.22%; Found: C, 71.27; H, 5.37.
8-Methoxy-3-methyl-6-phenyl-6,12-methanobenzo[d]
pyrano[3,4-g][1,3]dioxocin-1(12H)-one (7j): Colorless
crystals (217 mg, 60 % yield). M.p. 142-143 ○C. 1H NMR
(300 MHz, CDCl3): 7.72-7.68 (m, 2H, Ar-H), 7.48-7.43
(m, 4H, Ar-H), 6.93 (dt, J = 7.5 and 1.5 Hz, 1H, H-11), 6.81
(d, J = 8.1 Hz, 1H, H-9), 5.91 (s, 1H, H-4), 4.23 (t, J = 3 Hz,
6-Ethyl-3-methyl-6,12-methanobenzo[d]pyrano[3,4-g]-
[1,3]dioxocin-1(12H)-one (8b): Colorless crystals (168 mg,
77 % yield). M.p. 115-116 ○C. 1H NMR (300 MHz, CDCl3):
7.41 (br. d, J = 7.1 Hz, 1H, H-11), 7.11 (br. t, 1H, J = 6.0
Hz, H-9), 6.92-6.82 (m, 2H, H-8 and H-10), 5.80 (s, 1H, H-
4), 4.12 (br. s, 1H, H-12), 2.20-2.12 (m, 2H, CH2), 2.14 (s,
3H, 3-CH3), 1.43 (br. s, 1H, HB of 6-CH2-), 0.91 (br. s, 1H,
HA of 6-CH2-), 0.66 (br. s, 3H, CH3) ppm. 13C NMR (75
1H, H-12), 3.87 (s, 3H, 8-OCH3),2.38-2.23 (m, 2H, CH2),
2.17 (s, 3H, 3-CH3) ppm. 13C NMR (75 MHz, CDCl3): 
163.3, 163.0, 160.9, 148.1, 140.7, 139.7, 129.2, 128.5,
126.7, 125.8, 121.8, 119.9, 110.9, 103.1, 100.0, 99.9, 56.1,
32.9, 26.7, 19.9 ppm. HRMS (ESI): calcd. for C22H19O5
(M+H)+ 363.1232; found 363.1419.
MHz, CDCl3):
 = 163.4, 162.8, 160.7, 151.2, 127.9, 125.9,
8-Methoxy-3-methyl-6-(p-tolyl)-6,12-methanobenzo[d]-
pyrano[3,4-g][1,3]dioxocin-1(12H)-one (7k): Colorless
crystals (241 mg, 64 % yield). M.p. 145-146 ○C. IR (KBr): ν
= 1713, 1649, 1585, 1487, 1443, 1399, 1338, 1271, 1251,
121.6, 115.9, 103.3, 99.8, 99.7, 30.7, 26.3, 19.8, 18.5, 1.2,
0.8 ppm. HRMS (TOF): calcd. for C17H17O4 (M+H)+
285.1127; found 285.1107.
1207, 1184, 1140, 1116, 1018, 995, 944, 863, 816, 769 cm-
6-Isobutyl-3-methyl-6,12-methanobenzo[d]pyrano-[3,4-
g]-[1,3]dioxocin-1(12H)-one (8c): Colorless crystals (234
mg, 75 % yield). M.p. 114-115○C. 1H NMR (300 MHz,
1
1. H NMR (300 MHz, CDCl3):
 = 7.57 (d, J = 8.1 Hz, 2H,
H-2′,6′), 7.25 (d, J = 7.8 Hz, 2H, H-3′,5′), 7.09 (d, J = 7.5 Hz,
1H, H-11), 6.91 (t, J = 7.8 Hz, 1H, H-10), 6.79 (d, J = 8.1
Hz, 1H, H-9), 5.90 (s, 1H, H-4), 4.21 (br. t, J =2.4 Hz, 1H,
H-12), 3.85 (s, 3H, 8-OCH3), 2.39 (s, 3H, 4′-CH3), 2.36-2.24
(m, 2H, CH2), 2.16 (s, 3H, 3-CH3) ppm. 13C NMR (75 MHz,
CDCl3):  163.3, 163.1, 160.8, 148.1, 140.7, 139.1, 136.8,
129.1, 126.7, 125.6, 121.7, 119.9, 110.9, 103.1, 100.0,
99.9, 56.1, 32.9, 26.8, 21.2, 19.9 ppm. HRMS (ESI): calcd.
for C23H21O5 (M+H)+ 377.1389; found 377.1641.
CDCl3): = 7.36 br. d, J = 7.3 Hz, 1H, H-11), 7.05 (br. t, J =
7.8 Hz, 1H, H-9), 6.85-6.79 (m, 2H, H-8 and H-10), 5.69 (s,
1H,H-4), 4.05 (t, 1H, J = 2.5 Hz, H-12), 2.05 (s, 3H, 3-CH3),
2.14-1.98 (m,3H), 1.98 (br. s, 2H, 6-CH2-), 1.02 (br. s, 6H,
2×CH3) ppm. 13C NMR (75 MHz, CDCl3): = 163.3, 162.6,
160.7, 151.1, 127.94, 127.9, 125.7, 121.5, 116.0, 103.1,
101.3, 99.8, 47.6, 29.4, 26.0, 24.1, 23.9, 23.8, 19.8 ppm.
Anal. calcd for C19H20O4: C, 73.06; H, 6.45. Found: C,
73.11; H, 6.39.
3-Methyl-6-(thiophen-2-yl)-6,12-methanobenzo-
[d]pyrano-[3,4-g][1,3]dioxocin-1(12H)-one (7l): Colorless
crystals (302 mg, 78 % yield). M.p. 123-124 ○C. 1H NMR
6-Cyclopropyl-3-methyl-6,12-methanobenzo[d]pyrano-
[3,4g][1,3]dioxocin-1(12H)-one (8d): Colorless crystals
1
(300 MHz, CDCl3):

= 7.47 (br. d, J = 6.7 Hz, 1H, H-11),
(213 mg, 72 % yield). M.p. 110-111○C. H NMR (300 MHz,
7.40 (d, J = 4.3 Hz, 1H, H-5′), 7.30 (br. d, J = 2.7 Hz, 1H, H-
2′), 7.18 (t, J = 6.9 Hz, 1H, H-9), 7.07 (br. t, J = 3.9 Hz, 1H,
H-4′), 7.00 (d, J = 7. 1H, 6 Hz, H-8), 6.95 (t, J = 7.4 Hz,
CDCl3):  7.41 (1H, d, J = 6.6 Hz, Ar-H), 7.12 (1H, t, J =
7.0 Hz, Ar-H), 6.89 (2H, m, Ar-H), 5.80 (1H, s, H-4), 4.13
(1H, br. s, Aliph. CH), 2.14 (3H, s, 3-CH3), 2.15-2.02 (3H,
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201701224
European Journal of Organic Chemistry
FULL PAPER
m, CH of cyclopropyl unit and –CH2- of chroman unit), 1.11
(4H, t, J = 7.3Hz, -CH2-CH2- of cyclopropyl unit) ppm. 13C
NMR (75 MHz, CDCl3): = 163.4, 162.9, 160.6, 151.4,
128.0, 125.7, 121.5, 116.0, 103.3, 101.3, 99.9, 32.5, 28.5,
25.9, 19.8, 7.6 ppm. HRMS (TOF): calcd. for C18H17O4
(M+H)+ 297.1127; found 297.1110.
Docking Study
The molecular docking studies of 7b and 7c into the
three dimensional structures of ct-DNA and of 7c and 7k
with
package. The energy minimized conformation of each of
7b 7c and 7k, used for docking study, was obtained from
-lg were carried out using Auto Dock 4.2.0 software
,
DFT optimization. The docking study was done using an
efficient and durable algorithm, Lamarckian Genetic
Algorithm (LGA) for 126 x 126 x 126 grid box.
Recovery of the Catalyst
The catalyst amberlyst-15 separated from the reaction
mixture was washed successively with ethyl acetate and
acetone till the washings became colorless. It was then
dried in a hot air oven at 100°C for 10 h. The dried catalyst
was used in the next cycle.
Study of Antibacterial Properties
Antimicrobial activities of the compounds 7b, 7c, 7k and 8a
were tested using the well diffusion method. The liquid
Nutrient Agar (NA) medium was poured on Petri dishes and
it was allowed to solidify under laminar airflow chamber. A
sterile swab was used to evenly distribute 1mL microbial
culture over the agar surface. The plates were subjected to
drying for 15 minutes before two wells of 0.5 cm diameter
were made in the agar medium using a sterile cork borer. A
solution of each of the said compounds (50 μl) was placed
into each well. All the plates were then placed at 37°C
incubator for 24 h. The inhibition zones were measured with
a caliper.
UV-vis Absorption Spectroscopy
UV-Visible study of DNA/compound 7b and 7c system
was performed on
a
SIMADZU UV1700 at room
temperature (250C) with a four-clear-sided quartz cell (2
mL) of 1 cm path length. The respective spectra were
recorded with a matched pair of silica cuvettes over the
wavelength range 200-600 nm against a solvent blank
reference.
Fluorescence Spectroscopy
Acknowledgements
Fluorescence titrations were carried out at constant
concentration of 7b and 7c (10 μM) and with the gradual
addition of DNA (1 to 20 µM) at pH 7.4 using Shimadzu RF-
5301 PC at room temperature. Emission spectra were
recorded from 315 to 550 nm with an excitation wavelength
of 295 nm.
Financial support and spectral facilities from the PURSE and
FIST programs of the DST and CAS program of the UGC, New
Delhi to the Department of Chemistry, Jadavpur University is
gratefully acknowledged. The UPE-II program of the UGC is also
thanked for financial assistance. The authors are grateful to
Professor S. Das, Department of Physics, Jadavpur University
for extending the facility for fluorescence spectral studies and
antibacterial studies. N.S., C.G. and S.M. are thankful to the
UGC, New Delhi for their Research Fellowships.
Emission spectra for iodide quenching experiments
were recorded in presence and/or absence of 20
In this experiment, fluorescence spectra from 300–500 nm
of 50 7b and 7c in 10 M phosphate buffer (pH 7.4)
M DNA.

M

Keywords: new 2,8-dioxabicyclo[3.3.1]nonanes • amberlyst-15 •
ct-DNA • β-lactoglobiulin • binding studies
were taken with varying concentration of KI between 0-8
mM at 280 nm excitation wavelength.
In case of EB displacement assay, a solution of EB-
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of 7b and 7c (excited at 471 nm and emission recorded
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Author(s), Corresponding Author(s)*
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Title
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Key Topic* Bicyclic Ketals
Nayim Sepay, Chayan Guha, Sanhita
Maity, Asok K. Mallik*
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Synthesis of 6,12-Methanobenzo-
[d]pyrano[3,4-g][1,3]dioxocin-1(12H)-
ones and Study of Their Interaction
with DNA and β-Lactoglobiulin
Sixteen 6,12-methanobenzo[d]pyrano-[3,4-g][1,3]dioxocin-1(12H)-ones,
a
new
class of 2,8-dioxabicyclo[3.3.1]nonanes, have been synthesized and fully
characterized. Among them two show significant ct-DNA-binding property and all
show strong binding with β-lactoglobulin. Study done on four compounds, shows
that two of them exhibit moderate antibacterial properties.
*one or two words that highlight the emphasis of the paper or the field of the study
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