An efficient synthesis of 1,3-dimethyl-5-(2-phenyl-4: H -chromen-4-ylidene) pyrimidine-2,4,6(1 H,3 H,5 H)-triones and investigation of their interactions with β-lactoglobiulin

Article abstract of DOI:10.1039/c6ra13584f

An efficient method for synthesis of 1,3-dimethyl-5-(2-phenyl-4H-chromen-4-ylidene)pyrimidine-2,4,6(1H,3H,5H)-triones has been developed by treatment of 2-hydroxychalcones and 1,3-dimethylbarbituric acid in refluxing toluene in the presence of amberlyst-15 as catalyst in air. The process involves the occurrence of a domino sequence of Michael addition, cyclization and aerial oxidation. The compounds synthesized showed an interesting property of free rotation around the bond linking the two heterocyclic moieties and three of them were found to show binding property with the milk protein β-lactoglobulin (β-lg). DFT and docking (with β-lg) studies of one of the compounds have been done.

Full text of DOI:10.1039/c6ra13584f

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An efficient synthesis of 1,3-dimethyl-5-(2-phenyl-4H-chromen-4-
ylidene) pyrimidine-2,4,6(1H,3H,5H)-triones and investigation of
their interactions with β-lactoglobiulin
Received 00th January 20xx,
Accepted 00th January 20xx
Nayim Sepay, Sumitava Mallik, Chayan Guha and Asok K. Mallik*a
DOI: 10.1039/x0xx00000x
An efficient method for synthesis of 1,3-dimethyl-5-(2-phenyl-4H-chromen-4-ylidene)pyrimidine-2,4,6(1H,3H,5H)-triones
has been developed by treatment of 2-hydroxychalcones and 1,3-dimethylbarbituric acid in refluxing toluene in presence
of amberlyst-15 as catalyst in air. The process involves the occurrence of a domino sequence of Michael addition,
cyclization and aerial oxidation. The compounds synthesized showed an interesting property of free rotation around the
bond linking the two heterocyclic moieties and three of them were found to show binding property with the milk protein
β–lactoglobulin (β-lg). DFT and docking (with β-lg) studies of one of the compounds have been done.
www.rsc.org/
Construction of molecules by joining two or more biologically
active moieties has been growing as an important strategy for
Introduction
getting access to newer molecules of pharmaceutical interest10
.
Chromenes are well-known for their important biological
activities like anticancer, antitumor, antidiabetic, antioxidant, anti-
inflammatory, antianxiety, antitubercular, antitrypanosomal,
antiviral, antidyslipidemic, antihelmenthic, anticonvulsant, anti-
depressant, antiproliferative, anti-Alzheimer’s, anti-Perkinson’s
anti-Huntington’s, anti-HIV, anticoagulant, antianaphylatic,
antibacterial/antimicrobial, monoamineoxidase (MAO) inhibitor,
fungicidal, diuretic, hypothermal, vasodilatory, TNF-α inhibitor,
estrogenic, herbicidal and analgesic activitities1. Besides, some of
them have drawn attention as photochromic materials2. Though
both the 2H- and 4H-chromene moieties are often found to be
Recently, we have developed methods for synthesis of such
molecules where an indole, cyclopentan-1,3-dione or indane-1,3-
dione moiety is attached to 2-chromenes11 at the 4-position of the
latter. In case of synthesis of 2-aryl-4-(indol-3-yl)-4H-chromenes (1),
an acid-catalysed condensation of a 2-hydroxychalcone (3) and an
indole by use of amberlyst-15 as catalyst was the method11a
.
However, in the synthesis of 2-(2-phenyl-4H-chromen-4-ylidene)-
2H-indene-1,3-diones (2), analogous condensation of the two
constituent units was followed by an interesting aerial oxidation11b
.
Considering the versatile biological activities of 5-substituted
barbiturates5,6 and 2-chromenes1-4, we got interested to synthesise
compounds incorporating these two moieties. So, we undertook
present in the structural motif of a variety of natural products1a-c,3
,
development of synthetic routes to differently substituted
chromenes is still an important area of research for organic
R3
R2
R1
chemists1a-c,4
.
O
O
R2
R1
In the late 19th and early 20th centuries barbiturates (C-5
substituted barbituric acids) became very well-known for their
sedative and hypnotic properties5,6, though barbituric acid itself is
biologically inactive5,6. Throughout the 20th century, 50 out of more
than 2500 barbiturates synthesized were found to be used often for
clinical purpose and many of them are used till today7. The
pharmacological properties of barbiturates hugely depend on the
substituent(s) present at their C-57. There are a number of
strategies known in literature to get C-5 substituted barbiturates8.
One of the methylene hydrogens of barbituric acid is quite acidic
(pKa = 4.03)9. The acidic property of these methylene hydrogens is
commonly utilized to construct C-5 substituted barbiturates
through reactions like alkylation, Knoevenagel condensation,
Michael addition etc.8
O
O
R3
N
H
1
2
Fig. 1 Some 4H-chromenes attached to a biologically
important moiety at the 4-position.
a.Department of Chemistry, Jadavpur University, Kolkata 700 032, India. E-mail:
mallikak52@yahoo.co.in
Electronic Supplementary Information (ESI) available: See
DOI: 10.1039/x0xx00000x
Scheme 1 Novel synthesis of 1,3-dimethyl-5-(2-phenyl-4H-
chromen-4-ylidene)pyrimidine-2,4,6(1H,3H,5H)-triones.
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the present work, wherein a 2-hydroxychalcone (3) was made to
undergo reaction with barbituric acid or its 1,3-dimethyl derivative
in presence of amberlyst-15 as catalyst under aerial condition.
Though the reaction with barbituric acid did not give any impressive
result, that with its 1,3-dimethyl derivative yielded an interesting
class of compounds, viz., 1,3-dimethyl-5-(2-phenyl-4H-chromen-4-
ylidene)-pyrimidine-2,4,6(1H,3H,5H)-trione (6) in good yield
through the involvement an aerial oxidation in the last step
(Scheme 1). Three compounds of the series 6 have been found to
possess an interesting binding ability with the milk protein β–
lactoglobulin (β-lg). DFT and docking studies of one compound (6a)
with β-lg have been done to get an insight about its structural
features and its binding site with the said protein. All these results
are presented herein.
Table 1. Optimization of reaction condition for synthesis of 6aa
O
OH
O
O
Amberlystꢀ15
+
N
4
N
Toluene, reflux
O
O
Me
Me
in air
O
O
N
N
Me
Me
3a
O
6a
Entry
Solvent
Catalyst
Amt.
of
Time Yield
(h)
(%)b
Cat.a
10
15
20
5
1
2
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
Water
EtOH
i-PrOH
THF
MeCN
Dioxane
Amberlyst-15
Amberlyst-15
Amberlyst-15
Fused ZnCl2
8
8
8
8
8
8
8
8
8
8
8
8
8
8
58
65
70
35
39
41
38
33
-
25
30
51
57
68
3
Results and discussion
4
5
Fused ZnCl2
7
Barbituric acid, a highly polar compound, is insoluble in most of
the common organic solvents and also in water. The main problem
in carrying out the reaction between a 2-hydroxychalcone (3) and
barbituric acid was to get a suitable solvent where both the
components are soluble. Ethyl alcohol was found to be a solvent
where both of them are somewhat soluble. Thus, we carried out
reaction of each of the two 2-hydroxychalcones, (E)-3-(2-
hydroxyphenyl)-1-phenylprop-2-en-1-one (3a) and (E)-1-(4-
Fused ZnCl2
6
7
9
5
Anhy. FeCl3
Anhy. FeCl3
8
7
9
Amberlyst 15
Amberlyst 15
Amberlyst 15
Amberlyst 15
Amberlyst 15
Amberlyst 15
40
40
40
40
40
40
10
11
12
13
14
chlorophenyl)-3-(2-hydroxyphenyl)prop-2-en-1-one
(3d),
with
barbituric acid in ethanol using amberlyst-15 as catalyst, and here
we got highly polar non-crystalline materials (sparingly soluble even
in DMSO) which were difficult to purify and characterize. 1,3-
Dimethylbarbituric acid (4), which is comparatively less polar than
barbituric acid, was then chosen for the reaction and this study
started by exploring the reaction of 3a and 4 in toluene using
amberlyst-15 , fused ZnCl2 and anhy. FeCl3 as catalysts under aerial
condition (Table 1). Among these catalysts amberlyst-15 showed
superior activity, and hence scope of the reaction using this catalyst
in solvents of different polarities was investigated. Reaction in a
highly polar solvent like water did not lead to the product 6a, but
when solvents like ethanol, isopropanol, THF, acetonitrile, dioxane,
DMF and o-xylene were used, the yield of 6a gradually improved
(entries 9-16, Table 1). As toluene was found to be the best solvent
for the reaction, optimization of catalyst load was then done (Table
1). Thus, when 3a was allowed to react with 4 in the presence of
amberlyst-15 (40 mg) in toluene under reflux for 8 h in open air,
1,3-dimethyl-5-(2-phenyl-4H-chromen-4-ylidene)pyrimi-dine-2,4,6-
(1H,3H,5H)-trione (6a) was formed in 75% yield, without formation
of any 1,3-dimethyl-5-(2-phenyl-4H-chromen-4-yl)pyrimi-dine-2,4,6-
(1H,3H,5H)-trione (5a, 5 with R1=R2=H) (entry 18 in Table 1). The
reaction was found to take place also in the absence of any catalyst,
but significantly poor yield of product was obtained even in solvents
like toluene or xylene under refluxing condition (entries 21 & 22,
Table 1). After getting the optimised reaction conditions, the
15
16
17
DMF
Amberlyst 15
Amberlyst 15
Amberlyst-15
40
40
30
8
8
8
59
70
72
o-Xylene
Toluene
18
19
20
Toluene
Toluene
Toluene
Amberlyst-15
Amberlyst-15
Amberlyst-15
40
50
60
8
9
75
75
74
10
21
22
Toluene
-
-
-
-
10
10
42
o-Xylene
35
aReaction conditions: 2-hydroxychalcone (3a) (1.0 mmol)
dimethylbarbituric acid (4) (1.0 mmol), solvent, catalyst [amberlyst-15
(in mg mmol-1 of 3 or 4), fused ZnCl2 or anhy. FeCl3 (in mol % with
respect of 3 or 4), reflux under atmospheric oxygen. Yield of isolated
pure product.
,
1,3-
b
carbonyl carbons at the 4 and 6 positions of the barbiturate
moiety of each of 6a-i gave only one 13C NMR signal12. These
are very much indicative of the occurrence of free rotation
around the double bond linking the two heterocyclic moieties
in the compounds at room temperature. Thus, in the structure
of the interesting compounds being reported the canonical
form 6′ contributes to a great extent. This view is supported by
the calculated C4-C5′′ bond length for the compound 6a
(considering the optimized structure obtained from DFT
study), which is 1.421 Å, quite longer than a normal C=C bond
(e.g., 1.35 Å for ethene13). It may be commented here that a
gain of aromaticity by the oxacyclic ring of
reason for its remarkable contribution. Thus, the compounds
of the series behave as interesting examples of push-pull
developed methodology was tested on
a
series of 2-
hydroxychalcones (3) and 1,3-dimethylbarbituric acid (4) and the
results are summarized in Table 2. 2-Hydroxychalcones (3) used
contained both electron donating and electron withdrawing groups
in their phenyl rings. It was observed that the yield of compound 6
was significantly high in cases of 2-hydroxychalcones containing a
OMe group at different positions of the phenyl rings.
6′ may be the
6
alkenes which in general show low barrier of rotation around
the double bond14.
A thorough literature survey revealed that some fifty years
back reaction between flavones and 1,3-dimethylbarbituric
acid (4) by heating them in acetic anhydride was reported as a
It is very interesting to note that for each of the compounds
of the series
1
6
only one H NMR signal and only one 13C NMR
signal were observed for its two N-Me groups. Again, the
valuable colour reaction for the former group of compounds15.
2 | J. Name., 2012, 00, 1-3
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○
temperature, as two sharp 1H doublets at –60 C and as a 2H
doublet at 55 ○C. These observations indicated slow rotation
around the exocyclic double bond of such compounds at room
Table 2: Reaction of 2-hydroxychalcones (
dimethylbarbituric acid (4
)a.
3) and 1,3-
O
R1
N
O
H
N
O
Me
N
2'
O
O
O
N
N
N
Me
7'
O
O
R2
O
O
R
H
O
O
8
9
7
R1
N
Me
N
Me
OMe
N
O
O
O
O
O
O
N
N
N
Me
O
R2
O
R
O
R1 =H, R2 =Me R1 =H, R2 =Bu
;
O
O
O
O
O
O
;
R=Me/Et
R
1 =H, R2 =Ph
R1 = R2 =Me/Et/Ph
N
N
N
N
N
N
Me
Me
Me
Me
Me
Me
temperature. Our view about the occurrence of free rotation
around the C4–C5′′ bond of at room temperature as
O
O
O
6a, 75%
6b, 63%
6c, 81%
6
mentioned above thus gets support from these reported
results. It may be mentioned here that x-ray crystallographic
studies on any compound of the series 6 could not be done due to
non-availability of quality crystals.
Me
Cl
O
O
O
Cl
Cl
O
O
O
O
O
O
Reactions were carried out also with two 2-hydroxychalcone
analogues, viz., (E)-3-(2-hydroxyphenyl)-1-(thiophen-2-yl)prop-2-en-
1-one (10) and (E)-4-(2-hydroxyphenyl)but-3-en-2-one (11). The
optimized condition shown in Table 1 was found to be unsuitable
for 10 and a dark brown gummy material resulted from which no
pure product could be isolated. However, the expected product 6l
could be obtained in lower yield by carrying out the reaction at
lower temperature [40 mg catalyst, 85○C, 14 h, 21 %] or by use of
lesser proportion of catalyst [5 mg, reflux, 14 h, 25 %] or by avoiding
the catalyst [reflux, 14 h, 17%] using toluene as solvent (Scheme 2).
In the reaction of 11, no product analogous to 6 could be obtained,
instead, a 2,8-dioxabicyclo[3.3.1]nonane derivative 12 resulted in
moderate yield (40 mg catalyst, reflux, 8 h, 42 %) (Scheme 3).
N
N
N
N
N
N
Me
Me
Me
Me
Me
Me
O
O
O
6d, 60%
6e, 72%
6f, 69%
OMe
Me
Me
OMe
O
O
O
Cl
Me
O
O
O
O
O
O
N
N
N
N
N
N
Me
Me
Me
Me
Me
Me
O
O
O
6g, 80%
6h, 83%
6i, 78%
Me
OMe
OMe
O
O
The mechanistic aspect for formation of
rationalized in the following way. In an aprotic solvent toluene,
the enol content of may be increased in the presence of
amberlyst-15, a polymer-based sulfonic acid. Compound may
6 may be
O
O
O
O
4
N
N
N
N
3
Me
Me
Me
Me
O
O
also be activated by amberlyst-15 and it can take part in a
Michael reaction with the enol form of 411. The Michael adduct
13 undergoes cyclisation followed by water elimination to
afford 1,3-dimethyl-5-(2-phenyl-4H-chromen-4-yl)pyrimidine-2,4,6-
6j, 81%
6k, 85%
aReaction conditions: 2-hydroxychalcone (3a) (1.0 mmol) and 1,3-
dimethylbarbituric acid (4) (1.0 mmol), amberlyst-15 (40 mg), toluene,
reflux under atmospheric oxygen, 8 h.
(1H,3H,5H)trione
give the final product
(
5
). The compound
5 is then oxidised by air to
Here, this irreversible aerial oxidation
6.
Scheme 2 Outcome of the reaction of 10 and 4
The products of this reaction were assigned the general
structure
only15. Subsequently, condensations of tropolone, 4,9-
methano[11]annulenone and benzo[d]tropolone with and
related compounds under similar reaction conditions were
also studied16. The products were assigned the structures
6 on the basis of their analytical and UV spectral data
4
7-9,
all having significant contribution of their zwitterionic
canonical forms where barbituric acid part bears negative
charge, just like that of 6’. In the unsymmetrical compounds of
Scheme 3 Formation of a 2,8-dioxabicyclo[3.3.1]nonane
derivative 12 in the reaction of 11 and 4
series 7, H-2′ and H-7′ appeared as two broad signals at room
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Interaction with β–lactoglobulin (β-lg)
.
OH
R1
R2
β–Lactoglobulin (β-lg), one of the main lipocalin proteins of
whey, is of great interest in the food and dairy industry due to its
nutritional and functional properties17. Its properties can variously
be favorable or unfavorable in dairy products and their
O
3
SO3H
OH
OH
SO3H
R1
O
R1
R2
processing18
. High abundance, easy purification, high water
Me
O
O
O
SO3H
N
N
OH
OH
O
solubility, owning of natural nutrient binding sites and
unresponsiveness toward peptic digestion make β-lg an attractive
model protein as an encapsulator and carrier of various agents,
bioavailability enhancer and target protein for ligand-binding as
well as other studies17. Its binding property with small organic
molecules has been studied in a widespread way in naturally
occurring, isolated and chemically modified forms19. With a view to
understanding the protein-ligand binding of the compounds
synthesized by us, β–lg was used as a model protein. In this study,
the presence or absence of interaction was investigated using UV-
vis absorbance and fluorescence in 5% ethanolic aqueous
phosphate buffer (pH = 7.4). It was evident from fluorescence study
that among the compounds 6a-k, only 6a, 6b and 6i showed
significant interaction, 6a giving the best result. Interaction with the
chlorine containing compounds 6d-f was very insignificant. The
O
N
Me
N
O
13
Me
Me
O
SO3H
H2O
O
R2
R2
R2
O
O
R1
R1
Air
H
O
O
O
O
O
N
N
N
N
N
N
Me
Me
Me
Me
Me
O
5
O
6
O
14
Scheme 4 Plausible mechanistic pathway for formation of 1,3-
dimethyl-5-(2-phenyl-4H-chromen-4-ylidene)pyrimidine-2,4,6
(1H,3H,5H)-triones (6).
(all other steps are reversible) and poor solubility of the remaining compounds were only slightly soluble in ethanol, which
product in the solvent toluene may be responsible for the debarred the study of their interaction.
6
success of the reaction. To get a better understanding about
the role of aerial oxygen in the oxidation step, we repeated the
As evident from the topmost line (black in colour) of Fig. 2d, the
experiment under the optimised conditions employing 3a and UV-vis spectrum of pure 6i has three λmax values at 255.8, 383.7 and
in an argon atmosphere and found the formation of only a 484.9 nm in 5% ethanol-aqueous phosphate buffer (pH = 7.4). A
trace amount of 6a However, when the supernatant liquid of the gradual decrease and saturation (after certain level) of intensity of
4
.
reaction mixture was separated from the catalyst and kept at room the peaks at 383.7 and 484.9 with increasing concentration of
temperature for 12 h with an exposure to air, the product 6a was protein in the same buffer was observed. We performed the same
obtained as precipitate (yield: 41%). Again, when the same experiment by addition of water instead of the protein solution to
experiment was conducted in an oxygen atmosphere, it was investigate the dilution effect on the UV-vis spectrum, and a very
observed that the reaction was complete within 6 h affording 6a in insignificant decrease in intensity of the peaks at 383.7 and 484.9
a higher yield (83 %). These observations definitely indicated that was found. Indole moieties of tryptophan in β-lg are mainly
the presence of aerial oxygen was indispensable for formation of 6a responsible for its fluorescence property. This protein gets excited
and a plausible mechanism for the overall reaction may be at 295 nm to give fluorescence at λmax 340 nm in the said buffer
delineated (Scheme 4). The very good yield of the methoxy- system. The gradual decrease to
containing compounds (6c 6g-h 6j-k) may be attributed to
greater reactivity of the intermediate towards aerial
a
certain extent and then
,
,
5
oxidation in these cases. In the reaction involving 11, an
intermediate analogous to 14 fails to undergo elimination
reaction leading to a 2-chromene, rather it undergoes a
cyclisation reaction generating the bicyclic compound 12. The
suggested mechanism shows that the overall process restores
the catalyst, which is used in the next cycle of reactions. Thus,
it has been observed that the catalyst can be recovered after
completion of the reaction and then it can be used further
without any significant reduction of activity (Table 3).
(a)
(b)
Table 3 Recycling of the catalyst for the synthesis of 6a
Entry
Condition of
the catalysta
Fresh
Time (h)
Yieldb (%)
1
2
3
4
5
8
8
75
73
73
70
68
Run 1
Run 2
Run 3
Run 4
(c)
Fig. 2 Fluorescence titration of 10
M of 6a, (b) 1-9 M of 6b, (c) 1-10
Vis spectrum of 6i and its change with addition of protein
(d)
M of β-lg with (a) 1-9
M of 6i and (d) UV-
9
10
10
ꢀ
ꢀ
ꢀ
ꢀ
a Amberlyst-15, bIsolated yield
solution. Insets: Plot of F0/F vs [compound 6a or 6b or 6i
as per the Stern-Volmer equation.
]
4 | J. Name., 2012, 00, 1-3
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The molecule 6a having four oxygen and two nitrogen atoms
may show hydrogen bonding interaction with proteins through
these heteroatoms, and the rest part is hydrophobic in nature and
may be responsible for electrostatic interactions like π-π, π-H-C and
other interactions. The parts of the molecule which would interact
with β-lg can be predicted by exploiting MEP calculation (a tool for
predicting the chemical activity of a molecule when it interacts with
another molecule potentially)23
. In this electrostatic potential
surface of constant electron density the red colour represents the
electronegative region, yellow the less electronegative region and
green the neutral region (Figs. 3c and 3d). From the MEP map, it
was found that the chromene oxygen (O15) and the two nitrogen
atoms of pyrimidine which are buried in the hydrophobic
environment, show no red colour, and may not be able to form any
hydrogen bond with protein. Among the remaining oxygen atoms of
the pyrimidine moiety, oxygen-31 forms the most electronegative
region and it may interact with a protein through hydrogen bonding
(Figs. 3c and d). The compound 6a is a neutral molecule having
large hydrophobic part (green regions) which can interact with a
protein mainly through electrostatic interactions.
Fig. 3 (a) Optimized geometry of 6a. (b) Potential energy
curve for the rotation about the C=C bond in 6a. (c) & (d)
Molecular electrostatic potential map of 6a
.
Modeling of 6a Binding Site in β-lg: Docking Study.
The structure of the protein β-lg has been determined several
times. Its monomeric form is globular in nature with 18400 Da
molecular mass and having 162 amino acid residues. The A and B
genetic variants of β-lg differ just by amino acids at 64 and 118
positions. Eight long antiparallel β-strands build a β-barrel, called
calyx which is a pocket having ability to accommodate small
saturation of fluorescence intensity of β-lg at the λmax with
increasing concentration of 6a, 6b and 6i were found. The above
results of UV-vis and fluorescence studies (Fig. 2) definitely suggest
that the compounds 6a, 6b and 6i originally present in a hydrophilic
environment in aqueous ethanolic solution move toward more
hydrophobic environment inside the protein. Depending upon the
characteristics of ligands, the binding constant for ligand-binding
with β-lg varies widely (from 1.5×102 to 5×107 M-1)20. The binding
constant for the interaction can be calculated from the ratio of
intercept and slope of the plot of (F0/F) vs [ligand] (where F0 is the
fluorescence intensity at λmax without addition of ligand and F is the
fluorescence intensity at λmax with different concentrations of
ligand) by using Stern-Volmer equation.21 From this plot, shown in
the insets of Figs. 2a, b and c for the compounds 6a, 6b and 6i, the
binding constants were calculated as 7.8×105, 5.9×105 and 3.4×105
M-1, respectively. These binding constant values indicated a lofty
level of affinity of ligand towards the protein.
(a)
(b)
DFT study: Ground-state geometry optimization and molecular
electrostatic potential (MEP) calculation.
A theoretical investigation utilizing density functional theory
(DFT) was performed to get a better understanding of the structural
features and chemical properties of 6a, a representative member of
the series 6. The structure of the compound, shown in Fig. 3a, was
optimized by DFT at the B3LYP/6-31G level of theory using Gaussian
09W. The pyrimidine ring of the molecule is tilted away from the
plane of chromene moiety by an angle of 36.8○. The bond linking
the two heterocyclic moieties is somewhat longer (1.421 Å) than a
normal C=C bond (1.35 Å). Calculations for interconversion of the
conformers A and B shows that in between them the most unstable
point is C which is at 76.50 kJ mol-1 higher level than either of A and
B (Fig. 3b). It is therefore reasonable that the potential energy of
the conformer having the two heterocyclic moieties in coplanar
arrangement will be less than this value and free rotation around
the C4-C5′′ bond will be possible at room temperature22. In reality,
this is evident from the 1H and 13C NMR spectral features of 6a and
other compounds of the series.
(c)
Fig. 4 (a) Docking pose showing the interaction site of the
compound 6a inside the protein (b) A close-up to the
binding pocket of the protein (c) Mode of interaction of 6a
with some constituent amino acids at the binding site of
β
-lg.
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Journal Name
hydrophobic molecules inside it, along with this, there is one α-helix resulting crude product was purified by crystallization from DCM
at the outer surface of the barrel in both the varients17. This tertiary hexane solvent system.
framework has three potential binding pockets for ligands: the
1,3-Dimethyl-5-(2-phenyl-4H-chromen-4-ylidene)pyrimidine-
2,4,6(1H,3H,5H)-trione (6a): Red crystals (yield: 75 %), m.p. 171-
172 ○C, IR (KBr pellet): 1700, 1630, 1544, 1467, 1440, 1401, 1358,
calyx, a channel between α-helix and the barrel and the region near
Trp19-Arg124. The last two pockets are at the outer surface of the
protein24 and polar aromatic compounds prefer the outer surface
binding sites25. Generally, hydrophobic and amphiphilic compounds
interact with β-lg via electrostatic and hydrogen bonding
1
1129, 1060, 861, 765 cm-1, H NMR (300 MHz, CDCl3): δ 3.43 (6H, s,
2 × >NCH3), 7.43 ( 1H, t, J=8.2 Hz), 7.56-7.61(3H, m, Ar-H), 7.67 (1H,
d, J=8.4 Hz), 7.78 (1H, t, J=8.4 Hz), 8.05-8.11(3H, m, Ar-H), 9.13 (1H,
s, H-3 of 2-chromene moiety). 13C NMR (75 MHz, CDCl3): δ 28.4,
99.6, 111.9, 118.0, 121.4, 124.8, 127.2, 129.4, 131.0, 131.8, 132.7,
135.1, 152.0, 154.8, 161.6, 162.6, 162.8, ESMS: m/z calculated for
C21H17N2O4 [M + H]+: 361.1188, found 361.1180, Anal. Calcd for
C21H16N2O4: C, 69.99; H, 4.48; N, 7.77. Found: C, 70.13; H, 4.67; N,
7.52.
1,3-Dimethyl-5-(2-p-tolyl-4H-chromen-4-ylidene)pyrimidine-
2,4,6(1H,3H,5H)-trione (6b): Red crystals (yield: 63 %), m.p. 175-
176 ○C, IR (KBr pellet): 1699, 1630, 1544, 1509, 1463, 1440, 1356,
1279, 1192, 1128, 1057, 763, 721, 615; 1H NMR (300 MHz, CDCl3): δ
2.47 (3H, s, CH3), 3.43 (6H, s, 2 × >NCH3), 7.37 (2H, d, J = 8.1Hz, Ar-
H), 7.42 (1H, t, J = 7.5Hz, Ar-H), 7.66 (1H, d, J = 8.7Hz, Ar-H), 7.78
(1H, br. t, J=7.2 Hz Ar-H), 8.00 (2H, d, J =8.2 Hz, Ar-H), 8.05 (1H, br.
d, J = 8.4 Hz, Ar-H), 9.07 (1H, s, H-3 of 2-chromene moiety), 13C NMR
(75 MHz, CDCl3): δ 21.7, 28.3, 98.9, 111.8, 118.0, 121.5, 124.8,
127.3, 128.1, 130.2, 131.8, 135.0, 144.0, 152.1, 154.7, 162.2, 162.9;
Anal. Calcd for C22H18N2O4: C, 70.58; H, 4.85; N, 7.48. Found: C,
70.72; H, 5.02; N, 7.61.
5-(2-(4-methoxyphenyl)-4H-chromen-4-ylidene)-1,3-dimethylpyri-
midine-2,4,6(1H,3H,5H)-trione (6c) Red crystals (yield: 81 %), m.p.
199-200 ○C 1H NMR (500 MHz, CDCl3): δ 3.42 (6H, s, 2 × >NCH3),
3.92 (3H, s, OCH3), 7.05 (2H,d, J=8.5 Hz, H-3′, 5′), 7.42 (1H, t, J=8.0
Hz, H-6), 7.65 (1H, d, J=8.5 Hz, H-8), 7.76 (1H, t, J=7.5 Hz, H-7), 8.05
(1H, d, J=8.5 Hz, H-5), 8.08 (2H,d, J=8.5 Hz, H-2′, 6′), 9.00 (1H, s, H-3
of 2-chromene moiety). 13C NMR (75 MHz, CDCl3): δ 28.3, 55.7, 98.2,
111.5, 115.0, 117.9, 121.6, 123.1, 124.8, 129.5, 131.8, 134.8, 152.2,
154.6, 162.3, 162.9, 163.8; ESMS: m/z calculated for C22H19N2O5 [M
+ H]+: 391.1294, found 391.1214. Anal. Calcd for C22H18N2O5: C,
67.69; H, 4.65; N, 7.18. Found: C, 67.49; H, 4.79; N, 7.03.
5-(2-(4-Chlorophenyl)-4H-chromen-4-ylidene)-1,3-dimethylpyrimi-
dine-2,4,6(1H,3H,5H)-trione (6d): Red crystals (yield:60%), m.p. 217-
218 ○C, 1H NMR (300 MHz, CDCl3): δ 3.42 (6H, s, 2 × >NCH3), 7.42
(1H, t, J=6.9 Hz), 7.53 (2H, d, J=8.7 Hz, H-3′& H-5′), 7.64 (1H, d,
J=8.1Hz, Ar-H), 7.78 (1H, t, J=7.5 Hz), 8.01-8.05 (3H, m, Ar-H), 9.12
(1H, s, H-3 of 2-chromene moiety). 13C NMR (75 MHz, CDCl3): δ 28.5,
100.4, 111.6, 119.5, 122.3, 127.2, 129.4, 130.4, 130.7, 130.9, 132.9,
134.9, 151.8, 153.1, 160.8, 161.5, 162.7, Anal. Calcd for
C21H15ClN2O4: C, 63.89; H, 3.83; N, 7.10. Found: C, 64.03; H, 3.76; N,
6.81.
interactions21
.
The molecular docking study has drawn extraordinarily large
attention of theoretical and experimental researchers. This study
was carried out to get better understanding about the protein
environment where the molecule 6a interacts. The docked pose
shows that the molecule prefers the outer surface channel of β-lg
between α-helix and the barrel to bind in the fashion shown in Fig.
4a. It is involved in hydrogen bonding with amide hydrogen of the
side chain of GLN35 residue and electrostatic interaction with
SER21, GLN35, ARG40, SER150, LEU156, CYS160 and HIS161
residues as apparent from the docked pose represented in Fig. 4b.
The docking study shows that oxygen-31 of the molecule is involved
in hydrogen bonding, which was predicted from MEP calculation
also (Fig. 3c and 3d). The neutral nature, lack of effective polar
atoms and presence of large hydrophobic part in the molecule of 6a
may be responsible for its efficiency of interaction through the
hydrophobic part. Thus, the electrostatic interactions with the
molecule become very important for showing affinity toward the
protein. This is reflected in the high binding constant values
obtained from fluorescence experiment. Using the docking tools,
the binding constant of this ligand-protein interaction was
calculated to be 3.05×105 M-1.
Experimental
General
Standard literature procedures were used to dry the solvents
used in the experiments. An oven-dried round-bottomed flask was
used to perform the reactions and they were conducted under the
atmospheric oxygen. Thin-layer chromatography plates (Silica gel G)
were visualized by exposure to ultraviolet light and/ or iodine
vapor. IR spectra were recorded on
a Perkin Elmer FT-IR
Spectrophotometer (Spectrum BX II) as KBr pellets. 1H NMR and 13
C
NMR spectra were recorded in CDCl3 on a Bruker 300 MHz or a
Bruker 500 MHz NMR spectrometer. Chemical shifts (δ) were
reported in parts per million (ppm) taking CHCl3 peak at δ 7.26.
Coupling constants (J) are quoted in hertz (Hz). ESMS were recorded
on a Jeol MS Station 700 mass spectrometer by electron spray
ionization (ESI) and HRMS on
a Waters Xevo G2QT mass
5-(6-Chloro-2-phenyl-4H-chromen-4-ylidene)-1,3-dimethylpyrimi-
dine-2,4,6(1H,3H,5H)-trione (6e): Red crystals (yield: 72%), m.p.
215-206 ○C, IR (KBr pellet): 2920, 2850, 1701, 1649, 1627,1515,
1467, 1366, 1262, 1192, 1120, 1022, 836, 753, 702, 641 cm-1; 1H
NMR (300 MHz, CDCl3): δ 3.42 (6H, s, 2 × >NCH3), 7.54-7.61(4H, m,
Ar-H),7.69 (1H, dd, J=8.0 & 2.4 Hz, H-7), 8.01(1H, d, J=2.4 Hz, H-5),
8.06 (2H, dd, J=8.2 & 2 Hz, H-2′& H-6′), 9.16 (1H, s, H-3 of 2-
chromene moiety). 13C NMR (75 MHz, CDCl3): δ 28.5, 99.6, 111.8,
118.0, 119.4, 125.0, 128.3, 129.5,129.6, 131.9, 135.4, 139.1, 153.0,
154.8, 160.0, 161.3, 163.1, Anal. Calcd for C21H15ClN2O4: C, 63.89; H,
3.83; N, 7.10. Found: C, 63.61; H, 3.67; N, 7.03.
spectrometer. Elemental analyses were done using two Perkin-
Elmer 2400 Series II C, H, N analyzers.
General Procedure for the Synthesis of 1,3-Dimethyl-5-(2-phenyl-4H-
chromen-4-ylidene)pyrimidine-2,4,6(1H,3H,5H)-triones (6). To
a
solution of 2-hydroxychalcone (1) (1 mmol) and 1,3-dimethyl-
barbituric acid (1 mmol) in anhydrous toluene (10 mL) was added
amberlyst-15 (40 mg) at room temperature. The resulting mixture
was refluxed with stirring under atmospheric oxygen for 8 h. After
completion of reaction, sufficient amount of dichloromethane was
added to dissolve the product and then amberlyst-15 was filtered
off. The filtrate was concentrated by removal of solvent and the
6 | J. Name., 2012, 00, 1-3
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5-(6-Chloro-2-p-tolyl-4H-chromen-4-ylidene)-1,3-dimethylpyrimi-
(1H, s, H-3 of 2-chromene moiety), ESMS: m/z calculated for
dine-2,4,6(1H,3H,5H)-trione (6f): Red crystals (yield: 69%), m.p. C23H20N2O5 [M + H]+: 405.13, found 405.11
203-204○C;1H NMR (300 MHz, CDCl3): δ 2.46 (3H, s, CH3), 3.42 (6H,
1,3-Dimethyl-5-(2-(thiophen-2-yl)-4H-chromen-4-ylidene)pyrimidine
s, 2 × >NCH3), 7.36 (2H, d, J=9 Hz, H-3′, 5′), 7.58 (1H, d, J=9 Hz, H-8),
7.68 (1H, dd, J=9.0 &2.3 Hz, H-7), 7.96 (2H, d, J=8.2 Hz, H-2′, 6′),
8.01 (1H, d, J=2.3 Hz, H-5), 9.10 (1H, s, H-3 of 2-chromene moiety).
13C NMR (75 MHz, CDCl3): δ 21.7, 28.4, 99.7, 111.4, 119.4, 122.4,
127.3, 127.8, 130.2, 130.4, 130.9, 134.8, 144.2, 151.9, 153.0, 161.0,
162.0, 162.7. ESMS: m/z calculated for C22H18ClN2O4 [M + H]+:
409.09, found 409.39
-2,4,6(1H,3H,5H)-trione (6l): Red crystals (yield: 25%), m.p. 243-244
○C,1H NMR (300 MHz, CDCl3): δ 3.42 (6H, s, 2 × >NCH3), 7.26-7.28
(1H, m, ), 7.41 (1H, t, J =7.5 Hz, Ar-H), 7.61 (1H, d, J = 8.1 Hz), 7.72-
7.76 (2H, m, Ar-H), 7.96 (1H, d, J = 3.6 Hz, H-5 of thiophene moiety),
8.01 (1H, d, J = 8.3 Hz, Ar-H), 8.93 (1H, s, H-3 of 2-chromene moiety)
13C NMR (125 MHz, CDCl3): δ 28.5, 99.1, 111.3, 118.0, 121.6, 124.9,
129.4, 131.0, 132.0, 133.1, 135.1, 152.5, 154.3, 158.2, 162.1, 162.9.
HRMS: m/z calculated for C19H15N2O4S [M + H]+: 366.0674, found
5-(6-Chloro-2-(4-methoxyphenyl)-4H-chromen-4-ylidene)-1,3-
dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (6g): Red crystals (yield: 367.0747
80%), m.p. 209-210 ○C,1H NMR (300 MHz, CDCl3): δ 3.42 (6H, s, 2 ×
2,4,6-Trimethyl-4,12-dihydro-1H-6,12-methanobenzo[7,8][1,3]di-
>NCH3), 3.92 (3H, s, OCH3), 7.03 (2H, d, J=9.0 Hz, H-3′, 5′), 7.58 (1H,
d, J=9 Hz, H-8), 7.67 (1H, dd, J=9 & 2.4 Hz, H-7), 8.00 (1H, br.s, H-5),
8.05 (2H, d, J=9 Hz, H-2′, 6′), 9.03 (1H, s, H-3 of 2-chromene moiety).
13C NMR (75 MHz, CDCl3): δ 28.4, 55.7, 99.6, 111.2, 115.0, 119.3,
122.4, 122.8, 129.4, 130.4, 130.9, 134.7, 152.0, 152.9, 161.0, 162.2,
163.9. Anal. Calcd for C22H17ClN2O5: C, 62.20; H, 4.03; N, 6.59.
Found: C, 62.07; H, 4.18; N, 6.68.
oxocino[4,5-d]pyrimidine-1,3(2H)-dione (12): Colourless crystals
(yield: 42%), m.p. 98-99 ○C,1H NMR (300 MHz, CDCl3): δ 1.91 (3H, s,
CH3), 2.14 and 2.23 (each 1H, br. d, J =13.5 Hz, CH2), 3.28 (3H, s,
>NCH3), 3.34 (3H, s, >NCH3), 4.21 (1H, br. s, aliph. CH ), 6.85 (1H, d, J
= 7.4 Hz, Ar-H), 6.91 (1H, t, J = 7.4 Hz, Ar-H), 7.12 (1H, t, J = 7.4 Hz,
Ar-H), 7.40 (1H, d, J = 7.5 Hz, Ar-H). 13C NMR (75 MHz, CDCl3): δ
25.5, 26.7, 28.0, 28.7, 31.2, 91.8, 102.3, 116.0, 122.0, 126.3, 127.8,
128.0, 150.7, 155.1, 161.6. HRMS: m/z calculated for C16H17N2O4 [M
+ H]+: 301.1110, found 301.1295.
5-(2-(4-Methoxyphenyl)-6-methyl-4H-chromen-4-ylidene)-1,3-
dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (6h): Red crystals (yield:
83%), m.p. 217-218 ○C, IR (KBr pellet):1699, 1628, 1551, 1515, 1430,
1466, 1363, 1262, 1224, 1120, 1025, 904, 838, 862, 804, 776, 754,
719, 643 cm-1; 1H NMR (300 MHz, CDCl3): δ 2.47 (3H, s, CH3), 3.42
(6H, s, 2 × >NCH3), 3.92 (3H, s, OCH3), 7.04 (2H,d, J=9.0 Hz, H-3′, 5′),
7.56 (2H, br.s, H-7 & H-8), 7.82 (1H, br.s, H-5), 8.07(2H,d, J=8.9 Hz,
H-2′, 6′), 8.93 (1H, s, H-3 of 2-chromene moiety). 13C NMR (75 MHz,
CDCl3): δ 21.4, 28.3, 55.7, 97.7, 111.9, 114.9, 117.6, 121.5, 123.1,
129.4, 131.0, 134.9, 136.5, 152.3, 153.1, 162.2, 162.8, 136.0, 163.7
Anal. Calcd for C23H20N2O5: C, 68.31; H, 4.98; N, 6.93. Found: C,
68.14; H, 4.88; N, 7.11.
Recovery of the catalyst.
The catalyst separated from the reaction mixture was washed
thoroughly with ethyl acetate and acetone successively until the
washings became colorless. It was then dried in a hot air oven at
100 °C for 8 h. The dried catalyst was used further.
Sample Preparation for UV−Vis and Fluorescence Study.
The solutions of β−lg (Sigma Aldrich, St. Louis, MO), used without
further purification, were prepared in 10 mM phosphate buffer of
3% ethanol-water mixture at pH 7.4. This pH level was chosen to
replicate the physiological conditions in which β-lg exist its stable
conformation. β-lg was dissolved directly into this buffer, and the
final concentrations were found 108ꢀM for β-lg. The compound 6a,
6b and 6i was dissolved in absolute ethanol and the concentration
was 1 mM.
1,3-Dimethyl-5-(6-methyl-2-p-tolyl-4H-chromen-4-
ylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (6i): Red crystals (yield:
78%), m.p. 181-182 ○C, IR: 1701, 1630, 1546, 1546, 1511, 1358,
1275, 1192, 1131, 1054, 816, 778, 764, 719, 643; 1H NMR (300 MHz,
CDCl3): δ 2.46 (3H, s, CH3), 2.48 (3H, s, CH3), 3.42 (6H, s, 2 × >NCH3),
7.36 (2H, d, J=7.8 Hz, H-3′, 5′), 7.58 (2H, s, H-7,8), 7.82 (1H, s, H-5),
7.98 (2H, d, J=7.8 Hz, H-2′, 6′), 9.00 (1H, s, H-3 of 2-chromene
moiety), 13C NMR (75 MHz, CDCl3): δ 21.4, 21.7, 28.3, 98.3, 112.1,
117.7, 121.5. 127.3, 128.1, 130.1, 131.0, 134.9, 136.7, 143.9, 152.2,
153.2, 162.1, 162.8, 163.1 ESMS: m/z calculated for C23H20N2O4 [M +
H]+: 389.1501, found 389.1541.
UV−Vis Absorpꢀon Spectroscopy.
A solution of compound 6i was placed in a four-clear-sided
quartz cell (2 mL) of 1 cm path length using a Shimadzu UV1700
spectrophptometer. Spectra were recorded with a matched pair of
silica cuvettes against a solvent blank reference from 200 to 650
nm.
5-(8-Methoxy-2-phenyl-4H-chromen-4-ylidene)-1,3-
dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (6j): Red crystals (yield:
81%), m.p. 219-220 ○C, IR: 2923, 2852, 1692, 1550, 1501, 1402,
1450682, 701, 723, 755, 775, 802, 863, 1068, 1140, 122874,
1353;1H NMR (300 MHz, CDCl3): δ 3.42 (6H, s, 2 × >NCH3), 4.06 (3H,
s, OCH3), 7.22 (1H, d, J=7.9 Hz, H-7), 7.32 (1H, t, J=8.4 Hz, H-6), 7.53-
7.59 (4H, m, Ar-H), 8.14(2H, dd, J= 7.8 & 2.0 Hz, H-2′, 6′), 9.14 (1H, s,
H-3 of 2-chromene moiety). ESMS: m/z calculated for C22H18N2O5
[M + H]+: 391.12, found 391.20.
Fluorescence Spectroscopy.
The compounds 6a-k were successively added from 1 to 10 µM
to the 10 µM concentration of β-lg for fluorescence studies at room
temperature using Shimadzu spectrofluorimeter (model RF 5301)
with the bandwidths of excitation and emission slits at 3 and 5 nm.
The excitation wavelength of β-lg (10 µM, with phosphate buffer,
pH 7.4, with 5% ethanol) was 295nm and emission scans were
recorded in the range 310-400 nm.
5-(8-Methoxy-2-p-tolyl-4H-chromen-4-ylidene)-1,3-
dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (6k): Red crystals (yield:
85%), m.p. 231ꢀ232 ○C, 1H NMR (300 MHz, CDCl3): δ 2.46 (3H, s,
CH3), 3.41 (6H, s, 2 × >NCH3), 4.05 (3H, s, OCH3), 7.20-7.37 (4H, m,
Ar-H), 7.54 (1H, d, J=8.0 Hz, H-5), 8.04 (2H, d, J= 8.1Hz, H-2′, 6′), 9.08
DFT Calculations.
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For the DFT calculations in this paper GAUSSIAN 09 program
Journal Name
(b) G. A. Iacobucci and J. G. Sweeny, Tetrahedron, 1983, 39
,
3005; (c) K. M. Meepagala, W. Osbrink, C. Burandt, A. Lax
and S. O. Duke, Pest Manag. Sci., 2011, 67, 1446; (d) D.
Joulain and R. Tabacchi, Phytochemistry, 1994, 37, 1769; (e)
package was used. All the calculations was done using Becke three
parameters hybrid exchange and the Lee–Yang–Parr correlation
functional (B3LYP). To optimize the geometry of 6a, 6-31G basis set
was used. The same method was used also for calculation of MEP
for the compound 6a.
G. A. Iacobucci and J. G. Sweeny, Tetrahedron, 1983, 39
3005.
,
4
5
(a) Y.-L. Shi and M. Shi, Org. Biomol. Chem., 2007,
(b) T. J. A. Graham and A. G. Doyle, Org. Lett., 2012, 14
5, 1499;
,
Docking study.
1616; (c) W. A. L. van Otterlo, E. L. Ngidi, S. Kuzvidza, G. L.
Morgans, S. S. Moleele and C. B. de Koning, Tetrahedron,
2005, 61, 9996; (d) S. A. Worlikar, T. Kesharwani, T. Yao and
R. C. Larock, J. Org. Chem., 2007, 72, 1347; (e) C. C. Malakar,
The molecular docking of 6a into the three dimensional
structures of β-lg (downloaded from RCSB website, PDB ID: 2BLG)26
was carried out using AutoDock 4.2.0 software package. The lowest
energy conformation of 6a obtained from DFT optimisation was
used for docking study. It was done using an efficient and durable
algorithm, Lamarckian Genetic Algorithm (LGA). The conformation
with the lowest binding energy was used to analyze ligand
placement.
D. Schmidt, J. Conrad and U. Beifuss, Org. Lett., 2011, 13
1972.
,
J. T. Bojarski, J. L. Mokrosz, H. J. Barton, M. H. Paluchowska,
in: A. R. Katritzky (Ed.), Recent Progress in Barbituric Acid
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G.L. Patrick, An Introduction to Medicinal Chemistry, 4th ed.,
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F. López-Muñoz, R. Ucha-Udabe and C. Alamo,
Conclusions
In conclusion, we report a very simple and new methodology for
synthesis of 1,3-dimethyl-5-(2-phenyl-4H-chromen-4-ylidene)-
pyrimidine-2,4,6(1H,3H,5H)-triones, an interesting class of
compounds having novel structural features. The flavylium cation
part of the compounds is responsible for their intense red colour
and the zwitterionic structure for their high polarity. They have
potential to find important applications for their biological activities
and material properties.
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Acknowledgements
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. N.S., S.M. and C.G. are thankful to the UGC,
New Delhi for their Research Fellowships.
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An efficient synthesis of 1,3-dimethyl-5-(2-phenyl-4H-chromen-4-ylidene)
pyrimidine-2,4,6(1H,3H,5H)-triones and investigation of their interactions with
β-lactoglobiulin
Nayim Sepay, Sumitava Mallik, Chayan Guha and Asok K. Mallik*a
OH
R1
R2
Amberlyst-15
O
Toluene, reflux
air, 8 h
+
O
O
N
N
Me
Me
O