β-CD-catalyzed multicomponent domino reaction: synthesis, characterization, in silico molecular docking and biological evaluation of pyrano[2,3-d]-pyrimidinone derivatives

Article abstract of DOI:10.1007/s11164-018-3479-9

Abstract: Simple and green synthetic procedures constitute an important goal in organic synthesis. The combination of multicomponent reactions (MCRs) and unconventional solvents has become a new research direction, which enables simultaneous growth of bot

Full text of DOI:10.1007/s11164-018-3479-9

Res Chem Intermed
https://doi.org/10.1007/s11164-018-3479-9
b-CD-catalyzed multicomponent domino reaction:
synthesis, characterization, in silico molecular docking
and biological evaluation of pyrano[2,3-d]-
pyrimidinone derivatives
Asha V. Chate1 Ravindra M. Dongre1 Mahadeo K. Khaire1
•
•
•
Giribala M. Bondle1 Jaiprakash N. Sangshetti2 Manoj Damale3
•
•
Received: 14 March 2018 / Accepted: 11 May 2018
Ó Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract Simple and green synthetic procedures constitute an important goal in organic
synthesis. The combination of multicomponent reactions (MCRs) and unconventional
solvents has become a new research direction, which enables simultaneous growth of
both MCRs and green solvents toward ideal organic synthesis. In this paper, we have
summarized recent results of MCRs obtained in unconventional media using water and
b-cyclodextrin, as supramolecular catalyst, for the synthesis of pyrano[2,3-d]-pyrim-
idinone (4a–q) derivatives. The compounds were evaluated for their in vitro antimi-
crobial activity. Among the synthesized compounds, compounds 4h, 4m and 4p
exhibited higher antimicrobial activity than ciprofloxacin used as the reference drug.
Most of the synthesized compounds have good to excellent antimicrobial activity.
Furthermore, \ molecular docking study was performed to help understand binding
interactions of the most active analogs with C30 carotenoid dehydrosqualene synthase
enzyme.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11164-
018-3479-9) contains supplementary material, which is available to authorized users.
&
Asha V. Chate
chateav@gmail.com
1
2
3
Department of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad,
M.S. 431 004, India
Department of Quality Assurance, Y. B. Chavan College of Pharmacy, Rafiq Zakaria Campus,
Aurangabad 431 001, India
Shree Bhagwan College of Pharmacy, Aurangabad, India
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A. V. Chate et al.
Graphical Abstract
Hydrophilic medium
H2O
CN
H2O
H2O
H2O
O
H2O
H2O
H2O
H2O
H2O
H2O
H2N NH
R
β-CD
β-CD
CN
(2)
H2O
H2O
CHO
O
O
(3)
(1a-q)
H2O
H2O
H2O
H2O
H2O
H2O
cavity
H2O
H2O
80 °C
Hydrophobic
Binding Pose and molecular interactions of 4p and 4m into the
active site of C30 carotenoid dehydrosqualene synthase
37-45 min.
R
Influence on the antimcrobial activity
(4i, 4m & 4p) shows higher antibacterial activity
O
CN
HN
It is active unit for the
variable antimicrobial activity
O
N
H
NH2
(4a-q)
Keywords Pyrano[2,3-d]-pyrimidinones Á Multicomponent reaction Á
b-Cyclodextrin Á Supramolecular catalysis Á Antimicrobial activity Á
Molecular docking
Introduction
The development of new biomolecules of therapeutic interest by searching and
optimizing a simple, environmentally benign and established synthetic route has
been an interesting part of contemporary medicinal chemistry research [1–5].
Although nature is the best source of biomolecules, its inadequacy in supplying
desired quantities of materials sometimes becomes a limiting factor in the
widespread use of such valuable compounds (i.e., the isolation of taxol from
treebarks). The use of complicated, lengthy and expensive procedures to isolate
biomolecules from natural sources has been another drawback. Synthetic chemists
are therefore important for the development of molecular frameworks, analogous
either totally or partially to that of biomolecules which can be synthetically
accessed.
Pyrano[2,3-d]pyrimidinone(thione) derivatives are very important because of
their essential biological properties such as antihypertensive, antihepatotoxicity [6],
antitumor [7, 8], antibronchitic and anticardiotonic activities [9]. These compounds
are also available as important units of some natural products, such as polyethers,
antibiotics, carbohydrates, phormones, iridoids and alkaloids [10, 11].
There are several reports on the synthesis of 7-amino-6-cyano-5-aryl-5H-
pyrano[2,3-d]pyrimidine-(1H,3H)-2,4-diones in which arylidene malononitrile with
123

b-CD-catalyzed multicomponent domino reaction: synthesis…
barbituric acid have been reacted under either traditional thermal condition [12] or
microwave irradiation [13]. Also, several catalysts have been reported wihch are
capable of the promotion of the formation of pyrano[2,3-d]pyrimidinone (thione)
derivatives, including [DABCO](SO3H)2(HSO4)2 [14], nano-sawdust-OSO3H [15],
Al-HMS-20 [16], silica-bonded N-propyltriethylenetetramine [17], nano-Al2O3,
[18], CaHPO4 [19], DABCO [20], KBr [21], choline chloride. ZnCl2 [22], alum
[23], Zn[L-proline]2 [24], L-proline [25], succinimidinium hydrogensulfate [26],
nano-CuO/ZnO [27], trichloroisocyanuric acid [28], nano-titania sulfuric acid and
B(OH)3 [29], n-butyl-3-methylimidazolium tetra-fluoroborate [30], nanocatalysts
[31], and ionic liquid [H2-DABCO][H2PO4]2} [32]. Each of these methods has its
own merit, with at least one of the limitations of drastic conditions to apply novel
conditions, long reaction times and effluent pollution. Thus, new routes for the
synthesis of these molecules have attracted considerable attention in the search for a
rapid entry to those heterocycles and their diverse biological properties.
During the past two decades, scientists have paid more and more attentions to the
preparation of functional polyrotaxanes using various macrocyclic molecules, such
as cyclodextrins [33, 34], crown ethers [35–39], cucurbiturils [40–42], and so on.
Among them, cyclodextrins (CDs) have attracted increasing interest for their diverse
binding selectivities. CDs are cyclic oligosaccharides, first reported by Villiers in
1891, consisting of six or more D-glucopyranose units connected through a-(1,4)
glycosidic linkages [43]. There are three well-known, naturally produced CDs: a-
CD, b-CD, and c-CD, made up of six, seven, or eight glucopyranose units,
respectively [44]. b-CD with the advantages of space geometry, cavity size,
availability and low cost has gained widespread application and presently accounts
for more than 95% of the number of CDs [45]. b-CD takes the form of a truncated
cone or torus with the primary hydroxyl functions (C6-OH) oriented to the narrower
end of the torus, and the secondary hydroxyl functions (C2, 3-OH) oriented towards
the wider end [46, 47]. The outer surface of the cavity is hydrophilic due to the
assembly of hydroxyl groups, whereas the void cavity, composed of hydrogen atoms
of C-3, C-5 and oxygen atoms of a glycosidic linkage, shows lipophilic behavior
[48].
As part of our program for applying green catalysts to multi-component reactions
[49–51], we have employed a tandem Knoevenagel–Michael reaction in our
ongoing research for the one-pot synthesis of various pyrano[2,3-d]-pyrimidinone
derivatives. We report here on b-CD-catalyzed tandem Knoevenagel–Michael
reactions of various substituted aromatic aldehydse with malonitrile and barbituric
acid in aqueous medium as a new, robust, greener and highly efficient procedure for
the synthesis of pyrano[2,3-d]-pyrimidinone derivatives. The synthesized com-
pounds have also been tested for their antimicrobial activities and we also
performed molecular docking of the synthesized compounds with the dihydrofolate
reductase (DHFR) receptor.
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A. V. Chate et al.
Results and discussion
Chemistry
Initially, the readily available benzaldehyde 1a, malonitrile 2 and barbituric acid 3
were chosen as model substrate (Scheme 1), and extensive investigations were
carried out to define the optimal reaction conditions. As shown in Table 1, without
any catalyst and using ethanol as a solvent, the target product was afforded in 40%
yield (Table 1, entry 1). Then, the investigatory experiment was performed to
improve the yield of the product by varying the reaction conditions, i.e., solvent,
temperature and supramolecular catalyst. All the reaction products were purified by
recrystallization using ethanol as a solvent. The observations (Table 1) led us to the
conclusion that the supramolecular catalyst has an obvious effect on the reaction.
Among the selected catalysts (Table 1, entries 2–3, 9, 12–13), the commercially
available catalyst b-CD was proved to be the most suitable (Table 1, entry 9) as it
gives a single product. The reaction of benzaldehyde 1a, malonitrile 2 and barbituric
acid 3 in the presence of b-CD was completely specific in affording only the one
product, whereas the other reported catalysts also yielded byproducts.
Several solvents were investigated (Table 1, entries 4–11) of which water was
the best (Table 1, entry 9), although aq. ethanol also produced comparable results
(Table 1, entry 8). The stoichiometry ratio of 15 mol% of the catalyst was chosen to
be the best for performing the reaction since further increases had no impact on the
yield of the reaction (Table 2, entry 4). Thus, the best result was obtained when
Table 1 Optimization of the catalyst for the preparation of pyrano[2,3-d]-pyrimidinones
Entry
Solvent
Catalyst
Mole (%)
Temp (°C)
Time (min)
Yielda (%)
1.
EtOH
No catalyst
Et3N
15
15
15
15
15
15
15
15
15
15
15
15
15
70
70
135
80
80
80
80
80
80
80
80
80
80
5–6 h
110
6–8 h
40
NRb
73
71
65
78
81
76
85
90
82
71
65
55
2.
Aq. EtOH
CH3COOH
CH3CN
Toluene
Chloroform
DMF
3.
P2O5
4.
b-CD
b-CD
b-CD
b-CD
b-CD
b-CD
b-CD
b-CD
a-CD
c-CD
5.
35
6.
45
7.
50
8.
Eq. ethanol
Water
40
9.
37
10.
11.
12.
13.
Ethanol
Methanol
Water
45
56
65
Water
85
All reactions were performed using aldehyde (1 mmol), malonitrile (1 mmol), and barbituric acid
(1 mmol), and solvent at 80 °C
aIsolated yield after filtration
bNR No reaction
123

b-CD-catalyzed multicomponent domino reaction: synthesis…
Table 2 Optimization of catalyst (%) for the synthesis of pyrano[2,3-d] pyrimidinones
Entry
Catalyst (mol%)
Time (min)
Yielda (%)
1
2
3
4
5
10
15
20
40
60
65
72
65
72
90
90
All reactions were carried out using aldehyde (1 mmol), malonitrile (1 mmol), and barbituric acid
(1 mmol), and solvent at 80 °C
aIsolated yield
benzaldehyde 1a, and malonitrile 2 were heated for 30 min in the presence of
15 mol% of b-CD as a catalyst for the in situ generation of intermediates of the
Knoevenagel condensation, followed by the Michael addition of barbituric acid 3 to
provide a precipitate which was filtered off and recrystallized from ethanol to yield
the desired product 4a in 90% yield.
With these results in hand, we then investigated the substrate scopes and
limitations of the synthesis of pyrano[2,3-d]pyrimidinones. First, we started the
reaction with barbituric acid 3 by varying the aldehydes 4a–q and malonitrile 2 to
afford pyrano[2,3-d]pyrimidinones (Scheme 2); the results are listed in Table 3.
Aromatic aldehydes having electron-donating groups like 4-methoxy benzaldehydes
(Table 3, entry 8) reacted with lower rates and with lower yields compared with
those substituted with electron-withdrawing groups like 4-fluoro, 4-chloro and
4-bromo benzaldehydes giving higher yields (Table 3, entries 4–6). Various
heterocyclic aldehydes such as thiophene-2-carbaldehyde and 1H-pyrrole-2-car-
baldehyde were also examined (Table 3, entries 11–12). In addition, our method-
ology has been used successfully in orders of aliphatic materials such as
propionaldehyde, butyraldehyde and iso-butyraldehyde, and corresponding pyr-
ano[2,3-d]pyrimidinones were obtained in excellent yields without the formation of
any byproducts (Table 3, entries 9, 14–15). As is clear from the obtained results, the
presented methodology can be used in oxygen-, nitrogen- and sulfur-containing
heteroaromatic aldehydes. Thus, the reaction was found to be general and to have a
broad scope due to its applicability to a variety of substrates, and the products (4a–
Scheme 1 Standard model reaction
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A. V. Chate et al.
Table 3 Synthesis of 7-amino-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-pyrano[2,3-d]pyrimidine-6-
carbonitrilea 4(a–q) under optimized conditions
Entry
Aldehyde
Product
Time (min)
Yieldb (%)
M.P. (°C) (found) [27]
1.
-Ph
4a
4b
4c
4d
4e
4f
37
38
48
38
42
45
48
35
38
42
48
32
35
45
38
48
43
90
225–228
275–278
228–230
220–222
250–252
[ 300
2.
4-OH,3-OMe-Ph
4-Cl-Ph
78.5
75
3.
4.
4-F-Ph
95
5.
3-OH,4-OMe-Ph
95.2
88.5
87
6.
4-Br-Ph
2-NO2-Ph
7.
4g
4h
4i
228–230
140–142
260–262
238–240
280–282
280–283
178–180
260–262
255–258
280–283
140–142
8.
4-OMe-Ph
86.5
75
9.
CH3-CH2-CHO
2,4-di-OMe-Ph
Thiophene-2 carbaldehyde
1H-Pyrrole-2-carbaldehyde
2-OH-Ph
10.
11.
12.
13.
14.
15.
16.
17.
4j
82.5
78.5
89
4k
4l
4m
4n
4o
4p
4q
93.5
83.5
78.5
82.5
89.5
C3H8-CHO
C3H6-CHO
Ph-CH2-CHO
4-OH-Ph-CHO
aAll reactions were performed using aldehyde (1 mmol), malonitrile (1 mmol), and barbituric acid
(1 mmol), b-CD (15 mil%), and H2O (15 mL), at 80 °C
bIsolated yield after filtration
q) were isolated in excellent yields (75–95%) under milder reaction conditions. The
results are summarized in Table 3.
Analytical results
The structure of all the prepared compounds were elucidated with the aid of IR, 1H
and 13C NMR,and mass spectroscopy and elemental analysis data have been
discussed for a representative compound, 4a. The IR spectrum of 4a exhibited a
sharp peak at 3379 cm-1 which corresponds to the –NH stretching of the –NH2
group, and peaks at 2191 and 1679 cm-1 correspond to the nitrile and amide
1
carbonyl, respectively. The H NMR spectra of 4a shows three singlets at d 10.98,
10.80 and 6.82 for the –NH and –NH2 protons, clearly indicating the incorporation
of both moieties in the product. In the 13C NMR spectra, the two carbonyl carbon
atoms display a signal at d 160.39 and 151.36 and the amide carbonyl carbon atom
resonated at d 153.91 and the nitrile carbon at d 118.21. The mass spectra also
exhibit a distinguishing peak at m/z 283.08 [M?H?]. The elemental analysis data of
4a correlate with the calculated value which is useful in the assignment of the
structure of 4a, which is shown in supplementary material of 4a. The structures of
all pyrano[2,3-d]pyrimidinones were consistent with the above-mentioned data.
123

b-CD-catalyzed multicomponent domino reaction: synthesis…
The recyclability of b-CD was examined using the model reaction of benzalde-
hyde, malonitrile and barbituric acid. After each run, ethyl acetate was added to
dissolve the product, and the catalyst was separated by an extraction process. After a
simple wash with ethyl acetate and subsequent vacuum removal of residual solvent,
the catalyst was reused for the next run. To our delight, the catalyst was highly
reusable under the investigated conditions, preserving its initial catalytic activity
almost unaltered after three uses, as is shown in Fig. 1. The purification of the b-CD
catalyst after the recycling was confirmed by the IR spectra shown in Fig. 2.
To explain the mechanism of this tandem reaction, a postulated reaction course is
depicted in Scheme 3. In the first step, malonitrile 2 undergoes a condensation
reaction with aldehyde promoted by b-CD with the removal of water molecules and
affords the intermediate 2 benzylidenemalononitrile. In the second step, Michael
addition of barbituric acid 3 to the product gives the intermediate 2-(phenyl(2,4,6-
trioxopiperidin-3-yl)methyl)malononitrile. b-CD protonates the intermediate and,
after cyclization and intramolecular condensation, the product 4a is produced.
Pharmacology
In vitro antimicrobial activity
Finally, all synthesized compounds were screened for antimicrobial activity. The
microorganisms used in this study were Escherichia coli, Pseudomonas aeruginosa
(Gram-negative bacteria) Bacillus subtilis, and Staphylococcus aureus (Gram-
Scheme 2 General scheme for the synthesis of 7-amino-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-
pyrano[2,3-d]pyrimidine-6-carbonitriles
123

A. V. Chate et al.
Fig. 1 Reuse and recovery of
b-CD and its effect on yield
100
90
90
89
85
85
84
82
80
80
80
70
Fresh
Run 1
Run 2
Run 3
Catalyst recovery
Yield
3500
3000
2500
2000
1500
1000
Wavenumber cm-1
Fig. 2 FTIR spectral analysis of recycling of: a b-cyclodextrin fresh; b b-CD first run; c b-CD second
run; d b-CD third run
positive bacteria). The minimum inhibitory concentration (MIC) of the synthesized
compounds was determined by well diffusion method [52] and compared to two
commercial antibiotics (Table 4). In addition, the antifungal activity against three
fungal species, Candida albicans, Aspergillus flavus and A. niger, was tested against
Miconazole. The results are compiled in Table 3. From the screening results, the
MIC of the most active compounds toward the microorganisms showed that the
MICs ranged between 25 and 50 lg/mL, with most of compounds exhibiting good
to potent in vitro antimicrobial activity against both Gram-positive and Gram-
negative strains. The obtained results, depicted in Table 4, reveal that compounds
4c, 4h, 4i, 4k, 4m and 4p could effectively inhibit the growth of most tested
bacterial strains, while compound 4i exhibited good activities against all the tested
fungal strains. Compounds 4h, 4m and 4p with a 4-OCH3, 2-OH and –CH2
derivative attached to the pyrano[2,3-d]-pyrimidinones backbone gave stronger
antibacterial efficacies and broader bioactive spectra than ciprofloxacin with quite
low MIC values, (MIC = 25 0.25, 25 0.43, 25 0.49 and 25 0.88 lg/mL)
123

b-CD-catalyzed multicomponent domino reaction: synthesis…
Scheme 3 Proposed mechanism for the multicomponent reaction using b-CD catalyst for the synthesis
of pyrano[2,3-d]-pyrimidinones
against E. coli and P. aeruginosa, while compounds 4m and 4p also exhibited
excellent antibacterial activity with quite low MIC values, (MIC = 25 0.77, and
25 0.75 lg/mL) against S. aureus compare with ciprofloxacin. Almost all of the
compounds were found to be more active than ciprofloxacin against all the tested
strains. On the other hand, in other cases of each class of products, the types of
substitution on the aldehyde moiety have affected the antibacterial activity.
In silico molecular docking study
A molecular docking study was performed to predict the possible binding modes
and to rationalize the observed biological activity. The docking study was carried
out using the Surflex–Dock module of the Sybyl 2.1.1 package following the
standard procedure [53].
In silico pharmacokinetics evaluation
An in silico absorption, distribution, metabolism, and excretion–toxicity (ADMET)
study was also performed to predict the pharmacokinetic and toxicity profile of the
synthesized compounds. Using ADMET software. The FAF Drug2 tool was used for
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A. V. Chate et al.
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b-CD-catalyzed multicomponent domino reaction: synthesis…
in silico ADMET predictions. These programs computed pharmacokinetic param-
eters such as molecular percent absorption %ABS, MW, logP, N-ROTB, H/C, PSA,
HBA, and HBD, etc. The values obtained are summarized in (Table 5).
Biological section
In vitro antibacterial activity
All the synthesized compounds (4a–q) showed moderate to very good antibacterial
activity.
It has been noted that for E. coli, compounds 4h and 4p were found to be
equipotent, i.e., 25 0.25 and 25 0.43 lg/mL a,s compared to ciprofloxacin,
i.e., 25 1.00 lg/mL. The compounds 4a, 4c, 4m and 4q have been noted to be
highly potent as compared to ampicillin. Compounds 4b, 4i, 4l and 4o showed
maximum potency, i.e., 50 – 0.24 lg/mL, as compared to ampicillin, i.e.,
100 1.24 lg/mL, as well as against P. aeruginosa. Compounds 4m and 4p were
found to be equipotent against P. aeruginosa, i.e., 25 0.49 and 25 0.88 lg/mL,
compared to ciprofloxacin, i.e. 25 1.15 lg/mL, and compounds 4a, 4g, 4h, 4k, 4l,
4o and 4q showed higher potency compared to P. aeruginosa and compared to
ampicillin, i.e., MIC 100 2.14 lg/mL. Compounds 4c, 4i, 4m and 4p were highly
potent and compounds 4h, 4k, 4l and 4o showed maximum potency against S.
Table 5 Molecular docking
results of pyrano[2,3-d]-
pyrimidinones analogues (4a–q)
Compd.
Molecular docking score
Total score (- log Ki) Crash score Polar score
4a
4.9804
3.5209
4.3198
2.2607
4.0916
3.0113
4.0734
4.7445
5.0558
3.6243
4.6797
4.2783
5.1109
3.8353
4.133
- 1.2586
- 1.8443
- 1.0598
- 2.7613
- 0.9534
- 1.6559
- 1.0279
- 2.7954
- 1.6234
- 0.5049
- 1.9226
- 1.7266
- 0.5016
- 1.3838
- 0.5843
- 0.7034
- 1.4132
- 3.4237
- 3.3287
2.2293
1.2225
2.1235
2.885
4b
4c
4d
4e
2.0091
1.6576
2.1996
1.1486
1.4042
0.921
4f
4g
4h
4i
4j
4k
2.0557
1.5441
1.3076
2.3866
1.2786
2.5962
1.7355
0.3824
0.8515
4l
Total score total docking score;
Crash score degree of
4m
4n
inappropriate penetration by the
ligand into the protein and of
interpenetration between ligand
atoms that are separated by
rotatable bonds; Polar score
contribution of the polar non-
hydrogen bonding interactions
to the total score
4o
4p
5.2555
3.7112
1.9548
4q
Ampicillin
Ciprofloxacin 3.707
123

A. V. Chate et al.
aureus compared to ampicillin, i.e., 250 2.99 lg/mL. Compounds 4c and 4i
exhibited the same influence as compared to ciprofloxacin. Compounds 4m and 4p
were found to have excellent potency against S. aureus compared to that of
ciprofloxacin. Also, against B. subtilis, compounds 4a, 4c, 4g, 4h, 4k and 4m
showed maximum potency and compounds 4i and 4p were found to be more active
(25 0.39 and 37.5 0.14 lg/mL) than ampicillin (250 0.88 lg/mL). Com-
pounds 4i and 4p showed maximum potency compared with that of ciprofloxacin,
i.e., MIC 50 0.96 lg/mL, against B. subtilis. The results suggest that compound
4m may be a potent candidate for a new class of antibacterial agents in the future.
Structure–activity relationship (SAR) studies for antibacterial activity revealed
that the substitution at the aromatic ring positively affects the antimicrobial
activities of most of the compounds, and especially those with electron-withdrawing
and also electron-donating properties such as the methoxy and hydroxyl groups.
Replacement of heterocyclic aldehydes 4k and 4l also increases the antibacterial
activity. The introduction of the 2-hydroxy group 4m compared to the 4-hydroxy
group 4q on the benzaldehyde ring led to the most active compound of the
synthesized library and showed an increase in antibacterial activity. The introduc-
tion of a bromo group at the 4th position of benzaldehyde 4f led to a decrease in
activity compared with compound 4m.
In vitro antifungal activity
The synthesized compounds (4a–q) were screened for their antifungal activity. The
MIC (lg/mL) of all compounds was determined using Miconazole as a standard
drug. However, none of the compounds exhibited good activity against the fungal
strains. The results of the antifungal activity of all compounds (4a–q) are shown in
Table 4.
In silico molecular docking study
In order to explore the binding affinity, binding mode and molecular interactions of
the synthesized agent, a molecular docking study was carried out against its X-ray
crystal structure. Bacterial C30 carotenoid dehydrosqualene synthase was a potential
target in the antibacterial drug design and drug discovery process [54, 55]. In
bacterial systems, such as S. aureus formation, a pigment system such as
staphyloxanthin was carried out with the incorporation of two molecules of
farnesyl diphosphate in a condensation reaction by enzyme dehydrosqualene
synthase. Farnesyl diphosphate was again been converted into dehydrosqualene by
the enzyme dehydrosqualenesynthase which on conversion results in staphyloxan-
thin and acts as a virulence factor, and its inhibition results in the development of
potential antibacterial agents.
To identify the possible identical positions of synthesized pyrano[2,3-d]-
pyrimidinones analogues as they docked into active sites of the protein, they were
further ranked based on total docking score and interactions. To perform molecular
docking, a three-dimensional X-ray crystal structure of bacterial C30 carotenoid
˚
dehydrosqualene synthase enzyme (PDB ID: 3ACX 1.31 A) in halo form was used
123

b-CD-catalyzed multicomponent domino reaction: synthesis…
[56]. The number of compounds that have shown high MIC values calculated using
the well diffusion method and replicating their potential with a very high total
docking score, polar score and low crash score indicate their non-covalent
interactions such as hydrogen bond interaction and p interactions [52]. To represent
the details of the docking score, the following terms are used: (1) total score is the
total docking score, (2) crash score is the degree of inappropriate penetration by the
ligand into the protein and of interpenetration between ligand atoms which are
separated by rotatable bonds of compounds, and (3) polar score gives an idea about
the contribution of the polar non-hydrogen bonding interactions to the total score, as
shown in Table 5.
The detailed analysis of binding affinity (- logki) values and molecular
interactions of the most active molecules among the series (4p, 4m and 4i,
ampicillin, and ciprofloxacin) shows they inhibit AmpC. The molecules interact
with the active pockets of protein by forming H-bonds. The docking scores of
molecules 4p (5.2555), 4m (5.1109) and 4i (5.0558) were excellent as compared to
the standard drugs, ampicillin and ciprofloxacin. It has been found that these
analogues have a strong potential to inhibit AmpC. The most active pyrano[2,3-d]-
pyrimidinones, i.e., 4p (5.2555), 4m (5.1109) and 4i (5.0558) show efficient binding
modes and penetrate active site cavities by forming thhydrogen bond interactions
and p interactions with active site residues. The most active 4p (5.2555) analogue
interacts with the active site amino acid residue CYS44 to form conventional
hydrogen bond interactions, and it also interacts with the p electrons of the cloud of
˚
diazinane to form an p–sulfur bond interaction of 5.91 A. The amino acids
VAL137, LEU141 and TYR41 interact with the diazinane ring p electron cloud to
form p–p stacking, and p–r and p–p T-shaped interactions. Amino acid residues
TYR41, ARG45 and HIS18 interact with the p electrons of the benzyl ring to form
p–p stacking, and p–r and p–p T-shaped and p–alkyl interactions, as shown in
Fig. 3a.
The second most active pyrano[2,3-d]-pyrimidinones analogue 4m (5.1109)
forms conventional hydrogen bond interactions with amino acids CYS44 and
TYR41 where they interact with nitrogen-substituted hydrogen atoms and carbonyl
oxygen of the diazinane ring. Active site amino acid residue HIS18 forms a carbon–
hydrogen bond by interacting with the carbonyl oxygen of the diazinane ring with a
˚
distance of 3.69 A. Hydrophobic amino acid PHE22 interacts with hydroxyl oxygen
atoms of the phenyl ring to form p–lone pair interactions. Other active site amino
acid residues such as ARG45, VAL137 and LEU141 form various kinds of weak p
interactions with the p electron cloud of diazinane and the phenyl ring forming p
interactions such as p–p stacking, p–r and p–p T-shaped and p–alkyl, as shown in
Fig. 3b.
In silico pharmacokinetic evaluation
The drug likeness of the synthesized molecules was identified by predicting the
pharmacokinetic properties in silico. In particular, we calculated the compliance of
the synthesized compounds considering the basic parameters of Lipinski’s rule of
five, [57], BBB, Caco2, %HIA, etc. The pharmacokinetic parameters were obtained
123

A. V. Chate et al.
Fig. 3 a Binding position and molecular interactions of 4p into the active site of C30 carotenoid
dehydrosqualene synthase. b. Binding position and molecular interactions of 4m into the active site of C30
carotenoid dehydrosqualene synthase
by the ADMET predictor FAFDrugs2 which runs on the Linux OS. This tool is
freely available and used for in silico ADMET filtering [58, 59]. For an orally active
compound , two violations of Lipinski’s rule are accepted [60, 61]. The molecules
of the present study were found to follow the rule with maximum violation of two,
thus demonstrating their drug-likeness properties. We have assessed parameters like
molecular percent absorption (%ABS [ 100), weight (MW [ 500), partition
coefficient (logP [ 5), number of rotatable bonds ([ 10) and rthe atio of H/
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b-CD-catalyzed multicomponent domino reaction: synthesis…
123

A. V. Chate et al.
Fig. 4 SAR analysis of synthesized compounds (4a–q)
C ([ 1). All these parameters signify oral bio-availability and good intestinal
absorption [62]. The values obtained are depicted in Table 6.
Topological polar surface area (TPSA), i.e., the surface belonging to polar atoms,
and molecular weight are the descriptors which correlate with passive molecular
transport through membranes, allowing the prediction of the route of transport of
drugs through barrier membranes such as the intestine and the blood–brain barrier.
The percentage of absorption (%ABS) was calculated using TPSA by using the
formula %ABS = 109 - (0.345 9 TPSA) [62]. All the synthesized compounds
exhibited a very good %ABS, ranging from 62.93 to 78.64%. The values of the
partition coefficient (logP [ 5), the number of rotatable bonds ([ 10), the number
of rigid bonds ([ 25) and the ratio of H/C ([ 1) determines the absorption
performance through the lipophilic phospholipid membranes and the toxicity.
Moreover, none of the pyrano[2,3-d]-pyrimidinones violated Lipinski’s rule of five.
All the synthesized compounds, pyrano[2,3-d]-pyrimidinones (4a–q), followed the
criteria for orally active drugs and, therefore, these compounds can be further
developed as oral drug candidates. The results of this in silico ADME prediction
analysis suggest that the synthesized compounds follow the computational
assessment and thus represent a pharmacologically active framework that should
be considered for progressing their further potential against bacterial species and
may be good candidates for drugs (Fig. 4).
Conclusion
In conclusion, we have used b-cyclodextrin, a green supramolecular catalyst for the
synthesis of pyrano[2,3-d]pyrimidinone derivatives, as a very important biologically
active compound in a green media. No organic solvent was used during the reaction
and separation process, the catalyst was removed by simple filtration, no extra
purification was necessary for the obtained products, yields of the reaction were
high and the catalyst showed excellent reusability. The synthesized compounds
were evaluated for their antimicrobial activity against Ampicillin, Ciprofloxacin and
Miconazole in vitro. The bioassay results demonstrated that the title compounds
exhibited desirable antibacterial activity. A molecular docking study was performed
to investigate the possible binding mode of the compounds with the active site of the
enzyme. The molecular docking and MIC study revealed good binding energies and
potent antimicrobial values, respectively, for synthesized compounds having
electron-releasing substituents.
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