A reusable magnetic nickel nanoparticle based catalyst for the aqueous synthesis of diverse heterocycles and their evaluation as potential anti-bacterial agent

Article abstract of DOI:10.1016/j.bmc.2018.08.033

A library of biologically important heterocycles, viz. pyrazolyl pyrimidine-triones, bis(heterocyclyl)methanes were successfully synthesised by the condensation of barbituric acid, pyrazolone with an aldehyde and dimedone/4-hydoxy coumarin with various su

Full text of DOI:10.1016/j.bmc.2018.08.033

Accepted Manuscript
A reusable magnetic nickel nanoparticle based catalyst for the aqueous synthesis
of diverse heterocycles and their evaluation as potential anti-bacterial agent
Deboshikha Bhattacharjee, Sanjoy Kumar Sheet, Snehardinarayan Khatua, Koel
Biswas, Sataram Joshi, Bekington Myrboh
PII:
S0968-0896(18)30950-7
https://doi.org/10.1016/j.bmc.2018.08.033
BMC 14520
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 May 2018
15 August 2018
26 August 2018
Please cite this article as: Bhattacharjee, D., Sheet, S.K., Khatua, S., Biswas, K., Joshi, S., Myrboh, B., A reusable
magnetic nickel nanoparticle based catalyst for the aqueous synthesis of diverse heterocycles and their evaluation
as potential anti-bacterial agent, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2018.08.033
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A reusable magnetic nickel nanoparticle based catalyst for the aqueous
synthesis of diverse heterocycles and their evaluation as potential anti-
bacterial agent
Deboshikha Bhattacharjee,a Sanjoy Kumar Sheet,a Snehardinarayan Khatua,a Koel Biswas,b
Sataram Joshib and Bekington Myrboha*
aCentre for Advanced Studies in Chemistry, Department of Chemistry, North-Eastern Hill
University , Mawlai Shillong -793022 India.
bMicrobiology Laboratory, Department of Biotechnology and Bioinformatics, North-Eastern
Hill University, Shillong – 793022, India
Abstract
A library of biologically important heterocycles, viz. pyrazolyl pyrimidine-triones,
bis(heterocyclyl)methanes were succesfully synthesised by the condensation of barbituric
acid, pyrazolone with an aldehyde and dimedone/4-hydoxy coumarin with various substituted
aldehydes in aqueous medium at room temperature catalysed by nickel nanoparticles which
proved to be an efficient magnetically recyclable catalyst. The method is simple, eco-friendly
and gave excellent yields of the products without taking recourse to column chromatographic
separation procedures. Computational method was employed to elucidate the selective
formation of uncyclised product in reaction course. The biological activity of the synthesized
compounds were investigated and the results demonstrated profound antibacterial activity.
1. Introduction
The discovery of multi-component reaction has led the synthesis of library of diverse
heterocycles in a simple, efficient and concerted steps minimising the by-products and
maximising the atom economy in a more environment friendly manner. Keeping in mind the
green principles of chemistry, the use of harmful volatile organic solvents have been avoided
and in lieu of hazardous solvents many alternative reaction media have been introduced.1 One
such reaction medium is the ‘universal solvent’ i.e. water as it is the safest and most abundant
substance on our planet.2 Besides as a solvent, water tends to enhance the reaction rate due to
its polarity, hydrophobic packing and hydrogen bonding.3 The search for a catalyst which is
nontoxic, affordable, easy to recycle and having high catalytic efficiency is an ongoing

process. Thus, the development of multi-component reaction in safer reaction medium using
a catalyst having the above mentioned properties is highly desirable.
The application of nanoparticles as catalysts in synthetic chemistry has gained tremendous
interest in recent years.4 Lately nickel nanoparticles have been the focus of attention because
of their unique properties and potential applications in magnetism,5 energy technology,6
electronics,7 and biomedicine.8 In comparison with the other metals however, nickel
nanoparticles have found less application in catalysis with only few reports of their
application in organic reactions.9
Barbiturates are important medicinal scaffolds which demonstrate inhibition against protein
kinase C (PKC)10 and found presence in CNS drugs like sedative, anxiolytic, anesthetic,
antirpileptic and anticonvulsant.11-13 In similar manner pyrazolones are also well known
inhibitors of numerous oncogenic signal transduction kinases such as RTK c-Met, ALK,
VEGFR-2 TGF bR1, and HAT.14 Moreover, the pyrazolone nucleus are core structural
component of compounds having antipyretic, anti-inflammatory, antimicrobial, analgesic,
antifungal, and antiviral activity.15-19 The zwitterionic compounds on the other hand, are
excellent options for drug designing and delivery because of their specific protein absorption,
evasion of quick recognition by immune system, slow blood clearance from the body and
excellent water solubility.20 Inspite of their biological significance however, not many reports
are available in the literature which describes the synthesis of pyrazolyl pyrimidinetriones
derivatives in its zwitterionic form except for those by Bihani et al.20 On the other hand,
bis(heterocyclyl)methanes which are also building blocks in natural porphyrins21 are studied
extensively because of their diverse biological and pharmacological activities.22 A number of
methods have been developed for their synthesis using different catalyst like molecular
24
27
2- 29
iodine,23 Zn(proline)2, surfactants,25 ionic liquids,26 MnCl2, DBU,28 TiO2/SO4 , choline
34
hydroxide,30 TBAB,31 nanomaterial,32 Mn(pbdo)2Cl2/MCM-41,33 Zr(DP)2 by conventional
method as well as by microwave irradiation. The common drawbacks like longer reaction
time, high temperature, tedious work-up, non-recyclability of catalyst and use of hazardous
organic solvents and expensive reagents still plague many of these methods. As part of our
continuing effort in the of development of greener synthetic methodology,35 we decided to
employ nickel nanoparticles as a catalyst in the three component reactions involving
barbituric acid, an aldehyde, pyrazolone and an aldehyde with dimedone/4-hydroxy
coumarins respectively for generating heterocycles having potential biological activity. We
report herein result of our effort.

2. Results and discussion
2.1 Chemistry
Thus, when aldehyde
1 (2.1 mmol), pyrazolone 2a (2.0 mmol) and barbituric acid 3a
(2.0 mmol) are reacted at room temperature in aqueous medium in presence of Nickel
nanoparticles (NPs) we were gratified to note that the three component reaction
yielded pyrazolyl pyrimidinetriones. Similarly when pyrazolone and barbituric acid
are replaced by 4-hydoxy coumarin 2b / dimedone 2c (4 mmol) and condensed with an
aldehyde
1 (2.1 mmol) bis(heterocyclyl) methanes are formed under similar reaction
condition (Scheme 1).
Scheme 1. Synthesis of pyrazolyl pyrimidinetriones, bis(4-hydroxy-2H-chromen-2-one) and
bis(3-hydroxy-5,5-dimethylcyclohex-2-enone) derivatives
2.1.1 Preparation of Nickel nanoparticles and its Characterization
Nickel nanoparticles were synthesized by the chemical reduction method as per the reported
procedure.36 To a solution of nickel chloride hexahydrate in water hydrazine hydrate was
added followed by addition of NaOH to maintain the alkalinity. The reaction was stirred at
60οC till a black precipitate was formed. To ensure the complete conversion, the reaction
mixture was allowed to stir for another 2 hrs at 60οC. The precipitate thus formed was
filtered, washed several times with ethanol and then dried overnight in a hot air oven at 80οC.
The nanoparticles exhibited magnetic properties and were used as such to catalyse our
reactions. The reaction involved in the synthesis of nanoparticles (Nps) is generally
represented as

The synthesised dried powder was characterized using transmission electron microscope
(TEM), scanning electron microscope (SEM) energy-dispersive X-ray (EDX) analysis and
powder x-ray diffraction (PXRD).
Fig.1. (a,b) TEM images of Ni NPs. The scale bar in the TEM image is 100 nm and 50 nm
respectively. (c) SAED image at 21 nm.
Fig.2 (a) SEM image of Ni Nps (b) EDAX Image of Ni Nps.
From the TEM (Fig. 1) and SEM (Fig. 2) image it is clear that the prepared nickel
nanoparticles are nearly spherical in shape has an average size of 10–20 nm. The presence of
some larger particles must be due to aggregation or overlapping of smaller particles. The
EDAX data (Fig.2b) of Ni nanoparticles shows the presence of Ni peak with no trace of
oxygen, thus confirming the nanoparticle is composed of nickel only.
Fig.3. Powder XRD of Ni NPs showing shows characteristic dihedral angles at 44.12ο, 51.60ο

The powder XRD pattern of the nickel nanoparticles (Fig. 3) shows characteristic dihedral
angles at 44.12ο, 51.60ο corresponding to Nickel (0) nanoparticles. No obvious peak of nickel
oxide or hydroxide was detected.
The FTIR spectroscopy of nickel nanoparticles were measured between 400 and 4000 cm -1
showed peaks at 3440, 1633, 1384, 1110,752, 601 cm -1 (Fig.4).
Fig. 4. FTIR spectrum of Ni NPs
Thereafter the successful synthesis and characterisation of Ni Nps, the as synthesised
nanoparticles were employed as a catalyst in the multi-component reaction under various
reaction condition. At first, a neat reaction of 4-chloro benzaldehyde 1a (2.1 mmol),
pyrazolone 2a (2.0 mmol), barbituric acid 3a (2.0 mmol) ran in the presence of catalytic
amount of Ni nanoparticles which did not show any product formation. The subsequent
reactions were then carried out in different solvent systems as shown in Fig.5. It was
observed that the reactions carried out in ACN and DMF showed some product formation
after 12 hrs. When the solvent system was changed to ethanol, the yield of the product
increased dramatically to 75% in 6 hrs. The yield of the reaction further increased marginally
in ethanol: water (1:1) solvent system but the best result was obtained when reaction was
carried out in water affording 92% yield of the product in 3 hrs. Next, the catalyst loading
was varied and we found that 25mg/mmol of the catalyst is the required amount for optimum
yield of the product (Fig.6). When the reaction was carried out at room temperature, in
absence of the catalyst no product formation was observed. After prolonged heating the
reaction mixture for 16 hrs 70 % consumption of starting material was observed along with
desired product formation.

Fig.5. Effect of solvent on the rate of the reaction
Fig.6. Effect of Catalyst loading
and the yield of the product
1
The structure of the compound 4a formed was confirmed by H, 13C NMR, LC-MS, IR
analyses and by single crystal XRD. The single crystal data of 4a (Fig.7a) revealed that
contrary to our expectation, the uncyclised product (Scheme 2) was formed. This can be
attributed to the fact that both barbiturate and pyrazolone can exist in keto and enol forms to
give zwitterionic moieties in which one hydrogen atom of the barbiturate is transferred of to
pyrazolone imparting negative and positive charge respectively.20
Scheme 2. The product formation 4a
After the optimisation of the reaction was achieved, the generality of the method was studied
by carrying out the reactions with differently substituted aromatic aldehydes. All the
reactions proceeded smoothly to give the desired products in good to excellent yields with
high purity. The presence of electron withdrawing or releasing substituents in the ortho-meta
and para-positions do not seem to influence the reaction. All the pyrazolyl pyrimidinetriones
derivatives (4a–4k, Table 1) were obtained in pure form by simple filtration and
recrystallisation without purification by column chromatography and were characterised by
1H, 13CNMR, Mass spectral and IR analyses.

Table 1. Ni Nps catalyzed synthesis of pyrazol-4-yl-methyl-pyrimidine-2,4,6(1H,3H,5H)-
trione derivatives
Entry
4a
Aldehyde
Product (4)a
Time (hrs)
Yieldb/ (%)
3
92
3
3
90
94
4b
4c
3
3
3
88
93
90
4d
4e
4f
3
91
4g

3
89
4h
3
3
85
83
4i
4k
a
Products were characterized by 1H & 13C NMR Mass and IR analyses. Reaction condition:
aldehyde (2.1 mmol), pyrazolone (2.0 mmol), barbituric acid (2.0 mmol), Ni Nps
(25mg/mmol), Solvent H2O, room temperature
b Isolated yields
Encouraged by these results, the reactions involving active methylene compounds such as
dimedone and 4-hydroxy coumarins with substituted aldehydes were also studied. The same
optimised reaction condition was employed for the synthesis of bis(4-hydroxy-2H-chromen-
2-one) and bis(3-hydroxy-5,5-dimethylcyclohex-2-enone) derivatives. As expected the
desired products were formed in each case irrespective of different positional substitution
with excellent yield and purity. Even hetero and aliphatic substituted aldehydes were
explored and to our amaze, the substituent resulted in good yield. The products obtained (5a-
5k, 6a-6k) were purified by simple filtration, washing with water and crystallisation from
13
ethanol. All the synthesised products (Table 2) are fully characterized by 1HNMR, CNMR,
IR, and Mass spectral analyses. The derivatives 5j and 6a were characterised by single crystal
x-ray analysis (Fig. 7b and 7c).

(a)
(b)
(c)
Fig. 7. ORTEP plots of compound 4a, 5j and 6a with 30% thermal ellipsoid probability.
Table 2. Ni Nps catalyzed synthesis of bis(4-hydroxy-2H-chromen-2-one) and bis(3-
hydroxy-5,5-dimethylcyclohex-2- enone) derivatives

Active methylene
compound
Time
(hrs)
Yieldb/
(%)
Entry
Aldehyde
Product (5/6)a
5a
5b
5c
3
3
3
3
3
94
86
90
89
87
5d
5e
5f
3
3
3
3
3
3
92
89
93
91
92
86
5g
5h
5i
5j
5k
6a
3
93

6b
6c
6d
3
3
88
90
3
85
6f
3
3
91
88
6g
6h
6i
3
3
86
90
6j
3
3
89
80
6k
a
Products were characterized by 1H & 13C NMR Mass and IR analyses. Reaction condition:
aldehyde (2.1 mmol), Active methylene compounds (4.0 mmol), Ni Nps (25 mg/mol),
Solvent H2O , room temperature
b Isolated yields
The plausible mechanism is depicted in Scheme 3. The reaction presumably proceeds through
the initial activation of the carbonyl carbon atom of both active methylene compound and
aldehyde by co-ordination with the Ni nanoparticle which increases the electrophilicity of the
carbonyl carbon atom.32c Nucleophilic attack of the enolized pyrazolone / 4-hydroxy
coumarin / dimedone at the electrophilic carbon centre of the aldehydes takes place to form

Knoevenagel product followed by intermolecular cyclization with active methylene group
(barbituric acid/4-hydroxy coumarin/dimedone), dehydration and tautomerisation afforded
the final product 4/5/6.
Scheme 3. Plausible mechanism for the formation of the product 4, 5, 6
The reusability of the catalyst was studied by performing many runs of the model reaction for
the synthesis of 4a using recycled Ni nanoparticles, and to our delight it was found that the
yield of the desired product 4a was not affected much till the 4th cycle (Fig.8). The decrease
in the yield can be due to the fact that the nanoparticles might have aggregated which is
evident from the TEM images (Fig. S4. In supporting information). The nanoparticles were
recovered magnetically by a simple magnet from the reaction mixture. Then washed with
acetone and millipore water for several times and dried in oven. After complete drying the
recovered nanoparticles were reused as an efficient catalyst.
Fig.8. Reusability of the Ni nanoparticles for the synthesis of 4a

2.2 Theoretical studies
Density functional theory (DFT) calculations [37] were performed with the Gaussian 09
program package38 for compound 6a to explicate the selective formation of uncyclised
product over cyclised one. The geometry optimizations of cyclised and uncyclised product
was performed using Becke’s three-parameter exchange functional39 and the nonlocal
correlation functional of Lee, Yang, and Parr (B3LYP) in conjunction with the 6-31G(d,p)
basis set. The energy profile diagram reveals that uncyclised product 6a is energetically
favourable compare to cyclised product (6a′) as the total energy of 6a′ is significantly higher
than 6a (Fig. S5. In supporting information). This considerable energy difference (ΔE= -
20.6518 Kcal/mol) indisputably suggests the exclusive formation of uncyclised products.
2.3 Biological Studies
The pyrazolyl pyrimidinetriones, bis(4-hydroxy-2H-chromen-2-one) and bis(3-hydroxy-5,5-
dimethylcyclohex-2-enone) derivatives are known to have significant biological activity. All
the synthesized compounds were therefore screened for their antibacterial activity. The
antibacterial activities, given by the inhibition zone of all the synthesized compounds 4(a-k),
5(a-k), 6(a-k) against five common pathogens which include spoilage organisms viz.
Escherichia coli, Streptococcus pyogenes, Klebsiella pneumoniae, Bacillus cereus and
Salmonella enterica are shown in (Fig. S6, Fig. S7, Fig. S8 In supporting information)
respectively. Compound 4(a-k) and 5(a-k) exhibited distinctly high activity against the
tested Gram positive and Gram negative organisms. Compounds 6b and 6j however showed
mild activity against few of the organisms tested.
Pyrazolyl pyrimidinetriones and bis(4-hydroxy-2H-chromen-2-one) derivatives which
showed the best antimicrobial activity using the diffusion method were further tested by the
dilution method to determine the Minimum Inhibitory Concentration (MIC) (Table 3)
Compound 4a, 4d, 4g, 4h showed the best activity with MIC = 0.39 mg/mL against K.
pneumoniae and 4j with MIC = 0.39 mg/mL against B. cereus.
Compounds 4c, 4e, 4i with the range of MIC = 3.12 mg/ml and 6.25 mg/mL showed
antimicrobial activity against B.cereus and K. pneumoniae respectively at a relatively high
concentration. Compounds 5a, 5b, 5j exhibited better activity with MIC = 0.19 mg/mL
against K. pneumoniae while 5a, 5j with MIC = 0.39 mg/mL and 0.19 mg/ml against B.

cereus respectively. From the above results it can be concluded that compounds 4b, 4d, 4g,
4j, 5a, 5c, 5j are potential antibacterial agents for pharmaceutical application.
Table 3. Minimal inhibitory concentration (MIC) pyrazolyl pyrimidinetriones 4(a-k), bis(4-
hydroxy-2H-chromen-2-one) 5(a-k) against two tested reference strains
K.
K.
B. cereus
B. cereus
pneumoniae
pneumoniae
Compound
Number
Compound
Number
MIC*
(mg/ml)
MIC*
(mg/ml)
MIC*
(mg/ml)
MIC*
(mg/ml)
4a
4b
4c
4d
4e
4f
1.56 ± 0.03
0.39 ± 0.22
0.78 ± 0.22
3.12 ± 0
5a
5b
5c
5d
5e
5f
0.39 ± 0.22 0.19 ± 0.11
0.78 ± 0
3.12 ± 0
0.39 ± 0
3.12 ± 1.8
6.25 ± 0
0.19 ± 0.05
0.39 ± 0.11
0.39 ± 0.11
3.12 ± 1.8
6.25 ± 1.8
0.78 ± 0.45
6.25 ± 1.8
1.56 ± 0
0.39 ± 0
3.12 ± 0.90
0.78 ± 0.22
0.39 ± 0
0.78 ± 0.45 0.39 ± 0.22
4g
4h
0.78 ± 0.45
1.56 ± 0.90
5g
5h
0.78 ± 0
0.39 ± 0.11
0.78 ± 0
0.39 ± 0.22
3.12 ± 0.90
4i
4j
4k
3.12 ± 0
0.39 ± 0.22
6.25 ± 1.8
3.12 ± 0.90
0.78 ± 0.45
3.12 ± 0.90
5i
5j
5k
0.78 ± 0.22 0.78 ± 0.45
0.19 ± 0.05
3.12 ± 1.8
0.19 ± 0
0.78 ± 0.45
Chloramph-
Chloramph-
0.09 ± 0
0.09 ± 0
0.09 ± 0
0.09 ± 0
enicol
enicol
*MIC values are mean ± SD of three replicates

3. Conclusions
A novel and efficient three-component condensation reaction of aryl aldehydes, and C–H-
activated compounds has been developed for the synthesis of diverse heterocycles. In general,
improvements in the rates and product yields are observed when the reaction is carried out in
the presence of Ni NPs at room temperature. The DFT calculations were done for structure
elucidation. The catalyst can be recovered magnetically and reused up to four successive runs
without much noticeable change in the product yield. The protocol is eco-friendly, needs no
chromatographic methods of purification. In addition the antibacterial assay was performed
and it showed that pyarazolo pyrimidone (4) and bis(4-hydroxy-2H-chromen-2-one) (5) were
found to be potential antibacterial agent, while bis(3-hydroxy-5,5-dimethylcyclohex-2-enone)
(6) did not show much effect on the bacterial growth of inhibition.
4. Experimental
4.1 General Information
All commercially available chemicals and reagents were purchased from Sigma Aldrich,
Merck and were used without further purification. Purity of the products were confirmed by
infrared (IR), 1H-NMR, 13C-NMR and mass spectra. Melting points were determined in open
capillary tubes and are uncorrected. IR spectra were recorded in KBr pellets on a Perkin
1
Elmer Spectrum 400 FTIR instrument, and the frequencies are expressed in cm-1. H-NMR
and 13C-NMR spectra were recorded on Bruker Avance II-400 spectrometer in DMSO-
d6/CDCl3 (Chemical shifts in δ with TMS as internal standard). Mass spectral data were
obtained with a WATERS (ZQ–4000) mass spectrometer. Elemental analysis was done using
CHN-OS analyser model: Perkin Elmer 2400 series II. All reactions were monitored by thin
layer chromatography (TLC) using precoated aluminum sheets (silica gel 60 F254 0.2-mm
thickness). Powder XRD analysis was conducted with an X’Pert Pro instrument. The TEM
images were captured using a transmission electron microscope of JEM-2100 make, 200 kV
(JEOL) and SEM and EDS imaging were carried out with scanning electron microscope of
JSM-6360 (JEOL) make.

4.1.1 Synthesis of Nickel Nanoparticles
Nickel chloride hexahydrate (1.138 gm) was dissolved in 100 ml deionized water. To
this solution 1.2ml of hydrazine hydrate, which acts as a reducing agent was added
followed by addition of 1 ml of 1 M NaOH to maintain the alkalinity during the course
of reaction. The reaction was stirred in a capped vessel in a heating magnetic stirrer at
60°C till black precipitate was formed. To ensure the complete conversion, the
reaction mixture was allowed to stir for another 2 hrs at 60οC. The precipitate thus
formed was filtered, washed several times with ethanol and then dried overnight in a
hot air oven at 80οC.
4.1.2 General procedure for synthesis of compound pyrazolyl pyrimidinetriones
(4a-4k)
A mixture of aldehyde 1 (2.1 mmol), pyrazolone 2a (2.0 mmol) and barbituric acid 3a (2.0
mmol) in 5 mL milliprore water was stirred at room temperature for appropriate time in the
presence of Ni nanoparticles (25mg/mmol). The completion of the reaction was monitored by
TLC. After the completion of the reaction, the reaction mixture was filtered and then washed
with water. The residue was recrystallised from 5 mL ethanol to give the pure product.
4.1.3 General procedure for synthesis of compound bis(4-hydroxy-2H-chromen-
2-one) (5a-5k) and bis(3-hydroxy-5,5-dimethylcyclohex-2-enone) (6a-6k)
A mixture of aldehyde 1 (2.1 mmol), 4-hydroxy coumarin 2b/ dimedone 2c (4.0 mmol) in 5
mL milliprore water was stirred at room temperature for appropriate time in the presence of
Ni nanoparticles (25mg/mmol). The completion of the reaction was monitored by TLC. After
the completion of the reaction, the reaction mixture was filtered and then washed with water.
The residue was recrystallised from 5 mL ethanol to give the pure product.
4.2 Material and Methods for biological studies
To determine the activity of the supernatant, agar well-diffusion method was used. Reference
strains, Streptococcus pyogenes MTCC 1925, Enterococcus faecalis MTCC 2729, E.coli
MTCC 730, Klebsiella pneumoniae MTCC 109 and Bacillus cereus MTCC 430 used for the
study were obtained from Institute of Microbial Technology (Microbial Type Culture
Collection Centre, Chandigarh, India).

Muller Hilton Agar (HiMedia, Mumbai, India) plates were swabbed with bacterial cell
suspension of the indicator strains and clinical pathogens adjusted to 1.5 x 108 CFU/ml. 5 mm
diameter wells were cut in the agar plate using a sterile cork borer. 100 µl aliquot of each
compound was added to each well and the plates were incubated at 37oC for 24 hr. The
broad-spectrum antibiotic chloramphenicol (30 mcg) was used as a positive control and
DMSO solution was used as a negative control. After incubation, zones of inhibition around
the wells were measured manually. All observations were made in triplicates.
Determination of minimum inhibitory concentration (MIC): MIC of pyrazolyl
pyrimidinetriones 4(a-k), bis(4-hydroxy-2H-chromen-2-one) 5(a-k)
derivatives were
performed in triplicate using broth microdilution method in 96 multi-well microtiter plates.
0.5 McFarland standard suspension of the reference strains were inoculated in Muller-Hilton
broth (HiMedia, India). 50 µl of the inoculum was added to appropriate wells containing 50
µl of pyrazolyl pyrimidinetriones 4(a-k), bis(4-hydroxy-2H-chromen-2-one) 5(a-k). The final
concentrations of the 4(a-k) and 5(a-k) in the range 0.024 mg/ml - 400 mg/ml (in DMSO)
was used to evaluate the antibacterial activity. The 96 well plates were then sealed with
°
parafilm and incubated in aseptic condition at 37 C for 18 h. After incubation, 50 µl of 0.5%
solution of 2,3,5-triphenyl tetrazolium chloride (TTC) (Sigma, India) was added to the wells
and the plates were incubated for another hour in dark. The colorless tetrazolium salt gets
reduced to a red colored compound by biologically active organisms. Therefore, the
inhibition of growth can be detected when the solution in the well remains clear after the
incubation with TTC. The lowest concentration of each compound showing no visible growth
was recorded as the MIC.
4.3 X-ray Crystallography
The X-ray data of 4a, 5j and 6a were collected at 293 K with a Agilent Xcalibur (Eos,
Gemini) diffractometer using graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å).
The data was collected and reduced in CrysAlis PRO (Agilent, 2011) software and cell
refinement was done in CrysAlis PRO software.40 The absorption was corrected by SCALE3
ABSPACK multi-scan method in CrysAlisPro.40The structures were solved by direct
methods using the program SHELXS-201441 and refined by full matrix least-squares
calculations (F2) by using the SHELXL-201441 software. All non-H atoms were refined
anisotropically against F2 for all reflections. All hydrogen atoms were placed at their
calculated positions and refined isotropically. ORTEP image of 4a, 5j, 6a is shown in Fig. 6

and the details of data collection and refinements are given in Table S1, S2 and S3 in
electronic supplementary information. The .cif files for 4a, 5j and 6a were deposited with the
Cambridge Crystallographic Data Centre, and the following code was allocated: CCDC-
1562450, CCDC: 1562452 and CCDC: 1562451. This data can be obtained free of charge via
the Internet: www.ccdc.cam.ac.uk/ data_request/cif.
Acknowledgements
B. M. acknowledged financial assistance from SERB, DST (SB/EMEQ-006/2013). Authors
thank SAIF-NEHU and IIT-Guwahati for the spectral analyses. D.B. thanks the UGC Non-
Net fellowship for the financial assistance.
Conflict of interest
The authors have declared no conflict of interest.
References
1. Li X-T, Zhao, A-D, Mo L-P, Zhang, Z-H. Meglumine catalyzed expeditious four-
component domino protocol for synthesis of pyrazolopyranopyrimidines in aqueous medium.
RSC Adv. 2014;4:51580-51588.
2. (a) Breslow R. Green Chemistry Frontiers in Benign Chemical Syntheses and Processes.
In: Anastas P.T. Williamson TC, eds Oxford University Press, UK; 1998:225-233. (b) Li CJ,
Chen L, Organic chemistry in water. Chem. Soc. Rev. 2006;35:68–82.
3. Heravi MM, Mousavizadeh F, Ghobadi N, Tajbakhsh M, A green and convenient protocol
for the synthesis of novel pyrazolopyranopyrimidines via a one-pot, four-component reaction
in water, Tetrahedron Lett. 2014;55:1226–1228.
4. (a) Taran J, Ramazani A, Aghahosseini H, Gouranlou F, Tarasi R, Khoobi M, Joo SW.
One-pot three-component syntheses of α-aminophosphonates from a primary amine,
quinoline-4-carbaldehyde and a phosphite in the presence of MCM-41@PEI as an efficient
nanocatalyst. Phosphorus Sulfur Silicon Relat Elem.2017;192:776-781.(b) Das D,
Bhattacharjee M. Synthesis and structure of heterobimetallic Mo–M [M = Na, Co, Ni and Zn]
compounds and synthesis of nanostructured mixed metal oxides MMoO4 and MoO3[M = Co,
Ni and Zn] from the heterobimetallic complexes. Polyhedron 2015;99 :122-131 (c) Santra S,
AK Bagdi, Majee A, Hajra A. RSC Adv. 2013;3:24931-24935.(d) Song H. Metal Hybrid

Nanoparticles for Catalytic Organic and Photochemical Transformations. Acc. Chem. Res.
2015;48:491−499.
5.(a) Hyeon T. Chemical synthesis of magnetic nanoparticles. Chem. Commun. 2003; 0:927–
934. (b) Gubin SP, Koksharov YA, Khomutov GB, Yurkov GY.Magnetic nanoparticles:
preparation, structure and properties. Russ. Chem. Rev. 2005;74:489–520.
6. Karmhag R, Tesfamichael T, Wӓckelgård E, Nikalsson G, Nygren M. Oxidation kinetics
of nickel particles: comparison between free particles and particles in an oxide matrix. Sol.
Energy 2000;68:329–333.
7. Tseng W, Chen C. Dispersion and rheology of nickel nanoparticle inks. J. Mater. Sci.
2006;41:1213–1219.
8. Rodríguez - Llamazares S, Merchán J, Olmedo I, Marambio HP, Muñoz JP, Jara P, Sturm
JC, Chornik B, Peña O, Yutronic N, Kogan MJ. Ni/Ni Oxides Nanoparticles with Potential
Biomedical Applications Obtained by Displacement of a Nickel-Organometallic Complex. J.
Nanosci. Nanotechnol. 2008;8:3820–3827.
9. (a) Park J, Kang E, Son SU, Park HM, Lee MK, Kim J, Kim KW, Noh H-J, Park J-H, Bae
CJ, Park J-G, Hyeon T. Monodisperse Nanoparticles of Ni and NiO:Synthesis,
Characterisation, Self-Assembled Superlattices, and Catalytic Applications in the Suzuki
Coupling Reaction. Adv. Mater. 2005;17:429–434.(b) Kidwai M, Mishra NK, Bansal V,
Kumar A, Mozumdar S. Ni-nanoparticles usage for the reduction of ketones. Catal. Commun.
2008;9:612–617. (c) Saxena A, Kumar A, Mozumdar S. Ninanoparticles: A mild
chemoselective catalyst for synthesis of thioethers. Appl. Catal. A: Gen. 2007;317:210–215.
(d) Alonso F, Riente P, Sirvent JA, Yus M. Nickel nanoparticles in hydrogen-transfer
reductions: Characterisation and nature of the catalyst. Appl. Catal. A: Gen. 2010;378:42–51.
10. Goekjian PG, Jirousek MR. Protein kinase C in the treatment of disease: signal
transduction pathways, inhibitors, and agents in development. Curr. Med. Chem. 1999;6:877–
903.
11. Brunton LL, Lazo JS, Parker KL, Buxton I, Blumenthal D. Goodman and Gilman’s The
pharmacological basis of therapeutics, The McGraw-Hill Companies Inc, 11th edn, 2006.
12. Johns MW. Sleep and Hypnotic Drugs. Drugs 1975;9:448–478.

13. Uhlmann C, Frӧscher W. Low Risk of Development of Substance Dependence for
Barbiturates and Clobazam Prescribed as Antiepileptic Drugs: Results from a Questionnaire
Study. CNS Neurosci. Ther. 2009;15:24–31.
14. (a) Tripathy R, McHugh R. J, Ghose AK, Ott GR, Angeles TS, Albom M. S, Huang Z,
Aimone LD, Cheng M, Dorsey BD. Pyrazolone-based anaplastic lymphoma kinase (ALK)
inhibitors: Control of selectivity by a benzyloxy group. Bioorg. Med. Chem. Lett. 2011;21:
7261–7264. (b) Liu L, Norman MH, Lee M, Xi N, Siegmund A, Boezio AA, Booker S,
Choquette D, D’Angelo ND, Germain J, Yang K, Yang Y, Zhang Y, Bellon SF, Whittington
DA, Harmange J-C, Dominguez C, Kim T-S, Dussault I. Structure-Based Design of Novel
Class II c-Met Inhibitors: 2. SAR and Kinase Selectivity Profiles of the Pyrazolone Series. J.
Med. Chem. 2012;55:1868–1897.(c) Ray NC, Clark RD, Clark DE, Williams K, Hickin HG,
Crackett PH, Dyke HJ, Lockey PM, Wong M, Devos R, White A, Belanoff JK. Discovery
and optimization of novel, non-steroidal glucocorticoid receptor modulators. Bioorg. Med.
Chem. Lett. 2007;17:4901–4905.
15. Tewari AK, Mishra A. Synthesis and Anti-Inflammatory Activities of N4, N5-
disubstituted-3-methyl-1H-pyrazolo[3,4-c]pyradizines. Bioorg. Med. Chem. 2001;9:715–718.
16. Abadi AH, Eissa AA, Hassan GS. Synthesis of Novel 1,3,4-Trisubstituted Pyrazole
Derivatives and Their Evaluation as Antitumor and Antiangiogenic Agents. Chem. Pharm.
Bull. 2003;51:838–844.
17. Tanitame A, Oyamada Y, Ofuji K, Fujimoto M, Suzuki K, Ueda T, Terauchi H,
Kawasaki M, Nagai K, Wachi M, Yamagishi J. Synthesis and antibacterial activity of novel
and potent DNA gyrase inhibitors with azole ring. Bioorg. Med. Chem. 2004;12:5515–5524.
18. Mackie TJ, Collee JG, McCartney JE. in Mackie and McCartney Practical Medicinal
Microbiology, Churchill Livingstone: Edinburgh, 13th edn., 1989, pp. 87.
19. Hult K, Berglund P. Enzyme promiscuity: mechanism and applications. Trends
Biotechnol. 2007;25:231–238.
20. (a) Bihani M, Bora PP, Bez G, Askari H. A green four-component synthesis of
zwitterionic alkyl/benzyl pyrazolyl barbiturates and their photophysical studies. Mol. Divers.
2014;18:745–757. (b) Bihani M, Bora PP, Verma AK, Baruah R, H.P. D. Boruah and G. Bez,

PPL catalyzed four-component PASE synthesis of 5-monosubstituted barbiturates: Structure
and pharmacological properties. Bioorg. Med. Chem. Lett. 2015;25:5732–5736.
21. (a) Vogel E, Lux N, Dorr J, Pelster T, Berg T, Bohm H.-S, Behrens F, Lex J, Bremm D,
Hohlneicher G. Furan-Based Porphyrins: Tetraoxa[4n+2]porphyrin Dications with 18-, 22-
, or 26- Electron Systems. Angew. Chem. Int. Ed. 2000;39:1101–1105. (b) Sessler JL, Seidel
D. Synthetic Expanded Porphyrin Chemistry. Angew. Chem, Int. Ed. 2003;42:5134–5175.
22. (a) Sone T, Ohba Y, Watanabe R. Nontemplate Synthesis of Macrocyclic Tetraimine
Schiff Bases Incorporating Dithienylmethane Units. Bull. Chem. Soc. Jpn. 1989;62:1346–
1348. (b) Maresca A, Scozzafava A, Supuran, CT. 7,8-Disubstituted- but not 6,7-
disubstituted coumarins selectively inhibit the transmembrane, tumor-associated carbonic
anhydrase isoforms IX and XII over the cytosolic ones I and II in the low
nanomolar/subnanomolar range. Bioorg. Med. Chem. Lett. 2010;20:7255–7258. (c) Vukovic
N, Sukdolak S, Solujic S, Niciforovic N. Substituted imino and amino derivatives of 4-
hydroxycoumarins as novel antioxidant, antibacterial and antifungal agents: Synthesis and in
vitro assessments. Food Chem. 2010;120:1011–1018. (d) Madari H, Panda D, Wilson L,
Jacobs, RS. Dicoumarol: a unique microtubule stabilizing natural product that is synergistic
with Taxol. Cancer Res. 2003;63:1214–1220. (e) Carta F, Maresca A, Scozzafava A, Supuran
C.T. Novel coumarins and 2-thioxo-coumarins as inhibitors of the tumor-associated carbonic
anhydrases IX and XII. Bioorg Med. Chem. 2012;20:2266–2273. (f) Bacci JP, Kearney A.M,
Vranken, DLV. Efficient Two-Step Synthesis of 9-Aryl-6-hydroxy-3H-xanthen-3-one
Fluorophores. J. Org. Chem. 2005;70:9051–9053.
23. Kidwai M, Bansal V, Mothsra P, Saxena S, Somvanshi RK, Dey S, Singh TP. Molecular
iodine: A versatile catalyst for the synthesis of bis(4-hydroxycoumarin) methanes in water. J.
Mol. Catal. A 2007;268:76–81.
24. Siddiqui ZN, Farooq F, Zn(Proline)2: a novel catalyst for the synthesis of dicoumarols.
Catal. Sci. Technol. 2011;1:810–816.
25. Mehrabi H, Abusaidi H. Synthesis of Biscoumarin and 3,4-Dihydropyrano[c]chromene
Derivatives Catalysed by Sodium Dodecyl Sulfate (SDS) in Neat Water. J. Ira. Chem. Soc.
2010;7:890–894.
26. (a) Zhu A, Wang M, Li L, Wang J. Tetramethylguanidium-based ionic liquids as
efficient and reusable catalysts for the synthesis of biscoumarin at room temperature. RSC

Adv. 2015;5:73974–73979. (c) Khurana JM, Kumar S. ChemInform Abstract: Ionic Liquid:
An Efficient and Recyclable Medium for the Synthesis of Octahydroquinazolinone and
Biscoumarin Derivatives. Monatsh. Chem. 2010;141:561–564.
27. Sangshetti JN, Kokare ND, Shinde DB. Water mediated efficient one-pot synthesis of bis-
(4-hydroxycoumarin)methanes. Green Chem. Lett. Rev. 2009;2:233–235.
28. Hagiwara H, Fujimoto N, Suzuki T, Ando M. Synthesis of Methylenebis(4-hydroxy-2-
pyrone) or Methylenebis(4-hydroxycoumarin) Derivatives by Organic Solid State Reaction.
Heterocycles 2000;53:549–552.
29. Karmakar B, Nayak A, Banerji, J. Sulfated titania catalyzed water mediated efficient
synthesis of dicoumarols—a green approach. Tetrahedron Lett. 2012;53:4343–4346.
30. Zhu A, Bai S, Li L, Wang M, Wang J. Choline Hydroxide: An Efficient and
Biocompatible Basic Catalyst for the Synthesis of Biscoumarins Under Mild Conditions.
Catal. Lett. 2015;145:1089–1093.
31. Khurana JM, Kumar S. Tetrabutylammonium bromide (TBAB): a neutral and efficient
catalyst for the synthesis of biscoumarin and 3,4-dihydropyrano[c]chromene derivatives in
water and solvent-free conditions. Tetrahedron Lett. 2009;50:4125–4127.
32. (a) Kour M, Paul S. Sulfonated carbon/nano-metal oxide composites: a novel and
recyclable solid acid catalyst for organic synthesis in benign reaction media. New J. Chem.
2015;39:6338–6350. (b) Heravi MM, Nahavandi F, Sadjadi S, Oskooie HA, Bamoharram FF.
Efficient Synthesis of Bis-Coumarins Using Silica-Supported Preyssler Nanoparticles. Synth.
Commun. 2010;40:498–503. (c) Karami B, Hoseini SJ, Eskandari K, Ghasemi A, Nasrabadi
H. Synthesis of xanthene derivatives by employing Fe3O4 nanoparticles as an effective and
magnetically recoverable catalyst in water. Catal. Sci. Technol. 2012;2:331–338.
33. Heravi MM, Daraie M. Mn(pbdo)2Cl2/MCM-41 as a green catalyst in multi-component
syntheses of some heterocycles. Res. Chem. Intermed. 2016;42:2979–2988.
34. Ghassamipour S, Ghashghaei R. Zirconium dodecylphosphonate promoted synthesis of
xanthene derivatives by condensation reaction of aldehydes and β-naphthol or dimedone in
green media. Monatsh Chem. 2015;146:159–163.

35. (a) Bhattacharjee D, Kshair B, Myrboh B. L-Proline as an efficient enantioinduction
organo-catalyst in the solvent-free synthesis of pyrazolo[3,4-b]quinoline derivatives via one-
pot multi-component reaction. RSC Adv. 2016;6:95944–95950. (b) Bhattacharjee D,
Sutradhar D, Chandra AK. Myrboh B. l-proline as an efficient asymmetric induction catalyst
in the synthesis of chromeno[2,3-d]pyrimidine-triones, xanthenes in water. Tetrahedron
2017;73:3497–3504.
36. Pandey A, Manivannan R. A Study on Synthesis of Nickel Nanoparticles Using Chemical
Reduction Technique. Recent Pat. Nanomed. 2015;5:33–37.
37. (a) Hohenberg P, Kohn W. Inhomogeneous Electron Gas. Phys. Rev. 1964;136:B864–
B871. (b) Kohn W, Sham LJ. Self-Consistent Equations Including Exchange and Correlation
Effects. Phys. Rev. 1965;140:A1133–A1138.
38. Frisch MJ. et al. Gaussian 09, Revision E.01, Gaussian, Inc., Wallingford CT, 2009.
39. Becke AD. Density-functional thermochemistry. III. The role of exact exchange. J. Chem.
Phys. 1993;98:5648–5652.
40. CrysAlisPro Software System, 1.171.36.28, Agilent Technologies UK Ltd., Oxford, UK,
2013.
41. (a) Sheldrick GM. SHELXL-97: Program for Crystal Structure Refinement; University of
Gꢀttingen: Gꢀttingen, Germany, 1997. (b) Sheldrick GM. A short history of SHELX. Acta
Cryst. 2008;A64:112–122.
HIGHLIGHTS
 Magnetically recoverable & reusable catalyst easily prepared and utilised
 Aqueous Media reaction devoid of toxic reagent
 Non-chromatographic method of purification for synthesised compounds
 Number of prepared derivatives found to have antimicrobial activity