Bovine serum albumin catalyzed one-pot, three-component synthesis of dihydropyrano[2,3-c]pyrazole derivatives in aqueous ethanol

Article abstract of DOI:10.1039/c5ra13014j

Bovine serum albumin (BSA) catalyzed synthesis of dihydropyrano[2,3-c]pyrazole derivatives via a one pot, three component reaction of an aldehyde/ketone/isatin, malononitrile and 3-methyl-1H-pyrazol-5-(4H)-one in H₂O-EtOH (7: 3) at ambient temp

Full text of DOI:10.1039/c5ra13014j

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Bovine Serum Albumin catalyzed one-pot, three-component synthesis of
dihydropyrano[2,3-c]pyrazole derivatives in aqueous ethanol
Kiran S. Dalala, Yogesh A. Tayadeb, Yogesh B. Waghb, Darshak R. Trivedic, Dipak S. Dalalband
Bhushan L. Chaudharia*
a School of Life Sciences, North Maharashtra University, Jalgaon ꢀ 425 001 (MS) India.
b School of Chemical Sciences, North Maharashtra University, Jalgaon ꢀ 425 001 (MS) India.
c
Supramolecular Chemistry Laboratory, Department of Chemistry, National Institute of
Technology Karnataka (NITK), Srinivasnagar, Surathkal, Mangalore 575025 Karnataka, India.
*Corresponding author. Tel.: +91 2572257424; Fax: +91ꢀ2572258403
*Eꢀmail address: blchaudhari@nmu.ac.in, blchaudhari@hotmail.com
Abstract
Bovine serum albumin (BSA) catalyzed synthesis of dihydropyrano[2,3ꢀc]pyrazole derivatives
via one pot, three component reaction of aldehyde/ketone or isatin, malononitrile and 3ꢀmethylꢀ
1Hꢀpyrazolꢀ5 (4H)ꢀone in H2OꢀEtOH (7:3) at ambient temperature was developed in this work.
The catalyst was found to work efficiently for aldehydes, ketones and isatins to give the
corresponding dihydropyrano[2,3ꢀc] pyrazole and spiro[indolineꢀ3,4'ꢀpyrano[2,3ꢀc]pyrazole]
derivatives in high yields. The BSA showed a broad range of catalytic promiscuity towards
various aldehydes, aromatic/aliphatic ketones and substituted isatins. Use of environmentally
benign protocol, reusability of the catalyst, free from hazardous solvent, excellent yields, easy
work up and no byproducts formation make BSA an attractive candidate for expanding its
applications as biocatalyst.

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Keywords: Biocatalysis, Dihydropyanopyrazole, Bovine serum albumin, 3ꢀMethylꢀ1Hꢀpyrazolꢀ
5(4H)ꢀone.
Introduction
Dihydropyrano[2,3ꢀc]pyrazole is a promising class of heterocyclic compounds and a
structural unit of a variety of therapeutic agents.1The synthesis of dihydropyrano[2,3ꢀc]pyrazole
derivatives is receiving great attention among synthetic chemists due to their diverse bioactivity
profiles with various medicinal activities which include potential inhibitor of human Chk1
kinase,2a antiꢀinflammatory,2b anticancer,3a analgesic,3b molluscicidal4 and antimicrobial.5
Because of the abovementioned important properties of pyranopyrazoles, development of new
methods for the synthesis of this heterocyclic moiety has gained huge significance and
demanding task in organic synthesis. Generally, it is synthesized by multicomponent reaction of
aldehyde/ketones, malononitrile, ethyl acetoacetate, and hydrazine hydrate in the presence of
environmentally noncompatible base catalysts.6ꢀ9 Recently, some environmentꢀfriendly catalysts
such as Lꢀproline,10 γꢀalumina,11 and perꢀ6ꢀaminoꢀcyclodextrin,12Amberlyst A21,13 piperidine,14a
meglumine,14b cocamidopropyl betaine (CAPB),15a cetyltrimethylammonium chloride
(CTACl),15b molecular sieves, (MS 4Å)16a and saccharose16b were also used to achieve this
transformation. Their diverse biological, pharmacological properties and high cost necessitates
search for economically efficient and environmentally benign synthetic methodology for the
preparation of these heterocyclic compounds.
Biocatalysis is a green chemistry approach to chemical transformations because of its
high selectivity, mild reaction conditions, low energy requirements and few byꢀproducts17ꢀ20. The
biocatalytic promiscuity provides a new tool for extending the applications of enzymes in
organic synthesis.21 Hence, search for exploring the new possible biocatalyst to achieve

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environmental friendly reaction continues that avoids use of hazardous organic solvents, toxic
metals, and other harsh reaction conditions and producing different types of chemical and
biological substance with higher yields.22 Since few decades, several examples to exploit the
biocatalysts, like lipase,23a DNA,23b whole cell23c and laccase23d for catalyzing organic synthesis
have been set up bearing high catalytic activity and simple operation. It is important to increase
the promiscuity of enzyme and develop cost effective as well as green protocol for the synthesis
of dihydropyrano[2,3ꢀc]pyrazole compounds. The literature states that Bovine Serum Albumin
(BSA) was an efficient biocatalyst in many organic reactions, which includes aerobic oxidative
coupling of thiols into disulphides,24 nitroꢀaldol addition,25 Gewald reaction,26 Stereoselective
thioꢀMichael addition to chalcones,27 Knoevenagel condensation between diethylmalonate with
aldehydes,28 and Biginelli reaction29 for the synthesis of valuable compounds. The BSA or
“Fraction V” is the most abundant, low cost serum albumin protein derived from cows. It binds
several organic molecules due to presence of hydrophobic binding sites and basic characters
have attracted features.30ꢀ31 Recently Bora et al.32 have reported biocatalytic route for the
synthesis of dihydropyrano[2,3ꢀc]pyrazoles from aldehydes and ketones using Lipase from
Aspergillus niger. However, there is a scope left to explore better biocatalyst. Therefore, it is
obvious to search new biocatalysts with ecofriendly protocol, atom economy and broad substrate
range. In general, the ketones are less reactive than aldehydes and less explored, hence ketone
based dihydropyrano[2,3ꢀc]pyrazole synthesis was investigated.
In continuation of our work on protein isolation33 and biomimetic heterocycles
synthesis,34 recently we developed an efficient method for the synthesis of dihydropyrano[2,3ꢀ
c]pyrazoles and spirooxindoles by βꢀcyclodextrin in ethanolꢀwater mixture.35 Herein, we
envisioned the highly efficient and environmentally benign protocol for the synthesis of

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dihydropyrano[2,3ꢀc]pyrazoles and spiro[indolineꢀ3,4’ꢀpyrano[2,3ꢀc] pyrazole] derived from
aldehydes, ketones and isatins respectively by using BSA at ambient temperature as a biocatalyst
(Scheme 1).
Scheme 1 Synthesis of dihydropyrano[2,3ꢀc]pyrazoles and spiro[indolineꢀ3,4’ꢀpyrano[2,3ꢀ
c]pyrazoles in aqueous ethanol
Results and Discussion:
Screening of enzymes:
The different commercially available enzymes were selected to screen their role as a
catalyst in the synthesis of dihydropyrano[2,3ꢀc]pyrazole compounds. We choose the equimolar
mixture of acetone, malononitrile and pyrazolone. Acetone (2 mmol), malononitrile (2 mmol)
were mixed together in round bottom flask containing mixture of water and ethanol (7:3) and
stirred vigorously for 10 min to make homogeneous solution. Then pyrazolone (2 mmol) and
BSA (60 mg) were added to it. Reaction was carried out at temperature (40 °C) with continuous
stirring for 1 h. The transformation of product was monitored by thin layer chromatography

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(TLC). The product obtained was solid and insoluble in water. So, the reaction mixture was
diluted with 10 mL water and separated by filtration. Different enzymes were used to catalyze
synthesis of 4a and results were summarized in Table 1.
Table 1 Screening of the catalytic activities of different enzymesa
Entry Enzyme
Yieldb (%)
1
2
3
4
5
6
7
8
No catalyst
20
13
35
57
30
40
25
94
Lipase from Candida rugosa
Lipase from Porcine pancreas
Trypsin from Bovine
Trypsin from Porcine Pancreas
Papain
Diastase αꢀAmylase
Bovine Serum Albumin
a Reaction conditions: Acetone (2 mmol), malononitrile (2
mmol), pyrazolone (2 mmol), BSA(60 mg) and H2OꢀEtOH
(7:3, 10 ml) at 40 oC for 1 h, bIsolated yield.
When Lipase from Candida rugosa used as a catalyst, only 13% yield was obtained in 1h
while Lipase from Porcine pancreas gave 35% yield. We also used some digestive enzymes like
trypsin from bovine, surprisingly 57% yield was found but in case of trypsin from Porcine

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pancreas, the yield was decreased (30%). When Papain was used 40% product formation
observed while Diastase αꢀAmylase gave lower yield (25%). If no catalyst added, only 20%
yield was detected. When BSA was used, the yield increased dramatically to 94% probably
bringing catalysis due to rich diversity of amino acids present on surface of BSA.36
Optimization of solvent
The reaction medium plays an important role in enzyme catalyzed reactions owing to its
effects on enzyme activity as well as substrate solubility.37a The effects of different solvents
having different polarities on the model reaction were investigated. The results in Table 2 exhibit
diverse effects of different solvent on activity of BSA in the reaction. Independently water
showed ~39% yield after 1 h whereas rest of the solvents such as ethanol, methanol, acetonitrile,
THF, DMF showed trace yields. However, it was observed that the mixture of water and ethanol
at different ratios enhances the yield of reaction. The best result was obtained by using mixture
of water and ethanol (7:3) with highest yield of 94 %. Therefore, the BSA in water and ethanol
mixture (7:3) was used to carry out further reactions.
Table 2 Optimization of solvent a
Entry
Solvent
Water
Yieldb (%)
39
1
2
Ethanol
Trace

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3
4
Methanol
Trace
Trace
Trace
Trace
Trace
43
Acetonitrile
5
THF
6
DMF
7
H2O : EtOH (1:1)
H2O : EtOH (9:1)
H2O : EtOH (8:2)
H2O : EtOH (7:3)
8
9
50
10
94
a Reaction conditions: Acetone (2 mmol), malononitrile (2
mmol), pyrazolone (2 mmol), BSA(60 mg) and H2OꢀEtOH
(7:3, 10 mL), Reaction Time: 1 h at 40 0C, bIsolated yield.
Effect of catalyst loading:
In order to optimize the catalyst loading of BSA, the model reaction was performed with
varied quantity of BSA as 20 to 70 mg per mmol of substrate (Figure 1) while rest of the reaction
conditions was kept same. It was observed that increase in enzyme concentration up to 60 mg,
increased the yield of reaction, however, no significant improvement in the yield of reaction
observed further. The best yield 94% was obtained with 60 mg BSA per 2 mmol of substrate in
1 h of duration.

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Figure 1. Study of the amount of catalyst on reaction yields
Effect of temperature:
Temperature is an important factor in every biocatalytic reactions. When the reaction
temperature was increased, the collision between enzyme and substrate molecules might have
accelerated to form enzymeꢀsubstrate complexes resulting in improved reaction rate.38 In the
present study, the reaction temperature was changed from 20 to 50 °C to investigate its effect.
The results shown in Fig. 2 indicated that the yield was greatly improved by raising the
temperature from 20 to 30 °C, and reached to highest of 94% at 40 °C in one hour duration,
further increase in the temperature up to 50 °C resulted in same yield.
Figure 2. Study of the effect of temperature on reaction yields

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Substrate Scope:
Having set optimized parameters, the substrate scope for BSAꢀcatalyzed
dihydropyrano[2,3ꢀc]pyrazole was investigated by varying the nature of ketones. As shown in
Table 3, the BSAꢀcatalyzed reaction proceeded smoothly with aliphatic, cyclic and aromatic
ketones. The chain length of ketone has shown great influence on the reaction time. In the case
of acetone (Entry 1, Table 3), compound 4a was obtained with excellent yield of 94% in one
hour, while when 2ꢀbutanone was used as ketone under the same condition, a slightly decreased
yield 90% was observed (Entry 2, Table 3) in 15 h. When ethyl acetoacetate and methyl
acetoacetate (Entries 3ꢀ4, Table 3) was used as ketone, 94 % and 95 % yields in 12 h and 15 h
respectively were obtained for compound 4c and 4d. This indicates that BSA was chemo
selectively reacts with ketone carbonyl in the presence of ester carbonyl group. Furthermore, the
ring size of cyclic ketones might be exerting an obvious influence. For example, the yield of
cyclohexanone, malononitrile and pyrazolone after 21 h came up to 80% (Entry 5, Table 3). But,
when cyclopentanone was used as a substrate, the yield dramatically increased to 86% (Entry 6,
Table 3) in only 10 h. This phenomenon might be ascribed to the higher internal ring strain.
When the methodology was tested to aromatic ketones (Entries 7ꢀ12, Table 3) having electron
donating and electron withdrawing substituents, reaction time for complete conversion to
products were found to be higher with good yields than previously reported method.32
The efficiency of protocol in multigram scale was established by using equimolar (100
mmol) acetone, malononitrile and pyrazolone. Acetone (100 mmol, 5.8 g), malononitrile (100
mmol, 6.6 g) were mixed together in round bottom flask containing mixture of water and ethanol
200 mL (7:3) and stirred vigorously for 10 min to make a homogeneous solution. Then

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pyrazolone (100 mmol, 9.8 g) and BSA (3 g) were added to it and stirred at 40 °C for 1 h. The
reaction promptly proceeded to afford 4a in 95 % yields (19.4 g).
Table 3 Synthesis of dihydropyrano[2,3ꢀc] pyrazoles from ketones
M.P. (oC)[Lit.]
Time Yield
Entry
Ketone
Product
(h)
(%)
Found
Reported
1
2
1
94
174ꢀ176 176ꢀ177[14b]
4a
4b
15
12
90
94
164ꢀ166
204ꢀ206
ꢀꢀ
ꢀꢀ
3
4c
4
5
15
21
95
80
200ꢀ202
ꢀꢀ
4d
4e
146ꢀ148 147ꢀ148[14b]

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6
7
10
30
86
94
164ꢀ166 164ꢀ165[14b]
4f
175ꢀ176 174ꢀ175[32]
4g
8
9
36
24
48
36
86
95
76
83
210ꢀ212 220ꢀ221[14b]
182ꢀ184 181ꢀ182[13]
193ꢀ194 194ꢀ195[13]
4h
Cl
CN
N
N
H
O
NH2
4i
10
11
4j
208ꢀ210
ꢀꢀ
4k

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12
48
86
182ꢀ183 183ꢀ184[13]
4l
13
14
24
24
72
85
140ꢀ142
158ꢀ160
ꢀꢀ
ꢀꢀ
4m
4n
a Reaction conditions: Ketone (2 mmol), malononitrile (2 mmol), pyrazolone (2 mmol), BSA (60
mg) and H2OꢀEtOH (7:3, 10 mL) at 40 °C, bIsolated yield.
Isatin is an important molecule for designing potential pharmacological agents, and its
derivatives have shown a broad range of bioactivity and involved in synthesis of various
heterocyclic compounds.39 Spiro compounds are naturally occurring compounds present in many
pharmacological agent and natural alkaloids.40 The indole nucleus show antibacterial and
antifungal activities and it is important heterocyclic agent present in diverse types of medicinal
agents and natural products.41 These interesting properties promoted us to widen the applicability
of this procedure with isatins. To explore the scope, we used isatin as a ketone substrate to react
with malononitrile, and 3ꢀmethylꢀ1Hꢀpyrazolꢀ5(4H)ꢀone (1a) under previously optimized
conditions. Interestingly, the reactions with isatin proceed speedily at room temperature to
spiro[indolineꢀ3,4'ꢀpyrano[2,3ꢀc]pyrazole] with excellent yields (Table 4). The reactions for
isatin, 5ꢀchloroisatin and 5ꢀbromoisatin (Entries 1ꢀ3, Table 4) were completed in 15 minutes

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where as Nꢀalkyl isatins (Entries 4ꢀ7, Table 4) required slightly longer reaction time. Overall,
reactions with isatin resulted in excellent yields with 35 minutes except 5ꢀbromoisatin resulted in
somewhat lower yields 84%. However, when the four component one pot reaction was attempted
by using acetone, malononitrile, hydrazine and ethyl acetoacetate, the reaction was completed in
longer reaction time with lower yields. In order to explore substrate scope, the carbonyl
compounds, malononitrile and 3ꢀmethylꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ5(4H)ꢀone (1b) were reacted in
aqueous ethanol medium to give the products 4m-n and 6h-j in good yields. Xꢀray studies and
molecular structure of pyranopyrazole (4c and 6e) revealed that it exists in the crystal phase as a
2H, instead of 1H tautomer as shown in Figure S1 and S2 (See Supporting information), which is
in congruence with the literature reports on pyranopyrazoles.43
Table 4 Synthesis of spiro[indolineꢀ3,4ꢀpyrano[2, 3ꢀc]pyrazole] derivatives from Isatins
M.P.
Reference
Sr.
Time Yield
Isatin
Product
No.
(min)
(%)
Found
1
15
94
284ꢀ286 286ꢀ287[14b]
6a

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2
3
15
15
98
84
296ꢀ298 298ꢀ299[14b]
6b
6c
278ꢀ280 283ꢀ284[14b]
4
5
6
7
30
35
20
25
90
93
94
97
284ꢀ286
267ꢀ268
274ꢀ276
274ꢀ276
284ꢀ286[35]
6d
6e
6f
ꢀꢀ
276ꢀ278[35]
ꢀꢀ
6g

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8
9
30
30
35
98
93
89
226ꢀ228 228ꢀ230[37b]
230ꢀ232 232ꢀ234[37c]
230ꢀ232 232ꢀ234[37c]
6h
6i
10
6j
a Reaction conditions: Isatin (2 mmol), malononitrile (2 mmol), pyrazolone (2 mmol), BSA(60 mg)
and H2OꢀEtOH (7:3, 10 mL) at rt., bIsolated yield.
Inspired from the above initial success, we generalized the applicability of this method
for the synthesis of dihydropyrano[2,3ꢀc]pyrazoles from various aromatic aldehydes.
Interestingly, a variety of aromatic aldehydes including electron withdrawing and donating
abilities participated well in this reaction and brought good to excellent yield (Table 5).
Table 5 Synthesis of dihydropyrano[2,3ꢀc]pyrazoles from aldehydes
M.P.
Reference
Sr.
Time Yield
(h) (%)
Aldehyde
Product
No.
Found

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1
2
CN
1.5
94
88
244ꢀ246
244ꢀ246[35]
N
N
H
O
NH2
8a
8b
8c
8d
8e
8f
Cl
2
236ꢀ238
246ꢀ248
178ꢀ180
218ꢀ220
210ꢀ212
234ꢀ236[35]
CN
N
N
N
N
N
N
H
O
NH2
NO2
3
4
5
6
2.5
82
95
97
94
248ꢀ250[35]
178ꢀ180[35]
223ꢀ224[35]
210ꢀ212[35]
CN
N
H
O
NH2
Br
2
CN
N
H
O
NH2
OH
2
CN
N
H
O
NH2
OCH3
OCH3
3
CN
H
O
N
H
O
NH2
a Reaction conditions: Aldehyde (2 mmol), malononitrile (2 mmol), pyrazolone (2 mmol), BSA (60
mg) and H2OꢀEtOH (7:3, 10 mL) at rt, bIsolated yield.

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Test of recyclability:
The enzyme BSA remains in aqueous medium after the reaction. This aqueous medium
was reused to recycle for three runs while the product isolated was good enough with potential to
make it cost effective as shown in Figure 3. Slight decrease in the yield of product was observed
in the first, second and third reaction runs (92%, 88% and 82% respectively).
Figure 3 Recycle studies of BSA and its effect on yield.
Plausible Mechanism:
On the basis of the above results and previous studies, a plausible mechanism is proposed
for the synthesis of dihydroprano[2,3ꢀc]pyrazole from ketones. According to the literature
reports,40 the catalytic activity of BSA is based on the basic character of the amino groups
present in the side chain of some amino acid residues, especially the lysine residue. It is proposed
that firstly, the lysine’s amine attacks on the carbonyl group of a ketone leading to the formation
of iminium ion 3. At same time, the removal of proton from malononitrile by the free basic
amino group of BSA forms a carbanion which is subsequently condensed with iminium ion to

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give α, βꢀunsaturated nitrile 5. This step is known as Knoevenagel condensation. Then activated
3ꢀmethylꢀ1Hꢀpyrazolꢀ5(4H)ꢀone 7 and α, βꢀunsaturated nitrile 5 reacts with each other through
Michael addition to form 8 which undergoes cyclization to the desired product 9 as shown in
Scheme 2.
BSA
N
H
H
H
H
H
N
N
O
O
N
N
BSA
NH2
BSA
NH
H
H2O
O
7
6
H2N
Knoevenagel
condensation
R1
R2
R1
R1
R2
R2
BSA
NH2
BSA
3
1
fast
N
N
H
H
H
H
BSA
NH2
N
NC
CN
5
BSA
NC
CN
H
4
2
BSA
NH3
Slow
Michael Addition
BSA
NH2
R1
R1
R2
R2
CN
H
CN
NH2
N
N
H
N
Cyclization
N
O
O
H
HN
H
H
N
BSA
H
9
8
Scheme 2 Plausible mechanism for the synthesis of dihydropyrano[2,3ꢀc]pyrazole derivatives in
the presence of BSA in aqueous ethanol medium.

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Conclusion
In conclusion, we described simple, efficient and biocatalytic route for the synthesis of various
dihydropyrano[2,3ꢀc] pyrazoles and spiro[indolineꢀ3,4'ꢀpyrano[2,3ꢀc] pyrazoles using
inexpensive BSA. The advantages of this protocol include good to high yields, operational
simplicity, simple filtration and needing no metal catalyst or activation, and no extraction or
separation by column chromatography is necessary. Overall approach towards the green
chemistry and the use of BSA as biocatalyst in reaction is of practical significance in expanding
its applications in biocatalysts.
Acknowledgements
The authors are thankful to the University Grants Commission (UGC) New Delhi, India for
financial assistance under Special Assistance Programme (SAP) and Department of Science and
Technology for infrastructural facilities under FIST programme to NMU. We also thank
Department of Science and Technology (DST), Government of India for providing the SCXRD
facility under the FIST program to Department of Chemistry, NITK Surathkal, Mangaluru and
SAIFꢀPunjab University, Chandigarh for characterization support to this work.
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Graphical Abstract
Bovine Serum Albumin