An efficient practical chemo-enzymatic protocol for the synthesis of pyrazoles in aqueous medium at ambient temperature

Article abstract of DOI:10.1016/j.molcatb.2015.05.020

An expeditious oxidative cyclocondensation reaction of hydrazines/hydrazides with 1,3-dicarbonyl compound was efficiently developed in aqueous medium using Saccharomyces cerevisae (baker's yeast) as a whole cell biocatalyst at room temperature. The method has been assigned using green chemistry measures and found to give a range of N-substituted pyrazoles with moderate to excellent yields (70-92%). The reaction progress was monitored by gas chromatography.

Full text of DOI:10.1016/j.molcatb.2015.05.020

Journal of Molecular Catalysis B: Enzymatic 121 (2015) 75–81
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
An efficient practical chemo-enzymatic protocol for the synthesis
of pyrazoles in aqueous medium at ambient temperature
Ananda Mane, Prabha Salokhe, Pallavi More, Rajashri Salunkhe∗
Department of Chemistry, Shivaji University, Kolhapur 416004, M.S., India
a r t i c l e i n f o
a b s t r a c t
Article history:
An expeditious oxidative cyclocondensation reaction of hydrazines/hydrazides with 1,3-dicarbonyl com-
pound was efficiently developed in aqueous medium using Saccharomyces cerevisae (baker’s yeast) as a
whole cell biocatalyst at room temperature. The method has been assigned using green chemistry meas-
ures and found to give a range of N-substituted pyrazoles with moderate to excellent yields (70–92%).
The reaction progress was monitored by gas chromatography.
Received 15 November 2014
Received in revised form 17 May 2015
Accepted 19 May 2015
Available online 22 July 2015
© 2015 Elsevier B.V. All rights reserved.
Keywords:
Baker’s yeast
Aqueous medium
Pyrazoles
Biocatalysis
1. Introduction
various organic transformations [5,7]. Baker’s yeast has been exten-
sively used in the synthesis of library of heterocyclic compounds
It is widely accredited that there is an increasing impor-
tance for more environmentally viable synthetic routes in organic
synthesis. This progressive development known as ‘Sustainable
Technology’ necessitates a change in scenario from traditional
concepts of process efficiency to more economic and greener
approaches [1]. In recent years, water as reaction medium has cap-
tured high priority as green media because it is safe to handle,
environment-friendly, easily available, and enhances the reactivity
and selectivity of reactions [2]. In this context, use of biocata-
lysts in aqueous media has emerged as a green synthetic strategy,
to construct structurally diverse scaffolds from simple molecules.
Biocatalysis highlights an effective and preferable alternative to
the standard synthesis of potent chemicals as they can accept
un-natural compounds as substrates. As whole-cell biocatalysts
regenerate their own respective cofactors, they are frequently more
advantages than isolated enzymes [3]. Among the various possible
biocatalysts, baker’s yeast (Saccharomyces cerevisae) is renowned
catalyst, due to its low cost, easy handling, high bioavailability,
and its growth does not require the assistance of a specialist in
microbiology [4]. Baker’s yeast projects better catalytic behaviour
in aqueous medium [5] and has the ability to accelerate the
transformations under mild reaction conditions such as temper-
ature, light, stirring etc. [6] and is known to play vital role in
[8] such as, benzimidazoles, quinoxalines, polyhydroquino-
lines, 4H-pyranes, 1,4-dihydropyridines, 3,4-dihydro-pyrimidine-
2-(1H)-ones, isoindolo[2,1-a]quinazolines, 1,4-benzothiazines etc.
Recently Singh et al. [9] reported the synthesis of indolyl chromenes
and bisindolyl alkanes in water using baker’s yeast as the catalyst.
Pyrazole derivatives constitute the core structure of natu-
rally occurring and biologically active heterocyclic compounds.
Pyrazole nucleus containing compounds represent important
building blocks for luminophores, dyes, insecto-acaricides, antibac-
terial, antidepressant, analgesic and antiphlogistic drugs [10].
Pyrazole derivatives are of great interest due to their pharma-
cological properties for instance, pyrazole diimide (Fig. 1, I)
acts as anticancer drug [11]. Celecoxib {4-[5-(methylphenyl)-
3-trifluoromethyl) pyrazol-1-yl] benzene sulphonamide} (Fig. 1,
II) acts as inhibitor of cyclo-oxygenase-2 (COX-2) and reduces
side effects in the gastrointestinal tract [12]. Methylthiopyrazole
epothilone B (Fig. 1, III) shows strong antitumor activity through
the stabilisation of microtubules by binding with tubulin [13]. Due
to unique biological activities of pyrazole derivatives in medicinal
chemistry, the development of elegant and efficient ways enabiling
facile access to this heterocycle is desirable.
In recent years, a large number of protocols [14] for prepa-
ration of pyrazoles have been developed in different ways
using polystyrene supported sulfonic acid (PSSA) [15], Al2O3/clay
(montmorillonite K10) [16], Amberlyst-70 [17], polymer bound-p-
toluene sulphonic acid (PTSA) [18], silica supported sulphuric acid
(H2SO4·SiO2) [19], Sc(OTf)3 [20], Zn[(L)-proline]2 [21], sulphamic
∗
Corresponding author.
E-mail address: rsschem1@gmail.com (R. Salunkhe).
http://dx.doi.org/10.1016/j.molcatb.2015.05.020
1381-1177/© 2015 Elsevier B.V. All rights reserved.

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A. Mane et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 75–81
Fig. 1. Biologically active compounds containing pyrazole as a core structure.
acid [22] as catalysts. These reported methodologies produced
good results in many instances. However most of the methods
reported suffer from certain limitations such as use of expen-
sive reagents, tedious procedure for preparation of catalysts, low
selectivity, generation of acidic and metallic waste and tedious
workup conditions. Hence, the development of an efficient, sim-
ple, easy workup and environmentally benign protocol using green
solvent for the construction of pyrazole derivatives is of significant
interest. In view of the above valid points, we disclose herein an
environmentally benign and effective protocol for the synthesis of
pyrazole derivatives employing baker’s yeast (S. cerevisae) in fer-
menting medium at an ambient temperature. To the best of our
knowledge, the role of baker’s yeast in the cyclocondensation of
hydrazines/hydrazides and 1, 3-diketones has not been previously
reported.
Shimadzu QP 2010 GCMS. Baker’s yeast was purchased from local
market.
2.2. Experimental procedure
2.2.1. Fermentation of baker’s yeast
In a round bottom flask containing 5 mL of 0.01 M phosphate
buffer (pH 7.0), bakers’ yeast (400 mg) and d-glucose (750 mg) were
added and resulting solution was stirred for 12 h at 32 ◦C for fer-
mentation.
2.2.2. Synthesis of pyrazoles
To the fermented baker’s yeast 1,3-dicarbonyl compound
(1 mmol) and hydrazine/hydrazide (1.2 mmol) were added and the
reaction mixture was stirred at room temperature for indicated
time (Table 1). The progress of the reaction was monitored by
thin layer chromatography. After completion of the reaction, it was
extracted with dichloromethane and finally dried over anhydrous
sodium sulphate. The organic layer was concentrated in vacuo. The
resulting crude product was purified by column chromatography
using petroleum ether:ethyl acetate (7:3) as eluent.
2. Experimental
2.1. Materials and instruments
Solvents and reagents involved in the synthesis were sourced
from commercial suppliers and used as such. Progress of all
reactions and purity of dicarbonyl compound, hydrazines and
hydrazides were monitored by thin layer chromatography (TLC)
carried out on silica gel G 60 F254plates (Merck). Chromatograms
were developed using petroleum ether: ethyl acetate (7:3) as sol-
vent system. The melting points of products were measured in open
capillary tubes and are uncorrected. GC analysis was performed
using a 25 m HP-5 column (crosslinked 5% PH ME Siloxane) with
an inner diameter of 0.25 mm and a film thickness of 0.25 m. GC
was operated using a temperature profile with a starting temper-
ature of 40 ◦C, then increased by 15 ◦C/min to an end temperature
of 300 ◦C Infrared spectra were recorded on Perkin Elmer FT-IR
spectrometer. The 1H and 13CNMR spectra were recorded on a
Brucker-Avance 300 MHz spectrometer using TMS as an internal
standard and CDCl3 as a solvent. Mass spectra were recorded on
Table 1
Effect of amount of catalyst on the reaction of acetyl acetone and phenyl hydrazine.a
Entry
Amount of catalyst (mg)
Reaction timeb (h)
Yield (%)c
1
2
3
4
5
100
200
300
400
500
00:15
00:15
00:15
00:15
00:15
78
83
85
92
89
Bold values mean that at 400 mg of catalyst yield of product is maximum.
a
Reaction conditions: acetyl acetone (1 mmol), phenyl hydrazine (1.2 mmol), d-
glucose, phosphate buffer (pH 7.0, 5 mL) at room temperature.
b
Reaction progress monitored by TLC.
Isolated yield.
c

A. Mane et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 75–81
77
O
O
Baker's yeast
R
NHNH2
R
N
Phosphate buffer
N
D-glucose, r.t.
1(a-l)
2a
3(a-l)
Scheme 1. Reaction of different hydrazines/hydrazides with 1,3-dicarbonyl compound in fermented baker’s yeast at room temperature (3a–3l).
2.3. Spectral data for representative compounds
3.1. Baker’s yeast mediated synthesis of pyrazoles
2.3.1. (4-Chlorophenyl)(3,5-dimethyl-1H-pyrazol-1-yl)
methanone (3)
To establish the optimal reaction conditions, we have carried out
different experiments as follows; (I) control experiment: a control
reaction was carried out using acetyl acetone (1 mmol) and phenyl
hydrazine (1.2 mmol) in 5 mL of 0.01 M phosphate buffer (pH = 7)
and d-glucose (Scheme 1). The reaction mixture was stirred at room
temperature for 24 h, after workup and purification of crude mix-
ture by column chromatography only 8% of 1-phenyl-3,5-dimethyl
pyrazole (3a) was obtained. (II) with dry yeast: baker’s yeast, d-
glucose (750 mg), acetyl acetone (1 mmol) and phenyl hydrazine
(1.2 mmol) were taken together in 0.01 M phosphate buffer (5 mL,
pH = 7) and stirred. After workup and purification, 44% of 1-phenyl-
3,5-dimethyl pyrazole (3a) was obtained. (III) with fermented yeast:
baker’s yeast (400 mg) and d-glucose (750 mg) were taken in 5 mL
of 0.01 M phosphate buffer and stirred for 12 h at room temperature
for fermentation. Acetyl acetone (1 mmol) and phenyl hydrazine
(1.2 mmol) were added to the fermented yeast. Surprisingly the
yield of product (3a) was increased to 90% after stirring the reac-
tion mixture for only 15 min at room temperature. (IV) with yeast
extract: we also carriedout a experimentin which, baker’syeast was
stirred in distilled water and supernatant solution thus obtained
after centrifugation was used as yeast extract in place of fermented
baker’s yeast for the model reaction. It was observed that 1-phenyl-
3,5-dimethyl pyrazole (3a) was formed in 20% yield after stirring
reaction mixture for 24 h. (V) with inactive yeast: the model reaction
was also run by employing inactivated baker’s yeast (inactivation
was carried out by boiling yeast in water and dead cells obtained
after centrifugation were used instead of active baker’s yeast) as
a catalyst. After 24 h stirring of reaction mixture only 8% yield of
product (3a) was isolated. Thus it was concluded that fermented
baker’s yeast plays a crucial role in efficient cyclocondensation
of hydrazines/hydrazides and 1,3-diketones. It is also clear that
addition of components (acetyl acetone and phenyl hydrazine) to
fermented yeast (experiment III) gives a good yield in comparison
to that in which all the components were added simultaneously
(experiment II).
Oil; IR (KBr): ꢀ = 3110, 2930, 1698, 1582, 1342, 1081, 910 cm−1
.
1H NMR (300 MHz, CDCl3): ı = 2.20 (s, 3H), 2.54 (s, 3H), 6.04 (s,
1H), 7.44 (d, J = 1.8, 6.6 Hz, 2H), 7.97 (dd, J = 1.8, 6.6 Hz, 2H) ppm.
13C NMR (75 MHz, CDCl3): ı = 13.8, 14.3, 111.2, 128.0, 131.5, 132.3,
138.7, 145.8, 152.3, 166.9 ppm. MS (EI): m/z = 234 (M+).
2.3.2. (4-Nitrophenyl)(3,5-dimethyl-1H-pyrazol-1-yl)
methanone (8)
Yellow solid; IR (KBr): ꢀ = 3060, 2850, 1620, 1562, 1340, 1071,
940 cm−1 1H NMR (300 MHz, CDCl3): ı = 2.24 (s, 3H), 2.52 (s, 3H),
.
6.19 (s, 1H), 8.38 (d, J = 1.8, 6.9 Hz, 2H), 8.77 (dd, J = 1.8, 6.6 Hz, 2H)
ppm. 13C NMR (75 MHz, CDCl3): ı = 14.3, 15.7, 112.3, 129.1, 133.5,
136.3, 140.7, 151.8, 158.3, 169.9 ppm. MS (EI): m/z = 245 (M+).
2.3.3. 1-(3,5-Dimethyl-1H-pyrazol-1-yl)-2-
(3-methylphenyl-oxy) ethanone (10)
White solid; IR (KBr): ꢀ = 3001, 2920, 2840, 1742, 1610, 1571,
1398, 1328, 1250, 1161, 1090, 964, 839, 774 cm−1 1H NMR
.
(300 MHz, CDCl3): ı = 2.25 (s, 3H), 2.35 (s, 3H), 2.59 (s, 3H), 5.37
(s, 2H), 5.98 (s, 1H), 6.75–6.80 (m, 3H), 7.12–7.18 (m, 1H) ppm.
13C NMR (75 MHz, CDCl3): ı = 13.7, 14.1, 21.5, 66.5, 110.9, 111.5,
116.0, 122.5, 128.3, 139.0, 144.2, 152.3, 158.1, 167.9 ppm. MS (EI):
m/z = 244 (M+).
3. Results and discussion
Herein, we report an efficient and economical protocol for
the synthesis of pyrazoles. The protocol involves the oxidative
cyclocondensation of hydrazines/hydrazides with 1,3-dicarbonyl
compounds, catalysed by baker’s yeast as a whole cell biocatalyst
at room temperature.
Table 2
Baker’s yeast catalysed synthesis of pyrazoles (3a–3l) at room temperature.a
Entry
R
Reaction time (h)
Product
M. P. (◦C)
Yield (%)b
1
2
3
4
5
6
7
8
9
C6H5
C6H5 CO
00:15
00:30
00:40
01:20
01:30
00:30
00:50
08:00
24:00
00:40
00:10
00:25
3a
3b
3c
3d
3e
3f
3g
3h
3i
Oil [23a]
228–230 [24a]
Oil [24a]
92
87
82
80
78
77
83
77
75
84
90
90
4-Cl C6H5 CO
2-Cl C6H5 CO
2-Br C6H5 CO
3-C5H4N CO
4-C5H4N CO
4-NO2 C6H5 CO
2,4 NO2 C6H5
3-Me C6H5 OCH2CO
H
Oil [23b]
52–55 [24a]
152–154 [24b]
Oil [24a]
134 [24a]
119–121 [20]
50–52 [24a]
106–108 [23a]
Oil [20]
10
11
12
3j
3k
3l
CH3CO
a
Reaction conditions: acetyl acetone (1 mmol), hydrazine/hydrazide (1.2 mmol), baker’s yeast (400 mg), d-glucose (750 mg), phosphate buffer (pH 7.0, 5 mL) at room
temperature.
b
Isolated (molar) yield after column chromatography.

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A. Mane et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 75–81
The effect of amount of biocatalyst on the reaction of acetyl
that electronic effects of substituent’s on hydrazine and hydrazide
components play a greater role in these reactions. The pyrazole
formation reaction proceeds smoothly with phenyl hydrazine
(entry 1) and hydrazine hydrate (entry 11) with 90–92% yield
in short time. Whereas slightly electron donating substituent’s
on hydrazides (entries 2–5) provides pyrazole products smoothly,
electron demanding hydrazide (entry 8) required more time for
product formation. In addition with respect to the substituent’s
on hydrazide component, it was observed that steric effects are
important. In the case of 4-chlorobenzhydrazide 40 min of stir-
ring was required to give desired product (entry 3), whereas in the
case of 2-chlorobenzohydrazide the pyrazole formation reaction
acetone with phenyl hydrazine was studied and results are summ-
arised in Table 1. It was observed that the 400 mg of catalyst
was sufficient for the condensation of acetyl acetone and phenyl
hydrazine to give 1-phenyl-3,5-dimethyl pyrazole (3a) within
15 min (Table 1, entry 4).
To delineate optimised reaction conditions, the methodology
was evaluated by using several structurally diverse, activated and
deactivated hydrazines/hydrazides. A series of pyrazole deriva-
tives was synthesised and results are recorded in Table 2 . From
these results it seems that baker’s yeast accepts broad array of sub-
strate combinations. The reaction time and reaction yield indicates
Fig. 2. Progress of pyrazole formation as monitored by gas chromatography at different time intervals. Gas chromatograms of: (a) phenyl hydrazine, (b) acetyl acetone, (c)
aliquot of reaction mixture after 3 min, (d) ∼ after 6 min (e) ∼ after 9 min (f) ∼ after 15 min.

A. Mane et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 75–81
79
Fig. 2. (Continued ).
was completed in 80 min (entry 4). Hydrazide with bromo sub-
stituent at ortho- position, requires more time to complete the
reaction with slight decrease in yield as compared to hydrazide
with chloro substituent at ortho- position (entries 4 and 5) As the
bulk of R group increases, the reaction requires more time. With 4-
nitrobenzohydrazide and 2,4-dinitrophenylhydrazine the pyrazole
formation reaction requires 8 h and 24 h respectively (entries 8 and
9). In the absence of the catalyst 2,4-dinitrophenyl hydrazine does
not form pyrazole even at 80 ◦C, but it gave satisfactory yield in the
presence of baker’s yeast (entry 9). The yield of pyrazole was found
to be good when 2-(m-tolyloxy) acetohydrazide was reacted with
1,3-dicarbonyl compound (entry 10). This is because m-tolyloxy
group is far away from reaction site resulting in less steric interac-
tion. Acetohydrazide was also found to afford N-acetyl pyrazole
in excellent yield (entry 12). Heteroaryl hydrazides, pyridine-
3-carbohydrazide and pyridine-4-carbohydrazide has also been
successfully condensed with 1,3-dicarbonyl compound in the pres-
ence of baker’s yeast to respective pyrazoles with 75–85% yield
(entries 6 and 7). The above results suggest that electron density of
hydrazines and hydrazides is essential for the success of pyrazole
formation reaction.
The formation of desired products (Table 2, entries 1–12) was
confirmed from IR, 1HNMR, 13CNMR spectra as well as by mass
spectroscopy and in accordance with literature. The 1HNMR of each

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A. Mane et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 75–81
cyclocondensation reactions [27] and is widely employed in organic
synthesis. In particular, amino acid residues like histidine, serine
and aspartate or glutamate enhances electrophilicity of carbonyl
carbon of 1, 3-dicarbonyl compound by forming 1, 3-dicarbonyl:
enzyme noncovalent complexes [27,28]. When the reaction was
run by employing thermally inactivated baker’s yeast (inactiva-
tion was carried out by boiling yeast in water) as a catalyst, the
yield of 1-phenyl-3,5-dimethyl pyrazole was 8%, but with active
baker’s yeast yield was increased up to 92%. Due to thermal inacti-
vation of baker’s yeast lipase is inactivated which results in lower
yield of product. It indicates that, components apart from enzymes
present in baker’s yeast are not responsible to catalyse reaction of
hydrazines/hydrazides with 1, 3-dicarbonyl compound. We have
also carried out a reaction by employing isolated lipase from S. cere-
visae as a catalyst and obtained 1-phenyl-3,5-dimethyl pyrazole
with 60% yield. Therefore we believe that the enzyme lipase avail-
able in baker’s yeast is likely to be responsible to accelerate the
addition of hydrazines/hydrazides on 1, 3-dicarbonyl compound
leading to the desired pyrazole derivatives.
Fig. 3. Product formation-time dependences for the condensation of acetyl acetone
and phenyl hydrazine in fermented baker’s yeast.
4. Conclusion
product exhibited a singlet in the range ı = 5.98–6.19 ppm due to
CH of the pyrazole ring. In IR spectra, existence of a strong inten-
sity peak in the range 1560–1600 cm−1 due to C N functionality,
indicates the complete conversion of hydrazines/hydrazides to cor-
responding pyrazoles.
To explore the potential value of fermented baker’s yeast as cat-
alytic system, a scaled-up version was performed. By treatment
of 6 mmol of phenyl hydrazine with 5 mmol 1, 3-dicarbonyl com-
pounds under the optimal reaction conditions, desired product was
produced in 88% yield.
We have reported for first time the use of baker’s yeast as whole
cell biocatalyst to accelerate the synthesis of pyrazole derivatives.
The biocatalyst is inexpensive, easily available and biodegradable
making the protocol cost effective and ecofriendly. Furthermore,
the described procedure allows general synthesis of inaccessible
pyrazoles offering attractive features such as ambient reaction
conditions, operational simplicity, clean reaction profile and tol-
erance of wide variety of functional groups. Ongoing studies are
focused on applying this strategy to synthesise more complex
molecules.
3.2. Kinetic study of pyrazole formation
Acknowledgements
The reaction progress for the condensation of compounds
1a and 2a catalysed by fermented baker’s yeast (Scheme 1)
was conveniently monitored by gas chromatography. Aliquots
of reaction mixture were collected at different time intervals,
extracted with dichloromethane and analysed by gas chromatogra-
phy {Fig. 2(c–f)}. Chromatograms of substrates are shown by Fig. 2a
and b. The peak at 22.738 min in Fig. 2a is due to phenyl hydrazine
and the peak at 8.697 min in Fig. 2b is due to acetyl acetone. Gas
chromatograms in Fig. 2(c–f) depicting the progress of reaction.
After 3 min of stirring of reaction mixture, we observed the peak at
28.705 min due to phenyl hydrazone intermediate (II) and a peak
at 20.896 min due to small quantity carbinolamine intermediate (I)
but we did not observed the peak due to pyrazoline intermediate.
This might be because of unstability of pyrazoline under GC con-
ditions (Fig. 2c). After 9 min formation of pyrazole was observed
due to rapid loss of intermediate I (Fig. 2e). Further we stirred the
reaction mixture to 15 min and observed the maximum conversion
of reactants into product. After 15 min, we found that there is no
noticeable increase in yield of product.
We gratefully acknowledge the financial support from the
Department of Science and Technology and University Grants Com-
mission, New Delhi, India.
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