Synthesis of new heterocyclic hybrids based on pyrazole and thiazolidinone scaffolds as potent inhibitors of tyrosinase

Article abstract of DOI:10.1016/j.bmc.2012.12.053

As a part of ongoing studies in developing new Tyrosinase inhibitors, a class of structurally novel 2-(2,4-dimethoxy phenylamino)-5 methylene-4- thiazolinone derivatives were synthesized by incorporating 2-(2,4-dimethoxy- phenylamino)-thiazol-4-one with various 1-(1-methyl-buta-1,3-dienyl)-3-phenyl- 1H-pyrazole-4-carbaldehyde. The results showed that some of the synthesized compounds exhibited significant inhibitory activities. Especially, 5-[3-(2-chloro-phenyl)-1-phenyl-1H-pyrazol-4-ylmethylene]-2-(2, 4-dimethoxy-phenylamino)-thiazol-4-one (5h) and 5-[3-(3-chloro-phenyl)-1-phenyl- 1H-pyrazol-4-ylmethylene]-2-(2,4-dimethoxy-phenylamino)-thiazol-4-one (5g) possessing 2-chloro-phenyl and 3-chloro-phenyl group exhibited the most potent tyrosinase inhibitory activity with an IC₅₀ value of 34.12 and 52.62 μM, respectively. The inhibition mechanism analysis of 5h and 5g thiazolidinone derivatives demonstrated that the inhibitory effects of the compounds on tyrosinase were reversible and competitive. Preliminary structure-activity relationships (SAR) analysis suggested that further development of such compounds might be of interest, as it manifests simple reversible slow binding inhibition against monophenolase and diphenolase.

Full text of DOI:10.1016/j.bmc.2012.12.053

Accepted Manuscript
Synthesis of new heterocyclic hybrids based on pyrazole and thiazolidinone
scaffolds as potent inhibitors of tyrosinase
Shrikant S. Gawande, Suchita C. Warangkar, Babasaheb P. Bandgar,
Chandrahasya N. Khobragade
PII:
S0968-0896(13)00032-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2012.12.053
BMC 10523
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
3 December 2012
13 December 2012
15 December 2012
Please cite this article as: Gawande, S.S., Warangkar, S.C., Bandgar, B.P., Khobragade, C.N., Synthesis of new
heterocyclic hybrids based on pyrazole and thiazolidinone scaffolds as potent inhibitors of tyrosinase, Bioorganic
& Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2012.12.053
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Synthesis of new heterocyclic hybrids based on pyrazole and thiazolidinone
scaffolds as potent inhibitors of tyrosinase
Shrikant S. Gawandea,*, Suchita C. Warangkarb, Babasaheb P. Bandgarc ,
Chandrahasya N. Khobragadeb
aMedicinal Chemistry Research Laboratory, School of Chemical Sciences, Bahirji Smarak
Mahavidyalaya, Basmathnagar-431512, Dist- Hingoli, Maharashtra, India
bBiochemistry Research Laboratory, School of Life Sciences, S.R.T.M. University,
Nanded-431606, Maharashtra, India
cMedicinal Chemistry Research Laboratory, School of Chemical Sciences, Solapur
University, Solapur- 413255, Maharashtra, India
Dedicated to my colleague late Prof. Yogesh Mundhe
* Corresponding author. Tel./fax: +91 2454 220118.
E-mail address: gawande.shri@gmail.com

A B S T R A C T
As a part of ongoing studies in developing new Tyrosinase inhibitors, a class of structurally
novel 2-(2,4- Dimethoxy phenylamino)-5 methylene-4-thiazolinone derivatives were
synthesized by incorporating 2-(2,4-Dimethoxy-phenylamino)-thiazol-4-one with various 1-
(1-methyl-buta-1,3-dienyl)-3-phenyl-1H-pyrazole-4-carbaldehyde. The results showed that
some of the synthesized compounds exhibited significant inhibitory activities. Especially, 5-
[3-(2-Chloro-phenyl)-1-phenyl-1H-pyrazol-4-ylmethylene]-2-(2,4-dimethoxy-phenylamino)-
thiazol-4-one (5h) and 5-[3-(3-Chloro-phenyl)-1-phenyl-1H-pyrazol-4-ylmethylene]-2-(2,4-
dimethoxy-phenylamino)-thiazol-4-one (5g) possessing 2-chloro-phenyl and 3-chloro-phenyl
group exhibited the most potent Tyrosinase inhibitory activity with an IC50 value of 34.12
and 52.62 µM, respectively. The inhibition mechanism analysis of 5h and 5g thiazolidinone
derivatives demonstrated that the inhibitory effects of the compounds on Tyrosinase were
reversible and competitive. Preliminary structure activity relationships (SAR) analysis
suggested that further development of such compounds might be of interest, as it manifests
simple reversible slow binding inhibition against monophenolase and diphenolase.
Keywords: Thiazolidinone scaffolds, Tyrosinase inhibitors, Competitive inhibition,
Heterocyclic hybrids

1. Introduction
Tyrosinase (monophenol or o-diphenol, oxygen oxidoreductase E.C. 1.14.18.1, syn
polyphenol oxidase), also known as polyphenol oxidase (PPO) is a multifunctional copper
containing enzyme from oxidase family, widely distributed in microorganisms, animals and
plants.1 Tyrosinase catalyzes melanin biosynthesis in two distinct reactions: in the o-
hydroxylation of monophenols to o-diphenols (monophenolase) and in the oxidation of o-
dipheols to o-quinones (Diphenolase).2 Such Quinines could polymerize spontaneously due
to high reactivity and can polymerize spontaneously to form high molecular weight melanin
pigment by a series of reactions with amino acids and proteins.3 Melanin is one of the most
important natural biopolymer responsible for pigmentation, colour patterns of mammalian
skin and protect skin from solar radiations.4 However, this beneficial trait is most widely
associated with some severe human maladies and threatening diseases due to the
overproduction of melanin such as senile lentigo, freckles, melasma, chloasama and
pigmented acne scars are of particular concern in women.5 The treatment usually involves the
use of medicine or medicinal cosmetics containing depigmenting agents or skin whitening
agents.6 Likewise Tyrosinase inhibitors were also used to treat the dermatological disorders
related to melanin hyperaccumulation viz age spots, actinic damages, freckles and essential in
cosmetics for depigmentation.7,8 Tyrosinase has been also linked to neurodegeneration in
patients suffering from Parkinson’s disease.9,10 In insects Tyrosinase is uniquely associated
with three different biochemical processes like sclerotisation of cuticle, defensive
encapsulation and melanisation of foreign organism and wound healing.11 Thus, involvement
of Tyrosinase in such key processes provide potential targets for developing safer and
effective Tyrosinase inhibitors as insecticides and for their control.
Previous reports confirmed that Tyrosinase was one of the main causes of most fruits
and vegetables quality loss during post harvest handling and processing, leading to faster

degradation and shorter shelf life.12,13 Therefore, inhibition of melanin formation by
Tyrosinase is also applicable to fruit and vegetable preservation by the alleviation of
browning. Thus, unregulated action of Tyrosinase and excessive production of melanin will
cause number of human disease aetiologies.14 Hence, therapies to such diseases can be
explored by Tyrosinase inhibition. Tyrosinase inhibitors had gain an insight as a therapeutic
agent, as insecticide and antibrowning agent from last couple of decades. Tyrosinase
inhibitors normally either render the copper within the active site inactive by chelation,
obviating the substrate enzyme interactions or inhibit oxidation via an electrochemical
process.15
Among the nitrogen containing heterocyclic skeletons, thiazolidin-4-ones, rigid
molecules, are considered as a biologically privileged scaffold well-tolerated in human
subjets16, 17 and could be perspective models for the designing of new hybrids.18 Keeping in
view the above facts and continuing our program on the development of efficient methods to
generate drug-like nitrogen-containing molecules.19-22 We were interested in a simple
synthesis of thiazolidinone analogues containing the pyrazole moiety and their evaluation as
potent inhibitors of tyrosinase.
2. Results and Discussions
2.1 Synthesis
In the present investigation 2-(2,4-Dimethoxy-phenylamino)-5-methylene-thiazol-4-one (5a-
5j) have been prepared by the condensation of 2-(2,4-Dimethoxy-phenylamino)-thiazol-4-
one (3) and substituted 1,3-diphenyl-1H-pyrazole-4-carbaldehydes (4a-4j) by the known
literature method23 (Scheme I). Substituted 1,3-diphenyl-1H-pyrazole-4-carbaldehydes (4a-
4j) were prepared by Vilsmeir–Haack reaction on acetophenonephenylhydrazones.24 The
resulting crud product was purified by recrystallization from acetic acid: DMF (2:1) to afford

1
title compound (5a-5j). The compounds structure was confirmed by spectral data (IR, H
NMR and MS).
2.2 Biological evaluation
The effect of thiazolidinone derivatives on Tyrosinase inhibition
For evaluating the tyrosinase inhibition, all the synthesised thiazolidinone scaffolds were
subjected to tyrosinase inhibition assay with L-DOPA as substrate, according to the method
reported by our groups with some slight modifications. The tyrosinase inhibition by kojic
acid was ever reported, therefore, it was selected as reference compound. The IC50 values of
thiazolidinone scaffolds against monophenolase and diphenolase were summarised in Table
1, and IC50 values of all these compounds were determined from logarithmic concentration–
inhibition curves and given as means of three different experiments. From results, it was
observed that compounds 5h, 5g, 5d and 5i exhibited potent inhibition of mushroom
tyrosinase with an IC50 values ranged from 34.0 to 82.0 µM. Especially, compounds 5h and
5g possessing 2-chloro phenyl and 3-chloro phenyl substituents showed more potent
inhibitory activities than the other compounds and kojic acid. Most of the thiazolidinone
scaffolds viz 5a and 5j showed lower or negligible activities against tyrosinase.
Taken as an ensemble, the following general features of SAR can be deduced from
compounds (5a–5j). When the activities of the scaffolds possessing different substituents viz
methoxy, methyl and fluro, bromo and chloro compared, then it was observed that substituted
thiazolidinones with halogen atom (-Cl, -F, -Br) exhibited more potent inhibitory activities
than the thiazolidinones substituted with other groups. In addition, the activities of
thiazolidinone derivatives substituted with chloro group at different positions on
phenylamino-thiazol-4-one have different activities. The highest inhibitory activity obtained
with derivative 5h, when derivative is substituted with 2-cholro phenyl group could form a
more tight interaction with an enzyme.
From the Table 1, it was observed that with the increase of lipophilicity of the
compound, the inhibitory activities increased gradually. These results showed that the
inhibitory activities of 5-[3-(2-Chloro-phenyl)-1-phenyl-1H-pyrazol-4-ylmethylene]-2-(2,4-
dimethoxy-phenylamino)-thiazol-4-one (5h), 5-[3-(3-Chloro-phenyl)-1-phenyl-1H-pyrazol-4-
ylmethylene]-2-(2,4-dimethoxy-phenylamino)-thiazol-4-one (5g) analogues related to the
lipophilicity showed the most potent inhibitory activity in all their homologues, and with the
increase of the radius of halogen atom, the inhibitory activities decreased gradually. The

inhibitory activities of the halo substituted homologues were more potent than the inhibitory
activities of other substituted thiazolidinones. This result might be related to different
inhibitory mechanism. Since the chloro substituted thiazolidinones mainly depended on
interaction with B site of Tyrosinase and may be due to the increase of the radius of halogen
atom might cause stereohindrance for the inhibitors approaching the active site of the
enzyme. However, substituted thiazolidinones with electron withdrawing groups are similar
to benzaldehyde-type inhibitors, the Tyrosinase inhibitory mechanism of this type of
inhibitors, come from the ability to form a Schiff base with a primary amino group in the
enzyme.26 The increase of lipophilicity might be benefited to the formation of Schiff base. As
shown in Table 1, all of the thiazolidinone derivatives exhibited certain inhibitory activities
against tyrosinase at the concentration of 200µM. The inhibitory activities of thiazolidinone
scaffolds also be related to the structure of tyrosinase, which contained a type-B site which is
other than a coupled binuclear copper active site in the catalytic core. Tyrosinase inhibition of
compounds 5h, 5g and 5d depended on the competency of the chlorine atom that binds with
B site, and tyrosinase would lose its catalysing ability after forming complex.25
Progress curve, a new simple graphical method described for the determination of
inhibition type and inhibition constants of an enzyme reaction without any replot. It may be
also helpful to detect the inhibition detection limit (IDL), which describes the limit of
detection of competitive inhibition for end point assays as a function of the proportion of
substrate converted into product, has been developed.27 The progress curve obtained for
oxidation of L-DOPA catalysed by mushroom Tyrosinase in presence of thiazolidinone
scaffolds. The progress curve depicted, as the concentration of inhibitors raised, the residual
enzyme activity drastically decreased showing inhibition of Tyrosinase (Fig.2). The enzyme
activity inhibition was manifested by all inhibitors shown similar relationship between the
enzyme activity and enzyme concentration. From the progress curve obtained, the
compounds showing solid lines below the line of enzyme activity has indicative of enzyme
inhibition and vice versa.
Inhibitory mechanism
The kinetic behaviors of the oxidation of L-tyrosine and L-DOPA, catalyzed by mushroom
tyrosinase at different concentrations of compounds (5h, 5g, 5d and 5i ) were studied. In this
experiment, the initial velocity of the enzyme was monitored via dopachrome formation at
475 nm. As shown in Figure 1, the Lineweaver–Burk plots of 1/V versus 1/[S] result in a
family of straight lines with the same y-axis intercept, as illustrated, respectively, for the four
tyrosinase inhibitors. In the figure, the abscissa 1/[L-DOPA] is the reciprocal of the

concentrations of L-tyrosine, whereas the ordinate 1/V is the reciprocal of the change of the
velocity, which reflects the reciprocal of tyrosinase activity. The arrangement of the family of
lines in each graph indicated that these are competitive inhibitors [(1, Ki = 11.2 lM), (3, 0.77
lM), (4, 0.29 lM), and (5, 15.6 lM)], with their inhibitory activity toward tyrosinase
decreasing with increasing concentration of the substrate. Most competitive inhibitors of
tyrosinase have molecular structures which closely resemble that of the product of each
respective step.
These results demonstrated that the inhibitory activity of compound 5h and 5g on the
tyrosinase were reversible and competitive, suggesting that chlorophenyl substituted scaffolds
more potent than inhibited the enzyme by binding to its binuclear active site reversibly. The
dissociation constant value of L-DOPA binding with Tyrosinase was higher in presence of
scaffold 5h and 5g, decreases in presence of other halide containing derivatives (5g, 5i, 5e
and 5f). The present investigation reported that thiazolidinone scaffolds had potent inhibitory
effects on the diphenolase activity of mushroom tyrosinase. Interestingly, compound 5h was
found to be the most potent inhibitor with IC50 value of 34.12 µM. Preliminary structure
activity relationships (SARs) analysis indicated that chlorophenyl moieties of 5h and 5g
might play an important role in determining their inhibitory activities, (1) because of the
chlorine atom might be binding with the B site other than the A active site, and Tyrosinase
would lose its catalysing ability after such binding25; (2) carboxyl group might be effective
group to the interaction of compound with the active site of tyrosinase.22 The inhibition
mechanism analysis of compounds 5h and 5g demonstrated that the inhibitory effects of the
two compounds on the tyrosinase were reversible and competitive.
3. Conclusion
In the present study, a series of thiazolidinone scaffolds were synthesized and their inhibitory
effects on mushroom Tyrosinase were evaluated. The results demonstrated that the analogues
possessing halide substituents with phenylamino linker exhibited more potent inhibitory
activities than the reference compound kojic acid. Analysis of the inhibition kinetics revealed
that thiazolidinone scaffolds were reversible competitive inhibitors.

4. Experimental
4.1. General
Melting points were recorded in open capillaries with electrical melting point apparatus and
were uncorrected. IR spectra (KBr disks) were recorded using a Perkin–Elmer 237
1
spectrophotometer. H NMR (400 MHz) spectra were recorded on a Bruker Avance
spectrometer using CDCl3 as solvent. Mass spectra were recorded on a Shimadzu LCMSQP
1000 EX. All the reagents and solvents used were of analytical grade and were used as
supplied unless otherwise stated. TLC was performed on silica gel coated plates for
monitoring the reactions.
4.1.1. 2-chloro-N-(2,4-dimethoxy-phenyl)-acetamide (2)
Yield: 97 %.
MF/FWt: C10H12ClNO3 /229
MP: 142 oC
IR cm -1: 3408, 3105, 2964, 1668, 1543, 1496, 1282.
1H NMR (400 MHz, CDCl3): δ 7.82 (d, 1H, J= 7.6 Hz), 6.61 (s, 1H), 6.52-6.50 (m, 1H), 4.21
(s, 2H), 3.87 (s, 1H), 3.86 (s, 3H), 3.79 (s, 3H).
MS: m/e 230 (M+1).
4.1.2. 2-(2,4-Dimethoxy-phenylamino)-thiazol-4-one (3)
Yield: 84 %.
MF/FWt: C11H12N2O3S /252
MP: 168 oC
IR cm -1: 3390, 3143, 3034, 1705, 1643.
1H NMR (400 MHz, CDCl3): δ 7.15 (d, 1H, J= 8.8 Hz), 6.50-6.44 (m, 2H), 3.97 (s, 1H), 3.86
(s, 2H), 3.83 (s, 3H), 3.80 (s, 3H).
MS: m/e 253 (M+1).
4.1.3. 1-(1-methyl-buta-1,3-dienyl)-3-phenyl-1H-pyrazole-4-carbaldehyde24 (4a-j)
4.1.4.1
2-(2,4-Dimethoxy-phenylamino)-5-methylene-thiazol-4-one (5a)
Yield: 72 %.

MF/FWt: C27H22N4O3S /482
MP: 175 oC
IR cm -1: 3120, 3053, 1710, 1666, 1340.
1H NMR (400 MHz, CDCl3): δ 8.06 (s, 1H), 7.90 (d, 2H, J= 8.3 Hz), 7.54 (d, 1H, J= 7.6
Hz), 7.66 (d, 2H, J=7.2 Hz), 7.51-7.46 (m, 2H), 7.43 (d, 2H, J= 6.8), 7.35 (t, 1H, J= 7.2 Hz),
7.26 (s, 1H), 7.09 (d, 1H, J= 8.3 Hz), 6.55 (t, 2H), 3.91 (s, 1H), 3.86 (s, 3H), 3.80 (s, 3H).
MS: m/e 483 (M+1).
4.1.4.2
2-(2,4-Dimethoxy-phenylamino)-5-(1-phenyl-3-p-tolyl-1H-pyrazol-4-ylmet
hyle- ne)-thiazol-4-one (5b)
Yield: 68 %.
MF/FWt: C28H24N4O3S /496
MP: 175 oC
IR cm -1: 3369, 3182, 3024, 1693, 1614.
1H NMR (400 MHz, CDCl3): δ 8.03 (s, 1H), 7.75 (d, 2H, J= 6.8 Hz), 7.73 (s, 1H), 7.54 (d,
2H, J= 7.6 Hz), 7.49-7.62 (m, 2H), 7.36 (s, 1H), 7.34-7.26 (m, 2H), 7.09 (d, 1H, J= 8.4 Hz),
6.99-6.52 (m, 2H), 3.91 (s, 1H), 3.86 (s, 3H), 3.83 (s, 3H), 2.42 (s, 3H).
MS: m/e 497 (M+1).
4.1.4.3
2-(2,4-Dimethoxy-phenylamino)-5-[3-(4-methoxy-phenyl)-1-phenyl-1H-
pyrazol-4-ylmethylene]-thiazol-4-one (5c)
Yield: 65 %.
MF/FWt: C28H24N4O4S /512
MP: 163 oC
IR cm -1: 3369, 3076, 1666, 1610.
1H NMR (400 MHz, CDCl3): δ 8.02 (s, 1H), 7.87 (s, 1H), 7.73 (d, 1H, J= 8 Hz), 7.58 (d, 1H,
J= 8 Hz), 7.49-7.46 (m, 2H), 7.31 (d, 2H, J= 6.8 Hz), 7.08 (d, 2H, J= 8.4 Hz), 7.01 (d, 1H, J=
8.4 Hz), 6.57-6.52 (m, 2H), 3.91 (s, 1H), 3.87 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H).
MS: m/e 513 (M+1).
4.1.4.4
5-[3-(4-Chloro-phenyl)-1-phenyl-1H-pyrazol-4-ylmethylene]-2-(2,4-
dimethoxy-phenylamino)-thiazol-4-one (5d)
Yield: 68 %.
MF/FWt: C27H21ClN4O3S /517

MP: 149 oC
IR cm -1: 3383, 3120, 1708, 1668, 1346.
1H NMR (400 MHz, CDCl3): δ 8.09 (s, 1H), 7.92 (d, 1H, J= 7.2 Hz), 7.84 (d, 1H, J=7.2 Hz),
7.56-7.50 (m, 3H), 7.47 (s, 1H), 7.45-7.32 (m, 2H), 7.06 (d, 2H, J= 8 Hz), 6.87 (d, 1H, J= 6.8
Hz), 6.62 (s, 1H), 6.51-6.47 (m, 1H), 3.91 (s, 1H), 3.86 (s, 3H), 3.83 (s, 3H).
MS: m/e 518 (M+1).
4.1.4.5 5-[3-(4-bromo-phenyl)-1-phenyl-1H-pyrazol-4-ylmethylene]-2-(2,4-dimethoxy-
phenylamino)-thiazol-4-one (5e)
Yield: 60 %.
MF/FWt: C27H21BrN4O3S /560
MP: 167 oC
IR cm -1: 3381, 3124, 1708, 1668, 134.
1H NMR (400 MHz, CDCl3): δ 8.39 (s, 1H), 7.92 (d, 1H, J= 7.2 Hz), 7.86 (d, 1H, J= 8.4 Hz),
7.58-7.51 (m, 3H), 7.48 (s, 1H), 7.40-7.31 (m, 2H), 7.09 (d, 2H, J= 7.6 Hz), 6.88 (d, 1H, J=
7.2 Hz), 6.60 (s, 1H), 6.50-6.48 (m, 1H), 3.91 (s, 1H), 3.88 (s, 3H), 3.83 (s, 3H).
MS: m/e 561 (M+1).
4.1.4.6
2-(2,4-Dimethoxy-phenylamino)-5-[3-(4-fluoro-phenyl)-1-phenyl-1H-
pyrazol-4-ylmethylene]-thiazol-4-one (5f)
Yield: 53 %.
MF/FWt: C27H21FN4O3S /500
MP: 167 oC
IR cm -1: 3387, 3121, 1703, 1662.
1H NMR (400 MHz, CDCl3): δ 8.37 (s, 1H), 7.93 (d, 1H, J= 7.2 Hz), 7.87 (d, 1H, J= 7.2 Hz),
7.55-7.50 (m, 3H), 7.45 (s, 1H), 7.42-7.34 (m, 2H), 7.10 (d, 2H, J= 7.6 Hz), 6.87 (d, 1H, J=
6.8 Hz), 6.62 (s, 1H), 6.52-6.49 (m, 1H), 3.91 (s, 1H), 3.87 (s, 3H), 3.83 (s, 3H).
MS: m/e 501 (M+1).
4.1.4.7
5-[3-(3-Chloro-phenyl)-1-phenyl-1H-pyrazol-4-ylmethylene]-2-(2,4-
dimethoxy-phenylamino)-thiazol-4-one (5g)
Yield: 50 %.
MF/FWt: C27H21ClN4O3S /517
MP: 120 oC

IR cm -1: 3380, 3051, 1710, 1655, 1317.
1H NMR (400 MHz, CDCl3): δ 8.04 (s, 1H), 7.86 (d, 1H, J= 7.2 Hz), 7.84 (d, 1H, J= 7.2
Hz), 7.55-7.53 (m, 3H), 7.45 (s, 1H), 7.37-7.33 (m, 2H), 7.11 (d, 2H, J= 7.6 Hz), 6.86 (d, 1H,
J= 6.8 Hz), 6.63 (s, 1H), 6.50-6.45 (m, 1H), 3.91 (s, 1H), 3.86 (s, 3H), 3.82 (s, 3H).
MS: m/e 518 (M+1).
4.1.4.8
5-[3-(2-Chloro-phenyl)-1-phenyl-1H-pyrazol-4-ylmethylene]-2-(2,4-
dimethoxy-phenylamino)-thiazol-4-one (5h)
Yield: 54 %.
MF/FWt: C27H21ClN4O3S /517
MP: 155 oC
IR cm -1: 3383, 3055, 1716, 1651, 1315.
1H NMR (400 MHz, CDCl3): δ 8.09 (s, 1H), 7.87 (d, 1H, J= 7.6 Hz), 7.84 (d, 1H, J= 7.8
Hz), 7.54-7.45 (m, 4H), 7.38-7.33 (m, 2H), 7.13 (d, 2H, J= 7.2 Hz), 6.85 (d, 1H, J= 7.6 Hz),
6.63 (s, 1H), 6.50-6.47 (m, 1H), 3.91 (s, 1H), 3.86 (s, 3H), 3.84 (s, 3H).
MS: m/e 518 (M+1).
4.1.4.9
5-[3-(2,4-Chloro-phenyl)-1-phenyl-1H-pyrazol-4-ylmethylene]-2-(2,4-
dimethoxy-phenylamino)-thiazol-4-one (5i)
Yield: 54 %.
MF/FWt: C27H20Cl2N4O3S /551
MP: 169 oC
IR cm -1: 3389, 3124, 1718, 1665.
1H NMR (400 MHz, CDCl3): δ 8.35 (s, 1H), 7.91 (d, 1H, J= 7.2 Hz), 7.89 (d, 1H, J= 7.6
Hz), 7.58-7.55 (m, 2H), 7.45 (s, 1H), 7.45-7.33 (m, 2H), 7.06 (m, 2H), 6.88 (m, 1H), 6.62 (s,
1H), 6.52-6.46 (m, 1H), 3.88 (s, 1H), 3.79 (s, 3H), 3.76 (s, 3H).
MS: m/e 552 (M+1).
4.1.4.10
2-(2,4-Dimethoxy-phenylamino)-5-[3-(2,4-dimethoxy-phenyl)-1-phenyl-
1H-pyrazol-4-ylmethylene]-thiazol-4-one (5j)
Yield: 61 %.
MF/FWt: C29H26N4O5S /542
MP: 177 oC
IR cm -1: 3372, 3070, 1668, 1615.

1H NMR (400 MHz, CDCl3): δ 8.04 (s, 1H), 7.89 (d, 1H, J= 7.6 Hz), 7.85 (d, 1H, J= 6.8
Hz), 7.54-7.51 (m, 2H), 7.47 (s, 1H), 7.39-7.32 (m, 2H), 7.10 (d, 2H, J= 7.2 Hz), 6.86 (d, 1H,
J= 7.6 Hz), 6.61 (s, 1H), 6.50-6.47 (m, 1H), 3.86 (s, 1H), 3.82 (s, 3H), 3.81 (s, 3H), 3.70 (s,
3H), 3.79 (s, 3H).
MS: m/e 543 (M+1).
4.2 Synthesis
4.2.1. General procedure for synthesis of 2-chloro-N-(2,4-dimethoxy-phenyl)-
acetamide (2)
To a stirred solution of 2, 4-Dimethoxy-phenylamine (1.53 g, 10 mmol) 1 in DMF (15 mL),
chloro-acetyl chloride (1.12 g, 10 mmol) was drop wise added under nitrogen and reaction
mixture was stirred for 3h. at room temperature. After reaction was completed the contents
were poured into ice-cold water. The solid mass was separated, filtered, washed with water
and then purified by recrystalization from ethanol to get 2-chloro-N-(2,4-dimethoxy-phenyl)-
acetamide 2.
4.2.2. General procedure for synthesis of 2-(2,4-Dimethoxy-phenylamino)-thiazol-4-one
(3)
To a stirred solution of 2-chloro-N-(2,4-dimethoxy-phenyl)-acetamide (2.29 g, 10 mmol) 2 in
ethanol (15 mL), ammonium thiocyanate (0.76 g, 10 mmol) was added and reaction mixture
was reflux for 1.5 h. After reaction was completed the contents were poured into ice-cold
water. The solid mass was separated, filtered, washed with water and then purified by
recrystallization from ethanol to get 2-(2,4-Dimethoxy-phenylamino)-thiazol-4-one 3.
4.2.3. General procedure for synthesis of 1-(1-methyl-buta-1,3-dienyl)-3-phenyl-1H-
pyrazole-4-carbaldehyde24 (4a-j)
4.2.4. General procedure for synthesis of 2-(2,4-Dimethoxy-phenylamino)-5-methylene-
thiazol-4-one (5a-j)
A well stirred solution of 2-(2,4-dimethoxy-phenylamino)-thiazol-4-one (0.504 g, 2
mmol) 3 in 17.5 mL of acetic acid was buffered with sodium acetate (4 mmol) and added
with the 1-(1-methyl-buta-1,3-dienyl)-3-phenyl-1H-pyrazole-4-carbaldehyde (0.744 g, 3
mmol) 4a. The solution was refluxed for 4 h and then poured into ice-cold water. The yellow

precipitate was filtered and washes with water, the resulting crud product was purified by
recrystallization from acetic acid: DMF (2:1) to afford title compound 5a.
4.3 Assay of tyrosinase activity
Mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.) was used as described
previously with some modifications, using either, L-DOPA (diphenolase) or L-tyrosine
(monophenolase) as substrate28. Briefly, all the synthesised compounds were screened for the
diphenolase inhibitory activity of tyrosinase using L-DOPA as substrate. All the compounds
were dissolved in DMSO. The final concentration of DMSO in the test solution was 2.0%.
Phosphate buffer, pH 6.8, was used to dilute the DMSO stock solution of test compounds.
Thirty units of mushroom tyrosinase (0.5 mg/ml) were first pre-incubated with the
compounds, in 50 mM phosphate buffer (pH 6.8), for 10 min at 25◦C. Then the L-DOPA (0.5
mM) was added to the reaction mixture and the enzyme reaction was monitored by measuring
the change in absorbance at 475 nm of formation of the DOPA chrome for 1 min.
The measurement was performed in triplicate for each concentration and averaged before
further calculation. IC50 value, a concentration giving 50% inhibition of tyrosinase activity,
was determined by interpolation of the dose–response curves. The percent of inhibition of
tyrosinase reaction was calculated as following:
Inhibition rate (%) = (B-S)/B × 100
Here, the B and S are the absorbances for the blank and samples. Kojic acid was used
as reference standard inhibitors for comparison.
4.4 Progress curves determination
All reactions were carried out using L-tyrosine as a substrate in 0.25 M phosphate
buffer (pH 6.8) at 30◦C. Enzyme activities were measured continuously for 15 min using a
UV spectrophotometer. The kinetic parameters associated with time-dependent inhibition of
tyrosinase, progress curves with 20 data points (30 s intervals) were obtained at different
inhibitor concentrations using fixed substrate concentrations.
Acknowledgment
The authors are thankful to the Director, NES Science College, Nanded, for providing the
necessary facilities.

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List of captions:
Scheme I. Synthesis of 2-(2,4-Dimethoxy-phenylamino)-5-methylene-thiazol-4-one (5a-j)
Figure 1. Lineweaver–Burk plots for the inhibition of the diphenolase activity of tyrosinase
by compounds (5h and 5g). (A) Concentrations of compound 5h for curves were 0, 10, 20,
and 30 µM; (B) compound 5g for curves were 0, 20, 30 and 40 µM; respectively. The final
enzyme concentration was 2.0 μg/ml. The data shown in the fig is mean of three different
experiments.
Figure 2. Progress curve for oxidation of L-DOPA catalyzed by mushroom tyrosinase in the
presence of thiazolidinone scaffolds (5h > 5g> 5d > 5e > 5f> 5c> 5i > 5b). Data shown in the
figure is mean of three different experiments.
Table 1. Tyrosinase inhibitory activities of different thiazolidinone scaffolds

Table 1. Tyrosinase inhibitory activities of different thiazolidinone scaffolds
Compounds CLog
Pc
IC50 (µM) a Percent of
inhibitionb
Type of Inhibition
Ki (µM)
5a
6.307
6.806
---
8.03
13.5
46.8
37.97
87.92
54.23
109.8
126.04
52.03
--
---
5b
194.02
---
5c
6.28147 114.05
7.03856 74.02
7.18856 131.08
6.46856 104.32
7.03856 52.62
6.78856 34.12
7.50384 81.68
5.77757 ---
---
5d
Competitive 5.70
competitive 9.46
competitive 7.18
competitive 21.52
competitive 29.08
competitive 16.92
5e
5f
5g
5h
5i
5j
Kojic acid
22.5
--
competitive 14.86
a Values were determined from logarithmic concentration–inhibition curves and are given as
means of three experiments.
b Percent of inhibition of tyrosinase reaction at the 200 µM.
c Value of CLog P was obtained by Chem BioDraw Ultra 12.0.
Data shown in the table is mean of three different experiments

O
N
b
a
NH2
NHCOCH2Cl
O
O
S
O
N
H
O
O
O
2
3
1
R
O
O
N
N
N
N
N
S
c
O
N
H
S
N
+
O
N
H
CHO
4a-j
O
R
O
3
5a-j
R= H, 4-CH3, 4-OCH3, 4-Cl, 4-Br, 4-F, 3-Cl, 2-Cl, 2,4-Cl, 2,4-OCH3
Scheme-I
Reagent and conditions:
a. ClCOCH2Cl, N,N-DMF, r.t., 2 h.
b. NH4SCN, ethanol, reflux, 2h.
c. CH3COONa, CH3COOH, reflux, 2-4 h.

Fig. 1 Lineweaver–Burk plots for the inhibition of the diphenolase activity of tyrosinase by
compounds (5h and 5g). (A) Concentrations of compound 5h for curves were 0, 10, 20, and
30 µM; (B) compound 5g for curves were 0, 20, 30 and 40 µM; respectively. The final
enzyme concentration was 2.0 μg/ml. The data shown in the fig is mean of three different
experiments.

Figure 2. Progress curve for oxidation of L-DOPA catalyzed by mushroom tyrosinase in the
presence of thiazolidinone scaffolds (5h > 5g> 5d > 5e > 5f> 5c> 5i > 5b). Data shown in the
figure is mean of three different experiments.

Synthesis of new heterocyclic hybrids based on pyrazole and thiazolidinone
scaffolds as potent inhibitors of tyrosinase
Shrikant S. Gawandea,*, Suchita C. Warangkarb, Babasaheb P. Bandgarc ,
Chandrahasya N. Khobragadeb
R
O
N
N
NH2
O
S
N
O
N
H
O
O
R= H, 4-CH3, 4-OCH3, 4-Cl, 4-Br, 4-F, 3-Cl, 2-Cl, 2,4-Cl, 2,4-OCH3