Synthesis, computational studies and enzyme inhibitory kinetics of substituted methyl[2-(4-dimethylamino-benzylidene)-hydrazono)-4-oxo-thiazolidin-5-ylidene]acetates as mushroom tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.09.009

The present article describes the synthesis and enzyme inhibitory kinetics of methyl[2-(arylmethylene-hydrazono)-4-oxo-thiazolidin-5-ylidene]acetates 5a–j as mushroom tyrosinase inhibitors. The title compounds were synthesized via cyclocondensation of thi

Full text of DOI:10.1016/j.bmc.2017.09.009

Accepted Manuscript
Synthesis, computational studies and enzyme inhibitory kinetics of substituted
methyl[2-(4-dimethylamino-benzylidene)-hydrazono)-4-oxo-thiazolidin-5-yli-
dene]acetates as mushroom tyrosinase inhibitors
Pervaiz Ali Channar, Aamer Saeed, Fayaz Ali Larik, Muhammad Rafiq, Zaman
Ashraf, Farukh Jabeen, Tanzeela Abdul Fattah
PII:
S0968-0896(17)31217-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.09.009
BMC 13969
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 June 2017
2 August 2017
6 September 2017
Please cite this article as: Channar, P.A., Saeed, A., Larik, F.A., Rafiq, M., Ashraf, Z., Jabeen, F., Fattah, T.A.,
Synthesis, computational studies and enzyme inhibitory kinetics of substituted methyl[2-(4-dimethylamino-
benzylidene)-hydrazono)-4-oxo-thiazolidin-5-ylidene]acetates as mushroom tyrosinase inhibitors, Bioorganic &
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.09.009
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Synthesis, computational studies and enzyme inhibitory kinetics of substituted methyl[2-(4-
dimethylamino-benzylidene)-hydrazono)-4-oxo-thiazolidin-5-ylidene]acetates as mushroom
tyrosinase inhibitors
Pervaiz Ali Channara, Aamer Saeed,a,* Fayaz Ali Larik,a Muhammad Rafiqb, Zaman Ashrafc,*,
Farukh Jabeend,e, Tanzeela Abdul Fattaha
aDepartment of Chemistry Quaid-i-Azam University-45320, Islamabad, Pakistan.
bDepartment of Biochemistry & Biotechnology (Baghdad-ul-Jadeed Campus), The Islamia
University of Bahawalpur, Bahawalpur, Pakistan,
cDepartment of Chemistry, Allama Iqbal Open University, Islamabad 44000, Pakistan
dFlorida Center of Heterocyclic Compounds, University of Florida, Gainesville, Fl, 32601, USA.
eCenter for Computationally Assisted Science and Technology, North Dakota State University,
Fargo, ND, 58102,
USA.
Keywords Thiazolidinone; Synthesis; Tyrosinase Inhibitors; Kinetic Mechanism; Molecular
Docking
* Address for Correspondence
Aamer Saeed
Email: asaeed@qau.edu.pk, aamersaeed@yahoo.com
Contact number: +92-51-9064-2128, Fax: +92-51-9064-2241
* Address for Correspondence
Zaman Ashraf,
Email: mzchem@yahoo.com
Contact number: +923215194461, Fax: +92512891471

Abstract
The present article describes the synthesis and enzyme inhibitory kinetics of methyl[2-
(arylmethylene-hydrazono)-4-oxo-thiazolidin-5-ylidene]acetates 5a–j as mushroom tyrosinase
inhibitors. The title compounds were synthesized via cyclocondensation of thiosemicarbazones
3a–j with dimethyl but-2-ynedioate (DMAD) 4 in good yields under solvent-free conditions. The
synthesized compounds were evaluated for their potential to inhibit the activity of mushroom
tyrosinase. It was unveiled that compounds 5i showed excellent enzyme inhibitory activity with
IC50 3.17µM while IC50 of standard kojic acid is 15.91µM. The presence of heterocyclic pyridine
ring in compound 5i play important role in enzyme inhibitory activity as rest of the functional
groups are common in all synthesized compounds. The enzyme inhibitory kinetics of the most
potent derivative 5i determined by Lineweaver-Burk plots and Dixon plots showed that it is non-
competitive inhibitor with Ki value 1.5 µM. It was further investigated that the wet lab results are
in good agreement with the computational results. The molecular docking of the synthesized
compounds was performed against tyrosinase protein (PDBID 2Y9X) to delineate ligand-protein
interactions at molecular level. The docking results showed that the major interacting residues
are His244, His85, His263, Val 283, His 296, Asn260, Val248, His260, His261 and Phe264
which are located in active binding site of the protein. The molecular modeling demonstrates that
the oxygen atom of the compound 5i coordinated with the key residues in the active site of
mushroom tyrosinase contribute significantly against inhibitory ability and diminishing the
human melanin synthesis. These results evident that compound 5i is a lead structure in
developing most potent mushroom tyrosinase inhibitors.

1. Introduction
Thiazolidin-4-ones is important scaffold in heterocyclic chemistry and the thiazolines ring is
present in many pharmacological active substances. Thiazolines derivatives have a great deal of
interest owing to their biological activities, such as anti-tuberculosis,1 anti-convulsant,2 and
fungistatic3 activities as well as inhibitory activity of gastrointestinal proliferation.4-5 The five
membered S- and S,N-heterocycles were synthesized by reacting dimethyl
acetylenedicarboxylate (DMAD) with esters and amides of dithiocarboxylic acids.6-7 Thioureas
react with DMAD to give 1:1 adducts with loss of methanol.8 A number of other more
convenient methods have been reported for the preparation of thiazolidinone derivatives. For
example, the reaction of thioamides or thiosemicarbazide derivatives with
dialkylacetylenedicarboxylates is an effective method to prepare 2-amino-5-methoxycarbonyl-
thiazolidin-4-ones.9-10 Also, 2-(dimethylamino)-thiazole reacted with DMAD to produce a
pyridine derivative via extrusion of a sulfur atom.11 In recent years, the use of microwave
irradiation has become popular among synthetic organic chemists, both to improve classic
organic reactions (shortening reaction times and/or improving yield), and to promote new
reactions.12 Aly et al. demonstrated a very convenient procedure to synthesize 1,3-thiazines by
the reaction of but-2-ynedioic acid, propynoic acid ethyl ester, and (E)-1,4-diphenyl-but-2-ene-
1,4-dione with aroyl-substituted thioureas in acetic acid.13 The same group changed the
conditions of the reaction by mixing a solution of DEAD and aroylthioureas together with
triphenyl phosphine.14 The products were identified as methyl-(2Z)-2-[(2Z)-2,3-diaryl-
carbonylimino-4-oxo-thiazolidin-5- ylidene]-acetates. Acylthiosemicarbazides represent versatile
synthon for various syntheses of nitrogen–sulfur heterocycles. The acylthiosemicarbazide moiety
provides an opportunity to perform cyclocondensations as well as addition-cyclization reactions.
The products of these reactions are thiazin-2-ylidene,15 thiazoline,16 triazole,17 and imidazolidine
derivatives.18-19 It has been reported that the reaction of dimethyl acetylenedicarboxylate with
thiocarbamides led to 4-oxathiazolidine derivatives.20
Owing to biological importance of the thiazolidinones we here describe the synthesis of title
thiazolidinones
5a-j
by
condensation
of
thiosemicarbazones
with
dimethyl
acetylenedicarboxylate. The presence of the thiazolidinone pharmacophore as reported in
previous studies, led us to investigate their mushroom tyrosinase inhibitory kinetics of the

synthesized compounds. The computational molecular docking studies has also been performed
to compare the wet lab results with dry lab findings.
2. Results and discussion
2.1 Chemistry
Thiazolidinone derivatives 5a-j were synthesized by condensation of substituted
thiosemicarbazones 3a–j with dimethyl acetylenedicarboxylate 4. Thiosemicarbazones 3a–j
were synthesized as intermediates by acid catalyzed condensation of thiosemicarbazide with a
range of substituted aromatic aldehydes (Scheme I). Thiosemicarbazones 3a–j were then reacted
with dimethyl acetylenedicarboxylate 4 in the absence of any solvent or catalyst, afforded
thiazolidinones 5a–j in good to excellent yields (Scheme I). DMAD is a highly electrophilic
reagent and widely employed as a dienophile in cycloaddition reactions, such as the Diels–Alder
reaction and behaving as Michael acceptor in organic transformations. The structures of 5a–j
were assigned on the basis of their FTIR, 1H and 13C-NMR spectral data. In the 1H-NMR spectra
of 5a-j, the down-field singlets in the region δ 13.04–8.06 ppm were assigned to NH ring proton,
while the azomethine protons (C2thiazolH=N) appeared in the region δ 9.38–7.49 ppm. The
olefinic protons (CH=C) resonated in the region δ 6.73–6.42 ppm, while the singlets in the
13
region δ 3.90–3.78 ppm were attributed to the protons acetoxy group. In the C NMR spectra,
carbonyl carbon atoms of the thiazole ring and the acetoxy group appeared in the regions δ
174.0–170.1 ppm and δ 168.5–164.5 ppm respectively, whereas C-2 of the thiazolidinone ring
resonated in the region δ 159.4–156.3 ppm. The presence of the signals in the region δ 149.6–
145.2 ppm were assigned to azomethine carbon atoms attached to the aromatic rings (ArCH=N),
except those of compounds 5a and 5i which appeared at δ 160.3 and158.4 ppm, respectively.
Olefinic carbon atoms (Cthiazol5=CH) were observed in the region δ 143.9–137.9 ppm, while C-
5 of the thiazolidinone ring appeared in the region δ 134.6–131.1 ppm.

Scheme I. Synthesis of 1-(Substituted benzylidene thiosemicarbazides 3a-j
2.2 Tyrosinase inhibitory activity
The synthesized thiazolidinones 5a-j have been screened for their inhibitory effects on
mushroom tyrosinase activity. The compound 5i showed excellent tyrosinase inhibitory activity
with IC50 value 3.17µM while IC50 value of standard kojic acid is 15.91µM. The presence of
heterocyclic pyridine ring in compound 5i play important role in enzyme inhibitory activity as
rest of the functional groups are common in all synthesized compounds Table 1.
Table 1 The inhibitory effects of compounds 5a-j on mushroom tyrosinase activity.
Tyrosinase Activity
Compounds
(IC50 SEM µM)
75.26 8.2
141.77 21.0
348.93 33.4
297.60 18.5
100.45 12.6
49.82 11.9
50.12 9.8
5a
5b
5c
5d
5e
5f
5g

208.25 20.2
3.17 0.37
ND
5h
5i
5j
15.91 2.5
Kojic acid
ND is not determined
2.3 Enzyme inhibitory Kinetics
Since 5i was the most potent inhibitor in hand, we therefore further study mechanism
underlying their inhibitory effect. The Lineweaver-Burk plot of 1/V versus (1/[S]) in the presence
of different concentrations of 5i gave a series of straight lines, all of which intersected at the
same point on the x-axis Figure.1 (a). The analysis showed that 1/Vmax increased to a new value
while that of Km remain same in the presence of increasing concentrations of 5i. This behavior
indicated that 5i inhibit tyrosinase non-competitively to form enzyme inhibitor (EI) Complex21.
To gain insightful the pathway in which 5i preferentially proceeded, binding affinities of EI were
determined. Ki value (the constant of dissociation of the enzyme-inhibitor complex into free
enzyme and inhibitor) was determined by the secondary re-plot of slope against concentration of
5i Figure. 1(b) and by the interpretation of Dixon plot by using initial velocities at 475nm Figure
2. Kinetic constants and inhibition constants are summarized in Table 2. It was observed that
compound 5i produced same enzyme inhibition at a lower dose than the reference kojic acid
which indicates its higher potency than kojic acid Figure 3.
Table 2 Kinetic constants and inhibition constants of compound 5i on mushroom tyrosinase
Dose
Vmax
(∆A475nm/Sec)
0.01
Km
(mM)
1
Inhibition Type
Ki
(µM)
IC50
(µM+SEM)
No inhibitor
1.06 µM
5.55 × 10-4
1
Non-competitive
1.5
3.17 0.37
8.50 µM
17.0 µM
1.00 × 10-3
3.33 × 10-3
1
1
Vmax is the velocity of reaction.
Km is the Michaelis-Menten constant.
Ki is the inhibition constant.
IC50 is the concentration at which 50% of the enzyme activity is inhibited.
SEM is standard error of the mean.

Figure 1. a) Lineweaver-burk plots for the inhibition of compound 5i on mushroom tyrosinase for
catalysis of L-DOPA. Inhibitor concentrations were 0.00, 1.06, 8.50 and 17.00 µM, L-DOPA
concentrations were 0.062, 0.125, 0.25, 0.5, 1 and 2 mM, respectively. b) Represents the secondary re-
plot of slope versus concentration of compound 5i to determine the inhibition constant (Ki).
Figure 2. Dixon plots for the inhibition of the diphenolase activity of mushroom tyrosinase by various
concentrations, 0.0, 1.06, 2.12, 4.25, 8.5, 17 and 34 µM of compound 5i in the presence of different
concentrations of L-DOPA (0.25, 0.5, 1 and 2 mM), respectively.
Figure 3. a) Effect of kojic acid and b) compound 5i on the diphenolase activity against mushroom
tyrosinase for the catalysis of L-DOPA at 25°C.

2.4 Docking Studies
The pharmacophore modeling and molecular docking studies has been introduced as a prevailing
way complementing traditional high through put screenings. Molecular docking is an
optimization problem that would define the ‘best-fit’ positioning of a compound that binds to a
specific protein of interest. The molecular docking studies thus play an important role in
preliminary drug research. In order to give an explanation and understanding of potent inhibitory
activity observed, molecular docking was performed against mushroom tyrosinase (PDBID
2Y9X). All the docked conformations for each compounds were analyzed and it has been found
that the most favorable docking poses out of 30 docked conformations for each compound, were
retained to investigate the interactions of the docked conformations within the active site. All the
top ranked docking conformations of synthesized compounds were stacked well inside the active
site cavity. It has been reported that His 244, His 85, His263, Val 283, His 296, Asn260, Val248,
His260, His261 and Phe264 are found to be key residues which are also reported in literature.22-
24
the active site was architecture with both the hydrophobic and hydrophilic amino acids along
with the two copper ions. The hydrophobic part was constructed by, Val 283, Met280, Ala246,
Phe90, Ala286, Val 248, Phe246 and His256 while hydrophilic portion contained amino acids
such as His244, His279, Gly86, His61, Ser282, His 85, His 263, Gly281, Glu256, Asn260,
His256, Glu322, Gly96, Ser282. Active site is a deep cavity with the wide opening and narrow
end. All the preferred docked poses stacked beautifully inside the cavity. The superimposition of
the best possible docked poses are shown in Figure 4.
Figure 4. a) Overlaying of all the minimum energy docked poses inside the pocket b) Close view,
(surfaces, line mode, are drawn around pocket to map the pocket). Ligands are shown in yellow color in
stick mode while key residues are shown in green color in stick mode.

An insight in to the binding mode analysis of the best possible binding mode of the most
preferred docked conformations revealed that ligands hydrogen bonding, hydrophobic, polar pi-
pi, pi-H and cationic interaction with Copper ion at different potential. Almost all the ligands
showed similar pattern of binding inside the active site. Figure S1 showed 2-D interactions of all
the ligands. The aromatic rings made similar stack of interactions with His 263, and Val283 in
some ligands. Experimental results and docking results complemented each other in terms of
interactions with key residues and inhibitory activity, the compounds which were found to be
active in experimental studies showed multiple binding interactions while the one which showed
fewer interactions showed decreased inhibitory potential. The docking model showed that 5i
applied distinct orientation to interact with the active site residues of mushroom tyrosinase. The
oxygen atom of carbonyl group in compound 5i made the close contacts with key amino acids
such as His 85 and His296. Figure 5 depicted the 3-D interaction of this ligand engaged the
active-site residues, using typical hydrogen bonds to anchor tightly within the backbone of the
enzyme. In addition, two hydrogen bonds were observed between the hydroxyl groups of 5i and
His85 and His296, respectively.
a)
b)
Figure 5. Molecular docking analyses of 5i toward mushroom tyrosinase. (a) The docking model of
molecule inside active site in 3D Interaction of ligand 5i with active site residues. (b) 2D schematic
representation of the interactions between 5i and active site residues of mushroom tyrosinase. (The green
dash lines in a and b panels donate the interactions, and the purple spheres represent two copper ions).

This indicated that the both the oxygen atom of acyl substitution of 5i is of great potential in
interacting with amino acids which are mandatory for the catalytic activities of the enzyme and
significantly contributed in mushroom tyrosinase inhibition. It has also been supported by the
investigation of the compounds 5g, 5h, 5i and 5j which are anchored through hydrogen bonding
in active site making this an evidence that the ester substitution at the this position is imperative
for inhibitory activities. The compounds 5g, 5h, 5i and 5j with ester substitution showed similar
orientation pattern and established significant hydrogen bonding with the catalytic residues His
85, His296 and Asn85. Compound 5g, 5h and 5i exhibited better activities (IC50, 49.82, 50.12
and 3.17 µmol respectively) as compared to the all other especially compound 5i much better
than the Kojic acid. (IC50 15.91 µmol). Compound 5j showed almost similar binding pattern to
that of compound 5i however experimental IC50 is not available but predicted IC50 proclaimed
this compound to be a potential surrogate for the tyrosinase inhibition.
Taking into account the peculiarities of the active-site residues in the tyrosinase catalysis, it can
be inferred that most of the synthesized compounds made contacts with the side chains of
mushroom tyrosinase His244, His 85, His263, Val 283, His 296, Asn260, Val248, His260,
His261 and Phe264 residues, which was reflected in their enhanced affinity to the residues Table
S1. Besides being directly involved in the architecture of the active site, the residue has a vital
role in positioning other key residues in the active site appropriately for the catalysis.
2.5 QSAR modeling
The best multi-linear regression (BMLR)-1 was utilized which is a stepwise search for the best
n-parameter regression equations (where n stands for the number of descriptors used), based on
the highest R2 (squared correlation coefficient), R2cvOO (squared cross-validation “leave one-
out, LOO” coefficient), R2cvMO(squared cross-validation “leave many-out, LMO” coefficient),
F (Fisher statistical significance criteria) values, and s2 (standard deviation). The QSAR models
up to 2 descriptor model describing the urease inhibitors against tyrosinase were generated
(obeying the rule of 5:1, which is the ratio between the data points and the number of QSAR
descript or model). The observed and predicted values of the training set compounds 5a-j
according to the multi-linear QSAR models are presented in Table 1.The established QSAR
model is statistically significant. The descriptors are sorted in the descending order of the
respective values of the Student’st-criterion, which is a widely accepted measure of statistical
significance of individual parameters in multiple linear regressions. Figure 6 shows the QSAR

multi-linear model plot of correlations representing the observed vs. predicted IC50 values for the
synthesized compounds. The scattered plots are uniformly distributed, covering ranges of IC50,
observed 3.17-348 and predicted 15.54-288.
Tyrosinase inhibition, Observed IC (µmol) vs Predicted IC (µmol)
50 50
282.1090
244.4670
188.0460
100.2180
46.4416
15.5408
3.17
100.45
208.25
348.93
49.82
141.77
297.60
R2=0.85
Observed IC50 (µmol)
Figure 6. Co-relation plot of observed IC50 vs experimental IC50 (QSAR) of compounds 5a-j.
2.6 Validation of 2D-QSAR model
The reliability and statistical relevance of the QSAR model is examined an internal validation
procedure. The dataset contains relatively few experimental data points but is homogeneous in
synthesized compounds. Therefore, application of the internal validation methodology is an
appropriate technique. Internal validation is applied by the CODESSA-Pro technique employing
both leave one out (LOO), which involves developing a number of models with one example
omitted at a time, and leave many out (LMO), which involves developing a number of models
with many data points omitted at a time (up to 20% of the total data points). The observed
correlations by the internal validation technique are R2cvLOO = 0.72, R2cvMO = 0.811,
respectively which are significantly correlated with the squared correlation coefficient of the
attained2D-QSAR model (R2 = 0.867). Standard deviation of the regression (s2 = 2354.08) is
also a measurable value for the attained model together with the Fisher test value (F = 26.709)
that reflects the ratio of the variance explained by the model and the variance due to their errors.
A high value of the F-test relative to the s2 value is also validation of the model. The
predicted/estimated tyrosinase inhibitory activity due to the attained BMLR-QSAR model of
most of the synthesized compounds are compatible with their experimentally observed values

preserving their relative potencies (Table 3). The estimated/predicted IC50 value of compound 5e
correlated well with its observed bio-activity (IC50 = 100.45, 100.21) for observed and estimated
bio-properties, respectively, error = 0.23). All of above cross validation results are good
indications of the predictive power of the attained 2D-QSAR model not only for validating the
observed bio-data but also for optimizing high potent hits having methyl [2-(4-Dimethylamino-
benzylidene)-hydrazono)-4-oxo-thiazolidin-5-ylidene]acetates scaffold.
Table 3 Observed and predicted IC50 values of synthesized compounds 5a-j against tyrosinase.
Entry
IC50 (obs.)
75.2 8.2
141.7 21.0
348.3 33.4
297.6 18.5
100.5 12.6
49.2 11.9
50.1 9.8
208.2 20.2
3.17 0.37
N/A
IC50 (pred.)
27.3281
188.046
288.612
244.467
100.218
67.2464
31.2712
282.109
15.5408
20.9611
46.4416
(error)
47.9319
-46.276
60.3185
53.133
5a
5b
5c
5d
0.231663
-17.4264
18.8488
-73.8592
-12.3708
N/A
5e
5f
5g
5h
5i
5j
15.91
-30.5316
Kojic acid
IC50 is the concentration of an inhibitor required to reduce the rate of an enzymatic reaction by 50%
3. Conclusion
We have described the synthesis of methyl[2-(arylmethylene-hydrazono)-4-oxo-thiazolidin-5-
ylidene]acetates 5a–j via cyclocondensation of thiosemicarbazones with dimethyl but-2-
ynedioate (DMAD) in good yields under solvent-free conditions. The title compounds were
screened for their mushroom tyrosinase inhibitory activity, compound 5i showed excellent
activity with IC50 value 3.17µM while IC50 value of standard kojic acid is 15.91µM. The enzyme
inhibitory kinetics of compound 5i determined by Lineweaver-Burk plots and Dixon plots
showed that it is non-competitive inhibitor with Ki value 1.5 µM. The computational molecular
docking performed against tyrosinase protein (PDBID 2Y9X) showed that the major interacting
residues are His244, His85, His263, Val 283, His 296, Asn260, Val248, His260, His261 and
Phe264 which are located in active binding site of the protein. The molecular modeling
demonstrates that the oxygen atom of the compound 5i coordinated with the key residues in the
active site of mushroom tyrosinase contribute significantly against inhibitory ability and

diminishing the human melanin synthesis. These results evident that compound 5i is a lead
structure in developing most potent mushroom tyrosinase inhibitors.
4. Experimental
Melting points were recorded using a digital Gallenkamp (SANYO) model MPD.BM 3.5
1
apparatus and are uncorrected. H-NMR spectra were determined as CDCl3 solutions at 300
MHz using a Bruker AM-300 spectrophotometer using TMS as an internal reference and 13C
NMR spectra were determined at 75 MHz using a Bruker 75 MHz NMR spectrometer in CDCl3
solution. FTIR spectra were recorded on an FTS 3000 MX spectrophotometer. Mass Spectra (EI,
70 eV) on a MAT 312 instrument, and elemental analyses were conducted using a LECO-183
CHNS analyzer.
General procedure for synthesis of 2-(hetero (aryl) methylene) hydrazine-1carbothioamides
(Schiff bases) (3a-3j)
To a stirred solution of 1 (0.138 g, 1.0 mM) in 25 mL absolute ethanol, suitably substituted
aldehydes or ketones(1.0 mM) along with 1–2 drops of conc. sulfuric acid were added, the
mixture was refluxed for 12 h, then cooled to room temperature, and the solids obtained were
filtered and recrystallized from ethanol to give compounds25,26
.
(3a) (Z)-2-(furan-2-ylmethylene)hydrazinecarbothioamide
(80%): m.p 210°C Rf :0.39; Petroleum ether : ethyl acetate (6:4) IR; (KBr,
cm-1): 3374 (NH2), 3240 (N-H), 3125 (sp2CH), 1603 (C=N), 1587 (Ar-C=C), 1090 (C=S),
1053(C-O) 1HNMR; (300MHz, DMSO): δ 11.42 (s, 1H, NH), 8.20 (s, 1H, CH=N), 7.96 (s, 1H),
7.80 (dd, J = 7.6 Hz, 1.6 Hz, 1H), 7.61 (t, J = 7.4 Hz, H). 7.96 (s, 2H NH2), 13CNMR (75
MHz,): δ 181.11, 148.31, 144.95, 141.59 120.79, 112.85.
(3b) (Z)-2-(4-(benzyloxy)benzylidene)hydrazinecarbothioamide

(95%), m.p 1710C; Rf: 0.53; IR; (KBr, cm-1): 3438 (NH2), 3305 (N-H), 1615
1
(CN), 1592(Ar-C=C), 1115 (C=S), cm-1; H NMR (Acetone-d6,300 MHz) δ: 10.8 (s, 1H, NH), 8.21 (s, 1H,
CH=N), 7.60-6.82 (m, 4H, ArH), 7.23 ( 5 H, ArH), 7.1 (s, 2H, -NH2), 5.70 (s, 1H, -OCH2);13C NMR (75 MHz) δ:
179. 0 (C=S), 146(C=N), 119 (Ar 2C), 122 (Ar 2C), 128 (Ar), 131(Ar), 153 (Ar), 159 (Ar), 146 (Ar), 134 (Ar,
2C), 139 (Ar, 2C), 135 (Ar), 78.5 (OCH2),
(3c) (E)-1-((5-bromothiophen-2-yl) methylene) thiosemicarbazide (3f)
Yield:75%: M.P 243 °C Rf : 0.42; Petroleum ether : ethyl acetate (6:4)
IR; (KBr, cm-1): 3417 (NH2), 3264 (N-H), 3126(sp2CH), 1609(C=N), 1587 (Ar-C=C), 1080
(C=S), 1H NMR (300 MHz, DMSO-d6): δ (ppm): 10.42 (s, 1H, NH), 7.63 (s, 1H, CH=N), 6.68 –
6.65 (m, 2H Ar-H). 6.52 (s, 2H -NH2), 13C NMR (75 MHz, DMSO-d6): δ (ppm): 178.4, 141.6,
138.8, 132.6, 131.6, 117.1.
(3d) (Z)-2-(4-((4-methoxybenzyl)oxy)benzylidene)hydrazinecarbothioamide
(92%) m.p 1780C; Rf: 0.58; IR; (KBr, cm-1): 3379 (NH2), 3235 (N-H), 1593
(CN), 1578(Ar-C=C), 1095 (C=S), cm-1; 1H NMR (Acetone-d6,300 MHz) δ: 10.3 (s, 1H, NH), 8.02 (s, 1H,
CH=N), 7.25-7.12 ( m, 4H, ArH), 7.20-6.79 (m, 4H, ArH), 7.1 (s, 2H, -NH2), 5.70 (s, 1H, -OCH2); 3.8 (s, 3H, -
OCH3); 13C NMR (75 MHz) δ: 179.0 (C=S), 146(C=N), 120 (Ar), 123 (Ar), 129 (Ar), 133(Ar), 154 (Ar), 158
(Ar), 149 (Ar), 136 (Ar, 2C), 141 (Ar, 2C), 137 (Ar), 75 (OCH2), 59 (OCH3).
(3e) (E)-2-(4-(Dimethylamino)benzylidene)hydrazine-1-carbothioamide (3a)

(70%): m.p 180°C; Rf : 0.46;( IR; (KBr, cm-1): 3374 (NH2), 3240 (N-
1
H), 3125 (sp2-CH), 2935 (sp3CH), 1603 (C=N), 1587 (Ar-C=C), 1090 (C=S), HNMR;
(300MHz, DMSO): δ 10.44 (s, 1H, NH), 7.07 (d, J = 7.5 Hz, 2H), 6.83 (d, J = 7.5 Hz, 2H), 6.75
13
(s, 2HNH2), 2.93 (s, 6H). CNMR (75 MHz,): δ 181.11, 153.88, 145.68, 128.76, 120.96,
110.95, 41.90.
(3f) (Z)-2-((5-methylfuran-2-yl)methylene)hydrazinecarbothioamide
(75%): m.p 243 °C Rf : 0.42;IR; (KBr, cm-1): 3414 (NH2), 3260 (N-H),
1
3125(sp2CH), 2935(sp3CH), 1603(C=N), 1587 (Ar-C=C), 1090 (C=S), 1053(C-O); HNMR;
(300MHz, DMSO): δ 10.52 (s, 1H, NH), 7.53 (s, 1H, CH=N), 7.48 (dd, J = 21.8, 7.5 Hz, 2H),
7.6 (t, J = 7.4 Hz, H), 7.3 (s, 2H, -NH2);2.20 (s, 3H). 13CNMR (75 MHz,): δ 181.11, 153.28,
146.59, 141.76, 117.91, 106.78, 14.49.
(3g) (Z)-2-((1H-pyrrol-2-yl)methylene)hydrazinecarbothioamide
(75%): m.p 232°C Rf : 0.42; Petroleum ether : ethyl acetate (6:4) IR; (KBr,
cm-1): 3414 (NH2), 3260 (N-H), 3125 (sp2CH), 1603 (C=N), 1587 (Ar-C=C), 1090 (C=S),
1HNMR; (300MHz, DMSO): δ 10.92 (s, 1H, NH), 7.53(s, 1H, CH=N), 7.05 – 6.96 (m, 2H),
6.59 – 6.51 (m, 3H), 6.26 (t, J = 7.5 Hz,1H) 6.1 (s,2H,-NH2); 6.0 (s, 1H, -NH);13C NMR (125
MHz,) δ 181.11,138.24 , 124.70, 119.49, 116.23, 110.84.
(3h) (E)-2-((E)-3-phenylallylidene)hydrazinecarbothioamide

Yield: 70%; Rf: Rf: 0.63; m.p: 240ºC; IR: (KBr) (λmax, cm−1):
3414 (NH2), 3260 (N-H), 3075 (Ar-CH), 2930 (C-H), 1739 1715 (2 C=O), 1675, 1619 (C=N,
1
C=C), HNMR; (300MHz, DMSO): δ 10.90 (s, 1H, NH), 7.53(s, 1H, CH=N), 6.59 – 6.51 (m,
5H), 6.26 (dd, 1H), 5.66 (dd, 1H) 6.1 (s,2H,-NH2); 6.0 (s, 1H, -NH); 13C NMR (125 MHz,) δ
181.6, 138.5 ,135.70, 134.6, 128.7, 127.2, 127.3 , 126.8.
(3i) (Z)-2-(pyridin-3-ylmethylene)hydrazinecarbothioamide
(85%): m.p 250°C Rf : 0.33; Petroleum ether : ethyl acetate (6:4) IR; (KBr,
cm-1): 3424 (NH2), 3260 (N-H), 3135 (sp2-CH), 1605 (C=N), 1583 (Ar-C=C), 1090 (C=S),
1HNMR; (300MHz, DMSO): δ 11.56 (s, 1H, NH), 8.90 (dd, J = 7.5, 1.3 Hz, 1H), 8.67 (d, J =
1.3 Hz, 1H), 8.26 (dt, J = 7.5, 1.4 Hz, 1H), 8.13 (s, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.40 (s, 2H
NH2). 13CNMR (75 MHz,): δ 178.2, 150.1, 148.0, 139.1, 133.9, 130.2, 123.7.
(3j) (E)-2-(2-hydroxybenzylidene)hydrazinecarbothioamide
Yield:75%: M.P 235°C Rf : 0.52; Petroleum ether : ethyl acetate (6:4) IR;
(KBr, cm-1): 3412 (NH2), 3250 (N-H), 3120 (sp2CH), 1608 (C=N), 1597 (Ar-C=C), 1085 (C=S),
1H NMR (300 MHz, DMSO-d6): δ (ppm): 8.05 (s, 1H, CH=N), 8.27-7.01 (m, ,4H Ar-H), 7.42
(s, 2H, -NH2); 6.02 (s, 1H, OH); 13C NMR (75 MHz, DMSO-d6):a δ (ppm):.178.5, 162.9, 159.6.
General method for the preparation of methyl 2-(2-(arylidenehydrazino)-4-oxo-thiazolidin-5-
ylidene)acetates (5a–j)
A thoroughly mixed uniform mixture of substituted benzylidenethiosemicarbazides 3a–j (1.0
mmol) and dimethyl acetylenedicarboxylate 4 (142 mg, 1.0 mmol) was stirred for 1–5 min under
MWI. The progress of reaction was monitored by TLC (hexane/ethyl acetate 4:1). After the
reaction was completed, the solution was diluted with EtOH and the mixture was filtered. The

precipitate was recrystallized from EtOH to afford the esters 5a–j in good yields27,28
.
2-(Furan-2-ylmethylene-hydrazono)-4-oxo-thiazolidin-5-ylidene]-acetic acid methyl ester
(5a)
Yellow Solid; Yield: 74% m.p: 276ºC; IR: (KBr) (λmax, cm−1): 3070 (Ar-CH), 2938 (C-H), 1740,
1
1725(2 C=O), 1673, 1630 (C=N, C=C), 1110 (C–O). H NMR (DMSO-d6, 300MHz); δ 12.89 (s,
1H, N-H), 8.36 (s, 1H, CH=N), 7.94 (dd, 1H, Furan- H-5), 7.08 (d, 1H furan-H-3), 6.70 (s, 1H),
6.67 (s, 1H furan-H-4), 3.79 (s, 3H).13C NMR (DMSO, 75MHz); δ166.80 (C-4), 165.40 (s,
CO2CH3), 160.00 (s, C-2), 159.50 (CH=N), 152.00 (C-2-furan), 144.00 (C-5-furan), 142.90 (C-
6), 120.00 (CH-3-furan), 114.30 (C-5), 113.00 (CH-4-furan), 61.30 (OCH3), 14.20 (CH3). Anal.
Calcd. For C11H9N3O4S: C, 47.31; H, 3.25; N, 15.05; S, 11.48 found: C, 47.29; H, 3.27; N,
15.04; S, 11.50
[2-[(4-Benzyloxy-benzylidene)-hydrazono]-4-oxo-thiazolidin-5-ylidene]-acetic acid methyl
ester (5b)
Yellow Solid; Yield: 70% m.p: 272ºC; IR: (KBr) (λmax, cm−1): 3080 (Ar-CH), 2948 (C-H),
1740, 1725 (2 C=O), 1658, 1636 (C=N, C=C), 1120 (C–O)1H NMR (DMSO-d6, 300MHz); δ
12.84(s, 1H, N-H), 8.34(s, 1H, CH=N), 7.30 – 7.19 (m, 7H), 7.14 – 6.99 (m, 2H), 6.92 (s, 1H),
5.21 – 5.17 (m, 2H), 3.87 (s, 3H).13C NMR (DMSO, 75MHz); δ 168.11, 166.5, 163.7, 163.3,
150.5, 142, 140.1, 131.5, 129.5, 130, 127.8, 126.5, 115, 114.5, 71.9, 53.Anal. Calcd. For
C20H17N3O4S: C, 60.75; H, 4.33; N, 10.63; S, 8.11 found:C, 60.73; H, 4.36; N, 10.61; S, 8.12
Methyl(Z)-2-((E)-2-(((E)-(5-bromothiophen-2-yl)methylene)hydrazono)-4-oxothiazolidin-5-
ylidene)acetate (5c)
Yellow Solid; Yield: 72% m.p: 278ºC; IR: (KBr) (λmax, cm−1): 3085 (Ar-CH), 2954 (C-H),
1
1735, 1720 (2 C=O), 1668, 1618 (C=N, C=C), 1020 (C–O) H NMR (DMSO-d6, 300MHz); δ
12.75 (s, 1H, N-H), 8.43 (s, 1H, CH=N), 7.46 (dd, 1H,), 6.95 (s, 1H), 6.74 (s, 1H), 3.93 (s, 3H),
13C NMR (DMSO, 75MHz); δ 170.3, 169.6, 165.3, 150.5, 145.5, 143.5, 142.5, 132.5, 131.0,
116.0, 63.5. Anal. Calcd. For C11H8BrN3O3S2: C, 35.31; H, 2.15; N, 11.23; S, 17.13 found:C,
35.30; H, 2.16; N, 11.24; S, 17.14
(2-{[4-(4-Methoxy-benzyloxy)-benzylidene]-hydrazono}-4-oxo-thiazolidin-5-ylidene)-acetic
acid methyl ester (5d)

Yellow Solid; Yield: 76% m.p: 232ºC; IR: (KBr) (λmax, cm−1): 3095 (Ar-CH), 2951 (C-H),
1730, 1718 (2 C=O), 1678, 1628 (C=N, C=C), 1032 (C–O) 1H NMR (DMSO-d6, 300MHz); δ
12.23 (s, 1H, N-H), 8.31(s, 1H, CH=N), 7.29 – 7.14 (m, 4H), 7.14 – 7.02 (m, 2H), 6.93 – 6.81
(m, 3H), 5.19 – 5.15 (m, 2H), 3.87 – 3.79 (m, 6H).13C NMR (DMSO, 75MHz); δ 169.11, 167.5,
166.7, 164.3, 151.5, 144, 143.1, 135.5, 134.5, 130, 128.8, 125.5, 115, 113.5, 71.9, 53, 14.5. Anal.
Calcd. ForC21H19N3O5S:C, 59.28; H, 4.50; N, 9.88; O, 18.80; S, 7.54 found:C, 59.26; H, 4.52; N,
9.86; S, 7.56
methyl(Z)-2-((E)-2-(((E)-4-(dimethylamino)benzylidene)hydrazono)-4-oxothiazolidin-5-
ylidene)acetate (5e)
Orange Solid Yield: 82%m.p: 245ºC; IR: (KBr) (λmax, cm−1): 3085 (Ar-CH), 2945 (C-H), 1733,
1715 (2 C=O), 1666, 1638 (C=N, C=C), 1035 (C–O) 1H NMR (DMSO-d6, 300MHz); 1H NMR
(DMSO-d6, 300MHz); δ 12.80 (s, 1H, N-H), 8.53 (s, 1H, CH=N), 7.46 (dd, 2H, H-o), 6.93 (m,
2H), 6.72 (s, 1H), 3.93 (m, 3H), 2.95 – 2.91 (m, 6H). 13C NMR (DMSO, 75MHz); δ170.4,
168.5, 164.5, 153.4, 150.7, 143.5, 131.5, 130.2, 125.4, 116.3, 55.3, 55.5 Anal. Calcd.
ForC15H16N4O3S: C, 54.20; H, 4.85; N, 16.86; S, 9.65 found: C, 54.18; H, 4.87; N, 16.83; S, 9.68
{2-[(5-Methyl-furan-2-ylmethylene)-hydrazono]-4-oxo-thiazolidin-5-ylidene}-acetic acid
methyl ester (5f)
Yellow precipitates; Yield: 70%m.p: 251ºC; IR: (KBr) (λmax, cm−1): 3081 (Ar-CH), 2947 (C-H),
1740, 1723 (2 C=O), 1675, 1622 (C=N, C=C), 1112 (C–O) 1H NMR (DMSO-d6, 300MHz); δ
12.77 (s, 1H, N-H), 8.32 (s, 1H, CH=N), 7.94 (dd, 1H, Furan- H-5)), 7.07 (d, 1H furan-H-3),
6.74 (s, 1H), 6.68 (s, 1H furan-H-4), 3.79 (s, 3H),2.27(s, 3H). 13C NMR (DMSO, 75MHz);
δ169.8 (C-4), 167.4 (s, CO2CH3), 163.0 (s, C-2), 159.5 (CH=N), 152.0 (C-2-furan), 146.0 (C-5-
furan), 145.9 (C-6), 123.0 (CH-3-furan), 117.30 (C-5), 114.0 (CH-4-furan), 65.30 (OCH3), 14.20
(CH3). 12.20 (CH3).Anal. Calcd. ForC12H11N3O4S: C, 49.14; H, 3.78; N, 14.33; S, 10.93 found:
C, 49.12; H, 3.79; N, 14.34; S, 10.92
{4-Oxo-2-[(1H-pyrrol-2-ylmethylene)-hydrazono]-thiazolidin-5-ylidene}-acetic acid methyl
ester (5g)
Brown Solid; Yield: 74% m.p: 282ºC; IR: (KBr) (λmax, cm−1): 3085 (Ar-CH), 2950 (C-H), 1742
1725 (2 C=O), 1665, 1620 (C=N, C=C), 1012 (C–O)1H NMR (DMSO-d6, 300MHz); 12.20 (s,
1H- NH), 8.34 (s, 1H, CH=N), 7.08 (d, J = 16.0 Hz, 2H), 6.72 (s, 1H), 6.43 (s, 1H), 6.28 (s, 1H),

3.63 (s, 3H).13C NMR (DMSO, 75MHz); δ173, 166.5, 165.5, 163.7,143.5, 135.5, 133.5, 122.5,
115.4, 110.5, 60.2Anal. Calcd. ForC11H10N4O3S: C, 47.48; H, 3.62; N, 20.13; S, 11.52 found: C,
47.47; H, 3.63; N, 20.14; S, 11.53
{4-Oxo-2-[(3-phenyl-allylidene)-hydrazono]-thiazolidin-5-ylidene}-acetic acid methyl ester
(5h)
Light orange Solid; Yield: 72% m.p: 248ºC; IR: (KBr) (λmax, cm−1): 3075 (Ar-CH), 2930 (C-H),
1
17391715 (2 C=O), 1675, 1619 (C=N, C=C), 1042 (C–O) H NMR (DMSO-d6, 300MHz); δ
12.30 (s, 1H N-H), 8.28(s, 1H, CH=N), 7.59 (s, 1H), 7.35 – 7.24 (m, 3H), 7.22 (s, 1H), 7.18 (s,
1H), 6.90 (s, 1H), 6.72 (s, 1H), 3.65 (s, 3H).13C NMR (DMSO, 75MHz); δ169.2, 167.3, 164.5,
159.0, 147.5, 145.2, 140.5, 137.4, 132.4, 130.5, 127.5, 120, 60. Anal. Calcd. ForC15H13N3O3S: C,
57.13; H, 4.16; N, 13.33; S, 10.17 found: C, 57.15; H, 4.18; N, 13.37; S, 10.13.
[4-Oxo-2-(pyridin-4-ylmethylene-hydrazono)-thiazolidin-5-ylidene]-acetic acid methyl ester
(5i)
White Solid; Yield: 78% m.p: 270ºC; IR: (KBr) (λmax, cm−1): 3092 (Ar-CH), 2950 (C-H), 1740,
1725 (2 C=O), 1665, 1629 (C=N, C=C), 1027 (C–O) 1H NMR (DMSO-d6, 300MHz); δ 12.81(s,
1H, N-H), 8.79 – 8.68 (m, 2H), 8.04 (s, 1H, CH=N), 7.86 – 7.72 (m, 2H), 6.91 (s, 1H), 3.87 (s,
3H).13C NMR (DMSO, 75MHz); δ168.5, 167.0, 165.0, 158.2, 153.0, 151.5, 145.2, 136.5, 128.4,
57.4 Anal. Calcd. ForC12H10N4O3S: C, 49.65; H, 3.47; N, 19.30; S, 11.04 found: C, 49.60; H,
3.52; N, 19.32; S, 11.02
{2-[(2-Hydroxy-benzylidene)-hydrazono]-4-oxo-thiazolidin-5-ylidene}-acetic acid methyl
ester (5j)
Light pink Solid; Yield: 70%m.p: 225ºC; IR: (KBr) (λmax, cm−1): 3438 (OH), 3095 (Ar-CH),
2940 (C-H), 1736 1720 (2 C=O), 1675, 1639 (C=N, C=C), 1037 (C–O)1H NMR (DMSO-d6,
300MHz); δ 12.06 (s, 1H N-H), 8.22 (s, 1H, CH=N), 7.40 (d, J = 17.3 Hz, 2H), 7.18 (d, J = 8.7
Hz, 2H), 6.91 (s, 1H), 3.85 (m, 3H), 2.96 (s, 1H).13C NMR (DMSO, 75MHz); δ173.2, 169.6,
165.7, 163.5, 155.4, 145.5, 138.5, 132.6, 131.5, 125.5, 120.5, 118.3, 56. Anal. Calcd.
ForC13H11N3O4S:C, 51.14; H, 3.63; N, 13.76; S, 10.50 found: C, 51.16; H, 3.62; N, 13.75; S,
10.52
Mushroom tyrosinase assay

The mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.) was used for in vitro
bioassays as described previously with some modifications.29 Briefly, 140 µL of phosphate
buffer (20 mM, pH 6.8), 20 µL of mushroom tyrosinase (30 U/mL) and 20 µL of the inhibitor
solution were placed in the wells of a 96-well micro plate. After pre-incubation for 10 min at
room temperature, 20 µL of L-DOPA (3,4-dihydroxyphenylalanine) (0.85 mM) was added and
the plate was further incubated at 25°C for 20 min. Subsequently the absorbance of dopachrome
was measured at 475 nm using a micro plate reader (OPTI Max, Tunable). Kojic acid was used as
a reference inhibitor and for negative tyrosinase inhibitor phosphate buffer was used instead of
the inhibitor solution. The extent of inhibition by the test compounds was expressed as the
percentage of concentration necessary to achieve 50% inhibition (IC50). Each concentration was
analyzed in three independent experiments run in triplicate. The IC50 values determined by the
data analysis and graphing software Origin 8.6, 64-bit.
Kinetic analysis of the inhibition of tyrosinase
A series of experiments were performed to determine the inhibition kinetics by following
method.30,31 Different inhibitor concentrations of compound 5i 0.00, 1.06, 8.50 and 17.00 µM,
with L-DOPA concentrations 0.062, 0.125, 0.25, 0.5, 1 and 2 mM, were taken respectively. Pre-
incubation and measurement time was the same as discussed in mushroom tyrosinase inhibition
assay. Maximal initial velocity was determined from initial linear portion of absorbance up to
five minutes after addition of mushroom tyrosinase at a 30s interval. The inhibition type on the
enzyme was assayed by Lineweaver–Burk plots of inverse of velocities (1/V) versus inverse of
substrate concentration1/[S] mM-1. The inhibition constant Ki was determined by two methods,
the second plots of the apparent slope versus the concentration of compound 5i, and with the
help of Dixon plot. In Dixon plot different concentrations 0.0, 1.06, 2.12, 4.25, 8.5, 17 and 34
µM of compound 5i plot versus inverse of velocities (1/V) with changing concentrations of L-
DOPA (0.25, 0.5, 1 and 2 mM), respectively.
Molecular docking
Preparation of receptor
Molecular Operating Environment (MOE 2014) was used for current studies32.The protein
preparation step involved 3D protonation, energy minimization and active site identification. The

crystal structure of was obtained from protein data bank with co-crystalized ligand33. Water
molecules were removed. Protein was energy minimized and 3-D protonated by using the
structure preparation module of MOE. Since, the protein contains the co-crystallized ligand, so
active site was identified around co-crystallized ligand by using LigX module of the MOE. The
pocket was found to be deep cavity lined with the key residues including both hydrophobic and
hydrophilic amino acids.
Preparation of Ligands
The ligand files for molecular docking studies were prepared in Molecular Operating
Environment (MOE-2014) by Chemical computing group (CCG) and were followed by energy
optimization at standard MMFF94 force field level, with a 0.0001 kcal/mol energy gradient
convergence criterion34. The optimized geometries were saved in molecular data base file for
further studies.
Docking studies
The optimized ligands were docked with the mushtroom tyrosinase (PDB code: 2Y9X) protein
using the MOE-Dock program. A total of 30 independent docking runs were performed using
MOE docking simulation program. The docked poses were analyzed and the best scored pose for
each compound was chosen for further studies of interaction evaluation. The 2D ligand-protein
interactions were visualized using the MOE ligand interactions program.
QSAR study
Methodology
The training set comprised of 10compounds, tyrosinase inhibitors were used in the
present QSAR study. Compound structures were optimized using molecular mechanics (MM2)
implemented in ChemBio Office 2012 (Chem3D Pro 13.0) and exported to CODESSA-Pro
where they were subjected to MOPAC calculation for refined final geometry optimization.
CODESSA-Pro calculated 507 constitutional, topological, geometrical, quantum chemical and
electrostatic descriptors for the exported bio-active scaffolds. The data set comprises training set
only. To validate the model generated internal cross validation criteria was used keeping in view
the small size data set.

Multi-linear modelling
BMLR (best multi-linear regression) was initiated stepwise to search for the best n-
parameter regression equations, based on the highest R2 (squared correlation coefficient) and F
(Fisher criteria) values. QSAR models with up to 2 descriptors were generated (the maximum
number allowed according to the 5:1 rule of thumb, the ratio between the data points and the
number of descriptors). The statistical parameters including the square of the correlation
coefficient (R2), the squared cross-validated correlation coefficient (R2cv), the Fisher criterion
(F) and the variance (s2) were used to select the best QSAR model.

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Synthesis, computational studies and enzyme inhibitory kinetics of substituted methyl[2-(4-
dimethylamino-benzylidene)-hydrazono)-4-oxo-thiazolidin-5-ylidene]acetates as mushroom
tyrosinase inhibitors
Graphical Abstract

Kinetic and in silico studies of novel hydroxy-based thymol analogues as inhibitors of
mushroom tyrosinase
ꢀ Hydroxylated thymol analogues (4a-4e) and (6a-6c) were efficiently synthesized.
ꢀ Compound (6b) showed highest tyrosinase inhibitory activity (IC50 15.20µM).
ꢀ Kinetic analysis revealed (6b) is mixed type inhibitor (Ki 25µM).
ꢀ Compound (6b) formed reversible enzyme-inhibitor complex.
ꢀ The compound (6b) also well docked with protein (PDBID 2ZMX).