Synthesis and biological activity of oxadiazole and triazolothiadiazole derivatives as tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.04.067

A series of 16 oxadiazole and triazolothiadiazole derivatives were designed, synthesized and evaluated as mushroom tyrosinase inhibitors. Five derivatives were found to display high inhibition on the tyrosinase activity ranging from 0.87 to 1.49 μM. Compound 5 exhibited highest tyrosinase inhibitory activity with an IC₅₀ value of 0.87 ± 0.16 μM. The in silico protein-ligand docking using autodock 4.1 was successfully performed on compound 5 with significant binding energy value of -5.58 kcal/mol. The docking results also showed that the tyrosinase inhibition might be due to the metal chelating effect by the presence of thione functionality in compounds 1-5. Further studies revealed that the presence of hydrophobic group such as cycloamine derivatives played a major role in the inhibition. Piperazine moiety in compound 5 appeared to be involved in an extensive hydrophobic contact and a 2.9 hydrogen bonding with residue Glu 182 in the active site.

Full text of DOI:10.1016/j.bmcl.2010.04.067

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3755–3759
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activity of oxadiazole and triazolothiadiazole
derivatives as tyrosinase inhibitors
Kok Wai Lam a, Ahmad Syahida b, Zaheer Ul-Haq c, Mohd. Basyaruddin Abdul Rahman d, Nordin H. Lajis a,d,
*
a Laboratory of Natural Products, Institute of Bioscience, University Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
b Department of Biochemistry, Faculty of Biotechnology and Molecular Sciences, University Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
c Dr. Panjwani Center for Molecular Medicine & Drug Research, International Center for Chemical & Biological Sciences, University of Karachi, Karachi 75270, Pakistan
d Department of Chemistry, Faculty of Science, University Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 16 oxadiazole and triazolothiadiazole derivatives were designed, synthesized and evaluated as
mushroom tyrosinase inhibitors. Five derivatives were found to display high inhibition on the tyrosinase
Received 26 January 2010
Revised 12 April 2010
Accepted 16 April 2010
Available online 18 April 2010
activity ranging from 0.87 to 1.49
lM. Compound 5 exhibited highest tyrosinase inhibitory activity with
an IC50 value of 0.87 0.16 M. The in silico protein–ligand docking using AUTODOCK 4.1 was successfully
l
performed on compound 5 with significant binding energy value of À5.58 kcal/mol. The docking results
also showed that the tyrosinase inhibition might be due to the metal chelating effect by the presence of
thione functionality in compounds 1–5. Further studies revealed that the presence of hydrophobic group
such as cycloamine derivatives played a major role in the inhibition. Piperazine moiety in compound 5
appeared to be involved in an extensive hydrophobic contact and a 2.9 Å hydrogen bonding with residue
Glu 182 in the active site.
Keywords:
Oxadiazole
Triazolothiadiazole
Piperazine
Mushroom tyrosinase inhibitor
Ó 2010 Elsevier Ltd. All rights reserved.
Tyrosinase is a copper containing enzyme which acts as a
catalyst in two different reactions involving the hydroxylation of
monophenols to o-diphenols and the oxidation of the o-diphenols
to o-quinones.1 This class of enzyme is widely distributed in plant,
animal and microorganism kingdoms.2 Studies have shown that
this enzyme was not only responsible for the browning of fruits
and vegetables but also caused some dermatological problems
such as flecks, melasma and ephelide due to overproduction of
melanin.1,3 Several known inhibitors such as hydroquinone, kojic
acid and arbutin were used in the treatment of skin diseases but
recently they were found to be unsafe to human health.4–6 Re-
cently, Khan et al. reported that 1,3,4-oxadiazole, to be active in
inhibiting the activity of tyrosinase enzyme.7 1,3,4-Oxadiazole
was also found to show a broad range of biological activities
including anti-microbial, antitumoral, anti-inflammatory and anal-
gesic activities.8–13 In this Letter, we describe the synthesis and
tyrosinase inhibitory activity of oxadiazole and triazolothiadiazole
derivatives. Compounds 1–5 were found actively inhibiting the
tyrosinase activity with compound 5 exhibited the highest inhibi-
The synthesis of title compounds was achieved by the reac-
tions in Scheme 1. The general method of preparing oxadiazole
and triazolothiadiazole derivatives were based on the work of
Mathew et al. and Almajan et al.14,15 The intermediates involved
for the synthesis of compounds 1–16 were 1,3,4-oxadiazole and
4-amino-5-mercapto-1,2,4-triazole. 1,3,4-Oxadiazole was pre-
pared in a stepwise manner involving esterification of the car-
boxylic group followed by the hydrazinolysis to give aroyl
hydrazides. Further reaction with carbon disulfide and potassium
hydroxide in ethanol afforded the potassium dithiocarbazates
salts. 1,3,4-Oxadiazole was obtained subsequently from acid
hydrolysis while reaction with hydrazine monohydrate afforded
4-amino-5-mercapto-1,2,4-triazole. Both of these intermediates
were used to prepare compounds 1–16. Compounds 1–5 were
prepared by the one-pot Mannich reaction consisting of oxadiaz-
ole condensation with secondary amine in the presence of form-
aldehyde. On the other hand, preparation of compounds 10–16
required strong dehydrating agent such as phosphorus oxychlo-
ride to complete the reaction. Compounds 6–9 required addi-
tional steps, first by nucleophilic substitution reaction with
ethane dibromides followed by the addition of secondary amine
such as piperidine and morpholine. The final products obtained
in Scheme 1 were in satisfactory yields. The structures of the
synthesized compounds were determined by spectroscopic tech-
niques, including 1H NMR, 13C NMR, EIMS, CHN elemental anal-
ysis and IR (see Scheme 1).
tion with an IC50 values of 0.87 0.16 lM. Molecular docking was
then performed using AUTODOCK 4.1 to examine the binding of the
active compound into the pocket site of the tyrosinase enzyme.
* Corresponding author. Tel.: +603 89468084; fax: +603 89468080.
E-mail addresses: nhlajis@ibs.upm.edu.my, nhlajis@gmail.com (N.H. Lajis).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.067

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K. W. Lam et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3755–3759
O
O
O
O
O
O
S
(c), (d)
R
(b)
R
SH
(a)
R
S
(e)
R
R
OH
O
R
OMe
R
O
NHNH2
N
N
N
N
N
NH
amine
1-5
S
S
O
SH
amine
(f)
Br
(g)
R
R
N
N
N
N
N
N
6-9
R
SH
N
O
N
H2N
(h), (i), (j)
(k)
N
R
N
S
R
NHNH2
N
R
N
N
10-16
Scheme 1. Synthesis of oxadiazole and triazolothiadiazole analogues. Reagents and reaction conditions: (a) methanol, H2SO4, reflux, 4 h; (b) NH2NH2ÁH2O, acetic acid,
ethanol, reflux, 4 h; (c) CS2, KOH, ethanol, reflux, 4–6 h; (d) HCl, cold distilled water; (e) respective secondary amines, formaldehyde, dioxane, ethanol; (f) 1,2-dibromoethane,
K2CO3, acetonitrile; (g) piperidine/morpholine, mixture of ethanol and acetone, rt, 4 h; (h) CS2, KOH, ethanol, reflux, 4–6 h; (i) NH2NH2ÁH2O, H2O, reflux, 4 h; (j) HCl, cold
distilled water; (k) respective carboxylic acid derivatives, POCl3, reflux 6 h.
Table 1
The docking and free energies calculated by using AUTODOCK 4.1 and comparison between the experimental and calculated IC50 values of tyrosinase inhibitory activities
Compound
Analogues
IC50
(l
M) (Mean SEMa)
Free binding energyb (kcal/mol)
Experiment
S
O
N
N
1
0.97 0.01
À4.91
N
S
O
O
N
2
3
4
1.34 0.14
1.33 0.18
1.49 0.17
À4.80
À4.56
À2.32
N
N
S
O
N
N
N
S
O
N
N
N
N
S
O
NH
N
N
5
6
0.87 0.16
À5.58
N
N
S
O
NAc
NAd
N
N
S
NAc
NAd
N
O
7
N
N

K. W. Lam et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3755–3759
3757
Table 1 (continued)
Compound
Analogues
IC50
(l
M) (Mean SEMa)
Free binding energyb (kcal/mol)
Experiment
Cl
S
S
O
8
9
NAc
NAd
N
N
N
N
N
Cl
O
NAc
NAd
O
N
10
NAc
NAd
N
N
S
N
N
N
N
N
S
N
N
N
11
12
NAc
NAd
N
N
N
S
N
N
N
NAc
NAd
Cl
Cl
N
S
N
N
N
13
14
15
NAc
NAc
NAc
NAd
NAd
NAd
N
N
N
N
N
S
N
N
N
N
N
N
S
N
Cl
16
NAc
NAd
NDd
N
N
N
N
N
S
N
Kojic Acide
9.18 0.28
a
SEM: standard error of the mean.
Free binding energy: values of binding energy generated by AUTODOCK 4.1.
b
c
NA: not active at 50
ND: not determined.
lM concentration.
d
e
Kojic acid: positive control used in the assay.

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K. W. Lam et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3755–3759
Inhibition activities of the synthesized oxadiazole and triazolo-
the catalytic core. Protein crystal structure with PDB code 1wx2
was obtained from the protein data bank followed by docking
studies using AUTODOCK 4.1.17 The ligands were constructed and en-
ergy minimized using MOPAC program with MMFF94 force field cal-
culation. The caddie protein (ORF 378) and water molecules were
removed before polar hydrogen atoms were added and Kollman
united atomic charges were assigned using the AUTODOCK 4.1. The
grid points were set as 40 Â 40 Â 40 with the spacing valued at
0.375 to the catalytic site of the tyrosinase while the grid center
were placed at À13.175, À14.234 and 16.952. The solvation param-
eters were added by the ADDSOL program. In default mode, parame-
ters were not given for the dicopper ions which were essentially
important for our docking studies. Therefore, the solvation of cop-
per ions was assigned to ‘0’ and the copper van der Waals param-
eters were assigned based on the Quanta 3.0 Parameters
Handbook. The type M equilibrium separation between the nucle-
uses of the two copper ions, Rij was set as 1.27 Å while the pairwise
potential energy epsij was adjusted to 0.06.18 Finally ADT, Chimera
and LIGPLOT software were used to evaluate the docking of ligand
in the active site of tyrosinase.19–21
thiadiazole analogues were tested on mushroom tyrosinase
according to the method reported by Hearing with slight modifica-
tion.16 The activities were compared to that of kojic acid as positive
inhibitor. The IC50 value of the compounds examined was summa-
rized in Table 1. Our results showed that compounds 1–5 exhibited
potent inhibitory on mushroom tyrosinase with the highest tyros-
inase inhibitory activity with an IC50 value of 0.87 0.16
lM, com-
pared to kojic acid which recorded IC50 of 9.18 0.28
lM. The
decrease in tyrosinase inhibitory activity of this group of com-
pounds, could be related to the nature of the cycloamine moiety at-
tached at N-3 of the oxadiazole ring (piperazine > piperidine >
pyrrolidine > morpholine > methylpiperazines). Compounds 6–16
did not show any significant activities at 50 lM concentration.
The discovery of compounds 1–5 as tyrosinase inhibitor has
encouraged us to further investigate the ligand–receptor interac-
tion of this group of compounds on mushroom tyrosinase (see
Table 1).
Tyrosinase is also known as polyphenol oxidase and contained a
type-3 copper center with a coupled dinuclear copper active site in
As shown in Figure 1, apparently the tyrosinase inhibition of
compounds 1–5 depended on the competency of the sulfur atom
to chelate with the dicopper nucleus in the active site. In fact,
Kim et al. proposed that the hydroxyl groups in flavonoids might
inhibit the tyrosinase enzyme by participating in a metal–ligand
binding interaction with the dicopper nucleus.22 Recent studies
found that higher number of hydroxyl groups in the benzene ring
of flavonoids played an important role in enzyme inhibition, while
methylated and glycosylated flavonoids exhibited less inhibitory
effects.23 Generally, these agreed well with our hypothesis that
compound 5 might display the same mode of inhibitory mecha-
nism through the metal chelating effects. In the absence of the
phenolic hydroxyl group, compound 5 was replaced by the thione
moiety which capable of acting as metal chelator. This was further
strengthened by the published article of tyrosinase crystal struc-
ture, 1 bug revealing the mechanism of phenylthiourea inhibitor,
where the sulfur atom was found to coordinate with both the cop-
per ions, and further increased the metal–metal separation be-
tween the ions through the sulfur ligand atom.24
The present docking studies revealed the fact that thione group
of compound 5, which resided 3.2–3.5 Å adjacent to the dicopper
nucleus are exposed to potential metal–ligand interaction. The LIG-
PLOT analyses were conducted to understand in depth interaction
pattern between the docked ligands and the active site residues
through hydrophobic interaction as well as hydrogen bonding pat-
tern.21 Figures 2 and 3 showed that compound 5 was involved in
an extensive hydrophobic contact with the nearby residues. The
Figure 1. Superimposition of five docking compounds (compound 5 in ball and
stick diagram, 1 = blue, 2 = yellow, 3 = red, 4 = cyan). Diagram above shows the
position of sulfur atom of the active compounds with the dicopper ions in the active
site.
Figure 2. (a) Binding mode of compound 5 into the binding site of tyrosinase (PDB code 1wx2).17 The amine group was involved in a hydrogen bonding with the uncharged
side chain carboxylate of Glu 182 with estimated free binding energy of À5.58 kcal/mol. (b) Interatomic contacts (yellow) of the ligand with the residues in active site based
on the van der Waals radii generated by Chimera software.20

K. W. Lam et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3755–3759
3759
cally through the binding energy related to interactions between
the inhibitor and the amino acid residues in the active site.18 In
our study, a good correlation between their IC50 of compounds
1–5 and AUTODOCK binding free energy was exhibited.
In conclusion, the present data showed promising initial results
in the rational design of anti-tyrosinase inhibitor. The results sug-
gested the capability of the thione group to chelate the dicopper
nucleus of the enzyme and the substitution pattern of cycloamine
moiety are the main contributors to the activity, indicate the sig-
nificance of the hydrophobic interaction surrounding the active
site. The information derived from the docking study of compound
5 provides a good starting platform for further structural modifica-
tions and synthesis of potent tyrosinase inhibitor.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.04.067.
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role of the secondary amine in hydrogen bonding interaction and
hydrophobic contact was further explored by substituting different
cycloamine derivatives including piperazine, piperidine, pyrroli-
dine, morpholine and N-methylpiperazine. From the docking re-
sults, the piperazine moiety was found to well occupy the pocket
consisting His 54, Trp 184, His 190 and Asn 191 residues, while
the piperazine side chain further formed a 2.9 Å hydrogen bonding
with the carboxylate side chain of Glu 182 (see Fig. 3). Replacing
the piperidine moiety with pyrrolidine, slightly reduced the inhib-
itory activity which could explain the importance of the hydropho-
bic interaction surrounding the active site. Chelation between the
sulfur atom and the metal nucleus could act as a bridge to link
the naphthyl and aminyl moieties with the hydrophobic pockets
surrounding the active site. In Figure 3, the naphthalene ring was
within close contact range with the adjacent residues of Val 195
and Asn 191. Compounds 6–16 did not show inhibitory effects
on the tyrosinase activity. Structurally these compounds lacked
the thione functionality and the shielding effects surrounding the
thioether group might be the reason for its inactivity. Based on
the bioactivity results and this structural observation, the presence
of thione is necessary as metal chelator (see Figs. 2 and 3). Previous
studies of tyrosinase inhibitors by Khatib et al. showed that the
performance of tyrosinase inhibitors could be predicted theoreti-