Hydroxyl substituted benzoic acid/cinnamic acid derivatives: Tyrosinase inhibitory kinetics, anti-melanogenic activity and molecular docking studies

Article abstract of DOI:10.1016/j.bmcl.2019.126722

The inhibition of tyrosinase is an established strategy for treating hyperpigmentation. Our previous findings demonstrated that cinnamic acid and benzoic acid scaffolds can be effective tyrosinase inhibitors with low toxicity. The hydroxyl substituted ben

Full text of DOI:10.1016/j.bmcl.2019.126722

Journal Pre-proofs
Hydroxyl substituted benzoic acid/cinnamic acid derivatives: tyrosinase inhib-
itory kinetics, anti-melanogenic activity and molecular docking studies
Yasir Nazir, Aamer Saeed, Muhammad Rafiq, Samina Afzal, Anser Ali,
Muhammad Latif, Johannes Zuegg, Waleed M. Hussein, Christian Fercher, Ross
T. Barnard, Matthew A. Cooper, Mark A.T. Blaskovich, Zaman Ashraf, Zyta
M. Ziora
PII:
S0960-894X(19)30680-8
DOI:
https://doi.org/10.1016/j.bmcl.2019.126722
BMCL 126722
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
17 June 2019
26 September 2019
27 September 2019
Please cite this article as: Nazir, Y., Saeed, A., Rafiq, M., Afzal, S., Ali, A., Latif, M., Zuegg, J., Hussein, W.M.,
Fercher, C., Barnard, R.T., Cooper, M.A., Blaskovich, M.A.T., Ashraf, Z., Ziora, Z.M., Hydroxyl substituted
benzoic acid/cinnamic acid derivatives: tyrosinase inhibitory kinetics, anti-melanogenic activity and molecular
docking studies, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.
2019.126722
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Bioorganic & Medicinal Chemistry Letters
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Hydroxyl substituted benzoic acid/cinnamic acid derivatives: tyrosinase
inhibitory kinetics, anti-melanogenic activity and molecular docking studies.
Yasir Nazira,b, Aamer Saeedc, Muhammad Rafiqd, Samina Afzale, Anser Alif, Muhammad Latif g, Johannes
Zuegga, Waleed M. Husseinh,i, Christian Fercherj, Ross T. Barnardh, Matthew A. Coopera, Mark A. T.
Blaskovicha, Zaman Ashrafb*, Zyta M. Zioraa*
aInstitute for Molecular Biosciences (IMB), The University of Queensland (UQ), St Lucia 4072, Qld, Australia.
bDepartment of Chemistry, Allama Iqbal Open University, Islamabad 44000, Pakistan.
cDepartment of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan.
dDepartment of Physiology and Biochemistry, Cholistan University of Veterinary and animal Sciences, Bahawalpur, Pakistan.
eFaculty of Pharmacy, Bahauddin Zakria University, Multan 60800, Pakistan.
fDepartment of Zoology, Mirpur University of Science and Technology (MUST), 10250, Mirpur, AJK, Pakistan.
gCollege of Medicine, Centre for Genetics and Inherited Diseases (CGID), Taibah University, Al-Madinah Al-Munawwarah, Saudi Arabia.
hSchool of Chemistry and Molecular Biosciences (SCMB) and ARC Training Centre for Biopharmaceutical Innovation, The University of Queensland (UQ), St
Lucia 4072, Qld, Australia.
iHelwan University, Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, EinHelwan, Helwan, Egypt.
jAustralian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland (UQ), St Lucia 4072, Qld, Australia.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The inhibition of tyrosinase is an established strategy for treating hyperpigmentation. Our
previous findings demonstrated that cinnamic acid and benzoic acid scaffolds can be effective
tyrosinase inhibitors with low toxicity. The hydroxyl substituted benzoic and cinnamic acid
moieties of these precursors were incorporated into new chemotypes that displayed in vitro
inhibitory effect against mushroom tyrosinase. The most active compound, (2-(3-
methoxyphenoxy)-2-oxoethyl (E)-3-(4-hydroxyphenyl) acrylate) 6c, inhibited tyrosinase with an
IC50 of 5.7 µM, while (2-(3-methoxyphenoxy)-2-oxoethyl 2, 4-dihydroxybenzoate) 4d had an
IC50 of 23.8 µM. In comparison, the positive control, kojic acid showed tyrosinase inhibition
with an IC50 =16.7 µM. Analysis of enzyme kinetics revealed that 6c and 4d displayed
noncompetitive reversible inhibition of the second tyrosinase enzymatic reaction with Ki values
of 11 µM and 130 µM respectively. In silico docking studies with mushroom tyrosinase (PDB
ID 2Y9X) predicted possible binding modes in the catalytic site for these active compounds. The
phenolic para-hydroxy group of the most active compound 6c is predicted to interact with the
catalytic site Cu++ ion. The methoxy part of this compound is predicted to form a hydrogen bond
with Arg 268. Compound 6c had no observable toxic effects on cell morphology or cell viability
at the highest tested concentration of 91.4 µM. When dosed at 91.4 µM onto B16F10 melanoma
cells in vitro 6c showed anti-melanogenic effects equivalent to kojic acid at 880 µM. 6c
displayed no PAINS (pan-assay interference compounds) alerts. Our results show that
compound 6c is a more potent tyrosinase inhibitor than kojic acid and is a candidate for further
development. Our exposition of the details of the interactions between 6c and the catalytic
pocket of tyrosinase provides a basis for rational design of additional potent inhibitors of
tyrosinase, built on the cinnamic acid scaffold.
Keywords:
Methoxy phenol,
tyrosinase inhibitors,
kinetic mechanism,
melanin quantification,
molecular docking studies
2009 Elsevier Ltd. All rights reserved.
Tyrosinase is widely distributed in nature, from
several factors such as UV exposure, α-melanocyte-stimulating
hormone, melanocortin-1 receptor and agouti-related protein
[10-11]. The process of melanogenesis represents a potential
cellular hazard and is confined to special melanosomes in
melanocytes, which synthesize pigments and transfer them to
recipient cells [12]. Melanoma is a type of skin cancer that
arises from the aberrant proliferation of melanocytes [13-14].
Malignant melanocytes tend to exhibit up-regulated
bacteria to plants and humans. It catalyses the o-hydroxylation
of monophenols to catechols and the subsequent two-electron
oxidation to quinones [1]. Its physiological function is the
conversion of tyrosine into dopaquinone, the first step of
melanin biosynthesis in specialized organelles called
melanosomes [2-3]. The non-essential aromatic amino acid L-
tyrosine serves as precursor to melanin biosynthesis [4]. L-
tyrosine and L-DOPA (or phosphorylated isomers of L-DOPA)
are key molecules in regulation of the melanin synthesis [5-7].
The distribution patterns of melanin in keratinocytes determine
the color of human skin [8-9]. Melanogenesis is modulated by
melanogenesis and defective melanosomes. Therefore,
controlling
a
tyrosinase-dependent
mechanism
of
melanogenesis might be the basis for a potential anti-
melanoma therapy. Specific tyrosinase inhibitors such as kojic
1

acid, arbutin, azelaic acid, hydroquinone and phenol have been
tested in pharmaceutical formulations for their ability to block
2

overproduction of melanin [15]. However, only kojic
acid has shown a slight inhibitory effect against
pigmentation within melanocytes [16].
evaluated their tyrosinase inhibitory activity with a view
to discovering potent tyrosinase inhibitors that bind to
the active site of tyrosinase more strongly than the
natural substrates. From these studies, we concluded
that compounds with a phenolic moiety can exhibit
potent tyrosinase inhibitory activity. In our studies on
tyrosinase inhibitors we have synthesized hydroxyl
substituted benzoic and cinnamic acid derivatives to
explore their tyrosinase inhibitory activity and to
understand how these compounds interact with the
catalytic center of the protein with a view to engineering
more potent compounds.
Currently available tyrosinase inhibitors
possess undesirable side effects such as carcinogenicity
and low clinical efficacy [17]. Therefore, it is of great
interest for both medical and cosmetic applications to
synthesize novel inhibitors. Recently (2019) Wakana
Ishioka et al. described resorcinol derivatives, which are
used as active ingredients in food preservatives and
functional cosmetics, as potent tyrosinase inhibitors
[18]. In 2018 Sultan Ullah et al. reported that
cinnamamide derivatives showed much higher
inhibitory effect than kojic acid on mushroom tyrosinase
and melanin production in B16F10 murine skin
melanoma cells, without cytotoxicity at the inhibitory
doses [19].
The difficulty of isolating pure and
homogeneous human tyrosinase from endogenous
sources has hindered the studies. Due to the absence of a
high-resolution crystal structure of the human enzyme
and the fact that both proteins share high sequence
similarity in their active core region, mushroom
tyrosinase has frequently served as a model to assess the
inhibitory activity of compounds [35].
The methoxy phenol moiety commonly found
in natural phenolic antioxidants is responsible for a
variety of biological activities such as carbonic
anhydrase inhibitory activity [20], anti-inflammatory
activity [21] and the scavenging of free radicals [22-25].
3-methoxyphenol has been reported as a depigmenting
and therapeutic agent for melanoma therapy [26].
However, the tyrosinase inhibitory activities of
compounds possessing a methoxy phenol moiety have
only rarely been discussed in the literature [27].
Menezes et al. 2011 and Miliovsky et al. 2013 reported
that cinnamic acid and benzoic acid analogues
Previous work by Abbas et al. (2017) has
validated this approach by showing that the potency of
inhibition of mushroom tyrosinase follows the trend in
potency against human tyrosinase [36]. Therefore, the
initial screening of the synthesized compounds as anti-
melanogenic agents was carried out using mushroom
tyrosinase. The in vitro mushroom tyrosinase inhibitory
activity of the target compounds with different hydroxy
substituted benzoic acids (4a-e) and cinnamic acids (6a-
c) were evaluated both experimentally and in silico.
The in-silico molecular docking of all the
synthesized compounds was performed against the
mushroom tyrosinase crystal structure (PDB ID: 2Y9X)
to ascertain the role of different functionalities in the
formation of a ligand-protein complex. All of these
compounds 4a-e and 6a-c passed the PAINS (pan-assay
interference compounds) filter [37], with the exception
of 4c. The most potent derivative, 6c, was selected for
further characterization of its effects on cellular
tyrosinase inhibition, cell toxicity, cell viability and
melanin biosynthesis in B16F10 melanoma cells.
possessing
a
substituted phenyl ring exhibited
antioxidant and tyrosinase inhibitory activity [28-29].
Cinnamic acid has been reported to have extensive
physiological actions including tyrosinase inhibitory
activity [30].
Our laboratory has synthesized a variety of
phenolic compounds, including thymol analogues,
vanillin and umbelliferone derivatives (Fig 1) [31-34]
that mimick the chemical structure of tyrosinase’s
natural substrates, L-DOPA and L-tyrosine. We have
OH
O
O
O
O
O
O
O
O
H
O
HO
Vanillin Derivative, IC50 = 16.13 uM O
Thymol analogue, IC50 = 15.2 uM
O
O
O
O
O
O
HO
Umbelliferone derivative, IC50 = 123.8 uM
Figure 1. Potent inhibitors of mushroom tyrosinase from previous studies.
3

O
O
i
+
Cl
Cl
Cl
O
OH
O
O
(1)
(2)
O
O
O
O
O
Cl
OH
(3a-e)
O
O
O
R
R
O
ii
(4a-e)
R=
a
b
c
d
e
=3-OH, = 4-OH, = 3,4-di-OH, = 2,4-di-OH, = 3,5-di-OH
Scheme 1.
(4a-e)
, Reagents and conditions,
Synthesis of compounds
o
(i)
(ii)
DMF/ (C2H5)3N/KI, r. t. stirring for 24 hrs.
CH2Cl2/ (C2H5)3N, 0_-5 C, stirring for 5 hrs,
O
O
O
O
O
OH
Cl
O
O
O
R
R
(5a-c)
O
ii
(6a-c)
R=
a
b
, c
= -H, = 2-OH = 4-OH
Scheme 2.
(6a-c) (ii)
, DMF/ (C2H5)3N/KI, r. t. stirring for 24 hrs.
Synthesis of compounds
The in-vitro mushroom tyrosinase inhibitory
activity of all the synthesized compounds (4a-e) and
(6a-c) were determined by following the previously
described method [38], with slight modifications as
detailed in the supplementary material. The synthesized
compounds (6a), (6b) and (6c) are all cinnamate esters
but differ regarding the substitution pattern on the
cinnamate phenyl ring. Compound (6a) possesses an
unsubstituted cinnamic acid component while
compound (6b) and (6c) possess 2-hydroxy and 4-
hydroxy substituted cinnamic acid, respectively. The
tyrosinase inhibitory activity of 4-hydroxy cinnamic
acid is already reported [39], its incorporation as an
ester in compound 6c results in an increase in the
activity. The replacement of hydrogen from the para
position of the cinnamic acid phenyl ring in compound
(6a) with a hydroxyl group (6c) led to a significant
increase in tyrosinase inhibitory activity (Table 1). The
compound (6a) (IC50 39.4 µM) was 7-fold less active
while (6b) (IC50 172.8 µM) was 30-fold less active than
(6c). It has already been well established that phenolic
hydroxyl plays a vital role in tyrosinase inhibitory
activity as the natural substrates tyrosine and L-DOPA
also bear the phenolic hydroxyls [40]. Here, we report
that the para-hydroxyl group of (6c) appears to play a
similar role in tyrosinase inhibitory activity.
Table 1. Tyrosinase inhibitory activity, substitution pattern, kinetics and % yield of compounds (4a-e) and (6a-c)
Mushroom tyrosinase
Compounds
R1
R2
Ki(µM)
Yield (%)
IC50 (µM)
4a
4b
4c
4d
4e
6a
6b
6c
314.5 ± 12.6
337.7 ± 17.2
298.7 ± 15.6
23.8 ± 3.7
192.7 ± 11.6
39.4 ± 4.5
172.8 ± 8.8
5.7 ± 0.3
16.7 ± 2.8
81
79
78
73
80
82
81
81
3-OH
4-OH
3,4-di-OH
2,4-di-OH
3,5-di-OH
n.d.*
-
-
130
n.d.*
-
-H
2-OH
4-OH
-
11
n.d.*
Kojic Acid
*n.d.= not determined
4

Kinetic analysis was conducted with
compounds (4d) and (6c) using the mushroom
tyrosinase enzyme and L-DOPA as substrate [41]. The
(1/V versus 1/ [S]) in Fig. 2, which showed for
type of inhibition was determined using Lineweaver-
Burk plots
competitive inhibition, with Ki values of 130 µM and 11
µM, for (4d) and (6c) respectively. Moreover, the
inhibitory mechanism of mushroom tyrosinase by
compound (6c) on the oxidation of L-DOPA was
studied. The plots of the remaining enzyme activity
versus the concentration of enzyme (4, 6, 8 and 10
µg/mL, respectively) in the presence of different
concentrations of compound (6c) for the catalysis of L-
DOPA gave a series of straight lines (Fig.3), which all
intersected at the same point on X-axis, indicating that
compound (6c) is a reversible inhibitor of tyrosinase
[43].
both compounds a slight preference of non-competitive
inhibition over competitive inhibition. The plots of 1/V
versus 1/ [S] gave a family of straight lines with
different slopes. The analysis showed that Vmax
decreased with changing Km in the presence of
increasing concentrations of compound. This result
indicates that both compounds (4d) and (6c) bind not
only to the free enzyme, but also to some degree to the
enzyme-substrate complex [42]. Similarly, analyzing the
kinetic inhibition using a Dixon plot (Fig. 2) indicates a
slight preference for noncompetitive inhibition over
Figure 2. Kinetic analysis of (4d and 6c) analyzing type of inhibition. a,c) Lineweaver-Burk plot b,d) Dixon plots. The concentration of
compound (4d) was 0.00 to 157 µM and that of compound 6c was 0.00 to 14.5 µM and the concentration of L-DOPA was 0.062 to 2.00 mM,
determining a slight preference for a non-competitive inhibition over competitive inhibition.
Figure 3. Relationship between the catalytic activity of tyrosinase at different concentrations of the enzyme and compound (6c).The lines
intersecting at the same point on X-axis indicates that 6c is a reversible inhibitor.
A cell viability assay in B16F10 melanoma
cells using MTT [3-(4, 5- dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide] was applied to assess
potential cytotoxic effects of (6c) [44]. Cell viability
was found to be higher than 95% and 99% when cells
were incubated with 40 and 30 μg/mL, respectively
(Figure 4). Consequently, we selected 30 μg/mL as the
highest concentration for further investigation as this
5

concentration had no considerable cytotoxic effects on
cell morphology (Figure S1) and cell viability.
Figure 4. Viability of B16F10 cells, treated with (6c) at different concentrations. Data is normalized against untreated samples (Con), displaying
means and standard deviation. No significance by Student’s t-tests to Control.
The inhibitory effects on tyrosinase and
cellular melanogenesis were also evaluated in B16F10
cells, normalizing results to treatment with kojic acid.
The effect of compound (6c) on the melanin content in
B16F10 cells was evaluated by treating the cells with
(6c) in the presence of 100 μM α-MSH at different
concentrations (20, 25 and 30 μg/mL). The inhibitory
effects of (6c) are more potent than that of kojic acid as
compound (6c) showed same inhibition at 30 μg/mL as
compared to kojic acid at 125 μg/mL (Fig. 5a). Within
the melanogenesis cascade of enzymatic reactions,
tyrosinase acts as the rate-limiting enzyme [45-46]. To
analyze the inhibition mechanism of melanin synthesis
by (6c), we examined the inhibitory effect of (6c) on
cellular tyrosinase activity. We found significant
intracellular tyrosinase inhibition at 25 and 30 μg/mL
concentration of (6c) in a dose-dependent manner (Fig.
5b).
Figure 5. (a) Melanin inhibition in B16F10 cells by (6c) at different concentrations, with MSH (100 μM) as a known stimulant of melanin
production and kojic acid (880 μM) as a known inhibitor. Intracellular melanin was monitored by absorbance at 405 nm. (b) Inhibition of
tyrosinase activity in B16F10 cells by (6c) at different concentrations. Cellular murine tyrosinase activity was monitored by transformation of L-
DOPA into dopachrome by absorbance at 490 nm. Data is normalized against untreated samples (Con), displaying means and standard deviation,
and significance by Student’s t-tests compared to control: *p<0.05, **p<0.005.
Computational
docking
studies
were
hydroxyphenol moiety shows consistent interactions
between the hydroxyl and the active site Cu ions. The
“tail” moiety of this compound occupies a distinct area
of the binding site close to Arg 268, picking up an H-
bond interaction between the oxygen of methoxy phenol
and the hydrogen of Arg 268. While the docking poses
have some degree of uncertainty, they give direction to
modifications that could improve the activity of the
compounds, and to the bonding features that will need to
be retained in new compounds based on this framework.
performed to predict the most favorable binding mode
of (6c) with 2Y9X inside the binding pocket of the
mushroom tyrosinase with proper orientation in term of
docking score –5.934 Kcal/ mol[47]. Such lower values
indicate good fitness of the compound in the binding
pocket of the protein and stable 6c-protein interaction
(Fig.6). The selected pose of the most active compound
(6c) was visualized in order to determine the amino
acids of tyrosinase that are involved in binding of the
ligand. For the most active compound (Fig. 6), the para
6

Figure 6. Ball and stick representation of the docking pose of the highly ranked pose for most active compound 6c in the binding site of
mushroom tyrosinase (2Y9X). Residues, in stick representation, that forming the binding site of the enzyme are labeled, Cu2+ ions are shown in
brown and labeled.
In summary, we have identified 2-(3-
methoxyphenoxy)-2-oxoethyl (2E)-3-(4-hydroxyphenyl)
prop-2-enoate (6c) as a novel anti-tyrosinase compound
that is more potent than the reference compound kojic
acid. It appears, based on kinetic analysis, that the mode
of inhibition by 6c is noncompetitive inhibition. This
leads us to suggest that the bulky side chains, distant
from the metallic center of the binding site are
contributing to the non-competitive component of
inhibition. The compound appears to reduce α-MSH
stimulated melanogenesis through noncompetitive and
reversible inhibition of tyrosinase in vitro, having a Ki
value of 11 µM. Docking studies suggested possible
binding poses and interactions for 6c in the active site of
mushroom tyrosinase. The absence of cytotoxic effects
of (6c) at the concentrations that inhibited
melanogenesis in B16F10 cells, supports the suggestion
that the suppression of melanogenesis by compound
(6c) can be attributed to the specific inhibition of
tyrosinase. The melanin inhibition results (Fig. 5a)
demonstrated that 6c has de-pigmenting activity at a
lower dose than kojic acid (91.4 µΜ compared to 880
μM) in the B16F10 cell system. It is evident from our in
vitro results that the presence of a hydroxyl-substituted
cinnamate ester scaffold conferred more potent
tyrosinase inhibitory activity than is possessed by kojic
acid. The compound (6c) by virtue of its anti-
melanogenic activity, is a promising regulator of
melanogenic activity and is a candidate for the further
development of agents that have inhibitory effects on
tyrosinase and melanin biosynthesis. Our exposition of
the interaction between 6c and the catalytic pocket of
tyrosinase provides a basis for rational design of
additional potent inhibitors of tyrosinase that are built
on the cinnamic acid scaffold.
Conflicts of Interest: The authors declare no conflict of
interest.
Acknowledgements MAC is a NHMRC principal
research fellow (APP1059354) and also holds a
fractional professorial research fellow appointment at
the University of Queensland, with his remaining time
as CEO of Inflazome Ltd, a company developing drugs
to address clinical unmet needs in inflammatory disease.
MATB is supported in part by Wellcome Trust Strategic
Grant WT1104797/Z/14/Z and NHMRC Development
grant APP1113719. JZG is supported in part by
Wellcome Trust Strategic Grant WT1104797/Z/14/Z.
RTB is a chief investigator in the ARC Training Centre
for Biopharmaceutical Innovation and received funding
from the University of Queensland. YN thanks the
Higher Education Commission HEC, Pakistan for
providing funding to undertake research at IMB-UQ,
Cooper group, Australia.
Appendix
A.
Supplementary
data
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Kinetic analysis of th
49.
Table 1. Tyrosinase inhibitory activity, substitution pattern,
kinetics and % yield of compounds (4a-e) and (6a-c)
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Compounds
R1
R2
Ki(µM)
Yi
IC50 (µM)
33. Ashraf Z, Rafiq M, Seo SY, Babar MM. Bioorg Chem.2015.
23(17):5870-80.
4a
4b
4c
4d
4e
6a
314.5 ± 12.6
337.7 ± 17.2
298.7 ± 15.6
23.8 ± 3.7
192.7 ± 11.6
39.4 ± 4.5
3-OH
4-OH
3,4-di-OH
2,4-di-OH
3,5-di-OH
n.d.*
-
-
130
n.d.*
-
34. Ashraf Z, Rafiq M, Seo SY, Babar MM, Zaidi NU. J Enzyme Inhib Med
Chem.2015, 30(6):874-83.
35. Parvez S, Kang M, Chung HS, Bae H. Phyther Res. 2007, 21(9):805-16.
36. Abbas Q, Ashraf Z, Hassan M, Nadeem H, Latif M, Afzal S, Seo SY.
Drug Des Devel Ther.2017; 11: 2029.
37. Baell JB, Nissink JW. Seven Year Itch: ACS Chem Bio. 2017, 13(1):36-
-H
44.
6b
6c
172.8 ± 8.8
5.7 ± 0.3
16.7 ± 2.8
38. Seo WD, Ryu YB, Curtis-Long MJ, Lee CW, Ryu HW, Jang KC, Park.
2-OH
4-OH
-
Eur J Med Chem. 2010, 45(5).
11
n.d.*
39. Loizzo, M. R.; Tundis, R.; Menichini, F. Compr. Rev. Food Sci. Food
Kojic Acid
Saf. 2012, 11, 378-398.
*n.d.= not determined
40. Parvez S, Kang M, Chung HS, Bae H. Phyther Res. 2008;22(4):544–
549.
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