Novel morpholine containing cinnamoyl amides as potent tyrosinase inhibitors

Article abstract of DOI:10.1016/j.ijbiomac.2019.05.201

Tyrosinase enzyme plays a crucial role in melanin biosynthesis and enzymatic browning process of vegetables and fruits. Hence, tyrosinase inhibitors are important in the fields of medicine, cosmetics and agriculture. In this study, novel N-(2-morpholinoethyl)cinnamamide derivatives bearing different substituents on phenyl ring were designed, synthesized and evaluated for their tyrosinase diphenolase inhibitory activity. The compounds were found to be better tyrosinase inhibitors (IC₅₀s were in micro molar range) than cinnamic acid. (E)-3-(3-chlorophenyl)-N-(2-morpholinoethyl)acrylamide (B6) exhibited the highest inhibition with IC₅₀ value of 15.2 ± 0.6 μM which was comparable to that of kojic acid. The inhibition kinetic analysis of B6 indicated that the compound was a mixed-type tyrosinase inhibitor. In silico ADME prediction indicated that B6 might show more skin penetration than kojic acid. Molecular docking analysis confirmed that the active inhibitors well accommodated in the mushroom tyrosinase active site and it was also revealed that B6 formed the most stable drug-receptor complex with the target protein. Therefore, cinnamamide B6 could be introduced as a potent tyrosinase inhibitor that might be a promising lead in cosmetics, medicine and food industry.

Full text of DOI:10.1016/j.ijbiomac.2019.05.201

Accepted Manuscript
Novel morpholine containing cinnamoyl amides as potent
tyrosinase inhibitors
Shahrzad Ghafari, Sara Ranjbar, Bagher Larijani, Mohsen Amini,
Mahmood Biglar, Mohammad Mahdavi, Maryam Bakhshaei,
Mahsima Khoshneviszadeh, Amirhossein Sakhteman, Mehdi
Khoshneviszadeh
PII:
S0141-8130(19)32555-3
DOI:
https://doi.org/10.1016/j.ijbiomac.2019.05.201
BIOMAC 12486
Reference:
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
8 April 2019
15 May 2019
27 May 2019
Please cite this article as: S. Ghafari, S. Ranjbar, B. Larijani, et al., Novel morpholine
containing cinnamoyl amides as potent tyrosinase inhibitors, International Journal of
Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.05.201
This is a PDF file of an unedited manuscript that has been accepted for publication. As
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ACCEPTED MANUSCRIPT
Novel morpholine containing cinnamoyl amides as potent tyrosinase inhibitors
a
b
c
a
d
Shahrzad Ghafari , Sara Ranjbar , Bagher Larijani , Mohsen Amini , Mahmood Biglar ,
e
e
f
Mohammad Mahdavi , Maryam Bakhshaei , Mahsima Khoshneviszadeh , Amirhossein
e
e,f
Sakhteman , Mehdi Khoshneviszadeh *
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences,
Tehran, Iran
b
Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
c
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences
Institute, Tehran University of Medical Sciences, Tehran, Iran
d
Drug Design and Development Research Center, Tehran University of Medical Sciences, Tehran, Iran
e
Department of Medicinal Chemistry, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz,
Iran
f
Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences,
Shiraz, Iran
Co-first authors:
Shahrzad Ghafari, Sara Ranjbar
(The authors contribute equally in this manuscript)
*
For correspondence:
Mehdi Khoshneviszadeh, Pharm D, PhD.
Department of Medicinal Chemistry, School of Pharmacy, Shiraz University of Medical Sciences, PO
Box 71345-3388, Shiraz, Iran. Tel: +98 713 230 3872; Fax: +98 713 230 2225, E-mail:
m.khoshneviszadeh@gmail.com
1

ACCEPTED MANUSCRIPT
Abstract
Tyrosinase enzyme plays a crucial role in melanin biosynthesis and enzymatic browning process
of vegetables and fruits. Hence, tyrosinase inhibitors are important in the fields of medicine,
cosmetics and agriculture. In this study, novel N-(2-morpholinoethyl)cinnamamide derivatives
bearing different substituents on phenyl ring were designed, synthesized and evaluated for their
tyrosinase diphenolase inhibitory activity. The compounds were found to be better tyrosinase
inhibitors (IC s were in micro molar range) than cinnamic acid. (E)-3-(3-chlorophenyl)-N-(2-
5
0
morpholinoethyl)acrylamide (B6) exhibited the highest inhibition with IC value of 15.2±0.6
50
μM which was comparable to that of kojic acid. The inhibition kinetic analysis of B6 indicated
that the compound was a mixed-type tyrosinase inhibitor. In silico ADME prediction indicated
that B6 might show more skin penetration than kojic acid. Molecular docking analysis confirmed
that the active inhibitors well accommodated in the mushroom tyrosinase active site and it was
also revealed that B6 formed the most stable drug-receptor complex with the target protein.
Therefore, cinnamamide B6 could be introduced as a potent tyrosinase inhibitor that might be a
promising lead in cosmetics, medicine and food industry.
Keywords: Tyrosinase, Cinnamic acid, Cinnamamide, Phenyl-N-(2-morpholinoethyl)acrylamide,
Docking, Kojic acid
2

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1
. Introduction
Tyrosinase is a multifunctional copper-containing oxidase abundantly distributed in
mammalians, plants, bacteria and fungi. The enzyme is found in the membrane of the
melanosome, and is produced only by melanocyte cells [1]. Tyrosinase enzyme involves in
melanin biosynthesis by catalyzing two distinct reactions; the hydroxylation of tyrosine
(monophenolase activity) and oxidation of 3,4-dihydroxyphenylalanine (L-Dopa) to o-
dopaquinone (diphenolase activity) [2]. Hence, tyrosinase is associated with some skin
pigmentation disorders [3]. Tyrosinase also catalyzes the neuro-melanin production by oxidation
of dopamine to dopaquinones. As excessive dopaquinones formation results in neuronal damage
and cell death, it is suggested that tyrosinase is responsible for the neuro-degeneration associated
with Parkinson and Huntington’s diseases [4, 5]. Tyrosinase has also a crucial role in enzymatic
vegetables and fruits browning which leads to fast degradation during the postharvest and
handing process [6]. Therefore, tyrosinase inhibitors are of great interest in medicinal and
cosmetic products, as well as in agriculture and food industry [7-9]. Many tyrosinase inhibitors
have been developed till now; however, due to some limitations such as toxicity, carcinogenicity,
low efficacy, poor bioavailability, instability during storage, causing off-flavor and off-donor
(especially in food industry) and allergic reactions, developing new tyrosinase inhibitors with
drug-like properties, is in great need [7, 10-12].
Cinnamic acid, 3-phenylprop-2-enoic acid, (Fig. 1, A) is one of the chief components of
Cinnamomum cassia BLUME which has been identified as an active plant ingredient with no
cytotoxic effect on human cells that inhibit melanogenesis [13-15]. It has been reported that
cinnamic acid showed mild tyrosinase inhibitory activity with IC s at millimolar values (0.21-
5
0
3

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2
.10 mM) [15-19]. It has been proved that natural and synthetic cinnamic acid derivatives
bearing 4-hydroxy, 3,4-dihydroxy and 4-hydroxy-3-methoxy, 4-chloro, 4-ethoxy and 4-nitro
substitutions on phenyl ring exhibit potent anti-tyrosinase activity [18-21]. Besides, some amide
derivatives of cinnamic acid are naturally present in plants [22-24]. Several cinnamamide
derivatives have been evaluated for their tyrosinase inhibitory activity and most of them were
stated to be more potent tyrosinase inhibitors than cinnamic acid [17, 25-27]. N-feruloylserotonin
and N-(p-coumaroyl)serotonin (Fig. 1, B) which were isolated from safflower seeds showed
significant inhibition on mushroom tyrosinase with IC50 values of 0.023 and 0.074 mM,
respectively [24]. Okombi et al. proved some synthetic N-hydroxycinnamoylphenalkylamides
had better inhibitory activities on tyrosinase than cinnamic acid did; for instance, trans-N-
dihydro-p-hydroxycinnamoyltyramine (Fig. 1, C) was reported to induce 100% inhibition at 0.1
mM [21]. Fan et al. reported some cinnamoyl amides with amino acid ester moiety (Fig. 1, D)
which demonstrated promising inhibition on mushroom tyrosinase with IC s in the range of
5
0
0
.18-460 μM [17]. Hoon Ha and Park developed some dimeric cinnamoylamide analogues with
different diamide-link chain lengths and various functional groups as depigmenting agents, and
indicated that compound E (Fig. 1, E) inhibited mushroom tyrosinase activity with IC value of
5
0
3
.4 μM [27]. Cinnamoyl cyclic amid derivatives, F and F (Fig. 1, F), were synthesized by
1 2
Ullah et al. and proved to have 93.72% and 78.97% inhibition on mushroom tyrosinase activity
at 25 μM concentration, respectively [26].
In this study, as a part of our ongoing efforts to find novel and effective tyrosinase
inhibitors, we synthesized a series of N-(2-morpholinoethyl)cinnamamide analogues and
investigated their anti-tyrosinase activity against mushroom tyrosinase. The most active analogue
was evaluated for its inhibition kinetic. Finally, in order to get insight about the interactions and
4

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binding mode of the active derivatives in the binding site of tyrosinase, molecular docking
analysis was performed.
Fig. 1. Structures of cinnamic acid and its natural/synthetic amide derivatives reported as tyrosinase
inhibitors.
2
2
. Materials and Methods
.1. Apparatus
The progress and completion of the reactions were checked by thin layer chromatography (TLC)
on MERCK precoated silica gel 60-F254 (0.5 mm) aluminum plates. Melting points were
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determined on a Kofler hot stage apparatus. The IR spectra obtained by using Nicolet FT-IR
1
13
Magna 550 spectrometer (KBr disks). H and C NMR spectra were recorded on a Bruker 500
MHz NMR instrument by using CDCl as solvent and TMS as an internal standard. The
3
chemical shifts (δ) and coupling constants (J) are expressed in parts per million (ppm) and Hertz
(Hz), respectively. All spectra affirmed the structure of the synthesized compounds. Elemental
analyses for C, H and N were performed with an Elementar Analysen system GmbH VarioEL.
The results of elemental analyses (C, H, N) were within ± 0.4% of the calculated value.
2
.2. Chemicals and reagents
All reagents and solvents were obtained from commercial suppliers and were used without
further purification. Methyl 3,4-dimethoxybenzoate, hydrazine hydrate, aldehydes and p-
toluenesulfonic acid (PTSA) were purchased from Merck. Analytical thin layer chromatography
(TLC) was performed on MERCK precoated silica gel 60-F254 (0.5 mm) aluminum plates.
Mushroom tyrosinase (EC1.14.18.1), Kojic acid, dimethyl sulfoxide (DMSO), L-3,4-
dihydroxyphenylalanine (L-Dopa) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were from Sigma
Chemical Co. (St. Louis, MO, USA).
2
.3. General procedure for the synthesis of cinnamic acid derivatives (A1-A19)
A solution of malonic acid (5.5 mmol) in pyridine (1.25 ml) was stirred at 110 ºC for several
minutes. Then, benzaldehyde derivatives (5 mmol), piperidine (0.05 ml) and ethanol (5 ml) were
added and heated at reflux condition for 24 h. After the reaction completion (monitored by TLC),
the mixture was cooled to room temperature and added to the HCl solution (1M) in an ice bath.
6

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The precipitated solid was isolated by filtration, washed with water and dried to give cinnamic
acid derivatives A1-A19.
2
.4. General procedure for the synthesis of morpholine containing cinnamamide derivatives
(B1-B19)
A mixture of corresponding cinnamic acid derivative (2 mmol), N-hydroxy benzotriazole
HOBT) (2.4 mmol) and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) (2.4 mmol) in
0 ml dry acetonitrile was stirred for 1h at room temperature. Then, 4-(2-Aminoethyl)
(
1
morpholine (2 mmol) was added and the reaction mixture was stirred for 48h at room
temperature. Reaction completion was monitored by TLC. The crude product was extracted
using CHCl (50 ml) and NaCl 10% (2×50 ml), NaHCO 10% (2×50 ml) and citric acid 10%
3
3
(
2×50 ml). The aqueous phase was neutralized with K CO and extracted with CHCl (3×50 ml).
2 3 3
The organic phase was dried by using Na SO and the solvent was evaporated under vacuum.
2
4
The residue recrystallized from ethyl acetate and petroleum ether to obtain final products in 39-
8
3% yields.
2
.5. Mushroom tyrosinase inhibition assay
Mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.), with specific activity of 1054.6
IU/mg solid that was determined according to a previous study [28], was assayed using L-Dopa
as the substrate as reported in our previous studies [29-32]. Enzyme activity was monitored by
observing dopachrome formation at 475 nm. All test samples were first dissolved in DMSO at 40
mM and diluted to the required concentrations. First, 10 ml of tyrosinase (0.5 mg ml) was mixed
7

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with 160 ml of 50 mM phosphate buffer (pH=6.8) in 96-well microplates and then 10 ml of the
test sample was added. After the mixture was pre-incubated at 28 °C for 20 min, 20 ml of L-
Dopa solution (0.5 mM) was added to the phosphate buffer. DMSO without test compounds was
used as the control, and kojic acid was used as the positive control. Each concentration was
analyzed in three independent experiments run in triplicate. The inhibitory activity of the tested
compounds was expressed as the concentration that inhibited 50% of the enzyme activity (IC ).
5
0
The percent inhibition ratio was calculated according to the following equation:
Inhibition (%) = 100 (Abscontrol - Abscompound)/Abscontrol.
2
.6. Determining the inhibition type
A series of experiments was performed to determine the inhibition kinetics of B6. The inhibitor
concentrations were: 0, 10, 25, 50 and 100 μM. Substrate (L-Dopa) concentrations were 0.2, 0.5,
0
.8, 1.0 and 1.4 mM in all kinetic studies. Pre-incubation and measurement time were the same
as discussed in mushroom tyrosinase inhibition assay protocol. The tyrosinase inhibition type
was then calculated using Lineweaver-Burk plots of inverse of velocities (1/V) versus inverse of
substrate concentrations 1/[S] mM. The Michaelis constant (Km) and maximal velocity (Vmax)
were also calculated by Lineweaver-Burk plots.
2
2
.7 Computational Details
.7.1 In silico physicochemical parameters and ADME profiling calculations
The molecular properties and Lipinski rule of five for the active compounds were calculated
using DruLiTo 1 software. Absorption, Distribution, Metabolism and Excretion (ADME)
8

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properties of the compounds were determined using the preADMET online server
(http://preadmet.bmdrc.org/).
2
.7.2 Molecular docking study
Docking was performed by AutoDock 4.2 and AutoDock Tools 1.5.4 (ADT). X-ray crystal
structure of Agaricus bisporus tyrosinase containing tropolone as the innate ligand in the binding
site (PDB ID: 2Y9X) was taken from protein data bank (http://www.rcsb.org). Before docking,
tropolone and water molecules were excluded from 2Y9X, then hydrogens were added and non-
polar hydrogens were merged. Finally, Gasteiger charges were calculated for protein 2Y9X. 3D
structures of ligands were sketched and minimized under Molecular Mechanics MMþ and then
semi-empirical AM1 methods using HyperChem software. PDBQT formats of the ligands were
prepared by adding Gasteiger charges and setting the degree of torsions. The grid maps were
constructed by AutoGride and grid box dimensions were set to 40×40×40 with 0.375 Å grid
spacing. The active site which comprises of copper metal ions was selected for docking and the
grids’ centers were placed on the tropolone's binding site. In order to determine the docking
parameter file, rigid macromolecule was chosen. Lamarckian genetic search algorithm was
applied and the number of GA runs was set at 100. The other parameters were left at program
default values. Validity of the docking procedure was tested using co-crystallized inhibitor as
ligand and the above-mentioned protocol.
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3
3
. Results and Discussions
.1 Design strategy
We designed the target compounds bearing N-(2-morpholinoethyl)cinnamamide scaffold based
on the structure of cinnamic acid as a well-studied natural tyrosinase inhibitor. In this vein, some
structural features were considered as follows:
-The amide moiety was chosen considering the fact that amide bond is an important functional
group in medicinal chemistry; because it is structurally stable and can act as hydrogen bond
donor and/or acceptor in the binding site of receptors. Moreover, several cinnamamide
derivatives have been reported to be potential tyrosinase inhibitors [21, 26, 27].
-With the aim of finding new and effective tyrosinase inhibitors, we brought the cinnamide
fragment with a morpholinoethyl moiety, assuming morpholine ring may provide some
additional hydrogen bonding and hydrophobic interactions with the enzyme.
-
In order to define a good structure–activity relationship (SAR), different substitutions were
inserted on different positions of the phenyl ring.
3
.2 Synthesis
The 19 novel N-(2-morpholinoethyl)cinnamamide derivatives were prepared according to the
procedure outlined in Scheme 1. Our strategy for cinnamic acid derivatization was to synthesize
stable morpholine containing cinnamamides with various substituents on the phenyl ring, which
could be easily prepared from Knoevenagel Condensation of various substituted benzaldehydes
with malonic acid followed by saponification and amine coupling reaction with 4-(2-
1
0

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1
13
Aminoethyl) morpholine. All the structures were confirmed by IR, H NMR, C NMR and
elemental analysis.
Scheme 1. The synthetic route to novel morpholine containing cinnamamide derivatives. Reagents and
conditions: (a) pyridine, piperidine and ethanol, reflux, 24 h. (b) HOBT, EDC, dry acetonitrile, stirring,
rt., 48 h.
3
3
.3 Biological evaluation
.3.1 Tyrosinase inhibitory activity
All the synthesized derivatives were screened for their inhibitory activity on mushroom
tyrosinase enzyme by evaluation of their potential to prevent enzymatic oxidation of L-Dopa.
Kojic acid was used as the positive control. The obtained results are presented in terms of IC s
5
0
in Table 1.
The most potent compound, B6, considerably inhibited tyrosinase with an IC value of
5
0
1
5.2 µM, which was comparable to that of kojic acid (IC = 14.4 µM). Compounds B1, B4, B7,
50
1
1

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B13, B15, B16, B17 and B19 exhibited inhibitory activity against mushroom tyrosinase (IC s =
5
0
2
1.3-75.8 µM). However, compounds B2, B3, B5, B8, B9, B10, B11, B12, B14, and B18 did not
inhibit the tyrosinase indicating that the inhibitory effect is dependent on the nature and position
of the phenyl ring substitutions. The most potent compound, B6, contained 3-chloro group on the
phenyl ring. Changing the position of chlorine atom to para (compound B7 with IC value of
5
0
2
8.6 µM) and ortho (compound B5) positions reduced and deleted the anti-tyrosinase activity,
respectively. In addition, it was found that compounds B8-B11 having methoxy group on
different positions of phenyl ring were completely inactive, however, increasing the number of
methoxy functions, as in B19 (IC = 50.0 μM) bearing three methoxy substituents at para and
5
0
meta positions, improved the activity. Introducing a 3-methyl group at the phenyl ring caused
B13 (IC = 21.3 μM) to be the second potent derivative, however, substituting the methyl
5
0
function to 2 and 4 positions led compounds B11 and B12 to be completely inactive. Introducing
a nitro moiety on any position of phenyl ring led to active analogues (B15-B17) with IC s in the
5
0
range of 25.2-36.0 μM, however, 4-nitro substitution was more favored.
It can be stated that N-(2-morpholinoethyl)cinnamamide derivative B1 (IC =75.8 μM) is
50
a considerably better tyrosinase inhibitor than cinnamic acid (0.2-2.1 mM) [15-18].
Consequently, the existence of an amide moiety as well as morpholine ring would improve the
anti-tyrosinase activity. The anti-tyrosinase activity of B6 is nearly equal to that of kojic acid and
we hope that the cinnamamid derivative would overcome some limitations of kojic acid such as
formulation stability and skin penetration difficulties [33].
1
2

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Table 1. Tyrosinase inhibitory effects of synthesized cinnamamide derivatives (6a-n) and kojic acid.
a
a
Compounds
R
H
IC (µM)
Compounds
B11
R
IC (µM)
50
50
B1
B2
B3
B4
B5
B6
B7
B8
B9
75.8±2.3
>100
3,4-OCH
3
>100
>100
2-F
B12
2-CH
3-CH
4-CH
3
3
3
3-F
>100
B13
21.3±2.0
>100
4-F
40.6±3.1
>100
B14
2-Cl
3-Cl
4-Cl
2-OCH
3-OCH
4-OCH
B15
2-NO
3-NO
4-NO
36.0±1.4
35.5±0.8
25.2±2.5
>100
2
2
2
15.2±0.6
28.6±1.9
>100
B16
B17
4-OH, 3-OCH
3
3
3
3
B18
>100
B19
3,4,5-OCH
-
50.0±4.4
3
B10
>100
Kojic Acid
14.4 ± 1.3
a
Values represent means ± SE of 3 independent experiments.
3
.3.2 Kinetic
The kinetic data on the inhibition of L-Dopa oxidation by the most potent derivative B6 was
expressed by Lineweavere-Burk double-reciprocal plots as shown in Fig. 2. The Lineweaver-
Burk plots (plot of 1/V versus 1/[S]) for the inhibition of tyrosinase by the selected compound
1
3

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give a group of lines that intercepted in the second quadrant, suggesting that Km value
increases, while Vmax value decreases with increasing concentrations of B6. The results indicate
that compound B6 demonstrated mixed-type inhibition towards tyrosinase enzyme.
90
80
70
60
50
40
30
20
10
0
0
1
μM
0 μM
25 μM
5
1
0 μM
00 μM
-
4
-2
0
2
4
6
-
10
-1
1
/[S] (mM )
Concentration (µM)
B6-0 µM
B6-10 µM
B6-25 µM
B6-50 µM
Vmax (mM/min)
K (mM)
m
0.13
0.11
0.10
0.09
0.08
0.50
0.62
0.83
0.91
1.22
B6-100 µM
Fig. 2. Lineweaver−Burk plot of mushroom tyrosinase enzyme inhibition by different
concentrations of B6 in the presence of L-Dopa as substrate. The reciprocal tyrosinase inhibitory
activity was plotted against the reciprocal substrate concentration (double reciprocal plot, n=3). Km is the
Michaelis-Menten constant and Vmax is the maximum reaction velocity.
3
3
.4 Computational section
.4.1 Drug-likeness and ADME properties
The physicochemical and ADME properties (including; human intestinal absorption, in vitro
Caco-2 (Caucasian colon adenocarcinoma) cell permeability, in vitro MDCK (Maden Darby
1
4

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Canine Kidney) cell permeability, in vitro plasma protein binding and in vivo BBB penetration)
were predicted for the derivatives that showed tyrosinase inhibition.
The physicochemical properties of the compounds were obtained using DruLiTo 1
software to assess their compliance with the Lipinski's rule of five and the Veber rule criteria
(Table 2). The analysis of data revealed that all molecules have molecular weight (MW) in the
range of 473–519 which is within the accepted limit of 500 and the logP values lower than 5.0
revealed that the compounds are not very lipophilic. The derivatives comply with hydrogen bond
properties and have total polar surface area (TPSA) value and rotatable bond number of lower
than 140 and 10, respectively. The aforementioned data show that the studied derivatives fulfill
the Lipinski's rule of five and the Veber rule.
ADME properties were calculated using the web-based application PreADMET and the
results obtained for compounds demonstrating tyrosinase inhibition are summarized in Table 3.
All the derivatives show well intestinal absorption. Moreover, the Caco and MDCK permeability
parameters indicate that the derivatives (except B15) belong to the group of compounds which
could relatively easily penetrate biological membranes. The compounds are predicted to bind
weakly to plasma proteins. Low blood–brain barrier permeation is observed for all the inhibitors,
therefore, it can be stated that they are less likely to cause neurotoxicity. The most potent
-1
tyrosinase inhibitor, B6, predicted to have better skin permeability (log Kp= -3.51 cm h )
-1
compared to kojic acid (log Kp= -4.51 cm h ). This can be an advantage of B6 over kojic acid, as
one of the limitations of kojic acid is its poor skin penetration [33].
Therefore, morpholine containing cinnamoyl amide tyrosinase inhibitors could serve as
suitable drug-like candidates with no violation of Lipinski's rule of five and Veber rule and
1
5

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according to ADMET properties of the derivatives, they can be proposed as effective candidates
for further drug development.
Table 2. In silico physicochemical parameters of cinnamamide derivatives possessing tyrosinase
inhibitory activity.
a
b
c
d
e
f
Code
Log P
HBA
HBD
TPSA
nRB
MW
Number of Lipinski
violations
B1
B4
1.12
1.29
1.80
1.80
1.46
0.95
0.95
0.95
1.17
4
4
4
4
4
7
7
7
7
1
1
1
1
1
1
1
1
1
41.57
41.57
41.57
41.57
41.57
81.88
81.88
81.88
69.26
5
5
5
5
5
6
6
6
8
260.15
278.14
294.11
294.11
274.17
306.14
306.14
306.14
350.18
0
0
0
0
0
0
0
0
0
B6
B7
B13
B15
B16
B17
B19
a
b
c
d
e
f
Logarithm of partition coefficient between n-octanol and water (LogP)
Number of hydrogen bond acceptors (HBA)
Number of hydrogen bond donors (HBD)
Topological polar surface area (TPSA)
Number of rotatable bonds (nRB)
Molecular weight (MW)
1
6

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Table 3. In silico ADME profiling of cinnamamide derivatives possessing tyrosinase inhibitory activity
and kojic acid.
Absorption
Distribution
In vitro plasma In vivo blood–brain
protein binding barrier penetration
Human intestinal
Absorption HIA
(percentage)
95.17
In vitro caco-2 cell In vitro MDCK Cell In vitro skin
permeability
Permeability
permeability
-
1
-1
-1
Code
B1
(nm s )
(nm s )
(log Kp, cm h )
(percentage)
(C.brain/C.blood)
53.23
198.96
241.02
112.42
107.08
202.97
13.59
28.76
33.02
88.58
9.77
-3.37
36.56
0.08
B4
95.18
95.66
95.66
95.16
92.10
92.10
92.10
96.18
55.19
52.31
53.34
52.90
18.34
22.22
20.40
56.11
3.79
-3.70
-3.51
-3.49
-3.31
-3.57
-3.58
-3.58
-4.09
-4.78
40.90
53.78
52.56
45.57
19.54
38.30
45.94
23.42
20.53
0.09
0.22
0.20
0.15
0.07
0.02
0.03
0.10
0.26
B6
B7
B13
B15
B16
B17
B19
Kojic 79.39
acid
3
.4.2 Molecular docking analysis
In order to gain a perception of the interactions and binding modes of the derivatives having
inhibitory effect on tyrosinase (B1, B4, B6, B7, B13, B15, B16, B17 and B19), cinnamic acid
and kojic acid in the active site of the enzyme, molecular docking analysis was performed. The
results are listed in Table 4. The values of estimated free energies of binding proved that all the
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docked N-(2-morpholinoethyl)cinnamamide derivatives formed more stable drug-receptor
complex than cinnamic acid and the known inhibitor kojic acid with the target protein. The most
active derivative (B6) possessed the lowest estimated free energy of binding (-6.84 kcal/mol) and
estimated inhibition constant (9.73 μM) (Table 4). All the docked derivatives well
accommodated within the active site of tyrosinase enzyme by hydrogen-bonding and
hydrophobic interactions.
The possible enzyme–inhibitor interactions of the four derivatives including B6, B7, B13 and
B17 with the active site of mushroom tyrosinase are shown in Fig. 3. Amide bond played an
important role by making some tough hydrogen bonds with Glu256, His244 and His259. Phenyl
and morpholine rings participated in hydrophobic interactions with His244, Val248, His263,
Phe264, Val283 and Ala286 residues. The nature of substitutions on phenyl ring determined the
binding orientations of these compounds. It appears that the binding pose of B6 and B7,
containing 4-Cl and 3-Cl on phenyl ring, respectively, are slightly different (Fig. 3a and Fig. 3b).
Consequently, B6 established some strong hydrophobic interactions between phenyl ring and
residues including Ala286, His263 and Val283 at the distances of 4.27, 3.66 and 3.83 Å,
respectively and also between morpholine ring and Val248 and His244 at the distances of 4.42
and 5.32 Å. Moreover, 4-Cl substitution formed two additional interactions with Phe264 and
His263 at the distances of 3.77 and 4.65 Å. However, in the case of B7, 3-chlorophenyl and
morpholine moieties did not accommodate in the hydrophobic pockets as well as 4-chlorophenyl
and morpholine ring in B6. Phenyl ring established two interactions with Val283 and Phe264 at
the distances of 5.38 and 5.03 Å, respectively and morpholine participated in three hydrophobic
interactions with His263, Val283 and Ala286 amino acids at the distances of 3.75, 4.48 and 5.26
Å, respectively. Furthermore, chlorine atom just interacted with Phe264 at the distance of 4.64
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Å. The docking results for compounds B6 and B7 were in good agreement with their biological
activities. 3-Methyl and 4-nitro substituents caused B13 and B17 to possess the second best
estimated free energies of bindings (-6.78 and -6.79 kcal/mol) by establishing some additional
hydrophobic interactions with His263, Val283 and Ala286 at the distances of 3.69, 3.14 and 4.55
Å (in the case of 3-methyl) and forming two strong hydrogen bonds with Arg268 at the distances
of 1.85 and 2.01 Å (in the case of 4-nitro) (Fig. 3c and Fig. 3d). Comparing the docking results
of the derivatives containing nitro group (B15, B16 and B17 revealed that the compound with
nitro moiety on para position (B17) demonstrated lower estimated free energy of binging than
ortho and meta substituted derivatives (B15 and B16). This is in accordance with the biological
evaluation results.
Table 4. Docking results of N-(2-morpholinoethyl)cinnamamide derivatives possessing tyrosinase
inhibitory activity and kojic acid into the mushroom tyrosinase binding site.
Compound
ΔG
kcal/mol)
Ki
(μM)
Interactions
Atom of ligand
Amino
acid
Distance
(
(Å)
B1
-6.35
22.22
H-bonding
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
H-bonding
C=O
Phenyl
Phenyl
Phenyl
Morpholine
Morpholine
Morpholine
NH
His244
Val283
Ala286
His263
Val283
His263
Phe264
Glu256
His259
His263
Val283
Ala286
Ser282
Val248
His244
Glu256
Ala286
1.80
4.18
4.63
3.89
5.47
4.67
4.24
1.98
3.02
3.97
3.53
4.43
2.36
5.29
5.34
1.75
4.27
B4
B6
-6.07
35.83
H-bonding
C=O
Hydrophobic
Hydrophobic
Hydrophobic
H-bonding
Hydrophobic
Hydrophobic
H-bonding
Phenyl
Phenyl
Phenyl
F
Morpholine
Morpholine
NH
-6.84
9.73
Hydrophobic
Phenyl
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Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
H-bonding
Phenyl
Phenyl
Morpholine
Morpholine
Cl
His263
Val283
Val248
His244
Phe264
His263
Glu256
His244
Val283
Phe264
His263
Val283
Ala286
Phe264
His244
His263
Val283
Ala286
Phe264
His263
His263
Val283
Ala286
His244
Val283
His263
Val283
Ala286
Val283
His244
Arg268
Arg268
Val248
Phe264
Val283
Ala286
His263
Glu256
His244
Arg268
Arg268
Val248
Phe264
His263
Val283
Ala286
His244
Arg268
Val248
Phe264
3.66
3.83
4.42
5.32
3.77
4.65
2.06
1.75
5.38
5.03
3.75
4.48
5.26
4.64
1.80
3.91
4.14
4.79
3.96
5.42
3.69
3.14
4.55
1.74
2.03
3.71
4.17
4.52
4.63
1.87
1.83
1.89
4.95
4.77
4.15
4.19
3.51
2.91
1.80
1.85
2.01
5.11
4.85
3.81
4.38
4.82
1.82
2.62
4.94
4.76
Cl
NH
C=O
Phenyl
B7
-6.00
-6.78
39.88
12.73
H-bonding
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
H-bonding
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
H-bonding
Phenyl
Morpholine
Morpholine
Morpholine
Cl
B13
C=O
Phenyl
Phenyl
Phenyl
Morpholine
Morpholine
Methyl
Methyl
Methyl
C=O
O (morpholine)
Phenyl
Phenyl
Phenyl
Morpholine
B15
B16
-5.49
-6.69
94.54
12.48
H-bonding
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
H-bonding
C=O
NO2
NO2
Phenyl
H-bonding
H-bonding
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
H-bonding
H-bonding
H-bonding
H-bonding
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
H-bonding
H-bonding
Hydrophobic
Hydrophobic
Phenyl
Morpholine
Morpholine
Morpholine
NH
B17
-6.79
11.97
C=O
NO2
NO2
Phenyl
Phenyl
Morpholine
Morpholine
Morpholine
C=O
B19
-5.67
69.78
OCH3
Phenyl
Phenyl
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ACCEPTED MANUSCRIPT
Hydrophobic
Hydrophobic
Hydrophobic
H-bonding
Morpholine
Morpholine
Morpholine
C=O (carboxyl)
OH (carboxyl)
Phenyl
His263
Val283
Phe264
Val283
Gly281
Phe264
Val283
Met260
Asn260
4.07
5.17
4.98
2.08
1.95
5.20
5.17
2.19
2.09
3
3
Cinnamic
acid
-3.40
-3.55
2.78×10
2.40×10
H-bonding
Hydrophobic
Hydrophobic
H-bonding
Phenyl
OH
C-OH
Kojic acid
H-bonding
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a
b
c
d
Fig. 3. The binding orientation of the most active compounds including B6 (a), B7 (b), B13 (c) and B17
d) within the active site of tyrosinase (PDB: 2Y9X). Ligands are displayed as yellow sticks while the
(
core amino acid residues are displayed as gray sticks. Hydrogen bonds and hydrophobic interactions are
displayed as green and cyan dashed, respectively.
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4
. Conclusions
Novel morpholine containing cinnamoyl amides were designed based on the natural product
cinnamic acid scaffold as tyrosinase inhibitors. The target derivatives were synthesized and
evaluated for their inhibitory effect on mushroom tyrosinase. Some of the compounds exhibited
better tyrosinase inhibitory activity (in low micromolar range) than cinnamic acid. Compound
B6, containing 3-Cl on phenyl ring, was the most potent derivative against tyrosinase with IC50
value of 15.2±0.6 μM which was comparable to that of kojic acid. In addition, inhibition kinetic
analysis revealed that B6 was a mixed-type inhibitor of mushroom tyrosinase. The in silico
physicochemical and pharmacological studies further indicated that the compounds are
compliant with the Lipinski and Veber rules and the designed molecules are good drug
candidates. Molecular docking analysis of the active derivatives in the binding site of mushroom
tyrosinase revealed that B6 possessed the most favorable estimated free energy of binding.
Docking results indicated that substitutions on phenyl moiety play an important role in forming
drug-receptor interactions and binding orientation of the compounds in the active site of
mushroom tyrosinase.
Acknowledgments
The authors wish to thank the support of the Vice-Chancellor for Research, Shiraz University of
Medical Sciences (Grant Number: 97-01-05-17464) and Vice-Chancellor for Research, Tehran
University of Medical Sciences.
Conflict of interest
The authors declare no competing interests.
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Graphical Abstract
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Highlights





Novel morpholine containing cinnamoyl amides were designed and synthesized.
B6 exhibited the highest tyrosinase inhibition comparable to kojic acid.
Kinetic analysis indicated that B6 is a mixed-type tyrosinase inhibitor.
Docking analysis confirmed that B6 well accumulated in the tyrosinase active site.
B6 could be a promising lead in cosmetics, medicine and food industry.
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