Development of highly potent melanogenesis inhibitor by in vitro, in vivo and computational studies

Article abstract of DOI:10.2147/DDDT.S137550

The present work describes the synthesis of few hydroxylated amide derivatives as melanogenesis inhibitors. In vitro, in vivo and computational studies proved that compound 6d is a highly potent melanogenesis inhibitor compared to standard kojic acid. The

Full text of DOI:10.2147/DDDT.S137550

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O r i g i n a l r e s e a r c h
Development of highly potent melanogenesis
inhibitor by in vitro, in vivo and computational
studies
This article was published in the following Dove Press journal:
Drug Design, Development andTherapy
5 July 2017
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Qamar abbas1
Abstract: The present work describes the synthesis of few hydroxylated amide derivatives as
melanogenesis inhibitors. In vitro, in vivo and computational studies proved that compound 6d
is a highly potent melanogenesis inhibitor compared to standard kojic acid. The title amides 4a–e
and 6a–e were synthesized following simple reaction routes with excellent yields. Most of the
synthesized compounds exhibited good mushroom tyrosinase inhibitory activity, but compound
6d showed excellent activity (IC50 0.15 µM) compared to standard kojic acid (IC50 16.69 µM).
Lineweaver–Burk plots were used for the determination of kinetic mechanism, and it was found
that compounds 4c and 6d showed non-competitive inhibition while 6a and 6b showed mixed-
type inhibition. The kinetic mechanism further revealed that compound 6d formed irreversible
complex with the target enzyme tyrosinase. The Ki values determined for compounds 4c, 6a, 6b
and 6d are 0.188, 0.84, 2.20 and 0.217 µM respectively. Results of human tyrosinase inhibitory
activity in A375 human melanoma cells showed that compound 6d exhibited 91.9% inhibi-
tory activity at a concentration of 50 µg/mL. In vivo cytotoxicity evaluation of compound 6d
in zebrafish embryos showed that it is non-toxic to zebrafish. Melanin depigmentation assay
performed in zebrafish indicated that compound 6d possessed greater potential in decreasing
melanin contents compared to kojic acid at the same concentration. Computational studies also
supported the wet lab findings as compound 6d showed a highest binding affinity with the target
protein (PDBID: 2Y9X) with a binding energy value of −7.90 kcal/mol. Molecular dynamic
simulation studies also proved that amide 6d formed the most stable complex with tyrosinase.
Based upon our in vitro, in vivo and computational studies, we propose that compound 6d is a
promising candidate for the development of safe cosmetic agent.
Zaman ashraf2
Mubashir hassan1
humaira nadeem3
Muhammad latif4
samina afzal5
sung-Yum seo1
1Department of Biology, college of
natural sciences, Kongju national
University, gongju, republic of
Korea; 2Department of chemistry,
allama iqbal Open University,
islamabad, 3riphah institute of
Pharmaceutical sciences, riphah
international University, islamabad,
Pakistan; 4center for genetics and
inherited Diseases,Taibah University,
almadinah almunawwarah, Kingdom
of saudi arabia; 5Faculty of Pharmacy,
Bahauddin Zakria University, Multan,
Pakistan
Keywords: melanogenesis, computational studies, tyrosinase inhibition, zebrafish, melanin
quantification
correspondence: sung-Yum seo
Department of Biology, college of
natural sciences, Kongju national
University, 56 gongjudehak-ro 56,
gongju, chungnam 32588, republic
of Korea
Introduction
Melanogenesis, the process of skin color formation, is controlled by a complex
molecular regulatory network embedded in the keratinocytes and melanocytes, which
results in the skin and hair pigmentation.1–3 Melanin biosynthesis in melanosomes is
regulated by a copper-containing metalloenzyme tyrosinase. Tyrosine in the presence
of tyrosinase is converted into melanin through a series of biochemical reactions.4,5
l-tyrosine and l-3,4-dihydroxyphenylalanine (l-DOPA) are the important substrates
involved in melanin biosynthesis.6–8 The presence of melanins in the surrounding
keratinocytes is the important factor that determines the color of the human skin.9,10
Ultraviolet exposure, α-melanocyte-stimulating hormone, melanocortin 1 receptor
and agouti-related protein are some of the other factors involved in melanogenesis.11,12
Tel +82 41 850 8503
Fax +82 41 850 8503
email dnalove@kongju.ac.kr
Zaman ashraf
Department of chemistry, allama iqbal
Open University, islamabad 44000,
Pakistan
Tel +92 32 1519 4461
Fax +92 51 2891471
email mzchem@yahoo.com
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abbas et al
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Melanogenesis corresponds to a possible cellular danger and Biospin, Switzerland). Chemical shifts (δ) were reported in
is confined to special melanosomes in melanocytes, which parts per million (ppm) downfield from the internal standard
synthesize pigments and transfer them to recipient cells.13 The tetramethylsilane. The purity of the compounds was checked
abnormal proliferation of melanocytes results in melanoma, by thin layer chromatography (TLC) on silica gel plate using
a type of skin cancer.14,15 Melasma and post-inflammatory n-hexane and ethyl acetate as mobile phase.
disorders are examples of hyperpigmentation disorders for
which patients pursue treatment.16
synthesis of 2-chloro-n-[2-methyl-5-
Higher quantity of tyrosinase activity can also be linked (propan-2-yl)phenyl]acetamide (2)
to neurodegenerative diseases due to o-quinones formation 2-amino-p-cymene 1 (0.01 mol) and triethylamine (0.01 mol)
by oxidation of dopamine results in neuronal damage and cell were reacted with chloroacetyl chloride (0.01mol) in
death.17–19 The agents that are clinically used for hyperpig- anhydrous dichloromethane (25 mL) at 0°C to −5°C. The
mentation disorders are usually associated with side effects. reaction mixture was stirred over a period of 1 h at constant
Therefore, there arises a need to develop some novel mel- temperature. The reaction mixture was then stirred at room
anogenesis inhibitors with decreased side effects. A number temperature for further 5 h and progress of the reaction was
of hydroxy-substituted aromatic acids and esters have been determined by TLC. The mixture was washed with 5% HCl
reported as potent melanogensis inhibitors.20–23 Takahashi and 5% sodium hydroxide solution; the organic layer was
and Miyazawa also reported the potential of hydroxylated washed with saturated aqueous NaCl, dried over anhydrous
amides and analogues to potentially inhibit melanogenesis.24 magnesium sulfate, filtered and the solvent was removed
The present work describes the synthesis of hydroxylated under reduced pressure. The crude 2-chloro-N-[2-methyl-5-
amides and their melanogenesis potency.
(propan-2-yl)phenyl]acetamide 2 was recrystallized in etha-
nol as needle-like crystals: melting point 94°C–96°C; yield
82%; Rf=0.54 (n-hexane: ethyl acetate 2:1); FTIR νmax cm−1:
3,354 (N–H), 2,987 (sp2 C–H), 2,893 (sp3 C–H), 1,656 (C=O
Materials and methods
chemicals
2-amino-p-cymene, mushroom tyrosinase, l-DOPA and ester), 1,598 (C=C aromatic), 1,140 (C–O, ester).
other required chemicals were purchased from Sigma-
synthesis of amide derivatives (4a–e)
Aldrich, St Louis, MO, USA. Melting points were deter-
mined using a Digimelt MPA 160 melting point apparatus and (6a–e)
and are reported uncorrected. FTIR spectra were recorded The hydroxy-substituted benzoic acids 3a–e (0.01 mol),
using Shimadzu FTIR–8400S spectrometer (Kyoto, Japan; triethyl amine (0.01 mol), potassium iodide (0.01 mol) in
υ, cm−1). 1H NMR and 13C NMR spectra (DMSO-d6) were dimethyl formamide (25 mL) and intermediate 2 (0.01 mol)
recorded using a Bruker 400 MHz spectrometer (Brüker were stirred overnight at room temperature (Scheme 1).
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Abbreviation: DMF, dimethylformamide.
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Development of highly potent melanogenesis inhibitor
After the completion of reaction, the mixture was poured 1,732 (C=O ester), 1,645 (C=O amide), 1,593 (C=C aro-
1
into finely crushed ice by stirring and extracted with ethyl matic), 1,148 (C–O, ester); H NMR (DMSO-d6, δ ppm):
acetate (4×25 mL). The combined organic layer was washed 8.64 (s, 1H, –NH), 8.12 (dd, J=7.6, 2.4 Hz, 2H, H-2, H-6),
with 5% HCl, 5% sodium hydroxide and finally with aqueous 7.34 (d, J=2.2 Hz, 1H, H-2′), 7.19 (dd, J=7.6, 2.2 Hz, 1H,
NaCl solution. The organic layer was dried over anhydrous H-4′), 7.04 (d, J=7.6, 1H, H-6′), 6.92 (dd, J=7.6, 2.2 Hz,
magnesium sulfate, filtered and the solvent was removed 2H, H-3, H-5), 5.01 (s, 2H, –CH2), 4.93 (s, 1H, –OH), 2.72
under reduced pressure to afford the crude products 4a–e. (sept, J=6.4 Hz, 1H, H-1″), 2.22 (s, 3H, –H-3″), 1.24 (d,
The title compounds 4a–e were purified by silica gel column J=6.4 Hz, 6H, H-2″); 13C NMR (DMSO-d6, δ ppm); 164.5
chromatography (n-hexane: ethyl acetate 3:1). The com- (C=O ester), 160.2 (C=O, amide), 154.5 (C-4), 149.7 (C-1′),
pounds 6a–e were prepared by reacting intermediate 2 with 139.5 (C-2′), 136.5 (C-5′), 133.6 (C-3, C-5), 130.4 (C-1),
substituted cinnamic acids 5a–e (Scheme 2). The reaction 128.3 (C-3′), 126.4 (C-4′), 123.5 (C-6′), 119.4 (C-2, C-6),
conditions were same as described for the preparation of 61.4 (–CH2), 28.5 (C-1″), 26.8 (C-3″), 21.9 (C-2″).
compounds 4a–e.
2-[2-methyl-5-(propan-2-yl)anilino]-2-
oxoethyl 2,4-dihydroxybenzoate (4c)
Yield, 78%; melting point, 136°C–138°C; Rf=0.44 (n-
2-[2-methyl-5-(propan-2-yl)anilino]-2-
oxoethyl 3-hydroxybenzoate (4a)
Yield, 85%; melting point, 116°C–118°C; Rf=0.56 (n- hexane:ethyl acetate 2:1); FTIR νmax cm−1: 3,364 (N–H),
hexane:ethyl acetate 2:1); FTIR νmax cm−1: 3,354 (N–H), 3,166 3,163 (O–H), 2,966 (sp2 C–H), 2,895 (sp3 C–H), 1,728
(O–H), 2,987 (sp2 C–H), 2,913 (sp3 C–H), 1,735 (C=O ester), (C=O ester), 1,651 (C=O amide), 1,590 (C=C aromatic),
1
1,657 (C=O amide), 1,590 (C=C aromatic), 1,149 (C–O, 1,160 (C–O, ester); H NMR (DMSO-d6, δ ppm): 8.64
ester); 1H NMR (DMSO-d6, δ ppm): 8.63 (s, 1H, –NH), 7.76 (s, 1H, –NH), 7.64 (d, J=7.4 Hz, 1H, H-6), 7.19 (d, J=7.4
(d, J=6.8 Hz, 1H, H-6), 7.52 (s, 1H, H-2), 7.42 (dd, J=6.8, 7.6 Hz, 1H, H-5), 7.11 (d, J=2.2 Hz, 1H, H-6′), 6.89 (s, 1H, H-3),
Hz, 1H, H-5), 7.36 (d, J=7.6 Hz, 1H, H-4), 7.14 (d, J=2.4 Hz, 6.42 (d, J=7.6 Hz, 1H, H-3′), 6.31 (dd, J=7.6, 2.2 Hz, 1H,
1H, H-3′), 7.05 (d, J=7.6 Hz, 1H, H-4′), 6.96 (s, 1H, H-6′), H-4′), 5.11 (s, 2H, –CH2), 4.67 (s, 2H, –OH), 2.850 (sept,
4.98 (s, 2H, –CH2), 3.06 (sept, 1H, J=6.5 Hz, H-1″), 2.32 J=7.1 Hz, 1H, H-1″), 2.09 (s, 3H, H-3″), 1.13 (d, J=7.1 Hz,
(s, 3H, H-3″), 1.36 (s, 1H, –OH), 1.25 (d, J=6.5 Hz, 6H, 6H, H-2″); 13C NMR (DMSO-d6, δ ppm); 166.5 (C=O ester),
H-2″); 13C NMR (CDCl3, δ ppm); 165.6 (C=O ester), 162.5 162.1 (C=O amide), 160.3 (C-2), 155.6 (C-4), 148.2 (C-1′),
(C=O, amide), 152.4 (C-3), 144.7 (C-1′), 138.6 (C-2′), 136.4 138.5 (C-2′), 136.3 (C-5′), 134.6 (C-6), 129.9 (C-4′), 127.7
(C-5′), 133.6 (C-6), 131.3 (C-2), 129.6 (C-1), 127.6 (C-3′), (C-3′), 120.8 (C-6′), 112.3 (C-3), 108.3 (C-5), 105.1 (C-1),
124.5 (C-4′), 122.3 (C-6′), 120.8 (C-4), 116.4 (C-5), 60.3 59.3 (–CH2), 29.1 (C-1″), 27.5 (C-3″), 20.5 (C-2″).
(–CH2), 27.4 (C-1″), 26.5 (C-3″), 20.3 (C-2″).
2-[2-methyl-5-(propan-2-yl)anilino]-2-
oxoethyl 3,4-dihydroxybenzoate (4d)
Yield, 76%; melting point, 126°C–128°C; Rf=0.48 (n-
2-[2-methyl-5-(propan-2-yl)anilino]-2-
oxoethyl 4-hydroxybenzoate (4b)
Yield, 80%; melting point, 121°C–123°C; Rf=0.53 (n- hexane:ethyl acetate 2:1); FTIR νmax cm−1: 3,376 (N–H),
hexane:ethyl acetate 2:1); FTIR νmax cm−1: 3,332 (N–H), 3,171 (O–H), 2,973 (sp2 C–H), 2,905 (sp3 C–H), 1,730 (C=O
3,153 (O–H), 2,966 (sp2 C–H), 2,901 (sp3 C–H), ester), 1,671 (C=O amide), 1,592 (C=C aromatic), 1,156
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Scheme 2 synthesis of amide derivatives (6a–e).
Abbreviation: DMF, dimethylformamide.
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(C–O, ester); 1H NMR (DMSO-d6, δ ppm): 8.45 (s, 1H, –NH), 2-[2-methyl-5-(propan-2-yl)anilino]-2-
7.69 (d, J=2.4 Hz, 1H, H-2), 7.52 (dd, J=7.6, 2.4 Hz, 1H, H-6),
7.36 (d, J=7.4 Hz, 1H, H-5), 7.16 (d, J=1.4 Hz, 1H, H-5′), 6.89
(d, J=7.6 Hz, 1H, H-3′), 6.78 (dd, J=7.6, 1.4 Hz, 1H, H-4′),
oxoethyl (2e)-3-(2-hydroxyphenyl)prop-
2-enoate (6b)
Yield, 82%; melting point, 190°C–192°C; Rf=0.54 (n-hexane:
4.96 (s, 2H, –CH2), 4.40 (s, 2H, –OH), 2.80 (sept, J=5.8 Hz,
1H, H-1″), 2.19 (s, 3H, H-3″), 1.19 (d, J=5.8 Hz, 6H, H-2″);
13C NMR (DMSO-d6, δ ppm); 169.6 (C=O ester), 162.7 (C=O
amide), 152.3 (C-3), 146.7 (C-4), 142.5 (C-1′), 138.5 (C-2′),
134.5 (C-5′), 131.9 (C-6), 128.6 (C-4′), 123.7 (C-3′), 122.5
(C-6′), 119.6 (C-2), 116.7 (C-5), 114.8 (C-1), 60.2 (–CH2),
29.5 (C-1″), 24.5 (C-3″), 22.3 (C-2″).
ethyl acetate 2:1); FTIR νmax cm−1: 3,365 (N–H), 3,138 (O–H),
2,965 (sp2 C–H), 2,895 (sp3 C–H), 1,738 (C=O ester), 1,673
(C=O amide), 1,595 (C=C aromatic), 1,162 (C–O, ester);
1H NMR (DMSO-d6, δ ppm): 8.24 (s, 1H, –NH), 7.83
(d, J=16.0 Hz, 1H, H-2), 7.62 (d, J=7.6, Hz, 2H, H-6′),
7.58 (dd, J=7.6, 2.2 Hz, 1H, H-4′), 7.42 (dd, J=7.6, 2.4 Hz,
2H, H-5′), 7.38 (d, J=7.6 Hz, 1H, H-3′), 7.18 (d, J=7.4 Hz,
1H, H-3″), 7.09 (d, J=7.4 Hz, 1H, H-4″), 6.91 (s, 1H,
H-6″), 6.56 (d, J=16.0 Hz, 1H, H-1), 5.02 (s, 2H, –CH2),
3.13 (sept, J=7.1 Hz, 1H, H-1′″), 2.31 (s, 3H, H-3′″), 1.21
(d, J=7.1 Hz, 6H, H-2′″); 13C NMR (DMSO-d6, δ ppm);
165.9 (C=O ester), 162.2 (C=O, amide), 156.8 (C-1″), 147.2
(C-2), 141.3 (C-2″), 138.9 (C-5″), 129.4 (C-3′, C-5′), 127.3
(C-4′), 125.2 (C-1′), 121.5 (C-3″), 119.4 (C-4″), 114.3 (C-2′,
C-6′), 111.1 (C-6″), 62.4 (–CH2), 28.4 (C-1′″), 25.4 (C-3′″),
21.8 (C-2′″).
2-[2-methyl-5-(propan-2-yl)anilino]-2-
oxoethyl 3,5-dihydroxybenzoate (4e)
Yield, 84%; melting point, 143°C–145°C; Rf=0.45 (n-
hexane:ethyl acetate 2:1); FTIR νmax cm−1: 3,385 (N–H),
3,154 (O–H), 2,954 (sp2 C–H), 2,893 (sp3 C–H), 1,731
(C=O ester), 1,643 (C=O amide), 1,590 (C=C aromatic),
1
1,160 (C–O, ester); H NMR (DMSO-d6, δ ppm): 8.49
(s, 1H, –NH), 7.33 (d, J=7.4 Hz, 1H, H-3′), 7.19 (d, J=7.4 Hz,
1H, H-4′), 6.98 (d, J=1.8 Hz, 2H, H-3, H-5), 6.87 (d, J=1.8
Hz, 1H, H-4), 6.67 (s, 1H, H-6′), 5.02 (s, 2H, –CH2), 4.62
(s, 2H, –OH), 2.83 (sept, J=6.2 Hz, 1H, H-1″), 2.20 (s, 3H,
H-3″), 1.12 (d, J=6.2 Hz, 6H, H-2″); 13C NMR (DMSO-d6,
δ ppm); 169.1 (C=O ester), 161.2 (C=O amide), 158.3 (C-3,
C-5), 155.3 (C-1′), 149.0 (C-2′), 139.1 (C-5′), 133.9 (C-2,
C-6), 130.1 (C-4′), 126.3 (C-3′), 123.1 (C-6′), 109.3
(C-4), 108.4 (C-1), 62.7 (–CH2), 25.5 (C-1″), 22.6 (C-3″),
19.6 (C-2″).
2-[2-methyl-5-(propan-2-yl)anilino]-2-
oxoethyl (2e)-3-(4-hydroxyphenyl)prop-
2-enoate (6c)
Yield, 84%; melting point, 206°C–208°C; Rf=0.52 (n-
hexane:ethyl acetate 2:1); FTIR νmax cm−1: 3,363 (N–H), 3,193
(O–H), 2,975 (sp2 C–H), 2,898 (sp3 C–H), 1,725 (C=O
ester), 1,652 (C=O amide), 1,596 (C=C aromatic), 1,166
(C–O, ester); 1H NMR (DMSO-d6, δ ppm): 8.78 (s, 1H, –NH),
7.62 (d, J=16.0 Hz, 1H, H-2), 7.53 (dd, J=7.4, 2.2 Hz, 2H,
H-2′, H-6′), 7.39 (dd, J=7.4, 2.2 Hz, 2H, H-3′, 5′), 7.19
(d, J=7.6 Hz, 1H, H-3″), 7.08 (d, J=7.6 Hz, 1H, H-4″),
6.98 (s, 1H, H-6″), 6.68 (d, J=16.0 Hz, 1H, H-1), 4.99
(s, 2H, –CH2), 2.98 (sept, J=6.2 Hz, 1H, H-1′″), 2.31 (s, 3H,
H-3′″), 1.21 (d, J=6.2 Hz, 6H, H-2′″); 13C NMR (DMSO-d6,
δ ppm); 166.3 (C=O ester), 161.3 (C=O, amide), 156.5
(C-1″), 142.8 (C-2), 139.5 (C-2″), 136.4 (C-5″), 132.5 (C-3′,
C-5′), 130.4 (C-4′), 127.3 (C-1′), 121.6 (C-3″), 119.4 (C-4″),
117.5 (C-2′, C-6′), 112.7 (C-6″), 62.4 (–CH2), 28.4 (C-1′″),
25.5 (C-3′″), 21.4 (C-2′″).
2-[2-methyl-5-(propan-2-yl)anilino]-2-
oxoethyl (2e)-3-phenylprop-2-enoate (6a)
Yield, 86%; melting point, 138°C–140°C; Rf=0.62 (n-
hexane:ethyl acetate 2:1); FTIR νmax cm−1: 3,381 (N–H),
2,951 (sp2 C–H), 2,898 (sp3 C–H), 1,729 (C=O ester),
1,665 (C=O amide), 1,597 (C=C aromatic), 1,148 (C–O,
ester); 1H NMR (DMSO-d6, δ ppm): 8.21 (s, 1H, –NH), 7.62
(d, J=16.0 Hz, 1H, H-2), 7.48 (dd, J=7.2, 2.4 Hz, 2H, H-2′,
H-6′), 7.35–7.39 (m, 3H, H-3′, H-4′, H-5′), 7.15 (d, J=7.2 Hz,
1H, H-3″), 7.01 (d, J=7.2 Hz, 1H, H-4″), 6.91 (s, 1H,
H-6″), 6.61 (d, J=16.0 Hz, 1H, H-1), 4.97 (s, 2H, –CH2),
2.81 (sept, J=6.8 Hz, 1H, H-1′″), 2.28 (s, 3H, H-3′″), 1.21
(d, J=6.8 Hz, 6H, H-2′″); 13C NMR (DMSO-d6, δ ppm);
166.8 (C=O ester), 160.5 (C=O, amide), 151.3 (C-1″), 140.9
2-[2-methyl-5-(propan-2-yl)anilino]-2-
oxoethyl (2e)-3-(2,4-hydroxyphenyl)prop-
(C-2), 137.2 (C-2″), 135.3 (C-5″), 130.3 (C-2′, C-6′), 128.4 2-enoate (6d)
(C-3′, C-5′), 124.8 (C-4′), 123.5 (C-1′), 121.2 (C-3″), 119.2 Yield, 76%; melting point, 224°C–226°C; Rf=0.44 (n-
(C-4″), 115.2 (C-6″), 60.5 (–CH2), 27.8 (C-1′″), 24.6 (C-3′″), hexane:ethyl acetate 2:1); FTIR νmax cm−1: 3,387 (N–H), 3,158
19.5 (C-2′″).
(O–H), 2,958 (sp2 C–H), 2,890 (sp3 C–H), 1,735 (C=O ester),
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Development of highly potent melanogenesis inhibitor
1,648 (C=O amide), 1,598 (C=C aromatic), 1,168 (C–O, percentage of concentration necessary to achieve 50% inhibi-
ester); 1H NMR (DMSO-d6, δ ppm): 8.89 (s, 1H, –NH), 7.78 tion (IC50). Each concentration was analyzed in three inde-
(d, J=16.0 Hz, 1H, H-2), 7.49 (d, J=7.2 Hz, 1H, H-6′), 7.43 pendent experiments. IC50 values were determined by data
(d, J=7.2 Hz, 1H, H-5′), 7.39 (s, 1H, H-3′), 7.19 (d, J=7.2 Hz, analysis and by a graphing software, Origin 8.6, 64-bit.
1H, H-3″), 7.02 (d, J=6.6 Hz, 1H, H-4″), 6.94 (s, 1H, H-6″),
Percent inhibition of tyrosinase was calculated using the
6.62 (d, J=16.0 Hz, 1H, H-1), 5.01 (s, 2H, –CH2), 3.21 (sept, following equation:
J=6.8 Hz, 1H, H-1′″), 2.35 (s, 3H, H-3′″), 1.31 (d, J=7.2 Hz,
6H, H-2′″); 13C NMR (DMSO-d6, δ ppm); 166.3 (C=O ester),
B−S
B


Inhibition(%) =
× 100




160.2 (C=O, amide), 157.6 (C-1″), 147.9 (C-2), 146.2 (C-2″),
137.5 (C-5″), 130.5 (C-3′), 127.1 (C-5′), 126.4 (C-4′), 127.2
(C-1′), 125.5 (C-3″), 123.2 (C-4″), 121.6 (C-2′), 118.5
(C-6′), 112.7 (C-6″), 62.4 (–CH2), 27.3 (C-1′″), 26.3 (C-3′″),
21.3 (C-2′″).
Here, B and S are the absorbance of the blank and
samples.
elastase inhibition assay
2-[2-methyl-5-(propan-2-yl)anilino]-2-
Elastase (elastase from porcine pancreas) inhibition assay
was performed based on the methods reported by Kraunsoe
et al26 and Kim et al27 with a few modifications. In order to
perform elastase inhibition assay, the amount of released
p-nitroaniline, which was hydrolyzed from the substrate
(N-succinyl-Ala-Ala-Ala-p-nitroanilide) by elastase, was
determined by measuring the absorbance at 410 nm. In
detail, 0.8 mM solution of N-succinyl-Ala-Ala-Ala-p-
nitroanilide was prepared in a 0.2 M Tris–HCl buffer (pH
8.0) and this buffer (130 µL) was added to the test sample
(10 µL) in a 96-well microplate. The microplate was pre-
incubated for 10 min at 25°C before elastase (0.0375 unit/
mL) stock solution (10 µL) was added. After the addition of
the enzyme, the microplate was kept at 25°C for 30 min and
the absorbance was measured at 410 nm using microplate
reader. All experiments were carried out in triplicate and
elastase inhibitory activity was calculated according to the
following formula:
oxoethyl (2e)-3-(4-chlorophenyl)prop-2-
enoate (6e)
Yield, 86%; melting point, 139°C–141°C; Rf=0.62 (n-
hexane:ethyl acetate 2:1); FTIR νmax cm−1: 3,365 (N–H), 2,967
(sp2 C–H), 2,898 (sp3 C–H), 1,735 (C=O ester), 1,656 (C=O
amide), 1,598 (C=C aromatic), 1,148 (C–O, ester); 1H NMR
(DMSO-d6, δ ppm): 8.24 (s, 1H, –NH), 7.82 (d, J=16.0 Hz,
1H, H-2), 7.51 (d, J=7.6 Hz, 2H, H-2′, 6′), 7.36 (d, J=7.6 Hz,
1H, H-3″), 7.16 (d, J=7.6 Hz, 1H, H-4″), 6.98 (s, 1H, H-6″),
6.77 (d, J=7.6 Hz, 2H, H-3′, H-5′), 6.48 (d, J=16.0 Hz, 1H,
H-1), 5.01 (s, 2H, –CH2), 3.11 (sept, J=5.8 Hz, 1H, H-1′″),
2.48 (s, 3H, H-3′″), 1.19 (d, J=5.8 Hz, 6H, H-2′″); 13C NMR
(CDCl3, δ ppm); 167.6 (C=O ester), 161.3 (C=O, amide),
153.4 (C-1″), 148.8 (C-2), 137.6 (C-2″), 134.6 (C-5″), 131.8
(C-2′, C-6′), 129.5 (C-3′, C-5′), 127.9 (C-4′), 124.5 (C-1′),
122.5 (C-3″), 120.4 (C-4″), 118.3 (C-6″), 62.9 (–CH2), 29.4
(C-1′″), 25.6 (C-3′″), 22.3 (C-2′″).
Mushroom tyrosinase inhibition assay
ODcontrol − ODsample
Elastaseinhibitionactivity(%) =
× 100
Mushroom tyrosinase (Sigma-Aldrich, St Louis, MO, USA)
inhibition was performed following our previously reported
methods.25 In detail, 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 microplate. After preincubation for 10 min at room
temperature, 20 µL of l-DOPA (3,4-dihydroxyphenylalanine,
ODcontrol
where ODcontrol is the optical density of the control and ODsample
is the optical density of the sample. Oleanolic acid was used
as the standard inhibitor for elastase.
Sigma-Aldrich) (0.85 mM) was added and the assay plate Free radical scavenging assay
was further incubated at 25°C for 20 min. Afterwards, the Free radical scavenging activity of amide derivatives was
absorbance of dopachrome was measured at 475 nm using determined based on previously reported methods with few
a microplate reader (Optimax Max Tunable, Sunnyvale, modifications.28,29 The assay solution consisted of 100 µL of
CA, USA). Kojic acid was used as a reference inhibitor and DPPH (150 µM) and 20 µL of increasing concentration of test
phosphate buffer was used as a negative control. The extent compounds, and the volume was adjusted to 200 µL in each
of inhibition by the test compounds was expressed as the well with DMSO. The reaction mixture was then incubated
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for 30 min at room temperature. Ascorbic acid (vitamin C) The supernatant containing tyrosinase was used for the
was used as a reference inhibitor. The assay measurements measurement of the inhibitory effects.
were carried out using a microplate reader (OPTIMax, Tunable)
at 517 nm. The reaction rates were compared and the percent
inhibition caused by the presence of tested inhibitors was
calculated. Each concentration was analyzed in three inde-
pendent experiments run in triplicate.
Tyrosinase inhibition assay
The tyrosinase inhibitory activity of the synthesized amides
was determined following the previously reported method
with few modifications.31,32 The assay reaction mixture
(200 µL) contained 3.3 mM l-DOPA in 0.33 M phosphate
buffer (pH 7.0) and the enzyme in the presence and absence
of inhibitors. Fifteen or 20 units of tyrosinase was used to
determine the % inhibition. The reaction mixture was incu-
bated at 37°C for 10 min and the absorbance was recorded
at 475 nm using a microplate reader (OPTI Max, Tunable).
One unit of enzyme was defined as the amount of enzyme
that increases the absorbance value by 0.001 at 475 nm per
minute under the same conditions as described earlier.
Kinetic analysis of the inhibition
of tyrosinase
A series of experiments was performed to calculate the
inhibitory kinetics of compounds 4c, 6a, 6b and 6d fol-
lowing the previously reported method.30 The compounds’
concentrations were as follows: 0, 3.125, 6.25 and 12.5 µM
for 4c; 0, 0.3915 and 0.783 µM for 6a; 0, 0.453, 0.965, 1.813
and 3.626 µM for 6b; and 0, 0.08, 0.16 and 0.32 µM for 6d.
Substrate l-DOPA concentration was between 0.0625 and
2 mM in all kinetic studies. Preincubation and measurement
time was the same as discussed in mushroom tyrosinase
inhibition assay protocol. Maximal initial velocity was
determined from initial linear portion of absorbance up to
5 min after addition of enzyme at a 30 s interval. The type
of inhibition was determined by using Lineweaver–Burk
plots. The enzyme inhibitor (EI) dissociation constant Ki
was determined by secondary plot of 1/V versus inhibitor
concentrations, while enzyme–substrate–inhibitor (ESI)-
dissociation constant Ki′ was determined by intercept versus
inhibitor concentrations. The reversible kinetics of the EI
complex was also determined for different concentrations
of compound 6d versus the enzyme concentration (4, 6, 8,
10, 15 and 20 µg/mL).
In vivo depigmentation assay in zebrafish
In vivo depigmentation assay was performed in zebrafish
according to the previously described method.33
Zebrafish husbandry
Adult wild-type zebrafish (Danio rerio) were purchased
from a commercial dealer and were acclimated for 1 month
in standard laboratory conditions using a photoperiod of
14 h light and 10 h dark at a temperature of 28.5°C. Fish
were fed two times daily with dry food and live brine shrimp
larvae. They were kept in thermostatic tanks under constant
chemical, biological and mechanical water filtration and
aeration. Embryos were obtained from natural spawning that
was induced at the morning by turning on the light. Collection
of embryos was completed within 30 min. All procedures
were performed as described in the “Principles of Laboratory
Animal Care” (NIH publication no 85-23, revised 1985), and
the study was approved by the Institutional Review Board
of Kongju National University (IRB No 2011-2).
human tyrosinase inhibition assay
cell culture and preparation of tyrosinase
A375 human melanoma cells were obtained from American
Type Culture Collection (ATCC, Rockville, MD, USA).
A375 cells were grown in Dulbecco’s Modified Eagle’s
Medium (Invitrogen, Burlington, ON, Canada) supplemented
compound treatment and phenotype-
with l-glutamine, 10% (v/v) fetal bovine serum (Invitrogen), based evaluation
50 µg/mL streptomycin (Sigma-Aldrich), 50 units/mL The collected synchronized embryos were arrayed by
penicillin (Sigma-Aldrich) and supplemented with 200 µM pipette into a 96-well plate: two to three embryos per well
of l-tyrosine for tyrosinase induction. Cell cultures were with 200 µL of E3 medium (NaCl 5 mM, KCl 0.17 mM,
incubated at 37°C, in a humidified atmosphere of 5% CO2. CaCl2 0.33 mM and MgSO4 0.33 mM). The most potent
Cells were scraped out from the tissue culture plate with compound 6d solution (in 0.1% DMSO) was added to the
phosphate-buffered saline (PBS) and were homogenized at E3 medium from 9 to 72 hpf (hours post-fertilization, a total
4°C in PBS. The homogenate was centrifuged at 1,000× g exposure time of 63 h). The positive control was treated with
for 10 min. The precipitate was sonicated in PBS on ice kojic acid. Dechorionated embryos were anesthetized in
and the mixture was centrifuged at 10,000× g for 30 min. tricaine methanesulfonate MS-222 solution (Sigma-Aldrich),
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Development of highly potent melanogenesis inhibitor
mounted in 1% methyl cellulose on a depression slide and from Protein Data Bank (PDB) (http://www.rcsb.org).
photographs were taken using stereomicroscope (SMZ745T; The energy minimization of target protein was carried out
Nikon, Shinagawa, Japan) for observation. Pixel measure- by employing conjugate gradient algorithm and Amber force
ments were carried out using ImageJ software package field in UCSF Chimera 1.10.1.37 Stereochemical proper-
(National Institutes of Health, Bethesda, MD, USA).
ties and Ramachandran graph and values38 of mushroom
tyrosinase were assessed by Molprobity server,39 while the
hydrophobicity graph was generated by Discovery Studio 4.1
Client.40 The protein architecture and statistical percentage
values of helices, beta-sheets, coils and turns were accessed
by VADAR 1.8.41
Determination of melanin contents
in zebrafish
Melanin contents in zebrafish were determined following
the previously reported method.34,35 For melanin contents
determination, 50 µM dose was selected on the basis of
in vivo depigmentation results. The collected synchronized
embryos were arrayed by dropper into the dish: 20±2 embryos
were used in the dish and treated with compound 6d and
50 µM of reference drug kojic acid in 3 mL of E3 medium.
After 72 hpf, embryos were anesthetized in tricaine meth-
anesulfonate MS-222 solution. After anesthetizing, embryos
were washed three times with E3 medium and eyes were
removed from all the treated and non-treated embryos.
Embryos extract (pellet) was prepared by homogenization
and centrifugation, and the pellet was dissolved in 1 mL of
1N NaOH at 100°C for 10 min. The level of melanin was
measured at an absorbance of 405 nm, and the result was
compared with a standard curve of synthetic melanin. All
experiments were repeated three times.
candidate structures
The synthesized amide derivatives 4a–e and 6a–e were
drawn by ACD/ChemSketch tool. The designed ligands were
further visualized and minimized by UCSF Chimera 1.10.1.
The biochemical properties of these compounds 4a–e and
6a–e were predicted by various computational tools such
as Molinspiration (http://www.molinspiration.com/) and
Molsoft (http://www.molsoft.com/). Lipinski’s rule of five
(RO5) was justified by Molsoft and Molinspiraion tools.
Molecular docking
Molecular docking experiment was employed on all synthe-
sized ligands 4a–e and 6a–e against mushroom tyrosinase
using diverse PyRx tool.42 The preferred orientation of ligands
within the active region of target proteins was determined in
docking studies. To run the docking experiment, grid box
parametric dimension values were adjusted as X =61.0781,
Y =56.3001 and Z =63.1015, whereas the centered values
were fixed as X =−2.9272, Y =22.0164 and Z =−32.4883,
respectively. The default exhaustiveness value was set as 8
to obtain the finest binding conformational pose of protein-
ligand docked complexes. All compounds were docked
separately against the crystal structure of mushroom tyro-
sinase. The docked complexes were further evaluated on
lowest binding energy (Kcal/mol) values and hydrogen and
hydrophobic bond analysis using Discovery Studio (4.1) and
UCSF Chimera 1.10.1. 2D graphical depictions of all other
compounds were carried out by LIGPLOT.43
Zebrafish toxicity assay
Zebrafish toxicity assay was performed according to the
previously reported method.36 Prior to compounds treatment,
48 hpf embryos were enzymatically dechorionated using
pronase (protease from Streptomyces griseus, 3 mg/mL;
Sigma-Aldrich) for 15 min, then rinsed with E3 medium three
times and two to three embryos were dispensed in 96-well
plate with 200 µL of E3 medium. Embryos were treated
with 10, 20 and 50 µM doses of compounds and pates were
maintained at 28°C for further 72 h. The survival rate of
larvae was assessed at day 5, and during the experiment no
food was provided to larvae. In order to evaluate any extent
of apoptotic cellular death, after 3 days of treatment (total
5 days), embryos were stained with 1 µg/mL of acridine
orange in E3 medium for 30 min. Excess dye was removed
by washing three times with E3 medium. The embryos were
anesthetized and photographs were taken as described in
section “In vivo depigmentation zebrafish assay”.
Molecular dynamics (MD) simulations
Based on in vitro analysis, docking energies and confor-
mational pose analysis, four best complexes were selected
for MD simulation. Groningen Machine for Chemicals
Simulations (GROMACS) 4.5.4 package44 with GROMOS
53A6 force field was applied on all complexes to interpret
computational studies
retrieval of protein structure
The three dimensional (3D) crystal structure of mushroom the backbone residual flexibility.45 The receptor and ligands
tyrosinase (Agaricus bisporus) (PDBID: 2Y9X) was retrieved topology files were created using GROMOS 53A6 force
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field and online PRODRG Server,46 respectively. Moreover,
the receptor–ligand complexes were solvated and placed
in a cubic box center adjusted at a distance of 9Å. Ions
were added to neutralize the charge of the system. Energy
minimization (nsteps =50,000) was done by steepest descent
method (1,000 ps). Energy calculation was done by Particle
Mesh Ewald method,47 whereas covalent bond constraints
were calculated by linear constraint solver algorithm.48 For
final MD setup in md.mdp file, time step for integration
was adjusted as 0.002 ps, while the cut-off distance for
short range neighbor list was adjusted as 0.8 nm. The MD
run was set to 15,000 ps with nsteps =750,000 for each
protein–ligand complexes, and trajectories files analysis
was done by Xmgrace tool (http://plasma-gate.weizmann.
ac.il/Grace/).
Table 1 inhibitory effects of amide derivatives 4a–e and 6a–e on
mushroom tyrosinase and porcine pancreas elastase
Compounds
Tyrosinase activity
Elastase activity
IC50 ± SEM (µM)
IC50 ± SEM (µM)
4a
10.97±1.3
17.98±2.5
1.80±0.31
10.96±1.8
20.20±4.9
0.78±0.09
1.81±0.4
1.41±0.1
0.15±0.01
16.71±3.1
17.20±2.6
–
41.88±7.5
81.85±15.2
32.93±9.4
24.31±5.7
33.73±7.2
5.01±1.8
13.32±3.2
23.32±4.2
3.73±0.5
35.80±8.2
–
4b
4c
4d
4e
6a
6b
6c
6d
6e
Kojic acid
Oleanolic acid
1.69±0.05
Note: Values are expressed as ic50 ± seM.
Abbreviation: seM, standard error of mean.
Results and discussion
chemistry
substitution of hydroxyl groups at phenyl ring is the decisive
factor of inhibitory activity.
The title amide analogues 4a–e and 6a–e were prepared
following the previously described method49 with slight
modification shown in Schemes 1 and 2. The 2-amino-p-
cymene in the first step is converted into 2-chloro-N-[2-
methyl-5-(propan-2-yl)phenyl]acetamide (2) by treating
with chloroacetyl chloride in the presence of (C2H5)3N
and anhydrous methylene chloride. The structure of the
intermediate 2 was ascertained by the presence of amide
carbonyl (–NHC=O) stretching at 1,656 cm−1 and –NH
stretching at 3,354 cm−1 in FTIR spectra. The compounds
4a–e and 6a–e were synthesized by simple nucleophilic
replacement of chloro group from intermediate 2 by car-
boxylic group (–COOH) of substituted benzoic acids 3a–e
and cinnamic acids 5a–e, respectively. The structures of
In vitro human tyrosinase inhibition assays showed
that compound 6d exhibited 91.9% inhibitory capacity at
50 µg/mL (Table 2). This shows that the amide 6d has a
greater potential to inhibit human tyrosinase compared to
standard kojic acid (72.9% inhibitory capacity). Hence, we
propose that substitution of hydroxyl group in phenyl ring in
case of compound 6d impedes the molecule to interact well
with the active sites of enzyme. The kinetic analysis and
docking studies helped us to explore the mode of binding
and residual interactions between the enzyme and synthe-
sized inhibitors.
1
Table 2 inhibitory effects of amide derivatives 4a–e and 6a–e on
human tyrosinase (from melanoma cells) free radical scavenging
the final products were confirmed by FTIR, HNMR and
13CNMR spectral data.
Compounds
Human tyrosinase
% inhibition
Free radical scavenging
% inhibition
in vitro enzymes inhibition assay
4a
61.25±3.9
53.83±5.3
66.67±6.4
56.85±6.5
54.62±5.8
82.35±8.5
53.70±4.2
55.83±5.7
91.87±8.43
57.40±4.92
72.94±5.92
–
25.23±1.0
1.70±0.5
2.4±0.6
Mushroom tyrosinase and elastase inhibitory activities were
determined and it was found that most of the synthesized
amides showed better tyrosinase inhibition than standard
drug. The hydroxylated amides with cinnamic acid moiety
showed higher tyrosinase inhibitory activity than those with
benzoic acid residue. The IC50 value of compound 6d bearing
2,4-dihydroxy substituted cinnamic acid functionality was
0.15 µM, which explains its excellent tyrosinase inhibitory
activity, whereas the IC50 value of standard kojic acid was
17.20 µM. The same compound (6d) also displayed higher
elastase inhibitory potential compared to all other synthesized
amide derivatives (Table 1). Bioassay results showed that the
4b
4c
4d
91.78±2.0
7.12±0.8
1.39±0.5
1.15±0.4
8.51±1.0
23.51±2.0
7.50±1.0
–
4e
6a
6b
6c
6d
6e
Kojic acid
Oleanolic acid
95.60±1.0
Notes: For human tyrosinase, the concentration of all inhibitors and kojic acid was
50 µg/ml, and for free radical scavenging, the concentration of all inhibitors and
vitamin c was 100 µg/ml. Values are expressed as mean ± standard error of mean.
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Development of highly potent melanogenesis inhibitor
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Figure 2 lineweaver–Burk plots for inhibition of tyrosinase in the presence of amide 6a.
Notes: (A) concentrations of 6a were 0, 0.3915, and 0.783 µM and those of substrate l-DOPa were 0.125, 0.25, 0.5, 1 and 2 mM. (B) The plot of the slope and (C) of the
vertical intercepts versus inhibitor 6a concentrations to determine inhibition constants. The lines were drawn using linear least squares fit.
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Figure 3 lineweaver–Burk plots for inhibition of tyrosinase in the presence of amide 6b.
Notes: (A) concentrations of 6b were 0, 0.453, 0.965, 1.813 and 3.626 µM and those of substrate l-DOPa were 0.125, 0.25, 0.5, 1 and 2 mM. (B) The plot of the slope and
(C) of the vertical intercepts versus inhibitor 6b concentrations to determine inhibition constants. The lines were drawn using linear least squares fit.
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Notes: (A) concentrations of 6d were 0, 0.08, 0.16 and 0.32 µM and those of substrate l-DOPa concentrations were 0.125, 0.25, 0.5, 1 and 2 mM. (B) The plot of the
slope. The lines were drawn using linear least squares fit.
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Development of highly potent melanogenesis inhibitor
Table 3 Kinetic parameters of mushroom tyrosinase for l-DOPa activity in the presence of different concentration of compounds 4c,
6a, 6b and 6d
Code
4c
Dose (µM)
Vmax (∆A/sec)
Km (mM)
Inhibition type
Inhibition
Ki (µM)
Ki’ (µM)
5.090×10−6
2.545×10−6
1.696×10−6
1.288×10−6
5.621×10−6
4.294×10−6
3.909×10−6
5.555×10−7
4.819×10−7
3.947×10−7
3.067×10−7
2.592×10−7
1.161×10−5
9.598×10−6
6.233×10−6
4.787×10−6
0.138
0.138
0.138
0.138
0.112
0.126
0.136
0.089
0.108
0.119
0.123
0.131
0.217
0.217
0.217
0.217
non-competitive
–
0.188
–
0.0
3.125
6.25
12.5
0.0
6a
6b
Mixed inhibition
Mixed inhibition
–
–
0.84
2.2
1.4
3.8
0.3915
0.783
0.0
0.453
0.965
1.813
3.626
0.0
6d
non-competitive
irreversible
0.217
–
0.08
0.16
0.32
of compounds 6a and 6b indicated that it inhibits tyrosinase effect of 6d on tyrosinase was irreversible (Figure 5).
by two different pathways: competitively forming EI These results suggest that the compound 6d effectively
complex and non-competitively interrupting ESI complex. inhibited the enzyme by binding to its binuclear active
The EI dissociation constants Ki (Figures 2B and 4B) of site irreversibly.50
compounds 6a and 6b are shown by the secondary plots of
slope versus concentration of compounds, whereas the ESI
In vivo depigmentation assay in zebrafish
dissociation constants Ki′ are shown by the secondary plots
of intercept versus concentration of compounds (Figures 2C
it possesses similar gene sequence system to human.51
and 3C). A lower value of Ki than Ki′ pointed out stronger
Zebrafish is a very important vertebrate animal model because
Zebrafish embryos were used to determine the depigmentation
binding between enzyme and compounds 6a and 6b, which
activity of compound 6d through in vivo assay. The inhibitory
suggested preferred competitive over non-competitive man-
ners (Table 3).
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In case of compounds 4c and 6d, Lineweaver-Burk plot
gave a family of straight lines, all of which intersected at
the same point on the x-axis (Figures 1A and 4A). The
analysis showed that 1/Vmax increased to a new value
but Km remained the same as a result of increase in the
concentrations of compounds 4c and 6d. This behavior
indicates that compounds 4c and 6d inhibit tyrosinase
non-competitively to form EI complex. Secondary plot
of slope against concentration of 4c and 6d shows the EI
dissociation constant (Ki) (Figures 1B and 4B). The inhibi-
tory mechanism of mushroom tyrosinase by compounds 6d
at different concentrations (0.0, 0.08, 0.16 and 0.32 µM)
was investigated. The plots of the remaining enzyme
activity versus the concentration of enzyme (4, 6, 8, 10,
15 and 20 µg/mL) at different inhibitor concentrations for
the catalysis of l-DOPA gave a series of parallel straight
lines with the same slopes indicating that the inhibitory
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Figure 5 effect of various doses of mushroom tyrosinase on its activity for the
catalysis of l-DOPa against different concentration of inhibitor 6d.
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Figure 6 effect of inhibitor 6d on pigmentation of zebrafish.
Notes: embryos were treated with 5, 10, 20 and 50 µM of 6d and positive control kojic acid. (A) Representation of the pigmentation levels of zebrafish treated with inhibitor
6d and kojic acid. (B) Pixels comparison of the depigmenting potency of 6d and kojic acid. *P,0.05; **P,0.01.
effects of 6d on the pigmentation of zebrafish were evaluated zebrafish normally without any evidence of apoptotic defor-
by adding 6d at concentrations of 5, 10, 20 and 50 µM to the mities. This absence of acute toxicity was encouraging for
embryos, and same concentrations were used for positive safer drug development. Importantly, no death was recorded
control kojic acid. Compound 6d significantly decreased in any treatment.
(P,0.05, Figure 6) the pigmentation level to about 57.3%,
while positive control kojic acid decreased the pigmentation
level to 24.2% at 50 µM. Moreover, inhibitor 6d showed bet-
ter depigmenting effects than kojic acid at 10 and 20 µM.
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Determination of melanin contents
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Melanin contents were measured using the extract of
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significantly (P,0.001) decreased in the 6d-treated zebrafish
embryos at 50 µM compared to non-treated embryos and
reference drug. Kojic acid only slightly reduced the melanin
contents in zebrafish embryos, whereas 6d highly reduced
the melanin contents (Figure 7).
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Zebrafish toxicity assay
In addition to the depigmenting capacity, other factors are
also important in selecting the lead compound for skin
whitening and related disorders. For that purpose, additional
in vivo zebrafish toxicity was also performed. In recent years,
zebrafish has emerged as an important tool for early screening
of toxicity of drugs. Acute toxicity is determined by treating
the larvae after 48 hpf with 10, 20 and 50 µM of inhibitor 6d,
and fish were examined after 3 days of treatment by staining
with acridine orange (Figure 8). The larvae developed into
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Figure 7 inhibitory effects of 6d and kojic acid on melanin contents.
Notes: Zebrafish embryos were treated with 50 µM of 6d and kojic acid. after
homogenation and centrifugation, pellets were prepared and dissolved with 1 n
naOh at 100°c, and absorbances were recodred at 405 nm and compared with
synthetic melanin. Values are expressed as % of control. *P,0.05; ***P,0.001.
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Development of highly potent melanogenesis inhibitor
&RQWURO
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study established a standard value for molecular weight
(160–480 g/mol) and PSA (x).52 Results showed that all
the predicted values of synthesized compounds 4a–e and
6a–e were comparable with standard values. The obtained
drug score is very important for various properties such as
physicochemical, pharmacokinetics and pharmacodynamics
of chemical compound. Higher drug scores mean good drug
characteristics.53 The drug scores obtained emphasize that
all the synthesized amides 4a–e and 6a–e exhibited drug-
like behavior. The highest and lowest drug scores of both 4c
and 6a are 1.41 and 0.35, respectively. Moreover, RO5 did
not explain anything about the specific chemistry or struc-
tural features of the drugs or non-drugs. The computational
results showed that all the synthesized compounds possessed
,10 HBA, ,5 HBD and ,5.0 Log P-values, which signifi-
cantly justified its drug-like behavior. Moreover, their molec-
ular weight was also ,500 g/mol, which also justified the
compounds’ better drug-like behavior. Rule of 5 indicates that
molecules with poor absorption are more likely to have more
than 5 HBD, MWT over 500, Log P over 5 and more than
10 HBA. However, there are plenty of examples that explain
RO5 violation among the existing drugs.54 In summary, the
values obtained justify the significance of the synthesized
compounds as good candidate molecules (Table 4).
F
G
D
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H
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Figure 8 Zebrafish embryos (48 hpf) were treated with 10, 20 and 50 µM of 6d.
Notes: normal development was observed after acridine orange staining. some
key features were labeled for reference. a, eyes; b, otic capsule; c, heart; d, yolk;
e, melanocytes.
Abbreviation: hpf, hours post-fertilization.
Molecular docking analyses
computational studies
The docked complexes of all amide derivatives 4a–e and 6a–e
were analyzed on the basis of lowest binding energy values
(kcal/mol) and hydrogen/hydrophobic bonding analyses.
Results showed that 6d was the most active compound
with best binding energy value (−7.90 kcal/mol) compared
to other derivatives. The docking pose was selected on the
basis of energy value and interaction patterns of ligand within
the active region of the target protein. The conformational
position within the active region of the target protein was
structural assessment of target protein
Mushroom tyrosinase is a class of oxidoreductase copper-
containing protein that comprises 391 amino acids. The
structure architecture of mushroom tyrosinase showed that
it consists of 39% α-helices (154 residues), 14% β-sheets
(57 residues) and 46% coils (180 residues). The crystal
structure showed its resolution 2.78Å, R-value 0.238 and
unit cell crystal dimensions such as length and angles of
coordinates. The unit cell length values were observed
for a =103.84, b =104.82 and c =119.36 with angles 90°,
110.45° and 90° for all α, β and γ dimensions, respectively.
The Ramachandran plots and values indicated that 95.9% of
protein residues were present in favored region and 100.0%
residues were lie in allowed region. The Ramachandran graph
values showed a good accuracy of phi (φ) and psi (ψ) angles
among the coordinates of receptor, and most of residues were
plunged in acceptable region.
Table 4 chemo-informatics evaluation of the synthesized
compounds
Properties 4a 4b 4c 4d 4e 6a 6b 6c 6d 6e
MW
hBa
hBD
log P
Psa
327 327 343 343 343 337 353 353 369 371
4
2
4
2
5
3
5
3
5
3
3
1
4
2
4
2
5
3
3
1
2.8 3.8 3.4 3.4 3.5 4.8 4.4 4.5 4.1 4.5
61 61 77 76 78 42 59 60 77 42
328 328 339 341 339 361 372 372 382 378
MV
chemo-informatics properties and lipinski’s rO5
The predicted chemo-informatics properties such as molecu-
lar weight (g/mol), molar volume (A3) and polar surface area
(PSA) were evaluated by computational tools. The literature
Drug score 1.09 1.11 1.41 1.30 0.85 0.35 0.42 0.68 0.69 0.83
rO5 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Abbreviations: MW, molecular weight (g/mol); hBa, hydrogen bond acceptor;
hBD, hydrogen bond donor; log P, lipophilicity of partition coefficient; PSA, polar
surface area; MV, molar volume (a3); RO5, rule of five.
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abbas et al
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confirmed by superimposition. The docked complex of 6d
This comparative analysis shows that 6d directly interacts
was superimposed against the experimental crystal structure with the active residues of the target protein. The structure–
of tyrosinase. Results showed that the ligand 6d actively activity relationship analysis showed that 6d formed two
bound within the binding pocket of the target protein. The hydrogen bonds and one π–π interaction at specific residues
docking energy values of all the docking complexes were His244, Met280 and His363 of target protein. The ester
calculated by the following equation:
carbonyl oxygen in 6d interacts with His244, while benzyl
oxygen moiety interacts with Met280 and forms strong
hydrogen bonds with lengths of 1.98Å and 2.87Å, respec-
tively. Another π–π interaction between the benzene and
metal-bonded His363 residue was formed with a bond length
of 4.07Å. The literature study also justified that these inter-
acted residues are significant in the downstream signaling
pathways.55 The binding interactions of 6d with the amino
acids of the target protein are presented in Figure 10, and the
2D-generated docking complexes of all ligands are presented
in Figures S3–S11.
∆Gbinding = ∆Ggauss + ∆Grepulsion + ∆Ghbond
+ ∆Ghydrophobic + ∆Gtors
where,
∆Ggauss: attractive term for the dispersion of two Gaussian
functions,
∆Grepulsion: square of the distance if closer than a threshold
value,
∆Ghbond: ramp function – also used for interactions with
metal ions,
∆Ghydrophobic: ramp function,
MD simulations analyses
∆Gtors: proportional to the number of rotatable bonds.
Docking results justified that the synthesized amides 6b,
6d and 6e exhibited good binding energy values (−8.0, −7.9
and −7.9 kcal/mol, respectively). The standard error for
Autodock is reported as 2.5 kcal/mol. Though the basic
nucleus of all the synthesized compounds was the same, most
of the ligands possessed efficient energy values and did not
show any significant difference in energy fluctuations. The
comparative binding energies of all the docking complexes
are presented in Figure 9.
Root mean square deviation and fluctuations of
target protein
To evaluate the mushroom tyrosinase flexibility and overall
stability of docking complexes, we executed time-dependent
MD simulation at 15 ns using Gromacs 4.5.4. The residual
deviations and fluctuation of the complexes 6a and 6d were
determined by root mean square deviation and fluctuation
(RMSD/F) graphs generated by Xmgrace software. Both
docked complexes were run separately in simulation to
check the fluctuating differences of the residual backbone
of the target protein. An increasing trend was observed in
the graph lines of both complexes with an RMSD value of
0–0.2 nm at time 0–1,250 ps, after which both complexes
showed slight fluctuations and attained a little stability from
1,250 to 5,000 ps with a constant RMSD value of 0.2 nm.
From 5,000 to 75,000 ps, 6d graph line remained steadily
constant and showed no fluctuations, whereas 6a graph line
showed an increasing trend and depicted higher RMSD
value of 0.25 nm. Comparative analysis showed that 6d
had more stable behavior compared to 6a complex. These
results justified that the binding of 6d toward the target
protein is more stable compared to all other 6a deriva-
tive (Figure 11).
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The overall RMSD results justified that 6d did not change
the conformational symmetry of the target protein compared
to 6a. The RMSF results also reflected that both C- and
N-terminal lobes of mushroom tyrosinase are fluctuated
throughout the simulation period. Comparative analysis justi-
fied that 6d is more stable and less fluctuated throughout the
simulation period compared to 6a (Figure 12). MD simula-
tion graphs showed stable behaviors of 6d docked complex
ꢀD ꢀE ꢀF ꢀG ꢀH ꢅD ꢅE ꢅF ꢅG ꢅH
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Figure 9 Docking energies of the synthesized amide derivatives 4a–e and 6a–e
calculated using Pyrx.
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Development of highly potent melanogenesis inhibitor
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Figure 10 Docking interactions between 6d and target protein.
Notes: (A) The 6d docking complex with ligand is in blue color and embedded functional groups such as oxygen and amino are highlighted in red and dark blue colors,
respectively. The protein structure is represented in purple color while interior helices are depicted in brown color. (B) six copper-interacted residues lie within the
active region of target protein and are represented in purple color. (C) The binding pocket of target protein in surface format is represented in dark purple color with
conformational position of ligand. (D) closer view of docking interaction. The active binding site amino acids are highlighted in brown color. Two copper ions are also
represented in gray color. Two hydrogen bonds and one π–π interaction were observed between 6d and receptor amino acids such as his244, Met280 and his363 with
bonding distances 1.98, 2.87, and 4.07Å, respectively. interacted residues are in red color labels.
throughout MD trajectories thus increasing the efficacy of surface area and dihedral pattern (Chi) of the target protein
docking results.
are given in Figures S12 and S13.
The compactness of the target protein was measured by
radius of gyration (Rg). The results obtained for both 6a Conclusion
and 6d showed that the Rg value underwent a little fluctua- Hydroxylated amide derivatives 4a–e and 6a–e were syn-
tion between 2 and 2.03 nm throughout the simulation time thesized following simple reaction routes with excellent
0–15 ns. These results justified that the residual backbone yields as melanogenesis inhibitors. The in vitro, in vivo and
and folding of the receptor protein 6d was comparatively computational studies proved that compound 6d is a highly
stable compared with 6a. The Rg values and graph lines of potent melanogenesis inhibitor compared to standard kojic
6a and 6d are shown in Figure 13. The solvent accessible acid. Among the synthesized amides, 6d showed excellent
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abbas et al
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Figure 11 rMsD graph of 6a and 6d at 15 ns.
Note: The graph lines with purple and green colors represent 6a and 6d complexes,
respectively.
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Abbreviation: rMsD, root mean square deviation.
Figure 13 radius of gyration (rg) graphs of 6a and 6d: bound and unbound
formats.
Note: The graph lines with green and purple represent the bound and unbound
target protein, respectively.
mushroom tyrosinase inhibitory activity (IC50 0.15 µM)
compared to standard kojic acid (IC50 16.69 µM). The
enzyme inhibition kinetics determined by Lineweaver–Burk
plots showed that compounds 4c and 6d exhibited non-
competitive inhibition, while 6a and 6b exhibited mixed-type
inhibitions. The kinetic mechanism further revealed that
compound 6d formed irreversible complex with the target
enzyme tyrosinase. The Ki values of compounds 4c, 6a, 6b
and 6d were 0.188 µM, 0.84 µM, 2.20 µM and 0.217 µM,
which shows that compound 6d possessed stronger binding
capacity. The same compound 6d also showed an inhibitory
activity of 91.9% on human tyrosinase. The in vivo cyto-
toxicity evaluation of 6d in zebrafish embryos showed that
it is non-toxic to zebrafish. The melanin depigmentation
assay in zebrafish indicated that compound 6d had a greater
potential in decreasing melanin contents compared to kojic
acid at same concentration. The computational studies also
supported the wet lab findings as the compound 6d exhibited
highest binding affinity with the target protein (PDBID:
2Y9X) with a binding energy value of −7.90 kcal/mol. The
MD simulation studies also proved that amide 6d formed
the most stable complex with tyrosinase. Based upon our
in vitro, in vivo and computational studies, we propose that
compound 6d is a promising candidate for the development
of safe cosmetic agent.
Disclosure
The authors report no conflicts of interest in this work.
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