Veratric acid derivatives containing benzylidene-hydrazine moieties as promising tyrosinase inhibitors and free radical scavengers

Article abstract of DOI:10.1016/j.bmc.2019.04.016

Tyrosinase enzyme plays a crucial role in melanin biosynthesis and enzymatic browning process of vegetables and fruits. A series of veratric acid derivatives containing benzylidene-hydrazine moieties with different substitutions were synthesized and their inhibitory effect on mushroom tyrosinase and free radical scavenging activity were evaluated. The results indicated that N′-(4-chlorobenzylidene)-3,4-dimethoxybenzohydrazide (D5)and N′-(2,3-dihydroxybenzylidene)-3,4-dimethoxybenzohydrazide (D12)showed the highest tyrosinase inhibitory activity with IC₅₀ values of 19.72 ± 1.84 and 20.63 ± 0.79 μM, respectively, that were comparable with the IC₅₀ value of kojic acid (19.08 ± 1.21 μM). D12 was also a potent radical scavenger with EC₅₀ value of 0.0097 ± 0.0011 mM. The free radical scavenging activity of D12 was comparable with the standard quercetin. The inhibition kinetic analyzed by Lineweaver-Burk plots revealed that compound D5 was a competitive tyrosinase inhibitor. Molecular docking study was carried out for the derivatives demonstrating tyrosinase inhibitory activity. D5 and D12 possessed the most negative estimated free energies of binding in mushroom tyrosinase active site. Therefore, D5 and D12 could be introduced as potent tyrosinase inhibitors that might be promising leads in medicine, cosmetics and food industry.

Full text of DOI:10.1016/j.bmc.2019.04.016

Accepted Manuscript
\Veratric acid derivatives containing benzylidene-hydrazine moieties as prom-
ising tyrosinase inhibitors and free radical scavengers
Zahra Dehghani, Mahsima Khoshneviszadeh, Mehdi Khoshneviszadeh, Sara
Ranjbar
PII:
S0968-0896(19)30406-7
https://doi.org/10.1016/j.bmc.2019.04.016
BMC 14870
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 March 2019
7 April 2019
9 April 2019
Please cite this article as: Dehghani, Z., Khoshneviszadeh, M., Khoshneviszadeh, M., Ranjbar, S., \Veratric acid
derivatives containing benzylidene-hydrazine moieties as promising tyrosinase inhibitors and free radical
scavengers, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.04.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Veratric acid derivatives containing benzylidene-hydrazine moieties as promising
tyrosinase inhibitors and free radical scavengers
Zahra Dehghania,c, Mahsima Khoshneviszadehb, Mehdi Khoshneviszadeha,b*, Sara Ranjbarc**
aDepartment of Medicinal Chemistry, School of Pharmacy, Shiraz University of Medical
Sciences, Shiraz, Iran
bMedicinal and Natural Products Chemistry Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran
cPharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
Corresponding Authors:
*Mehdi Khoshneviszadeh
Department of Medicinal Chemistry, School of Pharmacy, Shiraz University of Medical
Sciences, Shiraz, Iran
Tel: +98 713 230 3872; Fax: +98 713 230 2225; E-mail: m.khoshneviszadeh@gmail.com
**Sara Ranjbar
Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
Tel: +98 713 242 4128; Fax: +98 713 230 2225; E-mail: ranjbar18@yahoo.com
ranjbar90156@gmail.com
1

Abstract
Tyrosinase enzyme plays a crucial role in melanin biosynthesis and enzymatic browning
process of vegetables and fruits. A series of veratric acid derivatives containing benzylidene-
hydrazine moieties with different substitutions were synthesized and their inhibitory effect on
mushroom tyrosinase and free radical scavenging activity were evaluated. The results indicated
that
N'-(4-chlorobenzylidene)-3,4-dimethoxybenzohydrazide
(D5)
and
N'-(2,3-
dihydroxybenzylidene)-3,4-dimethoxybenzohydrazide (D12) showed the highest tyrosinase
inhibitory activity with IC50 values of 19.72±1.84 and 20.63±0.79 μM, respectively, that were
comparable with the IC50 value of kojic acid (19.08±1.21 μM). D12 was also a potent radical
scavenger with EC50 value of 0.0097±0.0011 mM. The free radical scavenging activity of D12
was comparable with the standard quercetin. The inhibition kinetic analyzed by Lineweaver–
Burk plots revealed that compound D5 was a competitive tyrosinase inhibitor. Molecular
docking study was carried out for the derivatives demonstrating tyrosinase inhibitory activity. D5
and D12 possessed the most negative estimated free energies of binding in mushroom tyrosinase
active site. Therefore, D5 and D12 could be introduced as potent tyrosinase inhibitors that might
be promising leads in medicine, cosmetics and food industry.
Keywords: tyrosinase inhibitor, benzohydrazide, kojic acid, diphenolase activity, veratric acid.
2

1. Introduction
Tyrosinase enzyme (EC 1.14.18.1) is a dicopper oxidase which is widely distributed in
bacteria, fungi, insects, plants, and animals.1,2 Tyrosinase is involved in melanin biosynthesis by
catalyzing rate-limiting steps of overall melanogenesis including the hydroxylation of L-tyrosine
to 3,4-dihydroxy-L-phenylalanine (L-DOPA) and subsequent oxidation of L-DOPA to
dopaquinone.3,4 As, tyrosinase contributes a significant role in the brain neuromelanin
development and too much formation of dopaquinone leads to neuronal damage and cell death, it
has been proved that the enzyme is associated to neurodegenerative disorders including
Parkinson’s and Huntington’s diseases.5-7 Moreover, tyrosinase is responsible for enzymatic
browning of fruits and vegetables due to polyphenol compounds oxidation which results in
quality loss and deterioration of nutritional value of fruits and vegetables during postharvest-
handling and processing.8,9 Therefore, inhibition of tyrosinase catalytic activity has proved to be
an effective approach to treat hyperpigmentation and neurodegenerative disorders, and also to
prevent food deterioration.10,11 Consequently, tyrosinase inhibitors have broad applications in the
fields of medicine, cosmetics, agriculture and food industry. Several compounds belonging to
different classes are reported as tyrosinase inhibitors.12,13 Unfortunately, these molecules have
adverse side effects and low efficacy. Thus, it is of great importance to develop new, effective
and non-toxic tyrosinase inhibitors.
3,4-Dimethoxybenzoic acid, known as veratric acid and derived from plants and fruits,
has been reported to have some biological activities such as anti-oxidant, anti-inflammation, and
blood pressure-lowering effects.14-18 It has been reported that veratric acid and its methyl ester do
not show significant inhibitory effect on tyrosinase.19 Hydrazides, acylated derivatives of
hydrazine, are a class of organic compounds containing azomethine group (–N=CH) linked to an
3

amide group. Hydrazide derivatives are pharmaceutically important, as they have been reported
to possess a number of biological effects such as anti-inflammatory, antitumoral, antitubercular,
anticonvulsant,
antioxidant,
analgesic,
antiplatelet,
antimalarial,
antimicrobial,
antimycobacterial, vasodilator, antiviral and antiulcer activities.20-28
In this study, we designed veratric acid derivatives containing different benzylidene-
hydrazine moieties. After the synthesis, we evaluated their tyrosinase inhibitory activity as well
as their free radical scavenging effect. The most active derivative was investigated for its
inhibition kinetic. Finally, molecular docking analysis was performed to gain a perception of the
ligand-receptor interactions of the active derivatives.
2. Materials and Methods
2.1. Apparatus
Melting points were determined using a hot stage apparatus (Electrothermal, Essex, UK)
and were uncorrected. Mass spectra were recorded on an Agilent spectrometer (Agilent
technologies 9575c inert MSD, USA). NMR spectra were done on a Burker-Avance DPX-300
MHz in DMSO-d6. All spectra affirmed the structure of the synthesized compounds. Elemental
analysis was performed by Microanalytical Department, Central Laboratories for Research,
Shiraz University of Medical Sciences and it was 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 and aldehydes
were purchased from Merck. Analytical thin layer chromatography (TLC) was performed on
4

MERCK pre-coated 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. Synthesis of 3,4-dimethoxybenzohydrazide (B)
A mixture of methyl 3,4-dimethoxybenzoate (20 mmol) and hydrazine hydrate (30 mL)
were refluxed in ethanol for 3 hours. The reaction proceeding was monitored by TLC. After
completion of the reaction, the mixture was cooled to room temperature, the solvent was reduced
and n-hexane was added to the reaction mixture. The resulted precipitate was collected and
washed with cold water to give us pure 3,4-dimethoxybenzohydrazide as the intermediate (yield:
88%).
2.4. Synthesis of benzylidene-3,4-dimethoxybenzohydrazide derivatives (D1-D16)
A mixture of methyl 3,4-dimethoxybenzoate (1 mmol) and corresponding benzaldehyde
(1 mmol) was refluxed in ethanol (7 ml) for 6 hours. Reaction completion was checked by TLC.
The mixture was cooled to room temperature and the resulted precipitate was collected and
washed with diethyl ether to yield the final pure products. Moreover, in some cases,
recrystallization in appropriate solvents was done in order to obtain the pure derivatives.
2.4.1. (E)-3,4-dimethoxy-N'-(4-nitrobenzylidene)benzohydrazide (D1)
1
Yellow solid; yield: 82%; M.P: 208-210 °C. H NMR (300 MHz, DMSO-d6): δ (ppm)
3.85 (s, 6H, OCH3), 7.09 (d, 1H, Ar-H-5, J=6.5 Hz), 7.42-7.58 (m, 2H, Ar-H-2, 6), 7.96 (d, 2H,
Ar-H-2´,6´), 8.28 (d, 2H, Ar-H-3´,5´), 8.55 (s, 1H, N=CH), 12.00 (s, 1H, NH). 13C NMR (75
5

MHz, DMSO-d6): δ (ppm) 56.10 (OCH3), 56.14 (OCH3), 111.46, 121.66, 121.70, 124.52,
125.53, 128.32, 141.26, 145.06, 148.20, 148.86, 152.45, 163.24 (C=O). MS (EI, 70 eV): m/z
(%)ꢀ= 329 (M+, 10), 165 (100). Anal. Calcd for C16H15N3O5: C 58.36, H 4.59, N 12.76%, found:
C 58.44, H 4.61, N 12.72%.
2.4.2. (E)-N'-(4-(dimethylamino)benzylidene)-3,4-dimethoxybenzohydrazide (D2)
Recrystallized in ethyl acetate. Cream solid; yield: 76%; M.P: 201-203 °C. 1H NMR
(300 MHz, DMSO-d6): δ (ppm) 2.97 (s, 6H, NCH3), 3.83 (s, 6H, OCH3), 6.75 (d, 2H, Ar-H-
3´,5´, J=7.2 Hz), 7.07 (d, 1H, Ar-H-5, J=7.5 Hz), 7.49-7.55 (m, 4H, Ar-H-2, 6, 2´,6´), 8.33 (s,
13
1H, N=CH), 11.42 (s, 1H, NH). C NMR (75 MHz, DMSO-d6): δ (ppm) 40.24 (NCH3), 56.09
(OCH3), 111.33, 111.45, 112.29, 121.25, 122.22, 126.37, 128.80, 148.63, 148.81, 151.92, 152.0,
162.62 (C=O). MS (EI, 70 eV): m/z (%)ꢀ= 327 (M+, 50), 181 (20), 165 (100), 146 (75). Anal.
Calcd for C18H21N3O3: C 66.04, H 6.47, N 12.84%, found: C 66.15, H 6.46, N 12.76%.
2.4.3. (E)-N'-(4-bromobenzylidene)-3,4-dimethoxybenzohydrazide (D3)
White solid; yield: 61%; M.P: 193-194 °C. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 3.84
(s, 6H, OCH3), 7.09 (d, 1H, Ar-H-5, J=8.4 Hz), 7.49 (s, 1H, Ar-H-2), 7.58 (d, 1H, Ar-H-6, J=8.4
Hz), 7.63-7.73 (m, 4H, Ar-H-2´, 3´,5´,6´), 8.44 (s, 1H, N=CH), 11.78 (s, 1H, NH). 13C NMR (75
MHz, DMSO-d6): δ (ppm) 65.142 (OCH3), 111.46, 121.51, 123.59, 125.82, 129.30, 132.30,
134.22, 146.40, 148.85, 152.29, 163.09 (C=O). MS (EI, 70 eV): m/z (%)ꢀ= 364 ([M++2], 6), 362
(M+, 5), 181 (33), 165 (100). Anal. Calcd for C16H15BrN2O3: C 52.91, H 4.16, N 22.00%, found:
C 53.00, H 4.18, N 21.96%.
6

2.4.4. (E)-N'-(3,4-dimethoxybenzylidene)-3,4-dimethoxybenzohydrazide (D4)
White solid; yield: 79%; M.P: 198-201 °C. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 3.83
(s, 12H, OCH3), 7.02-7.09 (m, 2H, Ar-H-5, 5´), 7.19 (d, 1H, Ar-H-6´, J=7.2 Hz), 7.34 (s, 1H,
Ar-H-2´), 7.51 (s, 1H, Ar-H-6), 8.57 (d, 1H, Ar-H-2, J=8.1 Hz), 8.43 (s, 1H, N=CH), 11.66 (s,
1H, NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 55.92 (OCH3), 56.04 (OCH3), 56.12 (OCH3),
108.72, 111.39, 111.47, 112.00, 121.44, 122.20, 126.09, 127.65, 148.03, 148.80, 149.54, 151.12,
152.13, 162.94 (C=O). MS (EI, 70 eV): m/z (%)ꢀ= 344 (M+, 17), 181 (38), 165 (100). Anal.
Calcd for C18H20N2O5: C 62.78, H 5.85, N 8.13%, found: C 62.96, H 5.83, N 8.15%.
2.4.5. (E)-N'-(4-chlorobenzylidene)-3,4-dimethoxybenzohydrazide (D5)
White solid; yield: 66%; M.P: 186-189 °C. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 3.84
(s, 12H, OCH3), 7.08 (d, 1H, Ar-H-5, J=9 Hz), 7.50-7.59 (m, 4H, Ar-H-2, 6, 3´, 5´), 7.75 (d, 2H,
Ar-H-2´, 6´, J=8.1 Hz), 8.46 (s, 1H, N=CH), 11.78 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6):
δ (ppm) 56.10 (OCH3), 56.13 (OCH3), 111.46, 121.52, 125.84, 129.06, 129.39, 133.88, 134.82,
146.24, 146.32, 148.84, 152.30, 163.10 (C=O). MS (EI, 70 eV): m/z (%)ꢀ= 318 (M+, 5), 181
(20), 165 (100). Anal. Calcd for C16H15ClN2O3: C 60.29, H 4.47, N 8.79%, found: C 60.36, H
4.47, N 8.81%.
2.4.6. (E)-3,4-dimethoxy-N'-(4-methoxybenzylidene)benzohydrazide (D6)
White solid; yield: 73%; M.P: 168-171 °C. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 3.81
(s, 3H, OCH3), 3.84 (s, 6H, OCH3), 7.01-7.09 (m, 3H, Ar-H-5, 3´, 5´), 7.50-7.68 (m, 4H, Ar-H-
2, 6, 2´, 6´), 8.41 (s, 1H, N=CH), 11.59 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm)
55.75 (OCH3), 56.12 (OCH3), 111.38, 111.44, 114.80, 121.37, 126.11, 127.48, 129.05, 130.44,
7

147.64, 148.82, 152.13, 162.88 (C=O). MS (EI, 70 eV): m/z (%)ꢀ= 314 (M+, 14), 181 (40), 165
(100). Anal. Calcd for C17H18N2O4: C 64.96, H 5.77, N 8.91%, found: C 64.02, H 5.76, N 8.88%.
2.4.7. (E)-3,4-dimethoxy-N'-(2-methoxybenzylidene)benzohydrazide (D7)
1
White solid; yield: 76%; M.P: 183-185 °C. H NMR (300 MHz, DMSO-d6): δ (ppm)
3.83-3.87 (m, 9H, OCH3), 7.00-7.12 (m, 3H, Ar-H-5, 3´, 4´), 7.41 (t, 1H, Ar-H-5´, J=7.2 Hz),
7.54 (s, 1H, Ar-H-2), 7.60 (dd, 1H, Ar-H-6, J=8.4/1.8 Hz), 7.88 (d, 1H, Ar-H-6´, J=7.2 Hz),
8.83 (s, 1H, N=CH), 11.73 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 56.10 (OCH3),
111.33, 111.47, 112.29, 121.20, 121.54, 122.93, 125.89, 125.93, 131.85, 143.03, 148.80, 152.18,
158.15, 162.85 (C=O). MS (EI, 70 eV): m/z (%)ꢀ= 314 (M+, 5), 181 (54), 165 (100). Anal. Calcd
for C17H18N2O4: C 64.96, H 5.77, N 8.91%, found: C 65.15, H 5.79, N 8.94%.
2.4.8. (E)-3,4-dimethoxy-N'-(3-nitrobenzylidene)benzohydrazide (D8)
1
Cream solid; yield: 91%; M.P: 189-192 °C. H NMR (300 MHz, DMSO-d6): δ (ppm)
3.85 (s, 6H, OCH3), 7.09 (d, 1H, Ar-H-5, J=8.1 Hz), 7.51 (s, 1H, Ar-H-2), 7.59 (d, 1H, Ar-H-6,
J=7.8 Hz), 7.75 (t, 1H, Ar-H-5´, J=7.2 Hz), 8.13 (d, 1H, Ar-H-4´, J=6.0 Hz), 8.53-8.56 (m, 2H,
Ar-H-6´ and N=CH), 11.96 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 56.09 (OCH3),
56.14 (OCH3), 111.45, 121.20, 121.65, 124.55, 125.60, 130.91, 133.76, 136.80, 145.18, 148.70,
148.86, 152.40, 160.93, 163.25 (C=O). MS (EI, 70 eV): m/z (%)ꢀ= 329 (M+, 9), 181 (5), 165
(100). Anal. Calcd for C16H15N3O5: C 58.36, H 4.59, N 12.76%, found: C 58.49, H 4.60, N
12.71%.
8

2.4.9. (E)-3,4-dimethoxy-N'-(3,4,5-trimethoxybenzylidene)benzohydrazide (D9)
Recrystallized in chloroform-ethanol. White solid; yield: 89%; M.P: 170-173 °C.
1H NMR (300 MHz, DMSO-d6): δ (ppm) 3.71 (s, 3H, OCH3), 3.84 (s, 12H, OCH3), 7.02 (s, 2H,
Ar-H-2´, 6´), 7.09 (d, 1H, Ar-H-5, J=8.1 Hz), 7.49 (s, 1H, Ar-H-2), 7.57 (d, 1H, Ar-H-6, J=7.8
13
Hz), 8.41 (s, 1H, N=CH), 11.71 (s, 1H, NH). C NMR (75 MHz, DMSO-d6): δ (ppm) 56.09
(OCH3), 56.13 (OCH3), 56.40 (OCH3), 60.58 (OCH3), 104.67, 111.43, 111.48, 121.48, 126.00,
130.42, 139.63, 147.86, 148.82, 153.67, 163.06 (C=O). MS (EI, 70 eV): m/z (%)ꢀ= 374 (M+, 18),
181 (33), 193 (10), 165 (100). Anal. Calcd for C19H22N2O6: C 60.95, H 5.92, N 7.48%, found: C
61.12, H 5.91, N 7.46%.
2.4.10. (E)-N'-(4-hydroxy-3,5-dimethoxybenzylidene)-3,4-dimethoxybenzohydrazide (D10)
1
Cream solid; yield: 68%; M.P: 158-161 °C. H NMR (300 MHz, DMSO-d6): δ (ppm)
3.83 (s, 12H, OCH3), 6.98 (s, 2H, Ar-H-2´, 6´), 7.08 (d, 1H, Ar-H-5, J=8.1 Hz), 7.49 (s, 1H, Ar-
H-2), 7.57 (d, 1H, Ar-H-6, J=7.5 Hz), 8.36 (s, 1H, N=CH), 8.92 (s, 1H, OH), 11.59 (s, 1H, NH).
13C NMR (75 MHz, DMSO-d6): δ (ppm) 55.08 (OCH3), 56.13 (OCH3), 56.49 (OCH3), 105.06,
106.28, 111.48, 121.40, 125.15, 126.17, 138.36, 148.57, 148.63, 148.82, 152.12, 162.93 (C=O).
MS (EI, 70 eV): m/z (%)ꢀ= 360 (M+, 29), 344 (12), 207 (5), 181 (41), 165 (100). Anal. Calcd for
C18H20N2O6: C 59.99, H 5.59, N 7.77%, found: C 60.20, H 5.57, N 7.76%.
2.4.11. (E)-N'-(4-hydroxybenzylidene)-3,4-dimethoxybenzohydrazide (D11)
1
Cream solid; yield: 30%; M.P: 182-183 °C. H NMR (300 MHz, DMSO-d6): δ (ppm)
3.84 (s, 6H, OCH3), 6.83 (d, 1H, Ar-H-5), 7.09 (d, 2H, Ar-H-3´, 5´), 7.22 (d, 2H, Ar-H-2´, 6´),
7.50 (s, 1H, Ar-H-2), 7.57 (d, 1H, Ar-H-6, J=7.8 Hz), 8.38 (s, 1H, N=CH), 9.65 (s, 1H, OH),
13
11.66 (s, 1H, NH). C NMR (75 MHz, DMSO-d6): δ (ppm) 56.12 (OCH3), 111.46, 113.01,
9

117.78, 119.18, 121.46, 125.95, 130.34, 147.79, 148.84, 152.21, 158.15, 163.00 (C=O). MS (EI,
70 eV): m/z (%)ꢀ= 300 (M+, 9), 181 (26), 165 (100). Anal. Calcd for C16H16N2O4: C 63.99, H
5.37, N 9.33%, found: C 63.31, H 5.38, N 7.36%.
2.4.12. (E)-N'-(2,3-dihydroxybenzylidene)-3,4-dimethoxybenzohydrazide (D12)
Recrystallized in ethyl acetate. Cream solid; yield: 32%; M.P: 206-207 °C. 1H NMR (300
MHz, DMSO-d6): δ (ppm) 3.85 (s, 6H, OCH3), 6.75 (t, 1H, Ar-H-5´, J=7.2 Hz), 6.86 (d, 1H, Ar-
H-4´, J=6.9 Hz), 6.95 (d, 1H, Ar-H-5, J=6.9 Hz), 7.11 (d, 1H, Ar-H-6´, J=7.8 Hz), 7.53 (s, 1H,
Ar-H-2), 7.60 (d, 1H, Ar-H-6, J=7.5 Hz), 8.60 (s, 1H, N=CH), 9.22 (s, 1H, OH), 11.26 (s, 1H,
OH), 11.97 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 56.10 (OCH3), 56.15 (OCH3),
111.38, 111.54, 117.76, 119.26, 119.60, 120.53, 121.54, 125.25, 146.06, 146.51, 148.92, 152.43,
162.72 (C=O). MS (EI, 70 eV): m/z (%)ꢀ= 316 (M+, 23), 181 (11), 165 (100). Anal. Calcd for
C16H16N2O5: C 60.75, H 5.10, N 8.86%, found: C 60.89, H 5.09, N 8.89%.
2.4.13. (E)-N'-(3-ethoxy-4-hydroxybenzylidene)-3,4-dimethoxybenzohydrazide (D13)
White solid; yield: 59%; M.P: 193-196 °C. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 1.37
(t, 3H, CH2-CH3, J=6.9 Hz), 3.83 (s, 6H, OCH3), 4.05-4.08 (m, 2H, CH2-CH3), 7.08 (d, 1H, Ar-
H-4´, J=8.4 Hz), 7.24-7.30 (m, 2H, Ar-H-5, 5´), 7.44 (s, 1H, Ar-H-2´), 7.50 (s, 1H, Ar-H-2), 7.56
(d, 1H, Ar-H-6, J=8.4 Hz), 8.35 (s, 1H, OH), 8.56 (s, 1H, N=CH), 11.54 (s, 1H, NH). 13C NMR
(75 MHz, DMSO-d6): δ (ppm) 15.18 (CH2–CH3), 56.08 (OCH3), 56.12 (OCH3), 64.35 (CH2–
CH3), 110.81, 111.47, 111.91, 116.04, 121.37, 123.76, 125.98, 126.30, 147.60, 147.65, 149.62,
150.55, 152.10, 162.85 (C=O). MS (EI, 70 eV): m/z (%)ꢀ= 344 (M+, 4), 328 (100), 299 (22), 272
(37), 191 (33), 181 (8), 165 (24), 135 (19). Anal. Calcd for C18H20N2O5: C 62.78, H 5.85, N
8.13%, found: C 62.91, H 5.85, N 8.12%.
10

2.4.14. (E)-N'-(3-methoxy-4-hydroxybenzylidene)-3,4-dimethoxybenzohydrazide (D14)
1
White solid; yield: 53%; M.P: 171-173 °C. H NMR (300 MHz, DMSO-d6): δ (ppm)
3.81-3.83 (m, 9H, OCH3), 6.96-7.09 (m, 3H, Ar-H-4´, 5´, 5), 7.29 (s, 1H, Ar-H-2´), 7.49 (s, 1H,
Ar-H-2), 7.57 (d, 1H, Ar-H-6, J=7.8 Hz), 8.32 (s, 1H, OH), 9.33 (s, 1H, N=CH), 11.55 (s, 1H,
NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 56.03 (OCH3), 56.08 (OCH3), 111.35, 111.45,
112.35, 112.73, 120.60, 121.35, 126.13, 127.77, 147.36, 147.91, 148.82, 150.16, 152.10, 162.82
(C=O). MS (EI, 70 eV): m/z (%)ꢀ= 330 (M+, 14), 181 (39), 165 (100). Anal. Calcd for
C17H18N2O5: C 61.81, H 5.49, N 8.48%, found: C 61.98, H 5.50, N 8.45%.
2.4.15. (E)-N'-(2,4-dihydroxybenzylidene)-3,4-dimethoxybenzohydrazide (D15)
White solid; yield: 61%; M.P: 271-273 °C. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 3.83
(s, 6H, OCH3), 6.33-6.38 (m, 2H, Ar-H-3´, 5´), 7.09 (d, 1H, Ar-H-5, J=8.4 Hz), 7.30 (d, 1H, Ar-
H-6´, J=8.1 Hz), 7.50 (s, 1H, Ar-H-2), 7.57 (d, 1H, Ar-H-6, J=8.1 Hz), 8.51 (s, 1H, N=CH), 9.96
(s, 1H, OH), 11.55 (s, 1H, OH), 11.78 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm)
56.08 (OCH3), 56.12 (OCH3), 103.14, 108.11, 111.07, 111.31, 111.51, 121.39, 125.48, 131.75,
148.87, 149.16, 152.27, 159.93, 161.07, 162.46 (C=O). MS (EI, 70 eV): m/z (%)ꢀ= 316 (M+, 9),
181 (38), 165 (100). Anal. Calcd for C16H16N2O5: C 60.75, H 5.10, N 8.86%, found: C 60.61, H
5.11, N 8.88%.
2.4.16. (E)-4-((2-(3,4-dimethoxybenzoyl)hydrazono)methyl)-2-methoxyphenyl acetate (D16)
White solid; yield: 33%; M.P: 114-118 °C. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 2.28
(s, 3H, O(CO)CH3), 3.84 (s, 9H, OCH3), 7.09 (d, 1H, Ar-H-5, J=5.4 Hz), 7.19 (d, 1H, Ar-H-5´,
J=7.2 Hz), 7.29 (d, 1H, Ar-H-6´), 7.47-7.60 (m, 3H, Ar-H-2, 6, 2´), 8.47 (s, 1H, N=CH), 11.78
(s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 20.86, 56.07 (OCH3), 56.12 (OCH3), 56.29
11

(OCH3), 110.75 111.46, 115.91, 120.89, 121.51, 123.77, 125.86, 133.82, 141.18, 147.14, 148.82,
151.68, 152.24, 163.11 (C=O), 168.93(O-C=O). MS (EI, 70 eV): m/z (%)ꢀ= 373 (M+, 6), 330
(8), 181 (34), 165 (100). Anal. Calcd for C19H20N2O6: C 61.28, H 5.41, N 7.52%, found: C
61.30, H 5.41, N 7.54%.
2.5. Mushroom tyrosinase inhibition assay
Mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.) was assayed using L-DOPA
as the substrate as reported in our previous studies.29-31 The enzyme diphenolase activity was
monitored spectrophotometrically by observing dopachrome formation at 475 nm. All the test
samples were first dissolved in DMSO at 40 mM and diluted to the required concentrations.
Initially, 10 ml of tyrosinase (0.5 mg ml-1) was mixed with 160 ml of 50 mM phosphate buffer
(pH=6.8) and then 10 ml of the test sample in 96-well microplates 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 a positive control. Each assay was conducted as three separate replicates. The inhibitory
activity of the tested compounds was expressed as the concentration that inhibited 50% of the
enzyme activity (IC50). The percentage inhibition ratio was calculated according to the following
equation:
Inhibition (%) = 100 (Abs control- Abs compound)/Abs control
.
12

2.6. Determination of the inhibition type
The most potent derivative, compound D5, was considered for kinetic analysis. The
inhibitor concentrations were: 0, 10, 25, 50 and 100 mM. Substrate (L-DOPA) concentrations
were 0.25, 0.5, 0.75 and 1 mM in all kinetic studies. Pre-incubation and measurement time were
the same as discussed in mushroom tyrosinase inhibition assay protocol. Maximum initial
velocity was determined from initial linear portion of absorbance up to 10 min after addition of
L-DOPA with 1 min interval. The Michaelis constant (Km) and the maximal velocity (Vmax) of
the tyrosinase activity were determined by the Lineweavere-Burk plot at various concentrations
of L-DOPA as a substrate. The inhibition type of the enzyme was assayed by Lineweavere-Burk
plots of inverse of velocities (1/V) versus inverse of substrate concentrations 1/[S] mM-1.
2.7. Free radical scavenging assay
Radical scavenging activity was determined by DPPH assay. A mixture of test
compounds (in different concentrations) and methanolic solution of DPPH (110 μM) was shaken
in the dark at 37 °C for 30 min. The mixture absorbance was measured at 517 nm. Quercetin was
used as the positive control. The percent scavenging activity was calculated using the following
equation:
Radical Scavenging activity (%) = 100 × (Abscontrol - Abscompound)/Abscontrol
.
The EC50 values were obtained from linear regression plot between concentrations of test
compounds and percent radical scavenging activity. Each concentration was analyzed in three
independent experiments run in triplicate.
13

2.8 Computational Details
2.8.1 In silico physicochemical parameters and pharmacokinetics calculations
The drug likeness and pharmacokinetics properties of the compounds were determined
using the preADMET online server (http://preadmet.bmdrc.org/).
2.8.2 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 (PDBID: 2Y9X) was retrieved from the RCSB Protein Databank
(http://www.rcsb.org). Before docking, tropolone and water molecules were removed from
2Y9X. Using ADT, the crystal structure was then prepared for docking. For this purpose,
hydrogens were added and non-polar hydrogens were merged and finally, Gasteiger charges
were calculated for protein 2Y9X. 3-D structures of ligands were sketched and minimized under
Molecular Mechanics MM+ and then Semi-empirical AM1 methods in HyperChem software.
PDBQT formats of the ligands were prepared by adding Gasteiger charges and setting the degree
of torsions by using ADT. The grid maps were constructed by AutoGrid and grid box dimensions
were set to 40×40×40 with 0.375 Å grid spacing. The active site which comprises the amino acid
residues surrounding the two copper ions was selected for docking and the grids’ center were
placed on the tropolone’s binding site. In order to determine the docking parameter file, rigid
macromolecule and Lamarckian genetic search algorithm were chosen and the number of GA
runs was set at 100. Default values were retained for the rest of the parameters. Docking validity
was tested using co-crystallized inhibitor as ligand and the above-mentioned procedure.
14

3. Results and discussion
3.1. Design strategy
The target N'-benzylidene-3,4-dimethoxybenzohydrazide scaffold was designed based on
the structures of some potent tyrosinase inhibitors reported in the literature (Figure 1). Some
benzamide derivatives (structure I in Figur 1) have been found to exhibit superior inhibitions on
tyrosinase activity (IC50=0.3-0.9 μM) than kojic acid did.32 Moreover, it has been reported that
some hydrazine-containing compounds, such as compound II, were effective tyrosinase
inhibitors (IC50=1.93 μM) which showed higher inhibitory activity than kojic acid.33 Considering
these structural features and principles of bioisoterism, we designed a series of benzylidene-3,4-
dimethoxybenzohydrazide derivatives as tyroinase inhibitors by applying fragment-based drug
design approach. Different substituents were inserted on benzylidene moiety to define beneficial
structure–activity relationships (SARs).
15

Figure 1. Fragment-based design of proposed tyrosinase inhibitors.
3.2. Synthesis
Target compounds were synthesized via a two-step reaction outlined in Scheme 1.
Initially, methyl 3,4-dimethoxybenzoate (A) was refluxed with hydrazine hydrate (B) in ethanol
for 3 hours. The resultant 3,4-dimethoxybenzohydrazide (B) was reacted with various aryl-
aldehydes (C) in ethanol and at reflux condition for about 6 hours to obtain final benzylidene-
3,4-dimethoxybenzohydrazide derivatives (D1-D16). All the target compounds were synthesized
in good yield and high purity. Their chemical structures were characterized and confirmed by
using 1H NMR, 13C NMR and MS analysis.
Scheme 1. Synthesis of benzylidene-3,4-dimethoxybenzohydrazide derivatives (D1-D16).
16

3.3. Effect of N'-benzylidene-3,4-dimethoxybenzohydrazide derivatives on the diphenolase
activity of mushroom tyrosinase
The inhibitory effect of the synthesized derivatives on mushroom tyrosinase activity has
been evaluated and the results are presented in Table 1. Compounds D7, D9, D12, D15 and D16
showed good to excellent inhibition on mushroom tyrosinase with IC50 ranging from 19.72 μM
to 37.41 μM, while rest of the derivatives displayed no remarkable inhibitory activity.
Compounds D5 and D12 exhibited the highest tyrosinase inhibitory activity with IC50 values of
19.72 ± 1.84 μM and 20.63±0.79 μM, respectively, which were comparable to that of the
standard kojic acid with IC50 value of 19.08±1.21 μM.
The bioassay results showed that substitutions on benzylidene played a major role in
tyrosinase inhibitory activity of 3,4-dimethoxybenzohydrazid derivatives as compound D5
bearing 4-chloro substitution exhibited excellent tyrosinase inhibitory activity (IC50=19.72 μM).
Moreover, the substitution pattern of hydroxyl and methoxy groups at phenyl ring was the
decisive factor of the inhibitory activity. Considering IC50 values reported in Table 1, it could be
stated that the compounds which possess hydroxyl or methoxy groups at the ortho and/or meta
positions were more active than those having these substitutions at the para position and
existence of an acetoxy group at para position improved the anti-tyrosinase effect.
Compounds D12 and D16 bearing 2,3-dihydroxy and 4-acetoxy-3-methoxy residues exhibited
the second and third best tyrosinase inhibitory activities (IC50s=20.63 and 28.26 μM,
respectively). Replacing 3-hydroxyl with 4-hydroxyl as in D15 (IC50=31.53 μM), led to a
considerable decrease in the activity as compared to D12.
17

Table 1. Antioxidant properties and tyrosinase inhibitory activities of (D1–D16).
Tyrosinase inhibitory
activity a
DPPH radical
scavenging activity a
Compound
c
Tyrosinase inhibition b
(%)
IC50
EC50
(μM)
˃50
(mM)
D1
D2
17.68 ± 1.14
13.60 ± 0.18
19.49 ± 2.62
13.37 ± 0.33
69.16 ± 1.75
28.51 ± 1.60
59.34 ± 2.44
15.61 ± 5.01
57.17 ± 2.97
25.17 ± 3.39
22.49 ± 4.10
69.04 ± 1.85
38.33 ± 2.06
39.94 ± 4.11
58.70 ± 3.25
60.21 ± 1.03
71.74 ± 2.08
-
>1
˃50
0.4482 ± 0.0018
D3
˃50
>1
D4
˃50
>1
D5
19.72 ± 1.84
˃50
>1
D6
>1
D7
35.16 ± 1.08
˃50
>1
D8
>1
D9
37.41 ± 5.22
˃50
>1
D10
D11
D12
D13
D14
D15
D16
Kojic acid
Quercetin
>1
˃50
>1
20.63 ± 0.79
˃50
0.0097 ±0.0011
0.0498 ± 0.0028
0.4260 ± 0.0063
0.0829 ± 0.0045
0.1535 ± 0.0052
-
˃50
31.53 ±2.17
28.26 ± 0.94
19.08 ± 1.21
-
0.0089 ± 0.0014
a Values represent means ± SE of 3-4 independent experiments.
b Tyrosinase inhibition for synthesized compounds and kojic acid was measured at 50 μM.
c
50 % inhibitory concentration (IC50).
18

3.4. Determining the inhibitory type on mushroom tyrosinase for D5 derivative
The enzyme inhibition mode by the most potent derivative, D5, was studied by
Lineweaver–Burk plot analysis. The results are presented in Figure 2. Lineweaver–Burk plots
(plot of 1/V versus 1/[S]) for the inhibition of tyrosinase was obtained with several
concentrations of D5 (as the inhibitor) and L-DOPA (as the substrate). The plots of 1/V versus
1/[S] presented straight lines which crossed the y-axis at similar points. It was found that as the
inhibitor concentration elevated, the value of Km increased, but Vmax was not affected by the
concentration. Therefore, the results represented that compound D5 is a competitive inhibitor of
mushroom tyrosinase.
120
100
80
0 μM
50 μM
60
40
20
0
-0.5
0
0.5
1
1.5
2
2.5
1/[S] mM-1
-20
Concentration (μM)
Vmax (mM/min)
Km (mM)
4.34
0
0.15
0.15
50
5.88
19

Figure 2. Lineweaver-Burk plot for mushroom tyrosinase enzyme inhibition by different concentrations
of D5 in the presence of L-DOPA. The reciprocal tyrosinase inhibitory activity was plotted against the
reciprocal substrate concentration (double reciprocal plot, n = 3).
3.5. Free radical scavenging activity
Radical scavenging of all compounds against DPPH was investigated and the IC50 values
were determined. The results are shown in Table 1. Compound D12, having 2,3-
dihydroxyphenyl substitution, exhibited the highest activity with EC50 value of 0.0097±0.0011
mM, comparable to that of quercetin as the positive control (EC50=0.0089±0.0014 mM). D13
and D15 derivatives (bearing 3-ethoxy-4-hydroxyphenyl and 2,4-dihydroxyphenyl substituents)
were found to be effective radical scavengers with EC50 values of 0.0498±0.0028 and
0.0829±0.0045 mM, respectively. Compounds D2, D14 and D16, possessing 4-
dimethylaminophenyl, 3-methoxy-4-hydroxyphenyl and 4-acetoxy-3-methoxyphenyl moieties,
respectively, demonstrated lower radical scavenging activities with EC50 values ranging from
0.1535 mM to 0.4482 mM.
3.6. Computational section
3.6.1. Drug-likeness and ADME properties
The drug likeness scores (including; Lipinski rule of five, CMC like rule, lead like rule,
MDDR like rule, and WDI like rule) and pharmacokinetics properties (including; human
intestinal absorption, in vitro Caco-2 (Caucasian colon adenocarcinoma) cell permeability, in
vitro MDCK (Maden Darby Canine Kidney) cell permeability, in vitro plasma protein binding
and in vivo BBB penetration) were predicted for the derivatives that showed tyrosinase
20

inhibition. Calculations were carried out using the web-based application PreADMET and the
results obtained for compounds demonstrating tyrosinase inhibition are summarized in Table 2
and Table 3. The data revealed that all molecules fulfill the Lipinski's rule of five, CMC like
rule, MDDR like rule, and WDI like rule (Table 2). As it is reported in Table 3, all the
derivatives show well intestinal absorption. The compounds are predicted to bind weakly to
plasma proteins. Moreover, low blood–brain barrier permeation is observed for all the inhibitors
(except D15), therefore, it can be stated that they are less likely to cause neurotoxicity.
Therefore, benzylidene-3,4-dimethoxybenzohydrazide tyrosinase inhibitors could serve
as suitable drug-like candidates and according to ADME properties of the derivatives, they can
be proposed as effective candidates for further drug development.
Table 2. In silico Lipinski rule of five, lead like rule, CMC like rule, MDDR like rule, and WDI
like rule prediction for veratric acid derivatives possessing tyrosinase inhibitory activity..
Code
CMC like
rule
Lead like
rule
MDDR like
rule
WDI like
rule
Lipinskiʼ s rule
of five
Qualified
Qualified
Qualified
Qualified
Qualified
Qualified
Violated
Suitable
Violated
Suitable
Suitable
Violated
Mid-structure
Mid-structure
Mid-structure
Mid-structure
Mid-structure
Mid-structure
Suitable
Suitable
Suitable
Suitable
Suitable
Suitable
In 90% cutoff
In 90% cutoff
In 90% cutoff
In 90% cutoff
In 90% cutoff
In 90% cutoff
D5
D7
D9
D12
D15
D16
21

Table 3. In silico ADME profiling of veratric acid derivatives possessing tyrosinase inhibitory activity.
Absorption
Distribution
In vitro plasma In vivo blood–brain
protein binding barrier penetration
Human intestinal
Absorption HIA
(percentage)
95.75
In vitro caco-2 cell In vitro MDCK Cell In vitro skin
permeability
(nm s-1)
Permeability
(nm s-1)
permeability
(log Kp, cm h-1)
-2.97
Code
D5
(percentage)
(C.brain/C.blood)
30.66
3.94
88.20
0.22
96.02
96.70
87.64
87.65
96.42
28.49
47.03
17.21
11.88
29.32
0.32
0.39
0.30
0.54
0.28
-3.06
-3.38
-3.95
-3.95
-3.17
82.17
81.51
80.56
78.36
81.26
0.14
0.18
0.22
0.25
0.14
D7
D9
D12
D15
D16
3.6.2. Molecular docking analysis
Molecular docking analysis of the derivatives showing tyrosinase inhibitory activity was
performed in order to theoretically describe the difference found in the antityrosinase activity of
the compounds and also to inquire the ligands binding modes and possible interactions into the
binding pocket of tyrosinase. The enzyme–inhibitor complexes binding affinities are presented in
Table 4. The lower value indicated the more stable complex formed between the ligand and
target enzyme. The most active benzohydrazide derivatives D5 and D12, containing 2-
chlorophenyl and 2,3-dihydroxyphenyl groups, respectively, possessed the most negative
estimated free energies of binding (-6.91 and -6.66 kcal/mol, respectively), however, their
binding modes were completely different. The binding orientations and interactions of D5 and
D12 with the active site of mushroom tyrosinase are illustrated in Figure 3a and Figure 3b,
respectively. The binding energies of compounds D7, D9, D11, D15 and D16 were -5.95, -5.89,
22

5.77, 6.23 and 5.93 kcal/mol, respectively. The estimated free energies of binding proved that
compound D5 and D12 made the most stable complex with the target protein. Docking
validation was done by extracting the structure of the co-crystallized ligand and re-docking it
into the receptor (self-docking). The root mean square deviation (RMSD) between the best pose
of co-crystallized ligand docked into the binding site of tyrosinase and the one in the crystal
structure was 1.63 Å
D5 is well accommodated in the binding site of tyrosinase by hydrogen-bond and Pi-H
interactions. As it is illustrated in Figure 3a, oxygen atoms of carbonyl and 4-methoxy moieties
established strong hydrogen bonds with Val283 (distance: 2.03 Å) and Arg268 (distance: 2.03
Å), respectively. Furthermore, 4´-Cl substituent interacted with Cu (distance: 2.67 Å) and
4-chlorophenyl ring involved in a Pi-H interaction with Val283. Compound D12 accommodated
within the active site through three hydrogen bonds (Figure 3b). Nitrogen atom of hydrazine
group (=N-NH) formed a hydrogen bond interaction with the residue Val283 at a distance of
1.94 Å. The two other hydrogen bond interactions were established between 2´-OH and 3´-OH
substitutions and Glu265 residue at distances of 1.71 and 1.97 Å, respectively.
The docking results are in good agreement with the experimental outcomes. These results
confirmed that the substitutions on benzylidene moiety had a significant effect in binding
orientations of N'-benzylidene-3,4-dimethoxybenzohydrazide within the active site of tyrosinase.
Copper atoms and some residues including Val283, Arg268, Gly231, His244, His263, His259
Met280, Glu265 and Phe264 played an influential role in the formation of stable ligand-target
complexes.
23

Table 4. Docking results of the presented tyrosinase inhibitors into the tyrosinase binding site.
ΔG
Ki
Amino
acid
Distance
(Å)
Compound
Interactions
Atom of ligand
(kcal/mol)
(μM)
H-bonding
H-bonding
C=O
4-Methoxy
Val283
Arg268
Cu
2.03
2.03
2.67
D5
-6.91
8.64
Cl
Pi-H
H-bonding
H-bonding
H-bonding
Pi-H
4-Cholorophenyl
C=O
Val283
Val283
Gly231
Val283
Val283
His244
Glu265
Glu265
D7
D9
-5.95
-5.89
43.78
47.77
1.83
1.83
2.39
NH of hydrazine (=N-NH)
N of Hydrazine (=N-NH)
3,4-Dimethoxyphenyl
N of hydrazine (=N-NH-)
2´-OH
H-bonding
H-bonding
H-bonding
H-bonding
H-bonding
Coordinate-
bond
1.94
1.71
1.95
D12
D15
-6.66
-6.23
18.61
26.98
3´-OH
C=O
2´-OH
4´-OH
Val283
Met280
Cu
1.79
2.03
2.56
Pi-Pi
3,4-Dimethoxyphenyl
4-Acetoxy-3-methoxyphenyl
4-Acetoxy-3-methoxyphenyl
3´-Methoxy
His244
His259
Val283
Phe264
His263
Pi-Pi
D16
-5.93
44.99
Pi-H
Alkyl-Pi
Alkyl-Pi
4´-Acetoxy
24

a
b
Figure 3. The binding orientation of the most active derivatives D5 (a) and D12 (b) within the active site
of tyrosinase (PDB: 2Y9X). Ligands are shown as cyan sticks while the amino acid residues are displayed
as gray sticks.
4. Conclusions
We developed a series of veratric acid derivatives containing benzylidene-hydrazine
moieties as novel tyrosinase inhibitors. While, veratric acid and its methyl ester did not show
25

significant inhibitory effect on tyrosinase, the results showed that some of our target analogues
demonstrated remarkable tyrosinase inhibitory activities with IC50 values of micro molar range.
Compounds D5 and D12 were found to be promising tyrosinase inhibitors with the IC50 values of
19.72±1.84 and 20.63±0.79 μM, respectively. The kinetic analysis disclosed a competitive mode
of inhibition for D5. Docking studies were performed to delineate the binding affinity of the
active derivatives in the binding site of tyrosinase. Docking studies showed good correlation with
experimental results. Some derivatives also indicated good free radical scavenging activity. D12
demonstrated the highest activity with EC50 value of 0.0097±0.0011 mM, comparable to that of
quercetin. The in silico studies further indicated that the active compounds are compliant with
most of the rules which lie under drug likeness and pharmacokinetics analysis. It can be
concluded that the compounds can serve as structural outlines and promising leads in order to
design and develop potential tyrosinase inhibitors.
Conflicts of interest
The authors confirm that this article content has no conflict of interest.
Acknowledgments
The authors wish to thank Vice-Chancellor for Research of Shiraz University of Medical
Sciences for supporting this study with project No. 97-01-36-18236.
References
26

1.
the hemolymph of the slipper lobster Scyllarides latus. J Comp Physiol B. 2005;175:405-11.
2. Sánchez-Ferrer A, Rodríguez-López JN, García-Cánovas F, García-Carmona F.
Olianas A, Sanjust E, Pellegrini M, Rescigno A. Tyrosinase activity and hemocyanin in
Tyrosinase: a comprehensive review of its mechanism. Biochimica et Biophysica Acta (BBA)-
Protein Structure and Molecular Enzymology. 1995;1247:1-11.
3.
basis and molecular mechanism. Trends Biochem Sci. 2000;25:392-7.
4. Kanteev M, Goldfeder M, Fishman A. Structure–function correlations in tyrosinases.
Protein. Science. 2015;24:1360-9.
5. Hasegawa T. Tyrosinase-expressing neuronal cell line as in vitro model of Parkinson’s
disease. Int J Mol Sci. 2010;11:1082-9.
6. Asanuma M, Miyazaki I, Ogawa N. Dopamine-or L-DOPA-induced neurotoxicity: the
Decker H, Tuczek F. Tyrosinase/catecholoxidase activity of hemocyanins: structural
role of dopamine quinone formation and tyrosinase in a model of Parkinson’s disease. Neurotox
Res. 2003;5:165-76.
7.
Tessari I, Bisaglia M, Valle F, Samorì B, Bergantino E, Mammi S, et al. The Reaction of
α-Synuclein with Tyrosinase possible implications for parkinson disease. J Biol Chem.
2008;283:16808-17.
8.
McEvily AJ, Iyengar R, Otwell WS. Inhibition of enzymatic browning in foods and
beverages. Crit Rev Food Sci Nutr. 1992;32:253-73.
9.
Chang T-S. An updated review of tyrosinase inhibitors. Int J Mol Sci. 2009;10:2440-75.
10.
Parvez S, Kang M, Chung HS, Bae H. Naturally occurring tyrosinase inhibitors:
mechanism and applications in skin health, cosmetics and agriculture industries. Phytotherapy
27

Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of
Natural Product Derivatives. 2007;21:805-16.
11.
hyperpigmentation: a comprehensive review. J Clin Aesthet Dermatol. 2016;9:49.
12. Zolghadri S, Bahrami A, Hassan Khan MT, Munoz-Munoz J, Garcia-Molina F, Garcia-
Sarkar R, Ranjan R, Garg S, Garg VK, Sonthalia S, Bansal S. Periorbital
Canovas F, et al. A comprehensive review on tyrosinase inhibitors. Enzyme Inhib Med Chem.
2019;34:279-309.
13.
Enzyme Inhib Med Chem. 2016;31:1-13.
14. Narasimhan B, Ohlan S, Ohlan R, Judge V, Narang R. Hansch analysis of veratric acid
derivatives as antimicrobial agents. Eur J Med Chem. 2009;44:689-700.
15. Saravanakumar M, Raja B. Veratric acid, a phenolic acid attenuates blood pressure and
oxidative stress in L-NAME induced hypertensive rats. Eur J Pharmacol. 2011;671:87-94.
16. Choi W-S, Shin P-G, Lee J-H, Kim G-D. The regulatory effect of veratric acid on NO
production in LPS-stimulated RAW264. 7 macrophage cells. Cell Immunol. 2012;280:164-70.
17. Wang Q-b, Sun L-y, Du Y. Veratric acid inhibits LPS-induced IL-6 and IL-8 production
in human gingival fibroblasts. Inflammation. 2016;39:237-42.
18. Saravanakumar M, Raja B, Manivannan J, Silambarasan T, Prahalathan P, Kumar S, et
Lee SY, Baek N, Nam T-g. Natural, semisynthetic and synthetic tyrosinase inhibitors.
al. Oral administration of veratric acid, a constituent of vegetables and fruits, prevents
cardiovascular remodelling in hypertensive rats: a functional evaluation. Br J Nutr.
2015;114:1385-94.
19.
Miyazawa M, Oshima T, Koshio K, Itsuzaki Y, Anzai J. Tyrosinase inhibitor from black
rice bran. J Agric Food Chem. 2003;51:6953-6.
28

20.
synthesis of hydrazide derivatives. Curr Med Chem. 2012;19:569-612.
21. Abdel-Aziz M, Abdel-Rahman HM. Synthesis and anti-mycobacterial evaluation of some
pyrazine-2-carboxylic acid hydrazide derivatives. Eur J Med Chem. 2010;45:3384-8.
22. Gurkok G, Altanlar N, Suzen S. Investigation of antimicrobial activities of indole-3-
aldehyde hydrazide/hydrazone derivatives. Chemotherapy. 2009;55:15-9.
23. Kajal A, Bala S, Kamboj S, Saini V. Synthesis, characterization, and computational
Narang R, Narasimhan B, Sharma S. A review on biological activities and chemical
studies on phthalic anhydride-based benzylidene-hydrazide derivatives as novel, potential anti-
inflammatory agents. Med Chem Res. 2014;23:2676-89.
24.
Dimmock JR, Vashishtha SC, Stables JP. Anticonvulsant properties of various
acetylhydrazones, oxamoylhydrazones and semicarbazones derived from aromatic and
unsaturated carbonyl compounds. Eur J Med Chem. 2000;35:241-8.
25.
Silva AG, Zapata-Sudo G, Kummerle AE, Fraga CA, Barreiro EJ, Sudo RT. Synthesis
and vasodilatory activity of new N-acylhydrazone derivatives, designed as LASSBio-294
analogues. Bioorg Med Chem. 2005;13:3431-7.
26.
Taha M, Ismail NH, Zaki HM, Wadood A, Imran S, Yamin BM, et al. 3, 4-
Dimethoxybenzohydrazide derivatives as antiulcer: Molecular modeling and density functional
studies. Bioorg Chem. 2017;75:235-41.
27.
Deng Y, Kang D, Shi J, Zhou W, Sun A, Ju J, et al. The semi-synthesis, biological
evaluation and docking analysis of the oxime, hydrazine and hydrazide derivatives of
platensimycin. MedChemComm. 2018;9:789-94.
29