Design, synthesis, in vitro and in silico studies of novel Schiff base derivatives of 2-hydroxy-4-methoxybenzamide as tyrosinase inhibitors

Article abstract of DOI:10.1002/ddr.21771

Due to the fact that tyrosinase is responsible for biosynthesis and regulation of melanins and browning food products, tyrosinase inhibitors can be favorable agents in cosmetics and medicinal industries. A series of novel 2-hydroxy-4-methoxybenzohydrazide were designed, synthesized, and their new application as tyrosinase inhibitors was also disclosed. Based on in vitro tyrosinase inhibitory assay, 4d as the strongest inhibitor of tyrosinase with an IC₅₀ value of 7.57 μM showed approximately 2.5-fold better inhibition than kojic acid as positive control followed by two compounds 4b (IC₅₀?= 8.19 ± 0.25 μM) and 4j (IC₅₀?= 8.92 ± 0.016) which displayed preferable tyrosinase inhibitory activity. Detailed investigations on the mechanism of action of the 4d reported mix type of inhibition. More importantly, molecular modeling assessments proposed the ability of 4d for potential interaction with Cu (metal)-His (residue) within tyrosinase active site. Overall, 4d is a promising candidate for the development of anti-tyrosinase agents.

Full text of DOI:10.1002/ddr.21771

Received: 20 May 2020
DOI: 10.1002/ddr.21771
Accepted: 29 November 2020
R E S E A R C H A R T I C L E
Design, synthesis, in vitro and in silico studies of novel Schiff
base derivatives of 2-hydroxy-4-methoxybenzamide as
tyrosinase inhibitors
Aida Iraji1
Mahsima Khoshneviszadeh1
| Zahra Panahi2
| Najmeh Edraki1
| Mehdi Khoshneviszadeh1,2
|
1Medicinal and Natural Products Chemistry
Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran
Abstract
Due to the fact that tyrosinase is responsible for biosynthesis and regulation
of melanins and browning food products, tyrosinase inhibitors can be favorable
agents in cosmetics and medicinal industries. A series of novel 2-hydroxy-
4-methoxybenzohydrazide were designed, synthesized, and their new application
as tyrosinase inhibitors was also disclosed. Based on in vitro tyrosinase inhibitory
assay, 4d as the strongest inhibitor of tyrosinase with an IC50 value of 7.57 μM
showed approximately 2.5-fold better inhibition than kojic acid as positive control
followed by two compounds 4b (IC50 = 8.19 0.25 μM) and 4j (IC50 = 8.92 0.016)
which displayed preferable tyrosinase inhibitory activity. Detailed investigations on the
mechanism of action of the 4d reported mix type of inhibition. More importantly, molec-
ular modeling assessments proposed the ability of 4d for potential interaction with Cu
(metal)-His (residue) within tyrosinase active site. Overall, 4d is a promising candidate for
the development of anti-tyrosinase agents.
2Department of Medicinal Chemistry, Faculty
of Pharmacy, Shiraz University of Medical
Sciences, Shiraz, Iran
Correspondence
Mehdi Khoshneviszadeh, Medicinal and
Natural Products Chemistry Research Center,
Shiraz University of Medical Sciences, Shiraz,
Iran.
Email: m.khoshneviszadeh@gmail.com
K E Y W O R D S
2-hydroxy-4-methoxybenzamide, kinetic study, molecular docking, synthesis, tyrosinase
inhibitor
1
|
INTRODUCTION
Tyrosinase hydroxylates monophenols compounds (e.g., l-tyrosine as pre-
cursor substrate) to o-diphenols (e.g., L-dopa as the second substrate)
Melanins are the pigments responsible for hair and skin color. The
abnormal accumulation of pigment in the body can cause hyper-
pigmentation problems and skin disorders including age spots,
melasma, and melanoma (Loizzo et al., 2012). Tyrosinase (TYR)
oxidoreductase enzyme (EC 1.14.18.1) widely distributed in microor-
ganisms, humans, animals, and plants. TYR and other tyrosinase-like
protein have an initial, and critical role in melanin synthesis and cataly-
sis rate-limiting step of melanin biosynthesize (Zolghadri et al., 2019).
and catalyzes o-diphenols to the o-quinones (e.g., dopaquinone)
(Hałdys & Latajka, 2019). Besides, the action of TYR has increased enzy-
matic browning reaction results in deteriorating food during commercial
or domestic processing and storage (Singh et al., 2018). It seems that
tyrosinase inhibitors (TYRI) could have potential applications in diverse
areas of agriculture, cosmetics, and pharmaceuticals (Yuan et al., 2020).
Different studies also confirmed the role of TYR in neurodegenerative
and autoimmune disorders (Lavezzo et al., 2016; Neagu et al., 2018;
Piechowska et al., 2019).
Abbreviations: 13C NMR, carbon-13 nuclear magnetic resonance; 1H NMR, proton nuclear
magnetic resonance; Ala, alanine; Arg, arginine; DMSO-d6, deuterated dimethyl sulfoxide;
His, histidine; L-dopa, L-dihydroxyphenylalanine; LGA, Lamarckian genetic algorithm; L-Tyr,
L-tyrosinase; Lue, Lucine; Pro, proline; Val, Valine.
TYR is a tetrameric Cu-containing metalloenzyme belonging to
the type-3 copper protein family. TYR composes two heavy subunits
(H subunits) with around about 43 kDa and two light subunits of
Drug Dev Res. 2020;1–10.
wileyonlinelibrary.com/journal/ddr
© 2020 Wiley Periodicals, LLC.
1

2
IRAJI ET AL.
about 14 kDa (L subunits). In the TYR active site, two copper ions
bound to three conserved essential histidine residues (Choi
et al., 2015; Ismaya et al., 2011; Matoba et al., 2006).
165.4, 171.7; MS (EI), m/z (%):151.0 (100), 166.0 (40), 315.0 (M+, 15);
I.R (KBr) Ʋmax (cm−1): ArO-H: 3497, N-H: 3293, C-H-aromatic: 3093,
C-H-aliphatic: 2970, C-H-aliphatic: 2927, C-H-aliphatic: 2840, C=O
amide:1679, C=N:1643, N-O nitro:1523, N-O nitro: 1332, C=C aro-
matic:1477, CH3:1387, C-N: 1289, C-O:1135.
2
|
MATERIAL AND METHOD
| Chemistry
Synthesis of 2-hydroxy-4-methoxy-N0-(4-nitrobenzylidene)
benzohydrazide (4b)
2.1
Yellow crystal; Yield: 54%; M.P: 264–266ꢀC; 1H NMR (500 MHz,
DMSO-d6) δH (ppm): 3.81 (s, 3H, OCH3), 6.51 (d, J = 2 Hz, 1H, H3-
2-hydroxy-4-methoxy-N0-methylenebenzohydrazide), 6.57 (dd, J = 8.8,
2.0 Hz, 1H, H5-2-hydroxy-4-methoxy-N0-methylenebenzohydrazide),
7.91 (d, J = 8.8 Hz, 1H, Ar-H), 7.99 (d, J = 8.4 Hz, 2H, Ar-H), 8.30 (d,
J = 8.6 Hz, 2H, Ar-H), 8.55 (s, 1H, CH=N), 11.98 (s, 1H, OH), 12.22 (s,
1H, NH); 13C NMR (126 MHz, DMSO) δc (ppm): 54.9, 100.8, 105.98,
107.0, 123.5, 127.5, 129.3, 139.9, 145.1, 147.4, 161.5, 163.5, 164.8;
MS (EI), m/z (%): 151.0 (100), 180.1 (47), 315.1 (M+, 17); I.R(KBr) Ʋmax
(cm−1): ArO-H: 3331, N-H: 3321, C-H-aromatic: 3027, C-H-aromatic:
3016, CH-aliphatic: 2971, CH-aliphatic: 2931, C=O amide: 1656, C=N:
1593, C=C aromatic: 1553, C=C aromatic: 1491, N-H: 1511, CH3: 1362,
C-N: 1258, C-O: 1156, C-Cl: 847.
All reagents were purchased from commercial sources and were used
without further. Solvents were procured from Merck and Samchane.
Thin-layer chromatography (TLC) was performed on Silica gel
60 F254 coated plates (Merck) to check the purity of compounds. 1H
and 13C NMR were recorded in Bruker NMR 500 and 125 MHz spec-
trophotometer. All the chemical shifts were reported as (δ) values
(ppm). Melting points were determined by Gallenkamp melting point
apparatus. FT-IR Perkin Elmer spectrophotometer was used. Mass
spectra were obtained on the Agilent 7890A spectrometer at 70 eV.
2.1.1
|
Synthesis of 2-hydroxy-
4-methoxybenzohydrazide (2)
Synthesis of N0-(4-bromobenzylidene)-2-hydroxy-
The methyl 2-hydroxy-4-methoxybenzoate (Compound 1, 2 mmol)
and hydrazine hydrate (3 mmol) were refluxed in ethanol (50 ml) for
4 h in the presence of the catalytic amount of acetic acid. The mixture
was cooled and kept for 3–4 h at room temperature. The solid sepa-
rated was filtered, dried and recrystallized from ethanol. The product
was carefully checked by TLC (Khoshneviszadeh et al., 2013; Yazdani
et al., 2019).
4-methoxybenzohydrazide (4c)
White crystal; yield: 57%; M.P: 224–227ꢀC; 1H NMR (500 MHz,
DMSO-d6) δH (ppm): 3.80 (s, 3H, OCH3), 6.51 (s, J = 2.8 Hz, 1H, H3-
2-hydroxy-4-methoxy-N0-methylenebenzohydrazide), 6.56 (dd, J = 9.0,
2.8 Hz, 1H, H5-2-hydroxy-4-methoxy-N0-methylenebenzohydrazide),
7.68 (q, J = 8.5 Hz, 4H, Ar-H), 7.90 (d, J = 8.9 Hz, 1H, H6-2-hydroxy-
4-methoxy-N0-methylenebenzohydrazide), 8.43 (s, 1H, CH=N), 11.85 (s,
1H, OH), 12.46 (s, 1H, NH); 13C NMR (126 MHz, DMSO) δc (ppm): 54.9,
100.8, 105.9, 106.9, 122.9, 128.5, 129.0, 131.3, 132.9, 146.51, 161.7,
163.4, 164.8; MS (EI) m/z (%): 151(100), 167 (57), 182 (18), 269 (33),
349.1 (M+, 5), 351.0 (M+2, 5); I.R (KBr) Ʋmax (cm−1): ArO-H: 3331, N-H:
3300, C-H-aromatic: 3046, C-H-aromatic: 3017, CH-aliphatic: 2970,
CH-aliphatic: 2929, CH-aliphatic: 2839, C=O amide: 1654, C=N: 1593,
C=C aromatic:1504, C=C aromatic:1488, C=C aromatic:1460, N-H:
1520, CH3: 1384, CH3:1256, C-H: 1361, C-N: 1203, C-O: 1156,
C-Br: 633.
2.1.2
|
General procedure for the synthesis of
compounds (4a–j)
The final step was performed according to the reported literature. For
this purpose, 2-hydroxy-4-methoxybenzohydrazide (Compound 2,
1 mmol) was dissolved in 50 ml ethanol at room temperature. The
reaction mixture was stirred for 10 min and different selected alde-
hyde (Compound 3, 1.2 mmol) were added. After 12 h string at 50ꢀC,
the mixture was cooled to room temperature and filtered. The residue
was recrystallized from ethanol to give the pure product. In the case
of some compounds, they were purified using plate chromatography
with EtOH/CHCl3 as eluent (Iraji et al., 2018).
Synthesis of N0-(4-chlorobenzylidene)-2-hydroxy-
4-methoxybenzohydrazide (4d)
White powder; yield: 58%; M.P: 210–214ꢀC; 1H NMR (500 MHz,
DMSO-d6) δH (ppm): 3.80 (s, 3H, OCH3), 6.51 (d, J = 2.2 Hz, 1H, H3-
2-hydroxy-4-methoxy-N0-methylenebenzohydrazide), 6.56 (dd, J = 8.9,
2.2 Hz, 1H, H5-2-hydroxy-4-methoxy-N0-methylenebenzohydrazide),
7.54 (d, J = 8.4 Hz, 2H, Ar-H), 7.77 (d, J = 8.2 Hz, 2H, Ar-H), 7.90 (d,
J = 8.9 Hz, 1H, H6-2-hydroxy-4-methoxy-N0-methylenebenzohydrazide),
8.45 (s, 1H, CH=N), 11.84 (s, 1H, OH), 12.45 (s, 1H, NH); 13C NMR
(126 MHz, DMSO) δc (ppm): 54.9, 100.8, 105.9, 106.9, 128.3, 128.4,
129.0, 132.6, 134.1, 146.4, 161.7, 163.4, 164.8; MS (EI), m/z (%): 151.0
(100), 166.9 (14), 304.7 (M+, 10), 306.0(M+2, 4); I.R (KBr) Ʋmax (cm−1):
ArO-H: 3331; N-H: 3327, C-H-aromatic: 3027, C-H-aromatic 3016,
Synthesis of 2-hydroxy-4-methoxy-N0-(3-nitrobenzylidene)
benzohydrazide (4a)
Yellow crystal; Yield: 60%; M.P: 260–264ꢀC; 1H NMR (500 MHz,
DMSO-d6) δH (ppm): 2.23 (s, 3H, CH3), 7.71 (m, 2H, Ar-H), 8.08 (s, 1H,
Ar-H) 8.10 (d, J = 7.6 Hz, 1H, Ar-H), 8.21 (dt, J = 7.7, 3.8 Hz, 2H, Ar-
H), 8.42 (s, 1H, Ar-H), 8.48 (s, 1H, N=CH), 11.47 (s, 1H, OH), 11.62 (s,
1H, N-H); 13C NMR (126 MHz, DMSO) δc (ppm): 19.7, 120.2, 120.3,
123.3, 123.5, 129.8, 132.2, 132.6, 135.6, 135.74, 139.6, 142.6, 147.6,

IRAJI ET AL.
3
CH-aliphatic: 2971, CH-aliphatic: 2931, C=O amide: 1656; C=N: 1593;
C=C aromatic:1553, C=C aromatic: 1491, N-H: 1511, CH3: 1362, C-N:
1258, C-O: 1156, C-Cl: 847.
aliphatic: 2845, C=O amide: 1637, C=N: 1589, C=C aromatic:1504, C=C
aromatic: 1444, N-H: 1520, CH3:1371, C-N: 1200, C-O:1155.
Synthesis of 2-hydroxy-N0-(3-hydroxy-4-methoxybenzylidene)-
4-methoxybenzohydrazide (4h)
Synthesis of 2-hydroxy-N0-(4-hydroxybenzylidene)-
4-methoxybenzohydrazide (4e)
White powder; Yield: 54%; M.P: 236–240ꢀC; 1H NMR (500 MHz,
DMSO-d6) δH (ppm): 3.80 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 6.50 (d,
J = 2.6 Hz, 1H, H3-2-hydroxy-4-methoxy-N0-methylenebenzohydrazide),
6.55 (dd, J = 9.0, 2.6 Hz, 1H, H5-2-hydroxy-4-methoxy-N0-methy-
lenebenzohydrazide), 6.98 (d, J = 8.3 Hz, 1H, Ar-H), 7.09 (dd, J = 8.4,
2.0 Hz, 1H, Ar-H), 7.30 (d, J = 2.1 Hz, 1H, Ar-H), 7.89 (d, J = 9 Hz, 1H,
H6-2-hydroxy-4-methoxy-N0-methylenebenzohydrazide), 8.32 (s, 1H,
Ar-H), 9.36 (s, 1H, N=CH), 11.65 (s, 1H, OH), 12.63 (s, 1H, NH); 13C
NMR (126 MHz, DMSO) δc (ppm): 54.9, 55.0, 100.8, 105.8, 106.8,
111.3, 111.8, 119.9, 126.4, 128.7, 146.3, 148.1, 149.4, 161.9, 163.3,
164.70; MS (EI), m/z (%): 151.0 (100), 167 (9), 301 (10), 316.0 (M+, 12);
I.R (KBr) Ʋmax (cm−1): ArO-H: 3390, N-H: 3316, C-H-aromatic: 3087,
CH-aliphatic: 2992, CH-aliphatic: 2949, CH-aliphatic: 2915, CH-ali-
phatic: 2838, C=O amide: 1642, C=N: 1598, C=C aromatic: 1555, C=C
aromatic:1440, N-H: 1503, CH3:1326, C-N: 1212, C-O:1108.
White crystal; yield: 41%; M.P: 215–220ꢀC; 1H NMR (500 MHz,
DMSO-d6) δH (ppm): 3.80 (s, 3H, OCH3), 6.49 (d, J = 2.3 Hz, 1H, H3-
2-hydroxy-4-methoxy-N0-methylenebenzohydrazide), 6.55 (dd, J = 8.7,
2.3 Hz, 1H, H5-2-hydroxy-4-methoxy-N0-methylenebenzohydrazide),
6.85 (d, J = 8.5 Hz, 2H, Ar-H), 7.58 (d, J = 8.5 Hz, 2H, Ar-H), 7.89 (d,
J = 8.7 Hz, 1H, H6-2-hydroxy-4-methoxy-N0-methylenebenzohydrazide),
8.36 (s, 1H, C=N), 10.00 (s, 1H, OH), 11.64 (s, 1H, OH), 12.66 (s, 1H,
NH); 13C NMR (126 MHz, DMSO) δc (ppm): 54.9, 100.8, 105.8, 106.8,
115.2, 124.5, 128.5, 128.7, 148.3, 159.1, 161.9, 163.3, 164.7; MS (EI),
m/z (%): 151(100), 196.1(35), 286.1(M+, 18); I.R(KBr) Ʋmax (cm−1):
ArO-H: 3521, ArO-H: 3405, N-H: 3196, C-H-aromatic: 3024, CH-ali-
phatic: 2870, C=O amide: 1646, C=N: 1605, C=C aromatic: 1516, C=C
aromatic: 1451, CH3: 1377, C-H: 1304; C-N: 1223, C-O:1166.
Synthesis of 2-hydroxy-N0-(4-hydroxy-3,5-dimethoxybenzylidene)-
4-methoxybenzohydrazide (4f)
Synthesis of N0-(3,4-dimethoxybenzylidene)-2-hydroxy-
Gray powder; Yield: 98%; M.P: 237–245ꢀC; 1H NMR (500 MHz,
DMSO-d6) δH (ppm): 3.80 (s, 3H, OCH3), 3.83 (s, 6H, OCH3), 6.49 (d,
J = 2.2 Hz, 1H, H3-2-hydroxy-4-methoxy-N0-methylenebenzohydrazide),
6.56 (dd, J = 8.9, 2.2 Hz, 1H, H5-2-hydroxy-4-methoxy-N0-methy-
lenebenzohydrazide), 7.01 (s, 2H, Ar-H), 7.90 (d, J = 8.9 Hz, 1H, H6-
2-hydroxy-4-methoxy-N0-methylenebenzohydrazide), 8.34 (s, 1H, Ar-H),
8.98 (s, 1H, N=CH), 11.68 (s, 1H, OH), 12.54 (s, 1H, NH); 13C NMR
(126 MHz, DMSO) δc (ppm): 54.9, 55.5, 100.8, 104.2, 105.8, 106.9,
123.8, 128.8, 137.6, 147.6, 148.5, 161.7, 163.3, 164.5; MS (EI), m/z (%):
151.0 (100), 180.1 (85), 330.1(28), 346.1 (M+); I.R (KBr) Ʋmax (cm−1):
ArO-H: 3521, ArO-H: 3434, N-H: 3227, C-H: aromatic: 3082, C-H: aro-
matic: 3059; CH-aliphatic: 2965, CH-aliphatic: 2942, CH-aliphatic:
2845, C=O amide: 1643, C=N: 1596, C=C aromatic: 1547, C=C aro-
matic: 1458, N-H: 1514, CH3: 1377, C-N: 1228, C-O:1115.
4-methoxybenzohydrazide (4i)
White powder; Yield: 90%; M.P: 162–167ꢀC; 1H NMR (500 MHz,
DMSO-d6) δH (ppm): 3.85–3.79 (m, 9H, OCH3), 6.50 (d, J = 2.3 Hz, 1H,
H3-2-hydroxy-4-methoxy-N0-methylenebenzohydrazide), 6.55 (dd, J = 8.8,
2.3 Hz, 1H, H5-2-hydroxy-4-methoxy-N0-methylenebenzohydrazide), 7.04
(d, J = 8.3 Hz, 1H, Ar-H), 7.23 (d, J = 8.2 Hz, 1H, Ar-H), 7.36 (s, 1H, Ar-H),
7.90 (d,
J
= 8.8 Hz, 1H, H6-2-hydroxy-4-methoxy-N0-methy-
lenebenzohydrazide), 8.39 (s, 1H, N=CH), 11.66 (s, 1H, OH), 12.53 (s, 1H,
NH); 13C NMR (126 MHz, DMSO) δc (ppm): 54.9, 54.9, 55.0, 100.7,
105.8, 106.9, 107.8, 110.9, 121.5, 126.3, 128.8, 148.1, 148.5, 150.4,
161.8, 163.3, 164.7; MS (EI), m/z (%): 151.0 (100), 315 (10), 330.1 (M+,
15); I.R (KBr) Ʋmax (cm−1): ArO-H: 3313, N-H: 3293, C-H-aromatic: 3077,
C-H-aromatic: 3009, CH-aliphatic: 2964, CH-aliphatic: 2935, CH-aliphatic:
2916, C=O amide: 1639, C=N: 1597, N-H: 1556, C=C aromatic: 1513,
C=C aromatic: 1461, CH3: 1367, C-H: 1361, C-N: 1258, C-O: 1168.
Synthesis of N0-(3-ethoxy-4-hydroxybenzylidene)-2-hydroxy-
4-methoxybenzohydrazide (4g)
White powder; Yield: 60%; M.P: 108–112ꢀC; 1H NMR (500 MHz,
DMSO-d6) δH (ppm): 1.37 (t, J = 6.9 Hz, 3H, CH3, 2-ethoxyphenol), 3.80
(s, 3H, OCH3), 4.07 (q, J = 6.9 Hz, 2H, 2-ethoxyphenol), 6.49 (d,
J = 2.3 Hz, 1H, H3-2-hydroxy-4-methoxy-N0-methylenebenzohydrazide),
Synthesis of N0-(4-acetyl-3-methoxybenzylidene)-2-hydroxy-
4-methoxybenzohydrazide (4j)
White powder; Yield: 42%; M.P: 180–185ꢀC; 1H NMR (500 MHz,
DMSO-d6) δH (ppm): 2.29 (s, 3H, C=O-CH3), 3.81 (s, 3H, OCH3), 3.85
(s, 3H, OCH3), 6.51 (d, J = 2.3 Hz, 1H, H3-2-hydroxy-4-methoxy-N0-
6.55 (dd,
J
= 8.9, 2.3 Hz, 1H, H5-2-hydroxy-4-methoxy-N0-methy-
methylenebenzohydrazide), 6.57 (dd,
2-hydroxy-4-methoxy-N0-methylenebenzohydrazide),
J
= 8.9, 2.3 Hz, 1H, H5-
7.20 (d,
lenebenzohydrazide), 6.87 (d, J = 8.1 Hz, 1H, Ar-H), 7.11 (d, J = 8.9 Hz,
1H, Ar-H), 7.31 (s, 1H, Ar-H), 7.90 (d, J = 8.9 Hz, 1H, H6-2-hydroxy-4-met-
hoxy-N0-methylenebenzohydrazide), 8.34 (s, 1H, N=CH), 9.55 (s, 1H, OH),
11.65 (s, 1H, OH), 12.63 (s, 1H, NH); 13C NMR (126 MHz, DMSO) δc
(ppm): 14.2, 54.8, 63.3, 100.8, 105.8, 106.8, 109.7, 115.0, 121.7, 124.9,
128.7, 146.6, 148.5, 148.9, 161.8, 163.3, 164.6; MS (EI), m/z (%): 43(20),
89(23), 151.0(100), 193(14), 207 (5), 330.1 (M+, 18); I.R (KBr) Ʋmax (cm−1):
ArO-H: 3524, N-H: 3301, C-H-aromatic: 3080, CH-aliphatic: 2975, CH-
J = 8.0 Hz, 1H, Ar-H), 7.32 (d, J = 8.9 Hz, 1H, H6-2-hydroxy-4-met-
hoxy-N0-methylenebenzohydrazide), 7.48 (s, 1H, Ar-H), 7.91 (d,
J = 8.7 Hz, 1H, Ar-H), 8.45 (s, 1H, N=CH), 11.83 (s, 1H, OH), 12.51 (s,
1H, NH); 13C NMR (126 MHz, DMSO) δc (ppm): 19.8, 54.9, 55.3,
100.8, 105.9, 106.9, 109.4, 120.1, 122.8, 129.0, 132.5, 140.4, 147.2,
150.7, 161.7, 163.4, 164.7, 167.9; MS (EI), m/z (%): 57.1(33), 149.0
(100), 152 (74), 192.2 (95), 342.3 (M+, 20); I.R (KBr) Ʋmax (cm−1):

4
IRAJI ET AL.
ArO-H: 3582, 3582, 3534, N-H: 3300, C-H-aromatic: 3092, C-H-aro-
matic: 3049, CH-aliphatic: 2975, CH-aliphatic: 2952, CH-aliphatic:
2855, C=O ketone: 1675, C=O amide: 1653, C=N: 1595, C=C aro-
matic: 1547, C=C aromatic: 1458, N-H: 1514, CH3:1357, C-N: 1228,
C-O: 1115.
was added to each well of 96-well polystyrene photometric microplates
and then 10 μL tyrosinase (0.5 mg/ml) was added to wells. Ten microli-
ters of the compounds at different concentrations (prepared in DMSO)
was added to the test well and preincubated for 20 min. Finally, 20 μL of
the L-dopa solution as substrate was added and the absorbance was
measured at 475 nm using a microplate reader. For the positive control,
tested compounds were replaced with a positive drug. In the blank
group, 190 μL phosphate buffer was added without positive control or
tested derivatives and substrate. Each group was measured three times
in parallel to average. The IC50 values of the compounds showing half of
the maximal inhibitory concentration, the measurements and the calcula-
tions were determined with GraphPad Prism 6. Results were expressed
as mean SE and all experiments were performed in triplicate.
2.2
|
Tyrosinase inhibition assay
The anti-tyrosinase activity was performed using L-dopa as a substrate.
All the assays were under 50 mM M KH2PO4/K2HPO4 buffer, pH 6.8, in
200 μL total volume using commercially available tyrosinase
(EC 1.14.18.1, 5771 units/mg solid). At first, 160 μL phosphate buffer
Carbonyl linker regarded as
important fragment in TYRIs
R
O
O
Ar
O
N
N
N
H
N
R: Various aryl substitution
including halogen, hydroxy,
methoxy, ...
H
HO
O
OH
(B
) TYRI IC50= 1.58 µM
hydrazone was proven to be
effective in TYR inhibition and
H-bound interaction with TYR
Bio-replacement of aromatic
substitution with 2-hydroxy-4-
methoxybenzamide
R
O
O
O
N
N
H
A
( ) TYRI IC50= 1.02 µM
H
HO
C
HO
( ) TYRI IC50= 9.9 µM
FIGURE 1 Design of new 2-hydroxy-4-methoxybenzohydrazide analogs
SCHEME 1 Synthesis of N0-benzylidene-2-hydroxy-4-methoxybenzohydrazide derivatives 4a–j

IRAJI ET AL.
5
2.3
|
Kinetic assay
added to the assay solution containing various concentrations of L-
dopa (0.1, 0.25, 0.5, 0.75, and 1.5 mM) as the substrate and pre-
incubated with the enzyme at 37 ꢀC for 20 min. Kinetic characteri-
zation catalyzed by enzymes was achieved spectrometrically at
475 nm. The obtained data including michaelis constant (Km) and
the maximal velocity (Vmax) were transformed to Lineweaver–Burk
The mechanism of action of 4d was obtained by Lineweaver–Burk
plot in which the reaction rate and substrate concentration were
used to determine the type of inhibition of the TYRI by the men-
tioned compound. Compound 4d at several concentrations was
TABLE 1 Chemical structures and
physical properties of the synthesized
compounds
Compound
4a
Chemical formula
C15H13N3O5
R
Yield (%)a
Melting pointa
92
260–264
4b
4c
4d
4e
4f
C
15H13N3O5
54
57
58
41
98
264–266
224–227
210–214
215–220
241–245
C15H13BrN2O3
C15H13ClN2O3
C15H14N2O4
C17H18N2O6
4g
4h
C17H18N2O5
60
54
108–112
236–240
C16H16N2O5
4i
4j
C
17H18N2O5
90
42
162–167
180–185
C18H18N2O5
aYield of isolated purified compounds.

6
IRAJI ET AL.
plots of the inverse of velocities (1/V) versus the inverse of sub-
strate concentrations 1/[S] mM.
TABLE 2 IC50 values of N0-benzylidene-2-hydroxy-
4-methoxybenzohydrazide against tyrosinase enzyme in the presence
of L-dopa as the substrate
2.4
|
Molecular docking
The molecular docking studies were performed with the Autodock
4.2package with the pymol program. The crystal structure of tyrosi-
nase (PDB code: 2Y9X) was downloaded from the RCSB protein data
bank and prepared with Autodock 4.2 package. Briefly, water mole-
cules were removed, missing hydrogens and residues were added,
nonpolar hydrogens were merged and Gasteiger charges were calcu-
lated for protein. The glide grid center was setting according the geo-
metrical center of the original inhibitor (tropolone) and the grid size
Compounds
4a
R
IC50 (μM)a
9.43 0.43
3
was 60 × 60 × 60 Aꢀ with the default grid spacing of 0.375 Aꢀ. The
4b
8.19 0.25
other parameters were left at program default values. The 3D struc-
tures of the ligands were drawn by using the ChemDraw software,
minimized energy using semiempirical force field and prepared using
the LigPrep platform. Lamarckian genetic algorithm was applied to
model the interaction/binding between then ligand and the tyrosinase
active site. To choose and validate the docking algorithm, the
tropolone as cognate ligand inside the 2Y9X was redocked. Finally,
the best docking confirmations with minimum binding energy were
visualized using Discovery studio (Edraki et al., 2015).
4c
4d
10.01 0.52
7.57 0.01
4e
4f
56.05 0.71
50.10 0.68
3
|
RESULTS AND DISCUSSION
| Design of novel 2-hydroxy-
3.1
4-methoxybenzamide derivatives
Literature survey highlighted natural and synthetic TYRIs including
terpenoids (Ashraf et al., 2017; Lehbili et al., 2018; Revoltella
et al., 2019) chalcones, hydrochalcones (Huang et al., 2010; Niesen
et al., 2015; Takahashi et al., 2012), oxadiazole, triazolothiadiazole
4g
4h
10.0 0.56
(Ghani
& Ullah, 2010; Ha et al., 2012; Lam et al., 2010),
81.25 1.15
thiosemicarbazone (Lee et al., 2010; Yi et al., 2011) derivatives.
Among them phenolic contacting structures exerted the stimulating
anti-TYR properties with low toxicity by forming compound-Cu2+-his-
tidine complex.
4i
4j
9.20 0.18
•
In previous studies, 4-hydroxy-3-methoxy benzaldehyde analogs
(compounds A and B, Figure 1) demonstrated promising anti-
tyrosinase activity (Iraji et al., 2020; Walton et al., 2003). In this
study 2-hydroxy-4-methoxybenzaldehyde as the core backbone
was selected due to potential binding ability to TYR enzymes via
strong hydrogen bond interactions using OH and OCH3 motifs.
Moreover, the benzene aromatic ring could exert weak interactions
such as ion-dipole, Van der Waals force, and so on.
8.92 0.016
Kojic acidb
–
20.24 4.28
aData presented here are the mean SEM. of three to six independent
•
Carbonyl-based compounds and related analogs (compound C,
Figure 1) are known by their copper chelating ability (Iraji
et al., 2018; Iraji et al., 2019) and H-bond interactions with amino
experiments.
bUsed as positive control.

IRAJI ET AL.
7
acids in TYR, leading to inactivation and loss of proteins function
3.3
|
Structure–activity relationship studies
as efficient anti-TYR agents.
•
Hydrazone linker (compounds B and C, Figure 1) as an active
nucleophilic motif could covalently bind to electron-deficient
groups; therefore, various aryl groups could bind into hydrazone
linker to investigate the effects of substitutions on TYR inhibitory
activities. Furthermore, hydrazone linker also exerts a pivotal role
The synthesized compounds (4a–j) were evaluated for their anti-TYR
potential against mushroom TYR. The concentrations of compounds
required for 50% inhibition of TYR enzyme (IC50) were determined
and listed in Tables 2.
All the compounds exhibited moderate to significant anti-TYR
activity with IC50 values ranging from 7.57 to 81.25 μM compared to
standard drug Kojic acid with IC50 = 20.24 4.28 μM.
According to these data, the preliminary SARs of these novel N0-
benzylidene-2-hydroxy-4-methoxybenzohydrazide derivatives were
summarized in Figure 2.
via H-bound interactions with
a TYR enzyme (Dehghani
et al., 2019; Iraji et al., 2019; Iraji et al., 2020).
As a result, the newly prepared 2-hydroxy-4-methoxybenzohydrazide
analogs were designed and screened for their in vitro anti-tyrosinase activ-
ities. Mechanism of inhibition and molecular docking studies of the highly
active compound was also evaluated.
1. The activities comparison of compounds 4a and 4b containing
NO2 motif demonstrated that 4-NO2 substitution (4b,
IC50 = 8.19 0.25 μM) had better potency than 3-NO2 one (4a,
IC50 = 9.43 0.43 μM).
3.2
|
Chemistry
2. The inhibitory activity of synthetic derivatives depicted that 4d
(para-chlorinated compound) was the best TYRI with IC50 of
An efficient synthesis of the new N0-benzylidene-2-hydroxy-
4-methoxybenzohydrazide analogs 4a-j has been performed starting
with methyl 2-hydroxy-4-methoxybenzoate (1) according to literature
methods (Schemes 1.).
90
80
70
60
50
40
30
20
10
0
0 μM
Commercially available methyl 2-hydroxy-4-methoxybenzoate
(1, Schemes 1) was reacted with hydrazine hydrate via nucleophilic
substitution reaction in refluxing ethanol to produce intermediate
2 in an excellent yield of 83%. Intermediate 2 was stirred with cata-
lytic amounts of acetic acid in refluxing ethanol and then
substituted aromatic aldehydes were added to the mixture. Com-
pletion of the reaction was monitored by TLC. Then, the mixture
was concentrated and the obtained precipitate was filtered and
crystallized from ethanol. 1H and 13C NMR confirmed all the hydro-
gen and carbon atoms of the synthesized compounds. Mass
spectroscopy confirmed the molecular weights of compounds by
their molecular ion peaks. Some physical properties of synthesized
derivatives are presented in Table 1.
10 μM
25 μM
1.2
1
0.8
0.6
0.4
0.2
0
-0.2
-0.4
-10
1/[S] (mM )-1
FIGURE 3 Lineweaver–Burk plot obtained through the kinetics
study of the compound 4d on TYR
FIGURE 2 General representation of 2-hydroxy-4-methoxybenzamide for SAR analysis

8
IRAJI ET AL.
7.57 μM. It seems that the para substituent on the benzyl ring is
0.5, 0.75, and 1 mM). A parallel control experiment was carried out
without compound 4d in the mixture. Lineweaver–Burk plot was
made based on the reaction rate and substrate concentration to
determine the type of inhibition of the TYR by the compound. The
graphical analysis and inhibitory data of the 4d against TYR is shown
in Figure 3 and Table 3, respectively. According to the Figure 3, it can
be judged that the inhibition mode of compound 4d against TYR is
mixed inhibition.
crucial to improve the anti-TYR activities.
3. The effects of OH motif on the benzyl ring were also considered.
Compound 4e with the 4-OH-benzyl group at R position depicted
the IC50 of 56.05 μM. Adding two extra methoxy groups with
electron-donating properties slightly increases the inhibitory activ-
ity like 4f with IC50 of 50.10 μM. Interestingly, compounds 4g with
4-hydroxy-3-ethoxybenzyl motif at R position demonstrated sig-
nificant improvement against tyrosinase (IC50 = 10.0 0.56 μM).
This might be attributed to the presence of extra bulky sites of
hydrogen bonding interaction in 4g with TYR.
3.5
|
Molecular modeling
4. In the case of OMe-substituted compounds (4f, 4h, 4i, and 4j), it
was found that the least activity belong to 4h with 3-hydroxy-4-
With the aim of obtaining useful information about the binding inter-
actions between the most potent compound (4d) and inactive com-
pound (4h) molecular modeling study was carried out using a docking
program AUTODOCK 4.0 package with PyMOL program. The free
binding energy of the mentioned compounds plus their interactions
with amino acid residues in the TYR active site were presented in
Table 4. The self-docking validation process resulted in RMSD <2.0.
3D interaction patterns of compound 4d have been shown in
Figure 4. Compound 4d as the most potent TYR inhibitor with IC50
value 7.57 μM demonstrated critical metal acceptor interaction with
one of Cu2+ ion plus H-bound and pi-pi interactions with His263. Fur-
ther four π-aryl stacking interactions were also constructed which
methoxy benzyl derivative at
R position. Unexpectedly, the
isosteric substitution of a meta-OH group with a meta-OMe sub-
stituent of phenyl ring can increase the inhibitory activity signifi-
cantly (4h, IC50 = 81.25 1.15 μM vs. 4i, IC50 = 9.20 0.18 μM).
The replacement of para methoxy group of 4i with acetyl one exerted
another excellent TYR inhibitor (4j) with IC50 of 8.92 μM. These data
were contributed to the anti-tyrosinase activities of OMe-containing
compounds in the order of 4j (3-methoxy,4-hydroxy) > 4i (3,4-
dimethoxy)
> 4f (4-hydroxy-3,5-dimethoxy) > 4h (3-hydroxy,
4-methoxy) analogs.
3.4
|
The mechanism of tyrosine inhibition
A study for the inhibitory mechanism of action was carried out with
the active TYRI compound, 4d. For this purpose, the rate of the
enzyme activity was carried out without the inhibitor and in 10 and
20 μM concentrations of the inhibitor in the presence of tyrosinase
enzyme using different concentrations of L-dopa as substrate (0.25,
TABLE 3 Kinetic parameters for the compounds 4d against TYR
Compound
4d (0 μM)
Km (mM)
11.64
Vm (OD/min)
0.23
4d (10 μM)
4d (25 μM)
12.18
0.22
FIGURE 4 3D interaction pattern of 4d in the active site of TYR
enzyme
12.91
0.21
TABLE 4 Docking scores and
interactions of 4d and 4h against TYR
Compound
4d
ΔGb (kcal/Mol)
−9.95
Ki
Interactions
50.75 (nM)
Hydrogen bond: Val283
enzyme
Pi-pi interaction: His263
Pi-Aryl interaction: Val283
Pi-Aryl interaction: Ala286
Pi-Aryl interaction: Pro277
Pi-Aryl interaction: Lue275
Metal acceptor interactions: one Cu2+
4h
−6.71
12.06 (μM)
Pi-Aryl interaction: Pro277
Pi-pi interactions: His263
Metal acceptor interactions: one Cu2+

IRAJI ET AL.
9
DATA AVAILABILITY STATEMENT
The data sets used and analyzed during the current study are available
from the corresponding author on reasonable request. We have pres-
ented all data in the form of tables and figures.
ORCID
Aida Iraji
https://orcid.org/0000-0002-8442-2205
Zahra Panahi
Najmeh Edraki
https://orcid.org/0000-0002-4967-1361
https://orcid.org/0000-0001-8306-2642
Mahsima Khoshneviszadeh
4624
https://orcid.org/0000-0002-6993-
Mehdi Khoshneviszadeh
https://orcid.org/0000-0003-1458-2964
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How to cite this article: Iraji A, Panahi Z, Edraki N,
Khoshneviszadeh M, Khoshneviszadeh M. Design, synthesis,
in vitro and in silico studies of novel Schiff base derivatives of
2-hydroxy-4-methoxybenzamide as tyrosinase inhibitors. Drug
Dev Res. 2020;1–10. https://doi.org/10.1002/ddr.21771