ORIGINAL ARTICLES
Dihydo p-methoxy cinnamic acid (3a): Light yellow powder; Yield 95.4%;
m.p. 96–98 ◦C. IR max (KBr): 2931, 1701, 1613, 1513, 821 cm−1. 1H-NMR
(400 MHz, CDCl3) δ 7.12 (2H, d, 8.4 Hz, H-2 and H-6), 6.83 (2H, d, 8.4 Hz,
H-3 and H-5), 3.78 (3H, s, 4-OMe), 2.89 (2H, t, 8.0 Hz, H-7), 2.64 (2H, t,
8.0 Hz, H-8); 13C NMR (100 MHz, CDCl3) δ 29.7 (C-7), 35.9 (C-8), 55.2(4-
OMe), 113.9 (C-3 and C-5), 129.2 (C-2 and C-6), 132.2(C-1), 158.1 (C-4),
178.9 (C-9).
Dihydro caffeic acid (4a): Light yellow crystals; Yield 98.0%; m.p. 130–
131 ◦C. IR
(KBr) 3367, 1681, 1604, 1526, 820 cm−1 1H NMR
.
max
(400 MHz, DMSO) δ 6.62 (1H, d, 8.0 Hz, H-5), 6.59 (1H, d, 2.0 Hz, H-
2), 6.44 (1H, dd, 2.0 Hz, 8.0 Hz, H-6), 2.64 (2H, t, 7.2 Hz, H-7), 2.42 (2H, t,
7.2 Hz, H-8); 13C NMR (100 MHz, DMSO) δ 29.8 (C-7), 35.8 (C-8), 115.5
(C-5), 115.7 (C-2), 118.8 (C-6), 131.7 (C-1), 143.4 (C-3), 145.0 (C-4), 174.0
(C-9).
Dihydro ferulic acid (5a): Light beige crystals; Yield 98.0%; m.p. 86–87 ◦C.
IR
(KBr): 3423, 2938, 1702, 1605, 819 cm−1 1H NMR (400 MHz,
.
max
DMSO) δ 6.84 (1H, d, 7.2 Hz, H-5), 6.71 (1H, d, 2.0 Hz, H-2), 6.70 (1H, dd,
2.0 Hz, 7.2 Hz H-6), 3.87 (3H, s, 3-OMe), 2.89 (2H, t, 8.0 Hz, H-7), 2.65
(2H, t, 8.0 Hz, H-8); 13C NMR (100 MHz, DMSO) δ 30.1 (C-7), 35.8 (C-8),
55.5 (3-OMe), 112.5 (C-2), 115.3 (C-5), 120.3 (C-6), 131.7 (C-1), 144.7
(C-3), 147.4 (C-4), 174.0 (C-9).
Dihydro isoferulic acid (6a): White crystals; Yield 94.6%; m.p. 142–
143 ◦C. IR max (KBr): 3396, 2939, 1702, 1590, 1518, 808 cm−1. 1H NMR
(400 MHz, CDCl3) δ 6.79 (1H, d, 1.6 Hz, H-2), 6.77 (1H, d, 8.4 Hz, H-5),
6.68 (1H, dd, 8.4 Hz, 1.6 Hz, H-6), 3.86 (3H, s, 4-OMe), 2.87 (2H, t, 7.6 Hz,
H-7), 2.64 (2H, t, 7.6 Hz, H-8); 13C NMR (100 MHz, CDCl3) δ 30.0 (C-
7), 35.6 (C-8), 56.0 (4-OMe), 110.7 (C-5), 114.5 (C-2), 119.6 (C-6), 133.5
(C-1), 145.1 (C-3), 145.6 (C-4), 178.2 (C-9).
Fig. 4: Inhibitory effects of dihydro cinnamic acid derivatives 1a-8a against
tyrosinase activity, Dihydro cinnamic acid (1a), Dihydro-p-coumaric
acid (2a), Dihydro-p-methoxy cinnamic acid (3a) Dihydro caffeic acid
(4a), Dihydro ferulic acid (5a), Dihydro isoferulic acid (6a),
3,4-Dimethoxy-dihydro cinnamic acid (7a), Dihydro sinapic acid (8a),
Arbutin, Kojic acid
3,4-Dimethoxy dihydro cinnamic acid (7a): Light beige crystals; Yield
78.7%; m.p. 89–90 ◦C. IR
(KBr): 2934, 1699, 1591, 1517, 841 cm−1
.
max
1H NMR (400 MHz, CDCl3) δ 6.80 (1H, d, 8.8 Hz, H-5), 6.75 (1H, dd,
8.8 Hz, 2.0 Hz, H-6), 6.74 (1H, d, 2.0 Hz H-2), 3.86 (3H, s, 4-OMe), 3.87
(3H, s, 3-OMe), 2.91 (2H, t, 8.0 Hz, H-7), 2.67 (2H, t, 8.0 Hz, H-8); 13C
NMR (100 MHz, CDCl3) δ 30.2 (C-7), 35.8 (C-8), 55.8 (4-OMe), 55.9 (3-
OMe) 111.3 (C-2), 111.7 (C-5), 120.0 (C-6), 132.8 (C-1), 147.5 (C-4), 148.9
(C-3), 178.2 (C-9).
antioxidants may inhibit the oxidation step, without interact-
ing with tyrosinase. Compounds 2 and 6 showed the highest
tyrosinase inhibitory activity with IC50 values of 115.6 M and
114.9 M. but these compounds showed almost no free radi-
cal scavenging activity. The inhibition data combined with the
free radical scavenging activity indicated that two activities are
not directly correlated. However, the substituent groups of cin-
namic acid, and C=C double bond of cinnamic acid derivatives
effect both tyrosinase inhibitory activity and radical scavenging
activity. The results obtained have provided a useful clue to the
design and development of new tyrosinase inhibitors.
Dihydro sinapic acid (8a): Beige crystals; Yield 93.2%; m.p. 97–98 ◦C.
IR
(KBr): 3457, 2948, 1703, 1621, 804 cm−1 1H NMR (400 MHz,
.
max
CDCl3) δ 6.43 (2H, s, H-2, 6), 3.85 (6H, s, 3-OMe and 5-OMe), 2.88 (2H, t,
8.0 Hz, H-7), 2.65 (2H, t, 8.0 Hz, H-8)13C-NMR (100 MHz, CDCl3) δ 30.8
(C-7), 36.0 (C-8), 56.2 (3-OMe and 5-OMe), 104.9 (C-2,6), 131.3 (C-1),
133.1 (C-4), 147.0 (C-3,5), 178.9 (C-9).
3.2.2. Synthesis of dihydro p-coumaric acid (2a)
Compound 2 (200 mg, 1.2 mmol) was dissolved in 4 ml of methanol and
then p-toluene sulfonic acid monohydrate (8.0 mg, 0.05 mmol) was added
and the mixture was refluxed for 2 h. After addition of H2O, the mixture
was made neutral by adding prydine and extracted with EtOAc. The extract
was washed with brine, dried over Na2SO4, and evaporated under reduced
pressure to leave white crystals, which were column-chromatographed on
SiO2 with hexane – EtOAc (6:1, v/v) to give p-methyl coumarate (172 mg,
79.2%). To a stirred solution of p-methyl coumarate (142 mg, 0.8 mmol) and
CuCl (238 mg, 2.4 mmol) in methanol (15 ml) was added NaBH4 (304 mg,
8 mmol) in small portions over a period of 30 min at 0 ◦C (Narisada et al.
1989). Then the resulting black residue was removed by filtration, and the
filtrate was acidified with 5% aqueous HCl and extracted with EtOAc. The
extract was washed successively with saturated aqueous NaHCO3 and brine
and dried over Na2SO4. The solvent was evaporated under reduced pressure,
to give white crystals. The white crystals were identified as dihydro p-methyl
coumarate (134 mg, 93.3%) by NMR data. The base hydrolysis of dihydro
p-methyl coumarate gave compound 2a (87 mg, 70.2%).
3. Experimental
3.1. Materials
Thin layer chromatography (TLC) was performed on precoated plates
(silica gel 60 F254, 0.25 mm, Merck, Darmstadt, Germany). Column chro-
matography was carried out using 70 – 230 mesh silica gel (Kieselgel 60,
Merck, Germany). Melting points (m.p.) were measured on a MP-5000D
melting-point apparatus. UV spectra were measured on a Hitachi U-1500
spectrophotometer. 1H and 13C NMR data were all obtained on a JEOL
ECA-400 (400 MHz) in CDCl3 and DMSO-d6 with TMS as internal stan-
dard. Tyrosinase (E.C. 1.14.18.1) was purchased from Sigma-Aldrich (St.
Louis, Mo), l-tyrosine, cinnamic acid, p-coumaric acid, ferulic acid, caf-
feic acid, sinapic acid, p-methoxy-cinnamic acid, 3,4-dimethoxy cinnamic
acid, CuCl, NaBH4, DPPH (1,1-diphenyl-2-picrylhydrazyl), BHT(2,6-di-
tert-butyl-4-methyl- phenol), and p-toluenesulfonic acid monohydrate were
purchased from Wako Pure Chemistry (Osaka, Japan), arbutin, kojic acid
and palladium 10% on carbon (wetted with ca. 55% water) were purchased
from Tokyo Kasei Kogyo (Tokyo, Japan), and isoferulic acid and all solvents
were purchased from Kanto Chemical (Tokyo, Japan).
Dihydro p-coumaric acid (2a): White crystals; Yield 51.9%; m.p. 118–
120 ◦C. IR
(KBr): 3393, 3026, 1703, 1599, 828 cm−1 1H NMR
.
max
(400 MHz, CDCl3) δ 7.07 (2H, d, 8.0 Hz, H-2 and H-6), 6.76 (2H, d, 8.0 Hz,
H-3 and H-5), 2.89 (2H, t, 8.0 Hz, H-7), 2.64 (2H, t, 8.0 Hz, H-8); 13C NMR
(100 MHz, CDCl3) δ 29.8 (C-7), 35.5 (C-8), 115.4 (C-3 and C-5), 129.4
(C-2 and C-6), 131.1 (C-1), 155.6 (C-4), 179.9 (C-9).
3.2. Synthesis of dihydro cinnamic acid derivatives 1a - 8a
3.2.1. Synthesis of dihydro cinnamic acid derivatives 1a, 3a – 8a
Compounds 1, 3–8 (2 mmol) were dissolved in 8 ml of methanol, palladium
on carbon (10%, wet, 200 mg) was added and the mixture was stirred under
H2 at atmospheric pressure at room temperature for 2 h. The catalyst was
filtered through a bed of Celite® and solvent was evaporated under reduced
pressure to yield 78.7%-98.0%.
3.3. Methods
3.3.1. DPPH radical scavenging activity
The scavenging activity of cinnamic acid derivatives for the DPPH radical
was monitored according to the method of Gaspar et al. (2009). A quantity
of 500 L of a 0.5 mM methanoic DPPH solution was mixed in a cuvette
with 500 L of cinnamic acid derivatives at different concentration levels.
These cuvettes were shaken vigorously. The cuvettes were allowed to stand
at 27 ◦C for 30 min, and the absorbance was measured at 517 nm using a U-
1500 spectrophotometer. The percentage of radical scavenging activity was
Dihydro cinnamic acid (1a): White crystals; Yield 95.0%; m.p. 46–49 ◦C.
IR max (KBr): 3029, 1698, 1454, 754 cm−1. 1H NMR (400 MHz, CDCl3) δ
7.19 – 7.31 (5H, m, aromatic), 2.96 (2H, t, 8.0 Hz, H-7), 2.68 (2H, t, 8.0 Hz,
H-8); 13C NMR (100 MHz, CDCl3) δ 30.6 (C-7), 35.6 (C-8), 126.3 (C-4),
128.2 (C-3 and C-5), 128.5 (C-2 and C-6), 140.1 (C-1), 179.2 (C-9).
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Pharmazie 65 (2010)