H. T. Aung et al. / Bioorg. Med. Chem. 19 (2011) 2392–2396
2395
Cinnamic acid, 2-hydroxy cinnamic acid, 4-hydroxy cinnamic acid,
caffeic acid, 3-(4-hydroxy-3-methoxyphenyl)lactic acid, 4-hydro-
xy-3-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, 3,4-
dihydroxyphenyllactic acid, 3,4,5-trimethoxycinnamic acid, 2,4,5-
trimethoxycinnamic acid, 2,3,4-trimethoxycinnamic acid, crotonic
acid and sorbic acid were obtained from Tokyo Chemical Industry
Co., Ltd (Tokyo, Japan). 3-Hydroxy cinnamic acid was purchased
from Wako Pure Chemical Industries, Ltd (Osaka, Japan). 2,4-
Dihydroxycinnamic acid was purchased from Sigma–Aldrich Co.
(St. Louis, MO, U.S.A.). Other chemicals used were of analytical
grade. Rosmarinic acid (1, Z and E-mixture) and rosmarinic methyl
ester (5) were isolated from methanolic extract of A. argentea as
described in Ref. 5.
Saturated caffeic acid (= 3-(3,4-Dihydroxyphenyl)propanoic
acid) (22): IR mmax (film) 1715 cmꢁ1 1H NMR (CD3OD, 500 MHz):
;
d 6.62 (1H, d, J = 7.6 Hz; H-50), 6.59 (1H, d, J = 1.5 Hz; H-20), 6.48
(1H, dd, J = 7.6, 1.5 Hz; H-60), 2.71 (2H, t, J = 7.6 Hz; H-3), 2.47
(2H, t, J = 7.6 Hz; H-2); EIMS m/z 182 [M]+, 123 (base).
4.7. Methylation of caffeic acid (2)
Trimethylsilyldiazomethane (174 lL) was added to caffeic acid
(2, 215 mg, 1.19 mmol) in 10 mL of acetone, and the solution was
stirred at 0 °C for 20 min. The solution was evaporated, and the res-
idue was purified by SiO2 column chromatography to afford
methyl caffeate (6, 145 mg, 67.4%).
Methyl caffeate (6): IR m
max (film) 3363 (br), 1684 cmꢁ1; 1H NMR
4.3. Separation of Z- and E-isomers of rosmarinic acid
(CD3OD, 500 MHz): d 7.52 (1H, d, J = 15.9 Hz; H-3), 7.01 (1H, d,
J = 1.5 Hz; H-20), 6.92 (1H, dd, J = 8.3, 2.2 Hz; H-60), 6.75 (1H, d,
J = 8.3 Hz; H-50), 6.24 (1H, d, J = 15.9 Hz; H-2), 3.73 (3H, s;
COOCH3); EIMS m/z 194 [M]+.
A mixture of Z- and E-isomers of rosmarinic acid were separated
using HPLC equipped with COSMOSIL Cholester (/20 ꢀ 250 mm)
eluted by a mixed solvent of acetonitrile–water–trifluoroacetic
acid (2:8:0.05) at a flow rate of 5 mL/min. The column was main-
tained at a constant temperature of 30 °C. UV monitoring was done
at 220 nm. (Z)- and (E)-Rosmarinic acid (1a and 1b) was eluted at
68 min and 76 min, respectively.
4.8. Preparation of (E)-3,4,5-trihydroxycinnamic acid (12)
Aniline (50 lL) was added to a solution of 3,4,5-trihydroxybenz-
aldehyde (250 mg) and malonic acid (250 mg) in anhydrous pyri-
dine (1.25 mL) and the reaction mixture was stirred at 50 °C for
12 h. After cooling, diethylether (20 mL) was added to the solution.
The organic phase was washed with 2 M hydrochloric acid and
water, and dried over anhydrous magnesium sulfate. The product
was purified by HPLC, which afforded the desired product (12,
160 mg, 64%) as a brown crystal.
4.4. Anti-hemorrhagic activity assay
Anti-hemorrhagic activity was examined under the modified
method reported previously.5,10 Male ddY mice of 20 g average
weight were used for this experiment. Test solutions were pre-
pared as follows: Crude snake venom solution of P. flavoviridis
(E)-3,4,5-Trihydroxycinnamic acid (12 ): 1H NMR (500 MHz,
CD3OD): d 7.42 (1H, d, J = 15.9 Hz; H-2), 6.58 (2H, s; H-20 and H-
60), 6.15 (1H, d, J = 15.9 Hz; H-3); EIMS m/z 196 [M]+, 122 (base).
(0.14 mg/mL in saline, 50
ylsulfoxide (DMSO)–saline, 50
37 °C for 10 min. The test solution (100
l
L) and sample solution in 10% dimeth-
L) were mixed and incubated at
L) was injected subcuta-
l
l
neously into the abdomen of the mice. Mice injected the vehicle
solution only served as a control group. After 24 h, mice were
euthanized by inhalation of chloroform, the skin covering the
abdomen was removed, and hemorrhagic lesions were determined
as follows. We evaluated the lesions by calculating a value of major
axes x minor axes, just as that of an ellipse, since the shapes of the
lesions are always amorphous.
4.9. Preparation of Z-trihydroxycinnamic acid
E-Trimethoxycinnamic acid was dissolved to dry CH2Cl2 under
an argon atmosphere and cooled to ꢁ78 °C. 1 M BBr3 in CH2Cl2
solution (2.8 equiv) was added dropwise to the solution and was
stirred at ꢁ78 °C for 1 h. Then the reaction temperature was grad-
ually raised to 0 °C. When the starting material disappeared on TLC,
methanol was added to the solution, and the reaction mixture was
evaporated. Methanol was added again to the residue and the solu-
tion was evaporated to remove methylborate. The product was
purified by SiO2 chromatography, and the desired product was
obtained.
(Z)-2,3,4-Trihydroxycinnamic acid (13) from E-2,3,4-trimethoxy-
cinnamic acid (8.9% yield): 1H NMR (CD3OD, 500 MHz): d 7.4 (1H,
d, J = 9.8 Hz; H-3), 6.87 (1H, d, J = 8.3 Hz; H-60), 6.47 (1H, d,
J = 8.3; H-50), 6.15 (1H, d, J = 9.8 Hz; H-2); 13C NMR (CD3OD,
125 MHz): d 162.1, 149.8, 145.3, 143.6, 132.1, 118.8, 112.6,
112.4, 110.8; EIMS m/z 178 [M–H2O]+, 150 (base).
4.5. Preparation of saturated rosmarinic acid (4)
Rosmarinic acid (1, 105 mg) was dissolved in acetone (1 mL),
and 5% palladium on activated carbon (20 mg) was added to the
solution. The heterogeneous mixture was stirred under a hydrogen
atmosphere at room temperature for 3 h. The mixture was filtered
through celite, affording a saturated rosmarinic acid (4) as a yellow
crystal (98 mg, 93%).
Saturated rosmarinic acid (4): IR mmax (film) 3364 (br) cmꢁ1
;
1H NMR (CD3OD, 500 MHz): d 6.71 (1H, d, J = 1.5 Hz; H-2), 6.61
(1H, d, J = 7.6 Hz; H-5), 6.55 (1H, dd, J = 7.6, 1.5 Hz; H-6), 4.97
(1H, dd, J = 9.8, 3.0 Hz; H-8), 2.84 (1H, dd, J = 14.4, 9.8 Hz; H-7),
3.04 (1H, dd, J = 14.4, 3.0 Hz; H-7), 6.58 (1H, d, J = 1.5 Hz; H-20),
6.66 (1H, d, J = 8.3 Hz; H-50), 6.36 (1H, dd, J = 8.3, 1.5 Hz; H-60),
2.65 (2H, t, J = 7.6 Hz; H-70), 2.52 (2H, t, J = 7.6 Hz; H-80); FAB-MS
m/z 385 [M+Na]+.
(Z)-2,4,5-Trihydroxycinnamic acid (14) from E-2,4,5-trimethoxy-
cinnamic acid (5.7% yield): 1H NMR (CD3OD, 500 MHz): d 7.75 (1H,
d, J = 8.3 Hz; H-3), 6.91 (1H, s, H-60), 6.73 (1H, d, J = 8.3, H-50),
6.15 (1H, d, J = 8.3 Hz; H-2); 13C NMR (CD3OD, 125 MHz): d 162.9,
150.7, 149.1, 144.7, 143.2, 111.7, 111.5, 111.1, 102.3; EIMS m/z
178 [M–H2O]+, 150 (base).
(Z)-3,4,5-Trihydroxycinnamic acid (15) from E-3,4,5-trimethoxy-
cinnamic acid (0.5% yield): 1H NMR (CD3OD, 500 MHz): d 7.6 (1H,
d, J = 9.1 Hz; H-3), 6.91 (1H, s; H-60), 6.72 (1H, s; H-20), 6.14 (1H,
d, J = 9.1 Hz; H-2); EIMS m/z 178 [M–H2O]+, 150 (base).
4.6. Preparation of saturated caffeic acid (22)
Five percentage palladium on activated carbon (20 mg) was
added to caffeic acid (2, 100 mg) in acetone (l mL). The heteroge-
neous mixture was stirred under a hydrogen atmosphere at room
temperature for 3 h. The mixture was filtered through celite which
afforded a saturated caffeic acid (22, 95 mg, 95%) as a brown
crystal.
4.10. Preparation of methyl cinnamate (20)
To a solution of (E)-cinnamic acid (7, 5.1 g) in 50 mL of acetone,
23.7 g of K2CO3 and 3.25 mL of dimethyl sulfate were added. The