successively with 4 M HCl solution, saturated NaHCO solution, and water, and then dried over Na SO and evaporated.
3
2
4
The crude products were purified by column chromatography on silica gel with petroleum ether–ethyl acetate (4:1) as eluent.
The structures of the synthesized esters 1–15 were established by spectroscopic methods. Among them, 6, 8, and 12 are new
compounds, and the others are known compounds.
Hexan-2-yl 3,4,5-trihydroxybenzoate (6), C H O , obtained in 45% yield as a colorless solid. EI-MS (m/z, Irel.,
1
3 18 5
+
1
%
): 254 [M] (26), 224 (10), 197 (4), 170 (100), 153 (69), 125 (14), 107 (3), 79 (8), 56 (19). H NMR (400 MHz, CDCl , δ,
3
ppm, J/Hz): 7.25 (2H, s, H-1, 5), 5.05 (1H, m, CH), 1.86, 1.30, 1.28 (each 2H, m, CH ), 1.27 (3H, d, J = 6.1, CH ), 0.86 (3H,
2
3
1
3
t, J = 7.3, CH3). C NMR (100 MHz, CDCl , δ, ppm): 167.3 (CO), 143.9 (C), 143.8 (C), 136.8 (C), 121.9 (C), 109.9 (CH),
3
1
09.9 (CH), 72.4 (OCH), 35.6 (CH ), 27.5 (CH ), 22.5 (CH ), 19.9 (CH ), 13.9 (CH ).
2 2 2 3 3
Benzyl 3,4,5-trihydroxybenzoate (8), C H O , obtained in 52% yield as a colorless solid. EI-MS (m/z, Irel., %):
1
4 12 5
+
1
2
60 [M] (16), 224 (33), 153 (40), 143 (34), 125 (5), 107 (4), 99 (43), 83 (10), 70 (23), 56 (100). H NMR (400 MHz, CDCl ,
3
1
3
δ, ppm): 7.13 (2H, s, H-2, 6), 7.37–7.29 (5H, m, ArH), 5.24 (2H, s, OCH2). C NMR (100 MHz, CDCl , δ, ppm): 167.0 (CO),
3
1
(
44.5 (C), 144.5 (C), 137.4 (C), 136.1 (C), 128.4 (CH), 128.4 (CH), 128.0 (CH), 128.0 (CH), 128.0 (CH), 120.9 (C), 109.4
CH), 109.4 (CH), 66.4 (OCH2).
-Isopropyl-5-methylcyclohexyl 3,4,5-trihydroxybenzoate (12), C H O , obtained in yield 54% as a colorless
2
1
7 24 5
–
1
solid. ESI-MS m/z 307 [M – 1] . H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 7.11 (2H, s, ArH), 3.41, 2.02 (each 1H, m, CH),
3
1
.87 (2H, br, CH ), 1.84 (1H, m, CH), 1.66 (1H, m, CH), 1.62, 1.27 (each 2H, m, CH ), 0.85 (6H, d, J = 7.7, CH ), 0.72 (3H,
2
2
3
1
3
d, J = 6.9, CH3). C NMR (100 MHz, CDCl , δ, ppm): 166.8 (C), 144.5 (C), 144.5 (C), 137.0 (C), 121.7 (C), 109.3 (CH),
3
1
2
09.3 (CH), 74.1 (CH), 47.2 (CH), 40.9 (CH ), 33.7 (CH ), 23.5 (CH ), 31.4 (CH), 26.3 (CH), 23.5 (CH ), 22.0 (CH ),
2 2 2 3 3
0.7 (CH3).
The known compounds 1–5, 7, 9–11, 13–15 were characterized as methyl 3,4,5-trihydroxybenzoate (1, C H O ,
8
8 5
yield 93%) [8, 10], ethyl 3,4,5-trihydroxybenzoate (2, C H O , yield 86%) [8, 10], isopropyl 3,4,5-trihydroxybenzoate
9
10 5
(
3, C H O , yield 81%) [9], isobutyl 3,4,5-trihydroxybenzoate (4, C H O , yield 87%) [9], isopentyl 3,4,5-trihydroxybenzoate
10 12 5 11 14 5
(
5, C H O , yield 77%) [8], heptyl 3,4,5-trihydroxybenzoate (7, C H O , yield 63%) [8, 10, 11], octyl 3,4,5-trihydroxybenzoate
12 16 5 14 20 5
(
9, C H O , yield 71%) [7, 10, 11], nonyl 3,4,5-trihydroxybenzoate (10, C H O , yield 80%) [8, 10, 11], decyl
15 22 5 16 24 5
3
,4,5-trihydroxybenzoate (11, C H O , yield 68%) [8, 10, 11], dodecyl 3,4,5-trihydoxybenzoate (13, C H O , yield 72%)
17 26 5 19 30 5
[
(
7, 10, 11], hexadecyl 3,4,5-trihydroxybenzoate (14, C H O , yield 73%) [10], and octadecyl 3,4,5-trihydroxybenzoate
23 38 5
1
13
15, C H O , yield 61%) [10] by comparison of their H NMR, C NMR, and MS data with those reported.
2
5 42 5
Biological Assay. Mycelial Growth Inhibition Test in vitro. The synthesized compounds were dissolved in acetone
and tested for antifungal activities in vitro by a poison food technique [26]. Potato dextrose agar (PDA) was used as the
–
1
medium for all test fungi. The media incorporating test compounds at a concentration of 50 μg mL was inoculated at the
center of the test fungi in agar discs (4 mm diameter). Three replicate plates for each fungi were incubated at 26 ± 2°C.
Control plates containing media mixed with acetone (1 mL) were included. After incubation for 2–6 days, the mycelial growth
of fungi (mm) in both treated (T) and control (C) Petri dishes were measured diametrically in three different directions until the
fungal growth in the control dishes was almost complete. The percentage of growth inhibition (I) was calculated using the
formula
I (%) = [(C – T)/C] × 100 (1)
The corrected inhibition (IC) was then calculated as follows:
IC = [(I – CF)/(100 – CF)] × 100 (2),
where CF = [(90 – C )/C × 100], 90 is the diameter (mm) of the Petri dish, and C is the growth (mm) of the fungus in the control.
0
0
0
Analysis of variance was performed on the data with the PROC GLM procedure (SAS Institute, Cary, NC, USA).
If the value of P > F was less than 0.01, the means were separated with the least significant different (LSD) test at the
p = 0.05 level.
In vivo Assay. In order to further investigate the in vivo antifungal activities of the synthesized compounds, such as
the duration of protection and curative activity, the plant disease of wheat powdery mildew (E. graminis) was used in the test.
The effects of the test compounds on disease development and spread were determined using potted plants in a greenhouse.
The potted plants were arranged randomly in two groups in a greenhouse and watered twice daily with tap water. The potted
–1
plant seedlings were sprayed with solutions of the test compounds in water–acetone (95:5) that contained Tween 20 (250 g mL )
as wetter and allowed to stand for 24 h.
For the test of preventive effects, the plants in the first group were inoculated with the pathogen of the plant disease
–
1
one day after being sprayed with either the test compounds or a standard fungicide at a dose 500/250 μg mL . For the test of
4
1