A New Diglycoside of Diterpene from Ageratina vacciniaefolia

From leaves and flowers of Ageratina vacciniaefolia [1-7] we have isolated several compounds: a flavonoid, a diterpene [8] and a new compound identified as b-D-glucopyranosil ester of (-)17-(b- glucopiranosyloxyl)-16-hydroxy-kauran-19-oic acid (see the formula). The structure was established by using 1HNMR, 13CNMR, COSY, NOESY and HMBC spectroscopic techniques (

Table 1. The assignment of the NMR data [2-6].

No. C	Type of C	d -ppm
19	-COO	178.2
1*	CH(anomeric)	98.5
1**	CH(anomeric)	95.6
16	C-O	91.4
	CH –O	78.7
	CH -O	78.7
	CH -O	78.3
	CH -O	77.7
	CH-O	75.2
	CH -O	74.1
	CH -O	71.4
	CH -O	71.1
17	CH2-O	63.6
	CH2OH	62.5
	CH2-O	62.4
9	CH	58.5
5	CH	57.3
15	CH2	51.4
solv.	solv.	49.8
8	C	46.0
4	C	45.1
13	CH	44.2
7	CH2	43.1
1	CH2	41.8
10	C	40.9
3	CH2	39.1
14	CH2	37.6
18	CH3	29.1
12	CH2	26.8
6	CH2	23.2
11	CH2	20.1
2	CH2	19.7
20	CH3	16.4

) [1,7].

The 13CNMR spectrum showed totally 32 signals, 12 of them at ca. 62-99 ppm which are characteristic of glycoside. The rest 20 signals are attributed to the aglicone which is a compound of diterpene kaurane type, and the structure has been confirmed by both 13CNMR and 1HNMR spectra.

The signals from 1HNMR at d 4.48 ppm (d, J=7.2 Hz) was assigned to the anomeric proton, whcih has a direct correlation with C of d 98.45 ppm indicating a b-glycoside; while 1HNMR signals appear at d 5.434 ppm (d, J= 7.2 Hz) coupling directly with C at d 95.56 ppm can be attributed to a b-glucose ester.

Furthermore, the absence of signals to vinilic protons and the absence of signals to C9 and C15 connected to oxygen, similar to those in the other compound [8], indicate that this aglicone compound is a Kaurane derivative. The signal from 13CNMR spectrum at d 63.60 ppm showed a -CH2OH connected to C17 which was confirmed by correlation observed on HMBC and NOESY spectra. A signal at d 91.55 ppm indicating a glucose connected to C16 is confirmed by a long range correlation on HMBC spectrum. An enzymatic hydrolysis of this compound with b-glucosidase also confirmed the structure purposed.

In summary, this compound was identified as b-D-glucopyranosil ester of (-)17-(b-

glucopiranosyloxyl)-16-hydroxykauran-19-oic acid by using HNMR and 13CNMR spectra analysis and enzymatic hydrolysis with b-glucosidase [7].

The ethanolic extract from leaves and flowers of A. vacciniaefolia yielded white crystals after column chromatography and eluted with CH2Cl2, EtOAc and mixtures of these solvents. The compound was purified further by column chromatography on RP-18 eluted with MeOH-H2O (2:1).

M.p. 198ºC.

[a]20D = -52.5 (0.0043 MeOH).

DCIMS [isobutane] m/z: 499; 481; 463; 419; 392; 391; 361; 319; 273; 163; 145 (100%); 127.

1HNMR (360 MHz, CD3OD): d in ppm, 0.96 (s, 3H, CH3); 1.20 (s, 3H, CH3);1.45 (m, 1H); 1.85 (m, 1H);

2.25 (m, 1H); 3.18 (m, 1H); 3.33 (m, 1H); 3.37 (m, 2H); 3.7 (dd, 1H, J=12.3Hz); 3.8 (dd, 1H, J=12.2 Hz);

4.48 (d, 1H, J=7.2 Hz, anomeric proton); 5.43 (d, 1H J=7.2, anomeric proton).

13CNMR (90.5 MHz, CD3OD): d 16.38 (-CH3, C); 19.71; 29.05 (-CH3, C); 20.12; 25.17; 26.82; 37.62;

39.05; 40.92; 41.84; 43.07; 44.21; 45.08; 45.97; 51.40; 57.29; 58.54; 62.40; 62.52 (-CH2OH); 63.60

(-CH2-O-); 71.10; 71.42; 74.08; 75.18; 77.73; 78.29; 78.64 (2C); 91.55; 95.56; 98.45; 178.16 (-COO,

C19). For details, see the following table (Table 1).