The absolute configuration of prunioside A from Spiraea prunifolia and biological activities of related compounds Meng Que

Content 1. Abstract 2. Introduction 3. Experimental 4. Discussion

Phytochemistry, H Oh, G S Oh, W G S, et al. Prunioside A: A New Terpene Glycoside from Spiraea prunifolia [J]. J. Nat. Prod. 2001, 64:

1. Abstract The configurations at C-5 and C-6 were determined. The modified Mosher’s method, CD analysis, and 13 C NMR spectroscopic data analysis of an acetonide derivative. Other compounds related to prunioside A have inhibitory effects on the synthesis of nitric oxide in LPS-stimulated macrophage-like RAW cells.

2. Introduction Prunioside A (1) has been isolated from the methanol extract of Spiraea prunifolia var. simpliciflora. The roots of this plant have been used traditionally for the treatment of malaria, fever, and emetic conditions.

3. Experimental 3.1. Extraction and isolation of prunioside A (1) Collected from Iksan City, Chonbuk Province, Korea in May Fresh roots were dried in a well-ventilated darkroom. Symmetry Pre C 18 column (1.9 × 30 cm; 7-μm particle size; flow rate of 4 ml/min)

3.2. Determination of the absolute configurations of Prunioside A (1) The relative configurations of C-5 and C-6 could not be established from the NMR data, and exhaustive efforts to obtain crystals of 1 or 1a were unsuccessful.

3.2. Determination of the absolute configurations of Prunioside A (1) Enzymatic hydrolysis of 1 Mosher’s method Hydrolysis of 3 under mild alkaline condition Formation of acetonide 9 It has been reported that acetonides of syn and anti- 1,2-diols can be unambiguously distinguished by the 13 C NMR chemical shifts of the acetonide methyl groups. (Dana and Danechpajouh, 1980; Solladie′ et al., 1997) Formation of 8

Preparation of (S)-MTPA ester 5 and (R)-MTPA ester 6

Mosher’s method

The  SR values must be sufficiently large and be above the level of experimental error. The distribution of the signs of the parameter  SR must be uniform for a given substituent. If the sign of  SR is negative for one substituent, then the sign of  SR for the other substituent must be positive.

Determination of the configuration at C-6

Hydrolysis of 3 under mild alkaline condition

Enzymatic hydrolysis of prunioside A (1) with tannase

Formation of acetonide 9 A smaller non-equivalence between the gem- dimethyl groups 3 for the syn diol, 0.8 ppm, than for the anti diol, 3 ppm. The chemical shifts for the two acetonide methyl groups (26.4 and 26.8) in the 13 C NMR spectrum of 9 were typical of gem- dimethyl groups of erythro-diol acetonides (Dana and Danechpajouh, 1980; Solladie′ et al., 1997).

Formation of 8

Exciton chirality method

3.2. Determination of the absolute configurations of Prunioside A (1) The CD spectrum of di-p-bromobenzoate (8) showed a clear positive exciton split [first Cotton effect at 256 nm (Δε=+4.48), and a second Cotton effect at 239 nm (Δε =-5.95)]. Afford clear evidence for the 5S and 6R configurations.

3.3. Biological activities Nitric oxide (NO) is produced by nitric oxide synthases in certain cells, and has been implicated in a wide range of physiological and pathological processes. NO synthases can be classified into two major groups. The inducible isoform of NO synthase (iNOS) plays important roles in macrophage-mediated cytotoxicity.

3.3. Biological activities Compounds 4 and 11 showed dose-dependent inhibition of NO production, with IC50 values of 2.2 and 5.1 mg/ml, respectively, while compound 10 showed a very weak inhibitory effect (12% inhibition at 10 mg/ml).

RAW cells LPS Different dose of 3 Mesurement of nitrite concentration Dose-dependent inhibition of NO production was observed with an IC50 value of 3.0 mg/ml.

4. Discussion We can try to obtain crystals of 10 or 11. CD spectrum could be used to determine the absolute configurations of Prunioside A. The biological test has been a hotspot, but it’s very complicated and difficult to administrate.