The absolute configuration of prunioside A from Spiraea prunifolia
and biological activities of related compounds
Meng Que
• Phytochemistry, 2003.• 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: 942-944.
OHO
HOOH
O
OH
O
O
O OH
Oprunioside A
1
2
34 5
68
10
9
1'' 4''
1' OHO
HOOH
O
OH
O
O
O OH
O OH
ASE-20
1. Abstract
• The configurations at C-5 and C-6 were determined.• The modified Mosher’s method, CD analysis, and
13C 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 264.7 cells.
OHO
HOOH
O
OH
O
O
O OH
Oprunioside A
1
2
34 5
68
10
9
1'' 4''
1'
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)
The air-dried roots of S. prunifolia (1 kg)
Extracted with MeOH for 24 h
ResidueMeOH extract Extract
Concentrated
Suspended in H2O
Partitioned withn-hexane
Partitioned withEtOAc
Partitioned withCH2Cl2
SPE (4.6 g) SPH SPC
C18 flash column chromatography (CC)40%~100% MeOH : H2O
Fraction eluted at 60% MeOH-H2O (1.2 g)
Reversed-phase HPLC20%~30% CH3CN : H2O over 50 min100% CH3CN for 10 min
Compound 1 (648 mg)
Collected from Iksan City, Chonbuk Province, Korea in May 2000. Fresh roots were dried in a well-ventilated darkroom.
Symmetry Pre C18 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.
ORO
ROOR
O
OR
O
O
O OR
O
1 R = H1a R = Ac
1
2
34 5
68
10
9
1'' 4''
1'
3.2. Determination of the absolute configurations of Prunioside A (1)
OHO
HOOH
O
OH
O
O
O OH
O
1
2
34 5
68
10
9
1'' 4''
1'
1
HOO
O
OH
3 R = H
1
2
34 5
68
10
9
Enzymatic hydrolysis of 1
ROO
O
OR
5 R = (S)-MTPA6 R = (R)-MTPA
1
2
34 5
68
10
9Mosher’s method
Hydrolysis of 3 under mild alkaline condition
OR
7 R = H
34 5
68
10
OR
O
O
1Formation of acetonide 9O
9
34 5
68
10
O
O
O
1
1'
2'
3'
It has been reported that acetonides of syn and anti-1,2-diols can be unambiguously distinguished by the 13C NMR chemical shifts of the acetonide methyl groups. (Dana andDanechpajouh, 1980; Solladie′ et al., 1997)
O
8
56 O
O
O
OBr
O
Br
Formation of 8
Preparation of (S)-MTPA ester 5 and (R)-MTPA ester 6
Compound 3 (2.5 mg)dissolved in CH2Cl2 (0.5 ml)
+4-N,N-dimethylaminopyridine (0.5 mg) triethylamine (35 ul) (S)-MTPA-Cl (20 ul)
Stirred for 12 hConcerntrated Reversed-phase HPLC
Compound 5 (2.7 mg)
Compound 3 (2.5 mg)dissolved in CH2Cl2 (0.5 ml)
+4-N,N-dimethylaminopyridine (0.5 mg) triethylamine (35 ul) (R)-MTPA-Cl (20 ul)
Stirred for 12 hConcerntrated Reversed-phase HPLC
Compound 6 (2.7 mg)
Mosher’s method
• The DdSR values must be sufficiently large and be above the level of experimental error.
• The distribution of the signs of the parameter DdSR must be uniform for a given substituent.
• If the sign of DdSR is negative for one substituent , then the sign of DdSR for the other substituent must be positive.
Hydrolysis of 3 under mild alkaline condition
Compound 3 (5 mg)dissolved inTHF (2 ml)
+ NaOH (1 N, 2 ml)
Stirred at 25 ℃for 80 min
The reaction mixture
2 Partitioned between CH2Cl2 and H2O (1N HCl)1 Concentrated
The organic phase The water phase
1 Dried 2 Reversed-phase HPLC
Compound 7 (3.4 mg)
Enzymatic hydrolysis of prunioside A (1) with tannase
Compound 1 (22.4 mg) + tannase (15 mg) dissolved in H2O (5 ml)
30 ℃ for 12 h Extracted with EtOAc (3 × 5 ml)
EtOAc extract
Reversed-phase HPLC
Compound 3 (5.9 mg)
Formation of acetonide 9 Compound 7 (2 mg)dissolve in anhydrous acetone (1 ml)
+ 2,2-dimethoxypropane (10 ul)p-toluenesulfonic acid (0.2 mg)
Stirred at 25 ℃ for 6 h
The mixture
1 Remove the organic solvents in vacuo 2 Extracted with ether (3 × 3 ml)
The dried organic residue
Semi-preparative reversed-phaseHPLC
Aacetonide 9 (1.4 mg)
OR
7 R = H
34 5
68
10
OR
O
O
1
9
34 5
68
10
O
O
1
1'
2'
3'
• 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 13C 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 Compound 7 (2.2 mg)dissolved in CH3CN (2 ml)
+ Triethylamine (100 ul)4-N,N-dimethylaminopyridine (0.5 mg)
Cl
O
BrOR
7 R = H
56 OR
O
O
(5 mg)
1 Stirred at room temperaturefor 2 h
2 Evaporated under N2
The residue
1 Redissolved in 2 ml of EtOAc
2 Extractedwith H2O (2 ml)
The organic phase The water phase
1 Dried 2 Reversed-phase HPLC
Compound 8 (1.2 mg)O
8
56 O
O
O
OBr
O
Br
+
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.
OHO
HOOH
O
OH
O
O
O OH
Oprunioside A
1
2
34 5
68
10
9
1'' 4''
1'
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 O
RORO
ORO
OR
O
O
O OR
O
1 R = H1a R = Ac
1
2
34 5
68
10
9
1'' 4''
1' HOO
O
OH
3
1
2
34 5
68
10
9
AcOO
O
OAc
4
1
2
34 5
68
10
9
ROO
O
O OR
O
10 R = H11 R = Ac
1
2
34 5
68
10
9
1'' 4''
Compounds 4 and 11showed dose-dependent inhibition of NO production,with IC50 values of 2.2 and 5.1 mg/ml, respectively, whilecompound 10 showed a very weak inhibitory effect(12% inhibition at 10 mg/ml).
RAW 264.7 cells
LPS
Different dose of 3
Mesurement of nitrite concentration
Dose-dependentinhibition of NO production was observedwith 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.