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, 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

Mosher’s method

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.

Determination of the configuration at C-6

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

+

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.

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.

Thanks for your attention