1374
Vol. 52, No. 11
8-(R)-MTPA ester (3a) was prepared by adding (S)-(ꢂ)-a-methoxy-(tri-
fluoromethyl)phenylacetyl chloride (MTPA chloride) (50mg) to 3 (5 mg) in
dry pyridine (0.1 ml) under anhydrous conditions. The mixture was stirred at
RT for 5 h. Completion of the reaction was determined by TLC monitoring.
Chromatographic purification (EtOAc–hexane, 3 : 2) yielded the ester 3a, as
ity to convert dihydroxykawain (1) into 3ꢀ-hydroxydihy-
drokawain (2) and (8S)-hydroxydihydrokawain (3) in quanti-
ties sufficient to fully characterize the compounds and for fu-
ture pharmacological studies.
1
an amorphous white solid (8 mg). H- and 13C-NMR: see Table 2. HR-ESI-
MS [MꢂNa]ꢂ: (m/z) 487.1321 (Calcd for C24H23F3O6ꢂNa: 487.1344).
Ester derivative (3b) was obtained as a white solid using (R)-(ꢁ)-a-
methoxy-(trifluoromethyl)phenylacetyl chloride (MTPA chloride) under the
same experimental conditions as above. 1H- and 13C-NMR: see Table 2. HR-
ESI-MS [MꢂNa]ꢂ: (m/z) 487.1314 (Calcd for C24H23F3O6ꢂNa: 487.1344).
Experimental
General Experimental Procedures IR spectra were run in CHCl3 using
an ATI Mattson Genesis series FTIR spectrophotometer. UV spectra were
recorded on a Hewlett Packard 8452A diode array spectrometer. Optical ro-
tations were measured with a Jasco DIP-370 digital polarimeter and CD
measurements on a Jasco J-710 instrument in MeOH. 1H- and 13C-NMR
were recorded in CDCl3 on a Varian Unity Inova 600 spectrometer. HR-ESI-
MS data were obtained using a Bruker GioApex 3.0.
Substrate Dihydrokawain was isolated from a methanol extract of the
aerial parts of Piper methysticum. Its authenticity was established by physi-
cal methods including NMR and HPLC.
Acknowledgements The authors thank Dr. Chuck Dunbar for conduct-
ing HR-ESI-MS analysis and Mr. Frank Wiggers for assistance in obtaining
2D NMR spectra. This work was supported, in part, by the United States De-
partment of Agriculture, Agricultural Research Specific Cooperative Agree-
ment No. 58-6408-2-00009.
Organisms and Metabolism 31 Microorganisms from the collection of
the National Center for Natural Products Research, University of Missis-
sippi, were used to identify organisms capable of metabolizing dihy-
drokawain (1). A medium (referred to as medium-a)17) consisting of dex-
trose, 20 g; NaCl, 5 g; K2HPO4, 5 g; bacto-peptone (Difco Labs, Detroit,
MI), 5 g and yeast extract (Difco Labs), 5 g per liter of distilled water was
used to carry out fermentations. Initial fermentations were conducted in
125 ml Erlenmeyer flasks containing 25 ml medium-a. The usual two-stage
fermentation procedure was adopted.22) 1 was added in dimethylformamide
(0.5 mg/ml) to 24 h old stage II cultures. Incubation was at room temperature
on a rotary shaker (New Brunswick Model G10-21) at 100 rpm for a period
of 14 d. Sampling was carried out at seven-day intervals. Monitoring was by
TLC using precoated Si gel 60 F254 plates (E. Merck) with EtOAc–hexane
(3 : 2) as the solvent system. Spots were visualized by UV light (254 and
365 nm) and p-anisaldehyde as the spray reagent. Scale-up fermentations
were performed under the same conditions with five 2 l flasks, containing
500 ml of medium-a and 100 mg of substrate, each. Extractions of the cul-
ture filtrates and residues were carried out with EtOAc. The solvent was
evaporated in vacuo at 40°C to obtain the residues. The isolation of metabo-
lites was by column (Silica gel 230—400 mesh: E. Merck) and preparative
thin layer (Silica gel 60 F254) chromatography. Culture and substrate controls
were run simultaneously with the above experiments.17)
Microbial Metabolism of Dihydrokawain (1) by R. arrhizus The fil-
trate of the combined fermentation broth was exhaustively extracted with
EtOAc. Evaporation of the solvent gave a light brown gummy mixture
(500 mg). It was column chromatographed (Si gel 230—400 mesh: E.
Merck, 30 g, column diameter: 20 mm.) using CHCl3 gradually enriched
with MeOH.
The fractions were combined and further purified by column and prepara-
tive layer chromatography (EtOAc–hexane, 3 : 2). 3ꢀ-hydroxydihydrokawain
(2) was isolated as a white amorphous solid (20 mg, 4% yield). [a]2D6 ꢂ0.05°
(cꢃ0.74, MeOH). Rf 0.4; 1H- and 13C-NMR: see Table 1. UV lmax (MeOH)
nm (log e): 206 (4.41), 226 (4.47), 276 (2.84). CD (MeOH) [q]250ꢃꢂ52.7;
IR nmax (CHCl3) cmꢁ1: 3310, 2925, 1677, 1619, 1516, 1456, 1397, 1227,
1041, 823. HR-ESI-MS [MꢂNa]ꢂ: (m/z) 271.0958 (Calcd for C14H16O4ꢂ
Na: 271.09482).
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(8S)-Hydroxydihydrokawain (3) was purified as a white amorphous solid
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1
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