Studies with tissue cultures of tripterygium wilfordii. Isolation of metabolites and biotransformation studies
Creators: name: Roberts, Malcolm
Publisher: name: University of British Columbia
Year: 2011

description: In a program aimed at the identification of compounds responsible for the immunosuppressive and antifertility activities of the perennial twining vine, Tripterygium wilfordii. 5 new and 13 known compounds were isolated from the TRP-4a tissue culture cell line developed from Tripterygium wilfordii. The structures of the new compounds were determined by a combination of spectral analysis, chemical correlation and single crystal X-ray diffraction analysis. 22β-Hydroxy-3-oxoolean-12-en-29-oic acid (137), 22α-hydroxy-3-oxoolean- 12- en-29-oic acid (138) and 3β, 22β-dihydroxyolean-12-en-29-oic acid (139) are new triterpenes possessing an oleanene-type skeleton and were chemically correlated with 3β, 22α-dihydroxyolean-12-en-29-oic acid (51), the structure of which was confirmed by single crystal X-ray diffraction analysis. Oleanolic acid (127), β-sitosterol (128) and polpunonic acid (55), were isolated previously from the TRP-4a cell line in earlier studies in this laboratory. α-Amyrin (145), β-amyrin (146), 3β, 29-dihydroxyolean-12-ene (151) and 3β, llα-dihydroxyolean-12-ene (152) are known triterpenes possessing an oleanene-type skeleton and are isolated for the first time from the TRP-4a cell line. Tingenone (148) and 22β-hydroxytingenone (150) are quinone methide triterpenes, also isolated for the first time from the TRP-4a cell line. Similarly, the novel diterpene, 12-methoxyabieta-8, 11, 13- trien-3α-ol (147) and the novel triterpene, methyl-22β-hydroxy-3, 21-dioxo-D:A-friedo-29-noroleanan-24-oate (149), a member of the friedelane family, are isolated for the first time. A biosynthetic pathway, based on the isolation of 149 and its structural similarity to polpunonic acid (55) and 22β-hydroxytingen6ne (150), is postulated for the quinone methides. The cytotoxic diterpenes, tripdiolide (1) and triptolide (2) and the hydroxy acid, 160, isolated as the methyl ester, 124, have been previously reported from this laboratory. Tripdiolide (1) and triptolide (2) have been shown to possess strong antifertility and immunosuppressive activities. In another aspect of our program, biotransformation studies of the synthetic precursors, 19 (4➙3)abeo-abieta-2, 8, 11, 13-tetraen-19-ol (171) and 19-hydroxy-18(4➙3)abeo-abieta-3, 8, 11, 13-tetraen-18-oic acid lactone (91), and the radioactive congeners, 182 and 209, were carried out using the TRP-4a cell line. It was hoped that the data obtained might shed some light on the "late stage" biosynthetic pathway of the diterpene triepoxides, tripdiolide (1) and triptolide (2). Synthesis of 171 was achieved in 5 steps from dehydroabietic acid (80). The radioactive congener, 182, was synthesised using ¹⁴C-paraformaldehyde with 0.4% incorporation of the radiolabel. Biotransformation of 171 using the TRP-4a cell line yielded 19(4➙3)abeo-abieta-2, 8, 11, 13-tetraen-19-al (185) and 19(4➙3)abeo-abieta-2, 8, 11, 13-tetraen-19-oic acid (186) for spectral identification. Biotransformation of 182 yielded the aldehyde, 183 (33.2%) and the acid, 184 (51.9%), the radioactive congeners of 185 and 186 respectively. Synthesis of 91 was achieved in 4 steps from dehydroabietic acid (80). The radioactive congener, 209, was synthesised using ¹⁴C-methyl iodide via ¹⁴C-dimethylsulphonium methylide, with 0.6% incorporation of the radiolabel. Biotransformation of 91 using TRP-4a tissue cultures yielded 19-hydroxy-7-oxo-18(4➙3)abeo-abieta-3, 8, 11, 13-tetraen-18-oic acid lactone (214), 2β, 19-dihydroxy-7-oxo-18(4➙3)abeo-abieta-3, 8, 11, 13-tetraen-18-oic acid lactone (215), 7β, 19-dihydroxy-18(4➙3)abeo-abieta-3, 8, 11, 13-tetraen-18-oic acid lactone (216) and 2β 19-dihydroxy-18(4➙3)abeo-abieta-3, 8, 11, 13-tetraen-18-oic acid lactone (96), for spectral identification. Biotransformation of 209 yielded the ketone, 210 (56.7%), the hydroxy ketone, 211 (5.9%), the benzylic alcohol, 212 (9.6%) and the C2 alcohol, 213 (6.8%), the radioactive congeners of 214,215,216 and 96 respectively. A biosynthetic pathway to the diterpene triepoxides is postulated based on the oxygenated biotransformation products. [formulas omitted]