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hSTRACT.-~rcduction of alkaloids with both canthin-6-one and P-carboline structure by Adantbur altissima cell cultures is reported. Besides canthin-6-one (l), l-methoxycanthin-6-one (2), canthin-6-one-3-oxide 0, 1-methoxycanthin-6-one-3-oxide (8), l-hydroxycanthin-6-one (3), 5-hydroxycanthin-6-one (6), P-carboline-1-propionic-acid (ll), and 4-methoxy-Pcarboline-I-carboxylic acid methylester (lo), three new alkaloids are described. These are 2-hydroxycanthin-6-one (4), 4-hydroxycanthin-6-one (5), and 4,5-dihydrocanthin-6-one (9).Ailanthus altissima Swingle (Simaroubaceae) is known to be a producer of indole alkaloids and has been extensively studied (1-4). These alkaloids belong to the class of either the simple P-carboline or the more complex canthin-6-one derivatives. The literature has recently reported (5) cell cultures of A. altissima that produced canthin-6-one (1) and 1-methoxycanthin-6-one (2), which are the two most abundant alkaloids in the intact plant.We investigated the production of some minor alkaloids whose presence had been suspected during a previous study undertaken in order to clarify the different steps of their biosynthesis (6). Eleven alkaloids were identified and isolated in satisfactory yields from a batch of 50 liters of cell suspension cultures ofA. altissima. Besides the main alkaloids l and 2, cell cultures produced their corresponding 3-oxides, 7 and 8, respectively, as well as P-carboline-1-propionic acid (ll), 4-methoxy-P-carboline-l-carboxylic acid methylester (lo), 5-hydroxycanthin-6-one (6) previously found in A. altissima (1,2,4), and 1-hydroxycanthin-6-one (3) found in Ailanthus giraldii (7). Furthermore, the following three alkaloids never before found in nature, 2-hydroxycanthin-6-one (4), 4-hydroxycanthin-6-one (5), and 4,5-dihydrocanthin-6-one (9), have been extracted. In spite of the predominant alkaloid canthin-6-one (1) produced in the amount of 300 Fglml of cell suspension, each of the new compounds 4,5, and 9 represent 0.3% of the total alkaloids, and their production is 2 pg/ml or less. RESULTS AND DISCUSSIONIn order to obtain a good amount of crude alkaloids and to identify all the alkaloids present in it, a batch of about 50 liters of cell suspension culture was prepared by pooling 900 Erlenmeyer flask cultures. Cells only were extracted as reported in the Experimental section, the medium being discarded because of its negligible content in alkaloids and high concentration of nutrients. The toluene extract of the cells was loaded on a silica gel column in toluene and eluted with increasing amounts of EtOAc. By this method pure 1 and 2 were obtained, while alkaloids 2,7,8, and 10 required further purification. Compound 9 submitted to eims showed a molecular ion at mlz 222, corresponding to a dihydrocanthin-6-one. The fragmentation pattern was similar to that

Dermorphin, DM, (H‐Tyr‐d‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH2) and its [l‐Ala2]‐analog, LDM, have been investigated in aqueous and DMSO solutions by means of 1H‐ and 13C‐n.m.r., CD and u.v. with the aim of establishing possible differences in the conformational preferences of the two peptides. A study of the pH dependence of 13C chemical shifts resulted in the determination of the pKa of the α‐amino group (6.97 0.02 for DM and 7.13 0.02 for LDM). A significantly higher value (7.75) is obtained for LDM from the pH dependence of CD spectra. This difference is attributed to the presence of associates at the high concentrations required for the 13C‐n.m.r. measurements. The average pKa's of the two phenol groups have also been obtained and the values determined from u.v. absorption spectra (9.79 for DM, 9.85 for LDM) are in fair agreement with those determined from the CD spectra (9.94 for DM and 10.11 for LDM). An analysis of titration shifts in 13C‐n.m.r. spectra indicates that the α‐amino group is not involved in interactions stabilizing folded conformers in aqueous solution. Temperature coefficients and exchange times of the NH protons suggest that both peptides exist in DMSO‐d6 as a mixture of rapidly interconverting conformers. A model is proposed in terms of an extended β‐sheet conformation stabilized by intermolecular association at the high concentrations required for the n.m.r. measurements. CD spectra are dominated by contributions of the aromatic chromophores and cannot be used as a source of information about the main chain conformation. However, differences between the spectra of the two peptides indicate that the conformational freedom of one tyrosine side chain (probably Tyr1) is slightly more restricted in DM than in LDM.

1. Metabolites of the antimycobacterial agent 4-deoxo-3,4-[2-spiro-(N-isobutyl-4-piperidyl)]-(1H)-imidazo-(2,5-dihydro )- rifamycin S (rifabutin) were isolated from human urine after administration of a single oral dose of the drug. Some of these metabolites were identified by direct inlet mass spectrometry, 1H-n.m.r. spectrometry and, in two cases, by chromatographic comparison with reference compounds. 2. Unchanged drug, 25-O-deacetyl rifabutin and four other metabolites were identified in human urine. 25-O-Deacetyl rifabutin was the main urinary metabolite, other metabolites were characterized as oxidized, and oxidized-deacetylated derivatives. 3. Routes of metabolic transformation were: (a) deacetylation at position 25, (b) oxidation of methyl groups 31 or 32 or at the piperidine nitrogen, and (c) combination of these.

The isolation and structure elucidation of a new ergot peptide alkaloid, ergobine [1], from submerged cultures of Claviceps purpurea strain 231 FI, are described. Its purification and identification were achieved by feeding experiments with a labelled precursor.FEEDING experiments.-[ 1 -uC]-Aminobutyric acid (Amersham, GB) (250 µ-Ci, 9.25 MBq) was fed to a seven-day-old 50 ml submerged culture of C. purpurea 231 FI (ATCC 20106). Medium and experimental 'Present address: Pharmaton, 6934 Bioggio (Switzerland).