and from the leaves of Podocarpus macrophyllus var. maki (Japanese name: Rakanmaki), norditerpenes and totarols having cytotoxic activities against P388 murine leukemia cells have been reported by us. [5][6][7] In the present study, from the bark of Podocarpus macrophyllus D. DON, we isolated 8 new diterpenes along with known ones. This paper describes the isolation and structural elucidation of these new diterpenes and their antibacterial activities against oral pathogenic microorganisms.
Results and DiscussionBy sequential Diaion ® HP-20 column chromatography (H 2 O-MeOH) and silica gel column chromatography (CHCl 3 -MeOH) and the following repeated reversed-phase HPLC (MeCN-H 2 O, MeOH-H 2 O), a hot methanol extract of the bark of Podocarpus macrophyllus D. DON gave 8 new diterpene compounds (1-8) along with known lambertic acid (9), 8) 4b-carboxy-19-nortotarol (10), 8) totaradiol (11), 8) and macrophyllic acid (12). 9) Compound 1 was isolated as a colorless amorphous solid with [a] D ϩ262.6°(CHCl 3 ) and mp 124-126°C. The molecular formula was determined to be C 20 H 28 O 3 from the [M ϩ ϩNa] ion peak at m/z 339.1927 in HR-ESI-MS, implying the presence of 7 degrees of unsaturation. The IR absorption spectrum showed bands of carbonyl (1694 cm Ϫ1 ) and hydroxyl groups (3376 cm Ϫ1 ). The 1 H-and 13 C-NMR spectra of 1 (Table 1) . These data and the molecular formula suggested that 1 was an isomer of a known diterpene, 4b-carboxy-19-nortotarol (10) obtained also from this material. The heteronuclear multiple bond coherence (HMBC) correlations observed between the hydrogens H-15, H16, H-17 and the carbon C-12, and between the hydrogens H-14, H15, H-16 and the carbon C-13 indicated that the isopropyl group was at C-12 and the phenolic hydroxyl group at C-13. Further HMBC correlations between the hydrogens H-3 and H-18 and the carbonyl carbon C-19 showed that the carboxyl group was at C-4. The nuclear Overhauser effect (NOE)s between the methine proton H-5 and both the methyl protons H-18 and H-1a, respectively, and between the methyl protons H-20 and H-1b implied that the ring junction of A/B was trans and the stereochemistry of the methyl group attached to C-4 was a-oriented (Fig. 2). Hence, 1 was concluded to be 13-hydroxy-12-isopropyl-8,11,13-podocarpatrien-19-oic acid and was named inumakiol A. Only very few 12-isopropyl diterpenes are known and 1 was shown to be an analogue of such 12-isopropyl diterpene, sempervirol, 10) whose methyl group at C-4 is replaced by a carboxy group (Fig. 1).2 was isolated as a colorless amorphous solid with [a] D ϩ11.0°(CHCl 3 ) and mp 228-233°C. The molecular formula was determined to be C 20 H 28 O 3 from the [M ϩ ϩNa] ion peak at m/z 339.1895 in HR-ESI-MS, implying the presence of 7 degrees of unsaturation. The IR absorption spectrum showed bands of carbonyl (1646 cm Ϫ1 ) and hydroxyl groups (3432 cm Ϫ1 ). The 1 H-and 13 C-NMR spectra of 2 showed the presence of an isopropyl group [d C 28.5 (d), 22.7 (q), 22.6 (q) and d Table 1). The HMBC correlations observed between the hyd...
