Chemical investigation of the roots of Aconitum forrestii Stapf resulted in the isolation of two novel C₁₉-diterpenoid alkaloids, forestine (6) and foresticine (7), together with three known alkaloids, chasmanine (2), talatizamine (3), and yunaconitine (4). The structure derivation of the new alkaloids is based mainly on spectroscopic evidence and correlation of foresticine with chasmanine (2). The roots of Aconitum forrestii Stapf are used in Chinese traditional medicine for the treatment of rheumatism. Chen and Breitmaier have reported the isolation of a "new" alkaloid, foresaconitine (actually vilmorrianine C), from Aconitum forrestii Stapf var. albo-villosum (Chen et Liu) W.T. Wang (1). As part of a program to investigate the crude drugs of China (2-5), a careful examination of the roots of A. forrestii Stapf has shown that this plant contains five diterpenoid alkaloids. These were isolated from the 90% EtOH extract by a combination of pH gradient separation, column and thick-layer chromatographic techniques. Four of the compounds were isolated from the crude alkaloid fraction (pH 8; 0.9% of the dry roots), of which three were identified as chasmanine (2) (6), talatizamine (3) (7), and yunaconitine (4) (8-9) by direct comparison with authentic samples. A new alkaloid designated as forestine (6) was obtained in an amorphous form, and its molecular formula C₃₃H₄₇NO₉ was derived from mass spectral (M⁺ 601) and ¹³C NMR data. In the infrared absorption spectrum, forestine shows hydroxyl (3460 cm⁻¹), aromatic (1610, 1580 cm⁻¹), and ester carbonyl (1715 cm⁻¹) bands. The ¹H NMR spectrum of the base shows a methyl group at δ 1.1 (t, J=7 Hz, N-CH₂-CH₃), four methoxyl singlets at δ 3.3, 3.32, 3.33, and 3.4. The spectrum also exhibits one-proton doublets at δ 4.06 (J=6 Hz) and 5.12 (J=5 Hz) attributed to C-6 and C-14 β-protons, respectively. Aromatic protons with an A₂B₂ pattern at δ 6.95 (2H, J=9 Hz), 8.05 (2H, J=9 Hz) together with an aromatic methoxyl at δ 3.86 and a UV maximum at λ 255 nm (log ε 4.04) indicated the presence of an anisoyl group (10). Considering a total of 14 carbon atoms accounted for so far, forestine should be a C₁₉-diterpenoid alkaloid. Biogenetic considerations and the oxygenation pattern of alkaloids belonging to this group lead to the partial structure (5). ¹³C NMR spectral values (cf. 1, 2, 4, 9 in Table 1) and the normal oxygenation of this class of alkaloids indicate that C-1 and C-6 are substituted by methoxyl groups (11). The C-6 methoxyl is α-oriented, as seen from the ¹H NMR signal at δ 4.06, which appears as a broad doublet (J=6 Hz, ω₁/₂ 1.5 Hz) showing maximum coupling with the C-5 proton and negligible coupling with the C-7 proton (-92° dihedral angle). In all probability, the methoxyl at C-1 is present in the α-configuration as in the case of a large number of C₁₉ diterpenoid alkaloids (12). The ¹³C NMR spectrum of forestine showed 31 signals corresponding to 33 carbon atoms of the molecule (Table 1). The signals at 131.8 and 113.8 ppm represent two carbons each and are assigned to the 2',6'- and 3',5'-carbons, respectively, of the anisoyl group. According to known substitution patterns, the remaining hydroxyl groups in the partial structure (5) can be assigned to two of the five possible sites, viz: C-7, C-8, C-9, C-10, or C-13, to accommodate the observed singlets at 73.7 and 76.1 ppm. Chemical shifts due to C-7, C-9, and C-10 bearing a hydroxyl group appear significantly downfield in the region 78-88 ppm. The two hydroxyls can therefore be placed at C-8 and C-13, leading to the assignment of structure (6) for forestine. The structure appears to show consistent ¹³C NMR signals when compared with the related alkaloids yunaconitine (4) (8-9) and foresaconitine (vilmorrianine C) (1) (1,8,9,13). The alkaloid extracted at pH 10 (0.05%) after column and thick-layer chromatography on alumina afforded foresticine (7), mp 79-80°C, [α]ᴰ -1.9° (c 1%, CHCl₃) as colorless plates. High-resolution mass spectrometry, 437.2785, indicated a molecular formula C₂₄H₃₇NO₆ for the compound. The IR spectrum showed a hydroxyl band and no peaks in the carbonyl region. The ¹H NMR spectrum (90 MHz; CDCl₃) exhibited the following signals: δ 1.12 (3H, t, J=7 Hz; N-CH₂-CH₃), 3.32, 3.4, 3.42 (3H each, s, OCH₃), 4.2 (1H, t, J=4.5 Hz; H-14β), 4.9 (1H, d, J=7 Hz, H-6β). These data suggested that foresticine is also a C₁₉-diterpenoid alkaloid containing an N-ethyl group, three aliphatic methoxyls, and three hydroxyl groups. The noise-decoupled ¹³C NMR spectrum exhibited 23 signals for the 24 carbon atoms of the molecule (Table 1). The modulated off-resonance decoupled spectrum showed three singlets at 39.1, 50.6, and 74.0 ppm, which can be attributed to the quaternary carbon atoms C-4, C-11, and C-8 (bearing an oxygen function), respectively. The methoxyls whose signals appear at 56.1, 56.4, and 59.2 ppm can be assigned to carbons C-1, C-16, and C-18, respectively, on the basis of known substitution patterns in this class of alkaloids (11). Two of the remaining three hydroxyls should be placed at C-8 and C-14 on biogenetic considerations, consistent with the ¹³C chemical shift assignments. The third hydroxyl group can occupy either the C-3 or C-6 position in conformity with the aconitine class, although the former alternative is less likely from the observed coupling constant of 7 Hz at δ 4.9. Foresticine on acetylation with Ac₂O-pyridine afforded the 6,14-diacetate (8). In the ¹H NMR spectrum, 8 showed the following signals: δ 1.03 (3H, t, J=7.5 Hz, N-CH₂-CH₃), 2.03, 2.1 (each 3H, OAc), 3.29, 3.31 (9H, s, OCH₃), 4.85 (1H, t, J=4.5 Hz, H-14β), 5.74 (1H, d, J=7 Hz, H-6β). The structure (7) assigned to foresticine was confirmed by methylation to give 6,14-dimethoxyforesticine (9) as an amorphous solid identical with the methylation product of chasmanine (2). The ¹³C NMR spectra of the two methylation products were identical (Table 1).