New alkaloids from the fruiting bodies of Ganoderma sinense

Four new alkaloids, sinensines B–E (1–4), together with one known alkaloid, sinensine (5), were isolated from the fruiting bodies of Ganoderma sinense. Their structures were elucidated on the basis of 1D and 2D NMR spectra analysis. The structure of sinensine E was confirmed by X-ray crystallographic analysis of its acetyl product (4a). Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-011-0026-4 and is accessible for authorized users.


Introduction
The fruiting bodies of Ganoderma spp., commonly known as the Reishi mushroom, were widely used in China, Japan, and Korea as valuable crude drug, especially in the treatment of chronic hepatitis, nephritis, hepatopathy, neurasthenia, arthritis, bronchitis, asthma, gastric ulcer, and insomnia. 1 The chemical constituents of Ganoderma spp. comprised polysaccharides, fatty acids, alkaloids, nucleotides, proteins and peptides, trace elements, sterols, and triterpenoids. [2][3][4] Among them, triterpenoids were mainly responsible for the biological activities. It was reported that triterpenoids played an important role in antitumor, 5 inhibiting HIV-1 protease, 6 antiplasmodial, 7 antiviral, 8 and antimicrobial. 9 Alkaloids were another chemical constituents obtained from Ganoderma spp. In 1990, ganoderma alkaloids A and B were isolated from the fruiting bodies of G. capense for the first time. 10 Researchers had detected ganoderma alkaloid by means of HPLC, but they did not find the trace of ganoderma alkaloid. 11 Until 2010, from the fruiting bodies of G. sinense, a novel alkaloid, named sinensine was isolated and exhibited activity in protecting the injury induced by hydrogen peroxide oxidation on HUVEC. 12 To discover ganoderma alkaloids and evaluate their biological activities, amounts of the fruiting bodies of G. sinense conducted. On our progress of investigating the chemical constituents of G. sinense, three new triterpenoids with fourmember ring displaying anti-tumor activity had been reported. 13 In this paper, the isolation and identification of four new alkaloids, sinensines BE (14), together with one known alkaloid, sinensine (5) were reported. Their structures were identified mainly by 1D, 2D NMR spectra and X-ray analysis.
The molecular formula of compound 1 was assigned as C 14 H 13 (Table 1) showed fourteen carbon signals, including three methylene siganls at δ C 32.9, 30.0 and 25.0; five sp 2 methine signals at δ C 141.8, 119.6, 119.3, 115.9 and 113.5; six sp 2 quaternary-carbon signals at δ C 156.2, 156.1, 153.6, 151.1, 138.5 and 120.4. These data of 1 were similar to those of sinensine (5) 12 . However, the 1D spectra of 1 displayed one more sp 3 methylene and sp 2 methine, as well as less a methyl and an oxymethine than those of 5.
Sinensine C (2) possessed a molecular formula C 14 H 13 NO 3 by HRESIMS ([M + H] + ; m/z 244.0966, calcd 244.0973), which differed from 1 for one more oxygen atom. By detail comparison of 1D NMR data of 2 and 1, they were similar, except for the absence of a methylene and the presence of an oxymethine. Above data indicated that an additional hydroxyl group in 2 was attached to cyclopentenyl group. The further proof was established from the HMBC correlations from H-4 (δ H 2.93 and 2.66) to C-9 (δ C 141.5), C-8 (δ C 157.2) and C-3 (δ C 116.3), from H-5 (δ H 2.42 and 2.13) to C-9, C-8, C-4 (δ C 30.4), and C-6 (δ C 73.7), and from H-6 (δ H 5.48) to C-7 (δ C 142.8), C-9 and C-8. This illustrated that the hydroxyl group was located at C-6. Thus, the plantar structure of compound 2 was identified.
Molecular formula C 14 H 11 NO 3 of compound 3 was determined by HRESIMS. Its IR spectrum showed presence of hydroxyl group (3445 cm −1 ) and aromatic rings (1614 and 1482 cm −1 ). On comparison of the 1D NMR data between 3 and 2, the signals due to the phenyl group and the group of cyclopenta[b]pyridine could be observed. However, the 1D NMR spectra of 3 showed a down-field sp 2 quaternary carbon signal (δ C 160.8) instead of an up-field sp 2 methine signal in 2, which suggested that an sp 2 methine in 2 was oxidized to an sp 2 quaternary carbon in 3.
Since there were two oxidized olefinic carbons at the phenyl group, the additional oxidized olefinic carbon could be located at the pyridine. Meanwhile, in the HMBC spectrum, the correlations of H-7 with C-8, C-9 and C-6 indicated that the additional oxidized olefinic carbon was at C-3. Base on the molecular formula C 14 H 11 NO 3 (10 degree of unsaturation) of 3, we deduced that C-2' and C-3 formed an ether bond. Furthermore, ChemDraw 3D model of 3 (Figure 1) showed that the phenyl group, the furan and the pyridine were formed a conjugate  In order to further verify the structure of 4 deduced above and to describe the configuration of 4, the single-crystal X-ray would be helpful. However, 4 was not easy to crystallize in most kinds of organic solvent. Fortunately, acetylization of 4 afforded 5',6-diacetyl sinensine E (4a) crystallized in MeOH/H 2 O solvent. Consequently, a single X-ray crystallographic diffraction 14 of 4a was conducted as shown in Figure 2. The relative configuration of OH-6 was deduced as β-oriented. Based on above evidence, the structure of 4 was assigned.
On the basis of the specific rotations of 2, 3 and 4, it was illustrated that the relative configurations of OH-6 in 2 and 3 were determined to be β, the same as that of 4.

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-011-0026-4 and is accessible for authorized users.