[20(22)E]-Lanostane Triterpenes from the Fungus Ganoderma australe

Twelve new lanostane triterpenoids (1–5, 7–13) were isolated from the fruiting bodies of the fungus Ganoderma australe. The structures of the new compounds were elucidated by extensive 1D and 2D NMR, and HRESIMS spectroscopic analysis. All the triterpenes are featured by 20(22)E configurations which are uncommon in the Ganoderma triterpene family. The absolute configuration of the C-25 of compounds 1, 2, and 6 were determined by the phenylglycine methyl ester (PGME) method. A postulated biosynthetic pathway for compound 1 was discussed. This study opens new insights into the secondary metabolites of the chemically underinvestigated fungus G. australe.


Introduction
Mushrooms are popular in the food market due to their delicious taste and nutrition values. Mushroom-derived secondary metabolites have contributed lots of lead compounds for medical and agricultural use. Psilocybin, a specialized compound from the genus Psilocybe, is a naturally occurring hallucinogenic prodrug for treating psychiatric disorders [1]. Strobilurins, firstly originated from the mushroom Strobilurus tenacellus, are a group of natural products and their synthetic analogs are used in agriculture as fungicides [2,3]. More and more attention has been paid to mining promising lead compounds from the mushroom natural product reservoir in recent years.
Ganoderma, called "lingzhi" in China, is a group of wood-decaying mushrooms with hard fruiting bodies which grow mostly in spare scatting sunshine, on the trees, and on open grounds [4]. It is a genus of notable medicinal fungi and traditional herbal medicine for the treatment of diseases such as hepatopathy, nephritis, neurasthenia, and asthma [5][6][7][8][9][10]. The Shennong Ben Cao Jing, an ancient Chinese medicinal book, documented that Ganoderma was effective for maintaining health, prolonging life, boosting memory, and relieving stress. Ganoderma lucidum and G. sinense are two registered species recorded in the Chinese Pharmacopoeia (2015). Many studies show that triterpenoids and polysaccharides are the main bioactive substances in Ganoderma [11][12][13][14][15][16][17]. Ganoderma australe is a species used in folk medicine as the alternative of G. lucidum. However, this fungus has rarely been chemically investigated compared to other Ganoderma species, such as G. lucidum, G. cochlear, and G. sinense. Previous studies on this fungus have led to the isolation of lanostane triterpenes [18][19][20][21], meroterpenoids [22,23], and alkaloids [22]. The lanostanoids from this species are over-oxygenated compared to the ones isolated from other species of Ganoderma, especially the position of C-20 [18,20]. The quaternary hydroxy group substituted at C-20 led to the introduction of an additional chiral carbon of which the stereochemistry was difficult to be assigned even by chemical derivatization. Moreover, this substituted pattern of the C-20 hydroxy group always triggered to dehydration between C-21 to produce the

Synthesis of the Phenylglycine Methyl Ester (PGME) Derivatives
To a solution of 1 (2.0 mg, 3.9 µmol) in DMF (0.5 mL) on ice add PyBOP (2.5 mg, 4.8 µmol), HBTU (1.9 mg, 5.0 µmol), N-methylmorpholine 100 µL, and (S)-PGME (1.0 mg, 4.9 µmol). The reaction mixture was stirred at room temperature for 3 h. The reaction was stopped by adding 1 mL of EtOAc and then washed with H 2 O. The EtOAc layer was concentrated under reduced pressure to obtain a pale yellow oil sample, which was purified by HPLC to furnish (S)-PGME amide product 1a. Similarly, (R)-PGME amide product 1b was prepared from 1 (2.0 mg) and (R)-PGME (1.0 mg). NMR assignments of the protons for 1a and 1b were achieved by analysis of the 1 H-1 H COSY spectra.
To a solution of 6 (2.5 mg) in THF (1 mL) was added 1 mL of LiOH (1 mol/L). The reaction mixture was stirred at room temperature overnight. The reaction was stopped by concentrating under reduced pressure to obtain a pale yellow oil sample, which was purified by HPLC to obtain 6H (0.4 mg). Then 6Ha was prepared from 6H (0.2 mg) with (S)-PGME, 6Hb was prepared from 6H (0.2 mg) with (R)-PGME. NMR assignments of the protons for 6Ha and 6Hb were achieved by analysis of the 1 H-1 H COSY spectra.

Results and Discussion
Compound 1 (Figure 1), obtained as a yellow oil, gave an [M + Na] + ion peak at m/z 535.26685 in the HRESIMS (calcd for C 30   . The chemical shifts of 1D NMR of 1 indicated that it was a lanostane triterpenoid similar to resinacein N, except for the substitutions at C-3, C-7, and C-15 [29]. In the HMBC spectrum of 1, the correlations from Me-29 to the carbonyl C-3, from Me-30 to the methylene carbon C-15, and from H-5 (δ H 2.38) and H-6 (δ H 2.36, 2.68) to the carbonyl C-7, along with the 1 H-1 H COSY correlation of H-15 (δ H 2.24, 1.87)/H-16 (δ H 2.04, 1.91) (Figure 2), suggested that C-3 and C-7 were ketone carbons and C-15 was a methylene instead of being a hydroxylated methine in resinacein N. Therefore, the planar structure of 1 was elucidated as shown in Figure 1.
The key ROESY correlations between H-22 (δ H 6.51) and H-16a/b (δ H 2.04, 1.91) allowed the assignment of the E configuration of the C-20-C-22 double bond ( Figure 3). The absolute configuration of the chiral center C-25 was determined by the PGME method ( Figure 4). The (R)-and (S)-PGME amide derivatives were chemically synthesized, and the ∆δ H (δ S − δ R ) values indicated that C-25 was the S configuration. The attempt to assign the absolute configuration of C-24 by Mosher's method failed, probably due to the bulky groups around the hydroxy group. Therefore, the configuration of C-24 remained unassigned. Compound 1 was elucidated as [20(22)E,24R,25R]-24-hydroxy-3,7,11,23-tetraoxolanosta-8,20-dien-26-oic acid, and was given the trivial name ganoaustralenone A.
Compound 2, obtained as a white powder, displayed an [M + Na] + peak at m/z 551.26422 in the HRESIMS (calcd for C 30 H 40 O 8 Na, 551.26209). The 1D NMR data of 2 (Tables 1 and 2) showed a resemblance to those of compound 1, implying the analogous structures of the two compounds. Analysis of the 1D NMR data suggested that the only difference between 1 and 2 was C-6. The HMBC correlation from H-5 to a hydroxymethine at δ C 72.2 (C-6), as well as the 1 H-1 H COSY correlation of H-5 and the proton at δ H 4.44 (H-6) (Figure 2), revealed that the C-6 in 2 attached to a hydroxy group compared to that of 1. These assignments are consistent with the HRESIMS result. The absolute configuration of C-25 was determined by the PGME method, as in the case of compound 1 (Figure 4). Therefore, compound 2 was determined as shown in Figure 1, and trivially named ganoaustralenone B.  Compound 3, obtained as a yellow oil, displayed an [M + Na] + ion peak at m/z 537.28180 in the HRESIMS analysis (calcd for C 30 H 42 O 7 Na, 537.28227). The 1 H NMR and 13 C NMR data of 3 (Tables 1 and 2) highly resemble those of 1, except for the chemical shift of C-7. The key 1 H-1 H COSY correlations H-5 (δ H 2.11)/H-6 (δ H 1.70)/H-7 (δ H 4.47), as well as the HMBC correlation from H-7 (δ H 4.47) and C-8 (δ C 160.3) (Figure 2), implied the presence of a hydroxyl group at C-7. The key ROESY correlations of H-7/H-15β/H 3 -18 indicated the α orientation of 7-OH ( Figure 3). The absolute configurations of C-25 were determined by the PGME method, as in the case of compound 1 (Figure 4). Therefore, compound 3 was determined as shown in Figure 1, and identified as ganoaustralenone C.
The yellowish oil compounds 4 and 5 gave the sodium adduct ion peaks of m/z 565.27728 and m/z 549.28210 in the HRESIMS analysis, corresponding to the molecular formulas of C 31 (Tables 1 and 2) showed characteristic signals of triterpene, indicating the same skeletons of 1-5. Analysis of the 1D NMR spectra of 4 and 5 suggested that the two compounds were highly similar to those of 1 and 2, respectively. The differences between these two pairs of compounds (1 vs. 4, 2 vs. 5) were the status of C-26 carboxylic group. The correlations from the methoxy singlets to the carbonyl group (C-26) in the HMBC spectra of 4 and 5 ( Figure 2) indicated that C-26 of 4 and 5 have been methyl esterified instead of being free carboxylic groups in 1 and 2. Therefore, compounds 4 and 5 were elucidated as the C-26 methyl ester derivatives of 1 and 2, respectively. However, these changes hampered the absolute configuration determination of C-25 of 4 and 5 by the PGME method. The relative configurations of C-24 and C-25 were assigned as R* and S*, respectively, by analysis of the Newman projections of Compound 6 was determined to be methyl gibbosate O by comparison with the NMR spectroscopic data (Supplementary Materials) [30,31]. However, the chemical shifts of C-13 and C-14 of methyl gibbosate O have been erroneously assigned previously [30].
The key HMBC correlation of H-11 (δ H 5.66, s) to an sp 3 -quaternary carbon at δ C 58.0, together with the HMBC correlation from H-7 (δ H 6.50, m) to an sp 3 -quaternary carbon at δ C 52.5 enabled the correct assignment of the chemical shifts of C-13 (58.0 ppm) and C-14 (52.5 ppm). Moreover, the absolute configuration of C-25 of gibbosic acid O was assigned as S without any evidence [31], while for methyl gibbosate O, the C-25 configuration was assigned to be same with gibbosic acid O only by comparison with the chemical shifts [30]. However, C-25 is far away from any other chiral centers in the structure, so the chemical shift deviation is inadequate to discriminate the S and R configuration of C-25. Therefore, more solid evidence should be presented to corroborate the real configuration of C-25. In order to determine the absolute configuration of the chiral center C-25, compound 6 was firstly hydrolyzed by LiOH to obtain the previously reported compound gibbosic acid O (6H). Then, the (R)-and (S)-PGME amide derivatives of 6H were chemically synthesized (Scheme 1), and the ∆δ H (δ S − δ R ) values indicated that C-25 was the S configuration ( Figure 4). Therefore, the absolute configuration of compound 6 has been fully assigned. Scheme 1. Alkaline hydrolysis and PGME derivatization of compound 6.
The HRESIMS analysis of 7, a yellow oily compound, gave a sodium adduct ion peak at m/z 547.30286, corresponding to the molecular formula of C 32 H 44 O 6 (calcd for C 32 H 44 O 6 Na, 547.30356) with 11 double bond equivalences. Comparing the 1D NMR data of 7 (Tables 2 and 3) with those of 6 suggested that 7 differed from 6 with the presence of an oxygenated methylene and a triplet methyl group with the absence of the methoxy group. These signals were assigned to be ethyl ester moiety of the C-26 carbonyl group instead of the methyl ester moiety in 6. The 1 H-1 H COSY correlation of OCH 2 CH 3 (δ H 4.13)/OCH 2 CH 3 (δ H 1.25), and the HMBC correlation from OCH 2 CH 3 (δ H 4.13) to C-27 (δ C 176.3) (Figure 2), confirmed the above assignments. Notably, 15-OH was assigned to be β orientation by the key ROESY correlation of H-15 (δ H 4.31)/Me-30 (δ H 1.00) (Figure 3). Therefore, compound 7 was named ganoaustralenone F.
Compound 10, a pale yellow oil, gave an [M + Na] + ion peak at m/z 563.29749 (C 32 H 44 O 7 Na) in the HRESIMS (calcd for C 32 H 44 O 7 Na, 563.29847). The 1 H and 13 C NMR spectroscopic data of 10 (Tables 3 and 4) showed high similarity to those of the structure 15α-hydroxy-3,11,23-trioxolanosta-8,20E(22)-dien-26-oic acid methyl ester, a lanostane triterpenoid isolated from the G. lucidum [33]. Further analysis of the 2D NMR spectra revealed that the only difference between these two structures was C-7. The diagnostic HMBC correlations from the protons at δ H 2.26 (H-5), 2.49 (H-6a), 2.62 (H-6b) to a carbonyl group at δ C 204.4 ( Figure 2) suggested that C-7 was a carbonyl group in 10 instead of being a methylene group in 15α-hydroxy-3,11,23-trioxolanosta-8,20E(22)-dien-26-oic acid methyl ester. In addition, the alcohol for forming the C-26 ester group was ethanol in 10 instead of methanol in the reported structure, as supported by the two chemical shifts at δ C 60.6 (-OCH 2 CH 3 ) and 14.2 (-OCH 2 CH 3 ). Therefore, compound 10 was identified as ethyl 20 (22) (Tables 4 and 5) showed 30 carbon resonances with high resemblance to those of compound 10. Further analysis of the 2D NMR data (Figures 2 and 3) suggested that 11 differed from 10 by the presence of the methyl ester group. The significant HMBC correlation from the methoxy group (δ H 3.68) to the carbonyl group C-26 (δ C 176.6) (Figure 2) verified the terminal carboxylic group in 11 has been methyl esterified instead of being ethyl esterified in 10. Therefore, compound 11 was identified as ganoaustralenone J.
The pale yellow oil compound 12 exhibited an [M + Na] + ion peak at m/z 565.31287 in the HRESIMS analysis (calcd for C 32 H 46 O 7 Na, 565.31412). The NMR spectroscopic data of 12 (Tables 4 and 5) highly resemble those of 9, except for the chemical shifts of C-24 and the alcoholic part of the C-26 ester. The important HMBC correlations from Me-27 (δ H 1.30) to a hydroxymethine at δ C 78.6 (C-24) (Figure 2), together with the chemical shifts of the alcoholic part [δ C 61.0 (-OCH 2 CH 3 ) and 14.3 (-OCH 2 CH 3 )], indicated that a hydroxy group situated at C-24 and the presence of ethyl ester of C-26 in 12 compared to those of 9. Therefore, compound 12 was identified as ganoaustralenone K.
Compound 13, obtained as a yellow oil, displayed an [M + Na] + ion peak at m/z 563.29688 in the HRESIMS analysis (calcd for C 32 H 44 O 7 Na, 563.29792). Analysis of the 1 H and 13 C NMR data (Tables 4 and 5) revealed that this compound was a structural analog to 12. The main difference between the NMR data of the two analogs was the position C-7 (δ C 201.2), which indicated that C-7 was a carbonyl carbon. In the HMBC spectrum of compound 13, significant correlations from H 2 -6 (δ H 2.48, 2.34) to C-7 (δ C 201.2) ( Figure 2) indicated that C-7 was a carbonyl carbon. Therefore compound 13 was identified as ganoaustralenone L.
The identification of a series of 20(22)E-lanostanes from this species of Ganoderma inspired a proposal of the possible biosynthetic pathways. Take compound 1 as an example, as shown in Scheme 2, the common precursor squalene, which was derived from two molecules of farnesyl pyrophosphate, which was oxygenated and followed by function migration to give the lanostane scaffold. The lanosterol was oxygenated at the positions of C-3, C-7, C-11, C-20, C-23, and C-26 to give the key intermediate A, which underwent an elimination reaction by the E1cb mechanism to yield B. Finally, a nucleophilic attack at C-24 by a hydroxy group produced compound 1.