Irlactane and Tremulane Sesquiterpenes from the Cultures of the Medicinal Fungus Irpex lacteus HFG1102.

A new irlactane-type, namely irlactin K (1), and 22 tremulane-type sesquiterpenes including fourteen previously undescribed ones, namely irpexolactins A-N (2-15), and a known irlactane-type sesquiterpenoid, were isolated from the fermentation broth of the medicinal fungus Irpex lacteus HFG1102. The structures of all the isolates were characterized by extensive spectroscopic methods, including 1D and 2D NMR and MS spectroscopic analysis. The absolute configurations of irlactin K and the known compound conocenol B (20) were established by single-crystal X-ray diffraction analysis. The vasorelaxant effects of irlactin K (1), irpexolactins A (2), C (4), K (12), and irlactam (22) were evaluated.


Introduction
Medicinal fungi are dominate resources of natural products with diverse structural scaffolds and promising biological activities [1]. The wood-decaying fungus Irpex lacteus widely distributed throughout temperate areas globally is traditionally used as a medicinal fungus for diuretic, antibacterial, and anti-inflammatory function. The Yishenkang capsule made from the fermentated polysaccharide fraction of this fungus is clinically used as a remedy for chronic glomerulonephritis in China [2]. However, the fungus has not yet been fully chemically explored.
Tremulanes are a class of 5/7-ring-fused sesquiterpenoid which are typically found from fungi [1], and were originally isolated from the wood-decaying fungus Phellinus tremulae [3], and later from the medicinal fungus Phellinus igniarius, and the mushroom Conocybe siliginea. Recently, we reported ten tremulane/tremulanerelated sesquiterpenoids, namely irlactins A-J from the cultures of the fungus I. lacteus, among which irlactins A-D featured by an unprecedented skeleton in sesquiterpenoid class [4][5][6]. Herein, as part of our ongoing research for secondary metabolites as promising drug leads from higher fungi, we reported fifteen previously undescribed irlactane/tremulanes, namely irlactin K (1), and irpexolactins A-N (2-15) ( Fig. 1), along with nine known ones from the culture broth of the fungus I. lacteus.

Structure Elucidation of Previously Undescribed Fungal Metabolites (1-15)
Irlactin K (1) was isolated as colorless crystals. The 1 H NMR spectroscopic data (Table 1) presented three methyl singlets (δ H 0.88, 1.00, 1.23) and a methoxy group (δ H 3.44). The 13 C NMR spectrum (Table 4) showed sixteen carbon resonances ascribable to four methyls, four methylenes (one oxygenated), four methines (three oxygenated), and four quaternary carbons. All these data are reminiscent of those of irlactins C and D which were isolated from the cultures of the same fungus but different strains [4]. The presence of a methoxy group rather than a hydroxy group at C-12 in 1 as suggested by the HMBC correlation from the methoxy at δ H 3.44 to the ketal carbon at δ C 111.3 (C-12) discriminated its structure from those of irlactins C/D. The above assignments led to the determination of the planar structure of 1 as depicted in Fig. 2. The absolute configuration of 1 was determined to be 3R,5S,6R,7S,12R via X-ray single crystal diffraction analysis with the small Flack parameter 0.15(6) (Fig. 3 (Tables 1, 4) revealed the presence of three methyls (two singlets at δ H 1.07, 1.07, one doublet at δ H 1.03), five methylenes (one oxygenated at δ C 60.4), four methines (one oxygenated at δ C 74.7), and five quaternary carbons (two carbonyls at δ C 172.7, C-11; 173.2, C-12) and a tetrasubstituted double bond at δ C 145.5 (C-2), 139.4 (C-3), indicating three double bond equivalences. All these data bore striking similarity with those of coriolopsin A, a tremulane sesquiterpene isolated from the endophytic fungus Coriolopsis sp. J5 [7]. The structure of compound 2 was further corroborated by 2D NMR analysis, which suggested that the Δ 1 double bond in coriolopsin A shifted to Δ 2 in 2 by HMBC correlation from H-1 (δ H 2.83), H 2 -4 (δ H 2.81, 2.65) to C-2 and C-3 (Fig. 2). Furthermore, the oxygenated methine at δ C 74.7 was assigned to C-5 by the HMBC correlation from H 3 -13 (δ H 1.03) to C-5 and 1 H-1 H COSY correlation between H-5 (δ H 3.63) and H-6 (δ H 1.98) (Fig. 2). The remaining two methylenes was assigned to an aminoethanol group same as that harbored in vibralactamide A [8] according to the chemical shifts of C-1′ (δ C 41.3), C-2′ (δ C 60.4) and the 1 H-1 H COSY correlations between H-1′ (δ H 3.56)/H-2′ (δ H 3.63). The HMBC correlations from H-1′ to two carbonyls at C-11 and C-12, revealed a succinamide moiety.
Compounds 6 and 7 were inseparable mixture with a ratio of 1.9:1 according to the integration of 1 H NMR spectrum. The HRESIMS analysis demonstrated that they had the same molecular formula of C 15 H 24 O 3 (m/z 275.1621 [M + Na] + , calcd for 275.1618). The doubled signals in the 1 H and 13 C NMR spectra and similar proton peak types indicated that the two compounds were epimers. The 1D NMR spectra of the mixture showed signals which were categorized into three methyls, four methylenes (one oxygenated), five methines (two oxygenated), and three quaternary carbons (two olefinic) (Tables 2, 4). The data showed a resemblance to those of ceriponol G [12], except an absence of a methoxy signal at C-12. Thus, the planar structures of 6 and 7 were elucidated as shown in Fig. 2. The inseparable feature of 6 and 7 implied that they were semiacetal tautomers. The 12-OH of 6 and 7 were assigned as α and β orientation, respectively, by the key ROESY cross peaks between H-3 (δ H 2.54)/H-12 (δ H 5.23) of 6, and the absence of ROESY signals between H-3 (δ H 2.27) and H-12 (δ H 4.74) of 7 (Fig. 4). The 5-OH of 6 and 7 were α orientation as suggested by the ROESY correlations between H 3 -13/H-5 (Fig. 4). Therefore, compounds 6 and 7 were identified as irpexolactins E and F.
The molecular formula of irpexolactin L (13) was assigned as C 17 1 and 171.1). The HMBC correlation from H-12 (δ H 4.29 and 4.23) to carbonyl of acetyl group, suggesting an acetoxy group substituted at C-12. Therefore, compound 13 was elucidated as shown in Fig. 1.
Compound 14 was obtained as a colorless oil and found to possess the molecular formula of C 15 (Tables 3, 5) were highly similar to those of conocenol A [11]. The most significant deviation of the 13 C NMR data between conocenol A and 14 located on the hydroxymethyl (δ C 68.7 for conocenol A (C-15), δ C 71.6 for 14 (C-14)) which were recorded in the same deuterium solvent (CD 3 OD), implying the only difference involved in the configuration of hydroxymethyl group attached to C-9. The crucial ROESY correlations of H-7/H-8α, H-15\H-8β helped to determine the β orientation of 15-CH 2 OH, while it was α in conocenol A when its relative configuration was drawn as same as 14. Thus, compound 14 was established as irpexolactin M.

Biological Activities
Compounds 1, 2, 4, 12, and 22 were evaluated on vasorelaxant effect on KCl precontracted thoracic aorta rings. Nifedipine was used as the positive control. However, none of them showed significant vasorelaxant effect.

Conclusions
The

Animals
Adult male Wistar rats (250-300 g) were kept in an animal room with a constant temperature of 22 ± 2 ℃, a humidity of 60 ± 5% and had free access to food and water. Experiments were performed in accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experimental procedures were approved by the Research Ethics Committee of the Kunming Institute of Botany, Chinese Academy of Sciences.

Methods
Vasorelaxant effects of 100 µmol/L of the compounds was evaluated on endothelium-intact thoracic aorta rings precontracted with KCl. Rat aortic rings were prepared according to that described [16,17]. Nifedipine, a calcium channel blocker, was used as the positive control. Aortic rings were mounted on stainless steel hooks in organ baths containing 37 ℃ Krebs solution continuously bubbled with 95% O 2 and 5% CO 2 , then equilibrated for 60 min under a resting tension of 1.5 g. After equilibration, the vessels were exposed to 1 μmol/l phenylephrine (PE), followed by 10 μmol/l acetylcholine (Ach) to check functional endothelial integrity, more than 80% relaxation of the rings was considered to be an endothelium-intact ring. Endothelium-intact rings precontracted with 60 mmol/l KCl were treated with different compounds, DMSO or nifedipine for 30 min or 60 min, the changes in tension of aortic rings were recorded. The vasorelaxant effect was calculated as a percentage of the relaxation in response to KCl on the aortic rings. Data were presented as mean ± SD and evaluated by one-way ANOVA followed by Bonferroni's test using SPSS 19.0. P < 0.05 was regarded to be statistically significant.