Four Meroterpenoids with Novel Aminoglycoside Moiety from the Basidiomycete Clitocybe clavipes with Cytotoxic Activity

Four new meroterpenoids, Clavilactone M-P, possessing novel aminoglycoside moiety (1–4) and a 10-membered carbocycle fused with an α,β-epoxy-γ-lactone, were isolated from Clitocybe clavipes, a basidiomycete. Their structures with absolute configurations were determined by extensive analysis of their spectroscopic data, and the ECD method. All the isolated compounds (1–4) were evaluated for their antitumor activity against three human cancer cell lines using the MTT assay. Compound 1 and 2 exhibited a significant suppression of cell viability in the Hela (IC50 = 22.8 and 19.7 μM) cell line.

So far, no more than 40 naturally occurring meroterpenoids containing benzo-fused 10-membered carbocycle fragments have been found from natural sources. Our group has long been interested in this type of structure and discovered several kinds of novel clavilactones from Clitocybe clavipes. For instance, clavilactones G-K were isolated, which all possessed a 10-membered carbocycle connected to a hydroquinone with moderate cytotoxic activities [17,18]. Previously, we have reported the isolation of two kinds of novel nitrogen-containing meroterpenoids, clavipyrrine A [19] and clavipines A-C [20], from the basidiomycete Clitocybeclavipes. These unique structures with promising anti-tumor activity led to our ongoing investigation of nitrogenous meroterpenoids. As a result, four new aminoglycoside meroterpenoids, clavilactone M-P (1-4) (Figure 1), were obtained. To the best of our knowledge, this is the first report on aminoglycoside meroterpenoids in nature. Herein, their isolation and structural elucidation, as well as their cytotoxic activities, are described. 2

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Molecules 2023, 28, x FOR PEER REVIEW 2 of 10 in nature. Herein, their isolation and structural elucidation, as well as their cytotoxic activities, are described.
The relative configuration of 1 was revealed by the proton coupling and the NOE data. H-6(δ H 6.76, s) and H-7(δ H 4.08, s) protons form a dihedral angle of about 90°as there is no vicinal coupling between the two adjacent protons. This suggests that the relative configuration of C-6, C-7, and C-8 is either 6R, 7R, 8R or 6S, 7S, 8S. Meanwhile, the Z geometry of C-11/C-12 olefin was identified by the key ROESY correlations of H-11/H-15 labelled in Figure 2B.
The ECD spectra were calculated using density functional theory (DFT) at the APFD/6-311+g (2d, p) level. The aglycone of 1 was used to conduct the ECD experiment. The comparison of the theoretically calculated and experimental ECD curves showed that the calculated ECD spectrum of (6R,7R,8R)-1 fitted better with the experimental one than the ECD spectrum of (6S,7S,8S)-1 ( Figure 3). Finally, the stereoabsolute configurations of the C-6, C-7, and C-8 positions of compound 1 are determined to be 6R, 7R, and 8R, and the compound was named Clavilactone M.
which combined with one oxygenated methylene carbon (δC 62.7) and four oxygenated methine carbons (δC 79.6, 78.7, 74.1, 71.5) indicated the existence of a glucose fragment in the molecule. In addition, the HMBC correlations of H-1′ to C-3 (δC 148.1) and C-2′ revealed the glucose fragment was attached to the C-3 position (Figure 2). The acid hydrolysis of 1 liberated the D-glucopyranose, which was in agreement with the remaining 1 H, 13 C NMR data and HMBC correlations. With overall consideration of these signals, the planar structure of compound 1 was considered an unusual β-D-aminoglycoside meroterpenoid containing a benzoquinone fused to a ten-membered carbocycle with α, βepoxy-γ-lacton.
The relative configuration of 1 was revealed by the proton coupling and the NOE data. H-6 (δH 6.76, s) and H-7 (δH 4.08, s) protons form a dihedral angle of about 90• as there is no vicinal coupling between the two adjacent protons. This suggests that the relative configuration of C-6, C-7, and C-8 is either 6R, 7R, 8R or 6S, 7S, 8S. Meanwhile, the Z geometry of C-11/C-12 olefin was identified by the key ROESY correlations of H-11/H-15 labelled in Figure 2B.
The ECD spectra were calculated using density functional theory (DFT) at the APFD/6-311+g (2d, p) level. The aglycone of 1 was used to conduct the ECD experiment. The comparison of the theoretically calculated and experimental ECD curves showed that the calculated ECD spectrum of (6R,7R,8R)-1 fitted better with the experimental one than the ECD spectrum of (6S,7S,8S)-1 ( Figure 3). Finally, the stereoabsolute configurations of the C-6, C-7, and C-8 positions of compound 1 are determined to be 6R, 7R, and 8R, and the compound was named Clavilactone M.  Compound 2 was obtained as a red powder. Its molecular formula was determined to be C 22 H 25 NO 10 Table 1). The only difference was the anomeric proton C-1 shifted to δ H 5.11 compared with compound 1 at δ H 4.52, and the coupling constant of the anomeric proton became 4.8 Hz, which suggested that the sugar moiety in 2 was α-D-N-glucose. The sugar unit was identified as D-glucose by TLC in comparison with authentic D-sugar (visualization with ethanol 5%H 2 SO 4 spraying) followed by gas chromatography. Meanwhile, the HMBC correlations of H-2 to C-13(δC 27.6) and H-1 to C-3(δC 147.5)/C-2 (δC 74.0) revealed the α-D-N-glucose was contained in the molecular and the amino was substituted at the C-3 position (Figure 4). The planar structure of 2 was shown in Figure 1. On the basis of biogenetic consideration, the absolute configuration at the C-6, C-7, and C-8 positions of 2 was proposed to be same as that of 1. Meanwhile, the experimental ECD spectra of their aglycones showed the identical tendency (supplementary Figures S29). Thus, the absolute configuration of 2 was identified as 6R, 7R, 8R, and the compound was given the name Clavilactone N. difference was the anomeric proton C-1′ shifted to δH 5.11 compared with compou δH 4.52, and the coupling constant of the anomeric proton became 4.8 Hz, which sug that the sugar moiety in 2 was α-D-N-glucose. The sugar unit was identified as D-g by TLC in comparison with authentic D-sugar (visualization with ethanol 5%H2SO4 ing) followed by gas chromatography. Meanwhile, the HMBC correlations of H-2 t (δC 27.6) and H-1′ to C-3 (δC 147.5)/C-2′ (δC 74.0) revealed the α-D-N-glucose wa tained in the molecular and the amino was substituted at the C-3 position (Figure planar structure of 2 was shown in Figure 1. On the basis of biogenetic considerati absolute configuration at the C-6, C-7, and C-8 positions of 2 was proposed to be s that of 1. Meanwhile, the experimental ECD spectra of their aglycones showed the cal tendency (supplementary Figures S29). Thus, the absolute configuration of 2 wa tified as 6R, 7R, 8R, and the compound was given the name Clavilactone N.   (Figure 4). The acid hydrolysis of 3 liberated glucopyranose; the absolute configurations of the sugars were also determined chromatography as β-D-glucose. Finally, the planar structure of 3 was establis shown in Figure 1. In the ECD experiment, the trend experimental data of aglycon 3 was consistent with that of 1 (Supplementary Figure S29). From the perspective biosynthetic pathway and ECD experiment, the absolute configuration of 3 was mined to be 6R, 7R, 8R, and the compound was given the name Clavilactone O.
Compound 4 obtained as a red powder, was determined to be with the mo formula of C22H25NO11 by the positive HR-ESI-MS ion peak at m/z 502.1335 [M+N culated as 502.1325), corresponding to 10 degrees of unsaturation. Overall consid of 1 H-and 13 C-APT NMR spectral data (Table 2) suggested that compound 4 was a pine-type meroterpenoid with an α, β-epoxy-ɤ-lactone ring, similar to Clavilacton 183.9, 181.9, 172.1, 149.2, 146.9, 135.1, 134.1, 133.3, 123.4, 72.7, 62.9, 60.6, 26.6, 24. 21.8). In detail, the two carbonyl carbons (δC 183.9, 181.9) and the four olefinic carb 149.2, 146.9, 133.3, 135.1) indicated the presence of a quinone skeleton. Furthermo APT NMR signals of 72.7 (C-6), 62.9 (C-7), 60.6 (C-8), and 172.1 (C-16) and their re HSQC correlations indicated an α,β-epoxy ɤ-lactone moiety contained in the str which was also proved by HMBC between H-7 (4.5, s) and C-6 (72.7) (Figure 4). Th difference was a downfield shift carbon at δC 149.2, which suggested the carbo   (Figure 4). The acid hydrolysis of 3 liberated the D-glucopyranose; the absolute configurations of the sugars were also determined by gas chromatography as β-D-glucose. Finally, the planar structure of 3 was established as shown in Figure 1. In the ECD experiment, the trend experimental data of aglycone from 3 was consistent with that of 1 (Supplementary Figure S29). From the perspective of the biosynthetic pathway and ECD experiment, the absolute configuration of 3 was determined to be 6R, 7R, 8R, and the compound was given the name Clavilactone O.
-lactone moiety contained in the structure, which was also proved by HMBC between H-7(4.5, s) and C-6(72.7) (Figure 4). The only difference was a downfield shift carbon at δ C 149.2, which suggested the carbon was substituted by oxygen. The anomeric signal δ C 83.1(C-1 ), oxygenated methylene carbon (δ C 61.2) and four oxygenated methine carbons (δ C 78.6, 77.2, 72.1, 70.2) indicated the existence of a glucose fragment. The acid hydrolysis of 4 confirmed the sugar moiety as β-D-glucopyranose. The anomeric proton H-1 (4.39, t, 7.2) and its HMBC correlations to C-3(δ C 146.9) revealed the attachment of a β-D-aminoglycoside moiety at the C-3 position. The four olefinic carbons were assigned by the HMBC correlations between H-6 (δ H 5.87) and C-5(133.3), C-14(135.1) (Figure 4). The HMBC correlations between H-15(δH, 1.47, 3H, s) and C-14(135.1) also confirmed the assignment of olefinic carbons (Figure 4). Accordingly, the entire structure of compound 4 is elucidated in Figure 4. The absolute configuration of 4 was established by comparing the experimental curve of ECD with that of compound 1 (Supplementary Figure S29). The ECD spectrum of their aglycones matched well, which confirmed the 6R,7R,8R configuration, and the compound was given the name Clavilactone P. In addition, the antitumor activity was measured by IC 50 value against three human cancer cells (Hela, SGC-7901, and SHG-44) using the MTT assay [22]. Cisplatin was used as a standard for comparison. The antitumor effects are displayed in Table 3. Compound 1 and 2 exhibited moderate cytotoxic activity against Hela cell lines, with IC 50 values of 22.8 and 19.7 µM, respectively.

Discussion
In summary, four meroterpenoids with a novel aminoglycoside moiety were isolated for the first time from the fungus C. clavipes, and they represent the first group of aminoglycoside meroterpenoids possessing a 10-membered carbocycle fusing α,β-epoxy-γ-lactone. The novel aminoglycoside is a rare in nature, and this kind of nitrogenous meroterpenoid not only enriched the structural diversity, but also provided potent activity. Meanwhile, the glycosylation of natural products and drugs can often effectively change their physical and chemical activities, so natural glycoside compounds have great advantages in drug discovery, for example, the modification of digitoxigenin by glycosidation with neogluco/xylosides revealed sugar amine regiochemistry and had a dramatic effect upon anti-tumor activity [23]. The screening of cytotoxic activity proved that 1 and 2 exhibited a significant suppression of cell viability in the Hela (IC 50 = 22.8 and 19.7 µM) cell line. The relationship between glycosidation and the cytotoxic activity of this scaffold need to be further studied.

General Experimental Procedures
NMR spectra were obtained with a Bruker AV 600 NMR spectrometer (chemical shift are presented as δ values with TMS as the internal standard) (Bruker, Billerica, Germany). Abbreviations are as follows: s (singlet), d (doublet), dd (doublet of doublet) t (triplet), q