Rhytidhylides A and B, Two New Phthalide Derivatives from the Endophytic Fungus Rhytidhysteron sp. BZM-9

Two new phthalide derivatives, rhytidhylides A (1) and B (2), together with ten known compounds (3–12) were isolated from cultures of Rhytidhysteron sp. BZM-9, an endophyte isolated from the leaves of Leptospermum brachyandrum. Their structures were identified by an extensive analysis of NMR, HRESIMS, ECD, and through comparison with data reported in the literature. In addition, the cytotoxic activities against two human hepatoma cell lines (HepG2 and SMMC7721) and antibacterial activities against MRSA and E. coli were evaluated.


Introduction
Endophytic fungi plays a role not only in supplying plants with the basic nutrients indispensable for their growth and helping them in the mechanisms of adaptation to various environmental stresses (i.e., salinity, drought), but they can also produce various bioactive natural products [1]. Phthalide, widely found in several higher and lower plant and fungal genera, is a naturally occurring benzobutyrolactones [2]. Many of the naturally occurring phthalides display different biological activities including antibacterial [3], antifungal [4], insecticidal [5], cytotoxic [6], and antioxidant [7] effects, which has also attracted widespread attention from other researchers.
During our search for new bioactive, secondary metabolites of fungi isolated from various medicinal plants, the strain Rhytidhysteron sp. BZM-9 was obtained and identified, which was isolated from Leptospermum brachyandrum. Rhytidhysteron sp. is a clinically pathogenic fungus [8]. The genus Rhytidhysteron includes two species: R. rufulum and R. hysterinum, which has a worldwide distribution and occurs particularly in the tropics and subtropics [8]. As far as we know, only a few chromones and Spirobisnaphthalenes have been reported from the genus Rhytidhysteron [9][10][11][12]. In our previous work, some chlorinated cyclopentene and isocoumarin derivatives from this strain were reported [13,14]. In the course of our continued search for bioactive compounds from endophytes, two new phthalide derivatives, rhytidhylides A (1) and B (2), along with ten known compounds (3)(4)(5)(6)(7)(8)(9)(10)(11)(12), were isolated from cultures of Rhytidhysteron sp. BZM-9 ( Figure 1). Herein, we describe the isolation, structure elucidation, and biological activities of these compounds.  , and three methyls [δH 2.06 (H-11), 1.57 (H-10), and 1.08 (d, J = 6.5 Hz, H-9); δC 6.4 (C-11), 20.7 (C-10), 16.1 (C-9)]. Comparison of the 1 H and 13 C NMR data (Table 1) of 1 with those of known compound 3 suggested that both compounds shared similar structural features, with the difference being that one proton of C-8 was replaced by a hydroxyl group. The HSQC correlation of C-8 with a methine proton (δH 3.87) rather than a methylene proton and obvious down-field chemical shift of C-8 (δC 71.1) verified this conclusion. Thus, the planar structure of 1 was completely established, which was further supported by HMBC and 1 H-1 H COSY correlations, as present in Figure 2.

Results and Discussion
Compound 1 was obtained as a yellow solid, and the molecular formula was deduced as C 12 (Table 1) of 1 revealed the presence of one ester carbonyl [(δ C 171.6 (C-1)], one five substituted benzene ring [δ H 6.41 (H-4); δ C 99.9 (C-4), 163.3 (C-5), 111.2 (C-6), 155.4 (C-7), 151.6 (C-3a), 102.8 (C-7a)], one oxymethine [δ H 3.87 (q, J = 6.5 Hz, H-8); δ C 71.1 (C-8)], one oxygenated sp 3 non-protonated carbon [(δ C 89.5 (C-3)], and three methyls [δ H 2.06 (H-11), 1.57 (H-10), and 1.08 (d, J = 6.5 Hz, H-9); δ C 6.4 (C-11), 20.7 (C-10), 16.1 (C-9)]. Comparison of the 1 H and 13 C NMR data ( Table 1) of 1 with those of known compound 3 suggested that both compounds shared similar structural features, with the difference being that one proton of C-8 was replaced by a hydroxyl group. The HSQC correlation of C-8 with a methine proton (δ H 3.87) rather than a methylene proton and obvious down-field chemical shift of C-8 (δ C 71.1) verified this conclusion. Thus, the planar structure of 1 was completely established, which was further supported by HMBC and 1 H-1 H COSY correlations, as present in Figure 2.   (Table 1) of 1 with those of known compound 3 compounds shared similar structural features, with the difference be of C-8 was replaced by a hydroxyl group. The HSQC correlation of proton (δH 3.87) rather than a methylene proton and obvious down-fi C-8 (δC 71.1) verified this conclusion. Thus, the planar structure of 1 tablished, which was further supported by HMBC and 1 H-1 H COSY sent in Figure 2.    Compound 1 has two asymmetric centers. The NOESY correlations were not conclusive in the case of 1. Thus, to determine the relative configuration of 1, gauge-independent atomic orbital (GIAO) DFT 13 C NMR calculations were performed at the ωB97x-D/6-31G* level using MeOH as the solvent, and the calculations data were compared with their experimental values, following the reported STS protocol. According to linear regression analysis of 13 C NMR chemical shifts, the values of the correlation coefficient (R 2 ) were 0.9983 for 1a and 0.9987 for 1b (Figure 3. Moreover, the resulting P mean and P rel parameters as well as MAE and RMS values further showed 1b or its enantiomers are correct structures for 1 (Table 2). Subsequently, the absolute configurations of 1 were determined to be 3R,8S on the basis that the experimental ECD perfectly matched with the calculated ECD ( Figure 4). Therefore, compound 1 was named rhytidhylide A. Compound 1 has two asymmetric centers. The NOESY correlations were not conclusive in the case of 1. Thus, to determine the relative configuration of 1, gauge-independent atomic orbital (GIAO) DFT 13 C NMR calculations were performed at the ɷB97x-D/6-31G* level using MeOH as the solvent, and the calculations data were compared with their experimental values, following the reported STS protocol. According to linear regression analysis of 13 C NMR chemical shifts, the values of the correlation coefficient (R 2 ) were 0.9983 for 1a and 0.9987 for 1b (Figure 3. Moreover, the resulting Pmean and Prel parameters as well as MAE and RMS values further showed 1b or its enantiomers are correct structures for 1 (Table 2). Subsequently, the absolute configurations of 1 were determined to be 3R,8S on the basis that the experimental ECD perfectly matched with the calculated ECD ( Figure 4). Therefore, compound 1 was named rhytidhylide A.   analysis of 13 C NMR chemical shifts, the values of the correlation coefficient (R 2 ) were 0.9983 for 1a and 0.9987 for 1b (Figure 3. Moreover, the resulting Pmean and Prel parameters as well as MAE and RMS values further showed 1b or its enantiomers are correct structures for 1 (Table 2). Subsequently, the absolute configurations of 1 were determined to be 3R,8S on the basis that the experimental ECD perfectly matched with the calculated ECD ( Figure 4). Therefore, compound 1 was named rhytidhylide A.    (Table 1) of 2 and 1 suggested that they shared closely similar NMR resonances. The main difference between them was the exhibition of a methoxy group at the C-7 position of 1 and a hydroxyl group C-7 position of 1. This conclusion could be further verified by the HMBC correlation from -OCH 3 (δ H 3.86) to C-7 (δ C 157.5) (Figure 2). Hence, the planar structure of 2 was corroborated.

Results and Discussion
Similarly, the relative configuration of 2 was assigned by (GIAO) DFT 13 C NMR calculations. By comparing the calculated 13 C NMR data with the corresponding experimental values, the possible configuration of 2 was distinguished ( Figure 3) (Table 2). Finally, the absolute configuration was established as 3S, 8R based on the experimental ECD, which was highly similar to the calculated ECD ( Figure 4). Thus, compound 2 was named rhytidhylide B.

General Experimental Procedures
Optical rotations were measured on a Rudolph Research Analytical Autopol IV automatic polarimeter. HRESIMS spectra were recorded on an Agilent 6500 series Q-TOF mass spectrometer (Agilent, Singapore) analyser with a positive ion mode. Experimental ECD spectra were performed on a chirascan plus Circular Dichroism spectrometer. NMR spectra (1D and 2D) were obtained using Bruker 500MHz spectrometers and using TMS A review has reported that phthalide derivatives are present in nature with an enormous spectrum of bioactivities extending from bactericidal to cytotoxic [2]. Additionally, methicillin-resistant Staphylococcus aureus (MRSA) is endemic in hospitals worldwide and causes substantial morbidity and mortality, which is becoming an important public health problem [25]. Therefore, all compounds were evaluated for their cytotoxic activities inhibited by two human hepatoma cell lines (HepG2 and SMMC7721) and antibacterial activities against MRSA and E. coli. Among them, compound 12 displayed weak antibacterial activity against MRSA with a MIC value of 62.5 ug/mL (Table 3

General Experimental Procedures
Optical rotations were measured on a Rudolph Research Analytical Autopol IV automatic polarimeter. HRESIMS spectra were recorded on an Agilent 6500 series Q-TOF mass spectrometer (Agilent, Singapore) analyser with a positive ion mode. Experimental ECD spectra were performed on a chirascan plus Circular Dichroism spectrometer. NMR spectra (1D and 2D) were obtained using Bruker 500MHz spectrometers and using TMS as an internal standard. The single crystal data were collected on an Agilent Xcalibur Novasingle-crystal diffractometer equipped with CuKα radiation. Preparative HPLC was done using an Agilent 1100 prep-HPLC system with a YMC-peak ODS-A column (5 µm, 250 × 10 mm). Sephadex LH-20 (GE Healthcare, Uppsala, Sweden), silica gel (200-300 and 60-100 mesh, Qingdao Marine Chemical Factory, Qingdao, China), and C 18 reversed-phase silica gel (40-75 µm, Fuji, Kasugai, Japan) were used for column chromatography (CC). All solvents were of analytical grade.

Fungal Material
The fungal strain Rhytidhysteron sp. BZM-9 was isolated from the leaves of Leptospermum brachyandrum, collected in the South China Botanical Garden in Guangzhou city, Guangdong province of China, in September 2016. The strain was deposited at the Xiangya School of Pharmaceutical Sciences, Central South University. The fungus was identified as Rhytidhysteron sp. BZM-9 (GenBank accession number: MN788611).

Fermentation, Extraction and Isolation of Compounds
The fungus was inoculated on solid rice medium, which was prepared by autoclaving 250 g of rice in 300 mL of demineralized water in a 500 mL Erlenmeyer flask. The fermentation was performed in 15 flasks under static conditions at room temperature for a month [26]. The fungal culture was extracted with ethyl acetate added to each flask, and the extract was subsequently dried under a vacuum to afford 50 g.

X-ray Crystallographic Analysis
Crystal data for 4.

Quantum Chemistry Calculations
The conformation optimization, ECD spectrum calculation, and DFT GIAO 13 C NMR calculation were performed as previously described [14].

Antimicrobial Activity Assay
Antimicrobial activities of all compounds were evaluated by calculating MIC values against MRSA and E. coli using the broth microdilution method according to CLSI guidelines [27]. Vancomycin (MIC = 1.25 µg/mL) was used as a positive control.

Conclusions
In the course of our continued exploration of the fungal strain Rhytidhysteron sp. BZM-9 for biologically active metabolites, two new phthalide derivatives, rhytidhylides A (1) and B (2), together with ten known compounds (3-), were isolated and identified. The cytotoxic activities and antimicrobial activities of 1-12 were also evaluated, but only compound 12 showed weak inhibitory activity against MRSA. All in all, the activities of these compounds were not thoroughly investigated, just the evaluation of the cytotoxicity and antibacterial assays, and only a few cells and strains have been tested. Other activities and their mechanisms are still worthy of further exploration.