Polysubstituted Phenyl Glucosides Produced by the Fungus Metarrhizium anisopliae*

Wen-jing WANG, E-mail: wangwj0122@163.com #Corresponding author, Chun-mei CHEN, E-mail: chenchunmei @hust.edu.cn;Yong-hui ZHANG, E-mail:zhangyh@mails. tjmu.edu.cn *This work was financially supported by the Program for Changjiang Scholars of Ministry of Education of the People’s Republic of China (No. T2016088), the National Natural Science Foundation for Distinguished Young Scholars (No. 81725021), the National Natural Science Foundation of China (No. 31900288), the Program from the China Postdoctoral Science Foundation (No. 2019M652660), and the Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College (HUST, China). Polysubstituted Phenyl Glucosides Produced by the Fungus Metarrhizium anisopliae*

Marine-derived fungi have recently been proved to be a significant source of secondary metabolites with structural and biological diversity [1,2] . According to the literature, more than 4000 natural compounds have been isolated from marine-derived fungi since 1945, which have attracted the attention of chemists and biologists because of their unique chemical structure and various biological activity, and are playing an increasingly important role in drug discovery and development process [3][4][5][6][7][8][9][10][11] . As part of our ongoing investigation on new bioactive secondary metabolites from marine-derived microorganisms, the strain Metarrhizium anisopliae (M. anisopliae), isolated from the mangrove soil, attracted our attention. M. anisopliae, an insecticidal fungus, has been reported to produce various secondary metabolites including destruxin, cytochalasin, aurovertin and cyclic hetapeptide, which exhibited extensive bioactivities [12][13][14][15][16][17] . The ethyl acetate (EtOAc) extract of the fungal culture exhibited moderate anti-inflammatory activity, furthermore, chemical investigation on this strain has resulted in the isolation of seven new polysubstituted benzene glucosides metarhizosides A-G (1-7), together with four known compounds 8-11 ( fig. 1). These new structures were confirmed by extensive nuclear magnetic resonance (NMR) spectroscopy and chemical method, and they all feature a 4′-O-methyl-β-glucopyranose unit. The known compounds were identified as phellinignincisterol C (8) [18] , lepistamide B (9) [19] , cyclotryprostatin E (10) [20] , 6-methoxyspirotryprostatin B (11) [21] . In the bioassay, compounds 1 and 2 showed inhibitory activity against lipopolysaccharides (LPS)induced nitric oxide (NO) production. Herein, details of isolation, structural elucidation, and biological activities are presented.
Lambda 35 spectrophotometer (PerkinElmer, Inc., USA). ECD data were measured with a JASCO-810 instrument (JASCO Co., Ltd., Japan). HRESIMS data were acquired on Bruker micrOTOF Ⅱ spectrometer. An Agilent 1260 HPLC system semi-preparative HPLC equipped with a DAD detector was used to purify the compounds. Chemical shifts are expressed in ppm with reference to the solvent peaks CD 3 OD (δ H 3.31/δ C 49.0) and DMSO-d 6 (δ H 2.50/δ C 39.52).

Fungal Material
The fungus M. anisopliae was isolated from a mangrove mud sample collected in Xiamen, China. The mud sample was suspended and diluted with sterile water, and then coated individually on potato dextrose agar (PDA) medium containing chloramphenicol. Followed by the routine microbiological methods, the single colonies were obtained [22] . The internal transcribed spacer (ITS) region of this fungus was submitted to the GenBank and identified as M. anisopliae with accession no. MH079423. The voucher sample MA414 was preserved in the Culture Collection Center of Tongji Medical College, Huazhong University of Science and Technology.

Fermentation and Isolation
The fungus MA414 was incubated on PDA at 28°C for 7 days. The agar was cut into small pieces and then inoculated in 100 × 1 L Erlenmeyer flasks, each one contained 200 g rice and 200 mL distilled water. After incubation at 28°C for 21 days, the solid medium was concentrated and then distilled with CH 3 CH 2 OH repeatedly. After that, the EtOAc was used to extract and 80 g of the extraction was finally obtained. The separation of the EtOAc extract was subjected to column chromatography on silica gel (CC, 80-120 mesh) eluting with CH 2 Cl 2 -MeOH (100:1-0:1, v/v) to obtain four fractions (Fr. A-Fr. D).

Hydrolysis of 4
Compound 4 (10 mg) was hydrolyzed in 3 mL 5% aqueous hydrochloric acid and heated up to 90°C for 14 h. The reaction fluid was extracted by 3 mL EtOAc three times, and then the solvent of the aqueous layer was evaporated under reduced pressure to get a mixed production. The mixture was further purified by semipreparative HPLC to obtain an anomeric mixture of 4′-O-methyl-glucopyranose (2.3 mg) and the optical rotation was [α] D 25 +53.7(c 0.1,MeOH). The EtOAc layer (7.0 mg) was concentrated in vacuo and then identified as 2-(2-hydroxyethyl) phenol. 1.5 Biological Assays 1.5.1 Anti-inflammatory Activity Anti-inflammatory activity of the new compounds was evaluated using the ELISA with indomethacin and dexamethasone serving as the positive controls [23] . RAW 264.7 cells were cultured in DMEM (HyClone, Logan, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS) (HyClone) at 37°C in a humidified atmosphere with 5% CO 2 . Then the cells were inoculated in 48-well microtiter plates at 1×10 4 cells/ well for 24 h, then the test compounds (40 µmol/L) were added to the wells. Three hours later, the LPS with a concentration of 100 ng/mL was added, and the incubation was continued for 24 h at 37°C. The NO level in the supernatant was determined according to the manufacturer's instructions (Boster Biological Technology Co., Ltd., China) using ELISA.

Cytotoxicity against Cancer Cell Lines
Cytotoxicity of the selected compounds against four cancer cell lines (HepG2, HL-60, A549 and MCF-7), was evaluated using the MTT method with doxorubicin as positive controls [24] . All the cells were cultured in RPMI.1640 medium (Hangzhou Jinuo Biology Technology Co., Ltd., China), supplemented with 10% FBS (ExCell Biology Co., Ltd., China), 100 μg/mL streptomycin (Solarbio) and 100 units/mL penicillin at 37°C in a humidified atmosphere with 5% CO 2 . Then the tumor cells were inoculated in 96-well plates at a density of 5000 cells/well to incubate for 24 h. After that, test compounds (40 µmol/L) were added to the wells and incubated for another 48 h, 20 µL 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Solarbio) was added, and the incubation was continued for 4 h at 37°C. The MTT assay results were obtained using a microplate spectrophotometer (SpectraMax® I3, Molecular Devices, USA).  [25] . At the meantime, the 4′-OH was methylated on the glucose unit that was identified by heteronuclear multiple bond correlation (HMBC) signals from H-7′ to C-4′, and H-4′ to C-7′. Based on the chemical shift (δ H 4.81) and coupling constant (J = 7.6 Hz) of the anomeric proton, the glycosyl unit was identified as 4′-O-methyl-βglucopyranose [26] . The observed correlations from H 3 -7 to C-3, C-4 and C-5, from H-3 to C-2 and C-6 and from H-5 to C-1 and C-3 in HMBC spectrum suggested the presence of the 1,3,4-trisubstituted benzene ring. Further HMBC correlation from the anomeric proton (H-1′) to C-1 revealed the location of the glycosyl at C-1 of the benzene ring. Combined with the molecular formula, the structure of 1 was ultimately concluded as shown in fig. 2.

Metarhizoside
Metarhizoside B (2) was also isolated as pale yellow oil. Its molecular formula was determined to be C 15 H 22 O 8 based on its HRESIMS data. Subsequent comparison of the 1 H and 13 C NMR data of 2 (tables 1 and 2) with those of 1 confirmed that 2 was also a glycoside derivative. Further analysis of their 2D NMR spectroscopic data revealed the same glycosyl moiety as 4′-O-methyl-β-glucopyranose, while the methyl at C-6 of the aglycone was replaced by hydroxyethyl in 2. Furthermore, key HMBC correlation from H-1′ to C-1 demonstrated the same location of the glycosyl moiety. Therefore, the structure of 2 was established as shown.
Compound 3 was called metarhizoside C and obtained as pale yellow oil. The molecular formula of C 17 H 24 O 9 with 6 degrees of unsaturation was deduced from the HRESIMS spectrum. The similar NMR data of 3 and 2 (tables 1 and 2) revealed the resembled structure of them. Detailed analysis of their 2D NMR spectroscopic data showed that compound 3 was an acetylization derivative of 2, which was further confirmed by the molecular formula. Thus, compound 3 was deduced as shown.
Metarhizoside D (4) possessed the molecular formula of C 15 H 22 O 7 based on its HRESIMS spectrum. Analysis of the 1D NMR spectra revealed that compound 4 features the same sugar unit of 4′-O-methylβ-glucopyranose as those of 1, 2, and 3. The rest 1 H NMR data (table 1) of 4 showed signals corresponding to four olefinic protons at δ H 7.15 (1H, d, J = 7.1 Hz ), 7.13 (1H, t, J = 7.3 Hz), 7.05 (1H, d, J = 7.1 Hz) , 9.91 (1H, td, J = 7.3, 1.2 Hz), which suggested the presence of an ortho substituted benzene ring. Observed HMBC correlation from H-1′ to C-1 revealed that the sugar moiety was connected to the aglycone unit at C-1 of the benzene ring. Another substitution on the benzene ring was identified as hydroxyethyl and further confirmed by HMBC correlations from H-7 to C-1 and C-3 and COSY correlation between H-7/H-8. As a result, the structure of 4 was established as shown.
The molecular formula of metarhizoside E (5) was identified as C 16 H 24 O 8 from the HRESIMS data; this formula contains 30 mass units (OCH 2 ) more than that of 4. The 1 H NMR data (table 1) of 5 showed three olefinic protons at δH 7.03 (1H, d, J=8.3 Hz ), 6.67 (1H, d, J=2.5 Hz), 6.49 (1H, dd, J=8.3, 2.5 Hz), which were presumed to be an ABX spin-coupled system on the benzene ring. Subsequent comparison of the 1D NMR data of 5 (tables 1 and 2) with those of 4 confirmed that they shared the same skeleton, except for an additional methoxy group substitution on the benzene ring of 5. Furthermore, the location of the methoxy group at C-5 was identified by key HMBC correlation from 5-OCH 3 to C-5. Accordingly, the structure of 5 was deduced as shown.
Metarhizoside F (6) was also isolated as pale yellow oil. The positive-mode HRESIMS spectrum of 6 exhibited a [M+Na] + ion peak at m/z 337.1268 that is consistent with a molecular formula of C 15 H 22 O 7 , which was the same as that of 4. Similar to that observed for 4, the 1D NMR data (tables 1 and 2) recorded for 6 revealed the existence of the same sugar unit as 4′-O-methyl-β-glucopyranose and the substituted benzene ring. However, the chemical shift of the sugar anomeric proton in 6 was reduced by 0.6 ppm while the chemical shift of C-8 and C-1′ was increased by 7.4 and 2.0 ppm respectively, suggesting that the connection between the sugar moiety and aglycone unit has changed. Observed HMBC correlations from H-1′ (δ H 4.15) to C-8 (δ C 68.4) and H-8 (δ H 3.87, 3.56) to C-1′ (δ C 102.8) ( fig. 3) revealed the sugar unit was incorporated into the benzene ring through the -CH 2 CH 2 O-substitution at C-2. Therefore, compound 6 was deduced as shown. Metarhizoside G (7) gave a [M+Na] + ion peak in the HRESIMS spectrum at m/z 367.1373 (calcd. for C 16 H 24 NaO 8 , 367.1369) appropriate for a molecular formula of C 16 H 24 O 8 , which contains 30 mass units (OCH 2 ) more than that of 6. The 1H NMR data (table 1) of 7 showed the sugar anomeric proton at δ H 4.15, suggesting the same connection of the sugar moiety and aglycone unit. At the meantime, the 1H NMR spectrum gave three olefinic protons at δH 6.99 (1H, d, J=8.3 Hz), 6.37 (1H, d, J=2.6 Hz) and 6.29 (1H, dd, J=8.3, 2.6 Hz), which led to another methoxyl substitution on the benzene ring at C-5 and was further confirmed by the key HMBC correlation from 5-OCH 3 (δ H 3.65) to C-5 (δ C 158.8). Thus, the structure of 7 was ultimately concluded as shown.
To determine the absolute configuration of the sugar unit in these new structures, compound 4 was selected to be acid hydrolysis [27] . The sugar fragment obtained from the hydrolysate aqueous layer gave positive specific rotation sign of [α] D 25 +53.7 (c 0.1, MeOH), which indicated the D-configuration of the 4-O-methylglucopyranose as the consistency of experimental data and literature data [α] D 26 +80 (c 1.3, MeOH) [26,28] .
In the bioassay, new compounds (1-7) were investigated for their anti-inflammatory activity by using LPS-stimulated murine macrophage RAW 264.7 cells and the cytotoxicity against four human cancer cell lines (HepG2, HL-60, A549, and MCF-7). As a result, compounds 1 and 2 showed moderate inhibitory activity against NO production at a concentration of 40 µmol/L with indomethacin and dexamethasone as the positive control ( fig. 4). However, none of these glycoside compounds showed cytotoxicity against selected cancer cell lines including HepG2, HL-60, A549, and MCF-7 at 40 µmol/L.

DISCUSSION
Metarhizosides A-G (1-7), seven new polysubstituted benzene glucosides featured a 4′-O-methyl-β-glucopyranose unit, were isolated from the EtOAc extracts of marine-derived fungus M. anisopliae. Their structures were elucidated by NMR spectroscopy and chemical method. Compounds 1 and 2 showed moderate anti-inflammatory effect on the inhibition of the production of NO.