Meyeroguilline E, a New Isoindolinone Alkaloid from the Poisonous Mushroom Chlorophyllum molybdites, and Identification of Compounds with Multidrug Resistance (MDR) Reversal Activities

Three isoindolinone alkaloids (1–3), including one new isoindolinone-type alkaloid, meyeroguilline E (1), and six other known compounds (4–9) were isolated from the poisonous mushroom Chlorophyllum molybdites (Agaricaceae). The structure of the new compound was determined using extensive spectroscopic analyses via one-dimensional (1D) and two-dimensional (2D) NMR data interpretation and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). To the best of our knowledge, compound 1 is the first example of a natural isoindolinone with a butanoic acid moiety, and this study is the first to detect the other known compounds (2–9) in C. molybdites. The isolated compounds (1–9) were examined for their multidrug resistance (MDR) reversal activity against MES-SA, MES-SA/DX5, HCT15, and HCT15/CL02 human cancer cells. Based on the results, 20 μM of compounds 3 and 6 slightly potentiated paclitaxel (TAX)-induced cytotoxicity in MES-SA/DX5, HCT15, and HCT15/CL02 cells; however, the compounds had no effect on the cytotoxicity against MES-SA and nonMDR cells.


INTRODUCTION
Chlorophyllum molybdites (Meyer. ex Fr.) Mass. (Agaricaceae), a false parasol or green-spored parasol, is a widespread poisonous mushroom that usually grows on lawns and parks in tropical and subtropical regions. 1 This poisonous mushroom is also distributed in a restricted area throughout Korea and Japan. 1 Owing to a mushroom poisoning accident, C. molybdites has been reported to be a gastrointestinal irritant for over a century in many countries, including North America. 2 Further, there are increasing reports of C. molybdites poisoning worldwide each year. 3,4 The representative toxin of C. molybdites, which causes serious gastrointestinal discomfort that can last 1−6 h after ingestion, is a toxic protein called molybdophylysin. 5 Previous chemical investigation of this mushroom revealed the presence of two pyrrolidine alkaloids, lepiothins A and B, 6 α-methylglyceric acid, 7 fatty acid derivatives, 8 simple alkaloids, 8 and steroid derivatives. 9 In particular, some steroids isolated from C. molybdites were reported to exert cytotoxicity against human gastric carcinoma Kato III cells, with IC 50 values ranging from 2.69 to 14.27 μg/ mL. 9 As part of the continuing research to isolate bioactive novel natural products and elucidate their structures from diverse natural resources, 10−16 we investigated potentially bioactive metabolites from poisonous mushrooms. 17,18 In the present study, the bioactive compounds from a methanol (MeOH) extract of the fruiting bodies of C. molybdites were assessed using liquid chromatography−mass spectrometry (LC/MS)-guided analysis. The MeOH extract of C. molybdites fruiting bodies was subjected to solvent partitioning and intensive chemical analysis using repeated column chromatography and semipreparative HPLC. This separation process led to the isolation of three isoindolinone alkaloids (1−3) and six other known compounds (4−9). The structure of the new compound (1) was established via detailed one-dimensional (1D) and two-dimensional (2D) NMR and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analyses. Herein, the isolation and structural determination of compounds 1−9 have been described, and their multidrug resistance (MDR) reversal activity has been determined.

Isolation and Structural
Elucidation of Compounds 1−9. The MeOH extract of the fruiting bodies of C. molybdites was subjected to solvent partitioning using four organic solvents, namely, hexane, CH 2 Cl 2 , EtOAc, and BuOH. The four main fractions derived from solvent partitioning, i.e., hexane-, CH 2 Cl 2 -, EtOAc-, and BuOH-soluble fractions, were subjected to LC/MS analysis, which revealed that the CH 2 Cl 2and EtOAc-soluble fractions contained metabolites of interest, including N-containing compounds. Accordingly, the CH 2 Cl 2and EtOAc-soluble fractions were subjected to intensive chemical analysis by repeated column chromatography and semipreparative HPLC under the guidance of LC/MS analysis ( Figure 1). This analysis led to the isolation of three isoindolinone alkaloids (1−3) (Figure 2), including one new isoindolinone-type alkaloid, meyeroguilline E (1), and six other known compounds (4−9).  Figure S1), which aligns with the molecular formula C 13 H 15 NO 5 . Of note, this molecular formula was corroborated by the NMR spectroscopic data ( Table 1). The IR spectrum of 1 exhibited absorption bands for the hydroxyl (3440 cm −1 ) and carbonyl (1712 and 1620 cm −1 ) groups, and its 1 H NMR spectrum (Table 1 and Figure S2 Figure 3). The heteronuclear multiple bond correlation (HMBC) correlations from H 2 -9 to C-11 and OCH 3 to C-11 confirmed the methoxyl group at C-11, indicating the presence of butanoic acid methyl ester as a side chain ( Figure S5). The HMBC correlations from H-5 to C-3a and C-7; from H-7 to C-1, C-3a, and C-5; and from H 2 -3 to C-3a and C-7a constructed an isoindolinone moiety, and the linkage of the butanoic acid methyl ester to N-2 of the isoindolinone core was established by the HMBC correlation from H 2 -8 to C-3 and C-1 ( Figure 3). Hence, the chemical structure of 1 was elucidated, as shown in Figure 2, and named meyeroguilline E. To the best of our knowledge, compound 1 is the first example of a natural isoindolinone with a butanoic acid moiety.
The known compounds were identified as 4,6-dihydroxy-2,3-dihydro-1H-isoindol-1-one (2), 20 meyeroguillines D (3), 19 5′-methylthioadenosine (4), 21 (E)-2-decenedioic acid (5), 22 pantheric acid C (6), 23 5-phenylisoxazole (7), 24 benzoic acid (8), 25 and 5,10-dihydrobenzo[g]pteridine-2,4(1H,3H)-dione (9) 26 ( Figure 4) based on a comparison of their NMR spectroscopic data with reported values and the results of LC/ MS analysis. To the best of our knowledge, all known compounds (2−9) were isolated for the first time from C. molybdites. Among the substances isolated from C. molybdites, 5′-methylthioadenosine (4) is called 5′-methylthioadenosine (MTA) and is the S-methyl derivative of adenosine. MTA is an intermediate in the methionine salvage pathway, also called the 5′-methylthioadenosine (MTA) cycle, which recycles the sulfur of MTA, a byproduct of the biosynthesis of polyamine and the plant hormone, ethylene. 27 As the source of the sulfur atom in MTA, methionine (MET) was first identified in 1923, and the methyl group of MTA is derived from Sadenosylmethionine, which is synthesized from MET and adenosine triphosphate (ATP) through the catalysis of MET adenosyltransferases. 28 2.2. Evaluation of the Multidrug Resistance (MDR) Reversal Activity of the Isolated Compounds. According to our previous study, 29 isoindolinone alkaloids isolated from the mushroom Hericium erinaceum showed cytotoxic activity against the human cancer cell lines, A549, SK-OV-3, SK-MEL-2, and HCT-15. Based on these results, the cytotoxic activity of the isoindolinone alkaloids and the other known compounds identified in C. molybdites was investigated. All compounds tested in this study had little effect on the growth of A549, SK-OV-3, SK-MEL-2, and HCT-15 human cancer cells up to 30 μM (data not shown).
The MDR reversal activities of these compounds were tested in the human colorectal cancer cell line (HCT15) Figure 5). However, all compounds (20 μM) or VER (10 μM) had no effect on the cytotoxicity against MES-SA and nonMDR cells.

CONCLUSIONS
In this study, a chemical investigation of the MeOH extract from the fruiting bodies of the poisonous mushroom C. molybdites resulted in the isolation and identification of a new isoindolinone-type alkaloid, meyeroguilline E (1), and eight known compounds (2−9). In the present study, the new compound (1) was reported to be the first example of a natural isoindolinone with a butanoic acid moiety, and the other known compounds 2−9 were isolated from C. molybdites for the first time. Among the isolated compounds, 20 μM of compounds 3 and 6 slightly potentiated the paclitaxel (TAX)induced cytotoxicity to MES-SA/DX5, HCT15, and HCT15/ CL02 cells. Further, compounds 3 and 6 enhanced the survival fraction at 1000 nM of TAX from −11.66 to −33.93 and −41.46, respectively, in HCT15/CL02 cells.

Fungal Material.
Fruiting bodies of Chlorophyllum neomastoideum were collected from a mixed forest in Pocheon, GyeongGi-do, Korea, in August 2020. The samples were identified by one of the authors (R. Ryoo) via DNA analysis; fungal-specific PCR primers, ITS1 and ITS4, were used to amplify the internal transcribed spacer (ITS) region. The DNA sequence of this material was analyzed according to a modified method. 30 The homology of the ITS sequence was matched with that of C. neomastoideum (syn. Macrolepiota neomastoidea) with the highest score in the NCBI GenBank database. A voucher specimen (SKKU 2020-08-CN) was deposited at the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.

Extraction and Isolation.
Dried C. molybdite fruiting bodies (1.7 kg) were extracted with 100% methanol (MeOH, 3.0 L × 3) and filtered. The resulting extract was vacuumconcentrated using a rotary evaporator to obtain the MeOH extract (449.3 g). The concentrated MeOH extract was suspended in distilled water (700 mL) and solvent-partitioned three times each with hexane, dichloromethane (CH 2 Cl 2 ), ethyl acetate (EtOAc), and n-butanol (n-BuOH) to obtain four fractions: hexane-soluble (24.   31 Cell cultures were conducted in Falcon T-25 (Becton Dickinson, Lincoln Park, NJ) flasks containing 10 mL of RPMI-1640 medium with glutamine, sodium bicarbonate, gentamycin, amphotericin, and 5% fetal bovine serum (FBS). The cells were dissociated using 0.25% trypsin and 3 mM CDTA solution for passaging. The cells were maintained in an incubator at 37°C in a humidified atmosphere of 5% CO 2 .
4.5. MDR Reversal Assay. The ability of the samples to potentiate the cytotoxicity of paclitaxel (TAX) was evaluated in the MES-SA, MES-SA/DX5, HCT15, and HCT15/CL02 cell lines as previously described. 32 The cells were then inoculated over a series of standard 96-well flat-bottom microplates and preincubated for 24 h to allow attachment to the microtiter plate. The attached cells were incubated with serial dilutions of TAX in the absence or presence of each sample or verapamil. After continuous exposure to the compounds for 72 h, the culture medium was removed from each well and the cells were fixed with 10% cold TCA at 4°C for 1 h. After washing with distilled water, the cells were stained with 0.4% SRB solution and incubated for 30 min at room temperature. The cells were washed again and solubilized with 10 mM unbuffered Tris base solution (pH 10.5). The absorbance was measured spectrophotometrically at 520 and 690 nm using a microtiter plate reader (Molecular Devices, Sunnyvale, CA). To eliminate the effects of nonspecific absorbance, the absorbance at 690 nm was subtracted from that at 520 nm. The cell survival fractions were calculated via three basic measurements: time zero (Tz) at the beginning of drug incubation, cell control (CC) at the end of incubation without drug, and drug treatment (DT) at the end of the drug incubation period. CC is the fraction of cells incubated with the MDR reversal sample alone. If DT ≥ Tz, the net perfect cell growth inhibition was calculated as (DT − Tz)/(CC − Tz) × 100. If DT < Tz, the net percentage of cell-killing activity was calculated as (DT − Tz)/Tz × 100. All data represent the average values of three wells in each experiment.