marine drugs

: Three new catecholic compounds, named meirols A–C ( 2 – 4 ), and one known analog, ar-govin ( 1 ), were isolated from the marine-derived fungus Meira sp. 1210CH-42. Their structures were determined by extensive analysis of 1D, 2D NMR, and HR-ESIMS spectroscopic data. Their absolute configurations were elucidated based on ECD calculations. All the compounds exhibited strong antioxidant capabilities with EC 50 values ranging from 6.01 to 7.47 µ M (ascorbic acid, EC 50 = 7.81 µ M), as demonstrated by DPPH radical scavenging activity assays. In the α -glucosidase inhibition assay, 1 and 2 showed potent in vitro inhibitory activity with IC 50 values of 184.50 and 199.70 µ M, respectively (acarbose, IC 50 = 301.93 µ M). Although none of the isolated compounds exhibited cytotoxicity against one normal and six solid cancer cell lines, 1 exhibited moderate cytotoxicity against the NALM6 and RPMI-8402 blood cancer cell lines with GI 50 values of 9.48 and 21.00 µ M, respectively. Compound 2 also demonstrated weak cytotoxicity against the NALM6 blood cancer cell line with a GI 50 value of 29.40 µ M.


Introduction
Small-molecule natural products, such as catechol and β-lactam, are widely utilized in the drug discovery process in various ways [1,2].Because of their low weight, smallmolecule drugs can traverse cell membranes, interact with proteins and enzymes within cells, and disrupt specific processes.Alternatively, a biologically active natural product may inspire the discovery of clinically useful drug agents by offering insights into types of structural features that may prove valuable.Catechols, benzene derivatives containing a 3,4-dihydroxyphenyl group, have recently been discovered as diverse derivatives in microorganisms, plants, insects, and marine organisms [3].Their ubiquity can be attributed to their rich redox chemistry, ability to cross-link through complex and irreversible oxidation mechanisms, excellent chelating properties, and the various ways in which vicinal hydroxyl groups interact with surfaces of significantly diverse chemical and physical characteristics [4,5].Aligned with their structural variety, catecholamines have been reported to exhibit a broad spectrum of biological activities, encompassing antiparasitic, antibacterial, metal-chelating, anti-inflammatory, immune-modulation, wound-healing, antioxidant, Mar.Drugs 2024, 22, 87 2 of 10 neuroprotective, nephroprotective, and metabolic regulation activities [3].Hence, novel catechol compounds derived from natural sources will serve as a promising foundation for broadening their potential biological and medical applications.
In 2023, the first marine-derived Meira sp. was described in our previous report as Meira sp.1210CH-42 [12].We identified new thiolactones and steroids from the Meira sp.strain, which exhibited α-glucosidase inhibitory activities [12].In our ongoing efforts to find new bioactive small compounds, we have paid attention to the fungus Meira because the genus has not been studied well, and the strain was the first marine-derived species showing good bioactivities in our preliminary screening.Therefore, a mass culture was conducted to isolate bioactive secondary metabolites from the strain.As a result, we discovered novel bioactive catecholic compounds from the culture extract of the fungus Meira sp.1210CH-42.Herein, we describe the structure determination of catecholic compounds (1-4), along with their antioxidant, α-glucosidase inhibitory, cytotoxic, tyrosinase inhibitory, and antimicrobial activities.

Structure Elucidation of New Compounds
Compound 1 was isolated as a white amorphous powder, and its molecular formula was determined to be C 9 H 8 O 3 based on an HR-ESIMS analysis at m/z 165.0552 [M + H] + (calcd for C 9 H 9 O 3 + , 165.0552), with six degrees of unsaturation.The 1 H and 13 C NMR data of 1 are summarized in Tables 1 and 2. The 1 H NMR spectrum of 1 in CD 3 OD revealed two olefinic protons (δ H 7.15 and 6.84) and two methylene protons (δ H 3.01 and 2.63).The 13 C NMR and HSQC spectra showed the presence of nine signals, including one carbonyl carbon (δ C 209.1), two oxygen-bearing sp 2 (δ C 153.1 and 143.1), two non-protonated sp 2 (δ C 144.7 and 130.8), two protonated sp 2 (δ C 117.2 and 116.9), and two methylene (δ C 37.6 and 23.3) carbons.The structure of 1 was identified as argovin (4,5-dihydroxyindan-1-one) by an analysis of its NMR and HRMS data and a comparison of its spectroscopic data with those previously reported in the literature (Figure 1) [11].
NMR and HSQC spectra showed the presence of nine signals, including one carbonyl carbon (δC 209.1), two oxygen-bearing sp 2 (δC 153.1 and 143.1), two non-protonated sp 2 (δC 144.7 and 130.8), two protonated sp 2 (δC 117.2 and 116.9), and two methylene (δC 37.6 and 23.3) carbons.The structure of 1 was identified as argovin (4,5-dihydroxyindan-1-one) by an analysis of its NMR and HRMS data and a comparison of its spectroscopic data with those previously reported in the literature (Figure 1) [11].Compound 2 was purified as a white amorphous powder.The molecular formula of 2 was determined to be C8H7NO3 by HR-ESIMS analysis at m/z 188.0324 [M + Na] + (calcd for C8H7NO3Na + , 188.0324), which was determined to possess six degrees of unsaturation.The 1 H NMR spectrum of 2 displayed the signals for two olefinic protons (δH 7.19 and 6.90) and two methylene protons (δH 4.34, overlapped) (Table 1).The 13 C NMR and HSQC spectra exhibited one carbonyl (δC 174.5), two oxygen-bearing sp 2 (δC 150.4 and 141.6), two  non-protonated sp 2 (δC 132.4 and 125.2), two protonated sp 2 (δC 116.9 and 116.5), and one methylene (δC 44.5) carbons (Table 2).The indicated the presence of a 1-indanone ring system.Additionally, the HMBC correlation of the singlet at H-3 (δH 4.34, s) with a carbonyl carbon C-1 (δC 174.5) confirmed the con nectivity of an amide carbonyl to the benzene ring (Figure 2).Thus, the structure of 2 wa elucidated as a previously unreported catecholic compound, 4,5-dihydroxyisoindolin-1 one, and 2 was named meirol A. Compound 3 was obtained as a purple amorphous powder.The molecular formula of 3 was analyzed for C9H8O4 on the basis of its parent ion in the HR-ESIMS analysis a m/z 203.0321 [M + Na] + (calcd for C9H8O4Na + , 203.0320), which required six degrees o

Bioactivity Evaluation of Compounds
The antioxidant activities of 1-4 were assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay.As indicated in Table 3, 1-4 exhibited considerable free radical scavenging activities with EC50 values ranging from 6.01 ± 0.07 to 7.47 ± 0.13 µM, showing better activities than the positive control, ascorbic acid (EC50 = 7.81 ± 0.25 µM).Also, 1-4 were evaluated for α-glucosidase inhibitory activities (Table 3).Compounds 1 and 2 exhibited significant inhibitory effects with IC50 values of 184.50 ± 2.93 and 199.70 ± 1.87 µM, respectively.Meanwhile, 3 showed weaker activity (IC50 = 367.43± 3.01 µM) than the positive control, acarbose (IC50 = 301.93± 3.55 µM).A structure-activity relationship analysis of 1-4 indicated that the hydroxy groups at C-2 or C-3 in diol-indanone impacted their antioxidant properties but did not have a significant impact on their αglucosidase inhibitory activity.Furthermore, all the compounds were screened for their cytotoxic activity against six solid and seven blood cancer cell lines (Table 3).Compounds 1 and 2 showed selective cytotoxicity against two out of the seven blood cancer cell lines

Fungal Strain and Fermentation
The fungal strain Meira sp.1210CH-42 was obtained from a seawater sample collected at the Chuuk Islands, Federated States of Micronesia, in 2010, as described previously [12].The strain was identified as Meira sp.(GenBank accession number OQ693946) through DNA amplification and by sequencing the ITS region of the rRNA gene, as described earlier [12].The cultures of strain 1210CH-42 were performed in modified Bennett's broth medium (1% D-glucose, 0.2% tryptone, 0.1% yeast extract, 0.1% beef extract, 0.5% glycerol, sea salt 10 g/L, pH 7.0).A seed culture was prepared from a spore suspension of strain 1210CH-42 by inoculating it into 2 L flasks and incubating it at 28 • C for 7 days in a rotary shaker at 120 rpm.The seed culture was inoculated aseptically into a 100 L fermenter containing 70 L of sterilized culture medium (0.1% v/v).Large-scale fermentation was conducted at 28 • C, 40 rpm, and with an airflow rate of 10 L/min (LPM) for a duration of 21 days before being harvested.

Computational Analysis
The initial geometry optimization and conformational searches were generated using Conflex 8 (rev.B, Conflex Corp., Tokyo, Japan).The optimization and calculation for electronic circular dichroism (ECD) were conducted utilizing the Gaussian 16 program (rev.B.01, Gaussian Inc., Wallingford, CT, USA).Conformational searches were executed through MMFF94s force field calculations, with a search limit set at 5 kcal/mol.The conformers were optimized using the ground state method at the CAM-B3LYP/6-31 G+ (d, p) level in MeOH with an IEFPCM model for ECD.The theoretical calculations of ECD spectra were performed using TD-SCF at the CAM-B3LYP /6-31 G+ (d, p).The ECD spectrum was derived by calculating the Boltzmann-weighted sum of conformer spectra.The final ECD spectra were simulated using SpecDis (v.1.71) with σ values ranging from 0.20 to 0.30 eV.All calculated curves were UV-shifted by +10 to +15 nm to better simulate experimental spectra.

Antioxidant Activity Assay
The DPPH radical scavenging activities of 1-4 were determined by the reported method [13].In a 96-well plate, 100 µL of sample solution (in MeOH) was mixed with 100 µL of DPPH solution (0.16 mM in MeOH), shaken several times, and then incubated at room temperature for 30 min.The absorbance at 517 nm was recorded.Ascorbic acid was used as the positive control, and the experiments were performed in triplicate.

α-Glucosidase Inhibitory Activity Assay
The α-glucosidase inhibitory activity was determined by measuring the absorbance increase resulting from the hydrolysis of p-nitrophenyl-α-D-glucopyranoside (pNPG, TCI) by α-glucosidase at 405 nm using a microplate reader, according to the reference to previously reported literature [12].The 130 µL sample solution (in 0.1 mM PBS) with the 30 µL α-glucosidase solution (0.2 U/mL) was incubated at 37 • C for 10 min.Subsequently, 40 µL of 5 mM pNPG was added.The reaction mixture was further incubated at 37 • C for 20 min.The α-glucosidase inhibitory activity was determined using a microplate reader at 405 nm.The negative control was prepared by substituting PBS buffer for the sample in the same way as the test.Acarbose served as the positive control, and the experiments were conducted in triplicate.

Cytotoxicity Assay
The cytotoxic activities of 1-4 were measured using the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI, USA) and conducted by the SRB (sulforhodamine B) assay, as previously described [14,15].The luminescence signal was quantified using a GloMax-Multi Detection System (Promega, Madison, WI, USA), and GI 50 values were determined utilizing a relative GI 50 model in GraphPad Prism (GraphPad, San Diego, CA, USA).Doxorubicin was used as the positive control.

Tyrosinase Inhibitory Activity Assay
The tyrosinase inhibitory activity was assessed using L-DOPA and the 96-well microplate method, as previously reported [16].Briefly, 140 µL of 20 mM phosphate buffer (pH 6.8) and 20 µL of mushroom tyrosinase (480 U/mL) in the same buffer were added to wells containing 20 µL of the test compounds.After incubation for 10 min at 25 • C, 20 µL of L-DOPA (0.85 mM) was added to the 200 µL reaction system.The incubation was continued for another 20 min at 25 • C; then, the colored end product's absorbance was measured at 475 nm using a microplate reader.Kojic acid was utilized as a positive control in the reference sample experiment.All experiments were performed in triplicate for each compound.

Antibacterial Assay
The antibacterial activities were determined using the 96-well microplate method, as described in a published report, against three Gram-positive and three Gram-negative bacteria [17].

Conclusions
In summary, four catecholic compounds (1−4), including three new ones (2-4), were identified from Meira sp.1210CH-42.Their structures were elucidated by a detailed analysis of NMR and HRESIMS data.The absolute configurations of 3 and 4 were determined by calculating the ECD spectra of their possible isomers.The antioxidant-DPPH assay showed that all compounds exhibited more significant free radical scavenging activity (EC 50 = 6.01-7.47µM) than ascorbic acid.Additionally, 1 and 2 exhibited moderate αglucosidase inhibitory activity (IC 50 = 184.50 and 199.70 µM, respectively) and selective cytotoxicity against blood cancer cell lines (RPMI-8402 and NALM6, GI 50 = 9.47-29.40µM).As a result, the catecholic indanones from Meira sp.1210CH-42 could serve as a potential agent for antioxidative, α-glucosidase inhibitory, and anticancer leads.Further investigation is needed to determine the biological mechanism of compounds from the marine-derived fungus Meira.

Figure 3 .
Figure 3. Experimental and calculated ECD spectra of 3 and 4.