p-Terphenyl and Diphenyl Ether Derivatives from the Marine-Derived Fungus Aspergillus candidus HM5-4

Two undescribed p-terphenyl derivatives, asperterphenylcins A–B (1–2), and two undescribed diphenyl ether derivatives, asperdiphenylcins A–B (3–4), together with three previously described p-terphenyl derivatives—4″-deoxyterprenin (5), terphenyllin (6), and 3″-hydroxyterphenyllin (7)—were obtained from the solid-rice culture of the marine-derived fungus Aspergillus candidus HM5-4, which was isolated from sponges from the South China Sea. Their structures were elucidated by HRESIMS data and NMR spectroscopic analysis. Compound 1 showed a strong inhibitory effect on Neoscytalidium dimidiatum, with an inhibition circle diameter of 31.67 ± 2.36 mm at a concentration of 10.0 µg/disc. Compounds 5 and 7 displayed cytotoxic activity against human chronic myeloid leukemia cells (K562), human liver cancer cells (BEL-7402), human gastric cancer cells (SGC-7901), human non-small cell lung cancer cells (A549) and human HeLa cervical cancer cells, with IC50 values ranging from 3.32 to 60.36 µM, respectively. Compounds 2, 6 and 7 showed potent inhibitory activity against α-glucosidase, with IC50 values of 1.26 ± 0.19, 2.16 ± 0.44 and 13.22 ± 0.55 µM, respectively.


Introduction
Terphenyls are an important family of natural products, including o-terphenyls, mterphenyls, and p-terphenyls [1].Among these, p-terphenyls are the most frequently occuring natural terphenyls, and the most conspicuous feature of their structure is that two terminal benzene rings and a central ring make up their para arrangement [2,3].Their structural diversity results from the different substituted functional groups on the three aromatic rings and the different connection modes between the rings [4].Over 250 p-terphenyl derivatives have been identified, mainly from actinomyces, mosses and macrofungi [1].In most cases, these secondary metabolites exhibit extensive biological activity, such as antimicrobial [5,6], cytotoxicity [7,8], free radical scavenging [9,10], phosphodiesterase inhibitory [11] and α-glucosidase inhibitory activities [12].The unique marine environment, with its high pressure, low temperature and high salinity, leads to the generation of secondary metabolites with different marine fungi biological activities, which are an important source of natural products with pharmacological effects [13].Recently, a fair number of p-triphenyl derivatives with novel structures and good biological activity have been found from the Aspergillus candidus that is widely distributed in marine environments [7,[14][15][16].Almost all of these marine-derived p-triphenyls have cytotoxic effects, but some have neuroprotective and antibacterial effects.For this reason, in the process of searching for new bioactive natural products from marine-derived fungi [17][18][19][20], we carried out chemical studies on A. candidus, a fungus from a marine sponge which was collected from the coast of Lingao County, Hainan Province.Two undescribed p-terphenyl derivatives, asperterphenylcins A-B (1-2), and two undescribed diphenyl ether derivatives, asperdiphenylcins A-B (3-4), together with three previously described p-terphenyl derivatives-4 ′′ -deoxyterprenin (5), terphenyllin (6), and 3 ′′ -hydroxyterphenyllin (7; Figure 1)-were isolated from the crude EtOAc extract of a solid-rice culture of the marine sponge-derived fungus A. candidus HM5-4.Based on the extensive biological activity of p-terphenyls, all compounds were screened for antifungal, cytotoxic and α-glucosidase inhibitory activities.In this paper, we discuss the separation, structure identification and biological activities of these obtained compounds in detail.
Mar. Drugs 2024, 22, x FOR PEER REVIEW 2 of 11 such as antimicrobial [5,6], cytotoxicity [7,8], free radical scavenging [9,10], phosphodiesterase inhibitory [11] and α-glucosidase inhibitory activities [12].The unique marine environment, with its high pressure, low temperature and high salinity, leads to the generation of secondary metabolites with different marine fungi biological activities, which are an important source of natural products with pharmacological effects [13].Recently, a fair number of p-triphenyl derivatives with novel structures and good biological activity have been found from the Aspergillus candidus that is widely distributed in marine environments [7,[14][15][16].Almost all of these marine-derived p-triphenyls have cytotoxic effects, but some have neuroprotective and antibacterial effects.For this reason, in the process of searching for new bioactive natural products from marine-derived fungi [17][18][19][20], we carried out chemical studies on A. candidus, a fungus from a marine sponge which was collected from the coast of Lingao County, Hainan Province.Two undescribed p-terphenyl derivatives, asperterphenylcins A-B (1-2), and two undescribed diphenyl ether derivatives, asperdiphenylcins A-B (3-4), together with three previously described p-terphenyl derivatives-4′′-deoxyterprenin (5), terphenyllin (6), and 3′′-hydroxyterphenyllin (7; Figure 1)-were isolated from the crude EtOAc extract of a solid-rice culture of the marine sponge-derived fungus A. candidus HM5-4.Based on the extensive biological activity of p-terphenyls, all compounds were screened for antifungal, cytotoxic and α-glucosidase inhibitory activities.In this paper, we discuss the separation, structure identification and biological activities of these obtained compounds in detail.

Structure Elucidation of New Compounds 1-4
Asperterphenylcin A (1) was isolated as a yellow film.Its molecular formula C21H20O6 was deduced by the HRESIMS ion peak at m/z 391.1139 (calcd.391.1152 for C21H20O6Na, [M + Na] + ; Figure S8), revealing twelve indexes of hydrogen deficiency.The characteristic IR spectrum absorption peak at 3415 cm −1 suggested the existence of a hydroxy group.The 1 H NMR spectrum showed eight aromatic protons (from δH 6.38 to δH 7.43) and three methoxy groups (δH 3.29, δH 3.64 and δH 3.73) (Table 1).The 13 C NMR spectrum revealed twenty-one carbon signals with the aid of the DEPT and HSQC spectra, which were attributed to eight protonated sp 2
Asperdiphenylcin B (4) was obtained as a white crystal.Its molecular formula was determined to be C 16 H 12 O 7 by the HRESIMS data at m/z 655.1028 (calcd.655.1058 for C 32 H 24 O 14 Na, [2M + Na] + ; Figure S38), indicating the presence of two carbon and two hydrogen atoms less than 3, with eleven degrees of unsaturation.The IR spectrum of 4 also showed the typical absorption bands of hydroxyl (3422 cm −1 ) and ester carbonyl (1736 cm −1 ) groups.A comprehensive analysis of the 1 H NMR and 13 C NMR spectra of 4 revealed that the NMR data of 4 almost resembled those of 3, indicating that they almost shared an identical structure skeleton (Table 2).Specifically, the characteristic peaks in the 13 C NMR spectrum of five protonated sp 2 carbons (δ C 110.8, 115.2, 119.4,122.8 and 134.5), seven non-protonated sp 2 carbons (δ C 116.6, 127.9, 128.9, 148.3, 149.8, 152.9 and 158.3), one oxygenated methylene (δ C 69.0) and one ester-carbonyl (δ C 166.4) in 4 indicated the same diphenyl ether skeleton with an eight-membered lactone ring as in 3, which was further confirmed by the similar key HMBC correlations in Figure 2. The only difference between 4 and 3 was the side chain at C-11, in the light of HMBC correlations between H-10 (δ H 7.65) and C-15 (δ C 166.6) and between H-12 (δ H 7.39) and C-15 (δ C 166.6), suggesting that there was a carboxyl group attached to the C-11 in 4 instead of the acetoxymethyl group observed in 3. Thus, the structure of 4 was assigned (Figure 1).

α-Glucosidase Inhibitory Activity
All the above-mentioned compounds were subjected to an α-glucosidase inhibitory activity test by a reported method [24], using genistein as a positive control.The results indicated that compounds 2 and 6 displayed more potent inhibitory activity (IC 50 values were 1.26 ± 0.19 and 2.16 ± 0.44 µM) than genistein (IC 50 value was 13.04 ± 2.56 µM; Table 4).The inhibitory activity of compound 7 was comparable to that of the positive drug genistein.Compound 1 had the weakest inhibitory activity, but it was still 6.7-times more active than acarbose.As shown in Table 4, all the diphenyl ether derivatives (3 and 4) exhibited no α-glucosidase inhibitory activity and all the p-terphenyl derivatives (1, 2, 6 and 7) except 5 exhibited α-glucosidase inhibitory activity.Comparing the structure of p-terphenyl derivatives 1, 2, 5, 6 and 7 revealed that the α-glucosidase inhibitory activity of 5 completely disappeared, probably because the hydrogen in the hydroxyl group was substituted by an isopentene group in 5.

Antifungal Activity
The antifungal activities of three known compounds (5-7) were determined by the filter paper disc agar diffusion method.The results revealed that only compound 5 showed inhibitory activity against N. dimidiatum, which can cause a serious disease of stem canker in red-fleshed dragon fruit (Hylocereus polyrhizus); the diameter of its inhibition zone was 31.67 ± 2.36 mm.Carbendazim, as a positive control, displayed inhibitory activity against N. dimidiatum; the diameter of its inhibition zone was 55.00 ± 0.82 mm.

Fungal Material and Fermentation
The marine fungus A. candidus HM5-4 was isolated from sponges collected from the coast of Lingao County, Hainan Province.The molecular sequence of the strain was cloned using the primers ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCT-TATTGATATGC), and the ITS sequence of the strain was determined.The sequences were submitted to the BLAST database in NCBI (sequence number: OP550289) and were found to be 100% homologous to A. candidus (MH398542.1).Therefore, the fungus was identified as Aspergillus candidus, together with the colony morphology of the fungus.The fungal strain was preserved in the South China Sea Marine Fungus Bank, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agriculture Sciences.
For chemical investigations, the marine fungal strain A. candidus HM5-4 was incubated on PDA medium (consisting of 200.0 g/L potato, 20.0 g/L glucose, 20.0 g/L agar, and 1000.0 mL deionized water) at 28 • C after being obtained from the South China Sea Marine Fungus Bank (maintained at −80 • C in an ultra-low temperature freezer).After three days, three pieces of media with fungi were transferred aseptically to three Erlenmeyer flasks with PDB media (consisting of 200.0 g/L potato, 20.0 g/L glucose, and 1000.0 mL deionized water) and incubated on a rotary shaker (180 rpm) at 28 • C for 72 h.Then, 2.0 mL of seed solution was added to one hundred 1000 mL Erlenmeyer flasks with rice solid media (each flask contained 80.0 g rice and 120.0 mL artificial seawater) for fermentation, respectively.These Erlenmeyer flasks were cultivated under static conditions at 28 • C for 30 days.
a Recorded in DMSO-d 6 .
a 1 H NMR (125 MHz) and
Values represent means ± SD based on three parallel experiments; b positive control-no activity at a concentration of 20 µg/mL. a