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Hemiacetalmeroterpenoids A–C and Astellolide Q with Antimicrobial Activity from the Marine-Derived Fungus Penicillium sp. N-5

Four new compounds including three andrastin-type meroterpenoids hemiacetalmeroterpenoids A-C (1–3), and a drimane sesquiterpenoid astellolide Q (15), together with eleven known compounds (4–14) were isolated from the cultures of the marine-derived fungus Penicillium sp. N-5, while compound 14 was first isolated from a natural source. The structures of the new compounds were determined by analysis of detailed spectroscopic data, and the absolute configurations were further decided by a comparison of the experimental and calculated ECD spectra. Hemiacetalmeroterpenoid A (1) possesses a unique and highly congested 6,6,6,6,5,5-hexa-cyclic skeleton. Moreover, the absolute configuration of compound 14 was also reported for the first time. Compounds 1, 5 and 10 exhibited significant antimicrobial activities against Penicillium italicum and Colletrichum gloeosporioides with MIC values ranging from 1.56 to 6.25 μg/mL.

Hemiacetalmeroterpenoid C (3) was also purified as a white powder. The molecular formula was specified as C 28 H 38 O 7 (ten degrees of unsaturation) by HRESIMS (m/z 509.25015 [M+Na] + ), which is 42 mass units higher than that of 2 ( Figure S17). Analysis of its NMR data (Table 1) revealed the presence of the same partial structure as that found in compound 2. The only difference was 3 has an additional acetyl fragment. Finally, a weak HMBC correlation from Ac-CH 3 to C-15 suggested that the acetyl fragment was attached to C-15 ( Figure 2).
Because compound 3 has the same chiral center as 2, the NOESY correlation and experimental ECD spectrum of compound 3 were in agreement with those of 2 (Figures 3 and 4). Thus, the absolute configuration of 3 was identified as 3R, 5S, 8S, 9R, 10S, 13R and 14R.
Compound 14 was obtained as a yellow powder. Analysis of its 1 H NMR and 13 C NMR data showed that the planar structure of 14 was the same as compound V, which was the product of the alkaline hydrolysis of parasiticolide A [27]. However, the absolute configuration of compound V was ambiguous.
The relative configuration of 14 was also defined by the NOESY correlation. The correlations of H 3 -14 with H 1 -5 and H 1 -6, and H 2 -13 with H 2 -15 were found in the NOESY spectrum, which means H 1 -5, H 1 -6, and H 3 -4 were on the same side, and H 2 -13 and H 2 -15 were on the opposite face ( Figure 3). Thus, the absolute configuration of the stereogenic centers in 14 was assigned as 4R, 5R, 6S, 10S by comparing its experimental ECD spectrum with that of the calculated model molecule (Figure 4). Finally, compound 14 was named as astellolide J.
Astellolide Q (15) was also acquired as a yellow powder. It molecular formula was determined as C 17 13 C NMR data showed the presence of 17 carbon signals, including two ester carbonyl carbons (δ C 173.1, 177.0) and two olefinic carbons (δ C 124.0, 169.0), one methyl, seven methylenes (three oxygenated), two methines (one oxygenated), two aliphatic quaternary carbons ( Table 2). Analysis of its 1 H NMR and 13 C NMR data in association with the 2D NMR data established a nucleus of drimane sesquiterpenoid characterized by an astellolide scaffold, structurally similar to the co-isolated compound 14 (Figures S26 and S27). It can be clearly observed that compound 15 has an additional acetyl fragment. Furthermore, the HMBC from H 2 -13 to Ac-OCO indicated that the acetyl fragment was linked to C-13 ( Figure 3). Finally, the NOESY correlation and experimental ECD spectrum of compound 15 were identical to those of 14 (Figures 3 and 4). Thus, the absolute configuration of 15 was also assigned as 4R, 5R, 6S, 10S.

Antimicrobial Assay
Compounds 1-15 were investigated for their antimicrobial activities against two phytopathogenic fungi and four bacterial strains. As shown in Table 3, andrastin-type meroterpenoids have better antimicrobial activities against phytopathogenic fungus than against bacteria. Most of all the tested compounds (9 compounds out of total 15 compounds) displayed potent antimicrobial activities (MIC < 50 µg/mL). Among them, compounds 1, 5 and 10 exhibited remarkable antimicrobial activities against Penicillium italicum and Colletrichum gloeosporioides with MIC values of 6.25, 1.56, 6.25 and 6.25, 3.13, 6.25 µg/mL. Moreover, compound 1 showed inhibitory activities against Bacillus subtilis under concentration of 6.25 µg/mL. Compound 10 also displayed significant antimicrobial activity against Salmonella typhimurium with an MIC value of 3.13 µg/mL. Notably, compound 5 revealed potential antimicrobial activity against all the strains, the MIC values were lower than 25 µg/mL.  As for the study of the structure-activity relationship (SAR), it was found that the degree of oxidation at C-21 had different effects on the activities of the compounds. The compound with methyl (10) at C-21 has significantly antimicrobial activity, followed by the aldehyde group (7), and hydroxymethyl (13) was the weakest. In-depth analysis showed that apart from the degree of oxidation at C-21, keto-enol tautomerism at the cyclopentane ring also had obvious influences on the antimicrobial activities of compounds. Compared to compounds 7 and 13 (enol form), compounds 8 and 9 (keto form) showed no activities against all strains (Table 3).

Fungal Material
Fungus N-5 was isolated from the rhizosphere soil of mangrove plant Avicennia marina (collected in October 2021 from Nansha Mangrove National Nature Reserve in Guangdong Province, China). It was identified as Penicillum sp. by the ITS region (deposited in GenBank, accession no ON926808), and fungus N-5 was deposited at Sun Yat-sen University, China.

Fermentation
The fungus Penicillum sp. N-5 was cultured in one hundred 1000 mL Erlenmeyer flasks at 25 • C for 30 days; these contained autoclaved rice solid-substrate medium composed of 50 g rice and 50 mL 3‰ saline water.

Extraction and Purification
After incubation, the mycelia and solid rice medium were extracted four times with EtOAc, and 75 g of residue was obtained. Next, the residue was separated by a gradient of petroleum ether/EtOAc from 9:1 to 0:10 (v/v) on silica gel CC and divided into six fractions (Fr.1-Fr.6). Fr. 2 (10 g) was separated to Sephadex LH-20 (methanol) to yield three sub-fractions (SFrs.

Bioassays Antimicrobial Activity
Antimicrobial activity assay was performed as previously described in [31,32].

Conclusions
In summary, three new andrastin-type meroterpenoids (1-3), one new drimane sesquiterpenoid (15) and one sesquiterpenoid J (14) that was first isolated from a natural source, together with ten known compounds (4)(5)(6)(7)(8)(9)(10)(11)(12)(13) were isolated from the cultures of the rhizosphere soil of mangrove plant Avicennia marina fungus Penicillium sp. N-5. Their structures were determined by the analysis of NMR, HR-MS and ECD spectra. All the isolated compounds were investigated for their antimicrobial activities against two phytopathogenic fungi and four bacterial strains. Among them, compounds 1, 5 and 10 exhibited significant inhibition against Penicillium italicum and Colletrichum gloeosporioides with MIC values of 6.25, 1.56, 6.25 and 6.25, 3.13, 6.25 µg/mL. Notably, compound 5 showed potential antimicrobial activity against all the strains and the MIC values were lower than 25 µg/mL. Moreover, andrastin-type meroterpenoid antimicrobial activity against phytopathogenic fungi was reported for the first time.