New Isocoumarin Analogues from the Marine-Derived Fungus Paraphoma sp. CUGBMF180003

Nine new secondary metabolites, including six isocoumarin analogues, 7-hydroxyoospolactone (1), 7-methoxyoospolactone (2), 7-methoxy-9-hydroxyoospolactone (3), 10-acetoxy-9-hydroxyoospolactone (4), 6-dehydroxysescandelin (5), parapholactone (6), and three compounds with a rare skeleton of isocoumarin coupled with phenylethylamine, namely paraphamide A (12), paraphamide B (13), and paraphamide C (14), together with five known compounds, oospolactone (7), 8-O-methyloospolactone (8), 10-hydroxyoospolactone (9), 9,10-dihydroxyoospolactone (10), and oospoglycol (11), were isolated and identified from the marine-derived fungus Paraphoma sp. CUGBMF180003. Their chemical structures were determined using spectroscopic data, including HRESIMS and 1D and 2D NMR techniques. Furthermore, the stereogenic carbons in 5 and 14 were determined by comparing the experimental and calculated electronic circular dichroism (ECD) spectra. The carbon skeleton of 12–14 was identified as the first example of isocoumarin coupled with phenylethylamine derivatives. All of these compounds were examined for antimicrobial activities against Candida albicans and Staphylococcus aureus. Both 1 and 6 showed antibacterial activity against S. aureus with MIC values of 12.5 μg/mL.


Structure Elucidation
Compound 1 was isolated as a light yellow powder. The molecular formula of 1 was determined to be C11H10O4 based on the HRESIMS spectrum (m/z [M + H] + 207.0652, calcd. for C11H11O4, 207.0652), accounting for seven degrees of unsaturation ( Figure S1). The 1 H NMR data of 1 ( Table 1, Figure S2) demonstrated two aromatic proton signals [(δH 6.93, d, J = 8.5 Hz, H-5), (δH 7.31, 1H, d, J = 8.5 Hz, H-6)], two methyl singlets at δH 2.24 (3H, s, H-9) and 2.06 (3H, s, H-10), and two phenolic hydroxyl groups at δH 9.69 (1H, brs, 7-OH) and 11.12 (1H, brs, 8-OH). The 13 C and HSQC spectra of 1 ( Figures S3 and S4) indicated 11 carbon signals (Table 1) which were categorized as one carboxyl at δC 166.2 (C-1), three oxygenated sp 2 carbons at δC 146.7 (C-3), 143.8 (C-7), and 148.6 (C-8), two nonprotonated sp 2 carbons at δC 129.6 (C-4a) and 105.9 (C-8a), and two protonated sp 2 carbons at δC 113.3 (C-5) and 124.2 (C-6). Detailed analysis of the 2D NMR data ( Figures S4-S6) revealed the existence of an isocoumarin skeleton for 1. The HMBC correlations from H-5 to C-7 and C-8a, from H-6 to C-4a, C-7 and C-8 revealed the o-dihydroxyl substituted benzene moiety. The long-range HMBC correlation from H-5 to C-1 indicated the presence of a carboxyl substituent on C-8a. Furthermore, the HMBC correlations from H-9 and H-10 to C-3 and C-4 indicated that the methyl groups were on C-3 and C-4. The connection from C-4 to C-4a was evidenced by the HMBC correlation from H-10 to C-4a. With the downfield chemical shifts of C-1 and C-3, it was revealed that C-1 and C-3 were connected by an oxygen atom. Therefore, the structure of 1 was assigned as shown in Figure 1 and named 7-hydroxyoospolactone.   Compound 2 was isolated as a light yellow powder. The molecular formula of 2 was determined to be C 12 Figure S8) displayed similar signals to those of 1. Comparison of the NMR data of the two compounds revealed the presence of one methoxyl signal at δ H 3.85, s (δ C 56.1) in 2. The HMBC spectrum (Figure S12) exhibited correlations from H-5 and -OMe to C-7, indicating that the methoxyl group was on C-7. Therefore, the structure of 2 was elucidated as shown in Figure 1 and named 7-methoxyoospolactone.  Figure S14) displayed similar signals to those of 2, except for the presence of the signal of hydroxymethyl protons δ H 4.36 (2H, s, H-9; δ C 57.6 C-9). Detailed analysis of 13 C and 2D NMR data (Figures S15-S17) revealed the structure of 3. The HMBC spectrum ( Figure 2, Figure S17) exhibited correlations from H-9 to C-3 and C-4, and from H-10 to C-3, C-4, and C-4a indicating that the hydroxymethyl group was on C-3. Therefore, the structure of 3 was elucidated as shown in Figure 1 and named 7-methoxy-9-hydroxyoospolactone.  Figure S7). The 1 H NMR data of 2 (Table 1, Figure S8) displayed similar signals to those of 1. Comparison of the NMR data of the two compounds revealed the presence of one methoxyl signal at δH 3.85, s (δC 56.1) in 2. The HMBC spectrum ( Figure S12) exhibited correlations from H-5 and -OMe to C-7, indicating that the methoxyl group was on C-7. Therefore, the structure of 2 was elucidated as shown in Figure 1 and named 7-methoxyoospolactone.
Compound 3 was isolated as a light yellow powder. The molecular formula of 3 was determined to be C12H12O5 based on the HRESIMS spectrum (m/z [M + H] + 237.0758, calcd. for C12H13O5, 237.0757), accounting for seven degrees of unsaturation ( Figure S13). The 1 H NMR data of 3 (Table 1, Figure S14) displayed similar signals to those of 2, except for the presence of the signal of hydroxymethyl protons δH 4.36 (2H, s, H-9; δC 57.6 C-9). Detailed analysis of 13 C and 2D NMR data (Figures S15-S17) revealed the structure of 3. The HMBC spectrum ( Figure 2, Figure S17) exhibited correlations from H-9 to C-3 and C-4, and from H-10 to C-3, C-4, and C-4a indicating that the hydroxymethyl group was on C-3. Therefore, the structure of 3 was elucidated as shown in Figure 1 and named 7-methoxy-9-hydroxyoospolactone.     Figures S20 and S21) showed 13 carbon signals, including those of the isocoumarin skeleton similar to those of oospolactone, and the two methyls in oospolactone were replaced by two hydroxymethyls at δ C 57.1 (C-9) and 60.1 (C-10), as well as two signals for acetyl groups at δ C 171.8 (C-11) and 21.0 (C-12). The presence of the hydroxymethyl group on C-3 was confirmed by the HMBC correlations ( Figure S23) from H-9 to C-3 and C-4. Additionally, the HMBC correlations from H-10 to C-3, C-4, C-4a, and C-11 and from H-12 to C-11 revealed an acetoxy group on C-10. Thus, the structure of 4 was elucidated as shown in Figure 1 and named 10-acetoxy-9-hydroxyoospolactone.  (oxygenated, C-8), and 106.8 (C-8a), as well as one signal for oxygenated methine sp 3 at δ C 65.0 (C-9) and one methyl carbon at δ C 23.3 (C-10). Detailed analysis of the 2D NMR data (Figures S27-S29) revealed that 5 is an analogue of the isocoumarin oospolactone. However, the methyl group at C-3 was replaced by a proton, and another methyl group at C-4 was replaced by a 1-hydroxyethyl group. The proposed structure was confirmed by the HMBC correlations from H-3 to C-1, C-4a, C-9, and C-10, and from H-10 to C-4 and C-9. Thus, the planar structure of 5 was elucidated. The absolute configuration of C-9 was assessed by comparison of experimental and calculated ECD spectra ( Figure 3A), confirming the S configuration of C-9, and the compound was named 6-dehydroxysescandelin.
comparison of experimental and calculated ECD spectra ( Figure 3A), confirming the S configuration of C-9, and the compound was named 6-dehydroxysescandelin.   Figures S33-S35) revealed an isocoumarin analogue. HMBC correlations from H-10 to C-3, C-4, and C-11, and from H-9 to C-3 and C-4 suggested that the 4-hydroxycyclopentenone ring was fused with the chromone ring through C-3 and C-4. Compound 6 showed weak optical rotation value of +0.91 (c 0.11, MeOH) and did not display ECD absorptions; therefore, 6 was elucidated as a racemic mixture. Therefore, the structure of 6 was defined as shown in Figure 1 and named parapholactone.
Compound 12 was isolated as a light yellow powder. The molecular formula of 12 was determined to be C 19 Figure  S36). The 1 H and 13 C NMR data of 12 (Table 3 H-9), δ C 45.6 (C-9) and one conjugated amide carbonyl at δ C 160.9 (C-10). Detailed analysis of 2D NMR data ( Figures S39-S41) confirmed the connections of the phenylethyl moiety to the isocoumarin core. The molecular formula (C 19 H 15 NO 4 ) of 12 and downfield shift of C-9 (δ C 45.6), combined with the HMBC correlations from H-9 to C-3, C-4, C-10, and C-1 , and from H-1 to C-9 and C-10 confirmed the connection of C-1 , C-9, and C-10 to N, forming a cyclopentenamide ring. Therefore, the structure of 12 was elucidated as shown in Figure 1 and named paraphamide A.  Figure  S42). The 1 H and 13 C NMR spectra of 13 (Table 3, Figures S43 and S44) resembled those of 12. Detailed analysis of the 1 H and 13 C NMR data ( Figures S43 and S44) revealed that H-6 of 12 was replaced by a hydroxyl group in 13. The structure of 13 was confirmed by the downfield shift of C-6 (δ C 155.8) and molecular formula. Thus, the structure of 13 was elucidated as shown in Figure 1 and named Figure S48). The 1 H and 13 C NMR spectra of 14 (Table 3, Figures S49 and S50) resembled those of 12. Detailed analysis of 2D NMR spectra (Figures S51-S53) revealed that one of the protons attached to C-1 of 12 was replaced by a methyl formate group with resonances at δ C 171.1 (C-1"), δ H 3.76 (3H, s, H-2"), and δ C 52.9 (C-2"). This moiety was confirmed by HMBC correlations from H-1 , H-2 , and H-2" to C-1". The absolute configuration of C-1 was also determined by comparison of experimental and calculated ECD spectra ( Figure 3B), confirming the S configuration of C-1 . Thus, the structure of 14 was elucidated as shown in Figure 1 and named as paraphamide B.

Biological Activity
All of the isolated compounds were subjected to tests of antibacterial activities against Candida albicans ATCC 10231 and Staphylococcus aureus ATCC 25923. Both 1 and 6 showed an inhibitory effect against S. aureus with minimum inhibitory concentration (MIC) values of 12.5 µg/mL; however, none of the isolates inhibited the growth of C. albicans.

Biological Activity
Compounds 1-14 were evaluated for their antimicrobial activities in 96-well plates according to the antimicrobial susceptibility testing standards outlined by the Clinical and Laboratory Standards Institute document M07-A7 (CLSI) [15]. Briefly, C. albicans ATCC 10231 was inoculated on potato dextrose agar plate and cultured for 24 hours at 35 • C. Five colonies of about 1 mm in diameter were picked and suspended in 5 mL of physiological saline. The suspension was then adjusted to approximately 10 6 CFU/mL with RPMI 1640. For the antibacterial assay, S. aureus ATCC 25923 was inoculated on a Mueller-Hinton broth agar plate and cultured for 24 hours at 37 • C. Five colonies of about 1 mm in diameter were then picked and suspended in 5 mL of physiological saline. The suspension was then adjusted to approximately 10 6 CFU/mL with Mueller-Hinton broth and 2 µL of 2-fold serial dilution of each compound (in DMSO) was added to each row in the 96-well microplate, which contained 78 µL of microbe suspension in each well. Amphotericin B and vancomycin were used as positive controls for fungi and bacteria, respectively; DMSO was used as negative control. The 96-well plates were incubated at 35 • C aerobically for 24 hours. The MIC was defined as the minimum concentration of the compound that prevented visible growth of the microbes.