Secondary metabolites from a marine sponge-associated fungus Xenomyrothecium sp. IMBC-FP2.11

Cite this paper: Vietnam J. Chem., 2020, 58(6), 752-758 Article DOI: 10.1002/vjch.202000067 752 Wiley Online Library © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Secondary metabolites from a marine sponge-associated fungus Xenomyrothecium sp. IMBC-FP2.11 Tran Hong Quang 1* , Pham Thi Mai Huong 1 , Nguyen Thi Thanh Ngan 2 , Tran Thi Hong Hanh 1 , Nguyen Xuan Cuong 1 , Nguyen Hoai Nam 1 , Chau Van Minh 1 1 Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam 2 Institute of Genome Research, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam Received April 30, 2020; Accepted July 9, 2020 Abstract In the study, seven compounds were isolated from an EtOAc extract of the marine-derived fungal strain Xenomyrothecium sp. IMBC-FP2.11, including (3R,4R)-4-hydroxymellein (1), (3R,4S)-4-hydroxymellein (2), trans-3,4- dihydro-3,4,8-trihydroxynaphtalen-1(2H)-one (3), (3S)-6-hydroxy-8-methoxy-3,5-dimethyl-isochroman (4), 8-hydroxy- 6-methyl-9-oxo-9H-xanthene-1-carboxylate (5), TMC-256A1 (6), and -hydroxyemodin (7). Their chemical structures were elucidated by detailed analysis of the 1D and 2D NMR and mass spectra as well as comparison with those of the previously reported compounds. This is the first case to report the chemical profile of a Xenomyrothecium fungal strain. Keywords. Xenomyrothecium, marine fungus, isochroman, naphtalenone, xanthone, naphtho--pyrone, anthraquinone. 1. INTRODUCTION Marine-derived fungi have been demonstrated as a rich source of bioactive metabolites with high diverse chemical structures. Xenomyrothecium (Stachybotriaceae family) is a monotypic genus derived from Myrothecium, [1] the fungal genus has been shown to produce various types of secondary metabolites, including cyclopentenones, [2] macrocyclic trichothecenes, [3-6] and meroterpenoids and isocoumarinoids. [7] However, chemical profile of the Xenomyrothecium genus has yet to be discovered so far. In our continuing search for secondary metabolites from the Vietnamese marine- derived fungi, [8,9] we conducted a chemical investigation of a fungal strain Xenomyrothecium sp. IMBC-FP2.11 isolated from an unidentified sponge. The present study dealt with isolation and structural determination of seven compounds from the EtOAc extract of the fermentation culture of the fungal strain. 2. MATERIAL AND METHODS 2.1. Fungal material The fungal strain IMBC-FP2.11 was isolated from an unidentified sponge collected from Quang Nam sea, Vietnam during May, 2019. After sterilizing the surface, a small slice of the sponge sample was homogenized in sterile seawater. A part of the mixture was transferred to a potato dextrose agar (PDA) plate using the spread method and incubated at 25 o C over 15 days. The fungal isolates were sub- cultured and purified to give pure fungal strain which was preserved at -80 o C. Taxonomic classification of the fungal strain was done by analyzing the ITS region of the fungal rDNA. A GenBank search using the ITS rDNA gene of IMBC-FP2.11 (GenBank accession number MT367734) revealed that Xenomyrothecium tongaense (NR_154511.1) is the closest match, with 99 % similarity. Consequently, the fungal strain IMBC-FP2.11 was identified as Xenomyrothecium sp. 2.2. General experimental procedures Optical rotations were measured by a Jasco P-2000 digital polarimeter. The NMR spectra were obtained on Bruker AVANCE III HD 500 FT-NMR and JEOL JNM ECP-400 FT-NMR spectrometers. ESIMS were acquired by an ESI Q-TOF MS/MS system (AB SCIEX Triple). Thin layer Vietnam Journal of Chemistry Tran Hong Quang et al. © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 753 chromatography (TLC) was carried out using Kieselgel 60 F254 and RP-18 F254s plates (Merck). Column chromatography (CC) was performed using silica gel (Kieselgel 60, 230-400 mesh, Merck) and YMC*Gel (ODS-A, 12 nm, S-150 m, YMC Co., Ltd.) resins. Preparative high-performance liquid chromatography (HPLC) was performed on an Agilent 1200 Preparative HPLC System. 2.3. Fermentation and extraction The fungal strain Xenomyrothecium sp. IMBC- FP2.11 was grown on 50 replicate 2L-Erlenmeyer flasks (each flask contained 100 mL of PDA and 3.0 % NaCl). The seed cultures (2 mL) of the fungal strain was added to the media and incubated at 25 o C for 20 days. Subsequently, the combined agar media and fungal biomass were extracted three times with EtOAc (10 L/time) to yield the extract which was further concentrated in vacuo to give a residue (4.8 g). 2.4. Isolation and identification The EtOAc extract of Xenomyrothecium sp. IMBC- FP2.11 was fractionated by reversed phase (RP) C18 flash column chromatography (CC), using a stepwise gradient elution of 20, 40, 60, 80, and 100 % MeOH in H2O to provide fractions F1-F6, respectively. Next, fraction F2 was subjected to sephadex LH-20 CC, eluting with CH2Cl2-MeOH (15:1, v/v) to yield for subfractions (F2.1-F2.4). F2.4 was then purified by silica gel prep. TLC, using CH2Cl2-EtOAc (5:1, v/v) to obtain 3 (8 mg). F3 was introduced to sephadex LH-20 CC, eluting with MeOH-H2O (4:1, v/v) to provide five subfractions (F3.1-F3.5). F3.2 was then purified by prep. RP C18 HPLC, using a gradient elution of MeOH (40 to 100 %) in H2O over 60 min to give 4 (2 mg). Using the similar method, F3.4 was separated by prep. RP C18 HPLC, eluting with 20 % acetonitrile in H2O over 60 min to give 1 (14 mg) and 2 (2 mg). Fraction F4 was introduced to an RP C18 fractionation, eluting with MeOH in H2O (1:1 to 2:1, v/v) to provide subfractions F4.1-F4.4. F4.2 was further purified by RP C18 prep. HPLC, using a gradient elution of 50- 100 % MeOH in H2O over 60 min to give 7 (5 mg). By the similar method, compound 6 (2 mg) was isolated from F4.3 by RP C18 prep. HPLC, using a gradient elution of MeOH in H2O (60-100 %) over 60 min. Finally, compound 5 (3 mg) was obtained from F4.4 by silica gel prep. TLC, using n-hexane- EtOAc (1:6, v/v) as a mobile phase. (3R,4R)-4-hydroxymellein (1): white, amorphous powder; []D 25 = -21.5 (c = 0.1, MeOH); C10H10O4, M = 194; ESIMS: m/z 217 [M+Na] + ; 1 H (CD3OD, 500 MHz) and 13 C NMR details (CD3OD, 125 MHz), see table 1. (3R,4S)-4-hydroxymellein (2): white, amorphous powder; []D 25 = -16.0 (c = 0.1, MeOH); C10H10O4, M = 194; ESIMS: m/z 217 [M+Na] + ; 1 H (CD3OD, 500 MHz) and 13 C NMR details (CD3OD, 125 MHz), see table 1. trans-3,4-Dihydro-3,4,8-trihydroxynaphtalen- 1(2H)-one (3): white, amorphous powder; []D 25 = +31.3 (c = 0.1, MeOH); C10H10O4, M = 194; ESIMS: m/z 195 [M+H] + ; 1 H (CD3OD, 400 MHz) and 13 C NMR details (CD3OD, 100 MHz), see table 2. (3S)-6-Hydroxy-8-methoxy-3,5-dimethyl- isochroman (4): white, amorphous powder; []D 25 = +121.6 (c = 0.2, MeOH); C12H16O3, M = 208; ESIMS: m/z 209 [M+H] + ; 1 H (DMSO-d6, 400 MHz) and 13 C NMR details (DMSO-d6, 100 MHz), see table 2. 8-hydroxy-6-methyl-9-oxo-9H-xanthene-1- carboxylate (5): yellow, amorphous powder; C16H12O5, M = 284; ESIMS: m/z 285 [M+H] + ; 1 H (CDCl3, 400 MHz) and 13 C NMR details (CDCl3, 100 MHz), see table 3. TMC-256A1 (6): yellow, amorphous powder; C15H12O5, M = 272; ESIMS: m/z 295 [M+Na] + ; 1 H (DMSO-d6, 400 MHz) and 13 C NMR details (DMSO-d6, 100 MHz), see table 3. -Hydroxyemodin (7): yellow, amorphous powder; C15H10O6, M = 286; ESIMS: m/z 287 [M+H] + ; 1 H (DMSO-d6, 400 MHz) and 13 C NMR details (DMSO-d6, 100 MHz), see table 3. 3. RESULTS AND DISCUSSION The molecular formula of compound 1, C10H10O4 was deduced by a quasi-molecular ion [M+Na] + at m/z 217 in the ESIMS in combination with an analysis of its NMR spectroscopic data. Its 1 H NMR spectrum was shown to contain proton signals characteristic of an ABC spin system [H 7.00 (d, J = 8.0 Hz, H-5), 7.57 (t, J = 8.0 Hz, H-6), and 7.00 (d, J = 8.0 Hz, H-7)], suggesting the presence of an 1,2,3-trisubstituted aromatic ring. The 1 H NMR spectrum additionally showed signals of two oxymethine groups [H 4.74 (qd, J = 2.0, 6.5 Hz, H- 3) and 4.58 (d, J = 2.5 Hz, H-4)] and one secondary methyl group at H 1.54 (d, J = 6.5 Hz, H3-9). Analysis of the 13 C NMR and HSQC spectra of 1 revealed 10 signals, of which one carbonyl group at C 171.0 (C-1), three non-protonated signals at C 143.2 (C-4a), 162.9 (C- 8), and 108.4 (C-8a), two oxymethine at C 80.0 (C-3) and 67.7 (C-4), and a methyl at C 16.3 (C-9) were recognized (table 1). With the observed 1 H and 13 C NMR spectroscopic Vietnam Journal of Chemistry Secondary metabolites from © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 754 Figure 1: Structures of compounds 1-7 from Xenomyrothecium sp. IMBC-FP2.11 Table 1: 1 H and 13 C NMR spectroscopic data for 1 and 2 Position C #1 1 C #2 2 C a,b H a,c (J in Hz) C a,b H a,c (J in Hz) 1 169.7 171.0 168.5 170.2 3 67.5 80.0 4.74 (qd, 2.0, 6.5) 79.9 81.6 4.56 (qd, 4.5, 6.5) 4 78.8 67.7 4.58 (d, 2.0) 69.1 69.5 4.57 (d, 4.5) 4a 141.0 143.2 141.2 144.1 5 118.9 119.8 7.00 (d, 8.0) 116.2 117.7 7.10 (d, 8.5) 6 137.2 137.7 7.57 (t, 8.0) 136.8 137.8 7.59 (t, 8.5) 7 118.7 118.4 7.00 (d, 8.0) 117.8 117.8 6.96 (d, 8.5) 8 162.3 162.9 161.9 162.9 8a 107.2 108.4 106.6 108.0 9 16.4 16.3 1.54 (d, 6.5) 17.9 18.2 1.49 (d, 6.5) a Recorded in CD3OD, b 125 MHz, c 500 MHz; #1δC of (3R,4R)-4-hydroxymellein in CDCl3; [10] #2δC of (3R,4S)-4- hydroxymellein in CDCl3. [13] evidence, compound 1 was suggested to be an isocoumarin derivative. Comparison of the NMR data of 1 with those of the previously reported isocoumarin, (3R,4R)-4-hydroxymellein revealed a good agreement, implying that these compounds have the identical structures. [10] The small coupling (J = 2.0 Hz) between two vicinal protons (H-3 and H-4) indicated that these protons have a cis- relationship. [10-12] This was supported by the agreement between the specific optical rotation of 1 with the previously reported value: 1: []D 25 = -21.5 (c = 0.1, MeOH) vs. (3R,4R)-4-hydroxymellein: []D 25 = -39.2 (c = 0.25, MeOH). In addition, the structure of 1 was confirmed by HMBC correlations from H3-9 to C-3 and C-4, from H-3 to C-1, C-4, and C-4a, and from H-7 to C-8 and C-8a (figure 2). Consequently, compound 1 was identified as (3R,4R)-4-hydroxymellein. Compound 2's molecular formula was established as C10H10O4 by a sodium adduct ion [M+Na] + at m/z 217 observed in the ESI mass spectrum. Comparison between the NMR data of 2 and 1 resulted in a good agreement, except that the carbon chemical shifts of C-3, C-4, and C-9 of 2 are Figure 2: Selected HMBC correlations of compounds 1, 2, and 4-6 more down-field shifted, at C 81.6, 69.5, and 18.2, respectively (table 1). In addition, the significant higher coupling between H-3 and H-4 of 2 (J = 4.5 Hz) compared with that of 1 (J = 2.0 Hz) revealed that these protons are trans- configured. Thus, compound 2 was suggested to be adiastereoisomer of 1. This was further supported by the close similarity of the 1 H and 13 C NMR data and the Vietnam Journal of Chemistry Tran Hong Quang et al. © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 755 specific optical rotation of 2 compared with those of the previously reported analogs. [11-13] Consequently, the overall structure of 2 was confirmed by detailed analysis of the HSQC and HMBC spectra (figure 2). On the basis of thespectroscopic evidence obtained, compound 2 was elucidated to be (3S,4R)-4- hydroxymellein. Compound 3 possessed the molecular formula C10H10O4, as established by the present of a protonated molecular ion [M+H] + at m/z 195 in the ESIMS. Its 1 H NMR spectrum showed proton signals for an ABC spin system [H 6.85 (d, J = 8.0 Hz, H-5), 7.53 (t, J = 8.0 Hz, H-6), and 7.13 (d, J = 8.0 Hz, H-7)], two oxymethine groups at H 4.07 (m, H-3) and 4.60 (d, J = 6.8 Hz, H-4), and a methylene group [H 2.70 (dd, J = 8.0, 17.2 Hz, Ha-2) and 3.10 (dd, J = 4.0, 17.2 Hz, Hb-2)]. In the 13 C NMR spectrum, signals of three non-protonated carbons at C 145.8 (C-4a), 163.2 (C-8), and 116.7 (C-8a) and three aromatic methine carbons at C 120.1 (C-5), 137.9 (C-6), and 117.7 (C-7) were observed, characterizing the presence of a 1,2,3-trisubstituted benzene ring. The 13 C NMR spectrum additionally exhibited signals of a carbonyl functional group at C 204.3 (C-1), two oxymethine carbons at C 71.6 (C-3) and 73.2 (C-4), and a methylene carbon at C 44.3 (C-2). These aforementioned 1 H and 13 C NMR values allowed to suggest that compound 3 is a naphthalenone derivative. Accordingly, the 1 H NMR spectroscopic data of 3 was identical with those of the previously reported naphthalenone, trans-3,4- dihydro-3,4,8-trihydroxynaphtalen-1(2H)-one, suggesting the identical structures of both compounds (table 2). [14] The trans-relationship between H-3 and H-4 of 3 was deduced from their relative large vicinal couplings (J = 6.8 Hz) which were consistent with those of the reported compound (Table 2). [14] On the basis of the aforementioned analysis, compound 3 was identified as trans-3,4- dihydro-3,4,8-trihydroxynaphtalen-1(2H)-one. The molecular formula of compound 4, C10H14O4 was deduced by a quasi-molecular ion [M+H] + at m/z 209 in the ESIMS. The 1 H NMR spectrum of 4 exhibited a singlet of an aromatic proton at H 6.31 (H-7), implying the presence of a penta-substituted benzene ring. The 1 H NMR spectrum additionally displayed two methyl signals at H 1.25 (d, J = 6.4 Hz, C-3) and 1.91 (s) and one methoxy signal at H 3.65 (s, 8-OCH3). The 13 C NMR and DEPT spectra of 4 contained six sp 2 [including three aromatic non-protonated carbons at C 112.7 (C-5), 153.8 (C-6), and 153.0 (C-8)] and six sp 3 carbons [including one oxymethine at C 69.8 (C-3), one oxymethylene at C 63.7 (C-1), and one methylene at C 33.5 (C-4)] (table 2). This spectroscopic evidence led to a hypothesis that 4 has the isochroman skeleton type. Accordingly, the 1 H and 13 C NMR data of 4 were in a good match compared with those of the reported isochroman derivative, (3S)-6-hydroxy-8-methoxy-3,5- dimethylisochroman, indicating that their structures are identical. [15] Subsequently, the structure of 4 was confirmed by HMBC spectrum. An HMBC correlation observed from H 3.65 to C 153.0 (C-8) revealed that the methoxy group is located at C-8 (figure 2). Furthermore, HMBC correlations from Table 2: 1 H and 13 C NMR data for compounds 3 and 4 Position H #1 (J in Hz) 3 C #2 4 C a,b H a,c (J in Hz) C d,b H d,c (J in Hz) 1 204.3 64.6 63.7 4.66 (d, 14.4) 4.40 (d, 14.4) 2 2.72 (dd, 8.0, 17.2) 3.11 (dd, 4.0, 17.2) 44.3 2.70 (dd, 8.0, 17.2) 3.10 (dd, 4.0, 17.2) 3 4.05-4.11 (m) 71.6 4.07 (m) 70.6 69.8 3.60 (m) 4 4.63 (d, 7.0) 73.2 4.60 (d, 6.8) 34.0 33.5 2.58 (dd, 2.8, 16.0) 2.26 (dd, 10.0, 16.0) 4a 145.8 134.1 133.6 5 6.88 (dd, 0.6, 8.6) 120.1 6.85 (d, 8.0) 112.9 112.7 6 7.56 (dd, 7.4, 8.4) 137.9 7.53 (t, 8.0) 152.7 153.8 7 7.14 (dt, 0.6, 7.4) 117.7 7.13 (d, 8.0) 96.2 95.9 6.31 (s) 8 163.2 153.9 153.0 8a 116.7 115.2 113.7 9 21.7 21.6 1.25 (d, 6.4) 10 10.0 10.0 1.91 (s) 8-OCH3 55.0 54.8 3.65 (s) a Recorded in CD3OD, b 100 MHz, c 400 MHz, d in DMSO-d6; #1δH of trans-3,4-dihydro-3,4,8-trihydroxynaphtalen- 1(2H)-one in CD3OD; [14] #2δC of (3S)-6-hydroxy-8-methoxy-3,5-dimethylisochroman in CDCl3. [15] Vietnam Journal of Chemistry Secondary metabolites from © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 756 H3-9 to C-3 and C-4 and from H3-10 to C-4a, C-5, and C-6 enabled to confirm the position 9-CH3 and10-CH3, respectively. Thus, compound 4 was identified as (3S)-6-hydroxy-8-methoxy-3,5- dimethylisochroman. The molecular formula of compound 5, C16H12O5 was established based on a protonated molecular ion [M+H] + at m/z 285 in the ESIMS. In the 1 H NMR spectrum, aromatic proton signals for a 1,2,3-trisubstituted benzene ring [H 7.31 (d, J = 8.0 Hz, C-2), 7.73 (t, J = 8.0 Hz, C-3), and 7.52 (d, J = 8.0 Hz, C-4)] and a 1,2,3,5-tetrasubstituted aromatic ring [H 6.74 (br s, H-5) and 6.63 (br s, H-7)], along with singlet signals of a methyl at H 2.42 (6-CH3) and a methoxy at H 4.02 (1-COOCH3) (Table 3). Analysis of the 13 C NMR and DEPT spectra pointed out 16 signals, including two carbonyl carbons at C 180.4 (C-9) and 169.7 (1-COOCH3), seven aromatic non-protonated carbons [including three oxygenated carbons at C 161.5 (C-8), 156.0 (C-4a), and 155.7 (C-4b) ]. This 1 H and 13 C NMR evidence suggests that 5 belongs to the xanthone skeleton type. This was supported by comparing the NMR data of 5 with those of the previously reported xanthone, 8- hydroxy-6-methyl-9-oxo-9H-xanthene-1- carboxylate (table 3). [16,17] In the HMBC spectrum, cross-peaks from H 12.14 (8-OH) to C-7, C-8, and C-8a, from H 4.02 (1-COOCH3) to C-1, from H-2 to C-1, and from H 2.42 (6-CH3) to C-6 were observed, thus confirming the gross structure of 5 (figure 2). Therefore, compound 5 was determined to be 8-hydroxy-6-methyl-9-oxo-9H-xanthene-1- carboxylate. Compound 6 had the molecular formula C10H10O4, as determined by the ESIMS. In the 1 H NMR spectrum of 6, proton signals of two meta- coupled protons were observed at H 6.42 and 6.64 (each d, J = 2.0 Hz, H-7 and H-9), implying the existence of a 1,2,3,5-tetrasubstituted aromatic ring. In addition, a singlet signal at H 7.03 (s, H-10) implying a penta-substituted benzene ring, one methyl group at H 2.36 (s, 2-CH3), one methoxy group at H 3.85 (s, 6-OCH3), and one olefinic proton at H 6.15 (s, H-3) were also recognized. The 13 C NMR and DEPT spectra displayed signals of 15 carbons, including one carbonyl carbon at C 183.6 (C-4) and eight aromatic non-protonated carbons [including five carbons-bearing oxygen at C 168.4 (C-2), 162.3 (C-5), 160.6 (C-6), 160.0 (C-8), and 152.4 (C-10a)]. On the basis of the observed 1 H and Table 3: 1 H and 13 C NMR data for compounds 5-7 Position C #1 5 C #2 6 C #3 7 C a,b H a,c (J in Hz) C d,b H d,c (J in Hz) C d,b H d,c (J in Hz) 1 133.6 133.6 162.6 161.4 2 122.5 122.4 7.31 (d, 8.0) 168.3 168.4 120.9 120.7 7.25 (s) 3 134.8 134.7 7.73 (t, 8.0) 106.5 106.5 6.15 (s) 153.1 152.8 4 119.4 119.4 7.52 (d, 8.0) 183.5 183.6 117.3 117.1 7.65 (s) 4a 156.0 156.0 102.8 102.8 131.2 132.9 4b 155.7 155.7 5 107.4 107.4 6.74 (br s) 162.2 162.3 109.0 108.8 7.14 (d, 2.0) 5a 106.5 106.5 6 149.4 149.4 160.5 160.6 165.5 165.6 7 111.7 111.7 6.63 (br s) 97.4 97.5 6.42 (d, 2.0) 108.1 107.8 6.61 (d, 2.0) 8 161.5 161.5 159.9 160.0 165.7 164.4 8a 107.0 107.0 109.7 109.0 9 180.4 180.4 101.0 101.0 6.64 (d, 2.0) 191.0 189.7 9a 117.6 117.6 140.8 140.8 114.4 114.1 10 99.7 99.8 7.03 (s) 181.4 181.4 10a 152.4 152.4 133.6 135.2 1-COOCH3 169.7 169.7 1-COOCH3 53.1 53.2 4.02 (s) 1-OH 12.09 (s) 2-CH3 20.0 20.1 2.36 (s) 3-CH2OH 62.9 62.0 4.61 (s) 6-CH3 22.6 22.6 2.42 (s) 6-OCH3 55.6 55.7 3.85 (s) 8-OH 12.14 (s) a Recorded in CDCl3, b 100 MHz, c 400 MHz, d in DMSO-d6; #1δC of