Two Additional New Compounds from the Marine-Derived Fungus Pseudallescheria ellipsoidea F42-3

Two additional new compounds, pseudellone D (1) and (5S,6S)-dihydroxylasiodiplodin (3), along with the two known compounds lasiodipline F (2), (5S)-hydroxylasiodiplodin (4) were isolated from the marine-derived fungus Pseudallescheria ellipsoidea F42-3 associated with the soft coral Lobophytum crassum. Their structures, including absolute configurations, were elucidated on the basis of the corresponding spectroscopic data and electronic circular dichroism (ECD) spectra.


Introduction
In recent years, secondary metabolites from marine-derived fungi have drawn considerable attention for their unique structures and interesting biological properties [1]. In our continued exploration of the chemical potential of fungi from special niches, such as soft corals and sponges, a fungal strain characterized as Pseudallescheria ellipsoidea was isolated from the soft coral Lobophytum crassum. This fungus can cause invasive infections with a high mortality rate and is difficult to treat [2], but its chemistry is little explored.
Previously, we obtained three novel alkaloids, named pseudellones A-C, from the culture broth in glucose-peptone-yeast extract (GluPY) medium of the fungus Pseudallescheria ellipsoidea F42-3 [3]. The ongoing investigation on the secondary metabolites of this fungus afforded two additional new compounds: a diketopiperazine pseudellone D (1) and a 12-membered lactone derivative (5S,6S)-dihydroxylasiodiplodin (3), as well as two known related analogues: lasiodipline F (2), and (5S)-hydroxylasiodiplodin (4) (Figure 1) by silica gel column chromatography and RP-HPLC of the EtOAc extract of the culture broth. The structure elucidation was accomplished by MS, 1D, 2D NMR and ECD calculation. Herein, we describe the isolation and structure determination of the new compounds 1 and 2.

Results and Discussion
Pseudellone D (1) was obtained as a pale yellow solid. The molecular formula was established as C15H17N3O2S from a positive HREIMS peak at m/z 303.1029 [M] + (calcd. 303.1041). The 1 H-and 13 C-NMR spectra (Table 1) displayed characteristic signals for a diketopiperazine. Two carbonyl groups at δC 166.5 and 169.3 along with the two α-amino acid carbon resonances at δC 51.9 and 68.1 and one α-amino acid methine proton at 3.44 (qd, 7.2, 2.4) implied the existence of two amino acid residues. The presence of an indole nucleus in the structure was deduced from the chemical shifts and the splitting pattern of the five aromatic protons at δH 7.76 (d, H-10), 6.99 (td, H-11), 7.07 (td, H-12), 7.35 (d, H-13), 7.24 (d, H-16) in the 1 H-NMR spectrum, which was further confirmed from the 1 H-1 H COSY data and the HMBC crosspeaks from H-10 to C-9, from H-13 to C-14, from H-16 to C-8, C-9 and C-14 ( Figure 2). Additionally, the 1 H-NMR spectrum exhibited three exchangeable protons at δH 7.17, 7.50 and 10.18 attributed to two amide protons and an indole amino, and two methylene protons at δH 3.78 (d, H-7a), 3.21 (d, H-7b) together with S-and C-bonded methyl groups at δH 2.29(s, H3-18) and 1.35 (d, H3-17). Accordingly, the 13 C-NMR and DEPT spectra showed fifteen carbon signals consisting of two carbonyl groups, eight aromatic carbons, one sp 3 quaternary carbon, one sp 3 methine, one sp 3 methylene and two methyls. The 1 H-1 HCOSY correlation between H-2 and H3-17 and the HMBC crosspeaks from H-2 to C-1, H-7 to C-5 and C-8, and from H3-18 to C-5 determined the planar structure of the compound. The NOESY experiment didn't provide enough information to assign the relative configuration of the compound. The ECD spectra with the four configuration options (2S,5R)-, (2R,5S)-, (2R,5R)-and -(2S,5S) resulting from the two chiral centers in the structure were calculated and compared with the experimental CD curve. As shown in Figure 3, the spectrum calculated for the (2S,5R)-stereochemistry agreed with the experimental one. Therefore, the absolute configuration of the compound was established to be (2S,5R). The known compound lasiodipline F (2) [4], was identified as the cyclization product of C-2 and C-16 of pseudellone D. The experimental CD spectrum of 2 reproduced ( Figure S39) the experimental and computed CD curves of lasiodipline F in the literature. It was notable that the absolute stereochemistry of the two chiral centers of lasiodipline F was determined as (2R,5S), opposite of the (2S,5R) one of pseudellone D because of the configurational transformation occurring during the cyclization process.

Results and Discussion
Pseudellone D (1) was obtained as a pale yellow solid. The molecular formula was established as C 15 H 17 N 3 O 2 S from a positive HREIMS peak at m/z 303.1029 [M] + (calcd. 303.1041). The 1 H-and 13 C-NMR spectra (Table 1) displayed characteristic signals for a diketopiperazine. Two carbonyl groups at δ C 166.5 and 169.3 along with the two α-amino acid carbon resonances at δ C 51.9 and 68.1 and one α-amino acid methine proton at 3.44 (qd, 7.2, 2.4) implied the existence of two amino acid residues. The presence of an indole nucleus in the structure was deduced from the chemical shifts and the splitting pattern of the five aromatic protons at δ H 7.76 (d, H-10), 6.99 (td, H-11), 7.07 (td, H-12), 7.35 (d, H-13), 7.24 (d, H-16) in the 1 H-NMR spectrum, which was further confirmed from the 1 H-1 H COSY data and the HMBC crosspeaks from H-10 to C-9, from H-13 to C-14, from H-16 to C-8, C-9 and C-14 ( Figure 2). Additionally, the 1 H-NMR spectrum exhibited three exchangeable protons at δ H 7.17, 7.50 and 10.18 attributed to two amide protons and an indole amino, and two methylene protons at δ H 3.78 (d, H-7a), 3.21 (d, H-7b) together with S-and C-bonded methyl groups at δ H 2.29(s, H 3 -18) and 1.35 (d, H 3 -17). Accordingly, the 13 C-NMR and DEPT spectra showed fifteen carbon signals consisting of two carbonyl groups, eight aromatic carbons, one sp 3 quaternary carbon, one sp 3 methine, one sp 3 methylene and two methyls. The 1 H-1 HCOSY correlation between H-2 and H 3 -17 and the HMBC crosspeaks from H-2 to C-1, H-7 to C-5 and C-8, and from H 3 -18 to C-5 determined the planar structure of the compound. The NOESY experiment didn't provide enough information to assign the relative configuration of the compound. The ECD spectra with the four configuration options (2S,5R)-, (2R,5S)-, (2R,5R)-and -(2S,5S) resulting from the two chiral centers in the structure were calculated and compared with the experimental CD curve. As shown in Figure 3, the spectrum calculated for the (2S,5R)-stereochemistry agreed with the experimental one. Therefore, the absolute configuration of the compound was established to be (2S,5R). The known compound lasiodipline F (2) [4], was identified as the cyclization product of C-2 and C-16 of pseudellone D. The experimental CD spectrum of 2 reproduced ( Figure S39) the experimental and computed CD curves of lasiodipline F in the literature. It was notable that the absolute stereochemistry of the two chiral centers of lasiodipline F was determined as (2R,5S), opposite of the (2S,5R) one of pseudellone D because of the configurational transformation occurring during the cyclization process.

Results and Discussion
Pseudellone D (1) was obtained as a pale yellow solid. The molecular formula was established as C15H17N3O2S from a positive HREIMS peak at m/z 303.1029 [M] + (calcd. 303.1041). The 1 H-and 13 C-NMR spectra (Table 1) displayed characteristic signals for a diketopiperazine. Two carbonyl groups at δC 166.5 and 169.3 along with the two α-amino acid carbon resonances at δC 51.9 and 68.1 and one α-amino acid methine proton at 3.44 (qd, 7.2, 2.4) implied the existence of two amino acid residues. The presence of an indole nucleus in the structure was deduced from the chemical shifts and the splitting pattern of the five aromatic protons at δH 7.76 (d, H-10), 6.99 (td, H-11), 7.07 (td, H-12), 7.35 (d, H-13), 7.24 (d, H-16) in the 1 H-NMR spectrum, which was further confirmed from the 1 H-1 H COSY data and the HMBC crosspeaks from H-10 to C-9, from H-13 to C-14, from H-16 to C-8, C-9 and C-14 ( Figure 2). Additionally, the 1 H-NMR spectrum exhibited three exchangeable protons at δH 7.17, 7.50 and 10.18 attributed to two amide protons and an indole amino, and two methylene protons at δH 3.78 (d, H-7a), 3.21 (d, H-7b) together with S-and C-bonded methyl groups at δH 2.29(s, H3-18) and 1.35 (d, H3-17). Accordingly, the 13 C-NMR and DEPT spectra showed fifteen carbon signals consisting of two carbonyl groups, eight aromatic carbons, one sp 3 quaternary carbon, one sp 3 methine, one sp 3 methylene and two methyls. The 1 H-1 HCOSY correlation between H-2 and H3-17 and the HMBC crosspeaks from H-2 to C-1, H-7 to C-5 and C-8, and from H3-18 to C-5 determined the planar structure of the compound. The NOESY experiment didn't provide enough information to assign the relative configuration of the compound. The ECD spectra with the four configuration options (2S,5R)-, (2R,5S)-, (2R,5R)-and -(2S,5S) resulting from the two chiral centers in the structure were calculated and compared with the experimental CD curve. As shown in Figure 3, the spectrum calculated for the (2S,5R)-stereochemistry agreed with the experimental one. Therefore, the absolute configuration of the compound was established to be (2S,5R). The known compound lasiodipline F (2) [4], was identified as the cyclization product of C-2 and C-16 of pseudellone D. The experimental CD spectrum of 2 reproduced ( Figure S39) the experimental and computed CD curves of lasiodipline F in the literature. It was notable that the absolute stereochemistry of the two chiral centers of lasiodipline F was determined as (2R,5S), opposite of the (2S,5R) one of pseudellone D because of the configurational transformation occurring during the cyclization process.   a These data may be interchanged. (5S,6S)-Dihydroxylasiodiplodin (3) was isolated as a white solid. The molecular formula was established as C17H24O6 by the [M] + peak 324.1562 in high-resolution EI mass spectrometry, indicating six degrees of unsaturation. Careful inspection of its 1 H-, 13 C- (Table 1), DEPT and HMQC spectra disclosed the existence of seventeen carbons, including one carbonyl, four sp 2 quaternary carbons, two sp 2 methines, three oxygenated sp 3 methines, five sp 3 methenes, one oxygenated sp 3 methyl and one sp 3 methyl. An aliphatic eight-carbon chain (C3-C4-C5-C6-C7-C8-C9-C10) was detected on the basis of 1 H-1 H COSY experiment. HMBC correlations from H-14 to C-13, C-15, C-12 and C-16, from H-12 to C-13, C-14 and C-16, from H3-17 to C-15 indicated the presence of a tetrasubstituted benzene ring and two aromatic carbons were oxygenated, consistent with the chemical shifts pattern of the aromatic carbons and coupling constants of aromatic protons (Figure 2). The ester carbonyl must attach to C-16, and ester oxygen and C-11 was linked to C-3, C-10 respectively to form a dodecane-membered ring, satisfying the unsaturation degrees of the molecular formula. This connectivity was further confirmed by the distinct HMBC crosspeaks of H-10 with C-11, C-12 and C-16, of H-3 with C-1. The    (5S,6S)-Dihydroxylasiodiplodin (3) was isolated as a white solid. The molecular formula was established as C 17 H 24 O 6 by the [M] + peak 324.1562 in high-resolution EI mass spectrometry, indicating six degrees of unsaturation. Careful inspection of its 1 H-, 13 C- (Table 1), DEPT and HMQC spectra disclosed the existence of seventeen carbons, including one carbonyl, four sp 2 quaternary carbons, two sp 2 methines, three oxygenated sp 3 methines, five sp 3 methenes, one oxygenated sp 3 methyl and one sp 3 methyl. An aliphatic eight-carbon chain (C3-C4-C5-C6-C7-C8-C9-C10) was detected on the basis of 1 H-1 H COSY experiment. HMBC correlations from H-14 to C-13, C-15, C-12 and C-16, from H-12 to C-13, C-14 and C-16, from H 3 -17 to C-15 indicated the presence of a tetrasubstituted benzene ring and two aromatic carbons were oxygenated, consistent with the chemical shifts pattern of the aromatic carbons and coupling constants of aromatic protons (Figure 2). The ester carbonyl must attach to C-16, and ester oxygen and C-11 was linked to C-3, C-10 respectively to form a dodecane-membered ring, satisfying the unsaturation degrees of the molecular formula. This connectivity was further confirmed by the distinct HMBC crosspeaks of H-10 with C-11, C-12 and C-16, of H-3 with C-1. The remaining two hydroxyl groups were bonded to C-5 and C-6 respectively to complete the planar structure of the compound 3.
Firstly, we tried to obtain some information about the absolute configuration of (5S,6S)-dihydroxylasiodiplodin by CD spectroscopy due to the small quantity available. Careful inspection of the eight computed ECD curves resulting from the three chiral centers of the compound 3, showed that the experimental CD spectrum matched well with the ECD curves of the 3S-stereochemistry ( Figure S38). Actually, the ECD curves of four configuration options of the 3S stereochemistry were identical, which indicated the different configurations at C-5 and C-6 positons didn't affect the ECD absorbance. From the biosynthetic standpoint, the absolute stereochemistry of chiral centers C-3 and C-5 of compound 3 should be consistent with that of the known compound (5S)-hydroxy-lasiodiplodin [5]. For the 12-membered ring lactone 5-hydroxylasiodiplodin, the carbon chemical shift of 5S at δ C 66.7 and 5R at δ C 70.6 in CDCl 3 solvent were distinctly different. The chemical shift at the C-5 position of compound 3 was at δ C 64.5 in DMSO-d 6 and δ C 67.1 in CD 3 OD, respectively. Apart from the solvent effect, the chemical shift at C-5 of compound 3 was consistent with that of (5S)-hydroxylasiodiplodin. Correspondingly, the stereochemistry of C-3 and C-5 of compound 3 was determined as 3S, 5S. For the other analogue (3S),(6R)-6-hydroxylasiodiplodin [6], the carbon chemical shift of 6R at δ C 70.5 in CDCl 3 /CD 3 OD solvent was distinctly different from the carbon chemical shift at C-6 position of compound 3 at δ C 76.3 in CD 3 OD solvent. Furthermore, the NOESY spectrum of compound 3 displayed a NOE correlation between H-5 and H-6, therefore, the absolute configuration of (5S,6S)-dihydroxylasiodiplodin was proposed to be (3S,5S,6S).

Fungal Material
The marine fungus Pseudallescheria ellipsoidea F42-3 was isolated from the inner tissue of the soft coral Lobophytum crassum collected from Hainan Sanya National Coral Reef Reserve, China. This fungal strain was maintained in 15% glycerol aqueous solution at´80˝C. A voucher specimen was deposited in the School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China. Analysis of the ITS rDNA by BLAST database screening provided a 99.7% match to Pseudallescheria ellipsoidea (compared with JQ690937).