α-Pyrone and decalin derivatives from the marine-derived fungus Trichoderma harzianum PSU-MF79

Abstract Two new compounds, one α-pyrone (trichoharzianone) and one decalin (trichoharzianin), along with eight known compounds including three decalins, two δ-lactones, two carboxylic acids and one isochroman were isolated from the marine-derived fungus Trichoderma harzianum PSU-MF79. The structures were determined by spectroscopic methods. The relative configuration of trichoharzianin was assigned based on NOEDIFF data and coupling constants whereas the absolute configurations were established by comparison of electronic circular dichroism data with those of the co-metabolites. Known (-)-massoia lactone exhibited mild antifungal activity against Cryptococcus neoformans ATCC90113 flucytosine-resistant, Candida albicans ATCC90028 and C. albicans NCPF3153 with MIC values of 128, 200 and 200 µg/mL, respectively, and weak cytotoxic activity against HCT-116 and MCF-7 cell lines with the respective IC50 values of 17 and 32 µM. In addition, it was noncytotoxic against noncancerous Vero cells with an IC50 value of >100 µM. Graphical Abstract


Introduction
Trichoderma spp. have received considerable attention for their production of a wide range of secondary metabolites (Javaid and Ali 2011;Faedda et al. 2012;Ahluwalia et al. 2015;Han et al. 2019;Harwoko et al. 2021). Marine-derived fungi are recognized as rich sources of new secondary metabolites with pharmaceutical potential (Blunt et al. 2018). Although the marine-derived fungus T. harzianum was first studied in 1993 (Kobayashi et al. 1993), chemical investigation of secondary metabolites from the marine isolates of this fungus was limited. Up to the present, more than 60 compounds with diverse structures and interesting biological activities have been isolated from the marine-derived T. harzianum (Shi et al. 2020;Yu et al. 2021). These included antimicroalgal diterpenes and sesquiterpenes (Song et al. 2018), antibacterial sesquiterpenes (Fang et al. 2019), cytotoxic decalins (Suzue et al. 2016;Yamada et al. 2015) and diterpenes (Yamada et al. 2017;Liang et al. 2020), and a b-amyloid fibrillization inhibitory cyclopentenone (Fang et al. 2017). In our ongoing search for bioactive secondary metabolites from marine-derived fungi, T. harzianum PSU-MF79 was isolated from an unidentified tunicate collected from Phuket Province, Thailand. The crude broth extract of the fungus PSU-MF79 displayed antifungal activity against Cryprococcus neoformans ATCC90113 flucytosine-resistant with an MIC value of 128 mg/mL. Herein, the isolation, structure elucidation, and antimicrobial as well as cytotoxic activities of some isolated compounds which were obtained in sufficient amount are described.
Trichoharzianone (1) was obtained as a colorless gum with the molecular formula C 11 H 14 O 5 determined by the HRESIMS peak at m/z 249.0723 [M þ Na] þ . The UV spectrum showed an absorption band at 267 nm for a conjugated carbonyl chromophore . The IR spectrum displayed absorption bands for hydroxy and two carbonyl functional groups at 3367, 1752 and 1617 cm À1 , respectively. The 1 H NMR spectroscopic data (Table S1) contained signals for one olefinic proton of a trisubstituted alkene (d H 5.56, s, 1H), four sets of equivalent methylene protons [d H 2.12 (t, J ¼ 7.2 Hz, 2H), 1.49 (quint, J ¼ 7.2 Hz, 2H), 1.34 (m, 2H) and 1.28 (m, 2H)] and one methyl group (d H 0.88, t, J ¼ 6.6 Hz, 3H). The 13 C NMR spectrum (Table S1) (Table S1) of H 3 -11 with C-9 (d C 32.4) and C-10 (d C 23.1) constructed a n-pentyl unit. In addition, the HMBC correlations of H-3 (d H 5.56) with C-2 (d C 157.6), C-4 (d C 170.7) and C-5 (d C 99.2) and those of H 2 -7 of the n-pentyl unit with C-5 and C-6 (d C 171.7) as well as the chemical shifts of C-2, C-4 and C-6 established an a-pyrone ring with a hydroxy group and the n-pentyl unit at C-4 and C-6, respectively. The substituent at C-5 was assigned as a carboxyl group based on the molecular formula and the chemical shift of 5-CO 2 H (d C 170.5). Therefore, trichoharzianone had the structure 1.
Trichoharzianin (2) was obtained as a colorless gum with the molecular formula C 24 H 38 O 5 determined by the HRESIMS peak at m/z 429.2612 [M þ Na] þ . The UV spectrum showed an absorption band at 216 nm for a conjugated carbonyl chromophore (Kobayashi et al. 1993). The IR spectrum was similar to those of 5 with an additional absorption band at 1655 cm À1 for an unsaturated carbonyl functionality (Kobayashi et al. 1993). The 1 H and 13 C NMR spectroscopic data (Table S2) were similar to those of 5 except for additional signals for a 3-methylcrotonic acid unit [d H 5.75 (sept, J ¼ 1.2 Hz, 1H), 2.18 (d, J ¼ 1.2 Hz, 3H) and 1.92 (d, J ¼ 1.2 Hz, 3H) and d C 167.2, 158.2, 116.1, 27.6 and 20.5] in 2. This unit was established on the basis of the HMBC correlations (Table S2) of H-2 0 (d H 5.75) with C-1 0 (d C 167.2), C-4 0 (d C 27.6) and C-5 0 (d C 20.5) and those of H 3 -4 0 (d H 1.92) and H 3 -5 0 (d H 2.18) with C-2 0 (d C 116.1) and C-3 0 (d C 158.2) as well as the chemical shift of C-1 0 . It was attached at C-2 (d C 72.4) on the basis of the HMBC cross peak of H-2 (d H 5.24) with C-1 0 . The NOEDIFF data (  et al. 2017) indicated that 2 and 5 had an identical relative configuration of the decalin moiety. This assigned relative configuration was confirmed based on these vicinal coupling constants (J 1,8a ¼ J 4a,8a ¼ J 4,4a ¼ 10.8 Hz, J 1,2 ¼ J 2,3a ¼ J 2,3b ¼ 3.3 Hz and J 6,7 ¼ 4.5 Hz). In addition, the relative configuration at C-1 0 '' in 2 was identical to that of 5 based on the NOEDIFF data of H-4a/H a -2 0 '' (d H 1.47) and 1 0 ''-Me (d H 0.93)/H-7 (d H 5.70) (Nakadate et al. 2007;Li et al. 2017). The absolute configurations of 2 were assigned as 1S, 2 R, 4 R, 4aS, 5S, 6S, 8aR and 1 0 ''R, identical to those of the co-metabolites 3-5, based on biogenesis consideration and the ECD spectrum of 2 which displayed the same negative and positive Cotton effects at 247 and 301 nm, respectively, as those of 3-5 ( Figure S30). In addition, 2 showed the same sign of specific rotation as those of 3-5 (Table S3). Therefore, 2 was identified as a 3-methylcrotonate derivative of 5.
In conclusion, one new a-pyrone (1) and one new decalin (2) together with eight known compounds (3-10) were obtained from the marine-derived fungus T. harzianum PSU-MF79. These decalin-type analogues were produced by fungi in the genera Eupenicillium (Nakadate et al. 2007;Li et al. 2014Li et al. , 2017 and Paecilomyces (Rahbaek et al. 1998). In addition, the known compounds 6-9 were isolated from this fungus for the first time. This is also the first report on antifungal activity against C. neoformans ATCC90113 flucytosine-resistant and cytotoxicity against HCT-116 cell lines of 6.

General experimental procedures
The infrared (IR) spectra were recorded neat using a Perkin-Elmer spectrum BX FT-IR spectrometer. The ultraviolet (UV) absorption spectra were measured in MeOH on a Shimadzu UV-2600 UV-Vis spectrophotometer. Electronic Circular Dichroism (ECD) spectra were recorded on a JASCO J-815 polarimeter. The 1 H and 13 C NMR spectra were recorded on a 300 or a 500 MHz Bruker FTNMR Ultra Shield TM spectrometer using tetramethylsilane (TMS) as an internal standard. The 1 D and 2 D NMR spectra were recorded using standard Bruker pulse sequences. The specific rotations were recorded on a JASCO P-2000 polarimeter. Mass spectra were obtained on a Bruker MicrOTOF mass spectrometer and Liquid chromatograph-mass spectrometer (2090, LCT, water, micromass). Thin-layer chromatography (TLC) was performed on silica gel 60 GF 254 (Merck). Column chromatography (CC) was carried out on Sephadex LH-20, silica gel (Merck) type 60 (70-230 mesh ASTM), or on reverse phase C 18 silica gel.

Fungal material
The marine-derived fungus PSU-MF79 was isolated from an unidentified tunicate collected from Phuket Province, Thailand. This fungus was deposited as BCC83157 at BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. The fungus PSU-MF79 was identified based on its morphological and molecular characteristics. Colonies on potato dextrose agar (PDA) at 25 C grew very fast, reaching 4.5 cm in 3 days. The molecular analysis of the internal transcribed spacers (ITS) ribosomal DNA revealed that PSU-MF79 closely matched with several ITS nucleotides of Trichoderma harzianum strains comprising T. harzianum KY225642, KY764866, KY764853 and KY750449 (100% nucleotide similarity). The GenBank accession numbers of ITS and large subunit (LSU) rDNA are KY397993 and KY398004, respectively. Therefore, this fungus was identified as Trichoderma harzianum.

Antimicrobial assays
The activity was conducted using the procedure described by the Clinical and Laboratory Standards Institute (Phongpaichit et al. 2006). MIC values of positive controls are shown in Table S4.
Following the removal of supernatant, DMSO was added to dissolve the remaining crystals with their absorbance being quantified at 570 nm using a Synergy Neo2 Multi-Mode Microplate Reader (BioTek Instruments, VT, USA). IC 50 value of afatinib in Calu-3 cells was 30 mM whereas IC 50 values of ellipticine in HCT-116 and MCF-7 cells were 10 and 5 mM, respectively.