Sesquiterpenes from the soil-derived fungus Trichoderma citrinoviride PSU-SPSF346

Two new sesquiterpenes, trichocitrinovirenes A (1) and B (2), and five known compounds including four structurally related sesquiterpenes and one γ-lactone were isolated from the soil-derived fungus Trichoderma citrinoviride PSU-SPSF346. The structures were identified by analysis of their spectroscopic data. The relative configuration was assigned based on NOEDIFF data. The absolute configuration of compound 1 was established according to specific rotations and ECD data while that of compound 2 was proposed based on biosynthetic considerations. Compound 2 possesses a rare bicyclic sesquiterpene skeleton. The antimicrobial and cytotoxic activities of the isolated compounds were evaluated.


Introduction
The fungus Trichoderma citrinoviride produces structurally diverse secondary metabolites including diterpenes [1,2], alkaloids [3], sorbicillinoids [4,5], long chain alcohols [6], and cyclonerane sesquiterpenes [7]. Some of them display antibacterial [1,2,7], cytotoxic [3], anti-inflammatory [4], and antimicroalgal [7] activities. Based on these data, the investigation of secondary metabolites from this fungus is still limited. In our ongoing search for antimicrobial secondary metabolites from soil-derived fungi, T. citrinoviride PSU-SPSF346 was isolated from a soil sample collected from the Sirindhorn Peat Swamp Forest, Narathiwat Province, Thailand. The crude mycelial extract of T. citrinoviride PSU-SPSF346 displayed antimicrobial activities against Staphylococcus aureus, methicillin-resistant S. aureus, and Cryptococcus neoformans ATCC90113 with MIC values of 128, 200 and 64 μg/mL, respectively. Herein, we report the isolation and structure elucidation as well as antimicrobial and cytotoxic activities of some isolated compounds which were obtained in sufficient amount.

Results and Discussion
Chemical investigation of the broth and mycelial extracts of T. citrinoviride PSU-SPSF346 by various chromatography techniques led to the isolation of two new sesquiterpenes, trichocitrinovirenes A (1) and B (2), four known structurally related sesquiterpenes including gliocladic acid (3), hydroheptelidic acid (4), and xylaric acids B (5) and D (6) [8], as well as one known γ-lactone, fusidilactone A (7) [9] (Figure 1). Their structures were elucidated on the basis of analysis of their spectroscopic data including IR, UV, NMR, and MS. The relative configuration was assigned based on NOEDIFF data. Furthermore, the absolute configuration of compound 1 was determined by comparison of its specific rotation and electronic circular dichroism (ECD) data with those of compound 3, whereas that of compound 2 was proposed based on biosynthetic considerations. The structures of the known compounds were further confirmed by comparison of their 1 H and 13 C NMR spectroscopic data, specific rotations, and ECD data with those previously reported.
Trichocitrinovirene A (1) was isolated as a colorless gum and had the molecular formula C 15  . These NMR spectroscopic data were similar to those of compound 3 except for the replacement of signals for the nonequivalent oxymethylene protons (δ H 4.37 and 4.32, H ab -3; δ C 57.4, C-3) in compound 3 with signals of the nonequivalent methylene protons resonating at higher field (δ H 3.40 and 3.30, each d, J = 16.8 Hz, 1H; δ C 33.6, C-3), and an additional signal for a carboxyl carbonyl carbon (δ C 175.1) in compound 1. The HMBC cross peaks of these nonequivalent methylene protons with C-1 (δ C 171.1), C-2 (δ C 128.1), C-4 (δ C 175.1), and C-5 (δ C 149.7) ( Figure 2) together with the chemical shift of C-3 indicated that the 3-OH group in compound 3 was replaced by a carboxyl group in compound 1. The relative configuration was determined by the NOEDIFF data ( Figure 2). A signal enhancement of H-7, but not H-5, after irradiation of H ab -3, indicated an E-configuration of the trisubstituted α,β-unsaturated carboxylic acid. In addition, a trans relationship between H-6 and H-11 was established according to signal enhancement of H-12 (δ H 1.71), H 3 -13, and H 3 -14 after irradiation of H-6. The absolute configuration at C-6 was assigned as R based on the experimental ECD spectrum of compound 1 which showed a   Trichocitrinovirene B (2) was isolated as a colorless gum. The molecular formula C 15 H 22 O 6 was determined on the basis of the HRESIMS peak at m/z 321.1320 [M + Na] + . The IR spectrum was similar to that of compound 1, indicating that they possessed similar functional groups. The 1 H NMR spectrum (Table 1)  3) as well as the chemical shifts of C-7 and C-15 (δ C 69.2) constructed a cyclohexane ring with both a hydroxymethyl group and an oxy substituent at C-7, the isopropyl group at C-10 and other substituents at C-5 (δ C 47.0) and C-6. The substituent at C-6 was assigned as a carboxyl group on the basis of the HMBC correlation of H-6 with the carboxyl carbonyl carbon (C-14, δ C 179.6). The HMBC correlations of the geminal olefinic protons (H ab -3, δ H 5.93 and 5.52) to the remaining carboxyl carbonyl carbon (C-1, δ C 174.5), C-2 (δ C 150.2) and C-4 (δ C 84.6) and H-4 (δ H 4.72) to C-2 and C-3 (δ C 118.7) and the chemical shift of C-4 constructed a propenoic acid unit with a 1,1-disubstituted oxymethyl substituent at C-2. The HMBC correlations of H-4 of this unit with C-5, C-6, C-7, and C-10 as well as the chemical shifts of C-4 and C-7 established an ether bond between C-4 and C-7 and a C-C bond between C-4 and C-5 to form a bicyclic skeleton. The relative configuration was determined by the NOEDIFF data ( Figure 2). The signal enhancement of H-5 and H-10 upon irradiation of H-4 indicated their close proximity and the orientation of the isopropyl group at an α-position. Irradiation of H-6 enhanced the signal intensities of H b -3, H-5, and H ab -15, indicating that the carboxyl moiety was α-orientated. Biosynthetically, compound 2 might be derived from compound 4 or compound 5 by oxa-Michael reaction of 7-OH to the α,β-unsaturated carboxylic acid moiety to form a tetrahydrofuran unit followed by ring opening of the lactone moiety and demethylation, respectively ( Figure 4). Subsequent dehydration would afford compound 2 with an α,β-unsaturated carboxylic acid moiety. Alternatively, the ring opening of compound 4 and demethylation of compound 5 would occur prior to the oxa-Michael reaction. Accordingly, the absolute configurations at C-5, C-6, C-7, and C-10 of compound 2 were proposed to be 5S, 6S, 7S, and 10R identical to those of the co-metabolites 4 and 5. The absolute configuration at C-4 was thus assigned to be R. Therefore, compound 2 is a rare bicyclic sesquiterpene.
The isolated compounds 1 and 3-6 with sufficient amount were evaluated for their antibacterial activity against S. aureus ATCC25923 and methicillin-resistant S. aureus SK1, antifungal activity against C. neoformans ATCC90113 as well as cytotoxic activity against KB, MCF-7, and noncancerous Vero (African green monkey kidney fibroblast) cells. None of them showed antimicrobial activity against the tested pathogenic microorganisms at the concentration of 200 μg/mL. These results indicate either a synergistic effect and/or that the active components were not isolated. In addition, these compounds were inactive against the tested cell lines at the concentration of 50 μg/mL.

Conclusion
The investigation of the crude extracts of the soil-derived fungus T. citrinoviride PSU-SPSF346 resulted in the isolation of seven compounds including two new (1 and 2) and four known sesquiterpenes (3)(4)(5)(6), and one known γ-lactone (7). Sesquiterpenes of this type were previously isolated from T. virens [11,12], T. reesei [10], and Xylaria sp. [8,13] whereas compound 7 was obtained from Fusidium sp. [9] and T. hypoxylon [14]. Therefore, this is the first report on the isolation of these types of compounds from the fungus T. citrinoviride. In addition, compound 2 is a rare bicyclic sesquiterpene. Unfortunately, none of the tested compounds 1 and 3-6 displayed antimicrobial and cytotoxic activities.

Experimental
General experimental procedures Infrared (IR) spectra were recorded with a Perkin-Elmer spectrum BX FTIR spectrometer. Ultraviolet (UV) spectra were obtained using a Shimadzu UV-2600 UV-vis spectrophotometer in MeOH. ECD spectra were recorded on a JASCO J-815 polarimeter. 1 H and 13 C nuclear magnetic resonance ( 1 H and 13 C NMR) spectra were recorded on 300 and 500 MHz Bruker FTNMR Ultra Shield TM spectrometers. Specific rotations were measured with a JASCO P-2000 polarimeter. ESI-TOF mass spectra were obtained using a TOF/Q-TOF Mass spectrometer. Thin-layer chromatography (TLC) and preparative TLC were performed on silica gel 60 GF 254 (Merck). Column chromatography (CC) was conducted on silica gel (Merck) type 100 (70-230 mesh ASTM) and type 60 (230-400 mesh ASTM), Sephadex LH-20, or reversed-phase C 18 silica gel.

Fungal material
The fungus PSU-SPSF346 was isolated from a soil sample collected from the Sirindhorn Peat Swamp Forest, Narathiwat Province, Thailand. This fungus was deposited as BCC88125 at BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. The fungus SPSF346 was identified based on its morphological and molecular characteristics. The molecular analysis of the internal transcribed spacers (ITS) (GenBank accession number MH997885) and partial large subunit (LSU) (GenBank accession number MH997897) ribosomal RNA gene revealed that the fungus PSU-SPSF346 had close relationships with several strains of Trichoderma citrinoviride with 99% nucleotide identity for both DNA regions. Therefore, this fungus can be identified as Trichoderma citrinoviride.

Antimicrobial assay
Antimicrobial activity was evaluated according to the Clinical and Laboratory Standards Institute [16]. Vancomycin was used as a positive control for S. aureus and methicillin-resistant S. aureus and exhibited MIC values of 0.25 and 1.0 μg/mL, respectively. Amphotericin B was used as a positive control for C. neoformans ATCC90113 and displayed a MIC value of 0.25 μg/mL.

Cytotoxicity assay
The activity assay against African green monkey kidney fibroblast (Vero) cells was performed in triplicate employing the method described by Hunt and co-workers [17]. Ellipticine, the standard drug, displayed an IC 50 value of 4.06 μM. The activities against KB and MCF-7 cell lines were evaluated using the resazurin microplate assay [18]. Doxorubicin was used as a standard drug for KB and MCF-7 cell lines and displayed IC 50 values of 1.21 and 15.84 μM, respectively.

Supporting Information
Supporting Information File 1 HRESIMS profiles for compounds 1 and 2 and copies of NMR spectra for compounds 1-7.