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Cyclonerane sesquiterpenes and an isocoumarin derivative from the marine-alga-endophytic fungus Trichoderma citrinoviride A-WH-20-3

Two new cyclonerane sesquiterpenes, (10E)-isocyclonerotriol (1) and (10Z)-isocyclonerotriol (2), and one new isocoumarin derivative, trichophenol A (3), were isolated from Trichoderma citrinoviride A-WH-20-3, an endophyte from the marine red alga Laurencia okamurai. Their structures and relative configurations were assigned by spectroscopic techniques, highlighted by using Sarotti's DP4+ sheet to confirm the cyclopentane ring. The absolute configuration of 1 was established by comparison of experimental and calculated specific rotation data. Compounds 1 and 2 represent the first occurrence of ring-isomerized cycloneranes. Compound 3 is the first 3phenylisocoumarin derivative from Trichoderma and features inhibition of several marine-derived bacteria and phytoplankton species.


General
Optical rotations and UV spectra were measured on a Chirascan CD spectrophotometer. IR spectra were acquired on a Nicolet iS50 FT-IR spectrometer. NMR spectra were recorded on a Bruker Avance III 500 NMR spectrometer (500 and 125 MHz for 1 H and 13 C, respectively) using tetramethylsilane (TMS) as an internal standard. Low and high resolution EI mass spectra were obtained on an Autospec Premier P776 mass spectrometer. Low and high resolution ESI + mass spectra were determined on an Agilent G6230 TOF mass spectrometer. HPLC separation was operated on an Agilent HPLC system (1260 infinity quaternary pump, 1260 infinity diode-array detector) using an Eclipse SB-C18 (5 μm, 9.4

Fungal material and fermentation
Trichoderma citrinoviride A-WH-20-3 was isolated from the inner tissue of the surface-sterilized red alga Laurencia okamurai collected from the coast of Weihai, China, in August 2016. The species was identified by morphology and by analysis of the ITS regions of its rDNA, whose sequence data have been deposited in GenBank with the accession number MN088387. Its fermentation was carried out statically at room temperature for 30 days in 200 × 1 L Erlenmeyer flasks, each containing 50 g of rice, 0.55 g of peptone, 0.55 g yeast extract powder, 0.033 g calcium bromide, 0.33 g monosodium glutamate, 55 mL of pure H 2 O, and 55 mL of natural seawater from the coast of Yantai, China.

Results and discussion
Compound 1 was purified as a colorless oil. Its molecular formula was established to be C 15 H 28 O 3 by interpretation of HRESI + MS data, requiring two degrees of unsaturation. The IR spectrum exhibited a broad absorption band at 3386 cm −1 , indicating the presence of hydroxy groups. In combination with HSQC data, the 1 H NMR spectrum (Table 1) showed notable signals including one methyl doublet, three methyl singlets, one singlet ascribable to an oxymethylene, and one broad triplet attributable to an olefinic proton. Aided by DEPT experiments, 15 resonances in the 13 C NMR spectrum (Table 1) were classified as four methyls, five methylenes, three methines, and three nonprotonated carbons. HMBC correlations from H 3 -1 to C-2, C-3, and C-6 and from H 3 -13 to C-2, C-3, and C-4 and COSY correlations of H 3 -1/H-2/H-6/H 2 -5/H 2 -4 confirmed the connectivity around ring A, which then extended to C-10 as seen from the HMBC correlations from H 3 -14 to C-6, C-7, and C-8 and COSY correlations of H 2 -9 with H 2 -8 and H-10 ( Fig. 1). Additionally, the connectivity of the side chain terminus was confirmed by the HMBC correlations from H 2 -12 to C-10, C-11, and C-15 and from H 3 -15 to C-10, C-11, and C-12. In view of the deshielded NMR signals of C-3, C-7, and C-12, they were deduced to be hydroxylated. Thus, the planar structure of 1 was assigned to be the same as that of (10E)-cyclonerotriol [17], and the E configuration of the double bond at C-10 was supported by the NOE correlations between H-10 and H 2 -12 and between H 2 -9 and H 3 -15.
In order to ascertain the absolute configuration of compound 1, its specific optical rotation was determined (−66). On the other hand, the specific optical rotation for the 2S, 3S, 6R, and 7R configuration was calculated to be −114 at the B3LYP/aug-cc-pVDZ level in MeOH [29]. The agreement of experimental and calculated values and sign suggested the absolute configuration of 1, trivially named (10E)-isocyclonerotriol, to be 2S, 3S, 6R, and 7R.
Compound 2 was obtained as a colorless oil. HRESI + MS analysis gave the molecular formula C 15 H 28 O 3 , the same as for 1. The IR absorption at 3385 cm −1 suggested the presence of hydroxy groups. A detailed comparison of the 1 H and 13 C NMR spectra (Table 1) revealed that 2 differed from 1 mainly at the side chain terminus. HMBC and COSY correlations (Fig. 1) indicated the same planar structure of 2 as that of 1. However, the double bond at C-10 was deduced to feature a Z configuration based on the NOE correlations between H-10 and H 3 -15 and between H 2 -9 and H 2 -12. The relative and absolute configurations around ring A were proposed to be the same as those of 1 on the basis of their identical NMR and specific rotation data. Compound 3 was isolated as a white powder. The molecular formula C 16 H 12 O 6 was determined by HREIMS, implying eleven degrees of unsaturation. Its IR spectrum showed a broad absorption band at 3420 cm −1 , corresponding to one or more hydroxy groups. The 1 H NMR spectrum (Table 2) displayed one methyl singlet, four doublets and one singlet attributable to five aromatic protons, and three singlets ascribable to four exchangeable protons. The 13 C NMR spectrum ( Table 2) exhibited 16 resonances, sorted into one methyl, five methines, and ten nonprotonated carbons by DEPT and HSQC data. The NMR data partially resembled those for thunberginol B [30], which indicated the presence of a 6,8-dihydroxyisocoumarin moiety. It was further supported by the HMBC correlations ( Fig. 1) from H-4 to C-4a, C-5, and C-8a, from H-5 to C-7 and C-8a, from H-7 to C-5 and C-8a, and from OH-8 to C-7, C-8, and C-8a. A comparison of the remaining NMR data with those for 4-methoxy-6-(2′,4′-dihydroxy-6′-methylphenyl)pyran-2-one revealed the presence of a 2′,4′-dihydroxy-6′-methylphenyl moiety [31], which was verified by the HMBC correlations from H-3′ to C-1′ and C-5′, from H-5′ to C-1′ and C-3′, and from H-7′ to C-1′, C-5′, and C-6′. The linkage of these two units was established by the HMBC correlation from H-4 to C-1′. The above information evidenced the structure of 3, trivially named trichophenol A.
Compounds 1-3 were assayed for inhibition of four marine phytoplankton species (Chattonella marina, Heterosigma akashiwo, Karlodinium veneficum, and Prorocentrum donghaiense) that can cause red tides [25]. The results (Table 3) showed that 1 was more active against K. veneficum than the other two, but 3 was more active against C. marina, H. akashiwo, and P. donghaiense. Compared to 1 and 2, (10E)-and (10Z)cyclonerotriol were previously reported to possess high inhibition of P. donghaiense (IC 50 1.1 and 3.4 μg/mL, respectively) [32], which suggested that the isomerization of ring A greatly affected their activities. As analogs of 3, thunberginols A and B exhibited inhibition of oral bacteria [30]. During an antibacterial assay against the marine-derived bacteria Vibrio parahaemolyticus, V. anguillarum, V. harveyi, V. splendidus, and Pseudoalteromonas citrea [3], 3 displayed inhibition of the five bacteria tested (Table 3), and the MIC against P. citrea was determined to be 16 μg/mL.

Conclusion
Chemical survey on the marine-alga-endophytic fungus Trichoderma citrinoviride A-WH-20-3 has resulted in the discovery of two new cyclonerane sesquiterpenes (1 and 2) and one new isocoumarin derivative (3). Of those, 1 and 2 are the first ring-isomerized cyclonerane derivatives, and 3 features a 3-phenylisocoumarin unit that has never occurred in Trichoderma metabolites. These new isolates diversify the molecular structures of sesquiterpenes and polyketides from Trichoderma, and they possess inhibitory effects on some marine-derived bacteria and phytoplankton species.

Declaration of Competing Interest
The authors declare no conflict of interest.