Trypethelone and Phenalenone derivatives isolated from the mycobiont culture of Trypethelium eluteriae Spreng. and their anti-mycobacterial properties

The metabolites of the mycobiont culture of the lichen Trypethelium eluteriae were isolated by column chromatography and preparative TLC. Nine compounds ( 1 – 9 ) including two new trypethelones, 8-methoxytrypethelone ( 6 ) and 5’-hydroxy-8-ethoxytrypethelone ( 9 ), together with four known trypethelones ( 3 – 4 , 7 – 8 ), and two known phenalenones ( 1 – 2 ) were characterized. It is the first report of 8-methoxytrypethelone methyl ether ( 5 ) purification as a racemic mixture in T. eluteriae . Earlier, 7-hydroxyl-8-methoxyltrypethelone ( 10 ) was reported as new compound with erroneous spectroscopic data. This compound was identified later as 8-hydroxytrypethelone methyl ether ( 4 ). X-ray crystallographic structures of compounds 5 – 7 were elucidated for the first time. Phenalenones ( 1 – 2 ) and trypethelones ( 5 – 6 and 9 ) were the additional compounds discovered in the cultured mycobiont of T. eluteriae . Six compounds ( 1 – 2 , 5 – 8 ) were screened against Mycobacterium tuberculosis H37Rv and two compounds ( 7 – 8 ) against non-tuberculosis mycobacteria and other human pathogenic bacteria. Compound ( 7 ) inhibited M. tuberculosis H37Rv strain with an MIC of 12.5µg/mL.


General Experimental Procedures
All melting points were determined by the open capillary method on a Dolphin micro melting point apparatus and were uncorrected. Optical rotations were obtained in a Rudolph Polarimeter (A22109) Autopol I automatic connected with the 3.03 software version using CH 3 OH. UV spectra were recorded on a Shimadzu UV-1800 spectrophotometer installed with a 2.03 UV probe, and ECD spectra were obtained on a JASCO J-815 spectropolarimeter using CH 3 OH. FT-IR was performed on a Spectrum One FT-IR spectrometer, Perkin-Elmer using KBr disc. 1 H (500 MHz) and 13 C (125 MHz) NMR spectra were recorded in CDCl 3 or CD 3 OD using a Bruker AVANCE III with tetramethylsilane as an internal reference standard. HRESIMS was performed on a Thermo Scientific, Orbitrap Elite Mass spectrometer. X-ray diffraction was performed using a Bruker AXS Kappa Apex II CCD Diffractometer. Analytical HPLC was conducted using the Shimadzu HPLC system with an (254 and 366 nm) followed by spraying with 10% aqueous H 2 SO 4 then derivatized at 110 °C for 20 min. Preparative TLC was performed for fractions containing two closely-associated major spots. The sample solution was prepared using a suitable solvent and applied onto the TLC plate (20 × 20 cm) at 15 mm from the bottom of the plate using a Linomat 5 applicator with a 500 µL syringe as a streak ranging from 10 to 190 mm in length. The spotted TLCs were developed in the respective solvent system and allowed to air-dry thoroughly at room temperature. TLCs were redeveloped again in the same mobile phase to achieve the complete and compact separation of spots as distinct bands. The silica gel at the spot of interest was scraped separately from the TLC plate and loaded immediately on the ideal glass column.
The compound was desorbed from the silica gel using a suitable solvent, concentrated and analyzed in TLC prior to spectroscopic analysis.

Extraction and isolation of compounds
The mycobiont cultures were harvested from the medium using a scalpel and 148.2 g of the harvested material was dried in a hot air oven at 45 °C for two days, and powdered using a mixer grinder, yielding dry weight of 64.4 g. The mycelial powder was subjected to cold extraction at room temperature (28 ±1 °C) using acetone, and repeated four times (250 mL × 4) every 48 h for complete extraction of the compounds. The crude extract was filtered with Whatman No.1 filter paper, and the filtrate was concentrated using a rotary evaporator under reduced pressure yielding 5.62 g of solid residue. In addition, the metabolites that diffused from the mycelium and were deposited on the upper surface of the medium were removed carefully, and dried at 50 °C for four days to eliminate moisture and prevent fungal growth, resulting in a dry weight of 119.8 g. The dried metabolites were powdered and extracted with acetone (500 mL × 4) as described above, producing a gummy substance (4.89 g). The solid residue and gummy substance were combined for subsequent purification.

Crystallization
The compounds were dissolved in a single or a combination of two solvents (v/v = 10:1), and heated in a water bath at 65 °C. The solutions were cooled and left undisturbed at room temperature (28 ±1 °C) to allow slow evaporation of the solvent and the development of crystals. This process was repeated continuously until good diffraction-quality crystals were obtained. Compounds 5, 6 and 7 yielded diffraction-quality crystals and the following solvents were suitable for the crystallization of these compounds: Chloroform-ether mixred plates (compound 5); tetrahydrofurandark purple-red crystals (compound 6); chloroformether mixdark violet crystals (compound 7).
The automatic cell determination routine with 36 frames at three different orientations of the detector was employed to collect reflections for unit cell determination. Furthermore, intensity data for structure determination were collected through an optimized strategy which gave an average 4-fold redundancy. The APEX2-SAINT program (Bruker 2004) was used for integrating the frames. Four-fold redundancy per reflection was utilized for achieving good multi-scan absorption correction using the SADABS program (Bruker 2004). In addition to absorption, Lorentz, polarization and decay corrections were applied to intensity during data reduction. The structures were solved by direct methods using SIR92 (Altomare et al. 1993), and refined by full-matrix least squares technique using SHELXL-2014/7 (Sheldrick 2015) program. Molecular graphics were drawn using ORTEP3 (Farrugia 1997).

The crystallographic data have been deposited in the Cambridge Crystallographic Data
Centre under the reference numbers 1411890 (compound 5), 1411971 (compound 6) and 1520256 (compound 7). The CCDC data are freely available in the database. (Compounds 1, 2, 5-8)

Antimycobacterial assay
Minimum inhibitory concentration (MIC) was determined against mycobacterial strains using the 7H11 Middlebrook agar dilution method (Hacek 1992). Standardized inocula of mycobacterial strains were grown on drug-free media and media containing graded concentrations of the drugs to be tested, and incubated at 37 o C under 5-10% CO 2 . Colonyforming units (CFU) were determined after eight weeks of incubation. The concentration of the compound that inhibited growth of mycobacteria was referred to as the MIC of that drug.
Rifampicin was included in the assay as a positive control and its MIC was determined to be 8 µg/mL, which is satisfactory according to WHO standards for testing rifampicin.

Antibacterial assay
The biological activity of the isolated compounds against pathogenic Gram-positive