A new 1,4-benzoxazine derivative produced by endophytic Colletotrichum gloeosporioides B-142

Abstract Colletotrin A (1), a new 1,4-benzoxazine derivative, and eight known compounds, including two alkaloids (2-3), one acylamide (4), one benzaldehyde (5), and four sterols (6-9) were obtained from endophytic fungus Colletotrichum gloeosporioides B-142, which was isolated from Dracaena cochinchinensis. Among them, 1,2,3,4-tetrahydroquinoline-4,8-diol (2) was reported for the first time as natural product. The structure of compound 1 was elucidated with help of spectroscopic data including IR, UV, electronic circular dichroism (ECD) calculation, HRESIMS, 1D and 2D NMR (COSY, HSQC and HMBC). The antimicrobial activities were evaluated by disc diffusion method. Graphical Abstract

In the present study, strain B-142 was isolated from the stem of Dracaena cochinchinensis, a traditional Chinese folk medicine collected at Xishuangbanna, Yunnan province of China, and characterized identified as Colletotrichum gloeosporioides by Dr. Xiao-mei Zhang.Nine compounds were isolated from the culture of Colletotrichum gloeosporioides B-142 and identified to be a new 1, 4-benzoxazine derivative (1), as well as eight known compounds (2-9).In the study, the fermentation, isolation, structural elucidation and antimicrobial activities of compounds 1-9 were described.

Results and discussion
Compound 1 was obtained as yellow solid.Its molecular formula was determined to be C 13 H 15 NO 3 by the HRESIMS data (m/z 256.0946 C 13 H 15 NO 3 Na ＋ 256.0950), which indicated 7 degrees of unsaturation.The 1 H NMR spectrum of 1 showed signals for four aromatic protons signals [d 7.37 (1H,d,J ¼ 7.5 Hz,7.26 (1H,d,J ¼ 7.5 Hz, H-10), 7.05 (1H, t, J ¼ 7.5 Hz, H-9), 6.96 (1H, t, J ¼ 7.5 Hz, H-8)], two methylene proton signals [d 4.01 (2H, t, J ¼ 5.5 Hz, H-3), 2.73 (2H, t, J ¼ 5.5 Hz, H-4)], two methyl groups [d 1.54 (6H, s, H-13, 14)].It could be preliminarily judged that the compound structure includes a symmetric benzene ring, and the two adjacent hydrogen groups on the benzene ring are replaced by other different groups.The 13 C NMR and DEPT data (Table S2) implied the presence of 13 carbon signals that were attributed to six aromatic carbons (d C 137.7, 128.1, 122.0, 119.7, 118.7, 111.8)C-12 (d C 140.1).Therefore, substituent of compound 1 was identified 5,5-dimethyl tetrahydro-2H-pyran-3-ol.The absolute configuration is determined by optical rotation calculation, where the compound has only one chiral carbon, so the relative configuration for 1 was considered to be 5R, 5S.Conformational searches were carried out for the relative configurations R and S, and there were two conformations, respectively.Among them, the lower-conformer energy, Boltzmann specific gravity and OR value of configuration R and S are shown in Table S3.According to the weight and OR value of each conformation, the OR value of configuration 5R and 5S were calculated to be À33.88 and 33.88 respectively, compared with the experimental value of 16.12 (Figure S2).The calculated ECD spectra of 1 (5S) and ent-1 (5R) were conducted, from which the calculated spectrum of 1 at the MacroModel/B3LYP/6-311G level coincided well with the experimental ECD spectrum (Figure S5), which confidently corroborated the absolute stereochemistry of the stereo center of 1 to be 5S.Therefore, the absolute configuration of the chiral carbon in the compound was determined to be S. On the basis of all the foregoing evidence, compound 1 was determined to be (5S)-1,1-dimethyl-3,4-dihydro-1H,4aH-benzo-pyrano [3,4-e] [1,4] oxazin-4a-ol, namely colletotrin A.
The antimicrobial activities of compounds 1-9 were measured using disc diffusion assay against ten pathogens (Table S1).Compound 1 showed antimicrobial activities against Klebsiella pneumonia ATCC13883 with inhibition zone of 10 mm at 50 mg.Compound 6 showed antimicrobial effects on the 9 indicator microbes.Compounds 6 and 9 inhibited the growth of Staphylococcus aureus ATCC25923, Mycobacterium smegmatis 1037, Acinetobacter baumanii ATCC 19606 and Klebsiella pneumonia ATCC13883.Compounds 2-4 and 8 didn't show obvious activities against the test pathogens at the dose of 50 lg.

Fungal material
The fungus was isolated from the stem of a traditional Chinese folk medicine, Dracaena cochinchinensis collected from Xishuangbanna, Yunnan Province of China, and identified as Colletotrichum gloeosporioides based on the sequencing of the internal transcribed spacer (ITS) region sequencing and morphology.A voucher sample was deposited in Laboratory of Microbial Natural Products, Yunnan University of Chinese Medicine with the number of B-142.The ITS gene sequence of strain B-142 was submitted to GenBank and assigned the accession number of ON679646.

Fermentation of endophytic Colletotrichum gloeosporioides B-142
Endophytic Colletotrichum gloeosporioides B-142 was maintained on PDA media (peeled and cut potato, 200 g/L, glucose, 20 g/L, and agar, 15 g/L) at 4 C.A slant culture of the strain B-142 was inoculated in PDB media (peeled potato, 200 g/L; glucose, 20 g/L) for 7 days at 28 C on a rotary shaker at 180 rpm as seed liquid, and then the seeds were cultured in rice media with the a medium inoculation of 10% at 28 C for 31 days.

Extraction and isolation of compounds 1-9
A total of 9 kg rice fermentation products of strain B-142 was extracted with acetone (AC) three times and methanol (MeOH) three times.These two extracts were combined and extracted with ethyl acetate (EAC).Then, the EAC extract was degreased with MeOH/PE (1:1) to obtain a methanol crude extract (39 g) and a petroleum ether crude extract (31 g).

ECD calculation
Conformational analyses were carried out with conformation searching for MacroModel module in Maestro 10.2 program using the OPLS3 force field method to calculate the conformational energy, and those conformations whose energy were less than 3.0 kcal/mol were selected for quantum chemical optimization.The conformers were reoptimised using DFT at the B3LYP/6-311G (2d, p) level with the Gaussian 09 program.The 60 excitations states of the dominant conformation (boltzmann specific gravity greater than 1) were calculated with the TD-DFT methodology at the B3LYP/6-311G (2d, p) level.The ECD profiles of compounds were fitted by SpecDis software and compared with the experimental profiles.

Antimicrobial assay
Pathogenic bacteria was inoculated into liquid LB medium and cultured at 37 C at 200 r/min for 12 h, Candida albicans was inoculated into SAB medium and cultured at 28 C at 200 r/min for 24 h, then diluted to 1 Ã 10 6-7 cfu/mL in liquid medium for later use.
The antimicrobial activities of the 9 compounds were carried out with the paper diffusion method (Digdem et al. 2015;Selemani et al. 2021).Kanamycin (30 lg), ampicillin (10 lg) and amphotericin B (30 lg) were used as positive control.The diameters of the inhibition zones including the 5 mm disc diameter were measured after 24 h of incubation at 37 C.With 50 lg as the measured dose, 14 pathogens were selected as of indicators for antimicrobial activities test.

Conclusion
In summary, chemical investigation of Colletotrichum gloeosporioides B-142 afforded a new compound colletotrin A (1), one new natural product 1,2,3,4-tetrahydroquinoline-4,8-diol (2), and seven known compounds (3-9).The results further enriched the structure types of active secondary metabolites of Colletotrichum gloeosporioides.Among them, five compounds (1, 5-7, 9) demonstrated antimicrobial activities against more than two pathogen.Compounds 6 and 9 have broad spectrum antimicrobial activities against more than six pathogenic microbes, which can be selected as lead compounds in the development of antimicrobial drugs for external use.