A new cyclohexenone derivative from phomopsis sp. XM-01

Abstract A new cyclohexenone derivative, phomopine (1), together with five known compounds (2-6) were isolated from Phomopsis sp. XM-01. The structure of 1 was determined by extensive spectroscopic analyses and electronic circular dichroism (ECD) calculation. In vitro bioassays, compounds 1 and 2 exhibited potent antimicrobial activities against Candida albicans and Staphylococcus aureus with their corresponding minimum inhibitory concentrations (MICs) of 64 μg/mL and 16 μg/mL, respectively. Graphic Abstract


Introduction
Medicinal plants are an essential resource for natural product research.However, since the natural resources of medicinal plants were being exploited and destroyed without restraint, many of them are on the verge of extinction (Yang et al. 2004).Exploring substitutes for medicinal plants to discover leading compounds has become a new direction in natural medicinal chemistry research.Endophytic fungi coevolve with their hosts and are likely to produce novel and bioactive natural products (Gupta et al. 2020;Zhang et al. 2006;Qian et al. 2022).Moreover, endophytic fungal secondary metabolites have long been considered as a reservoir of structurally diverse compounds with a wide range of pharmacological properties (Strobel et al. 2004).Thence, endophytic fungi have been regarded as a new frontier in natural product research (Gupta et al. 2020) Figure 1.

Morphology and taxonomy
Strain XM-01 was isolated from the typical stalk of I. rhodantha, and it was incubated at 28 C on the PDA plates in the dark to promote mycelial growth.After four days of cultivation, mycelia were dense and slightly raised; some developed prominent growth rings, with margins becoming gray with age.Colonies were initially thin-white to gray, becoming gray-black from the middle within seven days, and black with age (Mostert et al. 2001) shown in Figure S11.Through molecular identification, a phylogenetic tree was constructed using the homologous sequences in GenBank by the Maximum Likelihood method (ML) (Udayanga et al. 2011).The strain was identified by analysis of the internal transcribed spacer (ITS) region of the rRNA sequence, which was most closely related to Phomopsis sp.(Phomopsis mali B2 (MN486557.1)shown in Figure S12.

Structural elucidation
Compound 1 was isolated as a white amorphous powder.Its molecular formula was determined as C 8 H 12 O 4 by high resolution electrospray ionization mass spectroscopy (HR-ESI-MS) at m/z 195.0625 [M þ Na] (Calcd for C þ 8 H 12 O 4 Na, 195.0628), indicating 3 degrees of unsaturation.Its infrared spectrum (IR) showed the absorbance of hydroxyl (3423 cm þÀ1 ) and carbonyl (1679 cm À1 ) groups.The 13 C NMR spectrum of 1 indicated the existence of eight carbons, including two methyl groups (d C 17.8, 16.9), one methylene group (d C 37.1), one methine group (d C 73.3), and four quaternary carbons (d C 199.1, 143.7, 129.1, 77.8).One carbonyl carbon and a double bond accounted for 2 degrees of unsaturation, indicating that compound 1 should possess a ring in the structure.Detailed comparison of the nuclear magnetic resonance (NMR) data suggested that 1 shared the same cyclohexanone skeleton with leptosphaerone A (Ayer et al. 1993).The main difference between 1 and leptosphaerone A was that 1 possessed an additional hydroxyl group at C-2, further supported by the chemical shifts of C-2 moved towards downfield.The HMBC correlations from H-5 (d H 3.86 dd, J ¼ 9.7, 5.6 Hz) to C-6 (d C 77.8) and C-7 (d C 17.8), from H-4 (d H 2.53 dd, J ¼ 17.8, 5.6 Hz; d H 2.36 ddq, J ¼ 17.7, 9.7, 1.7 Hz) to C-3 (d C 129.1), C-2 (d C 143.7) and C-8 (d C 16.9), from H-8 (d H 1.88 d, J ¼ 1.8 Hz) to C-2 (d C 143.7), and from H-7 (d H 1.22 s) to C-1 (d C 199.1) were consistent with the above structural elucidation.Thus, the planar structure of 1 was demonstrated, as shown in Figure S7.
The relative configuration of 1 was pointed out by examination of its NOESY spectrum.The NOE correlation between H-5 (d H 3.86 dd, J ¼ 9.7, 5.6 Hz) and H-7 (d H 1.22 s) demonstrated that these protons should be on the same face of the six-membered ring.Therefore, two possible isomers (5S,6S) and (5 R,6R) were excluded.The theoretical electrostatic circular dichroism (ECD) data of the two isomers were calculated and compared with the experimental spectrum (Figure S10).The experimental ECD spectrum was in good accordance with that of the calculated (5S,6S)-isomer, which suggested the absolute configuration of 1 was 5S, 6S.Therefore, the structure of 1 was established as (5S,6S)-2,5,6-trihydroxy-3,6-dimethylcyclohex-2-en-1-one and named phomopine (1).

Assessment of antimicrobial activity
Compound 1 exhibited potent antimicrobial activity against Candida albicans with a minimum inhibitory concentration (MIC) of 64 lg/mL, and nystatin was used as positive control (MIC 16 lg/mL).Compound 2 illustrated moderate antimicrobial activity against Staphylococcus aureus with a minimum inhibitory concentration (MIC) of 16 lg/ mL, and kanamycin was used as positive control (MIC 32 lg/mL).The remaining compounds were inactive under all tested strains.

Conclusion
In summary, six compounds (1-6), including one new cyclohexenone derivative phomopine (1), were isolated from Phomopsis sp.XM-01.The structure of phomopine (1) was determined by extensive spectroscopic analyses and ECD calculations.Compound 1 exhibited antimicrobial activity against C. albicans with a MIC of 64 lg/mL, and compound 2 showed antimicrobial activity against S. aureus with a MIC of 16 lg/mL.These findings will enrich the chemical diversity of cyclohexenone derivative.