Novel Antimicrobial Compounds as Ophiobolin-Type Sesterterpenes and Pimarane-Type Diterpene From Bipolaris Species TJ403-B1

Six previously undescribed ophiobolin-type sesterterpenes, namely, bipolatoxins A–F (1–6); and one previously undescribed pimarane-type diterpene, namely, 1β-hydroxy momilactone A (7); together with three known compounds, namely, 25-hydroxyophiobolin I (8), ophiobolin I (9), and ophiobolin A lactone (10); were isolated and identified from the endophytic fungus Bipolaris species TJ403-B1. Their structures with absolute configurations were elucidated on the basis of extensive spectroscopic analyses (including 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy data), single-crystal X-ray diffraction analyses, and comparison of experimental circular dichroism data. All compounds (except for 5) were evaluated for antimicrobial potential, which indicated that bipolatoxin D (4) showed significant inhibitory activity against Enterococcus faecalis with a minimum inhibitory concentration (MIC) value of 8 μg/mL, and ophiobolin A lactone (10) showed significant inhibitory activity against Acinetobacter baumannii and E. faecalis with MIC values of 8 and 8 μg/mL, respectively.


INTRODUCTION
Microbial natural products and their derivatives have been an important source of new antibiotics required for the treatment of infectious diseases (Wright, 2017). Since the first antibiotic, penicillin, was discovered from the fungus Penicillium notatum in 1928 (Wright, 2017), multiple classes of anti-infectives have been isolated from a variety of fungi, such as gliotoxin, beauvericin, and roquefortine C (Jakubczyk and Dussart, 2020). However, the rapid evolution of antimicrobial resistance in both hospital and community settings is decreasing the efficacy of our current therapies and causing a serious global public health crisis (Baker, 2015;Brown and Wright, 2016). One strategy to combat antimicrobial resistance is to discover and develop novel antimicrobial drugs that are not subject to existing resistance mechanisms (Demirci et al., 2013;Ziemert et al., 2016). Fungi-derived natural products hold great promise in the search for new therapies.

Fungus Material
The fungal strain in our project was obtained from the leaves of wheat, which was collected from Wuhan City of Hubei Province, China, in May 2016. Sequence data for this fungal strain have been submitted to the DDBJ/EMBL/GenBank under accession no. MH545913. A voucher sample has been preserved in the culture collection center of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China).

X-Ray Crystal Structure Analysis
The suitable crystals of compounds 1, 4, and 8 were acquired from MeOH-H 2 O (20:1, vol/vol) at room temperature. The intensity data were recorded on an XtaLAB PRO MM007HF diffractometer (Cu Kα). Using Olex2 (Dolomanov et al., 2009), the structures were solved via direct methods with SHELXL-2014/7 (Sheldrick, 2008). Refinements were executed by the SHELXL-2014/7 refinement package via means of full-matrix least squares on F 2 , and the anisotropic displacement parameters were applied for all the non-hydrogen atoms. All the hydrogen atoms were placed on the calculated positions and refined by a riding model. The crystallographic data for these structures were deposited in the Cambridge Crystallographic Data Center (CCDC 1971181 for 1, CCDC 1913829 for 4, and CCDC 1913832 for 8). Copies of the data can be obtained free of charge on application to CCDC (Cambridge, United Kingdom; e-mail: deposi@ccdc.cam.ac.uk).

Determination of the Minimum Inhibitory Concentrations
Determination of the minimum inhibitory concentrations (MICs) was conducted according to our previously reported broth microdilution method (Gao et al., 2019;Yang et al., 2019). In short, the inoculum was standardized to approximately 5 × 10 5 colony-forming units/mL. The plates were incubated at 37 • C for 16 h, and the MIC values were recorded as the lowest concentration of antibiotic, at which no visible microbial growths were observed. Each experiment was performed three times.

Statistical Analysis
GraphPad Prism 5.0 software (GraphPad, San Diego, United States) was used to carry out statistical analysis of the data. The data were expressed as the means ± SD. Values were analyzed with SPSS version 12.0 software (Softonic, Barcelona, Spain) by one-way analysis of variance, and p < 0.05 was considered statistically significant.
Compound 1 was obtained as colorless needle crystals, and its molecular formula was assigned as C 25 H 38 O 3 , based on the HRESIMS data at m/z 409.2729 ([M+Na] + , calcd for C 25 H 38 O 3 Na + , 409.2713) in conjunction with the NMR data analyses, which was indicative of an index of hydrogen deficiency of seven. The 13 C NMR data ( Table 3) together with the DEPT spectrum of 1 showed a total of 25 resonances that could be assigned as five methyls, six methylenes (including one oxygenated), nine methines (including four olefinic), and five non-protonated carbons (including one ketone, one oxygenated, and two olefinic). Apart from four indices of hydrogen deficiency attributed to three double bonds (δ C 183.1/130.6, 135.1/130.7, 126.3/140.6) and a carbonyl (δ C 212.0), the remaining indices of hydrogen deficiency suggested that 1 has a tricyclic ring system.
Compound 2 was obtained as a colorless oil, which had a molecular formula of C 26 H 40 O 3 , according to its HRESIMS data at m/z 423.2882 ([M+Na] + , calcd for 423.2870). The 1D NMR data (Tables 1 and 3) showed close similarities to those of 1, except for the C-19 hydroxy group in 1 being replaced by a methoxy group (δ C 50.5) in 2, as supported by the key HMBC correlation (Figure 2) from δ H 3.14 (3H, s, OMe-19) to C-19 (δ C 76.4). The close resemblance of the NOESY data (Supplementary Data Sheet S5) and experimental CD curves (Figure 5) of 1 and 2 suggested their identical absolute configuration. Accordingly, the structure of 2 was defined and named bipolatoxin B.
Compound 3 was also purified as a colorless oil. The HRESIMS analysis of 3 displayed a sodium adduct ion at m/z 407.2550 [M+Na] + (calcd for 407.2557), suggesting a molecular formula of C 25 H 36 O 3 . Subsequent comparison of the 1 H and 13 C NMR data (Tables 1 and 3) of 3 with those of 1 suggested that 3 contained an additional double bond (δ C 121.1 and 151.2). Further analyses of the HMBC data (Figure 2) of 3  revealed that the double bond was located between C-13 and C-14, as supported by the correlations from H 3 -23 (δ H 1.09) to C-14 (δ C 151.2) and from H-13 (δ H 5.32) to C-10 (δ C 52.1) and C-11 (δ C 46.2). The stereochemical configuration of 3 was established to be the same as 1 by their closely resembled NOE data (Supplementary Data Sheet S5) as well as shared biogenesis. Accordingly, the structure of 3 was defined and named bipolatoxin C.
The HRESIMS ion at m/z 425.2653 ([M+Na] + , calcd for 425.2662), together with the 13 C NMR and DEPT data for 4, revealed its molecular formula to be C 25 H 38 O 4 , requiring an index of hydrogen deficiency of seven. The 1 H and 13 C NMR data (Tables 1 and 3) of 4 closely resembled those of ophiobolin X (Zhu et al., 2018), indicating that both compounds shared the same A/B/C rings and corresponding substituents, with the only difference being on the side chain that the conjugated double bonds (C-16/C-17 and C-18/C-19) were replaced by an sp 3 methylene (δ C 41.1, C-16), a double bond between C-17 (δ C 127.0) and C-18 (δ C 140.4), and a hydroxylated quaternary carbon (δ C 71.1, C-19). These conclusions were further confirmed by the 1 H-1 H COSY correlations of H-14/H-15 (H 3 -23)/H 2 -16/H-17/H-18 and an obvious HMBC correlation from H 3 -24 to C-19 (Figure 2). The ROESY correlations (Figure 3)  at room temperature, 4 furnished a crystal suitable for X-ray diffraction analysis (Figure 4). The Flack parameter of 0.05(10) allowed an unambiguous assignment of the complete absolute configurations of all chiral centers as 2S, 3R, 5S, 6S, 10S, 11R, 14R, and 15S, as well as an E geometry of the 17,18 double bond. Accordingly, the structure of 4 was defined and named bipolatoxin D.
Compound 7 was isolated as a colorless oil, with the molecular formula of C 20 H 26 O 4 , as determined by its HRESIMS data ([M+H] + ion peak at m/z 331.1913). Its 1 H and 13 C NMR data (Tables 2 and 3) were similar to those of momilactone A (Germain and Deslongchamps, 2002), with the replacement of an sp 3 methylene in momilactone A by an oxygenated sp 3 methine (δ C /δ H 68.2/4.18) in 7, which was supported by analysis of its 2D NMR data (Figure 2). In the 1 H-1 H COSY spectrum, the correlation of H-1 (δ H 4.18)/H 2 -2 (δ H 2.41 and 2.86), together with HMBC correlations from H-5 (δ H 2.52) and H 3 -20 (δ H 0.93) to C-1 (δ C 68.2), suggested that a hydroxylated methine was located at C-1. In the NOESY spectrum (Figure 3), the crosspeaks of H-1/H-5/H 3 -18 suggested that the OH-1 group should be β-oriented. Accordingly, the structure of 7 was established as shown and named 1β-hydroxy momilactone A.
The three known compounds were identified as 25hydroxyophiobolin I (8) (Sugawara et al., 1987), ophiobolin I (9) (Sugawara et al., 1987), and ophiobolin A lactone (10) (Li et al., 1995), by comparison of their NMR data and specific rotations with literature. Remarkably, it is the first time that the absolute structure of compound 8 was defined by the single-crystal X-ray diffraction analysis (Figure 4).

Biological Evaluation
Because of the limited amount of compound 5, only compounds 1-4 and 6-10 were evaluated for the antimicrobial activity against six drug-resistant microbial pathogens, including ESBLproducing E. coli, A. baumannii, P. aeruginosa, K. pneumoniae, methicillin-resistant S. aureus (MRSA), E. faecalis, and one fungus C. albicans. As shown in Table 4, except for compound 8, all the other test compounds showed antimicrobial activity against certain microbial pathogens (MIC = 8-64 µg/mL), of which compound 4 showed significant inhibitory activity against E. faecalis with an MIC value of 8 µg/mL, and compound 10 showed significant inhibitory activity against A. baumannii and E. faecalis with MIC values of 8 and 8 µg/mL, respectively.

CONCLUSION
A total of 10 secondary metabolites (1-10), incorporating six new ophiobolin-type sesterterpenes (1-6) and one new pimarane-type diterpene (7), were isolated and identified from the solid cultures of fungus Bipolaris species TJ403-B1. The antimicrobial activity assay revealed that compound 4 showed significant inhibitory activity against E. faecalis with an MIC value of 8 µg/mL, and 10 showed significant inhibitory activity against A. baumannii and E. faecalis with MIC values of 8 and 8 µg/mL, respectively. Our current work not only replenishes new members to ophiobolintype sesterterpenes, but also furnishes potential antimicrobial lead compounds that are necessary to check further for their synergistic and efflux pump inhibition properties.

DATA AVAILABILITY STATEMENT
The crystallographic data for these structures were deposited in the Cambridge Crystallographic Data Centre (CCDC 1971181 for 1, CCDC 1913829 for 4, and CCDC 1913832 for 8).