A new cytotoxic 12-membered macrolactone from the endophytic fungus Exserohilum rostratum LPC-001

Abstract A new oxacyclododecindione-type macrolactone, (13R,14S,15R)-13-hydroxy-14-deoxyoxacyclododecindione (1), has been obtained from the solid cultures of the fungus Exserohilum rostratum, a fungal strain endophytic in Gymnadenia conopsea. Its structure, including the absolute configuration, was extensively established by 1D and 2D NMR data, the modified Mosher method, and a combination of experimental and theoretically calculated electronic circular dichroism (ECD) spectra. Compound 1 showed weak selective cytotoxicity against the A549 lung cell line with an IC50 value of 9.2 µM.


Introduction
In recent years, the cultivation of endophytic fungi has proven to be one of the most productive sources of secondary metabolites with novel structures and significant bioactivities [1][2][3][4][5]. Exserohilum rostratum has been separated from both terrestrial and marine sources and its chemical investigation has resulted in the identification of a variety of structures with biological properties, such as disulfides with cytotoxicity, antifungal monocerins and polyketides, and oxacyclododecindione-type macrolactones as inhibitor of TGF-b and IL-4 signaling in mammalian cells [6][7][8][9][10]. In an effort to broaden our research in new bioactive metabolites from different resources, we investigated the chemistry of the fungus E. rostratum LPY-001 isolated from a leaf tissue of Gymnadenia conopsea, a Tibet medicinal plant known as "wangla" (Chinese) which has been traditionally used for the treatment of cough, asthma, and other syndromes [11]. This led to the discovery of one new oxacyclododecindione-type macrolactone named (13R,14S,15R)-13-hydroxy-14-deoxyoxacyclododecindione (1) (Figure 1). Herein, we report its isolation, structure elucidation, and cytotoxic activity. The absolute configuration of the new compound was established by the modified Mosher method and a combination of experimental and theoretically calculated electronic circular dichroism (ECD) spectra.

Results and discussion
Compound 1 was obtained as a colorless solid with [a] 20 D þ31.0 (c 0.20, MeOH), and its IR spectrum pointed out the absorption for hydroxyl (3394 cm À1 ), carbonyl (1715 cm À1 ), and conjugated carbonyl (1646 cm À1 ) moiety. During positive highresolution ion electrospray, 1 showed the typical isotope pattern of monochlorinated compound due to two protonated ion at m/z 369.1107 and m/z 371.1083 in a ratio of 3:1, consistent with the molecular formula C 18 H 21 O 6 Cl. This molecular formula, requiring nine degrees of unsaturation, was corroborated by NMR spectroscopic data of 1 (Table 1). The 1 H and 13 C NMR data associated with DEPT and HSQC data of 1 in DMSO-d 6 14)]. Also observed in the 1 H and 13 C NMR spectra of 1 was two carbonyls at d C 167.7 and 197.6 (C-1 and C-9) and three hydroxyl protons at d H 9.66 (1H, s, OH-5), 10.22 (1H, brs, OH-7), and 4.71 (1H, d, J ¼ 4.2 Hz, OH-13). The structure of 1 was finally assembled once the COSY and HMBC data sets had been collected.  and the diagnostic HMBC correlations from H 3 -16 to C-9 and C-11, from H 3 -17 to C-15 and C-13, from H 3 -18 to C-14, from H-11 to C-16 and C-9, and from OH-13 to C-13 and C-12 ( Figure 2). HMBC correlations observed from H-15 to the carbonyl group (C-1) which in turn showed HMBC cross peaks with the benzylic CH 2 group (d H 3.41 and 3.02) suggested a 12-member macrolactone ring skeleton. This conclusion was quickly affirmed by the HMBC correlations from benzylic CH 2 group to C-3, C-4 and C-8. A question immediately arises as to how to determinate the location of the Cl atom and the two aromatic hydroxyl groups due to only one aromatic proton in 1. Fortunately, key HMBC cross peaks were observed from the hydroxyl proton at d H 9.66 (1H, s, OH-5) to C-5 and C-4 in the HMBC spectrum of 1 ( Figure  2). This observation, coupled with the strong HMBC correlations from H-6 to C-8, C-4 and C-5, and from benzylic CH 2 to C-3, C-4 and C-8, as well as consideration of the lower field chemical shift of C-4 attributed to the heavy-atom effect, confirmed the Cl atom and one of the hydroxyl groups were positioned at C-4 and C-5,  respectively, leaving another hydroxyl group to be located at C-7. Thus, the planar structure was established as shown in Figure 1.
The configuration of 1 was deduced from the NOESY spectrum, the modified Mosher method, and a combination of experimental and theoretically calculated ECD spectra. An E-geometry double bond was assigned according to the NOESY correlation of H 3 -16/H 2 -12 and the absent NOESY correlation of H 3 -16/H-11. Compound 1 was converted to (R)-and (S)-MTPA for determination of the absolute configuration at C-13. According to the Mosher arguments, C-13 was assigned with R absolute configuration, since the signs of Dd (d S -d R ) were negative for H-14, H-15, H 3 -17, and H 3 -18, and positive for H 2 -12 based on analysis of the 1 H-NMR and 1 H-1 H COSY spectra of (R)-and (S)-MTPA monoesters 1a and 1b, respectively (Scheme 1) [12]. The NOESY spectrum of 1 showed identical NOE correlations with (14S,15R)deoxyoxacyclododecindione isolated from the same fungus (Figure 3), for which absolute configuration has been proved by X-ray crystallography [8,13,14]. On the basis of these data and biosynthetic considerations, the 13R,14S,15R absolute configurations of 1 were established. This was confirmed by the calculation of ECD using time-dependent density functional theory (TDDFT) [15]. The calculated ECD data of 1 and its enantiomer in MeOH are shown in Figure 4. The calculated ECD of 1 matches well with the experimental CD, while the calculated ECD of its enantiomer is opposite to  the experimental value, allowing the assignment of the 13R,14S,15R configurations of 1. Therefore, the structure of 1 was characterized as (13R,14S,15R)-13-hydroxy-14deoxyoxacyclododecindione Compound 1 was examined in a cytotoxicity assay against the HCT-8 colon, A2780 ovary, BGC-823 stomach, Bel-7402 hepatoma, and A549 lung cell lines. As a result, compound 1 exhibited weak selective cytotoxicity against the A549 lung cell line with an IC 50 value of 9.2 mM. The positive control, paclitaxel, gave IC 50 value of 0.11 mM.

Fungal material
The fungus strain LPC-001 was isolated from the leaves of Gymnadenia conopsea collected from Diebu County, Tibetan Autonomous Prefecture of Ganan, China. It was identified according to its morphological characteristics and 18S RNA. A voucher specimen is deposited in our laboratory at À80 C.

Fermentation and extraction
Large-scale growth of the fungus for the isolation and identification of new metabolites was carried out in Erlenmeyer flasks (1L each). The fungus was cultured in PDA medium containing glucose (20 g/L), potato extract (200 g/L) at 28 C on a shaker platform at 155 rpm for 3 days to get seed solution (100 ml). Seed solution was diluted 100 times with sterile rice medium. Solid fermentation was statically performed at 28 C for 40 days. The mycelia and solid rice medium were extracted with EtOAc. The organic solvent was evaporated under reduced pressure to yield 2.1 g of crude extract. This crude extract was subjected to column chromatography over silica gel employing a PE-acetone gradient system (100:1, 50:1, 30:1, 10:1, 5:1, 1:1, 0:1) to yield five fractions (F1-F5). Fraction F3 was fractionated by Sephadex LH-20 column chromatography eluting with CHCl 3 -MeOH (1:1), to yield five subfractions (F31-F35). Subfraction F33 was further purified by reversed-phase preparative HPLC (RP 18

Calculated ECD of 1
Conformational analysis of 1 was performed by using the MMFF94 molecular mechanics force field via the Discovery studio 2018 software package [16]. The lowest-energy conformers having relative energies within 9.0 kcal/mol were optimized with the Gaussian 16 program package at the B3LYP/6-31G(d) level, and the frequency was calculated at the same level of theory. The ECD calculations of stable conformers without imaginary frequencies were calculated using the TDDFT method at the CAM-B3LYP/6-311 þ G(2d,p) level in MeOH [17]. The final ECD spectrum of 1 was obtained according to Boltzmann weighting of each conformer simulated using SpecDis v1.70.1 with a half-bandwidth of 0.3 eV [18].

Disclosure statement
No potential conflict of interest was reported by the authors.