Mono- and Dimeric Sorbicillinoid Inhibitors Targeting IL-6 and IL-1β from the Mangrove-Derived Fungus Trichoderma reesei BGRg-3

Four new sorbicillinoids, named trichodermolide E (1), trichosorbicillin J (2), bisorbicillinolide B (3), and demethylsorbiquinol (5), together with eight known compounds (4, 6–12), were isolated from the cultures of the mangrove-derived fungus Trichoderma reesei BGRg-3. The structures of the new compounds were determined by analyzing their detailed spectroscopic data, while the absolute configurations were further determined through electronic circular dichroism calculations. Snatzke’s method was additionally used to determine the absolute configurations of the diol moiety in 1. In a bioassay, compounds 7 and 10 performed greater inhibitory activities on interleukin-6 and interleukin-1β than the positive control (dexamethasone) at the concentration of 25 μM. Meanwhile, compounds 5 and 6 showed potent effects with stronger inhibition than dexamethasone on IL-1β at the same concentration.


Introduction
The mangrove is a complex ecosystem occurring in tropical and subtropical intertidal estuarine zones [1].The special geographical location of mangroves provides a special environment with high salt, hypoxia, frequent tides, and strong solar radiation, which nourishes a diverse group of microorganisms.Among them, mangrove-derived fungi play an essential role in creating structurally unique and diverse bioactive secondary metabolites, which attract the significant attention of organic chemists and pharmacologists [1,2].
The relative configuration of compound 1 was determined using coupling constants and a NOESY (nuclear overhauser effect spectroscopy) spectrogram (Figure 3).The double bonds of the side chain were supposed to be E geometries according to the coupling constants (15.6 Hz for J H-12/H-13 ) and the NOESY correlations of H-13/H-15 and H-12/H-14.Due to the heterocycle skeleton, the configuration of C-2 and C-6 was assumed to be 2S, 6S, or 2R, 6R.Additional NOESY correlations of H 3 -8/H-9 and H 3 -7/H-9 indicated that the relative configuration of the skeleton was 2R*, 6R*, 9R*.
The absolute configuration of compound 1 was confirmed based on the ECD (electronic circular dichroism) calculation and Snatzke's method [17,18].In order to determine the absolute configuration of the skeleton, ECD calculations were performed in B3LYP functional with the 6-311+G(d,p) basis set, using the B3LYP/6-31G(d) optimized geometries which were found through the conformer distribution calculation in the MMFF force field.Supported by the comparison of the experimental ECD spectrum with the calculated one (Figure 4a), the absolute configuration of the skeleton was confirmed to be 2R, 6R, 9R.Then, Snatzke's method was used to determine the absolute configuration of acyclic 1,2-diols.Based on the Mo 2 (OAc) 4 -induced ECD measurement (Figure 4b), the 17S configuration was confirmed by the positive cotton effect at 360 nm [18].Thus, the absolute configuration of compound 1 was defined as 2R, 6R, 9R, and 17S.The absolute configuration of compound 1 was confirmed based on the ECD (electronic circular dichroism) calculation and Snatzke's method [17,18].In order to determine the absolute configuration of the skeleton, ECD calculations were performed in B3LYP   The absolute configuration of compound 1 was confirmed based on the ECD (electronic circular dichroism) calculation and Snatzke's method [17,18].In order to determine the absolute configuration of the skeleton, ECD calculations were performed in B3LYP tries which were found through the conformer distribution calculation in the MMFF force field.Supported by the comparison of the experimental ECD spectrum with the calculated one (Figure 4a), the absolute configuration of the skeleton was confirmed to be 2R, 6R, 9R.
Then, Snatzke's method was used to determine the absolute configuration of acyclic 1,2diols.Based on the Mo2(OAc)4-induced ECD measurement (Figure 4b), the 17S configuration was confirmed by the positive cotton effect at 360 nm [18].Thus, the absolute configuration of compound 1 was defined as 2R, 6R, 9R, and 17S.
Bisorbicillinolide B (3) showed the appearance of a red oil and its molecular formula was C28H36O8 based on the HR-ESIMS (m/z 499.2346 [M−H] − , calcd.for C28H35O8, 499.2326, Figure S17).According to the 13 C NMR spectrum (Table 2), 28 carbon signals were found and were then classified into six methyls, four methylenes, three methines, six quaternary carbons, four alkene carbons, one enol carbon, one carboxyl, and three carbonyls based on the DEPT and HSQC spectra, thus revealing a similar structure to the known compound bisorbicillinolide (4) [12].Analyzing the NMR data (Table 2), in association with the indices of hydrogen deficiency, 3 was assumed to be structurally related to known compound 4 with a difference in the reduced Δ 2′ -and Δ 2″ -double bonds.The COSY correlations (Figure 2) proved the existence of two side chains from H-2′ to H-6′ and from H-2″ ).Then, 14 protons were connected with their associated carbons according to the HSQC spectrum (Table 1).The COSY correlations (Figure 2) showed the existence of a side chain from C-8 to C-13.The COSY correlation of H-4/H-5, together with the coupling constant (8.9 Hz for J H-4/H-5 ), showed the ortho arrangement of them.The HMBC correlation (Figure 2 S17).According to the 13 C NMR spectrum (Table 2), 28 carbon signals were found and were then classified into six methyls, four methylenes, three methines, six quaternary carbons, four alkene carbons, one enol carbon, one carboxyl, and three carbonyls based on the DEPT and HSQC spectra, thus revealing a similar structure to the known compound bisorbicillinolide (4) [12].Analyzing the NMR data (Table 2), in association with the indices of hydrogen deficiency, 3 was assumed to be structurally related to known compound 4 with a difference in the reduced ∆ 2 -and ∆ 2 -double bonds.The COSY correlations (Figure 2) proved the existence of two side chains from H-2 to H-6 and from H-2 to H-6 .According to the HMBC correlations, together with the remaining COSY correlations (Figure 2), the constitutional formula of 3 was allowed to be confirmed.
The double bonds in the side chains were both proved to be E geometries according to the NOESY signals ( The comparison between the experimental spectrum (Figure 5) with the calculated one could give the absolute configuration of compound 3 as 1S, 4R, 5R, 7S, 8S, 9S, 10S, 11R.Demethylsorbiquinol (5) showed its molecular formula as C27H30O7, confirmed through HR-ESIMS (m/z 465.1925 [M−H] − , calcd.for C27H29O7, 465.1908, Figure S25).Its 13 C NMR data showed 27 carbons, including five methyls, three methines, two quaternary carbons, seven alkene carbons, six aromatic carbons, one enol carbon, and three carbonyls.A similar compound, sorbiquinol (6) [13], was obtained in the same fraction.A comparison of the NMR data is shown in Table 3, showing the absence of a methyl in 5.

Anti-Inflammatory Activities
The bioactivities of 12 sorbicillinoids (compounds 1-12) were evaluated by measuring the effects of each compound on IL-6-and IL-1β-induced luciferase expression in RAW264.7 cells, testing under the concentration of 25 µM for 4 h.Their cytotoxicity was also evaluated at the same concentration level.The results are shown in Table 4.Among them, compounds 2, 5-7, and 10 exhibited inhibition against IL-6 and IL-1β.Compounds 7 and 10 presented their remarkable anti-inflammatory activities, respectively, with 47% and 67% inhibition of IL-6, 85% and 87% inhibition of IL-1β, which were even more effective than the dexamethasone.Compound 5 also showed its potent inhibition of IL-6 and IL-1β (27% and 58%, respectively), which was mildly weaker than the similar compound 6.However, compound 5 exhibited the advantage of less cytotoxicity.Moreover, compound 2 presented a moderate inhibition of both targets with little cytotoxicity.

Fungal Material
Fungus BGRg-3 was isolated from the root of mangrove plant Avicennia marina, which was collected from the Nansha Mangrove National Nature Reserve in Guangdong Province, China.The strain was then identified as Trichoderma reesei based on the analysis of its ITS sequence (deposited in GenBank, accession no OR353740).The fungus is now deposited at Sun Yat-sen University, China.

Fermentation
The fungus Trichoderma reesei BGRg-3 was cultivated on solid cultured medium in 1 L erlenmeyer flasks, containing 50 g of rice and 80 mL of 3‰ saline water.A total of 100 flasks were fermented for 30 days at room temperature.

Extraction and Purification
When the fermentation was carried out, the mycelia, as well as the rice medium, was extracted four times with MeOH and EtOAc.Eventually, a 95.5 g extract was collected (Figure S37).Then, the crude extract was eluted with a gradient elution with petroleum ether (PE) and EtOAc (from 9:1 to 0:10) on silica gel CC to obtain six fractions (Fr.A-F).

ECD Calculation
Conformational analyses were carried out via random searching with an MMFF force field using Spartan'14 (v1.1.0)[20].Subsequently, the conformers were re-optimized with density functional theory (DFT) methods at the B3LYP/6-31+G (d, p) level, solvated in methanol, with the CPCM method using the Gaussian 09 [21].Then, TDDFT calculations were performed at the B3LYP/6-311+G(d, p) level (nstates = 50) in methanol.The ECD spectra were obtained by overlapping Gaussian functions (σ = 0.3 eV).To obtain the final spectra, the simulated spectra of each conformer were averaged weighted with Boltzmann distribution and their relative Gibbs free energy, using the program SpecDis (v1.64) [22].The theoretical ECD spectra of the corresponding enantiomers were obtained through direct inversions of the ECD spectra of the abovementioned conformers.

Absolute Configurations of the 17,18-Diol Moiety in 1
A modified version of Snatzke's method was performed according to the published literature [17,18].Mixtures of 1:1.2 diol-Mo 2 (OAc) 4 for 1 were subjected to ECD measurement at a compound concentration of 1.0 mg/mL in DMSO for each.After mixing, the first ECD was recorded immediately, and the time evolution was surveyed until stationary (about 10 min after mixing).The inherent ECD was subtracted.In the induced ECD spectra,

Figure 5 .
Figure 5. (a) Experimental and calculated ECD spectrum of 3; (b) experimental and calculated ECD spectrum of 5.

Table 4 .
The cytotoxicity and the inhibitory effects on IL-6 and IL-1β of the tested compounds.Data are presented as the mean ± SD.2Positive control; NA: no activity (inhibition ≤ 20%); NT: not tested.