Anti-inflammatory butenolide derivatives from the coral-derived fungus Aspergillus terreus and structure revisions of aspernolides D and G, butyrolactone VI and 4′,8′′-diacetoxy butyrolactone VI

Chemical investigation of the coral-derived fungus Aspergillus terreus led to the discovery of ten butenolide derivatives (1–10), including four new ones (1–4). The new structures were characterized on the basis of comprehensive spectroscopic analysis, including 1D and 2D NMR and HRESIMS data. Compounds 1 and 2 were a pair of rare C-8′′ epimers with vicinal diol motifs. The absolute configurations of 1–4 were determined via [Mo2(AcO)4] induced circular dichroism (ICD) spectra and comparison of their experimental ECD spectra. Importantly, the structures of reported aspernolides D and G, butyrolactone VI and 4′,8′′-diacetoxy butyrolactone VI have been correspondingly revised via a combined strategy of experimental validations, 13C NMR predictions by ACD/Labs software, and 13C NMR calculations. Herein we provide valuable referenced 13C NMR data (C-7′′, C-8′′, and C-9′′) for the structure elucidations of butenolide derivatives with 1-(2-hydroxyphenyl)-3-methylbutane-2,3-diol, 2-(2,3-dihydrobenzofuran-2-yl)propan-2-ol, or 2,2-dimethylchroman-3-ol motifs. Additionally, all the isolates (1–10) were assessed for anti-inflammatory activity by measuring the amount of NO production in lipopolysaccharide (LPS)-induced RAW 264.7 mouse macrophages, and compound 10 showed an even stronger inhibitory effect than the postive control indomethacin, presenting it as a promising lead compound for the development of new anti-inflammatory agents.


Introduction
Microorganisms have been regarded as an under-explored source of structurally interesting and bioactive natural products with the potential to provide attractive lead compounds for drug discovery. 1 As one of the most useful fungi, the Aspergillus genus was found to have powerful clusters to biosynthesize plenty of complex secondary metabolites, 2 including lignans, alkaloids, terpenes, polyketides, peptides, etc., showing intriguing pharmaceutical activities, upon which some groundbreaking research has been nished by our research group. For example, Aspergillus avipes produces several bioactive merocytochalasans (anti-tumor agents), namely asperchalasine A, 3 epicochalasines A and B, 4 asperavipine A, 5 and aspergilasines A-D, 6 which were characterized by architecturally complex polycyclic rings and multiple chiral centers; Aspergillus terreus produces two unprecedented meroterpenoids, namely asperterpenes A and B, 7 showing potent BACE1 inhibitory activities for Alzheimer's disease treatment; Aspergillus sp. TJ23 produces a bridged spirocyclic meroterpenoid, namely spiroaspertrione A, 8 which was found to be a PBP2a inhibitor and act as a potent potentiator of oxacillin against methicillin-resistant Staphylococcus aureus. Inspired by these structurally unexpected natural products with tempting pharmacological activities from the Aspergillus genus, we are devoted to the investigation of Aspergillus species from different origins for chemical and pharmacological diversity.
In our efforts to explore bioactive natural products from marine-derived fungi, 9 we performed a chemical investigation on the fermented rice substrate of a coral-derived fungus Aspergillus terreus, resulting in the isolation of ten butenolide derivatives (1-10), including four new ones (1-4), wherein 1 and 2 were a pair of rare C-8 00 epimers with vicinal diol motifs. Importantly, the NMR data of 5 and 7 were closely similar to those of reported aspernolide D 10 and butyrolactone VI, 11 which inspired us to perform the structure reassignments of reported aspernolides D and G, butyrolactone VI and 4 0 ,8 00diacetoxy butyrolactone VI, as assisted by a combined strategy of experimental validations, 13 C NMR predictions by ACD/Labs soware, and 13 C NMR calculations. Herein, we report the isolation, structure elucidation, structure reassignments, and anti-inammatory activity of these butenolide derivatives (Fig. 1).   , indicating the presence of a mono-substituted benzene motif. These characteristic data suggested that compound 1 was a butenolide derivative. Detailed analysis of the 1D and 2D NMR data of 1 implied that its structural features were closely related to those of the known compound versicolactone B (10), 12 whose absolute structure was conrmed by single-crystal X-ray diffraction analysis, with the only difference that a D 8 00 ,9 00 double bond in 10 was replaced by an oxygenated methine carbon (d C 81.2, C-8 00 ) and an oxygenated tertiary carbon (d C 74.0, C-9 00 ) in 1, as supported via the molecular formula C 24 H 26 O 8 required by its HRESIMS data and the HMBC correlations from H 3 -10 00 to C-8 00 and C-9 00 . The gross structures of 1 and 2 were further dened as shown via 2D NMR analysis, including 1 H-1 H COSY and HMBC spectral data (Fig. 2).
To determine the absolute congurations, the experimental ECD spectra of compounds 1 and 2 were measured in MeOH ( Fig. 3), which were identical to that of versicolactone B, 12 showing positive Cotton effects at approximately 203 and 307 nm and a negative Cotton effect at approximately 230 nm that were ascribed to the conjugated functionality of an a,bunsaturated carboxylic ester motif linked to a benzene group. Thus, the C-4 in 1 and 2 were both dened to be R-congurations. Accordingly, compounds 1 and 2 should be a pair of C-8 00 epimers.
The absolute congurations of 8 00 ,9 00 -diol motifs in 1 and 2 were determined on the basic of in situ dimolybdenum CD method. 13 Compound 1 was mixed with Mo 2 (AcO) 4 in DMSO to provide a metal complex, which showed a negative Cotton effect at approximately 305 nm (Fig. 4), permitting assignment of the 8 00 R-conguration for 1, according to the empirical helicity rule relating the Cotton effect sign of the diagnostic O-C-C-O moiety. 13 Just using the same method like 1, compound 2 showed a positive Cotton effect at approximately 305 nm ( Fig. 4), thus suggesting the 8 00 S-conguration for 2. 14 Therefore, the absolute structures of 1 and 2 were dened and named 8 00 R,9 00 -diol versicolactone B and 8 00 S,9 00 -diol versicolactone B, respectively.   Compound 3 was obtained as a white, amorphous powder. The HRESIMS data showed a sodium adduct ion at m/z 531.1986 [M + Na] + (calcd for C 29 H 32 O 8 Na, 531.1995), indicating a molecular formula of C 29 H 32 O 8 . A direct comparison of its 1D NMR data ( Table 1) with those of 5 indicated that a 1,4-disubstituted benzene motif in 5 was replaced by a 1,2,4trisubstituted benzene group in 3 with an isopentene group positioned at C-3 0 , as supported by the 1 H-1 H COSY correlation of H 2 -7 0 and H-8 0 and HMBC correlations from H 3 -10 0 and H 3 -11 0 to C-8 0 and C-9 0 and from H 2 -7 0 and H-8 0 to C-3 0 (d C 128.4) (Fig. 2). Moreover, the experimental ECD spectrum of 3 was related to those of 1 and 2 (Fig. 3), suggesting a 4R-conguration for 3. Hence, the structure of 3 was dened and named 3 0 -isoamylene butyrolactone IV.
Compound 4, also puried as a white, amorphous powder, was assigned the molecular formula C 24 H 24 O 6 based on HRE-SIMS data at m/z 431.1464 [M + Na] + (calcd for C 24 H 24 O 6 Na, 431.1471). The 1 H and 13 C NMR data of 4 (Table 1) were similar to those of 6, with the only difference being that a 1,4-disubstituted benzene motif in 6 was replaced by a mono-substituted benzene group linked to C-3 in 4, as supported via the 1 H-1 H COSY correlations of H-2 0 /H-3 0 /H-4 0 /H-5 0 /H-6 0 and HMBC correlation from H-2 0 to C-3 (Fig. 2). Furthermore, the experimental ECD spectrum (Fig. 3) of 4 coincided well with those of 1 and 2, suggesting that a 4R-conguration should also exist for 4. Hence, the absolute structure of 4 was dened and named 4 0dehydroxy aspernolide A.
In our screening of anti-inammatory agents from natural products, 21 all the isolates (1-10) were evaluated for inhibitory effects against NO production in RAW264.7 mouse macrophages induced by lipopolysaccharide (LPS) at a concentration of 20 mM, with indomethacin (50 mM) as the positive control. Among them (Fig. 8), the inhibitory effect of compound 10 (***p < 0.001) was even stronger than that of indomethacin. Additionally, compounds 3 and 9 also exerted modest inhibitory effect (*p < 0.05) on NO production with inhibition ratios of nearly 25.1% and 25.3%, respectively. The remaining seven compounds (1, 2 and 4-8) were inactive against NO production.

Conclusions
In conclusion, ten butenolide derivatives (1-10), including four new ones (1-4), were isolated from the coral-derived fungus Aspergillus terreus. Remarkably, compounds 1 and 2 were a pair of rare C-8 00 epimers with vicinal diol motifs, and the absolute congurations of 1-4 were determined via [Mo 2 (AcO) 4 ] induced circular dichroism (ICD) spectra and comparison of their experimental ECD spectra. Importantly, the structures of reported aspernolides D and G, butyrolactone VI and 4 0 ,8 00 -diacetoxy butyrolactone VI have been correspondingly revised via a combined strategy of experimental validations, 13 C NMR predictions by ACD/Labs soware, and 13 C NMR calculations. Remarkably, compounds 3, 9 and 10 showed remarkable inhibitory effects against NO production, of which compound 10, was even stronger than that of indomethacin (a positive control), endowing 10 as a promising lead compound for the development of new anti-inammatory agents. Our ndings in this report not only enrich our knowledge about the chemical and pharmacological diversities of butenolide derivatives in the Aspergillus genus, but also provide a valuable referenced 13 C NMR data (C-7 00 , C-8 00 , and C-9 00 ) for structure elucidations of the butenolide derivatives with 1-(2-hydroxyphenyl)-3-methylbutane-2,3-diol, 2-(2,3-dihydrobenzofuran-2-yl)propan-2-ol, or 2,2-dimethylchroman-3-ol motifs.

Experimental section
General experimental procedures

Fungus material
The fungal strain Aspergillus terreus was isolated from a piece of tissue from the inner part of the so coral Sarcophyton subviride collected from the Xisha Island (16 45

Fermentation, extraction, and isolation
The fungal strain Aspergillus terreus was grown on PDA medium at 28 C for 7 days, which was inoculated statically in 300 Â 500 mL Erlenmeyer asks (each containing 200 g rice and 200 mL water) for 28 days. The whole rice solid medium was extracted seven times in 95% aqueous EtOH at room temperature, and the solvent was concentrated under reduced pressure to afford a total residue, which was then suspended in water and partitioned successfully with EtOAc. The EtOAc extract (1.5 kg) was subjected to silica gel CC eluted with a stepwise gradient of petroleum ether-ethyl acetate-MeOH (10 : 1 : 0, 7 : 1 : 0, Fraction E (186 g) was chromatographed on silica gel CC (CH 2 Cl 2 -MeOH, 1 : 0-50 : 1, v/v) to yield ve main fractions (E1-E5). Fraction E4 (4.6 g) was applied to Sephadex LH-20 using CH 2 Cl 2 -MeOH (1 : 1, v/v), and followed by semipreparative HPLC using CH 3  [Mo 2 (AcO) 4

] induced circular dichroism
[Mo 2 (AcO) 4 ] (1 mg) dissolved in DMSO (1 mL) was prepared as the stock solution, to which compounds 1 and 2 (each 0.5 mg) were added, respectively. The circular dichroism (CD) spectra were recorded immediately aer mixing and scanned every 10 min for 30 min, to afford the stationary [Mo 2 (AcO) 4 ] induced circular dichroism spectra for each compound.

C NMR calculations
The conformations generated by BALLOON were subjected to semiempirical PM3 quantum mechanical geometry optimizations using the Gaussian 09 program. 22 Duplicate conformations were identied and removed when the root-mean-square (RMS) distance was less than 0.5Å for any two geometryoptimized conformations. The remaining conformations were further optimized at the B3LYP/6-31G(d) level in chloroform with the IEFPCM solvation model using Gaussian 09, and the duplicate conformations emerging aer these calculations were removed according to the same RMS criteria above. The number of conformers from the conformational search and nal optimization for compounds 1, 5, and 7 were 400 to 9, 259 to 10, and 160 to 11, respectively. The harmonic vibrational frequencies were calculated to conrm the stability of the nal conformers. The NMR chemical shis were calculated for each conformer at the B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) level with chloroform as solvent by the IEFPCM solvation model implemented in Gaussian 09 program, which were then combined using Boltzmann weighting according to their population contributions.

Anti-inammatory assay
The anti-inammatory activity of compounds 1-10 was assessed by measuring the amount of NO production in LPS-induced RAW 264.7 mouse macrophages (positive control, indomethacin), according to the previously described method. 23

Conflicts of interest
There are no conicts to declare.