Three New Compounds from the Actinomycete Actinocorallia aurantiaca

Aurantiadioic acids A (1) and B (2), two new furan-containing polyketides, and aurantoic acid A (3), a new natural product, were isolated from the liquid fermentation of the sika deer dung-derived actinomycete Actinocorallia aurantiaca. The structures of the new compounds were established by extensive spectroscopic methods, including 1D & 2D NMR, HRESIMS spectroscopic analysis. The absolute configuration of 3 was assigned by comparison of the specific optical rotations with the reported derivatives. Biological activity evaluations suggested that compounds 1–3 showed weak inhibition on NO production in the murine monocytic RAW 264.7 macrophages with IC50 values of 35.8, 41.8, 45.2 μM, respectively. Compound 3 showed weak inhibition on influenza A virus (A/PuertoRico/8/1934, H1N1) with an EC50 value of 35.9 μM, and a selective index higher than 13.3. Electronic supplementary material The online version of this article (10.1007/s13659-019-00217-0) contains supplementary material, which is available to authorized users.


Introduction
The actinomycetous secondary metabolites, which have attracted great attention from natural product research community in past decades, are considered to be a promising reservoir of new bioactive natural products for drug discovery [1]. The examples of secondary metabolites from actinomycetes, such as streptomycin, actinomycin, tetracycline, rifamycin, vancomycin and mitomycin, etc., have great influence on the treatment of human diseases. The gut microbes from insects have emerged to be fruitful resources for drug leads in recent years [2][3][4]. However, the gut actinomycetes associated with wild animals have long been underexplored for their potential in drug discovery [5].
In this study, we examined the secondary metabolites of the actinomycete Actinocorallia aurantiaca which was isolated from the feces of sika deer. The actinomycete A. aurantiaca belongs to the family Thermomonosporaceae, and it has never been chemically investigated. Herein, we report the isolation, structural elucidation, and anti-NO activity of three compounds from the cultures of A. aurantiaca. Exhaustive analysis of the 2D NMR spectra furnished the establishment of the structure of 1. The 1 H-1 H COSY correlations allowed the connection of C-2-C-3, and C-8-C-9. The HMBC correlations from Me-11 to C-4 (δ C 147.2), C-5 (δ C 129.2), and C-6 (δ C 112.2) indicated the methyl connected to C-5. Furthermore, the trans-olefinic protons (H-2, H-3) correlated to a carboxylic group at δ C 171.2 (C-1), and C-4 in the HMBC spectrum, indicative of the connection of C-1-C-2-C-3-C-4. Besides, the HMBC correlations from two methylene protons (H-8, H-9) to C-7 and C-10 enabled the connection of C-7-C-8-C-9-C-10 ( Fig. 2). Therefore, compound 1 was established to be a furan derivative with two carboxylic groups ( Fig. 1), and was given the name aurantiadioic acid A. Compound 2 was obtained as a yellow oil. The molecular formula of C 11 H 10 O 5 was determined by the (+)-HRESIMS protonated ion peak at m/z 223.06007 [M+H] + , and sodiumadduct ion peak at m/z 245.04192 [M+Na] + , corresponding to seven degrees of unsaturation. The 1 H and 13 C NMR spectroscopic data of 2 (Table 1) showed highly similarities to those of 1, indicating that it was a congener of 1. The presence of an additional carbon double bond at δ C 119.7 (C-9), 131.5 (C-8) of 2 compared to those of 1 was assigned by the HMBC correlations from the trans-double bond protons H-8 and H-9 to C-7 (δ C 153.2) and C-10 (δ C 170.5) (Fig. 2). Thus, the structure of 2 was established as shown in Fig. 1, and was trivially named as aurantiadioic acid B. The yellow oil compound 3, possessed the molecular formula of C 11 H 13 O 5 N as determined by the (+)-HRESIMS sodium-adduct ion peak at m/z 262.06842 [M+Na] + (calcd for C 11 H 13 O 5 NNa, 262.06914), indicating six indices of hydrogen deficiency. The 1 H NMR spectroscopic data of 3 (Table 1)  The HMBC correlations from H-2 to a carbonyl at δ C 169.6 (C-7) indicated the attachment of a carbonyl at C-1. The down-field chemical shifts of C-6 (δ C 160.4) suggested the presence of a hydroxy substituent. Furthermore, the methyl singlet at δ H 1.60 showed HMBC correlations to the hydroxymethyl at δ C 66.1 (C-3′), the quaternary carbon at δ C 62.9 (C-1′), and the carboxylic group at δ C 177.0 (C-2′) suggested the presence of an isolated unit assembled by C-1′ to C-4′. The nitrogen atom was assigned between C-7 and C-1′ based on the chemical shifts of C-7 and C-1′ to satisfy the element composition of the molecular formula. Thus, compound 3 was established as shown in Fig. 1. However, although this compound was recorded in SciFinder database, but there was no literature information available. We herein reported the chemical shifts, and first origin organism of this compound.

Results and Discussion
When examining the structure of 3, it was possibly generated by dehydration of an anthranilic acid and an unusual amino acid 2-methylserine. Since compound 3 harbored a sole chiral center, it was subjected to the chiral-phase HPLC analysis to investigate the optical purity. As depicted in Fig. 3, the analysis result suggested that it presented in enantiomerically pure form. Thus, the absolute configuration of 3 was assigned as 1′S according to the specific optical rotatory

Actinomycete Material
The strain was identified as Actinocorallia aurantiaca by Prof. Shen Qin of Yunnan University. A voucher strain (YIM 111109) was deposited at the School of Pharmaceutical Sciences, South-Central University for Nationalities, China.
The actinomycete A. aurantiaca strain was cultured in 50 500-mL Erlenmeyer flasks with the liquid culture medium consist of glucose 20 g, peptone 2 g, yeast extract 2 g, soluble starch 5 g, K 2 HPO 4 0.5 g, MgSO 4 0.5 g, NaCl 4 g, CaCO 3 2 g in 1 L of deionized water, the pH was adjusted to 7.8 before autoclaving. All flasks were incubated at 25 °C and shaking at 150 rpm for 25 days.

Extraction and Isolation
The total liquid culture (20 L) was evaporated to 5 L, then extracted with EtOAc for four times to obtain a total extract 19.2 g. The crude extract was eluted on MPLC with a stepwise gradient of MeOH/H 2 O (0-100%) to afford eight fractions (A-H).

Nitric Oxide Inhibitory Assay
The procedures of nitric oxide inhibitory assay were similar with those in previously reported literature [7]. Moreover, PDTC (ammonium pyrrolidine dithiocarbamate) was used as positive control in this research.

Viral Replication Inhibition Assay
The viral replication inhibition assay against the influenza virus strain A/PuertoRico/8/1934 (H1N1) was performed with the procedures that similar with those reported in the literature [8].