p-Aminoacetophenonic Acids Produced by a Mangrove Endophyte Streptomyces sp. (strain HK10552)

Four new p-aminoacetophenonic acids, named (2E)-11-(4′-aminophenyl)-5,9-dihydroxy-4,6,8-trimethyl-11-oxo-undec-2-enoic acid (1), 9-(4′-aminophenyl)-3,7-dihydroxy-2,4,6-trimethyl-9-oxo-nonoic acid (2), (2E)-11-(4′-aminophenyl)-5,9-O-cyclo-4,6,8-trimethyl-11-oxo-undec-2-enoic acid (3) and 9-(4′-aminophenyl)-3,7-O-cyclo-2,4,6-trimethyl-9-oxo-nonoic acid (4), were isolated from an endophyte Streptomyces sp. (strain HK10552) of the mangrove plant Aegiceras corniculatum. The structures of 1–4 were elucidated by using spectroscopic analyses. The relative stereoconfigurations of compounds 3 and 4 were determined by NOESY experiments. In the bioassay test, 1–4 showed no cytotoxicity against the Hela cell lines. Compound 4 also showed no inhibitory bioactivity on HCV protease and SecA ATPase and wasn’t active against VSVG/HIV-luc pseudotyping virus.

In general, the production of secondary metabolites is potentially useful for pharmaceutical and agricultural applications [3]. Our previous investigation of endophytic microorganisms from mangrove plants resulted in a number of bioactive and structurally unique metabolites: new pyrrole and indole alkaloids from an endophytic Fusarium incarnatum isolated from A. corniculatum [4], new cyclopentenone derivatives from another endophytic Streptomyces sp. [5], indole triterpenoids [6], polyketides [7], and penicillenols [8] were isolated from endophytic Penicillium sp. associated with A. corniculatum. The indole triterpenoids showed blocking activity on large-conductance calciumactivated potassium channels [6,9].

Results and Discussion
Compound 1 was isolated as a yellow oil and its molecular formula was determined to be C 20 H 29 NO 5 on the basis of HRESIMS analysis (364.21266 [M+H] + , calcd. for C 20 H 30 NO 5 , m/z 364.21185), which indicated seven degrees of unsaturation. In the 1 H NMR spectrum three methyl groups at δ H 0.76 (d, J = 6.36 Hz), 0.80 (d, J = 6.55 Hz) and 0.97 (d, J = 6.32 Hz), and two exchangeable protons at δ 4.35 (brs, OH-9) and 4.54 (brs, OH-5) were detected (See Table 1). Carbon signals of the 13 C-NMR spectrum at δ C 153.9 and 168.0 indicated an olefinic bond, which was supported by the correlation signal of two relevant protons at δ 5.73 (1H, d, J = 15.73 Hz) and δ 6.85 (1H, m) in the 1 H-1 H COSY spectrum (see Figure 2.). This olefinic bond was assigned to be Econfigurated on the basis of its large vicinal coupling constant of 15.73 Hz [11]. The substituted position of the double bond in the aliphatic chain was deduced by the correlation from H-2 to C-4 in HMBC spectrum and the correlation signal between H-3 and H-4 in 1 H-1 H COSY spectrum. An attached carboxylic acid group at the double bond was assigned on the basis of the high-field carboxy signal at δ 168.0 and the HMBC correlation from H-2 to C-1. Quarternary carbon signals at δ 197.3 and 168.0 implied the presence of keto-carbonyl acid groups. The 1 H-1 H COSY correlations confirmed the assembly of the aliphatic chain between H-2 and H 2 -10 and the position of Me-4, OH-5, Me-6, Me-8 and OH-9. Aromatic proton signals at δ 6.55 (2H, d, J = 8.41 Hz), 7.68 (2H, d, J = 8.42 Hz) were attributed to a p-disubstituted phenyl moiety, the assignment of which was supported by carbon signals at δ 125.7 (C-1′), 131.0 (C-2′), 112.9 (C-3′), and 153.9 (C-4′) in the 13 C-NMR spectrum. One of the substituted groups of the aromatic ring was 4´-NH 2 (6.02, s), which was deduced from the HMBC correlation between C-3´ and 4´-NH 2 (6.02, s) and the aminophenonic moiety was linked to C-11 as deduced from the HMBC correlation between H-2´ and C-11 (see Figure 2). Thus, compound 1 was determined as (2E)-11-(4-aminophenyl)-5,9-dihydroxy-4,6,8-trimethyl-11-oxo-undec-2-enoic acid (1).   5 , m/z 338.19620), which indicated the molecule with six degrees of unsaturation. Comparison of 1 H-and 13 C-NMR data of 2 with those of 1 showed that 2 was a homologue of 1 with the olefinic bond missing. The newly found HMBC correlations from H-2 (2.36, m) to C-1 (176.5, s) confirmed the suggested structure. Therefore, 2 was elucidated to be 9-(4-aminophenyl)-3,7-dihydroxy-2,4,6-trimethyl-9-oxo-nonoic acid (2).
Compound 3, a yellow oil, was determined to be C 20 H 27 NO 4 on the basis of its HRESIMS data (346.20109 [M+H] + , calcd for C 20 H 28 NO 4 , m/z 346.20128), requiring eight degrees of unsaturation. According to the comparison of mass spectral data between 3 and 1, there is 18 amu missing suggesting that 3 was the product of dehydration-condensation from 1. Compound 3 contained similar structural elements as 1, expect for the two missing hydroxyl groups, as observed in the 1 H-NMR spectrum. The carbon signals at δ 29.3 (d, C-6), 30.2 (d, C-8) were shifted remarkably to highfield by 4-8 ppm as compared with 1, while signals at δ 82.8 (d, C-5), 81.7 (d, C-9) were moved downfield for 6-9 ppm, indicating that the attached two hydroxyl groups of 1 were condensed to form a pyran-ring in In cytotoxicity testing against Hela cells compounds 1-4 showed no cytotoxicity (IC 50 > 100 μg/mL). In further testing, compound 4 proved to be inactive against HCV protease and SecA ATPase and did not inhibit VSVG/HIV-luc pseudotyping virus on 293T cell line.
Compounds 1 and 2 show certain structural similarity to the polyene antibiotics, e.g. candicidin produced by Streptomyces griseus, which consists of the aromatic moiety, the macrolide ring and the amino sugar moiety. Compounds 1 and 2 have different polyketide chain lengths which may be due to different biosynthetic polyketide assembly procedures. It was reported the the p-aminoacetophenone moiety of candicidin is synthesized from chorismic acid via the aromatic amino acid pathway and paminobenzoic acid (PABA) has been identified as the immediate precursor [12]. The isolation of 4methylaminobenzoic acid from the crude extract in our study suggested the presence of this biosynthetic pathway Streptomyces sp. (strain HK10552).
Polyene macrolide antibiotics are the most effective antifungal agents due to their potent broad spectrum fungicidal activity and relatively low frequency of resistance among the fungal pathogens [13]. Therefore, the novel derivatives of polyene antibiotics discovered in our study would be a potential target for further chemical and microbiological investigations in this respect.

General
1 H-and 13 C-NMR spectra were measured on a Bruker Avance DRX 500 spectrometer using TMS as an internal standard. Chemical shifts (δ) expressed in parts per million (ppm) and coupling constants (J) are reported in Hertz (Hz). Optical rotations were recorded on a Perkin-Elmer 341 LC polarimeter. ESIMS spectra were measured on a Quattro. Premier XE tandem mass spectrometer (Micromass, UK), while HRESIMS spectra were measured on a LTQ Orbitrap X1 Thermo Scientific and a FT-MS-Bruker APEX IV (7.0T).Column chromatography was carried with silica gel (200-300 mesh), and GF 254 Silica gel for TLC was provided by Qingdao Marine Chemistry Co.. Semipreparative HPLC was performed on an Alltech-HPLC (USA) using a Kromasil column (ODS, 10 μm, 10 × 250 mm). The chemical reagents used for chromatography were purchased from Beijing Chemical Works Co. Ltd. (Beijing).

Strain Isolation, Characterization, and Cultivation
The strain was isolated from the mangrove plant A. corniculatum. Pieces of the leaves were rinsed with sterile water, sterilized by soaking in 70% ethanol (1 min) and 3.5% NaCl solution (3 min), and rinsed again with sterile water. Tiny pieces obtained by cutting with a sterile knife were placed on agar plates with GPY agar, which was supplemented with streptomycin (0.1 g/L) and chloramphenicol (0.2 g/L), and incubated at 22 ºC until growth appeared. Colonies were transferred to fresh agar plates for further growth. General laboratory cultivation was performed on malt agar [malt extract 20.0 g/L, yeast extract 2.0 g/L, glucose 10.0 g/L, (NH 4 ) 2 HPO 4 0.5 g/L, pH 6.0] or the respective liquid medium at 22 ºC. For long-term preservation, cultures grown on agar plates supplemented with 5% glycerol were maintained in the vapor phase of liquid nitrogen. The strain isolate has been deposited in the strain collection of the Leibniz Institute for Natural Products Research and Infection Biology.
For screening purposes, the strain was grown in 250 mL Erlenmeyer flasks with 100 mL of the production culture medium, consisting of saccharose 20 g/L, soybean flour10 g/L, cornsteep 10 g/L, and KCl 8 g/L, adjusted to pH 6.5 prior to sterilization. It was inoculated with pieces (1 × 1 cm 2 from an agar plate of a well grown culture and cultivated as resting culture for 17 days at 22 ºC. The production culture was carried out in a 300 L fermentor filled with 200L of the above production medium; it was grown for 10 days at 22 ºC (aeration 50 L/min, pH 6.5) and stirred at 125 rpm. The inoculum (2 L) was obtained after several steps of resting cultures with increasing cultivation volume in a modified malt extract medium [malt extract 10.0 g/L, yeast extract 4.0 g/L, glucose 4.0 g/L, (NH 4 ) 2 HPO 4 0.5 g/L, pH 5.5].