Introduction

In the course of our screening for apoptosis inducers in Ras-dependent Ba/F3-V12 cells, we isolated ammocidin A (formerly named ammocidin) from the culture broth of Saccharothrix sp. AJ9571.1, 2 Ammocidin A induced apoptotic cell death in Ras-dependent Ba/F3-V12 cells with an IC50 of 66 ng ml−1, and no cell death was observed in IL-3-dependent Ba/F3-V12 cells at less than 100 μg ml−1 of ammocidin A. Further searching for congeners from the ammocidin-producing strain resulted in the isolation of ammocidins B, C and D (Figure 1). In this paper, we report the isolation, structure elucidation and cytotoxic activities of the ammocidins.

Figure 1
figure 1

Structures of ammocidin A, B, C and D.

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Results

Production and isolation

The seed medium was composed of yeast extract 0.1%, beef extract 0.1%, N-Z amine type A 0.2% and glucose 1.0% (pH 7.3). Saccharothrix sp. AJ9571 was cultured in flasks containing the seed medium on a rotary shaker at 30 °C for 4 days. The resultant seed culture at 2.0% was transferred into 500-ml Erlenmeyer flasks containing 100 ml of a production medium consisting of glycerol 2.0%, starch 1.0%, rape seed meal 2.0%, soytone 0.3% and calcium carbonate 0.3% (pH 7.0). The fermentation was carried out on a rotary shaker at 30 °C for 7 days. The culture filtrate (10 l) was extracted with ethyl acetate and the extract was subjected to silica gel column chromatography with chloroform–methanol (8 : 1). The active eluate was chromatographed on a Sephadex LH-20 column with methanol. The active fraction was purified by HPLC using a Senshu Pak PEGASIL ODS column with 70% methanol. Further purification was carried out on the same column with 33% acetonitrile to give ammocidins A (387 mg), B (24 mg), C (15 mg) and D (24 mg).

Physico-chemical properties

The physico-chemical properties of ammocidins B, C and D are summarized in Table 1. The molecular formulae of ammocidins B, C and D were determined by high-resolution electrospray ionization mass spectra to be C60H98O22, C53H86O18 and C46H74O16, respectively. The IR spectra indicated the presence of hydroxyl (3430 cm−1) and conjugated carbonyl groups (1670–1720 cm−1). The UV spectra of ammocidins B, C and D are characterized by a maximum absorption around 320 nm.

Table 1 Physico-chemical properties of ammocidin B, C, and D
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Structure elucidation

The structures of ammocidins B, C and D were established by 1H- and 13C-NMR and two-dimensional NMR experiments (Correlation Spectroscopy (COSY), Heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple-bond correlation (HMBC)) on a 600-MHz spectrometer. The 1H and 13C-NMR assignments of ammocidins B, C and D in CD3OD are listed in Table 2.

Table 2 13C- and 1H-NMR data for ammocidin B, C and D in methanol-d4
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The 1H-NMR spectrum of ammocidin B showed the presence of 12 methyl groups between 1.10 and 2.08 p.p.m. and four methoxy groups between 3.24 and 3.53 p.p.m. The 13C-NMR spectrum confirmed the presence of 60 carbons. 1H spin networks from 7-H to 9-H, from 15-H to 20-H, from 22-H to 23-H and from 25-H to 28-H, and 1H–13C long-range correlations as shown in Figure 2 revealed the aglycone structure of ammocidin B, which is the same as ammocidin A. Moreover, a 1H spin network from an anomeric proton (1′-H) to 6′-H and an heteronuclear multiple-bond correlation between 1′-H and C-5′ revealed the presence of 6-deoxyglucose, which is the same component as that of ammocidin A. Two 2,6-dideoxy-3-methylpyranose moieties were constructed by 1H spin networks from an anomeric proton 1″-H (1′″-H) to 2″-H (2′″-H) and from H-4″ (H-4′″) to 6″-CH3 (6′″-CH3), and heteronuclear multiple-bond correlation from 3″-CH3 (3′″-CH3) to C-2″ (C-2′″), C-3″ (C-3′″) and C-4″ (C-4′″). Vicinal proton coupling constants (Table 2) and an NOE between 1″-H (1′″-H) and 3″-CH3 (3′″-CH3) identified these sugars as β-olivomycose. The three glycoside linkages were formed on the basis of long-range couplings between 1′-H and C-9, between 1″-H and C-24, and between 1′″-H and C-4″. These data indicate that ammocidin B is an ammocidin A derivative containing the second β-olivomycose residue in place of the β-digitoxose residue (Figure 1).

Figure 2
figure 2

Key correlations in 2-D NMR of ammocidin B.

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The 1H- and 13C-NMR spectra of ammocidin C were similar to those of ammocidin B, although signals due to the terminal olivomycose residue (C-1′″–C-6′″) were absent in ammocidin C. In the aglycone, signals indicative of a methylene group (δC 32.2, δH 2.58, 1.98) appeared in place of those for the oxymethine (C-16) in ammocidin B. A 1H spin network from 15-H to 20-H identified ammocidin C as a 16-deoxyaglycone metabolite. The molecular formula and the upfield shift of C-4″ (δC 90.3 in ammocidin B and δC 80.1 in ammocidin C) indicated that ammocidin C is the 16-deoxy-4″-O-deolivomycosyl derivative of ammocidin B. Finally, two-dimensional NMR analyses established the planar structure of ammocidin C as shown in Figure 1.

The 1H- and 13C-NMR spectra indicated that ammocidin D consists of the same aglycone as ammocidin B and 6-deoxyglucose as a sole sugar moiety. The molecular formula of ammocidin D and the upfield shift of 24-C (δC 82.2 in ammocidin B and δC 75.0 in ammocidin D) showed the loss of two olivomycose residues from ammocidin B. The planar structure of ammocidin D was confirmed as shown in Figure 1 by two-dimensional NMR data.

Cytotoxic activities of ammocidins

To determine the cytotoxic activities of the ammocidins, A549 human lung carcinoma cells, MCF-7 human breast carcinoma cells and HCT116 human colon carcinoma cells were tested. The cell proliferation assay showed potent cytotoxic activities of ammocidins A and B (Table 3). The IC50’s of ammocidins A and B were 0.06–0.4 μM.

Table 3 Anti-proliferative activities of ammocidins against human cancer cell lines
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Discussion

During our efforts to obtain congeners from the ammocidin-producing strain, we isolated new ammocidin derivatives, ammocidins B, C and D. The main structural differences of these compounds are the number of olivomycose residues attached to 24-O. Whereas ammocidin A contains a digitoxose residue and an olivomycose residue, the sugar chain of ammocidin B consisted of two olivomycose residues. The ammocidins are considered to be good tools to investigate the importance of sugar moieties for the biological activity of 20-membered macrolides.

We evaluated the anti-proliferative effects of ammocidins on human cancer cell lines. Although all ammocidins showed cytotoxic activities, ammocidins A and B were more potent than ammocidins C and D. Thus, deoxysugar moieties attached to 24-O play an important role in the anti-proliferative activities of ammocidins. Human cancer cell lines A549 and HCT116 harbor oncogenic ras mutation (A549: K-ras-G12S, HCT116: K-ras-G13D). MCF-7 cells, however, have no ras mutation (but harbor PI3KCA mutation). Ammocidins showed potent anti-proliferative activities against all the three cell lines, implying that their anti-proliferative activities might not be due to Ras inhibition. Further biological studies on ammocidins are in progress.

Methods

Cell culture

The cancer cell lines A549 (lung adenocarcinoma), MCF-7 (breast adenocarcinoma) and HCT-116 (colon carcinoma) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 °C under 5% CO2 atmosphere.

Assay of anti-proliferative activity

Anti-proliferative activities were measured by using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega Corporation, Madison, WI, USA), which determines the number of viable cells in a culture based on quantitation of ATP. In brief, cancer cells were seeded at 1 × 103 cells per well in 96-well microplates and cultured overnight. The cells were treated with various concentrations of ammocidins for 48 h. After 50 μl per well of CellTiter-Glo Reagent was added to the cell culture, the luminescence was measured by a ARVO.sx multilabel counter (Wallac Berthold, Turku, Finland). IC50 values were determined from the dose–response curves of growth inhibition.

General experimental procedures

NMR spectra were obtained on a Varian NMR System 600 NB CL (Varian NMR Systems, Palo Alto, CA, USA). Methanol-d4 was used as a solvent and an internal reference. Melting points were determined with a Yanagimoto (Seisakusyo, Japan) micro melting point apparatus. High-resolution electrospray ionization mass spectra were recorded on a Waters LCT-Premier XE mass spectrometer (Waters, Milford, MA, USA). Optical rotations were recorded on a HORIBA SEPA-300 polarimeter (Horiba, Tokyo, Japan). UV spectra were obtained on a HITACHI U-3200 spectrophotometer (Hitachi, Tokyo, Japan). IR spectra were obtained using a HORIBA FT-720 spectrophotometer.