Nidulin (1), nornidulin (2), and related depsidones are secondary metabolites specifically produced by fermentation of the common fungi Aspergillus unguis and Aspergillus nidulans (Fig. 1). There have been several reports regarding biological activities of nidulin and closely related Aspergillus depsidones. Unguinol (2,4,7-trisdechloro-nornidulin) was reported to be a potential herbicide candidate due to its inhibitory activity of the C4 plant enzyme pyruvate phosphate dikinase [1]. Unguinol and its derivatives are claimed to be animal growth permittants in a US patent [2]. Kittakoop and co-workers reported aromatase inhibitory, radical scavenging, and cytotoxic activities of nidulin and co-metabolites, aspergillusidones, from A. unguis CRI-282-03 [3, 4]. Recently, antibacterial, antifungal, antimalarial, and cytotoxic activities of depsidones and depsides from A. unguis have been reported [5, 6]. During the preparation of this paper, a report on isolation of many depsidones from A. unguis and evaluations of their activities against Gram-positive bacteria was published [7].

Fig. 1
figure 1

Structures of nidulin and nornidulin

Several years ago, we isolated nidulin, nornidulin and three related depsidones from cultures of a mangrove-derived fungus Halosarpheia kandeliae BCC 16551. However, based on the checking of the culture collection deposit of the strain, and the failure of the reproduction by new fermentations, it was later concluded that these depsidones were most likely produced due to the contamination of Aspergillus unguis into a seed culture in the large-scale fermentation process. Evaluation of biological activities of the isolated depsidones revealed that nidulin exhibits activity against Gram-positive bacteria, Bacillus cereus (MIC 1.56 μg ml−1) and Enterococcus faecium (MIC 3.13 μg ml−1). Nornidulin showed weaker activity when compared with nidulin. As preliminary study on structure-activity relationship (SAR), we examined preparation of 3-O-methyl-nornidulin by alkylation of 2 with one equivalent of MeI (K2CO3, DMF, room temperature) expecting the production of a mixture of 3-O-methyl-2 and 8-O-methyl-2 (nidulin, 1). Interestingly, the O-methylation underwent in a highly regioselective manner giving only nidulin (1). 3-O-Methyl-nidulin, prepared by O-methylation of 1 with excess MeI, was inactive against these bacteria, which suggested that 3-OH group is crucial for antibacterial activity. On the basis of these preliminary results on SAR and regioselectivity of O-alkylation, we had planned semisynthesis of nidulin derivatives by selective alkylation of nornidulin 8-OH to evaluate the potential of the skeleton as antibacterial agents. We report here synthesis of various nidulin derivatives and evaluation of their antibacterial activity.

As substrate for the semisynthesis, nornidulin was produced by fermentation of the commercial strain, A. unguis ATCC 10032, which is a known to producer of nidulin derivatives. Fermentation using a common media, potato dextrose broth (PDB), under static condition gave nornidulin but as a minor metabolite with unguinol as the major product. This result was probably due to the lack of chloride ion source in the liquid culture media. Similarly to the fermentation of a marine-derived A. unguis CRI-282-03 [3, 4], feeding NaCl worked fruitfully for ATCC 10032. Thus, addition of 2% (w/v) NaCl in PDB resulted in the enhanced production of nornidulin and nidulin as two major metabolites, while production of unguinol and other dechloro derivatives were reduced. Higher composition of NaCl (5%) resulted in serious inhibition of the mycelial growth. Gram-quantity of nornidulin was obtained using this simply optimized fermentation conditions and chromatographic separation/purification.

Reaction of nornidulin with one equivalent of alkyl, allylic, or benzylic halide (R-X; X = I, Br, Cl) and K2CO3 (2 equiv) in DMF at room temperature underwent in highly regioselective manner to give 8-O-substituted derivatives (3–17) (Fig. 2). Regioselectivity of the O-alkylation was confirmed by the NOESY correlation between 9-CH3 and H2-1ʺ for compound 5, and by comparison of the 1H and 13C NMR spectroscopic data of the alkylated compounds with those of nidulin and nornidulin. A dimeric nidulin derivative 18 was synthesized by the reaction of 2 with a half equivalent of 1,4-diiodobutane. Nidulin derivatives with a pyridine ring (19 and 20) or a hydroxyl group (21) were also synthesized using the same procedure. A tert-butyl ester derivative 22 was converted into a carboxylic acid derivative 23 by deprotection with trifluoroacetic acid. Two 8-O-acylated derivatives, 24 and 25, were obtained by the reaction of 2 with acetic anhydride and benzoyl chloride, respectively. Several nornidulin 8-O-aryl ethers (26–31) were synthesized by the reactions of 2 with 1 equivalent of fluorobenzenes bearing an electron-withdrawing group (–NO2 or –CN) and K2CO3 (2 equiv), heating at 60 °C. Regioselectivity of the O-arylation was confirmed by the NOESY correlation between 9-CH3 and H-2ʺ/H-6ʺ for compound 28, and by comparison of the 1H and 13C NMR spectroscopic data of the O-arylated compounds with those of nidulin and nornidulin. Similarly, a pyridyl ether derivative 32 was synthesized from 2 and 2-chloro-5-nitropyridine.

Fig. 2
figure 2

Structures of the semisynthetic nidulin derivatives

The semisynthetic nidulin derivatives were tested for activities against Gram-positive bacteria, B. cereus ATCC 11778, E. faecium ATCC 51559, and Staphylococcus aureus ATCC 29213 [8], and cytotoxicity to nonmalignant Vero cells (African green monkey kidney fibroblasts) [9] (Table 1). Most of the 8-O-alkyl derivatives with short side-chain showed more potent antibacterial activity than nidulin. Among these derivatives, compound 5 (8-O-butyl) exhibited highest activity against B. cereus (MIC 0.391 μg ml−1). Alkyl derivatives with long side chain, 8 and 9, showed much weaker antibacterial activity. It should also be noted that certain level of steric bulkiness of the alkyl group may be accepted (15, 16, and 22). On the other hand, weak antibacterial activities of compounds 19–21, and 23 suggested that polar functional group on the alkyl side-chain significantly reduces the activity. Acylated derivatives (24 and 25) exhibited weaker activity. All of the O-aryl derivatives (26–32) showed potent antibacterial activity.

Table 1 Antibacterial activities and cytotoxicity of nidulin derivatives

To further demonstrate the antibacterial potential, nidulin (1, lead compound) and five high-activity derivatives were selected and tested for activity against Methicillin-resistant S. aureus SK1 (MRSA) [10] by one of the authors (S.P.). In particular, compounds 26, 30, and 31, were significantly active also against MRSA (MIC 0.5 μg ml−1).

Many of the synthetic compounds displayed weak to moderate cytotoxicity to Vero cells. Since the SAR pattern for cytotoxicity was different from that for antibacterial activities, they could potentially be distinguishable through synthetic design.

In conclusion, the present results demonstrate a possibility of the Aspergillus depsidone (nidulin) as a core structure for antibacterial agents. In particular, 8-O-aryl ether derivatives have been shown to exhibit significant activities against Gram-positive bacteria, including MRSA.