Ergosterol is an essential component for fungal growth because it is included in fungal cell membranes and controls membrane fluidity. Antifungal drugs that target ergosterol, such as amphotericin B [1] and nystatin [2] (both polyene drugs), have been widely used to treat serious systemic fungal infections. In particular, amphotericin B has been used clinically for more than five decades due to its potent fungicidal activity, and relatively few examples of drug resistance to this antibiotic have been reported [3]. Amphotericin B has been a mainstay of antifungal therapy, despite there being problems with potential adverse events after intravenous administration and the drug’s poor aqueous solubility [4]. New antifungal drugs will hopefully minimize or overcome these problems and improve antifungal therapy safety.

Based on this concept, we developed silica-binding ergosterol (Si-ES) resin and searched for natural products interacting with ergosterol from secondary metabolites of Actinomycetes. Physico–chemical properties of compounds interacting with Si-ES resin were analyzed by LC-UV and LC/MS as previously described [5]. First, a culture broth including amphotericin B produced by an Actinomycete strain was run through an Si-ES resin column. After washing with water, amphotericin B could be eluted by methanol. Polyenes as well as diketopiperazines and flavonoids without antifungal activity into the culture broths were also found to interact with Si-ES resin (data not shown). In our tests, a compound interacting with Si-ES resin from a culture broth of Amycolatopsis sp. K16-0194 was detected by HPLC (Figure S1). The MS and UV spectra were compared with those of known compounds using an existing database, the Dictionary of Natural Products (http://dnp.chemnetbase.com/), and the compound was deduced to be a new compound. An analog that does not interact with Si-ES resin was also found from the same cultured broth using data of MS analysis and UV spectra. These compounds possess a 2,2′-dipyridine core skeleton and were named dipyrimicin A (1) and B (2).

Si-ES resin was prepared via a two-step reaction: silica gel surface modification with 3-(trimethoxysilyl)propanethiol and thiol-ene reaction of thiolated silica gel with ergosterol. Si-ES resin was manufactured as follows; 20 g of silica gel (Chromatorex MB100-75/200, Fuji Silysia Chemical Ltd., Aichi, Japan) was put in a 500-ml separable flask and 300 ml of toluene was added. The slurry was agitated and reacted following the addition of 5.9 g of 3-(trimethoxysilyl)propanethiol for 8 h at 80 °C under a nitrogen gas atmosphere. After cooling, the reaction mixture was washed with toluene and methanol. The resin was harvested by drying at 80 °C for 12 h. The yield of resin with thiol groups on the surface of silica gel was 21.5 g. Subsequently, 20 g of silica gel with thiol groups was put in a 500 ml separable flask and mixed with 300 ml of 1,4-dioxane. The slurry was agitated with 4.0 g of ergosterol. Subsequently, 80 mg of azobisisobutyronitrile as a radical initiator was added and reacted for 14 h at 85 °C under a nitrogen gas atmosphere. After cooling, the resin was washed with toluene, tetrahydrofuran and methanol. The resin was harvested by filtration and dried at 80 °C for 12 h. The yield of resin with ergosterol on the surface of the silica gel was 20.7 g.

Fermentation, isolation and purification of 1 and 2 are shown in Scheme S1 of the Supplementary Information. Amycolatopsis sp. K16-0194 was cultured at 27 °C for 9 days with rotary shaking in 500 ml Erlenmeyer flasks each containing 100 ml of a production medium. The mycelial cake was extracted with MeOH and concentrated in vacuo. The MeOH extract was subjected to flash column chromatography on Diaion HP-20. The fractions with 1 were collected and concentrated in vacuo. Finally, 1 was purified by ODS flash column chromatography. The yield of 1 was 86.7 mg. The supernatant was extracted with EtOAc and concentrated in vacuo. The EtOAc extract was subjected to flash column chromatography on Si-ES resin. Compound 2 was easily purified using Si-ES resin and the yield of 2 was 16.6 mg.

The Physico-chemical properties of 1 and 2 are shown in Table S1. Compound 1 was isolated as a yellow powder ([α]D25.0 −9.74, c 0.1, MeOH) that gave a [M + H]+ ion at m/z 247.0714 in the HR-ESIMS. This was consistent with the molecular formula C12H10N2O4 (calculated for C12H11N2O4, 247.0719). Compound 2 was isolated as a yellow powder ([α]D25.0 −2.74, c 0.1, MeOH) that gave a [M + H]+ ion at m/z 246.0884 in the HR-ESIMS. This was consistent with the molecular formula C12H11N3O3 (calculated for C12H12N3O3, 246.0879). The 1H and 13C nuclear magnetic resonance (NMR) spectral data of 1 and 2 are shown in Table S2. The 1H NMR spectrum of 2 in DMSO-d6 displayed five olefin protons from 8.96 to 7.55 ppm, one methoxy (δH 3.93), one hydroxyl group (δH 14.9), and one primary amide (δH 8.30 and 7.50 ppm). The 13C NMR spectrum showed the resonances of 12 carbons, which were classified into one carbonyl carbon at 165.9 ppm, ten olefinic and aromatic carbons from 156.9 to 106.0 ppm, and one oxygenated sp3 methyl carbon (δ c 55.8 ppm) by HSQC spectra (Figures S9-S11). As shown in Fig. 1, the 1H–1H COSY of 2 indicated the presence of one partial structure from C-3 to C-6. The 1H-13C HMBC analysis confirmed the presence of a hydroxyl at the 3′ position, a methoxy at the 4′ position and an amide at the 6′ position. Finally, the structure of two pyridines linked to C-2 and C-2′ was confirmed by the HMBC correlation from H-3 to C-2′ (Figure S13) and the 1H-15N HMBC (Figure S14). In addition, the structure of 2 was supported by the chemical shifts of cyanogriside F that are compounds with a 2,2′-bipyridine moiety [6]. The low-field chemical shift (δH 14.9) of the hydroxyl proton at the 3′ position indicates the presence of strong intramolecular hydrogen-bonding of this hydroxyl group with the nitrogen atom, as well as cyanogriside [6] and caerulomycin B [7, 8].

Fig. 1
figure 1

Structures of dipyrimicin A (1) and B (2) isolated from a cultured broth of Amycolatopsis sp. K16-0194

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The NMR chemical shifts (Figures S3-S8) and the physico-chemical properties (Table S2) of 1 were similar to those of 2. The differences between compounds were determined by molecular formula and the HMBC correlation from the amino group of 2 (from δH 8.30 and 7.51 to δc 141.8 ppm). The 1H NMR spectrum of 1 in DMSO-d6 displayed five olefin protons from 8.73 to 7.58 ppm, one methoxy (δH 3.93) and two hydroxyl groups (δH 15.0 and 12.7). The 13C NMR spectrum showed the resonances of 12 carbons, which were identified as one carbonyl carbon at 165.6 ppm, ten olefinic and aromatic carbons from 156.6 to 108.6 ppm, and one oxygenated sp3 methyl carbon (δc 56.0 ppm) by HSQC spectra (Figures S3–S5). As shown in Fig. 1, the 1H-1H COSY of 1 indicated the presence of one partial structure from C-3 to C-6. The structure of two pyridines linked to C-2 and C-2′ was confirmed by the 1H-13C HMBC correlation from H-3 to C-2′ (Figure S7).

Antimicrobial and cytotoxic activities of dipyrimicin A (1) and B (2) were evaluated by the paper disk method and Cell Counting Kit-8 assays (see Supplementary Information S1. Biological activites). As shown in Table 1, dipyrimicin A (1) showed potent antimicrobial activity, except against Aspergillus niger, and was cytotoxic against all cell lines tested. Conversely, dipyrimicin B (2), with an amide group instead of the carboxy group of 1, demonstrated remarkably poor antimicrobial and cytotoxic activity, with the  being active only against Escherichia coli among the eight microbes tested. In addition, the cytotoxic effect of 2 on H1299 (a human non-small cell lung carcinoma cell line) was equal to or more than that of 1. Although these structures were described in the SciFinder® chemical data base as CAS numbers 1235020-43-5 and 1332747-97-3, only structure details are present and no data on physico–chemical properties, NMR chemical shifts, bioactivity, etc. Therefore, we named them dipyrimicin A and B. A number of 2,2′-dipyridine compounds have been discovered as secondary metabolites of Actinomycetes, such as the caerulomycins, including glycosides, from Streptomyces caeruleus [7] and Actinoalloteichus cyanogriseus [6, 9], and the collismycins [10]. However, the 2,2′-dipyridine core structures are the first reported from secondary metabolites in Amycolatopsis species.

Table 1 Biological activities of dipyrimicin A (1) and B (2)
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In conclusion, we isolated dipyrimicin B (2) which interacts with Si-ES resin from the culture broth of Amycolatopsis sp. K16-0194. Dipyrimicin A (1), which does not interact with the resin was also isolated from the same broth. Dipyrimicin A (1) and B (2) contain a unique 2,2′-dipyridine core skeleton. It was strongly suggested that an amide group with 2 facilitates the interaction with the Si-ES resin. Dipyrimicin A (1) showed potent antimicrobial and cytotoxic activities but dipyrimicin B (2) displayed only weak activities. It is suggested that the different in carboxyl and amide groups of dipyrimicins is the primary factor involved in the interaction with Si-ES resin and in bestowing antimicrobial and cytotoxic properties. Although the interaction mechanism between Dipyrimicin B (2) and Si-ES resin is unclear, we are continuing to search for new natural products with antifungal activity using Si-ES resin.