A new furan carboxamide and two potential precursors from a terrestrial streptomycete

1 Introduction

Streptomyces spp. are widespread in nature and play a significant role in the production of bioactive metabolites [1], [2], [3]. Many of their secondary products are having a great bio-functional diversity, e.g. as antibiotics, antifungal, antiviral, anticancer, immunosuppressant agents, insecticides, herbicides, etc., and are playing an important role as potentially useful pharmacological and agricultural agents [4], [5], [6], [7], [8], [9], [10].

Extracts of the terrestrial Streptomyces sp. strain ANK245 exhibited several diversely staining bands (dark red to brown) on thin layer chromatography (TLC) after spraying with anisaldehyde-sulfuric acid and heating. A large-scale fermentation of the strain and separation of its extract using a series of chromatographic techniques led to the isolation of three new products, 1,6-dihydroxy-2-methyl-heptan-4-one (1), 4-hydroxy-1-(2-methyl-oxiranyl)-pentan-2-one (2) and 2-(2-hydroxy-propyl)-4-methylfuran-3-carboxylic acid amide (3) (see Fig. 1). Additionally, p-vinylanisol (4a), p-vinylphenol (4b) and 2-phenylethanol were isolated. In the non-polar part of the mycelium extract, tetradecanoic acid, 9-octadecenoic acid, hexadecanoic acid, 2-methoxy-4-vinylphenol (4c), 4-hydroxy-3-methoxy-benzaldehyde, o-hydroxybiphenyl, p-vinylphenol (4b), 1,5,9-trimethyl-4,8,13-cyclotetradecatrien-1,3-diol (5), and 1,1′-(p-chloro)-bisphenyl sulfone (a widespread pollutant) were identified by gas chromatography/mass spectrometry (GC-MS). The structures of the new compounds 1–4 were solved using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry [chemical ionization (CI), electron ionization (EI), electrospray ionization (ESI) and ESI-HRMS].

Fig. 1:

Structural formulae of compounds 1–5.

2 Results and discussion

Compound 1 was obtained as a moderately polar colorless oil, which was not UV absorbing or fluorescent, but developed on TLC with anisaldehyde-sulfuric acid a dark green color, later changing to dark blue. Pseudomolecular ions of 1 were found by CI-MS and (+)-ESI-MS at 160 [(M−H₂O)+NH₄]⁺ and 183 Dalton [M+Na]⁺, respectively, and the molecular formula was established as C₈H₁₆O₃ by ESI-HRMS of the [M+Na]⁺ ion, indicating one double bond equivalent (DBE) (Table 1).

The ¹³C NMR spectrum displayed eight signals, which were identified by their HSQC data as two methyls, three methylene and two methine groups, and one carbonyl; according to the shifts, one methylene and methine group was hydroxylated. The ¹H NMR and COSY data of 1 revealed the existence of two separate spin systems, identified as a 2-hydroxypropyl (–CH₂–CHOH–CH₃) and a 3-hydroxy-2-methyl-propyl fragment (HO–CH₂–CH(CH₃)–CH₂–). According to the number of carbon atoms and the empirical formula, it is evident that these fragments are connected via a remaining carbonyl group. This was confirmed by the ¹³C NMR data and the HMBC couplings of the carbonyl with the methylene protons CH₂-3 and CH₂-5 and also the methines CH-2 and CH-6 (Fig. 2). Based on these data, 1 was identified as 1,6-dihydroxy-2-methyl-heptan-4-one, a compound, which had not been described before [1], [2], [11].

Fig. 2:

HMBC (→) and H,H COSY (—) connectivities of 1,6-dihydroxy-2-methylheptan-4-one (1).

A closer inspection of the ¹³C NMR spectrum showed that all carbons were represented by a narrow pair of signals in the ratio of about 0.83:1, so that a mixture of diastereomers was expected. Nevertheless, the determination of the configuration was possible by means of Mosher derivatives (see Table S1 in the Supporting Information) [12]. For a mixture of the rel-(2R/S, 6R) diastereomers, only one doublet (or a close pair of doublets) should be visible for Me-7, which would give a positive Δδ_(S−R) value for the (6R) diastereomer, while for the two (2R/S)-Me signals a negative difference is expected: this is the case for the mixture of diastereomers isolated here (see Figure S7 in the Supporting Information). In contrast, for rel-(2R, 6S/R)-1, two separated Me-7 signals must be visible, one with a positive, the other one with a negative (δ_(S)–δ_(R)) value, which is not found here. This result is confirmed by the carbon shift difference of the signal pairs: while for C-4 to C-8 shift differences between 0.00 and 0.06 ppm are observed, these differences are substantially higher for C-1 (0.07), C-2 (0.25), and C-3 (0.20), so that the epimerisation had certainly occurred at C-2, and 1 has the absolute (2R/S, 6R) configuration. This result was confirmed by the density functional theory (DFT)-calculated optical rotation of – 93° for a 1:1 mixture of the (2R/S, 6R) diastereomers, which is therefore unequivocally identified as (2R/S, 6R)-1,6-dihydroxy-2-methyl-heptan-4-one.

A compound 2, closely related to 1, was obtained as higher polar yellow oil that gave an orange coloration with anisaldehyde-sulfuric acid, which later turned to violet. No UV activity was visible, an indication for the absence of aromatic and unsaturated system. The molecular weight was determined as 158 Dalton by CI- and ESI-MS, and ESI-HRMS deduced the molecular formula as C₈H₁₄O₃, corresponding to two DBEs (Table 1).

The ¹H and HMQC spectra of 2 (Table 2) revealed a high similarity with the previous compound, and a 2-hydroxy-n-propyl group was identified as in 1 (Fig. 1). In addition, an isobutyl fragment was found; the signal of 2-Me appeared here, however, as a singlet at δ=1.20 ppm, along with a 2H singlet at δ=3.44 ppm, corresponding to an oxygenated methylene. The remaining methylene protons (H₂-3) appeared at δ=2.80 (dd, 16.0, 9.2 Hz) and 2.56 ppm (m), respectively. In accordance, the sole difference between compounds 2 and 1 was attributed to the dehydrogenation during a ring closure between C-1 and C-2 in 1 via oxygen, giving an oxirane ring in 2. According to this conclusion, the methine carbon C-2 in 1 (δ=31.9 ppm) had changed into a quaternary oxygenated carbon at δ=72.6 ppm. Correspondingly, the methyl doublet of 2-CH₃ in 1 (δ=0.87 ppm) appeared here as singlet (δ_H=1.20, δ_C=24.3 ppm), where the downfield shift indicated its neighborhood to the oxygenated C-2. The structure of 2 was finally confirmed by HMBC correlations (Fig. 3), showing for the central 4-CO (δ=212.6 ppm) the same correlations from both sides. So, the structure of 2 was finally elucidated as 4-hydroxy-1-(2-methyl-oxiranyl)-pentan-2-one, a new natural product. Also here, the ¹³C NMR spectrum indicated a mixture of diastereomers. A further determination of the configuration as for 1 was, however, not possible due to rapid decomposition. If the structural similarity of 2 with 1 and 3 is reflecting also a biosynthetic relation, C-6 in 2 may be (R)-configured as well, and C-2 is racemic.

Fig. 3:

HMBC (→), and H,H COSY (—) correlations of 4-hydroxy-1-(2-methyl-oxiranyl)-pentan-2-one (2).

Compound 3 was obtained as colorless solid of moderate polarity, showing UV absorbance at 254 nm and a pink color with anisaldehyde-sulfuric acid. The UV spectrum of 3 showed an intense peak at λ_max=242 nm, indicating a conjugated system, other than in 1 and 2 (Table 1). The molecular weight of 3 was determined as 183 Dalton by ESI-MS, indicating the existence of an odd number of nitrogen atoms in the structure. The corresponding molecular formula was derived as C₉H₁₃NO₃ by HRESI-MS, which indicated four DBEs. On EI-MS, the parent molecule lost a fragment with a mass of 44 amu, which was identified by EI-HRMS as a carboxamido group (CONH₂) and confirmed by a ¹³C NMR signal at δ=167.2 ppm.

The ¹H NMR spectrum of 3 displayed a 1H quartet at δ=7.09 ppm (J ~ 1.1 Hz), pointing to a long-range coupling via a double bond with the doublet at δ=2.13 ppm (J ~ 1.2 Hz) of an sp²-bound methyl group. Two broad 1 H signals due to the amide group were visible at δ=7.24 and 6.19 ppm. In the aliphatic region, three signals were visible: a methyl doublet at δ=1.26 ppm (J ~ 7 Hz) and a methylene multiplet (δ=2.95 ppm) both gave COSY correlations (Fig. 4) with the oxymethine multiplet at δ=4.14 ppm, so that a 2-oxypropyl fragment was established as in 1 and 2.

Fig. 4:

HMBC (→) and H, H COSY (—) correlation of 2-(2-hydroxy-propyl)-4-methylfuran-3-carboxylic acid amide (3).

In addition to signals of the methyl, carboxamido and propyl fragments as discussed above, four olefinic carbons were identified in the ¹³C NMR spectrum (Table 2), an oxymethine carbon (δ=138.4 ppm) and three quaternary atoms. One of the latter was also oxygenated (δ=155.9 ppm), the other two were found at δ=119.6 and 118.6 ppm. Based on the remaining oxygen and the left DBE, compound 3 should be a trisubstituted furan, decorated with a methyl, a carboxamido, and a 2-hydroxy-propyl group.

In the HMBC spectrum, the sp² methine (δ_Η= 7.09/δ_C=138.4 ppm) and the propyl protons CH₂-6, and CH-7 all coupled with the low-field carbon at δ=155.9 ppm; consequently, the latter signal was assigned as C-2, and the methine as C-5 of the furan ring (Fig. 4). Among the atoms seen by 4-CH₃, only that at δ=118.6 ppm showed also a cross signal with CH₂-6. The resulting structure 3 of 2-(2-hydroxy-propyl)-4-methylfuran-3-carboxamide was confirmed unequivocally by further HMBC correlations (Fig. 4).

In contrast to compounds 1 and 2, the furan 3 gave a CD spectrum (Fig. 5) of suitable quality to determine the absolute configuration. As for previous natural products [13], CD and ORD data were calculated with DFT methods using Spartan’14 [14] and Gaussian 09 [15] (see Section 3). The CD spectrum obtained for (R)-3 (Fig. 5) agreed sufficiently well with the experimental data. This result was confirmed by the optical rotation, which delivered a Boltzmann-weighted value of –35° at 589 nm, while –42° was measured in methanol (Table 1).

Fig. 5:

Experimental (bold line, MeOH) CD spectrum of 2-(2-hydroxy-propyl)-4-methylfuran-3-carboxylic acid amide (3), and CD data calculated for (R)-3 (dotted line).

For an additional control of the ab initio results, we also synthesized the Mosher derivatives (see Table S1 in the Supporting Information) [12]. The ¹H NMR shift differences between the (S)- and (R)-MTPA esters resulted in a positive Δδ_(S−R) value for the methyl protons at C-8, and negative values for the remaining protons on the other side of the molecule (Fig. 6). All the results above assigned the configuration of 3 unequivocally as (7R).

Fig. 6:

NMR shift differences Δδ_(S−R) of the MTPA esters of 1 and 3; the data were measured in CDCl₃ at 600 MHz. Due to the mixture of diastereomers, for some atoms of 1, two differences were obtained.

Furan containing compounds (e.g. albafuran A and roseophilin) are widespread in nature and are exhibiting diverse biological activities [1], [2], [16], [17], [18], [19], [20]. The physico-chemical properties of the new compounds (1–3) are listed in Table 1.

Three further compounds, 4-vinylanisole (4a), phenethyl alcohol [21] and p-vinylphenol (4b) [22], [23] were identified by NMR and MS, and by comparison with literature data; 4a was previously obtained by synthesis, but is a new natural product. GC-MS analysis of the nonpolar extract of the mycelia indicated further the presence of tetradecanoic acid, hexadecanoic acid, 9-octadecenoic acid, 2-methoxy-4-vinylphenol (4c) [24], 4-hydroxy-3-methoxybenzaldehyde, o-hydroxybiphenyl, 1,5,9-trimethyl-4,8,13-cyclotetradecatrien-1,3-diol (5, tentatively), and 1,1′-(p-chloro)-bisphenyl sulfone, a widespread pollutant (Table 3).

The crude extract and the isolated compounds 1–3 were tested against Bacillus subtilis, Staphylococcus aureus, Streptomyces viridochromogenes (Tü 57), Escherichia coli, Candida albicans, Mucor miehei, Chlorella vulgaris, Chlorella sorokiniana, Scenedesmus subspicatus, Rhizoctonia solani and Pythium ultimum, using the agar diffusion method with paper disks (40 μg per disc). No activity was found for the extract and the isolated metabolites. Compounds (1–3) exhibited, however, weak brine shrimp toxicity (3%–8.3%, 10 μg mL⁻¹, Table 4).

3 Experimental section

4 Supporting information

NMR spectra and other supplementary data associated with this article are given as supporting information available online (DOI: 10.1515/znb-2016-0202).