Two new lankacidin-related metabolites from Streptomyces sp. HS-NF-1178

Abstract

Two new lankacidin-related metabolites, 2,18-seco-lankacidinol A (1), 2,18-seco-lankacidinol B (2) and a known compound, lankacidinol (3), were isolated from the fermentation broth of Streptomyces sp. HS-NF-1178. Their structures were determined on the basis of spectroscopic analysis, including 1D and 2D NMR techniques as well as ESI-MS and comparison with data from the literature. These two new compounds, especially compound 1, exhibited potent antitumor activity.

Lankacidin-group antibiotics, produced by organism Streptomyces rochei, are a class of unique 17-membered macrocyclic antibiotics different from traditional even-membered macrolides^{1, 2}. These antibiotics and their derivatives showed antimicrobial activity against various Gram-positive bacteria, Neisseria gonorrhoeae, Vibrio cholerae and Xanthomonas oryzae³. More importantly, they exhibited strong effect on staphylococcal infection in mice by oral or intraperitoneal administration⁴. In addition, they also displayed considerable in vivo antitumor activity against certain cell line models such as L1210 leukemia, melanoma B16 and 6C3 HED/OG lymphosarcoma⁵. In the course of hunting for new microbe-derived bioactive secondary metabolites, two new lankacidin-related metabolites, designated as 2,18-seco-lankacidinols A and B (1-2) and a known compound, lankacidinol (3) (Fig. 1), were isolated from the fermentation broth of Streptomyces sp. HS-NF-1178. In this paper, the details of fermentation, isolation, structure characterization and bioactivity of these two new compounds are described.

Fig. 1

Chemical structures of 2,18-seco-lankacidinol A (1), 2,18-seco-lankacidinol B (2) and lankacidinol (3)

Strain Streptomyces sp. HS-NF-1178 was isolated from a soil sample collected from a pine forest in Tianmu Mountain of Hangzhou, Zhejiang province, China. The strain was identified as the genus Streptomyces because its 16S rRNA sequence (accession no: KY884722 in the GenBank) exhibited a high-sequence similarity of 99.93% with that of Streptomyces sp. NRRL-16374 (T) (accession no: DQ026660).

This strain was incubated for 6–8 days at 28 °C on YMS medium containing malt extract (Becton, Dickinson and Company, Franklin Lake, NJ, USA) 10.0 g, yeast extract (Oxoid, Basingstoke, UK) 2.0 g, KNO₃ 1.0 g and agar (Becton, Dickinson and company, Franklin Lake, NJ, USA) 20.0 g in 1.0 l tap water at pH 7.2–7.4. The strain of stock culture was transferred into 1 l Erlenmeyer flasks containing 25% volume of the seed medium and incubated at 28 °C for 48 h, shaken at 250 r.p.m. The seed medium consisted of glucose (Sinopharm Chemical Reagent, Shanghai, China) 4.0 g, malt extract 10.0 g, yeast extract 4.0 g and CaCO₃ 2.0 g in 1.0 l tap water, pH 7.2–7.4. All of the media were sterilized at 121 °C for 30 min. Then, 1 l of the culture was transferred into a 50 l fermentor containing 30 l of producing medium consisting of glucose 10 g, soluble amylum (Haiyan Liuhe Starch Chemical Co, Ltd., Haiyan, China) 40 g, yeast extract 4 g, malt extract 10.0 g, CaCO₃ 2 g, MgSO₄·7H₂O 1 g, NaCl 1 g, KH₂PO₄ 2 g, pH 7.2–7.4. The fermentation was carried out at 28 °C for 6 days and stirred at 100 r.p.m. with an aeration rate of 700 l of air per hour.

The fermentation broth (30 l) was centrifuged to separate mycelial cake and supernatant. The mycelial cake was extracted with MeOH (5 l) and the supernatant was subjected to a Diaion HP-20 resin (Mitsubushi Chemical Co., Ltd., Tokyo, Japan) column eluting with 95% EtOH (5 l). The MeOH extract and the EtOH eluents were evaporated under reduced pressure at 50 °C to yield a mixture (36.6 g). The crude extract was chromatographed on a silica gel (Qingdao Haiyang Chemical Group, Qingdao, Shandong, China; 100–200 mesh) column and successively eluted with a stepwise gradient of CHCl₃/MeOH (100:0, 95:5, 90:10, 85:15, 80:20 and 70:30, v/v) to give four fractions (Fr.1–Fr.4) based on the TLC profiles. TLC was performed on silica-gel plates (HSGF254, Yantai Chemical Industry Research Institute, Yantai, China) with solvent system of CHCl₃/MeOH (9:1, v/v). The Fr.2 eluted with CHCl₃/MeOH (85:15, v/v) was subjected to a Sephadex LH-20 (GE Healthcare, Glies, UK) column eluted with CHCl₃/MeOH (1:1, v/v) and detected by TLC to obtain three subfractions (Fr.2–1–Fr.2–3). Fr.2-2 was further isolated by preparative HPLC (Shimadzu LC-8A, Shimadzu-C18, 5 µm, 250 × 20 mm inner diameter; 20 ml min⁻¹; 220 nm/254 nm; Shimadzu, Kyoto, Japan) eluting with a stepwise gradient CH₃CN/H₂O (15–33%, v/v, 25 min) to give four subfractions (Fr.2-2-1 to Fr.2-2-4) based on the retention time. Then Fr.2-2-3 (t_R 13.6 min) was purified by semi-preparative HPLC (Agilent 1100, Zorbax SB-C18, 5 μm, 250 × 9.4 mm inner diameter; 1.5 ml min⁻¹; 220 nm; Agilent, Palo Alto, CA, USA) eluting with CH₃CN/CH₃OH/H₂O (20:20:60, v/v) to obtain compound 1 (t_R 18.3 min, 22 mg). Fr.2-2-4 (t_R 17.9 min) was separated by semi-preparative HPLC to yield compound 2 (t_R 30.3 min, 12.3 mg). Fr.2-2-2 (t_R 11.2 min) was isolated by semi-preparative HPLC to give compound 3 (t_R 10.4 min, 25.3 mg). ¹H and ¹³C NMR spectra were measured with a Bruker DRX-400 (400 MHz for ¹H and 100 MHz for ¹³C) spectrometer (Rheinstetten, Germany). The ESI-MS and ESI-HRMS spectra were taken on a Q-TOF Micro LC-MS-MS mass spectrometer (Milford, MA, USA).

Compound 1 was obtained as white powder with $\left[ \alpha \right]_{\mathrm{D}}^{25}$ +9 (c 0.2, EtOH) and UV (EtOH) λ_max nm (log ε): 229 nm (3.97). It exhibited a molecular formula of C₂₅H₃₅NO₇ as deduced from the ESI-HRMS at m/z 462.2477 [M + H]⁺ (calcd for C₂₅H₃₆NO₇ 462.2486) in combination with the NMR data (Tables 1 and 2). The IR spectrum of 1 displayed absorption bands for hydroxyl (at 3395 cm⁻¹) and carbonyl (at 1712 cm⁻¹) groups. Analysis of ¹H NMR spectrum (Table 1) of 1 revealed the presence of two aliphatic methyl doublets (δ_H 1.26, 1.36), three olefinic methyls (δ_H 1.73, 1.78, 1.89), four oxygenated methine protons (δ_H 4.17, 4.22, 4.30, 4.39) in addition to seven downfield proton signals. The ¹³C NMR and DEPT135 spectra (Table 2) of 1 showed 25 resonances attributable to three downfield carbons (δ_C 170.8, 172.2, 177.5), six sp² methines, two sp² quaternary carbons, five oxygen bearing methines, two methylenes, one methine, five methyl carbons in addition to a carbon signal at δ_C 97.6. The complete assignment of all ¹H and ¹³C NMR spectral data of 1 was subsequently accomplished by the ¹H-¹H COSY, HMQC and HMBC spectra. The ¹H-¹H COSY correlations (Fig. 2) of H₃−20/H-4/H-5/H-6/H-7/H-8/H-9, H-11/H-12/H-13/H-14/H-15, H-17/H-18, H-24/H-25 indicated the four structural fragments (shown by thick lines) of C-20-C-9, C-11-C-15, C-17-C-18, C-24-C-25. The observed HMBC correlations (Fig. 2) from H₃-21 to C-9, C-10, C-11, from H₃-22 to C-15, C-16, C-17 established the linkage of C-20-C-18. The HMBC correlations of H₃-19 with C-1, C-2, C-3, H-5 with C-1 and H₃-20 with C-3 established the C-1-C-5 six-membered lactone ring moiety as shown in Fig. 2. The linkage of C-3 and C-18 through an oxygen atom was supported by the HMBC correlation from H-18 to C-3 and the downfield carbon resonance of C-3 (δ_C 172.2). The connection of C-18 and C-23 through a NH group was evident from the correlation of H-18 to C-23 in the HMBC spectrum and NMR data of C-18 (δ_H 6.05; δ_C 83.6). Taken the molecular formula of C₂₅H₃₅NO₇ into account, two hydroxyl groups were situated at C-7 and C-13, respectively. On the basis of the above spectroscopic analysis, a gross structure of 1 was elucidated as shown in Fig. 1. Thus, compound 1 was named 2, 18-seco-lankacidinol A with a different skeleton from lankacidinol A (3). The downfield shifting of C-2 and C-18 (δ_C 97.6 and 83.6, respectively, in 1; δ_C 56.9 and 50.5 in 3) as well as the upfield shifting of C-3 and C-19 (δ_C 172.2 and 8.6, respectively, in 1; δ_C 211.7 and 20.5 in 3) further confirmed the structural assignment of 1. The olefin conformations of 1 were determined based on the J values and NOESY experiment. The coupling constants of H-9 (δ_H 6.31, d, J = 15.4 Hz) and H-15 (δ_H 6.29, d, J = 15.5 Hz) unambiguously revealed double bond geometry at C-8 and C-14 to be both trans. In the NOESY spectrum, the crossing signals (Fig. 2) of H-17 to H-15, H₃-22 to H-18, H₃-21 to H₂-12 indicated the double bonds at C-10 and C-16 were both trans. The other chiral centers of 1 were assigned as described for lankacidinol.

Table 1 ¹H NMR data of compounds 1, 2 and 3

Table 2 ¹³C NMR data of compounds 1, 2 and 3

Fig. 2

Key ¹H-¹H COSY, HMBC and NOESY correlations of 2,18-seco-lankacidinol A (1)

Compound 2 was isolated as colorless oil with UV (EtOH) λ_max nm (log ε): 238 nm (4.34) and $\left[ \alpha \right]_{\mathrm{D}}^{25}$ + 1 (c 0.1, EtOH). Its molecular formula was determined to be C₂₂H₃₂O₆ on the basis of the ESI-HRMS at m/z 391.2118 [M–H]⁻ (calcd for C₂₂H₃₁O₆ 391.2126). The IR spectrum of 2 showed absorption bands for hydroxyl (at 3418 cm⁻¹) and carbonyl (at 1711 cm⁻¹) groups. The ¹H NMR spectrum (Table 1) of 2 displayed a aliphatic methyl doublet (δ_H 1.12), three olefinic methyls (δ_H 1.70, 1.77, 1.77), three oxygenated methine protons (δ_H 4.18, 4.30, 4.37), one oxygenated methylene protons (δ_H 4.21) in addition to six downfield olefinic proton signals. The ¹³C NMR and DEPT135 spectra (Table 2) of 2 showed 22 resonances attributable to two downfield carbons, six sp² methines, three sp² quaternary carbons, four methines (three oxygenated), three methylenes (one oxygenated) and four methyl carbons. Comparison of the ¹H and ¹³C NMR data of 2 with those of 1 suggested that 2 had the same C-1 to C-22 structural unit as 1. Obviously, one of the difference between 2 and 1 was that the 2-hydroxy-propionamido moiety in 1 was absent in 2 and one downfield methine of C-18 (δ_C 83.6) in 1 was replaced by one oxygenated methylene (δ_C 59.4) in 2. Considered the molecular formula C₂₂H₃₂O₆ of 2, the connection between C-3 and C-18 in 1 was cleaved in 2 and two hydroxyl groups were attached at C-3 and C-18, respectively. Consequently, the planar structure of 2 was established and compound 2 was named 2, 18-seco-lankacidinol B. The correlations of H₃-20/H-4/H-5/H-6/H-7/H-8/H-9, H-11/H-12/H-13/H-14/H-15, H-17/H-18 in the ¹H-¹H COSY spectrum (Fig. 3) and the observed HMBC correlated signals from H₃-19 to C-1, C-2, C-3, from H₃-20 to C-3, from H₃-21 to C-9, C-10, C-11 and from H₃-22 to C-15, C-16, C-17 further confirmed the above structure assignment of 2. The coupling constants of H-9 (δ_H 6.28, d, J = 16.0 Hz) and H-15 (δ_H 6.24, d, J = 15.7 Hz) showed the double bond geometries at C-8 and C-14 to be trans. The highfield ¹³C NMR chemical shifts of C-21 (δ_C 12.9), C-22 (δ_C 12.6), were very similar to those reported for lankacidinol A, lankacidin C and 2,18-seco-lankacidinol A (1), indicated the double bonds at C-10 and C-16 both being trans. The other relative stereochemistry of 2 was assigned by analogy with 1.

Fig. 3

Key ¹H-¹H COSY and HMBC correlations of 2,18-seco-lankacidinol B (2)

Compound 3 was obtained as white powder. Its structure was elucidated as lankacidinol by the analysis of its spectroscopic data (Tables 1 and 2) and comparison with literature values⁶.

The antimicrobial activities of compounds 1, 2 and 3 were measured by disk diffusion method using gentamicin as a positive control⁷. Both compounds 1 and 3 were found to be active against Micrococcus luteus with broadness of the clear ring of 15 mm and 17 mm at 100 μg per 7 mm paper disks. Compound 3 showed activity against Staphylococcus aureus with broadness of the clear ring of 13 mm. Compound 2 exhibited weak inhibitory activity against Bacillus subtilis and Staphylococcus aureus with broadness of the clear ring of 3 and 2 mm (Table 3), respectively.

Table 3 Antimicrobial activities of compounds 1, 2 and 3

The cytotoxicity of compounds 1, 2 and 3 were assayed for growth-inhibition activity in vitro against two human tumor cell lines, human lung tumor cells A549 and human prostate cancer cells PC-3 according to CCK8 colorimetric method as reported in our previous papers^{8, 9} using doxorubicin as positive control. The results (Table 4) showed that the two new compounds exhibited potent antitumor activities against two cancer cell lines (PC-3, A549).

Table 4 Cytotoxic activity of compounds 1, 2 and 3 against selected human tumor cell lines

From a biosynthetic view, 2,18-seco-lankacidinol A (1) seems to be formed by a different cyclization route from lankacidin. It was reported that a nucleophilic attack of an enolate anion at C-3 on an imide at C-18 occurred through the C-2 and C-3 double bond in an oxidized metabolite of LC-KA05, which resulted in a lankacidin carbon skeleton¹⁰. In the case of 2,18-seco-lankacidinol A (1), a direct nucleophilic attack of an enolate anion at C-3 on an imide at C-18 formed a different skeleton with an ether linkage. This result has opened a way to create lankacidin-group antibiotics with ether linkage.