Streptospirodienoic acids A and B, 6,6-spiroketal polyketides from Streptomyces sp.

Abstract

Three new 6,6-spiroketal polyketides, streptospirodienoic acids A (1), B (2) and streptospirodienoate A (3), were obtained from the fermentation broth of Streptomycetes sp. (no. 0950134). Their structures were constructed by one 6,6-spiroketal core coupled with hexadienoic acid, in which the starter unit might be 2-methylpropionyl-CoA. Their chemical structures were determined by detailed analyses of UV, IR, HRESIMS, 1D, 2D-NMR spectroscopic data and the X-ray crystallography technique. The biosynthetic pathway for 1 and 2 was proposed. Compound 3 showed moderate cytotoxic activity against HCT-116 cells (IC₅₀ = 5.2 μM), while compounds 1 and 2 showed no activity (IC₅₀ > 10.0 μM).

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Actinomycetes, in particular the members of the genus Streptomyces, have proved to be a particularly rich source of secondary metabolites with extensive industrial applications.¹ Recently, because the rate of new compound identification from terrestrial actinomycetes is rapidly decreasing, more and more people are focusing their attention on the secondary metabolites of marine actinomycetes.² In our ongoing search for bioactive secondary metabolites from actinomycetes,^3–5 three new 6,6-spiroketal polyketides, streptospirodienoic acids A (1)‡, B (2) and streptospirodienoate A (3), were obtained from the fermentation broth of Streptomyces sp. (no. 0950134) derived from mangrove soil (Wenchang, Hainan, China). Herein, we report the isolation and structural elucidation of compounds 1–3 and their cytotoxic activity.

The EtOAc extract (3.2 g) of the fermentation broth (40 L) of Streptomyces sp. (no. 0950134) was chromatographed on ODS medium pressure liquid chromatography (MPLC) and preparative HPLC to obtain compounds 1 (15.0 mg), 2 (11.6 mg), and 3 (2.6 mg) (Fig. 1).

Fig. 1 Chemical structures of 1–3.

Compound 1 was obtained as colorless needle crystals (MeOH). The ESI-MS gave a pseudo-molecular ion at m/z 449.3 [M + Na]⁺ and 425.5 [M − H]⁻. The analysis of high resolution ESI-TOF mass spectrometry revealed the molecular formula C₂₃H₃₈O₇ (m/z 449.2514 [M + Na] ⁺, calcd 449.2515), indicating 5 degrees of unsaturation. The ¹H NMR spectrum showed three olefinic proton signals at δ_H 7.19 (1H, d, 11.4), 6.66 (1H, dd, 15.3, 11.4), and 5.90 (1H, dd, 15.3, 8.0), one methoxyl proton signal at δ_H 3.31 (3H, s), and six methyl proton signals at δ_H 1.95 (3H, s), 1.02 (3H, d, 9.2), 0.94 (3H, d, 4.0), 0.92 (3H, d, 4.4), 0.85 (3H, d, 6.0), and 0.83 (3H, d, 6.0) (Table 1). The ¹³C NMR spectrum and DEPT135 displayed twenty carbon signals including seven methyl carbon signals, one methylene carbon signal, eleven methine carbon signals, and one quaternary carbon signals. Analyses of ¹H, ¹³C NMR, HSQC, and HMBC spectra revealed three additional carbon signals at δ_C 172.3 (CO), 138.5 (CH), and 131.1 (C), which were not detected in the ¹³C NMR spectrum. This was consistent with HRESIMS data. Since four olefinic carbons and one carbonyl carbon accounted for three unsaturations, it was implied that 1 should contain two rings.

Table 1 NMR data of 1 and 3 in CD₃OD (δ in ppm, J in Hz)

No.	1		3
	δC	δH	δC	δH
1	172.3		170.6
2	129.5		129.1
3	138.5	7.19, d (11.4)	138.7	7.19, d (11.4)
4	131.1	6.66, dd (15.3, 11.4)	130.6	6.66, dd (15.6, 12.0)
5	142.4	5.90, dd (15.3, 8.0)	142.8	5.95, dd (15.6, 7.8)
6	81.8	3.73, t (8.0)	81.7	3.74, (t, 8.4)
7	73.9	3.97, dd (9.3, 2.1)	74.1	3.97, dd (9.6, 2.4)
8	37.1	2.18, m	37.1	2.19, m
9	73.2	3.68, dd (11.1, 5.1)	73.2	3.68, dd (11.4, 4.8)
10	42.7	1.64, m	42.8	1.63, m
11	103.3		103.3
12	38.4	2.30, dd (15.2, 6.9); 1.59, m	38.4	2.33, dd (15.0, 7.2); 1.61, m
13	73.5	3.37, (t, 6.8)	73.5	3.37, t (7.2)
14	40.8	1.75, m	40.7	1.77, m
15	81.2	3.08, dd (11.1, 2.1)	81.2	3.07, dd (9.0, 2.4)
16	30.4	1.79, m	30.4	1.78, m
17	20.9	1.02, d (9.2)	20.9	1.03, d (7.2)
18	15.7	0.85, d (6.0)	15.6	0.85, d (6.6)
19	15.5	0.83, d (6.0)	15.5	0.82, d (7.2)
20	12.3	0.94, d (6.5)	12.3	0.94, d (6.6)
21	5.6	0.92, d (6.5)	5.6	0.93, d (7.2)
22	13.1	1.95, s	13.1	1.97, s
1-OCH3			52.6	3.77, s
6-OCH3	57.2	3.31, s	57.3	3.30, s

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Partial structures of C3–C4–C5–C6–C7–C8–C9–C10–C20, C21–C8, C12–C13–C14–C15–C16–C17, C14–C19, and C16–C18 were deduced based on detailed analyses of ¹H–¹H COSY (Fig. 2). In the HMBC spectrum, correlations from H21/δ 0.92 (3H, d, 4.4) to C7/δ 73.9 (CH), C8/δ 37.1 (CH), and C9/δ 73.2 (CH) suggested that C8 was attached to C7. Correlations from H22/δ 1.95 (3H, s) to C1/δ 172.3 (CO), C2/δ 129.5 (C), and C3/δ 138.5 (CH) indicated that the C1–C2–C22 unit was attached to C3. The HMBC correlations from H20/δ 0.94 (3H, d, 4.0), H12/δ 2.30 (1H, dd, 15.2, 6.9) and H13/δ 3.37 (1H, t, 6.8) to C11/δ 103.3 indicated the presence of spiroketal structure in the structure of 1, which was further confirmed by the chemical shift of C-11 (δ 103.3 ppm) and degrees of unsaturation. The HMBC correlation from δ_H 3.31 (3H, s) to C6/δ 81.8 suggested that the methoxy group was attached to C6. Thus, the planar structure of 1 was determined.

Fig. 2 Key correlations of 1 h–1 h COSY and HMBC for compound 1.

The geometry of the double bonds at Δ² and Δ⁴ were both defined as E-configurations based on the J values of 15.3 and 11.4 Hz in the ¹H NMR spectrum, which was confirmed by ROESY correlation from H-4 (δ 6.66, 1H, dd, 15.3, 11.4) to H-22 (δ 1.95, 3H, s). The relative configuration of the 6,6-spiroketal core structure was determined by analyses of J values in the ¹H NMR and ROESY experiments. In the ROESY spectrum, correlations between δ_H 3.97 (H-7) with δ_H 3.68 (H-9) and δ_H 1.64 (H-10) with δ_H 0.92 (H-21) defined the relative configurations of C7, C8, C9, and C10 in the ring A, which was further confirmed by the J value of H-7 and H-8 at 2.1 Hz. In the ROESY spectrum, correlations between δ_H 2.30 (H-12a) with δ_H 1.64 (H-10), δ_H 0.94 (H-20), and δ_H 3.08 (H-15) suggested that C-12 was in an α-orientation in the ring A. In the ring B, the 15-isopropyl residue and 14-CH₃ were both defined in equatorial orientations based on the J value of 11.1 Hz between H-14 and H-15 in the ¹H NMR spectrum. ROESY correlation between H-13 and H-15 revealed that they were in axial orientations. Thus, the relative configuration of the 6,6-spiroketal core structure in 1 was determined.

The absolute configuration of 1 was determined by X-ray crystallographic analysis (Fig. 3). The X-ray crystal structure analysis of 1 was conducted using Cu Kα radiation. The diffraction pattern established the absolute configuration of 1 to be 6S, 7R, 8S, 9R, 10S, 11R, 13R, 14S, and 15R by the small Flack parameter 0.00 (2). Thus compound 1 was determined as 6-[(2R,3S,4R,5S,6R,8R,9S,10R)-8-isopropyl-4-dihydroxy-3,5,9-trimethyl-1,7-dioxaspiro[5,5]-undecan-2-yl]-(2E,4E,6S)-6-methoxy-2-methyl-2,4-hexadienoic acid named streptospirodienoic acid A.

Fig. 3 X-ray structure of 1 drawn by ORTEP.

Compound 2 was obtained as white, amorphous powder. The high resolution ESI-TOF mass spectrometry gave the molecular formula C₂₃H₃₈O₇ (m/z 449.2511 [M + Na]⁺, calcd 449.2515), the same as that of 1. The ¹H and ¹³C NMR spectra showed closely similar to that of 1 (Table 2). Analyses of ¹H, ¹³C NMR, DEPT 135, ¹H–¹H COSY, HSQC, and HMBC data revealed that 2 had the same planar structure as that of 1. A comparison of the ¹H and ¹³C NMR data of 2 with those of 1 showed differences of the chemical shifts of C-12, C-13, C-15, and C-19, indicating that the relative configurations of C6, C7, C8, C9, and C10 in 2 were the same to those of 1. In the ROESY spectrum, H-12a (δ 2.10, dd, 13.2, 4.0) correlated with H-7 (δ 3.35, dd, 9.6, 2.0) and H-9 (δ 3.54, dd, 11.6, 4.8) instead of H-10 (δ 1.69, dq, 11.6, 6.4), indicating that the stereochemistry of C-11 in 2 was inverted. The large J_H-14–H-15 value of 10.4 Hz suggested a trans relationship of the two protons. In addition, in the ROESY spectrum, H-13 (δ 3.24, td, 10.0, 4.4) and H-15 (δ 3.38, dd, 10.4, 2.0) correlated with H-5 (δ 5.89, dd, 15.2, 7.6), indicating that the substituents at C-13 and C-15 were assigned a cis orientation. Based on the biogenetic similarity, compound 2 was determined as 6-[(2R,3S,4R,5S,6S,8R,9S,10R)-8-isopropyl-4,10-dihydroxy-3,5,9-trimethyl-1,7-dioxaspiro[5,5]-undecan-2-yl]-(2E,4E,6S)-6-methoxy-2-methyl-2,4-hexadienoic acid named streptospirodienoic acid B.

Table 2 NMR data of 2 in CD₃OD (δ in ppm, J in Hz)

No.	δC	δH
1	172.1
2	129.2
3	138.6	7.21, d (11.2)
4	131.2	6.60, dd (15.2, 11.2)
5	142.0	5.89, dd (15.2, 7.6)
6	82.4	3.74, t (8.4)
7	75.2	3.35, dd (9.6, 2.0)
8	37.4	2.17, m
9	74.4	3.54, dd (11.6, 4.8)
10	42.5	1.69, dq (11.6, 6.4)
11	103.1
12	33.7	2.10, dd (13.2, 4.0); 1.21
13	70.4	3.24, td (10.0, 4.4)
14	42.1	1.27, m
15	79.1	3.38, dd (10.4, 2.0)
16	29.4	1.80, 7d (6.5, 2.0)
17	21.3	0.95, d (6.8)
18	14.7	0.82, d (6.4)
19	13.1	0.77, d (6.4)
20	12.9	0.95, d (6.4)
21	5.8	0.93, d (6.4)
22	12.7	1.94, s
6-OCH3	57.3	3.32, s

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Compound 3 was obtained as white, amorphous powder. It had the molecular formula of C₂₄H₄₀O₇ on the basis of the high resolution ESI-TOF mass spectrometry (m/z 463.2664 [M + Na]⁺, calcd 463.2672), indicating 5 degrees of unsaturation. The ¹H and ¹³C NMR data were almost identical with those of 1 except for the presence of a methoxyl signals at δ_H 3.77 (3H, s)/δ_C 52.6 (Table 1). The HMBC correlation from the methoxyl proton signal at δ_H 3.77 (3H, s) to the carbonyl carbon signal at δ_C 170.6 suggested that 3 was the methyl esterified derivative of 1, which were confirmed by 1D and 2D NMR spectra. Thus, compound 3 was identified as methyl [(2R,3S,4R,5S,6R,8R,9S,10R)-8-isopropyl-4,10-dihydroxy-3,5,9-trimethyl-1,7-dioxaspiro[5,5]-undecan-2-yl]-(2E,4E,6S)-6-methoxy-2-methyl-2,4-hexadienate.

The structures of compounds 1–3 were constructed by one 6,6-spiroketal core coupled with hexadienoic acid. Among them, compound 3 might be an artifact of the isolation process since it was the methyl esterified derivative of 1. The proposed biosynthetic pathway for streptospirodienoic acids A (1) and B (2) was proposed in Scheme 1. The carbon skeleton is likely constructed of one 2-methylpropionyl-CoA, three malonyl-CoA, and four methylmalonyl-CoA. The starter group is 2-methylpropionyl-CoA derived from L-valine. The 6,6-spiroketal core structure may be constructed by cyclization of epoxide/ketone intermediate (Scheme 1) or through dehydrative cyclization.^6–8 It is very interesting that streptospirodienoic acids may have close relationship with bafilomycin A,⁹ a 16-membered macrolide isolated from the broth of Streptomyces griseus, from the view of biogenetic pathway. Both of them were derived from the polyketide pathway and the starter group and extend units of them might be similar. The differences between them lay in the numbers of the extend unit and the way of cyclization. Bafilomycin A has inspired much interest in chemical synthesis of natural products since it was a potent H⁺-ATPase inbibitor and exhibited broad antibacterial and antifungal activities.^10–14 The core 6,6-spiroketal skeleton of streptospirodienoic acids was firstly constructed during the total synthesis of bafilomycin A, and their difference lay in the aliphatic side chain. Due to their structural similarity, streptospirodienoic acid analogs may also exhibit strong antibacterial and antifungal activities which need to be evaluated.

Scheme 1 Proposed biosynthetic pathway for compounds 1 and 2.

The cytotoxic activity of compounds 1–3 against HCT-116 cancer cells was evaluated by MTT method. Doxorubicin was used as a positive control (IC₅₀ = 0.041 μM). The result revealed that compound 3 showed moderate cytotoxic activity against HCT-116 cancer cells with an IC₅₀ value of 5.2 μM, while 1 and 2 showed no cytotoxic activity (IC₅₀ > 10.0 μM).

Acknowledgements

This work was financially supported by grants from the Ministry of Science and Technology of China (no. 2012ZX09301002003), the National Natural Science Foundation of China (81422054, 81373306), the Guangdong Natural Science Funds for Distinguished Young Scholar (S2013050014287), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (Hao Gao, 2014), and Fok Ying Tung Education Foundation (no. 121039) from the Ministry of Education of China.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: The general experimental procedure, strain fermentation, extraction and isolation, spectroscopic data of 1–3, single-crystal X-ray data of 1, MTT assay, and NMR spectra of compounds 1–3. CCDC 1007092. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra10672e

‡ Crystal data for streptospirodienoic acid A (1): data collection was performed on a SMART CCD using graphite monochromated Cu Kα radiation (λ = 1.54184 Å) under low temperature (nitrogen gas); 2869 unique reflections were collected to θ_max = 62.65, in which 3870 reflections were observed [F² > 4σ(F²)]. Crystal data: colorless needles, C₂₃H₃₈O_7.50, monoclinic, P2₁, a = 9.9993(2), b = 15.1269(3), c = 9.5353(2) Å, β = 96.722(2), V = 1243.06(5) ų, Z = 2, d_x = 1.161 Mg m⁻³, F (000) = 472. The structures were solved by direct methods (SHELXTL version 5.1) and refined by full-matrix least-squares on F². In the structure refinements, non-hydrogen atoms were refined anisotropically. Hydrogen atoms bonded to carbons were placed on the geometrically ideal positions by the ‘ride on’ method. Hydrogen atoms bonded to oxygen were located by the difference Fourier method and were included in the calculation of structure factors with isotropic temperature factors. The final R = 0.0341, R_w = 0.0905 and S = 1.031.

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