Sporormiellin A, the first tetrahydrofuran-fused furochromone with an unprecedented tetracyclic skeleton from Sporormiella minima

Abstract

Sporormiellin A (1), the first tetrahydrofuran-fused furochromone with an unprecedented tetracyclic skeleton, has been obtained from the fungal strain Sporormiella minima. The structure with absolute configuration was elucidated by NMR data, X-ray crystallography, and quantum chemical ECD calculations.

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2H-Furo[2,3-b]chromen-4(3H)-ones are a subclass of furochromenones with diverse biological activities, such as inhibitory activity on nitric oxide-release,¹ anti-α-glucosidase,² anthelminthic,³ antiplatelet aggregation,⁴ anti-HIV replication,⁴ cytotoxic⁵ and neuroprotective⁶ activities, and so on. Up to now, around 150 2H-furo[2,3-b]chromen-4(3H)-ones have been reported. Natural 2H-furo[2,3-b]chromen-4(3H)-ones possess a tricyclic skeleton (6/6/5, such as pallidones I and J,⁷ fukanefurochromones,¹ and coniofurol A⁸) or a benzene-fused tetracyclic skeleton (6/6/5/6, such as aervins A–C,⁹ ayamenins,¹⁰ and euchretins⁴).

During our ongoing research on bioactive secondary metabolites from fungi,¹¹ a chemical investigation of metabolites from Sporormiella minima (no. 66-3-4-2) isolated from the lichen Nephromopsis pallescens (Schaer.) Y. S. Park was carried out, which led to the isolation of three novel linear pyran–furan fused furochromenones, sporormiellins A–C (1–3), and three biogenetically related compounds, sporormiellones A (4), B (5), and microsphaeropsone A (6) (Scheme 1). Interestingly, sporormiellin A (1) features an unprecedented tetracyclic skeleton core (6/6/5/5) and is the first case of a tetrahydrofuran-fused furochromone. Herein details of the structure elucidation for 1–3 (Fig. 1) are reported. In addition, a plausible biogenetic pathway of 1–3 is proposed.

Scheme 1 The plausible biogenetic pathway of 1–3.

Fig. 1 The chemical structures of 1–3.

The fermented material (70 g × 20 of rice) of Sporormiella minima (no. 66-3-4-2) was extracted with EtOAc, and the organic solvent was evaporated under vacuum to afford a dry crude extract (30.6 g). The crude extract was then fractionated using silica gel column chromatography (CC) with cyclohexane–MeOH (100 : 0 and 0 : 100, v/v) to afford a cyclohexane fraction (C, 17.6 g) and a methanol fraction (W, 12.1 g). The methanol fraction was successively subjected to repeated column chromatography over ODS and reversed-phase HPLC (RPHPLC) to obtain compounds 1–6.

Sporormiellin A (1) was established as C₁₆H₁₄O₇ using HRESIMS, with 10 degrees of unsaturation. Analyses of its ¹H, ¹³C and HSQC data (Table 1) revealed one exchangeable proton (δ_H 12.50), two methyl groups (including one methoxyl), one oxygenated methylene, two sp³ methines (including one oxygenated methine), one sp³ oxygenated quaternary carbon, eight olefinic/aromatic carbons (including three protonated), one ester carbonyl (δ_C 169.5), and one α,β-conjugated carbonyl (δ_C 179.5). The ¹H-¹H COSY experiment revealed two isolated spin-systems (C-1–C-2–C-3/C-15 and C-7–C-8–C-9) as shown in Fig. 2. The HMBC correlations from the exchangeable proton (δ_H 12.50) to C-9/C-10/C-11, from H-7 (δ_H 6.87) to C-6/C-9/C-11, from H-8 (δ_H 7.47) to C-6/C-10, and from H-9 (δ_H 6.84) to C-7/C-10/C-11 indicated the existence of a 1,2,3-trisubstituted benzene ring in 1. Furthermore, the weak ⁴J_CH HMBC correlations from H-7/H-9 to C-12 suggested the connection of the α,β-conjugated carbonyl (δ_C 179.5, C-12) to C-11. The methoxyl is connected to C-5 based on the HMBC correlation from H₃-16 (δ_H 3.90) to C-5. Combined with the ¹H-¹H COSY analysis, the HMBC correlations from H-3 (δ_H 5.09) to C-1/C-4/C-5/C-15, from H₂-15 (δ_H 3.96, 3.91) to C-1/C-3/C-4, and from H₃-1 (δ_H 1.20) to C-2/C-3/C-15 revealed the existence of a methyl-4-methyltetrahydrofuran-2-carboxylate moiety in 1. By considering the chemical shifts of C-13 (δ_C 94.6) and C-14 (δ_C 170.1) (β-oxygenated enone system¹²) and the unsaturation requirement for 1, the planar structure of 1 was established (Fig. 2), and the assignments of all of the proton and carbon resonances are shown in Table 1.

Table 1 1D and 2D NMR data of 1 in CDCl₃ (δ in ppm, J in Hz)

No.	δC	δH	^1H-^1H COSY	HMBC	ROESY
1	15.6	1.20, d (7.3)	2	2, 3, 15	3, 15b
2	39.9	2.64, m	1, 3, 15a,15b
3	97.3	5.09, d (0.9)	2	1, 4, 5, 15	1, 15b
4	89.5
5	169.5
6	153.8
7	106.9	6.87, dd (8.3, 0.6)	8, 9	6, 9, 11, 12
8	134.3	7.47, t (8.3)	7, 9	6, 10
9	113.4	6.84, dd (8.3, 0.6)	8, 7	7, 10, 11, 12
10	161.4
11	109.5
12	179.5
13	94.6
14	170.1
15	73.0	3.96, dd (9.5, 5.0), a	2	1, 3, 4	1, 3
		3.91, dd (9.5, 2.8), b	2	1, 3, 4
16	53.5	3.90, s		5
10-OH		12.50, s		9, 10, 11

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Fig. 2 Key 2D NMR correlations of 1.

In the ROESY experiment, the observed correlations between H₃-1 and H-15_b, H₃-1 and H-3, and H-3 and H-15_b showed that H₃-1, H-3 and H-15_b are on the same face of the tetrahydrofuran ring (Fig. 2). The single-crystal X-ray crystallography of 1 (Fig. 3) confirmed the above deduction, and the absolute configuration of 1 was assigned as 2S,3R,4R. In addition, the conformational analysis of a pair of enantiomers ((2S,3R,4R)-1 and (2R,3S,4S)-1) was carried out using OMEGA version 2.3 via the MMFF94s force field, which provided the five lowest energy conformers which differed in the dihedral angles of C-1–C-2–C-15–H-15_a and C-13–C-4–C-5–O (0–5 kcal mol⁻¹). The lowest energy conformers (Fig. 4) were used in the ECD calculations at a [B3P86/6-311++G (2d, p)] level, and the predicted ECD curve of (2S,3R,4R)-1 was similar to the experimental curve (Fig. 5, for details see the ESI†), which was consistent with the deduction from the X-ray crystallography analysis.§ Therefore, the absolute configuration of 1 was established as 2S,3R,4R.

Fig. 3 The X-ray structure of 1.

Fig. 4 The most stable conformers of (2S,3R,4R)-1 (the relative populations are in parentheses).

Fig. 5 The experimental ECD spectra of 1–3 and the calculated ECD spectra of (2S,3R,4R)-1 and (2R,3S,4S)-1.

The molecular formula of sporormiellin B (2) (a pale brown oil) was C₁₆H₁₆O₈ (9 degrees of unsaturation), established using HRESIMS (m/z 359.0472, [M + Na]⁺), which was one degree of unsaturation less than 1. In addition, the molecular weight was 18 atomic mass units more than 1, which indicated that one ether ring in 1 should be cleaved by H₂O. The comparative analyses of the ¹H and ¹³C NMR data (in CDCl₃, Table 2) with those of 1 revealed that 1 and 2 should have a similar furochromenone skeleton and differ in the tetrahydrofuran ring moiety, which was confirmed by the analyses of ¹H-¹H COSY and HMBC. Furthermore, all of the exchangeable proton signals were detected in DMSO-d₆ using NMR spectroscopy (for details see the ESI†). Therefore, the deduction was confirmed by the observations of the exchangeable proton signal for 15-OH (δ_H 4.78) in the ¹H NMR spectrum (in DMSO-d₆) and the key HMBC correlations (in DMSO-d₆) from the exchangeable proton 4-OH (δ_H 6.30) to C-4/C-5/C-13. Therefore, the planar structure of 2 was established as shown in Fig. 6.

Table 2 NMR data of 2 and 3 in CDCl₃ (δ in ppm, J in Hz)

No.	2		3
	δC	δH	δC	δH
1	13.4	1.18, d (6.8)	13.4	1.23, d (6.9)
2	34.1	2.61, m	32.1	2.66, m
3	94.4	5.09, d (9.4)	92.4	5.03, d (8.4)
4	78.8		78.5
5	172.6		172.3
6	153.8		153.9
7	107.0	6.87, d (8.3)	107.0	6.88, dd (8.3, 0.6)
8	134.2	7.45, t (8.3)	134.3	7.47, t (8.3)
9	113.2	6.81, d (8.3)	113.3	6.83, dd (8.3, 0.6)
10	161.3		161.4
11	109.5		109.6
12	179.5		179.3
13	98.3		98.3
14	169.8		169.9
15	64.8	3.53, dd (10.2, 7.7), a	65.6	4.16, dd (11.5, 4.5), a
		3.78, dd (10.2, 3.5), b		4.07, dd (11.5, 5.6), b
16	54.0	3.90, s	54.3	3.92, s
17			170.5
18			20.7	2.07, s
10-OH		12.46, s		12.38, s

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Fig. 6 Key HMBC correlations of 2 and 3.

In the selective ge-1D NOESY experiment (in DMSO-d₆), an enhancement was observed for H₃-16 in the irradiation of H-3, and an enhancement was observed for H-2 in the irradiation of 4-OH, which indicated the cis relationship of H-3 and C-5. Thus, the relative configuration between C-3 and C-4 was the same as that in 1. In the ECD experiment, the ECD curve of 2 was similar to that of 1 (Fig. 5), which indicated that the absolute configurations of C-3 and C-4 in 2 should be same as those in 1.

Furthermore, the derivatization of 2 was carried out. 2 was treated with SOCl₂ in CDCl₃ for 4 hours at 40 °C, and then the reaction mixture and 1 were compared using HPLC (Fig. 7) (for details see the ESI†). The results showed that the peak of the major reaction product prepared from 2 was identical to that of 1 isolated from fungal broth. The derivatization indicated that the configurations of C-2, C-3 and C-4 in 2 were the same as those in 1. Therefore, the absolute configuration of 2 was assigned as 2S,3R,4R.

Fig. 7 The products prepared from 2 and 1 were compared using HPLC with three eluting systems ((a) MeCN–H₂O (40 : 60, v/v); (b) MeCN–H₂O (35 : 65, v/v); and (c) MeOH–H₂O (50 : 50, v/v)).

Sporormiellin C (3) was established as C₁₈H₁₈O₉ (10 degrees of unsaturation) using HRESIMS (m/z 401.0844 [M + Na]⁺). The molecular weight of 3 was 42 atomic mass units more than 2, which indicated that 3 may be an acetylated derivative of 2. Except for the signals detected for one ester carbonyl (δ_C 170.5) and one methyl group (δ_C 20.7/δ_H 2.01), the NMR data (in CDCl₃) of 3 were similar to the data of 2, which indicated that 3 should also have a furochromenone skeleton. The key HMBC correlations (in DMSO-d₆, for details see the ESI†) from H₂-15 (δ_H 4.16, 4.07) to the additional ester carbonyl (δ_C 170.2, C-17) and from the additional methyl protons (δ_H 2.01, H₃-18) to C-17 indicated the existence of an acetyl group and the acetylation at C-15 (Fig. 6).

In the selective ge-1D NOESY experiment (in DMSO-d₆), an enhancement was observed for H₃-16 in the irradiation of H-3, which indicated the cis relationship of H-3 and C-5. In the ECD experiment, the ECD curve of 3 was similar to that of 2 (Fig. 5), which suggested that the absolute configurations of C-3 and C-4 in 3 should be the same as those in 2.

Since 2 and 3 coexist in Sporormiella minima (no. 66-3-4-2), the configurations of C-2, C-3 and C-4 in 3 should be the same as those in 2. To confirm the deduction, the derivatization of 3 was carried out. The products prepared from 3 and 2 were compared using HPLC (Fig. 8, for details see the ESI†). The results showed that the peak of the major product prepared from 3 was identical to that of 2 isolated from fungal broth. Therefore, the absolute configuration of 3 was assigned as 2S,3R,4R, which was the same as that of 2.

Fig. 8 The products prepared from 3 and 2 were compared using HPLC with three eluting systems ((a) MeCN–H₂O (40 : 60, v/v); (b) MeCN–H₂O (30 : 70, v/v); and (c) MeOH–H₂O (50 : 50, v/v)).

In addition, three biogenetically related compounds, sporormiellones A (4), B (5), and microsphaeropsone A (6)¹³ were also obtained from the fermented material of Sporormiella minima (no. 66-3-4-2) (Scheme 1). Among them, sporormiellone A (4) is a new benzophenone, whose structure was established using the analysis of 1D and 2D NMR spectra (for details see the ESI†). The NMR data of 5 (a known benzophenone) was firstly reported (for details see the ESI†), and 5 was named as sporormiellone B.

In summary, sporormiellin A (1) is a new member of the furochromenone class of metabolites, possessing an unprecedented tetrahydrofuran-fused tetracyclic skeleton core (6/6/5/5). On the basis of previous biosynthetic studies of the xanthones from fungi^8,13 and the structures of obtained compounds (1–6) reported by us, sporormiellins A–C (1–3) could originate from chrysophanol by oxidative cleavage, ring-expansion, and dehydration/cyclization steps as shown in the hypothetical biosynthetic pathway (Scheme 1).

This work was financially supported by grants from the Ministry of Science and Technology of China (2012ZX09301002003), the National Natural Science Foundation of China (81373306, 81202441), the Guangdong Natural Science Funds for Distinguished Young Scholar (S2013050014287), the program for New Century Excellent Talents in University (NCET-10-0120) from the Ministry of Education of China, and the high-performance computing platform of Jinan University.

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Footnotes

† Electronic supplementary information (ESI) available: The general experimental procedure, fungus material, extraction and isolation, spectroscopic data of compounds 1–5, single-crystal X-ray data of 1, quantum chemical ECD calculations of 1, derivatizations of 2 and 3, and NMR spectra of compounds 1–5. CCDC 990532. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra02491e

‡ These authors have contributed equally to this work.

§ Crystal data for sporormiellin A (1): data were collected using a Sapphire CCD with graphite monochromated Cu Kα radiation, λ = 1.54178 Å at 173.0 (3) K. Crystal data: C₁₆H₁₄O₇, M = 318.27, orthorhombic, space group P2₁2₁2₁; unit cell dimensions were determined to be a = 7.2831(2) Å, b = 12.4744(3) Å, c = 16.0184(4) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1455.31(6) ų, Z = 4, Dx = 1.453 g cm⁻³, F (000) = 664, μ (Cu K_α) = 0.983 mm⁻¹. 11 414 reflections were collected until θ_max = 62.70°, in which independent unique 2189 reflections were observed [F² > 4σ (F²)]. The final refinement gave R = 0.0363, R_W = 0.0991, S = 1.064, and Flack = −0.1(2). Crystal data of 1 were deposited in the Cambridge Crystallographic Data Centre (CCDC 990532).

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