Phomeketales A–F, six unique metabolites from the endophytic fungus Phoma sp. YN02-P-3

Abstract

Phomeketales A–F (1–6), six new xyloketals, with unprecendented carbon substitution at C-16 and C-17 simultaneously, were isolated from the endophytic fungus Phoma sp. YN02-P-3. Their structures were elucidated on the basis of 1D and 2D NMR spectral data and ECD analysis. Compound 3 exhibited moderate anti-AChE activity and cytotoxicity against HL-60.

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Over the last few decades, endophytic fungus has been recognized as a rich source for pharmacologically active metabolites.¹ Members of the genus Phoma were reported to produce a high variety of secondary metabolites, exhibiting diverse and remarkable biological properties such as antifungal,² antitubercular,³ anticancer,⁴ antibacterial⁵ and farnesyl protein transferase inhibitory activity.⁶ Therefore, as a promising source of naturally bioactive metabolites, Phoma sp. has attracted much attention.⁷ In our ongoing search for new bioactive secondary metabolites from endophytic fungus, a fungus Phoma sp. YN02-P-3 isolated from healthy leaf tissue of the plant Sumbaviopsis J. J. Smith from Yunnan Province, China was undertaken for cultivation in solid culture of mature rice. Chemical investigation of its 95% EtOH extract led to the isolation of six new ketals, phomeketales A–F (1–6), which represented a novel carbon skeleton with the co-occurrence of C-16 and C-17 at the aromatic ring, showing modest cytotoxic effects against a series of cancer cell lines and inhibitory activity against acetylcholine esterase. Details of the structural elucidation and biological evaluation of these metabolites are reported herein.

Phomeketale A (1) was obtained as yellow oil from methanol. [α]²⁰_D +7.25° (c 0.40, MeOH). Its molecular formula was established to be C₁₆H₂₀O₅ on the basis of HRESIMS at m/z 315.1216 [M + Na]⁺ (calcd 315.1203). Inspection of ¹H and ¹³C NMR spectra (Table 1) indicated one penta-substituted aromatic ring (δ_H 6.30, δ_C 97.8, 109.3, 153.5, 133.9, 118.7 and 154.8), a carboxyl group (δ_H 12.67, δ_C 169.1), an aromatic methoxy group (δ_H 3.67, δ_C 55.5), two methylene groups (δ_H 3.41/4.13, δ_C 73.1 and δ_H 2.69, δ_C 20.9), two methine groups (δ_H 1.89, δ_C 35.0 and δ_H 1.98, δ_C 47.2), and three methyl groups (δ_H 2.10, δ_C 15.9; δ_H 1.40, δ_C 22.6 and δ_H 1.02, δ_C 21.9). The HMBC spectrum corroborated the presence of the tetrahydrofuropyran moiety based on correlations from H₂-4, H₃-11 and H-6 to the methine carbon C-5 and from H₂-4, H-6 and H₂-7 to the oxygenated carbon C-2 (δ_C 106.9). The correlations from H₂-7 and H-6 to C-8 and from H₂-7 to C-9 suggested that the aromatic ring and the tetrahydrofuropyran moiety attached to C-8 and C-9. Further HMBC correlations (Fig. 2) from H₃-16 to C-8, C-12 and C-13 and from H-15 to C-8, C-9, C-12, C-13 and C-14 established the planar structure of 1. The NOESY correlations (Fig. 2) of H-6 with H₃-11 and H₃-10 suggested that these protons are close to each other in space. The relative configuration of C-2, C-5 and C-6 in 1 indicated there were only two possible structures, with the absolute configuration of (2R,5R,6R) or (2S,5S,6S). The absolute configurations of 1 were established by theoretical calculations of its ECD (ESI†). The calculated ECD spectrum of (2S,5S,6S)-1 agreed well with the measured spectrum. Therefore, the absolute configurations at C-2, C-5 and C-6 of 1 were determined as 2S, 5S, 6S (Fig. 3).

Table 1 ¹H and ¹³C NMR data of 1 and 2^a

Position	1		2
	δC	δH (m, J in Hz)	δC	δH (m, J in Hz)
a Measured in DMSO-d6 at 400 MHz for ^1H and 100 MHz for ^13C.
2	106.9		107.7
4	73.1	3.14 (1H, t, 8.1)	73.3	3.44 (1H, t, 8.3)
		4.13 (1H, t, 8.1)		4.02 (1H, t, 8.3)
5	35.0	1.89 (1H, m)	34.8	1.87 (1H, m)
6	47.2	1.98 (1H, m)	46.9	2.04 (1H, m)
7	20.9	2.69 (2H, m)	20.7	2.73 (2H, m)
8	109.3		110.8
9	153.5		158.3
10	22.6	1.40 (3H, s)	22.6	1.44 (3H, s)
11	15.4	1.02 (3H, d, 6.4)	15.4	1.01 (3H, d, 6.4)
12	133.9		141.3
13	118.7		117.0
14	154.8		162.8
15	97.8	6.30 (1H, s)	97.9	6.41 (1H, s)
16	15.9	2.10 (3H, s)	15.5	2.44 (3H, s)
17	169.1		190.1	10.36 (1H, s)
14-OCH3	55.5	3.67 (3H, s)	55.9	3.80 (3H, s)

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Fig. 1 Structures of compounds 1–6.

Fig. 2 Key HMBC and Noesy correlations of compounds 1–6.

Fig. 3 Experimental and calculated ECD spectra of 1 and 2.

Phomeketale B (2) was isolated as yellow amorphous powder. [α]²⁰_D −22.25° (c 0.40, MeOH). Its positive HRESIMS afforded an [M + Na]⁺ ion peak at m/z 299.1252 (calcd. 299.1254), indicating the molecular formula of C₁₆H₂₀O₅. The ¹H and ¹³C NMR spectra (Table 1) displayed a great deal of similarity with those of 1 except for the presence of an aldehyde group (δ_H 10.36, δ_C 190.1) instead of the carboxylic group in 1, which was assigned on the basis of the HMBC correlations (Fig. 2) from H-17 to C-12 and C-13. The relative configuration of 2 was determined to be the same as that of 1 according to their identical NOESY correlations (Fig. 2) from H₃-10 to H-6 and from H₃-11 to H-6. The absolute configuration of 2 was determined as 2R, 5R, 6R by comparison of its experimental electronic ECD curve with that calculated (Fig. 3).

Phomeketale C (3) was obtained as yellow amorphous powder. [α]²⁰_D −2.87° (c 0.50, MeOH). The molecular formula of 3 was established as C₂₄H₂₈O₈ on the basis of the quasi-molecular peak appearing at m/z 449.1554 [M − H₂O + Na]⁺ (calcd 449.1571) in the HRESIMS spectrum. The ¹H and ¹³C NMR data (Table 2) of 3 showed that it was another analogue of 1 possessing an additional penta-substituted aromatic ring. The HMBC correlations (Fig. 2) observed from H₂-17 to the aromatic carbon C-12, C-13, C-14, C-1′ and C-2′ demonstrated two penta-substituted aromatic rings were connected by the methylene group. HMBC correlations from H₂-17 to the aromatic carbon C-12, C-13, C-14, C-1′ and C-2′ and from H₂-8′ to C-3′ further established the planar structure of 3 as shown in Fig. 1. The NOESY correlations (Fig. 2) of the tetrahydrofuropyran moiety were completely identical to those of 1, implying the same relative configuration. The experimental ECD curve of 3 matched well with the calculated ECD spectrum, which confirmed the absolute configuration of 3 as 2S, 5S, 6S (Fig. 4).

Table 2 ¹H and ¹³C NMR data of 3 and 6^a

Position	3		6
	δC	δH (m, J in Hz)	δC	δH (m, J in Hz)
a Measured in DMSO-d6 at 400 MHz for ^1H and 100 MHz for ^13C.
2	106.2		108.5
4	72.9	3.35 (1H, t, 7.9)	73.1	3.42 (1H, t, 8.2)
		3.97 (1H, t, 7.9)		3.98 (1H, t, 8.2)
5	35.2	1.88 (1H, m)	35.1	1.87 (1H, m)
6	47.7	1.90 (1H, m)	46.7	2.04 (1H, m)
7	21.9	2.64 (2H, m)	19.3	2.93 (1H, dd, 17.8, 6.2)
				3.36 (1H, d, 17.8)
8	108.8		109.8
9	151.0		152.0
10	22.8	1.36 (3H, s)	22.6	1.47 (3H, s)
11	15.6	0.99 (3H, d, 5.7)	15.4	0.99 (3H, d, 6.2)
12	136.2		121.4
13	120.0		127.0
14	157.0		146.1
15	97.6	6.14 (1H, s)	113.5
16	15.2	2.09 (3H, s)	170.5
17	20.0	4.27 (1H, d, 17.8)	66.9	5.15 (2H, s)
		4.23 (1H, d, 17.8)
14-OCH3	55.2	3.53 (3H, s)
1′	118.8
2′	123.7		107.7
3′	124.4
4′	149.7		73.2	3.44 (1H, t, 8.4)
				4.03 (1H, t, 8.4)
5′	107.9	5.10 (1H, s)	35.1	1.87 (1H, m)
6′	157.2		46.4	2.04 (1H, m)
7′	171.1		19.2	2.76 (2H, m)
8′	66.2	5.10 (2H, s)	23.1	1.43 (3H, m)
9′			15.6	1.00 (3H, m)

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Fig. 4 Experimental and calculated ECD spectra of 3 and 4.

Phomeketale D (4) was obtained as yellow oil from methanol, [α]²⁰_D −9.45° (c 0.40, MeOH), and had a molecular formula of C₂₄H₂₈O₈ as determined by HRESIMS (m/z 385.1628 [M + Na]⁺, calcd 385.1622), corresponding to eight degrees of unsaturation. The ¹H NMR and ¹³C NMR spectroscopic data of 4 (Table 3) indicated the presence of an additional trans-disubstituted double bond (δ_H 6.65, δ_C 125.4 and δ_H 5.95 δ_C 125.4), an oxymethine group (δ_H 4.72, δ_C 71.8) and a methyl groups (δ_H 3.66, δ_C 51.7) compared with those of 1. The HMBC correlations from H-2′ to C-1′ and C-3′, from H-17 to C-14 and from H-4′ to C-3′ established the planar structure of 4 as shown in Fig. 1. The reliability of the absolute configuration assignment at the C-2′ tertiary alcohol was deduced by the CD data of the in situ formed [Rh₂(OCOCF₃)₄] complex with the inherent contribution subtracted. The sign of the E band (at ca. 350 nm) can be used to correlate the absolute configuration of a tertiary alcohol by applying the bulkiness rule.⁸ In this case, the positive Cotton effect at 350 nm in the Rh₂(OCOCF₃)₄-induced CD spectrum (Fig. S41, ESI†) indicated the 2′S absolute configuration on the basis of the bulkiness rule of 4.⁹ The NOESY correlations (Fig. 2) from H₃-10 to H-6 and from H₃-11 to H-6 indicated that these protons located on the same side of the tetrahydrofuropyran ring. The experimental ECD spectrum of 4 was consistent with the corresponding calculated ECD spectrum (Fig. 4), thus the absolute configurations of 4 were determined as 2R, 5R, 6R, 2′S.

Table 3 ¹H and ¹³C NMR data of 4 and 5^a

Position	4		5
	δC	δH (m, J in Hz)	δC	δH (m, J in Hz)
a Measured in DMSO-d6 at 400 MHz for ^1H and 100 MHz for ^13C.
2	106.6		107.7
4	73.1	3.40 (1H, t, 8.3)	73.1	3.40 (1H, t, 8.3)
		4.00 (1H, t, 8.3)		4.00 (1H, t, 8.3)
5	35.1	1.90 (1H, m)	34.9	1.87 (1H, m)
6	47.5	1.95 (1H, m)	46.7	1.98 (1H, m)
7	21.7	2.70 (2H, m)	19.0	2.90 (1H, dd, 17.9, 6.1)
				3.30 (1H, d, 17.9)
8	109.6		108.7
9	152.4		154.2
10	22.7	1.39 (3H, s)	22.9	1.42 (3H, s)
11	16.1	1.01 (3H, d, 6.3)	15.4	0.97 (3H, d, 6.2)
12	135.9		123.8
13	117.4		127.0
14	156.6		150.9
15	97.7	6.26 (1H, s)	108.6	6.49 (1H, s)
16	15.5	2.18 (3H, s)	170.8
17	125.4	6.65 (1H, dd, 16.0, 1.0)	67.3	5.15 (2H, s)
14-OCH3	55.3	3.68 (3H, s)
1′	130.4	5.95 (1H, dd, 16.0, 6.0)
2′	71.8	4.72 (1H, d, 5.6)
3′	173.2
4′	51.7	3.66 (3H, s)

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Phomone E (5) was obtained as white needles. [α]²⁰_D −44.80° (c 0.78, MeOH). It had the molecular formula of C₁₅H₁₈O₆ determined by HRESIMS at m/z 299.0918 [M − H₂O + Na]⁺ (calcd 299.0918). The 1D (Table 3) and 2D NMR data of 5 clearly revealed that it had the same tetrahydrofuranobenzopyran moiety as 1, the major difference between 5 and 1 occurred in the aromatic ring. The signals at δ_H 5.15 (2H, s), δ_C 67.3 (CH₂-17) and HMBC correlations (Fig. 2) from H₂-17 to C-12, C-13, C-14 and C-16 implied an oxymetheneyl group at C-17 and a carboxyl group at C-16. In addition, the methyl group of 1 was replaced by a hydroxyl group in 5. By combining all this evidence and data, the planar structure of 5 was determined as shown in Fig. 1. The NOESY spectrum (Fig. 2) gave diagnostic correlations of H₃-10 with H-6 and H₃-11 with H-6, which illustrated the same orientation of H-6, H₃-10 and H₃-11. Therefore, the absolute configurations of 5 were limited to two enantiomers of (2R,5R,6R)-5 or (2S,5S,6S)-5 based on the above established relative configurations, and determined by the calculated ECD spectra to be 2R, 5R, 6R (Fig. 5).

Fig. 5 Experimental and calculated ECD spectra of 5 and 6.

Phomone F (6) was obtained as white needles. [α]²⁰_D −32.55° (c 0.65, MeOH). Its molecular formula was determined to be C₂₂H₂₈O₇ as deduced from the HRESIMS at m/z 409.1623 [M − H₂O + Na]⁺ (calcd 409.1622). 1D and 2D NMR spectra (Table 2) analysis furnished compound 6 with the addition of a tetrahydrofuropyran “arm” and the loss of a hydroxyl group compared with 5. The additional tetrahydrofuropyran moiety was obviously attached to C-14 and C-15 due to the confirmation of the HMBC correlations (Fig. 2) from H₂-7′ to C-9, C-14 and C-15 as shown in Fig. 1. The NOESY correlations (Fig. 2) from H₃-10 and H₃-11 to H-6 and from H₃-8′ and H₃-9′ to H-6′ indicated H₃-10/H₃-11/H-6 and H₃-8′/H₃-9′/H-6′ were both in the same side of the ring system. Finally, the absolute configuration of 6 was verified by comparing the experimental CD spectra (Fig. 5) with the predicted. Therefore, compound 6 was determined as 2R, 5R, 6R, 2′S, 5′S, 6′S (Fig. 1).

As phomeketales shared a common tetrahydrofuranobenzopyran substructure, a close biosynthetic relationship could be speculated. A biosynthetic pathway involving orsellinic acid and orsellinic aldehyde, as confirmed for depsides and depsidones could be suggested (Scheme 1).¹⁰

Scheme 1 Suggested biosynthetic pathway of 1–6.

All the compounds were tested for their cytotoxicity against HL-60, Molm 13 and PC-3 cell lines with 5-fluorouracil used as positive control. As shown in Table 4, compound 3 showed selective cytotoxic activity against HL-60 cell line with IC₅₀ value of 12.39 μM. In addition, the AChE inhibitory activities were evaluated for all the isolated compounds, and compound 3 exhibited moderate inhibitory acetylcholine esterase activity with the IC₅₀ value of 40.0 μM with (−)-Huperzine A used as positive control.

Table 4 The cytotoxic activities and anti-AChE activity in vitro of 1–6

Compounds	HL-60 IC50 (μM)	Molm 13 IC50 (μM)	PC-3 IC50 (μM)	anti-AChE IC50 (μM)
1	46.27	80.68	>80	>100
2	37.48	52.82	>80	>100
3	12.39	37.81	48.40	40.00
4	>80	41.41	>80	>100
5	>80	>80	>80	>100
6	57.15	60.79	>80	>100
5-Fluorouracil	6.38	0.11	7.77
(−)-Huperzine A				0.27

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Phomeketales A–F (1–6) belong to the xyloketals family which are a rare group of metabolites originated from the mangrove fungus sp. Xylaria sp. 2508.¹¹ Over the decades, the structural novelty and associated biological properties¹² of xyloketals have triggered intense interest within the synthetic community.¹³ To the best of our knowledge, phomeketales bearing co-occurrence of C-16 and C-17 at the aromatic ring were reported for the first time, and xyloketals were firstly isolated from the fungi Phoma sp. In this contribution, our findings would both enrich chemical context of the genus Phoma and expand the variety of xyloketals.

Acknowledgements

This work was supported by National Natural Science Foundation of China (No. 81202425). The ECD calculations were carried out in the High Performance Computing Center (HPCC) of Shenyang Pharmaceutical University. The computing staff at HPCC is acknowledged for computational support.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: 1D and 2D NMR, HRESIMS, UV, IR, and ECD spectra of phomeketale and detailed experimental procedures. See DOI: 10.1039/c6ra12509c

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