The Cytochalasins and Polyketides from a Mangrove Endophytic Fungus Xylaria arbuscula QYF

Abstract

Twelve compounds, including four undescribed cytochalasins, xylariachalasins A–D (1–4), four undescribed polyketides (5–8), and four known cytochalasins (9–12), were isolated from the mangrove endophytic fungus Xylaria arbuscula QYF. Their structures and absolute configurations were established by extensive spectroscopic analyses (1D and 2D NMR, HRESIMS), electronic circular dichroism (ECD) calculations, ¹³C NMR calculation and DP4+ analysis, single-crystal X-ray diffraction, and the modified Mosher ester method. Compounds 1 and 2 are rare cytochalasin hydroperoxides. In bioactivity assays, Compound 2 exhibited moderate antimicrobial activities against Staphylococcus aureus and Candida albicans with MIC values of 12.5 μM for both Compound 10 exhibited significant cytotoxic activity against MDA-MB-435 with an IC₅₀ value of 3.61 ± 1.60 μM.

1. Introduction

The mangrove forest ecosystem, located in tropical and subtropical intertidal estuarine zones, is one of the most productive ecosystems at the junction of land and sea [1,2]. In order to adapt to this special marine environment, mangrove endophytic fungi have evolved unique biological metabolic pathways to produce abundant structurally novel and diverse bioactive secondary metabolites [3,4], which have attracted significant attention from organic chemists and pharmacologists [5]. The Xylaria genus, belonging to the family of Xylariaceae, is generally found among saprophytic and endophytic fungi [6]. Previous studies have proved that the genus Xylaria can produce various kinds of secondary metabolites including cytochalasins [7], polyketides [8], terpenes [9], alkaloids [10], etc. Moreover, most of them possess promising pharmacological activities associated with drug discovery, such as cytotoxic [11], antimalarial [12], antimicrobial [13], and α-glucosidase inhibitory activities [14].

During our ongoing exploration of new bioactive natural products from mangrove endophytic fungi [3,15,16,17,18,19], the chemical investigation of the fungus Xylaria arbuscula QYF collected from the mangrove plant Kandelia candel, led to the isolation of four undescribed cytochalasins, xylariachalasins A–D (1–4), four undescribed polyketides (5–8), and four known cytochalasins (9–12) (Figure 1). The known cytochalasins were identified as zygosporin E (9) [20], cytochalasin D (10) [21], cytochalasin C (11) [22], and cytochalasin O (12) [23]. Antimicrobial and cytotoxic activities of all isolated compounds were tested. Herein, the details of isolation, structure elucidation, and bioactivities of these compounds are reported.

2. Results and Discussion

2.1. Structure Identification

Xylariachalasin A (1) was gained as a white powder. Its molecular formula C₃₀H₃₇NO₈ with thirteen degrees of unsaturation was deduced by the ion peak of HR-ESI-MS m/z [M + Na]⁺ 562.2435 (calcd. for 562.2411). The ¹H and ¹³C NMR data (

Table 1. ¹H and ¹³C NMR data of 1 and 2 (δ ppm).

Position	1 a		2 b
	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)
1	176.7, C		174.6, C
3	61.8, CH	3.36, m	60.6, CH	3.35, m
4	50.3, CH	2.52, br.s	50.3, CH	2.50, overlapped
5	134.9, C		128.9, C
6	137.0, C		137.6, C
7	83.0, CH	4.07, d (10.0)	83.2, CH	4.11, d (10.2)
8	44.8, CH	3.09, t (10.2)	44.6, CH	3.00, overlapped
9	54.4, C		53.3, C
10	44.7, CH2	α: 2.85, dd (13.2, 9.9) β: 3.06, dd (13.2, 5.3)	44.4, CH2	2.98, overlapped
11	16.7, CH3	1.19, s	17.4, CH3	1.46, s
12	58.7, CH2	α: 4.04, d (11.5) β: 4.22, d (11.5)	14.2, CH3	1.75, s
13	131.0, CH	5.69, dd (15.5, 10.0)	132.3, CH	6.01, dd (15.7, 10.4)
14	134.1, CH	5.34, ddd (15.4, 10.7, 5.0)	131.3, CH	5.95, overlapped
15	39.6, CH2	α: 2.04, m β: 2.40, m	38.3, CH2	α: 2.02, m β: 2.52, overlapped
16	43.4, CH	2.85, m	42.3, CH	2.72, m
17	211.6, C		210.2, C
18	79.4, C		77.8, C
19	129.4, CH	5.29, dd (15.4, 2.0)	127.9, CH	5.16, dd (15.7, 2.4)
20	133.0, CH	5.96, overlapped	131.9, CH	5.34, m
21	76.5, CH	5.91, overlapped	75.2, CH	5.94, overlapped
22	19.8, CH3	1.15, d (6.8)	19.5, CH3	1.21, d (6.8)
23	24.6, CH3	1.50, s	24.4, CH3	1.52, s
24	172.1, C		170.2, C
25	20.7, CH3	2.33, s	21.1, CH3	2.32, s
1′	138.9, C		137.6, C
2′/6′	129.8, CH	7.32, d (7.0)	129.0, CH	7.32, d (7.7)
3′/5′	130.7, CH	7.25, m	129.2, CH	7.19, m
4′	128.0, CH	7.26, m	127.2, CH	7.25, m

^{a 1}H (400 MHz) and ¹³C (100 MHz) NMR data in CD₃OD. ^{b 1}H (400 MHz) and ¹³C (100 MHz) NMR data in CDCl₃.

) as well as the HSQC spectrum of 1 showed one methyl doublet at δ_H 1.15 (d, J = 6.8 Hz, H₃-22), three methyl singlets at δ_H 1.19 (s, H₃-11), δ_H 1.50 (s, H₃-23), and δ_H 2.33 (s, H₃-25), one oxygenated methylene at δ_H 4.04 (d, J = 11.5 Hz, H-12α), and δ_H 4.22 (d, J = 11.5 Hz, H-12β), two pairs of trans carbon–carbon double bonds at δ_H 5.29 (dd, J = 15.4, 2.0 Hz, H-19), δ_H 5.96 (overlapped, H-20), and δ_H 5.34 (ddd, J = 15.4, 10.7, 5.0 Hz, H-14), δ_H 5.69 (dd, J = 15.5, 10.0 Hz, H-13), one ketone carbonyl (δ_C 211.6), one amide group (δ_C 176.7), one ester group (δ_C 172.1), and a mono substituted phenyl group (δ_C 128.0, 129.8, 130.7, and 138.9), which suggested it was a typical 10-phenyl cytochalasin.

The ¹H-¹H COSY experiment of 1 displayed four proton spin–spin systems in the structure, namely H-2′/H-3′/H-4′/H-5′/H-6′, H-10/H-3/H-4, H-7/H-8/H-13/H-14/H-15/H-16/H₃-22, and H-19/H-20/H-21 (Figure 2). The HMBC correlations from H-10 and H-2′ to C-1′ indicated that the phenyl group substitutes at C-10 (Figure 2). According to the HMBC correlations from H-4 to C-1, C-5, and C-9, H-7 to C-5 and C-6, H-8 to C-9, H₃-11 to C-5, and H-12 to C-6, the fusing pattern of rings A and B can be determined. According to the HMBC correlations from H-16, H₃-22, and H₃-23 to C-17, H-19 and H₃-23 to C-18, H₃-25 to C-21 and C-24, and H-21 to C-9, ring C fused with ring B was elucidated. Therefore, the planar structure of Compound 1 was established. The rare hydroperoxyl at C-7 of Compound 1 was affirmed by the ¹³C NMR data of the oxygenated methine C-7 at δ_C 83.0 and the ion peak of HR-ESI-MS m/z [M + H − H₂O]⁺ 522.2493 (calcd. for 522.2486) of 1 [24]. To the best of our knowledge, this is the second report of typical 10-phenyl cytochalasin possessing a hydroperoxyl group.

In the NOESY experiment, the NOE correlations (Figure 3) from H-4 to H-8, H-10, and H-21, from H-14 to H-8 and H-15β, and from H-16 to H-15β and H₃-23, combined with the coupling constant of H-7/H-8 (³J_7,8 = 10.0 Hz), H-14/H-15α (³J_14,15α = 10.7 Hz), and H-14/H-15β (³J_14,15β = 5.0 Hz) suggested the Hα-3, Hβ-4, Hα-7, Hβ-8, Hβ-16, H₃β-23 orientations as well as the α-position for the 1-amide group. The ¹³C NMR calculation and DP4+ analysis were used to support the absolute configuration of C-21 of Compound 1 (21R-1, or 21S-1). The calculated result of 21R-1 (R² = 0.9983) was a better match with the experimental data than that of 21S-1 (R² = 0.9960) (Figure S78). Moreover, according to the DP4+ probability analysis, 21R-1 was assigned with a 100.00% (Figure S79) probability. Combined with the comparison of the experimental ECD spectrum (Figure 4) of 1 with the calculated one, the absolute configuration of 1 was determined as 3S, 4R, 7S, 8R, 9R, 16S, 18R, and 21R.

Fortunately, we got the qualified single-crystal of the known Compound 11 [Flack parameter of −0.1(2)] (Figure 5). The ¹³C NMR data in CD₃OD of Compound 11 (Figure S80) was similar to that of Compound 1. The main difference was that the methyl (δ_C 14.4) at C-6 and the hydroxyl at C-7 (δ_C 69.8) in Compound 11 was replaced by a hydroxymethyl (δ_C 58.7) and a hydroperoxyl (C-7, δ_C 83.0) in 1, respectively. The single crystal of Compound 11 further proved the absolute configuration of Compound 1.

From the biogenetic and structural point of view, the single-crystal of Compounds 10 and 11 (Figure 5) could be used as model compounds to support the assignment of the absolute configuration of new Compounds 1–4.

Xylariachalasin B (2) was isolated as a white powder. Its molecular formula C₃₀H₃₇NO₇ with thirteen degrees of unsaturation was deduced by the ion peak of HR-ESI-MS m/z [M + Na]⁺ 546.2456 (calcd. for 546.2462). The ¹H and ¹³C NMR data (Table 1) displayed one methyl doublet [δ_H 1.21 (d, J = 6.8 Hz, H₃-22)], four methyl singlets [δ_H 1.46 (s, H₃-11), δ_H 1.52 (s, H₃-23), δ_H 1.75 (s, H₃-12), and δ_H 2.32 (s, H₃-25)], two pairs of trans carbon-carbon double bonds [δ_H 5.16 (dd, J = 15.7, 2.4 Hz, H-19), δ_H 5.34 (m, H-20), and δ_H 5.95 (overlapped, H-14), δ_H 6.01 (dd, J = 15.7, 10.4 Hz, H-13)], one ketone carbonyl (δ_C 210.2), one amide group (δ_C 174.6), one ester group (δ_C 170.2), and a mono-substituted phenyl group (δ_C 127.2, 129.0, 129.2, and 137.6), which suggested that Compound 2 was also a typical 10-phenyl cytochalasin.

The ¹H-¹H COSY signals of H-2′/H-3′/H-4′/H-5′/H-6′ and the HMBC correlations from H-10 and H-2′ to C-1′ indicated that the phenyl group substitutes at C-10 (Figure 2). The ¹H-¹H COSY correlations of H-10/H-3/H-4, H-7/H-8/H-13/H-14/H-15/H-16/H₃-22, and H-19/H-20/H-21, combined with the HMBC correlations from H-4 to C-1, C-5, and C-9, H-7 to C-5 and C-6, H-8 and H-21 to C-9, H₃-11 to C-5, H₃-12 to C-6, H-16, H₃-22 and H₃-23 to C-17, H-19 and H₃-23 to C-18, H₃-25 to C-21 and C-24 manifested the three rings A/B/C fused system. According to the ¹³C NMR data of C-7 (δ_C 83.2) and the ion peak of HR-ESI-MS m/z [M + H − H₂O]⁺ 506.2536 (calcd. for 506.2537) of 2, a rare hydroperoxyl at C-7 of Compound 2 was also confirmed. Thus, Compounds 2 and 1 were structurally similar; they are both rare cytochalasin hydroperoxides, with the main difference between them being that the hydroxymethyl at C-6 in 1 was replaced by methyl in 2.

Though the overlapped chemical shifts [such as H-4 (δ_H 2.50) and H-15β (δ_H 2.52), H-8 (δ_H 3.00) and H-10 (δ_H 2.98)] influenced the determination of the relative configuration through NOE correlation, we could rule out the impossible case by the spatial structure of the Compound 2. For example, the NOESY spectrum showed correlations of δ_H 2.50 (H-4, H-15β) and δ_H 3.00 (H-8, H-10). According to the space structure of Compound 2, the NOE correlation was between H-4 and H-8 (H-10), not between H-15β and H-8 (H-10). Combined with the biosynthesis of cytochalasins [25], the relative configuration of H-4, H-8, and H-10 can be confirmed as Hα-3, Hβ-4, and Hβ-8 orientations. Excluding the impact of overlapped chemical shifts through the spatial structure of Compound 2, the NOE correlations from H-4 to H₃-25, from H-13 to H-7, and H-15α, from H-16 to H-15β, H-19, and H₃-23, from H-20 to H-21, and from H-19 to H₃-25, combined with the large coupling constant of H-7/H-8 (³J_7,8 = 10.2 Hz), suggested the Hα-7, Hβ-16, H₃β-23, Hα-21 orientations and α-position for 1-amide group. Hence, Compounds 1 and 2 had the same relative configuration. The absolute configuration of 2 was elucidated by comparing the experimental ECD spectrum with 1 (Figure 4). So, the absolute configuration of 2 was defined as 3S, 4R, 7S, 8R, 9R, 16S, 18R, and 21R.

Xylariachalasin C (3) was obtained as a white powder. Its molecular formula C₃₀H₃₅NO₇ with fourteen degrees of unsaturation was deduced by the ion peak of HR-ESI-MS m/z [M + Na]⁺ 544.2309 (calcd. for 544.2306). The ¹H and ¹³C NMR data of compound 3 (

Table 2. ¹H and ¹³C NMR data of 3 and 4 (δ ppm).

Position	3 a		4 b
	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)
1	175.1, C		177.1, C
3	61.2, CH	3.49, m	62.1, CH	3.29, m
4	50.7, CH	2.91, m	50.2, CH	2.45, m
5	154.5, C		129.6, C
6	136.3, C		131.2, C
7	199.7, C		70.1, CH	3.72, d (10.4)
8	53.2, CH	3.58, d (9.7)	43.3, CH	2.84, overlapped
9	54.2, C		50.6, C
10	44.9, CH2	α: 2.98, dd (13.2, 9.9) β: 3.14, dd (13.2, 5.2)	45.0, CH2	α: 2.85, overlapped β: 3.00, dd (13.2, 5.4)
11	18.0, CH3	1.40, s	17.2, CH3	1.04, s
12	56.0, CH2	α: 4.04, d (11.5) β: 4.25, d (11.5)	14.4, CH3	1.61, s
13	130.4, CH	5.35, dd (15.7, 10.4)	132.1, CH	5.82, overlapped
14	128.2, CH	5.86, overlapped	134.8, CH	5.19, m
15	39.4, CH2	α: 2.02, m β: 2.42, m	38.7, CH2	α: 1.97, m β: 2.32, m
16	43.3, CH	2.87, m	47.8, CH	2.65, m
17	211.9, C		210.4, C
18	79.5, C		45.9, CH2	α: 2.88, overlapped β: 3.26, m
19	131.7, CH	5.81, overlapped	117.9, CH	5.25, m
20	135.1, CH	5.21, m	135.3, CH	5.85, m
21	75.9, CH	5.98, t (2.4)	76.8, CH	5.81, overlapped
22	19.6, CH3	1.16, d (6.8)	18.9, CH3	1.08, d (6.9)
23	24.6, CH3	1.49, s	172.2, C
24	171.9, C		20.7, CH3	2.27, s
25	20.7, CH3	2.36, s
1′	138.5, C		139.0, C
2′/6′	130.0, CH	7.35, d (7.5)	129.7, CH	7.30, d (7.6)
3′/5′	130.7, CH	7.27, m	130.7, CH	7.23, m
4′	128.3, CH	7.29, m	127.9, CH	7.22, m

^{a 1}H (400 MHz) and ¹³C (100 MHz) NMR data in CD₃OD. ^{b 1}H (600 MHz) and ¹³C (150 MHz) NMR data in CD₃OD.

) were similar to that of 1, which indicated that 3 was also a typical 10-phenyl cytochalasin. Further analysis of the ¹H-¹H COSY and the HMBC correlations (Figure 2) of 3 showed that Compound 3 had a ketone carbonyl (δ_C 199.7) in Position 7 instead of an oxygenated methine compared to Compound 1, which was verified by the HMBC correlations from H-12 to C-6, and from H-8 and H-12 to C-7. Therefore, the planar structure of 3 was established, as shown in Figure 2.

The relative configuration of 3 was established by the coupling constant of H-8/H-13 (³J_8,13 = 9.7 Hz) and H-20/H-21 (³J_20,21 = 2.4 Hz) as well as the key NOESY correlations (Figure 3) between H-4/H-8, H-10, H-21, H-13/H-15β, H-20, H-16/H-15α, H₃-23, H-20/H-21. Hence, Compounds 3 and 1 had similar relative configurations. The absolute configuration of 3 was determined as 3S, 4R, 8R, 9R, 16S, 18R, and 21R by comparing the experimental ECD spectrum (Figure 6) with the calculated one and the ECD spectra of 7-oxo-cytochalasin C [26].

Xylariachalasin D (4) was isolated as a white powder. Its molecular formula C₂₉H₃₅NO₅ with thirteen degrees of unsaturation was deduced by the ion peak of HR-ESI-MS m/z [M + Na]⁺ 500.2400 (calcd. for 500.2407). Comparing the 1D and 2D NMR data (Table 2, Figure 2) of 4 with 1 indicated the absence of a methyl and a hydroxyl at C-18 in 4. The hydroperoxyl [δ_H 4.07 (d, J = 10.0 Hz), δ_C 83.0] at C-7 in 1 was replaced by a hydroxyl [δ_H 3.72 (d, J = 10.4 Hz), δ_C 70.1] in 4, and the hydroxymethyl [δ_H 4.04 (d, J = 11.5 Hz), δ_H 4.22 (d, J = 11.5 Hz), δ_C 58.7] at C-6 in 1 was replaced by a methyl [δ_H 1.61, δ_C 14.4] in 4. Therefore, the planar structure of 4 was established. Excluding the impact of overlapped chemical shifts through the spatial structure of Compound 4, combined with the biosynthesis of cytochalasins [25], the relative configuration of 4 was established by the large coupling constant of H-7/H-8 (³J_7,8 = 10.4 Hz) and the NOE correlations (Figure 3) of H-4/H-8, H-10, H₃-24, H-14/H-8, H-19, and H-19/H-16, H₃-24. The calculated ECD curve of 3S, 4R, 7S, 8R, 9R, 16S, and 21R was well matched with the experimental data of 4 (Figure 6), combined with the common biosynthetic pathway of cytochalasins in Xylaria arbuscula QYF, the absolute configuration of 4 was defined.

Compound 5 was obtained as a yellow oil. Its molecular formula C₁₄H₁₀O₄ with ten degrees of unsaturation was deduced by the ion peak of HR-ESI-MS m/z [M + Na]⁺ 265.0467 (calcd. for 265.0471). The ¹H NMR spectrum (

Table 3. ¹H (600 MHz) and ¹³C (150 MHz) NMR data of 5 and 6 (δ ppm) in CD₃OD.

Position	5		6
	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)
2	171.4, C		169.7, C
3	117.3, C		98.0, C
4	123.6, C		159.1, C
5	126.4, CH	8.58, d (8.0)	104.0, CH	6.41, s
6	125.3, CH	7.26, t (8.0)	156.1, C
7	131.4, CH	7.40, t (8.0)	139.4, C
8	111.3, CH	7.17, overlapped	121.4, C
9	155.4, C		70.8, CH	5.12, s
10	124.4, CH	7.47, s	113.8, C
11	152.0, C		43.5, CH2	α: 2.08, dd (12.8, 9.5) β: 2.73, dd (12.8, 6.2)
12	124.1, CH	7.17, overlapped	76.3, CH	4.39, m
13	112.2, CH	6.65, d (3.4)	53.5, CH3	3.50, s
14	162.0, C		22.1, CH3	1.38, d (6.2)
15	57.8, CH2	4.74, s

) showed one oxygenated methylene [δ_H 4.74 (s, H-15)], and seven unsaturated protons [δ_H 6.65 (d, J = 3.4 Hz, H-13), 7.17 (overlapped, H-8 and H-12), 7.26 (t, J = 8.0 Hz, H-6), 7.40 (t, J = 8.0 Hz, H-7), 7.47 (s, H-10), and 8.58 (d, J = 8.0 Hz, H-5)]. The ¹³C NMR and HSQC spectrum displayed a total of 14 carbon signals, including one ester carbonyl (δ_C 171.4), five non-protonated sp² carbons [δ_C 117.3, 123.6, and three oxygenated (δ_C 152.0, 155.4, and 162.0)], seven sp² methine carbons (δ_C 111.3, 112.2, 124.1, 124.4, 125.3, 126.4, and 131.4), and one oxygenated methylene (δ_C 57.8).

The ¹H-¹H COSY correlations (Figure 2) of H-5/H-6/H-7/H-8, together with the HMBC from H-5 and H-6 to C-4, from H-7 and H-8 to C-9, from H-5 and H-10 to C-3, and from H-10 to C-2 indicated the existence of a 3-methylenebenzofuran-2(3H)-one moiety. Under the assistance of degrees of unsaturation, the ¹H-¹H COSY signals of H-12/H-13 together with HMBC correlations from H-12 to C-11, and from H-13 and H-15 to C-14 led to the identification of a 2-hydroxymethylfuran moiety. And the above two moieties were connected through C-10, according to HMBC correlations from H-10 to C-11. Thus, the planar structure of 5 was established. The stereochemistry of 5 was determined by NOESY correlations (Figure 7) of H-5/H-15, and the double bond at 3(10) was E isomer [27].

Compound 6 was isolated as a light-yellow oil. Its molecular formula C₁₃H₁₄O₇ with seven degrees of unsaturation was deduced by the ion peak of HR-ESI-MS m/z [M − H]⁻ 281.0665 (calcd. for 281.0667). The ¹H NMR spectrum (Table 3) revealed one methyl [δ_H 1.38 (d, J = 6.2 Hz, H₃-14)], one methylene [δ_H 2.08 (dd, J = 12.8, 9.5 Hz, H-11α), and 2.73 (dd, J = 12.8, 6.2 Hz, H-11β)], one singlet methoxyl [δ_H 3.50 (s, H₃-13)], two oxygenated methines [δ_H 4.39 (m, H-12), and 5.12 (s, H-9)], and one aromatic proton [δ_H 6.41 (s, H-5)]. The ¹³C NMR and HSQC spectrum indicated an ester carbonyl at δ_C 169.7, three oxygenated olefinic carbons at δ_C 139.4, 156.1, and 159.1, two non-protonated sp² carbons at δ_C 98.0 and 121.4, a protonated olefinic carbon at δ_C 104.0, a ketal carbon at δ_C 113.8, two oxymethines at δ_C 70.8 and 76.3, a methoxyl at δ_C 53.5, a methylene at δ_C 43.5, and a methyl at δ_C 22.1.

The planar structure of 6 was determined by comprehensive analysis of its 2D NMR data as follows (Figure 2). The ¹H-¹H COSY correlations of H-11/H-12/H₃-14, combined with the HMBC correlations from H-9, H-11, and H₃-13 to C-10, and from H-11 to C-9, established a 4-methoxy-2-methyltetrahydrofuran fragment. The HMBC correlations from H-5 to C-2, C-3, C-4, C-6, C-7, and C-8, and from H-9 to C-3 and C-8 revealed the presence of a 5,6,8-trihydroxyisochroman-1-one moiety, sharing C-9 and C-10 with the tetrahydrofuran fragment. Therefore, the planar structure of 6 was confirmed.

The relative configuration of 6 was identified by key NOESY correlations (Figure 7) of H-9/H₃-13 and H₃-14, indicating these protons were co-facial. Therefore, the relative configuration of 6 was assigned to be (9S*,10S*,12S*). Further, ECD calculations were used to clarify the absolute configuration of 6. As shown in Figure 8, the calculated ECD curve of (9S, 10S, 12S)-6 was similar to that of the experimental one, indicating that the absolute configuration of 6 was 9S, 10S, and 12S.

Compound 7 was acquired as a brown oil. Its molecular formula C₁₁H₁₆O₄ with four degrees of unsaturation was deduced by the ion peak of HR-ESI-MS m/z [M + Na]⁺ 235.0936 (calcd. for 235.0941). The ¹H NMR spectrum (

Table 4. ¹H and ¹³C NMR data of 7 and 8 (δ ppm).

Position	7 a		8 b
	δC, Type	δH (J in Hz)	δC, Type	δH (J in Hz)
2	72.0, CH	4.31, m	169.8, C
3	34.1, CH2	α: 1.38, m β: 2.11, m	117.1, CH	5.73, s
4	67.1, CH	4.19, br.s	157.8, C
5	113.3, C		39.7, CH2	2.51, t (6.5)
6	197.9, C		62.7, CH2	4.31, t (6.5)
7	46.7, CH2	α: 2.45, dd (15.8, 9.0) β: 2.60, dd (15.8, 4.4)	167.0, C
8	65.5, CH	4.15, m	41.4, CH2	3.38, s
9	38.7, CH2	α: 2.48, dd (17.4, 8.4) β: 2.72, dd (17.4, 5.0)	166.5, C
10	174.0, C		52.7, CH3	3.74, s
11	56.7, CH3	3.35, s	19.0, CH3	2.20, s
12	20.8, CH3	1.40, d (6.4)

^{a 1}H (600 MHz) and ¹³C (150 MHz) NMR data in CD₃OD. ^{b 1}H (600 MHz) and ¹³C (150 MHz) NMR data in CDCl₃.

) showed one doublet methyl [δ_H 1.40 (d, J = 6.4 Hz, H₃-12)], three methylenes [δ_H 1.38 (m, H-3α), δ_H 2.11 (m, H-3β), δ_H 2.45 (dd, J = 15.8, 9.0 Hz, H-7α), δ_H 2.60 (dd, J = 15.8, 4.4 Hz, H-7β), δ_H 2.48 (dd, J = 17.4, 8.4 Hz, H-9α), and 2.72 (dd, J = 17.4, 5.0 Hz, H-9β)], one singlet methoxyl [δ_H 3.35 (s, H₃-11)], and three oxygenated methines [δ_H 4.15 (m, H-8), δ_H 4.19 (br.s, H-4), and 4.31 (m, H-2)]. The ¹³C NMR and HSQC spectrum revealed a ketone carbonyl (δ_C 197.9), two non-protonated sp² carbons [δ_C 113.3, and one oxygenated (δ_C 174.0)], three oxygenated methines (δ_C 65.5, 67.1, and 72.0), a methoxyl (δ_C 56.7), three methylenes (δ_C 34.1, 38.7, and 46.7), and a methyl (δ_C 20.8).

The ¹H-¹H COSY correlations of H-7/H-8/H-9 combined with the HMBC correlations from H-7 to C-5, C-6, from H-9 to C-10 established a cyclohexenone fragment. The ¹H-¹H COSY correlations of H₃-12/H-2/H-3/H-4 combined with the HMBC correlations from H₃-11 to C-4, from H-4 to C-5, C-6, C-10, indicated a 4-methoxy-2-methyltetrahydro-2H-pyran moiety was connected with the cyclohexenone fragment. Thus, the planar structure of 7 was confirmed, as shown in Figure 2.

The absolute stereochemistry on C-8 of 7 was determined by the modified Mosher’s method [28]. The differences in ¹H NMR chemical shifts between (S)- and (R)-MTPA esters (Δδ = δS − δR) (Figure 9) were calculated, which proved that the configuration of C-8 was S. The NOE correlations of H-3α/H-4, and H-3β/H-2, H₃-11 revealed that H-4 and H₃-12 were co-facial (Figure 7). We calculated all the possible absolute configurations of 7 (Figure 8). The computed ECD curve of (2S, 4S, 8S)-7 was well matched with the experimental one, so the absolute configuration of 7 was defined as 2S, 4S, and 8S.

Compound 8 was isolated as a brown oil. Its molecular formula C₁₀H₁₄O₆ with four degrees of unsaturation was deduced by the ion peak of HR-ESI-MS m/z [M + Na]⁺ 253.0682 (calcd. for 253.0683). The ¹H NMR spectrum (Table 4) showed one singlet methyl [δ_H 2.20 (s, H₃-11)], three methylenes [δ_H 2.51 (t, J = 6.5 Hz, H-5), δ_H 3.38 (s, H-8), δ_H 4.31 (t, J = 6.5 Hz, H-6)], one singlet methoxyl [δ_H 3.74 (s, H₃-10)], and one unsaturated proton [δ_H 5.73 (s, H-3). The ¹³C NMR and HSQC spectrum indicated three ester carbonyls (δ_C 166.5, 167.0, and 169.8), two olefinic carbons (δ_C 117.1, and 157.8), three methylenes [δ_C 39.7, 41.4, and one oxygenated (δ_C 62.7)], one methoxyl (δ_C 52.7), and one methyl (δ_C 19.0).

The ¹H-¹H COSY signal of H-5/H-6 with HMBC correlations from H-5 to C-3 and C-4, from H-3 to C-2, from H₃-11 to C-4, from H-6 and H-8 to C-7, and from H-8 and H₃-10 to C-9 led to the determination of the planar structure of 8 (Figure 2). The NOESY correlation (Figure 7) of H-3/H-5 manifested that the double bond at 3(4) was an E isomer. Therefore, the structure of 8 was established as (E)-5-((3-methoxy-3-oxopropanoyl)oxy)-3-methylpent-2-enoic acid.

2.2. Antimicrobial Activity Assays

The isolated Compounds 1–12 were evaluated for antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, Salmonella typhimurium, and Pseudomonas aeruginosa, and antifungal activities against Candida albicans. The antimicrobial activity assays were carried out in 96-well plates by a serial dilution test in the range of 0.1–100 μM. Results showed that the cytochalasins (1–3, and 10–12) exhibited promising inhibitory activities against the fungi, with a minimal inhibition concentration (MIC) value in the range of 12.5 to 50 μM (

Table 5. MIC for antibacterial and antifungal activities of compounds.

	MIC of Compounds/μM
	1	2	3	10	11	12	Amp. 1	Ket. 2
MRSA	>100	25	50	>100	50	50	0.25	NT
S. aureus	>100	12.5	>100	>100	>100	>100	0.25	NT
S. typhimurium	>100	>100	25	>100	25	>100	0.25	NT
P. aeruginosa	>100	25	>100	>100	>100	12.5	0.13	NT
C. albicans	25	12.5	>100	25	50	50	NT	0.13

¹ Ampicillin, positive control toward bacteria. ² Ketoconazole, positive control toward fungi.

). Wherein, Compound 2 showed moderate antimicrobial activities against S. aureus and C. albicans with MIC both for 12.5 μM. And Compound 12 displayed moderate inhibitory activity against P. aeruginosa with an MIC value of 12.5 μM. No obvious antimicrobial effect was observed under the concentration of 100 μM for Compounds 4–9.

2.3. Cytotoxic Activity Assays

The isolated Compounds 1–12 were also evaluated for cytotoxic activities against six human cancer cell lines: MDA-MB-435, MDA-MB-231, HCT116, A549, SNB19, and PC-3. Results showed that Compound 10 displayed significant inhibitory activity against MDA-MB-435 with an IC₅₀ value of 3.61 ± 1.60 μM. Moreover, the new Compound 1 exhibited moderate cytotoxic activity against SNB19 with an IC₅₀ value of 39.18 ± 0.71 μM (

Table 6. Cytotoxic activities of compounds (IC₅ ± SD, μM).

Compound	MDA-MB-435	MDA-MB-231	HCT116	A549	SNB19	PC3
1	>50	>50	>50	>50	39.18 ± 0.71	>50
10	3.61 ± 1.60	>50	>50	>50	23.76 ± 0.54	>50
11	>50	>50	>50	20.11 ± 2.71	>50	22.92 ± 0.43
Cisplatin 1	44.43 ± 3.25	38.16 ± 5.93	14.68 ± 1.34	26.42 ± 7.58	26.54 ± 1.53	33.00 ± 0.19

¹ Cisplatin, positive control.

). No obvious cytotoxic effect was observed under the concentration of 50 μM for Compounds 2–9, and 12. Moreover, no obvious cytotoxic effect was observed under the concentration of 50 μM for Compounds 1 and 10–11 toward normal human cell line HLF.

3. Materials and Methods

3.1. General Experimental Procedures

HRESIMS spectral data were obtained on a ThermoFisher LTQ–Orbitrap–LC–MS spectrometer (Palo Alto, CA, USA). The 1D and 2D NMR spectra were obtained on Bruker Advance 400 MHz and 600 MHZ spectrometer at room temperature. Optical rotations were measured with an MCP300 (Graz, Austria). UV–Vis and ECD curves were acquired using an Applied Photophysics Chirascan spectropolarimeter (Surrey, UK). Silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Amersham Pharmacia, Stockholm, Sweden) were used for column chromatography. Semi-preparative HPLC was performed on a Hitachi Primaide 1430 HPLC system combined with a DAD detector (HITACHI, Tokyo, Japan), and a Phenomenex Cellulose-2 column (10 × 250 mm, 5 μm) was applied for separation.

3.2. Fungal Material

The endophytic fungus QYF was isolated from the leaf of the mangrove plant Kandelia candel, which was collected from Hainan Dongzhai Harbor Mangrove Reserve in 2014. The fungus was identified as Xylaria arbuscula. (GenBank accession No. MT123074) based on the analysis of ITS sequence data of the rDNA gene. The strain has been preserved in our laboratory at Sun Yat-sen University, China.

3.3. Fermentation, Extraction, and Isolation

The fungus was cultivated on solid cultured medium (sixty 1 L Erlenmeyer flasks, each containing 50 g of rice and 50 mL of 2% saline water) for 30 days at 25 °C. After that, the fermented materials were extracted with EtOAc to yield 60 g of residue. Then, the crude extracts were subjected to silica gel column chromatography using step gradient elution with petroleum ether/ethyl acetate from 9:1 to 0:1 (v/v) to get seven fractions (Frs.1–Frs.7).

Frs. 6 (20 g) was subjected to Sephadex LH-20 (CH₂Cl₂/MeOH, v/v, 1:1) to yield six subfractions (Frs. 6.1–6.6). Frs. 6.1 (10 g) was applied to silica gel column chromatography (CC) (CH₂Cl₂/MeOH, v/v, 40:1) to give a mixture of 1, 2, and 3 (60 mg). The mixture was isolated using reverse phase C18 silica gel column (40–60 μm, MeOH/H₂O, v/v, 8:2) to yield 1 (5 mg), 2 (6 mg), and 3 (7 mg). Frs. 4 (7 g) was subjected to Sephadex LH-20 (CH₂Cl₂/MeOH, v/v, 1:1) to yield four subfractions (Frs. 4.1–4.4). Frs. 4.2 (1.2 g) was applied to silica gel CC (CH₂Cl₂/MeOH, v/v, 100:1) to give 5 (2 mg) and 6 (4 mg). Frs. 4.3 (1 g) was applied to silica gel CC (CH₂Cl₂/MeOH, v/v, 50:1) to give 7 (8 mg). Frs. 5 (10 g) was subjected to Sephadex LH-20 (CH₂Cl₂/MeOH, v/v, 1:1) to yield three subfractions (SFrs. 5.1–5.3). Frs. 5.1 (4 g) was applied to silica gel CC (CH₂Cl₂/MeOH, v/v, 100:1) to give a mixture of 4 and 8 (50 mg). The mixture was isolated utilizing HPLC with the Phenomenex Cellulose-2 column, respectively, at t_R = 15.6 and 10.2 min (the gradient was MeOH/H₂O, v/v, 8:2; flow rate: 1.5 mL/min) to give 4 (4 mg) and 8 (2 mg).

Xylariachalasin A (1): C₃₀H₃₇NO₈; white powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ = +16.4 (c 0.1, MeOH); UV (MeOH) λ_max (log ε): 230 (2.90), 270 (2.52) nm; ECD (MeOH) λ_max (Δ ε) 230 (+2.86), 290 (−1.67) nm; HRESIMS: m/z [M + Na]⁺ 562.2435 (calcd. for 562.2411), m/z [M + H − H₂O]⁺ 522.2493 (calcd. for 522.2486); ¹H and ¹³C NMR data (Table 1).

Xylariachalasin B (2): C₃₀H₃₇NO₇; white powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ = +20.5 (c 0.1, MeOH); UV (MeOH) λ_max (log ε): 230 (2.88), 270 (2.52) nm; ECD (MeOH) λ_max (Δ ε) 230 (+1.89), 290 (−3.38) nm; HRESIMS: m/z [M + Na]⁺ 546.2456 (calcd. for 546.2462), m/z [M + H − H₂O]⁺ 506.2536 (calcd. for 506.2537); ¹H and ¹³C NMR data (Table 1).

Xylariachalasin C (3): C₃₀H₃₅NO₇; white powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ = +40.0 (c 0.1, MeOH); UV (MeOH) λ_max (log ε): 224 (2.11), 270 (1.31) nm; ECD (MeOH) λ_max (Δ ε) 226 (+1.32), 287 (−0.47) nm; HRESIMS: m/z [M + Na]⁺ 544.2309 (calcd. for 544.2306); ¹H and ¹³C NMR data (Table 2).

Xylariachalasin D (4): C₂₉H₃₅NO₅; white powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ = +13.5 (c 0.1, MeOH); UV (MeOH) λ_max (log ε): 225 (2.07), 270 (1.37) nm; ECD (MeOH) λ_max (Δ ε) 228 (−1.71), 289 (−0.63) nm; HR-ESI-MS: m/z [M + Na]⁺ 500.2400 (calcd. for 500.2407); ¹H and ¹³C NMR data (Table 2).

(E)-3-((5-(hydroxymethyl)furan-2-yl)methylene)benzofuran-2(3H)-one (5): C₁₄H₁₀O₄; yellow oil; HR-ESI-MS: m/z [M + Na]⁺ 265.0467 (calcd. for 265.0471); ¹H and ¹³C NMR data (Table 3).

Xylariachromanone A (6): C₁₃H₁₄O₇; light-yellow oil; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ = +21.4 (c 0.1, MeOH); UV (MeOH) λ_max (log ε): 210 (2.31), 222 (2.00), 232 (2.07), 249 (1.66), 269 (1.87), 289 (1.29), 332 (1.86) nm; ECD (MeOH) λ_max (Δ ε) 226 (−0.26), 243 (+0.37), 270 (+0.54) nm; HR-ESI-MS: m/z [M − H]⁻ 281.0665 (calcd. for 281.0667); ¹H and ¹³C NMR data (Table 3).

Xylariaone A (7): C₁₁H₁₆O₄; brown oil; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ = −109.5 (c 0.1, MeOH); UV (MeOH) λ_max (log ε): 216 (1.58), 254 (2.38) nm; ECD (MeOH) λ_max (Δ ε) 215 (+0.36), 252 (−1.27) nm; HR-ESI-MS: m/z [M + Na]⁺ 235.0936 (calcd. for 235.0941); ¹H and ¹³C NMR data (Table 4).

(E)-5-((3-methoxy-3-oxopropanoyl)oxy)-3-methylpent-2-enoic acid. (8): C₁₀H₁₄O₆; brown oil; HR-ESI-MS: m/z [M + Na]⁺ 253.0682 (calcd. for 253.0683); ¹H and ¹³C NMR data (Table 4).

3.4. ECD and Optical Rotation Computation Methods

Initial conformational analysis was carried out by using Spartan 14′ software (Wavefunction Inc., Irvine, CA, USA). The conformers with a Boltzmann distribution larger than 1% were selected for optimization and calculation at B3LYP/6-31 + G(d,p) level in methanol with the density functional theory (DFT) executed by Gaussian 09 [29]. The calculated ECD spectra were extracted and generated by SpecDis 1.6 software (University of Würzburg, Würzburg, Germany).

3.5. Antimicrobial Assays

Antimicrobial tests for Compounds 1–12 were carried out against pathogenetic strains, including methicillin-resistant Staphylococcus aureus (A7983, clinical isolate donated by Zhijun Yu in Dalian Friendship Hospital, Dalian, China), Staphylococcus aureus (ATCC 6538), Salmonella typhimurium (ATCC 6539), Pseudomonas aeruginosa (ATCC 9027), and Candida albicans (ATCC 10231). Muller Hinton agar (MHA) and Sabouraud agar (SA) were used for antibacterial and antifungal tests, respectively.

The compounds tested were dissolved individually in dimethyl sulfoxide (DMSO), and the antimicrobial activity assays were carried out in 96-well plates by a serial dilution test in the range of 0.1–100 μM [30,31]. Ampicillin and ketoconazole were used as positive controls for antibacterial and antifungal assays, respectively, and DMSO was utilized as a blank control. The experiments were repeated three times.

3.6. Cytotoxic Assays

The cytotoxicities of the isolated Compounds 1–8 against six human cancer cell lines— MDA-MB-231 (breast cancer cells), MDA-MB-435 (breast cancer cells), HCT116 (colon cancer cells), A549 (lung cancer cells), SNB19 (glioma cells), PC3 (prostate cancer cells), and normal human cell line HLF (human lung fibroblasts)—were evaluated using the MTT method as previous described. Cisplatin was used as the positive control.

Human cancer cell lines MDA-MB-231, MDA-MB-435, HCT116, A549, SNB19, PC3 cells, and normal human cell line HLF were obtained from the Shanghai Institute of Biological Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carls-bad, CA, USA) supplemented with 5% fetal bovine serum (Hyclone, Logan, UT, USA), 2 mM L-glutamine, 100 mg⋅mL⁻¹ streptomycin, and 100 units⋅mL⁻¹ penicillin (Invitrogen, Carlsbad, CA, USA). The cultures were maintained at 37 °C in a humidified atmosphere of 5% CO₂. The time of cell cultivation with tested compounds was 36 h.

3.7. Preparation of MTPA Esters of 7 by the Modified Mosher Ester Method

Compound 7 (2.0 mg) was reacted with (S)-α-methoxy-α-(trifluoromethyl)-phenylacetyl chloride [(S)-MTPA-Cl, 50 μL] in pyridine-d₅ (500 μL) for 12 h at room temperature. Through the same procedure, (R)-MTPA-Cl was used for the reaction. The ¹H NMR and HSQC spectra of the (R)- and (S)-MTPA esters were recorded. The chemical shifts were assigned based on the HSQC spectrum in order to calculate the Δδ_S−R values.

(S)-MTPA ester for 7a: ¹ H NMR (pyridine-d₅, 600 MHz) δ_H 2.92 (Hα-7), δ_H 2.95 (Hβ-7), δ_H 2.68 (Hα-9), δ_H 2.98 (Hβ-9).

(R)-MTPA ester for 7b: ¹ H NMR (pyridine-d₅, 600 MHz) δ_H 2.82 (Hα-7), δ_H 2.93 (Hβ-7), δ_H 2.84 (Hα-9), δ_H 3.05 (Hβ-9).

3.8. X-ray Crystallographic Analyses

Compounds 10 and 11 were obtained as colorless crystals from a methanol solution evaporated at room temperature. Crystal X-ray diffraction data for 10 and 11 were measured on an Agilent Gemini Ultra diffractometer using graphite-monochromated Cu Kα radiation (λ = 1.54178 Å). The crystallographic data of 10 and 11 have been deposited in the Cambridge Crystallographic Data Centre (CCDC number: 2379563, and 2379564, respectively).

Crystal data of 10: C₃₀H₃₇NO₆, M_r = 507.60, monoclinic, space group: C2, Z = 4, a = 28.9239(5) Å, b = 7.38262(13) Å, c = 13.1203(3) Å, α = γ = 90°, β = 98.9524(17)°, V = 2767.50(9) ų, D_c = 1.218 g/cm³, μ = 0.682 mm⁻¹, and F(000) = 1088.0. Independent reflections: 5592 (R_int = 0.0655, R_sigma = 0.0416). The final R₁ value was 0.0476, wR₂ = 0.1213 [I ≥ 2σ (I)]. The goodness of fit on F² was 1.044. The Flack parameter value was 0.14(15).

Crystal data of 11: C₃₀H₃₇NO₆, M_r = 507.60, monoclinic, space group: C2, Z = 4, a = 29.0003(9) Å, b = 7.2996(2) Å, c = 13.5642(5) α = γ = 90°, β = 98.693(3)°, V = 2838.45(16) ų, D_c = 1.188 g/cm³, μ = 0.665 mm⁻¹, and F(000) = 1088.0. Independent reflections: 5538 (R_int = 0.0857, R_sigma = 0.0672). The final R₁ value was 0.0571, wR₂ = 0.1349 [I ≥ 2σ (I)]. The goodness of fit on F² was 1.039. The Flack parameter value was −0.1(2).

4. Conclusions

In conclusion, twelve compounds, including four undescribed cytochalasins, xylariachalasins A–D (1–4), four undescribed polyketides (5–8), and four known cytochalasins (9–12), were isolated from the mangrove endophytic fungus Xylaria arbuscula QYF. The planar structure of compounds was elucidated through detailed HRESIMS and 1D and 2D NMR analysis. The absolute configurations of the new compounds were confirmed by ECD calculation, ¹³C NMR calculation and DP4+ analysis, single-crystal X-ray diffraction, and the modified Mosher ester method. Compounds 1 and 2 are rare cytochalasin hydroperoxides. In bioassays, antimicrobial and cytotoxic activities were carried out for compounds 1–12. Compound 2 showed moderate antimicrobial activities against MRSA, S. aureus, P. aeruginosa, and C. albicans, with MIC values in the range of 12.5–25 μM. Compound 10 exhibited significant cytotoxic activity against MDA-MB-435 with an IC₅₀ value of 3.61 ± 1.60 μM.