Antimicrobial activity and cytotoxicity of polyketides isolated from the mushroom Xerula sp. BCC56836

Abstract

Twelve new compounds, including (−)-oudemansin A acid (1), (−)-oudemansin A ethyl ester (3) and (−)-oudemansin X ethyl ester (5), (+)-oudemansin A lactone (6) and (+)-oudemansin X lactone (7), (+)-dihydrooudemansinol (8), (−)-11-epidihydrooudemansinol (9), (+)-xeruhydrofuranol (10), (+)-9-epixeruhydrofuranol (11), xerucitrinic acids A (14) and B (15), and (2R,3R,8aR)-2-(E-hept-5-en-1-yl)-3-methyltetrahydro-2H-pyrrolo[2,1-b][1,3]oxazin-4(3H)-one (16) and one naturally new compound, strobilurin A acid (12), together with nine known compounds such as (−)-oudemansins A (2) and X (4), (3Z,5E)-3-methyl-6-phenylhexa-3,5-dien-1-ol (13), 2-(E-hept-5-en-1-yl)-3-methyl-6,7,8,8a-tetrahydro-4H-pyrrolo[2,1-b][1,3]oxazin-4-one (17), scalusamides A–C (18–20), phenol A acid (21), and dihydrocitrinone (22), were isolated from the mushroom Xerula sp. BCC56836. Their chemical structures were established based on the information from NMR spectroscopic and mass spectrometric analyses, specific rotation values, and chemical means. A plausible biosynthesis of xerucitrinic acids A (14) and B (15), the first citrinin dimers with spiro skeletons, was also proposed. In addition, the isolated compounds were evaluated for antimicrobial activity, including antimalarial, antifungal, and antibacterial activities, and their cytotoxicity against both cancerous (MCF-7, KB, NCI-H187) and non-cancerous (Vero) cells. Compounds 2, 4, and 5 possessed antimalarial activity against Plasmodium falciparum, K1 strain (IC₅₀ 1.19–13.70 μM) and compounds 3–5, 12–14, and 17 exhibited anti-Bacillus cereus activity (MIC 12.5–25.0 μg mL⁻¹). Most isolated compounds showed low cytotoxicity against both cancerous and non-cancerous cells.

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1. Introduction

Basidiomycetes, especially mushrooms, have received considerable attention as a source of diverse secondary metabolites, which are sometimes unique in chemical structure, including sterostreins A–E from Stereum ostrea BCC22955,¹ nambinones A–D and 1-epi-nambinone B from Neonothopanus nambi,² terreumols A–D from Tricholoma terreum kib20111212,³ ganodermalactones A–G from Ganoderma sp. KM01,⁴ hexagonins A–E from Hexagonia apiaria,⁵ laxitextines A and B from Laxitextum incrustatum STMA 14285,⁶ and pyristriatins A and B from Cyathus cf. striatus MFLU15-1416.⁷ These compounds also possess a wide range of biological activities such as antimalarial (against Plasmodium falciparum),^1,4 anti-inflammatory,⁵ antibacterial, and antifungal activities⁷ and display a broad range of cytotoxicities (against cancerous and non-cancerous cell lines).^1–3,6,7 In addition, the genus Xerula is a member of the class Basidiomycetes and to the best of our knowledge, only few bioactive compounds from this genus have been documented. For example, antimicrobial (E)-β-methoxyacrylates, strobilurin C, and (−)-oudemansin B were isolated from the cultures of Xerula longipes and X. melanotricha, respectively.⁸ Moreover, γ-alkylidenebutenolides (such as xerulin, dihydroxerulin, and xerulinic acid) reported as cholesterol biosynthesis inhibitors were isolated from the culture of Xerula melanotricha.⁹

In our continuing screening programme in the search for bioactive compounds from Thai microorganisms, we came across the crude extracts from broth and cells of Xerula sp. BCC56836, exhibiting antimalarial activity against Plasmodium falciparum K1 strain, multidrug-resistant strain, with IC₅₀ values in a range of 3.47 to >10 μg mL⁻¹ and also giving prolific chemical profiles from HPLC analysis. Therefore, the chemical constituents from the crude extracts were conducted. The investigation led to the isolation of twelve new compounds, which included (−)-oudemansin A acid (1), (−)-oudemansin A ethyl ester (3) and (−)-oudemansin X ethyl ester (5), (+)-oudemansin A lactone (6) and (+)-oudemansin X lactone (7), (+)-dihydrooudemansinol (8), (−)-11-epidihydrooudemansinol (9), (+)-xeruhydrofuranol (10), (+)-9-epixeruhydrofuranol (11), xerucitrinic acids A (14) and B (15), (2R,3R,8aR)-2-(E-hept-5-en-1-yl)-3-methyltetrahydro-2H-pyrrolo[2,1-b][1,3]oxazin-4(3H)-one (16), and one naturally new compound, strobilurin A acid (12), along with nine known compounds, which were (−)-oudemansins A (2) and X (4), (3Z,5E)-3-methyl-6-phenylhexa-3,5-dien-1-ol (13), 2-(E-hept-5-en-1-yl)-3-methyl-6,7,8,8a-tetrahydro-4H-pyrrolo[2,1-b][1,3]oxazin-4-one (17), scalusamides A–C (18–20), phenol A acid (21), and dihydrocitrinone (22). The isolated compounds were then evaluated for antimicrobial activity, including antimalarial (against Plasmodium falciparum, K1 – multidrug resistant strain), antibacterial (against Bacillus cereus), antifungal (against Candida albicans), anti-phytopathogenic fungal (against Alternaria brassicicola, Colletotrichum capsici, C. gloeosporioides) activities, as well as cytotoxicity against both cancerous (MCF-7, KB, NCI-H187) and non-cancerous (Vero) cells.

2. Results and discussion

2.1. Structure elucidation

Chemical structures of twelve new secondary metabolites and one naturally new compound were determined on the basis of spectroscopic information including NMR, mass, FT-IR, UV spectroscopic data together with the specific rotations and chemical methods. The ¹H and ¹³C NMR spectroscopic data of the known compounds were compared with the previously published data for (−)-oudemansin A (2),^10,11 (−)-oudemansin X (4),^12,13 (3Z,5E)-3-methyl-6-phenylhexa-3,5-dien-1-ol (13),¹⁴ 2-(E-hept-5-en-1-yl)-3-methyl-6,7,8,8a-tetrahydro-4H-pyrrolo[2,1-b][1,3]oxazin-4-one (17),¹⁵ scalusamides A–C (18–20),¹⁶ phenol A acid (21),¹⁷ and dihydrocitrinone (22).¹⁸

Compound 1 was obtained as a yellow oil. Its molecular formula was deduced to be C₁₆H₂₀O₄ from the mass ion peak at m/z 299.1259 [M + Na]⁺ by HRESIMS data. The IR spectrum showed a broad hydroxyl absorption at ν 2500–3600 cm⁻¹ and a strong absorption at ν_max 1676 cm⁻¹ for a conjugated carboxylic acid carbonyl. Thus, the carbonyl signal resonating at δ_C 167.9 in the ¹³C NMR spectrum must be a carboxylic carbon. The ¹H and ¹³C NMR spectroscopic data (Table 1) of compound 1 were similar to those of previously identified oudemansin A (2),^10,11 except that one methoxy signal was absent in compound 1. The HMBC spectrum showing correlations from H-10 to C-11, C-12, C-13, and C-14; from H-12 to C-10, C-11, C-13, and C-15; and from H-15 to C-12 indicated the presence of a β-methoxyacrylate moiety linking to C-10 at the α-position of the carboxylic group. The spectroscopic evidence suggested that a methyl ester of oudemansin A (2) was replaced with an acid group. The rest of the molecule was also reassured by 2D NMR spectroscopic analyses including HMQC, HMBC, and COSY data. The configurations of two double bonds at C-7 and C-11 were assigned as E, indicated by a large coupling constant of 15.9 Hz between H-7 and H-8 and by comparison of the chemical shift with the previously reported data for oudemansins, respectively.^8,10–13 Moreover, the specific rotation value of compound 1 ([α]²⁶_D −27.0) were in good agreement with that of (−)-oudemansin A ([α]²²_D −17.0),^10,11 (−)-oudemansin B ([α]²²_D −8.3),⁸ and (−)-oudemansin X ([α]²⁵_D −20.0).^12,13 Therefore, the absolute configurations at C-9 and C-10 was assigned as 9S, 10S, same as those assigned for (−)-oudemansins. Compound 1 possess the chemical structure as shown in Fig. 1 and is given the trivial name of (−)-oudemansin A acid.

Table 1 The ¹H and ¹³C NMR assignments for compounds 1, 3, and 5

Position	1^a		3^b		5^c
	δC, type	δH, mult. (J in Hz)	δC, type	δH, mult. (J in Hz)	δC, type	δH, mult. (J in Hz)
a Recorded in acetone-d6, 400 MHz for ^1H NMR and 100 MHz for ^13C NMR.b Recorded in CDCl3, 400 MHz for ^1H NMR and 100 MHz for ^13C NMR.c Recorded in acetone-d6, 500 MHz for ^1H NMR and 125 MHz for ^13C NMR.d Data given from ^1H decoupling experiment recorded in acetone-d6.
1,5	126.3, CH	7.38, d (7.3)	126.4, CH	7.42, d (6.9)	127.5, CH	7.30, d (8.7)
2,4	128.4, CH	7.30(3), t (7.3)	128.4, CH	7.38, dd (6.9, 7.0)	113.9, CH	6.87, d (8.7)
3	127.2, CH	7.22, t (7.3)	127.3, CH	7.30, t (7.0)	159.4, C
6	137.3, C		137.2, C		129.7, C
7	132.2, CH	6.47, d (15.9)	132.4, CH	6.50, d (15.9)	132.0, CH	6.38, d (15.9)
8	129.9, CH	5.93, dd (15.9, 8.5)	129.8, CH	6.01, dd (15.9, 8.6)	127.3, CH	5.74, dd (15.9, 8.7)
9	84.7, CH	3.99, dd (9.7, 8.5)^d	85.1, CH	4.04, dd (9.7, 8.6)^d	85.0, CH	3.92, dd (9.7, 8.7)^d
10	35.8, CH	2.94, dq (9.7, 6.9)	35.8, CH	3.08, dq (9.7, 6.9)	35.7, CH	2.94, dq (9.7, 6.9)
11	112.0, C		112.8, C		112.4, C
12	159.6, CH	7.30, s	159.1, CH	7.26, s	159.2, CH	7.24, s
13	167.9, C		167.8, C		167.0, C
14	15.1, CH3	1.23, d (6.9)	15.7, CH3	1.35, d (6.9)	15.3, CH3	1.22, d (6.9)
15	60.8, CH3	3.85, s	61.2, CH3	3.85, s	60.9, CH3	3.83, s
3-OCH3					54.7, CH3	3.77, s
9-OCH3	55.7, CH3	3.26, s	56.5, CH3	3.40, s	55.5, CH3	3.24, s
16			59.6, CH2	4.20, q (7.1)	59.0, CH2	4.05, q (7.1)
17			14.3, CH3	1.30, t (7.1)	13.8, CH3	1.16, t (7.1)

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Fig. 1 The chemical structures of compounds 1–22 isolated from the mushroom BCC56836.

Compound 3 was obtained as a pale yellow oil. The ¹H and ¹³C NMR spectra were similar to those of compound 1, apart from two additional signals of a methyl at δ_H 1.30 (t, J = 7.1 Hz) and of two methylene protons at δ_H 4.20 (q, J = 7.1 Hz). The IR spectrum indicated the presence of an ester carbonyl at ν_max 1700 cm⁻¹ and the extra methylene signal attributed in HMQC spectrum to the carbon at δ_C 59.6, indicating the attachment to an oxygen. The spectroscopic information suggested that the acid in compound 1 was replaced with an ethyl ester group. HRESIMS spectrum confirmed the molecular formula of C₁₈H₂₄O₄ by showing the mass ion peak at m/z 327.1579 [M + Na]⁺. In addition, the negative specific rotation observed for compound 3 confirmed the absolute configuration at C-9 and C-10 as 9S, 10S, as shown in the Fig. 1. Compound 3 is given the trivial name of (−)-oudemansin A ethyl ester.

Compound 5 was obtained as a pale yellow oil and revealed the molecular formula C₁₉H₂₆O₅ by showing the mass ion peak at m/z 357.1670 [M + Na]⁺ in HRESIMS spectrum. The ¹H and ¹³C NMR spectroscopic data (Table 1) of compound 5 were similar to those of oudemansin X (4),^12,13 except the absence of a methoxy signal and the presence of a triplet methyl at δ_H 1.16 and a quartet methylene protons at δ_H 4.05. The similar pattern of the additional signals to those appeared for compound 3 together with the negative specific rotation ([α]²⁵_D −16.8) indicated that compound 5 had the absolute configuration at C-9 and C-10 as “S” and “S”, respectively (Fig. 1). Thus, compound 5 is named (−)-oudemansin X ethyl ester.

Compound 6 was obtained as a pale yellow oil and gave the molecular formula C₁₅H₁₆O₃, determined from the mass ion peak at m/z 267.0992 [M + Na]⁺ in HRESIMS spectrum. The IR spectrum showed a strong carbonyl absorption at ν_max 1746 cm⁻¹, suggesting a presence of a γ-lactone ring. The ¹H and ¹³C NMR spectroscopic data (Table 2) were almost identical to those of γ-lactone of noroudemansin A, which was previously synthesized by Engler-Lohr and coworkers.¹⁹ The extensive 2D NMR spectroscopic analyses, including HMQC, HMBC, and COSY data, confirmed the structural identity of compound 6 to that of documented γ-lactone derived from noroudemansin A, except that the smaller coupling constant of J_9H,10H (5.1 Hz) was observed, compared with the reported data (J_9H,10H = 7.3 Hz) for noroudemansin A.¹⁹ The evidence suggested the difference of the stereogenic centers at C-9 and C-10 in the γ-lactone moiety. Moreover, NOESY spectrum showed the cross-peak correlations from H-8 to H-10 and from H-9 to H-7 and H₃-14, indicating the cis-relationship between H-9 and H₃-14. Compound 6 should derive from the same biosynthetic pathway as those of compounds 1–5, the absolute configuration at C-10 was then assigned as “S”. The absolute configuration at C-9 was then determined to be “R”, suggested by aforementioned NOESY spectroscopic data. Thus, the chemical structure of compound 6 with absolute configurations at C-9 and C-10 as 9R, 10S was depicted in the Fig. 1. As suggested by chemdraw, the dihedral angle of approx. 125 degree between H-9 and H-10 of compound 6 resulted in a smaller coupling constant than that of γ-lactone from noroudemansin A, which had approx. 25 degree of dihedral angle. The result was assured by the Karplus equation²⁰ and the conformational study on α-methylene-γ-butyrolactones.²¹ In addition, the positive specific rotation ([α]²⁸_D +96.9) was observed for compound 6, while the negative specific rotation ([α]²⁵_D −56.5) was reported for γ-lactone derived from noroudemansin A with the absolute configurations at C-9 and C-10 as S and S, respectively.¹⁹ Therefore, compound 6 is given the trivial name of (+)-oudemansin A lactone and could possibly derive from compound 1.

Table 2 The ¹H and ¹³C NMR assignments for compounds 6–9

Position	6^a		7^a		8^a		9^b
	δC, type	δH, mult. (J in Hz)	δC, type	δH, mult. (J in Hz)	δC, type	δH, mult. (J in Hz)	δC, type	δH, mult. (J in Hz)
a Recorded in acetone-d6, 400 MHz for ^1H NMR and 100 MHz for ^13C NMR.b Recorded in acetone-d6, 500 MHz for ^1H NMR and 125 MHz for ^13C NMR.c Data given from ^1H decoupling experiment recorded in acetone-d6.
1,5	127.6, CH	7.50, d (7.3)	127.9, CH	7.43, d (8.7)	127.4, CH	7.48, d (7.4)	126.5, CH	7.49, d (7.6)
2,4	129.5, CH	7.35, dd (7.7, 7.3)	114.1, CH	6.91, d (8.7)	129.5, CH	7.34, t (7.4)	128.5, CH	7.34, t (7.6)
3	128.9, CH	7.28, t (7.7)	159.9, C		128.5, CH	7.25, t (7.4)	127.6, CH	7.25, t (7.6)
6	137.3, C		129.0, C		137.9, C		136.9, C
7	132.9, CH	6.71, d (15.9)	131.8, CH	6.64, d (15.8)	133.4, CH	6.60, d (16.0)	132.9, CH	6.64, d (16.0)
8	128.6, CH	6.35, dd (15.9, 7.1)	125.2, CH	6.17, dd (15.8, 7.4)	129.7, CH	6.21, dd (16.0, 7.3)	128.5, CH	6.21, dd (16.0, 7.6)
9	85.6, CH	4.59, dd (7.1, 5.1)^c	85.0, CH	4.54, ddd (7.4, 5.3, 0.9)^c	85.1, CH	3.69, dd (7.3, 4.3)	83.9, CH	3.80, dd (7.6, 4.6)^c
10	39.2, CH	3.00, ddq (6.9, 5.1, 2.4)^c	38.3, CH	2.97, ddq (6.9, 5.3, 2.4)^c	39.3, CH	2.09, ddq (7.5, 7.0, 4.3)^c	38.0, CH	2.02, ddq (7.0, 6.5, 4.6)^c
11	109.3, C		108.5, C		51.5, CH	2.68, dt (7.5, 6.6)^c	50.8, CH	2.75, dt (6.5, 6.4)^c
12	158.2, CH	7.29, d (2.4)	157.1, CH	7.28, d (2.4)	62.0, CH2	3.78, d (6.6)	61.7, CH2	3.77–3.82, m
13	171.7, C		170.8, C		175.5, C		173.9, C
14	17.5, CH3	1.30, d (6.9)	16.4, CH3	1.28, d (6.9)	12.3, CH3	0.99, d (7.0)	11.5, CH3	0.95, d (7.0)
15	62.5, CH3	3.94, s	61.5, CH3	3.93, s
3-OCH3			54.7, CH3	3.80, s
9-OCH3					57.0, CH3	3.23, s	55.9, CH3	3.27, s
13-OCH3					51.5, CH3	3.63, s	50.4, CH3	3.63, s

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Compound 7 was obtained as a colorless solid and had a melting point of 94.0–95.8 °C. HRESIMS data established the molecular formula C₁₆H₁₈O₄, corresponding to the mass ion peak at m/z 297.1093 [M + Na]⁺. The IR spectrum also displayed a strong carbonyl absorption of a γ-lactone ring at ν_max 1745 cm⁻¹. The ¹H and ¹³C NMR spectroscopic data (Table 2) were similar to those of compound 6, apart from the pattern of para-substituted benzyl ring and an extra methoxy signal at δ_H 3.80 (δ_C 54.7). The HMBC spectrum showed correlation from the additional methoxy signal to one of the aromatic carbon at δ_C 159.9 (C-3). The cis-relationship between H-9 and H₃-14 of compound 7 was disclosed by a cross peak correlation in the NOESY spectrum. In addition, compound 7 showed similar CD spectrum to that of compound 6 and gave the specific rotation of +133.0, versus specific rotation of +96.9 reported for the absolute configuration as 9R, 10S of lactone 6. Thus, the absolute configurations at C-9 and C-10 of compound 7 were assigned to be the same as those for compound 6 (9R and 10S). (+)-Oudemansin X lactone is given as the trivial name for compound 7.

Compounds 8 and 9 were obtained as pale yellow oil. Both gave the same molecular formula C₁₆H₂₂O₄, established by HRESIMS data. Their ¹H NMR spectra were almost identical, except that of the methylene protons (H₂-12) in compound 8 which appeared as a doublet, whereas those in 9 were shifted slightly downfield and superimposed with H-9. From HPLC analyses, compound 8 possessed higher polarity than compound 9 in the same solvent system. Both ¹H and ¹³C NMR spectroscopic data (Table 2) were similar to those of oudemansin A (2), except for the absence of an olefinic methine proton and a methoxyl signal and the presence of an additional sp³ methine proton and oxymethylene protons. The additional signals attributed in HMQC spectrum to methine (δ_C 51.5 and 50.8, C-11) and methylene carbons (δ_C 62.0 and 61.7, C-12). In the COSY spectrum, the methine at H-11 was coupled to H-10 and H₂-12. The HMBC spectrum showed partial correlations from H-11 to C-9, C-10, C-12, C-13, and C-14; from H₂-12 to C-10, C-11, and C-13; and from H₃-14 to C-9, C-10, and C-11. The spectroscopic information suggested that compounds 8 and 9 were reduced forms of oudemansin A (2). The methine signal at H-10 in compounds 8 and 9 resonated at higher field than that of compound 2 because H-10 in compound 2 was at the allylic position. The configuration of double bond at C-7 of both compounds was trans- because of the same coupling constant of 16.0 Hz. The significant difference between compounds 8 and 9 was that the coupling constant between H-10 and H-11 as verified by the ¹H decoupling experiment at H-12. The coupling constants between H-10 and H-11 in compounds 8 and 9 were 7.5 Hz and 6.5 Hz, indicating trans- and cis-relationship, respectively, due to a result of small difference in the dihedral angles. Reaction of both compounds (8 and 9) with p-TsCl in a presence of NEt₃ and DMAP, and then followed by treatment with DBU yielded compound 23 (Scheme 1), confirmed by ¹H NMR and HRESIMS data. Compound 23 gave the negative specific rotation ([α]²⁵_D −43.5), indicating that the absolute configurations at C-9 and C-10 were both S, same as those reported for (−)-oudemansins.^8,10–13 In addition, the opposite specific rotations given by compounds 8 ([α]²⁶_D +12.1) and 9 ([α]²⁶_D −11.9) were observed for the difference of stereochemistry at C-11. Together with the difference in the coupling constants between H-10 and H-11 of both compounds, the absolute configurations at C-11 in compounds 8 (J = 7.5 Hz) and 9 (J = 6.5 Hz) were assigned as “S” and “R”, respectively. Therefore, the chemical structures of compounds 8 and 9 with the absolute configurations were assigned as 9S, 10S, 11S and 9S, 10S, 11R (Fig. 1). (+)-Dihydrooudemansinol and (−)-11-epidihydrooudemansinol are given as trivial names for compounds 8 and 9, respectively.

Scheme 1 Semi-synthesis of compound 23.

Compound 10 was obtained as a colorless oil. HRESIMS established the molecular formula C₁₃H₁₆O₂, deduced from the mass ion peak at m/z 227.1054 [M + Na]⁺ in HRESIMS spectrum. The ¹H NMR spectrum gave signals of a methyl at δ_H 1.33 (s), a methylene at δ_H 2.02 (dd, J = 8.1, 6.7 Hz), two non-equivalent oxymethylene protons at δ_H 3.81 (dt, J = 13.6, 6.7 Hz) and 4.04 (dt, J = 13.6, 8.1 Hz), an oxymethine at δ_H 4.02 (d, J = 7.2 Hz), two methine at δ_H 6.35 (dd, J = 16.0, 7.2 Hz) and 6.62 (d, J = 16.0 Hz), and five aromatic protons at δ_H 7.23 (1H, t, J = 7.6 Hz), 7.32 (2H, t, J = 7.6 Hz), and 7.45 (2H, d, J = 7.6 Hz). The ¹³C NMR spectrum (Table 3) gave 11 signals, which differentiated by DEPT-135 spectrum, consisting of one methyl, two methylene, one oxymethine, five sp² methine, and two quaternary carbons. The COSY spectrum showed three spin systems from H-1–H-5, H-7–H-9, and H₂-11–H₂-12. The HMBC spectrum showed the correlations from H-8 to C-6, C-9, and C-10; from H-9 to C-7, C-12, and C-14; from H₂-11 to C-9, C-10, C-12, and C-14; from H₂-12 to C-9, C-10, and C-11; from H₃-14 to C-9, C-10, and C-11; and from 10-OH to C-9, C-10, C-11, and C-14. The coupling constant of 16.0 Hz between H-7 and H-8 indicated the configuration of the double bond as trans-. The relative stereochemistry of compound 10 was proposed based on the NOEDIFF experiments; the signals of the olefinic proton at H-7 and methyl protons at H₃-14 were enhanced upon irradiation at H-9, indicating H₃-14 and H-7 were syn-facial. Therefore, compound 10 has the chemical structure as shown in Fig. 1 and is named (+)-xeruhydrofuranol.

Table 3 The ¹H and ¹³C NMR assignments for compounds 10 and 11

Position	10^a		11^a
	δC, type	δH, mult. (J in Hz)	δC, type	δH, mult. (J in Hz)
a Recorded in acetone-d6, 400 MHz for ^1H NMR and 100 MHz for ^13C NMR.b Data given from ^1H decoupling experiment recorded in acetone-d6.
1,5	127.4, CH	7.45, d (7.6)	127.3, CH	7.44, d (7.5)
2,4	129.4, CH	7.32, t (7.6)	129.4, CH	7.32, t (7.5)
3	128.2, CH	7.23, t (7.6)	128.2, CH	7.23, t (7.5)
6	138.4, C		138.3, C
7	133.1, CH	6.62, d (16.0)	131.4, CH	6.59, d (15.9)
8	127.9, CH	6.35, dd (16.0, 7.2)	129.4, CH	6.24, dd (15.9, 6.5)
9	88.3, CH	4.02, d (7.2)	89.0, CH	4.21, d (6.5)
10	79.2, C		80.1, C
11	42.2, CH2	2.02, dd (8.1, 6.7)^b	41.5, CH2	2.05 (overlapped with acetone-d6 signal), 1.98, dt (11.8, 8.2)^b
12	66.4, CH2	4.04, dt (13.6, 8.1)^b, 3.81, dt (13.6, 6.7)^b	66.6, CH2	3.95, dd (8.2, 6.0)^b
14	24.4, CH3	1.33, s	24.1, CH3	1.26, s
10-OH		3.63, s		3.98, s

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Compound 11 was obtained as a colorless oil and had the same molecular formula as that of compound 10 by revealing the mass ion peak of m/z 227.1047 [M + Na]⁺ in the HRESIMS spectrum. The ¹H NMR spectrum (Table 3) was similar to that of compound 10, except that the pattern of methylene signals of H₂-11 and H₂-12 was significantly different. From HPLC analyses, compound 10 was slightly more polar than compound 11 in the same solvent system. However, the spectroscopic evidences, including COSY, HMQC, and HMBC data, confirmed the structural identity of compounds 10 and 11. From NOEDIFF experiment, upon irradiation at H-9, the signals at H-7 and 10-OH were enhanced, while H-8 was affected when H₃-14 was irradiated. The latter indicated the different stereogenic center at C-9. Therefore, compound 11 was an epimer of compound 10 and was given the trivial name of (+)-9-epixeruhydrofuranol.

Compound 12 was obtained as a yellow oil. Its molecular formula was determined to be C₁₅H₁₆O₃ based on the mass ion peak at m/z 267.0994 [M + Na]⁺ in HRESIMS spectrum. The spectroscopic data were in a good agreement with the data previously reported for synthetic strobilurin A acid.²² However, this is the first report of compound 12 isolated from a natural source.

Compound 14 was obtained as a yellow oil. The molecular formula was determined to be C₂₆H₃₀O₈, deduced from the mass ion peak at m/z 493.1843 [M + Na]⁺ in the HRESIMS spectrum. The IR spectrum showed broad hydroxyl absorption at ν 2500–3600 cm⁻¹ and a carbonyl absorption at ν_max 1681 cm⁻¹, suggesting a presence of carboxylic group. The ¹H NMR spectrum (Table 4) showed signals of six methyl, two sp³ methine, two sp³ oxymethine, one methylene, one oxymethylene, and three hydroxyl protons. Moreover, twelve quaternary, one sp³ oxygenated quaternary, and one carbonyl carbons were observed in the ¹³C NMR spectrum, which differentiated by DEPT-135 spectrum. The COSY spectrum showed cross-peak correlations of four isolated spin-systems, which included H-1/H₂-11, H-3/3-CH₃, H-4/4-CH₃, and H-7′/7′-CH₃. The HMBC spectrum showed correlations from H-3 to C-1, 3-CH₃, 4-CH₃, and C-4a; from H-4 to 3-CH₃, 4-CH₃, C-4a, C-5, and C-8a; from 3-CH₃ to C-3 and C-4; from 4-CH₃ to C-3, C-4, and C-4a; from 5-CH₃ to C-4a, C-5, and C-6; from 6-OH to C-5, C-6, and C-7; from H₂-11 to C-1, C-8a, and C-10; from H₂-2′ to C-2′a, C-3′, C-6′a, and C-10; from 4′-CH₃ to C-3′, C-4′, and C-5′; from 6′-CH₃ to C-5′, C-6′, and C-6′a; from H-7′ to C-6′, C-6′a, 7′-CH₃, and C-10; from 7′-CH₃ to C-6′a, C-7′, and C-10; from 3′-OH to C-2′a and C-4′; and from 5′-OH to C-4′ and C-6′ (Fig. 2). The spectroscopic data suggested that there were two subunits linking at C-10, one of which was closely related to penicitrinol K, isolated from a marine-derived Penicillium sp.²³ The remaining carboxylic carbon (δ_C 171.0) was placed at C-7 by comparison with the spectroscopic data of penicitrinol K.²³ The zero coupling constant between H-3 and H-4 (J = 0 Hz) indicated a dihedral angle of 90 degree (Fig. 3). In addition, the key NOESY correlations (Fig. 3) from 3-CH₃ to H-1 and H-4; from H-1 to H-11α; and from H-7′ to H-11β and 6′-CH₃ led to the relative configurations at C-1, C-3, C-4, C-10, and C-7′. Furthermore, the co-metabolite 21 (phenol A acid) was isolated from this mushroom and could be a precursor of compound 14. The absolute configurations at C-2 and C-3 of compound 21 were identically assigned to those of the related compound, threo(2R,3S)-phenol A, by comparison of ¹H NMR spectroscopic data and the specific rotations ([α]²¹_D −14.6 of compound 21 versus [α]²³_D −36.8 of phenol A).¹⁷ Therefore, the complete stereochemical assignment for compound 14 could be established as 1R, 3R, 4S, 10S, 7′S (Fig. 1). The absolute configurations at C-3, C-4, and C-7′ were identical with those of previously reported citrinin and redoxcitrinin, respectively.²⁴ Compound 14 is given the trivial name of xerucitrinic acid A.

Table 4 The ¹H and ¹³C NMR assignments for compounds 14–16

Position	14^a		15^a		16^b
	δC, type	δH, mult. (J in Hz)	δC, type	δH, mult. (J in Hz)	δC, type	δH, mult. (J in Hz)
a Recorded in acetone-d6, 500 MHz for ^1H NMR and 125 MHz for ^13C NMR.b Recorded in acetone-d6, 400 MHz for ^1H NMR and 100 MHz for ^13C NMR.c Exchangeable.
1	58.6, CH	4.91, dd (12.3, 4.7)	66.9, CH	4.74, dd (11.4, 6.2)
2					78.7, CH	3.41, dt (9.0, 6.8)
3	73.9, CH	4.14, q (6.8)	79.4, CH	3.76, q (6.2)	40.3, CH	2.35, quint (6.8)
4	34.9, CH	2.83, q (6.9)	38.8, CH	2.85–2.92, m	170.1, C
4a	144.0, C		146.7, C
5	117.1, C		118.2, C
6	160.4, C		162.0, C		44.2, CH2	3.67, dt (11.1, 6.0), 3.19, dt (11.1, 6.1)
7	97.7, C		98.2, C		21.5, CH2	1.84–1.92, m, 1.74–1.84, m
8	148.6, C		148.1, C		32.2, CH2	2.13–2.22, m, 1.84–1.92, m
8a	110.7, C		113.1, C		83.0, CH	5.22, dd (4.9, 3.4)
9
10	105.7, C		105.3, C
11	35.9, CH2	2.58, dd (12.3, 4.7), H-11α, 2.00, dd (12.3, 12.3), H-11β	35.3, CH2	2.72, dd (13.0, 6.2), H-11β, 1.91, dd (13.0, 11.4), H-11α
1′					33.5, CH2	2.13–2.22, m, 1.52–1.67, m
2′	61.6, CH2	4.97, d (15.3), 4.85, d (15.3)	62.5, CH2	4.93, d (15.3), 4.70, d (15.3)	25.1, CH2	1.52–1.67, m
2′a	110.7, C^c		111.3, C
3′	148.1, C		149.5, C		29.0, CH2	1.31–1.44, m
4′	110.4, C^c		111.1, C		32.5, CH2	1.96–2.03, m
5′	152.9, C		153.8, C		131.3, CH	5.35–5.47, m
6′	113.7, C		114.5, C		124.4, CH	5.35–5.47, m
6′a	131.7, C		132.6, C
7′	38.4, CH	3.32, q (6.9)	38.5, CH	3.32, q (7.0)	17.0, CH3	1.61, br s
3-CH3	17.6, CH3	1.36, d (6.8)	21.8, CH3	1.35, d (6.2)	12.0, CH3	1.07, d (6.8)
4-CH3	21.2, CH3	1.22, d (6.9)	19.5, CH3	1.24, d (7.0)
5-CH3	9.2, CH3	2.08, s	11.2, CH3	2.11, s
6-OH		12.32, s		12.4, s
7-COOH	171.0, C		172.0, C
3′-OH		7.36^c, s		7.32^c, s
4′-CH3	8.3, CH3	2.19, s	9.2, CH3^c	2.17(5), s
5′-OH		7.25^c, s		7.25^c, s
6′-CH3	9.6, CH3	2.16, s	10.5, CH3^c	2.17(9), s
7′-CH3	16.8, CH3	1.24, d (6.9)	17.4, CH3	1.26, d (7.0)

---

Fig. 2 COSY and selected HMBC correlations of compound 14.

Fig. 3 Key NOESY correlations of compounds 14 and 15.

Compound 15 was obtained as a yellow oil. HRESIMS spectrum revealed the same mass ion peak as that of compound 14. Compound 15 had a lower polarity than compound 14 in the same solvent system. The ¹H NMR spectrum (Table 4) was almost identical to that of compound 14, except the upfield methine at δ_H 3.76 (H-3), 4.74 (H-1), and a slight shift of the non-equivalent methylene signals at δ_H 1.91 and 2.72. The evidences suggested they were distinct. However, 2D NMR spectroscopic data including COSY, HMQC, and HMBC provided the same information to those of compound 14 as well as the cross-peak correlations in NOESY spectrum from H-3 to 4-CH₃ and from H-4 to 3-CH₃. Moreover, the different cross-peak correlations in the NOESY spectrum from H-3 to H-1; and from H-11β to H-1 and 7′-CH₃ suggested the different stereogenic centers at C-1 and C-10 (Fig. 3). Thus, the chemical structure of compound 15 could be depicted as 1S, 3R, 4S, 7′S, 10R (Fig. 1). Compound 15 was given a trivial name of xerucitrinic acid B. However, it should be noted that compounds 14 and 15 were not quite stable, they slowly decomposed.

A plausible biosynthetic pathway of compounds 14 and 15 was proposed via citrinin biosynthesis (Scheme 2).^25–29 A biogenetic precursor of citrinin, known as redoxcitrinin,^28,30 would presumably be involved in the biosynthesis by a conjugate addition with citrinin to provide intermediate A. Redoxcitrinin was earlier isolated as co-metabolite from the marine fungus Penicillium sp., along with citrinin and phenol A acid.³⁰ Then, intermediate A underwent aldehyde reduction (intermediate B) and ring cyclization³¹ to form spiroketal compounds 14 and 15, which explained the possibility of having different configurations at C-1 and C-10 of both compounds. Both compounds 14 and 15 can be categorized in the azaphilones family that has citrinin as a core structure. Azaphilones containing citrinin moiety have been widely produced from various fungal genera such as Penicillium, Monascus, and Aspergillus.³²

Scheme 2 A plausible biosynthetic pathway of compounds 14 and 15.

Compound 16 was obtained as a yellow oil. HRESIMS data revealed the mass ion peak at m/z 274.1767 [M + Na]⁺ for the molecular formula to be C₁₅H₂₅NO₂. The ¹H and ¹³C NMR spectroscopic data (Table 4) of compound 16 were similar to those of 2-(E-hept-5-en-1-yl)-3-methyl-6,7,8,8a-tetrahydro-4H-pyrrolo[2,1-b][1,3]oxazin-4-one (17),¹⁵ apart from the absence of a singlet methyl signal and the presence of a double methyl signal at δ_H 1.07, a quintet methine at δ_H 2.35, and a doublet of triplets oxymethine signal at δ_H 3.41. In the HMQC spectrum, two extra methine signals attributed to the carbons at δ_C 40.3 and 78.7. The spectroscopic information indicated the absence of double bond at C-2/C-3 in compound 17. The rest of the structure was secured by 2D NMR spectroscopic analyses (COSY, HMQC, and HMBC). The E-configuration of the double bond at C-5′ was suggested by the chemical shift of an allylic carbon at C-7′ (δ_C 17.0), compared to the previously reported data for trans-allylic carbons.^16,33,34 The NOESY spectrum showed cross-peak correlations from H-2 to 3-CH₃; and from H-8a to H-3 and H₂-1′. Therefore, the relative stereochemistry of compound 16 was assigned as 2R*, 3R*, 8aR* (Fig. 1). Thus, compound 16 was (2R,3R,8aR)-2-(E-hept-5-en-1-yl)-3-methyltetrahydro-2H-pyrrolo[2,1-b][1,3]oxazin-4(3H)-one.

2.2. Biological activity

All isolated compounds, except 15 which was obtained in insufficient amount, were subjected to biological tests for antimalarial against Plasmodium falciparum (K1, multidrug-resistant strain), for antibacterial against Bacillus cereus, for antifungal against Candida albicans, for anti-phytopathogenic against Alternaria brassicicola, Colletotrichum capsici, and C. gloeosporioides activities, and for cytotoxicity against cancerous (MCF-7, KB, NCI-H187) and non-cancerous (Vero) cells (Table 5).

Table 5 Biological activities of the isolated compounds 2–6, 10, 12–14, and 17

Compounds	Anti-P. falciparum^b (IC50, μM)	Anti-B. cereus^c (MIC, μg mL^−1)	Anti-C. albicans^d (IC50, μM)	Anti-phytopathogenic^d (MIC, μg mL^−1)			Cytotoxicity^d (IC50, μM)
				A. brassicicola	C. capsici	C. gloeosporioides	MCF-7	KB	NCI-H187	Vero
a Antimicrobial activity against other organisms using a different method was reported in the literature.b Maximum tested concentration was done at 10 μg mL^−1.c Maximum tested concentration was done at 25 μg mL^−1.d Maximum tested concentration was done at 50 μg mL^−1.
2^a	9.23	>25	137.45	50.00	25.00	>50	120.13	160.18	63.30	26.73
3	>32.85	25.00	>164.26	>50	25.00	>50	>164.26	>164.26	57.03	108.18
4^a	1.19	25.00	129.78	25.00	25.00	>50	>156.06	>156.06	99.07	85.90
5	13.70	25.00	>149.52	50.00	12.50	25.00	>149.52	>149.52	65.46	67.19
6	>40.93	>25	>204.67	>50	>50	>50	>204.67	>204.67	198.33	>204.67
10	>48.95	>25	>244.77	>50	>50	>50	>244.77	>244.77	203.85	>244.77
12	>40.93	25.00	>204.67	>50	>50	>50	>204.67	>204.67	>204.67	>204.67
13	>53.12	25.00	>265.58	>50	>50	>50	>265.58	231.10	>265.58	>265.58
14	>21.25	12.50	>106.27	>50	>50	>50	>106.27	>106.27	>106.27	>106.27
17	>40.10	25.00	191.62	>50	>50	>50	>200.52	>200.52	191.26	117.71
Dihydroartemisinin	2.83 × 10^−3	—	—	—	—	—	—	—	—	—
Mefloquine	0.04	—	—	—	—	—	—	—	—	—
Vancomycin	—	2.00	—	—	—	—	—	—	—	—
Amphotericin B	—	—	0.21	1.56	1.56–3.13	1.56–3.13	—	—	—	—
Ellipticine	—	—	—	—	—	—	—	5.48	7.23	5.56
Doxorubicin	—	—	—	—	—	—	13.56	0.96	0.20	—
Tamoxifen	—	—	—	—	—	—	18.06	—	—	—

---

Compounds 1, 7–9, 11, 16, and 18–22 were inactive in our biological assays at maximum tested concentration. Compounds 2, 4, and 5 exhibited anti-P. falciparum with IC₅₀ values in a range of 1.19–13.70 μM and showed anti-phytopathogenic activity against A. brassicicola and C. capsici with MIC values ranging from 12.5–50.0 μg mL⁻¹. Compound 3 was active against C. capsici, while compound 5 was active against C. gloeosporioides with the same MIC value of 25.0 μg mL⁻¹. Compounds 2, 4, and 17 displayed weak antifungal activity against C. albicans with IC₅₀ values in a range of 129.78–191.62 μM (Table 5). All compounds (except compounds 2, 6, and 10) exhibited anti-B. cereus with MIC values ranging from 12.5–25.0 μg mL⁻¹ and showed weak cytotoxicity against both cancerous and non-cancerous cells (Table 5).

(−)-Oudemansin A (2) and (−)-oudemansin X (4) were originally isolated from Basidiomycete Oudemansiella mucida¹⁰ and O. radicata,¹² respectively. The biological activity of both compounds was suggested to be due to the presence of an (E)-β-methoxyacrylate moiety. Both compounds exhibited a broad range of antifungal properties towards several fungi such as Paecilomyces variotii, Rhodotorula glutinis, Saccharomyces cerevisiae.^10,12 (3Z,5E)-3-Methyl-6-phenylhexa-3,5-dien-1-ol (13) was formerly isolated from the fungus Bolinea lutea and proposed to be a precursor or a side-product during the biosynthesis of strobilurin A.¹⁴ 2-(E-Hept-5-en-1-yl)-3-methyl-6,7,8,8a-tetrahydro-4H-pyrrolo[2,1-b][1,3]oxazin-4-one (17) was previously isolated from Penicillium brevicompactum Dierckx.¹⁵ Compound 17 displayed insecticidal activity against the fourth-instar milkweed bug, Oncopeltus fasciatus (Dallas), with a LD₅₀ value of 20 μg mL⁻¹ and also inhibited more than 40% mycelial growth at 100 μg mL⁻¹ of various plant phytopathogens e.g., Fusarium culmorum, Colletotrichum coccodes, Alternaria tenuis, Penicillium italicum.³⁵ Scalusamides A–C (18–20) were originally isolated from the marine-derived fungus Penicillium citrinum N055.¹⁶ Only scalusamide A had antifungal against Cryptococcus neoformans and antibacterial against Micrococcus luteus activities with MIC values of 16.7 and 33.3 μg mL⁻¹, respectively.¹⁶ Moreover, phenol A acid (21) and dihydrocitrinone (22) were previously isolated from Penicillium citrinum MST-F10130 ³⁶ and Aspergillus terreus Thom,³⁷ respectively. Compounds 21 and 22 displayed enzyme-inhibitory activity against cathepsin B with IC₅₀ values of 20.4 ± 1.9 and 28.5 ± 1.7 μM, respectively, while compound 21 also exhibited antifouling activity against Bugula neritina larvae settlement with an EC₅₀ value of 14.35 ± 1.72 μg mL⁻¹.³⁸

3. Conclusions

Thirteen naturally new compounds (1, 3, 5−12, and 14–16), together with nine known compounds (2, 4, 13, 17–22), have been isolated and identified from the mushroom Xerula sp. BCC56836. These new compounds included oudemansins 1, 3, 5–9, furanol derivatives 10 and 11, strobilurin derivative 12, the rare citrinin dimers with spiro skeleton 14 and 15, and compound 16. These isolated compounds exhibited various biological activities, which included compounds 2, 4, and 5 showing antimalarial activity against P. falciparum (IC₅₀ 1.19–13.70 μM) and anti-phytopathogenic activities against A. brassicicola and C. capsici (MIC 12.5–50.0 μg mL⁻¹), compounds 2, 4, and 17 having antifungal activity against C. albicans (IC₅₀ 129.78–191.62 μM), and compounds 3–5, 12–14, and 17 exhibiting antibacterial against B. cereus (MIC 12.5–25.0 μg mL⁻¹). Moreover, compounds 3 displayed activity against C. capsici, while compound 5 showed anti-phytopathogenic activity against C. gloeosporioides with the same MIC value of 25.0 μg mL⁻¹. All compounds, except compound 2, displayed low cytotoxicity against both cancerous and non-cancerous cells. Only compound 2 had strong cytotoxicity against non-cancerous cells (Vero) with an IC₅₀ value of 26.73 μM. From our results, compounds 4 and 5 showed broad spectrum of antimicrobial activity with relatively low cytotoxicity. Also, it should be noted that the instability of the spiro compounds might affect in the biological activity.

4. Experimental

4.1. General experimental procedures

A melting point was determined by using a melting point M565 apparatus from Buchi. Specific rotations were obtained by using JASCO P-1030 digital polarimeter. The CD spectra were measured in MeOH on a JASCO J-810 spectropolarimeter. UV spectra were taken in MeOH on a Spekol 1200 from Analytik Jena UV-Vis spectrophotometer. FTIR spectra were performed on a Bruker ALPHA spectrometer. NMR spectra were detected on either Bruker Avance-III 400 (400 MHz for ¹H and 100 MHz for ¹³C) or Bruker Avance 500 (500 MHz for ¹H and 125 MHz for ¹³C) NMR spectrometers. HRESIMS data were recorded on a Bruker MicrOTOF mass spectrometer. Semi-preparative HPLC was done by a reverse phase column (SunFire C₁₈ OBD, 5 μm, diam. 19 mm × 150 mm) at the flow rate of 9 mL min⁻¹ and preparative HPLC was performed by a reverse phase column (SunFire C₁₈ OBD, 10 μm, diam. 19 mm × 250 mm) at the flow rate 15 mL min⁻¹. Both semi-preparative and preparative HPLC were done on a Dionex – Ultimate 3000 series equipped with a binary pump, an autosampler, and a diode array detector. Preparative thin layer chromatography (PLC) was performed by using silica gel 60 GF₂₅₄ from Merck.

4.2. Fungal material

The fungus was isolated from soil in a forest in Chiang Rai province, Thailand. It was deposited and registered as BCC56836 at BIOTEC Culture Collection (BCC), the National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. The fungus was identified based on the analysis of the internal transcribed spacer (ITS1-5.8S – ITS2) rDNA and the partial nuclear large subunit ribosomal DNA (nc28S). The sequences (ITS and nc28S) were registered at GenBank with accession numbers KX755407 and KX755408, respectively. The ITS and nc28S sequence analyses, compared with the fungal taxa from GenBank and CBS-KNAW Fungal Biodiversity Centre, confirmed that the fungus was in the family Physalacriaceae, order Agaricales. This fungus also had 97–98% ITS similarity, compared with Xerula furfuracea strain JM98/155 (GenBank accession number AF321484). Therefore, the fungus strain BCC56836 was then identified as Xerula sp., and classified in Physalacriaceae, Agaricales, Agaricomycetidae, Agaricomycetes, Agaricomycotina, Basidiomycota.

4.3. Fermentation, extraction, and isolation

The fungus Xerula sp. BCC56836 was maintained on potato dextrose agar (PDA) plates at 25 °C and then cut into small pieces (1 cm × 1 cm each). The pieces were transferred into 8 × 250 mL Erlenmeyer flasks, which each contained 25 mL of potato dextrose broth (PDB, containing potato starch 4.0 g L⁻¹, dextrose 20.0 g L⁻¹ in distilled water). The seed culture was inoculated at 25 °C on a rotary shaker at 200 rpm for 6 days, then each flask was equally transferred into 4 × 1 L Erlenmeyer flasks, containing 250 mL of PDB medium. The production culture (20 L) was cultivated at 25 °C under shake condition at 200 rpm for 8 days.

After the cultivation period, the broth and cells were separated by simple filtration. The culture broth was then extracted three times with equal volume of EtOAc. The combined organic layer was dried over Na₂SO₄ and evaporated to dryness to provide a brown gum (3.4 g). The cells were macerated in MeOH (1 L) for 3 days and then in CH₂Cl₂ (1 L) for 3 days. Organic solvents were combined and concentrated under reduced pressure. Water (100 mL) was added and the mixture was then extracted three times with equal volume of EtOAc. The combined organic layer was dried over Na₂SO₄ and then evaporated in vacuo to give a brown gum (2.1 g).

The crude extract from broth (3.4 g) was passed through a Sephadex LH20 column (4.5 cm × 40 cm) using 100% MeOH as an eluent to give 8 fractions. The first and sixth–eighth fractions were discarded due to an absence of organic material of interest. The second fraction (1.6 g) was further fractionated by a Sephadex LH20 column (4.5 cm × 40 cm) eluted with 100% MeOH to provide 8 subfractions (2.1–2.8). Purification of subfraction 2.4 (0.6 g) by a preparative HPLC using a linear gradient elution of 30–70% aqueous MeCN over 40 min obtained compounds 18 (0.2 g), 19 (17.5 mg), and 17 (11.0 mg), respectively. Subfraction 2.5 (0.3 g) was purified by a preparative HPLC using a linear gradient elution of 30–70% aqueous MeCN over 40 min to give compounds 18 (67.0 mg), 19 (7.0 mg), 8 (3.4 mg), 20 (8.7 mg), 16 (6.1 mg), 17 (11.0 mg), 2 (2.5 mg), and 3 (10.4 mg), respectively. The third fraction (2.2 g) was passed through a Sephadex LH20 column (4.5 cm × 40 cm) eluted with 100% MeOH to yield 7 subfractions (3.1–3.7). Subfraction 3.3 (0.5 g) was further purified by a preparative HPLC using a linear gradient elution of 30–70% aqueous MeCN over 40 min to obtain compounds 18 (0.2 g), 19 (13.8 mg), 20 (14.7 mg), 16 (7.7 mg), 17 (17.8 mg), 2 (4.7 mg), and 3 (10.4 mg), respectively. Subfraction 3.4 (1.2 g) was subjected to a Sephadex LH20 column (4.5 cm × 40 cm) eluted with 100% MeOH to give 3 subfractions (3.4.1–3.4.3). Compound 18 (0.1 g) was obtained from subfraction 3.4.2 and subfraction 3.4.3 (1.0 g) was further purified by a preparative HPLC using a linear gradient elution of 30–70% aqueous MeCN over 45 min to provide 25 subfractions (3.4.3.1–3.4.3.25), from which compounds 8 (37.4 mg), 9 (53.7 mg), 1 (12.9 mg), 17 (28.0 mg), 2 (91.9 mg), and 3 (43.7 mg) were obtained in subfractions 3.4.3.12, 3.4.3.13, 3.4.3.14, 3.4.3.17, 3.4.3.18, and 3.4.3.21, respectively. Subfraction 3.4.3.8 (76.8 mg) was then purified by a preparative thin layer chromatography (PLC) eluted with 25% EtOAc in n-hexane to afford compounds 10 (33.5 mg) and 11 (7.3 mg). Subfractions 3.5 (0.4 g) was separated by a preparative HPLC using a linear gradient elution of 30–70% aqueous MeCN over 40 min to give 37 subfractions (3.5.1–3.5.37). Compounds 8 (7.1 mg), 9 (10.2 mg), 13 (8.3 mg), 7 (26.7 mg), 6 (13.7 mg), and 2 (8.1 mg) were obtained from the subfractions 3.5.17, 3.5.18, 3.5.22, 3.5.24, 3.5.25, and 3.5.30, respectively. Subfraction 3.5.10 (19.8 mg) was further purified by a PLC eluted with 25% EtOAc in n-hexane to furnish compounds 10 (8.8 mg) and 11 (4.7 mg), respectively. The fourth fraction (0.3 g) was applied to a preparative HPLC using a linear gradient elution of 30–70% aqueous MeCN over 40 min to provide 24 subfractions (4.1–4.24). Compounds 12 (9.1 mg) and 13 (6.7 mg) were obtained from subfractions 4.15 and 4.17, respectively. Purification of subfraction 4.8 (11.3 mg) by a PLC eluted with 25% EtOAc in n-hexane furnished compounds 10 (2.7 mg) and 11 (1.9 mg). After purification by a PLC eluted with 20% EtOAc in n-hexane, compounds 6 (2.6 mg) and 7 (5.6 mg) were respectively obtained from subfraction 4.18 and compound 2 (1.5 mg) was obtained from subfraction 4.22. The fifth fraction (0.1 g) was subjected to the same protocol as for the fourth fraction to provide compounds 7 (1.4 mg), 14 (6.3 mg), and 15 (1.9 mg), respectively. The sixth fraction (54.7 mg) was purified by a preparative HPLC using a linear gradient elution of 5–70% aqueous MeCN over 40 min to furnish compounds 21 (5.9 mg), 22 (1.9 mg), and 14 (1.3 mg), respectively.

The crude extract from cells (2.1 g) was passed through a Sephadex LH20 column (4.5 cm × 40 cm) eluted with 100% MeOH to afford 9 fractions. Fractions 1–6 were discarded due to no compounds of interest. The seventh fraction (0.6 g) was applied to a preparative HPLC using a linear gradient elution of 30–70% aqueous MeCN over 40 min to provide 14 subfractions (7.1–7.14). Compounds 18 (9.9 mg), 8 (2.2 mg), 9 (3.4 mg), 17 (3.3 mg), 4 (6.4 mg), 2 (32.7 mg), and 3 (33.5 mg) were given from subfractions 7.4, 7.5, 7.6, 7.8, 7.9, 7.10, and 7.12, respectively. Subfraction 7.11 (17.8 mg) was further purified by a PLC eluted with 15% EtOAc in n-hexane to give 5 (7.4 mg). After purification of the eighth fraction (0.5 g) by a preparative HPLC using a linear gradient elution of 30–70% aqueous MeCN over 40 min, compounds 18 (5.6 mg), 8 (4.9 mg), 9 (3.9 mg), 1 (2.6 mg), 7 (2.5 mg), 4 (23.3 mg), 2 (73.3 mg), 5 (16.5 mg), and 3 (32.7 mg) were respectively obtained. The ninth fraction (0.3 g) was subjected to the same protocol as for the eighth fraction to furnish compounds 8 (2.1 mg), 9 (1.9 mg), 1 (2.0 mg), 7 (18.1 mg), 4 (10.3 mg), 2 (30.8 mg), 5 (5.3 mg), and 3 (15.4 mg), respectively.

(−)-Oudemansin A acid (1). Yellow oil; [α]²⁶_D −27.0 (c 0.07, EtOH); UV (MeOH) λ_max (log ε) 215 (3.68), 242 (3.81) nm; FTIR (ATR) ν_max 2500–3600 (br), 2934, 2853, 1676, 1639, 1451, 1248, 1135, 1085, 967, 751, 695 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 299.1259 [M + Na]⁺ (calcd for C₁₆H₂₀O₄Na, 299.1254).

(−)-Oudemansin A ethyl ester (3). Pale yellow oil; [α]²⁵_D −16.8 (c 0.09, EtOH); UV (MeOH) λ_max (log ε) 215 (4.00), 243 (4.23) nm; FTIR (ATR) ν_max 2978, 2933, 2853, 1700, 1640, 1451, 1245, 1134, 1114, 1085, 966, 751, 694 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 327.1579 [M + Na]⁺ (calcd for C₁₈H₂₄O₄Na, 327.1567).

(−)-Oudemansin X ethyl ester (5). Pale yellow oil; [α]²⁴_D −18.3 (c 0.08, EtOH); UV (MeOH) λ_max (log ε) 215 (4.14), 245 (4.30), 260 (4.33) nm; FTIR (ATR) ν_max 2931, 2854, 1700, 1640, 1608, 1511, 1463, 1249, 1174, 1134, 1109, 1084, 966, 853, 814, 772 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 357.1670 [M + Na]⁺ (calcd for C₁₉H₂₆O₅Na, 357.1672).

(+)-Oudemansin A lactone (6). Pale yellow oil; [α]²⁸_D +96.9 (c 0.07, EtOH); UV (MeOH) λ_max (log ε) 215 (3.91), 248 (4.20); FTIR (ATR) ν_max 2953, 2932, 2854, 1746, 1672, 1596, 1450, 1235, 1146, 1087, 1022, 967, 753, 693 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 267.0992 [M + Na]⁺ (calcd for C₁₅H₁₆O₃Na, 267.0992); CD (MeOH, c 4.09 × 10⁻⁵ M) Δε (nm): +10.8 (215), −29.6 (237), +40.9 (259).

(+)-Oudemansin X lactone (7). Colorless solid; mp 94.0–95.8 °C; [α]²⁷_D +133.0 (c 0.09, EtOH); UV (MeOH) λ_max (log ε) 217 (4.10), 263 (4.37); FTIR (ATR) ν_max 2958, 2931, 2852, 1745, 1672, 1607, 1512, 1460, 1235, 1176, 1146, 1088, 1023, 967, 855, 822, 750 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 297.1093 [M + Na]⁺ (calcd for C₁₆H₁₈O₄Na, 297.1097); CD (MeOH, c 3.64 × 10⁻⁵ M) Δε (nm): +10.1 (217), −38.1 (245), +52.4 (267).

(+)-Dihydrooudemansinol (8). Pale yellow oil; [α]²⁶_D +12.1 (c 0.09, EtOH); UV (MeOH) λ_max (log ε) 214 (3.88), 248 (4.09); FTIR (ATR) ν_max 3443 (br), 2981, 2945, 2892, 1730, 1600, 1450, 1437, 1275, 1261, 1196, 1172, 1084, 972, 750, 695 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 301.1394 [M + Na]⁺ (calcd for C₁₆H₂₂O₄Na, 301.1410).

(−)-11-Epidihydrooudemansinol (9). Pale yellow oil; [α]²⁶_D −11.9 (c 0.10, EtOH); UV (MeOH) λ_max (log ε) 215 (3.92), 247 (4.12); FTIR (ATR) ν_max 3443 (br), 2974, 2939, 2893, 1730, 1600, 1450, 1437, 1380, 1367, 1196, 1169, 1083, 971, 749, 694 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 301.1404 [M + Na]⁺ (calcd for C₁₆H₂₂O₄Na, 301.1410).

(+)-Xeruhydrofuranol (10). Colorless oil; [α]²⁷_D +13.9 (c 0.07, EtOH); UV (MeOH) λ_max (log ε) 215 (3.81), 248 (4.08); FTIR (ATR) ν_max 3429 (br), 3025, 2965, 2925, 2886, 2855, 1637, 1600, 1578, 1494, 1450, 1375, 1281, 1156, 1116, 1037, 967, 747, 694 cm⁻¹; ¹H and ¹³C NMR data, see Table 3; HRESIMS m/z 227.1054 [M + Na]⁺ (calcd for C₁₃H₁₆O₂Na, 227.1043).

(+)-9-Epixeruhydrofuranol (11). Colorless oil; [α]²⁷_D +19.3 (c 0.15, EtOH); UV (MeOH) λ_max (log ε) 215 (3.98), 245 (4.21); FTIR (ATR) ν_max 3411 (br), 3026, 2970, 2925, 2885, 2853, 1636, 1600, 1494, 1450, 1377, 1273, 1143, 1107, 1034, 970, 745, 693 cm⁻¹; ¹H and ¹³C NMR data, see Table 3; HRESIMS m/z 227.1047 [M + Na]⁺ (calcd for C₁₃H₁₆O₂Na, 227.1043).

Strobilurin A acid (12). Yellow oil; UV (MeOH) λ_max (log ε) 229 (4.00), 296 (4.04) nm; FTIR (ATR) ν_max 2700–3600 (br), 2925, 2855, 1679, 1610, 1452, 1378, 1243, 1125, 965, 751, 694 cm⁻¹; ¹H NMR (400 MHz, acetone-d₆) δ_H 1.94 (3H, s, H₃-14), 3.89 (3H, s, H₃-15), 6.19 (1H, d, J = 10.8 Hz, H-9), 6.49 (1H, d, J = 15.7 Hz, H-7), 6.75 (1H, dd, J = 15.7, 10.8 Hz, H-8), 7.18 (1H, t, J = 7.6 Hz, H-3), 7.29 (2H, t, J = 7.6 Hz, H-2 and H-4), 7.39 (2H, d, J = 7.6 Hz, H-1 and H-5), 7.51 (1H, s, H-12); ¹³C NMR (100 MHz, acetone-d₆) δ_C 23.9 (CH₃, C-14), 62.1 (CH₃, C-15), 111.3 (C, C-11), 127.0 (CH, C-1 and C-5), 129.4 (CH, C-2 and C-4), 127.9 (CH, C-3), 128.3 (CH, C-8), 130.2 (CH, C-9), 131.1 (CH, C-7), 133.1 (C, C-10), 139.1 (C, C-6), 160.2 (CH, C-12), 168.0 (C, C-13); HRESIMS m/z 267.0994 [M + Na]⁺ (calcd for C₁₅H₁₆O₃Na, 267.0992).

Xerucitrinic acid A (14). Yellow oil; [α]²⁶_D +23.2 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 228 (4.20), 255 (4.09), 317 (3.77), 424 (3.07); FTIR (ATR) ν_max 2500–3600 (br), 2975, 2928, 2874, 2856, 1681, 1620, 1452, 1423, 1398, 1381, 1348, 1303, 1280, 1267, 1160, 1117, 1081, 1044, 959, 900, 811, 736, 704 cm⁻¹; ¹H and ¹³C NMR data, see Table 4; HRESIMS m/z 493.1843 [M + Na]⁺ (calcd for C₂₆H₃₀O₈Na, 493.1833).

Xerucitrinic acid B (15). Yellow oil; [α]²⁵_D −25.9 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 224 (4.13), 255 (3.99), 316 (3.71), 422 (3.22); FTIR (ATR) ν_max 2500–3600 (br), 2959, 2927, 2856, 1681, 1621, 1454, 1423, 1399, 1378, 1355, 1266, 1174, 1115, 1081, 1030, 965, 909, 814, 737, 702 cm⁻¹; ¹H and ¹³C NMR data, see Table 4; HRESIMS m/z 493.1857 [M + Na]⁺ (calcd for C₂₆H₃₀O₈Na, 493.1833).

(2R,3R,8aR)-2-(E-Hept-5-en-1-yl)-3-methyltetrahydro-2H-pyrrolo[2,1-b][1,3]oxazin-4(3H)-one (16). Yellow oil; [α]²¹_D −22.9 (c 0.22, CHCl₃); UV (MeOH) λ_max (log ε) 220 (3.23), 234 (3.27), 262 (3.12); FTIR (ATR) ν_max 2931, 2854, 1723, 1658, 1441, 1378, 1342, 1246, 1180, 1118, 1072, 965 cm⁻¹; ¹H and ¹³C NMR data, see Table 4; HRESIMS m/z 274.1767 [M + Na]⁺ (calcd for C₁₅H₂₅NO₂Na, 274.1778).

4.4. Preparation of compound 23

To a solution of compound 8 (5.00 mg, 0.0180 mmol, 1.00 eq.) in CH₂Cl₂ (500 μL) was added triethylamine (3.01 μL, 0.0216 mmol, 1.20 eq.) and p-toluene sulfonyl chloride (3.57 mg, 0.0187 mmol, 1.04 eq.) at 0 °C. After stirring for 15 min, DMAP (2.20 mg, 0.0180 mmol, 1.00 eq.) was added and the mixture was left overnight stirring at 0 °C. The solution was then quenched with a saturated aqueous NH₄Cl (2 mL) at 0 °C. The aqueous phase was then extracted with EtOAc (2 mL × 3) and EtOAc was then combined, dried over Na₂SO₄ and concentrated under reduced pressure. The resulting tosylate was obtained as yellow oil (4.57 mg, 59%) and used without further purification. To a solution of tosylate (4.57 mg, 0.0106 mmol, 1.0 eq.) in DME (400 μL) was added NaI (4.77 mg, 0.0318, 3.0 eq.) at room temperature. DBU (3.50 μL, 0.0233 mmol, 2.2 eq.) was then added and the mixture was left stirring overnight at room temperature. After addition of water (2 mL), the mixture was extracted with diethyl ether (2 mL × 3). The combined organic layer was dried over Na₂SO₄ and evaporated to dryness. The mixture was then purified by a semi-preparative HPLC, using a linear gradient elution of 30–70% aqueous MeCN over 40 min to give compound 23 (1.46 mg, 35% over two steps) as a yellow oil; [α]²⁵_D −43.5 (c 0.04, EtOH); ¹H NMR (500 MHz, acetone-d₆) δ_H 1.19 (3H, d, J = 7.0 Hz, H₃-14), 2.94 (1H, dq, J = 7.0, 6.8 Hz, H-10),^a 3.25 (3H, s, 9-OCH₃), 3.64 (3H, s, 13-OCH₃), 3.73 (1H, dd, J = 8.0, 6.8 Hz, H-9),^a 5.68 and 6.15 (each 1H, s, H₂-12), 6.06 (1H, dd, J = 17.0, 8.0 Hz, H-8), 6.52 (1H, d, J = 17.0 Hz, H-7), 7.24 (1H, t, J = 7.5, H-3), 7.32 (2H, t, J = 7.5 Hz, H-2 and H-4), 7.42 (2H, d, J = 7.5 Hz, H-1 and H-5); ¹³C NMR (125 MHz, acetone-d₆) δ_C 15.8 (CH₃, C-14), 41.2 (CH, C-10), 51.9 (CH₃, 13-OCH₃), 56.8 (CH₃, 9-OCH₃), 86.2 (CH, C-9), 125.3 (CH₂, C-12), 127.3 (CH, C-1 and C-5), 128.4 (CH, C-3), 129.4 (CH, C-2 and C-4), 129.9 (CH, C-8), 133.6 (CH, C-7), 138.0 (C, C-6), 144.2 (C, C-11), 168.3 (C, C-13); HRESIMS m/z 283.1309 [M + Na]⁺ (calcd for C₁₆H₂₀O₃Na, 283.1305).

^aThe coupling constants were determined by ¹H decoupling experiments recorded in acetone-d₆.

Compound 23 was also prepared from compound 9 by using the same protocol as for compound 8.

4.5. Biological assays

Antimalarial activity against P. falciparum (K1, multidrug-resistant strain) was performed by using the microculture radioisotope technique.³⁹ Dihydroartemisinin and mefloquine were used as standard references. Antibacterial activity against B. cereus (ATCC 11778), antifungal activity against C. albicans (ATCC 90028), and cytotoxicity against cancerous cells including MCF-7 (human breast cancer, ATCC HTC-22), KB (human oral epidermoid carcinoma, ATCC CCL-17), and NCI-H187 (human small-cell lung cancer, ATCC CRL-5804) cells were done by using the resazurin microplate assay (REMA).^40,41 Vancomycin and amphotericin B were used as standard references for anti-B. cereus and for anti-C. albicans, respectively. Doxorubicin and tamoxifen were used as standard references for anti-MCF-7, while doxorubicin and ellipticine were used as standard references for cytotoxicity against KB and NCI-H187 cells. The 5(6)-carboxyfluorescein diacetate (CFDA) fluorometric assay was employed to evaluate anti-leaf spot disease pathogen towards A. brassicicola (BCC42724)^42,43 and amphotericin B was used as a standard reference. Anti-plant pathogenic fungi⁴⁴ against C. capsici (BMGC106) and C. gloeosporioides (BMGC107) and cytotoxicity against non-cancerous cells (Vero, African green monkey kidney fibroblasts, ATCC CCL-81)⁴⁵ were carried out by using the green fluorescent protein microplate assay (GFPMA). Amphotericin B was used as a standard reference for anti-C. capsici and C. gloeosporioides, and ellipticine was used as a standard reference for cytotoxicity against Vero cell. Maximum tested concentration for all tests was at 50 μg mL⁻¹, except those for P. falciparum and B. cereus were at 10 and 25 μg mL⁻¹, respectively.

Acknowledgements

Financial support from the Thailand Research Fund (grant number TRG5880037) is gratefully acknowledged. The facilities provided by BIOTEC are kindly acknowledged.

Notes and references

1. M. Isaka, U. Srisanoh, W. Choowong and T. Boonpratuang, Org. Lett., 2011, 13, 4886.
2. S. Kanokmedhakul, R. Lekphrom, K. Kanokmedhakul, C. Hahnvajanawong, S. Bua-art, W. Saksirirat, S. Prabpai and P. Kongsaeree, Tetrahedron, 2012, 68, 8261.
3. X. Yin, T. Feng, Z.-H. Li, Z.-J. Dong, Y. Li and J. K. Liu, J. Nat. Prod., 2013, 76, 1365.
4. W. Lakornwong, K. Kanokmedhakul, S. Kanokmedhakul, P. Kongsaeree, S. Prabpai, P. Sibounnavong and K. Soytong, J. Nat. Prod., 2014, 77, 1545.
5. T. D. Thang, P.-C. Kuo, N. T. B. Ngoc, T.-L. Hwang, M.-L. Yang, S.-H. Ta, E.-J. Lee, D.-H. Kuo, N. H. Hung, N. N. Tuan and T.-S. Wu, J. Nat. Prod., 2015, 78, 2552.
6. C. M. Mudalungu, C. Richter, K. Wittstein, M. A. Abdalla, J. C. Matasyoh, M. Stadler and R. D. Süssmuth, J. Nat. Prod., 2016, 79, 894.
7. C. Richter, S. E. Helaly, B. Thongbai, K. D. Hyde and M. Stadler, J. Nat. Prod., 2016, 79, 1684.
8. T. Anke, H. Besl, U. Mocek and W. Steglich, J. Antibiot., 1983, 36, 661.
9. D. Kuhnt, T. Anke, H. Besl, M. Bross, R. Herrmann, U. Mocek, B. Steffan and W. Steglich, J. Antibiot., 1990, 43, 1413.
10. T. Anke, H. J. Hecht, G. Schramm and W. Steglich, J. Antibiot., 1979, 32, 1112.
11. H. Akita, H. Koshiji, A. Furuichi, K. Horikoshi and T. Oishi, Tetrahedron Lett., 1983, 24, 2009.
12. T. Anke, A. Werle, M. Bross and W. Steglich, J. Antibiot., 1990, 43, 1010.
13. I. Umezawa, M. Nozawa, S. Nagumo and H. Akita, Chem. Pharm. Bull., 1995, 43, 1111.
14. A. Fredenhagen, P. Hug and H. H. Peter, J. Antibiot., 1990, 43, 661.
15. Á. Cantín, P. Moya, M.-A. Castillo, J. Primo, M. A. Miranda and E. Primo-Yúfera, Eur. J. Org. Chem., 1999, 221.
16. M. Tsuda, M. Sasaki, T. Mugishima, K. Komatsu, T. Sone, M. Tanaka, Y. Mikami and J. Kobayashi, J. Nat. Prod., 2005, 68, 273.
17. T. Rödel and H. Gerlach, Liebigs Ann., 1995, 885.
18. Z.-H. Xin, L. Tian, T.-J. Zhu, W.-L. Wang, L. Du, Y.-C. Fang, Q.-Q. Gu and W.-M. Zhu, Arch. Pharmacal Res., 2007, 30, 816.
19. M. Engler-Lohr, T. Anke, V. Hellwig and W. Steglich, Z. Naturforsch., C: J. Biosci., 1999, 54, 163.
20. M. Karplus, J. Chem. Phys., 1959, 30, 11.
21. A. Kolmer, L. J. Edwards, I. Kuprov and C. M. Thiele, J. Magn. Reson., 2015, 261, 101.
22. M. Kettering, O. Sterner and T. Anke, Z. Naturforsch., C: J. Biosci., 2004, 59, 816.
23. M. L. Wang, C. H. Lu, Q. Y. Xu, S. Y. Song, Z. Y. Hu and Z. H. Zheng, Molecules, 2013, 18, 5723.
24. P. P. Mehta and W. B. Whalley, J. Chem. Soc., 1963, 3777.
25. J. Barber, R. H. Carter, M. J. Garson and J. Staunton, J. Chem. Soc., Perkin Trans. 1, 1981, 2577.
26. L. Colombo, C. Gennari, D. Potenza, C. Scolastico, F. Aragozzini and C. Merendi, J. Chem. Soc., Perkin Trans. 1, 1981, 2594.
27. U. Sankawa, Y. Ebizuka, H. Noguchi, Y. Isikawa, S. Kitaghawa, Y. Yamamoto, T. Kobayashi and Y. Iitak, Tetrahedron, 1983, 39, 3583.
28. B. Balakrishnan, R. Chandran, S.-H. Park and H.-J. Kwon, Bioorg. Med. Chem. Lett., 2016, 26, 392.
29. Y. He and R. J. Cox, Chem. Sci., 2016, 7, 2119.
30. D. Zhang, X. Li, J. S. Kang, H. D. Choi, J. H. Jung and B. W. Son, J. Microbiol. Biotechnol., 2007, 17, 865.
31. P. Sun, Q. Zhao, F. Yu, H. Zhang, Z. Wu, Y. Wang, Y. Wang, Q. Zhang and W. Liu, J. Am. Chem. Soc., 2013, 135, 1540.
32. J.-M. Gao, S.-X. Yang and J.-C. Qin, Chem. Rev., 2013, 113, 4755.
33. M. Sasaki, M. Tsuda, M. Sekiguchi, Y. Mikami and J. Kobayashi, Org. Lett., 2005, 7, 4261.
34. Z.-F. Zhou, T. Kurtán, X.-H. Yang, A. Mándi, M.-Y. Geng, B.-P. Ye, O. Taglialatela-Scafati and Y.-W. Guo, Org. Lett., 2014, 16, 1390.
35. M.-A. Castillo, P. Moya, A. Cantín, M. A. Miranda, J. Primo, E. Hernández and E. Primo-Yúfera, J. Agric. Food Chem., 1999, 47, 2120.
36. B. R. Clark, R. J. Capon, E. Lacey, S. Tennant and J. H. Gill, Org. Biomol. Chem., 2006, 4, 1520.
37. C. H. Hassall and D. W. Jones, J. Chem. Soc., 1962, 4189.
38. X.-H. Nong, Z.-H. Zheng, X.-Y. Zhang, X.-H. Lu and S.-H. Qi, Mar. Drugs, 2013, 11, 1718.
39. R. E. Desjardins, C. J. Canfield, J. D. Haynes and J. D. Chulay, Antimicrob. Agents Chemother., 1979, 16, 710.
40. J. O'Brien, I. Wilson, T. Orton and F. Pognan, Eur. J. Biochem., 2000, 267, 5421.
41. S. D. Sarker, L. Nahar and Y. Kumarasamy, Methods, 2007, 42, 321.
42. E. A. Aremu, T. Furumai, Y. Igarashi, Y. Sato, H. Akamatsu, M. Kodama and H. Otani, J. Gen. Plant Pathol., 2003, 69, 211.
43. R. P. Haugland, Handbook of Fluorescent Probes and Research Products, Molecular Probes, Eugene, OR, 9th edn, 2002.
44. C. Chutrakul, P. Khaokhajorn, P. Auncharoen, T. Boonruengprapa and O. Mongkolporn, Biosci., Biotechnol., Biochem., 2013, 77, 259.
45. C. Changsen, S. G. Franzblau and P. Palittapongarnpim, Antimicrob. Agents Chemother., 2003, 47, 3682.

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Footnote

† Electronic supplementary information (ESI) available: 1D, 2D NMR and mass spectra of all new isolated compounds (1, 3, 5−12, and 14–16). See DOI: 10.1039/c6ra21898a

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