Note pubs.acs.org/jnp
Gymnopalynes A and B, Chloropropynyl-isocoumarin Antibiotics from Cultures of the Basidiomycete Gymnopus sp. Benjarong Thongbai,† Frank Surup,‡ Kathrin Mohr,‡ Eric Kuhnert,‡ Kevin D. Hyde,† and Marc Stadler*,‡ †
Institute of Excellence in Fungal Research and School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand Department Microbial Drugs, Helmholtz Centre for Infection Research GmbH, Inhoffenstraße 7, 38124 Braunschweig, Germany
‡
S Supporting Information *
ABSTRACT: A chlorinated isocoumarin with an acetylenyl side chain and its 3,4-dihydro derivative, named gymnopalynes A (1) and B (2), were isolated from cultures of a basidiomycete originating from the rain forest of Northern Thailand. The producing organism was identified as a species of Gymnopus (Marasmiaceae). Their structures were elucidated by spectroscopic methods including UV/vis and NMR spectroscopy as well as high-resolution mass spectrometry as 3-(3-chloroprop-1-yn-1-yl)-1H-isochromen-1-one (1) and 3(3-chloroprop-1-yn-1-yl)-3,4-dihydro-1H-isochromen-1-one (2). The absolute stereochemistry of 2 was assigned as S by CD spectroscopy. Both compounds showed weak to moderate antimicrobial and pronounced cytotoxic activities.
N
ovel antibiotics with unusual scaffolds and new modes of actions are urgently needed, since the resistance against antibiotics in use is constantly increasing.1 As most of the known antibiotics are derived from fungal or microbial cultures, screening of previously untapped groups of these organisms appears promising to discover novel bioactive metabolites. Cultures of basidiomycetes (subdivision Basidiomycota of the kingdom Fungi) have been screened for novel antibiotics since the late 1940s.2 Among other compounds, these early screening programs resulted in the discovery of pleuromutilin. However, it took several decades until the pleuromutilin derivative retapamulin was approved for clinical use against bacterial pathogens, and this represents the first such agent derived from a basidiomycete.3 In addition, most of the novel compounds from basidiomycetes were derived from species collected in the temperate zones, and a systematic screening for bioactive compounds from these organisms has not been carried out because they are more difficult to isolate and handle than the more commonly used bacteria and fungi. In the course of our ongoing search for novel antibiotics, we have focused on tropical basidiomycetes from Asia. A basidiomycete was collected in the rainforest of Chiang Mai Province, Northern Thailand. In an antimicrobial screen, extracts prepared from submerged cultures of this strain showed prominent activity. Bioactivity-guided fractionation by RP-HPLC, with Mucor hiemalis as the indicator organism, led to the isolation of two unprecedented antimicrobial metabolites that constitute the active principles. Their physicochemical and biological characterization is reported here. Gymnopalyne A (1) was obtained as a colorless oil. The presence of a chlorine atom was indicated by its characteristic isotopic pattern in the HRESIMS that provided the molecular formula of 1 as C12H7O2Cl, implying 9 degrees of unsaturation. © 2013 American Chemical Society and American Society of Pharmacognosy
The 1H NMR spectra revealed the presence of five aromatic protons (δH 8.24, 7.82, 7.63, 7.60, 7.02) besides a methylene signal (δH 4.55), which were connected to their corresponding carbon atoms (δC 136.5, 130.8, 130.4, 127.6, 113.8, 30.6) by HSQC correlations. Furthermore, the 13C spectrum showed one carbonyl (δC 163.1), three quaternary sp2-hybridized carbons (δC 138.2, 137.8 122.7), and two quaternary sphybridized carbons (δC 89.9, 79.1) (Table 1). While the COSY spectrum connected a spin system of a 1,2-disubstituted benzene ring, the HMBC correlations shown in Figure 1 established the carbon skeleton. The chemical shift (δC 30.6) and the remaining open valence of the methylene group indicated its connection to the chlorine atom, completing the structure of 3-(3-chloroprop-1-yn-1-yl)-1H-isochromen-1-one (1). 1 presented UV absorption maxima at λmax 232, 285, 297, and 326 nm, which are consistent with an isocoumarin backbone conjugated with an acetylenic side chain.4 The molecular formula C12H9O2Cl of gymnopalyne B (2) specified two additional protons compared to 1. The UV spectrum of 2 with maxima at 235 and 281 nm indicated a shorter conjugated π-system. The NMR characteristics were highly similar to those of 1, except that the NMR resonances assigned to olefinic carbons C-3 and C-4 were replaced by sp3hybridized methine (δC 69.2, δH 5.52) and methylene (δC 34.4, δH 3.39, 3.32) signals. HMBC correlations emanating from 3-H Received: July 26, 2013 Published: October 31, 2013 2141
dx.doi.org/10.1021/np400609f | J. Nat. Prod. 2013, 76, 2141−2144
Journal of Natural Products
Note
Table 1. NMR Data (1H 700 MHz, 13C 175 MHz) of Gymnopalynes A (1) and B (2) in Methanol-d4 1 pos
δC, type
1 3
163.1, C 138.2, C
4
113.8, CH
4a 5 6
137.8, C 127.6, CH 136.5, CH
7
130.8, CH
8 8a 1′ 2″ 3″
130.4, 122.7, 79.1, 89.8, 30.6,
CH C C C CH2
δH, m (J in Hz)
2 δC, type 166.1, C 69.2, CH
7.02, s
7.60, br d (7.6) 7.82, ddd (7.6, 7.6, 1.2) 7.62, ddd (7.6, 7.6, 1.2) 8.24, br d (7.6)
4.55, s
34.4, CH2
139.7, C 129.1, CH 135.5, CH 129.0, CH 130.4, 125.7, 83.1, 83.1, 30.3,
CH C C C CH2
δH, m (J in Hz) 5.52, ddd (7.1, 4.5, 1.7) 3.39, dd (16.6, 4.5) 3.32, dd (16.6, 7.1)
panel of bacteria and fungi (Table 2), showing weak to moderate, apparently nonselective activities against some of the
7.39, d (7.7) 7.64, ddd (7.7, 7.7, 1.2) 7.46, br dd (7.7, 7.7) 8.02, dd (7.7, 1.2)
Table 2. Minimal Inhibitory Concentrations (MIC) of Compounds 1 and 2 MIC (μg/mL) test strain/strain number Bacteria Chromobacterium violaceum DSM 30191 Escherichia coli DSM 1116 Micrococcus luteus DSM 20030 Mycobacterium diernhoferi DSM 43524 Nocardia sp. DSM 43069 Nocardioides simplex DSM 20130 Paenibacillus polymyxa DSM 36 Pseudomonas aeruginosa DSM 50071 Staphylococcus aureus DSM 346 Fungi Aspergillus clavatus DSM 816 Candida albicans DSM 1665 Hormoconis resinae DSM 1203 Mucor hiemalis DSM 2656 Nematospora coryli DSM 6981 Penicillium capsulatum DSM 2210 Pichia anomala DSM 6766 Rhodotorula glutinis DSM 10134 Schizosaccharomyces pombe DSM 70572 Trichosporon oleaginosus DSM 11815
4.24, d (1.7)
Figure 1. Key HMBC correlations for gymnopalynes A (1) and B (2) indicated by arrows.
and 4-Ha,b (Figure 1) proved 2 to be 3-(3-chloroprop-1-yn-1yl)-3,4-dihydro-1H-isochromen-1-one. By comparison of the CD spectrum and the optical rotation of 2 with data reported for dihydrocoumarins from Ginkgo biloba, whose stereochemistry had been demonstrated by total synthesis,5 it was possible to assign the absolute configuration of gymnopalyne B (2): The compound has a negative optical rotation in chloroform, and its CD spectrum shows a negative maximum at 235 nm. These data are is in accordance with an S absolute configuration.5,6 Numerous isocoumarin and 3,4-dihydroisocoumarin derivatives have been isolated from fungi, bacteria, plants, and insects.7 However, only a few of these contain halogen atoms and/or acetylenic side chains,8 and the combination of both seems to be unprecedented. Remarkably, gymnopalyne A (1) has a striking structural similarity to corfin (3), isolated from roots of Chamaemelum mixtum.4 The formal replacement of the chlorine atom of 1 by a methyl group converts the structure to that of corfin. The production of corfin by a plant vs gymnopalyne A (1) by a basidiomycete presents a nice example of convergent evolution leading to similar bioactive metabolites produced by organisms from different kingdoms. Aromatic acetylenic antibiotics with chlorine have previously also been reported from other basidiomycete genera, including Gymnopilus,9 which produces 7-chloro-hepta-4,6-diyn-3-ol (4), and Mycena, which produces mycenon (5).10 Isocoumarins are well known to exhibit a wide range of biological activities, including antimicrobial, anti-inflammatory, phytotoxic, and cytotoxic properties.11 The bioactivity of 1 and 2 was evaluated by determining their MIC against a broad test
1
2
control
33.3 n.i.d n.i. n.i. 67 33.3 n.i. 67 n.i.
33.3 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i.
1.0a 1.0a 2.1a ≤0.25a 0.25a 16.6a 6.7a 67b ≤0.25a
16.6 n.i. 33.3 16.6 16.6 n.i. n.i. 33.3 16.6 33.3
n.i. n.i. n.i. 33.3 n.i. n.i. n.i. n.i. n.i. n.i.
2.1c 4.2c
Gymnopalynes A and B, Chloropropynyl-isocoumarin Antibiotics from Cultures of the Basidiomycete Gymnopus sp. Benjarong Thongbai,† Frank Surup,‡ Kathrin Mohr,‡ Eric Kuhnert,‡ Kevin D. Hyde,† and Marc Stadler*,‡ †
Institute of Excellence in Fungal Research and School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand Department Microbial Drugs, Helmholtz Centre for Infection Research GmbH, Inhoffenstraße 7, 38124 Braunschweig, Germany
‡
S Supporting Information *
ABSTRACT: A chlorinated isocoumarin with an acetylenyl side chain and its 3,4-dihydro derivative, named gymnopalynes A (1) and B (2), were isolated from cultures of a basidiomycete originating from the rain forest of Northern Thailand. The producing organism was identified as a species of Gymnopus (Marasmiaceae). Their structures were elucidated by spectroscopic methods including UV/vis and NMR spectroscopy as well as high-resolution mass spectrometry as 3-(3-chloroprop-1-yn-1-yl)-1H-isochromen-1-one (1) and 3(3-chloroprop-1-yn-1-yl)-3,4-dihydro-1H-isochromen-1-one (2). The absolute stereochemistry of 2 was assigned as S by CD spectroscopy. Both compounds showed weak to moderate antimicrobial and pronounced cytotoxic activities.
N
ovel antibiotics with unusual scaffolds and new modes of actions are urgently needed, since the resistance against antibiotics in use is constantly increasing.1 As most of the known antibiotics are derived from fungal or microbial cultures, screening of previously untapped groups of these organisms appears promising to discover novel bioactive metabolites. Cultures of basidiomycetes (subdivision Basidiomycota of the kingdom Fungi) have been screened for novel antibiotics since the late 1940s.2 Among other compounds, these early screening programs resulted in the discovery of pleuromutilin. However, it took several decades until the pleuromutilin derivative retapamulin was approved for clinical use against bacterial pathogens, and this represents the first such agent derived from a basidiomycete.3 In addition, most of the novel compounds from basidiomycetes were derived from species collected in the temperate zones, and a systematic screening for bioactive compounds from these organisms has not been carried out because they are more difficult to isolate and handle than the more commonly used bacteria and fungi. In the course of our ongoing search for novel antibiotics, we have focused on tropical basidiomycetes from Asia. A basidiomycete was collected in the rainforest of Chiang Mai Province, Northern Thailand. In an antimicrobial screen, extracts prepared from submerged cultures of this strain showed prominent activity. Bioactivity-guided fractionation by RP-HPLC, with Mucor hiemalis as the indicator organism, led to the isolation of two unprecedented antimicrobial metabolites that constitute the active principles. Their physicochemical and biological characterization is reported here. Gymnopalyne A (1) was obtained as a colorless oil. The presence of a chlorine atom was indicated by its characteristic isotopic pattern in the HRESIMS that provided the molecular formula of 1 as C12H7O2Cl, implying 9 degrees of unsaturation. © 2013 American Chemical Society and American Society of Pharmacognosy
The 1H NMR spectra revealed the presence of five aromatic protons (δH 8.24, 7.82, 7.63, 7.60, 7.02) besides a methylene signal (δH 4.55), which were connected to their corresponding carbon atoms (δC 136.5, 130.8, 130.4, 127.6, 113.8, 30.6) by HSQC correlations. Furthermore, the 13C spectrum showed one carbonyl (δC 163.1), three quaternary sp2-hybridized carbons (δC 138.2, 137.8 122.7), and two quaternary sphybridized carbons (δC 89.9, 79.1) (Table 1). While the COSY spectrum connected a spin system of a 1,2-disubstituted benzene ring, the HMBC correlations shown in Figure 1 established the carbon skeleton. The chemical shift (δC 30.6) and the remaining open valence of the methylene group indicated its connection to the chlorine atom, completing the structure of 3-(3-chloroprop-1-yn-1-yl)-1H-isochromen-1-one (1). 1 presented UV absorption maxima at λmax 232, 285, 297, and 326 nm, which are consistent with an isocoumarin backbone conjugated with an acetylenic side chain.4 The molecular formula C12H9O2Cl of gymnopalyne B (2) specified two additional protons compared to 1. The UV spectrum of 2 with maxima at 235 and 281 nm indicated a shorter conjugated π-system. The NMR characteristics were highly similar to those of 1, except that the NMR resonances assigned to olefinic carbons C-3 and C-4 were replaced by sp3hybridized methine (δC 69.2, δH 5.52) and methylene (δC 34.4, δH 3.39, 3.32) signals. HMBC correlations emanating from 3-H Received: July 26, 2013 Published: October 31, 2013 2141
dx.doi.org/10.1021/np400609f | J. Nat. Prod. 2013, 76, 2141−2144
Journal of Natural Products
Note
Table 1. NMR Data (1H 700 MHz, 13C 175 MHz) of Gymnopalynes A (1) and B (2) in Methanol-d4 1 pos
δC, type
1 3
163.1, C 138.2, C
4
113.8, CH
4a 5 6
137.8, C 127.6, CH 136.5, CH
7
130.8, CH
8 8a 1′ 2″ 3″
130.4, 122.7, 79.1, 89.8, 30.6,
CH C C C CH2
δH, m (J in Hz)
2 δC, type 166.1, C 69.2, CH
7.02, s
7.60, br d (7.6) 7.82, ddd (7.6, 7.6, 1.2) 7.62, ddd (7.6, 7.6, 1.2) 8.24, br d (7.6)
4.55, s
34.4, CH2
139.7, C 129.1, CH 135.5, CH 129.0, CH 130.4, 125.7, 83.1, 83.1, 30.3,
CH C C C CH2
δH, m (J in Hz) 5.52, ddd (7.1, 4.5, 1.7) 3.39, dd (16.6, 4.5) 3.32, dd (16.6, 7.1)
panel of bacteria and fungi (Table 2), showing weak to moderate, apparently nonselective activities against some of the
7.39, d (7.7) 7.64, ddd (7.7, 7.7, 1.2) 7.46, br dd (7.7, 7.7) 8.02, dd (7.7, 1.2)
Table 2. Minimal Inhibitory Concentrations (MIC) of Compounds 1 and 2 MIC (μg/mL) test strain/strain number Bacteria Chromobacterium violaceum DSM 30191 Escherichia coli DSM 1116 Micrococcus luteus DSM 20030 Mycobacterium diernhoferi DSM 43524 Nocardia sp. DSM 43069 Nocardioides simplex DSM 20130 Paenibacillus polymyxa DSM 36 Pseudomonas aeruginosa DSM 50071 Staphylococcus aureus DSM 346 Fungi Aspergillus clavatus DSM 816 Candida albicans DSM 1665 Hormoconis resinae DSM 1203 Mucor hiemalis DSM 2656 Nematospora coryli DSM 6981 Penicillium capsulatum DSM 2210 Pichia anomala DSM 6766 Rhodotorula glutinis DSM 10134 Schizosaccharomyces pombe DSM 70572 Trichosporon oleaginosus DSM 11815
4.24, d (1.7)
Figure 1. Key HMBC correlations for gymnopalynes A (1) and B (2) indicated by arrows.
and 4-Ha,b (Figure 1) proved 2 to be 3-(3-chloroprop-1-yn-1yl)-3,4-dihydro-1H-isochromen-1-one. By comparison of the CD spectrum and the optical rotation of 2 with data reported for dihydrocoumarins from Ginkgo biloba, whose stereochemistry had been demonstrated by total synthesis,5 it was possible to assign the absolute configuration of gymnopalyne B (2): The compound has a negative optical rotation in chloroform, and its CD spectrum shows a negative maximum at 235 nm. These data are is in accordance with an S absolute configuration.5,6 Numerous isocoumarin and 3,4-dihydroisocoumarin derivatives have been isolated from fungi, bacteria, plants, and insects.7 However, only a few of these contain halogen atoms and/or acetylenic side chains,8 and the combination of both seems to be unprecedented. Remarkably, gymnopalyne A (1) has a striking structural similarity to corfin (3), isolated from roots of Chamaemelum mixtum.4 The formal replacement of the chlorine atom of 1 by a methyl group converts the structure to that of corfin. The production of corfin by a plant vs gymnopalyne A (1) by a basidiomycete presents a nice example of convergent evolution leading to similar bioactive metabolites produced by organisms from different kingdoms. Aromatic acetylenic antibiotics with chlorine have previously also been reported from other basidiomycete genera, including Gymnopilus,9 which produces 7-chloro-hepta-4,6-diyn-3-ol (4), and Mycena, which produces mycenon (5).10 Isocoumarins are well known to exhibit a wide range of biological activities, including antimicrobial, anti-inflammatory, phytotoxic, and cytotoxic properties.11 The bioactivity of 1 and 2 was evaluated by determining their MIC against a broad test
1
2
control
33.3 n.i.d n.i. n.i. 67 33.3 n.i. 67 n.i.
33.3 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i.
1.0a 1.0a 2.1a ≤0.25a 0.25a 16.6a 6.7a 67b ≤0.25a
16.6 n.i. 33.3 16.6 16.6 n.i. n.i. 33.3 16.6 33.3
n.i. n.i. n.i. 33.3 n.i. n.i. n.i. n.i. n.i. n.i.
2.1c 4.2c
