HETEROCYCLES

Paper | Regular issue | Vol. 87, No. 7, 2013, pp. 1481-1491
Received, 15th March, 2013, Accepted, 13th May, 2013, Published online, 17th May, 2013.
DOI: 10.3987/COM-13-12707

■ Palmaerins A-D, New Chlorinated and Brominated Dihydroisocoumarins with Antimicrobial and Plant Growth Regulating Activities from Discomycete Lachnum palmae

Yuka Tanabe, Takunori Matsumoto, Tsuyoshi Hosoya, Hiroyasu Sato, and Hideyuki Shigemori*

Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Japan

Abstract

A new chlorinated dihydroisocoumarin, palmaerin A (1), and three new brominated dihydroisocoumarins, palmaerins B (2), C (3), and D (4), were isolated from the supernatant of the culture broth containing KCl and KBr of discomycete Lachnum palmae, respectively. The structures of 1-4 were elucidated by spectroscopic data and X-ray diffraction analysis. Compounds 2 and 3 exhibited weak antibacterial activity against Bacillus subtilis and Staphylococcus aureus, while compounds 1-3 showed plant growth regulating activity against Lepidium sativum.

INTRODUCTION
In previous reports, several new bioactive substances such as scyphostatin1 and F-10863s2,3 which have enzyme inhibition activity, have been isolated from fungi of the order Leotiales, Discomycetes. Nevertheless, the members of it have been underutilized for microbial screenings.4 Therefore, we noticed discomycetes as research source for new bioactive substances. In our screening for antimicrobial activity of various strains of discomycetes in the National Museum of Nature and Science, Lachnum palmae was selected. The genus of Lachnum is known to embrace about 250 species and yet more members have been added to science,5 and the antimicrobial and nematicidal pentaketide compounds have been isolated from Lachnum papyraceum.6-8
In our previous investigation, we found two new chlorinated dibenzo-α-pyrones, palmariols A and B, and two new chlorinated cyclopentenones, palmaenones A and B which have antimicrobial activity, have been isolated from the mycelial extracts and culture broth of L. palmae, respectively.9,10 On the other hand, chlorinated or brominated compounds have been isolated from some discomycetes. For example, chloromonilicin and bromomonilicin as self-growth inhibitors,11,12 and chloromonilinic acids A and B13 as antifungal compounds were isolated from Monilinia fructicola. KS-504a-e containing seven chlorine atoms were isolated from Mollisia ventosa.14,15 Furthermore, chlorinated antifungal compounds, mollisins A and B were isolated from Mollisia caesia, and this discomycete has been shown to have chloroperoxidase which catalyze the chlorination.16,17 These reports suggest that L. palmae which belongs to the same order as them has also haloperoxidases for halogenation.
So in order to research other halogenated compounds from L. palmae, KCl or KBr was added to the culture media. In this paper, we describe the isolation and structure elucidation of four new halogenated dihydroisocoumarins 1-4 from the supernatant of the culture broth containing KCl or KBr of L. palmae and their antimicrobial activity and plant growth regulating activity.

RESULTS AND DISCUSSION
The KCl- or KBr-containing culture broth of L. palmae was centrifuged. The supernatants were extracted with EtOAc. The EtOAc-soluble portions were subjected to silica gel column chromatography and reversed-phase HPLC. As results of these, a new chlorinated dihydroisocoumarin, palmaerin A (1) was obtained together with known chlorinated cyclopentenes, palmaenones A and B10 from the KCl-containing culture media, while three new brominated dihydroisocoumarins, palmaerins B-D (2-4) were obtained from the KBr-containing culture media.
Palmaerin A (1) showed pseudomolecular ion peaks at m/z 275 (M-H)-, 277 (M+2-H)-, and 279 (M+4-H)- (10:6:1) in the ESI-MS, indicating the presence of two chlorine atoms in 1. The molecular formula of 1 was deduced as C11H1035Cl2O4 from HRESI-MS [m/z 274.9885 (M-H)-, Δ +0.7 mmu]. 1H NMR spectrum of 1 was almost the same as 5,7-dichloro-3,4-dihydro-6,8-dihydroxy-3-methylisocoumarin18 except a methoxy group at δH 3.86 (3H, s). The IR spectrum indicated the presence of hydroxy group (3241 cm-1) and α,β-unsaturated lactone (1698 and 1266 cm-1), and UV absorptions at 226, 252, and 311 nm were observed. These data were almost similar to those of 5,7-dichloro-3,4-dihydro-6,8-dihydroxy- 3-methylisocoumarin.19 The gross structure of 1 was deduced from detailed analysis of the 13C and 1H NMR data (Tables 1 and 2) aided by 2D NMR experiments (1H-1H correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond connectivity (HMBC)). 1H NMR spectrum at δH 1.42 (3H, d, J = 6.3 Hz) implied the presence of one methyl group. Two double doublet with J value of 16.9 Hz between δH 2.73 and δH 3.20 indicated the existence of nonequivalent methylene protons. The 13C NMR data indicated that 1 possessed one unsaturated carbonyl carbon, six aromatic carbons, one oxymethine carbon, one methoxy carbon, one methylene carbon, and one methyl carbon. The 1H-1H COSY connectivities between H-3 (δH 4.55) and methyl proton (δH 1.42) at C-3 as well as the HMBC correlations of the methyl proton to C-3 (δC 74.4) and C-4 (δC 34.6) indicated that the methyl group was connected to C-3, which was connected to C-4. The HMBC correlations of H2-4 (δH 2.73 and 3.20) to C-4a (δC 140.2), C-5 (δC 116.3), and C-8a (δC 117.5) revealed that C-4 was connected to C-4a which is one of aromatic carbons. The HMBC correlations of the methoxy proton (δH 3.86) to C-8 (δC 159.5) indicated that the methoxy group was connected to C-8 (Figure 2). Comparison of the 13C NMR data of compound 1 with 3,4-dihydro-6-hydroxy-8-methoxy-3-methylisocoumarin20 revealed the presence of two chlorine atoms at C-5 and C-7. The configuration of methyl group at C-3 was determined by comparison of the optical rotation data of the previous report,21 and revealed that compound 1 has R-configuration. Thus the structure of palmaerin A was assigned as 1.

Palmaerin B (2) showed no methoxy group in the 1H NMR spectrum (Table 2), but the signal pattern was the same as 1. In the ESI-MS, specific pseudomolecular ion peaks at m/z 349 (M-H)-, 351 (M+2-H)-, and 353 (M+4-H)- (1:2:1) were observed, indicating the presence of two bromine atoms in 2. The molecular formula of 2 was deduced as C10H879Br2O4 from HRESI-MS [m/z 348.8717 (M-H)-, Δ +0.6 mmu]. The IR and UV spectra of 2 were similar to those of 1. The gross structure of 2 was deduced from detailed analysis of the 2D NMR experiments (1H-1H COSY, HMQC, and HMBC). As a result of it, it was revealed that bromine atoms were connected to C-5 and C-7. The absolute configuration of methyl group at C-3 was R-configuration by the single-crystal X-ray diffraction analysis of 2 (Figure 3). Thus the structure of palmaerin B was determined as 2.
Palmaerin C (3) showed pseudomolecular ion peaks at m/z 363 (M-H)-, 365 (M+2-H)-, and 367 (M+4-H)- (1:2:1) in the ESI-MS, indicating the presence of two bromine atoms in 3, and the molecular formula of 3 was deduced as C11H1079Br2O4 from HRESI-MS [m/z 362.8877 (M-H)-, Δ +0.9 mmu]. Considered that the 1H NMR spectrum (Table 2) of 3 indicated the presence of methoxy group at δH 3.85 (3H, s), it was suggested that 3 possesses a methoxy group instead of a hydroxy group of 2. The detailed structure of 3 was determined from analysis of 13C (Table 1) and 2D NMR experiments (1H-1H COSY, HMQC, and HMBC). As a result of it, the HMBC correlation of the methoxy proton (δH 3.85) to C-8 (δC 161.1) was observed, indicating methoxy group was connected to C-8 (Figure 4). Furthermore, chemical shift values at C-5 (δC 107.9) and C-7 (δC 107.1) were shifted to higher field than those of 1, indicating existence of two bromine atoms at C-5 and C-7. The configuration of methyl group at C-3 was determined by comparison of the optical rotation data of the previous report21 as R-configuration. Thus the structure of palmaerin D which is the brominated derivative of 1 was determined as 3.
Palmaerin D (4) showed one singlet peak at δH 6.47 (1H, s) in the 1H NMR spectrum (Table 2). Also the presence of one bromine atom in 4 was indicated by ESI-MS (m/z 271 [M-H]- and 273 [M+2-H]- (1:1)), and the molecular formula was deduced as C10H979BrO4 from HRESI-MS [m/z 270.9610 (M-H)-, Δ +0.4 mmu]. These results suggest that 4 is the debromoderivative of 2. The gross structure of 4 was

determined from detailed analysis by 2D NMR experiments (1H-1H COSY, HMQC, and HMBC) (Figure 5). The HMBC correlations of H-7 (δH 6.47) to C-5 (δC 103.3), C-6 (δC 162.5), C-8 (δC 164.7), and C-8a (δC 102.4) and comparison of the 13C NMR data of 4 with (R)-(-)-5-chloro-3,4-dihydro-6,8-dihydroxy-3-methyl-1H-2-benzopyran-1-one21 revealed that compound 4 has one bromine atom at C-5. The configuration of methyl group at C-3 was determined as R-configuration by comparison of optical rotations with compounds 1-3 and the previous report.22 Thus the structure of palmaerin D was assigned as 4.
Palmaerins A-D (1-4) should be pentaketides, and it is suggested that the origin of these dihydroisocoumarins and cyclopentenes such as palmaenones A and B is a common six-membered intermediate.18 A lot of isocoumarins have been obtained from various plants and fungi as secondary metabolites, and some of them possess interesting bioactivities. For example, 5-methylmellein obtained from Fusicoccum amygdari Del. inhibits the germination of spores of fungi, while it showed no phytotoxic activity. 6-Methoxymellein was isolated from fungi Sporormia bipartis Cain and Sporormia affinis Sacc., and was also produced by a carrot inoculated with fungi, is thought to be a phytoalexin. Sclerin, sclerotinins A and B promote remarkably both germination and elongation of rice, caster bean, mung bean, and other plants.23

Hence, antimicrobial activity and plant growth regulating activity of palmaerins A-D (1-4) were evaluated. As a result, compounds 1-3 showed antimicrobial activity against Bacillus subtilis and Staphylococcus aureus (Table 3). Compounds 2 and 3 exhibited stronger activity than 1, indicating that bromine atom plays an important role in antibacterial activity. On the other hand, compounds 1-3 exhibited plant growth regulating activity against Lepidium sativum (Figure 6): at high concentrations, the growth of roots of L. sativum was significantly inhibited, contrary to this, at low concentrations, 1-3 promoted the growth of those. But this auxin-like activity of palmaerin D (4) was weaker than those of 1-3. These results suggest that halogen atoms play an important role in showing plant growth regulating (auxin-like) activity against L. sativum.
Together with the fact that L. palmae was isolated from decaying leaves of Livistona, this result implied that dihydroisocoumarins effect on the host plant positively. Furthermore, it was suggested that production of potent antimicrobial palmaenones enable L. palmae itself and host plant to protect from other fungi’s threat. So it is possible that L. palmae is endophytic fungus which has another role as latent saprotroph. Actually, several fungi which have dual roles as endophyte and saprotroph have been already reported.24

EXPERIMENTAL
General Procedures
Optical rotations were measured with a Jasco DIP-370 with a cell path of 1 cm. UV spectra were obtained with a HITACHI U-2000A spectrometer. IR spectra were obtained with a JASCO FT/IR-300 spectrometer. 1H and 13C NMR spectra were obtained with a Bruker Avance-500 spectrometer in acetone-d6. The resonances of acetone-d6 at δH 2.00 and δC 30.3 were used as internal references for the 1H and 13C NMR spectra, respectively. ESI-MS and HRESI-MS spectra were recorded with a Waters Synapt G2 mass spectrometer.

Fungal Material
The isolated NBRC-106495, deposited in the National Institute of Technology and Evaluation, Biological Resource Center (NBRC) was obtained by a single ascospore isolation using a Skerman’s micromanioulator from an apothecium of Lachnum palmae (Kanouse). Spooner produced on the decaying leaves of Livistona, collected in Suzaki, Shimoda, Shizuoka Prefecture in July, 2004. The specimen was preserved as TNS-F-11197 in the National Museum of Nature and Science. The isolated TNS-F-11197 was grown and kept on potato dextrose agar (PDA, Nissui).

Fermentation
L. palmae was inoculated in 100 mL Erlenmeyer flasks (×5) containing 30 mL of seed medium (PYG: polypeptone (1%), yeast extract (0.5%), glucose (2%), and KCl or KBr (0.5%) in deionized water, pH was adjusted to 7.5 before autoclaving). After incubation on a rotary shaker at 120 rpm, 25 °C for 14 days, each seed culture was transferred into 500 mL Sakaguchi flasks (×20) containing 300 mL of PYG medium and incubated on rotary shaker at 120 rpm, 25 °C for 1 week. The culture broth was centrifuged at 7,500 rpm for 15 min.

Extraction and Isolation
The supernatant filtrate of L. palmae culture broth (6 L) was extracted with EtOAc and evaporated to dryness in vacuo at 37 °C. When KCl was added to the culture media, the EtOAc–soluble portion (462.9 mg) was subjected to silica gel column chromatography (φ1.1×35 cm, n-hexane/acetone, 99:1→0:100). A fraction (38 mg) in which characteristic peaks of palmaenone derivatives were observed, was applied to silica gel column chromatography (φ1.1×22 cm, n-hexane/acetone, 80:20→0:100) to yield palmaerin A (1) (18.7 mg).　When KBr was added to the culture media, the EtOAc–soluble portion (253.7 mg) was subjected to silica gel column chromatography (φ1.1×22 cm, n-hexane/acetone, 99:1→0:100). A fraction (127.6 mg) in which characteristic peaks of dihydroisocoumarin derivatives were observed by the 1H NMR spectra, was applied to silica gel column chromatography (φ1.1×22 cm, n-hexane/acetone, 95:5→0:100) and the fraction (63.6 mg) eluted with n-hexane/acetone (80:20→75:25) was further separated by reversed-phase HPLC (TSK-gel ODS-80Ts, TOSOH φ7.8×300 mm, flow rate 2.0 mL/min; MeOH:H2O, 70:30 (0 min)→90:10 (30 min)→100:0 (30 min)) to yield palmaerin B (2) (0.9 mg), palmaerin C (3) (11.9 mg), and palmaerin D (4) (5.0 mg).

Palmaerin A (5,7-dichloro-3,4-dihydro-6-hydroxy-8-methoxy-3-methylisocoumarin) (1): Colorless amorphous solid; [α]D24 -114° (c 1.0 in acetone); UV λmax (MeOH) nm (log ε) 226 (4.09), 252 (3.92), 311 (3.84); IR (KBr) νmax cm-1 3241, 1698, 1266; 1H and 13C NMR (Table 1); HMBC correlations (acetone-d6, H/C): 4/3, 4/4a, 4/5, 4/8a, 4/Me-3, Me-3/3, Me-3/4, MeO-8/8; ESI-MS (negative ion) m/z: 275 [M-H]-, 277 [M+2-H]-, 279 [M+4-H]- (10:6:1); HRESI-MS (negative ion) m/z: 274.9885 [M-H]- (Calcd for C11H935Cl2O4: 274.9878).
Palmaerin B (5,7-dibromo-3,4-dihydro-6,8-dihydroxy-3-methylisocoumarin) (2): Colorless crystal; [α]D24 -47.7° (c 1.0 in acetone); UV λmax (MeOH) nm (log ε) 224 (4.69), 250 (4.50), 317 (4.65); IR (KBr) νmax cm-1 3332, 1713, 1249; 1H and 13C NMR (Table 1); HMBC correlations (acetone-d6, H/C): 4/3, 4/4a, 4/5, 4/8a, 4/Me-3, Me-3/3, Me-3/4; ESI-MS (negative ion) m/z: 348 [M-H]-, 350 [M+2-H]-, 352 [M+4-H]- (1:2:1); HRESI-MS (negative ion) m/z: 348.8717 [M-H]- (Calcd for C10H779Br2O4: 348.8711).
Palmaerin C (5,7-dibromo-3,4-dihydro-6-hydroxy-8-methoxy-3-methylisocoumarin) (3): Colorless amorphous solid; [α]D24 -99.4° (c 1.0 in acetone); UV λmax (MeOH) nm (log ε) 220 (4.42), 246 (4.27), 313 (4.63); IR (KBr) νmax cm-1 3244, 1697, 1259; 1H and 13C NMR (Table 1); HMBC correlations (acetone-d6, H/C): 4/3, 4/4a, 4/5, 4/8a, 4/Me-3, Me-3/3, Me-3/4, MeO-8/8; ESI-MS (negative ion) m/z: 363 [M-H]-, 365 [M+2-H]-, 367 [M+4-H]- (1:2:1); HRESI-MS (negative ion) m/z: 362.8877 [M-H]- (Calcd for C11H979Br2O4: 362.8868).
Palmaerin D (5-bromo-3,4-dihydro-6,8-dihydroxy-3-methylisocoumarin) (4): Colorless amorphous solid; [α]D24 -61.4° (c 1.0 in acetone); UV λmax (MeOH) nm (log ε) 218 (4.35), 267 (3.99), 314 (4.15); IR (KBr) νmax cm-1 3254, 1697, 1245; 1H and 13C NMR (Table 1); HMBC correlations (acetone-d6, H/C): 4/3, 4/4a, 4/5, 4/8a, 4/Me-3, 7/5, 7/6, 7/8, 7/8a, Me-3/3, Me-3/4; ESI-MS (negative ion) m/z: 271 [M-H]-, 273 [M+2-H]- (1:1); HRESI-MS (negative ion) m/z: 270.9610 [M-H]- (Calcd for C10H879BrO4: 270.9606).

Single-crystal X-ray crystallography of palmaerin B (2) Suitable colorless platelets of 2 were obtained from a solution of acetone. The crystal (0.200×0.150×0.030 mm) belongs to the monoclinic system, space group P21 (#4), with a = 8.7403(7) Ǻ, b = 7.0111(6) Ǻ, c = 9.0846(7) Ǻ, β = 97.334(2)°, V = 552.14(8) Ǻ3, Z = 2, Dcalcd = 2.117 g/cm3, and (MoKα) = 73.568 cm-1. Intensity data were measured on a Rigaku RAXIS-RAPID diffractometer up to 2θmax of 55.0°. All 6774 reflections were collected. The structure was solved by direct methods (SHELX97) and refined with full-matrix least-squares on F2 procedure. Non-hydrogen atoms were refined with anisotropic thermal parameters. Hydrogen atoms were located by different Fourier techniques and refined with isotropic thermal parameters. The refined structural model converged to a final R1 = 0.0447; wR2 = 0.1223; Flack parameter (Friedel pairs = 1155) = 0.06(2) for 2524 observed reflections [I > 2.00σ(I)] and 148 variable parameters. Crystallographic data of 2 have been deposited with the Cambridge Crystallographic Data Centre (deposit No. CCDC 929142). Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2, 1EZ, UK (e-mail: deposit@ccdc.cam.ac.uk).

Plant growth regulating activity assay
Filter discs (33 mm diameter) bearing test solutions (final concentration, 10-4-10-7 mol/L) were placed in dishes, and wetted with 500 µL of water containing 0.01% Tween 20. Seven seeds of the plant (Lepidium sativum) were put on each disc. After incubation at 25 °C for 24 h in the dark, length of the roots was measured, and compared with a negative control. The assays were carried out in duplicate.

Antimicrobial assay
Antimicrobial activity against two gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), one gram-negative bacteria (Escherichia coli), three fungi (Penicillium sp., Botrytis sp., and Mucor rouxii), and one yeast (Candida utilis) were tested by plate diffusion assay using 8 mm paper disc. Palmaerins A-D (1-4) solutions (1 μmol/50 µL) were prepared by dissolving each compound in acetone. Each adjusted solution (50 µL) was added to paper disc, and the paper discs were dried. The paper discs were set on the agar plate suspended tested microorganisms. After cultivating microorganisms at 37 °C for 24 h (bacteria) or at 24 °C (fungi and yeast) for 72 h, the strength of antimicrobial activity was evaluated by measuring the diameter length of inhibition zone (mm).

ACKNOWLEDGEMENT
This work was partly supported by Grants-in-Aid for the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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