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Dibenzofurans from the cultured lichen mycobionts of Lecanora cinereocarnea
Phytochemistry 58 (2001) 1129–1134 www.elsevier.com/locate/phytochem
Dibenzofurans from the cultured lichen mycobionts of Lecanora cinereocarnea Takao Tanahashia,*, Yukiko Takenakaa, Naotaka Nagakuraa, Nobuo Hamadab a Kobe Pharmaceutical University, 4-19-1, Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan Osaka City Institute of Public Health and Environmental Sciences, 8-34, Tojo-cho, Tennouji-ku, Osaka 543-0026, Japan
b
Received 7 June 2001; received in revised form 23 August 2001
Abstract From the cultures of the spore-derived mycobionts of the lichen Lecanora cinereocarnea, five dibenzofurans, 3,7-dihydroxy-1,9dimethyldibenzofuran, 2-chloro-3,7-dihydroxy-1,9-dimethyldibenzofuran, 2,8-dichloro-3,7-dihydroxy-1,9-dimethyldibenzofuran, 3hydroxy-7-methoxy-1,9-dimethyldibenzofuran, and 2-chloro-7-hydroxy-3-methoxy-1,9-dimethyldibenzofuran, were isolated. Their structures were determined by spectroscopic methods. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Lecanora cinereocarnea; Lichen; Isolated mycobiont; 3,7-Dihydroxy-1,9-dimethyldibenzofuran; Structure elucidation
1. Introduction
2. Results and discussion
Lichens, a symbiotic association of mycobiont and photobiont partners, produce diverse secondary metabolites, some of which show a wide range of potentially useful biological activities (Yamamoto, 1991). One of the intriguing questions in lichenology is the role of mycobiont and photobiont partners in the biosynthesis of such substances. Our recent studies demonstrated that cultures of spore-derived lichen mycobionts have an ability to produce certain lichen substances or novel metabolites in large amounts under osmotically stressed conditions (Tanahashi et al., 1997, 1999, 2000; Takenaka et al., 2000). It was also pointed out that cultures of lichen mycobionts could be good systems for investigating secondary metabolism in lichens. In the course of our studies on cultured lichen mycobionts, we cultivated mycobionts of the lichen Lecanora cinereocarnea and isolated from their cultures five dibenzofurans, four of which are new. The isolation and structural determination of these compounds are reported.
The polyspore-derived mycobionts of Lecanora cinereocarnea collected in Japan were cultured on conventional malt-yeast extract medium supplemented with 10% sucrose at 18 C in the dark. After cultivation over 11–12 months, the colonies and agar medium were harvested and extracted with cold Et2O and Me2CO. The extract was separated by a combination of preparative TLC and preparative HPLC to afford five compounds, 1–5. Compound 1 was isolated as colorless needles, mp. 242 C. Its HR–EIMS spectrum indicated a molecular formula of C14H12O3. Its 1H NMR spectrum (Table 1) exhibited a broad singlet for a methyl group and a pair of meta-coupled doublets. The 13C NMR spectrum of 1 showed a methyl carbon, two aromatic CH carbons and four quaternary carbons, two of which were oxygenated (Table 2). These NMR spectral features together with its structural formula suggested a symmetrical dibenzofuran structure for 1. The presence of two phenolic hydroxyl groups in 1 was indicated by its acetylation to a diacetate 1a. The substitution pattern on the aromatic rings was demonstrated by the HMBC spectrum of 1 where an aromatic proton at 6.70 was coupled with two oxygenated carbons at 156.9 and 159.2, a methine carbon at 114.8 and a quaternary carbon at d 117.6, the latter two carbons were correlated with the methyl
* Corresponding author. Tel.: +81-78-441-7546; fax: +81-78-4417546. E-mail address: [email protected] (T. Tanahashi).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00394-6
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protons at 2.76. Final confirmation was obtained by direct comparison with an authentic sample prepared from 3,5-dimethoxytoluene (Asahina and Aoki, 1941). Although this compound has already been known as a synthetic product, this constitutes the first instance of it’s isolation and characterization as a natural product. The HR–EIMS spectrum of compound 2 established the composition C14H11ClO3. Conventional acetylation of 2 gave a diacetate 2a, C18H15ClO5. The 1H NMR spectrum of 2 exhibited signals for two methyl groups at 2.76 and 2.86, a pair of meta-coupled aromatic protons at 6.57 and 6.70 and an aromatic proton at 6.86. These findings suggested the isolated compound to be a chlorinated derivative of 1. Significant HMBC correlations between the aromatic proton at 6.86 and two oxygenated carbons at 152.5 and 156.3 showed that the chlorine atom was substituted at C-2 rather than C4. This was further confirmed by HMBC experiments with 2a. Thus, the structure of 2 was determined as 2chloro-3,7-dihydroxy-1,9-dimethyldibenzofuran.
Table 1 1 H NMR spectral data for compounds 1–5 and 1a–5a H
1b
2b
3b
4b
2 4 6 8 1-Me 9-Me 3-OMe 7-OMe
6.55 br d (2.0)a 6.70 d (2.0) 6.70 d (2.0) 6.55 br d (2.0) 2.76 br s 2.76 br s
6.86 s 6.70 d (2.5) 6.57 br d (2.5) 2.86 s 2.76 br s
6.82 s 6.82 s 6.66 m 2.75 s 2.75 s
6.57 m 6.72 d (2.0) 6.88 d (2.0) 6.61 m 2.79 br s 2.81 br s
2 4 6 8 1-Me 9-Me 3-OMe 7-OMe 3-OAc 7-OAc
6.86 br d (2.0) 7.18 d (2.0) 7.18 d (2.0) 6.86 br d (2.0) 2.91 br s 2.91 br s
7.24 s 7.20 d (2.0) 6.89 br d (2.0) 2.99 s 2.90 br s
2.34 s 2.34 s
2.40 s 2.34 s
c
Compound 3, C14H10Cl2O3, also showed NMR spectral features suggesting a symmetrical structure. The 13C NMR spectral data of 3 and its acetate 3a were in good agreement with those of chlorinated aromatic ring of 2 and 2a, respectively, suggesting chlorination at C-2 and C-8 in 3 and 3a. This proposal was further supported by
c
1a
b
7.08 s 6.75 d (2.0) 2.92 s 2.81 br s 3.93 s
3.83 s c
H
a
5b
2a
c
3a
4ac
6.71 m 7.25 s 6.91 d (2.0) 7.25 s 7.14 d (2.0) 6.82 m 2.93 s 2.87 br s 2.93 s 2.89 br s 3.98 s 3.88 s 2.41 s 2.41 s 2.41 s
5ac 7.00 s 7.17 d (2.0) 6.86 m 2.97 s 2.89 br s
2.34 s
Values in parentheses are coupling constants in Hz. Measured in CD3OD. Measured in CDCl3.
significant correlations between the aromatic proton signal and two oxygenated carbon signals in the HMBC spectra of 3 and 3a. Accordingly, the isolated compound was characterized as 2,8-dichloro-3,7-dihydroxy1,9-dimethyldibenzofuran. A comparison of the 1H and 13C NMR spectra of the new compound 4 with those of 1 suggested a close relationship between their structures. The NMR spectral features of 4 resembled those of 1 except for the presence of an aromatic methoxyl signal [H 3.83, C 56.0]. The HR–EIMS of 4, showed a molecular ion peak at m/z 242.0961, indicating a molecular formula of C15H14O3, an increase of CH2 when comparing with 1. Conventional acetylation of 4 yielded the monoacetate 4a. These findings suggested that a hydroxyl group of 1 was methylated in 4, and this received further support from methylation of 1 with dimethylsulfate, which afforded 4 and the dimethyl ether 6. Thus, 4 was also elucidated. Compound 5 was isolated in minute amount. Its HR– EIMS spectrum indicated a molecular formula of C15H13ClO3. Its 1H NMR spectrum exhibited signals for two methyl groups at 2.81 and 2.93, a methoxyl group at 3.93, a pair of meta-coupled aromatic protons at 6.61 and 6.75, and an aromatic proton at 7.08 (s). Acetylation of 5 yielded an acetate 5a, indicating the presence of a phenolic hydroxyl group in 5. These findings suggested that 5 is a chlorinated derivative of 4. The detailed 2D-NMR studies confirmed that the substitution of chlorine atom was at C-2, and not at C-4, C-5, nor C-7. The NOESY spectrum showed a throughspace connectivity between the methoxyl group and a
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T. Tanahashi et al. / Phytochemistry 58 (2001) 1129–1134 Table 2 13 C NMR spectral data for compounds 1–5 and 1a–5a C
1a
2a
3a
4a
5a
1ab
2ab
3ab
4ab
5ab
1 2 3 4 4a 5a 6 7 8 9 9a 9b 1-Me 9-Me 3-OMe 7-OMe 3-OAc
132.9 114.8 156.9 96.6 159.2 159.2 96.6 156.9 114.8 132.9 117.6 117.6 25.1 25.1
130.5 118.9c 152.5 97.4 156.3 159.6 96.7 157.4 115.4 133.2 117.2 118.5c 21.2 25.7
130.9 119.0d 153.1 97.4 156.6 156.6 97.4 153.1 119.0d 130.9 118.4d 118.4d 22.0 22.0
133.2 115.0 157.2 96.6 159.1e 159.6e 94.7 159.4 114.3 133.0 118.5 117.4 25.1 25.1
130.9 119.5f 155.0 94.5 156.4 159.7 96.7 157.7 115.7 133.5 117.0 119.4f 21.2 25.7 56.9
133.0 119.3 149.1 103.1 157.2 157.2 103.1 149.1 119.3 133.0 121.5 121.5 24.9 24.9
131.3 122.9g 145.6 104.7 154.5 157.5 103.3 149.5 119.9 133.2 121.2 123.1g 21.2 25.5
131.6 122.7h 146.1 104.8 154.8 154.8 104.8 146.1 122.7h 131.6 123.5h 123.5h 22.0 22.0
132.8 118.9 148.2 102.8 156.6 158.5 93.7 159.0 114.0 131.9 116.7 122.1 24.8 24.8
131.1i 117.6 154.5j 93.3 155.8j 156.8 103.0 148.5 119.6 132.3i 121.9 119.0 21.1 25.5 56.5
21.2 169.5 21.2 169.5
20.7 168.7 21.2 169.3
20.7 168.6 20.7 168.6
56.0
7-OAc
55.5 21.2 168.6 21.2 169.5
a
Measured in CD3OD. Measured in CDCl3. cj Values with the same letters may be interchangeable. b
singlet for a aromatic proton. In the HMBC spectrum of 5, the aromatic proton signal was coupled with two oxygenated carbons at 155.0 and 156.4, and two quaternary carbons at 119.4 and 119.5; the latter two carbon resonances were correlated with the methyl singlet at 2.92, implying that a methoxyl group and a chlorine atom were located at C-3 and C-2, respectively, on the same benzene ring of the dibenzofuran. Further, HMBC correlations among the signals due to another benzene ring, as well as comparison of the 1H and 13C NMR spectral data of 5 and 5a with those of 1 and 1a suggested the same substitution pattern on a benzene ring of 1 and 5. These observations thus allowed us to formulate the structure 5 for the isolated compound. The location of the chlorine atom at C-2 in 5 was further supported by comparative studies of the 13C NMR spectral data of 1, 2, 4, 5, 1a, 2a, 4a and 5a. When a chlorine atom was introduced at C-2 in 1 and 1a, C-2 was shifted downfield whereas C-1, C-3, C-4a and 1-Me were shifted upfield. Similar trends were observed with 4, 5, 4a and 5a. Accordingly the structure of 5 was elucidated as 2-chloro-7-hydroxy-3-methoxy-1,9-dimethyldibenzofuran. It is noteworthy that the isolated dibenzofurans from the cultured mycobionts have no carboxyl group in the structure by contrast with dibenzofurans from natural lichens such as pannaric acid (Huneck and Yoshimura, 1996). According to Culberson and Ahamdjian, most lichen substances arise from phenolic acid precursors such as orsellinic acid. In non-lichen fungi these precursors lead to phycotoxic compounds, the biosynthesis
of which includes a phenolic acid decarboxylation involving fungal decarboxylases. In lichens the algal partner may inhibit such decarboxylases to divert the phenolic acid precursors into secondary metabolites that are nontoxic to photobionts and accumulate in large quantities in the thallus (Culberson and Ahmajian, 1980). This hypothesis was consistent with our results that dibenzofurans with no carboxyl group were obtained from the cultured mycobionts. These results indicated that dormant fungal metabolism was induced in the cultures of the isolated mycobiont.
3. Experimental 3.1. General Melting points were measured on a Yanaco micro melting point apparatus and are uncorr. The UV spectra were recorded on a Shimadzu UV-240 spectrophotometer and the IR spectra on a Shimadzu FTIR8200 infrared spectrophotometer. HR–EIMS were obtained with a Hitachi M-4100 mass spectrometer. The NMR spectroscopic experiments were performed with Varian VXR-500, Varian Gemini-300 and Varian Gemini-200 spectrometers, with tetramethylsilane as internal standard. HPLC was performed using a Waters system (600E Multisolvent Delivery System, 486 Tunable Absorbance Detector). Thin-layer chromatography was performed on precoated Kieselgel 60F254 plates (Merck) and spots were visualized under UV light.
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3.2. Plant material Specimens of Lecanora cinereocarnea (Eschw.) Vain. were collected from the bark of trees in Okinawa Prefecture, Japan (27 N, ca. 100 m alt.) in 1996. The voucher specimen was identified by Dr. H. Miyawaki, Saga University, Japan and was deposited at Osaka City Institute of Public Health and Environmental Sciences with the registration No. NH96112944. Mycobionts of L. cinereocarenea were obtained from the spores discharged from apothecia of a thallus, and were cultivated in test tubes containing modified MY10 medium (malt extract 10 g, yeast extract 4 g, sucrose 100 g, agar 15 g, H2O 1 l, pH 7) at 18 C in the dark. After cultivation for 11–12 months, the colonies and slants with crystals were harvested. 3.3. Extraction and isolation (1) The harvested colonies (101 test tubes, fresh cell weight 12.3 g) were extracted continuously with Et2O and then with Me2CO. The slants were collected and extracted in the same way. The Et2O extracts were concentrated under reduced pressure to give a residue (550 mg). The extracts were repeatedly subjected to preparative TLC (toluene–acetone 9:1) and preparative HPLC (mBondasphere 5 mC18-100 A˚, MeCN–H2O, 7:13), giving 1 (1.2 mg), 2 (14.7 mg), 3 (20.3 mg), 4 (4.9 mg) and 5 (1.6 mg). The Me2CO extracts (898 mg) were also repeatedly purified by preparative TLC and preparative HPLC to afford 2 (5.0 mg), 3 (7.5 mg), 4 (2.5 mg) and 5 (1.4 mg). (2) The harvested colonies (37 test tubes, dry cell weight 6.86 g) and slants were extracted continuously with Et2O and then with Me2CO. The Et2O extracts (380 mg from the colonies and 1.02 g from the slants) were repeatedly subjected to preparative TLC with (toluene–acetone, 19:1, Me2CO–CHCl3, 3:1), giving 1 (3.3 mg), 2 (7.9 mg) and 3 (11.4 mg). The Me2CO extracts (898 mg from the colonies and 3.72 g from the slants) were also repeatedly purified by preparative TLC with (toluene–acetone, 19:1) to afford 1 (5.0 mg), 2 (11.6 mg) and 3 (24.5 mg). 3.3.1. 3,7-Dihydroxy-1,9-dimethyldibenzofuran (1) Colorless crystalline solid, mp 242 C (MeOH). UV MeOH lmax nm (log "): 219 (4.51), 226 (4.52), 239.5 (4.35), 257 sh (4.09), 263 (4.12), 302 (4.23), 310 (4.23); IR KBr max cm1: 3275, 1616, 1585, 824; 1H NMR: see Table 1; 13C NMR: see Table 2; HMBC correlations: 1-CH3/9CH3!C-1/C-9, C-2/C-8, C-9a/C-9b; H-2/H-8!C-3/C7, C-4/C-6, C-9a/C-9b; H-4/H-6!C-2/C-8, C-3/C-7, C4a/C-5a, C-9a/C-9b; HR-EIMS m/z: calc. for C14H12O3 [M]+: 228.0787. Found: 228.0781.
243 sh (4.35), 263 (4.09), 306.5 (4.25), 314.5 (4.25); IR 1 1 KBr max cm : 3324, 1611, 1576, 830; H NMR: see Table 1; 13 C NMR: see Table 2; NOESY correlation: H-8$9CH3; HMBC correlations: 1-CH3!C-1, C-2, C-9b; H4!C-2, C-3, C-4a, C-9b; H-6! C-5a, C-7, C-8, C-9a; H-8!C-6, C-7, C-9a; 9-CH3!C-8, C-9, C-9a; HREIMS m/z: calcd. for C14H1135ClO3 [M]+: 262.0397. Found: 262.0374; calc. for C14H1137ClO3 [M]+: 264.0368. Found: 264.0357. 3.3.3. 2,8-Dichloro-3,7-dihydroxy-1,9dimethyldibenzofuran (3) Colorless crystalline solid, mp 217.5–218.5 C (MeOH). UV lMeOH nm (log "): 231 (4.60), 245 (4.35), max 1 262.5 (4.02), 309 sh (4.27), 317.5 (4.32); IR KBr max cm : 1 13 3422, 1607, 1583, 812; H NMR: see Table 1; C NMR: see Table 2; HMBC correlations: 1-CH3/9-CH3!C-1/ C-9, C-2/C-8, C-9a/C-9b; H-4/H-6!C-2/C-8, C-3/C-7, C-4a/C-5a, C-9a/C-9b; HR–EIMS m/z: calc. for C14H1035Cl2O3 [M]+: 296.0007. Found: 296.0016; calc. for C14H1035Cl37ClO3 [M]+: 297.9978. Found: 297.9962; calc. for C14H1037Cl2O3 [M]+: 299.9948. Found: 299.9936. 3.3.4. 3-Hydroxy-7-methoxy-1,9-dimethyldibenzofuran (4) Colorless crystalline solid, mp 139–139.5 C (MeOH). UV lMeOH nm (log "): 219 (4.45), 225.5 (4.46), 239 sh max (4.30), 257 sh (4.04), 263 (4.07), 300.5 (4.19), 308.5 1 1 (4.23); IR KBr max cm : 3368, 1612, 1582, 808; H NMR: 13 see Table 1; C NMR: see Table 2; NOESY correlations: 1-CH3$H-2; OCH3$H-4; H-8$9-CH3; HMBC correlations: 1-CH3!C-1, C-2, C-9b; H-4!C-4a; H6!C-5a, C-6; H-8!C-7; 9-CH3!C-8, C-9, C-9a; HR– EIMS m/z: calc. for C15H13O3 [M]+: 242.0944. Found: 242.0961. 3.3.5. 2-Chloro-7-hydroxy-3-methoxy-1,9dimethyldibenzofuran (5) Colorless crystalline solid, mp 198–199 C (MeOH). UV lMeOH nm (log "): 227 (4.26), 241 sh (4.18), 255 max 1 (3.88), 263 (3.91), 305 (4.07), 312.5 (4.06); IR KBr max cm : 1 13 3110, 1638, 1609, 1589, 814; H NMR: see Table 1; C NMR: see Table 2; NOESY correlations: H-4$OCH3, H-8$9-CH3; HMBC correlations: 1-CH3!C-1, C-2, C-9b; OCH3!C-3; H-4!C-2, C-3, C-4a, C-9b; H6!C-5a, C-8, C-9a; H-8!C-6, C-9a; 9-CH3!C-8, C-9, C-9a. HR–EIMS m/z: calc. for C15H1435ClO3 [M]+: 276.0554. Found: 276.0577; calc. for C15H1437ClO3 [M]+: 278.0524. Found: 278.0503. 3.4. Acetylation of 1–5
3.3.2. 2-Chloro-3,7-dihydroxy-1,9-dimethyldibenzofuran (2) Colorless crystalline solid, mp 193–194 C (MeOH). UV lMeOH nm (log "): 205 (4.37), 223 (4.55), 228 (4.57), max
Compound 1 (3.4 mg) was acetylated with Ac2O-pyridine (each 0.1 ml) and the crude acetate was purified by preparative TLC (CHCl3) to yield 1a (3.9 mg). Com-
T. Tanahashi et al. / Phytochemistry 58 (2001) 1129–1134
pound 2 (7.7 mg) and 3 (7.8 mg) were acetylated and purified in the same as for 1 to give 2a (8.0 mg) and 3a (7.6 mg), respectively. A mixture (19.1 mg) of 4 and 5 was acetylated and purified by preparative HPLC (mBondasphere 5 mC18–100 A˚, MeCN–H2O, 5:3) to give 4a (2.9 mg) and 5a (4.6 mg). 3.4.1. 3,7-Diacetoxy-1,9-dimethyldibenzofuran (1a) Colorless crystalline solid, mp. 162–162.3 C (MeOH). UV lMeOH nm (log "): 213 (4.66), 227 (4.73), 252 (4.21), max 1 260 (4.30), 287 (4.46), 296 sh (4.34); IR KBr max cm : 1753, 1 13 1609, 1585, 1499; H NMR: see Table 1; C NMR: see Table 2; HMBC correlations: 1-CH3/9-CH3!C-1/C-9, C-2/C-8, C-9a/C-9b; H-2/H-8!C-1/C-9, C-3/C-7, C-4/ C-6, C-9a/C-9b; H-4/H-6!C-2/C-8. EIMS m/z (rel. int.%): 312 (21.6) [M]+, 270 (17.8), 228 (100); HR– EIMS m/z: calc. for C18H16O5 [M]+: 312.0998. Found: 312.1015. 3.4.2. 3,7-Diacetoxy-2-chloro-1,9-dimethyldibenzofuran (2a) Colorless crystalline solid, mp 171.5–172.0 C (MeOH). UV lMeOH nm (log "): 216 (4.51), 230 (4.61), max 253 (4.02), 261 (4.12), 289 (4.29), 299 sh (4.11), 312 1 1 (3.74); IR KBr max cm : 1765, 1601, 1587; H NMR: see 13 Table 1; C NMR: see Table 2; HMBC correlations: 1CH3!C-1, C-2, C-9b; H-4!C-2, C-3, C-4a, C-9b; H6!C-7, C-8; H-8!C-6, C-7, C-9a; 9-CH3!C-8, C-9, C-9a; EIMS m/z: (rel. int.%): 348 (3.5), 346 (9.8) [M]+, 306 (5.5), 304 (15.4), 264 (37), 262 (100); HR-EIMS m/z calc. for C18H1535ClO5 [M]+: 346.0609. Found: 346.0602; calc. for C18H1537ClO5 [M]+: 348.0579. Found: 348.0583. 3.4.3. 3,7-Diacetoxy-2,8-dichloro-1,9dimethyldibenzofuran (3a) Colorless crystalline solid, mp 192–193 C (MeOH). UV lMeOH nm (log "): 218 (4.52), 233 (4.66), 253 (4.03), max 1 261 (4.12), 293 (4.29), 304 sh (4.00); IR KBr max cm : 1771, 1 13 1593; H NMR: see Table 1; C NMR: see Table 2; HMBC correlations: 1-CH3/9-CH3!C-1/C-9, C-2/C-8, C-9a/C-9b; H-4/H-6!C-2/C-8, C-3/C-7, C-4a/C-5a, C9a/C-9b; EIMS m/z: (rel. int.%): 384 (1.1), 382 (4.9), 380 (7.9) [M]+, 342 (1.4), 340 (4.9), 338 (9.3), 300 (12), 298 (67), 296 (100); HR–EIMS m/z calc. for C18H1435Cl2O5 [M]+: 380.0219. Found: 380.0203; calc. for C18H1435Cl37ClO5 [M]+: 382.0189. Found: 382.0143; calc. for C18H1437Cl2O5 [M]+: 384.0160. Found: 384.0108. 3.4.4. 3-Acetoxy-7-methoxy-1,9-dimethyldibenzofuran (4a) Colorless crystalline solid, mp 116–117 C (MeOH). UV lMeOH nm (log "): 215.5 (4.35), 226 (4.49), 237 sh max (4.35), 256 sh (4.01), 262 (4.09), 295 (4.26), 301 (4.26), 1 1 305.5 (4.26); IR nKBr max cm : 1759, 1607, 1578, 829; H 13 NMR: see Table 1; C NMR: see Table 2; EIMS m/z:
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(rel. int.%): 284 (37) [M]+, 242 (100). HR–EIMS m/z: calc. for C17H16O4 [M]+: 284.1049. Found: 284.1060. 3.4.5. 7-Acetoxy-2-chloro-3-methoxy-1,9dimethyldibenzofuran (5a) Colorless crystalline solid, mp 169 C (MeOH). UV MeOH lmax nm (log "): 229.5 (4.41), 254.5 (3.88), 262 (3.95), 1 291 sh (4.04), 301 (4.10), 313.5 (3.95); IR KBr max cm : 1 13 1747, 1607, 1541, 810; H NMR: see Table 1; C NMR: see Table 2; EIMS m/z: (rel. int.%): 320 (11), 318 (30) [M]+, 278 (36), 276 (100), 262 (12), 260 (35); HR–EIMS m/z: calc. for C17H1535ClO4 [M]+: 318.0659. Found: 318.0675; calc. for C17H1537ClO4 [M]+: 320.0630. Found: 320.0637. 3.5. Synthesis of 1 and 4 Compound 1 was prepared from 3,5-dimethoxytoluene according to Asahina. (Asahina and Aoki, 1941). The physical and spectral data of the product were identical to those of the isolated compound. To a solution of 1 (19.8 mg) in acetone (20 ml) were added K2CO3 (600 mg) and Me2SO4 (20 ml), and the whole was heated under reflux for 4 h. The reaction mixture was diluted with 5% NaHCO3 and extracted with CHCl3. The washed and dried CHCl3 layers were concentrated in vacuo and the residue was subjected to prep. TLC with (toluene–acetone 19:1) to afford a monomethyl ether (3.3 mg) and a dimethyl ether (6) (17.9 mg). The monomethyl ether was identified with 4. 6: needles, mp. 153.7–154 C (MeOH). 1H NMR (CDCl3): 2.83 (6H, s, 1-CH3 and 9-CH3), 3.84 (6H, s, 3-OCH3 and 7-OCH3), 6.69 (2H, m, H-2 and H-8), 6.92 (2H, m, H-4 and H-6). EIMS m/z (%): 330 [M]+ (35), 315 (100), 300 (7.3), 285 (28). HR–EIMS m/z: calc. for C16H16O3 [M]+: 256.1100. Found: 256.1095. Acknowledgements This research was financially supported by Grant-inAid for Scientific Research (C) (No. 09672179 and No. 11672129) from the Ministry of Education, Sciences, Sports and Culture of Japan and Kobe Pharmaceutical University Collaboration Fund. We are grateful to Dr. H. Miyawaki (Saga University, Japan) for identification of the voucher specimen. Thanks are also due to Dr. M. Sugiura (Kobe Pharmaceutical University) for 1H and 13 C NMR spectra, and to Dr. K. Saiki (Kobe Pharmaceutical University) for mass spectra measurements. References Asahina, Y., Aoki, M., 1941. On oxy-diphenyleneoxide derivatives. Yakugaku Zasshi 64, 41–47. Culberson, C.F., Ahmajian, V., 1980. Artificial reestablishment of lichens. II. Secondary products of resynthesized Cladonia cristatella and Lecanora chrysoleuca. Mycologia 72, 90–109.
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Huneck, S., Yoshimura, I., 1996. Identification of Lichen Substances. Springer Verlag, Berlin, Heidelberg. Takenaka, Y., Tanahashi, T., Nagakura, N., Hamada, N., 2000. 2,3Dialkylchromones from mycobiont cultures of the lichen Graphis scripta. Heterocycles 53, 1589–1593. Tanahashi, T., Kuroishi, M., Kuwahara, A., Nagakura, N., Hamada, N., 1997. Four phenolics from the cultured lichen mycobiont of Graphis scripta var. pulverulenta. Chem. Pharm. Bull. 45, 1183–1185. Tanahashi, T., Takenaka, Y., Nagakura, N., Ikuta, Y., Tani, K.,
Hamada, N., 1999. Xanthones from the cultured lichen mycobionts of Pyrenula japonica and Pyrenula pseudobufonia. Phytochemistry 52, 401–405. Tanahashi, T., Takenaka, Y., Nagakura, N., Hamada, N., Miyawaki, H., 2000. Two isocoumarins from the cultured lichen mycobiont of Graphis sp. Heterocycles 53, 723–728. Yamamoto, Y., 1991. Production of lichen substances. In: Komamine, A., Misawa, M., DiCosmo, F. (Eds.), Plant Cell Culture in Japan. CMC, Tokyo, pp. 58–71.
Dibenzofurans from the cultured lichen mycobionts of Lecanora cinereocarnea Takao Tanahashia,*, Yukiko Takenakaa, Naotaka Nagakuraa, Nobuo Hamadab a Kobe Pharmaceutical University, 4-19-1, Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan Osaka City Institute of Public Health and Environmental Sciences, 8-34, Tojo-cho, Tennouji-ku, Osaka 543-0026, Japan
b
Received 7 June 2001; received in revised form 23 August 2001
Abstract From the cultures of the spore-derived mycobionts of the lichen Lecanora cinereocarnea, five dibenzofurans, 3,7-dihydroxy-1,9dimethyldibenzofuran, 2-chloro-3,7-dihydroxy-1,9-dimethyldibenzofuran, 2,8-dichloro-3,7-dihydroxy-1,9-dimethyldibenzofuran, 3hydroxy-7-methoxy-1,9-dimethyldibenzofuran, and 2-chloro-7-hydroxy-3-methoxy-1,9-dimethyldibenzofuran, were isolated. Their structures were determined by spectroscopic methods. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Lecanora cinereocarnea; Lichen; Isolated mycobiont; 3,7-Dihydroxy-1,9-dimethyldibenzofuran; Structure elucidation
1. Introduction
2. Results and discussion
Lichens, a symbiotic association of mycobiont and photobiont partners, produce diverse secondary metabolites, some of which show a wide range of potentially useful biological activities (Yamamoto, 1991). One of the intriguing questions in lichenology is the role of mycobiont and photobiont partners in the biosynthesis of such substances. Our recent studies demonstrated that cultures of spore-derived lichen mycobionts have an ability to produce certain lichen substances or novel metabolites in large amounts under osmotically stressed conditions (Tanahashi et al., 1997, 1999, 2000; Takenaka et al., 2000). It was also pointed out that cultures of lichen mycobionts could be good systems for investigating secondary metabolism in lichens. In the course of our studies on cultured lichen mycobionts, we cultivated mycobionts of the lichen Lecanora cinereocarnea and isolated from their cultures five dibenzofurans, four of which are new. The isolation and structural determination of these compounds are reported.
The polyspore-derived mycobionts of Lecanora cinereocarnea collected in Japan were cultured on conventional malt-yeast extract medium supplemented with 10% sucrose at 18 C in the dark. After cultivation over 11–12 months, the colonies and agar medium were harvested and extracted with cold Et2O and Me2CO. The extract was separated by a combination of preparative TLC and preparative HPLC to afford five compounds, 1–5. Compound 1 was isolated as colorless needles, mp. 242 C. Its HR–EIMS spectrum indicated a molecular formula of C14H12O3. Its 1H NMR spectrum (Table 1) exhibited a broad singlet for a methyl group and a pair of meta-coupled doublets. The 13C NMR spectrum of 1 showed a methyl carbon, two aromatic CH carbons and four quaternary carbons, two of which were oxygenated (Table 2). These NMR spectral features together with its structural formula suggested a symmetrical dibenzofuran structure for 1. The presence of two phenolic hydroxyl groups in 1 was indicated by its acetylation to a diacetate 1a. The substitution pattern on the aromatic rings was demonstrated by the HMBC spectrum of 1 where an aromatic proton at 6.70 was coupled with two oxygenated carbons at 156.9 and 159.2, a methine carbon at 114.8 and a quaternary carbon at d 117.6, the latter two carbons were correlated with the methyl
* Corresponding author. Tel.: +81-78-441-7546; fax: +81-78-4417546. E-mail address: [email protected] (T. Tanahashi).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00394-6
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protons at 2.76. Final confirmation was obtained by direct comparison with an authentic sample prepared from 3,5-dimethoxytoluene (Asahina and Aoki, 1941). Although this compound has already been known as a synthetic product, this constitutes the first instance of it’s isolation and characterization as a natural product. The HR–EIMS spectrum of compound 2 established the composition C14H11ClO3. Conventional acetylation of 2 gave a diacetate 2a, C18H15ClO5. The 1H NMR spectrum of 2 exhibited signals for two methyl groups at 2.76 and 2.86, a pair of meta-coupled aromatic protons at 6.57 and 6.70 and an aromatic proton at 6.86. These findings suggested the isolated compound to be a chlorinated derivative of 1. Significant HMBC correlations between the aromatic proton at 6.86 and two oxygenated carbons at 152.5 and 156.3 showed that the chlorine atom was substituted at C-2 rather than C4. This was further confirmed by HMBC experiments with 2a. Thus, the structure of 2 was determined as 2chloro-3,7-dihydroxy-1,9-dimethyldibenzofuran.
Table 1 1 H NMR spectral data for compounds 1–5 and 1a–5a H
1b
2b
3b
4b
2 4 6 8 1-Me 9-Me 3-OMe 7-OMe
6.55 br d (2.0)a 6.70 d (2.0) 6.70 d (2.0) 6.55 br d (2.0) 2.76 br s 2.76 br s
6.86 s 6.70 d (2.5) 6.57 br d (2.5) 2.86 s 2.76 br s
6.82 s 6.82 s 6.66 m 2.75 s 2.75 s
6.57 m 6.72 d (2.0) 6.88 d (2.0) 6.61 m 2.79 br s 2.81 br s
2 4 6 8 1-Me 9-Me 3-OMe 7-OMe 3-OAc 7-OAc
6.86 br d (2.0) 7.18 d (2.0) 7.18 d (2.0) 6.86 br d (2.0) 2.91 br s 2.91 br s
7.24 s 7.20 d (2.0) 6.89 br d (2.0) 2.99 s 2.90 br s
2.34 s 2.34 s
2.40 s 2.34 s
c
Compound 3, C14H10Cl2O3, also showed NMR spectral features suggesting a symmetrical structure. The 13C NMR spectral data of 3 and its acetate 3a were in good agreement with those of chlorinated aromatic ring of 2 and 2a, respectively, suggesting chlorination at C-2 and C-8 in 3 and 3a. This proposal was further supported by
c
1a
b
7.08 s 6.75 d (2.0) 2.92 s 2.81 br s 3.93 s
3.83 s c
H
a
5b
2a
c
3a
4ac
6.71 m 7.25 s 6.91 d (2.0) 7.25 s 7.14 d (2.0) 6.82 m 2.93 s 2.87 br s 2.93 s 2.89 br s 3.98 s 3.88 s 2.41 s 2.41 s 2.41 s
5ac 7.00 s 7.17 d (2.0) 6.86 m 2.97 s 2.89 br s
2.34 s
Values in parentheses are coupling constants in Hz. Measured in CD3OD. Measured in CDCl3.
significant correlations between the aromatic proton signal and two oxygenated carbon signals in the HMBC spectra of 3 and 3a. Accordingly, the isolated compound was characterized as 2,8-dichloro-3,7-dihydroxy1,9-dimethyldibenzofuran. A comparison of the 1H and 13C NMR spectra of the new compound 4 with those of 1 suggested a close relationship between their structures. The NMR spectral features of 4 resembled those of 1 except for the presence of an aromatic methoxyl signal [H 3.83, C 56.0]. The HR–EIMS of 4, showed a molecular ion peak at m/z 242.0961, indicating a molecular formula of C15H14O3, an increase of CH2 when comparing with 1. Conventional acetylation of 4 yielded the monoacetate 4a. These findings suggested that a hydroxyl group of 1 was methylated in 4, and this received further support from methylation of 1 with dimethylsulfate, which afforded 4 and the dimethyl ether 6. Thus, 4 was also elucidated. Compound 5 was isolated in minute amount. Its HR– EIMS spectrum indicated a molecular formula of C15H13ClO3. Its 1H NMR spectrum exhibited signals for two methyl groups at 2.81 and 2.93, a methoxyl group at 3.93, a pair of meta-coupled aromatic protons at 6.61 and 6.75, and an aromatic proton at 7.08 (s). Acetylation of 5 yielded an acetate 5a, indicating the presence of a phenolic hydroxyl group in 5. These findings suggested that 5 is a chlorinated derivative of 4. The detailed 2D-NMR studies confirmed that the substitution of chlorine atom was at C-2, and not at C-4, C-5, nor C-7. The NOESY spectrum showed a throughspace connectivity between the methoxyl group and a
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T. Tanahashi et al. / Phytochemistry 58 (2001) 1129–1134 Table 2 13 C NMR spectral data for compounds 1–5 and 1a–5a C
1a
2a
3a
4a
5a
1ab
2ab
3ab
4ab
5ab
1 2 3 4 4a 5a 6 7 8 9 9a 9b 1-Me 9-Me 3-OMe 7-OMe 3-OAc
132.9 114.8 156.9 96.6 159.2 159.2 96.6 156.9 114.8 132.9 117.6 117.6 25.1 25.1
130.5 118.9c 152.5 97.4 156.3 159.6 96.7 157.4 115.4 133.2 117.2 118.5c 21.2 25.7
130.9 119.0d 153.1 97.4 156.6 156.6 97.4 153.1 119.0d 130.9 118.4d 118.4d 22.0 22.0
133.2 115.0 157.2 96.6 159.1e 159.6e 94.7 159.4 114.3 133.0 118.5 117.4 25.1 25.1
130.9 119.5f 155.0 94.5 156.4 159.7 96.7 157.7 115.7 133.5 117.0 119.4f 21.2 25.7 56.9
133.0 119.3 149.1 103.1 157.2 157.2 103.1 149.1 119.3 133.0 121.5 121.5 24.9 24.9
131.3 122.9g 145.6 104.7 154.5 157.5 103.3 149.5 119.9 133.2 121.2 123.1g 21.2 25.5
131.6 122.7h 146.1 104.8 154.8 154.8 104.8 146.1 122.7h 131.6 123.5h 123.5h 22.0 22.0
132.8 118.9 148.2 102.8 156.6 158.5 93.7 159.0 114.0 131.9 116.7 122.1 24.8 24.8
131.1i 117.6 154.5j 93.3 155.8j 156.8 103.0 148.5 119.6 132.3i 121.9 119.0 21.1 25.5 56.5
21.2 169.5 21.2 169.5
20.7 168.7 21.2 169.3
20.7 168.6 20.7 168.6
56.0
7-OAc
55.5 21.2 168.6 21.2 169.5
a
Measured in CD3OD. Measured in CDCl3. cj Values with the same letters may be interchangeable. b
singlet for a aromatic proton. In the HMBC spectrum of 5, the aromatic proton signal was coupled with two oxygenated carbons at 155.0 and 156.4, and two quaternary carbons at 119.4 and 119.5; the latter two carbon resonances were correlated with the methyl singlet at 2.92, implying that a methoxyl group and a chlorine atom were located at C-3 and C-2, respectively, on the same benzene ring of the dibenzofuran. Further, HMBC correlations among the signals due to another benzene ring, as well as comparison of the 1H and 13C NMR spectral data of 5 and 5a with those of 1 and 1a suggested the same substitution pattern on a benzene ring of 1 and 5. These observations thus allowed us to formulate the structure 5 for the isolated compound. The location of the chlorine atom at C-2 in 5 was further supported by comparative studies of the 13C NMR spectral data of 1, 2, 4, 5, 1a, 2a, 4a and 5a. When a chlorine atom was introduced at C-2 in 1 and 1a, C-2 was shifted downfield whereas C-1, C-3, C-4a and 1-Me were shifted upfield. Similar trends were observed with 4, 5, 4a and 5a. Accordingly the structure of 5 was elucidated as 2-chloro-7-hydroxy-3-methoxy-1,9-dimethyldibenzofuran. It is noteworthy that the isolated dibenzofurans from the cultured mycobionts have no carboxyl group in the structure by contrast with dibenzofurans from natural lichens such as pannaric acid (Huneck and Yoshimura, 1996). According to Culberson and Ahamdjian, most lichen substances arise from phenolic acid precursors such as orsellinic acid. In non-lichen fungi these precursors lead to phycotoxic compounds, the biosynthesis
of which includes a phenolic acid decarboxylation involving fungal decarboxylases. In lichens the algal partner may inhibit such decarboxylases to divert the phenolic acid precursors into secondary metabolites that are nontoxic to photobionts and accumulate in large quantities in the thallus (Culberson and Ahmajian, 1980). This hypothesis was consistent with our results that dibenzofurans with no carboxyl group were obtained from the cultured mycobionts. These results indicated that dormant fungal metabolism was induced in the cultures of the isolated mycobiont.
3. Experimental 3.1. General Melting points were measured on a Yanaco micro melting point apparatus and are uncorr. The UV spectra were recorded on a Shimadzu UV-240 spectrophotometer and the IR spectra on a Shimadzu FTIR8200 infrared spectrophotometer. HR–EIMS were obtained with a Hitachi M-4100 mass spectrometer. The NMR spectroscopic experiments were performed with Varian VXR-500, Varian Gemini-300 and Varian Gemini-200 spectrometers, with tetramethylsilane as internal standard. HPLC was performed using a Waters system (600E Multisolvent Delivery System, 486 Tunable Absorbance Detector). Thin-layer chromatography was performed on precoated Kieselgel 60F254 plates (Merck) and spots were visualized under UV light.
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3.2. Plant material Specimens of Lecanora cinereocarnea (Eschw.) Vain. were collected from the bark of trees in Okinawa Prefecture, Japan (27 N, ca. 100 m alt.) in 1996. The voucher specimen was identified by Dr. H. Miyawaki, Saga University, Japan and was deposited at Osaka City Institute of Public Health and Environmental Sciences with the registration No. NH96112944. Mycobionts of L. cinereocarenea were obtained from the spores discharged from apothecia of a thallus, and were cultivated in test tubes containing modified MY10 medium (malt extract 10 g, yeast extract 4 g, sucrose 100 g, agar 15 g, H2O 1 l, pH 7) at 18 C in the dark. After cultivation for 11–12 months, the colonies and slants with crystals were harvested. 3.3. Extraction and isolation (1) The harvested colonies (101 test tubes, fresh cell weight 12.3 g) were extracted continuously with Et2O and then with Me2CO. The slants were collected and extracted in the same way. The Et2O extracts were concentrated under reduced pressure to give a residue (550 mg). The extracts were repeatedly subjected to preparative TLC (toluene–acetone 9:1) and preparative HPLC (mBondasphere 5 mC18-100 A˚, MeCN–H2O, 7:13), giving 1 (1.2 mg), 2 (14.7 mg), 3 (20.3 mg), 4 (4.9 mg) and 5 (1.6 mg). The Me2CO extracts (898 mg) were also repeatedly purified by preparative TLC and preparative HPLC to afford 2 (5.0 mg), 3 (7.5 mg), 4 (2.5 mg) and 5 (1.4 mg). (2) The harvested colonies (37 test tubes, dry cell weight 6.86 g) and slants were extracted continuously with Et2O and then with Me2CO. The Et2O extracts (380 mg from the colonies and 1.02 g from the slants) were repeatedly subjected to preparative TLC with (toluene–acetone, 19:1, Me2CO–CHCl3, 3:1), giving 1 (3.3 mg), 2 (7.9 mg) and 3 (11.4 mg). The Me2CO extracts (898 mg from the colonies and 3.72 g from the slants) were also repeatedly purified by preparative TLC with (toluene–acetone, 19:1) to afford 1 (5.0 mg), 2 (11.6 mg) and 3 (24.5 mg). 3.3.1. 3,7-Dihydroxy-1,9-dimethyldibenzofuran (1) Colorless crystalline solid, mp 242 C (MeOH). UV MeOH lmax nm (log "): 219 (4.51), 226 (4.52), 239.5 (4.35), 257 sh (4.09), 263 (4.12), 302 (4.23), 310 (4.23); IR KBr max cm1: 3275, 1616, 1585, 824; 1H NMR: see Table 1; 13C NMR: see Table 2; HMBC correlations: 1-CH3/9CH3!C-1/C-9, C-2/C-8, C-9a/C-9b; H-2/H-8!C-3/C7, C-4/C-6, C-9a/C-9b; H-4/H-6!C-2/C-8, C-3/C-7, C4a/C-5a, C-9a/C-9b; HR-EIMS m/z: calc. for C14H12O3 [M]+: 228.0787. Found: 228.0781.
243 sh (4.35), 263 (4.09), 306.5 (4.25), 314.5 (4.25); IR 1 1 KBr max cm : 3324, 1611, 1576, 830; H NMR: see Table 1; 13 C NMR: see Table 2; NOESY correlation: H-8$9CH3; HMBC correlations: 1-CH3!C-1, C-2, C-9b; H4!C-2, C-3, C-4a, C-9b; H-6! C-5a, C-7, C-8, C-9a; H-8!C-6, C-7, C-9a; 9-CH3!C-8, C-9, C-9a; HREIMS m/z: calcd. for C14H1135ClO3 [M]+: 262.0397. Found: 262.0374; calc. for C14H1137ClO3 [M]+: 264.0368. Found: 264.0357. 3.3.3. 2,8-Dichloro-3,7-dihydroxy-1,9dimethyldibenzofuran (3) Colorless crystalline solid, mp 217.5–218.5 C (MeOH). UV lMeOH nm (log "): 231 (4.60), 245 (4.35), max 1 262.5 (4.02), 309 sh (4.27), 317.5 (4.32); IR KBr max cm : 1 13 3422, 1607, 1583, 812; H NMR: see Table 1; C NMR: see Table 2; HMBC correlations: 1-CH3/9-CH3!C-1/ C-9, C-2/C-8, C-9a/C-9b; H-4/H-6!C-2/C-8, C-3/C-7, C-4a/C-5a, C-9a/C-9b; HR–EIMS m/z: calc. for C14H1035Cl2O3 [M]+: 296.0007. Found: 296.0016; calc. for C14H1035Cl37ClO3 [M]+: 297.9978. Found: 297.9962; calc. for C14H1037Cl2O3 [M]+: 299.9948. Found: 299.9936. 3.3.4. 3-Hydroxy-7-methoxy-1,9-dimethyldibenzofuran (4) Colorless crystalline solid, mp 139–139.5 C (MeOH). UV lMeOH nm (log "): 219 (4.45), 225.5 (4.46), 239 sh max (4.30), 257 sh (4.04), 263 (4.07), 300.5 (4.19), 308.5 1 1 (4.23); IR KBr max cm : 3368, 1612, 1582, 808; H NMR: 13 see Table 1; C NMR: see Table 2; NOESY correlations: 1-CH3$H-2; OCH3$H-4; H-8$9-CH3; HMBC correlations: 1-CH3!C-1, C-2, C-9b; H-4!C-4a; H6!C-5a, C-6; H-8!C-7; 9-CH3!C-8, C-9, C-9a; HR– EIMS m/z: calc. for C15H13O3 [M]+: 242.0944. Found: 242.0961. 3.3.5. 2-Chloro-7-hydroxy-3-methoxy-1,9dimethyldibenzofuran (5) Colorless crystalline solid, mp 198–199 C (MeOH). UV lMeOH nm (log "): 227 (4.26), 241 sh (4.18), 255 max 1 (3.88), 263 (3.91), 305 (4.07), 312.5 (4.06); IR KBr max cm : 1 13 3110, 1638, 1609, 1589, 814; H NMR: see Table 1; C NMR: see Table 2; NOESY correlations: H-4$OCH3, H-8$9-CH3; HMBC correlations: 1-CH3!C-1, C-2, C-9b; OCH3!C-3; H-4!C-2, C-3, C-4a, C-9b; H6!C-5a, C-8, C-9a; H-8!C-6, C-9a; 9-CH3!C-8, C-9, C-9a. HR–EIMS m/z: calc. for C15H1435ClO3 [M]+: 276.0554. Found: 276.0577; calc. for C15H1437ClO3 [M]+: 278.0524. Found: 278.0503. 3.4. Acetylation of 1–5
3.3.2. 2-Chloro-3,7-dihydroxy-1,9-dimethyldibenzofuran (2) Colorless crystalline solid, mp 193–194 C (MeOH). UV lMeOH nm (log "): 205 (4.37), 223 (4.55), 228 (4.57), max
Compound 1 (3.4 mg) was acetylated with Ac2O-pyridine (each 0.1 ml) and the crude acetate was purified by preparative TLC (CHCl3) to yield 1a (3.9 mg). Com-
T. Tanahashi et al. / Phytochemistry 58 (2001) 1129–1134
pound 2 (7.7 mg) and 3 (7.8 mg) were acetylated and purified in the same as for 1 to give 2a (8.0 mg) and 3a (7.6 mg), respectively. A mixture (19.1 mg) of 4 and 5 was acetylated and purified by preparative HPLC (mBondasphere 5 mC18–100 A˚, MeCN–H2O, 5:3) to give 4a (2.9 mg) and 5a (4.6 mg). 3.4.1. 3,7-Diacetoxy-1,9-dimethyldibenzofuran (1a) Colorless crystalline solid, mp. 162–162.3 C (MeOH). UV lMeOH nm (log "): 213 (4.66), 227 (4.73), 252 (4.21), max 1 260 (4.30), 287 (4.46), 296 sh (4.34); IR KBr max cm : 1753, 1 13 1609, 1585, 1499; H NMR: see Table 1; C NMR: see Table 2; HMBC correlations: 1-CH3/9-CH3!C-1/C-9, C-2/C-8, C-9a/C-9b; H-2/H-8!C-1/C-9, C-3/C-7, C-4/ C-6, C-9a/C-9b; H-4/H-6!C-2/C-8. EIMS m/z (rel. int.%): 312 (21.6) [M]+, 270 (17.8), 228 (100); HR– EIMS m/z: calc. for C18H16O5 [M]+: 312.0998. Found: 312.1015. 3.4.2. 3,7-Diacetoxy-2-chloro-1,9-dimethyldibenzofuran (2a) Colorless crystalline solid, mp 171.5–172.0 C (MeOH). UV lMeOH nm (log "): 216 (4.51), 230 (4.61), max 253 (4.02), 261 (4.12), 289 (4.29), 299 sh (4.11), 312 1 1 (3.74); IR KBr max cm : 1765, 1601, 1587; H NMR: see 13 Table 1; C NMR: see Table 2; HMBC correlations: 1CH3!C-1, C-2, C-9b; H-4!C-2, C-3, C-4a, C-9b; H6!C-7, C-8; H-8!C-6, C-7, C-9a; 9-CH3!C-8, C-9, C-9a; EIMS m/z: (rel. int.%): 348 (3.5), 346 (9.8) [M]+, 306 (5.5), 304 (15.4), 264 (37), 262 (100); HR-EIMS m/z calc. for C18H1535ClO5 [M]+: 346.0609. Found: 346.0602; calc. for C18H1537ClO5 [M]+: 348.0579. Found: 348.0583. 3.4.3. 3,7-Diacetoxy-2,8-dichloro-1,9dimethyldibenzofuran (3a) Colorless crystalline solid, mp 192–193 C (MeOH). UV lMeOH nm (log "): 218 (4.52), 233 (4.66), 253 (4.03), max 1 261 (4.12), 293 (4.29), 304 sh (4.00); IR KBr max cm : 1771, 1 13 1593; H NMR: see Table 1; C NMR: see Table 2; HMBC correlations: 1-CH3/9-CH3!C-1/C-9, C-2/C-8, C-9a/C-9b; H-4/H-6!C-2/C-8, C-3/C-7, C-4a/C-5a, C9a/C-9b; EIMS m/z: (rel. int.%): 384 (1.1), 382 (4.9), 380 (7.9) [M]+, 342 (1.4), 340 (4.9), 338 (9.3), 300 (12), 298 (67), 296 (100); HR–EIMS m/z calc. for C18H1435Cl2O5 [M]+: 380.0219. Found: 380.0203; calc. for C18H1435Cl37ClO5 [M]+: 382.0189. Found: 382.0143; calc. for C18H1437Cl2O5 [M]+: 384.0160. Found: 384.0108. 3.4.4. 3-Acetoxy-7-methoxy-1,9-dimethyldibenzofuran (4a) Colorless crystalline solid, mp 116–117 C (MeOH). UV lMeOH nm (log "): 215.5 (4.35), 226 (4.49), 237 sh max (4.35), 256 sh (4.01), 262 (4.09), 295 (4.26), 301 (4.26), 1 1 305.5 (4.26); IR nKBr max cm : 1759, 1607, 1578, 829; H 13 NMR: see Table 1; C NMR: see Table 2; EIMS m/z:
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(rel. int.%): 284 (37) [M]+, 242 (100). HR–EIMS m/z: calc. for C17H16O4 [M]+: 284.1049. Found: 284.1060. 3.4.5. 7-Acetoxy-2-chloro-3-methoxy-1,9dimethyldibenzofuran (5a) Colorless crystalline solid, mp 169 C (MeOH). UV MeOH lmax nm (log "): 229.5 (4.41), 254.5 (3.88), 262 (3.95), 1 291 sh (4.04), 301 (4.10), 313.5 (3.95); IR KBr max cm : 1 13 1747, 1607, 1541, 810; H NMR: see Table 1; C NMR: see Table 2; EIMS m/z: (rel. int.%): 320 (11), 318 (30) [M]+, 278 (36), 276 (100), 262 (12), 260 (35); HR–EIMS m/z: calc. for C17H1535ClO4 [M]+: 318.0659. Found: 318.0675; calc. for C17H1537ClO4 [M]+: 320.0630. Found: 320.0637. 3.5. Synthesis of 1 and 4 Compound 1 was prepared from 3,5-dimethoxytoluene according to Asahina. (Asahina and Aoki, 1941). The physical and spectral data of the product were identical to those of the isolated compound. To a solution of 1 (19.8 mg) in acetone (20 ml) were added K2CO3 (600 mg) and Me2SO4 (20 ml), and the whole was heated under reflux for 4 h. The reaction mixture was diluted with 5% NaHCO3 and extracted with CHCl3. The washed and dried CHCl3 layers were concentrated in vacuo and the residue was subjected to prep. TLC with (toluene–acetone 19:1) to afford a monomethyl ether (3.3 mg) and a dimethyl ether (6) (17.9 mg). The monomethyl ether was identified with 4. 6: needles, mp. 153.7–154 C (MeOH). 1H NMR (CDCl3): 2.83 (6H, s, 1-CH3 and 9-CH3), 3.84 (6H, s, 3-OCH3 and 7-OCH3), 6.69 (2H, m, H-2 and H-8), 6.92 (2H, m, H-4 and H-6). EIMS m/z (%): 330 [M]+ (35), 315 (100), 300 (7.3), 285 (28). HR–EIMS m/z: calc. for C16H16O3 [M]+: 256.1100. Found: 256.1095. Acknowledgements This research was financially supported by Grant-inAid for Scientific Research (C) (No. 09672179 and No. 11672129) from the Ministry of Education, Sciences, Sports and Culture of Japan and Kobe Pharmaceutical University Collaboration Fund. We are grateful to Dr. H. Miyawaki (Saga University, Japan) for identification of the voucher specimen. Thanks are also due to Dr. M. Sugiura (Kobe Pharmaceutical University) for 1H and 13 C NMR spectra, and to Dr. K. Saiki (Kobe Pharmaceutical University) for mass spectra measurements. References Asahina, Y., Aoki, M., 1941. On oxy-diphenyleneoxide derivatives. Yakugaku Zasshi 64, 41–47. Culberson, C.F., Ahmajian, V., 1980. Artificial reestablishment of lichens. II. Secondary products of resynthesized Cladonia cristatella and Lecanora chrysoleuca. Mycologia 72, 90–109.
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