paraconfuranone a-h, eight new furanone … · web viewcolumn chromatography was conducted on...

Paraconfuranone A-H, eight new furanone analogues from the insect-associated fungus Paraconiothyrium brasiliense MZ-1

New Eremophilane and Dichlororesorcinol Derivatives Produced by Endophytes Isolated from Ficus ampelas

Yoshihito Shiono1[footnoteRef:-1]*, Niken Istikhari Muslihah1,2, Takuma Suzuki1, Nanang Rudianto Ariefta1,3, Chairil Anwar2, Handojo Hadi Nurjanto4, Takako Aboshi1, Tetsuya Murayama1, Keitaro Tawaraya1, Takuya Koseki1, Jun Yoshida5, Narandulam Usukhbayar3, Shota Uesugi3, Ken-ichi Kimura3 [-1: * Corresponding author. Tel./fax: +81-235-28-2873; e-mail:[email protected] ]

1Department of Food, Life, and Environmental Science, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan

2Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia

3 The United Graduate School of Agricultural Sciences, Iwate University, Morioka, Iwate 020-8550, Japan

4Department of Silviculture, Faculty of Forestry, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia

5Center for Liberal Arts and Sciences, Iwate Medical University, Yahaba, Iwate, 028-3694, Japan

Experimental Section.

1. General experimental procedures

Optical rotation values were measured with a Horiba SEPA-300 polarimeter (HORIBA, Kyoto, Japan), and UV and IR spectra were respectively recorded with Shimadzu UV mini-1240 (SHIMADZU, Kyoto, Japan) and Jasco J-20A (JASCO Cooperation, Tokyo, Japan) spectrophotometers. Mass spectra were obtained with a Synapt G2 mass spectrometer instrument (Milford, Waters, USA). NMR data were recorded on a Jeol ECZ-600 spectrometer at 600 MHz for 1H and 125 MHz for 13C (JEOL, Tokyo, Japan). Chemical shifts are given on a (ppm) scale with TMS as an internal standard. 1H, 13C, DIFNOE, DEPT, COSY, HMQC and HMBC spectra were recorded using standard Jeol standard pulse sequences. Semi-preparative HPLC was carried out with Shimadzu pump (SHIMADZU, Kyoto, Japan) and UV LC-10A detector (SHIMADZU, Kyoto, Japan) on Mightysil ODS column (250 x 6.0 mm i.d.) at the flow rate of 1.5 mL min-1 (Kanto Chemical Co., Inc., Japan). Column chromatography was conducted on silica gel 60 (Kanto Chemical Co., Inc., Japan) and ODS (Fuji Silysia, Aichi, Japan). TLC was carried out on Merck precoated silica gel plates (silica gel 60 F254, 20 x 20 cm, Merck, Darmstadt, Germany), and spots were detected by spraying with 10% vanillin in H2SO4 followed by heating, or by UV irradiation.

2. Biological material

The fungal strains of Penicillium sp. N-175-1 and Phomopsis sp. N-125 waere isolated from a surface sterilized branch of Ficus ampelas collected in Wanagama forest, Indonesia (southern latitude: 75337; east longitude: 110 32 49). Fungal strains N-175-1 and N-125 were identified as Penicillium sp. and Phomospis sp. respectively, by the using a DNA analysis of the 18S rDNA regions. They have been deposited at our laboratory in the Faculty of Agriculture of Yamagata University.

3. Fermentation of Penicillium sp. N-175-1, extraction and isolation of 1, 2, 3 and 4

The strain was grown under static conditions at 25 C for 30 days in 1 L Erlenmeyers containing 100 g of the steamed unpolished rice and 150 ml of water. The moldy unpolished rice (900g) was extracted with methanol, and the methanol extract was concentrated. The resulting aqueous concentrate was partitioned into n-hexane and EtOAc layers. The purification of the EtOAc layer was guided by the intense blue characteristic coloration with vanillin-sulfuric acid solution on TLC plates. The EtOAc layer (11 g) was chromatographed on a silica gel column using first a stepwise of n-hexaneEtOAc (100:0-0:100) and then a mixture of EtOAcMeOH (50:50) as eluting solvents to give 13 fractions (Fr. 1-1 - 1-13). Fraction 1-7 (n-hexaneEtOAc 40: 60, 2.6 g) was chromatographed on silica gel using a stepwise gradient of CHCl3EtOAc to give fractions 1 12 (Fr. 1-7-1 to 1-7-12). Fraction 1-7-4 (CHCl3EtOAc 70: 30, 1.2 g) was subjected to ODS column chromatography by eluting stepwise with H2O and an increasing ratio of MeOH to afford 12 fractions (1-7-4-1 to 1-7-4-12). Frs. 1-7-4-5 and 1-7-4-6 (H2O-MeOH 60:40, 50:50, 270 mg) were subject to flash silica gel chromatography (CHCl3-MeOH, 50:1) to afford sporogen-AO1 (2, 20 mg) and petasol (3, 5.0 mg). Fr. 1-7-4-7 (H2O-MeOH 40:60, 110 mg) was applied to ODS column chromatography using with H2O and an increasing ratio of MeOH to afford fractions (10 mg) including 4 which were finally purified by preparative TLC (CHCl3-MeOH, 199:1) to obtain 6-dehydropetasol (4, 13 mg). Fraction 1-11 (100% EtOAc, 470 mg) was chromatographed on silica gel using a gradient of CHCl3EtOAc to afford fractions (11.5 mg) including 1 which were finally purified by ODS column chromatography using with H2O and an increasing ratio of MeOH to obtain 11-O-methylpetasitol (1, 5.0 mg).

4. Fermentation of Phomopsis sp. N-125, extraction and isolation of 5 and 6

The strain was grown under static conditions at 25 C for 30 days in 1 L Erlenmeyers containing 100 g of the steamed unpolished rice and 150 ml of water. The moldy unpolished rice (900g) was extracted with methanol, and the methanol extract was concentrated. The resulting aqueous concentrate was partitioned into n-hexane and EtOAc layers. The purification of the EtOAc layer was guided by the intense blue characteristic coloration with vanillin-sulfuric acid solution on TLC plates. The EtOAc layer (16.5 g) was chromatographed on a silica gel column using a 10% stepwise of n-hexaneEtOAc and then a mixture of EtOAcMeOH (50:50) as eluting solvents to give 13 fractions (Fr. 2-1 - 2-13). Fr. 2-7 (n-hexaneEtOAc 40 : 60, 984 mg) was chromatographed on silica gel using a gradient of CHCl3EtOAc to afford crude fractions (390 mg) including 5 and 6 which were finally purified by ODS HPLC with a mixture of MeOH - H2O (70 : 30) to yield cosmochlorins A (5, 3.5 mg, tR = 11.0 min) and B (6, 3.0 mg, tR = 13.5 min).

4.1. (3R)-11-O-Methylpetasitol (1)

White amorphous powder; []D20 +143.0 (c 0.01, MeOH); UV (MeOH) max (log ) : 243 (4.3), 314 (3.9) nm; IR (KBr) max cm-1: 3325, 2913, 2715, 1720, 1643, 1268, 1187; 1H and 13C NMR see Table 1; HRESITOFMS m/z 265.1779 [M+H]+ (calcd. for C16H25O5, 265.1804).

4.2. Cosmochlorin A (5)

White amorphous powder; UV (MeOH) max (log ) : 234 (4.1), 297 (2.0) nm; IR (KBr) max cm-1: 2983, 2933, 1680, 1583, 1176, 999; 1H and 13C NMR see Table 2; HRESITOFMS m/z 289.0396 [M+H]+ (calcd. for C13H1535Cl2O3, 289.0398), m/z 291.0366 [M+H]+ (calcd. for C13H1535Cl37ClO3, 289.0369), and m/z 293.0336 [M+H]+ (calcd. for C13H1537Cl2O3, 293.0339) .

4.3. Cosmochlorin B (6)

White amorphous powder; UV (MeOH) max (log ) : 287(4.0) nm; IR (KBr) max cm-1: 2994, 2926, 1681, 1555, 1177, 996; 1H and 13C NMR see Table 2; HRESITOFMS m/z 329.0710 [M+H]+ (calcd. for C16H1935Cl2O3, 329.0711), m/z 331.0689 [M+H]+ (calcd. for C16H1935Cl37ClO3, 331.0682) and m/z 333.5658 [M+H]+ (calcd. for C16H1937Cl2O3,333.0652).

5. Preparation of MTPA ester derivatives (1a and 1b) from 1.

To 1 (1.0 mg) in CH2Cl2 (0.5 mL) were added (S)-() or (R)-(+)--methoxy--trifluoromethylphenylacetic acid (MTPA, 2.0 mg), dicyclohexylcarbodiimide (2.0 mg) and 4-(dimethylamino)pyridine (2.0 mg), and the mixture was stirred at room temperature for 12 h. EtOAc was added to the reaction mixture, before the resulting solution was washed with a saturated solution of aqueous NaHCO3 and brine, and concentrated in vacuo. Purification by column chromatography on silica gel (n-hexane-EtOAc) gave the (S)-()-MTPA esters (1a, 0.9 mg) or (R)-(+)-MTPA esters (1b, 0.9mg) of 1.

1a: HRESITOFMS m/z: 503.2061 [M+H]+ (calcd. for C26H31F3NaO5, 503.2021); 1H (600 MHz, CDCl3) H 0.96 (3H, d, J = 6.7 Hz, Me-14), 1.43 (3H, s, Me-12), 1.45 (3H, s, Me-13), 1.67 (2H, m, H-1a, H-4), 1.19 (3H, s, Me-15), 2.41-2.44 (2H, m, H-1b, H-2a), 2.58 (1H, td, J = 12.7, 5.2 Hz, H-2b), 3.19 (3H, s, 11-OMe), 3.50 (3H, OMe of MTPA), 5.08 (1H, td, J = 11.1, 4.0 Hz, H-3), 6.03 (1H, s, H-9), 7.05 (1H, s, H-6), 7.40-7.42 (3H, m, Ph of MTPA), 7.47-7.49 (2H, m, Ph of MTPA).

1b: HRESITOFMS m/z: 503.2088 [M+H]+ (calcd. for C26H31F3NaO5, 503.2021); 1H (600 MHz, CDCl3) H 1.12 (3H, d, J = 6.7 Hz, Me-14), 1.20 (3H, s, Me-15), 1.43 (3H, s, Me-12), 1.45 (3H, s, Me-13), 1.67 (2H, m, H-1a, H-4), 2.35-2.41 (2H, m, H-1b, H-2a), 2.56 (1H, td, J = 12.5, 5.0 Hz, H-2b), 3.20 (3H, s, 11-OMe), 3.54 (3H, OMe of MTPA), 5.06 (1H, td, J = 11.0, 4.1 Hz, H-3), 6.01 (1H, s, H-9), 7.08 (1H, s, H-6), 7.39-7.42 (3H, m, Ph of MTPA), 7.49-7.51 (2H, m, Ph of MTPA).

6. Cell culture and cytotoxicity

HL60 cells (RCB0041, RIKEN BioResource Center, Tsukuba, Japan) and Hela cells (RCB0007) were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FBS (BioWest, Canada) and penicillin (50 units/ml)-streptomycin (50 g/ml) (Gibco Corp., Carlsbad, USA) in a humidified atmosphere at 37C under 5% CO2. The cytotoxicity of the compounds were examined by MTT assay, as described previously (Aburai et al., 2010). Positive control camptothecin was used as positive control for HL60 and doxorubicin for HeLa with IC50 = 23.6 nM and 1.9 M respectively.

7. Growth restoring activity of samples against Saccharomyces cerevisiae (cdc2-1 rad9D) mutant strain.

Screening was performed according to previous described method (Kimura et al., 2012). Each sample was dissolved in MeOH and two-fold dilutions of them were used. Difco yeast-peptone-dextrose (YPD) broth and YPD agar were purchased from Becton Dickinson Biosciences (Franklin Lakes, NJ, USA). The mutant yeast (cdc2-1 rad9) was derivatives of strain W303-1A (Tsuchiya et al., 2001a). A 5 l aliquot of samples was spotted on a YPD agar medium containing the mutant strain. After 3 days of incubation at 37C for 6 h and then 28C for 2 days, the intensity of the growth spot were observed as the result of inhibition of the DNA damage check point pathway. Hydroxyurea (1.25 mg/spot) was used as a positive control. Hydroxyurea was purchased from MP Biomedicals LLC, Irvine USA.

References

Aburai, N., Yoshida, M., Ohnishi, M., Kimura, K., 2010. Pisiferdiol and pisiferic acid isolated from Chamaecyparis pisifera activate protein phosphatase 2C in vitro and induce caspase-3/7-dependent apoptosis via dephosphorylation of Bad in HL60 cells. Phytomedicine 17, 782-788.

Kimura, K., Sakamoto, Y., Fujisawa, N., Uesugi, S., Aburai, N., Kawada, M., Ohba, S., Yamori, T., Tsuchiya, E. and Koshino, H., 2012. Cleavage mechanism and anti-tumor activity of 3,6-epidioxy-1,10-bisaboladiene isolated from edible wild plants. Bioorg. Med. Chem. 20, 3887-3897.

Tsuchiya, E., Yukawa, M., Miyakawa, T., Kimura. K., Takahashi, H., 2010a. Borrelidin inhibits a cyclin-dependent kinase (CDK), Cdc28/Cln2, of Saccharomyces cerevisiae. J. Antibiot. 54, 84-90.

List of supporting information

Figure S1. The 1H-NMR spectrum of 1 (150 MHz, CDCl3).

Figure S2. The 13C-NMR spectrum of 1 (150 MHz, CDCl3).

Figure S3. The 1H-1H COSY spectrum of 1 (600 MHz, CDCl3).

Figure S4. The HMQC spectrum of 1 (600 MHz, CDCl3).

Figure S5. The HMBC spectrum of 1 (600 MHz, CDCl3).

Figure S6. The 1H-NMR spectrum of 5 (600 MHz, CD3OD).

Figure S7. The 13C-NMR spectrum of 5 (150 MHz, CD3OD).

Figure S8. The HMQC spectrum of 5 (600 MHz, CD3OD).

Figure S9. The HMBC spectrum of 5 (600 MHz, CD3OD).




Figure S13. The HMBC spectrum of 6 (600 MHz, CD3OD)

Figure S14. Growth restoring activities of 5 (a) and 6 (b) against S. cerevisiae YNS17 (zds1 erg3 pdr1/3) in the presence of 0.3 M CaCl2.

Figure S15. Growth restoring activities of AG1024 (a), TX-1918 (b), staurosporine (c), wortmannin (d) and rapamycin (e) against S. cerevisiae (cdc2-1 rad9D).

Figure S1. The 1H-NMR spectrum of 1 (600 MHz, CDCl3).

Figure S2. The 13C-NMR spectrum of 1 (150 MHz, CDCl3).

Figure S3. The 1H-1H COSY spectrum of 1 (600 MHz, CDCl3).






Figure S9. The HMBC spectrum of 5 (600 MHz, CD3OD).





1: 5 mg/spot sample, 2-6 : Dilution 2 fold with MeOH, 7: FK506 20 ng/spot for 5 and 6.

Figure S14.

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(a) Compound 5 (b) Compound 6

Growth restoring activities of 5 and 6 against S. cerevisiae YNS17 (zds1 erg3 pdr1/3) in the

presence of 0.3 M CaCl2. 1: 5 g/spot sample, 2-6 : Dilution 2 fold with MeOH, 7: FK506 20 ng/spot for 5 and 6.

Supplementary Fig.

(a) AG1024 (b) TX-1918 (c) Staurosporine

(d) Wortmannin (d) Rapamycin

Supplementary Fig. Growth restoring activities of AG1024 (a), TX-1918 (b),

staurosporine (c), wortmannin (d) and rapamycin (e) against S. cerevisiae (cdc2-1

rad9).

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paraconfuranone a-h, eight new furanone … · web viewcolumn chromatography was conducted on...

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