Arch. Pharm. Res. DOI 10.1007/s12272-014-0418-1
RESEARCH ARTICLE
Chemical constituents from the fungus Chaetomium cupreum RY202 Natcha Panthama • Somdej Kanokmedhakul Kwanjai Kanokmedhakul • Kasem Soytong
•
Received: 14 February 2014 / Accepted: 26 May 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Two new angular types of azaphilones, isochromophilonol (1) and ochrephilonol (2), together with ten known compounds (3–12), were isolated from Chaetomium cupreum RY202. Their structures were established on the basis of spectroscopic data and the absolute configurations of 1 and 3 were determined by the modified Mosher’s method. Compounds 1, 2 and 4 showed moderated cytotoxicity against KB and NCI-H187 cell lines (IC50 9.63–32.42 lg/mL). Keywords Cheaetomium Cheaetomium cupreum Azaphilone Cytotoxicity
Introduction Chaetomium is one of the largest genera of saprophytic ascomycetes. It belongs to the family Chaetomiaceae and this family includes more than 300 species worldwide of which 24 species have been found in Thailand (Pornsuriya et al. 2008, 2011). Investigations on Chaetomium species
have been previously reported in numerous types of compounds such as benzoquinone derivatives (Brewer et al. 1968), tetra-S-methyl derivatives (Safe and Taylor 1972), azaphilones (Takahashi et al. 1990; Kanokmedhakul et al. 2006; Phonkerd et al. 2008), bis-azaphilones (Phonkerd et al. 2008), indo-3-yl-[13]cytochalasans and chaetogobosin analogs (Sekita et al. 1976; Udagawa et al. 1979; Probst and Tamm, 1981; Sekita et al. 1982, 1983; Kanokmedhakul et al. 2002; Ding et al. 2006; Thohinung et al. 2010), anthraquinone-chromanone (Kanokmedhakul et al. 2002), globosumones (Bashyal et al. 2005), chaetochalasin A (Oh et al. 1998), depsidones (Oh et al. 1998; Li et al. 2008; Khumkomkhet et al. 2009) and longirostrerones A-D (Panthama et al. 2011). Bioassay results indicated that the MeOH extract from C. cupreum RY202 possesses cytotoxicity against KB cell lines with IC50 value of 3.24 lg/mL. We report herein the isolation, structural determination, and cytotoxicity assay of two new azaphilones, 1 and 2, together with ten known compounds (3–12) from C. cupreum RY202.
Materials and methods N. Panthama Department of Applied Chemistry, Faculty of Sciences and Liberal Arts, Rajamangala University of Technology Isan, Nakhon Ratchasima 3000, Thailand N. Panthama S. Kanokmedhakul (&) K. Kanokmedhakul Natural Products Research Unit, Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: [email protected] K. Soytong Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Ladkrabang, Bangkok 10520, Thailand
General experimental procedure Optical rotations were obtained using a JASCO P-1030 digital polarimeter, and CD spectra were obtained using a JASCO J-810 apparatus. UV spectra were measured on an Agilent 8453 UV–visible spectrophotometer. IR spectra were taken on a BRUKER TENSOR 27 FT-IR spectrophotometer. NMR spectra were recorded in CDCl3 and CD3OD on a Varian Mercury Plus 400 spectrometer, using residual CHCl3 and MeOH as internal standards. HRESITOFMS were recorded on a Micromass Q-TOF-2TM
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N. Panthama et al.
spectrometer. Column chromatography was carried out on MERCK silica gel 60 (230–400 mesh) and Sephadex LH20. TLC was performed with precoated MERCK silica gel 60 PF254 aluminum sheets; the spots were visualized under UV light (254 and 366 nm) and further by spraying with anisaldehyde, and heating until charred.
Table 1 1H and 13C NMR spectral data of 1 and 2 in CDCl3 (1H: 400 MHz, 13C: 100 MHz)a position
1 dH
2 dC
dH
dC
1
7.38 s
145.6
7.30 s
146.2
3
–
158.4
–
156.8
Fungal materials
4 4a
6.58 s –
105.1 140.4
6.10 s –
107.5 144.5
The fungus C. cupreum RY202 was collected from a pineapple plantation at Rayong province, Thailand, in 2007 and was identified by K. Soytong. A voucher specimen (Ch-RY202) was deposited at the Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand. The fungus was cultured in conical flasks (1 L, 80 flasks) with potato dextrose broth (PDB) (200 mL/flask) and incubated in a standing condition at 28–30 °C for 50 days. The culture broth was filtered to give a fresh fungal biomass and then air-dried at room temperature.
5
–
109.3
5.43 s
106.4
6
–
185.0
–
191.9
7
–
83.6
–
83.1
8
3.45 d (12.0)
44.1
3.37 d (12.4)
8a
–
114.3
–
114.8
9
6.08 d (15.6)
116.1
5.95 d (15.6)
116.0
10
7.02 d (15.6)
142.6
6.96 d (15.6)
141.3
11
–
131.9
–
131.9
12
5.65 d (9.6)
148.3
5.61 d (9.6)
147.3
13
2.45 m
35.0
2.46 m
14
1.41 m, 1.30 m
30.0
1.41, 1.26 m
30.1
15
0.86 t (7.2)
11.9
0.84 t (7.2)
11.9
16
1.0 d (6.6)
20.2
0.99 d (6.8)
20.2
17 18
1.82 s 1.58 s
12.3 22.7
1.81 s 1.55 s
12.3 22.7
19
–
20
2.85 dd (3.6, 12.0)
49.5
2.85 dd (3.6, 12.0)
21
4.30 m
65.7
4.25 m
65.6
22
1.35 d (6.6)
21.1
1.35 d (6.6)
20.9
Extraction and isolation Air-dried biomass of C. cupruem RY202 (195 g) was ground and extracted successively with EtOAc (400 mL 9 3) and MeOH (400 mL 9 3) at room temperature to give EtOAc (16.4 g) and MeOH (16.7 g) extracts. The EtOAc extract was applied to silica gel flash column chromatography (FCC), gradually eluted with EtOAchexane and EtOAc–MeOH by increasing polarity of solvents to give 5 fractions, E1–E5. Purification of E2 using FCC, eluted with a gradient system of 90 % CH2Cl2-hexane to 50 % EtOAc-CH2Cl2 afforded a white solid of 5 (70 mg) and a red amorphous powder of 7 (150 mg). Fraction E3 was subjected to silica gel FCC, gradually eluted with 3 % EtOAc-CH2Cl2 to give six subfractions, E3.1–E3.6. Subfraction E3.3 was then purified by preparative TLC using 3 % EtOAc-CH2Cl2 (developed 9 2) as eluent, to provide a red amorphous powder of 8 (15 mg), a red amorphous powder of 9 (15 mg) and red crystals of 10 (20 mg). Fraction E4 was applied to silica gel FCC, isocratic eluting with 50 % EtOAc-hexane to generate three subfractions, E4.1–E4.3. Subfraction E4.1 was then separated by using Sephadex LH20 CC, eluted with MeOH to yield an additional amount of 10 (200 mg). Purification of E4.2 using Sephadex LH20 CC, eluted with MeOH gave a white solid of 6 (50 mg) and a red amorphous powder of 1 (230 mg). Fraction E5 was purified by preparative TLC, developed with 30 % acetone-hexane to provide a yellow powder of 2 (20 mg). Fraction E5 was purified using Sephadex LH 20 to afford a white solid of 4 (45 mg). The MeOH extract (16.7 g) was applied to silica gel FCC,
123
174.1
–
44.5
35.0
174.7 49.5
a Chemical shift values in ppm, and J values (in Hz) are presented in parentheses
eluted with a gradient system of EtOAc-hexane, and MeOH-EtOAc to give 9 fractions, M1–M9. Fraction M2 was purified by FCC, eluted with an isocratic system of 5 % acetone-CH2Cl2 to obtain an additional amount of 10 (400 mg). Fraction M3 was separated by Sephadex LH 20, eluted with MeOH to yield an additional quantity of 1 (30 mg) and a red amorphous powder of 11 (10 mg). Fraction M5 was separated by FCC, eluted with a gradient system of 5–15 % acetone–hexane to obtain a red amorphous powder of 12 (5 mg). Fraction M8 was separated by FCC, eluted with 30 % acetone–hexane and then 40 % acetone–hexane to yield colorless crystals of 3 (400 mg). Isochromophilonol (1) Yellow amorphous powder (260 mg, 0.133 %); UV (MeOH) kmax (log e), 409 (4.2), 360 (4.1), 227 (4.2); [a]27 D -141.6 (c 0.12, EtOH); CD (MeOH) nm: 315 (-59.6), 232 (-28.3), 257 (?8.1), 209 (?48.2); IR (neat) mmax cm-1; 3402, 2964, 2927, 1773, 1654, 1617, 1516, 1096; 1H and 13 C NMR data, see Table 1. HRESIMS m/z 441.1432
Chemical constituents from the fungus
[M ? Na]? (calcd for C23H27ClO5 ? Na, 441.1445), m/z 443.1400 [M ? Na ? 2]? (calcd for C23H27ClO5 ? Na ? 2, 443.1415). Ochrephilonol (2) Yellow amorphous powder (20 mg, 0.010 %); UV (MeOH) kmax (log e) 386 (3.6), 227 (3.7); [a]27 D -137.0 (c 0.12, EtOH); CD (MeOH) nm: 323 (-56.7), 313 (-58.2), 236 (-22.0), 266 (?6.8), 210 (?36.2); IR (neat) mmax cm-1; 3403, 2964, 2926, 1772, 1661, 1610, 1540, 1454, 1378, 1178, 1091; 1H and 13C NMR data, see Table 1; HRESIMS m/z 385.2000 [M ? H]? (calcd for C23H28O5 ? H, 385.2015). Preparation of the (S) and (R)-a-methoxy-a(trifluoromethyl)phynyl acetate of 1 To a solution of isochromophilonol (1) (10 mg, 23.9 lmol) in dry CH2Cl2 (2 mL), DMAP (2 mg, 16.4 lmol) and (R)MTPACl (25 lL, 133.6 lmol) were added. The mixture was stirred under N2 at room temperature for 24 h, and the solvent was removed in vacuo. The product was purified by preparative TLC using 4 % MeOH-CH2Cl2 as eluent, to yield (S)-MTPA ester (1a, 3.5 mg). The (R)-MTPA acetate of 1 was prepared using the same method as described above [alcohol 1 (10 mg), CH2Cl2 (2 mL), DMAP (2.0 mg), and (S)-MPTA-Cl (25 lL)] to yield (R)-MTPA ester (1b, 2 mg). Preparation of the (S) and (R)-a-methoxy-a(trifluoromethyl)phynyl acetate of 3 To a solution of 3 (10 mg, 44.13 lmol) in dry CH2Cl2 (2 mL), DMAP (2 mg, 16.4 lmol) and (R)-MTPACl (25 lL, 133.6 lmol) were added. The mixture was stirred under N2 at room temperature for 24 h, and the solvent was removed in vacuo. The product was purified by TLC using CH2Cl2 as eluent, to yield (S)-MTPA ester (3a, 2 mg). The (R)-MTPA acetate of 3 was prepared using the same method as described above [alcohol 3 (10 mg), CH2Cl2 (2 mL), DMAP (2.0 mg), and (S)-MPTA-Cl (25 lL)] to yield (R)-MTPA ester (3b, 3 mg). Cytotoxicity assay Cytotoxic assays against human epidermoid carcinoma (KB), human breast adenocarcinoma (MCF7), and human small cell lung cancer (NCI-H187) were performed employing the colorimetric method as described by Skehan and coworkers (Skehan et al. 1990). The reference substances were doxorubicine and ellipticine.
Results and discussion The EtOAc and MeOH extracts of air dried fungal biomass of C. cupreum RY202 were purified by chromatographic techniques to yield eight azaphilones (1, 2 and 7–12), two a-pyrone derivatives (3 and 4), as well as two steroids (5 and 6) (Fig. 1). By physical and spectroscopic data measurements (IR, 1H and 13C NMR, 2D NMR, and MS) as well as by comparing the data obtained with published values, structures of the known compounds were identified as clearanols A and B (3 and 4) (Gerea et al. 2012), ergosterol (5) (Bok et al. 1999), ergosterol epoxide (6) (Ro¨secke and Ko¨nig 2000), rubrorotiorin (7) (Gray and Whalley 1971), isochromophilone II (8) (Omura et al. 1993), (-)-rotiorin (9) (Kanokmedhakul et al. 2006), rotiorinols A–C (10–12) (Kanokmedhakul et al. 2006) (Fig. 1). Compound 1 was obtained as a red solid, and its molecular formula, C23H27ClO5, was deduced from HRESIMS m/z 441.1432 [M ? Na]?, 443.1400 [M ? Na ? 2]?, indicating 10 degrees of unsaturation. The intensity ratio 3:1 of isotope peaks ([M ? Na]?:[M ? Na ? 2]?) supports the presence of a chlorine atom in the molecule. The IR spectra of 1 showed the characteristic absorption band of hydroxyl (3,402 cm-1), carbonyl lactone (1,773 cm-1) and a,b-conjugated ketone (1,654 cm-1) groups. The NMR spectra of 1 were similar to those of an angular type of azaphilone, isochromophilone I (Matsuzaki et al. 1995), except for a missing of carbonyl carbon at C-21 and the appearance of signals of an oxymethine proton (dH 4.30 m) and carbon (dC 65.7). The complete interpretation of the NMR data of 1 was established as a result of conclusive DEPT, COSY, HMBC, and NOESY experiments. The COSY spectrum of 1 showed correlations of H-8/ H-20/H-21/H-22 also the HMBC correlations of H-8 to C-1, C-18, C-19, C-20, and C-21; H-20 to C-8, C-19, C-21, C-22; H-21 to C-8, C-19, C-22; and H-22 to C-20, C-21 confirmed the lactone ring and side chain C-20-C-21. The complete HMBC correlation of 1 is shown in Fig. 2. The NOESY spectrum exhibited the correlations of H-8 to methyl protons (H3-18), and H-20 to H3-18, indicating that these protons were located on the same side (Fig. 3). The coupling constant between H-8 and H-20 (12.0 Hz) suggested that the dihedral angle of these protons is close to zero. The absolute stereochemistry at C-7 was suggested to be S from its CD spectrum, which showed a negative Cotton effect at 315 nm (De -59.6), as those reported for chaetomugilin B (323 nm, De -4.2), and chaetomugilin L (323 nm, De -4.9) (Muroga et al. 2009). Assignment of the absolute configuration at C-21 was carried out using the modified Mosher’s ester method (Ohtani et al. 1991). Reaction of 1 with (R)- and (S)-a-methoxy-a-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) provided the Sand R-MTPA esters of 1, respectively. The difference in the 1 H NMR chemical shifts between the S- and R-MTPA derivatives (DdH = dS-dR) near C-21 determined the
123
N. Panthama et al. Fig. 1 Structures of compounds 1-12
O 18
7
O 19
8a
8
20
O
4
5 4a
6
16
17
R1
H H
21
1
9
11 10
O
12
13
O O
14
7
MeO
22
R1 = Cl, R2 = H R1 = Cl, R2 = R-MTPA R1 = Cl, R2 = S-MTPA R1 = H, R2 = H
O 4
OO 6
Cl
Cl
O
O O
O O
O
HO
O
O 8
7 O
O
O
O
O
O O
R1
R2
O
O OH
9
Cl O O
OH
Fig. 2 HMBC (arrow line) and COSY (bold line) correlations of isochromophilonol (1)
absolute configuration at this position to be R (Fig. 4). Thus, compound 1 was deduced to be a new azaphilone and was named as isochromophilonol.
123
MeO
3: R1 = R2 = H 3a: R1 = R2 = R-MTPA 3b: R1 = R2 = S-MTPA
5
O
OR2
HO
HO
HO
O
O
R1O
OR2 1: 1a: 1b: 2:
O
15
10: R1 = H, R2 = CH3 11: R1 = OH, R2 = CH3 12: R1 = H, R2 = CH2OH
Compound 2 was obtained as a yellow powder, and its molecular formula, C23H28O5 was deduced from HRESIMS m/z 385.2000 [M ? H]?. The IR spectra of 2 showed the characteristic absorption bands of hydroxyl (3,403 cm-1), carbonyl lactone (1,772 cm-1) and a,bconjugated ketone (1,661 cm-1) groups. The 1H and 13C NMR spectra of 2 were similar to those of 1, except for the appearance of an olefinic proton at dH 5.43 and carbon dC 106.4 which were assigned to H-5 and C-5, respectively. This was supported by the HMBC correlations of H-5 to C-6, C-7, C-4a, C-8a and C-1. The NOESY spectrum of 2 displayed the correlations of H-8, H-20 and H-9 as in 1. In addition, the absolute configuration at C-7 was also assigned to be S from the negative Cotton effect at 336
Chemical constituents from the fungus
O O MeO R1O
+0.0560
OR2 Hb
Ha -0.1288
-0.6070
3a: R1 = R2 = R-MTPA 3b: R1 = R2 = S-MTPA Fig. 5 Dd values (Dd = dS - dR in ppm) obtained for MTPA esters 3a and 3b
Fig. 3 NOESY correlations of 1
Cl
lines with IC50 values of 9.63 and 27.18 lg/mL, respectively. Compound 2 showed weak cytotoxicity against KB cell lines with IC50 value of 30.2 lg/mL. However, compound 4 displayed weak cytotoxicity against KB and MCF7 cell lines with IC50 values of 32.42 and 13.01 lg/ mL, respectively.
O Me O
O H -0.115 H -0.039 +0.007
O
OR 1a: R = R-MTPA 1b: R = S-MTPA
Acknowledgments Financial support from the Thailand Research Fund (Grant no. DBG5380047) for S.K. and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Advanced Functional Materials Cluster of Khon Kaen University are gratefully appreciated. We thank the Center of Excellence for Innovation in Chemistry (PERCH-CIC) and Rajamangala University of Technology Isan for partial support. We are indebted to the National Center for Genetic Engineering and Biotechnology via the Bioresources Research Network (BRN) for bioactivity tests.
Fig. 4 Dd values (Dd = dS - dR in ppm) obtained for MTPA esters 1a and 1b
References
(De -17.5) in the CD spectrum (Muroga et al. 2009). Thus, the structure of 2 was determined as a new azaphilone, which was named ochrephilonol. Clearanol A (3), the a-pyrone derivative, has been reported from the fungus Glomeromycete (possible Entrophospora sp.) and the absolute configuration at C-7 was identified by the X-crystallographic technique (Gerea et al. 2012). We then confirmed the absolute stereochemistry at C-7 by the modified Mosher ester method (Ohtani et al. 1991). Clearanol A (3) was treated with (R) and (S)-amethoxy-a-(trifluoromethyl) phenylacetyl chloride (MTPACl) to yield esters 3a and 3b. The differences in the 1H NMR chemical shifts between (S)-MTPA (3a) and (R)-MTPA esters (3b) around the C-7 position were analyzed to determine the absolute configuration of this position which was concluded to be R (Fig. 5). The results of the biological activities of two new isolated azaphilones, 1 and 2 indicated that 1 exhibited moderated cytotoxicity against KB and NCIH-187 cell
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RESEARCH ARTICLE
Chemical constituents from the fungus Chaetomium cupreum RY202 Natcha Panthama • Somdej Kanokmedhakul Kwanjai Kanokmedhakul • Kasem Soytong
•
Received: 14 February 2014 / Accepted: 26 May 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Two new angular types of azaphilones, isochromophilonol (1) and ochrephilonol (2), together with ten known compounds (3–12), were isolated from Chaetomium cupreum RY202. Their structures were established on the basis of spectroscopic data and the absolute configurations of 1 and 3 were determined by the modified Mosher’s method. Compounds 1, 2 and 4 showed moderated cytotoxicity against KB and NCI-H187 cell lines (IC50 9.63–32.42 lg/mL). Keywords Cheaetomium Cheaetomium cupreum Azaphilone Cytotoxicity
Introduction Chaetomium is one of the largest genera of saprophytic ascomycetes. It belongs to the family Chaetomiaceae and this family includes more than 300 species worldwide of which 24 species have been found in Thailand (Pornsuriya et al. 2008, 2011). Investigations on Chaetomium species
have been previously reported in numerous types of compounds such as benzoquinone derivatives (Brewer et al. 1968), tetra-S-methyl derivatives (Safe and Taylor 1972), azaphilones (Takahashi et al. 1990; Kanokmedhakul et al. 2006; Phonkerd et al. 2008), bis-azaphilones (Phonkerd et al. 2008), indo-3-yl-[13]cytochalasans and chaetogobosin analogs (Sekita et al. 1976; Udagawa et al. 1979; Probst and Tamm, 1981; Sekita et al. 1982, 1983; Kanokmedhakul et al. 2002; Ding et al. 2006; Thohinung et al. 2010), anthraquinone-chromanone (Kanokmedhakul et al. 2002), globosumones (Bashyal et al. 2005), chaetochalasin A (Oh et al. 1998), depsidones (Oh et al. 1998; Li et al. 2008; Khumkomkhet et al. 2009) and longirostrerones A-D (Panthama et al. 2011). Bioassay results indicated that the MeOH extract from C. cupreum RY202 possesses cytotoxicity against KB cell lines with IC50 value of 3.24 lg/mL. We report herein the isolation, structural determination, and cytotoxicity assay of two new azaphilones, 1 and 2, together with ten known compounds (3–12) from C. cupreum RY202.
Materials and methods N. Panthama Department of Applied Chemistry, Faculty of Sciences and Liberal Arts, Rajamangala University of Technology Isan, Nakhon Ratchasima 3000, Thailand N. Panthama S. Kanokmedhakul (&) K. Kanokmedhakul Natural Products Research Unit, Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: [email protected] K. Soytong Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Ladkrabang, Bangkok 10520, Thailand
General experimental procedure Optical rotations were obtained using a JASCO P-1030 digital polarimeter, and CD spectra were obtained using a JASCO J-810 apparatus. UV spectra were measured on an Agilent 8453 UV–visible spectrophotometer. IR spectra were taken on a BRUKER TENSOR 27 FT-IR spectrophotometer. NMR spectra were recorded in CDCl3 and CD3OD on a Varian Mercury Plus 400 spectrometer, using residual CHCl3 and MeOH as internal standards. HRESITOFMS were recorded on a Micromass Q-TOF-2TM
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N. Panthama et al.
spectrometer. Column chromatography was carried out on MERCK silica gel 60 (230–400 mesh) and Sephadex LH20. TLC was performed with precoated MERCK silica gel 60 PF254 aluminum sheets; the spots were visualized under UV light (254 and 366 nm) and further by spraying with anisaldehyde, and heating until charred.
Table 1 1H and 13C NMR spectral data of 1 and 2 in CDCl3 (1H: 400 MHz, 13C: 100 MHz)a position
1 dH
2 dC
dH
dC
1
7.38 s
145.6
7.30 s
146.2
3
–
158.4
–
156.8
Fungal materials
4 4a
6.58 s –
105.1 140.4
6.10 s –
107.5 144.5
The fungus C. cupreum RY202 was collected from a pineapple plantation at Rayong province, Thailand, in 2007 and was identified by K. Soytong. A voucher specimen (Ch-RY202) was deposited at the Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand. The fungus was cultured in conical flasks (1 L, 80 flasks) with potato dextrose broth (PDB) (200 mL/flask) and incubated in a standing condition at 28–30 °C for 50 days. The culture broth was filtered to give a fresh fungal biomass and then air-dried at room temperature.
5
–
109.3
5.43 s
106.4
6
–
185.0
–
191.9
7
–
83.6
–
83.1
8
3.45 d (12.0)
44.1
3.37 d (12.4)
8a
–
114.3
–
114.8
9
6.08 d (15.6)
116.1
5.95 d (15.6)
116.0
10
7.02 d (15.6)
142.6
6.96 d (15.6)
141.3
11
–
131.9
–
131.9
12
5.65 d (9.6)
148.3
5.61 d (9.6)
147.3
13
2.45 m
35.0
2.46 m
14
1.41 m, 1.30 m
30.0
1.41, 1.26 m
30.1
15
0.86 t (7.2)
11.9
0.84 t (7.2)
11.9
16
1.0 d (6.6)
20.2
0.99 d (6.8)
20.2
17 18
1.82 s 1.58 s
12.3 22.7
1.81 s 1.55 s
12.3 22.7
19
–
20
2.85 dd (3.6, 12.0)
49.5
2.85 dd (3.6, 12.0)
21
4.30 m
65.7
4.25 m
65.6
22
1.35 d (6.6)
21.1
1.35 d (6.6)
20.9
Extraction and isolation Air-dried biomass of C. cupruem RY202 (195 g) was ground and extracted successively with EtOAc (400 mL 9 3) and MeOH (400 mL 9 3) at room temperature to give EtOAc (16.4 g) and MeOH (16.7 g) extracts. The EtOAc extract was applied to silica gel flash column chromatography (FCC), gradually eluted with EtOAchexane and EtOAc–MeOH by increasing polarity of solvents to give 5 fractions, E1–E5. Purification of E2 using FCC, eluted with a gradient system of 90 % CH2Cl2-hexane to 50 % EtOAc-CH2Cl2 afforded a white solid of 5 (70 mg) and a red amorphous powder of 7 (150 mg). Fraction E3 was subjected to silica gel FCC, gradually eluted with 3 % EtOAc-CH2Cl2 to give six subfractions, E3.1–E3.6. Subfraction E3.3 was then purified by preparative TLC using 3 % EtOAc-CH2Cl2 (developed 9 2) as eluent, to provide a red amorphous powder of 8 (15 mg), a red amorphous powder of 9 (15 mg) and red crystals of 10 (20 mg). Fraction E4 was applied to silica gel FCC, isocratic eluting with 50 % EtOAc-hexane to generate three subfractions, E4.1–E4.3. Subfraction E4.1 was then separated by using Sephadex LH20 CC, eluted with MeOH to yield an additional amount of 10 (200 mg). Purification of E4.2 using Sephadex LH20 CC, eluted with MeOH gave a white solid of 6 (50 mg) and a red amorphous powder of 1 (230 mg). Fraction E5 was purified by preparative TLC, developed with 30 % acetone-hexane to provide a yellow powder of 2 (20 mg). Fraction E5 was purified using Sephadex LH 20 to afford a white solid of 4 (45 mg). The MeOH extract (16.7 g) was applied to silica gel FCC,
123
174.1
–
44.5
35.0
174.7 49.5
a Chemical shift values in ppm, and J values (in Hz) are presented in parentheses
eluted with a gradient system of EtOAc-hexane, and MeOH-EtOAc to give 9 fractions, M1–M9. Fraction M2 was purified by FCC, eluted with an isocratic system of 5 % acetone-CH2Cl2 to obtain an additional amount of 10 (400 mg). Fraction M3 was separated by Sephadex LH 20, eluted with MeOH to yield an additional quantity of 1 (30 mg) and a red amorphous powder of 11 (10 mg). Fraction M5 was separated by FCC, eluted with a gradient system of 5–15 % acetone–hexane to obtain a red amorphous powder of 12 (5 mg). Fraction M8 was separated by FCC, eluted with 30 % acetone–hexane and then 40 % acetone–hexane to yield colorless crystals of 3 (400 mg). Isochromophilonol (1) Yellow amorphous powder (260 mg, 0.133 %); UV (MeOH) kmax (log e), 409 (4.2), 360 (4.1), 227 (4.2); [a]27 D -141.6 (c 0.12, EtOH); CD (MeOH) nm: 315 (-59.6), 232 (-28.3), 257 (?8.1), 209 (?48.2); IR (neat) mmax cm-1; 3402, 2964, 2927, 1773, 1654, 1617, 1516, 1096; 1H and 13 C NMR data, see Table 1. HRESIMS m/z 441.1432
Chemical constituents from the fungus
[M ? Na]? (calcd for C23H27ClO5 ? Na, 441.1445), m/z 443.1400 [M ? Na ? 2]? (calcd for C23H27ClO5 ? Na ? 2, 443.1415). Ochrephilonol (2) Yellow amorphous powder (20 mg, 0.010 %); UV (MeOH) kmax (log e) 386 (3.6), 227 (3.7); [a]27 D -137.0 (c 0.12, EtOH); CD (MeOH) nm: 323 (-56.7), 313 (-58.2), 236 (-22.0), 266 (?6.8), 210 (?36.2); IR (neat) mmax cm-1; 3403, 2964, 2926, 1772, 1661, 1610, 1540, 1454, 1378, 1178, 1091; 1H and 13C NMR data, see Table 1; HRESIMS m/z 385.2000 [M ? H]? (calcd for C23H28O5 ? H, 385.2015). Preparation of the (S) and (R)-a-methoxy-a(trifluoromethyl)phynyl acetate of 1 To a solution of isochromophilonol (1) (10 mg, 23.9 lmol) in dry CH2Cl2 (2 mL), DMAP (2 mg, 16.4 lmol) and (R)MTPACl (25 lL, 133.6 lmol) were added. The mixture was stirred under N2 at room temperature for 24 h, and the solvent was removed in vacuo. The product was purified by preparative TLC using 4 % MeOH-CH2Cl2 as eluent, to yield (S)-MTPA ester (1a, 3.5 mg). The (R)-MTPA acetate of 1 was prepared using the same method as described above [alcohol 1 (10 mg), CH2Cl2 (2 mL), DMAP (2.0 mg), and (S)-MPTA-Cl (25 lL)] to yield (R)-MTPA ester (1b, 2 mg). Preparation of the (S) and (R)-a-methoxy-a(trifluoromethyl)phynyl acetate of 3 To a solution of 3 (10 mg, 44.13 lmol) in dry CH2Cl2 (2 mL), DMAP (2 mg, 16.4 lmol) and (R)-MTPACl (25 lL, 133.6 lmol) were added. The mixture was stirred under N2 at room temperature for 24 h, and the solvent was removed in vacuo. The product was purified by TLC using CH2Cl2 as eluent, to yield (S)-MTPA ester (3a, 2 mg). The (R)-MTPA acetate of 3 was prepared using the same method as described above [alcohol 3 (10 mg), CH2Cl2 (2 mL), DMAP (2.0 mg), and (S)-MPTA-Cl (25 lL)] to yield (R)-MTPA ester (3b, 3 mg). Cytotoxicity assay Cytotoxic assays against human epidermoid carcinoma (KB), human breast adenocarcinoma (MCF7), and human small cell lung cancer (NCI-H187) were performed employing the colorimetric method as described by Skehan and coworkers (Skehan et al. 1990). The reference substances were doxorubicine and ellipticine.
Results and discussion The EtOAc and MeOH extracts of air dried fungal biomass of C. cupreum RY202 were purified by chromatographic techniques to yield eight azaphilones (1, 2 and 7–12), two a-pyrone derivatives (3 and 4), as well as two steroids (5 and 6) (Fig. 1). By physical and spectroscopic data measurements (IR, 1H and 13C NMR, 2D NMR, and MS) as well as by comparing the data obtained with published values, structures of the known compounds were identified as clearanols A and B (3 and 4) (Gerea et al. 2012), ergosterol (5) (Bok et al. 1999), ergosterol epoxide (6) (Ro¨secke and Ko¨nig 2000), rubrorotiorin (7) (Gray and Whalley 1971), isochromophilone II (8) (Omura et al. 1993), (-)-rotiorin (9) (Kanokmedhakul et al. 2006), rotiorinols A–C (10–12) (Kanokmedhakul et al. 2006) (Fig. 1). Compound 1 was obtained as a red solid, and its molecular formula, C23H27ClO5, was deduced from HRESIMS m/z 441.1432 [M ? Na]?, 443.1400 [M ? Na ? 2]?, indicating 10 degrees of unsaturation. The intensity ratio 3:1 of isotope peaks ([M ? Na]?:[M ? Na ? 2]?) supports the presence of a chlorine atom in the molecule. The IR spectra of 1 showed the characteristic absorption band of hydroxyl (3,402 cm-1), carbonyl lactone (1,773 cm-1) and a,b-conjugated ketone (1,654 cm-1) groups. The NMR spectra of 1 were similar to those of an angular type of azaphilone, isochromophilone I (Matsuzaki et al. 1995), except for a missing of carbonyl carbon at C-21 and the appearance of signals of an oxymethine proton (dH 4.30 m) and carbon (dC 65.7). The complete interpretation of the NMR data of 1 was established as a result of conclusive DEPT, COSY, HMBC, and NOESY experiments. The COSY spectrum of 1 showed correlations of H-8/ H-20/H-21/H-22 also the HMBC correlations of H-8 to C-1, C-18, C-19, C-20, and C-21; H-20 to C-8, C-19, C-21, C-22; H-21 to C-8, C-19, C-22; and H-22 to C-20, C-21 confirmed the lactone ring and side chain C-20-C-21. The complete HMBC correlation of 1 is shown in Fig. 2. The NOESY spectrum exhibited the correlations of H-8 to methyl protons (H3-18), and H-20 to H3-18, indicating that these protons were located on the same side (Fig. 3). The coupling constant between H-8 and H-20 (12.0 Hz) suggested that the dihedral angle of these protons is close to zero. The absolute stereochemistry at C-7 was suggested to be S from its CD spectrum, which showed a negative Cotton effect at 315 nm (De -59.6), as those reported for chaetomugilin B (323 nm, De -4.2), and chaetomugilin L (323 nm, De -4.9) (Muroga et al. 2009). Assignment of the absolute configuration at C-21 was carried out using the modified Mosher’s ester method (Ohtani et al. 1991). Reaction of 1 with (R)- and (S)-a-methoxy-a-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) provided the Sand R-MTPA esters of 1, respectively. The difference in the 1 H NMR chemical shifts between the S- and R-MTPA derivatives (DdH = dS-dR) near C-21 determined the
123
N. Panthama et al. Fig. 1 Structures of compounds 1-12
O 18
7
O 19
8a
8
20
O
4
5 4a
6
16
17
R1
H H
21
1
9
11 10
O
12
13
O O
14
7
MeO
22
R1 = Cl, R2 = H R1 = Cl, R2 = R-MTPA R1 = Cl, R2 = S-MTPA R1 = H, R2 = H
O 4
OO 6
Cl
Cl
O
O O
O O
O
HO
O
O 8
7 O
O
O
O
O
O O
R1
R2
O
O OH
9
Cl O O
OH
Fig. 2 HMBC (arrow line) and COSY (bold line) correlations of isochromophilonol (1)
absolute configuration at this position to be R (Fig. 4). Thus, compound 1 was deduced to be a new azaphilone and was named as isochromophilonol.
123
MeO
3: R1 = R2 = H 3a: R1 = R2 = R-MTPA 3b: R1 = R2 = S-MTPA
5
O
OR2
HO
HO
HO
O
O
R1O
OR2 1: 1a: 1b: 2:
O
15
10: R1 = H, R2 = CH3 11: R1 = OH, R2 = CH3 12: R1 = H, R2 = CH2OH
Compound 2 was obtained as a yellow powder, and its molecular formula, C23H28O5 was deduced from HRESIMS m/z 385.2000 [M ? H]?. The IR spectra of 2 showed the characteristic absorption bands of hydroxyl (3,403 cm-1), carbonyl lactone (1,772 cm-1) and a,bconjugated ketone (1,661 cm-1) groups. The 1H and 13C NMR spectra of 2 were similar to those of 1, except for the appearance of an olefinic proton at dH 5.43 and carbon dC 106.4 which were assigned to H-5 and C-5, respectively. This was supported by the HMBC correlations of H-5 to C-6, C-7, C-4a, C-8a and C-1. The NOESY spectrum of 2 displayed the correlations of H-8, H-20 and H-9 as in 1. In addition, the absolute configuration at C-7 was also assigned to be S from the negative Cotton effect at 336
Chemical constituents from the fungus
O O MeO R1O
+0.0560
OR2 Hb
Ha -0.1288
-0.6070
3a: R1 = R2 = R-MTPA 3b: R1 = R2 = S-MTPA Fig. 5 Dd values (Dd = dS - dR in ppm) obtained for MTPA esters 3a and 3b
Fig. 3 NOESY correlations of 1
Cl
lines with IC50 values of 9.63 and 27.18 lg/mL, respectively. Compound 2 showed weak cytotoxicity against KB cell lines with IC50 value of 30.2 lg/mL. However, compound 4 displayed weak cytotoxicity against KB and MCF7 cell lines with IC50 values of 32.42 and 13.01 lg/ mL, respectively.
O Me O
O H -0.115 H -0.039 +0.007
O
OR 1a: R = R-MTPA 1b: R = S-MTPA
Acknowledgments Financial support from the Thailand Research Fund (Grant no. DBG5380047) for S.K. and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Advanced Functional Materials Cluster of Khon Kaen University are gratefully appreciated. We thank the Center of Excellence for Innovation in Chemistry (PERCH-CIC) and Rajamangala University of Technology Isan for partial support. We are indebted to the National Center for Genetic Engineering and Biotechnology via the Bioresources Research Network (BRN) for bioactivity tests.
Fig. 4 Dd values (Dd = dS - dR in ppm) obtained for MTPA esters 1a and 1b
References
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