Polyketide anthraquinone, diphenyl ether, and xanthone derivatives from the soil fungus Penicillium sp. PSU-RSPG99

Accepted Manuscript Polyketide anthraquinone, diphenyl ether and xanthone derivatives from the soil fungus Penicillium sp. PSU-RSPG99 Vatcharin Rukachaisirikul , Siwaporn Satpradit , Saranyoo Klaiklay , Souwalak Phongpaichit , Kawitsara Borwornwiriyapan , Jariya Sakayaroj PII:

S0040-4020(14)00811-4

DOI:

10.1016/j.tet.2014.05.105

Reference:

TET 25654

To appear in:

Tetrahedron

Received Date: 12 April 2014 Revised Date:

16 May 2014

Accepted Date: 27 May 2014

Please cite this article as: Rukachaisirikul V, Satpradit S, Klaiklay S, Phongpaichit S, Borwornwiriyapan K, Sakayaroj J, Polyketide anthraquinone, diphenyl ether and xanthone derivatives from the soil fungus Penicillium sp. PSU-RSPG99, Tetrahedron (2014), doi: 10.1016/j.tet.2014.05.105. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

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Graphical Abstract Polyketide anthraquinone, diphenyl ether and xanthone derivatives from the soil fungus Penicillium sp. PSU-RSPG99

Leave this area blank for abstract info.

H3CO

O

O

OCH3

O

H3CO

O

COOH

Cl CH 3

HO

O

OH

O

CH3

Cl

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SC

O

TE D EP AC C

O

Cl

HO H3CO

H3CO

OH

OH

HO

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Vatcharin Rukachaisirikul,* Siwaporn Satpradit, Saranyoo Klaiklay, Souwalak Phongpaichit, Kawitsara Borwornwiriyapan, Jariya Sakayaroj

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Polyketide anthraquinone, diphenyl ether and xanthone derivatives from the soil fungus Penicillium sp. PSU-RSPG99 a

a

Vatcharin Rukachaisirikul,*, Siwaporn Satpradit, Saranyoo Klaiklay,

b

Souwalak Phongpaichit,

c

a

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Kawitsara Borwornwiriyapan, Jariya Sakayarojd

Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of

b

SC

Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

Faculty of Science and Industrial Technology, Prince of Songkla University, Surat Thani

Campus, Surat Thani 84100, Thailand

Natural Products Research Center of Excellence and Department of Microbiology, Faculty

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c

of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand d

National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science

AC C

EP

TE D

Park, Klong Luang, Pathumthani 12120, Thailand

* Corresponding author. Tel.: +66-74-288-435; Fax: +66-74-558-841; e-mail address: [email protected] (V. Rukachaisirikul)

c

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ACCEPTED MANUSCRIPT Abstract

Three new polyketides including one new diphenyl ether, penicillither (1), one new anthraquinone, penicilliquinone (2), and one new xanthone, penicillixanthone (3), together with twelve known compounds were isolated from the soil fungus Penicillium sp. PSURSPG99. Their structures were identified by spectroscopic evidence. In addition, the position

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of a chlorine atom in 1 was confirmed by the plausible biosynthetic pathway from the isolated chlorobenzophenone. Known GKK1032B displayed mild antimycobacterial and moderate cytotoxic (against human oral carcinoma, KB, human breast cancer, MCF-7, and

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noncancerous Vero cell lines) activities.

Keywords: Penicillium sp.

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Diphenyl ethers Anthraquinones Xanthones Antimycobacterial

AC C

EP

TE D

Cytotoxic

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ACCEPTED MANUSCRIPT 1. Introduction

The fungi of the genus Penicillium produce secondary metabolites with diverse structures and interesting biological activities including, the antitrypanosomal trichodimerol,1 cytotoxic chloctanspirone2 and antioxidant citrinins.3 Recently, polyketides including anthraquinones, benzophenones and xanthones were isolated from the soil fungus P. citrinum

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PSU-RSPG95 in our laboratory.4 We describe herein the isolation of one new polyketide biphenyl ether, penicillither (1), and seven known compounds including methyl asterrate (4),5 asterric acid (5),5 questinol (6),6 dihydrogeodin (7),7 2-(3-chloro-4-methyl-γ-resorcyloyl)-5−)-asperpentyn (10)10 from the hydroxy-m-anisic acid methyl ester (8),8 sulochrin (9),9 and (−

SC

broth extract of the soil fungus Penicillium sp. PSU-RSPG99. In addition, one new anthraquinone, penicilliquinone (2), and one new xanthone, penicillixanthone (3), and five

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known compounds, methyl dichloroasterrate (11),5 emodin (12),11 methyl 8-hydroxy-6-methyl9-oxo-9H-xanthene-1-carboxylate (13),12 GKK1032B (14),13 and coniochaetone B (15),14 were isolated from the mycelial extract of this fungus. Some of the isolated compounds were evaluated for antimycobacterial activity against Mycobacterium tuberculosis and cytotoxic activity against human oral carcinoma, KB, human breast cancer, MCF-7, and noncancerous

9'

9

H3CO

O

OR1

O 8'

8

O 3

OH 1'

B1

3' A 5'

5

H3CO 7

R1 8

R2

3

R CH 3

HO

EP

HO

TE D

Vero cell lines.

AC C

H3CO

OH

10

9a

1

8 3

4a

11

R2

H3C

OH

O

H3CO HO R2 7: R1 = R2 = Cl 8: R1 = H, R2 = Cl 9: R1 = R2 = H

R1

8a

O 9

H3C H3C H

9a

R2

1 3

5 10a

O

4a

CH3

11

3: R1 = OH, R2 = R3 = Cl 13: R1 = R2 = R3 = H

CH3 OH O

O H O CH3

CH3

OH

R3

R1

HO

O

12

2: R1 = OCH3, R2 = COOH 6: R1 = OCH3, R2 = CH2OH 12: R1 = OH, R2 = CH3

O

O

10a

9

H3CO

OH

O

7'

1: R1 = CH3, R2 = H, R3 = Cl 4: R1 = CH3, R2 = R3 = H 5: R1 = R2 = R3 = H 11: R1 = CH3, R2 = R3 = Cl

5

8a

13

O

H

NH

H O H

O H3C

O

OH 10

14

15

Fig. 1. Compounds 1-15 isolated from the fungus Penicillium sp. PSU-RSPG99.

OH

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ACCEPTED MANUSCRIPT 2. Results and discussion

The extracts from the culture broth and mycelia of the fungus PSU-RSPG99 were purified using various chromatographic techniques to yield four diphenyl ethers (1, 4-5 and 11), three anthraquinones (2, 6 and 12), two xanthones (3 and 13), three benzophenones (7-9), one epoxycyclohexene (10), one tricyclicdecahydrofluorene (14) and one benzopyranone (15)

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(Fig. 1). Their structures were identified on the basis of spectroscopic analyses, including 2D NMR spectroscopic data. For known compounds, their NMR data were compared with those previously reported. The relative configurations of 10 and 14 were determined by NOEDIFF data while the absolute configurations of these compounds and 15 were assigned by

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comparison of their specific rotations with those reported previously.

Penicillither (1) was obtained as a colorless gum. The HREIMS showed the molecular

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formula C18H17ClO8. The IR spectrum displayed absorption bands at 3380 and 1726 cm-1 for hydroxy and conjugated ester carbonyl functionalities, respectively. The

1

H NMR

spectroscopic data (Table 1) showed signals for one hydroxy group (δH 10.95, s), two metacoupled aromatic protons (δH 6.79 and 6.57, each d, J = 2.7 Hz), one aromatic proton of a pentasubstituted benzene (δH 6.68, s), three methoxy groups (δH 3.77, 3.74 and 3.55, each s) and one aromatic methyl group (δH 2.33, s). The HMBC data shown in Table 1 indicated that

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1 contained a B-ring identical to that of compounds 4, 5 and 11. For the A-ring, the location of the methyl group was assigned at C-5' (δC 144.2), identical to compounds 4, 5 and 11. The C-5' methyl protons (H3-7', δH 2.33) displayed HMBC correlations to the methine carbon (δC 114.4) and quaternary carbon (δC 117.7) which were assigned as C-4' and C-6', respectively.

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For compounds 4 and 5, the assignment of H-4' and H-6' was performed using HMBC correlations from 3'-OH to C-2', C-3' and C-4' as well as the HMQC cross peak from C-4' to

AC C

an aromatic proton. We found that H-4' in 4 (δH 6.42) and 5 (δH 6.47) appeared at much lower field than H-6' in 4 (δH 5.75) and 5 (δH 5.89). In addition, C-4' in 4 (δC 111.0) and 5 (δC 111.6) resonated at lower field than C-6' in 4 (δC 105.9) and 5 (δC 104.9). However, these assignments were different from those previously reported for compounds 4 and 5.5 Unfortunately, 3'-OH in 1 showed none of HMBC correlations due to the appearance as a weak signal. The singlet aromatic proton (δH 6.68) and its methine carbon (δC 117.7) in 1 were assigned as H-4' and C-4', respectively, by comparison of these data with those of compounds 4 and 5. Accordingly, the substituent at C-6 was a chlorine atom. In addition, the methoxyl protons, H3-9' (δH 3.77), gave a HMBC cross peak to C-8'. These results established

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a methyl ester moiety at C-2'. Therefore, 1 was assigned as a C-6' chloro regioisomer of methyl chloroesterate.6

Table 1 H, 13C NMR and HMBC data for 1 in CDCl3

δH, J in Hz

δC

1

139.4, qC

2

138.9, qC

3

6.79, d, 2.7

6.57, d, 2.7

6

105.0, CH 151.1, qC

7

3.55, s

56.7, CH3

8

166.3, qC

9

3.77, s

52.2, CH3

1'

159.8, qC

2'

105.9, qC

3'

159.8, qC

3'-OH

10.95, brs

4'

6.68, s

114.4, CH 144.2, qC

6'

117.7, qC

2.33, s

21.0, CH3

C-1, C-3, C-4, C-6

C-6

C-8

C-2', C-3', C-6', C-8'

C-4', C-5', C-6'

170.4, qC

AC C

8'

EP

5'

9'

C-1, C-4, C-5, C-8

151.1, qC

5

7'

HMBC

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4

108.2, CH

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1

SC

Position

TE D

1

3.74, s

52.5, CH3

C-8'

Penicilliquinone (2) was obtained as an orange gum with the molecular formula

C16H9O7 on the basis of HRESIMS. The UV and IR spectra were almost identical to those of 6. The 1H NMR spectroscopic data (Table 2) were similar to those of 66 except for the absence of signal for the hydroxymethylene protons (δH 4.75) in 2. The substituent at C-3 (δC 147.1) was assigned as a carboxyl group (C-11, δC 173.4) on the basis of HMBC correlations of both H-2 (δH 7.75, d, J = 1.8 Hz) and H-4 (δH 8.24, d, J = 1.8 Hz) with C-11. Therefore, 2 was a new 3-carboxylanthraquinone derivative.

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ACCEPTED MANUSCRIPT Table 2 H, 13C NMR and HMBC data for 2 in CD3OD and 3 in CDCl3 Position 2 δH, J in Hz

δC

HMBC

164.6, qC 12.98, s 7.75, d, 1.8

8.24, d, 1.8

121.4, CH

7.31, d, 2.4

109.7, CH

6.82, d, 2.4

106.9, CH

8a

115.3, qC

9

189.8, qC

9a

120.1, qC

10

185.2, qC

10a

135.0, qC

11

173.4, qC

C-6, C-7, C-8a, C-10

C-5, C-6, C-8, C-8a

57.9, CH3

7.01, d (1.0)

6.87, d (1.0)

104.9, CH

C-6, C-7, C-10a

162.8, qC

113.6, CH

C-5, C-8a, C-9, C-12

135.9, qC 110.8, qC 180.3, qC 106.6, qC

157.6, qC 2.64, s

C-8

EP

3.97, s

111.7, qC 149.5, qC

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166.5, qC

13

C-2, C-9a, C-10, C-11

168.1, qC

8

12

143.9, qC

140.2, qC

6 7

116.3, qC

147.1, qC

4a 5

C-1, C-4, C-9a, C-11

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4

HMBC

C-1, C-2, C-9a

SC

3

126.5, CH

δC 155.5, qC

1-OH 2

δH, J in Hz

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1

3

18.8, CH3

C-2, C-3, C-4, C-4a

168.8, qC 4.02, s

53.3, qC

C-12

Penicillixanthone (3) was obtained as a yellow gum. The UV and IR spectra were

AC C

1

almost identical to those of 13. The HREIMS showed the molecular formula C16H12Cl2O6. The 1H NMR spectroscopic data (Table 3) were similar to those of 1312 except for the absence of signals for two meta-coupled aromatic protons (δH 6.70 and 6.58) in 3. These data together the above molecular formula indicated the replacement of these aromatic protons in 13 with two chlorine atoms in 3. The methyl protons resonating at δH 2.64 (H3-11) displayed HMBC correlations with C-2 (δC 116.3), C-3 (δC 143.9) and C-4 (δC 111.7). The chemical shifts of C-2 and C-4 supported the attachment of the chlorine atoms at these carbons. In addition, three aromatic protons of the 1,2,3-trisubstituted benzene (δH 7.68, 7.46 and 7.24) in 13 were replaced, in 3, by two meta-coupled aromatic protons (δH 7.01 and 6.87, each d, J =

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1.0 Hz, 1H). The aromatic proton resonating at 6.87 was assigned as H-7 according to the HMBC correlation with C-12 (δC 168.8). Thus, the remaining aromatic proton was attributed to H-5 which exhibited the HMBC correlations with C-6 (δC 162.8), C-7 (δC 113.6) and C-10a (δC 157.6). The substituent at C-6 was identified as a hydroxy group on the basis of the 13C

OH

H3CO Emodin O -methyltransferanse

HO

CH3

O

Oxidation CH3 HO O Oxidation Methanolysis

HO

O 12

Oxidative cyclization

O O

CH3

H3CO2C

HO

A

O

H3CO

H3CO2C

5

Methylation

O

CO2CH3 OH

CH3

O

H3CO

H3CO HO Cl 8

H3CO O

CH3

AC C

4

O 3

H3CO2C

O

OH

CH3

HO

CH3

OH Cl

H3CO HO Cl 7

OH

H3CO O

Cl O

O CO2CH3 Cl

O CH3

CH3

CO2CH3 Cl

C

B

Oxidative cleavage

Oxidative cleavage

Methylation

Methylation H3CO2C

CO2CH3 OH

O HO

CH3

Oxidative cyclization

OH

O

HO

HO

Oxidative cyclization

EP

H3CO2C

OH

Chlorination

HO CH3

O

Chlorination

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HO

R

O 2 : R = COOH 6 : R = CH 2OH

H3CO2C

H3CO HO 9

Oxidative cleavage CO2H OH

OH

OH

Cyclization

CO2CH3

H3CO2C

O

O

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OH

H3CO O

H3CO

OH

SC

OH O

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NMR chemical shift of C-6. Therefore, penicillixanthone had the xanthone structure 3.

H3CO2C

HO H3CO

Cl 1

CH3

CO2CH3 OH

O

H3CO

Cl 11

Cl CH3

Scheme 1. Proposed biosynthesis pathway of the isolated compounds 1-9 and 11-12.

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ACCEPTED MANUSCRIPT

The proposed biosynthesis pathway for the isolated diphenyl ethers (1, 4-5 and 11), anthraquinones (2 and 6), xanthone (3) and benzophenones (7-9) from the precursor 12, an octaketide anthraquinone,15 is shown in Scheme 1. Methylation and subsequent oxidation would provide 2 and 6. The benzophenone 9 could be derived from the methylated 12 by Baeyer-Villiger oxidation to give a depsidone intermediate and subsequent methanolysis.

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Compound 9 could be an important precursor for most of the isolated secondary metabolites. The chloro derivatives 8 and 7 would be obtained from 9 upon mono- and dichlorination, respectively. Cyclization of 9 would afford the xanthone 3 while oxidative cyclization of 9 and subsequent cleavage of a spirofuran-3-one intermediate A16-17 would yield 5 which would

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undergo methylation to afford 4. Compound 1 would be transformed from 8 via a spirofuran3-one intermediate B followed by oxidative cleavage and subsequent methylation. This proposed biosynthetic pathway supported the position of the chlorine atom in 1 at C-6'.

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Finally, 11 would be obtained from 7 using the same pathway as 1 via a spirofuran-3-one intermediate C.

The antimycobacterial (against Mycobacterium tuberculosis H37Ra), and cytotoxic (against KB, MCF-7, and Vero cells) activities of compounds 5, 7, 9 and 14 which were obtained in sufficient amount, were evaluated. Compound 14 was weakly active against M. tuberculosis (MIC 48.35 µM) and was mildly cytotoxic to KB, MCF-7, and Vero cells with

showed no activity.

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3. Conclusion

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the IC50 values of 19.26, 14.71 and 29.55 µM, respectively. The remaining compounds

Chlorinated diphenyl ethers are rare fungal secondary metabolites.5 In general,

AC C

diphenyl ethers with chlorine atom(s) in the A ring were isolated from Penicillium and Aspergillus spp. whereas Xylaria ad Pestalotiopsis spp. produced compounds of the same type but having chlorine atom(s) in the B ring. These conclusions are in agreement with our recent report on the isolation of diphenyl ethers with B ring chlorination from the endophyte Pestalotiopsis sp.17 and the present work on the new diphenyl ether carried the chlorine atom at the A ring from Penicillium sp.

4. Experimental 4.1 General experimental procedures

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Optical rotations were measured on a JASCO P-1020 polarimeter. The ultraviolet (UV) absorption spectra were measured in MeOH on a Perkin-Elmer Lambda 45 spectrophotometer. The infrared (IR) spectra were recorded neat using a Perkin-Elmer 783 FTS165 FT-IR spectrometer. 1H and

13

C NMR spectra were recorded on a 300 or 500 MHz

Bruker FTNMR Ultra Shield spectrometer. Chemical shifts are expressed in δ (ppm) referring

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to the tetramethylsilane peak. Mass spectra were obtained on a MAT 95 XL mass spectrometer (Thermo Finnigan) except for that of compound 2, which were recorded on a Bruker MicrOTOF mass spectrometer. Thin-layer chromatography (TLC) and preparative TLC were performed on silica gel 60 GF254 (Merck). Column chromatography was carried

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out on Sephadex LH-20 with MeOH, silica gel (Merck) type 60 (230-400 mesh ASTM) or type 100 (70-230 mesh ASTM), or on reversed phase C18 silica gel.

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4.2 Fungal material

The fungus PSU-RSPG99 was isolated from soil samples collected from the Rajjaprabha Dam, Surat Thani province, Thailand. The pure culture was deposited as BCC56879 at the BIOTEC Culture Collection, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park and its DNA sequences was submitted to

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NCBI database to receive the accession number of KC478542. The colony on PDA was white, velvety, moderate growth and the media changed to yellow. As the result of ITS rDNA analysis, this isolate grouped with several strains of Penicillium sp. supported by 61% bootstrap value but without any closely associated taxa. Therefore, RSPG99 was identified

EP

molecular data to be Penicillium sp.

AC C

4.3. Fermentation and isolation

The soil fungus PSU-RSPG99 was grown on potato dextrose agar at 25 °C for 5 days.

Five pieces (0.5 x 0.5 cm2) of mycelial agar plugs were inoculated into 500 mL Erlenmeyer flasks containing 300 mL of potato dextrose broth at room temperature for three weeks. The flask culture was filtered to separate filtrate from wet mycelia. The filtrate was was extracted three times with EtOAc. The combined EtOAc extract was dried over anhydrous Na2SO4 and evaporated to dryness under reduced pressure to give a broth extract (6.25 g). The mycelial cake was extracted with MeOH (1.0 L). The MeOH layer was concentrated under reduced pressure. H2O was added to the extract, and the mixture was washed with hexane. The aqueous residue was extracted three times with EtOAc. The EtOAc layer was then dried over anhydrous Na2SO4 and evaporated to dryness to obtain a brown gum (3.6 g). The broth

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ACCEPTED MANUSCRIPT

extract was subjected to column chromatography (CC) over Sephadex LH-20 using MeOH as a mobile phase to give five fractions (A-D). Fraction B (819.2 mg) was fractioned by CC over Sephadex LH-20 using MeOH/CH2Cl2 (1:1) to afford four fractions (B1-B4). Fraction B2 (180.7 mg) was purified by CC over reversed phase C18 silica gel to provide six fractions (B21-B26). Fraction B22 (21.6 mg) was purified using the same procedure as fraction B to

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afford 1010 (2.5 mg, a colorless gum, [α]D25 -18.0, c 0.1, acetone). Fraction B25 (40.2 mg) was subjected to CC over silica gel using MeOH/CH2Cl2 (1:99→100:0) followed by preparative TLC using acetone/CH2Cl2 (1:99) as a mobile phase to provide 1 (1.4 mg, a colorless gum) and 45 (1.4 mg, a colorless gum). Fraction C (3,953.9 mg) was divided into two portions. The first portion (CR, 547.0 mg) was further purified using the same method as

SC

fraction B2 followed by CC over silica gel using acetone/CH2Cl2 (1:99→100:0) to afford 77 (6.5 mg, a yellow solid, mp 230-232 oC). The second portion (CS, 3,405.9 mg) was purified

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using the same method as fraction B to give nine fractions (CS1-CS9). Compound 55 (7.8 mg, a white solid, mp 192-194 oC) was obtained from fraction CS3 after purification by CC using acetone/CH2Cl2 (1:4→1:0). Fraction CS7 (871.0 mg) was fractionated by CC over silica gel using MeOH/CH2Cl2 (1:49→100:0) to provide six fractions (CS71-CS76). Fraction CS73 (141.8 mg) was purified using the same method as fraction CS7 followed by preparative TLC

TE D

using acetone/CH2Cl2 (1:99) to afford 88 (2.6 mg, a pale yellow solid, mp 248-250 oC). Fraction CS74 was dissolved with MeOH to provide a MeOH soluble part (106.4 mg) which was purified using the same procedure as fraction B2 followed by preparative TLC using acetone/CH2Cl2 (5:95) as a mobile phase to give 99 (8.2 mg, a pale yellow solid, mp 248-250 o

EP

C). Fraction D (844.4 mg) was purified using the same as fraction B to give five fractions

(D1-D5). Fraction D4 (188.6 mg) was subjected to CC over reversed phase C18 silica gel

AC C

followed by CC over Sephadex LH-20 using MeOH as a mobile phase to give 66 (1.0 mg, an orange gum). The mycelial extract was purified using the same method as the broth extract to give seven fractions (CE1-CE7). Fraction CE4 (640.9 mg) was purified using the same procedure as fraction CS7 to give eight fractions (CE41-CE48). Compound 1413 (141.1 mg, a colorless gum, [α]D25 +174.8, c 0.2, MeOH) was obtained from fraction CE43. Fraction CE45 (97.5 mg) was purified using the same procedure as fraction CS7 to give four fractions. The third fraction (37.6 mg) was purified using the same procedure as fraction CS7 to provide 115 (2.8 mg, a colorless gum) and 1514 (2.5 mg, a colorless gum, [α]D25 +82.6, c 0.1, MeOH). Fraction CE5 (732.5 mg) was dissolved with MeOH to give a MeOH soluble part (251.4 mg) which was subjected to CC over silica gel using acetone/CH2Cl2 (1:19→100:0) to give five fractions. The first fraction (8.9 mg) was dissolved with MeOH/CH2Cl2 (1:1) to give a

12

ACCEPTED MANUSCRIPT

soluble part (6.2 mg) which was purified using the same method as fraction B to give 1312 (2.0 mg, a yellow gum). Fraction CE7 (201.0 mg) was purified using the same method as fraction B2 to give five fractions. The first fraction (69.6 mg) was purified using the same method as fraction B2 followed by dissolving with MeOH to provide 2 (1.6 mg, an orange gum) from an insoluble part. The third fraction (8.7 mg) was dissolved with acetone to give

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1211 (5.2 mg, an orange gum) from a soluble part. The fourth fraction (2.9 mg) was purified using the same method as the third fraction to give 3 (2.0 mg, a yellow gum).

4.3.1. Penicillither (1): Colorless gum. UV λmaxMeOH nm (log ε): 222 (3.57), 249 (2.13), 322 (2.57). FT-IR νmaxneat cm−1: 3380, 1726, 1662; 1H and

13

C NMR (CDCl3) data see Table 1;

SC

HREIMS m/z 396.0604 [M]+ (calcd. for C18H17ClO8, 396.0612).

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4.3.2. Penicilliquinone (2): Orange gum. UV λmaxMeOH nm (log ε): 225 (2.27), 275 (1.92), 291 (1.82), 436 (1.38). FT-IR νmaxneat cm−1: 3400, 1725, 1662, 1587; 1H and

13

C NMR (CD3OD)

data see Table 2; HRESIMS m/z 313.0354 [M-H]+ (calcd. for C16H9O7, 313.0348). 4.3.3. Penicillixanthone (3): Yellow gum. UV λmaxMeOH nm (log ε): 230 (3.25), 294 (3.19), 369 (2.69). FT-IR νmaxneat cm−1: 3327, 1728, 1642; 1H and 13C NMR (CDCl3) data see Table

4.4 Bioassay activity

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2; HREIMS m/z 367.9848 [M]+ (calcd. for C16H10Cl2O2, 367.9849).

EP

The cytotoxic assay against Vero cells was performed employing the colorimetric method18 while the cytotoxic assays against MCF-7 and KB cells were conducted using the method described by O'Brien and coworkers.19 The standard compound for Vero and KB cell

AC C

lines was ellipticine, exhibiting IC50 values of 1.97 and 3.49 µM against Vero and KB cell lines, respectively. The standard compounds for the MCF-7 cell lines were tamoxifen and doxorubicin, exhibiting IC50 values of 23.61 and 15.34 µM, respectively. Antimycobacterial activity was determined against M. tuberculosis H37Ra using a green fluorescent protein (GFP)-based fluorescent detection.20 Standard drugs, rifampicin and isoniazid, exhibited MIC values of 0.06 and 3.51 µM, respectively.

Acknowledgements

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V.R. thanks the Thailand Research Fund (TRF) for the TRF Senior Research Scholar (Grant No. RTA5480002). S.S. thanks the Science, Mathematics and Technology Excellence Development Project; Teacher Professional Development Project (TPDP) for a scholarship. The Center of Excellence for Innovation in Chemistry (PERCH-CIC), the Office of Higher Education Commission, and Prince of Songkla University are acknowledged for partial

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support. We thank Dr. Pattama Pittayakhajonwut for HRESIMS data. Finally, the National Center for Genetic Engineering and Biotechnology (BIOTEC) is acknowledged for antimycobacterial, anticancer and cytotoxic assays.

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