Allahabadolactones A and B from the endophytic fungus, Aspergillus allahabadii BCC45335

Accepted Manuscript Allahabadolactones A and B from the endophytic fungus, Aspergillus allahabadii BCC45335 Karoon Sadorn, Siriporn Saepua, Nattawut Boonyuen, Pattiyaa Laksanacharoen, Pranee Rachtawee, Samran Prabpai, Palangpon Kongsaeree, Pattama Pittayakhajonwut PII:

S0040-4020(15)30240-4

DOI:

10.1016/j.tet.2015.11.056

Reference:

TET 27313

To appear in:

Tetrahedron

Received Date: 7 August 2015 Revised Date:

10 November 2015

Accepted Date: 24 November 2015

Please cite this article as: Sadorn K, Saepua S, Boonyuen N, Laksanacharoen P, Rachtawee P, Prabpai S, Kongsaeree P, Pittayakhajonwut P, Allahabadolactones A and B from the endophytic fungus, Aspergillus allahabadii BCC45335, Tetrahedron (2015), doi: 10.1016/j.tet.2015.11.056. 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 Graphical Abstract Allahabadolactones A and B from the

Leave this area blank for abstract info.

endophytic fungus, Aspergillus allahabadii BCC45335 Karoon Sadorna,b,*, Siriporn Saepuac, Nattawut Boonyuenc, Pattiyaa Laksanacharoenc, Pranee Rachtaweec, Samran

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Prabpaid,e, Palangpon Kongsaereed,e, Pattama Pittayakhajonwutc

ACCEPTED MANUSCRIPT

Allahabadolactones A and B from the endophytic fungus, Aspergillus allahabadii BCC45335

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Karoon Sadorna,b,*, Siriporn Saepuac, Nattawut Boonyuenc, Pattiyaa Laksanacharoenc, Pranee

a

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Rachtaweec, Samran Prabpaid,e, Palangpon Kongsaereed,e, Pattama Pittayakhajonwutc

Department of Chemistry, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Chalongkrung

Road, Ladkrabang, Bangkok 10520, Thailand

Department of Chemistry, School of Science, University of Phayao, Paholyothin Road, Muang, Phayao 56000,

Thailand c

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

Development Agency (NSTDA),

Thailand Science Park, Paholyothin Road, Klong Luang, Pathumthani 12120,

Thailand

Department of Chemistry, Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University,

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b

Rama 6 Road, Bangkok 10400, Thailand e

Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Rama 6 Road,

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Bangkok 10400, Thailand

* Corresponding author. Tel.: +66 2 329 8400x290; fax: +66 2 329 8428; e-mail address: [email protected].

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ACCEPTED MANUSCRIPT Abstract Two new compounds, allahabadolactones A (1) and B (2), along with ten known compounds

including

16-amino-isopimar-7-en-19-oic

acid

(3),

16-α-D-

glucopyranosyloxyisopimar-7-en-19-oic acid (4), 16-α-D-mannopyranosyloxyisopimar-7-en-19-oic (5),

ergosterol,

(22E)-5α,8α-epidioxyergosta-6,22-dien-3β-ol,

cerevisterol,

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acid

(R)-(-)-

methoxycarbonylmellein, (-)-piliformic acid, 7-dechlorogriseofulvin, and cytochalasin D, were isolated from the endophytic fungus, Aspergillus allahabadii BCC45335. Their chemical structures

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were determined based on NMR spectroscopic and mass spectrometric analyses. The absolute

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stereochemistry of compound 1 was established by an X-ray crystallographic analysis and the reactions with Mosher’s reagents. A plausible biosynthesis of allahabadolactones A (1) and B (2) was also proposed. Antibacterial activity against B. cereus and cytotoxicity against MCF-7, KB,

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NCI-H187, and Vero cells of the isolated compounds were also evaluated.

Keywords. allahabadolactone A; allahabadolactone B; Aspergillus allahabadii; endophytic fungus; antibacterial activity

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ACCEPTED MANUSCRIPT 1. Introduction Aspergillus is a common genus of fungi in the family Trichocomaceae and has long been known as a prolific source of bioactive compounds with unique chemical structures.

Many

secondary metabolites with various biological activities have recently been isolated from

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Aspergillus spp. For example, prenylated dihydroquinolone derivative, 22-O-(N-Me-L-valyl)-21epi-aflaquinolone B, from Aspergillus sp. XS-20090B15 showed promising antiviral activity against respiratory syncytial virus (IC50 42 nM).1 Alkaloids, aspergillines A − E, from A. versicolor

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YNCA1266 displayed interesting anti-tobacco mosaic virus activity (IC50 15.4 − 48.6 µM) and also showed a broad range of cytotoxicity towards NB4 (human acute promyelocytic leukemia), A549

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(human lung epithelial carcinoma), SHSY5Y (human neuroblastoma), PC3 (human prostate cancer), and MCF-7 (human breast adenocarcinoma) cells (IC50 1.2 − >10 µM).2 Moreover, cyclic tetrapeptide, asperterrestide A, from A. terreus SCSGAF0162 exhibited cytotoxic activity against U937 (human leukemic monocyte lymphoma) and MOLT-4 (acute lymphoblastic leukemia) cells

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with IC50 values of 6.4 and 6.2 µM, respectively, and also inhibited influenza virus strains H1N1 and H3N2 with IC50 values of 15 and 8.1 µM, respectively.3 Indole-benzodiazepine-2,5-dione derivatives, asperdiazapinones A – F, were isolated from Aspergillus sp. PSU-RSPG185.4

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Furthermore, dimeric 1,3-dihydroisobenzofurans, flavimycins A and B, from A. flavipes

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KCTC10880BP showed antibacterial activity towards Staphylococcus aureus peptide deformylase with IC50 values of 35.8 and 100.1 µM, respectively.5 As part of our continuing search for new bioactive secondary metabolites from

microorganisms collected from various parts of Thailand, we encountered the crude extract of the endophytic fungus BCC45335, exhibiting antibacterial activity against Bacillus cereus with IC50 value of 6.50 µg/mL and providing a good chemical profile from HPLC analysis. Chemical investigation was then conducted. Both cell and broth led to the isolation of two new compounds, allahabadolactones A (1) and B (2), together with ten known compounds, 16-amino-isopimar-7-en-

3

ACCEPTED MANUSCRIPT 19-oic

acid

(3),

16-α-D-glucopyranosyloxyisopimar-7-en-19-oic

acid

(4),

16-α-D-

mannopyranosyloxyisopimar-7-en-19-oic acid (5), ergosterol, (22E)-5α,8α-epidioxyergosta-6,22dien-3β-ol,

cerevisterol,

(R)-(-)-5-methoxycarbonylmellein,

(-)-piliformic

acid,

7-

dechlorogriseofulvin, and cytochalasin D. Antibacterial activity as well as their cytotoxicity of the

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isolated compounds was also reported.

2. Results and discussion

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Chemical structures of the isolated secondary metabolites were elucidated based on

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spectroscopic information including NMR, mass, FT-IR, UV spectroscopic data and specific rotations. The absolute configuration of compound 1 was confirmed by X-ray crystallographic data and Mosher’s reactions. The 1H and

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C NMR spectral data of the known compounds were

compared to those previously published data for 16-amino-isopimar-7-en-19-oic acid (3),6 16-α-Dglucopyranosyloxyisopimar-7-en-19-oic acid (4),7 16-α-D-mannopyranosyloxyisopimar-7-en-19-

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oic acid (5),7 ergosterol,8 (22E)-5α,8α-epidioxyergosta-6,22-dien-3β-ol,9 cerevisterol,10 (R)-(-)-5methoxycarbonylmellein,11 (-)-piliformic acid,12 7-dechlorogriseofulvin,13 and cytochalasin D.11

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Compound 1 was obtained as colourless crystals. Its molecular formula was determined to be C18H26O3, deduced from HRESIMS (m/z 313.1764, [M+Na]+) indicating six degrees of

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unsaturation. The 13C NMR spectral data together with DEPT-135 spectrum revealed 18 signals, which were three methyl, three methylene, four sp3 methine, two oxymethine, four olefinic methine, one sp3 quaternary, and one carbonyl carbons (Table 1). The chemical structure of compound 1 was disclosed by interpretations of the COSY and HMBC correlations (Fig. 1). The COSY spectral data of compound 1 showed the presence of two spin-systems, which were C-5 – C10 with the methyl at δH 0.91 (11-CH3) attached to C-8 and C-1 – C-3 / C-14 – C-18. The HMBC showed the correlations from the methyl at H3-11 to the methine carbon at δC 32.8 (C-8) and two methylene carbons at δC 35.1 (C-7) and 41.6 (C-9); from the methylene at δH 1.76 − 1.87 (H-7 and

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ACCEPTED MANUSCRIPT H2-6) to the methine carbon at δC 40.2 (C-5); from the methylene at δH 0.81 (H-9) to two sp3 methine carbons at δC 36.2 (C-10) and C-5; from the methine at δH 1.22 − 1.38 (H-5) to the methyl at δC 16.3 (C-13) and to the sp3 methine carbon at C-10; from the methyl at δH 1.14 (H3-13) to two methine carbons at δC 51.5 (C-3), C-5, and to two quaternary carbons at δC 44.7 (C-4), 179.2 (C-

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12); from the methine at δH 5.62 (H-1) to three methine carbons at C-3, C-5, and C-10; from the methine at δH 2.32 − 2.38 (H-3) to the oxymethine carbon at δC 82.6 (C-14).

The spectral

information indicated two six-membered rings fused at C-5 and C-10, which possessed an ester

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group (δC 179.2) at position 4. The IR absorption at νmax 1770 cm-1 suggested the presence of a γ-

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carbonyl lactone. Moreover, in the HMBC spectrum, the correlations from the methine at δH 5.72 (H-15) and 5.92 (H-16) to two oxymethine carbons at δC 67.7 (C-17) and C-14; from the methine at

δH 4.47 (H-14) to two methine carbons at δC 120.8 (C-2) and 138.9 (C-16); from the oxymethine at δH 4.39 (H-17) to the methyl at δC 23.3 (C-18) and sp2 methine carbon at δC 125.9 (C-15); from the

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methyl at δH 1.30 (H3-18) to two methine carbons at C-16 and C-17 indicated a side-chain connected at the position 14. The double bond at C-15 and C-16 was assigned as E-configuration, indicated by a coupling constant of 15.4 Hz. The NOESY spectrum revealed cross-peak

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correlations from 13-CH3 to H-3 and H-10; from H-3 to H-15; from H-10 to H-8; from H-14 to H-5 and H-16 (Fig. 1). The structure of compound 1 was confirmed on the basis of a single-crystal X-

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ray diffraction analysis (Fig. 2). Its relative stereochemistry was also reassured by X-ray crystallographic data. Treatment of compound 1 with the Mosher’s reagents, (R)- and (S)MTPACl14 confirmed the absolute configuration at C-17 as “S” configuration (Fig. 3). Together with the evidence from the X-ray crystallographic information, the absolute configuration of compound 1 was therefore established as 3R,4S,5R,8R,10R,14S,17S. Thus, compound 1 had the chemical structure as shown in Fig. 4 and its trivial name was given as allahabadolactone A.

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Fig. 2. X-ray crystal structure of allahabadolactones A (1).

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Fig. 1. COSY, key HMBC and selected NOESY correlations of allahabadolactone A (1).

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Fig. 3. ∆δ Values (δS − δR) of (S)- and (R)-MTPA esters, 1a and 1b.

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ACCEPTED MANUSCRIPT Compound 2 was obtained as a colourless semi-solid. The mass ion peak at m/z 313.1784 [M+Na]+ in HRESIMS spectrum suggested that compound 2 was an isomer of compound 1. The 1

H NMR spectrum of compound 2 was also similar to that of compound 1. The significant

differences were the downfield shifts of the methine signal at H-3 (δH 2.65), H-17 (δH 5.87), and the

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methyl doublet at H3-18 (δH 1.75) together with the upfield shifts of the methine signals at H-14 (δH 4.14), H-15 (δH 4.39 − 4.44), and H-16 (δH 5.49) (see Table 1). The data pointed to a difference in the side chain moiety, compared to that of 1. The HMBC spectrum showed correlations from the

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oxymethine at H-15 to the methine carbons at δC 50.0 (C-3), 84.3 (C-14), 127.6 (C-16), and 129.8 (C-17); from the sp2 methine signals at H-16 and H-17 to the methyl at δC 17.8 (C-18) and to the

17.

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methine carbon at δC 72.2 (C-15); from the methyl at H3-18 to the methine carbons at C-16 and CThe spectral information indicated the double bond at positions 16 and 17, which was

determined as E-configuration, based on its coupling constant of 15.4 Hz.

The NOESY

correlations were consistent with those of compound 1. In addition, compound 2 should derive

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from the same biosynthetic pathway as that of 1 (Scheme 1), thus its absolute stereochemistry, except at position 15, was then assigned the same as that of 1. Due to an inadequate material of

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compound 2 for the Mosher’s reactions, the absolute configuration at C-15 has not yet been

4.

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determined. Therefore, allahabadolactone B (2) possessed the chemical structure as shown in Fig.

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ACCEPTED MANUSCRIPT Fig. 4. Chemical structures of compounds 1 – 5 from the endophytic Aspergillus allahabadii BCC45335. 12

14

10

16

2

8

11

OH

H

5

H

O

H

15

3

4 7

O O

1

H

17

H

18

1

2 R

OH O 4 R=

5 R=

OH OH O OH OH

O

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OH O

OH OH

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HOOC

3 R = NH2

OH

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O 13

H

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ACCEPTED MANUSCRIPT Table 1. 1H (500 MHz) and 13C (125 MHz) NMR assignments for allahabadolactones A (1) and B (2) in CDCl3. Compound 2 C

134.9 120.8 51.5 44.7 40.2 25.5 35.1

10 11 12 13 14 15 16

5.62 (1H, br d, J = 10.1 Hz) 5.53 (1H, ddd, J = 10.1, 4.5, 2.6 Hz) 2.32−2.38 (1H, m) − 1.22−1.38 (1H, m) 1.76−1.87 (2H, m) 0.87−0.98 (1H, m) 1.76−1.87 (1H, m) 1.43−1.57 (1H, m) 0.81 (1H, q, J = 12.2 Hz) 1.87−1.97 (1H, m) 1.87−1.97 (1H, m) 0.91 (3H, d, J = 6.6 Hz) − 1.14 (3H, s) 4.47 (1H, dd, J = 9.9, 7.3 Hz) 5.72 (1H, ddd, J = 15.4, 7.3, 1.0 Hz) 5.92 (1H, dd, J = 15.4, 5.4 Hz)

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4.39 (1H, dq, J = 6.4, 5.4 Hz)a

67.7

18

1.30 (3H, d, J = 6.4 Hz)

23.3

8 9

36.2 22.3 179.2 16.3 82.6 125.9 138.9

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5.57 (1H, br d, J = 10.2) 5.53−5.55 (1H, m) 2.65 (1H, dd, J = 9.8, 4.0 Hz) − 1.20−1.32 (1H, m) 1.78−1.87 (2H, m) 0.85−0.95 (1H, m) 1.78−1.87 (1H, m) 1.43−1.53 (1H, m) 0.78 (1H, q, J = 12.1 Hz) 1.85−1.93 (1H, m) 1.85−1.93 (1H, m) 0.91 (3H, d, J = 6.6 Hz) − 1.12 (3H, s) 4.14 (1H, dd, J = 9.8, 3.6 Hz) 4.39−4.44 (1H, m) 5.49 (1H, ddd, J = 15.4, 6.5, 1.6 Hz) 5.87 (1H, dq, J = 15.4, 6.6 Hz) 1.75 (3H, d, J = 6.6 Hz)

C

133.9 122.7 50.0 45.0 40.9 25.5 35.1 32.8 41.6 36.0 22.3 179.3 15.9 84.3 72.2 127.6 129.8 17.8

H Homonuclear decoupling experiments recorded in CDCl3

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a1

32.8 41.6

1

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H

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1 2 3 4 5 6 7

Compound 1 1

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position

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ACCEPTED MANUSCRIPT Compounds 1 and 2 may be derived from polyketide and the biosynthetic pathway of the decalin moiety could be derived similarly to that reported for lovastatin,15 apiosporamide,16 solanapyrone D,17 and ZG-1494α.18 The formation of trans-fused decalin was involved in the biological intramolecular Diels–Alder reaction of the triene via endo cyclization.17,19 After

compounds 1 and 2 as shown in scheme 1.

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cyclization, it could undergo oxidation, lactonization, methylation, and dehydration to afford

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Scheme 1. Plausible biosynthetic pathway of compounds 1 and 2.

All isolated compounds were subjected to biological tests for antibacterial activity against B. cereus and cytotoxicity against cancerous (MCF-7, KB, NCI-H187) and non-cancerous (Vero) cells. Compounds 2 and (22E)-5α,8α-epidioxyergosta-6,22-dien-3β-ol exhibited anti-B. cereus with IC50 values of 12.50 and 3.13 µg/mL, respectively. Both compounds showed cytotoxicity

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ACCEPTED MANUSCRIPT against NCI-H187 and Vero cells with IC50 values of 30.51, 24.24 and 21.00, 15.76 µg/mL, respectively. Compound 1 was inactive against B. cereus at maximum tested concentration (50 µg/mL) but displayed cytotoxicity against NCI-H187 and Vero cell lines with IC50 values of 17.78 and 31.50 µg/mL, respectively. Cytochalasin D showed strong cytotoxicity against Vero cell (IC50

for all biological assays at maximum tested concentration.

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0.58 µg/mL) but weak cytotoxicity against NCI-H187 (IC50 23.51 µg/mL). The rest were inactive

Isopimarane derivatives (3 – 5), (R)-(-)-5-methoxycarbonylmellein, (-)-piliformic acid, and

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cytochalasin D have generally been distributed in the Xylariaceae. 16-Amino-isopimar-7-en-19-oic acid (3) was originally obtained from the fungus Xylaria sp. 290 and was inactive against HeLa

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(human cervical carcinoma) cell lines.6

Isopimarane diterpene glycosides, 16-α-D-

glucopyranosyloxyisopimar-7-en-19-oic acid (4) and 16-α-D-mannopyranosyloxyisopimar-7-en19-oic acid (5), were formerly isolated from the culture of Xylaria polymorpha NBRC33288 and showed antitumor activity against several cell lines, including HL60 (human promyelocytic

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leukemia), K562 (human erythromyeloblastoid leukemia), HeLa (human cervical carcinoma), LNCaP (human prostate adenocarcinoma), A549 (human lung cancer) and SGC7901 (human

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gastric cancer) cell lines, with IC50 ranging from 71 to >200 µM.7,20 Ergostane-type sterols, including ergosterol, (22E)-5α,8α-epidioxyergosta-6,22-dien-3β-ol,

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cerevisterol, and 7-dechlorogriseofulvin were previously isolated from various species of genus Aspergillus such as A. fumigatus,21 A. awamori,22 A.versicolor.23,24 They possessed a variety of biological activities, for instance, ergosterol derivatives exhibited a broad range of antibacterial activity (MIC 50 − >200 µg/mL) against Agrobacterium tumefaciens, Escherichia coli, Pseudomonas lachrymans, Ralstonia solanacearum, Xanthomonas vesicatoria, Bacillus subtilis, Staphylococcus aureus, and Staphylococcus haemolyticus, antifungal activity (IC50 82.0 − 126.9 µg/mL) against Magnaporthe oryzae, and antituberculosis (MIC 1 − >128 µg/mL) against Mycobacterium tuberculosis strain H37Rv.25-27 Ergostane-type sterols were active against various 11

ACCEPTED MANUSCRIPT human cancer cell lines (IC50 7 − 80 µM) such as HT-29 (human colon carcinoma), HepG-2 (human hepatoblastoma), PC-3 (human prostate cancer) cell lines.28,29

(22E)-5α,8α-

epidioxyergosta-6,22-dien-3β-ol was also weakly inhibited growth of phytopathogenic fungus Alternaria solani (MIC 50 µM).30

In addition, cerevisterol also had a broad range of anti-

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phytopathogenic activity (MIC 12.5 − 50.0 µM) against Botrytis cinerea, Glomerella cingulate, Fusarium graminearum, Alternaria solani, and Gibberella saubinetti, displayed significant antioxidant activity against DPPH radical (IC50 11.4 µM) and showed promising anti-inflammatory

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activity on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in mice (ID50 0.67 µM/ear).30-32 Cerevisterol was also strongly active against menogaril-resistant mouse leukemia

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(P388) cell lines (IC50 0.12 µM).33 Moreover, 7-dechlorogriseofulvin displayed a broad range of antifungal activity (EC50 28.5 − >100 µg/mL) towards several plant pathogenic fungi.34

3. Conclusion

known

compounds,

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Two new polyketide derivatives, allahabadolactones A (1) and B (2), together with ten including

16-amino-isopimar-7-en-19-oic

acid

(3),

16-α-D-

acid

(5),

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glucopyranosyloxyisopimar-7-en-19-oic acid (4), 16-α-D-mannopyranosyloxyisopimar-7-en-19-oic ergosterol,

(22E)-5α,8α-epidioxyergosta-6,22-dien-3β-ol,

(R)-(-)-5-

(-)-piliformic acid, 7-dechlorogriseofulvin, and cytochalasin D, were

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methoxycarbonylmellein,

cerevisterol,

isolated from the endophytic fungus, Aspergillus allahabadii BCC45335. Only allahabadolactone B (2) and (22E)-5α,8α-epidioxyergosta-6,22-dien-3β-ol showed antibacterial activity against B. cereus with respective IC50 values of 12.50 and 3.13 µg/mL, while others were inactive at the maximum tested concentration (50 µg/mL). Compounds 1, 2, (22E)-5α,8α-epidioxyergosta-6,22dien-3β-ol, and cytochalasin D displayed cytotoxicity against NCI-H187 and Vero cells with IC50 values in a range of 0.58 − 30.51 µg/mL.

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ACCEPTED MANUSCRIPT 4. Experimental section 4.1. General experimental procedures Melting points were obtained from a melting point M565 apparatus from Buchi. Specific rotations were measured on JASCO P-1030 digital polarimeter. UV spectra were taken in MeOH

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on a Spekol 1200, Analytik Jena, spectrophotometer. FTIR spectra were done on a Bruker ALPHA spectrometer. NMR spectra were recorded on either Bruker Avance-III 400 (400 MHz for 1H and 100 MHz for

C) or Bruker Avance 500 (500 MHz for 1H and 125 MHz for

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C) NMR

X-ray diffraction data were collected on a Bruker–Nonius kappaCCD

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spectrometers.

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diffractometer with graphite monochromated MoKα radiation (λ = 0.71073 Å) at 298(2) K.

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HRESIMS data were detected on a Bruker MicrOTOF mass spectrometer. Analytical reversed phase HPLC (Merck LiChroCART C18, 3 µm, diam. 55 mm × 2 mm, eluted with a linear gradient system of 0 − 100% MeCN in water, containing 0.05% formic acid, at the flow rate 0.5 mL/min over 15 min) was used to analyze purity of pure compounds. Preparative HPLC was carried out by

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using reverse phase column (SunFire C18 OBD, 10 µm, diam. 19 mm × 250 mm) at the flow rate 15 mL/min on a Dionex – Ultimate 3000 series equipped with a binary pump, an autosampler, and

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Merck.

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diode array detector. Preparative thin layer chromatography was done on silica gel 60 GF254 from

4.2. Fungal material

The endophytic fungus was isolated from the root of Cinnamomum subavenium Miq. at

Khao Yai National Park, Nakhon Ratchasima Province, Thailand by Ms. Sujinda Sommai (Mycology Laboratory, BIOTEC).

The fungus was later identified by Dr. Nattawut Boonyuen

(Mycology Laboratory, BIOTEC) as Aspergillus allahabadii based on the analyses of the partial sequence 18S ribosomal RNA gene, internal transcribed spacer 1, 5.8S ribosomal RNA gene, internal transcribed spacer 2, and partial sequence 28S ribosomal RNA gene. Its taxonomic identity

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ACCEPTED MANUSCRIPT was in agreement with Ascomycota, Pezizomycotina, Eurotiomycetes, Eurotiomycetidae, Eurotiales, Trichocomaceae, with 99% identity of nucleotide BLAST scores from GenBank (NCBI+EMBL+DDBJ+PDB). The fungus was registered as BCC45335 and deposited at BIOTEC Culture Collection (BCC), the National Center for Genetic Engineering and Biotechnology

GenBank with accession number KR189024.

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4.3. Fermentation, extraction, and isolation

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(BIOTEC), Thailand. The sequences of the fungal strain BCC45335 have been submitted to

The fungus was grown on potato dextrose agar (PDA) plates at 25 oC. The agar was then

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cut into pieces (1 x 1 cm2) and then transferred into 8 × 250 mL Erlenmeyer flasks, which each contained 25 mL of potato dextrose broth (PDB; containing potato starch 4.0 g/L, dextrose 20.0 g/L in distilled water). The seed culture was inoculated at 25 oC on a rotary shaker at 200 rpm for 7 days. Each 250 mL Erlenmeyer flask was then transferred equally into 4 × 1 L Erlenmeyer flasks,

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which each contained 250 mL of peptone-yeast extract-glucose medium (PYGM; containing bactopeptone 5.0 g/L, yeast extract 20 g/L, glucose 10 g/L, KH2PO4 1.0 g/L, MgSO4·7H2O 0.5 g/L in

25 oC.

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distilled water). The production culture (20 L) was cultivated under static condition for 26 days at

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After cultivation period, the mycelium and broth were separated by simple filtration. The cell was macerated in MeOH (1 L) for 3 days and consecutively in CH2Cl2 (1 L) for 3 days. The organic solvents were then combined and evaporated using rotary evaporator. Water (100 mL) was added and the mixture was then extracted three times with equal volume of n-hexane, followed by three times with equal volume of EtOAc. n-Hexane and EtOAc were separately dried over Na2SO4 and concentrated by a rotary evaporator to provide crude extracts from n-hexane (a brown gum, 2.5 g) and from EtOAc (a brown gum, 1.4 g).

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ACCEPTED MANUSCRIPT The crude extract from n-hexane (2.5 g) was fractionated on a Sephadex LH20 column (3.5 × 28 cm2), eluted with 100% MeOH, to provide 9 fractions. The third fraction (1.5 g) was precipitated from MeOH to give ergosterol (17.5 mg) and the filtrate was then purified by a preparative HPLC, using a linear gradient system of 80 − 100% aqueous MeCN in 40 min, to afford

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14 fractions (3.1 – 3.14). Cerevisterol (42.7 mg) and (22E)-5α,8α-epidioxyergosta-6,22-dien-3β-ol (74.3 mg) were obtained from the fractions 3.10 and 3.14, respectively. Purification of the fraction 3.2 (43.1 mg) by a preparative HPLC, using a linear gradient system of 30 − 70% aqueous MeCN

SC

in 40 min furnished compound 1 (21.4 mg). The fourth fraction (0.1 g) was precipitated from MeOH to give ergosterol (13.1 mg) and the filtrate was further separated by a preparative HPLC,

M AN U

eluted with a linear gradient system of 40 − 100% aqueous MeCN in 50 min to furnish 16-aminoisopimar-7-en-19-oic acid (3, 4.9 mg), compounds 1 (2.5 mg), 2 (3.5 mg), and (22E)-5α,8αepidioxyergosta-6,22-dien-3β-ol (7.8 mg), respectively. Ergosterol (13.0 mg) was also obtained from the fifth fraction (63.8 mg) by precipitation with MeOH and the filtrate was further subjected

TE D

to a preparative HPLC, using linear gradient elution of 10 − 70% aqueous MeCN in 40 min to yield (R)-(-)-5-methoxycarbonylmellein (2.1 mg) and 16-amino-isopimar-7-en-19-oic acid (3, 3.8 mg).

EP

Moreover, (R)-(-)-methoxycarbonylmellein (1.6 mg) was also obtained from the sixth fraction (29.1 mg) after purified by a preparative HPLC, eluted with a linear gradient system of 45 − 55%

AC C

aqueous MeCN in 30 min.

The crude from EtOAc (1.4 g) was passed through a Sephadex LH20 column (3.5 × 28

cm2), using 100% MeOH as an eluent to provide 13 fractions.

The fourth fraction (0.4 g) was

precipitated from MeOH to afford cerevisterol (12.0 mg) and the filtrate was then purified by a preparative HPLC, using linear gradient elution of 10 − 60% aqueous MeCN in 50 min to furnish 23 fractions (4.1 – 4.23).

Cerevisterol (1.6 mg) was also obtained from the fraction 4.2.

Purification of the fraction 4.18 (33.3 mg) by a preparative HPLC, eluted with 40% aqueous MeCN for 30 min provided 2 fractions (4.18.1 – 4.18.2). (-)-Piliformic acid (3.2 mg) was obtained from 15

ACCEPTED MANUSCRIPT the fraction 4.18.1. Cytochalasin D (2.3 mg) was given from the fraction 4.18.2 after further purification by a preparative HPLC, using isocratic elution (35% aqueous MeCN) for 50 min. The fifth fraction (0.2 g) was subjected to a preparative HPLC, using a linear gradient system of 10 − 55% aqueous MeCN in 40 min, to afford 16-α-D-glucopyranosyloxyisopimar-7-en-19-oic acid (4,

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6.0 mg), 16-α-D-mannopyranosyloxyisopimar-7-en-19-oic acid (5, 3.7 mg), and (-)-piliformic acid (13.5 mg), respectively. The sixth fraction (0.2 g) was further purified by the same protocol as for the fifth fraction to yield 7-dechlorogriseofulvin (1.5 mg), 16-α-D-mannopyranosyloxyisopimar-7-

SC

en-19-oic acid (5, 3.2 mg), and (-)-piliformic acid (1.6 mg), respectively. 4.3.1. Allahabadolactone A (1)

M AN U

Colourless crystals (from CH2Cl2/hexane); m.p. 117.3 − 118.7 °C; tR 7.15; [α]27D +26.6 (c 0.13, CHCl3); UV (CHCl3) λmax nm (log ε): 260 (2.88); FTIR (ATR) νmax: 3413, 3023, 2922, 2851, 1770, 1455, 1377, 1312, 1156, 1094 cm-1; 1H and

13

C NMR data see Table 1; HRESIMS at m/z

313.1764 [M+Na]+ (calcd for C18H26O3Na, 313.1774).

TE D

4.3.2. Allahabadolactone B (2)

Colourless semi-solid; tR 7.63; [α]27D +55.7 (c 0.18, CHCl3); UV (CHCl3) λmax nm (log ε):

cm-1; 1H and

13

EP

252 (2.72); FTIR (ATR) νmax: 3439, 3025, 2921, 2851, 1769, 1633, 1455, 1378, 1312, 1157, 1092 C NMR data see Table 1; HRESIMS at m/z 313.1784 [M+Na]+ (calcd for

AC C

C18H26O3Na, 313.1774).

4.3.3 X-ray crystallographic analysis of 1. C18H26O3, M = 290.39, tetragonal, space group P41212 (No. 92), a = 8.1100(1) Å, b = 8.1100(1) Å, c = 50.8500 (1) Å, V = 3344.51(11) Å3, Z = 8, T = 298(2) K, µ(Mo Kα) = 0.077 mm-1, Dx = 1.153g/cm3, reflections measured/unique: 5326/1953, number of observations [I > 2σ (I)] 1533. The final R indices [I > 2σ (I)]: R1 = 0.0634 and wR2 was 0.1978 (all data). The structure was solved using the direct methods with SIR9735 and refined with SHELXL refinement package36. Crystallographic data have been deposited at the Cambridge Crystallographic Data Centre under the reference number CCDC 1431334. Copies of the data can

16

ACCEPTED MANUSCRIPT be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (e-mail: [email protected]).

4.4. Preparation of the MTPA esters (1a and 1b)

RI PT

Compound 1 (1.0 mg) was treated with (R)-MTPACl (30 µL) in CH2Cl2 (0.2 mL) and pyridine (0.2 mL) at room temperature and left stirring for 16 h. The mixture was diluted with EtOAc and then washed with H2O. The organic layer was dried over MgSO4 and then evaporated

SC

to dryness under reduced pressure. The crude mixture was purified by semi-preparative HPLC (SunFire C18 OBD, diam. 5 µm, diam. 19 × 150 mm2), eluted with a linear gradient system of 25 −

M AN U

95% aqueous MeCN in 40 min at the flow rate 9 mL/min, to provide (S)-MTPA ester (1a, 2.2 mg). Using the same protocol as for 1a, (R)-MTPA ester (1b, 2.3 mg) was also prepared from compound

4.5. Biological assays

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1 (1.0 mg) and (S)-MTPACl (30 µL).

Cytotoxicity against cancer cells including MCF-7 (human breast cancer), KB (human oral epidermoid carcinoma), and NCI-H187 (human small-cell lung cancer) cells and antibacterial

EP

activity against B. cereus were performed by using the resazuin microplate assay (REMA).37

AC C

Ellipticine and doxorubicin were used as the standard references for cytotoxicity against KB and NCI-H187 cells and displayed IC50 values of 2.34 – 2.97, 2.32 – 2.70 and 1.22 – 1.70, 0.070 – 0.095 µg/mL, respectively. Doxorubicin and tamoxifen as the standard references for anti-MCF-7 showed IC50 values of 8.28 – 9.55 and 8.97 – 9.73 µg/mL, respectively. Vancomycin as the standard reference for antibacterial activity against B. cereus exhibited IC50 value of 1.00 – 2.00 µg/mL. Cytotoxicity against non-cancerous cell (Vero, African green monkey kidney fibroblasts) was evaluated by using the green fluorescent protein microplate assay (GFPMA)38 and ellipticine as

17

ACCEPTED MANUSCRIPT the standard reference exhibited IC50 value of 0.87 – 2.38 µg/mL. Maximum tested concentration was done at 50 µg/mL for all tests.

Acknowledgements

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Financial support from the Bioresources Research Network (Grant number BRN 003 G-56), National Center for Genetic Engineering and Biotechnology (BIOTEC) is gratefully acknowledged.

SC

PK thanks Mahidol University and Center for Innovation in Chemistry for financial support.

Supplementary data

M AN U

Supplementary data include NMR spectra of compounds 1 and 2, described in the article.

AC C

EP

TE D

These data can be found in online version at http://dx.doi.org/ xxx.

18

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