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Cytotoxic cochlioquinone derivatives from the endophytic fungus Bipolaris sorokiniana derived from Pogostemon cablin
Cytotoxic cochlioquinone derivatives from the endophytic fungus Bipolaris sorokiniana derived from Pogostemon cablin Mo Wang, Zhang-Hua Sun, Yu-Chan Chen, Hong-Xin Liu, Hao-Hua Li, Guo-Hui Tan, Sai-Ni Li, Xiao-Ling Guo, Wei-Min Zhang PII: DOI: Reference:
S0367-326X(16)30022-3 doi: 10.1016/j.fitote.2016.02.005 FITOTE 3352
To appear in:
Fitoterapia
Received date: Revised date: Accepted date:
27 December 2015 3 February 2016 4 February 2016
Please cite this article as: Mo Wang, Zhang-Hua Sun, Yu-Chan Chen, Hong-Xin Liu, Hao-Hua Li, Guo-Hui Tan, Sai-Ni Li, Xiao-Ling Guo, Wei-Min Zhang, Cytotoxic cochlioquinone derivatives from the endophytic fungus Bipolaris sorokiniana derived from Pogostemon cablin, Fitoterapia (2016), doi: 10.1016/j.fitote.2016.02.005
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ACCEPTED MANUSCRIPT Cytotoxic Cochlioquinone Derivatives from the Endophytic Fungus Bipolaris sorokiniana Derived
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from Pogostemon cablin
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Mo Wanga,b,†, Zhang-Hua Suna,†, Yu-Chan Chena, Hong-Xin Liua, Hao-Hua Lia,
a
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Guo-Hui Tana, Sai-Ni Lia, Xiao-Ling Guob, Wei-Min Zhanga,*
State Key Laboratory of Applied Microbiology Southern China, Guangdong
Provincial Key Laboratory of Microbial Culture Collection and Application,
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Guangdong Open Laboratory of Applied Microbiology, Guangdong Institute of Microbiology, Xianlie Road 100, Guangzhou 510070, China Guangdong Pharmaceutical University, Guangzhou 510006, China
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b
ABSTRACT
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Chemical investigation of the liquid culture of the endophytic fungus Bipolaris
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sorokiniana A606, which was isolated from the medicinal plant Pogostemon cablin
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resulted in the isolation of four new cytotoxic compounds, named isocochlioquinones DE (12) and cochlioquinones GH (34), along with five known cochlioquinone analogues (59). Their structures were determined on the basis of extensive
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spectroscopic analysis. Isocochlioquinone D (1) possessed a rare benzothiazin-3-one moiety and cochlioquinone G (3) was the first example of cochlioquinones bearing an indole-4,7-dione fragment. All of the isolates (19) were evaluated for their cytotoxic activities against MCF-7, NCI-H460, SF-268 and HepG-2 tumor cell lines by the sulforhodamine B (SRB) assay. Compounds 4 and 69, featuring a cochlioquinone core, exhibited potent cytotoxicities in vitro against the four tumor cell lines, and a preliminary structure-activity relationship of these compounds was also discussed. Keywords: Bipolaris
sorokiniana;
endophytic fungus;
Pogostemon
cochlioquinones; cytotoxic activity
Corresponding author. tel: +86 020 87688309. fax: +86 020 87688612 (W.M. Zhang) E-mail address: [email protected] (W.M. Zhang) † These authors contributed equally to this work.
cablin;
ACCEPTED MANUSCRIPT 1. Introduction Endophytic fungi that reside in plants are promising sources of a variety of bioactive metabolites. These metabolites are usually structurally novel and display
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important biological or pharmaceutical properties, such as antimicrobial or cytotoxic
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activities [1-3]. In the past decades, less than 30 cochlioquinones have been isolated
These
cochlioquinones,
characterized
by
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from phytopathogenic fungi, mainly the genera Bipolaris and Stachybotrys [4-5]. a
1-alkylated-3,5-dihydroxyphenyl
derivative couple with a farnesyl-OPP unit [6], were key fungal phytotoxins
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associated with severe diseases of cereal crops [7] and exhibited interesting biological properties such as inhibition of cholesterol acyltransferase [8], phytotoxic [9, 10],
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cytotoxic [11], anti-angiogenic [12] and antibiotic activities [13]. During our continuing search for structurally diverse and biologically significant metabolites from endophytic fungal strains [12-14], a fraction of the broth extract of
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the fungus Bipolaris sorokiniana A606 derived from the medicinal plant Pogostemon
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cablin showed an antiproliferative activity against HepG-2 cell line with an inhibition
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rate of 96.2% at a concentration of 100 g/mL1. Subsequent chemical investigation on the endophytic fungal strain A606 resulted in the isolation of four new cochlioquinones, named isocochlioquinones DE (12) and cochlioquinones GH
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(34) (Figure 1), as well as five known analogues. Isocochlioquinone D (1) possessed a rare benzothiazin-3-one moiety and cochlioquinone G (3) was the first example of cochlioquinones bearinganindole-4,7-dione fragment. All of the isolates (19) were evaluated for their cytotoxic activities against MCF-7, NCI-H460, SF-268 and HepG-2 tumor cell lines by the sulforhodamine B (SRB) assay. Herein, the isolation, structural elucidation, cytotoxic activity, and a preliminary structure-activity relationship (SAR) of these compounds are described. 2. Materials and methods 2.1. General experimental procedures Optical rotation was measured on an Anton Paar MCP-500 spectropolarimeter. The IR spectrum was recorded on an IRAffinity-1 spectrophotometer in cm-1. UV spectra were measured on a SHIMADZU UV-2600 UV-visible spectrophotometer. 1D
ACCEPTED MANUSCRIPT and 2D NMR spectra were recorded on a Bruker Avance-500 spectrometer with TMS as internal standard, δ in ppm, J in Hz. HRESIMS was measured on a Thermo MAT95XP high-resolution mass spectrometer and ESIMS was measured on an
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Agilent Technologies 1290-6430A Triple Quad LC/MS. All solvents were of analytical grade (Guangzhou Chemical Reagents Company, Ltd.). Silica gel (200-300
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mesh) was used for column chromatography, and precoated silica gel GF254 plates (Qingdao Marine Chemical Inc. Qingdao, China) were used for TLC spotting. C18 reversed-phase silica gel (40-63 µm, Merck, German), and Sephadex LH-20 gel
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(Pharmacia Fine Chemical Co. Ltd., Sweden) were also used for column chromatography (CC).A Shimadzu LC-20AT equipped with a SPD-M20A PDA
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detector was used for HPLC, and a YMC-pack ODS-A column (250×10 mm, S-5μm, 12nm) was used for semi-preparative HPLC separation. TLC spots were visualized
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under UV light and by dipping into 10% H2SO4 in alcohol followed by heating.
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2.2. Fungal material
The endophytic fungal strain Bipolaris sorokiniana A606 was isolated from the
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plant of Pogostemon cablin, which was collected at Gaoyao, Guangdong province, China, in October, 2012. The strain was identified by sequence analysis of rDNA ITS (internal transcribed spacer) region. The sequence of the ITS region of the
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B.sorokiniana A606 has been submitted to GenBank (Accession No. KF494823). By using BLAST (nucleotide sequence comparison program) to search the GenBank database, B.sorokiniana A606 has 99.6% similarity to B.sorokiniana B41 (Accession No. KF725816). The strain was preserved at the Guangdong Provincial Key Laboratory of Microbiol Culture Collection and Application, Guangdong Institute of Microbiology. 2.3. Fermentation, extraction and isolation B.sorokiniana was grown on potato-dextrose agar (PDA) medium at 28 C for 5 days and then inoculated into 5 flasks (500 mL) containing potato-dextrose (PD) medium (250 mL). After 5 days of incubation at 28 C on a rotary shaker at 120 r/m, a portion of the liquid culture was aseptically transferred into each of a total of 240 flasks (1000 mL) containing PDA medium (500 mL). Following 7 days of cultivation
ACCEPTED MANUSCRIPT at 28 C and 120 r/m on a rotary shaker, the culture (total of 120 L) was filtered to give the filtrate and mycelia. The broth was exhaustively extracted with EtOAc (3 4.5 L), and then the
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EtOAc layers were combined and evaporated under reduced pressure at a temperature
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not exceeding 40 C to yield a crude extract (18.8 g). The EtOAc extract was
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separated into twenty-six fractions (Fr.1-Fr.26) by silica column chromatography eluted with a gradient of PE/EtOAc (v/v, 10:0→0:10) based on TLC monitoring. Fr.8 was further separated on a reversed phase ODS column with a gradient of MeOH/H2O
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(v/v, 3:7→10:0) to yield compound 9 (3.7 mg). Fr.9 was further separated on a reversed phase ODS column with a gradient of MeOH/H2O (v/v, 4:6→10:0) to yield
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compound 6 (4.2 mg) and 4 (6.3 mg). Fr.10 was separated by Sephadex LH-20 chromatography eluting with CH2Cl2/MeOH (1:1, v/v), followed by further
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purification on a semi-preparative HPLC (MeCN/H2O, 4:6→ 10:0, 3 mL/min) to
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yield compound 3 (3.0 mg), 5 (4.6 mg), and 7 (7.3 mg). Fr.11 was separated by Sephadex LH-20 chromatography eluting with CH2Cl2/MeOH (1:1, v/v), followed by
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further purification on a semi-preparative HPLC (MeCN/H2O, 3:7→ 10:0, 3 mL/min) to yield compound 1 (3.2mg), 8 (4.5mg), and 2 (3.0 mg). 2.4. Spectroscopic data
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Isocochlioquinone D (1): yellow oil; [α]25D 236.9 (c 0.1, EtOH); UV (EtOH) λmax (log ε) 220 (4.28), 260 (4.00), 306 (3.58) nm; IR (KBr) νmax 3352, 3275, 2947, 2935, 2874, 1661, 1016 cm–1. 1H (500 MHz) and
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C (125 MHz) NMR data, see Tables 1
and 2; HRESIMS m/z 560.2681 [M H] (calcdfor C30H42NO7S, 560.2682). Isocochlioquinone E (2): yellow oil; [α]25D 253.9 (c 0.1, EtOH); UV (EtOH) λmax (log ε) 214 (4.25), 284 (4.02), 383 (3.44) nm; IR (KBr) νmax 3402, 2972, 2931, 2870, 1643, 1634, 1361, 1188, 1097, 1047 cm–1. 1H (500 MHz) and
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C (125 MHz) NMR
data, see Tables 1 and 2; HRESIMS m/z 485.2543 [M H] (calcd for C28H37O7, 485.2539). Cochlioquinone G (3): red oil; [α]25D 142.5 (c 0.1, EtOH); UV (EtOH) λmax (log ε) 203 (4.22), 238 (4.42), 294 (4.25) nm; IR (KBr) νmax 3483, 3321, 2957, 2926, 2855, 1674, 1622, 1599, 1205, 1093 cm–1. 1H (500 MHz) and
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C (125 MHz) NMR data,
ACCEPTED MANUSCRIPT see Tables 1 and 2; HRESIMS m/z 486.2878 [M H] (calcd for C28H40NO6, 486.2856). Cochlioquinone H (4): red oil; [α]25D 69.4 (c 0.1, EtOH); UV (EtOH) λmax (log ε)
13
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1095 cm–1. 1H (500 MHz) and
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203 (3.92), 268 (3.79) nm; IR (KBr) νmax 3456, 2961, 2926, 2855, 1713, 1651, 1099, C (125 MHz) NMR data, see Tables 1 and 2;
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HRESIMS m/z 471.2753 [M H] (calcd for C28H39O6, 471.2747). 2.5. Cytotoxicity assay
The cell growth inhibitory activities of compounds 19 against human cancer
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cell lines SF-268, MCF-7, NCI-H460, and HepG-2, were tested using the previously published method [17].
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3. Results and discussion
The broth of B.sorokiniana A606 was extracted with EtOAc. The EtOAc extract
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was subjected to various column chromatography protocols to afford compounds 19.
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The structures of new compounds were identified by spectroscopic analysis and physicochemical properties, while the known compounds were assigned as
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isocochlioquinone C (5) [7], cochlioquinone C (6) [7], cochlioquinone D (7) [13], cochlioquinone E (8) [18] and cochlioquinone B (9) [8] by comparison of their spectroscopic data with those in the literatures.
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Compound 1 was obtained as a yellow oil and gave a [M H] peak at m/z 560.2681 in the HRESIMS for a molecular formula of C30H43NO7S (calcd for C30H42NO7S 560.2682), requiring ten sites of unsaturation. The 1H NMR spectrum of 1 displayed signals for seven methyl groups [δH 1.31 (d, J = 6.9 Hz, H3-27), 1.23 (s, H3-26), 1.18 (s, H3-24), 1.17 (s, H3-23), 1.05 (s, H3-25), 0.91 (d, J = 6.9 Hz, H3-28) and 0.83 (t, J = 7.4 Hz, H3-1)], three oxymethine protons [δH 4.95 (t, J = 10.2 Hz, H-12), 3.18 (dd, J = 3.8,12.0 Hz, H-17), and 3.25 (m, H-21)], and a series of aliphatic methylene multiplets. The 13C NMR spectrum, in combination with DEPT experiment, showed 30 carbon resonances attributable to two carbonyls, seven methyls, six sp3methylenes, six methines (three oxygenated), and nine quaternary carbons (two oxygenated sp3 and six sp2). Analysis of the NMR spectra (Tables 1 and 2), including the 1H−1H COSY, HMBC, and NOESY spectra, implied that 1 consisted of two
ACCEPTED MANUSCRIPT partial structures (units A and B), which were presumed to be structurally related to cochlioquinone C (6) and a rare benzothiazin-3-one moiety [19-21], respectively. The gross structures of units A and B were elucidated as follows (Figure 2). The unit A
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(C-12−C-26) was identified to be the same as that part of cochlioquinone C (6). In unit B (C-1−C-11 and C-27C-30), the existence of a persubstituted benzene ring
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(C-6−C-11) was deduced by the 1D NMR data (δC 139.7, 139.5, 129.1,125.5,113.5, 113.1), while two aliphatic methylene signals at δH 3.37 and 3.25 and a carbonyl signal at δC 165.3 implied the presence of a benzothiazin-3-one moiety (C-6−C-11,
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and C-29C-30). This was also supported by HMBC correlations from a methylene (δH 3.37 and 3.23, H2-29) to a sp2-hybridized quaternary carbon (δC 113.1, C-11) and
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the carbonyl group (δC 165.3, C-30), and from NH (δH 9.52) to C-29 (δC 30.3), C-9 (δC 113.5), and C-11 (δC 113.1), as well as from OH-7 (δH 5.80) to C-6 (δC 125.5), C-7 (δC
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139.7), and C-8 (δC 139.5) and from H-5 to C-6, C-7, and C-11. The connectivity of
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A/B moieties was assigned as depicted by HMBC correlations from H-12 to C-8, C-9, and C-10. Thus, the planar structure of 1 was assigned as depicted.
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The relative configuration of 1 was deduced on the basis of NOESY experiment (Figure 3). The NOE correlations of H-21/H-19α, H-17/H-19α and H-17/H-13 suggested that the four protons were cofacial, and arbitrarily assigned as α-orientation.
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Moreover, the informative NOE interactions observed between H3-25/H-12, H-12/H3-26, H-12/H-19β, indicated the β-configurations of Me-25 and Me-26, and the moiety at C-21. Considering the same biosynthetic pathway for this kind of compound, the relative configuration in the aliphatic side chain C-1−C-5 of 1 was assumed to be identical to those of known analogues 59. Thus, the structure of 1 was established and given a trivial name isocochlioquinone D. Compound 2 was isolated as a yellow oil that gave a [M H] ion at m/z 485.2543 in the HRESIMS consistent with a molecular formula of C28H38O7 (calcd for C28H37O7, 485.2539), which was 2 mass units less than that of 5. The 1H and 13C NMR spectra of 2 were similar to those of isocochlioquinone C (5) except for the presence of double bond at C-2 [δC 138.2 and 138.1; δH 6.87 (1H, d, J = 6.8 Hz)] in 2, indicating that 2 was a 2,3-didehydrogenated derivative of 5. This was supported by
ACCEPTED MANUSCRIPT the significant downfield-shifted H-2 signal in 2 with respect to that in 5 (δH 6.87 in 2; δH 1.76 and 1.65 in 5) and the upfield-shifted carbonyl at C-4 (15 ppm). The planar structure of 2 was further established by detailed interpretation of its 2D NMR data.
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The relative configuration of 2 was identified to be the same as that of 5 on the basis of NOESY correlations and comparison of their 1D NMR data. Thus, compound 2
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was assigned as depicted and named isoochlioquinone E.
Compound 3 was obtained as a red oil that gave a [M H] ion at m/z 486.2878 in the HRESIMS consistent with a molecular formula of C28H39NO6 (calcd for
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C28H40NO6, 486.2856), requiring ten sites of unsaturation. The 1D NMR spectra of 3 were similar to those of 6 except for the presence of an additional tetrasubstituted
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double bond (δC 143.1 and 118.8) and absence of both the carbonyl (δC 212.2 at C-4 in 6) and aromatic proton (δH 6.48 at H-11 in 6), implying 3 was an analogue of 6. As
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nine of the ten degrees of unsaturation were accounted for a cochlioquinone core and
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a tetrasubstituted double bond, the remaining unsaturation unit required that 3 had one more ring than that of 6. The aforementioned information implied that 3 was bearing
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an indole-4,7-dione unit [22]. This was confirmed by the COSY correlations of H-1/H-2/H-3/H-28 and HMBC correlations from H-3 (δH 2.83) to C-4 (δC 143.1), C-5 (δC 118.8), and C-27 (δC 10.0), and from H-27 (δH 2.21) to C-4, C-5, and C-6 (δC
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122.5). The planar structure of 3 was further confirmed by molecular formula of C28H39NO6.
The relative configuration of 3 was deduced by analyzing its NOESY spectrum. Diagnostic NOE correlations of H-17/H-19α, H-21 and H-13 positioned them on the same side and established the cis relationships of these protons. On the other hand, NOE correlations of H3-25/H-19β, and H-12 and H-12/H3-26 indicated that these protons were on the other face of the molecule (Figure 4). The structure of 3 was established and given the trivial name cochlioquinone G. Compound 4, a red oil, had the molecular formula C28H38O6, as determined by HRESIMS. The 1H and 13C NMR data of 4 were similar to those of co-isolated known compound cochlioquinone B (9), except for the presence of ∆12,13 double bond (δC 147.7 and 110.5) in 4. The trisubstituted double bond at C-12C-13 was deduced from
ACCEPTED MANUSCRIPT HMBC correlations for both two methyl protons [δH 1.09 (s, H3-25) and 1.50 (s, H3-26)] to the olefinic carbon [δC 147.7 (C-13)]. Diagnostic ROESY correlations positioned H-17 and H-21 on the same () face of the heterocyclic scaffold, and both
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H3-25 and H3-26 on the face, and defined the relative configuration for 4. Therefore, the structure of 4 was established and given the trivial name cochlioquinone H.
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Compounds 19 were evaluated for their in vitro cytotoxic activities against four human tumor cell lines, including SF-268 (human glioma cell line), MCF-7 (human breast adenocarcinoma cell line), NCI-H460 (human non-small cell lung cancer cell
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line) and HepG-2 (human hepatoma cell line) by the SRB method, and cisplatin was used as the positive control (Table 3). As a result, all compounds showed obvious
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cytotoxic effects against the four tumor cell lines, and compounds 69, sharing the quinone structural features, showed broad inhibitory effects against the cell lines, with
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the activities comparable to those of cisplatin (most IC50s<10 μM). However, selective
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cytotoxicity was observed within this sub-class. Compounds 7 showed excellent inhibitory activities against SF-268 and HepG-2 cell lines with IC50 values of 1.5 and
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1.2 μM, respectively, implying that the dehydrogenation between C-2 and C-3 might contribute the selectivity to SF-268 and HepG-2 in 7 and 9, while hydroxy at C-12 was important to the selectivity to NCI-H460 in 6 and 9. Compared to 69, the
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introduction of a hydroxy or other substituent group at C-11 (3) greatly decreased their activities.
Interestingly, despite of generally low activity, the isocochlioquinones 12, 5 reserved their anti-proliferative activities and showed selective cytotoxicities against certain tumor cell lines. Compounds 2 selectively inhibited SF-268 and HepG-2 with IC50 values of 17.2 and 13.6 μM, respectively. Further analysis showed the dehydrogenation between C-2 and C-3 and the presence of hydroxy at C-10 in 2 led to more selective cytotoxicity against SF-268 and HepG-2. Previous bioassay of cochlioquinone derivatives indicated the moderate lipophilicity at C-4, a hydroxy residue at C-12 and C-19, and the replaceable at 7,10-quinone part were important for the activity against methicillin-resistant Staphylococcus aureus (MRSA) [23]. In this study, the inhibitory activities of
ACCEPTED MANUSCRIPT cochlioquinones against the four human tumor cell lines also implied the above-mentioned moieties may be important influencing factors on cytotoxic activity. In summary, nine cochlioquinones (19) were isolated from the endophytic
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fungus Bipolaris sorokiniana. Their structures were determined by comprehensive spectroscopic analysis. The isolates were evaluated for their cytotoxicities against
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four human tumor cell lines. Compound 7, possessing the C-2,3 double bond but lacking the C-12 hydroxy, showed excellent activity against SF-268, MCF-7 and HepG-2 cell lines with IC50 values of 1.5, 2.4, and 1.2 μM, respectively, being
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stronger than cisplatin, while 2 and 4 showed selective inhibitory activities against SF-268 and HepG-2. This study not only enriches the chemodiversity of the genus
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Bipolaris by the isolation of a series of cochlioquinones, but also provides scientific basis for the usage of the phytopathogenic fungus B.sorokiniana as a source of
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antineoplastic agents in future.
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Acknowledgments
This work was financially supported by the National Basic Research Program of
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China (973 Program, No. 2014CB460613), National Natural Science Foundation of China (No. 81203006), Natural Science Foundation of Guangdong Province (No. 2015A030313710), and Guangdong Provincial Project for Science and Technology
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(No. 2015A030302060).
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of
some
2H-1,4-benzothiazol-3-one derivatives, Heterocycl Commun. 6 (2000) 49-54. [21] S.D. Cho, J.W. Chung, S.K. Kim, D.H. Kewon, K.H. Park, Y.J. Yoon. Synthesis of
novel
n-acyclonucleosides:
benzotriazole,
benzothiazinone
pyridooxazinone acyclonucleosides, J Heterocycl Chem. 33 (1996) 315-318.
and
ACCEPTED MANUSCRIPT [22] A.S. Eastabrook, C. Wang, E.K. Davison, J. Sperry. A procedure for transforming indoles into indolequinones, J Org Chem. 80 (2015) 1006-1017. [23] H. Yamazaki, N. Koyama, S. Omura, H. Tomoda. Structure-activity relationships
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of stemphones, potentiators of imipenem activity against methicillin-resistant
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Staphylococcus aureus, J Antibiot. 61 (2008) 426-441.
ACCEPTED MANUSCRIPT Table 1. 1H NMR data for compounds 14 (500 MHz, δ in ppm, J in Hz). 1a
2a
3b
4a
1 2
0.83, t (7.4) 1.57, m 1.36, m 2.45, m 4.09, q (6.9)
1.80, d (6.8) 6.87, d (6.8)
0.83, t (7.4) 1.64, m 1.24, m 2.83, dq (13.8, 7.0)
0.83, t (7.4) 1.71, m 1.35, m 2.67, m 4.08, qd (7.2, 1.2) 6.45, d (1.2) 6.29, s
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NH 7-OH 10-OH a
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2.02, m 1.86, m 1.76, m 1.59, m 3.19, dd (11.9, 3.7) 2.41, m 1.46, m 1.64, m 1.43, m 3.25, dd (11.9, 2.7) 1.16, s 1.18, s 1.05, s 1.31, s 2.21, s 1.25, d (7.0)
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20
2.70, m 1.28, m 1.82, m 1.68, m 3.15, dd (11.9, 3.9) 2.10, m 2.03, m 1.64, m 1.45, m 3.25, dd (12.0, 2.7) 1.18, s 1.18, s 1.12, s 1.48, s 1.36, d (6.9) 1.75, s
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17 19
1.95, m 1.85, m 1.60, m 1.46, m 3.18, dd (12.0, 3.8) 2.42, m 1.82, m 1.79, m 1.49, m 3.25, m 1.17, s 1.18, s 1.04, s 1.23, s 1.30, d (6.9) 0.91, d (6.9) 3.37, d (14.7); 3.23, d(14.7) 9.52, brs 5.80, brs
2.76, s
4.94, d (10.2) 1.69, d (10.2)
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4.95, t (10.2) 1.89, d (10.2)
16
21 23 24 25 26 27 28 29
4.78, q (6.9) 6.21, s
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3 5 11 12 13 14 15
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no.
5.52, s 10.78, s
Recorded in CDCl3; brecorded in CD3OD.
2.26, dt (13.3, 3.3) 1.98, dd (13.3, 4.5) 1.77, m 1.71, m 3.14, d (3.2) 2.04, m 1.53, t (3.3) 1.65, dd (5.0, 3.3) 1.56, dd (5.0, 2.2) 3.16, d (3.3) 1.15, s 1.16, s 1.09, s 1.50, s 1.26, d (7.2) 1.11, d (6.9)
ACCEPTED MANUSCRIPT Table 2. 13C NMR data for compounds 14 (125 MHz, δ in ppm, J in Hz). 1a
2a
3b
4a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
11.5, CH3 27.0, CH2 44.7, CH 213.8, C 46.7, CH 125.5, C 139.7, C 139.5, C 113.5, C 129.1, C 113.1, C 65.4, CH 55.3, CH 81.0, C 37.7, CH2 25.3, CH2 83.8, CH 36.7, C 38.6, CH2 21.5, CH2 85.0, CH 71.9, C 23.7, CH3 26.0, CH3 12.6, CH3 21.9, CH3 13.8, CH3 15.5, CH3 30.3, CH2 165.3, C
11.4, CH3 138.2, CH 138.1, C 201.5, C 39.7, CH 133.9, C 137.2, C 144.4, C 107.3, C 153.7, C 106.8, CH 198.2, C 60.3, CH 83.5, C 37.3, CH2 24.9, CH2 83.6, CH 35.4, C 37.6, CH2 21.3, CH2 85.3, CH 71.9, C 23.7, CH3 26.0, CH3 12.3, CH3 22.0, CH3 17.4, CH3 15.0, CH3
12.5, CH3 30.5, CH2 33.7, CH 143.1, C 118.8, C 122.5, C 180.8, C 153.6, C 120.0, C 179.0, C 131.4, C 64.0, CH 54.0, CH 83.5, C 38.6, CH2 26.3, CH2 85.4, CH 37.7, C 39.9, CH2 22.5, CH2 86.5, CH 72.8, C 25.5, CH3 25.5, CH3 13.1, CH3 21.1, CH3 10.0, CH3 20.2, CH3
11.7, CH3 25.7, CH2 47.3, CH 212.6, C 43.0, CH 146.0, C 181.0, C 148.7, C 117.5, C 184.4, C 132.4, CH 110.5, CH 147.7, C 80.9, C 37.7, CH2 24.4, CH2 81.5, CH 38.6, C 34.7, CH2 21.5, CH2 84.4, CH 71.8, C 26.0, CH3 23.7, CH3 20.1, CH3 27.0, CH3 14.8, CH3 16.5, CH3
Recorded in CDCl3; brecorded in CD3OD.
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no.
ACCEPTED MANUSCRIPT Table 3. Antiproliferative activity of the compounds (19) towards cancer cells Cell lines/IC50 SD (μM) Compounds MCF-7
NCI-H460
HepG-2
1
32.8 0.5
28.3 1.1
42.6 2.5
38.7 1.1
2
17.2 1.1
28.1 0.7
31.1 1.1
13.6 0.5
3
35.9 1.3
21.1 0.6
26.9 2.8
11.3 1.9
4
6.4 0.2
8.6 0.2
5
38.4 1.9
32.3 1.0
6
5.3 1.7
5.6 3.4
7
1.5 0.1
2.4 0.2
9.2 0.2
1.2 0.2
8
5.2 0.6
7.3 0.9
24.1 0.9
8.4 0.9
9
7.1 0.2
13.1 1.3
42.8 8.4
7.7 0.1
Cisplatin
4.1 0.2
2.9 0.5
2.9 0.2
2.5 0.2
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7.6 0.1
46.3 0.4
50.6 5.2
13.2 1.7
4.1 3.8
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SF-268
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Figure 1. Structures of compounds 19 isolated from Bipolaris sorokiniana A606.
) and HMBC (
) correlations for compounds 14.
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Figure 2. Key COSY (
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Figure 3. Key NOESY correlations for compounds 14.
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S0367-326X(16)30022-3 doi: 10.1016/j.fitote.2016.02.005 FITOTE 3352
To appear in:
Fitoterapia
Received date: Revised date: Accepted date:
27 December 2015 3 February 2016 4 February 2016
Please cite this article as: Mo Wang, Zhang-Hua Sun, Yu-Chan Chen, Hong-Xin Liu, Hao-Hua Li, Guo-Hui Tan, Sai-Ni Li, Xiao-Ling Guo, Wei-Min Zhang, Cytotoxic cochlioquinone derivatives from the endophytic fungus Bipolaris sorokiniana derived from Pogostemon cablin, Fitoterapia (2016), doi: 10.1016/j.fitote.2016.02.005
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ACCEPTED MANUSCRIPT Cytotoxic Cochlioquinone Derivatives from the Endophytic Fungus Bipolaris sorokiniana Derived
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from Pogostemon cablin
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Mo Wanga,b,†, Zhang-Hua Suna,†, Yu-Chan Chena, Hong-Xin Liua, Hao-Hua Lia,
a
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Guo-Hui Tana, Sai-Ni Lia, Xiao-Ling Guob, Wei-Min Zhanga,*
State Key Laboratory of Applied Microbiology Southern China, Guangdong
Provincial Key Laboratory of Microbial Culture Collection and Application,
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Guangdong Open Laboratory of Applied Microbiology, Guangdong Institute of Microbiology, Xianlie Road 100, Guangzhou 510070, China Guangdong Pharmaceutical University, Guangzhou 510006, China
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b
ABSTRACT
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Chemical investigation of the liquid culture of the endophytic fungus Bipolaris
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sorokiniana A606, which was isolated from the medicinal plant Pogostemon cablin
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resulted in the isolation of four new cytotoxic compounds, named isocochlioquinones DE (12) and cochlioquinones GH (34), along with five known cochlioquinone analogues (59). Their structures were determined on the basis of extensive
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spectroscopic analysis. Isocochlioquinone D (1) possessed a rare benzothiazin-3-one moiety and cochlioquinone G (3) was the first example of cochlioquinones bearing an indole-4,7-dione fragment. All of the isolates (19) were evaluated for their cytotoxic activities against MCF-7, NCI-H460, SF-268 and HepG-2 tumor cell lines by the sulforhodamine B (SRB) assay. Compounds 4 and 69, featuring a cochlioquinone core, exhibited potent cytotoxicities in vitro against the four tumor cell lines, and a preliminary structure-activity relationship of these compounds was also discussed. Keywords: Bipolaris
sorokiniana;
endophytic fungus;
Pogostemon
cochlioquinones; cytotoxic activity
Corresponding author. tel: +86 020 87688309. fax: +86 020 87688612 (W.M. Zhang) E-mail address: [email protected] (W.M. Zhang) † These authors contributed equally to this work.
cablin;
ACCEPTED MANUSCRIPT 1. Introduction Endophytic fungi that reside in plants are promising sources of a variety of bioactive metabolites. These metabolites are usually structurally novel and display
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important biological or pharmaceutical properties, such as antimicrobial or cytotoxic
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activities [1-3]. In the past decades, less than 30 cochlioquinones have been isolated
These
cochlioquinones,
characterized
by
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from phytopathogenic fungi, mainly the genera Bipolaris and Stachybotrys [4-5]. a
1-alkylated-3,5-dihydroxyphenyl
derivative couple with a farnesyl-OPP unit [6], were key fungal phytotoxins
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associated with severe diseases of cereal crops [7] and exhibited interesting biological properties such as inhibition of cholesterol acyltransferase [8], phytotoxic [9, 10],
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cytotoxic [11], anti-angiogenic [12] and antibiotic activities [13]. During our continuing search for structurally diverse and biologically significant metabolites from endophytic fungal strains [12-14], a fraction of the broth extract of
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the fungus Bipolaris sorokiniana A606 derived from the medicinal plant Pogostemon
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cablin showed an antiproliferative activity against HepG-2 cell line with an inhibition
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rate of 96.2% at a concentration of 100 g/mL1. Subsequent chemical investigation on the endophytic fungal strain A606 resulted in the isolation of four new cochlioquinones, named isocochlioquinones DE (12) and cochlioquinones GH
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(34) (Figure 1), as well as five known analogues. Isocochlioquinone D (1) possessed a rare benzothiazin-3-one moiety and cochlioquinone G (3) was the first example of cochlioquinones bearinganindole-4,7-dione fragment. All of the isolates (19) were evaluated for their cytotoxic activities against MCF-7, NCI-H460, SF-268 and HepG-2 tumor cell lines by the sulforhodamine B (SRB) assay. Herein, the isolation, structural elucidation, cytotoxic activity, and a preliminary structure-activity relationship (SAR) of these compounds are described. 2. Materials and methods 2.1. General experimental procedures Optical rotation was measured on an Anton Paar MCP-500 spectropolarimeter. The IR spectrum was recorded on an IRAffinity-1 spectrophotometer in cm-1. UV spectra were measured on a SHIMADZU UV-2600 UV-visible spectrophotometer. 1D
ACCEPTED MANUSCRIPT and 2D NMR spectra were recorded on a Bruker Avance-500 spectrometer with TMS as internal standard, δ in ppm, J in Hz. HRESIMS was measured on a Thermo MAT95XP high-resolution mass spectrometer and ESIMS was measured on an
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Agilent Technologies 1290-6430A Triple Quad LC/MS. All solvents were of analytical grade (Guangzhou Chemical Reagents Company, Ltd.). Silica gel (200-300
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mesh) was used for column chromatography, and precoated silica gel GF254 plates (Qingdao Marine Chemical Inc. Qingdao, China) were used for TLC spotting. C18 reversed-phase silica gel (40-63 µm, Merck, German), and Sephadex LH-20 gel
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(Pharmacia Fine Chemical Co. Ltd., Sweden) were also used for column chromatography (CC).A Shimadzu LC-20AT equipped with a SPD-M20A PDA
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detector was used for HPLC, and a YMC-pack ODS-A column (250×10 mm, S-5μm, 12nm) was used for semi-preparative HPLC separation. TLC spots were visualized
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under UV light and by dipping into 10% H2SO4 in alcohol followed by heating.
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2.2. Fungal material
The endophytic fungal strain Bipolaris sorokiniana A606 was isolated from the
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plant of Pogostemon cablin, which was collected at Gaoyao, Guangdong province, China, in October, 2012. The strain was identified by sequence analysis of rDNA ITS (internal transcribed spacer) region. The sequence of the ITS region of the
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B.sorokiniana A606 has been submitted to GenBank (Accession No. KF494823). By using BLAST (nucleotide sequence comparison program) to search the GenBank database, B.sorokiniana A606 has 99.6% similarity to B.sorokiniana B41 (Accession No. KF725816). The strain was preserved at the Guangdong Provincial Key Laboratory of Microbiol Culture Collection and Application, Guangdong Institute of Microbiology. 2.3. Fermentation, extraction and isolation B.sorokiniana was grown on potato-dextrose agar (PDA) medium at 28 C for 5 days and then inoculated into 5 flasks (500 mL) containing potato-dextrose (PD) medium (250 mL). After 5 days of incubation at 28 C on a rotary shaker at 120 r/m, a portion of the liquid culture was aseptically transferred into each of a total of 240 flasks (1000 mL) containing PDA medium (500 mL). Following 7 days of cultivation
ACCEPTED MANUSCRIPT at 28 C and 120 r/m on a rotary shaker, the culture (total of 120 L) was filtered to give the filtrate and mycelia. The broth was exhaustively extracted with EtOAc (3 4.5 L), and then the
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EtOAc layers were combined and evaporated under reduced pressure at a temperature
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not exceeding 40 C to yield a crude extract (18.8 g). The EtOAc extract was
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separated into twenty-six fractions (Fr.1-Fr.26) by silica column chromatography eluted with a gradient of PE/EtOAc (v/v, 10:0→0:10) based on TLC monitoring. Fr.8 was further separated on a reversed phase ODS column with a gradient of MeOH/H2O
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(v/v, 3:7→10:0) to yield compound 9 (3.7 mg). Fr.9 was further separated on a reversed phase ODS column with a gradient of MeOH/H2O (v/v, 4:6→10:0) to yield
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compound 6 (4.2 mg) and 4 (6.3 mg). Fr.10 was separated by Sephadex LH-20 chromatography eluting with CH2Cl2/MeOH (1:1, v/v), followed by further
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purification on a semi-preparative HPLC (MeCN/H2O, 4:6→ 10:0, 3 mL/min) to
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yield compound 3 (3.0 mg), 5 (4.6 mg), and 7 (7.3 mg). Fr.11 was separated by Sephadex LH-20 chromatography eluting with CH2Cl2/MeOH (1:1, v/v), followed by
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further purification on a semi-preparative HPLC (MeCN/H2O, 3:7→ 10:0, 3 mL/min) to yield compound 1 (3.2mg), 8 (4.5mg), and 2 (3.0 mg). 2.4. Spectroscopic data
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Isocochlioquinone D (1): yellow oil; [α]25D 236.9 (c 0.1, EtOH); UV (EtOH) λmax (log ε) 220 (4.28), 260 (4.00), 306 (3.58) nm; IR (KBr) νmax 3352, 3275, 2947, 2935, 2874, 1661, 1016 cm–1. 1H (500 MHz) and
13
C (125 MHz) NMR data, see Tables 1
and 2; HRESIMS m/z 560.2681 [M H] (calcdfor C30H42NO7S, 560.2682). Isocochlioquinone E (2): yellow oil; [α]25D 253.9 (c 0.1, EtOH); UV (EtOH) λmax (log ε) 214 (4.25), 284 (4.02), 383 (3.44) nm; IR (KBr) νmax 3402, 2972, 2931, 2870, 1643, 1634, 1361, 1188, 1097, 1047 cm–1. 1H (500 MHz) and
13
C (125 MHz) NMR
data, see Tables 1 and 2; HRESIMS m/z 485.2543 [M H] (calcd for C28H37O7, 485.2539). Cochlioquinone G (3): red oil; [α]25D 142.5 (c 0.1, EtOH); UV (EtOH) λmax (log ε) 203 (4.22), 238 (4.42), 294 (4.25) nm; IR (KBr) νmax 3483, 3321, 2957, 2926, 2855, 1674, 1622, 1599, 1205, 1093 cm–1. 1H (500 MHz) and
13
C (125 MHz) NMR data,
ACCEPTED MANUSCRIPT see Tables 1 and 2; HRESIMS m/z 486.2878 [M H] (calcd for C28H40NO6, 486.2856). Cochlioquinone H (4): red oil; [α]25D 69.4 (c 0.1, EtOH); UV (EtOH) λmax (log ε)
13
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1095 cm–1. 1H (500 MHz) and
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203 (3.92), 268 (3.79) nm; IR (KBr) νmax 3456, 2961, 2926, 2855, 1713, 1651, 1099, C (125 MHz) NMR data, see Tables 1 and 2;
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HRESIMS m/z 471.2753 [M H] (calcd for C28H39O6, 471.2747). 2.5. Cytotoxicity assay
The cell growth inhibitory activities of compounds 19 against human cancer
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cell lines SF-268, MCF-7, NCI-H460, and HepG-2, were tested using the previously published method [17].
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3. Results and discussion
The broth of B.sorokiniana A606 was extracted with EtOAc. The EtOAc extract
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was subjected to various column chromatography protocols to afford compounds 19.
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The structures of new compounds were identified by spectroscopic analysis and physicochemical properties, while the known compounds were assigned as
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isocochlioquinone C (5) [7], cochlioquinone C (6) [7], cochlioquinone D (7) [13], cochlioquinone E (8) [18] and cochlioquinone B (9) [8] by comparison of their spectroscopic data with those in the literatures.
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Compound 1 was obtained as a yellow oil and gave a [M H] peak at m/z 560.2681 in the HRESIMS for a molecular formula of C30H43NO7S (calcd for C30H42NO7S 560.2682), requiring ten sites of unsaturation. The 1H NMR spectrum of 1 displayed signals for seven methyl groups [δH 1.31 (d, J = 6.9 Hz, H3-27), 1.23 (s, H3-26), 1.18 (s, H3-24), 1.17 (s, H3-23), 1.05 (s, H3-25), 0.91 (d, J = 6.9 Hz, H3-28) and 0.83 (t, J = 7.4 Hz, H3-1)], three oxymethine protons [δH 4.95 (t, J = 10.2 Hz, H-12), 3.18 (dd, J = 3.8,12.0 Hz, H-17), and 3.25 (m, H-21)], and a series of aliphatic methylene multiplets. The 13C NMR spectrum, in combination with DEPT experiment, showed 30 carbon resonances attributable to two carbonyls, seven methyls, six sp3methylenes, six methines (three oxygenated), and nine quaternary carbons (two oxygenated sp3 and six sp2). Analysis of the NMR spectra (Tables 1 and 2), including the 1H−1H COSY, HMBC, and NOESY spectra, implied that 1 consisted of two
ACCEPTED MANUSCRIPT partial structures (units A and B), which were presumed to be structurally related to cochlioquinone C (6) and a rare benzothiazin-3-one moiety [19-21], respectively. The gross structures of units A and B were elucidated as follows (Figure 2). The unit A
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(C-12−C-26) was identified to be the same as that part of cochlioquinone C (6). In unit B (C-1−C-11 and C-27C-30), the existence of a persubstituted benzene ring
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(C-6−C-11) was deduced by the 1D NMR data (δC 139.7, 139.5, 129.1,125.5,113.5, 113.1), while two aliphatic methylene signals at δH 3.37 and 3.25 and a carbonyl signal at δC 165.3 implied the presence of a benzothiazin-3-one moiety (C-6−C-11,
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and C-29C-30). This was also supported by HMBC correlations from a methylene (δH 3.37 and 3.23, H2-29) to a sp2-hybridized quaternary carbon (δC 113.1, C-11) and
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the carbonyl group (δC 165.3, C-30), and from NH (δH 9.52) to C-29 (δC 30.3), C-9 (δC 113.5), and C-11 (δC 113.1), as well as from OH-7 (δH 5.80) to C-6 (δC 125.5), C-7 (δC
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139.7), and C-8 (δC 139.5) and from H-5 to C-6, C-7, and C-11. The connectivity of
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A/B moieties was assigned as depicted by HMBC correlations from H-12 to C-8, C-9, and C-10. Thus, the planar structure of 1 was assigned as depicted.
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The relative configuration of 1 was deduced on the basis of NOESY experiment (Figure 3). The NOE correlations of H-21/H-19α, H-17/H-19α and H-17/H-13 suggested that the four protons were cofacial, and arbitrarily assigned as α-orientation.
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Moreover, the informative NOE interactions observed between H3-25/H-12, H-12/H3-26, H-12/H-19β, indicated the β-configurations of Me-25 and Me-26, and the moiety at C-21. Considering the same biosynthetic pathway for this kind of compound, the relative configuration in the aliphatic side chain C-1−C-5 of 1 was assumed to be identical to those of known analogues 59. Thus, the structure of 1 was established and given a trivial name isocochlioquinone D. Compound 2 was isolated as a yellow oil that gave a [M H] ion at m/z 485.2543 in the HRESIMS consistent with a molecular formula of C28H38O7 (calcd for C28H37O7, 485.2539), which was 2 mass units less than that of 5. The 1H and 13C NMR spectra of 2 were similar to those of isocochlioquinone C (5) except for the presence of double bond at C-2 [δC 138.2 and 138.1; δH 6.87 (1H, d, J = 6.8 Hz)] in 2, indicating that 2 was a 2,3-didehydrogenated derivative of 5. This was supported by
ACCEPTED MANUSCRIPT the significant downfield-shifted H-2 signal in 2 with respect to that in 5 (δH 6.87 in 2; δH 1.76 and 1.65 in 5) and the upfield-shifted carbonyl at C-4 (15 ppm). The planar structure of 2 was further established by detailed interpretation of its 2D NMR data.
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The relative configuration of 2 was identified to be the same as that of 5 on the basis of NOESY correlations and comparison of their 1D NMR data. Thus, compound 2
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was assigned as depicted and named isoochlioquinone E.
Compound 3 was obtained as a red oil that gave a [M H] ion at m/z 486.2878 in the HRESIMS consistent with a molecular formula of C28H39NO6 (calcd for
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C28H40NO6, 486.2856), requiring ten sites of unsaturation. The 1D NMR spectra of 3 were similar to those of 6 except for the presence of an additional tetrasubstituted
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double bond (δC 143.1 and 118.8) and absence of both the carbonyl (δC 212.2 at C-4 in 6) and aromatic proton (δH 6.48 at H-11 in 6), implying 3 was an analogue of 6. As
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nine of the ten degrees of unsaturation were accounted for a cochlioquinone core and
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a tetrasubstituted double bond, the remaining unsaturation unit required that 3 had one more ring than that of 6. The aforementioned information implied that 3 was bearing
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an indole-4,7-dione unit [22]. This was confirmed by the COSY correlations of H-1/H-2/H-3/H-28 and HMBC correlations from H-3 (δH 2.83) to C-4 (δC 143.1), C-5 (δC 118.8), and C-27 (δC 10.0), and from H-27 (δH 2.21) to C-4, C-5, and C-6 (δC
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122.5). The planar structure of 3 was further confirmed by molecular formula of C28H39NO6.
The relative configuration of 3 was deduced by analyzing its NOESY spectrum. Diagnostic NOE correlations of H-17/H-19α, H-21 and H-13 positioned them on the same side and established the cis relationships of these protons. On the other hand, NOE correlations of H3-25/H-19β, and H-12 and H-12/H3-26 indicated that these protons were on the other face of the molecule (Figure 4). The structure of 3 was established and given the trivial name cochlioquinone G. Compound 4, a red oil, had the molecular formula C28H38O6, as determined by HRESIMS. The 1H and 13C NMR data of 4 were similar to those of co-isolated known compound cochlioquinone B (9), except for the presence of ∆12,13 double bond (δC 147.7 and 110.5) in 4. The trisubstituted double bond at C-12C-13 was deduced from
ACCEPTED MANUSCRIPT HMBC correlations for both two methyl protons [δH 1.09 (s, H3-25) and 1.50 (s, H3-26)] to the olefinic carbon [δC 147.7 (C-13)]. Diagnostic ROESY correlations positioned H-17 and H-21 on the same () face of the heterocyclic scaffold, and both
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H3-25 and H3-26 on the face, and defined the relative configuration for 4. Therefore, the structure of 4 was established and given the trivial name cochlioquinone H.
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Compounds 19 were evaluated for their in vitro cytotoxic activities against four human tumor cell lines, including SF-268 (human glioma cell line), MCF-7 (human breast adenocarcinoma cell line), NCI-H460 (human non-small cell lung cancer cell
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line) and HepG-2 (human hepatoma cell line) by the SRB method, and cisplatin was used as the positive control (Table 3). As a result, all compounds showed obvious
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cytotoxic effects against the four tumor cell lines, and compounds 69, sharing the quinone structural features, showed broad inhibitory effects against the cell lines, with
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the activities comparable to those of cisplatin (most IC50s<10 μM). However, selective
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cytotoxicity was observed within this sub-class. Compounds 7 showed excellent inhibitory activities against SF-268 and HepG-2 cell lines with IC50 values of 1.5 and
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1.2 μM, respectively, implying that the dehydrogenation between C-2 and C-3 might contribute the selectivity to SF-268 and HepG-2 in 7 and 9, while hydroxy at C-12 was important to the selectivity to NCI-H460 in 6 and 9. Compared to 69, the
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introduction of a hydroxy or other substituent group at C-11 (3) greatly decreased their activities.
Interestingly, despite of generally low activity, the isocochlioquinones 12, 5 reserved their anti-proliferative activities and showed selective cytotoxicities against certain tumor cell lines. Compounds 2 selectively inhibited SF-268 and HepG-2 with IC50 values of 17.2 and 13.6 μM, respectively. Further analysis showed the dehydrogenation between C-2 and C-3 and the presence of hydroxy at C-10 in 2 led to more selective cytotoxicity against SF-268 and HepG-2. Previous bioassay of cochlioquinone derivatives indicated the moderate lipophilicity at C-4, a hydroxy residue at C-12 and C-19, and the replaceable at 7,10-quinone part were important for the activity against methicillin-resistant Staphylococcus aureus (MRSA) [23]. In this study, the inhibitory activities of
ACCEPTED MANUSCRIPT cochlioquinones against the four human tumor cell lines also implied the above-mentioned moieties may be important influencing factors on cytotoxic activity. In summary, nine cochlioquinones (19) were isolated from the endophytic
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fungus Bipolaris sorokiniana. Their structures were determined by comprehensive spectroscopic analysis. The isolates were evaluated for their cytotoxicities against
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four human tumor cell lines. Compound 7, possessing the C-2,3 double bond but lacking the C-12 hydroxy, showed excellent activity against SF-268, MCF-7 and HepG-2 cell lines with IC50 values of 1.5, 2.4, and 1.2 μM, respectively, being
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stronger than cisplatin, while 2 and 4 showed selective inhibitory activities against SF-268 and HepG-2. This study not only enriches the chemodiversity of the genus
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Bipolaris by the isolation of a series of cochlioquinones, but also provides scientific basis for the usage of the phytopathogenic fungus B.sorokiniana as a source of
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antineoplastic agents in future.
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Acknowledgments
This work was financially supported by the National Basic Research Program of
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China (973 Program, No. 2014CB460613), National Natural Science Foundation of China (No. 81203006), Natural Science Foundation of Guangdong Province (No. 2015A030313710), and Guangdong Provincial Project for Science and Technology
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(No. 2015A030302060).
Appendix A. Supplementary data 1D, 2D NMR and HRESIMS spectra of 14 are available free of charge via the Internet at References [1] A.H. Aly, A.Debbab, P Proksch. Fungal endophytes: Unique plant inhabitants with great promises, Appl Microbiol Biot. 90 (2011) 1829–1845. [2] A.H. Aly, A.Debbab, J. Kjer, P Proksch. Fungal endophytes from higher plants: A prolific source of phytochemicals and other bioactive natural products, Fungal Divers. 41 (2010) 1–16. [3]. M.E. Amrani, D. Lai, A. Debbab, A.H. Aly, K. Siems, C. Seidel, M. Schnekenburger, A. Gaigneaux, M. Diederich, D. Feger. Protein kinase and
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[6] M. Arayama, T. Nehira, H. Maeda, K. Tanaka, H. Miyagawa, T. Ueno, S. Hosokawa, M. Hashimoto. Isolation, ECD assisted structural analyses,
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Haruyama, S. Sato, T. Koga, Y Tsujita. Epi-cochlioquinone A, a novel acyl-CoA:cholesterol acyltransferase inhibitor produced by Stachybotrys bisbyi, J Antibiot. 49 (1996) 409-413. [9] H. Miyagawa, S. Nagai, T. Tsurushima, M. Sato, T. Ueno, H. Fukami. Phytotoxins produced by the plant pathogenic fungus Bipolaris bicolor El-1, Biosci Biotech Bioch. 58 (1994) 1143-1145. [10] C.H. Lim, H. Ueno, H. Miyoshi, H. Miyagawa, H. Iwamura, T. Ueno. Phytotoxic compounds cochlioquinones are inhibitors of mitochondrial NADH-ubiquinone reductase, J Pesticide Sci. 21 (1996) 213-215. [11]
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[13] N. Koyama, T. Nagahiro, Y. Yamaguchi, R. Masuma, H. Tomoda, S. Omura. Stemphones, novel potentiators of imipenem activity against methicillin-resistant
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benzopyrans from the endophytic fungus Nodulisporium sp. A4 from Aquilaria sinensis, Helv Chim Acta. 93 (2010) 920-924.
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[15] M.H. Tao, J. Yan, X.Y. Wei, D.L. Li, W.M. Zhang, J.W. Tan. A novel sesquiterpene alcohol from Fimetariella rabenhorstii, an endophytic fungus of
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Aquilaria sinensis, Nat Prod Commun. 6 (2011) 763-766.
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[16] D.L. Li, Y.C. Chen, M.H. Tao, H.H. Li, W.M. Zhang. Two new octahydronaphthalene derivatives from Trichoderma spirale, an endophytic
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fungus derived from Aquilaria sinensis, Helv Chim Acta. 95 (2012) 805-809. [17] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J.T. Warren, H. Bokesch, S. Kenney, M. R. Boyd. New colorimetric cytotoxicity
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assay for anticancer-drug screening, J Nat Cancer Inst. 82 (1990) 1107-1112. [18] H. Seijiro, M. Kaoru, T. Shinpei, T. Tatsuya, S. Yasuaki, T. Kuniaki. Structures and biological activities of phytotoxins produced by the plant pathogenic fungus Bipolaris cynodontis cynA, Tetrahedron Lett. 51 (2010) 5532-5536. [19]
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of stemphones, potentiators of imipenem activity against methicillin-resistant
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ACCEPTED MANUSCRIPT Table 1. 1H NMR data for compounds 14 (500 MHz, δ in ppm, J in Hz). 1a
2a
3b
4a
1 2
0.83, t (7.4) 1.57, m 1.36, m 2.45, m 4.09, q (6.9)
1.80, d (6.8) 6.87, d (6.8)
0.83, t (7.4) 1.64, m 1.24, m 2.83, dq (13.8, 7.0)
0.83, t (7.4) 1.71, m 1.35, m 2.67, m 4.08, qd (7.2, 1.2) 6.45, d (1.2) 6.29, s
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NH 7-OH 10-OH a
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2.02, m 1.86, m 1.76, m 1.59, m 3.19, dd (11.9, 3.7) 2.41, m 1.46, m 1.64, m 1.43, m 3.25, dd (11.9, 2.7) 1.16, s 1.18, s 1.05, s 1.31, s 2.21, s 1.25, d (7.0)
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2.70, m 1.28, m 1.82, m 1.68, m 3.15, dd (11.9, 3.9) 2.10, m 2.03, m 1.64, m 1.45, m 3.25, dd (12.0, 2.7) 1.18, s 1.18, s 1.12, s 1.48, s 1.36, d (6.9) 1.75, s
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1.95, m 1.85, m 1.60, m 1.46, m 3.18, dd (12.0, 3.8) 2.42, m 1.82, m 1.79, m 1.49, m 3.25, m 1.17, s 1.18, s 1.04, s 1.23, s 1.30, d (6.9) 0.91, d (6.9) 3.37, d (14.7); 3.23, d(14.7) 9.52, brs 5.80, brs
2.76, s
4.94, d (10.2) 1.69, d (10.2)
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4.95, t (10.2) 1.89, d (10.2)
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21 23 24 25 26 27 28 29
4.78, q (6.9) 6.21, s
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5.52, s 10.78, s
Recorded in CDCl3; brecorded in CD3OD.
2.26, dt (13.3, 3.3) 1.98, dd (13.3, 4.5) 1.77, m 1.71, m 3.14, d (3.2) 2.04, m 1.53, t (3.3) 1.65, dd (5.0, 3.3) 1.56, dd (5.0, 2.2) 3.16, d (3.3) 1.15, s 1.16, s 1.09, s 1.50, s 1.26, d (7.2) 1.11, d (6.9)
ACCEPTED MANUSCRIPT Table 2. 13C NMR data for compounds 14 (125 MHz, δ in ppm, J in Hz). 1a
2a
3b
4a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
11.5, CH3 27.0, CH2 44.7, CH 213.8, C 46.7, CH 125.5, C 139.7, C 139.5, C 113.5, C 129.1, C 113.1, C 65.4, CH 55.3, CH 81.0, C 37.7, CH2 25.3, CH2 83.8, CH 36.7, C 38.6, CH2 21.5, CH2 85.0, CH 71.9, C 23.7, CH3 26.0, CH3 12.6, CH3 21.9, CH3 13.8, CH3 15.5, CH3 30.3, CH2 165.3, C
11.4, CH3 138.2, CH 138.1, C 201.5, C 39.7, CH 133.9, C 137.2, C 144.4, C 107.3, C 153.7, C 106.8, CH 198.2, C 60.3, CH 83.5, C 37.3, CH2 24.9, CH2 83.6, CH 35.4, C 37.6, CH2 21.3, CH2 85.3, CH 71.9, C 23.7, CH3 26.0, CH3 12.3, CH3 22.0, CH3 17.4, CH3 15.0, CH3
12.5, CH3 30.5, CH2 33.7, CH 143.1, C 118.8, C 122.5, C 180.8, C 153.6, C 120.0, C 179.0, C 131.4, C 64.0, CH 54.0, CH 83.5, C 38.6, CH2 26.3, CH2 85.4, CH 37.7, C 39.9, CH2 22.5, CH2 86.5, CH 72.8, C 25.5, CH3 25.5, CH3 13.1, CH3 21.1, CH3 10.0, CH3 20.2, CH3
11.7, CH3 25.7, CH2 47.3, CH 212.6, C 43.0, CH 146.0, C 181.0, C 148.7, C 117.5, C 184.4, C 132.4, CH 110.5, CH 147.7, C 80.9, C 37.7, CH2 24.4, CH2 81.5, CH 38.6, C 34.7, CH2 21.5, CH2 84.4, CH 71.8, C 26.0, CH3 23.7, CH3 20.1, CH3 27.0, CH3 14.8, CH3 16.5, CH3
Recorded in CDCl3; brecorded in CD3OD.
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ACCEPTED MANUSCRIPT Table 3. Antiproliferative activity of the compounds (19) towards cancer cells Cell lines/IC50 SD (μM) Compounds MCF-7
NCI-H460
HepG-2
1
32.8 0.5
28.3 1.1
42.6 2.5
38.7 1.1
2
17.2 1.1
28.1 0.7
31.1 1.1
13.6 0.5
3
35.9 1.3
21.1 0.6
26.9 2.8
11.3 1.9
4
6.4 0.2
8.6 0.2
5
38.4 1.9
32.3 1.0
6
5.3 1.7
5.6 3.4
7
1.5 0.1
2.4 0.2
9.2 0.2
1.2 0.2
8
5.2 0.6
7.3 0.9
24.1 0.9
8.4 0.9
9
7.1 0.2
13.1 1.3
42.8 8.4
7.7 0.1
Cisplatin
4.1 0.2
2.9 0.5
2.9 0.2
2.5 0.2
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7.6 0.1
46.3 0.4
50.6 5.2
13.2 1.7
4.1 3.8
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Figure 1. Structures of compounds 19 isolated from Bipolaris sorokiniana A606.
) and HMBC (
) correlations for compounds 14.
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Figure 2. Key COSY (
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Figure 3. Key NOESY correlations for compounds 14.
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