New cytotoxic tricycloalternarenes from fungus Alternaria brassicicola

Bioorganic Chemistry 92 (2019) 103279

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Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg

New cytotoxic tricycloalternarenes from fungus Alternaria brassicicola a,1

b,1

a

b

b

a

Fengli Li , Ying Tang , Weiguang Sun , Jiankun Guan , Yuanyuan Lu , Sitian Zhang , ⁎ ⁎ Shuang Lina, Jianping Wanga, Zhengxi Hua, , Yonghui Zhanga,

T

a

Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China

b

ARTICLE INFO

ABSTRACT

Keywords: Alternaria brassicicola Tricycloalternarenes Structural elucidation Cytotoxicity

Seven previously undescribed metabolites, designated as tricycloalternarenes Q–W (1–7), were isolated and identified from the fermented rice substrate of fungus Alternaria brassicicola. The planar and absolute structures of all new compounds were determined on the basis of extensive NMR spectroscopic data, a modified Mosher’s method, X-ray crystallographic analysis, and electronic circular dichroism (ECD) spectral analyses. All the isolates were evaluated for in vitro cytotoxicity against five human tumor MM231, MM468, HeLa, SW1990, and SW480 cell lines, and compounds 1, 2, 5, and 7 showed selective cytotoxicity against certain human tumor cell lines with IC50 values ranging from 12.83 to 32.87 μM, with no obvious cytotoxicity to the normal LO2 cell.

1. Introduction

2. Experiment

Phytopathogenic fungi constitute one of the major infectious agents in plants, thus resulting in multiple alterations during growth stages including post-harvest, absorbing nutritional ingredients from the host plants, and therefore, causing huge economic losses [1]. Universally, phytopathogenic fungi owned many virulence factors that were countered by plant defense mechanisms, and thus bioactive secondary metabolites produced by phytopathogenic fungi emerged to defend against the stress ecological conditions [2]. Nevertheless, in the past decades, chemical investigations on the phytopathogenic fungi attracted no much attention, which suggested that these unique microbial resources were a promising reservoir for exploring structurally novel and bioactive natural products for drug discovery. In previous studies, we have identified several novel fusicoccanederived diterpenoids from the phytopathogenic fungus Alternaria brassicicola [3–6]. As a part of our on-going program to search for more architecturally complex and bioactive constituents from this genetically powerful strain, the ethyl acetate extracts were chemically investigated, which resulted in the isolation of seven previously undescribed tricycloalternarenes, designated as tricycloalternarenes Q–W (1–7). In this paper, we describe the isolation, structural elucidation, and in vitro cytotoxicity evaluation of these compounds (Fig. 1).

2.1. General Optical rotations were measured with a PerkinElmer 341 polarimeter (Waltham, MA, USA). Ultraviolet (UV) spectra were measured with a Varian Cary 50 (Santa Clara, CA, USA) spectrophotometer. Experimental CD data were recorded with a JASCO-810 CD (Oklahoma City, OK, USA) spectrometer instrument. IR spectra were measured with a Bruker Vertex 70 instrument (Billerica, MA, USA) with KBr pellets. 1D and 2D NMR spectra were recorded on a Bruker AM-400 spectrometer (Bruker, Karlsruhe, Germany). All chemical shifts (δ) were expressed in ppm with reference to the solvent signals. The high-resolution electrospray ionization mass spectra (HRESIMS) data were recorded with a Thermo Fisher LTQ XL LC/MS instrument (Thermo Fisher, Palo Alto, CA, USA). Semi-preparative HPLC was performed on an Agilent 1200 liquid chromatograph with Zorbax SB-C18 (9.4 mm × 250 mm) column. Column chromatography (CC) was performed with silica gel (100–200 mesh and 200–300 mesh, Qingdao Marine Chemical, Inc., Qingdao, People’s Republic of China), Lichroprep RP-C18 gel (40–63 μm, Merck, Darmstadt, Germany), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Thin-layer chromatography (TLC) was carried out on precoated TLC plates (200–250 μm thickness, silica gel 60 F254, Qingdao Marine Chemical, Inc.), and fractions were monitored by TLC and spots were

Corresponding authors. E-mail addresses: [email protected] (Z. Hu), [email protected] (Y. Zhang). 1 These authors contributed equally to this work. ⁎

https://doi.org/10.1016/j.bioorg.2019.103279 Received 17 April 2019; Received in revised form 9 September 2019; Accepted 11 September 2019 Available online 11 September 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved.

Bioorganic Chemistry 92 (2019) 103279

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Fig. 1. Chemical structures of compounds 1–7.

visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH.

2.4. Spectroscopic data Tricycloalternarene Q (1): colorless crystals; [α]20 D : +283 (c 0.09, MeOH); UV (MeOH) λmax (log ε) = 202 (3.90), 264 (4.08) nm; CD (MeOH) λmax (Δε) = 260 (+14.48) nm; IR (KBr) νmax = 3436, 2968, 2933, 1656, 1617, 1396, 1164, 1094, 1032, 889, 570 cm−1; HRESIMS m/z 401.2304 [M + Na]+ (calcd. for C22H34O5Na+, 401.2298); For 1H and 13C NMR data, see Table 1. Tricycloalternarene R (2): colorless oil; [α]20 D : +290 (c 0.9, MeOH); UV (MeOH) λmax (log ε) = 202 (3.86), 262 (4.03) nm; CD (MeOH) λmax (Δε) = 258 (+10.67) nm; IR (KBr) νmax = 3439, 2968, 2932, 1651, 1611, 1382, 1294, 1250, 1161, 1093, 1074, 903, 672, 583 cm−1; HRESIMS m/z 387.2156 [M + Na]+ (calcd. for C21H32O5Na+,

2.2. Fungal material The strain Alternaria brassicicola was isolated from the leaves of Siegesbeckia pubescens Makino (Compositae), which were collected from Huoshan County, Anhui Province, P. R. China, in September 2012. This fungus was authenticated by one of the authors (J. Wang) based on the morphology and sequence analysis of its ITS region of rDNA, and the sequence data for this fungal strain have been submitted to the GenBank database with accession no. KR779774. A voucher fungal strain has been deposited in the culture collection of Tongji Medical College, Huazhong University of Science and Technology.

Table 1 1 H and 13C NMR spectroscopic data (δ in ppm, J in Hz) for compounds 1–3.

2.3. Fermentation, extraction, and purification

No.

The strain A. brassicicola was cultivated on potato dextrose agar (PDA) plates at 28 °C for 10 days. Then the agar was cut into pieces (0.4 × 0.4 × 0.4 cm3) and inoculated into 150 Erlenmeyer flasks [composition: rice (200 g) in the distilled water (200 mL)]. All flasks were incubated at 28 °C for 21 days. Following incubation, the fungi were killed by adding 300 mL of EtOAc to each flask, and the culture was homogenized. The suspension was extracted repeatedly with EtOAc (4 × 9 L), and the organic solvent was evaporated under reduced pressure to provide a crude extract (210 g). The total crude extract (210 g) was loaded onto RP-C18 silica gel column chromatography (CC) eluted with CH3OH–H2O (20%, 40%, 50%, 60%, 80%, and 100%) to get six fractions (A–F). Fraction D (25 g) was fractionated by silica gel CC using a gradient elution of CH2Cl2–MeOH system (30:0 → 10:1) to obtain five main fractions (D1–D5) by the TLC analysis. Fraction D3 (3 g) was subjected to Sephadex LH-20 (CH2Cl2–MeOH, 1:1, v/v), subsequently, by repeated purification using semi-preparative HPLC [CH3CN–H2O, 50:50, v/v, 2.0 mL/min, 210 nm, tR = 22 min, for 6 (3.2 mg); CH3CN–H2O, 44:56, v/v, 2.0 mL/min, 210 nm, tR = 33 min, for 4 (147 mg); CH3CN–H2O, 30:70, v/v, 2.0 mL/min, 210 nm, tR = 20 min, for 5 (22.7 mg)]. Fraction E (10 g) was applied to Sephadex LH-20, eluted with CH2Cl2–MeOH (1:1, v/v) to get three main fractions (E1–E3). Subsequently, the fraction E2 (2.5 g) was subjected to silica gel CC (200–300 mesh) using a gradient of petroleum ether (PE)/EtOAc system (5:1 → 1:1), further purified by semi-preparative HPLC [CH3CN–H2O, 42:58, v/v, 2.0 mL/min, 210 nm, tR = 36 min, for 2 (116.5 mg); tR = 38 min, for 3 (10.5 mg); CH3CN–H2O, 65:35, v/v, 2.0 mL/min, 210 nm, tR = 36 min, for 7 (18.7 mg)]. Fraction E3 (2 g) was loaded onto silica gel CC (200–300 mesh) eluted with a gradient of PE/EtOAc system (5:1 → 1:1), then applied to Sephadex LH-20 eluted with MeOH, and further purified by RP-HPLC [CH3CN–H2O, 50:50, v/v, 2.0 mL/ min, 210 nm, tR = 30 min, for 1 (126.5 mg)].

1 (in methanol-d4) δH

δC

2 (in methanol-d4)

c

δH

a,b

δC

3 (in methanol-d4)

c

1

1.11 s

25.5, CH3

1.11 s

25.7, CH3

2 2′ 3

– 1.07 s 3.15 dd (2.5, 10.2) 1.75 m; 2.25 m 5.49 m – 1.68 s 2.80 m 1.65 m; 1.73 m 1.87 m; 2.08 m – 1.29 s 1.99 m 2.00 m; 2.28 m – – 2.48 m; 2.50 m 1.88 m; 2.25 m 3.79 dd (4.7, 11.4) – 3.48 s

73.7, C 25.1, CH3 80.5, CH

– 1.07 s 3.16 dd (2.6, 10.2) 1.75 m; 2.24 m 5.48 m – 1.69 s 2.82 m 1.68 m; 1.73 m 1.86 m; 2.08 m – 1.29 s 1.99 m 2.00 m; 2.31 m – – 2.43 m; 2.53 m 1.83 m; 2.23 m 4.08 dd (5.2, 12.7) – –

73.7, C 25.1, CH3 80.4, CH

4 5 6 6′ 7 8 9 10 10′ 11 12 13 14 15 16 17 18 OMe a b

c

2

a,b

30.6, CH2 127.0, CH 136.8, C 18.6, CH3 42.9, CH 26.0, CH2 38.7, CH2 88.8, 22.7, 44.3, 16.7,

C CH3 CH CH2

107.3, C 171.7, C 28.5, CH2 28.0, CH2 81.1, CH 199.0, C 58.3, CH3

30.7, CH2 127.0, CH 136.8, C 18.6, CH3 42.9, CH 26.0, CH2 38.8, CH2 88.7, 22.6, 44.3, 16.8,

C CH3 CH CH2

106.6, C 171.9, C 28.8, CH2 30.6, CH2 72.4, CH 200.5, C –

δHa,b

δCc

4.77 m; 4.83 m – 1.64 s 3.79 t (6.9) 2.07 m

112.1, CH2

5.23 m – 1.65 s 2.76 m 1.68 m; 1.75 m 1.89 m; 2.15 m – 1.34 s 2.00 m 2.04 m; 2.18 m – – 4.28 t (4.9) 1.98 m; 2.18 m 2.30 m; 2.63 m – –

Recorded at 400 MHz. “m” means overlapped or multiplet with other signals. Recorded at 100 MHz.

148.0, C 17.4, CH3 77.2, CH 34.0, CH2 124.9, CH 137.5, C 18.5, CH3 43.1, CH 26.2, CH2 38.5, CH2 88.8, 22.8, 44.3, 16.8,

C CH3 CH CH2

108.4, C 171.2, C 66.8, CH 30.5, CH2 33.5, CH2 200.8, C –

Bioorganic Chemistry 92 (2019) 103279

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Table 2 1 H and 13C NMR spectroscopic data (δ in ppm, J in Hz) for compounds 4–7. No.

1 2 2′ 3 4 5 6 6′ 7 8 9 10 10′ 11 12 13 14 15 16 17 18 OMe a b

c

4 (in methanol-d4)

5 (in CDCl3)

δHa,b

c

δC

1.11 s – 1.05 s 3.14 dd (2.5, 10.1) 1.74 m; 2.25 m 5.47 m – 1.68 s 2.82 m 1.71 m; 1.77 m 1.91 m; 2.14 m – 1.35 s 2.00 m 2.06 m; 2.24 m – – 3.86 t (3.9) 2.14 m 2.25 m; 2.62 m – 3.49 s

26.1, CH3 73.5, C 24.7, CH3 80.2, CH 30.5, CH2 127.0, CH 136.7, C 18.6, CH3 42.9, CH 26.0, CH2 38.7, CH2 88.9, C 23.0, CH3 44.5, CH 16.7, CH2 109.4, C 169.8, C 76.2, CH 27.8, CH2 33.1, CH2 201.0, C 58.6, CH3

δH

a,b

1.15 s – 1.04 s 3.25 dd (3.0, 9.9) 1.89 m; 2.00 m 5.29 m – 1.64 s 2.67 m 1.61 m; 1.72 m 1.82 m; 2.11 m – 1.32 s 1.90 m 2.00 m; 2.35 m – – 4.37 m 1.94 m; 2.22 m 2.29 m; 2.59 m – –

6 (in methanol-d4) δC

c

26.7, CH3 72.8, C 24.0, CH3 78.5, CH 30.2, CH2 125.7, CH 137.0, C 18.6, CH3 41.8, CH 25.3, CH2 38.0, CH2 88.3, C 23.2, CH3 43.5, CH 15.7, CH2 107.6, C 168.7, C 66.4, CH 29.0, CH2 33.6, CH2 199.1, C –

7 (in methanol-d4)

δHa,b

c

δC

1.10 s – 1.02 s 3.24 dd (2.5, 10.1) 1.70 m; 2.19 m 5.47 m – 1.68 s 2.78 m 1.71 m 1.89 m; 2.14 m – 1.34 s 1.97 m 2.01 m; 2.23 m – – 4.27 t (4.9) 1.98 m; 2.18 m 2.28 m; 2.63 m – 3.17 s

21.5, CH3 78.5, C 20.5, CH3 78.3, CH 30.3, CH2 127.0, CH 136.7, C 18.6, CH3 42.9, CH 26.0, CH2 38.6, CH2 88.8, C 22.8, CH3 44.6, CH 16.7, CH2 108.6, C 171.3, C 66.8, CH 30.5, CH2 33.6, CH2 200.7, C 49.6, CH3

δHa,b

δCc

1.62 s – 1.52 s 4.90 m 2.50 m 5.23 m – 1.64 s 2.80 m 1.78 m 1.89 m; 2.16 m – 1.34 s 1.99 m 2.07 m; 2.17 m – – 4.27 t (4.8) 1.99 m; 2.12 m 2.27 m; 2.65 m – –

25.7, CH3 132.0, C 17.8, CH3 124.4, CH 27.4, CH2 127.9, CH 135.5, C 18.4, CH3 43.0, CH 26.2, CH2 38.6, CH2 88.7, C 22.8, CH3 44.3, CH 16.8, CH2 108.5, C 171.2, C 66.7, CH 30.5, CH2 33.5, CH2 200.7, C –

Recorded at 400 MHz. “m” means overlapped or multiplet with other signals. Recorded at 100 MHz.

387.2142); For 1H and 13C NMR data, see Table 1. Tricycloalternarene S (3): colorless oil; [α]20 D : +214 (c 0.4, MeOH); UV (MeOH) λmax (log ε) = 202 (3.95), 263 (4.08) nm; CD (MeOH) λmax (Δε) = 258 (+9.32) nm; IR (KBr) νmax = 3434, 2965, 2930, 1616, 1442, 1388, 1240, 1161, 1078, 1018, 894, 671 cm−1; HRESIMS m/z 369.2050 [M + Na]+ (calcd. for C21H30O4Na+, 369.2036); For 1H and 13 C NMR data, see Table 1. Tricycloalternarene T (4): colorless oil; [α]20 D : +247 (c 1, MeOH); UV (MeOH) λmax (log ε) = 202 (3.96), 264 (4.06) nm; CD (MeOH) λmax (Δε) = 257 (+11.78) nm; IR (KBr) νmax = 3434, 2967, 2932, 1624, 1447, 1392, 1164, 1094, 1023, 564 cm−1; HRESIMS m/z 401.2304 [M + Na]+ (calcd. for C22H34O5Na+, 401.2298); For 1H and 13C NMR data, see Table 2. Tricycloalternarene U (5): colorless oil; [α]20 D : +474 (c 1, MeOH); UV (MeOH) λmax (log ε) = 203 (3.46), 264 (3.62) nm; CD (MeOH) λmax (Δε) = 259 (+14.75) nm; IR (KBr) νmax = 3427, 2968, 2932, 1617, 1389, 1164, 1078, 1013, 540 cm−1; HRESIMS m/z 387.2141 [M + Na]+ (calcd. for C21H32O5Na+, 387.2142); For 1H and 13C NMR data, see Table 2. Tricycloalternarene V (6): colorless oil; [α]20 D : +232 (c 1, MeOH); UV (MeOH) λmax (log ε) = 202 (3.98), 263 (4.10) nm; CD (MeOH) λmax (Δε) = 259 (+11.70) nm; IR (KBr) νmax = 3427, 2968, 2933, 1618, 1387, 1164, 1082, 1029, 570 cm−1; HRESIMS m/z 401.2301 [M + Na]+ (calcd. for C22H34O5Na+, 401.2298); For 1H and 13C NMR data, see Table 2. Tricycloalternarene W (7): colorless oil; [α]20 D : +252 (c, 1 MeOH); UV (MeOH) λmax (log ε) = 200 (4.28), 261 (4.21) nm; CD (MeOH) λmax (Δε) = 259 (+29.4) nm; IR (KBr) νmax = 3435, 2966, 2930, 1643, 1611, 1391, 1256, 1164, 1074, 1012 cm−1; HRESIMS [M + Na]+ m/z 353.2103 (calcd. for C21H30O3Na+, 353.2087); For 1H and 13C NMR data, see Table 2.

radiation. The structure was solved by direct methods with SHELXS-97 and refinement was carried out with SHELXL-97 using full-matrix leastsquares, in which anisotropic displacement parameters were used for all the non-hydrogen atoms [7]. The hydrogen atoms were fixed at the calculated positions and refined with a riding model. Molecular graphic was computed with PLATON. The crystallographic data (excluding structure factor tables) have been deposited in the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 1,869,928 for 1. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. +44 (0) (1223) 336 033; e-mail: [email protected]]. Crystallographic data for 1: C22H34O5, M = 378.49, monoclinic, a = 5.582 Å, b = 25.21120(10) Å, c = 15.05540(10) Å, α = 90°, β = 100.5470(10)°, γ = 90°, V = 2082.822(17) Å3, T = 100.01(10) K, space group P21, Z = 4, μ(Cu Kα) = 0.676 mm−1, 48,649 reflections measured, 8297 independent reflections (Rint = 0.0354, Rsigma = 0.0198). The final R1 values were 0.0265 (I > 2σ(I)). The final wR(F2) values were 0.0688 (I > 2σ(I)). The final R1 values were 0.0266 (all data). The final wR(F2) values were 0.0689 (all data). The goodness of fit on F2 was 1.033. Flack parameter = 0.02(3). 2.6. Preparation of the (S)-MTPA and (R)-MTPA esters of 1–3 and 5 Compound 1 (0.5 mg) was dissolved in 1 mL of anhydrous CH2Cl2. Dimethylaminopyridine (25 mg), triethylamine, and (S)-MTPA chloride (20 μL) were consecutively added. The reaction mixture was stirred at room temperature for 1 h, and then quenched by the addition of 1 mL of aqueous MeOH. The solvents were concentrated under reduced pressure, affording a residue which was then applied to a small silica gel column using petroleum ether–EtOAc (10:1) as elution to provide the (R)-MTPA ester of 1 (0.9 mg). And the (S)-MTPA ester of 1 (0.7 mg) was also prepared with (R)-MTPA chloride and purified in the same manner. For the preparation of (S)-MTPA and (R)-MTPA esters of 2–3 and 5, the same experimental procedures were conducted. (S)-MTPA ester of 1 (1a). 1H NMR (CDCl3, 400 MHz) δ: 1.04 (3H, s, H3-1), 1.07 (3H, s, H3-2′), 2.07 (1H, m, H-4a), 2.34 (1H, m, H-4b), 5.20 (1H, m, H-5), 1.58 (3H, s, H3-6′).

2.5. X-ray crystallographic analysis A suitable crystal of compound 1 was obtained in MeOH (with two drops of water). The intensity data were acquired on a Bruker APEX DUO diffractometer equipped with an APEX II CCD with Cu Ka 3

Bioorganic Chemistry 92 (2019) 103279

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followed by the removal of the medium and adding 100 μL of dimethyl sulfoxide. The optical density of the lysate was measured at 570 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by Reed and Muench’s method [9]. 3. Results and discussion 3.1. Structure elucidation Tricycloalternarene Q (1) was obtained as colorless crystals. Its molecular formula was determined to be C22H34O5 based on the HRESIMS analysis at m/z 401.2304 [M + Na]+ (calcd. for C22H34O5Na+, 401.2298). The 1H NMR spectrum (Table 1) of 1 revealed the obvious signals for four methyl singlets at δH 1.07, 1.11, 1.29, and 1.68), one olefinic proton at δH 5.49 (1H, m), two oxymethine protons at 3.15 (1H, dd, J = 2.5, 10.2 Hz) and 3.79 (1H, dd, J = 4.7, 11.4 Hz), and two methine protons at 1.99 (1H, m) and 2.80 (1H, m). The 13C NMR data (Table 1) along with DEPT and HSQC spectra demonstrated the presence of five methyls (one oxygenated), six methylenes, five methines (one olefinic and two oxygenated), and six nonprotonated carbons (three olefinic, two oxygenated, and one ketone). A detailed comparison of the 1D NMR data of 1 with those of TCA 9b [10] unlocked their structural similarity, with the diagnostic differences being that 1 possessed a –OCH3 group at C-17 rather than a –OH group at the same position in TCA 9b, and a C-2/C-3 double bond in TCA 9b was replaced by a C-2–C-3 vicinal diol motif in 1, as supported by the 1 H–1H COSY correlations (Fig. 2) of H-3/H2-4/H-5 and H2-15/H2-16/H17 and HMBC correlations (Fig. 2) from H3-2′ (δH 1.07) to C-1/C-2/C-3 (δC 80.5), from H-17 (δH 3.79) to C-18 (δC 199.0), and from a methoxy proton (δH 3.48) to C-17 (δC 81.1). Therefore, the planar structure of 1 was determined. In the NOESY experiment (Fig. 3), the correlations of H3-10′/H-11, H-11/H3-6′, H3-6′/H-5, and H2-4 (δH 1.75/2.25)/H-7 suggested that H310′ and H-11 were all β-oriented, while H-7 was α-oriented, and the double bond between C-5 and C-6 was Z-configuration. However, the configurations of C-3 and C-17 were unable to be determined by analyzing the observed NOESY data alone. Fortunately, after repeated recrystallization, a suitable crystal of 1 was collected from MeOH (with two drops of water), which was then subjected to a single-crystal X-ray diffraction experiment with Cu Kα radiation (Fig. 4). The Flack parameter of 0.02(3) (CCDC 1869928) permitted our definition of its absolute configuration to be 3R,7R,10R,11S,17R. Furthermore, a modified Mosher’s method was conducted, and the significant ΔδH values (ΔδH = δS-MTPA-ester – δR-MTPA-ester) of the proton signals adjacent to C-3 were observed in Fig. 5. Judging from the rule of a modified Mosher’s method [11], C-3 was confirmed to be an R-configuration, which further supported our above conclusion. Tricycloalternarene R (2) was obtained as colorless oil with the molecular formula C21H32O5, based on the HRESIMS data at m/z 387.2156 [M + Na]+ (calcd. for C21H32O5Na+, 387.2142). Detailed analyses of the 1D NMR data of 2 (Table 1) revealed that its structure highly resembled that of 1, with both compounds sharing the identical carbon skeletons and substitution patterns, except for that a methoxy group at C-17 (δC 81.1) in 1 was replaced by a hydroxyl group at the

Fig. 2. Key 1H–1H COSY and HMBC correlations of 1–7.

(R)-MTPA ester of 1 (1b). 1H NMR (CDCl3, 400 MHz) δ: 1.11 (3H, s, H3-1), 1.15 (3H, s, H3-2′), 2.04 (1H, m, H-4a), 2.26 (1H, m, H-4b), 5.05 (1H, m, H-5), 1.52 (3H, s, H3-6′). (S)-MTPA ester of 2 (2a). 1H NMR (CDCl3, 400 MHz) δ: 0.99 (3H, s, H3-1), 0.99 (3H, s, H3-2′), 2.15 (1H, m, H-4a), 2.30 (1H, m, H-4b), 5.19 (1H, m, H-5), 1.58 (3H, s, H3-6′). (R)-MTPA ester of 2 (2b). 1H NMR (CDCl3, 400 MHz) δ: 1.03 (3H, s, H3-1), 1.05 (3H, s, H3-2′), 2.10 (1H, m, H-4a), 2.19 (1H, m, H-4b), 5.06 (1H, m, H-5), 1.53 (3H, s, H3-6′). (S)-MTPA ester of 3 (3a). 1H NMR (CDCl3, 400 MHz) δ: 4.92 (1H, s, H-1a), 4.93 (1H, s, H-1b), 1.58 (3H, s, H3-2′), 2.23 (1H, m, H-4a), 2.35 (1H, m, H-4b), 5.13 (1H, m, H-5), 1.61 (3H, s, H3-6′). (R)-MTPA ester of 3 (3b). 1H NMR (CDCl3, 400 MHz) δ: 4.97 (1H, s, H-1a), 5.02 (1H, s, H-1b), 1.68 (3H, s, H3-2′), 2.13 (1H, m, H-4a), 2.33 (1H, m, H-4b), 5.04 (1H, m, H-5), 1.59 (3H, s, H3-6′). (S)-MTPA ester of 5 (5a). 1H NMR (pyridine‑d5, 400 MHz) δ: 1.34 (3H, s, H3-1), 1.31 (3H, s, H3-2′), 2.38 (2H, m, H2-4), 5.58 (1H, m, H-5), 1.65 (3H, s, H3-6′). (R)-MTPA ester of 5 (5b). 1H NMR (pyridine‑d5, 400 MHz) δ: 1.43 (3H, s, H3-1), 1.40 (3H, s, H3-2′), 2.25 (2H, m, H2-4), 5.34 (1H, m, H-5), 1.64 (3H, s, H3-6′). 2.7. Cytotoxicity assay Five human tumor cell lines, including MM231 (breast cancer), MM468 (breast cancer), HeLa (cervical cancer), SW1990 (pancreatic cancer), and SW480 (colon cancer), and one normal hepatic cell, LO2, were used in the cytotoxicity assays. All cells were cultured in RPMI1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere containing 5% CO2. The cell survival assay was carried out with the previously reported MTT method.[8] Briefly, 100 μL of the adherent cells was seeded into each well of the 96-well culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before addition of the test compound, both with an initial density of 1 × 105 cells/mL in 100 μL of medium. Each human tumor cell line was exposed for 48 h in triplicate to the test compounds at concentrations of 0.0625, 0.32, 1.6, 8, and 40 μM, with DDP (cisplatin, Sigma) as the positive control. After incubation, culture supernatants were removed and exchanged with medium containing 0.5 mg/ mL MTT. Then, the cells were incubated for 4 h at 37 °C in darkness,

Fig. 3. Key NOESY correlations of 1. 4

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Therefore, the absolute structure of 2 was determined. Tricycloalternarene S (3) was determined to have a molecular formula of C21H30O4 on the basis of its HRESIMS data at m/z 369.2050 [M + Na]+ (calcd. for C21H30O4Na+, 369.2036). The 1H and 13C NMR spectroscopic data (Table 1) of 3 were closely similar to those of tricycloalternarene F,[10] suggested that 3 was also a tricycloalternarene derivative, with the differences being that a double bond from C-2/C-3 in tricycloalternarene F migrated to C-1 (δC 112.1)/C-2 (δC 148.0) in 3 and an additional hydroxyl group existed at C-3 (δC 77.2) in 3, as confirmed by the HMBC correlations (Fig. 2) from H2-1 to C-2, C-2′, and C-3. The similar NOESY data of 3 and 2 suggested that both compounds possessed the identical absolute configurations for C-7, C-10, and C-11 and Z-geometry of the double bond between C-5 and C-6. The absolute configuration of C-15 in 3 was determined to be the same (S-configuration) as that of tricycloalternarene F and tricycloalterfurene A[12] by comparison of their experimental CD spectra showing an identical positive Cotton effect at approximately 258 nm (Fig. 6). The absolute configuration of C-3 was deduced as R by interpretation of the 1H NMR chemical shift differences (ΔδS − δR) between Mosher esters (3a vs 3b) (Fig. 5). Therefore, the absolute structure (3R,7R,10R,11S,15S) of 3 was determined as depicted. Tricycloalternarene T (4) was deduced to have the same molecular formula C22H34O5 as that of 1, according to its HRESIMS analysis at m/z 401.2304 [M + Na]+ (calcd. for C22H34O5Na+, 401.2298). Comparison of the 1D and 2D NMR data (Table 2) of 1 and 4 suggested that they were a pair of epimers, differing only in the replacement of a methoxy group at C-17 (δC 81.1) in 1 by a methoxy group at C-15 (δC 76.2) in 4, which as evidenced by the HMBC correlations (Fig. 2) from H-15 (δH 3.86) to C-13 (δC 109.4) and C-14 (δC 169.8) and from a methoxy proton (δH 3.49) to C-15 as well as the key 1H−1H correlations (Fig. 2) of H-15/H2-16/H2-17. The experimental CD spectrum of 4 matched well with that of 3 (Fig. 6), suggesting the configuration of C15 to be S. Therefore, the absolute configuration of 4 was determined as 3R,7R,10R,11S,15S. Tricycloalternarene U (5) was determined to have a molecular formula of C21H32O5 based on its HRESIMS data at m/z 387.2141 [M + Na]+ (calcd. for C21H32O5Na+, 387.2142), which was 14 mass units less than that of 4. Analysis of the 1D NMR data (Table 2) of 5 revealed that the structure of 5 resembled that of 4, with the only difference occurring at C-15. A deshielded proton at δH 4.37 was bonded to a shielded carbon (C-15) at δC 66.4 through the HSQC correlation in 5, suggesting that a hydroxyl group was linked to C-15 in 5 rather than a methoxy group at the same position in 4. This conclusion was further confirmed by the HMBC correlations (Fig. 2) from H-15 (δH 4.37) to C13 (δC 107.6) and C-14 (δC 168.7). Similar NOESY spectra and experimental CD curves (Fig. 6) of 4 and 5 implied that both compounds shared the identical absolute configuration (3R,7R,10R,11S,15S). Moreover, a modified Mosher’s method (Fig. 5) supported our conclusion regarding its absolute configuration of C-3. Tricycloalternarene V (6), also obtained as colorless oil, gave a molecular formula of C22H34O5, according to the HRESIMS anslysis at m/z 401.2301 [M + Na]+ (calcd. for C22H34O5Na+, 401.2298), which was 14 mass units more than that of 5. Detailed analysis of its 1D and 2D NMR data (Table 2) suggested that 6 was a methoxy product of 5. The methoxy group was determined to be linked to C-2 by a key HMBC correlation (Fig. 2) from a methoxy proton (δH 3.17) to C-2 (δC 78.5) as well as comparison of the closely similar chemical shifts (δH 3.25/δC 78.5 for 5; δH 3.24/δC 78.3 for 6). Similar NOESY and experimental CD curves (Fig. 6) implied that 6 possessed the same absolute configuration as 5. Tricycloalternarene W (7) was determined to have a a molecular formula of C21H30O3, based on a sodium adduct at m/z 353.2103 ([M + Na]+, calcd. for C21H30O3Na+, 353.2087) in the HRESIMS analysis. Comparison of the 1D NMR data (Table 2) of 7 with those of 5 suggested that the C-2–C-3 vicinal diol motif in 5 was replaced by a double bond in 7, as supported by the HMBC correlations (Fig. 2) from H3-1 (δH

Fig. 4. ORTEP drawing of compound 1.

Fig. 5. ΔδH(S–R) values (in ppm) for the (S)- and (R)-MTPA esters of compounds 1–3 and 5.

same position (δC 72.4, C-17) in 2, as supported by the key HMBC correlations (Fig. 2) from H2-15 to C-14, from H2-16 to C-14 and C-18, and from H-17 to C-18. Accordingly, the planar structure of 2 was determined. The similar NOESY data of 1 and 2 as well as the corresponding biogenesis suggested that 2 also possessed the same R-, R-, and S-configuration for C-7, C-10, and C-11, respectively. Moreover, a modified Mosher’s analysis (Fig. 5) just like 1 determined the configuration of C3 to be R. To determine the absolute configuration of C-17, the experimental CD curve of 2 was recorded in MeOH (Fig. 6), which was in accord with the experimental CD spectrum of 1 with a positive Cotton effect at approximately 262 nm, suggesting C-17 to be R-configuration.

Fig. 6. Experimental CD spectra of compounds 1–7. 5

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potential biosynthetic capacities to produce structurally novel and bioactive natural products.

Table 3 Cytotoxicity of 1–7 against several human cell lines. no.

1 2 3 4 5 6 7 Cis-platina a

IC50 (µM) MM231

MM468

HeLa

SW1990

SW480

LO2

12.83 14.67 > 40 > 40 23.15 > 40 28.68 3.27

> 40 20.7 > 40 > 40 > 40 > 40 > 40 1.43

> 40 > 40 > 40 > 40 > 40 > 40 > 40 2.51

22.81 > 40 > 40 > 40 27.94 > 40 > 40 0.94

17.69 > 40 > 40 > 40 > 40 > 40 32.87 1.72

> 40 > 40 > 40 > 40 > 40 > 40 > 40 1.28

Acknowledgments We greatly thank the Analytical and Testing Center at Huazhong University of Science and Technology for CD, UV, and IR analyses. The generous supports from the Program for Changjiang Scholars of Ministry of Education of the People's Republic of China (No. T2016088), the National Natural Science Foundation of China (Nos. 21702067, 81573316, 31870326, and 81602986), the National Science Fund for Distinguished Young Scholars (No. 81725021), and the Innovative Research Groups of the National Natural Science Foundation of China (No. 81721005) are also gratefully acknowledged.

Cis-platin was used as the positive control.

Declaration of Competing Interest

1.62) to C-2 (δC 132.0), C-2′ (δC 17.8), and C-3 (δC 124.4) and 1H−1H correlations (Fig. 2) of H-3 (δH 4.90)/H2-4 (δH 2.50)/H-5 (δH 5.23). The similar NOESY spectra and experimental CD curves (Fig. 6) of 5 and 7 suggested both compounds shared the identical absolute configurations.

The authors of the present manuscript have declared that no competing interests exist. Appendix A. Supplementary material

3.2. Biological activity assessment

Supplementary data associated with this article including HRESIMS, 1D and 2D NMR, UV, and IR spectra of 1–7 can be found online at https://doi.org/10.1016/j.bioorg.2019.103279.

Compounds 1–7 were evaluated for cytotoxic activity against five human tumor cell lines, including MM231, MM468, HeLa, SW1990, and SW480 cells, and one normal LO2 cell. As shown in Table 3, compounds 1, 2, 5, and 7 showed selective cytotoxic activity against certain human tumor cell lines with IC50 values in the range of 12.83–32.87 μM, with no obvious cytotoxicity to the normal LO2 cell, while other compounds (3, 4, and 6) were inactive with IC50 values of > 40 μM.

References [1] T. Pusztahelyi, I.J. Holb, I. Pócsi, Front. Plant Sci. 6 (2015) 573. [2] S. Kusari, C. Hertweck, M. Spiteller, Chem. Biol. 19 (2012) 792–798. [3] Y. Tang, Y. Xue, G. Du, J. Wang, J. Liu, B. Sun, X.N. Li, G. Yao, Z. Luo, Y. Zhang, Angew. Chem., Int. Ed. 55 (2016) 4069–4073. [4] Z. Hu, W. Sun, F. Li, J. Guan, Y. Lu, J. Liu, Y. Tang, G. Du, Y. Xue, Z. Luo, J. Wang, H. Zhu, Y. Zhang, Org. Lett. 20 (2018) 5198–5202. [5] F. Li, W. Sun, J. Guan, Y. Lu, S. Zhang, S. Lin, J. Liu, W. Gao, J. Wang, Z. Hu, Y. Zhang, Org. Lett. 20 (2018) 7982–7986. [6] F. Li, W. Sun, J. Guan, Y. Lu, S. Lin, S. Zhang, Weixi Gao, Junjun Liu, Du Guang, Jianping Wang, Hucheng Zhu, Changxing Qi, Hu Zhengxi, Yonghui Zhang, Org. Biomol. Chem. 16 (2018) 8751–8760. [7] W. Gao, Y. He, F. Li, C. Chai, J. Zhang, J. Guo, C. Chen, J. Wang, H. Zhu, Z. Hu, Y. Zhang, Bioorg. Chem. 83 (2019) 98–104. [8] B. Yang, W. Sun, J. Wang, S. Lin, X.N. Li, H. Zhu, Z. Luo, Y. Xue, Z. Hu, Y. Zhang, Mar. Drugs 16 (2018) 110. [9] L.J. Reed, H. Muench, Am. J. Hyg. 27 (1938) 493–497. [10] Q.X. Wang, L. Bao, X.L. Yang, H. Guo, B. Ren, L.D. Guo, F.H. Song, W.Z. Wang, H.W. Liu, L.X. Zhang, Fitoterapia 85 (2013) 8–13. [11] I. Ohtani, T. Kusumi, Y. Kashman, H. Kakisawa, J. Am. Chem. Soc. 113 (1991) 4092–4096. [12] Z.Z. Shi, F.P. Miao, S.T. Fang, X.H. Liu, X.L. Yin, N.Y. Ji, J. Nat. Prod. 80 (2017) 2524–2529.

4. Concluding remarks In conclusion, seven previously undescribed meroterpenoids, tricycloalternarenes Q–W (1–7), were characterized from fungus Alternaria brassicicola. Their structures including absolute configurations were determined by extensive NMR spectroscopic data, a modified Mosher’s method, X-ray crystallographic analysis, and experimental CD spectral analyses. Additionally, compounds 1, 2, 5, and 7 showed selective cytotoxic activity against certain human tumor cell lines with IC50 values in the range of 12.83–32.87 μM, with no obvious cytotoxicity to the normal LO2 cell. These findings not only expanded the new members of tricycloalternarenes, but also implied that phytopathogenic fungi should receive much more attention from scientific community for their

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