Polyoxygenated Cyclohexenoids with Promising α-Glycosidase

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Cite This: J. Agric. Food Chem. 2018, 66, 1140−1146

Polyoxygenated Cyclohexenoids with Promising α‑Glycosidase Inhibitory Activity Produced by Phomopsis sp. YE3250, an Endophytic Fungus Derived from Paeonia delavayi Rong Huang,‡ Bo-Guang Jiang,† Xiao-Nian Li,§ Ya-Ting Wang,† Si-Si Liu,† Kai-Xuan Zheng,† Jian He,*,⊥ and Shao-Hua Wu*,† †

Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, China ‡ School of Chemical Science and Technology, Yunnan University, Kunming 650091, China § State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China ⊥ Group of Peptides and Natural Products Research, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China S Supporting Information *

ABSTRACT: Seven new polyoxygenated cyclohexenoids, namely, phomopoxides A−G (1−7), were isolated from the fermentation broth extract of an endophytic fungal strain Phomopsis sp. YE3250 from the medicinal plant Paeonia delavayi Franch. The structures of these compounds were established by spectroscopic interpretation. The absolute configurations of compounds 1 and 4 were confirmed by X-ray crystallographic analysis and chemical derivative approach. All isolated compounds showed weak cytotoxic activities toward three human tumor cell lines (Hela, MCF-7, and NCI-H460) and weak antifungal activities against five pathogenic fungi (Candida albicans, Aspergillus niger, Pyricularia oryzae, Fusarium avenaceum, and Hormodendrum compactum). In addition, compounds 1−7 showed a promising α-glycosidase inhibitory activity with IC50 values of 1.47, 1.55, 1.83, 2.76, 2.88, 3.16, and 2.94 mM, respectively, as compared with a positive control of acarbose (IC50 = 1.22 mM). KEYWORDS: Phomopsis, Paeonia delavayi, polyoxygenated cyclohexenoids, α-glucosidase inhibitor

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INTRODUCTION Endophytic fungi have gained much more attention from natural product chemists because of their extraordinary abilities in producing diverse and structurally unprecedented secondary metabolites.1−6 Most of these metabolites are potential candidates for drug discovery. Among them, Phomopsis is a creative genus known for making a large number of exclusive and structurally significant bioactive compounds.7 Interestingly, many Phomopsis species are symbiotic prevalently inside their host plant growing in temperate and tropical regions,7 which intrigued us to search for novel metabolites from these materials. As a consequence, in our previous work, several new compounds including ten-membered lactones and steroids have been isolated and identified from endophytic species of Phomopsis.8,9 The medicinal plant Paeonia delavayi Franch. is an important source of traditional Chinese medicine “mudanpi”. It is not only a major herbal material used as an anti-inflammatory and sedative agent, but also curing cardiovascular and female diseases in oriental traditional medicine.10−12 To our surprise, up to now, minimal work has been performed on the endophytic fungi from this plant except for our previous report on new sesquiterpenes from an endophytic Trichoderma sp.13 As a result of our continuous bioactive screening of the endophytic fungi isolated from P. delavayi, the EtOAc extract of the fermentation broth from the strain Phomopsis sp. YE3250 exhibited potent α-glucosidase inhibitory activity with an IC50 © 2018 American Chemical Society

value of 1.08 mg/mL, meanwhile the IC50 value of positive control (acarbose) was 0.79 mg/mL. In this paper, we report the isolation and structure elucidation of bioactive secondary metabolites from this strain, as well as their α-glycosidase inhibitory activity. Besides that, the in vitro cytotoxic and antifungal activities of the metabolites are also addressed.

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MATERIALS AND METHODS

General. An XRC-1 apparatus was used for measuring melting points (mp), and the reported data were uncorrected. A HORIBA SEPA-300 polarimeter was used to obtain optical rotations. A Bio-Rad FTS-135 infrared spectrophotometer was used for recording infrared (IR) spectra with KBr pellets. Ultraviolet (UV) spectra were obtained on Shimadzu double-beam 210A spectrophotometer. Circular dichroism (CD) spectra were measured with an Applied Photophysics Chirascan spectrometer. Nuclear magnetic resonance (NMR) spectra were acquired on a Bruker DRX-500 spectrometer with tetramethylsilane (TMS) as internal standard. High-resolution electrospray ionization mass spectrometry (HR-ESIMS) was performed on an Agilent G3250AA LC/MSD TOF spectrometer. Silica gel (200−300 mesh, Qingdao Marine Chemical Inc., China), reversed-phase silica gel C18 (40−63 μm, Merck), and Sephadex LH-20 (Pharmacia) were employed for column chromatography (CC). Received: Revised: Accepted: Published: 1140

October 27, 2017 January 5, 2018 January 15, 2018 January 15, 2018 DOI: 10.1021/acs.jafc.7b04998 J. Agric. Food Chem. 2018, 66, 1140−1146

Article

1141

2.49, t (7.4) 1.53−1.54, m 1.23−1.30, m 0.88, t (6.6) 5.84, dt (15.1, 6.4) 2.05, d (6.7) 1.36−1.38, m 1.26−1.35, m 0.88, t (6.6) 4.06, q (6.7) 1.53−1.57, m 1.47−1.50, m 1.31−1.40, m 0.87, t (6.5) 5.81, dt (15.0, 7.0) 2.09, q (7.0) 1.41−1.43, m 1.32−1.40, m 0.92, t (6.5) 4.13, q (6.4) 1.53−1.57, m 1.40−1.42, m 1.33−1.40, m 0.92, t (6.5) a

Measured in CD3OD bMeasured in CDCl3.

5.82, dt (15.3, 6.8) 2.08, q (6.8) 1.40−1.42, m 1.32−1.40, m 0.92, t (6.3) 4.01, q (6.4) 1.31−1.33, m 1.20−1.28, m 1.20−1.28, m 0.80, t (6.3) 4.13, q (6.5) 1.55−1.59, m 1.40−1.42, m 1.33−1.40, m 0.92, t (5.3)

5.95, d (11.3) 5.53, dd (11.3, 6.4) 6.45, d (15.9) 5.94, dd (15.9, 6.5) 1′ 2′

3′ 4′ 5′ 6′−10′ 11’

4.46, br s 3.57, br s 3.54, br s 4.49, br s 4.86, br d (9.8) 4.53, d (9.8) 5.28, br s 5.39, dd (15.1, 7.8) 4.37, br s 3.57, t (2.8) 3.52, t (2.5) 4.61, br s 4.34, d (11.7) 3.99, d (11.7) 6.14, d (11.3) 5.69, dd (11.3, 8.8) 4.20, d (7.5) 3.57, dd (11.0, 7.5) 3.82, dd (11.0, 8.0) 4.24, d (8.0) 4.79, dd (13.0, 4.0) 4.62, d (13.0) 5.27, br s 5.44, dd (15.5, 7.5) 4.66, br s 3.52, t (3.4) 3.45, t (2.6) 4.58, br s 4.39, d (12.1) 4.22, d (12.1) 6.38, d (16.0) 5.98, dd (16.0, 6.6) 4.18, d (6.7) 3.46, dd (10.1, 7.2) 3.50, dd (10.1, 7.0) 4.06, d (7.0) 4.78, dd (12.8, 3.9) 4.61, dd (12.8, 4.5) 5.27, br s 5.46, dd (15.3, 7.5) 3.83, d (5.5) 3.36, dd (10.4, 7.6) 3.31, dd (10.4, 7.3) 4.15, d (7.1) 4.10, br s 4.22, d (6.1) 3.55, dd (9.3, 6.9) 3.48, dd (9.3, 7.4) 4.26, d (6.6) 4.37, br s 3 4 5 6 7

4a 3a 2a 1a position

Table 1. 1H NMR Data of Compounds 1−7 and 4a (δ in ppm, J in Hz, 500 MHz)

4aa

5b

6b

7b

Fungal Material. The endophytic strain YE3250 was isolated from the fresh stems of Paeonia delavayi in Songming County, Yunnan, China, following an isolation protocol described previously.13 The strain was deposited in our institute. It was identified to be Phomopsis sp. on the basis of morphological characteristic and analysis of its internal transcribed spacer (ITS) sequences (GenBank accession no. KF733183) according to the reported method previously.8 Fermentation, Extraction, and Isolation. The fresh mycelium of the strain was grown on potato dextrose agar (PDA) medium at 28 °C for 7 days. Then, it was inoculated into 500 mL Erlenmeyer flasks (×50) containing 120 mL of potato dextrose broth (PDB) medium (200 g of potato and 20 g of dextrose in 1 L of H2O). After the culture solutions were incubated at 28 °C with shaking (200 rpm) for 4 days, 30 mL of each seed culture solution was added into 1 L Erlenmeyer flasks (×150) containing 300 mL of PDB medium. The following cultivation was carried out at 28 °C with shaking (200 rpm) for 7 days. The culture broth was filtered and concentrated to 10 L and then sufficiently partitioned with EtOAc (10 L) for three times. After evaporating the organic solvent under vacuum, the final brown gum (32.6 g) was obtained and chromatographed on silica gel column, eluting gradiently with a mixture solvents of CHCl3/MeOH from 1:0 to 0:1 (v/v) to obtain seven fractions (Frs. 1−7). The α-glucosidase inhibitory activity test showed that fractions 3−6 were active. Fr. 3 (2.1 g) was purified by CC over reversed-phase silica gel C18, eluting with MeOH/H2O (3:7, 4:6, 1:1, 6:4), and then on silica gel, eluting with petroleum ether (PE)/ acetone (7:3, 6:4) afforded compound 6 (3.9 mg) and compound 7 (6.3 mg). Further separation of Fr. 4 (2.9 g) by CC over reversed-phase silica gel C18 (MeOH/H2O, 1:1, 6:4, 7:3) and Sephadex LH-20 (MeOH) obtained compound 4 (50.6 mg) and compound 5 (8.7 mg). Purification of Fr. 5 (1.8 g) by CC over Sephadex LH-20 (MeOH) and silica gel (CHCl3/MeOH, 9:1) yielded compound 3 (5.7 mg). Fr. 6 (2.6 mg) was separated by CC over silica gel (CHCl3/MeOH, 9:1, 8:2) and reversed-phase silica gel C18 (MeOH/H2O, gradiently from 2:8 to 4:6) to give compound 1 (15.5 mg) and compopund 2 (7.4 mg). Phomopoxide A (1). The title compound was obtained as colorless crystals; TLC Rf 0.36 (CHCl3/MeOH, 85:15); mp 186−187 °C; [α]20 D − 14.8 (c 0.31, MeOH); UV (MeOH) λmax (log ε) 238 (4.28) nm; CD (c 0.29, MeOH) λmax (Δε) 233 (−3.26) nm; IR (KBr) νmax 3377 (br), 3331, 2955, 2922, 2852, 1643, 1463, 1406, 1384, 1217, 1040, 996 cm−1; HRESIMS m/z 367.2095 [M + Na]+ (calcd for C18H32O6Na, 367.2097); 1H and 13C NMR spectral data, see Tables 1 and 2. Phomopoxide B (2). The title compound was obtained as a white powder; TLC Rf 0.39 (CHCl3/MeOH, 85:15); [α]20 D + 25.7 (c 0.21, MeOH); UV (MeOH) λmax (log ε) 197 (4.02) nm; CD (c 0.64, MeOH) λmax (Δε) 196 (+7.49), 222 (+2.48) nm; IR (KBr) νmax 3457 (br), 3211, 2958, 2928, 2857, 1666, 1462, 1266, 1106, 996 cm−1; HRESIMS m/z 367.2094 [M + Na]+ (calcd for C18H32O6Na, 367.2097); 1H and 13C NMR spectral data, see Tables 1 and 2. Phomopoxide C (3). The title compound was obtained as a white powder; TLC Rf 0.48 (CHCl3/MeOH, 85:15); [α]20 D − 35.1 (c 0.23, MeOH); IR (KBr) νmax 3405 (br), 3377, 2955, 2926, 2856, 1642, 1403, 1251, 1090, 994 cm−1; HRESIMS m/z 349.1993 [M + Na]+ (calcd for C18H30O5Na, 349.1991); 1H and 13C NMR spectral data, see Tables 1 and 2. Phomopoxide D (4). The title compound was obtained as a white powder; TLC Rf 0.23 (PE/acetone, 6:4); [α]20 D − 68.3 (c 0.38, MeOH); UV (MeOH) λmax (log ε) 237 (4.25) nm; CD (c 0.27, MeOH) λmax (Δε) 234 (−9.87) nm; IR (KBr) νmax 3366 (br), 2957, 2925, 2855, 1638, 1465, 1257, 1087, 1030, 966 cm−1; HRESIMS m/z 349.1985 [M + Na]+ (calcd for C18H30O5Na, 349.1991); 1H and 13C NMR spectral data, see Tables 1 and 2. Phomopoxide E (5). The title compound was obtained as a white powder; TLC Rf 0.37 (PE/acetone, 6:4); [α]20 D + 9.1 (c 0.24, MeOH); UV (MeOH) λmax (log ε) 196 (4.02) nm; CD (c 0.64, MeOH) λmax (Δε) 204 (−4.65), 233 (−1.08) nm; IR (KBr) νmax 3346 (br), 3194, 2962, 2932, 2858, 1633, 1495, 1252, 1098, 1012, 960 cm−1; HRESIMS m/z 349.1997 [M + Na]+ (calcd for C18H30O5Na, 349.1991); 1H and 13C NMR spectral data, see Tables 1 and 2. Phomopoxide F (6). The title compound was obtained as a colorless gum; TLC Rf 0.51 (PE/acetone, 6:4); [α]20 D − 28.7 (c 0.17, MeOH); IR

4.56, br s 3.57, t (3.2) 3.52, t (2.8) 4.48, br s 4.76, ddd (9.4, 3.2, 2.4) 4.51, d, (9.4) 5.27, br s 2.93, dd (15.8, 5.2) 2.73, dd (15.8, 5.7)

Journal of Agricultural and Food Chemistry

DOI: 10.1021/acs.jafc.7b04998 J. Agric. Food Chem. 2018, 66, 1140−1146

Article

Journal of Agricultural and Food Chemistry Table 2. 13C NMR Data of Compounds 1−7 and 4a (δ in ppm, 125 MHz)

a

position

1a

2a

3a

4a

4aa

5b

6b

7b

1 2 3 4 5 6 7 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′

136.8 135.9 74.6 76.7 76.3 74.5 59.7 126.3 139.0 74.4 38.8 27.0 31.5 31.2 30.8 33.5 24.1 14.9

135.9 135.6 76.9 77.3 77.1 75.2 61.4 128.4 138.2 69.2 38.3 26.9 31.2 31.1 30.8 33.5 24.1 14.8

136.1 137.5 71.6 78.7 79.2 71.4 75.5 88.4 129.9 135.5 33.6 30.6 31.1 31.0 30.8 33.5 24.1 14.8

134.6 134.1 66.7 56.6 55.9 65.3 60.7 126.1 139.5 74.4 38.8 27.0 31.2 31.2 30.8 33.5 24.1 14.9

135.8 135.0 69.8 76.8 68.5 70.5 73.6 86.7 127.9 134.0 31.8 28.7 29.1 29.0 28.8 31.6 22.3 13.1

132.0 133.2 67.7 54.5 55.6 67.1 64.2 128.5 136.9 68.4 37.4 25.9 30.1 30.0 29.9 32.3 23.0 14.5

135.1 132.6 62.8 55.3 58.3 61.7 75.0 87.3 127.9 135.9 32.9 30.1 29.5 29.5 29.3 32.8 23.1 14.5

133.1 134.6 63.2 54.9 57.3 61.4 75.3 82.5 46.9 211.5 44.6 23.9 32.2 29.7 29.5 29.5 23.0 14.4

Measured in CD3OD bMeasured in CDCl3.

Figure 1. Chemical structures of compounds 1−7 and 4a. νmax 3358, 2953, 2916, 2852, 1629, 1469, 1217, 1128, 1100, 963 cm−1; HRESIMS m/z 367.1652 [M + Na]+ (calcd for C18H29O4ClNa, 367.1652); 1H and 13C NMR spectral data, see Tables 1 and 2. X-ray Crystallography of Compounds 1 and 4a. The crystallographic data of 1 and 4a were collected with Cu Kα radiation (λ = 1.54178 Å) and graphite-monochromated Mo Kα radiation (λ = 0.71073 Å), respectively, at T = 100 (2) K on the Bruker APEX DUO diffractometer, equipped with an Oxford Cryostream 700+ cooler. The structures were solved using direct method with SHELXS-97 and then refined using full-matrix least-squares on F2 with SHELXL package software.14 The absolute configurations were determined by refinement of the Flack parameter15 on the basis of resonant scattering of the light atoms. Crystallographic Data for 1. C18H32O6·H2O, Mr = 362.45, orthorhombic, crystal dimensions 0.09 × 0.12 × 0.90 mm, space group P212121, a = 4.668 Å, b = 9.78500(10) Å, c = 41.7773(5) Å, V = 1908.24(3) Å3, α = 90.00°, β = 90.00°, γ = 90.00°, Z = 4, Dcalcd = 1.262 mg/m3, F(000) = 792, μ(CuKα) = 0.790 mm−1, Tmin/Tmax = 0.54/0.93, 2.11° ≤ θ ≤ 69.62°, 11914 reflections measured, 3359 unique reflections (Rint = 0.0229), final R1 = 0.0278 and wR(F2) = 0.0742 (I > 2σ(I)), final

(KBr) νmax 3446, 3367 (br), 2950, 2924, 2851, 1464, 1412, 1347, 1258, 1119, 1090, 971 cm−1; HRESIMS m/z 331.1887 [M + Na]+ (calcd for C18H28O4Na, 331.1885); 1H and 13C NMR spectral data, see Tables 1 and 2. Phomopoxide G (7). The title compound was obtained as a colorless gum; TLC Rf 0.47 (PE/acetone, 6:4); [α]20 D − 15.1 (c 0.21, MeOH); IR (KBr) νmax 3451 (br), 3387, 2956, 2927, 2856, 1711, 1466, 1408, 1351, 1271, 1120, 1038, 943 cm−1; HRESIMS m/z 347.1837 [M + Na]+ (calcd for C18H28O5Na, 347.1834); 1H and 13C NMR spectral data, see Tables 1 and 2. Chemical Transformation of 4 to 4a. In the initial step, 50 μL of concentrated HCl (37%) was added into the MeOH solution (5 mL) of compound 4 (10 mg). The transformation was performed by stirring for 1 h at 38 °C. The reaction mixture was concentrated and then added to 10 mL with CHCl3. The obtained solution was washed sufficiently with 10 mL of saturated NaHCO3 for three times and neutralized to pH 7. The CHCl3 extract was concentrated and purified by CC over silica gel eluting with PE/acetone (8:2) and then crystallized in MeOH/H2O (1:1) to afford 4a (8.3 mg). Compound 4a was obtained as colorless crystals; TLC Rf 0.56 (PE/ acetone, 6:4); mp 173−174 °C; [α]20 D −54.7 (c 0.24, MeOH); IR (KBr) 1142

DOI: 10.1021/acs.jafc.7b04998 J. Agric. Food Chem. 2018, 66, 1140−1146

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Journal of Agricultural and Food Chemistry R1 = 0.0280 and wR(F2) = 0.0744 (all data), goodness of fit on F2 = 1.096. The Flack parameter is 0.06(13). Crystallographic Data for 4a. C18H29ClO4·H2O, Mr = 362.88, monoclinic, crystal dimensions 1.14 × 0.45 × 0.09 mm, space group P2(1), a = 4.6368(6) Å, b = 7.8718(9) Å, c = 25.885(3) Å, α = 90°, β = 93.657(2)°, γ = 90°, V = 942.9(2) Å3, Z = 2, Dcalcd = 1.278 mg/m3, F(000)= 392, μ(MoKα) = 0.226 mm−1, Tmin/Tmax = 0.78/0.98, 10463 reflections measured, 5362 unique reflections (Rint = 0.0217), final R1 = 0.0275 and wR(F2) = 0.0737 (I > 2σ(I)), final R1 = 0.0307 and wR(F2) = 0.0811 (all data), goodness of fit on F2 = 1.133. The Flack parameter is 0.016(17). The above crystallographic data have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication numbers CCDC 966947 for compound 1 and CCDC 1577168 for compound 4a. These data can be acquired free of charge via www.ccdc. cam.ac.uk/data_request/cif. α-Glucosidase Inhibitory Effect Assay. Evaluations of all isolated compounds for enzyme inhibitory activity against α-glycosidase were conducted following the method reported previously,16,17 by using acarbose as a positive control. Cytotoxicity Assay. Three cancer cell lines, human cervical carcinoma (Hela), human breast carcinoma (MCF-7), and human nonsmall cell lung carcinoma (NCI-H460), were employed in this experiment. The in vitro cytotoxic activities of compounds 1−7 and 4a were assessed by using tetrazolium-based colorimetric MTT (3-(4,5dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, as described in the literature.18 Each cell line was tested in triplicates with paclitaxel (Sigma, U.S.A.) as a positive control. Cytotoxic effects were estimated by comparing the cell survival rate of compound-treated with that of solvent-treated. The IC50 values were calculated by using Reed and Muench’s method.19 Antifungal Assays. Five pathogenic fungal strains, Candida albicans (YM 2005), Aspergillus niger (YM 3029), Pyricularia oryzae (YM 3051), Fusarium avenaceum (YM 3065), and Hormodendrum compactum (YM 3077) were used as indicators in this experiment. The minimal inhibitory concentrations (MICs) for antifungal activity were measured by the broth microdilution method described previously9 with nystatin as positive control.

Figure 2. Selected HMBC and 1H−1H COSY correlations of compound 1.

multiple bond correlations (HMBCs) for both oxymethine protons at δH 4.22 (H-3) and 4.26 (H-6) to C-1 (δC 136.8), C-2 (δC 135.9), C-4 (δC 76.7), and C-5 (δC 76.3) established construction of a cyclohexene moiety (Figure 2). The tracking cross-peaks between H-2′ (δH 5.94) and H-1′ (δH 6.45) and H-3′ (δH 4.13) in the 1H−1H COSY spectrum deduced that the transdouble bond was connected with an aliphatic linear chain through an oxymethine group. The double bond was linked to C2 in the monocyclic system based on the HMBC correlations from H-2′ to C-2 and from H-1′ to C-1 and C-2. Similarly, the hydroxy methylene group at δC 59.7 (C-7) was attached to C-1 in the cyclohexene ring by the HMBC correlations from δH 4.37 (H-7) to C-1, C-2 and C-6. Thus, the planar structure of 1 was assigned. The 6.1−9.3 Hz coupling constants observed for the contiguous protons of H-3/H-4, H-4/H-5, and H-5/H-6 suggested that these four hydrogens are trans relationship to each other, which was also supported by selective 1D nuclear Overhauser effect spectroscopy (NOESY) experiment. The selective irradiation of H-4 (δH 3.55) caused an NOE enhancement only for the signal of H-6 (δH 4.26). In a similar way, the selective irradiation of H-5 (δH 3.48) led to NOE intensification only for the signal of H-3 (δH 4.22). A single colorless crystal of compound 1 was obtained from a mixed solvent of methanol and acetone (1:5) after several crystallization attempts. Its absolute configuration was ultimately established by a low-temperature single crystal X-ray diffraction experiment performed with Cu Kα. The last Flack parameter 0.06(13) and Hooft parameter 0.03(3) unambiguously confirmed the stereochemistry of 3R, 4S, 5S, 6R in the cyclohexene moiety, and C-3′ as R in the side chain (Figure 3). Consequently, the structure of compound 1 was formulated as in Figure 1 and given its name of phomopoxide A.

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RESULTS AND DISCUSSION Structural Elucidation. The current chemical investigations on fermentation product of Phomopsis sp. YE3250 resulted in the isolation of seven new polyoxygenated cyclohexenoids, named as phomopoxides A−G (1−7, Figure 1). Their structures were respectively determined by one-dimensional (1D) and twodimensional (2D) NMR experiments, combined with X-ray crystallographic analysis. The molecular formula of phomopoxide A (1) was determined as C18H32O6 according to its positive-ion HRESIMS at m/z 367.2095 [M + Na]+ (calcd 367.2097) and NMR spectroscopic data. The 1H NMR spectrum (Table 1) exhibited a primary methyl signal at δH 0.92 (t, J = 5.3 Hz), five oxymethine protons (δH 3.48, 3.55, 4.13, 4.22, 4.26), an oxymethylene singlet at δH 4.37, and two olefinic protons at δH 6.45 (d, J = 15.9 Hz) and 5.94 (dd, J = 15.9, 6.5 Hz), which indicated a trans-double bond. The 13 C NMR spectrum of compound 1 (Table 2) displayed 18 signals including four olefinic carbons, five oxymethines, one oxymethylene, seven aliphatic methylenes, and a methyl group. A monocyclic structure was proposed for 1 by three degrees of unsaturation deduced from the molecular formula except for two double bonds. Furthermore, a broad IR absorption band at 3377 cm−1, together with the signals of five oxygenated carbons in the 13 C NMR spectrum, indicated the presence of multiple hydroxy groups. Interpretation of the 1H−1H correlation spectroscopy (COSY) spectrum (Figure 2) revealed that four adjacent oxymethine groups were linked together. The heteronuclear

Figure 3. X-ray crystallographic structure of compound 1.

Phomopoxide B (2) was isolated as a white powder and determined to have the same molecular formula of C18H32O6 as 1 because of its HRESIMS at m/z 367.2094 [M + Na]+. The NMR spectroscopic data of 2 showed only slight differences in chemical shifts with those of 1, suggesting that they were stereoisomers. On the basis of the similar coupling constants for each of H-3/H4, H-4/H-5, and H-5/H-6 with those of 1, together with the view of their biogenetic consideration, the chiral centers of cyclohexene moiety in 2 could be assigned as the same 3R, 4S, 5S, 6R1143

DOI: 10.1021/acs.jafc.7b04998 J. Agric. Food Chem. 2018, 66, 1140−1146

Article

Journal of Agricultural and Food Chemistry

disubstituted epoxide20 in compound 4. The 1H−1H COSY spectrum showed the couplings of each epoxide proton with one of the other two oxymethine protons at δH 4.66 (H-3) and 4.58 (H-6) for a small coupling constant. Thus, the substructure of C3−C6 unit could be confirmed. Further analysis of 2D NMR spectra indicated that the aliphatic side chain was linked to C-2 in a same manner with 1. The planar structure of 4 was accordingly determined, containing an epoxy unit between C-4 and C-5. Chlorination of compound 4 was carried out in the hope of introducing an extra heavy atom in 4 to confirm its absolute configuration by single crystal X-ray analysis. Compound 4 was transformed into 4a as the major product after adding hydrochloric acid to its methanol solution. The structure of 4a could be determined by the elucidation of NMR spectra. A chlorine atom and a hydroxyl group were connected with C-4 and C-5, respectively in 4a, instead of an epoxy moiety. With the similarity of compound 3, the existence of a 2,5-dihydrofuran unit in 4a could be deduced by the weak couplings between the nonequivalent oxymethylene protons H2-7 (δH 4.62 and 4.79) and the oxymethine proton H-1′ (δH 5.27) in the 1H−1H COSY spectrum. A colorless crystal of 4a was obtained in the MeOHH2O (1:1, v/v) mixture solution. It was unambiguously feasible to use single crystal X-ray diffraction analysis for assignment of the absolute configurations of all chiral centers as 3R, 4S, 5S, 6R, 1′R (Figure 4) with the Flack parameter 0.016(17). The X-ray

configuration. A careful comparison of their NMR data indicated that the most obvious difference is the chemical shift of C-3′ at δC 69.2 in 2 and δC 74.4 in 1. This was indicated that compound 2 was an epimer of 1 at C-3′. In the CD spectra of 1 and 2 (Figures S10 and S20 of the Supporting Information), a negative Cotton effect appeared at 233 (Δε − 3.26) nm in 1, while a positive Cotton effect was observed at 196 (Δε + 7.49) and 222 (Δε + 2.48) nm in 2. Furthermore, the opposite optical rotation 20 value of 2 ([α]20 D +25.7) to that of 1 ([α]D −14.8) indicated that compound 2 possessed the different configuration of C-3′ with that in 1. As the absolute configuration of C-3′ in 1 was R, 3′S configuration was accordingly established in 2. The relatively small coupling constant (J = 11.3 Hz) for H-1′ with H-2′ clearly confirmed Z-geometry of the Δ1′-double bond in the aliphatic side chain. Thus, the structure of compound 2 was formulated as in Figure 1 and given its name of phomopoxide B. Phomopoxide C (3) was isolated as a white powder, and possessed the molecular formula of C18H30O5 due to its HRESIMS at m/z 349.1993 [M + Na]+ (calcd 349.1991), which was 18 mass units less than that of 1. The NMR spectrometry of 3 showed resemblance to that of 1. Comprehensive analysis of its 2D NMR spectra led to the determination of the 3,4,5,6-tetrahydroxycyclohexene moiety. The main difference in 13C NMR spectrum between 3 and 1 was the downfield chemical shift of the oxymethylene group at C-7 to δ 75.5 and one of the oxymethine group to δ 88.4 in 3. These two carbons were directly correlated to the downfield shifted signals of two nonequivalent protons at δH 4.61 (dd, J = 12.2, 4.5 Hz) and 4.78 (dd, J = 12.2, 3.9 Hz), and the oxymethine proton at δH 5.27 (br s), respectively in the 1H NMR spectrum, according to heteronuclear multiple-quantum coherence (HMQC) experiment. Its 1H−1H COSY spectrum showed the weak couplings from two nonequivalent oxymethylene protons at δH 4.61 and 4.78 to H-6 (δH 4.06) and the oxymethine proton at δH 5.27. In addition, the weak correlation between this oxymethine proton and H-3 (δH 4.18) could also be observed. The presence of an additional ring in 3 was supported by four degrees of unsaturation deduced from the molecular formula except for a cyclohexane ring and two double bonds. Based on the above data, it could be easily deduced that the oxymethine group at δC 88.4 (C-1′) was linked not only to C-7 through an oxygen atom but also to the olefinic quaternary carbon C-2 (δC 137.5). Thus, a five-membered ring was formed, though it could not be sufficiently elucidated from the HMBC spectrum. The correlation between the oxymethine proton (δH 5.27) and the olefinic proton (δH 5.46) in 1H−1H COSY spectrum established that the decyl terminated double bond was connected to C-1′. The large coupling constant of J = 15.3 Hz between two olefinic protons at δH 5.46 and 5.82 confirmed the trans-double bond between C-2′ and C-3′. The absolute configuration of cyclohexene moiety in 3 could be also determined as 3R, 4S, 5S, 6R according to its homologues 1 and 2. The stereochemistry of the remaining chiral center C-1′ in compound 3 was assigned to be R on the basis of its negative optical rotation ([α]20 D − 35.1). Thus, the structure of compound 3 was formulated as in Figure 1 and given its name of phomopoxide C. Phomopoxide D (4) was also isolated as a white powder. The quasi-molecular ion peak at m/z 349.1985 [M + Na]+ in HRESIMS suggested a molecular formula of C18H30O5. Its NMR signals displayed resemblance with those of 1, indicating the similar oxygenated cyclohexene ring and the same side chain with 1. The chemical shifts of two oxymethine groups (δH 3.45, 3.52; δC 55.9, 56.6) clearly demonstrated the presence of a 1,2-

Figure 4. X-ray crystallographic structure of compound 4a.

crystallographic analysis of 4a also established that 3-OH and 5OH were β-oriented, whereas 6-OH was in α-orientation, which were identical with those of 1−3. The chlorine atom was added to C-4 from α-orientation due to its less steric hindrance. The relative configuration of epoxy unit in 4 could be easily confirmed as β-orientation based on the structure and configuration of its derivative 4a. The configurations at the chiral centers of C-3 and C-6 were identical to those in 4a. The chemical shift of C-3′ at δC 74.4 in compound 4 was identical to that in compound 1. The CD spectrum of 4 (Figure S38 in Supporting Information) resembled that of 1 with the negative Cotton effect at 234 (Δε − 9.87) nm. These data, combined with the similar negative optical 20 rotation value of 4 ([α]20 D − 68.3) as that of 1 ([α]D − 14.8), implied that both compounds possessed the same 3′R configuration. The absolute configuration of 4 was accordingly established as 3R, 4R, 5S, 6R, 3′R. Consequently, the structure of compound 4 was formulated as in Figure 1 and given its name of phomopoxide D. Phomopoxide (5), isolated as a white powder, possessed the same molecular formula of C18H30O5 as 4 based on its HRESIMS at m/z 349.1997 [M + Na]+. The NMR spectral data of 5 were closely resembled with those of 4. The major difference between these two compounds was the chemical shift of C-3′. In a similar manner with 1 and 2, compound 5 was also the C-3′ epimer of 4. In its 13C NMR spectrum, the chemical shift of C-3′ at δC 68.4 in 5 was close to the signal of C-3′ at δC 69.2 in 2. The CD spectrum 1144

DOI: 10.1021/acs.jafc.7b04998 J. Agric. Food Chem. 2018, 66, 1140−1146

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Journal of Agricultural and Food Chemistry Table 3. α-Glucosidase Inhibitory, Cytotoxic, and Antifungal Activities of Compounds 1−7 and 4a cytotoxicity (IC50, μM)

antifungal (MIC, μg/mL)

compds

α-glucosidase inhibitions (IC50, mM)

H460

Hela

MCF-7

Caa

Anb

Poc

Fad

Hce

1 2 3 4 4a 5 6 7 acarbose paclitaxel nystatin

1.47 1.55 1.83 2.76 2.24 2.88 3.16 2.94 1.22 NT NT

11.22 19.34 6.73 7.37 6.26 15.11 9.06 15.38 NTf 0.06 NT

13.29 9.10 >40 8.15 13.70 16.14 6.25 8.87 NT 0.04 NT

>40 >40 >40 12.77 >40 11.88 8.57 14.24 NT 0.05 NT

256 32 128 64 16 128 128 64 NT NT 8

128 64 256 64 32 256 >512 512 NT NT 8

256 256 128 256 256 512 256 256 NT NT 8

512 >512 512 512 256 >512 512 >512 NT NT 16

128 64 256 256 128 128 256 128 NT NT 8

a

Candida albicans YM 2005. bAspergillus niger YM 3029. cPyricularia oryzae YM 3051. dFusarium avenaceum YM 3065. eHormodendrum compactum YM 3077. fNT not tested.

4.51 and 4.76) in its 1H−1H COSY spectrum indicated the same five-membered dihydrofuran ring formed through the linkage of C1’−O-C7 as that in 6. The carbonyl group was positioned at C-3′ due to HMBC correlations from H-1′ (δH 5.27) and H2-2′ (δH 2.73 and 2.93) to the quaternary carbon at δC 211.5. The close optical rotation value of 7 (−15.1) to that of 6 (−28.7) implied the same 3R, 4R, 5S, 6R, 1′R configuration, which could be also supported by a shared biogenesis for both compounds. Consequently, the structure of compound 7 was formulated as in Figure 1 and given its name of phomopoxide G. Biological Activities. All compounds were tested for their enzyme inhibitory activity against α-glycosidase. As a consequence, compounds 1−7 and 4a showed a promising αglycosidase inhibition with IC50 values of 1.47, 1.55, 1.83, 2.76, 2.88, 3.16, 2.94, and 2.24 mM, respectively, by employing acarbose as a positive control (IC50 = 1.22 mM) (Table 3). It is noted that the inhibitory effects of compounds 4−7, which forming an epoxy moiety, were weaker than those of 1−3, suggesting the tetrahydroxyl substitution in cyclohexene ring is likely to be crucial for α-glycosidase inhibition. Compounds 1−7 and 4a were further assayed in vitro for their cytotoxic activities toward Hela, MCF-7, and NCI-H460 cell lines. Each of them displayed weak activities against the tested tumor cell lines (Table 3). Moreover, these compounds were also evaluated for their antifungal activities toward A. niger, C. albicans, F. avenaceum, H. compactum, and P. oryzae by the broth microdilution method. Among them, compound 4a bearing a chlorine atom possessed the most potent activity against C. albicans and A. niger with MIC values of 16 and 32 μg/mL, respectively. In addition, compound 4 displayed moderate inhibitory activity toward C. albicans and A. niger with MIC value of 64 μg/mL. Compound 2 inhibited the growth of C. albicans, A. niger, and H. compactum with MIC values of 32, 64, and 64 μg/mL, respectively. The remaining five compounds showed only weak antifungal activities toward the tested pathogenic fungal strains. Structurally, this group of new polyoxygenated cyclohexenoids is typical of fungal polyketide metabolites. The cyclohexene ring seems to be formed by the cyclization of a polyketide intermediate. Thus far, these molecules have drawn more and more attention by chemists owing to their characteristics of extensive oxygenation pattern and stereochemical variations.21 Recently, several compounds with similar skeleton had also been isolated from the genus Phoma, 22 Eupenicillium, 23 and Aspergillus.16 Particularly, this group of polyoxygenated cyclo-

of 5 (Figure S56 of the Supporting Information) showed the negative Cotton effect at 204 (Δε − 4.65) and 233 (Δε − 1.08) nm. In addition, the similar positive optical rotation value of 5 20 ([α]20 D + 9.1) to that of 2 ([α]D + 25.7), while the negative optical 20 rotation value of 4 ([α]D − 68.3), indicated an absolute configuration of 3′S in compound 5. The stereochemistry of the other chiral centers in 5 was the same as those of 4 to be 3R, 4R, 5S, 6R from biogenetic consideration. Z-geometry of the Δ1’double bond in side chain could be deduced by the relatively small coupling constant (J = 11.5 Hz) for H-1′ with H-2′. Therefore, the structure of compound 5 was formulated as in Figure 1 and given its name of phomopoxide E. Phomopoxide F (6) was isolated as a colorless gum. The quasimolecular ion peak at m/z 331.1887 [M + Na]+ in its HRESIMS confirmed a molecular formula of C18H28O4, suggesting the presence of five degrees of unsaturation. Comparison of its 1H and 13C NMR spectra with those of 5 indicated the existence of the same 4,5-epoxy cyclohexenoid moiety in 6. Due to five degrees of unsaturation calculated for compound 6, one more ring should present in the structure except for two double bonds, a cyclohexene system, and an epoxy unit. With the similar structural elucidation of 3, a five-membered dihydrofuran ring was formed by the linkage of C-7 with C-1′ through an oxygen atom. This was deduced by the correlations between two nonequivalent oxymethylene protons H2-7 (δH 4.53 and 4.86) and the oxymethine proton H-1′ (δH 5.28) in the 1H−1H COSY spectrum, whereas H2-7 were only showed HMBC correlations with C-1 and C-2. A double bond was located between C-2′ and C-3′ based on the coupling sequence from H-1′ to H-4′ by tracking correlations in the 1H−1H COSY spectrum. A large coupling constant of J = 15.1 Hz between two olefinic protons at δH 5.39 and 5.84 indicated E-geometry of the double bond between C-2′ and C-3′. Interestingly, compound 6 could be also transformed into 4a by adding hydrochloric acid to its methanol solution. Based on the determination of 4a, the absolute configuration of 6 was definitely confirmed to be 3R, 4R, 5S, 6R, 1′R. Thus, the structure of compound 6 was formulated as in Figure 1 and given its name of phomopoxide F. Phomopoxide G (7) was also isolated as a colorless gum. A molecular formula of C18H28O5 was confirmed by the quasimolecular ion peak at m/z 347.1837 [M + Na]+ in the HRESIMS. Its NMR spectroscopic data were resemble with those of 6, except for an additional carbonyl group (δC 211.5) and one more aliphatic methylene group instead of a double bond in 7. The cross-peaks between H-1′ (δH 5.27) and H-7 (δH 1145

DOI: 10.1021/acs.jafc.7b04998 J. Agric. Food Chem. 2018, 66, 1140−1146

Article

Journal of Agricultural and Food Chemistry hexenoids also occur in genus Streptomyces.24 Nevertheless, in addition to contribute more new homologues to this type of compounds, our current work indicates that these cyclohexenoids might be a promising source for new α-glycosidase inhibitors.

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(11) Kang, S. S.; Shin, K. H.; Chi, H. J. Galloylpaeoniflorin, a new acylated monoterpene glucoside from Paeony root. Arch. Pharmacal Res. 1991, 14, 52−54. (12) Kubo, M.; Tani, T.; Kosoto, H.; Kimura, Y.; Arichi, S. Studies on Moutan Cortex (I): historical analysis. Shoyakugaku Zasshi 1979, 33, 155−157. (13) Wu, S. H.; Zhao, L. X.; Chen, Y. W.; Huang, R.; Miao, C. P.; Wang, J. Sesquiterpenoids from the endophytic fungus Trichoderma sp. PR-35 of Paeonia delavayi. Chem. Biodiversity 2011, 8, 1717−1723. (14) Sheldrick, G. M. A short history of SHELX. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, A64, 112−122. (15) Flack, H. D. On enantiomorph-polarity estimation. Acta Crystallogr., Sect. A: Found. Crystallogr. 1983, A39, 876−881. (16) Kong, F. D.; Zhao, C. Y.; Hao, J. J.; Wang, C.; Wang, W.; Huang, X. L.; Zhu, W. M. New α-glucosidase inhibitors from a marine spongederived fungus, Aspergillus sp. OUCMDZ-1583. RSC Adv. 2015, 5, 68852−68863. (17) Wei, J.; Zhang, X. Y.; Deng, S.; Cao, L.; Xue, Q. H.; Gao, J. M. αGlucosidase inhibitors and phytotoxins from Streptomyces xanthophaeus. Nat. Prod. Res. 2017, 31, 2062−2066. (18) Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55−63. (19) Reed, L. J.; Muench, H. A simple method of estimating fifty percent endpoint. Am. J. Epidemiol. 1938, 27, 493−497. (20) Hussain, H.; Akhtar, N.; Draeger, S.; Schulz, B.; Pescitelli, G.; Salvadori, P.; Antus, S.; Kurtán, T.; Krohn, K. New bioactive 2,3epoxycyclohexenes and isocoumarins from the endophytic fungus Phomopsis sp. from Laurus azorica. Eur. J. Org. Chem. 2009, 2009, 749− 756. (21) Mehta, G.; Roy, S.; Davis, R. A. On the stereostructures of (+)-eupenoxide and (−)-3′,4′-dihydrophomoxide: a caveat on the spectral comparisons of oxygenated cyclohexenoids. Tetrahedron Lett. 2008, 49, 5162−5164. (22) Liu, Z. M.; Jensen, P. R.; Fenical, W. A cyclic carbonate and related polyketides from a marine-derived fungus of the genus Phoma. Phytochemistry 2003, 64, 571−574. (23) Davis, R. A.; Andjic, V.; Kotiw, M.; Shivas, R. G. Phomoxins B and C: Polyketides from an endophytic fungus of the genus Eupenicillium. Phytochemistry 2005, 66, 2771−2775. (24) Wei, J.; Liu, L. L.; Dong, S.; Li, H.; Tang, D.; Zhang, Q.; Xue, Q. H.; Gao, J. M. Gabosines P and Q, new carbasugars from Streptomyces sp. and their α-glucosidase inhibitory activity. Bioorg. Med. Chem. Lett. 2016, 26, 4903−4906.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b04998. 1D and 2D NMR, HRESIMS, and IR spectra of compounds 1−7 and 4a; UV and CD spectra of compounds 1, 2, 4, and 5 (PDF) X-ray crystallographic data of compound 1 (CIF) X-ray crystallographic data of compound 4a (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail for S.-H.W.: [email protected]. Phone/Fax: +86-87165034264. *E-mail for J.H.: [email protected]. Phone/Fax: +86-2061648717. ORCID

Shao-Hua Wu: 0000-0003-4972-7449 Funding

This research was financially supported by the National Natural Science Foundation of China (81460545 and 81773556), and Program for Excellent Young Talents, Yunnan University. Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.jafc.7b04998 J. Agric. Food Chem. 2018, 66, 1140−1146