2.2. Fermentation, Extraction, and Isolation

The fungus Bipolaris sp. was isolated from fresh and healthy stems of kiwifruit plants (Actinidia chinensis Planch., Actinidiaceae), which were collected from the Cangxi county of the Sichuan Province (GPS: N 31°12′, E 105°76′) in July 2018. Each fungus was obtained simultaneously from at least three different healthy tissues. The fungus was identified as one species of the genus Bipolaris by observing the morphological characteristics and analysis of the internal transcribed spacer (ITS) regions. A living culture (internal number HFG-20180727-HJ32) has been deposited at the School of Pharmaceutical Sciences, South-Central University for Nationalities, China.

This fungal strain was cultured on a potato dextrose agar (PDA) medium at 24 °C for 10 days. The agar plugs were inoculated in 500 mL Erlenmeyer flasks, each containing 100 mL potato dextrose media. Flask cultures were incubated at 28 °C on a rotary shaker at 160 rpm for two days as the seed culture. Four hundred 500 mL Erlenmeyer flasks, each containing 150 mL potato dextrose broth (PDB), were individually inoculated with 25 mL of seed culture and were incubated at 25 °C on a rotary shaker at 160 rpm for 25 days.

The cultures of Bipolaris sp. (60 L) were extracted four times by EtOAc to afford a crude extract (32.0 g) which was subjected to CC over silica gel eluted with a gradient of CHCl₃-MeOH (a gradient from 1:0 to 0:1) to give six fractions, A–F. Fraction B (13.0 g) was fractionated by MPLC CC over RP-18 eluted with MeOH–H₂O (from 10:90 to 100:0, v/v) to give twelve sub-fractions (B₁–B₁₂). Fraction B₃ (1.2 g) was applied to Sephadex LH-20 eluting with CHCl₃–MeOH (1:1, v/v) and was further purified by preparative HPLC with MeCN–H₂O (19:81, v/v, 4.0 mL/min) to obtain compounds 9 (18.6 mg, retention time (t_R) = 40 min), 18 (22.6 mg, t_R = 15.8 min), 2 (3.3 mg, t_R = 32 min), and 1 (5.4 mg, t_R = 36 min). Fraction B₅ (2.1 g) was separated by CC over silica gel with a gradient elution of the CHCl₃–MeOH system (50:1→0:1) and was prepared by HPLC with MeCN–H₂O (12:88, v/v, 4.0 mL/min) to obtain 3 (4.9 mg, t_R = 36 min), 4 (14.4 mg, t_R = 46 min), 17 (28.3 mg, t_R = 43 min), and 5 (2.1 mg, t_R = 40 min). Fraction B₆ (1.8 g) was purified over Sephadex LH-20 eluted with MeOH to give four subfractions (B_6.1–B_6.4). Fraction B_6.2 (210 mg) was purified using semipreparative HPLC with MeOH-H₂O (28:72, v/v, 3.0 mL/min) to afford 8 (8.8 mg, t_R = 17.8 min) and 7 (9.6 mg, t_R = 21.1 min). Fraction B_6.3 (170 mg) was purified by preparative HPLC with MeCN–H₂O (23:77, v/v, 4 mL/min) to yield 6 (4.3 mg, 26 min). Fraction C (4.3 g) was separated by CC over silica gel with a gradient elution of PE-acetone (50:1→0:1) to afford subfractions C₁–C₈. Fraction C₂ (340 mg) was purified by preparative HPLC with MeCN-H₂O (55:45, v/v, 4 mL/min) to give 12 (10.3 mg, t_R = 38 min), 13 (3.7 mg, t_R = 39 min), 14 (3.1 mg, t_R = 36 min) and 15 (3.4 mg, t_R = 34 min). Fraction C₅ (230 mg) was isolated by CC over Sephadex LH-20 (MeOH) and was prepared by HPLC (32:68, v/v, 4 mL/min) to give 10 (3.7 mg, t_R = 28 min), 11 (4.2 mg, t_R = 29 min), and 16 (5.1 mg, t_R = 24 min).

Bipolarisorokin A (1): colorless crystals; mp 145–148 °C; [α]²⁰_D + 67.8 (c 0.01, MeOH); UV (MeOH) λ_max (log ε) 205 (3.30); IR (KBr) ν_max 3360, 2947, 2833, 1651, 1454, 1114, 1031 cm⁻¹; ¹H NMR (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Table 1; positive ion HRESIMS m/z 251.16624 [M–H]⁺, (calculated for C₁₅H₂₃O₃⁻ 251.16527).

¹H (600 MHz) and ¹³C (150 MHz) NMR Spectroscopic Data for 1–3.

^a Measured in CDCl₃; ^b Measured in methanol-d₄.

Bipolarisorokin B (2): colorless oil; [α]²²_D − 100.1 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 210 (3.23); ¹H NMR (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Table 1; positive ion HRESIMS m/z 275.16166 [M+Na]⁺, (calculated for C₁₅H₂₄O₃Na⁺ 275.16177).

Bipolarisorokin C (3): colorless, needle-like crystals (MeOH); mp 135–138 °C; [α]²²_D − 21.8 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 265 (3.49) nm; ¹H NMR (600 MHz, methanol-d₄) and ¹³C NMR (150 MHz, methanol-d₄) data, see Table 1; positive ion HRESIMS m/z 253.17971 [M+H]⁺ (calculated for C₁₅H₂₅O₃⁺ 253.17982).

Bipolarisorokin D (4): colorless oil; [α]²⁵_D + 32.0 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 255 (3.65); ¹H NMR (600 MHz, methanol-d₄) and ¹³C NMR (150 MHz, methanol-d₄) data, see Table 2; positive ion HRESIMS m/z 275.16153 [M+Na]⁺ (calculated for C₁₅H₂₄NaO₃⁺ 275.16177). Bipolarisorokin E (5): colorless oil; [α]²⁵_D − 22.7 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 210 (3.24); ¹H NMR (600 MHz, methanol-d₄) and ¹³C NMR (150 MHz, methanol-d₄) data, see Table 2; positive ion HRESIMS m/z 221.15529 [M–H]⁻ (calculated for C₁₄H₂₁O₂⁻ 221.15470).

¹H (600 MHz) and ¹³C (150 MHz) NMR Spectroscopic Data for 4–6.

^a Measured in CDCl₃; ^b Measured in methanol-d₄.

Bipolarisorokin F (6): white powder; [α]²⁰_D − 3.3 (c 0.04, MeOH); UV (MeOH) λ_max (log ε) 215 (3.72); ¹H NMR (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Table 2; positive ion HRESIMS m/z 225.18506 [M+H]⁺ (calculated for C₁₄H₂₅O₂⁺ 225.18491).

Bipolarisorokin G (7): colorless oil; [α]²⁰_D + 17.2 (c 0.02, MeOH); UV (MeOH) λ_max (log ε) 230 (3.21); ¹H NMR (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Table 3; positive ion HRESIMS m/z 275.20059 [M+H]⁺ (calculated for C₁₈H₂₇O₂⁺ 275.20056).

¹H (600 MHz) and ¹³C (150 MHz) NMR Spectroscopic Data for 7–9.

^a Measured in CDCl₃; ^b Measured in methanol-d₄.

Bipolarisorokin H (8): colorless oil; [α]²⁵_D − 136.9 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 225 (3.93); ¹H NMR (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Table 3; positive ion HRESIMS m/z 277.17984 [M+H]⁺ (calculated for C₁₇H₂₅O₃⁺ 277.17982).

Bipolarisorokin I (9): colorless crystals; mp 191–194 °C; [α]²²_D + 8.8 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 210 (3.46); ¹H NMR (600 MHz, methanol-d₄) and ¹³C NMR (150 MHz, methanol-d₄) data, see Table 3; positive ion HRESIMS m/z 251.16621 [M–H]⁻, (calculated for C₂₁H₂₃O₃⁻ 251.16527).

Bipolarithone A (10): colorless oil; [α]²³_D + 136.0 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 245 (3.30); ¹H NMR (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Table 4; positive ion HRESIMS m/z 349.09143 [M+H]⁺, (calculated for C₁₇H₁₇O₈⁺ 349.09179).

¹H (600 MHz) and ¹³C (150 MHz) NMR Spectroscopic Data for 10 and 11.

^a Measured in CDCl₃; ^b Measured in methanol-d₄.

Bipolarithone B (11): colorless oil; [α]²³_D − 24.2 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 245 (3.30); ¹H NMR (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Table 4; positive ion HRESIMS m/z 349.09157 [M+H]⁺, (calculated for C₁₇H₁₇O₈⁺ 349.09179).

Bipolarithone C (12): colorless oil; [α]²⁵_D + 52.9 (c 0.5, MeOH); UV (MeOH) λ_max (log ε) 245 (4.06); ¹H NMR (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Table 5; positive ion HRESIMS m/z 541.24310 [M+H]⁺, (calculated for C₃₀H₃₇O₉⁺ 541.24321).

¹H (600 MHz) and ¹³C (150 MHz) NMR Spectroscopic Data for 12 and 13.

^a Measured in CDCl₃; ^b Measured in methanol-d₄.

Bipolarithone D (13): colorless oil; [α]²⁵_D + 10.2 (c 0.5, MeOH); UV (MeOH) λ_max (log ε) 245 (3.88); ¹H NMR (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Table 5; positive ion HRESIMS m/z 541.24316 [M+H]⁺, (calculated for C₃₀H₃₇O₉⁺ 541.24321).

Crystal data for Cu_1_0m: C₁₅H₂₄O₃, M = 252.34, a = 9.7038(6) Å, b = 13.7866(8) Å, c = 16.6333(10) Å, α = 95.329(3)°, β = 104.898(2)°, γ = 102.525(3)°, V = 2073.0(2) ų, T = 100(2) K, space group P 1, Z = 6, μ(Cu Kα) = 1.54178 mm⁻¹, F(000) = 828, 82979 reflections measured, 16831 independent reflections (R_int = 0.0695). The final R₁ values were 0.0437 (I > 2σ(I)). The final wR(F²) values were 0.1047 (I > 2σ(I)). The final R₁ values were 0.0531 (all data). The final wR(F²) values were 0.1143 (all data). The goodness of fit on F² was 1.039. Flack parameter = −0.10(7). CCDC: 2124305. Available online: https://www.ccdc.cam.ac.uk (accessed on 11 December 2021).

Crystal data for Cu_3_0m: C₁₅H₂₄O₃, M = 252.34, a = 7.0044(5) Å, b = 10.1468(8) Å, c = 20.1433(14) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V= 1431.63(18) ų, T = 295(2) K, space group P 21 21 21, with Z = 4, μ(Cu Kα) = 1.54178 mm⁻¹, F(000) = 552, 6263 reflections measured, 2527 independent reflections (R_int = 0.0500). The final R₁ values were 0.0519 (I > 2σ(I)). The final wR(F²) values were 0.1538 (I > 2σ(I)). The final R₁ values were 0.0719 (all data). The final wR(F²) values were 0.2087 (all data). The goodness of fit on F² was 1.117. Flack parameter = −0.40(17). CCDC: 2124306. Available online: https://www.ccdc.cam.ac.uk (accessed on 11 December 2021).

Crystal data for Cu_9_0m: C₁₅H₂₄O₃, M = 252.34, a = 6.8634(2) Å, b = 15.0872(4) Å, c = 13.5156(3) Å, α = 90.00°, β = 90.4010(10)°, γ = 90.00°,V = 1399.50(6) ų, T = 295(2) K, space group P 1 21 1, with Z = 4, μ(Cu Kα) = 1.54178 mm⁻¹, F(000) = 552, 32232 reflections measured, 5982 independent reflections (R_int = 0.0279). The final R₁ values were 0.0300 (I > 2σ(I)). The final wR(F²) values were 0.0808 (I > 2σ(I)). The final R₁ values were 0.0304 (all data). The final wR(F²) values were 0.0812 (all data). The goodness of fit on F² was 1.057. Flack parameter = −0.01(3). CCDC: 2124307. Available online: https://www.ccdc.cam.ac.uk (accessed on 11 December 2021).

Crystal data for Cu_17_0m: C₁₄H₂₄O₂, M = 224.33, a = 13.6388(2) Å, b = 13.6388(2) Å, c = 13.0174(2) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 2097.04(7) ų, T = 296(2) K, space group P 31 2 1, with Z = 6, μ(Cu Kα) = 1.54178 mm⁻¹, F(000) = 744, 39026 reflections measured, 3033 independent reflections (R_int = 0.0459). The final R₁ values were 0.0353 (I > 2σ(I)). The final wR(F²) values were 0.0988 (I > 2σ(I)). The final R₁ values were 0.0366 (all data). The final wR(F²) values were 0.1003 (all data). The goodness of fit on F² was 1.047. Flack parameter =0.01(5). CCDC: 2126101. Available online: https://www.ccdc.cam.ac.uk (accessed on 11 December 2021).

Crystal data for Cu_18_0m: C₁₅H₂₆O₂, M = 238.36, a = 13.1977(2) Å, b = 13.1977(2) Å, c = 8.49040(10) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1478.85(5) ų, T = 297(2) K, space group P 43, with Z = 4, μ(Cu Kα) = 1.54178 mm⁻¹, F(000) = 528, 14568 reflections measured, 3063 independent reflections (R_int = 0.0269). The final R₁ values were 0.0534 (I > 2σ(I)). The final wR(F²) values were 0.1525 (I > 2σ(I)). The final R₁ values were 0.0541 (all data). The final wR(F²) values were 0.1539 (all data). The goodness of fit on F² was 1.051. Flack parameter =0.12(7). CCDC: 2126105. Available online: https://www.ccdc.cam.ac.uk (accessed on 11 December 2021).