3.4. Extraction and Isolation

The fungal strain Pseudogymnoascus sp. HSX2#-11 was cultivated in a PDA liquid medium in 200 Erlenmeyer flasks (300 mL in each 1000 mL flask) at 16 °C for 45 days. The broth and mycelia were separated through two layers of gauze. Then the mycelia were first extracted by ethyl acetate (EA) three times (3 × 4000 mL) and then with dichloromethane (DCM)/MeOH (MO) (v/v, 1:1) three times (3 × 4000 mL). The organic extractive broth was obtained through repeated extraction with EA (3 × 60 L). All of the fungal crude extracts were put together and evaporated to dryness under reduced pressure to provide a residue (71.5 g). The residue was subjected to vacuum liquid chromatography (VLC) eluted with EA-petroleum ether (PE) (0–100%) and MO-EA (0–100%) on silica gel to obtain eight fractions (Fr.1–Fr.8). Fr.4 was separated through column chromatography (CC) on Sephadex LH-20 eluted with DCM/MO (v/v, 1:1) to afford two fractions (Fr.4.1, Fr.4.2). Fr.4.1 was subjected to silica gel CC eluting with EA–PE (0–50%), then purified by using semi-preparative HPLC on an ODS column (Kromasil C₁₈, 250 × 10 mm, 5 µm, 2 mL/min) eluted with 65% MO–H₂O to give compound 3 (2.6 mg). Fr.4.2 was separated on silica gel CC eluted with EA–PE (0–50%) to give compound 7 (3.9 mg). Fr.6 was separated on silica gel Sephadex LH-20 eluted with DCM/MO (v/v, 1:1) to afford three fractions (Fr.6.1–Fr.6.3). Fr.6.3 was the pure compound 5 (17.6 mg). Fr.6.1 was first eluted with EA–PE (20–100%) on silica gel CC, and then purified through HPLC with 60% and 70% MO–H₂O for 2 (5.3 mg) and 1 (5.6 mg), respectively. Fr.7 was separated on Sephadex LH-20 CC eluted with DCM/MO (v/v, 1:1) to get three fractions (Fr.7.1–Fr.7.3). Fr.7.2 and Fr.7.3 were subjected to HPLC with 50% MO–H₂O to gain 4 (7.0 mg) and 6 (8.7 mg), respectively.

Pseudophenone A (1): colorless crystals; UV (CH₂Cl₂) λ_max (log ε): 224 (5.38), 261 (5.10), 325 (4.83); ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 690.1583 [M]⁺ (calcd for C₃₅H₃₀O₁₅, 690.1579), [M + H]⁺ m/z 691.1540 (calcd for C₃₅H₃₁O₁₅, 691.1657).

X-ray crystallographic analysis of 1: C₃₅H₃₀O₁₅, Mr = 690.59, triclinic crystals, space group P⁻¹, a = 11.8213(4) Å, b = 11.9228(4) Å, c = 16.4176(6) Å, α = 69.674 (2)°, β = 70.338 (2)°, γ = 81.306 (2)°, V = 2041.80(13) ų, Z = 2, D_calcd = 1.123 mg/cm³, T = 173 (2) K, λ (Cu Kα) = 1.54184 Å, F(000) = 720, crystal size 0.180 × 0.160 × 0.150 mm³, Final R₁ value was 0.1135, wR₂ = 0.3457 (I > 2σ(I)). Crystallographic data for 1 were given the number CCDC 2064117 after being deposited in the Cambridge Crystallographic Data Centre (CCDC).