Hieracium calophyllum, R. Uechtr.

4.3. Preparation of MeOH extract of H. calophyllum View in CoL flowering heads and isolation of SLs

Dried and powdered flowering heads of H. calophyllum ( 13 g) were macerated with CH 2 Cl 2 (1:15 w/v) at room temperature for 48 h. The extract was filtered, and the residual material dried at room temperature and re-extracted with MeOH (1:15 w/v) by bimaceration procedure (2 × 48 h). Combined MeOH extracts were evaporated under reduced pressure using a rotary evaporator (Büchi Rotavapor R- II, Flawil, Switzerland). The thus obtained dried MeOH extract ( 3 g) was then suspended in H 2 O (300 ml), extracted successively with CH 2 Cl 2 (300 ml), EtOAc (600 ml) and n -BuOH (600 ml), and then lyophilised. The thus obtained dry residue ( 0.5 g) was redissolved in H 2 O ( 8 mg /ml) and subjected to preparative HPLC with MS detection, on an Agilent LC/MS System, equipped with Zorbax SB-C 18 column (250 × 9.4 mm; 5 μm particle size). SLs were eluted using 0.1% (v/v) HCO 2 H/H 2 O (mobile phase A) and MeOH (mobile phase B), and selected gradient program: 10–20% B (5 min), 20–30% B (5 min), isocratic 30% B (3 min), 30-20% B (7 min), 20–50% B (4 min), 50–90% B (6 min), with the total run time of 30 min, post run time of 5 min, flow rate of 2 ml/min, and the injection volume ( Vinj) of 100 μl. DAD was operating at 210 nm, 254 nm, 320 nm and 350 nm wavelengths. MSD was recording in the positive ion and full-scan mode in the range of m/ z 80–700 to acquire Total Ion Chromatogram (TIC). Optimized ion source parameters were as follows: fragmentor voltage of 200 V, nebulising drying gas flow of 9 L/min at 350 ̊C, nebulizer pressure of 40 psi and capillary voltage of 3000 V. Splitter was set at 1000:1. Time-based fractionation afforded eleven fractions, F 1–11, which were collected during 0.5 min (F 1–3), 1 min (F 4–6, 9), or 3 min (F 7, 8, 10, 11). Fraction F 4 ( tR = 14.32 min) afforded compound 1 ( 1 mg), F 6 ( tR = 15.55 min) compound 2 ( 1.5 mg), F 7 ( tR = 16.72 min) compound 3 ( 3 mg) and F 9 ( tR = 31.25 min) compound 4 ( 5 mg). Compounds 2 and 3 gradually decomposed upon one-week standing in D 2 O solution at temperature above 303 K. The mixture of 2 and its degradation products was further separated on Zorbax SB-C 18 column (50 × 9.4 mm; 5 μm particle size) affording 2a ( 0.5 mg). For this purpose, mobile phases: 0.1% (v/v) HCO 2 H/H 2 O (A) and MeOH (B), and fast gradient program: 20–30% B (5 min), 50–90% B (6 min), 90- 20% B (3 min), with the total run time of 15 min, flow rate of 2 ml/ min, and Vinj of 60 μl were used. The mixture of 3 and its degradation products was fractioned using the same column, mobile phases, flow rate and Vinj, with slightly different gradient program, i.e. 20–30% B (5 min), 30-20% B (5 min), 50–90% B (2 min), 90-20% B (1 min), and the total run time of 14 min, affording 4.

4.3.1. Calophyllamine B (1, 3 β -( β -glucopyranosyl)-oxy-8 α -hydroxy-13 α - (N-prolyl)-eudesma-1,4(15)-dien-5 α, 6 β, 7 α, 11 β H-12,6-olide)

White solid [ α] D +106 ( c 0.5, H 2 O). 1 H, 13 C, DEPT, HMBC, and NOESY spectroscopic data are presented in Table 2 View Table 2 . HRMS m/z: 540.2456 [M + H] + (calcd 540.2439 for [M + H] + C 26 H 38 NO 11), qTOF-MS/MS (15 eV) m/z (rel. int.): 540.2456 (100), 494.2445 (22), 316.2084 (3), 128.0718 (4), 100.0759 (4).

4.3.2. Calophyllamine A (2, 3 β, 8 α -dihydroxy-13 α -(N-prolyl)-guaia- 4(15),10(14)-dien-5 α, 6 β, 7 α, 11 β H-12,6-olide)

White solid. 1 H, 13 C, HMBC, ROESY spectroscopic data are presented in Table 2 View Table 2 . HRMS m/z: 378.1916 [M + H] + (calcd 378.1911 for [M+H] + C 20 H 28 NO 6), qTOF-MS/MS (15 eV) m/z (rel. int.): 378.1892 (100), 332.1865 (22), 128.0710 (10), 100.0729 (4).

4.3.3. 8-Epiixerisamine A (3, 3 β -( β -glucopyranosyl)-oxy-8 α -hydroxy-13 α - (N-prolyl)-guaia-4(15),10(14)-dien-5 α, 6 β, 7 α, 11 β H-12,6-olide)

White solid [ α] D −3.6 ( c 1.2, H 2 O). 1 H, 13 C, DEPT, ROESY and HMBC spectroscopic data are presented in Table 2 View Table 2 . HRMS m/z: 540.2456 [M + H] + (calcd 540.2439 for [M + H] + C 26 H 38 NO 11), qTOF-MS/MS (15 eV) m/z (rel. int.): 540.2456 (100), 494.2414 (9), 378.1885 (22), 360.1806 (5), 332.1909 (5), 316.1909 (3), 128.0734 (10), 100.0886 (6).

4.3.4. Crepiside E (4, 3 β -( β -D-glucopyranosyl)-oxy-8 α -hydroxy-guaia- 4(15),10(14),11(13)-trien-5 α, 6 β, 7 α -12,6-olide)

Brownish gum. [ α] D +24.3 (c 1.2, MeOH). 1 H, 13 C, COSY, NOESY,

TOCSY, HMBC spectroscopic data are presented in Table S1 View Table 1 . HRMS m/ z: 447.1645 [M + Na] + (calcd 447.1625 for [M + Na] + C 21 H 28 O 9 Na), qTOF-MS/MS (25 eV) m/z (rel. int.): 263.1304 (29); 245.1204 (16); 227.1058 (100); 217.1268 (57); 209.1308 (14); 201.1261 (14); 199.1140 (62); 183.1078 (11); 181.104 (72); 175.0810 (5); 171.1170 (55); 143.0926 (12); 131.0869 (19).

4.3.5. Desacylcynaropicrin (2a, 3 β, 8 α -dihydroxy-guaia-4(15),10(14), 11(13)-trien-5 α, 6 β, 7 α -12,6-olide)

White solid, [ α] D = +34 ( c 0.5, H 2 O). 1 H NMR (500 MHz, D 2 O, TMS): δ H 6.25 (d, J = 3.4 Hz, H-13b); 6.16 (d, J = 3.0 Hz, H-13a); 5.45 (s, H-15b); 5.34 (s, H-15a); 5.16 (s, H-14b); 5.04 (s, H-14a); 4.64 (t, J = 6.2 Hz, H-3); 4.36 (t, J = 9.8 Hz, H-6); 4.06 (dd, J = 7.6, 4.7 Hz, H- 8); 3.06 (m, H-1); 2.99 (q, J = 9.9 Hz, H-5); 3.00 (t, J = 9.8 Hz, H-7); 2.72 (dd, J = 13.6, 4.9 Hz, H-9β); 2.33 (dd, J = 14.6, 3.8 Hz, H-9α); 2.22 (td, J = 13.5, 7.0 Hz, H-2β); 1.76 (td, J = 11.9, 9.2 Hz, H-2α). 13 C NMR (125 MHz, D 2 O): δ C 173.0 (C-12); 151.6 (C-4); 141.6 (C-10); 123.7 (C-13); 116.8 (C-14); 112.4 (C-15); 79.6 (C-6); 72.6 (C-3); 71.2 (C-8); 50.1 (C-7); 49.3 (C-5); 44.7 (C-1); 40.2 (C-9); 37.6 (C-2).