2.1 Isolation and structural elucidation
The fractions 49 to 56 from column exhibited one large spot with minor contamination throughout TLC silica plates after employing dragendorff reagent. Impurities were removed by hexane washing. The fractions (49-56) were blended and squeezed together. After that, it was cleaned with hexane once more. A total of 0.075g of product was created. The bioactive eluent was separated by HPTIC at 450 nm using solvent solutions of methanol, ethyl acetate, chloroform, and formic acid (1:7:2:0.2). The Rf value, height, and area were 0.59, 2.8, and 111, respectively (Supplementary file: Figure S1). The m.p is 465-468 0C. The elemental analysis (%) of the isolated compound (C21H29N3O4) is as follows (Calculated: C, 65.09; H, 7.54; N, 10.84; O, 16.52. Found: C, 65.17; H, 7.55; N, 10.86; O, 16.54).
The isolated compound showed following spectral characteristics. 1HNMR (CDCl3): δ 4.336 (d, J=9.6, H-3), 1.971 (d, J=6.4, H-4), 2.803 (s, H-5), 8.516 (d, H-6), 4.632 (d, J=14.8, H-7), 8.475 (s, H-9), 4.761 (d, J=11.2, H-10), 8.452 (s, H-12), 7.530 (t, J=6.0 Hz, H-13), 7.380 (d, J=4.4 Hz, H-14), 6.977 (d, J=15.2 Hz, H-15), 6.742, (d, J=10.4 HZ, H-17), 6.904 (d, J=16.4 Hz, H-18), 7.214 (s, J=4.8 Hz, H-19), 3.817 (s, J=4.0 Hz, H, of -OCH3), 1.712 (s, H-10-1'), 1.298 (s, J=4.0, H-10-2'), 1.987(d, J=6.4, H-10-3'), 0.947 (d, J=13.6 Hz, H-10-4') (Supplementary file: Figure S2 to S5).
13CNMR (CDCl3): δ 150.240 (-CH=, C-1 / 16), 77.017 (CH-, C-3), 39.176 (CH2-, C-4 / 5), 171.196 (-C=O, C-8 / 11), 55.874 (-CH-, C-10), 32.432 (CH2-, C- 10-1', 10-2'), 29.706 (CH- C-10-3'), 14.771(-CH3, C-10-4'), 118.17 (CH=, C-13 /14 ), 128.766 (CH- C-15), 60.369 (-OCH3, O-CH3), 113.674 (CH-, C-17 / 18 / 19), (Supplementary file: Figure S6).
IR (KBr) vmax 2955, 2869, 1686, 1648, 3065, 3394, 1221, 1033, 883, 1454, 1376 cm−1(Supplementary file: Figure S7)
MS (FAB; m/z): 387. [M+1]; (Supplementary file: Figure S8). The orbitrap high-resolution MS showed the mass of isolated compound is 378.4711 (Supplementary file: Figure S9). The structure of isolated compound (IC) was given in Figure 1. To the best of our knowledge this is the first-time reporting of the isolated compound, i.e., [(16-methoxy-10-(3-methyl-butyl)-2-oxa-6, 9, 12-triaza-tricyclo [13.3.1.03, 7] nonadeca-1(18), 13, 15 (19), 16-tetraene-8, 11-dione] from this species.