20-hydroxyecdysone (20E) was purchased from Shaanxi KingSci Biotechnology (Xi’an, China) in a purity of 90%. By applying recrystallization from ethyl acetate–methanol (2:1, v/v), 20E was afforded in an RP-HPLC purity of 97.8%.

Ecdysteroid derivatives 20E-EOx and 20E-ZOx were prepared via semi-synthesis from 20E, following our previously described procedure [24]. Briefly, an aliquot of 1 g of hydroxylamine hydrochloride (14.39 mmol) was dissolved in 15 mL of freshly distilled ethanol. An ethyl alcoholic solution of potassium hydroxide (807.4 mg, 14.39 mmol) was added to the solution to liberate the hydroxylamine free base from its salt, and, subsequently, 1 g of 20E was added. The reaction mixture was stirred for 2 weeks at the boiling point. Following this, silica gel (4 g) was added to the solution, which was then evaporated to dryness under reduced pressure to prepare the sample for dry loading flash chromatographic purification. The normal-phase chromatographic separation was performed on a 24 g silica column (flow rate 35 mL/min, run time: 30 min) with a gradient of dichloromethane (A) and methanol (B), from 0% to 35% of solvent B in A. The eluted product mixture consisted of 20E-EOx and 20E-ZOx in an approximately 2:1 ratio. Subsequently, the ecdysteroid oxime isomers were separated using preparative RP-HPLC (16% aq. acetonitrile, flow rate: 15 mL/min), affording 20E-EOx in 49% yield (509 mg) and 20E-ZOx in 18% yield (181 mg).20EL was synthesized from 20E-EOx, by slightly modifying our previously published procedure [20]. Briefly, a 300 mg aliquot of 20E-EOx (0.61 mmol) was dissolved in 20 mL of acetone. Subsequently, 83.3 mg of Na2CO3 (0.786 mmol, 3 equiv.) and 461.6 mg of p-toluenesulfonyl chloride (TsCl, 2.42 mmol, 4 equiv.) were added to the solution, and the mixture was stirred for 24 h at room temperature. Following this, silica gel (3 g) was added to the solution, which was then evaporated to dryness on a rotary evaporator to prepare the sample for dry loading flash chromatographic purification. The product was purified on a 24 g silica column (flow rate 35 mL/min, run time: 30 min) with a gradient of dichloromethane (A) and methanol (B), from 0% to 35% of solvent B in A. In a second chromatographic step, the product’s concentrated sample (approx. 230 mg) was further purified by preparative RP-HPLC (55% aq. methanol, flow rate: 15 mL/min), to afford ecdysteroid lactam 20EL in a yield of 61.3% (183.9 mg).20EL. White solid; Isolated yield: 61.3% (183.9 mg); RP-HPLC purity: 99.7%; HR-MS: C27H45NO7, [M + H]+ Calculated 496.32743, found: 496.32814. 1H NMR (500 MHz, [D6]DMSO) δ = 7.60 ppm (1H, d, J = 7.93 Hz), 5.48 ppm (1H, s), 4.45 ppm (1H, s), 4.37 ppm (1H, d, J = 5.95 Hz), 4.30 ppm (1H, d, J = 5.04 Hz), 4.22 ppm (1H, d, J = 2.75 Hz), 4.03 ppm (1H, s), 3.80 ppm (1H, m), 3.69 ppm (1H, m), 3.50 ppm (1H, s), 3.22 ppm (1H, m), 3.20 ppm (1H, m), 3.13 ppm (1H, m), 2.28 ppm (1H, m), 1.93 ppm (1H, m), 1.90 ppm (1H, m), 1.85 ppm (1H, m), 1.84 ppm (1H, m), 1.73 ppm (1H, dd, J = 13.12, 4.73), 1.67 ppm (1H, m), 1.65 ppm (1H, m), 1.60 ppm (1H, m), 1.56 ppm (1H, m), 1.52–45 ppm (3H, m), 1.41 ppm (1H, m), 1.29 ppm (1H, m), 1.26 ppm (1H, m), 1.12 ppm (1H, m), 1.08 ppm (3H, s), 1.06 ppm (3H, s), 1.05 ppm (3H, s), 0.85 ppm (3H, s), 0.78 ppm (3H, s); 13C NMR (125 MHz, [D6]DMSO) δ = 167.4 ppm, 153.5 ppm, 119.0 ppm, 85.8 ppm, 76.1 ppm, 75.6 ppm, 68.6 ppm, 68.4 ppm, 66.3 ppm, 53.3 ppm, 48.9 ppm, 47.8 ppm, 41.3 ppm, 40.8 ppm, 39.4 ppm, 38.4 ppm, 34.0 ppm, 32.3 ppm, 31.2 ppm, 29.7 ppm, 29.0 ppm, 26.0 ppm, 23.4 ppm, 23.0 ppm, 20.7 ppm, 20.2 ppm, 17.1 ppm. One- and two-dimensional NMR spectra of 20EL is provided as Supplementary Materials, Figures S1–S5, and its high-resolution mass spectrum as Figure S7.

Stock solutions for the bioactivity tests were prepared in methanol at 40 mg/mL concentration. Verapamil and quinidine were dissolved in DMSO at 5.0 (verapamil) and 2.5 (quinidine) mg/mL concentration, indomethacin stock solution was prepared in ethanol at 7.0 mg/mL concentration.