5.1. General
Reagents were obtained from Sigma-Aldrich, Germany. Silica gel for column chromatography were of 100-200 mesh. Solvents were purified by following standard procedures. Thin layer chromatography (TLC) was carried using silica gel F254 pre-coated plates. UV-light and I2 stain were used to visualize the spots. The 1H and 13C NMR spectra were recorded on NMR spectrometer (Bruker: 600 MHz for 1H, 150 MHz for 13C and 564 MHz for 19F) using CDCl3 as a solvent. The high-resolution electrospray ionization mass spectra (HR-ESI-MS) were recorded on Agilent 6530 LC Q-TOF instrument. Organic extracts and solutions of pure compounds were dried over anhydrous MgSO4.
5.2. (3-Hydroxypropyl) 3β-acetyloxy-urs-12-en-24-oate (3)
To a solution of 1 (350 mg, 0.702 mmol) in DMF (10 mL), K2CO3 (97 mg, 0.702 mmol) and 3-bromo-1-propanol (0.07 mL, 0.773 mmol) were added. After stirring the reaction mixture at room temperature for 18 h, the mixture was diluted with H2O (45 mL) and extracted with EtOAc (3 × 40 mL). The combined organic layers were washed successively with H2O (2 × 20 mL), saturated aqueous sodium bicarbonate (NaHCO3), brine (1 × 20 mL), dried over anhydrous magnesium sulfate (MgSO4), filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel, n-hexane/EtOAc, 85:15) to yield 3 (296 mg, 76%) as a white amorphous solid; 1H NMR (600 MHz, chloroform-d): δ = 5.30 (d, J = 3.4 Hz, 1H), 5.12 (s, 1H), 4.26 (dt, J = 12.1, 6.3 Hz, 1H), 4.16 (dt, J = 11.7, 6.2 Hz, 1H), 3.70 (t, J = 6.2 Hz, 2H), 2.10 (dd, J = 12.0, 3.8 Hz, 1H), 2.07 (d, J = 5.0 Hz, 3H), 1.98 (dt, J = 13.2, 6.8 Hz, 1H), 1.91–1.87 (m, 3H), 1.87–1.66 (m, 6H), 1.66–1.44 (m, 5H), 1.39 (td, J = 13.8, 13.0, 6.9 Hz, 4H), 1.33–1.29 (m, 2H), 1.25 (d, J = 7.2 Hz, 1H), 1.17 (s, 5H), 1.10 (s, 2H), 1.01 (s, 2H), 0.98 (d, J = 6.1 Hz, 2H), 0.90 (d, J = 6.0 Hz, 2H), 0.86 (d, J = 11.4 Hz, 3H), 0.81 (d, J = 6.9 Hz, 3H), 0.78 (d, J = 7.2 Hz, 5H) ppm; 13C NMR (150 MHz, chloroform-d) δ 176.4, 170.3, 145.0, 139.5, 124.5, 121.7, 73.4, 61.3 , 59.3, 59.1, 50.5, 46.8, 46.7, 42.2, 41.9, 41.5, 40.0, 39.7, 39.6, 37.2, 34.5, 33.8, 33.0, 31.6, 31.2, 28.7, 28.1, 26.5, 23.7, 23.6, 23.3, 23.2, 21.3, 21.3, 19.7, 17.4, 16.8, 13.4 ppm; HRMS (ESI+): Found (M+Na+): 579.4056 C35H56O5Na required 579.4054.
5.3. (3-Hydroxypropyl) 3β-acetyloxy-11-oxo-urs-12-en-24 oate (4)
Following the same procedure as described for the synthesis of 3, from 2 (350 mg, 0.683 mmol) in DMF (10 mL), K2CO3 (94 mg, 0.683 mmol) and 3-bromo-1-propanol (0.07 mL, 0.756 mmol) followed by flash column chromatography (silica gel, n-hexane/EtOAc, 85:15) compound 4 (287 mg, 74%) was obtained as a white amorphous solid; 1H NMR (600 MHz, chloroform-d): δ = 5.53 (s, 1H), 5.30 (s, 1H), 4.26 (dd, J = 11.4, 6.0 Hz, 1H), 4.17 (dd, J = 11.4, 5.9 Hz, 1H), 3.70 (t, J = 6.1 Hz, 2H), 2.51 (dt, J = 13.5, 3.5 Hz, 1H), 2.39 (s, 1H), 2.21–2.15 (m, 1H), 2.07 (s, 4H), 1.89 (q, J = 6.3 Hz, 3H), 1.84–1.81 (m, 1H), 1.66 (dd, J = 12.9, 3.7 Hz, 1H), 1.61–1.58 (m, 1H), 1.52 (d, J = 11.1 Hz, 1H), 1.47 – 1.41 (m, 3H), 1.39–1.36 (m, 2H), 1.33 (s, 3H), 1.30–1.20 (m, 6H), 1.16 (d, J = 5.8 Hz, 6H), 1.04 (s, 3H), 0.93 (s, 3H), 0.87 (q, J = 6.3, 5.9 Hz, 1H), 0.81–0.77 (m, 6H) ppm; 13C NMR (150 MHz, chloroform-d): δ = 199.2, 175.9, 170.2, 164.9, 130.4, 73.2, 61.4, 60.2, 59.2, 59.0, 50.4, 46.7, 45.0, 43.7, 40.9, 39.3, 39.2, 37.2, 34.6, 33.9, 32.8, 31.5, 30.9, 28.8, 27.5, 27.2, 24.0, 23.6, 21.3, 21.1, 20.5, 18.7, 18.3, 17.4, 13.3 ppm; HRMS (ESI+): Found (M+H+): 571.3751 C35H55O6 required 571.3753.
5.4. (3-(Tosyloxy)propyl) 3β-acetyloxy-urs-12-en-24-oate (5)
To a solution of compound 3 (270 mg, 0.485 mmol) in dry DCM (25.0 mL), Et3N (0.14 mL, 0.970 mmol) was added at 0 °C. Then tosyl chloride (102 mg, 0.534 mmol) and a catalytic amount of DMAP (6 mg, 0.0485 mmol) were added. The resulting mixture was allowed to warm to room temperature and stirred for 5 h. The reaction mixture was treated with aqueous 1 N HCl (10 mL), stirred for 10 min and extracted with DCM (3 × 40 mL). The organic layer was washed with saturated NaHCO3 (20 mL) and H2O (25 mL). The combined organic phases were dried over anhydrous MgSO4 and concentrated under reduced pressure. Flash column chromatography (silica gel, n-hexane/EtOAc, 90:10) of the crude product afforded 5 (275 mg, 80%) as a white gummy solid; 1H NMR (600 MHz, chloroform-d): δ = 7.77 (d, J = 7.9 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 5.23 (q, J = 2.7 Hz, 1H), 5.17 (d, J = 3.8 Hz, 1H), 4.12 (dt, J = 12.0, 5.7 Hz, 3H), 4.04 (dt, J = 11.7, 6.1 Hz, 1H), 2.43 (s, 3H), 2.06 (d, J = 5.0 Hz, 3H), 2.00 (p, J = 6.0 Hz, 4H), 1.91–1.83 (m, 2H), 1.67–1.58 (m, 7H), 1.43–1.35 (m, 5H), 1.33–1.26 (m, 4H), 1.23 (s, 3H), 1.09 (s, 5H), 0.98 (s, 3H), 0.94 (s, 1H), 0.90 (d, J = 6.0 Hz, 2H), 0.86 (d, J = 4.1 Hz, 3H), 0.82 (s, 1H), 0.79–0.76 (m, 4H), 0.72 (d, J = 9.4 Hz, 2H) ppm; 13C NMR (150 MHz, chloroform-d): δ = 175.7, 170.1, 144.8, 139.5, 132.9, 129.9, 127.8, 124.4, 121.7, 73.3, 66.8, 60.2, 59.1, 50.5, 46.8, 46.7, 42.2, 41.5, 40.0, 39.7, 39.6, 37.2, 34.5, 33.8, 33.0, 31.2, 29.6, 28.7, 28.2, 28.1, 26.5, 23.6, 23.6, 23.5, 23.3, 23.2, 21.6, 21.3, 21.2, 19.7, 17.4, 16.8, 13.3 ppm; HRMS (ESI+): Found (M+Na+): 733.4103 C42H62O7SNa required 733.4101.
5.5. (3-(Tosyloxy)propyl) 3β-acetyloxy-11-oxo-urs-12-en-24 oate (6)
Following the same procedure as described for the synthesis of compound 5, from 4 (260 mg, 0.456 mmol) in dry DCM (25.0 mL), Et3N (0.13 mL, 0.912 mmol), TsCl (95 mg, 0.501 mmol) and cat. amount of DMAP (6 mg, 0.0456 mmol), followed by flash column chromatography (silica gel, n-hexane/EtOAc, 90:10) 6 (273 mg, 83%) was obtained as a white amorphous solid; 1H NMR (600 MHz, chloroform-d): δ = 7.76 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H), 5.52 (s, 1H), 5.22 (s, 1H), 4.11 (td, J = 13.9, 12.8, 5.9 Hz, 3H), 4.07–4.03 (m, 1H), 2.47 (dt, J = 13.4, 3.6 Hz, 1H), 2.42 (s, 3H), 2.37 (s, 1H), 2.05 (s, 4H), 1.99 (p, J = 6.1 Hz, 3H), 1.89–1.85 (m, 1H), 1.54–1.50 (m, 2H), 1.48–1.41 (m, 3H), 1.39–1.34 (m, 2H), 1.31 (s, 3H), 1.28 (d, J = 13.9 Hz, 2H), 1.23 (s, 3H), 1.18 (d, J = 4.1 Hz, 1H), 1.13 (s, 3H), 1.09 (s, 3H), 1.00 (dd, J = 14.9, 4.1 Hz, 1H), 0.96 (s, 3H), 0.92 (s, 4H), 0.85 (dd, J = 10.0, 4.4 Hz, 2H), 0.80 (s, 3H), 0.78 (d, J = 6.4 Hz, 2H) ppm; 13C NMR (150 MHz, chloroform-d): δ = 199.1, 175.3, 170.0, 164.9, 144.9, 132.8, 130.47, 129.9, 127.8, 73.0, 66.8, 60.4, 60.2, 59.0, 50.3, 46.6, 45.0, 43.7, 40.9, 39.3, 39.2, 37.1, 34.5, 33.9, 32.8, 30.9, 29.6, 28.8, 28.2, 27.5, 27.2, 23.7, 23.5, 21.6, 21.3, 21.1, 20.5, 18.8, 18.3, 17.4, 13.3 ppm; HRMS (ESI+): Found (M+H+): 725.4007 C42H61O8S required 725.4009.
5.6. (3-Azidopropy) 3β-acetyloxy-urs-12-en-24-oate (7)
A stirred mixture of 5 (260 mg, 0.366 mmol) and NaN3 (72 mg, 1.098 mmol) in DMF (10 mL) was heated for 3 h at 70 °C, cooled and then treated with ice-water (200 mL). The reaction mixture was extracted with diethyl ether (2 × 150 mL), and the combined extracts were washed successively with water (2 × 40 mL) and brine (1 × 30 mL), dried over anhydrous MgSO4, and concentrated in vacuo. The crude product was purified by flash column chromatography (silica gel, n-hexane/EtOAc, 92:8) to yield 7 (156 mg, 74%) as a white gummy solid; 1H NMR (600 MHz, chloroform-d): δ = 5.29 (q, J = 2.5, 2.0 Hz, 1H), 5.15 (dt, J = 32.6, 3.7 Hz, 1H), 4.18 (dt, J = 12.0, 6.2 Hz, 1H), 4.10 (dt, J = 11.6, 6.2 Hz, 1H), 3.41 (t, J = 6.7 Hz, 2H), 2.07 (d, J = 5.0 Hz, 4H), 2.00 (dt, J = 13.2, 6.5 Hz, 1H), 1.92 (p, J = 6.0, 5.3 Hz, 3H), 1.86–1.82 (m, 1H), 1.70 (d, J = 4.0 Hz, 2H), 1.67–1.50 (m, 6H), 1.47 (dt, J = 13.4, 3.6 Hz, 1H), 1.39 (ddd, J = 24.4, 11.1, 4.2 Hz, 4H), 1.31 (d, J = 2.9 Hz, 2H), 1.28–1.22 (m, 4H), 1.20 (d, J = 4.4 Hz, 1H), 1.17 (s, 3H), 1.10 (s, 2H), 1.04–0.99 (m, 3H), 0.95–0.87 (m, 3H), 0.85 (s, 2H), 0.79 (q, J = 8.3, 7.1 Hz, 7H) ppm; 13C NMR (150 MHz, chloroform-d) δ 175.9, 170.2, 139.5, 124.4, 121.7, 73.3, 61.3, 59.1, 50.5, 48.3, 46.8, 46.7, 42.2, 41.5, 40.0, 39.7, 39.6, 37.2, 34.5, 33.8, 33.0, 31.2, 28.7, 28.1, 28.0, 26.5, 23.6, 23.3, 23.2, 21.3, 19.7, 17.4, 16.8, 13.3 ppm; HRMS (ESI+): Found (M+Na+): 604.4102 C35H55N3O4Na required 604.4105.
5.7. (3-Azidopropyl) 3β-acetyloxy-11-oxo-urs-12-en-24-oate (8)
Following the same procedure as described for the synthesis of 7, from 6 (250 mg, 0.345 mmol) in DMF (10 mL) and NaN3 (68 mg, 1.035 mmol), followed by flash column chromatography (silica gel, n-hexane/EtOAc, 92:8) 8 (145 mg, 71%) was obtained as a white gummy solid; 1H NMR (600 MHz, chloroform-d): δ = 5.53 (s, 1H), 5.29 (d, J = 2.9 Hz, 1H), 4.19 (dt, J = 12.1, 6.2 Hz, 1H), 4.10 (dd, J = 11.5, 6.0 Hz, 1H), 3.40 (t, J = 6.7 Hz, 2H), 2.51 (dt, J = 13.5, 3.5 Hz, 1H), 2.39 (s, 1H), 2.16 (ddd, J = 14.1, 11.2, 3.2 Hz, 1H), 2.06 (s, 4H), 1.92 (t, J = 6.5 Hz, 2H), 1.86 (dd, J = 13.7, 5.2 Hz, 1H), 1.82–1.77 (m, 1H), 1.74 (d, J = 3.3 Hz, 1H), 1.66 (s, 3H), 1.60–1.57 (m, 1H), 1.52 (d, J = 11.0 Hz, 1H), 1.48–1.42 (m, 3H), 1.39–1.36 (m, 2H), 1.32 (s, 3H), 1.30–1.25 (m, 2H), 1.21 (s, 1H), 1.16 (d, J = 2.8 Hz, 6H), 1.03 (s, 3H), 0.92 (s, 3H), 0.87 (t, J = 7.3 Hz, 1H), 0.81–0.77 (m, 6H) ppm; 13C NMR (150 MHz, chloroform-d): δ = 199.1, 175.4, 170.1, 164.9, 130.4, 73.1, 61.5, 60.2, 59.0, 50.4, 48.3, 46.7, 45.0, 43.7, 40.9, 39.3, 39.2, 37.2, 34.5, 33.9, 32.8, 30.9, 28.8, 27.9, 27.5, 27.2, 23.9, 23.6, 21.3, 21.1, 20.5, 18.8, 18.3, 17.4, 13.3 ppm; HRMS (ESI+): Found (M+H+): 596.3653 C35H54N3O5 required 596.3651.
5.8. General procedure for synthesis of 1H-1,2,3-triazolyl analogues of 3-O-acetyl-β-boswellic acid (10a-d) and 3-acetyl-11-keto-β-boswellic acid (11a-d)
To a solution of 7 (30 mg, 0.052 mmol) or 8 (30 mg, 0.051 mmol) and alkyne 9a-d (0.062 mmol, 1.2 eq) in acetonitrile (10 mL) were added CuI (20 mg, 0.104 mmol) and Et3N (0.022 mL, 0.156 mmol) at room temperature, and the mixture was stirred for 3 h. The reaction mixture was diluted with EtOAc (30 mL), 20 mL of aqueous NH4Cl was added, and the aqueous layer was extracted with EtOAc (3 × 30 mL), and the combined organic layer was washed with brine (1 × 20 mL), dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo. The crude residue was purified by flash column chromatography (silica gel, n-hexane/EtOAc, 85:15) to yield 10a-d (68-75%) or 11a-d (72-76%), respectively.
5.8.1. (3-(4-Phenyl-1H-1,2,3-triazol-1-yl)propyl) 3β-acetyloxy-urs-12-en-24-oate (10a)
Pale yellow gummy solid; yield = 72%; 1H NMR (600 MHz, chloroform-d): δ = 7.81 (d, J = 7.6 Hz, 2H), 7.76 (s, 1H), 7.40 (t, J = 7.6 Hz, 2H), 7.31 (d, J = 7.5 Hz, 1H), 5.32 (d, J = 3.9 Hz, 1H), 5.21–5.09 (m, 1H), 4.49 (t, J = 6.9 Hz, 2H), 4.17 (dq, J = 9.8, 4.9, 3.7 Hz, 1H), 4.13–4.05 (m, 2H), 2.33 (q, J = 6.6 Hz, 2H), 2.08 (d, J = 5.0 Hz, 4H), 2.02 (s, 1H), 1.98 (dt, J = 13.2, 7.1 Hz, 1H), 1.93–1.81 (m, 3H), 1.77–1.72 (m, 2H), 1.62 (d, J = 8.9 Hz, 1H), 1.59–1.46 (m, 3H), 1.45–1.38 (m, 4H), 1.31 (s, 2H), 1.24 (q, J = 6.4 Hz, 4H), 1.19 (s, 3H), 1.17 (s, 1H), 1.10 (s, 2H), 1.04–1.00 (m, 3H), 0.98 (s, 1H), 0.90 (d, J = 6.0 Hz, 2H), 0.85 (s, 3H), 0.81 (s, 3H), 0.79 (d, J = 8.4 Hz, 3H) ppm; 13C NMR (150 MHz, chloroform-d): δ = 175.8, 170.3, 147.9, 139.5, 130.4, 128.8, 128.2, 125.7, 124.4, 119.7, 73.2, 60.9, 60.3, 59.1, 50.5, 47.2, 46.9, 46.7, 42.2, 41.5, 40.0, 39.7, 39.5, 37.2, 34.5, 33.8, 33.0, 32.5, 31.2, 31.0, 29.4, 28.7, 28.1, 26.5, 23.7, 23.3, 23.2, 21.3, 19.8, 17.4, 16.9, 14.1, 13.4 ppm; HRMS (ESI+): Found (M+H+): 684.4752 C43H62N3O4 required 684.4754.
5.8.2. (3-(4-(4-(Trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)propyl) 3β-acetyloxy-urs-12-en-24-oate (10b)
White gummy solid; yield = 75%; 1H NMR (600 MHz, chloroform-d): δ = 7.93 (d, J = 8.0 Hz, 2H), 7.86 (s, 1H), 7.67 (d, J = 8.0 Hz, 2H), 5.32 (d, J = 4.7 Hz, 1H), 5.12 (d, J = 3.7 Hz, 1H), 4.51 (q, J = 4.9, 2.8 Hz, 2H), 4.19 (dt, J = 11.8, 6.0 Hz, 1H), 4.12-4.08 (m, 1H), 2.36–2.33 (m, 2H), 2.08 (d, J = 4.6 Hz, 5H), 1.99 (q, J = 8.2, 7.8 Hz, 1H), 1.93–1.86 (m, 2H), 1.84–1.78 (m, 1H), 1.65 (d, J = 13.8 Hz, 2H), 1.61–1.58 (m, 1H), 1.50–1.47 (m, 1H), 1.41 (dt, J = 12.6, 5.8 Hz, 3H), 1.31 (s, 2H), 1.24 (d, J = 6.9 Hz, 3H), 1.19 (s, 3H), 1.10 (s, 3H), 1.00 (d, J = 24.5 Hz, 6H), 0.90 (d, J = 6.0 Hz, 2H), 0.85 (s, 3H), 0.81 (s, 3H), 0.79 (d, J = 8.6 Hz, 6H) ppm; 13C NMR (150 MHz, chloroform-d): δ = 175.8, 170.2, 146.5, 145.0, 139.5, 133.9, 128.8, 125.8, 125.7, 124.4, 120.5, 73.2, 60.8, 59.1, 50.6, 47.4, 46.9, 46.7, 42.2, 41.5, 40.0, 39.7, 39.6, 37.2, 34.5, 33.8, 33.0, 31.2, 29.4, 28.7, 28.1, 26.5, 23.7, 23.3, 23.2, 21.3, 19.8, 17.4, 16.9, 13.5 ppm; 19F NMR (564 MHz, chloroform-d): δ = -62.61ppm; HRMS (ESI+): Found (M+H+): 752.4488 C44H61F3N3O4 required 752.4486.
5.8.3. (3-(4-(4-Fluorophenyl)-1H-1,2,3-triazol-1-yl)propyl) 3β-acetyloxy-urs-12-en-24-oate (10c)
White amorphous solid; yield = 73%; 1H NMR (600 MHz, chloroform-d): δ = .78 (dd, J = 8.4, 5.4 Hz, 2H), 7.73 (s, 1H), 7.10 (t, J = 8.5 Hz, 2H), 5.32 (d, J = 3.8 Hz, 1H), 5.13 (s, 1H), 4.49 (t, J = 6.9 Hz, 2H), 4.18 (dt, J = 12.0, 6.1 Hz, 1H), 4.09 (dt, J = 11.8, 6.1 Hz, 1H), 2.35–2.31 (m, 2H), 2.08 (d, J = 4.9 Hz, 4H), 1.99 (q, J = 8.1, 7.4 Hz, 1H), 1.93–1.86 (m, 2H), 1.73 (t, J = 8.8 Hz, 2H), 1.67–1.60 (m, 5H), 1.54–1.47 (m, 2H), 1.41 (ddd, J = 14.0, 10.9, 5.9 Hz, 4H), 1.31 (s, 2H), 1.24 (d, J = 5.6 Hz, 5H), 1.19 (s, 4H), 1.10 (s, 2H), 1.02 (s, 3H), 0.90 (d, J = 6.0 Hz, 2H), 0.87–0.85 (m, 3H), 0.81 (s, 3H), 0.79 (d, J = 6.9 Hz, 3H) ppm; 13C NMR (150 MHz, chloroform-d) δ 175.8, 170.2, 163.5, 161.8, 147.0, 139.5, 127.5, 127.4, 124.4, 119.4, 115.8, 115.7, 73.2, 60.8, 59.1, 50.6, 47.2, 46.9, 46.7, 42.2, 41.5, 40.0, 39.7, 39.6, 37.2, 34.5, 33.8, 33.0, 31.2, 29.6, 29.4, 28.7, 28.1, 26.5, 23.6, 23.3, 23.2, 21.3, 21.2, 19.8, 17.4, 16.9, 13.5 ppm; 19F NMR (564 MHz, chloroform-d): δ = -113.49 ppm; HRMS (ESI+): Found (M+H+): 702.4751 C43H61FN3O4 required 702.4753.
5.8.4. Methyl 1-(3-((3β-acetyloxy-urs-12-en-24carbonyl)oxy)propyl)-1H-1,2,3-triazole-4-carboxylate (10d)
White gummy solid; yield = 68%; 1H NMR (600 MHz, chloroform-d): δ = 8.10 (s, 1H), 5.30 (d, J = 3.5 Hz, 1H), 5.15 (dt, J = 32.5, 3.7 Hz, 1H), 4.50 (t, J = 7.0 Hz, 2H), 4.14 (dt, J = 12.1, 6.1 Hz, 1H), 4.08–4.04 (m, 1H), 3.94 (s, 3H), 2.33–2.30 (m, 2H), 2.08 (d, J = 5.0 Hz, 4H), 1.99 (dt, J = 12.9, 6.3 Hz, 1H), 1.90 (dt, J = 10.2, 4.7 Hz, 2H), 1.85–1.81 (m, 1H), 1.66 (s, 2H), 1.75–1.70 (m, 2H), 1.62 (d, J = 6.0 Hz, 1H), 1.49 (s, 1H), 1.41 (d, J = 10.5 Hz, 2H), 1.38 (s, 1H), 1.31 (s, 2H), 1.24 (d, J = 15.2 Hz, 6H), 1.18 (s, 3H), 1.10 (s, 3H), 1.02 (s, 3H), 0.90 (d, J = 5.9 Hz, 2H), 0.85 (d, J = 4.2 Hz, 3H), 0.80 (s, 3H), 0.78 (d, J = 6.1 Hz, 5H) ppm; 13C NMR (150 MHz, chloroform-d): δ = 175.8, 170.2, 161.0, 140.1, 139.5, 127.5, 124.4, 73.2, 60.5, 59.1, 52.2, 50.5, 47.6, 46.9, 46.7, 42.2, 41.5, 40.0, 39.7, 39.6, 37.2, 34.5, 33.8, 33.0, 31.2, 29.6, 29.3, 28.7, 28.1, 26.5, 23.6, 23.3, 23.2, 21.3, 21.2, 19.8, 17.4, 16.9, 13.5 ppm; HRMS (ESI+): Found (M+H+): 666.4444 C39H60N3O6 required 666.4442.
5.8.5. (3-(4-Phenyl-1H-1,2,3-triazol-1-yl)propyl) 3β-acetyloxy-11-oxo-urs-12-en-24-oate (11a)
White amorphous solid; yield = 72%; 1H NMR (600 MHz, chloroform-d): δ = 7.81 (d, J = 7.7 Hz, 2H), 7.77 (s, 1H), 7.40 (t, J = 7.6 Hz, 2H), 7.32 (d, J = 7.5 Hz, 1H), 5.53 (s, 1H), 5.33 (s, 1H), 4.49 (t, J = 6.8 Hz, 2H), 4.20 (dt, J = 12.1, 6.1 Hz, 1H), 4.09 (dd, J = 11.5, 5.7 Hz, 1H), 2.53 (dt, J = 13.4, 3.4 Hz, 1H), 2.40 (s, 1H), 2.34 (t, J = 6.5 Hz, 2H), 2.21–2.15 (m, 1H), 2.07 (s, 4H), 1.90–1.79 (m, 3H), 1.62 (dd, J = 15.4, 3.6 Hz, 2H), 1.52 (d, J = 11.1 Hz, 1H), 1.48–1.42 (m, 3H), 1.40–1.37 (m, 2H), 1.32 (s, 3H), 1.29 (s, 1H), 1.23 (d, J = 3.1 Hz, 3H), 1.19 (s, 3H), 1.16 (s, 3H), 1.05 (s, 3H), 1.01 (d, J = 5.7 Hz, 1H), 0.92 (s, 3H), 0.89–0.85 (m, 1H), 0.81–0.76 (m, 6H) ppm; 13C NMR (150 MHz, chloroform-d): δ = 199.1, 175.4, 170.2, 165.0, 130.4, 128.8, 128.2, 125.7, 119.8, 73.0, 61.1, 60.2, 59.0, 50.4, 47.3, 46.7, 45.0, 43.7, 40.8, 39.3, 39.2, 37.2, 34.5, 33.9, 32.7, 30.9, 29.6, 29.4, 28.8, 27.5, 27.2, 23.9, 23.6, 21.3, 21.1, 20.5, 18.8, 18.3, 17.4, 13.4 ppm; HRMS (ESI+): Found (M+H+): 698.4556 C43H60N3O5 required 698.4558.
5.8.6. (3-(4-(4-(Trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)propyl) 3β-acetyloxy-11-oxo-urs-12-en-24-oate (11b)
White amorphous solid; yield = 76%; 1H NMR (600 MHz, chloroform-d): δ = 7.92 (d, J = 8.0 Hz, 2H), 7.87 (s, 1H), 7.65 (d, J = 8.1 Hz, 2H), 5.51 (d, J = 2.6 Hz, 1H), 5.30 (dt, J = 17.7, 2.8 Hz, 1H), 4.50 (td, J = 6.9, 4.0 Hz, 2H), 4.19 (dt, J = 12.0, 6.1 Hz, 1H), 4.09 (q, J = 3.9, 2.9 Hz, 1H), 2.52 (dt, J = 13.2, 3.5 Hz, 1H), 2.38 (d, J = 6.1 Hz, 1H), 2.35 (q, J = 6.6 Hz, 2H), 2.16 (td, J = 11.4, 5.9 Hz, 1H), 2.06 (d, J = 8.0 Hz, 4H), 1.89–1.75 (m, 6H), 1.68–1.58 (m, 3H), 1.51 (d, J = 11.1 Hz, 1H), 1.46–1.36 (m, 6H), 1.31 (s, 3H), 1.17 (s, 3H), 1.14 (s, 3H), 1.03 (s, 3H), 1.00 (d, J = 4.7 Hz, 1H), 0.91 (s, 3H), 0.79–0.76 (m, 6H) ppm; 13C NMR (150 MHz, chloroform-d): δ = 199.1, 175.4, 170.2, 165.1, 146.5, 133.8, 130.4, 125.8, 120.6, 72.9, 61.0, 60.1, 59.0, 50.4, 47.4, 46.7, 45.0, 43.7, 40.8, 39.3, 39.2, 37.2, 34.5, 33.9, 32.7, 30.8, 29.3, 28.8, 27.4, 27.2, 23.9, 23.7, 21.3, 21.1, 20.5, 18.8, 18.3, 17.4, 14.1, 13.4 ppm; 19F NMR (564 MHz, chloroform-d): δ = -62.59 ppm; HRMS (ESI+): Found (M+H+): 766.0004 C44H59F3N3O5 required 766.0002.
5.8.7. (3-(4-(4-Fluorophenyl)-1H-1,2,3-triazol-1-yl)propyl) 3β-acetyloxy-11-oxo-urs-12-en-24-oate (11c)
White gummy solid; yield = 74%; 1H NMR (600 MHz, chloroform-d): δ = 7.78 (dd, J = 8.5, 5.2 Hz, 2H), 7.74 (s, 1H), 7.10 (t, J = 8.3 Hz, 2H), 5.53 (s, 1H), 5.32 (d, J = 2.8 Hz, 1H), 4.49 (s, 2H), 4.20 (dt, J = 11.4, 5.8 Hz, 1H), 4.10 (q, J = 6.2 Hz, 1H), 2.56–2.50 (m, 1H), 2.40 (s, 1H), 2.34 (s, 2H), 2.17 (t, J = 14.5 Hz, 1H), 2.07 (s, 5H), 1.87 (dd, J = 13.4, 5.2 Hz, 1H), 1.83 (d, J = 11.2 Hz, 1H), 1.63 (d, J = 3.4 Hz, 2H), 1.52 (d, J = 11.1 Hz, 1H), 1.48–1.43 (m, 3H), 1.40–1.37 (m, 2H), 1.32 (s, 3H), 1.29 (s, 1H), 1.23 (d, J = 3.1 Hz, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.04 (s, 3H), 1.01 (d, J = 3.8 Hz, 1H), 0.92 (s, 3H), 0.87 (d, J = 8.7 Hz, 1H), 0.80 (s, 3H), 0.78 (d, J = 6.5 Hz, 3H) ppm; 13C NMR (150 MHz, chloroform-d): δ = 199.0, 175.4, 170.1, 164.9, 163.5, 147.0, 130.4, 127.5, 127.4, 126.6, 119.5, 115.8, 115.7, 72.9, 61.1, 60.2, 59.0, 50.4, 47.3, 46.8, 45.0, 43.7, 40.8, 39.3, 39.2, 37.2, 34.5, 33.9, 32.8, 30.8, 29.3, 28.8, 27.5, 27.2, 23.9, 23.7, 21.3, 20.5, 18.8, 18.3, 17.4, 13.4 ppm; 19F NMR (564 MHz, chloroform-d): δ = -113.50 ppm; HRMS (ESI+): Found (M+H+): 716.4799 C43H59FN3O5 required 716.4801.
5.8.8. Methyl 1-(3-((3β-acetyloxy-11-oxo-urs-12-en-24carbonyl)oxy)propyl)-1H-1,2,3-triazole-4-carboxylate (11d)
White gummy solid; yield = 72%; 1H NMR (600 MHz, chloroform-d): δ = 8.10 (s, 1H), 5.53 (s, 1H), 5.30 (d, J = 2.8 Hz, 1H), 4.50 (td, J = 6.9, 3.8 Hz, 2H), 4.17 (dd, J = 11.7, 5.9 Hz, 1H), 4.09–4.04 (m, 1H), 3.94 (s, 3H), 2.53 (dt, J = 13.4, 3.5 Hz, 1H), 2.40 (s, 1H), 2.32 (t, J = 6.5 Hz, 2H), 2.15 (t, J = 14.4 Hz, 1H), 2.08 (s, 4H), 1.89 (dd, J = 13.7, 5.1 Hz, 1H), 1.78 (dd, J = 24.9, 13.3 Hz, 3H), 1.67 (d, J = 17.0 Hz, 2H), 1.54 (s, 1H), 1.49–1.41 (m, 3H), 1.42–1.36 (m, 2H), 1.33 (s, 3H), 1.23 (s, 3H), 1.17 (d, J = 10.8 Hz, 6H), 1.04 (s, 3H), 0.93 (s, 3H), 0.87–0.84 (m, 2H), 0.80 (s, 3H), 0.78 (d, J = 6.4 Hz, 3H) ppm; 13C NMR (150 MHz, chloroform-d): δ = 199.0, 175.4, 170.1, 164.9, 161.0, 130.4, 127.5, 72.9, 60.8, 60.2, 59.0, 52.2. 50.4, 47.6, 46.8, 45.0, 43.7, 40.9, 39.3, 39.2, 37.2, 34.5, 33.9, 32.8, 30.9, 29.6, 29.3, 28.8, 27.5, 27.2, 23.9, 23.6, 21.3, 21.1, 20.5, 18.8, 18.3, 17.4, 13.4 ppm; HRMS (ESI+): Found (M+Na+): 702.3879 C39H57N3O7Na required 702.3877.
5.9. In-vitro assay for CA II
In this assay, colorless 4-nitrophenyl acetate (4-NPA) is hydrolyzed to yellow 4-nitrophenol27. The assay was carried out at 25 °C in 20 mM HEPES-Tris buffer of pH 7.4 in 96-well plate. Each well of 96-well plate comprised 140 µL of HEPES-Tris buffer solution, 20 µL of fresh enzyme solution (0.1 mg/mL in buffer) of purified bovine erythrocyte CA II and 20 µL of test compound in DMSO (10% final concentration). The mixture of enzyme and inhibitor was pre-incubated for 15 min at room temperature to allow the formation of the EI complex. After incubation, the reaction was initiated by adding of 20 µL substrate 4-NPA (0.7 mM). For kinetics studies 0.8, 0.4, 0.2 and 0.1 mM of substrate were used. It was followed by continuous measurement of amount of product formed at = 400 nm for 30 min at 1 min intervals in 96-well flat bottom plates, using ELISA Reader xMARK Microplate spectrophotometer, BIORAD (USA). The activity of control (in the absence of inhibitor) was taken as 100%. The measurements were taken in triplicates at each used concentration 26,28.
The % inhibition was calculated by using the following formula:
% Inhibition = 100-(OD test well/OD control) ×100
5.10. Molecular Docking
The X-ray crystallographic structure of CA II in complex with carbon dioxide and bicarbonate ion (PDB code: 2VVB, resolution: 1.66Å) was used in docking. The structures of all the compounds were prepared on ChemDraw software and saved in mol format, then imported into MOE database where each molecule was minimized with MMFF94x force field until an RMSD gradient of 0.1 kcal∙mol−1Å−1 was achieved, and the partial charges were automatically applied on each molecule during the minimization process. Hydrogen atoms and partial charges were added on the enzyme structure with the default settings of the Protonate 3D protocol in MOE. The binding site was defined by selecting the residues of the allosteric site. Triangle Matcher placement method and London dG scoring function were used for docking. Later, thirty docked conformations of each compound were saved for conformational sampling. The best docked pose of each compound with respect to the docking score and binding interactions, was selected for SAR analysis.
The logP, logS, and TPSA values of active compounds were calculated by MOE using the 3D-structures of compounds, while gastro-intestinal absorption and blood brain barrier permeability was calculated through SwissADME webserver (http://www.swissadme.ch/index.php).