(+)-Betulonic acid

Betulonic acid belongs to the pentacyclic triterpenic derivative class, has antitumor activities.In vitro: BEA-NP is found over three-times more permeable than that solubilized by DMSO in Caco-2 cell monocultures.[1]In vivo: The tumor growth in the S180 berry mice orally doses with BEA-NP at 75 mg/kg is inhibited by 50%. Rubusoside is effective in solubilizing BEA, maintaining its cytotoxicity, enhancing its permeability and reducing tumor growth when orally administered.[1] antitumor ac

Betulonic acid is a triterpenoid. It has a role as an anticoronaviral agent.

Jones’ reagent: CrO3 (26.72 g) was dissolvedin H2SO4 (23 ml); then, the solutionwas diluted to 100 ml with water. Asolution of BA (500 mg, 1.09mmol) inCH2Cl2 (12 ml) and acetone (12 ml) wascooled, to which Jones’ reagent (1 ml) wasslowly added. The mixture was stirred atroom temperature for 15 min; then, MeOH(5 ml) was added. After another stirring for10 min, CH2Cl2 (20 ml) was added and theorganic layer was washed with water andsaturated NaCl solution sequentially, driedover anhydrous Na2SO4, and concentratedin vacuo. The product was recrystallized inMeOH for three times as a white solid(462 mg, 93percent).To a cooled solution of BA (500 mg, 1.09 mmol) in CHTo a solution of betulinic acid (5.0g, 10.9mmol) in DMSO (50mL), IBX (6.2g, 22.0mmol) was added. The reaction mixture was stirred for 6h at room temperature and then ice water (100mL) and AcOEt (50mL) were added. The mixture was filtered and the filtrate was extracted with AcOEt (30mL×3). The combined organic extract was washed with brine (30mL×2), dried over anhydrous NaBetulinic acid (5 g, 10.9 mmol) was dissolved in DMSO (50 mL) andTHF (50 mL)IBX (6.16 g, 22 mmol) was added, Reaction at room temperature for 6 hours.After TLC detection reaction is complete, H20O (100 mL) and ethyl acetate (50 mL) were added. Insoluble material was filtered off with suction and the filtrate was extracted with ethyl acetate (30 mL x 3). The combined organic phases were dried over anhydrous Na2SO4, Concentrated to give a betulinic acid as a white solid (4.5 g, 90percent), Used directly in the next step without purification.To a solution of betulinic acid (1 gr, 2.19 mmol) in acetone/EtOAc 5:5 (10 mL) was added Jones reagent until the disappearance of the starting material (control by TLC). The reaction was washed with brine and extracted with EtOAc. The organic phases were dried over Na2SO4 and evaporated under vacuum and the crude was purified over silica gel (PE/EtOAc 9:1), 3-Oxo-lup-20(29)-en- 28-acid (compound XVI; CAS num.: 448 1-62-3) (89percent) as an off-white solid.Step 1 Preparation of (1R, 3aS, 5aR, 5bR, 7aR, 11aR, 11bR, 13aR, 13bR)-5a, 5b, 8, 8, 11a-pentamethyl-9-oxo-1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-3a-carboxylic acid To a chilled solution of (1R, 3 aS, 5aR, 5bR, 7aR, 9S, 11aR, 11bR, 13 aR, 13bR)-9-hydroxy-5a, 5b, 8, 8, 11a-pentamethyl-1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-3a-carboxylic acid (2.35 g, 5.15 mmol) in DMF (45 mL) at ˜4° C. under nitrogen was added pyridinium dichromate (PDC 3.87 g, 10.29 mmol) in a single portion. The suspension was rapidly stirred, forming an orange solution, but rapidly changed into dark brownish with all the PDC dissolved into the reaction mixture. The reaction was kept in an ice bath, and was allowed to warm to RT slowly over 8 hours. Stirring continued for 48 hrs at RT thereafter. The PDC reaction mixture acquired a dull dark brownish appearance but no PPT was observed. The crude DMF reaction solution was poured into vigorously stirred ethyl acetate (400 mL) causing a PPT of a light brownish solid. The suspension was filtered through a short bed (˜1' thick) of silica gel type-H in a large diameter filter funnel. 3-Deoxy-betulinic acid (31): To a solution of 1 (2 g, 1 eq) in DMF was added pyridium dichromate (PDC, 2 eq). The mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with EtOAc (30 mL) and the precipitate was filtered through a short pack of Florisil. The solution was washed with 20percent HCl and distilled water. The organic layer was dried over anhydrous NaPrepared from 1 (13.1 g, 28.7 mmol) by Jones oxidation (4 h, 25 C) as previously reported [38], followed by chromatographicworkup (silica gel, hexanes/ethyl acetate, 9:1), and 4 (10.6 g, 81percent)was obtained as a colorless solid; RF 0.79 (silica gel, hexanes/ethylacetate, 7:3); m.p.: 242e244 C (lit.: [38] 245e247 C;[a]D 39.42 (c 0.64, CHCl3) (lit.: [57] [a]D 32 (c 0.37, CHCl3), [a]D 40 (c 0.86, CHCl3) [58]; MS (ESI, MeOH): m/z 455.2 (100percent, [MH]), 508.9 (24percent, [M Na MeOH]), 909.3(16percent, [2M H]), 931.3 (46percent, [2M Na]).The compound was prepared by oxidation of compound 1 (2.2mmol) in DCM using pyridinium chlorochromate (PCC) (2.4mmol) as a catalyst [27]. Upon completion, the reaction mixture was passed through celite, the filtrate was concentrated on rota evaporator and subjected to column chromatography with elution in 10percent ethyl acetate in hexane to furnish betulonic acid 9 yield: 70percent; mp: 250°C; HRESIMS m/z calcd for CTo a solution of compound 1 (5g, 11mmol) in DCM was added PCC (3.54g, 16mmol) dissolved in DCM dropwise till dark colour appears and kept it at r. t. for 2h. After completion, reaction mixture was passed through celite and filtrate was concentrated at rotavapour. Purification was done through column chromatography with EtoAc: Hexane (1: 13) as the eluent to afford product 7 colourless solid (3.5g, 70percent yield). A solution of 1 (0.52 g, 1.14 mmol) in acetone (20 mL) was cooled, and 10 mL of Jones reagent (27 g CrOTo a solution of pyridine (25.47 mL, 315.29 mmol) in CHGeneral procedure: A suspension of 1a-4a (4.4 mmol) in 100 mL ofCH2Cl2/acetone (1:1, v/v) was cooled to 0 °C, a solution ofJones’ reagent (2 mL, ca 1.2 equiv) was added dropwise over30 min maintaining the bath temperature at 0 °C. The mixturewas stirred for an additional 15 min, prior to the additionof i-PrOH (1 mL) and H2O (4 mL). The resulting mixturewas stirred at room temperature for 10 min, and then water(30 mL) and CH2Cl2 (20 mL) were added and organic layersseparated. The combined organic layers were washed withsaturated aq. NaCl (20 mL), dried, and concentrated invacuo. 1b was obtained by crystallization in methanol. Compounds2b-4b were purified by silica gel chromatography.The yields of compounds 1b-4b were 99percent, 98percent, 42percent and64percent, respectively. The spectral data (see supporting information)of compounds 1b [37, 38], 2b [39], 3b [39] and4b [40] were consistent with previous reported in literature(1) Weigh 12.00 g of betulin into 600 mL of acetone solvent, Stir at 40 ° C for 30 min and then cool to -10 ° C.The newly prepared 144.0 g of Jones reagent was then added dropwise to the above reaction flask.The reaction temperature is maintained at -10 ° C and the reaction time is maintained for 4 h.After the reaction was completed, it was quenched by dropwise addition of 600 mL of ethanol reagent at -10 ° C.Then continue stirring for 60 min. Finally, add 600 mL of water and stir for 30 min.The reaction solution was subjected to reduced pressure rotation to remove dark green precipitate.The residue was extracted with tetrahydrofuran at least 3 times, then washed with saturated brine 3 times, Finally, dry over anhydrous Na2SO4 overnight.Filtration, rotary steaming pale green solid that is crude betulonic acid. The crude betulonic acid was dissolved in a small amount of methylene chloride and the upper column was separated. The eluent was eluted with a gradient mixture of dichloromethane: ethyl acetate = 2: 1, 1: 1, The target fractions of keto acid were combined and pulled dry, and then placed in a vacuum oven at 45 for 24 h, yielding 10.80 g of betulinic acid product with a purity of 99.5percent. The yield was 87.6percent.The bark (100 g) of the white birch (Betula verrucosa), collected in Poland, was cut into small pieces, soaked in dichloromethane (1 L) and refluxed for 8 h. To a solution of betulinol (20) (340 mg, 0.77 mmol) in acetone (20 mL) was added Jones reagent [freshly prepared from CrO3 (877 mg, 0.81 mmol) and sulfuric acid (0.6 mL) in water (5 mL) dropwise within 10 min at 0 °C. The reaction mixture was allowed to warm to room temperature and stirring was continued for 4 h, then quenched by the addition of methanol (10 mL) in an ice bath. The acetone was distilled off and the residue extracted with ethyl acetate (3 × 15 mL). The combined organic phases were washed with water (20 mL), saturated aqueous NaHCO3 solution (20 mL) and brine (20 mL). The solvent was removed in vacuo. The residue was subjected to flash chromatography (eluent: Petroleum ether/EtOAc = 5:1) to give 220 mg (63percent) of compound 7 as a white solid. To a solution of betulonic acid (21) (265 mg, 0.58 mmol) in THF (20 mL) was added NaBH4 (93 mg, 2.33 mmol). The reaction mixture was stirred at room temperature for 5 h, then quenched by the addition of 2 N HCl solution and THF was removed under vacuum down to 50percent volume. The solution was diluted with EtOAc (30 mL) and was washed with H2O (3 × 10 mL) and brine (10 mL). The organic layer was dried (Na2SO4), filtered, and the solvent was removed in vacuo. The residue was dissolved in hot ethanol and cooled to 0oC to induce recrystallization of betulinic acid (22, 162 mg, 61percent) as a white crystal. Rf = 0.26 (PE/EtOAc = 2:1); mp 289-290 °C; [lit.21 291-292 °C]. 1H NMR (400 MHz, pyridine-d5): δ 5.03 (s, 1H, H29.), 4.86 (s, 1H, H29.), 3.61 (m, 1H, H19), 3.54 (t, 1H, J = 7.8 Hz, H3), 2.82 (m, 1H, H13), 2.71 (m, 1H, H16.), 2.33 (m, 2H), 1.88, 1.31, 1.16, 1.15, 1.10, 0.92 (s, each 3H, 6 × CH3), 0.91 (m, 1H, H5); 13C NMR (100 MHz, pyridine-d5): δ 179.08, 151.56, 110.20, 78.33, 56.86, 56.15, 51.18, 49.99, 48.01, 43.07, 41.34, 39.76, 39.51, 38.84, 37.82, 37.75, 35.06, 33.10, 31.43, 30.51, 28.90, 28.54, 26.34, 21.44, 19.70, 19.02, 16.65, 16.59, 15.13.In a 1000 mL three-necked flask, betulinic acid (20.0 g, 45.2 mmol) and acetone (400 mL) were added, and then freshly prepared Jones reagent (100 mL) was added dropwise in an ice bath. After continuing the reaction for 30 minutes in an ice bath, the ice bath was removed and stirred at room temperature for 8 h. The reaction was quenched by the addition of methanol (250 mL) and water (250 mL). Add water, extract with ethyl acetate, combine the organic phases, dry and concentrate.Column chromatography gave 2 white solid (12.2 g, 26.8 mmol, 59.3percent).In a 1000 mL three-necked flask, add betulin (20.0 g, 45.2 mmol) and acetone (400 mL).Then freshly prepared Jones reagent (100 mL) was added dropwise under ice bath.The reaction was continued for 30 minutes in an ice bath and the ice bath was removed.Stir at room temperature for 8 h, The reaction was quenched by the addition of EtOAc (EtOAc) (EtOAc)A white solid 2 (12.2 g, 26.8 mmol, 59.3percent) was obtained.a) To a solution of betulin 1 (50 g, 113 mmol) in acetone (1500 ml), a Jones reagent was added during 1 hour in an ice bath. The reaction mixture was allowed to warm to room temperature, and agitation was continued for 21 hours. Methanol (700 ml) and water (1000 ml) were added to the reaction mixture. The precipitate was filtered, dried in vacuum, taken up in diethyl ether (600 ml) and washed with water, 7.5 percent hydrochloric acid, water, saturated NaHCOA 2-L four-necked flask equipped with a stirrer, thermometer, reflux condenser, and dropping funnel was charged with bark (50 g) ground to particle size 1–3 mm and Me2CO (1 L). The mixture was stirredvigorously, treated slowly with freshly prepared Jones reagent [25 mL, CrO3 (6.7 g) dissolved in H2SO4 (5.8 mL, 98percent) andthen diluted with H2O to 25 mL], stirred for 3 h at 20°C, and filtered of Cr salts and residual bark. The filtrate was concentratedin vacuo. The residue was diluted with H2O (300 mL). The resulting precipitate was filtered off, rinsed on the filter with H2O, and dried. The dry precipitate was purified by dissolving in C6H6 (150 mL), adding activated charcoal, filtering through alayer (7 mm) of Al2O3, and treating with KOH solution (10percent) until the potassium salt of 2 was fully precipitated. The solidwas filtered off, rinsed on the filter with C6H6 (30–40 mL), dried at room temperature to constant weight, and dissolved inEtOH (60 mL). The solution was poured into a beaker containing HCl solution (200 mL, 10percent). The resulting precipitate of2 was filtered off, rinsed with H2O, dried, and purified by recrystallization from MeOH. Rf 0.56, mp 245–248°C, lit. [8]mp 247–249°C, C30H46O3. Elemental analyses agreed with those calculated.Step 2 Preparation of (1R, 3aS, 5aR, 5bR, 7aR, 11aR, 11bR, 13aR, 13bR)-5a, 5b, 8, 8, 11a-pentamethyl-9-oxo-1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-3a-carboxylic acid To a solution of (1R, 3aS, 5aR, 5bR, 7aR, 11aR, 11bR, 13aR, 13bR)-5a, 5b, 8, 8, 11a-pentamethyl-9-oxo-1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-3a-carbaldehyde (17.6 g, 36.1 mmol) in t-BuOH (100 mL) was added 161 2-methyl-2-butene (40 mL, 476 mmol). A solution of sodium chlorite (15 g, 133 mmol) and sodium phosphate monobasic monohydrate (25 g, 181 mmol) in water (200 mL) was added drop wise over 1.25 h and the mixture was stirred at rt for an additional 45 minutes. The mixture was diluted with saturated aqueous ammonium chloride (100 mL) and extracted with ethyl acetate (3×125 mL). The combined organic layers were washed with brine and dried over sodium sulfate. The drying agent was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was purified by using a 300 g Thomson silica gel column and a 10-50percent 126 ethyl acetate in hexanes gradient. The fractions containing the expected product were combined and concentrated under reduced pressure to give (1R, 3aS, 5aR, 5bR, 7aR, 11aR, 11bR, 13aR, 13bR)-5a, 5b, 8, 8, 11a-pentamethyl-9-oxo-1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-3a-carboxylic acid as a white foam (16.4 g, 36.1 mmol, 100percent). LCMS: m/e 453.2 (M−H)Step 2: Preparation of (1R, 3aS, 5aR, 5bR, 7aR, 11aR, 11bR, 13aR, 13bR)-5a, 5b, 8, 8, 11a-pentamethyl-9-oxo-1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-3 a-carboxylic acid [0380] (1R, 3aS, 5aR, 5bR, 7aR, 11aR, 11bR, 13aR, 13bR)-5a, 5b, 8, 8, 11a-pentamethyl-9-oxo-1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-3 a-carbaldehyde (17.6 g, 36 1 mmol) in t-BuOH (100 mL) was added 2-methyl-2-butene (40 mL, 476 mmol). A solution of sodium chlorite (15 g, 133 mmol) and sodium phosphate monobasic monohydrate (25 g, 181 mmol) in water (200 mL) was added drop wise over 1.25 h and the mixture was stirred at rt for an additional 45 minutes. The mixture was diluted with saturated aqueous ammonium chloride (100 mL) and extracted with ethyl acetate (3×125 mL). The combined organic layers were washed with brine and dried over sodium sulfate. The drying agent was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was purified by using a 300 g Thomson silica gel column and a 10-50percent ethyl acetate in hexanes gradient. The fractions containing the expected product were combined and concentrated under reduced pressure to give (1R, 3aS, 5aR, 5bR, 7aR, 11aR, 11bR, 13 aR, 13bR)-5a, 5b, 8, 8, 11a-pentamethyl-9-oxo-1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-3a-carboxylic acid as a white foam (16.4 g, 36.1 mmol, 100percent). LCMS: m/e 453.2 (M−H)To a solution of (1R, 3aS, 5aR, 5bR, 7aR, 11aR, 11bR, 13aR, 13bR)-5a, 5b, 8, 8, 11a-pentamethyl-9-oxo-1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-3a-carbaldehyde (17.6 g, 36.1 mmol) in t-BuOH (100 mL) was added 2-methyl-2-butene (40 mL, 476 mmol). A solution of betulonic aldehyde (2, 10.0 g, 22.3 mmol) that wasprepared from betulin (1) via Swern oxidation [10] in t-BuOH (100 mL) at 0–10°C was treated simultaneously with solutionsof NaH2PO4 (16.2 g, 136.8 mmol) in H2O (60 mL) and NaClO2 (12.4 g, 136.8 mmol) in H2O (50 mL) and then with H2O2(30percent, 12 mL), stirred at 10°C for 15 min (TLC monitoring), diluted with H2O (200 mL), and extracted with MTBE (3 100 mL).The organic layer was washed with saturated NaCl solution, dried over Na2SO4, and evaporated. The solid (10.5 g) wasdissolved in benzene (150 mL), treated with KOH solution (75 mL, 15percent), and refluxed for 1 h. The precipitate was filteredoff, rinsed sequentially with H2O and MTBE, dissolved in HCl solution (10percent, 200 mL), and refluxed for 1 h. The precipitatewas filtered off, rinsed with H2O, and dried to afford 3 (8.62 g, 85percent). []D20 +43° (c 0.65, CHCl3); lit. []D25 +45° (c 0.51, CHCl3) [10]. PMR and 13C NMR spectral parameters were identical to those reported earlier [10].In a mixture of 20 mL of glacial acetic acid and 3 mL of water was dissolved 0.1 g of chromic anhydride. The solution was cooled, and at stirring 0.2 g (0.46 mmol) of betulonic aldehyde was added (TLC monitoring). After 45–60 min the reaction product was precipitated by adding 10percent water solution of sodium chloride, the precipitate was filtered off, washed with water solution of sodium chloride, and dried. The dry residue was dissolved in a mixture of 20 mL of methanol and 5 mL of dichloromethane and 0.1 g of potassium hydroxide was added. The separated precipitate was filtered off, the filtrate was evaporated by half, to the residue 10 mL of acetic acid and water was added till an amorphous precipitate formed. The latter was filtered off and dried in air. Yield 0.16 g (80percent). White amorphous powder, mp 243–245°C [7].Betulonic aldehyde (1 , 00 g, 2, 3 mmol), NaOCIA solution of 1 (5.00 g, 11.29 mmol) in CH2Cl2 (500 mL)was treated with PCC (12.17 g, 56.47 mmol), stirred for 2 h, diluted with Et2O (500 mL), stirred for 15 min, and filtered through a layer of Al2O3. The filtrate was concentrated. The residue was purified by flash chromatography over SiO2[C6H6–methyl-tert-butylether (MTBE), 4:1] to afford betulonic aldehyde (4.20 g, 85percent) (PMR and 13C NMR spectra agreedwith the literature [16]). A solution of the aldehyde (4.20 g, 9.57 mmol) in t-BuOH (200 mL) was treated with 2-methyl-2-butene (4 mL, 37.75 mmol) and simultaneously dropwise with NaClO2 (5.20 g, 57.44 mmol) in H2O (22 mL) and NaH2PO4(6.89 g, 57.44 mmol) in H2O (21 mL). The reaction mixture was stirred for 15 min, diluted with H2O (200 mL), and extracted with CHCl3. The organic layer was separated, washed with H2O, dried over Na2SO4, and evaporated. The resulting residue was dissolved in C6H6 (150 mL), treated with KOH (0.58 g, 10.36 mmol, 15percent solution in H2O), and refluxed for 1 h.The resulting precipitate of the potassium salt of betulonic acid was filtered off and dissolved in a mixture of MTBE (200 mL) and HCl (10percent, 50 mL). The organic layer was separated, washed with H2O, dried over Na2SO4, and evaporated to afford 2(3.26 g, 75percent). The PMR and 13C NMR spectra agreed with the literature data [16].synthesis of 3-oxo-lup-20(29)-en-28-oic acid (betulonic acid) 1; In a 250 ml round-bottomed flask, equipped with a magnetic stirring bar, a 100 ml addition funnel and a septum fitted with a nitrogen inlet, was placed 1 g (2.258 mmol) betulin in 150 ml acetone. The flask was cooled with an ice-acetone bath and the suspension of betulin was treated dropwise with Jones' reagent [L. F. Fieser, M. Fieser, Reagents for Organic Synthesis, vol. 1, 1967, p. 143] over 15 minutes. The reaction mixture was stirred at 0EXPERIMENTAL SECTIONTo a stirred suspension of betulin (5.0 g, 11.29 mmol) in acetone (150 mL) at 0° C. under NGeneral procedure: A modified procedure from the literature36 was used. Each of the 3-oxo derivatives 3, 4, and 8 (20 g, 45 mmol) was dissolved in dry dioxane (400 mL) and excess NaH (22 g) was added slowly to the solution, under argon. The mixture was heated slowly to boiling and then ethyl formate (6 mL) was slowly added over 3 h. The mixture was cooled to room temperature and any remaining NaH was quenched with 30 mL of EtOH in dioxane (100 mL). The crude product was precipitated out of the solution by adding 10% HCl (1 L) and was then extracted into chloroform. The product was purified by chromatography on silica gel (200 g) in toluene (for compound 11) or by using gradient from toluene to 10% diethyl ether in toluene (for acids 12 and 13) and was subsequently crystallized from CHCl3/MeOH to give the pure aldehyde.To a solution of 9 (0.24mmol) in 20ml of acetone was added K2CO3 (0.24mmol), CH3I (0.3mmol) and stirred at room temperature. After completion, the reaction mixture was dried under vacuum and product 14 was crystallized in methanol as white solid (yield 98%). HRESIMS m/z calcd for C31H49O3 [M+H]+ 469.3681, found 469.3686.To a suspension of General procedure: Iodomethane (1.606mmol) was added to a stirred solution of 1a (0.143mmol) and anhydrous potassium carbonate (0.352mmol) in dry CH2Cl2(2 mL), in an N2atmosphere at 55 C. The mixture was stirred for 24h and iodomethane (1.606mmol) was added once again and stirred for another 24h. After 48h, the reaction mixture was diluted in water (50 mL) and the aqueous phase was extracted with dichloromethane (3 x 50 mL). The resulting organic phase was washed with brine, dried over Na2SO4, filtered and evaporated. In order to produce a white solid, the organic phase was purified by silica gel chromatography using a gradient elution of C6H12/CH2Cl2(Yield: 24%).

Molecular Weight:454.7
XLogP3:7.9
Hydrogen Bond Donor Count:1
Hydrogen Bond Acceptor Count:3
Rotatable Bond Count:2
Exact Mass:454.34469533
Monoisotopic Mass:454.34469533
Topological Polar Surface Area:54.4
Heavy Atom Count:33
Complexity:902
Defined Atom Stereocenter Count:9
Covalently-Bonded Unit Count:1
Compound Is Canonicalized:Yes