4.3. Preparation of Betulin and Characterization of Betulin and Its Derivatives

The betulin and novel compounds were obtained in high yields and were identified by ¹H and ¹³C-NMR, moreover, the partition coefficient for all compounds was calculated in the MestReNova program (Figure 1, Table 1).

The structures of betulin and the obtained compounds were confirmed based on the analysis of nuclear magnetic resonance (NMR) spectroscopy spectra. Measurements were performed using a Bruker DPX-400 spectrometer. ¹H-NMR spectra were recorded under the following conditions: 400.13 MHz, a 12 kHz spectrum width, 65.5 K data points, a 0.488 Hz/point resolution, a data acquisition time of 4.09 s, a repetition time of 1 s, a 7.8 μs pulse width, and 16–32 scans. ¹³C-NMR spectra were as follows: 100.62 MHz, a spectrum width of 24 kHz, 65.5 K data points, a resolution of 1.46 Hz/point, a data acquisition time of 1.37 s, a repetition time of 1 s, a pulse width of 9.2 μs, and 1–8 scans.

The bark was boiled in a 1% aqueous KOH solution for 2 h. The bark was then filtered on a Büchner funnel and washed with hot water. The bark was dried in a vacuum dryer at 80 ℃ for 2 days.

The isolation of betulin was carried out from the shredder birch bark of the Betula verrusoca tree. An amount of 100 g of dried bark was first extracted with hexane (500 mL) in a Soxhlet apparatus, for 24 h (2 × 12 h), then with methylene chloride (500 mL) for 24 h (2 × 12 h). After evaporation to dryness, the hexane extract yielded 3.25 g of dry extract, while the methylene chloride extract yielded 11.17 g of dry extract.

The 11.17 g of dry extract obtained was heated under a reflux condenser in methanolic KOH 2% solution (100 mL) for 10 h. The flask was allowed to stand overnight at room temperature. The precipitate was drained on a Büchner funnel and washed copiously with hot water. The obtained impure betulin (8.4 g) was dried in a vacuum oven at 80 °C overnight. The betulin was then purified on a chromatography column in the system hexane:ethyl acetate (H:OAc) 40:1→5:1 v/v, monitoring the extraction with TLC (H:OAc 5:1). Finally 7.23 g of pure betulin was obtained (1).

An amount of 1 g (2.26 mmol) of betulin (1) was placed in 3-neck round-bottomed flask, in 150 mL of acetone, at 0 °C (ice bath) under N₂. Freshly prepared Jones reagent (3.9 mL, 0.39 M, 7.68 mmol) was added dropwise from an additional funnel to the stirred suspension of betulin (1) over 15 min. After 1 h, stirring was continued at room temperature for 2 h, then 25 mL of methanol was added until a dark green color appeared. The reaction mixture was then poured onto 100 mL of water with ice. Methanol and acetone were removed under reduced pressure. The aqueous residue was washed 3 times with 40 mL of ethyl acetate. The combined organic layers were first washed with brine (20 mL) then with water (20 mL) and finally dried with anhydrous MgSO4. The solvent was evaporated under reduced pressure to give a crude white solid, which was purified by column chromatography to deliver first 59.7 mg (6%) of betulonal as a white solid, then 860 mg (86%) of betulonic acid as a white solid.

An amount of 5 g (11.3 mmol) of betulin (1) and DMAP (3.05 g, 25 mmol) were placed in a round-bottomed flask, in 200 mL of dry pyridine, at 0 °C (cooling bath). Then, 24.5 mL (0.25 mmol) of Ac₂O was slowly added dropwise (over 1 h) to the stirred solution from the additional funnel. The solvent was evaporated under reduced pressure to give a crude dark solid, which was purified by column chromatography in the system hexane:ethyl acetate (H:OAc) 40:1→5:1 v/v, to deliver first 0.55 g (11%) of betulin 3,28-diacetate 3 as a w white crystalline powder, then 1.86 g (37%) of betulin 28-acetate as a white crystalline powder.

A solution of 5 (0.5 mmol, 265 mg) in dry CCl₄ (5 mL) was treated with freshly recrystallized NBS (0.15 g, 0.85 mmol,) and refluxed for 3 h. The solid was filtered off and the mother liquor was evaporated under vacuum. The dark solid was recrystallized from EtOH to obtain 0.183 g (69%) as brown needles.

To a solution of 4 (3.09 mmol, 1.5 g) in 60 mL mixture of MeOH/THF (2:1 v/v), 10% Pd/C (167 mg) was added and hydrogenated under H₂ (40 psi) for 24 h. Next, it was filtered, and the solvent was evaporated under reduced pressure to give 1.49 g, (99+%) as a white crystalline solid.

To a cooled (ice bath) solution of monoacetate 7 (1.40 g, 2.88 mmol) in 50 mL of DCM, PCC (3.68 g, 17.07 mmol) was slowly added in small portions over 5 min. The reaction was monitored by TLC. After 4 h, the flask with the reaction mixture was placed in the freezer overnight. The solvent was evaporated under reduced pressure and the obtained residue was purified by column chromatography (H:OAc 1:1 v/v) to give 1.30 g (92%) of the ketone 8 as a white solid.

To a solution of ketone 8 (1.20 g, 2.48 mmol) in 50 mL of chloroform was added 70%-mCPBA (3.23 g, 15 mmol). The reaction mixture was heated to 30–35 °C and after 4 h another portion of 70%-mCPBA (0.91 g, 4.22 mmol) was added. The reaction was monitored by TLC, there was still presence of the substrate. After 24 h (the substrate was still present on TLC), another portion of 70%-mCPBA (0.58 g, 2.69 mmol) was added. After 12 h (there was only a trace of the substrate, TLC), 30 mL solution of 10%-Na₂S₂O₃ was slowly added (the temperature of the mixture increases). The mixture was transferred to a separatory funnel and the organic layer was washed with 30 mL of saturated NaHCO₃ solution. The solvent was evaporated under reduced pressure and the obtained residue was purified by column chromatography (H:OAc 20:1→3:1 v/v) to give 1.06 g (85%) of 9 as a white solid.

To a cooled 20 mL amount of THF (about −40 °C acetonitrile with dry ice), LiAlH₄ (606 mg, 16 mmol) was added followed by 9 (800 mg, 1.6 mmol). The reaction was stirred for 1.5 h until the substrate disappeared (TLC). Once the reaction was complete, 10 mL of ethyl acetate was added followed by slowly added 30 mL of saturated NH₄Cl solution (until bubbling disappeared). The organic layer was separated and filtered through a thin layer of silica gel. The aqueous layer was extracted with 20 mL of ethyl acetate. The solvents were evaporated under reduced pressure and the obtained residue was purified by column chromatography (H:OAc 1:1 v/v) to give 736 mg (96%) of 10 as a white solid.

To a cooled solution (about −70 °C isopropyl alcohol and dry ice) of 10 (500 mg, 1.08 mmol) in 20 mL THF was added imidazole (235.39 mg, 3.46 mmol) and TBDPSCl (445.48 mg, 1.62 mmol, d = 1.057 g/mL). When the temperature reached ambient temperature, the mixture was further stirred for 24 h. Next, the solvent was evaporated under reduced pressure and the obtained oily residue was purified by column chromatography (H:OAc 20:1→5:1 v/v) to give 370 mg (49%) of ketone 11 as a light straw-colored oil.