Betulin

Phosphorus oxytrichloride (7.56 mL, 81.6 mmol; Sigma Aldrich, Moscow, Russia, 99%) in dioxane (60 mL) was added dropwise to a solution of betulin (6.0 g, 13.56 mmol) in a mixture of dioxane (120 mL) and pyridine (7.08 mL, 81.6 mmol) at 10–20 °C into a three-necked flask. The reaction mixture was stirred for 24 h at room temperature and then treated by water and ice mixture (1000 g) in the beaker. White precipitate was filtered off and washed by water many times. A wet sediment contained 3–25% of water that corresponds to the hydrate of betulin-3,28-diphosphate × H₂O, where x = 1–9 (7.90 g, 94% as dried product): mp: 147–148 °C (BDP-1), 156–160 °C (BDP-2); UV (EtOH): λ_max (log ε) 256 (2.64) nm; FTIR (KBr): ν_max 3421, 2331, 2342, 1641-1700, 1240, 1031, 973, 501 cm⁻¹; ¹H- NMR (DMSO-d₆, 400 MHz) δ 0.68–1.99 (42H, m, 6CH₃, (CH₂)₁₀, (CH)₄), 2.35–2.42 (1H, m, H-19), 2.97 (0.25H, wide t, α-H-3, J = 7.7 Hz), 3.69 (0.75H, ddd, β-H-3 m, J = 4.6, 7.8, 11.2 Hz), 3.96 (1H, dd, H-28, J = 9.7, 4.5 Hz) and 3.52 (H, dd, H-28′, J = 9.5, 4.5 Hz), 4.55, 4.69 (2H, two s, H-29), 5.69 (protons in the phosphate groups O-P(O)(OH)₂, wide blurred s); ¹³C-NMR (DMSO-d₆, 101 MHz) δ 149.93 (C, C-20), 109.92 (CH₂, C-29), 82.96 (CH, =CHOH), 63.22 (CH₂, CH₂OH), 54.90 (CH, C-5), 49.71 (CH, C-9), 48.11 (CH, C-19), 47.26 (CH, C-18), 46.75 (C, C-17), 42.30 (C, C-14), 40.48 (C, C-8), 38.57 (C, C-4), 38.35 (CH₂, C-1), 37.99 (C, C-10), 37.07 (CH, C-13), 36.57 (CH₂, C-7), 33.79 (CH₂, C-22), 29.11 (CH₂, C-21), 28.96 (CH₂, C-16), 28.17 (CH₃, C-23), 27.90 (CH₂, C-2), 26.57 (CH₂, C-15), 24.83 (CH₂, C-12), 20.43 (CH₂, C-11), 18.84 (CH₃, C-30), 18.01 (CH₂, C-6), 16.14 (CH₃, C-26), 15.91 (CH₃, C-24), 15.70 (CH₃, C-25), 14.56 (CH₃, C-27); ³¹P-NMR (DMSO-d₆, 202.46 MHz) δ −0.4 (d, J = 8.2 Hz, phosphoric acid residue at C-3β), 0.48 ppm (t, J = 4.6 Hz, phosphoric acid residue at C-28).
In the ¹³C-NMR spectrum the signals of the C-28 and C-3 atoms of BDP (60 and 80 ppm, respectively) were shifted by 3 ppm in comparison with botulin, which is characteristic for phosphoric acid esters.
The ³¹P-NMR spectra of BDP in DMSO-d₆ were obtained in the presence of Ph₃P (δ = −6.0 ppm) and H₃PO₄ (δ = −0.14 ppm) which was added just before recording the spectrum at 30 °C. The phosphoric acid residue at C-3β of BDP in the spectrum without decoupling of the protons was recorded as a doublet (δ = −0.4 ppm), while the coupling constant J_H-P ~ 8 Hz is typical for CH-O-P (Figure S1). The phosphoric acid residue at C-28 of BDP is represented as a triplet at δ = +0.48 ppm, with a coupling constant J_H-P ~ 4.6 Hz that is characteristic of the CH₂-O-P fragment of phosphatidic acids (alkylacyl glycerophosphates), where the signal (δ = +0.55 ppm, the coupling constant J_H-P ~ 6.9 Hz) corresponds to the -CH₂-O-P(O)(OH)₂ fragment [29 ].
Phosphorus content determined by spectrophotometric analysis [30 (link)] was equal to 9.59–10.32% (Calc. content for C₃₀H₅₂O₆P₂ is equal to 10.30%).
BDP assay was performed by reversed phase HPLC analysis: 210 nm, 40 °C, mobile phase A30%-B70% v/v (A—acetonitrile (grade 0), B—buffer solution of KH₂PO₄, pH = 6.36), flow 1.0 mL∙min⁻¹. The retention time is 5.19 min.
BDP-1 and BDP-2 separation. The wet BDP crude (9 g) was dissolved in ethanol (600 mL) and the insoluble part (BDP-2) was filtered off. BDP-1 was isolated by precipitation from BDP ethanol solution with acetone, and BDP-1 was recrystallized from ethanol.

Betulin was isolated from the nanoemulsion by employing methylene chloride for extraction, followed by centrifugation and subsequent evaporation of the organic layer to yield a desiccated residue that was reconstituted in HPLC-grade methanol. The betulin content was determined using an Agilent 1290 Infinity chromatographic system (Agilent, Carpinteria, CA, USA) on a reversed stationary phase column (ZORBAX Eclipse Plus C18 RRHD with dimensions of 2.1 × 50 mm and 1.8 µm) with an operating pressure of up to 1200 bar. The mobile phase consisted of a mixture of water and acetonitrile (20:80 (v/v)) with a flow rate of 0.35 mL/min, an injection volume of 2 µL at 35 °C, a detection wavelength of 210 nm, and a slit width of 4 nm. Identification and quantification of betulin were performed using a calibration curve of 24–56 µg/mL, and 2 µL of betulin was dispensed into the chromatographic system in triplicate. To determine the concentration of betulin in the emulsion, samples packed in vials were enclosed in a constant climate chamber “Binder KBF 240” (Binder, Tuttlingen, Germany) at a temperature of 55 °C. Every 180 h, the vial was removed, cooled, and visually assessed for the degree of emulsion separation, as well as the mass concentration of betulin using HPLC.

Betulin (BET) was separated through extraction from birch tree bark using the standard procedure [11 (link)]. The obtained substance was recrystallized from isopropyl alcohol and characterized using IR and NMR spectroscopy.
The IR specter (KBr, ν, cm^–1) of betulin is characterized by absorption bands in the 3368 cm^–1 area, which is typical for –OH groups, 1646 cm^–1 (C=C) and others: 2968–2864 (C–H), 1450, 1372, 1105, 1029, 878.
¹H NMR (400,17 MHz, CDCl₃, δ, ppm): the weaker fields area has isopropenyl group proton signals (4.51 and 4.61 ppm); 3.26 and 3.73 (p, 2H, C₂₈H₂-OH); 3.10–3.14 (m, 1H, C₃H-OH); 2.28–2.35 (m, 1H, C₁₉–H); in the 0.79–2.00 ppm area there are lupane skeleton proton signals, where C-H group proton signals are found in betulin structure (6CH₃, 10CH₂, 5CH).
¹³C NMR (100,63 MHz, CDCl₃, δ, m.f.): 150,51 (C-20); 109,71 (C-29); 78,98 (C-3); 64,46 (C-17); 60,51 (C-28); 55,28 (C-5); 50,38 (C-9); 48,74 (C-18); 47,79 (C-19); 42,71 (C-14); 40,91 (C-8); 38,87 (C-4); 38,69 (C-1); 37,30 (C-13); 37,15 (C-10); 34,22 (C-22); 33,98 (C-7); 29,74 (C-16); 29,16 (C-21); 27,99 (C-15); 27,38 (C-2); 27,04 (C-12); 25,37 (C-23); 25,19 (C-24); 20,83 (C-11); 19,09 (C-30); 18,30 (C-6); 16,12 (C-25); 15,98 (C-26); 15,38 (C-27).