Sulfonation of BA by NH SO H in the presence of urea in DMF or 1,4-dioxane formed the ammonium salt of BA
2
3
3-sulfate (2), which was treated with H SO (10%) to give BA 3-sulfonic acid (3a); with KOH or NaOH solution (3–5%),
2
4
the stable salts 3b or 3c.
–1
IR spectra of 3b and 3c, in contrast to that of BA, showed an absorption band at 830 cm (SO), and a strong band at
1221 (SO ), which confirmed that 3b and 3c contained sulfate groups [11] in addition to 2 and 3a.
2
13
C NMR spectra of the natural 3ꢀ- and 3ꢁ-stereomers of BA and 3ꢀ- and 3ꢁ-stereomers of BA sulfates isolated from
Schefflera octophylla leaves were reported [11] and showed that the C-3 resonance in 3ꢀ-BA was observed at 75.5 ppm; in
3ꢁ-BA, at 78.2 ppm. The C-3 resonance of BA 3ꢀ-sulfate had a chemical shift of 82.9 ppm; of BA 3ꢁ-sulfate, 86.0 ppm.
13
A comparison of the C NMR spectra of starting BA and sulfates 3b and 3c showed that replacing the OH by SO H
3
shifted the C-3 resonance from 79.0 to 86.5 ppm.
Thus, it was established that reaction of BA and NH SO H in the presence of urea in DMF and 1,4-dioxane occurred
2
3
in 2.5–3.5 h at 65–75°C. The yield of 2 was 96%; 3a, 94; 3b, 93; 3c, 95.
The advantage of BA sulfonation by NH SO H is the use of more available and less aggressive reagents. The nature
2
3
of the solvent (DMF and 1,4-dioxane) did not substantially affect the course of BA sulfonation.
EXPERIMENTAL
General. IR spectra were taken from KBr pellets (3 mg of sample/300 mg of KBr) on a Tensor 27 FTIR spectrometer
–1 13
(Bruker, Germany) in the range 400–4000 cm . C NMR spectra were recorded in CD OD with TMS = 0 on a Bruker
3
Avance III spectrometer (600 MHz). Elemental analysis with simultaneous determination (%) of C, H, N, S, and O was
TM
performed on a FlashEA 1112 analyzer (Thermo Quest, Italy). The K and Na contents were determined on an AAnalyst-
400 atomic absorption spectrometer (PerkinElmer) using an acetylene/nitrous-oxide flame. Melting points were measured on
an Electrothermal A9100 apparatus.
Betulinic acid (1) was prepared from betulin by the known method in two steps. The first consisted of betulin
oxidation by CrO in AcOH to betulonic acid (61% yield, mp 247–249°C) [1]. The second involved betulonic acid reduction
3
by NaBH in THF to betulinic acid (94% yield, mp 290–292°C) [1, 12, 13]. The BA used for sulfonation was the single
4
ꢁ-stereomer. This was confirmed by the full agreement of its physicochemical properties with those published [7, 11–13].
DMF was dried by shaking with KOH and distilled over CaO. 1,4-Dioxane was dried over KOH and distilled over Na [14].
Salts 3b and 3c were purified by recrystallization from MeOH (75%).
Sulfonation of BA in DMF and 1,4-Dioxane. Ammonium 3ꢁ-O-Sulfate-lup-20(29)-en-28-oate (2). DMF or
1,4-dioxane (50 mL) in a 100-mL three-necked flask equipped with a stirrer and thermometer was stirred vigorously; treated
with NH SO H (1.46 g, 0.015 mol), urea (0.90 g), and 1 (4.56 g, 0.01 mol); heated on a water bath at 65–75°C for 2.5–3.5 h,
2
3
cooled, diluted with H O (100 mL), transferred to a separatory funnel, and extracted with BuOH (130–150 mL). The BuOH
2
extract was washed with H O and evaporated to dryness in vacuo. Yield of 2, 96%, mp (dec.) 147–150°C. C H N O S.
2
30 54 2 6
3ꢁ-O-Sulfate-lup-20(29)-en-28-oic Acid (3a). The BuOH extract containing 2 was acidified with H SO solution
2
4
(10%) to pH 2–3. The BuOH layer was separated and concentrated in vacuo to afford 3a in 94% yield, mp (dec.) 139–143°C,
lit. mp 140–142°C [14]. C H O S.
30 48
6
Potassium 3ꢁ-O-Sulfate-lup-20(29)-en-28-oate (3b). The BuOH extract containing 2 was worked up with KOH
solution (3–4%) to pH 8–9. The BuOH layer was separated and concentrated in vacuo to afford 3b in 93% yield; 74% after
–1
recrystallization, mp (dec.) 312–315ꢂC. C H O SK . IR spectrum (KBr, ꢃ, cm ): 838 (C–O–S), 1221 (O=S=O).
30 46
6
2
13
C NMR spectrum (CD OD, ꢄ, ppm): 38.5 (C-1), 24.1 (C-2), 86.4 (C-3), 38.6 (C-4), 56.1 (C-5), 18.1 (C-6), 34.7 (C-7), 40.1
3
(C-8), 51.1 (C-9), 37.2 (C-10), 20.9 (C-11), 26.5 (C-12), 38.5 (C-13), 42.4 (C-14), 31.4 (C-15), 32.0 (C-16), 56.5 (C-17), 47.1
(C-18), 48.2 (C-19), 151.1 (C-20), 31.3 (C-21), 37.6 (C-22), 28.4 (C-23), 16.6 (C-24), 16.2 (C-25), 16.1 (C-26), 14.7 (C-27),
179.0 (C-28), 20.1 (C-29), 109.3 (C-30).
Sodium 3ꢁ-O-Sulfate-lup-20(29)-en-28-oate (3c) was prepared analogously to 3b using NaOH solution (3–4%) in
13
95% yield, 76% after recrystallization, mp (dec.) 314–317°C. C H O SNa . The IR and C NMR spectra of 3c were
30 46
6
2
identical to those published [15].
895