Synthesis of Conjugates of Lupane-Type Pentacyclic Triterpenoids with 2-Aminoethane- and N-Methyl-2-Aminoethanesulfonic Acids. Assessment of in vitro Toxicity

Conjugates of betulin and betulinic and betulonic acids with 2-aminoethane- and N-methyl-2-aminoethanesulfonic acids were synthesized for the first time and were interesting as potential biologically active compounds. Experiments in vitro in MDCK cell culture using the MTT assay found that betulin and betulinic-acid derivatives with aminoethanesulfonic acid bound to triterpene C-3 or C-28 through an ester linker were less toxic than the native compounds.

Lupane-type pentacyclic triterpenoids are interesting as platforms for developing new drugs with various biological activities [1,2,3,4]. In continuation of research on the modification of lupane triterpenoids, we developed approached to conjugates containing aminoalkanesulfonic acids. The endogenous metabolite taurine (2-aminoethanesulfonic acid), its derivatives, and natural compounds containing taurine possess broad spectra of biological activity [5,6,7] including antiviral [8,9,10,11,12], antibiotic [13], and antitumor [14, 15]. Introduction of taurine or its homologs into lupane-type pentacyclic triterpenoids would expand the spectrum of biologically active compounds in this series and provide new information about the structure–activity (biological) relationship.

Herein, conjugates containing C-28/C-3 2-aminoethane- or N-methyl-2-aminoethanesulfonic acid (N-methyltaurine) as the ammonium or sodium salt or the free aminoethanesulfonic acids bound to the triterpene skeleton via an ester linker were synthesized from betulin (1) chloroacetates, betulonic acid (2) bromoalkyl esters, and betulinic acid (3a) methyl chloroacetates or benzyl esters. Experiments in vitro assessed the toxicity of the synthesized compounds in MDCK cell culture (canine kidney cell line).

Starting chloroacetates 4 and 5a,b were obtained from 1 or 3b,c and chloroacetic acid using the carbodiimide method. Betulonic acid 2-bromoethyl and 3-bromopropyl esters 6a,b were synthesized via O-alkylation of acid 2 with the appropriate dibromoalkane (DMF, K₂CO₃). Acid 2 was prepared by a modified literature method [16] using sequential oxidation of 1 by pyridinium chlorochromate (PCC) (2 h) and treatment of the obtained betulone aldehyde with NaClO₂–NaH₂PO₄ in the presence of 2-methyl-2-butene for 15 min with an aldehyde–NaClO₂–NaH₂PO₄ ratio of 1:6:6. The yield of acid 2 after purification through the potassium salt was 75%. Acid 3a and methyl 3b and benzyl 3c esters were synthesized by the reported methods [17,18,19]. N-Methyltaurine was prepared by N-alkylation of methylamine with sodium 2-bromoethanesulfonate in 73% yield [20].

Conjugates of taurine 7 and 8 or N-methyltaurine 9 and 10a,b were synthesized by N-alkylation of the tetrabutylammonium salt of taurine [21] or N-methyltaurine chloroacetates 4 and 5a or 5b in DMF in the presence of K₂CO₃ at 95°C.

Betulonic acid derivatives with sodium N-methyl-2-aminoethanesulfonate 11a and 11b were prepared by reacting bromoalkyl esters 6a and 6b with the sodium salt of N-methyltaurine [22] in the presence of K₂CO₃ in DMF at 95°C for 50 h.

The yields of conjugates 7–9, 10a,b, and 11a,b were 64–90%. The structures of these compounds were established using PMR and ¹³C NMR spectroscopy and mass spectrometry. PMR and ¹³C NMR spectra of conjugates 8, 9, 10b, and 11a were fully assigned using 2D experiments (Table 1).

Table 1. ¹³C NMR Spectra of 6a,b–9, 10a,b, and 11a,b (δ, ppm)*

Toxicity in vitro of 1, 2, 3a, 7–9, 10a, and 11a. Assessment of the toxicity of the compounds was an important step in preclinical studies of their biological activity. The toxicity of synthesized 7–9, 10a, and 11a was screened in vitro using MDCK cell culture and the MTT assay. The median cytotoxic dose (CTD50, dose leading to the death of 50% of the cells) and minimum toxic dose (MTD, dose causing toxic effects to appear in the cells) were determined. The MTD became an independent important toxicity characteristic if the CTD₅₀ value exceeded the upper limit of the tested concentrations. According to this criterion, conjugates 7, 9, and 10a were slightly toxic compounds (Table 2). A criterion reflecting the rate of toxicity increase with increasing concentration (CTD₅₀/MTD) served as a guide for comparing the toxicity of compounds with similar CTD₅₀ values, e.g., 8 (CTD₅₀ 30 μg/mL) and 11a (CTD₅₀ 37.5 μg/mL). According to this criterion, 11a (CTD₅₀/MTD = 1.3; for 8, 4.8) was the most toxic compound.

Table 2. Toxicity Screening of 1, 2, 3a, 7–9, 10a, and 11a

The toxicity results for 7–9 and 10a showed that adding aminoethanesulfonic acids through ester linkers to triterpene C-3 or C-28 reduced considerably the toxicity of the conjugates as compared with native precursors 1 and 3a (Table 2, compounds 1, 7, 9, 3a, 8, 10a). The exception was betulonic acid derivative 11a, the toxicity of which was the same as acid 2 itself (CTD₅₀/MTD = 1.5 and 1.6, respectively). An analysis of the toxicities of betulin and betulinic acid derivatives with the same substituents (pairs 7,8 and 9,10a) (Table 2) or derivatives within each series 7,9 and 8,11a with different substituents showed that the toxicity depended on both the triterpenoid structure and the fine differences in the substituents.

Thus, methods for preparing conjugates of betulin and betulinic and betulonic acids with aminoethanesulfonic acids that were interesting as potential biologically active compounds were developed. They were also intermediates for synthesizing various sulfonamide and peptide derivatives of lupane-type pentacyclic triterpenoids. The in vitro experiments provided seminal information on the effects of aminoethanesulfonic acid substituents on the toxicity of these compounds that was valuable for planning further syntheses of biologically active compounds based on aminoethanesulfonic acids.

Experimental

IR spectra were taken in mineral oil on a Shimadzu Prestige-21 IR spectrophotometer. PMR and ¹³C NMR spectra were recorded on a Bruker Avance III pulsed spectrometer at operating frequencies 500.13 MHz for ¹H and 125.47 MHz for ¹³C using a Z-gradient PABBO probe at 298 K or a Bruker AM-300 spectrometer at operating frequencies 300.13 MHz for ¹H and 75.47 MHz for ¹³C. Chemical shifts in PMR and ¹³C NMR spectra were given in ppm relative to solvent (CDCl₃) resonances (δ_H 7.27 and δ_C 77.1) or TMS internal standard. 2D spectra were recorded using standard multi-pulse sequences of the Bruker Avance III spectrometer software. Mass spectra were obtained using electrospray ionization (ESI) or chemical ionization at atmospheric pressure (APCI) on an LCMS-2010EV liquid chromatograph-mass spectrometer (Shimadzu) (sample injection via syringe, 0.1 mL/min, MeCN–H₂O eluent, 95:5) in positive- and negative-ion modes at capillary potential 4.5 and –3.5 kV. The APCI interface temperature was 250°C, heater 200°C, vaporizer 230°C. The spray-gas (N₂) flow rate was 1.5 and 2.5 L/min for ESI and APCI, respectively. Rotation angles were measured on a PerkinElmer 341C instrument. Column chromatography used SiO2 (PTSKh-AF-A, Imid, Krasnodar, Russia). Melting points were determined on a Kofler apparatus. Betulin (1) was isolated from birch bark by the literature method [23]. Spectral characteristics (PMR and ¹³C NMR) of betulinic acid 3a and its esters 3b and 3c that were prepared according to the literature [17,18,19] agreed with those reported. Commercial 2-aminoethanesulfonic acid was used. DMF was stored over MgSO₄ and vacuum distilled under Ar from 4Å molecular sieves. CH₂Cl₂ was distilled from CaH₂. K₂CO₃ was calcined before the reactions.

3-Oxolup-20(29)-en-28-oic Acid (betulonic acid) (2). A 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, 85%) (PMR and ¹³C NMR spectra agreed with 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, 15% 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 (10%, 50 mL). The organic layer was separated, washed with H₂O, dried over Na₂SO₄, and evaporated to afford 2 (3.26 g, 75%). The PMR and ¹³C NMR spectra agreed with the literature data [16].

3 β -Hydroxylup-20(29)-en-28-yl 2-Chloroacetate (4). A solution of 1 (3.00 g, 6.78 mmol) and Et₃N (2.83 mL, 20.33 mmol) in CH₂Cl₂ (300 mL) was stirred, treated dropwise with a solution of chloroacetic chloride (1.3 mL, 16.26 mmol) in CH₂Cl₂ (50 mL), stirred at 20°C for 30 min, washed with HCl (10%, 100 mL) and H₂O, dried over Na₂SO₄, and evaporated.

The residue was chromatographed over SiO₂ (C₆H₆–MTBE, 8:1) to afford chloroacetate 4 (2.18 g, 63%), mp 152–154°C (lit. [24]: 154–156°C). IR spectrum (ν, cm^–1): 1660, 1740, 3325. ¹H NMR spectrum (300 MHz, CDCl₃, δ, ppm, J/Hz): 0.67 (1H, d, J = 8.9, H-5), 0.76 (3H, s, CH₃-24), 0.82 (3H, s, CH₃-25), 0.96 (3H, s, CH₃-23), 0.98 (3H, s, CH₃-26), 1.03 (3H, s, CH₃-27), 1.68 (3H, s, CH₃-30), 2.43 (1H, td, J = 10.5, 5.5, H-19), 3.18 (1H, dd, J = 11.0, 5.3, H-3), 3.86 (2H, s, OC(O)CH₂), 3.94, 4.37 (each 1H, d, J = 11.0, H-28), 4.60, 4.70 (each1H, br.s, H-29) (only characteristic resonances are given). The ¹³C NMR spectrum agreed with the literature data [24].

Preparation of 5a and 5b. A solution of ester 3b or 3c (1.06 mmol) in CH₂Cl₂ (60 mL) was treated under Ar with chloroacetic chloride (2.13 mmol) and DMAP (2.13 mmol), stirred for 5 min, treated with DCC (2.13 mmol), and stirred for 20 min. The precipitate of N,N-dicyclohexylurea was filtered off. The organic layer was washed with HCl (10%, 20 mL) and H₂O, dried over Na₂SO₄, and evaporated. The residue was purified by flash chromatography over SiO₂ (C₆H₆–MTBE, 4:1).

Methyl Ester of 3 β -(2-Chloroacetoxy)lup-20(29)-en-28-oic Acid (5a). Yield 0.54 g (94%), amorph., ${\left[\upalpha \right]}_{\mathrm{D}}^{20}$ +4° (c 0.15, CHCl₃). IR spectrum (ν, cm^–1): 1650, 1734. ¹H NMR spectrum (300 MHz, CDCl₃, δ, ppm, J/Hz): 0.68 (1H, d, J = 10.0, H-5), 0.84 (3H, s, CH₃-24), 0.85 (3H, s, CH₃-25), 0.86 (3H, s, CH₃-23), 0.91 (3H, s, CH₃-27), 0.95 (3H, s, CH₃-26), 1.68 (3H, s, CH₃-30), 1.87 (2H, m, H-21, 22), 2.19 (1H, m, H-13), 2.22 (1H, m, H-16), 3.00 (1H, td, J = 11.0, 4.5, H-19), 3.66 (3H, s, C(O)OCH₃), 4.05 (2H, s, CH₂Cl), 4.56 (1H, dd, J = 10.0, 6.0, H-3), 4.60, 4.73 (each 1H, s, H-29). ¹³C NMR spectrum (75.47 MHz, CDCl₃, δ, ppm): 14.64 (C-27), 16.13 (C-25), 16.40 (C-26), 15.92 (C-24), 18.10 (C-6), 19.31 (C-30), 20.86 (C-11), 23.52 (C-2), 25.41 (C-12), 27.88 (C-23), 29.63 (C-15), 30.55 (C-21), 32.12 (C-16), 34.17 (C-7), 36.92 (C-22), 37.06 (C-10), 37.97 (C-4), 38.18 (C-13), 38.28 (C-1), 40.65 (C-8), 41.23 (CH₂Cl), 42.37 (C-14), 49.41 (C-19), 51.26 (C(O)OCH₃), 46.95 (C-18), 50.40 (C-9), 55.35 (C-5), 56.51 (C-17), 83.35 (C-3), 109.62 (C-29), 150.52 (C-20), 167.14 (C=O), 176.64 (C-28). Mass spectrum (ESI), m/z 453 [M+H-ClCH₂CO₂H]⁺ (calcd for C₃₃H₅₁ClO₄, 546.5).

Benzyl Ester of 3 β -(2-Chloroacetoxy)lup-20(29)-en-28-oic Acid (5b). Yield 0.61 g (92%), mp 57–59°C, ${\left[\upalpha \right]}_{\mathrm{D}}^{20}$ –9° (c 0.22, CHCl₃). IR spectrum (ν, cm^–1): 700, 1650, 1730, 1762. ¹H NMR spectrum (300 MHz, CDCl₃, δ, ppm, J/Hz): 0.76 (3H, s, CH₃-24), 0.83 (3H, s, CH₃-25), 0.84 (3H, s, CH₃-26), 0.85 (3H, s, CH₃-23), 0.94 (3H, s, CH₃-27), 1.68 (3H, s, CH₃-30), 1.88 (2H, m, H-21, 22), 2.18 (1H, td, J = 11.0, 5.4, H-13), 2.28 (1H, d, J = 11.8, H-16), 3.02 (1H, td, J = 10.5, 4.2, H-19), 4.02, 4.05 (each1H, d, J = 18.5, CH₂Cl), 4.55 (1H, dd, J = 10.5, 4.3, H-3), 4.60, 4.72 (each 1H, s, H-29), 5.08, 5.18 (each 1H, d, J = 19.6, CH ₂Ph), 7.36 (5H, m, Ph). ¹³C NMR spectrum (75.47 MHz, CDCl₃, δ, ppm): 14.64 (C-27), 15.83 (C-25), 16.19 (C-26), 16.43 (C-24), 18.14 (C-6), 19.35 (C-30), 20.90 (C-11), 23.56 (C-2), 25.46 (C-12), 27.94 (C-23), 29.55 (C-15), 30.55 (C-21), 32.10 (C-16), 34.18 (C-7), 36.94 (C-22), 37.07 (C-10), 38.00 (C-4), 38.15 (C-13), 38.31 (C-1), 40.65 (C-8), 41.28 (CH₂Cl), 42.40 (C-14), 46.96 (C-19), 49.41 (C-18), 50.43 (C-9), 55.37 (C-5), 56.53 (C-17), 65.74 (CH₂Ph), 83.35 (C-3), 109.68 (C-29), 128.08, 128.26, 128.50 (Ph), 136.50 (Ph: C-1), 150.55 (C-20), 167.17 (C=O), 175.83 (C-28). Mass spectrum (ESI), m/z 529 [M + H – ClCH₂CO₂H]⁺ (calcd for C₃₉H₅₅ClO₄, 622).

Preparation of 6a and 6b. A suspension of acid 3 (4.40 mmol) and K₂CO₃ (4.40 mmol) in DMF (18 mL) was treated dropwise with 1,2-dibromoethane or 1,3-dibromopropane (4.40 mmol) in DMF (2 mL) and stirred for 4 h. The K₂CO₃ was filtered off. The filtrate was evaporated. The residue was filtered through a layer of Al₂O₃. The filtrate was evaporated.

2-Bromoethyl Ester of 3-Oxolup-20(29)-en-28-oic Acid (6a). Yield 1.9 g (77%), amorph., ${\left[\upalpha \right]}_{\mathrm{D}}^{20}$ +4° (c 0.25, CHCl₃). IR spectrum (ν, cm^–1): 1650, 1726. ¹H NMR spectrum (300 MHz, CDCl₃, δ, ppm, J/Hz): 0.92 (3H, s, CH₃-25), 0.97 (3H, s, CH₃-26), 0.98 (3H, s, CH₃-27), 1.02 (3H, s, CH₃-24), 1.07 (3H, s, CH₃-23), 1.69 (5H, s, CH₃-30, m, H-15, H-18), 1.91 (3H, m, H-1, 21, 22), 2.24 (1H, td, J = 12.0, 4.8, H-13), 2.28 (1H, m, H-16), 2.44 (H, m, H-2), 3.02 (1H, td, J = 11.5, 5.3, H-19), 3.54 (2H, t, J = 6.0, CH₂Br), 4.40, 4.42 (each 1H, dt, J = 12.0, 6.0, C(O)OCH ₂CH₂), 4.61, 4.74 (each 1H, s, H-29). ¹³C NMR spectrum (Table 1). Mass spectrum (APCI), m/z 561 [M + H]⁺ (calcd for C₃₂H₄₉BrO₃, 560).

3-Bromopropyl Ester of 3-Oxolup-20(29)-en-28-oic Acid (6b). Yield 1.9 g (76%), mp 80–83°C, ${\left[\upalpha \right]}_{\mathrm{D}}^{20}$ –6° (c 0.40, CHCl₃). IR spectrum (ν, cm^–1): 1630, 1730. ¹H NMR spectrum (300 MHz, CDCl₃, δ, ppm, J/Hz): 0.92 (3H, s, CH3-25), 0.96 (3H, s, CH3-26), 0.97 (3H, s, CH₃-27), 1.01 (3H, s, CH₃-24), 1.06 (3H, s, CH₃-23), 1.68 (5H, s, CH₃-30, m, Hb-15, H-18), 1.88 (3H, m, H-1, 21, 22), 2.20 (2H, m, H-13, 16), 2.43 (2H, m, H-2), 3.00 (1H, m, H-19), 3.48 (2H, t, J = 6.5, CH₂Br), 4.21 (2H, m, C(O)OCH₂CH₂), 4.64, 4.73 (each 1H, s, H-29) (only characteristic resonances are given). Table 1 lists the ¹³C NMR spectrum. Mass spectrum (APCI), m/z 575 [M + H]⁺ (calcd for C₃₃H₅₁BrO₃, 574).

Tetrabutylammonium 2-{2-[3 β -Hydroxylup-20(29)-en-28-yloxy]-2-oxoethylamino}ethanesulfonate (7). A solution of NH₂CH₂CH₂SO₃ ^–Bu4N⁺ (0.16 g, 0.44 mmol) in DMF (4 mL) was treated with K₂CO₃ (0.06 g, 0.44 mmol), stirred for 10 min, treated dropwise with betulin chloroacetate (4, 0.23 g, 0.44 mmol) in DMF (5 mL), and stirred at 95°C for 3 h. The K₂CO₃ was filtered off. The filtrate was evaporated. The residue was chromatographed over SiO₂ (C₆H₆–MTBE, 4:1; CHCl₃–MeOH, 10:1) to afford 7 (0.25 g, 68%), amorph. IR spectrum (ν, cm^–1): 1035, 1192, 1640, 1740, 3378. ¹H NMR spectrum (500 MHz, CDCl₃, δ, ppm, J/Hz): 0.67 (1H, d, J = 9.1, H-5), 0.76 (3H, s, CH₃-24), 0.82 (3H, s, CH₃-25), 0.96 (6H, s, CH₃-23, 27), 1.01 (12H, t, J = 7.3, butyl: CH₂CH₂CH₂CH ₃), 1.03 (3H, s, CH₃-26), 1.45 (10H, m, butyl: CH₂CH₂CH ₂CH₃, 2H-2), 1.67 (8H, m, butyl: CH₂CH ₂CH₂CH₃), 1.69 (3H, s, CH₃-30), 1.76 (1H, dd, J = 12.2, 8.8, H-22), 1.83 (1H, d, J = 12.4, H-16), 1.95 (1H, m, H-21), 2.42 (1H, td, J = 10.5, 5.5, H-19), 3.03 (2H, t, J = 6.3, NCH₂CH ₂SO₃), 3.13 (2H, t, J = 6.3, NCH ₂CH₂SO₃), 3.18 (1H, dd, J = 11.2, 4.5, H-3), 3.32 (8H, t, J = 6.5, butyl: CH ₂CH₂CH₂CH₃), 3.48 (2H, s, OC(O)CH₂), 3.88, 4.29 (each 1H, d, J = 11.0, H-28), 4.58, 4.68 (1H, s, H-29). Table 1 lists the ¹³C NMR spectrum. Mass spectrum (ESI), m/z 606 [M – N⁺Bu₄]^– (calcd for C₅₀H₉₂N₂O₆S, 848).

Tetrabutylammonium 2-{2-[17 β -Methoxycarbonyllup-20(29)-en-3 β -yloxy]-2-oxoethylamino}ethanesulfonate (8) was prepared analogously to 7. Yield 0.30 g (82%), mp 78–80°C, ${\left[\upalpha \right]}_{\mathrm{D}}^{20}$ –8.5° (c 0.43, CHCl₃). IR spectrum (ν, cm^–1): 1034, 1166, 1190, 1200, 1640, 1729, 3420. ¹H NMR spectrum (500 MHz, CDCl₃, δ, ppm, J/Hz): 0.76 (1H, d, J = 9.5, H-5), 0.82 (3H, s, CH₃-25), 0.83 (3H, s, CH₃-26), 0.91 (3H, s, CH₃-24), 0.82 (3H, s, CH₃-23), 0.95 (3H, s, CH₃-27), 0.96 (1H, m, H-1), 1.01 (12H, t, J = 7.3, butyl: CH₂CH₂CH₂CH ₃), 1.14 (1H, m, H-15), 1.28 (1H, m, H-11), 1.36 (4H, m, 1H-6, 2H-7, 1H-21), 1.45 (4H, m, 1H-11, 15, 16, 22), 1.46 (2H, m, H-2), 1.47 (8H, sextet, J = 7.3, butyl: CH₂CH₂CH ₂CH₃), 1.49 (1H, m, H-6), 1.58 (1H, t, J = 11.3, H-18), 1.65 (9H, m, butyl: CH₂CH ₂CH₂CH₃, H-1), 1.65 (8H, m, butyl: CH₂CH ₂CH₂CH₃), 1.68 (3H, s, CH₃-30), 1.88 (2H, m, H-21, 22), 2.21 (1H, td, J = 12.0, 5.5, H-13), 2.23 (1H, dd, J = 10.0, 3.3, H-16), 2.90 (1H, br.s, NH), 3.00 (1H, m, H-19), 3.01 (2H, t, J = 6.2, NCH₂CH ₂SO₃), 3.12 (2H, t, J = 6.2, NCH ₂CH₂SO₃), 3.31 (8H, t, J = 6.5, butyl: CH ₂CH₂CH₂CH₃), 3.45 (2H, s, OC(O)CH₂), 3.67 (3H, s, C(O)OCH₃), 4.50 (1H, dd, J = 11.0, 5.5, H-3), 4.60, 4.73 (each 1H, s, H-29). Table 1 lists the ¹³C NMR spectrum. Mass spectrum (ESI), m/z 634 [M – N⁺Bu₄]^– (calcd for C₅₁H₉₂N₂O₇S, 876).

Preparation of 9, 10a, and 10b. General Method. A suspension of N-methyltaurine (0.19 mmol) in DMF (3 mL) was treated with K₂CO₃ (0.19 mmol), stirred for 15 min, treated dropwise with chloroacetate 4, 5a, or 5b (0.19 mmol) in DMF (2 mL), and stirred at 95°C for 50 h (for 9) or 6 h (for 10a and 10b). The K₂CO₃ was filtered off. The filtrate was evaporated to dryness. The resulting oily residue was triturated with hexane. The resulting solid was filtered off. Solid compound 9 was rinsed with MTBE and dried in vacuo (KOH, 80°C). Compounds 10a and 10b that were obtained after hexane work-up were chromatographed over SiO₂ (C₆H₆–MTBE, 4:1; CHCl₃–MeOH, 4:1).

2-({2-[3 β -Hydroxylup-20(29)-en-28-yloxy]-2-oxoethyl}(methyl)amino)ethanesulfonic Acid (9). Yield 0.11 g (90%), amorph., ${\left[\upalpha \right]}_{\mathrm{D}}^{20}$ +9.5° (c 0.64, CH₃OH). IR spectrum (ν, cm^–1): 1038, 1192, 1213, 1637, 1653, 1748, 3420. ¹H NMR spectrum (500 MHz, DMSO-d₆, δ, ppm, J/Hz): 0.62 (1H, d, J = 9.8, H-5), 0.65 (3H, s, CH₃-24), 0.76 (3H, s, CH₃-25), 0.83 (1H, m, Ha-1), 0.87 (3H, s, CH₃-23), 0.94 (3H, s, CH₃-27), 0.99 (5H, s, CH₃-26, m, H_a-12, 15), 1.09 (1H, m, H_a-22), 1.20 (1H, m, H_a-11), 1.26 (2H, m, H-9, H_a-16), 1.34 (5H, m, H_a-6, 21, 2H-7, H_b-11), 1.44 (3H, m, 2H-2, H_b-6), 1.54 (1H, m, H_b-1), 1.57 (1H, m, H-18), 1.60 (2H, m, H-13, H_b-12), 1.65 (4H, s, CH₃-30, m, H_b-15), 1.72 (1H, m, H_b-22), 1.74 (1H, m, H_b-16), 1.91 (1H, m, H_b-21), 2.48 (1H, m, H-19), 2.66 (3H, s, NCH₃), 2.84 (2H, t, J = 7.5, NCH₂CH ₂SO₃), 2.96 (1H, dd, J = 10.0, 5.3, H-3), 3.21 (2H, m, NCH ₂CH₂SO₃), 3.28 (1H, d, J = 10.8, H_a-28), 3.98 (2H, s, OC(O)CH₂), 4.37 (1H, d, J = 10.8, H_b-28), 4.57, 4.71 (each 1H, s, H-29), 8.73 (1H, br.s, SO₃H). Table 1 lists the ¹³C NMR spectrum. Mass spectrum (ESI), m/z 620 [M – H]^–(calcd for C₃₅H₅₉NO₆S, 621).

2-({2-[17 β -Methoxycarbonyllup-20(29)-en-3 β -yloxy]-2-oxoethyl}(methyl)amino)ethanesulfonic Acid (10a). Yield 0.08 g (66%), amorph., ${\left[\upalpha \right]}_{\mathrm{D}}^{20}$ –8° (c 0.65, CH₃OH). IR spectrum (ν, cm^–1): 1045, 1174, 1188, 1201, 1640, 1729, 3446. ¹H NMR spectrum (300 MHz, DMSO-d₆, δ, ppm, J/Hz): 0.79 (10H, m, H-5, s, CH₃-23, 24, 25), 0.83 (3H, s, CH₃-26), 0.94 (3H, s, CH₃-27), 1.64 (3H, s, CH₃-30), 2.12 (1H, m, H-16), 2.24 (3H, s, NCH₃), 2.63 (2H, m, NCH₂CH ₂SO₃), 2.75 (2H, m, NCH ₂CH₂SO₃), 2.90 (1H, m, H-19), 3.23 (2H, s, OC(O)CH₂), 3.58 (3H, s, C(O)OCH₃), 4.40 (1H, dd, J = 10.5, 5.3, H-3), 4.56, 4.69 (each 1H, s, H-29). Table 1 lists the ¹³C NMR spectrum. Mass spectrum (ESI), m/z 648 [M – H]^– (calcd for C₃₆H₅₉NO₇S, 649).

2-({2-[17 β -Benzyloxycarbonyllup-20(29)-en-3 β -yloxy]-2-oxoethyl}(methyl)amino)ethanesulfonic Acid (10b). Yield 0.09 g (64%), amorph., ${\left[\upalpha \right]}_{\mathrm{D}}^{20}$ +11° (c 0.38, CH₃OH). IR spectrum (ν, cm^–1): 1041, 1129, 1149, 1191, 1231, 1640, 1719, 1724, 3300. ¹H NMR spectrum (500 MHz, DMSO-d₆, δ, ppm, J/Hz): 0.69 (3H, s, CH₃-24), 0.78 (3H, s, CH₃-25), 0.79 (7H, m, H-5, s, CH₃-23, 26), 0.92 (4H, m, Ha-1, s, CH₃-27), 0.97 (1H, m, H_a-12), 1.03 (1H, m, H_a-15), 1.12 (1H, m, H_a-11), 1.22 (2H, m, H_a-7, H_b-15), 1.27 (2H, m, H_a-6, H-9), 1.32 (3H, m, H_b-7, 11, H_a-21), 1.42 (2H, m, H_b-6, H_a-16), 1.47 (1H, m, H_a-22), 1.55 (1H, m, H_a-2), 1.57 (2H, m, H_b-2, H-18), 1.60 (2H, m, H_b-1, 12), 1.64 (3H, s, CH₃-30), 1.75 (1H, m, H_b-21), 1.80 (1H, m, H_b-22), 2.11 (1H, td, J = 11.0, 4.0, H-13), 2.17 (1H, d, J = 13.0, H_b-16), 2.26 (3H, s, NCH₃), 2.62 (2H, m, NCH₂CH ₂SO₃), 2.78 (2H, m, NCH ₂CH₂SO₃), 2.94 (1H, td, J = 11.0, 5.5, H-19), 3.22, 3.26 (each 1H, d, J = 17.3, OC(O)CH₂), 4.41 (1H, dd, J = 10.5, 5.3, H-3), 4.56, 4.68 (each 1H, s, H-29), 5.08, 5.12 (each 1H, d, J = 12.3, CH ₂Ph), 7.35 (5H, s, Ph). Table 1 lists the ¹³C NMR spectrum. Mass spectrum (ESI), m/z 724 [M – H]^– (calcd for C₄₂H₆₃NO₇S 725).

Preparation of 11a and 11b. A suspension of the Na-salt of N-methyltaurine (0.14 g, 0.89 mmol) and K₂CO₃ (0.12 g, 0.89 mmol) in DMF (8 mL) was stirred under Ar for 15 min, treated dropwise with the appropriate bromoalkyl ester 6a or 6b (0.50 g, 0.89 mmol) in DMF (12 mL), and stirred for 50 h at 95°C. The K₂CO₃ was filtered off. The filtrate was evaporated. The oil residue was triturated with hexane. The solution was decanted. The solid was rinsed with MTBE (TLC monitoring) and dried (KOH, 80°C).

Sodium 2-({2-[3-Oxolup-20(29)-en-17 β -carbonyloxy]ethyl}(methyl)amino)ethanesulfonate (11a). Yield 0.42 g (74%), mp 170–172°C, ${\left[\upalpha \right]}_{\mathrm{D}}^{20}$ +15.3° (c 0.45, CH₃OH). IR spectrum (ν, cm^–1): 1200, 1720, 3420. ¹H NMR spectrum (500 MHz, CD₃OD, δ, ppm, J/Hz): 0.93 (3H, s, CH₃-25), 0.98 (3H, s, CH₃-26), 1.01 (6H, s, CH₃-24, 27), 1.05 (3H, s, CH₃-23), 1.10 (1H, qd, J = 12.5, 5.0, H-12), 1.20 (1H, m, H_a-15), 1.30 (1H, dt, J = 12.5, 5.0, H_a-11), 1.40 (1H, m, H-5), 1.46 (10H, m, 1H-1, 6, 16, 21, 22, 2H-7, H-9, 1H-11, 15), 1.50 (1H, m, H-6), 1.66 (1H, t, J = 12.5, H-18), 1.68 (3H, s, CH₃-30), 1.73 (1H, m, H-12), 1.91 (2H, m, H-1, 22), 2.28 (2H, m, H-16, 21), 2.30 (1H, m, H-13), 2.33 (3H, s, NCH₃), 2.48 (2H, m, H-2), 2.70 (2H, m, C(O)OCH₂CH ₂)), 2.93 (2H, m, NCH ₂CH₂SO3), 3.00 (1H, m, H-19), 3.03 (2H, m, NCH₂CH ₂SO₃), 4.18, 4.24 (each 1H, dt, J = 11.5, 5.3, C(O)OCH ₂CH₂), 4.59, 4.71 (each 1H, s, H-29). Table 1 lists the ¹³C NMR spectrum. Mass spectrum (ESI), m/z 618 [M – Na]^– (calcd for C₃₅H₅₆NNaO₆S, 641).

Sodium 2-({2-[3-Oxolup-20(29)-en-17 β -carbonyloxy]propyl}(methyl)amino)ethanesulfonate (11b). Yield 0.40 g (69%), mp 162–164°C, ${\left[\upalpha \right]}_{\mathrm{D}}^{20}$ +15.5° (c 0.54, CH₃OH). ¹H NMR spectrum (300 MHz, CD₃OD, δ, ppm, J/Hz): 0.95 (3H, s, CH₃-25), 0.99 (3H, s, CH₃-26), 1.02 (3H, s, CH₃-24), 1.03 (3H, s, CH₃-27), 1.06 (3H, s, CH₃-23), 1.70 (5H, s, CH₃-30, m, 1H-12, H-18), 1.88 (4H, m, 1H-1, 22, C(O)OCH₂CH ₂CH₂), 2.29 (3H, m, H-13, 1H-16, 21), 2.38 (3H, s, NCH₃), 2.51 (4H, m, 2H-2, C(O)OCH₂CH₂CH ₂), 2.92 (2H, m, NCH ₂CH₂SO₃), 3.00 (3H, m, NCH₂CH ₂SO₃, H-19), 4.12 (2H, m, C(O)OCH ₂CH₂CH₂), 4.61, 4.73 (each 1H, s, H-29) (only characteristic resonances are given). Table 1 lists the ¹³C NMR spectrum. Mass spectrum (ESI), m/z 632 [M – Na]^– (calcd for C₃₆H₅₈NNaO₆S, 655).

Screening for Toxicity in vitro . The toxicity of the compounds was studied using a continuous culture of Madin-Darby canine kidney cells (MDCK) that was obtained initially from the Centers for Disease Control and Prevention (CDC, Atlanta, USA). Toxicity in vitro was assessed using the MTT assay, i.e., reduction of the dye thiazolyl blue tetrazolium bromide (MTT) by cells in culture. The experiments with the cell culture were conducted as follows. A weighed portion (5 mg) of compound in a sterile tube was diluted with DMSO to a concentration of 1 mg/mL (stock solution). Then, six sequential two-fold dilutions (100, 50, 25, 12.5, 6, and 3 μg/mL) with MDCK growth medium (Biolot, St. Petersburg) produced the solutions used to determine the toxicity. The test was performed in triplicate for each concentration. A 1-d culture of MDCK cells grown in 96-well plates was inspected visually using an inverted microscope for the integrity of the monolayer. The plates were rinsed twice with medium without serum. The test compound (100 μL per well) of the appropriate concentration was placed into each well. The plates were incubated for 72 h at 37°C with 5% CO₂. Test results were recorded visually by evaluating the integrity of the cell monolayer as compared with a control.

The MTT assay used 96-well plates and the standard protocol [25]. Optical density was recorded at 550 nm on a Varioscan microplate reader (Thermo Fisher). The median cytotoxic dose (CTD₅₀) was determined by linear regression of the photometric data using the Excel 2010 program. The regression equations were valid because R² > 0.9.