Synthesis of Sulfobetaines Based on Betulinic Acid and its Esters

Methods for preparing sulfobetaines of betulinic acid that contained N,N-(dimethylammonium)butane-1-sulfonate, a new type of Zwitter-ion in a series of lupane-type pentacyclic triterpenoids, were developed in order to prepare new betulinic acid derivatives and to study the structure–biological-activity relationship.

Betulinic acid is the lead compound among lupane-type pentacyclic triterpenoids and is interesting as a platform for developing drugs with antitumor, antiviral, antimicrobial, and other types of pharmacological activity [1–5]. Recently, a series of ionic betulinic-acid derivatives such as ammonium and triphenylphosphonium salts were synthesized. They exhibited significantly higher antitumor and antimicrobial activity than the acid itself [6–13]. Sulfobetaine derivatives of betulinic acid and other lupane-type pentacyclic triterpenoids have not been reported despite the frequent use of the biomimetic sulfobetaine group to design biologically active compounds with antibacterial, antiviral, and antiproliferative properties; to increase the solubility of hydrophobic compounds; and also to construct nanocarriers for targeted drug delivery [14–21].

New derivatives of betulinic acid (1) were prepared and their structure–biological-activity relationship was studied using the synthetic method developed by us for betulinic-acid sulfobetaines 2–4, a new class of lupane-type pentacyclic triterpenoids containing N,N-(dimethylammonium)butane-1-sulfonate bonded to the C-3 or C-28 position of the triterpene backbone.

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Sulfobetaines 2–4 were prepared by treating the corresponding triterpene C-3 or C-28 tertiary amine of 5–7 with commercially available 1,4-butane sultone [22, 23]. Betulinic acid C-3 tertiary amine 5 was synthesized by acylating the C-3-OH of betulin 28-O-vinyl ether with chloroacetic acid (DCC, DMAP) and oxidizing in two steps the resulting betulin 3β-chloroacetate (9) with pyridinium chlorochromate (PCC) to give the corresponding aldehyde, which was reacted immediately after flash chromatography with NaClO₂ to obtain the 3β-chloroacetate of acid 10. Amination of 10 with an excess of N,N-dimethylamine (40% aqueous) gave tertiary amine 5 in 93% yield. C-28-O-Vinyl ether 8, which was prepared by us earlier via vinylation of betulin with acetylene in KOH–DMSO superbase [24], turned out to be a convenient intermediate for synthesizing betulinic acid 3β-chloroacetate 10.

Protection of the primary hydroxyl in betulin by a vinyl group, its removal by treating the reaction mixture with HCl (10%), and subsequent purification of the product by flash chromatography gave the target compound in a satisfactory yield of 65% and was a good alternative to tetrahydropyranyl or dimethyl-t-butylsilyl protecting groups that are usually used for this [25–29].

3β-(2-Dimethylamino)acetate 6 was prepared via O-chloroacylation of betulinic acid methyl ester 12 with chloroacetic acid (DCC, DMAP) followed by amination of the resulting chloroacetate 11 with N,N-dimethylamine. C-28-Tertiary amine 7 was synthesized via amination of bromomethylate 13 [8] analogously to that for amines 5 and 6.

Sulfobetaines 2–4 were obtained after refluxing the corresponding tertiary amines 5–7 with an excess of 1,4-butane sultone under Ar in anhydrous MeCN. The precipitate of sulfobetaine that formed during the reaction was filtered off, rinsed several times with MeCN, and dried in vacuo. The structures of 2–4 were confirmed by mass, PMR, and ¹³C NMR spectral data (Table 1).

Table 1 13C NMR Spectra of 2–4, 5–7, 9–11, and 13 (δ, ppm)*

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Experimental

IR spectra were recorded in mineral oil on a Prestige-21 IR spectrophotometer (Shimadzu). PMR and ¹³C NMR spectra were recorded on an Avance III pulsed spectrometer (Bruker) at operating frequency 500.13 MHz for ¹H and 125.47 MHz for ¹³C using a Z-gradient PABBO probe at constant sample temperature 298 K or on an AM-300 spectrometer (Bruker) at operating frequency 300.13 and 75.47 MHz, respectively. Chemical shifts δ in PMR and ¹³C NMR spectra were measured in ppm vs. TMS internal standard. Spin–spin coupling constants were measured in Hz. PMR and ¹³C NMR spectra of 2–4 were assigned using standard 1D and 2D programs embedded in the Avance III spectrometer. Positive- and negative-ion mass spectra were recorded using electrospray ionization (ESI) on an LCMS-2010EV GC-MS (Shimadzu) (heater temperature 200°C, vaporizer temperature 230°C, sprayer flow rate 1.5 L/min, ion-source potential 3.5 kV, injected sample volume 2.5 μL, MeCN–H₂O eluent). Rotation angles were measured on a PerkinElmer 341C instrument. Column chromatography used SiO₂ (L grade, 40/60 μm, Russia). TLC was performed on Sorbfil plates (PTSKh-AF-A, ZAO Sorbpolimer, Krasnodar, Russia). Melting points were determined on a Kofler apparatus. Betulin was isolated from Betula pendula bark by the literature method [30]. Betulin O-vinyl ether 8 was prepared as before [24]; bromomethyl ester 13, by the literature method [8]; betulinic acid methyl ester, by the literature method [31]. Anhydrous MeCN was prepared by distillation over P₂O₅ and then over CaH₂.

3 β -(2-Chloroacetoxy)-28-hydroxylup-20(29)-ene (9). A solution of vinyl ether 8 (0.78 g, 1.667 mmol) and chloroacetic acid (0.32 g, 3.333 mmol) in anhydrous CH₂Cl₂ (30 mL) was treated under Ar with DMAP (0.41 g, 3.333 mmol), stirred for 5 min, treated in one portion with DCC (0.69 g, 3.333 mmol), and stirred for 30 min. The precipitate of N,N-dicyclohexylurea was filtered off. The filtrate was washed with HCl (10%, 10 mL) and water and dried over Na₂SO₄.

The solvent was evaporated. The residue was purified by flash chromatography over SiO₂ (C₆H₆–MTBE, 4:1) to afford 9 (0.56 h, 65%) [32]. Amorphous, [α] ²⁰_D +4.8° (c 0.37, CHCl₃). IR spectrum (ν, cm^–1): 3400, 1758, 1645. ¹H NMR spectrum (300 MHz, CDCl₃, δ, ppm, J/Hzö): 0.79 (1H, d, J = 9.4, H-5), 0.84 (6H, s, CH₃-24, 25), 0.86 (3H, s, CH₃-23), 0.96 (3H, s, CH₃-26), 1.01 (3H, s, CH₃-27), 1.67 (3H, s, CH₃-30), 1.80–2.00 (3H, m, H_b-16, 21, 22), 2.37 (1H, dt, J = 5.7, 11.0, H-19), 3.30 (1H, d, J = 10.8, H_a-28), 3.79 (1H, d, J = 10.8, H_b-28), 4.04 (2H, s, CH₂Cl), 4.55 (1H, m, H-3), 4.57 (1H, s, H_a-29), 4.67 (1H, s, H_b-29). ESI-MS, m/z 425 [M – ClCH₂CO₂]⁺ (calcd 518 for C₃₂H₅₁ClO₃).

3 β -(2-Chloroacetoxy)lup-20(29)-en-28-oic Acid (10). A solution of 9 (0.56 g, 1.088 mmol) in anhydrous CH₂Cl₂ (30 mL) was stirred under Ar, treated in one portion with PCC (0.70 g, 3.263 mmol), stirred for 1 h, diluted with MTBE (30 mL), stirred for 15 min, and filtered through a layer of Al₂O₃. The solvent was evaporated. The residue was purified by flash chromatography over SiO₂ (C₆H₆–MTBE, 4:1) to afford the aldehyde (0.56 g, 98%), which was dissolved in t-BuOH (33 mL), treated with 2-methyl-2-butene (0.5 mL) and then simultaneously dropwise with solutions of NaClO₂ (0.74 g, 6.505 mmol) in H2O (5 mL) and NaH₂PO₄ (0.78 g, 6.505 mmol) in H₂O (3 mL), stirred for 40 min, diluted with H₂O (100 mL), and extracted with CHCl₃ (5 × 30 mL). The organic layer was dried over Na₂SO₄. The solvent was evaporated. The residue was chromatographed over SiO₂ (C₆H₆, C₆H₆–MTBE, 4:1) to afford 10 (0.40 g, 70%) [32]. Amorphous, [α] ²⁰_D +2.5° (c 0.6, CHCl₃). IR spectrum (ν, cm^–1): 1728 (C=C, C=O), 1693, 1638. ¹H NMR spectrum (300 MHz, CDCl₃, δ, ppm, J/Hz): 0.79 (1H, d, J = 9.4, H-5), 0.85 (6H, s, CH₃-24, 25), 0.86 (3H, s, H-23), 0.92 (3H, s, CH₃-27), 0.96 (3H, s, CH₃-26), 1.68 (3H, s, CH₃-30), 1.96 (2H, m, H_b-21, 22), 2.17 (1H, t, J = 10.0, H-13), 2.27 (1H, d, J = 12.0, H_b-16), 3.02 (1H, dt, J = 5.5, 10.5, H-19), 4.05 (2H, s, CH₂Cl), 4.54 (1H, dd, J = 6.4, 10.0, H-3), 4.61 (1H, s, H_a-29), 4.74 (1H, s, H_b-29). ESI-MS, m/z 531 [M – H]–(calcd 532 for C₃₂H₄₉ClO₄).

Methyl 3 β -(2-Chloroacetoxy)lup-20(29)-en-28-oate (11). A solution of methyl ester 12 (0.50 g, 1.064 mmol) in anhydrous CH₂Cl₂ (60 mL) was treated with chloroacetic acid (0.20 g, 2.128 mmol) and DMAP (0.26 g, 2.128 mmol), stirred for 5 min, treated with DCC (0.44 g, 2.128 mmol), and stirred under Ar for 20 min. The precipitate of N,N-dicyclohexylurea was filtered off. The organic layer was washed with HCl (10%, 20 mL) and water and dried over Na₂SO₄. The solvent was evaporated. The residue was purified by flash chromatography over SiO₂ (C₆H₆–MTBE, 4:1) to afford 11 (0.55 g, 94%). Amorphous, [α] ²⁰_D 4° (c 0.15, CHCl₃). IR spectrum (ν, cm^–1): 1734, 1650. 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_b-21, 22), 2.19 (1H, m, H-13), 2.22 (1H, m, H_b-16), 3.00 (1H, dt, J = 4.5, 11.0, H-19), 3.66 (3H, s, COOCH₃), 4.05 (2H, s, CH₂Cl), 4.56 (1H, dd, J = 6.0, 10.0, H-3), 4.60 (1H, s, H_a-29), 4.73 (1H, s, H_b-29). ESI-MS, m/z 453 [M – ClCH₂CO₂]⁺ (calcd 546 for C₃₃H₅₁ClO₄).

3 β -[2-(Dimethylamino)acetoxy]lup-20(29)-en-28-oic Acid (5). A suspension of 10 (0.30 g, 0.498 mmol) in EtOH (15 mL) was treated with dimethylamine (0.6 mL, 4.975 mmol, 40% aq.) and refluxed for 4 h. The solvent was evaporated. The residue was purified by flash chromatography over SiO₂ (CHCl₃–MeOH, 10:1) to afford 5 (0.25 g, 93%). Amorphous, [α] ²⁰_D +8.6° (c 0.33, CHCl3). IR spectrum (ν, cm^–1): 3389, 1734, 1707, 1641. 1H NMR spectrum (300 MHz, CDCl₃, δ, ppm, J/Hz): 0.80 (1H, m, H-5), 0.82 (3H, s, CH₃-24), 0.83 (3H, s, CH₃-23), 0.85 (3H, s, CH₃-25), 0.92 (3H, s, CH₃-27), 0.97 (3H, s, CH₃-26), 1.68 (3H, s, CH₃-30), 1.90 (2H, m, H_b-21, 22), 2.21 (2H, m, H-13, H_b-16), 2.38 (6H, s, N(CH₃)₂), 2.98 (1H, dt, J = 5.6, 11.0, H-19), 3.21 (2H, s, OC(O)CH₂N), 4.56 (1H, dd, J = 5.3, 11.0, H-3), 4.58 (1H, s, H_a-29), 4.71 (1H, s, H_b-29). ESI-MS, m/z 542 [M + H]⁺ (calcd 541 for C₃₄H₅₅NO₄).

Methyl 3 β -[2-(Dimethylamino)acetoxy]lup-20(29)-en-28-oate (6). A solution of 11 (0.20 g, 0.338 mmol) in EtOH (10 mL) was treated with dimethylamine (1.0 mL, 3.384 mmol, 40% aq.) and stirred for 1 h at 20°C. The solvent was evaporated. The residue was purified by flash chromatography over SiO₂ (C₆H₆–MTBE, 4:1) to afford 6 (0.11 g, 60%) amine 6. Mp 147–148°C, [α] ²⁰_D + 4° (c 0.39, CHCl₃). IR spectrum (ν, cm^–1): 1732, 1708, 1641. ¹H NMR spectrum (300 MHz, CDCl₃, δ, ppm, J/Hz): 0.83 (10H, s, H-5, CH₃-23, 24, 25), 0.91 (3H, s, CH₃-27), 0.95 (3H, s, CH₃-26), 1.68 (3H, s, CH₃-30), 1.88 (2H, m, H_b-21, 22), 2.22 (2H, m, H-13, H_b-16), 2.36 (6H, s, N(CH₃)₂), 2.97 (1H, dt, J = 5.3, 11.0, H-19), 3.16 (2H, s, OC(O)CH₂N), 3.66 (3H, s, COOCH₃), 4.57 (1H, dd, J = 5.3, 10.5, H-3), 4.59 (1H, s, H_a-29), 4.73 (1H, s, H_b-29). ESI-MS, m/z 556 [M + H]⁺ (calcd 555 for C₃₅H₅₇NO₄).

2-Bromoethyl 3_-Hydroxylup-20(29)-en-28-oate (13). A suspension of 1 (1.0 g, 2.193 mmol) in anhydrous DMF (24 mL) under Ar was treated with calcined K₂CO₃ (0.30 g, 2.13 mmol), stirred for 20 min, treated dropwise with dibromoethane (0.2 mL, 2.193 mmol) in DMF (1 mL), and stirred at 20°C for 5 h. The K₂CO₃ was filtered off. The filtrate was evaporated. The residue was chromatographed over SiO₂ (C₆H₆, C₆H₆–MTBE, 16:1) to afford 13 (0.57 g, 50%). Mp 182–183°C (lit. [8] 185°C (MeOH)). ¹H NMR spectrum (300 MHz, CDCl₃, δ, ppm, J/Hz): 0.68 (1H, d, J = 8.0, H-5), 0.75 (3H, s, CH₃-24), 0.81 (3H, s, CH₃-25), 0.91 (3H, s, CH₃-26), 0.95 (6H, s, CH₃-23, 27), 1.68 (3H, s, CH₃-30), 1.92 (2H, m, H_b-21, 22), 2.17 (1H, dt, J = 5.5, 11.0, H-13), 2.29 (1H, dd, J = 8.8, 1.8, H_b-6), 3.01 (1H, dt, J = 5.5, 10.5, H_b-19), 3.18 (1H, dd, J = 5.2, 11.0, H-3), 3.53 (2H, t, J = 6.0, CH₂Br), 4.39 (1H, dt, J = 15.0, 6.0, COOCH₂: H_a), 4.41 (1H, dt, J = 15.0, 6.0, COOCH₂: H_b), 4.60 (1H, s, H_a-29), 4.73 (1H, s, H_b-29).

2-(Dimethylamino)ethyl 3 β -Hydroxylup-20(29)-en-28-oate (7). A solution of 13 (0.26 g, 0.443 mmol) in EtOH (15 mL) was treated with dimethylamine (0.2 mL, 4.434 mmol, 40% aq.) and stirred for 1 h at 20°C. The solvent was evaporated. The residue was purified by flash chromatography over SiO₂ (C₆H₆–MTBE, 4:1) to afford 7 (0.24 g, 98%). Mp 181–182°C, [α] ²⁰_D –2.4° (c 0.3, CHCl₃). IR spectrum (ν, cm^–1): 3440, 1726, 1650. ¹H NMR spectrum (300 MHz, CDCl₃, δ, ppm, J/Hz): 0.69 (1H, m, H-5), 0.75 (3H, s, CH₃-24), 0.81 (3H, s, CH₃-25), 0.91 (3H, s, CH₃-26), 0.95 (6H, s, CH₃-23, 27), 1.67 (3H, s, CH₃-30), 1.91 (2H, m, H_b-21, 22), 2.23 (1H, m, H_b-6), 2.28 (7H, s, H-13, N(CH₃)₂), 2.56 (2H, t, J = 6, CH₂N), 3.01 (1H, m, H-19), 3.17 (1H, dd, J = 5.2, 10.5, H-3), 4.20 (2H, m, COOCH₂), 4.59 (1H, s, H_a-29), 4.72 (1H, s, H_b-29). ESI-MS, m/z 529 [M + H]⁺ (calcd 528 for C₃₄H₅₇NO₃).

4-({2-[17 β -Carboxylup-20(29)-en-3 β -yloxy]-2-oxoethyl}dimethylammonium)butane-1-sulfonate (2). A suspension of 5 (0.10 g, 0.185 mmol) in anhydrous MeCN (3 mL) under Ar was treated dropwise with a solution of 1,4-butane sultone (0.17 mL, 1.661 mmol) in anhydrous MeCN (1.0 mL) (three equal portions every hour), and refluxed for 30 h. The precipitate of 5 was filtered off, rinsed with MeCN (5 × 5 mL), and dried in vacuo (KOH, 80°C) to afford 2 (0.06 g, 48%, white powder). Mp 288–290°C, [α] ²⁰_D +11° (c 0.31, HCOOH). IR spectrum (ν, cm^–1): 3440, 1743, 1725, 1640, 1204, 1193, 1151, 1307, 794. ¹H NMR spectrum (500 MHz, CD₃COOD, δ, ppm, J/Hz): 0.86 (1H, m, H-5), 0.89 (3H, s, CH₃-24), 0.91 (6H, s, CH₃-23, 25), 0.99 (3H, s, CH₃-26), 1.04 (3CH, s, CH₃-27), 1.06 (1H, m, H_a-1), 1.10 (1H, m, H_a-12), 1.20 (1H, d, J = 12.0, H_a-15), 1.31 (1H, m, H_a-11), 1.40 (1H, m, H-9), 1.42 (1H, m, H_a-21), 1.45 (2H, m, 2H-7), 1.48 (3H, m, H_a-6, Hb-11, H_a-16), 1.51 (1H, m, H_a-22), 1.56 (2H, m, H_b-6, 15), 1.65 (1H, m, H-18), 1.72 (5H, s, 2H-2, CH₃-30), 1.75 (2H, m, H_b-1, 12), 1.90 (2H, m, NCH₂CH₂CH ₂CH₂SO₃), 2.00 (2H, m, H_b-21, 22), 2.07 (2H, m, NCH₂CH ₂CH ₂ CH₂SO₃), 2.30 (2H, m, H-13, H_b-16), 3.06 (3H, m, H-19, NCH₂CH₂CH₂CH ₂SO₃), 3.37 (6H, s, N(CH₃)₂), 3.65 (2H, m, NCH ₂CH₂CH₂CH₂SO₃), 4.36 (1H, d, J = 17.0, OC(O)CH₂N: H_a), 4.42 (1H, d, J = 17.0, OC(O)CH₂N: H_b), 4.63 (1H, s, H_a-29), 4.68 (1H, dd, J = 5.3, 10.5, H-3), 4.77 (1H, s, H_b-29). ESI-MS, m/z 678 [M + H]⁺ (calcd 677 for C₃₈H₆₃NO₇S).

4-({2-[17 β -Methoxycarbonyllup-20(29)-en-3 β -yloxy]-2-oxoethyl}dimethylammonium)butane-1-sulfonate (3) was prepared analogously to 2 from 6 (0.11 g, 0.202 mmol) and 1,4-butane sultone (0.20 mL, 2.02 mmol). Yield 0.054 g (39%) (white powder). Mp 283–286°C, [α] ²⁰_D +13° (c 0.28, HCOOH). IR spectrum (ν, cm^–1): 1729, 1650, 1203, 1166, 1136, 1040. ¹H NMR spectrum (500 MHz, CD₃CO₂D, δ, ppm, J/Hz): 0.86 (1H, d, J = 10, H-5), 0.88 (3H, s, CH₃-24), 0.90 (6H, s, CH₃-23, 25), 0.95 (3H, s, CH₃-26), 1.02 (3H, s, CH₃-27), 1.05 (1H, m, H_a-1), 1.10 (1H, m, H_a-12), 1.20 (1H, m, H_a-15), 1.30 (1H, m, H_a-11), 1.39 (2H, m, H-9, H_a-21), 1.45 (7H, m, H_a-6, 2H-7, H_b-11, H_b-15, H_a-16, H_a-22), 1.52 (1H, m, H_b-6), 1.65 (1H, m, H-18), 1.71 (3H, s, CH₃-30), 1.73 (4H, m, H_b-1, 2H-2, H_b-12), 1.90 (4H, m, NCH₂CH₂CH ₂CH₂SO₃, H_b-21, 22), 2.06 (2H, m, NCH₂CH ₂CH₂CH₂SO₃), 2.24 (2H, m, H-13, H_b-16), 3.03 (3H, m, H-19, NCH₂CH₂CH₂CH ₂SO₃), 3.34 (6H, s, N(CH₃)₂), 3.64 (2H, m, NCH ₂CH₂CH₂CH₂SO₃), 3.68 (3H, s, COOCH₃), 4.36 (1H, d, J = 17.0, OC(O)CH₂N: H_a), 4.39 (1H, d, J = 17.0, OC(O)CH₂N: H_b), 4.61 (1H, s, H_a-29), 4.66 (1H, dd, J = 5.3, 10.5, H-3), 4.73 (1H, s, H_b-29). ESI-MS, m/z 714 [M + Na]⁺ (calcd 691 for C₃₉H₆₅NO₇S).

4-({2-[3 β -Hydroxylup-20(29)-en-17 β -carbonyloxy]ethyl}dimethylammonium)butane-1-sulfonate (4) was prepared analogously to 2 from 7 (0.12 g, 0.224 mmol) and 1,4-butane sultone (0.20 mL, 2.02 mmol). Yield 0.04 g (27%) (white powder). Mp 265–267°C, [α] ²⁰_D +9.5° (c 0.4, HCOOH). IR spectrum (ν, cm^–1): 3414, 1726, 1650, 1212, 1166, 1037, 1036. ¹H NMR spectrum (500 MHz, CD₃CO₂D, δ, ppm, J/Hz): 0.68 (1H, d, J = 10.0, H-5), 0.78 (3H, s, CH₃-24), 0.86 (3H, s, CH₃-25), 0.95 (4H, s, CH₃-26, m, H_a-1), 0.96 (3H, s, CH₃-23), 1.02 (3H, s, CH₃-27), 1.08 (1H, m, H_a-12), 1.25 (2H, m, H_a-11, 15), 1.40 (1H, m, H-9), 1.43 (6H, m, H_a-6, 2H-7, H_b-15, H_b-11, H_a-21), 1.54 (3H, m, H_b-6, H_a-16, H_a-22), 1.65 (1H, m, H-18), 1.68 (2H, m, H-2), 1.70 (4H, m, H_b-1, s, CH₃-30), 1.74 (1H, m, H_b-12), 1.92 (4H, m, H_b-21, 22, NCH₂CH ₂CH₂CH₂SO₃), 2.05 (2H, m, NCH₂CH₂CH ₂CH₂SO₃), 2.23 (1H, d, J = 13.7, H_b-16), 2.28 (1H, dt, J = 3.5, 12.5, H-13), 3.02 (1H, dt, J = 5.5, 11.5, H-19), 3.07 (2H, t, J = 7.5, NCH₂CH₂CH₂CH ₂SO3), 3.23 (6H, s, N(CH₃)₂), 3.26 (1H, dd, J = 5.5, 10.5, H-3), 3.54 (2H, m, NCH ₂CH₂CH₂CH₂SO), 3.78 (2H, br.s, C(O)OCH₂CH₂N), 4.59 (1H, d, J = 14.0, C(O)OCH₂CH₂N: H_a), 4.63 (1H, s, H_a-29), 4.65 (1H, d, J = 14.0, C(O)OCH₂CH₂N: H_b), 4.76 (1H, s, H_b-29). ESI-MS, m/z 664 [M + H]⁺ (calcd 663 for C₃₈H₆₅NO₆S).