Table 9. HPLC gradient profile to monitor deacylation of QA saponin.
Time (min) % H2O %ACN Flow (mL/min)
0 90.0 10.0 1.0
30.0 60.0 40.0 1.0
40.0 30.0 70.0 1.0
42.0 90.0 10.0 1.0
50.0 90.0 10.0 1.0
Column: Vydac 214TP C4 5μ UV Detector: Wavelength = 210 nm
Figure 11. Change of %concentration of saponin starting material and product.
3.7． Global deprotection and amide formation of hexasaccharide saponins
With compound 46 in hands, our initial strategy to finish saponin target B was to conjugate with amines at final stage before removing all the protections (Table 10). The fully protected 46 was proceeded under hydrogenolysis of benzyl group, then followed by amide coupling. However, the resulting residue was a mixture of saponins with different numbers of deprotection of TES groups. Furthermore, the mixture became even more messy after conjugating with amine on TLC analysis (Table 10, Entry 2).
-20
Table 10. Global deprotection and amide formation steps.
With the experience of entry 2, the deprotection step was arranged prior to the amide
bond formation step (Table 10, Entry 3). After the debenzylation, the resulting residue was proceeded under acidic hydrolysis the isopropylidene, triethylsilyl groups, and then methanolysis the remaining acetyl groups. Because C-28 ester bond was easily to be hydrolyzed, the concentration of TFA was adjusted from 80% to 75%, and the yield was improved from 13% to 28% for three steps. Followed by HPLC purification and concentration, compound 50 was obtained in 28% ready for late stage amine conjugation with 4-phenoxyphenyl-octanoic acid and 4-methoxyphenyl-octanoic acid by using HBTU/DIPEA in DMA to give final compound 51a (80%) and 51b (30%), respectively (Scheme 11).
Scheme 11. Global deprotection then amide formation.
4. Conclusion
In order to get various GPI-0100 analogues for SAR study, as well as development of efficient strategy to get these compounds, two series of GPI-0100 analogues which contained the same trisaccharide moieties at C-28 of quillaic acid, but different modifications on 3-O-linkage were synthesized.
Target A was designed to replace 3-glucuronic acid of GPI-0100 analogues by 6-N-glucosyl moiety. Synthesizing target A was concise and efficient, two glycosylation steps were catalyzed by different promotors, TBDMSOTf was applied to build the linkage between 6-N3-glycoside 20 and quillaic ester 3 which resulted in β-selective compound 36 in 71% yield. BF3•OEt was employed to promote glycosylation of 39 with trisaccharide 21 to obtain tetrasaccharide sapnin 40 in 88% yield (α/β = 1/10). Amide bond formation and deprotection steps to give final A was tedious, first removal of benzyl of 40 was achieved under 55 psi H2 and 10% Pd(OH)2/C. Amide bond formation of 41 with arylalkyl acids by using HBTU/DIPEA as coupling reagents, and then global deprotection of the remaining protecting groups, the final 42a and 42b were obtained by purification of using HPLC.
Target B was designed to take advantage of Q. Saponaria extract to give 3-O-trisaccharirde containing quillaic acid which avoided notorious glucuronidation step. To
our grateful, conditions of isolation of the desired monoacid prosapogenin from ultra Q-100 was fine-tuned to have 38% yield. Again, glycosylation of monoacid 45 with
trisaccharide 21 was achieved by using BF3•OEt2 as a promotor to give hexasaccharide saponin 46 in 87% yield. The hexasaccharide saponin finals 51a and 51b were carried out by global deprotection prior to amide formation with arylalkyl amines by HBTU/DMA system. This procedure allowed us to achieve late stage modification in synthesizing derivatives. However, the global deprotection steps could be further improved by adjusting the concentration of acid or the reaction temperature.
The adjuvant capability of these compounds will be evaluated afterward.
5. Experimental section
5.1. General Procedures
All reagents and solvents were reagents grade and used without further purification.
For those reagents which were stored in fridge, were opened and used after materials were recovered to rt. Molecular sieves were activated at 200 °C and cooled down to rt prior to use. Reaction progress was monitored by RP-HPLC or analytical TLC on 0.25 mm Merck Milipore silica gel 60 F254 using p-anisaldehyde and ceriumammonium molybdate as staining agents. Flash column chromatography was performed using 230-400 mesh silica gel.
5.2. Chemical reagents
Acros Organics:
Acetic acid, acetonitrile (ACN), acetone, amberlite IR-120 resin, benzyl chloroformate (CbzCl), boron trifluoride diethyl etherate (BF3·OEt2), N-bromosuccinimide (NBS), 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), dichloromethane (CH2Cl2), 4-dimethylamino pyridine (DMAP), dimethylforamide (DMF), DIPEA, N-iodosuccinimide (NIS), Na2S2O3, NH4Cl, pyridine, Pd(OH)2/C, t-Butyldimethylsilyl trifluoromethane (TBDMSOTf), tetrahydrofuran (THF), TFA, p-thiocresol, Triethylsilyl
trifluoroethanesulfonate (TESOTf), Trimethylsilyl trifluoromethanesulfonate (TMSOTf), triethylamine, L-arabinose, D-fucose, D-xylose, L-rhamnose
Alfa Aesar:
CDCl3, Kieselgrl 60 silica gel 40-63 μm (230-400 mesh)
Nanjing Spring & Autumn Biological Engineering Co., Ltd. (南京春秋生物工程有限 公司):
Quillaic acid
Sigma-Aldrich:
Ac2O, CD3OD, 2,4,6-tri-tert-butylpyridine (TBP)
Uni-onward corp. (友和貿易股份有限公司):
ACS-CHCl3
5.3. Instruments
NMR spectra were acquired by employing AV-400 (400 MHz) and Bruker-AV-600 (600 MHz). Chemical shifts (δ) are presented in ppm relative to 1H: 7.26 ppm,
13C: 77.0 ppm for CDCl3; 1H: 3.31 ppm, 13C: 49.0 ppm for methanol-d4. Splitting patterns
are reported as s (singlet), brs (broad singlet), d (double), t (triplet), q (quartet), dd (double doublet), m (multiplet). Coupling constant (J) are given in Hertz (Hz).
Reversed phase HPLC purification and analyses were performed on HITCHI D-2000 Elite HPLC system equipped with auto-sampler L-2200, UV detector L-2420 and pump L-2130.
Exact mass measurements were carried out on VG platform electrospray ESI/MS or BioTOF II.
5.4. Synthetic Procedures
Trichloroacetimidoyl 2,3,4-tri-O-acetyl-α/β-D-xylopyranoside (25).
Imidate 25 was prepared by known procedures and confirmed by 1H NMR spectrum.78 25α:1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 6.47 (d, J = 3.4 Hz, 1H, H-1), 5.56 (t, J = 9.9 Hz, 1H, H-3), 5.15–4.97 (m, 2H, H-2, H-4), 3.98 (dd, J = 11.0, 5.9 Hz, 1H, H-5a), 3.80 (t, J = 11.0 Hz, 1H, H-5b), 2.05 (s, 6H, CH3), 2.01 (s, 3H, CH3).
Benzyl 3,4-O-isopropylidene-α/β-D-fucopyranoside (23α/β).
To a stirred suspension of D-fucose (3.0 g, 18mmol) in BnOH (35 mL) was added camphorsulfonic acid (0.42 g, 1.8 mmol) and heated to 50 °C for 3 h. After cooling down to rt, the reaction mixture was diluted with EtOAc, washed by H2O and brine, dried over MgSO4, and then concentrated under reduced pressure. The residue was filtered by flash column (silica gel, MeOH/CH2Cl2 = 1/5) to obtain α/β-benzylated mixture as a white solid. To a stirred solution of α/β-benzylated mixture in ACN was added 2,2-dimethoxypropane (2.2 mL, 17 mmol) and TsOH·H2O (0.21 g, 1.1 mmol) under N2
atmosphere at rt. The reaction mixture was stirred for 1.5 h, quenched by Et3N and then concentrated under reduced pressure. The residue was diluted with CH2Cl2, washed by saturated NaHCO3, brine, dried over MgSO4, and then concentrated under reduced pressure. The residue was purified by column (silica gel, EtOAc/hexanes = 1/3 to 1/2) to afford 23α/β (2.0 g, 58%) as yellow syrups. 23α: Rf 0.28 (EtOAc/hexanes = 1/2) ; 1H
Ph-CH2), 4.58 (d, J = 11.6 Hz, 1H, Ph-CH2), 4.23 (d, J = 8.3 Hz, 1H, H-1), 4.06–3.97 (m, activated 4 Å molecular sieve powder in anhydrous CH2Cl2 (12 mL) was added TMSOTf (0.13 mL, 0.72 mmol) at -50 °C under N2 atmosphere. Upon completion of the reaction after 2 h, the reaction was quenched by addition of Et3N, warmed up to rt, filtered and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; EtOAc//hexanes = 1/3) to give 28 (582 mg, 90%) as a yellow syrup: Rf 0.48 (EtOAc/hexanes = 1/1); 1H NMR (400 MHz, CDCl3) δ 7.34 (d, J = 7.9 Hz, 2H), 7.12 (d, J = 7.9 Hz, 2H), 5.64 (s, 1H, H-1), 5.20 (t, J = 8.5 Hz, 1H, H-3′), 5.00 (d, J
= 6.8 Hz, 1H, H-1′), 4.97–4.88 (m, 2H, H-4′, H-2′), 4.29 (d, J = 5.4 Hz, 1H, H-2), 4.17–
°C for 12 h. The residue mixture was evaporated and then azeotrope distilled with toluene
(50 mL) twice under reduced pressure. After drying by high vacuum, the crude syrup was treated with Ac2O (2.2 mL, 23 mmol), Et3N (5.2 mL, 38 mmol) and DMAP (9 mg, 0.074 mmol) in CH2Cl2 under N2 atmosphere at rt. Upon completion of the reaction after 2 h, the mixture was diluted with CH2Cl2, washed by H2O, brine, dried over MgSO4, and then
concentrated under reduced pressure, The residue was purified by column chromatography (silica gel; EtOAc/hexanes = 2/3) to give 22 (3.8 g, 76%) as a white solid:
Rf 0.19 (EtOAc/hexanes = 1/2); 1H NMR (400 MHz, CDCl3) δ 7.34 (d, J = 7.9 Hz, 2H),
Benzyl 2,3,4-tri-O-acetyl-β-D-xylopyranosyl-(1→4)-2,3-di-O-acetyl-α-L -rhamnopyranosyl-(1→2)-3,4-O-isopropylidene-α-D-fucopyranoside (29α).
To a stirred suspension of 23 (111 mg, 0.21 mmol), 22 (214 mg, 0.35 mmol), and activated 4 Å molecular sieves powder in anhydrous CH2Cl2 (1 mL) was added NIS (130 mg, 0.58 mmol) under N2 atmosphere at -50 °C. Upon completion of the reaction after 2 h, the reaction was quenched by addition of saturated NaHCO3 and 10% Na2S2O3 aqueous solution, the reaction mixture was warmed to rt, stirred for 1 h and then filtered. Filtrate was diluted with CH2Cl2, washed by 10% Na2S2O3, saturated NaHCO3, brine, dried over
H-6), 1.32 (s, 3H), 1.14 (d, J = 5.6 Hz, 3H, H-6′); BBD 13C NMR (151 MHz, CDCl3) δ atmosphere. After being stirred for 3 days, the mixture was filtered through celite and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; EtOAc/hexanes = 1/2 to 1/1) to afford hemiacetal (30 mg, 20%) as a white foam solid. To a stirred solution of hemiacetal (110 mg, 0.16 mmol) in anhydrous CH2Cl2 (1 mL) was added Cl3CCN (48 μL, 0.48 mmol), and DBU (9 μL, 0,064 mmol) at rt under N2 atmosphere. After being stirred for 2 h, the reaction was complete
as indicated by TLC analysis, the mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel; EtOAc/hexanes = 1/3 to 1/1, contained 0.1% Et3N) to give 21. 21α: Rf 0.46 (EtOAc/hexanes = 1/1); 1H NMR 169.8, 169.5, 169.3, 161.1, 109.3, 101.0 (C-1′′), 98.0 (C-1′), 95.2 (C-1), 91.0, 76.2, 75.7, 75.1, 74.9, 72.1, 71.3, 71.0, 69.9, 69.3, 67.3, 65.9, 62.5, 28.3, 26.4, 21.0, 20.9, 20.7, 20.6, 20.4, 17.6, 16.3.
28-O-Allyl-quillaic ester (3).
To a stirred suspension of quillaic acid (1.0 g, 2.1 mmol), allyl bromide (0.54 mL, 6.0 mmol), K2CO3 (852 mg, 6.0 mmol), and TBAI (7.4 mg, 0.02 mmol), in THF/H2O = 10/1 (20 mL) was heated to reflux temperature 65 °C. The reaction mixture was stirred for 6 h, and then cooled to rt. After removal of the solvent under reduced pressure, the residue was diluted with CH2Cl2, washed by H2O, brine, dried over MgSO4, and then concentrated under reduced pressure, the residue was purified by column chromatography (silica gel, EtOAc/hexanes = 1/3 to 1/2) to afford 3 (909 mg, 84%) as white foam: Rf 0.33
Trichloroacetimidoyl 2-O-Benzoyl-3,4-di-O-benzyl-6-azido-α-D-glucopyranoside (20).
To a stirred solution of 35 (1.2 g, 2.01 mmol) in acetone/H2O = 10/1 (12 mL) was added NBS (1.5 g, 8.42 mmol) at rt. After being stirred for 2 h, the mixture was quenched by addition of saturated NaHCO3 and 10% NaS2O3(aq). The resulting mixture was stirred at rt for 1 h then he solvent was removed under reduced pressure. The residue was diluted with CH2Cl2, washed by 10% NaS2O3(aq), NaHCO3 and brine, dried over MgSO4, and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, EtOAc/hexanes = 1/5) to afford hemiacetal (884 mg, 87%) as a white solid. To a stirred solution of hemiacetal (0.30 g, 0.61 mmol) in anhydrous CH2Cl2 (3 mL) was added Cl3CCN (187 μL, 1.86 mmol) and DBU (3 μL, 0.24 mmol) at rt under N2. After being stirred for 2 h, the reaction was complete as indicated by TLC analysis, the mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, EtOAc/hexanes = 1/6, contained 0.1% Et3N) to afford 20 (274 mg, 71%) as yellow syrup: Rf 0.62 (EtOAc/hexanes = 1/4); 1H NMR
(400 MHz, CDCl3) δ 8.58 (s, 1H, Bz), 8.00 (d, J = 7.6 Hz, 2H, Bz), 7.61 (t, J = 7.4 Hz, 133.5, 129.9, 129.2, 128.7, 128.5, 128.4, 128.3, 128.1, 127.9, 93.7, 91.0, 79.4, 77.6, 75.7 (Bn CH2), 75.6 (Bn CH2), 73.0, 72.6, 50.8 (C-6) ppm.
3-O-(2-O-Benzoyl-3,4-di-O-benzyl-6-azido-β-D-glucopyranosyl)-28-O-Allyl-quillaic ester (36).
To a stirred suspension of 20 (713 mg, 1.12 mmol) and 3 (495 mg, 0.94 mmol), and activated 4 Å molecular sieve powder in anhydrous CH2Cl2 (15 mL) was added TBDMSOTf (21.6 μL, 0.09 mmol) at rt under N2 atmosphere. Upon completion of the reaction after 1 h, the reaction was quenched by addition of Et3N, filtered and then
concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, EtOAc/hexanes = 1/8 to 1/4) to afford 36 (782 mg, 83%) as white solid: Rf 0.53 (EtOAc/hexanes = 1/4); 1H NMR (600 MHz, CDCl3) δ 9.17 (s, 1H, 129.8, 129.7, 128.5, 128.3, 128.2, 128.1, 128.0, 127.8, 122.6 (C-12), 118.1 (All terminal alkenyl CH2), 101.9 (C-1′), 82.5 (C-3), 78.3 (C-5′), 75.2 (Bn CH2), 75.1 (Bn CH2), 74.9 (C-16), 74.6 (C-4′), 73.7 (C-2′), 65.2, 54.5, 51.3 (C-6′), 49.6, 48.7, 46.5, 46.3, 41.4, 40.6, 39.7, 38.0, 36.0, 35.5, 35.4, 32.8, 32.2, 30.7, 30.4, 26.9, 25.0, 24.6, 23.2, 20.1, 17.0, 15.6,
10.4 ppm; HRMS+ (ESI-TOF) calcd. for C60H75N3O10Na [M+Na]+ 1020.5345, found 1020.5350.
3-O-(2-O-triethylsilyl-3,4-di-O-benzyl-6-azido-glucopyranosyl)-16-O-triethylsilyl-28-O-Allyl-quillaic ester (37).
To a stirred solution of 36 (0.78 g 78 mmol) in THF/MeOH = 4/1 (20 mL), was added Na (18 mg, 0.78 mmol). Upon string for 4 h at 40 °C water bath, the mixture was cooled down to rt then quenched by Amberlyst IR-120H+, filtered, concentrated, and then dried under reduced pressure. The residue was subsequently dissolved in anhydrous CH2Cl2 (20 mL) under N2 atmosphere, and then treated with 2,6-lutidine (0.45 mL, 390 mmol) and TESOTf (0.53 mL, 234 mmol). After being stirred for 12 h at rt, the reaction mixture was diluted with CH2Cl2, washed by NaHCO3, H2O, brine, dried over MgSO4, and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, EtOAc/hexanes = 1/20) to afford 37 (220 mg, 25%) as colorless syrup: Rf 0.83 (EtOAc/ hexanes = 1/3); 1H NMR (400 MHz, CDCl3) δ 9.45 (s, 1H, H-23), 7.61-7.56 (m, 1H, Bz), 7.48-7.44 (m, 2H, Bz), 7.36-7.26 (m, 8H, Bn),
7.18-7.12 (m, 2H, Bn), 5.92–5.80 (m, 1H, All internal alkenyl CH), 5.40-5.26 (m, 2H, H-12, (C-28), 143.4 (C-13), 138.6, 137.6, 132.2, 128.4, 128.2, 127.9, 126.8, 121.9 (C-12), 117.8 (All terminal alkenyl CH2), 101.1 (C-1′), 85.5 (C-3), 78.8 (C-5′), 75.2 (Bn CH2), 75.1 (Bn CH2), 74.9 (C-16), 74.0 (C-4′), 65.0, 54.5, 51.5 (C-6′), 48.9, 47.9, 46.6, 46.4, 41.4, 40.4, 39.6, 38.1, 35.9, 35.2, 34.5, 32.7, 32.3, 31.6, 30.5, 26.4, 24.5, 24.2, 23.2, 20.0, 16.9, 15.6, 10.1, 7.1, 7.0, 5.0, 4.9 ppm; HRMS+ (ESI-TOF) calcd. for C65H100N3O9Si2 [M+H]+ 1233.6993, found 1122.7010.
3-O-(2-O-triethylsilyl-3,4-di-O-benzyl-6-N-benzylcarbamoyl-β-D
-glucopyranosylcarbamate)-16-O-triethylsilyl-28-O-Allyl-quillaic ester (38).
To a stirred solution of 37 (160 mg, 0.14 mmol) in THF (3 mL) was added PPh3 (112 mg, 0.43 mmol). After stirring the mixture for 12 h, it was cooled down to 0 °C, and then treated with CbzCl (24 μL, 0.17 mmol). Upon the completion of the reaction after 30 minutes of stirring at ice bath, the residue was quenched by addition of saturated NaHCO3, concentrated under reduced pressure to remove THF. The residue was diluted with CH2Cl2, washed by NaHCO3, brine, dried over MgSO4, and then concentrated under pressure. The residue was purified by column chromatography (silica gel, EtOAc/CH2Cl2/hexanes = 1/1/10) to afford 38 (114 mg, 65%) as colorless syrup: Rf 0.15 (EtOAc/ CH2Cl2/hexanes = 1/1/10); 1H NMR (400 MHz, CDCl3) δ 9.41 (s, 1H, H-23), 7.61-7.56 (m, 1H, Bn), 7.40-7.28 (m, 12H), 7.24-7.21 (m, 2H), 5.93–5.82 (m, 1H, internal alkenyl CH), 5.39-5.27 (m, 2H, H-12, terminal alkenyl CHa ), 5.24-5.13 (m, 3H, Cbz NH,
0.71–0.65 (m, 14H); BBD 13C NMR (100 MHz, CDCl3) δ 207.0 (C-23), 176.4 (C-28), 156.2 (Cbz CO), 143.4 (C-13), 143.3, 138.7, 137.6, 136.4, 132.2, 128.6, 128.5, 128.4, 128.2 (2C), 128.1 (2C), 127.9, 127.2, 126.8, 121.8 (C-12), 117.8 (All terminal alkenyl CH2), 101.2 (C-1′), 85.5 (C-3), 79.2, 78.7 (C-5′), 75.1 (Bn CH2), 75.1, 75.0 (Bn CH2), 1,4-dioxane (1 mL) was added pre-mixed formic acid (44 μL, 1.2 mmol)/Et3N (154 μL, 1.1 mmol) in 1,4-dioxane (1 mL) and Pd(OAc)2 (6 mg, 0.03 mmol) in 1,4-dioxane (1 mL) at
rt. The reaction mixture was stirred for 12 h, and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, EtOAc/CH2Cl2/hexanes = 1/1/10 to 1/1/6) to afford 39 (50 mg, 76%) as white solid: Rf
3-O-(2-O-triethylsilyl-3,4-di-O-benzyl-6-N-benzylcarbamoyl-β-D
reaction was quenched by Et3N, and warmed to room temperature. The resulting mixture was diluted with CH2Cl2 and filtered through 5 μm filter paper. The resulting filtrate was
concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; EtOAc/ hexanes = 1/3 to 1/2) to give 37 (50 mg, 88%, β/
= 10:1) as white solid foams. 40: Rf 0.50 (EtOAc/hexanes = 1/1);1H NMR (600 MHz, CDCl3) δ 9.40 (s, 1H, H-23), 7.36-7.27 (m, 11H), 7.23-7.17 (m, 4H), 5.39 (d, J = 7.6 Hz,
1H, H-1′′), 5.31 (m, 1H, H-12), 5.24-5.22 (m, 1H, H-3′′′), 5.19-5.17 (m, 1H, H-4′′′), 3H), 1.49-1.46 (m, 1H), 1.45-1.41 (m, 2H), 1.34-1.31 (m, 7H), 1.28-1.22 (m, 10H), 1.19-1.16 (m, 2H), 1.13 (s, 3H), 1.12–1.09 (m, 1H), 0.97-0.94 (m, 12 H), 0.91-0.87 (m, 12H), 0.71 (s, 3H), 0.66-0.63 (m, 5H), 0.59-0.53 (m, 5H); BBD 13C NMR (150 MHz, CDCl3) δ 207.3 (C-23), 175.5 (C-28), 170.3, 170.0, 169.8, 169.4, 169.3, 182.6, 156.3 (CBz CO), 143.1 (C-13), 138.7, 137.7, 136.4, 128.5, 128.4, 128.3, 128.2 (2C), 128.1, 128.0, 127.9, 127.2, 126.9, 121.0 (C-12), 101.3 (C-1′), 101.3 (C-1′′′′), 97.0 (C-1′′′), 93.1 (C-1′′), 85.5 (C-3), 79.3 (C-2′), 78.8 (C-5′), 77.9 (C-3′′′), 76.5, 76.3 (C-2′′), 76.1 (C-4′′), 75.2 (Bn CH2), 75.2 (C-16), 75.0, (C-2′′′′), 74.9 (Bn CH2), 73.2 (C-4′), 72.4 (C-3′′′′), 71.2 (C-2′′′), 70.9 (C-4′′′), 70.1 (C-3′′′), 69.3 (C-5′′), 69.2 (C-4′′′′), 67.9 (C-3′), 66.9 (Bz CH2), 62.6 (C-5′′′′),
54.6, 48.9 (C-5), 48.5 (C-17), 46.6 (C-19), 46.5 (C-9), 41.4 (C-6′), 40.7 (C18), 39.8, 38.3, 36.0, 35.0 (C-21), 34.6 (C-15), 32.7 (C-29), 32.4 (C-6), 31.0, 30.5 (C-22), 26.4, 26.3 (isopropylidene CH3), 26.1 (isopropylidene CH3), 24.8, 24.5 (C-30), 23.3 (C-11), 21.0, 20.9, 20.7, 20.6, 20.5, 20.1 (C-7), 17.9 (C-6′′′), 17.1 (C-26), 16.2 (C-6′), 15.6 (C-25), 10.3, 7.2, 7.0, 5.1, 5.0 ppm; HRMS+ (ESI-TOF) calcd. for C100H145N1O28Si2Na [M+Na]+ 1886.9384, found 1886.9389.
3-O-(6-amino-6-deoxy-β-D-glucopyranosyl)-28-O-[β-D-xylopyranosyl-(1→4)-α-L -rhamnoyranosyl-(1→2)-β-D-fucopyranosyl] quillaic ester (41).
To a suspension of 40 (28 mg, 15 μmol) and 10% Pd(OH)2/C (5 mg, 4 μmol) in THF/MeOH = 4/1 (1.5 mL) was stirred at rt under 55 psi H2 atmosphere. The reaction mixture was stirred for 36 h. The resulting mixture was filtered through celite, concentrated under reduced pressure. To a stirred solution of crude acid intermediate in
CH2Cl2 (0.5 mL) was added pre-cooled TFA/H2O = 4/1 (0.5 mL) at 0 °C, and stirred for 30 min. The solvent was evaporated under reduced pressure (<1 torr) at 0 °C, and then dried under high vacuum at rt for 1 h. To a stirred solution of the residue in MeOH (1mL) was added K2CO3 (30 mg, 433 μmol) and stirred for 12 h. The suspension was filtered, and the filtrate was purified by HPLC to afford product 41 (2 mg) in 11% three steps yield as a white solid (HPLC column: SUPELCO Ascentis C18 25 cm × 10 mm, 5μm; mobile phase: 20% ACN/H2O (contained 0.05% TFA) gradient to 90% ACN/H2O in 20 min, and then 90% ACN/H2O isocratic for 15 min; flow rate: 2.4 mL/min): HRMS+ (ESI-TOF) calcd. for C53H86NO21 [M+H]+ 1072.5687, found 1072.5685.
3-O-(6-N-[10-(4-(4-fluorophenoxy)phenyl)decanyl]-6-deoxy-β-D
-glucopyranosylamide)-28-O-[β-D-xylopyranosyl-(1→4)-α-L
-rhamnoyranosyl-(1→2)-β-D-fucopyranosyl] quillaic ester (42a).
To a suspension of 40 (28 mg, 15 μmol) and 10% Pd(OH)2/C (5 mg, 4 μmol) in THF/MeOH = 4/1 (1.5 mL) was stirred at rt under 55 psi H2 atmosphere. The reaction mixture was stirred for 36 h. The resulting mixture was filtered through celite, concentrated under reduced pressure. To a stirred suspension of residue, 10-(4-(4-fluorophenoxy)phenyl)decanoic acid 17 (5 mg, 15 μmol), HBTU (5 mg, 15 μmol) in anhydrous DMA (0.3 mL) was added DIPEA (3 μL, 15 μmol) under N2 atmosphere. Upon the completion of the reaction after 12 h, the reaction mixture was concentrated under reduced pressure, diluted with CH2Cl2, washed by H2O for two times, brine, dried over MgSO4, and then concentrated under pressure. To a stirred solution of crude tetrasaccharide saponin in CH2Cl2 (0.5 mL) was added pre-cooled TFA/H2O = 4/1 (0.5 mL) at 0 °C, and stirred for 30 min. The solvent was evaporated under reduced pressure (<1 torr) at 0 °C, and then dried under high vacuum at rt for 1 h. To a stirred solution of the residue in MeOH (1mL) was added K2CO3 (40 mg, 300 μmol) and stirred for 12 h.
The suspension was filtered, concentrated then purified by HPLC to afford product 40a
(1 mg) in 3% as a white solid (HPLC column: SUPELCO Ascentis C18 25 cm × 10 mm, 5μm; mobile phase: 35% ACN/H2O gradient to 80% ACN/H2O in 20 min, and then 90%
ACN/H2O isocratic for 15 min; flow rate: 5 mL/min): HRMS+ (ESI-TOF) calcd. for C76H113FNO23 [M+H]+ 1426.7683, found 1426.7682.
3-O-(6-N-[(E)-10-(4-(4-fluorophenoxy)phenyl)dec-9-enyl]-6-deoxy-β-D
- glucopyranosylamide)-28-O-[β-D-xylopyranosyl-(1→4)-α-L-rhamnoyranosyl-(1→2)-β-D-fucopyranosyl] quillaic ester (42b).
To a suspension of 40 (30 mg, 15 μmol) and 10% Pd(OH)2/C (5 mg, 4 μmol) in THF/MeOH = 4/1 (1.5 mL) was stirred at rt under 55 psi H2 atmosphere. The reaction mixture was stirred for 36 h. The resulting mixture was filtered through celite, concentrated under reduced pressure. To a stirred suspension of residue,
(E)-10-(4-(4-fluorophenoxy)phenyl)dec-9-enoic acid 16 (5 mg, 15 μmol), HBTU (5 mg, 15 μmol) in anhydrous DMA (0.3 mL) was added DIPEA (3 μL, 15 μmol) under N2 atmosphere. Upon
the completion of the reaction after 12 h, the reaction mixture was concentrated under reduced pressure, diluted with CH2Cl2, washed by H2O for two times, brine, dried over MgSO4, and then concentrated under pressure. To a stirred solution of crude
tetrasaccharide saponin in CH2Cl2 (0.5 mL) was added pre-cooled TFA/H2O = 4/1 (0.5 mL) at 0 °C, and stirred for 30 min. The solvent was evaporated under reduced pressure (<1 torr) at 0 °C, and then dried under high vacuum at rt for 1 h. To a stirred solution of the residue in MeOH (1mL) was added K2CO3 (40 mg, 300 μmol) and stirred for 12 h.
The suspension was filtered, concentrated then purified by HPLC to afford product 42b
(1 mg) in 3% as a white solid (HPLC column: SUPELCO Ascentis C18 25 cm × 10 mm, 5μm; mobile phase: 35% ACN/H2O gradient to 80% ACN/H2O in 20 min, and then 90%
ACN/H2O isocratic for 15 min; flow rate: 5 mL/min) : HRMS+ (ESI-TOF) calcd. for C76H111FNO23 [M+H]+ 1424.7436, found 1426.7435.
3-O-(2,3,4,6-tetra-O-triethylsilyl-β-D
-galactopyranosyl-(1→2)-[2,3,4-tri-O-triethylsilyl-β-D-xylopyranosyl-(1→3)]-(3-O-triethylsilyl-β-D
-glucuropyranosyl))-16-O-triethylsilylquillaic acid (44).
To a stirred suspension of Ultra Q-100 (1.72 g) in anhydrous pyridine (25 mL) was added TESOTf (5.0 mL, 22.1 mmol) at room temperature under N2 atmosphere. The reaction mixture was stirred for 2 days, then TESOTf (1.3 mL, 5.8 mmol) was added, followed by 1 further addition (1.0 mL, 4.4 mmol) after 24 h later. The reaction mixture was stirred for 5 days in total. The resulting mixtures was concentrated and passed through a short plug of silica gel eluted with Hexanes/EtOAc (2:1). The eluate was concentrated and dried under reduced pressure to afford yellow oil. The resulting yellow oil was dissolved in MeOH/THF (1:1) (80 mL), and the solution was stirred for 3 days at room temperature. The reaction mixture was concentrated under reduced pressure, and then purified by column chromatography (silica gel; EtOAc/Hexanes = 1/6 to 1/4) to afford (TES)9-protected prosapogenin diacid 44 (0.66 g, ~38%) as a white solid foams.
44: Rf 0.47 (EtOAc/Benzene = 1/4); 1H NMR (600 MHz, CDCl3) δ 9.68 (s, 1H), 5.35 (br.
1.84-1.71 (m, 4H), 1.68(t, J = 8.9 Hz, 1H, H-19a), 1.63-1.31 (m, 1H, H-1), 1.57-1.50 (m, 1H), (C-18), 39.60 (C-8), 37.73 (C-1), 36.13 (C-10), 35.12 (C-21), 34.79 (C-15), 32.67 (C-29), 32.18 (C-6), 31.41(C-22), 30.47 (C-20), 26.5 (C-27), 24.6 (C-2), 24.3 (C-11), 20.3 (C-7), 17.0 (C-26), 15.9 (C-25), 11.9 (C-24), 7.5, 7.4 7.2, 7.1,7.1, 7.1, 6.9, 6.8, 6.8, 5.8, 5.6, 5.4, 5.3,5.3, 5.3, 5.3, 5.0, 4.4 ppm; HRMS+ (ESI-TOF) calcd. for C101H200O20Si9 [M+2H]2+
993.6288, found 993.6375.
3-O-{(2,3,4,6-tetra-O-triethylsilyl-β-D
-galactopyranosyl-(1→2)-[2,3,4-tri-O-triethylsilyl-β-D-xylopyranosyl-(1→3)]-(benzyl 3-O-triethylsilyl-β-D -glucuropyranosyloxyuronate))}-16-O-triethylsilylquillaic acid (45).
To a stirred suspension of 44 (253 mg, 127 μmol), TBP (319 mg, 1.29 mmol) and anhydrous pyridine (94 μL, 1.2 mmol) in CH2Cl2 (2.2 mL) was added CbzCl (47 μL, 0.33 mmol) under N2 atmosphere. Upon the completion of the reaction after 14 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; EtOAc/hexanes = 1/20 to 1/10) to give 45 (207 mg, 65%) as white solid foams. 45: Rf 0.74 (EtOAc/Benzene = 1/9); 1H NMR (600 MHz, CDCl3) δ 9.68 (s, 1H), 7.33-7.29 (m, 5H), 5.31 (br. s, 1H, H-12), 5.23 (d, J = 12.0 Hz, 1H Bn CH2), 5.07 (d, J=12.0 Hz, 1H Bn CH2), 4.53 (d, J = 7.6 Hz, 1H, H-1'''), 4.51 (br. s, 1H, H-16), 4.40 (d, J = 7.2 Hz, 1H, 1''), 4.12 (d, J = 7.2 Hz, 1H, 1'), 3.93-3.79 (m, 4H, 4'',
H-3', H-5', H-3''), 3.878-3.74 (m, 2H, H-5a''', H-2'), 3.72 (t, J = 9.1 Hz, 1H, H-6a''), 3.61-3.52 (C-6'), 143.3 (C-13), 135.3, 128.5 (2C), 128.3, 128.1 (2C), 122.2 (C-12), 103.5 (C-1'), 101.4 1''), 100.8 1'''), 86.1 3), 78.8 5'), 78.7 3'''), 76.4 2''), 76.97
3-O-{14-tetra-O-triethylsilyl-β-D -galactopyranosyl-(1→2)-[2,3,4-tri-O-triethylsilyl-β-D-xylopyranosyl-(1→3)]-(benzyl 3-O-triethylsilyl-β-D -glucuropyranosyloxyuronate)}-28-O-[2,3,4-tri-O-acetyl-β-D -xylopyranosyl-(1→4)-2,3-di-O-acetyl-α-L-rhamnoyranosyl-(1→2)-3,4-O-isopropylidene-β-D
-fucopyranosyl]-16-O-triethylsilylquillaic ester (46).
To a stirred suspension of 21 (68.8 mg, 83.6 μmol), 45 (130 mg, 62.3 μmol) and activated 4 Å molecular sieve powder in anhydrous CH2Cl2 (3.0 mL) was added BF3·OEt2
(ca. 48%, 4 μL, 24 μmol) at -75 °C under N2 atmosphere. Upon completion of the reaction after 0.5 h, the reaction was quenched by Et3N, warmed to room temperature. The resulting mixture was diluted with CH2Cl2 and filtered through 5 μm filter paper. The resulting filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; EtOAc/CH2Cl2/hexanes = 1/1/6 to 1/1/4) to give 46
(160 mg, 87%) as white solid foams. 46: Rf 0.63 (EtOAc/hexanes = 1/1); 1H NMR (600 (C-23), 175.6 (C-28), 170.3 (C-6'), 170.0, 169.8, 169.3, 169.2, 168.4, 143.1 (C-13), 135.3,
128.5, 128.5, 128.3, 128.1, 128.1, 122.0 (C-12), 110.1, 103.5 (C-1'), 101.4 (C-1''), 101.2 26.5 (isopropylidene CH3), 26.1 (isopropylidene CH3), 25.3 2), 24.5 30), 23.3 11), 21.0, 20.1, 20.7, 20.4, 20.3 7), 17.7 6''''') 17.2 26), 16.2 6''''), 15.8 (C-25), 12.2 (C-24), 7.5, 7.4 7.2, 7.1, 7.1, 7.1, 7.0, 6.8, 6.8, 6.6, 5.9, 5.8, 5.6, 5.4, 5.4, 5.3, 5.3,5.2, 5.2, 5.0, 4.9, 4.4 ppm; HRMS+ (ESI-TOF) calcd. for C138H246O37Si9Na [M+Na]+ 2772.5224, found 2772.5586.
3-O-{β-D-galactopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]- [β-D
-glucuropyranosyl]}-28-O-[β-D-xylopyranosyl-(1→4)-α-L
-rhamnoyranosyl-(1→2)-β-D-fucopyranosyl] quillaic ester (50).
To a suspension of 46 (36 mg, 26 μmol) and 10% Pd/C (5 mg, 5 μmol) in THF/MeOH = 4/1 (1.5 mL) was stirred at rt under H2 atmosphere (balloon). The reaction mixture was stirred for 16 h. The resulting mixture was filtered through celite, concentrated under reduced pressure. To a stirred solution of crude acid intermediate in CH2Cl2 (0.5 mL) was added pre-cooled TFA/H2O = 3/1 (0.5 mL) at 0 °C, and stirred for 30 min. The solvent was evaporated under reduced pressure (<1 torr) at 0 °C, and then dried under high vacuum at rt for 1 h. To a stirred solution of the residue in MeOH (1mL) was added K2CO3 (30 mg, 433 μmol) and stirred for 12 h. The suspension was filtered, and the filtrate was purified by HPLC to afford product 50 (5 mg) in 28% three steps yield
as a white solid (HPLC column: SUPELCO Ascentis C18 25 cm × 10 mm, 5μm; mobile phase: 20% ACN/H2O (contained 0.05% TFA) gradient to 90% ACN/H2O (contained 0.05% TFA) in 20 min, and then 90% ACN/H2O (contained 0.05% TFA) isocratic for 15 min; flow rate: 2.4 mL/min): 1H NMR (600 MHz, CD3OD) δ 9.44 (s, 1H, H-23), 5.37 (d,
71.3 2'''''), 71.0 3''), 70.8 4'''), 68.8 5'''''), 67.3 5'''), 67.2 5''''''), 62.2 (C-6'), 56.3 (C-4), 49.9 (C-17), 48.0 (C-19, C-9), 42.7 (C-14), 42.2, 41.0, 37.1 (C-10), 36.5 10), 35.0 15, C-21), 34.5 15), 33.5 6), 33.4 29), 32.1, 31.3, 30.8, 27.2 27), 25.7 2), 24.8 30), 24.5 11), 21.5 7), 32.4 6), 31.0 22), 30.4 20), 27.7 27), 25.3 2), 24.5 30), 23.3 11), 21.4 7), 18.3 6'''''), 17.7 (C-26), 16.5 (C-6''''), 16.3 (C-25), 11.0 (C-24) ppm; HRMS- (ESI-TOF) calcd. for C64H99O32
[M-H]- 1379.6145, found 1379.6114.
3-O-{β-D-galactopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]- [N-(8-(4-phenoxyphenyl)octyl)-β-D-glucuropyranosyluroamide]}-28-O-[β-D -xylopyranosyl-(1→4)-α-L-rhamnoyranosyl-(1→2)-β-D-fucopyranosyl] quillaic ester (51a).
To a stirred suspension of 50 (5 mg, 4 μmol), 8-(4-phenoxyphenyl)octan-1-amine
(10 mg, 40 μmol), and HBTU (12 mg, 40 μmol) in anhydrous DMA (0.5 mL) was added DIPEA (6 μL, 40 μmol) under N2 atmosphere. Upon the completion of the reaction after
24 h, the reaction mixture was concentrated under reduced pressure, diluted with MeOH then filtered through 5 μm filter paper. The filtrate was concentrated then purified by
HPLC to afford product 51a (2 mg) in 80% as a white solid (HPLC column: SUPELCO Ascentis C18 25 cm × 10 mm, 5μm; mobile phase: 20% ACN/H2O gradient to 90%
3H, H-6'''''), 1.16 (s, 3H, H-23), 1.15 (m, 1H), 1.08 (m, 3H), 0.98 (s, 3H, H-25), 0.92 (s, 3H, H-30), 0.90 (m, 2H), 0.86 (s, 3H, H-24), 0.74 (s, 3H, H-26); BBD 13CNMR (150 MHz, CD3OD) δ 221.0 (C-23), 177.1 (C-28), 170.7 (C-6'), 159.2, 156.5, 145.0, 139.2, 130.8, 130.7, 124.0, 123.1 12), 120.1, 119.4, 107.1 1''''''), 105.01'''), 104.5 (C-1'), 103.7 (C-1''), 101.3 (C-1'''''), 95.0 (C-1''''), 86.9 (C-2'''), 86.3 (C-3), 84.3 (C-2''''''), 78.2
3-O-{β-D-galactopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]- [N-(8-(4-methoxyphenyl)octyl)-β-D-glucuropyranosyluroamide]}-28-O-[β-D -xylopyranosyl-(1→4)-α-L-rhamnoyranosyl-(1→2)-β-D-fucopyranosyl] quillaic ester (51b).
To a stirred suspension of 50 (8 mg, 6 μmol), 8-(4-methoxyphenyl)octan-1-amine (13 mg, 58 μmol), and HBTU (22 mg, 58 μmol) in anhydrous DMA (0.5 mL) was added DIPEA (10 μL, 58 μmol) under N2 atmosphere. Upon the completion of the reaction after 24 h, the reaction mixture was concentrated under reduced pressure, diluted with MeOH then filtered through 5 μm filter paper. The filtrate was concentrated then purified by HPLC to afford product 51b (2 mg) in 30% as a white solid (HPLC column: SUPELCO Ascentis C18 25 cm × 10 mm, 5μm; mobile phase: 30% ACN/H2O gradient to 80%
ACN/H2O in 20 min, and then 90% ACN/H2O isocratic for 15 min; flow rate: 5 mL/min);
1H NMR (600 MHz, CD3OD) δ 9.45 (s, 1H, H-23), 8.55 (s, 1H, amide NH), 7.01 (m, 2H),
6.82 (m, 2H), 5.38 (d, J = 1.2 Hz, 1H, H-1'''''), 5.28 (m, 2H, H-12, H-1''''), 4.79 (d, J = 7.2
Hz, 1H, H-1''), 4.57 (d, J = 7.2 Hz, 1H, H-1'''), 4.50 (s, 1H, H-16), 4.47 (d, J = 7.8 Hz, (C-6'), 161.1, 159.2, 135.9, 130.3, 123.1 (C-12), 114.7, 107.1 (C-1''''''), 105.0(C-1''''), 104.5 (C-1'), 103.7 (C-1''), 101.3 (C-1'''''), 95.0 (C-1''''), 86.9 (C-2'), 86.3 (C-3), 84.3 (C-2'''''')
(C-25), 14.4, 11.0 ppm; HRMS+ (ESI-TOF) calcd. for C79H123NO32Na [M+Na]+ 1620.7920, found 1620.7920.
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