Facile Synthesis of Saikosaponins

Saikosaponin A (SSa) and D (SSd) are typical oleanane-type saponins featuring a unique 13,28-epoxy-ether moiety at D ring of the aglycones, which exhibit a wide range of biological and pharmacological activities. Herein, we report the first synthesis of saikosaponin A/D (1–2) and their natural congeners, including prosaikosaponin F (3), G (4), saikosaponin Y (5), prosaikogenin (6), and clinoposaponin I (7). The present synthesis features ready preparation of the aglycones of high oxidation state from oleanolic acid, regioselective glycosylation to construct the β-(1→3)-linked disaccharide fragment, and efficient gold(I)-catalyzed glycosylation to install the glycans on to the aglycones.


Introduction
The roots of Radix Bupleuri, one of the most common traditional Chinese medicines, have been frequently used for the treatment of common cold with fever, influenza, inflammation, and infectious diseases [1,2]. Saikosaponin A (SSa, 1) and D (SSd, 2) are the major compounds in Radix Bupleuri and are used as the markers for evaluation of the quality of the medicine (Figure 1) [3]. Indeed, these two triterpene glycosides together with their congeners exhibit a variety of pharmacological activities, including anti-inflammatory, antitumor, neuro-regulation, immuno-regulation, hepatoprotection, and antiviral effects [1,4]. Structurally, SSa (1) and SSd (2) are a pair of the oleanane-type disaccharide stereoisomers, with the aglycones, namely saikogenin F (SGF) and saikogenin G (SGG), respectively, featuring a 13,28-epoxy-ether moiety at the D ring and an opposite chirality at 16-OH. Interestingly, SSa (1) was reported to show a strong anti-inflammatory effect, whereas SSd (2) showed a strong antitumor effect [1]. The corresponding monosaccharides, named prosaikogenin F (PSF, 3) and G (PSG, 4) were identified as metabolites of SSa and SSd [5], which showed hemolytic activity and the absorbability on red blood cells [6,7]. Saikosaponin Y (5) is likely a biogenetic precursor/product of SSa and SSd, which has a higher oxidation state at C-16; this congener displayed potent cytotoxic and antiviral activities [8][9][10]. Monosaccharide 6, named posaikogenin, was found as an artifact which might be derived from disaccharide 5. A number of saikosaponin-like compounds have also been identified from Clinopodium gracile and other plants, among which Clinoposaponin I (7) bearing a trisaccharide residue is a typical one [11][12][13]. All these seven saponins (1-7) belong to type I saikosaponins, which bear the 13,28-epoxy-ether moiety, and this epoxy-ether moiety is found crucial to their cytotoxic activities. Notwithstanding, these naturally extracted saikosaponins are associated with systemic toxicities and side effects [14,15]. Thus, saikosaponins could induce obvious hepatoxicity and necrosis upon 15 day administration to rats [14]. Furthermore, SSd (2) alone was found to stimulate mitochondrial apoptosis in human hepatocyte cells [15]. On the other hand, these saikosaponins are difficult to purify from plants; therefore, in-depth studies have not been carried out on their structure-activity relationship (SAR) and mode of action. Chemical synthesis of saikosaponins is also a challenging task, given the scarce availability of the triterpene aglycones that bear high oxidation state at the D/E rings [16][17][18][19]. Very recently, we disclosed a site-selective C-H hydroxylation reaction at the D/E rings of pentacyclic triterpenoids, thus paving a venue for the synthesis of saikosaponins [18,20]. Herein, we report the synthesis of saikosaponin A (1), D (2), and their congeners (3)(4)(5)(6)(7), using the largely available oleanolic acid as a starting material.
To a stirred solution of the above product (50 mg, 0.07 mmol) in CH 3 CN/H 2 O (0.8 mL/0.2 mL), ceric ammonium nitrate (CAN) (89 mg, 0.16 mmol) was added. The mixture was stirred at room temperature for 2 h before it was quenched with Na 2 S 2 O 3 and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under vacuum. The residue was purified by flash column chromatography (petroleum ether/EtOAc, 1:1) to give the corresponding lactol product (33 mg, 80%) as an orange foam. The α/β anomers were difficult to separate.

Synthesis of Compound 12
To a stirred solution of compound 10 (830 mg, 1.4 mmol) in pyridine (5 mL), benzoyl chloride (0.48 mL, 4.14 mmol) and DMAP (50 mg, 0.4 mmol) were added. The mixture was stirred at room temperature for 8 h before it was quenched with NaHCO 3 and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 , and filtered. The filtrate was concentrated under vacuum. The residue was purified by flash column chromatography on silica gel (petroleum ether/EtOAc = 3/1) to give the corresponding benzoylated product (1.07 g, 96%) as a white solid.
To a stirred solution of the above product (800 mg, 0.99 mmol) in CH 3 CN/H 2 O (12 mL/3 mL), ceric ammonium nitrate (1.35 g, 2.5 mmol) was added. The mixture was stirred at room temperature for 2 h before it was quenched with Na 2 S 2 O 3 and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under vacuum. The residue was purified by flash column chromatography (petroleum ether/EtOAc = 1:1) to give the corresponding lactol product (560 mg, 81%) as an orange foam. The α/β anomers were difficult to separate.

Synthesis of Compound 14
Compound 13 (100 mg, 0.17 mmol) was dissolved in pyridine (5 mL), to which was added HF·pyr (0.2 mL) at room temperature. After stirring at 90 • C for 24 h, the mixture was quenched with saturated NaHCO 3 and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under vacuum. The residue was purified by flash column chromatography (petroleum ether/EtOAc = 2:1) to give the 3-ol product (76 mg, 88%) as a white foam. R f = 0.2 (silica, PE/EtOAc = 5:1).
The above product (3.6 g, 7.2 mmol) was dissolved in CH 2 Cl 2 (40 mL), to which Dess-Martin periodinane (DMP) (3.7 g, 8.6 mmol) was added. After being stirred for 2 h at room temperature, the reaction mixture was quenched with a saturated aqueous Na 2 S 2 O 3 solution, and the aqueous phase was extracted with CH 2 Cl 2 . The combined organic extracts were dried over anhydrous Na 2 SO 4 and concentrated in vacuo.
The crude product was dissolved in MeOH/CH 2 Cl 2 (80 mL/40 mL), to which NH 2 OH·HCl (692 mg, 10.8 mmol) and NaOAc (1.76 g, 21.6 mmol) were added at room temperature. After stirring two hours at reflux, the reaction mixture was diluted by addition of brine, then extracted with CH 2 Cl 2 . The extract was dried over anhydrous Na 2 SO 4 , followed by filtration and concentration in vacuo. The residue was purified by silica gel flash column chromatography (petroleum ether/EtOAc = 6/1 to 3/1) to give compound 14 (3.

Synthesis of Compound 15
To a solution of compound 14 (1.9 g, 3.62 mmol) in HOAc (170 mL), NaOAc (350 mg, 4.3 mmol) and Na 2 PdCl 4 (1.3 g, 4.3 mmol) were added at room temperature. The mixture was stirred at room temperature for 3 d, and then ice water (300 mL) was added. The resulting mixture was stirred for an additional 20 min and then filtered. The filtered residue was washed with water several times, dried, and used without further purification.
The residue was dissolved in CH 2 Cl 2 (20 mL) at room temperature, to which NEt 3 (1 mL, 7.24 mmol), DMAP (88 mg, 0.72 mmol), and Ac 2 O (0.68 mL, 7.24 mmol) were added. The resulting mixture was stirred at room temperature for 3 h and then diluted with EtOAc. The mixture was thoroughly washed with water and brine, and the organic layer was dried with anhydrous Na 2 SO 4 . After filtration, the solution was concentrated under vacuum to give a residue. The residue was dissolved in THF (120 mL), and anhydrous pyridine (0.3 mL) was added dropwise. After the mixture was stirred for 40 min, a solution of Pb(OAc) 4 (1.5 mg, 3.2 mmol) in HOAc (51 mL) was added dropwise at −78 • C. The mixture was warmed to room temperature and then stirred for 24 h. The reaction mixture was cooled to 0 • C, and a solution of NaBH 4 (136 mg, 1.7 mmol) in NaOH (1M, 46 mL) was slowly added. The resulting solution was stirred at 0 • C for an additional 1 h and diluted with EtOAc. The mixture was washed with a saturated NaHCO 3 solution and then brine, and the organic layer was dried with anhydrous Na 2 SO 4 . After filtration, the solution was concentrated to give a residue, which was purified by flash column chromatography on silica gel (petroleum/EtOAc = 7:1) to give compound 15 (1.4 g, 63% for 3 steps) as a white foamy solid.  13

Synthesis of Compound 16
A solution of compound 15 (1.0 g, 1.6 mmol) and Na 2 CO 3 (866 mg, 8.2 mmol) in MeOH (120 mL) was stirred at room temperature for 24 h. After removing MeOH in vacuo, the resulting colorless solid was dissolved in Et 2 O. The organic layer was washed with 1 N HCl solution, saturated NaHCO 3 solution and brine, dried over anhydrous Na 2 SO 4 , and filtered. The filtrate was evaporated in vacuo to give a solid.
To a solution of the above product in THF (55 mL), NH 4 OAc (1.5 g, 20 mmol) and a buffered solution of TiCl 3 (3.4 mL of HCl solution containing 15% TiCl 3 , 4 mmol) were added. The mixture was stirred at room temperature for 8 h. The mixture was washed with saturated NaHCO 3 solution and brine, dried over anhydrous Na 2 SO 4 , filtered, and evaporated in vacuo to give a solid.  13  To a stirred solution of compound 16 (90 mg, 0.2 mmol) in pyridine (4 mL), pivaloyl chloride (0.5 mL, 4 mmol) and DMAP (6 mg, 0.06 mmol) were added. The mixture was stirred at room temperature for 8 h before it was quenched with NaHCO 3 and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under vacuum. The residue was purified by flash column chromatography on silica gel (petroleum ether/EtOAc = 8/1) to give the acylated product (115 mg, 94%) as a white solid.  To a stirred solution of the above product (90 mg, 0.14 mmol) in MeOH (3 mL) at 0 • C, NaBH 4 (23 mg, 0.5 mmol) was added. The stirring continued for 15 min, the solution was quenched by addition of saturated NH 4 Cl solution, then diluted with brine and extracted with CH 2 Cl 2 . The extract was dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 4/1) to give compound 17 (76 mg, 85%) as a white foam.  13

Synthesis of Compound 18
To a stirred mixture of compound 11 (180 mg, 0.24 mmol), compound 17 (120 mg, 0.18 mmol), and 4Å molecular sieves (400 mg) in CH 2 Cl 2 (4 mL), PPh 3 AuNTf 2 (26 mg, 0.04 mmol) was added. The mixture was stirred at room temperature for 1 h before it was quenched with NEt 3 . The mixture was filtered through a pad of celite and washed with EtOAc. The filtrate was concentrated under vacuum.  13

Synthesis of Compound 19
Compound 16 (290 mg, 0.6 mmol) was dissolved in CH 2 Cl 2 (10 mL), to which DMP (636 mg, 1.5 mmol) was added. After being stirred for 40 min at room temperature, the reaction mixture was quenched with a saturated Na 2 S 2 O 3 solution and the aqueous phase was extracted with CH 2 Cl 2 . The combined organic extracts were dried over anhydrous Na 2 SO 4 and concentrated in vacuo.
To a solution of the above product in MeOH (10 mL) under stirring at 0 • C, NaBH 4 (95 mg, 2.5 mmol) was added. The mixture was stirred for 15 min before it was quenched by addition of saturated NH 4 Cl solution. The mixture was diluted with brine and extracted with CH 2 Cl 2 . The combined organic phases were dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 2/1) to give compound 19 (190 mg, 65%) as a white foam.  13

Synthesis of Compound 20
To a stirred solution of compound 19 (187 mg, 0.4 mmol) in CH 2 Cl 2 (4 mL), pivaloyl chloride (58 µL, 0.48 mmol) and pyridine (0.57 mL, 7.2 mmol) were added. The mixture was stirred at −10 • C for 8 h before it was quenched with NaHCO 3 and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under vacuum.  13

Synthesis of Compound 21
A solution of tetramethylammonium triacetoxyborohydride (1.9 g, 7.2 mmol) in MeCN (27 mL) and AcOH (1.12 mL) was cooled to −40 • C. To the mixture was added a solution of the compound 16 (680 mg, 1.4 mmol) in CH 2 Cl 2 (9 mL). The mixture was stirred at room temperature for 0.5 h before it was quenched with Rochelle salt (sat. aq.) and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under vacuum.
The above product was dissolved into DMF (5 mL), to which 2,6-lutidine (2.5 mL) was added, and the solution was cooled to 0 • C. Di-tert-butylsilylbis(trifluoromethansulfonate) (0. 6 mL, 1.7 mmol) was added. The mixture was stirred at room temperature for 2 h before it was quenched with NaHCO 3 and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under vacuum.
The above product was dissolved in CH 2 Cl 2 (5 mL), then the solution was added DMP (736 mg, 1.7 mmol). After being stirred for 40 min at room temperature, the reaction mixture was quenched with a saturated Na 2 S 2 O 3 solution, and the aqueous phase was extracted with CH 2 Cl 2 . The combined organic extracts were dried over anhydrous Na 2 SO 4 and concentrated in vacuo.  13

Synthesis of Compound 22
The compound 21 was dissolved in THF (5 mL), to which HF·pyr (1 mL) was added at room temperature. After stirring at room temperature for 1 h, the mixture was quenched with saturated NaHCO 3 and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under vacuum. The residue was purified by flash column chromatography (petroleum ether/EtOAc = 2:1) to give the diol product (500 mg, 99%) as a white foam.  13  To a stirred solution of the above product (470 mg, 1 mmol) in CH 2 Cl 2 (10 mL), pivaloyl chloride (0.15 mL, 1.2 mmol) and pyridine (0.8 mL, 12 mmol) were added. The mixture was stirred at −10 • C for 30 h before it was quenched with NaHCO 3 and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under vacuum. The residue was purified by flash column chromatography on silica gel (petroleum ether/EtOAc = 9/1) to give compound 22

Synthesis of Compound 23
To a stirred mixture of compound 22 (50 mg, 0.09 mmol), compound 12 (102 mg, 0.11 mmol), and 4Å molecular sieves (200 mg) in CH 2 Cl 2 (2 mL), PPh 3 AuNTf 2 (13 mg, 0.02 mmol) was added. The mixture was stirred at room temperature for 1 h before it was quenched with NEt 3 . The mixture was filtered through a pad of celite and washed with EtOAc. The filtrate was concentrated under vacuum.  13

Synthesis of Saikosaponin D (2)
To a solution of compound 23 (102 mg, 0.08 mmol) in i-PrOH (2 mL) at −20 • C, NaBH 4 (15 mg, 0.4 mmol) was added. The mixture was stirred for 3 h before it was quenched by addition of saturated NH 4 Cl solution and diluted with brine and extracted with CH 2 Cl 2 . The extract was dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 6/1) to give the 16-OH product (90 mg, 89%) as a white foam.
To a solution of the above product (49 mg, 0.04 mmol) in MeOH (3 mL), KOH (110 mg, 2 mmol) was added at room temperature. The mixture was stirred at 55 • C for 24 h before it was quenched with acetic acid. The mixture was concentrated under vacuum. The residue was purified by reversed-phase silica gel column chromatography (ODS RP-18) (CHCl 3 /MeOH = 5:1 to 7:1) to give saikosaponin D (2) (26 mg, 83%) as a white powder.  13

Synthesis of Compound 24
To a stirred solution of compound 9 (290 mg, 1.1 mmol) in pyridine (5 mL), benzoyl chloride (0.8 mL, 4.9 mmol) and DMAP (39 mg, 0.3 mmol) were added. The mixture was stirred at room temperature for 8 h before it was quenched with NaHCO 3 and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under vacuum. The residue was purified by flash column chromatography on silica gel (petroleum ether/EtOAc = 9/1) to give corresponding benzoylated product (625 mg, 99%) as a white solid.
To a stirred solution of the above product (582 mg, 1.0 mmol) in CH 3 CN/H 2 O (8 mL/2 mL), ceric ammonium nitrate (1.2 g, 2.1 mmol) was added. The mixture was stirred at room temperature for 2 h before it was quenched with Na 2 S 2 O 3 and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under vacuum. The residue was purified by flash column chromatography (petroleum ether/EtOAc = 2:1) to give the lactol (375 mg, 81%) as an orange foam. The α/β anomers were difficult to separate.

Synthesis of Compound 26
To a stirred mixture of compound 24 (97 mg, 0.15 mmol), compound 22 (70 mg, 0.13 mmol), and 4Å molecular sieves (300 mg) in CH 2 Cl 2 (3 mL), PPh 3 AuNTf 2 (19 mg, 0.03 mmol) was added. The mixture was stirred at room temperature for 1 h before it was quenched with NEt 3 .The mixture was filtered through a pad of celite and washed with EtOAc. The filtrate was concentrated under vacuum. The residue was purified by flash column chromatography (petroleum ether/EtOAc = 5:1) to give compound 26 (106 mg, 84%) as a white foam.  13  To a stirred solution of compound 26 (90 mg, 0.09 mmol) in i-PrOH (3 mL) at −20 • C, NaBH 4 (7 mg, 0.18 mmol) was added. The mixture was stirred for 3 h, the solution was quenched by addition of saturated NH 4 Cl solution and diluted with brine and extracted with CH 2 Cl 2 . The extract was dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 8:1) to give the 16-OH product (86 mg, 95%) as a white foam.
To a stirred solution of the above product (70 mg, 0.07 mmol) in MeOH (3 mL), KOH (193 mg, 3.5 mmol) was added at room temperature. The mixture was stirred at 55 • C for 24 h before it was quenched with acetic acid. The mixture concentrated under vacuum.   (6) To a stirred solution of the compound 26 (100 mg, 0.1 mmol) in MeOH (3 mL), KOH (360 mg, 5 mmol) was added at room temperature. The mixture was stirred at 55 • C for 24 h before it was quenched with acetic acid. The mixture was concentrated under vacuum. The residue was purified by reversed-phase silica gel column chromatography (ODS RP-18) (MeOH/H 2 O = 5:1) to give compound prosaikogenin (6) 13

Synthesis of Compound 27
To a stirred solution of the compound 10 (225 mg, 0.4 mmol) in MeOH (3 mL), CH 3 ONa (12 mg, 0.2 mmol) was added. The mixture was stirred at room temperature for 8 h before it was quenched with acid resin until pH = 7. The mixture was filtered through a pad of celite and washed with MeOH. The filtrate was concentrated under vacuum.
To a stirred mixture of the above product in pyridine (5 mL), triphenylmethyl chloride (1.04 g, 3.8 mmol) and DMAP (45 mg, 0.4 mmol) were added. The mixture was stirred for 8 h at 75 • C, then benzoyl chloride (0.43 mL, 3.8 mmol) was added in the flask at 0 • C. The mixture was stirred for 4 h at room temperature, the solution was quenched by addition of

Synthesis of Compound 31
To a stirred mixture of compound 30 (220 mg, 0.14 mmol), compound 17 (68 mg, 0.1 mmol), and 4Å molecular sieves (300 mg) in CH 2 Cl 2 (3 mL), PPh 3 AuNTf 2 (16 mg, 0.03 mmol) was added. The mixture was stirred at 0 • C for 1 h before it was quenched with NEt 3 . The mixture was filtered through a pad of celite and washed with EtOAc. The filtrate was concentrated under vacuum. The residue was purified by flash column chromatography (petroleum ether/EtOAc = 4:1) to give compound 31 (171 mg, 79%) as a white foam.  To a stirred solution of the compound 31 (51mg, 0.02 mmol) in MeOH (3 mL), KOH (111 mg, 2.0 mmol) was added. The mixture was stirred at 55 • C for 24 h before it was quenched with acetic acid. The mixture concentrated under vacuum.  13

Conclusions
Here, we report the facile synthesis of seven saikosaponins; namely, saikosaponin A (1), D (2), prosaikosaponin F (3), G (4), saikosaponin Y (5), prosaikogenin (6), and clinoposaponin I (7), which have been identified from Radix Bupleuri, a common traditional Chinese medicine and relevant plants. These mono-, di-, and trisaccharide saponins feature triterpene aglycones of high oxidation states, which bear 13,28-epoxy-ether moiety and oxo groups at C16 and C23. The methods previously developed for the selective hydroxylation of the triterpenes of low oxidation states, such as oleanolic acid, have enabled the present synthesis. In addition, the gold(I)-catalyzed glycosylation with o-alkynylbenzoates as donors has been applied successfully in the present synthesis. Given the conserved nature of the structures of saikosaponins, the work reported herein offers the prospect of being able to access many more members of saikosaponins and their natural and synthetic analogs, thus facilitating in-depth studies on biological and pharmacological activities of these components of folk medicines.