Facile Synthesis of Oleanolic Acid Monoglycosides and Diglycosides

Oleanolic acid and its glycosides are important natural products, possessing various attractive biological activities such as antitumor, antivirus and anti-inflammatory properties. In the present work, fifteen oleanolic acid saponins bearing various saccharide moieties, including 3-monoglycoside, 28-monoglycoside and 3,28-diglycoside, were easily synthesized in high yields. Benzyl was chosen as the protective group for the COOH(28) group, instead of commonly used methyl and allyl, to avoid difficulties in the final deprotection. Alkali-promoted condensation of the carboxylic acid with bromo-glycosides was found to be more efficient in the synthesis of 28-glycosides. Two approaches were investigated and proved practicable in the preparation of 3,28-diglycosides. This method is suitable for preparing oleanolic acid glycosides with structural diversity for extensive biological evaluation and structure-activity relationship study, and it also apply new idea for the corresponding synthetic methods to the glycoside derivatives of other triterpenoid.

Although OA has become an auxiliary drug in the treatment of liver disease, many bioactivities of OA glycosides are too weak to be utilized in clinical therapy. Chemical synthesis and modification is known to be a powerful tool in preparation of novel compounds with diversity for pharmacology studies and new chemical entity development. In fact, the synthesis of OA glycosides has attracted much attention from researchers for a long time. As early as 1952, Hardegger et al. reported the glycosylation of OA esters with acetylated bromoglycosides [7]. Later a few studies were reported on the glycosylation of OA by the Koenigs-Knorr method [8][9][10][11][12][13], which was usually inefficient and resulted in uneven yields. Although methyl was most commonly used as protective group for the carboxylic acid in the early synthesis work, alkaline hydrolysis of OA esters was very difficult due to the high steric hinderance [14]. Therefore, halolysis (usually LiI/DMF, reflux) was often employed to cleave OA esters, but was of low efficiency and too harsh for many groups. In 1999, Deng et al. reported a highly efficient glycosylation of triterpenoids and steroids via the Schmidt method [15,16], in which the allyl ester of OA was glycosylated with benzoylated trichloroacetimidates in very high yields (>90%). Based on this method, Zang et al. accomplished in 2005 the synthesis of four 3-monoglycosides of OA [17]. The glycosylation of allyl oleanolate was carried out in 80~90% yield, however, removal of allyl in the final step by treatment with PdCl 2 for 24 h afforded relatively lower yields (only 41~47%). A few syntheses of some bioactive OA saponins bearing complex saccharide moieties were accomplished in recent years [18][19][20], nevertheless more facile and efficient synthetic methods towards this interesting structure require further investigation. Based on our experience in triterpenoid saponin synthesis, we started with the highly efficient preparation of OA glycosides on a large scale for extensive bioactivity evaluation and structure-activity relationship study.

Results and Discussion
OA has two glycosylation sites, i.e. C(3)-OH and COOH(28), which can be glycosylated to form 3-monoglycosides, 28-monoglycosides and 3,28-diglycosides. The present work describes the initial glycosylation study of OA, which was carried out with some commercially available sugars, such as Dglucose, L-arabinose, lactose and maltose.

Preparation of OA 3-glycosides
Since common alkyl esters of OA are difficult to retransform into free carboxylic acids, selection of a suitable protective group for COOH(28) is the primary problem. Trityl (Tr) and tert-butyldiphenylsilyl (TBDPS) had been used in the previous work [21,22], however, they are too sensitive to act as permanent protective groups in the preparation of complex glycosides. As mentioned above, the deprotection was usually inefficient when allyl was employed [17,18]. In our previous work on triterpenoid saponin synthesis [19,23], benzyl was chosen and proved to be an excellent permanent protective group for COOH(28), as it can be conveniently removed through catalytic hydrogenolysis in nearly quantitive yields, while the double bond between C(12) and C (13) would not be affected [24]. Therefore, we adopted benzyl in the present work. As shown in Scheme 1, OA was first converted into its benzyl ester 2 in 98% yield [23], and C(3)-OH was then glycosylated with trichloroacetimidates 3 under promotion with TMSOTf. The benzyl moiety was then removed under catalytic hydrogenolysis to give free carboxylic acids 5, which would be subjected to further glycosylation to give 3,28-diglycosides in the later work. Removal of benzoyl on 5 through ester exchange in NaOMe-MeOH afforded target OA 3-glycosides 6. According to this method, seven 3-glycosides of OA were easily prepared in overall yields of 58%~79%.

Preparation of OA 28-glycosides
Two approaches were attempted to prepare OA 28-glycosides. In the first approach, the C(3)-OH of OA was acetylated in Ac 2 O-pyridine and glycosylation of COOH(28) was carried out with benzoylated trichloroacetimidates. Benzoyl and acetyl groups were then removed by NaOMe-MeOH to give OA 28-glycosides (Scheme 2). However, removal of Ac on C(3)-OH was found to be more difficult than that of benzoyl groups on the saccharide moieties due to the steric hinderance from C(23) and C (24). Stronger alkaline condition usually led to the partial breakage of acyl glycoside linkage at C(28). By this approach OA ester of β-D-galactose (9a) and α-L-rhamnose (9b) were synthesized in 18% and 21% total yields, respectively.

Scheme 2.
Synthesis of OA 28-glycosides (the first approach).   Alternatively, alkaline-promoted condensation of free OA with benzoylated bromoglycosides (R-Br, R=P 2 , P 3 , P 6 , and P 7 ) under phase transfer catalysis was attempted (Scheme 3) [25]. However, no significant reaction was observed under the reported conditions, possibly because benzoylated bromoglycosides are more stable than the acetylated donors employed in the literature. When modified conditions (K 2 CO 3 , Bu 4 NBr, CH 2 Cl 2 -H 2 O, reflux) were used [22], the reaction proceeded very slowly and the decomposed bromoglycoside was the main by-product mixed with the desired compound. As improvements, the solvent dichloromethane was replaced by chloroform and the turbid system was heated up to 50°C and stirred vigorously to successfully give the glycosylation products. All the benzoyl groups on the sugar parts were removed by NaOMe/MeOH, while the acyl glycoside linkages were not affected under these conditions. By this approach, glycosides 9a and 9b were synthesized once more in higher yields of 80% and 78%. Furthermore, other two 28-glycosides (12b, 12c) were readily prepared from OA in 74% and 70% yields, respectively. Understandably, due to the relatively less efficiency of Schmidt glycosylation of carboxylic acid and low selectivity in the deprotection in the first approach, the second approach was more preferable for the preparation of OA 28-glycosides.

Preparation of OA 3,28-diglycosides
Many natural occurring saponins of OA with important biological activities are 3,28-diglycosides. Two approaches were also investigated in the preparation of this type of structures. The first approach was to introduce one saccharide moiety at C(3)-OH after protection of COOH(28) with benzyl, and to attach another saccharide moiety to the carboxylic acid after removal of the benzyl. The second approach was to introduce one saccharide to COOH(28) using a bromoglycoside under alkaline conditions, then C(3)-OH was to be glycosylated with a trichloroacetimidate. In the present work, intermediates 5 and 11 in the preparation of 3-glycosides and 28-glycosides were utilized in the synthesis of OA 3,28-diglycosides through these two approaches, respectively. Scheme 4. Synthesis of OA 3,28-diglycosides (the first approach).
It is obvious that the second approach to 3,28-diglycosides is preferable to the first one, not only due to its conciseness in avoiding introduction and removal of benzyl group, but also because alkalinepromoted condensation was more efficient in the glycosylation of carboxylic acid. However, the first approach also affords good yields and it is applicable in certain cases. For instance, in the synthesis of glycoside analogues with the same 3-O-saccharide moiety, or in preparation of glycosides in large amounts, it can make use of various intermediates with free carboxylic acids.
The 15 glycosides prepared in the present work and the overall yields from OA are listed in Table 1. All glycosidic linkages formed in the glycosylation are 1,2-trans-, due to the neighboring participating effect of benzoyl at C(2)-OH of saccharide donors, which can also be confirmed by the J values of hydrogen on the anomeric carbon of the saccharide moiety. In 1 H-NMR spectra of the synthetic saponins, the 1,2-trans-configuration of the anomeric carbons of all glucosyl, gallactosyl, arabinosyl and xylosyl units were determined by the J H1-H2 values (above 6.0 Hz), while the J H1-H2 values (1.0-1.5 Hz) of all rhamnosyl units indicated the alpha-configuration of their anomeric carbons. For compound 6a, its 7.7 Hz J value of the hydrogen on anomeric carbon with characterized 4.93 ppm chemical shift indicates that two hydrogens on C(1') and C(2') of glucose are 1,2-trans-configuration. And in the case of 9b, its 1.4 Hz J value of the hydrogen on anomeric carbon (5.43 ppm) also indicates that two hydrogens on C(1') and C(2') of rhamnose are 1,2-trans-configuration.

Conclusions
In conclusion, 15 OA glycosides, including 3-monoglycosides, 28-monoglycosides and 3,28diglycosides, were easily synthesized in high yields. This method is suitable for preparing OA glycosides on a large scale and analogues for extensive biological evaluation, mechanism research and structure-activity relationship study. Moreover, this work may also provide an efficient method in preparing glycoside derivatives of other pentacyclic triterpenoid carboxylic acids, such as ursolic acid, glyrrhetinic acid and boswellic acid. Related work in this field is currently in progress and will be reported in due course.

General
Commercial reagents were used without further treatment unless specialized. Solvents were dried and distilled prior to use in the usual way. Boiling range of petroleum ether was 60~90°C. Analytical TLC was performed with silica gel GF 254 . Preparation column chromatography was performed with silica gel H. Saccharide donors (trichloroacetimidates and bromoglycosides) were prepared according to the reported methods [16,25,26]. 1 H-NMR and 13 C-NMR spectra were recorded on a Bruker ARX 300 MHz instrument. J values were given in Hz. ESI-MS were obtained on an Agilent 1100 mass spectrometer. HRMS was detected on High resolution ESI-FTICR mass spectrometry (Ion spec 7.0T).