Facile Synthesis of the Naturally Cytotoxic Triterpenoid Saponin Patrinia-Glycoside B-II and Its Conformer

The first chemical synthesis of the natural triterpenoid saponin Patrinia-glycoside B-II, namely oleanolic acid 3-O-α-l-rhamnopyranosyl-(1→2)-[β-d-gluco-pyranosyl-(1→3)]-α-l-arabinopyranoside, has been accomplished in a linear 11-step sequence 11 with 9.4% overall yield. The abnormal 1C4 conformation of the arabinose residue was found to occur via conformational fluctuation during preparation of the intermediates. Molecular mechanism and quantum chemistry calculations showed that Patrinia-glycoside B-II and its conformer 1 cannot interconvert under normal conditions. Preliminary structure-activity relationships studies indicated that the 4C1 chair conformation of the arabinose residue in the unique α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl disaccharide moiety is one of the chief positive factors responsible for its cytotoxic activity against tumors.

Patrinia-glycoside B-II (PB-II, Figure 1), a typical triterpenoid saponin isolated from the seeds of Patrinia scabiosaefolia Fischer [11], contains the unique oligosaccharide substructure and also displays prominent inhibitory activity against many tumor cell lines [7]. To investigate the structure-activity relationships (SARs) of this type of saponin, we have developed a convenient method for the chemical preparation of Patrinia-glycoside B-II.

Synthesis
The target compound consists of a trisaccharide moiety and oleanolic acid as the aglycone. With regard to the trisaccharide moiety, there is no neighboring participating group but rather a rhamnose residue connected to the C2-OH of arabinose. Because of this, the standard strategy of preparing a trisaccharide donor followed by coupling with the aglycone is not appropriate, as it would result in a mixture of α-and β-anomers. Accordingly, a stepwise glycosylation strategy was adopted in this work, as this strategy generally affords the 1,2-trans-glycoside linkage exclusively. Based on our experience, we chose isopropylidene, acetyl, and benzoyl groups as the temporary protecting groups for the hydroxyl groups of different sugars, and a benzyl group for the carboxylic acid of the oleanolic acid. In addition, perbenzoylated or peracetylated glycosyl trichloroacetimidates (SD-1 to SD-5) were used as sugar donors, which were readily prepared from L-arabinose, L-rhamnose and D-glucose according to the reported methods [12,13].
As shown in Scheme 1, the benzyl ester of oleanolic acid 2 [9] was glycosylated with perbenzoylated arabinosyl trichloroacetimidate SD-1 under trimethylsilyl trifluoromethanesulfonate (TMSOTf) catalysis to give compound 3 in excellent yield. Debenzoylation of compound 3 in a MeOH solution of NaOMe afforded saponin 4 without affecting the benzyl ester at C-28 of the aglycone. Selective protection of the 3 I -OH and 4 I -OH groups was successfully carried out using 2,2-dimethoxypropane (Me 2 C(OMe) 2 ) to furnish product 5. Then, the coupling of compound 5 with perbenzoylated rhamnosyl trichloroacetimidate SD-2 under the same glycosylation conditions generated compound 6 in 79% yield. The isopropylidene moiety of compound 6 was removed with para-toluenesulfonic acid (p-TsOH), and the 3 I -OH and 4 I -OH were deprotected to afford compound 7. However, products 8 and 9 were generated (in a ratio of 3:7) when we attempted to selectively protect the 4 I -OH using Bu 2 SnO/BzCl [14]. This interesting phenomenon inspired us to check the structure of the former product 7. Based on the 1 H-NMR spectrum of compound 7, the J 1'-2' value of the arabinose residue was adjusted to 1.3 Hz, which is much smaller than the normal value of the α-arabinosyl conformation (usually no less than 5.0 Hz). Moreover, the chemical shift of the anomeric carbon (δ 109.3) is larger than that of the natural product (δ 104.8). Hence, we realized that the 1 C 4 chair conformer of the arabinose residue was formed when compound 7 was produced. Based on our knowledge, the aglycone and perbenzoylated rhamnose (connected to the 1 I -OH and 2 I -OH of the arabinose residue, respectively) increase the steric bulk, resulting in the chair inversion of arabinose from the naturally stable 4 C 1 conformation to the 1 C 4 conformation. As expected, the 4 I -OH of compound 7 is on the equatorial bond, which is favorable for generating the thermodynamically more stable compound 9 as the major species. Similar regioselectivity in 3-OH and 4-OH of arabinose has been reported previously [10,[15][16][17][18], with the reduced activity of 3-OH rationalized by the presence of a bulky group at 2-OH. The polarities of compounds 8 and 9 were so similar that it was difficult to separate them by silica gel column chromatography. Fortunately, we found that compound 8 could be slowly converted into compound 9 in the presence of boron trifluoride ethyl etherate (BF 3 ·Et 2 O), via a benzoyl group migration [19]. Compound 9 was then reacted with peracetylated glucosyl donor SD-3 under the promotion of BF 3 ·Et 2 O at 0 °C to give trisaccharide compound 10 in 78% yield. Finally, facile removal of the benzyl group through catalytic hydrogenation followed by debenzoylation and deacetylation in one pot yielded the conformer 1. Interestingly, this novel saponin displayed a very robust structure; the 1 C 4 conformation of arabinose did not convert to the 4 C 1 form even under reflux conditions at 150 °C for 24 h.

Scheme 1.
Considering the negative influence of the bulky group at 2 I -OH on the arabinose residue, we changed the perbenzoylated rhamnosyl trichloroacetimidate SD-2 sugar donor to the much smaller SD-4. This time, compound 5 was glycosylated with SD-4 at room temperature to give disaccharide saponin 11 in 73% yield. The isopropylidene group of compound 11 was removed by p-TsOH to generate the key intermediate 12. Based on the 1 H-NMR spectrum of compound 12, the chemical shift (δ 4.73) and coupling constant (J 1'-2' 5.0 Hz) of the anomeric 1 I -H atom suggested that the arabinose ring maintained the normal 4 C 1 conformation. Next, compound 12 was converted into a cyclic ortho ester and was then cleaved in 50% HOAc to furnish product 13 with the 3 I -OH on the equatorial bond. Compound 13 was able to couple with a bulkier sugar donor, such as perbenzoylated glucosyl trichloroacetimidate SD-5, under the promotion of BF 3 ·Et 2 O at a low temperature. Finally, removal of the benzyl group and all the acyl groups generated the target saponin (Scheme 2). In general, the natural Patrinia-glycoside B-II was synthesized in 11 linear steps from oleanolic acid with a 9.4% overall yield. The 1 H-NMR and 13 C-NMR spectra are consistent with those of the natural product. Therefore, Patrinia-glycoside B-II and its conformer were both prepared.

Computational Calculations
To determine the conditions in which the two compounds could interconvert, the Sybyl 6.9 software package (Tripos, st. Louis, MO, USA) was used to analyze structural conformations of compound 1. The structure of compound 1 was built with the Sketcher module, and the energy was minimized by Powell's method using the Tripos force field. Gasteiger-Hückel charges were used for electrostatic field computations. The minimization was terminated after 1000 steps at a maximum value of the gradient at 0.05 kcal/mol −1 ·Å −1 . The structure was then subjected to a simulated annealing, in which it was heated from 200 K to 1,000 K and annealed slowly to 200 K. The results showed that the 1 C 4 and 4 C 1 conformations are stable and unable to interconvert at 1000 K. In addition to the two-chair conformations, some boat conformations with irregular conformation are also stable conformations. Next, a 300 K to 500 K simulated annealing was used to study the structural conformations: besides the original 1 C 4 conformation, the boat with irregular conformations also has low energy, but the 4 C 1 conformation could not be found. This suggests that the arabinosyl conformation of compound 1 might not be able to convert to a 4 C 1 conformation at 500 K.
Further tautomerism studies were carried out using the Amsterdam Density Functional (ADF) program [20]. The geometry optimizations for compound 1, Patrinia-glycoside B-II and the transition state were performed in a vacuum. The total energy values were obtained from the optimization output and are listed in Table 1. From the results, it can be observed that both compound 1 and Patriniaglycoside B-II are energetically stable. Although the energy gap between them is only 0.314 kcal/mol, interconversion would be difficult given the large energy barrier of 185.79 kcal/mol ( Figure 2). Further, the C 1 -C 2 chemical bond needs to be broken and created, as shown by the transition state ( Figure 3). Therefore, both molecular mechanism and quantum chemistry calculations showed that compound 1 and Patrinia-glycoside B-II cannot interconvert under normal conditions.

Biological Evaluation
A preliminary in vitro pharmacology assay was then performed to evaluate the cytotoxicity of these saponins against eight human cancer cell lines using a standard MTT method, in which 5-fluorouracil (5-FU) was chosen as the positive control. As shown in Table 2, Patrinia-glycoside B-II showed effective inhibitory activity against all of those tumor cell lines at micromolar concentrations. Whereas compound 1 did not exhibit significantly cytotoxicity against any tumor cell lines compared to Patrinia-glycoside B-II and 5-FU. This result indicated that the 4 C 1 chair conformation of the arabinose residue in the unique α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl disaccharide moiety is one of the chief positive factors responsible for its cytotoxic activity against tumors.

General
Commercial reagents were used without further purification unless specialized. Solvents were dried and redistilled prior to use in the usual way. Boiling range of petroleum ether was 60-90 °C. Analytical TLC was performed with silica gel HF 254 . Preparation column chromatography was performed with silica gel H. Melting points were detected with a Büchi B-540 Melting Point apparatus. Optical rotations were measured at the sodium D-line at room temperature with a Perkin-Elmer 241MC polarimeter. 1 H and 13 C-NMR spectra were recorded on an Avance AV 600 MHz spectrometer using Me 4 Si as the internal standard. The high resolution mass spectrometry (HRMS) measurements were performed using a high resolution Fourier Transform ion cyclotron resonance (FT-ICR) instrument (Bruker, Germany) operated in electrospray ionization mode. (5) The benzyl ester of oleanolic acid 2 (1.1 g, 2.01 mmol), trichloroacetimidate SD-1 (1.27 g, 2.10 mmol) and powdered 4Å molecular sieve (MS 4Å, 500 mg) were mixed in dry dichloromethane (DCM, 25 mL) and allowed to stir at r.t. for 20 min. A dry DCM solution (2 mL) of TMSOTf (0.20 mL, 0.10 mmol) was then added dropwise. The mixture was stirred for about 1 h until the starting materials were completely consumed to form a new compound. The mixture was neutralized with triethylamine (Et 3 N, 0.2 mL, 1.43 mmol) and filtered through a pad of Celite. The filtrate was concentrated and purified by silica gel column chromatography (8:1, petroleum ether-ethyl acetate (EtOAc)) to afford compound 3 (1.87 g, 97%) as a white foam. To a solution of compound 3 (1.5 g, 1.50 mmol) in DCM-MeOH (1:2, v/v, 40 mL) was added a solution of NaOMe in MeOH (1.00 mol/L, 1.70 mL). The mixture was allowed to stir at r.t. for 2 h and neutralized with Dowex H + resin and then filtered. The filtrate was concentrated and the residue was subjected to a silica gel column chromatography (EtOAc) to give saponin 4 (991 mg, 96%) as a white amorphous solid. To a solution of compound 4 (679 mg, 1.00 mmol) in dry acetone (10 mL) was added Me 2 C(OMe) 2 (0.31 mL, 2.50 mmol) and p-TsOH (17.20 mg, 0.10 mmol). The mixture was stirred for 4 h before Et 3 N (0.20 mL, 1.43 mmol) was added. The solution was concentrated and the residue was purified by silica gel column chromatography (6:1, petroleum ether-EtOAc) to afford a white foam 5 (634 mg, 89%) with R f 0.45 (4:1, petroleum ether-EtOAc).

Computational Methods
All computational calculations were performed using the Sybyl software package and Amsterdam Density Functional (ADF) program. PBE functional and standard DZP basis set with large frozen core are used in geometry optimization and transition state search.

Cell Culture
HeLa human cervical cancer cells; HepG2 human hepatoma cells; HT1080 human fibrosarcoma cells; A549 human lung cancer cells; A375-S2 human melanoma cell; K562 human malignant myeloid cell; HL-60 human promyelocytic leukemia cell and U937 human lymphoma cell were obtained from the American Type Culture Collection (Rockville, MD, USA). The cancer cells were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS); 2% glutamine. Cultures were maintained in a humidified atmosphere incubator at 37 °C in 5% CO 2 .

Cell Viability Assay
The cell viability was evaluated using MTT assay. After diluting to 5 × 10 4 cells mL −1 with the complete medium, 100 μL of the obtained cell suspension was added to each well of 96-well culture plates. The subsequent incubation was permitted at 37 °C, 5% CO 2 atmosphere for 24 h before the cytotoxicity assessments. Tested samples at pre-set concentrations were added to each well, and then the cells were incubated for 48 h. The MTT solution (100 μL, 0.5 mg/mL) was added to each well, and the cells were incubated for another 4 h. The formazan crystals were dissolved in 150 μL of DMSO. Cell viability was assessed by measuring the absorbance at a 492 nm wavelength using an EMax Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).

Conclusions
In summary, the natural cytotoxic triterpenoid saponin Patrinia-glycoside B-II and its novel conformer 1 were synthesized by a stepwise glycosylation strategy. The abnormal 1 C 4 conformation of the arabinose residue was found to occur via conformational fluctuation during preparation of the intermediates. Molecular mechanism and quantum chemistry calculations showed that compound 1 and Patrinia-glycoside B-II cannot interconvert under normal conditions. Preliminary structure-activity relationships studies indicated that the 4 C 1 chair conformation of the arabinose residue in the unique α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl disaccharide moiety is one of the chief positive factors responsible for its cytotoxic activity against tumors.