Synthesis and Tumor Cytotoxicity of Novel Amide Derivatives of β-Hederin

Thirteen novel triterpenoid saponins, designed as amide derivatives of the natural cytotoxic saponin β-hederin, were synthesized by a stepwise glycosylation strategy. The in vitro cytotoxic activity of these compounds was evaluated against five different tumor cell lines. Most of the evaluated compounds showed effective inhibitory activity against at least one tumor cell line at micromolar concentrations. The preliminary structure-activity relationships (SAR) indicate that mide derivatization at C-28 resulted in highly cytotoxic derivatives on specific tumor cell lines, and also resulted in an increase in the antitumor selectivity of β-hederin.


Introduction
Oleanolic acid (OA, see Figure 1) is a pentacyclic triterpenoid widely distributed in Nature and possessing various important bioactivities, such as antitumor, anti-HIV, hepatoprotection, and anti-
Recently, some researchers have explored the structure-activity relationships of other pentacyclic triterpenic compounds, such as ursolic acid [9,10] and betulonic acid [11]. Those studies led to similar conclusions that the derivatives with a substitution of amino groups at C-28 often showed stronger cytotoxic antitumor activity. Therefore, we were inspired to investigate the possibility of similar properties in the medicinal chemistry of triterpenoid saponins. The present work describes the initial study of the synthesis of 13 novel amide derivatives of β-hederin and the evaluation of their cytotoxic activity against tumors.

Synthesis
As far as β-hederin is concerned, the hydroxyl group at C3 is connected with a disaccharide moiety to form the so-called OA 3-glycosides. Therefore, only if the sugar substructure was well protected, can the free carboxyl group at C-28 be modified by various acylations. Based on our previous work [7,8,12], we developed a facile method to prepare the amide derivatives of β-hederin using OA, Larabinose, L-rhamnose and some commercially available amines as the starting materials.
The compounds were synthesized as depicted in Scheme 1. To protect the carboxyl group of OA, benzyl ester 1 was prepared by the combination of OA, BnBr and K 2 CO 3 in THF-H 2 O with Bu 4 NI as a phase transfer catalyst. Compound 1 was glycosylated with perbenzoylated arabinosyl trichloroacetimidate SD-1 [13] under the promotion of TMSOTf to produce an excellent yield of compound 2. Debenzoylation of compound 2 in the MeOH solution of NaOMe yielded saponin 3 without affecting the benzyl ester at C-28. Selective protection of 3-OH and 4-OH of the arabinose residue was successfully accomplished using 2,2-dimethoxypropane to furnish compound 4, which was combined with perbenzoylated rhamnosyl trichloroacetimidate SD-2 [14] under the same glycosylation conditions to generate compound 5 with a 79% yield. When the isopropylidene moiety was removed to prepare the intermediate 6, the 1 C 4 conformation of the arabinose residue resulted produced via conformational inversion. Based on the 1 H-NMR spectrum of compound 6, the J 1'-2' value of the arabinose residue was changed to 1.3, much smaller than the normal value of the α-arabinosyl conformation (usually not less than 5.0). Moreover, the chemical shift (δ) of anomic C atom (109.3) is larger than that of the natural product (104.8). From the point of view of the chemical structure, the aglycone and perbenzoylated rhamnose connected with 1-OH and 2-OH of the arabinose residue, respectively, increased steric hindrance to such an extent that a chair inversion of arabinose resulted. Next, the two free hydroxyl groups were shielded by acetylation in pyridine solution to furnish compound 7, whose benzyl group was easily removed through catalytic hydrogenation to produce compound 8 without affecting the double bond between C-12 and C-13 of OA. Compound 8 was treated with oxalyl chloride to yield compound 9, which then reacted with the appropriate amino compounds in the presence of Et 3 N to generate corresponding compounds 10. Last, rapid and complete removal of all acyl groups in basic solution yielded the final products TS1-TS13. Fortunately, the arabinose ring returned to the normal 4 C 1 conformation after the final deprotection, which was confirmed by the 1 H-NMR data.

Biological evaluation
The preliminary in vitro biological evaluation was performed by a standard MTT assay to investigate the cytotoxicity of these saponins against five tumor cell lines: HeLa (cervical), MCF-7 (breast), HL-60 (leukemia), HT1080 (fibrosarcoma), and HepG2 (liver). The synthetic β-hederin was used as a reference compound. As shown in Table 1, although most of the evaluated compounds were found to be less active than β-hederin, they showed effective inhibitory activity against at least one tumor cell line at micromolar concentrations. It is worth emphasizing that compounds with a substitution of piperazine or methylpiperazine (TS8 and TS9) displayed more potent activity than βhederin. However, none of the evaluated compounds showed any toxicity towards the HT1080 or HepG2 cell lines within the investigated concentration range. The SAR indicated that the conversion of β-hederin into an amide yielded a moderately active derivative. The modified saponins with the aliphatic amine substructure seemed to display stronger activity than those bearing aromatic amines. Generally, the amide derivatization at C-28 resulted in highly cytotoxic derivatives on specific tumor cell lines, which means the antitumor selectivity of β-hederin was increased.

General
Commercial reagents were used without further purification unless otherwise stated. Solvents were dried and redistilled prior to use in the usual way. Analytical TLC was performed on silica gel HF 254 plates. Preparative column chromatography (CC) was performed with silica gel H. Melting points were measured with a Büchi B-540 Melting Point apparatus. Optical rotations were measured at the sodium D-line at room temperature (RT) with a Perkin-Elmer 241MC polarimeter. 1 H-and 13 C-NMR spectra were recorded on a Bruker Avance AV600 MHz spectrometer using Me 4 Si as the internal standard. HRMS spectra were recorded on a high resolution ESI-FTICR mass spectrometer.

General procedure for the synthesis of oleanolic amide 3-O-α-L-rhamnopyranosyl-(1→2)-α-Larabinopyranosides
Oxalyl chloride (0.37 mL, 4.40 mmol) was added dropwise to a solution of compound 8 (500 mg, 0.44 mmol) in redistilled DCM (10 mL). The mixture was stirred at RT for 6 h. Then, the solvents were co-evaporated with toluene for complete removal of the excess oxalyl chloride to yield compound 9 as a yellow foam. A solution of compound 9 in redistilled DCM (10 mL) was treated with the corresponding amine (0.80 mmol) and 5 drops of Et 3 N and stirred for 4 h under nitrogen. The mixture was washed with satd aq NaCl (10 mL × 3), dried over MgSO 4 , and concentrated in vacuo to yield the crude product 10 which was dissolved in dry 1:2 DCM-MeOH (12 mL) and treated with a fresh solution of NaOMe in MeOH (1.0 mol/L, 1.00 mL). The solution was stirred at RT for 2 h, neutralized with Dowex H + resin to pH 7, and filtered. The filtrate was concentrated and subjected to CC (20:10:1 CHCl 3 -MeOH-H 2 O) to give the target saponins in yields of 67%-85% (for the 3 steps).

Conclusions
In summary, 13 novel amide derivatives of β-hederin were synthesized and evaluated in vitro for tumor cytotoxicity. The results from the antitumor screening showed that compounds TS8 and TS9 possessed potent antitumor activity against HeLa and MCF-7 cell lines. The preliminary structureactivity relationships indicated that the conversion of the C-28 carboxylic acid group of β-hederin into an amide derivative often resulted in a loss of broad spectrum antitumor activity, but also resulted in an increase in the antitumor selectivity. To provide more clarity about the structure-activity relationship, further studies on additional systematic structural variations are underway.