Synthesis and Anti-Tumor Activity of Oleanolic Acid Derivatives

Zhenyu Kuai,Lei Li,Shenwen Zhan,Chunlin Li,Yuwei Zhao,Chen,Hongrong,Yanqiu Meng* Department of Pharmaceutical Engineering, Shenyang University of Chemical Technology, Shenyang 110142, China Abstract Using the techniques of computer-aided drug design, the docking of Survivin and known active small molecules was simulated and then the key amino acid residue fragment of the target protein was analyzed. It led to the discovery of active groups capable of binding to the critical sites. Through the use of the natural product, Oleanolic Acid, as a lead compound, the introduction of the active groups onto the Aring, and the modification of the carboxyl group at the C-28 position using esterification or amidation, twenty new Oleanolic acid derivatives had been designed and synthesized.A549 and SGC-7901 cells were used to screen the antitumor activity in vitro through the standard MTT method. The compounds, II3, III5 and IV4, exhibited more potent cytotoxicity than positive drugs.


Introduction
Oleanolic acid (OA) is a pentacyclic triterpenoid compound widely presenting in plants in the form of dissociation or in the combination with sugars [1] .Its effective ingredients were mainly extracted from Prunella vulgaris, Honeysuckle, Olive, Forsythia, Aloe Vera and Ligustrum lucidum and other plants [2] .Oleanolic acid has a multitude of important pharmacological functions, such as anti-virus [3][4][5] , anti-diabetic and hypoglycemic, anti-HBV, and anti-tumor.Our research group has carried out a research on the anti-tumor activity of the pentacyclic triterpenoid analogues since 2000, with main focus on the extensive structural modification of inactylactic acid, ursolic acid, asiatic acid, and on the evaluation of vitro antitumor activity [6][7][8][9] .In addition, some progress has been made in guiding the structural transformation of oleanolic acid, ursolic acid and asiatic acid, using computer-aided design to simulate the combination of analogue analyte with protein target [10][11] .
Survivin, a member of the family of apoptotic proteins, plays a key role in cell division and inhibition of apoptosis and is considered an important target for anticancer therapy [12] .Survivin protein can inhibit the apoptosis and regulate the mitotic function of cells by inhibiting various endogenous and exogenous apoptosis-related factors such as Caspase3, Caspase7 and P53.Survivin is overexpressed in a variety of tumor tissues and hardly expressed in the normal cells.It is also closely related to poor prognosis and drug resistance.However,Inhibiting Survivin as a new strategy for the treatment of cancer and to overcome the drug resistance of tumor cells [13] .Oleanolic acid (OA) and 5-fluorouracil(5-FU) in combination inhibit Survivin which led to the significant increase in human pancreatic cell (Panc-28) apoptosis [14] .OA can improve the proapoptotic protein Bax, change the balance of Bcl-2/Bax, and significantly reduce the expression of anti-tumor apoptotic protein, Survivin [15] .YM155 has a naphthoquinolidazole structure that inhibits the transformation of Survivin gene through inhibiting the promoter activity [16][17] , Their research level nearly reaches the clinical stage I, II study.SC144, with the structure of a quinolide hydrazine, can inhibit the IL-6/gp130/Stat3 signal axis, bind gp130, induce gp130 phosphorylation and deglycosylation, prevent Stat3 phosphorylation and nuclear migration, and ultimately inhibit the downstream expression of Survivin [18] .It is the first oral active inhibitor of gp130.By combining the three-dimensional crystal structure (PDB: 3UIH) of Survivin protein in the PDB database, analyzing the interaction between known Survivin small molecule inhibitor and target enzyme using molecular simulation docking method, and analyzing the key amino acid residue fragment, we were able to determine the active groups that can bind to the critical site (Figure 1).We further introduced the active group fragments in the partial inhibitor structure were introduced into the tricyclic triterpenoid matrix.The main transformation was concentrated in the parallel nitrogencontaining heterocyclic ring of the A-ring (Figure 2).As a result, twenty new oleanolic acid analogs were designed, synthesized and tested for in vitro activity.In this essay, we combined the structural modification of pentacyclic triterpene compounds suggested by Meng's research team with the antitumor compounds which have entered the clinical study (Figure 1) and the computer simulation to analyze the structure of the key active groups (Figure 2).We introduced these groups to A ring of oleanolic acid.In the computer simulation of molecular docking, the combination mode of II 3 、Ⅲ 5 and Ⅳ 4 with Survivin targeting protein has displayed a strong combining ability.The antitumor activities have been tested in vitro by MTT method.The results showed that compounds Ⅲ 5 and Ⅳ 4 have more outstanding antitumor activities on A549 and SGC-7901 cells than gefitinib.
Scheme1 Synthetic routes of target compounds.

Molecular docking
Oleanolic acid derivatives I-Ⅳ have the combined affinity with Survivin protein (PDB code: 3UIH).Molegro Virtual Docker (MVD) is able to predict how protein interacts with macromolecular ligands and its interaction energy.The connection between 3UIH and molecules was assessed by MVD.Scores are expressed as binding free energy (Escore kcal/mol).Using MVD 6.0, the binding scores showed that compound II 3 : -76.432, compound III 5 : -86.021, compound Ⅳ 4 :-78.851, in comparison with protein's small molecule ligands (-75.542).The lower the energy score is, the stronger the binding affinity becomes.Through intuiting analysis of the combination of compounds and targets by the Discovery Studio 4.0, we found that some compounds interacted with Survivin protein and closely connected with the surrounding amino acids.Based on Figure 3-5, compounds II 3 , III 5 and Ⅳ 4 were firmly fixed in the hydrophobic pocket and they interacted with key amino acids through hydrophobic bond and H-bond.Key energy could not be measured in the process of the molecular docking.MolDock score of all compounds are shown in Table 2.In this step, we cooperated with Shenyang Pharmaceutical University.

Conclusions
In summary, four new series of OA derivatives were designed and synthesized.Their antitumor activities on A549 and SGC-7901 cell lines were evaluated.All the tested compounds showed some anticancer activity against A549 and SGC-7901 cell lines.Molecular docking studies demonstrated that twenty OA derivatives were obtained through structural optimization of the lead compound(OA) and they docked into Survivin protein-tyrosine kinase.Molegro Virtual Docker (MVD) was able to tell if there was good binding affinity of the synthesized all compounds with Survivin protein.Specifically, compounds II 3 、III 5 and Ⅳ 4 exhibited outstanding inhibitory activities on A549 cells (IC50 =8.31µM, IC50 =7.82µM, IC50 =5.31µM) and SGC-7901 cells (IC50=6.22µM,IC50=4.27µM,IC50 =7.92µM).As the ester chain of OA increases, the anticancer activity increases.The structure-activity relationships of newly synthesized compounds have shown in Figure 6.Our data indicated that proper structural modification at A ring and C-28 position of OA was necessary to enhance the anticancer activity of Oleanolic acid.

General experimental procedures
The melting points were determined on a Büchi B-540 melting point apparatus produced by Broker Corporation (Flawil, Switzerland) and are uncorrected. 1HNMR spectra were recorded on Bruker a ARX-300 MHz spectrometers from Bruker Corporation (Ettlingen, Germany) and the solvent is CDCl3, using trimethylsilane as an internal standard.ESI-MS were measured on a Thermo-Finnigan LCQ equipment from Thermo Finnigan (San Francisco, CA, USA).Thin-layer chromatography (TLC) ware carried out with GF 254, column chromatograph with silica gel (200-300 mesh) obtained from Qing-dao Marine Chemical Factory (Qingdao, China).The reagents were all of analytical grade or chemically pure.

Preparation of the compounds 4.2.1. 3-Oxo-olean-12-ene-28-oic acid (OA-1)
OA (0.500g) was dissolved in 50 mL of acetone,and allowed to react with the newly prepared Jones' reagent (0.64mL) under ice bath.The end of the reaction was detected by TLC. 15 mL of isopropanol was added to quench its oxidizing property and stirred at room temperature for 30 min.A small amount of saturated sodium chloride solution and moderate ethyl acetate were added to the reaction mixture to extract for three times.The organic layer was dried with anhydrous magnesium sulfate for 4 h .The crude product was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate =12/1(V/V)).Then, the solvent were removed to give a powder OA-1, with a yield of 98.0%.m.p. 200.4~202.1 o C.

Cell Proliferative Assay
The antiproliferative activities of the title compounds were evaluated in vitro using the MTT method against A549 and SGC-7901 cell lines, with gefitinib and Adriamycin as the positive control.The negative control contains cells, culture medium, MTT and DMSO.The two tumor cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS).Tumor cells were detached by trypsinisation, seeded at 1.0~2.0× 10 4 cells each well in 96-well culture plates and incubated in 5% CO2 at 37 o C overnight, the test compounds was added at different indicated concentration of 10 -5 , 10 -6 , 10 -7 , and 10 -8 mol/L for 72 h.Then MTT solution(100 µL per well) was added and incubated at 37 o C for 4 h.The MTT-formazan formed by metabolically viable cells was dissolved in 150 µL DMSO each well, and monitored by a microplate reader at dual-wavelength of 490 nm, IC50 was defined as the drug concentrations that inhibited the cell number to 50% after 72 h.Each experiment was repeated at least three times and the results averaged.

Figure 3 .
Figure 3. Binding of compound II 3 to the active site of Survivin, it exhibited 1 H-bond with ASP71, the hydrogen bonds formed colored in green.

Figure 4 .
Figure 4. Binding of compound III 5 to the active site of Survivin, it exhibited 2 H-bonds with LYS62 and ASP71, the hydrogen bonds formed colored in green.

Figure 5 .
Figure 5. Binding of compound Ⅳ 4 to the active site of Survivin, it exhibited 1 H-bonds with GLU65, the hydrogen bonds formed colored in green.

Figure 6 .
Figure 6.Summarized structure-activity relationships of novel compounds with regard to cancer inhibition.

Table 1 .
Antitumor activity of the target compounds on A549 and SGC-7901 cell lines.

Table 2 .
Comparison of energy scores for different compounds with Survivin protein.