Design and synthesis of novel oridonin analogues as potent anticancer agents

Abstract To identify anticancer agents with higher potency and lower toxicity, a series of oridonin derivatives with substituted benzene moieties at the C17 position were designed, synthesised, and evaluated for their antiproliferative properties. Most of the derivatives exhibited antiproliferative effects against AGS, MGC803, Bel7402, HCT116, A549, and HeLa cells. Compound 2p (IC50 = 1.05 µM) exhibited the most potent antiproliferative activity against HCT116 cells; it was more potent than oridonin (IC50 = 6.84 µM) and 5-fluorouracil (5-FU) (IC50 = 24.80 µM). The IC50 value of 2p in L02 cells was 6.5-fold higher than that in HCT116 cells. Overall, it exhibited better selective antiproliferative activity and specificity than oridonin and 5-FU. Furthermore, compound 2p arrested HCT116 cells at the G2 phase of the cell cycle and increased the percentage of apoptotic cells to a greater extent than oridonin.


Introduction
Over the last several decades, cancer has been one of the leading causes of death in the world 1 . Cytotoxic agents are the mainstay of anticancer therapy. However, due to their inability to differentiate between normal and cancerous cells, they cause severe adverse effects, leading to poor patient compliance 2 . Therefore, the identification of novel anticancer agents with higher potency and lower toxicity is needed for the treatment of aggressive and refractory cancers. More than 60% of all clinically used anticancer drugs originate from natural sources 3 , highlighting the success of natural products.
Compounds with substituted benzene rings display a broad spectrum of biological activities; therefore, structural modification by adding substituted benzene rings is widely used in medicinal chemistry. It increases receptor binding by promoting hydrophobic interactions and enhances lipid solubility to facilitate drug permeation. In addition, an arylating a,b-unsaturated ketone system is commonly present in several anticancer agents, such as compounds 3-5 [19][20][21] (Figure 1). In a previous experiment, the arylated a,b-unsaturated ketone was added to the structure of costunolide; the resultant compound 3 was found to have better antiproliferative activity against A549 cells (IC 50 ¼ 15.65 mM) than costunolide (IC 50 ¼ 39.80 mM) 19 (Figure 1). This substitution in the D ring was also found to be responsible for the antiproliferative activity of oridonin; the reduction or expansion of this ring is expected to significantly reduce efficacy. Inspired by these reports, we synthesised novel oridonin analogues, containing the arylated a,b-unsaturated ketone system in its D ring, to evaluate whether these analogues display better anticancer activity than oridonin. In this study, we designed, synthesised, and evaluated novel oridonin derivatives (2a-2y) containing a substituted benzene at the C-17 position ( Figure 1) for their in vitro antitumour efficacy.

Materials and methods
Chemistry IR spectra were recorded (in KBr) on IR Prestige-21. 1 H-NMR and 13 C-NMR spectra were measured on an AV-300 (Bruker BioSpin, Switzerland), 13 C-NMR spectra were measured on an AV-500 (Bruker BioSpin, Switzerland), and all chemical shifts were given in ppm relative to tetramethylsilane (TMS). High-resolution mass spectra were measured using a matrix-assisted laser desorption/ ionisation-time of flight (TOF)/TOF mass spectrometer (Bruker Daltonik, Bremen, Germany). The major chemicals were purchased from Aldrich Chemical Corporation. All other chemicals were of analytical grade.
General procedure for the synthesis of compound (2a-2y) To a stirred solution of oridonin (0.19 mmol, 70.00 mg), substituted iodobenzene (0.23 mmol) in N, N-dimethylformamide (5.00 ml), triethylamine (0.58 mmol, 58.18 mg), palladium acetate (0.01 mmol, 2.24 mg) and triphenylphosphine (0.020 mmol, 5.24 mg) was added. The reaction mixture was stirred at 90 0 (oil bath temperature) for 20-24 h monitored by TLC and then concentrated under reduced pressure. The residue was added water (10.0 ml) and extracted with dichloromethane. The organic layer was washed with saturated NaHCO 3 , saturated NaCl and several portions of 15% hydrochloric acid solution, and dried over anhydrous Na 2 SO 4 . Then it can be purified by chromatography on silica eluting with a gradient of methanol/dichloromethane (1:80-1:30) to obtain the compounds (2a-2y) as white solids.

Cell lines and cell culture
Human gastric cancer (AGS), human differentiation of advanced gastric cancer (MGC803), human colorectal cancer (HCT116), human lung cancer (A549), human hepatocellular carcinoma (Bel7402) and human cervical cancer (HeLa) cell lines were obtained from the State Key Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain (Yanbian University) and maintained in Dulbecco's modified Eagle's medium and RPMI Media 1640 (RPMI1640), supplemented with 10% foetal bovine serum 100 IU/ml penicillin, 100 mg/ml streptomycin and 2 mmol/l L-glutamine (Sigma) at 37 7 in a humidified atmosphere containing 5% CO 2 .

Determination of anticancer activity in vitro
All six human cancer cell lines and normal L02 cells were seeded in 96-well plates at the appropriate densities to ensure exponential growth throughout the experimental period (9 Â 10 3 cells per well) and then allowed to adhere for 24 h. The cells were then treated for 48 h with four serial dilutions (100, 50, 10, and 1 mM) of each compound. 5-Fluorouracil (5-FU) was used as the positive control. After incubation for 48 h, 10 ml MTT solution was added to each well to give a final concentration of 2 mg ml À1 . The plates were then incubated for further 4 h. After incubation, the MTT solution was removed and 150 ml DMSO was added to each well to solubilise the formed product. To ensure complete solubilisation, the plates were shaken vigorously for 10 min at room temperature. The optometric density (OD) was read on a microplate reader (ELx800, BioTek, Highland Park, Winooski, VT) at a wavelength of 492 nm and the data were subsequently analysed. The percentage of cell growth inhibition was calculated from the following equation: inhibitory rate (%) ¼

Biological evaluation
MTT assay and structure-activity relationship (SAR) studies Antiproliferative activities of the synthesised compounds were evaluated against six human cancer cell lines (AGS, MGC803, Bel7402, HCT116, A549, and HeLa) and compared with those of oridonin and 5-FU. The MTT assay was performed using a standard protocol 23 .
Based on these preliminary results, we identified the following structure-activity relationships: (1) the introduction of benzene with hydrophobic groups, such as alkoxy, methyl, and halogen, at the C17 position of oridonin, significantly improves antitumour activity with the order of positive potency as alkoxy > methyl > halogen; and (2) the introduction of benzene with hydrophilic groups, such as -OH and -NH 2 , reduces the antitumour activity of oridonin. Based on this interpretation, we suspected that the lipid partition coefficient crucially affects the antitumour activity of oridonin derivatives.

Selective inhibition of cancer cell growth by compounds 2l and 2q
Lack of selective cytotoxicity is the main factor that restricts the dose of most conventional chemotherapeutic agents 24 . We compared the toxicity of compounds 2l and 2p with oridonin or 5-FU on human normal liver cells (L-02). Selectivity indexes between cancer cells and L-02 cells were calculated. As shown in Table 2, compound 2p exhibited 6.5-fold higher selectivity for HCT116 cells than for normal L-02 cells; this selectivity displayed by 2p was significantly higher than that displayed by oridonin. Therefore, compound 2p was further analysed to identify its mechanism of selective cytotoxicity.

Cell cycle regulation by compound 2p
Numerous cytotoxic compounds exert their antiproliferative effect by inducing cell cycle arrest (at a particular cell cycle checkpoint), apoptosis, or both 25 . These mechanisms are considered to be effective anticancer strategies 26 . We used fluorescence-activated cell sorting analysis to explore the mechanism by which compound 2p reduced the viability of HCT116 cells. Compound 2p and oridonin were selected and tested against HCT116 cell lines at a concentration of 5 mM. As shown in Figure 2, compound 2p significantly increased the percentage of G2 cell population from 19.35 to 41.46% after 48 h of incubation, whereas oridonin increased it to 37.80%. This finding suggests that compound 2p induces cell cycle arrest at the G2 phase.

Apoptotic effects of compound 2p
Because compound 2p arrested the cells in the G2 phase, we investigated whether it also causes apoptosis. HCT116 cells were treated with the vehicle, compound 2p (5 mM), or oridonin (5 mM) for 48 h, and then stained with annexin V-FITC and PI. As shown in Figure 3, the percentage of total apoptotic cells (right quadrants, Q 2 þ Q 3 ) increased to 60.94% after treatment with compound 2p, Figure 3. Apoptosis induction in HCT116 cancer cell after treatment with 2p (5.00 lM), oridonin (5.00 lM) and no treatment (Ctrl) as a reference control for 48 h.
whereas it was 24.84% after oridonin treatment; vehicle treatment induced apoptosis in 6.20% of the cells. This result indicated that compound 2p is a more potent inducer of apoptosis than oridonin.

Conclusion
A series of oridonin derivatives with substituted benzene analogues at the C17 position were designed, synthesised, and evaluated for their antiproliferative properties against six human cancer cell lines (AGS, MGC803, Bel7402, HCT116, A549, and HeLa), and the noncancerous human L02 cells. Most of the synthesised oridonin derivatives displayed significant antiproliferative effects in these cancer cell lines. SAR analysis indicated that the alkoxyphenyl ring at the C17 position of oridonin effectively improves its antitumour efficacy. Compound 2p possessed the highest antiproliferative activity against HCT116 cells; it was 6.8-fold more potent than oridonin. The IC 50 value of 2p in L02 cells was 6.5-fold higher than that in HCT116 cells, indicating that it exhibits selective antiproliferative effects.
In addition, cell cycle analysis revealed that compound 2p arrested HCT116 cells at the G2 phase. It increased the percentage of apoptotic cells to a greater extent than oridonin. Therefore, compound 2p could serve as a promising lead candidate for further studies.