Synthesis and in vitro antitumour activities of lupeol derivatives

Abstract Nine lupeol derivatives were synthesised and assayed in vitro for their antitumour activities against three human tumour cells lines, A549, LAC and HepG2. Of lupeol derivaties, six were new compounds, and five compounds against A549 cells, four compounds against HepG2 cells and three compounds against LAC cells were effective in reducing viability, and the most promising compounds 5, 6 and 9 exhibited high activities against lung and liver cancer cells, even higher activities than those of adriamycin.

In the present study, to systematically evaluate the antitumour effect of the 3β-hydroxy group of 1, nine lupeol derivatives (2-10, Figure 1) were synthesised. Of lupeol derivatives, six (2-6 and 9) were new compounds. All the synthesised compounds were investigated in vitro for their antitumour activities against A549, LAC and HepG2 cells.

Chemistry
Esterification of various acid anhydrides or acids with various alcohols was carried out using N, N′-dicyclohexylcarbodiimide (DCC) as condensing agent and 4-dimethylaminopyridine (DMAP) as catalyst under mild conditions (Yu et al. 2006;Li et al. 2013). Here, 1 reacted with chloroacetic acid in anhydrous dichloromethane to afford new compound 2 in high yield at ambient temperature in the presence of DCC as dehydrating agent and DMAP as catalyst ( Figure 2), 2 reacted with dimethylamine in 33% aqueous solution or diethylamine in dichloromethane to give new compound 3 or 4, respectively in good yields at room temperature ( Figure 2). Compound 1 reacted with 3, 4-dimethoxycinnamic acid or 3, 4, 5-trimethoxycinnamic acid in anhydrous dichloromethane to give new compound 5 or 6, respectively with mild yields in the presence of DCC and DMAP at room temperature and the above reactions required strictly anhydrous condition (Figure 3).

In vitro antitumour activity
All the synthesised compounds were tested on three human cancer cells lines, A549, LAC and HepG2, according to the method of MTT assay. Adriamycin had been proven one of the most effective single antitumour agents with an impressively broad spectrum of activity encompassing many of the solid tumours. It was widely used as a   Figure 3. synthesis of compounds 5 and 6. (a). 3, 4-dimethoxycinnamic acid or 3, 4, 5-trimethoxycinnamic acid, dcc, dMaP, ch 2 cl 2 , r.t., 24-48 h, 50.5%-65.3%.
was expressed as the concentration of compound that achieved 50% inhibition (IC 50 ) to tumour cells. Data were expressed as mean ± SD (standard deviation) of three different experiments. Statistical analysis was carried out by SPSS 12.0 software. The results were summarised in Table 1. If IC 50 value was less than 50 μM, the sample was evaluated to have antitumour activity. Based on the IC 50 values, adriamycin had good inhibitory abilities against A549 and LAC growth, but it had no antitumour activity against HepG2 cells. Lupeol (1) was not capable of inhibiting liver cancer cells growth and showed less resistant activity for lung cancer than adriamycin. Among lupeol derivatives, lupeol chloroacetate (2) was stronger resistant to A549 growth than lupeol and its antitumour activity against HepG2 cells exceeded adriamycin. When chlorine of 2 was displaced by dimethylamine or diethylamine to form compound 3 or 4, respectively, the antitumour activities of the latter compounds decreased without even activities. Lupeol cinnamate analogues 5 and 6 exhibited good antitumour activities, even exceeded adriamycin. In surprise, lupenone hydrazone (9) as an imine derivative of lupeol possessed good antitumour activities, but lupenone (7), lupenone oxime (8) and lupenone 2, 4-dinitrophenylhydrazone (10) did not possess antitumour activities against lung and liver cancers.

Conclusions
A series of lupeol derivatives were synthesised and evaluated in vitro for their antitumour activities against A549, LAC and HepG2 cells. Based on the above study for the antitumour effect of 3β-hydroxy group of lupeol, the following conclusion can be drawn: (1) The 3β-hydroxyl group of lupeol is esterified with cinnamoyl group, the antitumour activities of the derivatives are enhanced. (2) The 3β-hydroxyl group is converted to carbonyl group, the antitumour activities of the derivatives thereof disappear. (3) After the imidisation of carbonyl  group, only imine derivative 9 possesses good antitumour activities. (4) Of lupeol derivatives, the most promising compounds 5, 6 and 9 present high activities against lung and liver cancer cells, even higher activities than those of adriamycin.

Supplementary material
All experimental sections relating to this article are available online, alongside Figures S1-S9.

Disclosure statement
No potential conflict of interest was reported by the authors.