Synthesis and in vitro antiproliferative activities of lupanol derivatives towards human esophageal squamous carcinoma cells

Abstract A series of lupanol derivatives were synthesized and evaluated in vitro for their inhibitory activities against three human esophageal squamous carcinoma cells lines, Eca-109, TE-1 and EC-9706. Among lupanol derivatives, seven were new compounds, and lupanol cinnamate analogues 5 and 6, hydrazone analogues 9 and 10 presented high activities towards all the tested tumour cells, even higher activities than those of doxorubicine. Graphical Abstract


Introduction
Lupanol (1) (Figure 1) is a natural pentacyclic triterpenoid with biological and pharmacological activities (Salem et al. 2016;Wang et al. 2020), its natural contents are relatively small and there are fewer reports in the literatures. Recent studies have shown that lupanol dicarboxylic acid monoester derivatives had good anti-HIV activities because they were capable of inhibiting HIV-1 replication in lymphocytes (Yu et al. 2006) and had good anticancer activities against lung and liver cancers (Li et al. 2013). Therefore, the study of lupanol derivatives has been paid more attention.

Chemistry
Lupanol (1) reacted with excess chloroacetyl chloride in anhydrous dichloromethane to obtain new compound 2 in high yield at room temperature (Figure 2), and the latter reacted with dimethylamine in 33% aqueous solution or diethylamine in dichloromethane to afford new compound 3 or 4, respectively with mild yields at ambient temperature ( Figure 2).
Esterification of various acid anhydrides or acids with various alcohols was performed in good yields 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 3, 4-dimethoxycinnamic acid in anhydrous dichloromethane to obtain new compound 5 with mild yield at room temperature in the presence of DCC as condensing agent and DMAP as catalyst and the above reaction required strictly anhydrous condition ( Figure 3).
3, 4, 5-Trimethoxycinnamic acid was refluxed with excess thionyl chloride to lead to the formation of the corresponding acyl chloride, and the latter reacted with 1 in anhydrous dichloromethane to give new compound 6 in good yield at room temperature ( Figure 3).
Compound 7 is a known natural product, and its oxime (8) had been reported in the literatures ( Kumar et al. 2008;Kuriata-Adamusiak et al. 2012;Banerjee et al. 2014 ). Here, 7 and 8 were prepared according to the literature's method (Kumar et al. 2008). Compound 7 was refluxed with hydrazine hydrate in 80% aqueous solution or   2, 4-dinitrophenylhydrazine in ethanol to afford new compound 9 or 10, respectively with mild yields (Figure 4).

In vitro inhibitory activities on proliferation of human esophageal squamoous carcinoma cells
Using MTT assay, the in vitro anticancer activities of all the synthesized lupanol derivatives were evaluated against three human esophageal squamous carcinoma cells lines, Eca-109, TE-1 and EC-9706. Here, doxorubicine was used as a positive control. It had been proven one of the most effective single anticancer agents with an impressively broad spectrum of activity encompassing many of the solid tumors. It was widely used as a positive control for in vitro screening of anticancer compounds in the literatures ( Cortes-Funes et al. 2007;Hao et al. 2011;Li et al. 2013 ). According to evaluation criteria of in vitro anticancer activity, the anticancer activity of each compound was expressed as IC 50 value to cancer cells. The IC 50 value represented the concentration of a compound that was required for 50% inhibition towards cancer cells growth in vitro. Here, all data were presented as mean ± SD (standard deviation) of three different experiments. Statistical analysis was performed using SPSS 12.0 software. The results were recorded in Table S1.
If IC 50 value was less than 50 mM, the sample was evaluated to have anticancer activity. The smaller IC 50 value was, the better the anticancer activity of the tested sample was. According to the IC 50 values, doxorubicine had good inhibitory abilities against all the tested human esophageal squamous carcinoma cells growth. Lupanol (1), its chloroacetate (2) and glycine ester derivatives 3 and 4 had no anticancer activities against all the tested cancer cells. However, the IC 50 values of lupanol cinnamate analogues 5 and 6 were less than 8 mM towards Eca-109 cells and TE-1 cells, and less than 33 mM against EC-9706 cells. Their anticancer activities surpassed those of doxorubicine. The 3b-hydroxyl group of 1 was oxidized to form carbonyl group derivative 7, the latter reacted with hydroxylamine to give lupanone oxime 8, and they did not possess anticancer activities. However, hydrazone analogues 9 and 10 presented significant inhibitory effects against all the tested three human esophageal squamous carcinoma cells lines, even higher activities than those of doxorubicine.

Conclusions
Nine lupanol derivatives were synthesized and evaluated in vitro for their antiproliferative activities against Eca-109, TE-1 and EC-9706 cells. According to the above study for the anticancer effect of 3b-hydroxy group of lupanol, the following conclusions can be obtained: (1) The 3b-hydroxyl group of lupanol is esterified with 3, 4-dimethoxycinnamoyl or 3, 4, 5-trimethoxycinnamoyl group, the anticancer activities of the derivatives are promoted. (2) The 3b-hydroxyl group is converted to carbonyl group, the anticancer activities of the derivatives disappear. (3) After the imidization of carbonyl group, only hydrazone analogues 9 and 10 exhibit good inhibitory abilities against Eca-109, TE-1 and EC-9706 cells proliferation. (4) Among lupanol derivatives, the most promising compounds 5, 6, 9 and 10 exhibit significant antiproliferative activities against all the tested human esophageal squamous carcinoma cells, even higher activities than those of doxorubicine.

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