Synthesis of the vitamin E amino acid esters with an enhanced anticancer activity and in silico screening for new antineoplastic drugs

Abstract

Tocopherols and tocotrienols belong to the family of vitamin E (VE) with the well-known antioxidant properties. For certain α-tocopherol and γ-tocotrienol derivatives used as the lead compounds in this study, antitumor activities against various cancer cell types have been reported. In the course of the last decade, structural analogs of VE (esters, ethers and amides) with an enhanced antiproliferative and proapoptotic activity against various cancer cells were synthesized. Within the framework of this study, seven amino acid esters of α-tocopherol (4a–d) and γ-tocotrienol (6a–c) were prepared using the EDC/DMAP reaction conditions and their ability to inhibit proliferation of the MCF-7 and MDA-MB-231 breast cancer cells and the A549 lung cancer cells was evaluated. Compound 6a showed an activity against all three cell lines (IC₅₀: 20.6 μM, 28.6 μM and 19 μM for the MCF-7, MDA-MB-231 and A549 cells, respectively), while compound 4a inhibited proliferation of the MCF-7 (IC₅₀ = 8.6 μM) and A549 cells (IC₅₀ = 8.6 μM). Ester 4d exerted strong antiproliferative activity against the estrogen-unresponsive, multi-drug resistant MDA-MB-231 breast cancer cell line, with IC₅₀ value of 9.2 μM. Compared with the strong activity of compounds 4a, 4d and 6a, commercial α-tocopheryl succinate and γ-tocotrienol showed only a limited activity against all three cell lines, with IC₅₀ values > 50 μM. Investigation of the cell cycle phase distribution and the cell death induction confirmed an apoptosis of the MDA-MB-231 cells treated with 4d, as well as a synergistic effect of 4d with the known anticancer drug doxorubicin. This result suggests a possibility of a combined therapy of breast cancer in order to improve the therapeutic response and to lower the toxicity associated with a high dose of doxorubicin. The stability study of 4d in human plasma showed that ca. 83% initial concentration of this compound remains in plasma in the course of six hours incubation. The ligand based virtual screening of the ChEMBL database identified new compounds with a potential antiproliferative activity on MCF-7 and on multi-drug resistant MDA-MB 231 breast cancer cells.

Introduction

Vitamin E (VE) is a generic term that represents two families of compounds, i.e., tocopherols (T) and tocotrienols (T₃). Both families share similar chemical structure characterized by a saturated (T) or unsaturated (T₃) phytyl chain attached to the chromane ring. Moreover, each family has four isomers (α, β, γ, δ) that differ in the number and positions of methyl groups in the chromanol ring (Eitenmiller and Lee, 2004). Although the VE family members are the best known for their antioxidant activity (Jiang, 2014), recent studies have shown that they exhibit many antioxidation-independent effects such, as inhibition of protein kinase C, 5-lipoxygenase and phospholipase A2, activation of protein phosphatase 2A and diacylglycerol kinase, inhibition of cell proliferation, platelet aggregation and monocyte adhesion (Zingg and Azzi, 2014). Nowadays, special attention is paid to the antiproliferative effect of VE. Tocopherols and tocotrienols exert antitumor activity on various types of cancer cells, at the same time being selective to normal cells (Moya-Camarena and Jiang, 2012, Ling et al., 2012). Direct comparison between the two VE subclasses showed that tocotrienols are generally more potent than tocopherols in reducing proliferation of tumor cells, with δ-tocotrienol being the most potent and α-tocopherol the least potent isomer. (McIntyre et al., 2000, Guan et al., 2012, Campbell et al., 2006).

Although an exact mechanism by which the VE family members inhibit carcinogenesis remains unknown, several models are proposed, including apoptosis via the cleavages of poly (ADP-ribose) polymerase (PARP) (Park et al., 2010, Patacsil et al., 2012), or inhibition of the nuclear factor kappa-light-chain-enhancer of the activated B cells (NF-κB) signaling pathway (Sun et al., 2014). Modulation of angiogenesis is now recognized as a strategy for preventing various angiogenesis-mediated disorders, including solid tumors (Zhao and Adjei, 2015). Recent studies confirmed that the palm tocotrienols exhibit anti-angiogenic properties through a decreased expression of vascular endothelial growth factor in human umbilical vein endothelial cells (Selvaduray et al., 2012), human hepatocellular carcinoma cells (Siveen et al., 2014) and murine mammary cancer cells (Selvaduray et al., 2010). Synthetic analogs of tocopherols showed more potent effects than the natural forms in inducing dephosphorylation of the protein kinase B (Akt) and thereby in inhibiting the Akt-mediated signals that promote cell metabolism, proliferation, and motility (Huang et al., 2013). The experimental findings showed that the combined treatment with γ-T3 and the antihyperlipidemic drugs such, as erlotinib and gefitinib, resulted in a synergistic inhibition in vitro of highly malignant + SA mouse mammary epithelial cells, due to a down-regulation of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase activity, which is elevated in cancer cells (Goldstein et al., 2006, Sylvester, 2012). Furthermore, a combined treatment with a low dose of γ-T₃ and an anti-inflammatory drug celecoxib can greatly improve a therapeutic response in the treatment of breast cancer through the inhibition of the cyclooxygenase-2 (COX-2) activity (Shirode and Sylvester, 2011).

In the course of the last decade, the VE derivatives with a modified hydroxyl group have been synthesized and tested for their anticancer activity. These redox-silent analogs that lost their antioxidative potency showed an improved antiproliferative activity and stability relative to the parent compounds (Behery et al., 2010, Birringer et al., 2003, Djuric et al., 1997, Elnagar et al., 2010). The most prominent VE analog, α-tocopheryl succinate (α-TOS), was shown to exhibit anticancer properties in several cancer models, while retaining low toxicity to healthy cells (Huang et al., 2009, Dong et al., 2008). Selectivity of the VE analogs to cancer cells can be explained by higher esterase activity in normal cells that cleave α-TOS and similar agents, to produce the nonapoptogenic α-T (Neuzil et al., 2004). Due to their lesser susceptibility to oxidation, the succinate and acetate ester of α-tocopherol became commercially available for use in the supplements and cosmetics (Zingg, 2007). In order to enhance stability of the VE derivatives in vivo, the ester bond was replaced by an amide bond or by an even more resistant ether bond. The amide and ether VE derivatives showed an additional enhancement of the proapoptotic activity on the MDA-MB-453 breast cancer, Jurkat T lymphoma, the U937 leukemic and the Meso-2 malignant mesothelioma cells (Tomic-Vatic et al., 2005, Nishikawa et al., 2003).

Derivatization of the phenolic hydroxyl of α-T as an amino acid ester conjugate (Arya et al., 1998) introduces the NH₂ group that is protonated in an acidic medium of the cancer tissue (Montcourrier et al., 1994). The protonated amino group together with the long alkyl chain of α-T acts as a cationic detergent and interacts with the lysosomal and mitochondrial membranes in cancer cells, causing an increased membrane permeability and destabilizing lysosomes (Neuzil et al., 2001). Contrary to α-T and α-TOS, the α-tocopheryl-lysine ester exerts an antiproliferative activity against the human MCF-7 breast cancer cell line (Arya et al., 1998).

Within the framework of this study, we synthesized four amino acid esters of α-T (1) with lysine (4a), proline (4b), glutamine (4c), and asparagine (4d), and three amino acid esters of γ-T₃ (2) with lysine (6a), proline (6b) and glutamine (6c). Based on the results from our previous QSAR studies (Nikolic and Agbaba, 2009, Gagic et al., 2016), esterification of the phenol group at position C6 of the chromane ring with at least one hydrogen bond donor and/or hydrogen bond acceptor present in the ester chain, and an increased polarity of the molecule, exert positive effect on the antiproliferative activity of the synthesized compounds. This activity was tested on the human MCF-7 and MDA-MB-231 breast cancer cell lines and the A549 lung cancer cell lines, and compared with the commercially available α-TOS and γ-T₃. Since an exact anticancer mechanism and drug targets of these compounds are not known, we could only perform a ligand based virtual screening (LBVS), employing the ChEMBL database to identify the new compounds with a potential antiproliferative activity on human MCF-7 and MDA-MB-231 breast cancer cell lines.