New derivatives of silybin and 2,3-dehydrosilybin and their cytotoxic and P-glycoprotein modulatory activity

https://doi.org/10.1016/j.bmc.2006.01.035 Get rights and content

Abstract

Large series of O-alkyl derivatives (methyl and benzyl) of silybin and 2,3-dehydrosilybin was prepared. Selective alkylation of the silybin molecule was systematically investigated. For the first time we present here, for example, preparation of 19-nor-2,3-dehydrosilybin. All prepared silybin/2,3-dehydrosilybin derivatives were tested for cytotoxicity on a panel of drugs sensitive against multidrug resistant cell lines and the ability to inhibit P-glycoprotein mediated efflux activity. We have identified effective and relatively non-cytotoxic inhibitors of P-gp derived from 2,3-dehydrosilybin. Some of them were more effective inhibitors at concentrations lower than a standard P-gp efflux inhibitor cyclosporin A. Another group of 2,3-dehydrosilybin derivatives also had better inhibitory effects on P-gp efflux but a cytotoxicity comparable with that of parent 2,3-dehydrosilybin. Structural requirements for improving inhibitory activity and reducing toxicity of 2,3-dehydrosilybin were established. Effect of E-ring substitution as well as an influence of the substituent size at the C-7-OH position of A-ring on P-gp-inhibitory activity was evaluated for the first time in this study.

Introduction

Silymarin obtained from the seeds of Silybum marianum (milk thistle) has been used since the time of ancient physicians and herbalists to treat a range of liver and gallbladder disorders, including hepatitis, cirrhosis, and jaundice, and to protect the liver against poisoning from chemical and environmental toxins, including snakebites, insect stings, mushroom poisoning, and alcohol. These are typical applications, for which silymarin, its main component silybin (1), and their preparations have been used and prescribed until now.1, 2

Recently, however, silybin received attention due to its alternative beneficiary activities that are not directly bound to its hepatoprotective and/or cytoprotective actions.3

One of the most promising activities of this compound is its anticancer activity that results at least partially from its cytoprotective, antioxidant, and chemopreventing properties. Moreover silybin has the potential to be applied as an adjuvant in the anti-cancer chemotherapy since it diminishes carcinogenic potential of a number of chemicals.4

However, silybin also acts on the receptor level affecting various processes involved either in cancerogenesis and/or tumor proliferation. Modulations of various mitogenic, signaling, apoptotic, and cell-cycle regulators by silybin and silymarin were observed.5, 6, 7, 8, 9, 10, 11, 12, 13 In a human prostate cancer, silymarin inhibits mitogenic signaling pathways and alters cell cycle regulators, leading to growth inhibition and death of advanced and androgen-independent prostate carcinoma cells.14, 15 Some steroid hormone-dependent tumors are also inhibited by silybin, since both the silymarin and silybin potentiated activity of antiandrogens in androgen dependent prostate cancer cell line LNCaP.16, 17

Anticarcinogenic and anti-inflammatory effects of silymarin and silybin might be also related to the inhibition of the transcription factor NF-κB, which regulates and coordinates the expression of various genes involved in the inflammatory process, in cytoprotection and carcinogenesis. Silymarin was highly active in the inhibition of NF-κB factor activation by various signals.18, 19, 20, 21 Newly discovered effects of silymarin and silybin, for example, on the cell cycle regulation, apoptosis, nuclear and estrogenic receptors, and many recently reviewed by Křen and Walterová.3

Silybin was also investigated as cytoprotective agent and/or sensitizer for anticancer chemotherapy using classical cytostatics such as cisplatin or adriamycin without compromising antitumor activity.22, 23, 24 On the contrary, potentiation of anticancer activity of those drugs by silybin was clearly demonstrated.22

One of the plausible explanations for synergistic effects of silybin and anti-cancer drugs is inhibition of efflux function of dominant multidrug resistance (MDR) transporter P-glycoprotein.25 P-gp is a prominent member of ABC transporters protein family, that has a similar architecture plan comprising four major domains: two membrane bound domains, each with six transmembrane segments, and two cytosolic ATP binding motifs, commonly known as the Walker A and B domains, that bind and hydrolyze ATP (also known as the nucleotide-binding domains, or NBDs). Both ATP binding sites are essential for drug efflux. P-gp binds its substrates in the cytosolic membrane leaflet most likely via H-bond formation and moves them to the extracellular leaflet or to the extracellular aqueous environment at the expense of one to two molecules of ATP.

Modulation or inhibition of P-gp activity can be achieved by: (i) an inhibition of ATP binding, ATP hydrolysis or coupling of ATP hydrolysis to the translocation of substrates, (ii) inhibition of conformational changes required for drug extrusion via antibody binding to certain extracellular loops of the transporter, and (iii) non-competitive or competitive inhibition of P-gp achieved by direct interaction of a compound with one or more binding sites on P-gp.26

Besides ABC transport proteins there is another MDR mechanism represented for instance by the lung resistance-related protein (LRP). It is located intracellularly in vaults and transports toxic agents from the nucleus of neoplastic cells to the endosomal compartments.27, 28

Silymarin increased daunomycin accumulation in P-gp-positive cells, but not in P-gp-negative cells, in a drug concentration and P-gp expression level-dependent manner.29 Silymarin potentiated doxorubicin cytotoxicity in P-gp-positive cells, while it inhibited P-gp ATPase activity and azidopine photoaffinity labeling of P-gp, suggesting a direct interaction with P-gp substrate binding.29 The 1.5 h accumulation of digoxin and vinblastine in Caco-2 cells was significantly increased by silymarin, and this effect was concentration dependent.30 In the human prostate carcinoma DU145 cells, silybin potentiated doxorubicin-induced growth inhibition and apoptosis.31 This P-gp inhibitory activity was pronounced in the 2,3-dehydrosilybin (2) derivatives carrying prenyl or geranyl substituents.32 More recently, the capacity of silybin derivatives to chemosensitize drug-resistant cells with specific MDR transporter profile has been investigated.33 In addition to P-gp, some flavonoids related to silybin were found to inhibit another important MDR-associated protein, the multidrug resistance protein 1 (MRP1).33, 34

The aim of our study was to investigate in vitro anti-cancer and P-gp inhibitory activity of oxidized and selectively alkylated silybin derivatives in order to identify the potential anti-cancer and/or chemosensitizing compounds. Selective alkylation of the silybin hydroxyls was systematically investigated and a series of novel silybin and 2,3-dehydrosilybin derivatives were prepared (see Chart 1).

Section snippets

Chemistry

Silybinic acid (3) and 2,3-dehydrosilybinic acid (4) were prepared by CrO3/H5IO6 oxidation of selectively acetylated derivatives of silybin (1) and 2,3-dehydrosilybin (2) in acetonitrile followed by deacetylation of the oxidized products.35

General methods

The reactions were monitored by TLC on silica gel F254 (Merck) and the spots were visualized by UV light and by charring with 5% H2SO4 in ethanol.

NMR spectra were recorded on a Varian INOVA-400 spectrometer (399.89 MHz for 1H, 100.55 MHz for 13C) in CDCl3 or DMSO-d6 (see text) at 30 °C. Chemical shifts were referenced to the residual solvent signal (δH 7.265, δC 77.00; δH 2.50, δC 39.60). Digital resolution used justified reporting the proton and carbon chemical shifts to three and two decimal

Acknowledgments

This work was supported by the Grant Agency of the Czech Republic (Grant No. 303/02/1097), the Czech Ministry of Education (Research concepts No. MSM 6198959216, MSM 0021620835, and AV0Z50200510) and KONTAKT project No. 7-2006-16. Authors are indebted to Drs. K. Hofbauerová and V. Kopecký Jr., Institute of Physics, Charles University, Prague, for calculation of molecular models of some compounds involved in this study.

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