Gravante, Raffaele (2017) Synthesis and characterization of new modified metabolites, molecules with strong pharmacological activities. [Tesi di dottorato]


Download (5MB) | Preview
[error in script] [error in script]
Item Type: Tesi di dottorato
Resource language: English
Title: Synthesis and characterization of new modified metabolites, molecules with strong pharmacological activities
Date: 10 April 2017
Number of Pages: 98
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Chimiche
Dottorato: Scienze chimiche
Ciclo di dottorato: 29
Coordinatore del Corso di dottorato:
Di Fabio, GiovanniUNSPECIFIED
Date: 10 April 2017
Number of Pages: 98
Keywords: organic chemistry, synthesis, natural metabolites
Settori scientifico-disciplinari del MIUR: Area 03 - Scienze chimiche > CHIM/06 - Chimica organica
Date Deposited: 03 May 2017 17:23
Last Modified: 14 Mar 2018 10:04
DOI: 10.6093/UNINA/FEDOA/11775

Collection description

Polyphenols are the most widely distributed class of plant secondary metabolites and several thousand different compounds have been identified. They play many different roles in plant biology and human life, as UV protective agents, defensive compounds against herbivores and pathogens, they contribute to plant colors and to the taste of food and drink. Moreover it is widely known that natural metabolites have played a crucial role in the identification of many of the drugs that are on the market today. Nowadays natural products used pure in pharmacological preparations represent only 5% of the total, but their semi-synthesis derivatives represent more than 50% of the drugs in use.(1) My PhD project is focused on the chemistry of natural substances, and I developed some strategies to synthetize new silibinin conjugates. Silibinin is the major biologically active component of the seeds extract of the milk thistle (Silybum marianum) also known as silymarin.(2) Structurally, silibinin is a diastereoisomeric mixture of two flavonolignans, silybin A and silybin B in a ratio of approximately 1:1.(3) Silibinin is a metabolite with multiple biological activities operating at various cell levels.(4) Unfortunately, its therapeutic efficiency is rather limited by its low bioavailability due to its very low water solubility. Aiming to improve the solubility, during the first year of my PhD project, we have developed an efficient synthetic procedure to obtain new 9”-phosphodiester silibinin conjugates with different mono- and di-saccharide labels through the anomeric hydroxyl group.(5) In our approach 9’’-phosphoramidite has been used as silibinin substrate and 1-OH full protected mono- and disaccharide derivatives as sugar starting materials. We initially converted full acetylated mono and di-saccharides into 1-OH derivatives and these compounds were coupled with 9’’-phosphoramidite silibinin. The oxidation and the deprotection treatments led to the desired phosphodiester derivatives in good yields. The crude materials were then subjected to the purification by reverse phase analysis (RP-18 HPLC), using a variety of columns and elution conditions, but unfortunately it was too difficult to purify the mixture of diastereoisomers. In the end new silibinin analogues were obtained as a mixture of diastereoisomers, observed by 31P NMR analysis. The NMR analysis has proved to be very complex, in fact 1H and 31P NMR spectra of all compounds showed a dramatic complexity, due to the presence of a lot of diastereoisomers. This drawback has not allowed a complete and detailed NMR characterization of the new derivatives. The structures were confirmed by 31P NMR and ESI-MS mass spectra signals. In preliminary study, new derivatives were subjected to DPPH free radical scavenging and Xanthine Oxidase inhibition assays to evaluate their antioxidant activities. Independently of the sugar moiety present, all compounds exhibited a radical scavenging activities slightly higher than that of the silibinin, and Xanthine Oxidase inhibition at least as that of the silibinin. On the other hand the new derivatives showed a water solubility well above that of silibinin, in fact it was possible to prepare solutions of about 70 mg/mL in water. These two data encouraged our studies during my second year of PhD to improve this synthetic strategy and to realize libraries of optically pure glyco-conjugated silibinins. The 9''-phosphoramidite has been used as silibinin substrate and fully protected 6-OH mono- and di-saccharide derivatives as sugar starting materials.(6) We initially converted opportunely protected mono- and di-saccharides (Glucose, Mannose, Galactose, N- Acetylglucosamine, Trehalose and Lactose) into 6-OH derivatives and then they were coupled with 9"-phosphoramidite silibinin following typical phosphoramidite chemistry procedure. The crude materials were purified by reverse phase chromatography (RP-18 HPLC) and characterized by NMR and MALDI- TOF/TOF-MS analyses. All compounds were obtained in good yields and as a mixture of two silibinin diastereoisomers (A and B). Finally new phosphodiester derivatives were converted into the corresponding sodium salt by cation exchange resin carrying crystalline samples. New derivatives showed water solubility well above that of silibinin, with the possibility to prepare solutions of about 70 mg/mL in water. The stability of new glyco-conjugates was investigated in human serum by HPLC analyses and the 50% disappearance of the peak corresponding to the intact glyco-conjugate was observed after ca. 40-68 hours. All derivatives were subjected to DPPH free radical scavenging assay and they exhibited radical scavenging activities slightly higher than that of the silibinin. In order to verify the potential biological properties of these derivatives, a biological assay was used to evaluate the cytotoxicity of the new glyco-conjugates, compared with that of the silibinin, on human liver cancer cell line (Hep G2). No significant changes in the viability of treated cells were observed when they were subjected to the action of glyco-conjugates, also considering the longest incubation (72 hours) at the maximum dose taken (30 µM). Also, in order to investigate the role of the different OH groups in the antioxidant activity of new glyco-conjugates in comparison with silibinin, their acidity and redox capacity have been evaluated. A redox-deep characterization was carried out in collaboration with Prof. Mauro Iuliano and Dr Gaetano De Tommaso of our Department, using potentiometric and voltammetric techniques. The determination of the acidity constants was conducted by potentiometric titrations measuring the concentration of hydrogen ions with a glass electrode. By processing data, it was possible to define three equilibrium constants. Analysis of the data shows that the conjugation in position 9" does not affect the redox behaviour of the silibinin scaffold. As a part of our continuing research effort towards the synthesis of new natural product analogues exploiting the phosphoramidite chemistry, in the last year of my PhD project the attention was focused on the development of synthetic methods to obtain oligoflavonoids based on silibinin, and to investigate their anti-radical activity. Exploiting the selective protection to hydroxyl groups of the silibinin we have developed an efficient strategy for the synthesis of new 3-9", 3-3 and 9"-9" dimers of silibinin in good yields.(7) In order to obtain suitable building blocks for the dimers synthesis, we have developed a selective protective reaction for the different hydroxyl groups with isobutyric anhydride. The building blocks obtained were coupled in the three different provisions with 3- and 9”-silibinin phosphoramidite using the well known phosphoramidite chemistry. After oxidation, deprotection and RP-18 HPLC purification the products were converted into the corresponding sodium salts by cation exchange on a DOWEX (Na+ form) resin, leading to the desired phosphodiester dimers derivatives in good yields. The structures of new analogues were confirmed by 31P NMR and MALDI-TOF/TOF-MS analyses. The stability of new silibinin dimers was investigated in human serum by HPLC analysis and the 50% disappearance of the peak corresponding to the intact molecule (t1/2) was observed after ca. 80 hours. All derivatives were subjected to DPPH free radical scavenging assay and they exhibited activities quite higher than silibinin. Moreover, the solubility in water of silibinin and its dimers as well as their ability to react with reactive oxygen species (ROS) were determined by estimating their second order rate constant with singlet oxygen (1O2) and hydroxyl radical (HO∙) in solution. This data were obtained in the laboratories of Prof. Marcello Brigante during my experience work in his research team at the University Blaise Pascal, Institute of Chemistry of Clermont-Ferrand (France). Solubility experiments indicate that dimers were completely dissolved (0.1 mg in 10 mL) in water and solubility can be estimated to be ≥ 19.5 µM corresponding to > 20 mg/L. Reactivity between silibinin and dimers with 1O2 and HO∙ was determined by use of Rose Bengal (RB) and hydrogen peroxide as respective ROS sources. For this purpose, 545 nm centered excitation of RB and laser flash photolysis (LFP) experiments were coupled with a kinetic competition approach. Dimers reactivity toward singlet oxygen results to be close to the value determined for silibinin, or about 35% lower. Second order rate constant fare in the same order of magnitude reported in literature for molecules with similar structure. Morales and co-workers8 reported a reactivity ranging from 2.4 to 13.4 × 107 M-1s-1 for flavonoid derivative such as quercetin and morin. Estimation of second order rate constant reactivity with HO∙ indicates that some dimers showed a second order rate constant ≥ 1.5 × 1010 M–1s–1. Wang and co-workers (9) investigated the reactivity of hydroxyl radical with phenolic compounds in order to estimate their anti-oxidative ability using aqueous pulse radiolysis. The value of 1.5 × 1010 M–1s–1 was found for quercetin that is close to those estimates for green tea polyphenols. Interestingly, Husain at al.10 reported that reactivity of flavonoids toward photo-generated hydroxyl radical increases with the number of hydroxyl groups in the aromatic ring. References 1 Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2012, 75, 311. 2 Gažák, R.; Walterová, D.; Křen, V. Curr. Med. Chem. 2007, 14, 315. 3 Napolitano, J. G.; Lankin, D. C.; Graf, T. N.; Friesen, J. B.; Chen, S-N.; McAlpine, J. B.; Oberlies, N. H.; Pauli, G. F. J. Org. Chem. 2013, 78, 2827. 4 Zhan, T.; Digel, M.; Küch, E.-M. J. Cell. Biochem. 2011, 112, 849 and references therein. 5 Zarrelli, A.; Romanucci, V.; Tuccillo, C.; Federico, A.; Loguercio, C.; Gravante, R.; Di Fabio, G. Bioorg. Med. Chem. Lett. 2014, 24, 5147. 6 Romanucci, V.; Gravante, R.; Di Marino, C.; Iuliano, M.; De Tommaso, G.; Caruso, T.; Zarrelli, A.; Di Fabio, G. submitted for publication. 7 Gravante, R .; Romanucci, V.; Cimafonte, M.; Di Marino, C.; Mailhot, G.; Brigante, M.; Zarrelli, A.; Di Fabio, G. submitted for publication. 8 Morales, J.; Günther, G.; Zanocco, A. L.; Lemp, E. PLoS One 2012, 7, e40548. 9 Wang, W. F.; Luo, J. S.; Yao, D.; Lian, Z. R.; Zhang, J. S.; Lin, N. Y. Radiat. Phys. Chem. 1993, 42, 985. 10 Rafat Husain, S.; Cillard, J.; Cillard, P. Phytochemistry 1987, 26, 2489.


Downloads per month over past year

Actions (login required)

View Item View Item