Dottorato in SCIENZE FARMACEUTICHE

Titolo della tesi: Original characterization of silica surface, and kinetic and thermodynamic studies concerning stereolability, self-association or redox processes of bioactive compounds

ABSTRACT CHAPTER 1 In the first chapter it is described the development of an original experimental procedure conceived to promote high degrees of deprotonation of the silanol groups (≡Si-OH) bound to the surface of silica matrices, in the perspective to strongly increase the nucleophilicity of silanol oxygens (generation of silanolate groups, ≡Si-O-) in view of a their subsequent fruitful chemical derivatization. Indeed, it is expected that the generated ≡Si-O- sites may, much more easily and effectively, establish bonds of both covalent and coordination (mediated by metals) nature with organic molecules. Typically, such materials of derivatized silica may found interesting and useful application as either chromatographic selectors or promoters in catalysis processes to be carried out in the heterogeneous phase. In order to determinate the degree of deprotonation achieved on silica matrices submitted to the above procedure, two expressly dedicated analytical methods have been developed, based on an initial coordination of metal ions on the ≡Si-O- groups (Ag+, in one method, Cu+2 in the other). This step is then followed by detachment of such metal ions as Ag-EDTA or Cu-EDTA complexes, which are next dosed by UV procedure. Further studies were also directed towards the development of original analytical methodologies focused to the quantification of amino groups bound on the surface of solid siliceous matrices (a determination that, however, could also be generalized to the case of other types of basic sites, provided they are characterized by pKa values not lower than 5). This quantification was based on the salification of these groups by reaction with 3,5 dinitrobenzoic acid (DNBA), which is characterized by pKa=2.7. In this way, the dosage of the amino functionalities is next obtained by UV spectrophotometric back-titration of the DNBA detached from the salified solid matrix by treatment with a suitable either acid (release of undissociated DNBA) or basic (release of the conjugate base of DNBA) species. Alternatively, the DNBA displaced in the previously step can be quantified by reverse-phase HPLC determination. The procedure is fast, easy to perform and non-destructive for the analyzed material. Thanks to the mild operating conditions required by the method, this was also used to quantify residual basic/amino sites chemically linked to amino-ureidic stationary phases already packed into HPLC columns, thus allowing to clarify the fine composition possessed by the selector. ABSTRACT CHAPTER 2 In the second chapter they are discussed the studies conducted in the field of Dynamic High Performing Liquid Chromatography (D-HPLC) on biologically active compounds. An experimental and theoretical study was carried out aimed at analyzing the configurational lability possessed by the enantiomers of some of the most common drugs endowed with a central non-planar seven-membered benzo-cycloheptadiene ring, which confers chirality to these compounds (it represents a case of "chiral plane"). The energy barrier that opposes the inversion of the chiral tricyclic scaffold characterizing such species (also taking into account the presence of different substituents on the cycles ), has been studied by using D-HPLC, Dynamic-HNMR and Off-column racemization techniques (monitored, in this case, by the decay of the CD signal), as well as by evaluations based on computational calculations. Interesting indications were obtained by making targeted structural changes to allow accurate control of the stereolability characterizing the tricyclic ring. The study carried out on Nevirapine, an important antiretroviral drug used in anti-HIV-1 therapy, which is capable of binding to the allosteric site of reverse transcriptase, was of considerable interest. As demonstrated in a previous work by our research group, at room temperature it is not possible to observe the two atropoisomeric forms of the molecule, as the enantiomerization process (butterfly effect) is extremely fast. However, a decrease in the temperature of the chiral chromatographic column (values close to -50 °C) allowed to observe two base-resolved peaks relative to the two isomeric forms. As a consequence of this observation, structural changes were made to the Nevirapine molecule in order to modulate the interconversion phenomena, making much more slow the chiral-flipping. The performed studies have envisaged the use of the following techniques and analytic methodologies: enantioselective liquid chromatography (e-HPLC); dynamic chromatography (D-HPLC); off-column racemization analyses; circular dichroism and optical rotary dispersion analyses; computational analyses. Finally, the obtained molecules were isolated as single enantiomers, and the biological activity of each of these evaluated on cell cultures. Staying in the field of the dynamic chromatography, the reduction reaction of α-Lipoic acid (ALA) was studied by innovative employment of this methodology. ALA is an organosulfuric compound, naturally present in the human body, in which it is enzymatically synthesized inside the mitochondria starting from cysteine and octanoic acid. Being present in many physiological processes, as a cofactor in numerous enzymatic complexes, ALA has been the subject of numerous studies, some of which focused on its possible application as an antioxidant agent in preventive medicine, for events related to aging. Exogenous ALA is partially reduced in vivo to α-dihydrolipoic acid (DHALA). The DHALA/ALA pair is characterized by a very negative redox potential (-320 mV), which justifies the behavior of ALA as an effective oxidizing agent. In the here reported work, the irreversible reduction process of ALA was studied by dynamic-HPLC approach (D-HPLC). The originality of this investigation lies in the fact that it represents the first example in which the D-HPLC technique was used in the execution of a kinetic analysis not concerning a classic conformational or constitutional isomerization process. The reduction reaction ALADHALA was promoted during the HPLC analysis of ALA by the presence in the mobile phase of dithiothreitol (DTT), which is able to acts as effective reducing agent. The dynamic chromatograms obtained under different conditions of pH, temperature and concentrations of DTT in the mobile phase, were successfully simulated by use of the computer program Auto-DHPLC-y2k. In this way, from the obtained dynamic-chromatograms, it was possible to extrapolate the desired kinetic data related to the redox process studied. ABSTRACT CHAPTER 3 In the last chapter, studies conducted in the field of supramolecular chemistry are discussed. These involved an accurate analysis (based on UV-Vis spectroscopy) of the tendency to self-aggregation that can be induced on macrocyclic resorc[4]arenes structures (three different species) with a defined and large lipophilic portion in their lower-rim and a polar portion, characterized by two kind of ionizable frameworks (carboxylic and phenolic groups), in their upper-rim. In particular, the studies were conducted by progressively varying the hydrophilicity of the upper-rim through the use as the solvent of a water/tetrahydrofuran mixture, in which both composition of the employed mixture and pH of the aqueous phase have been suitably modified, step by step. In this way, the tendency to self-associate manifested by these compounds in such solutions of progressive different polarity has been evaluated by UV-Vis spectroscopic analyses, as well as by DLS (Dynamic Light Scattering) determinations, which allowed to establish the dimensions of the formed supramolecular aggregates. In addition, a possible use of these macrocycles as carriers of pharmacologically active substances endowed with a predominantly lipophilic structure, such as, for example, Glabrescione (GlaB), has also been evaluated. GlaB is a natural isoflavone promising candidate for the treatment of Hedgehog (Hh) dependent tumors.