From Folke Medicine to Medicine Chemistry: Study, Using in Vitro and Cellular Assays, of Receptors Mechanism Involved in the Activities of Natural Compounds

My Doctorate Research has been focused on the evaluation of the pharmacological activity of a natural extract of chestnut wood (ENC) towards the cardiovascular and gastrointestinal system and on the identification of the active compounds. The ENC has been shown to contain more than 10% (w/w) of phenolic compounds, of which tannins as Vescalgin and Castalgin are the more representative. ENC cardiovascular effects have been investigated in guinea pig cardiac preparations; furthermore its activity has been evalueted in guinea pig aorta strips. ENC induced transient negative chronotropic effect in isolated spontaneously beating right atria and simultaneously positive inotropic effect in left atria driven at 1 Hz. Cardiac cholinergic receptors are not involved in the negative chronotropic effect and positive inotropic effects are not related to adrenergic receptors. In vascular smooth muscle, natural extract of chestnut did not significantly change the contraction induced by potassium (80 mM) or that induced by noradrenaline (1μM). In guinea pig ileum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. In guinea pig ileum, the antispasmodic activity of ENC showed a significant antispasmodic activity against a variety of different spasmogenic agents including histamine, KCl, BaCl2. In guinea pig proximal colon, stomach and jejunum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. ENC contracted gallbladder guinea pig in a reversible and concentration-dependent manner. This effect does not involve cholinergic and cholecystokinin receptors and it is reduced by nifedipine. ENC relaxed Oddi sphincter smooth muscle. The cholecystokinetic and Oddi sphincter relaxing activities occurred also in guinea pigs fed a lithogenic diet. The cholecystokinetic occurred also in human gallbladder. The Fractionation of the extract led to the identification of the active fraction.

Stereoselective Catalytic Processes for the Synthesis of Polycyclic Aromatic Compounds

In this PhD-thesis, two methodologies for enantioselective intramolecular ring closing reaction on indole cores are presented. The first methodology represents a highly stereoselective alkylation of the indole N1-nitrogen, leading to 3,4-dihydro-pyrazinoindol-1-ones – a structural class which is known for its activity on the CNS and therefore of high pharmacological interest concerning related diseases. In this approach, N-benzyl cinchona-alkaloids were used for the efficient catalysis of intramolecular aza-Michael reactions. Furthermore, computational studies in collaboration with the research group Prof. Andrea Bottoni (Department of Chemistry “G. Ciamician”, Bologna) were accomplished in order to get insight into the key interactions between catalyst and substrate, leading to enantiomeric excesses up to 91%. The results of the calculations on a model system are in accordance with the experimental results and demonstrate the high sensibility of the system towards structural modifications. The second project deals with a metal catalyzed, intramolecular Friedel-Crafts (FC)-reaction on indolyl substrates, carrying a side chain which on its behalf is furnished with an allylic alcohol unit. Allylic alcohols are part of the structural class of “π-activated alcohols” – alcohols, which are more easily activated due to the proximity to a π-unit (allyl-, propargyl-, benzyl-). The enantioselective intramolecular cyclization event is catalyzed efficiently by employment of a chiral Au(I)-catalyst, leading to 1-vinyl- or 4-vinyl-tetrahydrocarbazoles (THCs) under the formation of water as byproduct. This striking and novel process concerning the direct activation of alcohols in catalytic FC-reactions was subsequently extended to similar precursors, leading to functionalized tetrahydro-β-carbolines. These two methodologies represent highly efficient approaches towards the synthesis of scaffolds, which are of enormous pharmaceutical interest and amplify the spectra of enantioselective catalytic functionalisations of indoles.

Design and development of new green catalytic methodologies for the synthesis of enantiomerically enriched molecules

The following Ph.D work was mainly focused on catalysis, as a key technology, to achieve the objectives of sustainable (green) chemistry. After introducing the concepts of sustainable (green) chemistry and an assessment of new sustainable chemical technologies, the relationship between catalysis and sustainable (green) chemistry was briefly discussed and illustrated via an analysis of some selected and relevant examples. Afterwards, as a continuation of the ongoing interest in Dr. Marco Bandini’s group on organometallic and organocatalytic processes, I addressed my efforts to the design and development of novel catalytic green methodologies for the synthesis of enantiomerically enriched molecules. In the first two projects the attention was focused on the employment of solid supports to carry out reactions that still remain a prerogative of omogeneous catalysis. Firstly, particular emphasis was addressed to the discovery of catalytic enantioselective variants of nitroaldol condensation (commonly termed Henry reaction), using a complex consisting in a polyethylene supported diamino thiopene (DATx) ligands and copper as active species. In the second project, a new class of electrochemically modified surfaces with DATx palladium complexes was presented. The DATx-graphite system proved to be efficient in promoting the Suzuki reaction. Moreover, in collaboration with Prof. Wolf at the University of British Columbia (Vancouver), cyclic voltammetry studies were reported. This study disclosed new opportunities for carbon–carbon forming processes by using heterogeneous, electrodeposited catalyst films. A straightforward metal-free catalysis allowed the exploration around the world of organocatalysis. In fact, three different and novel methodologies, using Cinchona, Guanidine and Phosphine derivatives, were envisioned in the three following projects. An interesting variant of nitroaldol condensation with simple trifluoromethyl ketones and also their application in a non-conventional activation of indolyl cores by Friedel-Crafts-functionalization, led to two novel synthetic protocols. These approaches allowed the preparation of synthetically useful trifluoromethyl derivatives bearing quaternary stereocenters. Lastly, in the sixth project the first γ-alkylation of allenoates with conjugated carbonyl compounds was envisioned. In the last part of this Ph.D thesis bases on an extra-ordinary collaboration with Prof. Balzani and Prof. Gigli, I was involved in the synthesis and characterization of a new type of heteroleptic cyclometaled-Ir(III) complexes, bearing bis-oxazolines (BOXs) as ancillary ligands. The new heteroleptic complexes were fully characterized and in order to examine the electroluminescent properties of FIrBOX(CH2), an Organic Light Emitting Device was realized.

Use of solvents and environmental friendly materials for applications in Green Chemistry

The present Thesis studies three alternative solvent groups as sustainable replacement of traditional organic solvents. Some aspects of fluorinated solvents, supercritical fluids and ionic liquids, have been analysed with a critical approach and their effective “greenness” has been evaluated from the points of view of the synthesis, the properties and the applications. In particular, the attention has been put on the environmental and human health issues, evaluating the eco-toxicity, the toxicity and the persistence, to underline that applicability and sustainability are subjects with equal importance. The “green” features of fluorous solvents and supercritical fluids are almost well-established; in particular supercritical carbon dioxide (scCO2) is probably the “greenest” solvent among the alternative solvent systems developed in the last years, enabling to combine numerous advantages both from the point of view of industrial/technological applications and eco-compatibility. In the Thesis the analysis of these two classes of alternative solvents has been mainly focused on their applicability, rather than the evaluation of their environmental impact. Specifically they have been evaluated as alternative media for non-aqueous biocatalysis. For this purpose, the hydrophobic ion pairing (HIP), which allows solubilising enzymes in apolar solvents by an ion pairing between the protein and a surfactant, has been investigated as effective enzymatic derivatisation technique to improve the catalytic activity under homogeneous conditions in non conventional media. The results showed that the complex enzyme-surfactant was much more active both in fluorous solvents and in supercritical carbon dioxide than the native form of the enzyme. Ionic liquids, especially imidazolium salts, have been proposed some years ago as “fully green” alternative solvents; however this epithet does not take into account several “brown” aspects such as their synthesis from petro-chemical starting materials, their considerable eco-toxicity, toxicity and resistance to biodegradation, and the difficulty of clearly outline applications in which ionic liquids are really more advantageous than traditional solvents. For all of these reasons in this Thesis a critical analysis of ionic liquids has been focused on three main topics: i) alternative synthesis by introducing structural moieties which could reduce the toxicity of the most known liquid salts, and by using starting materials from renewable resources; ii) on the evaluation of their environmental impact through eco-toxicological tests (Daphnia magna and Vibrio fischeri acute toxicity tests, and algal growth inhibition), toxicity tests (MTT test, AChE inhibition and LDH release tests) and fate and rate of aerobic biodegradation in soil and water; iii) and on the demonstration of their effectiveness as reaction media in organo-catalysis and as extractive solvents in the recovery of vegetable oil from terrestrial and aquatic biomass. The results about eco-toxicity tests with Daphnia magna, Vibrio fischeri and algae, and toxicity assay using cultured cell lines, clearly indicate that the difference in toxicity between alkyl and oxygenated cations relies in differences of polarity, according to the general trend of decreasing toxicity by decreasing the lipophilicity. Independently by the biological approach in fact, all the results are in agreement, showing a lower toxicity for compounds with oxygenated lateral chains than for those having purely alkyl lateral chains. These findings indicate that an appropriate choice of cation and anion structures is important not only to design the IL with improved and suitable chemico-physical properties but also to obtain safer and eco-friendly ILs. Moreover there is a clear indication that the composition of the abiotic environment has to be taken into account when the toxicity of ILs in various biological test systems is analysed, because, for example, the data reported in the Thesis indicate a significant influence of salinity variations on algal toxicity. Aerobic biodegradation of four imidazolium ionic liquids, two alkylated and two oxygenated, in soil was evaluated for the first time. Alkyl ionic liquids were shown to be biodegradable over the 6 months test period, and in contrast no significant mineralisation was observed with oxygenated derivatives. A different result was observed in the aerobic biodegradation of alkylated and oxygenated pyridinium ionic liquids in water because all the ionic liquids were almost completely degraded after 10 days, independently by the number of oxygen in the lateral chain of the cation. The synthesis of new ionic liquids by using renewable feedstock as starting materials, has been developed through the synthesis of furan-based ion pairs from furfural. The new ammonium salts were synthesised in very good yields, good purity of the products and wide versatility, combining low melting points with high decomposition temperatures and reduced viscosities. Regarding the possible applications as surfactants and biocides, furan-based salts could be a valuable alternative to benzyltributylammonium salts and benzalkonium chloride that are produced from non-renewable resources. A new procedure for the allylation of ketones and aldehydes with tetraallyltin in ionic liquids was developed. The reaction afforded high yields both in sulfonate-containing ILs and in ILs without sulfonate upon addition of a small amount of sulfonic acid. The checked reaction resulted in peculiar chemoselectivity favouring aliphatic substrates towards aromatic ketones and good stereoselectivity in the allylation of levoglucosenone. Finally ILs-based systems could be easily and successfully recycled, making the described procedure environmentally benign. The potential role of switchable polarity solvents as a green technology for the extraction of vegetable oil from terrestrial and aquatic biomass has been investigated. The extraction efficiency of terrestrial biomass rich in triacylglycerols, as soy bean flakes and sunflower seeds, was comparable to those of traditional organic solvents, being the yield of vegetable oils recovery very similar. Switchable polarity solvents as been also exploited for the first time in the extraction of hydrocarbons from the microalga Botryococcus braunii, demonstrating the efficiency of the process for the extraction of both dried microalgal biomass and directly of the aqueous growth medium. The switchable polarity solvents exhibited better extraction efficiency than conventional solvents, both with dried and liquid samples. This is an important issue considering that the harvest and the dewatering of algal biomass have a large impact on overall costs and energy balance.

Quali-quantitative study of the phenolic and polyphenolic compounds and their antioxidant capacity in vegetal matrix processed with different technologies

Food technologies today mean reducing agricultural food waste, improvement of food security, enhancement of food sensory properties, enlargement of food market and food economies. Food technologists must be high-skilled technicians with good scientific knowledge of food hygiene, food chemistry, industrial technologies and food engineering, sensory evaluation experience and analytical chemistry. Their role is to apply the modern vision of science in the field of human nutrition, rising up knowledge in food science. The present PhD project starts with the aim of studying and improving frozen fruits quality. Freezing process in very powerful in preserve initial raw material characteristics, but pre-treatment before the freezing process are necessary to improve quality, in particular to improve texture and enzymatic activity of frozen foods. Osmotic Dehydration (OD) and Vacuum Impregnation (VI), are useful techniques to modify fruits and vegetables composition and prepare them to freezing process. These techniques permit to introduce cryo-protective agent into the food matrices, without significant changes of the original structure, but cause a slight leaching of important intrinsic compounds. Phenolic and polyphenolic compounds for example in apples and nectarines treated with hypertonic solutions are slightly decreased, but the effect of concentration due to water removal driven out from the osmotic gradient, cause a final content of phenolic compounds similar to that of the raw material. In many experiment, a very important change in fruit composition regard the aroma profile. This occur in strawberries osmo-dehydrated under vacuum condition or under atmospheric pressure condition. The increment of some volatiles, probably due to fermentative metabolism induced by the osmotic stress of hypertonic treatment, induce a sensory profile modification of frozen fruits, that in some way result in a better acceptability of consumer, that prefer treated frozen fruits to untreated frozen fruits. Among different processes used, a very interesting result was obtained with the application of a osmotic pre-treatment driven out at refrigerated temperature for long time. The final quality of frozen strawberries was very high and a peculiar increment of phenolic profile was detected. This interesting phenomenon was probably due to induction of phenolic biological synthesis (for example as reaction to osmotic stress), or to hydrolysis of polymeric phenolic compounds. Aside this investigation in the cryo-stabilization and dehydrofreezing of fruits, deeper investigation in VI techniques were carried out, as studies of changes in vacuum impregnated prickly pear texture, and in use of VI and ultrasound (US) in aroma enrichment of fruit pieces. Moreover, to develop sensory evaluation tools and analytical chemistry determination (of volatiles and phenolic compounds), some researches were bring off and published in these fields. Specifically dealing with off-flavour development during storage of boiled potato, and capillary zonal electrophoresis (CZE) and high performance liquid chromatography (HPLC) determination of phenolic compounds.

Abiotic and prebiotic phosphorus chemistry

The chief obstacle to understand the metabolic origin of life or RNA-based life is to identify a plausible mechanism for overcoming the clutter wrought by abiotic chemistry. Probably trough simple abiotic and then prebiotic reactions we could arrive to simple pre-RNA molecules. Here we report a possible preibiotic synthesis for heterocyclic compounds, and a self-assembling process of adenosine phosphates a constituent of RNA. In these processes we use a simple and prebiotic phosphorus cyclic compounds, as P4O10 and its derivatives. The processes are driven by the formation of hypercoordinated species that activate the processes by a factor of 106-8.

Design, Synthesis and Characterization of N-Containing Organic Compounds

The needed of new intermediates/products for screening in the fields of drug discovery and material science is the driving force behind the development of new methodologies and technologies. Organic scaffolds are privileged targets for this scouting. Among them a priority place must be attributed to those including nitrogen functionalities in their scaffolds. It comes out that new methodologies, allowing the introduction of the nitrogen atom for the synthesis of an established target or for the curiosity driven researches, will always be welcome. The target of this PhD Thesis’ work is framed within this goal. Accordingly, Chapter 1 reports the preparation of new N-Heteroarylmethyl 3-carboxy-5-hydroxy piperidine scaffold, as potential and selective α-glucosidase inhibitors. The proposed reversible uncompetitive mechanism of inhibition makes them attractive as interesting candidate for drug development. Chapter 2 is more environmentally method-driven research. Eco-friendly studies on the synthesis of enantiomerically pure 1,4-dihydropyridines using “solid” ammonia (magnesium nitride) is reported via classical Hantzch method. Chapter 3 and Chapter 4 may be targeted as the core of the Thesis’s research work. Chapter 3 reports the studies addressed to the synthesis of N-containing heterocycles by using N-trialkylsilylimine/hetero-Diels–Alder (HAD) approach. New eco-friendly methodology as MAOS (Microwave Assisted Organic Synthesis) has been used as witness of our interest to a sustainable chemistry. Theoretical calculations were adopted to fully clarify the reaction mechanism. Chapter 4 is dedicated to picture the most recent studies performed on the application of N-Metallo-ketene imines (metallo= Si, Sn, Al), relatively new intermediates which are becoming very popular, in the preparation of highly functionalized N-containing derivatives, accordingly to the Thesis’ target. Derivatives obtained are designed in such a way that they could be of interest in the field of drug and new material chemistry.

Bioconjugation and synthetic approach towards enantioenriched gem-difluoromethylene compounds through carbenium ions

Bioconjugation of peptides and asymmetric synthesis of gem-difluoromethylene compounds are areas of the modern organic chemistry for which mild and selective methods continue to be developed. This thesis reports new methodologies for these two areas based on the use of stabilized carbenium ions. The reaction that makes the bioconjugation of peptides possible takes place via the direct nucleophilic substitution of alcohols and is driven by the spontaneous formation of stabilized carbenium ions in water. By reacting with the thiol group of cysteine in very mild conditions and with a high selectivity, these carbenium ions allow the site-specific ligation of polypeptides containing cysteine and their covalent derivatization with functionalized probes. The ligation of the indole ring of tryptophan, an emerging target in bioconjugation, is also shown and takes place in the same conditions. The second area investigated is the challenging access to optically active gem-difluoromethylene compounds. We describe a methodology relying on the synthesis of enantioenriched 1,3-benzodithioles intermediates that are shown to be precursors of the corresponding gem-difluoromethylene analogues by oxidative desulfurization-fluorination. This synthesis takes advantage of the highly enantioselective organocatalytic α-alkylation of aldehydes with the benzodithiolylium ion and of the wide possibilities of synthetic transformations offered by the 1,3-benzodithiole group. This approach allows the asymmetric access to complex gem-difluoromethylene compounds through a late-stage fluorination step, thus avoiding the use of fluorinated building blocks.

Cancer and aging: a multidisciplinary medicinal chemistry approach on relevant biological targets such as proteasome, sirtuins and interleukin 6

It is well known that ageing and cancer have common origins due to internal and environmental stress and share some common hallmarks such as genomic instability, epigenetic alteration, aberrant telomeres, inflammation and immune injury. Moreover, ageing is involved in a number of events responsible for carcinogenesis and cancer development at the molecular, cellular, and tissue levels. Ageing could represent a “blockbuster” market because the target patient group includes potentially every person; at the same time, oncology has become the largest therapeutic area in the pharmaceutical industry in terms of the number of projects, clinical trials and research and development (R&D) spending, but cancer remains one of the leading causes of mortality worldwide. The overall aim of the work presented in this thesis was the rational design of new compounds able to modulate activity of relevant targets involved in cancer and aging-related pathologies, namely proteasome and immunoproteasome, sirtuins and interleukin 6. These three targets play different roles in human cells, but the modulation of its activity using small molecules could have beneficial effects on one or more aging-related diseases and cancer. We identified new moderately active and selective non-peptidic compounds able to inhibit the activity of both standard and immunoproteasome, as well as novel and selective scaffolds that would bind and inhibit SIRT6 selectively and can be used to sensitize tumor cells to commonly used anticancer agents such gemcitabine and olaparib. Moreover, our virtual screening approach led us also to the discovery of new putative modulators of SIRT3 with interesting in-vitro and cellular activity. Although the selectivity and potency of the identified chemical scaffolds are susceptible to be further improved, these compounds can be considered as highly promising leads for the development of future therapeutics.

Synthesis of new bioactive β-lactam compounds

New biologically active β-lactams were designed and synthesized, developing novel antibiotics and enzymatic inhibitors directed toward specific targets. Within a work directed to the synthesis of mimetics for RGD (Arg-Gly-Asp) sequence able to interact with αvβ3 and α5β1-type integrins, new activators were developed and their Structure-Activity Relationships (SAR) analysis deepened, enhancing their activity range towards the α4β1 isoform. Moreover, to synthesize novel compounds active both against bacterial infections and pulmonary conditions of cystic fibrosis patients, new β-lactam candidates were studied. Among the abundant library of β-lactams prepared, mainly with antioxidant and antibacterial double activities, it was identified a single lead to be pharmacologically tested in vivo. Its synthesis was optimized up to the gram-scale, and pretreatment method and HPLC-MS/MS analytical protocol for sub-nanomolar quantifications were developed. Furthermore, replacement of acetoxy group in 4-acetoxy-azetidinone derivatives was studied with different nucleophiles and in aqueous media. A phosphate group was introduced and the reactivity exploited using different hydroxyapatites, obtaining biomaterials with multiple biological activities. Following the same kind of reactivity, a small series of molecules with a β-lactam and retinoic hybrid structure was synthesized as epigenetic regulators. Interacting with HDACs, two compounds were respectively identified as an inhibitor of cell proliferation and a differentiating agent on steam cells. Additionally, in collaboration with Professor L. De Cola at ISIS, University of Strasbourg, some new photochemically active β-lactam Pt (II) complexes were designed and synthesized to be used as bioprobes or theranostics. Finally, it was set up and optimized the preparation of new chiral proline-derived α-aminonitriles through an enantioselective Strecker reaction, and it was developed a chemo-enzymatic oxidative method for converting alcohols to aldehydes or acid in a selective manner, and amines to relative aldehydes, amides or imines. Moreover, enzymes and other green chemistry methodologies were used to prepare Active Pharmaceutical Ingredients (APIs).

Study of medicinal and food plants as a source of biologically active compounds.

In the last few decades, scientific evidence has pointed out the health-beneficial effects of phenolic compounds in foods, including a decrease in risk of developing degenerative and chronic diseases, known to be caused by oxidative stress. In this frame can be inserted research carried out during my PhD thesis, which concerns the phytochemical investigation of phenolic composition in sweet cherries (Prunus avium L.), apple fruits (Malus domestica L.) and quinoa seeds (Chenopodium quinoa Willd.). The first project was focused on the investigation of phytochemical profile and nutraceutical value of fruits of new sweet cherry cultivars. Their phenolic profile and antioxidant activity were investigated and compared with those of commonly commercialized cultivars. Their nutraceutical value was evaluated in terms of antioxidant/neuroprotective capacity in neuron-like SH-SY5Y cells, in order to investigate their ability to counteract the oxidative stress and/or neurodegeneration process The second project was focused on phytochemical analysis of phenolic compounds in apples of ancient cultivars with the aim of selecting the most diverse cultivars, that will then be assayed for their anti-carcinogenic and anti-proliferative activities against the hepato-biliary and pancreatic tumours. The third project was focused on the analysis of polyphenolic pattern of seeds of two quinoa varieties grown at different latitudes. Analysis of phenolic profile and in vitro antioxidant activity of seed extracts both in their free and soluble-conjugated forms, showed that the accumulation of some classes of flavonoids is strictly regulated by environmental factors, even though the overall antioxidant capacity does not differ in quinoa Regalona grown in Chile and Italy. During the internship period carried out at the Department of Organic Chemistry at Universidad Autónoma de Madrid (UAM), it was achieved the isolation of two pentacyclic triterpenoids, from an endemic Peruvian plant, Jatropha macrantha Müll. Arg., with bio-guided fractionation technique.

Photoredox Catalysis and Nucleophilic Organometallic Reagents – Application in Organic Synthesis

The main purpose of my PhD was the combination of the principles of transition metal catalysis with photoredox catalysis. We focused our attention on the development of novel dual catalytic protocols for the functionalization of carbonyl compounds through the generation of transient nucleophilic organometallic species. Specifically, we focused on the development of new methodologies combining photoredox catalysis with titanium and nickel in low oxidation state. Firstly, a Barbier-type allylation of aromatic and aliphatic aldehydes –catalytic in titanium– in the presence of a blue photon-absorbing dye was developed. Parallelly, we were pleased to observe that the developed methodology could also be extended to the propargylation of aldehydes under analogous conditions. After an extensive re–optimization of all the reaction parameters, we developed an enantioselective and diastereoselective pinacol coupling of aromatic aldehydes promoted by non-toxic, cheap and easy to synthetize titanium complexes. The key feature, that allows the complete (dia)stereocontrol played by titanium, is the employment of a red-absorbing organic dye. The tailored (photo)redox properties of the red-absorbing organic dye [nPr–DMQA+][BF4–] promote the selective reduction of Ti(IV) to Ti(III). Moreover, even if the major contribution in dual photoredox and nickel catalysis is devoted to the realization of cross-coupling-type reactions, we wanted to evaluate different possible scenarios. Our focus was on the possibility of exploiting intermediates arising from the oxidative addition of nickel complexes as transient nucleophilic species. The first topic considered regarded the possibility to perform allylation of aldehydes by dual photoredox and nickel catalysis. In the first instance, a non–stereocontrolled version of the reaction was presented. Finally, after a long series of drastic modification of the reaction conditions, a highly enantioselective variant of the protocol was also reported. All the reported methodologies are supported by careful photophysical analysis and, in some cases, computational modelling.

Novel developments of atropisomeric organic molecules. Design of atropisomeric drugs, fluorescent compounds and investigation of reaction mechanisms.

In recent years, the study of restricted rotation bonds in organic compounds has aroused increasing interest. The reason is that this characteristic can lead to obtaining new properties in organic compounds. In this research thesis, an intense investigation was carried out using DFT calculations and experimental evaluation of the barriers to rotational energies, in order to discover new properties deriving from the restricted rotation bonds. Research has been developed in various fields of organic chemistry, ranging from drugs (the atropisomeric atorvastatin in Chapter 3) to luminescent compounds (aryls amino borane in Chapter 4). Furthermore, an organocatalytic central to axial conversion mechanism was investigated through DFT calculations, finding out interesting outcomes (Chapter 5). Finally, a project in collaboration with Dr. Farran and Prof. Vanthuyne of the Aix-Marseille University was done to investigate the interactions in transition states of rotational barriers.

Drug of abuse analysis: new methods in herbal and biological matrices for medicinal chemistry and forensic investigation

In this Ph.D. project, original and innovative approaches for the quali-quantitative analysis of abuse substances, as well as therapeutic agents with abuse potential and related compounds were designed, developed and validated for application to different fields such as forensics, clinical and pharmaceutical. All the parameters involved in the developed analytical workflows were properly and accurately optimised, from sample collection to sample pretreatment up to the instrumental analysis. Advanced dried blood microsampling technologies have been developed, able of bringing several advantages to the method as a whole, such as significant reduction of solvent use, feasible storage and transportation conditions and enhancement of analyte stability. At the same time, the use of capillary blood allows to increase subject compliance and overall method applicability by exploiting such innovative technologies. Both biological and non-biological samples involved in this project were subjected to optimised pretreatment techniques developed ad-hoc for each target analyte, making also use of advanced microextraction techniques. Finally, original and advanced instrumental analytical methods have been developed based on high and ultra-high performance liquid chromatography (HPLC,UHPLC) coupled to different detection means (mainly mass spectrometry, but also electrochemical, and spectrophotometric detection for screening purpose), and on attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) for solid-state analysis. Each method has been designed to obtain highly selective, sensitive yet sustainable systems and has been validated according to international guidelines. All the methods developed herein proved to be suitable for the analysis of the compounds under investigation and may be useful tools in medicinal chemistry, pharmaceutical analysis, within clinical studies and forensic investigations.