Innovative Strategies for the Synthesis of Biologically Active Small Molecules

The post genomic era, set the challenge to develop drugs that target an ever-growing list of proteins associated with diseases. However, an increase in the number of drugs approved every year is nowadays still not observed. To overcome this gap, innovative approaches should be applied in drug discovery for target validation, and at the same time organic synthetic chemistry has to find new fruitful strategies to obtain biologically active small molecules not only as therapeutic agents, but also as diagnostic tools to identify possible cellular targets. In this context, in view of the multifactorial mechanistic nature of cancer, new chimeric molecules, which can be either antitumor lead candidates, or valuable chemical tools to study molecular pathways in cancer cells, were developed using a multitarget-directed drug design strategy. According to this approach, the desired hybrid compounds were obtained by combining in a single chemical entity SAHA analogues, targeting histone deacetylases (HDACs), with substituted stilbene or terphenyl derivatives able to block cell cycle, to induce apoptosis and cell differentiation and with Sorafenib derivative, a multikinase inhibitor. The new chimeric derivatives were characterized with respect to their cytotoxic activity and their effects on cell cycle progression on leukemia Bcr-Abl-expressing K562 cell lines, as well as their HDACs inhibition. Preliminary results confirmed that one of the hybrid compounds has the desired chimeric profile. A distinct project was developed in the laboratory of Dr Spring, regarding the synthesis of a diversity-oriented synthesis (DOS) library of macrocyclic peptidomimetics. From a biological point of view, this class of molecules is extremely interesting but underrepresented in drug discovery due to the poor synthetic accessibility. Therefore it represents a valid challenge for DOS to take on. A build/couple/pair (B/C/P) approach provided, in an efficient manner and in few steps, the structural diversity and complexity required for such compounds.

Microwave-Mediated Hetero Diels-Alder reaction: Synthesis of biologically active compounds

Heterocyclic compounds represent almost two-thirds of all the known organic compounds: they are widely distributed in nature and play a key role in a huge number of biologically important molecules including some of the most significant for human beings. A powerful tool for the synthesis of such compounds is the hetero Diels-Alder reaction (HDA), that involve a [4+2] cycloaddition reaction between heterodienes and suitable dienophiles. Among heterodienes to be used in such six-membered heterocyclic construction strategy, 3-trialkylsilyloxy-2-aza-1,3-dienes (Fig 1) has been demonstrated particularly attractive. In this thesis work, HDA reactions between 2-azadienes and carbonylic and/or olefinic dienophiles, are described. Moreover, substitution of conventional heating by the corresponding dielectric heating as been explored in the frame of Microwave-Assisted-Organic-Synthesis (MAOS) which constitutes an up-to-grade research field of great interest both from an academic and industrial point of view. Reaction of the azadiene 1 (Fig 1) will be described using as dienophiles carbonyl compounds as aldehyde and ketones. The six-membered adducts thus obtained (Scheme 1) have been elaborated to biologically active compounds like 1,3-aminols which constitutes the scaffold for a wide range of drugs (Prozac®, Duloxetine, Venlafaxine) with large applications in the treatment of severe diseases of nervous central system (NCS). Scheme 1 The reaction provides the formation of three new stereogenic centres (C-2; C-5; C-6). The diastereoselective outcome of these reactions has been deeply investigated by the use of various combination of achiral and chiral azadienes and aliphatic, aromatic or heteroaromatic aldehydes. The same approach, basically, has been used in the synthesis of piperidin-2-one scaffold substituting the carbonyl dienophile with an electron poor olefin. Scheme 2 As a matter of fact, this scaffold is present in a very large number of natural substances and, more interesting, is a required scaffold for an huge variety of biologically active compounds. Activated olefins bearing one or two sulfone groups, were choose as dienophiles both for the intrinsic characteristic flexibility of the “sulfone group” which may be easily removed or elaborated to more complex decorations of the heterocyclic ring, and for the electron poor property of this dienophiles which makes the resulting HDA reaction of the type “normal electron demand”. Synthesis of natural compounds like racemic (±)-Anabasine (alkaloid of Tobacco’s leaves) and (R)- and (S)-Conhydrine (alkaloid of Conium Maculatum’s seeds and leaves) and its congeners, are described (Fig 2).

Stereoselective synthesis of heterocycles as intermediates of biologically active compounds

The aim of this thesis was to investigate the synthesis of enantiomerically enriched heterocycles and dehydro-β-amino acid derivatives which can be used as scaffolds or intermediates of biologically active compounds, in particular as novel αvβ3 and α5β1 integrin ligands. The starting materials of all the compounds here synthesized are alkylideneacetoacetates. Alkylidene derivates are very usefull compounds, they are usually used as unsaturated electrophiles and they have the advantage of introducing different kind of functionality that may be further elaborated. In chapter 1, regio- and stereoselective allylic amination of pure carbonates is presented. The reaction proceeds via uncatalyzed or palladium-catalyzed conditions and affords enantiopure dehydro-β-amino esters that are useful precursor of biologically active compounds. Chapter 2 illustrates the synthesis of substituted isoxazolidines and isoxazolines via Michael addition followed by intramolecular hemiketalisation. The investigation on the effect of the Lewis acid catalysis on the regioselectivity of the addition it also reported. Isoxazolidines and isoxazolines are interesting heterocyclic compounds that may be regarded as unusual constrained -amino acids or as furanose mimetics. The synthesis of unusual cyclic amino acids precursors, that may be envisaged as proline analogues, as scaffolds for the design of bioactive peptidomimetics is presented in chapter 3. The synthesis of 2-substituted-3,4-dehydropyrrole derivatives starting from allylic carbonates via a two step allylic amination/ring closing metathesis (RCM) protocol is carried out. The reaction was optimized by testing different Grubbs’ catalysts and carbamate nitrogen protecting groups. Moreover, in view of a future application of these dehydro-β-amino acids as central core of peptidomimetics , the malonate chain was also used to protect nitrogen prior to RCM. Finally, chapter 4 presents the synthesis of two novel different classes of integrin antagonists, one derived from dehydro-β-amino acid prepared as described in chapter 1 and the other one has isoxazolidines synthesized in chapter 2 as rigid constrained core. Since that these compounds are promising RGD mimetics for αvβ3 and α5β1 integrins, they have been submitted to biological assay. and to interpret on a molecular basis their different affinities for the αvβ3 receptor, docking studies were performed using Glide program.

The Domon Family of Plant Plasma Membrane B-Type Cytochromes: Heterologous Expression, Biochemical Characterization and Physiological Roles in Vivo

The DOMON domain is a domain widespread in nature, predicted to fold in a β-sandwich structure. In plants, AIR12 is constituted by a single DOMON domain located in the apoplastic space and is GPI-modified for anchoring to the plasma membrane. Arabidopsis thaliana AIR12 has been heterologously expressed as a recombinant protein (recAtAIR12) in Pichia pastoris. Spectrophotometrical analysis of the purified protein showed that recAtAir12 is a cytochrome b. RecAtAIR12 is highly glycosylated, it is reduced by ascorbate, superoxide and naftoquinones, oxidised by monodehydroascorbate and oxygen and insensitive to hydrogen peroxide. The addition of recAtAIR12 to permeabilized plasma membranes containing NADH, FeEDTA and menadione, caused a statistically significant increase in hydroxyl radicals as detected by electron paramagnetic resonance. In these conditions, recAtAIR12 has thus a pro-oxidant role. Interestingly, AIR12 is related to the cytochrome domain of cellobiose dehydrogenase which is involved in lignin degradation, possibly via reactive oxygen species (ROS) production. In Arabidopsis the Air12 promoter is specifically activated at sites where cell separations occur and ROS, including •OH, are involved in cell wall modifications. air12 knock-out plants infected with Botrytis cinerea are more resistant than wild-type and air12 complemented plants. Also during B. cinerea infection, cell wall modifications and ROS are involved. Our results thus suggest that AIR12 could be involved in cell wall modifying reactions by interacting with ROS and ascorbate. CyDOMs are plasma membrane redox proteins of plants that are predicted to contain an apoplastic DOMON fused with a transmembrane cytochrome b561 domain. CyDOMs have never been purified nor characterised. The trans-membrane portion of a soybean CyDOM was expressed in E. coli but purification could not be achieved. The DOMON domain was expressed in P. pastoris and shown to be itself a cytochrome b that could be reduced by ascorbate.

The role of the NadR regulator during infection and its implication for the coverage of a new Meningococcus B vaccine

Background: Neisseria meningitides represents a major cause of meningitis and sepsis. The meningococcal regulator NadR was previously shown to repress the expression of the Neisserial Adhesin A (NadA) and play a major role in its phase-variation. NadA is a surface exposed protein involved in epithelial cell adhesion and colonization and a major component of 4CMenB, a novel vaccine to prevent meningococcus serogroup B infection. The NadR mediated repression of NadA is attenuated by 4-HPA, a natural molecule released in human saliva. Results: In this thesis we investigated the global role of NadR during meningogoccal infection, identifying through microarray analysis the NadR regulon. Two distinct types of NadR targets were identified, differing in their promoter architectures and 4HPA responsive activities: type I are induced, while type II are co-repressed in response to the same 4HPA signal. We then investigate the mechanism of regulation of NadR by 4-HPA, generating NadR mutants and identifying classes or residues involved in either NadR DNA binding or 4HPA responsive activities. Finally, we studied the impact of NadR mediated repression of NadA on the vaccine coverage of 4CMenB. A selected MenB strains is not killed by sera from immunized infants when the strain is grown in vitro, however, in an in vivo passive protection model, the same sera protected infant rats from bacteremia. Finally, using bioluminescent reporters, nadA expression in the infant rat model was induced in vivo at 3 h post-infection. Conclusions: Our results suggest that NadR coordinates a broad transcriptional response to signals present in the human host, enabling the meningococcus to adapt to the relevant host niche. During infectious disease the effect of the same signal on NadR changes between different targets. In particular NadA expression is induced in vivo, leading to efficient killing of meningococcus by anti-NadA antibodies elicited by the 4CMenB vaccine.

Synthesis of Biologically Active Small Molecules: Different Approaches to Drug Design

In the past years, genome biology had disclosed an ever-growing kind of biological targets that emerged as ideal points for therapeutic intervention. Nevertheless, the number of new chemical entities (NCEs) translated into effective therapies employed in the clinic, still not observed. Innovative strategies in drug discovery combined with different approaches to drug design should be searched for bridge this gap. In this context organic synthetic chemistry had to provide for effective strategies to achieve biologically active small molecules to consider not only as potentially drug candidates, but also as chemical tools to dissect biological systems. In this scenario, during my PhD, inspired by the Biology-oriented Synthesis approach, a small library of hybrid molecules endowed with privileged scaffolds, able to block cell cycle and to induce apoptosis and cell differentiation, merged with natural-like cores were synthesized. A synthetic platform which joined a Domino Knoevenagel-Diels Alder reaction with a Suzuki coupling was performed in order to reach the hybrid compounds. These molecules can represent either antitumor lead candidates, or valuable chemical tools to study molecular pathways in cancer cells. The biological profile expressed by some of these derivatives showed a well defined antiproliferative activity on leukemia Bcr-Abl expressing K562 cell lines. A parallel project regarded the rational design and synthesis of minimally structurally hERG blockers with the purpose of enhancing the SAR studies of a previously synthesized collection. A Target-Oriented Synthesis approach was applied. Combining conventional and microwave heating, the desired final compounds were achieved in good yields and reaction rates. The preliminary biological results of the compounds, showed a potent blocking activity. The obtained small set of hERG blockers, was able to gain more insight the minimal structural requirements for hERG liability, which is mandatory to investigate in order to reduce the risk of potential side effects of new drug candidates.