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Inhibidores de fosfodiesterasas como potenciales fármacos para el tratamiento de enfermedades neurodegenerativas

AutorGarcía Fernández, Ana María
DirectorGil Ayuso-Gontán, Carmen; Martínez Gil, Ana
Palabras claveQuinazoline
Fecha de publicación10-jul-2015
EditorUniversidad Complutense de Madrid
CSIC - Centro de Investigaciones Biológicas (CIB)
CSIC - Instituto de Química Médica (IQM)
ResumenNeurodegenerative diseases are characterized by the progressive loss of neurons in a specific part of the brain. These pathologies share an unknown aetiology, the progressive destruction of specific areas of the brain and the lack of an effective treatment. Although their aetiology is not well understood, it is likely to involve both genetic and environmental factors. In this context, some common mechanisms which could trigger neuronal death have been identified, as excitotoxicity, oxidative stress, the accumulation of intracellular or extracellular protein aggregates, due to a missfolding of certain proteins; and neuroinflammation, among others. Since cAMP levels play an important role in neuroprotection and neuroinflammation, PDEs have emerged as new potential targets for the regulation of inflammatory process, because their inhibition could control the levels of this nucleotide. PDEs selectively degrade cyclic purine nucleotides cAMP and cGMP to their inactive 5’-monophosphate forms. This family of enzymes has been classified into 11 groups, according to their sequence homology, cellular distribution and sensitivity to different PDEs inhibitors. PDE4, PDE7 and PDE8 are cAMP-specific PDEs while PDE3, PDE5 and PDE9 are cGMP-specific PDEs. The rest of them are dual ones. According to their distribution in the central nervous system, PDE7, PDE8 and PDE10 could be considered attractive therapeutic targets to control the inflammatory process which trigger neuronal death. That means inhibition of high-expressed in brain PDEs could be considered a novel and promising strategy in order to find a neurodegenerative-modified treatment for these unmet pathologies. Objectives Since the treatments available for central nervous system diseases are palliative, there is an urgent needed of new treatments able to stop or delay the underlined neurodegenerative process. Having into account the possibility to control cAMP levels by inhibition of PDEs high-expressed in central nervous system, the aim of this work is the design and synthesis of new PDE7, PDE10 and PDE8 inhibitors, which could validate the important role of these enzymes in central nervous system (CNS) diseases, as well as be used as effective treatment for these pathologies.
Results and discussion The thesis is divided into three chapters: Chapter 1. Phosphodiesterase 7 as therapeutic target for the treatment of neurodegenerative diseases. In the first chapter, PDE7 inhibitors have been developed following two different approaches. The first one consists on the development of new analogues of the quinazoline S14, a PDE7 inhibitor previously described, whose clinical development is ongoing. The synthesis of 18 quinazoline derivatives have been carried out by microwaveassisted organic synthesis (MAOS), and after their isolation and characterization, they were evaluated against PDE7A. Most of these derivatives have submicromolar potency against PDE7A enzyme. Furthermore, they have shown a good pharmacological profile: most of them are able to cross the blood-brain barrier (BBB), to increase the survival of cells treated with 6-OHDA, and have shown neuroprotective properties in primary cell cultures treated with LPS, a lipopolysaccharide which induces inflammation. That means these new PDE7 quinazoline-type inhibitors might be used as replacement compounds of S14 for the treatment of Parkinson’s disease, which guarantees the final success of the clinical trial program. Moreover, since PDE7 inhibition has shown a great potential in multiple sclerosis, derivative 19 has been tested in a murine model of EAE, commonly used to study drug candidates for multiple sclerosis, showing a significant decrease into the clinical score of the animals. Secondly, we used a chemical genetic approach in a cellular model of Parkinson disease in order to find new candidates for the treatment of this pathology. As a result, a new diarylsulfide derivative, SC072, was identified as a hit, and subsequently we developed a new PDE7 inhibitor family by the introduction of some chemical modifications in order to study structure-activity relationship of these kinds of compounds. In general, these new derivatives were synthesized by nucleophilic aromatic substitution of the corresponding aryl halides, using thiol or alcoxy derivatives as nucleophilic agents. More than 50 compounds were synthesized and they were tested for their inhibitory potencies against PDE7A. Some of them presented IC50 values in the low micromolar range, so we decided to select the most potent ones for further pharmacological characterization. In this context, they showed efficacy in cellular model of Parkinson’s disease and inflammation, selectivity among other PDEs and good permeability to BBB. Moreover, compounds 48 and 73 were selected to study their binding mode, both theoretically and experimentally, showing they are competitive with cAMP. Moreover, compound 48 was used to demonstrate the absence of emetic effects associated with this sulfide-like family of PDE7 inhibitors, and in addition, the same results were obtained with other chemically diverse families of PDE7 inhibitor, which enhances the therapeutic potential of this enzyme because their inhibitors are both effective and safe drugs (figure 1).
Finally, for further characterization of ADME properties, the corresponding metabolites from in vitro metabolic process using rat hepatic microsomes were identified by comparison to chemical oxidated compounds, which were previously isolated and characterized by NMR and mass spectroscopy. It is noteworthy to mention that all the new quinazoline and sulfide-like PDE7 inhibitors were fully characterized according to analytic and spectroscopic techniques. Figure 1. Sulfide-like PDE7 inhibitors identified by phenotypic screening and validation of chemically diverse PDE7 inhibitors as non-emetogenic drugs. Chapter 2. Identification and development of new PDE10A inhibitors for their study in models of Parkinson’s disease. PDE10A is a dual PDE highly expressed in striatum, an area of brain which represents the input and output of information related to basal ganglia circuit. Since this circuit is associated with motor functions, PDE10A inhibitors could play an important role in neurological diseases which also present motor symptoms, such as Hungtington’s disease and Parkinson’s disease. In this chapter, computational studies have been used to identify new PDE10A inhibitors chemically diverse. First of all, carrying out a virtual screening on the catalytic domain of this enzyme, some imidazole-based compounds were identified as hits. Some more derivatives based on imidazole core were synthesized and their theoretical binding mode was studied. Afterwards, they were tested in cellular models of Parkinson’s disease and additionally, one of these derivatives has shown efficacy in animal models of this pathology, which has allowed us to validate PDE10A as target for Parkinson’s disease (figure 2A). Moreover, a computational study to look for new cavities in the catalytic domain of the enzyme was carried out by using F-Pocket program, and it allowed us to identify some important differences between PDE7 and PDE10, which could be useful to modify their catalytic activities in an allosteric way. Finally, since PDE10A has a GAF domain in its regulatory one, which could be used as an allosteric modulation cavity, we developed a virtual screening based on this domain, and we identified some maleidimide-based compounds which inhibit PDE10A in micromolar range. Further experimental studies are ongoing to confirm our theoretical hypothesis (figure 2B). Figure 2. Families of compounds identified as PDE10A inhibitors. (A) Imidazole-based compound as catalytic site inhibitors. (B) Maleidimide derivatives as allosteric modulators. Chapter 3. Design of PDE8 inhibitors and synthesis of new 3-acylindazole derivatives using solid-phase organic synthesis. PDE8 has recently been identified as a new attractive target for the treatment of neurological disease, because it is overexpressed in Alzheimer’s disease patient brain and also it is involved in movement disorders, as Parkinson’s disease and Hungtington’s disease. As starting point of our work in PDE8 as a target for central nervous system disease, we have designed, based on the enzyme structure, some new indazole derivatives which could be potential PDE8 inhibitors. The new indazoles were synthesized by solid phase organic synthesis, using triazene linker T1. The required nitrogen atoms are originated from diazonium salts being cleaved from triazene resins.
We have tried some different carboxylic acids/choride acids and 2-aminobenzonitriles as starting materials in order to study the structural requirements for indazol formation (scheme 1). We have found that 3-substitued-2-aminobenzonitriles are necessary as starting materials for heterocycle formation and that, only when we used methyl chloride acid, benzotriazine is obtained instead of indazole. The employment of different 2- aminobenzonitriles yield, after cleavage, side products corresponding to traceless or hydrolyzed ones. These new indazole compounds are going to be tested as PDE8A inhibitors in near future. Scheme 1. Products obtained using different 2-aminobenzonitriles as starting materials. Conclusions In this work, new PDE7 and PDE10A inhibitors were designed as potential therapeutic agents for the treatment of central nervous system diseases. Regarding PDE7, we have obtained some quinazoline-type inhibitors based on S14 structure, whose clinical trial development is ongoing, to guarantee the success of the program. Moreover, a sulfide-like family of inhibitors have been developed, which has allowed us to demonstrate the absence of emetogenic effects related to PDE7 inhibition. Related to PDE10A, it has been identified a new imidazole-type inhibitors, which have demonstrated to be effective in a cellular model of Parkinson’s disease, and are being tested in an animal mod of allosteric modulators based on new identified cavities in the catalytic domain of the enzyme, as well as based on the GAF domain structure of PDE10A. Regarding PDE8, we have designed some potential inhibitors to this enzyme based on an indazole core, and we have obtained them by a new synthetic solid phase pathway based on triazene linker T1.el of the disease. Furthermore, we have initiated the identification
Descripción230 p.-72 fig.-25 esq.
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