Design, synthesis and pharmacological evaluation of new potential tools for the treatment of central nervous system diseases involving dopamine receptors

Schizophrenia is a CNS pathology first described over one century ago. Since that time many discoveries have been done, but still several pathophysiology details are unclear, however, the dopamine hypothesis, emerged forty years ago, still remain one of the main possibility. Since the discovery of chlorpromazine the antipsychotic drugs have been the treatment of choice for schizophrenia and their activity appears to be accounted to the high affinity and antagonistic activity at D2 receptors. However, they are responsible of severe side effects mainly related to the dopamine receptor antagonistic activity itself. A newer approach seems to be represented by the use of D2-like receptor partial agonists. Drugs with such a mechanism of action could act as antagonists where the dopaminergic activity is oversized, and, at the same time, as agonists where the dopaminergic activity is reduced. Some molecules showing dopaminergic D2 partial agonist activity are based on the template moiety 2-(3-hydroxyphenoxy)ethylamine 1. Based on the beneficial role on D2-like receptor affinity played by the reported conformational restrictions given by benzodioxane and benzodihydropyran rings, we have designed and synthesized (chapter 2) a series of compounds embedding the template moiety in a partially (tetrahydroisoquinoline) or fully rigid (tetrahydrochromenoisoquinoline) molecules. The receptor binding affinities toward DA receptor subtypes of the new compounds were evaluated. The obtained results show that in the tetrahydroisoquinoline series only compound 6b displays low affinities for D2 (3.93 mM) and D3 (2.10 mM) receptor subtypes. Compounds 7a,b, belonging to the tetrahydrochromenoisoquinoline series, show affinity values comparable to those of 6b, but a selectivity toward DA D3 receptor subtype may be underlined. Although lower affinity values, the results are in agreement with the binding profile of the lead compound, which shows quite good selectivity for the D2-like receptor family, especially toward the D3 receptor subtype. Compounds 6b and 7a,b embed the structure of the lead in a partially or fully rigid conformation, respectively. Such a feature, reduces or annuls, respectively, its conformational freedom, locking the conformation in a non-effective one. The behavioral activity and the effects on brain neurochemistry of the synthesized compounds 6b and 7a,b were also evaluated in vivo in rats. No statistically significant changes in behavioral activity and no statistically significant changes in rat brain neurochemistry were observed after s. c. administration of the compounds 6b and 7a,b in the dose range 3.7-100 µmol/kg. The pharmacological data led us to conclude that even if the template structure is endowed with dopaminergic activity, a high level of conformational freedom seems to be crucial for a high affinity binding to the dopamine receptors. Several studies demonstrated the versatility of the imidazoline ring. In fact, depending on the particular kind of substituent inserted in position 2 of imidazoline nucleus, it was possible to modulate the ligand profile, both with regard to different systems (a2-Adrenergic Receptors, Imidazoline Binding Sites, Nicotinic and Muscarinic Receptors, MAO-A inhibitors) and inside the same system, with resultant enhanced subtype selectivity. Imidazoline compounds have not been yet explored as DA receptor ligands, but the well known prototype of I2 receptor ligands 2-(benzofuran-2-yl)-4,5-dihydro-1H-imidazole (2-BFI) behaved also as DA indirect agonist and displayed a 47 mM binding to the DA D2 receptors. Therefore, with the aim to improve the DA D2-like receptor binding affinity, novel molecules based on the 2-BFI scaffold were designed and synthesized (chapter 3). The 5 and/or 6 substituents R1-R3 were chosen among those reported to be beneficial for the binding of DA D2-like receptor agonists. Competitive binding of the new compounds at DA D2-like receptors has been evaluated on porcine striatal membranes. The affinity at I2-IBS and a2-ARs has also been evaluated on rat brain membranes. All values are expressed as Ki (mM) values. The most interesting compounds have also been tested for D2-like potency and intrinsic activity. Interestingly, it has observed that the 2-BFI is able to induce a partial activation of D2-like receptor (EC50 = 37.7 mM; i.a.= 0.57). The modest and conservative modifications performed on the basic structure of the lead modulated its biological profile. The introduction of one hydroxyl group in position 6 of the aromatic ring (compound 3f ) slightly enhances, the D2-like profile of the lead. Moreover, 3f behaves as partial agonist (EC50=5.7 mM, i.a.=0.48) displays a potency comparable to that of DA. Moreover, it maintains significant I2-IBS affinity (Ki = 0.076 mM). On the contrary, the isomer 5-hydroxy substituted 3a, still endowed with good I2-IBS, was unable to bind at the D2-like receptors. It appears that only the maintenance of a meta relationship between the OH group with respect to the benzofuranic oxygen atom, proved to be endowed with D2 affinity. The negative influence of the 5-hydroxy substitution is also observed in the catechol derivative 3i that showed strongly reduced I2 character compared to the lead, and was not able to interact with D2 like receptors. Interestingly, the N-substitution seems to impart a more decisive modulation of the biological profile to the lead. In fact, such a modification caused general decrease of the I2-affinity, with, in some cases, accompanying significant D2-like intrinsic activity enhancement. In particular, the N-benzyl derivatives behaved as a nearly D2 full agonists (3e: i.a.=0.85; 3h: i.a. = 0.82), with affinity (3e: Ki = 5.00 mM; 3h: Ki = 5.66 mM), and potency (3e: 6.3 mM; 3h: 5.8 mM) comparable to those of DA (Ki = 3.9 mM; EC50 = 4.8 mM). By our results it can be argued that the degree of receptor activation, is favored by the N-substitution, and might be affected by peculiar features of the pendant group (i.e. steric hindrance) and, sometimes, by the structural characteristics of the parent compound. Parkinson's disease (PD) is a progressive, neurodegenerative disorder which involves the loss of dopaminergic neurons of the substantia nigra pars compacta. Current therapy is essentially symptomatic, and L-Dopa (LD), the direct precursor of dopamine (DA), is the treatment of choice in more advanced stages of the disease. Substitution therapy with LD is, however, associated with a number of acute problems mainly correlated to the poor bioavailability strictly connected with the drug's physical-chemical properties, such as low water and lipid solubility, and the high susceptibility to chemical and enzymatic degradation. With the aim to ameliorate the bioavailability of LD we designed and synthesized some its imidazolinones prodrugs (chapter 4). The choice to embed the aminoacidic group of L-DOPA into the imidazolinone structure derives from reported studies concerning pharmacologically active peptides. Thus, conjugation of LD with glycine gives a dipeptide that can be converted in an imidazolinone protection system that, compared to LD, is more lipophilic and less sensitive to metabolic degradation, meanwhile able to release the drug after spontaneous or enzyme catalyzed chemical hydrolysis. The physical-chemical parameters water solubility and octanol/water partition coefficient were determined on the new compounds (see chapter 4). The chemical stability of the new prodrugs was assayed in vitro in aqueous buffer solutions of pH 1.3 (non enzymatic simulated gastric fluid) and pH 7.4 and the products of the reaction were characterized. Their enzymatic stability was also studied at 37 °C in 80% rat and in 80% human plasma. Taken together, the results indicate that the inclusion of the aminoacidic moiety of LD in a imidazoline-4-one ring give prodrugs that might be able to across unchanged the acidic environment of the stomach, stable enough to be absorbed from the intestine, and then release LD in human plasma after enzymatic hydrolysis. However, further modifications of the prodrug molecules to increase their lipophilicity seems to be necessary in order to boost the possibility of a good intestinal adsorption. Further biochemical and pharmacological studies have been developed in order to test the capability of prodrugs to increase the basal level of striatal DA and influence the brain neurochemistry strictly connected with the dopaminergic activity. Concerning the basal level of striatal dopamine compound 6a showed a long lasting effect. The obtained profile may indicate a slow release of LD to the brain that might represent a positive feature for the reduction of the frequency of drug administration. The measurement of the basal level of the metabolite 3,4-dihydrophenyl acetic acid (DOPAC) confirms the previous statement being the incremental profile of the basal level of DOPAC comparable to that of DA. It has been reported that chronic oxidative stress induces progressive irreversible damage affecting the normal physiological functions of the central nervous system, and seems to play a crucial role in several CNS diseases, including Parkinson's disease. Phenolic compounds are known to be active antioxidants quenching oxygen-derived free radicals by donating hydrogen atom or an electron to the free radical. The DPPH (diphenyl-P-picrylhydrazyl) radical assay measures the most common natural antioxidants, especially phenols. Thus the radical-scavenging activity against DPPH was evaluated on compounds 6a,b and (S)-15a and on vitamin E, caffeic acid and LD as reference compounds. From the obtained results it can be observed that the compound 6b display an high antioxidant activity comparable to that of the reference compounds even at low concentration. The di-acetyl derivative (S)-15a proved to be the less active in the series probably because it lacks the catecholic or phenolic function. Finally, we have synthesized a new LD prodrug based on an imidazolinone nucleus. Our catecholic compounds might be able to across unchanged the acidic environment of the stomach, are stable enough to be absorbed from the intestine, and they release LD in human plasma after enzymatic hydrolysis. However, work are in progress to evaluate whether an improvement of their lipophilicity might be useful for ameliorate their possibility of adsorption after oral administration and their distribution to the central nervous system.