Baronissi, Giuliana (2021) Chemistry and Pharmacology of Steroidal and Non-steroidal Modulators of Human Receptors. [Tesi di dottorato]

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Metabolic and inflammatory diseases, affecting the liver and gastrointestinal system, are very widespread and often their pathogenesis is still unknown. Because these disorders represent a growing global public health problem and the present therapies expose the patients to several side effects, there is increased interest in the development of new pharmacological tools that could provide new opportunities in the treatment of complex metabolic disorders in which several target pathways are involved. The main liver manifestation of metabolic disorders are: NASH (Non-Alcoholic SteatoHepatitis), caused by the accumulation of fat in the liver, and PBC (Primary Biliary Cholangitis) an autoimmune disease that causes damage to the small bile ducts. In these pathologies, alterations of bile acid pool regulation have revealed a link between bile acid and metabolic homeostasis. The bile acid receptors farnesoid X receptor (FXR) and GPBAR1 both regulating lipid, glucose and energy metabolism, are today recognized promising targets for NASH and PBC. My research project was mainly focused on the design, synthesis and biological evaluation of small molecules as new modulators of human receptors involved in hepatic and metabolic diseases. Specifically, my research activity was addressed to the investigation of three major targets: the bile acids receptors (FXR and GPBAR1) and the cysteinyl leukotriene receptor 1 (CysLT1R). The obtained results can be summarized in the three main sections reported below according to the target of interest:  Discovery of 6-ethylcholane derivatives as potent bile acid receptor agonists with improved pharmacokinetic properties The nuclear receptor FXR has been proposed as a potential target for the treatment of various pathologies, such as cholestasis, hepatic steatosis, atherosclerosis, dyslipidaemias, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Based on the previous results, extensive ligand/receptor binding studies, using the hGPBAR1 homology model and FXR crystal structure, allowed to elucidate the structural requirements for GPBAR1 and FXR recognition. These outcomes have paved the way for the rational design of a new generation of potent FXR ligands. GPBAR1 has been recently demonstrated the physiological mediator of itching, a common symptom observed in cholestasis and the severity of this side effect limits the pharmacological utility of dual FXR/GPBAR1 agonists in the treatment of different cholestatic disorders. In this context, the discovery of new chemical entities endowed with selective agonistic activity on FXR represents a promising approach in the identification of new pharmacological protocols for the treatment of metabolic disorders. My project concerned the synthesis, starting from the 6-ethylcholane scaffold, of new small molecules as modulators of the nuclear receptor FXR. These studies led us to identify compound 6 as a selective FXR agonist even if with reduced potency respect to 6-ECDCA (EC50=0.5 M) and compounds 1 and 3 which showed a dual activity (FXR/GPBAR1) but with improved pharmacodynamic and pharmacokinetic capabilities.  Synthesis of novel isoxazole derivatives with FXR agonistic activity In vivo acetaminophen (APAP) is one of the most prescribed drugs worldwide, but the misuse causes acute liver failure. Since FXR ligands have shown effective in reducing liver injury in some experimental, I have decided to elaborate the chemical structure of GW4064, the first non-steroidal agonist for FXR, in order to obtain a new library of isoxazoles endowed with FXR agonistic activity. Compound 28 was the most effective FXR agonist of the library (EC50 = 0.30 ± 0.006 M). This compound was orally active and rescued mice from acute liver toxicity caused by APAP overdose.  Development of dual CysLT1R and GPBAR1 modulators In order to develop multitarget drugs for the treatment of various metabolic diseases such as type 2 diabetes, fatty liver disease, dyslipidemia and inflammatory states affecting the enterohepatic system, we have decided to explore if there is a possible cross-talk between CysLT1R antagonists and the two bile acid receptors FXR and GPBAR1. New evidence suggests that cysteinyl leukotriene receptor type 1 (CysLT1R) is a critical signaling molecules implicated in the immune response, cell proliferation, inflammation regulation and intestinal barrier maintenance. Recently, a selected group of CysLT1R antagonists was tested on FXR and GPBAR1 by my research group. Results showed that REV5901 was effective as GPBAR1 agonist attenuating inflammation and immune dysfunction with an EC50 of 2.5 µM. None of the tested compounds exhibited activity on FXR. Therefore, in order to obtain a new library of compounds with improving CysLT1R antagonist/GPBAR1 agonist dual modulation, we have decided to synthetize a new library of compounds preserving the quinoline ring of REV5901 and modifying the substituents on the benzene ring. In vitro and in vivo assays showed that the most effective in modulating the two receptors, were compounds 44 (IC50 = 2.8M and EC50 = 3M, respectively) and 45 (IC50 = 1.2M and EC50 = 7.4M, respectively). These results could be a starting point for the development of new drugs for the treatment of metabolic and inflammatory diseases.

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