Design, Synthesis and Structure-Activity-Relationships of Potent Mas-Related G Protein-Coupled Receptor X (MRGPRX) Modulators

Abstract

The G protein-coupled receptor (GPCR) superfamily with more than 800 members is one of the most important target classes for drug development. After an agonist binds to the receptor, it induces a conformational change leading to the activation of a heterotrimeric guanine nucleotide-binding protein (G protein). G proteins play a key role in signal transduction from GPCRs activated by extracellular signals to intracellular second messenger systems. GPCRs are involved in almost all physiological and pathophysiological processes. About 100 of the more than 800 human GPCRs are so-called orphan receptors, whose endogenous ligands remain to be identified or confirmed. <br /> In 2001, Dong <em>et al</em>. discovered a subfamily of orphan receptors comprising over 50 rodent and human GPCRs, which were termed Mas-related gene receptors (MRGs). Later, in 2002, Lembo <em>et al</em>. discovered the same GPCR family to be expressed in rat primary cultures of dorsal root ganglia (DRG) and designated them sensory neuron-specific receptors (SNSRs). The current nomenclature declared by the International Union of Pharmacology (IUPHAR) of these orphan receptors is Mas-related G protein-coupled receptors (MRGPR). <br /> This receptor family can be divided into nine distinct subfamilies (MRGPRA-H and -X). They belong to the δ-branch of class A, rhodopsin-like GPCR subgroup. Among the nine subfamilies of MRGPRs, four members (MRGPRX1-4) are primate-specific. Due to their expression profile, they are supposed to be involved, e.g., in pain transmission, immune responses, itching and wound healing. <br /> The present study was aimed at developing potent, selective ligands as pharmacological tool compounds for investigating the poorly studied subtypes MRGPRX2 and -4 based on previously discovered hit compounds. <br /> To achieve this, we initially developed an optimized synthesis of carboxamides of 5,6- diaminouracils, which are crucial intermediates for the synthesis of xanthines, a novel scaffold for MRGPRX4 agonists. Subsequently, their physicochemical properties were examined in detail. These studies led to two publications (Marx, D., Wingen, L.M., Schnakenburg, G., Müller, C.E., Scholz, M.S. Synthesis of 6-amino-5-carboxamidouracils as precursors for 8- substituted xanthines. <em>Front. Chem</em>., 2019, 7, 56, 1-15 and Marx, D., Schnakenburg, G., Grimme, S., Müller, C.E. Structural and conformational studies on carboxamides of 5,6-diaminouracils-precursors of biologically active xanthine derivatives. <em>Molecules</em> 2019, 24, 2168). <br /> The optimization of the synthesis of key precursors for MRGPRX4 ligands provided the basis for the development of a large number of diverse MRGPRX4 agonists, and part of this study was submitted as a patent application (Marx, D.; Müller, C.; Alnouri, W.; Riedel, Y.; Namasivayam, V.; Pillaiyar, T.; Thimm, D.; Hockemeyer, J. MRGPRX4 agonists and antagonists. <em>UBN0005(EP)</em> 2020). Since no synthetic procedure for the synthesis of these selective and highly potent MRGPRX4 agonists was known in the literature, a broad range of chemical methods had to be investigated to finally obtain the desired target compounds in 6 to 9 synthesis steps. This reaction usually starts with 6-aminouracils, which can be selectively alkylated at the N3 position, followed by nitrosylation and subsequent reduction to obtain 5,6-diaminouracil derivatives. A further regioselective amide coupling reaction yields the 6-amino- 5-carboxamidouracil derivatives described above. With diethyl(4-iodobutyl)phosphonate as alkylating agent, followed by a ring closure reaction using 2N NaOH, the desired xanthine derivatives bearing a alkyle phosphonate group in the N3 position can be obtained after cleavage of the protective group of the phosphonic acid ester. The compounds were designed supported by molecular modeling, synthesized and structure- activity relationship (SARs) were analyzed. Homology models were established, and molecular docking studies were performed to support the drug development efforts. In cooperation with pharmacologists, the potencies of the newly synthesized compounds were determined in calcium mobilization and β-arrestin assays. <br /> A further patent application describes the design, synthesis and structure-activity relationships of a series of novel tricyclic benzimidazole derivatives as antagonists for MRGPRX2 (Müller, C.; Alnouri, W.; Riedel, Y.; Thimm, D.; Marx, D.; Namasivayam, V.; Gattner, S.; Herdewyn, P.; De Jonghe, S.; Leonczak, P.; Verdonck, S. MRGX receptor antagonists. <em>US16/670,149</em> 2019). The new MRGPRX2 antagonists were shown to inhibit mast cell degranulation and thus constitute potential novel therapeutics for asthma, allergic rhinitis, anaphylaxis, chronic urticaria, atopic dermatitis and other inflammatory diseases. <br /> The final part of the present cumulative thesis describes the development of MRGPRX2 antagonists that show significantly improved water solubility compared to the compounds disclosed in the above-mentioned patent. <br /> The present thesis constitutes a significant advance in the field of MRGPRX receptors and their modulators, which have great potential as future drugs.