Design, synthesis and optimization of receptor ligands: tools for studying purine-activated and related orphan G protein-coupled receptors

Abstract

<strong>Adenosine receptors</strong> <br /> The A_2A and A_2B adenosine receptors (ARs) are promising drug targets in Alzheimer´s and Parkinson´s disease, inflammatory diseases, autoimmune diseases, diabetes and, most importantly, in cancer (immuno)therapy. The A_2AAR is expressed on a variety of immune cells including natural killer cells and T-lymphocytes, and its stimulation decreases cytokine production causing immunosuppression. Stimulation of A_2BARs supports cancer cells by inducing tumor proliferation, metastasis, angiogenesis, and it mediates immunosuppressive effects by activation of A2BARs on immune cells. Consequently, blocking of A_2A- and A_2BARs is a promising strategy in cancer (immuno)therapy. In this thesis, a series of xanthine-based receptor antagonists was synthesized, their SARs were analyzed, and fluorescence-labeled probes for biological studies were developed. In another sub-project, a series of A2AAR antagonists was developed and explored with regard to its SARs. <br /> <strong>Exploration of N-acyl-8-(4-piperazine-1-sulfonyl)phenylxanthines as potent and selective adenosine A2B receptor antagonists </strong> <br /> Potent and selective A2BAR antagonists based on the xanthine scaffold have been reported in literature. On the basis of parent compound 7, the new scaffold 8-(4-acylpiperazine-1-sulfonyl)phenyl-1-propylxanthine was explored by straightforward acylation of the piperazine moiety of precursor 12 with a variety of carboxylic acids, yielding 19 novel A2BAR antagonists (see Figure 1). N-Acylxanthines can be formed by employing the coupling reagent HBTU at rt. An SAR analysis of the new compounds revealed that benzoyl moieties bearing lipophilic substituents (such as halogens or CF3) and substituted 2-phenylfuran moieties attached to the piperazine residue lead to A2BAR antagonists with subnanomolar affinities and high selectivities versus the other AR subtypes. Derivative 29 turned out to be the most potent (K_i A_2B 0.246 nM) and selective (>4,000-fold) compound in this series (see Figure 1), and can be considered as a new lead structure for further optimization, e.g. regarding pharmacokinetic properties. <br /> <strong>Functionalized potent and selective 8-(4-acylpiperazinyl-1- sulfonyl)phenylxanthines providing a platform for fluorescence-labeling of adenosine A_2B receptor antagonists</strong> <br /> Fluorescence-labeled AR antagonists are versatile biological probes for a variety of applications including microscopy, flow cytometry, and ligand binding assays. In this project, a functionalized cyanine fluorophore was combined with 8-substituted xanthine derivatives forming novel potent and selective A_2BAR antagonists as biological probes. Xanthine precursor 12 was conjugated with a variety of benzoic acid derivatives that exhibited reactive groups. Cyanine dyes bearing functionalized linkers were coupled to these xanthines forming fluorescent A2BAR ligands. Compounds 39 (PSB-20077) and 40 (PSB-20104) showed high potency (K_i A_2BAR 149 nM and 159 nM) and subtype-selectivity, and they were investigated in confocal microscopy studies, revealing specific binding to the A2BAR. Currently, 39 and 40 will be evaluated as pharmacological probes, e.g. for BRET-based binding assays. <br /> <strong>Synthesis of 3 amino-N-benzyl-1-phenyl-1H-pyrazole-4-carboxamides as A2AAR antagonists</strong> <br /> 5-Amino-2-phenyl-1,2,3-triazole derivatives have been reported as A2AAR antagonists in literature. In this study, closely related 3-amino-N-benzyl-1-phenyl-1H-pyrazole-4-carboxamide derivatives were explored as A_2AAR antagonists. Therefore, a regioselective synthesis, starting from the appropriate phenylhydrazines, was employed and improved. A protecting group strategy could exclude the formation of undesired regioisomers. Moreover, copper-catalyzed coupling reactions were explored with regard to the formation of N1-substituted 3-aminopyrazoles. A compound library of 53 new A2A-antagonists was prepared and analyzed with regard to SARs at the four AR subtypes. Figure 3 illustrates the most potent compound 36a as well as the general insights in the SARs observed for this scaffold. As a result, the 1H-indazol-5-yl residue turned out to be the most favorable moiety at the N1-position leading to compounds with affinities in the low nanomolar range between 5.07 and 68.2 nM (36a-36f). Small residues (such as a hydroxy group) at the meta- and/or para-position were very well tolerated by the A_2AAR. 2-Chloro-6-fluorobenzylamine stood out as the best substituent for the benzylamine moiety. However, small substituents in the ortho-position (such as halogens or hydroxy groups) showed good results as well. Compounds 9a, 21a and 21c were submitted to metabolic stability studies, and water-solubility of 9a was measured. Our studies suggested drug-like properties for this scaffold. Compounds 21e and 36a are planned to be tested as therapeutics in cancer models. <br /> <strong>Synthesis of 2,4,6,8-tetrasubstituted pyrimido[5,4-d]pyrimidines as MRGPRX3 agonists</strong> The MRGPRX3 is an orphan GPCR that is predicted to be involved in pain and itch perception. In the present study, the first agonist screening hit, dipyridamole, was the prototype for the synthesis of various MRGPRX3 agonists on the basis of 2,4,6,8-tetrasubstituted pyrimido[5,4 d]pyrimidines. For a rational synthesis, 2,4,6,8-tetrachloropyrimido[5,4-d] pyrimidine (TCPP) was stepwise substituted with nucleophiles under controlled conditions. This yielded 40 compounds with defined substitution patterns. An analysis of their biological activity revealed steep SARs; only very specific substitution patterns led to potent compounds. SARs indicated that alternative binding modes are likely. Derivative 96 was the most potent compound with an EC50 value of 0.942 nM. In addition, 8 compounds based on a purine scaffold and 4 compounds based on a 2,4-disubstituted pyrimidine scaffold were prepared, however, none of those compounds showed any significant activity for MRGPRX3. All derivatives of this project were additionally tested as antagonists but no compound showed antagonistic activity. For the future, 96 can be considered as the new lead structure. Furthermore, new scaffolds should be tested as ligands for MRGPRX3, especially antagonists would be of great interest due to their expected biological activities, e.g. in pain therapy.