The M2 muscarinic acetylcholine receptor (M2R) belongs to a highly conserved five-membered family within the class A of G protein-coupled receptors (GPCRs).5-7 The five subtypes are ubiquitously expressed and play a key role in numerous physiological processes.15, 17, 18 Upon activation, the M2R causes, among other things, negative chronotropy and inotropy, central analgesia, and functions as a presynaptic autoreceptor, which modifies effects of other subtypes in the peripheral parasympathetic nervous system.5, 15, 17, 18 The agonists applied hitherto lack subtype-selectivity and thus trigger typical muscarinic effects as adverse events, e.g., bronchospasm, sweating, and diarrhea.1, 51-53 However, recent progress in the structural elucidation of the M2R stimulates the search for selective ligands.7, 14, 19-21 The objective of this thesis was to develop M2R agonists in ligand- and structure-based approaches. A series of hybrids derived from known M2R agonists sheds light on their ligand-receptor interactions. By taking advantage of insights from molecular dynamics (MD) simulations, conducted by Jonas Kaindl (FAU Erlangen-Nürnberg), and docking studies employing the first active M2R crystal structure19, we sought to develop novel M2R ligands. Finally, we present an irreversibly binding derivative of the most intriguing hybrid agonist. Design and synthesis of hybrids derived from M2R agonists To investigate the highly diverse binding affinities55, activation profiles55, and the underlying ligand-receptor interactions of the “superagonist” iperoxo44, the endogenous ligand acetylcholine1, and the full-agonist carbachol1, hybrid derivatives of the three ligands were designed (Figure 44). A set of iperoxo derivatives with varying chain lengths, including two to four carbons, was synthesized based on the convergent synthesis of iperoxo71. The set revealed that the binding affinity improved with increasing chain length, probably due to stronger interactions with Asn6.52. The hybrid consisting of the dihydroisoxazole of iperoxo, and the ethylene spacer of acetylcholine and carbachol, respectively (PR10), displays very weak binding to the M2R. MD simulations indicate that the water-mediated interaction between the carbonyl oxygen of acetylcholine and Asn6.52 seems to be significantly more durable than for the dihydroisoxazole of PR10. The exchange of the butynyl spacer by a more flexible butylene unit (PR13) reveals the importance of its rigidity, which helps to adopt the bent conformation observed in the M2R crystal structure19. The replacement of the dihydroisoxazole moiety of iperoxo by the acetate unit of acetylcholine led to another hybrid derivative (PR15) of the endogenous ligand and the “superagonist”.70 PR15 was easily accessible through the acetylation and N-methylation of dimethylaminobutynol, which is also a central building block in the iperoxo synthesis71.70 Compared to acetylcholine, hybrid PR15 exhibited augmented binding affinity and M2R-selectivity.70 Further functional biological experiments, MD simulations, and solution NMR experiments on ligand-induced conformational changes of the M2R proved that PR15 is a true hybrid, as it features similar characteristics of iperoxo in G protein activation and acetylcholine in β-arrestin recruitment.70 MD simulations revealed that the introduction of the acetylene spacer led to a deeper binding into the M2R.70 Thus, enhanced stability of the water-mediated hydrogen bond between the carbonyl function of PR15 and Asn6.52 and the active rotamer of the “toggle-switch” Trp6.48 was observed.70 In brief, the interaction between the ligand and TM6 is intensified, which could explain the iperoxo-like supraphysiological efficacy in G protein activation of PR15. The NMR studies indicated similar conformational changes of the M2R induced by iperoxo and PR15, which primarily differ from the endogenous ligand acetylcholine near the orthosteric binding pocket and the G protein binding site.70 As a complement to the series of hybrids, structural elements of the two artificial agonists, iperoxo and carbachol, were combined to form PR33. The synthesis was accomplished by transformation of dimethylaminobutynol to a phenyl carbonate, following ammonolysis and N-methylation. Interestingly, the introduction of an acetylene spacer to carbachol resulted in a compound with a diminished binding affinity towards the M2R. The strengthened water-mediated interaction with Asn6.52 detected for PR15 seems not to occur for the hybrid derivative of carbachol. Further structure-activity relationships of the M2 receptor hybrid agonist PR15 The exceptional biological profile of hybrid compound PR15 merited further exploration. For this reason, a series of PR15 derivatives was developed in which the crucial trimethyl butynylammonium unit and the carbonyl oxygen remained untouched. An exchange of the acetate by a ketone gave PR29, whose PR15-like binding affinity supports the finding that the water-mediated interaction exists between Asn6.52 and the carbonyl oxygen rather than the ether oxygen. Remarkably, the selectivity of PR29 for the M2R over M3R even increased from 100- to around 150-fold compared to PR15. Even though amides are generally stronger hydrogen bond acceptors than the analogous esters, the replacement of the acetate of PR15 by an acetamide (PR152) led to a drastic reduction in binding affinity, which could be due to a less favorable geometry. By developing the different ester analogs PR127, PR130, PR138, and PR144, we revealed that prolonged or branched alkyl chains occupy too much space in the binding pocket since these analogs feature diminished binding affinities. Moreover, we designed a conformatively restricted analog of PR15, which showed enantioselective binding of (-) PR90 for the M2R. Retrospective docking indicated that the eutomer is R configured. The synthesis routes of all compounds either base on acylation of dimethylaminobutynol (PR127, PR138, PR144) or aminoalkylation of the prepared terminal alkynes in a Mannich-like reaction71, 149 (PR29, PR90, PR130, PR152) and contain a final methylation step to form the quaternary ammonium salts. Development of novel M2 receptor ligands based on findings from MD simulations The first crystal structure of an acetylcholine receptor in an active conformation (pdb entry 4MQS)19 and MD simulations with the latter provided valuable insights into the architecture of the binding pocket of the M2R. The co-crystallized agonist iperoxo interacts mostly with Asn6.52, Asp3.32, and the “tyrosine-lid” comprising Tyr3.33, Tyr6.51, and Tyr7.39.19 We aimed to develop conformatively fixated compounds that could benefit from a direct interaction to Asn6.52, observed exclusively for iperoxo.64, 70 The design process resulted in tetrahydropyrazolo[1,5-a]pyridines, which demonstrated promising binding poses in docking experiments with the M2R. In a first synthesis approach, a tetrahydropyrazolo[1,5-a]pyridinone was assembled based on 1,3-dipolar cycloaddition103, subsequently reacted by nucleophilic addition with a terminal alkyne and N-methylated to form PR42. A modified synthesis approach allows the introduction of alkyl substituents in the 5-position of tetrahydropyrazolo[1,5-a]pyridines for the first time. Again, 1,3-dipolar cycloaddition103 was applied to form a tetrahydropyrazolo[1,5-a]pyridine-5-carbaldehyde, which could be transformed into a terminal alkyne in a Seyferth-Gilbert homologation116. Aminoalkylation by Mannich-like reaction71, 149, optional ester hydrolysis and decarboxylation as well as N methylation yielded the final compounds PR62, PR67, and PR72. Still, the development of tetrahydropyrazolo[1,5-a]pyridines did not lead to strong binding ligands. Either the docking did not result in compounds that can form a direct hydrogen bond to Asn6.52 or the water-mediated interaction detected for iperoxo and PR15,70 which cannot be formed by the tetrahydropyridine nitrogen, is even more critical for strong ligand binding. Moreover, MD simulations predicted a hydrophilic pocket beneath Asp3.32, which we intended to address with an additional hydroxymethyl function, introduced next to the quaternary ammonium ion of iperoxo. The synthesis was inspired by the established iperoxo synthesis71, but a protected hydroxyacetaldehyde was used in the Mannich-like reaction instead of formaldehyde to implement the additional hydroxymethyl moiety in PR123. The introduction of a polar functionality in the acetylene spacer resulted in a compound with a decreased binding affinity towards the M2R, which indicates that the additional moiety is too bulky for the small hydrophilic pocket. Development of novel M2 receptor ligands based on docking of known agonists In the search for novel M2R agonists, docking experiments based on the M2R crystal structure19 in the active state were conducted. To validate the experiments, we initially docked known agonists such as iperoxo and oxotremorine M, and ideas for novel ligands were generated from the resulting binding poses. First, a series of heteroaryl carboxamides synthesized by amide coupling and different γ lactones did not result in an adequate alternative for the dihydroisoxazole of iperoxo. Moreover, oxotremorine M was modified by the introduction of an oxazolidinone to allow direct hydrogen bonding with Asn6.52. This attempt led to diminished M2R affinity but improved M2R/M3R-selectivity. The results gained from the structure-guided approaches concur with previous studies55. The development of novel M2R agonists remains challenging even when exploiting the structural insights of the active-state M2R. The conformation of the M2R induced by iperoxo appears unique. Besides the stable salt bridge between Asp3.32 and the ammonium head, which is also present for acetylcholine, PR15, and other ligands55, 70, the double hydrogen bond by direct and water-mediated interaction between Asn6.52 and the dihydroisoxazole70 stays inimitable. Development of an irreversible derivative of the M2 receptor hybrid agonist PR15 The final part of this thesis covers the development of a chloroethylamine derivative of hybrid compound PR15 resulting in the covalent agonist PR135. Docking experiments applying the active M2R crystal structure19 prior to the synthesis suggested a binding mode for the active aziridinium ion of PR135 similar to that of PR15 seen in dockings and molecular dynamics simulations70. Inspired by the synthesis of the chloroethylamine derivative of iperoxo19, we synthesized an aminoalcohol precursor, which can be transformed into the reactive chloroethylamine in a final clean reaction step by an Appel-reaction146. PR135 features a submicromolar binding affinity towards the M2R, and the binding affinity towards the M1R and M3R increased significantly compared to the reversible lead analog PR15. These improvements could be beneficial regarding the therapy of xerostomia and glaucoma, as in this case, activation of the M1R and M3R receptor is desired.15 These indications represent exciting fields of application for covalent ligands since the latter need to be administered less frequently than their reversible analogs.129 Experiments on the blocking of activation with the M2R and M3R revealed that PR135 is capable of rapid covalent binding and activating both receptors. Whereas the chloroethylamine derivative of acetylcholine fails to activate the receptor after permanent binding, the irreversible analog of iperoxo features an exceptionally high affinity and efficacy.58, 139 This may require safety precautions limiting its therapeutic use.58 Hence, we present PR135 with its decent agonistic profile for the M2R and M3R as a milder alternative.