Structure–Activity Relationships of Privileged Structures Lead to the Discovery of Novel Biased Ligands at the Dopamine D₂ Receptor

Biased agonism at GPCRs highlights the potential for the discovery and design of pathway-selective ligands and may confer therapeutic advantages to ligands targeting the dopamine D₂ receptor (D₂R). We investigated the determinants of efficacy, affinity, and bias for three privileged structures for the D₂R, exploring changes to linker length and incorporation of a heterocyclic unit. Profiling the compounds in two signaling assays (cAMP and pERK1/2) allowed us to identify and quantify determinants of biased agonism at the D₂R. Substitution on the phenylpiperazine privileged structures (2-methoxy vs 2,3-dichloro) influenced bias when the thienopyridine heterocycle was absent. Upon inclusion of the thienopyridine unit, the substitution pattern (4,6-dimethyl vs 5-chloro-6-methoxy-4-methyl) had a significant effect on bias that overruled the effect of the phenylpiperazine substitution pattern. This latter observation could be reconciled with an extended binding mode for these compounds, whereby the interaction of the heterocycle with a secondary binding pocket may engender bias

Homobivalent Ligands of the Atypical Antipsychotic Clozapine: Design, Synthesis, and Pharmacological Evaluation

To date all typical and atypical antipsychotics target the dopamine D₂ receptor. Clozapine represents the best-characterized atypical antipsychotic, although it displays only moderate (submicromolar) affinity for the dopamine D₂ receptor. Herein, we present the design, synthesis, and pharmacological evaluation of three series of homobivalent ligands of clozapine, differing in the length and nature of the spacer and the point of attachment to the pharmacophore. Attachment of the spacer at the N4′ position of clozapine yielded a series of homobivalent ligands that displayed spacer-length-dependent gains in affinity and activity for the dopamine D₂ receptor. The 16 and 18 atom spacer bivalent ligands were the highlight compounds, displaying marked low nanomolar receptor binding affinity (1.41 and 1.35 nM, respectively) and functional activity (23 and 44 nM), which correspond to significant gains in affinity (75- and 79-fold) and activity (9- and 5-fold) relative to the original pharmacophore, clozapine. As such these ligands represent useful tools with which to investigate dopamine receptor dimerization and the atypical nature of clozapine

Development of a Photoactivatable Allosteric Ligand for the M₁ Muscarinic Acetylcholine Receptor

The field of G protein-coupled receptor drug discovery has benefited greatly from the structural and functional insights afforded by photoactivatable ligands. One G protein-coupled receptor subfamily for which photoactivatable ligands have been developed is the muscarinic acetylcholine receptor family, though, to date, all such ligands have been designed to target the orthosteric (endogenous ligand) binding site of these receptors. Herein we report the synthesis and pharmacological investigation of a novel photoaffinity label, MIPS1455 (<b>4</b>), designed to bind irreversibly to an allosteric site of the M₁ muscarinic acetylcholine receptor; a target of therapeutic interest for the treatment of cognitive deficits. MIPS1455 may be a valuable molecular tool for further investigating allosteric interactions at this receptor

Synthesis and Pharmacological Evaluation of M₄ Muscarinic Receptor Positive Allosteric Modulators Derived from VU10004

The M₄ mAChR is implicated in several CNS disorders and possesses an allosteric binding site for which ligands modulating the affinity and/or efficacy of ACh may be exploited for selective receptor targeting. We report the synthesis of a focused library of putative M₄ PAMs derived from VU10004. These compounds investigate the pharmacological effects of target thieno­[2,3-<i>b</i>]­pyridines assembled from primary cycloalkanamines and cyclic secondary amines providing useful estimates of affinity (<i>K</i>_B), cooperativity (αβ), and direct agonist properties (τ_B)

Probing Structural Requirements of Positive Allosteric Modulators of the M₄ Muscarinic Receptor

The M₄ mAChR is implicated in several CNS disorders and possesses an allosteric binding site for which ligands modulating the affinity and/or efficacy of ACh may be exploited for selective receptor targeting. We report the synthesis of a focused library of putative M₄ PAMs derived from VU0152100 and VU10005. These compounds investigate the pharmacological effects of previously identified methoxy and fluoro substituents, providing useful estimates of affinity (<i>K</i>_B), cooperativity (αβ), and direct agonist properties (τ_B)

Proof of Concept Study for Designed Multiple Ligands Targeting the Dopamine D₂, Serotonin 5‑HT_2A, and Muscarinic M₁ Acetylcholine Receptors

Herein we describe the hybridization of a benzoxazinone M₁ scaffold with D₂ privileged structures derived from putative and clinically relevant antipsychotics to develop designed multiple ligands. The M₁ mAChR is an attractive target for the cognitive deficits in key CNS disorders. Moreover, activity at D₂ and 5-HT_2A receptors has proven useful for antipsychotic efficacy. We identified <b>9</b> which retained functional activity at the target M₁ mAChR and D₂R and demonstrated high affinity for the 5-HT_2AR

Subtle Modifications to the Indole-2-carboxamide Motif of the Negative Allosteric Modulator <i>N</i>‑((<i>trans</i>)‑4-(2-(7-Cyano-3,4-dihydroisoquinolin-2(1<i>H</i>)‑yl)ethyl)cyclohexyl)‑1<i>H</i>‑indole-2-carboxamide (SB269652) Yield Dramatic Changes in Pharmacological Activity at the Dopamine D₂ Receptor

SB269652 (<b>1</b>) is a negative allosteric modulator of the dopamine D₂ receptor. Herein, we present the design, synthesis, and pharmacological evaluation of “second generation” analogues of <b>1</b> whereby subtle modifications to the indole-2-carboxamide motif confer dramatic changes in functional affinity (5000-fold increase), cooperativity (100-fold increase), and a novel action to modulate dopamine efficacy. Thus, structural changes to this region of <b>1</b> allows the generation of a novel set of analogues with distinct pharmacological properties

A Structure–Activity Analysis of Biased Agonism at the Dopamine D2 Receptor

Biased agonism offers an opportunity for the medicinal chemist to discover pathway-selective ligands for GPCRs. A number of studies have suggested that biased agonism at the dopamine D₂ receptor (D₂R) may be advantageous for the treatment of neuropsychiatric disorders, including schizophrenia. As such, it is of great importance to gain insight into the SAR of biased agonism at this receptor. We have generated SAR based on a novel D₂R partial agonist, <i>tert</i>-butyl (<i>trans</i>-4-(2-(3,4-dihydroisoquinolin-2­(1<i>H</i>)-yl)­ethyl)­cyclohexyl)­carbamate (<b>4</b>). This ligand shares structural similarity to cariprazine (<b>2</b>), a drug awaiting FDA approval for the treatment of schizophrenia, yet displays a distinct bias toward two different signaling end points. We synthesized a number of derivatives of <b>4</b> with subtle structural modifications, including incorporation of cariprazine fragments. By combining pharmacological profiling with analytical methodology to identify and to quantify bias, we have demonstrated that efficacy and biased agonism can be finely tuned by minor structural modifications to the head group containing the tertiary amine, a tail group that extends away from this moiety, and the orientation and length of a spacer region between these two moieties

Synthesis and Pharmacological Evaluation of Analogues of Benzyl Quinolone Carboxylic Acid (BQCA) Designed to Bind Irreversibly to an Allosteric Site of the M₁ Muscarinic Acetylcholine Receptor

Activation of the M₁ muscarinic acetylcholine receptor (mAChR) is a prospective treatment for alleviating cognitive decline experienced in central nervous system (CNS) disorders. Current therapeutics indiscriminately enhance the activity of the endogenous neurotransmitter ACh, leading to side effects. BQCA is a positive allosteric modulator and allosteric agonist at the M₁ mAChR that has high subtype selectivity and is a promising template from which to generate higher affinity, more pharmacokinetically viable drug candidates. However, to efficiently guide rational drug design, the binding site of BQCA needs to be conclusively elucidated. We report the synthesis and pharmacological validation of BQCA analogues designed to bind irreversibly to the M₁ mAChR. One analogue in particular, <b>11</b>, can serve as a useful structural probe to confirm the location of the BQCA binding site; ideally, by co-crystallization with the M₁ mAChR. Furthermore, this ligand may also be used as a pharmacological tool with a range of applications

Structure–Activity Study of <i>N</i>‑((<i>trans</i>)‑4-(2-(7-Cyano-3,4-dihydroisoquinolin-2(1<i>H</i>)‑yl)­ethyl)­cyclohexyl)‑1<i>H</i>‑indole-2-carboxamide (SB269652), a Bitopic Ligand That Acts as a Negative Allosteric Modulator of the Dopamine D₂ Receptor

We recently demonstrated that SB269652 (<b>1</b>) engages one protomer of a dopamine D₂ receptor (D₂R) dimer in a bitopic mode to allosterically inhibit the binding of dopamine at the other protomer. Herein, we investigate structural determinants for allostery, focusing on modifications to three moieties within <b>1</b>. We find that orthosteric “head” groups with small 7-substituents were important to maintain the limited negative cooperativity of analogues of <b>1</b>, and replacement of the tetrahydroisoquinoline head group with other D₂R “privileged structures” generated orthosteric antagonists. Additionally, replacement of the cyclohexylene linker with polymethylene chains conferred linker length dependency in allosteric pharmacology. We validated the importance of the indolic NH as a hydrogen bond donor moiety for maintaining allostery. Replacement of the indole ring with azaindole conferred a 30-fold increase in affinity while maintaining negative cooperativity. Combined, these results provide novel SAR insight for bitopic ligands that act as negative allosteric modulators of the D₂R