Clinical development of catechol-based orthosteric agonists of the dopamine D1 receptor has thus far been unsuccessful due to multiple challenges. To address these issues, we identified LY3154207 (3) as a novel, potent, and subtype selective human D1 positive allosteric modulator (PAM) with minimal allosteric agonist activity. Conformational studies showed LY3154207 adopts an unusual boat conformation, and a binding pose with the human D1 receptor was proposed based on this observation. In contrast to orthosteric agonists, LY3154207 showed a distinct pharmacological profile without a bell-shaped dose-response relationship or tachyphylaxis in preclinical models. Identification of a crystalline form of free LY3154207 from the discovery lots was not successful. Instead, a novel cocrystal form with superior solubility was discovered and determined to be suitable for development. This cocrystal form was advanced to clinical development as a potential first-in-class D1 PAM and is now in phase 2 studies for Lewy body dementia.

The D1 dopamine receptor is linked to a variety of neuropsychiatric disorders and represents an attractive drug target for the enhancement of cognition in schizophrenia, Alzheimer disease, and other disorders. Positive allosteric modulators (PAMs), with their potential for greater selectivity and larger therapeutic windows, may represent a viable drug development strategy, as orthosteric D1 receptor agonists possess known clinical liabilities. We discovered two structurally distinct D1 receptor PAMs, MLS6585 and MLS1082, via a high-throughput screen of the NIH Molecular Libraries program small-molecule library. Both compounds potentiate dopamine-stimulated G protein- and β-arrestin-mediated signaling and increase the affinity of dopamine for the D1 receptor with low micromolar potencies. Neither compound displayed any intrinsic agonist activity. Both compounds were also found to potentiate the efficacy of partial agonists. We tested maximally effective concentrations of each PAM in combination to determine if the compounds might act at separate or similar sites. In combination, MLS1082 + MLS6585 produced an additive potentiation of dopamine potency beyond that caused by either PAM alone for both β-arrestin recruitment and cAMP accumulation, suggesting diverse sites of action. In addition, MLS6585, but not MLS1082, had additive activity with the previously described D1 receptor PAM "Compound B," suggesting that MLS1082 and Compound B may share a common binding site. A point mutation (R130Q) in the D1 receptor was found to abrogate MLS1082 activity without affecting that of MLS6585, suggesting this residue may be involved in the binding/activity of MLS1082 but not that of MLS6585. Together, MLS1082 and MLS6585 may serve as important tool compounds for the characterization of diverse allosteric sites on the D1 receptor as well as the development of optimized lead compounds for therapeutic use.

The ability to rapidly assess the preferred conformation of key fragments in a structure "by visual inspection" is a very useful starting point in the process of drug design. With the ability to do so, one could address questions like: "How could we avoid planarity in a molecule?", "Will a molecule change its conformational preference if we make it more or less basic?" or "How does this electronic repulsion affect the conformational preference in the system?" in timely fashion. In this paper, we describe how the conformational energy profile (CEP, plot of energy as a function of dihedral bond angle) of a fragment can be interpreted through the understanding the interplay between resonance stabilization, steric effects and electrostatic interactions. Fifty-nine biaryl and aryl carbonyl fragments present in oral drugs or which are close derivatives thereof were selected. Calculation of their CEPs using ab initio methodology allowed us to conclude the relative importance of these factors in the conformational preference of these fragments as follows: "steric repulsion > lone pair-lone pair repulsion > lone pair-fluorine repulsion > resonance stabilization" and to formulate "rules of thumb" that the practicing medicinal/organic chemist can apply when analysing molecules that contain these fragments.

DETQ, an allosteric potentiator of the dopamine D1 receptor, was tested in therapeutic models that were known to respond to D1 agonists. Because of a species difference in affinity for DETQ, all rodent experiments used transgenic mice expressing the human D1 receptor (hD1 mice). When given alone, DETQ reversed the locomotor depression caused by a low dose of reserpine. DETQ also acted synergistically with L-DOPA to reverse the strong hypokinesia seen with a higher dose of reserpine. These results indicate potential as both monotherapy and adjunct treatment in Parkinson's disease. DETQ markedly increased release of both acetylcholine and histamine in the prefrontal cortex, and increased levels of histamine metabolites in the striatum. In the hippocampus, the combination of DETQ and the cholinesterase inhibitor rivastigmine increased ACh to a greater degree than either agent alone. DETQ also increased phosphorylation of the AMPA receptor (GluR1) and the transcription factor CREB in the striatum, consistent with enhanced synaptic plasticity. In the Y-maze, DETQ increased arm entries but (unlike a D1 agonist) did not reduce spontaneous alternation between arms at high doses. DETQ enhanced wakefulness in EEG studies in hD1 mice and decreased immobility in the forced-swim test, a model for antidepressant-like activity. In rhesus monkeys, DETQ increased spontaneous eye-blink rate, a measure that is known to be depressed in Parkinson's disease. Together, these results provide support for potential utility of D1 potentiators in the treatment of several neuropsychiatric disorders, including Parkinson's disease, Alzheimer's disease, cognitive impairment in schizophrenia, and major depressive disorder.

Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a Kb of 26 nM. The maximum response to DETQ alone was ∼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was ∼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3-20 mg/kg orally) caused a robust (∼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30-240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists.

Allosteric potentiators of the dopamine D1 receptor could be useful for treatment of Parkinson's disease, schizophrenia, depression, ADHD, and narcolepsy. We identified a novel D1 potentiator (“DETQ”)by high‐throughput screening followed by iterative chemical optimization. In a HEK293 cell line expressing the human D1 receptor, agonist‐stimulated cAMP production was measured in the presence of an EC20 concentration of dopamine using homogeneous time resolved fluorescence (HTRF) technology. DETQ potentiated the dopamine response with a mean EC50 of 5.8 nM and mean efficacy corresponding to 95% of the maximum response to dopamine. In the absence of exogenous dopamine (agonist mode), the maximum efficacy of DETQ was 11.6% with an EC50 of 30 nM; based on the initial slopes of the two curves (Ehlert R‐ratio), DETQ was thus 43‐fold less potent as an allosteric agonist than as a potentiator. When concentration‐response curves for dopamine were carried out at multiple DETQ concentrations in the cAMP assay, DETQ shifted the dopamine curve 21‐fold to the left with a KB of 26 nM. In experiments measuring binding of the D1 antagonist 3H‐SCH23390 to the human D1 receptor, 100 nM DETQ caused a 5‐fold leftward shift in the concentration‐inhibition curve for dopamine, indicating adirect effect of DETQ on the D1 receptor. The potency of DETQ in the cAMP assay was about 50‐fold lower at the mouse and rat D1 receptors than at human, rhesus, and dog D1 receptors. DETQ was inactive at concentrations up to atleast 10 uM when tested in agonist and potentiator modes at related receptors, including D2, D5, β1, β2, β3, and 5HT6. DETQ was also inactive in binding and/or functional assays at a largeset of unrelated targets.These results suggest that DETQ could be a useful tool to probe applications of dopamine D1 potentiators for CNS disorders.

Allosteric potentiators increase the affinity of endogenousagonist, in effect amplifying physiological control circuits. Because a potentiator should depend onendogenous tone, its effects are predicted to be self‐limiting and less proneto rapid tolerance development compared to direct‐acting agonists. Our objective was to test this hypothesis using DETQ, a novel allosteric potentiator of the dopamine D1 receptor.Although DETQ has high affinity for the human D1 receptor, it is 70‐fold less potent at the mouse and rat D1 receptors, limiting its use as a pharmacological tool in rodents. To overcome this limitation, we created a transgenic knock‐in mouse expressing the human D1 receptor (hD1). Homozygotes showed normal behavior and breeding.After oral dosing, DETQ caused a dose‐dependent 10‐fold increase in locomotor activity in habituated hD1 mice but not in wild‐type mice, implying a requirement for the human D1 receptor. The increase in locomotor activity was blocked by the D1 antagonist SCH39166 and by pretreatment with a high dose of reserpine, indicating that the behavioral response is dependent on endogenous dopamine release. At higher doses, the response to DETQ reached a plateau even though brain concentrations of unbound drug continued to rise. In contrast, the D1 agonists SKF82958 and A‐77636 showed bell‐shaped dose‐response curves, with a profound decrease in locomotor activity at the highest doses. The suppression of locomotor activity at high doses was due to engagement of competing stereotyped behaviors such as intense grooming. The stereotyped behaviors were not seen with DETQ, providing evidence that the response to DETQ is less liable to cause over stimulation. In repeated dosing over four days, the locomotor response to DETQ was maintained, whereas the response to A‐77636 showed rapid tolerance.In hD1 mice treated with a low dose of reserpine, DETQ restored locomotor activity to untreated control levels; this model is relevant to stand‐alone therapy in mild‐to‐moderate Parkinson's disease. Versus a higher dose of reserpine, DETQ acted synergistically with L‐DOPA in restoring locomotor activity. DETQ also increased wakefulness and decreased sleep and was found to be effective in a behavioral despair model. We also compared DETQ with SKF82958 for efficacy in the Y‐maze. While both compounds enhanced the number of arm entries in a dose–dependent fashion, mice treated with DETQ maintained spontaneous alternation even at high doses, whereas mice treated with higher doses of SKF82958 showed an increased prevalence to return to the same arm (asign of cognitive dysfunction). Finally, DETQ increased spontaneous eye blink rate in the rhesus monkey, a response related to central D1 activation.These results confirm that D1 potentiators may possess advantages over D1 agonists for the treatment of Parkinson's disease and other CNS disorders.

Preclinical experiments and clinical observations suggest the potential effectiveness of selective 5-HT1F receptor agonists in migraine. Identifying compounds with enhanced selectivity is crucial to assess its therapeutic value. Replacement of the indole nucleus in 2 (LY334370) with a monocyclic phenyl ketone moiety generated potent and more selective 5-HT1F receptor agonists. Focused SAR studies around this central phenyl ring demonstrated that the electrostatic and steric interactions of the substituent with both the amide CONH group and the ketone CO group play pivotal roles in affecting the adopted conformation and thus the 5-HT1F receptor selectivity. Computational studies confirmed the observed results and provide a useful tool in the understanding of the conformational requirements for 5-HT1F receptor agonist activity and selectivity. Through this effort, the 2-F-phenyl and N-2-pyridyl series were also identified as potent and selective 5-HT1F receptor agonists.

Positron emission tomography (PET) imaging has become a useful noninvasive technique to explore molecular biology within living systems; however, the utility of this method is limited by the availability of suitable radiotracers to probe specific targets and disease biology. Methods to identify potential areas of improvement in the ability to predict small molecule performance as tracers prior to radiolabeling would speed the discovery of novel tracers. In this retrospective analysis, we characterized the brain penetration or peak SUV (standardized uptake value), binding potential (BP), and brain exposure kinetics across a series of known, nonradiolabeled PET ligands using in vivo LC-MS/MS (liquid chromatography coupled to mass spectrometry) and correlated these parameters with the reported PET ligand performance in nonhuman primates and humans available in the literature. The PET tracers studied included those reported to label G protein-coupled receptors (GPCRs), intracellular enzymes, and transporters. Additionally, data for each tracer was obtained from a mouse brain uptake assay (MBUA), previously published, where blood–brain barrier (BBB) penetration and clearance parameters were assessed and compared against similar data collected on a broad compound set of central nervous system (CNS) therapeutic compounds. The BP and SUV identified via nonradiolabeled LC-MS/MS, while different from the published values observed in the literature PET tracer data, allowed for an identification of initial criteria values we sought to facilitate increased potential for success from our early discovery screening paradigm. Our analysis showed that successful, as well as novel, clinical PET tracers exhibited BP of greater than 1.5 and peak SUVs greater than approximately 150% at 5 min post dose in rodents. The brain kinetics appeared similar between both techniques despite differences in tracer dose, suggesting linearity across these dose ranges. The assessment of tracers in a CNS exposure model, the mouse brain uptake assessment (MBUA), showed that those compound with initial brain-to-plasma ratios >2 and unbound fraction in brain homogenate >0.01 were more likely to be clinically successful PET ligands. Taken together, early incorporation of a LC/MS/MS cold tracer discovery assay and a parallel MBUA can be an useful screening paradigm to prioritize and rank order potential novel PET radioligands during early tracer discovery efforts. Compounds considered for continued in vivo PET assessments can be identified quickly by leveraging in vitro affinity and selectivity measures, coupled with data from a MBUA, primarily the 5 min brain-to-plasma ratio and unbound fraction data. Coupled utilization of these data creates a strategy to efficiently screen for the identification of appropriate chemical space to invest in for radiotracer discovery.

Positron emission tomography (PET) imaging has become a useful noninvasive technique to explore molecular biology within living systems; however, the utility of this method is limited by the availability of suitable radiotracers to probe specific targets and disease biology. Methods to identify potential areas of improvement in the ability to predict small molecule performance as tracers prior to radiolabeling would speed the discovery of novel tracers. In this retrospective analysis, we characterized the brain penetration or peak SUV (standardized uptake value), binding potential (BP), and brain exposure kinetics across a series of known, nonradiolabeled PET ligands using in vivo LC-MS/MS (liquid chromatography coupled to mass spectrometry) and correlated these parameters with the reported PET ligand performance in nonhuman primates and humans available in the literature. The PET tracers studied included those reported to label G protein-coupled receptors (GPCRs), intracellular enzymes, and transporters. Additionally, data for each tracer was obtained from a mouse brain uptake assay (MBUA), previously published, where blood-brain barrier (BBB) penetration and clearance parameters were assessed and compared against similar data collected on a broad compound set of central nervous system (CNS) therapeutic compounds. The BP and SUV identified via nonradiolabeled LC-MS/MS, while different from the published values observed in the literature PET tracer data, allowed for an identification of initial criteria values we sought to facilitate increased potential for success from our early discovery screening paradigm. Our analysis showed that successful, as well as novel, clinical PET tracers exhibited BP of greater than 1.5 and peak SUVs greater than approximately 150% at 5 min post dose in rodents. The brain kinetics appeared similar between both techniques despite differences in tracer dose, suggesting linearity across these dose ranges. The assessment of tracers in a CNS exposure model, the mouse brain uptake assessment (MBUA), showed that those compound with initial brain-to-plasma ratios >2 and unbound fraction in brain homogenate >0.01 were more likely to be clinically successful PET ligands. Taken together, early incorporation of a LC/MS/MS cold tracer discovery assay and a parallel MBUA can be an useful screening paradigm to prioritize and rank order potential novel PET radioligands during early tracer discovery efforts. Compounds considered for continued in vivo PET assessments can be identified quickly by leveraging in vitro affinity and selectivity measures, coupled with data from a MBUA, primarily the 5 min brain-to-plasma ratio and unbound fraction data. Coupled utilization of these data creates a strategy to efficiently screen for the identification of appropriate chemical space to invest in for radiotracer discovery.

MePPEP ((3R,5R)-5-(3-methoxy-phenyl)-3-((R)-1-phenyl-ethylamino)-1-(4-trifluoromethyl-phenyl)-pyrrolidin-2-one) is an inverse agonist shown to be an effective PET ligand for labeling cannabinoid CB1 receptors in vivo. [11C]MePPEP and structurally related analogs have been reported to specifically and reversibly label cannabinoid CB1 receptors in rat and non-human primate brains, and [11C]MePPEP has been used in human subjects as a PET tracer. We have generated [3H]MePPEP, an ortholog of [11C]MePPEP, to characterize the molecular pharmacology of the cannabinoid CB1 receptor across preclinical and clinical species. [3H]MePPEP demonstrates saturable, reversible, and single-site high affinity binding to cannabinoid CB1 receptors. In cerebellar membranes purified from brains of rat, non-human primate and human, and cells ectopically expressing recombinant human cannabinoid CB1 receptor, [3H]MePPEP binds cannabinoid CB1 receptors with similar affinity with Kd values of 0.09 nM, 0.19 nM, 0.14 nM and 0.16 nM, respectively. Both agonist and antagonist cannabinoid ligands compete [3H]MePPEP with predicted rank order potency. No specific binding is present in autoradiographic sections from cannabinoid CB1 receptor knockout mouse brains, demonstrating that [3H]MePPEP selectively binds cannabinoid CB1 receptors in native mouse tissue. Furthermore, [3H]MePPEP binding to anatomical sites in mouse and rat brain is comparable to the anatomical profiles of [11C]MePPEP in non-human primate and human brain in vivo, as well as the binding profiles of other previously described cannabinoid CB1 receptor agonist and antagonist radioligands. Therefore, [3H]MePPEP is a promising tool for translation of preclinical cannabinoid CB1 receptor pharmacology to clinical PET ligand and cannabinoid CB1 receptor inverse agonist therapeutic development.