Synthesis of enantiomerically pure [14 C]-labelled morpholine derivatives for a class of trace amine-associate receptor 1 agonists.

Various agonists of the trace amine-associate receptor 1, under consideration as potential clinical development candidates, were labelled with carbon-14 for use in preclinical in vitro and in vivo drug metabolism studies. Herein, the [14 C]-radiosynthesis of 2-phenyl-substituted morpholines 1 is described. After evaluating and optimizing different synthetic routes, 4-iodonitrobenzene 3 was selected as starting material for the 14-step synthesis. Incorporation of carbon-14 into the acetyl moiety allowed a safe and efficient synthesis of [14 C]-labelled 4-nitroacetophenone 2 in five steps and 38% yield. Further transformation of 2 to the target compounds 1 was achieved in a 9-step synthesis. In a representative example, [14 C]-labelled 1 was obtained in an overall yield of 11% and was isolated in >99% radiochemical purity and a specific activity of 47 mCi/mmol.

Discovery and Characterization of 2-Aminooxazolines as Highly Potent, Selective, and Orally Active TAAR1 Agonists.

2-Aminooxazolines were discovered as a novel structural class of TAAR1 ligands. Starting from a known adrenergic compound 1, structural modifications were made to obtain highly potent and selective TAAR1 ligands such as 12 (RO5166017), 18 (RO5256390), 36 (RO5203648), and 48 (RO5263397). These compounds exhibit drug-like physicochemical properties, have good oral bioavailability, and display in vivo activity in a variety of animal models relevant for psychiatric diseases and addiction.

Incretin-like effects of small molecule trace amine-associated receptor 1 agonists.

OBJECTIVE: Type 2 diabetes and obesity are emerging pandemics in the 21st century creating worldwide urgency for the development of novel and safe therapies. We investigated trace amine-associated receptor 1 (TAAR1) as a novel target contributing to the control of glucose homeostasis and body weight. METHODS: We investigated the peripheral human tissue distribution of TAAR1 by immunohistochemistry and tested the effect of a small molecule TAAR1 agonist on insulin secretion in vitro using INS1E cells and human islets and on glucose tolerance in C57Bl6, and db/db mice. Body weight effects were investigated in obese DIO mice. RESULTS: TAAR1 activation by a selective small molecule agonist increased glucose-dependent insulin secretion in INS1E cells and human islets and elevated plasma PYY and GLP-1 levels in mice. In diabetic db/db mice, the TAAR1 agonist normalized glucose excursion during an oral glucose tolerance test. Sub-chronic treatment of diet-induced obese (DIO) mice with the TAAR1 agonist resulted in reduced food intake and body weight. Furthermore insulin sensitivity was improved and plasma triglyceride levels and liver triglyceride content were lower than in controls. CONCLUSIONS: We have identified TAAR1 as a novel integrator of metabolic control, which acts on gastrointestinal and pancreatic islet hormone secretion. Thus TAAR1 qualifies as a novel and promising target for the treatment of type 2 diabetes and obesity.

A UGT2B10 splicing polymorphism common in african populations may greatly increase drug exposure.

RO5263397 [(S)-4-(3-fluoro-2-methyl-phenyl)-4,5-dihydro-oxazol-2-ylamine], a new compound that showed promising results in animal models of schizophrenia, is mainly metabolized in humans by N-glucuronidation. Enzyme studies, using the (then) available commercial uridine 5'-diphosphate-glucuronosyltransferases (UGTs), suggested that UGT1A4 is responsible for its conjugation. In the first clinical trial, in which RO5263397 was administered orally to healthy human volunteers, a 136-fold above-average systemic exposure to the parent compound was found in one of the participants. Further administration in this trial identified two more such poor metabolizers, all three of African origin. Additional in vitro studies with recombinant UGTs showed that the contribution of UGT2B10 to RO5263397 glucuronidation is much higher than UGT1A4 at clinically relevant concentrations. DNA sequencing in all of these poor metabolizers identified a previously uncharacterized splice site mutation that prevents assembly of full-length UGT2B10 mRNA and thus functional UGT2B10 protein expression. Further DNA database analyses revealed the UGT2B10 splice site mutation to be highly frequent in individuals of African origin (45%), moderately frequent in Asians (8%) and almost unrepresented in Caucasians (<1%). A prospective study using hepatocytes from 20 individual African donors demonstrated a >100-fold lower intrinsic clearance of RO5263397 in cells homozygous for the splice site variant allele. Our results highlight the need to include UGT2B10 when screening the human UGTs for the enzymes involved in the glucuronidation of a new compound, particularly when there is a possibility of N-glucuronidation. Moreover, this study demonstrates the importance of considering different ethnicities during drug development.

Optimisation of imidazole compounds as selective TAAR1 agonists: discovery of RO5073012.

A series of imidazole compounds has been identified which affords potent and selective partial and full agonists of the TAAR1 receptor. Starting from 2-benzyl-imidazoline screening hits, a series of structurally related 2-benzyl- and 4-benzyl-imidazoles was investigated first, but it proved highly challenging to obtain compounds having sufficient selectivity against the adrenergic alpha 2 receptor. This issue could be successfully addressed by modification of the linker region and SAR exploration led to the discovery of highly selective isopropyl-substituted 4-aminomethyl-imidazole compounds. The work culminated in the identification of the selective TAAR1 partial agonist RO5073012 (4-chlorophenyl)-(1H-imidazol-4-ylmethyl)-isopropyl-amine, 24), which has a good pharmacokinetic profile after oral administration in rodents. RO5073012 has been found to be active in a behavioural rat model which is considered indicative for schizophrenia.

Brain-specific overexpression of trace amine-associated receptor 1 alters monoaminergic neurotransmission and decreases sensitivity to amphetamine.

Trace amines (TAs) such as ß-phenylethylamine, p-tyramine, or tryptamine are biogenic amines found in the brain at low concentrations that have been implicated in various neuropsychiatric disorders like schizophrenia, depression, or attention deficit hyperactivity disorder. TAs are ligands for the recently identified trace amine-associated receptor 1 (TAAR1), an important modulator of monoamine neurotransmission. Here, we sought to investigate the consequences of TAAR1 hypersignaling by generating a transgenic mouse line overexpressing Taar1 specifically in neurons. Taar1 transgenic mice did not show overt behavioral abnormalities under baseline conditions, despite augmented extracellular levels of dopamine and noradrenaline in the accumbens nucleus (Acb) and of serotonin in the medial prefrontal cortex. In vitro, this was correlated with an elevated spontaneous firing rate of monoaminergic neurons in the ventral tegmental area, dorsal raphe nucleus, and locus coeruleus as the result of ectopic TAAR1 expression. Furthermore, Taar1 transgenic mice were hyposensitive to the psychostimulant effects of amphetamine, as it produced only a weak locomotor activation and failed to alter catecholamine release in the Acb. Attenuating TAAR1 activity with the selective partial agonist RO5073012 restored the stimulating effects of amphetamine on locomotion. Overall, these data show that Taar1 brain overexpression causes hyposensitivity to amphetamine and alterations of monoaminergic neurotransmission. These observations confirm the modulatory role of TAAR1 on monoamine activity and suggest that in vivo the receptor is either constitutively active and/or tonically activated by ambient levels of endogenous agonist(s).

Trace amine-associated receptor 1 partial agonism reveals novel paradigm for neuropsychiatric therapeutics.

BACKGROUND: Trace amines, compounds structurally related to classical biogenic amines, represent endogenous ligands of the trace amine-associated receptor 1 (TAAR1). Because trace amines also influence the activity of other targets, selective ligands are needed for the elucidation of TAAR1 function. Here we report on the identification and characterization of the first selective and potent TAAR1 partial agonist. METHODS: The TAAR1 partial agonist RO5203648 was evaluated for its binding affinity and functional activity at rodent and primate TAAR1 receptors stably expressed in HEK293 cells, for its physicochemical and pharmacokinetic properties, for its effects on the firing frequency of monoaminergic neurons ex vivo, and for its properties in vivo with genetic and pharmacological models of central nervous system disorders. RESULTS: RO5203648 showed high affinity and potency at TAAR1, high selectivity versus other targets, and favorable pharmacokinetic properties. In mouse brain slices, RO5203648 increased the firing frequency of dopaminergic and serotonergic neurons in the ventral tegmental area and the dorsal raphe nucleus, respectively. In various behavioral paradigms in rodents and monkeys, RO5203648 demonstrated clear antipsychotic- and antidepressant-like activities as well as potential anxiolytic-like properties. Furthermore, it attenuated drug-taking behavior and was highly effective in promoting attention, cognitive performance, and wakefulness. CONCLUSIONS: With the first potent and selective TAAR1 partial agonist, RO5203648, we show that TAAR1 is implicated in a broad range of relevant physiological, behavioral, and cognitive neuropsychiatric dimensions. Collectively, these data uncover important neuromodulatory roles for TAAR1 and suggest that agonists at this receptor might have therapeutic potential in one or more neuropsychiatric domains.

Selective antagonists of mouse trace amine-associated receptor 1 (mTAAR1): discovery of EPPTB (RO5212773).

High throughput screening of the Roche compound library identified benzanilides such as 1 and 2 as antagonists of TAAR1. Optimisation of this hit series led to the first selective TAAR1 antagonist (N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide EPPTB (RO5212773, 9f) having IC(50) of 28 nM at mouse TAAR1.

Discovery of benzoylisoindolines as a novel class of potent, selective and orally active GlyT1 inhibitors.

Benzoylisoindolines were discovered as a novel structural class of GlyT1 inhibitors. SAR studies and subsequent lead optimization efforts focused primarily on addressing hERG liability and on improving in vivo efficacy resulted in the identification of potent GlyT1 inhibitors displaying excellent selectivity and in vivo PD and PK profiles.

4-Substituted-7-N-alkyl-N-acetyl 2-aminobenzothiazole amides: drug-like and non-xanthine based A2B adenosine receptor antagonists.

7-N-Acetamide-4-methoxy-2-aminobenzothiazole 4-fluorobenzamide (compound 1) was chosen as a drug-like and non-xanthine based starting point for the discovery of A(2B) receptor antagonists because of its slight selectivity against A(1) and A(2A) receptors and modest A(2B) potency. SAR exploration of compound 1 described herein included modifications to the 7-N-acetamide group, substitution of the 4-methoxy group by halogens as well as replacement of the p-flouro-benzamide side chain. This work culminated in the identification of compound 37 with excellent A(2B) potency, modest selectivity versus A(2A) and A(1) receptors, and good rodent PK properties.

Selective GlyT1 inhibitors: discovery of [4-(3-fluoro-5-trifluoromethylpyridin-2-yl)piperazin-1-yl][5-methanesulfonyl-2-((S)-2,2,2-trifluoro-1-methylethoxy)phenyl]methanone (RG1678), a promising novel medicine to treat schizophrenia.

The GlyT1 transporter has emerged as a key novel target for the treatment of schizophrenia. Herein, we report on the optimization of the 2-alkoxy-5-methylsulfonebenzoylpiperazine class of GlyT1 inhibitors to improve hERG channel selectivity and brain penetration. This effort culminated in the discovery of compound 10a (RG1678), the first potent and selective GlyT1 inhibitor to have a beneficial effect in schizophrenic patients in a phase II clinical trial.

The selective antagonist EPPTB reveals TAAR1-mediated regulatory mechanisms in dopaminergic neurons of the mesolimbic system.

Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) that is nonselectively activated by endogenous metabolites of amino acids. TAAR1 is considered a promising drug target for the treatment of psychiatric and neurodegenerative disorders. However, no selective ligand to identify TAAR1-specific signaling mechanisms is available yet. Here we report a selective TAAR1 antagonist, EPPTB, and characterize its physiological effects at dopamine (DA) neurons of the ventral tegmental area (VTA). We show that EPPTB prevents the reduction of the firing frequency of DA neurons induced by p-tyramine (p-tyr), a nonselective TAAR1 agonist. When applied alone, EPPTB increases the firing frequency of DA neurons, suggesting that TAAR1 either exhibits constitutive activity or is tonically activated by ambient levels of endogenous agonist(s). We further show that EPPTB blocks the TAAR1-mediated activation of an inwardly rectifying K(+) current. When applied alone, EPPTB induces an apparent inward current, suggesting the closure of tonically activated K(+) channels. Importantly, these EPPTB effects were absent in Taar1 knockout mice, ruling out off-target effects. We additionally found that both the acute application of EPPTB and the constitutive genetic lack of TAAR1 increase the potency of DA at D2 receptors in DA neurons. In summary, our data support that TAAR1 tonically activates inwardly rectifying K(+) channels, which reduces the basal firing frequency of DA neurons in the VTA. We hypothesize that the EPPTB-induced increase in the potency of DA at D2 receptors is part of a homeostatic feedback mechanism compensating for the lack of inhibitory TAAR1 tone.

Discovery of benzoylpiperazines as a novel class of potent and selective GlyT1 inhibitors.

Screening of the Roche compound library led to the identification of the benzoylpiperazine 7 as a structurally novel GlyT1 inhibitor. The SAR which was developed in this series resulted in the discovery of highly potent compounds displaying excellent selectivity against the GlyT2 isoform, drug-like properties, and in vivo efficacy after oral administration.

The stereocontrolled total synthesis of altohyrtin A/spongistatin 1: the AB-spiroacetal segment.

The convergent synthesis of the C1-C15 AB-spiroacetal subunit of altohyrtin A/spongistatin 1 is described. This highly stereocontrolled synthesis relies on matched boron aldol reactions of chiral methyl ketones, under Ipc(2)BCl mediation, to establish the C5, C9 and C11 stereocentres, and formation of the desired thermodynamic spiroacetal under acidic conditions. The scalable synthetic sequence developed provided access to multi-gram quantities of , thus enabling the successful completion of the total synthesis of altohyrtin A/spongistatin 1, as reported in Part 4.

Stereocontrolled Total Synthesis of (+)-Altohyrtin A/Spongistatin 1.

As an exceptionally potent antimitotic macrolide, altohyrtin A/spongistatin 1 shows great promise in cancer chemotherapy but its extreme scarcity in the natural sponges has halted its further preclinical development. A highly stereocontrolled total synthesis, which exploits boron-mediated aldol bond constructions, has been realized to provide, for the first time, a useful amount of synthetic material.