Potent and Selective Tetrahydroisoquinoline Kappa Opioid Receptor Antagonists of Lead Compound (3 R)-7-Hydroxy- N-[(1 S)-2-methyl-1-(piperidin-1-ylmethyl)propyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (PDTic).

Past studies have shown that it has been difficult to discover and develop potent and selective κ opioid receptor antagonists, particularly compounds having potential for clinical development. In this study, we present a structure-activity relationship (SAR) study of a recently discovered new class of tetrahydroisoquinoline κ opioid receptor antagonists which led to (3 R)-7-hydroxy- N-{(1 S)-2-methyl-1-[(-4-methylpiperidine-1-yl)methyl]propyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (12) (4-Me-PDTic). Compound 12 had a Ke = 0.37 nM in a [35S]GTPγS binding assay and was 645- and >8100-fold selective for the κ relative to the µ and δ opioid receptors, respectively. Calculated log BB and CNS (central nervous system) multiparameter optimization (MPO) and low molecular weight values all predict that 12 will penetrate the brain, and pharmacokinetic studies in rats show that 12 does indeed penetrate the brain.

Potent and Selective Tetrahydroisoquinoline Kappa Opioid Receptor Antagonists of Lead Compound (3 R)- N-[1 R)-1-(Cyclohexylmethyl)-2-methylpropyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (CDTic).

Animal pharmacological studies suggest that potent and selective κ opioid receptor antagonists have potential as pharmacotherapies targeting depression, anxiety, and substance abuse (opiates, alcohol, nicotine, cocaine). We recently reported lead compound 1 as a new class of κ opioid receptor antagonists with only one basic amine group. Analogues were synthesized and evaluated for their in vitro opioid receptor antagonist properties using a [35S]GTPγS binding assay. All analogues were pure opioid receptor antagonists with no agonist activity. Compounds 1, 8, 9, 13, and 14 ( Ke values 0.058-0.64 nM) are highly potent and highly selective for the κ relative to the µ and δ opioid receptors. Favorable calculated physiochemical properties were confirmed in rat PK studies, demonstrating brain penetration for selected compounds 1, 9, and 13. High κ opioid receptor potency and selectivity and highly favorable calculated physiochemical and PK properties for brain penetration suggest these compounds should be considered for further development.

Simple Tetrahydroisoquinolines Are Potent and Selective Kappa Opioid Receptor Antagonists.

Potent and selective κ opioid receptor antagonists have been derived from the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine class of pure opioid receptor antagonists. In order to determine if the 3-hydroxyphenyl and/or the piperidine amino groups are required for obtaining the pure opioid antagonists, (3R)-7-hydroxy-N-[(1S)-2-methyl-1-(piperidine-1-ylmethyl)propyl]-1,2,3,4-tetrahydroiosquinoline-3-carboxamide (1), which does not have a 4-(3-hydroxyphenyl) group, and (3R)-N-(1R)-1-(cyclohexylmethyl)-2-methylpropyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (2), which does not have a 4-hydroxylphenyl or a piperidine amino group, were synthesized and evaluated for their [35S]GTPγS binding properties at the µ, δ, and κ opioid receptors. Surprisingly compound 1 remained a pure opioid antagonist with a Ke = 6.80 nM at the κ opioid receptor and is 21- and 441-fold selective for the κ receptor relative to the µ and δ opioid receptors, respectively. Even more unexpected and novel is the finding that 2 has a Ke = 0.14 nM at κ and is 1730- and 4570-fold selective for κ relative to the µ and δ opioid receptors, respectively.

Identification of 2-({[1-(4-Fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazol-3-yl]carbonyl}amino)tricyclo[3.3.1.13,7]decane-2-carboxylic Acid (NTRC-844) as a Selective Antagonist for the Rat Neurotensin Receptor Type 2.

Neurotensin receptor type 2 (NTS2) compounds display analgesic activity in animal pain models. We have identified the first high-affinity NTS2-selective antagonist (8) that is active in vivo. This study also revealed that the NTS2 FLIPR assay designation for a compound, agonist, partial agonist, and so forth, did not correlate with its in vivo activity as observed in the thermal tail-flick acute model of pain. This suggests that calcium mobilization is not the signaling pathway involved in NTS2-mediated analgesia as assessed by the thermal tail-flick model. Finally, we found a significant bias between rat and human for compound 9 in the NTS2 binding assay.

Discovery of a novel small molecule agonist scaffold for the APJ receptor.

The apelinergic system includes a series of endogenous peptides apelin, ELABELA/TODDLER and their 7-transmembrane G-protein coupled apelin receptor (APJ, AGTRL-1, APLNR). The APJ receptor is an attractive therapeutic target because of its involvement in cardiovascular diseases and potentially other disorders including liver fibrosis, obesity, diabetes, and neuroprotection. To date, pharmacological characterization of the APJ receptor has been limited due to the lack of small molecule functional agonists or antagonists. Through focused screening we identified a drug-like small molecule agonist hit 1 with a functional EC50 value of 21.5±5µM and binding affinity (Ki) of 5.2±0.5µM. Initial structure-activity studies afforded compound 22 having a 27-fold enhancement in potency and the first sub-micromolar full agonist with an EC50 value of 800±0.1nM and Ki of 1.3±0.3µM. Preliminary SAR, synthetic methodology, and in vitro pharmacological characterization indicate this scaffold will serve as a favorable starting point for further refinement of APJ potency and selectivity.

Design, synthesis, and pharmacological evaluation of JDTic analogs to examine the significance of replacement of the 3-hydroxyphenyl group with pyridine or thiophene bioisosteres.

The potent and selective KOR antagonist JDTic was derived from the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine class of pure opioid antagonists. In previous studies we reported that compounds that did not have a hydroxyl on the 3-hydroxyphenyl group and did not have methyl groups at the 3- and 4-position of the piperidine ring were still potent and selective KOR antagonists. In this study we report JDTic analogs 2, 3a-b, 4a-b, and 5, where the 3-hydroxyphenyl ring has been replaced by a 2-, 3-, or 4-pyridyl or 3-thienyl group and do not have the 3-methyl or 3,4-dimethyl groups, remain potent and selective KOR antagonists. Of these, (3R)-7-hydroxy-N-(1S)-2-methyl-[4-methyl-4-pyridine-3-yl-carboxamide (3b) had the best overall binding potency and selectivity in a [(35)S]GTPγS functional assay, with a Ke=0.18nM at the KOR and 273- and 16,700-fold selectivity for the KOR relative to the MOR and DOR, respectively. Calculated physiochemical properties for 3b suggest that it will cross the blood-brain barrier.

ML314: A Biased Neurotensin Receptor Ligand for Methamphetamine Abuse.

Pharmacological treatment for methamphetamine addiction will provide important societal benefits. Neurotensin receptor NTR1 and dopamine receptor distributions coincide in brain areas regulating methamphetamine-associated reward, and neurotensin peptides produce behaviors opposing psychostimulants. Therefore, undesirable methamphetamine-associated activities should be treatable with druggable NTR1 agonists, but no such FDA-approved therapeutics exist. We address this limitation with proof-of-concept data for ML314, a small-molecule, brain penetrant, ß-arrestin biased, NTR1 agonist. ML314 attenuates amphetamine-like hyperlocomotion in dopamine transporter knockout mice, and in C57BL/6J mice it attenuates methamphetamine-induced hyperlocomotion, potentiates the psychostimulant inhibitory effects of a ghrelin antagonist, and reduces methamphetamine-associated conditioned place preference. In rats, ML314 blocks methamphetamine self-administration. ML314 acts as an allosteric enhancer of endogenous neurotensin, unmasking stoichiometric numbers of hidden NTR1 binding sites in transfected-cell membranes or mouse striatal membranes, while additionally supporting NTR1 endocytosis in cells in the absence of NT peptide. These results indicate ML314 is a viable, preclinical lead for methamphetamine abuse treatment and support an allosteric model of G protein-coupled receptor signaling.

Design, synthesis, and pharmacological evaluation of JDTic analogs to examine the significance of the 3- and 4-methyl substituents.

The design and discovery of JDTic as a potent and selective kappa opioid receptor antagonist used the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine pharmacophore as the lead structure. In order to determine if the 3-methyl or 4-methyl groups were necessary in JDTic and JDTic analogs for antagonistic activity, compounds 4a-c, and 4d-f which have either the 3-methyl or both the 3- and 4-methyl groups removed, respectively, from JDTic and analogs were synthesized and evaluated for their in vitro opioid receptor antagonist activities using a [(35)S]GTPγS binding assay. Other ADME properties were also assessed for selected compounds. These studies demonstrated that neither the 3-methyl or 3,4-dimethyl groups present in JDTic and analogs are required to produce potent and selective κ opioid receptor antagonists.

The amide linker in nonpeptide neurotensin receptor ligands plays a key role in calcium signaling at the neurotensin receptor type 2.

Compounds acting via the GPCR neurotensin receptor type 2 (NTS2) display analgesia in relevant preclinical models. The amide bond in nonpeptide NTS1 antagonists plays a central role in receptor recognition and molecular conformation. Using NTS2 FLIPR and binding assays, we found that it is also a key molecular structure for binding and calcium mobilization at NTS2. We found that reversed amides display a shift from agonist to antagonist activity and provided examples of the first competitive nonpeptide antagonists observed in the NTS2 FLIPR assay. These compounds will be valuable tools for determining the role of calcium signaling in vitro to NTS2 mediated analgesia.

Identification of N-{[6-chloro-4-(2,6-dimethoxyphenyl)quinazolin-2-yl]carbonyl}-l-leucine (NTRC-808), a novel nonpeptide chemotype selective for the neurotensin receptor type 2.

Compounds acting via the GPCR neurotensin receptor type 2 (NTS2) display analgesic effects in relevant animal models. Using a pharmacophore model based on known NT receptor nonpeptide compounds, we screened commercial databases to identify compounds that might possess activity at NTS2 receptor sites. Modification of our screening hit to include structural features known to be recognized by NTS1 and NTS2, led to the identification of the novel NTS2 selective nonpeptide, N-{[6-chloro-4-(2,6-dimethoxyphenyl)quinazolin-2-yl]carbonyl}-l-leucine (9). This compound is a potent partial agonist in the FLIPR assay with a profile of activity similar to that of the reference NTS2 analgesic nonpeptide levocabastine (5).

Design, synthesis, and biological evaluation of (3R)-1,2,3,4-tetrahydro-7-hydroxy-N-[(1S)-1-[[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl]-2-methylpropyl]-3-isoquinolinecarboxamide (JDTic) analogues: in vitro pharmacology and ADME profile.

JDTic analogues 4-15 which have the hydroxyl groups replaced with other groups were synthesized and their in vitro efficacy at the µ, δ, and κ opioid receptors determined and compared to JDTic using [(35)S]GTPγS assays. Compounds 4, 5, 6, 13, 14, and 15 had Ke = 0.024, 0.01, 0.039, 0.02, 0.11, and 0.041 nM compared to the Ke = 0.02 nM for JDTic at the κ receptor and were highly selective for the κ receptor relative to the µ and δ opioid receptors. Unexpectedly, replacement of the 3-hydroxyl substituent of the 4-(3-hydroxyphenyl) group of JDTic with a H, F, or Cl substituent leads to potent and selective KOR antagonists. In vitro studies to determine various ADME properties combined with calculated TPSA, clogP, and logBB values suggests that the potent and selective κ opioid receptors 4, 5, 13, and 14 deserve consideration for further development toward potential drugs for CNS disorders.

Identification of N-[(5-{[(4-methylphenyl)sulfonyl]amino}-3-(trifluoroacetyl)-1H-indol-1-yl)acetyl]-l-leucine (NTRC-824), a neurotensin-like nonpeptide compound selective for the neurotensin receptor type 2.

Compounds acting via the neurotensin receptor type 2 (NTS2) are known to be active in animal models of acute and chronic pain. To identify novel NTS2 selective analgesics, we searched for NTS2 selective nonpeptide compounds using a FLIPR assay and identified the title compound (NTRC-824, 5) that, to our knowledge, is the first nonpeptide that is selective for NTS2 versus NTS1 and behaves like the endogenous ligand neurotensin in the functional assay.

Identification of 1-({[1-(4-fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazol-3-yl]carbonyl}amino)cyclohexane carboxylic acid as a selective nonpeptide neurotensin receptor type 2 compound.

Compounds active at neurotensin receptors (NTS1 and NTS2) exert analgesic effects on different types of nociceptive modalities, including thermal, mechanical, and chemical stimuli. The NTS2 preferring peptide JMV-431 (2) and the NTS2 selective nonpeptide compound levocabastine (6) have been shown to be effective in relieving the pain associated with peripheral neuropathies. With the aim of identifying novel nonpeptide compounds selective for NTS2, we examined analogues of SR48692 (5a) using a FLIPR calcium assay in CHO cells stably expressing rat NTS2. This led to the discovery of the NTS2 selective nonpeptide compound 1-({[1-(4-fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazol-3-yl]carbonyl}amino)cyclohexane carboxylic acid (NTRC-739, 7b) starting from the nonselective compound 5a.

Effect of the 3- and 4-methyl groups on the opioid receptor properties of N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines.

N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines (2a,b) are opioid receptor antagonists where the antagonist properties are not due to the type of N-substituent. In order to gain a better understanding of the contribution that the 3- and 4-methyl groups make to the pure antagonist properties of 2a,b, we synthesized analogues of 2a,b that lacked the 4-methyl (5a,b), 3-methyl (6a,b), and both the 3- and 4-methyl group (7a,b) and compared their opioid receptor properties. We found that (1) all N-methyl and N-phenylpropyl substituted compounds were nonselective opioid antagonists (2) all N-phenylpropyl analogues were more potent than their N-methyl counterparts, and (3) compounds 2a,b which have both a 3- and 4-methyl substituent, were more potent antagonists than analogues 5a,b, 6a,b, and 7a,b. We also found that the removal of 3-methyl substituent of N-methyl and N-phenylpropyl 3-methyl-4-(3-hydroxyphenyl)piperazines (8a,b) gives (4a,b), which are opioid antagonists.

Imidazole-derived agonists for the neurotensin 1 receptor.

A scaffold-hop program seeking full agonists of the neurotensin-1 (NTR1) receptor identified the probe molecule ML301 (1) and associated analogs, including its naphthyl analog (14) which exhibited similar properties. Compound 1 showed full agonist behavior (79-93%) with an EC50 of 2.0-4.1µM against NTR1. Compound 1 also showed good activity in a Ca mobilization FLIPR assay (93% efficacy at 298nM), consistent with it functioning via the Gq coupled pathway, and good selectivity relative to NTR2 and GPR35. In further profiling, 1 showed low potential for promiscuity and good overall pharmacological data. This report describes the discovery, synthesis, and SAR of 1 and associated analogs. Initial in vitro pharmacologic characterization is also presented.

4ß-Methyl-5-(3-hydroxyphenyl)morphan opioid agonist and partial agonist derived from a 4ß-methyl-5-(3-hydroxyphenyl)morphan pure antagonist.

In previous studies we reported that addition of 7α-acylamino groups to N-phenylpropyl-4ß-methyl-5-(3-hydroxyphenyl)morphan (4) led to compounds that were pure opioid receptor antagonists. In contrast to these findings we report in this study that addition of a 7α-amino (5a), 7α-alkylamino (5b-e), or 7α-dialkylamino (5f-h) group to 4 leads to opioid receptor ligands with varying degrees of agonist/antagonist activity. The 7α-amino and 7α-methylamino analogues were full agonists at the µ and δ receptors and antagonists at the κ receptor. The 7α-cyclopropylmethylamino analogue 5h was a full agonist at the µ receptor with weaker agonist activity at the δ and κ receptors. Whereas the addition of a 7α-acylamino group to the pure nonselective opioid receptor antagonist N-phenylpropyl-4ß-methyl-5-(3-hydroxyphenyl)morphan (4) led to κ selective pure opioid receptor antagonist, the addition of a 7α-amino, 7α-alkylamino, or 7α-dialkylamino group to 4 leads to opioid ligands that are largely µ or δ agonist with mixed agonist/antagonist properties.

Discovery of N-{4-[(3-hydroxyphenyl)-3-methylpiperazin-1-yl]methyl-2-methylpropyl}-4-phenoxybenzamide analogues as selective kappa opioid receptor antagonists.

There is continuing interest in the discovery and development of new κ opioid receptor antagonists. We recently reported that N-substituted 3-methyl-4-(3-hydroxyphenyl)piperazines were a new class of opioid receptor antagonists. In this study, we report the syntheses of two piperazine JDTic-like analogues. Evaluation of the two compounds in an in vitro [(35)S]GTPγS binding assay showed that neither compound showed the high potency and κ opioid receptor selectivity of JDTic. A library of compounds using the core scaffold 21 was synthesized and tested for their ability to inhibit [(35)S]GTPγS binding stimulated by the selective κ opioid agonist U69,593. These studies led to N-[(1S)-1-{[(3S)-4-(3-hydroxyphenyl)-3-methylpiperazin-1-yl]methyl}-2-methylpropyl]-4-phenoxybenzamide (11a), a compound that showed good κ opioid receptor antagonist properties. An SAR study based on 11a provided 28 novel analogues. Evaluation of these 28 compounds in the [(35)S]GTPγS binding assay showed that several of the analogues were potent and selective κ opioid receptor antagonists.

Discovery of ML314, a Brain Penetrant Non-Peptidic ß-Arrestin Biased Agonist of the Neurotensin NTR1 Receptor.

The neurotensin 1 receptor (NTR1) is an important therapeutic target for a range of disease states including addiction. A high throughput screening campaign, followed by medicinal chemistry optimization, led to the discovery of a non-peptidic ß-arrestin biased agonist for NTR1. The lead compound, 2-cyclopropyl-6,7-dimethoxy-4-(4-(2-methoxyphenyl)- piperazin-1-yl)quinazoline, 32 (ML314), exhibits full agonist behavior against NTR1 (EC50 = 2.0 µM) in the primary assay and selectivity against NTR2. The effect of 32 is blocked by the NTR1 antagonist SR142948A in a dose dependent manner. Unlike peptide based NTR1 agonists, compound 32 has no significant response in a Ca2+ mobilization assay and is thus a biased agonist that activates the ß-arrestin pathway rather than the traditional G q coupled pathway. This bias has distinct biochemical and functional consequences that may lead to physiological advantages. Compound 32 displays good brain penetration in rodents, and studies examining its in vivo properties are underway.

1-Substituted 4-(3-Hydroxyphenyl)piperazines Are Pure Opioid Receptor Antagonists.

This report describes the discovery that 1-substituted 4-(3-hydroxyphenyl)piperazines are pure opioid receptor antagonists. Compounds in this new series include N-phenylpropyl (3S)-3-methyl-4-(3-hydroxyphenyl)piperazine and (3R)-3-methyl-4-(3-hydroxyphenyl)piperazine, both of which diaplay low nanomolar potencies at µ, δ, and κ receptors and pure antagonist properties in a [(35)S]GTPγS assay.

Discovery and biological evaluation of potent, selective, orally bioavailable, pyrazine-based blockers of the Na(v)1.8 sodium channel with efficacy in a model of neuropathic pain.

Na(v)1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons. It has been implicated in the pathophysiology of inflammatory and neuropathic pain, and we envisioned that selective blockade of Na(v)1.8 would be analgesic, while reducing adverse events typically associated with non-selective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 6-aryl-2-pyrazinecarboxamides, which are potent blockers of the human Na(v)1.8 channel and also block TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons. Selected derivatives display selectivity versus human Na(v)1.2. We further demonstrate that an example from this series is orally bioavailable and produces antinociceptive activity in vivo in a rodent model of neuropathic pain following oral administration.