SYK-623, a δ Opioid Receptor Inverse Agonist, Mitigates Chronic Stress-Induced Behavioral Abnormalities and Disrupted Neurogenesis.

The δ opioid receptor (DOR) inverse agonist has been demonstrated to improve learning and memory impairment in mice subjected to restraint stress. Here, we investigated the effects of SYK-623, a new DOR inverse agonist, on behavioral, immunohistochemical, and biochemical abnormalities in a mouse model of imipramine treatment-resistant depression. Male ddY mice received daily treatment of adrenocorticotropic hormone (ACTH) combined with chronic mild stress exposure (ACMS). SYK-623, imipramine, or the vehicle was administered once daily before ACMS. After three weeks, ACMS mice showed impaired learning and memory in the Y-maze test and increased immobility time in the forced swim test. SYK-623, but not imipramine, significantly suppressed behavioral abnormalities caused by ACMS. Based on the fluorescent immunohistochemical analysis of the hippocampus, ACMS induced a reduction in astrocytes and newborn neurons, similar to the reported findings observed in the postmortem brains of depressed patients. In addition, the number of parvalbumin-positive GABA neurons, which play a crucial role in neurogenesis, was reduced in the hippocampus, and western blot analysis showed decreased glutamic acid decarboxylase protein levels. These changes, except for the decrease in astrocytes, were suppressed by SYK-623. Thus, SYK-623 mitigates behavioral abnormalities and disturbed neurogenesis caused by chronic stress.

Discovery of 7-Azanorbornane-Based Dual Agonists for the Delta and Kappa Opioid Receptors through an In Situ Screening Protocol.

In medicinal chemistry, the copper-catalyzed click reaction is used to prepare ligand candidates. This reaction is so clean that the bioactivities of the products can be determined without purification. Despite the advantages of this in situ screening protocol, the applicability of this method for transmembrane proteins has not been validated due to the incompatibility with copper catalysts. To address this point, we performed ligand screening for the µ, δ, and κ opioid receptors using this protocol. As we had previously reported the 7-azanorbornane skeleton as a privileged scaffold for the G protein-coupled receptors, we performed the click reactions between various 7-substituted 2-ethynyl-7-azanorbornanes and azides. Screening assays were performed without purification using the CellKeyTM system, and the putative hit compounds were re-synthesized and re-evaluated. Although the "hit" compounds for the µ and the δ receptors were totally inactive after purifications, three of the four "hits" for the κ receptor were true agonists for this receptor and also showed activities for the δ receptor. Although false positive/negative results exist as in other screening projects for soluble proteins, this in situ method is effective in identifying novel ligands for transmembrane proteins.

Evaluation of the Intracellular Signaling Activities of κ-Opioid Receptor Agonists, Nalfurafine Analogs; Focusing on the Selectivity of G-Protein- and ß-Arrestin-Mediated Pathways.

Opioid receptors (ORs) are classified into three types (µ, δ, and κ), and opioid analgesics are mainly mediated by µOR activation; however, their use is sometimes restricted by unfavorable effects. The selective κOR agonist nalfurafine was initially developed as an analgesic, but its indication was changed because of the narrow safety margin. The activation of ORs mainly induces two intracellular signaling pathways: a G-protein-mediated pathway and a ß-arrestin-mediated pathway. Recently, the expectations for κOR analgesics that selectively activate these pathways have increased; however, the structural properties required for the selectivity of nalfurafine are still unknown. Therefore, we evaluated the partial structures of nalfurafine that are necessary for the selectivity of these two pathways. We assayed the properties of nalfurafine and six nalfurafine analogs (SYKs) using cells stably expressing κORs. The SYKs activated κORs in a concentration-dependent manner with higher EC50 values than nalfurafine. Upon bias factor assessment, only SYK-309 (possessing the 3S-hydroxy group) showed higher selectivity of G-protein-mediated signaling activities than nalfurafine, suggesting the direction of the 3S-hydroxy group may affect the ß-arrestin-mediated pathway. In conclusion, nalfurafine analogs having a 3S-hydroxy group, such as SYK-309, could be considered G-protein-biased κOR agonists.

Morphinan derivatives with an oxabicyclo[3.2.1]octane structure as dual agonists toward δ and κ opioid receptors.

The κ opioid receptor (KOR) is one of the promising targets to develop analgesics lacking morphine like side effects. To seek a novel KOR agonist we designed 6-amide derivatives with an oxabicyclo[3.2.1]octane structure based on a proposed active conformation of a selective KOR agonist nalfurafine. All the synthesized compounds strongly bound to the KOR and some compound showed KOR selectivities. 6R-Amides were more potent and efficacious KOR agonists than the corresponding 6S-isomers. However, most 6-amide derivatives were partial KOR agonist. Conformational analyses of 6R- and 6S-amide derivatives and nalfurafine well accounted for the difference of KOR agonistic activities between two diastereomers. Surprisingly, the tested N-H amides were full δ opioid receptor (DOR) agonists. Among the tested compounds 7a with benzamide moiety was the most potent dual DOR/KOR agonist. On the other hand, 6S-phenylacetamide 8b was potent full DOR agonist with less efficacious agonist activity for the µ receptor and KOR. 6-Amide derivatives with an oxabicyclo[3.2.1]octane structure were expected to be a promising fundamental skeleton for the dual DOR/KOR agonists and/or selective DOR agonists.

Oxytocin Is a Positive Allosteric Modulator of κ-Opioid Receptors but Not δ-Opioid Receptors in the G Protein Signaling Pathway.

Oxytocin (OT) influences various physiological functions such as uterine contractions, maternal/social behavior, and analgesia. Opioid signaling pathways are involved in one of the analgesic mechanisms of OT. We previously showed that OT acts as a positive allosteric modulator (PAM) and enhances µ-opioid receptor (MOR) activity. In this study, which focused on other opioid receptor (OR) subtypes, we investigated whether OT influences opioid signaling pathways as a PAM for δ-OR (DOR) or κ-OR (KOR) using human embryonic kidney-293 cells expressing human DOR or KOR, respectively. The CellKeyTM results showed that OT enhanced impedance induced by endogenous/exogenous KOR agonists on KOR-expressing cells. OT did not affect DOR activity induced by endogenous/exogenous DOR agonists. OT potentiated the KOR agonist-induced Gi/o protein-mediated decrease in intracellular cAMP, but did not affect the increase in KOR internalization caused by the KOR agonists dynorphin A and (-)-U-50488 hydrochloride (U50488). OT did not bind to KOR orthosteric binding sites and did not affect the binding affinities of dynorphin A and U50488 for KOR. These results suggest that OT is a PAM of KOR and MOR and enhances G protein signaling without affecting ß-arrestin signaling. Thus, OT has potential as a specific signaling-biased PAM of KOR.

Identification of a Putative ß-Arrestin Superagonist of the Growth Hormone Secretagogue Receptor (GHSR).

Ghrelin is a pleiotropic feeding hormone which also has a pivotal role in the central nervous system. Upon the activation of its receptor, growth hormone secretagogue receptor (GHSR), the Gαq/11 -mediated and the ß-arrestin-mediated signaling pathways are activated. As the ß-arrestin pathway is a potential drug target, there is a strong need for ß-arrestin-biased GHSR modulators. Activation of the ß-arrestin pathway should inhibit the Gαq/11 -mediated calcium flux through internalization of the receptor. Hence, we used the antagonistic activity in the calcium assay as the first screening for the ß-arrestin activation. By conducting the second screening assay for the ß-arrestin activation based on extracellular signal regulated kinase (ERK) 1/2 phosphorylation, we discovered a putative ß-arrestin-biased superagonist. The activity of the compound was not completely blocked with the competitive antagonist, which implies that the effect is mediated, at least partly, by allosteric binding of the compound.

Visible-Light-Induced Synthesis of 1,2,3,4-Tetrahydroquinolines through Formal [4+2] Cycloaddition of Acyclic α,ß-Unsaturated Amides and Imides with N,N-Dialkylanilines.

1,2,3,4-Tetrahydroquinolines should be applicable to the development of new pharmaceutical agents. A facile synthesis of 1,2,3,4-tetrahydroquinolines that is achieved by a photoinduced formal [4+2] cycloaddition reaction of acyclic α,ß-unsaturated amides and imides with N,N-dialkylanilines under visible-light irradiation, in which a new IrIII complex photosensitizer, a thiourea, and an oxidant act cooperatively in promoting the reaction, is reported. The photoreaction enables the synthesis of a wide variety of 1,2,3,4-tetrahydroquinolines, while controlling the trans/cis diastereoselectivity (>99:1) and constructing contiguous stereogenic centers. A chemoselective cleavage of an acyclic imide auxiliary is demonstrated.

Enhancement of the rewarding effects of 3,4-methylenedioxymethamphetamine in orexin knockout mice.

Orexinergic neurons, which are closely associated with narcolepsy, regulate arousal and reward circuits through the activation of monoaminergic neurons. Psychostimulants as well as 5-HT-related compounds have potential in the treatment of human narcolepsy. Previous studies have demonstrated that orexin receptor antagonists as well as orexin deficiencies affect the pharmacological effects of psychostimulants. However, little information is available on the consequences of psychostimulant use under orexin deficiency. Therefore, the present study was designed to investigate the abuse liability of psychostimulants in orexin knockout (KO) mice. In the present study, conditioned place preferences induced by methamphetamine and methylphenidate were not altered in orexin KO mice. Interestingly, we found that MDMA induced a conditioned place preference in orexin KO mice, but not in wild type (WT) mice. In addition, MDMA produced methylphenidate/methamphetamine-like discriminative stimulus effects in orexin KO mice, but not WT mice. Increases in 5-HT and dopamine release in the nucleus accumbens induced by MDMA were not altered by knockout of orexin; the steady-state level of G protein activation was higher in the limbic forebrain of orexin KO mice. In substitution tests using a drug discrimination procedure, substitution of 5-HT1A receptor agonist for the discriminative stimulus effects of methylphenidate was enhanced in orexin KO mice. These findings indicate that the orexinergic system is involved the rewarding effects of psychostimulants. However, there is a risk of establishing rewarding effects of psychostimulants even under orexin deficiency. On the other hand, deficiencies in orexin may enhance the abuse liability of MDMA by changing a postsynaptic signal transduction accompanied by changes in discriminative stimulus effects themselves.

Effects of N-Substituents on the Functional Activities of Naltrindole Derivatives for the δ Opioid Receptor: Synthesis and Evaluation of Sulfonamide Derivatives.

We have recently reported that N-alkyl and N-acyl naltrindole (NTI) derivatives showed activities for the δ opioid receptor (DOR) ranging widely from full inverse agonists to full agonists. We newly designed sulfonamide-type NTI derivatives in order to investigate the effects of the N-substituent on the functional activities because the side chain and S=O part in the sulfonamide moiety located in spatially different positions compared with those in the alkylamine and amide moieties. Among the tested compounds, cyclopropylsulfonamide 9f (SYK-839) was the most potent full inverse agonist for the DOR, whereas phenethylsulfonamide 9e (SYK-901) showed full DOR agonist activity with moderate potency. These NTI derivatives are expected to be useful compounds for investigation of the molecular mechanism inducing these functional activities.

δ Opioid Receptor Inverse Agonists and their In Vivo Pharmacological Effects.

The discovery of δ opioid receptor inverse agonist activity induced by ICI-174,864, which was previously reported as an δ opioid receptor antagonist, opened the door for the investigation of inverse agonism/constitutive activity of the receptors. Various peptidic or non-peptidic δ opioid receptor inverse agonists have since been developed. Compared with the reports dealing with in vitro inverse agonist activities of novel compounds or known compounds as antagonists, there have been almost no publications describing the in vivo pharmacological effects induced by a δ opioid receptor inverse agonist. After the observation of anorectic effects with the δ opioid receptor antagonism was discussed in the early 2000s, the short-term memory improving effects and antitussive effects have been very recently reported as possible pharmacological effects induced by a δ opioid receptor inverse agonist. In this review, we will survey the developed δ opioid receptor inverse agonists and summarize the possible in vivo pharmacological effects by δ opioid receptor inverse agonists. Moreover, we will discuss important issues involved in the investigation of the in vivo pharmacological effects produced by a δ opioid receptor inverse agonist.

Discovery of δ opioid receptor full agonists lacking a basic nitrogen atom and their antidepressant-like effects.

We have recently reported that the elaboration of the N-substituent in the δ opioid receptor (DOR) antagonist naltrindole (NTI) enabled the regulation of the DOR activities from full inverse agonists to weak partial agonists. The investigations of amide-type NTI derivatives revealed that N-phenylacetyl and N-dihydrocinnamoyl derivatives 3a and 3b were DOR full agonists. The same transformations were applied to a DOR agonist KNT-127 to provide the more potent DOR agonists 6a and 6b. Among the tested compounds, the most efficacious compound 6a showed dose-dependent antidepressant-like effects in the mouse forced swim test. The antidepressant-like effects by 6a seemed to be more potent than those of KNT-127, which is a more potent DOR agonist in in vitro assays. The amide-type compound like 6a may more fully penetrate into the central nervous system.

A New Lead Identification Strategy: Screening an sp3 -rich and Lead-like Compound Library Composed of 7-Azanorbornane Derivatives.

Although the advantages of sp3 -rich, sterically complicated molecules in drug development have been pointed out, modern screening libraries are filled with planar, sp2 -rich components. Compounds that are sp3 -rich are difficult to synthesize, and thus we aimed to invent an efficient method to construct sp3 -rich libraries. By modifying sp3 -rich 7-azanorbornane scaffolds through click chemistry, we efficiently prepared a small set of compounds. These compounds were not only sp3 -rich, but also had sufficient "lead-like" properties in view of molecular weights and hydrophobicity. Screening assays of this library provided weak κ opioid receptor agonists and growth hormone secretagogue receptor agonists with high hit rates. These results indicate that the 7-azanorbornane scaffold may be a "privileged structure" for lead identification in drug discovery.

Development of Novel δ Opioid Receptor Inverse Agonists without a Basic Nitrogen Atom and Their Antitussive Effects in Mice.

Our previous results showed that naltrindole (NTI) derivatives with certain types of electron-withdrawing groups as an N-substituent showed δ opioid receptor (DOR) inverse agonistic activities. We therefore synthesized N-acylated NTI derivatives 3a-e and observed that N-benzoyl and N-cyclopropanecarbonyl derivatives SYK-736 (3b) and SYK-623 (3c) were DOR full inverse agonists and the N-acryloyl derivative 3d was a DOR partial inverse agonist. SKY-623 was over 110-fold more potent than the reference compound ICI-174,864. Both naltriben (NTB) and 7-benzylidenenaltrexone (BNTX) derivatives with N-benzoyl and N-cyclopropanecarbonyl groups were also DOR full inverse agonists. These N-acylated inverse agonists are interesting compounds because they have no basic nitrogen atom, which has been demonstrated to be an important pharmacophore. NTI and BNTX-type DOR inverse agonists SYK-623 and SYK-723 (12c) showed dose-dependent antitussive effects in a mouse cough model induced by citric acid exposure. The antitussive effects by SYK-623 and SYK-723 were significantly attenuated by pretreatment with DOR agonist SNC80.

Discovery of δ Opioid Receptor Full Inverse Agonists and Their Effects on Restraint Stress-Induced Cognitive Impairment in Mice.

The cyclopropylmethyl group in classical δ opioid receptor (DOR) antagonist NTI, BNTX, and NTB was replaced with various electron-withdrawing groups to develop DOR inverse agonists. N-Benzyl NTB derivative SYK-657 was a potent DOR full inverse agonist and its potency was over 10-fold potent than that of a reference compound ICI-174,864. Intraperitoneal administration of SYK-657 induced the short-term memory improving effect in mice without abnormal behaviors.

Neuropeptide oxytocin enhances µ opioid receptor signaling as a positive allosteric modulator.

Oxytocin (OT) is a 9-amine neuropeptide that plays an essential role in mammalian labor, lactation, maternal bonding, and social affiliation. OT has been reported to exert an analgesic effect in both humans and animals, and the results of certain animal experiments have shown that the analgesic effect of OT is partially blocked by opioid receptor antagonists. To investigate the relationship between OT and µ opioid receptor (MOR), we evaluated how OT affects MOR in vitro by performing an electrical impedance-based receptor biosensor assay (CellKey™ assay), an intracellular cAMP assay, and a competitive receptor-binding analysis by using cells stably expressing human MOR and OT receptor. In both the CellKey™ assay and the intracellular cAMP assay, OT alone exerted no direct agonistic effect on human MOR, but treatment with 10-6 M OT markedly enhanced the MOR signaling induced by 10-6 M endomorphin-1, ß-endorphin, morphine, fentanyl, and DAMGO. Moreover, in the competitive receptor-binding assay, 10-6 M OT did not alter the affinity of endomorphin-1 or morphine for MOR. These results suggest that OT could function as a positive allosteric modulator that regulates the efficacy of MOR signaling, and thus OT might represent a previously unrecognized candidate analgesic agent.

C-Homomorphinan Derivatives as Lead Compounds to Obtain Safer and More Clinically Useful Analgesics.

Buprenorphine shows strong analgesic effects on moderate to severe pain. Although buprenorphine can be used more safely than other opioid analgesics, it has room for improvement in clinical utility. Investigation of compounds structurally related to buprenorphine should be an approach to obtain novel analgesics with safer and improved profiles compared to buprenorphine. In the course of our previous studies, we observed that derivatives obtained by cyclizing C-homomorphinans were structurally related to buprenorphine. Hence, we synthesized cyclized C-homomorphinan derivatives with various oxygen functionalities on the side chains and evaluated their in vitro pharmacological profiles for the opioid receptors. Among the tested compounds, methyl ketone 2a with an N-methyl group showed full agonistic activities for the µ and the δ receptors and partial agonistic activity for the κ receptor. These properties were similar to those of norbuprenorphine, a major metabolite of buprenorphine, which reportedly contributes to the antinociceptive effect of buprenorphine. From these results, we concluded that cyclized C-homomorphinan would be a possible lead compound to obtain novel analgesics with buprenorphine-like properties.

Design and synthesis of novel δ opioid receptor agonists with an azatricyclodecane skeleton for improving blood-brain barrier penetration.

We designed and synthesized novel δ opioid receptor (DOR) agonists 3a-i with an azatricyclodecane skeleton, which was a novel structural class of DOR agonists. Among them, 3b exhibited high values of binding affinity and potent agonistic activity for the DOR that were approximately equivalent to those of 2 which bore an oxazatricyclodecane skeleton. In vitro assays using the blood-brain barrier (BBB) permeability test kit supported the idea that 3b achieved an excellent BBB permeability by converting an oxygen atom of 2 to a carbon atom (methylene group) in the core skeleton. As a result, 3b showed potent antinociceptive effects.

Novel delta opioid receptor agonists with oxazatricyclodecane structure showing potent agonistic activities.

We recently reported oxazatricyclodecane derivatives 1 as δ opioid receptor (DOR) agonists having a novel chemotype, but their DOR agonistic activities were relatively low. Based on the working hypothesis that the dioxamethylene moiety in 1 may be an accessory site and that it may interfere with the sufficient conformational change of the receptor required for exerting the full agonistic responses, we designed and synthesized new oxazatricyclodecane derivatives 2-4 lacking the dioxamethylene moiety. As we expected, the designed compounds 2-4 showed pronouncedly improved agonistic activities for the DOR. Compound 2a with the 17-cyclopropylmethyl substituent was a potent agonist with the highest selectivity for the DOR and was expected to be a lead compound for novel and selective DOR agonists.

Design and Synthesis of Potent and Highly Selective Orexin 1 Receptor Antagonists with a Morphinan Skeleton and Their Pharmacologies.

Nalfurafine, a κ-selective opioid receptor agonist, unexpectedly showed a selective antagonist activity toward the orexin 1 receptor (OX1R) (Ki = 250 nM). Modification of the 17-amino side chain of the opioid ligand to an arylsulfonyl group and the 6-furan acrylamide chain to 2-pyridyl acrylamide led to compound 71 with improvement of the antagonist activity (OX1R, Ki = 1.36 nM; OX2R, not active) without any detectable affinity for the opioid receptor. The dihydrosulfate salt of 71, freely soluble in water, attenuated the physical dependence of morphine. Furthermore, all of the active nalfurafine derivatives in this study had almost no activity for OX2R, which led to high OX1R selectivity. These results suggest that nalfurafine derivatives could be a useful series of lead compounds to develop highly selective OX1R antagonists.

Narcolepsy-like sleep disturbance in orexin knockout mice are normalized by the 5-HT1A receptor agonist 8-OH-DPAT.

RATIONALE: Orexin knockout (KO) mice exhibit a phenotype that is similar to human narcolepsy, and monoamine-related compounds, such as psychostimulants and 5-HT uptake inhibitors, have been used for the treatment of narcoleptic disorders. However, little information is available regarding the pathophysiological features of orexin KO mice, particularly with respect to their narcoleptic-like disorder and how it is affected by monoamine-related compounds. OBJECTIVES: The present study was designed to investigate both the nature of the neuronal changes in orexin KO mice and the therapeutic effects of monoamine-related compounds on the sleep disorder in orexin KO mice. RESULTS: A decrease in locomotor activity in the dark phase was observed in orexin KO mice, and psychostimulants and 5-HT-related compounds, such as 8-OH-DPAT (5-HT1A receptor agonist) and DOI (5-HT2 receptor agonist), inhibited this hypolocomotion. We also found that 5-HT1A receptor mRNA levels, but not those for 5-HT2 or dopamine receptors, were significantly decreased in the prefrontal cortex of orexin KO mice in the dark period and were accompanied by compromising the increase in 5-HT metabolite levels. In addition, the sleep disorder in orexin KO mice, as analyzed by a polysomnography during the dark period, was completely normalized by 8-OH-DPAT. CONCLUSION: These results suggest that a dysfunction of 5-HT1A receptors is involved in the narcoleptic-like sleep dysfunction in orexin KO mice, and such dysfunction may participate in orexin deficiency-induced sleep disorders. Further, the use of 5-HT1A receptor agonist could be useful for treating the sleep disorder under a deficiency of orexin.