Synthesis and biological evaluation of conformationally restricted derivatives of tryptophan as NK1/NK2 ligands

Summary Chemical modifications on the NK₁ competitive antagonist L-732,138, with a view to creating a dual NK₁/NK₂ ligand, led to the tryptophan derivative 1 possessing the protected Gly-Leu sequence of the C-terminus of substance P and neurokinin A. Modifications in the nature of the carbamate function increased the selectivity for the NK₁ receptor, whereas the inclusion of the indole moiety in β-carboline or carbazole rings decreased the affinity for both receptors. Free indolylmethyl and Cbz carbamate groups were shown to be essential for NK₂ affinity.     

Synthesis and biological evaluation of conformationally restricted derivatives of tryptophan as NK1/NK2 ligands

Chemical modifications on the NK₁ competitive antagonist L-732,138, with a view to creating a dual NK₁/NK₂ ligand, led to the tryptophan derivative 1 possessing the protected Gly-Leu sequence of the C-terminus of substance P and neurokinin A. Modifications in the nature of the carbamate function increased the selectivity for the NK₁ receptor, whereas the inclusion of the indole moiety in -carboline or carbazole rings decreased the affinity for both receptors. Free indolylmethyl and Cbz carbamate groups were shown to be essential for NK₂ affinity.     

Synthesis and biological evaluation of tripeptide derivatives of Cbz-Gly-Leu-Trp-OBzl(CF3)2 as NK1/NK2 ligands

Chemical modifications were obtained on the dual NK₁/NK₂ ligand Cbz-Gly-Leu-Trp-OBzl(CF₃)₂ with a view to optimizing affinities for both NK₁ and NK₂ receptors. Replacement of the Gly residue by other amino acids increased affinities for NK₁/NK₂ receptors or induced selectivity for the NK₁ receptor.     

Synthesis and biological evaluation of tripeptide derivatives of Cbz-Gly-Leu-Trp-OBzl(CF3)2 as NK1/NK2 ligands

Summary Chemical modifications were obtained on the dual NK₁/NK₂ ligand Cbz-Gly-Leu-Trp-OBzl(CF₃)₂ with a view to optimizing affinities for both NK₁ and NK₂ receptors. Replacement of the Gly residue by other amino acids increased affinities for NK₁/NK₂ receptors or induced selectivity for the NK₁ receptor.     

Identification,biological characterization and pharmacophoric analysis of a new potent and selective NK1 receptor antagonist clinical candidate

The last two decades have provided a large weight of preclinical data implicating the neurokinin-1 receptor (NK₁) and its cognate ligand substance P (SP) in a broad range of both central and peripheral disease conditions. However, to date, only the NK₁ receptor antagonist aprepitant has been approved as a therapeutic and this is to prevent chemotherapy-induced nausea & vomiting (CINV). The belief remained that the full therapeutic potential of NK₁ receptor antagonists had yet to be realized; therefore clinical evidence that NK₁ receptor antagonists may be effective in major depression disorder, resulted in a significant further investment in discovering novel CNS penetrant druggable NK₁ receptor antagonists to address this condition. At GlaxoSmithKline after the discovery of casopitant, that went on to demonstrate efficacy as a novel antidepressant in the clinic, additional novel analogues of this NK₁ receptor antagonist were designed to further enhance its drug developability characteristics. Herein, we therefore describe the discovery process and the vivo pharmacological and pharmacokinetic profile of the new NK₁ receptor antagonist 3a (also called orvepitant), selected as clinical candidate and further progressed into clinical studies for major depressive disorder. Moreover, molecular modeling studies enabled us to improve the pharmacophore model of the NK₁ receptor antagonists with the identification of a region able to accommodate a variety of heterocycle moieties.     

Design,optimization, and in vivo evaluation of a series of pyridine derivatives with dual NK1 antagonism and SERT inhibition for the treatment of depression

A series of substituted pyridines, ether linked to a phenylpiperidine core were optimized for dual NK₁/SERT affinity. Optimization based on NK₁/SERT binding affinities, and minimization of off-target ion channel activity lead to the discovery of compound 44. In vivo evaluation of 44 in the gerbil forced swim test (a depression model), and ex-vivo NK₁/SERT receptor occupancy data support the potential of a dual acting compound for the treatment of depression.     

Identification of a new series of non-peptidic NK3 receptor antagonists

The identification and structure-activity relationships of 2-aminomethyl-1-aryl cyclopropane carboxamides as novel NK₃ receptor antagonists are reported. The compound series was optimized to give analogues with low nanomolar binding to the NK₃ receptor and brain exposure, leading to activity in vivo in the senktide-induced hypoactivity model in gerbils.     

New quinoline NK3 receptor antagonists with CNS activity

Lead optimisation starting from the previously reported selective quinoline NK₃ receptor antagonists talnetant 2 (SB-223412) and 3 (SB-222200) led to the identification of 3-aminoquinoline NK₃ antagonist 10 (GSK172981) with excellent CNS penetration. Investigation of a structurally related series of sulfonamides with reduced lipophilicity led to the discovery of 20 (GSK256471). Both 10 and 20 are high affinity, potent NK₃ receptor antagonists which despite having different degrees of CNS penetration produced excellent NK₃ receptor occupancy in an ex vivo binding study in gerbil cortex.     

Tetrahydroindolizinone NK1 antagonists

A new class of potent NK₁ receptor antagonists with a tetrahydroindolizinone core has been identified. This series of compounds demonstrated improved functional activities as compared to previously identified 5,5-fused pyrrolidine lead structures. SAR at the 7-position of the tetrahydroindolizinone core is discussed in detail. A number of compounds displayed high NK₁ receptor occupancy at both 1 h and 24 h in a gerbil foot tapping model. Compound 40 has high NK₁ binding affinity, good selectivity for other NK receptors and promising in vivo properties. It also has clean P₄₅₀ inhibition and hPXR induction profiles.     

Role of the tachykinin NK1 receptor in a murine model of cigarette smoke-induced pulmonary inflammation

Background

The tachykinins, substance P and neurokinin A, present in sensory nerves and inflammatory cells such as macrophages and dendritic cells, are considered as pro-inflammatory agents. Inflammation of the airways and lung parenchyma plays a major role in the pathogenesis of chronic obstructive pulmonary disease (COPD) and increased tachykinin levels are recovered from the airways of COPD patients. The aim of our study was to clarify the involvement of the tachykinin NK₁ receptor, the preferential receptor for substance P, in cigarette smoke (CS)-induced pulmonary inflammation and emphysema in a mouse model of COPD.

Methods

Tachykinin NK₁ receptor knockout (NK₁-R^-/-) mice and their wild type controls (all in a mixed 129/sv-C57BL/6 background) were subjected to sub acute (4 weeks) or chronic (24 weeks) exposure to air or CS. 24 hours after the last exposure, pulmonary inflammation and development of emphysema were evaluated.

Results

Sub acute and chronic exposure to CS resulted in a substantial accumulation of inflammatory cells in the airways of both WT and NK₁-R^-/- mice. However, the CS-induced increase in macrophages and dendritic cells was significantly impaired in NK₁-R^-/- mice, compared to WT controls, and correlated with an attenuated release of MIP-3α/CCL20 and TGF-β1. Chronic exposure to CS resulted in development of pulmonary emphysema in WT mice. NK₁-R^-/- mice showed already enlarged airspaces upon air-exposure. Upon CS-exposure, the NK₁-R^-/- mice did not develop additional destruction of the lung parenchyma. Moreover, an impaired production of MMP-12 by alveolar macrophages upon CS-exposure was observed in these KO mice. In a pharmacological validation experiment using the NK₁ receptor antagonist RP 67580, we confirmed the protective effect of absence of the NK₁ receptor on CS-induced pulmonary inflammation.

Conclusion

These data suggest that the tachykinin NK₁ receptor is involved in the accumulation of macrophages and dendritic cells in the airways upon CS-exposure and in the development of smoking-induced emphysema. As both inflammation of the airways and parenchymal destruction are important characteristics of COPD, these findings may have implications in the future treatment of this devastating disease.     

Discovery of a cyclopentylamine as an orally active dual NK1 receptor antagonist–serotonin reuptake transporter inhibitor

Cyclopentylamine 4 was identified as a potent dual NK₁R antagonist–SERT inhibitor. This compound demonstrated significant oral activity in the gerbil forced swimming test, suggesting that dual NK₁R antagonists–SERT inhibitors may be useful in treating depression disorders.     

Discovery of a novel, potent and orally active series of gamma-lactams as selective NK1 antagonists

Strategic replacement of the nitrogen of the lead compound 1 in the original cyclic urea series with a carbon resulted in the discovery of a novel, potent and orally more efficacious γ-lactam series of selective NK₁ antagonists. Optimization of the lactam series culminated in the identification of compounds with high binding affinity and excellent oral CNS activity.     

SR 142801, The first potent non-peptide antagonist of the tachykinin NK3 receptor

SR 142801 is the first potent and selective non-peptide antagonist of the tachykinin NK₃ receptor. It inhibited [MePhe⁷]NKB binding to its receptor from various species, including humans. SR 142801 was a competitive antagonist of [MePhe⁷]NKB-mediated contractions of guinea-pig ileum and inhibited the acetylcholine release following the activation of the guinea-pig ileum tachykinin NK₃ receptor. In vivo, SR 142801 potently inhibited the turning behaviour induced by intrastriatal injection of senktide in gerbils, and appears as a powerful tool for investigation of the physiological and pathological role of NKB and its NK₃ receptor.     

Neurons bearing NK3 tachykinin receptors in the guinea-pig ileum revealed by specific binding of fluorescently labelled agonists

The localisation of NK₃ tachykinin receptors in guinea-pig ileum was studied using the fluorescently labelled agonists, Cy3.5-neurokinin A and Cy3.5-kassinin. Binding to nerve cell bodies in the myenteric and submucosal plexuses was visualised using confocal microscopy. Binding to NK₁ receptors was blocked by the NK₁ receptor antagonist, CP-99994. NK₃ receptors, demonstrated by binding in the presence of CP-99994, occurred in 72% of myenteric and 38% of submucosal neurons. Colocalisation with other markers was examined to deduce the classes of neurons with NK₃ receptors. In myenteric ganglia, NK₃ receptors were present on the following: 73% of calbindin-immunoreactive (IR) intrinsic primary afferent neurons, 63% of calretinin-IR excitatory motor neurons and ascending interneurons, 63% of nitric oxide synthase-IR inhibitory motor neurons and descending interneurons, 79% of strongly neuropeptide Y (NPY)-IR secretomotor neurons, 67% of weakly NPY-IR descending interneurons and motor neurons, and 46% of NK₁ receptor-IR neurons. In submucosal ganglia, NK₃ receptors were on 65% of calretinin-IR secretomotor/vasodilator neurons, 81% of NPY-IR cholinergic secretomotor neurons, 2% of vasoactive intestinal peptide-IR non-cholinergic secretomotor neurons and were completely absent from substance P-IR intrinsic primary afferent neurons. The results support physiological studies suggesting that NK₃ receptors mediate tachykinin transmission between myenteric sensory neurons and to interneurons and/or motor neurons in descending inhibitory and ascending excitatory pathways. Accepted: 22 June 1999     

Biochemical Characterization and Solubilization of Human NK2 Receptor Expressed in Chinese Hamster Ovary Cells

Abstract

The human ileum neurokinin NK₂ receptor has been stably expressed in Chinese hamster ovary (CHO) cells using the dihydrofolate reductase (DHFR) expression system. Amplified cell populations expressing approximately 7×10⁵ NK₂ receptors/cell were selected in the presence of the DHFR inhibitor methotrexate. Cross-linking of [¹²⁵I]NKA to NK₂ receptor transfected cells revealed a specifically labeled protein of apparent molecular weight 64 kDa by SDS-polyacrylamide gel electrophoresis. This protein was deglycosylated by the enzymes N-glycosidase F and endoglycosydase F to a protein of apparent molecular weight of 39 kDa. The NK₂ receptor was solubilized in an active form from CHO cell membranes using the zwitterionic detergent CHAPS. This method represents a valuable approach for the production of significant amounts of NK₂ receptor protein from mammalian cells.     

Discovery of disubstituted piperidines and homopiperidines as potent dual NK1 receptor antagonists–serotonin reuptake transporter inhibitors for the treatment of depression

This report describes the synthesis, structure–activity relationships and activity of piperidine, homopiperidine, and azocane derivatives combining NK₁ receptor (NK₁R) antagonism and serotonin reuptake transporter (SERT) inhibition. Our studies culminated in the discovery of piperidine 2 and homopiperidine 8 as potent dual NK₁R antagonists-SERT inhibitors. Compound 2 demonstrated significant activity in the gerbil forced swimming test, suggesting that dual NK₁R antagonists-SERT inhibitors may be useful in treating depression disorders.     

Bioactive conformations of peptides and mimetics as milestones in drug design: Investigation of NK1 receptor antagonists

Summary Nine potent and selective substance P receptor antagonists (NK₁ ^-) were analyzed with respect to their conformational space, with the aim to suggest probable conformations adopted at the receptor site and superposition rules for each structure (pharmacophore mapping). Key atoms within the ligands as well as receptor site points were considered in order to identify acceptable solutions (DISCO program). The results obtained allowed the suggestion or probable peptidic pharmacophores based on the structure of 1 (FK888). This knowledge was used to search commercial databases. The number and diversity of known retrieved NK₁ antagonists allowed a general evaluation of the proposed pharmacophores. Moreover, a search in our proprietary database detected a short peptide with modest affinity but high selectivity for the NK₁ receptor. The combination of molecular modeling with database searches is useful in a strategy aiming to develop new NK₁ antagonists starting from existing knowledge.     

Convergent QSAR studies on a series of NK3 receptor antagonists for schizophrenia treatment

The dopamine hypothesis states that decreased dopaminergic neurotransmission reduces schizophrenia symptoms. Neurokinin-3 receptor (NK₃) antagonists reduce dopamine release and have shown positive effects in pre-clinical and clinical trials. We employed 2D and 3D-QSAR analysis on a series of 40 non-peptide NK₃ antagonists. Multivariate statistical analysis, PCA and HCA, were performed to rational training/test set splitting and PLS regression was employed to construct all QSAR models. We constructed one highly predictive CoMFA model (q^2?=?0.810 and r^2?=?0.929) and acceptable HQSAR and CoMSIA models (HQSAR q^2?=?0.644 and r^2?=?0.910; CoMSIA q^2?=?0.691, r^2?=?0.911). The three different techniques provided convergent physicochemical results. All models indicate cyclopropane, piperidine and di-chloro-phenyl ring attached to cyclopropane ring and also the amide group attached to the piperidine ring could play an important role in ligand–receptor interactions. These findings may contribute to develop potential NK₃ receptor antagonists for schizophrenia.     

Further Identification of Neurokinin Receptor Types and Mechanisms of Calcium Signaling Evoked by Neurokinins in the Murine Neuroblastoma C1300 Cell Line

Abstract: It has been suggested that murine neuroblastoma C1300 cells express endogenous neurokinin NK₂ receptors with features that differ from those of NK₂ receptors characterized in other systems. In this study, we have further characterized the neurokinin receptor types present in this cell line. RNA blots showed that mRNAs of NK₂ and NK₃ receptors, but not of NK₁ receptors, were expressed in C1300 cells. The increase in the cytosolic calcium concentration ([Ca²⁺]_i) induced by 0.33 µM neurokinin A was completely inhibited by SR 48968, an NK₂ receptor antagonist, whereas the partial response to 0.33 µM neurokinin B was unaffected, and the response was completely inhibited by SR 142801, an NK₃ receptor antagonist. In addition, the [Ca²⁺]_i increase by 0.33 µM senktide, an NK₃ receptor agonist, was inhibited by SR 142801 but not by SR 48968. These findings indicated that C1300 cells endogenously express functional NK₂ and NK₃ receptors. It was also demonstrated that NK₂ and NK₃ receptors can be activated independently by 3.3 µM neurokinin A in the presence of 1.0 µM SR 142801 or 1.0 µM senktide, respectively. Therefore, the mechanisms of Ca²⁺ signaling mediated by endogenous NK₂ and NK₃ receptors were investigated. The independent activation of NK₂ or NK₃ receptors induced not only the [Ca²⁺]_i increase, but also stimulated the formation of inositol trisphosphates; both these responses were inhibited by U73122, a phospholipase C (PLC) inhibitor. In addition, NK₂ and NK₃ receptor-mediated [Ca²⁺]_i increase was partially attenuated in the absence of extracellular Ca²⁺ or in the presence of nickel, an inorganic Ca²⁺ influx blocker, but was unaffected by nifedipine and ω-conotoxin, L- and N-type voltage-dependent Ca²⁺ channel blockers, respectively. Furthermore, the depolarization by 60 mM K⁺ did not affect the [Ca²⁺]_i. These findings suggested that the NK₂ and NK₃ receptor-mediated [Ca²⁺]_i increase was due to the activation of PLC and was dependent on the mobilization of internal Ca²⁺ and the entry of extracellular Ca²⁺ through voltage-independent channels. This study showed that the C1300 cell line is a useful system with which to investigate pharmacological functions and signaling pathways of endogenous NK₂ and NK₃ receptors.     

New substance P receptor antagonists. Conception, synthesis and biological evaluation of spirolactam derivatives and their tripeptide Cbz-Pro-Leu-Trp-OBzl(CF3)2 and lactam pseudopeptide derivatives

New chemical entities were designed from the C-terminal sequence of GR71251, an NK₁ pseudopeptide antagonist. Three new NK₁ antagonists were identified with high affinity and selectivity for NK₁ receptors.