Jörg Holenz

Synthesis and Biological Evaluation of the 1-Arylpyrazole Class of σ1 Receptor Antagonists: Identification of 4-{2-[5-Methyl-1-(naphthalen-2-yl)-1H-pyrazol-3-yloxy]ethyl}morpholine (S1RA, E-52862)

The synthesis and pharmacological activity of a new series of 1-arylpyrazoles as potent σ(1) receptor (σ(1)R) antagonists are reported. The new compounds were evaluated in vitro in human σ(1)R and guinea pig σ(2) receptor (σ(2)R) binding assays. The nature of the pyrazole substituents was crucial for activity, and a basic amine was shown to be necessary, in accordance with known receptor pharmacophores. A wide variety of amines and spacer lengths between the amino and pyrazole groups were tolerated, but only the ethylenoxy spacer and small cyclic amines provided compounds with sufficient selectivity for σ(1)R vs σ(2)R. The most selective compounds were further profiled, and compound 28, 4-{2-[5-methyl-1-(naphthalen-2-yl)-1H-pyrazol-3-yloxy]ethyl}morpholine (S1RA, E-52862), which showed high activity in the mouse capsaicin model of neurogenic pain, emerged as the most interesting candidate. In addition, compound 28 exerted dose-dependent antinociceptive effects in several neuropathic pain models. This, together with its good physicochemical, safety, and ADME properties, led compound 28 to be selected as clinical candidate.

Design and synthesis of β-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors with in vivo brain reduction of β-amyloid peptides.

The evaluation of a series of aminoisoindoles as β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors and the discovery of a clinical candidate drug for Alzheimers disease, (S)-32 (AZD3839), are described. The improvement in permeability properties by the introduction of fluorine adjacent to the amidine moiety, resulting in in vivo brain reduction of Aβ40, is discussed. Due to the basic nature of these compounds, they displayed affinity for the human ether-a-go-go related gene (hERG) ion channel. Different ways to reduce hERG inhibition and increase hERG margins for this series are described, culminating in (S)-16 and (R)-41 showing large in vitro margins with BACE1 cell IC(50) values of 8.6 and 0.16 nM, respectively, and hERG IC(50) values of 16 and 2.8 μM, respectively. Several compounds were advanced into pharmacodynamic studies and demonstrated significant reduction of β-amyloid peptides in mouse brain following oral dosing.

Efficacy of selective 5‐HT6 receptor ligands determined by monitoring 5‐HT6 receptor‐mediated cAMP signaling pathways

1 Two novel selective 5‐HT6 receptor ligands E‐6801 (6‐chloro‐N‐(3‐(2‐(dimethylamino)ethyl)‐1H‐indol‐5‐yl)imidazo[2,1‐b]thiazole‐5‐sulfonamide) and E‐6837 (5‐chloro‐N‐(3‐(2‐(dimethylamino)ethyl)‐1H‐indol‐5‐yl)naphthalene‐2‐sulfonamide) were investigated and compared to the putative 5‐HT6 receptor antagonists SB‐271046 (5‐chloro‐N‐(4‐methoxy‐3‐(piperazin‐1‐yl)phenyl)‐3‐methylbenzo[b]thiophene‐2‐sulfonamide) and Ro 04‐06790 (N‐(2,6‐bis(methylamino)pyrimidin‐4‐yl)‐4‐aminobenzenesulfonamide) using a cAMP‐mediated pathway. 2 Forskolin stimulation, to increase the magnitude of agonist cAMP responses, and site‐directed mutagenesis of the 5‐HT6 receptor, in order to yield constitutively active receptor, were applied. 3 5‐HT (Emax, % over basal: 200), E‐6801 (120) and E‐6837 (23) induced cAMP formation at the rat 5‐HT6 receptor. In the copresence of forskolin, cAMP responses were more potent and enhanced to 294 (5‐HT, % over forskolin), 250 (E‐6801) and 207 (E‐6837), respectively. 5‐HT‐mediated cAMP formation was dose‐dependently blocked by SB‐271046 (pA2: 8.76±0.22) and Ro 04‐6790 (pA2: 7.89±0.10) and not affected by the copresence of forskolin. Both E‐6801 and E‐6837 yielded partial antagonism of the 5‐HT response in the absence of forskolin, whereas antagonism was either completely absent (E‐6801) or attenuated (E‐6837) in the copresence of forskolin. Intrinsic activity of these 5‐HT6 receptor ligands at a constitutively active human S267K 5‐HT6 receptor in Cos‐7 cells indicated similar efficacy (Emax, % over basal) for 5‐HT (97), E‐6801 (91) and E‐6837 (100), while Ro 04‐6790 (‐33) and SB‐271046 (‐39) were equi‐efficacious inverse agonists. 4 The use of either forskolin or a constitutively active S267K 5‐HT6 receptor enhances the resolution for monitoring the efficacy of 5‐HT6 receptor ligands. E‐6801 and E‐6837 are potent partial agonists at the 5‐HT6 receptor. Ro 04‐6790 and SB‐271046 appear to act as inverse agonists/antagonists.

Aminoimidazoles as Bace-1 Inhibitors: The Challenge to Achieve in Vivo Brain Efficacy

The evaluation of a series of bicyclic aminoimidazoles as potent BACE-1 inhibitors is described. The crystal structures of compounds 14 and 23 in complex with BACE-1 reveal hydrogen bond interactions with the protein important for achieving potent inhibition. The optimization of permeability and efflux properties of the compounds is discussed as well as the importance of these properties for attaining in vivo brain efficacy. Compound (R)-25 was selected for evaluation in vivo in wild type mice and 1.5h after oral co-administration of 300μmol/kg (R)-25 and efflux inhibitor GF120918 the brain Aβ40 level was reduced by 17% and the plasma Aβ40 level by 76%.

Sulfonimidamides as Sulfonamides Bioisosteres: Rational Evaluation through Synthetic, in Vitro, and in Vivo Studies with γ-Secretase Inhibitors

The proof of the pudding: A proof-of-concept study using γ-secretase inhibitors as a model has shown that sulfonimidamides act as bioisosteres for sulfonamides. Detailed in vitro and in vivo profiling reveal that the sulfonimidamide motif imparts desirable properties such as decreased lipophilicity and plasma protein binding, accompanied by increased solubility. Our data support a wider use of this unique functional group in the design of new pharmacologically active agents.

Pharmacological and metabolic characterisation of the potent σ1 receptor ligand 1′-benzyl-3-methoxy-3H-spiro[[2]benzofuran-1,4′-piperidine]

Objectives The pharmacology and metabolism of the potent σ1 receptor ligand 1′‐benzyl‐3‐methoxy‐3H‐spiro[[2]benzofuran‐1,4′‐piperidine] were evaluated.

A Medicinal‐Chemistry‐Guided Approach to Selective and Druglike Sigma 1 Ligands

Based on a medicinal‐chemistry‐guided approach, three novel series of druglike cycloalkyl‐annelated pyrazoles were synthesized and display high affinity (pKi >8) for the σ1 receptor. Structure–affinity relationships were established, and the different scaffolds were optimized with respect to σ1 binding and selectivity versus the σ2 receptor and the hERG channel, resulting in selective compounds that have Ki values (for σ1) in the subnanomolar range. Selected compounds were screened for cytochrome P450 inhibition (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4), metabolic stability (rat and human liver microsomes), and cell‐membrane permeability (Caco‐2). They showed favorable in vitro ADME properties as well as favorable calculated druglike and experimental physicochemical properties. Furthermore, compounds 7 f and 17 a, for example, displayed high selectivity (affinity) for the σ1 receptor against a wide range of other receptors (>60). With these valuable tool compounds in hand, we are further exploring the role of the σ1 receptor in relevant animal models corresponding to such medicinal indications as drug abuse, pain, depression, anxiety, and psychosis.

Identification of Novel Indanylsulfonamide Guanylhydrazones as Potent 5-HT6 Serotonin Receptor Antagonists

Changing the N,N-(dimethylamino)ethyl side chain in the N-[3-(aminoethyl)inden-5-yl]sulfonamide 5-HT(6) serotonin receptor agonists 1 by a conformationally rigid guanylhydrazone moiety at the indene 3-position led to the identification of the title indanylguanylhydrazones 6, which exhibited excellent binding affinities and an antagonistic response at the 5-HT(6) receptor, with K(i) and IC(50) values in the nanomolar range (K(i) >or= 1.2 nM, IC(50) >or= 47 nM, and I(max) <or= 173%).

Indene-based frameworks targeting the 5-HT6 serotonin receptor: Ring constraint in indenylsulfonamides using cyclic amines and structurally abbreviated counterparts

Further studies in quest of 5-HT(6) serotonin receptor ligands led to the design and synthesis of a few selected examples of N-(inden-5-yl)sulfonamides with a ring-constrained aminoethyl side chain at the indene 3-position, some of which exhibited a high binding affinity, such as the pyrrolidine analogue 28 (K(i)=3nM). Moreover, the structurally abbreviated N-(inden-5-yl)sulfonamides showed K(i) values > or = 43 nM, which indicates that neither the N,N-aminoethyl nor the conformationally restricted aminoethyl side arm at the indene 3-position are required for binding. Selected compounds were then tested in a functional cAMP stimulation assay and found to act as 5-HT(6) antagonists, although with moderate potency at the micromolar level.

The small molecule CLP257 does not modify activity of the K + –Cl − co-transporter KCC2 but does potentiate GABA A receptor activity

To the Editor: The neuronal K+–Cl– co-transporter KCC2 (also known as solute carrier family 12 member 5, or SLC12A5) critically maintains the neuronal Cl– gradient to establish hyperpolarizing signaling by GABAA receptors, the primary mediators of fast synaptic inhibition in the brain. These receptors are also the principal targets of benzodiazepines, neurosteroids, and intravenous general anesthetics1. The downregulation of KCC2 in neuropathic pain models and seizurerelated disorders indentifies it as a putative therapeutic target. Through a small-molecule screen in neuroblastoma/glioma-derivative NG108-15 cells, Gagnon et al.2 identified CLP257 as a ‘KCC2 activator’. We therefore sought to confirm and further determine its mechanism of action. We began our investigation by using gramicidin perforated-patch recordings to measure the levels of intracellular Cl– ([Cl]i) in NG108-15 cells that were cultured as detailed in the original report. We observed no change in intracellular Cl– values after a 5-h exposure to CLP257, which according to the original paper would produce an easily detectable ~23 mM reduction in Cl– (Fig. 1a). Furthermore, acute administration of the selective KCC2 inhibitor VU0463271 did not affect Cl– levels in either vehicleor CLP257-treated cells (Supplementary Fig. 1)3. In parallel experiments, we analyzed the actions of CLP257 on KCC2 function using fluorescence resonance energy transfer (FRET)-based Cl– imaging that reproduced the conditions of the original publication2. Importantly, upon dissolution, CLP257 formed a bright yellow solution, confounding interpretation of CFP-YFP conjugate–based Cl– imaging, which was not explicitly described in the original report. In contrast to the original findings, after stimulation of NG108-15 cells with medium containing increased extracellular Cl–, CLP257 increased intracellular Cl–. This effect was further potentiated by VU0463271 and therefore is not mediated by KCC2 (Fig. 1b and Supplementary Fig. 2a,b). In addition, we confirmed a previously reported resting Cl– current in NG10815 cells (Supplementary Fig. 2c,d)4, which further complicates data interpretation in these cells5. To further investigate the expression of KCC2 in NG108-15 cells, we used standard biochemical analyses. To match the original report2, we performed immunoprecipitations with a KCC2-specific antibody and control IgG in NG108-15 cell lysates and rat cortical neuronal extracts. KCC2 protein was specifically detected in lysates from rat cortical neurons. In contrast, no KCC2 protein was found in NG108-15 cells obtained from a commercial source or in NG108-cl cells (stably expressing the Cl−-sensitive indicator Clomeleon) from the cell stock obtained from Y. De Koninck at Laval University and used in the original CLP257 report (Fig. 1c and Supplementary Fig. 3a,b). This result is in agreement with a previous report indicating a lack of KCC2 protein in NG108-15 cells6. We also did not detect KCC2 protein after a 5-h exposure to CLP257 (Supplementary Fig. 3c,d). Collectively, our electrophysiological, imaging, and biochemical experiments suggest that NG108-15 cells do not express detectable levels of KCC2 protein or activity, calling into question the use of these cells in a screen to identify KCC2 activators. To further examine whether CLP257 functions through KCC2, we exogenously expressed human KCC2 in HEK293 cells. We used fluorescence-based Tl+ influx assays that have repeatedly been used to determine KCC2 activity7. Five-hour exposure of HEK-KCC2 cells to CLP257 did not accelerate Tl+ transport. The well-established KCC2 potentiator N-ethylmaleimide (NEM) substantially increased Tl+ uptake, and VU0463271 reduced Tl+ uptake (Fig. 1d and Supplementary Fig. 4a)8. Additionally, CLP257 did not modify the activity of NKCC1 (also known as solute carrier family 12 member 2, or SLC12A2), whereas exposure to bumetanide, a known NKCC1 inhibitor, resulted in a clear reduction in activity (Supplementary Fig. 4b). We then used perforated-patch electrophysiological assays to assess Cl– homeostasis. Baseline Cl– levels were unaffected by CLP257 even though the calculated flux reversal point of KCC2 under these recording conditions was lower than the values we observed8, indicating that there is a window to potentiate KCC2 function (Supplementary Fig. 4c). After a 2-min exposure to bathing solution containing high K+, the rate of Cl– uptake was not affected by CLP257 (Fig. 1e). Likewise, after switching back to solutions with normal K+ levels for a 2-min period, the rate of Cl– extrusion was not affected by CLP257 (Fig. 1e). Isolation of the KCC2 component of the Cl– flux with furosemide again revealed no effect of CLP257 on Cl– transport (Supplementary Fig. 4d)8,9. Thus, CLP257 does not modulate KCC2 activity. CLP257 was stated to activate KCC2 by increasing cell-surface levels in the original report2. To examine this claim, we exogenously expressed tagged KCC2 constructs together with previously published KCC2 mutants that exhibit either increased (KCC2 A/A) or decreased (KCC2 E/E) cell-surface expression in N2a cells (Fig. 1f and Supplementary Fig. 5)10. At low concentrations, CLP257 did not modify cell-surface levels of KCC2, whereas at higher concentrations of CLP257 these levels were reduced. Thus, CLP257 does not increase the cell-surface expression of KCC2 as originally stated. We then employed backscattering interferometry technology to determine whether CLP257 binds to KCC2 (ref. 11). Whereas VU0463271 robustly bound to human KCC2 in this assay with an affinity close to its half-maximal inhibitory concentration (IC50), no CLP257 binding was observed (Supplementary Table 1). Given this observation, we sought to identify other targets of CLP257 that could explain the observed benefits in animal models. CLP257 demonstrated potent inhibition of MAO-B with nanomolar efficacy and binding to other targets in the low micromolar concentration range including PPARg, the 5-HT1A recepThe small molecule CLP257 does not modify activity of the K+–Cl– co-transporter KCC2 but does potentiate GABAA receptor activity