Quinolone bioisosteres of phenolic GluN2B-selective NMDA receptor antagonists.

Cyclopenta[g]quinolones of type 4 were designed with the aim to bioisosterically replace the phenol of potent GluN2B ligands such as ifenprodil and Ro 25-6981 by the quinolone system and to restrict the conformational flexibility of the aminopropanol substructure in a cyclopentane system. The designed ligands were synthesized in an eight-step sequence starting with terephthalaldehyde (5). Key steps pf the synthesis were the intramolecular Friedel-Crafts acylation of propionic acids 10 to yield the cyclopenta[g]quinolinediones 11 and the Mannich reaction of diketone 11a followed by conjugate addition at the α,ß-unsaturated ketone 12a. Although the quinolones 13a, 15a, and 16a contain an H-bond donor group (secondary lactam) as ifenprodil and Ro 25-6981, they show only moderate GluN2B affinity (Ki > 410 nM). However, the introduction of lipophilic substituents at the quinolone N-atom resulted in more than 10-fold increased GluN2B affinity of the benzyl and benzyloxymethyl derivatives cis-13c (Ko = 36 nM) and 13e (Ko = 27 nM). All compounds are selective over the phencyclidine (PCP) binding site of the N-methyl-D-aspartate (NMDA) receptor. The benzyl derivative 13c showed six- and threefold selectivity over σ1 and σ2 receptors, respectively.

Chemical, pharmacodynamic and pharmacokinetic characterization of the GluN2B receptor antagonist 3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol - starting point for PET tracer development.

GluN2B-NMDA receptors play a key role in several neurological and neurodegenerative disorders. In order to develop novel negative allosteric GluN2B-NMDA receptor modulators, the concept of conformational restriction was pursued, i.e. the flexible aminoethanol substructure of ifenprodil was embedded into a more rigid tetrahydro-3-benzazepine system. The resulting tetrahydro-3-benzazepine-1,7-diol (±)-2 (WMS-1410) showed promising receptor affinity in receptor binding studies (K i = 84 nM) as well as pharmacological activity in two-electrode-voltage-clamp experiments (IC 50 = 116 nM) and in cytoprotective assays (IC 50 = 18.5 nM). The interactions of (R)-2 with the ifenprodil binding site of GluN2B-NMDA receptors were analyzed on the molecular level and the "foot-in-the-door" mechanism was developed. Due to promising pharmacokinetic parameters (logD7.4 = 1.68, plasma protein binding of 76-77%, sufficient metabolic stability) F-substituted analogs were prepared and evaluated as tracers for positron emission tomography (PET). Both fluorine-18-labeled PET tracers [18F]11 and [18F]15 showed high brain uptake, specific accumulation in regions known for high GluN2B-NMDA receptor expression, but no interactions with σ 1 receptors. Radiometabolites were not observed in the brain. Both PET tracers might be suitable for application in humans.

In vitro ADME characterization of a very potent 3-acylamino-2-aminopropionic acid-derived GluN2C-NMDA receptor agonist and its ester prodrugs.

The GluN2C subunit exists predominantly, but not exclusively in NMDA receptors within the cerebellum. Antagonists such as UBP1700 and positive allosteric modulators including PYD-106 and 3-acylamino-2-aminopropionic acid derivatives such as UA3-10 ((R)-2-amino-3-{[5-(2-bromophenyl)thiophen-2-yl]carboxamido}propionic acid) represent promising tool compounds to investigate the role of GluN2C-containing NMDA receptors in the signal transduction in the brain. However, due to its high polarity the bioavailability and CNS penetration of the amino acid UA3-10 are expected to be rather low. Herein, three ester prodrugs 12a-c of the NMDA receptor glycine site agonist UA3-10 were prepared and pharmacokinetically characterized. The esters 12a-c showed higher lipophilicity (higher logD 7.4 values) than the acid UA3-10 but almost the same binding at human serum albumin. The acid UA3-10 was rather stable upon incubation with mouse liver microsomes and NADPH, but the esters 12a-c were fast hydrolyzed to afford the acid UA3-10. Incubation with pig liver esterase and mouse serum led to rapid hydrolysis of the esters 12a-c. The isopropyl ester 12c showed a promising logD 7.4 value of 3.57 and the highest stability in the presence of pig liver esterase and mouse serum. These results demonstrate that ester prodrugs of UA3-10 can potentially afford improved bioavailability and CNS penetration.

A novel NMDA receptor test model based on hiPSC-derived neural cells.

N-Methyl-D-aspartate receptors (NMDARs) are central for learning and information processing in the brain. Dysfunction of NMDARs can play a key role in the pathogenesis of neurodegeneration and drug addiction. The development of selective NMDAR modulators represents a promising strategy to target these diseases. Among such modulating compounds are ifenprodil and its 3-benzazepine derivatives. Classically, the effects of these NMDAR modulators have been tested by techniques like two-electrode voltage clamp (TEVC), patch clamp, or fluorescence-based assays. However, testing their functional effects in complex human systems requires more advanced approaches. Here, we established a human induced pluripotent stem cell-derived (hiPSC-derived) neural cell system and proved its eligibility as a test system for investigating NMDAR modulators and pharmaceutical effects on human neurons.

The second PI(3,5)P2 binding site in the S0 helix of KCNQ1 stabilizes PIP2-at the primary PI1 site with potential consequences on intermediate-to-open state transition.

The Phosphatidylinositol 3-phosphate 5-kinase Type III PIKfyve is the main source for selectively generated phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a known regulator of membrane protein trafficking. PI(3,5)P2 facilitates the cardiac KCNQ1/KCNE1 channel plasma membrane abundance and therewith increases the macroscopic current amplitude. Functional-physical interaction of PI(3,5)P2 with membrane proteins and its structural impact is not sufficiently understood. This study aimed to identify molecular interaction sites and stimulatory mechanisms of the KCNQ1/KCNE1 channel via the PIKfyve-PI(3,5)P2 axis. Mutational scanning at the intracellular membrane leaflet and nuclear magnetic resonance (NMR) spectroscopy identified two PI(3,5)P2 binding sites, the known PIP2 site PS1 and the newly identified N-terminal α-helix S0 as relevant for functional PIKfyve effects. Cd2+ coordination to engineered cysteines and molecular modeling suggest that repositioning of S0 stabilizes the channel s open state, an effect strictly dependent on parallel binding of PI(3,5)P2 to both sites.

Validation of TREK1 ion channel activators as an immunomodulatory and neuroprotective strategy in neuroinflammation.

Modulation of two-pore domain potassium (K2P) channels has emerged as a novel field of therapeutic strategies as they may regulate immune cell activation and metabolism, inflammatory signals, or barrier integrity. One of these ion channels is the TWIK-related potassium channel 1 (TREK1). In the current study, we report the identification and validation of new TREK1 activators. Firstly, we used a modified potassium ion channel assay to perform high-throughput-screening of new TREK1 activators. Dose-response studies helped to identify compounds with a high separation between effectiveness and toxicity. Inside-out patch-clamp measurements of Xenopus laevis oocytes expressing TREK1 were used for further validation of these activators regarding specificity and activity. These approaches yielded three substances, E1, B3 and A2 that robustly activate TREK1. Functionally, we demonstrated that these compounds reduce levels of adhesion molecules on primary human brain and muscle endothelial cells without affecting cell viability. Finally, we studied compound A2 via voltage-clamp recordings as this activator displayed the strongest effect on adhesion molecules. Interestingly, A2 lacked TREK1 activation in the tested neuronal cell type. Taken together, this study provides data on novel TREK1 activators that might be employed to pharmacologically modulate TREK1 activity.

Diastereoselective synthesis and structure-affinity relationships of σ1 receptor ligands with spirocyclic scaffold.

Spirocyclic scaffolds play an increasing role in drug discovery as they define a rigid three-dimensional space to increase specific interactions with protein binding sites. Herein, a spirocyclic center was introduced into the lead compound 1 to rigidify its flexible benzylaminoethyl side chain. The key step of the synthesis was the reaction of different α,ß-unsaturated amides 6 and 13-16 with methyl acrylate in the presence of TBDMSOTf. DFT calculations explain the mechanism of this transformation as concerted Diels-Alder reaction (functionals B3LYP and TPSS) or double (aza)-Michael addition (functionals PBE and wB97X-D). After separation of the diastereomeric spirocyclic products 8 and 17-20, LiAlH4 reduction provided the spirocyclic hydroxymethyl piperidines 21a,b-25a,b showing low nanomolar σ1 affinity (Ki < 100 nM). trans-Configured ligands (a-series) showed higher or equal σ1 affinity and higher selectivity over σ2 receptors and GluN2B-NMDA receptors than their cis-configured analogs (b-series). The additional hydroxymethyl moiety brings the log D7.4 value in a promising range. The high σ1 affinity (Ki = 3.6 nM) and the low lipophilicity result in the highest lipophilic ligand efficiency for the dispiro compound 23a (LLE = 6.0). The spirocyclic compounds reported herein and in particular the dispiro compound 23a demonstrate that ligands containing a large number of sp3 C-atoms possess favorable pharmacological (σ1 receptor affinity, receptor selectivity) and physicochemical properties (log D7.4 value) resulting in promising LLE.

Negative allosteric modulators of NMDA receptors with GluN2B subunit: synthesis of ß-aminoalcohols by epoxide opening and subsequent rearrangement.

In order to obtain novel antagonists of GluN2B subunit containing NMDA receptors, aryloxiranes were opened with benzylpiperidines. Phenyloxiranes 6 and (indazolyl)oxirane 15 were opened regioselectively at the position bearing the aryl moiety. Reaction of the resulting ß-aminoalcohols 7 and 16 with carboxylic acids under Mitsunobu conditions (DIAD, PPh3) led to rearrangement and after ester hydrolysis to the regioisomeric ß-aminoalcohols 9 and 18. This strategy allows the synthesis of amino-ifenprodil 12 as well using phthalimide in the Mitsunobu reaction. Unexpectedly, the isomeric (indazolyl)oxirane 21 reacted with benzylpiperidines to afford both regioisomeric ß-aminoalcohols 22 and 23. In radioligand receptor binding studies, the indazolyl derivative 18a, which can be regarded as indazole bioisostere of ifenprodil, showed high GluN2B affinity (Ki = 31 nM). Replacement of the benzylic OH moiety of ifenprodil by the NH2 moiety in amino-ifenprodil 12 also resulted in low nanomolar GluN2B affinity (Ki = 72 nM). In TEVC experiments, 18a inhibited the ion flux to the same extent as ifenprodil proving that the phenol of ifenprodil can be replaced bioisosterically by an indazole ring maintaining affinity and inhibitory activity. Whereas 10-fold selectivity was found for the ifenprodil binding site over σ1 receptors, only low preference for the GluN2B receptor over σ2 receptors was detected. The log D7.4 value of 18a (log D7.4 = 2.08) indicates promising bioavailability.

Negative allosteric modulators of NMDA receptors with GluN2B subunit: Alanine-derived benzoxazolone bioisosteres of 2-methyl-3-benzazepine-1,7-diols.

Inspired by besonprodil, the phenol of potent negative allosteric modulators of GluN2B-N-methyl-d-aspartate (NMDA) receptors was replaced by a benzoxazolone system. To increase the similarity to the lead compounds, an additional methyl moiety was installed in the 8-position of tricyclic oxazolobenzazepines, resulting in compounds 6. The additional methyl moiety originates from alanine, which was introduced by a Mitsunobu reaction of benzoxazolylethanol 7 with N-triflyl-protected alanine methyl ester. A crucial feature of the synthesis was the protection of the oxazolone ring by an allyl moiety, which was cleaved off at the end of the synthesis by RhCl3 -catalyzed isomerization. Due to the additional methyl moiety, the intramolecular Friedel-Crafts acylation of acid 10 to afford ketone 11 required careful optimization to minimize the formation of the side product tetrahydroisoquinoline 16. Alkylation or reductive alkylation of secondary amine 13 led to diastereomeric oxazolobenzazepines cis-14 and trans-14, which were separated by flash chromatography. Phenylbutyl derivatives cis-6a and trans-6a revealed twofold higher GluN2B affinity than analog 5a without 8-CH3 group. The methylated oxazolobenzazepines 6 and 14 did not interact with the phencyclidine binding site of NMDA receptors and σ2 receptors. However, the σ1 receptor preferred cis-configured oxazolobenzazepines. The highest σ1 receptor affinities were obtained for cis-14a (Ki = 26 nM) and cis-6b (Ki = 30 nM).

Synthesis of oxazolo-annulated 3-benzazepines designed by merging two negative allosteric NMDA receptor modulators.

To improve the metabolic stability and receptor selectivity of ifenprodil (1), the benzoxazolone moiety of besonprodil (2) and the 3-benzazepone moiety of WMS-1410 (3) were merged to obtain oxazolobenzazepines of type 4. The 5-(hydroxyethyl)benzoxazolone 7 representing the first key intermediate was prepared in four steps starting with the 4-(2-hydroxyethyl)phenol (8). Mitsunobu reaction of primary alcohol 7 with N-sulfonylated glycine esters established the necessary side chain. The intramolecular Friedel-Crafts acylation of acid 12a containing the N-tosyl protective group led upon decarbonylation exclusively to the tricyclic tetrahydroisoquinoline 14. Protection of the amino moiety by the stronger electron-withdrawing triflyl group resulted in the desired 3-benzazepine 15 without the formation of analogous isoquinoline. The triflyl protective group was cleaved off by K2 CO3 -induced elimination of trifluoromethanesulfinate. In a one-pot three-step procedure, various oxazolobenzazepinediones 15 were obtained, which were reduced to afford the desired secondary alcohols 18.

Phenol-Benzoxazolone bioisosteres: Synthesis and biological evaluation of tricyclic GluN2B-selective N-methyl- d-aspartate receptor antagonists.

Tricyclic tetrahydrooxazolo[4,5-h]-[3]benzazepin-9-ols 22 were designed as phenol bioisosteres of tetrahydro-3-benzazepine-1,7-diols. Key features of the synthesis are the introduction of the trifluoromethylsulfonyl and allyl protective groups at the heterocyclic N-atoms. Two methods were developed to convert the triflyl-protected ketone 16 into tricyclic alcohols 21 bearing various N-substituents. According to the first method, trifluoromethanesulfinate was removed by K2 CO3 . Following the selective reduction of the imino moiety of 17 with NaBH(OAc)3 afforded the aminoketone 18, which was reductively alkylated and reduced. According to the second method, both the imine and the ketone of the iminoketone 17 were reduced with NaBH4 to yield the aminoalcohol 20, which was alkylated or reductively alkylated to form tertiary amines 21f-21r. In the last step, the allyl protective group of 21 was removed with RhCl3 and HCl to obtain oxazolones 22. In receptor binding studies using [3 H]ifenprodil as radioligand ketone, 22m showed the highest GluN2B affinity (Ki = 88 nM). However, a reduced affinity toward GluN2B subunit-containing N-methyl- d-aspartate (NMDA) receptors was observed for oxazolones 22 compared to bioisosteric 3-benzazepine-1,7-diols. High selectivity of 22m for the ifenprodil binding site of GluN2B-NMDA receptors over the 1-(1-phenylcyclohexyl)piperidine binding site and σ2 receptors was observed, but only negligible selectivity over σ1 receptors. In two-electrode voltage clamp experiments, the 4-phenylbutyl derivative 22d (Ki = 422 nM) demonstrated 80% inhibition of ion flux at a concentration of 1 µM. The differences in GluN2B affinity and inhibitory activity are explained by docking studies. In conclusion, 22d is regarded as a novel scaffold of highly potent GluN1/GluN2B antagonists.

Phenol-benzoxazolone bioisosteres of GluN2B-NMDA receptor antagonists: Unexpected rearrangement during reductive alkylation with phenylcyclohexanone.

Negative allosteric modulators of N-methyl- d-aspartate receptors containing the GluN2B subunit represent promising drug candidates for the treatment of various neurological disorders including stroke, epilepsy, and Parkinson's disease. To increase the bioavailability and GluN2B affinity, the phenol of the potent benzazepine-based inhibitor, WMS-1410 (3), was replaced bioisosterically by a benzoxazolone moiety and the phenylbutyl side chain was conformationally restricted in a phenylcyclohexyl substituent. A four-step, one-pot procedure transformed the oxazolo-benzazepine 7 into the phenylcyclohexyl derivative 11. The same protocol was applied to the methylated analog 12, which unexpectedly led to ring-contracted oxazolo-isoquinolines 18. This rearrangement was explained by the additional methyl moiety in the 8-position inhibiting the formation of the planar intermediate iminium ion with phenylcyclohexanone. The allyl protective group of 11 and 18 was removed with RhCl3 and HCl to obtain the tricyclic compounds 5 and 19 without substituent at the oxazolone ring. The structures of the rearranged products 18 and 19 were elucidated by X-ray crystal structure analysis. The oxazolo-isoquinoline trans-18 with allyl moiety (Ki = 89 nM) and the oxazolo-benzazepine 5 without substituent at the oxazolone ring (Ki = 114 nM) showed GluN2B affinity in the same range as the lead compound 3. In two-electrode voltage clamp measurements, 5 displayed only weak inhibitory activity.

Imaging of the calcium activated potassium channel 3.1 (KCa 3.1) in vivo using a senicapoc-derived positron emission tomography tracer.

The calcium-activated potassium channel 3.1 (KCa 3.1) is overexpressed in many tumor entities and has predictive power concerning disease progression and outcome. Imaging of the KCa 3.1 channel in vivo using a radiotracer for positron emission tomography (PET) could therefore establish a potentially powerful diagnostic tool. Senicapoc shows high affinity and excellent selectivity toward the KCa 3.1 channel. We have successfully pursued the synthesis of the 18 F-labeled derivative [18 F]3 of senicapoc using the prosthetic group approach with 1-azido-2-[18 F]fluoroethane ([18 F]6) in a "click" reaction. The biological activity of the new PET tracer was evaluated in vitro and in vivo. Inhibition of the KCa 3.1 channel by 3 was demonstrated by patch clamp experiments and the binding pose was analyzed by docking studies. In mouse and human serum, [18 F]3 was stable for at least one half-life of [18 F]fluorine. Biodistribution experiments in wild-type mice were promising, showing rapid and predominantly renal excretion. An in vivo study using A549-based tumor-bearing mice was performed. The tumor signal could be delineated and image analysis showed a tumor-to-muscle ratio of 1.47 ± 0.24. The approach using 1-azido-2-[18 F]fluoroethane seems to be a good general strategy to achieve triarylacetamide-based fluorinated PET tracers for imaging of the KCa 3.1 channel in vivo.

Photocatalytic Isomerization of (E)-Anethole to (Z)-Anethole.

Natural product (E)-anethole was isomerized to (Z)-anethole in a photocatalytic reaction. For this purpose, a self-designed cheap photoreactor was constructed. Among 11 photosensitizers (organo and metal complex compounds), Ir(p-tBu-ppy)3 led to the highest conversion. Triplet energies of (E)- and (Z)-anethole were predicted theoretically by DFT calculations to support the selection of appropriate photosensitizers. A catalyst loading of 0.1 mol% gave up to 90% conversion in gram scale. Further additives were not required and mild irradiation with light of 400 nm overnight was sufficient. As a proof of concept, (E)- and (Z)-anethole were dihydroxylated diastereoselectively to obtain diastereomerically pure like- and unlike-configured diols, respectively.

Synthesis and Antioxidative Properties of 1,2,3,4-Tetrahydropyridine Derivatives with Different Substituents in 4-Position.

Natural products are an excellent source of inspiration for the development of new drugs. Among them, betalains have been extensively studied for their antioxidant properties and potential application as natural food dyes. Herein, we describe the seven-step synthesis of new betalamic acid analogs without carboxy groups in the 2- and 6-position with an overall yield of ~70%. The Folin-Ciocalteu assay was used to determine the antioxidant properties of protected intermediate 21. Additionally, the five-step synthesis of betalamic acid analog 35 with three ester moieties was performed. Using NMR techniques, the stability of the obtained compounds towards oxygen was analyzed.

Deconstruction - Reconstruction: Analysis of the Crucial Structural Elements of GluN2B-Selective, Negative Allosteric NMDA Receptor Modulators with 3-Benzazepine Scaffold.

BACKGROUND/AIMS: The NMDA receptor plays a key role in the pathogenesis of neurodegenerative disorders including Alzheimer's and Huntington's disease, as well as depression and drug or alcohol dependence. Due to its participation in these pathologies, the development of selective modulators for this ion channel is a promising strategy for rational drug therapy. The prototypical negative allosteric modulator ifenprodil inhibits selectively GluN2B subunit containing NMDA receptors. It was conformationally restricted as 2-methyl-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol, which showed high GluN2B affinity and inhibitory activity. For a better understanding of the relevance of the functional groups and structural elements, the substituents of this 3-benzazepine were removed successively (deconstruction). Then, additional structural elements were introduced (reconstruction) with the aim to analyze, which additional modifications were tolerated by the GluN2B receptor. METHODS: The GluN2B affinity was recorded in radioligand receptor binding studies with the radioligand [3H]ifenprodil. The activity of the ligands was determined in two-electrode voltage clamp experiments using Xenopus laevis oocytes transfected with cRNA encoding the GluN1-1a and GluN2B subunits of the NMDA receptor. Docking studies showed the crucial interactions with the NMDA receptor protein. RESULTS: The deconstruction approach showed that removal of the methyl moiety and the phenolic OH moiety in 7-positon resulted in almost the same GluN2B affinity as the parent 3-benzazepine. A considerably reduced GluN2B affinity was found for the 3-benzazepine without further substituents. However, removal of one or both OH moieties led to considerably reduced NMDA receptor inhibition. Introduction of a NO2 moiety or bioisosteric replacement of the phenol by a benzoxazolone resulted in comparable GluN2B affinity, but almost complete loss of inhibitory activity. An O-atom, a carbonyl moiety or a F-atom in the tetramethylene spacer led to 6-7-fold reduced ion channel inhibition. CONCLUSION: The results reveal an uncoupling of affinity and activity for the tested 3-benzazepines. Strong inhibition of [3H]ifenprodil binding by a test compound does not necessarily translate into strong inhibition of the ion flux through the NMDA receptor associated ion channel. 3-(4-Phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine- 1,7-diol (WMS-1410) shows high GluN2B affinity and strong inhibition of the ion channel. Deconstruction by removal of one or both OH moieties reduced the inhibitory activity proving the importance of the OH groups for ion channel blockade. Reconstruction by introduction of various structural elements into the left benzene ring or into the tetramethylene spacer reduced the NMDA receptor inhibition. It can be concluded that these modifications are not able to translate binding into inhibition.

Targeting Kca3.1 Channels in Cancer.

The Kca3.1 channels, previously designated as IK1 or SK4 channels and encoded by the KCNN4 gene, are activated by a rise of the intracellular Ca2+ concentration. These K+ channels are widely expressed in many organs and involved in many pathologies. In particular, Kca3.1 channels have been studied intensively in the context of cancer. They are not only a marker and a valid prognostic tool for cancer patients, but have an important share in driving cancer progression. Their function is required for many characteristic features of the aggressive cancer cell behavior such as migration, invasion and metastasis as well as proliferation and therapy resistance. In the context of cancer, another property of Kca3.1 is now emerging. These channels can be a target for novel small molecule-based imaging probes, as it has been validated in case of fluorescently labeled senicapoc-derivatives. The aim of this review is (i) to give an overview on the role of Kca3.1 channels in cancer progression and in shaping the cancer microenvironment, (ii) discuss the potential of using Kca3.1 targeting drugs for cancer imaging, (iii) and highlight the possibility of combining molecular dynamics simulations to image inhibitor binding to Kca3.1 channels in order to provide a deeper understanding of Kca3.1 channel pharmacology. Alltogether, Kca3.1 is an attractive therapeutic target so that senicapoc, originally developed for the treatment of sickle cell anemia, should be repurposed for the treatment of cancer patients.

Can Unlikely Neanderthal Chloride Channel CLC-2 Gene Variants Provide Insights in Modern Human Infertility?

BACKGROUND/AIMS: Neanderthals, although well adapted to local environments, were rapidly replaced by anatomically modern humans (AMH) for unknown reasons. Genetic information on Neanderthals is limited restricting applicability of standard population genetics. METHODS: Here, we apply a novel combination of restricted genetic analyses on preselected physiological key players (ion channels), electrophysiological analyses of gene variants of unclear significance expressed in Xenopus laevis oocytes using two electrode voltage clamp and transfer of results to AMH genetics. Using genetic screening in infertile men identified a loss of CLC-2 associated with sperm deficiency. RESULTS: Increased genetic variation caused functionally impaired Neanderthals CLC-2 channels. CONCLUSION: Increased genetic variation could reflect an adaptation to different local salt supplies at the cost of reduced sperm density. Interestingly and consistent with this hypothesis, lack of CLC-2 protein in a patient associates with high blood K+ concentration and azoospermia.

Selective Inhibition of N-Methyl-d-aspartate Receptors with GluN2B Subunit Protects ß Cells against Stress-Induced Apoptotic Cell Death.

Participation of N-methyl-d-aspartate (NMDA) receptors (NMDARs) in the failure of pancreatic ß cells during development of type 2 diabetes mellitus is discussed. Our study investigates whether ß cell mass and function can be preserved by selectively addressing the GluN2B subunit of the NMDAR. NMDAR activation by NMDA and its coagonist glycine moderately influenced electrical activity and Ca2+ handling in islet cells at a threshold glucose concentration (4-5 mM) without affecting glucose-mediated insulin secretion. Exposure of islet cells to NMDA/glycine or a glucolipotoxic milieu increased apoptosis by 5% and 8%, respectively. The GluN2B-specific NMDAR antagonist WMS-1410 (0.1 and 1 µM) partly protected against this. In addition, WMS-1410 completely prevented the decrease in insulin secretion of about 32% provoked by a 24-hour-treatment with NMDA/glycine. WMS-1410 eliminated NMDA-induced changes in the oxidation status of the islet cells and elevated the sensitivity of intracellular calcium to 15 mM glucose. By contrast, WMS-1410 did not prevent the decline in glucose-stimulated insulin secretion occurring after glucolipotoxic culture. This lack of effect was due to a decrease in insulin content to 18% that obviously could not be compensated by the preservation of cell mass or the higher percentage of insulin release in relation to insulin content. In conclusion, the negative effects of permanent NMDAR activation were effectively counteracted by WMS-1410 as well as the apoptotic cell death induced by high glucose and lipid concentrations. Modulation of NMDARs containing the GluN2B subunit is suggested to preserve ß cell mass during development of type 2 diabetes mellitus. SIGNIFICANCE STATEMENT: Addressing NMDA receptors containing the GluN2B subunit in pancreatic islet cells has the potential to protect the ß cell mass that progressively declines during the development of type 2 diabetes. Furthermore, this study shows that harmful effects of permanent NMDAR activation can be effectively counteracted by the compound WMS-1410, a selective modulator for NMDARs containing the GluN2B subunit.

Synthesis and pharmacological evaluation of enantiomerically pure endo-configured KOR agonists with 2-azabicyclo[3.2.1]octane scaffold.

Conformationally restricted bicyclic KOR agonists 10 with an endo-configured amino moiety were synthesized to analyze the bioactive conformation of conformationally flexible KOR agonists such as 2-5. A seven-step synthesis starting with (S)-configured 4-oxopiperidine-2-carboxylate 13 was developed. cis- and trans-configured diesters 12 were obtained in a 3 : 1 ratio via hydrogenation of the α,ß-unsaturated ester 14. After establishment of the bicyclic scaffold, a diastereoselective reductive amination of ketone 11 provided exclusively the endo-configured bicyclic amines 10a,b. The 3 : 1 mixtures of enantiomers were separated by chiral HPLC, respectively, leading to enantiomerically pure KOR agonists (1S,5S,7R)-10a,b and (1R,5R,7S)-10a,b (ent-10a,b). The KOR affinity was determined in receptor binding studies with the radioligand [3H]U-69 593. The high KOR affinity of endo-configured amines 10a (Ki = 7 nM) and 10b (Ki = 13 nM) indicates that the dihedral angle of the KOR pharmacophoric element N(pyrrolidine)-C-C-N(phenylacetyl) of 42° is close to the bioactive conformation of more flexible KOR agonists. It should be noted that changing the configuration of potent and selective KOR agonists 10a and 10b led to potent and selective σ1 ligands (e.g. ent-10aKi(σ1) = 10 nM).