Fluorinated piperidine acetic acids as γ-secretase modulators

We report herein a novel series of difluoropiperidine acetic acids as modulators of γ-secretase. Synthesis of 2-aryl-3,3-difluoropiperidine analogs was facilitated by a unique and selective β-difluorination with Selectfluor®. Compounds 1f and 2c were selected for in vivo assessment and demonstrated selective lowering of Aβ42 in a genetically engineered mouse model of APP processing. Moreover, in a 7-day safety study, rats treated orally with compound 1f (250 mg/kg per day, AUC_0–24 = 2100 μM h) did not exhibit Notch-related effects.     

PDE2 inhibition: Potential for the treatment of cognitive disorders

Phosphodiesterase inhibition has received much attention in the past 20 years for the potential treatment of CNS disorders. A primary focus of this work is the enhancement of memory and/or cognitive functioning. The role of PDEs in the augmentation of cyclic nucleotide signaling makes these enzymes attractive targets for enhancing the effects of neuronal communication. This review focuses on recent findings with respect to the role of PDE2 inhibition in cognitive functioning. Special attention is paid to recently disclosed, selective tool compounds and the use of these tool compounds to support the role of PDE2 inhibition in cognition. Recently reported SAR and modeling work will be presented along with discussion of the entry of new PDE2 inhibitors into the clinic.     

治疗阿尔茨海默病的早老素/γ-分泌酶抑制剂和调节剂

阿尔茨海默病（Alzheimer’s disease,AD）又称老年痴呆症,是一种中枢神经系统（central nervous system,CNS）退行性疾病。β-淀粉样蛋白（β-amyloid,Aβ42）被认为在阿尔茨海默病（AD）的发生、发展过程中起核心作用。Aβ42由APP经β-和γ-分泌酶相继切割产生。γ-分泌酶是一个蛋白酶复合体,早老素（presenilin,PS）是γ-分泌酶的催化组分。因此,抑制PS/γ分泌酶的活性是治疗AD的关键,因而PS/γ分泌酶也是治疗AD的主要靶点。根据这些理论,人们提出了一些治疗AD的新方法,其中PS/γ-分泌酶抑制剂和调节剂成为近年来人们关注的焦点。     

(S)-N-(5-Chlorothiophene-2-sulfonyl)-β,β-diethylalaninol a Notch-1-sparing γ-secretase inhibitor

Accumulation of beta-amyloid (Aβ), produced by the proteolytic cleavage of amyloid precursor protein (APP) by β- and γ-secretase, is widely believed to be associated with Alzheimer’s disease (AD). Research around the high-throughput screening hit (S)-4-chlorophenylsulfonyl isoleucinol led to the identification of the Notch-1-sparing (9.5-fold) γ-secretase inhibitor (S)-N-(5-chlorothiophene-2-sulfonyl)-β,β-diethylalaninol 7.b.2 (Aβ _40/42 EC₅₀ = 28 nM), which is efficacious in reduction of Aβ production in vivo.     

Novel chiral-diazepines function as specific,selective receptor agonists with variable coupling and species variability in human,mouse and rat BRS-3 receptor cells

Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein coupled receptor which is classified in the bombesin receptor (BnR) family with which it shares high homology. It is present widely in the central nervous system and peripheral tissues and primarily receptor-knockout studies suggest it is involved in metabolic-glucose-insulin homeostasis, feeding and other CNS behaviors, gastrointestinal motility and cancer growth. However, the role of BRS-3 physiologically or in pathologic disorders has been not well defined because the natural ligand is unknown. Until recently, no selective agonists/antagonists were available; however, recently synthetic high-affinity agonists, chiral-diazepines nonpeptide-analogs (3F, 9D, 9F, 9G) with low CNS penetrance, were described, but are not well-categorized pharmacologically or in different labarotory species. The present study characterizes the affinities, potencies, selectivities of the chiral-diazepine BRS-3 agonists in human and rodents (mice,rat). In human BRS-3 receptors, the relative affinities of the chiral-diazepines was 9G > 9D > 9F > 3F; each was selective for BRS-3. For stimulating PLC activity, in h-BRS-3 each of the four chiral diazepine analogs was fully efficacious and their relative potencies were: 9G (EC₅₀: 9 nM) > 9D (EC₅₀: 9.4 nM) > 9F (EC₅₀: 39 nM) > 3F (EC₅₀: 48 nM). None of the four chiral diazepine analogs activated r,m,h-GRPR/NMBR. The nonpeptide agonists showed marked differences from each other and a peptide agonist in receptor-coupling-stiochiometry and in affinities/potencies in different species. These results demonstrate that chiral diazepine analogs (9G, 9D, 9F, 3F) have high/affinity/potency for the BRS-3 receptor in human and rodent cells, but different coupling-relationships and species differences from a peptide agonist.     

Inhibition of γ-secretase by the CK1 inhibitor IC261 does not depend on CK1δ

CK1 and γ-secretase are interesting targets for therapeutic intervention in the treatment of cancer and Alzheimer’s disease. The CK1 inhibitor IC261 was reported to inhibit γ-secretase activity. The question is: Does CK1 inhibition directly influence γ-secretase activity? Therefore we analyzed the SAR of 15 analogues and their impact on γ-secretase activity. The most active compounds were investigated on CK1δ activity. These findings exclude a direct influence of CK1δon γ-secretase, because any change in the substitution pattern of IC261 diminished CK1 inhibition, whereas γ-secretase inhibition is still exerted by several analogues.     

Inhibition of serotonin outflow by nociceptin/orphaninFQ in dorsal raphe nucleus slices from normal and stressed rats: Role of corticotropin releasing factor

In the dorsal raphe nucleus (DRN) many inputs converge and interact to modulate serotonergic neuronal activity and the behavioral responses to stress. The effects exerted by two stress-related neuropeptides, corticotropin releasing factor (CRF) and nociceptin/orphaninFQ (N/OFQ), on the outflow of [³H]5-hydroxytryptamine were investigated in superfused rat dorsal raphe nucleus slices.Electrical stimulation (100 mA, 1 ms for 2 min) evoked a frequency-dependent peak of [³H]5-hydroxytryptamine outflow, which was sodium and calcium-dependent. Corticotropin releasing factor (1–100 nM), concentration-dependently inhibited the stimulation (3 Hz)-evoked [³H]5-hydroxytryptamine outflow; the inhibition by 30 nM corticotropin releasing factor (to 68 ± 5.7%) was prevented both by the non selective CRF receptor antagonist alpha-helicalCRF(9-41) (α-HEL) (300 nM) and by the CRF₁ receptor antagonist antalarmin (ANT) (100 nM). The CRF₂ agonist urocortin II (10 nM) did not modify [³H]5-hydroxytryptamine outflow, ruling out the involvement of CRF₂ receptors. Bicuculline (BIC), a GABA_A antagonist (10 μM), prevented the inhibitory effect of corticotropin releasing factor (30 nM), supporting the hypothesis that the inhibition was mediated by increased γ-aminobutyric acid (GABA) release. Nociceptin/orphaninFQ (1 nM–1 μM) exerted an antalarmin- and bicuculline-insensitive inhibition on [³H]5-hydroxytryptamine outflow, with the maximum at 100 nM (to 63 ± 4.2%), antagonized by the NOP receptor antagonist UFP-101 (1 μM). Dorsal raphe nucleus slices prepared from rats exposed to 15 min of forced swim stress displayed a reduced [³H]5-hydroxytryptamine outflow, in part reversed by antalarmin and further inhibited by nociceptin/orphaninFQ. These findings indicate that (i) both corticotropin releasing factor and nociceptin/orphaninFQ exert an inhibitory control on dorsal raphe nucleus serotonergic neurons; (ii) the inhibition by corticotropin releasing factor involves γ-aminobutyric acid neurons; (iii) nociceptin/orphaninFQ inhibits dorsal raphe nucleus serotonin system in a corticotropin releasing factor- and γ-aminobutyric acid-independent manner; (iv) nociceptin/orphaninFQ modulation is still operant in slices prepared from stressed rats. The nociceptin/orphaninFQ-NOP receptor system could represent a new target for drugs effective in stress-related disorders.     

Structural optimization of a CXCR2-directed antagonist that indirectly inhibits γ-secretase and reduces Aβ

Amyloid β (Aβ), a key molecule in the pathogenesis of Alzheimer’s disease (AD), is derived from the amyloid precursor protein (APP) by sequential proteolysis via β- and γ-secretases. Because of their role in generation of Aβ, these enzymes have emerged as important therapeutic targets for AD. In the case of γ-secretase, progress has been made towards designing potent inhibitors with suitable pharmacological profiles. Direct γ-secretase inhibitors are being evaluated in clinical trials and new strategies are being explored to block γ-secretase activity indirectly as well. In this regard, we have previously reported an indirect regulation of γ-secretase through antagonism of CXCR2, a G-protein coupled receptor (GPCR). We demonstrated that N-(2-hydroxy-4-nitrophenyl)-N′-(2-bromophenyl)urea (SB225002), a selective inhibitor of CXCR2 also plays a role in an indirect inhibition of γ-secretase. Furthermore, we reported a ～5-fold difference in the selective inhibition of APP versus Notch processing via γ-secretase following treatment with SB225002. Herein we describe the synthesis and optimization of SB225002. By determination of the structure–activity relationship (SAR), we derived small molecules that inhibit Aβ40 production with IC₅₀ values in the sub-micromolar range in a cell-based assay and also validated the potential of CXCR2 as a new target for therapeutic intervention in AD.     

Discovery of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides as selective antagonists of the kappa opioid receptor. Part 1

Initial high throughput screening efforts identified highly potent and selective kappa opioid receptor antagonist 3 (κ IC₅₀ = 77 nM; μ:κ and δ:κ IC₅₀ ratios >400) which lacked CNS exposure in vivo. Modification of this scaffold resulted in development of a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides showing potent and selectivity κ antagonism as well as good brain exposure. Analog 6c (κ IC₅₀ = 20 nM; μ:κ = 36, δ:κ = 415) was also shown to reverse κ-agonist induced rat diuresis in vivo.     

Further characterization of a putative serine protease contributing to the γ-secretase cleavage of β-amyloid precursor protein

The 3-alkoxy-7-amino-4-chloro-isocoumarins JLK-6 and JLK-2 have been shown to markedly reduce the production of Amyloid β-peptide (Aβ) by Amyloid-β Precursor Protein (APP) expressing HEK293 cells by affecting the γ-secretase cleavage of APP, with no effect on the cleavage of the Notch receptor. This suggested that these compounds do not directly inhibit the presenilin-dependent γ-secretase complex but more likely interfere with an upstream target involved in γ-secretase-associated pathway. The mechanism of action of these compounds is unknown and there are high fundamental and therapeutical interests to unravel their target. Isocoumarin compounds were previously shown to behave as potent mechanism-based irreversible inhibitors of serine proteases, suggesting that the JLK-directed target could belong to such enzyme family. To get further insight into structure–activity relationships and to develop more potent isocoumarin derivatives, we have synthesized and evaluated a series of isocoumarin analogues with modifications at positions 3, 4 and 7. In particular, the 7-amino group was substituted with either acyl, urethane, alkyl or aryl groups, which could represent additional interaction sites. Altogether, the results highlighted the essential integrity of the 3-alkoxy-7-amino-4-chloro-isocoumarin scaffold for Aβ-lowering activity and supported the involvement of a serine protease, or may be more generally, a serine hydrolase. The newly reported 7-N-alkyl series produced the most active compounds with an IC₅₀ between 10 and 30 μM. Finally, we also explored peptide boronates, a series of reversible serine protease inhibitors, previously shown to also lower cellular Aβ production. The presented data suggested they could act on the same target or interfere with the same pathway as isocoumarins derivatives.     

From NMDA receptor antagonists to discovery of selective σ2 receptor ligands

Following previous studies focused on the search for new molecules targeting GluN2B-containing NMDA, a small series of 1-(1H-indol-3-yl)-2-(4-phenylpiperidin-1-yl)ethanone derivatives has been synthesized by using Microwave Assisted Organic Synthesis (MAOS). Given that GluN2B ligands frequently exert off-target effects we also tested their affinity towards sigma receptors. Binding assay revealed that only the 1-(5-hydroxy-1H-indol-3-yl)-2-(4-phenylpiperidin-1-yl)ethanone (7a) retained GluN2B affinity. Interestingly, the 5-methoxyindoles 5a and 6a were efficient and selective ligands toward σ₂ receptor (K_i values of 10 nM and 20 nM, respectively). Thus, in this case the discovery of new σ₂ receptor selective ligands was an unexpected result emerging from the screening of cross-activity against other CNS receptors.     

Co-activation of synaptic and extrasynaptic NMDA receptors by neuronal insults determines cell death in acute brain slice

Overactivation of NMDA receptors is linked to cell death during neuronal insults. However the precise role of synaptic and extrasynaptic NMDA receptors remains to be further determined. In this study, we used the acute brain slice to examine the contributions of synaptic and extrasynaptic NMDA receptors to neuronal death. By activation of synaptic NMDA receptors with bath application of 100 μM bicuculline in acute brain slices, we observed a significant up-regulation in activation of neuronal survival-related signaling (p-CREB, p-ERK1/2 and p-AKT), without an obvious increase of LDH release and neuronal death. Interestingly, activation of extrasynaptic NMDA receptors alone by high dose of glutamate (200 μM) following blockade of synaptic NMDA receptors with co-application of 20 μM MK801 and 100 μM bicuculline, we failed to observe inhibition of neuronal survival signaling and neuronal damage. In contrast, co-activation of synaptic and extrasynaptic NMDA receptors by applying 200 μM glutamate or oxygen–glucose deprivation (OGD) to acute brain slices for 30 min, we observed a significant inhibition of CREB, ERK1/2 and AKT activation, an increase of LDH release and neuronal condensation. Together, co-activation of synaptic and extrasynaptic NMDA receptors by neuronal insults contributes to cell death in acute brain slice.     

A ‘conovenomic’ analysis of the milked venom from the mollusk-hunting cone snail Conus textile—The pharmacological importance of post-translational modifications

Cone snail venoms provide a largely untapped source of novel peptide drug leads. To enhance the discovery phase, a detailed comparative proteomic analysis was undertaken on milked venom from the mollusk-hunting cone snail, Conus textile, from three different geographic locations (Hawai’i, American Samoa and Australia's Great Barrier Reef). A novel milked venom conopeptide rich in post-translational modifications was discovered, characterized and named α-conotoxin TxIC. We assign this conopeptide to the 4/7 α-conotoxin family based on the peptide's sequence homology and cDNA pre-propeptide alignment. Pharmacologically, α-conotoxin TxIC demonstrates minimal activity on human acetylcholine receptor models (100 μM, <5% inhibition), compared to its high paralytic potency in invertebrates, PD₅₀ = 34.2 nMol kg⁻¹. The non-post-translationally modified form, [Pro]^2,8[Glu]¹⁶α-conotoxin TxIC, demonstrates differential selectivity for the α3β2 isoform of the nicotinic acetylcholine receptor with maximal inhibition of 96% and an observed IC₅₀ of 5.4 ± 0.5 μM. Interestingly its comparative PD₅₀ (3.6 μMol kg⁻¹) in invertebrates was ∼100 fold more than that of the native peptide. Differentiating α-conotoxin TxIC from other α-conotoxins is the high degree of post-translational modification (44% of residues). This includes the incorporation of γ-carboxyglutamic acid, two moieties of 4-trans hydroxyproline, two disulfide bond linkages, and C-terminal amidation. These findings expand upon the known chemical diversity of α-conotoxins and illustrate a potential driver of toxin phyla-selectivity within Conus.     

Gamma-melanocyte stimulating hormone regulates the expression and cellular localization of epithelial sodium channel in inner medullary collecting duct cells

Gamma₂-melanocyte-stimulating hormone (γ₂MSH) is a peptide hormone released by the pituitary gland which is thought to act directly on the renal inner medulla to promote increased sodium excretion into urine (natriuresis). The aim of this study was to determine if a stable analog, [Nle³, D-Phe⁶]-γ₂MSH (NDP-γ₂MSH), of the native peptide regulated the activity, expression and cellular localization of epithelial sodium channel (ENaC) in a murine inner medullary collecting duct (mIMCD-3) cell line. Our results indicate that expression of the γ₂MSH receptor, melanocortin receptor 3 receptor (MC3R), is up-regulated by culturing the cells in media with an increased osmolality (∼400 mOsm/kg). Furthermore, stimulation of cAMP signaling and sodium transport by 1 nM NDP-γ₂MSH occurs only in cells cultured in the high osmolality media. Finally, treatment of mIMCD-3 cells cultured in high osmolality medium for 1 h with 1 nM NDP-γ₂MSH causes a reduction in expression of serum- and glucocorticoid-induced kinase (sgk1) and a reduction in expression and cell surface abundance of the alpha subunit of ENaC. Collectively, this data suggest that γ₂MSH directly regulates both ENaC expression and cellular localization in the inner medulla to exert its natriuretic effect.     

Discovery of a selective M4 positive allosteric modulator based on the 3-amino-thieno[2,3-b]pyridine-2-carboxamide scaffold: Development of ML253, a potent and brain penetrant compound that is active in a preclinical model of schizophrenia

Herein we report a next generation muscarinic receptor 4 (M₄) positive allosteric modulator (PAM), ML253 which exhibits nanomolar activity at both the human (EC₅₀ = 56 nM) and rat (EC₅₀ = 176 nM) receptors and excellent efficacy by the left-ward shift of the ACh concentration response curve (fold shift, human = 106; rat = 50). In addition, ML253 is selective against the four other muscarinic subtypes, displays excellent CNS exposure and is active in an amphetamine-induced hyperlocomotion assay.     

Biotransformation of isoimperatorin and imperatorin by Glomerella cingulata and β-secretase inhibitory activity

Biotransformation studies conducted on the furanocoumarins isoimperatorin (1) and imperatorin (3) have revealed that 1 was metabolized by Glomerella cingulata to give the corresponding reduced acid, 6,7-furano-5-prenyloxy hydrocoumaric acid (2), and 3 was transformed by G. cingulata to give the dealkylated metabolite, xanthotoxol (4) in high yields (83% and 81%), respectively. The structures of the new compound 2 have been established on the basis of spectral data. The metabolites 2 and 4 were tested for the β-secretase (BACE1) inhibitory activity in vitro, and metabolite 2 slightly inhibited the β-secretase activity with an IC₅₀ value of 185.6 ± 6.8 μM. The metabolite 4 was less potent activity than compounds 1–3. In addition, methyl ester (2Me), methyl ether (2a) and methyl ester and ether (2aMe) of 2 were synthesized, and investigated for the ability to inhibit β-secretase. Compound 2aMe exhibited the best β-secretase inhibitory activity at the IC₅₀ value 16.2 ± 1.2 μM and found to be the 2aMe showed competitive mode of inhibition against β-secretase with K_i value 11.3 ± 2.8 μM.     

Design and synthesis of triarylacrylonitrile analogues of tamoxifen with improved binding selectivity to protein kinase C

The clinical selective estrogen receptor modulator tamoxifen is also a modest inhibitor of protein kinase C, a target implicated in several untreatable brain diseases such as amphetamine abuse. This inhibition and tamoxifen’s ability to cross the blood brain barrier make it an attractive scaffold to conduct further SAR studies toward uncovering effective therapies for such diseases. Utilizing the known compound 6a as a starting template and guided by computational tools to derive physicochemical properties known to be important for CNS permeable drugs, the design and synthesis of a small series of novel triarylacrylonitrile analogues have been carried out providing compounds with enhanced potency and selectivity for PKC over the estrogen receptor relative to tamoxifen. Shortened synthetic routes compared to classical procedures have been developed for analogues incorporating a β-phenyl ring, which involve installing dialkylaminoalkoxy side chains first off the α and/or α′ rings of a precursor benzophenone and then condensing the resultant ketones with phenylacetonitrile anion. A second novel, efficient and versatile route utilizing Suzuki chemistry has also been developed, which will allow for the introduction of a wide range of β-aryl or β-heteroaryl moieties and side-chain substituents onto the acrylonitrile core. For analogues possessing a single side chain off the α- or α′-ring, novel 2D NMR experiments have been carried out that allow for unambiguous assignment of E- and Z-stereochemistry. From the SAR analysis, one compound, 6c, shows markedly increased potency and selectivity for inhibiting PKC with an IC₅₀ of 80 nM for inhibition of PKC protein substrate and >10 μM for binding to the estrogen receptor α (tamoxifen IC₅₀ = 20 μM and 222 nM, respectively). The data on 6c provide support for further exploration of PKC as a druggable target for the treatment of amphetamine abuse.     

Identification of a new selective dopamine D4 receptor ligand

The dopamine D₄ receptor has been shown to play key roles in certain CNS pathologies including addiction to cigarette smoking. Thus, selective D₄ ligands may be useful in treating some of these conditions. Previous studies in our laboratory have indicated that the piperazine analog of haloperidol exhibits selective and increased affinity to the DAD₄ receptor subtype, in comparison to its piperidine analog. This led to further exploration of the piperazine moiety to identify new agents that are selective at the D₄ receptor. Compound 27 (K_iD₄ = 0.84 nM) was the most potent of the compounds tested. However, it only had moderate selectivity for the D₄ receptor. Compound 28 (K_iD₄ = 3.9 nM) while not as potent, was more discriminatory for the D₄ receptor subtype. In fact, compound 28 has little or no binding affinity to any of the other four DA receptor subtypes. In addition, of the 23 CNS receptors evaluated, only two, 5HT_1AR and 5HT_2BR, have binding affinity constants better than 100 nM (K_i <100 nM). Compound 28 is a potentially useful D₄-selective ligand for probing disease treatments involving the D₄ receptor, such as assisting smoking cessation, reversing cognitive deficits in schizophrenia and treating erectile dysfunction. Thus, further optimization, functional characterization and evaluation in animal models may be warranted.