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.

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.

Image-based screen capturing misfolding status of Niemann-Pick type C1 identifies potential candidates for chaperone drugs.

Niemann-Pick disease type C is a rare, fatal neurodegenerative disorder characterized by massive intracellular accumulation of cholesterol. In most cases, loss-of-function mutations in the NPC1 gene that encodes lysosomal cholesterol transporter NPC1 are responsible for the disease, and more than half of the mutations are considered to interfere with the biogenesis or folding of the protein. We previously identified a series of oxysterol derivatives and phenanthridine-6-one derivatives as pharmacological chaperones, i.e., small molecules that can rescue folding-defective phenotypes of mutated NPC1, opening up an avenue to develop chaperone therapy for Niemann-Pick disease type C. Here, we present an improved image-based screen for NPC1 chaperones and we describe its application for drug-repurposing screening. We identified some azole antifungals, including itraconazole and posaconazole, and a kinase inhibitor, lapatinib, as probable pharmacological chaperones. A photo-crosslinking study confirmed direct binding of itraconazole to a representative folding-defective mutant protein, NPC1-I1061T. Competitive photo-crosslinking experiments suggested that oxysterol-based chaperones and itraconazole share the same or adjacent binding site(s), and the sensitivity of the crosslinking to P691S mutation in the sterol-sensing domain supports the hypothesis that their binding sites are located near this domain. Although the azoles were less effective in reducing cholesterol accumulation than the oxysterol-derived chaperones or an HDAC inhibitor, LBH-589, our findings should offer new starting points for medicinal chemistry efforts to develop better pharmacological chaperones for NPC1.

Synthesis of multisubstituted cycloalkenes through carbomagnesiation of strained cycloalkynes.

An efficient synthetic method of seven- and six-membered cycloalkenes through the generation of strained cycloalkynes and following carbomagnesiation is described. Further bond formations of the resulting cycloalkenylmagnesium intermediates with a wide variety of electrophiles enabled us to prepare diverse cycloalkene derivatives including benzoxepine analogs having a fully substituted alkene structure.

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.

A facile preparation of functional cycloalkynes via an azide-to-cycloalkyne switching approach.

A facile method for preparing various functional cycloalkynes, including proteins incorporated with a cycloalkyne moiety, from the corresponding azides is developed. Treatment of diynes bearing strained and terminal alkyne moieties with a copper salt enabled terminal alkyne-selective click conjugation with azides, whereas a more azidophilic strained alkyne moiety was transiently protected from the click reaction via complexation with copper.

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.

Phenanthridin-6-one derivatives as the first class of non-steroidal pharmacological chaperones for Niemann-Pick disease type C1 protein.

Niemann-Pick disease type C is a fatal, progressive neurodegenerative disease mostly caused by mutations in Nieamnn-Pick type C1 (NPC1), a late endosomal membrane protein that is essential for intracellular cholesterol transport. The most prevalent mutation, I1061T (Ile to Thr), interferes with the protein folding process. Consequently, mutated but intrinsically functional NPC1 proteins are prematurely degraded via proteasome, leading to loss of NPC1 function. Previously, we reported sterol derivatives as pharmacological chaperones for NPC1, and showed that these derivatives can normalize folding-defective phenotypes of I1061T NPC1 mutant by directly binding to, and stabilizing, the protein. Here, we report a series of compounds containing a phenanthridin-6-one scaffold as the first class of non-steroidal pharmacological chaperones for NPC1. We also examined their structure-activity relationships.

Vildagliptin and its metabolite M20.7 induce the expression of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells.

Vildagliptin is a potent, orally active inhibitor of dipeptidyl peptidase-4 (DPP-4) for the treatment of type 2 diabetes mellitus. It has been reported that vildagliptin can cause hepatic dysfunction in patients. However, the molecular-mechanism of vildagliptin-induced liver dysfunction has not been elucidated. In this study, we employed an expression microarray to determine hepatic genes that were highly regulated by vildagliptin in mice. We found that pro-inflammatory S100 calcium-binding protein (S100) a8 and S100a9 were induced more than 5-fold by vildagliptin in the mouse liver. We further examined the effects of vildagliptin and its major metabolite M20.7 on the mRNA expression levels of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells. In HepG2 cells, vildagliptin, M20.7, and sitagliptin - another DPP-4 inhibitor - induced S100A9 mRNA. In HL-60 cells, in contrast, S100A8 and S100A9 mRNAs were significantly induced by vildagliptin and M20.7, but not by sitagliptin. The release of S100A8/A9 complex in the cell culturing medium was observed in the HL-60 cells treated with vildagliptin and M20.7. Therefore, the parental vildagliptin- and M20.7-induced release of S100A8/A9 complex from immune cells, such as neutrophils, might be a contributing factor of vildagliptin-associated liver dysfunction in humans.

Generation of cycloheptynes and cyclooctynes via a sulfoxide-magnesium exchange reaction of readily synthesized 2-sulfinylcycloalkenyl triflates.

Cycloheptynes and cyclooctynes were efficiently generated via a sulfoxide-magnesium exchange reaction of readily synthesized 2-sulfinylcycloalkenyl triflates. Cycloadditions between various ynophiles and the cycloalkynes generated by this method proceeded efficiently, providing an easy method to prepare a wide range of heterocycles fused with seven- or eight-membered carbocycles.

Structure-activity relationship study of non-steroidal NPC1L1 ligands identified through cell-based assay using pharmacological chaperone effect as a readout.

Niemann-Pick type C1-like 1 (NPC1L1) is an intestinal cholesterol transporter that is known to be the target of the cholesterol absorption inhibitor ezetimibe. We previously discovered steroidal NPC1L1 ligands by using a novel cell-based assay that employs pharmacological chaperone effect as a readout. Those steroid derivatives bound to a site different from both the sterol-binding domain and the ezetimibe-binding site, implying that they may be a novel class of NPC1L1 inhibitors with a distinct mode of action. As an extension of that work, we aimed here to find non-steroidal NPC1L1 ligands, which may be better candidates for clinical application than steroidal ligands, by using the same assay to screen our focused library of ligands for liver X receptor (LXR), a nuclear receptor that recognizes oxysterols as endogenous ligands. Here we describe identification of a novel class of NPC1L1 ligands with a ring-fused quinolinone scaffold, and an analysis of the structure-activity relationships of their derivatives as NPC1L1 ligands.

Structure-activity relationships of oxysterol-derived pharmacological chaperones for Niemann-Pick type C1 protein.

Niemann-Pick disease type C is a fatal neurodegenerative disease, and its major cause is mutations in NPC1 gene. This gene encodes NPC1 protein, a late endosomal polytopic membrane protein required for intracellular cholesterol trafficking. One prevalent mutation (I1061T) has been shown to cause a folding defect, which results in failure of endosomal localization of the protein, leading to loss-of-function phenotype. We have previously demonstrated that several oxysterols and their derivatives act as pharmacological chaperones; binding of these compounds to NPC1(I1061T) mutant protein corrects the localization/maturation defect of the mutant protein. Here, we disclose detailed structure-activity relationships of oxysterol derivatives as pharmacological chaperones for NPC1(I1061T) mutant.

Structure-activity relationship studies of Niemann-Pick type C1-like 1 (NPC1L1) ligands identified by screening assay monitoring pharmacological chaperone effect.

A number of hereditary diseases are caused by defective protein trafficking due to a folding defect resulting from point mutations in proteins. Ligands that bind to the folding intermediates of such mutant proteins and rescue their trafficking defects, known as pharmacological chaperones, have promise for the treatment of certain genetic diseases, including Fabry disease, cystic fibrosis, and Niemann-Pick disease type C. Here we show that this pharmacological chaperone effect can be used for ligand screening, that is, binding of candidate ligands can be detected by monitoring the ligand-mediated correction of a localization defect caused by artificially introduced point mutations of the protein of interest. Using this method, we discovered novel steroidal ligands of Niemann-Pick type C1-like 1 (NPC1L1), an intestinal cholesterol transporter that is the target of the cholesterol absorption inhibitor ezetimibe, and conducted structure-activity relationship studies. We also present data indicating that the binding site of the new ligands is distinct from both the N-terminal sterol-binding domain and the ezetimibe-binding site.

Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

Niemann-Pick type C1 (NPC1) is a polytopic endosomal membrane protein required for efflux of LDL-derived cholesterol from endosomes, and mutations of this protein are associated with Niemann-Pick disease type C, a fatal neurodegenerative disease. At least one prevalent mutation (I1061T) has been shown to cause a folding defect, which results in failure of endosomal localization, leading to a loss-of-function phenotype. Here, we show that several oxysterols and their derivatives act as pharmacological chaperones; binding of these compounds to I1061T NPC1 corrects the localization/maturation defect of the mutant protein. Further, these compounds alleviate intracellular cholesterol accumulation in patient-derived fibroblasts, suggesting that they may have therapeutic potential. These oxysterol derivatives bind to a domain of NPC1 that is different from the known N-terminal sterol-binding domain; i.e., there is an additional sterol-binding site on NPC1.

Visible-light-triggered release of nitric oxide from N-pyramidal nitrosamines.

Although many organic/inorganic compounds that release nitric oxide (NO) upon photoirradiation (phototriggered caged-NOs) have been reported, their photoabsorption wavelengths mostly lie in the UV region, because X-NO bonds (X=heteroatom and metal) generally have rather strong π-bond character. Thus, it is intrinsically difficult to generate organic compounds that release NO under visible light irradiation. Herein, the structures and properties of N-pyramidal nitrosamine derivatives of 7-azabicyclo[2.2.1]heptanes that release NO under visible light irradiation are described. Bathochromic shifts of the absorptions of these nitrosamines, attributed to HOMO (n)-LUMO (π*) transitions associated with the nonplanar structure of the N-NO moiety, enable the molecules to absorb visible light, which results in N-NO bond cleavage. Thus, these compounds are innate organic caged-NOs that are uncaged by visible light.

7-azabicyclo[2.2.1]heptane as a structural motif to block mutagenicity of nitrosamines.

Nitrosamines are potent carcinogens and toxicants in the rat and potential genotoxins in humans. They are metabolically activated by hydroxylation at an α-carbon atom with respect to the nitrosoamino group, catalyzed by cytochrome P450. However, there has been little systematic investigation of the structure-mutagenic activity relationship of N-nitrosamines. Herein, we evaluated the mutagenicity of a series of 7-azabicyclo[2.2.1]heptane N-nitrosamines and related monocyclic nitrosamines by using the Ames assay. Our results show that the N-nitrosamine functionality embedded in the bicyclic 7-azabicylo[2.2.1]heptane structure lacks mutagenicity, that is, it is inert to α-hydroxylation, which is the trigger of mutagenic events. Further, the calculated α-C-H bond dissociation energies of the bicyclic nitrosamines are larger in magnitude than those of the corresponding monocyclic nitrosamines and N-nitrosodimethylamine by as much as 20-30 kcal/mol. These results are consistent with lower α-C-H bond reactivity of the bicyclic nitrosamines. Thus, the 7-azabicyclo[2.2.1]heptane structural motif may be useful for the design of nongenotoxic nitrosamine compounds with potential biological/medicinal applications.