Discovery of a Novel Series of Potent, Selective, Orally Available, and Brain-Penetrable C1s Inhibitors for Modulation of the Complement Pathway.

A novel series of non-amidine-based C1s inhibitors have been explored. Starting from high-throughput screening hit 3, isoquinoline was replaced with 1-aminophthalazine to enhance C1s inhibitory activity while exhibiting good selectivity against other serine proteases. We first disclose a crystal structure of a complex of C1s and a small-molecule inhibitor (4e), which guided structure-based optimization around the S2 and S3 sites to further enhance C1s inhibitory activity by over 300-fold. Improvement of membrane permeability by incorporation of fluorine at the 8-position of 1-aminophthalazine led to identification of (R)-8 as a potent, selective, orally available, and brain-penetrable C1s inhibitor. (R)-8 significantly inhibited membrane attack complex formation induced by human serum in a dose-dependent manner in an in vitro assay system, proving that selective C1s inhibition blocked the classical complement pathway effectively. As a result, (R)-8 emerged as a valuable tool compound for both in vitro and in vivo assessment.

Development of human cGAS-specific small-molecule inhibitors for repression of dsDNA-triggered interferon expression.

Cyclic GMP-AMP synthase (cGAS) is the primary sensor for aberrant intracellular dsDNA producing the cyclic dinucleotide cGAMP, a second messenger initiating cytokine production in subsets of myeloid lineage cell types. Therefore, inhibition of the enzyme cGAS may act anti-inflammatory. Here we report the discovery of human-cGAS-specific small-molecule inhibitors by high-throughput screening and the targeted medicinal chemistry optimization for two molecular scaffolds. Lead compounds from one scaffold co-crystallize with human cGAS and occupy the ATP- and GTP-binding active site. The specificity and potency of these drug candidates is further documented in human myeloid cells including primary macrophages. These novel cGAS inhibitors with cell-based activity will serve as probes into cGAS-dependent innate immune pathways and warrant future pharmacological studies for treatment of cGAS-dependent inflammatory diseases.

Design, synthesis, and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitors-Part 2.

Peripherally selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of stress urinary incontinence to overcome adverse effects associated with central action. Herein, we describe our medicinal chemistry approach to discover peripheral-selective noradrenaline reuptake inhibitors to avert the risk of P-gp-mediated DDI at the blood-brain barrier. We observed that steric shielding of the hydrogen-bond acceptors and donors (HBA and HBD) of compound 1 reduced the multidrug resistance protein 1 (MDR1) efflux ratio; however, the resulting compound 6, a methoxyacetamide derivative, was mainly metabolized by CYP2D6 and CYP2C19 in the in vitro phenotyping study, implying the risk of PK variability based on the genetic polymorphism of the CYPs. Replacement of the hydrogen atom with a deuterium atom in a strategic, metabolically hot spot led to compound 13, which was mainly metabolized by CYP3A4. To our knowledge, this study represents the first report of the effect of deuterium replacement for a major metabolic enzyme. The compound 13, N-{[(6S,7R)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl]methyl}-2-[(2H(3))methyloxy]acetamide hydrochloride, which exhibited peripheral NET selective inhibition at tested doses in rats, increased urethral resistance in a dose-dependent manner.

Design, synthesis, and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitors - Part 3.

Peripheral-selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of stress urinary incontinence to overcome adverse effects associated with central action. Here, we describe our medicinal chemistry approach to discover a novel series of highly potent, peripheral-selective, and orally available noradrenaline reuptake inhibitors with a low multidrug resistance protein 1 (MDR1) efflux ratio by cyclization of an amide moiety and introduction of an acidic group. We observed that the MDR1 efflux ratio was correlated with the pKa value of the acidic moiety. The resulting compound 9 exhibited favorable PK profiles, probably because of the effect of intramolecular hydrogen bond, which was supported by a its single-crystal structure. The compound 9, 1-{[(6S,7R)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl]methyl}-2-oxo-1,2-dihydropyridine-3-carboxylic acid hydrochloride, which exhibited peripheral NET-selective inhibition at tested doses in rats by oral administration, increased urethral resistance in a dose-dependent manner.

Design, synthesis and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitor.

Centrally acting noradrenaline reuptake inhibitor (NRI) is reportedly effective for patients with stress urinary incontinence (SUI) by increasing urethral closure in the clinical Phase IIa study with esreboxetine. Noradrenaline transporters are expressed in both central and peripheral nervous systems and the contribution of each site to efficacy has not been clarified. This report describes the development of a series of peripheral-selective 7-phenyl-1,4-oxazepane NRIs to investigate the contribution of the peripheral site to increasing urethral resistance in rats. (6S,7R)-1,4-Oxazepane derivative 7 exhibited noradrenaline transporter inhibition with high selectivity against inhibitions of serotonin and dopamine transporters. A replacement of hydroxyl with acetamide group contributed to enhancement of peripheral selectivity by increasing molecular polarity. Compound 12, N-{[(6S,7R)-7-(3,4-dichlorophenyl)-1,4-oxazepan-6-yl]methyl}acetamide 0.5 fumarate, which showed effectively no brain penetration in rats, increased urethral resistance in a dose-dependent manner and exhibited a maximal effect on par with esreboxetine. These results demonstrate that the urethral resistance-increasing effects of NRI in rats are mainly caused by the inhibition of noradrenaline transporters in the peripheral sites.

Direct labeling of 5-methylcytosine and its applications.

Cytosine methylation is one of the most important epigenetic events, and much effort has been directed to develop a simple reaction for methylcytosine detection. In this paper, we describe the design of tag-attachable ligands for direct methylcytosine labeling and their application to fluorescent and electrochemical assays. The effect of the location of bipyridine substituents on the efficiency of osmium complexation at methylcytosine was initially investigated. As a result, a bipyridine derivative with a substituent at the C4 position showed efficient complexation at the methylcytosine residue of single-stranded DNA in a reaction mixture containing potassium osmate and potassium hexacyanoferrate(III). On the basis of this result, a bipyridine derivative with a tag-attachable amino linker at the C4 position was synthesized. The efficiency of metal complex formation in the presence of the osmate and the synthetic ligand was clearly changed by the presence/absence of a methyl group at the C5 position of cytosine. The succinimidyl esters of functional labeling units were then attached to the bipyridine ligand fixed on the methylcytosine. These labels attached to methylcytosine enabled us to detect the target methylcytosine in DNA both fluorometrically and electrochemically. For example, we were able to fluorometrically obtain information on the methylation status at a specific site by means of fluorescence resonance energy transfer from a hybridized fluorescent DNA probe to a fluorescent label on methylcytosine. In addition, by the combination of electrochemically labeled methylcytosine and an electrode modified by probe DNAs, a methylcytosine-selective characteristic current signal was observed. This direct labeling of methylcytosine is a conceptually new methylation detection assay with many merits different from conventional assays.

Evaluation of novel one-electron reduction with metal in DNA.

Indium(0) is known to work as a good one-electron reductant with a small first ionization potential. Additionally, this metal is very stable in aqueous media. Therefore, indium has a bright prospect of developing a new method for the site-selective reduction of biopolymers. In this paper, the reduction of 5-halouracils with indium in aqueous solution is reported. We prepared four halouracils, i.e., 5-fluorouracil, 5-chlorouracil, 5-bromouracil, and 5-iodouracil, and evaluated their reduction with indium in water. Incubation of 5-bromouracil and 5-iodouracil with sonication in the presence of indium efficiently caused dehalogenation to uracil. Natural nucleobases (G, A, C, T and U) were not damaged by indium under the same reaction condition.

Development of fluorescence-labeling method for methylcytosine with metal complexation.

We report a methylcytosine-selective fluorescence labeling. An amino-modified bipyridine was prepared and used as an osmium ligand for methylcytosine-selective oxidation. Different fluorophores were easily incorporated to the modified methylcytosine site through an amide formation of the ligand amino group after osmium complexation.

Development of electrochemical detection for methylcytosine and its application.

We report electrochemical detection of methylcytosine. We developed a bipyridine ligand possessing an amino linker. This ligand was used for methylcytosine-selective osmium oxidation and subsequent redox labeling. The DNA labeled methylcytosine selectively with anthraquinone showed the current signal through hybridization with a DNA probe fixed on a gold surface.

Sequence-selective osmium oxidation of DNA: efficient distinction between 5-methylcytosine and cytosine.

5-Methylcytosine was distinguished from cytosine using the large difference of their osmium oxidation rates, and this reaction was applied to detection of the cytosine methylation status at a specific site of a long sequence using the formation of a bulge structure by hybridization with a guide DNA.

DNA hole transport on an electrode: application to effective photoelectrochemical SNP typing.

A useful feature of DNA is that long-range hole transport through DNA is readily achieved. Photostimulated long-range hole transport through DNA has prospective use in the development of a conceptually new electrochemical single-nucleotide polymorphism (SNP) typing method for use as a versatile platform for gene diagnostics and pharmacogenetics. We have applied artificial DNAs designed for photostimulated long-range hole transport through DNA to SNP typing. By hybridizing photosensitizer-equipped DNA probes, immobilized on gold working electrodes, with a target DNA strand containing an SNP site, we observed a cathodic photocurrent, which markedly changed depending on the nature of the base at the specific site. The use of a combination of hole-transporting bases constitutes a very powerful method for a single-step electrochemical assay applicable to SNP typing of all types of sequences.

Electrochemical evaluation of alternating duplex-triplex conversion effect on the anthraquinone-photoinjected hole transport through DNA duplex immobilized on a gold electrode.

Amperometry was employed to characterize the anthraquinone (AQ)-photoinjected hole transport through a 20-mer oligodeoxynucleotide (ODN) duplex, as immobilized on the surface of a gold electrode, and its triplex forms converted by association with several third oligopyrimidine (OPD) short strands. While the cathodic photocurrent was observed upon irradiation at 365 nm of the AQ photosensitizer linked to the end of DNA duplex, a marked lowering of the current density was identified to occur by the triplex formation of a duplex with a given third OPD short strand. The photocurrent through the DNA duplex showed a reversible fall-rise response concomitant with alternating association-dissociation cycle of the OPD short-strand, as regulated by temperature change around the corresponding melting temperature of the DNA triplex. Both the switched photoirradiation and the thermally alternating duplex-triplex conversion could provide tools of regulating the DNA hole transport.

Genotyping with hole-transporting DNA self-assembled monolayer.

High-throughput DNA sensors capable of detecting single-base mismatches are required for the routine screening of genetic mutations and disease. A new strategy for the electrochemical detection of single-base mismatches in DNA has been developed based on hole transport through DNA self-assembled monolayers. We prepared hole-transporting DNA self-assembled monolayers. Their photocurrent measurements were carried out using amperometory. Large photocurrent was observed when the DNA duplex which constituted a hole-transporting DNA self-assembled monolayer had a full-matched sequence, whereas the photocurrent for a DNA duplex containing a mismatched base pair was much smaller.

Photostimulated hole transport through a DNA duplex immobilized on a gold electrode.

Photostimulated hole transport through DNA duplexes immobilized on gold electrodes has been investigated. By modifying a gold electrode with a DNA duplex containing a photosensitizer, we have observed a sequence-dependent cathodic photocurrent. DNA acts as a good mediator for cathodic photocurrent when appropriate sequences are selected.

Electrochemical behavior of gold electrodes modified with photosensitizer-tethered DNA.

We investigated electrochemical characteristics of gold electrodes modified with photosensitizer-tethered DNA. We synthesized 3'-thiol-modified DNA strands (ODN-SH) and 3'-anthraquinone-modified DNA strands (AQ-ODN). We prepared an AQ-ODN/ODN-SH duplex-modified electrode through hybridization of AQ-ODN with ODN-SH on the gold electrode. We used an amperometric technique for its photocurrent measurement. The large photocurrent was observed by irradiation of a DNA modified electrode at 365 nm. We also measured photocurrent with different DNA sequences on gold electrodes.