Discovery of an 8-methoxytetrahydroisoquinoline derivative as an orally active N-type calcium channel blocker for neuropathic pain without CYP inhibition liability.

In lead optimization efforts starting from the tetrahydroisoquinoline (S)-1, we identified 2-{[(2R)-2-hydroxypropyl]amino}-1-[(1S)-8-methoxy-1-phenyl-3,4-dihydroisoquinolin-2(1H)-yl]ethanone ((1S)-8t) as a novel orally active small-molecule N-type calcium channel blocker without CYP inhibition liability. CYP3A4 inhibition profile was improved by reducing the lipophilicity of compound (S)-1. Moreover, introduction of a methoxy group to the C-8 position of tetrahydroisoquinoline led to identification of (1S)-8t, which eliminated CYP2D6 inhibition liability. Oral administration of (1S)-8t exerted efficacy in a rat spinal nerve ligation (SNL) model of neuropathic pain with an ED50 value of 2.8 mg/kg.

Coexistence of TDP-43 and tau pathology in neurodegeneration with brain iron accumulation type 1 (NBIA-1, formerly Hallervorden-Spatz syndrome).

We report here an autopsy case of sporadic adult-onset Hallervorden-Spatz syndrome, also known as neurodegeneration with brain iron accumulation type 1 (NBIA1), without hereditary burden. A 49-year-old woman died after a 27-year disease course. At the age of 22, she suffered from akinesia, resting tremor, and rigidity. At the age of 28, she was admitted to our hospital because of worsening parkinsonism and dementia. Within several years, she developed akinetic mutism. At the age of 49, she died of bleeding from a tracheostomy. Autopsy revealed a severely atrophic brain weighing 460 g. Histologically, there were iron deposits in the globus pallidus and substantia nigra pars reticulata, and numerous axonal spheroids in the subthalamic nuclei. Neurofibrillary tangles were abundant in the hippocampus, cerebral neocortex, basal ganglia, and brain stem. Neuritic plaques and amyloid deposits were absent. Lewy bodies and Lewy neurites, which are immunolabeled by anti-α-synuclein, were absent. We also observed the presence of TDP-43-positive neuronal perinuclear cytoplasmic inclusions, with variable frequency in the dentate gyrus granular cells, frontal and temporal cortices, and basal ganglia. TDP-43-positive glial cytoplasmic inclusions were also found with variable frequency in the frontal and temporal lobes and basal ganglia. The present case was diagnosed with adult-onset NBIA-1 with typical histological findings in the basal ganglia and brainstem. However, in this case, tau and TDP-43 pathology was exceedingly more abundant than α-synuclein pathology. This case contributes to the increasing evidence for the heterogeneity of NBIA-1.

Design, synthesis, and pharmacological evaluation of N-bicyclo-5-chloro-1H-indole-2-carboxamide derivatives as potent glycogen phosphorylase inhibitors.

As a result of the various N-bicyclo-5-chloro-1H-indole-2-carboxamide derivatives with a hydroxy moiety synthesized in an effort to discover novel glycogen phosphorylase (GP) inhibitors, 5-chloro-N-(5-hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl)-1H-indole-2-carboxamide (5b) was found to have potent inhibitory activity. The introduction of fluorine atoms both at a position adjacent to the hydroxy group and in the central benzene moiety lead to the optically active derivative 5-chloro-N-[(5R)-1,3,6,6-tetrafluoro-5-hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl]-1H-indole-2-carboxamide (25e(alpha), which was the most potent compound in this series (IC(50)=0.020microM). This compound inhibited glucagon-induced glucose output in cultured primary hepatocytes with an IC(50) value of 0.69microM, and showed oral hypoglycemic activity in diabetic db/db mice at 10mg/kg. Compound 25e(alpha) also had an excellent pharmacokinetic profile, with high oral bioavailability and a long plasma half-life, in male SD rats. The binding mode of 25e(alpha) to this molecule and the role of fluorine atoms in that binding were speculated in an enzyme docking study.

Identification of a key element for hydrogen-bonding patterns between protein kinases and their inhibitors.

In this article, we report crystal structures for inhibitor-kinase complexes in which the inhibitor has different binding orientations and hydrogen-bonding patterns with extracellular-signal regulated kinase 2 and insulin receptor tyrosine kinase. Our crystallographic studies, and sequence and structural analyses of 532 coordinates of kinases held in the Protein Data Bank, suggest that the length of the "specificity linker" described here is a key structural element of the hydrogen-bonding patterns between protein kinases and their inhibitors.

Synthesis and pharmacological evaluation of bis-3-(3,4-dichlorophenyl)acrylamide derivatives as glycogen phosphorylase inhibitors.

During our research using a high-throughput screening system for discovery of a new class of human liver glycogen phosphorylase a (hLGPa) inhibitors, a series of 3-(3,4-dichlorophenyl)acrylamide derivatives were synthesized, and their inhibitory activities toward hLGPa were evaluated. Among the derivatives, (2E,2'E)-N,N'-pentane-1,5-diylbis[3-(3,4-dichlorophenyl)acrylamide] (6c) inhibited hLGPa with an IC(50) value of 0.023 microM. An X-ray crystallographic study of the enzyme-6c complex showed that the inhibitor is bound at the dimer interface site, where the 3,4-dichlorophenyl moiety interacts hydrophobically with the enzyme.

Synthesis of 5-chloro-N-aryl-1H-indole-2-carboxamide derivatives as inhibitors of human liver glycogen phosphorylase a.

A series of 5-chloro-N-aryl-1H-indole-2-carboxamide derivatives were prepared and evaluated as inhibitors of human liver glycogen phosphorylase a (hLGPa). One compound, 5-chloro-N-[4-(1,2-dihydroxyethyl)phenyl]-1H-indole-2-carboxamide (2f), inhibited hLGPa with an IC(50) of 0.90microM. The pyridine analogue of 2f showed inhibitory activity of glucagon-induced glucose output in cultured primary hepatocytes with an IC(50) of 0.62microM and oral hypoglycemic activity in diabetic db/db mice. Crystallographic determination of the complex of 2f with hLGPa showed binding of the inhibitor in a solvent cavity at the dimer interface, with the two hydroxyl groups making favorable electrostatic interactions with hLGPa.

D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi: molecular cloning of the enzyme gene and crystal structure of the enzyme.

The gene coding for d-3-hydroxybutyrate dehydrogenase (HBDH) was cloned from Pseudomonas fragi. The nucleotide sequence contained a 780 bp open reading frame encoding a 260 amino acid residue protein. The recombinant enzyme was efficiently expressed in Escherichia coli cells harboring pHBDH11 and was purified to homogeneity as judged by SDS-PAGE. The enzyme showed a strict stereospecificity to the D-enantiomer (3R-configuration) of 3-hydroxybutyrate as a substrate. Crystals of the ligand-free HBDH and of the enzyme-NAD+ complex were obtained using the hanging-drop, vapor-diffusion method. The crystal structure of the HBDH was solved by the multiwavelength anomalous diffraction method using the SeMet-substituted enzyme and was refined to 2.0 A resolution. The overall structure of P.fragi HBDH, including the catalytic tetrad of Asn114, Ser142, Tyr155, and Lys159, shows obvious relationships with other members of the short-chain dehydrogenase/reductase (SDR) family. A cacodylate anion was observed in both the ligand-free enzyme and the enzyme-NAD+ complex, and was located near the catalytic tetrad. It was shown that the cacodylate inhibited the NAD+-dependent D-3-hydroxybutyrate dehydrogenation competitively, with a Ki value of 5.6 mM. From the interactions between cacodylate and the enzyme, it is predicted that substrate specificity is achieved through the recognition of the 3-methyl and carboxyl groups of the substrate.

Synthesis and biological activity of novel 1,2-disubstituted benzene derivatives as factor Xa inhibitors.

Factor Xa (fXa) is a serine protease that plays a pivotal role in the coagulation cascade. High-throughput screening of the Yamanouchi compound library yielded lead compound 1 with the ability to inhibit fXa at micromolar concentrations. To improve its fXa inhibitory activity and its oral anticoagulant activity, the linker between benzamidine and the central benzene ring was modified and a carboxyl group was introduced at the central benzene ring. The resulting compounds 40b (YM-203552), 41a (YM-202054), and 41c (YM-203558) exhibited potent fXa inhibitory activity and oral anticoagulant activity. In particular, YM-203558 exhibited the most potent oral anticoagulant activity, prolonging PT more than 3-fold at 0.5 and 2.0 h. Additionally, these compounds showed a high degree of selectivity for other serine proteases.

Design, synthesis and biological activity of YM-60828 derivatives. Part 2: potent and orally-bioavailable factor Xa inhibitors based on benzothiadiazine-4-one template.

Compound YM-60828 was previously characterized in our laboratory as a potent, selective and orally-bioavailable Factor Xa (FXa) inhibitor. The L-shape conformation of this compound in the active site of FXa was recognized as an important factor in displaying its FXa inhibitory activity. This led to the exploration of conformationally restricted cyclic scaffolds bearing a similar active conformation. The current study investigated a novel series of benzothiadiazine-4-one based compounds as FXa inhibitors. Structure-activity relationship (SAR) investigations revealed some potent FXa inhibitors that were selected for further in vitro and ex vivo anticoagulant studies. Among them, compound 6j (YM-169920) was proved to be most effective anticoagulant in this series. The synthesis and SAR in addition to docking studies of this class of inhibitors are described.

Design, synthesis and biological activity of YM-60828 derivatives: potent and orally-bioavailable factor Xa inhibitors based on naphthoanilide and naphthalensulfonanilide templates.

Factor Xa (FXa) is a serine protease which plays a pivotal role in the coagulation cascade. The inhibition of FXa has received great interest as a potential target for the development of new antithrombotic drug. Herein we describe a series of novel 7-amidino-2-naphthoanilide and 7-amidino-2-naphthalensulfonanilide derivatives which are potent FXa inhibitors. These scaffolds are rigid and are allowed to adopt an L-shape conformation which was estimated as the active conformation based on a docking study of YM-60828 with FXa. Optimization of the side chain at the central aniline nitrogen of 7-amidino-2-naphthoanilide has led to several potent and orally active FXa inhibitors. 5h (YM-169964), the best compound of these series, showed potent FXa inhibitory activity (IC(50)=3.9nM) and effectively prolonged prothrombin time by 9.6-fold ex vivo at an oral dose of 3mg/kg in squirrel monkeys.

Macrophage migration inhibitory factor and the discovery of tautomerase inhibitors.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine released from T-cells and macrophages, and is a key molecule in inflammation. Although a detailed understanding of the biological functions of MIF has not yet been found, it is known that MIF catalyzes the tautomerization of phenylpyruvate and a non-physiological molecule, D-dopachrome. A potent tautomerase inhibitor would be expected, as a validation tool, to shed light on role of MIF activity and the relationship between its biological and enzymatic activity. Such tautomerase inhibitors would be useful in the treatment of MIF-related diseases, such as sepsis, acute respiratory distress syndrome (ARDS), asthma, atopic dermatitis, rheumatoid arthritis (RA), nephropathy and tumors. In this review, we have focused on (1) the biological and enzymatic activities of MIF, (2) the discovery of novel, drug-like tautomerase inhibitors of MIF using a structure-based computer-assisted search, and (3) a crystallographic and molecular modeling study of the MIF-tautomerase inhibitor complexes (A review with 133 references).

The discovery of YM-60828: a potent, selective and orally-bioavailable factor Xa inhibitor.

Since Factor Xa (FXa) is well known to play a central role in thrombosis and hemostasis, inhibition of FXa is an attractive target for antithrombotic strategies. As a part of our investigation of a non-peptide, orally available FXa inhibitor, we found that a series of N-[(7-amidino-2-naphthyl)methyl]aniline derivatives possessed potent and selective inhibitory activities. Structure--activity relationship (SAR) of the substituent (R(1)) on the central aniline moiety suggested that increasing lipophilicity caused a detrimental effect on anticoagulant activity (prothrombin time assay) in plasma. Several compounds bearing a hydrophilic substituent in R(1) showed not only potent FXa inhibitory activities but also high anticoagulant activities. The best compound in this series was sulfamoylacetic acid derivative (YM-60828) which was a potent, selective and orally bioavailable FXa inhibitor and was chosen for clinical development.