Discovery of a novel 2-(1H-pyrazolo[3,4-b]pyridin-1-yl)thiazole derivative as an EP1 receptor antagonist and in vivo studies in a bone fracture model.

We describe a medicinal chemistry approach to the discovery of a novel EP1 antagonist exhibiting high potency and good pharmacokinetics. Our starting point is 1, an EP1 receptor antagonist that exhibits pharmacological efficacy in cystometry models following intravenous administration. Despite its good potency in vitro, the high lipophilicity of 1 is a concern in long-term in vivo studies. Further medicinal chemistry efforts identified 4 as an improved lead compound with good in vitro ADME profile applicable to long term in vivo studies. A rat fracture study was conducted with 4 for 4 weeks to validate its utility in bone fracture healing. The results suggest that this EP1 receptor antagonist stimulates callus formation and thus 4 has potential for enhancing fracture healing.

Characterization of crystal water molecules in a high-affinity inhibitor and hematopoietic prostaglandin D synthase complex by interaction energy studies.

Hematopoietic prostaglandin D synthase (H-PGDS) is one of the two enzymes that catalyze prostaglandin D2 synthesis and a potential therapeutic target of allergic and inflammatory responses. To reveal key molecular interactions between a high-affinity ligand and H-PGDS, we designed and synthesized a potent new inhibitor (KD: 0.14 nM), determined the crystal structure in complex with human H-PGDS, and quantitatively analyzed the ligand-protein interactions by the fragment molecular orbital calculation method. In the cavity, 10 water molecules were identified, and the interaction energy calculation indicated their stable binding to the surface amino acids in the cavity. Among them, 6 water molecules locating from the deep inner cavity to the peripheral part of the cavity contributed directly to the ligand binding by forming hydrogen bonding interactions. Arg12, Gly13, Gln36, Asp96, Trp104, Lys112 and an essential co-factor glutathione also had strong interactions with the ligand. A strong repulsive interaction between Leu199 and the ligand was canceled out by forming a hydrogen bonding network with the adjacent conserved water molecule. Our quantitative studies including crystal water molecules explained that compounds with an elongated backbone structure to fit from the deep inner cavity to the peripheral part of the cavity would have strong affinity to human H-PGDS.

A series of novel indazole derivatives of Sirt 1 activator as osteogenic regulators.

A series of indazole derivatives were identified as Sirt 1 activators though high-throughput screening. Optimization of each substituent on the indazole ring led to the identification of compound 13. Compound 13 appeared to give the best Sirt 1 activity of the compounds tested and also showed osteogenesis activity in a cell assay. Sirt 1 activators are therefore potential candidates for the treatment of osteoporosis.

Discovery of 2-(1H-indazol-1-yl)-thiazole derivatives as selective EP(1) receptor antagonists for treatment of overactive bladder by core structure replacement.

We have designed a series of potent EP1 receptor antagonists. These antagonists are a series of 2-(1H-indazol-1-yl)-thiazoles in which the core structure was replaced with pyrazole-phenyl groups. In preliminary conscious rat cystometry experiments, two representative candidates, 2 and 22, increased bladder capacity. In particular, the increase using 22 was approximately 2-fold that of the baseline. More detailed profiling of this compound and further optimization of this series promises to provide a novel class of drug for treating overactive bladder (OAB).

Design, synthesis and SAR investigation of thienosultam derivatives as ADAMTS-5 (aggrecanase-2) inhibitors.

A series of 1,1-dioxothieno[2,3-d]isothiazole (thienosultam) derivatives were designed and synthesized as novel ADAMTS-5 inhibitors for an investigation into a side chain of thienosultam for the S1' pocket. The resulting compounds (19 and 24) show high ADAMTS-5 inhibition and other MMP selectivity, and these compounds show good oral bioavailability.

SAR-based optimization of 2-(1H-pyrazol-1-yl)-thiazole derivatives as highly potent EP1 receptor antagonists.

We describe a medicinal chemistry approach for generating a series of 2-(1H-pyrazol-1-yl)thiazoles as EP1 receptor antagonists. To improve the physicochemical properties of compound 1, we investigated its structure-activity relationships (SAR). Optimization of this lead compound provided small compound 25 which exhibited the best EP1 receptor antagonist activity and a good SAR profile.

Hit-to-lead optimization of 2-(1H-pyrazol-1-yl)-thiazole derivatives as a novel class of EP1 receptor antagonists.

We describe a medicinal chemistry approach to generate a series of 2-(1H-pyrazol-1-yl)thiazole compounds that act as selective EP1 receptor antagonists. The obtained results suggest that compound 12 provides the best EP1 receptor antagonist activity and demonstrates good oral pharmacokinetics.

A series of thiazole derivatives bearing thiazolidin-4-one as non-competitive ADAMTS-5 (aggrecanase-2) inhibitors.

A series of thiazole bearing thiazolidin-4-one was discovered via high-throughput screening as non-competitive inhibitors of ADAMTS-5. Compound 31 appeared to give the best ADAMTS-5 inhibition and good selectivity over other metalloproteases.

Knee extensor strength is associated with Mini-Mental State Examination scores in elderly men.

The present study examined whether the strength capability of knee extensor muscles is associated with global cognitive function, assessed by Mini-Mental State Examination (MMSE), in non-disabled elderly men. Isometric torques during maximal voluntary knee extension, plantar flexion, and elbow flexion and MMSE scores were determined in 39 non-disabled men aged 61-79 years and used for the cross-sectional analysis examining the associations among the measured variables. Moreover, 27 of the subjects participated in a training program consisted of body mass-based exercises (sitting down onto and standing up from a chair, hip joint extension and flexion, calf raises, side leg raises, and trunk flexion and extension) 6 days a week for 3 months. Isometric torques and MMSE scores were determined after the intervention. Among the data before intervention, only knee extension torque (KET) and KET relative to body mass (KET/BM) significantly correlated to the MMSE scores: r = 0.579 (P < 0.0001) for KET and r = 0.520 (P < 0.001) for KET/BM. After the intervention, KET and KET/BM increased significantly, but MMSE score did not. However, the absolute change in MMSE scores was significantly associated with that in KET (r = 0.381, P < 0.05) and KET/BM (r = 0.422, P < 0.05). These findings indicate that the strength capability of knee extensors is associated with global cognitive function in non-disabled elderly men, and provide a new perspective to a general concept that exercises strengthening knee extensor muscles should be included in resistance training programs for elderly individuals.

Receptor Transporter Protein 4 (RTP4) in the Hypothalamus Is Involved in the Development of Antinociceptive Tolerance to Morphine.

Receptor transporter protein 4 (RTP4), one of the receptor chaperone proteins, contributes to the maturation and membrane trafficking of opioid receptor heteromers consisting of mu (MOPr) and delta (DOPr) opioid receptors (MOPr-DOPr). Although MOPr-DOPr is known to mediate the development of morphine tolerance, the extent to which RTP4 plays a role in this process has not been elucidated. Given that RTP4 can be upregulated by repeated administration of morphine, especially in the hypothalamus, here we investigated the effect of hypothalamus-selective ablation of RTP4 on the development of antinociceptive tolerance to morphine. In this study, we generated RTP4flox mice and selectively knocked-out RTP4 using local injection of adeno-associated virus expressing Cre recombinase (AAV-Cre) into the hypothalamus. The AAV-Cre injection partially, but significantly, decreased the level of RTP4 expression, and suppressed the development of antinociceptive tolerance to morphine. Next, we examined the mechanism of regulation of RTP4 and found that, in neuronal cells, Rtp4 induction is via Gi and MAPK activation, while, in microglial cells, the induction is via Toll-like receptor 4. Together, these studies highlight the role of MOR activity in regulating RTP4, which, in turn, plays an important role in modulating morphine effects in vivo.

Discovery of 4,6- and 5,7-Disubstituted Isoquinoline Derivatives as a Novel Class of Protein Kinase C ζ Inhibitors with Fragment-Merging Strategy.

Two chemical series of novel protein kinase C ζ (PKCζ) inhibitors, 4,6-disubstituted and 5,7-disubstituted isoquinolines, were rapidly identified using our fragment merging strategy. This methodology involves biochemical screening of a high concentration of a monosubstituted isoquinoline fragment library, then merging hit isoquinoline fragments into a single compound. Our strategy can be applied to the discovery of other challenging kinase inhibitors without protein-ligand structural information. Furthermore, our optimization effort identified the highly potent and orally available 5,7-isoquinoline 37 from the second chemical series. Compound 37 showed good efficacy in a mouse collagen-induced arthritis model. The in vivo studies suggest that PKCζ inhibition is a novel target for rheumatoid arthritis (RA) and that 5,7-disubstituted isoquinoline 37 has the potential to elucidate the biological consequences of PKCζ inhibition, specifically in terms of therapeutic intervention for RA.

Discovery of selective PDE4B inhibitors.

In this study the first PDE4B selective inhibitor is described. Optimization of lead 2-arylpyrimidine derivatives afforded a series of potent PDE4B inhibitors with >100-fold selectivity over the PDE4D isozyme. With a good pharmacokinetic profile, a selected compound exhibited potent anti-inflammatory effects in vivo and showed less emesis compared with Cilomilast.

Discovery of Novel Transient Receptor Potential Vanilloid 4 (TRPV4) Agonists as Regulators of Chondrogenic Differentiation: Identification of Quinazolin-4(3 H)-ones and in Vivo Studies on a Surgically Induced Rat Model of Osteoarthritis.

Osteoarthritis (OA) is a degenerative disease characterized by joint destruction and loss of cartilage. There are many unmet needs in the treatment of OA and there are few promising candidates for disease-modifying OA drugs, particularly, anabolic agents. Here, we describe the identification of novel quinazolin-4(3 H)-one derivatives, which stimulate chondrocyte cartilage matrix production via TRPV4 and mitigate damaged articular cartilage. We successfully identified the water-soluble, highly potent quinazolin-4(3 H)-one derivative 36 and studied its intra-articular physicochemical profile to use in in vivo surgical OA model studies. Compound 36·HCl provided relief from OA damage in a rat medial meniscal tear (MT) model. Specifically, 36·HCl dose-dependently suppressed cartilage degradation and enhanced the messenger RNA expression of aggrecan and SOX9 in cartilage isolated from MT-operated rat knees compared with knees treated with vehicle. These results suggest that 36 induces anabolic changes in articular cartilage and consequently reduces OA progression.

Effect of Long-Term Body-Mass-Based Resistance Exercise on Cognitive Function in Elderly People.

The study examined the effect of a body-mass-based home exercise program on cognitive functioning among 170 male and female elderly people (52-81 years). This program comprised five kinds of resistance exercises that elderly people can perform at home without supervision or specialized equipment using only their body mass for resistance. Various cognitive tasks were used to assess cognitive functioning, including a simple reaction task, Go/No-Go reaction task, Stroop task, serial subtraction task, and coincident timing task. These tasks were performed before and after a 3-month body-mass-based home exercise program. Although there were no significant improvements in the simple reaction and coincident timing tasks, significant improvement was shown in the Go/No-Go reaction task and serial subtraction task. This study shows that even simple resistance exercise, using only body mass for resistance, may be an effective method for preventing age-related cognitive decline of inhibitory control and working memory among elderly people.

Can Handgrip Strength Improve Following Body Mass-Based Lower Body Exercise?

Knee extension strength (KES) improves following body mass-based lower body exercise training; however, it is unknown whether this type of exercise increases handgrip strength (HGS) as a result of a cross-education effect in older individuals. Our aim was to investigate the effect of a body mass-based exercise intervention on HGS and KES in older adults. At baseline, 166 subjects started a 12-week intervention program, and 160 (108 women and 52 men) subjects completed the study. A self-selected group of 37 older adults (21 women and 16 men) served as a control group. HGS, KES, and ultrasound-derived anterior thigh muscle thickness (anterior thigh MT) were measured at baseline and post-testing, and relative strength of the knee extensor (KES/anterior thigh MT) was calculated. A linear regression model controlling for baseline values of body-mass index, % body fat, fat-free mass, HGS, chair stand time, anterior thigh MT, and KES/body mass ratio found a significant difference between control and training groups for KES post-testing values (p = 0.001) and anterior thigh MT post-testing values (p = 0.012), but not for HGS post-testing values (p = 0.287). Our results suggest that increases in lower body strength and muscle size following a 12-week lower body mass-based exercise intervention fail to translate into improvements in HGS.

Relationship between performances of 10-time-repeated sit-to-stand and maximal walking tests in non-disabled older women.

AIM: Sit-to-stand (STS) test is extensively used to assess the functionality of the lower body in elderly people. This study aimed to examine how the score of STS can be associated with that of maximal walking (MW) tests through a cross-sectional as well as longitudinal analysis for non-disabled older women. METHOD: Times taken for a 10-time-repeated STS (STS time) and 5-m MW (MW time) were determined before (pre) and after (post) a 3-month body mass-based exercise program in 154 non-disabled women aged 60 to 79 years. In addition to the time scores, STS and MW power indexes (STS-PI and MW-PI, respectively) were calculated using the following equations: STS-PI = (body height - 0.4) × body mass × 10/STS time and MW-PI = body mass × 5/MW time. RESULTS: At pre- and post-intervention, STS-PI was significantly correlated to MW-PI, with higher correlation coefficients (r = 0.545-0.567, P < 0.0001) than those between the two time scores (r = 0.271-0.309, P < 0.001). The intervention significantly improved STS-time (13.6 ± 3.2 s at pre to 9.4 ± 1.8 s at post, P < 0.0001), MW time (2.4 ± 0.3 s to 2.2 ± 0.3 s, P < 0.0001), STS-PI (46.5 ± 12.5 to 65.7 ± 12.7, P < 0.0001), and MW-PI (112.1 ± 20.2 to 124.2 ± 24.4, P < 0.0001). There were significant correlations between the changes of STS and MW times (r = 0.281, P < 0.001) and between those of STS-PI and MW-PI (r = 0.366, P < 0.0001). CONCLUSION: In elderly women, the performance of sit-to-stand task and its training-induced gain are associated with those of the maximal walking task. In addition, the current results indicated that translation of the performance scores of the sit-to-stand and maximal walking tasks to power indexes may be a useful approach for examining the association between the two tasks.

Associations of sit-up ability with sarcopenia classification measures in Japanese older women.

To test the hypothesis that sit-up performance is associated with sarcopenia classification measures, 93 older women aged 53-78 years were divided into three groups based on achieved repetitions (30 s) for the sit-up performance test: Group 0 (G 0, n = 33) performed 0 repetitions, Group 1-9 (G 1-9, n = 30) performed between 1 and 9 repetitions, and Group 10+ (G 10+, n = 30) performed over 10 repetitions. Dual-energy X-ray absorptiometry-derived appendicular lean soft tissue mass (aLM), handgrip strength (HGS), usual walking speed, and chair stand were measured, and low muscle mass (aLM index) and poor physical function were defined according to previous studies. Age and body mass index were similar among the three groups. HGS was higher in G 10+ compared with G 0. The prevalence rate of low muscle mass was 30% for G 0, 20% for G 1-9, and 3% for G 10+. Low HGS was observed in both G 0 (24%) and G 1-9 (20%), but not in G 10+. Only two persons in G 0 were classified as slow walking speed. Our results suggest that sit-up performance may be a useful indicator to determine the extent of sarcopenia because low muscle mass and poor function were almost non-existent in individuals who could perform over 10 sit-ups.

Sex difference in age-related changes in knee extensor strength and power production during a 10-times-repeated sit-to-stand task in Japanese elderly.

BACKGROUND: For middle-aged and elderly women, age-related decline in an index representing power production during STS task (STS-PI), calculated by using an equation reported previously, has been shown to be greater than that in the force generation capability of lower extremity. Whether this is specific to women remains unclear. This study examined how the age-related changes in knee extensor strength and power production during STS differ between Japanese men and women aged 65 years or older. METHODS: The time taken for a 10-times-repeated STS test (STS time) and force developed during maximal voluntary isometric knee extension (KE-F) were determined in Japanese younger-old (262 men and 285 women) aged 65-74 years and older-old (96 men and 89 women) aged 75-90 years. STS-PI was calculated using the following equation: STS-PI = (body height - 0.4) × body mass × 10/STS time. RESULTS: KE-F and STS-PI were significantly greater in the younger-old than in the older-old group (p < 0.0001) and in men than in women (p < 0.0001). STS-PI and KE-F, expressed as the percentages of the mean value of the corresponding variable for the younger-old group (%STS-PI and %KE-F, respectively), were negatively correlated to chronological age in both men (r = -0.386 and r = -0.269, respectively, p < 0.0001) and women (r = -0.504 and r = -0.294, respectively, p < 0.0001). Regression slopes in the relationship between age and %KE-F were not significantly different between men (-1.521) and women (-1.618). However, regression slope in the relationship between age and %STS-PI was significantly steeper in women (-3.108) than in men (-2.170) (p < 0.05). In OOG, %KE-F had no significant effect of sex, but %STS-PI was significantly lower in women than in men (p < 0.001). CONCLUSIONS: In Japanese men and women aged 65 years or over, age-related loss in power production during STS is steeper in women than in men, with greater magnitude than that in knee extensor strength. This suggests a higher priority of improving power generation capability during whole-body movement such as STS in older women than in older men.

Synthesis of Methyl 5'-Thio-α-isomaltoside via an Acyclic Monothioacetal and its Behavior toward Glucoamylase.

Methyl 5'-thio-α-isomaltoside (1), which contains the ring-sulfur analogue of the nonreducing glucoside of isomaltose, was synthesized from gentiobiose through a novel ring opening-recyclization approach. The nonreducing glucoside of per-O-benzylated phenyl 1-thio-ß-gentiobioside underwent O-5'-C-1' bond cleavage with dimethyl-boron bromide and thiolacetic acid to give the acyclic monothioacetal 4 with the 1-thioglucopyranoside at the reducing end intact. The HO-5' group in 4 was inverted by a standard oxidation-reduction process with good efficiency. Recyclization under Mitsunobu condition allowed C-5'-S-1' bond formation with inversion of configuration at C-5', to give 1 after functional group interconversion. TLC analysis showed that 1, unlike isomaltose, was not hydrolyzed by glucoamylase from Rhizopus niveus. A fluorometric assay confirmed that the dissociation constant (Kd ) for 1 with the enzyme was 39 mM at 20°C, which is comparable with that for isomaltose. A binding assay involving fluorescence titration of the enzyme-1 complex with gluconolactone indicated that the disaccharide 1 was bound to the catalytic and noncatalytic subsites. Since isomaltose is known to bind only to the noncatalytic subsites, this result indicates a relatively high affinity of the 5-thioglucose moiety for the catalytic subsite.

Discovery of a benzimidazole series of ADAMTS-5 (aggrecanase-2) inhibitors by scaffold hopping.

We describe a medicinal chemistry approach to generate a series of benzimidazoles bearing thiazolidin-4-one using scaffold hopping from thiazole 1, our previously described thiazole. Our goal was to discover a potent and permeable small-molecule ADAMTS-5 inhibitor. The results suggest that small compound 22 shows promise as a potent small-molecule ADAMTS-5 inhibitor with good permeability.