Discovery of orally active sulfonylphenyl thieno[3,2-d]pyrimidine derivatives as GPR119 agonists.

G-protein-coupled receptor 119 (GPR119) has great potential as a therapeutic target for the treatment of type II diabetes. Novel thieno[3,2-d]pyrimidine derivatives were discovered as GPR119 agonists through a bioisosteric replacement strategy. The sulfonylphenyl thieno[3,2-d] pyrimidine scaffold was introduced, and its derivatives exhibited potent agonistic activity for GPR119 in cell-based assays. The representative derivative 43 displayed excellent pharmacokinetic profiles in rodents and significantly improved glucose tolerance in vivo. In OGTT study, compound 43 reduced significantly blood glucose levels in both mice and rats.

Belvarafenib penetrates the BBB and shows potent antitumor activity in a murine melanoma brain metastasis model.

Brain metastasis is a common complication in melanoma patients with BRAF and NRAS mutations and has a poor prognosis. Although BRAF inhibitors are clinically approved, their poor brain penetration limits their efficacy in brain metastasis. Thus, melanoma brain metastasis still requires better treatment. Belvarafenib, a pan-RAF inhibitor, has reported antitumor activity in melanoma with RAF and RAS mutations in animal models and patients. However, brain permeability and antitumor efficacy on brain metastasis have not been determined. This study confirmed the brain penetration of belvarafenib, the antitumor activity on BRAF and NRAS mutant melanoma, and the efficacy on melanoma within the brain. Belvarafenib strongly suppressed melanoma in BRAF V600E mutant A375SM tumor-bearing mice. It also significantly inhibited tumor growth in NRAS mutant SK-MEL-30 and K1735 tumor-bearing mice and synergized to enhance the antitumor activity combined with cobimetinib or atezolizumab. Belvarafenib was penetrated at considerable levels into the brains of mice and rats following oral administration. The exposure of belvarafenib in the brain was similar to or higher than that in plasma, and this high brain penetration differed significantly from that of other BRAF inhibitors with low brain penetration. Most importantly, belvarafenib strongly reduced tumor burden and markedly improved survival benefits in mice intracranially implanted with A375SM melanoma. These results demonstrated that belvarafenib, which has favorable BBB permeability, and potent antitumor activity on the tumors with BRAF/NRAS mutations, may be a promising therapeutic option for patients with BRAF/NRAS mutant melanoma brain metastasis.

Synergistic Effects of BTK Inhibitor HM71224 and Methotrexate in a Collagen-induced Arthritis Rat Model.

BACKGROUND/AIM: Using a rat model of collagen-induced arthritis (CIA), we evaluated the therapeutic effects of HM71224 (BTKi), as well as the drug-drug interactions in combined therapy with methotrexate (MTX) based on both drugs' pharmacological role in immune regulation and antiinflammation. MATERIALS AND METHODS: Arthritis in rats was induced using type II collagen and incomplete Freund's adjuvant. The therapeutic effects of HM71224 (alone or in combination with MTX) were evaluated by arthritis score, paw volume, body weight, and histopathological examination (H&E and Safranin-O staining). The drug-drug interactions between HM71224 and MTX were investigated by measuring plasma, liver enzyme and creatinine levels and blood cell counts. RESULTS: HM71224 reduced the clinical signs of arthritis, paw volume, and body weight loss in CIA rats. ED50 and ED90 were 1.0 and 2.5 mg/kg, respectively. HM71224 combined with MTX decreased the arthritis score, bone erosion, synovitis, and cartilage degradation without apparent interaction. CONCLUSION: The combination of HM71224 and MTX improved the therapeutic effect with no drug-drug interactions in RA.

Target modulation and pharmacokinetics/pharmacodynamics translation of the BTK inhibitor poseltinib for model-informed phase II dose selection.

The selective Bruton tyrosine kinase (BTK) inhibitor poseltinib has been shown to inhibit the BCR signal transduction pathway and cytokine production in B cells (Park et al. Arthritis Res. Ther. 18, 91, https://doi.org/10.1186/s13075-016-0988-z , 2016). This study describes the translation of nonclinical research studies to a phase I clinical trial in healthy volunteers in which pharmacokinetics (PKs) and pharmacodynamics (PDs) were evaluated for dose determination. The BTK protein kinase inhibitory effects of poseltinib in human peripheral blood mononuclear cells (PBMCs) and in rats with collagen-induced arthritis (CIA) were evaluated. High-dimensional phosphorylation analysis was conducted on human immune cells such as B cells, CD8 + memory cells, CD4 + memory cells, NK cells, neutrophils, and monocytes, to map the impact of poseltinib on BTK/PLC and AKT signaling pathways. PK and PD profiles were evaluated in a first-in-human study in healthy donors, and a PK/PD model was established based on BTK occupancy. Poseltinib bound to the BTK protein and modulated BTK phosphorylation in human PBMCs. High-dimensional phosphorylation analysis of 94 nodes showed that poseltinib had the highest impact on anti-IgM + CD40L stimulated B cells, however, lower impacts on anti-CD3/CD-28 stimulated T cells, IL-2 stimulated CD4 + T cells and NK cells, M-CSF stimulated monocytes, or LPS-induced granulocytes. In anti-IgM + CD40L stimulated B cells, poseltinib inhibited the phosphorylation of BTK, AKT, and PLCγ2. Moreover, poseltinib dose dependently improved arthritis disease severity in CIA rat model. In a clinical phase I trial for healthy volunteers, poseltinib exhibited dose-dependent and persistent BTK occupancy in PBMCs of all poseltinib-administrated patients in the study. More than 80% of BTK occupancy at 40 mg dosing was maintained for up to 48 h after the first dose. A first-in-human healthy volunteer study of poseltinib established target engagement with circulating BTK protein. Desirable PK and PD properties were observed, and a modeling approach was used for rational dose selection for subsequent trials. Poseltinib was confirmed as a potential BTK inhibitor for the treatment of autoimmune diseases.Trial registration: This article includes the results of a clinical intervention on human participants [NCT01765478].

Discovery of Encequidar, First-in-Class Intestine Specific P-glycoprotein Inhibitor.

Many chemotherapeutics, such as paclitaxel, are administered intravenously as they suffer from poor oral bioavailability, partly because of efflux mechanism of P-glycoprotein in the intestinal epithelium. To date, no drug has been approved by the U.S. Food and Drug Administration (FDA) that selectively blocks this efflux pump. We sought to identify a compound that selectively inhibits P-glycoprotein in the gastrointestinal mucosa with poor oral bioavailability, thus eliminating the issues such as bone marrow toxicity associated with systemic inhibition of P-glycoprotein. Here, we describe the discovery of highly potent, selective, and poorly orally bioavailable P-glycoprotein inhibitor 14 (encequidar). Clinically, encequidar was found to be well tolerated and minimally absorbed; and importantly, it enabled the oral delivery of paclitaxel.

Synthesis and biological activity of 2-cyanoacrylamide derivatives tethered to imidazopyridine as TAK1 inhibitors.

The importance of transforming growth factor beta-activated kinase 1 (TAK1) to cell survival has been demonstrated in many studies. TAK1 regulates signalling cascades, the NF-κB pathway and the mitogen-activated protein kinase (MAPK) pathway. TAK1 inhibitors can induce the apoptosis of cancerous cells, and irreversible inhibitors such as (5Z)-7-oxozeaenol are highly potent. However, they can react non-specifically with cysteine residues in proteins, which may have serious adverse effects. Reversible covalent inhibitors have been suggested as alternatives. We synthesised imidazopyridine derivatives as novel TAK1 inhibitors, which have 2-cyanoacrylamide moiety that can form reversible covalent bonding. A derivative with 2-cyano-3-(6-methylpyridin-2-yl)acrylamide (13h) exhibited potent TAK1 inhibitory activity with an IC50 of 27 nM. It showed a reversible reaction with ß-mercaptoethanol, which supports its potential as a reversible covalent inhibitor.

Chemosensitivity to HM90822, a novel synthetic IAP antagonist, is determined by p-AKT-inducible XIAP phosphorylation in human pancreatic cancer cells.

Inhibitor of apoptosis proteins (IAPs) are overexpressed in the majority of cancers and prevent apoptosis by inhibiting caspases. IAPs have therefore attracted considerable attention as potential targets for anticancer therapy. Here, we demonstrated that HM90822 (abbreviated HM822; a new synthetic IAP antagonist) induced apoptotic cell death via proteasome-dependent degradation of BIR2/3 domain-containing IAPs in human pancreatic cancer cells. HM822 inhibited the expression of XIAP and cIAP1/2 proteins in Panc-1 and BxPC-3 cells, which are sensitive to HM822. HM822 also induced IAP ubiquitination and promoted proteasome-dependent IAP degradation. However, cells expressing phospho-XIAP (Ser87) and AKT exhibited resistance to HM822. In other words, the overexpression of AKT-CA (constitutive active form for AKT) or AKT-WT induced resistance to HM822. In addition, in Panc-1 xenograft and orthotopic mouse models, we revealed that tumor growth was suppressed by the administration of HM822. Taken together, these results suggest that HM822 induces apoptosis through ubiquitin/proteasome-dependent degradation of BIR3 domain-containing IAPs. These findings suggest that phospho-XIAP and phospho-AKT may be used as biomarkers for predicting the efficacy of HM822 in pancreatic cancer patients.

Synthesis and biological evaluation of novel thienopyrimidine derivatives as diacylglycerol acyltransferase 1 (DGAT-1) inhibitors.

A novel series of thieno[3,2-d]pyrimidine derivatives were synthesised and their inhibitory effects against diacylglycerol acyltransferase 1 (DGAT-1) were assessed. cis-Isomer 17a showed potent and selective inhibitory activity against DGAT-1 in SF9 cells. In addition, 17a had an acceptable pharmacokinetic profile and accumulated mainly in the small intestine and liver. Oral administration of 17a led to a significant reduction in plasma triacylglycerol level during an oral lipid tolerance test (OLTT) in murine and canine models. Taken together, 17a is a high-quality candidate that deserves further investigation.

Pharmacological evaluation of HM41322, a novel SGLT1/2 dual inhibitor, in vitro and in vivo.

HM41322 is a novel oral sodium-glucose cotransporter (SGLT) 1/2 dual inhibitor. In this study, the in vitro and in vivo pharmacokinetic and pharmacologic profiles of HM41322 were compared to those of dapagliflozin. HM41322 showed a 10-fold selectivity for SGLT2 over SGLT1. HM41322 showed an inhibitory effect on SGLT2 similar to dapagliflozin, but showed a more potent inhibitory effect on SGLT1 than dapagliflozin. The maximum plasma HM41322 level after single oral doses at 0.1, 1, and 3 mg/kg were 142, 439, and 1830 ng/ml, respectively, and the T1/2 was 3.1 h. HM41322 was rapidly absorbed and reached the circulation within 15 min. HM41322 maximized urinary glucose excretion by inhibiting both SGLT1 and SGLT2 in the kidney. HM41322 3 mg/kg caused the maximum urinary glucose excretion in normoglycemic mice (19.32±1.16 mg/g) at 24 h. In normal and diabetic mice, HM41322 significantly reduced glucose excursion. Four-week administration of HM41322 in db/db mice reduced HbA1c in a dose dependent manner. Taken together, HM41322 showed a favorable preclinical profile of postprandial glucose control through dual inhibitory activities against SGLT1 and SGLT2.

WITHDRAWN: HM47118A, a novel insulinotropic GPR119 agonist and potential oral antidiabetic agent.

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Synthesis and anti-tumor activity of imidazopyrazines as TAK1 inhibitors.

Transforming growth factor-ß activated kinase-1 (TAK1) is a potential therapeutic target for cancers and inflammatory diseases. We synthesized a series of novel imidazopyrazine derivatives, which were found to exhibit potent inhibitory effect against TAK1. Compound 22a, which possesses a good pharmacokinetic profile, showed excellent in vitro kinase activity and significant in vivo efficacy in mice xenografted with SW620, a KRAS-dependent colon cancer cell line.

Synthesis and evaluation of thieno[3,2-d]pyrimidine derivatives as novel FMS inhibitors.

Colony stimulating factor-1 receptor (CSF-1R or FMS) and it ligand, CSF-1, signaling regulates the differentiation and function of tumor-associated macrophages (TAMs) that play an important role in tumor progression. Derivatives of thieno[3,2-d]pyrimidine were synthesized and evaluated as kinase inhibitors of FMS. The most representative compound 21 showed strong activity (IC50 = 2 nM) against FMS kinase and served as candidate for proof of concept. Anti-tumor activity alone and/or in combination with paclitaxel was examined via a tumor cell growth inhibition assay and via an in vitro tumor invasion assay using human breast adenocarcinoma cells.

HM71224, a selective Bruton's tyrosine kinase inhibitor, attenuates the development of murine lupus.

BACKGROUND: Systemic lupus erythematosus (SLE) is associated with B cell hyperactivity, and lupus nephritis (LN), in particular, is promoted by the production of autoantibodies and immune complex deposition. Bruton's tyrosine kinase (BTK) plays critical roles in B cell receptor-related and Fc receptor-related signaling. We aimed to investigate the impact of therapeutic intervention with HM71224 (LY3337641), a selective BTK inhibitor, on the development of murine SLE-like disease features. METHODS: We examined the therapeutic effects of HM71224 on SLE-like disease features in MRL/lpr and NZB/W F1 mice. The disease-related skin lesion was macroscopically observed in MRL/lpr mice, and the impact on splenomegaly and lymphadenopathy was determined by the weight of the spleen and cervical lymph node. The renal function was evaluated by measuring blood urea nitrogen, serum creatinine, and urine protein, and the renal damage was assessed by histopathological grading. Survival rate was observed during the administration period. The impact of B cell inhibition was investigated in splenocytes from both mice using flow cytometry. Autoantibody was measured in serum by ELISA. RESULTS: HM71224 effectively suppressed splenic B220+GL7+, B220+CD138+, and B220+CD69+ B cell counts, and anti-dsDNA IgG and reduced splenomegaly and lymph node enlargement. The compound also prevented skin lesions caused by lupus development, ameliorated renal inflammation and damage with increased blood urea nitrogen and creatinine, and decreased proteinuria. Furthermore, HM71224 also decreased mortality from lupus development in both mouse models. CONCLUSION: Our results indicate that inhibition of BTK by HM71224 effectively reduced B cell hyperactivity and significantly attenuated the development of SLE and LN in rodent SLE models.

HM71224, a novel Bruton's tyrosine kinase inhibitor, suppresses B cell and monocyte activation and ameliorates arthritis in a mouse model: a potential drug for rheumatoid arthritis.

BACKGROUND: Bruton's tyrosine kinase (Btk) is critical for activation of B cells and myeloid cells. This study aimed to characterize the effects of HM71224, a novel Btk inhibitor, both in vitro and in a mouse model of experimental arthritis. METHODS: The kinase inhibition profile of HM71224 was analyzed. The in vitro effects of HM71224 on B cells and monocytes were analyzed by examining phosphorylation of Btk and its downstream signaling molecules, along with cytokine production and osteoclast formation. The in vivo effects of HM71224 were investigated in a mouse model of collagen-induced arthritis (CIA). RESULTS: HM71224 irreversibly bound to and inhibited Btk (IC50 = 1.95 nM). The compound also inhibited the phosphorylation of Btk and its downstream molecules such as PLCγ2, in activated Ramos B lymphoma cells and primary human B cells in a dose-dependent manner. Furthermore, HM71224 effectively inhibited the production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß by human monocytes, and osteoclast formation by human monocytes. Finally, HM71224 improved experimental arthritis and prevented joint destruction in a murine model of CIA. CONCLUSIONS: HM71224 inhibits Btk in B cells and monocytes and ameliorates experimental arthritis in a mouse model. Thus, HM71224 is a potential novel therapeutic agent for rheumatoid arthritis in humans.

Discovery of novel pyrimidine and malonamide derivatives as TGR5 agonists.

Takeda G-protein-coupled receptor 5 (TGR5) is a promising molecular target for metabolic diseases. A series of 4-(2,5-dichlorophenoxy)pyrimidine and cyclopropylmalonamide derivatives were synthesized as potent agonists of TGR5 based on a bioisosteric replacement strategy. Several compounds exhibited improved potency, compared to a reference compound with a pyridine scaffold. The pharmacokinetic profile of the representative compound 18 was considered moderate.

Synthesis of proline analogues as potent and selective cathepsin S inhibitors.

Cathepsin S is a potential target of autoimmune disease. A series of proline derived compounds were synthesized and evaluated as cathepsin S inhibitors. We discovered potent cathepsin S inhibitors through structure-activity relationship studies of proline analogues. In particular, compound 19-(S) showed promising in vitro/vivo pharmacological activities and properties as a selective cathepsin S inhibitor.

Antitumor activity of HM781-36B, a highly effective pan-HER inhibitor in erlotinib-resistant NSCLC and other EGFR-dependent cancer models.

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases has been implicated in a variety of cancers. In particular, activating mutations such as the L858R point mutation in exon 21 and the small in-frame deletions in exon 19 of the EGFR tyrosine kinase domain are correlated with sensitivity to EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC) patients. Clinical treatment of patients is limited by the development of drug resistance resulting mainly from a gatekeeper mutation (T790M). In this study, we evaluated the therapeutic potential of a novel, irreversible pan-HER inhibitor, HM781-36B. The results from this study show that HM781-36B is a potent inhibitor of EGFR in vitro, including the EGFR-acquired resistance mutation (T790M), as well as HER-2 and HER-4, compared with other EGFR tyrosine kinases inhibitors (erlotinib, lapatinib and BIBW2992). HM781-36B treatment of EGFR DelE746_A750-harboring erlotinib-sensitive HCC827 and EGFR L858R/T790M-harboring erlotinib-resistant NCI-H1975 NSCLC cells results in the inhibition of EGFR phosphorylation and the subsequent deactivation of downstream signaling proteins. Additionally, HM781-36B shows an excellent efficacy in a variety of EGFR- and HER-2-dependent tumor xenograft models, including erlotinib-sensitive HCC827 NSCLC cells, erlotinib-resistant NCI-H1975 NSCLC cells, HER-2 overexpressing Calu-3 NSCLC cells, NCI-N87 gastric cancer cells, SK-Ov3 ovarian cancer cells and EGFR-overexpressing A431 epidermoid carcinoma cancer cells. On the basis of these preclinical results, HM781-36B is the most potent pan-HER inhibitor, which will be advantageous for the treatment of patients with NSCLC including clinical limitation caused by acquired mutation (EGFR T790M), breast cancer and gastric cancer.

New clopidogrel napadisilate salt and its solid dispersion with improved stability and bioequivalence to the commercial clopidogrel bisulphate salt in beagle dogs.

The purpose of this study was to develop a novel clopidogrel napadisilate-loaded solid dispersion with improved stability and bioequivalence to the clopidogrel bisulphate-loaded commercial product. Clopidogrel napadisilate prepared in this study appeared as a white crystalline powder unlike clopidogrel base. However, this salt did not improve the solubility of clopidogrel, even with improved stability compared to clopidogrel bisulphate. To improve the solubility of clopidogrel napadisilate, a novel clopidogrel napadisilate-loaded solid dispersion was prepared by the spray-drying technique using HPMC and colloidal silica, and the physicochemical properties, dissolution and bioavailability in beagle dogs were evaluated compared to the clopidogrel bisulphate-loaded commercial product. The solid dispersion composed of clopidogrel napadisilate, HPMC and colloidal silica at a weight ratio of 11.069/3/3.5 improved solubility by 6.5-fold compared to clopidogrel napadisilate, even if it did not improve drug solubility compared to clopidogrel bisulphate. However, unlike clopidogrel bisulphate, this formulation improved the stability of clopidogrel. Furthermore, the clopidogrel napadisilate solid dispersion-loaded tablet showed similar dissolution to the clopidogrel bisulphate-loaded commercial product and was bioequivalent to the commercial product in beagle dogs. Thus, this clopidogrel napadisilate-loaded solid dispersion could be a promising candidate for improving the stability and bioavailability of clopidogrel.

Comparative antihypertensive activities of losartan and HM70186 in rats with hepatic dysfunction.

HM70186, a medoxomil ester of EXP3174 which is an active metabolite of angiotensin II receptor blocker losartan, was synthesized, and its antihypertensive efficacy was evaluated in rats with hepatic dysfunction. Male Wistar rats were intraperitoneally injected with 0.5 mL/kg of carbon tetrachloride to cause hepatic injury, and implanted with an osmotic minipump containing angiotensin II (0.4 mg/kg/day) to induce hypertension. After confirmation of both hepatic damage and hypertension, the rats were orally administered losartan or HM70186, and then blood pressure and heart rate were monitored for 24 h. In normal animals, angiotensin II-induced hypertension was lowered by losartan, resulting in an ED(-30 mmHg) of 9.05 mg/kg. HM70186 also immediately decreased the blood pressure in a dose-dependent manner, exhibiting an ED(-30 mmHg) of 0.89 ng/kg (10,000 times the potency observed with losartan). Moreover, HM70186 (3 ng/kg) exerted a strong antihypertensive effect even in rats with hepatic injury, while losartan (10 microg/kg) was ineffective. These results suggest that HM70186 could be a promising candidate for the treatment of hypertension accompanied by hepatic dysfunction.

Safety pharmacology of sibutramine mesylate, an anti-obesity drug.

Sibutramine mesylate is a new anti-obesity drug. It is a crystalline salt of sibutramine developed to improve the solubility of sibutramine hydrochloride. Methanesulfonic acid was used as a salt-forming acid instead of hydrochloric acid, resulting in a greatly improved solubility of 1000 mg/mL in water. Sibutramine mesylate was administered orally to ICR mice, Sprague-Dawley rats, and beagle dogs at dose levels of 1.15, 3.45, and 11.50 mg/kg to measure its effects on the central nervous system (CNS), general behaviour, cardiovascular-respiratory system and the other organ systems. Following administration of sibutramine mesylate, spontaneous locomotor activity was significantly increased from 120 min to 24 hours at 3.45 mg/kg and from 30 min to 24 hours at 11.50 mg/kg. Furthermore, there were a decrease in hexobarbital-induced sleep time, an increase in respiratory rate at 120 min, increases in intestinal transport capacity and gastric pH at 11.50 mg/kg, and decreases in gastric volume and total acidity at 3.45 and 11.50 mg/kg. However sibutramine mesylate caused no effects on general behaviour, motor coordination, body temperature, analgesia, convulsion, blood pressure, heart rate, electrocardiogram, cardiac functions of the isolated rat heart, isolated smooth muscles and renal function. Based on the above results, it was concluded that sibutramine mesylate caused effects on the spontaneous locomotor activity, hexobarbital-induced sleep time, respiration, gastrointestinal transport, and gastric secretion at a dose level of 3.45 mg/kg or greater but caused no effects on other general pharmacological reactions.